INTRODUCTION:

Tea is the most widely consumed beverage made from the leaves of the Camellia sinensis plant, member of the Theaceae family¹. India and China are the first countries reported to cultivate tea and at present more than 30 countries globally participated in the cultivation². The production rate of green tea is gradually increased and China contributes approximately 75% of world green tea³. Numerous studies on the chemical components of tea and their biological characteristics such as such as antimutagenic⁴, anticarcinogenic⁵ and antioxidant^6,7, antibacterial⁸, antifungal⁹, antiviral¹⁰ and antiallergic activities¹¹have been spurred by the rising curiosity around the possible health advantages of tea additionally, its popularity as a beverage.

Plants are the important source of many currently available drugs that may be derived directly or indirectly from the plant sources. The tea plant is also a rich source of bioactive components and provides more than 4000 metabolites¹². Many researchers made an attempt to investigate phytochemical evaluation of Camellia sinensis¹³. The complex bioactive constituents of tea includes polyphenols, flavonoids, alkaloids, volatile compounds, minerals, trace elements, and many unidentified compounds. Among these, one-third constitutes the polyphenols, the most interesting group, and are the main bioactive molecules in tea¹⁴. The powerful scavenging and antioxidant activity of tea polyphenols inactivates direct carcinogens and inhibits the activation of indirect carcinogens extracellularly¹⁵. Common green and black tea consist of about 25-30% flavonoids, including kaempferol, quercetin, gallic esters, and myricetin¹⁶. Tea flavonoids play a protective role to maintain cardiovascular health, reduce inflammation, oxidative damage, blood pressure, and the risk of type 2 diabetes. Tea also plays a protective role to maintain cardiovascular health¹⁷.

The potential for utilizing tea extract in antimicrobial therapy and the prevention of diseases has been investigated by many researchers. The antimicrobial properties of tea extract was reported against Micrococcus luteus, Escherichia coli, Staphylococcus aureus, Salmonella typhi, Bacillus cereus, and Pseudomonas aeruginosa¹⁸. The tea extracts exhibited fungicidal activities against several fungi. The polyphenols in the tea extract in disc diffusion assay exhibit antifungal activity on Candida albicans and Cryptococcus neoformans¹⁹. The objective of the present study is to characterize the antioxidant and antimicrobial activity against selected bacteria and fungi from the methanol extract obtained from Camellia sinensis.

MATERIALS AND METHODS:

Sample Collection:

Leaves of Camellia sinesis were collected from Vaalparai (Poonthotam Estate), Tamilnadu. Fresh leaf materials were thoroughly washed with sterile distilled water, dried at room temperature in shade, and then grounded to make a fine powder using mechanical grinder and stored in air tight container.

Preparation of crude extracts:

The crude extract was prepared by adding 25g of shade dried powder to 150mL of methanol as solvent by the Soxhlet extraction method. The aqueous crude extract was transferred into Petri plates and allowed to remove methanol from the extract by evaporation at room temperature. The evaporated solid extract was collected aseptically and suspended in methanol (1mg/mL).

Assessment of overall content of phenols:

The overall phenolic components in the crude extract were assessed by Folin-Ciocalteau method²⁰. 1mL of the crude extract, 5mL of Folin-Ciocalteau reagent (1:10 v/v distilled water), and 4 mL (75g L^-1) of sodium carbonate were mixed in a test tube and vortexed for 15sec. The tube was incubated at 40^oC for 30min. The blue colour developed in the reaction mixture was read at 765nm using a UV spectrophotometer (Spectrophotometer UV100 cyber lab USA). The overall phenolic compounds from the crude plant extract was estimated from the standard curve of Catechol and the quantity was expressed as gallic acid g^-1.

Assessment of total flavonoid content:

The total flavonoid compound from the extract were estimated by Vanillin – Hydrochloride method proposed by Robert²¹. An aliquot (1mL) of crude extract of tea leaves was mixed with 5mL of freshly prepared vanillin hydrochloride reagent (equal volumes of 8% HCl in methanol) and 4% vanillin in methanol). The absorbance of the reaction mixture was compounded at 500nm using a UV spectrophotometer (Spectrophotometer UV100 cyber lab USA). The total amount of flavonoid from the extract of Camellia sinensis was compounded as catechin g^-1using standard curve constructed from the Catechin.

High-Performance Liquid Chromatography analysis for flavonoid:

The HPLC system, Shimadzu HPLC utilized for flavonoid analysis and is equipped with a Diode array detector (PDA). The column used was a block heating type C₁₈, (5μm, 4.6x150mm) and the mobile system was Toluene: Ethyl acetate (8:2). 5μL of the sample was injected, a low rate was maintained at 0.8mL/min and detection was carried out at 366nm using myricetin as standard.

Assessment of Antioxidant Activity:

The method proposed by Liyana-Pathiranan and Shahidi was employed to assess the antioxidant property of extract²². An equal volume of diluted crude extract (25 to 300mg) and a solution of 2, 2-Diphenyl-1-picrylhydrazyl (0.135mM DPPH in methanol) was prepared, vortexed, and incubated in the dark at condition at ambient temperature for 30 min. The optical density of the incubated reaction mixture was checked at 517nm by using ascorbic acid as the standard and distilled water as blank. Using the following formula, the crude extract's capacity to capture the DPPH radical was determined: Percentage of DPPH radical capture activity (%) = [(Abs_control − Abs_sample)]/(Abs_control)] × 100. IC50 is the parameter used in this study to measure the radical scavenging activity of the crude extract. IC50 is the appropriate concentration of antioxidant that scavenges 50% of DPPH radicals within 30 min of the incubation period.

Assessment of Antibacterial activity:

The antimicrobial activity of methanol extracts from Camellia sinensis against bacteria was tested with Bacillus sp., E.coli, Klebsiella sp., Mycobacterium philei, Salmonella typhi, Streptococcus sp. Antibacterial activity was measured using the disc diffusion method²³. The bacterial suspensions (100μL) with turbidity visually comparable to that of 0.5 McFarland standard were spread evenly onto Mueller-Hinton agar. Sterilized filter paper discs (6mm diameter) impregnated with crude extract (125, 250, 500, and 1000μg per disc), air dried aseptically, and placed firmly onto the inoculated Mueller-Hinton agar and incubated overnight at 37°C. A zone of inhibition due to the antibacterial activity of the plant was measured.

Assessment of Antifungal activity:

The antimicrobial activity of methanol extracts from Camellia sinensis against fungi was tested with Aspergillus flavus, Aspergillus niger, Collectotrichum sp., Mucor sp., and Penicillium sp. by well diffusion method²⁴. The spores of fungal strains were maintained on Potato Dextrose (PD) agar and inoculated with potato dextrose agar slants and incubated at 28°C for 7 days. The fungal spore suspension was prepared from the slants using PD broth and 100μL (approximately 1.0×10⁶ spores/ml) were spread evenly onto solidified potato dextrose agar plates. In each plate, 2 wells were made with sterile cork-borer and filled with crude plant extract (125, 250, 500, and 1000μg per well). Plates with inoculated fungal spores were incubated at 28°C for 7 days.

RESULT AND DISCUSSION:

Total phenolic content:

One of the oldest methods developed to estimate the total phenols is the Folin-Ciocalteu assay. The overall phenolic compounds from the plant extract was calculated from the standard graph and shown in Figure 1 (y = 0.0027x + 0.0024 where R² = 0.992). The total phenolic contents (Gallic acid equivalents, mg/g) from the Camellia sinensis extracts were assessed using the formula, C = C₁ × V/m (C = overall phenolic content in mg/g, in GAE (Gallic acid equivalent), C₁ = Gallic acid concentration in mg/ml, V = volume of Camellia sinensis extract in ml, and m = the weight of the Camellia sinensis extract in g). The overall phenolic contents from the Camellia sinensis extracts were compounded as 55.2±1.14mg/g. The amounts of total phenolics varied widely in methanolic tea extracts and ranged from 24.8 to 92.5mg mL^-1. This variation is expected in Camellia sinensis extracts due to other constituents in addition to the variation in the type of phenolic²⁵.

Figure 1: Calibration curve of gallic acid.

Total flavonoid content:

The concentration of total flavonoid content was quantified based on a spectrophotometric assay method. The overall flavonoid compound was assessed from the calibration curve of catechin using the calibration equation of y = 0.003x + 0.0241 (R²= 0.9844) and the overall flavonoid compound was reported as milligrams of catechin equivalents, per gram of dry weight sample (Figure 2). The total flavonoid contents (catechin equivalents per gram) from the obtained extracts were compounded as 33.88±0.54mg/g. The concentrations of flavonoids in the ethanol extraction of tea leaves were reported in the ranges from 12.3 to 136.3mg/g. The flavonoid content in the tea leaves is influenced by many factors such as geographical origin, soil composition, differences in the composition of different leaves, time of harvesting, postharvest treatments, and physical structure of the different leaves probably influence the composition of tea leaves²⁶.

Figure 2: Calibration curve of Catechin.

HPLC analysis for flavonoid:

HPLC analysis for flavonoid was performed using Shimadzu HPLC. The active ingredients from the obtained extract were separated that adhere to the separation column. Among the various compounds, myricetin has been identified according to the spectrum correlation with standard (myricetin). Retention time and peak area were observed as 1.437and 51.3 respectively (Figure 3). HPLC techniques are the most preferred method reveal the presence of various flavonoids in tea extract²⁷. HPLC method is sensitive, selective and provide acceptable precision, accuracy, and linearity²⁸.

Figure 3: HPLC analysis of crude extract for flavonoid.

DPPH radical capture activity:

The antioxidant compounds present in the crude extract decrease the absorbance during the incubation time. The radical capture capacity in this investigation was read after 30 min incubation time. The antioxidant properties obtained for the extract were found to vary from 14.16 to 81.66% (Figure 4). The peak antioxidant activity was recorded at 200μg of sample concentration. The value (IC₅₀) for the Camellia sinensis extract was 63.12mg/mL, which was comparatively higher than the IC₅₀ (21.8 µg/ml) of ascorbic acid.

Figure 4: Free radical scavenging activity of crude extract.

Antibacterial activity:

The antimicrobial activity of the methanolic extract against bacteria was measured using the disc-diffusion method. The zone of inhibition around the filter paper disk due to the antibacterial activity of the extract was measured after 24h of the incubation period (Table 1). Significant antibacterial activity was observed against E.coli, Klebsiella sp., Mycobacterium philei, and Streptococcus sp. The highest zone of inhibition, 18 mm was observed with 1000 µL crude methanol extract against Mycobacterium philei. The zone of inhibition was not recorded against Bacillus sp., and Salmonella typhi. A similar result except for Salmonella typhi was reported by Friedman et al.²⁹.

Table 1: Antibacterial activity of tea extract

S. No.	Bacteria	Zone of Inhibition (mm)
S. No.	Bacteria	1000 μg	500 μg	250 μg	125 μg
1.	Bacillus sp.	-	-	-	-
2.	E.coli	15	15	13	12
3.	Klebsiella sp.	13	12	12	10
4.	Mycobacterium philei	18	17	17	15
5.	Salmonella typhi	-	-	-	-
6.	Streptococcus sp.	15	12	11	10

Antifungal activity:

The antimicrobial activity against fungi from the obtained extract was measured using the well-diffusion method. A zone of inhibition around the well was measured after 7 days of the incubation period (Table 2). Significant antifungal activity was observed against Aspergillus flavus, Collectotrichum sp., Mucor sp., and Penicillium sp. The highest zone of inhibition, 15 mm was observed with 1000 µL crude methanol extract against Aspergillus flavus and Mucor sp. The zone of inhibition was not recorded against Aspergillus niger. The antimicrobial activity of Camellia sinensis was highly susceptibile³⁰. On other hand, the prolonged incubation period made all the fungal strains to show resistance to the extract.

Table 2: Antifungal activity of tea extract

S. No.	Fungus	Zone of Inhibition (mm)
S. No.	Fungus	1000 μg	500 μg	250 μg	125 μg
1.	Aspergilus flavus	15	12	11	10
2.	Aspergillus niger	-	-	-	-
3.	Collectotrichum sp.	10	09	08	06
4.	Mucor sp.	15	12	11	09
5.	Penicillium sp.	10	09	08	07

CONCLUSION:

Camellia sinensis is a potential source for many biologically active compounds of flavonoids and polyphenols and exhibits antioxidant, antibacterial, and antifungal activity. Identifying the unique component for each activity may explore the Camellia sinesis as a natural source to treat and prevent several chronic diseases, especially cardiovascular diseases and cancer.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

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Received on 07.05.2024 Revised on 11.09.2024

Accepted on 06.12.2024 Published on 12.06.2025

Available online from June 14, 2025

Research J. Pharmacy and Technology. 2025;18(6):2475-2479.

DOI: 10.52711/0974-360X.2025.00353

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