|Year : 2018 | Volume
| Issue : 4 | Page : 282-290
Larvicidal activity of Ricinus communis extract against mosquitoes
Nisha Sogan1, Neera Kapoor2, Himmat Singh1, Smriti Kala3, A Nayak1, BN Nagpal4
1 ICMR–National Institute of Malaria Research, New Delhi, India
2 Indira Gandhi National Open University (IGNOU), New Delhi, India
3 Institute of Pesticide Formulation Technology (IPFT), Gurugram, Haryana, India
4 World Health Organization, SEARO, New Delhi, India
|Date of Submission||02-Jul-2018|
|Date of Acceptance||03-Dec-2018|
|Date of Web Publication||18-Apr-2019|
B N Nagpal
WHO–SEARO, World Health House, Indraprastha Estate, Mahatma Gandhi Marg, New Delhi–110 002
Source of Support: None, Conflict of Interest: None
Background & objectives: Vector control strategies play significant role in reducing the transmission of malaria, dengue and other vector-borne diseases. The control of vector population using synthetic insecticides has resulted in development of insecticide resistance and negative effects on humans and environment. The present investigation evaluated the larvicidal potential of methanol, dichloromethane and hexane extracts of leaves and seeds of Ricinus communis (castor) plant against the early IV instar larvae of the dengue vector, Aedes aegypti, and malaria vector, Anopheles culicifacies.
Methods: Plant extracts were screened for their efficacy against Ae. aegypti and An. culicifacies using WHO standard larval susceptibility test method. Dose response bioassay was performed to get lethal concentrations. Further, gas chromatography-mass spectroscopy (GC-MS) analysis was carried out to identify the bioactive chemical constituents of the extracts of R. communis. Toxicity of the extracts towards non-target organism, Poecilia reticulata was also evaluated.
Results: The leaf and seed extracts of R. communis showed significant mortality against the larvae of Ae. aegypti and An. culicifacies at concentrations of 31.25, 62.5, 125, 250, 500 ppm; and 2, 4, 8, 16, 32, 64 ppm, respectively. At 24 h of the exposure period, the larvicidal activities were highest for the methanol extract of seeds with LC50 15.52 and 9.37 ppm and LC90 45.24 and 31.1 ppm for Ae. aegypti and An. culicifacies, respectively. The methanol extract of seeds and leaves was found to be safe towards non-target organism, P. reticulata. The GC-MS profile showed that seed extracts were having higher concentration of stigmasterol (7.5%), β-sitosterol (11.48%), methyl linoleate (2.5%), vitamin E (11.93%), and ricinoleic acid (34%) than the leaf extracts.
Interpretation & conclusion: The seed extract of R. communis has better larvicidal activity than the leaf extract and can be used as an effective larvicide against mosquitoes. The non-toxicity of the extracts towards P. reticulata further suggests that these plant extracts could be used along with predatory fishes in integrated vector control approaches.
Keywords: Aedes aegypti; Anopheles culicifacies; bioefficacy; GC-MS; non-target organism; Ricinus communis
|How to cite this article:|
Sogan N, Kapoor N, Singh H, Kala S, Nayak A, Nagpal B N. Larvicidal activity of Ricinus communis extract against mosquitoes. J Vector Borne Dis 2018;55:282-90
|How to cite this URL:|
Sogan N, Kapoor N, Singh H, Kala S, Nayak A, Nagpal B N. Larvicidal activity of Ricinus communis extract against mosquitoes. J Vector Borne Dis [serial online] 2018 [cited 2020 Apr 4];55:282-90. Available from: http://www.jvbd.org/text.asp?2018/55/4/282/256563
| Introduction|| |
Mosquitoes are responsible for the transmission of various parasite and pathogen-borne diseases like malaria, filariasis, dengue, chikungunya, Zika, etc. that have affected people around the world especially those inhabiting tropical countries. Drug prophylaxis and vector control are the only options available in case of malaria but in case of dengue there is no medicine available, hence prevention by vector control strategies remains the only cure. It plays an important role in reducing the incidence of malaria, dengue, Zika virus and filariasis. The various control programmes for mosquito larvae involve the use of synthetic larvicides like organophosphates (e.g. temephos, fenthion, etc.) and insect growth regulators (e.g. diflubenzuron, methoprene, etc.). The control of vector population using synthetic insecticides has resulted in insecticide resistance and negative effects on humans and environment. Therefore, the development of ecofriendly control tools is important for welfare of public health,,.
In this context, botanical pesticides can provide effective and ecofriendly tools against mosquitoes. Green pesticides are reported to be biodegradable, economical, non-toxic to non-target pests and have high specific activities towards the target pests. Plant extracts and essential oils have shown good larvicidal activity against the mosquitoes, which are biodegradable, economical and more sustainable option as compared to synthetic pesticides.
Ricinus communis (castor) is a widely distributed plant throughout the tropics and warm temperate regions of the world,. Various medicinal properties of R. communis are well-documented in the literature such as hepatoprotective, anti-inflammatory, diuretic, anticancer, antibacterial, insecticidal, hypoglycemic and free radical scavenging,,,,,,. Besides having medicinal values, R. communis is reported to be rich in secondary metabolites, especially phenolic compounds and alkaloids,. These compounds are involved in protection against the diseases. Flavonoids have been isolated from leaves such as kaempferol-3-O-beta-D-rutinoside and kaempferol-3-O-beta-D-xylopyranoide,. Tannins and alkaloids such as ricinine, N-demethylricinine have also been extracted from castor leaves,.
In this context present investigation was carried out to explore the larvicidal efficacy of leaf and seed extracts of R. communis against early IV instar larval stage of Aedes aegypti and Anopheles culicifacies.
| Material & Methods|| |
Different solvents of varying polarity, such as methanol, hexane and dichloromethane (DCM) were used for the extraction of plant material. All the solvents (analytical grade) were purchased from SD Fine-Chem Ltd., Mumbai. Tween-80 (Finar Ltd., Gujarat) and azadirachtin technical (Ozoneem®) (Ozone Biotech, Haryana) with 25% purity were used throughout the study.
Plant material and extraction
The study was carried out during February–September 2017. Leaves and seeds of the R. communis plants were collected from Dwarka region, New Delhi, India. The dried plant parts (200 g each of the leaves and seeds) were powdered mechanically using commercial electrical stainless steel blender and extracted with methanol in a Soxhlet apparatus, to obtain sufficient amount of toxic contents (active ingredient) from the plant materials. Cotton plug was used at the place of the thimble to stop the entry of the crude material into the siphoning tube. The required volume of the solvent (1–1.5 L) was filled up into the flask of the apparatus. The apparatus was then connected with the water supply to the condenser. The temperature of the heating mantle was maintained at 60–65 °C. The process was carried out for 6–8 h. The extracts were filtered through a Buchner funnel with Whatman No.1 filter paper. The solvents were removed using rotary evaporator. The residues obtained were stored at 4 °C till further analysis. Same procedure was followed to extract the phytochemicals from leaves and seeds of R. communis using other solvents such as, DCM and hexane except temperature for DCM was maintained at 36–39°C.
Laboratory reared III instar larvae of An. culicifacies were obtained from the Insectary of the ICMR-National Institute of Malaria Research, New Delhi. Aedes aegypti larvae were collected from the field and colonized in the laboratory. The larvae were maintained at room temperature (25 ± 2 °C) and kept in dechlorinated tap water in an enamel bowl. Larvae were fed on the dog biscuit and yeast powder in 3 : 1 ratio.
Dose response bioassay
The obtained extracts were screened for their efficacy against Ae. aegypti and An. culicifacies using WHO standard larval susceptibility test method. Stock solution was prepared at an initial concentration of 500 ppm (of leaf and seed extracts) for preliminary activity tests by solubilizing an appropriate aliquot in distilled water containing 0.1% tween-80 and methanol (with sonication, if required). The treatments that showed at least 100% mortality within 24 h were followedup for further bioassays (of stock solution) at different concentrations in order to determine the concentration required to kill 50% (LC50) and 90% (LC90) of the larvae population.
Various concentrations were prepared in triplicates from the stock solution, viz. 15.625, 31.25, 62.5, 125, 250, 500 ppm and 2, 4, 8, 16, 32, 64 ppm for the leaves and seeds, respectively with distilled water. A total of 20 early IV instar (in a 250-ml beaker with 200 ml of the extract) were used for each concentration for larvicidal assay and five replicates were maintained for each concentration. Azadirachtin was used as the positive control with the concentrations of (2, 4, 6, 8, 10) ppm. No food was offered to the larvae during the exposure period. The percent mortality was calculated at different exposure periods of 3, 6, 12, 24 h. All moribund mosquito larvae were considered as dead. Same concentration of solvents (water, methanol and tween-80) that was used in making stock solution was used as control. All the bioassays were performed at the room temperature (25 ± 2 °C).
Toxicity of crude plant extracts to Poecilia reticulata fish
To determine the toxicity of the plant extracts to a nontarget organism, P. reticulata (Guppy fish) was selected as the test organism. Poecilia reticulata were collected from a pond in Moti Nagar area of New Delhi and acclimatized in aquarium for five days at the room temperature of 27–28 °C and were provided artificial diet (Tetra Bits Complete, Tetra). Healthy fishes were used in the experiments according to methods described earlier by Promsiri et al, with slight modification. Assessment of toxicity was carried out at a LC50 value and then at a LC90 value of the leaf and seed extracts. A total of 20 P. reticulata fishes were placed in a rectangular, glass aquarium containing 400 ml of plant extract water solution in three replicates. Each group of 20 fishes was exposed to a test solution. A control, consisting of 20 fishes in dechlorinated tap water, was observed at the same time. The numbers of dead fishes were recorded at 24 and 48 h period, and the percentage mortalities were calculated. All these bioassay tests were conducted at a room temperature of 27–28 °C, without aeration or replenishment of water.
GC-MS of methanol extracts of seeds and leaves
Among all the extracts studied, only methanol extracts of leaf and seed showed larvicidal activity. Therefore, gas chromatography-mass spectroscopy (GC-MS) was carried out to identify the phyto-constituents which might be responsible for the observed larvicidal activity.
Around 1μl extracts of seeds and leaves were injected on a 30 m capillary column (0.25 mm ID × 0.25 μm film) (Shimadzu, Japan GC-MS QP2010). The GC-MS temperature was programmed with an initial oven temperature of 70 °C (hold time 5 min), which was increased at the rate of 10 °C/min to 300 °C (hold time 5 min) and sample injection temperature was 270 °C, with a split ratio of 10. The GC-MS was operated in electron ionization (EI) mode at 70 eV. Identification of the constituents was based on the retention indices and by comparison of their mass spectral fragmentation patterns matching against commercial library mass spectra, National Institute of Standards and Technology (NIST), Pfleger, Wiley.
The percentages of larval mortality and standard deviations were calculated for each concentration of the extracts. Lethal concentrations (LC50 and LC90) were determined at 95% confidence level using probit analysis. Statistical analysis was carried out using SPSS software version 22. Mortality in control was calculated using Abbott's formula whenever required.
Ethical statement: Not applicable.
| Results|| |
The study revealed that, methanol extract of R. communis (leaf and seed) was most potent against the larvae of Ae. aegypti and An. culicifacies. The DCM extract of leaf, and seed showed 35, and 45% mortality, respectively against An. culicifacies, but no mortality was observed against Ae. aegypti. Hexane extract of leaf and seed was found to be least effective larvicide as no mortality was observed for both the species—Ae. aegypti and An. culicifacies. The LC50 of leaf, and seed extracts of methanol against Ae. aegypti was 191.54, and 15.52 ppm and that of An. culicifacies was 65.629, and 9.37ppm, respectively. The in vitro bioassay revealed that the larvicidal efficacy of the extracts increased as the concentration and exposure time increased [Table 1]; and [Figure 1] and [Figure 2]. The LC50 values confirmed that seed extracts were more effective than the leaf extracts,.
|Figure 1: Percent mortality of methanol extract of leaf against Aedes aegypti and Anopheles culicifacies at different time intervals—(a) 3 h; (b) 6 h; (c) 12 h; and (d) 24 h.|
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|Figure 2: Percent mortality of methanol extract of seed against Aedes aegypti and Anopheles culicifacies at different time intervals—(a) 3 h; (b) 6 h; (c) 12 h; and (d) 24 h.|
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|Table 1: Percent mortality of different extracts of R. communis leaves and seeds against An. culicifacies and Ae. aegypti|
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Both the species of mosquitoes differ in their susceptibility to the treatments. Anopheles culicifacies larvae were found to be more susceptible than Ae. aegypti. These results were compared with positive control azadirachtin, which showed LC50 values of 4.96 and 2.16 ppm, and LC90 values of 9.99 and 4.52 ppm against Ae. aegypti and An. culicifacies, respectively. No mortality was observed in the control set up containing water, methanol and tween-80. This clearly indicated that methanol and tween-80 had no toxic effect on the larvae. Also, no mortality was recorded for both the species—Ae. aegypti and An. culicifacies during the exposure period of 3 h (31.25 ppm of leaf extract). On further increasing the exposure periods to 24 h (31.25 ppm of leaf extract) the percent mortality also increased to 10 and 23.3% for Ae. aegypti and An. culicifacies, respectively. For methanol leaf extract, result of 100% mortality was observed in larvae of An. culicifacies and Ae. aegypti at 250 and 500 ppm, respectively. For the seed extract, 100% mortality was observed at 32, and 64 ppm for An. culicifacies and Ae. aegypti, respectively.
Since, the methanol extract of the leaf and seed of R. communis was found more effective as a larvicide, it was further analysed in GC-MS to identify the phytoconstituents which might be responsible for their significant larvicidal activity. The GC–MS profile revealed that seeds were having higher concentration of stigmasterol (7.05%), ß-sitosterol (11.48%), methyl linoleate (2.38%), vitamin E (11.93%), and ricinoleic acid (34.10%) than the leaf extract. Besides these, the other components were ricinine, 13-Hexyloxacyclotridec-10-en-2-one, γ-sitosterol, fucosterol, ergosterol, methyl linolenate and fatty acids [Table 2].
The GC-MS profile of the leaf showed the presence of 1,7 dimethyl hypoxanthine as a major constituent; other compounds included α-linolenic acid, palmitic acid, ricinoleic acid, lupeol, pyrogallol, 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyranone, hydroxyquinole, ß-amyrin, ß-sitosterol, phytol, mome inositol, neophytadiene, 9-octadecanoic acid, linoleic acid, stearic acid, palmitic acid and stigmasterol [Table 2]. Ricinine was absent in the leaf extract.
Plant extracts were further screened for their toxic effect on a non-target organism, P. reticulata. The leaf and seed extracts of R. communis did not exhibited any noticeable effects on P. reticulata after 24 or 48 h of exposure at their LC50 and LC90 values against IV instar larvae of Ae. aegypti and An. culicifacies.
| Discussion|| |
Plant extracts exert myriads of biological activity on pests including larvicidal, repellent, ovicidal, insect growth regulator, etc. This can be due to various phytochemicals present in the plants that might be acting synergistically to produce such responses. Botanical pesticides are biodegradable and rarely develop resistance against the pests because of the synergistic activity of complex biomolecules thus moderating the long-term environmental effects of synthetic pesticide use. Ricinus communis is one such plant that has the potential to be used in vector control programme due to the rich phytochemicals that it harbours.
In the present investigation, R. communis plant extracts were screened for their larvicical efficacy against Ae. aegypti and An. culicifacies. The seed extract was found to have better larvicidal activity than the leaf extract as corroborated by various reports,. The parts of the plant and type of solvent used for extraction affect the larvicidal activity. It has been reported that non-polar (petroleum ether) extracts of the roots of Berberis lycium and leaves of Hedera nepalensis are more active against Aphis craccivora than that of the polar (aqueous methanol) extracts. Petroleum ether, carbon tetrachloride, and methanol extracts of fruit (Azadirachta indica) and extracts of seed (Momordica charantia and R. communis) have been evaluated for larvicidal activity against Culex quinquefasciatus. The findings revealed that methanol extract of A. indica had highest activity with LC50 at 74.04 and 58.52 ppm after 24 and 48 h of exposure period. In the present study also, methanol extract was found to be more potent than the extracts of hexane and DCM.
Among the extracts tested, hexane extract had no insecticidal activity against Ae. aegypti and An. culicifacies, while DCM extracts had lower insecticidal activity, i.e. 35 and 45% for leaf and seed, respectively against An. culicifacies, though no mortality was observed for Ae. aegypti. It might be because of the differential solubility of the phytochemicals present in the extract. Similar to our findings methanol seed extract has been reported to have better insecticidal activity than the other extracts (hexane and ethyl acetate),. The insecticidal activity of the methanol extract of R. communis seed could be attributed to the castor oil and ricinine. While hexane extract had no insecticidal activity due to insolubility of ricinine in hexane, which might be active larvicidal compound. Similarly, DCM had lower insecticidal activity which might be due to low solubility of ricinine.
Other compounds, such as 13-hexyloxacyclotridec-10-en-2-one, has been previously isolated and characterized, from Ambrosia maritima L by Dirar et al and found to possess antitumor activity. Ricinine is reported to act as anti-inflammatory, anticonvulsant, central nervous system stimulant, insecticidal, towards An. gambiae,,,,. Beta sitosterol is a plant found to have antimicrobial, anti-inflammatory, cytotoxic and insecticidal activities,,,. The fatty acids—linoleic acid, linolenic acid, palmitic acid and stearic acid have been reported from the leaf and seed extracts of R. communis,,,. Among these, linoleic and linolenic acids have been reported to have insectistatic and insecticidal activities against Spodoptera frugiperda The compound 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyranone has been previously isolated from onion and has anticancerous activity. This is the first report of compound 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyranone from castor leaves.
There are various reports on the efficacy of R. communis seed and leaf extracts against different pests. Okonkwo and Okoye showed the efficacy of R. communis leaf powder against Callosobruchus maculatus, a stored grain pest. Tounou et al have reported significant mortality of adults and larvae of Plutella xylostella L. (Lepidoptera: Plutellidae) under semi-field conditions when treated with aqueous extract of seed kernel and oil extracts of R. communis. Using aqueous extracts from leaves of R. communis, Aouinty et al have reported high larvicidal activity against II and IV instar larvae of four mosquito species, Cx. pipiens (L.), Ae. caspius (Pallas), Culiseta longiareolata (Aitken) and An. maculipennis (Meigen) with LC50 values of 600, 270, 200 and 1090 ppm, respectively.
Elimam et al have studied the activity of aqueous extracts from leaves of R. communis and the recorded LC50 value was 498.88 ppm against IV instar larvae of An. arabiensis, and 1445.44 ppm against IV larval instars of Cx. quinquefasciatus. In terms of the LC50 values, the methanol extract of R. communis seed in this study showed 10 times more toxicity. The presence of larvicidal activity against An. culicifacies and Ae. aegypti larvae may be due to the synergistic activity of the mixture of bioactive constituents like phenolics, terpenoides, flavonoids and alkaloids present in the extract. Sometimes isolation of pure active from crude extract may affect the activity. It has been reported that isolated neoprocurcumenol, from rhizomes of Curcuma aromatica exhibited less efficacy (LC50 = 13.69 ppm) than the parent petroleum ether extract (LC50 = 11.42 ppm). Larvicidal activity prediction against mosquito using computational tools are also reported to be efficient.
Mandal had studied the larvicidal and adult emergence inhibition activities of castor seed extract against An. stephensi, Cx. quinquefasciatus and Ae. albopictus. LC50 for Cx. quinquefasciatus was lowest while that for Ae. albopictus was the highest, in the order Cx. quinquefasciatus (7.10 ppm) < An. stephensi (11.64 ppm) <Ae. albopictus (16.84 ppm). In the present study, An. culicifacies was found to be more susceptible than Ae. aegypti with LC50 for Ae. aegypti (15.52 ppm) >An. culicifacies (9.37 ppm).
Methanolic extracts from fruits and seeds of Solanum xanthocarpum have been reported to exhibit larvicidal activity against An. culicifacies, An. stephensi, Ae. aegypti and Cx. quinquefasciatus. LC50 values for fruits were 51.6, 118.3, 66.9 and 123.8 mg/l and for seeds were 52.2, 157.1, 73.7and 154.9 mg/l against An. culicifacies, An. stephensi, Ae. aegypti and Cx. quinquefasciatus, respectively. In that study, An. culicifacies was found to be most susceptible which substantiates the results of present study.
The essential oil of Ipomoea cairica has shown 100% larval mortality against Cx. tritaeniorhynchus, Ae. aegypti, An. stephensi and Cx. quinquefasciatus mosquitoes at concentrations of 100–170 ppm. Similarly, it has been reported that Anacardium occidentale, Mammea siamensis, Phyllanthus pulcher, Anethum graveolens, Kaempferia galangal, Cinnamomum porrectum, Costus speciosus and Acorus calamus possess remarkable larvicidal activity at the concentration of 100 μg/ml after an exposure of 48 h. In the present study, 100% mortality was observed at concentration of 64 ppm against Ae. aegypti and at 32 ppm against An. culicifacies.
The leaf and seed extracts of R. communis were found to be safer towards non-target organism, i.e. P. reticulata as it did not exhibit any noticeable effects on P. reticulata after 24 or 48 h of exposure at their LC50 and LC90 values against IV instar larvae of Ae. aegypti and An. culicifacies. Present results are in agreement to a study carried out by Mandal, who has reported that R. communis seed extract are non-toxic towards non-target organism, O. niloticus. Further investigations are necessary to identify the pure active compound from the crude extract. This may further lead to optimization of the dose and may be formulated as commercial larvicide formulation.
| Conclusion|| |
Plant extracts have shown efficacy against the various pests including mosquitoes. This study shows that R. communis seed extract has potential to be developed as an effective larvicide against mosquitoes. The non-toxicity of methanol leaf and seed extract to P. reticulata suggests that these extracts could be used along with this predatory fish in integrated vector control programmes.
Further study on fractionations and isolation of active compounds for physicochemical stability is required to develop cost-effective formulations from the active fractions and for making substantial representation of plant-based insecticides in global insecticide market.
Conflict of interest
The authors declare no conflict of interest associated with this study.
| Acknowledgements|| |
The authors acknowledge the guidance from Late Dr Adarsh Shanker retired from the Institute of Himalayan Bioresource Technology (IHBT), Palampur and the Director, ICMR-National Institute of Malaria Research, New Delhi for providing all necessary facilities.
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[Figure 1], [Figure 2]
[Table 1], [Table 2]