Evaluation of Hepatocyteprotective and Anti-hepatitis B Virus Properties of Cichoric Acid from Cichorium intybus Leaves in Cell Culture

Abstract

Hepatitis B is the most common serious liver infection in the world. To date, there is still no complete cure for chronic hepatitis B. Natural caffeic acid analogues possess prominent antiviral activity, especially anti-hepatitis B virus (HBV) and anti-human immunodeficiency virus effects. Cichoric acid is a caffeic acid derivative from Cichorium intybus. In the study, the anti-hepatitis B property of cichoric acid was evaluated by the D-galactosamine (D-GalN)-induced normal human HL-7702 hepatocyte injury model, the duck hepatitis B virus (DHBV)-infected duck fetal hepatocytes and the HBV-transfected cell line HepG2.2.15 cells, respectively. The results showed that cichoric acid attenuated significantly D-GalN-induced HL-7702 hepatocyte injury at 10–100 µg/mL and produced a maximum protection rate of 56.26%. Moreover, cichoric acid at 1–100 µg/mL inhibited markedly DHBV DNA replication in infected duck fetal hepatocytes. Also, cichoric acid at 10–100 µg/mL reduced significantly the hepatitis B surface and envelope antigen levels in HepG2.2.15 cells and produced the maximum inhibition rates of 79.94% and 76.41%, respectively. Meanwhile, test compound at 50–100 µg/mL inhibited markedly HBV DNA replication. In conclusion, this study verifies the anti-hepatitis B effect of cichoric acid from Cichorium intybus leaves. In addition, cichoric acid could be used to design the antiviral agents.

Hepatitis B virus (HBV) is one of the most common causes of chronic liver disease worldwide. Chronic HBV can cause necroinflammation and over time can cause hepatic fibrosis and eventually cirrhosis, end-stage liver disease, and hepatocellular carcinoma.¹⁾ Of the 350 million to 400 million individuals worldwide infected with the HBV, one-third resides in China, with 130 million carriers and 30 million chronically infected.²⁾ However, the current anti-hepatitis B drugs such as interferon-α and lamivudine have many drawbacks such as rebound phenomenon of HBV replication after withdrawal of treatment, drug resistance during the long-term use and some side effects etc.³⁾ Therefore, there is a pressing need to develop more safe and effective anti-hepatitis B agents. Cichoric acid (synonyms: chicoric acid, dicaffeoyltartaric acid), as a derivative of both caffeic acid and tartaric acid, occurs in a variety of plant species. Cichoric acid was first isolated from Cichorium intybus but also occurs in significant amounts in Echinacea, particularly E. purpurea, dandelion leaves, basil, lemon balm and in aquatic plants, including algae and sea grasses.^4,5) Cichorium intybus is widely used medicinally and has been investigated for its potent hepatoprotective activity.^6–9) Cichoric acid has been shown to possess phagocytosis stimulatory activity in vitro and in vivo, to inhibit hyaluronidase and to protect collagen type III from free radical induced degradation.¹⁰⁾ What is more important is that chicoric acid also exhibits significant antiviral effects against human immunodeficiency virus type 1 (HIV-1) and herpes simplex virus (HSV-I).^11–15)

It is well known that some classical anti-HIV drugs such as interferon-α and lamivudine often possess potent anti-HBV effect in clinical practice. Furthermore, coinfection with HBV and HIV is common due to shared routes of transmission, with 70–90% of HIV-infected individuals having evidence of past or active infection with HBV.¹⁶⁾ And that chronic HBV infection occurs in 5–10% of HIV-infected individuals who are exposed to HBV.¹⁷⁾ In addition, caffeic acid analogues exhibits a variety of bioactivities, especially anti-HBV and anti-HIV effects.^18,19) Therefore, we initiate a program aiming at validating the anti-hepatitis B activity of cichoric acid. In this study, the anti-hepatitis B property of cichoric acid from Cichorium intybus was evaluated by the D-galactosamine (D-GalN)-induced normal human HL-7702 hepatocyte damage model, the duck hepatitis B virus (DHBV)-infected duck fetal hepatocytes and the HBV-transfected HepG2.2.15 cells. Silymarin from milk thistle is a hepatoprotective drug that was widely used in the world. Silybin is the strongest active component of silymarin. Lamivudine, as a potent nucleoside analog reverse transcriptase inhibitor, is used worldwide for treatment of hepatitis B. Hence, Silybin and Lamivudine were employed as the reference drugs of hepatoprotective and anti-HBV tests, respectively. The study is first to verify the hepatocyteprotective and anti-hepatitis virus effects of natural cichoric acid. Hence, we report the details here.

MATERIALS AND METHODS

Materials

Cichoric acid (HPLC Purity ≥98%, Batch No. 13033016) from chicory (Cichorium intybus LLINN) leaves was purchased from Chengdu Biopurify Phytochemicals Ltd. (China). Its sample was deposited at Department of Pharmacy, College of Life Sciences, China Jiliang University. Its chemical structure was shown in Fig. 1. Fetal bovine serum, 1640 medium and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from Gibco-BRL (Grand Island, NY, U.S.A.). D-Galactosamine, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), insulin, hydrocortisone and silybin were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.). HBV DNA polymerase chain reaction (PCR)-fluorescence quantitation kit, enzyme immunoassay (EIA) kits for the detection of hepatitis B surface antigen (HBsAg) and hepatitis B envelope antigen (HBeAg) were provided from Shanghai Kehua Bio-engineering Co., Ltd. Lamivudine was provided from GlaxoSmithKline (China) Investment Co., Ltd. G418 was provided from Invitrogen Corporation (U.S.A.). Plasmid mini preparation kit was provided from Axygen Biosciences. SYBR® Premix Ex Taq™ II, Taq DNA polymerase, PMD-18T vector kit and DNA marker and conventional PCR reagents were provided from TaKaRa Biotechnology (Dalian) Co., Ltd. (China). Viral DNA kit was provided from Omega Bio-Tek, Inc. (U.S.A.). All other reagents were of the highest commercial grade available. Other reference drugs were dissolved in the final concentration of 0.1% dimethyl sulfoxide (DMSO) solution in all tests.

Fig. 1. Structure of Cichoric Acid from Cichorium intybus

Isolation and Primary Culture of Duck Fetal Hepatocytes

Primary duck fetal hepatocytes were isolated from 20-d old embryonated, un-hatched, duck eggs layed by the DHBV-infected ducks as described previously with some modifications.²⁰⁾ DHBV infection was confirmed by PCR assay on the viral DNA obtained from the duck fetal allantoic fluid using DHBV specific primers. The forward primer is 5′-AAC CAT TGA AGC AAT CAC TAG AC-3′ and the reverse primer is 5′-ATC TAT GGT GGC TGC TCG AAC TA-3′. The size of the target gene fragment is 218 bp (Fig. 2). The cells were maintained at 1.0×10⁵ cells/mL in 1640-medium containing 2 mM glutamine, 1 µg/mL insulin, 7.5 µg/mL hydrocortisone and 10% (v/v) fetal bovine serum at 37°C in a 5% CO₂ atmosphere (Fig. 3).

Fig. 2. PCR Amplification Product of DHBV DNA from Duck Fetal Allantoic Fluid

Lanes 1, 2: the PCR products; lane 3: the marker (molecular weight standard); target gene fragment 218 bp.

Fig. 3. Primary Duck Fetal Hepatocytes Isolated from 20-d Old Embryonated, Un-hatched, Duck Eggs Layed by the Duck Hepatitis B Virus-Infected Ducks

(A) 20-d old embryonated duck egg; (B) duck fetal hepatocytes.

Effect of Cichoric Acid on D-GalN-Induced Hepatocytes Injury

HL-7702 hepatocytes were obtained from Cell Bank, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences and maintained in 1640-medium containing 2 mM glutamine and 10% (v/v) fetal bovine serum at 37°C (95% humidity, 5% CO₂). The cytotoxicity induced by cichoric acid was analyzed with the MTT assay as follows: HL-7702 hepatocytes were incubated in 1640-medium with a concentration of 1.0×10⁵ cells/mL in the presence of 10–100 µg/mL cichoric acid for 48 h and then 10 µL of MTT (5 mg/mL) was added to cells in each well. After 4 h of culture, the medium was removed, and the blue formazan crystals that had formed were dissolved in DMSO. The absorbency of formazan generated from MTT was measured at 570 nm using a multi-well plate reader (MULTISKAN MK3, Thermo Fisher Scientific Inc., U.S.A.). Cytotoxicity of cichoric acid was described according to the amount of formazan production relative to those of untreated cells.

To evaluate hepatocyteprotective effect of cichoric acid, HL-7702 hepatocytes were then cultured for another 8 h with 70 mM D-GalN after the cells were incubated for 48 h in culture medium containing 10–100 µg/mL cichoric acid. Silybin at concentrations of 10–100 µg/mL was used as the reference drug. Hepatocyte injury was detected using the MTT assay as described above. Hepatocyteprotective effect of test compound was assessed by cell viability and expressed as percentage protection.

Anti-DHBV Effect of Cichoric Acid in Primary Duck Fetal Hepatocytes

Cytotoxicity induced by cichoric acid treatment was measured using the MTT assay as follows: The DHBV-infected duck fetal hepatocytes were cultured in 1640-medium with a concentration of 1.0×10⁵ cells/mL in the presence of 1–100 µg/mL cichoric acid for 6 d and then the MTT assay was carried out as described above. Cytotoxicity was reported according to the amount of formazan production relative to those obtained from untreated cells. For measurement of DHBV DNA, the infected duck fetal hepatocytes were treated with different concentrations of test compound and the medium with test compound was replaced every 3 d. On the third and sixth days, the DHBV DNA levels in replaced culture supernatants were respectively detected with a SYBR green dye-based quantitative PCR assay as follows: In general, DHBV DNA was extracted and amplified with Bio-Rad iQ5 Real Time PCR system (Bio-Rad Laboratories Inc., Irvine, CA，U.S.A.). The thermal programme comprised of an initial denaturation at 94°C for 10 min followed by 40 amplification cycles of 94°C for 30 s, 55°C for 30 s, and then 72°C for 45 s. The forward primer is 5′-AGC TGG CCT AAT CGG ATT AC-3′ and the reverse primer is 5′-TGT CCG TCA GAT ACA GCA AG-3′. SYBR® Premix Ex Taq™ II was used to amplify and detect DNA during the reaction. A plasmid containing the DHBV genome was used to create the standard curve for quantifying DHBV level. Lamivudine was used as the reference drug.

Anti-HBV Effect of Cichoric Acid in HepG2.2.15 Cells

HepG2.2.15 cells were provided from State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Zhejiang University, China. The cells were maintained in DMEM containing 2 mM glutamine, 10% (v/v) heat-inactivated fetal bovine serum and 380 µg/mL of G418 at 37°C (95% humidity, 5% CO₂). Cellular cytotoxicity induced by cichoric acid treatment in HepG2.2.15 cells was analyzed with the MTT method. The cells were seeded in 96-well plate with a concentration of 1.0×10⁵ cells/mL. Different concentrations of cichoric acid were applied to culture wells in triplicate. After incubation for 6 d, the MTT assay was carried out as described above. Cytotoxicity was expressed according to the amount of formazan production compared with untreated control cultures.

To measure HBV antigens and HBV DNA levels, HepG2.2.15 cells were treated with various concentrations of test compound and the medium with test compound was replaced every 3 d. On the third day, the replaced medium was assayed for HBsAg and HBeAg. On the sixth day, the replaced medium was measured for HBsAg, HBeAg and HBV DNA. Lamivudine at concentrations of 1–100 µg/mL was used as the reference drug. HBsAg and HBeAg levels in replaced culture supernatants were respectively determined by HBsAg and HBeAg EIA kits according to the protocol provided with the kit. The results were read at 450 nm by a multi-well plate reader.

HBV DNA level in replaced culture supernatants was detected with a HBV DNA PCR-fluorescence quantitation kit. In sum, HBV DNA was extracted and amplified with Bio-Rad iQ5 Real Time PCR system. The thermal programme comprised of an initial denaturation at 94°C for 2 min followed by 40 amplification cycles each of two steps: 95°C for 5 s and 60°C for 30 s. The forward primer is 5′-CCG TCT GTG CCT TCT CAT CTG-3′, the reverse primer is 5′-AGT CCA AGA GTA CTC TTA TAG AAG ACC TT-3′ and the Taqman probe is FAM-CCG TGT GCA CTT CGC TTC ACC TCT GC. A plasmid containing the full-length insert of HBV genome was used to prepare the standard curve.

Statistical Analysis

Results are reported as mean±standard deviations (S.D.) and subjected to a one-way ANOVA and Student’s t-test. A p<0.05 was chosen as the criterion of statistical significance.

RESULTS

Hepatocyteprotective Effect of Cichoric Acid

The result of cytotoxicity test showed that cichoric acid exhibited no significant toxicity against HL-7702 hepatocytes at concentrations of 1–100 µg/mL (Table 1). To evaluate hepatocyteprotective effect of cichoric acid, hepatocyte injury was induced by exposure to 70 mM D-GalN for 8 h after the cells were pretreated with test compound. The results showed that cichoric acid improved significantly cell viability at concentrations of 10–100 µg/mL (Table 2). At a concentration of 100 µg/mL, it produced a maximum protection rate of 56.26%, while silybin used as the standard drug indicated protection rate of 59.89% on hepatocyte injury.

Table 1. Cytotoxicity of Cichoric Acid in HL-7702 Hepatocytes

Groups	Concentration (µg/mL)	Absorbency (570 nm)	Cell survival (%)
Vehicle	—	1.229±0.051	100
Silybin	100	1.186±0.073	96.50
	50	1.207±0.062	98.21
	10	1.211±0.037	98.53
Cichoric acid	100	1.201±0.048	97.72
	50	1.214±0.052	98.78
	10	1.222±0.033	99.43

Absorbency values are expressed as means±S.D. of five replicates. No significant difference compared with the vehicle control.

Table 2. Effect of Cichoric Acid on Survival of D-GalN-Injured HL-7702 Hepatocytes

Group	Concentration (µg/mL)	Absorbency (570 nm)	Protection rate (%)
Vehicle	—	1.314±0.022***	—
D-GalN-control	—	0.521±0.024	—
Silybin	100	0.996±0.080***	59.89
	50	0.981±0.104***	57.97
	10	0.810±0.040***	36.40
Cichoric acid	100	0.967±0.043***	56.26
	50	0.839±0.008***	40.04
	10	0.833±0.020***	39.33

Absorbency values are expressed as means±S.D. of five replicates. Protection rate=(A value in experimental group−A value in D-GalN-control group)/(A value in vehicle group−A value in D-GalN-control group)×100%. *** p<0.001 represent the significance of the difference from the D-GalN control.

Anti-DHBV Effect of Cichoric Acid

Cytotoxicity test indicated that cichoric acid was not significantly toxic to primary duck fetal hepatocytes at concentrations of 1–100 µg/mL (Table 3). Effect of cichoric acid on DHBV DNA level was shown in Table 4. After the cells were treated with test compound for 3 d, cichoric acid at concentrations of 25–100 µg/mL inhibited significantly the DHBV DNA replication in culture supernatants. On the sixth day, cichoric acid at a concentration of 1–100 µg/mL inhibited markedly DHBV DNA replication. Lamivudine used as the standard drug also indicated the similar effect.

Table 3. Cytotoxicity of Cichoric Acid in Infected Primary Duck Fetal Hepatocytes

Groups	Concentration (µg/mL)	Absorbency (570 nm)	Cell survival (%)
Vehicle	—	0.847±0.053	100.00
Lamivudine	100	0.829±0. 074	97.87
Cichoric acid	100	0.827±0.054	97.64
	50	0.836±0.071	98.70
	25	0.841±0.049	99.29
	10	0.845±0.066	99.76
	1	0.847±0.067	100.00

Absorbency values are expressed as means±S.D. of three replicates. No significant difference compared with the vehicle control.

Table 4. Anti-DHBV Effect of Cichoric Acid in Infected Primary Duck Fetal Hepatocytes

Compound	Concentration (µg/mL)	LogDHBV-DNA (copy/µL)
		3 d	6 d
Vehicle	—	7.263±0.021	7.282±0.014
Lamivudine	100	6.974±0.015***	6.961±0.010***
Cichoric acid	100	6.965±0.019***	6.915±0.013***
	50	7.066±0.030***	6.968±0.014***
	25	7.115±0.051**	7.103±0.012***
	10	7.182±0.069	7.174±0.033**
	1	7.248±0.012	7.220±0.011**

After the cells were treated with the test compound for 3 and 6 d. Log^DHBV-DNA values are expressed as means±S.D. of three replicates. ** p<0.01 and *** p<0.001 compared with the vehicle group.

Anti-HBV Effect of Cichoric Acid

Cellular toxicity test indicated that cichoric acid caused no significant harm to HepG2.2.15 cells at concentrations of 1–100 µg/mL (Table 5). The HBsAg and HBeAg levels in culture supernatants of HepG2.2.15 cells were assayed after the cells were incubated with test compound for 3 d (Table 6). The results indicated that cichoric acid at concentrations of 1–100 µg/mL had significant inhibitory effect on expression of HBsAg and HBeAg and produced the maximum inhibition rates of 67.88% and 55.92% at a concentration of 100 µg/mL, respectiverly. The HBsAg, HBeAg and HBV DNA levels in culture supernatants were measured after the cells were treated with test compound for 6 d (Table 7). At concentrations of 10–100 µg/mL, cichoric acid inhibited significantly the expressions of HBsAg and HBeAg. At a concentration of 100 µg/mL, it produced the maximum inhibition rates of 79.94% and 76.41% on expression of HBsAg and HBeAg, respectively. Moreover, cichoric acid inhibited markedly HBV DNA replication at concentrations of 50–100 µg/mL.

Table 5. Cytotoxicity of Cichoric Acid in HepG2.2.15 Cells

Groups	Concentration (µg/mL)	Absorbency (570 nm)	Cell survival (%)
Vehicle	—	0.954±0.042	100.00
Lamivudine	100	0.936±0.079	98.11
	50	0.945±0.069	99.06
	25	0.948±0.082	99.37
	10	0.951±0.074	99.68
	1	0.953±0.057	99.90
Cichoric acid	100	0.931±0.091	97.59
	50	0.939±0.073	98.43
	25	0.951±0.084	99.68
	10	0.954±0.058	100.00
	1	0.955±0.073	100.01

Absorbency values are expressed as means±S.D. of three replicates. No significant difference compared with the vehicle control.

Table 6. Anti-HBV Effect of Cichoric Acid in HepG2.2.15 Cells

Compound	Concentration (µg/mL)	HBsAg			HBeAg
		Absorbency (570 nm)	Inhibition (%)	IC50 (µg/mL)	Absorbency (570 nm)	Inhibition (%)	IC50 (µg/mL)
Vehicle	—	2.616±0.053	—		2.293±0.096	—
Lamivudine	100	1.876±0.038***	28.14	324.37	2.126±0.096	7.16	—
	50	2.023±0.062***	22.48		2.146±0.080	6.30
	25	2.224±0.052***	14.78		2.162±0.087	5.58
	10	2.422±0.090*	7.19		2.122±0.156	7.34
	1	2.748±0.170	—		2.120±0.135	7.44
Cichoric acid	100	0.838±0.100***	67.88	33.45	1.009±0.064***	55.92	71.12
	50	0.913±0.078***	65.03		1.130±0.050***	50.64
	25	1.646±0.110***	36.92		1.372±0.087***	40.07
	10	1.945±0.069***	25.47		1.746±0.046***	23.77
	1	2.237±0.060***	14.29		1.811±0.116**	20.90

After the cells were treated with the test compound for 3 d. Inhibition (%)=(the mean absorbency value in negative control group−the mean absorbency value in experimental group)/(the mean absorbency value in negative control group)×100%. Absorbency values are expressed as means±S.D. of three replicates. * p<0.05, ** p<0.01 and *** p<0.001 compared with the vehicle group.

Table 7. Anti-HBV Effect of Cichoric Acid in HepG2.2.15 Cells

Compound	Concentration (µg/mL)	HBsAg			HBeAg			LogHBV-DNA (copy/µL)
		Absorbency (570 nm)	Inhibition (%)	IC50 (µg/mL)	Absorbency (570 nm)	Inhibition (%)	IC50 (µg/mL)
Vehicle	—	1.828±0.074	—		2.364±0.038	—		5.077±0.068
Lamivudine	100	1.511±0.155*	16.96	—	2.020±0.031***	14.39	—	4.912±0.063*
	50	1.620±0.126	10.99		2.179±0.013**	7.68		5.018±0.206
	25	1.637±0.068*	10.05		2.184±0.116	7.44		5.023±0.174
	10	1.682±0.070	7.60		2.230±0.057*	5.51		5.028±0.158
	1	1.760±0.115	3.30		2.206±0.071*	6.52		5.115±0.030
Cichoric acid	100	0.365±0.016***	79.94	35.01	0.557±0.035***	76.41	23.35	4.769±0.103*
	50	0.559±0.072***	69.30		0.639±0.001***	72.94		4.891±0.091*
	25	1.153±0.072***	36.63		1.126±0.052***	52.30		5.031±0.072
	10	1.621±0.089*	10.95		1.842±0.137**	21.94		5.306±0.051
	1	1.799±0.017	1.15		2.169±0.085*	8.11		5.324±0.085

After the cells were treated with the test compound for 6 d. Inhibition (%)=(the mean absorbency value in negative control group−the mean absorbency value in experimental group)/(the mean absorbency value in negative control group)×100%. Absorbency and log^HBV-DNA values are expressed as means±S.D. of three replicates. * p<0.05, ** p<0.01 and *** p<0.001 compared with the vehicle group.

DISCUSSION

Chronic HBV infection remains a serious and lifethreatening disease worldwide, despite the availability of a safe and effective vaccine.²¹⁾ The frequent emergence of lamivudine-resistant variants and the poor response to interferon α reveal a need for more effective therapies.^22,23) Cichoric acid, as a natural caffeic acid derivative, stimulates cell-mediated immunity, exhibits anti-vesicular stomatitis virus and antioxidant activities, and inhibits hyaluronidase, an enzyme involved in infection and inflammation, has a stimulant effect on phagocytes and an inhibitive effect on the HIV infection.^13,24–28) Although cichoric acid exhibits a variety of bioactivities, its pharmacological properties (especially antiviral potential) are not yet completely clear. In the present study, the hepatocyteprotective, anti-DHBV and anti-HBV effects of cichoric acid were evaluated by the D-GalN-induced HL-7702 cells damage model, the DHBV-infected primary duck fetal hepatocytes and the HBV-transfected HepG2.2.15 cells, respectively.

Generally, the beneficial role of hepatoprotectors in viral hepatitis is achieved through their inhibitory action on the inflammatory and cytotoxic cascades induced by viral infection.²⁹⁾ Additionally, these agents can also improve the regeneration process and normalize liver enzymes through their effects on protein synthesis.²⁹⁾ D-GalN is a toxin causing damage of the liver and is known as a suitable experimental model of hepatocyte injury.³⁰⁾ Among the numerous models of experimental hepatitis, D-GalN induced liver damage is very similar to human viral hepatitis in its morphological and functional features.³¹⁾ Oxygen-derived free radicals released from activated hepatic macrophages are the primary cause of D-GalN-induced liver damage.³²⁾ In this study, cichoric acid attenuated significantly D-GalN-induced hepatocyte injury after the cells were pretreated with test compound. In addition, cichoric acid has been shown to possess phagocytosis stimulatory, anti-hyaluronidase and antioxidant activities,^10,26,27) thereby implying that its anti-hyaluronidase, phagocytosis stimulatory and antioxidative properties may have contributed to the amelioration of chemical-induced hepatocyte damage.

DHBV is highly similar to HBV in terms of the mechanism of replication and genome organization.^33–35) DHBV is widely accepted as a surrogate for HBV due to their similarities.^36,37) The natural route of transmission is from the bloodstream of persistently infected laying ducks to the egg, resulting in congenital infection.^36,37) Congenitally infected ducks are at risk of developing hepatoma or secondary amyloidosis due to chronic stimulation of the immune system.^38–40) The DHBV infected primary duck hepatocytes model is a valuable model of hepadnavirus infection with high reproducibility and efficiency for evaluating new agents directed against HBV.^37,41) In the study, cichoric acid inhibited significantly the DHBV DNA replication of the infected primary duck fetal hepatocytes. And that cichoric acid stimulates cell-mediated immunity and inhibits hyaluronidase activity and HIV infection,^13,24,27) thus indicating that the anti-DHBV effect of test compound was probably related to its immunomodulatory property and blocking viral DNA replication.

HBV is a small double stranded DNA virus composed of an outer envelope containing HBsAg and an inner nucleocapsid consisting of HBeAg and hepatitis B core antigen (HBcAg). The viral core also contains a double stranded DNA genome and DNA polymerase. The presence of HBsAg is the most common marker of HBV infection, whereas HBeAg is used as an ancillary marker primarily to indicate active HBV replication and associated progressive liver disease.⁴²⁾ HepG2.2.15 cells is derived from human hepatoblastoma HepG2 cells that were transfected with a plasmid containing HBV DNA. The cells can stably secrete viral particles in culture medium.⁴³⁾ In this study, cichoric acid at concentrations of 10–100 µg/mL reduced significantly the HBsAg and HBeAg levels in HepG2.2.15 cells. Meanwhile, it inhibited markedly HBV DNA replication of HepG2.2.15 cells at concentrations of 50–100 µg/mL. Additionally, cichoric acid stimulates cell-mediated immunity and inhibits hyaluronidase activity and HIV infection,^13,24,27) thereby further suggesting that the anti-HBV effect of test compound is probably associated with its immunomodulation and blocking the steps of viral protein synthesis and DNA replication.

In conclusion, this study verifies the anti-hepatitis B effect of cichoric acid in cell culture. The hepatocyteprotective effect of this compound could be achieved by its anti-hyaluronidase, phagocytosis stimulatory and antioxidative properties. The antiviral effect of test compound may contribute to its immunomodulation and blocking the steps of viral protein synthesis and DNA replication. In addition, cichoric acid could be used to design the antiviral agents.

Acknowledgment

This research was supported by the Key Project of Chinese Ministry of Education (No. 212073), the Public Welfare Technology Applied Research Project of Zhejiang Province—Experimental Animal Science and Technology Project (No. 2013C37020) and the Project for Supporting Xinjiang through Science and Technology (No. 201191260).

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