Rosanortriterpene C, a 3,24-Dinor-2,4-seco-ursane Triterpene from the Fruits of Rosa laevigata var. leiocapus

Abstract

One new 3,24-dinor-2,4-seco-ursane triterpene, rosanortriterpene C (1), together with four known compounds including 24-norursane-type nortriterpenes (2–3), 24-noroleanane-type nortriterpene (4), ursane-type triterpene (5), was isolated from the fruits of Rosa laevigata var. leiocapus. The new structure was elucidated through comprehensive spectroscopic analysis, including one dimensional (1D) and 2D NMR data, as well as electrospray ionization high resolution (HR-ESI) MS and IR spectrometry. Compounds 1–5 showed moderate anti-inflammatory activities against the production of nitric oxide (NO) in RAW264.7 cells stimulated by lipopolysaccharide (LPS) with IC₅₀ values of 10.35 ± 0.92, 14.28 ± 1.20, 5.04 ± 1.43, 29.29 ± 3.64, and 14.37 ± 0.59 µM, respectively.

Introduction

Rosa laevigata, known locally in China as “Jin Ying Zi,” is an evergreen climbing shrub mainly distributed over the south of mainland China.¹⁾ Famous for curative effect on a variety of urinary diseases like wet dreams, uterine prolapse, urinary frequency and incontinence, the fruits of R. laevigata, recorded in the Chinese Pharmacopoeia, are widely consumed for the bio-active ingredients of a huge amount of Traditional Chinese Medicines such as “San Jin Pian,” “Jin Ji Jiao Nang,” and “Fu ke Qian Jin Pian.”²⁾ In the past decades, plenty of studies on chemical constituents of R. laevigata were proposed and carried out owing to its notable pharmacological effects, resulted in the isolation of polysaccharides,³⁾ flavonoids,^4,5) triterpenoids,^2,6–8) phenols,⁸⁾ steroids,⁶⁾ and tannins.⁹⁾

Rosa laevigata var. leiocapus, a variant of R. laevigata found in Boluo county, Guangdong province, China, was firstly reported by Wang and Chen in 1995.¹⁰⁾ Interestingly, there are no thorns on its smooth fruits. This particular morphological feature makes it easier to be collected and prepared. Combined with the possible chemical and biological similarities in the species, it showed good potential for larger quantity production of new medicines.

However, phytochemical investigation of the title plant is scant. To date, only two new nortriterpenoids, rosanortriterpenes A–B, were reported in our previous studies.¹¹⁾ Therefore, an extensive research was conducted on the ethanol extract of R. laevigata var. leiocapus as part of our continuing search for additional bioactive nortriterpenoids with novel structures from natural resources. As a result, one new 3,24-dinor-2,4-seco-ursane triterpene, designated as rosanortriterpene C (1), along with four known compounds: rosanortriterpene B (2),¹¹⁾ diospyric acid C (3),¹²⁾ rosanortriterpene A (4),¹¹⁾ and 2α,19α-dihydroxyursolic acid (5)¹³⁾ (Fig. 1). In this study, the isolation, structural elucidation, and anti-inflammatory bioassay of 1–5 are discussed.

Fig. 1. Structures of Compounds 1–5

Results and Discussion

Compound 1 was purified as a yellow powder. The molecular formula, C₂₈H₄₂O₆, was determined on the basis of high resolution electrospray ionization (HR-ESI) MS measurement wherein a protonated molecular ion at m/z 475.30703 [M + H]⁺ (Calcd 475.30542) was measured, corresponding to eight indices of hydrogen deficiency in the molecule.

In the IR spectrum, three characteristic absorptions at 3483, 1725, and 1691 cm⁻¹, indicated the presence of hydroxyl, carbonyl, and double bond functionalities, respectively. The ¹³C-NMR data and the heteronuclear single quantum coherence (HSQC) spectrum of 1 displayed 28 carbons, attributed to six methyls, eight methylenes, five methines, and nine quaternary carbons. Among these resonances, two carboxyls (δ_C 175.0, 181.0), one carbonyl (δ_C 212.7) and one double bond group (δ_C 128.4, 140.1) could be readily identified. The ¹H-NMR data of 1 exhibited characteristic resonance signals for five singlet methyls at δ_H 2.39 (H₃-23), 1.26 (H₃-25), 1.18 (H₃-26), 1.78 (H₃-27), and 1.39 (H₃-29), as well as one doublet methyl at δ_H 1.09 (H₃-30). Additionally, an olefinic proton at δ_H 5.67 (t, J = 3.1 Hz, H-12) was displayed in the ¹H-NMR spectrum.

Based on heteronuclear multiple bond correlation (HMBC) data analysis, the location of the double bond was deduced to be assigned from C-12 to C-13, as a result of the correlations of H₃-27 (δ_H 1.78, s) to C-13 (δ_C 140.1), as well as of H-18 (δ_H 3.08, s) to C-13 (δ_C 140.1) (Fig. 2); while the positions of a carboxylic acid and the doublet methyl were also determined, as indicated from the HMBC results, to be on C-17 and C-20, respectively. All these above evidence provide the extremely useful indications for a skeleton of urs-12-ene triterpene. Moreover, the observed correlations from H-1a (δ_H 2.59, d, J = 14.4 Hz) to C-2 (δ_C 175.0), C-5 (δ_C 56.8), C-9 (δ_C 39.6), C-10 (δ_C 40.0), and C-25 (δ_C 18.8) in the HMBC spectrum, established the position of another carboxyl group as well as its relationship to ring B. Apart from the above signals, the remaining ones appearing at δ_C 212.7 (C-4) and 32.0 (C-23) and δ_H 2.39 (s, H₃-23) suggested the existence of an acetyl group, and its placement at C-5 could be determined from the correlations between H-5 (δ_H 3.42) and C-23 in the HMBC experiment. Rupture of bond between C-2 and C-3 was therefore assumed. Thus, the aforementioned data was indicative of a 2,4-seco-ursan-12-ene nortriterpenoid skeleton for 1, differing from the characteristic pentacyclic skeleton commonly found in ursane-type triterpenoids. Detailed interpretation of its NMR data indicated the structure of 1 to be high similar to that of diospyric acid D.¹²⁾ The structural difference between these two compounds was mainly due to the absence of a hydroxyl group at C-22 position in the molecule of 1. This deduction was further confirmed by the HMBC correlations from H-22α (δ_H 2.17, m) to C-17, C-18, C-20, and C-28, and H-22β (δ_H 2.06, m) to C-17, C-18, and C-20, as well as the ¹H–¹H correlation spectroscopy (COSY) correlations between H₂-21 and H₂-22. Thus, the gross structure of 1 as a 4-oxo-19-hydroxy-3,24-dinor-2,4-seco-urs-12-en-2,28-dioic acid could be furnished.

Fig. 2. Key ¹H–¹H COSY and HMBC Correlations (H→C) of 1

The co-facial of H₃-25 and the acetyl group could be inferred from the nuclear overhauser effect spectroscopy (NOESY) spectrum wherein the correlations between H₃-25/H₃-23, H-1a/H-5, and H-1b/H-5 were observed (Fig. 3). Therefore the H₃-25 and acetyl group were assigned to be β-orientation based on biogenetic considerations of similar ursane-type triterpenoids.^12,14) Additionally, cross-peaks of H-5/H-9, H₃-25/H₃-26, H₃-26/H₃-29, H-9/H₃-27, H₃-29/H-20, and H-20/H-18 were also observed by NOESY analysis, strongly indicated the β-orientation for H-18, H-20, H₃-26, and H₃-29, and the α-orientation for H-5, H-9, and H₃-27 like structures of many widely occurring 19-hydroxy-ursane-type triterpenes.^12,14) Consequently, the structure of 1 was established as 4-oxo-19α-hydroxy-3,24-dinor-2,4-seco-urs-12-en-2,28-dioic acid, named as rosanortriterpene C.

Fig. 3. Key NOESY Correlations of 1

In our previous study, nortriterpenoids disclosed their potential to be a promising source for the development of new anti-inflammatory agents,¹¹⁾ thus, all the compounds were tested for their anti-inflammatory activities. As a result, compounds 1–5 showed moderate anti-inflammatory activities against the production of nitric oxide (NO) in RAW264.7 cells stimulated by lipopolysaccharide (LPS) with IC₅₀ values of 10.35 ± 0.92, 14.28 ± 1.20, 5.04 ± 1.43, 29.29 ± 3.64, and 14.37 ± 0.59 µM, respectively, whereas indomethacin gave IC₅₀ value of 18.34 ± 0.81 µM.

In summary, four nortriterpenes (1–4), including one new 3,24-dinor-2,4-seco-ursane triterpene (1), two known 24-nor-ursane triterpenes (2–3), and one known 24-nor-oleanane triterpene (4), together with one known ursane-type triterpene (5) were isolated from the fruits of R. laevigata var. leiocapus, and their anti-inflammatory activities against the production of NO in RAW264.7 macrophages stimulated by LPS were evaluated. All these isolated compounds exhibited moderate anti-inflammatory activities in the assay.

Experimental

Optical rotation was recorded on a Perkin-Elmer 341 polarimeter. The IR spectrum was obtained on a Nicolet Magna-IR 750 spectrophotometer by using KBr disks. NMR spectra were measured on a Bruker Avance III 500 spectrometer using tetramethylsilane as internal standard. HR-ESI-MS data was measured on a Waters Xevo QTof mass detector. Analytical HPLC was applied on a Waters 2695 instrument (Milford, MA, U.S.A.) coupled with an Alltech 2000 ELSD and a Waters 3100 MS detector. Purification by HPLC was performed on a Varian PrepStar pump with an Alltech 3300 ELSD detector and Waters Sunfire^® RP C₁₈ columns (5 µm, 30 × 150 mm and 5 µm, 19 mm × 150 m). Silica gel (Qingdao Marine Chemical Industrials, Qingdao, People’s Republic of China), MCI (polystyrene) gel CHP20P (75–150 µm, Mitsubishi Chemical Industries, Tokyo, Japan) and Sephadex LH-20 (Pharmacia Biotech AB, Uppsala, Sweden) were used for column chromatography (CC). TLC was performed on precoated silica gel GF₂₅₄ plates (Yantai Chemical Industrials, Yantai, People’s Republic of China) and the spots in the TLC were viewed at 254 nm and then visualized by heating after spraying with 5% H₂SO₄ in anhydrous ethanol containing 10 mg/mL vanillin. All solvents used for CC and preparative HPLC were of analytical grade (Shanghai Chemical Reagents Co., Ltd., Shanghai, People’s Republic of China) and HPLC grade (Merck KGaA, Darmstadt, Germany), respectively.

Plant Material

Fruits of R. laevigata var. leiocapus were collected from Boluo County (Guangdong Province, China) during October 2018. The plant material was identified by Professor Guowen Xie, Guangzhou University. A voucher specimen (No. 20181001) was deposited in the Laboratory of Botany, Guangzhou University.

Extraction and Isolation

Dried and powdered fruits of R. laevigata var. leiocapus (3 kg) were extracted with 95% EtOH three times (each 7 d) at room temperature and the solvent was evaporated under vacuum to afford a crude extract (508 g). The obtained crude extract was suspended in H₂O (3 L) and further partitioned with petroleum ether (3 L × 3) and EtOAc (3 L × 3), successively. The EtOAc layer was concentrated and then subjected to CC over MCI gel (EtOH/H₂O, 50 to 95%) to afford fractions A–C. Fraction B (32 g) was then subjected to CC over silica gel eluting with n-hexane/EtOAc mixture of increasing polarity (5 : 1 to 1 : 1) to yield six subfractions (B1–B6). Subsequently, subfraction B3 (1.3 g) was subjected to CC over Sephadex LH-20 (MeOH) to afford three subfractions (B3A–B3C). B3B (118 mg) was then selected for a purification by preparative HPLC (CH₃CN/H₂O, 50 to 80%), giving 2 (2.1 mg) and 4 (2.0 mg). Subfraction B4 (4.3 g) was chromatographed on a silica gel column with a gradient of CH₂Cl₂/EtOAc (100 : 1 to 20 : 1) to yield five subfractions (B4A–B4E). B4B (1.1 g) was purified further by CC over Sephadex LH-20 (MeOH) to yield 5 (48 mg). B4C (345 mg) was subjected to CC over Sephadex LH-20 (MeOH) and then preparative HPLC (CH₃CN/H₂O, 50% to 80%), giving 3 (18 mg). Similarly, B4D (132 mg) was purified by CC over Sephadex LH-20 (MeOH) and then preparative HPLC (CH₃CN/H₂O, 50 to 80%), affording 1 (1.3 mg).

Compound 1

Yellow power; [α]_D²⁰+30.3 (c 0.1, MeOH); IR (KBr) ν_max 3482, 2933, 1725, 1691, 1459, 1383, 1360, 1322, 1271, 1237, 1173, 1137, 1111, 1094, 1052, 1037, 932, 889, 865 cm⁻¹; HR-ESI-MS: m/z 475.30703 [M + H]⁺ (Calcd for C₂₈H₄₃O₆, 475.30542); ¹H- and ¹³C-NMR, see Table 1.

Table 1. ¹H- and ¹³C-NMR Data for 1 (500 and 125 MHz, Respectively) in C₅D₅N

No.	1
	δH (mult., J in Hz)	δC
1a	2.59 (d, 14.4)	45.0
1b	2.66 (d, 14.4)
2		175.0
3	nor
4		212.7
5	3.42 (d, 11.8)	56.8
6α	1.86 m	22.7
6β	1.66 m
7α	1.70 m	32.1
7β	1.38 m
8		40.6
9	2.82 (dd, 11.1, 6.4)	39.6
10		40.0
11α	2.60 m	24.4
11β	2.19 m
12	5.67 (t, 3.1)	128.4
13		140.1
14		43.1
15α	2.31 m	29.7
15β	1.28 m
16α	3.09 m	26.8
16β	2.07 m
17		48.6
18	3.08 s	55.0
19		73.0
20	1.50 m	42.7
21α	2.07 m	27.3
21β	1.36 m
22α	2.17 m	38.8
22β	2.06 m
23	2.39 s	32.0
24	nor
25	1.26 s	18.8
26	1.18 s	17.5
27	1.78 s	24.8
28		181.0
29	1.39 s	27.5
30	1.09 (d, 6.6).	17.1

Anti-inflammatory Assay

RAW264.7 cells were cultured into 96-well plates (5 × 10⁵ cells/well) for 12 h, then the cells were treated with demanded concentrations of 1–5, together with 1 µg/mL LPS (Sigma-Aldrich Co. LLC, St. Louis, MO, U.S.A.) for 24 h. Afterwards, the removed supernatant within NO production was evaluated after adding 100 µL of Griess reagent (Reagent A & Reagent B, respectively, Biotine) to 100 µL each of them. Eventually, the absorbance was measured at 420 nm with a Multiscan Spectrum (Thermo Fisher Scientific, Waltham, MA, U.S.A.). Indomethacin (Sigma) was used as a positive control. Data were presented as means ± standard deviation (S.D.) of three replicates.

Acknowledgments

Our thanks are given to the financial support of the State key Laboratory of Drug Research [Grant number SIMM1803KF-09].

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

The online version of this article contains supplementary materials.

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