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Abstract

Three compounds were obtained from Sabia parviflora Wall., and their structures were identified through nuclear magnetic resonance (NMR) spectroscopy, particularly 2-dimensional (2D)-NMR. The molecular masses were determined using quadrupole-time-of-flight-mass spectrometry. Electronic circular dichroism spectra were used to determine the absolute configuration of compound 1. The 3 new compounds were identified as 2,2,7,7-tetramethyl-8S,10-dihydroxy-2,7,8,9-tetrahydro-2H-naphtha[2,3-b]pyran-6-one-10-O-β-D-glucopyranoside, (2Z)-4-(3-carboxy-4-hydroxyphenyl)-2-methylbut-2-enoic acid, and (2Z)-5-(3-carboxy-4-hydroxyphenyl)-5-oxo-2-methylpent-2-enoic acid. The liver protective activities of these compounds were tested by HepG2 and LO2 cell lines, which were induced using free fatty acids.

Keywords

extraction identification hepatoprotective activity

Introduction

Sabia parviflora Wall., family Sabiaceae, is a plant mainly distributed in southwest China. It is a traditional Chinese herbal medicine that is mainly used for the treatment of “damp heat jaundice, hepatitis and hemostasis.”^1,2 Numerous chemical studies have shown that the plant contains triterpenoids,^1,3 flavonoids,^4,5 alkaloids,^1,6 phenolic acids, phenylpropane, and polysaccharides.⁷ Previous pharmacological studies have revealed that the plant's compounds possess antiviral,⁸ antiinflammatory,^{5, 9} antiliver injury,^5,9 and antioxidant¹⁰ activities.

Based on the results of earlier experimental research,¹¹ our group has continued to explore the constituents of the plant and their biological activities. In this study, we examined the dried stems of the species and isolated 3 new compounds: 2,2,7,7-tetramethyl-8S,10-dihydroxy-2,7,8,9-tetrahydro-2H-naphtha[2,3-b]pyran-6-one-10-O-β-D-glucopyranoside (1), (2Z)-4-(3-carboxy-4-hydroxyphenyl)-2-methylbut-2-enoic acid (2), and (2Z)-5-(3-carboxy-4-hydroxyphenyl)-5-oxo-2-methylpent-2-enoic acid (3).The structures of the compounds are shown in Figure 1.

Figure 1.

The 3 new compounds (1, 2, 3).

Finally, the biological activities of these 3 compounds were determined, and their hepatoprotective effects were investigated using HepG2 and LO2 cell lines.

Results and Discussion

Compound 1, a yellow powder, had a relative molecular mass of C₂₃H₃₀O₉ based on its molecular ion peak at m/z 451.1943 [M + H]⁺(calcd. for C₂₃H₃₀O₉, 450.1890) in its quadrupole time-of-flight tandem mass spectrum (Q-TOF-MS). The ¹H nuclear magnetic resonance (NMR) spectrum of compound 1 exhibited signals for an aromatic proton of a penta-substituted benzene (δ_H 7.47 s, 1H), 2 olefinic protons of a 1,2-disubstituted alkene (δ_H 5.77, d, J = 10.0 Hz, 1H and δ_H 6.41, d, J = 10.0 H z, 1H), 1 methylene group (δ_H 3.21 m, 1H and 3.33 m, 1H), 1 methine proton (δ_H 3.91, dd, J = 3.5, 6.2 Hz, 1H), 4 methyl protons (δ_H 1.48, 1.49, 1.16, 1.17 s, each 3H), and 1 anomeric proton (δ_H 4.96, d, J = 7.7 H z, 1H). The ¹³C-NMR spectrum of compound 1 showed 23 signals: a penta-substituted benzene (δ_C 122.4, 122.7, 125.6, 137.9, 143.1, and 151.1), a carbonyl carbon (δ_C 203.6), 2 double-bond carbons (δ_C 132.4 and 122.6), 1 methylene carbon (δ_C 30.4), 1 methine carbon (δ_C 75.6), 2 quaternary carbons (δ_C 79.7 and 48.5), 4 methyl carbons (δ_C 28.8, 28.5, 24.1, and 23.1), and 6 glucosyl carbons (δ_C 105.3, 75.9, 78.2, 71.8, 78.4, and 62.7). Furthermore, C-10 was found to be connected to the oxygen atom, which was determined by the chemical shift of δ_C 143.1 (C-10). The ¹H–¹³C-NMR spectra of compound 1 revealed structural similarity between compound 1 and sabianin C,¹¹ except for subtle differences in the 7 position of the methyl or hydroxymethyl substituents. The heteronuclear multiple bond correlation (HMBC) cross peak (δ 4.96/143.1) indicated that the glucose moiety was connected to C-10. Accordingly, acid hydrolysis of compound 1 gave D-glucose as the sugar residue, which was determined through gas chromatographic (GC) analysis of its corresponding trimethylsilylated L-cysteine adduct. The sugar in compound 1 was a β-glucose moiety according to the proton signal at δ_H 4.96 (d, J = 7.7 Hz). The nuclear overhauser effect spectroscopy spectrum showed 2 configurations for 1:2,2, 7,7-tetramethyl-8S,10-dihydroxy-2,7,8,9-tetrahydro-2H-naphtha[2,3-b] pyran-6-one-10-O-β-D-glucopyranoside and 2,2,7,7-tetramethyl-8R,10-dihydroxy-2,7,8,9-tetrahydro-2H-naphtha[2,3-b]pyran-6-one-10-O-β-D-glucopyranoside. The experimental circular dichroism (CD) spectrum and calculated CD(8S-C) exhibited the same trend at the 200-280 nm wavelength. The result established the absolute configuration as 2,2,7,7-tetramethyl-8S,10-dihydroxy-2,7,8,9-tetrahydro-2H-naphtha[2,3-b]pyran-6-one-10-O-β-D-glucopyranoside. The ¹H and ¹³C NMR spectroscopic data of compound 1 are listed in Table 1, the HMBC spectrum in Figure 2, and the electronic circular dichroism (ECD) spectrum in Figure 3.

Figure 2.

Important heteronuclear multiple bond correlation (HMBC) information (h→c) of the 3 new compounds 1, 2 and 3.

Figure 3.

Electronic circular dichroism (ECD) spectra of compound 1.

Compound 2, isolated as a colorless oil, had a molecular formula of C₁₂H₁₂O₅ based on its molecular ion peak at m/z 235.0608 [M-H]⁻ (calcd. for C₁₂H₁₂O₅, 236.0685) in its Q-TOF-MS. The ¹H-NMR spectrum of compound 2 showed signals for 3 aromatic protons of a trisubstituted benzene (δ_H 7.70, dd, J = 8.2, 2.2 Hz, 7.71, d, J = 8.2 Hz, 6.90 d, J = 8.2 Hz, each 1H), 1 olefinic proton (δ_H 6.01, dd, J = 7.68 Hz, 1H), 1 methine (δ_H 3.70, d, J = 7.6 Hz, 2H), and 1 methyl (δ_H 1.85 s, 3H). The ¹³C-NMR spectrum of compound 2 showed 12 signals: 1 benzene ring (δ_C 131.7, 126.7, 129.8, 121.7, 160.0, and 115.2), 2 carboxyl carbons (δ_C 167.8 and 169.8), double-bond carbons (δ_C 138.3 and 129.0), 1 methine carbon (δ_C 30.0), and 1 methyl carbon (δ_C 21.1). The HMBC cross peak (δ 3.70/126.7) suggested that the methene was connected to C-1 (δ_H 6.01/δ_C 30.0), and the double bond to C-4′ (δ_H 6.01/δ_C 169.9 and δ_H 1.85/δ_C 129.0). Furthermore, the HMBC cross peak also indicated that the carboxyl carbon and methyl carbon were connected to C-2′. The HMBC cross peaks (δ_H 3.70/δ_C 131.8, 129.9) and (δ_H 7.70/δ_C 121.7), and the ABX coupling system suggested that carboxyl and hydroxyl carbons were connected to C-3 and C-4, respectively. In the ROESY experiment, the cross peak of δ_H 6.01, H-3′/δ_H1.85, and H-1′a supports the Z-configuration of C-2′-C-3′. Compound 2 was thus established as (2Z)-4-(3-carboxy-4-hydroxyphenyl)-2-methylbut-2-enoic acid.

Compound 3, isolated as a yellow oil, had a molecular formula of C₁₃H₁₂O₆, based on the molecular ion peak at m/z 263.0570 [M-H]⁻(calcd. For C₁₃H₁₂O₆, 264.0634) in its Q-TOF-MS. The ¹H-NMR spectrum of compound 3 showed signals for 3 aromatic protons of a trisubstituted benzene (δ_H 7.69 m, 6.93, d, J = 8.2 Hz, 7.67 d, J = 8.2 Hz, each 1H), 1 olefinic proton (δ_H 6.00, t, J = 6.0 Hz, 1H), 1 methene (δ_H 3.7, d, J = 6.0 Hz, 2H), and 1 methyl (δ_H 1.88 s, 3H). The ¹³C-NMR spectrum of compound 3 showed 13 signals: 1 benzene ring (δ_C 130.7, 127.3, 138.5, 115.4, 161.3, and 125.1), 2 carboxyl carbons (δ_C 169.5 and 169.6), 2 double-bond carbons (δ_C 131.5 and 128.9), 1 methene carbon (δ_C 30.1), and 1 methyl carbon (δ_C 21.1). The ¹H- and ¹³C-NMR spectra of compound 3 were similar to those of compound 2, except for subtle differences at C-5′. The signal at δ_C 192.1 was used to define the carbonyl group. The HMBC cross peak (δ 7.67/192.1) indicated that the carbonyl carbon was connected to C-1. Furthermore, in the ROESY experiment, the cross peak at δ_H 6.00, H-3′/δ_H 1.88, H-2′a supports the Z-configuration of the C-2′-C-3′ double bond. Therefore, compound 3 was established as (2Z)-5-(3-carboxy-4-hydroxyphenyl)-5-oxo-2-methylpent-2-enoic acid. The NMR data of compounds 2 and 3 are listed in Table 2, and the HMBC spectrum is shown in Figure 2.

Table 1.

Detailed ¹H- and ¹³C-NMR Spectroscopic Data for Compound 1 and Sabianin C (δ in ppm, in CD₃OD, 600 and 150 MHz).

No.	Sabianin C		Compound 1
No.	δ_H (J in Hz)	δ _C	δ_H (J in Hz)	δ _C	HMBC
2		78.9		79.7
3	5.74 d (10.2)	132.3	5.77 d (9.9)	132.4	C-2, C-4a
4	6.41 d (9.6)	122.6	6.41 d (10.0)	122.6	C-5, C-10a
4a		121.5		122.4
5	7.61 s	126.3	7.47 s	122.7	C-4, C-6
5a		126.1		125.6
6		202		203.6
7		52.3		48.5
8	4.22 dd (3.7, 4.7)	73.8	3.91 dd (3.5, 6.2)	75.6	C-6, C-9
9	3.01 dd (4.9, 17.5)	129.0	3.21 m	128.9	C-5a, C-9a
9	3.24 dd (3.5, 17.7)	129.0	3.33 m	21.1	C-8, C-10
9a		143.8		137.9
10	6.61 s	117.4		143.1
10a		159.6		151.1
11	3.81 d (10.8)	66.1	1.16 s	23.1	C-6, C-7, C-8
11	3.97 d (10.8)
12	1.01 s	18.9	1.16 s	24.1	C-6, C-7, C-8
13	1.48 s	27.5	1.48 s	28.5	C-2, C-3
14	1.49 s	28.8	1.49 s	28.8	C-2, C-3
1′			4.96 d (9.7)	105.3
2′			3.51 m	75.9
3′			3.44 m	78.2
4′			3.33 m	71.8
5′			3.19 m	78.4
6′			3.65 m	62.7
6′			3.78 m

Abbreviations: NMR, nuclear magnetic resonance; HMBC, heteronuclear multiple bond correlation.

Table 2.

¹H- and ¹³C-NMR Spectroscopic Data for Compounds 2 and 3 (δ in ppm, in DMSO-d₆, 600 and 150 MHz).

No.	2			3
No.	δ_H (J in Hz)	δ _C	HMBC	δ_H (J in Hz)	δ _C	HMBC
1		126.7			127.3
2	7.70 dd (8.2, 2.2)	129.8	C-3a	7.67 m	130.7	C-3a, C-4, C-5′
3		121.7			125.1
3a		167.8			169.5
4		160.0			161.3
5	6.90 d (8.2)	115.2		6.93 d (8.2)	115.4
6	7.71 d (8.2)	131.8		7.67 d (8.2)	131.5
1′		169.8			169.6
1′a	1.85 s	21.1	C-2′, C-3′
			C-11′
2′		129.0			128.9
2′a				1.88 s	21.1	C-1′, C-2′, C-3′
3′	6.01 t (7.6)	138.3	C-1′	6.00 t (6.0)	138.5	C-1′
4′	3.70 d (7.6)	30.0	C-1, C-2	3.72 d (6.0)	30.1	C-3′, C-1
			C-6, C-3′
5′					192.1

NMR, nuclear magnetic resonance; DMSO-d₆, dimethylsulifde-d₆; HMBC, heteronuclear multiple bond correlation.

Compounds 1-3 were evaluated for their hepatoprotective activities. The results demonstrated that the 3 compounds had no hepatoprotective activity with no significant difference being observed between the cells in the presence or absence of these 3 compounds.

Experimental

Materials

Analytical methanol and glacial acetic acid were purchased from Sinopharm Chemical Reagent Co., Ltd. Dimethylsulfide-d₆ (DMSO-d₆) was used as a solvent to dissolve monomers. All chemicals used in this study were of analytical grade. Deionized ultrapure water was obtained using a MillQ Millipore Ultrapure Water Purification System (USA).

Methanol and acetonitrile for separation and purification were from Shanghai McLean Company, silica gel (300-400 mesh) for chromatographic separation from Qingdao Haiyang Chemical Group, and DIAION HP-20 macroporous resin from Mitsubishi. The medium-pressure liquid chromatography system was equipped with a Lisui EZ Purify III system, which was from Shanghai Lisui Chemical Engineering. The RP-C18 (25 µm) chromatographic column was purchased from Merck Company. The Ryong C18 column (10 µm, 30 × 250 mm) was equipped with a preparative high performance liquid chromatography (HPLC) system. We also used a Ryong C18 column (5 µm, 20 × 25 0 mm) for further purification. A Cosmosil C18 column (5 µm, 4.6 × 50 mm) was used in the component purity test. A Shimadzu LC-30A HPLC was connected to a TOF™5600 + mass spectrometer. The important NMR data were obtained using a Bruker Avance spectrometer. Palmitic acid (33.3%) and oleic acid (66.6%) were purchased from Sigma.

The 2 cell lines used in this study, HepG2 and LO2, were purchased from American Tissue Culture Collection.

Plant Material

The stems of S parviflora were collected in Ziyun City and identified by Professor Guo-Yue Zhong, Jiangxi from University of Traditional Chinese Medicine. A voucher specimen is deposited in the corresponding author's laboratory, the Center of National Medicine Resource, Jiangxi University of Traditional Chinese Medicine, which is located on Yangming Road, Donghu District, Nanchang City, Jiangxi Province, China

Extraction and Isolation

The dried stems of S parviflora (dry weight: 17 kg) were extracted with 70% ethanol (40 L × 3) through hot reflux. After removal of the solvent, the dry extract (380 g) was separated using macroporous resin HP20 (20 L) and eluted with an ethanol/water (30%→95%) gradient, thereby obtaining 4 fractions (frs 1-4). Fr 1 (253 g) was placed on a silica gel column and eluted with different proportions of CH₂Cl₂/CH₃OH (20:1→1:1 v/v), and at the end of the process, 20 fractions were obtained (frs 1.1-1.20). Fr 1.1 was eluted with ethanol/water (10:90→0:100 v/v, 30 mL min⁻¹), and 11 subfractions were obtained (Frs 1.1.1→1.1.11). Subfraction fr 1.1.3 (2.4 g) was separated using silica gel column chromatography where different proportions of CH₂Cl₂/CH₃OH (20:1→1:1 v/v) were used. Finally, compound 2 (5.1 mg, TR 21.5 min) was eluted with 22% acetonitrile (5 mL min⁻¹) through a reversed-phase HPLC-C18 chromatography column. Fr 2 (94 g) was chromatographed on a silica gel column with CH₂Cl₂/CH₃OH (20:1→1:1 v/v), and 16 subcomponents (frs 2.1-2.16) were obtained. Using medium- and low-pressure chromatographic columns (10 cm × 60 cm), 10 subcomponents (frs 2.1.1-2.1.10) were obtained with ethanol/water elution (20:80→0:100 v/v, 30 mL min⁻¹). Then, compounds 1 (2.0 mg, TR 42.5 min) and 3 (8.9 mg, TR 23.6 min) were purified from fr 2.1.8 (1.2 g) by semipreparative HPLC-C1 column chromatography (15% acetonitrile 5 mL min⁻¹).

2,2,7,7-Tetramethyl-8S,10-dihydroxy-2,7,8,9-tetrahydro-2H-naphtho(2,3-b) pyran-6-one-10-O-β-D-glucopyranoside (yellow powder); Table 1 presents the ¹H- and ¹³C-NMR data. Q-TOF-MS: m/z 451.1943 [M + H]⁺ (calcd. for C₂₃H₃₀O₉, 450.1890).

(2Z)-4-(3-carboxy-4-hydroxyphenyl)-2-methylbut-2-enoic acid (colorless oil); Table 2 presents the ¹H- and ¹³C-NMR data. Q-TOF-MS: m/z 235.0608 [M−H]⁻ (calcd. for C₁₂H₁₂O₅, 236.0685).

(2Z)-5-(3-carboxy-4-hydroxyphenyl)-5-oxo-2-methylpent-2-enoic acid (yellow oil); Table 2 presents the ¹H- and ¹³C-NMR data. Q-TOF-MS: m/z 263.0570 [M − H]⁻ (calcd. for C₁₃H₁₂O₆, 264.0634).

Cell Viability Assay

HepG2 and LO2 cells were preserved in Roswell Park Memorial Institute 1640 (RPMI 1640) medium under a humidified atmosphere of 5% CO₂ at 37°C. The medium contained 5% fetal bovine serum, penicillin (100 μm L⁻¹), and streptomycin sulfate (100 μm L⁻¹). HepG2 and LO2 cells were seeded and inoculated on 96-well plates at a density of 1 × 10⁴ cells/well for 24 h. Then, 100 μL of medium containing different concentrations of the 3 compounds was added to each well, except for the blank and model wells. After 4 h, free fatty acids (1 mmol L⁻¹) were added to all groups except the blank. After 24 h, the supernatant was discarded and a 10% CCK8 reagent (100 μL/well) was added and incubated for 2 h. In our experiment, a microplate reader was used to measure the absorbance of the sample at 450 nm. Cell viability is presented as a relative percentage of the untreated control.^12,13

Hydrolysis of Compound 1 and GC Analysis

As reported in the previous literature,¹⁴ compound 1(3 mg) was hydrolyzed with 2 mol L⁻¹ HCl–dioxane (1:1, v/v, 5 mL) at 85°C for 8 h. The reaction mixture was extracted with CHCl₃ (2 mL × 4). The aqueous layer was neutralized with 2 mol L⁻¹ NaOH and dried to obtain the monosaccharides. Then, in the presence of pyridine (2 mL), the dry powder was added to L-cysteine methyl ester hydrochloride (1.5 mg) and kept at 60°C for 1 h. Next, trimethylsilylimidazole (1.5 mL) was added to the reaction mixture and kept at 60°C for 30 min. The supernatant (4 μL) was directly subjected to GC and compared with a glucose standard. GC was performed using the following experimental conditions: Agilent-7890b ECD detector, Chromatographic column: HP-5 (30 m × 0.25 nm, 0.25 μm), flow: 2 mL min⁻¹; split ratio 30:1. temperature: 250°C.

Conclusion

Three new compounds were isolated from S parviflora. These 3 compounds showed no significant activity regarding liver protection. Further research is needed in order to understand other possible biological activities of these compounds.

Footnotes

Acknowledgements

We would like to thank MogoEdit () for its English editing during the preparation of this manuscript.

Author Contributions

Wrote or contributed to revising the manuscript: Huan He, WeiDong Du. Conceived and designed the study: Huan He, WeiDong Du, ZhiFeng Li, Qiang Zhou. Do experiment: Huan He, WeiDong Du, Qiang Zhou.Experimental technical support: Yuanying Fang, Qi Wang, YuLin Feng.Data processing and analysis: Huan He, WeiDong Du, ZhiFeng Li.All authors have read and approved this version of the manuscript.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the National Natural Science Foundation of China (No. 81960701), the funding programs for academic and technical leaders of major disciplines in Jiangxi Province (20194BCJ22020), Jiangxi Province 5511 R&D projects (No. 20165BCB19009), Jiangxi Province talent Project (No. [2016]332), and Nanchang innovative talent team (No. [2018]274), and Grant for Nanchang innovative talent training project (Grant No. [2020]137). This work was also supported by the General project of Chongqing Natural Science Foundation (cstc2021jxyj-msxmx0665) titled “Studies on the active components and anti-hepatitis B virus activity of Sabia parviflora from Chongqing.”

Ethical Approval

Not applicable, because this article does not contain any studies with human or animal subjects.

ORCID iD

Huan He

Statement of Human and Animal Rights

Not applicable, because this article does not contain any studies with human or animal subjects.

Informed Consent

Not applicable, because this article does not contain any studies with human or animal subjects.

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Three New Phenolic Compounds from Sabia parviflora and Their Hepatoprotective Activity

Abstract

Keywords

Introduction

Results and Discussion

Experimental

Materials

Plant Material

Extraction and Isolation

Cell Viability Assay

Hydrolysis of Compound 1 and GC Analysis

Conclusion

Footnotes

Acknowledgements

Author Contributions

Declaration of Conflicting Interests

Funding

Ethical Approval

ORCID iD

Statement of Human and Animal Rights

Informed Consent

References