Zoology, Presidency College, Chennai- 600 005, Tamil Nadu, India. Correpondence and Reprint Requests: Ashok K

Received: S e p t e m b e r 2 5 , 2015 | Accepted: November 5, 2015 | Published Online: November 28 , 2015

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (creativecommons.org/licenses/by/3.0) Conflict of interest: None declared | Source of funding: Nil

OPEN ACCESS at journalijcir.com

Research Article ISSN: 2395 -5775

PHYTOCHEMICAL PROFILING, ANTIOXIDANT AND ANTIMICROBIAL ACTIVITY OF METHANOL EXTRACT IN RAMBUTAN FRUIT (NEPHELIUM LAPPACIUM) EPICARP AGAINST THE HUMAN PATHOGENS

Nethaji R, Thooyavan G, Mullai Nilla K and Ashok K*

Abstract

A large number of the plants are claimed to possess the antibiotic properties in the traditional system and are also used extensively by the tribal people throughout the world. It is now mostly thought that nature has given the cure of every disease in one form or another. Plants have been known to cure people from various diseases in Ayurveda. Effects of crude extract of the Nephelium lappaceum epicarp were studied for the antioxidant and antibacterial activity. The research showed that the methanolic extract were more effective. The antioxidant activity of the methanol extract were carried out using DPPH which showed better antioxidant properties which could be very useful against reactive oxygen species that are formed during oxidative stress.

Key Words: Phytochemical profiling, antimicrobial activity and antioxidant activity

INTRODUCTION Rambutan fruit is one of the most interesting of all tropical fruits widely known and revered in South East Asia as the” King of Fruits”. The fruits are distinctive for its large size, unique odor and a formidable thorn covered hersk. The custard-like flesh has an exquisite flavor and is at the same time aromatic and sweet with a strange balsamic taste.Rambutan has been claimed that the fruit possess great rejuvenating power. The Rambutan fruits from the genus Durio belonging to the Durionaceae family. In the 1920s, Rambutan fruit products, Inc.,of New York City launched a product (Dur-India) as a health food supplement selling at US$9 for a dozen bottles, each containing 63 tablets [1]. Several medicinal investigations on the validity of this belief have been conducted with varying conclusion [2-3]. The custard-like flesh has an exquisite flavor and is at the same time aromatic and sweet with a strange balsamic taste. Rambutan has been claimed that the fruit possess great rejuvenating power [4]. Traditional systems of medicine,ayurveda were the basis of the health care system in India until early years of twentieth century.According to an estimation of world health organization approximately 80% of people in developing countries rely on traditional

medicinesfor primary health care needs [5]. Rambutan fruit is one of the most interesting of all tropical fruits widely known and revered in South East Asia as the” King of Fruits”. The fruits are distinctive for its large size, unique odor and a formidable thorn covered hersk [6]. The presumption of world health organization is that the edibles, particularly of plant derived, empathetically indicates that these items have medicinal values [7]. The drugs are derived from the whole plant or from different organs, like leaves, stem, bark, root, flower, fruits, seed etc. Some drugs are prepared from excretory plant product such as gum, resin and latex. Not only, that plant-derived drug offers a stable market worldwide, but also plants continue to be an important source for new drugs [8]. The scientific study of traditional medicines, derivation of drugs through bio prospecting and systematic conservation of the concerned medicinal plants are thus of great important. Many phytochemical found in fruits act as powerful antioxidants that give them color, flavor, odor and protection against human diseases. Rambutan fruits were used as good sex tonic and its fruit sap is very useful in treating more than 50 common diseases. But there is no scientific study on the antimicrobial activity of the extracts of the Rambutan fruits [9].

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Nephelium lappaceum Linn., commonly known as rambutan which belongs to the family of Sapindaceae, is an attractive tropical fruit widely distributed in South-East Asia, especially in Malaysia and Indonesia [10]. It is a potential fruit to be commercialized since it is widely planted all over Malaysia. Rambutan has been used as traditional medicine for centuries especially as a remedy for diabetes and high blood pressure [11-12]. Rambutan contains a large variety of substances possessing antioxidant activity, such as vitamin C, vitamin E, carotenes, xanthophylls, tannins and phenolics [13]. They also exhibit a wide range of biological activities including antimicrobial, anticarcinogenic as well as antiproliferative, and many other biological activities have been attributed to their antioxidant properties [14].

Medicinal plants represent a rich source of antimicrobial agents. There is also an urgent need to

search for new antimicrobial compounds with novel mechanisms of action because there has been an alarming increase in the incidence of new and re-emerging infectious diseases [15]. A numeral commercial variety of rambutan (Nephelium lappaceum Linn.) exists. In Malaysia, red and yellow rambutan fruits are available in the market. The fruits are ovoid, with a red or yellow pericarp covered with soft spines that vary in colouring from yellow and red. They are different in taste. However, so far there is no comparative study have been reported in rambutan varieties, though various extracts of Nephelium lappaceum have been reported for antibacterial activity [16-17]. The main target of our study is finding of phytoconstituents from epicarp methanol extract and to determine the antioxidant and antimicrobial efficacy of Nephelium lappaceum epicarp methanol extract against the human pathogens. MATERIALS AND METHODS Collection and identification of Nephelium lappaceum Fresh Rambutan fruits (Nephelium lappaceum) were purchased from Koyambedu fruit market, Chennai, Tamilnadu, India and were authentically identified by Prof. P. Jayaraman, Institute of Herbal Science, Plant Anatomy Research Centre, West Tambarm, Chennai, India, as Sapindaceae with voucher specimen no: PARC/2015/2126. Extraction Nephelium lappaceum epicarp were cut in ½ inch pieces and dried in air seven days and finally in mechanical drier at 60-70ºC. The dried samples were grinded to course powder with a mechanical grinder and extracted with methanol for 7 days with occasional shaking in a beaker. The extract was filtered and the filtrate was dried at 50 to 60°C and the yielded percentage was calculated.

Table1 Phytochemical analysis of Nephelium lappaceum epicarp methanolic extract

S.No. Phytochemicals Nephelium lappaceum

epicarp methanolic extract

1 Carbohydrates Molisch’s test

Present

2 Alkaloids

Dragendorff’s test Present

3 Steroids and sterols

Liebermann Burchard’s and Salkowski test

Present

4 Glycosides Baljet’s test

Present

5 Flavonoids

Shinoda test. Present

6 Triterpenoids

Tin and thionyl chloride test Present

7 Tannins

Lead acetate test and gelatin test Present

8 Proteins and aminoacids Biuret test and Ninhydrin

Present

Table 2 Antibacterial activity of Nephelium lappaceum epicarp methanolic extract

S.No. Name of the

extract Name of the bacteria

Zone of inhibition in mm 10

µg/ml 15

µg/ml 20

µg/ml 25

µg/ml 10 µg/ml Norflaxin

10 µg/ml Penicillin

1

Metanol

E.coli 9mm 10mm 12mm 14mm 18mm 11mm 2 Klebsiella pneumonia 8mm 9mm 11mm 13mm 18mm 0mm 3 Pseudomonas auerogenousa 0mm 0mm 0mm 0mm 18mm 13mm 4 Proteus vulgarius 9mm 11mm 13mm 15mm 19mm 11mm

Table 3 Antifungal activity of Nephelium lappaceum epicarp methanolic extract

S.No. Name of

the extract

Name of the fungi

Zone of inhibition in mm 10

µg/ml 15

µg/ml 20

µg/ml 25

µg/ml Ketconazole 10 µg/ml

Nyastin 10µg/ml

5 Methanol A. fumigatus 10mm 11mm 13mm 15mm 7mm 0mm

Table 4 Percentage of scavenging activity of Nephelium

lappaceum epicarp methanolic extract

Concentration (µg/ml)

% of scavenging activity Ascrobic acid Methanolic extract

0 0 0 1 35.26 34.789

10 41.632 38.061 20 45.98 44.162 30 55.252 57.509 40 56.292 55.87 50 60.59 62.49

International Journal of Cur

Phytochemical Profiling, Antioxidant And Antimicrobial Activity of Methanol Extract In Rambutan Fruit (Nephelium Lappacium) E

Phytochemical analysis The phytochemical analysis of Actinidia deliciosathe fruit extracts were carried out to identify the constituents, using standard pytochemical methods as described by Gomathi et al., 2012 [18]. The screening involves detection of secondary metabolities such as carbohydrates, alkaloids, steroids and sterols, glycosides, flavonoids, triterpenoids, tannins, proteins and aminoacids.

Test microorganisms The test microorganisms used for antimicrobial analysis were clinical isolates of pneumonia, E. coli, Pseudomonas auerogenousaProteus vulgaris, and Aspergillus fumigatesbacterial strains were maintained on Nutrient Agar and fungi on Sabouraud Dextrose Agar at the Medical Education Department, King Institute of Preventive Medicine and Research, Guindy, Chennai, India.

In vitro antimicrobial activity

Determination of zone of inhibition by disc diffusion method In vitro antibacterial and antifungal activities were

Fig1 Antimicrobial activity of Nephelium lappaceummethanolic extract (1A- E.coli, B- Klebsiella pneumonia

Pseudomonas auerogenousa, D- Proteus vulgarius10µg/ml, b- 15µg/ml, c-20 µg/ml, d-25 µg/ml, e

µg/ml), f- Nyastin (10 µg/ml)

Fig 2 Radical scavenging activity of Nephelium lappaceummethanolic extract

International Journal of Current Innovation Research, Vol. 1, Issue 9, pp 201-206,

Phytochemical Profiling, Antioxidant And Antimicrobial Activity of Methanol Extract In Rambutan Fruit (Nephelium Lappacium) EAgainst The Human Pathogens

Actinidia deliciosa of the fruit extracts were carried out to identify the constituents, using standard pytochemical methods as described by Gomathi et al., 2012 [18]. The screening involves detection of secondary metabolities such as carbohydrates, alkaloids,

terols, glycosides, flavonoids, triterpenoids, tannins, proteins and aminoacids.

The test microorganisms used for antimicrobial analysis were clinical isolates of Klebsiella

Pseudomonas auerogenousa, Aspergillus fumigates. The

bacterial strains were maintained on Nutrient Agar Sabouraud Dextrose Agar at the

Medical Education Department, King Institute of Preventive Medicine and Research, Guindy, Chennai,

Determination of zone of inhibition by disc diffusion

antibacterial and antifungal activities were

examined for methanol extract of lappaceum epicarp. Antibacterial and antifungal activities of methanol extract of lappaceum epicarp against pathogenic bacterias (Gram-positive and negativeinvestigated by the agar disk diffusion method [19]. Antimicrobial activity testing was carried out by using agar cup method. Purified methanol extracts were dissolved in dimethyl sulfoxide, sterilized by filtration using sintered glass filter, and stored at 4°C. For the determination of zone of inhibition, pure Grampositive, Gram-negative, and fungal strains were taken as a standard antibiotic for comparison of the results. All the extracts were screened for their antibacterial and antifuthe Escherichia coli, Pseudomonas aeruginosa,fungi Aspergillus fumigatus(10, 15, 20 and 25μg/ml) ofepicarp methanol and standard drugsprepared in double-distilled water using nutrient agar tubes. Mueller-Hinton sterile agar plates were seeded with indicator bacterial strains (10allowed to stay at 37°C for 3 hours. Control experiments were carried out under similar conditiby using norfloxacin and penicillin for antibacterial activity and Ketconazole and Nysatin for antifungal activity as standard drugs. The zones of growth inhibition around the disks were measured after 18 to 24 hours of in incubation at 37°C for bacteri48 to 96 hours for fungi at 28°C. The sensitivities of the microorganism species to the methanol extract of Nephelium lappaceum epicarpmeasuring the sizes of inhibitory zones (including the diameter of disk) on the agar surface ardisks, and values < 8 mm were considered as not active against microorganisms. Antioxidant Activity using radical The antioxidant activity ofNephelium lappaceum antioxidant ascorbic acid was assessed on the basis of the radical scavenging effect of the stable 2, 2diphenyl-1-picrylhydrazyl (DPPH) free radical activity according to the method described by Chanwitheesuk et al., 2005 [20].extract with different concentrations (1, 10, 20, 30, 40 and 50 μg/ml) were prepared using methanol. Ascorbic acid was used as the standard in 1μg/ml solution. 0.004 % of DPPH solution was prepared in methanol and 5 ml of this solution wamixed with 5 ml of methanol extract solution and standard solution distinctly. These solution mixtures

Nephelium lappaceum epicarp

Klebsiella pneumonia, C- Proteus vulgarius, E- A. fumigatus), (a-

25 µg/ml, e- Ketconazole (10 Nyastin (10 µg/ml)

Nephelium lappaceum epicarp

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Phytochemical Profiling, Antioxidant And Antimicrobial Activity of Methanol Extract In Rambutan Fruit (Nephelium Lappacium) Epicarp

examined for methanol extract of Nephelium Antibacterial and antifungal

activities of methanol extract of Nephelium against pathogenic bacterias

positive and negative) and fungi were investigated by the agar disk diffusion method [19]. Antimicrobial activity testing was carried out by using agar cup method. Purified methanol extracts were dissolved in dimethyl sulfoxide, sterilized by filtration

lter, and stored at 4°C. For the determination of zone of inhibition, pure Gram-

negative, and fungal strains were taken as a standard antibiotic for comparison of the results. All the extracts were screened for their antibacterial and antifungal activities against

Escherichia coli, Klebsiella pneumonia, Pseudomonas aeruginosa, Proteus vulgaris and the

Aspergillus fumigatus. The sets of four dilutions g/ml) of Nephelium lappaceum

methanol and standard drugs were distilled water using nutrient

Hinton sterile agar plates were seeded with indicator bacterial strains (108cfu) and allowed to stay at 37°C for 3 hours. Control experiments were carried out under similar condition by using norfloxacin and penicillin for antibacterial activity and Ketconazole and Nysatin for antifungal activity as standard drugs. The zones of growth inhibition around the disks were measured after 18 to 24 hours of in incubation at 37°C for bacteria and 48 to 96 hours for fungi at 28°C. The sensitivities of the microorganism species to the methanol extract of

epicarp were determined by measuring the sizes of inhibitory zones (including the diameter of disk) on the agar surface around the disks, and values < 8 mm were considered as not active against microorganisms.

Antioxidant Activity using radical-scavenging method

The antioxidant activity of methanolic extract epicarp and the standard

antioxidant ascorbic acid was assessed on the basis of the radical scavenging effect of the stable 2, 2-

picrylhydrazyl (DPPH) free radical activity according to the method described by

., 2005 [20]. The methanol extract with different concentrations (1, 10, 20, 30,

g/ml) were prepared using methanol. Ascorbic acid was used as the standard in 1-50 g/ml solution. 0.004 % of DPPH solution was

prepared in methanol and 5 ml of this solution was mixed with 5 ml of methanol extract solution and standard solution distinctly. These solution mixtures

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Ashok K et al.,

were kept in dark for 30 min. The degree of DPPH purple decolourization to DPPH yellow indicated the scavenging effectiveness of the extract. The absorbance of the combination was determined at517 nm using UV-Visible Spectrophotometer and ascorbic acid was served as a positive control. Lower absorbance of the reaction mixture indicated higher free radical scavenging activity. % DPPH radical scavenging = (Absorbance of control

-Absorbance of test Sample) / (Absorbance Of control) X 100

RESULT AND DISCUSSION Phytochemical screening The phytochemical screening of the methanolic extracts of Nephelium lappaceum epicarp showed that the leaves are rich in, carbohydrates, proteins and aminoacids, steroids, alkaloids flavonoids, tannins, triterpenoid and glycosides. Flavonoids are most commonly known for their antioxidant activity. They are transformers which modify the body‘s reactions to carcinogens, viruses, and allergens. They show anticancer, anti-inflammatory, antimicrobial and anti-allergic activity [21-22], and may be useful in therapeutic roles [23]. Alkaloids are organic compounds that contain nitrogen, and are physiologically active with sedative and analgesic properties. They are used in relieving pains, anxiety and depression [24]. Alkaloids are toxic due to their stimulatory effects, leading to excitation of cells and neurological dysfunction [25]. Glycosides are compounds containing a carbohydrate and non-carbohydrate residue in the same molecule. In these compounds, the carbohydrate moiety is attached by an acetal linkage of carbon-1 to the aglycone. They all contain steroid as aglycone component in combination with sugar molecules. They are important in medicine because of their action on heart and are used in cardiac insufficiency [26]. Thus, cardiac glycosides are drugs and can be used in the treatment of congestive heart failure and cardiac arrhythmia. They work by inhibiting the Na+/Na+ pump, resulting in an increase in the levels of sodium ions in the myocytes, which then leads to a rise in calcium ions level. This inhibition raises the amount of Ca2+ ions available for concentration of the heart muscle, increases cardiac output and reduces distension of the heart [27-28]. These glycosides are found as secondary metabolites in several plants and animals [29]. However, some glycosides, such as ouabain, are toxic as it inhibits active transport of Na+ in cardiac

muscle (sodium pump inhibitor), which results in inhibition of translocases during electron transport chain, and leading to death [30]. Also phloridzin (toxic glycosides) blocks the transport of sugar across the mucosal cells of small intestine and also renal tubular epithelium; it displaces Na+ from the binding sites of carrier protein and prevents the binding of sugar molecule and produces glycosuria [31-32]. In vitro antimicrobial activity Antibacterial activity of the methanol extract of Nephelium lappaceum epicarp was tested against the different test microorganisms are shown in the table 2 and3. The methanolic extract of Nephelium lappaceum epicarp showed higher antibacterial property against Escherichia coli, Klebsella pneumoniae and Proteus vulgaris but no activity was observed in Pseudomonas auerogonesa and in antifungal activity methanol extract showed moderate zone of inhibition against Aspergillus fumigatus as shown in the figure 1A,B,C and D. From the primary phytochemical screening, we have been able to identify that methanol extract of Nephelium lappaceum epicarp has got phytomedical property it may be due to the nature of biologically active compounds present in it whose activity are enhanced in the presence on methanol and also methanol has an stronger extraction capacity which could have produced greater number of active constituents responsible for antibacterial activity aswell as antifungal activity. The antimicrobial activities can be enhanced if the active components are purified and adequate dosage determined for proper administration. In vitro antioxidant activity DPPH test is based upon the ability of DPPH, a stable free radical, to decolourize from purple in the presence of antioxidants. It is a direct and dependable method for determining the radical scavenging action. Ascorbic acid was chosen as the standard antioxidant for this test. The DPPH radical contains an odd electron, which is responsible for the absorbance at 517 nm and also for a noticeable deep purple color. When DPPH accepts an electron donated by an antioxidant compound the DPPH becomes colourless, which is quantitatively measured from the changes in absorbance. Highest scavenging was observed with Nephelium lappaceum epicarp methanolic extract followed by Ascrobic acid known antioxidant standard. Scavenging activity of DPPH radical was found to rise with increasing concentration of the methanolic

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extract of Nephelium lappaceum epicarp. Additionally, it has been determined that the antioxidant effect of plant products is mainly due to radical scavenging activity of phenolic compounds such as flavonoids, polyphenols and tannins [33]. The antioxidant activity of phenolic compounds is mainly due to their oxidation reduction properties, which can play an important role in adsorbing and neutralising free radicals, reducing singlet and triplet oxygen, or decomposing peroxides [34]. Oxidative injury now appears as the fundamental mechanism causing a number of human neurologic and other disorders such as autoimmune pathologies, inflammation, viral infections and digestive system disorders including gastrointestinal inflammation and ulcer [35]. The present results suggest that all the tested plant extracts have moderate to potent antioxidant activity. Subsequently a variety of constituents are known from the four crude extracts which we studied therefore, it becomes very difficult to ascribe the antioxidant properties selectively to any one group of constituents without further studies it is impossible. Thus further thorough investigations are necessary. With the increase in absorbance there is increase in reducing antioxidant power. So with high amount of extract there is increase in absorbance and with the decrease in extract quantity the absorbance is lowered. CONCLUSION In conclusion, the above results showed that methanolic extract of rambutan fruit epicarp are good candidate for further investigation of antimicrobial and antioxidant potentials. Furthermore, the isolation of active constituents which are responsible for the activity is necessary for broad investigation. Acknowledgment The authors are thankfull to Sterline Bioscience, Chennai for the technical support. Conflicts of Interests The authors declare no conflict of interest References 1. Anagnostopoulou MA , Kefalas P, Papageorgiou

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