Kinetics of α -glucosidase inhibitory activity and phytochemical analysis of Piper crocatum Ruiz & Pav. leaves ethanol extract

α-Glucosidase is an enzymes group that playing essential roles in the digestion of polysaccharide. Inhibitor of a-glucosidase can decrease polysaccharide digestion rate and therefore plays a significant function in preventing the development of diabetes (type 2). Piper crocatum Ruiz & Pav. is an essential herb applied traditionally in Indonesia to treat diabetes mellitus. This work evaluated the a-glucosidase inhibitory activity of P. crocatum leaves ethanol extract. Phytochemical component of the extract was also analyzed. The α-glucosidase inhibitory activity of the P. crocatum leaves ethanol extract was examined by reacting its different concentrations with α-glucosidase and p-nitrophenyl glucopyranoside. Kinetics of the α-glucosidase inhibition was determined using a Lineweaver-Burke plot. Phytochemical in the extract was determined using GC-MS. Ethanol extract of P. crocatum leaves exhibited moderate α-glucosidase inhibitory activity compared with acarbose. Phytochemical analyses showed the presence of stilbene, linolenic acid, phenol, phytosteroid, and α-tocopherol. The competitive action of P. crocatum  leaves ethanol extract is due to its inhibitory effects on α-glucosidase. The stilbene and phenol compounds indicated responsible for anti-diabetic activity from P. crocatum leaves ethanol extract.

Acarbose, Diabetics, GC-MS, Phytochemical, Piper crocatum A α-Glucosidase is an enzymes group that playing essential roles in the digestion of polysaccharide. Inhibitor of a-glucosidase can decrease polysaccharide digestion rate and therefore plays a signi icant function in preventing the development of diabetes (type 2). Piper crocatum Ruiz & Pav. is an essential herb applied traditionally in Indonesia to treat diabetes mellitus. This work evaluated the a-glucosidase inhibitory activity of P. crocatum leaves ethanol extract. Phytochemical component of the extract was also analyzed. The αglucosidase inhibitory activity of the P. crocatum leaves ethanol extract was examined by reacting its different concentrations with α-glucosidase and pnitrophenyl glucopyranoside. Kinetics of the α-glucosidase inhibition was determined using a Lineweaver-Burke plot. Phytochemical in the extract was determined using GC-MS. Ethanol extract of P. crocatum leaves exhibited moderate α-glucosidase inhibitory activity compared with acarbose. Phytochemical analyses showed the presence of stilbene, linolenic acid, phenol, phytosteroid, and α-tocopherol. The competitive action of P. crocatum leaves ethanol extract is due to its inhibitory effects on α-glucosidase. The stilbene and phenol compounds indicated responsible for anti-diabetic activity from P. crocatum leaves ethanol extract.

INTRODUCTION
Diabetes mellitus (DM) is a syndrome metabolite disease with signi icant mortality and characterized by a high level of blood glucose (hyperglycemia) and disfunction in insulin secretion and/or insulin action (Wilkinson et al., 2019). In 2019, the DM prevalence in the world was an estimated 463 million (Saeedi et al., 2019). It is also predicted that DM prevalence will reach 578 and 700 million in 2030 and 2045, respectively (Saeedi et al., 2019). Generally, the three DM types are type I diabetes with insulin de iciency, type II diabetes with insulin resistance, and gestational diabetes (Zhang et al., 2019). Hyperglycemia condition will produce diabetic complications, such as cardiovascular diseases (Khouja et al., 2019), chronic kidney disease (Brugnara et al., 2018), blurred vision (Sayin et al., 2015), nephropathy (Elsayed et al., 2019), anxiety and depression (Khalighi et al., 2019), and retinopathy (Gargiulo et al., 2004). Therefore, prevent the occurrence of hyperglycemia condition is an alternative strategy inpatient diabetes management.
The inhibition of the a-glucosidase enzyme (EC 3.2.1.20) is commonly applied to cure diabetes through a mechanism by reducing polysaccharides hydrolysis (digestion) so that glucose absorption is low and consequently does not occur hyperglycemia (Sangeetha and Devi, 2018). Acarbose is a commercial drug of DM which able to inhibit aglucosidase. Still, it has several more side effects, such as the liver and abdominal illnesses, diarrhoea, and latulence (Godbout and Chiasson, 2007). Thus, the ind of novelty a-glucosidase inhibitor with lest side effect is of enormous signi icance for diabetes management. Recently, several reports have shown that the medicinal plant as a source of a-glucosidase inhibitor (Nurcholis et al., 2018;Magaji et al., 2020;Trinh et al., 2020). Piper crocatum Ruiz & Pav. is a medicinal plant that belongs to the family Piperaceae and traditionally used to treat diabetes mellitus in Indonesia (Sa ithri and Fahma, 2008). As the reported literature shows, no articles about the in luence of different concentrations of P. crocatum ethanol extract leaves on a-glucosidase inhibitory, and its phytochemical identi ication has been presented. This property was evaluated in this study. Thus, the objective of this work is to investigate the mechanism of a-glucosidase inhibitory activity and phytochemical analysis from P. crocatum ethanol extract leaves.

Reagents and chemicals
Acarbose and p-nitrophenyl-a-D-glucopyranoside were purchased from Sigma (St. Louis, MO, USA). All other chemicals were of analytical grade.

Plant material and extraction
The P. crocatum was collected from Bogor, West Java, Indonesia, and identi ied by Dr Mega Sa ithri. The extraction used the maceration method at room temperature. Brie ly, 25 g of P. crocatum fresh leaves were macerated using 70% ethanol (100 ml). After 2x24 h, the solution iltered and then evaporated at 50 • C and inally dried using freeze dryer at -50 • C and 8 mBar.

a-Glucosidase inhibition assay
The a-glucosidase inhibition of the sample extract and acarbose were measured according to the assay described by (Dubey et al., 2017), with modi ications. The α-glucosidase (0.5 mg) dissolved in 0.1 M phosphate buffer (pH 7.0) containing bovine serum albumin (100 mg). Before used, the enzyme solution was diluted 25 times, with 0.1 M phosphate buffer pH 7.0. The sample (20 ml) at various concentrations (0.1% to 1.0%) in DMSO was mixed with 980 ml buffer phosphate pH 7.0 (0.1 M) and 500 ml p-nitrophenyl-α-D-glucopyranoside (20 mM). After incubated for 5 min at 37 • C, the mixture was added enzyme solution (500 ml) and incubated again for 15 min at 37 • C. Finally, the reaction was terminated with 2 ml Na 2 CO 3 solution (200 mM), and absorbance at 400 nm was measured with UV-Vis spectrophotometer. The blank was used DMSO with no extract. The a-glucosidase inhibition of the sample extract was calculated as follows: where A control is an absorbance without extract; A sample is an absorbance with the extract.

The kinetic of a-glucosidase inhibition
The mechanism of kinetic inhibition of the sample, in the presence or absence, on a-glucosidase was determined by using assay method in Section a-glucosidase inhibition assay with serial concentration p-nitrophenyl-α-D-glucopyranoside (5 -25 mM) for 15 min at 37 • C. Reactions were stopped, and absorption was measured and plotted by the Libewaver-Burk curve.

GC-MS analysis
Phytochemical analysis of P. crocatum ethanol extract leaves was performed using the GC-MS instrument. GC-MS equipped with capillary column HP-5 (Agilent 19091J-433; 0:25 mm x 30 m x 0.25 µm; 5% diphenyl, 95% dimethylpolysiloxane). The solution sample (1 ml) injected and used a low rate of 1 ml/min-the temperature and pressure used of 300 • C and 10.47 psi, respectively. Helium gas used as the eluent. Mass parameter detection with masses 50 -800 (MS quad temperature of 150-200 • C and MS source 250-300 • C) were used to detection of compounds. The identi ication of massspectrum GC-MS was evaluated using the database of NIST.

RESULTS AND DISCUSSION
a-Glucosidase inhibitory activity of P. crocatum ethanol extract leaves (PCE) are illustrated in Figure 1. The concentration PCE of 0.1% to 1% have signi icantly lower inhibition on a-glucosidase activity compared to 1% acarbose (80.97%) at p < 0.05. The percentage of inhibition value of PCE ranged from 1.29% to 40.80%. These results indicated that the percentage of a-glucosidase inhibition increased   The enzyme kinetics of the a-glucosidase in the presence of the PCE (inhibitor/extract) was determined from Lineweaver-Burk curve. The Lineweaver-Burk plot is a general technique for evaluation of the enzyme kinetics (Rouzbehan et al., 2017). The inhibition kinetics of PCE on a-glucosidase is shown in Figure 2. The line showed the same in Y-intercept between a-glucosidase with extract (PCE) and without extract. This result indicated that the PCE exhibited a competitive type of a-glucosidase inhibition. This mechanism action of competitive is similarly with acarbose on a-glucosidase inhibition (Prasad et al., 2019). Therefore, the higher concentration of PCE is required to decrease the hyperglycemia condition.
The chromatogram of GC-MS of the PCE presented in Figure 3. The metabolite was identi ied based on a comparison between NIST library with the mass spectra of the compounds; the ive metabolites were identi ied and characterized (Table 1). The stilbene was the primary compound on PCE (44.69%) followed by phenol (12.19%), linolenic acid (6.13%), phytosteroid (4.52%), and a-tocopherol (1.65%). Among the metabolites, stilbene and phenol were reported to have a-glucosidase inhibitory activity (Zhang et al., 2017;Figueiredo-González et al., 2019).

CONCLUSIONS
We concluded that P. crocatum ethanol extract leaves possess of a-glucosidase inhibitory activity with competitive mode for kinetic enzyme mechanism. The metabolite of stilbene and phenol of P. crocatum ethanol extract leaves may be a role for its anti-diabetic properties.