Studies On The Antiviral Activity Of Three Medicinal Plants: R.sieboldii, R.sceleratus And L.pterodonta

Viruses have caused some of the most devastating diseases to afflict humanity and today viral diseases continue to pose some of the greatest challenges to modern medicine. Viruses are obligate intracellular parasites, which contain little more than bundles of gene strands of either RNA or DNA, and may be surrounded by a lipid-containing envelope. Viruses utilize the host cell environment to propagate new viruses. Virus structure, enzyme and replication mechanism may be potential antiviral targets. Several drugs have been approved for treatment of viral infections;of most of them were synthetic nucleoside analogues. However, a delay in initiating treatment after infection or reactivation reduces their efficacy. Additionally, nucleoside-resistant virus infections are increasingly encountered in immunocompromised individuals.In addition to the development of synthetic drugs as mentioned above, another approach is based on the screening of plant-derived substances for antiviral activity. There is currently a large and ever-expanding global population base that prefers the use of natural products in treating and preventing medical problems. This has influenced many pharmaceutical companies to produce new antimicrobial formulations extracted from plants or herbs.This paper describes the antiviral activities of three medicinal plants: Ranunculus sieboldii Miq., Ranunculus sceleratus L. and L. pterodonta which all be used by the natives as traditional herbal medicine for their remarkable anti-inflammatory and antimicrobial properties.Part 1. Evaluation of anti-HBV activity of Ranunculus sieboldii Miq. and Ranunculus sceleratus L.Species of Ranunculaceae are widely distributed throughout the world and especially common in slow streams, ditches and shallow, muddy-bottomed ponds of water. Ranunculus sieboldii Miq. and Ranunculus sceleratus L. are two widespread species scattered in China. They were traditionally used in the treatment of various diseases such as jaundice, hepatitis, asthma, rheumatic arthritis, stomachache and toothache by local people. Pharmacological researches supported that these plants had antibacterial and antihistaminic activities. According to the application of the title plants on the treatment of hepatitis B, it was implied that these plants should possess certain inhibitory effects on hepatitis B virus (HBV) and other related viruses. However, the whole plants were, traditionally, directly external applied on the skin around patient's liver in a freshly rubbed form, which usually caused contact dermatitis or skin ulcers after long-term use by chronic hepatitis B patients. Thus, it is necessary to find the nontoxic antiviral components from the whole plant as the alternative of the external application of the whole plants. Systematic phytochemical examination of these two species led to the isolation of 19 compounds. To preliminarily evaluate the antiviral activity of compounds purified from R. sieboldii and R. sceleratus, anti-HBV in vitro assays was conducted and the results were reported.Nineteen compounds named apigenin-4'-Oa-L-rhamnopyranoside (1) , apigenin-7-O-/5-D-glucopyranosyl-4'-O-a-L-rhamnopyranoside (2) , apigenin-8-C-ot-L-arabinopyranoside (3) , apigenin-8-C-/?-D-galactopyranoside (4) , tricin-7-0-/?-D-glucopyranoside (5) , tricin (6), luteolin (7), scopoletin (8), esculetin (9) , scoparone (10) , ferulic acid (11) , protocatechuic acid (12) , tematolide(13) , stigmasta-4-ene-3,6-dione (14), stigmasterol (15), eicosanoic acid (16), ^-sitosterol (17), isoscopoletin (18) and protocatechuic aldehyde (19) were purified from R. sieboldii and R. sceleratus.Cell culture: HepG2.2.15 cells (clonal cells derived from HepG2 cells that were transfected with a plasmid containing HBV-DNA) that secrete hepatitis B virions werecultivated with Dulbecco's modified Eagle's medium (DMEM) containing 2 mM glutamine, 10% (v/v) heat-inactivated fetal bovine serum, penicillin (100 IU/mL), streptomycin (100 ug/mL) and 380 ug/mL of G418. The cells were cultured at 37°C in a moist atmosphere containing 5% CC>2/95% air.Anti-proliferation assay: Prior to investigation of antiviral activity, the cytotoxicity effect of the tested compounds was determined. The modified MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-trazolium bromide] assay was performed. The cytotoxic concentration 50% (CC50) was calculated, as the compound concentrations required reducing the MTT signal by 50% compared with untreated control cultures.Anti-HBV assay: The antiviral activity of these compounds was examined at a concentration, which was lower than the CC10 values. 3-day-old HepG2.2.15 cells were treated with the various concentrations of compounds and the medium with compounds was replaced for every 4 days. On the twelfth day, the medium were collected and asssayed for HBsAg, hepatitis B virus e antigen (HBeAg) and HBV-DNA. Lamivudine (3TC) was used as a reference substance.The replaced medium was collected and clarified by centrifugation at 1000xg for 5 min;the levels of HBsAg and HBeAg secretion were determined by ELISA kits and performed according to the protocol provided with the kit. The results were read at 450 nm by an enzyme-linked immunosorbent assay reader.Extracellular HBV-DNA was measured by real-time quantitative PCR. A plasmid containing the full-length insert of the HBV genome was used to prepare the standard curve. In the same time, HBV-DNA was assayed by blot dot.The assay indicated that isoscopoletin (18) had significant inhibitory effects on the expression of HBsAg and HBeAg secretion from HepG2.2.15 cells. Meanwhile, apigenin-4'-0-a-L-rhamnopyranoside (1), apigenin-7-0-/?-D-glucopyranosyl-4'-0-e(-L-rhamnopyranoside (2), tricin-7-0-/?-D-glucopyranoside (5) and tricin (6) decreased the number of HBV DNA in the culture medium. All of these compounds reduced HBV DNA more than 1 log unit compared with the control. The same results were obtained from hybridization of collected medium with Dig-labeled HBV DNA probe.Effect of compounds on mitochondrial DNA: Some agents used in the treatment of HBV were surmised to be able to influence the mitochondrial function in liver cells, a detailed analysis on the effects of studied compounds on mitochondrial DNA synthesis was therefore carried out. HepG2.2.15 cells were cultured as above and after 24 h in culture, treatment with the compounds was initiated. Compounds were added at desired concentrations in the medium. After twelfth day, cells were collected and DNA was isolated according to the standard protocols. Mitochondrial DNA was quantified by measured the ratio of a mitochondrial gene (mitochondrial hypervariable region 2, HV2) to a nuclear gene (/?-actin) by multiplex PCR. No toxicity on mitochondrial DNA was observed for the tested compounds.The results suggested that compounds 1, 2, 5, and 6 might be the main effective components of R. sieboldii and R. sceleratus in the traditional application of HBV therapy.Compounds with the activity of inhibiting the replication of HBV-DNA in this reported study were all flavone glycosides. This type of compounds was reported to have several pharmacological functions, such as antiviral effects, hepatoprotective and neuroprotective effects. According to our study results, it was supported that flavone glycosides can potentially inhibit the virus infection.All results showed that R. sieboldii and R. sceleratus remarkably inhibit the replication of hepatitis B virus and are highly potent and effective antiviral plants.Part 2. Evaluation of antiviral activity of Laggera pterodontaLaggera pterodonta (DC.) Benth, a perennial plant that belongs to the Compositae family, is widely distributed in southwestern China, especially in Yunnan Province. The plants are traditionally used as anti-inflammation and anti-bacterial herbal drugs. It was rather common in the yards of the local citizens for stock herbal medicine. Very recently, the water extract of Laggera pterodonta was found to possess remarkable antiviral activity against human respiratory syncytial Viru. This further urged our interest of our on-going antiviral research on the aqueous extract of the plant. The aim of this study presented here was to determine the antiviral activity of certain part of the aqueous extract of L. pterodonta.Plant material: The whole herbs of L. pterodonta were collected from Tengchong County, Yunnan province, China in 2003. The plants were identified by Prof. Hua Peng. The voucher specimens (No. 200308LP) was deposited in the State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, Yunnan, China.Preparation of aqueous extracts: The whole plants (2.0 Kg) of L. pterodonta were dried at 50°C for 3 days before grinding. The ground material was suspended 1:10 in 75% EtOH at 70°C for three times. The resulting suspension was filtered and the supernatant was lyophilized (380.0 g) for further use.Quantification of phenolic compounds in the aqueous extracts: The extracts (100 mg) were dissolved in 5 mL of 0.3% HC1 in methanol/water (60:40, v/v). The resulting solution (100 mL) was added to 2 mL of 2% Na2CO3. After 2 min, 50% Folin-Ciocalteu reagent (100 mL) was added to the mixture, which was then left for 30 min. Absorbance was measured at 750 nm using a spectrophotometer. Gallic acid was used as standard sample. The results of qualification indicated that the aqueous extract of L. pterodonta has a high content of phenolic compounds that made up half of the extract (54.4 g GAE/100 g extract).HPLC assay of aqueous extracts: L. pterodonta aqueous extracts was used forHPLC/DAD analyses that carried on a reversed-phase Symmetry C18 column. The mobile phase was C (CH3CN) and D (0.1% HAC). The gradient elution was modified as follows: initial 16% C and held for 15 min;linear gradient to 85% C in 35 min. Flow rate was 0.8 ml/min and 5 (iL sample were injected. The column temperature was set as 35°C. The results indicated that isochlorogenic acids were the major phenolic components in the aqueous extract whose content attached to 53.0%.Assay for antiviral activity in vitro: Cytopathic effect (CPE) reduction method was employed for antiviral assay. Acyclovir and ribavirin, which are used clinically anti-Herpes simplex virus (HSV) and anti-Influenza virus A (IVA) drugs were used as positive controls in the antiviral assay. CPE inhibition data were expressed as the 50% effective (viral CPE-inhibitory) concentration (IC50), 50% cytotoxicity (cell-inhibitory) concentration (CC50) and selectivity index (SI), determined as the CC50/IC50. Aqueous extract of the plant and isochlorogenic acids all display significant antiviral activity in vitro. Their concentrations to inhibit HSV-1 and HSV-2 by 50% ranged from 41.01 to 88.64 ug/mL and 58.64 to 118.24 ug/mL. Furthermore, both of the phenolic mixture of the aqueous extract and the pure isochlorogenic acids showed anti-IVA activity with IC50 values ranging from 104.32 to 321.47 ug/mL. In all of the above assays, the IC50 values were considerably less than those of the CC50 values thus resulting a high SI values, which approved the aqueous extract of L. pterodonta which containing mainly isochloro- genie acids type phenolic compounds possess a good efficacy and safety for medicinal utilization.In vivo anti-IVA experiments: Female BALB/c mice (4 weeks old, 15-17 g) were anesthetized with halothane and inoculated intranasal with viral suspension. Four hours after infection, mice were again anesthetized, and then oral gavages administered L. pterodonta aqueous extracts. Mice were then treated twice daily for days 1-4. Control mice were treated with saline alone. Mice were observed for 21 days after infection. Anti-influenza efficacy was assessed by survival rate;mean day to death of mice dying prior to day 21 and mean lung parameters. Oral gavage administrations of L. pterodonta aqueous extract to mice infected with influenza virus A were effective in preventing death, inhibition of lung consolidation.In conclusion, the aqueous extract form L. pterodonta and its principle components isochlorogenic acids have been identified to show antiviral activity, including some DNA-virus and RNA-virus. This result may help understanding the popular planted phenomenon and the long history of its medicinal application by the local people, as well it afforded some basic background of the structure-activity relationships for further detailed investigation. Our results demonstrated that L. pterodonta is a good candidate for the development of new antiviral medicine.