The Chemical Constituents of Diaphragma Juglandis Fructus and Their Inhibitory Effect on α-Glucosidase Activity

In our current investigation, 37 constituents (1–37), including 11 megastigmanes (1–11), 17 flavonoids (12–28) and 9 phenylpropanoids (29–37), were isolated from a 70%-EtOH extract of Diaphragma juglandis Fructus. Among them, compounds 1–3, 12 and 29 were new compounds and their structures were elucidated on the basis of physicochemical evidence and meticulous spectroscopic analysis (NMR, HRESIMS and CD). Compounds 13, 16, 21 and 28 showed moderate inhibitory effect on α-glycosidase inhibitory activities, with IC50 values being in the range of 29.47–54.82 µM and stronger than the positive control (acarbose, 60.01 ± 4.82 µM).


Introduction
The walnut (Juglans regia L.) is consumed globally as a high economic value crop [1]. The edible portion of walnuts (kernel) has been processed into many types of foods due to its unique and highly nutritious nature and health-related benefits [2]. However, Diaphragma juglandis Fructus, the dry wooden diaphragm inside the walnut, mainly consists of undigestible fiber and lignin and is usually discarded as waste during the processing of the walnut [3]. In fact, Diaphragma juglandis Fructus is a traditional Chinese medicine and has been used to treat several illnesses such as insomnia, diarrhea, kidney deficiency and reproductive diseases for a long time [4,5]. It has also been used as an herbal tea and a dietary supplement in folk culture [6]. The research shows that Diaphragma juglandis Fructus is rich in a variety of bioactive components, such as flavonoids, saponins, phenolic acids and polysaccharides [7].
Previously, our team focused on the antidiabetic effect of Diaphragma juglandis Fructus, and it was found to improve symptoms of diabetes via the AKT/FoxO1 signaling pathway [8]. As part of our continuous program to identify new potential candidates to control diabetes using natural products, the subsequent phytochemical study led to the isolation of 37 constituents (1-37) from Diaphragma juglandis Fructus, including 11 megastigmanes (1-11) (Figure 1), (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28) and 9 phenylpropanoids (29-37). Among them, compounds 1-3, 12 and 29 were new compounds. Their structures were elucidated on the basis of physicochemical evidence, in-depth NMR spectroscopic analysis and highresolution mass spectrometry. Meanwhile, the α-glucosidase inhibition activities of all isolates were evaluated. The isolation, structural identification and bioactivity evaluation of the obtained compounds are reported herein.   -13)]. In addition, the 13 C-NMR ( Figure S4) and DEPT spectra revealed a quaternary carbon bearing an oxygen function (δ C 76.8), a trisubstituted olefin [δ H 6.15 (1H, br s, H-4), δ C 121.0 (C-4), 168.2 (C-5)] and a conjugated carbonyl carbon (δ C 202.6). The planar structure of 1 was determined to be a bicyclic neomegastigmane skeleton through the interpretation of various NMR experiments [9], including HSQC, 1 H-1 H COSY and HMBC spectra. Namely, the 1 H-1 H COSY experiments of 1 indicated the presence of one partial written in red bold lines ( Figure 2). In addition, obvious long-range correlations were observed between the following proton and carbon pairs in the HMBC experiments: H 2-2 and C-1, 3, 6; H-4 and C-5, 6, 13; H-6 and C-5; H 2 -8 and C-6, 9, 13; H-13 and C-4, 5, 9, 10; H 3 -10 and C-8, 9, 13; H 3 -11 and C-1, 2, 6, 12; H 3 -12; and C-1, 2, 6, 11 (blue arrow in Figure 2). Next, the relative stereostructure of 1 was clarified to be 9β-OH and 13β-OH by the NOESY experiment, in which correlations were observed between the following proton pairs: H 3 -12 and H-6; H-6 and H-13; H-13; and H 3 -10 ( Figure 3). Based on the above-mentioned evidence, the structure of 1 was elucidated to be a bicyclic megastigmane named diamegastigmane A, as shown in Figure 1. Bicyclic megastigmanes are a small but growing group of natural products, and a possible biosynthetic pathway for 1 is further proposed in this paper ( Figure 4).      (Table 1) for 2 closely resembled those of apocynol B, and further analysis of the 2D NMR spectra revealed that the significant difference was the absence of one olefin in the side chain [10]. The 1 H-1 H COSY spectrum of 2 enabled the identification of the H 2 -7/H 2 -8/H-9/H 3 -10 unit. Further, HMBC correlations from H 2 -7 to C-1, C-5 and C-6 arranged the carbon chain that connected to C-6 ( Figure 2). Moreover, the CD spectrum indicated a 6S-configuration due to a positive Cotton effect at 323 nm and a negative Cotton effect at 272 nm [11]. However, the chirality of C-9 in the side chain was difficult to assign due to the lack of direct evidence and thus, needs to be further determined. Consequently, the structure of 2 was identified ( Figure 1) and named diamegastigmane B.

Results and Discussion
Compound 3 has a molecular formula of C 26 H 36 O 9 based on the HRESIMS ion at m/z 493.2442 ([M+H] + , calcd 493.2437). The 1 H-NMR spectrum (Table 1) of 3 showed two doublet signals at δ H 7.89 (2H, d, J = 8.8 Hz, H-2 ,6 ) and 6.83 (2H, d, J = 8.8 Hz, H-3 ,5 ), attributed to the AA BB system in a 1,4-substituted benzene ring, assigned to the 4-hydroxybenzoyl group. The location of the 4-hydroxybenzoyl group was established at C-6 in the pyranosyl moiety according to the long-range correlation from a proton signal at δ H 4.34 (1H, d, J = 7.7 Hz, H-1 ) to a carbon signal at δ C 167.9 (C-7 ) in the HMBC spectrum ( Figure 2).
The 1D NMR spectroscopic data (Table 1) of 3 showed significant similarity to those of hirtionoside C, except for the replacement of the gallic acid at the 6 -position by a 4hydroxybenzoic acid [12]. The absolute configuration at the 6-position was confirmed to be R by the CD spectrum (positive Cotton effect at 333 nm) [13], and those at the 9-position were also determined to be R by comparing 13 C NMR data according to the β-Dglycosylation-induced shift-trend rule [14]. Therefore, the structure of 3 was determined ( Figure 1) and named diamegastigmane C.

Glucosidase Inhibitory Assay
Alpha-glucosidase is an enzyme that hydrolyzes the carbohydrates to monosaccharides in the final step of carbohydrate digestion. Therefore, inhibiting the activity of αglucosidase can effectively inhibit sugar uptake, thereby achieving lowered blood sugar [46]. Diaphragma juglandis Fructus was found to improve symptoms of diabetes [8] and the total flavonoids from it showed significant α-glucosidase inhibitory activities [47]. In order to search for bioactive substances to treat type 2 diabetes using Diaphragma juglandis Fructus, all isolated constituents (1-37) were assessed for antidiabetic activity using an in vitro α-glycosidase inhibition assay. As shown in Table 4,   Most of the bioactive compounds were flavonoids, suggesting that flavonoids might be the main bioactive substances contributing to the α-glucosidase inhibitory activity of Diaphragma juglandis Fructus. Furthermore, the structures of the A, B and C rings in the flavonoids were closely related to the inhibitory activity. Consistent with the previous reports, comparison among quercetin (21) and luteolin (28) revealed that hydroxylation at the 3-position of flavone enhanced the inhibitory effect. Comparison among (+)-catechin (16), quercetin (21) and luteolin (28) suggested the saturation of the 2,3-double bond in the C ring seemed to decrease the inhibitory activity [48]. In addition, among the taxifolin (13) and derivatives (14,15), taxifolin showed the strongest inhibitory effect, indicating that the presence of a sugar moiety at C-3 may be responsible for the lowered activity [49]. In addition, galloyl moieties strengthen the inhibitory effects of flavonoids against the α-glucosidase (such as compound 26) [50].

General Experimental Procedures
Optical rotations were determined by a Jasco P-2000 digital polarimeter. CD spectra were recorded on a Bio-Logic MOS-450 spectropolarimeter. One-dimensional (1D) and twodimensional (2D) NMR spectra were measured on a Bruker 600 spectrometer. HRESIMS data were obtained using a Q Exactive Focus LC-MS/MS spectrometer (Thermo Fisher, MA, USA) or Triple TOF™ 5600 MS/MS system from Applied AB Sciex (Foster City, CA, USA). Medium-pressure liquid chromatography was performed with Buchi C610. To perform the preparative HPLC separation, a C 18 preparative HPLC colum (21.2 mm × 250 mm, 5 µm, Sharpsil-U C18) on a Shimadzu LC-16P instrument equipped with an RID-20A refractive index detector was used for purification. Silica gel (200-300 mesh, Qingdao Marine Chemical Ltd., Qingdao, China) and RP-18 reversed-phase silica gel (YMC Company Ltd., Tokyo, Japan) were used for column chromatography. All organic solvents were analytical grade (Tianjin zhiyuan Chemical Regents Co., Ltd., Tianjin, China).

Plant Material
Diaphragma juglandis Fructus was purchased from Anguo Chinese medicinal materials markets (Hebei Province) in March 2021 and identified by Professor Xiang-ping Pei (Shanxi University of Chinese Medicine). A voucher specimen (No. 20210301) was deposited at Shanxi Modern Chinese Medicine Engineering laboratory (Shanxi University of Chinese Medicine).

Acid Hydrolysis of Compounds 3, 12 and 29
In order to determine the absolute configuration of monosaccharides in the new compounds, the acid hydrolysis of compounds 3, 12 and 29 were performed according to the previous literature [51]. The n-hexane fractions were then detected by GC-MS with a DB-5 capillary column, and the absolute configuration of sugar components was confirmed to be D-glucose, L-arabinofuranose and D-glucose in compounds 3, 12 and 29, respectively, compared with standards.

α-Glucosidase Inhibitory Assay
The α-glucosidase inhibition assay was performed according to a previous report [52] with a slight difference: the concentration of α-glucosidase was diluted to 0.15 unit/mL. The volume of α-glucosidase added to the 96-well plate was 10 µL. Acarbose was used as the positive control.

Conclusions
In summary, a detailed phytochemical investigation on the EtOAc partition of 70% ethanol extract of Diaphragma juglandis Fructus was carried out to afford 37 constituents in this research, and five of them were new structures (1-3, 12, 29). Their structures were elucidated based on MS and NMR spectroscopic data and comparison with data reported in the literature. Compounds 1-3 were new megastigmanes. Among them, compound 1 was a bicyclic neomegastigmane, and a plausible biogenetic pathway for it was further discussed in this paper. Compound 12 was a new chromone with α-L-arabinofuranoside. Compound 29 was a new phenylpropanoid. The α-glucosidase inhibition activity was also investigated. Compounds 13, 16, 21 and 28 were found to be quite potent and most of them were flavonoids, suggesting that flavonoids might be the main bioactive substances contributing to the α-glucosidase inhibitory activity of Diaphragma juglandis Fructus. These findings also revealed that these compounds could be target compounds for the development of new antidiabetic agents.
Author Contributions: J.T. and Y.C. carried out the extraction and purification process. S.W. and J.L. performed the whole experiment process as assistants. H.R. contributed to the revision of the paper. Y.Q. assisted with the α-glucosidase inhibitory assay. Q.L. contributed to the elucidation of the chemical structures. Y.W. designed the whole research plan and wrote the paper. All authors have read and agreed to the published version of the manuscript.