International Journal of Drug Development and Research

Key words

Phytochemical, Bioactive substances, Flavonoids, Phenolic compounds, Green tea.

Introduction

Phytochemicals are the bioactive compounds which are present in the medicinal plants [1]. Tea (Camellia sinensis) is consumed worldwide and is second to water in its popularity as a beverage and has ascribed many health benefits such as reduction in cholesterol and protection against cardiovascular disease [2]. Green tea is generally safe, nontoxic and has no side effects after consumption [3]. The most important bioactive compounds present in Camellia sinensis leaves are alkaloid, flavonoids, steroids and terpenoids which serve as valuable starting material for the medicine development [4]. Camellia sinensis possess an antimicrobial activity because of the bioactive compounds [5]. The plant materials have shown the antimicrobial activities against various pathogenic microorganisms therefore consumption of tea has been associated with reduced risk of major diseases [6]. The beneficial effects of the tea have been attributed to the strong antioxidant activity due to the phenoloic compounds [7]. The carotenoids, flavonoids, benzonic acid, ascorbic acid, tocotrienols, cinnamic acid, folic acid, tocopherols are some antioxidents produced by the plants for their substance [8].

Materials and Methods

Collection of the sample

The fresh leaves of Camellia sinensis were collected [9] from the dense tea state garden at Ooty, Coimbatore district, Tamil Nadu South India.

Preparation of the extract

The leaves of fresh samples were cleaned and washed under running tap water [10]. The samples were dried in the oven at 37°C for 6 days. After drying the samples were weighed and blended with warring blender and soaked with methanol [in ratio methanol: plant (6:1)] for 2 days and filtered using Whatman No. 1 paper. The methanol was completely removed by vacuum evaporator at 50°C till it gave a viscous mass. The crude extracts were weighed and stored at 4°C before analysis.

Preliminary phytochemical screening

The tests were done to find the presence of the active chemical constituents such as alkaloids, glycosides, terpenoids and steroids, flavonoids, reducing sugar and tannin [11].

Alkaloid test

The alcoholic extract was evaporated to dryness and the residue was heated on a boiling water bath with 2% Hydrochloric acid. After cooling, the mixture was filtered and treated with a few drops of 5% Sodium Hydroxide solution. The samples were then observed for the presence of turbidity or yellow precipitation

Glycoside test

0.5grams of extract was dissolved in 2ml of glacial acetic acid and mixed well. To this few drops of ferric chloride and concentrated sulphuric acid were added and observed for a reddish brown coloration at the junction of two layers and the bluish green color in the upper layer.

Terpenoid test

Four mg of extract was treated with 0.5 ml of acetic anhydride and 0.5 ml of chloroform. Then concentrated solution of sulphuric acid was added slowly and red violet color was observed for the presence of terpenoid.

Steroid test

Four mg of extract was treated with 0.5 ml of acetic anhydride and 0.5 ml of chloroform. Then concentrated solution of sulphuric acid was added slowly and green bluish color was observed for presence of steroids.

Flavonoid test

2ml of extract solution was treated with 1ml of lead acetate solution and white colour was observed for the presence of flavonoids.

Gallic tannin test

0.5 ml of extract was dissolved in 1 ml of water, mixed uniformly and then 2 drops of ferric chloride solution were added and blue color was observed for presence of gallic tannin.

Catecholic tannin test

0.5 ml of extract was dissolved in 1 ml of water, mixed uniformly then 2 drops of ferric chloride solution were added and green black colour was observed for presence of catecholic tannin.

Saponins test

0.5ml of extract was treated with 5ml of distilled water and frothing persistence indicate the presence of saponins.

Determination of total phenolic content

Determination of total phenolic content was carried out by [12] taking one hundred micro liters in 1ml of water methanol extract was dissolved in 1ml (1:1ratio) of the Folin–Ciocalteu reagent. The solutions were mixed and incubated at room temperature for 1 minute. After 1 minute, 1ml of 35% sodium carbonate (Na2CO3) solution was added and makeup to 8 ml with distilled water. The final mixture was shaken and then incubated for 1 ½ hour in the dark at room temperature. The absorbance of all samples was measured at 725 nm using colorimeter. Gallic acid was used as standard for the calibration curve then plotted at 0.02, 0.04, 0.06, 0.08 and 0.10 mg/ml respectively. The gallic acid that was prepared in 80% (v/v) methanol. The absorbance was recorded at 725 nm using 80% (v/v) methanol as blank. Triplicate measurements were carried out and total phenolic content was expressed as milligram of gallic acid equivalents (GAE) per 100 gram of samples.

Determination of total flavonoids content

The flavonoid content was estimated by [12] taking 0.5 ml of the sample and added to a test tube containing 1.5 ml of methanol. Then added 0.3 ml of 5% sodium nitrite solution and allowed to stand for 5 min. Added 0.3 ml of 10% aluminum chloride, after 6 min 1 ml of 1 M sodium hydroxide was added and the mixture was diluted with distilled water. The absorbance of the mixture at 510 nm was measured immediately. The flavonoid content was expressed as milligram catechin equivalents /g sample.

Assay of reducing power

The reductive capability of the extract was quantified by [13] taking one ml of methanolic extract, mixed with 2.5 ml of 0.2 M phosphate buffer (pH 6.6) and 2.5 ml of 1% potassium ferricyanide [K3 Fe (CN) 6]. Similar concentrations of standard ascorbic acid were used as standard. The mixture was incubated at 50°C for 20 min. reaction was terminated by adding 2.5 ml of 10% trichloroacetic acid. Then it was centrifuged at 3000 rpm for 10 minutes. The upper layer of solution (2.5 ml) was mixed with distilled water (2.5 ml) and 0.5 ml of 0.1%FeCl3. Blank reagent was prepared as above without adding extract. The absorbance was measured at 700 nm in a colorimeter against a blank sample. Increased absorbance of the reaction mixture indicated greater reducing power.

Fourier Transform Infrared Spectroscopy Qualitative analysis

Camellia sinensis extracts was subjected [14] to Fourier Transform Infrared Spectroscopy (FT-IR) Qualitative analysis by using Perkin Elmer Fourier Transform Infrared Spectroscope (Model-spectra 100) instrument and the obtained spectra for the product was analyzed and interpreted with a chart for characteristics infrared absorption frequencies of organic functional groups and carbonyl containing functional groups.

Antimicrobial activity of Camellia sinensis leaves

Preparation of inoculum

The inoculum was prepared by culturing the microorganisms in nutrient broth at 37°C for 12 hours to a concentration of approximately 105 CFU/ml.

Agar-well diffusion method

The agar-well diffusion assay was adopted [10] for the present assay. Each bacterial suspension was spread over the surface of Mueller-Hinton agar (Himedia, India) plates containing 4 wells having 6 mm diameter. The wells were filled with 30μl each of the various concentrations (100μg, 200μg and 300μg) of extracts. The plates were incubated at 37°C for overnight. The results were expressed in terms of the diameter of the inhibition zone and Methanol used as control.

Result

Identification of leaves

The leaves belongs to the Kingdom-Plantae, Order- Ericales, Family-Theaceae, Genus-Camellia, Speciessinensis is shown in Figure 1

Qualitative analysis of phytochemicals

The extract showed the presence of phytochemicals namely alkaloids, flavonoids, steroids, gallic tannins and catecholic tannin by changing the colour of the solution to yellow, white, green bluish, blue, green black respectively. While indicated the absence of terpenoid, saponins, and glycosides as there was no colour change in the solution with respect to them.

Total Contents

The total phenolic content in one gram of leaf extracts was found 0.7grams while the total flavonoidic content was14mg/gram of leaf extract. The reducing power of Camellia sinensis were 0.11grams/gram of leaf extract.

FT-IR Qualitative analysis

The Fourier Transform Infrared Spectroscopy (FTIR) Qualitative analysis of Camellia sinensis obtained was analyzed and interpreted with a chart for characteristics infrared absorption frequencies of organic functional groups and carbonyl containing functional groups which showed the presence of alkene, alcohol, ester, amine acid, alkane, aromatic alkane, nitro compounds, aromatic amide, alkene amide, carbonyl anhydride, alkane hydroxyl group ketone, aromatic amine, alcohol amine and alcohol (table 1).

Antimicrobial activity

The Camellia sinensis leaf extracts of various concentrations (100μg/ml, 200μg/ml and 300μg/ml) concentrations showed the zone of inhibition against gram positive bacteria such as Bacillus subtilis (Figure 2). The zones of inhibition of gram positive bacteria were measured in millimeter (mm) and then compared with antibiotic standard Erythromycin, Tetracycline and Ampicillin thus were found susceptible. The susceptibility increased when the concentration was increased. Similarly in case of gram negative bacteria such as Enterococcus sp (Figure 3) the zones of inhibition were measured in millimeter (mm) and compared with antibiotic standard Erythromycin, Tetracycline and Ampicillin were found susceptible. The susceptibility increased when the concentration was increased.

Discussion

The presence of phytochemical namely alkaloids, flavonoids, steroids, gallic tannins, catecholic tannin plays the vital role in the plant defense mechanisms [15, 5]. The total phenolic content was found 0.7g/gram leaves extract. The total flavonoid content was 14mg/gram of extract and the reducing power was found 1g of leave extract sample carries 0.11g of reducing power. The active substance found in tea is supposed to reduce growth and development of microorganisms [16]. The highest antimicrobial activity of tea is due to presence of catechins and polyphones which damages bacterial cell membrane [17]. They also serve in plant defense mechanisms to counteract reactive oxygen species in order to survive and prevent molecular damage and caused by microorganisms, insects, and herbivores [18]. The antibacterial activity of Camellia sinensis leaf against Listeria monocytogenes by disc diffusion method, the methanolic extract had greater antibacterial property as compared to the water extract [9]. In this work, methanolic extract of Camellia sinensis had greater antibacterial activity against Staphylococcus aureus and Enterococcus sp. These observations are likely to be the result of the differences in cell wall structure between Gram-positive and Gram-negative bacteria [19]. In the Gram-negative outer membrane acting as a barrier to many environmental substances including antibiotics [20]. The green sorts of tea have shown higher antimicrobial activity than the black ones [21. The antibacterial activity of Camellia sinensis tea extracts was selective and depends upon the concentration, type of the extracts and bacterial species [17, 22]. In this work different concentration of leaf extracts were used against different pathogenic bacteria and highest zone of inhibition was observed against Bacillus subtilis and Enterococcus sp [23].

Conclusion

Camellia sinensis leaves contain the phytochemicals which plays duel benefits as medicinal values and food value. In this study we found that leaf extracts were found to be potential antibacterial agents against the gram positive Bacillus subtilis and Enterococcus sp as shown by the maximum zone of inhibition. Thus Camellia sinensis leaves can be used an alternative medicine against the bacterial infection.

Tables at a glance

Table 1

Figures at a glance

Figure 1 Figure 2

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