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Toxicity and Effects of Nimolcine (Neem Compound) and Acorus calamus on Cholinesterase Enzyme of Callosobruchus analis in Comparison with Chemical Pesticides.

Byline: Nighat Gul, M. Farhanullah Khan, Saif-Ud-Din and Maisoor Ahmed Nafees

Summary: In the present research study Nimolicine (neem compound), Acorus calamus(crude extract) Cypermethrin (pyrethroid) and Methamidophos (organophosphate) were examined against Callosobruchus analis (F) by Filter paper impregnation method. Presently, LC50 values were found as 19, 5.1, 2.2 and 16ug/cm2 for nimolicine, Acorus calamus crude extract, cypermethrin and methamidopos respectively after 24 hours of treatment. The order of effectiveness was observed as cypermethrin >Acorus calamus > methamidophos >Nimolicine. The cholinesterase % inhibition was observed as 37.5, 40, 12 and 65% for Nimolicine, Acorus calamus, cypermethrin and methamidopos respectively. This research determines that Acorus calamus and Nimolicine both have a valuable potential against the Callosobruchus analis. However, further investigation is indispensable to see the effectiveness of these compounds for the positive pest control acceptable by integrated pest management (IPM) in economic, environmental and social means.

Key Words: Nimolcine , Acorus calamus, Toxicity, Cholinesterase, Callosobruchus analis.


Amongst the most damaging pest of stored grains almost all of the coleopterans insect spp., are known damaging stored grains like paddy, wheat, corn, rice, and pulses. They cause a lot of loss to stored food grain not in developing countries only but they are also a big problem of developed countries. Millions of US dollars losses annually due to insect ravages in stored food [1]. According to estimation about 5 - 10 % of world stored food grains is lost due to insect's infestation. [2-4]. In Pakistan 2-6% loss of stored grains per year recorded in storage bins [5-6].

As soon as the age of science and technology has developed, it also invented synthetic chemicals, applicable equipment but these costly chemicals have adverse effects on our environment, and they are causing destructive consequences for human health. Chemical pesticides leave harmful residuals effects in food products and develop resistance in the target species [7].

They not only have direct effects on human health but also indirect effects as well. Therefore, battle with insects is not stop and human being is still struggling to manage these pests by safer means. The hazardous effects associated with pesticides usage suggest the importance of alternative resources [8]. Among them use of chemosterilants, biocides, allele chemicals, insect growth regulators and plant origin substances are growing up [9-10]. In this regard, many of biologists worked on pesticides of plant origin against various groups of insect species. [11-17]


All the experiments were conduct on Callosobruchus analis.

Rearing Technique

Initial strain of Callosobruchus analis was procure from PARC and further rearing was made at Toxicology Laboratory of Department of Zoology, University of Karachi where Callosobruchus analis were cultured at controlled laboratory conditions of known diet, temperature and humidity for the regular supply of diseased-free insects. The insects were kept in 500g glass jars covered with muslin cloth tied by means of rubber band. Grains of Vigna radiata were use as food and egg laying media when the adults they soon died laid the eggs and the new adults were transfer to the fresh grain containing jars.

Toxicity determination

All the compounds were tested by means of filter paper impregnation method [18]. For this purpose, six sets of Petri dishes of 2.5 cm diameter were taken and these dishes were washed, dried in air and sterilized in oven. Five dishes were marked for each dose whereas one was kept as control to determine the environmental effect, whereas for Nimolicine another one set was marked as check for determination of solvent effects of methanol. Then filter paper was placed at the bottom of petri dishes and by the help of pipette five different containers of the Cypermethrin , Acorus calamus, Methamidophos and Nimolicine were spread on the filter paper. After that, 10 adults of same age and size were released in each petri dish separately. After 24 hours of treatment, percent mortality was observed in all petri dishes including control and check.

Estimation of cholinesterase (EC activity

Biochemical Estimation

Preparation of homogenate

For the preparation of homogenate or sample 100 adult beetles of same size and age of Callosobruchus analis were treated with LC50of four under test compounds i.e., Cypermethrin (2.2 ug/cm2), Nimolicine (19 ug/cm2), Acorus calamus (5.1 ug/cm2) and Methamidophos (16 ug/cm2).

A control batch was also kept to check the environmental effects while in case of Nimolicine methanol batch was also kept as check for comparison as the Nimolicine was prepared in methanol. After 24 hours of treatment, the insects were crush in one ml of distilled water with the help of mortar and pestle, and then homogenized for five minutes at 10,000rpm. The homogenate were centrifuged in "Labofuge 2000 Heraeus Sepatech" for 5 minutes at 5000rpm. The supernatants were taken in separate micro tubes and were kept in refrigerator at 2-8AdegC. This supernatant was use for the estimation Activity of cholinesterase was determined by the colorimetric method of RandoxCat No. CE 190, which is based upon the method [19].


Two test tubes were taken and tagged as control and treated, a third one was also used as check for Nimolicine then 1.5 ml of buffer was added to each test tubes, then 0.01 ml of supernatant of control, treated, and check was added to their respective test tubes. Finally, 0.05 ml of substrate was added to each test tube. Mixed and read the initial absorbance started the timer simultaneously. The Readings were noted again after 30, 60 and 90 sec., against air blank at 405 nm on Schimadzu spectrophotometer UV-120. Finally, these values were kept in the following formula to calculate the cholinesterase activity:

U/L = 11730 x IA 405 nm/min.

Result and Discussion

In the current research 65% inhibition by 16ug/cm2 of Methamidophos, 12% inhibition by 2.2 ug/cm2 of cypermethrin, 40% inhibition by 5.1 ug/cm2 of Acorus calamus and 37.5% inhibition by 19 ug/cm2 of nimolicine in cholinesterase activity of Callosobruchus analis was observed after 24 hours of treatment.

The adults of Callosobruchus analis were treated with 3.3, 6.631, 13.261, 26.522 and 53.05 ug/cm2 of Methamidophos, which showed 06, 20, 30, 50 and 86 percent average mortalities respectively, and the LC50 value was observed as 16ug/cm2.

Table-1: Activity of Cholinesterase in Callosobruchus Analis.

###Compound###Treatment###Mean u/L###S.D. (+-)###S.E. (+-)###Range at 95% confidence limit###% Inhibition

###Control###156.00###22.2###12.83###130.86 - 181.14###-


###Treated###54.65###9.3###5.3###44.26 - 65.03###65

###Control###195.5###4.9###2.8###190.01 - 200.9###-


###Treated###172.04###4.8###2.7###166.74 - 177.33###12

###Control###195.5###5.4###20.46###155.4 - 235.6###-

Acorus calamus

###Treated###117.3###7.3###10.00###97.7 - 136.9###40

###Control###31.28###6.77###3.91###23.62 - 38.94###-


###Treated###19.55###4.4###2.54###14.58 - 24.52###37.5

The adults of Callosobruchus analis were treated with 0.33156, 0.663, 1.326, 2.652 and 5.305 ug/cm2 of Cypermethrin which showed 12, 22, 32, 64 and 72% average %mortalities respectively and the LC50 value was observed as 2.2ug/cm2.

The adults of Callosobruchus analis were treated with 0.8289, 1.657, 3.3153, 6.631 and 13.2625 ug/cm2 of Acorus calamus, which showed 10, 28, 40, 56 and 90% average % mortalities respectively and the LC50 value was observed as 5.1ug/cm2.

The adults of Callosobruchus analis were treated with 4.97, 9.94, 19.89, 39.78 and 79.5 ug/cm2 of Nimolicine which showed 10, 26, 48, 76 and 94% average % mortalities respectively and the LC50 value was observed as 19ug/cm2.

Insecticidal activity of some plant species (in powder form) have been studied against the Callosobruchus analis by Jilani [20]. Out of these only crude Acorus calamus and seed of "soya" were reported as effective as an insecticide. In the current research however cypermethrin was observed to be most effective according to its LC50 value, which is 2.2 ug/cm2 while Acorus calamus was observed as 5.1 ug/cm2, which is greater than nimolicine as well as methamidophos, consequently the current research on Acorus calamus is parallel to the previous findings.

On Callosobruchus analis (F.) the mortality percentage due to Margosan-OTM (neem compound) and Cyfluthrin was reported by Tabassum et. al. LC50 values for Margosan-O2 and cyfluthrinwas found to be 2.2 ug/cm and 0.09 ug/cm respectively [21]. The present study co-relates the above-mentioned results, as the LC50 for cypermethrin was 2.2 ug/cm2. In a comparative study of Cyfluthrin, Dimethoate, H-34 (neem fraction), Margosan-O, Nimolicine, and DDVP (organophosphate) on Musca domestica observed that OP pesticides were found more lethal than Pyrethroids and neem fractions, the order of effectiveness was found to be DDVP > dimethoate>Cyfluthrin> Margosan-OTM> nimolicine > H-34 [22].

In a toxicity study, calamus oil was examined on some stored product pests viz., Sitophilus oryzae Callosobruchus maculatus , Lesioderma serricorne and Tribolium confusum. It was observed that at 30 ug/insect calamus oil was highly toxic to C. maculates, L. serricorne and Sitophilus oryzae with 98.3,100 and 61.7%average mortality correspondingly [23]. In the current exploration, Acorus calamus caused about 90% mortality of Callosobruchus analis at the highest concentration of 13.2625 ug/cm2. It is comparable to the previous result. The efficacy of Nerium indicum. Carissa carandas and Calotropis procera, was compared with permethrin + PBO. The LC50 values were calculated as 22 ug/cm2 for N. indicum, 14.8 ug/cm2 for C. procera and 11.9ug/cm2 for C. carandas [24]. The LC50 for N. indicum in the previous case (22 ug/cm2), is in line to the LC50 for nimolicine (19 ug/cm2) in the current case.

Toxic effects of the Boscia senegalensi, was observed at 2-4% (w/w) cowpea .which caused 80-100% average mortality against Callosobruchus maculatus (F.) [25]. While in the present case cypermethrin, Methamidophos, Acorus calamus and nimolicine caused upto 72, 86, 90 and 94% highest mortalities at their highest concentrations respectively which is in line with the mortality rate of previous research.

Ahmed [26] studied the efficacy of cypermethrin and methyl parathion (OP) as compared to neem extract (plant product) against the Tribolium castaneum. The LC50 for neem extract, Methyleparathion and cypermethrin were observed as 1300 ug/cm2, 0.19 ug/cm2 and 2.3ug/cm2 respectively. LC50 for cypermethrin in the above study (2.3 ug/cm2) is quite similar to the current study (2.2 ug/cm2) although the insect was different.

Toxicity of Acorus calamus, lorsban, cypermethrin, methamidophos and shogun was studied on Rhyzopertha dominica and the order of efficacy was observed as cypermethrin (0.032 ug/cm2) > lorsban (0.056 ug/cm2) >shogun (0.08 ug/cm2) >Acorus calamus (0.29 ug/cm2)>methamidophos(0.45 ug/cm2) [27]. While the current study the order of efficacy observed as cypermethrin (2.2 ug/cm2) >Acorus calamus (5.1 ug/cm2) >methamidophos (16 ug/cm2) >nimolicine (19 ug/cm2). The order of efficacy of both studies is similar although the LC50 values are different. This difference is because of the different target pest.

Azmi [28] reported toxicological effects of Cyhalothrin and Clerodendsum inerme along with biochemical estimations against Rhizopertha dominica, it was observed that both the compounds have the potential to decreased the cholinesterase and acid phosphatase activity to a little bit. Rizvi [28] confirmed similar findings. Same pesticides were tested for same enzymes but the test insect was the Tribolium castaneum. Similarly in the present case cypermethrin, Acorus calamus, Nimolicine and Methamidophos all showed inhibition of cholinestease to a positive level.

Inhibition in cholinesterase activity was observed as 46.35%, 34.10% and 33.83 %( after 30, 60 and 90 seconds interval) after treatment with Bakayan root extract against Coccinella septumpunctata Lin [29]. In the present exploration cholinesterase, activity has been decreased at 65%, 12%, 40% and 37% levels after treatment with methamidophos, cypermethrin, Acorus calamus and Nimolicine respectively. The cholinesterase inhibition levels of Nimolicine and Acorus calamus are in line to the previous results while cypermethrin and methamidophos are away from this range. Methamidophos responded highest levels of cholinesterase inhibition because Organophosphates have the potential of inhibit the cholinesterase at higest levels whereas cypermethrin showed a minimum level inhibition of this enzyme as the pyrethroids could not inhibit cholinesterase activity as well.


According to the toxicological effects of the test compounds cypermethrin is the most toxic as its LC50 value is almost doubled to the nearest compound Acorus calamus where as nimolicine is the least toxic compound but its LC50 value was nearer to the methamidophos as well as both plant products inhibited cholinesterase level to a positive extent. Our research concludes that both plant products nimolicine and Acorus calamus have a valueable potential for defense of mung bean from pulse weevil Callosobruchus analis. However, extra research is needed in the future, which will evaluate that how successfully they achieve the target of positive pest control acceptable by IPM (integrated pest management) in economic, social and environmental areas.


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Author:Gul, Nighat; Khan, M. Farhanullah; Saif-Ud-Din; Nafees, Maisoor Ahmed
Publication:Journal of the Chemical Society of Pakistan
Article Type:Technical report
Geographic Code:9PAKI
Date:Apr 30, 2019
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