Dipeptidyl peptidase IV Inhibitory Activity of Fraction from White Tea Ethanolic Extract (Camellia sinensis (L.) Kuntze) ex vivo

Background: Treatment for type-2 diabetes mellitus focuses on the incretin hormone, Glucagon-Like Peptide-1 (GLP-1). However, it has a short half-life. Inhibition of the enzyme Dipeptidyl peptidase IV (DPP IV) required maintaining the active form of GLP-1. Based on the previous studies on the highest activity of DPP IV enzyme inhibition of white tea extract, this study conducted on the fraction of white tea extract using rat blood serum (ex vivo). Objectives: This study aims to evaluate the inhibitory activity of fraction from white tea extract. Methods: White tea leaves extracted with ethanol. The inhibitory activity determined by using rat blood serum as DPP IV enzyme source (ex vivo), AMC (7-amino 4-methyl coumarin) as fluorescence substrate of DPP IV and sitagliptin as the standard reference. The the cleavage of fluorescence reaction product observed by a microplate reader with λex = 360 nm and λem=460 nm at 37 oC. Data expressed as mean ± SD and the IC50 value determined by nonlinear regression curve and fit using Prism Graph 7. Result: methanol fraction (250 μg/mL) has the greater inhibition percentage (50.487%), and the fraction of n-hexane and ethyl acetate are 32.417% and 36.541%. The methanol fraction IC50 value is 227 μg /mL. Conclusion: The methanol fraction is the most active to inhibit DPP IV enzyme.


INTRODUCTION
Treatment for type-2 diabetes mellitus depends on the incretin hormone.Glucagon-Like Peptide-1 (GLP-1) and Glucose-Dependent Insulinotropic Polypeptide (GIP) are the main incretin hormones secreted in the intestine.GLP-1 plays a role in the body's metabolism, such as insulin secretion, increases the mass of β-pancreatic cells, glucagon secretion, reduces gastric emptying and satiety. 1 However, GLP-1 tends with a half-life about 1-2 min due to degradation by the Dipeptidyl peptidase IV (DPP IV). 2 The inhibition of DPP IV required to maintain endogenous GLP-1 inactive form and longer half-life. 3DPP IV inhibition may also reduce the side effects of hypoglycemia, weight gain, and gastrointestinal disorders. 4,5Sitagliptin has reported as a potential inhibitor of DPP IV, but this treatment has side-effects on the upper respiratory tract, and the price is relatively high and difficult to obtain. 6PP IV inhibitor from plants expected to reduce side effects, cheaper, and easier to produce.The active compounds in plants have mechanisms in various forms, pancreatic β-cell functions, and glucose absorption on the incretin pathway.Some plants reported as anti-hyperglycemia. 7,8Studies on different types of tea as anti-diabetic that has done on green tea, black tea and oolong tea, but white tea has not known well.White tea (Camellia sinensis (L.) Kuntze) is a tea bud that still rolls processed without fermentation. 9,10hite tea has the highest polyphenol content and highest of catechin derivatives compared with other types of tea. 11,12,13The differences of bioactive components of each plant provides different antidiabetic activity mechanisms.The content of flavonoids (quercetin and catechin), tannins and polyphenols, have a role in the mechanism of decreasing blood glucose levels against animal testing through inhibition of antibacterial processes. 14he study conducted by Elya reported that white tea extract had inhibition of DPP IV enzyme with the highest percentage of inhibition (30.57%) compared to the other plant extracts. 15Based on the high content of polyphenols and their inhibitory activity against DPP IV enzyme as proposed by Elya, it is necessary to conduct further research on the effect of ethanol extract of Camellia Sinensis (L.) Kuntze.Blood serum used as a source of method by increased polarity of the solvent, i.e., n-hexane, ethyl acetate, and methanol.One kilogram of viscous extract weighed and fractionated by separation funnel, by means of extract dissolved with distilled water until completely dissolved.The extract solution added with n-hexane (1:1), gently shake and released the gas and then kept until it completely separated.The n-hexane and water fractions are separated and collected and then repeated.The non-mixed fraction in the n-hexane solvent was added with ethyl acetate and then carried out the procedure as in n-hexane.Furthermore, the non-mixed fraction in the ethyl acetate solution added to methanol, shaken and then accommodated.Each fraction obtained was then concentrated by a rotatory evaporator until a thickened fraction was obtained.

Phytochemical screening
Parameters examined on phytochemical identification were Flavonoids, alkaloids, tannins, saponins, steroids and triterpenoids by using the specific reagent.

Blood Serum preparation
Blood serum prepared from Sprague-Dawley (SD) rats and obtained from the tail.Previously, tail disinfected with 96% alcohol.The blood sample stored in eppendorf for one h then centrifuged by using Scan Speed, 4000 rpm for ten min.The supernatant (blood serum) pipetted and inserted into a new eppendorf, labeled and stored at -20°C.

Determination of inhibition percentage
The test performed by using DPP IV Spectrofluorometry Activity Assay Kit from Sigma Aldrich (St. Louis, MO, USA) with modified. 17The DPP IV enzyme from the blood serum hydrolyzes the fluorogenic substrate bonds (H-Gly-Pro) conjugated to the AMC group (H-Gly-Pro-AMC) to release the fluorescence product, i.e., the 7-Amino-4-Methyl Coumarin group (AMC) on λ ex = 360 nm and λ em = 460 nm, at 37 0 C. The inhibitory activity of DPP IV enzyme analyzed by calculating the percent inhibition of each fraction in various concentrations.Tests carried out using microwell plate 96 specifically for fluorescence.The parameters observed in this trial is the amount of fluorescence product released on each sample test then calculated the percentage of inhibition.Data expressed as mean ± SD and the IC 50 value determined by nonlinear regression curve and fit using Prism Graph 7.

Phytochemical screening
The results of the classification of the compounds presented in Table 1.The identification of class compounds indicated by the color changes generated by the addition of specific reagents.
DPP IV enzyme and it has been reported has the identical enzyme specificity and no differences in hydrolysis compared to the kidneys. 16

Plant material
Buds of Camellia sinensis (Theaceae) were collected from the Research Center for Tea and Quinine, Gamboeng, Indonesia.Plant was authenticated in the Indonesian Institute of Sciences, Bogor, Indonesia.

Chemical used
All chemicals and Dipeptidyl peptidase IV assay kit purchased from Sigma Aldrich (St. Louis, MO, USA).

Plant extraction and fractionation
The white tea leaves powder was weighed 750 g and extracted by reflux method with 70% ethanol (1:10), the temperature maintained 60°C about three h.The filtrate of the first reflux filtered and repeated two times.The filtrate from the reflux and the repetition evaporated at 50°C (MOH, 2000).The ethanol extract fractionated by liquid-liquid partition

Inhibitory activity
The concentration of sitagliptin in the well is 100 mM and generating 93.494% of inhibition.The methanol fraction is more active on inhibiting the DPP IV enzyme with 50.487% of inhibition at 250 μg/mL greater than the n-hexane and ethyl acetate (32.417% and 36.541%).The IC 50 value of methanol fraction is 227 μg /mL and it shown on Table 2. Based on the other studies, the IC 50 value of DPP IV enzyme from green tea and black tea were 5.3 mg/mL and 6.4 mg/mL. 18

DISCUSSION
Phenolic components generally can bind to protein from the enzyme and form enzyme-inhibitor bonds.This condition leads to decrease the activity of the enzyme because of the inability bind its substrate and the enzyme active site has occupied.Based on study, the active compound which plays a role in providing inhibition is catechin (hydrolyzed tannin class).EGCG (Epigallocatechin gallate), which derived from catechin also reported having a mechanism as DPP IV inhibitors. 7However, high concentrations of tannins may provide the experiment bias because of their specific properties that can precipitate proteins.The enzyme is a class of protein that can react with tannins to bind strongly and form a precipitate, so that enzyme activity decreases.DPP IV catalytic structure can be inhibited strongly by the enzyme serine inhibitor with a covalent bond to modify the active site of the enzyme.Substrate specificity of DPP IV in a mammal relatively limited at P 1 position (proline or alanine).However, studies analogous Growth Releasing Hormone (GRH) indicates that the bond residues P 1 were also hydrolyzed such as serine, valine, glycine, leucine although with the low level. 3

CONCLUSION
The methanol fraction of the white tea ethanol extracts is more active to inhibit DPP IV enzyme from blood serum.