INTRODUCTION:

Indonesia is one of the tropical country that has suitable climatic conditions for growing many fruit species, indigenous and exotic. Fruits and vegetables are apart from being the main food sources for some essential nutrients and also to be rich source of bioactive compounds which might have other beneficial health effect¹ such as antimicrobial and antidiabetic activities. However, the majority of the abundance of largely species diversity, particularly indigenous fruit species, has not been explored to its full potential. Isem kembang (Mangifera quadrifida Jack) is one of them.

It is a local and extremely rare fruit from Lampung, Indonesia. Isem kembang (Mangifera quadrifida Jack) is a member of the Mangifera genus in the Anacardiaceae family.

In addition, isem kembang (Mangifera quadrifida Jack) has the same genus with mango (Mangifera quadrifida Jack). The resulted studies found that the major active constituent of mango was mangiferin², which reported on antidiabetic, antioxidant, antitumor and antiviral properties of mangiferin³. Isem Kembang fruit has been traditionally used by the Menggala, Tulangbawang, and Lampung people in combination with chili (serwit) to impart a pleasant aroma and flavor. This research will use a part of the Isem Kembang (Mangifera quadrifida Jack) tree which is the baby fruit in Fig 1. Previous studies have reported that ethyl acetate and methanol extracts of baby fruit and ripe fruit flesh of Mangifera quadrifida Jack have very strong DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical scavenging activity^4,5. The results of phytochemical screening also reported that flavonoid and phenolic compounds were identified to be very high in the ethyl acetate and methanol extracts of the baby fruit Mangifera quadrifida Jack⁴. Numerous studies have demonstrated that phenolic and flavonoid compounds have a variety of biological activities, including anticancer, antioxidant, anti-inflammatory, antimicrobial, and cardioprotective properties^6-12. Phenolic compounds can donate protons so that free radicals will become stable radicals. The formation of stable radicals is due to resonance in the aromatic ring so that the electrons will be delocalized^13-15.

To our knowledge, the antimicrobial and antidiabetic activities of Isem Kembang (Mangifera quadrifida Jack) have never been reported. The objective of this research was to evaluate the capability of antimicrobial and antidiabetic activities for isem kembang baby fruits (Mangifera quadrifida Jack).

This study was conducted in two step namely extraction with a maceration method using n-hexane solvent, ethyl acetate and methanol. Furthermore, antimicrobial and antidiabetic were tested with disk diffusion assay methode and antidiabetic activity test with α-glucosidase inhibition test.

Figure 1: The baby of Mangifera quadrifida Jack Plant

MATERIALS AND METHODS:

Plant material:

We harvested fresh isem kembang (Mangifera quadrifida Jack) baby fruits from a local market in Menggala, Tulang Bawang Lampung, Indonesia. The baby fruits of isem kembang (Mangifera quadrifida Jack) were drained in room temperature and then were powdered. The baby fruits of isem kembang is macerated using organic solvents methanol, ethyl acetate, n-hexane. Other reagents used such as p-nitrophenyl-α-D-glucopyranoside, α-glucosidase solution were purchased from Merck and they were in pure analysis condition.

Sample Extraction:

Maceration with a variety of organic solvents was used to prepare the samples. Powdered Isem Kembang seeds were steeped for three days in 100mL of n-hexane and filtered. A raw n-hexane extract was prepared by evaporating the filter until a dry sample was obtained. In order to obtain an ethyl acetate raw extract, the residue from the first immersion was entirely immersed in 100 mL of the solvent for three days. Methanolic extract was made by soaking the residue in a 100mL solution for three days after filtering and drying. It was possible to obtain a clear extract by repeating the maceration process numerous times.

Determination of Antimicrobial Activity:

Antimicrobial assay of the baby fruit of isem kembang extracts was performed by disc diffusion method. To Nutrient agar, a 200μL solution of bacteria (E. coli and B. cereus) was added. Then, filter paper disc (about 5 mm diameter) containing the test compound at a desired concentration was placed on the agar surface. Petri dishes were incubated under suitable conditions (37°C, for 24 h). Observations were made by measuring the zone of resistance After incubation the diameter of inhibitory zones formed around each disc was measured in mm and recorded.

Inhibition zone = Total diameter (i.e. the clear zone + disc) - Diameter of disc.

Alpha-Glucosidase inhibition activity test:

The alpha-glucosidase inhibition test was carried out on a blank solution (test solution without sample/standard), acarbose solution as a comparison standard (positive control), baby fruit of isem kembang extract as a sample. The comparison standard and the baby fruit of isem kembang extract sample were weighed and dissolved with a phosphate buffer pH 6.8. Samples that were not dissolved with phosphate buffer were first dissolved with DMSO at a maximum of 10%. The standard solution and sample were diluted into several concentrations. A total of 30μL of standard solution and 17μL of samples were added to the para-Nitrofenil-α-D-glucopyranoside (PNPG) substrate. The solution was incubated for 5 minutes at 37^oC, and 17μL of the alpha-glucosidase solution was added. The solution was incubated again at 37^oC for 15 minutes, then added 100 μL of 200 mM sodium carbonate. The absorbance of the solution was measured with a microplate reader at λ 405 nm²⁴. The same procedure was carried out for the control test, but with a difference after the first incubation, 100µL of 200mM sodium carbonate was added first, and 17µL of the alpha- glucosidase solution was added after the second incubation. The absorbance of the solution was measured with a microplate reader at λ 405nm.

A_blank- A_sample

% Inhibition = ------------------- × 100%

A_blank

A blank = Absorbance of blank

A sampel = Absorbance of sample

The IC₅₀ values were computed using non-linear regression analysis using plots of percent inhibition versus log inhibitor concentration.

( 50– a)

IC50 = -------------

(b)

RESULTS AND DISCUSSION:

Antimicrobial Activity:

All the baby fruits extract showed antimicrobial activity against the tested Escherichia coli (E.coli) and Bacillus cereus (B. cereus) (Table 1).

Table 1: Zone of inhibition of Mangifera quadrifida Jack baby fruit extracts on bacterial cultures.

No	*Extract¹**	Zone of Inhibition *E. coli* (mm)	Zone of Inhibition *B. cerreus* (mm)
1	n-Hexane	8	18
2	Ethyl acetat	28	18
3	Methanol	27	23
Standard*²		0 mm	13

*1 the extract concentration were the same for all type of extracts

*2 the standart used in the experiment was amoxillin for bacteria

Extract of baby fruit isem kembang (Mangifera quadrifida Jack) is believed to possess antibacterial properties against some micro-organisms. The antimicrobial activity of hexane extract, ethyl acetate extract and methanol extract of Baby Isem Kembang (Mangifera quadrifida Jack), against two (2) bacteria was shown in Table 1. The Table 1 showed the highest zone of inhibition observed by ethyl acetate extract of the baby Isem Kembang (Mangifera quadrifida Jack) was 28 mm (standard 0 mm) for Escherichia coli (E. coli) and then followed by the methanol extract was 27 mm as well as the lowest inhibition zone by the hexane extract was 8 mm. The study discovered that the ethyl acetate extract of baby isem kembang (Mangifera quadrifida Jack) had superior antibacterial activity to the methanol extract. Additionally, the methanol extract of baby isem kembang had the strongest inhibitory action against food-borne pathogens, with a zone of inhibition of (23 mm) for Bacillus cereus (B. cerreus). However, hexane extract and ethyl acetate extract have the same point (18 mm and 18 mm respectively) with the standard (13 mm). The present study revealed that methanol extract of the baby isem kembang (Mangifera quadrifida Jack) has shown better activity against Bacillus cereus. Interpretation of inhibition zones uses the culture test¹⁶. A zone of inhibition is an area of media that is devoid of test organisms (pathogenic bacterial strains). A diameter of zone inhibition of 10 or less indicates that the test product is resistant to the organisms (pathogens); a diameter of zone inhibition of 11 to 15 shows that the test product is resistant to the test organisms in an intermediate manner. A diameter of 16 or greater shows that the substance tested is extremely resistant to the test organisms. It has been previously reported that Mangifera quadrifida Jack baby fruit extract contains hexadecanoic acid, octadecadienoic acid, 9,12-octadecadienoic acid, g-sitosterol stigmasta-5-en-3-ol, caryophyllene, and other compounds^4,17. Fatty acids and caryophyllene have been reported have biological activities such as antimicrobials and antifungal¹⁸.

(A)

Plate 1: Zone of inhibition against B cereus of hexane extract of baby fruit isem kembang

(B)

Plate 2: Zone of inhibition against B. cereus of methanol extract of baby fruit isem kembang

(C)

Plate 3: Zone of inhibition against B. cereus of ethyl acetate extract of baby fruit isem kembang

(D)

Plate 4: Zone of inhibition against E coli of methanol extract of baby fruit isem kembang

(E)

Plate 5: Zone of inhibition against E coli of etyl acetate extract of baby fruit isem

(F)

Plate 6: Zone of inhibition against E coli of hexane extract of baby fruit isem kembang

Figure 2: Zone of inhibition agains. B. cerreus and E. Coli of methanol, Ethyl acetate, and hexane extract of baby fruit isem kembang.

α-Glucosidase Inhibition Activity:

Diabetes mellitus is a condition of hyper-production of glucose inindividuals and less utilization by the tissues¹⁹. One strategy for controlling postprandial glucose levels is to consume a diet high in α-amylase and α-glucosidase inhibitors. These enzyme inhibitors decrease the rate of glucose absorption by delaying carbohydrate digestion and lengthening the time required for digestion. Inhibitors derived from plants represent an appealing therapeutic strategy^20,21. Thus, in this study, we examined the in vitro anti-diabetic properties of various parts of Isem Kembang (Mangifera quadrifida Jack) baby fruits extracted using various solvents against both enzymes, specifically the inhibition of -glucosidase activities using methanol, ethyl acetat, and hexane extracts.

An ability of all extracts in inhibiting the α-glucosidase activities was determined by their IC₅₀. The IC₅₀value was defined as the concentration of α-glucosidase inhibitor that inhibited 50% of α-glucosidase activity²². The lower IC₅₀value indicates greater inhibitory activity of the plant extract toward the enzymes²³. The α-glucosidase inhibitory activity of different extracts of the baby fruits isem kembang (Mangifera quadrifida Jack) was expressed as IC₅₀ (µg/mL) values and is presented in Table 2. The percentage inhibition of α-glucosidase was dose dependently raised by increasing the concentration of all extracts methanol, ethyl acetat, and hexane extracts. For baby fruits Isem Kembang hexane extracts showed the highest α-glucosidase inhibitory activity compared to other extracts because it had the lowest IC₅₀ value (0,04µg/mL). Additionally, ethyl acetat extracts had lower IC₅₀ value (0,62µg/mL) than methanol extracts (0.89µg/mL). The high inhibitory activity alpha glucosidase in three extract was related to the content of the compounds previously reported. Compounds of g-sitosterol and stigmasta-5-en-3-ol have been reported to be contained in Mangifera quadrifida Jack baby fruit extract⁴. Both compounds have been reported to have antidiabetic activity^24,25.

Table 2: IC₅₀ Of isem kembang (Mangifera quadrifida Jack) Extraction

Sample	Concentration (mg/L)	% Inhibition	IC₅₀ (mg/L)
Methanol extracts	0.16	10.32	0.89
	0.31	26.52
	0.63	41.84
	1.25	57.54
Ethyl Acetat extracts	0.16	9.08	0.62
	0.31	33.59
	0.63	54.91
	1.25	65.81
n-Hexane extracts	0.16	61.26	0.04
	0.31	71.92
	0.63	78.38
	1.25	80.57
Acarbose	30	35.31	98.67
	60	41.50
	90	47.23
	120	56.53
	150	60.11

The relationship between the concentration and % inhibition of acarbose and methanol, ethyl acetate and n-hexane extract is made in graphical form (Figure 3), so that the regression equations are y = 0.2154x + 28.747, y = 22.634lnx + 52.547, y = 27.614lnx + 63.406, y = 9.2834lnx + 80.616, respectively. From this equation, the IC₅₀ value for acarbose was 98.67mg/L and for the methanol extract of baby fruit isem kembang was 0.89 mg/L. The ethyl acetate extract of isem kembang infant fruit contained 0.62mg/L. The n-Hexane extract of isem kembang baby fruit contained 0.04mg/L.

Based on these data, the methanol extract, ethyl acetate extract, and n-hexane extract of baby fruit isem kembang has higher alpha-glucosidase enzyme inhibiting activity than acarbose standard. The antidiabetic properties of baby fruit isem kembang extract are related to the presence of phenolic groups that can donate hydrogen atoms to free radicals to become less reactive. Phenolic compounds can also act as competitive inhibitors of carbohydrate digesting enzymes (alpha-glucosidase enzymes) through hydrophobic interactions. These carbohydrates are not quickly hydrolyzed into glucose molecules²⁶.

CONCLUSION:

Antimicrobial test proved that methanol extract, ethyl acetate extract, and n-hexane extract of baby fruit isem kembang (Mangifera quadrifida Jack) had inhibitory activity against the growth of Escherichia coli of 27, 28, and 8mm respectively. While inhibitory activity against the growth of Bacillus cereus is 18, 18, and 23mm respectively. This inhibition activity is higher than standard. Antidiabetic test proved that hexane extracts of baby isem kembang (Mangifera quadrifida Jack) had antidiabetic activity with IC₅₀ of 0.04mg/L. However, both methanol and ethyl acetate extracts also have very high activity because their IC₅₀ values are below 1mg/L. As a result of these findings, it can be concluded that the three extracts of the baby fruit isem kembang (Mangifera quadrifida Jack) have significant potential as alpha glucosidase inhibitors and antimicrobial agents.

ACKNOWLEDGEMENT:

Politeknik AKA Bogor provided financial support for the Laboratorium and instrumentation.

CONFLICT OF INTEREST:

The authors declare that they have no conflict of interest.

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Received on 17.11.2021 Modified on 10.05.2022

Research J. Pharm. and Tech 2023; 16(6):2709-2714.

DOI: 10.52711/0974-360X.2023.00445