The Major Phyto-Compounds Heptasiloxane, hexadecamethyl-and 1,1-Dimethylethyl 3-Phenyl-2-Propenoate Derived from Indigofera tinctoria Medicinal Flora Tested Against Various Target Medical and Agronomic Pests

Objectives: To determine the phytochemical screening and major phyto-compounds from Indigofera tinctoria


Introduction
Recently, medical and vector biology fields are facing plenty of challenges to protect public and cattle from infected bite of female mosquitoes (1) . In many countries, mosquitoes are vastly spreading many unsolved diseases to human and other blood yielding faunas as well as they making serious alarming to economic crises (2) . The Dipteran vector spreads many different pathogenic viruses as the results that severely threaten blood yielding many faunal lives (3) .
Spodoptera litura is a voracious feeder and consuming varieties of flora (including vegetables, fruits, seeds, flower, rhizomes, etc.,). It is polyphagous lepidopteron pest which feeding above 200 floral hosts and it includes 40 different families (4) . Recently, it is vastly distributed in many terrains of Asian country and it has gradually emerged as vital pest in many revenue crops (5) . Helicoverpa armigera is a globally distributed polyphagous pest which vastly consuming many revenues agronomic crops (6) . It consuming above 180 floral species includes 47 families, at initially feeds on tender leaves then later stage consuming all parts of flora (Fruits, seeds, seed coat, flower, stems, rhizome, etc.,). Most of the Indian villagers nearly above 80% mainly depend on agronomy and its byproducts. Insect's pests significantly damaged plenty of crops as the results cause the productivity and revenue crises (7). Recently (a half a century), most of the countries are enormously used unadvisable toxic pesticides in vector control and crop protection aspects as the result unpredicted damage caused on eco-system, pest resurgence, high-cost production and very hard to application, great loss of soil productivity, declining population size of beneficial microbes, pollinator, non-target fauna etc., (8). The continued as a long-term application of toxic pesticides in the pest control management (both medical and crops protection) to develops negative impact on biosphere and its related factors. Therefore, most of the research communities/ scientists were planned to rectify through organic pesticides especially by naturally available phyto-products (9) . Indigofera tinctoria is a greenish small herb and it can be used to treat as a medicine for diseases and infection on human and cattle (10,11) . However, the bio-toxic efficacy of I. tinctoria and its phytocompounds on insect pests were not explored intensively. Therefore, present examination, we planned to I. tinctoria derived phytoproducts were tested towards the eggs and larval toxicity of target pests species.

Collection and processing of floral material
The fresh, neatly cleaned and diseases free I. tinctoria leaves (Figure 1 A-C) were collected during flowering season in the month of January-April, 2020 from Cuddalore and Mayiladuthurai District. The floral sample was identified by well skilled botanist Dr. R. Janagan, the fully shade dried leaf was ground into fine powder, followed by extraction and condensation done it through standard protocol (12) . The condensed floral materials were stored at -10 0 C by using deep refrigeration for dimensional activities of various bioassays. https://www.indjst.org/

Phytochemical screening and GC-MS analysis
The various leaf extracts (Hexane, diethyl ether, dichloromethane, acetate and methanol extract) of I. tinctoria medicinal herb were subjected to qualitative analysis of various phyto-chemicals were described through proper methods (13) . Moreover, plants are holding hundreds of phyto-compounds it can be classified into major and minor phyto-compounds which based on the acquiring concentration phyto-compounds. The GC-MS analysis of leaf methanol extract was subjected to Agilent technologies and identified various phyto-compounds were compared with NIST/ WILEY library (14) .

Target pest rearing
Ae. aegypti and Cx. quinquefasciatus eggs, larvae and pupae were collected from sewage treatment plant near AVC College (Autonomous) and S. litura and H. armigera eggs and larvae were collected from Arachis hypogaea and Abelmoschus esculentus agronomic field near Cuddalore and Mayiladuthurai District., Tamilnadu, India. The collected medical pests' juvenile stage (eggs, larvae and pupae) was reared in separate container. The plastic container filled with drinking tap water which maintained appropriate temperature, humidity and photoperiod. The larvae allowed to the diet of yeast powder, biscuits crumble, algae bloom and natural honey in the ratio of 1:2:1:1, respectively. The juvenile stage of agronomic pests were reared in above mentioned condition, the S. litura larvae allowed to fed with Ricinus communis tender leaves and H. armigera larvae fed with Abelmoschus esculentu tender fruits.

Eggs toxicity of target pests
Eggs toxicity of medical pests: The eggs toxicity was assessed through standard methodology (15) . The freshly laid eggs (0-5 hrs aged) of selected ecto-parasitic vector were collected from mother culture. 25 Nos. eggs were exposed to various concentrations of I. tinctoria phyto-products in the glass container. The phyto-products were dissolved with 1ml of DMSO which prepared in to various concentrations (range between lower to higher concentration). After 24 hrs. post treatment eggs toxicity were concluded as well as in-between every 6 hrs. interval thoroughly observed death rate of eggs. As usual, control was maintained without phyto-products composites. Eggs toxicity of agronomic pests: The eggs of polyphagous pests were collected which setup into (One batch was 25 Nos. eggs) five batches for single concentration. The required phyto-products of I. tinctoria were sprayed which allowed into 24 hrs. post treatment and estimated the eggs mortality (16) . The experiment was done on under laboratory condition. As usual, control was maintained without phyto-products composites.

Larval toxicity of target pests
Larval toxicity of medical pests: The phyto-products of I. tinctoria were diluted with 1ml DMSO which mixed with 450 ml of chlorine free tap water and make into different concentration made into 500 ml beaker each experiment constantly replicated five times. Invariably 3 rd instar larvae 25 count released into respective concentration in which control was maintained without phyto-products. Top of the beakers were tightly closed with muslin cloth which may give the protection and entry of any intruder. The larval toxicity carefully noticed every 6 hours interval which monitored up to 24 hours of post treatment. While https://www.indjst.org/ conducting experiment, larvae were starved and it was followed by the standard protocol (15) . The total larval mortality was corrected by prescribed method (16) . Larval toxicity of agronomic pest: The 3 rd instar larvae of S. litura were allowed to phytoproducts of I. tinctoria different concentration treated with Ricinus communis leaf disk as well as same larval stage of H. armigera larvae allowed to phyto-products of I. tinctoria different concentration treated with Abelmoschus esculentu sliced disk of tender fruits. The larval toxic bioassay followed by (15) . Agronomic pests, each batch allowed 25 larvae which introduced in separate containers, the toxicity noticed and the percent mortality corrected by standard method (16) .

Statistical Analysis
The recorded larval and eggs toxic raw data were assessed through Probit analysis (17) for examining for mean ± S.D, DMRT test, LC 50 /LC 90 , UCL, LCL, regression, chi-square, etc., were estimated with the help of IBM-SPSS 26.0v. The observation of p ≤ 0.05 were considered to be statistically significant.

Phytochemical screening and GC-MS analysis
The selected medicinal herb, different leaf extracts of I. tinctoria (hexane, diethyl ether, dichloromethane, ethyl acetate and methanol) were assessed for phytochemical screening, the more numbers of phytochemicals (Alkaloids, anthraquinones, coumarins, flavonoids, glycosides, phenolics, saponins, steroids, tannins and triterpenes) were obtained from methanol extract which clearly enumerated in Table 1. The Indian medicinal flora, I. tinctoria leaf extract (methanol extract) was tested by GC-MS analysis for observing various phyto-compounds which apparently shown in Figure 2 and its peak, retention time, concentration, etc., were evidently noticed in Table 2. A total of 15 phyto-compounds gained 100% besides two phytocompounds were major constituents Heptasiloxane, hexadecamethyl-(Peak 6; Retention Time 37.435; Composition Area 99326; Composition Area% 22.69, etc.,) and 1,1-Dimethylethyl 3-phenyl-2-propenoate (Peak 12; Retention Time 39.845; Composition Area 85954; Composition Area% 19.64, etc.,). The noticed major floral-bioactive compounds were obviously authenticated through MS analysis which presented in Figures 3, 4, 5 and 6. The different leaf extracts of I. tinctoria were evaluated for detecting the availability of phytochemical in the respective solvents and their results were compared with selected all extracts but a greater number of phytochemicals groups were identified from high polarity solvent of methanol extract followed by other solvent extracts. Previously, many investigations were evidently done on several herbal parts as well as they were well effective and target specificity on different life stages of pests (18,19) .

Values of major bio-active compounds
The two different floral-bioactive compounds Heptasiloxane, hexadecamethyl-and 1,   Table 4). The statistical analysis of the data clearly indicates that a phyto-compounds of I. tinctoria induced outstanding larval lethality were observed on selected pests. Similarly, many different works previously done and it has been evidently supported with present examination, Enhalus acoroides and Halophila ovalis showed above 50% larval death recorded at 500ppm against Ae. aegypti and Cx. quinquefasciatus (23) . The larval toxicity of various phyto-compounds showed predominant toxicity against agronomic pests (24) . The I. tinctoria derived phyto-compounds were showed appreciable safety towards environment and higher toxicity observed on selected pests.

Conclusion
The I. tinctoria borne phyto-products showed efficiently succeed that selected medical and agronomic pests. The phyto-products relatively higher toxicity on harmful pests and it could be former friendlier, zero hazards to environment, at low concentration obtained undeniably eradicating target-fauna and it can be approached to pest management techniques.