Biocatalysis and Agricultural Biotechnology 23 (2020) 101491

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Biocatalysis and Agricultural Biotechnology

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Antibacterial and invitro antioxidant potential of Indian mangroves

A. Arulkumar a, b, *, K. Sampath Kumar c, S. Paramasivam a
a
Department of Oceanography and Coastal Area Studies, School of Marine Sciences, Alagappa University, Thondi Campus, 623 409, Tamil Nadu, India
b
Achariya Arts and Science College, Pondicherry University, Villianur Puducherry, 605 110, India
c
Sengunthar Arts and Science College, Thiruchengode, 637 205, Tamil Nadu, India

A R T I C L E I N F O A B S T R A C T

Keywords: Herbal tea is medicinally well-known for its biochemical composition and medicinal usages. The present study
Tea proved for the first time that tea would be extracted from mangroves, the plants other than commercial tea.
Rhizophora mucronata Mangroves tea exhibitd good quality in the terms of theaflavin (TF), thearubigins (TR), total liquor colour (TLC)
Phytochemical
and highly polymerized substantce (HPS). Phytochemical screening of tea revealed the presence of phyto­
Antioxidant and R. annamalayana
chemical compounds such as protein, phenol and flavonoids etc. The antioxidant properties of Rhizophora
mucronata, Rhizophora apiculata, and Rhizophora annamalayana showed dose dependent activities and were
comparatively much higher in R. mucronata. R. mucronata (86.50 � 0.35%) showed much higher total antiox­
idant activity followed by R. apiculata (73.34 � 0.90%) and R. annamalayana (69.35 � 0.56%) at 100 μg/mL
concentration. The antimicrobial activity was higher in R. mucronata and R. apiculata than R. annamalayana as
evident by the presences of phenolic N–H and OH components found in mangroves tea. Therefore mangroves are
rich source of tea, biochemical constituent, phytochemical, antioxidant and antibacterial properties. The extract
contained polyphenolic and other bioactive compounds can be used for herbal drug formulation and
nutraceutical.

1. Introduction microbes. Fascinatingly, researchers have isolated a variety of other

India has a rich and prestigious heritage of mangrove forest oriented
medicines among the South Asian countries. However, the majority of
these plants have not yet undergone chemical, pharmacological and
toxicological studies to investigate their bioactive compounds (Kathir­
esan, 2000; Vadlapudi and Chandrasekhr naidu, 2009; Singh, 2009).
Traditional records and ecological diversity indicate that Indian plants
represent an exciting resource for possible lead structures in drug design.
Plants are endowed with free radical scavenging molecules, such as vi­
tamins, terpenoids, phenolic acids, lignins, stilbenes, tannins, flavo­
noids, quinones, coumarins, alkaloids, amines, betalains, and other
metabolites, which are rich in antioxidant activity (Zheng and Wang,
2001). The mangrove plants naturally synthesis many bioactive sec­
ondary metabolites. Such plant-derived phytochemicals are highly un­
explored for therapeutics. Mangroves are biochemically unique,
producing a wide array of novel natural products. They are rich in
polyphenols and tannins (Kathiresan and Ravi, 1990). In recent years,
there is growing interest in the discovery of noval compounds from
mangroves resources to control the emerging human pathogenic
mangrove compounds including taraxerol careaborin and taraxeryl
cis-p-hydroxycinnamate from leaves of Rhizophora apiculata (Kokpal
et al., 1990); 2-nitro-4-(20 -nitroethenyl phenol) from leaves of Sonneratia
acida (Bose et al., 1992); alkanes (46.7–97.9% wax) and triterpenoids
(53.3% wax) from leaves of Rhizophora species (Dodd et al., 1995); and
iridoid glycosides from leaves of Avicennia officinalis and A. germinans
(Fauvel et al., 1995; Sharma and Garg, 1996). Phytoconstituent of
R. mucronata was identified as Ethanone, 1-(2-hydroxy-5-methylphenyl
as role in the antibiotic activity against human and fish pathogen
(Manilal et al., 2015).
In recent past years, mangrove plants have gained much importance
due to its usage in fisher-folk medicine to treat various diseases (Ban­
daranayake, 2002) and it has been studied for their medicinal proper­
ties. Coastal plant extracts have been tested against viruses that cause
human and animal diseases, such as human immunodeficiency virus
(HIV), new castle disease virus, vaccinia virus, semliki forest virus,
encephalomyocarditis virus, and hepatitis-B-virus (Kathiresan, 2000). A
few of the mangrove plants belonging to family- Rhizophoraceae are most
effective against the viruses (Premanathan et al., 1992). Bioactive

* Corresponding author. Department of Oceanography and Coastal Area Studies, School of Marine Sciences, Alagappa University, Thondi Campus, 623 409, Tamil
Nadu, India.
E-mail address: aruul3@gmail.com (A. Arulkumar).

https://doi.org/10.1016/j.bcab.2019.101491
Received 4 May 2019; Received in revised form 17 December 2019; Accepted 30 December 2019
Available online 31 December 2019
1878-8181/© 2020 Elsevier Ltd. All rights reserved.
A. Arulkumar et al.
compounds present in the mangrove plants and polysaccharides
composed of galactose, galactosamine, glucose and arabinose reported
to have potent anti-HIV activity (Premanathan et al., 1999). Scientific
studies on a number of medicinal plants indicated that promising
phytochemical compounds can be developed for many health problems
(Gupta, 1994).
These phytochemicals are indeed chemically complex and many
structurally related active compounds often produce a synergistic effect.
Medicinal plants are perhaps the most valuable sources of new bioactive
chemical entities to benefit mankind against various ailments. Almost
122 pure chemical substances extracted from higher plants are used in
medicine throughout the world (Fabricant and Farnsworth, 2001).
Therefore, it is very important to determine the phytochemical, bioac­
tive compound and biochemical constituent in Rhizophora species for
noval drug discovery, antibiotics and noval nutraceutical compounds.
However, the present comparative study carried out for the first time to
evaluate the phytochemical, antioxidant and antibacterial activity of
mangroves species viz. Rhizophora mucronata, R. apiculata and
R. annamalayana. The bioactive efficacy of tea, extracted from three
mangrove Rhizophora species and identification of the functional groups
present in the tea extracts by using Fourier transform-infrared spec­
troscopy (FT-IR) analysis.
2. Materials and Methods
2.1. Collection of mangrove leaves
The fresh mangrove leaves of Rhizophora mucronata, R. apiculata and
R.annamalayana were collected from the artificially developed. Vellar
estuaring mangroves at Parangipettai, Tamilnadu, India. The River
Vellar is flowing on the Southeast Coast of India, mixing with the sea at
Porto Novo (also known as Parangipettai; Lat. 11� 290 N; Long. 79� 47’
E). The estuary is connected to the Pichavaram mangroves through the
backwater channel. The mouth of the Vellar estuary opens to the sea
during moon season and a sand bar sometimes closes it completely
during summer season.
2.2. Preparation and extraction of tea
Black tea was extracted from the mangrove leaves, adopting the
method of Kathiresan (1995). Fresh leaves were allowed to dry using a
warming blender. A known weight of the macerated sample was placed
in a piece of cloth and distilled water was continuously trickled over it
for 1 hrs. The fermented sample was dried in hot air oven at 95 � C for 30
min. Weight 1.25% of mangrove black tea was added to the boiling
water and allowed to infuse for 5 min, after which the infusions were
filtered through sterile membrane filter. The filtrates were lyophilized
and used for further study.
2.3. Tea quality constituents and caffine content of tea
Theaflavin (TF), thearubigins (TR), total liquor colour (TLC), highly
polymerized substantce (HPS) and caffeine content were analyzed, for
determination of quality of tea ( Ronald and Ronald, 1991).
2.4. Qualitative analysis of phytochemical screening
The qualitative chemical constituents present in the tea extracts of
mangroves plants were determined by Trease and Evans (1989).
Proteins-xanthoprotein: to 1 mL of extract, 3–5 drops of nitric acid
was added by the sides of the test tube and observed for formation of
yellow colour indicated presence of proteins.
Steroids: two mL of acetic anhydride was added to 0.5 g of extract
and 2 mL of sulphuric acid was added by the sides of the test tube and
observed the colour change from violet or blue-green.
Tannins: about 0.5 g of the each extract was taken in a boiling tube
2
Biocatalysis and Agricultural Biotechnology 23 (2020) 101491
and boiled with 20 mL distilled water filtered and then added few drops
of 0.1% ferric chloride mixed well and allowed to stand for some time.
Brownish green or a blue-black coloration indicated presence of tannins.
Glycosides─Keller-Killani method: about 0.5 mL of alcoholic ex­
tracts was taken and subjected to the following test. One ml of glacial
acetic acid containing traces of ferric chloride and 1 mL of conc. sul­
phuric acid were added to extract. The formation of reddish brown
colour at the junction of two layers and the upper layer turned bluish
green indicate the presence of glycosides.
Reducing sugars─Fehling’s test: few drops of Fehling’s solution A
and B in equal volume were added in diluted extracts and heated for 30
min and observed the formation of brick red coloured precipitate.
Sterols-Liebermana-Buchard method: the insoluble residue was
dissolved in chloroform and few drops of anhydride were added along
with few drops of concentrated H2SO4 from the side of the test tube and
the formation of blue to blood red colour was observed.
Terpenoids ─ Salkowski test: to 0.5 g of the extract, 2 mL of
chloroform was added; concentrated H2SO4 (3 mL) was carefully added
to form a layer. A reddish brown coloration in the interface indicates the
presence of terpenoids.
Cardiac glycosides (Keller ─ Killiani’s test): 100 mg of extract was
dissolved in 1 mL of glacial acetic acid containing one drop of ferric
chloride solution. This was then underlayered with 1 mL of concentrated
H2SO4. A brown ring obtained at the interface indicated the presence of
a deoxy sugar characteristics of cardenolides.
Carbohydrates─Molisch’s test: small quantities of alcoholic and
aqueous extracts were dissolved in 5 mL of distilled water and filtered.
To this solution 2–3 drops of α-naphthol was added and 1 mL of
concentrated H2SO4 was added along the sides of inclined test tube so as
to form two layers and observed for formation of violet coloured ring at
the interface to detect the presence of carbohydrates.
Flavonoids: a portion of the aqueous extract (2 mL) was heated, and
metallic magnesium and concentrated hydrochloric acid (5 drops) were
added. A red or orange coloration indicated the presence of flavonoids.
Phenols: the extracts were taken in water and warmed. To this 2 mL
of ferric chloride solution was added and the formation of green or blue
colour indicated the presence of phenols.
2.5. Chemicals and reagents
Butylaled hydroxytoluene (BHT), DPPH (1, 1-diphenyl, 2-picrylhy­
drazyl), of Sigma Chemicals, (USA) and all others high purity chemical
reagents of Merck (Germany) were used.
2.6. Estimation of antioxidants assay
2.6.1. Estimation of total antioxidants
Total antioxidant activity was measured by the method of Mitsuda
et al. (1996). Briefly 7.45 mL sulphuric acid (0.6 M solution), 0.9942 g
sodium phosphate (28 mM solution) and 1.2359 g ammonium molyb­
date (4 mM) were mixed together in 250 mL with distilled water and
labeled as total antioxidant capacity (TAC). Extract (100 μL) was dis­
solved in 1 mL of TAC and the absorbance was read at 695 nm after 15
min. Butyled hydroxytoluene (BHT) was used as positive control.
2.6.1.1. Calculation of 50% inhibitory concentration (IC50). The con­
centration (μg/mL) of the fractions that was required to scavenge 50% of
the radicals was calculated by using the percentage scavenging activities
at three different concentrations of the fractions. Percentage inhibition
(%) was calculated using the following formula,
% Inhibition ¼ [(Ac–As) /Ac] X 100
Where, Ac ¼ absorbance of the control As ¼ absorbance of the sample.
A. Arulkumar et al.
2.6.2. Determination of total phenol content
Total phenolic content of tea were estimated by Folin-Ciocalteu’s
method (Singleton and Rossi, 1965). Black tea extracts (10 mg) were
dissolved in 10 mL of distilled water. An aliquot of 100 μL of appropriate
dilution of the samples were shaken for 1min with 500 μL of the
Folin-Ciocalteu reagent freshly prepared in the laboratory and 6 mL of
distilled water. After the mixture was shaken 2 mL of 15% (mass per
volume) sodium carbonate was added and the mixture was shaken again
for 0.5 min. Finally, the solution was brought up to 10 mL with distilled
water. After 2 h of reaction at ambient temperature, the absorbance was
measured at 750 nm. Using gallic acid as standard, the total phenolic
content of mangroves was expressed as gallic acid equivalents (GAE).
2.6.3. DPPH radical scavenging assay
The free radical scavenging activity of tea extracts were measured by
DPPH according to Blois (1958) method. One mL of DPPH (0.1 mM)
solution in methanol was added to 3 mL of water extract of tea (100
μg/mL), shaken vigorously, then allowed to stand at room temperature
for 30 min and the absorbance was measured at 517 nm in a UV–visible
spectrophotometer (U-2800 model, Hitachi, Japan). A low absorbance
of the reaction mixture indicated the high free radical scavenging ac­
tivity. Butylated hydroxytoluene (BHT) was used as positive control. The
DPPH scavenging effect was calculated.
DPPH radical scavenging activity ¼ {(Abs control – Abs sample) / (Abs
Control)} � 100
Where; Abs control ¼ absorbance of DPPH radical þ BHT; Abs sample ¼
absorbance of DPPH radical þ sample extract or Standard.
2.6.4. Total reducing power
Total reducing capacity of tea was determined according to the
method of (Oyaizu, 1986). The tea extracts (100 μg/mL) in distilled
water and 1% potassium ferricyanide were mixed with phosphate buffer
(0.2 M, pH 6.6) and the mixture was incubated at 50 � C for 20 min. Then
2.5 mL of 10% TCA was added to the reaction mixture which was
centrifuged at 1000�g for 5 min. The upper layer of the solution (2.5
mL) was mixed with distilled water (2.5 mL), FeCl2 (0.5 mL, 0.1%) and
the absorbance was measured at 700 nm. Ascorbic acid was used as a
positive control. The higher absorbance of the reaction mixture indi­
cated the greater reducing power.
2.6.5. Nitric oxide radical (NO*) scavenging activity
Nitric oxide radical scavenging activity was determined by the
method of (Garrat, 1964). Sodium nitroprusside in aqueous solution at
physiological pH spontaneously generates nitric oxide, which interacts
with oxygen to produce nitrite ions, which can be determined by the
Griess-Illosvoy reaction. Briefly, 3 mL of the reaction mixture containing
10 mM sodium nitroprusside and the tea extract (100 μg/mL) in phos­
phate buffer were incubated at 25 � C for 150 min. After incubation, 0.5
mL of the reaction mixture was mixed with 1 mL of sulfanilic acid re­
agent (0.33% in 20% glacial acetic acid) and allowed to stand for 5 min
for complete diazotization. Then 1 mL of naphthyl ethylene diamine
dihydrochloride (0.1%) was added and after mixing well, the solution
was allowed to stand for 30 min at 25 � C. A pink coloured chromophore
was formed in diffused light. The absorbance of these solutions was
measured at 540 nm against the corresponding blank solutions. BHT was
used as positive control. Nitric oxide scavenging activity of the tea
extract is reported as % inhibition and was calculated using following
formula
NO radical scavenging activity ¼ {(Abs control – Abs sample) / (Abs control)}
� 100
Where; Abs control ¼ absorbance of NO radical þ BHT; Abs.
Sample ¼ absorbance of NO radical þ sample extract or standard.
3
Biocatalysis and Agricultural Biotechnology 23 (2020) 101491
2.7. FT-IR analysis of tea
For this 2 mg of the tea extracts samples (R. mucronata, R. apiculata
and R. annamalayana) were mixed with 200 mg KBr (FT-IR grade) and
made into thick pellets in a hydraulic press by allying 500 kg/m3
pressure. The pellet was immediately placed into the sample holder and
FT-IR spectra recorded between ranges of 7800–350 cm 1 (Mid-IR) for
the compound under study. FT-IR spectra of the purified compound
were recorded using FT-IR spectrometer, Shimadzu ( Model No:
A21374801626) Japan FT-IR.
2.8. Bacterial strains and culture conditions
Bacterial cultures namely Staphylococcus aureus, Escherichia coli,
Salmonella typhi, and Proteus vulgaris were obtained from CAS in Marine
Biology, Annamalai University, Parangipettai, India. The above cultures
were stored at 20 � C in nutrient broth (Hi Media, Mumbai, India)
supplemented with 50% glycerol. Before experimental use each strain
was grown twice in Nutrient broth at 37 � C for 48 hrs.
2.8.1. Determination of in vitro antibacterial activity against human
pathogenic bacteria
The antibacterial activity of the tea extracts (R. mucronata, R. api­
culata and R. annamalayana) was performed by the well diffusion
method on Muller-Hinton agar (MHA, Hi media, India). About 100 μL of
105 CFU/mL diluted inoculum of bacterial culture was applied on the
surface of MHA plates and allowed to solidify. MHA well was made with
well borer under aseptic conditions and filled with mangroves tea
extract samples and DMSO as used as negative control and penicillin was
acted as positive control. The plates were incubated at 37 � C for bacterial
growth. The antibacterial activity of the mangrove samples was evalu­
ated by measuring the zone of inhibition against the test human path­
ogenic bacteria. All the experiments were done three times and the data
were expressed as the mean values of experiments.
2.9. Thin layer chromatography (TLC)
TLC method was (Abu et al., 2011) used to separate the bioactive
compounds present in the tea extract R. mucronata, R. apiculata and
R. annamalayana (Abu et al., 2011). Methanol and chloroform (1:9) was
used as mobile phase. The sample with the concentration of about (1
mg/mL) was spotted on the TLC plates and dried. The spots were
identified in long UV, short UV and also in the iodine chamber. Rƒ value
was calculated to find the active metabolites.
3. Statistical analysis
All determinations were given in terms of mean� standard de­
viations (SD). The results obtained were compared by a one-way anal­
ysis of variance (ANOVA). The significance of the difference between
means was determined by Duncan’s multiple range test (P < 0.05) using
SPPS version 14 (USA).
4. Results and discussion
4.1. Physical nature of tea
The present study evaluated that phytochemical constituent of tea
from the three mangroves species, R. mucronata, R. apiculata and
R. annamalayana. The black tea extract showed a golden brown colour
with pleasant aroma taste. These characters revealed the good quality of
the tea. It was observed that, the maximum yield of tea was obtained
from R. mucronata. The percentage yield of R. mucronata was 0.125%
followed by R. apiculata 0.121% and R. annamalayana 0.110% respec­
tively. The colour and the yield of the tea extraction might be attributed
to the levels of polyphenol content.
A. Arulkumar et al. Biocatalysis and Agricultural Biotechnology 23 (2020) 101491

Table 1 R. annamalayana were qualitatively analyzed and the results are pre­
Chemical constituent quality and caffeine content of tea extracts (R. mucronata, sented in Table 2. The tea extract of all the three species of mangroves
R. apiculata and R. annamalayana). R. mucronata, R. apiculata and R. annamalayana showed the presence of
Chemicals constituent R. mucronata R. apiculata R. annamalayana phytochemical active compounds such as xanthoprotein, steroids, tan­
Theaflavin 0.42 � 0.02a 0.18 � 0.02a 0.5 � 0.08a
nins, glycosides, reducing sugar, carbohydrates, sterols, terpenoids,
Thearubigin 4.60 � 0.24b 3.49 � 0.22 b 4.52 � 0.20 b phenol, cardioglycosides and flavonoids. Mangroves and mangrove as­
Highly polymerized 3.95 � 0.18 c 4.71 � 0.20 c 3.9 � 0.28 c sociates possess novel agrochemical products, compounds of medicinal
substance value and biologically active compounds (Bandaranayake, 2002). The
Total liquor colour 1.1 � 0.14 d 0.49 � 0.03 d 0.51 � 0.03 d
phytochemical screening of tea extracts of R. mucronata, R. apiculata and
Caffeine ND ND ND
R. annamalayana revealed the presence of tannins, flavonoids, steroids
Dates were expressed as mean � standard division; ND- Not Detectable. The and saponins. It is clearly indicates that, the presence of the functional
different letters a-c are statistically significant with P < 0.05. group –OH in the structure and positions of the flavonoid molecules to
determine the phytochemical capacity. Recently, Saranya et al., (2014)
find out the important active metabolities through the phytochemical
Table 2
Phyto-chemical characterization of tea extracts.
screening R. mucronata showed the presence of alkaloids, terpenoids,
steroids, tannins, quinines, sapnins, flavonoids, cardiac glycosides and
Phytochemical test R. mucronata R. apiculata R. annamalayana
phenol, which corroborates the results of the present study. Abdurah­
Proteins-xanthoprotein þ þ þ man et al. (2016) reported that, the Rhizophora species have various
Steroids þ þ þ phyto-chemical compounds includes alcohol, aldehydes, amino acids,
Tannins þ þ þ
Glycosides þ þ þ
aromatic carboxylic acid, carbohydrates, carboxylic acids, esters, fatty
Reducing sugar þ þ þ acids, flavonoid, ketone, lipids, phenol, saponins, steroids, tannin and
Sterols þ þ þ terpenoid for the biological activity.
Terpenoids þ þ þ These results support the present finding that mangrove plants are a
Cardioglycoside þ þ þ
rich source of steroids, triterpenes, saponins, flavonoids, alkaloid and
Carbohydrate þ þ þ
Flavanoids þ þ þ tannins (Agoramoorthy et al., 2008; Bandaranayake, 2002). In the
Phenol þ þ þ present study it is found that Rhizophora species contains secondary
metabolities of phenolic compounds (N–H and OH molecules) and other
þ; presence of phyto-chemical components.
substances. Hence, the beneficial medicinal effects of plant material may
result from the combinations of secondary products present in the plant.
4.2. Tea quality constituents of tea extracts
Secondary products play a role in a plant defense through cytotoxity
towards microbial pathogens (Briskin, 2000).
The chemical constituent and quality of tea determined theaflavin
Tannins are known to be useful in the folk medicine and treatment of
(TF), thearubigins (TR), total liquor colour (TLC), highly polymerized
inflamed or ulcerated tissue and their remarkable anticancer activity
substantce (HPS) and caffeine content statistically significance differ­
(Ruch et al., 1989). Thus, Rhizophora species containing bioactive
ence with P < 0.05 were presented in Table 1. The R. mucronata was
compounds and it may serve as a potential sources of bioactive com­
observed to possess higher TF 0.42 � 0.02% when compared to
pounds in the treatment of cancer. The presence of phenol compounds in
R. apiculata (0.18 � 0.02%) and R. annamalayana (0.5 � 0.08%) tea
the Rhizophora species may contribute to antioxidant and antibacterial
extracts. The higher thearubigins (TR) was observed in R. mucronata
properties, and it can be formulate herbal medicine and nutraceuticals.
(4.60 � 0.24%) followed by R. annamalayana and R. apiculata.
Earlier study reported that, the phenolic compounds may exhibit a widel
R. mucronata was observed to possess higher HPS 4.71 � 0.20% followed
range of physiological function, such as antibacterial, antifungal, anti­
by R. apiculata 3.9 � 0.29% and R. annamalayana 3.95 � 0.18%. The
oxidant, antiinflammatory, anti artherogenic, anti thrombotic effects
total liquire colour (TLC) was higher R. mucronata in (1.1 � 0.14%)
(Benavente et al., 1997; Samman and Cook, 1998; Puupponen-Pimia
followed by R. annamalayana (0.51 � 0.03%) and R. apiculata (0.49 �
et al., 2001; Manilal et al., 2015; Abdurahman et al., 2016).
0.03%). Coffien content from the three mangroves spieces was not
detected (ND). It was observed that, R. mucronata contains higher TF,
4.4. Antioxidant
TR, TLC and HPS content than R. apiculata and R. annamalayana. Ven­
katesan et al. (2005) have reported that, the polyphenol found in the tea
The antioxidant activities for the tea extracts of R. mucronata, R.
leaves is responsible for all the biochemical reaction to make a good
apiculata and R. annamalayana at different concentration are shown in
quality of tea. The chemical basis of the quality of mangrove black tea
(Fig. 1a–e). It was found that, of the three species of tea extracts
indicated theaflavin (TF), thearubigins (TR), total liquor colour (TLC),
screened, R. mucronata (100 μg/mL) showed significant levels of anti­
highly polymerized substantce (HPS) are the important factors to
oxidant properties, in terms of total antioxidants, total phenol, DPPH
determine the liquor quality of mangrove black tea. Liang et al. (2007)
assay, and total reducing power and nitric oxide radical scavenging
have reported that, the tea owing to its favorable benefits on human
activity. The total antioxidant activity of the tea extracts increased with
health drink currently enjoys a great popularity among other beverages
increase in concentration (P < 0.05). The result suggested that, the
throught out the worldwide. In this study, higher TF, TR, TLC and HPS
R. mucronata showed high total antioxidant activity of 85.50 � 0.35%
was observed in R. mucronata followed by other potential species
compared to R. apiculata 73.34 � 0.90% and R. annamalayana 69.35 �
(Table 1). Bagyalakshmi et al. (2012) have reported that, the quality of
0.56% at the concentration of 100 μg/mL respectively (Fig. 1a). It was
tea characteristics TF, TR, HPS, TLC and caffeine 0.8, 7.6, 7.2, 2.3 and
observed that, R. mucronata contains 12.16% and 16.15% time higher
2.1 respectively in Camellia sinensis. In the study first time proved that
total antioxidant activity than R. apiculata and R. annamalayana. Gao
HPS was higher in R. mucronata (4.71 � 0.20%) followed by R. apiculata
and Xiao (2012) reported that, the Rhizophora species contains lyonir­
(3.9 � 0.29%) and R. annamalayana (3.95 � 0.18%) and there is no
esinol-3α-o-β-arabinopyranoside, lyoniresinol-3α-O-β-rhamnoside,
caffeine content found all the Rhizophora species.
afzelechin-3-rahmnoside bioactive compounds for antioxidant and bet­
ter radical scavenging activity.
4.3. Qualitative analysis of phytochemical constituents The total phenol contents of the tea extracts are possibly propor­
tional to the concentration. The higher TPC was observed from
Phytochemical constituents of R. mucronata, R. apiculata and R. mucronata 84.80 � 1.47% followed by R. apiculata 79.23 � 0.70% and

4
A. Arulkumar et al. Biocatalysis and Agricultural Biotechnology 23 (2020) 101491

Fig. 1. Antioxidant assay of mangroves black tea (a) Total Antioxidant (b) Total Phenol (c) DPPH assay (d) Total Reducing Power (e) Nitric Oxide AssayAll
the values expressed mean and standard deviation. The statistically significance different letters (a–
e) with (P < 0.05).

5
A. Arulkumar et al. Biocatalysis and Agricultural Biotechnology 23 (2020) 101491

Table 3
Effect of tea extracts on the growth of Gram positive and Gram negative human pathogenic bacteria.
Bacteria Positive control Negative control Zone of inhibition in (mm)

Penicillin DMSO R. mucronata R. apiculata R. annamalayana

Escherichia coli 11 � 0.01 – 10 � 0.11 2.0 � 0.01 6.0 � 0.05

Salmonella typhi 13.1 � 0.02 – 8.0 � 0.07 7.0 � 0.06 2.0 � 0.03
Proteus vulgaris 9.0 � 0.04 – 6.0 � 0.05 5.0 � 0.03 10 � 0.09
Staphylococcus aureus 10.1 � 0.00 – 5.0 � 0.04 15 � 0.12 7.0 � 0.05

Mean values (n ¼ 3 with SD).- No zone of inhibition.

Table 4
Comparison of biological potential in mangrove species as reported in the literature.
Name of the mangroves Study area Biological properties Reference

R.apiculata Parangipettai, Tamilnadu,
India
R.mucronata Parangipettai, Tamilnadu,
India
R.mucronata, R.apiculata, Avicennia officinalis, Ceriops decandra Pichavaram and
Tamilnadu, India
Avicennia marina Gulf of Aqaba, Egypt
R.mucronata, R.apiculata, R.annamalayana, Avicennia marina Pichavaram and Thondi,
and Ceriops decandra Tamilnadu, India
Avicennia officinalis, A.marina, R.conjugata, R.mucronata, Andhra Pradesh, India
Salicornia brachiata, Salvodara persica, xylocarpus granatum
Kandelia candel China
R.mucronata Parangipettai, Tamilnadu,
India
R.mucronata Kerala, Kollam, India
Ceriops decandra Parangipettai, Tamilnadu,
India
C. decandra Parangipettai, Tamilnadu,
India
R.mucronata, R.apiculata, R.annamalayana Parangipettai, Tamilnadu,
India
Antiviral and anti larvicidal Premanathan et al. (1999)
Antiviral, anti- HIV Premanathan et al. (1999)
Polyphenol and antioxidant Agoramoorthy et al. (2008)
Antibacterial, anticandidal, anti bacteriophage, Khafagi et al. (2003)
cytotoxicity and plant growth grgulatar
Antioxidant activity and antibacterial properties Pandima Devi et al. (2009)
Antioxidant activity Vadlapudi and
Chandrasekhr Naidu, 2009
Antioxidant properties Wei et al. (2010)
Phytochemical and Bioactive compound Saranya et al. (2014)
Phytochemical and Bioactive compound Manilal et al. (2015)
Mangrove black tea buccal pouch carcinogenesis Sithranga Boopathy et al.
in hamsters (2011a), (2011b)
Mangrove black tea prevents the oral cancer Sithranga Boopathy et al.
incidences (2011a), (2011b)
Mangrove black tea rich in Phytochemical, Present study
antioxidant and antibacterial activity

R. annamalayana 74.35 � 0.90% at the concentration of 100 μg/mL
significance level (P < 0.05) (Fig. 1b). It was clearly noted that,
R. mucronata contained 5.59% and 10.45% times higher total phenol
content than R. apiculata and R. annamalayana. Pandima Devi et al.,
2009 have reported that, the TPC of R. mucronata was 720.7 mg GAE/g
for methanolic extracts which corrabotes the results of R. mucronata in
the present study. The higher content of phenol in mangroves tea ex­
tracts indicates a better free radical scavenging ability, which can pre­
vent lipid oxidation in food products. Screening of antioxidant
substances derived from natural resources is one of the basic tool for
drug design and development. Agoramoorthy et al., (2008) have re­
ported the higher TPC of R. mucronata and R. apiculata was 157.4 mg
GAE/g and 302 mg GAE/g for dry weight respectively, which was
comparatively much higher than the results of the present experiment
(Fig. 1b). Recently, Wei et al., (2010) reported that, the TPC of
mangrove plant (Kandelia candel) was 400.43 μg GAE/ml. But Haq et al.,
(2011) have reported total phenol content from Bruguiera gymnorrhiza
was 178.73 mg GAE/g of dry leaves. The present study indicates that
R. mucronata, R. apiculata, and R. annamalayana tea extracts were po­
tential free radical scavengers, which reacted with free radicals by
donating their hydrogen (H2) and act as primary antioxidants and pre­
vent oxidation.
Several free radical such as OH, O2, LOO. have different reactivities
are formed during lipid oxidation. Antioxidants are able to scavenge
these radicals by donating H ions. Relatively stable DPPH radical has
been widely used to test the ability of compounds to act as free radicals
scavengers or H irons donors and thus to evaluate the antioxidant ac­
tivity (Jao and Ko, 2002). In this study, evident that the ability of DPPH
radicals scavenging activity of tea extracts is shown in (Fig. 1c). The
activity of tea extract increased with increase in concentration (P <
0.05). The result suggested that, the IC50 value of R. mucronata showed
higher radical scavenging activity of 91.83 � 1.26% compared to 78.92
� 1.72% for R. apiculata and 90.87 � 1.56% for R. annamalayana at the
concentration 100 μg/mL respectively. Pandima Devi et al., 2009 have
reported that, the IC50 value of R. mucronata DPPH was 43.171 μg/ml,
which was comparatively much lower than the results of the present
study. Similarly, Agoramoorthy et al. (2008) have reported that, the
DPPH free radical scavengers in R. mucronata and R. apiculata were 79.9
and 64.7 μg/mL which was comparable to our results (93.39 and
90.31%). Wei et al. (2010) have reported that, the DPPH radical scav­
engers was 115.67 μg/mL in K. candel in 70% acetone extracts, which
was comparatively higher than the results of the present study. The high
radical scavenging activity of R. mucronata could be due to the presence
of flavonoids that can be perform radical scavenging activity on free
radical (DPPH, hydroxyl and hydrogen peroxide H2O2) for their reduc­
tion of hydroxide formation. In this study, the presence of the functional
group –OH in the structure and its position of flavonoid and or phenol
molecules determine the antioxidant activity (Fig. 1c). This suggests that
the mangrove extracts contain compounds that are capable of donating
hydrogen to a free radical in order to remove odd electron which is
responsible for radicals reactivity (Wang et al., 1998). Research also
indicates that the total antioxidant and DPPH scavenging potential are
particularly suitable for the evaluation of antioxidant activity of crude
extracts (Poli et al., 2003). The result of DPPH scavenging activity assay
in this study indicated that the mangroves species were potently activity.
Reducing capacity of tea extracts can serve as a significant indicator
of their potential antioxidant activity. The total reducing ability of the
mangrove black tea were evaluated at varied concentrations (50,100,

6
A. Arulkumar et al. Biocatalysis and Agricultural Biotechnology 23 (2020) 101491

Fig. 2a. FTIR spectrum for R. mucronata tea extract.

Fig. 2b. FTIR spectrum for R. apiculata tea extract.

500 mg/mL) are shown in (Fig. 1d). Among the three tea extract tested,
R. mucronata showed maximum reducing power at all the concentrations
(P < 0.05) and showed higher reducing activity R. mucronata 0.9733 �
0.55% when compared to R. apiculata 0.9276 � 1.84% and
R. annamalayana 0.8255 � 1.20% at the concentration of 500 μg/mL
(Fig. 1d). Similarly, Pandima Devi et al., 2009 have reported that, the
reducing capacity of R. mucronata, R. apiculata and R. annamalayana
were 0.360, 0.263 and 0.259 μg/mL, which was comparatively much
lower than the present study. The reducing capacity of a compound may
serve as an important indicator of its potential antioxidant activity. The
results of high reducing power of R. mucronata were in agreement with
highest DPPH activity of the present study. The reducing properties are
thought to be associated with the development of reductone, which have
been shown to exert antioxidant action by terminating the free radical

7
A. Arulkumar et al.
Fig. 2c. FTIR spectrum for R. annamalayana black tea extract.
chain reactions by donating a hydrogen atom. Reductones are also re­
ported to react with certain precursors of peroxide, thus preventing
peroxide formation (Singh and Rajini, 2004). The result of tea were
capable of reducing Fe3þ to Fe2þ, which was related to the capability of
donating electrons of the present phenolics, suggesting that tea may act
as the terminators of free radical chains, transforming reactive free
radical species into more stable non-radical products. The antioxidant
capacity of flavonoids may improve endothelial function by lowering
oxidative stress. Better endothelial function impacts on vasomotor tone,
platelet activity, leukocyte adhesion and vascular smooth muscle cell
function. Studies have shown that black tea flavonoids improve coro­
nary circulation Hirata et al. (2004) and attenuate endothelial
dysfunction in humans (Duffy et al., 2001). The results suggested that, a
reducing power of the compound appears to be related to degree of
hydroxylation and extent of conjugation in polyphenols which was seen
in tea extracts especially in R. mucronata when compare to R. apiculata
and R. annamalayana.
NO* causes ischemic injury, the toxicity due to increase in reaction
with O2*- to produce ONOO , wich reacts with biomolecules like DNA,
proteins, lipids (Halliwell, 1991). Nitric oxide was generated when so­
dium nitro preside reacts with oxygen to form nitrite. Suppression of
NO* release may be attributed to a direct NO* scavenging effect of tea
extracts decreasing the amount of nitrite generated from the decompo­
sition of sodium nitroprusside invitro as shown in (Fig. 1e). The nitric
oxide radical scavenging activity of three mangrove extracts increased
as the concentration increased (P < 0.05). The maximum activity was
observed from R. mucronata (75.33 � 0.91%) followed by R. apiculata
(72.53 � 1.65%) and R. annamalayana (62.8 � 1.53%) at the concen­
tration of 100 μg/mL. Similarly, Pandima Devi et al. (2009) found that
NO* scavenging values of R. mucronata, R. apiculata and R. annamalayan
were 58.14, 27.5 and 29.7 μg/mL in nitric oxide assay. NO is a reactive
free radical produced by phagocytes and endothelial cells, to yield more
reactive species such as peroxynitrite which can be decomposed to form
OH radical. The level of nitric oxide was significantly reduced in this
study by the black tea extracts. Since NO* plays a crucial role in the
pathogenesis of inflammation (Moncada et al., 1991), this may explain
the use of Rhizophoraceae members for the treatment of inflammation
and for wound healing. Table 4 showed biological potential i. e anti­
oxidant, antibacterial and phytochemical and others activities in the
8
Biocatalysis and Agricultural Biotechnology 23 (2020) 101491
mangroves species of this region was compared with different region in
this world.
The tea extracts was analyzed by FT-IR to identify the functional
groups in the bioactive components based on its peak values and elec­
tron transition of compounds shown in Fig. 2a, b and c. The FT-IR
spectrum for the methanolic extracts of R. mucronata, R. apiculata and
R. annamalayan with its peak values were observed at 441,665,754,
1028, 1228, 1450, 1664, 2044, 2222, 1255, 2599, 2833, 2943, 3348,
3350 and confirmed the presence of the chloro alkanes, flour alkenes/
phenol, alkenes, nitrile, amide, methylene, methyl, alcohol/phenol/
flavonoid.
4.5. Antibacterial activity
The antibacterial activity of tea from Rhizophoraceae members
(R. mucronata, R. apiculata and R. annamalayana) showed significant and
effective antimicrobial activity against human pathogenic bacterial
strains (Escherichia coli, Salmonella typhi, Proteus vulgaris and Staphylo­
coccus aureus) tested and the results are shown in (Table 3). No zone of
inhibition was observed in DMSO (negative control) and the positive
control, penicillin tested for antibacterial activity exhibited maximum
zone of inhibiton of 13.1 � 0.02 mm against Salmonella typhi. R.
mucronata showed higher antibacterial activity compared with
R. apiculata and R. annamalayan. R. mucronata was more effective
against E. coli, S. typhi and P. vulgaris compared to gram positive bacteria
like S. aureus. These results are in agreement with report of Kumar et al.,
(2011) in which the methanolic extract Avicinea marina, E. agallocha,
and C. decandra showed higher antibacterial activity against S. aureus,
Bacillus subtilis, S. epidermides, E. coli, P. aeruginosa and K. pneumoniae.
The antimicrobial properties of mangroves extracts could be due the
presence of core compounds Ethanone, 1-(2-hydroxy- 5- methylphenyl),
which might played an important role in their antibacterial activity
(Manilal et al., 2015). Extracts from different mangrove plants and
mangrove associates are active against human and plant pathogens
(Vadlapudi and Chandrasekhr naidu et al., 2009). This indicates that
R. mucronata, R. apiculata and R. annmalayana tea extracts showed more
antibacterial activity against Staphylococcus aereus, Proteus vulgaris and
E. coli than other strains. These results indicated that, the effective anti
bacterial activity due to the potential bioactive, functional group OH
A. Arulkumar et al.
and N–H group (Phenol and flavonoids) present of mangrove plants.
Abdurahman et al., 2016 documented that, the antimicrobial com­
pounds present in Rhizophora sp includes polyphenols, carbohydrates,
fatty acids, palmitic acid, cis and trans isomers of oleic acid, linolenic
acid miristic acid, tannin and sterols.
4.6. TLC
The methanol extract with a concentration of 1 mg/mL reported the
presence of three major compounds with Rf values of 0.50, 0.36 and 0.14
in R. mucronata, 0.49, 0.46 and 0.12 in R. appiculata and 0.52, 0.31, 0.11
in R. annmalayana as visualized under iodine chamber and UV light. The
larger Rf value of a compound, the larger the distance it travels on the
TLC plate.
5. Conclusion
The present study concluded that mangroves are rich source of tea
extracts. The extracts contained phenolic and polyphenolic compounds
that exhibited high antioxidant properties and considerable antibacte­
rial activities against human bacterial pathogens. The tea extracts had
stable scavenging activity against the free radial molecules prevent cell
and DNA damage. The most powerful antioxidant activity of Rhizophora
species can be accredited to neutralizing and scavenging ability of the
free radicals. The leaf extracts of mangroves in particular Rhizophora
species has potential to be developed as a herbal drug and nutraceutical
and thus in curing infective human diseases. Furthermore, research is
needed to elucidate the complete mode of action and application of
phenol compounds to prevent the human bacterial pathogenic diseases
and aquatic pathogenic bacteria.
Declaration of competing interest
All the authors declare that there is no potential conflict of interest.
Acknowledgements
The authors are thankful to the authorities of Alagappa University,
Tamilnadu, India, for providing necessary facilities to carry out this
research work. Dr.S.Paramasivam thanks to external funding by
RUSA–2. 0 grant sanctioned vide Letter No. F.24–51/2014–U, Policy
(TN Multi–Gen), Department of Education, Government of India.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bcab.2019.101491.
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