Chemical Constituents from Mentha haplocalyx Briq. (Mentha canadensis L.) and Their α-Glucosidase Inhibitory Activities

Abstract Mentha haplocalyx (Mentha canadensis) is widely used as a medicinal plant in traditional Chinese medicine, and the extracts of its aerial parts are found to significantly inhibit the activity of α-glucosidase with an IC50 value of 21.0 μg/mL. Bioactivity-guided isolation of the extracts afforded two new compounds (1 and 2), together with 23 known ones (3–25). Their structures were established by extensive spectroscopic analyses (1D and 2D NMR, MS, IR and UV). Compounds 1–17 and 21–25 were evaluated for their α-glucosidase inhibitory activities. Compound 11 was the most active ones with an IC50 values of 83.4 μM. These results verify the α-glucosidase inhibitory activity of M. haplocalyx (M. canadensis) and specify its active compounds for the first time. Graphical Abstract Electronic supplementary material The online version of this article (10.1007/s13659-019-0207-0) contains supplementary material, which is available to authorized users.


Introduction
Diabetes mellitus is a chronic disease caused by inherited or acquired deficiency in insulin secretion and by decreased responsiveness of the organs to secreted insulin [1]. Such a deficiency results in an increased blood glucose level and in turn damages body systems including blood vessels and nerves [2]. It is one of the most serious diseases worldwide and developing with an increase in obesity and ageing [3]. An efficiently therapeutic approach is to retard absorption of glucose through the inhibition of carbohydrate-hydrolysing enzymes such as α-amylase and α-glucosidase in the digestive organs [4]. Clinically, acarbose and voglibose have been 1 3 used as effective α-glucosidase inhibitors to delay glucose absorption [5].
Mentha haplocalyx Briq. (Mentha canadensis L.), a perennial herbaceous plant of the family Lamiaceae, is widely distributed in southwest of China and popularly used in food, cosmetics and medicines. As a traditional Chinese medicine, it is clinically used to treat diseases in the nerve center, breath, procreation and digestive systems [6]. Pharmacological studies of M. haplocalyx (M. canadensis) revealed various biological activities, such as antimicrobial, antiinflammatory, antioxidant, antitumor, gastrointestinal protective, and hepatoprotective activities [7]. A large number of volatile compounds were reported from M. haplocalyx (M. canadensis), as well as a few polyphenolic acids, flavonoids, monoterpenoids, and glycosides, which might contribute to the medicinal benefits of this plant [8].
Several findings have depicted the potential antidiabetic capability of genus Mentha. M. piperita could alleviate hyperglycemia induced by streptozotocin-nicotinamideinduced type 2 diabetes in rats, and cause a reduction of glycemia in human [9,10]. However, no report has referred to the active compounds of Mentha responsible for its antidiabetic capability. Our preliminary bioassay revealed that extracts of the aerial parts of M. haplocalyx (M. canadensis) exhibited significant α-glucosidase inhibitory activity with an IC 50 value of 21.0 μg/mL. Subsequently, two new compounds (1 and 2) (Fig. 1) and 23 known ones  were isolated and identified through bioactivity-guided fractionation. This paper described the isolation, identification, and α-glucosidase inhibitory activity evaluation of these compounds.
Comparing the chemical structures and activity of these compounds, it can be found that triterpenoids (2 and 21), jasmonoid glucosides (16 and 17) and N-containing compounds (22)(23)(24)(25) were not responsible for the α-glucosidase inhibitory activity of the extracts from M. haplocalyx (M. canadensis). Among the monoterpene glucosides (1 and 3-10), only compounds 3 and 4 manifested moderate inhibitory activity against α-glucosidase, while 5 and 6 did not. Thus, it might be assumed that the absence of cyclic double bond in 3 and 4 markedly decreased their inhibitory activity.

General Experimental Instruments and Procedures
LC-MS analyses were performed on a UFLC/MS-IT-TOF apparatus and the analytical conditions were set as previously reported [31]. Mass spectra were measured through a Waters AutoSpec Premier P776 (Waters, USA) mass spectrometer. Optical rotations were measured through a Jasco model 1020 digital polarimeter (Horiba, Tokyo, Japan). UV and IR (KBr) spectra were recorded on a Shimadzu UV2401PC spectrophotometer (Shimadzu, Kyoto, Japan) and a Bio-Rad FTS-135 spectrometer (Hercules, California, USA), respectively. NMR spectra were recorded on the DRX-500 or AdvanceIII-600 NMR (Bruker, Bremerhaven, Germany) spectrometers with TMS as an internal standard.

Plant Material
The

Extraction and Isolation
The aerial parts of fresh M. haplocalyx (M. canadensis) (20 kg) were powdered and extracted with 50% ethanol for three times at room temperature (each 200 L). The combined extraction was concentrated in vacuo to yield a residue. The residue was then suspended in water and extracted with ethyl acetate (Fr.A, 130 g). The aqueous phase was subjected to D101 macroporous adsorption resin using a step gradient elution with C 2 H 5 OH-H 2 O (0:100, 50:50 and 90:10, v/v) as the mobile phase to give three fractions (Fr.B-Fr.D).

Inhibitory Assay of α-Glucosidase
The α-glucosidase inhibitory activity was measured in a 96-well microtiter plate based on p-nitrophenyl-α-dglucopyranoside (PNPG, Yuanye Biosciences Co. Ltd., Shanghai, China) as a substrate following the reported method with slight modifications [32]. In brief, 5.0 mM PNPG (20 μL) and 20 μL tested compounds of dissolved in 10 μL DMSO and 990 μL phosphate buffer (PB, 0.1 M, pH = 6.8) were sequentially added to a 96-well plate to be mixed. The mixture was incubated at 37 °C for 5 min. Reactions were initiated by addition of 2.0 U/mL α-glucosidase (20 μL, Yuanye Biosciences Co. Ltd., Shanghai, China) in PB. The reaction mixture was incubated at 37 °C for 15 min. Then, the incubation solution was stopped the reaction by adding 0.2 M Na 2 CO 3 (40 μL). The absorbance was recorded at 405 nm by a Bio-Rad 680 microplate reader (Hercules, CA, USA). The negative control was set by adding PB instead of the sample using the same procedure for the tests. Acarbose (Bayer) dissolved in PB was utilized as the positive control. The blank was set by adding phosphate buffer instead of the α-glucosidase using the same method. Inhibition rate (%) = [(ODnegative control − ODblank) − (ODtest − ODtest blank)]/(ODnegative blank − ODblank) × 100%. All data were subjected to an analysis of variance using SPSS 18.0. The significant differences in inhibition rates between the treatment and blank control were calculated using one-way analysis of variance (ANOVA).

Conclusion
In this study, the extracts of the aerial parts of M. haplocalyx (M. canadensis) were firstly found to exhibit significantly inhibitory activity against α-glucosidase. Two new compounds (1 and 2) and 23 known ones were isolated and identified through bioactivity-guided fractionation. Among them, compounds 3-9 and 24 were reported from M. haplocalyx (M. canadensis) for the first time, while compounds 10-20, 22-23 and 25 were firstly isolated from the genus Mentha. Bioactivity assay further traced the active compounds (3, 4 and 11), whose inhibitory activity against α-glucosidase had not been reported before. It was noted that the monoterpene glucosides and the ionone glycosides endowed this plant with the α-glucosidase inhibitory activity.