Determination of Antioxidant activity Presentation.

A study of antioxidants found in tea leaves and Clitoria ternatea infusion was done by my group .

1. DETERMINATION OF ANTIOXIDANT ACTIVITY OF TISANES OF Clitoria ternatea & BLACK TEA COLLECTED FROM DIBRUGARH, ASSAM   By- ANKUSH ROY ARPAN GOGOI HALEE GOGOI JYOTISHMAN SONOWAL KISHUR BORMON NILUTPAL BHUYAN PARAS HANDIQUE SHIKHAMONI LAHON B. Sc. 6 th Semester Department of Chemistry DHSK College

2. Contents: 1. Introduction 1.1 Reactive oxygen species (ROS) 1.2 Antioxidants 1.3 Natural sources of antioxidants 1.4 Review of literature 2. Objective of the work 3. Materials and Methods 3.1 Materials 3.2 Sample description & Place of collection 3.3 Preparation of tea and clitoria ternatea infusions 4. Results and Discussion 4.1 UV-Visible Spectral Analysis 4.2 DPPH radical scavenging assay 5. Findings 6. Future Scope 7. References

3. Reactive Oxygen Species A type of unstable molecule that contains oxygen and one or more unpaired electron. Easily reacts with other molecules in a cell. A build up of reactive oxygen species in cells may cause damage to DNA, RNA, and proteins, and may cause cell death. Reactive oxygen species are free radicals. Also called oxygen radical.

4. Antioxidants Plays crucial role in cellular protection. Inhibits oxidation. Removes reactive oxygen species (ROS), and neutralizes free radicals. Helps in lowering the risks of chronic diseases like diabetes, cancer, cardiovascular and neurological disorders, and aging. Their accumulation can lead to oxidative stress and subsequent damage to cells. [1]

5. Natural antioxidants, such as tocopherol (vitamin E), rosemary extract, and ascorbic acid (vitamin C) can effectively inhibit oxidation in food products without concerns of toxicity unlike synthetic antioxidants like BHA(Butylated hydroxy anisole), BHT(Butylated hydroxy toluene), TBHQ(Tertiary butylhydroquinine ). Vitamin E Vitamin C TBHQ BHT BHA Natural sources of antioxidants

6. Chen and Ho [2] conducted a study on the antioxidant properties of polyphenols extracted from green and black teas. They focused on extracting plant phytochemicals such as theaflavins from black tea and studied their scavenging of DPPH radicals. Yashinet et al. [3] conducted a study to evaluate the antioxidant properties of different types of tea using modern techniques. The results showed the antioxidant activity of tea is influenced by several natural polyphenols, such as catechins, oxyaromatic acids, tannins, flavonols , thearubigins , and theflavins . The antioxidant activity of different tea grades follows the order : green > oolong > black > Pu-erh. Kamkaen and Wilkinson [4] conducted a study on antioxidant activity of Clitoria ternatea petal extracts. It was demonstrated that a water extract of C. ternatea has stronger antioxidant activity , as assayed by the reduction of DPPH, than that of an ethanol extract. Review of literature

7. Objective of the work We have undertaken to study the antioxidant activities of Clitoria ternatea and Black Tea by DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging assay.

8. Materials and methods Table: 1 Botanical Information of the plants: LOCAL NAME SCIENTIFIC NAME TRADITIONAL USES Saah paat Camellia sinensis Uses as beverage, beauty and skincare, hair care, medicinal purposes Aparajita Clitoria ternatea Used for its medicinal properties, natural dye, culinary applications Materials

9. Apparatus and Reagents Apparatus: UV-vis spectrophotometer (Lab-Junction) Reagents: Methanol ( SimSon ), DPPH (SRL) Materials used : Micro-pipette ( Kasablanka ), Beakers, Test tubes, Measuring Cylinder. Fig 1(a) : UV-VIS Spectrophotometer Fig 1(b) : Micro-pipette Fig. 1(c) : Prepared DPPH Solution

10. Sample description & place of collection SIP Black Tea obtained from nearby market. Clitoria ternatea Geographical location of Clitoria ternatea selected from Dibrugarh district of Upper Assam of India : Latitude 27.4566184 , Longitude 94.8859126 Preparation of tea and Clitoria t ernatea infusions Water extracts of tea and Clitoria ternatea was prepared by soaking them in hot water (100˚ C) for 10 minutes and then filtered using Whatman No. 1 filter paper. Fig. 2: Preparation of tea and Clitoria ternatea infusions

11. Determination of antioxidant properties of tea and Clitoria ternatea infusion The antioxidant activities of the tea leaves were determined by 2,2-diphenyl-1picrylhydrazyl (DPPH) radical scavenging assay. The DPPH assay has been widely used to test the ability of compounds to act as free radical scavengers or hydrogen donors and to evaluate the antioxidant activity of foods and plant extracts [6,7] . 0.2g made tea was mixed with 100 mL of boiling water for 10 min, with constant shaking and the samples were then filtered through Whatman No. 1 filter paper. However, 4g was mixed with 100 mL of the same in case of fresh Clitoria ternatea . Scavenging of DPPH free radicals by tea samples was determined by slightly modified method [8] using a UV-Visible spectrophotometer (Model: LJ-2373, Make: Lab Junction). 4mg of DPPH was dissolved in 10mL of methanol to prepare a standard solution of DPPH (4000 μg mL -1 ). For the control measurement, 0.2 mL or 200 µL of DPPH solution from this stock solution was made up to 3 mL by adding methanol to make a test solution. The absorbance of the test solution was recorded at 517 nm. All investigated samples were also tested for their absorbance at 517 nm. The radical scavenging activity of the tea samples was studied by adding different concentrations of made tea infusion to 200 μL of DPPH (4000 μg mL -1 ) and the volume was made up to 3 mL by adding methanol. The absorbance was recorded for the sample at 5 sec, 5 min, 15 min, 30 min, and 45 min. The reduced absorbance was measured at 517 nm and was compared with a control of DPPH solution in methanol.

12. % inhibition of DPPH activity = [(A -A x )/A ] ×100 where A is the absorbance of control A x is the absorbance of sample analysed The percentage inhibition of the radicals due to antioxidant properties was calculated by using the following equation - Fig 3 : Before scavenging(control) & after scavenging (tested sample)

13. Results and Discussion UV-VISIBLE SPECTRAL ANALYSIS Fig 4 : DPPH scavenging activity of tea samples at various time intervals

14. Table 1 . Percentage inhibition of DPPH w.r.t. time for SIP Black Tea (10 µL) SIP Black Tea Time Percentage inhibition (%) 10 µL 5 sec 3.98 5 min 5.66 15 min 6.97 30 min 10.08 45 min 12.46 Fig 5 : Graphical representation of DPPH scavenging activity of SIP Black tea (10µL) DPPH radical scavenging assay

15. Table 2 . Percentage inhibition of DPPH w.r.t. time for SIP Black Tea (20 µL) SIP Black Tea Time Percentage inhibition (%) 20 µL 5 sec 6.49 5 min 8.77 15 min 12.14 30 min 16.01 45 min 19.06 Fig 6 : Graphical representation of DPPH scavenging activity of SIP Black Tea (20µL)

16. SIP Black Tea Time Percentage inhibition (%) 30 µL 5 sec 9.63 5 min 10.04 15 min 12.84 30 min 16.16 45 min 22.81 Table 3 . Percentage inhibition of DPPH w.r.t. time for SIP Black Tea (30 µL) Fig 7 : Graphical representation of DPPH scavenging activity of SIP Black Tea (30µL)

17. SIP Black Tea Time Percentage inhibition (%) 40 µL 5 sec 33.24 5 min 37.33 15 min 42.39 30 min 46.65 45 min 49.93 Table 4 . Percentage inhibition of DPPH w.r.t. time for SIP Black Tea (40 µL) Fig 8 : Graphical representation of DPPH scavenging activity of SIP Black Tea (40µL)

18. Table 5. Percentage inhibition of DPPH w.r.t. time for sundried Clitoria ternatea (10 µL) Sundried Clitoria ternatea Time Percentage inhibition (%) 10 µL 5 sec 0.41 5 min 0.95 15 min 2.99 30 min 5.75 45 min 7.64 Fig 9 : Graphical representation of DPPH scavenging activity of sundried Clitoria ternatea (10µL)

19. Table 6. Percentage inhibition of DPPH w.r.t. time for fresh Clitoria ternatea (10 µL) Sundried Clitoria ternatea Time Percentage inhibition (%) 10 µL 5 sec 10.58 5 min 11.72 15 min 14.94 30 min 15.66 45 min 16.44 Fig 10 : Graphical representation of DPPH scavenging activity of fresh Clitoria ternatea (10µL)

20. Table 7 : Percentage inhibition of different concentrations of SIP Black Tea Name of sample Concentration (µL) Time (min) Absorbance of the control Absorbance of the tested sample Percentage inhibition (%) SIP Black Tea 10 45 0.6179 0.5409 12.46 20 45 0.6884 0.5572 19.06 30 45 0.8067 0.6227 22.81 40 45 0.9134 0.4573 49.93 Fig 11 : Comparison of different concentrations of SIP Black Tea

21. Table 8 : Percentage inhibition of 10 µL Clitoria ternatea (sundried and fresh) Name of sample Concentration (µL) Time (min) Absorbance of the control Absorbance of the tested sample Percentage inhibition (%) Sundried(0.2g) 10 45 0.4176 0.3857 7.64 Fresh (4g) 10 45 0.8338 0.6967 16.44 Fig 12 : Comparison of 10 µL Clitoria ternatea (sundried and fresh)

22. Summary of the findings SIP Black Tea and Clitoria ternatea as well shows appreciable antioxidant activity. The percentage of DPPH scavenging ranged from 0.41% to 49.93% for the tea samples. The highest scavenging activity was shown by 40µL SIP Black Tea (49.93%). The radical scavenging depends upon the concentration of the tea samples. We have seen as the concentration increases, DPPH radical scavenging activity increases. However, due to time constraints, we could not analyse the effect of concentration on antioxidant activity above 4 0µL of SIP. The free radical scavenging ability and hence the antioxidant activity of black tea sample is better than the Clitoria ternatea (sundried) infusion as seen from our study (10 µL of 0.2g in 100ml).

23. Future scope of the work From the study we have learnt the method of determination of the antioxidant activity of tea infusion and clitoria tisanes. Along with the DPPH radical scavenging method, Trolox equivalent antioxidant capacity (TEAC) and ferric reducing antioxidant power (FRAP) can also be employed for determining the antimicrobial and antioxidant activities of tea infusions with better yields. We can extend our study to determination of total phenolic content (TPC) and caffeine content in our samples.

24. References [1] Deng, W., Chen, Y., Sun, X. and Wang, L., 2023. AODB: A comprehensive database for antioxidants including small molecules, peptides and proteins. Food Chemistry, 418, p.135992. [2] CHEN, C.W. and HO, C.T., 1995. Antioxidant properties of polyphenols extracted from green and black teas. Journal of food lipids, 2(1), pp.35-46. [3] Yashin , A., Yashin , Y. and Nemzer , B., 2011. Determination of antioxidant activity in tea extracts, and their total antioxidant content. American journal of biomedical sciences, 3(4), pp.322335. [4] N. Kamkaen and J.M. Wilkinson,2009. The antioxidant activity of Clitoria ternatea flower petal extracts and eye gel. Phytotherapy Research 23(11), 1624-1625. [5] Gayan Chandrajith Vidana Gamage, Yau Yan Lim, Wee Sim Choo, 2021. Anthocyanins from Clitoria ternatea flower : Biosynthesis, extraction, stability, antioxidant acitivity , and applications. Frontiers in Plant Science 12, 792303, 2021. [6] Satoh, E., Tohyama , N. and Nishimura, M., 2005. Comparison of the antioxidant activity of roasted tea with green, oolong, and black teas. International journal of food sciences and nutrition, 56(8), pp.551-559. [7] Jayasekera, S., Molan, A.L., Garg, M. and Moughan , P.J., 2011. Variation in antioxidant potential and total polyphenol content of fresh and fully-fermented Sri Lankan tea. Food chemistry, 125(2), pp.536-541. [8] Brand-Williams, W., Cuvelier , M.E. and Berset, C.L.W.T., 1995. Use of a free radical method to evaluate antioxidant activity. LWT-Food science and Technology, 28(1), pp.25-30.