Quality Standardization of Ethanol Extract of Tea Leaves (Camellia sinensis L.) through Determination of Total Flavonoid Levels as Antidiabetic Erza Genatrika a, Warsinah b, Hanif Nasiatul Baroroh b a University of Muhammadiyah Purwokerto. Jl. Raya Dukuhwaluh PO. Box 202 Purwokerto. 53182. Indonesia (Email: erzagenatrika@gmail.com) b University of Jenderal Soedirman. Jl. Dr. Soeparno 53122. Purwokerto. Indonesia ABSTRACT Tea (Camellia sinensis L.) leaves in the form of steepings have been used by many people as traditional medicine to cure diarrhea, high cholesterol, hypertension, and diabetes mellitus. However, there was no scientific study about ethanol extract of tea leaves as antidiabetic. Furthermore, the traditional medicine should be useful and fulfill the standard of quality and safety. The aim of this research was to find out the quality of extract through total flavonoid levels and antidiabetic activity of ethanol extract of tea leaves. The determination of total flavonoid levels was done by hydrolysis method. Then, antidiabetic activity test of ethanol extract of tea leaves with the doses of 0.0675 g/200 g BW, 0.10125 g/200 g BW, and 0.135 g/200 g BW which were given orally to Wistar male white mouse using glucose tolerance method. Blood sample was taken from lateral vein of the tail and then the blood glucose level was measured using Gluco Dr every 30 minutes for a period of two hours. The result of the research showed that the total flavonoid levels in in ethanol extract of tea leaves as much as 8.497 ± 0.422 µg/ml and ethanol extract could reduce blood glucose level in rats that induced by glucose. The administration of ethanol extract of tea leaves with dose of 0.0675 g/200 g BW had the highest percentage of decrease in blood glucose level (26.67%), followed by doses 0.10125 g/200 g BW (24.04%) and 0.135 g/200 g BW (19.72%). Key words: antidiabetic, diabetic mellitus, flavonoid, standardization, tea leaves. INTRODUCTION Indonesia is a tropical country with a diverse plant species are about 40.000 species of plants. Nearly 1.000 species of them used as traditional medicine. Empirically communities have used traditional medicine to cure various diseases including diarrhea, headache, cholesterol, and triglycerides, high blood pressure, gastrointestinal infections, cancer, and diabetes mellitus (Herawati, 1997). Diabetes mellitus is a metabolic disease of carbohydrate metabolism disorders which characterized by hyperglycemia and glucosuria. The prevalence of diabetes mellitus in Indonesia from time to time is increase. The number of patients with diabetes mellitus in Indonesia in 1995 about 4.5 million people, then in 2025 is expected to increase to 12.4 million people with diabetes mellitus. One of the herbs that can be used for the treatment of diabetes mellitus is the tea (Camellia sinensis L.) leaf. Chemical content in them are polyphenols, methylxanthine, amino acids, organic acids, carotene, carbohydrates, proteins, lipids, chlorophyll which acts as an antibacterial, anti- 69
inflammatory, antihypertensive, antihiperkolesterol, antimigrain, antidepressants, antispasmodics, anticancer, antioxidant, and antidiabetic (Duke, 2008; Maulana, 2008). Steeping tea leaves are given orally to rat at doses 25x the normal human dose (1.35 g / 200 g BB) can provide a hypoglycemic effect after 1 hour treatment. Furthermore, leaf tea may lower blood glucose levels in people with diabetes mellitus and several studies in Japan reported that people with high comsumption of tea lowers the risk of diabetes mellitus as much as 33%. Until now, this has never been done research on quality standardization of ethanol extract of tea leaves by total flavonoid levels related to its potential as an antidiabetic. It is necessary to quality standardization of extracts and test of antidiabetic activity in experimental animals so that obtained standardized extract which has antidiabetic effects. EXPERIMENTAL 2.1 Materials Tea leaves taken from Wonosobo (PT Plantation of Tambi), male Wistar rats aged 2 to 3 months weighing 150 to 200 g, NaCMC, glucose, glibenclamide, AICl 3, aquadest, ethanol, concentrated hydrochloric acid, magnesium P, hexamethyltetraamine, acetone, ethyl acetate, methanol, glacial acetic acid, and quersetin. 2.2 Preparation of Extracts Tea leaves are dried and powdered. Five hundred grams of dry powder are extracted with the maceration until all the powders are submerged (2L ethanol) for 24 hours. Ethanol filtrate from this beaker glass then filtered, subsequently conducted remaceration 2 times (2L ethanol) each of them for 24 hours. Then filtered, squeezed and the result of first filter mixed with the others. The essence of ethanol was evaporated, until obtained viscous ethanol extracts of tea leaves (Anonymous, 1985). 2.3 Identification of Flavonoids Ethanol extract ± 2ml, added with 0.1 grams of magnesium P and 10 drops of concentrated hydrochloric acid, if happened the change of orange to red until red to purple it indicated flavonoids (Anonymous, 1995). 2.4 Quality Standardization of Extract Standard solutions of flavonoids (quersetin) with a concentration series 5; 7.5; 10; 12.5; 15; 17.5 µg/ml measured at maximum wavelength, so it is obtained regression equation y = a + bx from the correlation of standard solution concentration with the absorbance. Total flavonoid levels of ethanol extract of tea leaves is determined through the hydrolysis process. Extracts are used in hydrolysis process equivalent with 200 mg of simplicia as much as 21.08 mg. Then, the result of solution from hydrolysis process is measured its absorbance with the 70
spectrophotometer UV-VIS at wavelength of maximum (370 nm). Determination of total flavonoid levels is repeated three times (replication). Futhermore, it is calculated the level of flavonoids by using the standard curve from flavonoid standard solutions (quersetin) (Anonymous, 2000). 2.5 The Timing of Administration of Glucose as Diabetogen There are 6 male rats were fasted for 12-18 hours and still being given the drink ad libitum, subsequently divided into 2 groups. Group 1 is burdened glucose orally after 30 minutes and 60 minutes for group II that are calculated from administration of extract etanol of tea leaves (1.35 g/200 g BB) orally with volume giving half of the volume should be. The most optimum time for administration of glucose that produce the smallest value of AUC. 2.6 Antidiabetic Test Thirty of male rats were divided into 5 groups, that are group of positive control (glibenclamide in NaCMC with a dose of 0.9 mg/200 g BB), negative control (NaCMC), test group at a dose of 0.0675 g/200 g BB, 0.10125 g/200 g BB, and 0.135 g/200 g BB are administered orally. After treated, all groups then are given glucose orally at a dose of 2 g/kg with time intervals based on preelimenary experiments. The blood is taken from a vein lateral at minute 0; 30; 60; 90; 120 that are calculated after administration of glucose orally and measured blood glucose levels using device of Gluco Dr. Then, calculated the value of AUC and %DBGL from each group, subsequently data are tested statistically by using normality test of Kolmogorov Smirnov, ANOVA test or Kruskal Wallis test, Post Hoc Tukey HSD test with a confidence level of 95%. RESULTS AND DISCUSSION Tea leaves as much as 20 kg conducted sorting wet, then dried in the sun with a black cloth as a cover in order to reduce the change or damage the chemical content present in tea leaves (Anonymous, 1985). Simplicia of tea leaves have been produced ± 3.7 kg. Simplicia then being crushed and 500 grams conducted maceration, viscous extract that has been obtained as much as 52.7 g. Tea leaf extract which was originally colored dark green then change color into red or reddish orange. According to Robinson (1995) red color was formed due to the reduction of flavonoids by Mg and concentrated HCl. Determination of total flavonoid levels are one of the chemical standardization to the quality of the ethanol extract of tea leaves. Extracts are used equivalent with 200 mg as much as 21.08 mg. Then do the hydrolysis by adding HCl, heksametiltetramina and acetone. The purpose of 71
hydrolysis is to separate aglycone with sugar so that target compound can be separated and identified (Mursyidi, 1997). Filtrate from result of hydrolysis (brownish red) has performed fractionation by adding distilled water and ethyl acetate. The addition of ethyl acetate aims to attract flavonoid compounds that are contained in extracts. Ethyl acetate fraction (yellow) has been added AlCl 3 in glacial acetic acid 5% (v/v methanol) and wait 30 minutes, then the absorbance is measured by using spectrophotometry UV-VIS at a maximum wavelength (370 nm). The total flavonoid levels in the ethanol extracts can be calculated by inputting data of sample absorbance into value of Y on linear regression equation that is obtained from the flavonoid standards (quersetin) is y = 0.0344 + 0,0475x with linearity of 0.9976. Calculation results in a complete total flavonoids shown in Table 1. Table 1. Calculation of total flavonoid levels of ethanol extract of tea leaves Replication Value (µg/ml) ±SD Value (mg/kg) ±SD 1 8.75 11.08 2 8.01 10.14 3 8.73 11.05 Mean 8.497 ±0.422 10.757 ±0.534 The level of total flavonoids on average in the extract is 8.497 ± 0.422 µg/ml are equivalent to 10.757 mg/kg. Results of this study are non-specific parameters of an extract as bioactive compounds that are known to be responsible for the pharmacological effects that can be quantified and compared with previous studies that showing ethanol extract of tea leaves fulfill the quality extracts. According to Bhagwat et al. (2003) suggested flavonoid levels in tea extract ranges are from 0.7 to 2453.8 mg/kg. Furthermore, Princen et al. (1998) stated that the green tea flavonoid levels in the range of 0.0 to 304.7 mg/g, whereas black tea had levels of flavonoids in the range 0.0 to 108.8 mg/g. Research conducted by Samman et al. (2001) showed that the extract of tea leaves had a flavonoid levels of 0.84 to 9.32 mg/100 g. Levels of flavonoids from the results of this study different with previous research, this was because the quality and quantity is strongly influenced by genetic diversity factors and environment in where to grow such as soil, biotic factors, nutrition, water, temperature, light quality, light intensity, altitude grows, and harvest time. The amount of decrease in the most of blood glucose levels which are based on the value AUC 0-120. The smallest of AUC 0-120 value indicates the greatest of hypoglycemic effects. From the results of glucose loading time orientation have obtained AUC 0-120 showing that are the most effective time to do 30 minutes after administration of the test preparation. This is proven by a smaller 72
AUC 0-120 value was 13.665 minute.mg.dl -1 than compared to 60 minutes after administration of the test preparation as shown in Table 2. Table 2. Area Under Curve (AUC 0-120 ) of blood glucose levels on the orientation of administration of ethanol extract of tea leaves AUC N 0-120 (minute.mg.dl -1 ) I II 1 13980 13605 2 13020 14655 3 13995 14745 Mean±SD 13665±456.12 14335±517.49 Information: N : The number of test animals (N=3) I : Treatment loading glucose after 30 minutes II : Treatment loading glucose after 60 minutes Measurement of blood glucose levels are started at minutes 0 up to 120 minutes every 30 minutes. Results of blood glucose levels of rats as shown in Table 3. Table 3. The average of rat blood glucose levels (mg/dl) in each control and treatment groups. Group The Average of Blood Glucose Level (mg/dl) ± SD (n=6) 0 30 60 90 120 I 122.17±9.7 126.7±13.8 113.7±4.8 105.7±13.9 100±11.1 II 138.5±7.7 192.8±26.6 181.3±17.9 136.7±10.6 119.3±10.3 III 111.5±11.1 132.17±11.7 118±3.2 110.17±10.5 103.7±9.3 IV 119±9.4 137.17±4.0 120.3±4.5 116.17±5.2 106.3±4.0 V 124.7±17.0 137.5±11.5 129.17±10.9 128±13.0 114.3±9.6 Information : G : Group I : Positive controls with glibenclamide treatment (0.09 mg/200 g BB) II : Negative control with treatment of Na-CMC 0.5% III : Treatment of ethanol extract of tea leaves with a dose 0.0675 g/200 g BB IV : Treatment of ethanol extract of tea leaves with a dose 0.10125 g/200 g BB V : Treatment of ethanol extract of tea leaves with a dose 0.135 g/200 g BB Widyaningsih et al. (2004) reported normal blood glucose levels ranging from 50-135 mg/dl. Based on Table 3, group I (positive control) showed normal blood glucose levels are <135 mg/dl, group II (negative control) showed that blood glucose levels were abnormal every time measurement. Furthermore, data of blood glucose levels were calculated value of Area Under Curve (AUC) from 0 up to 120 minutes (AUC 0-120 )(Figure 1). 73
Fig 1. Curve average of blood glucose levels (mg/dl) versus time (minutes) at each control and treatment groups. AUC 0-120 value was used as an illustration of antidiabetic effects of tea leaves ethanol extract. The value of AUC 0-120 is getting smaller, so the effect of antiabetic is getting bigger. Negative control group has the biggest AUC 0-120 value and the positive control group has the lowest AUC 0-120 value. Test group that has the lowest AUC 0-120 value indicated by the group that was treated with a dose of 0.0675 g/200 g BB (Table 4). Table 4. Curve average of blood glucose levels (mg/dl) versus time (minutes) at each control and treatment groups Group AUC±SD (minute mg/dl) Positive control (Glibenklamid) 13532.5±423.46 Negative control (NaCMC) 19207.5±148.11 Extract treatment at a dose 0.0675 g/200 g BB 14065±377.05 Extract treatment at a dose 0.10125 g/200 g BB 14590±163.40 Extract treatment at a dose 0.135 g/200 g BB 15420±297.29 AUC 0-120 values were analyzed statistically. Data analysis begun with Kolmogorof-Smirnov normality test with a confidence level of 95% and obtained significance value> 0.05 of all the groups, which meant that the data were normally distributed. Furthermore, data were analyzed by one-way ANOVA showed that F count (333.963)> F table (2.75) (p<0.05), it meant average of AUC 0-120 value from five groups was significantly differences, then proceeded with post hoc Tukey HSD test. In the post hoc Tukey HSD test, all groups showed significance <0.05 then the one group with the others group there were significantly differences. This suggest that a dose level of ethanol extract of tea leaves were very influential on antidiabetic effects. Each treatment groups were then calculated with % DBGL (Decrease Blood Glucose Levels) [Table 6]. AUC 0-120 value inversely proportional with value of % DBGL. AUC 0-120 value is getting smaller. % DBGL value is getting greater. 74
Table 5. The result of statistical analysis of AUC 0-120 value using Tukey HSD Post Hoc test with a level of 95% Between group Value p Statistics Test Results I-II 0.0001 Significantly different I-III 0.039 Significantly different I-IV 0.0001 Significantly different I-V 0.0001 Significantly different II-III 0.0001 Significantly different II-IV 0.0001 Significantly different II-V 0.0001 Significantly different III-IV 0.043 Significantly different III-V 0.0001 Significantly different IV-V 0.001 Significantly different Information : I : Positive controls with glibenclamide treatment (0.09 mg/200 g BB) II : Negative control with treatment of Na-CMC 0.5% III : Treatment of ethanol extract of tea leaves with a dose 0.0675 g/200 g BB IV : Treatment of ethanol extract of tea leaves with a dose 0.10125 g/200 g BB V : Treatment of ethanol extract of tea leaves with a dose 0.135 g/200 g BB Table 6. Percentage Decrease of Blood Glucose Levels (% DBGL) of the control and treatment groups Group % PKGD±SD Positive control (Glibenclamide) 29.55±1.9 Negative control (NaCMC) 0 Extract treatment at a dose 0.0675 g/200 g BB 26.77±1.8 Extract treatment at a dose 0.10125 g/200 g BB 24.04±0.7 Extract treatment at a dose 0.135 g/200 g BB 19.72±1.3 Group III (dose 0.0675 g/200 g BB) has the highest ability to reduce the blood glucose levels as much as 26.77 ± 1.8%, then compared with a dose of 0.10125 g/200 g BB and 0.135 g/200 g BB. However, the effect of antidiabetic is lower than the positive control group (glibenclamide) which has DBGL as much as 29.55± 1.9%. Based on normality test of % DBGL is obtained of the data abnormally distributed then continued with Kruskal Wallis test, it is gained the significance <0.05 then there is a difference from five of treatment groups. It shows each group with different doses very influential on antidiabetic effects. One of causes that improvement of blood glucose levels in diabetic rats was ability of flavonoids stimulate pancreas to produce more insulin as well as glibenclamide. CONCLUSIONS Ethanol extract of tea leaves fulfill the quality of extract with total flavonoid levels that is obtained as much as 8.497 ± 0.422 µg/ml and has a antidiabetic activity in male Wistar rats. Dose 75
of ethanol extract of tea leaves were effective in lowering blood glucose levels are 0.0675 g/200 g BB with DBGL as much as 26.77%. REFERENCES Anonymous (1985) simplisia preparation method. Ministry of Health of the Republic of Indonesia, Directorate General of Drug and Food, Jakarta. Anonymous (1995) Materia medika Indonesia Volume VI. Ministry of Health of the Republic of Indonesia, Directorate General of Drug and Food, Jakarta, p 337. Anonymous (2000) Parameter of general standard of drugs plant extract. Ministry of Health of the Republic of Indonesia, Directorate General of Drug and Food, Jakarta. Bhagwat et al. (2003) Composition of tea: comparison of black and green tea. J Agricultural Research Service 1. Duke (2008) Phytochemical and ethnobotanical databases, http:/www.ars-grin.gov/duke [online]. Herawati (1997) Biomolecular approach to new drug discovery from natural resources in Indonesia perspective. Cermin Dunia Farmasi 31:9-11. Maulana M. (2008) Recognize of diabetes melitus: practical guide of handle diabetes disease, Publisher Kata Hati, Yogyakarta. Mursyidi A. (1997) Analysis of secondary metabolites, Gadjah Mada University Press, Yogyakarta. Princen et al. (1998) No effect of consumption of green and black tea on plasma lipid and antioxidant levels and on LDL oxidation in smokers. Journal of The American Heart Association 833-841. Robinson T. (1995) Organic ingredients of high plant, Publisher ITB, Bandung. Samman et al. (2001) Green tea or rosemary extract added to foods reduces nonheme-iron absorption. American Journal Clinical Nutrition 18:607-612. 76