Hong-Xing Liu, Jun-Zeng Ma, Yan-Song Ye, Jian-Jun Zhao, Shi-Jie Wan, Xin-Yue Hu,2 and Gang Xu*

Abstract Diabetes is an urgent health issue characterized by ethnic and regional variations, and is inseparable from the different dietary habits.It is worthy to note that the incidence of diabetes in Bai nationality has been reported to be much lower than Han in China.As a daily vegetable of Bai, the phytochemical and antidiabetic study of Ottelia acuminata var.acuminata had not been carried out.In this study, 41 metabolites with diverse diarylheptanoid (six new ones,Otteacumienes A–F), flavone, sesquiterpenoid, coumarin, lignan, polyacetylene, and alkaloid skeletons were characterized from O.acuminata var.acuminata.Among them, the racemic nature of 3 was characterized by chiral resolution and calculated ECD methods.The biological study revealed diarylheptanoids showed significant α-glucosidase inhibitory activities with 5 as the most effective one (60-fold stronger than acarbose).Molecular docking studies indicated that these structures have different binding cavities with acarbose.This study demonstrated that O.acuminata var.acuminata might correlated with the low incidence diabetes of Bai and the diarylheptanoids may have potential therapeutic value for diabetes mellitus.

Keywords: Ottelia acuminata var.acuminata, Bai nationality, Vegetable, Diarylheptanoids, α-glucosidase

1 Introduction

The latest research from IDF (International Diabetes Federation) revealed that about 8.8% of the world’s population suffers from diabetes disease.Among them, type 2 diabetes (T2DM) is the most common type, accounting for more than 90% of all diabetic cases worldwide [1].Treatment of T2DM requires both diet and exercise to address the overweight or obesity.A range of combination therapy options were available for T2DM such as sulfonylureas,α-glucosidase inhibitors (AGI), thiazolidinediones, dipeptidyl peptidase 4 (DPP-4) inhibitors,glucagon-like peptide 1 (GLP-1) agonists, and sodiumglucose co-transporter 2 (SGLT-2) inhibitors [2].Among them,α-glucosidase is one of the most important digestive enzymes in the human body involved in the final step of carbohydrate digestion, and its inhibitors contributes to control postprandial glucose for diabetes treatment[3].However, the common side effects of flatulence, diarrhea, and hepatotoxicity for the clinicalα-glucosidase inhibitors (including acarbose and miglitol) encouraged us to find new type of inhibitors with high safety and effi-ciency [4].

It’s well-know that the development of diabetes is closely relevant to the dietary and lifestyle.Interestingly,the investigation of the incidence of diabetes in China has showed the characteristics of ethnic and regional variations [5], especially for the Bai nationality (7.83%)in contrast to the Han nationality (11.83%) in rural Yunnan [6].As one of the major ethnic minorities in Yunnan,the Bai nationality has a long history and splendid culture, most of whom live in Dali Bai Autonomous Prefecture [7].Notably,O.acuminatavar.acuminatahad long been used as a traditional daily vegetable in Bai as record in the “Textual Research on Reality and Titles of Plants”published in 175 years ago [8].

As an edible unique plant in China,Ottelia acuminatavar.acuminatabelongs to the genusOtteliaof the Hydrocharitaceae family, and mainly distributed in Yunnan Province [9].This plant is also a famous aquatic ornamental plant, attracting millions of tourists to Erhai and Lugu Lakes every year during the blooming period from May to October.Additionally, the high requirements for water quality could be applied to monitor water pollution and environmental protection [10].In order to explore antidiabetic chemical constituents of a daily vegetable for Bai nationality,O.acuminatavar.acuminatawas selected for this study.However, except for a preliminary analytical investigation by HPLC–ESI–MS, the phytochemical and antidiabetic ingredients of this vegetable have not been reported so far [11].

In this study, the first study of phytochemistry onO.acuminatavar.acuminatawas conducted and 41 compounds including six new diarylheptanoids (Otteacumienes A-F, 1–6) and 35 known ones possessing diverse diarylheptanoid, flavone, sesquiterpenoid, coumarin, lignan, and polyacetylene skeletons (7–41) were obtained(Fig.1).Structurally, the six new diarylheptanoids could be classified into three different types: diarylether, biaryl,and linear types of diaryheptanoids.Being distinct from diarylheptanoids reported from families Officinarum,Katsumadai, Blepharocalyx, Zingiberaceae, and Betulaceae [12], these structures in this study are characterized by low degree of oxidation.Bioactive study of compounds 3–8 showed substantial inhibitory effects onα-glucosidase but negligible effects on PTP1B, suggesting that they might be selective inhibitors ofα-glucosidase.In addition, the molecular docking studies implied that different types diarylheptanoids are binding to the different sites ofα-glucosidase, and the phenolic hydroxyl groups on diaryheptanoids might play a key role for their inhibitory activity.

Fig.1 Structures of new diarylheptanoids (1–6) and other representative compounds

2 Result and discussion

The DCM fraction of the EtOAc extract ofO.acuminatavar.acuminatawas subjected to MCI-gel column, silica gel column chromatography, RP-C18column chromatography, and preparative HPLC to afford eight diarylheptanoids including six new diarylheptanoids(Otteacumienes A–F, 1–6) and 35 known compounds possessing different phenylpropionids, coumarins,lignins, flavonoids, polyacetylene, sesquiterpenoid, and alkaloid architectures.Their structures were elucidated by comprehensive methods including NMR, MS, X-ray diffraction analyses, and calculated ECD spectra.In the antidiabetic studies, the diarylheptanoids showed significantα-glucosidase inhibitory activities and negligible effects on PTP1B.Additionally, molecular docking study further illustrated the possible binding cavities of the diarylheptanoids.

2.1 Structural identification of compounds

Otteacumiene A (1) was isolated as a yellow crystal and its molecular formula was determined as C19H18O3by the HRESIMS atm/z293.1182 [M–H]–(calculated for 293.1178).The IR spectrum showed characteristic hydroxyl broad absorption band at 3442 cm–1and aromatic ring absorption band at 1632, 1590, 1519,1428 cm–1.The1H-NMR signals atδH5.36 (1H, d,J= 2.2 Hz, H-3), 6.57 (1H, dd,J= 8.0, 2.2 Hz, H-5), 6.68(1H, d,J= 8.0 Hz, H-6),δH7.23 (1H, dd,J= 8.2, 2.6 Hz,H-2′), 7.29 (1H, dd,J= 8.2, 2.3 Hz, H-3′), 7.34 (1H, dd,J= 8.3, 2.3 Hz, H-5′), and 7.05 (1H, dd,J= 8.3, 2.6 Hz,H-6′) suggested a 1,2,4-trisubstituted benzene of an ABX spin system and a 1′,4′-disubstituted aromatic rings of an AA′BB′ spin system (Table 1), respectively [13].In addition, acisand atranscarbon–carbon double bonds were evidenced by key1H NMR data ofδH5.40 (1H, t,J= 10.1 Hz, H-9), 5.90 (1H, t,J= 10.1 Hz, H-10), 5.33(1H, m, H-11), and 6.07 (1H, dt,J= 15.0, 4.0 Hz, H-12).Besides 16 carbon atoms of two benzene ring and two carbon–carbon double bonds, the remaining three carbon atoms were attributed to be two methylene (C-7 and C-13) and an oxygenated methine (C-8) according to the13C-NMR and DEPT spectra (Table 2).The above characteristic signals implied that 1 could be a diaryheptanoid derivatives [14].

Table 1 1H NMR data of compounds 1–6a

In the 2D NMR spectra, the key1H-1H COSY correlations of H-7 (δH2.34, d,J= 4.5 Hz)/H-8/H-9/H-10/H-11/H-12/H-13 (δH3.51, m), together with the obvious HMBC correlations from H2-7 to C-3/C-5 and H2-13 to C-3′/C-5′/C-11 confirmed the presence of oxygenated unsaturated heptane chain and the connection of two benzene ring.The linkage of C-2 and C-1′ through an oxygen atom was elucidated by the downfield chemical shift of C-1 (δC145.0), C-1′ (δC157.6), and C-2 (δC152.3),the key HMBC correlations from 1-OH to C-1/C-2/C-6,together with the degrees of unsaturation.(Fig.2).Finally,the absolute configuration of C-8 was undoubtedly determined to be 8Sby X-ray diffraction analysis using Cu Kαradiation.[Flack parameter = 0.08(4)] (Fig.3, CCDC 2156830).

Fig.2 Key 1H-1H COSY and HMBC correlations of 1, 3, 4, and 5

Fig.3 X-ray crystallographic structures for 1 and 3

Otteacumienes B (2) was isolated as yellow oil.The molecular formula was determined to be the same as 1 by HREIMS.Detailed analysis of the1H and13C NMR spectra indicated that the structure of 2 was similar with that of 1 (Tables 1, 2).For 2, the main difference from 1 was ascribed to the position of carbon–carbon bond (Δ8,9and Δ10,11) and hydroxyl (at C-12) in the heptane chain as deduced by the1H-1H COSY correlations of H-7 (δH2.96, m)/H-8 (δH5.52, m)/H-9 (δH5.69, dd,J= 15.6,11.0 Hz)/H-10 (δH5.96, t,J= 11.2 Hz)/H-11 (δH5.23, t,J= 11.2 Hz)/H-12 (δH4.46, td,J= 10.3, 3.2 Hz)/H-13 (δH3.15, dd,J= 12.0, 3.2 Hz).The absolute configuration of C-12 was identified as 12Sby comparison experimental with calculated ECD spectra (Fig.4).

Otteacumiene C (3) was isolated as a colorless needle crystal.Its molecular formula was identified as C20H22O4by the HRESIMS data atm/z349.1407 [M + Na]+(calcd for 349.1410).Detailed comparison of their 1D NMR data indicated 3 and 1 are structurally similar (Tables 1,2).The most obvious differences of 3 compared to that of 1 lie in the appearance of an additional methoxy signal atδH3.20/δC57.4 as well as the absence of signals for a double bond in the heptane chain.These deductions were confirmed by downfield chemical shifts C-7 (δC87.5) and C-8 (δC75.0), the HMBC correlations from H-7 to C-3/C-4/C-5/C-9 and H-13 to C-3′/C-4′/C-5′/C-11, and COSY correlations of H-7/H-8/H-9/H-10/H-11/H-12/H-13(Fig.2).In addition, the crystals for single-crystal X-ray diffraction (Fig.3, CCDC 2155136) were obtained, whichclarified the relative configuration and the racemic nature of 3 with the crystal space groupP2/n.After attempts with various chiral columns and conditions of mobile phase, the chiral separation of 3 was achieved on a chiral-phase HPLC apparatus using a DAICEL CORPORATION semi-preparative column (Fig.S2).To further determine the absolute configurations of enantiomers,quantum-chemical calculation method was used, which eventually assigned the absolute configurations of (+)-3 and (?)-3 to be 7S, 8Sand 7R, 8R, respectively (Fig.4).

Table 2 13C NMR and DEPT (150 MHz) data of 1–6a

Fig.4 Calculated and experimental ECD spectra of 2 and 3

Otteacumiene D (4) was also isolated as yellow oil.The molecular formula was established as C19H18O3from its HRESIMS data atm/z293.1182 [M–H]?(calculated 293.1187).The IR bands at 1508, 1612, 1703, 3390 cm?1suggested the existence of aromatic ring, carbonyl, and hydroxyl groups in the structure.The1H NMR spectrum showed two sets of 1,2,4-trisubstituted benzene rings signals atδH7.28 (1H, d,J= 2.4 Hz, H-3′), 6.98 (1H, dd,J= 8.3, 2.4 Hz, H-5′), 6.72 (1H, d,J= 8.3 Hz, H-6′) and 6.87 (1H, d,J= 2.5 Hz, H-3), 7.03 (1H, dd,J= 8.4, 2.5 Hz,H-5), 6.85 (1H, d,J= 8.4 Hz, H-6), together with a pair oftransdouble bond signals atδH5.54 and 5.79 (1H, dt,J= 14.5, 6.4 Hz, H-10 and 1H, dt,J= 14.5, 6.9 Hz, H-11)(Table 1).The13C NMR and DEPT spectra displayed 19 carbon signals attributing to seven quaternary carbons(one carbonyl), eight methine, and four methylene.The evidences mentioned above, conjugated with a pair of unusual high field aromatic quaternary carbon signals atδC128.3/ 126.6 (C-2/C-2′) suggest that 4 could be a biaryl type cyclic diaryheptanoid derivative (Tables 1, 2)[13, 14].The1H-1H COSY correlations of H-9 (δH3.48,d,J= 6.4 Hz)/H-10/H-11/H-12 (δH2.41, m)/H-13 (δH2.76,J= 7.3, 5.8 Hz) and HMBC correlations from H2-7 to C-3/C-5/C-9, from H-10 to C-8/C-12, and from H2-13 to C-3′/C-4′/C-5′ established its biaryl type cyclic diaryheptanoid architecture.Then, the connection between C-2 and C-2′ was confirmed by the degrees of unsaturation together with the HMBC correlations of H-3/C-2′and H-3′/C-2 (Fig.2).Therefore, the structure of 4 was elucidated.

Otteacumiene E (5) was obtained as green oil, its molecular formula was determined to be C19H20O2,based on HRESIMS spectrum atm/z279.1390[M–H]?(calculated 279.1385).The1H NMR spectrum revealed the presence of two sets of 1,4-disubstituted benzene ringsδH7.00 (2H, m, H-3, 5), 6.75 (2H, m, H-2,6), 7.04 (2H, m, H-3′, 5′), 6.74 (2H, m, H-2′, 6′), a pair oftranscarbon–carbon double bond (δH5.74, 1H, dt,J= 15.0, 7.0 Hz, H-8 and 6.38, 1H, m, H-9), and a pair ofciscarbon–carbon double bond (δH5.96 t,J= 11.0, Hz,H-10 and 5.35, 1H, dt,J= 11.0, 7.5 Hz, H-11) (Table 1)for a typical linear diaryheptanoid [13, 14].The13C NMR and DEPT spectroscopic data demonstrated 19 carbon signals attributing to four quaternary carbons, 12 methine, and three methylene (Table 2).In the 2D NMR spectra, the key1H-1H COSY correlations of H-7/H-8/H-9/H-10/H-11/H-12H-13 and HMBC correlations from H2-7 to C-3/C-5 and H2-13 to C-3′/C-5′/C-11 confirmed diaryheptanoid nature of 5.In addition, the location of two hydroxyl at C-1 and C-1′ was evidenced by the HMBC correlations from 1-OH to C-2/C-6 and from 1′-OH to C-2′/C-6′ (Fig.2).Therefore, the structure of 5 was established as shown.

Compound 6 was also isolated as green oil, showed a molecular ion atm/z293.1547 [M – H]–in the HRESIMS(calculated 293.1542), which correlates to the molecular formula C20H22O2.The1H and13C NMR spectra were similar with those of 5 (Tables 1, 2) except for an extra methoxy signal (δH3.75,δC55.3) in 6.And this methoxyl was deduced to be located at C-1′ by its HMBC correlation with C-1′ (δC158.9).Therefore, its structure was established as the 1’-O-methylated 5 and named otteacumiene F.

By comparing spectroscopic data with literatures, the structures of 35 known compounds were elucidated as(1E,4E)-1,7-di(4-methoxyphenyl)-1,4-heptadiene (7)[15], tedarenes A (8) [16],trans-cinnamic acid (9) [17],p-hydroxymethylcinnamate (10) [18],trans-p-hydroxyl ethyl cinnamate (11) [19 3-(4-hydroxyphenyl)acrylic acid benzyl ester (12) [20], (2E)-3-(4-hydroxyphenyl)-2-propenoic acid 2-phenylethyl ester (13) [21](E)-cinnamyl-(E)-p-coumarate (14) [22], (E)-cinnamyl-(Z)-p-Coumarate (15) [23], (E)-cinnamyl-(E)-ferulate(16) [23], bupleurumin (17) [24], marginatoxin (18)[25], 3-(3,4-dimethoxybenzyl)-2-(3,4-methylenedioxybenzyl) butyrolactone (19) [26], Suchilactone (20) [27],osthol (21) [28], micropubescin (22) [29], 8-(2,3-dihydroxy-3-methylbutyl)-7-methoxy-2H-1-benzopyran-2-one (23) [29, 30], murraol (24) [31] murrayacaurpin B (25) [32] 5,6-furanocoumarin (26) [33] xanthotoxin(27) [34], isopimpinellin (28) [35], sakuranetin (29) [36]7,8-dihydroxyflavanone (30) [37], 5,3′-dihydroxy-7,4′-dimethoxyflavanone (31) [38] pinostrobin (32) [39],tectochrysin (33) [40] 5,7-dihydroxy-flavone (34) [41]desmethylnobiletin (35) [42] (9Z)-heptadeca-1,9-diene-4,6-diyn-3-one (36) [43] (8E)-octadeca-1,8-diene-4,6-diyne-3,10-diol (37) [43]1H-indole-3-carboxylic acid methyl ester (38) [44]1H-indole-3-carboxaldehyde (39)[45] vanillin (40) [46] and litseagermacrane (41) [47]respectively.

2.2 α-Glucosidase and PTB1B inhibitory activity

To explore the antidiabetic chemical constituents fromO.acuminatavar.acuminata, theα-glucosidase and PTP1B of inhibitory activities of diaryheptanoids 1–8 were evaluated.The results revealed that these compounds exhibited different levels of inhibitory activity ranging from 38.29% to 103.55% at 50 μM (Table 3), in which 3–8 with inhibition rates more than 50.0% were screened for their IC50values (acarbose as a positive control).Interestingly,all these compounds exhibited more potential inhibitory activity with IC50values of 3.81–26.44 μM (Table 3).Especially, 5 represented most effective inhibitor with 60 times more potent than that of acarbose (228.95 μM),the first-line drug for diabetes treatment.It’s notable that these diaryheptanoids exhibited negligible effects on PTP1B (Table S11), suggesting that these active ingredients may serve as selective inhibitors onα-glucosidase.

Table 3 Inhibitory effects of 1–8 against α-Glucosidasea

2.3 Molecular docking studies

To further explore the potential antidiabetic mechanism of the diarylheptanoids, the molecular docking studies were performed by using PyMol program.Compounds 4, 5, and 8 were selected as representative structures of diarylether, linear, and biaryl types of diaryheptanoids for molecular docking againstα-glucosidase.The results showed that all the three types of diaryheptanoids act with different cavities mode with that of acarbose [48] which may have contributed to their substantialα-glucosidase inhibitory activities.The molecular docking study for acarbose againstα-glucosidase indicated that this first-line medicine formed six hydrogen bonds with ASP-242 (2.9 ?), SER-242 (3.5 ?), GLN-279 (2.8 ?),ARG-422 (2.8 ?), GLU-411 (2.8 ?), and AGR-315 (2.9 ?),respectively.Notably, the 1′-OH of 5 formed three hydrogen bonds with SEP-241 (2.9 ?, 3.2 ?) and ARG-422(3.0 ?), which might be the reason for its superior activity in contrast to 4 and 8.Correspondingly, 1-OH and 1′-OH of 4 formed two hydrogen bonds with TRY-158(2.7 ?, 2.8 ?) and 1-OH of 8 formed a hydrogen bond with MET-70 (3.3 ?).Although 4, 5, and 8 in the docking process tended to combine with the same cavity, the difference in the ability to form hydrogen bonds with amino acid residues might be the reason for their different activities (Fig.5).

Fig.5 Molecular docking studies of 4, 5, 8, and acarbose against α-glucosidase

3 Conclusion

To the best of our knowledge, this is the first study on the phytochemistry ofO.acuminatavar.acuminataand their antidiabetic activity.Totally, 41 metabolites ingcluding eight diarylheptanoids (six new ones), eight phenylpropanoids, four lignans, eight coumarins, seven flavonoids, two polyacetylenes, a sesquiterpenoid, two alkaloids, and a vanillin were characterized in this study.Among them, 3 was obtained as a pair of enantiomers whose absolute configurations were determined by calculated ECD method after chiral separation.The biological activity studies displayed that 3–8 exhibited substantial inhibitory activity onα-glucosidase as well as negligible effects on PTP1B, which indicated that these diarylheptanoids might be selective inhibitors ofα-glucosidase.Notably, compound 5 was 60-fold stronger than positive control, acarbose.This study implied a common vegetable of Bai,O.acuminatavar.acuminatamay reduce the incidence of diabetes by inhibitingα-glucosidase.In addition, this work also provides new lead molecules for antidiabetic disease and a reference for medicinal use ofO.acuminatavar.acuminata.

4 Experimental

4.1 General experimental procedures

IR spectra were measured on a Bruker FT-IR Tensor-27 infrared spectrophotometer with KBr disks.Optical rotations were recorded on a JASCO P-1020 polarimeter.UV spectra were obtained with a Shimadzu UV-2401PC spectrometer.1D and 2D NMR spectra were performed on a Bruker DRX-600 spectrometer using TMS as an internal standard.The chemical shifts (δ) were expressed in ppm with reference to the solvent signals.HREIMS and HRESIMS analysis were obtained from Waters Xevo TQS and Agilent G6230 TOF mass spectrometers, respectively.Single-crystal X-ray diffraction data were exhibited on a Bruker D8 QUEST diffractometer.Semi-preparative HPLC was performed on a Waters 1525 HPLC with a ZORBAX SB-C18(9.4 × 250 mm) column.Silica gel(100–200 and 200–300 mesh, Qingdao Marine Chemical Co., Ltd., People’s Republic of China), and MCI gel(75–150 μm, Mitsubishi Chemical Corporation, Tokyo,Japan) were used for column chromatography.Fractions were monitored by TLC (GF 254, Qingdao Marine Chemical Co., Ltd.), and spots were visualized by heating silica gel plates immersed in 10% H2SO4in ethanol.Methanol (HPLC grade) and acetonitrile (HPLC grade)was purchased from CINC High Purity Solvents Co., Ltd(Shanghai, China).4-Nitrophenyl-α-d-glucopyranoside(PNPG),α-glucosidase, PTP1B assay kit, quercetin, and acarbose were purchased from Sigma Chemical (Merck KGaA, Darmstadt, Germany).

4.2 Plant materials

The whole plants ofO.acuminatavar.acuminatawere collected in Dali Prefecture (Yunnan, China) on October 2019.It was identified by Prof.Yun-Heng Ji in Kunming Institute of Botany, Chinese Academy of Sciences.The specimen of this plant was deposited at the State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, and the voucher number was KIB L-20191001.

4.3 Extraction and isolation

The dried samples ofO.acuminatavar.acuminata(20.0 kg) were crushed and extracted four times with methanol for two days each time, and the solvent was recovered under reduced pressure to obtain a crude extract (2.5 kg).The crude extract was eluted with 90%methanol through macroporous resin to obtain 200 g of eluted fractions.The 90% methanol eluent was applied to silica gel column chromatography eluted with dichloromethane (DCM), to afford fractionO-DCM (30.5 g).FractionO-DMC (30.5 g) was separated over an MCIgel column (MeOH-H2O from 6:4 to 10:0, v/v) to obtain six fractions from small to large polarities Fr.A–F, which were successively on purified by macroporous resin, silica gel, MCI-gel, RP-C18, and preparative or semi-preparative HPLC chromatographic metheds to give the 41 isolates (detailed process, Fig.S1).

Otteacumiene A (1):Yellow crystals; [α] + 52.4 (c0.120, MeOH); UV (MeOH)λmax(logε) 205.0 (4.81),280.8 (3.50) nm; IR (KBr)νmax3442, 1632, 1590, 1519,1503, 1200, 1109, 1030, 983 cm–1; ECD (MeOH)λmax(Δε) 285 (+ 3.75), 271 (– 0.82), 241 (+ 13.52) nm;HRESIMSm/z293.1182 [M – H]–(calculated for C19H17O3, 293.1178); and1H and13C NMR spectroscopic data, Tables 1, 2.

Otteacumiene B (2):Yellow oil; [α] + 384.0 (c0.098,MeOH); UV (MeOH)λmax(logε) 206.0 (3.73), 285 (2.57)nm; IR (KBr)νmax3400, 1630, 1594, 1515, 1503, 1432,1212, 1135 cm–1; ECD (MeOH)λmax(Δε) 246 (+ 31.34),200 (– 17.13) nm; HREIMSm/z294.1262 [M]+(calculated for C19H18O3, 294.1256); and1H and13C NMR spectroscopic data were shown in Tables 1, 2.

Otteacumiene C (3):Colorless crystals; [α] + 1.8(c0.075, MeOH); [α] – 68.3 (c0.048, MeOH) for (–)-3; [α] + 96.8 (c0.050, MeOH) for (+)-3; UV (MeOH)λmax(logε) 278.5 (3.71), 196.0 (4.83), 258.0 (3.56) nm;IR (KBr)νmax3408, 1892, 1732, 1516, 1500, 1429, 1208,1159, 1017 cm–1; ECD (MeOH)λmax(Δε) 285 (+ 19.98),270 (– 0.21), 259 (+ 2.96), 241 (– 78.41), 224 (+ 30.54),213 (– 50.23), 204 (+ 1.22) nm for (+)-3; ECD (MeOH)λmax(Δε) 285 (– 13.99), 270 (+ 0.22), 259 (– 2.71), 241(+ 52.79), 224 (– 22.91), 213 (+ 31,32), 204 (– 2.74) nm for (–)-3; HRESIMSm/z349.1407 [M + Na]+(calculated for C20H22O4Na, 349.1410); and1H and13C NMR spectroscopic data were shown in Tables 1, 2.

Otteacumiene D (4):Yellow oil; [α] – 0.8 (c0.120,MeOH); UV (MeOH)λmax(logε) 297.5 (4.48), 196.0(5.09) nm; IR (KBr)νmax3390, 1703, 1612, 1587, 1508,1430, 1069, 1053, 994 cm–1; HRESIMSm/z293.1182 [M– H]–(calculated for C19H17O3, 293.1178); and1H and13C NMR spectroscopic data, Tables 1, 2.

Otteacumiene E (5):Green oil; [α] – 0.2 (c0.098,MeOH); UV (MeOH)λmax(logε) 200.0 (4.42), 235.4(4.52), 279.0 (3.69) nm; IR (KBr)νmax3417, 3017, 2923,1613, 1514, 1450, 1383, 1246, 1101, 826 cm–1; HRESIMSm/z279.1390 [M – H]–(calculated for C19H19O2,279.1385); and1H and13C NMR spectroscopic data were shown in Tables 1, 2.

Otteacumiene F (6):Green oil; [α] – 0.6 (c0.110,MeOH); UV (MeOH)λmax(logε) 200.2 (4.20), 229.6(4.24), 277.6 (3.39) nm; IR (KBr)νmax3425, 2924, 1612,1513, 1442, 1245, 1177, 1036, 826 cm–1; HRESIMSm/z293.1547 [M – H]–(calculated for C20H21O2293.1542);and1H and13C NMR spectroscopic data were shown in Tables 1, 2.

Crystal data for 1:C19H18O3,M= 294.33,a= 7.5455(4)?,b= 9.2259(4) ?,c= 21.7568(10) ?,α= 90°,β= 90°,γ= 90°,V= 1514.58(12) ?3,T= 100.(2) K, space groupP212121,Z= 4,μ(Cu Kα) = 0.695 mm?1, 27,150 reflections measured, 2871 independent reflections(Rint= 0.0429).The finalR1values were 0.0251 (I> 2σ(I)).The finalwR(F2) values were 0.0643 (I> 2σ(I)).The finalR1values were 0.0255 (all data).The finalwR(F2) values were 0.0645 (all data).The goodness of fit onF2was 1.060.Flack parameter = 0.08(4).(CDDC: 2156830).

Crystal data for 3:C20H22O4?CH4O,M= 358.42,a= 10.1053(9) ?,b= 18.5003(16) ?,c= 19.6923(17) ?,α= 90°,β= 90°,γ= 90°,V= 3681.5(6) ?3,T= 100.(2) K,space groupPbca,Z= 8,μ(Cu Kα) = 0.744 mm?1, 60,171 reflections measured, 3516 independent reflections(Rint= 0.0599).The finalR1values were 0.0366 (I> 2σ(I)).The finalwR(F2) values were 0.0943 (I> 2σ(I)).The finalR1values were 0.0386 (all data).The finalwR(F2) values were 0.0959 (all data).The goodness of fit onF2was 1.070.(CDDC: 2155136).

4.4 α-Glucosidase and PTB1B inhibitory activities assay

Theα-glucosidase and PTB1B inhibitory activity were conducted according to the previous reports with slight modifications [49, 50].Briefly, in theα-glucosidase inhibitory assay, after 50 min incubation at 37 °C, the absorbance value at 405 nm was detected and acarbose was used as positive control.While in the PTB1B inhibitory assay, after addition of phosphate-based detection reagent then incubation at 30 °C for 20 min, and absorbance was measured at 620 nm and suramin was used as positive control.The inhibition percentage was calculated as follows: inhibition rate (%) = (E–S)/E × 100% (Eis the OD of the control andSis the OD of the sample) and IC50(50% concentration of inhibition) was calculated by Reed and Muench method.

4.5 Molecular docking studies

To explore the structure–activity relationship, the molecular docking studies was conducted according to the previous reports with slight modifications [51].In brief, the AutoDock and PyMol software was used to blind docking between 3D structure ofα-glucosidase which is downloaded from RCSB PDB website (PDB ID: 3A4A) and compound ligands.

Supplementary Information

The online version contains supplementary material available at https:// doi.org/ 10.1007/ s13659- 022- 00341-4.

Below is the link to the electronic supplementary material.Supplementary file1 (DOCX 8783 KB)

Acknowledgements

This study was supported financially by the Second Tibetan Plateau Scientific Expedition and Research program (2019QZKK0502) and State Key Laboratory of Phytochemistry and Plant Resources in West China (E0230211Z1 and P2019-ZZ05).

Author contributions

HX Liu carried out the experiments and drafted the manuscript; JZ Ma participated in the experiments; YS Ye performed ECD calculations and revised the manuscript; JJ Zhao completed samples collection; SJ Wan and XY Hu participated in the revision of manuscript; G Xu designed the experiments, revised the manuscript.All authors read and approved the final manuscript.

Declarations

Competing interests

The authors declare no competing interests.

Author details

1State Key Laboratory of Phytochemistry and Plant Resources in West China and Yunnan Key Laboratory of Natural Medicinal Chemistry, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, China.2University of Chinese Academy of Sciences, Beijing 100049, China.

Received: 17 March 2022 Accepted: 4 April 2022