Larvicidal activities of family Anacardiaceae on Aedes mosquitoes (Diptera: Culicidae) and identification of phenolic compounds

1 Introduction

Mosquito-borne diseases have economic impacts in the form of health and commercial losses, specifically in countries within the subtropical and tropical regions (Panneerselvam and Murugan, 2013). More than 100 countries are reported to be endemic to mosquito-borne diseases, and every year about two million people die due to these diseases (Subramaniam et al., 2011). Four countries, particularly Philippines, Cambodia, Malaysia, and Vietnam are confronting annual epidemics, which are more than 90% of the total reported dengue cases in the region (Chang et al., 2011). Aedes albopictus and Aedes aegypti and are reported as potential vectors of several viruses, including dengue, Yellow Fever, Zika and Chikungunya. To date, these species have become the leading vectors in the transmission of dengue hemorrhagic fever and dengue in subtropical and tropical regions (Centers for Disease Control and Prevention, 2005). Dengue is endemic in Malaysia, spreading irrespective of the urbanization level (SaifurRahman, 2012) transmitted by Ae. albopictus and Ae. aegypti (Smith, 1956). Aedes aegypti is a domestic mosquito in urban areas, whereas Aedes albopictus is a semi-domestic mosquito (Mendenhall et al., 2017) and an anthropic in nature (Huber et al., 2008). Most of these dengue vectors species are common in natural and artificial containers such as gutters, pools, septic tanks, the tree holes, leaf axils, fruit peels, discarded and unused tires, water jars, old boats and others (Cadena, 2013).

With the introduction of organic insecticides in the 1940s, chemical control became the most popular approach for controlling mosquito (Yap and Zairi, 2003), and the control effort of mosquitoes is mainly relying on chemical insecticide. Long term exposure to the chemical insecticides will lead to selecting mutation, conferring a level of resistance to chemical insecticides, and indeed, the insecticide-resistance population of mosquitoes is now affecting the success of control programs (Dusfour et al., 2019). Biological products such as plant extracts offer boundless possibilities for a new phytochemical discoveries due to the higher availability of a vast range of chemicals (Cos et al., 2006). Family Anacardiaceae is comprised of economically important edible plants like cashew nut (Anacardium occidentale), pistachio nuts (Pistacia Vera), and mango (Mangifera indica), along with other regional imperative plants (Garcia et al., 2000). Anacardiaceae consists of 76 genera comprised of 600 species, with the majority of which are found in tropical and subtropical regions (Hartley, 1998) and affirmed for their medicinal properties (Eloff, 2001). Some of these plants, especially Mn. indica have been used as herbal medicine in some countries to cure warts, ulcers, fever, corns, dysentery, tumour, piles, insect repellent and fungicidal (Olotu et al., 2020).

Compounds and extracts derived from Mn. indica is numerously reported for antifungal, antifeedant, antioxidant, antimicrobial, antidiarrheal, hepatoprotective, hypoglycemic and larvicidal activities (Rahuman et al., 2009). Cashew nut shell liquid (CNSL) from Anacardium occidentale contains flavonoids, steroids, tannins, glycosides, phenols, terpenoids, triterpenoids, anacardic acid, cardanol, cardol, carbachol, orcinol and quercetin (Oliveira et al., 2011).

Four plants from this family Anacardiaceae, were selected for this study due to their natural abundance in tropical regions and potential as a larvicidal agent, namely, An. occidentale, Mn. indica, Gluta renghas and Melanochyla fasciculiflora. This study aimed to (i) evaluate larvicidal efficacy on Ae. albopictus and Ae. aegypti larvae, and (ii) detect the phenolic compounds. Till now, only Mn. indica and An. occidentale had been studied for their chemical compounds. This is the first report on the chemical compounds of two other endemic plants of Malaysia (Ml. fasciculiflora and Gl. renghas).

2 Methodology

3 Results

3.1

3.1 Larvicidal activity of family Anacardiaceae

The larvicidal activity of Family Anacardiaceae was tested against field strain mosquitoes collected from dengue hotspot areas has been found to exhibit the lowest 100% mortality when treated with Gl. renghas of 900 mg/L for Ae. albopictus and 1000 mg/L for Ae. aegypti (Fig. 1). Among the four stem extracts listed in Table 1, Gl. renghas showed the strongest larvicidal effects towards both Ae. albopictus and Ae. aegypti at 607 mg/L and 976 mg/L, respectively. Significantly lower concentration of Gl. renghas were needed for Ae. albopictus as compared to Ae. aegypti (MS = 24.26, df = 1, P = 0.002, Table 2). The weakest larvicidal effects were given by An. occidentale with LC50 of 1200 mg/L for Ae. albopictus and 1347 mg/L for Ae. aegypti. In terms of the order, Gl. renghas exhibited the best effectiveness, followed by Mn. indica, Ml. fasciculiflora and An. occidentale for both Aedes species. Significant differences were detected between the mortalities caused by the crude extracts of the four plants against both Aedes species (MS = 12.65, df = 1, P = 0.002, Table 2).

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Fig. 1

Larval mortality of (A) Aedes aegypti, and (B) Aedes albopictus after 24 h exposure to methanolic stem extracts of Anacardium occidentale, Gluta renghas, Mangifera indica and Melanochyla fasciculiflora.

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Table 1 Mean LC₅₀ and LC₉₅ (mg/L) values with 95% confidence limits of Anacardiaceae stem extracts against Aedes albopictus and Aedes aegypti. Three replicates has been performed for each concentration.

Species	Plant Extract	N	LC50 (mg/L)
95% CIs	LC95 (mg/L)
95% CIs	Slope	x2 (df)
Aedes
albopictus	An. occidentale	480	638
(605–971)	1200
(1095.72–1355.59)	6.00 ± 0.51	1.25
	Gl. renghas	300	240
(185.18–296.59)	607
(448.83–1191.32)	4.08 ± 0.41	5.39
	Mn. indica	480	431
(398.29–463.40)	1076
(944.02–1284.15)	4.14 ± 0.35	2.87
	Ml. fasciculiflora	300	535.59d
(506.78–562.64)	888.68
(810.82–1018.46)	7.48 ± 0.83	1.10
Aedes
aegypti	An. occidentale	480	792
(758.07–825.06)	1347
(1243.83–1500.46)	7.122 ± 0.6	1.47
	Gl. renghas	360	623
(597.01–649.92)	976
(904.69–1082.83)	8.45 ± 0.77	4.15
	Mn. indica	480	582
(548.63–615.63)	1198
(1074.67–1385.58)	5.25 ± 0.44	2.30
	Ml. fasciculiflora	360	696
(660.91–730.80)	1222
(1101.82–1429.12)	6.73 ± 0.75	3.23

Table 2 MANOVA analysis on the effects of different plant species and concentrations against Ae. aegypti and Ae. albopictus larvae mortality after 24hrs of exposure.

Source	df	MS	F-Ratio	P
Aedes species	1	24.26	9.72	0.002
Plant species	3	219.43	87.94	0.000
Concentration	11	922.77	369.80	0.000
Aedes species × Plant species	3	12.65	5.07	0.002
Aedes species × Concentration	10	0.929	0.37	0.957
Plant × Concentration	23	5.92	2.37	0.001
Aedes species × Plant species × Concentration	20	1.18	0.47	0.972
Error	144	2.50

*df = degree of freedom, MS = Mean Square values.

*Significant values are in bold at 5% significance level.

3.2

3.2 Phytochemical and identification of phenolic compounds

Phytochemical compounds found in methanolic stem extracts of An. occidentale, Mn. indica, Ml. fasciculiflora and Gl. renghas were presented in Table 3. Screening results indicated the presence of saponins, tannins, alkaloids, reducing sugar, flavonoids and steroids in all stem extract of An. occidentale, Gl. renghas, Mn. indica and Ml. fasciculiflora from the family Anacardiaceae. However, alkaloids were tested negative for all stem extracts. Phytochemical tests confirmed the existence of the phenolic compound of flavonoid existed in all methanolic stem extracts of An. occidentale, Gl. renghas, Mn. indica and Ml. fasciculiflora.

Table 3 Phytochemical analysis of Anacardium occidentale, Gluta renghas, Mangifera indica and Melanochyla fasciculiflora.

Phytochemical Constituents	An. occidentale	Gl. renghas	Mn. indica	Ml. fasciculiflora
Saponins	+	+	+	+
Tannins	+	+	+	+
Alkaloids	–	–	–	–
Reducing sugar	+	+	+	+
Flavonoids	+	+	+	+
Steroids	+	+	+	+

Further test under the adjusted UPLC conditions, separation of the methanolic extracts of An. occidentale, Gl. renghas, Mn. indica and Ml. fasciculiflora stem was obtained in 7 min with UV detector recorded at 320 nm wavelength. The stem extracts were run by an appropriate reversed-phase Acquity UPLC separation system. The extract constituents were elated as individual UPLC peaks were trapped on solid-phase extraction cartridges using a Prospekt-2 SPE device (Spark Holland), which handles two trays with 96 cartridges each, and thus enables trapping of a large number of individual UPLC peaks in automation. Phenolic and flavonoid compounds in all extracts were identified by comparing UV spectra detected by photo-diode array detected and retention time. The standard calibration curve used authentic standards of gallic acid for phenolic and quercetin methanolic solution for flavonoid.

The major groups of compounds detected were flavonols from quercetin and apigenin derivatives for all the stem extracts of Anacardiaceae, as listed in Table 4. The highest concentrations of apigenin derivatives were detected in Mn. indica, followed by An. occidentale and Gl. renghas, while in Ml. fasciculiflora with the lowest concentration. Similarly, the highest amount of quercetin derivatives was also found in Mn. indica followed by An. occidentale, Gl. renghas and Ml. fasciculiflora. Conclusively, the average amount of apigenin derivatives was found less than the quercetin derivative.

Table 4 Retention times and heights of the compounds of An. occidentale, Gl. renghas, Mn. indica and Ml. fasciculiflora detected by UPLC.

Fig.	Plants	Peak No	Retention Time	Height	Derivatives
	Anacardium occidentale	2	4.136	100,219	Quercetin
		5	4.218	31,719	Quercetin
A		8	4.255	16,833	Quercetin
		10	4.326	38,966	Quercetin
		12	4.373	60,589	Quercetin
		15	4.987	153,358	Apigenin
	Gluta renghas	3	4.171	77,783	Quercetin
		7	4.252	26,644	Quercetin
		9	4.291	13,392	Quercetin
B		11	4.358	28,591	Quercetin
		13	4.403	47,245	Quercetin
		18	5.010	125,544	Apigenin
	Mangifera indica	1	4.043	105,930	Quercetin
C		6	4.219	230,236	Quercetin
		17	5.007	388,191	Apigenin
		19	5.307	124,347	Apigenin
	Melanochyla fasciculiflora	4	4.217	35,177	Quercetin
D		14	4.883	10,428	Apigenin
		16	4.991	10,462	Apigenin

Methanolic extracts of family Anacardiaceae; An. occidentale, Gl. renghas, Mn. indica and Ml. fasciculiflora were fractioned by UPLC, as shown in Fig. 2. The chromatographic spectrum shows 19 peaks, each peak indicating separate compounds. Anacardium occidentale spectrum displayed six prominent peaks, derived from quercetin with retention time from 4.136 to 4.373 min and apigenin at 4.987 min. The UV spectrum of quercetin was detected at the peaks of 4.171 to 4.403 min and apigenin at 5.010 min for Gl. renghas. Similarly, four main peaks were observed in the spectrum of Mn. indica methanolic extracts were represented by two principal groups of derivative compounds of quercetin and apigenin. Melanochyla fasciculiflora depicted only three noticeable peaks at the retention time of 4.217 min as derivative of quercetin, and apigenin compound was at 4.883 and 4.991 min. Although the spectrum may contain plenty of impurities, however; major compounds were confirmed using their respective standards.

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Fig. 2

UPLC chromatograms (at 320 nm) of the stem extracts of (A) Anacardium occidentale, (B) Gluta renghas, (C) Mangifera indica and (D) Melanochyla fasciculiflora. The UV spectra of several peaks generated by a photodiode array detector are indicated in all plant samples.

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4 Discussion

Mosquito control using the plant-based insecticide has been known for a long time with their ability to kill the larvae, reduce the number of eggs through oviposition deterrence and ovicidal activity (Zuharah and Yousaf, 2016). Gluta renghas has exhibited the best toxicity effects against both Aedes larvae more than those possessed by Mn. indica. The toxicity of the plants depends on the various bioactive compounds, including phenolics, terpenoids, flavonoids, and alkaloids, either as single or joint compounds (Elumalai et al., 2012). Plant phytochemicals are produced as secondary metabolites which encounter toxic effect to protect the insects from predatory (Ghosh et al., 2012). Gluta renghas has been reported for excellent toxicity against laboratory strains of Ae. albopictus at 600 mg/L (Yousaf and Zuharah, 2015), which might contain flavonoids, phenolic lipids and tripertenes as reported in many Anacardiaceae families (Correia et al., 2006). The variety of larvicidal potentials depends on the composition of chemical compounds in the plant. As to our knowledge, no study has been identified the chemical compound of Gl. renghas. Phenolic identification in the current study found high concentrations of flavonol derivatives of quercetin and apigenin in this plant which might be responsible for the larvicidal activity. Whereas many studies have been reported the potential of Mn. indica (Rahuman et al., 2009) and An. occidentale on various mosquitoes’ species and their chemical compounds (Farias et al., 2009).

Several plant species have been tested for their activities against different vectors and found to be target-specific. Our study has shown a significant different larvicidal effect on Ae. albopictus and Ae. aegypti mosquitoes, even though both species are a sibling. Aedes albopictus was found to be prone to the toxicity of all Anacardiaciae plants as compared to Ae. aegypti. The toxicity of the plant as larvicides on particular mosquito species is due to the synergistic effects from combining compounds existed in the plant extracts (Tawatsin et al., 2006). Other than that, the quality of compounds extracted from the plant is influenced by the extraction method, solvent polarity and also the part of the plant (Oliveira et al., 2010), including the plant species, cultivating condition, plant preparation and plant storage (Tawatsin et al., 2006).

Recently, interest has been raised by the beneficial potential of plants due to their phenolic compound, particularly flavonoids (Pandey and Kumar, 2012). Flavonoids are extensively distributed and most common group of the plant phenolic compounds found in all plants parts, especially the photosynthesizing cells of plants (Kumar and Pandey, 2013) which contain a huge number of compounds, abundant in plants, imparting the antioxidant activities, including anti-inflammatory, anti-carcinogenic, anti-bacterial, anti-viral, and anti-allergic effects (Yong et al., 2018). Other compounds, such as the presence of carbohydrates, saponins, phytosterols, phenols and tannins, including flavonoids in plants having also reported for larvicidal activities against mosquitoes (Khanna and Kannabiran, 2007).

In our study, we found that Gl. renghas had caused mortality to both Aedes mosquitoes at a low concentration which probably associated with a synergist of a cumulative quercetin and apigenin compound found in this plant. Even though UPLC detected low concentration of both phenolic compounds than those detected in Mn. indica and An. occidentale, but this plant species possessed the best toxicity effects on Aedes larvae. Thus, suggested, the synergistic effects between all profound compound are necessary to elicit the larvicidal properties. The high amount of quercetin in the leaves of a locust tree, Robinia pseudoacacia (Nasir et al., 2005) reduced the population of Lymantria dispar (Barbosa and Krischik, 1987) and prefer to attack forest trees in spring when the leaves have a small amount of flavonoids including quercetin (Salminen et al., 2004). No comparative study can discuss as this is considered the first evaluation of phenolic compound in Gl. renghas and Ml. fasciculiflora.

Cashew nut shell liquid (CNSL) derived from An. occidentale was examined for its antioxidant activities against Ae. aegypti and the GC-MS analysis of its constituents detected quercetin as the most active compound, followed by orcinol, cardanol, cardol and anacardic acid (Oliveira et al., 2011). We also detected a higher number of quercetin compounds from the stem part of An. occidentale suggested it’s ability as a potential larvicidal agent. A rich source of flavonoid had been observed to have good larvicidal activity against Cx. pipiens Pallens, Ae. aegypti and Ae. albopictus derived from methanolic seed extracts of Millettia pinnata (Perumalsamy et al., 2015). Similarly, methanolic and ethanolic extract of flowers and leaves of Lantana camara Linn. have demonstrated their larvicidal activities against Cx. quinquefasciatus and Ae. aegypti, which reported in the presence of flavonoids (Kumar and Maneemegalai, 2008).

Peel and pulp of the fruit of Mn. indicia were also reported with the presence of quercetin (Atawodi et al., 2014), similar to the stem extract from our study found two derivatives of quercetin compounds. Few studies also indicated flavonoid potential to inhibit and caused lethal effects on other animals and insects. The flavonoid can act as antiparasitic and causing mortality to parasite due to swollen mitochondria with massive multilobe vacuoles (Yong et al., 2018). Interestingly, the myricetin and Scutellarein from the flavonoid group were found to inhibit SARS-CoV helicase (nsP13) by inhibiting ATPase activity (Mani et al., 2020). The presence of flavonoids and tannins in aqueous extracts of immature fruits of Mn. indica had a higher potential for larval development inhibition against the larvae of Strongyloides stercoralis (El-Sherbini and Osman, 2013).

Quercetin was also found toxic as it inhibited Glutathione S-transferases (GST) in Triatoma infestans (Sivori et al., 1999), which was found to be toxic to the insect. Not only death but the active compound of apigenin, quercetin, luteolin and volatile oils derived from P. amboinicus leaf extract would interrupt the development and movement of dengue vectors without interrupting the predation ability, development and survivability of the natural enemy copepod (Murugan et al., 1996). Quercetin compound also increases the mortality rate of L. dispar larvae by decreasing the midgut activities of larvae resulting in decreased body mass and, finally death (Perić-Mataruga et al., 2014). Besides quercetin, flavonoids apigenin, vitexin, isovitexin, sitosterol and campesterol was pointed out for positive larvicidal activities against Ae. aegypti, Cx. quinquefasciatus (Rahuman et al., 2008) and An. arabiensis (Tomass et al., 2011). Quercetin is an interesting compound as it is found safe for other non-target organisms, including human beings and other vertebrates (Perić-Mataruga et al., 2014).

Therefore, this plant is probably safe to be used as one of the larvicidal products with no harm to the human being. The positive response and growing trend and of the community towards phytochemicals and their environment-friendly behaviour builds an open ground for the research and innovation of plant-based insecticides. For future research, it is worth extracting active ingredients of apigenin and quercetin from these Anacardiaceae plants, which had been endorsed the importance of its biological control potential to reduce the use of plant sources. This will promote the utilization of these chemical compounds of botanical origin as a safer option, readily degradable and environmentally safe insecticide for the vector control program.

Authors contributions

WFZ contributed in conceived and designed the study, analyzed data and wrote and corrected the final manuscript. YA collected the field samples, performed the bioassays, analyzed data and helped draft the manuscript. KLO and SFS have helped in analyzing the phenolic compound of the plant extracts. The final manuscript was approved by all authors.

Funding

This study was supported by the Universiti Sains Malaysia under Research University Grant (1001/PBIOLOGI/815079) and Fundamental Research Grant Scheme, Ministry of Higher Education Malaysia (203/PBIOLOGI/6711629).