Millettia speciosa Champ., belonging to Leguminosae, is a sub-shrub plant commonly known as a tonic and mainly growing in Guangdong and Guangxi provinces, China. Its roots are used to treat lumbago and strengthen the bones and muscles(Editorial Board of Chinese Materia Medica in Guangdong, 1991; State Administration of Editorial Board of Chinese Materia Medica, 1999). It was widely used as an edible plant with the effect of strengthening bones and muscles in local places, such as stewed chicken or soup. Previous phytochemical studies reported that the root extract of M. speciosa contains various constituents including alkaloid, oleanane-type triterpene saponin, flavonol, and phenolic glycosides(Uchiyama et al, 2003; Wang et al, 2008; Yin et al, 2008; Yin et al, 2010; Zong et al, 2009; Zhang et al, 2011), etc. In order to know more bioactive compounds and safe use of this herbal medicine, 13 compounds were isolated from petroleum ether, ethyl acetate, and n-butanol soluble parts of ethanol extract from the roots of M. speciosa. The compounds were identified as docosanoic acid(1), tetracosane(2), octadecane(3), hexacosanoic acid(4), β-sitosterol acetate(5), β-sitosterol(6), syringin(7), maackiain(8), formononetin(9), ψ-baptigenin(10), rotundic acid(11), pedunculoside(12), and daucosterol(13). Compounds5, 7, and 10-12 were reported for the first time in this plant. In this paper, we report the isolation and structure elucidation of these compounds.

TLC was performed on silica gel GF₂₅₄ plates(Qingdao Marine Chemical Factory) and developed with 10% sulphuric acid in ethanol. Chromatography was carried out on silica gel(200-300 mesh, Qingdao Marine Chemical Factory), Sephadex LH-20(Pharmacia), and RP-18 gel(50 µm, YMC, Japan)columns. Agilent 1100 Series LC/MSD Trap(USA Agilent Technologies) and Nicolet Impact 410 FT-IR Spectrometer were used; HR-ESI-MS was recorded on an APEX II spectrometer. Bruker AV-300 NMRS(Swiss, Bruker)was used. All the NMR data were obtained at room temperature(TMS as internal st and ard).

The roots of Millettia speciosa Champ. were collected in Conghua, Guangdong province, China, in August 2010, and identified by Prof. Ping Ding. A voucher specimen was deposited at the herbarium of Research Laboratory of Chinese Medicinal Resources, Guangzhou University of Chinese Medicine.

The dried sliced roots of M. speciosa(10 kg)were extracted with 95% EtOH(reflux, 2 h, three times). The combined extracts were concentrated in vacuum to yield a dark residue suspended in water and partitioned with ether, EtOAc, and n-BuOH. The ether extract(208 g) and EtOAc extract(250 g)were respectively subjected to column chromatography. The ether extract was re-chromatographed on a silica gel column eluted with gradient mixture of EtOEt- EtOAc(1:0 to 0:1)to yield compounds 1(30 mg), 2(32 mg), 3(28 mg), and 4(40 mg). The EtOAc extract was re-chromatographed on a silica gel column eluted with gradient mixture of CHCl₃-EtOAc(16:1 to 0:1)to give four fractions. Fr. 1(3.47 g)was separated with silica gel column eluted with CHCl₃-EtOAc(1:1 to 0:1)to yield compounds 5(53 mg) and 6(20 mg). Fr. 2(4.93 g)was subjected to repeated column chromatography on silica gel, eluted with CHCl₃-MeOH(12:1 to 0:1)to yield compounds 7(43 mg), 8(48 mg), 9(64 mg), and 10(37 mg). Fr. 3(5.27 g)was subjected to repeated column chromatography on silica gel, eluted with EtOAc-MeOH(20:1 to 0:1)to yield compounds 11(56 mg), 12(60 mg), and 13(120 mg).

Compound 1: white powder(CHCl₃). EI-MS and ¹H-NMR data of compound 1 were nearly identical to those of tetracosanic acid in the literature(Li et al, 2000). The characteristic fragment ions at m/z: 340(M⁺), 312, 298, 284, 270, 256, 242, 228, 214, 200, 129, 115, 101, 87, 73, 59, 45; ¹H-NMR δ: 0.86(3H, t, J = 6.4Hz, CH₃), 1.27 [m, 36H, (CH₂)₁₈], 1.60(2H, t, J = 7.2 Hz), and 2.25(2H, t, J = 7.6 Hz). The molecular weight of major fragment ions of compound 1 in EI-MS spectrum was 28 lower than that of tetracosanic acid in the literature(Li et al, 2000). Thus, compound 1 was identified as docosanoic acid.

Compound 2: white powder(CHCl₃). EI-MS data of compound 2 were nearly identical to those of tricosane in the literature(Ren et al, 2000). The characteristic fragment ions at m/z: 338(M⁺), 85, 71, 57, 43. The molecular weight of major fragment ions of compound 2 in EI-MS spectrum was 14 higher than that of tricosane in the literature(Ren and Yang, 2000). Thus, compound 2 was identified as tetracosane.

Compound 3: white powder(CHCl₃). EI-MS data of compound 3 were nearly identical to those of compound 2. The characteristic fragment ions at m/z: 254(M⁺), 85, 71, 57, 43. The molecular weight of major fragment ions of compound 3 in EI-MS spectrum was 84 lower than that of compound 2. Thus, compound 3 was identified as octadecane.

Compound 4: white powder(CHCl₃). EI-MS, ¹H-NMR and ¹³C-NMR data of compound 4 were nearly identical to those of tetracosanic acid in the literature(Li et al, 2000). The characteristic fragment ions at m/z: 396(M⁺), 368, 354, 342, 129, 73, 57. ¹H-NMR(CDCl₃)δ: 0.87(3H, t, J = 6.4 Hz, CH₃), 1.28 [m, 44H, (CH₂)₂₂], 1.63(2H, t, J = 7.2 Hz), 2.30(2H, t, J = 7.6 Hz). ¹³C-NMR(CDCl₃)δ: 14.1(C-26), 22.7(C-25), 24.7(C-3), 31.9(C-22), 33.7(C-2), 178.3(C-1). The molecular weight of major fragment ions of compound 4 in EI-MS spectrum was 28 higher than that of tetracosanic acid in the literature(Li et al, 2000). Thus, compound 4 was identified as hexacosanoic acid.

Compound 5: white powder(CHCl₃). HR-ESI-MS showed a molecular ion at m/z: 457(M + H)⁺. ¹H-NMR(CDCl₃)δ: 0.67(3H, s, CH₃-18), 1.00(3H, s, CH₃-19), 0.91(3H, d, J = 6.4 Hz, CH₃-21), 0.84(3H, d, J = 7.6 Hz, CH₃-26), 0.81(3H, d, J = 7.2 Hz, CH₃-27), 0.85(3H, t, J = 7.6 Hz, CH₃-29), 4.60(1H, brs, H-3), 5.36(1H, d, J = 4.4 Hz, H-6). ¹³C-NMR(CDCl₃)δ:37.0(C-1), 31.9(C-2), 73.7(C-3), 42.3(C-4), 139.7(C-5), 122.6(C-6), 31.9(C-7), 31.9(C-8), 50.0(C-9), 37.0(C-10), 21.0(C-11), 40.0(C-12), 42.3(C-13), 56.7(C-14), 24.3(C-15), 27.8(C-16), 56.0(C-17), 11.8(C-18), 19.3(C-19), 36.2(C-20), 18.8(C-21), 33.9(C-22), 29.2(C-23), 45.8(C-24), 29.7(C-25), 19.0(C-26), 19.8(C-27), 23.1(C-28), 12.0(C-29), 173.3(Ac), 22.7(Ac). The spectral data were consistent with those of β-sitosterol acetate. Thus, compound 5 was identified as β-sitosterol acetate by compari son of the ¹H-NMR and ¹³C-NMR data with the reported data(Wang and Zou, 2008).

Compound 6: white powder(CHCl₃), mp 136-137 ^oC. Lieberman-Burchard reaction was positive and 5% sulfuric acid-ethanol system was purple. TLC Rf of compound 6 and β-sitosterol reference substance was the same as the β-sitosterol reference substance. Two compounds were mixed that the melting point did not drop. Thus, compound 6 was identified as β-sitosterol.

Compound 7: white needles crystal(MeOH), mp 190-192 ^oC. HR-ESI-MS showed a molecular ion at m/z: 373 [M + H]⁺, ¹H-NMR(MeOD)δ: 6.74(1H, s, 3-H), 6.74(1H, s, 5-H), 6.54(1H, d, J = 16.0 Hz, 7-H), 6.32(1H, dt, J = 16.0 Hz, 8-H), 4.20(1H, m, 9-H), 3.84(3H, s, 10-H), 3.84(3H, s, 11-H), 4.85(1H, d, J = 6.8 Hz, Glc-1), 3.40(1H, m, Glc-2), 3.38(1H, m, Glc-3), 3.30(1H, m, Glc-4), 3.28(1H, m, Glc-5), 3.67(3H, m, Glc-6). ¹³C-NMR(MeOD)δ: 135.3(C-1), 154.4(C-2), 105.5(C-3), 135.9(C-4), 62.6(C-5), 154.4(C-6), 131.3(C-7), 130.0(C-8), 105.5(C-9), 57.0(C-10), 57.0(C-11), 100.8(Glc-1), 73.4(Glc-2), 76.9(Glc-3), 70.1(Glc-4), 76.7(Glc-5), 61.1(Glc-6). The spectral data were consistent with those of syringin. Thus, compound 7 was identified as syringin by comparison of ¹H-NMR and ¹³C-NMR data with the reported data(Wu et al, 1999).

Compound 8: white needles crystal(MeOH), mp 180-182 ^oC. HR-ESI-MS showed a molecular ion at m/z: 285 [M+H]⁺, ¹H-NMR(CD₃OD)δ: 4.21(2H, dd, J = 6.0, 0.8 Hz, H-2), 3.44(1H, m, 3-H), 5.44(1H, d, J = 6.8 Hz, H-4), 7.26(1H, d, J = 8.0 Hz, H-5), 6.48(1H, dd, J = 8.0, 2.0 Hz, H-6), 6.29(1H, d, J = 2.4 Hz, H-8), 6.44(1H, s, H-2′), 6.79(1H, s, H-5′), 5.87(1H, dd, J = 1.2, 0.8 Hz, -O-CH₂-O-), ¹³C-NMR(CD₃OD)δ: 67.4(C-2), 41.6(C-3), 80.0(C-4), 133.1(C-5), 110.7(C-6), 160.1(C-7), 104.1(C-8), 158.0(C-9), 112.9(C-10), 119.8(C-1′), 105.9(C-2′), 143.1(C-3′), 149.6(C-4′), 94.2(C-5′), 158.0(C-6′), 102.4(-O-CH₂-O-). The spectral data were consistent with those of maackiain. Thus, compound 8 was identified as maackiain by comparison of ¹H-NMR and ¹³C-NMR data with the reported data(Wang and Zou, 2008).

Compound 9: yellow crystal(MeOH). HR-ESI-MS showed a molecular ion at m/z: 269 [M + H]⁺. ¹H-NMR(DMSO-d₆)δ: 8.31(1H, s, H-2), 7.98(1H, d, J = 8.8 Hz, H-5), 6.94(1H, dd, J = 8.8, 2.0 Hz, H-6), 10.81(1H, s, H-7), 6.87(1H, d, J = 2.0 Hz, H-8), 7.50(2H, d, J = 8.8 Hz, H-2′, 6′), 6.98(2H, d, J = 8.8 Hz, H-3′, 5′), 3.77(3H, s, -OCH₃). ¹³C-NMR(DMSO-d₆)δ: 153.1(C-2), 123.2(C-3), 174.5(C-4), 127.3(C-5), 115.2(C-6), 162.6(C-7), 102.1(C-8), 157.4(C-9), 116.6(C-10), 125.8(C-1′), 130.1(C-2′, 6′), 113.6(C-3′, 5′), 158.9(C-4′), 55.1(-OCH₃). The spectral data were consistent with those of formononetin. Thus, compound 9 was identified as formononetin by comparison of ¹H-NMR and ¹³C-NMR data with the reported data(Wang et al, 2008).

Compound 10: yellow crystal(MeOH). HR-ESI-MS showed a molecular ion at m/z: 283 [M + H]⁺. ¹H-NMR(DMSO-d₆)δ: 8.32(1H, s, H-2), 7.97(1H, d, J = 8.8 Hz, H-5), 6.94(1H, dd, J = 8.8, 2.0 Hz, H-6), 10.79(1H, s, H-7), 6.87(1H, d, J = 2.0 Hz, H-8), 7.14(1H, d, J = 1.6 Hz, H-2′), 6.95(1H, d, J = 8.8 Hz, H-5′), 7.05(1H, d, J = 1.6 Hz, H-6′), 6.03(2H, s, -O-CH₂-O-). ¹³C-NMR(DMSO-d₆)δ: 153.4(C-2), 123.2(C-3), 174.6(C-4), 127.3(C-5), 115.2(C-6), 162.6(C-7), 102.1(C-8), 157.4(C-9), 116.6(C-10), 124.2(C-1′), 109.4(C-2′), 147.0(C-3′), 146.9(C-4′), 108.7(C-5′), 122.4(C-6′), 147.0(-O-CH₂-O-). The spectral data were consistent with those of ψ-baptigenin. Thus, compound 10 was identified as ψ-baptigenin by comparison of ¹H-NMR and ¹³C-NMR data with the reported data(Gu and Zhou, 1988).

Compound 11: white needles crystal(MeOH). HR-ESI-MS showed a molecular ion at m/z: 489 [M + H]⁺. ¹H-NMR(DMSO-d₆)δ: 1.14, 0.69, 0.96, 0.78, 1.31(each 3H, s, -CH₃), 0.92(3H, d, J = 6.4 Hz, -CH₃), 3.60(1H, dd, J = 6.8, 10.8 Hz, H-3), 5.29(1H, brs, H-12). ¹³C-NMR(DMSO-d₆)δ: 39.5(C-1), 27.2(C-2), 78.1(C-3), 42.7(C-4), 48.8(C-5), 19.3(C-6), 33.7(C-7), 41.0(C-8), 48.4(C-9), 37.9(C-10), 24.7(C-11), 129.4(C-12), 140.0(C-13), 43.1(C-14), 29.6(C-15), 26.6(C-16), 48.6(C-17), 55.1(C-18), 73.6(C-19), 43.3(C-20), 27.1(C-21), 39.1(C-22), 67.5(C-23), 12.7(C-24), 17.5(C-25), 16.6(C-26), 24.9(C-27), 182.3(C-28), 27.3(C-29), 16.2(C-30). The spectral data were consistent with those of rotundic acid. Thus, compound 11 was identified as rotundic acid by comparison of ¹H-NMR and ¹³C-NMR data with th e reported data(Ma et al, 2005).

Compound 12: white needles crystal(MeOH). HR-ESI-MS showed a molecular ion at m/z: 651 [M + H]⁺. ¹H-NMR(DMSO-d₆)δ: 1.29, 1.19, 0.97, 0.92, 0.77, 0.69(each 3H, s, -CH₃), 0.91(3H, d, J = 6.4 Hz, -CH₃), 3.56(1H, dd, J = 5.60, 4.80 Hz, H-3), 5.30(1H, brs, H-12), 4.59(1H, d, J = 7.5 Hz, Glc-1). ¹³C-NMR(DMSO-d₆)δ: 39.4(C-1), 26.5(C-2), 74.1(C-3), 39.6(C-4), 48.8(C-5), 17.7(C-6), 33.5(C-7), 41.0(C-8), 48.4(C-9), 37.9(C-10), 24.7(C-11), 129.7(C-12), 139.6(C-13), 42.7(C-14), 29.6(C-15), 27.1(C-16), 48.8(C-17), 54.8(C-18), 73.7(C-19), 43.3(C-20), 27.0(C-21), 41.2(C-22), 67.6(C-23), 12.6(C-24), 17.6(C-25), 17.5(C-26), 26.4(C-27), 178.6(C-28), 27.2(C-29), 16.1(C-30), 95.8(Glc-1), 74.1(Glc-2), 78.6(Glc-3), 71.1(Glc-4), 78.3(Glc-5), 62.4(Glc-6). The spectral data were consistent with those of pedunculoside. Thus, compound 12 was identified as pedunculoside by comparison of ¹H-NMR and ¹³C-NMR data with the reported data(Wei and Chen, 1991).

Compound 13: white powder(MeOH), mp 290-295 ^oC. Lieberman-Burchard and Molish reaction was positive and 5% sulfuric acid-ethanol system was purple. TLC Rf of compound 13 and daucosterol reference substance were the same. Two compounds were mixed that the melting point did not drop. Thus, compound 13 was identified as daucosterol.