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\def\RunningAuthor{Waras Nurcholis}
\firstPage{2032}
\articleType{Original Article}
\receivedDate{05 Sep 2020}
\acceptedDate{08 Oct 2020}
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\title{\textbf{Kinetics of \textbf{\ensuremath{\alpha }}} \textbf{-glucosidase inhibitory activity and phytochemical analysis of \textit{Piper crocatum} Ruiz \& Pav. leaves ethanol extract}}
\author{Muhammad~Alfarabi\textsuperscript{1},  
            Maria~Bintang\textsuperscript{2},  
            Suryani\textsuperscript{2},  
            Mega~Safithri\textsuperscript{2}, 
            Waras~Nurcholis\textsuperscript{*}\textsuperscript{3}~\\[5pt]{\textsuperscript{1}Department of Biochemistry, Faculty Medicine, Christian University of Indonesia, Jakarta 13630, Indonesia}~\\{\textsuperscript{2}Department of Biochemistry, Faculty of Mathematics and Natural Sciences, Bogor Agricultural University, Bogor 16680, West Java, Indonesia}~\\{\textsuperscript{3}Tropical Biopharmaca Research Center, Bogor Agricultural University, Bogor 16680, West Java, Indonesia}}

\begin{abstract}
\ensuremath{\alpha }-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). \textit{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 \textit{P. crocatum }leaves ethanol extract. Phytochemical component of the extract was also analyzed. The \ensuremath{\alpha }-glucosidase inhibitory activity of the \textit{P. crocatum }leaves ethanol extract was examined by reacting its different concentrations with \ensuremath{\alpha }-glucosidase and \textit{p}-nitrophenyl glucopyranoside. Kinetics of the \ensuremath{\alpha }-glucosidase inhibition was determined using a Lineweaver-Burke plot. Phytochemical in the extract was determined using GC-MS. Ethanol extract of \textit{P. crocatum }leaves exhibited moderate \textit{\ensuremath{\alpha }}-glucosidase inhibitory activity compared with acarbose. Phytochemical analyses showed the presence of stilbene, linolenic acid, phenol, phytosteroid, and \ensuremath{\alpha }-tocopherol. The competitive action of \textit{P. crocatum } leaves ethanol extract is due to its inhibitory effects on \textit{\ensuremath{\alpha }}-glucosidase. The stilbene and phenol compounds indicated responsible for anti-diabetic activity from \textit{P. crocatum }leaves ethanol extract. 
\end{abstract}\def\keywordstitle{Keywords}

\begin{keywords}Acarbose,\newline Diabetics,\newline GC-MS,\newline Phytochemical,\newline Piper crocatum
\end{keywords}
\twocolumn[ \maketitle {\printKwdAbsBox}]
\makeatletter{\raggedright\textsuperscript{*}Corresponding Author\par Name:\ Waras~Nurcholis~\\ Phone:\ ~\\  Email:\ wnurcholis@apps.ipb.ac.id
        \par}\vspace*{-11pt}\hrulefill\par{\fontsize{12}{14}\selectfont ISSN: 0975-7538}\par% 
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\section{Introduction}
Diabetes mellitus (DM) is a syndrome metabolite disease with significant mortality and characterized by a high level of blood glucose (hyperglycemia) and disfunction in insulin secretion and/or insulin action \unskip~\citep{911818:20577273}. In 2019, the DM prevalence in the world was an estimated 463 million \unskip~\citep{911818:20577257}. It is also predicted that DM prevalence will reach 578 and 700 million in 2030 and 2045, respectively \unskip~\citep{911818:20577257}. Generally, the three DM types are type I~diabetes with insulin deficiency, type II diabetes with insulin resistance, and gestational diabetes \unskip~\citep{911818:20577356}. Hyperglycemia condition will produce diabetic complications, such as cardiovascular diseases \unskip~\citep{911818:20577268}, chronic kidney disease \unskip~\citep{911818:20577254}, blurred vision \unskip~\citep{911818:20577272}, nephropathy \unskip~\citep{911818:20577260}, anxiety and depression \unskip~\citep{911818:20577263}, and retinopathy \unskip~\citep{911818:20577360}. 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 \unskip~\citep{911818:20577271}. Acarbose is a commercial drug of DM which able to inhibit a-glucosidase. Still, it has several more side effects, such as the liver and abdominal illnesses, diarrhoea, and flatulence \unskip~\citep{911818:20577256}. Thus, the find of novelty a-glucosidase inhibitor with lest side effect is of enormous significance for diabetes management. Recently, several reports have shown that the medicinal plant as a source of a-glucosidase inhibitor \unskip~\citep{911818:20577365,911818:20577361,911818:20577357}. \textit{Piper crocatum} Ruiz \& Pav. is a medicinal plant that belongs to the family Piperaceae and traditionally used to treat diabetes mellitus in Indonesia \unskip~\citep{911818:20577264}. As the reported literature shows, no articles about the influence of different concentrations of \textit{P. crocatum} ethanol extract leaves on a-glucosidase inhibitory, and its phytochemical identification 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 \textit{P. crocatum} ethanol extract leaves.
    
\section{Materials and Methods}
\textbf{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.

\textbf{Plant material and extraction}

The \textit{P. crocatum }was collected from Bogor, West Java, Indonesia, and identified by Dr Mega Safithri. The extraction used the maceration method at room temperature. Briefly, 25 g of \textit{P. crocatum } fresh leaves were macerated using 70\% ethanol (100 ml). After 2x24 h, the solution filtered and then evaporated at 50 \ensuremath{^\circ}C and finally dried using freeze dryer at -50 \ensuremath{^\circ}C and 8 mBar.

\textbf{a-Glucosidase inhibition assay}

The a-glucosidase inhibition of the sample extract and acarbose were measured according to the assay described by \unskip~\citep{911818:20577253}, with modifications. The \ensuremath{\alpha }-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 \textit{p}-nitrophenyl-\ensuremath{\alpha }-D-glucopyranoside (20 mM). After incubated for 5 min at 37\ensuremath{^\circ}C, the mixture was added enzyme solution (500 ml) and incubated again for 15 min at 37\ensuremath{^\circ}C. Finally, the reaction was terminated with 2 ml Na\ensuremath{_{2}}CO\ensuremath{_{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: 
\begin{eqnarray*}\mathrm{inhibition}\;\left(\%\right)=\frac{({\mathrm A}_\mathrm{control}\;-{\mathrm A}_\mathrm{sample})}{{\mathrm A}_\mathrm{control}}\times100 \end{eqnarray*}
where A\ensuremath{_{\ control}} is an absorbance without extract; A\ensuremath{_{\ sample}} is an absorbance with the extract. 

\textbf{The kinetic of } \textbf{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 \textit{p}-nitrophenyl-\ensuremath{\alpha }-D-glucopyranoside (5 {\textendash} 25 mM) for 15 min at 37\ensuremath{^\circ}C. Reactions were stopped, and absorption was measured and plotted by the Libewaver-Burk curve.

\textbf{GC-MS analysis}

Phytochemical analysis of \textit{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 \ensuremath{\mu }m; 5\% diphenyl, 95\% dimethylpolysiloxane). The solution sample (1 ml) injected and used a flow rate of 1 ml/min{\textemdash}the temperature and pressure used of 300\ensuremath{^\circ}C and 10.47 psi, respectively. Helium gas used as the eluent. Mass parameter detection with masses 50 {\textendash} 800 (MS quad temperature of 150-200 \ensuremath{^\circ}C and MS source 250-300 \ensuremath{^\circ}C) were used to detection of compounds. The identification of mass-spectrum GC-MS was evaluated using the database of NIST.
    
\section{Results and Discussion}
a-Glucosidase inhibitory activity of \textit{P. crocatum} ethanol extract leaves (PCE) are illustrated in Figure~\ref{f-7f6d6597f499}.  The concentration PCE of 0.1\% to 1\% have significantly lower inhibition on a-glucosidase activity compared to 1\% acarbose (80.97\%) at \textit{p} \textless\ 0.05. 


\begin{table*}[!htbp]
\caption{\boldmath {Metabolite identified in the \textit{Piper} \textit{crocatum }ethanol extract by GC-MS} }
\label{tw-3c4b425997aa}
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\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack No & \cAlignHack Retention Time (min) & \cAlignHack Area (\%) & \cAlignHack Metabolite\\
\tblmidrule 
\cAlignHack 1 &
  \cAlignHack 12.07 &
  \cAlignHack 6.13 &
  \cAlignHack Linolenic acid\\
\cAlignHack 2 &
  \cAlignHack 24.89 &
  \cAlignHack 12.19 &
  \cAlignHack Phenol\\
\cAlignHack 3 &
  \cAlignHack 26.12 &
  \cAlignHack 4.52 &
  \cAlignHack Phytosteroid\\
\cAlignHack 4 &
  \cAlignHack 27.20 &
  \cAlignHack 44.69 &
  \cAlignHack Stilbene\\
\cAlignHack 5 &
  \cAlignHack 36.46 &
  \cAlignHack 1.65 &
  \cAlignHack a-Tocopherol\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

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\caption{\boldmath {a-glucosidase inhibition activity of \textit{Piper crocatum} ethanol extract (PCE) and acarbose at different concentrations. Different letter on the top of the bar chart showed a significant difference at p \textless\ 0.05}}
\label{f-7f6d6597f499}
\end{figure*}
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\caption{\boldmath {Lineweaver-Burk plots of a-glucosidase inhibition at different concentrations of \textit{p}-nitrophenyl-\ensuremath{\alpha }-D-glucopyranoside (PnP-\ensuremath{\alpha }-G) by \textit{Piper crocatum} ethanol extract}}
\label{f-00e1978cc412}
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\caption{\boldmath {GC-MS chromatogram of \textit{Piper crocatum} ethanol extract}}
\label{f-9c2fcdec6ab6}
\end{figure}
\egroup
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 in the rising concentration of PCE. 

These data were coherent with the results of previous work on the beverage formula of P.\textit{crocatum}\unskip~\citep{911818:20577266,911818:20578109}

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 \unskip~\citep{911818:20577369}. The inhibition kinetics of PCE on a-glucosidase is shown in Figure~\ref{f-00e1978cc412}.  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 \unskip~\citep{911818:20577259}. Therefore, the higher concentration of PCE is required to decrease the hyperglycemia condition.

The chromatogram of GC-MS of the PCE presented in Figure~\ref{f-9c2fcdec6ab6}.  The metabolite was identified based on a comparison between NIST library with the mass spectra of the compounds; the five metabolites were identified and characterized (Table~\ref{tw-3c4b425997aa}). 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 \unskip~\citep{911818:20577355,911818:20577258}.
    
\section{Conclusions}
We concluded that \textit{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 \textit{P. crocatum} ethanol extract leaves may be a role for its anti-diabetic properties.
    
\section{Acknowledgement}
The authors thank to Laboratory Tropical Biopharmaca Research Center, Bogor Agricultural University, Indonesia, for providing facilities.

\textbf{Conflict of Interest}

The authors declare that they have no conflict of interest for this study.

\textbf{Funding Support}

The authors declare that they have no funding support for this study.
    

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