Hepatoprotection and Phytochemistry of the Vietnamese Herbs Cleome chelidonii and Cleome viscosa Stems

Faculty of Chemistry, Graduate University of Science and Technology, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet Cau Giay, Hanoi, Vietnam Natural Products Laboratory, Institute of Chemical Technology, Vietnam Academy of Science and Technology, 01A (anh Loc 29 (anh Loc District 12, Ho Chi Minh City, Vietnam Faculty of Pharmacy, University of Medicine and Pharmacy, 41 Dinh Tien Hoang District 1, Ho Chi Minh City, Vietnam Saigon Pharmaceutical Sciences & Technologies Center, University of Medicine and Pharmacy, 41 Dinh Tien Hoang District 1, Ho Chi Minh City, Vietnam Can (o University, 3/2 Street Ninh Kieu District, Can (o City, Vietnam University of the Sunshine Coast, 90 Sippy Downs Drive, Sippy Downs 4556, Queensland, Australia Ho Chi Minh City Institute of Physics, Vietnam Academy of Science and Technology, 01 Mac Dinh Chi District 1, Ho Chi Minh City, Vietnam Department of Science, Dong Nai University, Bien Hoa City, Dong Nai, Vietnam Bioactive Compounds Laboratory, Institute of Chemical Technology, Vietnam Academy of Science and Technology, 01A (anh Loc 29 (anh Loc District 12, Ho Chi Minh City, Vietnam

In vivo study on rats against CCl 4 -induced liver injury indicated that hydroalcohol, methanol, ethyl acetate, and hexane extracts of C. chelidonii root revealed hepatoprotective activity [28]. 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 [24]. In vivo study on ethanol extract of C. viscosa's whole plant against CCl 4 -induced hepatotoxicity [29], as well as methanol extract against streptozotocin-(STZ-) induced diabetic rats [30], 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 [35] and the Cleome genus [36][37][38][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 CCl 4 -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.  (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. e supernatants were filtered, and the solvents were removed under vacuum to obtain crude extracts CCS (970 g) and CVS (770 g), respectively. ose extracts were subjected to solid-phase separation and successively fractionated into nhexane, 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.

Study on Hepatic Protective Effect in Mice Acute Liver
Injury Induced by CCl 4 . Mice were divided into six groups with six animals in each group.
Test groups (IV-VI) were administered orally with 100, 200, and 400 mg/kg TFs, respectively, for 5 days. e three test groups received CCl 4 -olive oil (1 : 1, 2 mL/kg, i.p.) on days 2 and 3, 30 min after TFs administration. e mice were killed after the 24 h treatment. Blood was collected via heart puncture and serum was separated for examination of various biochemical parameters. e 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 [15].

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 C 18 (Merck). Analytical TLC was carried out in silica gel plates (Merck DC-Alufolien 60 F 254 ). Compounds were visualized by spraying with 10% H 2 SO 4 in EtOH and heating for 3-5 min.

Protective Activity of Extracts against CCl 4 -Induced Hepatoxicity in HepG2 Cells.
e in vitro cytotoxic and hepatoprotective effects of extracts were shown in Tables 1  and 2.
e 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. e 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 nhexane extract of C. viscosa stems, after 72 h treatment, at 100 μg/mL, increased from 20% to 30% cell viability.
e results show that the methanol extracts of the stems of C. chelidonii and C. viscosa significantly revealed in vitro hepatoprotective activity.
us, these ones were further examined for in vivo hepatoprotection against CCl 4 -induced liver toxicity in mice.

Hepatic Protective Effect of Extracts against CCl4-induced Liver Injury in Mice.
e in vivo hepatoprotective effects of methanolic extracts of the stems of C. chelidonii and C. viscosa (Table 3 and Figure 1) were tested against CCl 4induced toxicity of liver 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 ALTand ASTconcentrations in comparison with untreated extracts and the hepatic protection of these extracts was comparable to that of silymarin.
is result warranted the CCSM and CVSM extracts to be further investigated on phytochemical components.

Phytochemical Components of the Most Hepatic Protective Effect Extracts.
e 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)(2)(3)(4)(5)(6)(7)(8) (Figure 2). e phytochemical study confirmed that flavonoids are the main components of two species, which might be representative of their hepatoprotective effect. erefore, the hepatoprotections of flavonoids (1-8) were screened using HepG2 cell line.

Cytotoxicity and Hepatoprotective Activity of Purified
Compounds.
e cytotoxicity (Table 4) and hepatoprotection      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). e hepatoprotective effects of compounds 1 and 4 were tested for the first time.

Conclusions
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. e phytochemical study evidenced that flavonoids are the main compounds of two species. Furthermore, the hepatoprotections of visconoside A (1) and kaempferol 3-Oβ-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. e 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.

Data Availability
All the data related to the study are available from the corresponding author and can be provided upon request.

Conflicts of Interest
e authors declare that there are no conflicts of interest regarding the publication of this paper.

Acknowledgments
is work was partially supported by the Vietnam Academy of Science and Technology, Project no. UDSXTN.03/19-20, and Binh Phu Pharma Ltd.