Brine shrimp cytotoxicity, phytochemical screening and larvicidal activities of plectranthus barbatus extracts

The genus Plectranthus (Lamiaceae) represents a large and widespread group of species with a diversity of traditional medicinal uses in the world. The new findings from Plectranthus barbatus ethanolic extracts revealed that cytotoxicity lethality based on Artemia salina Leach in roots are active with LC50=40.07 μg/mL followed by twigs and leaves with LC50= 66.12 μg/mL and 186.33μg/mL respectively. All plant parts contained Saponins,Terpenoids and Glycosides as a mojor principal group of secondary metabolites. Extract from Leaves are more active in Anopheles gambiae with LC50=55.65 μg/mL followed by twigs and roots with LC50=465 μg/mL and LC50 636 μg/mL at 72hours exposure respectively. Twig extracts showed moderate effect on both A.gambiae and A.eagyptiae after 48hours and 72hours exposure time with 100% (MRC) Mortality Rate Concentration of >1000μg/mL. Correspondence to: Kidukuli AW, The Open University of Tanzania, Department of Physical Sciences, P.O.Box 31608. Dar es Salaam, Tanzania, Tel: +255 755 861 283; E-mail: kidukuli@yahoo.com


Introduction
Mosquitoes cause more human suffering than any other organism, they transmit several diseases like malaria, dengue fever, yellow fever, filariasis, schistosomiasis and Japanese encephalitis [1]. Here, malaria parasites are transmitted largely by mosquitoes of the Anopheles gambiae complex, which is the most efficient vector system in the world. This complex consists of a number of mosquito species that vary in their ability to transmit falciparum malaria in Africa [2]. Aedes aegypti is very important vector, transmitting the arbvovirus causing dengue haemorrhagic fever (HDF) and chikungunya in human [3]. At present, no effective vaccine is available for both dengue and malaria; therefore, the only way of reducing the incidence of this disease is by mosquito control, which frequently depends on applications of conventional synthetic insecticides which is not biodegradable [4]. On the other side of nature, the plants powder and plant extracts are sometimes more effective and environmental friendly than the synthetic pesticides and these phytochemicals have major role in mosquito control program [5][6][7].
Malaria is a major global health disease that affects humans, and it predominates in Africa and other areas with tropical climate [8]. Globally, an estimated 3.3 billion people were at risk of malaria in 2011, worldwide there are 104 countries and territories in which malaria is presently considered endemic [9]. In countries where the disease is endemic, pregnant women and children are most at risk. In epidemic areas, both adults and children are at risk [8]. In Tanzania, over 93% of Tanzania mainland population lives in areas where malaria is transmitted. The level of transmission is high in Lake Zone regions, Coastal regions and Southern lowlands. Malaria is still a leading cause of outpatient, inpatient and hospital deaths with 60-80 thousands estimated death per year mainly in children [10].
All over the world an estimated 2.5 billion people are at risk of dengue in tropical and subtropical areas [11]. This disease infection causes a spectrum of symptoms from mild flu-like to severe life threatening haemorrhage [12]. In recent years, Dengue fever and chikungunya affect most parts of the coastal areas of Tanzania and hence increasing burden to the health sector [13,14].
The genus Plectranthus (Laminaceae) represents a large and widespread group of species with a diversity of traditional medicinal uses. The genus comprises a group of around 300 species, distributed in tropical and subtropical areas of Africa, Asia and Australia [15]. One of the interesting species of this genus is Plectranthus barbatus Andr.,which is well-known for the treatment of various ailments. A diversity of the traditional medicinal uses of P.barbatus in India (Hindu and Ayurvedic),East and Central Africa, China and Brazil have been reported to treat many diseases [16].The majority of uses are for intestinal disturbances and liver fatigue, respiratory disorders, heart diseases and certain central nervous system disorder [16][17][18][19][20]. Clinical studies of the plant and the forskolin constituent support these traditional uses and they have other benefit such as prevention of cancer metastases [21] and for the veterinary purposes [22]. Furthermore, its essential oils in tube has very attractive and delicate odour with spicy which act as an antimicrobial agent [23,24].
The aim of this study was to evaluate the efficacy of ethanolic extracts from P.barbatus for larvicidal activity and safety using Artemia salina Leach as a test organism in cytotoxity activity. Moreover, the preliminary screenings of phytochemical constituents are evaluated.

Materials
The plant materials parts (roots, leaves and twigs) of P.barbatus species were collected from Temeke District, Dar es Salaam, Tanzania in February, 2014. The samples were identified and authenticated in the Department of Botany, University of Dar es Salaam. The Brine Shrimps eggs were purchased from Aquaculture innovations (Grahamstown 6140.South Africa) and sea salt was prepared locally by evaporating water collected from the Indian Ocean, along the Dar es Salaam Coast. Mosquito larva Anopheles gambiae and Aedes eagyptiae., were obtained from Tanzania Pesticides Research Institute, TPRI-Arusha, Tanzania, whereas ethanol (absolute) was bought from Flika Chemie GmbH (Sigma-Aldrich®.Zwijndrecht,Netherlands) and Dimethyl sulfoxide (DMSO), were purchased from Sigma®. Potassium hydroxide (KOH), Sodium Hydroxide (NaOH), Acetone,Chloroform, were purchases in the Dar es salaam , Tanzania. Acids (sulphuric acid, Tartaric acid, Hydrochloric acid) were purchases from,Sigma-Aldrich®Zwijndrecht. Netherlands.

Extraction process
The roots, leaves and twigs of P.barbatus were separately dried, grinded to fine powders and later soaked twice in methanol for 48 hours. After that they were separately filtered and further concentrated using a rotary evaporator. The extracts were kept in refrigerator and later subjected to various chemical tests, bioassays and secondary metabolites screening to detect the presence of different useful natural product groups.

Cytotoxicity lethality assay
Artemia salina Leach larvae were used as indicator animals for preliminary cytotoxicity assay of the extracts [25]. The artificial seawater was prepared by dissolving sea salt (3.8 g) in distilled water to make a concentration of 3.8 g/L and then filtered to remove the undeserved particles. The media solution was filled into a tank that has been divided into two unequal compartments by perforated polythene wall. Shrimp eggs were later sprinkled into the covered part of the tank and a lamp was illuminated on the uncovered part in order to attract the hatched auto-tropism shrimps. The mature nauplii were collected in between 36 and 48 hours of hatching [25]. The P. barbatus extracts were dissolved in Dimethyl sulfoxide (DMSO) in vials in duplicate at an initial concentration 240 µg/mL and decreasing up to24 µg/mL. In every vial containing the extract in solution, 10 brine shrimp larvae were added. An additional set of vials containing only a solvent (DMSO) in 5 mL of artificial seawater and 10 shrimp larvae acted as control [25]. The number of survived larvae was established after 24 hours and the LC 50 values of the concentrations required to kill 50% of the shrimp larvae, the concentrations to give 100% mortality rates concentration (MRC) and confidence intervals (CI) were obtained using probit analysis [26].

Testing for larvicidal activity
The larvicidal test was performed according to World Health Organization (WHO) protocol with minor modification [27]. The stock solutions (50 mg/mL) of plant extracts were prepared by first dissolving in DMSO. The stock solutions were diluted with distilled water to make 100mL each, of 500,250,100 and 50 µg/mL solutions of plant extracts. Ten late third instars laboratory reared Anopheles gambiae and Aedes aegyptiae larvae were separately then introduced in the test solution and mortality was observed after 24h, 48h 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 ± 2ºC) 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. There after the mortality rate concentrations (MRC) were derived from the regression equations.

Phytochemical screening of natural product group
Phytochemical screening of natural product groups are performed according to the following standard protocol as described below; Screening for saponins: About 0.5 g of the plants extract was shaken with water in a test tube. Frothing which persist on warming was taken as a preliminary evidence for the presence of Saponins. Few drops of olive oil was added to 0.5 g of extract and vigorously shaken. Formation of soluble emulsion in the extract indicates the presence of saponin [28].
Screening for tannin: Into 10mL of freshly prepared 10% potassium hydroxide (KOH) in a beaker, 0.5g of extract was added and shaken to dissolve. A dirty precipitate observed indicate the presence of tannin [28][29].
Screening for alkaloids: The filtrate was shaken into 3 beakers: Three gram of extract was stirred with ethanol containing 3% tartaric acid. The filtrate was shared into 3 beakers and tasted for alkaloids as follows: Into the first beaker, Hagar's reagent was added into the second beaker; Mayer's reagent was added and into the third beaker Marquin's reagent was added. Precipitations in any of the 3 tests indicate the presence of alkaloid [28,30].

Screening for cardiac glycoside; (Keller Killiani test):
Total 100 mg of extract was dissolved in 1 mL of glacial acetic acid containing one drop of ferric chloride solution. This was then under layered with 1 mL of concentrated sulphuric acid (H 2 SO 4 ). A brown ring obtained at the interface indicates the presence of a de-oxysugar characteristic of cardenolides [28][29][30].
Terpenoids: 0.2 g of the each extract was mixed with 2 ml of chloroform and concentrated H 2 SO 4 (3 ml) was carefully added to form a layer. The formation of a reddish brown coloration at the interface indicates positive results for the presence of terpenoids [28][29][30].

Result and discussion
The phytochemical screening from Table 1 revealed that saponins are present in both roots and leaves; however the activity differs due to the difference in concentration of the compound responsible for the action. These findings are supported by the recent study on phytochemistry, ethanobotanical uses and pharmacology review on the same plant species [15,16]. On the other side the polar group of natural product that is normally water soluble played a big role on the activities. Since on leaves part revealed less or no activity due to lack of this group. Hence the presence of saponins and glycosides as a water soluble natural product group played a big role on these activities as indicated in the Table 1. Table 2; the larvicidal activity in leaves are more active than the other parts with LC 50 =55.65 µg/ml at 72 hours exposure time in Anopheles gambiae, this is followed by twigs and roots at LC 50 of 465.67 µg/ml and 636.01 µg/ml respectively. Generally, Aedes egyptiae are more resistance towards P.barbatus ethanolic extract than

Saponins
Tanins Cardiac glycosides Alkaloids Terpenoids Key: (√) = Presence, (X) = Absence, ND= not done A. gambiae. The findings revealed that, there is no activity towards A.egyptiae up to maximum tested concentration of 80 µg/ml, thus the activity trend are in the order of leaves followed by twigs and roots.
In the other lethality (Table 3), it revealed that roots are more toxic towards Artemia salina Leach cells with LC 50 of 40.07 µg/ml followed by twigs and leaves with LC 50 of 66.12 µg/ml and 186.33 µg/ml respectively. The activity trend of P. barbatus towards brine cells are in order of roots followed by twigs and leaves. These trend comply with the traditional uses of these parts of plant as indicated in the literature [16][17][18][19][20] .Generally, these findings reflect the recently research done in Tanzania in which the plant species showed the potency of mosquito larvicidal in the laboratory trials [31,32].
Thus, the correlation of both mosquito larvicidal and Brine shrimp lethality showed that twigs have mildly activity in both bio-assays while leaves showed more activity on mosquito larvae specifically A. gambiae. These findings are more interesting since leaves do not affect the diversity of plants species and consequently is in line with biodiversity conservation. In Brine shrimp test, roots showed remarkable activity correlated to the traditional used.

Conclusion
These findings revealed that there is no direct connection between cytotoxicity and mosquito larvicidal activity towards the plant parts of P. barbatus. Since, leaves showed remarkable mosquito larvicidal activity towards A. gambiae and roots possessed strong activity towards Artemia salina cells. Thus the plant demonstrate their pharmacological potentials as it has been used for treatments of various diseases since time memorial.