Tin Thu Thu Aung·Meng-Yuan Xia·Pyae Phyo Hein·Rong Tang·Dong-Dong Zhang·Jun Yang·Xue-Fei Yang·Dong-Bao Hu·Yue-Hu Wang

Abstract

Keywords Cuscuta reflexa ·Porcine pancreatic lipase·Platelet aggregation·2 H-pyran-2-one glucosides·Steroidal glucosides

1 Introduction

Cuscuta reflexaRoxb.(Convolvulaceae), a twining parasitic plant, is distributed in China, Afghanistan, India, Indonesia,Malaysia, Myanmar, Nepal, Pakistan, Sri Lanka, and Thailand [1].In Nujiang Prefecture, Yunnan Province, China, the Lisu people call it mu-gua-zhua and use its whole plant or seeds to treat soreness and weakness of the waist and knees,erectile dysfunction, spermatorrhea, diabetes, dizziness,hypopsia, and threatened abortion [2].Named shwe-new or shwe-nwe-pin (Hsay) in Myanmar, the whole plant ofC.reflexais used to treat inflammation, irregularities of the blood,and other diseases [3].It is also medically used in India,Nepal, Bangladesh, and Pakistan [4].Based on published studies, the major chemical constituents ofC.reflexainclude flavonoids, coumarins, phenylpropanoids, triterpenoids, and cardiac glycosides [4—7].

A previous study showed that extracts ofC.reflexapossess an antiobesity effect [8], but no further studies clarified the active compounds responsible for the activity.Pancreatic lipase inhibition is one of the most widely studied mechanisms for antiobesity treatment [9].Purified human lipase(HPL) and porcine pancreatic lipase (PPL) show similar specific activities [10].Recently, the extract ofC.refl exawas found to have in vitro thrombolytic activity [11].This finding may somewhat explain its traditional use in the treatment of irregularities of the blood.However, no active constituents were reported in this research.In the current study, we report the structural elucidation of three new compounds from the whole plant ofC.reflexaand the results of a bioassay for the inhibitory activities against PPL and rabbit platelet aggregation.

2 Results and Discussion

2.1 Structural Elucidation

Through chromatographic techniques, three new compounds(1—3, Fig.1) and 12 known compounds (4—15, Supplementary Material, Fig.S1) were obtained from the EtOH extract ofC.reflexa.

Based on13C NMR data (Table 1) and the HRESIMS ion peak atm/z457.1106 [M+Na]+(calcd for C21H22NaO10,457.1111), the molecular formula of 1 was deduced to be C21H22O10.The1H and13C NMR data (Table 1) indicated the presence of ap-coumaroyl moiety [δH7.60(1H, d,J=15.9 Hz), 7.48 (2H, br d,J=8.7 Hz), 6.79(2H, br d,J=8.7 Hz), and 6.36 (1H, d,J=15.9 Hz);δC169.0], one methyl group [δH2.20 (3H, s);δC19.8], and oneβ-glucopyranosyl group [δH5.05 (d,J=7.4 Hz)].By comparing its NMR data with those of 4-(β-Dglucopyranosyloxy)-6-methyl-2H-pyran-2-one (5) [12],it was implied that compound 1 might be ap-coumaroyl derivative of 4-glucopyranosyloxy-6-methyl-2H-pyran-2-one.Based on the 2D NMR spectra of 1, especially the HMBC correlations from H-1? to C-4 and H 2-6? to C-9″,the 4-glucopyranosyloxy-6-methyl-2H-pyran-2-one moiety was confirmed, and thep-coumaroyl group was located at 6?-OH through an ester bond.An acetylated derivative(1a) was obtained using pyridine and acetic anhydride.We tried to obtain a crystal of 1a, but unsuccessful.After acidic hydrolysis of 1, D-glucose was obtained.Thus, the chemical structure of 1 was determined as shown in Fig.1 and named cuscutaroside A.

The molecular formula of cuscutaroside B (2) was determined to be C17H24O9based on the13C NMR data (Table 1)and the HRESIMS ion peak atm/z395.1313 [M+Na]+(calcd for C17H24NaO9, 395.1318).The1H and13C NMR data of 2 (Table 1) indicated the presence of one 6-methyl-2H-pyran-2-one moiety (δC 171.3, 167.2, 164.8, 101.5,and 91.6) [12], oneβ-glucopyranosyl group [δH 5.05 (d,J=7.5 Hz)], and one 2-methylbutyryl group (δC178.2, 42.3,27.9, 17.0, and 11.9) [13].Compound 2 was deduced as a 2-methylbutyryl derivative of 4-(β-glucopyranosyloxy)-6-methyl-2H-pyran-2-one by the COSY and HMBCcorrelations (Fig.2).Based on the HMBC correlations from H2-6? to C-1″, the 2-methylbutyryl group was located at 6?-OH.Thus, the structure of 2 (cuscutaroside B) was elucidated to be 4-[β-6-O-(2-methylbutyryl)-glucopyranosyloxy]-6-methyl-2H-pyran-2-one.We were unable to calculate the ECD spectrum of 2.The absolute configuration of C-2″remains unknown.

Table 1 1 H and 13 C NMR data of 1 and 2 in methanol-d4 (δ in ppm, J in Hz)

Table 2 1 H (800 MHz) and 13 C(200 MHz) NMR data of 3 and 4 in methanol-d4 (δ in ppm, J in Hz)

Based on13C NMR (Table 2) and HRESIMS data, compound 3 was determined to have the molecular formula C36H62O7.The1H and13C NMR data (Table 2) of 3 indicated the presence of oneβ-glucopyranosyl group [δH4.39(d,J=7.8 Hz)], one methoxy group [δH3.28 (3H, s);δC54.9],six methyl groups [δH1.07 (3H, s), 0.96 (3H, d,J=6.5 Hz),0.84 (3H, d,J=6.9 Hz), 0.88 (3H, d,J=7.2 Hz), 0.87 (3H, t,J=7.0 Hz), and 0.72 (3H, s)], and one trisubstituted double bond [δH5.48 (t,J=1.7 Hz);δC145.7 and 122.5].Comparison of its NMR data with those of 7-oxo-β-sitosterol 3-O-β-D-glucopyranoside (4, Table 2) and 7β-hydroxysitosterol-3-O-β-D-glucopyranoside indicated that compound 3 might be a 7-oxygenated derivative of daucosterol [14—16], which was confirmed by the COSY and HMBC correlations (Fig.2).According to the HMBC correlations from the OMe group to C-7, H-3 to C-1?, and H-1? to C-3, the methoxy group and the glucopyranosyloxy group were located at C-7 and C-3,respectively.The relative configuration of compound 3 was partially determined by ROESY correlations (Fig.2) and coupling constants (Table 2).ROESY correlations of H-1β/H3-19, H3-19/H-8, H-8/H3-18, and H3-18/H-20 were observed,indicating that these protons are cofacial, and thus, H-1α and H-17 are α-oriented.H-7 and H-8 should be in atransaxial relationship because of a large coupling constant for H-7/H-8(J=8.5 Hz), and thus, H-7 is α-oriented.The ROESY correlations of H-1α/H-3, H-9/H-7, and H-7/H-14 indicated that H-3,H-9, and H-14 are also α-oriented.The configurations of C-20 and C-24 on the side chain could not be determined by the ROESY correlations.Because the NMR data of the side chain were highly consistent with those in the literature [14—17],the configurations of C-20 and C-24 were suggested to be the same as those in daucosterol.Finally, compound 3 was elucidated to be 7β-methoxy-β-sitosterol 3-O-β-glucopyranoside.

The NMR data of 7-oxo-β-sitosterol 3-O-β-Dglucopyranoside (4) in pyridine-d5and CDCl3have been reported previously [14, 15].Its NMR data in methanold4are presented in Table 2.The other known compounds,4-(β-D-glucopyranosyloxy)-6-methyl-2H-pyran-2-one (5)[12], 4-hydroxyacetophenone (6) [18], piceoside (7) [19],scrophenoside B (8) [20], methyl 4,5-di-O-caffeoylquinate(9) [21], methyl 3,5-di-O-caffeoylquinate (10) [21], methyl 3,4-di-O-caffeoylquinate (11) [21], (6S,9R)-roseoside (12)[22], methyltrans-p-hydroxycinnamate (13) [23], ethyltrans-p-hydroxycinnamate (14) [24], andN-trans-feruloyltyramine (15) [25], were determined by comparing their NMR data with those in the literature.

2.2 Porcine Pancreatic Lipase and Platelet Aggregation Inhibition Assay

Compounds 1—15 were evaluated for their inhibitory activity against PPL.7β-Methoxysitosterol 3-O-βglucopyranoside (3) showed weak PPL inhibitory activity(IC50=67.2 ± 1.7 μg/mL) compared with the positive control orlistat (IC50=0.40 ± 0.02 ng/mL).7-Oxo-β-sitosterol 3-O-β-D-glucopyranoside (4) showed 12 ± 2% inhibition at a concentration of 100 μg/mL.The other tested compounds were inactive, with inhibition values less than 10% at a concentration of 100 μg/mL.

Compounds 1—15 and 1a were also evaluated for their inhibitory activity against rabbit platelet aggregation induced by thrombin (1 U/mL), platelet-activating factor(PAF, 0.4 μg/mL), arachidonate (AA, 0.5 mM), or collagen (4 μg/mL).Cuscutaroside A (1), 1a, and scrophenoside B (8) showed weak inhibitory activity against rabbit platelet aggregation induced by collagen with IC50values of 291.4 ± 47.9 μg/mL, 63.8 ± 4.4 μg/mL, and 180.5 ± 6.7 μg/mL, respectively, compared with aspirin(IC50=33.3 ± 1.3 μg/mL).Compound 1a also showed inhibitory activity against rabbit platelet aggregation induced by AA with an IC50value of 72.6 ± 10.5 μg/mL compared with aspirin (inhibition 88.1 ± 1.1% at 40 μg/mL).The other tested compounds were inactive (IC50>300 μg/mL).

3 Experimental Section

3.1 General Experimental Procedures

Optical rotations were recorded using a JASCO P-1020 Polarimeter (Jasco Corp., Tokyo, Japan).UV spectra were obtained using a Shimadzu UV-2401 PC spectrophotometer(Shimadzu, Kyoto, Japan).ECD spectra were recorded on a Chirascan CD spectrometer (Applied Photophysics Ltd.,Leatherhead, UK).1 H and 13 C NMR spectra were collected on DRX-500, Avance III-600, and Ascend? 800 MHz spectrometers (Bruker Corp., Karlsruhe, Germany) with TMS as an internal standard.ESIMS and HRESIMS analyses were performed on an API QSTAR Pulsar 1 spectrometer(Applied Biosystems/MDS Sciex, Foster City, CA, USA).EIMS and HREIMS were performed on a Waters Auto-Spec Premier p776 spectrometer (Waters, Milford, MA,USA).Silica gel G (80—100 and 300—400 mesh, Qingdao Meigao Chemical Co., Ltd., Qingdao, China), C18silica gel(40—75 μm, Fuji Silysia Chemical Ltd., Aichi, Japan), and Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) were used for column chromatography, and silica gel GF254(Qingdao Meigao Chemical Co., Ltd.) on precoated plates was used for preparative thin layer chromatography (TLC).TLC spots were visualized under UV light at 254 nm and by dipping them into 5% H2SO4in alcohol followed by heating.Semipreparative high-performance liquid chromatography (HPLC) was performed with an Agilent 1200 series pump (Agilent Technologies, Santa Clara, USA)equipped with a diode array detector and an Agilent Zorbax SB-C18column (5.0 μm,φ9.4 × 250 mm) and a Welch Ultimate AQ-C18column (5.0 μm,φ4.6 × 300 mm).

3.2 Plant Material

The plant material, growing on aBauhiniaplant, was collected near Golden Cave (20° 55′ 44.25″ N and 96° 38′57.41″ E) in Pindaya Township, Southern Shan State, Myanmar, in December 2016.It was identified asCuscuta reflexaRoxb.by Dr.Jie Cai and Ms.Jun Yang at the Kunming Institute of Botany, Chinese Academy of Sciences.A voucher specimen (no.MMR631) was deposited at the Southeast Asia Biodiversity Research Institute, Chinese Academy of Sciences, Yezin, Nay Pyi Taw, Myanmar and a copy was placed at the Key Laboratory of Economic Plants and Biotechnology, Kunming Institute of Botany, Chinese Academy of Sciences, China.

3.3 Extraction and Isolation

The air-dried powdered whole plant ofCuscuta reflexa(3.3 kg) was ultrasonically extracted for 30 min with 70%EtOH at 60 °C.The EtOH extract (519.0 g) was suspended in H2O and further partitioned with petroleum ether, EtOAc,andn-butanol to the yield petroleum ether-soluble portion(6.4 g, A), the EtOAc-soluble portion (66.8 g, B), and then-butanol soluble portion (276.6 g, C), respectively.

Part B was subjected to column chromatography (silica gel;CH2Cl2—MeOH, 50:1 → 0:1, v/v) to yield four fractions(B1—B4).Fraction B1 was separated on an RP-C18silica gel column eluted with MeOH—H2O (10% → 100%) to yield three major subfractions.The 20% MeOH-eluted portion was purified by Sephadex LH-20 column chromatography(MeOH) and silica gel column chromatography (petroleum ether-EtOAc, 10:1) to yield 6 (8.9 mg).The 40% MeOHeluted portion was purified by column chromatography(Sephadex LH-20, MeOH), preparative TLC (petroleum ether-acetone, 2:1), and semipreparative HPLC [Agilent Zorbax SB-C18column, MeCN-H2O (containing 0.05% TFA),35:65, 2 mL/min] to yield 13 (5.0 mg,tR=19.388 min) and 14 (2.0 mg,tR=32.159 min).

Fraction B2 was separated on an RP-C18silica gel column eluted with MeOH—H2O (10% → 100%) to yield one main subfraction.The 30% MeOH-eluted portion was isolated by Sephadex LH-20 (MeOH) and silica gel column chromatography (CH2Cl2—MeOH, 30:1) and further purified by semipreparative HPLC [Welch Ultimate AQ-C 18 column,MeCN-H2O (containing 0.05% TFA), 20:80, 1 mL/min] to yield 15 (19.4 mg,tR=36.402 min).

Fraction B3 was separated on an RP-C18silica gel column eluted with MeOH-H2O (10% → 100%) to yield two main subfractions (B3-1 and B3-2).B3-1 (40% MeOH-eluted portion) was separated by silica gel column chromatography (CH2C l2—MeOH, 100:1 and 50:1) to yield B3-1-1 and B3-1-2.B3-1-1 was separated by Sephadex LH-20 (MeOH)and silica gel column chromatography (CH2C l2—MeOH, 3:1)followed by semipreparative HPLC (Agilent Zorbax SB-C18column, MeOH—H2O, 40:60, 2 mL/min) to yield 2 (3.0 mg,tR=24.052 min) and 8 (2.1 mg,tR=37.642 min).B3-1-2 was separated by Sephadex LH-20 (MeOH) and silica gel column chromatography (CH2C l2—acetone, 3:1 → 1:1) to yield 1 (571.5 mg) and a mixture.The mixture was further purified by semipreparative HPLC [Agilent Zorbax SB-C18 column, MeCN—H2O (containing 0.05% TFA), 28:72,2 mL/min] to yield 9 (5.9 mg,tR=13.042 min), 10 (5.8 mg,tR=17.294 min), and 11 (2.6 mg,tR=19.588 min).B3-2(90% MeOH-eluted portion) was separated by Sephadex LH-20 column chromatography (MeOH) and semipreparative HPLC (Welch Ultimate AQ-C18column, MeOH—H2O,87:13, 1 mL/min) to yield 4 (3.5 mg,tR=42.732 min) and 3(2.0 mg,tR=64.797 min).

Fraction B4 was separated on an RP-C18silica gel column eluted with MeOH—H2O (10% → 100%) to yield three main fractions.The 10% MeOH-eluted portion was purified by silica gel column chromatography (CH2C l2—MeOH,30:1 and 20:1) and Sephadex LH-20 column chromatography (MeOH) to yield 5 (1.1 g) recrystallized from MeOH.The 15% MeOH-eluted portion was isolated by silica gel column chromatography (CH2C l2—MeOH, 30:1 and 20:1)and Sephadex LH-20 column chromatography (MeOH) and further purified by semipreparative HPLC (Welch Ultimate AQ-C18column, MeOH—H2O, 15:85, 1 mL/min) to yield 12 (6.4 mg,tR=36.095 min).The 20% MeOH-eluted portion was isolated by Sephadex LH-20 column chromatography (MeOH) and semipreparative HPLC (Agilent Zorbax SB-C18column, MeOH—H2O, 15:85, 2 mL/min) to yield 7(1.6 mg,tR=22.265 min).

3.4 Spectroscopic Data of Compounds

3.4.1 Cuscutaroside A (1)

3.4.2 Cuscutaroside B (2)

3.4.3 7 β-Methoxy-β-sitosterol 3-O-β-glucopyranoside (3)

3.5 Acidic Hydrolysis of Compound 1

Compound 1 (20.0 mg, 0.046 mM) was dissolved in 15 mL of 6% aq.HCl and hydrolyzed under reflux (5 h) at 90 °C.Then, the acidic solution was evaporated in vacuo to dryness and separated by silica gel column chromatography eluted with CHCl3—MeOH (10:1) to yield D-glucopyranose(5.5 mg, 0.031 mM, 67% yield), which was identified based on its1H NMR spectrum and optical rotation value of+40.0 (c0.09, H2O) [26].

3.6 Acetylation of Compound 1

Compound 1 (20.6 mg, 0.048 mmol) was dissolved in 500 μL of pyridine and 500 μL of acetic anhydride and stirred for 24 h at room temperature.Then, water (2 mL) was added to the reaction mixture, followed by extraction with ethyl acetate (4 mL).The upper layer was dried under reduced pressure and purified by silica gel column chromatography (petroleum ether-EtOAc, 10:1) to yield 1a (20.4 mg,0.034 mmol, 71% yield).White solid;1H NMR (500 MHz,CDCl3)δ7.68 (1H, d,J=16.0 Hz, H-7″), 7.61 (2H, br d,J=8.6 Hz, H-2″ and H-6″), 7.12 (2H, br d,J=8.6 Hz, H-3″and H-5″), 6.46 (1H, d,J=16.0 Hz, H-8″), 5.80 (1H, br s, H-5), 5.63 (1H, d,J=2.2 Hz, H-3), 5.28 (2H, m), 5.16(2H, m), 4.37 (1H, dd,J=12.6, 2.3 Hz, H-6?a), 4.27 (1H,dd,J=12.6, 6.2 Hz, H-6?b), 3.94 (1H, m, H-5?), 2.30 (3H,s), 2.19 (3H, s), 2.06 (3H, s), 2.06 (3H, s), 2.03 (3H, s);13C NMR (125 MHz, CDCl3)δ170.2, 169.5, 169.3, 169.2,168.1, 166.4, 164.4, 163.3, 152.4, 145.2, 132.0, 130.5,129.7, 122.3, 117.1, 100.1, 96.9, 91.4, 73.0, 72.6, 70.7, 68.2,62.1, 21.3, 20.7, 20.7, 20.7, 20.0;ESIMS (positive)m/z625[M+Na]+;HRESIMS (positive)m/z625.1528 [M+Na]+(calcd for C29H30NaO14, 625.1533).

3.7 In vitro Porcine Pancreatic Lipase Inhibition Assay

For lipase inhibition tests of each compound, porcine pancreatic lipase was used.p-Nitrophenyl butyrate (p-NPB) was used as the substrate.First, 5 μL of the lipase solution (40 U/mL) in Tris—HCl buffer (100 mM Tris—HCl, 5 mM CaCl2;pH 7.0) was added to a 96-well microtiter plate.Each compound in 1 μL of DMSO and 184 μL of Tris—HCl buffer were added and mixed with the enzyme buffer to start the reaction.After incubation at 37 °C for 15 min, 10 μL of the substrate solution (10 mMp-NPB in dimethyl formamide) was added.The enzymatic reaction was carried out for 15 min at 37 °C.The hydrolysis ofp-NPB top-nitrophenol was monitored at 400 nm using a spectrophotometer [27].

3.8 In Vitro Platelet Aggregation Assay

The inhibitory effects of the compounds against rabbit platelet aggregation induced by thrombin (1 U/mL), PAF (0.4 μg/mL), AA (0.5 mM), or collagen (4 μg/mL) were evaluated according to published methods [28—30].

4 Conclusion

Three new and 12 known compounds were isolated from the whole plants ofCuscuta reflexa(Convolvulaceae) collected from Myanmar.7β-Methoxy-β-sitosterol 3-O-βglucopyranoside (3) showed weak PPL inhibitory activity(IC50=67.2 ± 1.7 μg/mL).Cuscutaroside A (1), its acetylated derivative (1a), and scrophenoside B (8) showed weak inhibitory activity against rabbit platelet aggregation induced by collagen (4 μg/mL) with IC50values of 291.4 ± 47.9 μg/mL, 63.8 ± 4.4 μg/mL, and 180.5 ± 6.7 μg/mL, respectively.Compound 1a also showed inhibitory activity against rabbit platelet aggregation induced by AA (0.5 mM) with an IC50value of 72.6 ± 10.5 μg/mL.These results support previous findings in pharmacological studies related to the traditional uses of the plant.

AcknowledgementsThis study was supported by the Southeast Asia Biodiversity Research Institute, Chinese Academy of Sciences (Grant Nos.2015CASEABRIRG001 and Y4ZK111B01), the International Partnership Program of Chinese Academy of Sciences (Grant No.153631KYSB20160004), the National Natural Science Foundation of China (31960480), the Joint Special Project of Local Undergraduate Universities in Yunnan Province, China (Grant No.2018FH001-024),and the Second Tibetan Plateau Scientific Expedition and Research(STEP) Program of Ministry of Science and Technology of the People’s Republic of China (Grant No.2019QZKK0502).

Compliance with Ethical Standards

Conflicts of interestThe authors declare that there are no conflicts of interest associated with this work.

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