Journal o f Ethnopharmaeology. 34 ( 1991 ) I-6 Elsevier Scientific Publishers Ireland Ltd.
I
Screening of medicinal plants from Yunnan Province in southwest China for antiviral activity L. Y i p a, S. Pei c, J.B. H u d s o n b a n d G . H . N . T o w e r s a Departments qf "Botany and hMedical Microbiology, Universi O' of British Columbia, Vancouver, B. C. I/6 T 2B I (Canada) and ' Kunming Institute ~[' Botany, Kunming, Yunnan (China) (Accepted July 20, 1990)
In an ethnopharmacological screening of medicinal plants used in Y unnan province of China, ethanol extracts from 31 plant species were assayed for inhibition of murine cytomegalovirus and Sindbis virus infections. Parallel assays were carried out with and without exposure to UVA radiation to test for photo-mediation of activity. Antiviral activity was observed with 16 of the plant extracts, Eight plant extracts have been selected for further studies, with the objective of characterizing the antiviral constituents.
K~:v words." ethnopharmacology; antiviral screening: photo mediated antiviral activity.
Introduction
The significance of ethnopharmacologicai research in discovering new therapeutic activities of natural products has been discussed extensiviely (Bruhn and Holmstedt, 1981; Delaveau, 1981; Malone, 1983; Kyerematen and Ogunlana, 1987). According to Farnsworth et ai. (1985), 74% of the 121 bioactive plant-derived compounds currently in worldwide use were identified via research based on leads from folk- or ethnomedicine. With the increasing knowledge of the pathogenicity of viruses, the search for effective antiviral phytochemicals presents a growing area of pharmacological research (Van Den Berghe et al., 1978; Hudson, 1989a). Cell culture assays have become established procedures for the evaluation of antiviral chemotherapeutic potential, and some antiviral chemicals are now undergoing clinical trials and even clinical applications (Becker, 1984; Hu and Hsiung, 1989). The different possible sites of action for antiviral agents in viral replication process have been described by Mitchell (1973) and an overview of the process of developing anCorrespondence to." L. Yip, Departments of Botany. University of British Columbia, Vancouver, B.C. V6T 2BI, Canada. 0378-8741/$03.50 © 1991 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
tivirai agents from plants has been presented by Van Den Berghe et al. (1985). This laboratory has initiated an antiviral screening of plants of Yunnan for several reasons. Yunnan Province in the southwest of China is an area noted for its botanical diversity (Pei, 1988). Six hundred and seven species are listed in the Chinese Medicine Plants of Yunnan (Kunming Reserve Unit Health Division, 1970) and its sequel volume (Kunming Institute of Botany, 1978). As regards the number of plants in the traditional Chinese pharmacopeia, a data bank at the Chinese University of Hong Kong has 4,941 species of higher plants listed as having been used in medicine (Duke and Ayensu, 1985). In addition, there are over 20 ethnic minority groups in Yunnan. The medicinal uses of plants by these groups further increase the number of candidates for pharmacological investigation. Ethnobotanical information from the minority groups is in the process of being recorded (Yunnan Institute of Medicine Inspection, 1983). Clearly, Yunnan has rich resources in both plant and ethnobotanical knowledge and is an ideal study area for antiviral screening. In this project, plants were collected primarily on the basis of having documented or current use
in the traditional medicine of Yunnan for the treatment Of diseases such as colds, influenza and hepatitis that are now linked to viral causes. General ethnobotanical information, not restricted to the areas of Yunnan from which collections were made, was also considered. Thus, the same species of widely-distributed plants may be used by a number of different peoples. Extracts of these plants were tested for antiviral activity using cell culture systems. Duplicate assays were done in the presence or absence of long wave ultraviolet radiation (.UVA, 320--~00 nm) to test for the possibility that the activity of some might be lightmediated. The biological activity of many plant constituents can be dependent on, or enhanced by, simultaneous irradiation with light (Towers, 1980), Antiviral plant photosensitizers, particularly those activated by UVA, have been reviewed by Hudson (1989b).
China to Canada. For testing, each residue was stirred in 10 ml of 95% ethanol and the soluble fraction used. Cell culture and cytotoxicity assays Bioassays were performed using a cell culture system of the monolayer-forming murine fibroblast 3T3-L1 cell line (American Type Culture Collection). Cell layers for the assays were grown in 96-well microtiter plates (Falcon 3072). Cytotoxicity of the extracts was first tested by exposure of the cells to dilutions of the extracts in the culture medium (Dulbecco's modified Eagle A, with 10% fetal bovine serum), with the cells in suspension and after the formation of monolayers. The highest concentration of extract tested was the equivalent of 100 mg of starting plant material per ml. Two-fold serial dilutions were made from this concentration. The cells were observed for cytopathic effects (CPE).
Materials and Methods Plant collection The 31 medicinal plants assayed are listed in Table 1. Plant samples were collected from the Kunming area in central Yunnan, the Dali-Lijiang region in northwestern Yunnan, and Xishuangbanna, a region with a distinct tropical flora in the south of the province. With the help of Dr. Fan Bingjun in Dali, and Drs. He Zhegao and He Shixiu in Lijiang, collections were also made of plants currently used by herbal medicine doctors to treat viral-related diseases. Identification of the voucher herbarium specimens was verified by Mr. Li Yenhui at the Ethnobotany Laboratory of the Kunming Institute of Botany in Kunming, Yunnan. These voucher specimens have been deposited at the Herbarium of the University of British Columbia. Extract preparation Extracts were made from air-dried samples of the whole plant or, in a few cases, from the portion specified as being used medicinally . Ten-gram samples of plant material were percolated exhaustively in 90% ethanol and dried in vacuo at below 40°C. Drying the extracts to residue was done for ease of transportation of samples from
Antiviral assays In the antiviral assays two-fold serial dilutions of the plant extracts were used starting with the non-cytotoxic concentrations. The infectivity of two test viruses was assayed qualitatively through microscopic observations of characteristic viral CPE. Antiviral activity was determined by the absence of CPE in a treatment. Murine cytomegalovirus (MCMV) is a double strand DNA virus of the Herpesvirus group, while Sindbis is a single strand RNA virus of the Togavirus group. In a comprehensive antiviral assay, three separate segments of the viral life cycle were exposed to the plant extracts successively. The cell preparations were incubated with the extract dilutions during monolayer formation. The viruses, at a concentration of 1000 plaque forming units (pfu) per ml, were exposed to the same extract dilutions in phosphate buffered saline (PBS) at 4"C. The cell layers were then exposed to the virus/extract mixture for 1 h of adsorption time at 37"C. After removal of the virus mixture, the cells were incubated in medium with the same extract dilutions. Control cells were subject to the same treatments without the plant extracts. In the multiple-treatment assay, 16 plant extracts showed inhibition of viral infection. These
TABLE I ANTIVIRAL ACTIVITY OF Y U N N A N PLANTS AGAINST MURINE CYTOMEGALOVIRUS (MCMV) AND SINDBIS VIRUS (SV) Species listed by plant family
Part used"
Minimum active concentration (~g/ml) enhancement b MCMV
SV
APIACEAE
Centella asiatica (L.) Urban
PL
APOCYNACEAE Plumreria rubra L. var. acutifolia (Poir.) Ball.
RT
125
65
BERBERIDACEAE
Mahonia nepalensis DC.
BR
COMBRETACEAE
Quisqualis mdica L.
PL
--
COMPOSITAE Bidens pilosa L.
PL
125
CONVOLVULACEAE Dichondra repens Forst.
PL
--
125
CYCADACEAE Cycas siamensis Miq.
LF
--
160
12.5
65
EBENACEAE
Diospyros kaki L.f.
BK
EUPHORBIACEAE
*Euphorbia prolifera
PL
--
Ehrenb. ex. Boiss. Ricinus communis L.
RT
--
--
GENTIANACEAE Halenia ellipt&'a D.Don
PL
GRAMINEAE Phyllostachy sp.
PL
BU
--
125
PL PL PL
65 -65
65
PL PL
---
-125
PL
--
65
65
IRIDACEAE
Belamcanda chinensis DC. LABIATAE
*Acrocephalus indicus Briq. Elsholtzia densa Briq. *EIsholt=ia rugulosa
30
(Hemsl.) Levi.
Perillafrutescens (L.) Britton Rabdosia phyllostachys (Diels) Hara
*Stachys kouyangensis (Vaniot) Dunn
UVA
4
TABLE 1 (Continued) Species listed by plant family
Part used a
Minimum active concentration (/zg/ml) enhancement b MCMV
SV
UVA
LEGUMINOSAE Kummerowia striata
PL
(Thunb.) Schindl. MYRSINACEAE * Embelia sessiliflora Kurz
RT
30
30
OXALIDACEAE Oxalis corniculata L.
PL
PTERIDACEAE Stenoloma chusanum (L.) Ching
RUBIACEAE Hedyotis uncinella Hook. et Arn.
PL
RUTACEAE * Boenninghausenia sesilicarpa
PL
80
+
Levi. SAURURACEAE Houttuynia cordata Thunb.
PL
SCHOPHULARIACEAE * Siphonostegia chinensis Benth. Scutellaria orthocalyx Hand.-Mazz.
PL PL
40
VERBENACEAE * Verbena of/~cinalis L.
PL
65
30
'LPart used: BK, bark; BR, branch; BU, bulb: LF, leaf: PL, whole plant: RT, root. bUVA = long wave ultraviolet radiation 1350 nm). * = active species selected for further assays.
extracts were a s s a y e d again following the same p r o c e d u r e but with three parallel p r e p a r a t i o n s o f cells. I n s t e a d o f one set o f cells being subjected to three successive extract t r e a t m e n t s , each set was e x p o s e d to one o f the treatments. O n e set was exp o s e d to the extract p r i o r to infection, the s e c o n d to the virus with extract d u r i n g a d s o r p t i o n time, a n d the third o n l y after the r e m o v a l o f virus particles. F o r the t r e a t m e n t d u r i n g a d s o r p t i o n , the virus s u s p e n s i o n was e x p o s e d to the extract for 30 min p r i o r to i n c u b a t i o n with the cells. F u r t h e r
assays were carried o u t with extracts o f eight species. T h e respective extracts in e t h a n o l were filtered ( W h a t m a n p a p e r N o . l ) , e v a p o r a t e d to dryness a n d the residue taken up in I00% m e t h a nol. T h e m e t h a n o l phases were assayed following the same p r o c e d u r e , with extract t r e a t m e n t o f the virus suspension a n d o f the cells d u r i n g virus adsorption. UVA
irradiation
In the assays, one set o f cells a n d the virus sus-
pension with the extracts were exposed to 30 min of UVA radiation (350 nm), while a parallel set was kept in the dark. An extract was considered to show enhancement of activity by UVA radiation when antiviral effects were observed at lower concentrations of the extract, compared to the parallel culture which was kept in the dark.
Results In the multiple treatment assay, 16 plant extracts showed qualitative antiviral activity at noncytotoxic concentrations (Table 1). The antiviral effects were observed in cells treated with concentrations of extract in the range equivalent to 30 /~g/ml to 1.0 mg/ml of original material. When the cell cultures were exposed to extract treatments separately at the pre-infection, virus/infection, and post-infection stages, the antiviral activity in all cases occurred only upon treatment of the virus and of the cells during infection time. The activity reported was always compared to control blank treatments of infected and uninfected cells. Eight plant extracts which showed the greatest antiviral activity were selected for further assays (marked by an asterisk in Table 1). The observed minimum effective concentrations were in the range of 30--80 /zg plant material per ml. In general, activity was observed at lower extract concentrations against the single-strand RNA Sindbis virus than against the double-strand DNA murine cytomegalovirus. In the extracts showing UVA enhancement of antiviral activity, only the virus particles in the presence of the extracts were exposed to UVA radiation before cellular infection.
Discussion and Conclusions The antiviral effect in these assays could have taken place at several different stages of the viral cycle. The extract could have inhibited viral infection by affecting the virus particle itself, by interfering with the virus-host cell recognition and entry process, or by interfering with the early steps of intracellular viral reproduction. Antiviral activity from a pre-infection treatment would indicate an interferon-stimulatory type effect. Wachsman
et al. (1987) have reported this type of effect against Sindbis virus from plant extracts of the Meliaceae. Activity from a post-infection treatment would suggest interference in the later steps of viral replication, or in viral packaging and release. Ethnopharmacological information from Yunnan was used as a guideline in the selection of plants to screen for antiviral activity. The two viruses used in the assays represent the major divisions in virus classification: RNA vs. DNA and single-strand vs. double-strand. Both viruses are membrane-enveloped. The method used comprises a general screening approach rather than one that is specific to the illnesses for which the plants were used. The usage of each plant in the traditional medicine of Yunnan served as a first indication for finding the potential biologically active components in the plants. In general, a plant is used for the treatment of several different illnesses and symptoms, usually in combination with other plants. The fact that 16 out of 31 plants tested demonstrated antiviral activity in cultured fibroblast cells emphasizes the important role of traditional medicine in the search for antiviral compounds from natural sources. Chemical separations of the active extracts coupled with bioassays of the fractions are continuing in our laboratory.
Acknowledgements The authors thank the Kunming Institute of Botany and the members of the Ethnobotany Laboratory for the kind assistance with logistics in Yunnan. We especially thank Lu Liping and Zhang Yen for being valuable guides on collection trips. Help and encouragement from Drs. He Zhegao, He Shixiu and Fan Bingjun are greatly appreciated. For assistance in plant collection and field identification, we thank Cui Jingyun in Xishuangbanna, Wang Chongyu in Kunming, and Xu Hua and Shi Zhaolong in Dali. Elizabeth Graham was most helpful with the bioassay techniques. Financial support for this research was provided by the Natural Sciences and Engineering Research Council of Canada.
References Becker, Y. (1984) Current trends in the research on antiviral drugs. In: Y. Becker (Ed.), Antiviral Drugs and Interferon. The Molecular Basis of Their Activity. Martinus Nijhoff, Boston, pp. I--9. Bruhn, J.G. and Holmstedt, B. (1981) Ethnopharmacology: Objectives, principles and perspectives. In: J.L. Beal and E. Reinhard (Eds.), Natural Products as Medicinal Agents. Hippokrates Verlag, Stuttgart, pp. 4 0 5 4 3 0 . Delaveau, P. (1981 ) Evaluation of traditional pharmacopoeias. In: J.L. Beal and E. Reinhard (Eds.), Natural Products as Medicinal Agents. Hippokrates Verlag, Stuttgart, pp. 395404. Duke, J.A. and Ayensu, E.S. (1985) Medicinal Plants of China Vol. I. Reference Publications, Algonac, Michigan, p. 40. Farnsworth, N.R., Akerele, O., Bingel, A.S., Soejarto, D.D. and Guo, Z. (1985) Medicinal plants in therapy. Bulletin qf the Worm Health Organization 63(6), 965--981. Hu, J.M. and Hsiung, G.D. (1989) Evaluation of new antiviral agents: I. In vitro perspectives. Antiviral Research 11, 217--232. Hudson, J.B. (1989a) Antiviral Compounds.[rom Plants. CRC Press, Boca Raton, FL, 200 pp. Hudson, J.B. (1989b) Plant photosensitizers with antiviral properties. Antiviral Research 12, 55--74. Kunming Institute of Botany (1978) Chinese Medicine Plants q f Yunnan. Tiangjing People's Publishing, Tiangjing, 535 pp. Kunming Reserve Unit Health Division (1970) Chinese Medicine Plants o f Yunnan. Tiangjing People's Publishing, Tiangjing, 777 pp.
Kyerematen, G.A. and Ogunlana, E.O. (1987) An integrated approach to the pharmacological evaluation of traditional materia medica. Journal of Ethnopharmacology 20, 191--207. Malone, M. (1983) The pharmacological evaluation of natural products - - general and specific approaches to screening ethnopharmaceuticals. Journal of Ethnopharmacolgy 8, 127--148. Mitchell, W.M. (1973) Active sites of the animal viruses: Potential sites of specific chemotherapeutic attack. In: W.D. Carter (Ed.), Selective Inhibitors of Viral Functions. CRC Handbook. CRC Press, Boca Raton, FL, pp. 51 -77. Pei, S. (1988) The Status of Ethnobotany in China. Paper presented to the first International Congress of Ethnobiology, Bel6m, Brazil, July 19--25, 1988. Towers, G.H.N. (1980) Photosensitizers in plants and their photodynamic action (a review). Progress in Phytochemistry 6, 183--202. Van Den Berghe, D.A., leven, M., Mertens, F. and Vlietinck, A.J. (1978) Screening of higher plants for biological activities: I1. Antiviral activity. Lloydia 41, 4 6 3 4 7 1 . Van Den Berghe, D.A., Vlietinck, A.J. and Van HooL L. (1985) Present and future status of plant products as antiviral agents. In: A.J. Vlietinck and R.A. Dommisse (Eds.), Advances in Medicinal Plant Research, Wissenschaftliche, Stuttgart, pp. 47--99. Wachsman, M.B., Damonte, E.B., Coto, C,E. and de Torre, R.A. (1987) Antiviral effects of Melia azedarach L. leaves extracts on Sindbis virus-infected cells. Antiviral Research 8, 1 12. Yunnan Institute of Medicine Inspection (1983) List of Yunnan Minority Tribe Medicines, Yunnan, 112 pp.
I
Screening of medicinal plants from Yunnan Province in southwest China for antiviral activity L. Y i p a, S. Pei c, J.B. H u d s o n b a n d G . H . N . T o w e r s a Departments qf "Botany and hMedical Microbiology, Universi O' of British Columbia, Vancouver, B. C. I/6 T 2B I (Canada) and ' Kunming Institute ~[' Botany, Kunming, Yunnan (China) (Accepted July 20, 1990)
In an ethnopharmacological screening of medicinal plants used in Y unnan province of China, ethanol extracts from 31 plant species were assayed for inhibition of murine cytomegalovirus and Sindbis virus infections. Parallel assays were carried out with and without exposure to UVA radiation to test for photo-mediation of activity. Antiviral activity was observed with 16 of the plant extracts, Eight plant extracts have been selected for further studies, with the objective of characterizing the antiviral constituents.
K~:v words." ethnopharmacology; antiviral screening: photo mediated antiviral activity.
Introduction
The significance of ethnopharmacologicai research in discovering new therapeutic activities of natural products has been discussed extensiviely (Bruhn and Holmstedt, 1981; Delaveau, 1981; Malone, 1983; Kyerematen and Ogunlana, 1987). According to Farnsworth et ai. (1985), 74% of the 121 bioactive plant-derived compounds currently in worldwide use were identified via research based on leads from folk- or ethnomedicine. With the increasing knowledge of the pathogenicity of viruses, the search for effective antiviral phytochemicals presents a growing area of pharmacological research (Van Den Berghe et al., 1978; Hudson, 1989a). Cell culture assays have become established procedures for the evaluation of antiviral chemotherapeutic potential, and some antiviral chemicals are now undergoing clinical trials and even clinical applications (Becker, 1984; Hu and Hsiung, 1989). The different possible sites of action for antiviral agents in viral replication process have been described by Mitchell (1973) and an overview of the process of developing anCorrespondence to." L. Yip, Departments of Botany. University of British Columbia, Vancouver, B.C. V6T 2BI, Canada. 0378-8741/$03.50 © 1991 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
tivirai agents from plants has been presented by Van Den Berghe et al. (1985). This laboratory has initiated an antiviral screening of plants of Yunnan for several reasons. Yunnan Province in the southwest of China is an area noted for its botanical diversity (Pei, 1988). Six hundred and seven species are listed in the Chinese Medicine Plants of Yunnan (Kunming Reserve Unit Health Division, 1970) and its sequel volume (Kunming Institute of Botany, 1978). As regards the number of plants in the traditional Chinese pharmacopeia, a data bank at the Chinese University of Hong Kong has 4,941 species of higher plants listed as having been used in medicine (Duke and Ayensu, 1985). In addition, there are over 20 ethnic minority groups in Yunnan. The medicinal uses of plants by these groups further increase the number of candidates for pharmacological investigation. Ethnobotanical information from the minority groups is in the process of being recorded (Yunnan Institute of Medicine Inspection, 1983). Clearly, Yunnan has rich resources in both plant and ethnobotanical knowledge and is an ideal study area for antiviral screening. In this project, plants were collected primarily on the basis of having documented or current use
in the traditional medicine of Yunnan for the treatment Of diseases such as colds, influenza and hepatitis that are now linked to viral causes. General ethnobotanical information, not restricted to the areas of Yunnan from which collections were made, was also considered. Thus, the same species of widely-distributed plants may be used by a number of different peoples. Extracts of these plants were tested for antiviral activity using cell culture systems. Duplicate assays were done in the presence or absence of long wave ultraviolet radiation (.UVA, 320--~00 nm) to test for the possibility that the activity of some might be lightmediated. The biological activity of many plant constituents can be dependent on, or enhanced by, simultaneous irradiation with light (Towers, 1980), Antiviral plant photosensitizers, particularly those activated by UVA, have been reviewed by Hudson (1989b).
China to Canada. For testing, each residue was stirred in 10 ml of 95% ethanol and the soluble fraction used. Cell culture and cytotoxicity assays Bioassays were performed using a cell culture system of the monolayer-forming murine fibroblast 3T3-L1 cell line (American Type Culture Collection). Cell layers for the assays were grown in 96-well microtiter plates (Falcon 3072). Cytotoxicity of the extracts was first tested by exposure of the cells to dilutions of the extracts in the culture medium (Dulbecco's modified Eagle A, with 10% fetal bovine serum), with the cells in suspension and after the formation of monolayers. The highest concentration of extract tested was the equivalent of 100 mg of starting plant material per ml. Two-fold serial dilutions were made from this concentration. The cells were observed for cytopathic effects (CPE).
Materials and Methods Plant collection The 31 medicinal plants assayed are listed in Table 1. Plant samples were collected from the Kunming area in central Yunnan, the Dali-Lijiang region in northwestern Yunnan, and Xishuangbanna, a region with a distinct tropical flora in the south of the province. With the help of Dr. Fan Bingjun in Dali, and Drs. He Zhegao and He Shixiu in Lijiang, collections were also made of plants currently used by herbal medicine doctors to treat viral-related diseases. Identification of the voucher herbarium specimens was verified by Mr. Li Yenhui at the Ethnobotany Laboratory of the Kunming Institute of Botany in Kunming, Yunnan. These voucher specimens have been deposited at the Herbarium of the University of British Columbia. Extract preparation Extracts were made from air-dried samples of the whole plant or, in a few cases, from the portion specified as being used medicinally . Ten-gram samples of plant material were percolated exhaustively in 90% ethanol and dried in vacuo at below 40°C. Drying the extracts to residue was done for ease of transportation of samples from
Antiviral assays In the antiviral assays two-fold serial dilutions of the plant extracts were used starting with the non-cytotoxic concentrations. The infectivity of two test viruses was assayed qualitatively through microscopic observations of characteristic viral CPE. Antiviral activity was determined by the absence of CPE in a treatment. Murine cytomegalovirus (MCMV) is a double strand DNA virus of the Herpesvirus group, while Sindbis is a single strand RNA virus of the Togavirus group. In a comprehensive antiviral assay, three separate segments of the viral life cycle were exposed to the plant extracts successively. The cell preparations were incubated with the extract dilutions during monolayer formation. The viruses, at a concentration of 1000 plaque forming units (pfu) per ml, were exposed to the same extract dilutions in phosphate buffered saline (PBS) at 4"C. The cell layers were then exposed to the virus/extract mixture for 1 h of adsorption time at 37"C. After removal of the virus mixture, the cells were incubated in medium with the same extract dilutions. Control cells were subject to the same treatments without the plant extracts. In the multiple-treatment assay, 16 plant extracts showed inhibition of viral infection. These
TABLE I ANTIVIRAL ACTIVITY OF Y U N N A N PLANTS AGAINST MURINE CYTOMEGALOVIRUS (MCMV) AND SINDBIS VIRUS (SV) Species listed by plant family
Part used"
Minimum active concentration (~g/ml) enhancement b MCMV
SV
APIACEAE
Centella asiatica (L.) Urban
PL
APOCYNACEAE Plumreria rubra L. var. acutifolia (Poir.) Ball.
RT
125
65
BERBERIDACEAE
Mahonia nepalensis DC.
BR
COMBRETACEAE
Quisqualis mdica L.
PL
--
COMPOSITAE Bidens pilosa L.
PL
125
CONVOLVULACEAE Dichondra repens Forst.
PL
--
125
CYCADACEAE Cycas siamensis Miq.
LF
--
160
12.5
65
EBENACEAE
Diospyros kaki L.f.
BK
EUPHORBIACEAE
*Euphorbia prolifera
PL
--
Ehrenb. ex. Boiss. Ricinus communis L.
RT
--
--
GENTIANACEAE Halenia ellipt&'a D.Don
PL
GRAMINEAE Phyllostachy sp.
PL
BU
--
125
PL PL PL
65 -65
65
PL PL
---
-125
PL
--
65
65
IRIDACEAE
Belamcanda chinensis DC. LABIATAE
*Acrocephalus indicus Briq. Elsholtzia densa Briq. *EIsholt=ia rugulosa
30
(Hemsl.) Levi.
Perillafrutescens (L.) Britton Rabdosia phyllostachys (Diels) Hara
*Stachys kouyangensis (Vaniot) Dunn
UVA
4
TABLE 1 (Continued) Species listed by plant family
Part used a
Minimum active concentration (/zg/ml) enhancement b MCMV
SV
UVA
LEGUMINOSAE Kummerowia striata
PL
(Thunb.) Schindl. MYRSINACEAE * Embelia sessiliflora Kurz
RT
30
30
OXALIDACEAE Oxalis corniculata L.
PL
PTERIDACEAE Stenoloma chusanum (L.) Ching
RUBIACEAE Hedyotis uncinella Hook. et Arn.
PL
RUTACEAE * Boenninghausenia sesilicarpa
PL
80
+
Levi. SAURURACEAE Houttuynia cordata Thunb.
PL
SCHOPHULARIACEAE * Siphonostegia chinensis Benth. Scutellaria orthocalyx Hand.-Mazz.
PL PL
40
VERBENACEAE * Verbena of/~cinalis L.
PL
65
30
'LPart used: BK, bark; BR, branch; BU, bulb: LF, leaf: PL, whole plant: RT, root. bUVA = long wave ultraviolet radiation 1350 nm). * = active species selected for further assays.
extracts were a s s a y e d again following the same p r o c e d u r e but with three parallel p r e p a r a t i o n s o f cells. I n s t e a d o f one set o f cells being subjected to three successive extract t r e a t m e n t s , each set was e x p o s e d to one o f the treatments. O n e set was exp o s e d to the extract p r i o r to infection, the s e c o n d to the virus with extract d u r i n g a d s o r p t i o n time, a n d the third o n l y after the r e m o v a l o f virus particles. F o r the t r e a t m e n t d u r i n g a d s o r p t i o n , the virus s u s p e n s i o n was e x p o s e d to the extract for 30 min p r i o r to i n c u b a t i o n with the cells. F u r t h e r
assays were carried o u t with extracts o f eight species. T h e respective extracts in e t h a n o l were filtered ( W h a t m a n p a p e r N o . l ) , e v a p o r a t e d to dryness a n d the residue taken up in I00% m e t h a nol. T h e m e t h a n o l phases were assayed following the same p r o c e d u r e , with extract t r e a t m e n t o f the virus suspension a n d o f the cells d u r i n g virus adsorption. UVA
irradiation
In the assays, one set o f cells a n d the virus sus-
pension with the extracts were exposed to 30 min of UVA radiation (350 nm), while a parallel set was kept in the dark. An extract was considered to show enhancement of activity by UVA radiation when antiviral effects were observed at lower concentrations of the extract, compared to the parallel culture which was kept in the dark.
Results In the multiple treatment assay, 16 plant extracts showed qualitative antiviral activity at noncytotoxic concentrations (Table 1). The antiviral effects were observed in cells treated with concentrations of extract in the range equivalent to 30 /~g/ml to 1.0 mg/ml of original material. When the cell cultures were exposed to extract treatments separately at the pre-infection, virus/infection, and post-infection stages, the antiviral activity in all cases occurred only upon treatment of the virus and of the cells during infection time. The activity reported was always compared to control blank treatments of infected and uninfected cells. Eight plant extracts which showed the greatest antiviral activity were selected for further assays (marked by an asterisk in Table 1). The observed minimum effective concentrations were in the range of 30--80 /zg plant material per ml. In general, activity was observed at lower extract concentrations against the single-strand RNA Sindbis virus than against the double-strand DNA murine cytomegalovirus. In the extracts showing UVA enhancement of antiviral activity, only the virus particles in the presence of the extracts were exposed to UVA radiation before cellular infection.
Discussion and Conclusions The antiviral effect in these assays could have taken place at several different stages of the viral cycle. The extract could have inhibited viral infection by affecting the virus particle itself, by interfering with the virus-host cell recognition and entry process, or by interfering with the early steps of intracellular viral reproduction. Antiviral activity from a pre-infection treatment would indicate an interferon-stimulatory type effect. Wachsman
et al. (1987) have reported this type of effect against Sindbis virus from plant extracts of the Meliaceae. Activity from a post-infection treatment would suggest interference in the later steps of viral replication, or in viral packaging and release. Ethnopharmacological information from Yunnan was used as a guideline in the selection of plants to screen for antiviral activity. The two viruses used in the assays represent the major divisions in virus classification: RNA vs. DNA and single-strand vs. double-strand. Both viruses are membrane-enveloped. The method used comprises a general screening approach rather than one that is specific to the illnesses for which the plants were used. The usage of each plant in the traditional medicine of Yunnan served as a first indication for finding the potential biologically active components in the plants. In general, a plant is used for the treatment of several different illnesses and symptoms, usually in combination with other plants. The fact that 16 out of 31 plants tested demonstrated antiviral activity in cultured fibroblast cells emphasizes the important role of traditional medicine in the search for antiviral compounds from natural sources. Chemical separations of the active extracts coupled with bioassays of the fractions are continuing in our laboratory.
Acknowledgements The authors thank the Kunming Institute of Botany and the members of the Ethnobotany Laboratory for the kind assistance with logistics in Yunnan. We especially thank Lu Liping and Zhang Yen for being valuable guides on collection trips. Help and encouragement from Drs. He Zhegao, He Shixiu and Fan Bingjun are greatly appreciated. For assistance in plant collection and field identification, we thank Cui Jingyun in Xishuangbanna, Wang Chongyu in Kunming, and Xu Hua and Shi Zhaolong in Dali. Elizabeth Graham was most helpful with the bioassay techniques. Financial support for this research was provided by the Natural Sciences and Engineering Research Council of Canada.
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