Antiviral activity of some plants used in Nepalese traditional medicine.

Methanolic extracts of 41 plant species belonging to 27 families used in the traditional medicine in Nepal have been investigated for in vitro antiviral activity against Herpes simplex virus type 1 (HSV-1) and influenza virus A by dye uptake assay in the systems HSV-1/Vero cells and influenza virus A/MDCK cells. The extracts of Astilbe rivularis, Bergenia ciliata, Cassiope fastigiata and Thymus linearis showed potent anti-herpes viral activity. The extracts of Allium oreoprasum, Androsace strigilosa, Asparagus filicinus, Astilbe rivularis, Bergenia ciliata and Verbascum thapsus exhibited strong anti-influenza viral activity. Only the extracts of A. rivularis and B. ciliata demonstrated remarkable activity against both viruses.

Introduction

Plants have long been used as a source of medicine from ancient time to today all over the world. In developing countries the availability of modern medicines is limited. So traditional medicine is still the mainstay of health care and most drugs come from plants. Although many plants have long been recognized and widely used in Nepalese traditional medicine, some are relatively unexplored and not arrived to mainstream medicine (1). Therefore, the search on new drugs must be continued and natural products from plants, microorganisms, fungi and animals can be the source of innovative and powerful therapeutic agents for newer, safer and affordable medicines (2,3). On the other hand the screening of plants as a possible source of antiviral drugs has led to the discovery of potent inhibitors of in vitro viral growth (4–11).

Therefore, the present investigation was carried out to assess the antiviral effects of some native plants used by the local people belonging to Gurungs and Thakalis of Manang and Mustang districts that lie in the Annapurna Conservation Area Project (ACAP). Permission for the field study as well as the collection of voucher specimens was received from the headquarters of ACAP in Pokhara. The plants were selected on the basis of ethnopharmacological records, so the prospect of finding new bio-active compounds is always promising.

Methods

Plant Materials and Preparation of Extracts

The plants were collected in the Manang and Mustang district of Nepal during summer 2004 and 2005 and dried in shady place. The plants were authenticated by Prof. Ram P. Chaudhary, Central Department of Botany, Tribhuvan University, Kathmandu, Nepal and voucher specimens were deposited in the Tribhuvan University Central Herbarium (TUCH), Kirtipur, Nepal. The name of the plants, respective families, the parts used for the extract preparation and traditional uses of the plants are listed in Table 1.

Table 1.

Name of the plants, respective families, parts used for extraction and major traditional use(s)

Name of plant	Family	Collected part(s)	Vernacular (Gurung) name	Voucher no.	Major traditional use(s)
Abies spectabilis Spach.	Pinaceae	Leaves	Kye	342	Bone fracture
Allium oreoprasum Schrenk	Alliaceae	Whole plant	Lungho	2104	Cough, cold, sore throat
Allium prattii C.H. Wright	Alliaceae	Whole plant	Banlasun	493	Vegetables
Anaphalis busua DC.	Asteraceae	Leaves	Phosorosan	463	Cough, cold, sore throat
Anaphalis busua DC.	Asteraceae	Flowers	Phorosan	463	Cough, cold, sore throat
Androsace strigilosa Franch.	Primulaceae	Whole plant	Gadhikanakyo	169	Fever, edema
Anemone rivularis Buch.-Ham. ex DC.	Ranunculaceae	Roots	Angsoup	492	Cough, cold, stomachache
Arisaema flavum Schott	Araceae	Tubers	Timtry	618	Skin disease, wounds
Artemisia caruifolia Roxb.	Asteraceae	Whole plants	Bajha	421	Incense
Asparagus filicinus Buch.-Ham. ex D. Don	Asparagaceae	Tubers	Nirshing	2125	Tonic, menstrual problem
Astilbe rivularis Buch.-Ham. ex D. Don	Saxifragaceae	Rhizomes	Bhadhangoo	2070	Headache, improve fertility
Bergenia ciliata (Haw.) Sternb.	Saxifragaceae	Rhizomes	Pakhanved	2075	Diarrhea, dysentery, stomachache
Bistorta affinis Greene	Polygonaceae	Root	Khaldi	203	Cough, cold, tonsillitis, fever
Cassiope fastigiata D. Don	Ericaceae	Aerial parts	Sunpathi	433	Incense
Clinopodium umbrosum Matsum	Lamiaceae	Aerial parts	Sarshang	155	High blood pressure, pain, inflammation of body
Cotoneaster integrifolius (Roxb.) Klotz	Rosaceae	Fruits	Tsharsin	168	Edible
Delphinium brunonianum Royle	Ranunculaceae	Whole plant	Ponmar	262	Fever, jaundice
Dicranostigma lactucoides Hook.f. & Thomson	Papaveraceae	Whole plant	Rhafendhi	105	Easy delivery of baby (animals only)
Euphorbia longifolia D. Don	Euphorbiaceae	Root	Dhurbi	2018	Cough, cold, fever, skin disease
Geranium donianum Sweet	Geraniaceae	Aerial part	Kagheshurti	153	Gingivitis, toothache
Hyoscyamus niger var. agrestis (Kit.) Beck	Solanaceae	Flower	Lantang	2236	Anti-inflammatory
Juniperus squamata Buch.-Ham. ex Lamb	Cupressaceae	Aerial part	Sukri	265	Fever, cough, cold, skin disease
Maharanga emodi DC.	Boraginaceae	Roots	Maharangi (Nepali)	2071	Ear pain
Morina longifolia Wall. ex DC.	Morinaceae	Roots	Changtser goepa		Edema, stomachache, headache
Neopicrorhiza scrophulariiflora (Pennell) D.Y. Hong	Scrophulariaceae	Roots	Kutki	431	Fever, cough, cold, tonsillitis
Oxytropis williamsii I. T. Vassilchenko	Fabaceae	Whole plants	Sinshi	329	Wound healing, coagulate blood
Primula involucrata Sw. ex Duby	Primulaceae	Whole plants	Chyonker	178	Vegetable
Rhododendron anthopogon D. Don	Ericaceae	Aerial part	Palu, Sangalin	210	Reduce blood pressure, fever, inflammation
Rhododendron lepidotum Wall. & G. Don	Ericaceae	Aerial part	Bhaiunakpo	2122	Fever, cough, cold, tonsillitis
Rosa macrophylla Lindl.	Rosaceae	Flower	Seghu	343	Fever, diarrhea, dysentery
Rosa macrophylla Lindl.	Rosaceae	Fruits	Seghu	343	Nutrition in cold, cough
Rosa sericea Lindl.	Rosaceae	Fruits	Sewa	102	Diarrhea, dysentery, stomachache, dyspepsia
Rubus foliolosus D. Don	Rosaceae	Root	Mapalan	2019	Fever, dyspepsis, cough, cold, vertigo
Salix serpyllum Andersson	Salicaceae	Aerial part	Langmanackpo	2015	Stomachache, diarrhea, dysentery
Saussurea auriculata (DC.) Sch. Bip.	Asteraceae	Whole plant	Ta	283	Blood circulation
Saussurea fastuosa (Decne) Sch. Bip	Asteraceae	Aerial part	Singamindro	303	Cut, bleeding
Swertia ciliata (G. Don) B. L. Burtt	Gentianaceae	Whole plant	Tiktha	311	Fever due to stomach and liver disorder
Thalictrum cultratum Wall.	Ranunculaceae	Roots and stem	Nagghunensa	121	Fever, diarrhea (for animal only)
Thymus linearis Benth.	Lamiaceae	Whole plant	Akhino	126	Eye infection
Urtica dioica L.	Urticaceae	Leaves	Polo	409	Cough, cold
Valeriana jatamansi Jones	Valerianaceae	Roots	Nappu	2072	Sedative, headache
Verbascum thapsus L.	Scrophulariaceae	Aerial part	Yugisingh	195	Wound healing, urinary disease, edema
Zanthoxylum armatum DC.	Rutaceae	Fruits	Prumo	2183	Cough, cold, tonsillitis

The dried and powdered plant material (each 10 g) was extracted successively with n-hexane, dichloromethane and methanol in a soxhlet extractor for each 8 h. Evaporation of the solvent followed by drying in vacuum gave the respective crude dry extract. Only methanol extract was used for the antiviral assay, n-hexane and dichloromethane extracts were not included because of their insolubility in medium and high toxicity to the cells. Each 2 mg of the extract was dissolved in 10 μl dimethylsulfoxide (DMSO) before adding tissue culture medium supplemented with 2% fetal calf serum (FCS, GIBCO Life science technologies, Paisley, UK) and stocked at a concentration of 2 mg ml−1.

Cells and Viruses

Madine–darby canine kidney (MDCK) and African green monkey kidney (Vero) cells (cell bank of the Friedrich-Loeffler-Institute, Federal Research Institute for Animal Health, Greifswald-Insel Riems, Germany) were maintained in Eagle's minimal essential medium (MEM) supplemented with 5% FCS (GIBCO, Paisley, UK). The exponentially growing cells were harvested and seeded at a cell density of 60 000/well in a 96 well microtiter plate (8 mm diameter, Falcon Plastic, NJ) and incubated for 24 h at 37°C with 5% carbondioxide in a 90% humidified chamber so as to form confluent monolayers.

Human influenza virus A/WSN/33 (H1N1) London was obtained from the strain collection of the Institute of Medical Microbiology, University Greifswald, Germany, and propagated in embryonated hen eggs for 72 h. The infected allantoic fluids were harvested, the hemagglutination (HA) titer and virus infectivity were determined on MDCK cells and the virus stock was stored at −70°C.

Herpes simplex virus type 1 (HSV-1, strain KOS) was obtained from the strain collection of the Consiliar and Reference Center for Alpha Herpes Virus Infection, Institute of Virology and Antiviral Therapy, University Jena, Germany and propagated in Vero cells. The virus infected cells were frozen and thawed and the virus suspension was titrated on Vero cells and stored at −70°C (7).

Cytotoxicity Assay

The cellular toxicity of extracts on Vero and on MDCK cells was assessed by dye uptake method using neutral red (12) in 96-well tissue culture plates (8 mm diameter, Falcon Plastic, NJ). Only living cells are able to manage the active uptake of neutral red. Confluent monolayers of cells were treated with 100 μl 2-fold serial dilutions of extracts prepared at concentrations of 200, 100, 50 and 25 μg ml−1 in four replicates and incubated at 37°C in a humidified atmosphere of 5% CO2 for 72 h. The supernatant was removed and 200 μl neutral red solution (0.005%) in optimum was added. The microtiter plate was further incubated for 3 h at 37°C. After removal of the supernatant, the dye incorporated by the viable cells was extracted with 100 μl ethanol/water/glacial acetic acid solution (50 : 50 : 1) by shaking for 15 min. The absorbance was measured on an ELISA reader using Ascent software at 540 nm. The cytotoxic concentration that caused the reduction of viable cells by 50% [CC50] was calculated from dose–response curve.

Antiviral Assay

Antiviral activity was determined by dye uptake assay using neutral red as described by Mothana et al. (7). Non-cytotoxic extracts were tested in concentrations of 100, 50, 25, 12.5 and 6.25 μg ml−1. The antiviral tests of cytotoxic extracts started with the half of the individual CC50. The extracts were diluted 1 : 2 by medium. Confluent monolayers of Vero and MDCK cells were treated with 100 μl of extracts in four replicates for 30 min. After that Vero cells were infected with 30 TCID50 of HSV-1 and MDCK cells with 30 TCID50 of influenza virus A and incubated for 72 h at 37°C. TCID50 (tissue culture infectious dose) is the virus dose that leads to the infection of 50% of the cells. The virus suspension and dilution medium without samples were added, respectively, to the cell cultures to serve as the virus control and cell control. The supernatant was replaced by 200 μl neutral red solution (0.005%) and the cells were incubated for 3 h at 37°C. After removal of the supernatant, the dye incorporated by viable cells was eluted with 100 μl ethanol/water/glacial acetic acid solution (50 : 50 : 1) by shaking for 15 min. The absorbance was measured at 540 nm and the percentage protection was calculated by the following formula (13): where, (ODT)V, (ODC)V and (ODC)M correspond to absorbances in virus infected cells with test compounds, virus infected cells without test compounds and the mock infected control (assay without viruses), respectively.

Amantadine HCl and acyclovir were used as reference compounds in concentrations of 0.1, 1, 10 and 100 μg ml−1.

Results

Cytotoxicity of Extracts for Vero Cells

In this study, 43 methanolic extracts from 41 different plant species belonging to 27 families (Table 1) were screened for their antiviral activity against herpes simplex virus and influenza virus A by dye uptake assay. By methanolic extraction, a broad spectrum of compounds with different polarity can be obtained. As prerequisite for antiviral tests, the cytotoxicity of the extracts against virus-host cells was investigated. The results are summarized in Table 2.

Table 2.

Antiviral activities of plants used in Nepalese ethnomedicine

Plant extracts	Percentage yield of MeOH extract	Antiviral activity HSV-1/Vero cells		Antiviral activity Influenza A/MDCK cells
		Cytotoxicity CC50 (μg/ml)*	Antiviral activity IC50 (μg/ml)†	Cytotoxicity CC50 (μg/ml)*	Antiviral activity IC50 (μg/ml)†
Abies spectabilis	23.4	>100	–	>100	17
Allium oreoprasum	17.8	>100	–	>100	8
Allium prattii	7.5	>100	–	>100	97
Anaphalis busua Leaves	12.2	>100	–	>100	–
Anaphalis busua Flower	13.6	>100	–	>100	–
Androsace strigilosa	18.2	12.5	–	40	10
Anemone rivularis	14.5	21	–	40	–
Arisaema flavum	14.1	>100	–	>100	–
Artemisia caruifolia	12.3	92	–	22	–
Asparagus filicinus	18.7	40	–	30	<6.25
Astilbe rivularis	52.1	67	<6.25	>100	<6.25
Bergenia ciliata	33.2	35	<6.25	>100	9
Bistorta affinis	14.3	>100	80	>100	50
Cassiope fastigiata	18.2	>100	<6.25	>100	78
Clinopodium umbrosum	14.0	76	19	>100	–
Cotoneaster integrifolius	22.0	>100	18	>100	44
Delphinium brunonianum	12.3	11	–	25	–
Dicranostigma lactucoides	21.1	72	–	50	–
Euphorbia longifolia	18.5	25	–	19	–
Geranium donianum	24.4	89	–	69	–
Hyoscyamus niger	18.7	>100	–	50	40
Juniperus squamata	16.7	>100	82	>100	–
Maharanga emodi	14.7	>100	–	>100	29
Morina longifolia	5.9	>100	–	>100	–
Neopicrorhiza scrophulariiflora	38.3	>100	–	>100	–
Oxytropis williamsii	27.5	>100	78	>100	33
Primula involucrata	31.7	50	–	63	–
Rhododendron anthopogon	22.1	>100	50	>100	44
Rhododendron lepidotum	18.9	100	–	>100	58
Rosa macrophylla Flower	11.2	86	–	>100	45
Rosa macrophylla Fruits	10.5	74	–	>100	–
Rosa sericea	14.2	>100	–	>100	–
Rubus foliolosus	21.2	>100	50	>100	–
Salix serpyllum	26.2	>100	–	>100	–
Saussurea auriculata	11.4	31	–	100	42
Saussurea fastuosa	8.3	>100	–	>100	–
Swertia ciliata	6.2	>100	–	>100	–
Thalictrum cultratum	18.7	23	–	86	32
Thymus linearis	5.2	69	12.5	45	–
Urtica dioica	7.8	>100	–	>100	–
Valeriana jatamansi	50.1	>100	–	>100	20
Verbascum thapsus	12.3	>100	–	>100	<6.25
Zanthoxylum armatum	6.7	>100	–	36	–
Acyclovir			0.7
Amantadine HCl					16.8

*CC50 = the concentration that causes the reduction of viable cells by 50%; †IC50 = the concentration that protects 50% of the cells against destruction by viruses; – No measurable effect.

The values are the mean of four experiments.

The extracts of Androsace strigilosa, Anemone rivularis, Delphinium brunonianum, Euphorbia longifolia and Thalictrum cultratum exhibited strong cytotoxicity in Vero cells with CC50 (the concentration that causes the reduction of viable cells by 50%) ranging from 12.5 to 25 μg ml−1. A moderate cytotoxicity was observed for the extracts of Asparagus filicinus, Bergenia ciliata, Primula involucrata and Saussurea auriculata with CC50 ranging from 30 to 50 μg ml−1. Other eight extracts showed very mild toxicity while rest of the extracts were non-toxic at 100 μg ml−1.

Cytotoxicity of Extracts for MDCK Cells

Similarly, in MDCK cells extracts of Artemisia caruifolia, D. brunonianum and E. longifolia showed strong toxicity with CC50 ranging from 19 to 25 μg ml−1. A moderate toxicity was exhibited by the extracts of A. strigilosa, A. rivularis, Asparagus filicinus, Dicranostigma lactucoides, Hyoscyamus niger, Thymus linearis and Zanthoxylum armatum with CC50 ranging from 30 to 50 μg ml−1. Other three extracts demonstrated very low toxicity while rest of the extracts were non-toxic at 100 μg ml−1.

Antiviral Activity of Extracts Against HSV-1

Antiviral activity against HSV-1 was shown by 11 extracts at non-cytotoxic concentrations. The IC50 values (the concentration that protects 50% of the cells against destruction by viruses) ranged from <6.25 to 82 μg ml−1. The highest activity against HSV-1 with IC50 values <6.25 μg ml−1 was observed for the extracts of A. rivularis, B ciliata, Cassiope fastigiata and T. linearis. Moderate activity was shown by Cotoneaster integrifolius (IC50 18 μg ml−1) and Clinopodium umbrosum (IC50 19 μg ml−1). Weak activity (IC50 50–82 μg ml−1) was found in the extracts of Bistorta affinis, Juniperus squamata, Oxytropis williamsii, Rhododendron anthopogon and Rubus foliolosus.

Antiviral Activity of Extracts Against Influenza Virus A

Antiviral activity against influenza virus A was shown by 20 extracts at non-cytotoxic concentrations. The IC50 values ranged from <6.25 to 97 μg ml−1. The highest activity was shown by the extracts of A. filicinus, A. rivularis and Verbascum thapsus with IC50 < 6.25 μg ml−1. In addition, the extracts of Allium oreoprasum, A. strigilosa and B. ciliata also exhibited high activity (IC50 values from 8 to 10 μg ml−1). Moderate activity (IC50 values from 17 to 50 μg ml−1) was demonstrated by 11 extracts. Weak activity (IC50 values from 78 to 97 μg ml−1) was shown by three extracts (Table 2).

The extracts of A. rivularis and B. ciliata were found to be highly active against both viruses.

Discussion

The results of this work justify the potential of some of the investigated plants for the production of bioactive compounds. The phytochemical knowledge about these plants is so far very limited. The active principles present in A. rivularis are still unknown. Phytochemical investigation of A. rivularis revealed the presence of flavonoids, terpenoids and bergenin (14,15).

Bergnia ciliata is known to contain phenolic compounds (16). Polyphenols, especially high polymeric procyanidines possess strong anti-influenza viral activity (17), which is in agreement with our previous study (18). In our previous study (19), methanol–water extract of Bergenia ligulata, which is taxonomically closely related to B. ciliata, inhibited the growth of influenza virus A in cell culture with IC50 of 10 μg ml−1. The extract also inhibited the viral protein and nucleic acid synthesis (18). In the present study, the methanol extract of B. ciliata inhibited the influenza virus A and HSV-1 indicating that the genus Bergenia could be the source of potent antiviral drugs. Again potent activity of A. rivularis against both viruses indicated the high prospect of finding antiviral drugs in Saxifragaceae family.

No antiviral compounds have previously been isolated from A. filicinus. The plant is known to contain steroidal saponins (20,21), furostanol glycosides (22) and furostanosides (23,24). The phytochemicals possibly responsible for the high activity of C. fastigiata against HSV are not described. Some Cassiope species are reported to contain flavonoid glycosides (25). Similarly, the compounds responsible for the high anti-influenza viral activity of A. oreoprasum and A. strigilosa are not reported elsewhere.

Likewise, no antiviral constituents have been isolated from C. integrifolius, C. umbrosum and T. linearis. Other members of the genus Cotoneaster, have been found to possess phenolic glycosides (Cotoneaster orbicularis, 26), flavonols and isoflavones (Cotoneaster simonsii, 27). From the other member of the genus Clinopodium, C. chinensis var. parviflorum, oleanane triterpene saponins have been isolated (28).

Whereas for the extract of V. thapsus, antiherpes activity has been reported (29); our study revealed only the strong anti-influenza viral activity. However, no antiviral compounds have previously been isolated. The plant is known to contain phenylethanoid and lignan glycosides (30). On the other hand, the phytochemicals responsible for anti-influenza viral activity could be different from anti-herpes activity and also the amount of active constituents present in the plants depends on the geographical distribution, season of collection and climatic and ecological condition at the collection site.

Looking at the chemical structures of the already identified compounds, most of these substances should be extracted by methanol. The foregoing extraction by more lipophilic solvents (n-hexane and dichlormethane) alleviates the methanolic extraction and the planned fractionation.

Comparing the use of plants in traditional medicine and their antiviral activity, a direct correlation could be established for some plants, e.g. A. oreoprasum, A. strigilosa (anti-influenza activity) and T. linearis (antiherpes activity). For other plants, e.g. C. fastigiata, which exhibited potent anti-herpes activity, this cannot be recognized till now.

The extracts that exhibited only medium and low activity, could also be the source of potential antiviral drugs because the bioactive compounds may be present in too low concentrations to show effective antiviral activity at non-toxic concentration. Further fractionation and separation of extract(s) may reveal potent antiviral activity (31).

Our results indicate that several plants used in Nepalese traditional medicine could be the lead to potential antiviral drugs, which possibly provide molecules with drug-like properties and with incredible structural diversity. Besides, the results are useful for rationalizing the use of medicinal plants in primary health care in Nepal. The phytochemical characterization of the extracts, the identification of the responsible bioactive compounds and the elucidation of the mode of action and quality standards are necessary.