Abstract

INTRODUCTION

Euphorbiaceae family is one of the widespread plants in Indonesia that has beneficial health properties that potential to be studied. Some of the Euphorbiaceae was reported for having a hypoglycemic activity, for example, Phyllanthus emblica Linn,[1] Phyllantus sellowianus,[2] Antidesma celebicum (Miq),[3] and Antidesma bunius (L.) Spreng.[4] Hypoglycemic activity is the main character for identifying the antidiabetic agent. Asian countries, including Indonesia, contribute to >60% of the world's diabetic population as the prevalence of diabetes is increasing in this country.[5] One of the widely used antidiabetic drugs is α-glucosidase inhibitor. A. bunius (L.) Spreng which commonly known as Buni, is an edible plant found in Asia, including Indonesia. Although some preliminary studies on the antidiabetic properties of this plant have been reported, the results were still conflicting. The previous study showed that the 80% EtOH extract of Buni stem bark strongly inhibited α-glucosidase with half maximal inhibitory concentration (IC₅₀) value of 3.90 μg/mL.[4] Another research in contrary stated that ethyl acetate (EtOAc) fraction of Buni stem bark has the highest activity with IC₅₀ value of 5.73 μg/mL.[6] Moreover, based on our knowledge, there is no information regarding the active chemical substance of Buni stem bark. Therefore, we designed this present study to determine the highest α-glucosidase inhibitor activity fraction of Buni stem bark and identify its chemical substances.

MATERIALS AND METHODS

RESULTS AND DISCUSSION

α-Glucosidase inhibitory activity assay was performed in n-hexane, EtOAc, and MeOH fraction of Buni stem bark [Figure 1]. Table 1 shows that the ethyl acetate fraction from the liquid chromatography has the highest inhibitory activity against α-glucosidase with IC₅₀ value of 19.33 μg/mL while acarbose and miglitol as standards have IC₅₀ value of 5.75 and 59.76 μg/mL, respectively. It concluded that ethyl acetate fraction has a higher α-glucosidase inhibitory activity than miglitol, in line with the previous study.[8]

Open in a separate window

Figure 1

Buni stem bark

Table 1

Half maximal inhibitory concentration value of standards and Buni stem bark fraction

Open in a separate window

EtOAc fraction was subfractionated by column chromatography and gave 11 subfraction. EtF II (8–11) and EtF IV (F17–18) produce white needles isolate that soluble in CHCl₃ on recrystallization, nammed B1 (11.43 mg) and B2 (10 mg). Further purification was done by repeated column chromatography in EtF V (F19–29) and produced white powder isolate that soluble in DMSO, named B3 (10.22 mg). Each isolate was tested for the inhibition of α-glucosidase and gave B3 as the most active isolate with IC₅₀ value of 18.49 μg/mL as shown in Table 2. The isolated compounds were identified by spectroscopic analysis as well as by comparison of their spectral data with previously reported values.

Table 2

Half maximal inhibitory concentration value of the isolates from subfractionated ethyl acetate fraction

Open in a separate window

IR spectrum of B1 exhibited carbonyl (νmax: 1716.7 cm⁻¹), methyl, methylene, and methine (νmax: 2920.4; 2868.6; 1462.09; 1388.79 cm⁻¹). The ¹H-NMR spectrum of B1 using CHCl3 as solvent revealed signals for 8 methyl of triterpenoid (0.71(s); 0.86(m); 0.94(s); 0.99(d); 1.26(s); 1.04(s) ppm). The ¹³C-NMR spectrum showed the existence of 30 carbons which included 7 quaternary carbons (28.35; 29.80; 37.60; 38.46; 39.86; 42.33; and 213.5 ppm), 4 methine groups (42.94; 53.26; 58.39; dan 59.63 ppm), and 8 methyl groups (7.01; 14.83; 18.13; 18.86; 20.44; 31.96; 32.26; and 35.21 ppm). DEPT 135° spectrum showed 11 methylene groups (18.41; 22.47; 30.10; 32.59; 32.93; 35.51; 35.79; 36.17; 39.42; 41.45; and 41.71 ppm). Table 3 shows that these data were identical to those reported friedelin (C₃₀H₅₀O).[8] 2D-NMR spectrum also reveals that it suggests triterpenoid typical friedelin [Figure 2].

Table 3

The nuclear magnetic resonance spectral of B1 and friedelin[8]

Open in a separate window

Open in a separate window

Figure 2

Friedelin

Friedelin is derived from lupane triterpene. The previous study showed that friedelin isolated from Ficus drupacea leaves which tested for α-glucosidase inhibitory activity had 20.1% inhibition value at 100 μM.[9]

IR spectrum of B2 exhibited hydroxyl (νmax: 2900 cm⁻¹), vinyl (1732 cm⁻¹), methyl, and methylene (νmax: 1391.79 cm⁻¹; 1462.09). The ¹³C-NMR spectrum showed the presence of 29 carbons which consists of 3 quaternary carbons (36.59 ppm; 42.10 ppm; and 140.89 ppm), 11 methylene groups (–CH₂) which was detected by DEPT 135° spectrums (21.15; 23.12; 24.38; 26.09; 28.34; 31.44; 32.80; 34.01; 37.32; 39.84; and 42.39 ppm), 9 methine groups (–CH) (which was showed on 29.19; 31.98; 36.59; 45.89; 50.20; 56.12; 56.84; 71.63; and 121,77 ppm), and 6 –CH₃ groups (11.9; 12.04; 18.84; 19.08; 19.4; and 19.89 ppm). Carbons on 140.8 and 121.7 ppm indicated the presence of C = C, while carbons on 71.6 ppm indicated the presence of –OH. The ¹H-NMR spectrum of B2 revealed signals for olefinic proton on δ H5.29 (m, 1 H) that similar with H₆, and signal on δ H 3.44 (m, 1H) similar with oxymethine proton H₃ typical sterol. Literature study showed the similarity between B2 and β-sitosterol (C₂₉H₅₀O),[10] supported by the correlation showed in 2D-NMR spectrums [Table 4].

Table 4

The nuclear magnetic resonance spectral of B2 and β-sitosterol[10]

Open in a separate window

Isolation and bioactivity test of β-sitosterol [Figure 3] was done in many kinds of plant. One of them is from the root of Acorus calamus that showed its activity in inhibit α-glucosidase. With IC₅₀ value of 18.92 μg/mL.[11]

Open in a separate window

Figure 3

β-sitosterol

IR spectrum of B3 exhibited carbonyl typical carboxylate (νmax: 3473–2500 cm⁻¹), vinyl (1677 cm⁻¹), and methylene (νmax: 1462.09 cm⁻¹). ¹³C-NMR spectrums showed 30 atom carbons which consist of 7 quatenary carbons (38.5; 36.7; 38.5; 41.9; 55.4; 150.3 (specific for alkena); and 177.2 ppm (specific for carbonyl typical carboxylate)), 6 methine (CH) (which was detected in 76.7 (specific for -HC-OH); 54.8; 49.9; 37.5; 48.5; and 46.9 ppm), and 6 methyl (CH₃) (which was detected in 28.1; 15.73; 15.8; 14.3; 15.9; and 18.9 ppm). DEPT 135° spectrums gave 11 methylene groups (38.2; 27.1; 17.9; 33.9; 20.4; 25.0; 29.2; 31.7; 36.30; 30.09; and 109.6 ppm [which specific for alkene double bond]). ¹H-NMR spectrums revealed two multiple signals in δH 4.68 and 4.55 which is similar with vinyl hydrogen (H-29). Signals δH 1.63; 0.92; 0.75; 0.86; and 1.2 are similar with methyl groups. Based on the spectral data [Table 5], the structure of B3 was assigned as betulinic acid (C₃₀H₄₈O₃) [Figure 4] further supported by the 2D-NMR spectrum and spectral data reported from the literature.[12]

Table 5

The nuclear magnetic resonance spectral of B3 and betulinic acid[12]

Open in a separate window

Open in a separate window

Figure 4

Betulinic acid

Betulinic acid and its derivates have been reviewed as one of natural constituents that showed potent α-glucosidase inhibitory activity.[13] Betulinic acid is also derived from lupane triterpene. The previous research stated that betulinic acid isolated from Morus alba root had a potentiality as antidiabetic agent under in vivo assay on streptozotocin-induced diabetic mice.[14] Another isolation of betulinic acid was done in Dillenia indica leaves, then tested by in vitro assay to inhibit α-glucosidase and α-amylase activity. The results were 52.2% inhibition to α-glucosidase and 47.4% inhibition to α-amylase at 50 μg/mL concentration of betulinic acid.[15] Another research reported that the betulinic acid gave 1.42 μmol/L EC₅₀ into TGR5 receptor. TGR5 is one of the insulin secretion mediator from β-cell pancreas.[16] Those previous reports supported our results on antidiabetic activity of β-sitosterol, friedelin, and betulinic acid isolated from Buni stem bark.

CONCLUSION

Ethyl acetate fraction of Buni stem bark has a higher α-glucosidase inhibitory activity than miglitol. This study also afforded three triterpenes: friedelin (B1), β-sitosterol (B2), and betulinic acid (B3) from the stem bark of A. bunius (L.) Spreng, in which betulinic acid (B3) showed the highest α-glucosidase inhibitory activity.

Acknowledgements

REFERENCES