4.0. COLLECTION, IDENTIFICATION, EXTRACTION AND PHYTOCHEMICAL ANALYSIS OF MANGROVE PLANTS

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1 4.0. COLLECTION, IDENTIFICATION, EXTRACTION AND PHYTOCHEMICAL ANALYSIS OF MANGROVE PLANTS 4.1. Introduction Herbal medicines have long been used for the remedies of human diseases because they contain components of therapeutic value (Nostro et al., 2000). About 75 80% of the herbal medicine in the world population, mainly in the developing countries, for primary health care because of better cultural acceptability, better compatibility with the human body and lesser side effects. The World Health Organization (WHO) has defined traditional medicine (including herbal drugs) as comprising therapeutic practices that have been in existence, often for hundreds of years, before the development and spread of modern medicine and are still in use today (WHO, 1991). The global trade in herbals has an estimated value of US$12 billion, with trade in crude medicinal plant exceeding US$800 million and trade in herbal extracts and semi finished raw material exceeding US$8 billion (Brower, 1998). A comprehensive instructions has focused on the herbal medicines (WHO, 2007) which includes definition (botanical source, plant part used and its state), character (qualitative statement about the organoleptic character), identification (Macroscopical characters, microscopical characters, chromatographic procedures, chemical reactions), impurities (Heavy metal 49

2 Picture showing the plant parts of Avicennia marina (A) Avicennia marina Taxonomic position Kingdom : Plantae Division : Magnoliophyta Class : Magnoliopsida Order : Lamiales Family : Avicenniaceae Genus : Avicennia Species : marina Bark Stem Fruit Whole tree with roots 63

Magnoliopsida Order : Malpighiales Family :

cylindrica Bark Hypocotyl (Single arrow),

3 Picture showing the plant parts of Bruguiera cylindrica (B) Bruguiera cylindrica Taxonomic position Kingdom : Plantae Division : Magnoliophyta Class : Magnoliopsida Order : Malpighiales Family : Rhizophoraceae Genus : Bruguiera Species : cylindrica Bark Hypocotyl (Single arrow), collar (Double arrow) and leaf (Triple arrow) Flower Whole tree 64

4 Picture showing the plant parts of Ceriops decandra (C) Ceriops decandra Taxonomic position Kingdom : Plantae Division : Magnoliophyta Class : Magnoliopsida Order : Malpighiales Family : Rhizophoraceae Genus : Ceriops Species : decandra Flower Bark Collar (Double arrow) Hypocotyl (Single arrow) 65

5 Whole tree 66

6 Picture showing the plant parts of Rhizophora apiculata (D) Rhizophora apiculata Taxonomic position Kingdom : Plantae Division : Magnoliophyta Class : Magnoliopsida Order : Malpighiales Family : Rhizophoraceae Genus : Rhizophora Species : apiculata Flower Collar (Single arrow) and hypocotyl (Double arrow) Leaf Whole plant 67

7 Picture showing the plant parts of Rhizophora mucronata (E) Rhizophora mucronata Taxonomic position Kingdom : Plantae Division : Magnoliophyta Class : Magnoliopsida Order : Malpighiales Family : Rhizophoraceae Genus : Rhizophora Species : mucronata Collar (Single arrow) Hypocotyl (Double arrow) Whole tree Flower Stilt root Bark 68

8 Picture showing the plant parts of Lumintzera racemosa (F) Lumintzera racemosa Taxonomic position Kingdom : Plantae Division : Magnoliophyta Class : Magnoliopsida Order : Myrtales Family : Combretaceae Genus : Lumintzera Species : racemosa Flower Leaf Fruit Whole tree 69

9 contamination) and microbial counts are involved in the development of the herbal preparation. Herbal substances are a diverse range of botanical materials including leaves, herbs, roots, flowers, seeds, bark etc. The industrial processing of medicinal and aromatic plants starts with the extraction of the active components using various extraction procedures viz., maceration, infusion, percolation, digestion, decoction, hot continuous extraction (Soxhlet) and aqueous alcohol extraction by fermentation, microwave assisted extraction, water distillation, steam distillation and molecular distillation techniques etc. The type, concentration and quantity of extraction solvent may affect the spectrum of components obtained from a given amount of herbal material. According to WHO guidelines (WHO, 1991) organic solvents that can be used for herbal preparation/ products and manufacturing process can be classified in to three categories viz., class 1 (Solvents to be avoided such as benzene), class 2 (Limited toxic potential such as methanol or hexane) and class 3 (Low toxic potential such as ethanol). Keeping this in mind the present study was aimed to standardize the collection, identification and extraction of bioactive and phytochemical constituent s protocols for mangrove plant parts. 50

10 4.2. Materials and methods Description of study area The present study collected plant samples from 2 different geographical locations viz., Karangadu and Pichavaram along South Indian coasts. The study area, Karangadu (Lat N; Long E) is a notable dry place in Tamil Nadu located at Ramnad district (Fig. 10) which is interestingly blessed with important marine habitats and rich living resources. The total area is 400 hectare in Km situated along the banks of estuary in Kottakarai. The mangrove habitat receives seawater up to a distance of 5 km towards the riverside during high tide. The mangrove habitat situated in a semi-arid zone with low rainfall and high rate of evapotranspiration is dominated with a single strand of Avicennia marina species, which is known for its extreme environment tolerance. The study area, Pichavaram mangrove forest (Lat N; Long E) is located between the Vellar and Coleroon estuaries (Fig. 11). The forest is separated by intricate waterways that connect the Vellar and Coleroon estuaries. The Southern part near the Coleroon estuary is predominantly of mangrove vegetation, while the Northern part near the Vellar estuary is dominated by mud flats. The Vellar estuary opens into the Bay of Bengal at Parangipettai and links with the Coleroon river, which is distributary to the river Cauvery. The Pichavaram mangrove is influenced by 51

11 mixing of three types of waters: 1. Neritic water from the adjacent Bay of Bengal through a mouth called Chinnavaikkal, 2. Brackish water from Vellar and Coleroon estuaries and 3. Fresh water from an irrigation channel ( Khan Sahib Canal ), as well from the main channel of the Coleroon river. The mangrove covers an area about 1100 ha, of which 50% is covered by forest, 40% by water ways and the remaining filled by sand flats and mud flats. Pichavaram is a big and old mangrove in Tamilnadu with diversity of 110 numbers of plant species Collection and identification of samples Various parts of mangrove samples viz., leaf, bark, collar, hypocotyls, flower, stem and stilt roots were collected. The taxonomic identities of these plants were identified by Prof. Dr. K. Kathiresan, Centre for Advance study in Annamalai University, Parangipettai, Tamilnadu, India. The vernacular name, plant species and parts used for the present study was mentioned in Table 1. 52

12 Fig. 10. Map showing the study area of Pichavaram mangrove forest Fig. 11. Map showing the study area of Karangadu mangrove forest 53

13 Table 1. Name of the mangrove plants chosen for hepatoprotective activity Vernacular name AUOCAS0071 AUOCAS0072 AUOCAS0073 AUOCAS0074 AUOCAS0075 AUOCAS0076 Plant species Parts used Collection sites Bruguiera cylindrica Ceriops decandra Lumintzera recemosa Rhizophora apiculata Avicennia marina Rhizophora mucronata Leaf and hypocotyl Leaf, collar and hypocotyl Leaf and stem Bark, collar, hypocotyl and flower Leaf, bark and flower Bark, collar, hypocotyl and stilt root Pichavaram (Lat N; Long E) Karangadu (Lat N; Long E) These plants and their plant parts are scientifically and traditionally proved to have therapeutic properties are listed here under. Name of the plant species Avicennia marina Plant parts Traditional proof Scientific proof Bark, flower, fruits, leaves, root, seed and whole plant Treatment of rheumatism, small pox, ulcers, fodder for livestock (Bandaranayake, 2002) Analgesic, antivirus (Bandaranayake, 2002), antibacterial (Abeysinghe et al., 2006), antimicrobial (Nishiyama et al., 1978) Bruguiera cylindrica Bark, fruits, leaves and whole tree Treatment for hepatitis (Bandaranayake, 2002) Antiviral and larvicidal, biotoxicity on tobacco mosaic virus and fingerlings of fish (Bandaranayake, 2002), free radical scavenging (Agoramoorthy et al., 2008) 54

14 Ceriops decandra Bark, flower, fruit, leaves, root, whole plant Cure for hepatitis and ulcers (Bandaranayake, 2002) Antiviral (Bandaranayake, 2002) and antibacterial (Chitra, 2001) Rhizophora apiculata Bark, flower, fruit, leaves, root and whole plant Astringent for diarrhoea, treatment of nausea, vomiting, typhoid, hepatitis, insecticide and antiseptic (Bandaranayake, 2002) Antiviral, larvicidal, antifungal, antifeedant, antimicrobial activity, antiviral properties against human immunodeficiency (Bandaranayake, 2002), free radical scavenging (Vijayavel et al., 2006), antibacterial and antiyeast (Lim et al., 2006) Rhizophora mucronata Bark, fruit, leaves, stem, flower, whole plant and roots Treatment of elephantiasis, haematoma, hepatitis, ulcer and febrigue Antiviral, anti HIV activity, bio toxicity on fingerlings of fish (Bandaranayake, 2002), growth hormone tests on plants (Ganguly and Sircar, 1974), antimicrobial and antioxidant (Suganthy et al., 2009) Lumintzera racemosa Bark, fruit, flower, leaves, seed, root and whole plant Antifertility, treatment of asthma, snake bite Antiviral activity (Premanathan et al., 1992) and antihypertensive (Lin et al., 1993) 55

15 Preparation of extracts Collected fresh plant parts of mangrove plants were washed thrice in sterile distilled water to remove adhering soil particles and salts. About 500 g of each sample was subjected for size reduction to coarse powder. The powder was defatted with petroleum ether (50-60 C) and then extracted with 1 L of 70% of ethanol: water mixture by percolation method. The ethanolic extract was concentrated by using rotary flash evaporator (BUCHI, JAPAN) to get the fine residues and further lyophilised (BENCHTOP 2K) to remove the excess organic residues. The residual extract was further used for the screening of hepatoprotective activity. The percentage of the extract was calculated by the following formula. Weight of the extract (g) Percentage of the extraction (%) = X 100 Weight of the plant material Phytochemical analysis Test (s) Observation Inference References 0.5 g of each mangrove extract was stirred with 5 ml of 1% aqueous hydrochloric acid on a steam bath. A few drops of Dragendorff s reagent were used to treat 1 ml of the filterate. Formation of turbidity or precipitation Presence of alkaloiids Siddiqui and Ali,

16 0.5 g of the extract was dissolved in distilled water and about 10 ml of bromine water added Decolourizatio n of bromine water Presence of tannins Iyengar, g of extract was treated with 1.5 ml of 50% methanol solution. The solution was warmed and metal magnesium was added. To this solution, 5-6 drops of concentrated hydrochloric acid was added Formation of red colour Presence of flavonoids Siddiqui and Ali, g of mangrove extract was shaken with benzene layer separated and half of its own volume of 10% ammonia solution added. Formation of pink or red coloration in ammoniacal phase Presence of anthroquinone Brinda et al., g of mangrove extract was mixed with 0.5 ml of acetic anhydride and 0.5 ml of chloroform. Then concentrated solution of sulphuric acid was added slowly Formation of red violet colour Presence of terpenoids Siddiqui and Ali, g of mangrove extract was mixed with 0.5 ml of acetic anhydride and 0.5 ml of chloroform. Then concentrated solution of sulphuric acid was added slowly Formation of green bluish colour Presence of steroids Siddiqui and Ali, g of ethanolic extract was mixed with distilled water and adds few drops of ferric chloride. Formation of violet colour presence of phenolic group. Brinda et al.,

17 0.5 ml of alcoholic extract was mixed with concentrated HCl. Formation of pink colour presence of catachin Brinda et al., ml of ethanolic extract was mixed with Fehlings I and II solutions and boiling for half an hour in water bath. Formation of red precipitation Presence of reducing sugars Brinda et al., ml of ethanolic extract was mixed with equal volume of 5% sodium hydroxide and copper sulphate. Formation of violet colour Presence of protein Brinda et al., Results The percentage extraction of mangrove plant extracts is summarized in Table 2. Of the 18 mangrove extracts, the maximum percentage of extract was found in leaf extracts of Ceriops decandra (17.71%) followed by Bruguiera cylindrica (15.84%), bark extract of R. mucronata (12.85%), flower extract of R. apiculata (12.51%), leaf extract of L. racemosa (12.34%), stilt root of R. mucronata (11.90%), leaf extract of A. marina (11.79%), collar extract of R. apiculata (11.77%), collar extract of R. mucronata (9.54%), flower extract of A. marina (9.34%), stem extract of L. racemosa (9.15%), hypocotyl extract of R. mucronata (8.59%) and minimum (5.22%) extraction was found in hypocotyl extract of R. apiculata. 58

18 Among the plant parts, the maximum percentage extraction was found in leaf (19.12%) followed by stilt root (16.2%), flower (14.49), bark (14.15%), collar (12.72%) and (11.57%) hypocotyls showed minimum percentage of extraction (Fig 12). It is interesting to notice that, among the mangroves plants species the maximum (18.99%) percentage extraction was found in the species of R. mucronata followed by B. cylindrica (17.87%), A. marina (16.48%), L. racemosa (16.36%) and R. apiculata (14.21%) showed minimum percentage of extraction (Fig 13). As a part of the chemical standardization of mangrove extracts preliminary phytochemical analysis of the plant extract were carried out by the present study. It reveals that, the extracts from mangrove plants have variety of phytochemical constituent s viz., total sugars, protein, phenolic group, tannin, terpenoids, flavonoids, catachin, anthroquinone, alkaloid and steroids. The results reveal that, the reducing sugars were present in all the mangrove extracts and the presence of proteins were not identified in the bark extract of A. marina. Moreover, the total phenolic groups were identified in all the mangrove extracts except A. marina leaf extract. However, the steroids were not present in any parts of the plant extracts. The other phytochemical constituents such as tannin, terpenoids, flavonoids, catachin, anthroquinone and alkaloids are present discriminately reported among the mangrove plant extracts (Table 3). 59

19 Table 2. Extractive values of mangrove plant parts Plant Parts Name of the plant species Percentage of extracts (%) Avicennia marina Leaf Bruguiera cylindrica Ceriops decandra Lumintzera recemosa Avicennia marina Bark Rhizophora apiculata 7.85 Rhizophora mucronata Ceriops decandra 7.48 Collar Rhizophora apiculata Rhizophora mucronata 9.54 Bruguiera cylindrica 7.63 Hypocotyl Ceriops decandra 6.48 Rhizophora apiculata 5.22 Rhizophora mucronata 8.59 Flower Avicennia marina 9.34 Rhizophora apiculata Stem Lumintzera recemosa 9.15 Stilt Root Rhizophora mucronata

20 Table 3. Phytochemical constituents of mangrove plant extracts Mangrove plants A. marina B. cylindrica C. decandra L. racemosa R. apiculata R. mucronata Phytochemical constituents Leaf Flower Bark Leaf Hypocotyls Hypocotyls Leaf Collar Stem Leaf Collar Hypocotyls Bark Flower Collar Bark Stilt root Hypocotyls Reducing sugar Protein Phenolic group Alkaloid Steroid Triterpenes Flavonoids Catachin Tannin Anthroquinone : Presence; -: Absence 61

21 Fig. 12. Percentage of extraction between mangrove plant part extracts Fig. 13. Percentage of extraction between mangrove plant species 62

CHAPTER 5 PHYTOCHEMICAL EVALUATION

76 CHAPTER 5 PHYTOCHEMICAL EVALUATION Phytochemistry is mainly concerned with enormous varieties of secondary plant metabolites which are biosynthesized by plants. The plant kingdom represents a treasure