ISOLATION OF XANTHINE} OXIDASE INHIBITOR AND IMMUNOSTIMULAT COMPOUNDS ROTORVANE ORTHODOX BLACK TEA

• Main Author: Dadan Rohdiana, Raden
• Format: Dissertations
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/79457
• Institution: Institut Teknologi Bandung

High levels of uric acid in the body has the potential to cause gouty arthritis that is a joint disease accompanied by pain that does not allow people with activity. Chronically excessive levels of uric acid in the body can lead to kidney failure. Uric acid is known to affect the immune system and is also a risk factor for cardiovascular disease and diabetes. Based on the etiology, in addition to genetic factors, the main causes of the rise in the levels of uric acid in the body is an abnormal diet, for example too many food rich in purines and less drinking. Currently, there are several types of drugs used to treat hypeturicemia, e.g. allopurinol which inhibit xanthine oxidase activity the enzyme that plays an important role in the formation of uric acid in human body; and probenecid; sulfinpyrazone that is used to increase the excretion of uric acid. However, these drugs are not free from side effects that harm to the body. Allopurinol for example, can cause kidney damage and bone narrow suppression resulting in a deficiency of blood elements that can lead to immune deficiencies. Given the life-threatening effects of hyperuricemia and there is no really safe anti hyperuricemic medication, it is necessary to find alternative drugs to cope with hyperuricemia. Traditionally, people use plants to treat various diseases including hyperuricemia; sidaguri herbs, salam leaf, soursop leaves, and tea leaf are just to name a few. However, traditional medicine use is generally not accompanied by a definite dose and scientific evidence proven experimentally. In this study, tea was selected to be studied further to get the exact dose and the Scientific evidence of its pharmacological properties. Tea is a drink that is consumed by virtually the entire population of the world. In addition to an everyday drink consumed by people and as an antioxidant, as well as to keep the immune system, tea leaves also contain active compounds that are thought to potentially overcome hyperuricemia. Based on the processing, tea is classified into that non enzymatic oxidation tea (green tea), partial enzymatic oxidation tea (oolong tea) and full enzymatic oxidation tea (black tea). Most of the tea produced in Indonesia is a black tea. In general, there are two tea processing system in Indonesia i.e., orthodox system and the new system. Orthodox system consists of two sub-systems, pure orthodox and rotorvane orthodox. The new system also consists of two subsystems, namely Crushing-Tearing-Curling (CTC) system and the Lawrie Tea Processig (LTP) system. Rotorvane orthodox black tea was chosen to be studied in this experiment. There are 15 grades that belong to this tea: Broken Orange Pekoe (BOP), Broken Orange Pekoe Fanning (BOPF), Pekoe Fanning (PF), Dust, Broken Pekoe (BP), Broken Tea (BT), Pekoe Fanning 11 (PF 11), Dust 11, Broken Pekoe 11 (BP 11), Broken Tea 11 (BT 11), Broken Mixed (BM), Pekoe Fanning 111 (PF 111), Dust 111, Bohea Bulu (BBL) and Bohea Tulang (BTL). These black tea grades are classified on the basis of differences in physical appearance that include color, shape, size, and density. Because of lack of pharmacological activity data of the 15 grades of black tea based on the quality type, this study aims to evaluate the potential of the 15 grades of black tea as an antioxidant and its effect against xanthine oxidase activity and effect on the Immune system, followed by the isolation and characterization of the active compounds of the black tea with the most potent pharmacological properties. This research also covered characterization of the 15 grades of black tea, screening for their chemical contents, extraction of the active compounds, guided by pharmacological activities test. This was then followed by the isolation and characterization of isolates of the most pharmacologically potent black tea grade. Characteristics of black tea which included water content, water soluble extract content, total ash, water-insoluble ash, acid insoluble ash content, alkalinity of water-soluble ash, and crude fiber were assessed by using the method specified in SNI 01-1902-1995/1S0-3720 in 2011 about black tea. Chemical content of black tea determined in this study included alkaloids, tannins, quinones, flavonoids, saponins and steroids/triterpenoids in accordance with the method specified in the Indonesian Medical Materia volume Il. Catechins, the main chemical constituents of tea leaves was also detennined in this study. Extraction of active compounds from the 15 grades of black tea was carried out as the traditional use, by brewing in hot water. Pharmacological activities of the 15 grades of black tea studied included antioxidant effects, in vitro effect on xanthine oxidase activity and in vivo immunostimulatory effects. Antioxidant activity was tested by using 1,1-diphenyl-2-pircryl hidrazyl (DPPH) method; antihyperurisemic effect was determined through testing activity against xanthine oxidase (XO). Immunostimulatory effects of the 15 grades of black tea extract were determined through phagocytic activity of Reticulo Edhotelial System (RES) tested using carbon clearance method and the determination of the titer of two cytokines, namely Interferon-gamma (IFN-Y) and Tumor Necrosis Factoralpha (TNF-u) with Enzyme Linked Immunosorbent Assay (ELISA). The extract which showed the highest activity in inhibiting the activity of XO and Immunostimulatory effect was selected for further research i.e., isolation, guided by XO inhibiton activity on each steps of isolation. Characteristics of isolates were determined through assessment of melting point, maximum wavelength (X) with ultraviolet visible spectrophotometry, molecular weight and formulas with mass spectrometry (MS), and the structure of isolates with nuclear magnetic resonance (NMR) spectrometry. Characterization of the 15 grade of black teas which was done according to SNI 01-1992-1995/1SO 3720 2011 showed that all the 15 grade of black teas met the quality criteria with the following results: water content of black tea ranged from 6.54-9.44% with the quality requirements of < 8%; extracted matter in water ranged from 32.87-39.12% with the quality requirements of 32%; crude fiber content ranged from 12.20-18.02% with the quality requirements of 16,5% • total ash content ranged from 5.17-6.37% with the quality requirements of 4-8% • water soluble ash content ranged from 2.89-3.51% with the quality requirements of 1.8%; acid insoluble ash ranged from 0.06-0.28% with the quality requirements of S 1% ; alkalinity levels of ash water soluble ranged from 0.791.41% with the quality requirements of 1-3%. Phytochemical screening revealed the presence of alkaloids, flavonoids, steroids, saponins and triterpenoids classes of compounds in all of the 15 grades of rotorvane orthodox black tea extracts. Catechin levels of the 15 grades of black tea determined ranged from 2.49-10.18% with the highest content of 10.18% found in the PF type black tea, followed by BOPF (10.13%), Dust BOP (7.11 and BT (6.03%) . Results on antioxidant effect tests showed that the highest ability to scavenge DPPH free radicals was shown by PF liquor, with IC50 of 218 pg/mL. This was followed by BOPF liquor (227 ug/mL), Dust liquor (240 yg/mL), BOP liquor (252 gg/mL) and BT liquor (260 gg/mL). Experiment on XO activity showed that the highest XO inhibiting activity was caused by BOP extract with an inhibition percentage of 61.58%, followed by BP extract (60.43%) and PF extract (57.01 at a concentration of 100 pg/mL. In immunostimulatory effect test, the highest phagocytic activity of RES was observed after administration of PF extract with phagocytic index of 1.41, followed by BP extract (1.38), BOP extract (l .36) and Dust extract (1.28) at doses equivalent to 20 mg/mL of black tea in water. Based on the pharmacological action results, PF extract that showed the most potent activity was then chosen for further fractionation, isolation, and characterization of its active compound. Further fractionation of PF extract produced three fractions i.e., n-hexane fraction (0.04%), ethyl acetate fraction (5.85%) and water fraction (86.3%). Through XO activity test, ethyl acetate fraction showed the highest potential in inhibiting the activity of XO with inhibition percentage of 71.38%, followed by water and nhexane fraction with 27.21% and 12:51%, respectively. Allopurinol used as a standard and to validate XO test methods gave the inhibition of XO activity by 93.46%. The ethyl acetate fraction that showed the highest inhibitory effect against XO activity was chosen for further fractionation. Further fractionation of ethyl acetate fraction yielded Il subfractions with weight ranged from 1.21 to vil 4.25%. On the test against XO activity, the subfraction-3 gave the highest effect in inhibiting XO activity (74.96%). This fraction was then further purified. Purification of this subfraction gave four isolates with yellow crystal and hereinafter referred to as A (11.87 mg), B (19.01 mg), C (7.12 mg), and D (6.16 mg) isolates. Based on melting point determination, the four isolates were revealed as pure compounds. Through measurement of melting point, maximum wavelength (X) with ultraviolet visible spectrophotometry, molecular weight and formulas with mass spectrometry (MS), and the structure of isolates by nuclear magnetic resonance (NMR) spectrometry, those four isolates were indicated as kaempferol (isolate A), quercetin (isolate B), luteolin (isolate C) and myricetin (isolate D). These four pure isolates were tested for their effect on the activity of XO, IFN-Y and TNF-u content in vivo in mice. Result on the effect on XO activity showed that the highest inhibition was given by luteolin with IC50 of 5.06 ytg/mL followed by kaempferol (10.24 pg/mL), myricetin (22.72 pg/mL) and quercetin (26.88 gg/mL). The effect of the four compounds on IFN-y and TNF-a content in mice showed that the highest IFN-Y content was observed in the group of mice treated with quercetin (14.45 pg/mL), followed by kaempferol (13.61 pg/mL), luteolin (9,15 pg/mL), and myricetin (9.15 pg/mL). Meanwhile the highest TNF-u content was observed in group of mice treated with luteolin (25.16 pg/mL), followed by quercetin (23.93 pg/mL), kaempferol (21.32 pg/mL), and myricetin (18.00 pg/mL). Based on these experiment results it can be concluded that all of the 15 quality types of black tea tested, met the quality requirements of SNI 01-1902-1995/ ISO3720-2011. Of the 15 quality types of black tea tested, PF, BOP, Dust, and BP have antioxidant properties, inhibit XO activity, and are potent to stimulate immune system. The main chemical compounds which are likely to contribute to the activity of PF are quercetin, kaempferol, luteolin, and myricetin.