Hepatoprotection and Phytochemistry of the Vietnamese Herbs Cleome chelidonii and Cleome viscosa Stems
The study aims to determine the hepatoprotective effect of n-hexane, ethyl acetate, and methanol extracts of the leaves and stems of two Cleome species against carbon tetrachloride- (CCl4-) induced liver toxicity both in vitro using human hepatoma (HepG2) cells and in vivo in rats as well as the hepatoprotective property of all isolated compounds on HepG2. After 72 h of treatment, at the concentrations of 25, 50, and 100 μg/mL, the methanol of C. chelidonii stems (CCSM) ranged from 18.6% to 20.8%, whereas the methanol of C. chelidonii stems (CVSM) increased from 12.3% to 17.2% cell viability. The results show that CCSM and CVSM significantly expressed in vitro hepaprotective activity on HepG2. Therefore, the animals were daily treated with these extracts at the doses of 15, 30, and 45 mg/kg body weight for 5 days, and CCl4 was injected (2 ml/kg body weight, i.p.) on the 2nd and 3rd days. Levels of aspartate aminotransferase (ALT) and alanine aminotransferase (AST) in the blood were measured and compared to the silymarin control. The treatments with CCSM and CVSM (30, and 45 mg/kg) possessed significant hepatoprotection and were comparable with the activity of silymarin. Further, phytochemical studies of these ones were conducted and led to the identification of eight flavonoids: visconoside A (1), visconoside B (2), quercetin 3-O-β-D-glucopyranoside 7-O-α-L-rhamnopyranoside (3), kaempferol 3-O-β-D-glucopyranoside 7-O-α-L-rhamnopyranoside (4), cleomeside A (5), cleomeside B (6), cleomeside C (7), and quercetin-3-O-[β-D-glucopyranosyl-(1⟶2)]-α-L-rhamnopyranoside 7-O-α-L-rhamnopyranoside (8). Two major flavonoids (1 and 4) displayed significant hepatoprotective property (at the concentration of 100 μM, the prevention percentage values were 66.5% and 74.2%, respectively, compared to the quercetin control, with value of 80.3%).
The genus Cleome belonging to the Cleomaceae family comprises about 170 species. Five species were found in Vietnam . In the traditional Vietnamese medicine, C. chelidonii is used for treatment of fever, flu, headache, cough, snake bite, and nephritis, whereas C. viscosa is used to treat diarrhea, fever, inflammation, liver diseases, bronchitis, skin diseases, and malarial fever . Pharmacological investigations proved that C. chelidonii possessed antipyretic , antihyperglycemic , and anthelmintic  properties, while C. viscosa expressed anticonvulsant , antitumor , cytotoxic [8–12], antiangiogenic , antimalarial , larvicidal , antiallergic, diuretic , analgesic, antipyretic , α-glucosidase, and α-amylase inhibitory  activities. Additionally, both species exhibited antimicrobial [9–21], antinociceptive [3, 10, 22, 23], anti-inflammatory [3, 21, 23, 24], and antioxidant activities [5, 8, 9, 12, 14, 25–27].
In vivo study on rats against CCl4-induced liver injury indicated that hydroalcohol, methanol, ethyl acetate, and hexane extracts of C. chelidonii root revealed hepatoprotective activity . Another study on rats against paracetamol- and ethanol-induced liver toxicity also confirmed that a methanol extract of C. chelidonii’s whole plant displayed hepatoprotective property . In vivo study on ethanol extract of C. viscosa’s whole plant against CCl4-induced hepatotoxicity , as well as methanol extract against streptozotocin- (STZ-) induced diabetic rats , C. viscosa leaves against thioacetamide-induced hepatotoxicity [31, 32], and C. viscosa seeds against paracetamol-induced hepatotoxicity [33, 34] also showed that C. viscosa possessed a hepatoprotective effect on rat models.
So far, there has been no report on the hepatoprotection and phytochemical constituents of the C. chelidonii and C. viscosa stems. Continuing our study on bioactive composition of traditional Vietnamese medicines  and the Cleome genus [36–39], this paper detailed the evaluation of the hepatoprotective effect of different extracts (n-hexane, ethyl acetate (EtOAc), and methanol (MeOH) extracts) from the stems of two Cleome species against CCl4-induced liver intoxication in both in vitro and in vivo assays. All compounds isolated from the most active extracts were also measured for the hepatoprotective activity using in vitro assay.
2. Materials and Methods
2.1. Plant Materials
C. chelidonii and C. viscosa stems were collected in Ben Cat, Binh Duong province, Vietnam, in May 2015 and certificated by Professor Vo Van Chi. The voucher specimens (No. VH/MINH-1012 and No. VH/MINH-0515, respectively) were deposited in the Institute of Chemical Technology, Vietnam Academy of Science and Technology.
Dried stems powders of C. chelidonii (8 kg) and C. viscosa (7 kg) were extracted with 96% EtOH for three times (3 × 30 L, total amount 90 L) at room temperature. The supernatants were filtered, and the solvents were removed under vacuum to obtain crude extracts CCS (970 g) and CVS (770 g), respectively. Those extracts were subjected to solid-phase separation and successively fractionated into n-hexane, EtOAc, and MeOH extracts, respectively, to afford six extracts: CCSH (155 g), CCSE (355 g), CCSM (420 g), CVSH (130 g), CVSE (310 g), and CVSM (330 g). Similar protocols were used for powdered leaves of C. chelidonii (5 kg) and C. viscosa (7.5 kg), resulting in six extracts: CCLH (120 g), CCLE (228 g), CCLM (170 g), CVLH (150 g), CVLE (260 g), and CVLM (400 g). All extracts were stored at 4°C for further studies.
2.3. Chemicals and Reagents
Eagle’s Minimum Essential Medium (EMEM), fetal calf serum (FCS), and trypsin-EDTA were purchased from Gibco, USA; L-glutamine, penicillin, streptomycin, phosphate buffer, 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), doxorubicin, and carbon tetrachloride (CCl4) were from Sigma-Aldrich, USA; dimethyl sulfoxide (DMSO) and isopropanol were from Merck, Germany. All chemicals met cell culture standards.
2.4. Cell Culture
HepG2 cells (the American Type Culture Collection, Manassas, Rockville) were seeded and cultured in EMEM containing 10% FCS (v/v), 2 mM L-glutamine, 100 IU/mL penicillin, and 100 μg/mL streptomycin at 5% CO2 at 37°C to attain confluency.
Swiss albino mice weighing 26–30 g were purchased from Pasteur Institute in Ho Chi Minh City, Vietnam Ministry of Health. The mice were housed in standard cages (48 cm × 35 cm × 22 cm) at room temperature and provided with pelleted food and water.
2.6. Evaluation of the In Vitro and In Vivo Hepatoprotective Activity
2.6.1. Cell Viability
HepG2 cells were harvested and seeded in 96-well plates at 4.0 × 10 cells/cm2. Then, cells were treated with EMEM containing 2 mM CCl4 and compounds 1–8 alone or combined at different concentrations. Cell viability was measured as mitochondrial succinate dehydrogenase activity, a marker of viable cells using MTT test. Doxorubicin was used as positive control for cytotoxicity. The assay was performed using the MTT test, as previously described [17, 18]. Doxorubicin was used as a positive control for cytotoxicity.
For the cytotoxicity, the percentage of control (%) was calculated = OD570 sample/OD570 control × 100% measured at the different concentrations (25, 50, and 100 μg/mL) by MTT assay.
2.6.2. Study on Hepatic Protective Effect in Mice Acute Liver Injury Induced by CCl4
Mice were divided into six groups with six animals in each group.
Group I (normal control) received distilled water with 0.3% sodium carboxymethylcellulose (CMC-Na) (1 mL/kg body weight, p.o.) for 5 days and olive oil (1 mL/kg body weight, i.p.) on days 2 and 3.
Group II (CCl4-intoxicated) received 0.3% CMC-Na (1 mL/kg body weight, p.o.) for 5 days and CCl4–olive oil (1 : 1, 2 mL/kg body weight, i.p.) on days 2 and 3.
Group III (positive group) was treated daily with the positive silymarin drug (100 mg/kg body weight, p.o.) for 5 days and CCl4–olive oil (1 : 1, 2 mL/kg body weight, i.p.) on days 2 and 3, 30 min after silymarin administration.
Test groups (IV–VI) were administered orally with 100, 200, and 400 mg/kg TFs, respectively, for 5 days. The three test groups received CCl4–olive oil (1 : 1, 2 mL/kg, i.p.) on days 2 and 3, 30 min after TFs administration.
The mice were killed after the 24 h treatment. Blood was collected via heart puncture and serum was separated for examination of various biochemical parameters. The liver was carefully dissected and cleaned of extraneous tissue. A portion of the liver tissue was immediately transferred into 10% formalin for histopathologic investigation. Levels of biochemical parameters ALT and AST were measured and compared with silymarin control .
2.7. General Experimental Procedures for Isolation and Structural Identification
Column chromatography was carried out using Merck Silica gel normal-phase (230–240 mesh) and reversed-phase C18 (Merck). Analytical TLC was carried out in silica gel plates (Merck DC-Alufolien 60 F254). Compounds were visualized by spraying with 10% H2SO4 in EtOH and heating for 3–5 min.
The high-resolution electrospray ionization mass spectra (HR-ESI-MS) were acquired on a Bruker MicrOTOF-QII spectrometer. The 1H-NMR (500 MHz), 13C-NMR (125 MHz), DEPT, COSY, HSQC, and HMBC spectra were recorded on a Bruker AM500 FT-NMR spectrometer using tetramethylsilane (TMS) as an internal standard.
2.8. Isolation of Pure Compounds
CVSM extract (330 g) was subjected to silica gel column chromatography and eluted with gradient solvent systems of chloroform and methanol (95 : 5 ⟶ 5 : 95, v/v) to collect six fractions: M1 (20 g), M2 (32 g), M3 (90 g), M4 (80 g), M5 (47 g), and M6 (62 g). The fraction M4 (80 g) was chromatographed on silica gel and eluted with CHCl3–MeOH (6 : 1⟶3 : 1, v/v) to give four subfractions (M4.1–M4.4). The subfraction M4.2 (18 g) was separated and further purified by RP-18 with MeOH–H2O (4 : 1, v/v) to deliver compounds 1 (150 mg), 3 (2 g), and 4 (250 mg). The fraction M5 (5 g) was applied on a silica gel chromatographic column and eluted with CHCl3–MeOH (2 : 1, v/v) to yield compound 2 (50 mg).
Similarly, CCSM extract (420 g) was subjected to silica gel column chromatography and eluted with CHCl3–MeOH (95 : 5–5 : 95, v/v) to get seven fractions: M1 (15 g), M2 (25 g), M3 (20 g), M4 (18 g), M5 (15 g), M6 (11 g), and M7 (52 g). The fraction M3 (20 g) was eluted with CHCl3–MeOH–H2O (5 : 1:0.1, v/v/v) by silica gel column chromatography to receive compound 8 (30 mg). The fraction M4 (18 mg) was loaded on silica gel column chromatography using CHCl3–MeOH–H2O (4 : 1:0.1, v/v/v) and furnished compound 5 (70 mg). The fraction M5 (15 g) was separated on a silica gel column with CHCl3–MeOH–H2O (3 : 1:0.1, v/v/v) to obtain compound 6 (45 mg). The fraction M6 (11 g) was eluted with CHCl3–MeOH–H2O (2 : 1:0.1, v/v/v) on a silica gel column chromatography to yield compound 7 (18 mg).
3. Results and Discussion
3.1. Protective Activity of Extracts against CCl4-Induced Hepatoxicity in HepG2 Cells
The ethyl acetate extract of C. chelidonii stems mostly increased cell viability. Particularly, after 24 h of treatment, it increased 25% and 26% at the concentrations of 50 and 100 μg/mL, respectively; after 48 h of treatment, at 50 μg/mL, it increased 25% and 50%, respectively; after 72 h of treatment, at 100 μg/mL, it increased 26% and 60%, respectively.
The n-hexane and methanol extracts of C. chelidonii stems, after 72 h of treatment, at 100 μg/mL, approximately increased 21.4%, while the n-hexane and methanol extracts of C. chelidonii leaves, after 72 h of treatment at 100 μg/mL, increased 26.9% and 30%, respectively. Meanwhile, the ethyl acetate and methanol extracts of C. viscosa leaves and the n-hexane extract of C. viscosa stems, after 72 h treatment, at 100 μg/mL, increased from 20% to 30% cell viability.
The results show that the methanol extracts of the stems of C. chelidonii and C. viscosa significantly revealed in vitro hepatoprotective activity. Thus, these ones were further examined for in vivo hepatoprotection against CCl4-induced liver toxicity in mice.
3.2. Hepatic Protective Effect of Extracts against CCl4-induced Liver Injury in Mice
At the doses of 30 mg/kg and 45 mg/kg, the methanol extracts of the stems of C. chelidonii and C. viscosa (CCSM and CVSM) significantly decreased ALT and AST concentrations in comparison with untreated extracts and the hepatic protection of these extracts was comparable to that of silymarin.
This result warranted the CCSM and CVSM extracts to be further investigated on phytochemical components.
3.3. Phytochemical Components of the Most Hepatic Protective Effect Extracts
The most in vitro and in vivo liver protection extracts of two species stems (CCSM and CVSM) were subjected to silica gel normal-phase and reversed-phase RP-18 chromatography to give eight known flavonoids (1–8) whose structures were confirmed by HR-ESI-MS, NMR experiments, and comparisons with the published data: visconoside A (1), visconoside B (2) , quercetin 3-O-β-D-glucopyranoside 7-O-α-L-rhamnopyranoside (3), kaempferol 3-O-β-D-glucopyranoside 7-O-α-L-rhamnopyranoside (4) , cleomeside A (5), cleomeside B (6) , cleomeside C (7), and quercetin-3-O-[β-D-glucopyranosyl-(1⟶2)]-α-L-rhamnopyranoside 7-O-α-L-rhamnopyranoside (8)  (Figure 2).
The phytochemical study confirmed that flavonoids are the main components of two species, which might be representative of their hepatoprotective effect. Therefore, the hepatoprotections of flavonoids (1–8) were screened using HepG2 cell line.
3.4. Cytotoxicity and Hepatoprotective Activity of Purified Compounds
At tested concentrations, samples did not show cytotoxicity, except compounds 3, 5, 6, and 8 at 25 μM (cell viability decreased, ranging from 25.0% to 30.0%).
At the concentration of 100 μM, compounds 1 and 4 significantly showed hepatoprotective effect (with prevention percentages of 66.5% and 74.2%, respectively), whereas compounds 5 and 8 disclosed weaker activity (with prevention percentages of 32.3% and 34.3%, respectively, compared to that of 80.3% of quercetin positive control).
The hepatoprotective effects of compounds 1 and 4 were tested for the first time.
In vitro and in vivo hepatoprotections using HepG2 and in mice of C. chelidonii and C. viscosa stems and their phytochemical constituents were investigated for the first time. The phytochemical study evidenced that flavonoids are the main compounds of two species. Furthermore, the hepatoprotections of visconoside A (1) and kaempferol 3-O-β-D-glucopyranoside 7-O-α-L-rhamnopyranoside (4) were identified for the first time. However, further clinical examinations are required to determine the molecular mechanisms of hepatoprotection as well as qualitative and quantitative identification of main biological flavonoid markers (1, 4, and 6) from these species.
The present study suggests that C. chelidonii and C. viscosa plants are good sources of natural hepatoprotective agents and contribute to understanding the biological activities of Cleome species in traditional Vietnamese medicine.
All the data related to the study are available from the corresponding author and can be provided upon request.
Conflicts of Interest
The authors declare that there are no conflicts of interest regarding the publication of this paper.
This work was partially supported by the Vietnam Academy of Science and Technology, Project no. UDSXTN.03/19–20, and Binh Phu Pharma Ltd.
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