PRINT ISSN 1119-8362 Full-text Available Online at J. Appl. Sci. Environ. Manage. Electronic ISSN 1119-8362 https://www.ajol.info/index.php/jasem Vol. 24 (8) 1483-1488 August 2020 http://ww.bioline.org.br/ja Isolation and Characterization of Larvicidal Phenolic Acids from Kotschya thymodora Leaves ⃰ 1,2DANIEL, IJ; 2INNOCENT, E; 1SEMPOMBE, J; 1MUGOYELA, V; 3FOSSEN, T *1Department of Medicinal Chemistry, School of Pharmacy, Muhimbili University of Health and Allies Sciences, P. O. Box 65013, Dar es salaam, Tanzania. 2Department of Biological and Pre-clinical Studies, Institute of Traditional Medicine, Muhimbili University of Health and Allies Sciences, P. O. Box 65001, Dar es salaam, Tanzania. 3Department of Chemistry and Centre for Pharmacy, University of Bergen, Allégt. 41, N-5007 Bergen, Norway. *Corresponding Author Email: [email protected] ABSTRACT: Malaria is a vector borne disease responsible for high morbidity, mortality and poverty in many tropical and subtropical countries. The disease is transmitted through a bite from an infected female Anopheles mosquito, amongst which Anopheles gambiae s.s serves as the most prevalent vector. The control of An. gambiae s.s population can therefore lead to a reduction in malaria spreading. Previous studies have reported the crude extracts of Kotschya thymodora to be active against the larvae of An. gambiae s.s and Culex quinquefasciatus. In this report the phytochemical study on the crude aqueous ethanolic extract of K. thymodora leaves led to isolation of vanillic acid (1) and protocatechuic acid (2). The structures of these compounds and mosquitoes larvicidal activity against An. gambiae s.s were established by using spectroscopic techniques and WHO protocol of 1996 respectively. The two phenolic acids exhibited a moderate mosquito larvicidal activity with LC50 of 77.35 µg/mL (vanillic acid, 1) and 62.4 µg/mL (protocatechuic acid, 2) after 48 hrs exposure time. This is the first report on the isolation of the two phenolic acids from plants belonging to the genus Kotschya and their larvicidal potential against An. gambiae s.s. DOI: https://dx.doi.org/10.4314/jasem.v24i8.26 Copyright: Copyright © 2020 Daniel et al. This is an open access article distributed under the Creative Commons Attribution License (CCL), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Dates: Received: 07 June 2020; Revised: 27 July 2020; Accepted: 26 August 2020 Keywords; larvicidal activity, vanillic acid, protocatechuic acid, Anopheles gambiae s.s and Kotschya thymodora Anopheles mosquitoes are the main vectors time the use of synthetic insecticides and antimalarial responsible for transmission of malaria. In the life drugs have served as the mainstay methods for malaria cycle of malaria parasites, these mosquitoes serve as control. Insecticides are normally used with the aim of definitive host permitting their sexual multiplication eliminating An. gambiae s.s vector whereas and maturation to infective forms (Sporozoite) (Cox, antimalarial drugs for inhibiting the passage of 2010). Anopheles gambiae s.s, is known to be the most Plasmodium gametocytes from infected to uninfected prevalent vector of malaria parasites and also a key individual (Kimbi, 2012). The main synthetic transmitter of Plasmodium falciparum, a protozoan insecticides used in the mosquito vector control that causes severe forms of malaria (Leong et al., includes those derived from pyrethroid, carbonates, 2003). The parasite P. falciparum is also the main organochlorines and organophosphates. However, cause of deaths due to malaria in endemic countries some current reports show that some mosquitoes have located in Africa, South-east Asia and Western Pacific developed resistance against these synthetic region (WHO, 2019). About 94% of the malaria deaths insecticides and therefore their efficiency in worldwide occur in sub-Saharan Africa where the controlling malaria transmission is reduced climatic conditions favor the survival and propagation (Yewhalaw et al., 2011). On the other hand, the of An. gambiae s.s (Leong et al., 2003). Many of the malaria parasites have also shown to develop countries located in malaria endemic regions are also resistance towards many commonly used antimalarial characterized by economic underdevelopment. This drugs, hence posing a threat for its continued can be partly contributed by high malaria burden transmission (Kokwaro, 2009). These two factors which imparts a significant economic loss to present the major challenges towards the eradication households and national at large. The economic loss of malaria and its vectors (WHO, 2019). In order to due to malaria is usually the result of direct and improve the control of malaria transmission, it is indirect costs incurred for its prevention, diagnosis and therefore important to overcome the resistance of treatment (Teklehaimanot and Mejia, 2008). For long malaria parasites and mosquito vectors. This calls for *Corresponding Author Email: [email protected] Isolation and Characterization of Larvicidal Phenolic Acids….. 1484 an effective malaria vector control method from other fractions were collected from the following elutions: alternative sources. Plants offer a wide range of Super distilled water (fractions 1), 20% chemical substances that have insecticidal properties. methanol/water (fractions 2), 50% methanol/water For instance, the ethanolic crude extracts of Kotschya (fractions 3), 80% methanol/water (fractions 4), and thymodora (Baker F) leaves and roots have previously 100% methanol (fractions 5). Fraction 2 was been reported to be potent larvicides against An. concentrated using a rotary evaporator and subjected gambiae s.s (Daniel et al., 2020) and Cx. to Sephadex LH-20 column chromatography. A total quinquefasciatus (Innocent et al., 2012). The leaves of 88 sub-fractions were collected using the following extract exhibited larvicidal activity against An. mobile phases: methanol/water/TFA at 30:70:0.2 gambiae s.s with LC50 of 16.35 µg/mL. The mean (sub-fractions 2.1 - 2.18), methanol/water/TFA at mortality recorded for the crude ethanolic leaves 60:40:0.2 (sub-fractions 2.19 - 2.25), and extract of K. thymodora at 50 µg/mL was ≥ 80% after methanol/water/TFA at 80:20:0.2 (sub-fraction 2.26 - 48 hrs exposure. Another phytochemical study on 2.88). All sub-fractions were subjected to analysis by crude ethanolic leaves extract of K. thymodora HPLC and each of fractions 2.42 and 2.48 contained afforded the isolation of cycloartenone. The one major compound. These fractions were compound exhibited a mild larvicidal activity against concentrated on a rotary evaporator at 25oC, and An. gambiae s.s with a mean of mortality of 40% at 50 thereafter dried under nitrogen gas to give a brownish µg/mL after 72 hrs exposure (Innocent et al., 2015). amorphous powder named as compound 1 (38 mg) and The higher larvicidal potential reported for the crude compound 2 (46 mg) respectively. extracts of K. thymodora compared to that of cycloartenone makes it interesting to carry out further Qualitative UV detection of compound 1 and 2 using isolation of other active compound(s). Therefore, this Analytical HPLC-DAD: The UV absorption and study reports the structural characterization of two retention time for vanillic acid (1) and protocatechuic phenolic acids from K. thymodora leaves extract and acid (2) were recorded online using an Agilent their larvicidal activity against An. gambiae s.s. technologies 1260 HPLC instrument coupled to a Diode Array Detector (190-600 nm). The analyses MATERIALS AND METHODS were performed on a reversed phase column (C-18). Collection and identification of plant materials: The The HPLC pump system was first purged with leaves of K. thymodora were collected from Njombe solutions A (Super distilled water with 0.2% V/V, region (GPS coordinates 9056᾿ 11.2 E 34034᾿ 52.6) trifluoracetic acid), B (acetonitrile with 0.2% V/V, with voucher specimen number FMM 3628. The trifluoracetic acid), C (70% methanol/water) and D collection and authentication were done by Mr. Haji (70% acetonitrile/water) at 3 mL/min for 5 minutes. A Seleman, a Botanist from the Department of Botany at 15 µL volume of each sample was injected by an auto the University of Dar es salaam. The specimen is injector. The flow rate was set at 1 mL/min. Analysis deposited in the Herbarium of Institute of Traditional was performed using gradient elution from 90 %A/B Medicine (ITM) at Muhimbili University of Health to 10 %A/B within 55 minutes. The major compounds and Allied Sciences (MUHAS). in the fractions 2.42 and 2.48 had retention times of 7.42 min and 6.80 min respectively. The compound in Preparation of plant materials and extraction fraction 2.42 displayed UV absorption maxima at 262 procedure: The plant materials were chopped into nm and 292 nm. The compound in fraction 2.48 small pieces, dried at room temperature (28 ± 2oC) and (protocatechuic acid) displayed UV absorption then milled into course powder. Extraction of maxima at λmax 260 nm and 294 nm. powdered plant materials was done in 20% aqueous ethanol for 72 hrs with occasional shaking. The NMR spectroscopic analyses: The structures of the extracts were filtered, concentrated using rotary isolated compounds were determined using 1D and 2D evaporator at 25oC and then stored in the refrigerator. NMR spectroscopic data recorded on a Bruker 850 MHz instrument. The NMR experiments performed Chromatographic isolation of compounds (1) and (2): were 1H-NMR, 13C-NMR, 1H-1H COSY, 1H-13C About 75 g of the dried aqueous ethanolic extract from Edited HSQC and 1H-13C HMBC. The spectrometer K. thymodora leaves was fractionated through vacuum frequencies (SF) was set at 850.13 MHz and 213.77 liquid chromatography (VLC) using silica gel 60 (230- MHz for 1H and 13C respectively. The samples were 400 mesh) to give fractions from petroleum ether, dissolved in deuterated DMSO and the experimental ethyl acetate, and methanol mobile phases.