Natural bioactive compounds and their characterization ... · PDF fileNatural bioactive compounds and their characterization isolated from terrestrial ... sources for their livelihood

Natural bioactive compounds and their characterization isolated from terrestrial plants and marine algae

Patil Meena

Department of Zoology, Dr.Babasaheb Ambedkar Marathwada University. Aurangabad-431 004, Maharashtra State, India

The protection of human health and search for healthy food are continuous struggles of all people in the world. The use of pesticides and synthetic chemicals is hazardous to living organisms even if they are effective to achieve a particular target however, their side effects cannot be overlooked. The natural toxins or biotoxins present in living organisms are considered as a solution to this problem. Drugs from aquatic weeds, algae a marine organism, terrestrial plants and animals are being developed and are crucial need of time with a priority for aquaculture, pharmaceutical and cosmetic industries [1, 2]

With this concept, attempts were made to identify, isolate and characterize bioactive compounds from marine algae and few terrestrial plants for selective purpose. Bioactive compounds are naturally occurring secondary metabolites, often termed as ‘biotoxin’. The bioactive compounds are potential source of new materials for commercial and biomedical purposes. Their potency, specificity, and usefulness help to revive the physiological functions by understanding the pharmacological and chemical properties. These compounds are effective individually or in combined form to evoke physiological response in the organism of interest. In this chapter information on bioactive compounds from marine algae species belonging to Chlorophyta, Pheaophyta, Rhodophyta and terrestrial plants, namely Acacia nilotica, Moringa oleifera, Azardichta indica, Cestrum noctrum and Cestrum diurnam is included. Extracts were tested with focus on finding antimicrobial activity using human pathogens, Piscicidal activity of aquaculture species, insecticidal property for cotton pest and molluscicidal activity to check infestation of trematode larva in mollusk species Lymnae accuminata. Few compounds of interest were further purified, retested and their chemical structure was identified.

Keywords Bioactive compounds; marine algae; human pathogens; antimicrobial insecticidal, piscicidal

1. Background

Mankind has been using natural sources for their livelihood since ancient time and presently researchers looking seriously for alternative remedies that could be without side effects as therapeutic drugs. The organic molecules isolated from natural sources, so far surveyed, are reported in marine animals [3, 4], algae [5, 6], terrestrial plants [7, 8] and terrestrial animals [9]. These organic molecules are termed as bioactive compounds; their biological and chemical properties in algae, in variety of the bacterial species and in some Porifers, Coelenterate, Mollusk , Echinoderms are studied as ‘ biotoxins’ having medicinal properties[10 ,11,12,13,14,15,16]. Significant zootoxin specifically collected from invertebrates and venoms mainly from vertebrates groups are well documented as neurotoxic and cytotoxic,(17).The toxic components are complex and are capable of affecting, if not all, biologic systems and cells in living organisms. The therapeutic purpose of toxins may add to the potential beneficial use of this otherwise they are highly complex group of chemicals (18) For many centuries the Asian countries particularly India, China and Japan have had been using the plant material in drug therapy in treating hypothyroidism, anemia, menstrual difficulties and intestinal disorders and many more (19). The data available as ancient literature revealed the knowledge about the sources of medicinal plants with the properties and their uses. This information has now widened the scope for exploration of ‘New Drugs’ therefore, many industries are taking interest in ethnopharmacological studies. The information of some reputed industries, few to mention here from India, China and US is available on internet manufacturing herbal pharmaceutical and health-care products that are widely used globally(20,21,22,23). Thus the valuable information is continuously being added on the topic ‘concept of drugs’ from natural sources. It all begun more intensely from 1960 onwards. The record on antimicrobial, antifungal, antiviral, anticancer compounds etc and recently antioxidant activity (24) from all possible sources is generated in due course of time. Recent studies show microorganisms are becoming resistant to current antibiotics and discovery of new potent antibiotics is need of time. The literature review shows algae are excellent source of high polysaccharide, proteins, lipids, vitamins, potash, iodine, trace elements and even they can be used as fodder and fertilizers (25,26) The wide spectrum of complex compounds is isolated from marine algae - for example the alginates extracted can be used in plastic material. Because of its non-toxicity and colloidal properties alginic acids are used in food industry and have number of applications in pharmaceutical industries. The product agar-agar, a alginic acid derivative of red algae, is vastly used in microbiological studies. Another product Carrageenin, being non toxic, used in stabilizing emulsions, suspension of solids in drugs synthesis in pharmaceutical and in brewing as well as in leather and textile industries. However, some species of algae are toxic to other organisms. This characteristic can be used in many drugs production. Some mangrove species found

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more effective against viral strains (27, 28, 29, 30, 31) showed cytotoxic and anti-platelet aggregation activity and antioxidant activity (32). Taking in to account the multiple uses of natural compounds, the present chapter is mainly focused on isolation of bioactive compounds or biotoxins from seven species of marine algae and five terrestrial plants, their biological properties and chemical nature of some compounds.

2. Natural bioactive compounds

The use of plants for medicinal purposes is documented in Indian Ancient literature volumes- Rigveda and Ayurveda (4500-1600BC). The famous Indian physician Charaka (1000-600BC) described the uses and mode of application of some 350 plants in Charak Samhita and in 2001 Council of Scientific& Industrial Research (CSIR), New Delhi, India opened up Traditional Knowledge Digital Library (TKDL) about herbal medicines from India. In general the parts of plant having medicinal contents are roots, stem, bark, leaves, flowers and seeds that are utilized as source of drugs. Unfortunately the progress in the subject was poor due to several reasons, few of them are- difficulties in procurement of material or plant species, its purification, and clinical trials to bring the products into the market etc. Another point is the chemical composition of biologically active compounds varies from species to species with their topographical distribution and the part of plant used for extraction. With pace of advancement in Medical Science, people prefer for the quick-result therapeutic remedies, so gradually the ancient knowledge of drugs or industries manufacturing them could not attract the user or catch the market. Apart these realities, the studies are still carried out on the bioactive compound to provide information on sources, its availability according to the season, and the biological properties of the isolated compounds. In present scenario the efforts are being exercised in the nick of time to explore the physiological and biochemical properties of the bioactive compounds. The first and large-scale antimicrobial activity screening dates back to 1943 testing active components from 2300 species against microbes with a note that “the toxins of algae and plant can be utilized for human benefits”(33). Later many studies have added information on herbal drugs (34, 35, 36, 37, 38). World Health Organization (WHO) has accepted the world wide population still uses herbs and other traditional medicines as therapeutic agents for liver diseases, cough remedies diabetics, arthritics, memory enhancers; so organization appealed the research fraternity to assess quality of drugs based on the concentration of their active principles and standardization of herbal drugs as an essential contribution to the existing data.

3. Natural compounds are primary and secondary metabolites

The plant chemicals are often classified as either primary or secondary metabolites (39). Primary metabolites are substances needed for physiological processes that are linked with basic cell metabolism and are accumulated in seeds and vegetative store organs. These metabolites are used as food, food additives and raw material (25) for industries such as vegetable oils, fatty acids (used for making soap and detergents) and carbohydrates (sugars, starch, pectin and cellulose). The secondary metabolites are compounds biosynthesized from primary metabolites and their storage locations varies in species or in a family of particular taxonomic group. Furthermore their synthesis occur at distinct growth stages of plant. In this context an antibiotic can be defined as a secondary metabolite substance produced by living organism, which inhibits the growth or activity of another microorganism .It is selectively toxic agent with selective action in a fashion the agent does not harm the producer but it is toxic to others. Now the spectrum of producers is broadened of adding the list of plants, algae and other sources too (40)

4. Algae and plant material as bioactive compounds (Antimicrobial, Insecticidal, Molluscicidal, Piscicidal)

In present study, the marine algae and terrestrial plants were explored to identify their biological properties with further purification of selected compounds of interest to know their chemical nature. Five terrestrial plants and seven species of marine algae were selected, extracted with organic solvents to isolate compound of our interest and few of them were purified to find out the chemical nature. Scientific nomenclature of algal species was confirmed at National Institute of Oceanography, Goa, India and terrestrial plants in Post-graduate department of Botany in parent University. The bioactive compounds tested, organic solvents used for extraction, pathogens and species of test animals utilized are mentioned in Table 1-3. The plants belonging to: Family Solanaceae - Cestrum nocternum, Cestrum diurnum; Family-Moringaceae - Moringa oilefera; Mimoceae -Acacia nilotica and, Meliaceae - Azarditcta indica were extracted to isolate the molluscicidal, Insecticidal, Piscicidal, and antimicrobial activity.

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Algae belonging to Chlorophyta-Ulva spe ; Phaeophyta-Spatoglossum asperum, Padina tetrastromatica, Sargassum cinereum; Rhodophyta-Gracilaria corticata, Hypnea musciformis, Porphyra vietnamensis were utilized.

Table 1 The list of plants with specific bioactive compounds isolated and tested with various organisms.

Scientific name of plants bioactive compounds isolated Test organisms Human pathogens

Cestrum nocturnum Cestrum diurnum Acacia nilotica Azadirachta indica Moringa oleifera

Molluscicidal Insecticidal Piscicidal Anti microbial* Antimicrobial* Antimicrobial*

Lymnea accuminata Dysdercus koninjii Labeo rohita Cyprinus carpio, Cirrhina mrigala Catla catla Poecilia reticulata Human pathogens Human pathogens Human pathogens

* See Table 3 for species of microbial pathogens used.

Aqueous and organic solvents (Table 2) extracts were prepared by taking 4 part powder and 1 part solvent; the supernatant was collected and LC50 in fishes and snail was calculated by exposing the batches of test animals to various concentrations for 96 hr and mortality was noted every day in each batch (41). A test for insecticidal activity was performed by topical application of isolated compound on the body of insect Dysdercus konjii. Antimicrobial properties tested by zone of inhibition of microbial growth on agar or suitable medium inoculated with American Type Culture Collection(ATCC), The National Collection of Type Cultures (NCTC) and clinical isolates collected by the Department of Microbiology, Government Medical College, Aurangabad, India (Table 3). Table 2 Polar and non-polar organic solvents used for extraction of bioactive compounds in plants and algae.

Hydrocarbons

Alcohol Ketones Esters Bianary solvent mixture

Chloroform Ethanol Acetone Ethyl acetate Chloroform: Methanol(1:2)

Toluene Benzene Petroleum ether

Methanol Toluene: Methanol (1:3)

Our previous study (42) have confirmed C. nocturnum and C.dirnum, leaves extracts in ethanol were more toxic and that of C.nocturnum was more potent; moreover it was observed that toxicity was directly related to exposure time in all species studied. This part of study was performed to test piscicidal dose that kill weed fishes from the culture ponds without much damage to economically valued fishes.

Table 3 The list of bacterial pathogens used to test bioactive compound susceptibility measured by diameter of zone of growth inhibition.

Sr No Species name Gram +ve or -ve Code number 1 Escherichia coli -ve clinical isolate* 2 Escherichia coli -ve NCTC9002 3 Shingella spe -ve clinical isolate 4 Proteus vulgaris -ve clinical isolate 5 Salmonella typhi -ve clinical isolate 6 Micrococus luteus +ve ATCC9341 7 Bacillus pumilus +ve ATCC14884 8 Bacillus subtilis +ve ATCC6633 9 Staphylococcus aureus +ve NCTC10788 10 Pseudomonas arruginosa -ve NCTC 6750 11 Salmonella abony -ve NCTC6017

The molluscicidal activity was tested to check population growth of snail Lymnea accuminata to curb down trematode larvae infestation. The experimental data confirmed the presence of piscicidal, insecticidal and molluscidial

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components in Cesternum species and it was predominant in C.nocternum (Table 4). Antimicrobial activity confirmed as diameter of zone of microbe growth inhibition, which was not similar in all samples tested. Basically the rate of diffusion varies in an individual medium that follows a principle of diffusion. Therefore, diameter of zone of inhibition is directly proportional to the minimal inhibitory concentration (MIC) and it is not similar in all samples (43). It was observed that methanol and ethanol and binary solvents were effective in extracting antimicrobial activity from all most all species studied. The zone of inhibition was prominent in gram –ve bacteria compared to gram +ve (Fig 1). The antimicrobial property of algal species was not observed as a specification of a group however, Ulva spe, classified under chlorophyta, all organic extracts except for water and chloroform showed susceptibility as antimicrobial compound. Table 4 The LC50 values (mg/lit) for Cestrum spe to evaluate piscicidal, insecticidal and molluscicidal activity in leaves extracts.

Activity & Exposure Time

Extract of C. nocturnum

Cyprinus carpio

Cirrhina mrigula

Labeo rohita Catla Catla

Poecilia reticulata

Piscicidal 48hr

ethanol aqueous

2.75±.0.037 8.56±.0.015

2.6±0.019 8.47±.0.043

2.63±0.044 8.72±.0.051

2.81±0.044 9.03±.0.060

2.29±0.051 8.27±.0.032

Insecticidal 48hr

ethanol C. nocturnum

D.koninjii 6.98±0.016

ethanol C. diurnum

D.koninjii 7.06±0.032

aqueous C. nocturnum C. diurnum

-

Molluscicidal 48hr

ethanol C. nocturnum

L.accuminata 4.89±0.022

ethanol C. diurnum

L.accuminata 5.03±0.002

aqueous C. nocturnum C. diurnum

-

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Fig. 1 The diameter of growth inhibition zone (mm) shown by Chlorophyta, Phaeophyta and Rhodophyta algae extracts with pathogens; the number on each bar corresponds serial number of pathogen mentioned in Table –3.

The terrestrial plants also showed prominent antimicrobial contents extracted with water and organic solvents; moreover they could exhibit grater zone of inhibition (Fig 2) for microbial growth. Cestrum nocturnum being effective toxicant was further purified .The extract sample was subjected to TLC to know its chemical nature with digitonin and standard saponin. 12 bands were represented in a range Rf value 0.025 to 0.960 at 256 and 340 nm under UV source and collected samples were retested to confirm the biological properties. The extracts of Sargassum cinerum, Azadirachta indica and Moringa oleifera purified on column chromatography and aliquots collected were again tested with pathogens. Potency of total 18 fractions was screened and aliquots (200µg/disc) that produced maximum zone of inhibition were compared with standard antibiotics Tetracycline, Ampicillin, Chloramphenicol (10µg/disc ,Himedia) and that found to be almost equivalent.

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Fig. 2 The diameter of growth inhibition zone (mm) shown by terrestrial plants extracts with pathogens; the number on each bar corresponds serial number of pathogen mentioned in Table –3

The results for antimicrobial activity shown in graph (Fig1&2 ) are of those samples that showed inhibition-zone in more than 5 pathogens tested and showing all 11pathogens test-results was out of scope of this chapter. Cestrum nocternum and Cestrum diurnum showed same pattern of mortality results, the only difference was more concentration of Cestrum diurnum compound required to bring in the effect; hence, the results mentioned in Table 4 are confined to Cestrum nocturnum aqueous and ethanol extracts.

5. Chemical nature of the compounds

Ethanol extract of C.nocturnum was separated on TLC and fractions were identified by Libermann Brouchard reagent to specify presence of saponin and Dragendroff reagent for alkaloids. The fraction 4th and 5th showed presence of steroidal and triterpenoid saponins, and 2nd fraction was alkaloid. The fractions separated by column chromatography were collected and again mortatlity test were carried out. The fractions exhibited mortality are- fraction 2nd 40%, 5th 60% and 4th 100%. The chromogenic tests confirmed the presence of three components steroidal saponin, triterpenoid saponin and alkaloid. On the basis of chemical and spectroscopic evidence steroidal glycoside constituents of the leaves of Cestrum nocturnum has reported in Japan. Eight steroidal glycosides , which were classified into a spirostanol saponin,

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a furostanol saponin , a pseudo-furostanol saponin, two pregnane glycosides , two cholestane glycosides , pregnane-carboxylic acid γ-lactone glycoside and of two known spirostanol glycosides(43) .This confirms that Cestrum spe plant leaves do contain saponins that have insecticide( 44) )properties. The 4th fraction collected on column chromatography of marine algae ,Sargassum cinereum was further analyzed by NMR, IR and UV spectrometer; the compound was chromophoric with OH phenolic aromatic rings having antimicrobial activity.

6. Conclusions

The plant leaves and algae contain chlorophyll pigments combined with the secondary metabolites which could be utilized as bioactive compounds. The current study confirms the presence of antimicrobial compounds in marine algae and terrestrial plants selected for this study. Cestrum noctrum and Cestrum diurnam do contain antimicrobial compound (45, 43) insecticidal, molluscicidal and piscicidal components. In general all organic solvents could extract antimicrobial compound from algae and plants that could produce inhibitory zone with microbial pathogens used in this study. Total 80 samples (07 algae and 03 plants extracted with 08 organic solvents) were tested with 11human pathogens and specifically aqueous and ethanol extracts of Cestrum species were tested for insecticidal, molluscicidal and piscicidal activity. Ethanol &methanol was effective in isolating antimicrobial, insecticidal, molluscicidal and piscicidal compounds from plants and marine algae. It concludes that in nature all living organisms- unicellular to highly evolved multicellular, show defense mechanism through production of secondary metabolites and these could be utilized for benefits of human beings.

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