Antibacterial and invitro antioxidant potential of Indian mangroves

Journal Pre-proof Antibacterial and invitro antioxidant potential of Indian mangroves A. Arulkumar, K. Sampath Kumar, S. Paramasivam PII:

S1878-8181(19)30339-1

DOI:

https://doi.org/10.1016/j.bcab.2019.101491

Reference:

BCAB 101491

To appear in:

Biocatalysis and Agricultural Biotechnology

Received Date: 4 May 2019 Revised Date:

17 December 2019

Accepted Date: 30 December 2019

Please cite this article as: Arulkumar, A., Kumar, K.S., Paramasivam, S., Antibacterial and invitro antioxidant potential of Indian mangroves, Biocatalysis and Agricultural Biotechnology (2020), doi: https://doi.org/10.1016/j.bcab.2019.101491. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

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Antibacterial and invitro antioxidant potential of Indian mangroves

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A. Arulkumar*1,2, K. Sampath Kumar3 and S. Paramasivam1

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Department of Oceanography and Coastal Area Studies, School of Marine Sciences, Alagappa University- Thondi Campus 623 409, Tamil Nadu, India. Achariya Arts and Science College, Villianur Puducherry- 605 110 (Affiliated Pondicherry University). Sengunthar Arts and Science College, Thiruchengode-637 205, Tamil Nadu, India. *

Email: [email protected]; Ph: +91-9597319098.

Abstract

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Herbal tea is medicinally well-known for its biochemical composition and medicinal

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usages. The present study proved for the first time that tea would be extracted from

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mangroves, the plants other than commercial tea. Tea showed a theaflavin (TF), thearubigins

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(TR), Total liquor color (TLC) and highly polymerized substantce (HPS). These characters

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revealed the good quality of the tea. Phytochemical screening of tea revealed the presence of

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phytochemical compounds such as protein, phenol and flavonoids etc. antioxidant properties

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of tea were tested in invitro at varied concentrations. The antioxidant properties of R.

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mucronata, R. apiculata, and R. annamalayana showed dose dependent activities and were

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comparatively much higher in R. mucronata. R. mucronata (86.50±0.35%) showed much

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higher total antioxidant activity followed by R. apiculata (73.34±0.90%) and R.

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annamalayana (69.35±0.56%) at 100 µg/ml concentration. The antimicrobial activity was

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highest in R. mucronata and R. apiculata than R. annamalayana as evident by the presences

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of phenolic N-H and OH components found in tea. Therefore, it can be concluded that,

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mangroves are rich source of tea, biochemical consititent, phytochemical, antioxidant and

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antibacterial properties. The extract contained polyphenolic and other bioactive compounds

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can be used as a development of herbal drug formuation and nutraceutical.

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Keywords: Tea, Rhizophora mucronata, Phytochemical, antioxidant and R. annamalayana.

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1. Introduction

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India has a rich and prestigious heritage of mangrove forest oriented medicines among

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the South Asian countries. However, the majority of these plants have not yet undergone

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chemical, pharmacological and toxicological studies to investigate their bioactive compounds

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(Kathiresan, 2000; Singh et al., 2009). Traditional records and ecological diversity indicate

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that India plants represent an exciting resource for possible lead structures in drug design.

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Plants are endowed with free radical scavenging molecules, such as vitamins, terpenoids,

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phenolic acids, lignins, stilbenes, tannins, flavonoids, quinones, coumarins, alkaloids, amines,

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betalains, and other metabolites, which are rich in antioxidant activity (Zheng and Wang,

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2001). The mangrove plants naturaly syntheses many bioactive secondary metabolites. Such

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plant-derived phytochemicals are highly unexplored for therapeutics. Mangroves are

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biochemically unique, producing a wide array of novel natural products. They are rich in

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polyphenols and tannins (Kathiresan and Ravi, 1990). In recent years, there is growing

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interest in the discovery of noval compounds in mangroves resoures to control the emerging

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human pathogenic microbes. Fascinatlingly, researchers have isolated a variety of other

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mangrove compounds including taraxerol careaborin and taraxeryl cis-p-hydroxycinnamate

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from leaves of Rhizophora apiculata (Kokpol et al., 1990); 2-nitro-4-(2'-nitroethenyl phenol)

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from leaves of Sonneratia acida (Bose et al., 1992); alkanes (46.7–97.9% wax) and

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triterpenoids (53.3% wax) from leaves of Rhizophora species (Dodd et al., 1995); and iridoid

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glycosides from leaves of Avicennia officinalis and A. germinans (Fauvel et al., 1995; Sharma

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and Garg, 1996). Phytoconstituent of R. mucronata was identified as Ethanone, 1-(2-

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hydroxy- 5- methylphenyl as role in the antibiotic activity against human and fish pathogen

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(Manilal et al., 2015).

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In recent past years, mangrove plants have gained much importance due to its used in

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fisher-folk medicine to treat various diseases (Bandaranayake, 2002) and it has been studied

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for their medicinal properties. Coastal plant extracts have been tested against viruses that

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cause human and animal diseases, such as human immunodeficiency virus (HIV), new castle

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disease virus, vaccinia virus, semliki forest virus, encephalomyocarditis virus, and hepatitis-

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B-virus (Kathiresan, 2000). A few of the mangrove plants belonging to family-

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Rhizophoraceae are most effective against the viruses (Premanathan et al., 1992). Bioactive

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compounds present in the mangrove plants and polysaccharides composed of galactose,

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galactosamine, glucose and arabinose reported to have potent anti-HIV activity (Premanathan

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et al., 1999). Scientific studies on a number of medicinal plants indicated that promising

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phytochemical compounds can be developed for many health problems (Gupta, 1994).

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These phytochemicals are indeed chemically complex and many structurally related

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active compounds often produce a synergistic effect. Therefore medicinal plants are perhaps

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the most valuable sources of new bioactive chemical entities to benefit mankind against

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various ailments. Almost 122 pure chemical substances extracted from higher plants are used

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in medicine throughout the world (Fabriant and Farnsworth, 2001). Therefore, it very

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important to determined the phytochemical screening, bioactive compound (FTIR) and

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biochemical constitutent in Rhizophora species for noval drug discovary, antibiotics and

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navol nutraceutical compounds. However, for first time in this present investigation, was

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evaluate the campartive study of phytochemical, antioxidant and antibacterial activity of

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mangroves species viz. Rhizophora mucronata, R. apiculata and R. annamalayana. The

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bioactive efficacy of tea, extracted from three mangrove Rhizophora species and

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identification of the functional groups present in the tea extracts by using Fourier transform-

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infrared spectroscopy (FT-IR) analysis.

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2. Materials and Methods

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2.1 Collection of mangrove leaves

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The fresh mangrove leaves of Rhizophora mucronata, Rhizophora apiculata and

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Rhizophora annamalayana were collected from the artificially developed Vellar Estuary

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mangroves vegetation at Parangipettai, Tamilnadu, India. The river Vellar is flowing on the

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Southeast Coast of India, mixing with the sea at Porto Novo (also known as Parangipettai;

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Lat. 11° 29’ N; Long. 79° 47’ E). The estuary is connected to the Pichavaram mangroves

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through the backwater channel. The mouth of the Vellar Estuary opens to the sea and a sand

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bar sometimes closes it completely during summer season.

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2.2 Preparation and extraction of tea

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Black tea was extracted from the leaves of Rhizophora species, adopting the method of

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Kathiresan (1995). Fresh leaves were allowed to dry using a warming blender. A known

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weight of the macerated sample was placed in a piece of cloth and distilled water was

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continuously trickled over it for 1hrs. The fermented sample was dried in hot air oven at 95°C

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for 30 minutes. 1.25% of mangrove black tea was added to the boiling water and allowed to

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infuse for 5 minutes, after which the infusions were filtered through sterile mambrean filter.

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The filtrates were lyophilized and used for further study.

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2.3 Tea quality constituents and Caffine content of Tea

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Theaflavin (TF), Thearubigins (TR), Total liquor color (TLC), highly polymerized

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substantce (HPS) and caffeine content were analysed, for determination of quality of tea

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(Thanraj and Shesadari, 1990; Ronald and Ronald, 1991).

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2.4 Qualitative analysis of phytochemical screening

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The qualitative chemical constituents present in the tea extracts of mangroves plants were

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determined by (Trease and Evans, 1989).

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Proteins-xanthoprotein: to 1mL of extract, 3-5 drops of nitric acid was added by the sides

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of the test tube and observed for formation of yellow colour indicated presence of proteins.

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Steroids: two mL of acetic anhydride was added to 0.5 g of extract and 2 mL of sulphuric

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acid was added by the sides of the test tube and observed the colour change from violet or

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blue-green.

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Tannins: about 0.5 g of the each extract was taken in a boiling tube and boiled with 20 mL

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distilled water filtered and then added few drops of 0.1% ferric chloride mixed well and

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allowed to stand for some time. Brownish green or a blue-black colouration was observed.

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Glycosides─Keller-Killani method: about 0.5 mL of alcoholic extracts was taken and

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subjected to the following test. One ml of glacial acetic acid containing traces of ferric

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chloride and 1mL of conc. sulphuric acid were added to extract and observed for the

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formation of reddish brown colour at the junction of two layers and the upper layer turned

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bluish green in the presence of glycosides.

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Reducing sugars─Fehling’s test: few drops of Fehling’s solution A and B in equal volume

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were added in dilute extracts and heated for 30 min and observed for the formation of brick

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red coloured precipitate.

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Sterols-Liebermana-Buchard method: the insoluble residue was dissolved in chloroform

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and few drops of anhydride were added along with a few drops of concentration H2SO4 from

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the side of the test tube and the formation of blue to blood red color was obderved.

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Terpenoids ─ Salkowski test: to 0.5 g of the extract, 2 mL of chloroform was added; conc.

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H2SO4 (3mL) was carefully added to form a layer. A reddish brown coloration of the

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interface indicates the presence of terpenoids.

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Cardiac glycosides (Keller ─ Killiani’s test): 100 mg of extract was dissolved in 1 mL of

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glacial acetic acid containing one drop of ferric chloride solution. This was then underlayered

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with 1 mL of concentrated H2SO4. A brown ring obtained at the interface indicated the

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presence of a de-oxy sugar charactersitcs of cardenolides.

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Carbohydrates─Molisch’s test: small quantities of alcoholic and aqueous extracts were

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dissolved in 5 mL of distilled water and filtered. To this solution 2−3 drops of α-naphthol was

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added and 1 mL of concentrated H2SO4 was added along the sides of inclined test tube so as

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to form two layers and observed for formation of violet coloured ring at the interface to detect

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the presence carbohydrates.

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Flavonoids: a portion of the acqueous extract (2 ml) was heated, and metallic magnesium

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and concentrated hydrochloric acid (5 drops) were added. A red or orange coloration

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indicated the presence of flavonoids.

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Phenols: the extracts were taken in water and warmed. To this 2 mL of ferric chloride

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solution was added and observed for formation of green or blue colour.

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2.5 Chemicals and reagents

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Butyled hydoxytoluene (BHT), DPPH (1, 1-diphenyl, 2-picrylhydrazyl), resazurin of

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Sigma Chemicals, (USA) and all others high purity chemical reagents of Merck (Germany)

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were used.

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2.6 Estimation of Antioxidants assay

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2.6.1 Estimation of total antioxidants

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Total antioxidant activity was measured by the method of (Mitsuda et al., 1996). 7.45 mL

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sulphuric acid (0.6 M solution), 0.9942 g sodium phosphate (28 mM solution) and 1.2359 g

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ammonium molybdate (4 mM) were mixed together in 250 mL with distilled water and

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labeled as total antioxidant capacity (TAC). 100 µL of extract was dissolved in 1mL of TAC

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and the absorbance was read at 695 nm after 15 min. Butyled hydoxtoluene (BHT) was used

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as positive control.

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Calculation of 50% inhibitory concentration (IC50)

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The concentration (µg/mL) of the fractions that was required to scavenge 50% of the

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radicals was calculated by using the percentage scavenging activities at three different

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concentrations of the fractions. Percentage inhibition (%) was calculated using the following

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formula,

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% Inhibition = [(Ac-As) /Ac] X 100

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Where, Ac = absorbance of the control As = absorbance of the sample.

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2.6.2 Determination of total phenol content

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Total phenolic content of tea were estimated with the Folin-Ciocalteu’s method

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(Singleton et al., 1999). Black tea extracts (10 mg) were dissolved in 10 mL of distilled

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water. An aliquot of 100 µL of appropriate dilution of the samples were shaken for 1min with

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500 µL of the folin-ciocalteu reagent freshly prepared in the laboratory and 6 mL of distilled

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water. After the mixture was shaken 2 mL of 15% (mass per volume) sodium carbonate was

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added and the mixture was shaken again for 0.5 min. Finally, the solution was brought up to

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10 mL with distilled water. After 2 hrs of reaction at ambient temperature, the absorbance

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was measured at 750 nm. Using gallic acid as standard, the total phenolic content of both

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mangroves was expressed as gallic acid equivalents (GAE).

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2.6.3 DPPH radical scavenging assay

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The free radical scavenging activity of tea extracts were measured by DPPH according to

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(Blois, 1958) method. 1 mL of DPPH (0.1mM) solution in methanol was added to 3 mL of

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water extract of tea (100 µg/mL), shaken vigorously, allowed to stand at room temperature

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for 30 min and the absorbance was measured at 517 nm in a UV-visible spectrophotometer

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(U-2800 model, Hitachi, Japan). A low absorbance of the reaction mixture indicated the high

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free radical scavenging activity. Butylated hytroxytoluene (BHT) was used as positive

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control. The DPPH scavenging effect was calculated.

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DPPH radical scavenging activity = {(Abs control – Abs sample) / (Abs Control)} × 100

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Where; Abs control = absorbance of DPPH radical +BHT;

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Abs sample = absorbance of DPPH radical + sample extract or Standard.

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2.6.4 Total reducing power

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Total reducing capacity of tea was determined according to the method of (Oyaizu,

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1986). The tea extracts (100 µg/mL) in distilled water and 1% potassium ferricyanide were

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mixed with phosphate buffer (0.2 M, pH 6.6) and the mixture was incubated at 50°C for 20

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min. 2.5 mL of 10% TCA was added to the reaction mixture which was centrifuged at

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1000×g for 5 min. The upper layer of the solution (2.5 mL) was mixed with distilled water

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(2.5 mL), FeCl2 (0.5 mL, 0.1%) and the absorbance was measured at 700 nm. Ascorbic acid

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was used as a positive control. The higher absorbance of the reaction mixture indicated the

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greater reducing power.

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2.6.5 Nitric oxide radical (NO*) scavenging activity

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Nitric oxide radical scavenging activity was determined by the method reported by

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(Garrat, 1964). Sodium nitroprusside in aqueous solution at physiological pH spontaneously

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generates nitric oxide, which interacts with oxygen to produce nitrite ions, which can be

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determined by the griess-illosvoy reaction. Briefly, 3 mL of the reaction mixture containing

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10 mM sodium nitroprusside and the tea extract (100 µg/mL) in phosphate buffer were

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incubated at 25°C for 150 min. After incubation, 0.5 ml of the reaction mixture was mixed

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with 1mL of sulfanilic acid reagent (0.33% in 20% glacial acetic acid) and allowed to stand

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for 5 min for complete diazotization. Then 1 mL of naphthyl ethylene diamine

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dihydrochloride (0.1%) was added and after mixing well the solution was allowed to stand

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for 30 min at 25°C. A pink coloured chromophore was formed in diffused light. The

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absorbance of these solutions was measured at 540 nm against the corresponding blank

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solutions. BHT was used as positive control. Nitric oxide scavenging activity of the tea

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extract is reported as % inhibition and was calculated.

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NO radical scavenging activity = {(Abs control – Abs sample) / (Abs control)} × 100

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Where; Abs control = absorbance of NO radical + BHT; Abs

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Sample = absorbance of NO radical + sample extract or standard.

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2.7 FT-IR analysis of tea

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For this 2 mg of the tea extracts samples of (R. mucronata, R. apiculata and R.

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annamalayana) were mixed with 200 mg KBr (FT-IR grade) and made into thick pellets in a

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hydraulic press by allying 500 Kg/m3 pressure. The pellet was immediately placed into the

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sample holder and FT-IR spectra were recorded between ranges of 7800–350cm-1 (Mid-IR)

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for the compound under study. FT-IR spectra of the purified compound were recorded on a

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Shimadzu, Model No: A21374801626, Japan FT-IR spectrometer.

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2.8 Bacterial strains and culture conditions

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Bacterial cultures namely Staphylococcus aereus, Escherichia coli, Salmonella typhi, and

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Proteus vulgaris were obtained from the CAS in Marine Biology, Annamalai University,

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Parangipettai, India. The above cultures were long term storage took place at -20 oC in

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Nutrient broth (Hi Media, Mumbai, India) supplemented with 50% glycerol. Before

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experimental use each strain was grown twice in Nutrient broth at 37 oC for 48 h.

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2.8.1 Determination of In vitro antibacterial activity against human pathogenic bacteria

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The antibacterial activity of the tea extracts (R. mucronata, R. apiculata and R.

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annamalayana) was performed by the well diffusion method on Muller-Hinton agar MHA

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(Hi media, India). About 100 µL of 105 CFU/ml diluted inoculum bacterial culture was

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applied on the surface of MHA plates and allowed to solidify. MHA well was made with well

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borer under aseptic conditions and filled with mangroves tea extract samples and DMSO as

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used as negative control and penicilin was act as positive control. The plates were incubated

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at 37oC for bacterial growth/ the antibacterial activity of the mangrove samples was evaluated

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by measuring the zone of inhibition against the test human pathogenic bacteria. All the

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experiments were done three times and the data were expressed as the mean values of

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experiments.

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2.9 Thin layer chromatography (TLC)

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apiculata and R. annamalayana (Abu et al., 2011). Methanol and chloroform (1:9) was used

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as mobile phase. The sample with the concentration of about (1 mg/mL) was spotted on the

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TLC plates and dried. The spots were identified in long UV, short UV and also in the iodine

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chamber. Rƒ value was calculated to find the active metabolites. Rƒ value is distance

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travelled by the solute to the distance traveled by the solvent.

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3.0 Statistical analysis

TLC is used to separate the bioactive compounds present in the tea extract R. mucronata, R.

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Datas were expressed in triplicates (n=3) and strandard devision. The data were obtained

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were analyzed by the one way analysis of variation (ANOVA), with a P values of < 0.05

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being significant. All the statistical were performed using the OriginPro version 8.0 statistical

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package.

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4. Results and Discusion

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4.1 Physical nature of tea

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The present study evaluated that phytochemical constituent of tea from the three

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mangroves species, R. mucronata, R. apiculata and R. annamalayana. The black tea extract

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showed a golden brown colour with pleasant aroma taste. These characters revealed the good

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quality of the tea. It was observed that, the maximum yield of tea was obtained for the R.

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mucronata. The percentage yield of R.mucronata (0.125%) followed by R. apiculata

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(0.121%) and R.annamalayana (0.110%) respectively. The colour and the yield of the tea

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extraction might be attributed to the levels of polyphenol content.

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4.2 Tea quality constituents of tea extracts

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The chemical constituent and quality of tea determined theaflavin (TF), Thearubigins

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(TR), Total liquor color (TLC), highly polymerized substantce (HPS) and caffeine content

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statistically significance different with P< 0.05 were presented in (Table 1). The R.

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mucronata was observed to possess higher TF 0.42±0.02% when compared to R.apiculata

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(0.18±0.02%) and R. annamalayana (0.5±0.08%) tea extracts. The higher thearubigins (TR)

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was observated in R. mucronata (4.60±0.24%) followed by R. annamalayana and

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R.apiculata. The R.mucronata was observed to possess higher HPS 4.71±0.20% followed by

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R.apiculata 3.9±0.29% and R.annamalayana 3.95±0.18%. The total liquire colour (TLC) was

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higher R. mucronata in (1.1±0.14%) followed by R. annamalayana (0.51±0.03%) and R.

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apiculata (0.49±0.03%). Coffien content all the three mangroves speices was not detected

260

(ND). It was observed that, R. mucronata contains higher TF, TR, TLC and HPS content than

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R. apiculata and R.annamalayana. Venkatesan et al. (2005) have repoted that, the polyphenol

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found in the tea leaves and it important responsible for all the biochemical reaction with

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contribution to make a good quality of tea. The chemical basis of the quality of mangrove

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black tea indicated theaflavin (TF), Thearubigins (TR), Total liquor color (TLC), highly

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polymerized substantce (HPS) are the important factors determine the liquor quality of

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mangrove black tea. Liang et al., (2007) have reported that, the tea owing to its favorable

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benefits on human health dring currently enjoys a great popularity among other beverages

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throught out the worldwide. In this study, higher TF, TR, TLC and HPS was oberverved in

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R.mucronata followed by other potential species (Table 1). Bagyalakshmi et al., 2012 have

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reported that, the quality of tea characterisitics TF, TR, HPS, TLC and caffeine 0.8, 7.6, 7.2,

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2.3 and 2.1 respectively in Camellia sinensis. In This study first time proved that HPS was

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higher in R.mucronata (4.71±0.20%) followed by R.apiculata (3.9±0.29%) and

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R.annamalayana (3.95±0.18%) and there is no caffien content in all the Rhizophora species.

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4.3 Qualitative analysis of phytochemical constituents

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Phytochemical constituents of R. mucronata, R. apiculata and R. annamalayana were

276

qualitatively analyzed and the results are presented in (Table 2). The tea extract of all the

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three species viz. R. mucronata, R. apiculata and R. annamalayana showed the presence of

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phytochemical active compounds such as xanthoprotein, steroids, tannins, glycosides,

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reducing sugar, carbohydrates, sterols, terpenoids, phenol, cardioglycosides and flavonoids.

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Mangroves and mangrove associates possess novel agrochemical products, compounds of

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medicinal value, and biologically active compounds (Bandaranayake, 2002). The

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phytochemical screening of tea extracts of R. mucronata, R. apiculata and R. annamalayana

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revealed the presence of tannins, flavonoids, steroids and saponins. Its clearly indicates that,

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the presence of the functional group –OH in the structure and positions of the flavonoid

285

molecules to determine the phytochemical capacity. Recently, Saranya et al. 2014 reported

286

that, the important active metabolities find out through the phytochemical screening

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R.mucronata showed the presence of alkaloids, terpenoids, steroids, tannins, quinines,

288

sapnins, flavonoids, cardiac glycosides and phenol, which corroborates the results of the

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present study. Abdurahman et al., (2016) reported that, the Rhizophora species have various

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phyo-chemical compounds includes alcohol, aldehydes, aminoacids, aromatic carboxylic

291

acid, carbohydrates, carboxlic acids, esters, fatty acids, flavonoid, ketone, lipids, phenol,

292

saponins, steroids, tannin and terpenoid for the biological acitivity.

293 294

These results support the finding that mangrove plants are a rich source of steroids,

295

triterpenes, saponins, flavonoids, alkaloid and tannins (Agramoorthy et al., 2008;

296

Bandarnayake, 2002). Alkaloids, tannins, flavonoids and other phytochemicals are present in

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other plants as well (Barnabas and Nagarajan 1988). In the present study it is found that

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Rhizophora species contain these secondary metabolities which showed phenolic compounds

299

(N-H and OH molecules) and other substance. Hence, the beneficial medicinal effects of

300

plant material may result from the combinations of secondary products present in the plant.

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Secondary products play a role in a plant defense through cytotoxity towards microbial

302

pathogens (Briskin, 2000).

303

Tannins are known to be useful in the medicine flok and treatment of inflamed or ulcerated

304

tissue and thery remarkable anticancer (Ruch et al., 1989). Thus, Rhizophora species

305

containing bioactive compounds and its may serve as a potential sources of bioactive

306

compounds in the treatment of cancer. The presence of phenol compounds in this Rhizophora

307

species may contribute to their antioxidant and antibacterial propertices, its very usefulness

308

developments of herbal medicine and nuetraceuticals. Earlier study reported that, the

309

phenolic compounds may exhibit a widal range of physiological function, such as anti

310

bacterial, anti fungual, antioxidant, anti inflammatory, anti artherogenic, anti thrombotic

311

effects (Benavente et al., 1997; Samman et al., 1998; Puupponen-Pimia et al., 2001; Manilal

312

et al., 2015; Abdurahman et al., 2016).

313

4.4 Antioxidant

314

The antioxidant activities of tea extracts of three mangrove species, R. mucronata, R.

315

apiculata and R. annamalayana at different concentration are shown in (Fig.1a-e). It was

316

found that, of the three species of tea extracts screened, R. mucronata (100 µg/ml) showed

317

significant levels of antioxidant properties, in terms of total antioxidants, total phenol, DPPH

318

assay, and total reducing power and nitric oxide radical scavenging activity. The total

319

antioxidant activity of the tea extracts increased with increase in concentration (P< 0.05). The

320

result suggested that, the R. mucronata showed highest total antioxidant activity of

321

85.50±0.35% when compared to R. apiculata 73.34±0.90% and R. annamalayana

322

69.35±0.56% at the concentration of 100 µg/ml respectively (Fig.1a). It was observed that,

323

R.mucronata contains 12.16% and 16.15 % time’s higher total antioxidant activity than

324

R.apiculata and R.annamalayana. Gao and Xiao, 2012 reported that, the Rhizophora species

325

contains lyoniresinol-3α-o-β-arabinopyranoside, lyoniresinol-3α-O-β-rhamnoside, afzelechin-

326

3-rahmnoside bioactive compounds for antioxidant and better radical scavenging activity.

327 328

The total phenol contents of the tea extracts are possibly proportional to the

329

concentration. The highest TPC was observed from R. mucronata 84.80±1.47% followed by

330

R. apiculata 79.23±0.70% and R. annamalayana 74.35±0.90% at the concentration of 100

331

µg/ml significance level (P< 0.05) (Fig.1b ). It was clearly noted that, R.mucronata contains

332

5.59% and 10.45% times higher total phenol content than R.apiculata and R.annamalayana.

333

Pandima Devi et al. 2009 have reported that, the TPC of R.mucronata was 720.7 mg GAE/g

11

334

for methanolic extracts which corrabotes the results of R.mucronata in the present study. The

335

higher content of phenolic in mangroves tea extracts indicates a better free radical scavenging

336

ability, which can prevent lipid oxidation in food products. Screening of antioxidant

337

substances derived from natural resources is one of the basic tool for drug design and

338

development. Agoramoorthy et al. 2008 have reported that, the TPC higher phenol content of

339

R. mucronata and R.apiculata was 157.4 mg GAE/g and 302 mg GAE/g for dry weight

340

respectively, which was comparatively much higher than the results of the present experiment

341

(Fig.1b). Recently, Wei et al. 2010 reported that, the TPC of mangrove plant (Kandelia

342

candel) was 400.43µg GAE /ml. Haq et al. 2011 have reported that, the total phenol content

343

of Bruguiera gymnorrhiza was 178.73 mg GAE/g of dry leaves. The present study indicates

344

that R.mucronata, R. apiculata, and R. annamalayana tea extracts were potential free radical

345

scavengers, wich reacted with free radicals by donating their hydrogen (H2) and act as

346

primary antioxidants and prevent oxidation.

347

Several free radical such as OH, O2, LOO have different reactivities are formed

348

during lipid oxidation. Antioxidants are able to scavenge these radicals by donating H iorns.

349

Relatively stable DPPH radical has been widely used to test the ability of compounds to act

350

as free radicals scaverngers or H irons donors and thus to evaluate the antixodant activity (Jao

351

and Ko, 2002). In this study, evident that the ability of DPPH radicals scavenging activity of

352

tea extracts is shown in (Fig.1c). The activity of tea extract increased with increase

353

concentration (P< 0.05). The result suggested that, the IC50 value of R. mucronata showed

354

highest radical scavenging activity was 91.83±1.26% when compared to 78.92±1.72% for R.

355

apiculata and 90.87±1.56% for R. annamalayana at the concentration 100 µg/ml

356

respectively. Pandima Devi et al. 2009 have reported that, the IC50 value of R.mucronata

357

DPPH was 43.171µg/ml, which was comparatively much lower than the results of the present

358

study. Similalry, Agoramoorthy et al., 2008 have reported that, the DPPH free radical

359

scavengers

360

comparable to our results (93.39 and 90.31%). Wei et al., 2010 have reported that, the DPPH

361

radical scavengers was 115.67 µg/ml in K.candel in 70% acetone extracts, which was

362

comparatively higher than the results of the present study. The high radical scavenging

363

activity of R. mucronata could be due to presence of flavonoids that can be perform radical

364

scavenging activity action of on free radical (DPPH, hydroxyl and hydrogen H2O2) for their

365

reduction of hydroxide formation. In this study, the presence of the functional group -OH in

366

the structure and its position of flavonoid and or phenol molecules determine the antioxidant

in R. mucronata and R.apiculata were 79.9 and 64.7 µg/ml, which was

12

367

activity (Fig.1c). This suggests that the mangrove extracts contain compounds that are

368

capable of donating hydrogen to a free radical in order to remove odd electron which is

369

responsible for radical's reactivity (Wang et al., 1998). Research also indicates that the total

370

antioxidant and DPPH scavenging potential are particularly suitable for the evaluation of

371

antioxidant activity of crude extracts (Poli et al., 2003). The result of DPPH scavenging

372

activity assay in this study indicated that the mangroves species were potently active.

373

Reducing capacity of tea extracts can serve as a significant indicator of their potential

374

antioxidant activity. The total reducing ability of the mangrove black teas were evaluvated at

375

different varied concentrations (50,100, 500 mg/mL) are shown in (Fig.1d). Among the three

376

tea extract tested, R. mucronata showed maximum reducing power at all the concentrations

377

(P< 0.05) and showed highest reducing activity R. mucronata 0.9733±0.55% when

378

comparted for R. apiculata 0.9276±1.84% and R. annamalayana 0.8255±1.20% at the

379

concentration of 500 µg/ml (Fig.1d). Similarly, Pandima Devi et al. 2009 have reported that,

380

the reducing capacity of R.mucronata, R.apiculata and R.annamalayana were 0.360, 0.263

381

and 0.259 µg/ml, which was comparatively much lower than the present study. The reducing

382

capacity of a compound may serve also as an important indicator of its potential antioxidant

383

activity. The results of high reducing power of R.mucronata were agreement with highest

384

DPPH activity of the present study. The reducing properties are thought to be associated with

385

the development of reductones, which have been shown to exert antioxidant action by

386

terminating the free radical chain reactions by donating a hydrogen atom. Reductones are also

387

reported to react with certain precursors of peroxide, thus preventing peroxide formation

388

(Singh and Rajini, 2004). The result of teas were capable of reducing Fe3+ to Fe2+, which was

389

related to the capability of donating electrons of the present phenolics, suggesting that teas

390

may act as the terminators of free radical chains, transforming reactive free radical species

391

into more stable non-radical products. The antioxidant capacity of flavonoids may improve

392

endothelial function by lowering oxidative stress. Better endothelial function impacts on

393

vasomotor tone, platelet activity, leukocyte adhesion and vascular smooth muscle cell

394

function. Studies have shown that black tea flavonoids improve coronary circulation Hirata et

395

al., (2004) and attenuate endothelial dysfunction in humans (Duffy et al., 2001). The results

396

suggested that, a reducing power of the compound appears to be related to degree of

397

hydroxylation and extent of conjugation in polyphenols wich was seen in tea extracts

398

especially in R.mucronata when comapare to R.apiculata and R. annamalayana.

13

399

NO* causes ischemic injury, the toxicity of the due to increases on reaction with O2*-

400

to produce ONOO-, wich reacts with biomolecules like DNA, proteins, lipids (Halliwell,

401

1991). Nitric oxide was generated when sodium nitro preside reacts with oxygen to form

402

nitrite. Suppression of NO* release may be attributed to a direct NO* scavenging effect of tea

403

extracts decreasing the amount of nitrite generated from the decomposition of sodium nitro

404

preside invitro as shown in (Fig.1e). The nitric oxide radical scavenging activity of three

405

mangrove extracts increased as the concentration increased (P< 0.05). The maximum activity

406

was observed from R. mucronata (75.33±0.91%) followed by R. apiculata (72.53±1.65%)

407

and R. annamalayana (62.8±1.53%) at the concentration of 100 µg/ml. Similarly, Pandima

408

Devi et al., 2009 found that NO* scavenging values of R.mucronata, R.apiculata and

409

R.annamalayan were 58.14, 27.5 and 29.7 µg/ml in nitric oxide assay. Nitric oxide (NO) is a

410

reactive free radical produced by phagocytes and endothelial cells, to yield more reactive

411

species such as peroxynitrite which can be decomposed to form OH radical. The level of

412

nitric oxide was significantly reduced in this study by the black tea extracts. Since NO plays a

413

crucial role in the pathogenesis of inflammation (Moncada et al., 1991), this may explain the

414

use of Rhizophoraceae members for the treatment of inflammation and for wound healing.

415

Table 4 showed biological potential i.e antioxidant, antibacterial and phytochemical and

416

others activities in the mangroves species of this region was compared with different region

417

in this world.

418

The tea extracts was analyzed by FT-IR to identifiy the functional groups in the

419

bioactive components based on its peack values and electron transition of compounds shown

420

in Fig 2a, 2b and 2c. The FT-IR spectrum for the methanolic extracts of R.mucronata,

421

R.apiculata and R.annamalayan with its peak values were observed at 441,665,754, 1028,

422

1228, 1450, 1664, 2044, 2222, 1255, 2599, 2833, 2943, 3348, 3350 and confirmed the

423

presence of the chloro alkanes, flour alkenes/ phenol, alkenes, nitrile, amide, methylene,

424

methyl, alcohol/phenol/ flavonoid.

425

4.5 Antibacterial activity

426

The antibacterial activity of tea from Rhizophoraceae members (R. mucronata, R.

427

apiculata and R. annamalayana) showed significant and effective antimicrobial activity

428

against human pathogenic bacterial strains (Escherichia coli, Salmonella typhi, Proteus

429

vulgaris and Staphylococcus aureus) tested and the results are shown in (Table 3). No zone

430

of inhibition was observed in DMSO (negative control) and the positive control of penicillin

14

431

tested for antibacterial activity exhibited maximum zone of inhibio of 13.1±0.02 mm against

432

Salmonella typhi. R.mucronata showed higher antibacterial activity compared with

433

R.apiculata and R.annamalayan, R.mucronata was more effective against E. coli, S. typhi and

434

P.vulgaris compared to gram positive bacteria like S. aureus. These results are in agreement

435

with reported given by Kumar et al. 2011 observed that, the Avicinea marina, E.agallocha,

436

and C.decandra showed higher antibacterial activity against S.aureus, Bacillus subtilis,

437

S.epidermides, E.coli, P.aeruginosa and K.pneumoniae at methanolic extract. The

438

antimicrobial properties of mangroves extracts could be due the presence of core compounds

439

Ethanone, 1-(2-hydroxy- 5- methylphenyl), which might play an important role in their

440

antibacterial activity (Manilal et al., 2015). Extracts from different mangrove plants and

441

mangrove associates are active against human and plant pathogens (Chandrasekaran et al.,

442

2009). This indicates that R. mucronata, R.apiculata and R. annmalayana tea extracts showed

443

more antibacterial activity against Staphylococcus aereus, Proteus vulgaris and E.coli than

444

other strains. These results indicated that, the effective anti bacterial activity due to the

445

potential bioactive, functional group OH and N-H group (Phenol and flavonoids) present of

446

mangrove plants. Recently Abdurahman et al., 2016 documented that, the antimicrobial

447

compouns present in Rhizophora sp includes polyphenols, carbohydrates, fatty acids, palmitic

448

acid, cis and trans isomers of oleic acid, linolenic acid miristic acid, tannin and sterols.

449

4.6 TLC

450

The methanol extract with a concentration of 1 mg/mL reported the presence of three major

451

compounds with Rf values of 0.50, 0.36 and 0.14 in R.mucronata, 0.49, 0.46 and 0.12 in

452

R.appiculata and 0.52, 0.31, 0.11 in R. annmalayana as visualized under iodine chamber and

453

UV light. The larger Rf value of a compound, the larger the distance it travels on the TLC

454

plate.

455

CONCLUSION

456

The present study concluted that mangroves are rich source of tea extracts. The extracts

457

contained phenolic and polyphenolic compounds that exhibited high antioxidant properties

458

and considerable antibacterial activities against human bacterial pathogens. The tea extracts

459

had stable scavenging activity against the free radial molecules prevent cell and DNA

460

demage. The most powerful antioxidant activity of Rhizophora species can be accredited to

461

neutralizing and scavenging ability of the free radicals. The leaf extracts of mangroves in

15

462

particular Rhizophora species has potential to be developed as an herbal drug and

463

nutraceutical and thus in curing infective human diseases. Furthermore, research is needed to

464

elucidate the complete mode of action and application of phenol compounds to prevent the

465

human bacterial pathogenic diseases and aquatic pathogenic bacteria.

466

Acknowledgements

467

The authors are thankful to the authorities of Alagappa University, Tamilnadu, India, for

468

providing necessary facilities to carry out this research work.

469

Conflicts of interest

470 471

All the authors declare that there is no potential conflict of interest. 6. References

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614 615 616

Figure Caption:

617

Fig. 1. Antioxidant assay of mangroves black tea (a) Total Antioxidant (b) Total Phenol (c)

618

DPPH assay (d) Total Reducing Power (e) Nitric Oxide Assay

20

50 100 500

90 80

Total antioxidant %

70 60 50 40 30 20 10 0

R.mucronata

R.apiculata

R. annamalayana

Different concentrations

619 620 621

(1a)

50 100 500

90 80

Total phenol %

70 60 50 40 30 20 10 0 R. mucornata

R. apiculata Different Concentrations

622 623

(1b)

21

R. annamalayana

50 100 500

100

DPPH activity %

80

60

40

20

0 R. mucornata

R. apiculata

R. annamalayana

Different concentrations

624 625 626

(1c) 50 100 500

1.1 1.0

Total Reducing Power %

0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 R. mucornata

R. apiculata

Different concentrations

627 628 629

(1d)

22

R. annamalayana

50 100 500

80 70

Nitric Oxide %

60 50 40 30 20 10 0 R. mucornata

R. apiculata

R. annamalayana

Different concentrations

630 631

(1e)

632

All the values expressed mean and standard division. The statistical significant different

633

letters (a-e) with (P< 0.05).

634

635 636 637

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

638

23

639 640

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

641 642 643 644

645 646

Fig. 2c FTIR spectrum for R. annamalayana black tea extract

647

24

648 649

Table 1 Chemical constituent quality and caffeine content of tea extracts (R. mucronata, R. apiculata and R. annamalayana).

650

Table 2 Phyto-chemical characterization of tea extracts.

651

Table 3 Effect of tea extracts on the growth of Gram positive and Gram negative human

652

pathogenic bacteria.

653

Table 4 Comparison of biological potential in mangrove species as reported in the literature.

654 655

25

Table 1 Chemical constituent quality and caffeine content of tea extracts (R. mucronata, R. apiculata and R. annamalayana). Chemicals constituent

R. mucronata

R. apiculata

R. annamalayana

Theaflavine

0.42±0.02a

0.18±0.02a

0.5±0.08a

Thearubigin

4.60±0.24b

3.49±0.22 b

4.52±0.20 b

Highly polymerized substane

3.95±0.18 c

4.71±0.20 c

3.9±0.28 c

Total liquor colour

1.1±0.14 d

0.49±0.03 d

0.51±0.03 d

Caffeine

ND

ND

ND

Dates were expressed as mean ± standard division; ND- Not Detectable. The different letters a-c are statistically significant with P<0.05.

Table 2 Phyto-chemical characterization of tea extracts. Phytochemical test

R. mucronata

R. apiculata

R. annamalayana

Proteins-xanthoprotein

+

+

+

Steroids

+

+

+

Tannins

+

+

+

Glycosides

+

+

+

Reducing sugar

+

+

+

Sterols

+

+

+

Terpenoids

+

+

+

Cardioglycoside

+

+

+

Carbohydrate

+

+

+

Flavanoids

+

+

+

Phenol

+

+

+

+; presence of phyto-chemical components.

Table 3 Effect of tea extracts on the growth of Gram positive and Gram negative human pathogenic bacteria. Bacteria

Positive

Negative

control

control

Penicillin Escherichia

DMSO

Zone of inhibition in (mm)

R. mucronata

R. apiculata

R. annamalayana

11±0.01

-

10±0.11

2.0±0.01

6.0±0.05

13.1±0.02

-

8.0±0.07

7.0±0.06

2.0±0.03

9.0±0.04

-

6.0±0.05

5.0±0.03

10±0.09

10.1±0.00

-

5.0±0.04

15±0.12

7.0±0.05

coli Salmonella typhi Proteus vulgaris Staphylococcus aureus Mean values (n=3 with SD).- No zone of inhibition. Table 4 Comparison of biological potential in mangrove species as reported in the literature. Name of the

Study area

Biological properties

Reference

Parangipettai,

Antiviral and anti larvicidal

Premanathan

mangroves R.apiculata

Tamilnadu, India R.mucronata

Parangipettai,

et al., 1999 Antiviral, anti- HIV

Tamilnadu, India R.mucronata,

Pichavaram and

R.apiculata,

Tamilnadu, India

Premanathan et al., 1999

Polyphenol and antioxidant

Agoramoorthy et al., 2008

Avicennia officinalis, Ceriops decandra Avicennia marina

Gulf of Aqaba,

Antibcaterial, anticandidal, anti

Khafagi et al.,

Egypt

bacteriophage, cytotoxicity and

2003

plant growth grgulatar R.mucronata,

Pichavaram and

Antioxidant activity and

Pandima Devi

R.apiculata,

Thondi, Tamilnadu,

R.annamalayana,

India

antibacterial properties

et al., 2009

Antioxidant activity

Vadlapudi and

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Andhra Pradesh,

officinalis,

India

Naidu, 2009

A.marina, R.conjugata, R.mucronata, Salicornia brachiata, Salvodara persica, xylocarpus granatum Kandelia candel

China

Antioxidant properties

Wei et al., 2010

R.mucronata

R.mucronata

Ceriops decandra

Parangipettai,

Phytochemical and Bioactive

Saranya et al.,

Tamilnadu, India

compound

2014

Kerala, Kollam,

Phytochemical and Bioactive

Manilal et al.

India

compound

2015

Parangipettai,

Mangrove black tea buccal pouch

Sithranga

Tamilnadu, India

carcinogenesis in hamsters

Boopathy et al., 2011

C. decandra

Parangipettai,

Mangrove black tea prevents the

Sithranga

Tamilnadu, India

oral cancer incidences

Boopathy et al., 2011

R.mucronata,

Parangipettai,

Mangrove black tea rich in

R.apiculata,

Tamilnadu, India

Phytochemical, antioxidant and

R.annamalayana

antibacterial activity

Present study