Fitoterapia 98 (2014) 143–148

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Fitoterapia

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Triterpenoids and α-glucosidase inhibitory constituents fromSalacia hainanensis

Mei-Hua Yu a, Zheng-Feng Shi a, Bang-Wei Yu b, En-Hao Pi a, He-Yao Wangb,Ai-Jun Hou a, Chun Lei a,⁎a School of Pharmacy, Fudan University, 826 Zhang Heng Road, Shanghai 201203, People's Republic of Chinab Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zu Chong Zhi Road, Shanghai 201203, People's Republic of China

a r t i c l e i n f o

⁎ Corresponding author. Tel.: +86 21 51980173; fax:E-mail address: chunlei@fudan.edu.cn (C. Lei).

http://dx.doi.org/10.1016/j.fitote.2014.07.0160367-326X/© 2014 Elsevier B.V. All rights reserved.

a b s t r a c t

Article history:Received 26 May 2014Accepted in revised form 17 July 2014Available online 27 July 2014

Thirteen triterpenoids (1–13), including twonew lupane triterpenoids, salacinins A andB (1 and 2),aswell as one new friedelane triterpenoid, salacinin C (3),were isolated from the roots and stemsofSalacia hainanensis. The structures of new compounds were elucidated by extensive spectroscopicanalysis including 1D and 2D NMR, and MS experiments. Compound 1 possesses rare 2,3-seco-lupane skeleton. Compounds 4, 6 and 7 showed inhibitory effects on α-glucosidase in vitro.

© 2014 Elsevier B.V. All rights reserved.

Keywords:Salacia hainanensisLupineFriedelaneα-Glucosidase

1. Introduction

The genus Salacia (Hippocrateaceae) is composed of about200 species distributed throughout the world in tropical areas,including India, Sri Lanka, southern China, and other SoutheastAsian countries [1]. Among of them, some species, includingS. reticulata, S. oblonga, and S. chinensis (syn. S. prinoides), havebeen widely used for prevention or remedy of diabetes in localcountries [1–4]. The extracts of their roots and stems showedpotent inhibitory effects on intestinal α-glucosidase and werereported to be effective on controlling serum glucose levels inoral sucrose- and maltose-loaded rats [5–7]. Glucosidase inhib-itors including α-glucosidase inhibitors have been considered aspowerful and promising therapeutic agents in carbohydratemetabolic disorders, especially for diabetes mellitus [8,9]. Thus,great interests have been instigating for searching antidiabeticleading compounds from Salacia plants.

Salacia hainanensis Chun & How is an endemic plantdistributed mainly over Hainan Province of China, and has a

+86 21 51980005.

long history as traditional folk medicine for treating rheumaticjoint pain, lumbar muscle strain, and asthenia [10]. Pharma-cology study revealed that its root extract also has hypoglyce-mic activity [11]. Previous phytochemical investigations on thisplant have led to isolating a few triterpenoids, and some ofthem showed inhibitory activities on α-glucosidase [12–14]indicating that this plant is a good resource for searchingantidiabetic natural compounds. In order to fully exploit thisresource, we reinvestigated the chemical constituents of thisplant.

Separation of the EtOAc-soluble fraction from an ethanolextract of the roots and stems of S. hainanensis afforded twonew lupane triterpenoids, salacinins A and B (1 and 2), andone new friedelane triterpenoid, salacinin C (3), togetherwith 10 known pentacyclic triterpenoids 2-oxo-20(29)-lupen-3β-ol (4) [15], 2β,3β-dihydroxylup-20(29)-ene (5)[13], lupeol (6) [16], salacianone (7) [17], lup-20(29)-ene-3,16-diol (8) [18], lup-20(29)-ene-3-on-28-ol (9) [19], 15-hydroxyfriedelan-3-one (10) [20], friedelane-3-one-29-ol(11) [21], 21-hydroxyfriedelan-3-one (12) [22], and friedel-1-en-3-one (13) [23] (Fig. 1). Compound 1 possesses rare2,3-seco-lupane skeleton. Compounds 4, 6 and 7 showed

Fig. 1. Structures of compounds 1–13.

144 M.-H. Yu et al. / Fitoterapia 98 (2014) 143–148

inhibitory effects on α-glucosidase in vitro. In this paper, wepresent the structure elucidation of compounds 1–3 and thebiological evaluation against α-glucosidase.

2. Experimental

2.1. General

Optical rotations were measured on a Jasco P1030 polarim-eter. UV Spectra were obtained using a Shimadzu UV-2401PCspectrophotometer. IR spectra were obtained using a NicoletAvatar-360 spectrometer. 1H, 13C, and 2D NMR spectra wererecorded on a Bruker DRX-400 spectrometer. EIMS andHREIMSweremeasured on a FinniganMAT 95mass spectrom-eter. Semipreparative HPLC was performed on an Agilent 1200(Sepax ODS column, 150 × 10 mm i.d., 5 μm) with a variablewavelength detector (VWD). Thin-layer and column chroma-tography was performed using silica gel (Yantai Institute ofChemical Technology, GF254, 200–300mesh) and ChromatorexRP-18 gel (Fuji Silysia Chemical, Ltd.).

2.2. Plant material

The roots of S. hainanensiswere collected in Baoting countyof Hainan province, PR China, in July 2008 and air-dried. Theplant was identified by Dr. Yun Kang, Fudan University. A

voucher specimen (TCM 2008-07 Hou) was deposited in theHerbarium of the Department of Pharmacognosy, School ofPharmacy, Fudan University.

2.3. Extraction and isolation

The extraction and isolation procedure was summarized inSch.1S in the Supplementary data. The dried and powderedroots and stems (4.4 kg) of S. hainanensis were extracted with95% EtOH (25 l × 4, each time for 2 days) at r.t. The filtrate wasevaporated in vacuo to give a residue (150 g), which wassuspended in H2O and partitioned successively with AcOEt andn-BuOH. The AcOEt extract (26.0 g) was subjected to columnchromatograph (CC) (SiO2; petroleum ether (PE): acetone =50:1, 20:1, 10:1, 3:1, and 3:7) to afford 11 fractions (Frs. 1–11).Fr.3 (1.50 g) was separated by CC (SiO2; PE: CHCl3 = 10:1 and7:1) to give 5 fractions (Frs. 3.1–3.5). Fr. 3.1 (50 mg) wasfractionated by preparative TLC (PE: AcOEt: HCOOH 10:1:1d, Rf

0.5), followed by prep. HPLC (MeOH: CH3CN = 19:1, 1.0 ml/min, 210 nm) to afford compound 4 (7 mg; tR 30 min). Fr. 3.2(40 mg) was isolated by prep.TLC (PE: AcOEt: HCOOH =10:1:1d, Rf 0.4), followed by prep. HPLC (MeOH: CH3CN 19:1,1.0ml/min, 210nm) to give compound 2 (10mg; tR 20min). Fr.3.3 (150mg)was isolated by CC (SiO2; PE: AcOEt=8:1) to give3 fractions (Frs. 3.3.1–3.3.3). Fr. 3.3.1 (10 mg) was fractionatedby prep. HPLC (MeOH: CH3CN 9:1, 1.0 ml/min, 210 nm) to

Table 113C NMR data of compounds 1–3 (CDCl3, δ □ in ppm).

Position 1 2 3

1 42.1 53.5 130.22 176.2 211.4 148.93 60.8 82.9 201.94 46.3 45.6 57.75 48.4 54.6 43.66 22.0 18.5 39.87 33.1 33.8 17.78 40.7 43.9 50.79 42.2 50.4 36.610 42.1 41.3 62.211 20.8 21.0 36.012 25.1 25.0 30.213 38.2 37.2 39.114 43.2 42.8 38.815 27.4 27.0 29.916 35.4 29.1 36.017 43.0 47.7 32.518 48.1 48.6 44.319 48.0 47.7 34.320 150.8 150.3 36.021 29.8 29.7 74.222 39.9 33.9 47.023 27.9 29.3 6.724 13.9 16.3 13.725 19.7 17.0 17.426 15.9 15.6 20.127 14.4 14.7 19.228 18.0 60.5 33.129 109.5 109.9 24.930 19.2 19.1 31.831 180.1 − −32 23.6 − −

145M.-H. Yu et al. / Fitoterapia 98 (2014) 143–148

afford compound 5 (7 mg; tR 30 min). Fr. 3.3.2 (100 mg) waspurified by CC (SiO2; PE: AcOEt= 8:1), followed by prep. HPLC(MeOH: CH3CN = 17:3, 1.0 ml/min, 210 nm) to yieldcompounds 6 (8 mg; tR 25 min) and 7 (5 mg; tR 30 min). Fr.3.4 (100mg) was isolated by CC (SiO2; PE: IPA= 15:1) to give 2fractions (Frs. 3.4.1–3.4.2). Fr. 3.4.1 (10 mg) was fractionated byprep. HPLC (MeOH: CH3CN=9:1, 1.0ml/min, 210 nm) to affordcompound 8 (18 mg; tR 25 min). Fr. 3.4.2 (45 mg) was purifiedby prep. TLC (PE: AcOEt: HCOOH = 8:1:1d, Rf 0.4), followed byprep. HPLC (MeOH: CH3CN=19:1, 1.0ml/min, 210 nm) to yieldcompound 12 (7 mg; tR 15 min). Fr.5 (1.63 g) was fractionatedby CC (SiO2; PE: acetone = 8:1 and 5:1) to give 4 fractions (Frs.5.1–5.4). Fr.5.1 (200mg)was separated by CC (SiO2; PE: acetone= 8:1) to afford 3 fractions (Frs. 5.1.1–5.1.3). Frs. 5.1.1 (45 mg)was purified by prep. TLC (PE: AcOEt: HCOOH= 7:1:1d, Rf 0.4),followed by prep. HPLC (MeOH: CH3CN = 19:1, 1.0 ml/min,210nm) to yield compound1 (6mg; tR 18min). Fr. 5.1.2 (50mg)was isolated by prep.TLC (PE: AcOEt: HCOOH = 6:1:1d, Rf 0.4),followed by prep. HPLC (MeOH: CH3CN = 9:1, 1.0 ml/min,210 nm) to give compounds 10 (10 mg; tR 40 min) and 11(10mg; tR 45min). Fr.5.2 (150mg)was fractionated byCC (SiO2;PE: CHCl3 = 1:1) to afford 3 fractions (Frs. 5.2.1–5.2.3). Frs.5.2.1 (45 mg) was purified by prep. HPLC (MeOH: CH3CN =19:1, 1.0 ml/min, 254 nm) to yield compounds 3 (6 mg; tR28 min) and 13 (7 mg; tR 25 min). Frs. 5.2.2 (45 mg) waspurified by CC (ChromatorexRP-18 gel; MeOH: H2O= 19:1) togive compound 9 (3 mg).

2.3.1. 2,3-Seco-20(29)-lupene-3-acetoxy-2-oic-acid(salacinin A, 1)

White amorphous powder (CHCl3); [α]D20 −6.8° (c 0.2,CHCl3); IR (KBr) νmax 3428, 1710, 1630, 1390, 1120 cm−1; EIMSm/z (%): 500 (1, [M]+), 385 (12), 325 (10), 231 (12), 189 (17),155 (36), 147 (12), 121 (19), 116 (100), 107 (14); HREIMS m/z500.3865 [M]+ (calcd. for C32H52O4, 500.3866); 1H NMR (CDCl3,400MHz): δH = 4.68 (1 H, br s, H–29a), 4.56 (1 H, br s, H–29b),4.17 (1 H, d, J = 6.3 Hz, H–3a), 4.00 (1 H, d, J = 6.3 Hz, H–3b),2.50 (1 H, br d, J = 17.6 Hz, H–1a), 2.42 (1 H, m, H–19β), 2.34(1 H, br d, J=17.6 Hz, H–1b), 2.26 (1 H, br d, J=7.8 Hz, H–9α),1.89 (1 H, m, H–21β), 1.67 (3 H, s, CH3–30), 1.66 (2 H, m,overlapped, H–12β andH–13β), 1.64 (1H,m,H–15β), 1.50 (2H,m, CH2–11), 1.48 (2 H, m, CH2–7), 1.47 (1 H, m, overlapped, H–16β), 1.46 (2 H, m, CH2–6), 1.38 (1 H, m, overlapped, H–22β),1.37 (1 H, br d, J= 7.8 Hz, H–5α), 1.37 (1 H, m, overlapped, H–16α), 1.36 (1 H, br d, J=8.0 Hz, H–18α), 1.34 (1 H, m, H–21α),1.24 (9 H, s, CH3–23, CH3–24, and CH3–32), 1.21 (1 H, m, H–22α), 1.08 (1 H, m, H–12α), 1.05 (1 H, m, H–15α), 1.03 (3 H, s,CH3–26), 0.93 (6 H, s, CH3–25 and CH3–27), 0.78 (3 H, s, CH3–

28); 13C NMR (CDCl3, 100 MHz), see Table 1.

2.3.2. 3β,28-dihydroxylup-20(29)-en-2-one (salacinin B, 2)White amorphous powder (CHCl3); [α]D20 −18.5° (c 0.4,

CHCl3); IR (KBr) νmax 3495, 1727, 1636, 1444, 1166 cm−1; EIMSm/z (%): 456 (29, [M]+), 425 (42), 203 (59), 189 (60), 109 (64),55 (92), 43 (91); HREIMS m/z 456.3605 [M]+ (calcd. forC30H48O3, 456.3603); 1H NMR (CDCl3, 400 MHz): δH = 4.69(1H, br s, H–29a), 4.59 (1H, br s, H–29b), 3.88 (1H, br s, H–3α),3.79 (1 H, d, J = 10.2 Hz, H–28a), 3.44(1 H, br s, 3–OH), 3.35(1 H, d, J=10.2 Hz, H–28b), 2.52 (1 H, br d, J=12.1 Hz, H–1a),2.40 (1 H, m, H–19β), 2.00 (1 H, br d, J = 12.1 Hz, H–1b), 1.95(1 H, m, H–16β), 1.86 (1 H, m, H–22β), 1.84 (1 H, m, H–21β),

1.73 (1 H, m, H–15β), 1.69 (3 H, s, CH3–30), 1.68 (1 H, m, H–12β), 1.66 (2 H, m, CH2–6), 1.65 (1 H, m, overlapped, H–13β),1.63 (1 H, br d, J=7.5 Hz, H–9α), 1.60 (1 H, br d, J=8.2 Hz, H–18α), 1.49 (2 H, m, CH2–7), 1.42 (1 H, br d, J = 8.2 Hz, H–5α),1.42 (1 H, m, H–21α), 1.29 (2 H, m, CH2–11), 1.24 (1 H, m, H–16α), 1.18 (3 H, s, CH3–23), 1.09 (2 H, m, overlapped, H–12αand H–15α), 1.05 (1 H, m, H–22α), 1.03 (6 H, s, CH3–26 andCH3–27), 0.80 (3 H, s, CH3–25), 0.68 (3 H, s, CH3–24); 13C NMR(CDCl3, 100 MHz), see Table 1.

2.3.3. 21α-Hydroxyfriedel-1-ene-3-one (salacinin C, 3)White amorphous powder (CHCl3); [α]D20 −52.5° (c 0.3,

CHCl3); IR (KBr) νmax 3436, 2921, 1710, 1627 cm−1; EIMS m/z(%): 422 (30, [M–H2O]+), 407 (17), 203 (31), 135 (43), 123(100), 107 (41), 55 (30); HREIMSm/z 440.3657 [M]+ (calcd. forC30H48O2, 440.3654). δH = 6.99 (1 H, d, J=10.2 Hz, H–2), 6.11(1 H, dd, J = 10.2, 2.9 Hz, H–1), 3.76 (1 H, dd, J = 12.2, 4.7 Hz,H–21β), 2.32 (1H, d, J=2.9Hz, H–10α), 2.29 (1H, q, J=4.9Hz,H–4α), 1.85 (1 H, m, H–6β), 1.76 (1 H, dd, J=12.7, 12.2 Hz, H–22α), 1.69 (2H,m, CH2–19), 1.61 (1H, br d, J=8.7 Hz, H–18β),1.60 (1 H, m, H–7α), 1.59 (4 H, m, CH2–11 and CH2-16),1.51 (1 H, br d, J = 7.9 Hz, H–8α), 1.49 (1 H, m, H–12β),1.45 (2 H, m, CH2–15), 1.43 (1 H, m, H–6α), 1.36 (1 H, m, H–

12α), 1.30 (1 H, dd, J = 12.7, 4.7 Hz, H–22β), 1.26 (3 H, s,CH3–28), 1.16 (3 H, s, CH3–27), 1.13 (3 H, s, CH3–30), 1.05(3 H, s, CH3–29), 1.05 (3 H, d, J=4.9 Hz, CH3–23), 0.99 (3 H,s, CH3–26), 0.97 (3 H, s, CH3–25), 0.84 (3 H, s, CH3–24), 0.83(1 H, m, H–7β); 13C NMR (CDCl3, 100 MHz), see Table 1.

146 M.-H. Yu et al. / Fitoterapia 98 (2014) 143–148

2.4. Assay of α-glucosidase

The procedure was assayed according to the reportedmethods with slight modifications [24,25]. Briefly, the sub-strate, 1 mM 4-nitrophenyl α-glucopyranoside (50 μl, p-NPG,Sigma), was hydrolyzed to 4-nitrophenol by α-glucosidase(from yeast Saccharomyces cerevisiae, 0.0125U/ml, 100 μl,Sigma). The substrate and enzyme were dissolved in 50 mMsodium phosphate buffer (pH 6.8). All tested compounds andthe positive control acarbose (Melonepharma) was dissolvedin DMSO and diluted with 50 μl sodium phosphate buffer.Samples with different concentrations andα-glucosidase werepre-incubated at 37 °C for 15min. The increment in absorptionat 405 nm due to the hydrolysis of p-NPG by glycosidase wasmonitored on Flexstation III instrument (Molecular Devices,CA). The extent of α-glucosidase inhibition is expressed as IC50,which was calculated with Graphpad prism 5.

3. Results and discussion

Salacinin A (1), a white amorphous powder, was assigned amolecular of C32H52O4 byHREIMS [M]+ atm/z 500.3865 (calcd.500.3866), corresponding to seven degrees of unsaturation.The IR spectrum indicated the occurrence of hydroxyl(3424 cm−1), carbonyl (1720 cm−1), and double bond(1627 cm−1) moieties. The 1H NMR and 13C NMR (Table 1)spectra displayed eight methyls [δH 0.78 (3H, s), 0.93 (6H, s),1.03 (3H, s), 1.24 (9H, s), and 1.67 (3H, s); δC 13.9, 14.4, 15.9,18.0, 19.2, 19.7, 23.6, and 27.9], eleven methylenes (δC 20.8,22.0, 25.1, 27.4, 29.8, 33.1, 35.4, 39.9, 42.1, 60.8, and 109.5), fivemethines (δC 38.2, 42.2, 48.0, 48.1, and 48.4), and eightquaternary carbons (δC 40.7, 42.1, 43.0, 43.2, 46.3, 150.8,176.2, and 180.1). In particular, an AcO group (δH 1.24; δC 23.6and 180.1), and an isopropenyl moiety [δH 1.67 (3H, s), and4.56, 4.68 (each 1H, br s); δC 19.2, 109.5, and 150.8] wereobserved. These data indicated that 1 was an acetoxylizedlupane triterpenoid [26]. The seven degrees of unsaturationimplied a tetracyclic system for 1.

Interpretation of the 1H–1H COSY, HSQC, HMBC and ROESYspectra of 1 led to the unambiguous assignments of all protonand carbon resonances (Table 1). In the 1H–1H COSY spectrumof 1, the correlations of four proton spin systems of H–5/CH2–6/CH2–7, H–9/CH2–11/CH2–12/H–13, CH2–15/CH2–16, and H–18/H–19/CH2–21/CH2–22 were observed (Fig. 2). In the HMBCspectrum of 1, five key groups of correlations were present as

Fig. 2. 1H–1H COSY and selected HMBC correlations of compound 1.

follows: fromCH3–25 (δH 0.93, s) to C–1 (δC 42.1), C–5 (δC 48.4),C–9 (δC 42.2), andC–10 (δC 42.1), fromCH3–26 (δH 1.03, s) to C–7 (δC 33.1), C–8 (δC 40.7), C–9 (δC 42.2), and C–14 (δC 43.2),from CH3–27 (δH 0.93, s) to C–13 (δC 38.2), C–14 (δC 43.2), andC–15 (δC 27.4), fromCH3–28 (δH 0.78, s) to C–16 (δC 35.4), C–17(δC 43.0), C–18 (δC 48.1), and C–22 (δC 39.9), and from CH2–29(br s, δH 4.56, 4.68) to C–19 (δC 48.0), C–20 (δC 150.8), and C–30(δC 19.2) (Fig. 2). These aforementioned 2D correlations andcomparison of the NMR data between 1 and 6 revealed theidentical rings B–E of 1 and 6, which indicated that 1was a seco-lupane triterpenoid with a cleavage on ring A. The seco-lupanestructure had a cleavage between C–2 and C–3 rather than C–3andC–4,which can be deduced from thekeyHMBC correlationsfrom CH3–25 to C–1 and C–2 (δC 176.2), from CH2–1 (δH 2.34,2.50, br d, J = 17.6 Hz) to C–2 and C–10, from CH3–23, 24(δH 1.24, s) to C–3 (δC 60.8), C–4, and C–5 (δC 48.4), and fromCH2–3 (δH 4.00, 4.17, d, J = 6.3 Hz) to C–24 (δC 13.9) (Fig. 2).The AcO group was located at C–3, as established by the HMBCcorrelations from CH2–3 to the carbonyl carbon at δC 180.1(Fig. 2). The relative configuration of 1 was assigned by theROESY correlations of Hα–7 with Hα–5, Hα–9, and CH3–27, ofCH3–27with Hα–9 and Hα–18, of CH3–26with Hβ–13 and CH3–

25, and of CH3–28with Hβ–19 andCH3–13,which indicated theα-orientations of H–5, H–9, H–18, and CH3–27, andβ-directionsof H–13, H–19, CH3–25, CH3–26, and CH3–28. Thus, thestructure of 1 was elucidated as 2,3-seco-20(29)-lupene-3-acetoxy-2-oic-acid and 1 was named salacinin A (Fig. 1).

Compound 1 is a rare 2,3-seco-lupane natural triterpenoid[12]. It might biosynthetically be derived from pentacyclictriterpenoid lupeol (6) (Scheme 1). Lupeol (6) is assumed to besuccessively oxygenated at C-2 to give compound 5 and then 4.Baeyer-Villiger oxidation of 4 (intermediate i) and subsequent-ly hydrolysis of the lactone generates the 2,3-seco-lupanetriterpenoid intermediate ii. Further reduction of the aldehyde(intermediate iii) and acetylation of the hydroxyl group fulfillthe conversion of compound 1.

Salacinin B (2), a white amorphous powder, was assigned amolecular of C30H48O3 by HREIMS [M]+ atm/z 456.3605 (calcd.456.3603). The 1H and 13C NMR spectra (Table 1) revealedsignals of six methyls (δH 0.68, 0.80, 1.03, 1.03, 1.18, and 1.69,δC 14.7, 15.6, 16.3, 17.0, 19.1 and 29.3), one terminal olefinicgroup (δH 4.59 and 4.69, δC 109.9 and 150.3), one sp3

oxymethine (δH 3.88, br s, δC 82.9), one oxymethene (δH 3.35and 3.79, d, J = 10.2 Hz, δC 60.5), and one carbonyl (δC 211.4).Besides, the 13C NMR and DEPT spectra exhibited additional 19carbon signals attributable to ninemethylenes groups (δC 18.5,21.0, 25.0, 27.0, 29.1, 29.7, 33.8, 33.9, and 53.5), five methinesgroups (δC 37.2, 47.7, 48.6, 50.4, and 54.6), and five quaternarycarbons (δC 41.3, 42.8, 43.9, 45.6, and 47.7). These data suggestthat 2 is a triterpenoid derived froma lupane skeletonwith twoOH groups [27].

Comparison of the 13CNMRspectroscopic data of2 and4 [15]showed almost the same signals except for the those around C–28:C–17 in 2 down-shifted 4.7 ppm, and C–16 and C–22 in 2 up-shifted 6.4 and 6.0 ppm, respectively. At the same time, theoxygenatedmethylene (δH 3.35 and 3.79, d, J=10.2Hz; δC 60.5)in 2 was observed, which indicated that 2 was a C–28hydroxylated derivative of 4. The α-orientation of H–3 wasconfirmed by the ROESY correlations of H–3with Hα–5 (δH 1.42,br d, J = 8.2 Hz) and CH3–23, and of 3–OH (δH 3.44, br s) withCH3–25 (Fig. 3). The other stereochemistry of 2was established

Scheme 1. Hypothetical biogenetic pathway of 1.

147M.-H. Yu et al. / Fitoterapia 98 (2014) 143–148

the same as that of 4 by the ROESY experiment (Fig. 3). Thus, thestructure of 2was elucidated as 3β,28-dihydroxylup-20(29)-en-2-one and was named salacinin C (Fig. 1).

Salacinin C (3) is a white amorphous powder. HREIMS of 5showed [M]+ atm/z 440.3657, corresponding to a molecular ofC30H48O2 (calcd. 440.3654). It showed the presence of hydroxyl(3436 cm−1), an α,β-unsaturated cyclohexenone ring (λmax

208 and 269 nm; νmax 1665 cm−1) in its UV and IR spectra [28].The 1H and 13C NMR spectra (Table 1) exhibited signals forseven quaternary methyls (δH 0.84, 0.97, 0.99, 1.05, 1.13, 1.16,and 1.26; δC 13.7, 17.4, 19.2, 20.1, 24.9, 31.8, and 33.1), onesecondary methyl (δH 1.05, d, J = 4.9 Hz; δC 6.7), a sp3

oxymethine (δH 3.76, dd, J = 12.2, 4.7 Hz; δC 74.2), a doublebond system (δH 6.11, dd, J = 10.2, 2.9 Hz, and 6.99, d, J =10.2 Hz; δC 130.2 and 148.9), and one carbonyl at δC 201.9. Onthe basis of these data, the spectroscopic data of 3 showed thesame rings A–D as that of 13 [23], while the signals of ring C–Ewere consistent with 12 [22]. Along with the seven degrees ofunsaturation, 3 was assumed to be a D:A-friedoolean-l-en-3-one skeleton with a hydroxyl at C–21, whichwas confirmed bythe HMBC correlations from CH3–29 (δH 1.05), CH3–30 (δH1.13), and Hβ–22 (δH 1.76) to C–21 (δC 74.2). The β-orientation

Fig. 3. Key ROESY correlations of compound 2.

of H–21 was assigned from the cross-peaks of H–21 with bothCH3–28 and CH3–30 in the ROESY spectrum. The other relativeconfigurations were the same as D:A-friedoolean-l-en-3-oneskeleton derivatives [28]. Thus, the structure of 3was elucidatedas 21α-hydroxyfriedel-1-ene-3-one, and 3was named salacininC (Fig. 1).

Compounds 4, 6, 7, 11, and 13were evaluated for inhibitoryeffects on α-glucosidase. The results are shown in Table 2.Compounds 4 and 6 showed significant inhibitory activitiesagainst α-glucosidase with IC50 values of 40.0± 5.0 and 71.2 ±4.9 μM, respectively. Compound 7 also exhibited inhibitoryactivity with IC50 value of 446.1 ± 88.5 μM. Acarbose was usedas the positive control (IC50 = 386.3 ± 93.1 μM). Compounds11 and 13were inactive.

Conflict of interest

The authors declare no conflict of interest.

Acknowledgments

This study was supported by the National Natural ScienceFoundation of China (Nos. 81222045, 21372049) and the ShuGuang project supported by the Shanghai Municipal EducationCommission and Shanghai Education Development Foundation(No. 12SG02).

Table 2Effects of compounds 4, 6, 7, 11, and 13 on α-glucosidase.

Compound IC50 (μM)

4 40.0 ± 5.06 71.2 ± 4.97 446.1 ± 88.511 NA13 NAAcarbose 386.3 ± 93.1

NA: not active.

148 M.-H. Yu et al. / Fitoterapia 98 (2014) 143–148

Appendix A. Supplementary data

Supplementary data to this article can be found online athttp://dx.doi.org/10.1016/j.fitote.2014.07.016.

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