GC-MS ANALYSIS AND IN-VITRO ANTI-DIABETIC ACTIVITY OF BIOACTIVE FRACTIONS OF FERONIA ELEPHANTUM FRUIT

GC-MS ANALYSIS AND IN-VITRO ANTI-DIABETIC ACTIVITY OF BIOACTIVE FRACTIONS OF FERONIA ELEPHANTUM FRUIT

G. Jyothi Reddy ^{* 1}, K. Bhaskar Reddy ², G. V. Subba Reddy ³

Discipline of Pharmacy ¹, JNTUA, Ananthapuramu - 515002, Andhra Pradesh, India.

Sri Venkateswara College of Pharmacy ², RVS Nagar, Chittoor - 517127, Andhra Pradesh, India.

Department of Chemistry ³, JNTUA College of Engineering, Pulivendula - 515002, Andhra Pradesh, India.

ABSTRACT: The present study was carried out to characterize the bioactive phytoconstituents from the fractions of F. elephantum fruit and to evaluate their in-vitro anti-diabetic activity. Column chromatography of methanol extract of F. elephantum fruit yielded  Hexane: Ethyl acetate (1:1 v/v) fraction (HEFE), Ethyl acetate fraction (EFE) and Ethyl acetate: Methanol (1:1 v/v) fraction (EMFE), which were subjected to GC-MS analysis. They were also tested for in-vitro α-amylase and α-glucosidase inhibitory potential. GC-MS analysis of EFE predominantly showed 2,5-Furandione, dihydro-3-methylene; n-Hexadecanoic acid; 5-Eicosene,(E)-; cis-13-Octadecenoic acid; and γ-Sitosterol; 2,5-Furandione, dihydro-3-methylene-; cis-Aconitic anhydride; Ethanol, 2,2'-[(1-methylethyl)imino]bis-; and Propanedioic acid, ethyl-, diethyl ester; were the major compounds in EMFE. HEFE showed 2,5-Furandione, dihydro-3-methylene (18.5%), Dodecanoic acid (4.48%), n-Hexadecanoic acid (15.18%) and cis-13-Octadecenoic acid (18.95%) which are biologically active. Moreover, the α-amylase IC₅₀ values of HEFE, EFE, and EMFE were 68.77, 52.59, and 40.28 μg/mL, respectively, while that of acarbose was 41.99 μg/mL. And the α-glucosidase IC₅₀ values of HEFE, EFE, and EMFE were 69.53, 35.08, and 42.49 μg/mL, respectively, which were comparable to that of acarbose (39.21 μg/ml). Findings of the present study clearly indicate that F. elephantum fruit possesses numerous bioactive components and potential in-vitro antidiabetic activity, thus justifies the use of this plant for different ailments by traditional medical practitioners.

GC-MS Analysis, In-vitro anti-diabetic, Feronia elephantum fruit, α-amylase, α-glucosidase

INTRODUCTION: Plants produce an extensive range of bioactive phytochemical compounds with significant applications in different sectors. These compounds occur naturally in small quantities and are considered as secondary plant metabolites with pharmacological or toxicological properties in living organisms ¹. Among the secondary metabolites, polyphenolic compounds have a wide range of biological and physiological activities and serve as chemotaxonomic marker compounds ².

Feronia is a monotypic genus belonging to the family Rutaceae. Feronia elephantum correa (Limonia acidissima Linn, Schinus limonia Linnor Feronia limonia) is a moderate-sized tree whose parts such as fruits, leaves, root, bark, and gums have been used in traditional medicine for many ailments. The fruit (wood apple) contains flavonoids, saponins, glycosides, tannins, and some coumarins and tyramine derivatives ³. Wood apple is a dry land fruit, which is a nutritious, rich in natural acids such as oxalic, tannic, mallic, and citric acid.

It is a source of calcium, phosphorus, iron and vitamins A, B and C. Seeds and fruits contained oil and protein; oil composed of palmitic, oleic, linoleic and linolenic acids besides traces of palmitoleic and stearic acids; β-sitosterol, β-amyrin, lupeol and stigmasterol from unsaponifiable matter of seed oil. Fruit pulp has been reported for glycoside - 5,4-dihydroxy-3-(3-methyl-but-2-enyl) 3,5,6-trimethoxyflavone7 O-b-D-glucopyranoside ⁴. According to Ayurveda, the fruits are used for heart diseases (cardiotonic), cough, vomiting, dysentery, removes biliousness, “tridosah”, “vata”, and blood impurities, thirst, fatigue, hiccough; tumors, asthma, leucorrhoea, ophthalmia. In Yunani, the fruits are cardiotonic, tonic to the lungs and the liver, diuretic, strengthening the gums; the juice is good for sore throat and stomatitis. The fruit pulp is also used by tribal of Rewa District of Madhya Pradesh against diabetes, boils and amoebiosis ⁵, and hence is regarded as one of the most valuable medicinal plants in India.

To the best of our knowledge, there have not been any earlier reports on fractions from the fruits of this plant. However, as part of our search for new natural products and bioactive compounds, an investigation of α-glucosidase and α-amylase inhibitory activity of crude methanolic and aqueous extracts of F. elephantum fruit was undertaken. We herein aim to examine the phytochemical constituents of different fractions of F. elephantum and their in-vitro antidiabetic activity using α-amylase and α-glucosidase inhibitory assay.

MATERIALS AND METHODS:

Chemicals and Reagents: P-Nitrophenyl-α-D-Glucopyranoside (pNPG), 3,5-DinitroSalicylicAcid (DNSA), α-amylase and α-glucosidase enzymes and acarbose, were purchased from M/S Sigma–Aldrich Chemicals Pvt., Limited, Bangalore. All other chemicals and reagents used were of high purity analytical grade.

Collection of Plant and Preparation of Fractions: Ripe fruits of F. elephantum were collected (voucher no. 1328) and processed as described in our previous work ⁶. F. elephantum fruits powder was extracted with methanol by soxhlation, filtered, and evaporated under reduced pressure for viscous extract using Rotavapor (Buchi R-200). It was fractionated using column chromatography on silica gel with n-hexane yielding insoluble fraction, which was further fractionated with a mixture of an equal ratio of n-hexane and Ethyl acetate (1:1) yielding a soluble fraction and an insoluble fraction. The insoluble fraction of n-hexane and Ethyl acetate was then fractionated using ethyl acetate, yielding the ethyl acetate soluble fraction and insoluble fraction, which was then fractionated using methanol. All the fractions were concentrated by rotary vacuum evaporator and labeled as follows; Hexane: Ethyl acetate (1:1 v/v) fraction of F. elephantum as HEFE, Ethyl acetate fraction of F. elephantum as EFE and Ethyl acetate: Methanol (1:1 v/v) fraction of F. elephantum as EMFE.

Phytochemical Analysis of Bioactive Fractions using Gas Chromatography-Mass Spectrometry (GC-MS): GC-MS analysis of the fractions of F. elephantum viz., HEFE, EFE, and EMFE was carried out using GC (Agilent 7890A) with DB 5 Ms Column (30m L × 0.25mm ID × 0.25um film thickness). Helium (99.9995%) was used as carrier gas (flow rate 1 mL/min), and an injection volume of 1 μL was employed in a splitless mode. The injection temperature was 250 °C, and the auxiliary temp was 290 °C. Mass Spectrophotometer (5975C MSD) with Electron Impact Ionization and Quadrupole Mass Analyzer was used with the Scan Mass range of 30m/z to 700m/z. The MS source temperature was 250 °C, and the MS quad temperature was 180 °C in Table 1.

Sample Preparation: Given a sample made up to 2 mL with the respective solvent. It again diluted with 20 μL in 980 μL of solvent and injected 1uL into the GCMS instrument.

TABLE 1: TEMPERATURE RAMP

The phytoconstituents of HEFE, EFE, and EMFE were identified by comparison of mass spectra with the national libraries (NIST - 11). The molecular formula, molecular weight, and structure of the identified compounds were ascertained.

In-vitro α-Amylase Inhibitory Activity: The α-amylase inhibitory potential of all fractions was evaluated using 3, 5-dinitrosalicylic acid (DNSA) which is based on the spectrophotometric method using acarbose as standard reference 6. Stock solutions (500 µg /mL in distilled water) of HEFE, EFE, EMFE, and positive control, acarbose were prepared.500 μL of different concentrations (10, 20, 40, 80 and 160 µg/mL) of each sample were added to a 500 μL solution of α-amylase (0.5 mg/mL in 0.02 M,pH 6.9 sodium phosphate buffer) and was incubated for 10 min. Then add 500 μL of starch solution 1% (w/v) and incubate for 10 min at 25 °C.

The coloring reagent, DNSA (1 mL)was added, and heat the reaction mixture in a boiling water bath for 5 min, cool to room temperature. Then dilute it with 10 mL of distilled water and measure the absorbance at 540 nm using a UV-VIS spectro-photometer (ELICO SL159). A blank solution was prepared by substituting the α-amylase enzyme solution with 500 μl of sodium phosphate buffer. The tests were repeated thrice with the same protocol.

In-vitro α-glucosidase Inhibitory Activity: The study was performed using α-glucosidase and p-nitrophenyl-α-D-glucopyranoside (pNPG) as per previously reported model.6Each of 100 μLof HEFE, EFE, EMFE and positive control, acarbose at different doses (10, 20, 40, 80 and 160 µg/mL) was added to 50 μL of α-glucosidase (1 U/mL) prepared in 0.1 M phosphate buffer (pH 6.9). Then, add 250 μL of 0.1 M phosphate buffer. The mixture was incubated at 37 °C for 20 min. Then, 10 μl of 10 mM pNPG (in 0.1 M phosphate buffer, pH 6.9) was added and incubated at 37 °C for 30 min. The reactions were stopped by adding 650 μl of 1 M sodium carbonate, and the absorbance was measured at 405 nm in triplicate against the blank solution with 100% enzyme activity

Method for Calculation of α-amylase and α-Glucosidase Inhibitory Concentration (IC₅₀): The percentage of enzyme inhibition was calculated using the formula:

% inhibition = (A_control – A_sample / A_control) × 100

Where A_control is the absorbance of the control (blank with 100% enzyme activity), and A_sample is the absorbance of the sample.

The concentration of the fraction required to inhibit 50% of α-amylase and α-glucosidase activity under the assay conditions is defined as the IC₅₀ value. IC₅₀ was calculated by using the percentage inhibition at five different concentrations of the fractions by plotting percentage inhibition against the concentrations. The IC₅₀ value was calculated by using Linear Regression analysis.

RESULTS AND DISCUSSION:

Characterization of the Phytochemical Compounds of HEFE, EFE and EMFE using GC-MS: Three fractions were separated from methanol extract of F. elephantum fruit viz., HEFE, EFE, and EMFE. GC-MS analysis of HEFE, EFE, and EMFE revealed the presence of various complex compounds. GC-MS analysis of EFE shown 2,5-Furandione, dihydro-3-methylene (44.78%), n-Hexadecanoic acid (6.62%), 5-Eicosene, (E)- (4.04%), cis-13-Octadecenoic acid (6.08%) and γ-Sitosterol (2.99) as prominent compounds as presented in Fig. 1 along with other phytoconstituents, as reported in Table 2.

2,5-Furandione, dihydro-3-methylene- (68.47%), cis-Aconitic anhydride (5.19%), Ethanol, 2,2'-[(1-methylethyl) imino]bis- (6.27%) and Propanedioic acid, ethyl-, diethyl ester (7.11%) are the major compounds with higher peak areas in EMFE as seen in Fig. 2, listed in Table 3.

GC-MS profiling of HEFE shown different compounds as presented in Table 4, out of which the prominent constituents with predominant peak area, as shown in Fig. 3 are 2,5-Furandione, dihydro-3-methylene (18.5%), Dodecanoic acid (4.48%), n-Hexadecanoic acid (15.18%) and cis-13-Octadecenoic acid (18.95%) which are biologically active. Other important bioactive compounds present in the fractions of F. elephantum fruit are L-Glutamic acid, dimethyl ester; E-15-Hepta-decenal; Phenol, 2, 4-bis (1, 1- dimethylethyl; cis-Linaloloxide; citric acid; 6-Isopropenyl-4, 8a-dimethyl-1,2,3,5,6,7,8,8a-octa-hydro-naphthalen-2-ol; 9,12-Octadecadienoic acid (Z,Z)-; Dichloro-acetic acid, heptadecyl ester etc., which have been reported for anti-oxidant, antiinflammatory, hypo-cholesterolemic, anti-diabetic, anti-cancer, anti-microbial, antitubercular, antibacterial, antifungal activities which are represented in respective tables. These bioactive compounds are reported for the first time in the fractions of F. elephantum fruit through the present study.

FIG. 1: GCMS CHROMATOGRAM OF EFE

FIG. 2: GCMS CHROMATOGRAM OF EMFE

FIG. 3: GCMS CHROMATOGRAM OF HEFE

TABLE 2: PHYTOCONSTITUENTS IDENTIFIED FROM EFE BY GC-MS ANALYSIS AND THEIR REPORTED BIOLOGICAL ACTIVITIES

RT = Retention Time, MF = Molecular formula and MW=Molecular Weight. *Same compound but appeared at two different RT (min) showing two distinct peaks on the spectrum and with different % compositions

TABLE 3: PHYTOCOMPONENTS IDENTIFIED FROM EMFE BY GC-MS ANALYSIS AND THEIR REPORTED BIOLOGICAL ACTIVITIES

RT = Retention Time, MF = Molecular formula and MW = Molecular Weight

TABLE 4: PHYTOCOMPONENTS IDENTIFIED FROM HEFE BY GC-MS ANALYSIS AND THEIR REPORTED BIOLOGICAL ACTIVITIES

RT = Retention Time, MF = Molecular formula and MW = Molecular Weight

In-vitro α-amylase Inhibitory Activity of HEFE, EFE and EMFE: Three fractions viz., HEFE, EFE, and EMFE were screened at different doses for in-vitro α-amylase inhibitory potential, which might be deduced to perceive their antidiabetic potential. The results showed a concentration-dependent rise in the percentage inhibitory activity against the α-amylase enzyme, as represented in Table 5. HEFE, EFE, and EMFE at the maximum tested concentration, 160 μg/mL showed a percentage inhibition of 90.21, 91.65 and 97.53 respectively, while acarbose showed 95.06, which infers the inhibitory potential of fractions is comparable to that of standard reference, acarbose.

The IC₅₀ values of HEFE, EFE, and EMFE were 68.77, 52.59, and 40.28 μg/mL, respectively, while that of acarbose was 41.99 μg/mL. Since α-amylase plays a vital role in starch absorption in human beings and animals, the presence of such inhibitors in plant extracts or foodstuffs may be responsible for impaired starch digestion and thus anti-hyperglycemic effect ²⁹.

In-vitro α-glucosidase Inhibitory Activity of HEFE, EFE and EMFE: The obtained results showed a dose-dependent escalation in the percentage inhibition of α-glucosidase enzyme as depicted in Table 5. HEFE, EFE, and EMFE at the highest tested concentration, 160 μg/mL showed a percentage inhibition of 89.34, 93.70 and 94.38, respectively, which were comparable to that of standard drug acarbose with 90.65. The IC₅₀ values of HEFE, EFE, and EMFE were 69.53, 35.08 and 42.49 μg/mL, respectively, which were comparable to that of acarbose (39.21 μg/ml), however, there was a significant difference between the IC₅₀ of acarbose and HEFE. The α-glucosidase inhibitory effect exhibited by all the fractions indicates their potential effectiveness at managing Diabetes Mellitus, possibly by reducing postprandial glycemic levels and the total range of postprandial glucose levels ³⁰.

HEFE, EFE, and EMFE efficiently inhibited α-amylase and α-glucosidase enzymes in-vitro, which might be due to the presence of bioactive phytoconstituents like L-Glutamic acid dimethyl ester11, Benzoic acid 4-hydroxy12, Dodecanoic acid, 132,5-Furandione, dihydro-3-methylene, n-Hexadecanoic acid, 5-Eicosene,(E)-, γ-Sitosterol, 9,12-Octadecadienoic acid (Z,Z)-,methyl ester that were earlier reported for antidiabetic activity.

These results indicate that F. elephantum fruit could be used to reduce post-prandial blood glucose levels and may be of worth as novel therapeutic agents in the treatment of Diabetes Mellitus.

TABLE 5: α-AMYLASE AND α-GLUCOSIDASE INHIBITION ASSAY OF HEFE, EFE AND EMFE

All determinations were carried out in the triplicate manner, and values are expressed as the mean ± SEM

CONCLUSION: Findings of the present study clearly indicate that F. elephantum fruit possesses considerable inhibitory activity against α-amylases and α-glucosidases, with remarkable activity in EFE and EMFE. This observed antidiabetic activity of EFE, EMFE, and HEFE might be attributed to the bioactive compounds like L-Glutamic acid dimethyl ester, Benzoic acid 4-hydroxy, Dodecanoic acid, 2,5-Furandione,dihydro-3-methylene, n-Hexadecanoic acid, 5-Eicosene,(E)-, γ-Sitosterol, 9,12-Octadecadienoic acid (Z,Z)-,methyl ester which are identified as prominent compounds by GC-MS analysis.

This present work discloses the goodness of F. elephantum with numerous bioactive components, potential in-vitro antidiabetic activity, and other earlier reported biological activities justifies the use of this plant for different ailments by traditional medical practitioners. However, isolation of specific phytoconstituents and studying their biological activity will certainly give productive results.

ACKNOWLEDGEMENT: The authors thank the Department of Science and Technology (DST), New Delhi, for providing support under the FIST Program of DST [SR/FST/COLLEGE-280]. The authors are also thankful to Mass Spectrometry Facility, Division of Biological Sciences, Indian Institute of Science, Bengaluru for GC-MS characterization.

CONFLICTS OF INTEREST: The authors have no conflicts of interest to declare.

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    How to cite this article:

    Reddy GJ, Reddy KB, Reddy GVS: GC-MS analysis and in-vitro anti-diabetic activity of bioactive fractions of Feronia elephantum fruit. Int J Pharm Sci & Res 2020; 11(5): 2415-24. doi: 10.13040/IJPSR.0975-8232.11(5).2415-24.

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