Desmodium triquetrum(L.)DC is widely distributed in sub-tropical and Pacific regions of the world. Its leaves have been traditionally used for the treatment of diabetes, obesity, flu fever, sore throat, nephritis, cholestatic hepatitis, enteritis, bacillary dysentery, pregnant vomiting, and prostatic hyperplasia worldwide(Wang et al, 2007). Previous chemical studies disclosed the presence of secondary metabolites belonging to terpenes, flavanoids, and phenolic compounds. For instance, ursolic acid, (+)catechin, ilexgenin A(Wen et al, 2000), and salicylic acid(Lv et al, 1995)were obtained from this plant, respectively. In addition, tadehaginoside, a unique phenolic compound, was isolated from this plant and exhibited interesting activities in heptoprotection(Tang et al, 2014). As part of an ongoing investigation on discovery of naturally occurring bioactive agents from the plants of genus Desmodium Desv., a new phenolic compound, analog of tadehaginoside, together with eight known compounds were obtained from D. triquetrum. Herein, this paper mainly deals with the isolation of the new compound as well as the lipid-lowering effects of the isolates.

Optical rotations were obtained on a Perkin-Elmer 341 Digital Polarimeter(USA). UV and IR spectra were recorded on Shimadzu UV2550 and FTIR-8400S Spectrometer(Japan), respectively. NMR spectra were obtained with a Bruker AV 600 NMR Spectrometer(with TMS as internal st and ard)(German). HR-ESI-MS was performed on an LTQ-Obitrap XL Spectrometer(Thermo Fisher Scientific, USA). Silica gel(100-200 and 300-400 mesh, Qingdao Marine Chemical Inc., China)was used for column chromatography. Precoated silica gel GF₂₅₄ plates(Zhifu Huangwu Pilot Plant of Silica Gel Development, China)were used for TLC analysis. Sephadex LH-20 was purchased from GE Healthcare Bio-Sciences Co., Ltd.(Swiss). All solvents used were of analytical grade(Beijing Chemical Works, China). HepG2 cells originally from the American Type Culture Collection(USA), were obtained from China Union Medical University.

The whole plants of Desmodium triquetrum(L.)DC were collected in July 2013 from Haikou, Hainan Province, China and identified by Prof. Jian-ping Tian, School of Pharmaceutical Science, Hainan Medical University. A voucher specimen has been deposited there(Voucher specimen No. DT20130810).

The whole plants of D. triquetrum(5.0 kg)were extracted with 70% ethanol for three times. Removal of the solvent under reduced pressure yielded the ethanol extract(500 g). The extract was subjected to chromatography on silica gel(100-200 mesh)column eluted with petroleum ether, ethyl acetate, and n-butanol, respectively. The n-BuOH fraction(200 g)was subjected to silica gel column chromatography using CHCl₃-MeOH(1:0→2:1)as eluent to yield five fractions(Frs. A-E). Fr. B was purified using Sephadex LH-20 and eluted with MeOH. The subfractions were further purified with semi-preparative HPLC(35% MeOH-H₂O)to yield compounds 1(7.5 mg) and 2(14.0 mg), Fr. C was purified firstly with Sephadex LH-20 column followed by semi-preparative HPLC(40% MeOH-H₂O)to yield compounds 3(4.5 mg), 4(4.0 mg), and 5(5.0 mg). Fr. D was purified by Sephadex LH-20 column and then further isolated with semi-preparative HPLC(45% MeOH-H₂O)to yield compounds 6(4.0 mg) and 7(5.0 mg). Fr. E was purified with preparative HPLC(35% MeOH-H₂O)to yield compounds 8(10.5 mg) and 9(10.0 mg).

HepG2 cells were grown to 70%-80% confluence and then incubated in 0.02% BSA(Sigma-Aldrich)/DMEM(Gibco-BRL, USA)for 12 h. Cells were then washed and incubated with 1 μmol/L compounds or 1 mmol/L AMPK activator AICAR(Sigma-Aldrich, China)in 0.02% BSA + 100 μmol/L oleic acid/DMEM or 0.02% BSA + 100 μmol/L oleic acid/DMEM alone for 6 h. Subsequently, the cells were subjected to oil-red O staining or TC and TG determination.

Repeated chromatography of n-BuOH-soluble extract over silica gel and Sephadex LH-20 columns, followed by semi- preparative HPLC purification afforded compounds 1-9 finally.

Compound 1: white powder, the molecular formula C₂₁H₂₂O₁₀, was deduced from quasimolecular ion peak at m/z 457.1101 [M + Na]⁺(Calcd. for 457.1111)in the positive HR-ESI-MS spectrum. The IR spectrum showed the presence of hydroxyl groups(3380 cm^-1), a conjugated carbonyl group(1690 cm^-1), and a benzene ring(1602, 1560 cm^-1). The ¹H-NMR(600 MHz, CD₃OD)spectrum displayed signals attributed to AA′BB′ type aromatic protons at δ_H 7.60(2H, d, J = 7.2 Hz, H-2″, 6″), 6.72(2H, d, J = 7.2 Hz, H-3″, 5″), and one glucose moiety. The anomeric proton was resonated at δ_H 4.86(1H, d, J = 7.8 Hz) and the anomeric carbon was observed at δ 102.3. The large coupling constant of anomeric proton(J = 7.8 Hz)suggested a β-configured configuration(unit A). The ¹H-NMR spectrum also exhibited signals at δ_H 6.08(2H, s, H-2, 6), 5.95(1H, s, H-4)attributing to one phloroglucinol moiety(unit B). Additionally, two low-field doublets observed at δ_H 6.85(1H, d, J = 12.0 Hz, H-7″), 5.84(1H, d, J = 12.0 Hz, H-8″)are characteristic of α and β protons of a cis-double bond conjugated to a carbonyl group. The ¹³C-APT(150 MHz, CD₃OD)exhibited signals which were clearly recognized as individual carbons by combination of HSQC and HMBC analyses as well as comparing with the reported data of tadehaginoside(Wen et al, 2000). These data were attributed to be δ 102.3(C-1′), 74.9(C-2′), 78.0(C-3′), 71.7(C-4′), 75.6(C-5′), 64.3(C-6′), 97.1(C-2, 6), 160.2(C-3, 5), 98.3(C-4), 127.8(C-1″), 133.8(C-2″, 6″), 116.9(C-3″, 5″), 160.2(C-4″), 145.7(C-7″), 116.0(C-8″), 168.3(C-9″). This elucidation unambiguously confirmed the presence of the three units of a glucosyl group, a phloroglucinol moiety, and a cis p-coumaroyl group. The above structural elucidation was further confirmed by HSQC, ¹H-¹H COSY, and HMBC spectral analysis. The HMBC correlations between the olifenic protons and the carbonyl and benzene ring showed the presence of a cis-p-coumaroyl moiety(unit C). The linkages of units A, B, and C were successfully established by HMBC correlations between the anomeric proton(H-1′)at δ_H 4.86 and C-1 at δ_C 160.9. The correlations from H-6′ at δ_H 4.31, 4.45 to C-9″ at δ_C 168.3 are depicted in Figure 1. The spectroscopic data of compound 1 were superposable upon those of tadehaginoside(Wen et al, 2000), apart from the configuration of Δ^7″(8″) double bond. The observed NOE correlation from H-7″ and H-8″ further confirmed the cis configuration of the double bond. Based on these extensive analyses, the structure of compound 1was fully established as 6′-O-cis-p-coumaroyl-3, 5-dihydroxyphenyl β-D-glucopyranoside, and was characterized as a new phenolic compound as shown in Figure 2.

Figure 1

Figure 1 Key HMBC() and NOE correlations()of compound 1

Figure 2

Figure 2 Chemical structure of compound 1

Compound 1: white powder(MeOH); [α]25 D-30°(c 0.1, MeOH). IR ν ^KBr_max(cm^-1): 3380, 1693, 1604, 1560. ¹H-NMR(600 MHz, CD₃OD)δ: 6.08(2H, s, H-2, 6), 5.95(1H, s, H-4), 7.60(2H, d, J = 7.2 Hz, H-2″, 6″), 6.72(2H, d, J = 7.2 Hz, H-3″, 5″), 6.85(1H, d, J = 12.0 Hz, H-7″), 5.84(1H, d, J = 12.0 Hz, H-8″), 4.86(1H, d, J = 7.8 Hz, H-1′), 4.31(1H, d, J = 10.8 Hz, H-6′a), 4.45(1H, dd, J = 10.8, 2.0 Hz, H-6′b), 3.61(1H, brs, H-5′), 3.40-3.44(3H, m, H-2′, 3′, 4′). ¹³C-NMR(150 MHz, CD₃OD)δ: 160.3(C-1)97.1(C-2, 6), 160.2(C-3, 5), 98.3(C-4), 102.3(C-1′), 74.9(C-2′), 78.0(C-3′), 71.7(C-4′), 75.6(C-5′), 64.3(C-6′), 127.8(C-1″), 133.8(C-2″, 6″), 116.9(C-3″, 5″), 160.2(C-4″), 145.7(C-7″), 116.0(C-8″), 168.3(C-9″).

Compound 2: white powder(MeOH). HR-ESI-MS m/z 457.1104 [M + Na]⁺. ¹H-NMR(600 MHz, CD₃OD)δ: 6.10(2H, s, H-2, 6), 5.97(1H, s, H-4), 7.47(2H, d, J = 7.2 Hz, H-2″, 6″), 6.80(2H, d, J = 7.2 Hz, H-3″, 5″), 7.62(1H, d, J = 16.2 Hz, H-7″), 6.40(1H, d, J = 16.2 Hz, H-8″), 4.28(1H, dd, J = 11.4, 6.6 Hz, H-6′a), 4.54(1H, dd, J = 11.4, 2.0 Hz, H-6′b), 3.67(1H, brs, H-5′), 3.41-3.48(3H, m, H-2′, 3′, 4′). ¹³C-NMR(150MHz, CD₃OD)δ: 160.9(C-1), 97.0(C-2, 6), 160.2(C-3, 5), 98.2(C-4), 102.2(C-1′), 74.9(C-2′), 78.1(C-3′), 71.9(C-4′), 75.6(C-5′), 64.8(C-6′), 127.4(C-1″), 131.4(C-2″, 6″), 116.9(C-3″, 5″), 160.9(C-4″), 147.0(C-7″), 115.1(C-8″), 169.4(C-9″). Compound 2 was identified as tadehaginoside by comparison of the NMR data with the literature value(Wen et al, 2000).

Compound 3: light yellow powder(MeOH). ¹H-NMR(600 MHz, DMSO-d₆)δ: 7.54(1H, d, J = 2.0 Hz, H-2′), 7.52(1H, dd, J = 8.4, 2.0 Hz, H-6′), 6.84(1H, d, J = 8.4 Hz, H-5′), 6.38(1H, d, J = 1.8 Hz, H-8), 6.19(1H, d, J = 1.8 Hz, H-6), 5.33(1H, d, J = 7.2 Hz, H-1″), 4.38(1H, s, H-1′′′), 3.05-3.70(m, sugar protons), 0.98(3H, d, J = 6.0 Hz, H-6′′′). ¹³C-NMR(150 MHz, DMSO-d₆)δ: 156.5(C-2), 133.2(C-3), 177.2(C-4), 161.2(C-5), 98.6(C-6), 164.1(C-7), 93.5(C-8), 156.3(C-9), 103.8(C-10), 121.3(C-1′), 115.1(C-2′), 144.6(C-3′), 148.3(C-4′), 116.2(C-5′), 121.0(C-6′), 101.1(C-1″), 74.0(C-2″), 76.4(C-3″), 69.9(C-4″), 75.8(C-5″), 66.9(C-6″), 100.6(C-1″′), 70.3(C-2″′), 70.4(C-3″′), 71.7(C-4″′), 68.1(C-5″′), 17.6(C-6″′). Compound 3 was identified as rutin by comparison of the NMR data with the literature value(Tang et al, 2000).

Compound 4: yellow powder(MeOH).¹H-NMR(600 MHz, CD₃OD)δ: 6.19(1H, brs, H-6), 6.37(1H, brs, H-8), 6.85(1H, d, J = 7.2 Hz, H-5′), 7.56(1H, d, J = 1.8 Hz, H-2′), 7.58(1H, dd, J = 7.2, 1.8 Hz, H-6′), 5.23(1H, d, J = 7.2 Hz, H-1″). ¹³C-NMR(150 MHz, CD₃OD)δ: 158.2(C-2), 135.7(C-3), 179.2(C-4), 162.6(C-5), 100.0(C-6), 165.8(C-7), 94.8(C-8), 159.2(C-9), 105.5(C-10), 122.6(C-1′), 115.9(C-2′), 145.5(C-3′), 149.6(C-4′), 117.8(C-5′), 122.7(C-6′), 101.2(C-1″), 74.9(C-2″), 77.8(C-3″), 69.6(C-4″), 76.7(C-5″), 61.6(C-6″). Compound 4 was identified as quercetin- 3-O-β-D-glucopyranoside by comparison of the NMR data with the literature value(Li et al, 2010).

Compound 5: brown yellow powder(MeOH). ¹H-NMR(600 MHz, CD₃OD)δ: 7.71(1H, dd, J = 7.2, 1.8 Hz, H-6′), 7.57(1H, d, J = 1.8 Hz, H-2′), 6.85(1H, d, J = 7.2 Hz, H-5′), 6.37(1H, brs, H-8), 6.19(1H, brs, H-6), 5.23(1H, d, J = 7.2 Hz, glu-H-1). ¹³C- NMR(150 MHz, CD₃OD)δ: 158.2(C-2), 135.7(C-3), 179.2(C-4), 162.6(C-5), 100.0(C-6), 165.8(C-7), 94.8(C-8), 159.1(C-9), 105.1(C-10), 122.6(C-1′), 115.5(C-2′), 145.5(C-3′), 149.6(C-4′), 115.9(C-5′), 122.7(C-6′), 101.4(C-1″), 75.3(C-2″), 73.0(C-3″), 70.8(C-4″), 69.6(C-5″), 61.6(C-6″). Compound 5 was identified as quercetin-3-O-β-D-galactopyranoside by comparison of the NMR data with the literature value(Tang et al, 2008).

Compound 6: white powder(MeOH). ¹H-NMR(600 MHz, CD₃OD)δ: 7.61(1H, d, J = 18.0 Hz, H-7), 7.44(2H, d, J = 7.8 Hz, H-3, 5), 6.79(2H, d, J = 7.8 Hz, H-2, 6), 6.30(1H, d, J = 18.0 Hz, H-8), 4.43(1H, d, J = 7.8 Hz, H-1′). ¹³C-NMR(150MHz, CD₃OD)δ: 127.3(C-1), 131.3(C-2, 6), 117.0(C-3, 5), 161.4(C-4), 146.8(C-7), 115.2(C-8), 169.3(C-9), 98.4(C-1′), 73.9(C-2′), 78.1(C-3′), 71.9(C-4′), 75.6(C-5′), 65.1(C-6′). Compound 6 was identified as 6-O-(E)-p-hydroxy- cinnamoyl-β-glucose by comparison of the NMR data with the literature value(Wang et al, 2007).

Compound 7: white powder(MeOH). ¹H-NMR(600 MHz, CD₃OD)δ: 7.61(1H, d, J = 18.0 Hz, H-7), 7.45(2H, d, J = 7.8 Hz, H-3, 5), 6.81(2H, d, J = 7.8 Hz, H-2, 6), 6.34(1H, d, J = 18.0 Hz, H-8), 5.10(1H, d, J = 2.4 Hz, H-1′). ¹³C-NMR(150 MHz, CD₃OD)δ: 127.3(C-1), 131.3(C-2, 6), 117.0(C-3, 5), 161.4(C-4), 146.9(C-7), 115.1(C-8), 169.4(C-9), 94.2(C-1′), 72.2(C-2′), 76.4(C-3′), 71.0(C-4′), 74.9(C-5′), 65.0(C-6′). Compound 7 was identified as 6-O-(E)-p- hydroxy-cinnamoyl-α-glucose by comparison of the NMR data with the literature value(Wang et al, 2000).

Compound 8: yellow powder(MeOH).¹H-NMR(600MHz, DMSO-d₆)δ: 7.97(2H, d, J = 7.8 Hz, H-2′, 6′), 6.87(2H, d, J = 7.2 Hz, H-3′, 5′), 6.41(1H, d, J = 1.8 Hz, H-8), 6.20(1H, d, J = 1.8 Hz, H-6), 5.30(1H, d, J = 7.2 Hz, H-1″), 4.37(1H, d, J = 1.2 Hz, H-1′′′), 3.05-3.70(m, sugar protons), 0.99(3H, d, J = 6.0 Hz, H-6′′′). ^13C-NMR(150 MHz, DMSO-d₆)δ: 156.7(C-2), 133.1(C-3), 177.2(C-4), 161.1(C-5), 98.6(C-6), 164.1(C-7), 93.6(C-8), 156.3(C-9), 103.8(C-10), 120.7(C-1′), 130.7(C-2′, 6′), 114.9(C-3′, 5′), 159.7(C-4′), 101.2(C-1″), 74.1(C-2″), 76.3(C-3″), 69.8(C-4″), 75.6(C-5″), 66.8(C-6″), 100.6(C-1′′′), 70.2(C-2′′′), 70.5(C-3′′′), 71.7(C-4′′′), 68.1(C-5′′′), 17.6(C-6′′′). Compound 8 was identified as kaempferol-3-O-β-D-rutinoside by comparison of the NMR data with the literature value of(Tang et al, 2000).

Compound 9: yellow powder(MeOH). ¹H-NMR(600 MHz, DMSO-d₆)δ: 1.06(3H, d, J = 6.0 Hz, rha-CH₃), 4.40(1H, d, J = 1.8 Hz, H-1′′′), 5.34(1H, d, J = 7.2 Hz, H-1″), 6.19(1H, brs, H-6), 6.38(1H, brs, H-8), 6.82(1H, d, J = 7.8 Hz, H-5′), 7.53(1H, brs, H-2′), 7.65(1H, dd, J = 7.8, 1.8 Hz, H-6′), 12.59(1H, s, 5-OH). ¹³C-NMR(150MHz, DMSO-d₆)δ: 156.3(C-2), 133.4(C-3), 177.3(C-4), 161.1(C-5), 98.6(C-6), 164.1(C-7), 93.5(C-8), 156.5(C-9), 103.8(C-10), 121.8(C-1′), 115.1(C-2′), 144.7(C-3′), 148.4(C-4′), 115.9(C-5′), 120.9(C-6′), 101.1(C-1″), 71.0(C-2″), 73.5(C-3″), 67.9(C-4″), 73.0(C-5″), 65.0(C-6″), 99.9(C-1′′′), 70.2(C-2′′′), 70.5(C-3′′′), 71.8(C-4′′′), 68.2(C-5′′′), 17.6(C-6′′′). Compound 9 was identified as 3-O-β-D-galacopyranosyl(6-1)-α-L-rhamnosyl quercetin by comparison of the NMR data with the literature value(Zhang et al, 2001).

All the isolates are evaluated for their anti- hyperlipidemic activities. Among them, compounds 1 and 2 significantly reduce the oil-red O staining TC and TG(Figure 3)accumulation in HepG2 cells, suggesting that they play a vital role in the anti-hyperlipidemic activity of D. triquetrum. Considering their potent lipid-modulating activity and high concentration in D. triquetrum, phenolic compounds, particular tadehaginoside, are assigned as the main active components accounting for its anti- hyperlipidemic effect.

Figure 3

Figure 3 TC(A) and TG(B)accumulation in HepG2 cells P < 0.05 vs normal group; †P < 0.05 vs OA group