Anti-hepatitis B virus activities of triterpenoid saponin compound from Potentilla anserine L.

Introduction

Hepatitis B virus (HBV) is a causative agent of both acute and chronic hepatitis, a major etiologic factor of primary hepatocellular carcinoma, and a major global health problem with over 350 million estimated chronically infected individuals (Maddrey 2001). Although there are several promising antiviral agents currently in clinical trials, substantial problems have been encountered in clinical treatments trials of chronic HBV infection (Raj 2001). Interferon trials has been shown to be effective against HBV, but was effective in achieving HBeAg seroconversion only in about 40% of the patients, and severe side-effects were also noted (Niederau et al. 1996). Toxicity is also usually associated with nucleoside analog therapy, although this problem has been apparently minimized in some recent clinical trials. HBV viremia relapse has been noted in patients shortly after the cessation of lamivudine (3TC) (Fig. 1) therapy (Honkoop et al. 1995). Using 3TC for long-term treatment may result in the emergence of 3TC-resistant mutants (Honkoop et al. 1997). Because there is no satisfactory therapeutic strategy available, it is necessary to find alternative anti-HBV agents with less side-effects.

[FIGURE 1 OMITTED]

Traditional Chinese medicinal herbs have been widely used to treat chronic liver diseases in China, and several controlled trials have been done to assess the efficacy and safety of traditional Chinese herbs for chronic hepatitis B infection. The results are promising. Potentilla anserina L. is distributed widely in the western areas of China, especially in Tibet. For thousands of years, it has been used frequently as a crude substance, taken orally as folk medicine in HBV treatment (Qiao et al.1992). It is documented in the Chinese Tibetan Materia Medica that rhizomes of Potentilla anserina L. contain carbohydrates, protein, fat, tannin, flavonoids, Iycine, histidine, etc. (Chen et al. 2000).

We isolated a triterpenoid saponin (TS) (Fig. I) from Potentilla anserina L. and report an ursane-type from Potentilla anserina in this paper. Also, we evaluated the cytotoxity and inhibitory activity of this compound on HBV in vitro, as well as its effect on DHBV DNA replication in vivo. Our results suggest that the structure of this compound derived from Potentilla anserina L. offered an important foundation for the future development of a new drug.

Materials and methods

Plant material

The dried rhizomes of Potentilla anserina L. were collected in the Tibet province of China in October of 2000 and were identified by Professor Zhu Shihua, a taxonomist at Lanzhou Medical University. A voucher specimen (no. P 525) was deposited in the Institute of Traditional Chinese Material Medica, People's Liberation Army.

Extraction and isolation

Dried rhizomes (5 kg) of Potentilla anserina L. were cut into pieces and extracted three times with EtOH for 6h at room temperature. The extract was dissolved in [dH.sub.2 O], purified on macroporous resin, successively eluted with gradient [EtOH-H.sub.2.O] for 6 h at room temperature. The extract was dissolved in [dH.sub.2 O], purified on macroporous resin, successively eluted with gradient [EtOH-H.sub.2 O] mixture solvents to yield three fractions (A-C). Each fraction was assayed for anti-HBV activity and the results showed that fraction B having anti-HBV activity. Fraction B was evaporated and dried under reduced pressure, and purified again with MeOH to yield 13g yellow powder. The powder was further separated using silica gel column chromatography, eluted with gradient [CH.sub.2] [Cl.sub.2]-MeOH mixture solvents. Compounds 1, 2 and 3 were obtained in pure form by re-chromatography on silica gel followed by re-crystallization. After bioassay-guided tests, compound 2 was determined to have antiviral effect and 155mg of compound 2 were isolated.

The melting point (mp) of compound 2, as an amorphous powder, was 231.7 [degrees]C. The IR spectrum showed the presence of hydroxyl groups (3423 [cm sup.-1], ester carbonyl (1732[cm.sup.-1]) and double bond (1632 [cm.sup.-1]). The characteristic structure of triterpenoid saponin was revealed by positive Liebermann-Burchared and Molish tests.

The FAB mass spectrum showed a peak at m/z 651 [[M + H].sup.+], the EI mass spectrum exhibited [[M].sup.+] at m/z 488. In addition, the characteristic fragment peaks at m/z 264 [RDA], 246 [[264-[H.sub.2] O].sup.+], 201 [[264-H .sub.2] O-COOH].sup.+] and 189, suggested that compound 2 was an urs-12-en-28-oic acid derivative with one free hydroxyl function in ring D or E. Elemental analysis showed that its molecular formula was [C sup.36] [H sup.580] [O sup.10] The ethylenic carbon signals at (delta) 126.9 and at 138.2 were attributed to C-12 and C-13, respectively, which indicated the presence of an urs-12-en-skeleton. Acid hydrolysis of compound 2 afforded D-glucose. Comparison of the (13)CNMR and DEPT spectral data showed six tertiary methyl and one secondary methyl (Table 1). This was further confirmed in 'H NMR [a doublet signal at (delta) 0.84 (3H, J= 9.0Hz, H-30)]. The [sup.1 H] NMR spectrum showed a anomeric proton of glucose at (delta) 5.15(1H, d, J = 8.4 Hz), indicative of the linkage of glucosyl moiety with the aglycone in the [beta]-configuration (Table .2). Consequently the structure of compound 2 was established as 2[alpha], 3[beta],19[alpha]-trihydroxyurs-12-en-28-oic acid beta)-D-glucopyranosyl ester, TS (Fig. 1).

Table 1. [.sup.13] C NMR spectral data of compound 2 in DMSO-[d sub 6]

C        2                C         2

1        47.1  (t)        16        25.1  (t)
2        67.1  (d)        17        47.3  (s)
3        82.3  (d)        18        53.2  (d)
4        40.0  (s)        19        71.7  (s)
5        54.8  (d)        20        41.2  (d)
6        18.1  (t)        21        26.4  (t)
7        33.5  (t)        22        36.6  (t)
8        37.5  (s)        23        28.8  (q)
9        46.7  (d)        24        16.4  (q)
10       38.9  (s)        25        17.1  (q)
11       23.2  (t)        26        16.4  (q)
12       126.9 (d)        27        23.8  (q)
13       138.2 (s)        28        175.5 (s)
14       41.1  (s)        29        26.7  (q)
15       28.0  (t)        30        16.2  (q)
1'       94.1  (d)
2'       72.2  (d)
3'       76.7  (d)
4'       69.5  (d)
5'       77.6  (d)
6'       60.6  (t)

Cell culture

The 2.2.15 cell line, a clonal derivative from human hepatocellular carcinoma cell line transfected with a plasmid containing multiple tandem copies of the HBV genome, was maintained in DMEM culture media containing 10% fetal bovine serum (FBS) and antibiotics (100% units/ml of penicillin G, 100 [mu]g/ml of streptomycin) at 37 [degrees]C in an atmosphere of 5% [CO sup.2] and 100% humidity. Cells were routinely checked for resistance to G418 (Korba and Gerin 1992).

Table 2. [.sup.1]H NMR spectral data of compound 2 in DMSO-d[sub.6]

H             2

1.            1.78, 0.75 (m)
2             3.40 (m)
3             2.72 (d, J =9.6 Hz)
5             0.73 (m)
9             1.60 (br, t)
11            1.88 (2H)
12            5.16
18            2.36 (s)
20            1.21 (m)
23            0.91 (3H, s)
24            0.89 (3H, s)
25            0.70 (3H, s)
26            0.66 (3H, s)
27            1.27 (3H, s)
29            1.08 (3H, s)
30            0.84 (3H, d, J = 9.0 Hz)
1'            5.15 (d, J = 8.4 Hz)
2'            3.06 (m)
3'            3.17 (m)
4'            3.08 (m)
5'            3.11 (m)
6'            3.58 (dd, 11.2, 2.5 Hz)
              3.43 (dd, 11.2, 6.0 Hz)

Toxicity measurements

Cells were grown to confluence in 96-well flat bottomed tissue culture plates with 3 x [10 sup.4] cells per well and treated with TS. Eight concentrations of TS ranging from 6.25 to 800 [mu]g/ml were added, each in triplicates cultures in two-fold steps. Four days after the final addition of compound, cultured medium was removed and TS was added for addition 4 days. MTT solution (10 [mu]l per 100 [mu]1 medium) was added to all wells of an assay. Then plates were incubated at 37 [degrees]C for 4h. Acidisopropanol (100 [mu]1 of 0.04 N HCI in isopropanol) was added to all wells and mixed thoroughly to dissolve the dark blue crystals. After a few minutes at room temperature to ensure that all crystals were dissolved, the plates were read with a Microelisa reader at a wavelength of 490 nm (Tim 1983).

HBsAg, HBeAg and HBVDNA assay in cell culture

For the antigen expression and anti-HBV activity assay, 2.2.15 cells were seeded into 24 well tissue culture plates at 4 x [10.sup.5] and grew to confluence. The culture medium was changed 24h prior to exposure to test compound. During the treatment, the culture medium was removed and test compound was added to the cultures in fresh culture medium at 4-day intervals at the final concentrations of 40, 20, 10, 5 and 2.5 [mu]g/ml, respectively. After 8 days of treatment, culture medium was collected and HBsAg and HBeAg were determined by radiommunoassay, according to the manufacturer's protocol. The percentage of HBsAg and HBeAg present in the treated medium was calculated by radioimmunoassay, according to the manufacturer's protocol. The percentage of HBsAg and HBeAg present in the treated medium was calcultated by comparing the cpm value of TS-treated medium to that of DMSO treated medium 1% (v/v) (Kruining et al. 1995).

[FIGURE 2 OMITTED]

After treatment with TS and TC, 2.2.15 cells were collected by centrifugation and intracellular HBV DNA was isolated as described by Korba and Gerin (1992). DNA was analyzed using a dot blot hybridization technique, using a [.sup.32] P-labeled EcoR I HBV DNA fragment (Korba and Milman 1991), and quantitated by comparison with HBV standards on a nitrocellulose filter.

Detection of DHBV DNA in Peking ducklings

The Peking ducklings carrying DHBV used in this study were divided randomly into five groups. Onegroup served as placebo control. TS was administered at 0.05, 0.1 and 0.2g/kg body wt/day, respectively, and was given orally in a liquid diet. 3TC (0.1 g/kg body wt/day) was used as positive control.

Serum samples were taken for analysis 1 week prior to the initiation of treatment, on the 1st day ([T sub.0]) through the 5th day ([T.sub.5]) and on the 10th day ([T.sub.10]) of treatment and the 3rd day following the end of treatment ([P.sub.3]). The general health of the Peking ducklings was assessed by daily observation at the time they received food and water, at the time of drug (or placebo) administration. DHBV DNA extracted from the serum sample was studied by dot blot hybridization technique (Cova and Zoulim 2004).

[FIGURE 3 OMITTED]

Statistical analysis

All treatments were conducted in triplicates or more times and the results were represented by their mean [+ or -] standard error (S.E.M.). The statistical significance of all treatment effects was evaluated by the Student's (-test at a probability limit of p<0.05.

Results and discussion

Effect of TS on 2.2.15 cell viability

The effects of TS on 2.2.15 cell viability were determined by a MTT assay. The results showed that TS did not exhibit cytotoxicity at concentrations lower than 50ug/ml as compared with DMSO-treated control. The concentrations required to cause a reduction in viability of 50% for TS and 3TC were 236.19 and 88.70ug/ml, respectively.

Effect of TS on HBsAg, HBeAg and HBV DNA expression in 2.2.15 cell culture

The results showed that TS could inhibit the expression of HBsAg with 38.46%, 25.33%, 20.12%, 15.26% and 10.29% at the concentrations of 40, 20, 10, 5 and 2.5ug/ml, respectively. At the same concentration, it could inhibit the secretion of HBeAg in 2.2.15 cell culture with inhibitory rates of 56.12%, 41.28%, 33.66%, 29.47% and 16.56%, respectively.

In the DNA assay by dot blot hybridization, the inhibitory rates of TS on HBV DNA replication in 2.2.15 cell culture were 59.29%, 50.33%, 42.16%, 36.71% and 29.83% at the concentrations of 40, 20, 10, 5 and 2.5ug/ml, whereas those viral markers were affected by 3TC with an inhibitory rate of 68.97% at 20ug/ml. But no inhibitory effect on expression of HBsAg and HBeAg secretion was seen in cells treated with 3TC. The results showed that TS exhibited anti-HBV activity against secretion of HBsAg [(IC.sub.50] value = 57.67 ug/ml, TI = 4.10), HBeAg [(IC.sub.50] value = 30.05ug/ml, TI = 7.86) and HBV [(IC.sub.50] value = 19.45ug/ml, TI = 12.14) in vitro (Fig.3).

Inhibition of TS on DHBV DNA in DHBV transfected Peking ducklings

The results from the DHBV DNA test in vivo showed that DHBV DNA replication was suppressed after treatment with TS. Five days of treatment with TS at the concentration of 0.2 and 0.1 g/kg/body wt could inhibit DHBV DNA at 30.30% and 22.16% vs. controls, and similar treatment with 3TC showed 47.96% inhibition. Viremia remained depressed 10 days following the treatment with both TS and 3TC. Thelevels of DHBV DNA in the Peking ducklings treated with TS (0.2g/kg body wt) for 10 days were markedly reduced, by 58.48%. Although 3TC reduced the DHBV DNA at 70.92% after treatment for 10 days, the levels of viremia rebounded quickly in the third day after treatment. Following treatment with TS, viremia showed dose-dependent suppression with slower rebound after treatment withdrawal (Table 3). Therefore, our results indicated that TS had a long-term effect of inhibiting hepatitis B virus.

Table 3. Inhibitory effects of triterpenoid saponin (TS) on
the level of DHBV DNA in duckling serum; x[+ or -]s

Agent         Dose (g/kg  Number of   Days of treatment/post-treatment
              body wt)    animals

                                     [T.sub.o]

DHBV control              8          1.482 [+ or -] 0.104
3TC           0.1         8          1.403 [+ or -] 0.120
TS            0.2         8          1.420 [+ or -] 0.102
TS            0.1         8          1.399 [+ or -] 0.182
TS            0.05        8          1.416 [+ or -] 0.183

Agent          Days of treatment/post-treatment

              [T.sub.5]                  [T.sub.10]

DHBV control  1.5344 [+ or -] 0.110 (a)  1.566 [+ or -] 0.105 (a)
3TC           0.730 [+ or -] 0.167 (c)   0.408 [+ or -] 0.106 (c)
TS            0.722 [+ or -] 0.080 (c)   0.428 [+ or -] 0.105 (c)
TS            0.946 [+ or -] 0.05l (c)   0.732 [+ or -] 0.088 (c)
TS            1.127 [+ or -] 0.098 (b)   0.906 [+ or -] 0.091 (c)

Agent         Days of treatment/post-treatment

              [P.sub.3]

DHBV control  1.552 [+ or -] 0.116 (a)
3TC           0.945 [+ or -] 0.147 (c)
TS            0.590 [+ or -] 0.086 (c)
TS            0.817 [+ or -] 0.077 (c)
TS            1.013 [+ or -] 0.055 (b)

Discussion

HBV is a major health problem in the world and a common cause of cirrhosis and hepatocellular carcinoma. It is becoming a more prevalent clinical problem, especially in HBV-endemic areas (Hanazaki 2004). In China, about 170 million people are infected chronically with HBV and 10% suffer from chronically with HBV and 10% suffer from chronic hepatitis. About half a million Chinese die from hepatitis B caused hepatocellular carcinoma and end-stage cirrhosis each year (Sun et al. 2002).

In spite of the existence of a preventative vaccine, Interferon, and synthetic nucleoside analogs, each agent has inherent limitations for use in the treatment of chronic hepatitis B (Karayiannis 2003). Interferon alfa is effective in a small number of patients and has serious side-effects that limit its tolerability. The efficacy of lamivudine is limited by the emergence of drug-resistant HBV mutants, restricting its use as a long-term therapy for chronic hepatitis B. As a result, a large proportion of chronic hepatitis B patients continue to be in need of a safer and more effective therapy (Marcellin 2002), and research leading to the development of new drugs is warranted.

China is well known for a variety of plants which are used as plant-based medicines to treat human diseases. Potentilla anserina L. is a kind of herb cultivated in the west of China and is commonly used for the HBV treatment and prevention. In this study, 2[alpha], 3[beta], 19[alpha]-trihydroxyurs-12-en-28-oic acid [beta]-D-glucopyranesyl ester, a compound named as triterpenoid saponin (TS) was isolated from Potentilla anserina L. following bioassay-guided fractionation. Using the 2.2.15 cell line in vitro and the Peking duckling model in vivo, we showed that TS has a dose-dependent inhibitory effect on hepatitis B virus, with a related long-term effect. The antiviral activity of some triterpenoic acids was also reported by Baglin et al. (2003).

2.2.15, as a human cell line transfected with cloned HBV DNA, could model all essential virologic features of chronic HBV infection, including: (1) a stable pattern of integrated HBV DNA; (2) the presence of high levels of replicating virus; (3) the appropriate sites and patterns of viral-specific RNA transcripts and proteins and (4) the release of high titers of infectious HBV virions. This cell line can be used for the primary evaluation of compounds for activity against HBV (Brent 1991).

In the absence of animal models for HBV, the DHBV-carrying ducks turned out to be a fruitful animal model. Therefore, data concerning the early, post-attachment steps in virus entry are based largely on studies performed with DHBV in primary duck liver hepatocytes. These studies are shedding light on the mechanisms of hepatitis B virus cell entry and will provide some new insights into the molecular basis of the strict species specificity of hepatitis B virus infection (Hantz and Zoulim 2004).

The positive control in this study was lamivudine (3TC; Fig. 2) ([C.sub.8][H sub.11][N.sub.3][O.sub.3]S, MW: 229.25), which has been widely used in clinical HBV treatment, although resistance usually develops with long-term use (Staschke and Colacino 2001; Gumina et al. 2002; Schmilovitz-Weiss et al. 2003; Kann 2004; Ozgenc et al. 2004). With inhibitory activities against HBV DNA replications in vitro and in vivo, 3TC is usually used as positive control in anti-HBV research with the effect of inhibiting hepadnavirus replication in 2.2.15 cells and in ducks chronically infected with the duck hepatitis B virus (Ketzinel-Gilad et al. 2006; Li et al. 2005; Weber et al. 2002; Marion et al. 2002; Shuangsuo et al. 2006).

In summary, based on the evaluation for HBV antigen expression and anti-hepatitis B virus (HBV) activities in vitro and HBV DNA replication inhibitory effect in vivo, we showed that the triterpenoid saponin (TS) could not only inhibit the replication of HBV DNA but also restrain the expression of antigen, indicating that TS could be a candidate therapeutic agent for HBV treatment. We are now focusing on studying the virusinhibitory mechanism and pharmacokinetics of TS.

Acknowledgments

This work was supported by grants from the National "Ninety-Five" Scientific Key Project (96-C02-04-02) and Beijing Capital Key Foundation (2003047).

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Y -L Zhao (a), (b),*,G -M.Cai (a),X.Hong (a),L -M.Shan (a),X -H.Xiao (a)

(a) Institue of Traditional Chinese Material Medica, People's Liberation Army, Beijing 100039, PR China

(b) Chinese Academy of Military Medical Sciences, Beijing 100039, PR China

* Corresponding author at:Institute of Traditional Chinese Material Medica, People's Liberation Army, Beijing 100039,PR China.

Tel :[phone](mobile).

E-mail address:[e-mail](Y -L.Zhao)