Phytochemical Screening, Acute Toxicity and AntiRabies Activities of Extracts of Selected Ethiopian Traditional Medicinal Plants

Background : Rabies, endemic in most African and Asian countries, is a viral zoonosis that causes an estimated 59,000 human deaths a year, despite the existence of safe and effective vaccines. In most developing countries people believe to cure rabies with different traditional and religious treatment rather than seeking effective post exposure prophylaxis. Purpose : To investigate the phytochemical constituents, acute toxicity and antirabies activity of crude extracts of the leaves of Justicia schimperiana and Ricinus communis and the stem bark of Croton macrostachyus . Methods: To test the presence of various phytochemicals, standard procedures were used. For the determination of acute toxicity and in vivo antirabies activities, Organization for Economic Corporation and Development (OECD) Guideline No.423 was used. Different concentrations of extracts (0.4, 0.8, 1.6, 3.2, 6.4 and 12.8 mg/ml) were tested for their cytotoxic effect on Vero cells through 3-(4, 5-Dimethylthiazol-2-yl)-2, 5Diphenyltetrazolium Bromide (MTT) assay. The in vitro antirabies assay was carried out based on the minimal cytotoxic concentration of extracts. Results: The phytochemical screening result has revealed the presence of alkaloids, flavonoids, phenols, steroids, tannins and terpenoids in all plant extracts screened but lack saponins. All the extracts were slightly toxic in Swiss albino mice model but non cytotoxic in Vero cell lines. The antirabies assay result showed that all plant extracts had a moderate to good antirabies potential. The methanol extracts exhibited higher antirabies activity compared to the other extracts under investigation. Conclusion: The present study concluded that the studied plants have possessed different phytochemicals that helps in their antirabies potential. Utilization of these pharmacological properties involves further investigation of these active ingredients by implementation techniques of extraction, purification, separation, crystallization and identification.


Introduction
Rabies neglected viral zoonosis, is still a major public health problem all over the world which causes more than 50, 000 human deaths annually primarily in developing countries of Asia and Africa (Zulu et al., 2009). It is caused by a neurotropic, negative sense, non-segmented, singlestranded RNA virus that belongs to the Lyssavirus genus of the Rhabdoviridae family and Mononegavirale order, which causes physiological disorders by infecting the neurons at the central nervous system (Vural et al. 2016). All mammals are susceptible to rabies, but only a limited number of species also act as reservoir hosts (Balcha and Abdela 2016). The disease is mainly associated with dog bites in Europe, Asia, and Africa and with bats in the Americas (Hemachudha et al. 2013). In developing countries, the majority of confirmed and reported cases and over 90% of human exposures are from domestic dogs (Kaare et al. 2009). Rabies associated with bat RABV variants has atypical features, like focal brainstem signs, myoclonus, hemichorea, and signs and symptoms of Horner's syndrome (Hemachudha et al. 2013). It is generally transmitted through the bite of an infected mammal, but non-bite exposures also may occur under unusual circumstances.
Exposure may occur by direct contact of saliva, nervous or other infected tissues from a rabid animal with mucous membranes, or from scratches. The disease is almost always fatal, once symptoms appear (Kuzmin and Rupprecht 2008).
Though rabies is prevalent throughout the world, many countries and islands like Australia and Antarctica, have got rabies-free status due to strict quarantine or by virtue of their water locked geographical location (Singh et al. 2017). Globally, it is estimated that approximately 50,000 to 100,000 people die from rabies each year, although the true number is probably much higher than what's being reported (Leung et al. 2007). In the United States, multiple RABV variants circulate in wild mammalian reservoir populations including raccoons, skunks, foxes, and bats. In the developing world like Ethiopia, it is recognized that the number of deaths due to rabies officially reported greatly underestimates the true incidence of disease (Knobel et al. 2005). Post exposure prophylaxis (PEP) consisting of rabies immune globulin and rabies vaccine is successful in preventing the disease if prompt intervention is initiated (Blanton et al. 2010).
Death is almost always inevitable in unimmunized patients; only supportive measures are recommended after the onset of neurological signs and symptoms (Crowcroft and Thampi 2016).
However, the disease is 100% preventable by either pre-exposure prophylaxis (PrEP) or post-4 exposure prophylaxis (PEP) which together effectively prevent approximately 372,000 deaths yearly. Specifically, prompt administration of vaccines in conjunction with rabiesimmunoglobulins and proper wound management after exposure prevent rabies even after highrisk exposure (Nie et al. 2017). In Africa and Asia, almost 8 million people a year receive costly post-exposure prophylaxis (PEP) following bite injuries from rabid animals (Knobel et al. 2005) Although, a vaccine preventable disease, the annual number of human deaths caused by rabies is estimated to be more than 50, 000 per year, mostly in Africa and Asia (Okonko et al. 2010). The endemicity of rabies in developing countries like Ethiopia could be attributed to lack of risk communication, Poor surveillance of rabies-related viruses and poor diagnostic capability, low vaccine coverage and failure to immunize domestic dogs, which transmit rabies to humans.
Older nerve tissue vaccine is still being manufactured and utilized for the majority of exposed patients in Ethiopia, even though this vaccine has been discouraged by the WHO as they may

Cell culture and virus
Vero cells obtained from Vaccine and Diagnostics production directorate, EPHI were cultured using the T-75 culture flasks following the procedure described in (Ammerman et al. 2008).
Briefly, Vero cells kept in liquid nitrogen were recovered from frozen stock in a 37°C water bath.
The cell suspension was transferred from the cryovial into a 15ml conical tube containing 10ml of DMEM supplemented with FBS. The cells were pelleted by centrifugation at 200× g for 5 minutes at room temperature and the supernatant was removed and discarded. The cells were resuspended in 5-10 ml DMEM supplemented with 10% FBS and transferred to tissue culture flask with vented cap. Flasks were incubated in 37°C incubator with 5% CO2 for 2-3 days until a monolayer was obtained. The growth medium from confluent monolayer of Vero cells was removed and cells were washed with 10 ml 1X DPBS. Five ml of 1X trypsin-EDTA was added and the cells were incubated at 37°C for 2-3 minutes, until cells start to streak as they detached from the flask. To inactivate the trypsin-EDTA, 5 ml DMEM with 10% FBS were added and cells were washed down in media. The cell suspension was removed from flask and transferred to a sterile 15 ml conical tube and centrifuged at 200 × g for 5 minutes at room temperature. The cells were resuspended in 6 of cell culture cells in a total of 15 ml DMEM with 10% FBS was prepared and added to 75 cm 2 cell culture flasks with vented caps. Flasks were incubated in 37°C incubator with 5% CO2 until a monolayer was obtained. Finally, cells were collected within a volume of 20-30 ml in cell culture medium with 10% heat-inactivated FBS. Cells were maintained at 37 °C under a humidified 5 % CO2 atmosphere for bioassay.
The virus (PV) was propagated in Vero cells as previously described by (Webster and Clow 1937) and titer of infectious virus was obtained by the limit-dilution method and expressed as 50% tissue culture infections dose per ml (TCID50/ml). Briefly, serial 10-fold dilutions (10-1 to 10-7) of virus in serum-free MEM were added into the confluent monolayer in 96-well tissue culture plate and were incubated for 72 h at 37°C. After incubation, the medium was decanted and the cells were fixed by adding cold acetone (50 µl per well) and kept at 4°C for 30 minutes. After discarding the acetone, cells were stained by direct polyclonal florescent-labeled antibody for 30 minutes and washed three times with 0.01 M phosphate buffer saline (PBS), air dried, and visualized under an inverted fluorescence microscope. The titer was calculated by using Spearman-Karber method and expressed as TCID50. The virus titration was also performed in mice through intracerebral inoculation using serial ten-fold dilutions at a volume of 0.03 ml. Six mice were inoculated per virus dilution, and the end point was death. The statistical method of Spearman-Karber was used and the mortality at each dilution was calculated to determine the 50% end point titer (Ramakrishnan 2016).

In vivo acute toxicity test:
The extracts from the leaves of Justicia schimperiana and Ricinus communis and stem bark of

In vitro antirabies assay:
The in vitro antirabies activity of the plant extracts were evaluated by FAT assay. Vero cell lines were trypsinized and 2 × 10 5 cells/ml were seeded in a 96-well tissue culture micro plate and 9 incubated at 37°C for 72 h. After the incubation period, 50μl of rabies virus suspension were added to confluent cell monolayers in a 96-well plate and allowed to stand for 1h to enable virus adsorption. Thereafter, different concentrations (2mg/ml, 4mg/ml and 8mg/ml) of each extract based on cytotoxicity test result were added in triplicate into all the wells with the exception of the negative control wells that contained only Vero cells and the virus control that contained an equal virus concentration but lacked the plant extract. The plates were incubated at 37°C in 5% CO2 humidified incubator for 72 h. The medium was decanted and the cells were fixed by adding cold acetone (50µl per well) and kept at 4°C for 30 minutes. After discarding the acetone, cells were stained by direct polyclonal florescent-labeled antibody for 30 minutes and then washed (3 times) with 0.01 M PBS, air dried, and visualized under an inverted fluorescence microscope. Reading was qualitative, every well that shows specific fluorescence was considered to be positive.

Phytochemical study result of extracts
In the present study the result of qualitative phytochemical analysis of all extracts from the three plants using different standard methods showed positive results for alkaloids, but negative for saponins as shown in Table 1

The result of acute oral toxicity test
The acute toxicity results showed no evidence of toxicity of the ethanol, 80 % methanol and water extracts of Justicia schimperiana (leaf), Croton macrostachyus (stem bark) and Ricinus communis (leaf) in mice administered at 1000 mg/kg, 2000 mg/kg and 3000 mg/kg. No abnormalities were recorded at three doses with regard to food consumption, water intake and body weight of the mice. This shows that the plant extracts could be well tolerated up to the dose of 3000 mg/kg body weight of Swiss albino mice. However, at higher doses, i.e. 4000 mg/kg and 5000 mg/kg, mice showed common signs of toxicity like low locomotion, weakness and erection of hairs including death in the course of acute study. As a result, the LD50 of the extracts could be greater than 3000mg/kg body weight. It suggested that the extracts may not be completely safe at a dose higher than 3000 mg/kg. According to the (Hodge and Sterner 1943) toxicity scale, the ethanol, methanol and aqueous extracts of Justicia schimperiana (leaf), Croton macrostachyus (stem bark) and Ricinus communis (leaf) were placed in category IV (500 mg/kg-5000 mg/kg, p.o.), and hence classified as slightly toxic.
The percentage of mice that died at each dose was transformed to probits using Finney's method.
According to Finney's method the log dose at probit 5.0 (Log LD50) for the ethanol, methanol and water extracts of Justicia schimperiana were found to be 3.60, 3.54 and 3.65 and hence, LD50 was calculated by taking antilog of the Log LD50 values of each extract and found to be 4000mg/kg, 3500mg/kg and 4500mg/kg body weight respectively ( Table 2). The acute toxicity study in LD50 determination showed that methanol leaf extract of Justicia schimperiana is more toxic than the ethanol and water leaf extracts of the plant. Where, A1= group of mice treated with ethanol extracts of Justicia schimperiana, B1= mice treated with 80 % methanol extracts of Justicia schimperiana and C1= mice treated with water extracts of Justicia schimperiana. Similarly, the lethal dose (LD50) of the ethanol, methanol and water extracts of the leaves of Ricinus communis was also greater than 3000 mg/kg body weight of mice.
The Log LD50 for the ethanol and water extracts of Ricinus communis was found to be 3.54 but, its methanolic extract was found to be 3.60. Thus, the LD50 values were found to be 3500 mg/kg for the ethanol and water extracts and 4000 mg/kg for the methanol extract of the plant which were calculated by taking antilog of 3.54 and 3.60. Therefore, the ethanol and water leaf extract of Ricinus communisis more toxic than the methanol extract of the plant.
The lethal dose (LD50) of the ethanol, methanol and water extracts of the stem bark of Croton macrostachyus were also greater than 3000 mg/kg body weight of mice. The Log LD50 for the ethanol and methanol extracts of Croton macrostachyus was found to be 3.54. Thus, LD50 was calculated by taking antilog of 3.54 and found to be 3500 mg/kg body weight. Whereas, the Log LD50 for the water extract was found to be 3.59 hence, the LD50 value was 3900 mg/kg. 12 Evaluation on Vero cell lines by using MTT assay showed that the 50% cytotoxic concentration (CC50) values of the ethanol and aqueous extracts of three plants; Justicia schimperiana, Croton macrostachyus and Ricinus communis were found to be above 12.8 mg/ml.

Cytotoxicity determination of plant extracts in Vero cell line
However, the 50% cytotoxic concentration (CC50) value of the methanol extracts from Justicia schimperiana and Croton macrostachyus were found to be 9.6mg/ml and 8mg/ml whereas, Ricinus communis showed above 12.8 mg/ml (Table 3). Generally, the percentage viability was found to be increasing with decreasing concentration of test extracts. The MTT assay results revealed that all extracts tested were non-cytotoxic and exhibited CC50 values above the cut-off point which is 30µg/ml. An extracts can be considered as non-cytotoxic if the CC50 is higher than 30µg/ml (Nondo et al. 2015).

In vivo antirabies potential of plant extracts
Group of mice infected with rabies virus but not treated with any of plant extracts showed 0% survival rate and a mean survival period of 9.5 days. However, oral treatment of mice infected with rabies virus with ethanol, methanol and water extracts of the leaves of Justicia schimperiana and Ricinus communis as well as the stem bark of Croton macrostachyus at a dose of 3000 mg/kg significantly (p<0.05) increased the mean survival time compared to those of negative control group (Table 4). Relatively higher mean survival time (22.16 days) was obtained when methanol extract of Ricinus communis was administered to mice at a dose of 3000 mg/kg. One mouse from the negative control group died within four days of inoculation with PV but, didn't show any antigen against rabies virus which indicate the death was due to accidental rather than the effect of the virus. Death from samples taken from those at moribund state was due to the action of rabies 13 virus PV strain because all the samples showed a viral antigen but, most of the samples from survivors indicate negative for antigen detection.

Conclusions
The phytochemical analysis showed that the ethanol, methanol and water extracts of the leaves of Justicia schimperiana and Ricinus communis as well as the stem bark Croton macrostachyus contains a mixture of phytochemicals as alkaloids, flavonoids, phenols, steroids, tannins and terpenoids but lack saponins. All the extracts were slightly toxic in animal model but non cytotoxic in Vero cell lines. All the Plant extracts had a moderate to good antirabies activity against PV strain. The methanol plant extracts gave more antirabies activity compared to ethanol and water extracts in mice model.