Six New Phenolic Glycosides from the Seeds of Moringa oleifera Lam. and Their α-Glucosidase Inhibitory Activity

Plant-derived phytochemicals have recently drawn interest in the prevention and treatment of diabetes mellitus (DM). The seeds of Moringa oleifera Lam. are widely used in food and herbal medicine for their health-promoting properties against various diseases, including DM, but many of their effective constituents are still unknown. In this study, 6 new phenolic glycosides, moringaside B–G (1–6), together with 10 known phenolic glycosides (7–16) were isolated from M. oleifera seeds. The structures were elucidated by 1D and 2D NMR spectroscopy and high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) data analysis. The absolute configurations of compounds 2 and 3 were determined by electronic circular dichroism (ECD) calculations. Compounds 2 and 3 especially are combined with a 1,3-dioxocyclopentane moiety at the rhamnose group, which are rarely reported in phenolic glycoside backbones. A biosynthetic pathway of 2 and 3 was assumed. Moreover, all the isolated compounds were evaluated for their inhibitory activities against α-glucosidase. Compounds 4 and 16 exhibited marked activities with IC50 values of 382.8 ± 1.42 and 301.4 ± 6.22 μM, and the acarbose was the positive control with an IC50 value of 324.1 ± 4.99 μM. Compound 16 revealed better activity than acarbose.


Introduction
Diabetes mellitus (DM) is a metabolic disorder characterized by persistent hyperglycemia due to insufficient insulin secretion or impairment of islet function, which is now one of the major threats to human health in the 21st century [1].Type II DM (T2DM, i.e., non-insulin-dependent DM) accounts for about 90% of the total DM patients in the current world [2].Glycemic control is considered an effective therapy for the treatment of T2DM [3].As is well-known, α-glucosidase is a carbohydrate hydrolase that acts on the terminal α (1→4) bonds of starch and disaccharides to release α-glucose in the brush border of the small intestine [4].Through inhibiting the activities of α-glucosidase, the absorption of glucose in the intestine is slowed down, and the blood sugar level can be well managed [5].Therefore, α-glucosidase inhibitors have become the focus of hypoglycemic drug research in recent years.At present, the most common α-glycosidase inhibitors are acarbose, voglibose, and miglitol.However, these inhibitors all have serious side effects, such as flatulence, abdominal cramping, and diarrhea [6].Natural products are a rich source of safe and highly effective α-glucosidase inhibitors.Most of these natural bioactive compounds not only reduce hyperglycemia but are also associated with fewer side effects than currently applied α-glycosidase inhibitors and offer nutritional benefits for DM patients [7].In recent years, a large number of studies have shown that compounds with α-glucosidase inhibitory activity have been screened from natural products [8].
Moringa oleifera Lam.belongs to the genus Moringa (family Moringaceae), native to the dry tropical forests of northwestern India [9].M. oleifera is referred to as a "miracle tree" because of its rich nutritional and pharmacological properties [10].It has high nutritional value, including protein, fiber, and a variety of vitamins, especially in seeds [11].The seeds are rich in oils and unsaturated fatty acids, which can be used as a potential source of edible oil [12].The seeds have many benefits for humans.These have aroused the interest of researchers.At present, there are few studies on the chemical constituents of the seeds; the biological activities are mainly directed to the crude extracts, and the pharmacodynamic material basis is not clear.To date, only several flavonoids, phenolic glycosides, and sterols [13][14][15] have been reported, which exhibit significant properties such as anti-hyperglycaemic, anti-inflammatory, anti-oxidation, and so on [16][17][18].
flatulence, abdominal cramping, and diarrhea [6].Natural products are a rich source of safe and highly effective α-glucosidase inhibitors.Most of these natural bioactive compounds not only reduce hyperglycemia but are also associated with fewer side effects than currently applied α-glycosidase inhibitors and offer nutritional benefits for DM patients [7].In recent years, a large number of studies have shown that compounds with α-glucosidase inhibitory activity have been screened from natural products [8].
Moringa oleifera Lam.belongs to the genus Moringa (family Moringaceae), native to the dry tropical forests of northwestern India [9].M. oleifera is referred to as a "miracle tree" because of its rich nutritional and pharmacological properties [10].It has high nutritional value, including protein, fiber, and a variety of vitamins, especially in seeds [11].The seeds are rich in oils and unsaturated fatty acids, which can be used as a potential source of edible oil [12].The seeds have many benefits for humans.These have aroused the interest of researchers.At present, there are few studies on the chemical constituents of the seeds; the biological activities are mainly directed to the crude extracts, and the pharmacodynamic material basis is not clear.To date, only several flavonoids, phenolic glycosides, and sterols [13][14][15] have been reported, which exhibit significant properties such as anti-hyperglycaemic, anti-inflammatory, anti-oxidation, and so on [16][17][18].
Herein, in the current study, we have studied the chemical constituents from an 85% ethanol extract of M. oleifera seeds and six new phenolic glycosides (1-6); ten known phenolic glycosides (7-16) (Figure 1) have been isolated and identified.All the secondary metabolites were evaluated for their inhibitory activities against α-glucosidase.
Compound 1 was obtained as a colorless viscous oil.Its molecular formula was determined as C 15 2).The chemical shifts and coupling constants of H-1 indicated that the sugar is linked to the aglycone with α-glycosidic linkage.The singlet methylene signal at C-4 was confirmed by the HMBC correlation from δ H 4.43 to C-4 and C-3.The HMBC correlations from δ H 3.53 to C-7 indicated that the ethoxyl group was attached to C-7 (Figure 2).The gas chromatography (GC) analysis showed that the derivative of acid hydrolysis from 1 had the same retention time (t R = 26.24min) as the derivative of authentic L-rhamnose.Thus, compound 1 was identified as a phenolic glycoside derivative and named 4-(α-L-rhamnosyl) benzyl ethyl ester, which we trivially named moringaside B. All the 1 H and 13 C-NMR data of compound 1 were assigned (Table 1).The location of the singlet methylene signal at C-4 was confirmed by the HMBC correlation from δ H 5.83 to C-4 , C-3 , and C-5 .Other sugars, C-2 and C-3, were attached to C-7 by oxygen atoms, respectively, which were confirmed by the HMBC correlation from δ H 4.68 to C-7 and δ H 4.89 to C-7 (Figure 2).The 1 H-1 H homonuclear chemical-shift correlated spectroscopy (COSY) spectrum correlations (Figure 2) of H-1/H-2, H-2/H-3, H-3/H-4, H-4/H-5, and H-5/H-6 showed the assignment in the protons of the sugar moiety.And this sugar was confirmed to α-configuration by the chemical shift and coupling constant of δ H 5.11 (1H, d, J = 1.5 Hz).The GC analysis spectrum showed that the acid hydrolysate of 2 had the same retention time (t R = 26.24min) with the derivative of authentic sample L-rhamnose.The HMBC correlation from δ H 3.70 (1H, m) and 3.50 (1H, m) to C-1, as well as the 1 H-1 H COSY correlation of H-8/H-9 was observed, suggesting that the ethoxyl group was located at C-1.The nuclear overhauser effect spectroscopy (NOESY) spectrum correlations of H-7 with H-1/H-5 and H-1 with H-3 revealed their co-facial relationship, and they were assigned arbitrarily as α-oriented, while the correlations of H-4 with H-6/H-2 indicated that these protons were β-oriented (Figure 3).The absolute configuration of C-1/C-2/C-3/C-4/C-5/C-7 was assigned as 1R/2R/3S/4R/5R/7R by comparing the calculated ECD data (Figure 4) with the experimental data.Consequently, compound 2 was identified as (1R, 2R, 3S, 4R, 5R, 7R)-O-ethly-2,3-di-O-(1 -O-α-L-Rha-phenylmethylene)α-L-rhamnopyranoside and trivially named moringaside C. All the 1 H and 13 C-NMR data of compound 2 were assigned (Table 1).

2.2.α-Glucosidase Inhibitory Activity Evaluation
α-glucosidase is a key catalytic enzyme for carbohydrate digestion and glucose release.Inhibition of α-glucosidase can delay glucose uptake and reduce postprandial blood glucose levels, which may inhibit the progression of DM [31].Thus, all the isolated phenolic glycosides were evaluated for their inhibitory activities of α-glucosidase.As shown in Table 3, compared to the positive drug acarbose with an IC50 value of 324.1 ± 4.99 µM, compound 16 revealed excellent inhibitory activity of α-glucosidase with an IC50 value of 301.4 ± 6.22 µM, while compound 4 showed moderate activity with an IC50 value of 382.8

α-Glucosidase Inhibitory Activity Evaluation
α-glucosidase is a key catalytic enzyme for carbohydrate digestion and glucose release.Inhibition of α-glucosidase can delay glucose uptake and reduce postprandial blood glucose levels, which may inhibit the progression of DM [31].Thus, all the isolated phenolic glycosides were evaluated for their inhibitory activities of α-glucosidase.As shown in Table 3, compared to the positive drug acarbose with an IC 50 value of 324.1 ± 4.99 µM, compound 16 revealed excellent inhibitory activity of α-glucosidase with an IC 50 value of 301.4 ± 6.22 µM, while compound 4 showed moderate activity with an IC 50 value of 382.8 ± 1.42 µM.Other compounds had low inhibitory activity against α-glucosidase and are not listed in Table 3.

Plant Material
The seeds of M. oleifera were collected from Yunnan Province, People's Republic of China, and identified by Shao-Huan Liu, a senior experimentalist at Guizhou Medical University.The voucher specimens were stored in the Engineering Research Center for the Development and Application of Ethnic Medicine and TCM (Ministry of Education), Guizhou Medical University, Guiyang, China.

Acid Hydrosis and Sugar Identification
The sugar was identified according to the established method [32].Compounds 1-6 (each 0.3 mg), respectively, were dissolved with 2 mol/mL Hcl solution (2 mL) at 95 • C for 3 h.After cooling to room temperature, the ethyl acetate was added to the reaction solution and extracted three times.The water-soluble layer was dried to obtain the sugar residual.The sugar residuals, D-rhamnose (0.5 mL) and L-rhamnose (0.5 mL), separately, were added pyridine (0.4 mL) and L-cysteine methyl ester hydrochloride (1.0 mg), then heated at 60 • C for 1 h.N-trimethylsilyllimidazole (0.15 mL) was added to the mixture and reacted at 60 • C for 1 h again.Next, the reaction solution was dried, then dissolved in water (1.0 mL) and extracted with n-hexane (0.5 mL) three times.The organic layer was directly analyzed by GC analysis.The peaks of the acid-hydrolyzed derivatives of compounds 1-6 coincide with the derivatives of the authentic sample L-rhamnose.

Electronic Circular Dichroism Calculation of Compounds 2-3
The theoretical calculations were carried out using Gaussian 09 [33].At first, all conformers were optimized at PM6. Room-temperature equilibrium populations were calculated according to the Boltzmann distribution law, based on which dominative conformers of population over 1% were kept.The chosen conformers were further optimized at B3LYP/6-31G(d) in the gas phase.Vibrational frequency analysis confirmed the stable structures.ECD calculations [34] were conducted at the B3LYP/6-311G(d,p) level in methanol with the IEFPCM model using the time-dependent density functional theory

Compound 5
was obtained as yellow oil.Its negative HR-ESI-MS showed an [M -H] − ion at m/z 424.1600, which is in accordance with the molecular formula C 20 H 27 O 9 N (calculated for C 20 H 26 O 9 N, 424.1602), indicating 8 degrees of unsaturation.The IR spectrum of 5 shows frequencies at 3394, 2933, 2252, 1612, 1510, 1236, 1064, and 1022 cm −1 and was assigned to a hydroxyl, a methylene group, a cyanogen group, a benzene ring, and an aromatic ether bond.The 1D and 2D NMR data of compound 5 showed a high degree of similarity to the compound 4-[(β-D-glucopyranosyl)-(

Table 1 .
The 1 H and13C NMR spectroscopic data of compounds 1

Table 1 .
The 1 H and13C NMR spectroscopic data of compounds 1

-3 in CD 3 OD. NO. 1 a NO. 2 b 3 b δ H (J in Hz) δ C δ H (J in Hz) δ C δ H (J in Hz) δ
a : NMR data (δ) were measured at 400 MHz for 1 H and 100 MHz for13C; b : NMR data (δ) were measured at 600 MHz for 1 H and 150 MHz for13C; ov: overlapping signals within the same column.