General experimental procedures
1D (1H-NMR, 13C-NMR and DEPT) and 2D (COSY, HMQC and HMBC) NMR spectra were measured on Varian Mercury Plus (400 MHz for 1H-NMR and 100 MHz for 13C-NMR) spectrometers with TMS as an internal standard found in Atatürk University, Faculty of Science. HRESIMS data were recorded on an Agilent 6530 Accurate-Mass apparatus and AB Sciex TripleTOF 4600 found in Atatürk University East Anatolia High Technology Application and Research Center (DAYTAM). UV spectra were obtained with a Thermo Scientific Multiscan Go UV‑Vis spectrometer. Open column chromatography (CC) was carried out on silica gel 60 (0.063-0.2 mm) (Merck, Germany). Solvents were purchased from Sigma-Aldrich (USA). TLC analyses were carried out on precoated silica gel 60 F254 plates (Merck) and spots were visualized by spraying with 1% solution of vanillin in concentrated sulfuric acid followed by heating at 110°C. Dinitrosalicylic acid, α-glucosidase enzyme (from Saccharomyces cerevisiae) (EC 18.104.22.168), α-amylase enzyme (from pig pancreas) (EC 22.214.171.124), acarbose, p-nitrophenol-α-D-glucopyranoside from Sigma-Aldrich (USA) were used in the bioactivity assays.
Paliurus spina-christi Mill. fruits were collected in Erzurum City, Uzundere province, Turkey, in July 2016. The plant material was authenticated by Forest Engineer, MSc Mehmet ÖNAL at the Eastern Anatolia Forestry Research Institute. A voucher specimen (No. AUEF 1348) was deposited at the Biodiversity Application and Research Center, Atatürk University, Erzurum, Turkey.
Paliurus spina-christi were dried under shade at room temperature until completely dried. After drying and removing the soil, the fruits are separated from plants and pulverized using a mechanical blender. The dried samples were then extracted with solvents of different polarity.
Extraction and isolation of pure compounds
Paliurus spina-christi Mill. dry fruits (800 g) were powdered and left to maceration overnight with 2 liters of 70% methanol and then extracted 3 times for 3 hours at 40°C using a mantle heater and reflux cooler. The filtered extracts were concentrated to dryness in the rotary evaporator at 40°C and 120 rpm. Methanol extract (33.6 g) in water was fractionated using n-hexane (4 × 500 mL), chloroform (4 × 500 mL), ethyl acetate (6 × 500 mL), and n-butanol (9 × 500 mL), respectively. Sub-extracts and the remaining water phase were concentrated. At the end of the condensation process, 2.2 g n-hexane, 4.1 g chloroform, 2.5 g ethyl acetate, 10.2 g n-butanol and 11.2 g water extracts were obtained. The isolation studies went on n-hexane extract, due to it showed best α-glucosidase inhibitory effect.
The n-hexane extract (2.18 g) was fractionated by a silica gel CC (70–230 mesh) with n-hexane:ethyl acetate (100:0→0:100, v/v) to yield six fractions, Fr. A-F. Fr. B (88.5 mg), Fr. D (161.5 mg) and Fr. F (303.4 mg) were crystallized to obtain compound 2 (19.5 mg), compound 3 (7.4 mg) and a mixture of compounds 1a and 1b (60.8 mg), respectively.
Betulin (1a) and Betulinic acid (1b)
Amorphous, white powder. HRESIMS m/z 425.38021 [(M-OH)+ C30H50O2; calcd. 442.3811) and 455.35860 (M-H)+ C30H48O3; calcd. 456.3603)], 1H NMR and 13C NMR data were identical to those reported in the literatures (Table 2) [13, 14, S1-S7].
Lupeol (Lup-20(29)-en-3β-ol) (2): It was isolated as a white amorphous powder and its molecular formula, C30H50O, was determined by positive HRESIMS ion observed at m/z 409.38285 (M-OH)+ (calcd. 426.3862). 1H NMR and 13C NMR data agree with the literature (Table 2) [15, S8-S13].
β-Sitosterol [(24R)-24Etilkolest-5-en-3β-ol] (3): It was isolated as a white amorphous powder and its molecular formula, C29H50O, was determined by positive HRESIMS ion observed at m/z 414 (M+) (calcd. 414.384). 1H NMR and 13C NMR data agree with the literature (Table 3) [16, S14-S19].
α-Glucosidase enzym inhibitory assay
The α-glucosidase inhibitory effect was determined as previously described method  with slight modifications as described before . All samples (20 µL), enzyme solution (10 µL, 1 Unite/mL) and potassium phosphate buffer (50 µL, 50 mM, pH 6.9) were mixed in the plate. Mixture was incubated at 37°C for 5 min. Then p-nitrophenyl-α-D-glucopyranoside as substrate (20 µL, 3mM) was added for initiation of reaction and mixture was incubated at 37°C for 30 min. After incubation, 0.1 M sodium carbonate (50 µL) was added to all wells to quench the reaction. All solutions were prepared in a buffer system. Acarbose was used as a positive control. Amount of released p-nitrophenol was measured using a 96-well microplate reader at 405 nm. Each assay for all samples was performed in triplicate. Results are given as percentage inhibition of enzyme activity and IC50 value. Percentage inhibition of all samples was calculated using the equation at below:
α-Amylase enzym inhibitory assay
The α-amylase inhibitory effect was determined as previously described method  with slight modifications as described before . All samples (100 mL) and 1% starch solution (100 mL) in 20 mM sodium phosphate buffer (pH 6.9 with 6 mM sodium chloride) were incubated at 25°C for 10 min in 24-well microplate. After incubation, 100 µL α-amylase solution (0.5 mg/mL) was added to each well and the reaction mixtures were incubated at 25°C for 10 min. After incubation, dinitrosalicylic acid color reagent (200 µL) was added to stop reaction. The microplate was then incubated in a boiling water bath for 5 min and cooled to room temperature. It was taken 50 µL from each well and then was added to 96-well microplate. The reaction mixture was diluted after adding 200 µL distilled water and absorbance was measured at 540 nm. Acarbose was used as a positive control. Each assay for all samples was performed in triplicate. Results are given as percentage inhibition of enzyme activity and IC50 value. Percentage inhibition of all samples was calculated using the equation at below:
All experiments were performed in triplicate. Kruskal-Wallis test was used to determine statistical significance. Results were analyzed using SPSS (IBM SPSS Statistics 20, IBM Corporation, Armonk, NY, USA) at the significance level of P = 0.05. The percent inhibition and IC50 value data for extracts and compounds are presented as means ± standard deviation.