Total phenolic and flavonoid contents of Aphanamixis polystachya (Wall.) R.Parker leaf extract and its potential as antioxidant and inhibitor of α-glucosidase

Indonesia is one of the countries with the highest plant diversity in the world, including the Aphanamixis genus, and they use the plants in this genus as traditional medicine. The genus Aphanamixis has a wide variety of compounds with different activities, such as insecticidal, leishmanicidal, antimicrobial, anticancer, cytotoxic against some cancer cells, and antioxidant The focus of this research is to find out about the anti-oxidant, anti-diabetic, and anti-bacterial activities of Aphanamixis polystachya leaf extract from the Bali Botanical Garden plant collection. The antioxidant activity assay was carried out by scavenging the free radical 1-(2,6-dimethylphenoxy)-2-(3,4-dimethoxyphenyl ethylamino) propane hydrochloride (DPPH). The antidiabetic assay was carried out by inhibiting the activity of the α-glucosidas eenzyme, while the antibacterial test was carried out by the agar diffusion method. Based on the data generated in this study, the active ingredients of A. polystachya leaf extracts have high potency as antioxidants and α-glucosidase enzyme inhibitors, but the extract was not effective in inhibiting bacterial growth.


Introduction
Medicinal plants have been viewed as a complementary healthcare system and an effective treatment for certain individuals because of their low price and reliability [1].The use of medicinal plants as raw plant materials, refined crude extracts, mixes, etc. in medicinal medicines is significant among natural sources.Several thousand plants with therapeutic properties have been found to cure a variety of illnesses [2].
Aphanamixis polystachya (Wall.) R. Parker is a big tree species.It is native to much of East and Southeast Asia, present from the Indian Subcontinent east through Indochina to western China and south through Malesia to Papua New Guinea.Occurring across continental space and also on many islands.The species wide native range suggests that its population is also large.The species occurs in mid-to high-elevation ranges; in India, it is recorded up to 1, 300 m asl [3].
The roots, leaves, and bark of A. polystachya have medicinal significance and have been used in traditional remedies for tumors, cancer, spleen illnesses, ulcers, liver disease, cough, enlarged spleen, rheumatism treatment, and jaundice.Recent studies revealed its efficacy focused mainly on its antiulcer, antimicrobial, hepatoprotective, analgesic, CNS depressant, antimutagenic, antiproliferative, and antioxidant activities.Bark is used in spleen diseases, liver diseases, tumors, and abdominal complaints [4].J aundice is a plant that has numerous bioactive chemicals, such as rohitukine, amooranin, aphamines, aphanamoxenes, aphananoid, aphananin, and others, which can be utilized to cure a variety of illnesses.
Despite all these studies proving the therapeutic benefit of A. polystachya, no research has been done to date to support its anti-diabetic properties.The purpose of this study was to assess the antioxidant, anti-diabetic, and anti-bacterial effects of A. polystachya leaf extract, particularly those from Indonesia.

1 Simplicia setup
The leaves of A. polystachya were obtained from the Bali Botanical Garden plant collection with code XIV. A. 81.This plant was collected in J ambi Province, Sumatra.The leaves underwent a twoweek process of drying outside without sunlight after being washed with water, gathered, and dried.Simplicia was weighed, blended, and then passed through a 40-mesh screen.Until extraction is done, simplicity powder is kept separate and shielded from sunlight in dry, locked, identifiable containers.

2 Extraction of simplicia
The leaf powder of A. polystachya was weighed to as much as 100 g and then extracted with 1000 mLof hexane, ethyl acetate, and methanol solvents.

3 Total Phenolic
By applying the Folin-Ciocalteu method [5] and using gallic acids as a reference, the total phenolic content of the extract was ascertained.Mg of gallic acid equivalent per gram of dry weight was used to express the results.

4 Total Flavonoid
The aluminum chloride colorimetric assay technique was used to calculate the total flavonoid component content [6].The regression equation derived from the calibration curve was used to convert the total flavonoid component concentration to quercetin equivalent (mg/g extract).

5 Free radical scavenging action of DPPH
The method developed by Yen and Chen [7] was used to assess the antioxidant properties of A. polystachya leaf extracts.Amounts from specimens in MeOH (2 ml) were combined with 0. 5 ml of DPPH (1 mM) in MeOH for each concentration.Each of the mixtures wes thoroughly stirred before being allowed to mature at ambient humidity for 30 minutes in the dark.Using a spectrophotometer (UV-VIS Thermo Genesys 30), the reaction fluid's sensitivity was determined at 517 nm.Ascorbic acid was employed as the reference standard chemical, and three copies of the experiment were run.The optimum dose required to scavenge 50% of the radicals produced by DPPH, or the IC50 value, was determined using quadratic analysis and regression approximation.Greater antioxidant activity was indicated by a lower IC50 value.The following equation can be used to calculate activity decline:

Details :
A blank = Abs orption in the abs ence of a s ample A s ample = Sample abs orbance A linear equation is used to solve the estimated value (Y = bX + a), with the abscissa representing the ppm concentration (mg/L) on the X-axis and the ordinate representing the percent value of the reduction on the Y-axis.When the percent reduction is 50%, the IC50 value is calculated.

6 α-Glucosidase inhibition
According to Kim et al.' s findings [8], the inhibiting effect of α-glucosidase was determined.In a nutshell, 5 μL of the sample were dissolved in DMSO at various concentrations, and then 250 μL of ρ-NPG (3 mM) and 495 μL of 100 mM phosphate buffer (pH 7. 0) were added to the tube.A preincubation period of 5 minutes at 37 degrees Celsius was followed by the addition of 250 μL of αglucosidase (0. 065 U/mL) and a 15minute incubation period.By adding 1 mL of 0. 2 M Na2CO3, the process was halted.
By measuring the amount of ρ-nitrophenol emitted at 400 nm, the inhibitory effect on αglucosidase activity was identified.To adjust background absorbance, specific blanks for test samples were created, substituting 250 μL of phosphate buffer for the enzyme.% inhibition = (A -B)/A × 100, where A represents the reference reaction absorption and B represents the absorption while the sample is present, the percentage was computed using inhibition of α-glucosidase inhibitory and antioxidant activities.Logarithmic regression analysis was used to derive the IC50 from the primary inhibitory values.Results were presented as mean values ±SDs after the assays were completed in triplicate.As a benchmark, quercetin was used.

7 Antibacterial activity assay
An antibacterial assay in vitro of the leaf of A. polystachya extract against E. coli strain ATCC 8739, Pseudomonas aeruginosa strain ATCC 15442, Salmonella typhimurium strain ATCC 14028, Bacillus subtilis strain ATCC 19659, Staphylococcus aureus strain ATCC 6538, and Streptococcus mutans strain ATCC 25175.The Research Center for Raw Materials and Traditional Medicines (BRIN), which houses all of the bacterial isolates used in this investigation, keeps them all safe.Utilizing the disc diffusion method outlined by Pratiwi et al. [9] and Schwalbe et al. [10], the antibacterial activity assay was carried out.At 37 o C for 18 hours, bacterial isolates that were pathogenic were cultivated on 50 mL of NB medium (Merck) in a 250 mL Erlenmeyer flask.On the surface of the NA plate (15 cm diam.), 200 L of each pathogenic bacterial culture with 105 CFU/mL were applied.At 75, 150, 300, 600, 1, 250, 2, 500, 5, 000, and 10, 000 μg/mL in DMSO, the extracts were produced.As a positive control, tetracycline (500 μg/mL) was utilized, and as a negative control, DMSO without extract.15 μL of every single concentration were dispersed onto a Fuoroni disc with a 6 mm diameter before being put on NA plates.24-48 hours of incubation at 37 °C resulted in the detection of antimicrobial activity.The inhibitory zone diameter was used to gauge inhibition activity.

1 Contents of total phenolic and flavonoid compounds
In this study, the Folin-Ciocalteu technique was used to measure the total phenol levels in A. polystachya leaf tissue.Phosphomolybdic and phosphotungstic acids are present in the Folin-Ciocalteu reagent, which phenolic compounds will reduce to create a complex molybdenum-tungsten alloy that is bluish-purple in color [11,12].Because it is stable and a derivative of hydroxybenzoic acid, gallic acid is employed as a standard [13].
The extracts of many different plant sources can be used to create phenolic-group chemicals with bioactive qualities.The amount of phenol in each plant and each of its components varies.Tables 1 and 2 show the A. polystachya leaf extracts' total phenol and flavonoid concentrations after being macerated in hexane, ethyl acetate, and methanol.In terms of gallic acid equivalent, the total phenol content was given as mg GAE/g dry ext ract.The highest total phenolic content in the leaf of A. polystachya was contained in the methanol extract, which was 94, 55±0, 24, followed by the ethyl acetate extract, which was 6, 55±0, 14, and the lowest was contained in the hexane extract, which was 3, 27±0, 22 mg/g EQ of the sample.
According to the species of compounds and the type of solvent employed, the overall phenolic content will vary.According to a comparative investigation of the phenol content in various types of plant parts using various solvents, the highest concentration of phenol compounds was discovered in plant extracts using highly polar solvents [14].
Antioxidant activity typically correlates with the overall phenol content of plants.The antioxidant activity increases with phenol content [16].Free radicals can be inhibited by phenolic compounds by donating protons, which causes the radicals to stabilize.The creation of this stable radical is caused by vibration in the aromatic ring, which results in electron delocalization [17,18].

3 α-Glucosidase Enzyme Activity Inhibition Test
Several attempts have been undertaken to identify effective and safe α-glucosidase inhibitors from natural materials for the production of helpful diets for diabetes.In this investigation, the methanol extract of A. polystachya exhibited potent inhibitory activity against α-glucosidase with an IC50 of 4. 72 0, 03 μg/ml.Antioxidants are substances that can reduce or eliminate damage caused by oxidation; as a result, they can shield the body from a number of illnesses, including diabetes and its complications [19].The functions of α-glucosidase inhibitors and the characteristics of antioxidants should be present in a perfect anti-diabetes molecule [20].In conclusion, the A. polystachya leaf extracts showed good α-glucosidase inhibitory action as well as antioxidant activity.
The antioxidant activity test results, phytochemical screening, alpha-glucosidase inhibitory activity, and total phenolic data were all interrelated.Due to the presence of phenolic groups, which can give free radicals hydrogen atoms to reduce their reactivity, the antioxidant and anti-diabetic activities of the stem bark ethanol extract are related to their existence.Through hydrophobic interactions, phenolic substances can also function as competitive inhibitors of the alpha-glucosidase enzymes, which break down carbohydrates.These carbohydrates do not hydrolyze into molecules of glucose right away [21,22].

4 Antibacterial activity assay
The disc diffusion assay of the leaf of A. polystachya extract exhibited that there is no antibacterial activity against all of the bacteria (Table 5).The results of this study are different from those of [23], who stated that A. polystachya extract is effective against a range of bacteria (gram-positive and gramnegative bacteria).
The extract proved inefficient at preventing bacterial growth, which could be related to changes in the extract' s chemical components.The quantity of chemical substances in plants can vary greatly based on parameters such as cultivar species genetics, season, and growth site [24].T here are significant changes in phytochemical profiles between genotypes and cultivars of the same species [25].Several other studies [26,27] have found that environmental variables and the growing season influence the concentration of plant chemical components.

Conclusions
According to the findings of this investigation, the active components of A. polystachya leaf extracts are highly effective antioxidants and α-glucosidase enzyme inhibitors.Purification of crude extracts will be required in future studies to get active materials with high activity.

Figure 1 .
Figure 1.Graph of the relations hip between concentration and % inhibition for IC50 d etermination.(a) Quercetin; (b) Hexane extract; (c) Ethyl acetate extract and (d) Methanol extract of A. polystachya

Table 1 .
The res ults of Total Phenol Content for A. polystachya leaf extracts .

Table 2 .
The res ults of Total Flavonoid Content for A. polystachya leaf extracts .

Table 3 .
The res ults of DPPH free radicals scavenging activity

Table 4 .
Alpha-Glucos idas e Enzyme Activity Inhibition Tes t Res ults

Table 5 .
Antibacterial activity as s ay res ults