Larvicidal efficacy of crude and fractionated extracts of Dracaena loureiri Gagnep against Aedes aegypti, Aedes albopictus, Culex quinquefasciatus, and Anopheles minimus mosquito vectors

Damrongpan Thongwat, Ratchanaporn Chokchaisiri, Lucksagoon Ganranoo, Nophawan Bunchu

1Department of Microbiology and Parasitology, Faculty of Medical Science, Naresuan University, Phitsanulok, Thailand

2Centre of Excellence in Medical Biotechnology, Faculty of Medical Science, Naresuan University, Phitsanulok, Thailand

3Department of Chemistry, School of Science, University of Phayao, Phayao, Thailand

1. Introduction

Mosquito-borne diseases remain the biggest health problem for humans worldwide. In Thailand, Aedes aegypti (Ae. aegypti)and Aedes albopictus (Ae. albopictus) are the primary vectors for transmitting dengue and dengue hemorrhagic fever[1], Anopheles minimus (An. minimus) is one of the primary vector for the seasonal outbreaks of malaria[2], and Culex quinquefasciatus (Cx.quinquefasciatus) transmits Japanese encephalitis[3]. In 2017, the Bureau of Epidemiology, Department of Disease Control Ministry of Public Health in Thailand reported that more than 30000 Thai were infected by those mosquitoes-borne diseases.

Insecticides have traditionally been the first option for controlling outbreaks of vector-borne diseases, owing to their outstanding efficacy[4]. Temephos, the most well-known larvicide, is widely used for controlling the mosquito larvae population[5]. However,continuous use of temephos has led to negative effects on humans.Moreover, reports of temephos-resistant mosquitoes are continuously being published[6-8]. Therefore, plant biosubstances have been the focus of replacement insecticides.

Plant extracts have been a challenging subject with regard to vector control because of the abundance of plant species and human safety issues. One potentially safer alternative is Dracaena loureiri Gagnep(D. loureiri), commonly known as “Chan Pha”, “Chan Daeng”,and “Lukka Chan”. D. loureiri is a folkloric medical plant with antipyretic and analgesic properties that is used in Thailand for the treatment of colds, fever, cough, in flammation, and gastrointestinal disturbances[9,10]. We previously reported on the larvicidal efficacy of crude extract from the endocarp of D. loureiri against thirdstage larvae of Ae. aegypti, in which the 24-h and 48-h lethal concentration 50 (LC50) values were 84.00 mg/L and ＜ 50.00 mg/L,respectively[11]. Thus, we aimed to assess the larvicidal efficacy of crude and fractionated extracts of D. loureiri against Ae. aegypti and other mosquito species (i.e., Ae. albopictus, Cx. quinquefasciatus, and An. minimus).

2. Materials and methods

2.1. Crude extracts

Crude extracts of D. loureiri (voucher number: DTNU008) endocarp were prepared according to the method outlined in the previous study[11]. Brie fly, the fruits were collected from naturally growing trees and cleaned with tap water. Their endocarps (2.36 kg) were completely dried in a hot air oven at 45 ℃. The dried endocarps(586.33 g) were ground with an electric blender at 22000 r/min,and the resulting dried powder was macerated with absolute ethanol at a ratio of 1:10 (powder:solvent, w/v) with 24 h of continuous shaking (180 r/min) on a rotary shaker. The suspension was then filtered through a WhatmanTM No.1 filter paper (GE Healthcare UK Limited, UK) via a Büchner funnel. Afterward, the extracts were evaporated to dryness under reduced pressure to yield crude extract(26.29 g), which was stored in a desiccator.

2.2. Column chromatographic fractionation

The crude extract was fractionated by column chromatography(Merck silica gel 60 PF254, 250 g) using a gradient solvent system of CH2Cl2, CH2Cl2–MeOH, and MeOH, with increasing amounts of the more polar solvent (mobile phase: 10% MeOH in dichloromethane).After heating at 90–110 ℃ for 4 min, the developing reagent(anisaldehyde reagent, consisting of 3 mL p-methoxybenzaldehyde,2 mL concentrated sulfuric acid, 2 mL water, and 90 mL absolute ethanol) caused organic compounds to emit specific colors, which were examined by thin-layer chromatography. From there, six groups of fractionated extracts were obtained: RC-DT 009 (1.23 g),RC-DT 010 (0.59 g), RC-DT 011 (0.75 g), RC-DT 012 (0.70 g),RC-DT 013 (3.80 g) and RC-DT 014 (1.31 g).

2.3. Mosquito colonization

Ae. aegypti and Ae. albopictus colonies were obtained from laboratory strains from the Department of Microbiology and Parasitology,Faculty of Medical Science, Naresuan University, Thailand. Cx.quinquefasciatus and An. minimus were obtained from laboratory colonies from the Department of Parasitology, Faculty of Medicine,Chiang Mai University, Chiang Mai, Thailand. The larvae were reared in tap water under laboratory conditions: (25±2) ℃, 70%–80%relative humidity, and 10:14 (light:dark) photoperiod. Larval food consisted of powdery dog biscuits (for Aedes and Culex) and fish food (for Anopheles). After pupation, the larvae were transferred into plastic cups filled with tap water that were placed in mosquito cages (30 cm × 30 cm × 30 cm). After emergence, the adults were provided solutions of 5% sugar mixed with 5% multivitamin syrup.After 5 d, the females were provided blood meal through an artificial membrane feeding method. After blood-feeding, female Aedes and Culex were reared until gravid and permitted to lay eggs. Meanwhile,blood-fed female Anopheles were mated though an artificial mating method[12], after which they were permitted to lay eggs. After the eggs hatched, the larvae were reared according to the above conditions until they were required for bioassays.

2.4. Larvicidal bioassay

The protocol for testing larvicidal activity followed that of our previous study[11]. Brie fly, a stock solution of crude and fractionated extracts (1%,w/v) were prepared with dimethyl sulfoxide as the diluent. From the stock solutions, a series of crude and fractionated extract concentrations were prepared (30–190 mg/L and 2–110 mg/L, respectively). Afterward,200 mL of each concentration of extract was placed into plastic bowls.Twenty-five of the late third-stage larvae were transferred into the extract solutions. Mortality rates were determined after 24 h and 48 h of exposure. Larvae confirmed dead when they were pricked by a needle and not moved. This experiment was performed in quadruplicate (total of 100 larvae for each concentration). Dimethyl sulfoxide in distilled water was used as the control.

2.5. Data analysis

Larval mortality data from the larvicidal bioassays were analyzed using a computerized probit analysis for determination of LC50and lethal concentration 90 (LC90)[13]. The chi-square values and 95%fiducial confidence intervals [lower confidence limit (LCL) and upper confidence limit (UCL)] were calculated. A commercial LdP Line®software (Plant Protection Research Institute, Egypt) was used.

3. Results

The larvicidal activities of D. loureiri crude endocarp extract against Ae. aegypti, Ae. albopictus, Cx. quinquefasciatus, and An. minimus mosquitoes were presented in Table 1. At 24 h, An. minimus larvae had the highest susceptibility to crude extract (LC5077.88 mg/L). Its 24-h LC50was significantly lower than that of Ae. aegypti (224.73 mg/L), Ae. albopictus (261.75 mg/L), and Cx. quinquefasciatus (282.86 mg/L). At 48 h, An. minimus was so highly susceptible to crude extract (＞ 90% mortality rate at 30 mg/L) that we did not calculate the 48-h LC50value, although it was estimated to be ＜ 30 mg/L.

Fractionated extraction by column chromatography produced 188 eluted fractions from the crude extract. The fractions were classified into six groups: RC-DT 009 to RC-DT 014 (Figure 1). All groups were preliminarily screened for larvicidal ability. One concentration(110 mg/L) from each group was tested against the third-stage Ae.aegypti larvae. After 24 h of exposure, the RC-DT 012 and RC-DT 013 fractions produced ＞ 90% mortality rates, while the remaining fractions produced 0%–3% mortality rates. For that reason, RC-DT 012 and RC-DT 013 were selected for the bioassays.

The results of larvicidal activity experiments on RC-DT 012 and RCDT 013 were presented in Tables 2 and 3, respectively. In contrast to results from crude extract, Cx. quinquefasciatus (as opposed to An.minimus) was extremely susceptible to both fractions. For RC-DT 012,the 24-h LC50and LC90values were 0.66 and 3.29 mg/L, respectively.For RC-DT 013, those values were 0.94 and 2.77 mg/L, respectively. An.minimus, Ae. aegypti, and Ae. albopictus larvae had minor susceptibility to the fractions. However, the mortality rates of all mosquito species were significantly higher for those exposed to fractionated extracts than for those exposed to crude extract.

The LC50and LC90values of the crude and fractionated extracts for each mosquito species were compared and statistically analyzed.Results showed that the larvicidal activities of fractionated extracts were statistically greater than that of the crude extract for all mosquito species. In fact, the only values that were not statistically significant were the 48-h LC90values for Ae. albopictus (crudeextract: 279.89 mg/L; and RC-DT 012: 224.29 mg/L). According to the results in this study, fractionated extracts were more effective than crude extract against all tested mosquito species.

Table 1 Larvicidal activities of crude ethanolic D. loureiri extracts against the third-stage larvae of 4 mosquito vectors.

Table 2 Larvicidal activities of RC-DT 012 fractionated D. loureiri extract against the third-stage larvae of 4 mosquito vectors.

Figure 1. Thin-layer chromatography spots of organic compounds from isolated fractions (RC-DT 009-014) of D. loureiri.

4. Discussion

Surprisingly, the crude ethanol endocarp extract of D. loureiri had lower activity against Ae. aegypti at 24 h (LC50224.73 mg/L) and 48 h (LC5093.37 mg/L) than in the previous study (24-h LC5084.00 mg/L and 48-h LC50＜ 50 mg/L)[11]. Both studies utilized the same protocol for producing crude extract, so the differences in larvicidal efficacy could be attributed to climate and seasonal difference. That is, the previous study used plants harvested in October 2013[11];this study used the same plants, but the plants were harvested in September 2016.

Of all mosquito species tested, An. minimus showed the greatest susceptibility to D. loureiri crude extract. Other species (Ae. aegypti, Ae.albopictus, and Cx. quinquefasciatus) demonstrated a significant, threefold greater tolerance than that of An. minimus. Similarly, other studies have found that Anopheles larvae are more susceptible to plant extracts than other mosquitoes. For example, Govindarajan et al. discovered that Anopheles stephensi is more susceptible (LC5061.65 μg/mL) to Origanum scabrum essential oil than Ae. aegypti (LC5067.13 μg/mL),Cx. quinquefasciatus (LC5072.45 μg/mL), and Culex tritaeniorhynchus(LC5078.87 μg/mL)[14]. In addition, Anopheles stephensi is more susceptible to Terminalia chebula extract than Ae. aegypti and Cx.quinquefasciatus, with LC50values of 87.13, 93.24, and 111.98 ppm,respectively[15].

Table 3 Larvicidal activities of RC-DT 013 fractionated D. loureiri extract against the third-stage larvae of 4 mosquito vectors.

While An. minimus was the species most susceptible to crude extract,this did not hold true for fractionated extracts. On the contrary, the mosquitoes most susceptible to RC-DT 012 (LC500.66 mg/L) and RC-DT 013 (LC500.94 mg/L) were Cx. quinquefasciatus, which had the lowest LC50values. Furthermore, Cx. quinquefasciatus had the highest tolerance (LC50282.86 mg/L) to crude extract compared to other species: Ae. aegypti (LC50224.73 mg/L), Ae. albopictus (LC50261.75 mg/L), and An. minimus (LC5077.88 mg/L). This outcome could not be explained because of the data limitations of this study.However, we hypothesize that both fractions (RC-DT 012 and RCDT 013) must contain compounds that are highly toxic only to Culex larvae.

The fractionated extracts of D. loureiri provided much better larvicidal efficacy against mosquito vectors than crude extract, which concurs with studies on Sphaeranthus indicus Linn. (Asteraceae)extracts. In those studies, steam-distilled crude extract of leaves were compared with the most effective fractionated ethyl acetate extract of the whole plant[16,17], revealing that fractionated extract is more effective than crude extract against Ae. aegypti (24-h LC5036.76 ppm vs 140 ppm, respectively) and Cx. quinquefasciatus (24-h LC5032.60 ppm vs 130 ppm, respectively).

Our findings suggest that the larvicidal activity of crude extract was not a synergistic action of all compounds in the extract, echoing another recent study that reported the same[18]. In that study, only two of seven groups of fractionated extracts of Acacia pennata (L.)Willd. subsp. insuavis shoot tips contained compounds active against Ae. aegypti larvae. The LC50values of the Fr-G2 and Fr-G3 fractions were 50.75 and 39.45 mg/L, respectively, while the LC50values of the other fractions (Fr-G1 and Fr-G4–Fr-G7) were ＞ 100 mg/L.Similarly, our study found that the active substances in D. loureiri extract were contained only in RC-DT 012 and RC-DT 013, which had the lowest LC50and LC90values for all tested mosquito species.Phytochemical studies have revealed several flavonoids isolated from stems of D. loureiri, including homoiso flavans[9], dihydrochalcone[19],and stilbene[20]. Of those, (2S)-pinocembrin, (3S)-7,4′-dihydroxy-3-(4-hydroxybenzyl)-chromane, and loureirin D have antibacterial activity against Staphylococcus aureus and Bacillus subtilis; and 7,4′-dihydroxyflavan is fungitoxic against Botrytis cinerea and Cladosporium herbarum[9]. Studies by Meksuriyen and Cordell and Ichikawa et al. have reported that retrodihydrochalcones and homoiso flavones isolated from stem wood are estrogen agonists[19,21].In addition, stilbenoids, isolated from stem wood are potent inhibitors of cyclooxygenase (COX)-1 and COX-2 enzymes[20]. Although some phytochemical constituents and their activities have been studied,the phytochemical compounds in the fruit endocarp of D. loureiri have never been investigated. Moreover, until our previous study of crude extract[11], the larvicidal activity of D. loureiri has never been elucidated. Thus, the results of this study could not be compared to the results of other studies. Further studies on the larvicidal activity of D. loureiri extract, phytochemical constituent analysis (e.g., gas chromatography-mass spectroscopy)[22], purification, and mosquito larvicide evaluation of substances purified from the RC-DT 012 and RC-DT 013 groups must be performed.

Conflict of interest statement

The authors declare that there is no conflict of interest.

Acknowledgements

The authors acknowledge the Naresuan University Research Fund (Reference Number: R2560B057) for the financial support and Department of Microbiology and Parasitology, Faculty of Medical Science, Naresuan University for the laboratory facilities.We would like to thank Asst. Prof. Dr. Anuluck Junkum, the staff of Department of Parasitology, Faculty of Medicine, Chiang Mai University, Thailand, and Dr. Danita Champakaew for their laboratory assistance.

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