Antioxidant and Larvicidal Activity of Areal Parts of Scrophularia striata against Malaria Vector Anopheles stephensi.

Background
Scrophularia striata is a perennial plant which is native in all parts of Iran, Turkey, and Azerbaijan. In this study, the total phenol content, antioxidant and larvicidal activities of total extract and different fractions of this plant were evaluated.


Methods
The aerial parts of S. striata were collected from Boli village, Illam Province, western Iran in Apr 2013. The total phenol content of total extract and different fractions were evaluated by Folin-Ciocalteu method. Moreover, antioxidant activity was tested by DPPH and FRAPS assays. Larvicidal activity was investigated according to standard method described by WHO.


Results
Ethyl acetate fraction (EF) had the highest content of total phenol (75.9±0.06mg Gallic acid equivalent/g dry extract). Furthermore, among the tested extract, methanol-water fraction (MWF), total methanol extract (TME) and water fraction (WF) showed the highest antioxidant activity in the DPPH assay (IC50= 226.8, 283.66 and 299.4 μg.ml-1, respectively). In FRAP assay MWF and WF and TME had the highest antioxidant activities (664.4±0.002, 565.3±0.003, 519.5±0.003mmol FeII/g dry extract, respectively). Ethyl acetate fraction had maximum larvicidal activity (LC50 49.1ppm) followed by TME (LC50 64.26ppm) and hexane fraction (HF) (LC50 89.69).


Conclusion
Scrophularia striata collected from west of Iran illustrated considerable antioxidant and larvicidal effects and further in vitro and in vivo experimental models for investigation would be required.


Introduction
According to the word malaria report, in 2014, 198 million cases of malaria occurred globally in 2013 and led to 584000 deaths (1)(2)(3). In southern parts of Iran including Sistan and Baluchistan, Hormozgan and some parts of Kerman provinces, this disease remains as a vital public health problem (4). Six Anopheles vectors exist in this area including An. culicifacies, An. stephensi, An. dthali, An. fluviatilis, An. superpictus and An. pulcherrimus (5). The present proliferation of malaria is due basically to increasing resistance of mosquitoes to current insecticides (6).
Plant materials have been investigated for their susceptibility in controlling the malaria vector due to their ovicidal, larvicidal and adulticidal activities. The most susceptible stage to attack mosquitoes is larval stage as they are concentrated in smaller areas. Therefore, interrupting mosquito life cycle at larval stage is one of the important methods for controlling malaria transmission (7).
The genus Scrophularia (Scrophulariaceae) comprises about 300 known species. The various species of Scrophularia genus have been traditionally used in medical conditions including skin inflammatory disease like scabies, tumors, eczema (8), and psoriasis, inflammatory disorders fever, constipation, pharyngitis, neuritis, laryngitis affections (9). Among these species, S. ningpoensis Hemsl has been used for treatment of fever laryngitis, pharyngitis, neuritis and constipation swelling in China. Moreover, S. grossheimi and S. nodosa are used as diuretic agents (10). Recent studies reported antimalarial, antiprotozoal and antimycobacterial activities of S. cryptophila (11).
Scrophularia striata Boiss is a perennial plant grown in all parts of Iran as well as Turkey and Azerbaijan. These species have been used to cure different inflammatory diseases such as allergy, rheumatics and chronic inflammatory disorders in Iranian folk medicine (18). So far, phytochemical investigation revealed the presence of cinnamic acid, flavonoids such as quercetine, isorhamnetin-3-O-rutinoside, and nepitrinandand, and one phenyl propanoid glycoside acteoside 1 in this plant (10). Studied showed the inhibitory effect of S. striata extract on matrix metalloproteinases and astrocyte cancer cell line (1321) (18,19).
In the present study, total extract and different fractions of S. striata were investigated for larvicidal properties against main malaria vector, An. stephensi. In addition, total phenol content and antioxidant activity (using DPPH and FRAP methods) of this plant were evaluated.

Plant material and extraction
The aerial parts of S. striata were collected from Boli village, Ilam Province, western Iran in Apr 2013. The voucher specimen was deposited in herbarium of faculty of Pharmacy, Tehran University of Medical Sciences (Herbarium number: 6748-TEH).

Antioxidant activity DPPH radical-scavenging activity assay
The antioxidant activity of extracts was investigated by the DPPH (2, 2′-diphenyl-1-picrylhydrazyl) free radical scavenging method according to an established protocol. Sample solutions (1ml) were prepared in methanol at different concentration. Then the solutions added to DPPH methanol solution (2ml, 40μg.ml -1 ) and incubated at room temperature for 30 min. The absorbance was measured at 517nm. Vitamin E and butyl hydroxyanisole (BHA) were used as positive controls. IC50 values (indicate the concentration of the test samples providing 50% radical scavenging) were calculated from graph-plotted scavenging percentage against extract concentration (22).

Determination of total phenolic contents
Total phenolic contents of extracts were determined by Folin-Ciocalteu method (26). Folin-Ciocalteu (Merk) reagent diluted to tenfold with distilled water. Then 5ml of this solution was added to 1ml of each extract (1 mg.ml -1 ) and allowed to stand at room temperature for 10min. A 4ml sodium bicarbonate solution (75g.l -1 ) was added to the mixture. After 30min at room temperature, absorbance was measured at 765nm using a UV spectrophotometer (Pharmacia Biotech). Total phenolic contents were quantified by calibration curve obtained by measuring the absorbance of a known concentration of Gallic acid (GA) standard (20-200mg.l -1 ). The concentrations are expressed as milligrams of Gallic acid equivalents (GA) per gr dry extract (22,27).

Bioassays and larval mortality
Fourth instar larvae of An. stephensi Bandar-Abbas strain was exposed to test concentrations of 20, 40, 80, 160 and 320ppm of each extract (solvent: Ethanol) for 24h according to standard method described by WHO (1981).
Briefly, 1ml of appropriate dilution of each extract with 224ml of water and 25 larvae in 25ml water mixed and total volume was 250 ml (2). For control, only 1ml of ethanol with 224ml of water and 25 larvae in 25ml water mixed and total volume was 250ml. The experiment was repeated four times on different days. The percentage of mortality was reported from the average for the four replicates after 24h exposure period. From the regression line between logarithmic dose and probit mortality, the LC50 was measured (28,29).
The investigation of larvicidal activity has been carried out in the insectarium of Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran.

Discussion
Scrophularia striata is a perennial plant which is native in all parts of Iran as well as Turkey and Azerbaijan. In this study total methanol extract and different fractions from aerial parts of S. striata were investigated for antioxidant and larvicidal activities. According to our results, EF and TME had the highest amount of total phenolic compounds. Scrophularia steriata, as highest amount of total phenolic compounds, was reported in ethanol 70% and ethyl acetate extracts (79.7 and 65.5mg Gallic acid equivalent/g dry extract, respectively). Moreover, water extract of this plant was reported to have better radical scavenging activity (IC50 195µg.ml -1 ) (30).
Furthermore, evaluation of larvicidal activity of S. striata revealed that LC50 value of total extract and fractions ranged between 49.15 to 1265.96ppm, and the EF had maximum larvicidal activity (LC50 49.15ppm). Essential oils and extracts of different plant species have been investigated for larvicidal activity. For instance, the essential oil prepared from seeds of Heracleum persicum had larvicidal effect with LC50 value of 104.80ppm. The essential oil of Cupressus arizonica revealed significant larvicidal activity against An. stephensi with LC50 79.30ppm (31). Moreover, for Coriandrum sativum LC50 value of 120.95ppm and for Cymbopogon olivieri 321.90ppm were reported (5), which their activities were low when compared with EF and TME of S. striata. The LC50 of essential oils from Tagetes minuta and Foeniculum vulgare were 1.05 and 20.10ppm, respectively (5), these plants were more effective than S. striata.
The methanol extract of T. minuta had better LC50 value (2.5ppm) followed by methanol and aqueous extract of Nelumbo nucifera (8.89 and 11.82ppm, respectively) and methanol extract of Cassia fistula (17.97ppm) when compared with the extract and fractions of S. striata (32)(33)(34).

Different extracts of fruits and leaves of
Centratherum anthelminticum were tested for larvicidal activity against An. stephensi and the petroleum ether extract of fruits (LC50 162.60) were more toxic than that of leaf extract (LC50 522.94) (7). Petroleum ether extract from leaves of Artemisia annua had larvicidal activity with LC50 value of 263 ppm (7). The leaf and seed methanol extracts of Clitoria ternatea showed dose-dependent larvicidal activity against An. stephensi with LC50 values of 555.6 and 116.8ppm, respectively (35). The leaves petroleum ether extract of Gymnema sylvestre exhibited the highest mortality in the concentration of 1000ppm against the larvae of An. subpictus (LC50 166.28 ppm) (36). Moreover, the leave methanol extract of Calotropis gigantea showed larvocidal activity (LC50 121.69ppm) (37). Larvicidal activity of aceton extract of Millingtonia hortensis (LC50 223.9ppm) and ethanol extract was from peels of Citrus sinensis (LC50 291.69ppm) (35,38).

Conclusion
EF and TME of S. striata possesses better larvicidal activity against An. stephensi than other fractions. Moreover, antioxidant activity of MWF, WF, and TME were higher than other fractions in both DPPH and FRAP assays. Finally, complete phytochemical investigation is suggested to reveal most effective compound in this native species.