INTRODUCTION:

Plant are widely used in the treatment or prevention of certain diseases or disorders and are considered beneficial to healthcare. The free radicals like superoxide anion, hydroxyl radicals, singlet oxygen caused the damage.¹ Plant antioxidant can protect cells from the damaging effects of ROS. A normal, human ROS induced disease is cancer, cardiovascular diseases, diabetes, swollen inflammation, degenerative illnesses, anaemia and ischemia². Antioxidant therapy has become extremely important for treatment of these diseases. However, factors such as high costs, lack of availability And side effects of synthetic against oxidative stress remain major set-backs. Natural antioxidants have been shown to be openly available, cheaper and abundant of secondary effects in many plant sources³. Many medicinal plants for their antioxidant properties have been investigated. The human body has an intrinsic antioxidant system and this process is the origin of many bio-functions such as anti-mutagene, anti-carcinogenic, and counterfeit responses⁴.

Antioxidants stabilize or disable free radicals often prior to attacking biological cell targets. More than 50% were isolated from or associated with natural sources in clinical studies for anticancer activity⁵. The most abundant polyphenols in plant which reduce the risk of cancer, act against allergies, ulcers, tumours platelet aggregations and are also effective in controlling hypertension are phenolic acids, flavonoids, stilbenes and lignans⁶. Then, a new source for noble antioxidants, particularly those that are safe and affordable for all population groups is needed. Tea, widely used non-alcoholic beverages⁷, is obtained from Camelia sinensis, an evergreen shrub generally trimmed to height of 6 feet, is a reservoir of different chemical constituents⁸. Different degrees of fermentation method are adopted to produce different types of tea like white tea, green tea, black tea, oolong tea which also varies in their chemical constituents⁹. Black tea, being used by most of the population, of our interest of study which is rich in containing polyphenols like theaflavin and thearubigins¹⁰. The limited information about the antioxidant profiling and phytochemical screening encourage us to carry on the study. The present study was designed to investigate the phytochemical components and evaluate the antioxidant activities of black tea extract obtained from the leaves of Camelia sinensis.

MATERIALS AND METHODS:

Plant material:

The fresh green leaves were collected from Ganguram Tea Garden, Darjeeling, fresh tea and authenticated by Dr. Ranjan Gogoi, Scientist D of Botanical Survey of India, India (Ref. No- CNH/TECH. II/2018/27 dated 20th March 2018). The collected plant materials were washed, dried and powdered.

Preparation of black tea extract:

The pulverised crude drug was boiled with water and cooled. Fresh fruit juice 1/3^rd of the quantity of water was mixed with the extract and allowed to be fully fermented in specially designed chamber. After 3 days the fermentation was stopped by pouring ethyl acetate into it and filtered. The extract was lyophilised to get powder extract¹¹.

Drugs and chemicals:

1,1-Diphenyl-2-picryl-hydrazyl (DPPH) was obtained from Sigma Chemicals, USA. nitroblue tetrazolium (NBT), phenazine methosulphate (PMS), reduced nicotinamide adenine dinucleotide (NADH), sodium nitroprusside, napthyl ethylene diamine dihydrochloride, ascorbic acid, trichloroacetic acid (TCA), thiobarbituric acid (TBA), ethylene diamine tetra acetic acid (EDTA), sodium hydroxide (NaOH), hydrogen peroxide (H2O2), butylated hydroxy anisole (BHA), deoxyribose, potassium ferricyanide [K3Fe(CN)6], were purchased from Sisco Research Laboratories Pvt. Ltd., Mumbai, India. All other chemicals were used are of high analytical grade.

Phytochemical screening:

The powdered drug was dissolved in suitable solvent and subjected to qualitative analysis for its chemical constituents according to standard protocols¹²^,¹³.

Test for proteins:

Millon’s test:

Crude extract after mixing with 2ml of Millon’s reagent, white precipitate appeared which turned red upon gentle heating that confirmed the presence of protein.

Ninhydrin test:

Crude extract if boiled with 2ml of 0.2% solution of Ninhydrin, violet colour appeared suggesting the presence of amino acids and proteins.

Test for carbohydrates:

Fehling’s test:

Same volume of Fehling A and Fehling B reagents were mixed. Then 2ml of it was added to crude extract and gently boiled. A brick red precipitate visible at the bottom of the test tube confirmed the presence of reducing sugars.

Benedict’s test:

Crude extract mixed with 2ml of Benedict’s reagent and boiled, a reddish brown precipitate appeared which indicated the presence of the carbohydrates.

Molisch’s test:

Black tea extract mixed with 2ml of Molisch’s reagent and the mixture was shaken intensely. After that, 2ml of concentrated H₂SO₄ was introduced carefully along the side of the test tube. Forming of a violet ring at the interphase indicated the presence of carbohydrate.

Iodine test:

Black tea extract was mixed with 2ml of iodine solution. A dark blue or purple coloration confirmed the presence of the carbohydrate.

Test for phenols and tannins:

The extract was mixed with 2ml of 2% solution of FeCl₃. A blue-green or black colour indicated the presence of phenols and tannins.

Test for flavonoids:

Shinoda test:

The black tea extract was mixed with few fragments of magnesium ribbon and concentrated HCl was added drop wise. Pink scarlet colour visible after few minutes which showed the presence of flavonoids.

Alkaline reagent test:

The extract was mixed with 2ml of 2% solution of NaOH. An intense yellow colour was appeared which turned colourless on addition of few drops of diluted acid which confirmed the presence of flavonoids.

Test for saponins:

The extract was mixed with 5ml of distilled water in a test tube and it was shaken intensely. The appearance of stable foam was taken as an indication for the presence of saponins.

Test for glycosides:

Liebermann’s test:

The black tea extract was mixed with 2ml of chloroform and 2ml of acetic acid. The mixture was cooled in ice. Carefully concentrated H₂SO₄ was added. A colour change from violet to blue to green indicated the presence of steroidal nucleus, i.e., glycone portion of glycoside.

Salkowski’s test:

The extract was mixed with 2ml of chloroform. Then 2ml of conc. H₂SO₄ was added carefully and shaken. A reddish brown colour confirmed the presence of steroidal ring, i.e., glycone portion of the glycoside.

Keller-killiani test:

The extract was mixed with 2ml of glacial acetic acid containing 1-2 drops of 2% solution of FeCl3. The mixture was then poured into other test tube containing 2ml of conc. H₂SO₄. A brown ring at the interphase confirmed the presence of cardiac glycosides.

Test for steroid:

The extract was mixed with 2ml of chloroform and concentrated H₂SO₄ was poured sidewise. A red colour produced in the lower chloroform layer is the indication of the presence of steroids.

Test for terpenoids:

The black tea extract was dissolved in 2ml of chloroform and evaporated to dryness. To this, 2ml of concentrated H₂SO₄ was added and warmed for about 2 minutes. A greyish colour confirmed the presence of terpenoids.

Test for alkaloids:

The extract was mixed with 2ml of 1% HCl and heated gently. Mayer’s and Wagner’s reagents were then added to the mixture. Change in colours of the resulting precipitate was taken as evidence for the presence of alkaloids.

Assessment of Antioxidant properties:

DPPH radical scavenging activity:

DPPH radical scavenging activity was measured using the described method with some modifications¹⁴. At least 2.8ml of test solution or standard ascorbic acid (in methanol), at different concentrations and 0.2ml of DPPH (100μM in methanol) were mixed and incubated at 37°C for 30 min and absorbance of the resulting solution was measured at 517nm using spectrophotometer. The percentage inhibition of DPPH radical was calculated by comparing the results of the test with the control (not treated with extract) using the following formula.

Percentage inhibition = [(C-T)/C]x100

Where,

C = Absorbance at 517 nm of the control and

T = Absorbance at 517 nm of the test

Nitric oxide scavenging activity:

In aqueous solution at physiological pH, sodium nitropruside generate nitric oxide, which interacts with oxygen to produce nitrite ions and which can be measured by Griess reaction. At first 0.1ml of 10mM sodium nitropruside was mixed with 1ml of test solution or standard ascorbic acid at different concentrations in phosphate buffer (100mM, pH-7.4) and the final mixture was incubated at 25°C for 150 min. After incubation, 1 ml mixture was taken, added 1ml of Griess reagent (a 1:1 mixture of 0.1% naphthyl ethylene diamine dihydrochloride in water and 1% sulphailamide in 2% O-phoshoric acid) and kept in the dark at room temperature for 10 min. Absorbance of the chromophore formed by the diazotization of nitrite with sulfanilamide and subsequent coupling with napthyl ethylene diamine dihydrochloride was read at 546nm and percentage inhibition was calculated by comprising the results of the test with the control using the above mentioned formula ⁶.

Superoxide radical scavenging activity:

Superoxide anion scavenging activity was measured according to the described method with some modifications. All the mentioned solutions were prepared in 100mM phosphate buffer (pH- 7.4). Reaction mixture contains 1ml of nitroblue tetrazolium (NBT, 156μM), 1ml of reduced nicotinamide adenine dinucleotide (NADH, 468μM) and 3ml of the test solution or standard ascorbic acid at different concentrations. The reaction was initiated by adding 100 μl of phenazine methosulphate (PMS, 60μM) and incubated at 25°C for 5 min. Then absorbance was measured at 560nm and the percentage inhibition was calculated by using the above mentioned formula¹⁵

Hydroxyl radical scavenging activity:

The scavenging capacity for hydroxyl radical was measured according to the modified method¹⁴ Stock solutions of EDTA (1mM), FeCl3 (10mM), ascorbic acid (1mM), H2O2 (10mM), and deoxyribose (10mM), were made in distilled deionized water. The assay was performed by adding 0.1ml EDTA, 0.01ml of FeCl3, 0.1 ml of H2O2, 0.36ml of deoxyribose and 1.0ml of the extract or standard ascorbic acid (at different concentrations in water) followed by the addition of 0.33 ml of phosphate buffer (50mM, pH-7.4) and 0.1ml of ascorbic acid in sequence. The mixture was then incubated at 37°C for 1 h. About 1ml of the incubated mixture was mixed with 1ml of TCA and 1ml of 0.5% TBA (in 0.025 M NaOH containing 0.025% BHA) to develop the pink chromogen which was measured at 532 nm. The hydroxyl radical scavenging activity of the extract is reported as % inhibition of deoxyribose degradation and is calculated as mentioned above.

Reductive activity:

Reducing power of the samples was determined on the basis of the ability of their antioxidants principles to form coloured complex with potassium ferricyanide, TCA, ferric chloride (FeCl3). About 1ml of different concentration of the extract and standard ascorbic acid were mixed with potassium ferricyanide (2.5ml, 1%) and 2.5ml of phosphate buffer (pH- 6.6). The mixture was incubated at 50°C for 20 min add 2.5ml of TCA (10%) and centrifuge at 3,000rpm for 10 min. 2.5ml of supernatant was mixed with 2.5ml of water and 0.5ml of FeCl3 (0.1%) and absorbance was measured at 700nm. Higher absorbance of the reaction mixture is an indication of higher reducing power.

RESULTS:

Phytochemical screening:

The phytochemical screening revealed the presence of proteins, carbohydrates, cardiac glycoside, tannins, flavonoids and phenolic compounds.

Antioxidant assessment:

All the data are given as the mean±SEM of three individual measurements. 50% inhibitory concentrations (IC50) were calculated by plotting the data in the graph as concentration versus percentage inhibition using Graph Pad Prism software, version 4.03.

DPPH scavenging activity, nitric oxide scavenging activity, superoxide scavenging activity, hydroxyl scavenging activity and reductase test of black tea extract found significant (p<0.05) when compared with standard ascorbic acid as shown in fig.1-5

Fig. 1: 1, 1-diphenyl-2-picrylhydrazil (DPPH) scavenging activity of black tea extract and the standard ascorbic acid.

Fig 2: Nitric oxide (NO) scavenging activity of black tea extract and the standard ascorbic acid.

Fig. 3. Superoxide (SO) s scavenging activity of black tea extract and the standard ascorbic acid.

Fig. 4. Hydroxyl Radical scavenging activity of black tea extract and the standard ascorbic acid.

Fig.5: Reductive test of black tea extract and the standard ascorbic acid.

Table 1: Mean ± SEM of IC₅₀ of black tea extract and standard ascorbic acid.

Name of the Experiment	Mean ± SEM of IC₅₀
Name of the Experiment	BTE	Standard
DPPH scavenging activity	146.3±1.76	22±2.51
SO scavenging activity	195.7±3.52	56.31±3.94
Reductive test	131.6±2.58	74.88±3.43
Hydroxyl radical scavenging activity	115.8±3.37	54.97±3.07
NO scavenging activity	26.40±2.75	18.67±1.42

The comparison of IC₅₀ value also confirm the better response of BTE than standard.

Fig. 6: The IC₅₀ values of DPPH scavenging activity of extract and standard, superoxide scavenging activity of extract and standard, reductive test of extract and standard, hydroxyl radical scavenging activity of extract and standard, nitric oxide scavenging activity of extract and standard

DISCUSSION:

The phytochemical screening revealed the presence of phenolic compounds and flavonoid which has a beneficial effect on lifestyle disorder like diabetes mellitus, cancer and anti-microbial disease¹⁶. Presence of flavonoid can reduce the oxidative stress of human body ¹⁷. The black tea extract shows a good capacity to scavenge all the tested reactive species and all the IC₅₀ values (mean±SEM) for individual experiments being found at the μg/ml level. Oxidative stress refers to a situation where in the production of oxidants exceeds the capacity to neutralize them, leading to damage to cell membranes, lipids, nucleic acids, proteins and constituents of the extracellular matrix such as proteoglycans and collagens¹⁸. DPPH is a potent stable nitrogen compound. It does not generate in our body. DPPH is stable free radical. So, it reacts to the compound which give positive ion. In the experiment when DPPH reacts with extract the pink colour is vanished after incubation. So, the extract of the black tea should have an anti-oxidant activity. The IC₅₀ value of DPPH scavenging activity is 146.3±1.76 of extract and 22±2.51 of standard which is quite similar with the previous study¹⁹. The most reactive nitrogen species (RNS) of the body is nitric oxide which is generated in-vitro from sodium nitro-prusside when reacts with oxygen. Nitric oxide is a cellular conduit of different cell functions. It acts as a signal molecule in immune, nervous and vascular systems. When it reacts with superoxide, the toxicity of nitric oxide increases significantly to form the highly reactive peroxynitrite anion (ONOO−). The IC₅₀ value of Nitric Oxide scavenging activity is 26.40±2.75 of extract and 18.67±1.42 of standard. The extract inhibits nitrite formation by directly competing with oxygen in the reaction with nitric oxide²⁰. The most important ROS produced by the inflammatory cells is superoxide and hydroxyl radical. Membrane bound NADPH oxidase reduce the molecular oxygen to produce the superoxide anions which intern converted to hydrogen peroxide and hydroxyl radical as well as by using different enzymatic reaction in our body. The IC₅₀ value of Superoxide scavenging activity is 195.7±3.52 of extract and 56.31±3.94 of standard²¹. Hydroxyl radicals are the key active oxygen species causing lipid peroxidation and enormous biological damage. When the test extract was added to the reaction mixture, it removed hydroxyl radicals from the sugar and prevented their degradation. The IC₅₀ value of hydroxyl radical scavenging activity is 115.8±3.37of extract and 54.97±3.07 of standard. The IC₅₀ value of reductive test is 131.6±2.58 of extract and 74.88±3.43 of standard. The reductive ability of the extract in a dose-dependent manner, which indicates the good reducing power as compared to standard ascorbic acid. The present study proved that the extract has good scavenging activity of ascorbic acid as standard.

CONCLUSION:

The antioxidant activity was studied by in vitro DPPH (1,1-diphenyl –2 picryl hydrazyl) free radical, hydroxyl radical, superoxide radical, nitric oxide scavenging assay and reductive activity using ascorbic acid as standard drugs. The percentage of inhibition was on a concentration dependent manner in all the models. In conclusion, the presence of flavonoid in the plant could responsible for observed antioxidant activity. In the context of phytomedicine and herbal preparations (wherein large numbers of compounds are present as a mixture) may lead to the development of novel drugs of 21^st century. The study was also an attempt towards the search of such lead molecules from the plant drugs, which could contribute a little in the development of some newer molecules, having therapeutic value.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 11.12.2019 Modified on 26.01.2020

Research J. Pharm. and Tech. 2020; 13(10):4539-4544.

DOI: 10.5958/0974-360X.2020.00800.8