- Email: [email protected]
13,27-Cycloursane, ursane and oleanane triterpenoids from the leaves of Lucuma nervosa
Fitoterapia 136 (2019) 104178
Contents lists available at ScienceDirect
Fitoterapia journal homepage: www.elsevier.com/locate/fitote
13,27-Cycloursane, ursane and oleanane triterpenoids from the leaves of Lucuma nervosa
T
Fu-Cai Rena,c, Guan-Yan Lia, Nuosu Namab, Zhen-Hua Liua, Liu Yanga, Jun Zhoua, ⁎ Jiang-Miao Hua, a
State Key Laboratory of Phytochemistry and Plant Resources in West China, Yunnan Key Laboratory of Natural Medicinal Chemistry, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, People's Republic of China b The Eberly College of Science, The Pennsylvania State University, State College, PA 16803, United States c University of Chinese Academy of Sciences, Beijing 100049, People's Republic of China
A R T I C LE I N FO
A B S T R A C T
Keywords: Lucuma nervosa 13,27-Cycloursane Ursane Lucumic acid α-Glucosidase
One hitherto unknown 24-nor-13,27-cycloursane-type triterpenoid, lucumic acid A (1), one new 24-nor-ursane triterpenoid, lucumic acid B (2), along with six known triterpenoids were isolated from the ethanol extract of the leaves of Lucuma nervosa. Their structures were established on the basis of spectroscopic data interpretation. Lucumic acid A (1) is the first example of a 24-nor-triterpenoid with a 13,27-cyclopropane ring.
1. Introduction The plant Lucuma nervosa as semi-deciduous tropical fruit tree belonging to the plant family Sapotaceae is mainly distributed in the warm and tropical regions of America and Southeast Asia. Fruit of the plant is known locally as yolk fruit or “Xian-Tao” in China [1]. L. nervosa is well known as a new fruit with trace elements, such as zinc, iron and manganese, as well as various amino acids necessary for human body, and has some beneficial effect on digestion, promoting physical, sedation, regulate blood lipids and other effects [2]. Thus, the species has been planted in many suitable areas for the food industries, whereas, phytochemical and pharmaceutical research of L. nervosa has been so limited. With our continual research of chemical constituents with pharmacological usage from Nature [3–5], phytochemical investigation of the leaves of L. nervosa was done and led to the isolation of eight triterpenoids including 13,27-cycloursane, ursane, and oleanane skeleton. All of the triterpenoids were evaluated α-glucosidase inhibitory activity. Details of these efforts will be described below. 2. Materials and methods 2.1. General experimental procedures Optical rotations were measured on an Autopol VI Polarimeter manufactured by Rudolph Research Analytical (Hackettstown, NJ,
⁎
USA). IR spectra (KBr) were obtained on a Bruker Tensor-27 infrared spectrophotometer. NMR spectra were carried out on a Bruker Avance III 600 or Bruker DRX-500 (Bruker BioSpin GmbH, Rheinstetten, Germany) spectrometer with deuterated solvent signals used as internal standards. ESIMS and HRESIMS were measured using an Agilent G6230 time-of-flight mass spectrometer. Semi-preparative HPLC was carried out on an Agilent 1100 HPLC with a ZORBAX SB-C18 (9.4 × 250 mm, Agilent, USA) column. Column chromatography (CC) was performed on silica gel (100–200 mesh and 200–300 mesh, Qingdao Marine Chemical Co., Ltd., Qingdao, China), Lichroprep RP-18 (43–63 μm, Merck, Darmstadt, Germany), YMC*-GEL ODS-A (12 nm, S-50 μm, YMC, Japan) and Sephadex LH-20 (Amersham Biosciences AB, Uppsala, Sweden). Fractions were monitored by TLC plates (Si gel G and GF254, Qingdao haiyang Chemical Co., Ltd., Qingdao, China) and spots were visualized by heating silica gel plates sprayed with 10% H2SO4 in EtOH. 2.2. Plant material The leaves of L. nervosa were collected from Dai Autonomous Prefecture of Xishuangbanna, Yunnan province, People's Republic of China, in October 2014, and identified by Dr. Ting Zhang of Kunming Institute of Botany, Chinese Academy of Sciences. A voucher specimen (KIBZJ-20141001) was deposited at the State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences.
Corresponding author. E-mail address: [email protected] (J.-M. Hu).
https://doi.org/10.1016/j.fitote.2019.104178 Received 4 April 2019; Received in revised form 18 May 2019; Accepted 18 May 2019 Available online 21 May 2019 0367-326X/ © 2019 Published by Elsevier B.V.
Fitoterapia 136 (2019) 104178
F.-C. Ren, et al.
2.3. Extraction and isolation
Table 1 1 H and 13C NMR data for compounds 1 and 2 in pyridine-d5 (δH 8.71, δC 149.9 ppm).
The air-dried and powdered leaves of L. nervosa (5.3 kg) were extracted three times with 95% EtOH (25 L × 3) at room temperature and then filtered. The filtrate was evaporated in vacuo to afford a crude extract (ca. 1200 g), which was suspended in H2O and then sequentially extracted with petroleum ether (4 L × 4), EtOAc (4 L × 4) and n-BuOH (4 L × 4). The EtOAc fraction (ca. 240 g) was chromatographed on a silica gel (200–300 mesh) column and eluted with CHCl3–MeOH gradient system (40:1–1:1, v/v) to afford fractions A–E. Fraction B (36 g) was further fractionated over silica gel column chromatography (CC) and eluted with CHCl3–MeOH (80:1–10:1, v/v) to afford three subfractions (B1–B3). Compound 7 (15 mg) and 8 (7 mg) were subsequently obtained by silica gel CC eluted with CHCl3–MeOH (100:1, v/v) from Fraction B1. Fraction B2 was isolated by Sephadex LH-20 CC (CHCl3/MeOH; 1:1) and repeated silica gel CC (CHCl3/MeOH; 100:0 → 50:1) to yield 1 (8 mg) and 5 (13 mg). Fraction B3 was isolated by repeated silica gel CC (CHCl3/MeOH; 100:0 → 50:1) to yield subfractions B31 and B32. Compound 4 (5 mg) and 6 (16 mg) were obtained by Sephadex LH-20 CC (CHCl3/MeOH; 1:1) from fractions B31 and B32, respectively. Fraction C was further separated by silica gel CC (CHCl3/ MeOH; 200:1 → 20:1), Sephadex LH-20 (MeOH) and preparative TLC (PE/EtOAc; 10:1) to provide 3 (4.9 mg). Compound 2 (3.2 mg) was eventually acquired by repeated silica gel CC (CHCl3/MeOH; 80:1 → 10:1) and Sephadex LH-20 (CHCl3/MeOH; 1:1) from fraction D.
No.
1 2 3 4 5 6 7 8 9 10 11
1.43 2.49 4.09 4.33
dd (12.9, 11.0) dd (12.9, 5.1) ddd (11.0, 8.7, 5.1) d (8.7)
48.0
4.22 d (9.2)
82.0
3.94 dd (9.2, 3.9) 4.73 d (3.9)
1.84 1.54 1.69 1.31 1.87
d (11.7) m m m m
73.8 79.6 152.2 50.7 21.7
74.8 76.4 152.6 44.6 21.3
dd (13.3, 5.5) td (13.1, 6.4) dd (12.7, 4.2) td (12.5, 5.7)
1.45 m 1.38, m 1.94, m 2.03 td (12.5, 3.9) 2.15 dt (13.1, 3.8) 4.92 s 5.78 s
24 25 26 27
0.80 1.12 1.61 2.40
28 29 30 OH-19
b
δC
2.84 s
23
a
δH (J in Hz)
1.74 2.52 2.02 2.71
22
The α-glucosidase inhibitory activity was evaluated according to the chromogenic method described by Watanabe [6], using quercetin as a positive control and 4-nitrophenyl-α-D-glucopyranoside (PNPG, Sigma, USA) as substrate. Briefly, the test compounds (final concentration 50 μM) and the enzyme solution (final concentration 0.025 U/mL), buffer, substrate (final concentration 1 mM) were sequentially added to the 96-well ELISA plate, then thoroughly mixed, and two wells were set to repeat. A drug-free blank and a quercetin (final concentration 10 μM) positive control were also set. After incubate at 37 °C for 50 min, determine the OD value at 405 nm with microplate reader, and the αglucosidase inhibitory activity was calculated as follows:
δC
6.28 dd (10.1, 2.9)
17 18 19 20 21
2.4. Assessment of α-glucosidase inhibitory activity in vitro
δH (J in Hz)
5.40 dd (10.1, 2.4)
16
2.3.2. Lucumic acid B (2) White amorphous powder; [α]25 D −4.9 (c 0.06, MeOH); IR (KBr): νmax = 3424, 2975, 2932, 1687, 1641, 1460, 1384, 1047 cm−1; 1H and 13 C NMR data: see Table 1; positive HRESIMS m/z 511.3033 [M + Na]+ (calcd for C29H44O6Na, 511.3030).
2b
2.05 br s
12 13 14 15
2.3.1. Lucumic acid A (1) White amorphous powder; [α]25 D +41.2 (c 0.14, MeOH); IR (KBr): νmax = 3432, 3026, 2970, 2870, 1695, 1651, 1632, 1450, 1384, 1287, 1216, 1174 1056 cm−1; 1H and 13C NMR data: see Table 1; negative HRESIMS m/z 469.2960 [M − H]− (calcd for C29H41O5, 469.2954). (See Fig. 1.)
1a
s s d (4.6) d (4.6)
1.61 s 1.06 d (6.7) 5.13 s
35.9 33.9 51.1 38.4 119.2 141.7 27.9 33.4 22.3 26.4 47.3 47.1 75.5 42.5 26.9 37.6 104.3
2.76 1.52 1.60 1.40 1.62
d (11.0) m m m m
2.47 dd (11.3, 5.7) 2.55 ddd (19.0, 11.3, 3.2) 3.46 ddd (19.0, 5.7, 3.5) 5.70 dd (3.5, 3.2)
1.25 2.30 2.03 3.08
m m m td (13.2, 4.6)
3.04 s 1.47 1.32 2.08 2.05 2.13 4.80 5.13
m m m m m s s
32.4 41.2 45.8 43.2 28.8 129.9 139.1 42.4 29.4 26.5 48.5 54.9 72.7 42.5 27.0 38.5 110.3
16.1 16.2 15.9
1.09 s 1.23 s 1.65 s
10.2 17.8 24.8
180.8 27.1 15.9
1.42 s 1.11 d (6.6)
181.0 27.2 16.9
500 (125) MHz. 600 (150) MHz.
at δH 5.40 (dd, J = 10.1, 2.4 Hz), 6.28 (dd, J = 10.1, 2.9 Hz). The 13C and DEPT spectra (Table 1) displayed 29 carbon resonances, in which a carboxylic acid group at δC 180.9 (s), four methyls at δC 15.9 (q), 16.1 (q), 16.2 (q) and 27.1 (q), two olefin methines at δC 119.2 (d) and 141.7 (d), the exocyclic methylene at δC 104.3 (t) and 152.2 (s), and three oxygenated carbons at δC 79.6 (d), 75.5(s), 73.8 (d) can be obviously assignable. Detailed analysis of 1D and 2D NMR spectra of 1 found that the NMR spectroscopic features of rings A, B, and E are compatible with those of 2α,3β,19α-trihydroxy-24-norursa-4(23),12-dien-28-oic acid (5) [7], and 2α,3α,19α-trihydroxy-24-norursa-4(23),12-dien-28-oic acid [8]. Furthermore, instead of the methyl-27 in a ursane skeleton, a newly appearing methylene at δH 1.61 (d, J = 4.6 Hz), 2.40 (d, J = 4.6 Hz) and δC 15.9 (t) along with the double-bond-shift from ene-12,13 to ene11,12 at δH 5.40 (dd, J = 10.1, 2.4 Hz)/δC 119.2 (d) and δH 6.28 (dd, J = 10.1, 2.9 Hz)/δC 141.7 (d) of 1 suggested the presence of a 13α,27cyclopropane ring [9,10]. This inference was subsequently confirmed by the HMBC correlations (Fig. 2) from H-27 (δH 1.61, 2.40) to C-8 (δC 33.9), C-12 (δC 141.7), C-13 (δC 27.9), and C-18 (δC 47.1), as well as the HMBC correlations from H-12 (δH 6.28) and H-18 (δH 2.84) to C-27 (δC 15.9). According to the axial-axial couplings between H-1α and H-2 (J = 11.0 Hz), between H-3 and H-2 (J = 8.7 Hz), and an axial-equatorial coupling between H-1β and H-2 (J = 5.1 Hz) (Fig. 3), the hydroxy
α − glucosidase inhibitory activity (%) = [Ablank − (A sample − Abackground)]/Ablank × 100%
3. Results and discussion Compound 1, obtained as a white, amorphous powder. Its molecular formula was determined to be C29H42O5 on the basis of the negative-ion HRESIMS, with a [M − H]− peak occurring at m/z 469.2960 (calcd for C29H41O5, 469.2954). The 1H NMR spectrum (Table 1) of 1 exhibited signals ascribed to three tertiary methyls at δH 0.80 (s), 1.12 (s), 1.61 (s), a secondary methyl at δH 1.06 (d, J = 6.7 Hz), two exocyclic methylene protons at δH 4.92 (s), 5.78 (s), and a pair of cis-olefin protons 2
Fitoterapia 136 (2019) 104178
F.-C. Ren, et al.
Fig. 1. Structures of compounds 1–8.
groups at C-2 and C-3 were deduced as equatorial α-orientation and βorientation, respectively [11]. In the ROESY spectrum (Fig. 4), the significant correlation of H-9/H-27α was observed, indicating α-orientation of the 13,27-cyclopropane ring. Remaining relative configurations were assumed to be identical to above analogues based on 1H and 13C NMR data and ROESY spectrum (Supplementary data S6). In addition, to the best of our knowledge, no more than 15 derivatives of the 13,27-cycloursane skeleton had been reported since the first one isolated from Phyllanthus engleri in 1951 [12], and the absolute configuration of 13,27-cycloursane skeleton was confirmed by a singlecrystal X-ray diffraction study [10]. Therefore, the structure of 1 was established as 2α,3β,19α-trihydroxy-24-nor-13α,27-cycloursa-4(23),11dien-28-oic acid and given the trivial name lucumic acid A. Lucumic acid B (2), also obtained as a white amorphous powder, had a molecular formula of C29H44O6 determined by positive-ion HRESIMS, in which the [M + Na]+ peak occurred at m/z 511.3033 (calcd for C29H44O6Na, 511.3030). The 1H and 13C NMR signals (Table 1) of 2 were generally similar to those of 1. In the view of the signals of a trisubstituted olefinic double bond occurring at δH 5.70 (dd, J = 3.5, 3.2 Hz), δC 129.9, 139.1, it was deduced that 2 has a ursane triterpenoid skeleton. By detailed analysis, the NMR data of 2 were generally comparable with those of 2α,3α,19α-trihydroxy-24-norursa4(23),12-dien-28-oic acid [8], except one more oxygen atom in 2 which was deduced from the HRESIMS and NMR spectra. The HMBC
Fig. 2. Key HMBC (blue arrow) correlations of compounds 1 and 2.
Fig. 3. Chair conformations and peak shapes of rings A in compounds 1 and 2.
Fig. 4. Key ROESY (red dashed double arrow) correlations of compounds 1 and 2. 3
Fitoterapia 136 (2019) 104178
F.-C. Ren, et al.
References
correlations (Fig. 2) from H-2 (δH 3.94), H-3 (δH 4.73), and Me-25 (δH 1.09) to C-1 (δC 82.0) confirmed that the one more hydroxyl group was located at C-1. In the ROESY spectrum (Fig. 4), the correlations of H-1/ H5 and H-1/H-9 indicated α-orientation of these protons. Similarly, the correlations of H-2/H-3 and H-2/Me-25 revealed that these protons were β-orientation. Thus, the structure of 2 was determined as 1β,2α,3α,19α-tetrahydroxy-24-norursa-4(23),12-dien-28-oic acid. In addition, except for the new substances described above, six known triterpenoids were also isolated from L. nervosa, which were identified as 2α,3α,19α,23-tetrahydroxy-13α,27-cyclours-11-en-28-oic acid (3) [13], 4(R),23-epoxy-2α,3α,19α-trihydroxy-24-norurs-12-en-28oic acid (4) [8], 2α,3β,19α-trihydroxy-24-norursa-4(23),12-dien-28-oic acid (5) [7], euscaphic acid (6) [14], corosolic acid (7) [15], and maslinic acid (8) [16] by comparing their NMR data with literature. All of the isolates were tested for their inhibitory activity against αglucosidase, the results showed that corosolic acid (7) and maslinic acid (8) were found to have weak α-glucosidase inhibitory activity with the inhibitory rate of 66.3% and 55.2%, respectively, at the concentration of 50 μM.
[1] J.S. Ma, M.A. Sheng, L.X. Wang, H. Liu, Analysis and evaluation of nutritional components of Pouteria campechiana seeds, Chin. J. Trop. Crops 40 (2019) 368–372. [2] H. Mehraj, R.K. Sikder, U. Mayda, T. Taufique, A.F.M. Jamal Uddin, Plant physiology and fruit secondary metabolites of canistel (Pouteria campechiana), World Appl. Sci. J. 33 (2015) 1908–1914. [3] D. Yang, Z.Q. Cheng, L. Yang, B. Hou, J. Yang, X.N. Li, C.T. Zi, F.W. Dong, Z.H. Liu, J. Zhou, Z.T. Ding, J.M. Hu, Seco-Dendrobine-Type alkaloids and bioactive phenolics from Dendrobium findlayanum, J. Nat. Prod. 81 (2018) 227–235. [4] J.Y. Li, L. Yang, B. Hou, F.C. Ren, X.B. Yang, Y.F. Lv, M.T. Kuang, J.M. Hu, J. Zhou, Poly p-hydroxybenzyl substituted bibenzyls and phenanthrenes from Bletilla ochracea Schltr with anti-inflammatory and cytotoxic activity, Fitoterapia 129 (2018) 241–248. [5] J.Y. Li, M.T. Kuang, L. Yang, Q.H. Kong, B. Hou, Z.H. Liu, X.Q. Chi, M.Y. Yuan, J.M. Hu, J. Zhou, Stilbenes with anti-inflammatory and cytotoxic activity from the rhizomes of Bletilla ochracea Schltr, Fitoterapia 127 (2018) 74–80. [6] J. Watanabe, J. Kawabata, H. Kurihara, R. Niki, Isolation and identification of αGlucosidase inhibitors from tochu-cha (Eucommia ulmoides), Biosci. Biotechnol. Biochem. 61 (1997) 177–178. [7] J.P.L. Ekon, A.N. Bissoue, M. Fomani, F.A.A. Toze, A.F.W. Kamdem, N. Sewald, J.D. Wansi, Cytotoxic 24-nor-ursane-type triterpenoids from the twigs of Mostuea hirsuta, Z. Naturforsch. B 70 (2015) 837–842. [8] D.S. Jang, J.M. Kim, J.H. Kim, J.S. Kim, 24-nor-Ursane type triterpenoids from the stems of Rumex japonicus, Chem. Pharm. Bull. 53 (2005) 1594–1596. [9] J.J. Cheng, L.J. Zhang, H.L. Cheng, C.T. Chiou, I.J. Lee, Y.H. Kuo, Cytotoxic hexacyclic triterpene acids from Euscaphis japonica, J. Nat. Prod. 73 (2010) 1655–1658. [10] Y.M. Chiang, J.K. Su, Y.H. Liu, Y.H. Kuo, New cyclopropyl-triterpenoids from the aerial roots of Ficus microcarpa, Chem. Pharm. Bull. 49 (2001) 581–583. [11] F.C. Ren, L.X. Wang, Q. Yu, X.J. Jiang, F. Wang, Lanostane-type triterpenoids from Scilla scilloides and structure revision of drimiopsin D, Nat. Prod. Bioprospect. 5 (2015) 263–270. [12] K.B. Alberman, F.B. Kipping, Phyllanthol. A new alcohol from the root bark of Phyllanthus engleri (Pax), J. Chem. Soc. (1951) 2296–2297. [13] T.H. Lee, S.H. Juang, F.L. Hsua, C.Y. Wua, Triterpene Acids From the Leaves of Planchonella duclitan (Blanco) Bakhuizan, 52 (2005), pp. 1275–1280. [14] G.Y. Liang, A.I. Gray, P.G. Waterman, Pentacyclic triterpenes from the fruits of Rosa sterilis, J. Nat. Prod. 52 (1989) 162–166. [15] H. Saimaru, Y. Orihara, P. Tansakul, Y.-H. Kang, M. Shibuya, Y. Ebizuka, Production of triterpene acids by cell suspension cultures of Olea europaea, Chem. Pharm. Bull. 55 (2007) 784–788. [16] I.L. Acebey-Castellon, L. Voutquenne-Nazabadioko, H. Doan Thi Mai, N. Roseau, N. Bouthagane, D. Muhammad, E. Le Magrex Debar, S.C. Gangloff, M. Litaudon, T. Sevenet, N.V. Hung, C. Lavaud, Triterpenoid saponins from Symplocos lancifolia, J. Nat. Prod. 74 (2011) 163–168.
Conflict of interest statement The authors declare no conflict of interest for this study. Acknowledgments This work was supported by grants from the National Key Research and Development Program of China (2017YFD0201402) and Yunnan Provincial Science and Technology Department (No. 2015HB093, 2017ZF003-04). Appendix A. Supplementary data 1D and 2D NMR, HRESIMS and IR spectra of compounds 1–3 are available as Supplementary data. Supplementary data to this article can be found online at https://doi.org/10.1016/j.fitote.2019.104178.
4
Contents lists available at ScienceDirect
Fitoterapia journal homepage: www.elsevier.com/locate/fitote
13,27-Cycloursane, ursane and oleanane triterpenoids from the leaves of Lucuma nervosa
T
Fu-Cai Rena,c, Guan-Yan Lia, Nuosu Namab, Zhen-Hua Liua, Liu Yanga, Jun Zhoua, ⁎ Jiang-Miao Hua, a
State Key Laboratory of Phytochemistry and Plant Resources in West China, Yunnan Key Laboratory of Natural Medicinal Chemistry, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, People's Republic of China b The Eberly College of Science, The Pennsylvania State University, State College, PA 16803, United States c University of Chinese Academy of Sciences, Beijing 100049, People's Republic of China
A R T I C LE I N FO
A B S T R A C T
Keywords: Lucuma nervosa 13,27-Cycloursane Ursane Lucumic acid α-Glucosidase
One hitherto unknown 24-nor-13,27-cycloursane-type triterpenoid, lucumic acid A (1), one new 24-nor-ursane triterpenoid, lucumic acid B (2), along with six known triterpenoids were isolated from the ethanol extract of the leaves of Lucuma nervosa. Their structures were established on the basis of spectroscopic data interpretation. Lucumic acid A (1) is the first example of a 24-nor-triterpenoid with a 13,27-cyclopropane ring.
1. Introduction The plant Lucuma nervosa as semi-deciduous tropical fruit tree belonging to the plant family Sapotaceae is mainly distributed in the warm and tropical regions of America and Southeast Asia. Fruit of the plant is known locally as yolk fruit or “Xian-Tao” in China [1]. L. nervosa is well known as a new fruit with trace elements, such as zinc, iron and manganese, as well as various amino acids necessary for human body, and has some beneficial effect on digestion, promoting physical, sedation, regulate blood lipids and other effects [2]. Thus, the species has been planted in many suitable areas for the food industries, whereas, phytochemical and pharmaceutical research of L. nervosa has been so limited. With our continual research of chemical constituents with pharmacological usage from Nature [3–5], phytochemical investigation of the leaves of L. nervosa was done and led to the isolation of eight triterpenoids including 13,27-cycloursane, ursane, and oleanane skeleton. All of the triterpenoids were evaluated α-glucosidase inhibitory activity. Details of these efforts will be described below. 2. Materials and methods 2.1. General experimental procedures Optical rotations were measured on an Autopol VI Polarimeter manufactured by Rudolph Research Analytical (Hackettstown, NJ,
⁎
USA). IR spectra (KBr) were obtained on a Bruker Tensor-27 infrared spectrophotometer. NMR spectra were carried out on a Bruker Avance III 600 or Bruker DRX-500 (Bruker BioSpin GmbH, Rheinstetten, Germany) spectrometer with deuterated solvent signals used as internal standards. ESIMS and HRESIMS were measured using an Agilent G6230 time-of-flight mass spectrometer. Semi-preparative HPLC was carried out on an Agilent 1100 HPLC with a ZORBAX SB-C18 (9.4 × 250 mm, Agilent, USA) column. Column chromatography (CC) was performed on silica gel (100–200 mesh and 200–300 mesh, Qingdao Marine Chemical Co., Ltd., Qingdao, China), Lichroprep RP-18 (43–63 μm, Merck, Darmstadt, Germany), YMC*-GEL ODS-A (12 nm, S-50 μm, YMC, Japan) and Sephadex LH-20 (Amersham Biosciences AB, Uppsala, Sweden). Fractions were monitored by TLC plates (Si gel G and GF254, Qingdao haiyang Chemical Co., Ltd., Qingdao, China) and spots were visualized by heating silica gel plates sprayed with 10% H2SO4 in EtOH. 2.2. Plant material The leaves of L. nervosa were collected from Dai Autonomous Prefecture of Xishuangbanna, Yunnan province, People's Republic of China, in October 2014, and identified by Dr. Ting Zhang of Kunming Institute of Botany, Chinese Academy of Sciences. A voucher specimen (KIBZJ-20141001) was deposited at the State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences.
Corresponding author. E-mail address: [email protected] (J.-M. Hu).
https://doi.org/10.1016/j.fitote.2019.104178 Received 4 April 2019; Received in revised form 18 May 2019; Accepted 18 May 2019 Available online 21 May 2019 0367-326X/ © 2019 Published by Elsevier B.V.
Fitoterapia 136 (2019) 104178
F.-C. Ren, et al.
2.3. Extraction and isolation
Table 1 1 H and 13C NMR data for compounds 1 and 2 in pyridine-d5 (δH 8.71, δC 149.9 ppm).
The air-dried and powdered leaves of L. nervosa (5.3 kg) were extracted three times with 95% EtOH (25 L × 3) at room temperature and then filtered. The filtrate was evaporated in vacuo to afford a crude extract (ca. 1200 g), which was suspended in H2O and then sequentially extracted with petroleum ether (4 L × 4), EtOAc (4 L × 4) and n-BuOH (4 L × 4). The EtOAc fraction (ca. 240 g) was chromatographed on a silica gel (200–300 mesh) column and eluted with CHCl3–MeOH gradient system (40:1–1:1, v/v) to afford fractions A–E. Fraction B (36 g) was further fractionated over silica gel column chromatography (CC) and eluted with CHCl3–MeOH (80:1–10:1, v/v) to afford three subfractions (B1–B3). Compound 7 (15 mg) and 8 (7 mg) were subsequently obtained by silica gel CC eluted with CHCl3–MeOH (100:1, v/v) from Fraction B1. Fraction B2 was isolated by Sephadex LH-20 CC (CHCl3/MeOH; 1:1) and repeated silica gel CC (CHCl3/MeOH; 100:0 → 50:1) to yield 1 (8 mg) and 5 (13 mg). Fraction B3 was isolated by repeated silica gel CC (CHCl3/MeOH; 100:0 → 50:1) to yield subfractions B31 and B32. Compound 4 (5 mg) and 6 (16 mg) were obtained by Sephadex LH-20 CC (CHCl3/MeOH; 1:1) from fractions B31 and B32, respectively. Fraction C was further separated by silica gel CC (CHCl3/ MeOH; 200:1 → 20:1), Sephadex LH-20 (MeOH) and preparative TLC (PE/EtOAc; 10:1) to provide 3 (4.9 mg). Compound 2 (3.2 mg) was eventually acquired by repeated silica gel CC (CHCl3/MeOH; 80:1 → 10:1) and Sephadex LH-20 (CHCl3/MeOH; 1:1) from fraction D.
No.
1 2 3 4 5 6 7 8 9 10 11
1.43 2.49 4.09 4.33
dd (12.9, 11.0) dd (12.9, 5.1) ddd (11.0, 8.7, 5.1) d (8.7)
48.0
4.22 d (9.2)
82.0
3.94 dd (9.2, 3.9) 4.73 d (3.9)
1.84 1.54 1.69 1.31 1.87
d (11.7) m m m m
73.8 79.6 152.2 50.7 21.7
74.8 76.4 152.6 44.6 21.3
dd (13.3, 5.5) td (13.1, 6.4) dd (12.7, 4.2) td (12.5, 5.7)
1.45 m 1.38, m 1.94, m 2.03 td (12.5, 3.9) 2.15 dt (13.1, 3.8) 4.92 s 5.78 s
24 25 26 27
0.80 1.12 1.61 2.40
28 29 30 OH-19
b
δC
2.84 s
23
a
δH (J in Hz)
1.74 2.52 2.02 2.71
22
The α-glucosidase inhibitory activity was evaluated according to the chromogenic method described by Watanabe [6], using quercetin as a positive control and 4-nitrophenyl-α-D-glucopyranoside (PNPG, Sigma, USA) as substrate. Briefly, the test compounds (final concentration 50 μM) and the enzyme solution (final concentration 0.025 U/mL), buffer, substrate (final concentration 1 mM) were sequentially added to the 96-well ELISA plate, then thoroughly mixed, and two wells were set to repeat. A drug-free blank and a quercetin (final concentration 10 μM) positive control were also set. After incubate at 37 °C for 50 min, determine the OD value at 405 nm with microplate reader, and the αglucosidase inhibitory activity was calculated as follows:
δC
6.28 dd (10.1, 2.9)
17 18 19 20 21
2.4. Assessment of α-glucosidase inhibitory activity in vitro
δH (J in Hz)
5.40 dd (10.1, 2.4)
16
2.3.2. Lucumic acid B (2) White amorphous powder; [α]25 D −4.9 (c 0.06, MeOH); IR (KBr): νmax = 3424, 2975, 2932, 1687, 1641, 1460, 1384, 1047 cm−1; 1H and 13 C NMR data: see Table 1; positive HRESIMS m/z 511.3033 [M + Na]+ (calcd for C29H44O6Na, 511.3030).
2b
2.05 br s
12 13 14 15
2.3.1. Lucumic acid A (1) White amorphous powder; [α]25 D +41.2 (c 0.14, MeOH); IR (KBr): νmax = 3432, 3026, 2970, 2870, 1695, 1651, 1632, 1450, 1384, 1287, 1216, 1174 1056 cm−1; 1H and 13C NMR data: see Table 1; negative HRESIMS m/z 469.2960 [M − H]− (calcd for C29H41O5, 469.2954). (See Fig. 1.)
1a
s s d (4.6) d (4.6)
1.61 s 1.06 d (6.7) 5.13 s
35.9 33.9 51.1 38.4 119.2 141.7 27.9 33.4 22.3 26.4 47.3 47.1 75.5 42.5 26.9 37.6 104.3
2.76 1.52 1.60 1.40 1.62
d (11.0) m m m m
2.47 dd (11.3, 5.7) 2.55 ddd (19.0, 11.3, 3.2) 3.46 ddd (19.0, 5.7, 3.5) 5.70 dd (3.5, 3.2)
1.25 2.30 2.03 3.08
m m m td (13.2, 4.6)
3.04 s 1.47 1.32 2.08 2.05 2.13 4.80 5.13
m m m m m s s
32.4 41.2 45.8 43.2 28.8 129.9 139.1 42.4 29.4 26.5 48.5 54.9 72.7 42.5 27.0 38.5 110.3
16.1 16.2 15.9
1.09 s 1.23 s 1.65 s
10.2 17.8 24.8
180.8 27.1 15.9
1.42 s 1.11 d (6.6)
181.0 27.2 16.9
500 (125) MHz. 600 (150) MHz.
at δH 5.40 (dd, J = 10.1, 2.4 Hz), 6.28 (dd, J = 10.1, 2.9 Hz). The 13C and DEPT spectra (Table 1) displayed 29 carbon resonances, in which a carboxylic acid group at δC 180.9 (s), four methyls at δC 15.9 (q), 16.1 (q), 16.2 (q) and 27.1 (q), two olefin methines at δC 119.2 (d) and 141.7 (d), the exocyclic methylene at δC 104.3 (t) and 152.2 (s), and three oxygenated carbons at δC 79.6 (d), 75.5(s), 73.8 (d) can be obviously assignable. Detailed analysis of 1D and 2D NMR spectra of 1 found that the NMR spectroscopic features of rings A, B, and E are compatible with those of 2α,3β,19α-trihydroxy-24-norursa-4(23),12-dien-28-oic acid (5) [7], and 2α,3α,19α-trihydroxy-24-norursa-4(23),12-dien-28-oic acid [8]. Furthermore, instead of the methyl-27 in a ursane skeleton, a newly appearing methylene at δH 1.61 (d, J = 4.6 Hz), 2.40 (d, J = 4.6 Hz) and δC 15.9 (t) along with the double-bond-shift from ene-12,13 to ene11,12 at δH 5.40 (dd, J = 10.1, 2.4 Hz)/δC 119.2 (d) and δH 6.28 (dd, J = 10.1, 2.9 Hz)/δC 141.7 (d) of 1 suggested the presence of a 13α,27cyclopropane ring [9,10]. This inference was subsequently confirmed by the HMBC correlations (Fig. 2) from H-27 (δH 1.61, 2.40) to C-8 (δC 33.9), C-12 (δC 141.7), C-13 (δC 27.9), and C-18 (δC 47.1), as well as the HMBC correlations from H-12 (δH 6.28) and H-18 (δH 2.84) to C-27 (δC 15.9). According to the axial-axial couplings between H-1α and H-2 (J = 11.0 Hz), between H-3 and H-2 (J = 8.7 Hz), and an axial-equatorial coupling between H-1β and H-2 (J = 5.1 Hz) (Fig. 3), the hydroxy
α − glucosidase inhibitory activity (%) = [Ablank − (A sample − Abackground)]/Ablank × 100%
3. Results and discussion Compound 1, obtained as a white, amorphous powder. Its molecular formula was determined to be C29H42O5 on the basis of the negative-ion HRESIMS, with a [M − H]− peak occurring at m/z 469.2960 (calcd for C29H41O5, 469.2954). The 1H NMR spectrum (Table 1) of 1 exhibited signals ascribed to three tertiary methyls at δH 0.80 (s), 1.12 (s), 1.61 (s), a secondary methyl at δH 1.06 (d, J = 6.7 Hz), two exocyclic methylene protons at δH 4.92 (s), 5.78 (s), and a pair of cis-olefin protons 2
Fitoterapia 136 (2019) 104178
F.-C. Ren, et al.
Fig. 1. Structures of compounds 1–8.
groups at C-2 and C-3 were deduced as equatorial α-orientation and βorientation, respectively [11]. In the ROESY spectrum (Fig. 4), the significant correlation of H-9/H-27α was observed, indicating α-orientation of the 13,27-cyclopropane ring. Remaining relative configurations were assumed to be identical to above analogues based on 1H and 13C NMR data and ROESY spectrum (Supplementary data S6). In addition, to the best of our knowledge, no more than 15 derivatives of the 13,27-cycloursane skeleton had been reported since the first one isolated from Phyllanthus engleri in 1951 [12], and the absolute configuration of 13,27-cycloursane skeleton was confirmed by a singlecrystal X-ray diffraction study [10]. Therefore, the structure of 1 was established as 2α,3β,19α-trihydroxy-24-nor-13α,27-cycloursa-4(23),11dien-28-oic acid and given the trivial name lucumic acid A. Lucumic acid B (2), also obtained as a white amorphous powder, had a molecular formula of C29H44O6 determined by positive-ion HRESIMS, in which the [M + Na]+ peak occurred at m/z 511.3033 (calcd for C29H44O6Na, 511.3030). The 1H and 13C NMR signals (Table 1) of 2 were generally similar to those of 1. In the view of the signals of a trisubstituted olefinic double bond occurring at δH 5.70 (dd, J = 3.5, 3.2 Hz), δC 129.9, 139.1, it was deduced that 2 has a ursane triterpenoid skeleton. By detailed analysis, the NMR data of 2 were generally comparable with those of 2α,3α,19α-trihydroxy-24-norursa4(23),12-dien-28-oic acid [8], except one more oxygen atom in 2 which was deduced from the HRESIMS and NMR spectra. The HMBC
Fig. 2. Key HMBC (blue arrow) correlations of compounds 1 and 2.
Fig. 3. Chair conformations and peak shapes of rings A in compounds 1 and 2.
Fig. 4. Key ROESY (red dashed double arrow) correlations of compounds 1 and 2. 3
Fitoterapia 136 (2019) 104178
F.-C. Ren, et al.
References
correlations (Fig. 2) from H-2 (δH 3.94), H-3 (δH 4.73), and Me-25 (δH 1.09) to C-1 (δC 82.0) confirmed that the one more hydroxyl group was located at C-1. In the ROESY spectrum (Fig. 4), the correlations of H-1/ H5 and H-1/H-9 indicated α-orientation of these protons. Similarly, the correlations of H-2/H-3 and H-2/Me-25 revealed that these protons were β-orientation. Thus, the structure of 2 was determined as 1β,2α,3α,19α-tetrahydroxy-24-norursa-4(23),12-dien-28-oic acid. In addition, except for the new substances described above, six known triterpenoids were also isolated from L. nervosa, which were identified as 2α,3α,19α,23-tetrahydroxy-13α,27-cyclours-11-en-28-oic acid (3) [13], 4(R),23-epoxy-2α,3α,19α-trihydroxy-24-norurs-12-en-28oic acid (4) [8], 2α,3β,19α-trihydroxy-24-norursa-4(23),12-dien-28-oic acid (5) [7], euscaphic acid (6) [14], corosolic acid (7) [15], and maslinic acid (8) [16] by comparing their NMR data with literature. All of the isolates were tested for their inhibitory activity against αglucosidase, the results showed that corosolic acid (7) and maslinic acid (8) were found to have weak α-glucosidase inhibitory activity with the inhibitory rate of 66.3% and 55.2%, respectively, at the concentration of 50 μM.
[1] J.S. Ma, M.A. Sheng, L.X. Wang, H. Liu, Analysis and evaluation of nutritional components of Pouteria campechiana seeds, Chin. J. Trop. Crops 40 (2019) 368–372. [2] H. Mehraj, R.K. Sikder, U. Mayda, T. Taufique, A.F.M. Jamal Uddin, Plant physiology and fruit secondary metabolites of canistel (Pouteria campechiana), World Appl. Sci. J. 33 (2015) 1908–1914. [3] D. Yang, Z.Q. Cheng, L. Yang, B. Hou, J. Yang, X.N. Li, C.T. Zi, F.W. Dong, Z.H. Liu, J. Zhou, Z.T. Ding, J.M. Hu, Seco-Dendrobine-Type alkaloids and bioactive phenolics from Dendrobium findlayanum, J. Nat. Prod. 81 (2018) 227–235. [4] J.Y. Li, L. Yang, B. Hou, F.C. Ren, X.B. Yang, Y.F. Lv, M.T. Kuang, J.M. Hu, J. Zhou, Poly p-hydroxybenzyl substituted bibenzyls and phenanthrenes from Bletilla ochracea Schltr with anti-inflammatory and cytotoxic activity, Fitoterapia 129 (2018) 241–248. [5] J.Y. Li, M.T. Kuang, L. Yang, Q.H. Kong, B. Hou, Z.H. Liu, X.Q. Chi, M.Y. Yuan, J.M. Hu, J. Zhou, Stilbenes with anti-inflammatory and cytotoxic activity from the rhizomes of Bletilla ochracea Schltr, Fitoterapia 127 (2018) 74–80. [6] J. Watanabe, J. Kawabata, H. Kurihara, R. Niki, Isolation and identification of αGlucosidase inhibitors from tochu-cha (Eucommia ulmoides), Biosci. Biotechnol. Biochem. 61 (1997) 177–178. [7] J.P.L. Ekon, A.N. Bissoue, M. Fomani, F.A.A. Toze, A.F.W. Kamdem, N. Sewald, J.D. Wansi, Cytotoxic 24-nor-ursane-type triterpenoids from the twigs of Mostuea hirsuta, Z. Naturforsch. B 70 (2015) 837–842. [8] D.S. Jang, J.M. Kim, J.H. Kim, J.S. Kim, 24-nor-Ursane type triterpenoids from the stems of Rumex japonicus, Chem. Pharm. Bull. 53 (2005) 1594–1596. [9] J.J. Cheng, L.J. Zhang, H.L. Cheng, C.T. Chiou, I.J. Lee, Y.H. Kuo, Cytotoxic hexacyclic triterpene acids from Euscaphis japonica, J. Nat. Prod. 73 (2010) 1655–1658. [10] Y.M. Chiang, J.K. Su, Y.H. Liu, Y.H. Kuo, New cyclopropyl-triterpenoids from the aerial roots of Ficus microcarpa, Chem. Pharm. Bull. 49 (2001) 581–583. [11] F.C. Ren, L.X. Wang, Q. Yu, X.J. Jiang, F. Wang, Lanostane-type triterpenoids from Scilla scilloides and structure revision of drimiopsin D, Nat. Prod. Bioprospect. 5 (2015) 263–270. [12] K.B. Alberman, F.B. Kipping, Phyllanthol. A new alcohol from the root bark of Phyllanthus engleri (Pax), J. Chem. Soc. (1951) 2296–2297. [13] T.H. Lee, S.H. Juang, F.L. Hsua, C.Y. Wua, Triterpene Acids From the Leaves of Planchonella duclitan (Blanco) Bakhuizan, 52 (2005), pp. 1275–1280. [14] G.Y. Liang, A.I. Gray, P.G. Waterman, Pentacyclic triterpenes from the fruits of Rosa sterilis, J. Nat. Prod. 52 (1989) 162–166. [15] H. Saimaru, Y. Orihara, P. Tansakul, Y.-H. Kang, M. Shibuya, Y. Ebizuka, Production of triterpene acids by cell suspension cultures of Olea europaea, Chem. Pharm. Bull. 55 (2007) 784–788. [16] I.L. Acebey-Castellon, L. Voutquenne-Nazabadioko, H. Doan Thi Mai, N. Roseau, N. Bouthagane, D. Muhammad, E. Le Magrex Debar, S.C. Gangloff, M. Litaudon, T. Sevenet, N.V. Hung, C. Lavaud, Triterpenoid saponins from Symplocos lancifolia, J. Nat. Prod. 74 (2011) 163–168.
Conflict of interest statement The authors declare no conflict of interest for this study. Acknowledgments This work was supported by grants from the National Key Research and Development Program of China (2017YFD0201402) and Yunnan Provincial Science and Technology Department (No. 2015HB093, 2017ZF003-04). Appendix A. Supplementary data 1D and 2D NMR, HRESIMS and IR spectra of compounds 1–3 are available as Supplementary data. Supplementary data to this article can be found online at https://doi.org/10.1016/j.fitote.2019.104178.
4