EXTRACTION, ISOLATION AND CHARACTERIZATION …shodhganga.inflibnet.ac.in/bitstream/10603/23467/4/04. chapter - ii... · 31 CHAPTER II EXTRACTION, ISOLATION AND CHARACTERIZATION OF

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CHAPTER II

EXTRACTION, ISOLATION AND CHARACTERIZATION OF

NATURALLY OCCURRING ANTIOXIDANTS

1. Introduction

Traditional medicines are used by approximately 60 per cent of the world's

population. These are used for primary health care not just in rural areas of developing

countries, but also in developed countries where modern medicines are predominantly

used. While traditional medicines are derived from plants, minerals, and organic matter,

the herbal drugs are prepared exclusively from medicinal plants [1, 2]. Adaptation to

environmental change is crucial for plant growth and survival. Environmental stress

enhances the generation of reactive oxygen species (ROS) such as superoxide anion,

hydrogen peroxide and hydroxyl radicals [3, 4]. ROS have been proposed as a central

component of plant adaptation to both biotic and abiotic stresses. ROS are produced by

leakage of electrons on to molecular oxygen from chloroplasts, mitochondria and plasma

membrane linked electron transport [5]. Different types of ROS based on their in vivo

concentration can perform beneficial or deleterious functions in the cell [6].

Many antioxidants such as vitamin C, vitamin E and carotenoids, occur as dietary

constituents. There are a lot of strong antioxidant compounds found in fruits, vegetables

[7-9] and in different beverages [10-16]. For example, fair antioxidants have been found

in berries [17-21], apples [22- 25], citrus fruits [26] and in fruit juices [27-29]. Highly

active antioxidants were found in olives [30-32] and olive oil [33-39]. Activity changes

during the processing of olive oil have also been evaluated [40, 41]. Many research

studies were carried out on antioxidants in fruits, and changes of antioxidants during fruit

processing [27, 42, 43]. The effects of processing have been evaluated also on the

changes of antioxidant activity in some roasted cereal products [44]. Red wines contain a

variety of polyphenolic compounds, the most abundant being anthocyanins, and they

have been shown to have high antioxidant activity [45-49].

However, not all polyphenols are extracted from grapes during the wine

production process. Among the best known and most biologically active are resveratrol,

32

quercetin and the catechins. It has been reported that grape seeds [50] and grape pomace

peels [51] continue to contain antioxidants, so wine production can be considered as a

source of antioxidants. Antioxidant activity was also reported in whiskys [52, 53]. Green

and black teas have been extensively studied for antioxidant properties [12, 14, 54-56].

The main compounds responsible for antioxidant activity were found to be catechins [16].

(–)- Epigallocatechin 3-gallate, (–)-epigallocatechin, (–)-epicatechin 3-gallate, (–)-

epicatechin, (+)-gallocatechin and (+)-catechin were identified and their antioxidant

activities have been studied [57- 59]. Herbs and spices are also good sources of

antioxidants [60-64]. Extensive research has been performed in this area, but only some

extracts from rosemary and sage are available as commercial antioxidants [65, 66].

The main problem in the application of such extracts is that usually they have a

characteristic odour, taste or colour, which in most cases is undesirable in the final

product [67]. Therefore there have been attempts to obtain odourless extracts having

antioxidative properties [68, 69]. A great number of different spices and aromatic herbs have

been tested for their antioxidant activity, with rosemary and sage being the most investigated

[70-75]. However many more herbs and spices have never been examined in this respect.

Nowadays, there is an increasing demand for natural products and therefore

research in the area of natural compounds is also growing. It should be noted however,

that natural is not always synonymous with safety. Therefore natural compounds must

also be tested for safety aspects before applying them in foods for human consumption.

There are various methods available to measure lipid oxidation in foods. Changes in

chemical, physical, or organoleptic properties of fats and oils during oxidation can be

monitored; however, there is no single method for assessing all oxidative changes in

different food systems. To determine primary oxidation of fats, various parameters like

changes of fatty acid composition [81], weight gain at different time intervals [82, 83],

amount of hydroperoxides [84], or conjugated dienes, which correlate well with peroxide

values [85], can be monitored. Addition of antioxidants decreases oxidation rates of

samples and the decrease can be expressed as the antioxidant activity. These methods

33

require a lot of time and therefore are not convenient for screening purposes. Nowadays

accelerated methods, such as Rancimat, active oxygen method (AOM), or OXIPRES

method, are used for assessing the oxidative stability of fats and oils [86].

This chapter describes the collection, identification and certification of plant

sources for antioxidant isolation. It also gives details of experimental procedures for

extraction, isolation, purification and spectral investigation of natural antioxidants. Use of

soxhlet extraction procedure with solvents of various polarities is also explained. This chapter

also deals with the purification protocol of isolated compounds. The characterization of the

isolated compounds using various techniques and structural elucidation based on spectral

investigation are described in this chapter. This chapter presents the details of

investigations using techniques such as TLC, UV-Vis spectrophotometery, FT-IR, 1H-

NMR, 13C-NMR, GC, GC-MS, HPLC and MS. All the plant selected were commonly

known and easily available. Out of the six plant selected four were edible and two were

non edible.

Sl.No Plant Name Identified Name Identification

Number Herbarium

Acc. Number

1 Curry Leaf Murraya koenigii L. BSI/SRC/5/23/2011-

12/Tech. -100 006152

2 Coriander Leaf Coriandrum sativum L. BSI/SRC/5/23/2011-

12/Tech. -101 006151

3 Mint Leaf Mentha arvensis L. BSI/SRC/5/23/2011-

12/Tech – 99 006150

4 Turmeric Curcuma lunga BSI/SRC/5/23/2011-

12/Tech-1330 006158

5 Bitter apple or Bitter cucumber

Citrullus colocynthis

(L.)

BSI/SRC/5/23/2011-12/Tech. -500

006157

6 Water hyacinth Eichhornia crassipes

(Mart.) Solms. – Laub. BSI/SRC/5/23/2011-

12/Tech. -145 006149

Table 2.1: Identification and certification of plant sources.

The six plants were collected from different places in Tamilnadu, and identified and

certified by the Botanical Survey of India, TNAU branch, Coimbatore, and were assigned

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identification number and the herbarium account number (Table 2.1). The identified plants are

stored at the herbarium at Botany Department, Bharathiyar University, Coimbatore.

2. Murraya Koenigii L. (curry leaf)

The plant Murraya koenigii (L) Spreng, belonging to the family Rutaceae, is

native to India and now distributed in most of southern Asia. The Murraya species has

also been used in traditional medicine in eastern Asia. Previous studies on the Murraya

species include reports of coumarins, terpenoids and many investigations on carbazole

alkaloids [87 - 90]. The leaves of Murraya koenigii are also used as an ingredient in

ayurvedic medicine. Their properties include much value as an antidiabetic [91] and

hepatoprotectant [92]. Studies on carbazoles isolated from Murraya koenigii leaves have

been shown to possess antioxidant and antimicrobial activity [93 - 95].

It is a small tree, growing 4-6 m tall, with a trunk up to 40 cm in diameter.

The leaves are pinnate, with 11-21 leaflets, each leaflet 2-4 cm long and 1-2 cm broad.

The flowers are small white, and fragrant. The small black, shiny berries are edible, but

their seeds are poisonous. The wood is greyish white, hard, even, close grained and

durable. It has been used as timber for manufacture of many types of products. The curry

plant therefore is a multi-product source. If it can be utilised in a planned manner a chain

of rural industrial units can be planned and implemented [96].

The free amino acids present in the curry leaves are asparagine, serine, aspartic

acid, glutamic acid, threonine, proline, alanine, tyrosine, tryptophan, histidine, etc [97].

The leaves also contain a crystalline glucoside, koenigin and a resin. The twigs and leaves

contain 0.8% potash on dry matter basis. Fresh leaves, on steam distillation under pressure

yield 2.6% of a volatile oil, which may find use as a fixative for heavy type of soap perfume;

distillation at ordinary pressure gives very poor yield of oil, while distillation with

superheated steam yields dark colored foul-smelling oil. Rectified curry leaf oil is deep

yellow in colour with a strong spicy odour and pungent clove like taste [97, 98].

The use of essential oil as flavor or perfume is not yet popularised. There is a

good scope to create demand considering its virtues. In fact, the oil also has medicinal

virtues. The leaves, bark and the root are used in indigenous medicine as a tonic,

35

stomachic, stimulant and carminative. The leaves when taken with pepper early in the

morning on an empty stomach are known to reduce blood sugar. An infusion of the

roasted leaves is used to stop vomiting. The dry leaves of are ingredients in many herbal

medicines. The juice of the root is taken to relieve pain associated with the kidney.

The leaves are highly valued as seasoning in South Indian and Sri Lankan cooking, much

like bay leaves and especially in curries with fish or coconut milk. They are also available

dried, though the aroma is much inferior [98, 99].

Curry leaves are also known to be good for hair, in keeping them healthy and

long. Although most commonly used in curries, it is also used in many other dishes to

add spice [99 - 101]. The Murraya koenigii leaf contains nearly 42 compounds according

to several reports [101]. Several researchers reported the isolation of carbazole alkaloids

from curry leaves. This study reports the isolation of simple compounds like cymene,

caryophylene, carvone and phellendrene from the leaves of Murraya koenigii. These four

bioactive compounds are simple in structure and were characterized using FT-IR, 1H-NMR, GC, and GC-MS.

2.1. Materials and Method

Fresh curry leaf was collected from Siruvani crop field, Coimbaotre, India.

Petroleum ether (Merck, Germany), hexane (Merck, Germany), chloroform (Merck,

Germany), absolute alcohol (Jiangsu Huaxi, China), sodium sulphate (Na2SO4)

(Qualigens, India), TLC plates (Merck, Germany). Acetone (Merck, Germany) Tris.Hcl

(Loba, India) were used.

2.2. Extraction with Soxhlet apparatus

A Soxhlet extractor is laboratory apparatus invented in 1879 by Franz von

Soxhlet. It was originally designed for the extraction of a lipid from a solid material,

thought it is not limited to the extraction of lipids. Typically, a Soxhlet extraction is

required only when the desired compound has high solubility in a solvent, and the

impurity is insoluble in the solvent. If the desired compound has a high solubility in a

solvent then a simple filtration can be used to separate the compound from the insoluble

substance. Normally a solid material containing some of the desired compound is placed

inside a thimble made from thick filter paper, which is loaded into the main chamber of

36

the Soxhlet extractor. The Soxhlet extractor is positioned into a flask containing the

extraction solvent. The Soxhlet is then fitted with a condenser [88, 94].

The solvent is heated to reflux. The solvent vapour travels up a distillation tube

and floods into the chamber housing the thimble holding the solid. The condenser ensures

that solvent vapour condenses and drips back down into the chamber housing the solid

material. The chamber containing the solid material slowly fills with warm solvent. Some

of the desired compound then dissolves in the warm solvent. When the Soxhlet chamber

is almost full, the chamber is automatically emptied by a siphon side arm, with the

solvent running back down to the distillation flask. This cycle may be allowed to repeat

many times, over hours or days.

During each cycle, a portion of the non-volatile compound dissolves in the

solvent. After many cycles the desired compound is concentrated in the distillation flask.

The advantage of this system is that instead of many portions of warm solvent being

passed through the sample just one batch of solvent is recycled. After extraction the

solvent is removed typically by means of a rotary evaporator yielding the extracted

compound. The non-soluble portion of the extracted solid remains in the thimble,

and is usually discarded.

Fresh curry leaves obtained from the Siruvani crop field, Coimbatore, India were

washed and cleaned thoroughly under running water. The excess water was drained and

the leaves were dried in vacuum for one week. Dried curry leaves were powdered in a

multi-mill fitted with sieve to obtain a coarse powder.

Figure.2.1: Rotary evaporator setup

37

Two solvents namely chloroform and ethanol was used for the extraction.

The extraction from 35 g curry leaf powder with 250 ml of the solvent was carried out at

60°C for a period of 10 h. The solvent was evaporated using a rotator evaporator.

The evaporator was maintained below 50oC. 7 gm of chloroform extract of crude and 5

gm of ethanol extract of crude were obtained. Totally 8 such fractions were analyzed by

TLC.

2.3. Isolation of compounds from the extract

Column chromatography (CC) is another common and useful separation

technique in organic chemistry. This separation method involves the same principle as

TLC, but can be applied to separate larger quantities of compounds compared to TLC.

Column chromatography can be used on both large and small scales. TLC is useful in

determining the type and number of ingredients in the mixture, but isolating individual

components at preparative scale is difficult. However, column chromatography allows

separation and isolation of individual compounds in bulk.

Solvent systems for use as mobile phases in CC can be determined from previous

TLC experiments, literature, or experimentally. Normally, the separation process begins

by using nonpolar or low polarity solvent, allowing the compounds to adsorb to the

stationary phase. The polarity of the solvent is then gradually increased which desorbs the

compounds and allows them to travel along with the mobile phase. On a macroscale, the

mixing of two solvents can create heat and crack the column leading to poor separation.

There are several methods available to pack a column. The slurry method which

normally achieves the best packing results is often used for macroscale separations.

The solid stationary phase is thoroughly mixed with a small amount of nonpolar solvent

taken in a beaker until a consistent paste is formed, capable of flowing. This homogeneous

mixture is poured into the column carefully using a spatula to scrape out the solid as the

liquid is poured. Care is taken in order to create an evenly distributed and tightly packed

stationary phase. Cracks, air bubbles and channelling in the stationary phase eventually

leads to a poor separation of the compound.

38

After the column is packed, the sample mixture is loaded directly to the top of the

column. Normally, a minimum amount of a polar solvent, about 5-10 drops, is used to

dissolve the mixture. The mixture is then carefully added to the top of the column using

a pipette without disrupting the surface of the column. A thin horizontal band of sample

over the packing material is best for an optimal separation. After the sample is loaded, a

small layer of white sand is added to the top of the column which helps to keep the top of

the column level when adding the eluent. Once the mixture is added and the protective

layer of sand is in place, solvent eluent is continuously added while collecting small

fractions at the bottom of the column. Using a pipette to add the first bit of solvent on top

of the packing material, and the addition of white sand minimizes the disturbance of the

column and dilution of the sample [23 - 25].

Figure. 2.1: Schematic diagram of extraction and isolation of antioxidants from plants.

39

2.4. Results and Discussion

The isolation and study of antioxidant activity of the four compounds -

caryophyllene, cymene, carvone and phellandrene isolated from Murraya koenigii are

given in the following text. These four bioactive compounds are simple in structure and

were characterized using FT-IR, 1H-NMR, GC, and GC-MS. These simple bioactive

compounds can be used as food supplements and as natural antioxidants.

2.4.1. Structural elucidation of compounds

2.4.1.1. Chloroform extract from Murrya koenigii

The chloroform extract was separated using column chromatography with silica

gel as packing material, and hexane as a solvent. The polarity of the solvent was

increased step up step using hexane and acetone. The first two fractions of the extract

were obtained using difference in polarity of the eluent. The first compound was isolated

using hexane and the second compound was isolated using a mixture of 99.5% hexane

and 0.5% acetone. Both compounds were analysed by FTIR, GC, GC MS, HPLC, and 1H-NMR as described below.

2.4.1.2. Compound 1 from chloroform extract

IR spectrum (ν, cm-1) of compound 1 revealed bands at 1259 cm-1, 1126 cm-1 and

1301 cm-1 (-C-C- str), 1058 cm-1, 1028 cm-1 (-C=C-), 873 cm-1 (-C=C- mono subs), 538,

718, 1514 (H-C-H bend), 1893, 2869, 2925, 2978, 3016 and 3049 cm-1 (H-C-H Asy str).

Compound 1 was tested by GC and this compound gave a single peak confirming

the presence of only one compound. The retention time of the compound was 6.33 min

with purity of 99.4%.

The spectral data of compound 1 was found to be in good agreement with the

reported value for cymene [102]. In the 1H-NMR spectrum, characteristic signals for

isopropyl group was observed at 2.85 δ, and 1.23 δ (singlet, 6 proton) while the benzylic

methyl group appeared at 2.32 δ.

40

GC-MS studies revealed a sharp peak with m/z value of 134.20, which

corresponds to the molecular weight of cymene (134.22). From the NMR and GC-MS

data (Figure 2.3) we conclude that the isolated compound is cymene (Figure 2.2).

H3C CH3

CH3

Figure. 2.2: Structure of cymene

a

41

c

b

42

Figure. 2.3: Characterization of cymene isolated from murrya koenigii leaf using

chloroform as solvent a) FTIR, b) GC, c) 1H NMR, d)GC MS

2.4.1.3. Compound 2 from chloroform extract

IR spectrum (ν, cm-1) of compound 2 revealed bands at 719, 813, 927.79 cm-1,

1020, 1055, 1107 cm-1 (-C=C-mono subs), 1278, 1303, 1363, 1380 cm-1 (-C-C- str),

1460, 1514 cm-1 (H-C-H bend), 2869, 2925, 2960, 3016, 3049 cm-1 (H-C-H Asy str).

The chloroform extract of compound 2 was tested using a GC and the compound

yielded a single peak confirming the presence of only one compound. The retention time

was 22.5 min., with 98.74% purity.

The spectral data of compound 2 was found to be in good agreement with the

reported value for caryophyllene [103]. In the 1H-NMR spectrum of compound 2, the

peaks appearing at 1.29 δ and 1.96 δ indicate the presence of the CH2 group in the

molecule. The peak appearing at 2.73 δ shows the presence of a cyclobutane linkage

between the five-membered rings and the six-membered rings. The peaks appearing in

the range of 4.75 and 5.25 δ are due to presence of ethylene linkage.

d

43

GC-MS studies revealed a sharp peak with a retention time of 8.4 minutes, with

m/z value of 221 which corresponds to the molecular weight of caryophyllene (220.35).

From the GC-MS and 1H-NMR data (given below) we conclude that the isolated

compound is caryophyllene, whose structure is given below in Figure 2.4.

Figure. 2.4: Chemical structure of caryophyllene

H3CCH3

H2C

CH3

HH

a

44

c

b

45

Figure 2.5: Characterization of caryophyllene isolated from Murrya koenigii leaf using

chloroform as solvent a) FTIR, b) GC, c) 1H NMR, d)GC MS

2.4.2. Ethanol extract from Murrya koenigii

UV spectrum of the ethanol extract of the Murraya koenigii leaf revealed the

presence of eight peaks at 671, 610, 510, 490, 417, 298, 263.4 and 223.8 nm,

corresponding to eight different compounds. Of these, we were interested in studying

only the two major compounds whose absorption peaks were at 223.8 nm and 263.4 nm.

The extract was separated using a silica gel column, employing hexane as the

eluent initially. The polarity of the solvent was increased gradually using a mixture of

hexane and acetone. The first two fractions of the extract were obtained using difference

in polarity of the eluent. The first compound isolated (using 100% hexane) was analyzed

by UV spectrophotometer, which gave an absorption maximum of 223 nm. Column

chromatography was continued using hexane:acetone (95:5 v/v) mixture for the isolation

of the second compound. On UV spectrophotometery, the second compound exhibited an

absorption maximum of 263.4 nm.

d

46

2.4.2.1. Compound 1 from ethanol extract

IR spectrum (ν, cm-1) of compound 1 revealed bands at 1690 - 1760 cm-1 (-C=O-),

1259 cm-1, 1126 cm-1 (-C-C- str), 1058 cm-1, 1028 cm-1 (-C=C-)

GC analysis of the ethanol extract of compound 1 revealed a single peak with a

retention time of 8.53 min and 99.465% purity.

1H-NMR spectra of the extracted compound was found to be in good agreement

with the reported data [104]. In the 1H-NMR spectrum of compound 1, the signal

appearing at 1.37 δ and 1.6 δ indicate the presence of the CH2 group in the compound.

The signal appearing at 3.93 δ shows the presence of a C-C linkage between the

five-membered rings and the six-membered rings. The spectrum also showed a signal at

6.75 δ corresponding to the α, β – unsaturated olefin while the terminal olefin displays

signals at 4.85 – 4.7 δ. Two methyl groups appeared as singlets at 1.8 δ and 1.75 δ.

GC-MS studies revealed a sharp peak with m/z value of 150, which corresponds

to the molecular weight of carvone (150.2). From the NMR and GC-MS data (Figure 2.6)

we conclude that the isolated compound is carvone.

O

H

Figure. 2.6: Chemical structure of carvone

47

a

b

48

Figure. 2.7: Characterization of carvone isolated from Murrya koenigii leaf using ethanol

as solvent a) FTIR, b) GC, c) 1H NMR, d)GC MS

d

c

49

2.4.2.2. Compound 2 from ethanol extract

The ethanol extract of compound 2 gave a single peak on analysis with GC,

confirming the presence of a single compound with a retention time of 5.916 minuntes

and 97.44% purity.

IR spectrum (ν, cm-1) of compound 2 revealed bands at: 927.79 cm-1, 1058 cm-1

(-C=C-mono subs), and 1126 cm-1 (-C-C- str).

In 1H-NMR spectrum of compound 2, the peaks at 1.50 δ clearly showed the

presence of the CH group in the molecule. The peak around 2.15 δ indicated the presence of

the CH group of an aliphatic molecule. The peak around 3.93 δ in the spectrum indicated the

presence of a C-C linkage between the five-membered ring and a six-membered ring.

The peak at 7.32 δ was due to the presence of an aromatic ring in the compound.

GC-MS studies revealed a sharp peak with a m/z value of 136.2 which

corresponds to the molecular weightt of phellandrene (136.2). The NMR and GC-MS

data taken together confirms that the compound is phellendrene (Figure. 2.8). The GC,

FTIR, NMR and GCMS spectra are shown in Figure 2.9.

Figure. 2.8: Chemical structure phellendrene

50

a

b

51

Figure. 2.9: Characterization of phellendrene isolated from Murrya koenigii leaf using

ethanol as solvent a) FTIR, b) GC, c) 1H NMR, d)GC MS

d

c

52

3. Mentha arvensis L. (mint leaves)

‘Mint’...just say the word and cool, refreshing images come to mind: frosty

glasses of lemonade garnished with curly springs of spearmint; the clean, chilling taste of

a mint candy cane. Even chewing gum, mouthwash, and toothpaste companies use

images of crisp, clean snowy slopes to let us know how refreshing their mint flavoured

products are. Delicious recipes for soups and deserts have Mint as an ingredient. Mint

based remedies are available for various health troubles and countless other uses [105].

The plant Mentha arvensis L. belonging to the family Rutaceae, is native to India

and now distributed in most of southern Asia and European countries. It is commonly

called ‘Pudina’ in most Indian vernacular languages.

3.1. Main constituents

The essential oil of mint (up to 2.5% in the dried leaves) is mostly made up from

menthol (ca. 50%), menthone (10 to 30%), menthyl esters (up to 10%) and monoterpene

derivatives (pulegone, piperitone, menthofurane). Traces of jasmone (0.1%) improve the

oil's quality remarkably. Menthol and menthyl acetate are responsible for the pungent and

refreshing odour; they are mostly found in older leaves and are preferentially formed

during long daily sunlight periods [106].

3.2. Materials and Methods

Fresh Mint leaf was collected from Siruvani crop fields, Coimbatore, India.


Germany), absolute alcohol (Jiangsu Huaxi, China), sodium sulphate (Na2SO4;

Qualigens, India), TLC plates (Merck, Germany), tris.HCl(Loba, India) were used. All

the other materials and experimental techniques are as described in the previous section.


3.3.1. Chloroform extract of mint leaf

Mint leaf extracted with chloroform contained three compounds, as confirmed by

TLC and UV-Vis spectrophotometry. The major peak with a λmax of 223.3 nm indicated

53

the presence of carvone as one of the major constituents in the extract. This was further

confirmed by IR, NMR, GC and GC-MS analyses. The second peak observed at λmax of

263.4 nm was that of phellendrene.

3.3.2. Ethanol extract of mint leaf

TLC analysis of the ethanol extract exhibited 5 spots and UV-Vis spectrophotometry

revealed 5 peaks. However, carvone peak was not seen.

The extract was separated using column chromatograpy using a mixture of hexane

and acetone (9.6:0.4). The eluent had one compound which did not show absorbance

under UV. This compound was solid, colourless and had a sweet odour. According to

literature, mint leaves have 76% menthol which is solid in nature, indicating that this

compound could be menthol.

The IR spectrum revealed sharp bands at 3580 cm-1 to 3650 cm-1 due to the

presence of phenol or alcohol group. The band at 1126.47 cm-1 was due to -C-C-

stretching. The band at 2931.90 cm-1 was due to -C-H stretching. The band at 1259.96

cm-1 was due to-C-C- stretching.

The 1H-NMR data of extracted menthol was found to be in good agreement with

the reported data [107]. In the 1H-NMR spectrum, the characteristic signals for isopropyl

group appeared as multiplet at δ 2.15 and as doublet at δ 0.92. The -CH- proton attached

to –OH group was deshielded and was observed in the downfield at δ 3.4.

GC-MS analysis revealed a sharp peak with a retention time of 13.48 minutes,

with m/z value of 156.2, which corresponds to the molecular weight of menthol (155.6).

GCMS data and other spectral information confirm the isolated compound to be menthol

(Figure 2.10). The HPLC, FTIR, NMR and GCMS spectrum is shown in Figure 2.11.

54

OH

Figure. 2.10: Chemical structure of menthol

0.0 2.5 5.0 7.5 10.0 min

-100

0

100

200

300

400

500

600

700

800

mAU

/8.509

a

b

55

Figure. 2.11: Characterization of menthol isolated from mint leaf using ethanol as solvent

a) FTIR, b) HPLC, c) 1H NMR, d)GC MS

c

d

56

4. Curcuma lunga (Turmeric)

India has a rich history of using plants for medicinal purposes. Turmeric (Curcuma

longa L.) is a medicinal plant extensively used in ayurveda, unani and siddha medicine,

and as home remedy for various diseases [108]. C. longa L., botanically related to ginger

(Zingiberaceae family), is a perennial plant having a short stem with large oblong leaves

and bears ovate, pyriform or oblong rhizomes, which are often branched and brownish-

yellow in colour. Turmeric is used as a food additive (spice), preservative and colouring

agent in Asian countries, including China and South East Asia. It is also considered as

auspicious and is a part of religious rituals. In ancient Hindu medicine, it was extensively

used for the treatment of sprains and swelling caused by injury [109].

In recent times, traditional Indian medicine uses turmeric powder for the

treatment of biliary disorders, anorexia, coryza, cough, diabetic wounds, hepatic

disorders, rheumatism and sinusitis. In China, C. longa is used for diseases associated

with abdominal pains. The colouring principle of turmeric is the main component of this

plant and is responsible for the anti-inflammatory property. For the last few decades,

extensive work has been done to establish the biological activities and pharmacological

actions of turmeric and its extracts. Curcumin (diferuloylmethane), the main yellow

bioactive component of turmeric has been shown to have a wide spectrum of biological

actions. These include its, antioxidant, antibacterial, and antifungal activity. Clinically,

curcumin has already been used to reduce post-operative inflammation. Safety evaluation

studies indicate that both turmeric and curcumin are well tolerated at a very high dose

without any toxic effects. Thus, both turmeric and curcumin have the potential for the

development of modern medicine for the treatment of various diseases [108].

Turmeric is the rhizome or underground stem of a ginger-like plant. It is usually

available ground, as a bright yellow, fine powder. The whole turmeric is a tuberous

rhizome, with a rough, segmented skin. The rhizome is yellowish-brown with a dull

orange interior that looks bright yellow when powdered. The main rhizome measures

2.5 - 7 cm in length with a diameter of 2.5 cm with smaller tubers branching off.

57


Fresh turmeric plant was collected from Siruvani crop fields, Coimbatore, India.


Germany), absolute alcohol (Jiangsu Huaxi, China), Na2SO4 (Qualigens, India), TLC

plates (Merck, Germany), acetone (Merck- Germany) Tris.HCl (Loba, India) were used.

All experimental techniques were as described the previous sections.


4.2.1. Petroleum ether extract of turmeric

Turmeric was first extracted with petroleum ether at 40 – 60 °C and the extract

was subjected to TLC and UV-Visible spectrometry. The petroleum ether extract gave

only one spot on TLC and only one peak in UV-Visible spectrometry. The λmax of this

peak was 420.1 nm.

Figure 2.12: UV spectrum of petroleum ether extract of C. longa containing curcumin.

According to literature turmeric is a yellow solid that contains curcumin and has a

λmax of 420 nm. The petroleum ether extract on drying resulted in a yellow coloured solid

Indicating that this compound could be curcumin.

IR spectrum of the extract revealed the presence of a broad peak from 3580 cm-1 to 3

650 cm-1 due to the presence of phenols or alcohol groups. The band at 1710 cm-1 to 1720 cm-1

58

could be due to -C=O stretching, and the band at 1900 cm-1 to 2000 cm-1 may be due to alkenes

stretching and the band at 1259.96 cm-1 could be due to -C-C- stretching band.

HPLC analysis of the chloroform extract exhibited a single peak confirming the

presence of a single compound with a retention time of 2.62 minutes and with 99.4% purity.

The 1H-NMR data of extracted compound was found to be in good agreement

with the reported data [110]. In the 1H-NMR spectrum, the signal at δ 3.82 corresponds to

the methyl (-O-CH3) protons while the two signals at δ 6.3 (-C=CH-OH) and at 9.64

(-CH=CH-OH) corresponds to the enol form. The α, β unsaturated olefins (-CH=CH-

C=O) displayed two signals at δ 6.74 and δ 7.52 with high coupling constant (J=16 Hz),

while the aromatic protons gave rise to three signals at δ 6.82 (d), 7.14 (d), and 7.31 (s).

MS analysis revealed a sharp peak with a m/z value of 368.97 corresponding to

the molecular weight of curcumin (368.38). All the results taken together confirm that the

compound isolated was curcumin, whose structure is given below (Figure 2.13). The GC,

FTIR, NMR and MS spectra of curcumin is shown in Figure 2.14.

O O

H3CO

HO

OCH3

OH

H

Figure 2.13: Chemical structure of curcumin

59

a

b

60

Figure. 2.14: Characterization of curcumin isolated from Curcuma longa using

chloroform as solvent a) FTIR, b) HPLC, c) 1H NMR, d) MS

d

c

61

4.3. Ethanol extract of turmeric

Turmeric was extracted with ethanol and analyzed by TLC and UV-Visible

spectrophotometry. The extract gave two spots on TLC and two peaks when analyzed by

UV-Visible spectrophotometry. The λmax of the two peaks were 420.1 nm (corresponding

to curcumin) and 221 nm (Figure 2.15).

Figure 2.15: UV spectrum of ethanol extract of Curcuma longa.

The peak at 221 nm could be another compound or impurity. The extract was

dried to yield a yellow powder, which was subsequently washed with chloroform to

finally yield pure curcumin powder.

5. Citrullus colocynthis (L.)

Citrullus colocynthis is the plant which produces colocynth apples and is very

similar to the common watermelon vine. The colocynth apples are small, hard fruit with a

bitter pulp. The plant bears solitary sterile flowers and branched tendrils. The colocynth

plant is a native of arid soils. It has a large, fleshy perennial root, which sends out

slender, tough, angular, scabrid vine-like stems. These usually lie on the ground for want

of something to climb over, but which, if opportunity presents, climb over shrubs and

herbs by means of axiliary branching tendrils. The leaves are angular, lobed and, as

already stated, almost the exact duplicate of watermelon leaves. The flowers are yellow,

long-peduncled, solitary in the axils of the leaves. They are monecious, the stamens and

pistils being borne in different flowers on the same plant. Each has a yellow campanulate,

five-lobed corolla and a five-parted calyx. The female flowers are readily distinguished

62

by a globose, hairy, inferior ovary. The fruit is globular, smooth, with a hard but thin

rind, something like a gourd. It is filled with a soft, white pulp, in which are imbedded

numerous seed. This pulp is the article used in medicine [111].

The colocynth, or bitter-apple, of commerce, when deprived of its rind, as is

mostly the case, presents a white, light and spongy pulp that readily breaks into three

wedge-shaped pieces, each holding imbedded near its outer rounded surface a number of

flat, ovate seeds. The proportion between pulp and seed varies according to different

authors, from 23 to 33 per cent of pulp and 67 to 77 per cent of seed. The intensely bitter

taste of colocynth resides in the pulp only, while the seeds at best contain only traces of it;

hence the inert seeds are removed before making pharmaceutical preparations of colocynth.

The bitter taste and the powerful medicinal virtues of the pulp are due to the presence of a

probably amorphous glucoside colocynthin, first identified and named by Meissner and by

Vauquelin (1818), and later investigated and obtained in a much purer form [112].

Colocynthin is soluble in water and alcohol, but insoluble in benzol, benzin,

carbon disulfide and ether. Dilute acids resolve it into dextrose and tasteless colocynthein,

acetic acid being likewise formed [113]. Walz obtained from an alcoholic extract of

colocynth an ether-soluble crystalline and tasteless substance insoluble in water, which he

called colocynthin [114]. The ash of the pulp varies from 8.6 to 14 per cent while that of

the seeds amounts to about 2.5 percent [112].

Citrullus colocynthis (L.) has been the object of innumerable investigations.

Citrullus colocynthis has been used for its medicinal properties since ancient times. The

oldest record of Citrullus colocynthis is supposed to be found in connection with Prophet

Elisha’s miracle. During the second half of the nineteenth century [115]. While Hexke

could not establish the glycosidic character of colocynthin, Johannson described its

hydrolysis products and reported the isolation of colocynthein and α-elaterin together

with other substances [116]. Later Naylor and Chappe obtained colocynthin in a

crystalline form [117]. Power and Moore, who doubted the purity of the substances obtained

by their predecessors, reinvestigated the plant and isolated citrullol, α-elaterin, an alkaloid

and various other components [118]. While Hamilton and Kermac could not repeat the

63

previous work and did not isolate aelaterin, Siddiqui et al., obtained a series of crystalline

compounds including α-elaterin [119]. A recent publication by Khadem and Rahman’O

reported the isolation of a glycoside using a procedure that is followed in this work [120].

The distribution of cucurbitacins among the various species of the Cucurbitaceae

has been studied extensively by Enslin and a group of South African workers [121]. They

have found that these substances occur in nature as glycosides or as aglycones according

to the presence or absence of an enzyme named elaterase, a glycoside hydrolase, of

undetermined specificity which is capable of rapidly hydrolysing the glycosides.

A comparative study of several species showed that elaterase is present in high

concentration in genera such as Cucumis and Lugenaria, whereas it is absent in Citrulius

and Cucurbita. The complicated structure of the cucurbitacins and their high sensitivity

to hydrolytic agents accounts for the difficulties encountered by several authors in the

identification and the study of the nature of the aglycones and their glycosides. This

identification could best be done when they were extracted and isolated from plants in

which the enzyme was present, as for instance in Ecbaliium elaterium or other species.

5.2. Materials and methods

Fresh Citrulus colocynthis plants were collected from coastal areas of Tuticorin,

India. Petroleum ether (Merck, Germany), hexane (Merck, Germany), chloroform

(Merck, Germany), absolute alcohol (Jiangsu Huaxi, China), Na2SO4 (Qualigens, India), TLC

plates (Merck, Germany)., acetone (Merck, Germany), Tris.HCl (Loba, India) were used as

obtained. Experimental techniques followed were as described in the previous sections.


The Citrullus colocynthis fruit pulp was taken for extraction using ethanol as

solvent. A paste like compound was obtained which was washed with chloroform and the

solvent was carefully removed in nitrogen atmosphere. TLC and UV-Visible

spectrophotometer analysis of this extract revealed the presence of a single compound

which was further analysed by FTIR, HPLC, NMR, MS.

The FTIR analysis of the extract gave a broad band at 3477 cm-1 which was due to

OH stretching, bands at 2974, 2931, 2879 cm-1 can be attributed to H-C-H asymmetric

64

stretching, bands at 1707 and 1637 cm-1 are attributed to C=O stretching, bands at 1512,

1458, 1429, 1379 cm-1 can be assigned to -C-C- stretching, bands at 1265, 1222 cm-1 could

be because of -C-C- stretching, and bands at 1039, 927, 615cm-1 due to -C=C- mono subs.

The ethanol extract of compound was analyzed by HPLC and the extract gave a

single peak confirming the presence of a single compound with a retention time of 5.68

min and 99.6% purity.

The 1H-NMR data of extracted compound was found to be in good agreement

with the reported data [122]. In the 1H-NMR spectrum, the signals at δ 2.232 (3, 1H, d,

J=5.590), 1.443 (10, 3H), 1.443 (11, 3H), 1.423 (14, 3H), 3.825 (15, 1H, ddd, J=7.320,

J=5.590, J=2.190), 1.878 (16, 1H, dd, J=10.698, J=7.320), 1.833 (16, 1H, dd, J=10.698,

J=2.190), 2.474 (18, 1H, d, J=16.680), 2.447 (18, 1H, d, J=16.680), 1.742 (21, 1H, dd,

J=10.190, J=3.380), 2.245 (22, 1H, ddd, J=13.283, J=10.190, J=7.129), 2.249 (22, 1H, ddd,

J=13.283, J=5.229, J=3.380), 5.580 (23, 1H, dd, J=7.129, J=5.229), 3.039 (30, 1H, d, J=6.777).

Mass spectra studies reveal a single sharp peak with a m/z value of 515.41 which

corresponds to the molecular weight of cucurbitacin I (514.70). The HPLC, FTIR, NMR

and MS spectra are shown in Figure 2.16.

a

65

9 8 7 6 5 4 3 2 1 0 ppm

0.8035

0.8208

0.8297

0.8325

0.8534

0.8556

0.8620

0.8852

0.8928

0.9061

0.9218

0.9304

0.9387

0.9541

0.9626

0.9665

0.9858

0.9940

0.9961

1.0175

1.0767

1.0841

1.0917

1.1017

1.1090

1.1149

1.1395

1.3474

1.3592

1.4165

1.4248

1.4329

1.5905

1.5985

1.6067

1.6225

1.6304

1.6380

1.6473

1.6550

1.6644

1.6731

1.6781

1.6862

1.9438

1.9489

1.9526

1.9577

1.9790

1.9827

1.9878

2.1503

2.1573

2.1678

2.1747

2.1852

2.1922

3.3987

3.4108

3.4229

6.90

0.90

13.56

3.00

2.08

1.91

2.04

4.85

2.03

1.99

2.00

Current Data ParametersNAME CRR-SMR-I-43EXPNO 1PROCNO 1

F2 - Acquisition ParametersDate_ 20110910Time 17.27INSTRUM spectPROBHD 5 mm BBO BB-1HPULPROG zg30TD 65536SOLVENT CDCl3NS 16DS 2SWH 8223.685 HzFIDRES 0.125483 HzAQ 3.9846387 secRG 128DW 60.800 usecDE 6.00 usecTE 294.4 KD1 1.00000000 secTD0 1

======== CHANNEL f1 ========NUC1 1HP1 14.00 usecPL1 -0.90 dBSFO1 400.1324710 MHz

F2 - Processing parametersSI 32768SF 400.1300012 MHzWDW EMSSB 0LB 0.30 HzGB 0PC 1.003.353.403.45 ppm

3.374

3.385

3.399

3.411

3.423

3.437

3.448

c

b

66

Figure. 2.16: Characterization of cucurbitacin I isolated from Citrullus colocynthys using

ethanol as solvent a) FTIR, b) HPLC, c) 1H NMR, d) MS

Based on all the spectral analysis, the isolated compound was identified as

cucurbitacin I whose structure is given below (Figure 2.17).

Figure 2.17: Chemical structure of cucurbitacin I

O

OOH

OH

OH

O

HO

d

67

6. Coriandrum sativum L.

Coriander (Coriandrum sativum) also known as cilantro, Chinese parsley or dhania, is

an annual herb belonging to the family Apiaceae. Coriander is native to regions spanning

from southern Europe and North Africa to southwestern Asia. It is a soft, hairless plant

growing to 50 cm tall. The leaves are variable in shape, broadly lobed at the base of the

plant, and slender and feathery higher on the flowering stems. The flowers are borne in

small umbels, white or very pale pink, asymmetrical, with the petals pointing away from

the centre of the umbel longer (5–6 mm) than those pointing towards it (only 1–3 mm

long). The fruit is a globular, dry schizocarp 3–5 mm (0.12–0.20 in) in diameter [123].

The plant is grown widely all over the world for seed, as a spice, or for essential

oil production. At one time, Coriander was one among the world’s leading essential oil

plants [124, 125]. The composition of the essential oil of Coriander fruits in some of the

world has been studied and found to differ from each other. It has been reported that

Coriander seed oil contains linalool (60-70%) and 20% hydrocarbons and the

composition of the herb oil completely differs from the seed oil [126]. Rastogi and

Mehrotra reported detection of α-pinene, limonene, β-phellandrene, eucalyptol, linalool,

borneol, β-caryophyllene, citronellol, geraniol, thymol, linalyl acetate, geranyl acetate,

caryophyllene oxide, elemol and methyl heptenol in seed oil by TLC [127]. Telci et al.,

reported that in the ripe fruits, the content of essential oil is comparably low (typically,

less than 1%); the oil consists mainly of linalool (50 to 60%) and about 20% terpenes

(pinenes, γ-terpinene, myrcene, camphene, phellandrenes, α-terpinene, limonene,

cymene) [128]. Asolkar et al., reported a type from Mysore that contained high geranyl

acetate [129]. Coriander oil is a valuable ingredient in perfumes. Its soft, pleasant,

slightly spicy note blends into scents of oriental character. Ghani reported the presence of

linalool, pinene, cymene, phellandrene, geraniol and borneol [130].


Fresh Coriander plant was collected from Siruvani crop fields, Coimbatore, India.


Germany), absolute alcohol (Jiangsu Huaxi, China), Na2SO4 (Qualigens, India), TLC

68

plates (Merck, Germany),, acetone (Merck, Germany) Tris.HCl (Loba, India) were used

as received. All the other techniques adopted were as described in the previous sections.


The chloroform extract was separated by silica gel column chromatography with

hexane as the solvent. The polarity of the solvent was increased step by step using

acetone. The first two fractions of the extract were obtained by the difference in polarity

of the eluent. The first compound was isolated using hexane (100%) and analysed by

FTIR, GC, GC MS, HPLC, 1H-NMR. The second compound was isolated using a

mixture of 99% hexane and 1% acetone. This compound too was analysed by FTIR, GC,

GC MS, HPLC, 1H NMR.

6.3.1. Chloroform extract of Coriander leaves

6.3.1.1. Compound 1

Compound 1 isolated using chloroform, gave the same characteristic peak as

cymene in Gas chromatography. Cymene is explained in detail under the curry leaf

section. The structure of compound 1 was confirmed by mass spectrophotometry and

GC-MS which gave values that were identical to that of cymene. The FTIR, GC, GC MS

and 1H-NMR characterization data is already presented in the section explaining the

extract analysis of curry leaf.

6.3.1.2. Compound 2

The FTIR analysis of the extract gave a broad band at 3477cm-1 to 3000 cm-1

which was due to OH stretching, bands at 2974, 2931, 2879 cm-1 can be attributed to H-

C-H asymmetrical stretching, bands at 1707 and 1728 cm-1 are due to C=O stretching,

bands at 1512, 1458, 1429, 1379 and 1599 cm-1 can be assigned to -C-C- stretching,

bands at 1265, 1222 cm-1 is because of -C-C- stretching, and bands at 1039, 927, 615cm-

1 is due to -C=C- mono subs.

The chloroform extract of compound 2 was tested by HPLC and it gave a single

peak with a retention time of 3.822 minutes, with 99.2% purity.

69

The 13C-NMR analysis of compound 2 displayed the characteristic signal of 1,4

dihydroquinone at 152.5 δ, 150.2 δ, 118.5 δ and 117.2 δ. The methane carbon groups

(-O CH-O-) appeared at 101.2 δ while all other methane carbons ( ) appeared

between 77.1 δ and 68.5 δ.

Mass spectral analysis revealed a sharp peak with a m/z value of 272.39 which

corresponded to the molecular weight of arbutin (272.25), confirming its presence.

The HPLC, FTIR, NMR and GCMS spectra are shown in Figure 2.18.

a

70

c

b

71

Figure. 2.18: Characterization of arbutin isolated from Coriandrum sativum using

chloroform as solvent a) FTIR, b) HPLC, c) C13 NMR, d) MS

Thus based on spectral analysis, the isolated compound was identified as Arbutin,

whose structure is given below (Figure 2.19).

Figure. 2.19: Chemical structure of arbutin

OH

OO

OH

HO

HO

OH

d

72

6.3.2. Ethanol extract of Coriander leaves

The ethanol extract was separated by silica gel column chromatography using

hexane as a solvent. The polarity of the solvent was increased step by step using acetone.

The first two fractions of the extract were obtained using difference in polarity of the

eluent. The first compound was isolated using hexane (100%) and analysed by FTIR, GC,

GC MS, HPLC, 1H NMR. The second compound was isolated using a mixture of 99%

hexane and 1% acetone, and analysed by FTIR, GC, GC MS, HPLC, 1H-NMR techniques.

6.3.2.1. Compound 1

TLC and UV-Vis spectroscopy data of compound 1 isolated using ethanol exactly

matched the results obtained for phellendrene (Section 2.4.2.2), thereby confirming

its presence.

6.3.2.2. Compound 2

The FTIR analysis of the second compound gave a broad band at 3482 cm-1 to

3000 cm-1 which was due to OH stretching, bands at 2974, 2931, 2879 and 2788 cm-1 can

be attributed to H-C-H asymmetrical stretching, bands at 1707, and 1728 cm-1 are due to

C=O stretching, bands at 1512, 1228, 1280, 1458, 1429, 1379 and 1599 cm-1 can be

assigned to -C-C- stretching, bands at 1265 and 1222 cm-1 is because of -C-C- streching,

and bands at 1039, 927, 615 cm-1 is due to -C=C- mono subs.

Analysis by HPLC revealed that compound 2 had a retention time of 8.509

minutes, with 98.2% purity. The HPLC analysis confirmed the presence of this

compound as the sole compound.

The 1H-NMR spectrum (Figure 2.20) displayed the two aromatic protons of the

fused rings of rutin and appeared as singlets at 6.13 δ and 6.35 δ, while that of attached

aromatic ring showed signals at 6.79 δ (d), 7.52 δ (d), 7.5 δ (s). The acetal protons

displayed two signals at 6.32, 6.37 and 6.31 δ while methine protons of carbohydrate unit

gave signals at 6.18 δ and 6.39 δ.

Mass spectral studies (Figure 2.20) revealed a sharp peak with a m/z value of

664.24 which corresponds to the molecular weight of of Rutin (664.57). Based on MS,

73

the major peak was confirmed to be that of Rutin. The HPLC, FTIR, NMR and MS

spectra are shown in Figure 2.20.

a

b

74

Figure. 2.20: Characterization of rutin isolated from Coriandrum sativum using ethanol

as solvent a) FTIR, b) HPLC, c) 1H NMR, d) MS

Thus based on spectral analysis, the isolated compound was identified as rutin

whose structure is given below (Figure 2.21).

d

c

75

Figure. 2.21: Chemical structure of rutin

7. Eichhornia crassipes (Mart.) Solms. – Laub.

Its habitat ranges from tropical desert to subtropical or warm temperate desert to

rainforest zones. It tolerates annual precipitations of 8.2 dm to 27.0 dm (mean of

8 cases = 15.8 dm), annual temperatures from 21.1°C to 27.2°C (mean of 5 cases = 24.9°C),

and its pH tolerance is estimated at 5.0 to 7.5. It does not tolerate water temperatures

>34°C. Leaves are killed by frost and salt water, the latter trait being used to kill some of

it by floating rafts of the cut weed in the sea. Water hyacinths do not grow when the

average salinity is greater than 15% of sea water. In brackish water, its leaves show

epinasty and chlorosis, and eventually die [131].

7.1. Phytoremediation, waste water treatment

The roots of Eichhornia crassipes naturally absorb pollutants, including lead,

mercury, and strontium-90, as well as some organic compounds believed to be carcinogenic, in

concentrations 10,000 times that in the surrounding water [132]

There are two types of water hyacinth available in the region

1) Long type 2) Dwarf type.

O

OH

OH

O

OH

HO

O

O

OH

O

HO OH

OOH

OH

HO

HO

76

The former type is mostly available in stagnant water polluted with high effluents

while the later is available mostly in the paddy field. Analysis indicated that these plants

contain lot of potassium and sodium salts which hamper the balances of magnesium and

causes the symptoms of diuretics. The other anti-metabolites are due to the presence of

oxalates which affects its utilization.

Tannin content was also recorded to be a little lower in long type compared to

dwarf type of water hyacinth. There was much difference in tannin content of leaf, stem.

The tannin content of fresh water hyacinth was found to be a little higher than the

naturally dried plants. Alkaloids and glycosides were not present.

7.2. Bioenergy

Because of its extremely fast growth rate, Eichhornia crassipes is an excellent source

of biomass. One hectare of standing crop can thus produce more than 70,000 m3 of biogas

[131]. According to Curtis and Duke, one kg of dry matter can yield 370 liters of biogas,

giving a heating value of 22,000 kJ/m3 compared to pure methane. Wolverton and

McDonald report only 0.2 m3 methane per kg, indicating requirements of 350 MT

biomass/ha to attain the 70,000 m3 yield projected by the National Academy of Sciences

[133]. Ueki and Kobayashi mention more than 200 MT/ha/yr [134]. Reddy and Tucker

found an experimental maximum of more than a half ton per day.[13] Bengali farmers collect

and pile up these plants to dry at the onset of the cold season; they then use the dry water

hyacinths as fuel [135].

In India, a ton of dried water hyacinth yield circa 50 liters ethanol and 200 kg

residual fiber. Bacterial fermentation of one ton yields 26,500 cu ft gas (600 Btu) with

51.6% methane, 25.4% hydrogen, 22.1% CO2, and 1.2% oxygen. Gasification of one ton

dry matter by air and steam at high temperatures (800°) gives circa 40,000 ft3 (circa 1,100 m3)

natural gas (143 Btu/cu ft) containing 16.6% H3, 4.8% methane, 21.7% CO, 4.1% CO2,

and 52.8% N. The high moisture content of water hyacinth, adding so much to handling

costs, tends to limit commercial ventures [136]

77

7.3. Materials and methods

Fresh Eichhornia crassipes plant was collected from Ukkadam pond, Coimbatore,

India. Petroleum ether (Merck, Germany), hexane (Merck, Germany), chloroform

(Merck, Germany), absolute alcohol (Jiangsu Huaxi, China), Na2SO4 (Qualigens, India), TLC

plates (Merck, Germany), Acetone (Merck, Germany), Tris.HCl (Loba, India) were used as

received. The experimental techniques were adopted as described in previous sections.


The ethanol extract was separated by silica gel column chromatography using hexane

and acetone as solvents. The polarity of the solvent was increased step by step using acetone.

Three fractions of the extract were obtained using difference in polarity of the eluent. The first

compound was isolated employing a mixture of hexane and acetone in the ratio of 99.5:0.5.

The extract was analyzed by FTIR, GC, GC-MS, HPLC, 1H-NMR. The second compound was

isolated using a mixture of hexane and acetone in the ratio 98.5: 1.5. The third compound was

isolated using a mixture of hexane and acetone in the ratio 95:5. The second and third

compounds were again analysed by FTIR, GC, GC-MS, HPLC and 1H-NMR.

7.4.1. Compound 1

IR spectrum (ν, cm-1) of compound 1 revealed a broad band at 3100 cm-1 to 3000 cm-1

which was due to OH stretching. Bands at 1721 and 1681 cm-1 can attributed to C=O

stretching, bands at 1259 cm-1, 1126 cm-1 and 1301 cm-1 can be attributed to

-C-C- stretching, bands at 1058 cm-1, and 1028 cm-1 can be because of -C=C- stretching,

band at 873 cm-1 may be due to -C=C- mono subs, and those at 538, 718, 1514 cm-1 may

be due to H-C-H bending.

HPLC analysis of the ethanol extract revealed a single peak with a retention time

of 2.38 minutes and 99.74% purity, confirming the presence of a single compound.

The 1H-NMR can be viewed better as pyranone fused with aromatic ring and

attached with another aryl group. In the 1H-NMR spectrum, the protons of fused ring

appeared as two singlets at 6.18 and 6.39 δ while the protons of the attached ring

appeared at 7.69 δ (s), 7.52 δ (d) and 6.85 δ (d).

78

Mass spectal studies (Figure 2.22) exhibited m/z value of 176.17, which

corresponds to the molecular weight of 7-hydroxyl-4-methylcoumarin (177.25),

confirming the presence of methyl hydroxyl coumarin.

a

b

79

Figure. 2.22: Characterization of methyl hydroxyl coumarin isolated from Eichhornia

crasipus leaf using ethanol as solvent a) FTIR, b) HPLC, c) 1H NMR, d) MS

c

d

80

7.4.2. Compound 2

IR spectrum (ν, cm-1) of compound 2 revealed a broad band at 3400 cm-1 to 3358 cm-1

which was due to OH stretching. Bands at 2721 and 2681 cm-1 can attributed to C=O

stretching, those at 1259 cm-1, 1126 cm-1 and 1301 cm-1 can be attributed to

-C-C- stretching, the ones at 1058 cm-1and 1028 cm-1 to -C=C- stretching, the one at

873 cm-1 to -C=C- mono subs, and those at 538, 718, 1514 to H-C-H bending.

HPLC analysis of the second ethanol extract fraction resulted in a single peak

with a retention time of 3.506 minutes and a purity of 98.4%, confirming the presence of

a single compound.

The 1H-NMR spectrum (Figure 2.23) displayed signals at 7.92 δ (dd, J = 8.0Hz,

3.2Hz, 2H), 7.56-7.60 δ (m, 2H), 7.52 δ (dd, J = 2.1Hz, 1.2Hz, 2H), 7.32 δ (dd, J =4.0Hz,

1.2Hz, 2H), 3.78 δ (s, 2H).

Mass spectra (Figure 2.23) of the second compound exhibited a m/z value of

338.26 which corresponds to the molecular weight of quercetin dehydrate (338.72).

The HPLC, FTIR, NMR and MS spectra are shown in Figure 2.23.

a

81

b

c

82

Figure 2.23: Characterization of quercetin dehydrate isolated from Eichhornia crasipus

leaf using ethanol as solvent a) FTIR, b) HPLC, c) 1H NMR, d) MS

7.4.3. Compound III

IR spectrum (ν, cm-1) of compound 3 revealed a broad band at 3500 cm-1 to

2950 cm-1 which was due to OH stretching. Bands at 1705 and 1681 cm-1 can be

attributed to C=O stretching, those at 1893, 2869, 2925, 2978, 3016 and 3049 cm-1 to

H-C-H asymmetrical stretching, those at 1259 cm-1, 1126 cm-1 and 1301 cm-1 to

-C-C- stretching, the ones at 1058 cm-1 and 1028 cm-1 to -C=C- stretching, the one at

873 cm-1 to -C=C- mono subs, and those at 538, 718 and 1514 cm-1 to H-C-H bending.

The 1H-NMR spectrum (Figure 2.24) displayed signals at 8.7 to 9.6 ppm broad

singlet seems to be presence of phenolic OH, 9.8 to 10.2 ppm broad singlet is

also phenolic OH, 6.8 ppm to 7.2 ppm corresponding peaks indicates presence of

aromatic ring, 5.3 ppm and 5.5 ppm which peaks corresponds to O-CH2 protons. All the

δ values are given below, 4.443 δ (4, 2H, d, J=3.764), 3.337 δ (5, 1H, td, J=3.764,

J=2.690), 5.30 δ (7, 1H, d, J=2.680), 7.385 δ (12, 1H, d, J=2.033), 7.213 (23, 1H, d,

J=0.000), 7.204 δ (30, 1H, d, J=0.000), 5.283 δ (31, 1H, dd, J=3.470, J=2.680), 7.390 δ

d

83

(36, 1H, d, J=2.044), 7.106 δ (47, 1H, d, J=0.000), , 4.498 δ (55, 1H, dd, J=3.470,

J=3.460), 7.114 δ (71, 1H, d, J=0.000), 7.098 δ (78, 1H, d, J=0.000), 5.280 δ (79, 1H, dd,

J=3.460, J=2.690), 7.274 δ (95, 1H, d, J=0.000), 2.495 (103, 3H).

HPLC analysis of the third fraction of ethanol extract resulted in a single peak

with a retention time of 4.58 minutes and a purity of 99.4%, confirming the presence of a

single compound.

Mass spectra (Figure 2.24), showed that the third compound had a m/z value of

1699, which corresponds to the molecular weight of tannic acid (1701). The HPLC,

FTIR, NMR and MS spectra are shown in Figure 2.24.

a

84

b

c

85

Figure. 2.24: Characterization of methyl hydroxyl coumarin isolated from Eichhornia

crasipus leaf using ethanol as solvent a) FTIR, b) HPLC, c) 1H NMR, d) MS.

Based on spectral analyses compound 1 was identified as coumarin, compound 2 was

identified as quercetin dehydrate and compound 3 was identified as tannic acid.

The structure of coumarin, quercetin and tannic acid are given in Figure 2.25.

.

Quercetin Methylene hydroxyl coumarine

O OO O

OHOH

O

OH

OH

OH

OH

HO

O

d

86

Tannic acid

Figure. 2.25 : Chemical structure of quercetin, coumarin and tannic acid.

4. Conclusion

This chapter describes the collection, identification and certification of plant

sources. Details of experimental procedures for extraction, isolation, purification and

spectral investigation of natural antioxidants are also presented. Use of Soxhlet extraction

procedure with solvents of various polarities is explained. This chapter also deals with the

purification protocol of isolated compounds. Six different plants were selected from

which twelve compounds were isolated.

The characterization of isolated compounds using various techniques and

structure elucidation based on spectral investigation are described in this chapter. This

chapter presents the details of spectral/chromatographic investigations such as TLC, UV-

Vis spectrophotometery, FT-IR, 1H-NMR, 13C-NMR, GC, GC-MS, HPLC and MS. Table

2.2 summarizes the details of plant sources, the isolated natural antioxidants and the

investigation carried out to confirm their chemical structures.

O

OO

OO

O

O H

O H

H O

H O

O

O

H O

H O O H

O

O

H O O H

O

O

O H

O H

O H

O

OH

H O

O H

O

O

O H

O HO

O

H O O H

H O

O

O

O HH O

O

O

O H

O H

O H

87

Sl. No

Plant Sources Isolated Natural

Antioxidant FTIR GC

GC MS

HPLC MS 1H

NMR

13C NMR

1 Curry Leaf & Mint leaf

Carvone √ √ √ - - √ -

2 Curry, Mint & Coriander leaf

Phellandrene √ √ √ - - √ -

3 Curry, Mint & Coriander leaf

Cymene √ √ √ - - √ -

4 Curry leaf Caryophyllene √ √ √ - - √ -

5 Mint leaf Menthol √ - √ √ - √ √

6 Turmuric Curcumin √ - - √ √ √ -

7 Citrulus

colocynthis Cucurbitacin I √ - - √ √ √ -

8 Coriander Arbutin √ - - √ √ - √

9 Coriander, Citrulus

colocynthis Rutin √ - - √ √ √ -

10 Eichhornia

crasipes

3,3-methylene-bis (4-hydroxy

coumarin √ - - √ √ √ -

11 Eichhornia

crasipes Quercetin √ - - √ √ √ -

12 Eichhornia

crasipes Tannic acid √ - - √ √ √ -

Table 2.2: Isolated antioxidants and the charecterization investigation carried out.

88

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