Phenolic Constituents from the Stems of Morus nigra and their α-Glucosidase Inhibitory Activities

Abstracts A new sanggenon-type flavanone, nigragenon F (1), together with 11 known compounds, trans-resveratrol (2), (E)-4-isopentenyl-3,5,2′,4′-tetrahydroxystilbene (3), notabilisin E (4), notabilisin A (5), morusin (6), petalopurpurenol (7), 8-geranyl-5,7-dihydroxycoumarin (8), 2,4-dihydroxybenzaldehyde (9), 4-ethoxy-2,6-dihydroxybenzoic acid (10), 3-hydroxy-4-methoxybenzaldehyde (11), and 4-hydroxybenzaldehyde (12), were isolated from the stems of Morus nigra. Compound 10 was a new natural product, compounds 3, 4, 7, and 8 were reported from the Morus genus for the first time. All of the isolated compounds were evaluated for their α-glucosidase inhibition activity. Among them, six compounds showed obvious inhibitory effects against α-glucosidase with IC50 values ranging from 1.24 to 19.00 µmol/L.


Introduction
As a metabolic chronic disease, diabetes has severely affected people's health. Evidence suggested that α-glucosidase inhibitors, such as acarbose, miglitol as well as voglibose, can lower the glucose levels in plasma by delaying the absorbance of carbohydrates, and are used clinically to treat diabetes; however, they also bring adverse reactions such as abdominal pain, flatulence, and diarrhea. 1 Thus, the discovery of natural, side-effect-free, and effective α-glucosidase inhibitors from widely sourced medicinal plants are of important value for the treatment of diabetes.
Hyperglycemia is the major symptom of diabetes. It is well known that Morus plants are famous for their antihyperglycemia effects, and has received much attention in diabetes treatment. The isolated alkaloids from Morus plants, such as 1-deoxynojirimycin, and phenolic compo-nents have demonstrated antidiabetes activity by exhibiting potent α-glucosidase inhibitory activity. 2,3 Thus, Morus plants may be a natural source for drug discovery for diabetes therapy.
Morus nigra Linn., as a deciduous shrub or tree, belongs to the Morus genus (Moraceae). The plant of M. nigra was introduced from western Iran in the 16th century and mainly distributed in southern Xinjiang province, China. 4 Our previous program for screening antidiabetic bioactive substances has afforded a series of active compounds from M. nigra. [5][6][7] As a continuous study, this research provided 12 compounds (1-12), including a new sanggenon-type flavanone (1) and a new natural product (10). All of them were evaluated for their α-glucosidase inhibition activity, and six compounds showed significant inhibitory activity with IC 50 values ranging from 1.24 to 19.00 µmol/L. Our article provided novel potential compounds for the treatment of diabetes in the future.

General Methods
Ultraviolet (UV) spectra were collected using a UV-2500 PC instrument (Shimadzu Corporation, Japan). Mass spectrometry was determined on a Waters Xevo G2-XS-Q-TOF. Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker AV III instrument. Electronic circular dichroism spectra were obtained using a JASCO-810 spectropolarimeter. Silica gel (200-300 mesh, Qingdao Haiyang Chemical Co, Ltd.), Sephadex LH-20 (GE Healthcare, Sweden), and RP-C18 (YMC Co., Ltd., Japan) were used for column chromatography (CC). Thin layer chromatography was performed on silica gel HF254 plates using 10% H 2 SO 4 in ethanol (v/v) spray reagents followed by heating. Semipreparative high-performance liquid chromatography (HPLC) was carried out on a LC3050N HPLC using a C18 column (10 Â 250 mm, 5 μm, Waters Corporation, United States) and characteristic UV absorption at 210 nm. Reagents were of analytical reagent grade (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) except for acetonitrile and methanol which were of chromatographic grade.

α-Glucosidase Activity Screening
Compounds 1-12 were evaluated for their α-glucosidase inhibitory activity. All of them were initially tested for their inhibitory rates at the concentration of 100 μmol/L. Preliminary result showed that compounds 3-8 exhibited obvious inhibitory effect with inhibition rates of more than 90%, while the rest of the compounds with inhibition rates of below 50%. Then, IC 50 values of 3-8 were further determined. They showed potent inhibitory activities with IC 50 values ranging from 1.24 to 19.00 µmol/L (►Table 2). Of these, compound 5 showed the highest α-glucosidase inhibitory effect with IC 50 value of 1.24 µmol/L, approximately 800 times stronger than the positive control drug acarbose.

Conclusion
In the present study, phytochemistry investigation on the stems of M. nigra afforded 12 compounds (1-12), including a new sanggenon-type flavanone, a new natural product, and four compounds firstly reported from the Morus genus. The α-glucosidase inhibitory effect test provided different struc-ture-type α-glucosidase inhibitors from M. nigra, not only enriching the library of natural α-glucosidase inhibitors, but also laying experimental basis for the development and utilization of M. nigra as hypoglycemic medicinal plant resources.    Phenolic Constituents from the Stems of Morus nigra Wang et al. e11