Mosquito repellant activity of extracts of Chandana and Sarshapa
Gazala Hussain, Vinayakumar R Kadibagil, A. Jebanesan
1Associate Professor, Dept. of Rasashastra and Bhaishajya Kalpana,
Sri Dharmasthala Manjunatheshwara College of Ayurveda and Hospital, Hassan.
2Professor, Dept. of Rasashastra and Bhaishajya Kalpana,
Sri Dharmasthala Manjunatheshwara College of Ayurveda and Hospital, Hassan.
3Professor, Vector Biology Laboratory, Department of Zoology, Annamalai University.
*Corresponding Author E-mail: drvinaykadibagil@gmail.com
ABSTRACT:
Simple herbs are a remedy for various problems and holds good even for mosquito menace. Mosquitoes are well known vectors of several disease-causing pathogens. They are a cause of concern for various diseases like malaria, filariasis, dengue haemorrhagic fever and chikungunya.DEET (N, N-diethyl-3-methylbenzamide) is so far the golden standard for mosquito repellant effect. Many synthetic mosquito repellants are available but question of safety is an issue. Thus the need for a safer alternative with herbs has aroused. In Ayurveda treatise the combination of Chandana (Santalum album) and Sarshapa (Brassica campestris) as dhupana (fumigation) has been said as krimighna (insecticidal). The extracts of these two drugs have proved to have mosquito repellant action against Aedes aegypti. The study was conducted by Cone bioassay and Cage Bioassay methods. The repellency activity of these plant extracts were tested at five different concentration (1.0, 2.0, 3.0, 4.0 and 5.0 mg/cm2) and at the density of 100 mosquitoes against 3-4 days old, blood starved female Ae.aegypti. The skin repellency activity of Santalum album and Brassica campestris plant extract gave maximum protection at 5.0 mg/cm2 concentration (6.30 hours).
KEYWORDS: Mosquito repellant, Herbs, Chandana, Sarshapa, Ayurveda.
INTRODUCTION:
The world is gradually turning to herbal formulations which are known to be effective against a large repertoire of diseases and ailments. Many of the herbs and shrubs are found to have promising medicinal properties, and mosquito repellent properties1. Mosquitoes are well known vectors of several disease-causing pathogens.Mosquito-borne diseases, such as malaria2,3, filariasis4, dengue5,6,7,8,9 haemorrhagic fever and chikungunya are still imposing to be major public health problems in the South east Asian countries because of the tropical or subtropical climate10,11,12,13,14. Prevention of mosquito bite is thus the main aim to control the occurrence of these diseases. Mosquitoes need to be exterminated using preferably non-hazardous chemicals.
Many the pest control make chemical choices for getting rid of mosquitoes are extremely harmful for humans. These chemicals are not a good alternative as these mosquito controlproducts are not a green choice15.
The majority of commercial mosquito repellent preparations contain the chemical DEET (N, N-diethyl-3-methylbenzamide) first synthesized in 1954. It has been reported that these chemicals are not safe for public use16. Bio based mosquito repellent are pest management tools that are based on safe, biologically based active ingredients derived from plants. Benefits of bio-products include effective control of mosquito as well as human and environmental safety.
Extracts or essential oils from plants may be alternative sources of mosquito control agents, since they constitute a rich source of bioactive compounds that are biodegradable into nontoxic products and potentially suitable for use to control mosquitoes17.
MATERIALS AND METHODS:
Plant Extract:
Santalum album bark and Brassica campestris were taken and kept in hot air oven for one hour to completely dry.Then 250gms was weighed and made into fine powder by using motor and pestle. This powder was dissolved into 100ml of double distilled water and boiled at 60ºC for one hour and this mixture was filtered by using vacuum filter and the extract was collected into the conical flask and kept at 4ºC.
The protection time was tested by the method described by (Schreck and McGovern 1989; WHO 1996). The repellency of the Santalum album against dengue vectors was evaluated using the human bait technique. For investigation, 100 laboratories reared, blood starved, adult female Aedes aegypti that were between 5-7 days old were placed into separate laboratory cages (45cm X 45cm X 45cm).
The hand was washed with tap water dried with towel and then the extract was applied on the external surface of the first of human hand over an area of about 150cm2 at the rate of 1mg/cm2. The treated surface was exposed to 100 non blood fed female mosquitoes (5-7 days old) in the cage. Tests were conducted during 06:00 hr to 06:00 hr in the light room at 27±2º C and 70±5% RH. Each evaluator the number of bites was counted over 3 minutes every 30minutes biting counts and the experiments were performed in triplicate (n=6). DEET (N, N- diethyl-3-methylbenzamide) was used as standard. Tests were repeated at interval of 30 min and the experiment were completed when 5 mosquitoes had bitten. The repellent activity of DEET (N, N- diethyl-3-methylbenzamide) was used as a standard. The number of insects landing or biting was recorded for two volunteers (WHO, 1996).
No. of bites received by control arm
Cone tests were conducted following WHO guidelines. The bioassay was performed according the WHOPES guidelines. The set-up was prepared by cutting 25cm× 25 cm piece of netting. Four plastic cones were attached to each piece of net and held together with two binder clips. The assembly was held at one of the test angles; 0º. Using an aspirator, five to eight non blood-fed females aged two to five days were introduced into each cone and the holes were plugged by pieces of cotton.
The mosquitoes were exposed for 3 min and subsequently transferred into labeled 150ml plastic holding cups and provided with 10% sugar solution. Knockdown and mortality were recorded every 1 hour and 24 hr after exposure, respectively. Mosquitoes were scored as alive if they were able to fly, irrespective of the number of legs still intact and dead, or knocked down, if immobile or incapable of flying or standing in a coordinated manner.
An untreated net was used as control and was tested each day the bioassay was performed. The bioassays were performed at temperature and relative humidity ranges of 25.9-29º C and 58-73%, respectively.
Bioassay for knocking- down and killing effects (Mat and Liquidizer):
The bioassay for knocking- down and killing effects was conducted by modifying the method of Jantan et al., (2005). It was performed in a glass chamber measuring 120 x 120 x 60cm at front and sides and wooden lamination with white colored interior at top bottom and backside. Sliding glass doors (60 x 30) were provided at both sides of the chamber. An electric plug point was provided at the interior back of the chamber. Mosquitoes (100) from the mosquito colony were introduced into this chamber through the sliding window at one side. The liquid mosquito insecticide extract formulation (LMIEF) was allowed to vaporize for 2 min by using the vaporizer equipment provided with commercial liquid mosquito insecticide formulation. Knock down mosquitoes (i.e., those that no longer maintained normal posture and were unable to fly or were on their backs) were recorded at 1-minute intervals up to 12 hours or until total knock down was achieved. Knock – down mosquitoes were placed in a clean container containing cotton wool soaked with 5% sucrose solution and the mortality of the mosquitoes was observed after 24 hr. Six replicate of the experiments were carried out for each liquid mosquito formulation. One control was performed by exposing the mosquitoes to the vapour of the solvent (deodorized kerosene). A commercial reference standard liquid mosquito insecticide formulation (allethrin 3.6% w/w was also tested for comparison. Knock-down times KD50 and KD 90, as the minutes needed to Knock-down 50% and 90% mosquitoes, respectively) were determined by the probit analysis (Finney, 1971).
Abbott’s formula was applied for Mortality
Test mortality % − Control mortality %
Mortality % = ------------------------------------------------
100−Control mortality
The values of effective dose KD50 were obtained through Probit Analysis and by use of SPSS 16. Software and the values were drawn from six replicates. Effectiveness of the extracts was determined by comparing the 95% confidence intervals of the KD50values.
Cone Bioassay for Synergistic activity of Knock down effect of Santalum album and Brassica campestris plant extract:
Synergistic activity of Santalum album and Brassica campestris plant extract for 12 hrs of exposure showed effective knockdown activity against Ae. aegypti mosquito. The statistical data are presented in Table 2. The highest concentration of 5.0% provided cent percent (100%) protection upto 5 hours. At 0.5%, concentration 1.0% concentration the knockdown was observed for 1 hour and 2.5% concentration the knockdown was observed for 3 hours at 2.5% concentration and the knock down effect was observed for 3 hours.
The lower concentration of 0.5% provided cent percent protection upto 1 hours the total percentage protection observed upto five hours exposure time increasing in exposure hours there is a reduction of knock down effect was noticed as 96.00, 86.02, 80.00, 76.08, 62.33, 57.02, 46.00, 39.08, 30.33, 24.02 and 16.00 for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12 hours exposure period respectively.
The concentration of 1.0% provided cent percent protection upto 2 hours the total percentage protection observed upto five hours exposure time increasing in exposure hours there is a reduction of knock down effect was noticed as 85.03, 74.02, 69.03, 54.02, 49.02, 38.04, 30.00, 28.06, 20.00 and 12.06 for 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 hours exposure period respectively.
The concentration of 2.5% provided cent percent protection upto 3 hours the total percentage protection observed upto five hours exposure time increasing in exposure hours there is a reduction of knock down effect was noticed as 90.00, 80.02, 76.00, 68.06, 57.02, 46.00, 32.06, 26.00 and 18.06 for 4, 5, 6, 7, 8, 9, 10, 11, and 12 hours exposure period respectively.
The higher concentration of 5.0% provided cent percent protection upto 5 hours the total percentage protection observed upto seven hours exposure time increasing in exposure hours there is a reduction of knock down effect was noticed as 70.00, 64.02, 56.04, 49.03, 40.00, 32.06 and 24.08 for 6, 7, 8, 9-, 10-, 11-, and 12-hours exposure period respectively.
Results of the synergistic repellent activity of plant extract Santalum album and Brassica campestris against Ae. aegypti mosquito recorded maximum of 100% protection time (6.30 hours) was obtained at the concentration of 5.0mg/cm2 this statistically data are presented in Table 2. The statistically (1.0mg/cm2, X2 = 0.496; 2.0mg/cm2, X2 = 0.726; 3.0mg/cm2, X2 = 0.453; 4.0mg/cm2 , X2 = 0.137 and 5.0mg/cm2, X2 = 0.206, p< 0.001) between treated and control groups.
Investigations resulted in 6-7 hr of complete protection time with 5.0mg/cm2 of extract applied on the arms of human volunteers. The plant extract of Sandalwood and Mustard shows significant repellency against Ae.aegypti adults. It shows that repellency depends on the extracts concentration. A higher concentration of 5.0 mg/cm2 provided 100% protection up to 7 hr.
Table 1: Synergistic knockdown effect for the 12 hr exposure of plant extract Santalumalbum and Brassica campestris against Aedes aegypti
|
Name of the sample |
Mortality (%) |
||||||||||||
|
% Con. |
1hr |
2hr |
3hr |
4hr |
5hr |
6hr |
7hr |
8hr |
9hr |
10hr |
11hr |
12hr |
|
|
Plant extract Santalum album + Brassica campestris |
0.5 |
100 ± 0 |
96.00 ± 1.22 |
86.02 ± 1.13 |
80.00 ± 2.30 |
76.08 ± 0.34 |
62.33 ± 1.80 |
57.02 ± 2.94 |
46.00 ± 0.69 |
39.08 ± 1.94 |
30.33 ± 0.59 |
24.02 ± 0.34 |
16.00 ± 1.06 |
|
1.0 |
100 ± 0 |
100 ± 0 |
85.03 ± 2.73 |
74.02 ± 2.23 |
69.03 ± 1.80 |
54.02 ± 0.34 |
49.02 ± 0.54 |
38.04 ± 2.39 |
30.00 ± 2.42 |
28.06 ± 2.21 |
20.00 ± 1.26 |
12.06 ± 1.24 |
|
|
2.5 |
100 ± 0 |
100 ± 0 |
100 ± 0 |
90.00 ± 1.02 |
80.04 ± 1.70 |
76.00 ± 0.34 |
68.06 ± 1.80 |
57.02 ± 2.64 |
46.00 ± 2.88 |
32.06 ± 2.49 |
26.00 ± 2.20 |
18.06 ± 1.79 |
|
|
5.0 |
100 ± 0 |
100 ± 0 |
100 ± 0 |
100 ± 0 |
100 ± 0.0 |
70.00 ± 2.73 |
64.02 ± 0.34 |
56.04 ± 2.44 |
49.03 ± 1.09 |
40.00 ± 2.56 |
32.06 ± 2.47 |
24.08 ± 2.21 |
|
Values are mean of six replicates ± SE
*Significant at p<0.001 level.
Table 2: Synergistic repellent activity of Plant extractsSantalum album and Brassica campestris against Aedes aegypti
|
Concentration of Plant extract (mg/cm2 ) |
Mean number of bites ± SE |
Mean number of 100% protection (hours) |
Total % of protection for 12 hours |
X2 (df=2) |
|
|
Control |
Treated |
||||
|
1.0 |
86.50 ± 2.39 |
40.00 ± 1.40 |
1.00 |
15.02 |
0.496 |
|
2.0 |
76.00 ± 2.05 |
33.00 ± 1.69 |
2.00 |
26.05 |
0.726 |
|
3.0 |
69.40 ± 2.21 |
25.00 ± 2.21 |
3.00 |
59.19 |
0.453 |
|
4.0 |
67.00 ± 2.88 |
20.00 ± 2.0 |
5.30 |
80.06 |
0.137 |
|
5.0 |
64.66 ± 1.26 |
0.0 ± 0.0 |
6.30 |
96.04 |
0.206 |
Cage Bioassay:
Results of the synergistic repellent activity of plant extract Santalum album and Brassica campestris against Ae. aegypti mosquito recorded maximum of 100% protection time (6.30 hours) was obtained at the concentration of 5.0mg/cm2 this statistically data are presented in Table 4. The statistically (1.0mg/cm2, X2 = 0.496; 2.0mg/cm2, X2 = 0.726; 3.0mg/cm2, X2 = 0.453; 4.0mg/cm2, X2 = 0.137 and 5.0mg/cm2, X2 = 0.206, p< 0.001) between treated and control groups.
The investigations resulted in 6-7hr of complete protection time with 5.0mg/cm2 of extract applied on the arms of human volunteers. The plant extract of Sandalwood and Mustard shows significant repellency against Ae. aegypti adults. It shows that repellency depends on the strength of extracts concentration. A higher concentration of 5.0mg/cm2 provided 100% protection up to 7 hr.
Bioassay for Liquid Mosquito Insecticide Extract Formulation
Table 5. Knock – down (KD) and killing effect of liquid mosquito insecticide formulation containing Santalum album and Brassica campestris extract (LMIEF) against Ae.aegypti
|
Formulation |
KD50 (minutes) |
KD90 (minutes) |
Mortality (%) |
|
Control |
25.00 |
52.00 |
9.31 (17.76) |
|
1 % |
4.02 |
9.74 |
49.00 (44.43) |
|
3 % |
3.57 |
8.20 |
77.00 (61.35) |
|
5 % |
2.51 |
4.30 |
86.00 (68.06) |
(Mortality (%) values in parentheses are arcsine transformed)
The results of the bioassay for knockdown effects at two different levels KD50 and KD90 and killing effects of Santalum album and Brassica campestris extract in liquid formulations (LMIEF) (Table 5). It was observed that, the liquid insecticide showed a significant knockdown effects and mortality percentage at three different concentration viz; 1% w/w, 2% w/w and 5% w/w levels against Ae. aegypti. As the concentration of LMIEF increased the mortality rate was also increased. The maximum knockdown effect LMIEF was observed within a period of 2.51 minutes for KD50 and 4.30 minutes for KD90 against Ae. aegypti at 5 % w/w concentration whereas the control recorded 25.00 minutes for KD50 and 52.00 minutes for KD90 against Ae. aegypti.
It was also observed the LMIEF showed a significant mortality of Ae. aegypti in all three concentrations tested the maximum mortality of 86.00% was recorded at 5% w/w of liquid insecticide against Ae. aegypti whereas the control recorded only 9.31%.
CONCLUSION:
In the cone bioassay, the synergistic repellency of Santalum album and Brassica campestris plant extract was tested against Ae. aegypti. The synergistic repellency activity of Santalum album and Brassica campestrise plant extract were tested at 0.5%, 1.0%, 2.5 % and 5.0% and the density of 100 mosquitoes against 3-4 days old, blood starved female mosquito of Ae. aegypti.In cone bioassay the maximum Knock down activity of Santalum album and Brassica campestris plant extract gave maximum Knock down effect at 5.0% concentration for 5 hours.Among the four concentration tested the higher concentration 5.0% gave better Knock down effect. The synergistic repellency of Santalum album and Brassica campestris plant extracts was tested against Ae.aegypti was evaluated by using the percentage of protection in relation to dose method.The repellency activity of these plant extracts were tested at five different concentration (1.0, 2.0, 3.0, 4.0 and 5.0mg/cm2) and at the density of 100 mosquitoes against 3-4 days old, blood starved female Ae. aegypti.The skin repellency activity of Santalum album and Brassica campestris plant extract gave maximum protection at 5.0 mg/cm2 concentration (6.30 hours). The repellent activity was very high at the initial stage (h) of exposure. Increase in the exposure period showed reduction in repellent activity. The repellent activity was dependent on the concentration of the plant extracts and the density of mosquitoes.It was also observed that LMIEF showed a significant mortality of mosquitoes in all the three-concentration tested. The maximum mortality of 86% was recorded 5 % concentration of liquid insecticide against Aedes aegypti, whereas control could record only 9.31%.
SOURCE OF SUPPORT AND ACKNOWLEDGEMENT:
A note of gratitude to Rajiv Gandhi University of Health Sciences, Bengaluru Karnataka. For funding the research project under faculty research grant.
REFERENCES:
1. Mullai K, Jebanesan A, Pushpanathan T. Mosquitocidal and repellent activity of the leaf extract of Citrullus vulgaris (cucurbitaceae) against the malarial vector, Anopheles stephensi liston (diptera culicidae). European Review for Medical and Pharmacological Sciences. 2008;12(1):1-7. https://www.europeanreview.org/article/469
2. Thillainayagam M, Ramaiah S. Mosquito, malaria and medicines-A Review. Research Journal of Pharmacy and Technology. 2016;9(8):1268.https://doi.org/10.5958/0974-360X.2016.00241.9
3. Wardani AK, Wahid AR, Rosa NS. in vitro antimalarial activity of ashitaba root extracts (angelica keiskei K.). Research Journal of Pharmacy and Technology. 2020;13(8):3771.https://doi.org/10.5958/0974-360x.2020.00667.8
4. Qamandar MA, Shafeeq MAA. Possible Mosquito Control by Silver Nanoparticles Synthesized by Entomopathogenic Fungus Beauveria bassiana. Research Journal of Pharmacy and Technology. 2018;11:1058.https://doi.org/10.5958/0974-360X.2018.00198.1
5. Patil R, Makhija T, Suryawanshi HP, Pawar S. A Review on – Dengue. Research Journal of Pharmacy and Technology. 2013;6(9):930-6.https://rjptonline.org/Issues.aspx?VID=6 and IID=9
6. Thant Z, Tengku MA, Azmi H, Norizhar K. Dengue Virus Infections: Global Scenario, Classification, and Clinical Manifestations– A Review. Research Journal of Pharmacy and Technology. 2016;9:83.https://doi.org/10.5958/0974-360X.2016.00014.7
7. Tamilventhan A, Jayaprakash A. Larvicidal Activity of Terminalia arjuna Bark extracts on Dengue Fever Mosquito Ades aegypti. Research Journal of Pharmacy and Technology. 2019;12:87.https://doi.org/10.5958/0974-360X.2019.00017.9
8. MM Baheerati. Natural Therapy for Dengue Fever. Research Journal of Pharmacy and Technology. 2014;7(2):269-7. https://rjptonline.org/AbstractView.aspx?PID=2014-7-2-20
9. Rouhullah Dehghani, Hamid Kassiri. A Review on Epidemiology of Dengue Viral Infection as an Emerging Disease. Research Journal of Pharmacy and Technology. 2021; 14(4):2296-1. https://doi.org/10.52711/0974-360X.2021.00406
10. Sudjarwo SA, Ngadino, Koerniasari, Setiawan, Wardani Sudjarwo G. Larvicidal Activity of Ethanol Leaf Extract of Pinus merkusii on Aedes aegypti larvae. Research Journal of Pharmacy and Technology. 2017;10(4):1011-4.https://doi.org/10.5958/0974-360X.2017.00182.2
11. Bahadur A, Chandrashekar KS, Pai V. Formulation and Development of Polyherbal mosquito Repellent Incense Sticks. Research Journal of Pharmacy and Technology. 2020;13:124.https://doi.org/10.5958/0974-360X.2020.00025.6
12. Sharma B, Vasudeva N, Sharma S. Anti-scabies and mosquito repellent activity of Crinum asiaticum Linn. leaves extracts. Research Journal of Pharmacy and Technology. 2020;13(2):895–900. https://doi.org/10.5958/0974-360X.2020.00169.9
13. Gupta DK, Gupta RA. Development of poly herbal mosquito repellent. Research Journal of Pharmacy and Technology. 2020;13:4871.https://doi.org/10.5958/0974-360X.2020.00857.4
14. Mukesh Y, Savitri P, Kaushik R, Singh NP. Studies on repellent activity of seed oils alone and in combination on mosquito, Aedes aegypti. Journal of Environmental Biology. 35(5):917–22.http://www.jeb.co.in/journal_issues/201409_sep14/paper_21.pdf
15. Trivedi A, Rai P, Kumar J. Formulation of low smoke herbal mosquito repellent sticks by using different essential oils. The Pharma Innovation Journal. 2018;7(4):173–5.https://www.thepharmajournal.com/archives/2018/vol7issue4/PartC/7-3-62-780.pdf
16. Chavare SD, Karande KM, Aloorkar NH, Kulkarni AS, Majumdar SH. Formulation of novel herbal mosquito repellent: A new approach in antimalarial management. Int J Med Pharm Res. 2015;1:78-85. file:///C:/Users/admin.BAN-74DWQ72-LT/Downloads/article_1436991028.pdf
17. Prabakar K, Jebanesan A. Larvicidal efficacy of some Cucurbitacious plant leaf extracts against Culex quinquefasciatus (Say). Bioresour Technol. 2004 Oct;95(1):113-4. doi: 10.1016/j.biortech.2003.05.001. PMID: 15207304.
Received on 01.09.2021 Modified on 29.05.2022
Accepted on 19.10.2022 © RJPT All right reserved
Research J. Pharm. and Tech 2023; 16(5):2120-2124.
DOI: 10.52711/0974-360X.2023.00348