MOSQUITO LARVICIDAL ACTIVITY OF HYPTIS SUAVEOLENS (L) POIT EXTRACTS PURIFIED FRACTION

MOSQUITO LARVICIDAL ACTIVITY OF EXTRACT’S PURIFIED FRACTION OF HYPTIS SUAVEOLENS (L) POIT.

Mragendra Singh, Ritu Namdeo and Kapil K. Soni ^*

Pharmacogenomics Lab, Bioscience University Teaching Department, Barkatullah University, Bhopal - 462026, Madhya Pradesh, India.

ABSTRACT: Bio-pesticides are playing a very important role in controlling mosquitoes and their larvae. In the present scenario, chemical pesticides are causing several side effects on non-target organisms, particularly in men. Moreover, mosquitoes and their larvae become resistant to chemical pesticides; therefore, chemical pesticides are not much more effective. The conventional use of temephos and pyrethrum creates several health hazards in men, especially in upper and lower respiratory disorders, including allergy, asthma, and rhinitis. The plant Hyptis suaveolens is well known for its aromatic properties. Therefore, it was proposed to apply its purified fraction on different strains of mosquitoes for the evaluation of mosquito larvicidal activities. In the present study, identified and authenticated shade-dried whole plant materials were pulverized to get powder, extracted in various solvents through Soxhletion, and percentage yields were noted down. The main phytoconstituents reported in the extract were alkaloids, flavonoids, terpenoids, and tannin in huge amounts. Due to the presence of the pungent smell of flavonoids, terpenoids, alkaloids, and tannin in Hyptis suaveolens, mosquito larvicidal activities were reported in the purified fraction.

Larvicidal, Aedes aegypti, Anopheles stephensi, Hyptis suaveolens

INTRODUCTION: Nowadays, mosquitoes are causing enormous public health problems. Of these, few are very common in men, including malaria, filaria, dengue, Japanese encephalitis, yellow fever, zika virus, and chikungunya. Aedes aegypti mosquito is a vector of Flavivirus virus that spreads yellow fever, dengue fever, and chikungunya fever which are endemic in tropical and sub-tropical regions around the world.

Similarly, various species of female Anopheles mosquito transmits various species of malaria parasites, including Plasmodium vivex, Plasmodium falciparum, Plasmodium malariae, and Plasmodium ovale, and among humans, approximately 91% of the 216 million malaria cases were reported in 2010, which were only due to Plasmodium falciparum transmitted through Anopheles stephensi ¹.

The major problem associated with the use of chemicals for the control of mosquito is the development of resistance to the chemicals and their undesirable side effects, so the purified fraction of the extract isolated from plants Hyptis suaveolens could be served as an alternative source of chemical pesticides, which are biodegradable into non-toxic products and are potentially suitable for use to control mosquitoes. Chemically, these are secondary metabolites found in plants for their defensive purposes only, and plant extracts, in general, have been an important natural resource of these bio-insecticides. These phytochemicals derived from plant sources can act as mosquito larvicidal, insect growth regulators, mosquito repellents, mosquito ovipositioning attractants. They can play an interrupted role in the transmission of mosquito-borne diseases at the individual and at the community level.

A number of extensive work has been reported on the effect of plant extracts against mosquito larvae. Bagavan and Rahuman ² have reported toxicities of different plant extracts, including crude hexane extract of flower heads of Spilanthes acmella, Spilanthes calva, and Spilanthes paniculata, partially purified extracts of leaves of Vitex negundo, Nerium oleander, seeds of Syzygium, leaves of Artemisia annua and Azadirachta indica, the crude acetone extracts of Fagonia indica, Arachis hypogaea, Nerium indicum and Thuja orientalis on the larvae population of different mosquito species. Chore et al., (2014) ³ isolated purified extract from 3 species of Aloe genus viz. A. Ngongensis, A. Turkanensis, and A. Fibrosa in hexane, chloroform, ethyl acetate, acetone and methanol solvents, respectively, and have applied all extract for larvicidal activity against mosquito Aedes aegypti.

Dengue haemorrhagic fever is also the most important public health problem in tropical and sub-tropical regions of the world, including India. Recent estimates of WHO indicate that about 50-100 million infections, including 0.5 million DHF cases and 24000 deaths (mostly among children) occur every year throughout the world due to Dengue as reported by Chore et al., 3 Dengue fever have been affected 19 states in India and Aedes aegypti mosquito is the principal vector for transmitting dengue by carrying flavivirus. This mosquito breeds in storage clean water in containers viz. jars, drums, tanks, coolers, cement tanks, and clay pots available in and around houses. The transmission of flavivirus by Aedes aegypti vectors can be controlled effectively by targeting immature larvae of this mosquito. Moreover, mosquito larvae are relatively immobile and remain more accumulated than their adult stage. Development of resistance in Aedes aegypti against temephos has also been reported. In this regard, bio-insecticide development has received much attention as they are considered to be efficient, safe to the environment, and biodegradable compared to synthetic insecticides.

Natural insecticide comprises of number of secondary metabolites which act on both behavioural and physiological processes and on the target species of mosquitoes have been reported by Subramaniam et al., ⁴ Hence, the present study was proposed to isolate a purified fraction of extracts of Hyptis suaveolens of family Lamiaceae for controlling mosquitoes and its larvae. Hyptis suaveolens is an herbaceous aromatic plant of height approximately 30-150 cm. Leaves are hispid, having a strong scented smell, broadly ovate 2-8 cm long, 2-6 cm wide with finely toothed edges which are grafted an oval limb and an inflorescence in axillaries cymes.

MATERIALS AND METHODS: The fresh plant materials of the whole plant Hyptis suaveolens (L.) Fig. 1 was collected from Barkatullah University Campus, Bhopal (M.P.) after identification and authentication from BSI, Allahabad (No.97880), which was shade dried at room temperature and pulverized to get powder of 50-70 mesh size and loss in weight of the material was measured, then extracted in various solvents viz. dichloromethane (DCM), chloroform (CHCl₃), methanol (MeOH) and finally in distilled water in increasing order of polarity by Soxhlet apparatus and has reported maximum percentage yield in distilled water as shown in Table 1.

FIG. 1: SHOWING PLANT HYPTIS SUAVEOLENS

TABLE 1: ISOLATION OF EXTRACTS FROM PLANT MATERIALS OF HYPTIS SUAVEOLENS BY SOXHLETION

The obtained crude extract was separated and further purified by thin layer and column chromatography as reported by Tswett (1906) ⁵, Dubey et al., ⁶ by applying different solvent systems to get purified fractions. The spots developed on TLC Lux plates (Merck) were visualized in UV chamber at 356 nm, in iodine chamber, and in visible light and R_f values as shown in Table 2 and Fig. 2A, B, C of all spots which were calculated by applying the formula of Brimley and Barrett (1953) ⁷ as described below:

R_f = (Distance travelled by solutes) / (Distance travelled by solvent)

TABLE 2: THIN LAYER CHROMATOGRAPHY OF HYPTIS SUAVEOLENS WATER EXTRACT

FIG. 2: SHOWING THIN LAYER CHROMATOGRAPHY OF HYPTIS SUAVEOLENS (L.) IN (A) UV CHAMBER (B) VISUAL LIGHT (C) IODINE CHAMBER

Then, column chromatography (Stock and Rice, 1974)8 of the water extract was performed in solvent system viz. petroleum ether: toluene: ethyl acetate (5:3:2) and two purified fractions were obtained (HS^-1 and HS^-2) as shown in Table 3. Both purified fractions of Hyptis suaveolens (L.) plant extracts were tested on the larvae of Aedes aegypti and Anopheles stephensi for this purpose, adult larvae of Aedes aegypti and Anopheles stephensi of order Diptera and family Culicidae were collected from Saket Nagar huts, Bhopal by the door to door visit.

TABLE 3: COLUMN CHROMATOGRAPHY OF HYPTIS SUAVEOLENS EXTRACT

Total 130 healthy larvae of Aedes aegypti and Anopheles stephensi both the species were reared in the glass vials. Total 10 larvae per vial were released in 500 ml normal water in glass vials capped with muslin cloth for ventilation. Cultures were maintained in the laboratory under controlled conditions viz. temperature (27 ± 1 ºC), relative humidity (75 ± 5% RH). Toxicity assays of the purified fraction of crude water extracts of Hyptis suaveolens (L.) were conducted by using larvae of Aedes aegypti and Anopheles stephensi, respectively. A stock solution of temephos was prepared by applying the standard formula of NVBDCP, and herbal extracts concentration were prepared by dissolving purified fraction of extracts from 50 mg to500 mg in 10% to 100% volume of solvents, and 10 IV instars larvae were released in it and mortality was scored after 24 h of treatments. Beakers were kept at 28 ºC ± 2 ºC room temperature, and the larvae were exposed to 500 ml water and kept as controlled. Experiments as shown in Fig. 3 were performed in triplicates, and in each experiment, early aged larvae were used. In the bio-assay protocol, groups I was served as a control in which larvae were reared in maintained laboratory conditions in a beaker of standard size, and no treatment was given to the larvae of this group.

FIG. 3: MOSQUITO LARVICIDAL EFFICACY OF 10%-100% CONCENTRATION OF HYPTIS SUAVEOLENS WATER EXTRACTS PURIFIED FRACTION ON MOSQUITO LARVAE

Group II was served as vehicle control, in which larvae were reared in a beaker with vehicle solvent (distilled water), which was used finally for extraction of plant extract to observe the consequences of vehicle solvent on animals.  Group III was served as treated, in which larvae were reared by treating it with different concentration of plant extract in increasing order from 10% (50 mg extracts in 500 ml water) to 100% (500 mg extracts in 500 ml water) to monitor the larvicidal efficacy of different extracts of the plant. Group IV was served as the standard in which larvae were reared by treating it with the standard.

Reference drug temephos was used by dissolving it 0.75 ml in 500 ml water, and concentration was maintained 100% to observe the efficacy of the standard drug for comparing it with herbal extracts. For observing the efficacy, a purified fraction of the plant extract of Hyptis suaveolens was tested on the larvae for larvicidal activity, and three replicates were placed. Mortality in Aedes aegypti and Anopheles stephensi was recorded after 24 h of the treatment. Finally, LC₅₀ values were calculated according to the method of Finney (1971) ⁹. The results were compared with the help of PROBIT analysis, standard deviation (SD); standard error (SE), and significant value (P-value) calculated as shown in Tables 4 and Graphs 1 and 2.

TABLE 4: PROBIT ANALYSIS OF HYPTIS SUAVEOLENS (L) WATER EXTRACT

(n=120; Positive cases = 56.67 %, Negative cases = 43.33%),*DF: Degree freedom; LC: Lethal Concentration

RESULTS AND DISCUSSION: In the present study, the percentage yield of the extract of Hyptis suaveolens whole plant was reported to be 19.74% in distilled water Table 1. Similarly, Edeoga et al., (2006) ¹⁰ reported the chemical composition of Hyptis suaveolens and found that Hyptis suaveolens had the highest percentage yield of alkaloids (14.32%) followed by flavonoids (12.54%) in distilled water extracts after de-fating with 100ml of diethyl ether in Soxhlet apparatus for 2 h, then boiled with 50ml of petroleum for the extraction of the bio-active compound i.e. alkaloids, flavonoids, tannins, and saponins. They have also noticed that alkaloids are well known to play some metabolic role and developmental control in living beings.

Moreover, alkaloids also possess a protective role in animals as reported by Edeoga and Eriata (2001) ¹¹. Therefore, in the present study, distilled water extracts of Hyptis suaveolens were isolated and purified by thin-layer chromatography, respectively. For this purpose, TLC of the extract was done by applying solvent system viz. toluene: ethyl acetate (70:30) and 5 spots (HS-1 to HS5) were visualized with RF value 0.26, 0.52, 0.67, 0.74, and 0.82, respectively, as shown in Table 2 and Fig. 2A, B, C. Obtained distilled water extract was again purified by applying solvents system petroleum ether: toluene: ethyl acetate (5:3:2) in column chromatography and the purified fraction was used for mosquito larvicidal activities. Grassi et al., ¹² have separated and isolated two main compounds suaveolol and methyl suaveolate, from leaves of Hyptis suaveolens Poit. (also known as Chichinguaste) of family Lamiaceae by means of repeated preparative thin layer and column chromatography by using five-step petroleum ether and ethyl acetate elution system (5% < 10% < 20% < 40% and finally 100% ethyl acetate in petroleum ether) and detected TLC plates by anisaldehyde reagent. Recently, Baranitharan et al., (2019) ¹³ have also reported the phytochemical analysis of Erythrina variegata and observed the efficacy of Erythrina variegata methanol extract purified fraction against malarial and filarial vector and their results were found to be very similar to the results obtained in the present study as shown in Table 4.

In the present study, mosquito larvicidal activity of whole plant Hyptis suaveolens water extract purified fraction was tested against larvae of Aedes aegypti and Anopheles stephensi, and distilled water was used as vehicle solvent alone for testing its efficacy, then water extract of this plant, when applied against larvae of both the species, it was noticed that the maximum efficacy was observed with 83.33 ± 16.77% mortality of the extract reported in triplicates at 100% concentration as shown in Table 4, Graphs 1 and 2 and Fig. 3. Kamraj et al., (2011) ¹⁴ have reported larvicidal activity of medicinal plant extracts against Anopheles subpictus and Culex tritaeniorhynchus. They have also assessed the role for larvicidal activities of hexane, chloroform, ethyl acetate, acetone, and methanol dried leaves and barks extracts of Annona squamosa L., Chrysanthemum indicum L., Tridax procumbens L., respectively against the 4^th instars larvae of Anopheles subpictus and Culex tritaeniorhynchus mosquitoes. They have noticed that all plant extracts showed moderate effects after 24 h of exposure. In the present study, PROBIT analysis of the bioassay results of the purified fraction of plant extracts was done with the help of Latha (1999) ¹⁵ for the testing of the goodness of fit, which was assessed by Chi-square test, Cox and Snell R² regression analysis Table 4. Bioassay result of purified fraction HS^-1 of water extract of Hyptis suaveolens whole plants showed significant effect 99% (P<0.0001) on mortality of larvae of Aedes aegypti and Anopheles stephensi mosquitoes. Chi-square value was reported 38.69 (P<0.001) at 100% concentration of the purified fraction of the extract of Hyptis suaveolens on larvae of Aedes aegypti and Anopheles stephensi, respectively. The findings of the present investigation revealed that the Hyptis suaveolens (L.) water extract purified fraction possess remarkable mosquito larvicidal activities against two important mosquito species Aedes aegypti and Anopheles stephensi, respectively. Recently, Scalvenzi et al., (2019) ¹⁶ have noticed larvicidal activity of Ocimum campechianum, Ocotea quixos and Piper adimicum essential oils against Aedes aegypti, and a moment ago, Goyal et al., (2019) ¹⁷ have also assessed the chemical composition and larvicidal efficacy of secondary metabolites from aromatic plant extracts against dengue fever vector Aedes aegypti of order Diptera and of family Culicidae.

Awosolu et al., ¹⁸ have noticed larvicidal effects of plant Codiaeum variegatum (Croton) and plant Azadirachta indica (Neem) aqueous extract against Culex quinquefasciatus mosquito. Desiyamani et al., ¹⁹ have tested the toxicity of plant Sargassum polycystum extract for mosquito larvicidal, mosquito repellent and mosquito adulticidal activities against dengue filarial vectors. Rahuman et al., ²⁰ have isolated and identified a compound from Abutilon indicum (L.) for mosquito larvicidal activities.

CONCLUSION: In the present study, the efficacy of water extract purified fraction (HS^-1) of the Hyptis suaveolens whole plants was reported against the larvae Aedes aegypti and Anopheles stephensi for mosquito larvicidal activity that is an eco-friendly and economical means for the management of mosquito strain by controlling their larvae of without side effects of the biopesticides on non-target organism including men.

ACKNOWLEDGEMENT: Authors are highly acknowledged with thanks to UGC, New Delhi, for awarding UGC/BSR/Start-Up grant, and also to MPCST, Madhya Pradesh Bhopal, for providing a grant to Project Investigator (KKS).

CONFLICTS OF INTEREST: Authors of the manuscript have no conflicts of interest with a financial or non-financial interest in the subject matter or materials discussed in the manuscript.

REFERENCES:

1. Mukhopadhyay A, Hati A, Tamizharasu W and Babu P: Larvicidal properties of cashew nut shell liquid on immature stages of two mosquito species. Journal of Vector Borne Disease 2010; 47: 257-60.
2. Bagavan A and Rahuman AA: Evaluation of larvicidal activity of medicinal plant extracts against three mosquito vectors. Asian Pacific Journal of Tropical Medicine 2011; 4(1): 29-34.
3. Chore JK, Obonyo M, Wachira FN and Mireji P: Larvicidal activity of selected Aloe species against Aedes aegypti (Diptera: Culicidae). Journal of Insect Science 2014; 14: 1-3.
4. Subramaniam J, Kovendan K, Kumar PM, Murugan K and Walton W: Mosquito larvicidal activity of Aloe vera (Liliaceae) leaf extract and Bacillus sphaericus against Chikungunya vector Aedes aegypti. Saudi Journal of Biological Science 2012; 19 (4): 503-09.
5. Tswett M: Invention of chromatography. Ber Dtsch Botan Ges 1906; 24: 316-23.
6. Dubey S, Sharma PK, Rajput J, Tomar R and Baghel A: Phytochemical analysis of seeds of medicinal plants. Intern Res J Pharm 2014; 5(2): 102-05.
7. Brimley RC and Barrette FC: Practical Chromatography, Chapman and Hall Publication, London 1953: 26.
8. Stock R and Rice CBF: Chromatographic methods, Chapman and Hall Publication, London 1974: 1-376.
9. Finney DJ: Probit analysis edition 3^rd Cambridge Univ. Press, London 1971.
10. Edeoga HO, Omosun G and Uche LC: Chemical composition of Hyptis suaveolens and Ocimum gratissimum hybrids from Nigeria. African Journal of Biotechnology 2006; 5(10): 892-95.
11. Edeoga HO and Eriata DO: Alkaloid, tannin and saponin contents of some Nigeria medicinal plants. J Med Aromatic Plant Sci 2001; 23: 344-49.
12. Grassi P, Reyes Urias S Tomas, Sosa S, Tubaro A, Hofer O and Zitterl-Eglseer: Anti-inflammatory activity of two diterpenes of Hyptis suaveolens from EI Salvador. Zeitschrift fur Naturforschung C 2006; 61(3-4): 165-70.
13. Baranitharan M, Sawicka B and Gokulakrashnan J: Phytochemical profiling and larval control of Erythrena variegata methanol fractions against malaria and filaria vector. Adv J Prev Med 2019: 1-9.
14. Kamraj C, Bagavan A, Elango G, Zahir AA, Rajkuamr G, Marimuthu S, Santoshkumar T and Rahuman AA: Larvicidal activity of medicinal plant extracts against Anopheles subpictus and Culex tritaeniorhynchus. Indian J Med Res 2011; 134: 101-06.
15. Latha C, Vijayakumar PD, Saleena Velayudhan and Ammini Joseph: Biological activity of indigenous plant extracts as mosquito larvicides. Indian J Exp Biol 1999; 37: 206-08.
16. Scalvenzi L, Radice M, Toma L, Severini F, Boccolini D, Bella A, Guerrini A, tacchini M, Saccheti G, Chiarato M, Romi R and Luca MD: Larvicidal activity of Ocimum campechianum, Ocotea quixos and Piper adimicum essential oils against Aedes aegypti. Para J 2019; (26): 23.
17. Goyal M, Shinde L and Bayas R: Study of chemical composition and larvicidal efficacy of secondary metabolites from aromatic plants extracts against Dengue vectors Aedes aegypti (Linn) (Diptera: Culicidae) Int Journal of Mosquito Research 2019; 6(1): 26-33.
18. Awosolu O, Adesina F and Weagu IM: Larvicidal effects of croton (Codiaeum variegatum) and Neem (Azadirachta indica) aqueous extract against Culex quinquefasciatus mosquito. Int Journal of Mosquito Res 2018; 5(2): 15-18.
19. Desiyamani P, Ali MS, Anuradha V, Yogenanth N and Sheeba H: Eco-toxicity of plant based mosquito larvicidal repellent and adulticidal activity of Sargassum polycystum extract against Dengue and Filarial vectors. Journal of Entomology and Zoology Studies 2018; 6(2): 108-17.
20. Rahuman AA, Gopalakrishnan G, Venkatesan P and Geetha K: Isolation and identification of mosquito larvicidal compound from Abutilon indicum (Linn.) Sweet. Parasitological Research 2008; 102: 981-88.
    

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    How to cite this article:

    Singh M, Namdeo R and Soni KK: Mosquito larvicidal activity of extracts purified fraction of Hyptis suaveolens (L) Poit. Int J Pharm Sci & Res 2021; 12(3): 1608-14. doi: 10.13040/IJPSR.0975-8232.12(3).1608-14.

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