Journal of Natural Medicines

, Volume 65, Issue 2, pp 370–374

Antifibrotic activity of coumarins from Cnidium monnieri fruits in HSC-T6 hepatic stellate cells

Authors

  • Eunjin Shin
    • College of PharmacyChungbuk National University
  • Chul Lee
    • College of PharmacyChungbuk National University
  • Sang Hyun Sung
    • College of Pharmacy and Research Institute of Pharmaceutical ScienceSeoul National University
  • Young Choong Kim
    • College of Pharmacy and Research Institute of Pharmaceutical ScienceSeoul National University
  • Bang Yeon Hwang
    • College of PharmacyChungbuk National University
    • College of PharmacyChungbuk National University
Note

DOI: 10.1007/s11418-010-0485-7

Cite this article as:
Shin, E., Lee, C., Sung, S.H. et al. J Nat Med (2011) 65: 370. doi:10.1007/s11418-010-0485-7

Abstract

The CHCl3 fraction of Cnidium monnieri fruits significantly inhibited the proliferation of hepatic stellate cells in an in-vitro assay system employing HSC-T6 hepatic stellate cell lines. Activity-guided fractionation of the CHCl3 fraction of C. monnieri led to the isolation of ten coumarins: osthol (1), meranzin (2), auraptenol (3), meranzin hydrate (4), 7-hydroxy-8-methoxy coumarin (5), imperatorin (6), xanthotoxol (7), xanthotoxin (8), bergapten (9) and isopimpinellin (10). Of these, compounds 1 and 6 significantly inhibited proliferation of HSCs in a time- and concentration-dependent manner. In addition, compounds 1 and 6 significantly reduced collagen content in HSC-T6 cells.

Keywords

Cnidium monnieriCoumarinHSC-T6Hepatic stellate cellsOstholImperatorin

Introduction

Liver injury and consequent hepatic fibrogenesis may be caused by toxic, infectious or metabolic agents. An early event in the development of hepatic fibrosis is the activation of hepatic stellate cells (HSCs), which play important roles in normal liver, such as retinoid storage, remodeling of extracellular matrix (ECM) and production of growth factors. During fibrosis, HSCs undergo a complex activation process characterized by increased proliferation and ECM deposition, which is the major pathological feature of hepatic cirrhosis [1, 2].Antifibrotic agents targeting HSC activation have therefore been proposed as a therapeutic target against hepatic fibrosis [3, 4].

Natural products have been used in the treatment of various diseases for a long time, especially in Asian countries. In relation to liver fibrosis, a diverse array of natural products including flavonoids, alkaloids and terpenoids have been suggested to have antifibrotic activity [58].There is therefore a growing interest in searching for antifibrotic compounds from natural products with lower adverse effects.

Cnidium monnieri Cusson (Umbelliferae) is a leafy annual with flowers in clusters. The dried fruits of C. monnieri have been widely used to treat skin problems such as acne and eczema. C. monnieri is often combined with other herbs to help infertility, impotence and osteoporosis [9, 10]. Coumarins are the major constituents of C. monnieri fruits and are responsible for the diverse effects such as anticancer and anti-inflammatory activity [11]. In the course of screening the antifibrotic activity of natural products by employing HSC-T6, a rat hepatic stellate cell line, as an in-vitro assay system, the CHCl3 fraction of C. monnieri showed significant inhibitory activity on HSC proliferation. Thus, in the present study, we have attempted to isolate the antifibrotic constituents from C. monnieri and evaluate their antifibrotic activity.

Results and discussion

Though fibrosis is a severe health problem, no effective antifibrogenic therapy is available for its treatment in chronic liver diseases. Recently, there has been a growing interest in searching for antifibrotic compounds, especially from natural products. We also searched for antifibrotic compounds from natural resources by employing HSC-T6, a rat hepatic stellate cell line, as an in-vitro assay system. In the course of screening, the methanolic extract of C. monnieri fruits significantly inhibited HSC proliferation. The methanolic extract was further fractionated into n-hexane, CHCl3, EtOAc and n-BuOH fractions. Among them, the CHCl3 fraction inhibited HSC viability up to 85.9% of control at a concentration of 100 µg/ml.Activity-guided fractionation of CHCl3 was therefore carried out for the isolation of active constituents. Further fractionation and separation of the CHCl3 fraction by various chromatographic methods yielded ten coumarins, which were identified as osthol (1), meranzin (2), auraptenol (3), meranzin hydrate (4), 7-hydroxy-8-methoxy coumarin (5), imperatorin (6), xanthotoxol (7), xanthotoxin (8), bergapten (9) and isopimpinellin (10) (Fig. 1), by the direct comparison of their physicochemical and spectroscopic data with those previously reported [1216]. Among the compounds isolated, compounds 2 and 5 are reported from this plant for the first time.
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Fig. 1

Coumarins isolated from the fruits of C. monnieri

The inhibitory activity of the isolated compounds (110) was also evaluated in our assay system. Compound 1 showed the most potent inhibitory activity, followed by compound 6. At a concentration of 100 µM, compounds 1 and 6 inhibited cell viability up to 31 and 47% of control, respectively (Fig. 2), in a time- and concentration-dependent manner (Fig. 3). Compounds 5, 8, 9 and 10 also showed significant inhibitory activity, whereas compounds 2, 3, 4 and 7 showed little activity.
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Fig. 2

Effect of compounds 110 isolated from C. monnieri on cell viability of HSC-T6 cells. HSC-T6 cells were incubated with compounds (100 µM) for 48 h. Cell viability was measured by the MTT assay. The percent cell viability was calculated as 100 × (absorbance of compound-treated/absorbance of control). Results are expressed as mean ± SD of three independent experiments, each performed using triplicate wells. *p < 0.05, **p < 0.01, ***p < 0.001 compared with control

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Fig. 3

Concentration- and time-dependent effects of compounds 1 and 6 on cell viability in HSC-T6 cells. HSC-T6 cells were incubated with compounds 1 or 6 at concentrations ranging from 1.0 to 100 µM for 48 h (a), or 100 µM for the time indicated (b). Cell viability was measured by the MTT assay. The percent cell viability was calculated as 100 × (absorbance of compound-treated/absorbance of control). Results are expressed as mean ± SD of three independent experiments, each performed using triplicate wells. *p < 0.05, **p < 0.01, ***p < 0.001 compared with control

Suppression of HSC activation generally can be achieved by various pathways, such as inhibition of cell proliferation and/or induction of cell death. As shown in Fig. 4, morphological changes suggested that compounds 1 and 6 may not exert a direct toxic effect on HSCs. In addition, treatment with compounds 1 and 6 did not increase lactate dehydrogenase release in the culture medium (data not shown). Thus, we cautiously suggest that compounds 1 and 6 decreased cell numbers by the interference in cell proliferation, not by cytotoxicity.
https://static-content.springer.com/image/art%3A10.1007%2Fs11418-010-0485-7/MediaObjects/11418_2010_485_Fig4_HTML.jpg
Fig. 4

Effects of compounds 1 and 6 on cell morphology in HSC-T6 cells. HSC-T6 cells were incubated with 100 µM of compound 1 or 6 for 48 h. Cells were observed with phase contrast microscopy (original magnification ×100)

Excessive production and deposition of ECM such as collagen are another characteristic of HSC activation which is responsible for liver dysfunction [1]. Therefore, we investigated the effects of compounds 1 and 6 on collagen production in activated HSC-T6 cells using Sircol dye assay. As shown in Fig. 5, treatment with compounds 1 and 6 significantly reduced collagen content in HSC-T6 cells. At a concentration of 100 µM, compounds 1 and 6 decreased the collagen content up to 24.5 and 43.0%, respectively, compared to activated control.
https://static-content.springer.com/image/art%3A10.1007%2Fs11418-010-0485-7/MediaObjects/11418_2010_485_Fig5_HTML.gif
Fig. 5

Effects of compounds 1 and 6 on the collagen content of HSC-T6 cells. HSC-T6 cells were incubated with 100 µM of compound 1 or 6 for 48 h. Collagen content was measured by Sircol dye assay. Results are expressed as the mean ± SD of three independent experiments, each performed using triplicate wells. ***p < 0.001 compared with control

All ten compounds isolated from C. monnieri fruits are coumarin derivatives and can be divided into two subtypes. Compounds 15 have a simple coumarin skeleton whereas compounds 610 are furanocoumarin type. Compounds 14 have similar structures and only differ in the substitution in the C-8 position. However, only compound 1 which has a prenyl group at C-8 showed strong inhibitory activity. Compounds 24, which also have a prenyl derivative at C-8, but with different hydroxylation, showed little activity. In the case of furanocoumarin-type compounds (610), compound 6 with prenyl substitution showed the strongest activity. Compounds 710 have similar structures and only differ in the number of methoxyl groups. Compounds 810, which have one or two methoxyl groups, showed significant activity, whereas compound 7 which has only a hydroxyl group showed little activity. Therefore, we cautiously propose that prenyl substitution might be helpful for the inhibitory activity of coumarins on HSC proliferation; this needs to be clarified with more diverse derivatives. Consistent with our results, the prenyl group of osthol has been reported to be important in anticancer activity [17].

In summary, we isolated ten coumarin derivatives from C. monnieri and evaluated their antiproliferative activity using HSC-T6 cells. Among the ten coumarins isolated, compounds 1 and 6 showed significant inhibitory activity on HSC proliferation and collagen production. This will provide further insight into the design of new approaches to liver fibrosis.

Methods and materials

General experimental procedures

NMR spectra were recorded on a Bruker DRX 500 MHz NMR spectrometer using CDCl3 and CD3OD as solvents. EI-mass spectra were obtained on VG Autospec Ultima (Micromass, Manchester, UK) mass spectrometers. Semipreparative HPLC was performed using a Waters HPLC system equipped with Waters 600 Q-pumps, a 996 photodiode array detector, and Waters Empower software using a Xterra ODS-column (5 µm, 10 × 150 mm). Silica gel (70–230 mesh, Merck, Germany) and Sephadex LH-20 (25–100 µM, Amersham Biosciences, Sweden) were used for open column chromatography (CC). Thin-layer chromatography (TLC) was performed on a precoated silica gel 60 F254 (0.25 mm, Merck). All other chemicals and reagents were analytical grade

Plant material

The dried fruits of C. monnieri were purchased from the local herbal market in Chungbuk, Korea, in November 2008. A voucher specimen (CBNU-2008-CM) has been deposited in the Herbarium of the College of Pharmacy, Chungbuk National University.

Extraction and isolation

The fruits of C. monnieri (500 g) were extracted 3 times with 80% MeOH, which yielded the methanolic extract (18.9 g). This was then suspended in H2O and partitioned successively with n-hexane, CHCl3, EtOAc and n-BuOH. The CHCl3 fraction (7.8 g), which showed most potent activity, was subjected to silica gel column chromatography (CC) with n-hexane–EtOAc mixture to give 6 fractions (C1–C6). Compound 1 (1800 mg) was obtained from C3 by recrystallization using MeOH. C2 was subjected to CC over silica gel eluted with the n-hexane–EtOAc mixture to give 3 subfractions (C21–C23). Compounds 2 (11 mg), 4 (12 mg) and 5 (4 mg) were obtained from C22 by semi-preparative HPLC eluting with acetonitrile–water (55:45, F = 2 min/ml, Rt = 6.37, 5.91 and 5.02 min, respectively). Compound 3 (9 mg) was isolated from C23 by semipreparative HPLC eluting with acetonitrile–water (55:45, F = 2 min/ml, Rt = 4.48 min). After recrystallization of compound 6 (600 mg) from C4 with MeOH, the residual C4 was then subjected to CC over silica gel eluted with the n-hexane–EtOAc mixture, to yield 9 subfractions (C41–C49). Compound 7 (8 mg) was obtained from C42 by recrystalization using MeOH. C41 was subjected to CC over Sephadex LH-20 eluted with n-hexane–CH2Cl2–MeOH (5:5:1) mixture to afford 4 subfractions (C411–C414). Compounds 8 (29 mg) and 9 (21 mg) were isolated from C412 by semipreparative HPLC elution with acetonitrile–water (60:40, F = 2 min/ml, Rt = 6.55 and 7.48 min). Compound 10 (45 mg) was isolated from C411 by semipreparative HPLC elution with acetonitrile–water (60:40, F = 2 min/ml, Rt = 7.15 min).

Culture of HSC-T6 hepatic stellate cells

An immortalized rat hepatic stellate cell line, HSC-T6, was kindly provided by Prof. S.L. Freidman (Columbia University, New York, USA). HSC-T6 cells were maintained in DMEM supplemented with 10% heat-inactivated fetal bovine serum, 100 IU/ml penicillin and 100 µg/ml streptomycin at 37°C in a humidified atmosphere of 95% air–5% CO2.

Measurement of cell viability

HSC-T6 cells were treated with vehicle or samples to be tested for 48 h or as indicated. Cell viability was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay [5].

Measurement of collagen content

Collagen content in HSC-T6 cells were measured using a Sircol™ Collagen Assay kit (Biocolor Ltd., UK) according to the manufacturer’s protocol. Briefly, HSC-T6 cells were treated with vehicle or samples to be tested for 48 h. Cells were then washed with PBS and extracted with 0.5 M acetic acid. Sircol dye reagent was added to the cell extract and placed in a gentle shaker for 30 min to form a collagen–dye complex. After unbound dye had been removed by centrifugation and washing, the collagen–dye complex was dissolved in alkali reagent. The absorbance was measured at 550 nm in an ELISA plate reader.

Statistical analysis

The evaluation of statistical significance was determined by a one-way ANOVA test with p < 0.05 considered to be statistically significant.

Acknowledgments

We thank Prof. S.L. Freidman (Columbia University, New York) for the kindly gift of the HSC-T6 cells. This work was supported by the Medical Research Center Program (2010-0029480), the Regional Core Research Program (Chungbuk BIT Research-Oriented University Consortium) and Basic Science Research Program (2010-0025054) through the National Research Foundation of Korea (NRF).

Copyright information

© The Japanese Society of Pharmacognosy and Springer 2010