Bioactive compounds, antidiabetic and antimicrobial potential of pinang seeds extract (Areca catechu l.)

Diabetes mellitus is a chronic metabolic disorder affecting people of all ages. The critical aspect of fruits is that rich sources of antioxidants may act in combination with each other phytochemicals to provide their protective effect. Pinang ( Areca catechu L.) fruit is edible as the local indigenous plant from West Irian Jaya (Papua) Indonesia. This study was aimed at investigating the biologically active compounds of seeds, fruits, and leaves, the α-glucosidase inhibitory and antibacterial activity of seeds of Pinang against Gram-positive bacteria Staphylococcus aureus ATCC 25923, and Gram-negative bacteria Escherichia coli ATCC 25922. Pinang fruits were extracted by using the maceration method and ethanol solvent. Identify the chemical compounds in seeds by GC-MS technique, test to a-glycosidase inhibitory effect was measured with spectrophotometric. Well, the diffusion method was employed in evaluating the antimicrobial property of extracts. The evaluation of the bioactive compound of Pinang fruits revealed the presence of Vitamin E (0.20%). The inhibition of α-glucosidase of seeds extract of IC 50 values was 82.74 ppm, and the global standard was 0.34 ppm. This study confirmed that Pinang seeds contain glucosidase activity that has the potency to inhibit glucose. The antimicrobial activity was performed against bacteria as it showed zone inhibition. These results indicated that Pinang seed extracts exerted potent inhibitory effects against α-glucosidase and inhibited the proliferation of Gram-negative microorganisms.


INTRODUCTION
Diabetes mellitus (DM) is a metabolic disorder in which a person has abnormally high blood glucose levels.It is a severe, significant chronic with potency life-threatening, in which the pancreas hormone improperly regulates the homeostasis of carbohydrates, which impacts the health status.Blood glucose control is critical for preventing or reversing diabetic complications (Doan et al., 2018;Demir et al., 2021).At least estimated in 2019 that 463 million people have diabetes, projected to reach 578 million by 2030 and 700 million by 2045 (IDF 2019).
In recent years, there has been a resurgence of interest in treating diabetes mellitus (DM) using herbal remedies, mainly because of their non-toxic nature.The World Health Organization has also recommended the assessment of the effectiveness of herbal plants, particularly in cases where safe and modern pharmaceuticals are lacking.Exploring bioactive compounds in plants with potential antidiabetic properties is of utmost importance.It is well known that the presence and effectiveness of these bioactive compounds in plants can vary depending on their location.Many plant species have been documented as having hypoglycemic properties and are traditionally used for preventing and managing diabetes.Despite Sefforts to develop hypoglycemic agents from both natural and synthetic sources, diabetes and its associated complications continue to pose significant medical challenges (Arya et al., 2015;Chen H, 2021;Ansari et al., 2022).Numerous medicinal plants and their products, including active principles and crude extracts, have been reported in the literature for their traditional use in diabetes control (Hegde et al., 2016;Saad B, Kmail A, Haq SZ, 2022;Nabi M, Tabassum N, Ganai BA, 2022).Our ongoing investigation focused on assessing the inhibitory effects of Pinang (Areca catechu L.) plants on the α-glucosidase enzyme.These enzymes play crucial roles in various biochemical processes related to metabolic disorders and diseases, including diabetes.As such, there has been a concerted effort to design efficient αglucosidase inhibitors with promising potential applications (Chipiti et al. 2015; Di Santo MC, D'Antoni CL, Rubio AP, Alaimo A, Pérez OE, 2021).Alpha-glucosidase inhibitors are carbohydrates that function as competitive antagonists for the enzymes necessary for carbohydrate digestion, specifically the α-glucosidase enzymes located in the brush border of the small intestine.These intestinal α-glucosidase enzymes break down oligosaccharides, trisaccharides, and disaccharides into glucose and other monosaccharides within the small intestine.Inhibiting these enzyme systems reduces the rate at which carbohydrates are digested, leading to decreased glucose absorption, as the carbohydrates are not broken down into glucose molecules.The short-term impact of these drug therapies in diabetic patients is a reduction in current blood glucose levels, and the longterm effect is a slight decrease in hemoglobin levels (Hussain et al. 2019: Jeong D, Priefer R, 2022;Schauer et al., 2017).

Regenerate
Plants produce secondary metabolites in the form of phenolic compounds, also known as polyphenols, which are known for their antioxidant properties and, as a result, offer beneficial physiological effects.Polyphenols are among the most common antioxidant phytochemicals, and they are recognized for their ability to quench singlet oxygen and scavenge free radicals, thereby retarding the oxidation of lipids.Previous research has indicated that Areca catechu L. fruits are rich in phenolics and tannins, although their antioxidant activities remain relatively unexplored.In traditional markets in Papua, Pinang seeds are frequently used to prepare beverages like coffee.
Extensive research has been conducted on the antimicrobial properties of specific plant species.The pharmacological properties of phytochemicals suggest their potential for antimicrobial and antifungal effects (Shah et al., 2018).The mechanism through which antioxidants act against bacteria involves damaging the integrity of the cell wall and cell membrane, inhibiting intracellular enzyme activity, increasing the levels of reactive oxygen species (ROS), influencing the expression of associated genes, ultimately leading to bacterial apoptosis (Hu et al., 2019;Zhang et al., 2020; Ren X, An P, Zhai X, Wang S, Kong Q, 2019).These findings offer valuable insights into identifying bioactive compounds within Pinang seeds (Areca catechu L.) using GCMS, and the inhibitory effect on α-glucosidase activity was quantified using spectrophotometry.Furthermore, the research suggests a potential antimicrobial effect against both Gram-positive Staphylococcus aureus ATCC 25923 and Gram-negative Escherichia coli ATCC 25922, as evidenced by the observed zones of inhibition.This study holds the promise of enhancing the utility of Pinang fruits across various domains, with a particular emphasis on their applications within medicine.

Plant Extraction
Dried and fully matured fruits of Areca catechu L., commonly known as Pinang, were sourced from a traditional market in Papua, Indonesia.The extraction process involved soaking the sample in ethanol for a period of 48 hours, followed by subsequent extraction.Chloroform was employed for the extraction process, yielding a chloroform-soluble extract, which was then subjected to centrifugation at 10,000 rpm for a duration of 20 minutes.The resulting clear supernatant oil was subsequently analyzed using GCMS.

Procedures for Gas Chromatography-Mass Spectrometry (GC-MS)
Chemical compound identification was carried out using the SHIMAZU Gas Chromatography 5890-11 from Japan, equipped with a fused GC column composed of polymethyl silicon, specifically OV 101, with dimensions of 0.25 mm x 50 m.The temperature programming ranged from 80°C to 200°C, with an initial hold at 80°C for 1 minute, followed by a rate of increase at 5°C/min, and a final hold at 200°C for 20 minutes.The Flame Ionization Detector (FID) was set at 300°C, while the injection temperature was maintained at 250°C.Nitrogen served as the carrier gas, flowing at a rate of 1 cm³/min, with a split ratio of 1:75.For mass spectrum analysis, a GCMS-QP 2010 Plus instrument from Shimazu Japan was used, featuring an injector temperature of 230°C and a carrier gas pressure of 100 kph.The column had a length of 30 m, a diameter of 0.25 mm, and a flow rate of 50 m/min.The eluents were automatically directed into the Mass Spectrometer, where the detector voltage was set at 1.5 kv, and data were collected at a sampling rate of 0.2 seconds.The Mass Spectrometer was also equipped with a computer-fed Mass Spectra data bank.The centrifuge employed in the study was the HERMCE Z 233 M-Z from Germany.Reagents and solvents, such as ethanol for analytics, were sourced from Merck Germany (Iwu et al., 2018;Shahvar A, Shamsaei D, Saraji M, 2020;Kachangoon R, Vichapong J, Santaladchaiyakit Y, Srijaranai S, 2020).

Assessment of α-Glucosidase Inhibition
The initial inhibition test involved a reaction mixture comprised of 50 µL of 0.1 M Phosphate buffer (pH: 7.0), 25 µL of 0.5 mM 4-nitrophenyl α-D-glucopyranoside, 10 µL of the sample with a concentration of 500 µg mL-1, and 25 µL of α-glucosidase solution.This reagent was incubated at 37°C for 30 minutes, and the enzymatic reaction was terminated by adding 100 µL of 0.2 M Na2CO3 (200 mM).To assess the enzymatic absorbance of each mixture, a spectrophotometric measurement was performed using a microplate reader, recording the absorbance at 400 nm.
The negative control involved the reaction between the substrate and enzyme without the presence of an inhibitor.Simultaneously, a blank solution represented the reaction system where the enzyme and inhibitor were absent.This set of actions was performed in triplicate for accuracy.Acarbose was dissolved in a solution containing buffer and 2 N HCL in a 1:1 ratio and was employed as a positive control, serving as an α-glucosidase inhibitor.The calculation of % inhibition was as follows: The formula for calculating the inhibition percentage is expressed as: % inhibition = [1 -(sample absorbance / control absorbance)] x 100%.The sample concentration and its corresponding percentage of inhibition were then plotted on a graph using the equation Y = a + bx.The term "inhibition activity value" (IC50) was defined as the concentration at which the sample could inhibit 50% of the enzyme's activity.The assessment of enzyme inhibition activities for the α-glucosidase assay was conducted with slight adjustments, as per the work of Yin et al. in 2014.

Determination of Antibacterial Activity
The antibacterial properties of the Pinang extract were assessed through the agar well diffusion method.Escherichia coli ATCC 25922 (Gram-negative) and Staphylococcus aureus ATCC 25923 (Gram-positive) bacteria strains were utilized to evaluate the antibacterial efficacy of these extracts.The extract solution was prepared by dissolving 0.1 g of the extract in 100 mL of solvent, consisting of a mixture of distilled water and absolute ethanol, resulting in a 100 mg/mL concentration.This solution was further diluted to achieve 5%, 10%, and 15% concentrations in distilled water.Subsequently, 25 µL of the extract solution was added to prepared discs measuring 6 mm in diameter, which were placed on Muller-Hilton agar as part of the sensitivity test, followed by the determination of the minimum inhibitory concentration.Chloramphenicol was employed as the positive control, and the inhibition zone produced by the extract was measured after 24 hours of incubation at 37°C (Arvind et al., 2014;Dan et

The Results of Phytochemical Screening of Pinang (Areca catechu L) Plant
Analytical Gas Chromatography-Mass Spectrometry (GC-MS) was employed to determine the phytochemical components within the ethanol extracts of Pinang seeds.The identification of these compounds was confirmed by examining the molecular formula, retention time, and peak area, as illustrated in Figure 2 and detailed in Table 1.

Figure 2: The components in Pinang seeds
There are 30 components metabolites in the results of Pinang seeds extract, using GCMS in  The observed of the ethanol extract Pinang fruits, there were 50 compounds identified.The identification was assured by observing the molecular formula, retention time, and peak area of the data shown in Figure 3 and Table 2.

Figure 3. The components in Pinang fruits
There are 50 component metabolites in the results of Pinang fruits, using GC-MS in Table 2.The identified of the ethanol extract Pinang leaves, there were 50 compounds identified.The observed was assured by observing the molecular formula, retention time, and peak area of the data shown in Figure 4 and Table 3.

Figure 4. The components in Pinang leaves
There are 60 components metabolites in the results of Pinang leaves extract, using GCMS in Table 3: The GC-MS analysis results of the extracts indicated variations in the percentage of each metabolite.The ethanol extracts of Pinang seeds contained thirty different compounds, while the fruits showed the presence of fifty compounds, and the leaves exhibited a total of sixty compounds.A noteworthy bioactive compound with antioxidant properties, Vitamin E, was detected at 0.20% in the fruits.In comparison, the analysis of Pinang leaves revealed a higher concentration of Vitamin E at 1.38%.Each of these bioactive compounds is associated with specific physiological functions.We conducted an α-glucosidase test using the Pinang seeds extract to explore the potential antidiabetic properties.

Alpha-Glucosidase Enzyme Inhibition
Numerous plant-based remedies have gained recognition for their potential in managing degenerative conditions such as diabetes, and indirectly, they serve as a source for developing prospective modern drugs, particularly for addressing hyperlipidemia.Bioactive compounds present in these plants often act as antioxidants, capable of impeding glucose absorption.In vitro analyses have confirmed the α-glucosidase inhibitory properties of ethanolic-maceration extracts.These liver glucosidase inhibitors target -1, 6-glucosidase, a glycogen-debranching enzyme within the liver.By doing so, they reduce the glycogenolytic rate, leading to increased glycogen storage in the liver.Consequently, inhibiting these enzyme systems results in decreased blood glucose levels, representing a short-term effect and a modest reduction in hemoglobin A1c levels.Given the various side effects associated with synthetic glucosidase inhibitors, researchers in the present era have a growing focus on herbal medicines (Papuc et al., 2017).
Therefore, the Pinang seeds extracts (in 80% ethanol) showed a potent α-glycosidase inhibitor in this study.The αglucosidase inhibition of seeds may be related to the bioactive compounds properties of the fruits and leaves of this plant (Yin et al., 2014).The benefit of these inhibitors would retard starch digestion, absorption, and lowering blood glucose levels.IC50 values of α-glucosidase inhibited seeds extract were 82.74 ppm, and the global standard was 0.34 ppm, presented in Figures 5 and 6.This research showed an inhibitory of the α-glucosidase activity of seeds of Pinang.Our results suggest that the extract of Pinang seeds is a potential candidate for developing antidiabetic agents.

Potential Antimicrobial Effect
The data indicates that the extract derived from Pinang seeds has demonstrated potential antimicrobial properties by inhibiting the growth of the Gram-negative bacterium Escherichia coli ATCC 25922, as presented in Table 4.Our findings suggest that the mechanism of action of the Pinang seeds extract against E. coli could involve membrane depolarization and increased membrane permeability, which affect intracellular enzyme activities and elevate intracellular levels of reactive oxygen species (ROS).These alterations may lead to cell apoptosis and, ultimately, the death of the bacteria (Shah et al., 2018;Zhang et al., 2020;Lin et al., 2019).The results of the GC-MS analysis of the seeds, fruits, and leaves of Pinang (Areca catechu L.) plants are essential to determine the organic compounds.Vitamin E in fruits and leaves acts as an antioxidant in this plant.Pinang seeds' concentration of bioactive compounds has potential α-glucosidase inhibited activity, impacting the delayed absorption of carbohydrates and the potent effect on diabetes treatment to increase healthy glucose blood levels.The Pinang seeds formula has the potential antimicrobial activity to inhibit the growth of E.coli bacteria as it showed zone inhibition.The bioactive compounds in this plant extract might prevent diabetes and antimicrobial and may serve as alternative drugs for treating various illnesses in human beings.

CONCLUSIONS AND SUGGESTIONS
The analysis results of bioactive components in seeds, fruits, and leaves of Areca catechu (Areca catechu L.) plants have antioxidant activity.Active components in Areca nut seeds have the ability to inhibit the activity of the enzyme alpha-glucosidase, which affects carbohydrate metabolism and has the potential for alternative treatment of chronic hyperglycemia.Areca nut seeds have antibacterial bioactivity against Gram-negative bacteria E.coli, with an inhibition zone.Further research is needed to determine the benefits of the chemical composition of the Areca nut plant as an alternative medicine to help maintain health.