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Physiological Responses of Field Grown Lemongrass (Cymbopogon citratus) to Seasonal Changes
BY ABIDA AZIZ
M. Phil (UAF)
A thesis submitted in partial fulfillment of the requirements for the degree of
DOCTOR OF PHILOSOPHY
In
Botany
DEPARTMENT OF BOTANY FACULTY OF SCIENCES
UNIVERSITY OF AGRICULTURE FAISALABAD
PAKISTAN
2014
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The Controller of Examinations, University of Agriculture, Faisalabad
We, the Supervisory Committee, certify that the contents and form of t h e s i s
submitted by Abida Aziz, 2002-ag-1392 have been found satisfactory. The
suggestions by external examiners have been incorporated, checked and found
satisfactory. It is submitted with the recommendations for further necessary action
and final award of the Ph.D. Degree.
Supervisory Committee
----------------------------------------------
1. Chairman (Prof. Dr. Abdul Wahid)
--------------------------------------------
2. Member (Dr. Farrukh Javed)
------------------------------------------ 3. Member (Dr. Muhammad Farooq)
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Declaration
I hereby declare that contents of the thesis, “Physiological responses of field grown
Lemongrass (Cymbopogon citratus) to seasonal changes” are product of my own research and
no part has been copied from any published source (except the references, standard mathematical
/formulae/ protocols etc.). I further declare that this work has not been submitted for award of
any other diploma/degree. The university may take action if the information provided is found
inaccurate at any stage. (In case of any default, the scholar will be proceeded against as per HEC
plagiarism policy).
Abida Aziz 2002-ag-1392
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Dedicated
TO
My Father
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Acknowledgements
Thanks to my Supervisor Prof. Dr. Abdul Wahid, Chairman, Department of Botany, University
of Agriculture, Faisalabad, for his kindred spirit, his belief in me and his scholastic approach, insight,
excellence cooperation and write up guidance throughout my study. I am really obliged and thankful to
other committee members Dr. Farukh Javeed Associate professor Department of Botany and Dr.
Muhammad Farooq, Associate professor, Department of Agronomy for their valuable advice and
practical help during the conduct of this research work.
I am highly indebted to Dr. Shahzad Maqsood Ahmad Basra, Professor, Department of Crop
Physiology, University of Agriculture, Faisalabad for his utmost help, guidance, advice and facilitation
during the whole study I am grateful and feel highly obliged to Prof. Dr. Mohammad Ashfaq
Director, Institute of Agricultural and Resource Economics, University of Agriculture, Faisalabad for
moral support and encouragement during my PhD.
Special thanks to my dear friends Asima Batool, Rumana Sadiq, Durr-i-Ashian, Bushra
Munir, Shumaila Firdos, Rabia Qadri, Madiha Adeel and Sara for their best wishes for my success
in carrier. Really there are too many names to list of the multitude that have helped make this a reality,
apologies to those whose names I have neglected or omitted here, please forgive. You are not forgotten
and your aid and friendship are most appreciated.
I am indebted to my Mother and brother Wajid Aziz who instilled the desire to explore creation,
till the soil and forage for foods. Thanks to my brothers (Abid, Majid and Adil) and sisters (Musrat,
Naila, Nusrat, Farhat, Samina, Ishrat and Aliya) for your love.
Last but not the least I am highly obliged to Higher Education Commission (HEC),
Islamabad for sponsoring this research project under Indigenous Scholarship Scheme 5000.
Abida Aziz
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C O N T E N T S
Chapter Title Page 1 INTRODUCTION 1 2 REVIEW OF LITERATURE 5 3 MATERIALS AND METHODS 22 4 RESULTS AND DISCUSSION 37 5 SUMMARY 115 LITERATURE CITED 117
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ABSTRACT
A population of lemongrass (Cymbopogon citratus) was investigated in this research for
changes in metabolite profiles in the leaves of different ages i.e. penultimate (second fully
expanded leaf from the top), middle (a leaf from the central position of a tiller) and bottom (a
lowermost green leaf on a tiller) with changing seasons round the year for two consecutive years
(2010-2011). Measurements were made for some primary and secondary metabolites while the
essential oils profile of leaves was analyzed using GC-MS. The photosynthetic pigments
displayed sharp variations in the leaves of different ages with changes in the environmental
conditions. Higher chlorophyll a, b and carotenoid were recorded during summer months. The
accumulation trends of osmolytes in lemongrass showed notable seasonal variation. The
accumulation of total free amino acids, free proline and GB were higher during summer months.
The antioxidant enzymes minimized the effect of oxidative damage by scavenging H2O2 and
reuced MDA contents in extreme environmental conditions especially during summer. The
younger leaves of lemongrass exhibited much higher concentrations of vitamins as compared to
bottom leaves. The secondary metabolites i.e. alkaloid, phenolic and flavonoid contents in
lemongrass leaves were markedly increased with increased temperature. Seasonal variation
affected the nutritional profile of lemongrass substantially. The production of NDF ADF, ADL,
cellulose and starch was greater during summer. Cellulose and silica contents were higher in
bottom leaf possibly due to plant aging. Starch content was higher in penultimate leaf, which
declined with leaf age. Mineral concentration of lemongrass appears to be largely dependent on
temperature and evapotranspiration. The GC-MS analysis of lemongrass oil carried out in the
leaf samples collected during January, April, July and October revealed that out of 54
compounds analyzed, neral, citral, geranial, allerthin, caryophyllene oxide were major ones
synthesized in various sesasons.
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CHAPTER-1
INTRODUCTION
Pakistan has diverse range of climates and biodiversity in four distinct phytogeographical
regions, Irano-Turanian (45% species), Sino-Himalayan (10%), Saharo-Sindian (9.5%) and
Indian element (6%) (Shinwari, 2010). Total flora of Pakistan comprises 6000 species (Shinwari
et al., 2000) out of which about 600 are medicinally important. These medicinal plants were
explored in different areas. For example, 70% species were reported in Mansehra (Haq and
Hussain, 1993), 83 taxa used locally in Chitral (Ali and Qaiser, 2009), 114 in Baluchistan
(Goodman and Gafoor, 1992), 171 species in Kharan (Shah and Shinwari, 1996), throughout
Lahore-Islamabad motorway (Ahmad, 2007) and salt range (Ahmad and Hussain, 2008). With
this exclusive biodiversity of medicinal plants in Pakistan, there is a need for precise research on
these plants. According to National Institute of Health (NIH), Tibbi Pharmacopoeia listed 900
single drugs and 500 compound drugs obtained from medicinal plants. Some indigenous
medicinal plants of Pakistan are Artemisia sp., Ephedra sp., Bunum persicum, Emblica
officinalis, Glycyrrhiza glabra, Atropa acuminate, Commiphora wightii (Reddy et al., 2012) and
Cymbopogon citratus (Duke, 1990), which belong to various plant families i.e. Poeaceae
Asteraceae, Solanaceae, Liliaceae, Apocynaceae, Caesalpinaceae, Sapotaceae, Rutaceae,
Piperaceae, etc.
Influences of seasonal changes on ecosystems are impinging tremendously and transition
in weather conditions, temperature variation, rainfall trends and concomitant phenomena are
associated to environmental changes (Root et al., 2003). Researchers opine that chemical
constituent and endurance of medicinal plants are affected greatly by the environmental changes.
Normally plants under stress conditions can accumulate more secondary metabolites due to
inhibition of growth and diversion of fixed carbon in the biosynthesis of phenolics and glycosidic
compounds instead of photosynthesis (Gairola et al., 2010). Depending upon environmental
variations, photosynthesis fluctuate among species and decrease steadily with increasing leaf age
(Herath and Ormrod, 1979). Many studies have demonstrated that rise in temperature enhanced
the secondary metabolites production (Litvak et al., 2002), although others think that these
compounds declined (Snow et al., 2003). Many researchers believed that increase in temperature
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only enhances the streaming of chemical compounds in plants (Loreto et al., 2006) and the
effects of high temperature are still under consideration (Wahid et al., 2007).
Enzyme activities are an important index to foretell the plant responses to the changing
environments (Sen and Mukherji, 2009). Antioxidants in plants act as a line of defense against
unfavorable conditions (Lohrmann et al., 2004). Defense mechanisms of antioxidant in plants
comprise various enzymes such as induction of glutathione reductase, peroxidase, superoxide
dismutase, catalase and ascorbate peroxidase (Keles and Oncel, 2002). Lipids and fatty acids
composition in medicinal plants is also influenced by seasonal variations. Similarly fluctuations
in plant lipid levels are associated with uncertain weather conditions. Essential oils, being
complex mixtures, primarily comprise of lower classes of terpenes (Langenheim, 1994).
Silvestre et al. (1997) studied the influence of seasonal variations in the composition and
accumulation of sequestered oils. There are several reports on terpenoids accumulation in
aromatic plants, which vary with seasons seasons (Hendriks et al., 1997). Essential oils
accumulation in plant might be due to influence of stress on plant metabolism rather than
structural adaptation (Emara and Shalaby, 2011). Phenolics like flavonoids, anthocyanins and
lignin are important secondary metabolites and are well known for their role in the adaptation to
abiotic stress tolerance (Wahid and Tariq, 2008).
Medicinal plants are potential natural source of new drugs and bioactive compounds for
researchers and medicinal manufactures (Gangwar et al., 2010). Herbal remedies are as old as
human life. Even presently 80% world population of the world believes in traditional herbal
health care (Ahmad, 2007). Out of 250,000 higher plants species worldwide, more than 70,000
synthesize the phytochemicals used as drugs to treat various diseases (Farnsworth, 1988).
Modern pharmacopoeia has been using 280 chemical compounds extracted from various plant
tissues like leaves, root, stem, bark, seed, and from various plant liquid components such as sap,
latex, mucilage, gums etc. Such biologically active ingredients of plants are very vital and
important sources of new medicines that are beneficial in the treatment of several diseases (Dev,
1997). In the past, aspirin, artimesinin, atropine, reserpine digoxin, colchicine, morphine
ephedrine, pilocarpine physostigmine, quinine, quinidine, taxol, tubocurarine, vincristine, and
vinblastine have been obtained from medicinal plants (Ramawat and Goyal, 2008). These drugs
are good therapeutic agents and used to cure diabetes, mental sickness, skin infections,
tuberculosis, jaundice, hypertension and cancer. Many plants possess antibacterial, antidiabetic
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(Arcamone et al., 1980), antimicrobial activites (Perumal-Samy et al., 2006). Similarly,
Lagenaria breviflora is known for its broad spectrum medicinal uses, since many phytochemical
assays exposed the presence of saponins, phenolic acids and cucurbitacins in it (Wakimoto et al.,
2008).
Lemongrass [Cymbopogon citratus (D.C.) Stapf] belongs to the family Poaceae, and is a
commercially important aromatic C4 grass. In tropical and subtropical areas about 140 species of
Cymbopogon are found (Chase and Niles, 1962). Among these, 52 species are scattered in
Africa, 45 in the subcontinent (out of which eight species grow in Pakistan), six in Australia and
South America, four in European countries, and two in western hemisphere (Kak and Kaul,
1997). It is endemic to India and also grows in West Indies, Guatemala, Haiti and Pakistan
(Hassan et al., 2007). It can grow in diverse environments but hot and humid climate with
enough sunshine is ideal for its growth. Total age of lemongrass is 548 to 730 days, while it is
requisite to revive its cultivation after 6 to 8 years (Atal and Kapur, 1982). Lemongrass is rich in
such compounds which are greatly demanded due to their use in drugs, flavors, perfumes and
pharmaceutical industry. Aromatic drinks, decoctions or infusions from dry leaves of lemongrass
have been used as antispasmodic, stomachic, carminative and antifungal agents (Borrelli and
Izzo, 2000). Leaves extracts of lemongrass have protection against oxidative stress (Melo et al.,
2002), hypotensive, vasorelaxating and cancer chemo-prevention (Puatanachokchai et al., 2002).
Lemongrass oil has a strong lemon-like aroma and is yellow or reddish-brown in color.
Its chemical composition differs due to genetic diversity, habitat and agronomic practices.
Lemongrass essential oil consists of 41% citral, 0.3% - 4.5% neral and 0.5%-40.2% geraninal
contents (Khanuja et al., 2005; Negrelle and Gomes, 2007), which is used in the preparation of
many aromatic compounds, pharmaceuticals, vitamin A and E, and in various scents (Abello et
al., 2007). In addition to citral, lemongrass contains variety of compounds like flavonoids,
alkaloids, saponins and terpenes according to habitats (Crowford et al., 1975). Oil extracted from
lemongrass leaves have diuretic, tranquilizing and anti-inflammatory properties. It possesses
antioxidant activities, which is beneficial for human health. The lemongrass juice extraction
shows inhibition activity against carcinogenesis promotion, which is induced by cotton oil. In
oral form it is administered as anti-tumor drug for cancer and for lengthening survival time in
combination with cyclodextrin (Zheng et al., 1993). It contains high vitamin C contents, which is
used most often in the treatment of many diseases. Lemongrass oil is used for dissolving
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gallstone (Igimi et al., 1991) and shows activity against phytopathogenic fungi. In combination,
it has uses in human and domestic animal pathogens (Kisaki and Yama, 1998).
In view of all this information available in literature, the production of chemical
substances in lemongrass does not remain the same throughout the year. It depends upon
lemongrass type and climates where it grows. The aim of the current research was to evaluate the
consequences of seasonal changes on leaf physiology and its role in the synthesis of primary and
secondary metabolites and their association to ambient meteorological conditions. Following
were the specific objectives of the present project.
1. Determination of the consequence of seasonal variation in the leaf physiological
attributes and repercussion to climatic aspects on lemongrass
2. Determination of the consequence of seasonal variation in the primary and secondary
metabolites accumulation, phytochemical relations and nutritional constituents on
lemongrass over the various seasons
3. Studying the possible associations of various meteorological factors and phytochemical
constituents and their role in climatic response
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CHAPTER-2
REVIEW OF LITERATURE
2.1 Preliminary
Environmental perturbations are such changes that may induce measurable changes in the
metabolic phenomena in the living organisms. Land plants as immobile living organisms on
the earth are always subjected to environmental perturbations and thus have developed
adaptive features to cope with these adversaries. These features are of multifarious nature.
The plants capable of synthesizing excessive amounts of secondary phytochemicals, mainly
referred to as medicinal plants, show the physiological adaptive features by showing
modulations in their metabolites levels parallel with the changing climatic conditions.
Lemongrass (Cymbopogon citratus) is a C4 grass, capable of growing at supra-optimal
temperatures, and shows the synthesis of medicinally important phytochemicals. The
determination of physiological and biochemical characteristics enabling lemongrass to adjust
to prevailing environmental conditions is important in view of being a high-value plant. An
account of relevant literature on the environmental changes and their influences on the
changes in plants with particular reference to medicinal plants is given below.
2.2 Plants Interaction with Seasonal Variations
Biosynthetic and metabolic pathways in plants are greatly influenced by the seasonal
changes, wherein the synthesis of some compounds is induced while those of others are curtailed
(Salminen et al., 2001). Low seasonal rainfall during winter is associated with tannin
accumulation in the aerial parts of the plants (Wang et al., 2007). In many plants, seasonal
variations in cuticle thickness occur, which increase in spring and summer and decrease in winter
(Emara and Shalaby, 2011). Mostly higher levels of phytochemical compounds were extracted in
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autumn leaf tissues than spring samples where equal distribution of the phytochemicals was
observed (Chokoe et al., 2008). In shoots and leaves of bamboo secondary metabolites contents
were lower at the initial stages of growth in spring and summer but in winter and autumn when
metabolism is slow the greater accumulation of flavonoids, phenolics and triterpenes get started.
This is parallel to general law of accumulation and transition of secondary metabolites in plants
(Raffo et al., 2006). Moreover, biosynthesis of phenolic is peculiarly susceptible to different
abiotic and biotic stresses (Dixon and Paiva, 1995). Various other studies explain chemical
profile of aromatic compounds and secondary metabolites synthesis in different plants in various
seasons for measuring seasonal changes (Celiktas et al., 2007; Hussain et al., 2008). Wildy et al.
(2000) in Australia observed 0.01 to 13.0% in Australia, while Zafar et al. (2003) in Pakistan
noted 0.58 to 1.47% variations in oil production in various Eucalyptus species, which was
associated with soil composition and various agro-climatic regions.
Plants respond to stress by showing resistance, avoidance and acclimation. However,
under harsh environmental conditions plant cellular organization collapses, which results in
altered geographical distribution and early crop maturity (Schoffl et al., 1999; Porter, 2005;
Howarth, 2005). Changed climates result in reduction in crop production worldwide (Hall,
2001). The acclimation approaches adopted by common plants vary from morpho-anatomical
to physiochemical (Zhu, 2001). Plants make use of special acclimation mode by the
collaboration of genetic characters and stress type (Bray et al., 2000). Plants contain many
compounds of undetermined biological activity, and the quality and quantity of these compounds
can vary substantially based on the region where they are grown, the season in which they are
harvested and the genotypes cultivated (Currier et al., 2000; Ma et al., 2003). The seasonality in
environmental condition fluctuates in time and space so it is challenging to anticipate the plant
responses to the changing environments. Tolerance to such seasonal fluctuations in plants is due
to their physiological plasticity as different enzyme actions and physiological processes are
important plant response indices (Sen and Mukherji, 2007).
Mediterranean climate is characterized by the incidence of two stress periods; summer
drought and winter cold (Nahal, 1981). It has been argued that the prevailing stressful conditions
are helpful in the optimization of the growing and harvesting times in the medicinal plant species
(Lee et al., 2005). Saliminen et al. (2001) reported that seasonal and meteorological changes
bring about variations in the metabolic phenomena of the plants leading to variation in the
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contents and fluxes of the bioactive compounds. Wang et al. (2007) argued that changed pattern
of rainfall in winter season led to tannins accumulation in the aboveground part of Toona
sinensis. According to Emara and Shalaby (2011), the cuticle thickness in four Eucalyptus spp.
was strongly dependent upon seasonal changes. It increased from spring to summer but
decreased in the winter season. In Carpobrotuse dulis, during spring harvest, equal distribution
of the phytochemicals was observed within the leaf debris and the filtrate, but high
phytochemicals were found in the extracts of the samples collected during autumn season
(Chokoe et al., 2008). The phenolics biosynthesis in plants is particularly sensitive to induction
due to various biotic and abiotic stresses (Achakzai et al., 2009).
2.2.1 Plant morphology, water and osmotic relations
Physiological, morphological and biochemical responses vary from crop to crop, in
different growing seasons and in the environments in which they are grown. Seasonal
variations help the plants to acclimate and adapt to new environments and in stress
avoidance. Most useful strategies to surmount seasonal variation ranges from morpho-
anatomical to physiological and biochemical during the plant life cycle (Zhu, 2001). Not a
single factor controls the physiological processes in plants. Environmental conditions may
change biochemical and physiological processes along with low uptake and absorption of
nutrients in plants (Garg, 2003). Research on medicinal plants under field conditions suggested
that high soil water contents enhanced the growth performance of plants by increasing number of
leaves, plant height (Bargali, 1997), shoot length, leaf weight (Van Schaik et al., 1997) and leaf
width (Pandey et al., 1998). Carrot (Daucus carota) indicated unpredicted seasonal changes,
which affect the leaf surface (Brooks et al., 1996). A substantial decline in the processes like
transpiration rate, active transport, membrane accessibility and photoassimilate quality has
been reported in response to environmental stresses (Gunes et al., 2006).
Disturbances in water relations due to environmental effects are counteracted with the
accumulation of compatible solutes. Not only the nature of the osmolytes, but also their
concentrations exhibit specific seasonal pattern in plants. Compatible solutes are liable for
osmotic adjustment since they are also compatible with the cells metabolism (Wahid et al.,
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2007). In addition to their main function of osmotic adjustment, compatible solutes can either
help in stabilizing macromolecules under adverse conditions or protect against oxidative
damage (Flowers and Yeo, 1988; Wahid and Shabbir, 2005). Bhowmik and Matsui (2003)
examined changes in carbohydrate contents in changing seasons in many plants. Chinnasamy
and Bal (2003) and Schaberg et al. (2000) reported high concentrations of total soluble sugar in
red spruce seedlings during mid-winter. In another study, it was reported that high concentration
of soluble sugars act as cryoprotectants during cold acclimation (Taulavuori et al., 2001).
Glycinebetaine (GB) is a derived from glycine and acts as an excellent
osmoprotectant (Flowers and Yeo, 1988). It is naturally found in many living organisms in a
sufficient quantity (Rhodes and Hanson, 1993). It is a dipolar molecule, soluble in water at
physiological pH and acts as neutral molecule (Sakamoto and Murata, 2002). Such dynamic
features of glycinebetaine are helpful in stabilization of three dimensional structures of
protein and enzymes as a result of their hydrophilic and hydrophobic domains interaction
(Sakamoto and Murata, 2002). GB plays an important role against stress defense in many
plants (Ashraf and Foolad, 2007; Chen and Murata, 2008; Wang et al., 2010). Boscaiu et al.
(2011) reported changes in GB contents due to seasonal variation, which were higher in
extreme dry summer conditions.
Proline acts as a compatible solute (not toxic under high concentration) and plays
vital role in osmotic adjustment (Yamada et al., 2005), removes the harmful effect of
reactive oxygen species, helps to maintain the three dimensional structure of proteins and
also protects chloroplast and mitochondrion under fatal environmental situation when
accumulated in free form (Ashraf and Foolad, 2007; Wahid et al., 2007). When the stress is
alleviated, it is converted into reducing agents, which plays important role in oxidative
phosphorylation (Hare et al., 1998). Proline also plays role in the induction of stress
responsive genes (Johari-pireivatlou et al., 2010). Accumulation of proline as a result of
abiotic stresses occurs in many species of plant families, but its quantity is based on species
type and severity of stress (Wahid et al., 2007; Lotfi et al., 2010). In Camphorosma annua
and Limonium gmelini leaves, proline levels were higher in early spring which decreased
subsequently (Murakeozy et al., 2003). In the autumn, a decrease in proline content occurred in
L. anatolicum and L. lilacinum, while an increase in L. iconicum (Furtana et al., 2013).
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2.2.2 Oxidative stress and antioxidant activities
The most deteriorating factor under stress at molecular level is overproduction of reactive
oxygen species (Xu et al., 2006). During the partial reduction of light-induced respiration, ROS
reduced oxygen in the atmosphere, which affects photosynthesis and respiration (Xu et al.,
2006). They include superoxide -O2-, OH- and H2O2 (Mittler, 2002). Reactions of active oxygen,
followed by the peroxidation products (Scandalios, 1993) eventually lead to imbalance in the
normal metabolism. Evidences show that membrane damage is due to the ROS generation,
which results in lipid peroxidation of plasmalemma or intracellular organelles (Stewart and
Bewley, 1980; Farooq et al., 2009). Similarly, denaturation of proteins, nucleic acid and
hormonal damage, ultimately distresses the plant homeostasis (Mittler, 2002). Xu et al. (2006)
showed that heat resistance in different turfgrass species was based on the increased membrane
thermostability.
Hydrogen peroxide (H2O2) is most dominant and stable ROS with ability to regulate the
basic acclimatory, developmental and defensive processes in plants (Ślesak et al., 2007). It is
fatal for cell when it reacts with lipids, nucleic acid and proteins (Mittler, 2002). In leaves
photochemical quenching of excess light by photorespiration is involved in H2O2 production. In
leaves of wheat, H2O2 production during photorespiration is 70% of the total H2O2 produced and
it gradually increased with a decrease in CO2 concentration. Production of photorespiratory H2O2
escalates in high light and low CO2 concentrations (Noctor et al., 2002). H2O2 and other ROS are
ineluctably produced with the electron leakage to O2 during aerobic metabolic processes like
photosynthesis and respiration. Higher H2O2 concentrations were recorded for Aesculus glabra,
Plantago major, Glechoma hederacea and Viola soraria in the warm days while there were no
significant change in H2O2 production in Quercus macrocarpa (Cheeseman, 2006).
In order to reduce the intensity of damage or to reduce ROS generation under stress
condition, plants widely adapt and develop defense strategies. These strategies include the
removal of ROS through enzymatic and non-enzymatic defenses, which are important to cope
with ROS (Allen, 1995). In harsh environmental conditions, the ROS attack on biologically
important molecules, interfere with cell metabolism (Liu and Huang, 2000), cause ionic
imbalance (Taiz and Zeiger, 2010), self-catalytic membrane lipid peroxidation and loss of
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membrane integrity. Hence, MDA concentration and relative electrolyte leakage is important
tool to evaluate heat damage in a variety of crops as trufgrass (Xu et al., 2006) and in
Phalaenopsis (Ali et al., 2005).
During environmental stress, superoxide dismutase (SOD) provides protection from
activated oxygen (Bowler et al., 1992; Scandalios, 1993; Smirnoff, 1993; Foyer et al., 1997). In
Retama raetam and Atriplex halimus, the SOD activity was much higher under harsh desert sites
than in non-desert areas (Streb et al., 1997). Peroxidase (POD) comprises of multifunctional
enzymes family that regulates ROS production (Passardi et al., 2005) and stimulates the
breakdown of H2O2 by utilizing different substrates (phenolic, lignin, auxins) within plant cells
(Hiraga et al., 2001; De Gara 2004; Passardi et al., 2005). POD plays important protective role in
plants against different abiotic stresses. CAT is a mono-functional protein which is especially
localized in peroxisomes (Engel et al., 2006) and has ability to scavenge the huge amounts of
H2O2 into water and O2 (Sofo et al., 2004).
Medicinal plants are potential source of antioxidant compounds, it is necessary to study
their constituent and mode of action, and thus validate their utilization. According to Korotkova
et al. (2003), flavonoids, phenolic acids, tannins are many of the compounds that have
antioxidant activity and frequently found in medicinal plants. Antioxidants can be classified as
enzymatic and non-enzymatic present in almost all plants (Mittler, 2002), and help in ROS
detoxification; the enzymatic ones including SOD, POD and CAT while non-enzymatic ones
include AsA, carotenoide, tcopherols, flavonoids, and phenolic compounds (Ashraf et al., 2010).
Swanberg and Verhoeven (2002) found that yew leaves had capability to respond to
changing seasonal variations with the induction of antioxidants. During winter in leaves of
different plants antioxidant systems were increased (Logan et al., 1998). According to Vuleta et
al. (2010), greater activity of POD in leaves of Iris pumila was observed during spring and
summer seasons. POD activity was declined during spring through winter while CAT activity
increased before rainfall and then decreased (Kashefi et al., 2010). In dry conditions, these
antioxidant enzyme activities increased in different plant species, amongst the species and within
a species (Csiszar et al., 2007; Nikolaeva et al., 2010). Extended stress situations may lead to a
boost in antioxidant ability in different parts of plants (Pastori et al., 2000).
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2.2.3 Reducing powers and vitamins
The reducing power is a measure of electron transfer capacity of the compounds, which
acts as a powerful tool of its antioxidant activity. Raja et al. (2012) reported that nut extract
showed high reducing power activity. Shimada et al. (1992) reported that in mushroom high
reducing power activity was due to its electron donating ability. Huang (2000) reported that the
methanolic extract of Antrodia camphorata exhibited greater reducing power activity than
Brazilian mushrooms.
Vitamins are exclusive sources of reducing powers in plants. Ascorbic acid (vitamin C;
AsA) is evidenced for its role as antioxidant in plants, acts as cofactor, involved in cell
signaling, regulator of cell wall biosynthesis and provides photoprotection to the cells and
tissues (Vaidyanathan et al., 2003). AsA reduced the lipid peroxidation (Sairam et al., 2000)
and decreased the harmful effect of stress and increased stress resistance (Iriti and Faoro,
2007). AsA showed sensitivity towards air, water and temperature, acted as an important
antioxidant source and provided protection against free radicals (Demmig-Adams and Adams
2002). In greenhouse grown tomato, AsA content increased as the temperature increased (Liptay
et al., 1986; Vanderslice et al., 1990).
Riboflavin, also called a growth vitamin, is formed in the plastids (Sandoval et al., 2008). It
is modified to flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) in the cytosol
(Sinclair et al., 2000). Riboflavin assists in regulating the cellular redox state and thus controls the
plant antioxidant defense system (Sandoval et al., 2008). Niacin is yet another vitamin of great
physiological significance. Temperature, humidity, photoperiod and rainfall of the growing season
affect the niacin biosynthesis. Niacin increased in plants with increase in the day length. In maize,
an increase in niacin content with increase in temperature was observed (Mahmood et al., 2012).
2.2.4 Secondary metabolites synthesis
Phytochemicals extracted from plants are classified as primary or secondary
metabolites. Sugars, purines, proteins, amino acids and pyrimidines of nucleic acids and
chlorophylls that are essential for growth are categorize as primary metabolite (PM) while
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plant secondary metabolites (SM) included alkaloids, cyanogenic glycosides, phenolics,
terpenes, saponins, glucosinolates, anthraquinones and polyacetylenes (Wink, 2003). Secondary
metabolites are found in all higher plants and protect them attack of bacteria, fungi and viruses
and herbivore. Limonene (monoterpene) exhibited insecticidal and preventative role in plants
while carvoneis is a good sprouting inhibitors (Aflatuni, 2003).
Plants are well known dynamic organisms, therefore it is predicted that concentrations of
secondary metabolites in plants change within a plant both by spatially and temporally over
successive years (Brooks and Feeny, 2004). However, changes in levels of plant secondary
metabolites interfere with the effectiveness of phytomedicines (Gurib-Fakim, 2006). Scientific
studies revealed that changes in active compounds as a consequence of biotic and abiotic factors
can help to optimize the harvest time of medicinal raw material or to obtain the largest amounts
of active compounds (Lee et al., 2005). Seasonal changes in the composition and level of active
compounds of sequestered oils have been observed (Silvestre et al., 1997). Similarly in aromatic
plants the levels of active compounds such as terpenoids change due to internal biological clock
and also around the seasons (Hendriks et al., 1997).
Phenolic compounds are very essential for plants and play important role for tolerance
against different stresses (Sgherri et al., 2004; Wahid and Tariq, 2008). Phenolics compound
are good electron carriers, involved in energy transfer with the help of their own electronic
configuration and, in this way, enhance the absorbance of light in visible and UV range
(Cockell, 1997). They also play active role in programmed cell death of plants (Beckman,
2000). In Polygonum acre concentrations of total phenolics remain the same (approximately
1.57%) around the year (Lima et al., 2010). The spatial, seasonal and inter-specific variation
trend of concentrations of total phenolics in the Apocynum venetum and Poacynum
hendersonii were observed maximum in April to July (Ma et al., 2003) and then an obvious
decline after July. Concentrations of carotenoids were changed due to seasonal variation
(Lamare and Hoffman, 2004). In leaves of Carpobrotus edulis, the phytochemicals showed a
similar ratio in spring season but it increased in autumn. However, antioxidants showed
different trend; a high activity was detected in autumn (Chokoe et al., 2008).
Flavonoids belong to a large group of phenolic secondary metabolites; the major ones
are flavanols, flavones, anthocyanins and isoflavonoids. In nature, all plants contain these
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compounds present mainly as glycoside structures. They provide protection by absorbing
ultraviolet (UV) radiations (Harborne and Williams, 2000) and their production was
restricted to UV exposed tissues (Chalker-Scott, 1999; Winkel-Shirley, 2002). In low
temperature months leaf flavonoid content production increases (Christie et al., 1994).
Luengas-Caicedo et al. (2007) reported concentrations of flavonoid in Cecropia glaziovi
young and mature leaves did not significantly differ in the same season whereas both young
and mature leaves exhibited higher concentration in rainy season as compared to dry period.
Anthocyanins are a subclass of flavonoids and highly soluble in water. They may work
with other antioxidants to protect vegetative tissues against photo-oxidative stress (Chalker-
Scott, 1999; Grace, 2005). They are good photo-protector having ability to absorb high energy
light, which harms the Chlorophyll b (Hughes and Smith 2007). They also act as antioxidants for
by scavenging the free radicals, which cause structural and functional damage to thylakoid
membranes (Tattini et al., 2005). Edreva et al. (2007) suggested that in cotton much
physiological chaos due to stress was reduced by antioxidant capacity of anthocyanins.
Wahid (2007) has reported the exclusive role of anthocyanins in heat tolerance of sugarcane
sprouts. In leaves of Iris pumila anthocyanin contents depended on course of growth season and
were higher when oxidative load on leaf was at peak in hot season (Vuleta et al., 2010).
Tannins are common polyphenols in all plants and synthesized approximately equal to 50%
of the dry weight of leaf. Tannins are widely used in industries and also provide protection against
herbivores. Wahid and Tariq (2008) observed that plants grown in acidic soil with low mineral
nutrients show high accumulation of tannin. In Polygonum acre the contents of condensed tannins
(CT) varied significantly; the amount increased from April to August, and declined from August to
November (Lima et al., 2010). In start of hot season a higher content of hydrolysable tannins (HT)
has been observed in Quercus robur (Tikkanen and Julkunen-Tiitto, 2003) and birch tree (Riipi et
al., 2002). Vaithiyanathan and Singh (1989) observed that different species of tall plant leaves
responded differently to seasonal variation, with respect to tannin concentrations. Some species
showed increased tannins in hot months while the others in winter. Gupta et al. (1992) reported an
increase in tannin concentration in hot season. Salaj and Karmutak (1995) found higher tannin
concentration in cooler months.
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Studied have been made to explore the medicinal value of alkaloids in many plants
(Robber and Tyler, 1996). More than three thousand alkaloids have been recognized in 4000
plant species and most of them were present in herbaceous dicots and some in fungi. The
alkaloids level was higher in leaves in hot weather than winter or in monsoon in Sesbania
bispinosa (Momin and Kadam, 2011). In Atropa belladonna, the accumulation of alkaloid was
two times higher in July as compared to June (Sporer et al., 1993). Their concentration in roots
of Macleaya microcarpa varied significantly during vegetative growth and was negligible during
the month of June and July (Pencikova et al., 2011).
The temperature, photoperiod, relative humidity, irradiance (environmental factors) and
management practices effect the secondary metabolites composition. The nature of essential oils
in different seasons varied from winter to during summer seasons. Best essential oils percentages
were estimated in E. camaldulensis and E. cinerea, which ranged from 2.5 to 1.95%, respectively
in hot months. This might be due to the physical and chemical stresses on the plants especially
during summer drought (Emara and Shalaby, 2011). The incidence of stress resulted in
production of different protective compounds especially terpenoid in plants (Samuelsson, 1999).
2.2.5 Leaf pigments
Leaf chlorophyll is an important biochemical attribute of plants. It is related to
seasonal water availability and nutritional level of plants and reflects plant health (Epstein
and Bloom, 2005). Normally, light-harvesting ability of C3 plants is inflated as compare to
the C4 plant species (Salvucci and Crafts-Brandner, 2004). Harsh seasonal changes have a
great influence on PS-II leading to inhibition of photochemical efficiency (Barber and
Andersson, 1991). A significant decrease in phytochemical efficiency of PS-II during the month
of October was due to decline of chlorophyll a content (Polle et al., 1999). Different accession
of sugarcane exhibited an increase in chlorophyll a/b ratio and decrease in “chlorophyll b”
under stress (Wahid and Ghazanfar, 2006).
Carotenoids are very important accessory photosynthetic pigments and act as receiver of
extra light and act as cellular protector during different abiotic and biotic stress periods (Iriti and
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Faoro, 2007; Wahid, 2007). Carotenoids not only provides a mechanism of photoprotection but
also act as antioxidants under different seasonal conditions (Krinsky, 1998). They magnify the
stability of membrane by lowering peroxidation of membrane lipids in stress conditions (Havaux,
1998). Carotenoids are hydrophobic in nature and defend lipophilic membrane surfaces of
macromolecules (Vasconsuelo and Boland, 2007). This property of carotenoids is due to their
efficiency to scavenge reactive oxygen species (Rodrigues et al., 2003).
Seasonal variations affected the carotenoid concentration in Piceasit chensis in winter
period (Lewandowska and Jarvis, 1977). Tomato plant when grown in the rainy season exhibited
lower carotenoid content, which were generally liable to oxidative damage due to low
scavenging and detoxification activity of the ROS (Sen and Mukherji, 2000). Ollykainen (1969)
found that the carotenoid content increased during the vegetative growth periods during summer
but decreased during the winter. Under high light intensity, accumulation of carotenoid increased
lipid droplets (Ben-Amotz et al., 1982). While studying seasonal variations in carotenoid
concentration, Raffo et al. (2006) reported that carotenoid did not show specific seasonal
patterns in relation to solar radiation and average temperature.
2.2.6 Nutrient concentrations and nutritional relations
Various environmental conditions affect the availability of soil nutrients (Fageria et
al., 2003). Normally a decline in the absorption of plant nutrients results in a reduction in
transpiration rate, which reduces the membrane accessibility, active transport (Baligar et al.,
2001; Ganesh et al., 2008) and reduced tissue nutrient contents (McWilliams, 2003).
Different macro- and micronutrients like potassium, calcium, nitrogen, phosphorus and sulfur
play a prominent role in plant growth, osmotic adjustment, enzyme activation (respiration
and photosynthesis), stomatal opening/closing, phloem transport and so many other
metabolic processes (Hepler, 2005; Lopez et al., 2008).
Mineral composition of plants depends upon various environmental factors such as
seasonal changes, geography, climate, soil minerals and plant capacity to uptake soil
nutrients (Ganskopp and Bohnert, 2003; Khan et al., 2006).). Huston et al. (1981) found
lower values of calcium and phosphorus (1.4 and 1.0 g/kg, respectively) in early summer in
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Panicum hallii. Humphreys (1984) reported that, K+ concentration in tropical grass ranged
up to 12 g/kg. Abiotic stresses induced a change in Ca2+ concentration which brought in
different physiological responses (Reddy and Reddy, 2004). Mirza et al. (2004) found
significant changes in trace elements including copper (Cu), zinc (Zn), manganese (Mn), iron
(Fe), cobalt (Co), nickel (Ni), cadmium (Cd), lead (Pb), chromium (Cr), silver (Ag), sodium
(Na) and potassium (K) in indigenous medicinal diuretic plants.
Plants absorb nitrogen in the form of nitrate (NO3-), which is influenced by various
environmental factors like drought (Younis et al., 1965; Huffaker et al., 1970), light (Schuphan et
al., 1967; Cantliffe, 1972a, b; Scaife and Schloemer, 1994), temperature (Cantliffe, 1972c) and soil
type (Raikova and Petkov, 1996). P, a macronutrient for plants, is a key component of nucleic
acids, phospholipids, ATP and regularizes metabolic pathways (Theodorou and Plaxton, 1993). In
Pennisetum pedicellatum the P level in wet season was much higher than in the dry season (Ziblim,
2012). K+ is important plant cation, and its concentrations ranges from 50–150 mM in plants
(Leigh and Wyn Jones, 1984). High K+ concentration can temper intense environmental condition
like cold, late season rains and heat waves through stomatal movement and osmotic adjustment
(Marschner, 1995). In Ceratonia siliqua leaf K+ contents decreased during winter and increased
from spring until autumn (Correia and Martins-Loucao, 1997). On the other hand in clover
monthly fluctuations were observed; high levels were observed in late winters and early spring
while the lowest levels were recorded during early summer (Metson, 1978). Seasonal variation had
a significant impact on the nutritive value of plants (Snyman, 2006). In plants production of
different nutritive constituents like nutrient detergent fiber (NDF) and acid-detergent fiber (ADF),
acid detergent lignin (ADL), silica, cellulose, starch and protein content, showed changes within
years and in various plant growth phases within a growing season (Ball et al., 2001).
2.2.7 Essential oils composition
Essential oils are a mixture of fragrant compounds including terpenoids, aldehydes
(geranial, citronellal), alcohols (geraniol, citronellol, nerol), esters (linalyl acetate, citronellyl
acetate, isobornyl acetate) (Burt, 2004), which accumulate in different organ like leaves, barks,
woods, roots, rhizomes, fruits, and seeds. Essential oils are synthesized and accumulated in
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specialized organs (trichomes, secretory cavities or canals) located near surfaces (Bruneton,
1995). So any change in seasons has direct influence on essential oil concentration and
component (Silvestre et al., 1997). Environmental factors (temperature, irradiance, relative
humidity, photoperiod etc.) and cultivation practices have great effect on the composition and
concentration of essential oils. Many authors have reported seasonal variation in essential oil of
aromatic plants (Hendriks et al., 1997). Such seasonal changes in essential oil component were
observed in many plants like Santolina rosmarinifolia (Pala-Paul et al., 2001), Thymus vulgaris
(McGimpsey et al., 2006) and Abies sachalinensis (Satou et al., 2009).
Many environmental, physiological and genetic factors influenced the chemical profile
and make up of essential oils in plants (Angioni et al., 2006). Microenvironments like sun and
shade also affect the oil yield from month to month in growing season (Juliani et al., 2002).
Moreover, this variation in oil depends on precipitation and temperature (Pala-Paul et al., 2001).
Atti-Santos et al. (2004) observed higher essential oil concentration in spring in Thymus
vulgaris. In leaves of Pilocarpus microphyllus higher percentage of essential oil was observed in
the rainy season (Taveira et al., 2003). The α-pinene content in leaves of Abies sachalinensis oil
was higher in April to June, while a steady decline observed in November to December and this
increase was attributed to high precipitation, temperature, sunshine (Satou et al., 2009).
In Eucalyptus the contents of essential oils in different seasons varied from 0.05% during
winter to 2.5% during summer season. Best essential oils percentages were estimated in E.
camaldulensis and E. cinerea (2.5 and 1.95% respectively) in the summer season (Emara and
Shalaby, 2011). Similarly the level of these components reached its maximum in all studied plant
species during the summer season and had the lowest value during winter, and this may be due to
the physical and chemical stress on plant especially during summer drought. This stress led to
plant secretion to different defense components called secondary metabolites, as protecting
agents, especially terpenoid compounds (Samuelsson, 1999). With regard to significance
estimates, more consistent trends in percentages variation in Eucalyptus spp. during different
seasons were present for essential oils than for lipids, and this further confirm that the former are
mainly due to the effect of environmental condition on plant synthesis. Wildy et al. (2000)
investigated four promising Eucalyptus species and reported 0.01 to 13.0% oil production from
Western Australia from six locations. Zafar et al. (2003) also reported 0.58 to 1.47% oil potential
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for the different Eucalyptus species. These variations might be attributed to different agro-
climatic regions and soil composition in the districts of Punjab.
2.3 Medicinal Plants
Since ancient times medicinal plants have been used in almost all cultures as a source of
medicine. Many archaeological records have revealed that medicinal plants were used by
humans in Paleolithic period, approximately 60,000 years ago (Sumner, 2000). These plants
occupied different habit and habitat covering wild altitudinal range, desert and seacoast including
aquatic and lower plants like algae (Solecki, 1975). Only 10% information about herbal remedy
of medicinal plant is available. Thus a lot of research is required to explore the roles of leaves,
roots, stem, flowers and whole plants, which are commonly used in phytoextraction (Shanker,
1998). A huge range of chemicals such as flavonoids, alkaloids, phenolic and tannins are present
in medicinal plants; Almost 12,000 active compounds have been extracted from medicinal plants
until now, which is approximately less than 10% of the total compounds (Lai and Roy, 2004).
All parts of Aegle marmelosis i.e., leaves, roots, fruits and barks have medicinal
importance (Duke and Jo, 2002; Das et al., 2006). Adesanwo et al. (2009) observed that leaves
of Melaleuca bracteata (family Myrtaceae), is also famous for curing wounds and skin disorders.
It stimulates glandular secretions and reduces vein congestion. Numerous medicinal herbs and
spices, which are part of our everyday food intake help keep away food borne pathogens
(Tapsell et al., 2006). Medicinal value of myrrh and opium were reported five thousands year
ago (Sumner, 2000). Ayurveda medicines, combination of herbs, including turmeric has been
used perhaps as early as 1900 BC in subcontinent. Ibn Sina described medicinal healing system
in his Medical Encyclopedia “The Canon of Medicine”. In his book Pen Tsao, Shen Nung
described 365 plants including hemp, chaulmoogra and ephedra for treatments of various
ailments (Sumner, 2000). Medicinal plants can be demarcated in two important sectors: (a)
modern medicines that utilize about 30 to 35 medicinal plants; (b) traditional medicines that are
organized and codified with written treatise texts such as Ayurveda, Siddha, Unani, Amchi, and
Tibetan systems of medicine that used about 1,200 to 2,000 medicinal plant species (Dahanukar
et al., 2000; Rajasekharan and Ganeshan, 2002).
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Conventional and traditional medicines are used since geneses in domestic recipes and also
as source of minerals (Meskin, 2002). Ayurveda is one the world’s oldest medicinal system. In
China, about 5000 traditional remedies have been reported, which make up one fifth of the whole
Chinese pharmaceutical market (Lee, 2000). Just like other parts of the world, local communities
in Pakistan also use medicinal plants to cure various diseases (Morgan, 2002). In traditional health
care system, drug production methods involved chopping and boiling of particular bark, roots and
leaf parts. For stress relief tea decoction orally or sometimes mixed with milk and sugar was used.
Elixirs wine and herbal extracts of alcohol, usually with 12-38% alcohol content can also be used.
Saba et al. (2009) revealed that Lagenaria breviflora fruit ethanolic extract has toxicological
effects and commonly used in the West African folk medicine. In Ayurvedic system, Hemidesmus
indicus is regarded good for rheumatism, leprosy, impotence, skin infections, anti-ulcerogenic
(Jegadeesan, 2009), anti-thrombotic and anti-oxidation activity (Mary et al., 2003). L. breviflora is
used in a wide range of gastrointestinal problems and measles in West Africa (Tomori et al., 2007).
About 120 phytochemicals have been derived from plants, which are being used in
medicines (Fabricant and Farnsworth, 2001). Among these morphine, deserpidine, camphor,
vincristine atropine, vinblastine and yohimbine are derived from higher plants. Many new drugs in
era of 1971-1995 such as phyllodulcin, emetine, teniposide, pinitol, lectinan, ouabain and
ginkgolides rose all over the world. These plants derived drugs had eminent contribution in modern
therapies for example: morphine isolated from the Papaver somniferum was a famous analgesic.
Vinblastine extracted from the Catharanthus rosesus is good in curing choriocarcinoma, Hodgkins,
neck cancer (Farnsworth et al., 1967), acute cervical cancer and lymphocytic leukemia
(Farnsworth and Bingel, 1977).
Lack of herbal medicines safety and efficacy standards is a big issue now-a-days
(Ramawat and Goyal, 2008). In 1991, the World Health Organization (WHO) developed
guidelines in the 6th International Conference in Ottawa for the evaluation of medicinal herbs.
Same year, the Drug Regulatory Authorities approved the same guidelines. It has been estimated
that 80% of the population in Asian and African countries used medicinal plants for health care
having income less than $2 a day because pharmaceuticals are very expensive (Da Silva et al.,
2002). In fact, WHO reported that in United States, about 25% of the drugs are derived from
medicinal plants. In European countries, the sale of medicinal plants is varied and estimated as
the $1.5 billion (Germany), $1.6 billion (France), $ 0.6 (Italy) and 1.5 billion (Japan) in 1996.
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Currently, the world market of medicinal plants is increasing and observed only in the United
States to US$ 250 billion (Brower et al., 1998).
2.4 Lemongrass: Origin and Nature
Lemongrass is a famous Graminaceous perennial plant. Morphologically, the plant grows
in thick cluster up to 2 meters in diameter, 1.2 to 1.8 meter in height with long narrow leaves up
to one meter long and mostly cultivated in tropic and subtropics regions (Simon et al., 1984). It
has very pleasant taste, like lemon juice with a hint of ginger flavor. Lemongrass is marvelous
medicinal plant with sweet flavor. East Indian lemongrass (Cymbopogon flexuosus) is endemic
to India, Burma, Sri Lanka and Thailand. Its leaves are excessively consumed in Brazilian
traditional medicine as infusions due to its analgesic, anti-inflammatory, anti-spasmodic,
sedative, antipyretic properties and as diuretic (Blanco et al., 2009).
Essential oils in lemongrass are of tremendous economic importance as good food
preservative, tonic and flavoring ingredient in cosmetics and perfumes (Ganjewala and Luthra,
2007). Many pharmacologic activities of essential oil have been illustrated, like anticonvulsant
and anxiolytic activities (Silva et al., 2010) and antibacterial, antifungal and anti protozoal
properties (Oliveira et al., 2009). Lemongrass essential oil is recognized by its high citral content
(>45%) (Khanuja et al., 2005) which is raw material for the production of vitamin A, ionone and
beta carotene (Paviani et al., 2006). Citral is a mixture of trans-citral (geranial) and cis-citral
(neral) (Negrelle and Gomes, 2007). Many scientist reported antifungal properties of lemon grass
(Fiori et al., 2000; Pedroso et al., 2006). Caccioni et al. (1998) and Wang et al. (2007) also
reported lemongrass juice to possess antifungal ability, which assists the idea that lemongrass
and its juice are better in treating oral thrash than 0.5% aqueous solution of gentian violet in
HIV/AIDS patients (Wright et al., 2009).
2.4.1 Response of lemongrass to seasonal variation
Lemongrass has C4 photosynthesis with low CO2 compensation values like other C4
grasses. Photosynthetic rate in lemongrass leaves was significantly affected by leaf age and
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photosynthetic activity declined with an increment in leaf age, and from the apex to the bottom
of the leaf blade (Maffei et al., 1988). Lemongrass shows optimum growth at 27/21°C day/night
temperatures respectively. It required 8 h photoperiod, which is appropriate for maximum
photosynthetic activity. Herath and Ormrod (1979) measured highest photosynthetic activity in
newly developed fully expanded leaves in both strains of C. flexuosus and C. citrates
(lemongrass). Keeping in view the ecological conditions, lemongrass cultivation has very high
values of accumulation, even when the summer temperature remains near or below 18-21/30-
35°C. The fastidious effectiveness of the carboxylating reserves also in accordance with higher
chlorophyll a/b ratio (5.49) showed a plausible enrichment of the light-harvesting reaction
centers (Maffei et al., 1988).
In Thymus vulgaris oil composition and yield both were critical to the time of harvest
(Badi et al., 2004). Although lemongrass (Cymbopogon citratus) is mostly being examined for
its phytochemical components, insufficient literature is published for its growth variation in
different seasons. In lemongrass tiller production is sensitive to seasonal variation and its
production varies with the level of irrigation and season of harvest (Singh at al., 2000).
Lemongrass oil yield affected when grown in different season. Generally, lemongrass yielded
0.2% and 0.35% essential oil in rainy and dry seasons respectively. In Pinienta racemosa
essential oil content varied between 1.32 to 3.40% and this yield decreased with an increase in
rainfall (Childers et al., 2006). Lemongrass oil showed great variations in chemical composition
in different seasons (Kulkarni et al., 1997). Sarma et al. (2011) recorded higher oil contents in
lemongrass leaves at the onset of monsoon as compare to post monsoon period.
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CHAPTER-3
MATERIALS AND METHODS
3.1 Experimental Plan
The current work was carried out to appraise the variations in physiological and
biochemical characteristics of leaves of lemongrass (Cymbopogon citratus) in spring, summer
winter and autumn seasons. Experimental field was selected from New Botanical Garden,
University of Agriculture, and Faisalabad. Propagules of a selected lemongrass population were
transplanted in plots measuring 2 × 2 m in the month of October, 2010 and 2011. In both the
years, after seed germination, 100 plants were retained in each plot with a plant to plant distance
of 20 cm (Fig. 3.1). The experimental design was randomized complete block design (RCBD).
All the plots were watered using irrigation water at fortnightly intervals during summer season
with at three weeks interval during winter season. There were three blocks; each block with three
replicates. From each replicates 500 g of the leaves were taken from three positions i.e.,
penultimate, middle and bottom (Fig. 3.2) on 10th of each month during the years 2010 and 2011.
Half of the samples of the leaves of three ages were oven dried, while the other half samples
were frozen for fresh analysis at -50ºC. Physiological parameters were analyzed using frozen
fresh material. Nutrient composition and Oil composition, Na, K and Ca, nitrate and phosphate
were estimated from dry material.
3.2 Meteorological Data
Meteorological data was obtained from the Weather Observatory of the Department of
Crop Physiology, University of Agriculture Faisalabad. These data were used to draw the
correlations of various physiological and biochemical attributes with the environmental
conditions prevailing during the course of experiment.
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Fig. 3.1: Field sown lemongrass population at early stage
Fig. 3.2: Leaves of various ages/positions used to perform the analytical work
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3.3 Analytical Procedures
The determinations were made for oxidative stress and antioxidants characteristics,
osmoprotectants, some important secondary metabolites, vitamins photosynthetic pigments,
nutrients, nutritional and fiber characteristics during both the years.
3.3.1 Determination of oxidative stress parameters
a. Hydrogen peroxide estimation
Hydrogen peroxide (H2O2) contents were determined by using method of Velikova et al.
(2000). Fresh leaf tissues (0.1 g) were extracted in 0.1% trichloroacetic acid (TCA) in ice bath.
Then this extract was centrifuged at 12,000 × g and supernatant was collected. A 0.5 mL of 10
mM potassium phosphate buffer (pH 7.0) and 1 mL of 1 M potassium iodide was added to 0.5
mL of supernatant. This mixture was vortexed and absorbance was noted at 390 nm.
b. Malondialdehyde (MDA) Determination
The (MDA) was measured with the method given by Heath and Packer (1968). For the
determination of MDA, 0.1 g of fresh leaf tissue was extracted in 1 mL of 5% (w/v) TCA and
centrifuged at 12000 × g and supernatant was taken. To 1 mL of supernatant equal volume of
thiobarbarturic acid [(TBA) 0.5% in 20% (w/v) in TCA] was added followed by heating at
100oC. Reaction mixture was centrifuged at 7500 × g for 5 min. Absorbance was noted at 532
and 600 nm. MDA was calculated using extinction coefficient of 155 mmol mL-1 as:
MDA equivalents (nmol mL-1) = [(A532-A600) / 155000] × 106
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3.3.2 Determination of enzymatic antioxidants
a. Enzyme extraction
Fresh plant material (0.1 g) was extracted in 0.9 mL of 50 mM cooled phosphate buffer
(pH 7.8) in the presence of protease inhibitors (Cocktail protease inhibitors, Sigma, USA). This
extract was centrifuged at 15000 × g at 4oC and supernatant was collected and stored at -30oC for
enzyme assay.
Prior to the determination of the activities of enzymatic antioxidants, the proteins
concentration of the extract was determined with dye-binding method of Bradford (1976), as
described below (section 3.3.7 g). The activities of the enzymes were expressed as U/g
protein/min.
b. Superoxide dismutase (SOD) activity
Activity of SOD was assayed by following the method of Giannopolitis and Ries (1977).
Enzyme extract (50 μL) from the above was taken and mixed with 50 mM phosphate buffer (pH
7.8). To this extract, 50 μM NBT (Nitro blue tetrazolium dissolved in ethanol) was added
followed by 1.3 μM riboflavin and 13 mM methionine and 75 mM EDTA were added (total
reaction solution including enzyme extract 1 mL) and this mixture was kept in dark chamber
coated with aluminum foil. Then the reaction mixture was illuminated under fluorescent lamps of
30 (W FPL30EX-D) for 5 min. The SOD activity was estimated by monitoring the inhibition of
photochemical reduction of NBT at 560 nm by using a UV-visible spectrophotometer (UV-
4000, ORI). One unit of SOD was defined as the amount of enzyme required to cause 50%
inhibition of the rate of NBT reduction at 560 nm. Blank was used for comparison.
c. Catalase (CAT) and peroxidase (POD) activities
The activities of CAT and POD were estimated by following the method of Chance and
Maehly (1955) with some modification. Enzyme extract (0.1 mL) was taken and mixed with 50
mM phosphate buffer (pH 7.8) and 5.9 m M H2O2 and diluted up to 3 mL. The absorbance was
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noted at 240 nm after each 20 sec. CAT activity was expressed in units as μM of H2O2
decomposed per min. One unit was defined as an absorbance change of 0.01 units per min.
For POD activity, reaction solution containing 50 mM phosphate buffer (pH 7.0), 20 mM
guaiacol, 40 mM H2O2.was taken. Enzyme extract (0.1 mL) was added and change in the
absorbance was noted at 470 nm after each 20 sec. One unit POD activity was defined as the
change of 0.01 absorbance unit per min per mg of protein.
3.3.3 Osmoprotectants
a. Free proline determination
Free proline was determined by using the protocol of Bates et al. (1973). Fresh plant
material was extracted in 3% aqueous sulphosalicylic acid. One mL of the extract was dissolved
with 1 mL of glacial acetic acid and 1 mL of acid-ninhydrin (1.25 g ninhydrin in 30 mL glacial
acetic acid) in a test tube and was go through vortex. This mixture was heated at 100oC in a water
bath. After heating it was placed in an ice bath. After cooling in an ice bath, toluene was added
followed by vortexing the mixture for 5 sec. After warming to room temperature, the chromophore
was aspirated and its absorbance was taken at 520 nm. For preparation of standard curve, proline
standard from 5 to 25 µg/mL was used. Amount of free proline was calculated with the formula:
µg proline/mL × mL of toluene µmoles proline/g fresh weight = -------------------------------------------------
(115.5 µg/mole)/g sample/5
b. Glycinebetaine
Glycinebetaine contents were estimated according to the method of Grieve and Grattan
(1983). Dry plant material (0.5 g) was extracted in 20 mL of deionized water. This extract was
filtered and diluted with 2N H2SO4 followed by cooling on ice for 1 h. This mixture was added
with 0.5 mL of cold IK-I2 (periodide reagent) and stored at 4oC for 16 h. The reaction mixture
was centrifuged at 10,000 × g and periodide crystals were collected. These crystals were
dissolved in 1, 2-dichloroethane and left at room temperature for 2 h. The absorbance of the
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colored complex was noted at 365 nm. For construction of standard curve, standard series of
glycinebetaine (50, 100, 150 and 200 µg/mL) was run along with samples.
c. Total free amino acids (TFAA)
TFAA were determined by using the method given by Hamilton and Van Slyke (1943).
Fresh plant material (0.1 g) was extracted in phosphate buffer (pH 7.0) and extract was mixed
with 1 mL of 10% pyridine solution and 1 mL of 2% ninhydrin solution followed by heating for
30 min. After heating, the mixture was diluted up to 50 mL. Absorbance of this mixture was
taken at 570 nm using a spectrophotometer. For standard curve, leucine was used and total free
amino acids were calculated by using following formula:
TFAA (mg/g fresh weight) = Graph reading of sample × volume of the sample × dilution factor Weight of the tissue × 1000
d. Soluble sugars
Soluble sugars were determined by the method of Yoshida et al. (1976). Fresh plant
material (0.1 g) was taken and boiled in 5 mL distilled water, filtered and diluted up to 50 mL
with distilled water. To 1 mL of the diluted filtrate, 5 mL of anthrone reagent (prepared by
dissolving 1 g anthrone in 1 L conc. H2SO4) was added followed by heating at 90oC for 20 min
and absorbance was noted at 620 nm using a spectrophotometer. Glucose series (0, 20, 40, 60, 80
and 100 µM) was used for the preparation of standard curve.
3.3.4 Secondary Metabolites
For the determination of soluble phenolics, anthocyanins and flavonoids, frozen fresh material
was used. For the estimation of alkaloids, saponins and tannins, powdered dry material was
defatted following the method of AOAC (1990). For this purpose, plant sample (3 g) was mixed
with 30 mL hexane. This mixture was left on a shaker at 100 rpm for 24 h. Then this mixture was
centrifuged at 3500×g for 18 min. The residue was taken and again mixed with hexane followed
by centrifugation and allowed to stand for 24 h. After centrifuging, the supernatant and residue
were separated. This residue was oven dried and used as fat free sample for further analysis.
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a. Alkaloids
Alkaloids were determined by using the method of Harborne (1973). Fat free sample (0.5
g) was added in 20 mL of 10% acetic acid (in ethanol) in 250 mL beaker and taken in test tubes.
The test tubes were covered and allowed to stand for 4 h. The mixture was filtered and filtrate was
heated on a water bath at 90oC till one quarter of the original volume of extract was obtained. After
heating, conc. NH4OH was added drop-wise until the formation of precipitates, which were
collected, washed with NH4OH and re-filtered. The residue was oven dried and weighed.
b. Saponins
The total saponin contents were evaluated according to the method of Chapagain and
Wiesman (2005). Fat free sample (0.1 g) was taken after drying for 24 h and added 30 mL of
methanol. The mixture was shaken on a shaker at 100 rpm for two days and centrifuged. After
that three consecutive extractions were carried out by using solvent methanol. The solvent was
evaporated and a yellowish crystalline residue was obtained, which was carefully weighed and
saponins were estimated.
c. Tannins
The extraction of tannins was carried out by following the procedure of Van-Burden and
Robinson (1981) with some modifications. Fat free sample was taken and mixed with 50 mL
distilled water. This mixture was shaken for 1 h on a shaker at 100 rpm, filtered and volume
made up to 50 mL. A 5 mL of the filtrate was taken and mixed with 0.1 M FeCl3 (in 0.1 N HCl)
and 0.008 M potassium ferrocyanide. Absorbance of the mixture was taken at 605 nm. For
construction of standard curve, 10, 20, 30, 40 and 50 µg/mL concentrations of tannic acid were
used and run along with the unknown samples.
d. Anthocyanins
Anthocyanins were determined according to the method of Stark and Wray (1989). For
this, fresh leaf material (0.1 g) was extracted in 1 mL acidified methanol (1% HCl v/v). The
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mixture was heated at 50oC for 1 h and extract was filtered. The absorbance of the filtrate was
measured at 535 nm by using a spectrophotometer.
e. Total soluble phenolics
Total soluble phenolics were evaluated by using the method of Julkunen-Tiitto (1985)
using Folin-Ciocalteu reagent. Fresh plant material (0.5 g) was extracted in 80% acetone; extract
centrifuged at 12,000 and supernatant collected. A 100 µL aliquot of the extract was mixed with
0.5 mL of Folin-Ciocalteu’s phenol reagent and 2.5 mL of 20% Na2CO3. The volume of mixture
was made up to 5 mL and vortexed. Absorbance of the reaction mixture was noted at 750 nm.
f. Flavonoids
Flavonoids contents were determined by the method of Zhishen et al. (1999). Plant
material (0.1g) was extracted in 80% acetone and 1 mL of extract was added in a 10 mL
volumetric flask containing 4 mL of distilled water. The reaction mixture was added with 0.6 mL
of 5% NaNO2, 0.5 mL of 10% AlCl3 after 5 min, and 2 mL of 1 M NaOH after 1 min. The
reaction mixture was diluted with 2.4 mL of distilled water and mixed. The absorbance was
taken at 510 nm. The quercetin was used as a standard for the calibration curve.
3.3.5 Vitamins
a. Niacin
Niacin was estimated by the method of Okwu and Josiah (2006). Fat free sample (0.5 g)
as given above (section 3.2.4.) was taken and mixed with 5 mL of 1 N H2SO4 with shaking. Then
three drops of NH3 solution were mixed and filtered. Filtrate (1 mL) was taken and mixed with
0.5 mL potassium cyanide solution and 0.5 mL of 0.02 N H2SO4. The absorbance was noted at
470 nm by using a spectrophotometer. Final quantities of unknown samples were determined
from a standard curve constructed from the niacin.
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b. Riboflavin
Riboflavin was determined by the method of Okwu and Josiah (2006). For this, 0.5 g fat
free sample was extracted in 10 mL of 50% ethanol for 1 h and filtered. To 1 mL of the filtrate, 1
mL of 5% potassium permanganate and 1 mL of 30% H2O2 was added followed by heating in a
water bath at 50oC for 30 min. After cooling, 0.2 mL of 40% sodium sulfate was added to it and
diluted the reaction mixture up to 5 mL. After 5 min, the absorbance of the colored complex was
measured at 510 nm. Standard curve was constructed by using riboflavin.
c. Ascorbic acid
Ascorbic acid was determined by the method described by Mukherjee and Choudhuri
(1983). Fresh plant material (0.25 g) was extracted in 10 mL solution of 6% TCA. To 4 mL
extract, 2 mL of 2% dinitrophenyl hydrazine solution (in acidic medium by dissolving 2 g of
compound in 100 mL of HCl (37%) was added followed by addition of one drop of 10%
thiourea solution. The reaction mixture was heated in a water bath for 20 min. After cooling to
room temperature, 5 mL of 80% H2SO4 (v/v) was added. The absorbance was taken at 530 nm.
Standard curve was constructed by using the ascorbic acid.
d. Reducing powers assay (RPA)
For RPA, the leaf samples were prepared following the method of Sofowora (1993). The
samples were air-dried at room temperature and blended to a mesh size of 1 mm. The blended
samples (5 g) were soaked in 20 mL of 98% methanol for 48 h, filtered and filtrate was
concentrated to dryness using rotary evaporator and refrigerated. The reducing power (RPA) of
leaves was quantified with the method of Perumal and Becker (2003) with some modification.
The refrigerated powder was re-dissolved in 80% methanol. One mL of this extract was mixed
with phosphate buffer (5.0 mL of 2.0 M, pH 6.6) and potassium ferricyanide (5.0 mL of 1%
solution) mixed and incubated at 50oC for 20 min. On cooling 5 mL of TCA (10%) was added
and the mixture was centrifuged at 3000 × g for 10 min. The upper 5 mL layer of the solution
was aspirated and was mixed with 5 mL of distilled water and 1 mL of 0.1% ferric chloride. The
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absorbance of the pink color mixture was measured spectrophotometrically at 700 nm. Increased
absorbance of the mixture indicated increased reducing power.
3.3.6. Photosynthetic pigments
Chlorophyll a and b was determined by using the method of Arnon (1949) and
carotenoids was determined by the method of Davis (1976). For this, 0.5 g fresh plant material
was completely homogenized with pestle and mortar in 80% acetone in the darkness, filtered and
made the volume of filtrate up to 10 mL by adding 80% acetone. For the chlorophylls estimation,
the absorbance was taken at 645 nm for chlorophyll a; at 663 nm for chlorophyll b and at 480 nm
for carotenoids using spectrophotometer. The quantities of chlorophyll a, b and total chlorophyll
were calculated by using following formulas given by Arnon (1949).
Chl. a (mg/g) = [12.7(OD663)-2.69(OD645) × V/1000 × W
Chl. b (mg/g) = [22.9(OD645)-4.68(OD663) × V/1000 × W
Total Chl. (mg/g) = [20.2 (OD645) + 8.02 (OD663) × V/1000 × W
Where V= Volume of acetone used in extract (mL)
W= Fresh weight of plant in g
For the estimation of carotenoids, the following formula given by Davis (1976) was used.
Carotenoids (mg/mL) = (Acar/Em × 100)
where Em × 100 = 2500 and ACar = [(OD480) +114(OD663)-0.638(OD645)]/2500
3.3.7 Nutrients, nutritional and fiber characteristics
a. Determination of K+ and Ca2+
Determination of K+ and Ca2+ was done by using the method of Yoshida et al. (1976).
Dry plant material (0.2 g) was digested in 2.5 mL of mixture of concentrated nitric acid and
perchloric acid (2:1 ratio) at 250oC on a heating block till the samples became clear. The volume
of the digest was diluted up to 50 mL with distilled water. Ca2+ and K+ were estimated by using
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flame photometer (Sherwood model, 410 UK). Standard curves were constructed by running the
different grade series (10, 20, 30, 40, and 50 ppm) of Ca2+ and K+.
b. Soluble nitrate (NO3--N)
Soluble Nitrate (NO3--N) was determined by using the method of Kowalenko and Lowe
(1973). Dried ground material (0.5 g) was put in a test tube containing 5 mL distilled water and
autoclaved for 15 min. Then solution was filtered and diluted up to 50 mL with distilled water.
To 3 mL of the extract, 7 mL of the chromotropic acid (CTA) solution (prepared by dissolving 1
g CTA in 100 mL of conc. HNO3) was added and let stand for 20 min. The absorbance was taken
at 430 nm on a spectrophotometer. Distilled water was used as blank. Standard curve was drawn
by using the KNO3 grade series 10, to 50 mg L-1 NO3- prepared by diluting 100 mg L-1 NO3
-
stock solution (prepared by dissolving 0.7216 g of pure dried KNO3 in 1 L of distilled water).
c. Soluble phosphate (PO43--P)
For soluble PO43-‒P determination, to 1 mL of extract from the above (section 3.2.7 b), 2
mL of 2N HNO3 was added followed by dilution up to 8 mL with distilled water in a test tube.
Then 1 mL of the molybdate-vanadate reagent was added and volume was made 10 mL with
distilled water and stand for 20 min. The absorbance was taken at 420 nm. Distilled water was
used as blank and standard curve was made using the phosphate grade series (2.5‒15 mg L-1).
For preparation of this series stock solution (25 mg L-1 PO43- ) was prepared by dissolving 0.110
g of dried monobasic phosphate (KH2PO4) in 1000 mL of distilled water then diluted to 2.5, 5.0,
7.5, 10.0, 12.5 and 15.0 mg L-1 PO43- standard series (Yoshida et al., 1976).
d. Sulfate (SO42--S)
Sulfate was determined following the method described by Tendon (1993). To 10 mL of
the HNO3-HClO4 extract in a conical flask, 1 mL of 6N HCl and 1 mL of 0.5% (w/v) gum-acacia
solution were added. The solution was swirled for 10‒15 sec followed by the addition of 0.5 g
barium chloride crystals and swirled again and again until the solution became clear with
dissolved crystals. Sample transmittance was read at 340 nm. For constructing the standard
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curve, 100 mg L-1 stock solution was prepared by dissolving 0.543 g of K2SO4 in distilled water.
The stock was diluted in 250 mL volumetric flasks, to make 0, 4, 8, 12, 16 and 20 mg L-1 sulfate
solution. To each flask, 25 mL of salt-buffer solution (prepared as 50 g MgCl2.6H2O, 4.1 g
KNO3 and 28 mL ethanol in a final volume of 1 L) was added. The remaining procedure was the
same as described above.
e. Starch contents
Starch contents were estimated by following the method of Malik and Srivastava (1985).
Methanolic extract of fresh plant material was taken and filtered. Then residue was dried and re
extracted in 5 mL of distilled water. To this extract, 52% HCL (1:1 v/v) was added, centrifuged
and supernatant collected. To 0.5 mL of this supernatant 2 mL of anthrone reagent was added
and heated for 30 min. After cooling, the absorbance was taken at 625 nm. The starch content
was calculated by formula:
Starch (mg/g dry weight) = standard reading (absorbance/mg) × sample reading × dilution factor
g. Total soluble proteins
Total soluble proteins were analyzed by the method of Bradford (1976). For extraction of
total soluble proteins, 0.5 g fresh plant material was extracted in phosphate buffer saline (PBS;
2.7 mM KCl, 10 mM Na2HPO4, 1.37 mM NaCl and 2 mM KH2 PO4, pH 7.2 adjusted with
HCl), centrifuged and supernatant collected. For this purpose, 1.0 mL of the extract was
centrifuged and the supernatant from each sample was mixed with 200 µL of Coommassie
Brilliant Blue (CBB) dye reagent (Bio-Rad, USA) in the Appendorf tube (1.5 mL). After waiting
for 20 min at room temperature, the absorbance of the samples was taken at 595 nm using a
spectrophotometer. Bovine Serum Albumin (BSA) was used as standard (10-50 µg/mL) for the
construction of standard curve. Final amount of proteins present in the extract was computed
from the unknown samples.
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h. Neutral detergent fiber (NDF)
NDF was estimated by the method of Van Soest (1963). For this, dried material (1.0 g)
and 0.25 g of Na2SO3 was added in each flask. Fifty mL of NDF reagent (EDTA, Na2HPO4,
Lauryl sulfate, Sodium borate decahydrate and ethylene glycol monoethyl ether) was added with
water cooling arrangement with it. Then heated it slowly (to avoid foaming) on heating plate for
1 h then allowed to cool and filtered the solution with the help of suction pump. Then washed the
residue with hot water (60‒70ºC) for 4‒5 times using 5‒7 mL warm water. The residue was
washed in crucible with 5 mL acetone twice on a filter paper and placed in the hot air oven at
105ºC for overnight. Percentage of NDF was calculated using following formula.
% NDF = [(crucible wt. with fiber wt.) – (crucible wt. without fiber)/ sample wt.] x 100
i. Acid detergent fiber (ADF)
ADF was determined by the method of Van Soest and Wine (1963). For this, 1.0 g of
dried plant material was added with 50.0 mL of ADF solution (Hexadecyl trimethyl ammonium
bromide, sulfuric acid and distilled water) and heated slowly on heating plate in flask and air
condenser fixed on it and reflux for 1 h. Washed the residue with hot water for 3‒4 times then
washed with 5.0 mL of acetone twice. Residue were transferred to dried crucible and kept in an
oven at a temperature 70 oC for 3‒4 h and then put in desiccator for cooling. The weight of the
residue was taken and amount of ADF was computed with the formula.
% ADF = [(crucible wt. + with fiber) – (crucible weight without fiber) / sample weight] × 100
j. Determination of acid detergent lignin (ADL)
For the measurement of ADL, 1 g of dry sample was taken in a conical flask and added
50 mL of 1 N H2SO4 and added ADF solution. The flask was fixed with air condenser and
reflexed for 1 h. The residue was taken and washed with boiling water for 3‒4 times and again
washed with 5.0 mL acetone twice. The washed residue was put in an oven at 105oC for 3 h. The
residue from crucible was transferred in 250 mL beaker, and add 25 mL 72% H2SO4 and stirred
for 3 h. Contents were diluted up to 200 mL. Residue was washed with distilled water to remove
acid and again washed with 95% ethanol to remove excess water. Then residue was transferred
to crucible and kept in oven for 3 h and weighed. This contains lignin, cutin and silica; then
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heated the crucible in muffle furnace at 600oC for 1 h. Then weigh the crucibles. ADL was
calculated using following formula (Goring and Van Soest, 1970).
ADL (%) = (Crucible weight after acid soak – Crucible weight after ignition) / (Sample weight)
f. Cellulose
The ADL was used to determine the cellulose content by further extraction with acid
detergent, permanganate and 72% H2SO4. The cellulose content was calculated as loss of weight
from the ADF as described by Goring and Van Soest (1970).
g. Silica
Silica was determined as the crude, insoluble residue remaining after dissolving the ash
(from ignition at 500oC) in 6N-HC1 followed by evaporation to dryness on a steam bath and re-
dissolving the soluble residue in boiling 6N-HC1 (AOAC, 1955).
3.3.8 Leaf essential oil determination
Finely air dried grounded lemongrass leaves samples (100 g) collected during four
seasons in a year were hydro-distilled for 3 h in Clevenger-type apparatus. The lemongrass
essential oil layer was dissolved in diethyl ether and then detached through distilled water in
separating funnel by solvent extraction. Then extracted lemongrass essential oil was stored at -
4°C for Gas Chromatograph-Mass spectrometer (GC-MS) analysis.
GC analysis of lemongrass leaf essential oils was done using GC-17A Model (Data Apex
Ltd. CSW32-Chromatography station, Shimadzu, Japan) equipped with DB-wax column (30 m ×
0.25 mm), flame ionization detector (FID). The temperature of Injector and detector were set at
250 and 260°C, respectively. Column temperature was prearranged from 90°C for 2 min to
180°C at 2°C/min to 240°C/min. Helium gas at flow rate of 30 mL/min at 150 psi was used as a
carrier gas. Oil sample (1 µL) was injected through the injector. The lemongrass oil percentage
composition was calculated as a relative percentage of the total peak area.
To recognize the oil components a comparison of their mass spectra was made with NIST
mass spectral library compounds (Mass Spectral Library, 2002) and through the comparison of
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their retention indices either with those of authentic compounds or with literature values (Adam,
2001; Mimica-Dukic et al., 2003; Vagionas et al., 2007).
3.4 Statistical Analysis
The design of the experiments in both the years was factorial randomized complete block,
with three blocks and three replications in each block for all the determinations. The data
presented here is the average of both the years. The differences between factors and their
interactions were ascertained with analysis of variance (ANOVA) using computer software
Statistix 8.1. Pearson’s correlation coefficients were derived among different attributes were
made using MS-Excel. Data was presented graphically by using MS-Excel.
3.5 Chemicals
All chemicals used in the analytical work were purchased from Sigma, Merck, Bio-Rad,
Reidle and BDH grade.
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CHAPTER-4
RESULTS AND DISCUSSION
4.1 Preliminary
In nature the environmental factors are the key drivers in the production of an array of
changes in plants starting from gene expression to the appearance of visible features. Among the
environmental conditions, the changes in light, temperature and humidity are the most important
ones. Being a C4 plant, lemongrass (Cymbopogon citratus) responds fairly well to the prevailing
conditions and shows the synthesis of a wide range of plant secondary products, which enable it
to withstand the changing conditions in all the seasons. In the current work, the response of a
selected vegetatively propagated population of lemongrass was studied in primary and secondary
metabolites and oil composition of leaves of three ages (penultimate, middle and bottom) for two
consecutive years for seasonal variations on monthly basis.
4.2 Meteorological Attributes
Fig. 4.1 indicates average meteorological data for the years 2010 and 2011. It is evident
from the data that respective average minimum and maximum temperatures were 4 and 16oC in
January, which begun to increase from February onwards and was maximum in the month of
June. From July onwards, the temperature indicated a decline and in December, the temperature
was quite low. During both the experimental years, the maximum average RH was noted in the
August and September (~75%) when precipitation was maximum as well as in cool and humid
months of winter season (~73%). However, hot and dry months (May) exhibited the lowest RH
(~43%). The pattern of rainfall was very erratic and sporadic; highest in September, July and
August (155, 118 and 93 mm, respectively) of both the years but with great variations (Fig. 4.1).
Contrarily, months of January, November and December in both the years did not receive any
precipitation. Trend of evapotranspiration was closely associated with seasonal changes in the
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temperature ranges. The evapotranspiration was more (~6 mm) in hot and dry months (May and
June) while lower (1.2 mm) in cool months (January and February).
The trend of seasonal changes was similar in both the years, but the changes in
temperature were more crucial. The changes in climatic conditions are mainly dependent upon
prevailing temperatures, which also trigger the survival of plants with the induction of requisite
survival mechanisms (IPCC, 2007). In response to changes in temperature, plant responses are
usually noticed in terms of biosynthesis of various primary and secondary compounds (Chalker-
Scott, 1999; Wahid, 2007; Wahid and Tariq, 2008). Such diversions from normal metabolism
lead to growth and yield decline especially in medicinally important plants including lemongrass
(Hussain et al., 2012). Lemongrass showed conspicuous fluctuations in its active compounds
during different seasons, which might be ascribed to variations in seasonal variables such as
temperature, rainfall, humidity and soil moisture etc. Being a tropical and subtropical plant,
lemongrass displays wide range of variations in physiological and biochemical attributes in
different seasons. The present study provides the most elaborated description of seasonal
variation in the biochemical and physiological contents of lemongrass leaves.
Fig. 4.1: Average monthly data of meteorological conditions during the year 2010 and 2011. The error bars indicate the average variation in rainfall during both the years
0
20
40
60
80
100
120
140
160
180
Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec
Rainfall (mm)
Max temp
Min temp
RH (%)
Evapotranspiration (mm)
Months 2010-11
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4.3 Membrane characteristic and antioxidants
Antioxidants have seasonality in their formation and mode of action in different seasons.
Excessive production of ROS is causing progressive oxidative damage to the cellular machinery.
In lemongrass, the adverse seasonal changes induce the formation of reactive oxygen species. In
this part of the manuscript, the influence of seasonal changes on the levels of hydrogen peroxide
(H2O2), MDA, CAT, POD and SOD activities of lemongrass penultimate, middle and bottom
leaves was studied.
4.3.1 Results
The statistical analysis of data and significance of variance sources of the oxidative stress and
pattern of antioxidants expression in lemongrass leaves over a measured in the present studies 12
months period are given in Table 4.1
Table 4.1: Analysis of variance (mean squares) of oxidative stress parameters and antioxidants activities in three leaves of lemongrass over a 12 months period
SOV df H2O2 MDA CAT POD SOD
Block (B) 2 2.50 1.90 14.49 263.53 0.81
Leaves (L) 2 44845.61** 1948.81** 15471.41** 39741.11** 112.03**
Months (M) 11 238.52** 43.18** 878.09** 17778.57** 5.15**
L × M 22 76.90** 4.58** 184.72** 1306.81** 1.53**
Error 70 14.53 0.76 16.38 42.30 0.71
Significant at: *, significant at P = 5%; **, P = 1% levels of probability, respectively
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a. Hydrogen peroxide (H2O2)
There was significant difference in the leaves and months with significant interaction of
these factors for H2O2 concentrations (Table 4.1). In penultimate leaf H2O2 remained fairly
constant during spring (Feb-Apr) season. It increased slightly in summer (May-Jul) months but
declined markedly in autumn (Aug-Oct) months. It increased again in winter (Nov-Jan) months.
In middle leaf, with the onset of spring season, H2O2 consistently declined but then attained the
greatest value in summer (maximum in July) and a decline was observed in autumn, and then a
rise in winter. In bottom leaf, except for late winter months, the tend of H2O2 accumulation was
similar to penultimate and bottom leaves. The highest H2O2 concentrations was observed in
bottom, moderate in middle and lowest in penultimate leaf (Fig. 4.2).
Fig. 4.2: Effect of seasonal variation on hydrogen peroxide accumulation in the penultimate,
middle and bottom leaves of lemongrass
bc c cd dab ab a
cde e
cdb
abc
bcde
ede
cde ab
c aab
cdcd
ecd
e abc ab
de cde
efcd
ede
fbc
a acd
ef fab
0
20
40
60
80
100
120
Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec
Penultimate Middle Bottom
Hyd
roge
n pe
roxi
de (
nmol
/g f
resh
wei
ght)
Leaf position/sampling months
Probability level:
Penultimate leaf = **Middle leaf = **Bottom leaf = **
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b. Malondialdehyde (MDA)
The MDA content in leaves of lemongrass over months varied significantly with a
significant interaction of these factors (Table 4.1). Both penultimate and middle leaves exhibited
similar contents and trend of MDA accumulation. In both these leaves, MDA contents were
lower in spring season, which indicated a sharp increase at the onset of summer and the
maximum values were noted in Jun and Jul. The MDA contents began to decrease sharply at the
start of autumn but again increased in winter season. Bottom leaf showed increased contents of
MDA contents from May and achieved an apical value in July, while a drop was detected in
August, which continued till October. However, a steady-state rise was observed from Oct to Jan
(winter season). In all leaves, the value of MDA increased during summer and winter seasons
while minimum value was found in spring and autumn seasons (Fig. 4.3).
Fig. 4.3: Effect of seasonal variation on MDA concentration in the penultimate, middle and
bottom leaf of lemongrass
bcfg fg
hgh
dea ab
efh gh
cd cd
bde cdde
bca a
bcde e
b b
bcgh
fgh
efb
acd
egh
hde
bcd
0
5
10
15
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25
30
Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec
Penultimate Middle Bottom
Mal
ondi
alde
hyde
(nm
ol/g
fre
sh w
eigh
t)
Leaf position/sampling months
Probability level:
Penultimate leaf = **Middle leaf = **Bottom leaf = **
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c. Superoxide dismutase (SOD) activity
Data indicated significant differences in the leaves of various ages and sampling months
on SOD activity with a significant interaction of leaves and sampling months (Table 4.1).
Seasonal changes produced a great impact on the SOD activity of lemongrass leaves. The SOD
activity of the penultimate was the lowest SOD in spring season, which increased greatly in
summer season, attained the highest value in June (21.66 U/g protein/min), while a decline was
observed in July and onward which continued till October, while a gradual increase was
observed from November to December. SOD activity of middle leaf was more or less similar to
the penultimate leaf. However, bottom leaf indicated quite reduced SOD activity in most of the
months except a rise was noted in May (Fig. 4.4).
Fig. 4.4: Effect of seasonal variation on SOD activity in the penultimate, middle and bottom
leaves of lemongrass
bef
bcde bc bc
da
bef cd
efde
ff ef
cdf
de decd
aab
efcd
deef
bc
bccd
ede e de
abc
de
bbc
cde bc
d0
5
10
15
20
25
Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec
Penultimate Middle Bottom
Sup
erox
ide
dism
utas
e (U
/g p
rote
in/m
in)
Leaf position/sampling months
Probability level:
Penultimate leaf = **Middle leaf = **Bottom leaf = **
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d. Catalase (CAT) activity
The CAT activity of lemongrass leaves of three ages varied significantly in different
seasons with a significant interaction of leaf ages and sampling months (Table 4.1). All leaves
indicated individualistic behavior of CAT activity. The penultimate leaf showed the highest CAT
activity in July (113.14 U/g protein/min). However, a reduction in CAT activity was noticed
from August which attained a steady-state level up to December. In the middle leaf, CAT in
activity increased from January to March (winter to spring seasons) and attained the maximum
value in July (87.84 U/g protein/min), while a decline was observed from August to December.
On the other hand, bottom leaf over the seasons (sampling months) indicated lower CAT activity
winter months, declined in summer months, again increased in autumn and declined thereafter.
Overall, CAT activity was the highest in penultimate leaf followed by middle leaf (Fig. 4.5).
Fig. 4.5: Effect of seasonal variation on CAT activity in the penultimate, middle and bottom
leaves of lemongrass
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de decd
ba
cd dde
cdc
fef
ccd c
ba
c cde
ef f
fef
ab abde
bca a ab
cdef f
0
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40
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140
Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec
Penultimate Middle Bottom
Cat
alas
e ac
tivity
(U
/g p
rote
in/m
in)
Leaf position/sampling months
Probability level:
Penultimate leaf = **Middle leaf = **Bottom leaf = **
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e. Peroxidase (POD) activity
The data indicated significant differences in the leaves and months as well as there was
significant interaction of these factors for POD activity (Table 4.1). Different lemongrass leaves
presented peroxidase (POD) activity with value ranging from as low as 17.46 protein/min in
bottom leaf to as high as 157.06 U/g protein/min in penultimate leaf. The POD activity of
penultimate and middle leaves was lower in winter and autumn seasons but higher in summer
season, which sequentially declined from August and attained steady-state level thereafter.
However, bottom leaf exhibited differential behavior of POD activity; three distinct peaks were
observed in Feb, Jul and Nov. Overall, penultimate leaf indicated the highest POD activity
followed by middle leaf (Fig. 4.6).
Fig. 4.6: Effect of seasonal variation on POD activity in the penultimate, middle and bottom
leaves of lemongrass
deef
ded
bca
abc
df
def
def
de ecd
e cdb
ab ab
ce de de
bca
de decd
eb
fbc
dee
bcd
ab
0
20
40
60
80
100
120
140
160
180
Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec
Penultimate Middle Bottom
Per
oxid
ase
activ
ity (
U/g
pro
tein
/min
)
Leaf position/sampling months
Probability level:
Penultimate leaf = **Middle leaf = **Bottom leaf = **
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f. Correlation
Parallels drawn of oxidative stress parameters with antioxidants activities revealed that
H2O2 accumulation was positively related to MDA contents of all leaves. Among the
antioxidants, SOD activity was positively correlated with H2O2 accumulation in penultimate and
middle leaves. MDA contents indicated a positive relationship in case of penultimate and middle
leaves with the activities of SOD and POD while only with penultimate leaf in case of CAT
activity. Bottom leaf did not exhibit any association with the antioxidants activities (Table 4.2).
Table 4.2: Correlation coefficient (r) of metrological attributes with the Secondary metabolites of lemongrass at three leaves positions penultimate, middle, bottom X-Variable Y-Variable Penultimate Middle Bottom a. Correlations of oxidative stress parameters with antioxidants H2O2 MDA 0.777** 0.761** 0.610* SOD 0.581* 0.594* -0.193ns CAT 0.554ns 0.014ns 0.347ns POD 0.548ns 0.309ns -0.355ns MDA SOD 0.753** 0.711** -0.009ns CAT 0.774** 0.430ns -0.083ns POD 0.610* 0.622* -0.034ns b. Correlations of environmental variable with oxidative stress parameters and antioxidant Max. temperature H2O2 -0.062ns -0.188ns 0.205ns MDA 0.023ns 0.107ns 0.038ns SOD 0.173ns 0.371ns 0.021ns CAT 0.424ns 0.759** 0.632* POD 0.707* 0.759** -0.474ns Min. temperature H2O2 -0.069ns -0.100ns 0.346ns MDA 0.043ns 0.163ns 0.136ns SOD 0.120ns 0.367ns 0.043ns CAT 0.488ns 0.831** 0.729** POD 0.756** 0.823** -0.470ns Relative humidity H2O2 -0.210ns 0.377ns 0.346ns MDA -0.019ns 0.027ns 0.187ns SOD -0.281ns -0.091ns 0.331ns CAT 0.002ns -0.145ns 0.273ns POD -0.205ns -0.145ns 0.273ns Evapotranspiration H2O2 0.158ns -0.169ns 0.146ns MDA 0.169ns 0.203ns 0.101ns SOD 0.163ns 0.241ns -0.178ns CAT 0.454ns 0.721** 0.474ns POD 0.758** 0.760** -0.513ns Average rainfall H2O2 -0.136ns 0.218ns 0.616* MDA 0.050ns 0.206ns 0.246ns SOD -0.031ns 0.265ns 0.315ns CAT 0.495ns 0.649* 0.671* POD 0.492ns 0.566ns -0.253ns
Significant at: ns, non-significant; * and **, significant at P<0.05 and P<0.01 levels
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46
As for the seasonal changes, POD activity of penultimate leaf was positively correlated
with maximum and minimum temperatures, and evapotranspiration. CAT and POD activities of
middle leaf were positively correlated with maximum and minimum temperatures,
evapotranspiration and average rainfall. In case of bottom leaf, the CAT activity was correlated
with maximum and minimum temperatures and average rain fall while H2O2 accumulation was
related to average rainfall (Table 4.2).
4.3.2 Discussion
Plants respond to the seasonal changes by showing adjustments in their metabolism. One
of the most common consequences of heat stress is the generation of ROS leading to membrane
damage and disruption of cellular phenomena (Wahid et al., 2013). In this experiment,
monitoring the leaf H2O2 contents indicated that its greater production took place in summer and
winter season in all leaves, although bottom leaf indicated the highest while penultimate leaf the
lowest amounts of H2O2 (Fig. 4.2). With the induction of oxidative stress, MDA is produced due
to β-oxidation of membrane lipids (Lu et al., 2009), which perturbs the plant phenomena
including photosynthesis and respiration (Malencic et al., 2004). In the present study, there was a
substantial increase in the MDA contents of all the studies leaves in the harsh conditions of
summer and winter, although leaf age had a large effect. The MDA contents indicated the pattern
more or less similar to H2O2 production (Fig. 4.2, 4.3). This was also evident from the positive
correlation of H2O2 and MDA for all leaves (Table 4.2). Savicka and Skute (2010) observed that
high temperature accelerates MDA production (up to 58%) at later growth stages as compared
with early seedling stage (by 27%). Enhanced lipid peroxidation during hot months could be
associated with the damage on cell membranes (Huang et al., 2004) and in rice it reduced
antioxidant enzyme activities (Cao et al., 2009). It was important to note here that both seasonal
changes and leaf age have great impacts on the production of MDA, where cooler months and
younger (penultimate) leaf exhibited lower MDA production.
Both plant age and seasonal variation influence antioxidant enzyme activities of plants,
which are important indices of plant responses (Sen and Mukherji, 2007). Temperature above
33°C induced oxidative stress, which damage cell membrane due to degradation of protein and
also decrease in enzyme activities in wheat (Bavita et al., 2012). To circumvent enhanced ROS
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produced by seasonal variation, plant cells activate the key antioxidant enzyme like SOD, CAT
and POD (Mittler, 2002; Ozden et al., 2009). These antioxidants are the first line of defense
against ROS, which catalyzes the dismutation of O2- and dousing of other activated ions
(Takahashi and Asada 1983; Scandalios, 1993; Wahid et al., 2013). In the present study, the
SOD, CAT and POD activities in lemongrass leaves were increased markedly in summer months
(Figs. 4.4-4.6). However, leaves of different ages indicated differential response to the induction
of antioxidant defense. Correlation data indicated that there was a close association of the MDA
and H2O2 accumulation with the SOD and CAT activities in penultimate and middle leaves
(Table 4.2). A conspicuous increase in ROS scavenging activity with the induction of antioxidant
systems was of great advantage to these leaves. Under higher temperatures, plant respiratory
rates are higher which enhance antioxidant response due to subsequent higher ROS level in the
mitochondria. According to Dizengremel (2001), SOD formation increases with increase in
NADH synthesis during higher respiration. Similarly, high temperature stress enhanced the
respiration rate along with a significant increase in SOD-manganese activity in Nicotiana
plumbagifolia (Bowler et al., 1992).
Among the environmental variables, changes in the ambient temperature and
evapotranspiration were of greater significance. These factors cause osmotic strain on the leaves
and induction of enzymatic antioxidants has been well reported under abiotic stresses including
cold stress (Wu et al., 1999; McKersie et al., 1999), heat stress (Wahid et al., 2007), high light
intensity (Sen Gupta et al., 1993) and drought stress (Farooq et al., 2009). In the present study,
CAT and POD activities increased in lemongrass leaves in summer period during the months of
June and July (Fig. 4.5-4.6), which has close association with high and low temperatures and
evapotranspiration (Table 4.2). Increased CAT under adverse conditions has been considered as
a prerequisite for plants against fatal H2O2 accumulation (Streb et al., 1997a, b; Engel et al.,
2006). This notion stands fast in the current study too because high and low temperatures and
evapotranspiration are great stressing factor for plant growth, while POD protects the membrane
from damage due to H2O2 (Farooq et al., 2009).
To conclude, seasonal variation has a great impact on lemongrass antioxidant activity.
These antioxidant enzymes are capable of minimizing the effect of oxidative damage by
scavenging H2O2 and reducing MDA production in extreme environmental conditions.
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4.4 Osmoprotectants
In osmotic adjustment of plant cell organic solutes play an important role. Seasonal
variations occur not only in the type of the organic solutes, but also in the pattern of their
accumulation. Seasonal variation in soluble sugars, free proline, glycinebetaine and total free
amino acids content were measured in penultimate, middle and bottom leaves of lemongrass.
The results are interpreted and discussed below.
4.4.1 Results
Results regarding statistical analysis of data for the changes in the osmoprotectants
accumulation in the leaves of lemongrass in various seasons are given in Table 4.3
Table 4.3: Analysis of variance of data (mean squares) for soluble sugar, free proline, total free amino acid, and glycinebetaine contents of lemongrass under influence of seasonal condition
SOV df Soluble sugar TFAA Free proline Glycinebetaine
Block (B) 2 4.80 18.49 2.91 0.54
Leaves (L) 2 3267.95** 57666.30** 8769.98** 2323.35**
Months (M) 11 46.32** 1857.01** 129.85** 30.17**
L × M 22 5.92ns 1627.28** 70.41** 11.74**
Error 70 4.32 26.47 2.17 1.44
Significant at: ns, non-significant; * and **, significant at P<0.05 and P<0.01 levels
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a. Soluble sugars
The soluble sugars contents in different leaf positions of lemon grass varied significantly
in different sampling months with a non-significant interaction of these factors (Table 4.3).
Although there were substantial differences in the accumulation of soluble sugars in the leaves of
different ages and seasons, the trend of their accumulation in all leaves was similar. The soluble
sugars accumulation was lower in the spring (Feb-Apr), which increased reasonably in the
summer and autumn seasons (May-Sep). However, this accumulation was the greatest especially
in the month of Jan. (34.47 mg/g fresh weight) in winter season. Nonetheless, penultimate leaf
displayed more explicit variations in the soluble sugars accumulation than middle and bottom
leaves (Fig. 4.7).
Fig. 4.7: Effect of seasonal variation on soluble sugar concentration in the penultimate, middle
and bottom leaves of lemongrass
ab
de
cb
bcb ab
c bcab
acd
de d
ab abab
cab
ccd
abc
a
bc cd cd cd
abc
abc
a aab
0
5
10
15
20
25
30
35
40
Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec
Penultimate Middle Bottom
Sol
uble
sug
ar (
mg/
g fr
esh
wei
ght)
Leaf position/sampling months
Probability level:
Penultimate leaf = **Middle leaf = **Bottom leaf = **
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b. Total free amino acids (TFAA)
Statistical analysis of data indicated significant difference in the leaves of various ages
and sampling months with a significant interaction of both the factors for TFAA levels (Table
4.3). The trend of TFAA accumulation was similar over the seasons in the leaves of all ages,
although there were great differences in their contents. In all leaves, TFAA accumulation was the
lowest in the spring season, which increased with the onset of summer season; however, this
increase was the highest in penultimate leaf followed by middle leaf. With the start of autumn
season, the TFAA levels again declined and indicated an increase in the winter season. These
changes were more explicit in the penultimate leaf as compared to middle leaf and bottom leaf
indicated fewer changes (Fig. 4.8).
Fig. 4.8: Effect of seasonal variation on total free amino acid in the penultimate, middle and
bottom leaves of lemongrass
cde
db
a a
bcde
de de
b
ab bef e
da a
de e
cb
cd bcd
e de cda ab bcd
e deab
c a0
20
40
60
80
100
120
140
160
180
200
Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec
Penultimate Middle Bottom
Tot
al f
ree
amin
o ac
ids
(μg/
g fr
esh
wei
ght)
Leaf position/sampling months
Probability level:
Penultimate leaf = **Middle leaf = **Bottom leaf = **
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c. Free proline
Analysis of variance in the free proline concentration revealed highly significant (P<0.01)
difference in the leaves of different ages and sampling months with significant interaction of
both these factors (Table 4.3). Data on free proline accumulation in penultimate, middle and
bottom leaves exhibited individualistic trends. In penultimate leaf, free proline content was
lowest in spring season, which sharply increased in the summer season, reaching highest value in
July (44.89 µg/g fresh weight). In the autumn season, free proline levels again declined and then
showed an increase in the winter months. In middle leaf, free proline level was low in spring
season, which increased in Jun and Jul (~19 µg/g fresh weight). In autumn the free proline level
decreased and became steady in winter season. In bottom leaf free line level was kept low and
showed virtually no change over the seasons (Fig. 4.9).
Fig. 4.9: Effect of seasonal variations on free proline accumulation in the penultimate, middle
and bottom leaves of lemongrass
abc
bcd
d
bcab
abc
dcd cd
bcd bc
cd d d
ea a
bc
dc c
abc
cde
bcde
cde
bcd
a de e bcd
de bcd
ab
0
10
20
30
40
50
60
Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec
Penultimate Middle Bottom
Fre
e pr
olin
e (μ
g/g
fres
h w
eigh
t)
Leaf position/sampling months
Probability level:
Penultimate leaf = **Middle leaf = **Bottom leaf = ns
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d. Glycinebetaine (GB)
The amount of GB in leaves of various ages differed significantly in various sampling
months with a significant interaction of leaves and months (Table 4.3). Among the leaves of
three ages, penultimate leaf indicated the highest and more explicit trend of GB accumulation
followed by middle leaf, while bottom leaf indicated no specific change in GB accumulation. For
sampling months, both penultimate and middle leaves in autumn season (especially in Sep-Oct
months) indicated the lowest GB accumulation followed by spring season. The GB accumulation
in these leaves was the highest in both summer (especially in Jun) and winter (especially in Jan)
seasons (Fig. 4.10).
Fig. 4.10: Effect of seasonal variation on Glycinebetains concentration in the penultimate,
middle and bottom leaves of lemongrass
abc
d deb
ac
dee e
cdb
bcbc
de cde
bcd
abc
dde
fg ef
g def
bc
a ab ab ab ab ba ab ab a ab a
0
5
10
15
20
25
30
Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec
Penultimate Middle Bottom
Gly
cine
beta
ins
(μg/
g fr
esh
wei
ght)
Leaf position/sampling months
Probability level:
Penultimate leaf = **Middle leaf = **Bottom leaf = ns
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e. Correlation
In penultimate and middle leaves soluble sugars indicated positive association with free
proline in penultimate leaf and with TFAA and free proline in middle leaf. TFAA was positively
correlated with free proline in middle leaf, with GB in penultimate and middle leaves and free
proline was positively paralleled with GB in penultimate leaf. Among the environmental
variables, TFAA, free proline and GB of penultimate and middle leaves were positively related
to maximum temperature; evapotranspiration with TFAA in penultimate leaf and free proline
accumulation with average rainfall in middle leaf.
Table 4.4: Correlation coefficient (r) of mutual relationships of osmoprotectants and those of metrological attributes with the Osmoprotectant of lemongrass leaves
X-Variable Y-Variable Penultimate Middle Bottom a. Mutual correlations of osmoprotectants Soluble sugars TFAA -0.014ns 0.665* 0.485ns Proline 0.589* 0.602* 0.249ns GB 0.381ns 0.362ns 0.273ns TFAA Proline 0.284ns 0.688* 0.400ns GB 0.621* 0.810* 0.025ns Proline GB 0.795** 0.412ns -0.399ns b. Correlations environmental variable with osmoprotectants Max. temperature Soluble sugars -0.379ns -0.402ns -0.315ns TFAA 0.583* -0.583* -0.543ns Proline -0.685* 0.637* -0.114ns GB -0.673* -0.584* -0.387ns Min. temperature Soluble sugars -0.259ns -0.274ns -0.281ns TFAA 0.382ns -0.515ns -0.515ns Proline 0.005ns 0.371ns -0.200ns GB -0.289ns -0.182ns -0.327ns Relative humidity Soluble sugars 0.565ns 0.547ns 0.385ns TFAA -0.472ns 0.308ns 0.308ns Proline 0.202ns 0.305ns -0.224ns GB -0.110ns -0.204ns 0.309ns Evapotranspiration Soluble sugars -0.391ns -0.424ns -0.400ns TFAA 0.767** -0.436ns -0.436ns Proline 0.026ns 0.582* 0.068ns GB -0.029ns 0.138ns -0.461ns Average rainfall Soluble sugars 0.179ns 0.195ns -0.146ns TFAA 0.022ns -0.223ns -0.223ns Proline 0.181ns 0.583* -0.081ns GB -0.279ns -0.197ns -0.238ns Significant at: ns, non-significant; * and **, significant at P<0.05 and P<0.01 levels
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4.4.2 Discussion
In response to seasonal changes, a number of low molecular weight compounds can
accumulate in lemongrass leaves like amino acids, soluble sugars and quaternary ammonium
compounds (QAC). The osmolytes accumulation has been very well related to changes in the
prevailing environmental conditions (Bhowmik and Matsui, 2003; Wahid et al., 2007). In the
present study, among the various osmoprotectants studied, soluble sugars accumulation
displayed seasonal variations, being higher in winter followed by summer conditions while lower
in spring and autumn seasons (Fig 4.7). The harsh conditions during winter and summer produce
an osmotic stress, which is counterbalanced by the accumulation of osmoprotectant; the soluble
sugars appear to be major amongst those in the lemongrass. This is due to their property of being
hydrophilic in nature and having ability to replace water on the surfaces of proteins and
membranes, and thus ensuring their biological functions (Hasegawa et al., 2000). According to
Chinnasamy and Bal (2003) and Schaberg et al. (2000) total soluble sugar content showed
maximum amount in mid-winter. In another study, it was observed that high concentration of
soluble sugars act as cryoprotectants during cold acclimation (Taulavuori et al., 2001).
Amino acids are building blocks of proteins but their accumulation in free form has great
implications in the stress tolerance (Mahmood et al., 2012). In the present case, all the relatively
younger (penultimate and middle) leaves of lemongrass indicated great seasonality; their contents
were specifically higher in summer season. However, they were accumulated the least in bottom
leaves despite harsh condition of summer and winter (Fig. 4.8). Main function of the accumulation
of amino acids in free form appears to protect the younger tissues from stress damage by
associating with the cytoplasmic membranes (Simon-Sarkadi and Galiba, 1996).
Proline plays a role in osmotic adjustment (Yamada et al., 2005) and protects the sub-
cellular structures under adverse conditions (Ashraf and Foolad, 2007). Free proline
accumulation in response to stressful conditions occurs in a number of plant species (Ashraf
and Foolad, 2007; Farooq et al., 2009; Nasir et al., 2010). In present study, free proline
accumulation was significantly different in penultimate, middle and bottom leaves of
lemongrass. Among these leaves, penultimate leaf had higher proline concentration followed
by middle leaf (Fig. 4.9). Khan and Beena (2002) reported that in the leaves of Calotropis
procera, Senna holosericea and Aerva javanica, proline accumulation was greater in the
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young leaves, as noted in the present case too. Free proline accumulation in various plants
has been reported to be under severity of the prevailing stress (Zhu, 2001; Kavi-Kishore et
al., 2005; Wie-Tao et al., 2011). In autumn a decrease was observed in proline content of
lemongrass. In lemongrass leaves, greater free proline levels in summer months and its drop
to normal levels in spring and autumn season strongly witnessed its accumulation as a
protective response. Upon relief from stressful conditions, it readily disappeared, which has
been assigned to its breakdown in oxidative phosphorylation (Hare et al., 1998) and use as a
reserve substance in chlorophyll synthesis (William and Sharon, 1981).
Glycinebetaine is another osmoprotectant of great biological significance to plants,
since it has been reported to improve the cell water balance during adverse environmental
conditions (Ashraf and Foolad, 2007; Wahid et al., 2007) and it stabilizes the enzyme and
proteins at higher temperature (Kishitani et al., 1994; Allard et al., 1998). In the present study,
seasonal changes had great impact on GB content of lemongrass. Higher concentration of GB
was detected in summer (May-June) and winter periods (Dec-Jan) preferentially in penultimate
leaf and then in middle leaf while no such seasonal changes were notable in the bottom leaf (Fig.
4.10). Fluctuations in the levels of GB with the changing environmental conditions showed that
its accumulation is predominantly in response to stress. Such changes make the GB as an
osmoprotectant of great value in the tissues showing its accumulation (Boscaiu et al., 2011).
The validity of above changes in the osmoprotectants accumulation was ascertained by
drawing their mutual correlations and with the meteorological attributes (Table 4.4). Close
correlations GB with TFAA and free proline indicated that these nitrogenous compounds co-
occurred to perform a concerted action in younger leaves. However, positive correlations of
TFAA, free proline and GB with high temperature indicated their specific implication. The
summer season in Pakistan is too harsh; sometimes shooting the temperatures to over 50oC,
while the winter months are relatively mild (winter temperature 2-4oC). So the accumulation of
these osmoprotectants is important to maintain the cell water balance and metabolic functions at
the required pace, especially in actively metabolizing tissues (Rasheed et al., 2011).
In crux, prevailing environmental conditions appear to be real determinant of
osmoprotectants accumulation in leaves of different ages. The patterns of osmolytes
accumulation in the younger leaves are of great physiological significance in the survival of
lemongrass in different seasons.
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4.5 Secondary metabolites
In plants the relative proportion and total content of secondary metabolites can vary due
to seasonal and daily variation. Seasonal variation may lead to the synthesis of specific
secondary metabolites. In this part of the manuscript, lemongrass leaves were studied for
modulation in the level of soluble alkaloids, saponins, tannins, soluble phenolics, flavonoids and
anthocyanins, and their possible associations with seasonal variations.
4.5.1 Results
Statistical significance for the changes in the accumulation of various secondary
metabolites determined in the leaves of three ages of lemongrass over months is presented in
Table 4.5
Table 4.5: Analysis of variance of data (mean squares) for secondary metabolites accumulation in the lemongrass leaves under the influence of seasonal condition
SOV df Alkaloids Saponin Tannin Phenolics Flavonoid Anthocyanins
Block (B) 2 476.67 0.31 7.61 25.96 0.08 0.001
Leaves (L) 2 79275.51** 872.32** 29974.02** 2343.09** 78.48** 0.253**
Months (M) 11 205.23ns 4.90** 451.63** 1132.26** 8.71** 0.193**
L × M 22 168.61ns 2.46** 307.31** 973.40** 1.14** 0.102**
Error 70 119.00 0.36 7.62 598.97 0.10 0.005
Significant at: ns, non-significant; * and **, significant at P<0.05 and P<0.01 levels, respectively
Page 64
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a. Alkaloids
The data revealed significant differences in the leaves but a non-significant difference in
the months as well as there was no interaction of these factors for alkaloids concentration (Table
4.5). The alkaloids contents were lowest in the penultimate leaf while the bottom leaf indicated
their highest synthesis. A comparison of data revealed that for penultimate and middle leaves
although there were not much fluctuations in the alkaloids accumulation across the seasons,
nevertheless summer season (May-Jul) induced the alkaloids accumulation. The bottom leaves
indicated greater changes in the alkaloids accumulation with the changes in the seasonal
conditions (Fig. 4.11).
Fig. 4.11: Effect of seasonal variation on alkaloids concentration in the penultimate, middle and
bottom leaves of lemongrass
bc c abc aba
ab abc
bc abc ab a ab
bc c bc aba ab
bc cbc bc abc
abbc
abc
bca
abab
cc
bc bcab
cab
0
20
40
60
80
100
120
140
160
180
200
Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec
Penultimate Middle Bottom
Alk
aloi
ds (
μg/g
fre
sh w
eigh
t)
Leaf position/sampling months
Probability level:
Penultimate leaf = **Middle leaf = **Bottom leaf = **
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b. Saponins
Analysis of variance revealed significant differences in the leaves of three ages and
sampling months along with a significant interaction of these factors for the saponin contents
(Table 4.5). A comparison of leaves indicated not much difference in the saponin contents in the
penultimate and middle leaves but there was a large difference in the bottom leaf, which
displayed 2-3 times higher saponin contents. A comparison of sampling months revealed that
penultimate and middle leaves indicated slightly increased saponin contents in summer months,
which declined in the autumn season. Contrarily, bottom leaf showed much of the saponins
accumulation in spring and autumn seasons (Fig. 4.12).
Fig. 4.12: Effect of seasonal variation on saponins concentration in the penultimate, middle and
bottom leaves of lemongrass
cab bc bc ab a abc
c dbc
d
ab ab ca a a a a
bcd
bc c
cdbc bc
dcd
cdcd
a bcd
dbc
cd0
2
4
6
8
10
12
14
16
18
Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec
Penultimate Middle Bottom
Sap
onin
s (μ
g/g
fres
h w
eigh
t)
Leaf position/sampling months
Probability level:
Penultimate leaf = **Middle leaf = *Bottom leaf = *
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c. Tannins
Statistical analysis of results indicated significant difference in the leaves of different
ages and sampling months with significant interactions of these factors for tannins content (Table
4.5). The leaves indicated differential accumulation of tannins over various sampling months. In
penultimate leaf, the tannins contents were higher in the spring and autumn months while
reduced in the summer months. In middle leaf, both spring and summer seasons indicated greater
tannins accumulation, which declined in the autumn and winter seasons. In bottom leaf, there
was no clear trend of tannins accumulation over the seasons since large fluctuations were
observed in their contents. Overall penultimate leaf indicated the lowest while the bottom leaf
the highest tannins accumulation (Fig. 4.13).
Fig. 4.13: Effect of seasonal variation on tannins concentration in the penultimate, middle and
bottom leaves of lemongrass
fg fgab
cde a
ef fgbc
abc
dde
fg
bcd
ab
a abc cd
bbc
cdb
bc bc bce
bcd
bbc cd
acd cd
0
10
20
30
40
50
60
70
80
90
Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec
Penultimate Middle Bottom
Tan
nins
(μg
/g f
resh
wei
ght)
Leaf position/sampling months
Probability level:
Penultimate leaf = **Middle leaf = **Bottom leaf = **
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d. Soluble phenolic
The soluble phenolic contents in different leaf positions of lemon grass varied
significantly (P<0.001) in different seasons. Also there was significant interaction of leaves and
sampling months for soluble phenolics accumulation (Table 4.5). In both penultimate and middle
leaves, the soluble phenolics were low in the spring season, which showed a substantial increase
in the summer season. In the autumn season, there was a decline in the contents of soluble
phenolics, which again showed an increase at the onset of winter season, although this increase
was much lesser than that observed in summer season. In bottom leaf the soluble phenolics
accumulation was substantially lower than the penultimate and middle leaf, although this leaf
also showed their marked accumulation in the summer and autumn season (Fig. 4.14).
Fig. 4.14: Effect of seasonal variation on soluble phenolics content in the penultimate, middle
and bottom leaves of lemongrass
bc cdcd
cda a
ab
bcd d d
bc
bcd
bccd d
a aba
bcd
cdcd
bc b
d dcd
a ab aa
abc
abc
abc
abc
cd0
10
20
30
40
50
60
Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec
Penultimate Middle Bottom
Sol
uble
phe
nolic
s (μ
g/g
fres
h w
eigh
t)
Leaf position/sampling months
Probability level:
Penultimate leaf = **Middle leaf = **Bottom leaf = **
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e. Flavonoids
Data indicated significant difference in the leaves and sampling months of lemongrass
with a significant interaction of these factors for the flavonoid contents (Table 4.5). Seasonal
variation in the concentration of flavonoids irrespective of leaf age were quite significant. The
highest (p<0.001) concentration of flavonoids were observed in the summer season i.e. in May-
Jul in case of penultimate and middle leaves while in Jun in bottom leaves. However, the lowest
values were observed in spring and autumn seasons. Overall, penultimate leaf exhibited the
greatest flavonoids accumulation followed by middle leaf (Fig. 4.15).
Fig. 4.15: Effect of seasonal variation on flavonoids content in the penultimate, middle and
bottom leaves of lemongrass
aab
cdd
abc ab ab
ce e
ded
abc
bcb
cef
da
b
def
ded
bc
a abbc
def
cde aab
cde
ffg g
def
ab
0
1
2
3
4
5
6
7
8
9
10
Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec
Penultimate Middle Bottom
Fla
vono
ids
(μg/
g fr
esh
wei
ght)
Leaf position/sampling months
Probability level:
Penultimate leaf = **Middle leaf = **Bottom leaf = **
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f. Anthocyanins
The anthocyanin contents in different leaves of lemon grass varied significantly in
different sampling times with a significant interaction of these factors (Table 4.5). Pattern of
anthocyanins synthesis was more or less similar in the penultimate and middle leaves, while
bottom leaf deviated considerably. The anthocyanins concentration showed a decrease from the
spring (Mar-Apr) season and reached the lowest value in summer (May-Jun) season. The
anthocyanins showed an increase with the onset of autumn (Sep-Oct) season and attained the
highest value in Oct (1.194 A535). On the other hand, bottom leaf although showed much lower
anthocyanins than the penultimate and middle leaves (Fig. 4.16).
Fig. 4.16: Effect of seasonal variation on anthocyanin’s concentration in the penultimate, middle
and bottom leaves of lemongrass
bcbc
dde
efe
f efc
ab
bc
cd cef
deg
hef
de
ab b
c
cbc bc
abcd
cdc
a abbc
bcc
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec
Penultimate Middle Bottom
Ant
hocy
anin
s (A
535)
Leaf position/sampling months
Probability level:
Penultimate leaf = **Middle leaf = **Bottom leaf = **
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g. Correlation
The interrelationships drawn of the secondary metabolites in three lemongrass leaves
indicated that in penultimate leaf, the saponins were positively related with phenolics but
negatively with anthocyanins. Tannins were negatively correlated with flavonoids, while soluble
phenolics were negatively correlated with flavonoids and anthocyanins. The phenolics were
positively correlated with flavonoids but negatively with tannins. For middle leaf, the saponins
were negatively related to anthocyanins while for bottom leaf alkaloids were negatively
associated with anthocyanins. In case of environmental attributes, for penultimate leaf,
anthocyanins indicated negative correlation with maximum and minimum temperature, relative
humidity was negatively related to alkaloids, while evapotranspiration was positively correlated
with alkaloids and soluble phenolics but negatively with anthocyanins. For middle leaf, relative
humidity was negatively correlated with alkaloids and evapotranspiration was positively
correlated with tannins and negatively with anthocyanins. In bottom leaf, soluble phenolics
contents were positively correlated with maximum and minimum temperature and
evapotranspiration (Table 4.6).
4.5.2 Discussion
Plant secondary products are formed via distinctive metabolic pathways and act as line of
defense against biotic and abiotic adversaries (Winkel-Shirley, 2002; Aflatuni, 2003; Wahid
and Tariq, 2008). However, thorough studies are scarce with respect to seasonal changes in the
concentrations of plant secondary products in plants, although sporadic reports are available (Ma
et al., 2003; Maknickiene and Asakaviciute, 2008). In the present research, lemongrass leaves of
three ages were investigated for nitrogen-containing and non-nitrogen containing secondary
metabolites on monthly intervals.
Alkaloids are a diverse group of nitrogen-containing compounds, which play role against
herbivores. Pencikova et al. (2011) reported a rise in the alkaloids contents in the leaves of
Maccleaya microcorpa during spring season as compared to autumn season. In the present
research the penultimate and middle leaves indicated a substantially reduced alkaloids and
saponins contents over the seasons (sampling months), whilst the bottom leaf indicated the
greatest accumulation. Despite that, leaves of all the ages exhibited higher contents of both
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64
Table 4.6: Correlation coefficient (r) of meteorological attributes with the secondary metabolites of lemongrass at three leaves positions penultimate, middle, bottom X –Variable Y-Variable Penultimate Middle Bottom a. Mutual correlations of secondary metabolites Alkaloid Saponin -0.474ns -0.311ns 0.056ns Tannin 0.046ns 0.039ns 0.190ns Phenolics 0.449ns -0.114ns 0.026ns Flavonoid -0.077ns -0.140ns -0.468ns Anthocyanins -0.217ns -0.283ns -0.890** Saponin Tannin -0.140ns 0.107ns -0.244ns Phenolics 0.742** 0.224ns 0.304ns Flavonoid 0.178ns 0.192ns 0.046ns Anthocyanins -0.776** -0.723** 0.182ns Tannin Phenolics -0.152ns -0.390ns -0.126ns Flavonoid -0.659* -0.152ns -0.300ns Anthocyanins -0.257ns 0.102ns -0.405ns Phenolics Flavonoid 0.610* 0.562ns -0.176ns Anthocyanins -0.850** -0.365ns 0.667ns Flavonoid Anthocyanins -0.289ns -0.412ns -0.159ns b. Correlations environmental variable with secondary metabolites Max. temprature Alkaloid 0.473ns 0.468ns 0.062ns Saponin -0.214ns 0.266ns 0.242ns Tannin 0.557ns -0.252ns -0.286ns Phenolics 0.343ns 0.148ns 0.873** Flavonoid -0.368ns -0.447ns -0.367ns Anthocyanins -0.642* -0.433ns -0.132ns Min. temprature Alkaloid 0.227ns 0.219ns 0.055ns Saponin -0.114ns 0.389ns 0.404ns Tannin 0.546ns -0.354ns -0.424ns Phenolics 0.291ns 0.173ns 0.852** Flavonoid -0.311ns -0.444ns -0.171ns Anthocyanins -0.691* -0.359ns -0.018ns Relative humidity Alkaloid -0.871** -0.773** -0.034ns Saponin 0.210ns -0.046ns 0.256ns Tannin -0.151ns -0.323ns -0.293ns Phenolics -0.460ns 0.031ns -0.277ns Flavonoid 0.091ns 0.021ns 0.012ns Anthocyanins 0.286ns 0.563ns -0.035ns Evapotranpiration Alkaloid 0.656* 0.511ns -0.289ns Saponin -0.301ns 0.424ns 0.109ns Tannin 0.411ns 0.626* -0.192ns Phenolics 0.698* 0.210ns 0.735** Flavonoid -0.138ns -0.207ns 0.009ns Anthocyanins -0.724** -0.670* -0.422ns Average rainfall Alkaloid -0.336ns -0.297ns -0.229ns Saponin 0.065ns 0.416ns 0.369ns Tannin 0.271ns -0.241ns -0.379ns Phenolics -0.026ns 0.133ns 0.520ns Flavonoid -0.090ns -0.312ns -0.089ns Anthocyanins -0.438ns 0.101ns 0.555ns Significant at: ns, non-significant; * and **, significant at P<0.05 and P<0.01 levels
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alkaloids and saponins during summer (Fig. 4.11-4.12). The differences seen in the accumulation
of these nitrogen-containing products revealed that the younger leaves of lemongrass do not
preferentially induce the synthesis of alkaloids and saponins because of their no special
contribution in the primary growth responses. On the other hand, old (bottom) leaf indicated
quite higher accumulation of both these metabolites, which might be related to their age, since
aging induced saponins synthesis in Tamarix aphylla (Achakzai et al., 2009).
Phenolics are a large class of compounds, and are synthesized via the shikimic acid
pathway. Major classes of phenolics include lignins, tannins, flavonoids and anthocyanins. Being
water soluble, the synthesis and accumulation of phenolics is of great physiological significance
(Taiz and Zeiger, 2010). Prevailing growth conditions have great influence on their synthesis
(Sgherri et al., 2004; Wahid and Tariq, 2008), while the accumulation in various plants are not
well studied. Determinations made for tannins, soluble phenolics, flavonoids and anthocyanins
on the leaves of three ages of lemongrass revealed that seasonal changes had great influence on
the patterns and levels of their accumulation. Summer season did not induced the synthesis of
tannins in young leaves, while their synthesis increased with advancing leaf age (Fig. 4.13),
while soluble phenolics (Fig. 4.14) and flavonoids (Fig. 4.15) indicated greater synthesis in the
summer season, which declined to normal levels in spring and autumn season. Contrarily, the
anthocyanins synthesis took place only in the winter season, but severely declined in the summer
months (Fig. 4.16). The phenolics are synthesized after the elimination of ammonia with the
phenylalanine ammonia lyase (PAL) enzyme, the activity of which is much dependent on the
biotic and abiotic stress conditions (Collinge and Slusarenko, 1987; Wu and Lin, 2002). The
synthesis of various phenolics compounds takes place with the branching of pathways from the
trans-cinnamic acid for the synthesis of a variety of phenolics compounds (Adeyemi, 2011). In
this research, the synthesis of higher amounts of soluble phenolics and flavonoids in the younger
leaves in the summer and winter seasons suggested that these metabolites have definitive
physiological roles under adverse conditions, and the anticipated roles might be the osmotic or
antioxidative. However, a higher synthesis of tannins (with complex structure and water
insoluble) in the lemongrass leaves with the advancing age indicated that they have no-specific
physiological roles and plausibly leaf aging induces their synthesis. The anthocyanins are
reported for their greater roles in the reddening during winter season (Chalker-Scott, 1999),
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Metabolic pathways in plants are regulated in a coordinated manner to protect plants
when adverse conditions occur during the year. To look into the possibility of their synthesis in
various seasons, mutual correlations of secondary metabolites and their association with the
seasonal conditions prevailing during various months of the year were established. It was noted
that for penultimate leaf most of the secondary metabolites were co-accumulated, which was not
the case for middle or bottom leaf, thereby showing that (actively metabolizing leaves
strategically synthesize the metabolites to ensure survival as a coordinated way. A greater
synthesis of the physiologically active phenolic compounds in summer seasons indicated that
accumulation of soluble phenolics and flavonoids is more important in dehydrating summer
conditions while anthocyanins synthesis was mainly related to the winter season.
In conclusion, seasonal variation has great impact on qualitative and quantitative contents
of secondary metabolites in lemongrass. The alkaloid, phenolic and flavonoid contents in
lemongrass leaves exhibited a substantial increase with increasing temperature while the
synthesis of anthocyanins was relatively better in winter season. These trends of secondary
metabolites accumulation have great implications in the survival of actively metabolizing tissues
under the adverse seasonal conditions.
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4.6 Vitamins
The seasonal variation is related to changes in rain fall, light and temperature that affect
vitamins metabolism in leaves of lemongrass. Vitamins are organic substances which act as
coenzyme (NAD, NADP and FAD) in many oxidation-reduction reactions. They also act as
antioxidant and protect membrane and other hydrophobic compartments from damage. In this
part of the thesis, the seasonal changes were studied in niacin, ascorbic acid, riboflavin and
reducing powers assay (RPA), in the lemongrass leaves of three ages, the results of which are
given below.
4.6.1 Results
Leaf age and seasonal variations indicated significant differences in the concentrations of
vitamins and reducing powers assay. Statistical analyses of results of these parameters are given
in Table 4.7.
Table 4.7: Analysis of variance of data (mean squares) for niacin, ascorbic acid, riboflavin and reducing power assay of lemongrass leaves under the influence of seasonal condition
SOV df Niacin ASA Riboflavin RPA
Block (B) 2 0.001 0.023 0.018 0.018
Leaves (L) 2 32.753** 84.161** 24.940** 6.867**
Months (M) 11 0.766** 0.297** 0.329** 1.012**
L × M 22 0.505** 0.526** 0.111** 0.152**
Error 70 0.005 0.062 0.021 0.027
Significant at: ns, non-significant and **, significant at P<0.01 levels
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a. Niacin
The data presented that there was a tendency of lemongrass niacin concentrations to
display changes in its levels during the sampling months and leaves of three ages with a
significant interaction of these factors (Table 4.7). The niacin exhibit that penultimate leaf had
higher levels (3.22 µg g-1) among the leaves. The niacin contents in penultimate leaf were
maximum in summer months (June and July, 3.11 and 3.22 µg/g, respectively) while remained
mimum in September (1.31 µg/g) and October (1.24 µg/g). In the middle leaf the highest value
of niacin was noticed in June and July whereas comparable to each other in the remaining
months (11.39 µg g-1). The bottom leaf exhibited entirly different behavior. Net accumulation of
niacin was very low and did not change much over the months. Overall, the niacin contents were
the greatest in penultimate leaf follwed by middle leaf (Fig. 4.17).
Fig. 4.17: Effect of seasonal variation on Niacin concentration in the penultimate, middle and
bottom leaves of lemongrass
bcc
ffg
ba a
fg g g
ed
cd
ed
bca
ab
ede
c bc
b b bc bcd
ab a ab bc bcd
bcd
bcd
bcd
0
0.5
1
1.5
2
2.5
3
3.5
Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec
Penultimate Middle Bottom
Nia
cin
(μg/
g fr
esh
wei
ght)
Leaf position/sampling months
Probability level:
Penultimate leaf = **Middle leaf = **Bottom leaf = ns
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b. Ascorbic acid (AsA)
There was significant difference in the leaves of different ages and sampling months with
significant interaction of these factors for the ascorbic acid contents (Table 4.7). The AsA
contents in penultimate leaves varied throughout the year. Penultimate leaf AsA contents
decreased from Jan to Apr and then sharply increased in May and remained same till Jul, while
gradually decreased during the rainy season and trend was continued till Oct followed by further
increased Nov and Dec. A similar trend was observed in middle leaf but the contents was quite
low throughout the years. As for bottom leaves, the AsA content was low and there were no
marked changes in AsA except a decline in the summer months (Fig. 4.18).
Fig. 4.18: Influence of seasonal condition on Ascorbic acid concentration in the penultimate,
middle and bottom leaves of lemongrass
ab bccd
cd
a aa
bcd
cd dbc
ab
abc
bccd
abc ab a
bccd cd
abc ab
bcd
bcd
edc
abcd
ef fde
a abc
abcd
ab bcd
0
1
2
3
4
5
6
Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec Jan
Feb
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Jul
Aug Sep Oct
Nov
Dec Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec
Penultimate Middle Bottom
Asc
orbi
c ac
id (
μg/g
fre
sh w
eigh
t)
Leaf position/sampling months
Probability level:
Penultimate leaf = **Middle leaf = **Bottom leaf = **
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c. Riboflavin
The leaf age and prevailing seasonal conditions indicated significant differences for the
contents of riboflavin with a significant interaction of these factors (Table 4.7). The riboflavin
contents indicated sharp changes during the entire year in penultimate leaf, being highest in
summer (May, Jun and Jul) and serially reduced thereafter and were the lowest in Oct. The
winter conditions again caused them to accumulate. The middle leaf also showed the changes
more or less similar to the penultimate leaf but the riboflavin contents were substantially low.
The bottom leaf indicated no marked changes in the riboflavin contents throughout the years
some increase in the spring and autumn months. Overall, the penultimate leaf synthesized the
highest amount of riboflavin followed by middle leaf (Fig. 4.19).
Fig. 4.19: Effect of seasonal variation on Riboflavin concentration in the penultimate, middle and
bottom leaves of lemongrass
bde
eff
da
bccd de
fde
ab
cde
f f def
ba
def
def
def
efcd
c
ef cd
acd
ede
fde
ff
cd def bc ab
f0
0.5
1
1.5
2
2.5
3
Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec
Penultimate Middle Bottom
Rib
ofla
vin
(μg/
g fr
esh
wei
ght)
Leaf position/sampling months
Probability level:
Penultimate leaf = **Middle leaf = **Bottom leaf = **
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d. Reducing power assay (RPA)
Results for the RPA indicated significant differences in the leaves of various ages and the
sampling months with a significant interaction of leaves and months (Table 4.7). For penultimate
and middle leaves, the value of RPA was relatively lower in the spring season (Mar-Apr), which
increased appreciably in the summer months and reached its maximum in Jun. This value
declined subsequently and was the lowest again in autumn season and exhibited a decline in the
winter months also. However, the middle leaf indicated relatively sharper changes in RPA value
than the penultimate leaf. In case of bottom leaf, no specific pattern of RPA value was evident
although it was relatively higher in spring season and lower in summer months. Overall, the
middle leaf indicated a higher RPA value followed by penultimate leaf (Fig. 4.20).
Fig. 4.20: Effect of seasonal variation on reducing power assay concentration in the penultimate,
middle and bottom leaves of lemongrass
bcbc
a
a
bbc
dcd
bc b bccd
cbc bc
aab
ce e
d
ab bce e
de
abb
acd
cde
e ecd c c
e0
0.5
1
1.5
2
2.5
3
Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec
Penultimate Middle Bottom
Red
ucin
g po
wer
s as
say
(A70
0)
Leaf position/sampling months
Probability level:
Penultimate leaf = **Middle leaf = **Bottom leaf = **
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e. Correlation
To find the implication of the changes in vitamin attributes some correlations were
established at different leaf positions of lemongrass. In the penultimate and middle leaves,
contents of all the vitamins were correlated with each other while for bottom leaf these
relationships were absent altogether. As for meteorological variables, in penultimate leaf, RPA
was positively related with maximum and minimum temperatures and evapotranspiration, while
the vitamins or RPA of middle and bottom leaves were correlated to none of the meteorological
attributes.
Table 4.8: Correlation coefficient (r) of meteorological attributes with the Vitamin content of lemongrass at three leaves positions penultimate, middle, bottom
X-Variable Y-Variable Penultimate Middle Bottom a. Mutual correlations of Vitamins Niacin AsA 0.909** -0.756** -0.404ns Riboflavin 0.804** 0.865** -0.516ns RPA 0.601* -0.706* 0.064ns AsA Riboflavin, 0.892** -0.898** 0.348ns RPA 0.586* -0.571* -0.133ns Riboflavin RPA 0.657* -0.750** 0.317ns b. Correlations environmental variable with Vitamins Max. temperature Niacin -0.007ns 0.378ns 0.160ns AsA 0.215ns -0.063ns -0.333ns Riboflavin 0.145ns 0.186ns -0.514ns RPA 0.656* 0.671* -0.236ns Min. temperature Niacin 0.026ns 0.376ns 0.327ns AsA 0.202ns -0.037ns -0.227ns Riboflavin 0.219ns 0.193ns -0.519ns RPA 0.587* 0.162ns -0.282ns Relative humidity Niacin -0.064ns -0.166ns 0.353ns AsA -0.249ns -0.039ns 0.543ns Riboflavin 0.073ns -0.134ns 0.009ns RPA 0.225ns -0.540ns -0.186ns Evapotranspiration Niacin 0.021ns 0.181ns 0.479ns AsA 0.058ns -0.037ns 0.075ns Riboflavin 0.297ns 0.165ns -0.531ns RPA 0.655* 0.005ns -0.330ns Average rainfall Niacin 0.210ns 0.448ns 0.208ns AsA 0.417ns 0.128ns -0.610ns Riboflavin 0.251ns 0.374ns -0.558ns RPA 0.438ns 0.488ns -0.102ns Significant at: ns, non-significant; * and **, significant at P<0.05 and P<0.01 levels
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4.6.2 Discussion
Vitamins are important enzymatic cofactors play important role in plant metabolism
because of their redox chemistry. However, there are various factors such as plant age and
prevailing conditions that greatly influence the vitamins contents and metabolic roles (Denslow
et al., 2007; Mahmood et al., 2012). In the current research, the determinations made on the
lemongrass leaves in various seasons revealed that young leaves showed greater contents of
niacin (Fig. 4.17), AsA (Fig. 4.18) and riboflavin (Fig. 4.19), while old (bottom) leaf did not
show their accumulation with the seasonal changes during two years. Furthermore, RPA
increased highly in the summer season followed by winter season while decreased markedly in
spring and autumn seasons (Fig. 4.20).
The vitamins play various metabolic roles such as coenzymes in enzyme activities (Taiz
and Zeiger, 2010) and alleviation of oxidative stress (Mahmood et al., 2012). The role of niacin
is to function as coenzyme in the regulation of carbohydrate metabolism. Riboflavin and its
derivatives FMN and FAD are indispensable components of photosynthesis, energy generation
and redox metabolism in plants under normal conditions (Sandoval et al., 2008). Under abiotic
stresses too, different vitamins are involved in alleviating the stress effects and better plant
survival (Demmig-Adams and Adams, 2002; Rapala-Kozik et al., 2008; Leuendorf et al., 2010).
A higher contents of these vitamins in the penultimate and to some extent in middle leaf of
lemongrass in the summer and winter seasons strongly suggests the maintenance of metabolic
activities under the changing seasons. Secondly, leaf age is a factor that also determines the
contents of vitamins and thus the operation of metabolic activities. The contents of all the
vitamins were substantially low in aged bottom leaf, which further suggested that as a metabolic
requirement, younger tissues have a greater demand for the vitamins than the aged tissues.
Generation of reducing powers in appropriate concentrations is important for regulation
of cellular metabolic functions. Therefore, availability of reducing powers under adverse
conditions is critical. A comparison of data drawing the correlations of the vitamins and RPA
values indicated that all were positively and tightly interrelated in penultimate and middle leaves
but not in bottom leaf (Table 4.8). This is important in view of the fact that younger tissues are
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the sites of major metabolic activities while bottom (aged) leaves have lived their life and are
heading towards senescence, where there is least requirement for these metabolites.
Among the environmental variables, a positive relationship of RPA with maximum and
minimum temperatures and evapotranspiration in case of penultimate leaf and a positive
relationship of RPA with maximum temperature in middle leaf (Table 4.8) revealed that
generation of reducing powers is much important under high temperature and evapotranspiration
load (again related to high temperature) in young actively metabolizing leaves of lemongrass. Its
plausible role may be the provision of energy for the enzymatic activities triggered by the
vitamins (Rapala-Kozik et al., 2008; Leuendorf et al., 2010).
It summarized that an increase in vitamin contents in the lemongrass leaves is strongly
dependent upon the prevailing conditions while the hot and dry conditions are more conducive to
their synthesis. This might also be possibly involved in the alleviation of oxidative load on the
lemongrass leaves especially on the younger leaves.
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4.7 Photosynthetic Pigment
Photosynthetic pigments are integral compound of light harvesting systems in plants. Any
distortion induced in the light harvesting ability due to ambient factors declines the
photosynthetic activity of plants. Results indicated considerable changes in the photosynthetic
pigments of lemongrass in various seasons. The results of chl a, chl b, their total chl and
carotenoids are described below:
4.7.1 Results
The results regarding the photosynthetic pigments contents in the leaves of three ages and
in different seasons have been presented in Table 4.9.
Table 4.9: Analysis of variance of data (mean squares) for chlorophyll a and chlorophyll b, total chlorophyll and carotenoid contents of lemongrass under influence of seasons SoV df Chl a Chl b Total Chl Car
Block (B) 2 0.02 0.01 0.38 0.010
Leaf (L) 2 20.92** 2.89** 16.62** 1.693**
Month (M) 11 1.45** 0.26** 3.89** 0.121**
L × M 22 0.28** 0.03* 0.90** 0.021**
Error 70 0.10 0.02 0.24 0.004
ns = non-significant; *, **, *** = significant at 0.05, 0.01 and 0.001 levels, respectively
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a. Chlorophyll (Chl) a
Significant differences were noted in the sampling months and leaves of lemongrass with
a significant interaction of these factors for Chl-a contents (Table 4.9). Penultimate and middle
leaves indicated a lower contents of Chl-a in the spring (Feb-Apr) and autumn season (Aug-Oct),
while it was relatively higher in summer and winter seasons. On the other hand, bottom leaf
indicated higher Chl-a in the cool season (Nov-Feb), which declined in spring season and again
slightly increased in the summer season. Overall, Chl-a content was substantially higher in
penultimate and middle leave while it was much lower in the bottom leaf (Fig. 4.21)
Fig. 4.21: Effect of seasonal variation on chlorophyll a concentration in the penultimate, middle
and bottom leaves of lemongrass
vcxz
cdab
acd
d dc
aa ab ab
c
a ab b ccd cd cd
aba a
cb
ab aab ab
cbc
dcd d cd
abc ab a
ab
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec
Penultimate Middle Bottom
Chl
orop
hyll
a (m
g/g
fres
h w
eigh
t)
Leaf position/sampling months
Probability level:
Penultimate leaf = **Middle leaf = **Bottom leaf = **
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b. Chlorophyll b (point 46 of 2nd reviewer?)
Results indicated significant difference in the leaves of various ages and sampling months
with significant interaction of the factors for leaf Chl-b contents (Table 4.9). In penultimate leaf,
the Chl-b contents was low in winter season (Nov-Jan), which indicated a slight increase in the
summer months (May-July) but declined in the autumn months (Aug-Sep). A trend more or less
similar to the penultimate leaf was noted in middle leaf. The bottom leaf indicated no significant
fluctuations in the Chl-b contents over all seasons. Overall, penultimate and middle leaf
exhibited similar Chl-b contents while it was substantially lower in bottom leaf (Fig. 4.22)
Fig. 4.22: Effect of seasonal variation on chlorophyll b concentration in the penultimate, middle
and bottom leaves of lemongrass
dbc ab
bcab
cd cda
abc
cd cd
bc bb
c cab
aab
bc c c
aab
bcc
cb
c c c c c c
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec
Penultimate Middle Bottom
Chl
orop
hyll
b (m
g/g
fres
h w
eigh
t)
Leaf position/sampling months
Probability level:
Penultimate leaf = **Middle leaf = **Bottom leaf = **
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c. Total chlorophyll (Chl-T)
The two-way analysis of variance of the data indicated significant differences in the
leaves of various ages and sampling months with significant interaction of these factors for total
chlorophyll contents of leaves (Table 4.9). The Chl-T of the penultimate leaf decreased from Jan
to Apr but increased thereafter up to Jul. It again decreased in Aug-Sep and displayed a gradual
increase up to Dec. The middle leaf indicated the changes in Chl-T similar to those of
penultimate leaf except the Chl-T was relatively lesser in Dec-Jan. The bottom leaf indicated a
higher Chl-T in Jan-Feb, which declined in March and was steadier up to Jul. It showed a slight
increase in Aug but reduced in Sep and remained steady up to Dec. Overall, the Chl-T was
comparatively higher in middle leaf followed by penultimate leaf but the lower in bottom leaf
(Fig. 4.23).
Fig. 4.23: Effect of seasonal variation on total chlorophyll contents in the penultimate, middle
and bottom leaves of lemongrass
cab
ac cd cdc
aab
bc bcd
bcd
abc
bcd
cdd cd
bcd
ab aab
cdbc
d
a abcd
efde
fgef
gef
gg fg
cdef
cdef
abc
bcde
0
1
2
3
4
5
6
7
Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec
Penultimate Middle Bottom
Tot
asl c
hlor
ophh
yll (
mg/
g fr
esh
wei
ght)
Leaf position/sampling months
Probability level:
Penultimate leaf = **Middle leaf = **Bottom leaf = **
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d. Carotenoids (Car)
Lemongrass leaves of different ages and sampling months showed significant differences,
also with a significant interaction of these factors for Car contents (Table 4.9). In penultimate
leaf the Car contents were lower from Jan up to Mar, which increased thereafter and attained the
highest value in June. Car showed a decreasing trend thereafter and was the lowest in Oct, and
then an increase in Nov and Dec. A more or less trend similar to penultimate leaf was observed
in middle leaf for changes in Car. On the other hand, bottom leaf indicated higher Car in Jan,
which declined up to March. It showed a steady Car contents up to Oct but a slight increase in
Nov-Dec. Overall, Car contents were relatively higher in penultimate and middle leaf while it
was quite low in the bottom leaf (Fig.4.24).
Fig. 4.24: Effect of seasonal variation on carotenoid concentration in the penultimate, middle and
bottom leaves of lemongrass
dbc
a abc
dcd
dbc
bcd
cd
decd
ba
cde e
decd
bcde de
cdb
acd
ef
def
efcd bc
def
ef f0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec
Penultimate Middle Bottom
Car
oten
oids
(m
g/g
fres
h w
eigh
t)
Leaf position/sampling months
Probability level:
Penultimate leaf = **Middle leaf = **Bottom leaf = **
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e. Correlation
The correlations of leaf pigments showed that in penultimate leaf, the Chl-a, Chl-T and
Car were positively interrelated. For middle leaf, Chl-a was positively related to Chl-T, while
Chl-b and Chl-T were positively associated to Car. For bottom leaf, no pigments were
interrelated. As for meteorological attributes, in penultimate leaf the maximum temperature and
evapotrasnspiration were positively related to Chl-a, Chl-T and Car. For middle and bottom
leaves, none of the meteorological attributes were correlated with pigment contents (Table 4.10).
Table 4.10: Correlation coefficient (r) of meteorological attributes with the Pigment content of lemongrass at three leaves positions penultimate, middle, bottom X-Variable Y-Variable Penultimate Middle Bottom a. Mutual correlations of pigments Chl-a Chl-b 0.274ns -0.079ns 0.451ns Chl-T 0.976** 0.910** -0.002ns Car 0.721** 0.421ns -0.126ns Chl-b Chl-T 0.476ns 0.341ns 0.212ns Car 0.418ns 0.580* -0.329ns Chl-T Car 0.753** 0.614* 0.208ns b. Correlations environmental variable with pigments Max. temperature Chl-a 0.593* -0.259ns -0.300ns Chl-b 0.223ns 0.237ns -0.248ns Chl-T 0.593* -0.145ns -0.358ns Car 0.622* 0.145ns -0.058ns Min. temperature Chl-a 0.553ns -0.264ns -0.179ns Chl-b 0.092ns 0.076ns -0.202ns Chl-T 0.527ns -0.218ns 0.227ns Car 0.650ns 0.063ns -0.183ns Relative humidity Chl-a -0.280ns 0.061ns -0.427ns Chl-b -0.524ns 0.549ns -0.289ns Chl-T -0.374ns -0.227ns 0.493ns Car -0.246ns -0.280ns -0.244ns Evapotranspiration Chl-a 0.590* 0.114ns -0.297ns Chl-b 0.213ns -0.514ns -0.412ns Chl-T 0.648* -0.106ns -0.397ns Car 0.691* 0.416ns -0.010ns Average rainfall Chl-a 0.302ns 0.076ns -0.059ns Chl-b -0.213ns 0.418ns -0.262ns Chl-T 0.230ns -0.246ns -0.125ns Car 0.504ns -0.030ns -0.131ns
Significant at: ns, non-significant; * and **, significant at P<0.05 and P<0.01 levels
Page 88
81
4.7.2 Discussion
Lemongrass is a C4 plant and can grow well under relatively higher ambient
temperatures. Although no report is available which may show any direct relationship with a
reduction in plant dry matter yield, this fact also cannot be delineated. This is because
photosynthetic rate of the plant is dependent upon the light harvesting efficiency (Ruban, 2009).
The leaves of different ages show differential photochemical efficiency and thus likely to have
differential contribution to the plant performance (Kitajima et al., 2002). In this research, the
determinations made the photosynthetic pigment concentration in the leaves of three ages
indicated that relatively younger (penultimate and middle) leaves indicated substantially greater
pigments of Chl-a (Fig. 4.21), Chl-b (Fig. 4.22), Chl-T (Fig. 4.23) and Car (Fig. 4.24) all round
the year, while older leaf was on a disadvantage. However, it is important to note that during
summer season there was an increase in the contents of all the photosynthetic pigments. The
results of this research are contrary to the earlier reports, wherein it has been reported that high
light intensity induced photo-oxidative damage is a major deterrent of the chlorophyll species
while the carotenoids are able to sustain their contents (Ramel et al., 2013). This appears to be a
better adaptation to the supra-optimal ambient temperatures in the summer month.
It is important to note that the Car contents of the young leaves remained fairly high
during the summer season while they were in the normal range during the autumn, winter and
spring seasons (Fig. 4.24). The Car in addition to their role as accessory light harvesting
pigments, are also effective scavenger of the ROS produced during adverse conditions (Ramel et
al., 2012). It is plausible that production of ROS is efficiently doused by the high contents of Car
especially in the chloroplast of younger (penultimate and middle) leaves, and protecting the
photosynthetic pigments in the photosystems. Furthermore, the association of both the
chlorophyll may be differential.
To appraise the validity of the above observations, some correlations were established of
the photosynthetic pigments mutually as well as with the prevailing meteorological conditions
round the year (Table 4.10). These correlation indicated that the contents of both the chlorophyll
species (Chl-a and b) were independent of each, rather Chl-a and Chl-T were closely associated
to the Car contents. These data further strengthened the notion that the Car was the important
element in rescuing the younger leaves from the harsh conditions of summer. Likewise, in the
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penultimate leaf the Chl-a, Chl-T and the Car were positively correlated with the maximum
temperature and evapotranspiration throughout the year, which further showed that the
maintenance of greater chlorophyll contents was mainly related to the maintenance of Car, while
the older (bottom) leaf was on a disadvantage due to being having low photosynthetic efficiency
and probably heading towards senescence.
In conclusion, seasonal variation in meteorological attributes affects the photosynthetic
pigments levels in lemongrass leaves. Greater contents of Car and Chl-T suggested that the Car
played a major role in encountering the photo-oxidative damage and rescuing the photosystems
from the adverse conditions like those of heat stress and high evapotranspiration load.
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4.8 Nutritional quality characteristics
Lemongrass shows a marked variation in nutritional profile during different seasons
which have been widely associated to variations in environmental variables. The variations in
such attributes usually appears to be greatly affected by rise in plant age as well as changes in
seasonal variation during different seasons. In this part of the dissertation, the seasonal changes
were studied in NDF, ADF, ADL, cellulose, silica, starch and protein contents in penultimate,
middle and bottom leafs of lemongrass.
4.8.1 Results
Various nutritional quality characteristics indicated significant changes in all the leaves
analyzed during the sampling months. The results regarding statistical analysis are given in Table
4.9.
Table 4.11: Analysis of variance of data (mean squares) for nutrient detergent fiber, acid detergent fiber, acid detergent lignin, cellulose, silica, starch and protein content of lemongrass under influence of seasonal condition
SoV df NDF ADL ADF Cellulose Silica Starch Protein
Block (B) 2 0.08 0.06 0.50 3.01 0.01 0.18 0.03
Leaf (L) 2 193.30** 67.42** 114.42** 4919.24** 21.70** 127.36** 381.17**
Month (M) 11 2.33** 1.09** 30.94** 46.68** 0.18** 9.69** 2.85**
L × M 22 2.01** 0.53* 7.43** 6.28** 0.04ns 2.66** 2.48**
Error 70 0.60 0.28 0.96 2.01 0.02 0.578 0.716
Significant at: ns, non-significant; * and **, significant at P<0.05 and P<0.01 levels
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a. Nutrient detergent fiber (NDF)
Stsatistical analysis of results showed significant difference in the lemongrass leaves of
three ages and the sampling months for NDF contents with a significant interaction of these
factors (Table 4.11). In penultimate and middle leaf the NDF decreased from Jan and was the
lowest in May. It showed an increase from Jun and reached its highest in Aug, declined in Sep-
Nov and then increased in Dec to almost the level of Jan. On the other hand, in bottom leaf, the
NDF remained steady up to Aug except a small decline in Jun. However, it was the lowest in
winter months (Oct-Nov) but increased in Dec. Overall, NDF was higher in middle leaf, while
penultimate leaf values more or less comparable to bottom leaf (Fig. 4.25)..
Fig. 4.25: Effect of seasonal variation on Nutrient detergent fiber content in the penultimate,
middle and bottom leaves of lemongrass
bccd d cd
ed
ba
abc cd
bc
b bccd
d dc
ba b
cd cd c
abc
c abc abc
abc
ca a a
c cab
0
2
4
6
8
10
12
14
16
18
20
Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec
Penultimate Middle Bottom
ND
F (
%)
Leaf position/sampling months
Probability level:
Penultimate leaf = **Middle leaf = **Bottom leaf = *
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b. Acid detergent fiber (ADF)
Analysis of variance of the data for ADF indicated significant difference in lemongrass
leaves and sampling months with a significant difference of these factors (Table 4.11). In the
acid detergent fiber (ADF) contents between the evaluated seasons for penultimate middle and
bottom leaves indicated highly significant results (Table 4.11). With small differences, the trend
of ADF contents of penultimate, middle and bottom leaves were similar. ADF was generally low
from Jan to Apr. The ADF substantially increased in May-Jul (summer season) but declined
steadily in autumn and winter months. Overall, middle leaf indicated a greater ADF followed by
penultimate leaf (Fig. 4.26).
Fig. 4.26: Effect of seasonal variation on Acid detergent fiber content in the penultimate, middle
and bottom leaves of lemongrass
d dcd
db
a abbc
c cd cdd e
fe e
ba ab
c bc bc
d
debc
ede
a a abc c cd cd cd
0
5
10
15
20
25
Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec
Penultimate Middle Bottom
AD
F (
%)
Leaf position/sampling months
Probability level:
Penultimate leaf = **Middle leaf = **Bottom leaf = **
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c. Acid detergent lignin (ADL)
Results revealed significant differnce in the leaves of lemongrass as well as sampling
months with significant interaction of both these factors for ADL (Table 4.11). It is evident from
the results that in all leaves, the ADL was low during winter season, which furher declined in the
spring season (Feb-Apr). The ADL increased from May and onward and was highest in Jul but
declined afterwards and was the lowest in Oct-Nov and increased again to the level dectected in
Jan. Overall, the ADL was the highest in bottom leaf but the lowest in penultimate leaf (Fig.
4.27).
Fig. 4.27: Effect of seasonal variation on Acid detergent lignin content in the penultimate,
middle and bottom leaves of lemongrass
c cd cd d cdbc
aab ab
cbc
dc
bc bc bcc
bca a a
bc bcb b
bcd
bcd
cdcd
bcd
ab abc
bcd
dbc bc
d
0
1
2
3
4
5
6
7
8
9
Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec
Penultimate Middle Bottom
AD
L (
%)
Leaf position/sampling months
Probability level:
Penultimate leaf = **Middle leaf = **Bottom leaf = **
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d. Cellulose
Statistical analysis of data indicated significant differences in the leaves of three ages and
sampling months with a significant interaction of both these factors for cellulose contents (Table
4.11). The penultimate leaf indicated the lowest contents of cellulose, which, with some
fluctuations remained steady throughout the year. The middle leaf showed lower cellulose
content during Jan, which declined up to Mar (spring season) but again showed an increase in
May and was the highest in Jul-Aug, but declined steadily during rest of the months/seasons. The
bottom leaf manifested the trend of cellulose synthesis similar to the middle leaf but the contents
were too low. Overall cellulose was highest in middle leaf followed by bottom leaf (Fig. 4.28).
Fig. 4.28: Effect of seasonal variation on Cellulose content in the penultimate, middle and
bottom leaves of lemongrass
cd cdd de
b b b bc bca ab
cd
dde e
cdab ab
a abb c c cd ab
cab
ccd c
aba ab
abc
cd c c d0
5
10
15
20
25
30
35
40
45
50
Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec
Penultimate Middle Bottom
Cel
lulo
se (
%)
Leaf position/sampling months
Probability level:
Penultimate leaf = **Middle leaf = **Bottom leaf = **
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e. Silica
Significant differences were observed in the lemongrass leaves of three ages and
sampling months with a non-significant interaction of these factors for the silica contents (Table
4.11). The silica content of bottom leaf was higher than penultimate and middle leaves, although
the trend of its accumulation was more or less similar in all leaves. A highest silica content in the
leaves of different ages was observed in May-Jul (summer season), which decreased steadily in
the autumn and winter seasons being the lowest in Dec. Overall, the silica content was in the
order: bottom leaf > middle leaf > penultimate leaf (Fig. 4.29)
Fig. 4.29: Influence of seasonal condition on silica content in the penultimate, middle and bottom
leaves of lemongrass
bcd
e de e
ba
aa
bcd
bcd
bc bc
cdbc
dd d
bcd
a ab
bc bc bc bcd
bcd
bcf ef
abc
aa
abc
bcbc
dcb
bcd
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec
Penultimate Middle Bottom
Sil
ica
(%)
Leaf position/sampling months
Probability level:
Penultimate leaf = **Middle leaf = **Bottom leaf = **
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f. Starch
The starch contents showed statistically significant results in lemongrass leaves of three
ages and the sampling months with a significant interaction of these factors for starch contents
(Table 4.11). All the leaves indicated distinctive behavior across the seasons for starch contents
The penultimate leaf showed higher starch contents in winter and summer months; the highest
being in Jun-Aug and in Jan, while the lowest in Nov. The middle leaf indicated the trend of
starch contents similar to the penultimate leaf but there was large variation in the trend of
accumulation across the seasons. In bottom leaf, the starch contents were low at the end of
autumn to the end of winter season while it was higher in spring and summer season. Overall,
starch content was greater in bottom and middle leaves while it was lower was observed in the
penultimate leaf (Fig. 4.30).
Fig. 4.30: Influence of seasonal condition on Starch content in the penultimate, middle and
bottom leaves of lemongrass
ab bc bab
a a abc
cd dc
ba
cd d cda
b bcd
dcd
bc cde
abbc
dde
f bcd ab
c a ag
defg
g defg
0
2
4
6
8
10
12
14
16
Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec
Penultimate Middle Bottom
Sta
rch
(%)
Leaf position/sampling months
Probability level:
Penultimate leaf = **Middle leaf = **Bottom leaf = **
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g. Total soluble proteins
The total protein contents in the leaves of different ages of lemongrass and sampling
months varied significantly with a significant interaction of both the factors (Table 4.11). In
penultimate leaf total protein contents were higher than in middle and bottom leaves. In
penultimate leaf, the total soluble proteins were higher in winter and summer season while
reduced in spring and autumn seasons. The middle leaf showed a lower total soluble proteins in
Jan-Mar, which were greater in summer and autumn seasons and again showed a little decline in
the winter season. The bottom leaf indicated virtually no change in the soluble proteins across
the seasons (Fig. 4.31).
Fig. 4.31: Effect of seasonal variation on Total Proteins content in the penultimate, middle and
bottom leaves of lemongrass.
b abd
bcd
bca a bc ab a
b ab
cde
e deab ab
cab
cbc ab
abc
dab bc
d
bcd a ab a abc
bcd
abc
a d bc bc bc
0
5
10
15
20
25
Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec
Penultimate Middle Bottom
Tot
al p
rote
ins
(%)
Leaf position/sampling months
Probability level:
Penultimate leaf = **Middle leaf = *Bottom leaf = ns
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h. Correlation
Possible relationships between different seasons and nutritional attributes indicated that
in penultimate leaf ADF was positively related to ADL, cellulose and Silica while ADL, silica
and cellulose was negatively related to starch. In middle leaf, ADF was positively related to
ADL, cellulose and silica. ADL was positively related to cellulose, silica and starch, while
cellulose was positively associated with silica. In bottom leaf, ADF was positively related to
ADL, cellulose and silica. ADL showed positive correlation with cellulose silica and starch;
cellulose was positively associated to starch and protein (Table 4.12).
The correlation of meteorological attributes with the nutritional attributes revealed that
maximum temperatures was positively related to ADF, ADL and silica; minimum temperature
was associated positively to ADF, ADL and starch contents; relative humidity to NDF;
evapotranspiration was positively correlated with ADF and starch and average rainfall with
ADL. For middle leaf, maximum temperature was positively correlated with ADF and proteins,
minimum temperature with ADF, cellulose, silica and proteins; relative humidity with NDF and
average rainfall with NDF, ADF, silica and proteins. For bottom leaf, maximum temperature was
positively correlated with ADF and cellulose; minimum temperature was positively related to
ADF, cellulose and silica; evapotranspiration was positively correlated with ADF, cellulose and
silica while average rainfall was associated to silica contents.
4.8.2 Discussion
Seasonal variations had a significant impact on the nutritional quality attributes of plants
(Snyman, 2006). Nutritive constituents such as NDF, ADF, ADL, cellulose, silica, starch and
protein vary between years and between plant growth stages within growing season (Ball et al.,
2001). Results of this study revealed a lot of variations in the nutritional quality attributes of
lemongrass leaves during the whole year (to year study). The most distinctive changes were
observed in the leaf fiber attributes, wherein the NDF was greater in middle leaf; ADF was
similar in penultimate and middle leaves while bottom leaf displayed lowest contents of these
attributes (Fig. 4.25-4.26). A higher fiber content is a quality attribute of nutrition for medical
purpose and animal feed, since this provides a rich source of energy (Turner and Lupton, 2011).
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Table 4.12: Correlation coefficient (r) of meteorological attributes with the Nutritive content of lemongrass at three leaves positions penultimate, middle, bottom
Y-Variable X-Variable Penultimate Middle Bottom a. Mutual correlations of nutritional attributes NDF ADF -0.077na 0.214ns 0.082ns ADL 0.345ns 0.458ns 0.315ns Cellulose 0.220ns 0.209ns 0.081ns Silica 0.318ns 0.501ns 0.275ns Starch -0.375ns 0.366ns 0.107ns Protein -0.468ns 0.123ns -0.044ns ADF ADL 0.812** 0.670* 0.630* Cellulose 0.642* 0.873** 0.904** Silica 0.863** 0.871** 0.885** Starch 0.511ns 0.367ns 0.528ns Protein -0.231ns 0.536ns -0.193ns ADL Cellulose 0.040ns 0.684* 0.609* Silica 0.887** 0.789** 0.762** Starch 0.400ns 0.618* 0.709** Protein -0.487ns 0.049ns -0.170ns Cellulose Silica 0.221ns 0.826** 0.877** Starch -0.600* 0.473ns 0.769** Protein -0.273ns 0.264ns -0.229ns Silica Starch 0.314ns 0.510ns 0.746* Protein -0.544ns 0.372ns -0.276ns Starch Protein -0.047ns -0.385ns 0.137ns b. Correlations environmental variable with nutritional attributes Max. temperature NDF -0.161ns -0.138ns 0.104ns ADF 0.786** 0.628* 0.605* ADL 0.594* 0.240ns 0.196ns Cellulose -0.099ns 0.519ns 0.554ns Silica 0.578* 0.536ns 0.620* Starch 0.380ns -0.181ns 0.040ns Protein 0.298ns 0.664* -0.062ns Min. temperature NDF 0.064ns 0.140ns 0.252ns ADF 0.807** 0.667* 0.602* ADL 0.685* 0.382ns 0.312ns Cellulose -0.093ns 0.592* 0.595* Silica 0.325ns 0.666* 0.707** Starch 0.659* -0.051ns 0.217ns Protein 0.164ns 0.655* -0.053ns Relative humidity NDF 0.785** 0.844** 0.299ns ADF -0.166ns 0.046ns -0.209ns ADL 0.086ns 0.315ns 0.223ns Cellulose 0.350ns 0.138ns -0.153ns Silica 0.152ns 0.302ns 0.066ns Starch -0.481ns 0.355ns 0.439ns Protein -0.459ns -0.109ns -0.074ns Evapotranspiration NDF -0.399ns -0.311ns 0.066ns ADF 0.745** 0.524ns 0.658* ADL 0.551ns 0.159ns 0.160ns Cellulose -0.311ns 0.442ns 0.659* Silica 0.463ns 0.389ns 0.597* Starch 0.602* -0.105ns 0.014ns Protein 0.256ns 0.476ns -0.065ns Average rainfall NDF 0.591* 0.619* 0.541ns ADF 0.537ns 0.584* 0.353ns ADL 0.656* 0.462ns 0.424ns Cellulose -0.070ns 0.532ns 0.335ns Silica 0.561ns 0.689* 0.612* Starch -0.068ns 0.158ns 0.419ns Protein -0.248ns 0.599* -0.238ns Significant at: ns, non-significant; * and **, significant at P<0.05 and P<0.01 level
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In addition, these attributes were detected in substantially greater amounts in the summer
months. It is important to note that lemongrass, as a C4 plant, has a higher tendency to grow and
sustain under relatively higher temperature (Henry et al., 2000). The maintenance of greater fiber
provides a great opportunity of using lemongrass when the availability of other plant sources is
meager in the summer season.
In addition to the fiber contents, ADL, cellulose, silica and proteins are among the other
important nutritionally important attributes. It has been reported that the ADL and cellulose
synthesis takes place as a stress reaction leading to wall thickening (Zhong et al., 2001). Silica
has been implicated in providing tolerance against biotic and abiotic factors and improving plant
health (Epstein, 1999), while starch is also accumulated as a long term storage product
(Geigenberger, 2011). These data indicated that young penultimate leaf indicated a reduced
amount of ADL, cellulose, silica and starch, while enhanced contents of soluble proteins as
compared to middle and bottom leaves. However, the contents of all these attributes were
substantially greater in the summer followed by winter months (Fig. 4.27-4.31). Such an
accumulation has great implications with physiological standpoint and is important to plant
survival during the harsh growth periods. The ADL, cellulose and silica appeared to provide the
structural support while soluble proteins are expected either to act as osmolyte or provide amino
acids for this purpose. The older leaf showing a differential trend for the accumulation of these
nutritional attributes plausibly due to ageing factor.
The correlations were drawn to validate the current findings. The interrelationships of the
nutritional quality variables revealed that ADL, cellulose and silica showed the most
conspicuous association in all the leaves, although these correlations were much stronger in the
case of older leaves. This indicated that older leaves have higher capacity to synthesize and
accumulate these metabolites since this trend is clearer and typical of leaf ageing (Bassey et al.,
2001).
Drawing the correlation of meteorological attributes with the nutritional quality attributes
provided useful information. The positive association of changes in minimum and maximum
temperatures, evapotranspiration and rainfall patterns with ADF, ADL, silica and starch in the
penultimate leaf indicated that their enhanced synthesis under higher temperatures or
dehydrating conditions is helpful in the plants survival. This survival appears to improve the cell
wall properties under such adverse conditions. Moreover, all these changes appear to be
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transitory since the contents of these metabolites diminished when the adverse conditions were
relieved, and the young leaves indicated the normal growth patterns. Such changes appeared to
be lacking in the older leaves.
In conclusion, although the nutritional attributes are important with respect to animal feed
point of view and makes lemongrass an important nutritional source especially during the
summer months, such an accumulation is important with respect to changes in the wall properties
and acting as osmoprotectants, thus making it better able to survive under adverse conditions.
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4.9 Ionic determination
Leaf mineral profile is good way to assess nutritional status of plants. Climatic changes in
temperature, rainfall and light intensity have great influence on mineral accumulation in
lemongrass. To understand the physiology of lemongrass, the knowledge of seasonal variation in
leaf nutrient concentrations is necessary. The results of soluble nitrate, soluble phosphate, sulfur,
potassium and calcium are given below:
4.9.1 Results
The statistical results of differences in the significance of variance sources i.e. leaves of
three ages and seasonal conditions, with respect to leaf minerals are presented in Table 4.13.
Table 4.13: Analysis of variance of data (mean squares) for soluble nitrate, soluble phosphate, sulfur, potassium and calcium contents of lemongrass under influence of seasonal condition
Source of Variation
Df Nitrate-N Phosphate-P Sulfate-S K Ca
Block (B) 2 0.03 0.12 0.59 2.56 0.67
Leaf (L) 2 312.86** 21.25** 127.55** 240.74** 55.07**
Month (M) 11 7.22** 1.96** 3.00** 26.40** 6.77**
L × M 22 4.05** 0.22ns 0.73** 3.83ns 4.57**
Error 70 0.67 0.22 0.24 4.90 0.52
Significant at: ns, non-significant; * and **, significant at P<0.05 and P<0.01 levels
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a. Soluble nitrate-N
For soluble nitrate-N, the experimental results showed significant difference in the leaves
of various ages and sampling months with a significant interaction of these factors (Table 4.13).
In penultimate and middle leaves, the soluble nitrate-N contents decreased from Jan and attained
the lowest value in Feb-Mar (spring season). It attained the greatest amount in summer sesason
and again showed a decline in the autumn season followed by a rise in the winter season. In
bottom leaf, there were no variation in their contents along the changing seasons. Overall, the
soluble nitrate-N was much lower than those observed in the penultimate and middle leaves (Fig.
4.32).
Fig. 4.32: Effect of seasonal variation on Soluble Nitrate ion concentration in the penultimate,
middle and bottom leaves of lemongrass
bccd
ee
cdb
a a ac cd
cde
bc
bc
bc bca
a ab abc
dcd
b
abbc b ab
abab b cb
a cd a0
2
4
6
8
10
12
14
16
18
Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec
Penultimate Middle Bottom
Nitr
ate-
N (
mg/
g dr
y w
eigh
t wei
ght)
Leaf position/sampling months
Probability level:
Penultimate leaf = **Middle leaf = **Bottom leaf = **
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b. Soluble phosphate-P
Data revealed significant difference in the leaves of different ages, and sampling months
but with a non-significant interaction of these factors for soluble phosphate-P contents (Table
4.13). In case of penultimate leaf, the soluble phosphate-P was relatively lower in Jan, which
increased in Feb, and displayed a seady state level up to Oct, followed by a decline in Nov-Dec.
For middle leaf the soluble phosphate-P increased from Jan and was the highest in Apr, which
showed a decline thereafter up to Dec. For bottom leaf there was no specific trend of the
accumulation of soluble phosphate-P. Overall, the soluble phosphate-P accumulation was the
lowest in bottom leaf and highest in the middle leaf (Fig. 4.33).
Fig. 4.33: Effect of seasonal variation on soluble phosphate ion concentration in the penultimate,
middle and bottom leaves of lemongrass
cab ab ab
cab
c a a aba a bc
c
cd aba a de
cdab
ecd cd
ecd
ee
cde
bcd
ef fab
cde ab ab
ccd
ebc a
ef
0
2
4
6
8
10
12
Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec Jan
Feb
Mar
Apr
May Jun
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Aug Sep Oct
Nov
Dec
Penultimate Middle Bottom
Pho
spha
te-P
(m
g/g
dry
wei
ght)
Leaf position/sampling months
Probability level:
Penultimate leaf = **Middle leaf = **Bottom leaf = **
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c. Sulfate-S
There were highly significant differences in the leaves of different ages of lemongrass
and sampling months for the changes in the sulfate-S contents with a significant interaction of
leaves and sampling months (Table 4.13). Being relatively higher in Jan in penultimate leaf, the
sulfate-S declined to its lowest value in Mar, which indicated a gradual increase and attained a
highest value in summer months (Jun-Aug). Thereafter, it indicated a decline up to Nov and an
increase in Dec. In middle leaf, the sulfate-S indicated a steady state value up to Mar, but a rise
thereafter attaining the highest value in Jun-Aug and a decrease afterwards. The bottom leaf
indicated no significant change in the sulfate-S contents throughout the sampling months. The
penultimate leaf exhibited the highest sulfate-S contents while bottom leaf the lowest (Fig. 4.34).
Fig. 4.34: Effect of seasonal variation on Sulfate ion concentration in the penultimate, middle
and bottom leaves of lemongrass
cdd
dcd
cdab
ca
abbc bc
dcd
de dee
bcd
bcd
a aa
b bcde cd
e
bc bc abc
bc c bc ab a bc bc bc bc0
2
4
6
8
10
12
14
Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec
Penultimate Middle Bottom
Sul
fate
-S (
mg/
g dr
y w
eigh
t)
Leaf position/sampling months
Probability level:
Penultimate leaf = **Middle leaf = **Bottom leaf = ns
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d. Potassium (K)
Results indicated significant differences in the leaves and sampling months with a
significant interaction of both these factors for K contents (Table 4.13). All the three selected
leaves showed significant seasonal changes for K contents. In penultimate leaf, the K content
was the highest in Jan which declined in Feb-Apr. It increased in the summer season (May-Aug)
and then indicated a decline in autumn season followed by an increase in the winter months. The
middle leaf showed a steady state level of K in the spring season. It increased in the summer
months and was the highest in July. From Aug and onward K content decreased up to Nov but
again increased in Dec. For bottom leaf, the pattern of K accumulation was similar to the
penultimate leaf but the content was lower than penultimate and middle leaf (Fig. 4.35).
Fig. 4.35: Influence of seasonal condition on Potassium ion concentration in the penultimate,
middle and bottom leaves of lemongrass
bccd
e cdde
b b a bc cd
ee
bcd ab
cdd
abcd ab
cab
cab
aab
cdbc
d dbc
bcd
cde
cde
abbc
da
abf
f def
abc
a0
5
10
15
20
25
30
35
40
Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec
Penultimate Middle Bottom
Pot
assi
um (
mg/
g dr
y w
eigh
t)
Leaf position/sampling months
Probability level:
Penultimate leaf = **Middle leaf = **Bottom leaf = **
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e. Calcium (Ca)
It was evident from the results that leaves of three ages and months of sampling differed
significantly but there was no interaction of both these factors for Ca content (Table 4.13). The
Ca contents of penultimate leaf were low during winter season, which decreased further during
spring season. In summer season, the Ca content increased and was at its highest in May-Jul, but
decreased in the autumn season, and then showed an increase. In middle leaf too, the Ca content
was quite low in spring and autumn season but markedly higher in the summer and winter
seasons. The bottom leaf, however, indicated a reduced Ca content in Jan-Feb but a steady Ca
content during rest of the sampling months. Overall, the Ca content was higher in penultimate
leaf but lower in the bottom leaf (Fig. 4.36).
Fig. 4.36: Influence of seasonal condition on calcium ion concentration in the penultimate,
middle and bottom leaves of lemongrass
abc
cbc bc
ab a abab
cbc
cab
cab
bcde bc
dbc
d bca ab ab
debc
de
bc
cde
ecd bc
dcd bc
dbc
dcd bc cdbc
d a0
2
4
6
8
10
12
14
Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec Jan
Feb
Mar
Apr
May Jun
Jul
Aug Sep Oct
Nov
Dec
Penultimate Middle Bottom
Cal
cium
(m
g/g
dry
wei
ght)
Leaf position/sampling months
Probability level:
Penultimate leaf = **Middle leaf = **Bottom leaf = **
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f. Correlation
Interrelationships of nutrients revealed that in both penultimate and middle leaf, nitrate-N
was positively correlated with sulfate-S, K and Ca; sulfate-S with K and Ca while K was
correlated with Ca (Table 4.14). The relationships of nutrients with meteorological variable
showed that maximum temperature was positively correlated with phosphate-P and sulfate-S,
minimum temperature and relative humidity were positively correlated with all nutrients except
K, while evapotranspiration and rainfall were positive correlated with sulfate-S and Ca. In case
of middle leaf, sulfate-S was correlated with maximum temperature; sulfate-S and Ca with
minimum temperature, evapotranspiration with nitrate-N, sulfate-S and Ca, while rainfall was
associated positively to sulfate-S and Ca. In case of bottom leaf, no other environmental variable
was correlated with any nutrient except sulfate-S and K (Table 4.14).
4.9.2 Discussion
Mineral nutrients are essential for plant growth and development. Seasonal variations in
tissue mineral concentration appear to be largely dependent on soil, light, temperature and
rainfall (Wells, 1996; Gent, 2002). As a part of present study, seasonal variations in chemical
composition of lemongrass leaves P, S, N, K, and Ca concentrations were determined on
monthly basis throughout the years in the penultimate, middle and bottom leaves of lemongrass.
The plant nutrients play both structural and functional roles in the plants, since they are the part
of macromolecules (Epstein and Bloom, 2005).
The fact remains that under relatively adverse conditions, the plants need greater
resources for growth and development; the availability of nutrients is pivotal in this regard. On
whole body basis, the plants generally show insufficient concentration of important nutrients in
different seasons (Epstein and Bloom, 2005). However, there may be differences in the leaves of
various ages for the acquisition of various nutrients. Results revealed that seasonal changes had
great impact on the concentration of soluble nitrate-N (Fig. 4.32), soluble phosphate-P (Fig.
4.33), sulfate-S (4.34), K (Fig. 4.35) and Ca (Fig. 4.36) in the leaves of all ages. Overall the
results indicated that generally the young (penultimate and middle) leaves had greater contents of
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Table 4.14: Correlation coefficient (r) of meteorological attributes with the ion content of lemongrass at three leaves positions penultimate, middle and bottom
Significant at: ns, non-significant; * and **, significant at P<0.05 and P<0.01 levels
Y-Variable X-Variable Penultimate Middle Bottom a. Mutual correlations of nutrient elements Nitrate-N Phosphate-P 0.130ns -0.207ns -0.138ns Sulfate-S 0.904** 0.676* -0.560ns K 0.906** 0.731** 0.392ns Ca 0.823** 0.892** 0.501ns Phosphate-P Sulfate-S 0.441ns -0.312ns -0.027ns K -0.058ns 0.359ns -0.188ns Ca -0.175ns -0.330ns -0.268ns Sulfate-S K 0.743** 0.584* -0.219ns Ca 0.580* 0.739** 0.004ns K Ca 0.820** 0.682* 0.413ns b. Correlations environmental variable with nutrient elements Max. temperature Nitrate 0.521ns 0.504ns -0.012ns Phosphate 0.663* 0.052ns 0.314ns Sulfur 0.683* 0.693* -0.134ns K 0.302ns 0.302ns 0.180ns Ca 0.258ns 0.353ns 0.232ns Min. temperature Nitrate 0.612* 0.575ns -0.243ns Phosphate 0.749** 0.016ns 0.351ns Sulfur 0.793** 0.810** 0.068ns K 0.424ns 0.419ns 0.007ns Ca 0.823** 0.892** 0.412ns Relative humidity Nitrate 0.612* -0.086ns -0.486ns Phosphate 0.749** -0.393ns 0.111ns Sulfur 0.793** 0.152ns 0.611* K 0.424ns -0.064ns -0.686* Ca 0.823** -0.330ns -0.065ns Evapotranspiration Nitrate 0.533ns 0.578* 0.126ns Phosphate 0.490ns 0.203ns 0.139ns Sulfur 0.588** 0.578* -0.336ns K 0.357ns 0.462ns 0.449ns Ca 0.590* 0.739** -0.042ns Average rainfall Nitrate 0.553ns 0.467ns -0.392ns Phosphate 0.533ns -0.156ns 0.084ns Sulfur 0.725** 0.769** 0.416ns K 0.498ns 0.430ns -0.246ns Ca 0.820** 0.682* 0.197ns
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all the nutrients while the older (bottom) were deprived of this characteristic. In addition to the
leaf age or position on the plant, prevailing weather conditions appeared to be the main
determinant of the nutrient concentrations.
As is evident from the results, a greater nutrient content in the younger leaves is of great
physiological significance. These nutrients being present in the soluble phase are available for
the maintenance of water balance by acting as osmotica during the summer season, when there is
excessive water loss by evapotranspiration, while in winter season essential nutrients may act as
the cryoprotectants of cellular membranes (Fuller, 2004). In addition to these roles, they are
assimilated in the synthesis of macromolecules and cellular structures at large. For all these
practical purposes, the younger leaves were on an advantage to show normal metabolism and
greater growth even under sub- or supra-optimal temperatures.
To substantiate the above findings, mutual correlations of the nutrients and those of
meteorological attributes with the nutrient contents separately for three leaves were established
(Table 4.14). These correlations were quite strongly evident with most of the environmental
variable in case of penultimate and middle leaves but not in the bottom leaf. These data
suggested that most of these nutrients were correlated tightly with the prevailing temperature and
evapotranspiration. Furthermore, quicker response of younger tissues to the nutrients
accumulation in making metabolic adjustments as compared to the older leaf is of great
significance for growth and survival standpoints.
In conclusion, greater amounts of soluble nutrient in the younger leaves are of advantage
to lemongrass for its sustainable growth in the stress environments. Moreover, seasonal patterns
of variation in mineral concentration of lemongrass appear to be largely dependent on
temperature and evapotranspiration.
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4.10 Essential oil composition
The essential oils were extracted from the lemongrass leaves samples (as composite
samples) four times in Jan (winter season), Apr (spring season), Jul (summer season) and Oct
(autumn season). The results are given below.
4.10.1 Results
The GC-MS chromatograms for the chemical composition of lemongrass essential oils
are presented in Figs. 4.37-Fig. 4.40 and quantitative estimates are given in Table 4.15. The yield
of essential oil (% v/w) during different seasons varied from 0.6 to 1% with the highest in rainy
season. It is evident from the results that seasons have marked effect on quality and quantity of
lemongrass leaf essential oil constituents. Essential oil content was higher in summer season
(1%) followed by winter (0.9%) and autumn season (0.7%), whereas it was 0.6% during spring
season. In this study, a total of 54 essential oil components were identified in four different
seasons. There were 33 compounds representing 88.58% of lemongrass essential oil when
sampling was done in month of January. Lemongrass leaves sampled in Apr manifested 32
chemical compounds, representing 69.30% of essential oil. However, in July and October, 26
and 30 chemical compounds were detected representing 89.67 and 74.21%, respectively of the
essential oil were detected (Table 4.15). The principal components of lemongrass essential oils
were neral (11.22 – 17.28%), geranial (8.29% – 15.64%) and citral (13.86%). In addition,
lemongrass essential oils also contained a wide range of minor compounds e.g., Nerolidol (1.3 –
2%), Caryophyllene oxide (2.2 – 5.5%), Epoxy-linalooloxide (1.7 – 3.9%), Epiglobulol (1.0 –
3.4%) and 1-Heptatriacotanol (0.7% – 1.1%) in four different seasons (Fig. 4.37-4.40).
4.10.2 Discussion
As evident from the results, there was a great diversity of essential oils biosynthesized in
the leaves of lemongrass in four seasons, with great differences (Figs. 4.37-4.40 and Table 4.15).
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Fig. 4.37: Typical GC-MS chromatogram of lemongrass essential oil collected during January
Fig. 4.38: Typical GC-MS chromatogram of lemongrass essential oil collected during Apr
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Fig. 4.39: Typical GC-MS chromatogram of lemongrass essential oil collected during July
Fig. 4.40: Typical GC-MS chromatogram of lemongrass essential oil collected during October
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Table 4.15: Chemical composition (%) of lemongrass essential oil in different seasons Probable match January April July October (−)-Myrtenal 2.296 0.784 1.93 1.249 (+)-α-Longipinene 3.337 3.03 ‒ 2.184 1,1 Bipheny 1,3,4-diethyl- ‒ 0.789 ‒ ‒ 1,4-Cineolep ‒ 0.272 ‒ ‒ 1-Heptatriacotanol 0.948 0.878 1.109 0.738 2-2,4-Trimethy 1-3-[3,8,12,16 tetramethyl-heptadeca-3,7,11,15-tetraenyl]- 0.561 2.171 0.36 2.385 23-Dimethoxy-5-methyl-6-dekaisoprenyl-chinon ‒ 0.863 ‒ ‒ 2-Tridecanon 2.293 ‒ 1.921 ‒ 3,7-Nonadien-2-ol,4,8-dimethyl- ‒ ‒ 1.663 ‒ 3-[5-benzyloxy-3-methylpent-3-enyl]-2,2 dimethyloxirane 0.757 ‒ ‒ ‒ 3-Methoxymethoxy-1,5,5-trimethyl-cyclohexene ‒ ‒ 1.18 ‒ 4-[2,2-dimethyl-6-methylenecychohexyl] butanol ‒ 2.3 1.229 1.213 9-Hexadecanoic acid ‒ 4.563 ‒ ‒ Allethrin 4.115 0 5.441 ‒ Alloaromadendrene oxide-[1]- 1.024 0 ‒ ‒ Arisol, p-aallyl- ‒ 1.055 ‒ ‒ Aromadendrene ‒ 1.592 ‒ ‒ Ascaridole epoxide 1.858 ‒ ‒ ‒ Aspidoalbine ‒ ‒ ‒ 4.719 Caryophyllene oxide 5.517 4.47 2.255 3.876 Cholestan-3-ol,2-methylene-,[3β5a]- 1.499 1.431 ‒ 1.107 Cis-Z- a – Bisabolene epoxide ‒ 0.851 1.45 ‒ Citral 13.846 ‒ ‒ ‒ DCP-LA ‒ 2.034 ‒ 1.317 Egrosteryl acetate ‒ 1.106 ‒ 0.88 Epiglobulol 3.411 1.031 3.015 3.294 Ethyliso-allocholate ‒ 2.035 2.911 ‒ Expoxy-Linaloolooxide 1.772 3.745 3.971 2.099 Farnesyl bromide 2.372 1.017 ‒ 3.066 Geranial ‒ ‒ 15.647 8.293 Globulol 1.534 1.464 6.418 ‒ Himachalol 1.291 ‒ ‒ 3.42 Ilicicolin F 1.113 ‒ ‒ 1.474 Ingol 12-acetate 0.65 ‒ ‒ ‒ Ingol 12-acetate ‒ 1.275 1.011 ‒ Isophorol ‒ ‒ 0.908 ‒ Linoleic acid ‒ ‒ 4.252 1.246 Longiborneol ‒ 4.188 ‒ 3.1 Mesityl oxide ‒ ‒ ‒ 0.656 Myristoleic acid ‒ ‒ 2.668 ‒ Neral 17.286 14.525 16.525 11.223 Neric acid 2.283 0.918 2.883 1.566 Nerol 3.401 ‒ ‒ ‒ Nerolidol 2.015 1.651 ‒ 1.331 Neryl acetate 1.343 ‒ 4.48 ‒ Oleic acid 2.058 ‒ ‒ 0.543 Viridiflorol ‒ ‒ ‒ 4.365 α- Muurolol ‒ 2.013 1.236 ‒ α-Bergamotene, cis- 3.01 ‒ ‒ ‒ α-Cadinol, 1.559 ‒ ‒ 3.05 α-Terpineol 3.253 ‒ 2.163 ‒ β-Acoradiene 0.433 2.337 1.196 1.063 β-Estradiol 1.105 3.484 2.211 ‒ γ-Cadinene 0.633 ‒ ‒ 1.392 ‒ indicates the detection of no oil
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Among the oils, neral (17.28 - 11.22 %), citral (13.86%) and geranial (8.29-15.64%). Khanuja et
al. (2005); Negrelle and Gomes, (2007) reported that the composition of essential oil obtained
from lemongrass leaves varies markedly in different season. Tajidin et al. (2012) reported that
compounds (neral, geranial, nerol, and geranyl acetate) had concentrations greater than 1%.
These oils have high anti-inflammatory and antioxidant components.
This pattern of oil accumulation in lemongrass showed that the dynamics of the essential
oil content appears to be metabolically regulated during different seasons. The variations in the
chemical compositions of lemongrass essential oil across the year can be attributed to the varied
climatic and seasonal conditions of the region and adaptive metabolism of plant. The content of
the essential oils of lemongrass was distributed unevenly among seasons. The highest yield of
the oil was found during rainy season (1%) and winter season (0.9%) which decreased
significantly in autumn to 0.7%. Photoperiod, temperature, intensity of light (Clark and Menary,
1980) and seasons had great impact on oil composition in aromatic plants (McGimpsey et al.,
2006). Neral, a very important compound, was present in high quantity (17.28%, 16.52%) in the
lemongrass essential oil collected during winter and summer (Table 4.15). Sarma et al. (2011)
reported that in lemongrass leaves oil content was higher at the onset of monsoon as compare to
post-monsoon period. In the present investigation, lemongrass oil yield also increased in July up
to 1% and then decreased in October 0.7%. Citral and geranial are also commercially important
compounds, especially used in Vitamin A and ionone synthesis (Efraim et al., 1998). Citral,
geranial, Nerolidol, and Allethrin showed same seasonal pattern like neral, higher in summer and
winter period. The quality of the oil in Jamrosa ‘RL‐931 (Cymbopogon nardus var. confertiflorus
× C. jwarancusa) was high in July due to high temperature and low humidity, which enhanced
the accumulation of geraniol, and geranyl acetate (Bhan et al., 2003). Guenther (1961) reported
that lemongrass oil content is lower during the month of heavy rainfall compared to the dry
months.
Seasonal variation play a great role in changing the essential oil composition (both
quality and quantity). A higher essential oil yield was observed in the month of January and
October. Thus increased oil content in these months can be taken as a criterion for good harvest
time of lemongrass essential oils for commercial pursuits.
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GENERAL DISCUSSION
Lemongrass is a plant of great economic importance by virtue of the oil contents in the
leaf. The oil has a number of uses with medical standpoint. The lemongrass possesses C4
pathway of photosynthesis and can withstand relatively higher ambient temperatures. It also has
the ability to grow in the cool season, which shows quite a high flexibility of this grass to adjust
in the changing climatic conditions. However, no report could be found showing the
physiological basis of success of this important plant species to thrive in different seasons. These
studies were therefore aimed at exploring the comparative physiological, biochemical and
nutritional mechanisms of lemongrass leaves of three ages over two years.
The adverse climatic conditions lead to the mounting of oxidative load with the enhanced
production of ROS (Mittler, 2000; Bavita et al., 2012). Although ROS production and quenching
is a physiological need of the plants under normal conditions (Taiz and Zeiger, 2010; Wahid et
al., 2013), their enhanced production versus low dousing under adverse conditions causes
damage to the cellular and organelle membranes (Huang et al., 2004; Lu et al., 2009). To
counteract such effects, the cells deploy the antioxidant system, which may be both enzymatic
and non-enzymatic in nature (Sairam et al., 2000; Sofo et al., 2004; Farooq et al., 2009). In the
lemongrass leaves of three different ages, it was found in this study that although there was
enhanced oxidative stress on the leaves as determined in terms of H2O2 and MDA generation, the
younger (penultimate and middle) leaves indicated low levels of ROS and MDA than the older
(bottom) leaf (Fig. 4.2-4.3). Furthermore, these effects were more pronounced in summer and
winter seasons. Determination of activities of the antioxidant enzyme in these leaves indicated
that younger leaves had substantially higher activities of SOD, CAT and POD especially in the
summer season (Fig. 4.4-4.6). Furthermore, the extent of oxidative stress and the activities of
three antioxidant enzymes were tightly associated in younger leaves (Table 4.2), which showed
that deployment of the antioxidant system was more efficient in these leaves.
In addition to the antioxidant enzymes, the plants synthesize a great variety of
compounds of primary and secondary nature, which show accumulation under stressful
conditions and are active in the protection of cytoplasmic and organelle membranes (Wahid et
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al., 2007; Farooq et al., 2009). The osmoprotectants synthesis is important in the maintenance of
cell water balance and protection of biological membranes from being damaged by the
dehydrating forces such as salinity, drought and temperature extremes (Zhu, 2001; Ashraf and
Foolad, 2007; Furtana et al., 2013). Commonly studies osmoprotectants are soluble sugars,
amino acids and quaternary ammonium compounds (QACs) including betains (Cayley and
Record, 2003; Wahid et al., 2007). In this study, the determinations made in the leaves of various
ages indicated that all the osmoprotectants showed greater accumulation in the winter and
summer season while their levels were quite low in spring and autumn season. Moreover,
younger (penultimate and middle) leaves indicated their greater accumulation (Fig. 4.7- 4.10).
The correlations of these osmolytes indicated their co-accumulation as well as with the high
temperature and evapotranspiration (Table 4.4). This suggested their greater roles in rescuing the
younger tissues from the adverse effects of high temperature and dehydration being low
molecular weight and highly water soluble (Rinne et al., 1994; Bhowmik and Matsui, 2003).
Seasonal variations in lemongrass biochemical constituent appeared to be associated with
plant maturity, temperature and soil moisture contents throughout different seasons. Variation in
environmental condition enhanced the biosynthesis of secondary metabolites, presumably due to
preferred reversal of photosynthates to secondary metabolites as related to primary one (Morales
et al., 1993). All the biochemical compounds in lemongrass analyzed in the present experiments
varied significantly during different seasons. Photosynthetic pigments, secondary metabolites,
antioxidants, total minerals contents, and micro-nutrients were the highest in active growth
period, decreased in winter the period of languid growth. Such seasonal effects on biochemical
compounds have been recorded in different plant species such as Cymbopogon citatus (Siribel et
al., 2001), Ocimum basilicum (Hussain et al., 2008), Spinacia oleracea (Howard et el., 2002),
Mentha spicata (Kofidis et al., 2004), Adiantum capillus-veneris (Ahmad and Husain, 2008), and
Toona sinensis (Wang et al., 2007).
It is well recognized that quantitative and qualitative contents of secondary metabolites
has great variation, which is controlled by intrinsic and a biotic factors (moisture, light, nutrient
availability). Secondary metabolites implicated in stress tolerance include flavonoides (Winkel-
Shirley, 2002) anthocyanin (Chalker-Scott, 1999), tannin (Lees et al., 1994). In this study the
determinations made for the accumulation of alkaloids, saponins, tannins, soluble phenolics,
flavonoids and anthocyanins (Fig. 4.11-4.16). It was found that the synthesis of alkaloids and
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saponins was the lowest in the young (penultimate and middle) leaves but higher in the older
(bottom leaf), whilst the synthesis of tannins was relatively greater in the middle leaf but surely
lower than the bottom leaf in different months. More notably, the accumulation of soluble
phenolics and flavonoids was much higher in the young tissues especially during the summer
season, while anthocyanins were accumulated more in the winter season. Such an accumulation
of these metabolites has great implication in the physiological terms. The accumulation of
phenolics, flavonoids and anthocyanins are helpful to plants in a number of way e.g., osmotic
and antioxidative roles under stressful conditions (Chalker-Scott, 1999; Wahid and Ghazanfar,
2006; Wahid, 2007), which was also substantiated in this study by the correlations of these
metabolites with the environmental attributes (Table 4.6). A lower accumulation of alkaloids and
saponins in the younger tissues but their greater accumulation appears to be due to their lesser
physiological role in the primary metabolic phenomena of plants (Wahid and Tariq, 2008).
Operation of normal metabolism in plants under the varying seasonal conditions is of
primary importance for plant survival. The regulation of activities of enzymes is carried out with
the help of vitamins, which act as enzymatic cofactors because of their redox chemistry. Climate
and seasons have great impact on vitamin constituents of green plants. Studies revealed that light
and temperature are major forces which involve in vitamin synthesis (Asensi-Fabado and
Munne-Bosch. 2010; Mahmood et al., 2012). The results from the present study exhibited a
notable seasonality in niacin (Fig. 4.17), ascorbic acid (Fig. 4.18) and riboflavin (Fig. 4.19)
contents of lemongrass leaves growing throughout the year. Moreover, the generation of
reducing powers, reflected by RPA was also substantially changed under the prevailing
conditions; in fact improved in summer season (Fig. 4.20). These fluctuating responses can be
assigned to seasonal changes imposed mainly by temperature stress and evapotranspiration
(Table 4.8). A correlation of vitamins contents and RPA in the younger leaves supports the
notion that vitamins are important in the normalization of the cellular functions under adverse
conditions.
Changes in the photosynthetic pigments are among the other important factors, which
determine the photochemical efficiency of leaves (Ruban, 2009; Taiz and Zeiger, 2010).
Stressful conditions are usually damaging to the photosynthetic pigments due to photo-oxidative
stress, but this tendency is relatively greater in the stress susceptible plants (Ramel et al., 2013).
Younger leaves of lemongrass, in this research, incurred a minimal loss of Chl-a (Fig. 4.21), Chl-
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b (Fig. 4.22) and their total (Fig. 4.23), while there was generally an increase in the carotenoids
(Car) contents (Fig. 4.24). The Car have dual functions in plants; they act as accessory light
harvesting pigments (Taiz and Zeiger, 2010) and also are active in quenching the ROS produced
in the chloroplast as a result of photo-oxidative stress (Krinsky, 1998). Close association of Car
with the photosynthetic pigments and with the high temperature and evapotranspiration load
indicated that Car are mainly involved in alleviating the effect of heat stress on metabolically
active leaves of lemongrass.
There are a number of advantages of growing lemongrass. This grass can be found
growing round the year, can sprout from cuttings in any season and produces a large biomass
(Shylaraj et al., 1993). It has been very much exploited for the medicinal purpose, while its
capability as forage for animal rearing has been rarely explored. Generally the seasonal
conditions have a significant impact on the nutritive value of plants (Ball et al., 2001; Snyman,
2006). These studies were also focused to explore the possibility of using this grass for
nutritional purpose. The determinations were made for NDF (Fig. 4.25), ADF (Fig. 4.26), ADL
(Fig. 4.27), cellulose (Fig. 4.28), silica (Fig. 4.29), starch (Fig. 4.30) and protein content (Fig.
4.31). Related to the nutritional attributes are mineral nutrients, which are vital for performing
structural and functional roles in plants. Seasonal patterns of variation in mineral concentration
appear to be largely dependent on soil, light, temperature and rainfall (Wells, 1996; Gent, 2002).
As a part of present study, influence of seasonal variations on concentrations of lemongrass
leaves were studied for soluble nitrate-N (Fig. 4.32) soluble phosphate-P (Fig. 4.33), sulfate-S
(Fig. 4.34), K (Fig. 4.35) and Ca (Fig. 4.36). The concentrations of all these nutrients were in
greater quantities in the summer season followed by winter season, except soluble phosphate-P
which remained steady throughout the years. These data revealed that fiber contents (NDF and
ADF), starch and protein contents of the young leaves were higher while lignin cellulose and
silica contents were low, while the tissue nutrient contents were in the range as reported for the
traditional forage species. Taken together, all these attributes make the younger leaves of
lemongrass as a good source of nutrition. However, nutrient digestibility trials have to be made
before recommending lemongrass for the animal feed purposes.
The most important attribute of the lemongrass is the essential oil extracted from leaves,
which is commercially used for various purposes. The studies pertaining to the profile of
essential oils in different seasons are not available in literature. As regards commercial value of
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lemongrass oil, Tajidin et al. (2012) reported that at different growth stages the concentrations of
essential oils including neral, geranial, nerol and geranyl acetate, having high anti-inflammatory
and antioxidant properties, were greater than 1%. The GC-MS analysis of essential oils in leaves
of lemongrass in four seasons revealed that out of 54 compounds detected, only seven were
detected in all four seasons (Fig. 4.37-4.40; Table 4.15). Some compounds present in quite high
amounts in one season were absent in the other seasons. The most predominant compounds
(having concentrations more than 4%) were neral, citral, caryophyllene oxide and allethrin in
winter (January) season; neral and 9-hexadecanoic acid in spring (April) season; neral and
geranial in summer (July) season and neral, geranial and caryophyllene oxide in autumn season.
These data showed that lemongrass provides a great opportunity for use in the herbal and
medicinal preparations. Furthermore, determination of oil constituents provide clues about
cultivating and harvesting of the lemongrass in order to extract their maximum oil contents for
medicinal purposes.
Lemongrass deploys quite a few physiological phenomena, which enables it to thrive
under relatively more stressful conditions, the younger leaves of lemongrass present a valuable
resource for being used as antioxidant, vitamins, plant secondary products, nutrition for the
ruminant animals. A great diversity of essential oils present in the leaves make lemongrass
ethnobotanically important plant, which has a great opportunity of its use in the medicinal
industry. However, more studies are required to find out its stress tolerance potential when
challenged with the stress factor such as salinity, drought, heat and chilling.
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FUTURE PROSPECTS OF RESEARCH
The present studies on lemongrass have provided fundamental clues of the physiological
mechanisms of its survival in various seasons, together with its antioxidative and nutritional
values. There is a need to carry out concerted efforts on the seasonal variations in the gene
expression patterns in various plant parts under stressful conditions whilst comparing with the
control sets. These studies will further enhance our understanding of the functional analysis of
various genes and proteins conferring abiotic stress tolerance in this economically important
plant species.
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CHAPTER-5
SUMMARY
Lemongrass [Cymbopogon citratus (D.C.) Stapf] is a C4 grass species which belongs to
family Poaceae. It can grow round the year and has a lot of economic benefits. However, no
studies are available on the seasonal physiological responses of this grass species for its survival
in the changing climatic conditions. During this two years study, the seasonal changes in the
antioxidative properties, vitamins biosynthesis, osmoprotectants accumulation, production and
accumulation of plant secondary products, nutritional attributes and essential oil profiles were
studied in the penultimate, middle and bottom leaves of lemongrass. This study elaborated the
suitable season for maximum oil contents which could be exploited for commercial purposes by
the lemongrass growers.
Results revealed that there was enhanced oxidative stress on the leaves as determined in
terms of H2O2 and MDA generation; the younger (penultimate and middle) leaves indicated their
low levels than the bottom leaf. The H2O2 and MDA synthesis was greater in summer and winter
seasons. The younger leaves indicated higher enzymatic antioxidant property as determined in
terms of SOD, CAT and POD activities in the summer season. Close association of H2O2 and
MDA with the antioxidant system in younger leaves indicated an effective ROS scavenging
system in them. The determination of soluble sugars, total free amino acids, free proline
accumulation and glycinebetaine in the leaves of various ages indicated that all the
osmoprotectants showed greater accumulation in the winter and summer seasons while their
levels were quite low in spring and autumn seasons. The correlations of these osmolytes
indicated their co-accumulation with high temperature and evapotranspiration. In this study the
determinations made for the accumulation of secondary metabolites revealed that the synthesis of
alkaloids and saponins was the lowest in the young (penultimate and middle) leaves but higher in
the older (bottom leaf), whilst the synthesis of tannins was relatively greater in the middle leaf
but surely lower than the bottom leaf in different months. More notably, the accumulation of
soluble phenolics, flavonoids was much higher in the young tissues especially during the summer
season, while anthocyanins were accumulated more in the winter season. The results from the
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present study exhibited a notable seasonality in niacin, ascorbic acid and riboflavin. The
generation of reducing powers, was also substantially changed under the prevailing conditions
especially in the summer season. As for photosynthetic pigments, the younger leaves incurred a
minimal loss of Chl-a, Chl-b and their total, while there was generally an increase in the
carotenoids contents. Among the nutritional attributes of leaves, NDF, ADF, ADL, cellulose,
silica, starch and protein content were determined. Likewise, essential nutrient contents of the
leaves were also determined. The concentrations of all these nutrients were in greater quantities
in the summer season followed by winter season, except soluble phosphate-P the contents of
which remained steady throughout the years. The data revealed that fiber contents (NDF and
ADF), starch and protein contents of the young leaves were higher; lignin cellulose and silica
contents were low, while the tissue nutrient contents were in the range as reported for the
traditional forage species. Regarding medicinal properties, the GC-MS analysis of essential oils
in leaves of lemongrass in four season revealed that out of 54 compounds detected, only seven
were detected in all four seasons. Some compounds present in quite high amounts in one season
were absent in the other seasons. Among the compounds, neral, citral, caryophyllene oxide,
allethrin, 9-hexadecanoic acid and geranial in different seasons were the most distinct essential
oils. These data proved that the lemongrass provides a great opportunity for use in the herbal and
medicinal preparations because of a great variety of compounds synthesized in its leaves.
In short, while growing in different seasons, lemongrass exhibited the operation of quite a
few physiological phenomena, which appeared to enable it adapt to seasonal changes. More
notably, the younger leaves of lemongrass presented a valuable resource for its use as
antioxidant, vitamins, plant secondary products, nutrition for the ruminant animals. A great
diversity of essential oils present in the leaves make lemongrass ethnobotanically important
plant, which has the great opportunity of it use in the medicinal industry.
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