Claims:1. A method of synthesizing a biological agrochemical composition that is effective against pests, said method comprising the steps of:
air drying Pachyrhizus erosus seeds at room temperature for 15 days;
grinding the air dried seeds to powder form with a mixture grinder;
microwave-assisted extracting with dichloromethane and methanol, said microwave-assisted extracting comprising the steps of:
mixing the powder with a 50:50 mixture of dichloromethane and methanol, with the ratio of the powdered seeds to the dichloromethane and methanol mixture being 1:15;
performing extraction with a microwave system at 400 W and 2,500 MHz for 10 minutes;
filtering the sample with Whatman filter paper; and
evaporating the water to obtain a condensed mass; and
dissolving the condensed mass in ethanol to obtain a final extract concentration that ranges between 0.1% and 1%.
2. The method of synthesizing a biological agrochemical composition that is effective against pests as claimed in claim 1, wherein said method comprises the steps of:
dissolving 50% w/w of the extract in 40% of a solvent;
adding 10% of a single emulsifier or a blend of emulsifiers to the extract solution, with: the individual percentage of emulsifiers in the 10% blend ranging between 1% and 9%; and the ratio of the solvent to the single emulsifier or the blend of emulsifiers being 1:1 or 2:1; and
mixing in a homogenizer at 500 rpm and 60 degrees Centigrade.
3. The method of synthesizing a biological agrochemical composition that is effective against pests as claimed in claim 1 or claim 2, wherein 200 grams of Pachyrhizus erosus seeds are air dried at room temperature for 15 days, and mixed with 300 ml of the 50:50 mixture of dichloromethane and methanol.
4. The method of synthesizing a biological agrochemical composition that is effective against pests as claimed in claim 1 or claim 2, wherein the condensed mass is dissolved in ethanol to obtain a final extract concentration of 1%.
5. The method of synthesizing a biological agrochemical composition that is effective against pests as claimed in claim 1 or claim 2, wherein the pest is Plutella xylostella.
6. The method of synthesizing a biological agrochemical composition that is effective against pests as claimed in claim 2, wherein the solvent is kerosene, hexane, or paraffin oil.
7. The method of synthesizing a biological agrochemical composition that is effective against pests as claimed in claim 2, wherein the emulsifier is one or a blend of: sorbitan monooleate; and polyoxyethylene (20) sorbitan monolaurate.
8. A biological agrochemical composition that is effective against pests, said biological agrochemical composition being synthesized through the method as claimed in claim 1 or claim 2. , Description:TITLE OF THE INVENTION: METHOD OF SYNTHESIZING A BIOLOGICAL AGROCHEMICAL COMPOSITION AND BIOLOGICAL AGROCHEMICAL COMPOSITION THEREOF
FIELD OF THE INVENTION
The present disclosure is generally related to a method of synthesizing an agrochemical composition. The present disclosure is particularly related to a method of synthesizing a biological agrochemical composition. More particularly, the present disclosure is related to a method of synthesizing a botanical pesticide composition and a botanical pesticide composition thereof.
BACKGROUND OF THE INVENTION
Crop loss due to pest attack is a significant agricultural issue that is yet to be solved. The losses of crops caused by insect pests are quite high, not only in developing countries, but also in developed countries as well.
In the last 50 years, the protection of crops has been achieved with synthetic pesticides. However, synthetic pesticides are highly toxic; persistent; have adverse residual effects on crops, groundwater, and soil; and their excessive application has led to increased resistance of pests.
On the other hand, botanical pesticides are less persistent, less toxic to non-target organisms, and are eco-friendly; but, the science behind botanical pesticides is still not well known completely. However, only few such pesticides are in commercial use, which suffer from the drawbacks of: slower rates compared to synthetic chemical pesticides, lesser perseverance in the atmosphere, and proneness to unfavourable environmental conditions.
There is, therefore, a need in the art for a method of synthesizing a biological agrochemical composition, and a biological agrochemical composition thereof, which overcome the aforementioned drawbacks and shortcomings.
SUMMARY OF THE INVENTION
A method of synthesizing a biological agrochemical composition that is effective against pests is disclosed. Said method of synthesis broadly comprises the steps of:
air drying Pachyrhizus erosus seeds at room temperature for about 15 days;
grinding the air dried seeds to coarse powder form with a mixture grinder;
microwave-assisted extracting with dichloromethane and methanol; and
dissolving the condensed mass in ethanol to obtain a final extract concentration that ranges between about 0.1% and about 1%.
Said microwave-assisted extracting comprises the steps of:
mixing the powder with a 50:50 mixture of dichloromethane and methanol, with the ratio of the powdered seeds to the dichloromethane and methanol mixture being about 1:15;
performing extraction with a microwave system at about 400 W and about 2,500 MHz for about 10 minutes;
filtering the sample with Whatman filter paper; and
evaporating the water to obtain a condensed mass.
A method of synthesizing an emulsifiable concentrate of the biological agrochemical composition, and biological agrochemical compositions that are synthesized through the disclosed method(s), are also disclosed.
In an embodiment, the pest is Plutella xylostella.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 illustrates the results of Pachyrhizus erosus Soxhlet extraction with petroleum ether, ethanol, and chloroform, in accordance with various embodiments of the present disclosure;
Figure 2 illustrates the results of Pachyrhizus erosus ultrasound extraction with dichloromethane and ethanol, in accordance with various embodiments of the present disclosure;
Figure 3 illustrates the results of Pachyrhizus erosus microwave-assisted extraction with water, and mixture of dichloromethane and methanol, in accordance with various embodiments of the present disclosure;
Figure 4 illustrates the results of Pachyrhizus erosus maceration with chloroform and methanol mixture, and ethanol, in accordance with various embodiments of the present disclosure;
Figure 5 illustrates the sample of larvae, against which the bio-efficacy of Pachyrhizus erosus crude extract solution was tested, in accordance with present disclosure;
Figure 6 illustrates the sample of larvae, against which the bio-efficacy of Pachyrhizus erosus crude extract solution was tested, after spraying with the crude extract, in accordance with present disclosure;
Figure 7 illustrates the dead larvae, after spraying with Pachyrhizus erosus crude extract solution, in accordance with present disclosure;
Figure 8 illustrates the results of the bio-efficacy studies of Pachyrhizus erosus crude extract solutions, in accordance with various embodiments of the present disclosure;
Figure 9 illustrates the details of synthesized emulsifiable concentrate formulations, in accordance with various embodiments of the present disclosure;
Figure 10 illustrates the results of evaluation of physical appearance, cold test, and storage stability test, in accordance with various embodiments of the present disclosure;
Figure 11 illustrates the dose calculation of synthesized emulsifiable concentrate formulations, in accordance with various embodiments of the present disclosure;
Figure 12 illustrates the results of mortality studies performed with a synthesized emulsifiable concentrate formulation, in accordance with an embodiment of the present disclosure;
Figure 13 illustrates the results of mortality studies performed with another synthesized emulsifiable concentrate formulation, in accordance with an embodiment of the present disclosure;
Figure 14 illustrates the results of mortality studies performed with yet another synthesized emulsifiable concentrate formulation, in accordance with an embodiment of the present disclosure;
Figure 15 illustrates the results of mortality studies performed with yet another synthesized emulsifiable concentrate formulation, in accordance with an embodiment of the present disclosure;
Figure 16 illustrates the results of mortality studies performed with yet another synthesized emulsifiable concentrate formulation, in accordance with an embodiment of the present disclosure;
Figure 17 illustrates the results of mortality studies performed with yet another synthesized emulsifiable concentrate formulation, in accordance with an embodiment of the present disclosure; and
Figure 18 illustrates the toxicity level of synthesized emulsifiable concentrate formulations, in accordance with various embodiments of the present disclosure.
DETAILED DESCRIPTION OF THE INVENTION
Throughout this specification, the use of the words "comprise", “have”, “contain”, and “include”, and variations such as "comprises", "comprising", “comprised”, “having”, “contains”, “containing”, “includes”, and “including” may imply the inclusion of an element or elements not specifically recited. The disclosed embodiments may be embodied in various other forms as well.
Throughout this specification, the use of the phrase “biological agrochemical” and its variations are to be construed as “an agricultural chemical product where the active constituent comprises or is derived from a living organism”.
Throughout this specification, the use of the phrase “biological agrochemical” and its variations are to be construed as being inclusive of pesticides.
Throughout this specification, the use of the phrase “biological agrochemical composition” and its variations are to be construed as being inclusive of synthesized crude extracts and synthesized biological agrochemical compositions.
Throughout this specification, the use of the acronym “DCM” is to be construed as dichloromethane.
Throughout this specification, the use of the acronym “MeOH” is to be construed as methanol.
Throughout this specification, the use of the acronym “EC” is to be construed as emulsifiable concentrate.
Throughout this specification, the use of the acronym “SM” is to be construed as sorbitan monooleate.
Throughout this specification, the use of the acronym “PSM” is to be construed as polyoxyethylene (20) sorbitan monolaurate.
Throughout this specification, the use of the word “formulation” and its variations are to be construed as synthesized emulsifiable concentrate formulations (i.e. synthesized biological agrochemical compositions).
Throughout this specification, the words “the” and “said” are used interchangeably with the same meaning.
Throughout this specification, the disclosure of a range is to be construed as being inclusive of the lower limit of the range and the upper limit of the range.
Also, it is to be noted that embodiments may be described as a method (sequential process). However, many of the operations may be performed in parallel, concurrently, or simultaneously. In addition, the order of the operations may be re-arranged. A method may be terminated when its operations are completed, but may also have additional steps.
A method of synthesizing a biological agrochemical composition is disclosed. In an embodiment of the present disclosure, the method of synthesis broadly comprises the following steps:
The following experiments were carried out:
200 grams of Pachyrhizus erosus seeds were collected from a field in Kharagpur, West Bengal. The collected seeds were air dried at room temperature for about 15 days.
Extraction of Pachyrhizus erosus:
The air dried seeds were ground to coarsely powdered form (also referred to as “powder” or “powdered seeds”) with a mixture grinder.
Petroleum Ether Extraction: The petroleum ether extraction comprised the following steps:
About 580 ml of petroleum ether was added to the powder in a Soxhlet apparatus i.e. about 1:2.9 ratio of the powdered seeds to the petroleum ether;
The system was run for about 8 hours at about 55 degrees Centigrade;
After about 8 hours, the solvent was collected in the bottom flask;
The sample was filtered with Whatman filter paper;
The solvent was removed with a rotary evaporator; and
The weight of the concentrated mass was measured.
Ethanol Extraction: The ethanol extraction comprised the following steps:
About 800 ml of ethanol was added to the dry mass in the Soxhlet apparatus i.e. about 1:1.4 ratio of petroleum ether to ethanol;
The system was run for about 8 hours at about 55 degrees Centigrade;
After about 8 hours, the solvent was collected in the bottom flask;
The sample was filtered with Whatman filter paper;
The solvent was removed with a rotary evaporator; and
The weight of the concentrated mass was measured.
Chloroform Extraction: The chloroform extraction comprised the following steps:
About 850 ml of chloroform was added to a fresh powder sample in a Soxhlet apparatus i.e. about 1:4.25 ratio of the powdered seeds to the chloroform;
The system was run for about 8 hours at about 55 degrees Centigrade;
After about 8 hours, the solvent was collected in the bottom flask;
The sample was filtered with Whatman filter paper;
The solvent was removed with a rotary evaporator; and
The weight of the concentrated mass was measured.
The results (weight %) of the above extractions are illustrated in Figure 1.
Ultrasound Extraction:
Dichloromethane Extraction: The dichloromethane extraction comprised the following steps:
About 10 grams of a fresh powder sample was taken;
Ultrasound extraction was performed with about 1:70 ratio of the powdered seeds to a solvent dichloromethane for about 40 minutes;
The sample was filtered with Whatman filter paper; and
The filtrate was condensed with a rotary evaporator.
Ethanol Extraction: The ethanol extraction comprised the following steps:
About 10 grams of a fresh powder sample was taken;
Ultrasonication with ultrasonicator extractor was performed with about 200 ml of ethanol for about 45 minutes i.e. about 1:20 ratio of the powdered seeds to the ethanol;
The sample was filtered with Whatman filter paper; and
The filtrate was condensed with a rotary evaporator.
The results (weight %) of the ultrasound extractions are illustrated in Figure 2.
Microwave-Assisted Extraction:
With Water: The microwave-assisted extraction with water comprised the following steps:
About 10 grams of a fresh powder sample was mixed with about 200 ml of water i.e. about 1:20 ratio of the powdered seeds to the water;
Extraction was performed with a microwave system at about 400 W and about 2,500 MHz for about 10 minutes;
The sample was filtered with Whatman filter paper;
Water was evaporated and condensed mass was obtained; and
The weight of the crude sample was measured.
With Dichloromethane and Methanol: The microwave-assisted extraction with dichloromethane and methanol comprised the following steps:
About 20 grams of a fresh powder sample was mixed with about 300 ml of a 50:50 mixture of dichloromethane and methanol i.e. about 1:15 ratio of the powdered seeds to the dichloromethane and methanol mixture;
Extraction was performed with a microwave system at about 400 W and about 2,500 MHz for about 10 minutes;
The sample was filtered with Whatman filter paper;
Water was evaporated and condensed mass (also referred to as “crude extract”, “crude sample” or “extract”) was obtained; and
The weight of the crude sample was measured.
The results (weight %) of the microwave-assisted extractions are illustrated in Figure 3.
Maceration:
With Chloroform and Methanol: The maceration with chloroform and methanol comprised the following steps:
About 100 grams of a fresh powder sample was taken in a 250 ml beaker;
About 200 ml of solvent mixture of chloroform and methanol (50:50) was added to the sample i.e. about 1:2 ratio of the powdered seeds to the solvent mixture of chloroform and methanol;
The sample was kept for about 24 hours;
The sample was filtered with Whatman filter paper;
The filtrate was concentrated with a rotary evaporator; and
The weight of the crude sample was measured.
With Ethanol: The maceration with ethanol comprised the following steps:
About 100 grams of a fresh powder sample was taken in a 250 ml beaker;
The sample was soaked in about 200 ml of ethanol and kept for about 24 hours i.e. about 1:2 ratio of the powdered seeds to the ethanol;
The sample was filtered with Whatman filter paper;
The filtrate was concentrated with a rotary evaporator; and
The weight of the crude sample was measured.
The results (weight %) of the macerations are illustrated in Figure 4.
From Figure 1 through Figure 4, it was observed that highest yield was obtained in microwave extraction method with the mixture of dichloromethane and methanol as solvent.
Bio-Efficacy Studies of the Crude Extract:
Small samples (culture) of insects (diamondback moth in an embodiment) from the field were maintained on natural diet of cabbage in conical flask plugged with cotton. The insects were released into the flask with soft brush. The P. xylostella larvae after the third generation were collected for testing the mortality.
Six petridishes were taken with 10 larvae of Plutella xylostella in each petridish. The experiments were conducted at about 25 degrees Centigrade and about 60% R.H. Solutions of five different concentrations of crude extracts of Pachyrhizus erosus (extracted through any of the techniques as explained above; extracted with the mixture of dichloromethane and methanol as solvent in an embodiment) were made by dissolving in ethanol solvent i.e. about 0.1%, about 0.3%, about 0.5%, about 0.7%, and about 1% (i.e. final extract concentration that ranges between about 0.1% and about 1%; about 1% in an embodiment).
These solutions were sprayed on the larvae on five different petridishes with an injection syringe, and one petridish was kept untreated (control treatment). Five replications were done for each concentration. The mortality was observed at two days, four days, and seven days after treatment.
Mortality % = (Number of insects dead/ total number of insects released) x 100
Figure 5 illustrates the sample of larvae that were tested, while Figure 6 and Figure 7 illustrate the larvae after spraying with the crude extract solutions, and the dead larvae, respectively.
The results of the bio-efficacy studies of the crude extracts are illustrated in Figure 8. The values are the mean of five values (n= mean ± SE).
Synthesis of Emulsifiable Concentrates:
The synthesis of emulsifiable concentrate (EC) formulations comprised the following steps:
About 50% w/w of the crude extract (extracted through any of the techniques as explained above; extracted with the mixture of dichloromethane and methanol as solvent in an embodiment) was dissolved in different solvents (kerosene, hexane, and paraffin oil, in various embodiments) separately.
About 10% of emulsifier (single or blend; for example SM and/or PSM) was added to the extract solutions and stirred thoroughly to synthesize the EC formulations. Emulsifiers were mixed in a homogenizer at about 500 rpm and about 60 degrees Centigrade. A total of six EC formulations were developed, as illustrated in Figure 9.
Hydrophilic lipophilic balance (HLB) in Figure 9:
1. Total RHLB value = fraction % of solvent x RHLB of the solvent;
2. A = 100 (X-HLBB)/(HLBA – HLBB); and
3. B = 100 – A.
A and B are the two emulsifiers in the blend. The HLB value of A i.e. HLBA is higher than the HLB value of B i.e. HLBB. X is the total RHLB value.
Stability Study and Physico-Chemical Properties of EC Formulations:
The six different EC formulations were tested for stability study and physico-chemical properties.
Emulsion Stability Test:
The emulsion stability test comprised the following steps:
About 4 ml of each formulation was taken in a 150 ml beaker;
At about 35 degrees Centigrade, standard hard water was added at the rate of 25 ml/min to 30 ml/min;
The mixture was stirred continuously with a glass rod to obtain a diluted emulsion; and
It was transferred to a measuring cylinder and kept for about 45 minutes at about 35 degrees Centigrade.
It was found that, in the case of EC2 and EC4, two different layers were formed. The thick layer was formed at the top and the sedimentation occurred at the bottom.
Cold Test:
The cold test comprised the following steps:
About 25 ml of each formulation was taken in a stoppered glass container;
It was placed in ice-cold water at about 4 degrees Centigrade; and
It was stirred for about 45 minutes to check whether any oily layer or turbidity layer or both were formed.
Storage Stability Test:
The storage stability test comprised the following steps
Firstly, the formulations were kept at three different temperatures (about 7 degrees Centigrade, about 30 degrees Centigrade, and about 50 degrees Centigrade) for about 15 days. After about 15 days, the formulations were observed for any change in their visible properties like colour change or appearance of some separated phases or creamy layers. This test was done at three replications for each formulation.
The results of the evaluation of physical appearance, cold test, and storage stability test are illustrated in Figure 10.
Dose Calculations of the Formulations:
Before mortality studies with the synthesized formulations, their water solutions were synthesized for applying on the larvae. As in all the developed formulations viz. EC1, EC2, EC3, EC4, EC5 and EC6, the content of crude was 50% (Figure 11).
Mortality studies were performed by following the same procedure as explained earlier under “Bio-Efficacy Studies of the Crude Extract”. The results of the same are illustrated in Figure 12 (EC1), Figure 13 (EC2), Figure 14 (EC3), Figure 15 (EC4), Figure 16 (EC5), and Figure 17 (EC6). Values are the mean of five values (n= mean ± SE)
It was observed that insect mortality was highest (i.e. 86%) with 1% concentration of EC5. The mortality rate with same concentration of EC1, EC2, EC3, EC4, and EC6 were 82, 74, 80, 78, and 84, respectively.
Figure 18 illustrates the toxicity level of the formulations.
The present disclosure also discloses synthesized biological agrochemical compositions that are synthesized through the disclosed method(s).
The disclosed method(s) of synthesis and synthesized biological agrochemical compositions offer the following advantages: good insecticidal activity, nutrient effects on crops, reduced pest resistance, and low toxicity to non-target organisms.
It will be apparent to a person skilled in the art that the above description is for illustrative purposes only and should not be considered as limiting. Various modifications, additions, alterations and improvements without deviating from the spirit and the scope of the disclosure may be made by a person skilled in the art. Such modifications, additions, alterations and improvements should be construed as being within the scope of this disclosure.