A Review on Extraction, Identification and Application of Pesticidal Active Phytoderived Metabolites

 Reena Jamunkar^1,*, Deepak Sinha¹, Tarun Kumar Patle², Kamlesh Shrivas³

¹Department of Chemistry, Government Nagarjuna Post Graduate College of Science, Raipur, CG-492010, India

²Department of Chemistry, Pt. Sundarlal Sharma (Open) University, Chhattisgarh, Bilaspur-495009, India

³School of Studies in Chemistry, Pt. Ravishanakar Shukla University, Raipur-492010, CG, India

 Abstract

Bioactive compounds obtained from plants, microorganisms and minerals show some specific properties like insecticidal, herbicidal, repellent, antifeedant and toxicant activities called bio pesticide. They have specific modes of action against different pests. Due to their environmental eco-friendly nature, low cost, economic effectiveness, less pollution, and target specific quality they are in high demand in agriculture compared to chemical or synthetic pesticides. Extraction, purification, identification and characterization of these compounds from the plants materials are found always challenging. There are various types of traditional and non-traditional methods of extraction have been proposed such as maceration, distillation, ultrasonic-assisted extraction, soxhlet extraction, enzyme assisted extraction, microwave assisted extraction, accelerated solvent extraction, etc. have been reported for extraction of bioactive ingredients from plants complex matrix samples. The chromatographic separation techniques like thin layer chromatography(TLC), high performance thin layer chromatography (HPTLC), high performance liquid chromatography (HPLC) and gas chromatographic (GC) are used for their separation followed by the identification in order to determine their structure with the help of UV-Vis, fluorescence, NMR spectrometry, Fourier transforms infra-red spectrometry (FTIR) and mass spectrometry (MS). This review summarized the extraction procedure, formulation of biopesticide, structural identification and their application in agriculture.

Key words: Biopesticides, Synthetic Pesticide, Extraction, Identification, Formulation, Pesticidal Active Components.

 1. Introduction

Pesticide is a chemical compound used to control harmful pests present in the soil and plants. Based on sources pesticides are categorized into chemical or synthetic pesticides and Biopesticides. Chemical pesticides contain various chemicals and polymers that act as carriers (Rakhimol et al., 2020). These carriers are specific for different pests. They are used to control weeds as herbicides, algae as algaecides, rodents as rodenticides, insects as insecticides, nematodes as nematicides, molluscs as molluscicides, termites as termiticides, mites as miticides, ticks as acaricides, fungi as fungicides, bacteria as bactericides etc.(Farooq et al., 2019). Synthetic pesticides are also classified based on active components present in them such as dichlorvos, organochlorines, diazinons, chlorpyrifos, diamides, carbamates etc. (Decool et al., 2024).  Herbicides are used to control weeds to facilitate crop management by preventing their growth and increasing crop yields and commerciality (Thomson et al.,2016 ). There are some herbicides such as bipyridyl, phosphomethyl, amino acids, chloroacetanilides, chlorophenoxy compounds etc. that act as active ingredients to eliminate the harmful targeted weeds (Ayilara et al., 2023). Fungicides are used to protect plants against diseases caused by fungi by incapacitating or killing them. There are some chemicals used as fungicides such as phthalamides, dithiocarbamates, hexachlorobenzene, pentachlorophenol etc.(Ullah et al., 2019). It is reported that dithiocarbamates and phthalamides are less phytotoxic, more active and easier to prepare than other fungicides(Kumar et al., 2015). Insecticides are active ingredients used to kill harmful insects and are usually used in agriculture, industries and medicines (Abdollahdokht et al., 2022). DDT was the most common insecticide produced during the Second World War (Garces et al., 2020). Some chemicals such as anticholinesterases, avermectins, organochlorine, pyrethroids and pesticidal active compounds isolated from  plants like azadirachtin have also been reported as insecticides (Abdollahdokht et al., 2022). Algaecides are used to eradicate algae from different surfaces. Fumigants such as phosphine, ethylene bromide show broad spectrum activity against bacteria, fungi, insects etc.(Dogara et al., 2022). Zinc phosphide, fluoroacetate derivatives, alpha naphthyl thiourea, and anticoagulants act as rodenticides to control rodents such as rats, mice, squirrels, chipmunks, woodchucks, nutria etc. Although these rodents play important roles in nature they can damage crops, transmit diseases and be accountable for ecological damage (Pathak et al., 2022). Organochlorines are chemical pesticides used to kill silkworms and armyworms by altering the electrophysiological properties and enzymatic properties of nerve cells (anikwe et al., 2021). Diazinon is used to control Bactrocera invadens by inhibiting the enzymatic acetylcholine sterase that is responsible for hydrolyzing the neurotransmitter acetylcholine in cholinergic synapses (Kumar et al., 2021). Urea derivatives interfere with the deposition, synthesis and polymerization of chitin in dicotyledonous weeds and broom corn cereals (Liu et al., 2017). Carbamic and thiocarbamide derivatives inhibit the choline sterases enzymes in thrips (Frankliniella sp.) (Gupta et al., 2011). Diamide misregulates the ryanodine receptor in Spodoptera exigua insects and mosquitoes (Teixeira et al., 2013). Although pesticides increase crop production by killing harmful pests, improper use of them in agriculture leads to changes in antioxidant levels and oxidative enzymes in human beings resulting in various diseases caused by oxidative stress(Troczka et al., 2017). Chemical pesticides face many drawbacks such as persistence in the soil, impact on living beings (humans, animals, birds etc.) and environment, pest resistance, cost of purchase and production, discarding of contaminated crops etc. that affect the organic farms(Laxmishree et al., 2017). When chemical pesticides are used on a large scale on the soil for agricultural purposes, they remain non-degradable. Because of this, they persist in the environment for a longer time and leach to the surfaces and underground water, resulting in loss of biodiversity and pollution (Sharma et al., 2017). When chemical pesticides are applied on soil most of the affected organisms are non-targeted. Reports show that organophosphate and carbamate pesticides negatively affect nutrients present in soil by chelating with some important metal ions and making them unavailable for plant intake (Aktar et al., 2009). As well as plant reproduction, seed production, photosynthesis phenomenon are adversely affected by chemical pesticides(Pathak et al., 2022). Residues of chemical pesticides that remain in food and crops are biomagnified in humans through food (fish, grains, vegetables etc.), drinking water, pores of the skin (during pesticide spray), post harvested crop preservation and breathing causes severe diseases such as Parkinson’s disease, cancer, eye irritation, kidney diseases, diabetes, hypertension, cardiovascular diseases, skin diseases, liver dysfunction etc. (Pathak et al., 2022). High levels of pesticides i.e. 25-30% can lead to an increase in mental problems and 50% cause brain cancer, leukaemia (Nicolopoulou-Stamati et al., 2016).The continuous use of synthetic pesticides causes loss of productivity of soil and quality of crop products. Since synthetic pesticides directly kill the pests or deactivate them however they are also accountable for soil pollution, loss of agricultural productivity(Rani et al., 2021). Harmful effects of various chemical pesticides on human health are summarised in table 1.

Table 1.  Harmful effect of chemical pesticides on human health

Biopesticides are obtained from natural origins like plants, animals, microorganisms (bacteria, viruses, fungi, protozoa, etc.) and also certain minerals (Copping et al., 2000, Moshi et al., 2017). Biopesticides are very specific and very small quantities of them are sufficient to inactivate the pests and cause less pollution (Mazid et al., 2011, Arora et al. 2012) compared to synthetic pesticides. Different parts of plants such as leaves, barks, flowers, seeds, roots, fruits etc. contain bioactive components which show diverse pesticidal activity like insecticidal, antifeedant, repellent, toxicant, etc (Singh et al., 2002, Tripathi et al., 2016). Nowadays an appropriate technique is required for controlling pests to minimize the environmental impacts. To decrease the dangerous effect of synthetic pesticides, botanical insecticides have proven one of the potential alternatives (Prakash and Rao, 1997).Biopesticides are comparatively safer to control natural enemies (Santoso et al. 2006).There are many advantages of biopesticides, they are target specific and ineffective to the beneficial insects, eco-friendly, biodegradable, easily decompose into small residues and do not show any negative impact on water resources, have high performance, show less poisonous effect, less toxic compared to chemical pesticides and they create difficulty for the insect to develop resistance (Salma et al., 2011).

For this many extraction techniques have been proposed over the years like soxhlet extraction, distillation, ultrasound-assisted extraction (UAE), enzyme-assisted extraction (EAE), maceration, microwave assisted extraction (MAE), cold pressing method, super critical fluid (SCF) extraction, accelerated solvent extraction (ASE) etc. (Mossa, 2016, Montanez et al., 2014, Coloma et al.,2019, Dong et al., 2010)

The chromatographic separation and identifications of pesticidal active compounds are carried out by using different techniques such as thin layer chromatography ( TLC), high performance thin layer chromatography( HPTLC), high performance liquid chromatography(HPLC), gas chromatography(GC) and structural identification is carried out by using UV-Vis spectrophotometry, Fourier transform infrared(FTIR),nuclear magnetic resonance (NMR) spectrometry, mass spectrometry(MS).Recently, the research and developments are putting more attention on the preparation of sustainable chemical substances for commercial and industrial purposes to explore biopesticidal properties to mankind.

2. Classification of Biopesticides

Biopesticides can be divided into three categories Microbial pesticides, Biochemical/ herbal pesticides and Plant-Incorporated Protectants (PIP)(Vinod kumar, 2015,  Nawaz et al., 2016 ). Recently nanobiopesticides have also been reported by many researchers.

2.1. Microbial pesticides

Microbial pesticides are produced by several microorganisms such as bacteria, viruses, algae, certain types of fungi and protozoans. Each type of microbial pesticide controls specific pests because they are target specific (Vinod kumar, 2015, Vitekari et al., 2012).For example, Trichoderma (A fungal antagonist) grow into disease causing fungal tissues and secrets various enzymes to disrupt the cell wall of that microbes and inactivate their activities (USEPA, 2008, Anonymous, 2009, Kawalekar, 2003, Vinod kumar, 2015). Baculoviruses are viral biopesticides that attack on insects and other plant pests (Vinod kumar, 2015).Nematodes are very small worms having parasitic activity therefore they are used as an insecticide (USEPA, 2008, Karen et al., 2009).

2.2. Biochemical pesticides                                                                                            

Biochemical/herbal  pesticides are the biopesticides naturally occurring in the environment such as plant(botanical)  extracts containing some active chemical constituents  having the capacity to trap or inactivate insect and insect pheromones which interfere with their mating (USEPA, 2008, Karen et al., 2009,  Vinod kumar, 2015,  Vitekari et al., 2012).There are varieties of plants and their bioactive components have been reported over the year like azadirachtin from the neem tree(Azadirachta indica) (Boadu et al., 2011,  soni et al. 2011,  khanam et al.,2017), oleandrin and cardenolides from Nerium oleander (Praveen et al., 2012), karanjin from Pongamia pinnata  Seed oil Katekhaye et al., 2011), alkaloids from Datura metel (Kuganathan et al., 2010) showed pesticidal activity. In the past 30 years, approx. 1500 species of plants have been reported to have insect control properties (Arnason et al., 1989, Grainge &Ahmed, 1980), Jacobson, 1990, Hedin et at., 1997, Prakash and Rao, 1997). However, there are still many plant species that have not been studied yet for their pesticidal activity.

2.3. Plant incorporated protectants (PIP)

PIPs are the genes and proteins that are introduced into a plant by some specific method known as genetic engineering that allows this genetically modified plant to protect itself from particular pests like insects, bacteria, viruses, fungi etc. (Hirashima, 2008, salma et al., 2011, Vitekari et al. 2012, Agbo et al. 2016). Production of  transgenic plants which were derived from Bacillus thuringiensis (Bt), first commercialized in the United States (Agbo et al., 2016), But incorporated plants have been used against caterpillars, corn rootworms and arbuscular mycorrhizal fungus, (Baker et al., 1991, Larraur et al., 1996, Sundararaj et al., 2004).

2.4. Nanobiopesticides

These are the engineered nanoparticles made by mixing biopesticide or natural origin like microbes, green extracts, essential oils with nanoparticles. There are various formulation methods by which nanopesticides are formed such as nano-emulsions, nano- vesicles, nano-fibres, nano-encaptulations etc. which are used to improve the efficiency of developed nanobiopesticides. Nanopesticide formulation has considerably attracted researchers in recent years. Different pesticides with their examples are shown in Fig 1.

                                         Figure 1. Classification of pesticides

3.Methods for extraction of biopesticide compounds

Biopesticides are found in many parts of a plant such as leaves, bark, seeds, flowers, fruits etc. Generally, seeds, flowers and fruits contain some essential oils that show pesticidal activity (Mossa, 2016), so it is necessary to extract these bioactive chemical constituents from these plant’s parts before the analysis or applying them to particular pests. There are many extraction procedures described as below:

3.1. Maceration

Maceration is the simplest technique for the extraction of biopesticide from plant material. In this process suitable amount of plant material is taken in a closed container with the appropriate amount of solvents like hexane, ethanol, methanol, chloroform etc. depending on the plant materials. This sample is allowed to stand at room temperature for some time about 3 days with occasional shaking to dissolve the sample completely in the solvent. Now the obtained mixture is filtered and filtrate is used for applications(Jha et al., 2022). (Jadeja et al., 2011)reported maceration extraction for natural insecticide azadirachtin from azadirachta indica seed kernels. Montanez et al., (2014) reported a cold maceration procedure by taking freeze dried sample with the appropriate amount of solvent (1:5 w/v %) in a 250 mL Erlenmayer flask and this solution was kept in an incubator shaker for 30h at 20^oC.Some plant materials are macerated in warm water before the release of essential oil, i.e. leaves of wintergreen which contain precursors like gaultherin and enzymes like primeverosidase. When they are macerated in warm water the enzyme acts on the precursor and produces methyl salicylate.

3.2. Distillation method of extraction

It is a very common method of extraction of essential oil pesticides in which a cleavenger apparatus is used (Mossa, 2016).Three distillation methods are used for the extraction of biopesticides. Water distillation: In this method plant material is boiled by applying heat by direct fire, closed steam jacket, closed steam coil or open steam coil. This method is used for the extraction of biopesticide from dried plant material. In this process, there is direct contact between boiling water and plant materials( Fig.2).Water and steam distillation: This method is used for the extraction of both fresh and dried plant material (Mossa, 2016). Direct steam distillation: This method is used for the extraction of fresh plant material (Mossa, 2016) where the plant part is used to extract essential oil placed in a glass column which contains the appropriate amount of solvent and connected with water bath and condenser. After condensation, the essential oil is separated from water by decantation (Boutekedjiret et al., 2003).

3.3. Solid phase extraction method

QuEChERS( Quick, Easy, Cheap, Effective, Rugged and Safe) is a common example of a solid phase extraction method that  is used for the extraction of biopesticides from some plants such as cucumber, tobacco, etc. and also used for detection of  pesticide residue in the food. In this method, the sample to be analysed is firstly homogenized using a homogenization instrument and centrifuged with an appropriate reagent. The reagent used for centrifugation depends on the type of sample to be analysed. The sample is agitated with the appropriate reagent before centrifugation. After centrifugation supernatant is filtered by using suitable filter paper and filtrate is stored for further analysis(Silva et al., 2020). (Prestes et al., 2011)used this extraction method for the separation and determination of biopesticides in soil samples by taking samples in a 50mL propylene centrifuge tube by using different chemicals (acetic acid in acetonitrile, anhydrous magnesium sulphate, NaCl, sodium citrate  and sodium citrate dehydrate) at appropriate amount and supernatant was filtered.  Coloma et al.,(2011) reported this extraction procedure for the extraction of pesticide from cucumber and red wine samples by using acetate buffer and the same extraction procedure was carried out for wheat samples.

3.4. Ultrasound/Ultra sonication assisted extraction

This is an inexpensive, simple, and efficient technique compared to the traditional technique. This is a commonly used solid-liquid extraction technique in which plant material is used with an appropriate liquid solvent. The advantages of this technique are that it follows fast reaction kinetics and produces a high extraction yield (Dong et al., 2010). In this method plant material to be extracted is taken in a sonicator reactor with suitable solvent. When sonication is introduced into the liquid at high intensity the sound wave passed through the liquid result the generation of alternating high and low pressure cycles. In the low pressure cycle, the ultra- sonic waves create small bubbles in the solution. Now in the high pressure cycle, these bubble reaches the specific volume point where they cannot absorb energy, at this point, they gently collapses with each other. These processes are carried out at high temperature (approx. 5000k) and high pressure (approx. 2000atm). Due to these high forces cell walls are ruptured and intracellular material is extracted (Suslick, 1998).This extract is filtered and used for further analysis The special equipment for high performance sonicators is the Hielscher probe ultra sonicator UP 200s (200w, 24kHz) in which the sample is immersed in a temperature controlled water bath (Rojas et al., 2019) Fig.2.

         Figure 2.  Schematic representation of Ultra sonication assisted extraction

This  is the best method for the quantitative extraction of capsaicinoids from chilli pepper in the presence of methanol as a solvent (Sganzeria et al. 2014). For the extraction of caffeine from coffee this is a very suitable method (Wang et al., 2011). Prestes et al., (2011) reported an ultrasonication assisted method for the extraction of biopesticides  from soil samples by using ethyl acetate/ methanol(3:1 v/v) as a solvent. This sample was sonicated for a certain period of time and then applied for centrifugation (5 min, 5000 rpm, 4136xg). The supernatant was concentrated by evaporation and again dissolved in the appropriate amount of ammonium formate solution/methanol (1:1 v/v). Xia  et al.(2011) used ultra sound assisted extraction of Phillyrin from Forsythia suspense. Xia et al., (2012) reported ultra sound assisted extraction of oxymatrine from Sophora flavescens.

3.5. Soxhlet extraction

This extraction technique was developed by Soxhlet in 1879. The Soxhlet extraction method is a standard and commonly used laboratory method of extraction of biopesticide or bioactive compounds from the plant material and is based upon solid–liquid extraction. The Soxhlet extractor consists of thimble, solvent chamber, distillation chamber and siphon(Fig.3). In this process, the sample is placed in a thimble which is designed in such a way that it holds the solid particle but allows the liquid to pass through them. This thimble is placed in a Soxhlet extractor which contains the appropriate extraction solvent. Now this solvent is heated and refluxed result of which is the production of vapour which is condensed by the condenser. This condensed solvent fills up the thimble, when the thimble is filled with solvent it automatically returns into the solvent chamber. This process is repeated again and again until the chemical compound is completely extracted into the organic solvent(Alara et al., 2018). This method is generally used when the compound has limited solubility in the solvent (Wang et al., 2006).

Figure 3.  Schematic representation of Soxhlet extraction

Bakavathiappan et al., (2012) reported the extraction of biopesticide from Calotropis procera leaves by soxhletion using the powdered sample with hexane, chloroform, ethyl acetate, methanol, acetone and ethanol solvent. Sharma and Gupta, (2009) used this method for the extraction of biopesticides from different plants like neem, wild sage, caturangi, kaner, bhang, castor, makai, saffeda (blue gum). Bobade  et al.,(2012) used this method for the extraction of Pongamia pinnata ( Karanja) plant’s material using hexane solvent. Adesina et al., (2015) reported this extraction procedure for Secamone afzelii leaf by using ethanol and n-hexane as a solvent. Soni et al., (2011) reported this method for extraction of Azadirachtin from azadirachta indica leaves powder using ethanol as a solvent and this solvent was recovered by distillation. Kuganathan et al., (2010) reported this method for biopesticide extraction from Datura metel leaves by using chloroform as a solvent and this solvent was further evaporated.

3.6. Supercritical fluid extraction (SFE)

This is the method of separating one component from another matrix using supercritical fluid as an extracting solvent. This extraction is usually carried out from a solid matrix but sometimes a liquid matrix can also be used as an extraction matrix (Wrona et al., 2017). Generally, CO₂ is used as a supercritical fluid but sometimes ethanol and methanol are used as a co-solvent. The extraction conditions for super critical CO₂ the temperature should be above the critical temperature of 31⁰C and critical pressure of 74 bar (Coloma et al., 2012). The supercritical fluid extractor system consists of a pump for CO₂, a pressure cell that contains samples with a special technique to maintain the pressure in the system and a collecting vessel. At first, the supercritical fluid is pumped to the heating zone where it is heated at a critical condition. After, it is passed to the extraction vessel, where it diffuses into the solid matrix (sample) and dissolves the material to be extracted. Now this dissolved material is passed through a separator at low pressure due to which extracted material is settled down. The CO₂ is cooled, recycled, or discharged to the atmosphere and the extracted material is collected into the collecting vessel (Wrona et al., 2017). (Gonzalez et al., 1990, 1992, Fraga and Terrero, 1990, Coloma et al. 2012) reported SFE in Persea indica plant’s material which shows insecticidal properties and contains Rganodane diterpenes and alkylY– lactones. The extraction is carried out in the plant’s leaves and stems in the presence of ethanol as a co-solvent. Lucia Baldino  reported SFE using CO₂ for extraction of rotenoid from Derris elliptica root by using a homemade laboratory apparatus equipped with a 200mL internal volume extractor. In this procedure the appropriate amount of plant material is taken with a fixed amount of 3mm glass beads to avoid caking and channelling of the particle which formed during the extraction. Johnson et al., (1997) reported SFE of oil and triterpenoids from neem seeds using a back pressure regulator. Ambrosino et al., (1999) reported extraction of azadirachtin- A from neem seed kernels by SFE and its evolution by HPLC and LC/MS.

3.7. Enzyme assisted extraction(EAE)

This is a novel, green, safer, eco-friendly and effective alternative extraction method compared to traditional methods and used to extract bioactive components from plant material. Since enzymes are a form of protein molecule present in living things and show specific catalytic activity, this concept is used in the extraction of bioactive substances from plant material where the concentration of enzymes remains unchanged (Puri et al., 2012). Enzymes are very sensitive to the pH value, temperature, time of incubation, the concentration of substrate and enzymes (Baby et al., 2013). In the extraction procedure, the plant material to be extracted is taken in a flask with the appropriate solvent and add suitable enzyme at the optimized condition of temperature, pH, after hydrolysis and centrifugation solution is filtered using Whatmann filter paper No. 1 (Munishpuri et al., 2012). Since different plants contain different types of chemical constituents that show different activities like insecticidal, repellent, fungicidal etc towards pests so appropriate enzymes and optimized operational conditions should be used for the selected plant materials during the extraction process. Wang et al., (2017) reported ultrasound assisted enzymatic hydrolysis extraction of Quinolizidine alkaloids from Sophora alopecuroides  seed by using different amount of cellulase as an enzyme at optimized operational conditions i.e. pH = 5, temperature = 50^oc, incubation time = 45 min, solvent to solvent ratio = 100ml/g. Sowbhagya et al., (2008) reported the effect of enzyme assisted extraction to determine the quality of garlic volatile oil and found that in the presence of cellulase, pectinase, protease and visozyme, the yield of the product was increased  twofold. Primo et al., (2018) reported enzyme assisted extraction of neem plant material into two steps first was the determination of optimal condition for enzymes cellulase 17600L, crystalzyme cran, crystalzyme 100XL and crystalzyme PML-MX   and second was the evolution of the Azadirachtin release kinetic under determined optimal condition. They found that cellulase 17600L, crystalzyme cran, and crystalzyme 100XL had showed higher activity at 50^oc while crystalzyme PML-MX shown higher activity at 45^oc  similarly optimal pH value  was found as pH = 5  for crystalzyme cran and pH = 4.5 for cellulose, crystalzyme  PML-MX and crystalzyme 100XL.

3.8. Microwave assisted extraction (MAE)

This is the recently used extraction method and is generally used for the extraction of biopesticides and other bioactive component. In this process, microwave energy is used to heat the solvent which is in contact with the sample result of which is the disruption of the sample into the analyte take place. After extraction analyte is found in the solvent which is used for extraction. This extraction process is carried out by different mechanisms i.e. in the first case sample is taken with a single solvent. In the second case sample is extracted in a combined solvent i.e. solvent having low and high electric losses. In the third case sample having high dielectric loss is extracted with a microwave transparent solvent (Jassie et al., 1997, C.  Eskilsson et al., 2000). A commercially used MAE system is a closed vessel consisting of a magnetic stirrer, an oven containing an extraction vessel, monitoring devices for controlling temperature and pressure and many electronic components (Eskilsson et al., 2000). In the extraction procedure, firstly sample is loaded into the extraction vessel by adding the appropriate solvent. After closing the vessel microwave radiation is applied to heat the solvent at a time less than 2 min, this is called the pre-extraction step. Again sample is irradiated and extracted for 10-30 min, this is called the static extraction step. When the extraction is completed the sample is allowed to cool down and after filtration the extract is used for further analysis (Eskilsson et al., 2000). Kullu et al.,(2014)  reported MAE for mangiferin from C. amada with 550w power of microwave radiation for extraction at a time period of 30 min.  Xia et al., (2011a) used MAE for the extraction of oxymatrine from Sophora flavescence by using X-100A microwave extraction device at 50^oc for 50 min. under 500w microwave radiation.  Xia et al.,(2011b) reported microwave assisted extraction for oleanolic acid and ursolic acid from Ligusrum lucidum at same condition as above. Microwaves have the property of penetrating biomolecules and interfering with the polar molecule to generate heat (Naboulsi et al., 2018).

3.9. Accelerated solvent extraction

This is a new technique of extraction of organic compounds from solid and semisolid samples with liquid solvents. This technique can replace the Soxhlet, sonication and other extraction methods and requires a small amount of solvent and a short time (Gan et al., 1999). This is also known as pressurized solvent extraction (PSE), high pressure solvent extraction( HPSE), high pressure high temperature solvent extraction (HPHTSE), pressurized hot solvent extraction( PHSE), pressurized hot water extraction (PHWE)and sub critical solvent extraction (SCSE) (Ritcher et al., 2015). Accelerated solvent extraction is carried out at elevated temperature and pressure with an appropriate amount of liquid solvent. In accelerated solvent extraction the ASE-300 dionex system is commonly used. At first appropriate amount of suitable organic solvent is filled in the solvent chamber at elevated temperature and pressure. This system consists of a hydrocarbon gas sensor that checks the solvent leakages in order to provide great care from the solvent spray. Now sample is filled in the extraction cell, when the cell is filled and tightened this is kept in the oven and heated at 30- 200^oc for 12- 15 min. This system also contains a static valve that is open during preheat time. In the heating up procedure pump valve is opened and the solvent is mixed into the sample, at this time static valve is closed and in the extraction cell extraction procedure is started at elevated pressure. Now static valve is reopened and fresh solvent is mixed into the sample to flush the cell and finally the extract is collected in the collection vial (Richer., 2015). Giergielwicz et al., (2001) reported ASE for the analysis of environmental solid samples. Rahmalia et al., (2015) reported extraction of Bixin from Bixaorellana by using cyclo hexane – acetone solution (6:4 v/v) at 50^oc with a time of 5 min. and got the highest yield of 68.16 . Praveen et al., (2012) used the ASE method for the extraction of cardenolides from Nerium oleander by using chloroform as a solvent at pressure = 150 psi, heat up time = 5 min., static time = 10 min., flush volume = 60% , purge time = 100 sec and static cycle = 2.Grimalt et al., (2011) reported the ASE method for the extraction of azadirachtin and 3- tigloylazadirachtol from hardwood tree species like green ash (Fraxinus pennsylvanica), white ash(Fraxinus americana), London plane tree (Plantanus acerifolia) etc. by using acetonitrile as an extraction solvent at the pressure of 2000psi and 5 cycle with a static time of 2 min at room temperature. Yunet al., (2006) reported ASE or pressurized liquid extraction  of rotenone from Derris elliptica and Derris Malaccensis using Dionex ASE-200 Accelerated solvent extractor by taking CHCl₃ and 95% EtOH as a solvent and varying temperature and pressure ranges, with a static time of 6 min. per 1 cycle.

4. Formulation of Biopesticides

In simple words, formulation is a form of a specific product that we use for controlling pests (Rasheed et al., 2017). Since all biopesticides contain some active constituents that show pesticidal activity and active ingredients in their raw or unformulated condition are not suitable for pest control, after extraction of this bioactive constituent from their parent plant’s material or microorganism, they are formulated or converted into usable form by mixing them with other ingredients at the appropriate amount (Herzfeld et al., 2011,  Rasheed et al., 2017). The purpose of the formulation is to explain the difference between an active ingredient and a formulated biopesticide and to identify the strengths and weaknesses of particular biopesticides (Herzfeld et al., 2011). A biopesticide formulation consists of an active ingredient and an inactive ingredient (adjuvant or additive) and the function of inactive ingredients is to increase the effectiveness of the active ingredients (Rasheed et al., 2017). Some ideal characteristics of formulated biopesticide should be maintained while the formulation is chosen such as it should not be toxic to the crop plant and only effective against the pest; it should tolerate adverse environmental conditions; should be cost-effective and dissolve well in solvent (water); handling and mixing should be easy (kumar et al., 2014, Rasheed et al., 2017). There are various biopesticide formulations have been reported and described below:

4.1.Dust formulation

It is prepared by mixing the fine powder mixture of active constituents (low concentration of 10% or less by weight) with solid mineral or inert powder of clay, talc, chalk, volcanic ash, etc. (Gasic et al., 2013, Rasheed  et al., 2017). Dust is generally used in dry form and should never be mixed with water. This formulation is commonly used for the treatment of the surface part of the crop like seed dressing or internal wall void and they control cockroaches, insects, and sometimes lice, fleas, and other pests (Rasheed et al., 2017). They cause’s creation of some health issues like irritation of eyes, nose, skin, etc. take place (Knowles, 2001, Herzfeld et al., 2011,  Gasic et al., 2013).

4.2. Granules formulation

It is similar to dust formulation but the difference is that the particle size of granules is larger (size range-100-1000 microns for granules and 100-600 microns for micro granules) and heavier and the concentration of active ingredients is 5-20% (Gasic et al., 2013, Rasheed et al., 2017). The coarse particles are generally made from clay, corncobs, walnut shells, kaolin, attapulgite, silica, starch, polymers, dry fertilizers, and ground plant residues (Tadros, 2005, Gasic et al., 2013, Rasheed et al., 2017). This is generally used to control weeds, nematodes and insects living in the soil.

4.3. Wettable powder formulation

Wettable powder are dried, finely ground formulation like dust but the difference is that in this formulation appropriate amount of water is added to maximize the effectiveness of the formulation. It is produced by blending the active constituent with a surfactant, wetting and dispersing agent and inert filler and followed by grinding to an appropriate particle size of about 5 microns (Gasic et al., 2013). It can be used to control most of the pest problems and has the best residual activity due to their specific physical properties and most of the pesticide remains on the surface of concrete, plaster and untreated wood (Gasic et al., 2013, Rasheed et al., 2017).

4.4. Emulsion formulation

When one liquid is dispersed in the form of droplet in another liquid, the formation of emulsion take place. In this formulation the active ingredients is dissolved in an oil based solvent and when this product is mixed with water in the presence of emulsifying agent or emulsifier, an oil in water type emulsion is formed. The emulsifier helps in the prevention of emulsion from separating in dispersed phase and dispersion medium (Gasic et al., 2013,  Rasheedet al., 2017). Since lower shelf stability may affect the performance of emulsion formulation. Recently research work are being conducted to investigate the different types of oils and emulsifier to improve the emulsion formulation for biopesticide (Verner, 2007,  Gasic et al., 2013).

4.5. Invert emulsion formulation

This formulation is opposite to the emulsion formulation. In this method, the water-soluble active ingredient (biopesticidal component) acts as a dispersed phase and is mixed with the oil (dispersion medium). This formulation needs a special type of emulsifier which allows the pesticide to be mixed with a large volume of oil (generally fuel oil) (Bharti et al., 2020). The advantage of this formulation is the use of oil as a dispersion medium which evaporates more slowly than water resulting in the emulsion droplets shrinking less compared to another method of formulation, therefore, more pesticide reaches the target (Rasheed et al., 2017). This formulation is generally used in sensitive areas where drift to susceptible non-target plants (Herzfeld et al., 2011).

5.  Identification and Characterization of bioactive compound from biopesticidal extract

Since plant extracts usually contain various types of bioactive compounds having different properties like medicinal, pesticidal, etc. and having different polarities their isolation is still a big challenge for the process of identification and characterization of bioactive constituents. There are different types of isolation techniques have been proposed for the isolation of these bioactive constituents chromatographic techniques such as thin layer chromatography (TLC), high-performance liquid chromatography (HPLC), high performance thin layer chromatography (HPTLC) and then identification by instrumental techniques like Fourier- transform infrared spectrometry (FTIR), nuclear magnetic resonance spectrometry (NMR), gas chromatography (GC), gas chromatography-mass spectrometry (GC-MS)etc. With the help of chromatographic techniques, the bioactive compounds are separated into the pure form followed by the structural analysis of the structure of unknown compounds and further used for the determination of their structure and their biological activity (Sasidharan et al., 2010).

5.1. Thin layer chromatography (TLC)

TLC is a commonly used, simple, rapid, and inexpensive, procedure for the isolation and identification of an active compound present in the extract by comparing the R_f of an unknown compound present in the extract with the R_f of a known compound (Sasidharan et al., 2010). The R_f value of the compound to be isolated or identified can be calculated by using the following formula:

 Recently, the bio-autography technique has also been used for the detection of antimicrobial compounds of extract which is separated on a TLC layer. This process is carried out in three ways: (i) Direct bioautography in which micro-organism is grown directly on the thin layer chromatographic plate; (ii) Contact bioautography in which antimicrobial compounds are transferred from the TLC plate to the inoculated agar plate directly; (iii) Agar overlay bioautography in which seeded agar medium is directly applied on TLC plate (Hamburger and Cordell, 1987, Sasidharan et al., 2010). In the process of TLC the analytical plate (thickness 1mm) is coated by silica gel and plant extract to be isolated or identified is applied on this plate with the help of a capillary tube and a suitable mobile phase is used for the development of a TLC compartment. Finally, after drying, appropriate phytochemical screening reagents are sprayed or by observing the plate under UV light to identify the phytochemicals present in the plant extract (Kagan et al., 2014). (Sasidharan et al., 2010, Soni et al., 2012) reported the qualitative analysis of azadirachtin using the TLC method by using silica gel coated F254 aluminum plates and different solvent systems at different ratios like diethyl ether: acetone( 2:1), isopropanol: n-hexane ( 11:9), diethyl ether: alcohol ( 99:1)and by comparing Rf value of the sample with standard, azadirachtin was identified. Katekhaye et al., (2011) reported the isolation and identification of karanjin from Pongamia pinnata seed oil using the TLC method which is carried on a 0.20 mm silica gel 60 (E Merck) aluminum plate and is visualized under UV light at 254nm. Zahari et al., (2018) reported the isolation of an antifungal compound from Catharanthus roseus L.( pink) by using a TLC plate (silica gel F254, layer thickness of 0.28mm) by using hexane and ethyl acetate with different ratios and plate was observed under UV absorbent at 360 nm. Chen et al., (2016), reported the identification and characterization of biopesticide from Acorus tatarinawii and A. calamus by using TLC and analysis of the extract of Acorus essential oil was performed with hexane and diethyl ether as a solvent and visualized by spraying vanillin sulphuric acid reagent followed by heating. Sathiyamorthy et al., (1996) reported the preparation of cyanobacterial peptide toxin as a biopesticide against cotton pest and identified by TLC using methane: chloroform (3:1)as an eluting solvent which is characterized by UV spectral analysis. Bock et al.,(2013) reported the identification of the antifungal compound trans-cinnamic acid which is produced by photorhabdus luminescens (bacteria) as a potential biopesticide against pecan scab by using bioautography technique and this biopesticide was applied on colletotrichum spp. (Fungus)Which acted as a test organism to identify the antifungal activity.

5.2. High performance liquid chromatography (HPLC)

HPLC is a versatile and commonly used technique for the isolation and identification of bioactive pesticidal components from plant extract (Cannell, 1998, Sasidharan et al., 2010). Since bioactive constituents are generally present in minor quantities HPLC is ideally capable to identify or isolate such constituents from the multi-compound extract. HPLC instruments generally consist of a solvent delivery pump, a sample introduction device like an autosampler or manual injection valve, an analytical column, a guard column, a detector and a recorder or printer (Sasidharan et al., 2010). In HPLC, column plays a very important role in the separation of different biopesticidal or bioactive component because it contains stationary phase which acts as a bad of polar or non-polar particle according to the type of column i.e. these polar and non-polar columns are used based on the nature the sample to be identified or analyzed. Pumps are used to pump the mobile phase into the system and the injector introduces the sample into the mobile phase (a carrier) in the whole process.  In the procedure of chromatography plant extract to be analyzed is entered into the column, this column separates the bioactive constituents based on their polarity i.e. if the stationary phase is non-polar then it attracts the non-polar compound result of which remaining polar compounds elute first then non-polar compound is eluted or vice - versa. Now detector is used to determine the separated compounds by UV-absorption which depends on the concentration of the compound present in the mobile phase (Bonta et al., 2017). Katekhaye et al., (2011) reported the isolation and identification of karanjin from Pongamia pinnata by using HPLC method which was performed on Jasco system by using 250mm x4.6 mm i.d., RP-18 column (particle size = 5 microns), an intelligent pump ( PU-1580, PU-2080), a high pressure mixer ( MX- 2080-31), a manual sample injection valve, injection volume loop 20microlitre. Soni et al., (2012) reported quantitative estimation of azadirachtin by HPLC which was performed using a LC-100 cyberlabTM, salo Torrace, Millburry MAO1527 system with LC-UV-100 UV detector and acetonitrile and water as a solvent. HPLC combined with a mass spectrometry detector has been used by researchers to study the azadirachtin content of neem oil which was extracted from insecticidal formulation (Ambrosino et al., 1999, Barrek et al., 2004, khanam et al., 2017). Coloma et al., (2012) reported supercritical extraction of Persea indica where the composition of the extract was analyzed by HPLC-MS on a finnigan surveyor pump with a quaternary gradient system coupled to a finnigan LCQ deca ion trap mass spectrometer using an ESI interface and found that this SFE extracts contain rynodol, cinnzeyladol and alkyl Y- lactone as a major component. Chen et al., (2016) reported identification and characterization of biopesticide from Acorus calamus and Acorus tatarinowii by using the HPCL method which was performed using the aligent 1100 system. Tanaje et al. (2017) reported the analysis of the concentration of azadirachtin in powder Neem formulation which was determined by the HPLC method by using a C-18 analytical column with water acetonitrile mixture (65:35) as a mobile phase (flow rate 1ml/min; UV detector at 214nm). (Deota et al., 2007, Ambrosino et al., 1999) reported the estimation and isolation of Azadirachtin-A from neem seed kernels using HPLC.

5.3. High performance Thin-layer chromatoghraphy (HPTLC)

The working principle of HPTLC is similar to TLC and it is a popular quantitative analysis method because of its visual chromatogram simplicity, multiple sample handling, low running and maintenance cost, disposable layer etc. It needs to be applied, Layers of HPTLC are available in the form of pre-coats silica gel of very fine particle size is widely used as an adsorbent (Karthika et al., 2015). Sethi (1996). Patel et al., (2010) developed a simple, sensitive and selective HPTLC method on silica gel 60 F245 layers by using methanol-ethyl acetate (8.0/2.0, v/v)as a mobile phase. Praveenet al.,(2012) reported the residue of cardenolides (biopesticide) of Nerium oleander by HPTLC in an autopsy sample which was performed on 20*10 cm aluminum foil HTPLC plates coated with silica gel 60 F245(Merck) by using chloroform: acetone: acetic acid (8.5:1:0.5v/v)as a mobile phase.

5.4. UV-Vis spectrometry

UV-Vis spectroscopy is a type of absorption spectroscopy in which light from the ultraviolet region (200-400nm) and visible region (400-800) are absorbed by the molecule which causes  excitation of electrons from the ground state to a higher state take place. This spectrometry is used to detect the various functional groups in the bioactive compound. It is also used to identify the unknown biopesticide by comparing the spectrum of the unknown compound with the spectrum of the reference compound and if both spectrum coincides then it confirms the identification of the unknown component(Zavoi et al., 2011). Akamal et al.,(2018) reported the characterization of biopesticide compounds from bacillus subtilis AAF2 ( bacterial stain which was developed as a biopesticide producer) fermented product where after the isolation of bioactive compound by TLC, The isolated compound was analyzed by UV-Vis. spectrometry which was performed to obtained by using a UV-visible spectrometer and a standard solution was prepared with different solvents dichloromethane, sterile distilled water, chloroform, and Ethyl acetate and they found that AAF2 compound has .

5.5. Fourier-transform infrared spectroscopy (FTIR)

FTIR analysis measures the wavelength underlying the infrared region which are absorbed by a component of the extract to be analysed. The absorbance of the IR light energy by the sample at different wavelength is measured to determine the molecular composition and structure of bioactive component or biopesticides. The unknown components are identified by searching the spectrum against the reference spectra. FTIR analysis can be used to identify the unknown component present in the plant extract as well as to quantify the component of extract. In FTIR measurement, a simple device which is known as interferometer is used to identify the samples component by producing the signal with IR frequencies and these signals can be measured quickly (Wongsa et al., 2022). Tanaje et al.,( 2017) reported characterization of powder Neem formulation by using FTIR spectrometry where FTIR spectra of Neem fruit showed the following bonds and corresponding functional groups.

Table 2. IR frequencies with their corresponding functional groups

Devi et al., (2017)reported the exploration of an antimicrobial compound from Streptomyces S9 against phytopathogens, Corynespora cassiicola which is a fungus that affects many economic crops by causing leaf spots, target spot and leaf fall disease (Li et al., 2014, Looi et al., 2011). They found that antifungal agents like azoxystrobin mancozeb and fumonate show excellent fungal control of C. cassiicola (Ken et al.,2002) after the identification of biopesticide by bioautography TLC and purification of HPLC ( using C-18 reverse phase column) the structure of the pure compound was carried out with the help of FTIR.FTIR spectrum of the compound showed the presence of peaks at 1718, 1565 and 1200-1120 which indicate the presence of ester primary amine and C-O group respectively. Djamann et al.,(2018) reported the characterization of biopesticides compound from bacillus subtilis AAF2 fermentation product by using the FTIR method for structural or functional group determine where Perkin–Elmer model spectrum 400 FTIR spectrometer was used to determine the group of fermented metabolite compound which had been isolated by TLC and found that AAf2 compound has wave number (cm^-1) at 3232, 2926, 2156,2042,1655,1447,1326,1232 and 1070 indicating the presence of corresponding functional group –OH (3232cm-1), C-H aliphatic (2926cm-1), -N=C=O or –C=N (2156-2042cm-1), C=O ( 1655cm-1), CH₃ (1447-1326cm-1) and C-O-C (1070cm-1).

5.6. Nuclear magnatic resonance (NMR)

It is an analytical  technique that is used to determine the content and purity of a sample as well as its molecular structure by determining the chemical shift value ( in ppm)of the compound to be analyzed which depends upon the chemical environment of H and C i.e. H having different chemical environment give different pea(Mahrous et al., 2015). In this technique, TMS is taken as a reference standard and chemical shift is measured concerning it, i.e. more electronegative nucleus gives a peak at a high field means away from the TMS signal which electropositive nucleus gives a signal at lower field means towards the TMS signal (Devi et al. 2017). The details of chemical shifts for functional groups present in biopesticide compounds are given in Table 3.

Table 3. The details of chemical shifts for functional groups present in biopesticide compounds

Similarly C¹³NMR ranges are (Devi et al., 2017):

Many chemical compounds were isolated from plant extract and characterized by 1H-NMR and 13C-NMR spectrometry. This technique is very useful for the structural determination of bioactive components present in the extract after extraction and separation by various techniques. Some chemical compounds are following which were identified by these techniques such as Karanjin from Pongamia pinnata seed oil (Katekhaye et al., 2011), antifungal compound 2,3-dihydroxy benzoic acid, 3,10 dinitrodiftalone and desmethylomifensin from Catharanthus roseus  (Zahari et al.,2017), Oleandrin from nerium oleander (Praveen et al.,2012), the structure of antimicrobial compounds like propyl ester of octadec-9-enoic acid and 17-hydroxy,27-methoxy natamycin from streptomyces S9  (Devi  et al., (2017), antifungal compound (trans cinnamic acid) produced by photorhabdus luminescent Bock et al., (2013), Calamusenone, cis-Methylisoeugenol, isocalamusenone, camphor,Asarone, shyobunone, isoshyobunone from Acorus tatarinowii and A. calamus  (Chen et al., 2016).

5.7. Gas chromatography- Mass spectrometry(GC-MS)

GC-MS is a very powerful and sensitive technique used to detect, quantify, identify and characterize the chemicals present in plant samples. It is a hyphenated technique combination of GC which is used to separate or isolate the bioactive component present in plant extract and MS which is used for structural identification and characterization and the amount of chemical components analyzed by m/z ratio. It contains an unknown chemical which is used to determine the unique chemical structure of the component. The fingerprint can be compared to the library of known compounds and if the chemical component is not present in the library, this fingerprint helps us to develop a good idea of the chemical structure (Gomathi et al., 2015). The basic difference between LC-MS and GC-MS is that LC-MS is used mainly for the analysis of non-volatile compounds like analysis of vitamins, amino acids, and proteins; but GC-MS is used for the analysis of volatile compounds like essential oils, biopesticides etc (Konappa et al., 2020). (Shahid, 2012).Susan et al., (2015) used GC-MS to study the repellency of marigolds by using a 5% phenyl methyl polysiloxane capillary column at injector temperature 250^oc, oven temperature 60^oc for 3 min.  singh et al., (2003) reported the GC- MS method for the study of chemical and biocidal investigation of Tagetes erecta leaf volatile oil by using Hewlett Packard HP 6890 series GC fitted with a Hewlett Packard Mass detector. Various pesticidal components with their extractions and identifications methods are summarised in table (4) as:

Table 4. Various biopesticidal active compounds with extraction and identification method applied previously.

6. Application of Biopesticide in Agriculture

Biopesticides are biologically derived agents that are generally applied in the same manner as chemical pesticides but in an environmentally friendly way, therefore biopesticides are in higher agricultural demand. Biopesticides are found in different sources such as micro-organisms, plants, minerals etc. so they control the biological pest in the following ways:

6.1. Micro-organism derived product as a biopesticide

Biopesticides are used as microbial biological pest control agents applied in the farming process to replace chemical pesticides. Bacterial and fungal agents are commonly used biopesticides like Trichoderma spp., Amelomyces, Quisquolis (control agent for grapes powdery mildew), Bacillus subtilis which is used to control plant pathogens (Santoso et al., 2006). Bacillus thuringiensis products are used to control the pest tobacco budworm( H. versions), grass looper (M. capites), diamondback moth (P. xylostell), maizeborer, cassava hornworm, potato leaf miners, citrus leaf miner, squash pickle worms and other lepidopteron defoliators in vegetables. The acaricide product is also used for mite control in citrus, potato and plantain (Santoso et al.,2006).Copping et al., (2000) reported some micro-organism-derived products as biopesticides which show different pesticidal activities such as bactericidal, fungicidal, herbicidal etc. Blasticidin was derived from the soil actinomycete, Streptomyces, griseochromogens and it shows fungicidal properties (Mistato et al., 1959). Kasugamycin was isolated from soil actenomycete streptomyces kasugaensis and it acts as both bactericidal and fungicidal and is used to control rice blast, leaf spots in sugar beet and celery, bacterial disease in rice and vegetables etc., Mildiomycin it was produced by soil actinomycete streptoverticilium rimofaciens and active against the pathogens which cause powdery mildew. Some micro-organisms product also show insecticidal properties like Avermectinacts against nematodes, Emamectin is effective against Lepidoptera. Milbemectin acts against mites, Bacillus thuringiensis –endotoxin acts against Lepidoptera as well as nematodes some micro-organisms also act as herbicides like Bilanafos (Copping et al., 2000).

6.2. Compound derived from higher plant as a biopesticide

Plants are the most common source of biopesticide because their different parts contain specific bioactive components having biopesticidal properties like Azadirachtin extracted from different parts of neem shows significant insecticidal properties as well as antifeedant and repellent properties. Neem oil shows larvicidal activity and is used against mosquitoes. Neem and its derivatives (neem oil with polyoxy ethylene ether, orbitan dioleate  and  epichlorohydrin) which is commonly used for agricultural and farming purposes (copping et al., 2000). Nicotine is produced from the genus Nicotina and shows insecticidal behavior against organosulphur and pyrethroid-resistant whitefly. Pelargonic acid and related fatty acids extracted from both plants and animals interfere with the cell membrane constituents of target organism and kill them. Pyrethrins produced from Chrysanthamum cineriaefolium  show insecticidal properties with contact action. Rotenoneis obtained from Derris, Lonchocarpus and Tephrosia species has respiration inhibitor property and is used as a selective, non-systemic insecticide(Copping et al., 2000). The plant extract also contains some chemical components having plant growth regulation properties. Copping et al. (2000) reported some plant growth regulators such as 6-Benzyl amino purine whichregulate the increase in cell division, increased lateral bud formation flowering In xerophytic species etc. (Skinner et al., 1958).Zeatin is a plant derived growth regulator and regulates the initiation of plant cell division. Gibberellic acid and Gibberellin (discovered by E kurusare 1926) regulate plant growth and development. Indol-3yl acetic acid is a plant growth regulator and very effective at initiating the formation of root. (Saxena et al.,2014, Sharma et al.,2009,  kandpal, 2014) reported the following botanical biopesticide showing different effects, such as insecticidal, repellent toxicant, antifeedant etc. Some biopesticidal plant resources with species name, active constituents and their biological activities are given in Table 5. 

Table 5. The details of plant species and their use of plant parts for biopesticide containing different chemical substances for biological activity

6.3. Animal derived products as biopesticides

Copping et al.,(2000) reported some animal-derived products having pesticidal effects like insect hormones which control the various physiological processes in the insects. Insect-specific toxins are used by many predatory animals to kill their prey like snakes scorpions, wasps bees, spiders and mites, etc. pheromones are volatile chemicals produced by insects as a means of communication. These show many different effects and the specificity of insect mating disruption, pheromones preserve the natural enemies such as parasites and predators.

7. Conclusions

Extraction of the biopesticidal component from the different plants and micro-organisms is always in demand in agriculture which inspires the continuous research of new biopesticides and appropriate extraction techniques for them. Botanical biopesticides play an important role in traditional pest management because they provide a good source of economical, environment-friendly, biodegradable, cost-effective pest control agents but there are a very limited number of biopesticides that have been commercially used like neem products, this may be due to the failure of the fully characterization the original plant material and its pesticidal component because after the extraction plant extract is identified by various techniques. Chromatography is a very useful and widely used method for this purpose, this generally includes TLC, HPLC, HPTLC, etc and the identified component is characterized and its structure is determined by various spectrometry techniques like 1HNMR and 13C NMR, FTIR, mass spectrometry etc. Since different plants contain some specific chemical components showing different properties like medicinal, pesticidal etc., so identification of the specific biopesticide depends upon the extraction, identification and characterization techniques. So this review summarized the extraction, identification, formulation methods and application of biopesticide in agriculture.