J Arthropod-Borne Dis, March 2017, 11(1): 116–123 M Khanavi et al.: Chemical Constitute and … 116 http://jad.tums.ac.ir Published Online: March 14, 2017 Original Article Chemical Constitute and Larvicidal Activity of Fractions of Ajuga chamaecis- tus tomentella Plant against Malaria Vector Anopheles stephensi Mahnaz Khanavi 1, Behnaz Najafi 1, Seyede Nargess Sadati 2, Mohammad Reza Abai 3, *Hassan Vatandoost 3 1Department of Pharmacognosy and Persian Medicine and Pharmacy Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran 2Department of Traditional Pharmacy, School of Traditional Iranian Medicine, Tehran University of Medical Sciences, Tehran, Iran 3Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran (Received 10 Jan 2016; accepted 8 Feb 2016) Abstract Backgrand: The genus Ajuga, belongs to Lamiaceae family, is one of the exclusive subspecies in the flora of Iran. The plants of this genus are used traditionally for treatment of joints pain, gout, jaundice, and as insecticide. Methods: larvicidal activity of methanol 80% extract and partition fractions of hexane, chloroform, and ethyl acetate obtained from aerial parts of Ajuga chamaecistus subspecies tomentella against malaria vector An. stephensi was evaluated. Phytochemical study of active fraction was analyzed using column chromatography and spectroscopy. Results: According to the results, among different fractions, hexane fraction has the most larvicidal activity with mortality rate of 100% in concentration of 102 ppm and LC50 of 95.66ppm. The structure of compound 1, main phy- toecdysteroid compound separated from hexane fraction, was determined to be ajugalide-E. Conclusion: The results suggested that the hexane fraction of Ajuga chamaecistus subsp tomentella could be used as a natural and biodegradable insecticide. Keywords: Ajuga chamaecistus subspecies tomentella, Larvicidal, Anopheles stephensi, Phytoecdysteroid Introduction Mosquitoes are the main vector in trans- mission of malaria that is still a major en- demic disease in foci located in south and southeast of Iran. These areas include the provinces of Sistan and Baluchistan, Hor- mozgan and Kerman. Among all species of Anopheles recognized in Iran, 8 of them are considered as malaria vectors including: An. culicifacies, An. stephensi, An. dthali, An. flu- viatilis, An. superpictus, An. pulcherrimus, An. sacharovi, and An. Maculipenni (Doosti et al. 2006). Malaria control is an important goal in developing tropical countries. Mosquito con- trols, using synthetic chemical insecticides have adverse effects on the environment and also cause growing of insecticide resistance in ar - thropods (Edrissian 2006, Khanavi et al. 2011). Plants, rich in bioactive phytochemicals, have been investigated as a source of alternative agents for control of mosquitoes. Several ex- tract and essential oil of certain plants showed toxic effect against some public health pests (Hadjiakhoondi et al. 2003, Vatandoost et al. 2004, Hadjiakhoondi et al. 2006, Govindara- jan et al. 2011, Sedaghat et al. 2011). The genus Ajuga (Lamiaceae) with com- mon name of Bugle is found in China, Ko- rea, Japan and throughout Europe. Five spe- cies of this annual and perennial genus are found in Iran. Ajuga chamaecistus contains several exclusive subspecies, including A. chamaecistus subspecies tomentella (Mozaf- *Corresponding author: Prof Hassan Vatandoost, E-mail: hvatandoost1@yahoo.com, vatando@tums.ac.ir J Arthropod-Borne Dis, March 2017, 11(1): 116–123 M Khanavi et al.: Chemical Constitute and … 117 http://jad.tums.ac.ir Published Online: March 14, 2017 farian et al. 2007). Some species belonging to this genus are used in traditional medicine of different countries in the world. Moreover in Iranian traditional medicine, the genus Ajuga (Kamaphytus, Jaadeh) has been used for treatment of joint pain, gout, and jaun- dice and as insecticide (Naghibi et al. 2005, Jorjani 2012). Several biological studies have been performed on many species of this genus which have confirmed their ethno pharma- cological properties such as hypoglycemic (Hilaly et al. 2002), anti-inflammatory (Gau- tam et al. 2011), anabolic, analgesic, anti- arthritis, antipyretic, hepatoprotective, anti- bacterial, antifungal, antioxidant, cardiotonic (Israili et al. 2009), treatment of joint dis- eases (Ono et al. 2009), and their application as anti-malarial (Kuria et al. 2001). Antifeed- ant activity of Ajuga iva and Ajuga pseudoi- va extract and their active compounds against larvae of Sodoptera littoralis (Egyptian cot- ton leafworm) have been shown in some lit- eratures (Bondì et al. 2000, Ben Jannet 2000, Ben Jannet et al. 2001). Prior to this study, some phytochemicals such as 20-hydroxyecdysone, cyasterone, aju- galactone, makisterone A, and 24-dehydropre- cyasterone (phytoecdysteroids), 8-acetylharp- agide (iridoid), cis- and trans-melilotoside, lavandulifolioside, leonoside B, and martyno- side (phenylethanoid glycosides), were iden- tified from diethyl ether and n-butanolic fractions of Ajuga chamaecistus ssp. tomen- tella. Cytotoxicity evaluation of some frac- tions of this plant showed the cytotoxicity of hexane fraction against normal and cancer cell lines (Sadati et al. 2012 a, b). The aim of this study was to evaluate lar- vicidal activity of a methanol 80% extract and partition fractions of hexane, chloro- form, and ethyl acetate obtained from aerial parts of Ajuga chamaecistus subsp tomen- tella against malaria vector An. stephensi. Furthermore, we performed a phytochemical investigation on the hexane fraction to iden- tify the main components. Materials and Methods Plant material Aerial parts of Ajuga chamaecistus ssp tomentella were collected from Tehran, Iran, in June 2008 and verified by Prof GH Amin. A voucher specimen (THE-6697) has been deposited in the herbarium of the Depart- ment of Pharmacognosy, Faculty of Phar- macy, Tehran University of Medical sci- ences, Tehran, Iran. Preparation of total extract and fractions The air-dried and ground plants of A. chamaecistus ssp tomentella (250g) were extracted with methanol 80% at room tem- perature and concentrated under reduced pressure to give a dark brown extract. The extract (30g) was loaded on Silica gel (mesh 230–400) column and eluted with 250mL of hexane, chloroform, ethyl acetate and meth- anol 80%, separately. Finally the whole col- lected fractions were dried by the rotary evaporator and then by a vacuum oven. Preparing stock solutions Primary tests were performed to determine the concentration of stock solutions. Accord- ing to the results, the concentration of 320 ppm, 160ppm, 2560ppm, was determined for total extract, hexane fraction and methanol fraction respectively. Next, the stock solu- tions were serially diluted to obtain logarith- mic concentrations of solution for the test. For better solubility, DMSO was used as the solvent for hexane fraction and methanol for total extract and methanol fraction. These two solvent are completely safe for larvae as it proved in controls. Larvicidal assays Larvicidal activity assays of Ajuga chamae- cistus subsp tomentella on the larvae of An. stephensi were performed on the basis of WHO protocol. The insectary condition was 30±1 °C, 60±5% relative humidity and 10:14, J Arthropod-Borne Dis, March 2017, 11(1): 116–123 M Khanavi et al.: Chemical Constitute and … 118 http://jad.tums.ac.ir Published Online: March 14, 2017 dark: light periods. The mosquitoes were collected from malarious areas of Iran and then maintained at the Department of Medi- cal Entomology and Vector Control, School of Public health, Tehran University Medical Sciences. 1ml of prepared solution was mixed thoroughly with 224ml water in 400ml glass beakers. 25ml water containing 25 late instar larvae was slowly added. Four replicates maintained for each concentration. A control was set for all series of tests (1 ml of solvent was used instead of 1 ml of extract). Mortal- ity was counted after 24 h recovery period. LC50 (lethal concentration to cause 50% mor- tality in the population) and LC90 (lethal con- centration to cause 90% mortality in the pop- ulation) were determined by the use of re- gression line employed by Finney (Finney 1971, WHO 2014). Chromatography The hexane fraction (4g) was selected for phytochemical studies. Thus, it was chro- matographed on silica gel (mesh 230–400) eluting with a gradient of chloroform- meth- anol (9–1) to 100% methanol to afford 4 frac- tions. Fraction 2 was purified with a few amount of methanol and compound 1 (36.5 mg) was obtained. General experimental procedures 1H- and 13C-NMR were measured in CDCL3 solution on a Bruker Avance spectrometer (500MHz, TMS as internal standard) for com- pounds 1. FT-IR spectra determined using a Nicolet 550-A spectrometer (KBr disks). Column chromatography was acchived on Silica gel 60 (230–400 mesh, Merck) and RP-18 (Merck). Spectroscopic data Ajugalide-E (1): White amorphous pow- der, FT-IR νmax cm-1: 3237, 2953, 1735, 1689, 1248, 1029. 1H and 13C NMR (CDCL3), see Table 3. Results Larvicidal activity of the methanol 80% extract and partition fractions of hexane, chloroform, and ethyl acetate obtained from aerial parts of Ajuga chamaecistus tomen- tella against malaria vector An. stephensi was examined in different concentrations. According to the results presented in Fig. 1, the regression line was plotted for each ex- tract and LC50 was calculated. Among the extracts, hexane fraction showed the most larvicidal effect with LC50 value of 95.66 ppm. LC50 for total extract and methanolic 80% fraction was 117.72 and 954.19ppm respectively. Also, other statistical parame- ters were calculated (Table 1, Fig. 1). Isolated compounds 1, 2 from the hexane fraction of total methanolic extract of aerial parts of Ajuga chamaecistus ssp tomentella were identified by comparison of their NMR (1H-, 13C-NMR) data with those reported in the literature. 1H and 13C NMR data of these compounds run in CDCL3 reported for the first time. δH and δC (ppm) of compound 1 was noted in Table 2. The isolated com- pound 1 (Fig. 2) were identified as ecdyster- oids, ajugalide-E in comparison with the lit- erature (Chan et al. 2005). Tables 1. Lethal concentrations and other associated statistic of bioassay tests of some Extracts A b ± SE LC50 (ppm) ± 95%C.L. LC90 (ppm) ± 95%C.L. λ2 (heterogeneity) λ2 table (df) p- Value Total extract -5.6408 2.7238 ± 0.252 103.6986 117.7283 133.4075 285.1837 309.0209 455.8304 10.704 * 13.345 (2) 0.01 J Arthropod-Borne Dis, March 2017, 11(1): 116–123 M Khanavi et al.: Chemical Constitute and … 119 http://jad.tums.ac.ir Published Online: March 14, 2017 Hexane fraction -8.3002 4.1820 ± 0.199 88.5023 95.6640 105.6703 170.0557 195.5219 236.1008 16.614 * 13.345 (2) 0.01 Methan olic 80% fraction -16.0984 5.4028 ± 0.643 848.9428 954.1945 1071.6982 1420.8157 1647.5819 2045.8799 5.002 * 9.210 (2) 0.01 *No heterogeneity Fig. 1. Comparison of regression lines and equations of and total extract two fractions of Ajuga chamaepitys sub- species tomentella against larvae of Anopheles stephensi O O HO OH OH OAc 1 2 3 4 5 6 7 89 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 O (1) HO Fig. 2. Chemical structure of Ajugalide-E (1), isolated from hexane fraction of Ajuga chamaepitys subspecies tomentella Table 1. Continued… J Arthropod-Borne Dis, March 2017, 11(1): 116–123 M Khanavi et al.: Chemical Constitute and … 120 http://jad.tums.ac.ir Published Online: March 14, 2017 Table 2. 1H NMR and 13C NMR data of Ajugalide E (500 MHz in CDCL3) position δH δC 2 1.98 (td) 3 2.80(dd) 55.71 7 5.25(brs) 122.9 8 - 144.0 13 - 47.92 14 - 81.34 18 0.88(s) 17.6 19 0.72(s) 18.5 21 1.10(s) 20.7 22 4.47(t) 75.95 26 - 184.7 27 0.91(d) 15.8 29 0.84(d) OAC 2.02(s) 171.4 Discussion The larvicidal activity of several plant ex- tracts and phytochemicals against mosquito larvae has been established. In a study re- ported by Sharma, et al. (2004) petroleum- ether extract of Ajuga remota was the most effective extract with LC50 values of 0.033% after 24 hours and 0.029% after 48 hours of treatment against the larvae of An. stephensi (Sharma et al. 2004). Govindarajan et al. (2011) have reported that benzene extract of E. coronaria showed the highest larvicidal effect on the larvae of An. stephensi, Ae. ae- gypti, and Cx. quinquefasciatus with the LC50 and LC90 values were 79.08, 89.59, and 96.15 ppm and 150.47, 166.04, and 174.10 ppm, respectively (Edrissian GhH 2006). Manjari et al. (2014) indicated that acetone leaf extracts of Clausena dentata showed the larval mortality against the fourth instar lar- vae of An. stephensi, Cx. quinquefasciatus, and Ae. aegypti (Diptera: Culicidae). Culex quin- quefasciatus (LC50= 0.150278mg/ml, LC90= 7.302613mg/ml), A. aegypti (LC50= 0.169495 mg/ml, LC90= 1.10034mg/ml), and An. ste- phensi (LC50=0.045684 mg/ml, LC90= 0.045684mg/ml) (Manjari et al. 2014). In this study hexane fraction of methanolic extract of Ajuga chamaecistus ssp tomentella showed the most larvicida activity against An. ste- phensi larvae. In order to find the active ingredient of effective fraction, hexane frac- tion was chromatographed on silica gel and RP-18 resulted in isolation and identification of two phytoecdysteroid epimers, Ajugalide- E and 22-acetylcyasterone. In plants of ge- nus Ajuga, a variety of phytoecdysteroids have been identified among them, 20-hy- droxyecdysone (β-ecdysone) and cyasterone are the most abundant (Ramazanov et al. 2005). This group of natural products pro- duces a wide range of pharmacological ac- tivities in mammals including adaptogenic, anabolic, antidiabetic, hepatoprotective, im- munoprotective, wound-healing, antioxidant and free radical scavenging activities (Sadati et al. 2012 b). Phytoecdysteroids, present in many plants, are analogues of insect moult- ing hormone (ecdysteroids) that control in- sect growth, development, and reproduction (Rharrabe et al. 2010). Toxicity of some ec- dysone agonists on larvae of three mosquito species, Ae. aegypti, An. gombiae, and Cx. quinquefasciatus have been stablished (Beck- age et al. 2004). Nyamoita et al. (2013) in- vestigated that four phytoecdysteroids iso- lated from aceton extract of Vitex Schiliebe- nii showed potent toxic effect against larvae of An. gambiae (Nyamoita et al. 2013). Ac- cording to the result of this study the hexane fraction of total methanolic extract of the aerial parts of Ajuga chamaecistus ssp. to- mentella exhibited the most toxicity on An. stephensi larvae than the other fractions. In previous study two major ecdysteroid in ad- dition to three minor ones identified from this plant (Sadati et al. 2012b). Thin layer chromatography of the hexane fraction showed two main compounds and analyzing of this fraction resulted in isolation and identifica- tion of a phytoecdysteroid, ajugalide-E. Com- parison of our result with other reports indi- cated that the hexane extract of the studied plant was effective to control An. stephensi. J Arthropod-Borne Dis, March 2017, 11(1): 116–123 M Khanavi et al.: Chemical Constitute and … 121 http://jad.tums.ac.ir Published Online: March 14, 2017 Further analysis of hexane fraction to isolate the active component for larval control re- sulted in identifying the major phytoecdys- teroid compound. Based on our results it can be concluded that phytoecdysteroids are in- teresting molecules that can be considered as natural and biodegradable insecticide. Conclusion More investigation is required to assess the larvicidal activity of the product at the field situation. Acknowledgements The authors would like to appreciate very much for kind collaboration of all staff of department of medical Entomology and Vec- tor Control. 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