Inhibitory effect of anti-pyretic and anti-inflammatory herbs on herpes simplex virus replication

Inhibitory effect of anti-pyretic and anti-inflammatory herbs on herpes simplex virus replication

Chien-Yun Hsiang

Abstract: The increasing clinical use of acyclovir, ganciclovir, and foscarnet against herpes simplex virus (HSV), varicella-zoster virus, and cytomegalovirus has been associated with the emergence of drug-resistant herpesvirus strains. To develop anti-HSV compounds from plants, 31 herbs used as antipyretic and anti-inflammatory agents in Chinese medicine were screened. Five different preparations (cold aqueous, hot aqueous, ethanolic, acid ethanolic, and methanolic) from 31 herbs were analyzed by plaque reduction assay, and 7 extracts, which showed significant antiviral activities, were further elucidated for their antiviral mechanisms. Our results showed that ethanolic extract of Rheum officinale and methanolic extract of Paeonia suffruticosa prevented the process of virus attachment and penetration. Aqueous extract of P. suffruticosa and ethanolic extract of Melia toosendan inhibited virus attachment to cell surface. Aqueous extract of Sophora flavescens and methanolic extract of M. toosendan showed no effect on virus attachment and penetration. These data indicated that these 4 herbs have a potential value as a source of new powerful anti-HSV compounds.

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Herpes simplex virus (HSV) is a common human pathogen which causes a broad spectrum of illness ranging from asymptomatic infection to fulminant disseminated diseases, such as labial herpes, genital herpes, keratitis, and encephalitis (Corey and Spear, 1986; Stagno and Whitley, 1985). A major advance in antiviral therapy has been the use of acyclovir to treat HSV infections, but the appearance of acyclovir-resistant HSV strains has become evident in immunosuppressed patients, such as organ transplant recipients and patients with acquired immunodeficiency syndrome (Hirsch and Schooley, 1989). Thus, the development of new therapeutic agents with different mode of anti-HSV action is required.

Screening of medicinal plants is a major approach to discover new therapeutic agents against HSV. Several researchers showed that varied ethnic herbs exhibited direct virucidal or prophylactic effects (Taylor et al., 1996; Kott et al., 1999; Sindambiwe et al., 1999). These data suggested that the herb extracts could denature or inactivate viral proteins directly and consequently interfere with virus attachment to cell membranes. Furthermore, Montanha et al. (1995) showed that the aporphine alkaloids obtained from medicinal plants could interfere with the viral replicative cycle by reducing viral DNA synthesis. Therefore, it is interesting to elucidate the antiviral mechanism of several medicinal herbs which had been used as anti-HSV agents in Taiwan for years.

The traditional medicine is widely used in Taiwan. In Chinese medicine, HSV infection is classified as heat patterns, which arise when “yang” evil invades the body or when “yin” humor becomes insufficient. Thus, heat patterns are caused by a surplus of “yang” or a deficit of “yin”, which then form the repletion of heat or vacuity of heat patterns. The principle of treatment for heat patterns is to clear heat and resolve toxin, and the medicinal herbs used are usually classified as “cold” properties. Thirty-one herbs, which are commonly used as antipyretic or anti-inflammatory agents in Chinese medicine, were analyzed in this study. The cold aqueous, hot aqueous, ethanolic, acid ethanolic, and methanolic extracts of herbs were examined by plaque reduction assay, and the herbs exhibiting a potential antiviral effect were further evaluated for their anti-viral mechanisms.

Materials and Methods

Viruses and Cells

African green monkey kidney cells (Vero cells) were grown in Dulbecco’s minimum essential medium (DMEM) supplemented with 10% fetal bovine serum. Herpes simplex virus type 1 (HSV-1) was isolated from clinical specimens and identified by fluorescein-conjugated monoclonal antibodies. After plaque purification, the virus was grown on Vero cells and the virus stocks were stored at -70 [degrees] C for further assay.

Preparation of Herb Extracts

The plant materials were gifts from Sun Ten Pharmaceutical corporation (Taiwan). The properties of plants used in this study are shown in Table 1. Plant samples were ground with homogenizer to a fine powder. The cold aqueous, ethanolic, acid ethanolic, and methanolic extracts were prepared by mixing 3 g of each herb powder with 10 ml of double deionized water (DDW), 95% ethanol, acid ethanol (0.5% HCl in 95% ethanol), and 100% methanol, respectively, and shaking at 4 [degrees] C overnight. The hot aqueous extract was prepared by adding 3 g of herb powder to 10 ml of boiled DDW, shaking at 4 [degrees] C overnight, and incubating at 60 [degrees] C for 10 min. All extracts were centrifuged at 10000 rpm for 5 min, and the supernatants were then evaporated under vacuum to dryness and resuspended in appropriate solvents to a final concentration of 1 mg/ml. The extracts were stored at -20 [degrees] C in small aliquots.

Cytotoxicity Assay

Cell toxicity was monitored by determining the effect of the herb extracts on cell morphology. Vero cells were cultivated in 96-well culture plates. After 24 hrs of incubation at 37 [degrees] C in a humidified C[O.sub.2] atmosphere (5% C[O.sub.2]), serial twofold dilutions of the extracts were added to confluent cell monolayers and incubated for another 72 hrs. The 50% toxicity concentration (T[C.sub.50]) causing visible morphological changes in 50% of cells with respect to cell control was determined.

Plaque Reduction Assay

Cell monolayers cultivated in 24-well culture plates were infected with 200 plaque-forming units of HSV-1 for 1 hr at room temperature and subsequently for 30 min at 37 [degrees] C. The viruses were then discarded, and the cells were overlaid with 1 ml of 1% methylcellulose medium containing serial twofold dilutions of the herb extracts and then cultivated at 37 [degrees] C for 3 days. The cells were fixed by methanol, stained by crystal violet, and the number of plaques was counted. The effective concentration for 50% plaque reduction (E[C.sub.50]) was determined as the lowest herb extract concentration, which reduced plaque number by 50% in the treated cultures compared to untreated ones.

Inhibition of Virus Attachment

Vero cells were grown to confluence in 24-well culture plates and incubated in DMEM containing herb extracts at 37 [degrees] C. After a 3-hour incubation, the medium was removed and the cells were inoculated with DMEM containing HSV-1 (1 multiplicity of infection; m.o.i.) and herb extracts at 4 [degrees] C for 1 hr to allow virus attachment. The cells were then washed twice with phosphate-buffered saline (PBS) and incubated in DMEM at 37 [degrees] C for virus penetration. After a 30-min incubation, the cells were washed with PBS, overlaid with 1 ml of DMEM containing 1% methylcellulose, and incubated at 37 [degrees] C for 3 days. The cells were stained with crystal violet to count the number of plaques.

Inhibition of Virus Penetration

Virus penetration was measured by inactivation of unpenetrated viruses with a low-pH buffer as described by Highlander et al. (1987). Briefly, Veto cells were grown to confluence in 24-well culture plates, and infected with HSV-1 (1 m.o.i.) at 4 [degrees] C for 1 hr. The cells were washed twice with PBS and incubated in 1 ml DMEM containing herb extracts at 37 [degrees] C. After a 30-min incubation, 0.8 ml citrate buffer (40 mM citric acid, 10 mM KCI, 135 mM NaCI, pH 3.0) was added to each well and incubated for 2 min at room temperature. The cells were then washed twice with PBS, overlaid with 1 ml of DMEM containing 1% methylcellulose, and incubated at 37 [degrees] C for 3 days. The cells were stained with crystal violet to count the number of plaques.

Results

Inhibitory Effect of Herb Extracts on HSV-1 Replication

The thirty-one medicinal herbs collected in this study were used traditionally to treat diseases that could be caused by viral pathogens. To evaluate the antiviral activity of the methanolic extracts of these herbs, we examined the inhibitory effects on the plaque formation of HSV-1 by plaque reduction assay. Of these 31 plants, the methanolic extracts of 4 herbs (R. officinale, S. flavescens, P. suffruticosa, and M. toosendan) exhibited inhibitory effects on plaque number or plaque size. These 4 herbs were further chosen to elucidate their antiviral mechanisms.

Selective Index of the Aqueous and Alcoholic Extracts of R. officinale, S. flavescens, P. suffruticosa, and M. toosendan

The cytotoxicity of herb extracts was determined using uninfected Vero cells to obtain the 50% toxicity concentration (T[C.sub.50]), and the antiviral activity was estimated by effective concentration for 50% plaque reduction (E[C.sub.50]).

The cold aqueous, hot aqueous, ethanolic, acid ethanolic, and methanolic extracts of R. officinale, S. flavescens, P. suffruticosa, and M. toosendan were analyzed for their cytotoxicity and antiviral activity. Among these herbs, ethanolic extract of R. officinale, hot aqueous extract of S. flavescens, aqueous and methanolic extracts of P. suffruticosa, and acid ethanolic and methanolic extracts of M. toosendan showed significant plaque reduction abilities (Table 2). The selective index (T[C.sub.50]/E[C.sub.50]) exceeding 8.0 is considered significant (Kujumgiev et al., 1999).

Effect of Herb Extracts on Virus Attachment

To determine whether these 7 extracts have any effect on virus attachment, inhibition of virus binding to cells was measured after pretreatment of cells with herb extracts in a concentration of 2 ng/ml, which cause no visible morphological changes in cells. After a 1-hour incubation at 4 [degrees] C, the cells were washed, and the residual viruses were allowed to be penetrated and subjected to plaque assay.

Table 3 shows the percentage inhibition of each extract. The cells pretreated with the aqueous and methanolic extracts of P. suffruticosa, ethanolic extract of R. officinale, and acid ethanolic extract of M. toosendan inhibited virus attachment by approximately 95%. These data suggested that these 5 extracts exerted their effects by preventing virus attachment to cell surfaces.

Effect of Herb Extracts on Virus Penetration

To investigate the effect of these extracts on HSV-1 penetration, a penetration assay was performed based on the resistance of internalized viruses to inactivation by acid treatment (Highlander et al., 1987). The cells were treated with 2 ng/ml of herb extracts after virus adsorption, and the citrate buffer was used to inactivate unpenetrated viruses.

Table 3 shows that the hot aqueous extract of S. flavescens, aqueous extract of P. suffruticosa, and alcoholic extract of M. toosendan did not interfere with fusion between virus envelope and cell membrane. The ethanolic extract of R. officinale and methanolic extract of P. suffruticosa exhibited an approximately 65% inhibition on virus penetration, suggesting that these 2 extracts exerted their effects in part by preventing virus penetration. Taken together, these results suggested that these extracts might interfere with the adsorption and/or the penetration of HSV-1, and also act throughout the viral replication cycle, especially at a later stage.

Discussion

Five different preparations from 31 herbs, which were used as antipyretic or anti-inflammatory agents in Chinese medicine, were screened for their antiviral activities. Based on the plaque reduction assay, the methanolic and aqueous extracts of P. suffruticosa, ethanolic and methanolic extracts of M. toosendan, ethanolic extract of R. officinale, and aqueous extract of S. flavescens exhibited significant antiviral activities with a selective index which exceeded 8.0. Among these extracts, the ethanolic extract of R. officinale and methanolic extract of P. suffruticosa exerted their effects by preventing virus attachment and penetration, while the aqueous extract of P. suffruticosa and acid-ethanolic extract of M. toosendan exerted their effects in part by preventing virus attachment to cell surfaces. The aqueous extract or S. flavescens and the methanolic extract of M. toosendan showed no effect on viral attachment and penetration. It is assumed that they may play a role in viral genome replication or assembly. Further studies are needed to define the mechanism of antiviral activities.

In the screening of medicinal plants used in Nepal, Argentine, and Rwanda for the treatment of infectious diseases, the aqueous extracts of Polygonurn punctaturn, Lithraea molleoides, Sebastiania brasiliensis and Sebastiania klotzschiana, methanolic extracts of Hypericum cordifolium, H. uralum, Lygodium japonicurn, Carissa carandas, Tridax procumbens, Terminalia alata, Bauhinia vahlii, Milettia extensa, Streblus asper and Rurnex hastatus, and ethanolic extracts of Cassia mimosoides and Ipomoea involucrata exhibited virucidal or antiviral activities against HSV (Taylor et al., 1996; Sindambiwe et al., 1999; Kott et al., 1999). In our study, the R. officinale, S. flavescens, P. suffruticosa, and M. toosendan showed significant antiviral activities. Based on the five-kingdom classification system, all of these herbs with antiviral activities belonged to different families, including Polygonaceae, Anacardiaceae, Euphorbiaceae, Caesalpiniaceae, Convolvulaceae, Hypericaceae, Lygodiaceae, Apocynaceae, Asteraceae, Combretaceae, Fabaceae, and Moraceae. Herbs were grouped according to their cytological and morphological features. It seems that the antiviral activities of herbs were not related to their classification.

Various solvents have been used to extract plant metabolites, and the choice of solvent depends on what is intended with the extract. The major compounds extracted by water were amino acids, peptides, saponins, phenol glycosides, tannins, and alkaloids. Different compounds were extracted by using solvents with decreasing polarity, e.g. ethanol. These compounds include flavonoids, anthraquinones, terpenes, sterols, and essential oils. The aqueous extracts of P. suffruticosa exhibited the highest potency in inhibiting the lipid peroxidation by strong superoxide- and hydroxyl radical-scavenging activities (Liu and Ng, 2000). Sydiskis et al. (1991) showed that anthraquinones, prepared from methanolic extract of R. officinale, acted directly on the envelope of the virus and resulted in the prevention of virus adsorption and subsequent replication. Meliacine, a peptide isolated from Melia azedarach, prevented the process of uncoating of foot and mouth disease virus in BHK-21 cells by inhibiting the vacuolar acidification (Wachsman et al., 1998). Another compound, 28-deacetylsendanin, prepared from methanolic extract of M. azedarach, inhibited the replication of HSV-1, reduced the synthesis of HSV-1 thymidine kinase, and led to the formation of defective nucleocapsids (Kim et al., 1999). In our study, the ethanolic extract of R. officinale inhibited the process of virus attachment and penetration. The methanolic extract of M. toosendan exhibited a significant antiviral activity, although it did not interfere with virus attachment and penetration. Therefore, further studies designed to define the active compounds responsible for antiviral activity of R. officinale, S. flavescens, P. suffruticosa, and M. toosendan are in progress.

Table 1. List of Plants, Their Common Names,

Properties, and Medical Uses

Species (common name) Plant part used Properties

ASTERALES

Artemisia annua L. Whole Cool

(Qinghao)

Taraxacum sinicum Whole Cool

Kitag. (Pugongying)

DIPSACALES

Lonicera japonica Flower bud Cool

Thunb. (Jinyinhua)

Thlaspi arvensis L. Whole Cool

(Baijiang)

GENTIANALS

Gentiana manshurica Root, Rhizome Cool

Kitag. (Longdan)

LAMIALES

Prunella vulgaris L. Fruits Cool

(Xiakucao)

Schizonepeta tenuifolia Stems, leaves, Hot

Briq. (Jingjie) flowers

Scutellaria baicalensis Root Cool

Georgi (Huangqin)

SCROPHULARIALES

Baphicacanthus cusia Leaves Cool

Bremek. (Daqingye)

Forsythia suspensa Vahl Fruits Cool

(Lianqiao)

Fraxinus chinensis Bark Cool

Roxb. (Qinpi)

POLYGONALES

Rheum officinale Baill. Rhizome Cool

(Dahuang)

CAPPARALES

Isatis indigotica Fort. Root Cool

(Banlangen)

ILLICIALES

Schisandra chinensis Fruits, seeds Hot

Baill. (Wuweizi)

PIPERALES

Houttuynia cordata Whole Cool

Thunb. (Yuxingcao)

RANUNCULALES

Aconitum kusnezoffii Rhizome Hot

Reichb. (Caowuton)

Cimicifuga heracleifolia Rhizome Cool

Komar. (Shengma)

Coptis chinensis Franch. Rhizome Cool

(Huanglian)

Paeonia suffruticosa Root Cool

Andr. (Mudanpi)

APIALES

Cnidium monnieri Cuss. Fruits Hot

(Shechuangzi)

FABALES

Pueraria lobata Ohwi Rhizome Hot

(Gegen)

Sophora flavescens Ait. Root Cool

(Kushen)

MYRTALES

Quisqualis indica L. Fruits, seeds Hot

(Shijunzi)

ROSALES

Potentilla chinensis Ser. Root Cool

(Baitouweng)

Prunus mume Sieb. Fruits Hot

(Wumei)

SAPINDALES

Melia toosendan Sieb. Fruit Cool

(Kulianzi)

Phellodendron amurense Bark Cool

Rupr. (Huangbai)

Zanthoxylum bungeanum Peel Hot

Maxim. (Huajiao)

ARECALES

Areca catechu L. Seeds Hot

(Binglang)

TYPHALES

Sparganium stoloniferum Rhizome Hot

Buch.-Ham. (Sanleng)

ZINGIBERALES

Curcuma wenyujin Rhizome Hot

(Ezhu)

Species (common name) Usage

ASTERALES

Artemisia annua L. Antipyretic, anti-inflammatory

(Qinghao)

Taraxacum sinicum Antipyretic, anti-inflammatory, diuretic

Kitag. (Pugongying)

DIPSACALES

Lonicera japonica Antipyretic, anti-inflammatory, diuretic,

Thunb. (Jinyinhua) blood purifying, anti-diarrheal

Thlaspi arvensis L. Antipyretic, anti-inflammatory,

(Baijiang) circulation promoting

GENTIANALS

Gentiana manshurica Antipyretic, diuretic

Kitag. (Longdan)

LAMIALES

Prunella vulgaris L. Anti-inflammatory, diuretic

(Xiakucao)

Schizonepeta tenuifolia Anti-rheumatic, hemostatic,

Briq. (Jingjie) anti-inflammatory

Scutellaria baicalensis Antipyretic, anti-inflammatory,

Georgi (Huangqin) hemostatic

SCROPHULARIALES

Baphicacanthus cusia Antipyretic, anti-inflammatory,

Bremek. (Daqingye) blood purifying

Forsythia suspensa Vahl Antipyretic, anti-inflammatory, diuretic

(Lianqiao)

Fraxinus chinensis Antipyretic, diuretic, antitussive

Roxb. (Qinpi)

POLYGONALES

Rheum officinale Baill. Antipyretic, anti-inflammatory,

(Dahuang) blood purifying, diuretic

CAPPARALES

Isatis indigotica Fort. Antipyretic, anti-inflammatory,

(Banlangen) blood purifying

ILLICIALES

Schisandra chinensis Sedative, tranquilizer, anti-inflammatory

Baill. (Wuweizi)

PIPERALES

Houttuynia cordata Antipyretic, anti-inflammatory, diuretic

Thunb. (Yuxingcao)

RANUNCULALES

Aconitum kusnezoffii Anti-rheumatic, diuretic, analgesic,

Reichb. (Caowuton) anti-inflammatory

Cimicifuga heracleifolia Antipyretic, anti-inflammatory

Komar. (Shengma)

Coptis chinensis Franch. Antipyretic, anti-inflammatory

(Huanglian)

Paeonia suffruticosa Antipyretic, blood purifying, circulation

Andr. (Mudanpi) promoting

APIALES

Cnidium monnieri Cuss. Anti-helminthic, anti-rheumatic,

(Shechuangzi) anti-pruritic, anti-inflammatory

FABALES

Pueraria lobata Ohwi Antipyretic, anti-diarrheal

(Gegen)

Sophora flavescens Ait. Antipyretic, anti-rheumatic,

(Kushen) anti-helminthic

MYRTALES

Quisqualis indica L. Anti-helminthic, anti-swelling,

(Shijunzi) absorption promoting,

anti-inflammatory

ROSALES

Potentilla chinensis Ser. Antipyretic, anti-inflammatory, blood

(Baitouweng) purifying, anti-diarrheal,

Prunus mume Sieb. anti-helminthic

(Wumei) Antitussive, anti-diarrheal, hemostatic,

anti-helminthic, anti-inflammatory

SAPINDALES

Melia toosendan Sieb.

(Kulianzi) Analgesic, anti-helminthic,

Phellodendron amurense anti-inflammatory

Rupr. (Huangbai) Antipyretic, anti-inflammatory

Zanthoxylum bungeanum

Maxim. (Huajiao) Analgesic, anti-helminthic,

anti-pruritic, anti-inflammatory

ARECALES

Areca catechu L.

(Binglang) Anti-helminthic, diuretic,

anti-inflammatory

TYPHALES

Sparganium stoloniferum

Buch.-Ham. (Sanleng) Diuretic, analgesic, anti-inflammatory

ZINGIBERALES

Curcuma wenyujin

(Ezhu) Diuretic, analgesic, anti-inflammatory

(Ezhu)

Table 2. Effect of Herb Extracts on HSV-1 Replication

Herb Solvent Cytotoxicity (a)

([TC.sub.50];

[micro]g/ml)

Rheum officinale Acid-ethanol 1.20 [+ or -] 0.51

Rheum officinale Methanol 0.78 [+ or -] 0.01

(b) Rheum officinale Ethanol 0.99 [+ or -] 0.36

Rheum officinale DDW 1.16 [+ or -] 0.98

Rheum officinale Hot DDW 0.75 [+ or -] 0.37

Sophora flavescens Acid-ethanol 0.36 [+ or -] 0.16

Sophora flavescens Methanol 0.35 [+ or -] 0.21

Sophora flavescens Ethanol 0.19 [+ or -] 0.10

Sophora flavescens DDW 2.69 [+ or -] 1.07

(b) Sophora flavescens Hot DDW 3.44 [+ or -] 0.65

Paeonia suffruticosa Acid-ethanol 0.53 [+ or -] 0.00

(b) Paeonia suffruticosa Methanol 0.74 [+ or -] 0.22

Paeonia suffruticosa Ethanol 0.45 [+ or -] 0.12

(b) Paeonia suffruticosa DDW 3.04 [+ or -] 1.62

(b) Paeonia suffruticosa Hot DDW 1.82 [+ or -] 0.55

(b) Melia toosendan Acid-ethanol 0.16 [+ or -] 0.06

(b) Melia toosendan Methanol 0.24 [+ or -] 0.19

Melia toosendan Ethanol 0.17 [+ or -] 0.06

Acyclovir 530 [+ or -] 42

Herb Solvent Antiviral

activity (a)

([EC.sub.50];

[micro]g/ml)

Rheum officinale Acid-ethanol 0.16 [+ or -] 0.06

Rheum officinale Methanol 0.16 [+ or -] 0.08

(b) Rheum offcinale Ethanol 0.12 [+ or -] 0.04

Rheum offcinale DDW 0.18 [+ or -] 0.07

Rheum officinale Hot DDW 0.20 [+ or -] 0.12

Sophora flavescens Acid-ethanol 0.12 [+ or -] 0.02

Sophora flavescens Methanol 0.12 [+ or -] 0.06

Sophora flavescens Ethanol 0.07 [+ or -] 0.01

Sophora flavescens DDW 0.35 [+ or -] 0.23

(b) Sophora flavescens Hot DDW 0.41 [+ or -] 0.26

Paeonia suffruticosa Acid-ethanol 0.08 [+ or -] 0.03

(b) Paeonia suffruticosa Methanol 0.06 [+ or -] 0.03

Paeonia suffruticosa Ethanol 0.06 [+ or -] 0.02

(b) Paeonia suffruticosa DDW 0.36 [+ or -] 0.20

(b) Paeonia suffruticosa Hot DDW 0.20 [+ or -] 0.03

(b) Melia toosendan Acid-ethanol 0.02 [+ or -] 0.01

(b) Melia toosendan Methanol 0.03 [+ or -] 0.03

Melia toosendan Ethanol 0.03 [+ or -] 0.01

Acyclovir 0.52 [+ or -] 0.05

Herb Solvent Selective index

([TC.sub.50]/

[EC.sub.50])

Rheum officinale Acid-ethanol 7.5

Rheum officinale Methanol 4.9

(b) Rheum offcinale Ethanol 8.3

Rheum offcinale DDW 6.4

Rheum officinale Hot DDW 3.8

Sophora flavescens Acid-ethanol 3.0

Sophora flavescens Methanol 2.9

Sophora flavescens Ethanol 2.7

Sophora flavescens DDW 7.7

(b) Sophora flavescens Hot DDW 8.4

Paeonia suffruticosa Acid-ethanol 6.7

(b) Paeonia suffruticosa Methanol 12.3

Paeonia suffruticosa Ethanol 7.5

(b) Paeonia suffruticosa DDW 8.4

(b) Paeonia suffruticosa Hot DDW 9.1

(b) Melia toosendan Acid-ethanol 8.0

(b) Melia toosendan Methanol 8.0

Melia toosendan Ethanol 5.7

Acyclovir 1019

(a): Each value is the mean [+ or -] SD of triplicate assays

(b): Selective index [greater than or equal to] 8.0

Table 3. Effect of Herb Extracts on HSV-1 Attachment and Penetration

Percentage inhibition (a)

Herb Solvent Attachment

Rheum officinale Ethanol 97 [+ or -] 4

Sophora flavescens Hot DDW 7 [+ or -] 8

Paeonia suffruticosa Methanol 97 [+ or -] 4

Paeonia suffruticosa DDW 91 [+ or -] 3

Paeonia suffruticosa Hot DDW 92 [+ or -] 7

Melia toosendan Acid-ethanol 99 [+ or -] 1

Melia toosendan Methanol 6 [+ or -] 4

Percentage inhibition (a)

Herb Solvent Penetration

Rheum officinale Ethanol 64 [+ or -] 2

Sophora flavescens Hot DDW 22 [+ or -] 9

Paeonia suffruticosa Methanol 66 [+ or -] 19

Paeonia suffruticosa DDW 35 [+ or -] 2

Paeonia suffruticosa Hot DDW 39 [+ or -] 6

Melia toosendan Acid-ethanol 5 [+ or -] 4

Melia toosendan Methanol 20 [+ or -] 3

(a): Each value is the mean [+ or -] SD of triplicate assays

Acknowledgment

This work was supported by the grants from National Science Council, Council of Agriculture, and China Medical College Hospital, Taiwan, ROC.

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Chien-Yun Hsiang (1) Ching-Liang Hsieh (2), Shih-Lu Wu (3), I-Lu Lai (1) and Tin-Yun Ho (2), *

(1) Department of Microbiology, China Medical College, Taichung, Taiwan

(2) Institute of Chinese Medical Science, China Medical College, 91 Hsueh-Shih Road, Taichung 404, Taiwan

(3) Department of Biochemistry, China Medical College, Taichung, Taiwan

* Corresponding author

(Accepted for publication April 21, 2001)

COPYRIGHT 2001 Institute for Advanced Research in Asian Science & Medicine

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