J Arthropod-Borne Dis, June 2022, 16(2): 136–147 S Ghafari et al.: Phytochemical Composition and … 136 http://jad.tums.ac.ir Published Online: June 30, 2022 Original Article Phytochemical Composition and Bioassay on Iranian Teucrium Polium Extracts against Anopheles Stephensi (Diptera: Culicidae) Saeedeh Ghafari1, *Azar Tahghighi2, Khadijeh Shamakhte3, Hamzeh Alipour4, Naseh Maleki- Ravasan5, Mehdi Nateghpour6 1Traditional Medicine and Materia Medica Research Center, Shahid Beheshti University of Medical Scienc- es, Tehran, Iran 2Medicinal Chemistry Laboratory, Clinical Research Department, Pasteur Institute of Iran, Tehran, Iran 3Department of Biochemistry, Payame Noor University, Tehran, Iran 4Research Center for Health Sciences, Institute of Health, Shiraz University of Medical Sciences, Shiraz, Iran 5Department of Parasitology, Pasteur Institute of Iran, Tehran, Iran 6Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran *Corresponding author: Dr Azar Tahghighi, E-mail: atahghighi2009@gmail.com (Received 26 Nov 2019; accepted 16 May 2022) Abstract Background: Anopheles stephensi is an important malaria vector mosquito in Iran and other western Asian countries. In many human communities, plant products have been used traditionally instead of synthetic pesticides for mosquito control due to their minimal hazardous effects. Teucrium polium, known popularly as felty germander, has been intro- duced in Persian Medicine (PM) as an insect repellent from a long time ago. Methods: The present study was undertaken to evaluate repellent and larvicidal activity of dichloromethane (DCME- TP) and ethanolic extracts (EE-TP) of T. polium against An. stephensi under laboratory conditions. The possible chemi- cal components of the extracts were also investigated through gas chromatography/mass spectrometry (GC-MS) tech- nique. Results: Based on the results, DCME-TP showed better repellent activity than EE-TP with 56.67 and 28.33 % protec- tion, respectively. Larvicidal activity of DCME-TP with 49.41% mortality was also higher than EE-TP (20.24%). The main identified constituents of DCME-TP were long chain alkanes, phenol, aromatic ester, oxaspiro and triterpenoid. While phenolic and aliphatic acid were only the identified components in EE-TP. It is notable that lupeol was detected in DCME of T. polium for the first time. Conclusion: DCME-TP can be considered as a new herbal candidate to control An. stephensi mosquitoes. Further stud- ies are required on this extract for the fractionation and identification of the active compounds, and the evaluation of their bioactivity in the laboratory and field. Keywords: Larvicidal; Repellent; Teucrium polium; Anopheles stephensi; Phytochemical Introduction According to World Health Organization (WHO) report, “no significant gains were made in reducing malaria cases in the period 2015 to 2017. The estimated number of malaria deaths in 2021, at 619000, remained virtually unchanged over the previous year” (1). Malaria is caused by five different species of Plasmodium: P. fal- ciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi, which are transmitted by mosquitoes of the genus Anopheles. Anopheles mosquitoes are bloodsucking insects and responsible for the transmission of malaria, filariasis and arbovirus- es. There are more than 30 species currently rec- ognized as Anopheles species, out of which sev- en of them have important roles in malaria trans- mission in Iran (2, 3). Anopheles stephensi is the Copyright © 2022 The Authors. Published by Tehran University of Medical Sciences. This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International license (https://creativecommons.org/licenses/by- nc/4.0/). Non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ J Arthropod-Borne Dis, June 2022, 16(2): 136–147 S Ghafari et al.: Phytochemical Composition and … 137 http://jad.tums.ac.ir Published Online: June 30, 2022 primary vector of malaria in the southern parts of Iran and other West Asian countries (2). While the centralized application of malar- ia control programs has strongly diminished ma- laria transmission in malarious regions in the past year, malaria elimination remains an un- attainable aim in high-endemic areas (1). There are various methods for malaria control, such as, drug chemotherapy, personal protection, and mosquito control using chemical insect repel- lents and insecticides (4–5). Control of mosquitoes by repellents is an excellent strategy to avoid biting mosquitoes which can decrease the incidence of mosqui- to-transmitted diseases. Another control strat- egy is killing mosquitoes and their larvae by chemical insecticides such as pyrethroids, or- ganophosphates and carbamates (5). Although, control of Anopheles mosquitoes with synthetic repellents and insecticides is possible, their en- vironmental effects and the emergence of re- sistance are the main concerns in the world- wide (5). In this situation, novel control tools can play an important role in the effort to con- trol and eventually eliminate malaria. Application of herbal preparations is a suit- able tool which can be used as an alternative to synthetic repellents and insecticides. Rapid action, minimal side effects on the skin and quick decomposition in the environment are minimum benefits of medicinal plants which encourage the scientists to assess their anti– insect activities (6). Several studies have shown that some ex- tracts and essential oils of plants presented re- pellency, ovicidal, larvicidal and pupicidal ac- tivities against mosquitoes (7–11). In mosqui- to control programs, products with botanical origin and traditional medicine may have the potential to be used successfully (12). Persian Medicine (PM), as one of the old- est types of complementary medicine, has a long history of using plants for treatment and prevention of diseases (13). PM has introduced many plants as repellents or insecticides which could be the suitable alternatives to mosquito control programs (14). One of these plants is Teucrium polium, traditionally named “Joadah” (15) or “kalpooreh” (16), which belongs to the family Lamiaceae. In this regard, PM manu- scripts recommended “Joadah” could be spread in the environment or smoke to repel insects (15). In a study, the repellency effect and fu- migant toxicity of T. polium essential oil against Callosobruchus maculatus F. (Cole- optera: Bruchidae) and Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae), as the stored-product insects, have been also evalu- ated (17). In recent years, the recognition of bioactive chemical agents from plants and oth- er natural sources has been noticed by re- searchers to control mosquitoes. The aims of this study were to evaluate the repellency and larvicidal activities of dichloromethane extract (DCME) and ethanolic extract (EE) of T. po- lium against An. stephensi mosquito and iden- tifying the chemical components of these ex- tracts using gas chromatography- mass spec- trophotometry (GC-MS). Materials and Methods Plant materials The aerial parts of T. poilium were col- lected in March 2008 from Sarkouh, one of the parts of Bandar Lengeh City in Hormozgan Province, located on the north coast of Persian Gulf, South of Iran. Originality of the plant was identified by H Moazeni and A Pirani, botanists of Traditional Medicine and Materia Medica Research Center (TMRC), Shahid Beheshti Uni- versity of Medical Sciences, Iran. The voucher specimen 2095 (TMRC) of the plant has been deposited in the TMRC herbarium. Extraction The dried aerial parts of T. polium (200g) were powdered and defatted with hexane, and then macerated in dichloromethane (5:1). DCME-TP was separated by filtration and the residue of the plant was dried at room tem- perature and then macerated in ethanol sol- http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2022, 16(2): 136–147 S Ghafari et al.: Phytochemical Composition and … 138 http://jad.tums.ac.ir Published Online: June 30, 2022 vent (5:1). All extraction operations have been performed for 24 hours at room temperature with constant shaking. Each extraction pro- cess was repeated three times and finally three replicate extracts were added to each other. The dichloromethane) and ethanol extracts of T. polium were concentrated by a vacuum ro- tary evaporator under reduced pressure at a maximum temperature of 50 °C to yield 4.1 and 19.88g of residue, respectively and stored at 4 °C. Sample preparation for repellency test Formulation of samples composed of DCME-TP and EE-TP were prepared using oil and aqueous phases (18). The oil phase con- sisted of stearic acid (1.5g), cetyl alcohol (0.4g) and isopropyl myristate (0.3g) separately. The aqueous phase consisted of DCME-TP and EE- TP (2.5g) solved in glycerin (1.5g) and water (7.8mL). Finally, oil and aqueous phases were added to each other under heating about 65– 70 °C in a water bath and mixed suitably. The base formulation without extracts was used as control. Mosquito rearing The laboratory bred An. stephensi strain of Chabahar were used for repellency tests which was reared and maintained at 27±3 °C and 70– 80% relative humidity with a photoperiod of 12h light and 12h dark in the Insectarium of the School of public Health, Shiraz University of Medical Sciences (SUM). Repellency assay Five to seven days old non-blood fed fe- males An. stephensi were used for repellency tests. The repellency study was conducted by a modified 4-celled Klun and Debboun (K and D) module (19) which is used for quantitative measurement of the efficacy of mosquito re- pellents on 6cm2 of the forearm skin of healthy male volunteers aged about 45 (Fig. 1). The module was built by Plexiglas to minimize vis- ual error such as: four cells with larger dimen- sions to hold 20 mosquitoes in each cell. On the test day, the volunteer had no contact with any cream, lotions, perfumes, or perfumed soaps. Before the application of the formulated sam- ples for repellency test, the arm of the volun- teer was cleaned by distilled water. After dry- ing, marked areas on the skin (6cm2) were cov- ered with 100mg of sample for three cells and a K and D module bioassay system was put on these areas. Only samples foundation served as a control in cell number 4. Twenty mosquitos were then release into each cell by an aspirator. Ob- servations on the number of bites of An. ste- phensi mosquitoes were recorded at 30 minutes post treatment. The percentage of repellency was calculated by the following equation: % protection= [Nm/Nt]× 100 Where Nm is the mean number of unfed females in the treatment group and Nt is the total number of mosquitoes. Larvicidal bioassay Based on the preliminary tests, fourth and third instar larvae of An. stephensi, Chabahar strain were exposed to serially diluted test con- centrations of 62.5, 125, 250, 500, 1000, and 2000ppm of ethanolic and dichloromethane extracts of T. polium for 24 hours according to WHO protocol with minor modifications such as: application a surfactant to distribute monotonous of extracts (20). As the extracts do not dissolve in water completely, EE-TP was dissolved in ethanol and DCME-TP was dissolved in acetone. Test solutions were pre- pared by adding 1mL of appropriate dilution of extracts in ethanol and mixed with 99mL of dechlorinated water containing 0.05% Tween 80 (v/v aq). In the control beaker for EE-TP, 1% ethanol was added into water and Tween 80 (0.05%). For DCME-TP, control beaker was including 1% acetone and 0.05% Tween 80. Also, dechlorinated water served as untreated control. A minimum of 25 healthy larvae per each concentration were used for all the ex- periments. The dead larvae were counted after 24 hours recovery period, and the percentage of mortality was reported as the average of the four replicates. http://jad.tums.ac.ir/ https://www.researchgate.net/publication/328303444_Klun_Debboun_modules_Uses_and_data_analysis J Arthropod-Borne Dis, June 2022, 16(2): 136–147 S Ghafari et al.: Phytochemical Composition and … 139 http://jad.tums.ac.ir Published Online: June 30, 2022 Gas chromatography-mass spectrometry (GC-MS) analysis of the extracts GC–MS analysis of the extracts was per- formed using an Agilent 7000, Triple Quad, GC 7890A system operating in EI mode (70ev) equipped with a capillary column (HP-5MS, 30m (length), 0.25mm (diameter), film thick- ness of 0.25µm) as the stationary phase (7). The column oven temperature was initially held at 50 °C for two minutes, and then in- creased to 290 °C at the ramp rate of 5 °C/min and held for 10 minutes at the same tempera- ture. The total run time was 60 minutes. The temperatures of the injector and detector were set at 250 and 300 °C, respectively. The flow rate of helium as a carrier gas was 1mL/min. Compounds were identified by comparison of mass spectral fragmentation patterns and re- tention indices with National Institute of Stand- ards and Technology (NIST) mass spectral li- brary. The relative percentages of the compo- nents were obtained according to the peak ar- ea in the chromatogram. Adverse effect of the formulations on the skin of human volunteers Tests in human volunteers performed to detect allergic contact sensitization following application of extracts formulations. The cu- taneous reactions were monitored for any ab- normalities including erythema, edema, pruri- tus and urticaria, skin allergy and irritation af- ter 15-minute, 1 hour and 24 hours. Results Repellency assay The results obtained from repellency test based on the K and D module bioassay with prepared formulations of T. polium extracts on An. stephensi are presented in Table 1. All formulations showed good results with the 18% concentration used to achieve protection from mosquito bites. Lower blood feeding rate was observed in dichloromethane extract of T. polium with the protection percent 56.67% whereas it was 28.33% for ethanolic extract of T. polium. The base of formulated sample without extract as a control did not exhibit t repellent activity (Table 1). Larvicidal bioassay The consequence of different concentra- tions of the DCME and EE of T. polium at 62.5, 125, 250, 500, 1000 and 2000ppm on the larvicidal activity against An. stephensi after 24h exposure is depicted in Table 2. The high- est and lowest larval mortality of 49.41% and 0% was observed at 2000 and 62.5ppm con- centrations, respectively. The control groups didn’t have any mortality. The result indicated the larvicidal activity of DCME-TP was high- ly dose dependent. Whereas EE-TP with weak larvicidal activity was not completely dose de- pendent. This study showed medium larvicidal activity of DCME of T. polium against fourth- and third-instar larvae of An. stephensi. The cutaneous reactions on the skin of hu- man volunteers Tests in human volunteers to detect aller- gic contact sensitization of extracts formula- tions proved negative. The results did not show any skin irritation, skin sensitization, and skin keratinization. Chemical composition of the extracts Different alkanes such as tetradecane, hex- adecane, octadecane and eicosane, as well as 2,4-di-tert-butylphenol and di-isooctyl phthalate as the aromatic compounds and 7,9-di-tert-bu- tyl-1-oxaspiro (4, 5) deca-6,9-diene-2,8-dione as a di-keton compound were identified in DCME-TP (Table 3). Lupeol was another chem- ical component identified by GC-MS in DCME- TP. Chemical components of 2,4-di-tert-bu- tylphenol and 1-methyl-pyrrolidine-2-carbox- ylic acid were only identified in EE-TP (Table 3). http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2022, 16(2): 136–147 S Ghafari et al.: Phytochemical Composition and … 140 http://jad.tums.ac.ir Published Online: June 30, 2022 Table 1. Repellency assay by the prepared formulation of dichloromethane and ethanolic extracts of Teucrium polium (18% concentration) against Anopheles stephensi Blood feeding/non blood feeding Sample Repeat 1 Repeat 2 Repeat 3 Control %Protection DCME-TP 3/17 4/16 4/16 15/5 56.67 EE-TP 8/12 6/14 8/12 13/7 28.33 Abbreviation: DCME; Dichloromethane extract, EE; Ethanolic extract, TP; Teucrium polium Table 2. Larvicidal activity of dichloromethane and ethanolic extracts of Teucrium polium against Anopheles stephensi Larvicidal activity of DCME-TP Concentration (ppm) Repeat 1 Dead/T* Repeat 2 Dead/T* Repeat 3 Dead/T* Repeat 4 Dead/T* Mortality%** 2000 12/21 10/21 11/21 9/22 49.41 1000 5/21 6/23 4/20 5/22 25.26 500 4/22 4/24 3/20 2/26 14.13 250 2/23 2/20 1/22 2/24 7.86 125 0/24 0/20 1/20 1/22 2.5 62.5 0/21 0/25 0/24 0/20 0 Larvicidal activity of EE-TP Concentration (ppm) Repeat 1 Dead/T* Repeat 2 Dead/T* Repeat 3 Dead/T* Repeat 4 Dead/T* Mortality%*** 2000 4/21 3/21 6/21 4/21 20.24 1000 6/23 4/22 4/25 4/24 19.15 500 4/22 4/23 5/25 4/22 18.48 250 7/21 2/25 3/20 3/25 16.48 125 3/22 2/21 4/22 4/26 14.23 62.5 3/22 2/21 2/21 1/18 9.5 *T: total number of Anopheles stephensi larvae. Abbreviation: DCME; Dichloromethane extract, EE; Etha- nolic extract, TP; Teucrium polium. **The control of DCME-TP containing 1% acetone, 0.05% Tween 80, and 99ml dechlorinated water did not have any mortality. ***The control of EE-TP containing 1% ethanol and 0.05% Tween 80, and 99ml dechlorinated water did not have any mortality Fig. 1. Modified 4-celled Klun and Debboun module used for the repellency assay of dichloromethane (DCME-TP) and ethanolic extracts (EE-TP) of T. polium against Anopheles stephensi http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2022, 16(2): 136–147 S Ghafari et al.: Phytochemical Composition and … 141 http://jad.tums.ac.ir Published Online: June 30, 2022 Table 3. The identified compounds in dichloromethane and ethanol extracts of Teucrium polium by GC-MS analysis The identified compounds in DCME-TP S. No Retention time (min) Area% Name Family Structure 2 21.864 6.25 Tetradecane Alkane (CH2)12 CH3CH3 4 24.585 1.49 2,4-Di-tert-butylphenol Phenol (CH3)3C C(CH3)3 OH 5 26.574 21.96 Hexadecane Alkane (CH2)14 CH3CH3 11 30.826 25.5 Octadecane Alkane (CH2)16 CH3CH3 14 33.264 10.02 7,9-Di-tert-butyl-1- oxaspiro(4,5)deca-6,9- diene-2,8-dione Oxaspiro O O O (CH3)3C (CH3)3C 20 34.695 21.08 Eicosane (Alkane) Alkane (CH2)18 CH3CH3 33 43.799 100 Di-isooctyl phtalate Aromatic ester O O O O (CH2)5CH(CH3)2 (CH2)5CH(CH3)2 49 57.088 2.78 Lupeol Triterpenoid HO H3C CH3 CH3 CH3 CH3 CH3 CH3 H2C The identified compounds in EE-TP S. No Retention time (min) Area% Name Family Structure 1 16.048 0.27 2,4-Di-tert-butylphenol Phenol (CH3)3C C(CH3)3 OH 2 48.525 100 1-Methyl-pyrrolidine-2- carboxylic acid Aliphatic acid N O OH CH3 Abbreviation: DCME; Dichloromethane extract, EE; Ethanolic extract, TP; Teucrium polium Discussion Various species of Anopheles mosquitoes showed resistance to synthetic anti-mosquito agents, whilst their environmental risk is wor- rying due to adverse effects on human, and non-target organisms (7). For these problems, the effectiveness of chemical control agents has been limited in malaria control and eradica- tion programs in recent years. Herbal anti-mosquito agents are environ- mentally friendly alternatives for synthetic in- http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2022, 16(2): 136–147 S Ghafari et al.: Phytochemical Composition and … 142 http://jad.tums.ac.ir Published Online: June 30, 2022 secticides with high biodegradability, low re- sidual effect, novelty in mechanism of action, with insignificant effect on the health of hu- mans and pets, and with negligible harm to the non-targeted occupants of the area. Given these facts, mosquitoes control agents from natural sources can be considered as safe and effective alternatives. Several studies introduced mosquito repellent and larvicidal activity of some ex- tracts and essential oils of plants (7–11, 21-22). According to these studies, the products of tra- ditional medicine have the potential to be used successfully in mosquito control programs. The control of adult malaria vectors with adulticides and repellents is a suitable method to effect on longevity, mosquito densities and other transmission agents. Personal protection is a common approach for preventing mosqui- to bites which helps indirectly in diminishing the mosquito population by depriving the blood meal which is vital for nourishment of the mos- quito eggs in the female Anopheles mosquito. People are mostly being bitten during peak mosquito biting hours, early in the evening, and sometimes throughout the night. Therefore, find- ing a way to protect people from malaria is es- sential during these hours which this gap can fill with mosquito repellents including spatial, and topical repellents. The use of repellents protects local people and travelers in endemic areas and consequently reduces the occurrence of mosquito-borne diseases. There are the var- ious repellents, such as N,N-diethyl-mtoluamide (DEET), para-methane-3,8-diol (PMD), icaridin, 3-[N-butyl-N-acetyl]-aminopropionic acid, ethyl ester (IR3535) (23). DEET is the most effective insect repellent available for human which has broad-spectrum activity on most mosquitoes, ticks and fleas (24). Today, application of DEET has been restricted due to the adverse effects. Icaridin was also classified as slightly hazardous by the WHO hazard classification category (25). IR3535 and PMD have eye irritation. Larvicides, pupicides and ovicides treat- ments can also help to control transmission parameters (7-11, 21-22). A larvicide is a type of insecticide that effects on the larval life stage of an insect and consequently, reduces the adult mosquito population in breeding areas. There are different formulations of larvicides which can be applied directly on water. Therefore, these should not make an unreasonable health risk to humans or other wildlife. Methoprene, an insect growth regulator agent prevents the normal maturation of insect larvae, has moderate and high toxicity on dif- ferent aquatic animals and organisms. Temeph- os, an organophosphate larvicide, affects the cen- tral nervous system through inhibition of cho- linesterase and results in death before reaching the adult stage. Similarly, there are the concerns about its toxicity on non-targeted aquatic spe- cies. In this situation, achievement to new al- ternatives especially, natural products, is one of the most important approaches of research groups. Teucrium polium is a traditional plant in Iran belonging to the family Lamiaceae which is recommended in Iranian traditional medi- cine manuscripts and some papers as an insect repellent (12, 17, 26-27). The present study is the first report of repellent and larvicidal ac- tivities of different extracts of T. polium against An. stephensi. DCME of T. polium showed 49.41% lar- vicidal activity against An. stephensi larvae and 56.67% repellency against mosquitoes. Repel- lent and larvicidal activity of DCME-TP was greater than EE-TP. GC-MS analysis of T. polium extracts iden- tified eight components in DCME containing alkanes (tetradecane, hexadecane, octadecane, and eicosane); 2,4-di-tert-butylphenol; 7,9-di- tert-butyl-1-oxaspiro (4, 5) deca-6,9-diene-2,8- dione; di-isooctyl phthalate; and lupeol (Table 3). 2,4-Di-tert-butylphenol and 1-methyl-pyr- rolidine-2-carboxylic acid were identified in EE-TP(Table 3). The various studies reported larvicidal and repellent activities of plants containing alkanes (28–30). DCME of T. polium was rich in al- http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2022, 16(2): 136–147 S Ghafari et al.: Phytochemical Composition and … 143 http://jad.tums.ac.ir Published Online: June 30, 2022 kanes which might be responsible for its anti- mosquito activity. Citrus hystrix and Kaempfe- ria galanga essential oils presented larvicidal activity against Aedes aegypti (28). Both were containing tetradecane. Good larvicidal and adul- ticidal efficacy of Acacia nilotica seed essen- tial oil has been reported against larval mos- quitoes; An. stephensi, Ae. aegypti and Culex quinquefasciatus (29). Hexadecane was identified in A. nilotica as a major component that prob- ably affects its insecticidal activity. The acetone leaf extract of Melia azedarach was tested by Ranchitha et al. (30) against larvae and pupae of Ae. aegypti. Eicosane was deter- mined as one of its components. The various extracts of Ocimum canum were evaluated against Ae. aegypti and its chloroform extract showed significant larvicidal, pupicidal and adulticidal activity. Eicosan was determined as a major component of this extract (31). Phthalates are used as plasticizers in the production of plastics which are widely dis- tributed in the environment. Fortunately, their rapid photochemical and biological degrada- tion has led to their low level (32). There are little reports of biological active plants con- taining phthalates. Di-isooctyl phthalate was identified as a major component of DCME of T. polium. Ramamurthy et al. (33) reported mod- erate larvicidal and pupicidal activity of etha- nolic leaf extract of Mukia maderaspatana con- taining di-isooctyl phthalate against Ae. aegypti. Based on Babu et al. report (34), di-isooctyl phthalate was identified as a major component in the crude extract of Pongamia pinnata. An- tifeedant and larvicidal activity of di-isooctyl phthalate against Spodoptera litura showed re- markable results. Lupeol was also one of com- pounds identified in this plant (34). The nanoparticles of Acalypha indica in- cluding di-isooctyl phthalate showed the mos- quito repellent and larvicidal properties against Ae. aegypti, An. stephensi and Cx. quinquefas- ciatus (35). 2,4-Di-tert butylphenol, another identified component of T. polium has been found in var- ious species of microorganisms, plants, and an- imals (36). Chen and Dai (37) reported the ovi- cidal, larvicidal, and adulticidal activities of 2,4- di-tert butylphenol against Tetranychus cin- nabarinus in a concentration-dependent manner after treatment. Lupeol, a pentacyclic triterpene also known as fagarsterol (38), identified in DCME of T. polium. According to the literature review, there are no reports about the identification of lu- peol in T. polium. Duan et al. (39) have claimed the acaricidal activity of lupeol, derived from Inula japonica, and its potential as a botanical pest control agent. Díaz et al. (40) isolated lu- peol from Dodonaea viscosa and reported its anti-insect activity against Myzus persicae (green peach aphid) and Epilachna paenulata (lady- bird beetle). Lupeol identified in Vernonia brasiliana showed a mild inhibition activity against P. falciparum growth (41), since the lupeol is a lupane type of triterpenoids, it was considered as an interesting template for derivatization and was led to identify more potent antiplas- modial compounds (42). Ajaiyeoba et al. (43) also reported the antiplasmodial activity lupeol isolated from ethyl acetate fraction of Cassia siamea against multi-resistant strain of P. fal- ciparum (K1). Dichloromethane extract of Den- dranthema grandiflorum containing lupeol pre- sented larvicidal activity against A. aegypti third instar larvae (44). Larvicidal activity of chloroform extract of Carica papaya latex and silver nanoparticles (CPAgNPs) of aqueous latex extract confirmed better activity of CPAgNPs in lower dose against Ae. aegypti and Cx. quinquefasciatus (45). Di- isooctyl phthalate, 2,4-di-tert-butylphenol, hex- adecane, and 7,9-di-tert-butyl-1-oxaspiro (4, 5) deca-6,9-diene-2,8-dione were some identified compounds in chloroform extract of C. papa- ya similar to T. polium in this study (45). According to the studies mentioned above, each of the main components of T. polium identified in the present study has displayed proper anti-insect activity. The larvicidal and http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2022, 16(2): 136–147 S Ghafari et al.: Phytochemical Composition and … 144 http://jad.tums.ac.ir Published Online: June 30, 2022 repellent activity of DCME of T. polium can be probably related to the synergistic effect between the mentioned components. Conclusion This study introduces an ideal eco-friend- ly mosquito repellent from the extract of Teu- crium polium which has a proper potential as a promising candidate for mosquito control. However, further complementary studies such as fractionation extracts to get the molecule(s), mostly responsible for repellent activity, other formulation of extracts, and field trials should be performed to confirm repellent activity. Acknowledgements The authors would like to thank the staff members of the Insectarium of the School of public Health, Shiraz University of Medical Sciences (SUM) to perform bioassay. Ethical considerations This study was performed based on ethi- cal considerations and national regulations in animal experiments (no. 1080). Conflict of interest statement Authors declare that there is no conflict of interest. References 1. WHO (2022) World Malaria Report 2021. World Health Organization. Geneva, Swit- zerland. 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