Chemical constituents and strong larvicidal activity of Solanum xanthocarpum among selected plants extracts against the malaria, filaria, and dengue vectors : Journal of Vector Borne Diseases

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Chemical constituents and strong larvicidal activity of Solanum xanthocarpum among selected plants extracts against the malaria, filaria, and dengue vectors

Kumar, Pawan1,2; Shakya, Rashmi3; Kumar, Vikram1; Kumar, Dinesh1; Chauhan, RPS2; Singh, Himmat1,

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Journal of Vector Borne Diseases 60(1):p 18-31, Jan–Mar 2023. | DOI: 10.4103/0972-9062.361177
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Mosquitoes act as a vector for the transmission of various vector-borne diseases (VBDs), such as malaria, dengue, filariasis, and Chikungunya. These diseases contribute significantly, 17% of the total burden of infectious diseases globally[1]. Malaria, perhaps the best known VBD, has been recognized as one of the most important tropical diseases transmitted by female Anopheles species of mosquitoes (such as An. stephensi and An. gambiae) infected with Plasmodium parasites. In 2020, about 241 million cases of malaria have been reported from 85 malaria-endemic countries which is a significant increase of about 14 million cases as compared to 2019 (World Malaria Report 2021)[2]. Dengue is a mosquito-borne viral disease that is rapidly emerging as a pandemic-prone viral disease. It is transmitted to humans through the bite of infected Aedes species of mosquitoes (Ae. aegypti or Ae. albopictus). They are also responsible for the transmission of yellow fever, Chikungunya and Zika viruses. World Health Organization (WHO) estimates the global incidence of dengue fever has also increased significantly in the past decade with a two-fold increase. In 2019, about 5.2 million cases were reported as compared to 2.4 million in 2010. The nematode Wuchereria bancrofti, the causal organism of lymphatic filariasis is transmitted by a wide range of mosquitoes depending on the geographical area. It is transmitted by the mosquito Culex quinquefasciatus, a ubiquitous vector responsible for about 120 million infections out of which 76 million people have damaged lymphatic and renal systems[3].

One of the greatest challenges of the 21st century is the control of VBDs for global public health. Several million people die due to diseases transmitted by mosquitoes every year. The methods to control the growth of these insects are crucial for reducing their risks to humans. Efforts to control mosquitoes through insecticides under Public Health Departments are going on across the world in endemic countries of vector-borne diseases. However, there are reports on resistance against the insecticides acquired by vectors for existing chemical control methods[4,5,6]. Serious concerns resulting from the use of chemical insecticides and lack of vaccines have provided an opportunity to develop eco-friendly approaches such as the development of plant-based insecticides to combat VBDs.

Though drug prophylaxis is available for the prevention of malaria, it is still facing major challenges. Antimalarial drugs have been used for the treatment of malaria. Reports are available showing different parasites, such as Plasmodium falciparum and P. vivax developing resistance against all classes of antimalarials[7]. Using plant-derived artemisinin along with partner drugs used as artemisinin-combination therapies (ACTs) have been employed effectively as a treatment for malaria worldwide[8]. However, with the emergence of the artemisinin-resistant P. falciparum parasite in 2008, the use of ACTs in malaria treatment has been jeopardized[9]. Triple artemisinin-based combination therapies (TACTs) which are now standard for the treatment of tuberculosis and HIV infections have been proposed to prolong the utility of existing anti-malarials[10]. The development of the first malaria vaccine RTS, S/AS01 led to falling in cases of malaria worldwide (Rts, s CTP, 2015) but it is not been found to be effective against P. vivax parasite[11]. Currently, there is no specific treatment available for dengue[12]. The inadequate health infrastructures and poor socioeconomic conditions have further worsened the control measures of malaria for the poor marginalized population in tropical and subtropical countries of the world. Currently, there is no single intervention that is sufficient for reducing the global disease burden caused by mosquitoes. The elimination and prevention of mosquito-borne diseases have become difficult with an increase in problems associated with extensive usage of synthetic compounds. The repertoire of effective drugs is limited and the emergence of multi-resistant strains have highlighted the search for a new novel insecticidal molecule as well as alternative methods to combat these parasites.

Plants contain biologically active chemical compounds, referred to as phytochemicals which confer defence against pathogenic microorganisms and insects. Insecticides of plant origin are preferable because they are target-specific, biodegradable, and eco-friendly[13]. They have different mechanisms of action which in turn reduces the chance of developing resistance in mosquito population[14]. Plants may be considered as an alternative for controlling mosquito-associated diseases because they are a rich source of various biologically active compounds which are very effective against mosquito vectors due to their different mode of action[15]. Different parts of the plants, such as roots, leaves, stems, fruits, a mixture of different plant parts, or the whole plant body of herbs can be effectively used for plant-based insecticide. A significant change in the activity of a phytochemical can be observed depending on the plant species, part used, solvent used for extraction, and target mosquito species[16]. Several plant-based compounds have been reported to have mosquitocidal activities[17].

Solanum xanthocarpum (family Solanaceae) has been reported to have various pharmacological activities such as antiasthmatic, hepatoprotective, hypoglycemic, and cardiovascular effects and insect repellent properties[18,19]. The fruit has been reported to contain steroidal alkaloids like solanacarpine and campesterol steroids diosgenin, daucosterol, campesterol, and triterpenes, such as cycloartanol. It also contains natural steroid glycoalkaloids which are present in a large number of food crops, such as potatoes, eggplants, and tomatoes[20]. Due to the presence of various phytoconstituents, it has larvicidal potential and can be used to control mosquito populations in the laboratory as well as in field conditions. Parthenium hysterophorus (family Asteraceae) is another plant that has medicinal properties to cure hepatic amoebiasis and tumors and is larvicidal, muscle relaxant, and hypoglycemic[21,22]. Manihot esculenta (known as cassava or tapioca), a woody shrub belonging to the family Euphorbiaceae, is one of the main staple food crops cultivated in tropical and subtropical Africa, Asia and Latin America. Its roots have been reported to be of nutritional importance and are the third-largest source of carbohydrates in the meal[23]. The leaves have been reported to have medicinal properties to cure fever, diarrhoea, headaches, and rheumatism[24,25]. Chamaecyparis obtusa (family Cupressaceae) has also been reported to have antibiotics, allopathic and insecticidal properties[26].

Biological control strategies for mosquito vectors are considered preferable as they circumvent issues associated with extensive usage of insecticides, such as harmful consequences for the environment, non-target species, development of insecticide resistance and public health. Effective biocontrol methods include vector killing, modification of vector activities to enhance selfmortality, development of vectors which are either sterile or incapable of transmitting disease and use of herbal extracts[27,28]. Plant-based larvicidal agents have been reviewed recently by several researchers emphasizing the reduction in overuse of synthetic pesticides which boosts resistance development in mosquitoes[29,30,31]. A few fragmented reports on the larvicidal activity of the above plants have been reported, but a detailed investigation is needed for further exploration[32,33,34]. Considering the immense medicinal importance of S. xanthocarpum, P. hysterophorus, M. esculenta and C. obtusa, present work has been carried out to evaluate the efficacy of their leaf extracts with the possibility of identifying larvicidal plants which might pave a way for the development of a larvicidal drug in the future. Furthermore, LC-MS and GC-MS analyses were also undertaken to identify the potential larvicidal compounds from the selected plants for designing larvicidal products in the future.


Plant materials

Several plants were screened carefully through literature survey[29]. Based on high bio-efficacy against mosquito vectors, four plant species namely, S. xanthocarpum, P. hysterophorus, M. esculenta and C. obtusa were selected which have minimal LC50 and LC90 values. Fresh leaves for the preparation of extract were collected from these plants growing in West Delhi Region, India (Latitude 28.57 N; Longitude 77.07 E).

Preparation of leaves extracts

The leaf extract was prepared according to previously published work by the authors[35]. Fresh leaves from all four plant species were plucked and washed properly under running tap water to remove the dust particles, pesticides, and other impurities for 5–10 mins. After washing, leaves were cut into small pieces [Figure 1]. For quantification of the dry weight of each sample, the small pieces of leaves were kept in the oven at 28°C temperature for 72 h. Each sample was ground to powder separately using a mortar and pestle. For the preparation of leaves extract with two different solvents i.e. methanol and petroleum ether, each powdered sample was divided into two parts. Each time 10 g of the powder was extracted with 100 ml methanol and petroleum ether solvent separately. The solution of each leaf extract was filtered through a single-layered muslin cloth followed by filtration through Whatman filter paper No. 1. Each filtrate was dried at room temperature and stored separately at 4°C.

Figure 1:
Diagrammatic representation of the bioassay performed with methanolic and petroleum ether extract of S. xanthocarpum, P. hysterophorus, M. esculenta and C. obtusa against the larvae of An. stephensi, Ae. aegypti and Cx. quinquefasciatus mosquito vectors.

Mosquito culture and larvicidal bioassay

Late third-stage instar or early four-stage instar larvae of An. stephensi, Ae. aegypti and Cx. quinquefasciatus and were obtained from the Insectary of National Institute of Malaria Research (ICMR, Government of India), Dwarka, New Delhi. These stages were maintained at 28±2°C temperature and relative humidity of 70–75%. Larvae were fed on larval food composed of fish food and dog biscuit mixed with yeast. Bioassays were performed according to the standard procedure embellished by WHO 2016[36]. For the preparation of the stock solution, 200 mg of leaf extract was dissolved in 200 ml acetone. A series of dilutions were done using de-chlorinated water. The bioassays were carried out at 20, 40, 60, 80,100, and 120 ppm tested concentrations. Twenty-five larvae were treated in a 500 ml beaker having 1 ml of prepared extract and 249 ml of water. Each experiment was carried out in triplicates with two separate controls i.e. solvent and dechlorinated water.

Liquid chromatography-mass spectrometry analysis

Based on minimum LC50 and LC90 values of S. xanthocarpum leaf extract as compared to others, the methanolic leaf extract of only S. xanthocarpum was subjected to LC-MS analysis to explore the potent bioactive compounds at the advanced instrumentation research facility, Jawaharlal Nehru University, New Delhi, India[35]. It was done using a 2D Nano ACQUITY system operated with a column oven, in-line degasser on Waters Synapt G2, autosampler, and binary pump. The system was coupled to a Synapt G2 Q-TOF system supported by an electro spray ionization (ESI) source. LC- MS data were analyzed and compared to the published literature for the identification of peaks and bioactive compounds.

Gas chromatography mass spectroscopy (GC-MS) analysis

The methanolic leaf extract of S. xanthocarpum was also subjected to GC-MS analysis for the identification of unknown compounds. For this, 1 mg of leaf sample was dissolved in 1 ml of methanol and subjected to GC-MS analysis using a GC-MS unit (Shimadzu QP 2010 Plus) equipped with MS detector function under the following conditions; carrier gas: helium; oven temperature: 70–280°C; Rtx-5MS capillary column (0.25 mm × 0.25 mm × 30 m); flow rate: 1.21 ml/min; injector temperature: 260°C. The compounds identification was done by comparing retention indices and mass spectra with the National Institute of Standards and Technology (NIST) library.

Data Recording

Mortality was assessed after 24 h, 48 h and 72 h exposure to plant extracts. The final mortality was recorded after 72 h. Only those larvae which survived were counted as healthy. Abbott's formula was applied if the mortality was found to be >5% and <20%. The standard state of mortality was adopted from WHO 2012 guidelines[36].

Statistical analysis

The mortality was reported after 24 h, 48 h and 72 h of treatment for exposure concentrations of 20, 40, 60, 80, 100, and 120 mg/l of the plant extract, separately. All the experiments were performed in triplicates. Log probit analysis was performed with a determination of the LC50 (lethal concentration causing 50% mortality in the population) and the LC90 (lethal concentration causing 90% mortality in the population) which were analyzed with a 95% confidence interval using the SPSS software version 21, window 16[37]. The results were shown in [Table 1 & Table 2]. Differences that presented probability levels p≤0.001 for 24 h and p≤0.013 were considered statistically significant.

Table 1:
Log probit and regression analysis of methanolic extract of different plant against larvae of Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus
Table 2:
Log probit and regression analysis of petroleum ether extract of different plant species against larvae of An. stephensi, Ae. aegypti and Cx. quinquefasciatus

Ethical statement: Not Applicable


In the preliminary screening, the larvicidal activity of methanol and petroleum ether leaf extracts of S. xanthocarpum, P. hysterophorus, M. esculenta and C. obtusa, were checked against the early four-stage instar larvae of An. stephensi, Ae. aegypti and Cx. quinquefasciatus at 20-120 ppm concentrations from 12 h –72 h. Each extract showed moderate activity after 24 h of exposure which increased with increasing concentration and time. Methanolic leaf extracts of M. esculenta, C. obtusa and P. hysterophorus showed moderate larvicidal activities after 72 h of treatment with LC50= 29.898, 28.382 and 27.498 ppm and LC90= 61.945, 54.176 and 65.705 ppm against An. stephensi; LC50= 24.560, 42.030 and 23.384 ppm and LC90= 50.287, 80.125 and 60.091 ppm against Ae. aegypti and LC50= 17.026, 57.450 and 27.827 ppm and LC90= 30.632, 96.328 and 63.439 ppm against Cx. quinquefasciatus, respectively (Table 1 & Fig. 2). The larvicidal activities recorded for petroleum ether leaf extracts of M. esculenta, C. obtusa and P. hysterophorus showed a similar trend with LC50= 17.026, 18.83 and 28.41 ppm and LC90= 32.63, 34.452 and 57.43 ppm against An. stephensi; LC50= 26.08, 18.85 and 20.63 ppm and LC90= 39.3, 38.5, 41.04 ppm against Ae. aegypti and LC50= 26.59, 18.83 and 30.22 ppm and LC90= 51.18, 34.45 and 59.98 ppm against Cx. quinquefasciatus, respectively (Table 1 & Fig. 2). The highest larvicidal activity was observed for the leaf extracts of S. xanthocarpum in methanol and petroleum ether after 72 h of treatment. For methanol, leaf extracts LC50 = 09.201 and LC90= 21.578 ppm against An. stephensi; LC50 = 12.435 and LC90= 27.418 ppm against Ae. aegypti and LC50 = 11.450 and LC90= 26.328 ppm against Cx. quinquefasciatus were observed. For leaf extracts of S. xanthocarpum in petroleum ether the LC50 and LC90 values for An. stephensi, Ae. aegypti and Cx. quinquefasciatus were found to be 10.026 and 22.632 ppm; 12.962 and 26.731 ppm and. 13.325 and 30.409 ppm, respectively.

Figure 2:
Toxicity (LC50 and LC90) of A. Manihot esculenta, B. P. hysterophorus, C. S. xanthocarpum and D. C. obtusa plant extracts prepared in methanol (1) and petroleum ether (2) solvents against the 3rd instar larvae of Ae. aegypti, Cx. quinquefasciatus and An. stephensi mosquito vector after 72h of treatments.

Since the crude leaf extract of S. xanthocarpum showed promising larvicidal activity against mosquito vectors, the methanolic leaf extract was further subjected to GC-MS and LC-MS analyses (as it was less toxic as compared to petroleum ether extract) to explore the chemical constituents. LC-MS spectra revealed the presence of 34 compounds in the leaves extract of S. xanthocarpum (Table 3 & Fig. 3) which have a wide range of biological activities, such as larvicidal, anticancer, antimicrobial, antibacterial, antimalarial, anti-inflammatory, antiviral, and antifungal. Picrasin G, isolated from Quassia amara has been shown to exhibit strong antimalarial and cytotoxicity[38]. Spectral data of GC-MS analysis revealed 43 compounds (Fig. 4) which have been described briefly in terms of their retention time, area, and biological activity (Table 4). Major compounds identified in the leaves extract of S. xanthocarpum are Phytol (13.09%), 3-allyl-2-methoxy phenol (9.55%), (9Z, 12Z)-9, 12-octadecadienoyl chloride (7.93%), linoleic acid (5.45%), alpha-tocospiro B (5.08%) and hexadecanoic acid (4.35%) and remaining compounds were in the range of 0.2 to 5% (Table 4).

Table 3:
LC-MS analysis of leave extract of Solanum xanthocarpum
Table 4:
GC–MS analysis of leaves extract of Solanum xanthocarpum
Figure 3:
LC-MS chromatographic representations of S. xanthocarpum leave extract in positive mode.
Figure 4:
GC-MS chromatogram of crude extract of S. xanthocarpum leaves.


Larviciding is a fruitful way of diminishing mosquito populations in their breeding places before their emergence as adults from the larval stage. Larviciding principally relies upon the use of manufactured bug sprays. They are powerful and their regular use as natural control frameworks have brought broad advancement of mosquito obstruction[39,40]. Several studies reported the strong efficacy of plant-derived products against mosquito larvae which can be utilized for controlling the lethal vector-borne diseases[41]. Candles of citronella and geranial used as mosquito repellents have been reported to reduce mosquito biting by up to 50% in mixed populations[42]. Larvicidal activities of aqueous extracts of some of the plants have already been reported to be significant against the vectors in the present study. The aqueous seed extract of citrus exhibit moderate larvicidal activity against Cx. quinquefasciatus and Ae. aegypti with LC50 and LC90 values of 319.40, 135, 411.88 and 127 ppm, respectively[43]. The aqueous root extract of Hibiscus abelmoschus has been reported to show moderate bio-efficacy against the larvae of An. stephensi, Cx. quinquefasciatus and An. culicifacies with LC50 values of 52.6, 43.8, and 52.3 ppm, respectively[44]. Higher LC50 values of 9.681 and 5.124 mg/l were also reported in Rhinacanthus nasutus extract against Ae. aegypti and Cx. quinquefasciatus, respectively[49]. The methanolic seed extract of Clitoria ternatea has been shown to have promising larvicidal activity with LC50 values of 65.2, 154.5, and 54.4 ppm against the larvae of An. stephensi, Ae. aegypti and Cx. quinquefasciatus[45].

In the present study, among the four plant species, the highest larvicidal activity has been found in the methanolic leaf extracts of S. xanthocarpum. The extracts from different parts of the S. xanthocarpum have been used as an agricultural pest repellant and as a contact poison. The aqueous fruit and root extracts have been shown to have larvicidal potential against two anopheline species, namely An. stephensi and An. culicifacies. The bio-efficacy for larvicidal activity of fruit (LC50=0.058%) and root extract (LC50 = 1.08%) of S. xanthocarpum against An. stephensi had also been reported earlier[46]. The moderate larvicidal activity of ethanolic leaf extracts of S. xanthocarpum against first to fourth instars larvae and pupa of Cx. quinquefasciatus has also been reported earlier. The LC50 and LC90 values for the fourth-instar larvae were reported as 377.44 and 1,058.85 ppm, respectively[47]. Strong efficacy of S. xanthocarpum leaves extract prepared in carbon tetrachloride and petroleum ether has been reported against An. stephensi (LC50, 5.1ppm) and Cx. quinquefasciatus (62.2 ppm)[48]. Though, there have been reports of larvicidal activities of S. xanthocarpum against the vectors in present study but no work has been reported on the methanolic and petroleum ether leaf extracts against these three vectors. However, antifungal activity of methanolic leaf extracts against Aspergillus niger and A. fumigatus has been reported[49]. Moreover, present work has reported the highest larvicidal activities for methanolic leaf extracts against An. stephensi, Ae. aegypti and Cx. quinquefasciatus with LC50 and LC90 values as 09.201, 11.450 and 12.962 ppm, and 21.578, 26.328 and 26.731, respectively. The bioefficacy of medicinal plant species towards mosquito vector depends on various factors, such as the solvent used for extraction, plant parts used, development stage of the plant, mosquito species and their developmental stages.

The highest larvicidal activity is generally attributed to the presence of bioactive compounds which are highly effective against mosquito vectors. Variation in larvicidal activity against mosquito vectors is due to the presence of larvae receptor-specific compounds. Presence of compounds, such as apigenin, scopletin, esculetin, coumarin, methyl caffeate, cycloartenol, campesterol, cholesterol, solasodine, diogenin and lenoleic acid could be responsible for larvicidal activity of S. xanthocarpum[50]. Bioactive compounds, such as terpenoids, alkaloids and phenolics are present in plant extracts which might be independently or synergistically contributing to the generation of bio-efficacy against mosquito vectors[51,52]. Bio-efficacy of plant extract also depends upon spatial and temporal variation of plants because particular bioactive compounds are synthesized at specific developmental stages and in specific seasons. Concomitant with previously published work on the insecticidal activity of quassinoids on Plutella xylostella (Diamondback moth)[53], the present work also reports the presence of natural triterpenoid, picrasin G in the leaves extract of S. xanthocarpum[57]. The present work also shows the presence of laxiflorin, a member of flavanones, in the leaf extract of S. xanthocarpum. Laxiflorin is a natural product found in Derris laxiflora. Laxiflorin J, obtained from Isodon eriocalyx var. laxiflora leaves has been evaluated as potential anticancerous activity against T-24 cells[54]. Orysastrobin which has been obtained from S. xanthocarpum has been reported to be highly effective against major fungal diseases in rice plants including blast (Magnaporthe grisea) and sheath blight (Thanatephorus cucumeris)[55]. Another compound, the aflavin-3, 3’-digallate (TF-3), reported in this work has been previously isolated from black tea exhibiting significant growth inhibition of human oral squamous carcinoma HSC-2 cells[56]. Phytol has been shown to exhibit strong larvicidal activity against mosquito larvae[61]. Phytol obtained from Azolla pinnata has strong larvicidal activity against Aedes mosquito larvae[57]. Compounds, such as 3-allyl-2-methoxyphenol, (9Z, 12Z)-9, 12-octadecadienoyl chloride, linoleic acid, alpha-tocospiro B and hexadecanoic acid reported from S. xanthocarpum leaves extract have been known to possess larvicidal, anticancer, antimicrobial and antifungal activities[59,60,61,62,63,64]. Thus, it can be proposed that the larvicidal activity of the leaf extract against mosquito larvae may be due to the synergistic effect of larvicidal agents present in the leave of S. xanthocarpum. Present work has highlighted the significant potential of bioactive compounds present in the leaf extract of S. xanthocarpum that can be effectively used for controlling the population of mosquitoes which acts as vectors for malaria, filariasis and dengue. Furthermore, this work has broadened the possibilities for the exploration of larvicidal compounds from plants which might be helpful for the development of larvicidal drugs in future in an eco-friendly way.


Present work has reported a few potent candidates for the production of plant-based larvicides. Among the four plant species tested, petroleum ether and methanolic leaf extracts of S. xanthocarpum were found to be significantly effective against Ae. aegypti and An. stephensi at minimum concentrations and moderately effective against Cx. quinquefasciatus. This study also provides a window for the development of new products considering S. xanthocarpum as a potential source for larvicide drugs in the future against these harmful VBDs. As being natural, stable, nontoxic and nonhazardous for human life and the environment, these do not have negative effects on non-targeted organisms. These are required in a very small quantity and are cost-effective. Plant-based compounds are useful for long-term mitigation of resistance among the larvae against synthetic chemical larvicide and can contribute significantly to the National Vector Borne Disease Control Program of India for mosquito control in coming future along with other strategies.

Conflict of interest: None

Ethical statement: Not applicable


The authors acknowledge Director, ICMR-National Institute of Malaria Research, Sector-8, Dwarka, New Delhi for providing necessary infrastructure and support during the research work of this study. Dinesh Kumar is indebted to the Director of ICMR-NIMR and ICMR, New Delhi for the ICMR-Post-Doctoral Research and Research Associate fellowship.


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    Vector-borne diseases; Larvicide; plant extract; eco-friendly; mosquitoes

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