Larvicidal, antimicrobial and brine shrimp activities of extracts from Cissampelos mucronata and Tephrosia villosa from coast region, Tanzania

Background The leaves and roots of Cissampelos mucronata A. Rich (Menispermaceae) are widely used in the tropics and subtropics to manage various ailments such as gastro-intestinal complaints, menstrual problems, venereal diseases and malaria. In the Coast region, Tanzania, roots are used to treat wounds due to extraction of jigger. Leaves of Tephrosia villosa (L) Pers (Leguminosae) are reported to be used in the treatment of diabetes mellitus in India. In this study, extracts from the roots and aerial parts of C. mucronata and extracts from leaves, fruits, twigs and roots of T. villosa were evaluated for larvicidal activity, brine shrimps toxicity and antimicrobial activity. Methods Powdered materials from C. mucronata were extracted sequentially by dichloromethane followed by ethanol while materials from T.villosa were extracted by ethanol only. The extracts obtained were evaluated for larvicidal activity using Culex quinquefasciatus Say larvae, cytotoxicity using brine shrimp larvae and antimicrobial activity using bacteria and fungi. Results Extracts from aerial parts of C. Mucronata exhibited antibacterial activity against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi, Vibrio cholera, Bacillus anthracis, Streptococcus faecalis and antifungal activity against Candida albicans and Cryptococcus neoformans. They exhibited very low toxicity to brine shrimps and had no larvicidal activity. The root extracts exhibited good larvicidal activity but weak antimicrobial activity. The root dichloromethane extracts from C. mucronata was found to be more toxic with an LC50 value of 59.608 μg/mL while ethanolic extracts from root were not toxic with LC50>100 μg/mL). Ethanol extracts from fruits and roots of T. villosa were found to be very toxic with LC50 values of 9.690 μg/mL and 4.511 μg/mL, respectively, while, ethanol extracts from leaves and twigs of T. villosa were found to be non toxic (LC50>100 μg/mL). Conclusion These results support the use of C. mucronata in traditional medicine for treatment of wounds. Extracts of C. mucronata have potential to yield active antimicrobial and larvicidal compounds. The high brine shrimp toxicity of T. villosa corroborates with literature reports that the plant is toxic to both livestock and fish. The results further suggest that T. villosa extracts have potential to yield larvicidal and possibly cytotoxic compounds. Further studies to investigate the bioactive compounds responsible for the observed biological effects are suggested.


Background
Cissampelos mucronata A. Rich (Menispermaceae) or kishiki cha buga (Swahili) is a dioecious liana found in tropical and subtropical areas of Africa, America and Asia [1][2][3]. It is widely used as traditional herbal medicine [2]. In the Coast region, Tanzania, the powdered roots of C. mucronata are mixed with coconut oil for treatment of fresh wounds after extraction of jigger (Tunga penetrans) while in other parts of Tanzania, C. mucronata is used for treatment of indigestion, fever due to malaria and wounds [4,5]. In South Africa, the root decoction is used for the treatment of schistosomiasis while in India roots are used as antisnake venom [6,7]. Pharmacologically root extracts of C. mucronata are reported to be active against chloroquine -sensitive and chloroquineresistant P. falciparum strains, and active against Trypanosoma cruzi and Trypanosoma rhodensiense [5,8]. Other pharmacological properties of root extracts include sedative effect and antimicrobial activity [9,10]. Extracts from leaves are reported to have antibacterial, anti-ulcer and hypoglycaemic activities as well as uterine relaxant properties [11][12][13].
Tephrosia villosa L. Pers (Fabaceae) is an annual or perennial bushy herb which is commonly found on sandy soil. Despite being reported to be toxic to livestock and fish [14], aqueous extract of T. villosa leaves is used as herbal remedy in the treatment of diabetes mellitus in India [15,16]. In Africa, the herb is used as green manure to improve the soil [14].
The aim of this study was to evaluate the ethanolic and dichloromethane extracts from roots and aerial parts of C. mucronata as well as ethanolic extracts from leaves, fruits, twigs and roots of T. villosa for larvicidal and antimicrobial activity. The brine shrimp toxicity test was used to evaluate the extracts for toxicity and potential cytotoxic activity.

Collection and Preparation of Plant Materials
The roots and aerial parts (twigs and leaves) of C. mucronata, voucher specimen no. 3960 were collected from Bagamoyo District, while roots, twigs, leaves and fruits of T. villosa, voucher specimen no. 3961 was collected from Kibaha District, Coast region, Tanzania. The plants were identified by Mr. Haji Selemani, a botanist from the Department of Botany, University of Dar es Salaam. The plant materials were dried under shade and then pulverized to get powders for extraction.

Extraction process
The plant powders from the roots and aerial parts of C. mucronata were extracted sequentially by maceration using dichloromethane and ethanol whereas fruits, leaves, twigs, and roots of T. villosa were separately extracted using ethanol only (100%). The extracts were dried under vacuo using rotary evaporator and stored at -20°C until the time for testing.

Testing for larvicidal activity
The larvicidal test was performed according to World Health Organisation (WHO) protocol with minor modification [17]. Briefly, stock solutions (50 mg/mL) of each plant extract were prepared by first dissolving them in DMSO. The stock solutions were diluted with distilled water to make 100 mL each, of 500, 250, 100 and 50 μg/mL solutions of each plant extract. Ten late third instar laboratory reared C. quinquefasciatus mosquito larvae were then introduced in the test solution and mortality was observed after 24 h, 48 h and 72 h. Negative control tests contained mosquito larvae, DMSO (0.5%) and water only. All tests were carried out in duplicate under controlled temperature (25 ± 2°C) and relative humidity of 75-85%. The number of dead larvae was recorded after 24 h, 48 h, and 72 h, and the mean percentage mortalities calculated for each concentration. The mean results of the percentage mortality were plotted against the logarithms of concentrations using the were calculated from the regression equations obtained from the graphs.

Testing for antimicrobial activity
Minimum inhibitory concentrations (MICs) were determined by microdilution method [18] using 96-well microtitre plates. The plates were first preloaded with 50 μL of the broth media in each well followed by an addition of 50 μL of the extract (100 mg/mL) into the first wells of each row tested to make a total volume of 100 μl in the first wells. After thorough mixing 50 μl were drawn from each of the first row wells and put into the next row wells. The process was repeated down the columns to the last wells at the bottom where 50 μL was discarded. Thereafter, 50 μL of the bacterial suspension (0.5 Mac Farhland standard turbidity) was then added in each well to make the final volume of 100 μL in each well. The rows containing Gentamicin sulphate (50 -0.024 μg/mL) was used as a standard positive drug, DMSO as negative control while the rows with broth and bacteria only was used to monitor bacterial growth. The plates were then incubated at 37°C for 24 h. For each extract, MICs were determined by adding 40 μLof 0.02% p-iodonitrotetrazolium (INT) chloride dye in each well followed by incubation for 1 h at 37°C. Bacterial growth was indicated by a change in pink colour. The lowest concentration which showed no bacterial growth was considered as MIC.

Brine shrimps lethality test
Brine shrimps lethality test (BST) was used to predict the presence of bioactive compounds in the extract [19]. Briefly, stock solutions (40 mg/mL) of all extracts were prepared by dissolving them in DMSO. Different levels of concentrations (240, 120, 80, 40, 24 and 8 μg/mL) were prepared by drawing different volumes from the stock solutions and then added into vials, each containing ten brine shrimps larvae. The volume was then adjusted to 5 mL with artificial sea water prepared by dissolving 3.8 g of sea salt in 1 L of distilled water. Each level of concentration was tested in duplicate. The negative control contained brine shrimp, artificial sea water and DMSO (0.6%) only. The vials were incubated under light for 24 h. The dead larvae were counted and mean was subjected to analysis using

Data analysis
The mean results of the percentage mortality were plotted against the logarithms of concentrations using the Fig P computer program. Regression equations obtained from the graphs were used to obtain LC 16 , LC 50 , LC 84 and the 95% CI values [20]. An LC 50 value greater than 100 μg/mL is considered to represent an inactive compound or extract.

Larvicidal and Brine shrimp Activity
Larvicidal activity of ethanol and dichloromethane extracts from both roots and aerial parts of C. mucronata were evaluated after incubating mosquito larvae with different concentrations of extracts for 72 h. After 24 h of larvae exposure, ethanolic and dichloromethane root extracts from C. mucronata, CMRE and CMRD, respectively were found to have larvicidal activity whereas ethanolic and dichloromethane extracts from aerials parts of C. mucronata, CMAD and CMAE, respectively were found to have no activity against C. quinquefasciatus larvae (Table 1). However, among the active extracts, the dichloromethane root extract, CMRD exhibited high larvicidal activity with an LC 50 value of 126.11 μg/mL than ethanolic root extracts, CMRE which had an LC 50 value of 219.99 μg/mL. Generally the larvicidal activity increased slightly with the increase in the time of exposure except for CMAD which was found to be inactive even after 72 h of larvae exposure to different concentrations of this extract. Among the extracts from C. mucronata, the highest larvicidal activity was observed with CMRD extract which had an LC 50 value of 117.09 μg/mL) after 72 h of exposure.
All extracts from T. villosa showed larvicidal activity (Table 1) against mosquito larvae after 24 h of incubation with LC 50 values ranging from 65 μg/mL to 220 μg/mL. However, after this time of exposure, ethanolic fruit extract from T. villosa, TVFE, exhibited the best larvicidal activity with LC 50 value of 65.75 μg/mL as compared to other extracts from T. villosa. Larvicidal activity was increased with the time of exposure. At day three, TVFE was found to be more active against C. quinquefasciatus larvae with an LC 50 value of 53.25 μg/mL while the ethanolic leaf extract from T. villosa (TVLE), was found to be less active with an LC 50 value of 161.61 μg/mL.
Brine shrimps results as indicated in Table 2 show that all extracts from aerial parts and ethanolic root extract from C. mucronata were not toxic to brine shrimp larvae, giving LC 50 values above 100 μg/mL. The root dichloromethane extracts of C.mucronata was found to be the most toxic to brine shrimp (LC 50 = 59.608 μg/mL). Ethanolic extracts of the fruits (TVFE) and roots (TVRE) of T.villosa, were highly toxic to brine shrimp larvae with LC 50 values below 10 μg/mL. The ethanolic root extract of T. villosa was however the most toxic extract with an LC 50 value of 4.5 μg/mL. Ethanolic extracts from leaves (TVLE) and twigs (TVTE) were non toxic with LC 50 values above 100 μg/mL (Table 2).

Antimicrobial Activity
All the extracts from C. mucronata and T. villosa exhibited activity against at least one organism tested (Table 3). C. mucronata aerial parts and root extracts were found to be active against both bacteria and fungi. The aerial parts dichloromethane and ethanolic extracts exhibited the highest activity. The ethanolic aerial part extracts of C. mucronata was most active against V. cholera with MIC value of 0.391 mg/mL while the dichloromethane of the same was more active against S. typhi, B. anthracis and C. neoformans with MIC values of 0.781 mg/mL, 0.195 mg/mL and 0.195 mg/mL, respectively. The ethanolic root extract from C. mucronata exhibited low activity against B. anthracis, V. cholera, S. faecalis, C. albicans and C. neoformans with MIC values ranging between 1.563 mg/mL and 3.125 mg/mL (Table 3).
Among the T.villosa extracts the best activity was exhibited by the fruit extracts against C.neoformans with MIC value of 0.195 mg/mL, leaf extract against E. coli with MIC value of 0.781 mg/mL and roots extract against B.anthracis with MIC value of 0.195 mg/mL. Most of the organisms tested were less sensitive to the ethanolic twig extract (TVTE) except C. neoformans (MIC = 1.563 mg/mL) and S. typhi (MIC = 3.125 mg/mL).

Discussion
Leaves and roots of C. mucronata are used by some communities to treat venereal diseases, wounds and gastrointestinal complaints such as diarrhoea and dysentery [1,8]. T. villosa is reported to be toxic to animals and fish; however, in India it is used in the treatment diabetes mellitus [16]. There is no larvicidal activity is reported before for C. mucronata and T. villosa. Therefore, in this study, larvicidal, antimicrobial and brine shrimp activities were evaluated to determine the medicinal value and safety of these plant species.
The larvicidal activity assay was used to predict the presence of bioactive compounds that are able to kill mosquito larvae (C. quinquefasciatus larvae) or disrupt their development towards the adult form which is    responsible for disease transmission [17]. Similarly, the brine shrimp lethality test is normally used to predict presence of toxic bioactive compounds but also possible presence of compounds with potential anticancer activity [21]. In this regard, ethanol and dichloromethane root extracts from C. mucronata exhibited high larvicidal activity, with the highest activity observed with dichloromethane extract which had the LC 50 value of 117.09 μg/mL. C. mucronata aerial part extracts did not exhibit any larvicidal activity. The extract is generally regarded as non-toxic if its LC 50 is greater than 100 μg/mL in the brine shrimp lethality assay [21]. In this case, ethanolic extract of the roots of C. mucronata had larvicidal activity but not toxic, while dichloromethane extract showed larvicidal activity but also observed to be toxic. The literature report that the root extract of C. mucronata is active against chloroquine-sensitive and chloroquine-resistant P. falciparum malaria parasites [5], which is transmitted by Anopheles mosquito. Mosquitoes are responsible for the spread of various vector born diseases including malaria which is found in more than 100 countries in the world [22]. Although there are several ways of preventing the mosquito borne-diseases, one of the best option to prevent the spread of those diseases is to control mosquito vector by using larvicides. However, resistance and environment damage caused by synthetic agents have prompted for the search for environmental friendly and effective larvicides from plant sources [23]. In this study, results have shown that ethanol extract of the roots of C. mucronata may be useful as larvicidal agent. Based on arguments by Aligiannis et al., 2001 [24], extracts from C. mucronata with promising antimicrobial activity were CMAE against V. cholera (MIC = 0.391 mg/mL) and CMAD against B. anthracis (MIC = 0.195 mg/mL), S.typhi (MIC = 0.781 mg/mL), and C. neoformans (MIC = 0.195 mg/mL). Antimicrobial activities of C. mucronata collected from other parts of the world have already been documented [10,11]. Although C. mucronata roots are used by the people of Coast region in Tanzania for treatment of fresh wounds, the antimicrobial activity of the extracts from roots was found to be generally lower than that of the extracts from aerial parts (Table 2). Therefore, the use of root extracts for treatment of fresh wound may suggests that, apart from the weak antibacterial properties, C. mucronata root extract might has other properties such as anti-inflammatory property. Furthermore, these results suggest that, the aerial parts of this plant species may substitute roots in the preparation of herbs needed in the treatment of wounds and other bacterial infection.
All ethanolic extracts of T. villosa exhibited larvicidal activity, with highest activity being in the fruit extract which had LC 50 value of 53.25 μg/mL. Furthermore, extracts from leaves and twigs were not toxic to brine shrimp (LC 50 > 100 μg/mL) whereas extracts from fruits and roots were very toxic to brine shrimp with LC 50 < 10 μg/mL (Table 1). Interestingly, the LC 50 value of twigs extract in the larvicidal activity was lower than the LC 50 for brine shrimp toxicity. This suggests that, ethanolic extract from twigs may be useful as larvicides in the control of the spread of mosquito-borne diseases. This observation corroborates the earlier reports on the seeds of T. vogelii which share the same genus as T. villosa. The seeds of T. vogelii are reported to have larvicidal activity against Aedes aegti larvae [25].
Brine shrimp results obtained from fruits and roots support previous claims of T. villosa toxicity to animals and fish [14] and medicinal use of leaf extract (16). Fruit and root extracts of T. villosa were found to be very toxic with their LC 50 values below that of the anticancer drug, cyclophosphamide whereas leaf and twig extracts were found to be non-toxic (Table 1). This suggests that the toxic characteristic of T. villosa is due to the fruits and roots, and strengthen the speculation for potential to yield anticancer agents (26). The ethanolic extracts from T. villosa were also evaluated for their antimicrobial properties. The extracts with promising antimicrobial activity were from fruits against C.neoformans with MIC = 0.195 mg/mL, roots against B. anthracis with MIC = 0.195 mg/mL and leaves against E. coli with MIC = 0.781 mg/mL. When compared with other species in the same genus, most of the species reported earlier showed no or weak antimicrobial properties [26]. Some of the reported antimicrobial activities from the genus Tephrosia are of methanolic extract from aerial parts of T. apollinea which was active against C. albicans and of aqueous extract from the entire T. purpurea which was active against S. aureus [27,28]. Although most of the extracts from T. villosa showed weak antibacterial and antifungal activity, the good activity observed from the fruit extract against C. neoformans suggests that further studies is needed to identify bioactive compound responsible for the antimicrobial activity.

Conclusion
These results support the use of C. mucronata in traditional medicine for treatment of wounds. Extracts of C. mucronata have potential to yield active antimicrobial and larvicidal compounds. The high brine shrimp toxicity of T. villosa corroborates with literature reports that the plant is toxic to both livestock and fish. The results further suggest that T. villosa extracts have potential to yield larvicidal and possibly cytotoxic compounds. Further studies to investigate the bioactive compounds responsible for the observed biological effects are suggested.