Sarcokontums A and B,Two New 13,27-Cyclo-Pentacyclic Triterpenes from the Stems and Leaves of Sarcosperma kontumense Gagnep. ex Aubrév

Abstract

Two new 13,27-cyclo pentacyclic triterpenes (1 and 2), together with ten pentacyclic triterpenoids (3-12), were characterized from the stems and leaves of Sarcosperma kontumense Gagnep. ex Aubrév. Their chemical structures were elucidated by spectroscopic analyses including HRESIMS, and 1D and 2D NMR spectra as 2α,3β,19α,23-tetrahydroxy-13,27-cyclours-11-en-28-oic acid (1), 2α,3β,19α,23-tetrahydroxy-13,27-cycloolean-11-en-28-oic acid (2), 2α,3α,19α,23-tetrahydroxy-13,27-cyclours-11-en-28-oic acid (3), corosolic acid (4), pomolic acid (5), masilinic acid (6), 1β,2α,3α,19α,23-pentahydroxyurs-11-en-28-oic acid (7), rotundic acid (8), euscaphic acid (9), jacoumaric acid (10), 3-O-trans-coumaroyltormetic acid (11), and 3-O-cis-coumaroyltormetic acid (12). Compounds 3, 4, 6-8, and 10-12 were isolated from the genus Sarcosperma for the first time.

Introduction

Sarcosperma kontumense Gagnep. ex Aubrév. (Sapotaceae) is native to Asia and distributed in the South of Vietnam. In the framework of the International Associated Laboratory (LIA “NATPROCHEMLAB”) between the Centre National de la Recherche Scientifique (CNRS, ICSN, France) and the Vietnam Academy of Science and Technology (Institute of Marine Biochemistry, VAST, Vietnam), the methanol extract of the leaves and stems of this plant showed significant cytotoxic activity on KB cells with percent inhibitions of 46.8 and 32.3%, respectively, at the concentration of 1 µg/mL. Therefore, this plant was selected for further study. There has been only one paper reported on the chemical constituents of Sarcosperma affinis with the discovery of some triterpenes, lignans, and polyphenols. ¹ However, up to date, there have not been any studies on the chemical constituents and bioactivity of S. kontumense. This paper reports the isolation and determination of two new (1 and 2) and ten known compounds (3-12) from the methanol extract of the stems and leaves of S. kontumense. The cytotoxic activities of the isolates against two tumor cell lines (HepG2 and LU-1) were determined and the results are discussed herein.

Results and Discussion

The stems and leaves of S. kontumense were ultrasonically extracted with MeOH. This crude extract was partitioned in turn with n-hexane, dichloromethane, ethyl acetate and then fractionated by flash column chromatography combined with semi-preparative HPLC to obtain twelve compounds, 1-12. The known compounds were identified as 2α,3α,19α,23-tetrahydroxy-13,27-cyclours-11-en-28-oic acid (3), ² corosolic acid (4), ² pomolic acid (5), ³ masilinic acid (6), ⁴ 1β,2α,3α,19α,23-pentahydroxyurs-11-en-28-oic acid (7), ⁵ rotundic acid (8), ² euscaphic acid (9), ³ jacoumaric acid (10), ⁶ 3-O-trans-coumaroyltormetic acid (11), ⁶ and 3-O-cis-coumaroyltormetic acid (12) ⁶ by comparing their 1D and 2D NMzR data and mass spectral data with those reported in the literature. To the best of our knowledge, compounds 3, 4, 6-8, and 10-12 are reported from the genus Sarcosperma for the first time.

Compound 1 (Figure 1) was obtained as a colorless solid. Its molecular formula was determined to be C₃₀H₄₆O₆ by the HRESIMS ion at m/z 503.3366 [M + H]⁺ (calcd. for [C₃₀H₄₇O₆]⁺: 503.3367, Δ = −0.1 ppm), indicating eight indices of hydrogen deficiency. The NMR spectra of 1 were very similar to those of 3 measured in the same solvent (CD₃OD) suggesting an ursane triterpene (Table 1). ² In the ¹H NMR spectrum of 1, one CH = CH double bond [δ_H 5.13 (dd, J = 10.2, 3.0 Hz) and 5.86 (dd, J = 10.2, 2.4 Hz)], five methyl groups [δ_H 0.71, 0.83, 0.99, 1.38 (each 3H, s), and 0.91 (3H, d, J = 6.6 Hz)], two methine carbinol protons [δ_H 3.74 (1H, ddd, J = 12.0, 9.6, 2.4 Hz, H-2), 3.37 (1H, d, J = 9.6 Hz, H-3)], and one oxygenated methylene group [δ_H 3.52 (1H, d, J = 10.8 Hz), 3.28 (1H, d, J = 10.8 Hz), H₂-23)] were identified.^{2, 3} The ¹³C NMR, HSQC, HMBC, and DEPT spectra (Table 1) showed 30 carbon signals, including a carbonyl (δ_C 183.0, identified by HMBC spectrum). The NMR assignments of 1 were evidenced by HSQC, HMBC, and ¹H-¹H COSY spectra. Two quaternary carbons resonances were observed at δ_C 28.4 (C-13) and δ_C 33.8 (C-14), one methylene signal at δ_C 16.5, and two olefinic signals at δ_C 119.4 (C-11) and δ_C 142.0 (C-12). The above data suggested the 13/17-cyclopropane ring and a double bond C-11/C-12 as in euscaphic acids A-F. ³ The HMBC correlations from H₃-24 (δ_H 0.71)/H₂-23 (δ_H 3.52/3.28) to C-3 (δ_C 78.4)/C-4 (δ_C 44.1)/C-5 (δ_C 48.2), from H₃-23 to C-24 (δ_C 66.4), from H-23 to C-24 (δ_C 13.3), and from H-3 (δ_H 3.37 to C-2 (δ_C 69.7), together with COSY correlations of H-1 (δ_H 2.07/0.82)/H-2 (δ_H 3.74)/H-3 (δ_H 3.37) deduced that the three hydroxy groups were at C-2, C-3, and C-23. It is noted that the 3β-OH group perfectly corresponds to the values of δ_C−23 = 66.4 and δ_C−24 = 13.3, while a 3α-OH group supported values of δ_C−23 = 71.3 and δ_C−24 = 17.0 (Figure 2).^{2, 3} Therefore, the 2α,3β,23-hydroxy skeleton was suggested for 1. In addition, the large proton coupling constant, J_H−2/H−3 = 9.6 Hz, indicated that H-2 and H-3 had an axial orientation. Furthermore, NOESY correlations H-3/H-5, H-3/H-23, and H-2/H-24 were observed confirming 2α-OH, 3β-OH, 5α-H, and 23-OH. The 19α-OH group was confirmed by the HMBC correlations from H-30 (δ_H 0.91) to C-19 (δ 76.7)/C-20 (δ_C 43.2)/C-21 (δ_C 27.3), from H-29 (δ_H 1.38) to C-18 (δ_C 47.9)/C-20/C-21, as well as by the observation of NOESY cross peaks of H-18 (δ_H 2.35)/H-29 (δ_H 1.38), H-29/H-20 (δ_H 1.35) (Figure 3). Thus, compound 1 was determined to be 2α,3β,19α,23-tetrahydroxy-13,27-cyclours-11-en-28-oic acid, a new compound and named sarcokontum A (Supplemental Figures S1-S12).

Figure 1.

Chemical structures of 1-12.

Figure 2.

Key ¹H-¹H COSY and HMBC correlations of 1 and 2.

Figure 3.

Key NOESY correlations of 1 and 2.

Table 1.

¹H NMR and ¹³C NMR Spectroscopic Data for 1 – 3.

Pos.	1		2		3
	a,b δ C	a,cδH (mult, J in Hz)	a,b δ C	a,cδH (mult, J in Hz)	a,b δ C	a,cδH (mult, J in Hz)
1	47.9	2.07 (dd, 12.0, 2.0) 0.82 (dd, 12.0, 12.0)	47.9	2.09 (dd, 12.0, 2.0) 0.82 (dd, 12.0, 12.0)	42.4	1.75 (dd, 12.0, 4.2) 1.23 (dd, 12.0, 12.0)
2	69.7	3.74 (dd, 12.0, 9.6)	69.7	3.76 (dd, 12.0, 9.6)	67.1	3.93 (ddd, 12.0, 4.2, 2.4)
3	78.4	3.37 (d, 9.6)	78.4	3.36 (d, 9.6)	78.8	3.63 (d, 2.4)
4	44.1	-	44.1	-	42.5	-
5	48.2	1.30 (m)	48.2	1.30 (m)	44.8	1.58 (m)
6	19.2	1.50 (m)/1.54 (m)	19.2	1.50 (m)/1.57 (m)	19.0	1.48 (m)/1.50 (m)
7	38.4	1.62 (m)/1.74 (m)	38.4	1.30 (m)/1.81 (m)	38.3	1.61 (m)/1.74 (m)
8	34.5	-	34.5	-	34.5	-
9	54.0	1.74 (d, 3.0)	54.0	1.74 (d, 2.4)	53.9	1.83 (d, 2.4)
10	38.4	-	38.5	-	38.6	-
11	119.4	5.13 (dd, 10.2, 3.0)	120.7	5.13 (dd, 10.2, 3.0)	119.5	5.15 (dd, 10.2, 2.4)
12	142.0	5.86 (dd, 10.2, 2.4)	138.6	5.86 (dd, 10.2, 2.4)	141.9	5.88 (dd, 10.2, 3.0)
13	28.4	-	26.8	-	28.4	-
14	33.8	-	33.5	-	33.8	-
15	22.8	1.49 (m)/2.11 (m)	22.5	1.51 (m)/1.88 (m)	22.7	1.49 (m)/2.09 (m)
16	26.7	1.52 (m)/2.13 (m)	30.9	1.70 (m)/1.80 (m)	26.7	1.52 (m)/2.14 (m)
17	48.5	-	47.0	-	48.1	-
18	47.9	2.35 (s)	38.5	2.75 (s)	47.8	2.35 (s)
19	76.7	-	79.6	3.65 (d, 1.8)	76.2	-
20	43.2	1.35 (m)	36.1	-	43.2	1.35 (m)
21	27.3	1.25 (m)/1.57 (m)	32.9	1.68 (m)/1.78 (m)	27.3	1.25 (m)/1.56 (m)
22	37.6	1.28 (m)/1.80 (m)	33.4	1.14 (m)/1.36 (m)	37.8	1.28 (m)/1.80 (m)
23	66.4	3.52 (d, 10.8) 3.28 (d, 10.8)	66.4	3.53 (d, 10.8) 3.28 (d, 10.8)	71.3	3.54 (d, 10.8) 3.41 (d, 10.8)
24	13.3	0.71 (s)	13.3	0.72 (s)	17.0	0.79 (s)
25	19.3	0.99 (s)	19.4	1.00 (s)	19.0	0.98 (s)
26	16.8	0.83 (s)	17.6	0.85 (s)	16.8	0.82 (s)
27	16.5	nd	16.4	nd	16.5	nd
28	183.0#	-	182.5#	-	182.5#	-
29	27.1	1.38 (s)	28.7	0.99 (s)	27.1	1.38 (s)
30	15.7	0.91 d, 6.6)	27.2	0.99 (s)	15.7	0.92 d, 6.6)

^a

Measured in CD₃OD.

^b

150MHz, ^c600MHz.

^#

Signal identified by HMBC spectrum, nd: none detected.

Compound 2 had the same molecular formula as that of 1, determined from the HRESIMS ion at m/z 503.3365 [M + H]⁺ (calcd. for [C₃₀H₄₇O₆]⁺: 503.3367, Δ = −0.2 ppm). The ¹H NMR and ¹³C NMR spectra of 2 exhibited a high similarity to those of 1, with the major differences being around C-18, C-19, C-20, and C-30 (Table 1). The methyl doublet signal (C-30) in 1 was replaced by a methyl singlet in 2 suggesting an oleane compound. Three hydroxy groups were indicated at C-2, C-3, and C-23, as in compound 1, confirmed by the HMBC correlations from H-24 to C-3, C-4, C-5, and C-23, from H-23 to C-3, C-4, C-5, and C-24, from H-2 to C-2 and C-3, as well as by COSY cross peaks of H-1/H-2/H-3. These hydroxy groups all had an equatorial orientation determined by the large J value between H-2 and H-3 and by the NOESY correlations of H-2/H-24 and H-3/H-5. The 19-OH group was evidenced by the COSY correlation of H-18/H-19 and by HMBC correlations from H-29 and H-30 (δ_H 0.99) to C-19 (δ_C 79.6)/C-20 (δ_C 36.1)/C-21 (δ_C 32.9). H-19 (δ_H 3.65) appeared as a small doublet (J = 1.8 Hz) implying that the 19-OH orientation was α/axial, which was further confirmed by NOESY cross peaks of H-18 (δ_H 2.75) and H-19 (δ_H 3.65), H-18 and H-29/H-30 (δ_H 0.99). ⁷ Moreover, HMBC correlations were observed between H-26 (δ_H 0.85) and C-14 (δ_C 33.5), between H-18 (δ_H 2.75) and C-12 (δ_C 138.6), C-13 (δ_C 26.8), C-14 (δ_C 33.5), C-17 (δ_C 47.0), C-27 (δ_C 16.4), and C-28 (δ_C 182.5) indicating a C-11/C-12 double bond, 13,27-cyclopropane and C-28. Thus, compound 2 was proposed as 2α,3β,19α,23-tetrahydroxy-13,27-cycloolean-11-en-28-oic acid, a new compound and named sarcokontum B (Supplemental Figures S13-S25).

The NMR spectra of the known compounds 3-12 are shown in Supplemental Figures S26-S47.

In continuation of our screening program for bioactive components from plants, the isolated compounds were evaluated for their cytotoxic activity towards three cancer cell lines, human carcinoma (KB), human hepatocellular carcinoma (Hep-G2), and (human lung carcinoma) LU-1. ⁸ Unfortunately, all the isolates showed no cytotoxic activity, with IC₅₀ values over 100 µM, compared to the positive control compound, ellipticine (IC₅₀ ∼ 0.33 −0.39 µM).

Materials and Methods

General Experimental Procedures

The used characterization equipment is the same as that described in our previous work. ⁹

Refer to Supplemental Material

Plant Materials

The stems and leaves of Sarcosperma kontumense were collected from the Bidoup-Nui Ba National Park, Lamdong Province, Vietnam during May 2021 and identified by botanist Nguyen Quoc Dat at the Southern Institute of Ecology, VAST. The voucher specimen (VN-1436) has been deposited at IMBC, VAST.

Extraction and Isolation

The dried stems and leaves of S. kontumense were minced (5.0 kg) and ultrasonically extracted with methanol at 50 ^oC (5 times, each 15 L) to obtain the MeOH crude extract (400 g), which was further partitioned in turn with n-hexane, dichloromethane, and ethyl acetate to obtain the corresponding extracts, SK1 (140 g), SK2 (37 g), SK3 (130 g), and a water layer. The ethyl acetate fraction (SK3) was chromatographed on a silica gel column, eluting with the gradient solvent system of dichloromethane/methanol (20/1, 10/1, 2/1, 1/1, v/v) to give four fractions, SK3A-SK3D. Fraction SK3A (32.0 g) was fractionated on a reversed-phase C18 column eluting with acetone/water (3/1, v/v) to yield three fractions, SK3A1-SK3A3. Fraction SK3A3 (17.6 g) was further fractionated on a silica gel column, eluting with CH₂Cl₂/acetone (4/1, v/v) to produce four fractions, SK3A3A-SK3A3D. Fraction SK3A3A was purified by semi-preparative HPLC using 45% ACN to yield compound 3 (6.6 mg, t_R 40.9 min). Fraction SK3A3D was purified by HPLC using 40% CAN to yield compounds 7 (2.5 mg, t_R 39.3 min), 1 (2.3 mg, t_R 42.5 min), and 2 (6.2 mg, t_R 47.1 min). Fraction SK3C (38 g) was separated on a silica gel column by eluting with CH₂Cl₂/EtOAc (8/1, v/v) to get four fractions, SK3C1-SK3C4. Fraction SK3C2 (5.9 g) was chromatographed on a YMC column, eluting with MeOH/H₂O (2/1, v/v) and then purified by HPLC (70% CAN) to obtain compounds 11 (6.0 mg, t_R 46.7 min) and 12 (3.0 mg, t_R 49.8 min). Fraction SK3C3 (204 mg) was separated on a YMC column, eluting with MeOH/H₂O, 2/1, v/v) to obtain compounds 6 (15.0 mg), 8 (15.6 mg), and 9 (7.3 mg). Fraction SK3D (25 g) was chromatographed on a silica gel column, eluting with CH₂Cl₂/EtOAc (20/1, v/v) to yield four fractions, SK3D1-SK3D4. Fraction SK3D1 was purified by HPLC (70% CAN) to yield 5 (10.0 mg, t_R 36.5 min). Fraction SK3D4 was purified by HPLC (90% CAN) to obtain 4 (3.4 mg, t_R 36.6 min). Compound 10 (45 mg) was obtained from fraction SK3D2 by using a YMC column, eluted with MeOH/H₂O (3/1, v/v).

Sarcokontum A (1)

Colorless solid, $[\alpha {]}_D^{25}$ : + 9.7 (c 0.03, MeOH); UV (MeOH) λ_max (log ɛ): 219 (1.90); IR ν_max (KBr) 3442, 2935, 2875, 1687, 1459 cm⁻¹; HRESIMS m/z 520.3635 [M + NH₄]⁺ (calcd. for [C₃₀H₅₀NO₆]⁺: 520.3633, Δ =  + 0.4 ppm); m/z 525.3186 [M + Na]⁺ (calcd. for [C₂₈H₃₈O₁₃Na]⁺: 525.3187, Δ = −0.1 ppm); m/z 503.3366 [M + H]⁺ (calcd. for [C₃₀H₄₇O₆]⁺: 503.3367, Δ = −0.1 ppm); ¹H NMR (CD₃OD, 600 MHz) δ (ppm), ¹³C NMR (CD₃OD, 150 MHz) δ (ppm) data shown in Table 1.

Sarcokontum B (2)

Colorless solid, $[\alpha {]}_D^{25}$ : + 12.2 (c 0.03, MeOH); UV (MeOH, c 0.1 mg/mL) λ_max: (log ɛ): 219 (2.17); IR ν_max (KBr) 3434, 2932, 2876, 1690, 1458 cm⁻¹; HRESIMS m/z 520.3629 [M + NH₄]⁺ (calcd. for [C₃₀H₅₀NO₆]⁺: 520.3633, Δ = −0.8 ppm); m/z 503.3365 [M + H]⁺ (calcd. for [C₃₀H₄₇O₆]⁺: 503.3367, Δ = −0.2 ppm); ¹H NMR (CD₃OD, 600 MHz) δ (ppm), ¹³C NMR (CD₃OD, 150 MHz) δ (ppm) data shown in Table 1.

Conclusions

Two new 13,27-cyclo pentacyclic triterpenes (1 and 2), together with ten pentacyclic triterpenoids (3-12), were characterized from the stem and leaves of Sarcosperma kontumense Gagnep. ex Aubrév. Their chemical structures were elucidated as 2α,3β,19α,23-tetrahydroxy-13,27-cyclours-11-en-28-oic acid (1), 2α,3β,19α,23-tetrahydroxy-13,27-cycloolean-11-en-28-oic acid (2), 2α,3α,19α,23-tetrahydroxy-13,27-cyclours-11-en-28-oic acid (3), corosolic acid (4), pomolic acid (5), masilinic acid (6), 1β,2α,3α,19α,23-pentahydroxyurs-11-en-28-oic acid (7), rotundic acid (8), euscaphic acid (9), jacoumaric acid (10), 3-O-trans-coumaroyltormetic acid (11), and 3-O-cis-coumaroyltormetic acid (12) by spectroscopic analyses, including HRESIMS, and 1D and 2D NMR spectra.

Supplemental Material

sj-docx-1-npx-10.1177_1934578X221134882 - Supplemental material for Sarcokontums A and B, Two New 13,27-Cyclo-Pentacyclic Triterpenes from the Stems and Leaves of Sarcosperma kontumense Gagnep. ex Aubrév

Supplemental material, sj-docx-1-npx-10.1177_1934578X221134882 for Sarcokontums A and B, Two New 13,27-Cyclo-Pentacyclic Triterpenes from the Stems and Leaves of Sarcosperma kontumense Gagnep. ex Aubrév by Nguyen Quoc Vuong, Vu Van Chien, Nguyen Thi Hue, Pham Thi Hang, Nguyen Hoang Nam, Nguyen Le Tuan, Pham Van Cuong, Duong Thi Hai Yen, Nguyen Xuan Nhiem, Bui Huu Tai and Phan Van Kiem in Natural Product Communications