The solvents used for the extraction and isolation process were 96% ethanol (Brataco Chemica, http://bratachem.com/), n-hexane (MilliporeSigma), ethyl acetate (MilliporeSigma), methanol (MilliporeSigma) and chloroform (MilliporeSigma). Thin layer chromatography (TLC) was carried out using pre-coated silica gel 60 F254 plates with a thickness of 0.20 mm (MilliporeSigma). The human α-Amylase Inhibitor Screening kit (cat. no. K482-100, BioVision, Inc.), which hydrolyzes the synthetic substrate and yields a chromophore, was used for in vitro testing. Nuclear magnetic resonance (NMR) analyses, which included proton (¹H)-NMR, carbon-13 (13C)-NMR and distortionless enhancement by polarization transfer (DEPT) were performed using deuterated solvents (acetone-d6, CD₃OD, and/or CDCl₃) on 400 mHz NMR (JNM-ECZ500R/S1, JEOL, Ltd.) with tetramethylsilane (MilliporeSigma) as an internal reference.

The fresh ripe fruits were harvested in August, 2019 (outside temperature, 25 to 30˚C) at Kendari (Google coordinates: -4.045723429369156; 122.57746055261806), Southeast Sulawesi, Indonesia. The fruits were taxonomically identified at the College of Life Sciences, Bandung Institute of Technology (Bandung, Indonesia), and were confirmed as M. citrifolia L. (sample no. 66/I1.CO2.2). The characteristics of the plant samples matched those described in the flowering plants' taxonomic references (12-14).

The fruits were washed under tap water to separate any soil, dust, and other foreign inorganic matter, and were cleaned using standard pharmacognosy laboratory procedures, e.g., medicinal plant materials should be entirely free from visible signs of contamination by molds or insects and other animal contamination, including animal excreta. No abnormal odor, discoloration, slime, or signs of deterioration should be detected (15). The clean fruits were dried at 40˚C without being exposed to sunlight. The dried samples were mashed using a pestle in a white porcelain mortar and sealed in a plastic container before being further processed.

Dried M. citrifolia coarse powder fruit (3,064 g) was macerated with 20 liters of 96% ethanol three times for 24 h to obtain 540 g extract (yield, 17.62% w/w). The extract was then thickened using a vacuum rotary evaporator (Buchi) and further diluted in ethyl acetate and divided into soluble (338 g or 62.59%) and insoluble (338 g or 37.40%) partitions. The insoluble ethyl acetate partition contains polar molecules (glycosides) that can disrupt the isolation process.

The ethyl acetate extract was then fractionated using vacuum liquid chromatography apparatus (consisting of a glass Buchner filter funnel and a 10-cm length glass column connected to a vacuum pump) using 21 mobile phases or eluents (the volume of each solvent is 150 ml) i.e., n-hexane, a combination of n-hexane and ethyl acetate in the ratios of (9:1), (8:2), (7:3), (5:5), (3:7), (2:8), ethyl acetate and methanol, on a 10-cm column with a silica gel stationary phase (Silica gel 60 Merck^® for column chromatography; CAS no. 7631-86-9; MilliporeSigma) (250 g), yielding 21 fractions observed in the chromatogram pattern using thin layer chromatography. The fractions with the same stain pattern were merged based on the chromatograms and were tested for their hypoglycemic activity following the bioactivity guide method.

A total of 30 male Swiss-Webster mice (Mus musculus), weighing 20-30 g, were adapted to a controlled temperature condition at 28-30˚C under a 12-h dark/light cycle (light was turned on from 6 a.m. to 6 p.m.), with daily standard food and water freely available, for 1 week prior to treatment. The health and behavior of the mice were observed daily and their cages were cleaned every 2 days to remove the feces and urine. No animals were found dead during the acclimatization and the in vivo experiments. Animal handling, maintenance and euthanasia procedures were performed as approved by the Ethics Committee of Halu Oleo University, Kendari, Indonesia. Following 1 week of acclimatization, the mice were fasted for 18 h and their fasting blood glucose levels were measured using a glucometer (ACCU-CHEK Inform II, Roche Diagnostics). All the mice were then administered STZ at a dose of 65 mg/kg body weight intraperitoneally to induce diabetes; this was followed by a period of modification (16). STZ [chemical name, 2-deoxy-2-(3-(methyl-3-nitrosourea)-D-glucopyranose] is produced by Streptomycetes acromogenes. Following an intraperitoneal or intravenous administration, this antibiotic enters the pancreatic β-cell through the Glut-2 transporter and induces the alkylation of DNA (17).

At 48 h following the STZ administration, the blood glucose levels of the mice were measured by obtaining blood from the tail vein, and mice with blood glucose levels >200 mg/dl were randomly assigned to 9 groups, with 3 mice in each group in one cage, as follows: The negative control group (treated with a suspension of sodium carboxymethylcellulose 0.5%); the drug control group (treated with exogenous insulin 1 IU/kg body weight subcutaneously); and seven treatment groups of fractions A-G (150 mg/kg body weight) in a suspension of sodium carboxymethylcellulose 0.5% by oral administration using oral gavage feeding every afternoon at 2 p.m. for 14 consecutive days.

The blood glucose levels of the animals were measured on days 1, 3, and 7 after treatment. The reduction in blood glucose levels was calculated using the following formula:

The results of the present study are expressed as the mean ± SEM. At the end of the study period, the mice were sacrificed using isoflurane 2% inhalation 1 liter/minute for >4 h following a procedure described elsewhere with a modification of the dose (18). Animal death was verified by the absence of respiration and heartbeat for a period of >5 min.

Active fractions with hypoglycemic activity were further purified using a radial chromatographic method (The Chromatotron™ model 7924T), followed by ¹H-NMR, 13C-NMR, and DEPT analyses using the JEOL JNM-ECZ500R/S1 (JEOL, Ltd.) instrument, and were validated using liquid chromatography-mass spectroscopy (LC-MS Waters ACQUITY UPLC I-Class in tandem with the Xevo G2-X2 Quadrupole Time-of-Flight Mass Spectrometer; Waters Corporation), to elucidate the chemical structure of the isolate.

The α-amylase inhibitory activity of M. citrifolia fruit extracts and the isolate were assayed using the α-Amylase Inhibitor Screening Kit (BioVision Inc.). Acarbose provided in the enzyme kit (BioVision Inc.) was employed as the control drug and dimethyl sulfoxide (DMSO) as a blank solvent. Each sample was dissolved in DMSO to generate a concentration of 150, 75, 37.5, 18.75, and 9.375 ppm for each sample. A total of 50 µl of the sample (5x) was added to designated wells of a clear 96-well microplate. The preparation of the reagents and the procedure of this assay were performed by following the instructions provided with the kit. The microplate was incubated at 37˚C for 25 min before being measured at 405 nm.

The α-amylase inhibitory activity was calculated using the following equation:

The concentration of sample required to inhibit 50% of α-amylase activity under the above condition was defined as the IC₅₀ value. The α-amylase inhibitory activity of the samples was calculated, and its IC₅₀ values were determined using GraphPad Prism software.

Animal handling, maintenance and euthanasia procedures were performed as approved by the Research Ethics Committee, Halu Oleo University, Kendari, Southeast Sulawesi, Indonesia (document no. 1404/UN29.20/PPM/2020).

The 21 fractions were observed in the chromatogram pattern using thin-layer chromatography (Fig. 1). The fractions with the same stain pattern were merged based on the similarity of the chromatograms, yielding 7 fractions (A-G). These fractions (A-G) were tested for their hypoglycemic activity following the bioactivity guide method.

To determine the active fraction of M. citrifolia fruit, fractions A-G were assayed for their hypoglycemic activity in Swiss-Webster mice using the glucose tolerance method. The results are presented in Table I. The findings demonstrated that the control drug (exogenous insulin) and all fractions exhibited notable differences (95% CI) compared with the negative control (Na-CMC). Fraction G (48.10%), fraction C (43.08%) and fraction E (41.27%) exhibited the most prominent suppressive effects on blood sugar levels.

By considering the TLC pattern compared to a previous study (19) and the hypoglycemic activity of the fraction, fraction C was selected and proceeded to be purified using a radial chromatographic method and yielded eight sub-fractions (C1-C8). The C1 sub-fraction exhibited a single spot (isolate), which was further characterized using ¹H-NMR, 13C-NMR, DEPT spectroscopy and LC-MS.

According to the ¹H-NMR spectrum data, the molecular structure of the C1 isolate consists of 48 protons, four of which have relatively large chemical shifts of 5.34, 5.14, 5.00 and 3.51 ppm, indicating that the protons may have low electron density. In addition, the similarity of the coupling constant (J=15.5 Hz) values between the protons at the chemical shifts of 5.00 and 5.14 ppm indicate that these two protons are in trans-position. Furthermore, proton stacking with very large integration is observed in the ¹H-NMR spectrum and the multiplicity data (m, br d and br s), that indicate the number of neighboring protons or the position of the protons are quite close together. This confirms the structure of the C1 isolate may belong to the steroid class.

Furthermore, the 13C-NMR spectrum of the C1 isolate shows the 29 carbons that make up the structure. The DEPT technique reveals that the isolate possesses three quaternary carbons, 11 methines, 9 methylene and 6 methyls. Carbons with chemical shifts >95.0 ppm belong to the olefinic carbon (alkene) or Csp² carbon. One olefinic quaternary carbon indicates a chemical shift of 140.9 ppm, while three olefinic methine carbons indicate chemical shifts of 121.9, 129.4 and 138.5 ppm, respectively. These four olefinic carbons enable the formation of two double bonds.

By considering the ¹H-NMR, 13C-NMR and DEPT data, the molecular formula of the C1 isolate is C₂₉H₄₈O with double bond equivalence (DBE)=6, of which 2 come from the alkene group created by 4 Csp² atoms and the other 4 are from cyclic carbon.

By comparing the NMR data with a Wiley library database., the hypothesized chemical structure of the C1 isolate is stigmasterol (Fig. 2). Further verification was obtained by comparing the C1 isolate data with the reference (20) (presented in Table II).

The estimated chemical structure of the C1 isolate was validated by LC-MS/MS electrospray ionization. According to the LC-MS/MS data, the C1 isolate exhibited [M+H]⁺ ion at m/z 413.38, with molecular ion fragmentation at m/z 395.37, 351.31, 273.22, 271.21 and 255.21. The [M+H]⁺ ion at m/z 413.38 represents the first ionization of the C1 isolate with a positive ion (H⁺). [M+H]⁺ ion at m/z 413.38 undergoes fragmentation by releasing H₂O molecules as confirmed by the presence of the m/z 395.37 peaks or the m/z 273.22 to the m/z 255.21. The ¹H-NMR spectrum of the C1 isolate is presented in Fig. 3. Additionally, bond breaking occurs in the branching area, such as C17 and C20 bonds or C24 and C27 bonds, since secondary ion radicals and secondary carbocations are more stable than the primary form (branching effect). The proposed mechanism of fragmentation is presented in Fig. 4. Thus, it was concluded that the molecular weight of the C1 isolate is 412.37.

M. citrifolia fruit extract exhibited potent inhibitory activity against α-amylase with an IC₅₀ value of 14.16±5.72 ppm. Similarly, stigmasterol (the C1 isolate) exhibited a prominent inhibitory activity against α-amylase with an IC₅₀ value of 10.29±0.76 ppm which is more potent than that of acarbose, a known inhibitor of the enzyme,with an IC₅₀ value of 43.37±1.56 ppm. The results are presented in Table III.