Chisocheton pentandrus (Blanco) Merr. leaf as a potential antioxidant and α-glucosidase inhibitory agent

Indonesia is one of the countries with the highest diversity of plants worldwide, including the Chisocheton genus. The Chisocheton genus has been widely used in traditional medicine due to a variety of compounds with different activities, such as antiplasmodial, antiviral, and anti-inflammatory. The focus of this research is to determine the antioxidant, anti-diabetic, and anti-bacterial activity of Chisocheton pentandrus leaf extract from the Bali Botanic Garden plant collection, as well as the chemical compounds contained in the extract. The antioxidant activity test was carried out by capturing DPPH-free radicals, the antidiabetic test was carried out by assaying its inhibitory activity toward α-glucosidase enzyme, the antibacterial test was carried out using the agar diffusion method, while LC-MS/MS was used to determine the compounds contained in the extract. The results showed that the C. pentandrus leaf methanol extract showed the highest inhibitory activity as an antioxidant and inhibitor of the α-glucosidase enzyme in the very strong category; however, the extract is not effective in inhibiting bacterial growth.


Introduction
Because of their low cost and dependability, plants with therapeutic properties have been identified as a supplemental healthcare system and a beneficial treatment for certain patients [1].Among natural sources, the use of medicinal plants as a source of basic materials, enriched raw extracts, and blends in medical medications is considerable.Several thousand plants with medicinal characteristics have been recognized to treat various kinds of illnesses [2].
Chisocheton pentandrus (Blanco) Merr. is a large tree that can reach a height of 25 m [3].This plant grows in the tropics and subtropics parts of the world, such as Bhutan, Malaysia, India, Indochina, Indonesia, Nepal, Southern China, and Papua New Guinea [4].This species has long been employed as a laxative, medicinal, cosmetic ingredient, and as a toxin in fish [5].Secondary metabolites of this genus include different alkaloids, sterols, and terpenoids having biological activities such as antiplasmodial [6], antiviral [7], and anti-inflammation [8].Although there have been numerous studies on the usage of C. pentandrus, none have looked at its anti-diabetic potential.Thus, this research aimed to look at the antioxidant, anti-diabetic, and antibacterial properties of C. pentandrus leaf extract.

Extract preparation
The leaves of C. pentandrus were gathered from the plant collection of the Bali Botanic Garden that had been identified by the Registration Unit with Accession Number E 19860246 and Garden Number XIV.A. 43.This plant was obtained in Sumatra's Jambi Province.After being washed with water, the leaves were air-dried for two weeks.Simplicia was powdered and put through a 40-mesh filter.Simplicia powder is then kept from sunlight in a dry and closed container.Furthermore, the leaf powder was then extracted in three different solvents: hexane, ethyl acetate, and methanol.

Total phenolic and flavonoid content
The Folin-Ciocalteu method was used to figure out the total phenolic content of the extract [9] and gallic acids as a reference.The results were expressed in milligrams of gallic acid equivalent per gram of dry weight.
The total flavonoid component content was determined using the aluminum chloride colorimetric test procedure [10].The calibration line of the regression formula was adopted to convert the levels of flavonoid components to quercetin equivalent (mg/g extract).

Free radical scavenging action of DPPH
The antioxidant activities of C. pentandrus leaf extracts were evaluated using the method described by Yen and Chen [11].A spectrophotometer (UV-VIS Thermo Genesys 30) at 517 nm was used to assess the sensitivity of the reaction fluid.The experiment was done three times with ascorbic acid as the reference standard chemical.Through a quadratic modeling estimation (Figure 1), the ideal dose required to scavenge 50% of the radicals generated by DPPH (IC50) is established.A lower IC50 indicated greater antioxidant activity.The data obtained were analyzed using two-way ANOVA and continued with the Duncan test.

α-Glucosidase inhibition
The inhibitory impact of α-glucosidase was assayed according to Kim et al. [12].The inhibitory impact on α-glucosidase activity was revealed by measuring the quantity of ρ-nitrophenol emitted at 400 nm.The inhibitory percentage was calculated by combining antioxidant activities and its α-glucosidase inhibition.The IC50 was calculated using logarithmic regression analysis using the principal inhibitory values.Quercetin was utilized as a control.This assessment was conducted in triplicate and the results were reported as mean values.The data obtained were analyzed using two-way ANOVA and continued with the Duncan test.
Pathogenic bacterial isolates were cultured in 50 mL of nutrient broth media (Merck) for 18 hours at 37°C.A total of 200 L of each pathogenic bacterial culture with 10 5 CFU/mL was placed on the surface of the nutrient agar (NA) plate (15 cm diam.).The extracts were made at 75, 150, 300, 600, 1,250, 2,500, 5,000, and 10,000 g/mL in dimethyl sulfoxide (DMSO).Tetracycline (500 g/mL) was used as a positive control, while DMSO without extract was used as a negative control.Before being placed on NA plates, 15 L of each concentration was spread over a Fuoroni disc (6 mm diam.).Antimicrobial activity was detected after 48 hours of incubation at 37°C.The diameter of the inhibitory zone was utilized to assess inhibition activity.

Identification of compounds contained in the active extract
Liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) (Water Acquity UPLC I-Class and XEVO G2-XS QTOF) was used to identify the chemicals in the active extract.Condition of measurement: column (ACQUITY UPLC® BEH C18 1.7 m 50 mm) with H2O + 0.1% FA as mobile phase (eluent) for solvent A and ACN + 0.1% FA for solvent B. With a complete scan at 100-1200 (m/z), a 1 L sample is injected.

Contents of total phenolic and flavonoid compounds
Through Folin-Ciocalteu procedure, when phosphomolybdic and phosphotungstic acids are reduced by phenolic compounds, they create a complex bluish-purple molybdenum-tungsten alloy [15,16].Furthermore, gallic acid is utilized as a standard since it is stable and a hydroxybenzoic acid derivative [17].
Plant extracts can be used to synthesize phenolic-group compounds with bioactive characteristics.The amount of phenol in each species and its parts differs.Table 1 indicates the total phenol and flavonoid concentrations of C. pentandrus leaf extracts following maceration in hexane, ethyl acetate, and methanol.The overall phenol concentration was expressed as mg GAE/g dry extract or gallic acid equivalent.C. pentandrus leaf methanol extract had the highest total phenolic content (58.55±0.15mg/g EQ), followed by the hexane and ethyl acetate extracts, at 6.56±0.07 and 3.93±0.18mg/g EQ, respectively.
The total phenolic content varies depending on the plant species and the kind of solvent utilized.Previously, [18] revealed that phenol content in various plant sections extracted with different solvents found that plant extracts extracted with highly polar solvents had the greatest concentration of phenol components.
Correspondingly, TFC levels in methanol extract were greater than in other solvents.TFC in methanol, hexane, and ethyl acetate extracts of C. pentandrus leaves was 156.17±0.99,57.16±1.27,and 51.34±1.03,respectively (Table 1).Flavonoid concentrations are widely different in each part of the plant, as demonstrated by the findings for Morinda citrifolia and Aloe vera [19].Based on these results, 4 methanol is the best solvent for macerating C. pentandrus leaves to gain high amounts of phenols and flavonoids.

DPPH free radical scavenging activity
In this study, the methanol extract of C. pentrandus leaves exhibited very high activity (33.638 μg/mL) against DPPH free radicals (Table 2).On the contrary, the extracts of hexane and ethyl acetate exhibited moderate activity (IC50 values of 188.12±0.68 and 237.31±3.73μg/mL, respectively).Plant phenol content generally correlates with antioxidant activity.Antioxidant activity increases with increasing phenol content [20].Phenolic compounds can inhibit free radicals by stabilizing the radicals through proton donation.This stable radical is produced as a result of electron delocalization brought on by aromatic ring movement [21,22].

α-Glucosidase Enzyme Activity Inhibition Test
Many endeavors have been undertaken to develop safe and efficient α-glucosidase inhibitors from naturally occurring substances to construct optimal diabetic diets.As presented in Table 3, C. pentandrus methanol extract showed strong inhibitory action against α-glucosidase, with an IC50 of 7.31±0.03μg/mL.Antioxidants are compounds that can lessen or completely eradicate the harm that oxidation causes; consequently, they can protect the body against various ailments, such as diabetes and its complications [23].A perfect anti-diabetes molecule should have both antioxidant properties and α-glucosidase inhibitor functions [24].To sum up, the leaf extracts from C. pentandrus exhibited strong antioxidant and α-glucosidase inhibitory properties.There was a relationship between the inhibitory action against α-glucosidase, TPC, TFC, phytochemical content, and antioxidant activity test results.According to [25], the existence of phenolic groups, which can donate hydrogen atoms to free radicals to reduce their responsiveness, is linked to the antioxidant and anti-diabetic properties of the plant extract.Furthermore, [25,26] stated that by employing hydrophobic connections, phenolic compounds can also operate as α-glucosidase selective competitors, an enzyme that breaks down carbs.These carbohydrates are not hydrolyzed directly into glucose molecules.

Antibacterial activity assay
The disc diffusion experiment of C. pentandrus leaf extract revealed that it had no antibacterial action against all targeted bacteria.In contrast, [27] previously claimed that C. pentandrus is efficient against a wide variety of bacteria.
The extract was shown to be ineffective in inhibiting the spread of bacteria, which might be attributed to alterations in the chemical constituents of the extract.The number of phytochemical compounds varies greatly depending on factors such as the plant's genetics, season, and growth environment, according to [28].In addition, [29] found that there are considerable inconsistencies in phytochemical profiles across genotypes and cultivars of the same species.Several additional research [30,31] discovered that external factors such as growing location and growth season affect the quantity of phytochemical substances.

Conclusions
The active compounds of C. pentandrus leaf extract are efficacious antioxidants and inhibitors of the αglucosidase enzyme.Further research, including purification of the crude extract, is required to obtain active ingredients that have high activity.