Evaluation of traditionally used medicinal plants for anticancer, antioxidant, anti-inflammatory and anti-viral (HPV-1) activity

The aim of this study was to determine the anticancer, antioxidant, anti-inflammatory and antiviral activities of traditionally used medicinal plants. The extracts were tested for cytotoxicity against human melanoma (A375), epidermoid carcinoma (A431), cervical epithelial carcinoma (HeLa) and human embryonic kidney cells (HEK-293). The antioxidant and anti-inflammatory activities were also determined. Gomphocarpus fruticosus, Helichrysum kraussii and Syzygium jambos were selected for activity against the herpes simplex virus type-1. The extracts exhibited low toxicity towards HEK-293 cells, and four extracts; namely Acacia mellifera, G. fruticosus, H. kraussii and S. jambos, were able to inhibit the A431 and HeLa cells with fifty percent inhibitory concentrations (IC50) ranging from 34.90-56.20 μg/ml. Arbutus unedo, Combretum molle, Dissotis princeps, Erythrophleum lasianthum, Harpephyllum caffrum, H. kraussii and S. jambos, showed high DPPH inhibitory activity, with IC50 values ranging from 2.41–5.25 μg/ml. The highest antioxidant activity was seen for S. jambos (DPPH) and A. unedo (NO) respectively with greater activity than ascorbic acid. D. princeps, H. caffrum, Leucas martinicensis and S. jambos, showed high inhibition of the cyclooxygenase-2 (COX-2) enzyme with IC50 values ranging from 3.79-25.80 μg/ml with S. jambos showing the highest activity. S. jambos further showed the highest anti-HSV-1 activity at 50.00 μg/ml against 100TCID50virus challenge dose. This is the first report of the selected plants for their cytotoxicity, anti-inflammatory and viral inhibitory activity. S. jambos was able to show high inhibition of the HPV type-1 virus and the COX-2 enzyme.


Introduction
Traditional medicine is still used by approximately 65-80 % of the world"s population in developing countries as a source of primary health care (Tag et al. 2012). The main reason for the use of traditional medicine is due to affordability, accessibility and cultural beliefs (Benzie and Wachtel-Galor, 2011). In South Africa it is estimated that approximately 27 million individuals still rely on traditional medicine. Approximately 20,000 tons of indigenous South African plants are used each year from at least 771 plant species (HST, 2015).
In this study plants were selected based on their traditional usage for the treatment of various types of cancer and/or skin ailments such as sores, wounds and infections which could lead to the development of skin or cervical cancer. Plants were also selected based on usages which could contribute towards the health of skin such as blood-cleansing and astringent properties.
An infusion of Acacia caffra bark is used by the Zulu community of South Africa as a bloodcleansing emetic (Hutchings et al. 1996). Water extracts from the Acacia mellifera plant is used by a Kenyan community, known as "Kipsigis", for the treatment of various skin diseases (Mutai et al. 2008). The leaves of Arbutus unedo, which is commonly known as the "Strawberry tree", are traditionally used for its astringent properties (Pabuçcuoğluetal., 2003).
The Tswana communities of South Africa use the fresh flowers of Buddleja salviifolia as a decoction for the treatment of sores (Hutchings et al. 1996). The leaves of Clematis brachiata are used by many South African communities, such as Xhosas, Zulus, Sothos and Tswana"s, as a soothing foot wash for cracking and blistering feet (Viljoen, 2002). Combretum molle, which is commonly known as the velvet bushwillow, is used by Zulu communities. A paste made from fresh or dried leaves of C. molle are directly applied to wounds (Hutchings et al. 1996). The roots of Dissotis princeps are used as a food source during times of famine.
However, there have been reports of other Dissotis species which are used for the treatment of skin diseases (Ndjateu et al. 2014). An infusion of Erythrophleum lasianthum bark is used as a blood-cleansing emetic (Hutchings et al. 1996). The roots of Euclea divinorum, known as the "magic guarri", are extracted and used for the treatment of cancers as well as ulcers, wounds and snake bites (Prota4U, 2005). Gomphocarpus fruticosus, or more commonly "milkweed", is used in Ethiopia as an ointment for sores, and in Namibia a tea is made from the leaves for the treatment of skin cancer (Prota4U, 2011). Decoctions from the bark of Harpephyllum caffrum is used as a blood purifier and is also used as a face wash for the treatment of acne and eczema (Van Wyk, 1997). The roots of Helichrysum kraussii are ground and mixed with salt to treat venereal diseases, which could lead to the development of cervical cancer (Lourens et al. 2007). A decoction of the leaves or aerial parts of Leucas martinicensis is used for the treatment of inflammation as well as rheumatism (Chouhan and Singh, 2011). In India the "Rose apple" tree, Syzygium jambos, is used for the treatment of skin problems such as rashes (Zheng et al. 2011). Tabernaemontana elegans is used by the Zulu community of South Africa where the dried powder of the inner wall of the fruit is boiled in water and used to treat cancer (Cheek, 2010). Warburgia salutaris is used for the treatment of sores where the powder of the plant is applied directly (Rabe and van Staden, 1997).
In the present study, traditionally used plants were tested for their anticancer activity against skin and cervical cancer. The antioxidant and anti-inflammatory (COX-2) activities of the extracts were also determined. Selected plant extracts were evaluated for antiviral activity against herpes simplex virus type-1 (HSV-1).

Extraction and collection of plant material
The plant material was collected at the Walter Sisulu National Botanical Gardens, Roodepoort; Venda, Limpopo and at the Manie van der Schijff Botanical Gardens, University of Pretoria. The plant material was identified and voucher specimens were deposited at the HGWJ Schweickerdt Herbarium, Pretoria, South Africa (Table 1). The plant material was a Fifty percent inhibitory concentration; b 2, 2-diphenyl-1-picrylhydrazyl radical; c Nitric oxide; d Cyclooxygenase-2; e Cytotoxicity positive control; f Antioxidant positive control; g Cyclooygenase-2 positive control; -Not tested; ENS -Extract not in sufficient amount for testing.
Results are reported as mean ± SD (n = 3). Statistical analysis was done using one-way ANOVA followed by Dunnett's Multiple Comparison Test using GraphPad Prism statistical software. Cancer cell cytotoxicity IC 50 values statistically similar to H. kraussii on HeLa cells (IC 50 : 34.90±1.00 µg/ml) were identified positive (+). The IC 50 values statistically similar to the positive controls used in the DPPH, NO and COX-2 assay were identified positive (+) and IC 50 values with statistically higher activity than the positive controls were identified *p<0.01 shade dried for two weeks and then ground to a fine powder. The dried powder (300 g) was macerated in distilled ethanol (2.5 L) and shaken for 48 h and thereafter filtered through Whatman No.1 filter paper using a Buchner funnel. The filtrate of each plant was collected and subjected to reduced pressure using a rotary evaporator (Büchi R-200) at 37-40 ºC. The extracts were kept in a cold room at -20 ºC until further use.

Cell culture
The human cervical epithelial carcinoma (HeLa), African green monkey kidney cells (Vero) and human embryonic kidney (HEK-293) cell lines were maintained in culture flasks which contained Eagle"s Minimum Essential Medium (EMEM), whereas the human epidermoid carcinoma (A431) and human malignant melanoma (A375) cells were maintained in Dulbecco"s Modified Eagles Medium (DMEM). All cell lines were supplemented with 1 % antibiotics (100 U/ml penicillin, 100 µg/ml streptomycin and 250 µg/L fungizone) and 10 % heat-inactivated foetal bovine serum. The cells were grown at 37 °C in a humidified incubator set at 5 % CO 2 . Cells were sub-cultured after an 80 % confluent monolayer had formed.

In vitro cytotoxicity assay
Cytotoxicity was measured by the 2,3-Bis-(2-methoxy-4-nitro-5-sulfophenyl]-2Htetrazolium-5-carboxyanilide salt (XTT) method using the Cell Proliferation Kit II. The method described by Berrington and Lall (2012) was used to perform the assay. Briefly, 100 µl cells were seeded in 96-well plates (1×10 5 cells/ml or 10,000 cells/well) and incubated for 24 h at 37 ºC in 5 % CO 2 for cell adherence. Stock concentrations of the extracts were prepared at 20 mg/ml in dimethylsulfoxide (DMSO) and further diluted in media to appropriate concentrations. Cells were treated with 100 µl of the plant extracts at final concentrations ranging from 1.56-200 µg/ml for 72 h. Controls included a 2 % DMSO vehicle control and the concentration of the positive control, "Actinomycin D" (stock concentration of 1 mg/ml in distilled water) ranged between 3.9×10 -4 -0.05 µg/ml. After treatment, XTT (50 µl) was added to a final concentration of 0.3 mg/ml for 2 h. Blank plates were included which were prepared in the same manner above, however did not contain cells.
The blank plates were used to compensate for the colour of the plant extract so as not to interfere with the absorbance of XTT. Absorbance was measured at 490 nm and 690 nm (reference wavelength) using a BIO-TEK Power-Wave XS multi-well plate reader (A.D.P, Weltevreden Park, South Africa). Each sample"s concentration was tested in triplicate to calculate a fifty percent inhibitory concentration (IC 50 ) of cell viability.

DPPH radical scavenging activity
The method as described Berrington and Lall (2012) was followed to determine the radical scavenging capacity (RSC) of the extracts. The stock solution of the positive control, ascorbic acid, and extracts were prepared at 2 mg/ml and 10 mg/ml in ethanol respectively. Twenty microlitres of the samples were added to the top wells of a 96-well plate and serially diluted to final concentrations ranging from 3.90-500 µg/ml and from 0.78-100 µg/ml for the extracts and ascorbic acid respectively. Ethanol at 10 % was used as the blank. Extracts which showed high inhibition at the lowest concentration tested (3.90 µg/ml) were tested at concentrations ranging from 0.78-100 µg/ml. Ninety microlitres of the ethanolic 2, 2diphenyl-1-picrylhydrazyl radical (DPPH) solution (0.04 M) was added to each well and incubated for 30 min covered in foil. Negative colour controls were also prepared in the same manner as above, however distilled water was added instead of DPPH. Absorbencies were measured at 515 nm using a BIO-TEK Power-Wave XS multi-plate reader using KC junior software. The IC 50 values were calculated and the ascorbic acid equivalents were calculated as follows: (IC 50 of extract X 200 mg ascorbic acid)/ IC 50 of ascorbic acid. Each sample"s concentration was tested in triplicate.

NO radical scavenging activity
The method as described by Mayur et al. (2010) was followed to determine the nitric oxide scavenging capacity of the extracts. The stock concentrations of the positive control, ascorbic acid, and extracts were prepared at 10 mg/ml in ethanol. Twenty microlitres of the extract"s and ascorbic acid were added to the top well of a 96-well plate and serially diluted to final concentrations ranging from 7.81-1000 µg/ml. Ethanol at 10 % was used as the blank. Fifty microlitres of sodium nitroprusside (10mM) was added to all the wells and incubated at room temperature for 90 min. Thereafter, 100 µl Griess reagent was added to all the wells except for the negative colour control wells, where distilled water was added. Absorbencies were read at 546 nm using a BIO-TEK Power-Wave XS multi-well reader using KC Junior software and the IC 50 value was calculated. Each sample"s concentration was tested in triplicate.

Cyclooxygenase-2 assay
The method was performed as described by Reininger and Bauer (2006) to determine whether the sample"s were able to inhibit human recombinant cyclooxygenase-2 (COX-2).
To each well of a 96-well plate, 5 µl of the COX-2 enzyme (0.5 units/ well) was added to 180 µl of 100 mM TRIS buffer (pH 8.0) containing 5 µM porcine hematin, 18 mM L-epinephrine, and 50 µM Na 2 EDTA as co-factors. Stock concentrations of the extracts were prepared at 10 mg/ml in DMSO. Ten microlitres of the extracts were added to the wells at a final concentration of 10 µg/ml. Ibuprofen was used as the positive control and tested at final concentrations of 10 µM (stock concentration), 2 µM and 0.4 µM. DMSO at 5 % was used as the vehicle control. The reaction was initiated after 5 min incubation by adding 5 µl of arachidonic acid (10µM). The extracts, which showed >65 % inhibition at 10 µg/ml, were retested at concentrations ranging from 2.50-160 µg/ml to determine their IC 50 values. The plates were incubated at room temperature for a further 20 min. Finally 10 µl of 10 % formic acid was added to stop the reaction. Samples were diluted in a ratio of 1:15 using assay buffer. Quantification of PGE 2 , which is the main product of the reaction, was determined using the PGE 2 ELISA kit and the absorbance was measured at 405 nm using a BIO-TEK Power-Wave XS multi-well plate reader (A.D.P, Weltevreden Park, South Africa). The IC 50 values of the four extract were calculated using Microsoft Excel.

Virus culture
The HSV-1 was obtained from the Christian Medical College and Hospital, Vellore, Tamil Nadu, India. A stock suspension of HSV-1 in Vero cells with titres of 1×10 -7 TCID 50 /ml (50% tissue culture infective dose) was prepared. The virus was diluted in serum-free media (EMEM) and used at final concentrations of 10 and 100 TCID 50 respectively.

In vitro anti-Herpes Simplex Virus type-1 assay
To determine the antiviral activity of the extracts, the cytopathic effect (CPE) inhibition assay against different virus challenge doses of 10 and 100 TCID 50 was used as described by Hu and Hsiung (1989). Briefly, 100 µl of Vero cells were seeded in a 96-well plate at

Statistical analysis
All results are reported as mean ± SD (n = 3). Statistical analysis was done using one way analysis of variance (ANOVA) followed by Dunnett"s Multiple Comparison Test using the  (Steenkamp & Gouws, 2006). Extracts statistically similar to H. kraussii were identified with (+) and therefore had good activity. In the DPPH, NO and COX-2 assays, extracts which were statistically similar in activity to the positive controls were identified (+).
Extracts which had statistically higher activity than the positive controls, were reported as *(p<0.01).

In vitro cytotoxicity
The sixteen ethanolic extracts were tested for cytotoxicity against cancerous A431, A375 and jambos, however showed activity with IC 50 values < 60.00 µg/ml against both the HeLa and A431 cell line (Table 1).
kraussii has not previously been described for its cytotoxicity against cancer cells. These results are comparable to the results obtained by the above mentioned plants in the present study.
The statistical significance of the plant extracts IC 50 values were compared to the IC 50 (34.90±1.00 µg/ml) of H. kraussiii. The cytotoxicity of H. kraussii against HeLa cells was similar to the guidelines set by the American Cancer Institute, which sets the limit for activity of an extract at an IC 50 < 30.00 µg/ml after 72 h exposure (Steenkamp & Gouws, 2006 were close to that of G. fruticosus against HeLa cells. These four extracts could therefore, be considered to screen for activity against other cancerous cell lines.

Antioxidant activity
The radical scavenging capacity of the plant extracts was determined using the DPPH and NO free radical scavenging assays. Free radicals are a major cause of DNA damage which results in the initiation of carcinogenesis. Therefore, the inhibition of free radicals could potentially aid in preventing carcinogenesis. Eight of the tested extracts showed good antioxidant activity (IC 50 < 10.00 µg/ml). Four extracts showed IC 50 values below 40.00 µg/ml and the remaining extracts showed low inhibition (IC 50 > 70.00 µg/ml) ( Table 1).
The highest DPPH scavenging activity was observed for S. jambos with an IC 50 of 1.17±0.30µg/ml, which had statistically similar activity to that of ascorbic acid with an IC 50 of 1.98±0.01 µg/ml. Earlier reports by researchers document the isolation of myricetin, myricitrin and gallic acid from the ethanolic leaf extract of S. jambos, which could contribute towards the high antioxidant activity of the extract (Jayprakasham, 2010). In a previous study by Islam et al (2012), the ethanolic leaf extract of S. jambos showed less DPPH inhibition with an IC 50 value of 14.10 µg/ml. In the NO scavenging assay, twelve extracts showed low activity with inhibition starting from 1000 µg/ml. Three extracts showed inhibition < 500 µg/ml, with the highest activity noted for A. unedo with an IC 50 of 85.90±10 µg/ml which has statistically higher (*P<0.01) activity than that of ascorbic acid with an IC 50 of 285.90±26 µg/ml. A. unedo, with an IC 50 value of 4.51±0.20 against DPPH, also had statistically similar activity to ascorbic acid.

Cyclooxygenase-2 activity
The extract"s activity against COX-2 was tested as it has been implicated in a number of cancer types. It has also been linked to increased cancer cell proliferation (Sobolewski et al. 2010 H. caffrum has also previously been tested for COX-2 inhibition and it was found that the non-polar extracts were more active than the polar extracts (Moyo et al. 2011). In this study however, the polar ethanolic extract of H. caffrum showed an IC 50 of 6.40±1.60 µg/ml, which was statistically similar to ibuprofen. L. martinicensis showed a high COX-2 inhibition of 72.62±12 % at 10 µg/ml, however when the IC 50 (16.03±80 µg/ml) was calculated it was not found to be statistically similar to ibuprofen. This is the first report of the COX-2 inhibitory activity of L. martinicensis and S. jambos.

In vitro anti-Herpes Simplex Virus type-1 assay
Due to the anticancer activity of G. fruticosus, H. kraussii and S. jambos on cervical cancer cells, these extracts were further tested for antiviral activity (Table 1). Herpes simplex virus type-2 (HSV-2) is generally considered to be the causative agent for the development of genital herpes and therefore can cause cervical cancer. However, HSV-1, which is highly contagious, can also cause genital herpes, which causes genital or anal blisters or ulcers (WHO, 2017). Recent studies have suggested that the incidence of genital infections by HSV-1 has increased (Pereira et al. 2012). Therefore, the inhibitory activity of the plant extracts against HSV-1 was determined.
S. jambos, at 50.00 µg/ml exhibited potential anti-viral activity with 100 % viral inhibition when tested at the highest viral dose (100TCID 50 ). At a viral dose of 10TCID50, S. jambos was able to inhibit 100 % of the virus at all four concentrations tested ( In a study by Sirivan et al. (2008) a plaque reduction assay was performed using different extracts of S. jambos against HSV-1 and HSV-2. The extracts were each tested at a concentration of 100 µg/ml and were found to inhibit both HSV-1 and HSV-2 by more than 50 % when using the hexane and dichloromethane leaf extracts, whereas the methanol leaf extract showed 11.20 and 30.60 % inhibition against HSV-1 and HSV-2 respectively. Table 2 The anti-HSV-1 (IC 50 in µg/ml) activity of the selected plant extracts  Light microscopy (20 × magnification) of A) Vero cells where 100% cell growth is observed, B) Vero cells are infected with the HSV-1 (0% cell growth) and C) HSV-1 infected Vero cells treated with 5 μg/ml anti-viral positive control, acyclovir where there is 100% cell growth.

Conclusion
In this study four extracts exhibited anti-cancer activity against the HeLa and A431 cell lines.
Three of these extracts were further tested for anti-viral activity against HSV-1 and the highest activity was observed by S. jambos which showed 100 % viral inhibition at 50.00 µg/ml at the highest viral challenge dose. S. jambos also showed the greatest COX-2 and DPPH inhibitory activity. This is the first report of the activity of the selected plants for their cytotoxicity on the selected cell lines. The collective data for the anticancer, antioxidant and anti-inflammatory activity of S. jambos shows the potential of the plant extract to be considered for its application as a chemopreventive and anticancer agent. Furthermore, S. jambos could be considered a promising extract for anti-viral activity. This study further shows the potential of H. caffrum as an anti-inflammatory agent and the opportunity to test A. mellifera, G. fruticosus, H. kraussii and S. jambos to be tested against other cancerous cell lines.