Computational selections of terpenes present in the plant Calotropis gigantea as mosquito larvicide’s by blocking the sterol carrying protein, AeSCP-2

The present study reports the phytochemical properties of Calotropis gigantea (Asclepiadaceae) commonly known as milk weed. In addition, in silico docking analysis was also carried out to assess the mosquito larvicidal potential of three terpene compounds isolated from C. gigantea . Considerable amount of primary metabolites, essential macro and micro nutrients were documented in the plant. The GC-MS analysis of the chloroform extract revealed the presence of eight terpenes in the plant. From the docking studies it is evident that α-amyrin has a great potential against AeSCP-2. The phytochemical screening and docking results gives strong baseline information for the posterity.

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Elemental analysis: Two grams of dried sample was digested in a mixture of nitric acid, sulfuric acid and perchloric acid in the ratio 11:6:3, for 24 hours to remove the organic matters. The digested sample was made up to 100 mL and used for the assay of the trace elements through atomic absorption spectrophotometer (AAS-Varion 200AA) using suitable hollow-cathode lamps. Appropriate working standard was prepared for each element. All elements were determined through this procedure. A blank reading was also obtained.

Extraction and GC-MS analysis of terpenes:
The crude drug was subjected to extraction with analytical grade solvent of chloroform for GC-MS analysis. 25 g of the crude drug was taken in a round bottom flask and 50 mL of analytical grade chloroform was added and refluxed for 8 hours. After completion of the 8 hours, the round bottom flask was cooled and the extract was filtered through the Buchner funnel. The extract was evaporated to dryness under nitrogen atmosphere using turbo evaporator. The residue obtained was dissolved in 2 mL chloroform and transferred into the GC vial and injected into the GC-MS port.
GC-MS analysis was performed on an Agilent gas chromatograph model 6890 N coupled to an Agilent 5973 N mass selective detector. Analytes were separated on an HP-5MS capillary column (30 m x 0.25 mm x 1.0 µL) by applying the following temperature program: 40°C for 5 min, 40-70°C at 2°C /min, 70°C for 2 min, 70-120°C at 3°C/min, 120-150°C at 5°C/min, 150-220°C at 10°C/min and then 220°C for 2 min. Transfer line temperature was 280°C. Mass detector conditions were: electronic impact (EI) mode at 70 eV; source temperature: 230°C; scanning rate 2.88 scan S -1 ; mass scanning range: m/z 29-540. Carrier gas was helium at 1.0 mL/min. The tentative identification of volatile components was achieved by comparing the mass spectra with the data system library (NIST) and other published spectra (Mass Spectrometry Data Centre., 1974), supported by retention index data, which were compared with available literature retention indices. All compounds were quantified as 3octanol equivalents.
Docking studies: The present biocomputational investigation was carried out to identify the candidate therapeutic terpene compounds having potential for inhibiting the growth of mosquito larvae. The 3-D crystal structure of the protein AeSCP-2 was retrieved from the protein data bank (PDB). AeSCP-2 protein is a low molecular weight with high levels of expression in the midgut of the larvae and high binding affinity to cholesterol in the Aedes aegypti mosquito species (Kitamura et al., 1996). Structural and active site enumeration were done by using pymol molecular visualization software and CASTP (Computed Atlas of Surface Topography of Proteins). Four phytochemicals namely α-amyrin, oleanolic acid, 5-norbornene-2carboxylic acid and pyrethrin were screened against the protein AeSCP-2. The details of these phytochemical were obtained from pubchem database and there chemical structures were generated from SMILES notation (Simplified Molecular Input Line Entry Specification) by using the Chemsketch Software (www.acdlabs.com). The molecular docking analysis was performed using Argus Lab 4.0 which is widely distributed public domain molecular docking software.

Results and Discussion
Leaf sample of C. gigantea was analyzed for the composition of ash materials and the organic carbon, primary metabolites, essential macro nutrient, essential micro nutrient and trace elements. The plant contains 1.69% of ash and 27.5% of organic carbon ( Table I). The primary metabolites, carbohydrate, protein and lipids were 0.97%, 0.55% and 0.29% respectively. The essential macronutrients such as nitrogen (2.16%), phosphorous (0.42%), potassium (2.97%), calcium (4.59%), magnesium (3.16%), and sulfur (0.48%) were found in considerable level. The essential micro nutrients were also found in considerable level. Among micronutrients the amount of iron (156.3 ppm) was the highest and molybdenum (0.1 ppm) was the lowest. The heavy   Compared to previous studies our results are also in considerable level. Similar study on C. procera showed varied concentrations of phytochemicals (Altaf, 1997) and similar variations were also found in our study. These differences could be due to ecological, time of collection, collection sites or may be because of increasing pollution or environmental factors. Previous studies have also report the presence of phytochemicals like terpenes, cardenolides, flavonoids, pregnanes, nonprotein amino acid and cardiac glycoside as major constituents in Calotropis sp. and presence of these phytochemicals sturdily acknowledge the medicinal property of this plant (Wang et al., 2008;Ali and Gupta, 1999).
Approximately 3 billion people, one half of the world's population, live in at-risk regions for malaria infection. This leads to about 250 million malaria cases every year and nearly one million deaths. One of the most crucial obstacles for eradicating malaria is a widespread resistance of malarial parasite to almost all chemotherapeutic agents (Snow et al., 2005). Considering the dreadful global issue of the health, in silico docking analysis was carried out. Four phytochemicals namely alpha-amyrin, oleanolic acid, 5norbornene-2-carboxylic acid and pyrethrin were selected for screening against the protein AeSCP-2. The compounds were docked in Argus lab 4.0. Docking energy was found to be -14.1213 Kcal/mol, -13.7876 Kcal/mol and -8.3558 Kcal/mol in α-amyrin, oleanolic acid and 5-norbornene-2-Carboxylic acid respectively ( Table III). Hence, from the docking studies it is evident that α-amyrin (C. gigantea derived terpene) has a great potential against AeSCP-2.
Medicinal plants are being probed as an alternate source to get therapeutic compounds based on their medicinal properties. C. gigantea is easily available in most of the agricultural and non-agricultural fields and the usage of this plant for medicinal purpose was reported by several researchers. We conclude that C. gigantea represents a rich source of valuable medicinal compounds and leaves of C. gigantea contain α-amyrin  which could be a potential source for inhibiting the AeSCP-2.