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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
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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
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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), Flower collar (Double arrow) and leaf (Triple arrow)
Whole tree
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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)
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Whole tree
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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 Leaf hypocotyl (Double arrow)
Whole plant
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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
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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
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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.
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4.2. Materials and methods
4.2.1. 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. 9° 36’ N; Long. 78° 83’ 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 evapo-
transpiration 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. 11° 20’ N; Long. 79°
47’ 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
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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.
4.2.2. 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.
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Fig. 10. Map showing the study area of Pichavaram mangrove forest
Fig. 11. Map showing the study area of Karangadu mangrove forest
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Table 1. Name of the mangrove plants chosen for hepatoprotective activity
Vernacular name Plant species Parts used Collection sites
AUOCAS0071 Bruguiera cylindrica Leaf and hypocotyl
Pichavaram (Lat. 11° 20’ N; Long. 79° 47’ E)
AUOCAS0072 Ceriops
decandra Leaf, collar and
hypocotyl
AUOCAS0073 Lumintzera
recemosa Leaf and stem
AUOCAS0074 Rhizophora apiculata
Bark, collar, hypocotyl and flower
AUOCAS0075 Avicennia
marina Leaf, bark and flower Karangadu (Lat. 9° 36’ N; Long.
78° 83’ E) AUOCAS0076 Rhizophora
mucronata Bark, collar, hypocotyl
and stilt root These plants and their plant parts are scientifically and traditionally
proved to have therapeutic properties are listed here under.
Name of the plant species Plant parts Traditional proof Scientific proof
Avicennia marina
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)
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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)
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4.2.3. 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
4.2.4. 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,
1997
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0.5 g of the extract was dissolved in distilled water and about 10 ml of bromine water added
Decolourization of bromine
water
Presence of tannins
Iyengar, 1995
0.5 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,
1997
0.5 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 anthro-quinone
Brinda et al., 1981
0.5 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,
1997
0.5 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,
1997
0.5 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., 1981
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0.5 ml of alcoholic extract was mixed with concentrated HCl.
Formation of pink colour
presence of
catachin
Brinda et al., 1981
0.5 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., 1981
0.5 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., 1981
4.3. 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.
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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).
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Table 2. Extractive values of mangrove plant parts
Plant Parts Name of the plant species Percentage of extracts (%)
Leaf
Avicennia marina 11.79
Bruguiera cylindrica 15.84
Ceriops decandra 17.71
Lumintzera recemosa 12.34
Bark
Avicennia marina 11.32
Rhizophora apiculata 7.85
Rhizophora mucronata 12.85
Collar
Ceriops decandra 7.48
Rhizophora apiculata 11.77
Rhizophora mucronata 9.54
Hypocotyl
Bruguiera cylindrica 7.63
Ceriops decandra 6.48
Rhizophora apiculata 5.22
Rhizophora mucronata 8.59
Flower Avicennia marina 9.34
Rhizophora apiculata 12.51
Stem Lumintzera recemosa 9.15
Stilt Root Rhizophora mucronata 11.90
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Table 3. Phytochemical constituents of mangrove plant extracts
+: Presence; -: Absence
Phytochemical constituents
Mangrove plants
A. marina B. cylindrica C. decandra L. racemosa R. apiculata R. mucronata
Leaf
Flow
er
Bark
Leaf
Hyp
ocot
yls
Hyp
ocot
yls
Leaf
Col
lar
Stem
Leaf
Col
lar
Hyp
ocot
yls
Bark
Flow
er
Col
lar
Bark
Stilt
root
Hyp
ocot
yls
Reducing sugar + + + + + + + + + + + + - + + + + +
Protein + + - + + + + + + + + + + + + + + +
Phenolic group + + + + + + + + + + + + + + + + + +
Alkaloid - + - - - + + + + + + + - + + + + +
Steroid - - - - - - - - - - - - - - - - - -
Triterpenes - - - - - + + + + + + + - - + + + +
Flavonoids - - - - - + + + + - + + - + + + + +
Catachin - - - - + + + + + - + + - + + + + +
Tannin + + + + - + + + + + + + + + + + + +
Anthroquinone - - - - - + + + - - + - - + + + + -
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Fig. 12. Percentage of extraction between mangrove plant part extracts
Fig. 13. Percentage of extraction between mangrove plant species
