Genital herpes, usually caused by Herpes Simplex Virus type-2 (HSV-2), is the commonest sexually transmitted disease especially amongst rural women in Southern Africa including Zimbabwe. This predisposes them to HIV/AIDS infection, cancer and opportunistic infections (OIs). Current antiviral treatments are often cytotoxic and/or ineffective. This motivates active research to find alternative safer drugs or lead drugs from traditional medicinal sources. Twenty six (26) methanol extracts from commonly used and often endangered plant species (14) used by communities and traditional medical practitioners for treating illnesses and sexually transmitted diseases from 5-selected districts of Zimbabwe were investigated for toxicity by Brine shrimp lethality test (BSLT) and by 50% Cytopathic effect on VERO cultured cells. The extracts were also tested for antiviral activity against Herpes Simplex Virus-2 (HSV-2) by the End Point Titration Technique (EPTT) and Neutralisation Test (NT) on VERO cells. Results from the BSLTs ranged 66.66 - 4304 μg/ml; 50% Cytopathic effect from 19.53 - 312 μg/ml whilst the NT ID 50 values ranged from 10.41 - 125 μg/ml. The antiviral EPTT reduction factor (RF) was 1 - 10 4 with 13 extracts showing RF ≥ 10 3 . All the plant extracts had moderate to high toxicity (LC 50 , 789 - 66 μg/ml) in the BSLT. Six extracts had LC 50 values greater than 1000 μg/ml. All 26 extracts were cytotoxic with CC 50 values < 500 ug/ml of which 19 were more toxic CC 50 < 100 ug/ml. Nine extracts had in vitro therapeutic indexes ≥ 3.7. Cassia abbreviata , Dichrostachys cinerea and Hypoxis hemerocallidea had therapeutic indexes (TI) 7.5 - 15.0. The more active plant extracts were from roots and root tubers. The results confirm the rationale for the use of traditional medicinal plants by traditional medical practitioners for treating various diseases and could bring awareness for their better use and improve conservation. The results also provide an opportunity to develop more efficacious drugs by isolating lead compounds and determining their mode of action.
Third world countries are often endowed with rich flora and fauna which are put to good use in their traditional medical practices. Zimbabwe has over 5000 plant species of which 500 are established in Traditional Medicine (TM) since being legalised in 1981 through an Act of Parliament [
Ethno-botanical surveys on traditional medicinal plants used for treating common ailments, those most traded and threatened were carried out in five districts: Bulilima, Chimanimani, Chipinge, Mangwe and Matobo and the plants identified and authenticated by the National Herbarium and Botanic Gardens, Harare, as previously reported [
Selected plant parts were powdered (30 g), macerated in methanol (200 ml) [
Bioactivity testing using the Brine Shrimp (Artemia salina) Lethality Test (BSLT) method was carried out in triplicate with different concentrations of the sterilized plant extracts in brine solution (10 µg/ml - 1000 µg/ml) and the percentage lethality of the nauplii determined [
The lyophilized methanol extracts were dissolved in DMSO to a concentration of 10 mg/ml. Vero cells (African green monkey kidney) (Highveld Ltd., South Africa) were grown and maintained in essential growth medium supplemented with 5% foetal calf serum and incubated under 5% carbon dioxide at 37˚C. The confluent cells were removed and trypsinated with phosphate buffered saline solution to achieve a cell concentration of 1 - 2 × 104 cells per well for VERO cells.
Herpes simplex virus 2 (HSV-2) (Highveld Ltd., South Africa) was propagated on Vero cells and the recovered virus suspension (1 ml) diluted by infecting Vero confluent monolayers grown on microtitre plates with 0.1 ml of serial tenfold dilutions of the virus suspension quadruplicated and observed for 7 days for cytopathic effect (CPE). The HSV-2 virus titre was obtained from the 50% tissue culture infections dose per ml (TCID50/ml) [
Cytotoxicity of the different plant extracts that could cause non-specific cytopathic effect (CPE) on confluent Vero cells in growth medium was determined by using 100 ml of serial two fold dilution of the plant extracts onto confluent Vero cells on microtitre plates in quadruplicate. The 50% cytotoxicity concentration (CC50) was defined as the plant extract concentration causing 50% CPE compared to uninfected cells [
Antiviral assays were carried out using the Neutralization test (NT) to determine non-cytotoxic concentration (ID50) that inhibits/protects 50% of the monolayer cells against destruction by the virus compared to uninfected cells using the Spearman-Karber formula [
The bioactivity of the plant extracts using the Brine shrimp lethality test (BSLT) indicated that plants in this category were relatively toxic with 15 of the 26 extracts recording LC50 < 500 µg/ml (
Cytotoxicity effects for the extracts on Vero cells showed all 26 extracts had CC50 < 500 ug/ml, 19 with CC50 < 100 ug/ml and 12 with CC50 < 50 ug/ml (
No | Species, family name, voucher number, status | Plant part | BSLT LC50 (ug/ml) | CC50 (50% CPE) (ug/ml) | NT-ID50 (ug/ml) | TI: CC50/ID50 | EPTT (RFb) |
---|---|---|---|---|---|---|---|
1 | Cassia abbreviate Oliv., Fabaceae (2721): Cm. Status: Cm | lf rt bk | 454.93 ± 18.60 445.72 ± 22.15 1319.37 ± 356.63 | 156.25 156.25 39.06 | 20.83 10.41 NA | 7.5 15.0 - | 102 103 1 |
2 | Dichrostachys cinerea (L.) Wight and Arn. Mimosaceae (32): Cm. | lf rt | 539.39 ± 78.24 4304.59 ± 685.69 | 78.13 312.50 | 10.41 83.33 | 7.5 3.7 | 104 102 |
3 | Elaedendron matabelicum (Loes). Steedman Celastraceae (2121): Th., Endemic | rt | 1012.31 ± 217.69 | 78.13 | NA | - | 1 |
4 | Elephantorrhiza goetzei Harms. Fabaceae (7136): Th. | rt | 356.55 ± 24.55 | 156.25 | 83.33 | 1.9 | 102 |
5 | Flacourtia indica (Burm. f.) Merr. Flacourticaceae (57/62): Th. | lf rt | 281.81 ± 26.14 467.31 ± 39.01 | 78.13 156.25 | 83.33 125.00 | 0.9 1.3 | 102 103 |
6 | Gymnosporia senegalensis (Lam.) Loes. Celastraceae (15): Cm. | lf rt tw | 789.37 ± 104.06 2185.61 ± 872.25 754.70 ± 182.57 | 39.06 78.13 19.53 | 10.41 20.83 15.63 | 3.8 3.8 1.2 | 103 103 103 |
7 | Hypoxis hemerocallidea Fisch. & Ave’-Lall. Hypoxidaceae (MTDV06): Th. | tb | 735.34 ± 89.39 | 156.25 | 10.41 | 15.0 | 103 |
8 | Khaya anthotheca (Welw.) DC. Meliaceae (892): Cm. | bk | 482.19 ± 43.49 | 39.06 | 31.25 | 1.2 | 102 |
9 | Kigelia africana (Lam.) Benth. Bignoniaceae (5990): Cm. | bk ft rt | 262.20 ± 25.07 117.41 ± 30.27 501.35 ± 34.88 | 39.06 39.06 39.06 | 31.25 31.25 NA | 1.2 1.2 - | 103 104 1 |
10 | Rhuschirindensis Baker f. Anacardiaceae (103/67): Cm. | lf rt | 1023.26 ± 161.69 316.60 ± 30.07 | 312.50 78.13 | NA 20.83 | - 3.8 | 1 102 |
11 | Sclerocarya birrea (A. Riich.) Hochst. Anacardiaceae (3114): Th, Cm. | bk | 1112.37 ± 210.04 | 39.06 | 20.83 | 1.9 | 103 |
12 | Securidaca longepedunculata Fresen. Polygalaceae (264/59): V Th. | rt | 351.89 ± 35.70 | 78.13 | 20.83 | 3.8 | 103 |
13 | Terminalia sericea Burch. Ex. DC. Combretaceae (5): Th., endemic | lf rt | 66.66 ± 49.31 295.33 ± 37.19 | 39.06 39.06 | 31.25 20.83 | 1.2 1.9 | 102 103 |
14 | Warburgia salutaris (Bertol.f.) Chiov. Canellaceae (CPDV06): V Th. | lf rt bk | 351.41 ± 29.58 426.10 ± 55.55 359.66 ± 14.33 | 78.13 39.06 19.53 | NA 31.25 NA | - 1.2 - | 1 103 1 |
15 | Nerium oleander (ref-positive control) | lf | 141.67 ± 68.15 | Acylor-1.50 |
Plant part investigated: lf = leaf; rt = root; tb = tuber; bk = bark; Environmental status S: Cm = common; Th = threatened; VTh = very threatened; BSLT, Brine shrimp lethality test LC50, lethal concentration that kills 50% of the shrimps with corresponding 95% confidence intervals (95% CI); CC50 (50% CPE), plant extract cytotoxicity concentration that kills 50% tissue cells; NT, neutralisation test: NA = no activity; ID50, non-cytotoxic concentration that inhibits/protects 50% uninfected cells, NA = no activity; TI: therapeutic index = CC50/ID50 EPPT, End point titration test. RFb antiviral reduction factor: ratio of viral titre of control in absence of extract over viral titre in presence of extract.
induced destruction with EPTT reduction factors (RFs) ≥ 103 (
Sub-Saharan Africa still suffers the great burden for HIV/AIDS with millions infected and affected. Though prevalence has fallen, availability of antiviral drugs poses a continuing challenge. The use of TMPs is often plagued with issues of safety and/or toxicity. The antimicrobial and antiviral activities demonstrated by the plant extracts are further proof to support claims by TPs of their ability to treat more serious illness including HIV/ AIDS and opportunistic infections [
The authors would like to thank GEF/UNDP through the Ministry of Environment and Tourism (MET) for supporting two postgraduate students on the project, the local communities and traditional practitioners from the districts involved, the School of Pharmacy and the Research Board of the University of Zimbabwe and other national institutions for making this work possible through many linkages and collaborations.
Deniz Iklim Viol,Lameck Shoriwa Chagonda,Sylvester Rodgers Moyo,Ali Hikmet Mericli, (2016) Toxicity and Antiviral Activities of Some Medicinal Plants Used by Traditional Medical Practitioners in Zimbabwe. American Journal of Plant Sciences,07,1538-1544. doi: 10.4236/ajps.2016.711145