Insecticidal Activity of Extracts, Fractions, and Pure Molecules of Cissampelos pareira Linnaeus against Aphid, Aphis craccivora Koch

Aphis craccivora Koch is a polyphagous and major pest of leguminous crops causing significant damage by reducing the yield. Repeated application of synthetic insecticides for the control of aphids has led to development of resistance. Therefore, the present study aimed to screen the insecticidal activity of root/stem extracts/fractions, and pure molecules from Cissampelos pareira Linnaeus against A. craccivora for identification of lead(s). Among root extract/fractions, the n-hexane fraction was found most effective (LC50 = 1828.19 mg/L) against A. craccivora, followed by parent extract (LC50 = 2211.54 mg/L). Among stem extract/fractions, the n-hexane fraction (LC50 = 1246.92 mg/L) was more effective than the water and n-butanol fractions. Based on GC and GC-MS analysis, among different compounds identified in the n-hexane fraction of root and stem, ethyl palmitate (known to possess insecticidal activity) was present in the highest concentration (24.94 to 52.95%) in both the fractions. Among pure molecules, pareirarineformate was found most effective (LC50 = 1491.93 mg/L) against A. craccivora, followed by cissamine (LC50 = 1556.31 mg/L). Parent extract and fractions of C. pareira possess promising activity against aphid. Further, field bio-efficacy studies are necessary to validate the current findings for the development of botanical formulation.


Introduction
Aphis craccivora Koch (Hemiptera: Aphididae) is one of the most common polyphagous pest [1] reported in 50 host plants (19 families) and is considered as global threat of leguminous plants [2,3]. The nymphs and adults suck the sap from leaves, flowers, and pods of cowpea plants. The aphid also transmits plant viruses [4,5] and affects the yield [6]. In severe infestation, A. craccivora secrete honeydew on the plants, which serves as a medium for the growth of sooty mold, there by leaves became black and affect photosynthesis [7] and reported significant reduction in the seed yield to the extent of 12.8 to 61.1% [8].
The plant extracts and their formulations are normally less harmful to the environment, have low cost, and are less persistent, and safer to natural enemies and humans, and easily biodegradable than synthetic insecticides [9,10]. Numerous studies have already been done on plant-derived extracts/essential oils and their isolated compounds against insect pests [11]. Due to less availability of biopesticides, farmers/growers often spray synthetic insecticides (imidacloprid, thiamethoxam, acetamiprid, thiacloprid, diafenthiuron, chlorfenapyr, spiromesifen, and dimethoate) to control aphids [12,13], and other

Characterization of Isolated Molecules
The chemical structures of isolated molecules were elucidated by nuclear magnetic resonance spectroscopy (NMR), HRESI-MS, and finally by comparison with those reported in the literature [33,34]. Proton and carbon NMR spectra are shown in the supplementary information.

Quantification of Isolated Molecules
Isolated molecules namely curine (1), pareirarineformate (2), and cissamine (3) were quantified by UPLC-DAD method in different extracts and fractions of C. pareira (Table 1). Quantification results clearly depicted that the above-mentioned isoquinoline alkaloids were present in almost all extracts and fractions, albeit in variable quantities.

Discussion
Despite using chemical pesticides, there is considerable evidence indicating the loss of agriculture production and the potential threat of these chemical pesticides on humankind and biodiversity. Similarly, the resistance shown by insect pest is also a major concern, which emphasizes looking for alternative bio-tools for which role of plants comes into play. Plants have potential to serve as a greener alternative to chemical pesticides which is further proved by evaluating the insecticidal potential of C. pareira which showed promising activity against A. craccivora. Parent extracts, their fractions, and pure molecules of C. pareira against A. craccivora were tested. In the present study, the n-hexane fractions of root and stem were found more effective against A. craccivora followed by the parent extract of root, n-butanol, and chloroform fraction compared to stem fractions of water and n-butanol. Then-hexane fraction (LC 50 = 1828.19 mg/L) and parent extract of C. pareira root (LC 50 = 2211.54 mg/L) were more effective against A. craccivora in this study as compared to n-hexane fraction of Eupatorium adenophorum (LC 50 = 2881 mg/L) and Ageratum houstonianum (LC 50 = 2590 mg/L) [41,42]. GC analysis of n-hexane fractions of both root and stem confirmed the presence of esters of fatty acids. The n-hexane fraction of stem has high content of ethyl palmitate (52.95%), ethyl oleate (10.53%), and methyl linoleate (9.24%), whereas the n-hexane fraction of the root contains high contents of 8-octadecenoic acid (36.76%), ethyl oleate (30.37%), and ethyl palmitate (24.94%). These compounds are already known for their insecticidal potential. Ethyl oleate and ethyl palmitate were found in the extract of Eupatorium odoratum which act as oviposition repellent [43,44]. Ethyl palmitate is reported to have larvicidal activity [45,46]. Therefore, the presence of these compounds in n-hexane fractions of root and stem could be attributed to the potent insecticidal activity against A. craccivora.
In a similar study, the n-hexane fraction from tubers of Corydalis turtschaninovii at 2000 mg/L showed less efficacy (85% mortality) against Aphis gossypii [28] as compared to the present study. In another study, the ethanol root extract of Cissampelosmu cronata found more effective against larvae of Culex quinquefasciatus (LC 50 = 207.1 µg/mL) after 72 h [27] as compared to the present study. Similarly, in this study, the ethanol stem extract of C. pareira at a lower concentration (LC 50 = 1466.98 mg/L) was more effective against A. craccivora after 72 h as compared to aerial parts of ethanol extract of C. mucronata (LC 50 = 8000 µg/mL) against larvae of C. quinquefasciatus after 72 h [27]. In another study C. awariensis leaf and root slurries at 1% showed 96-100% mortality against Prostephanustruncatus horn and Sitophilus oryzae L. [29] as compared to the present study where C. pareira root and stem extract showed more promising activity at a lower dose.

Collection and Authentication of Plant Material
The root and stem part of C. pareira were collected from Palampur, Himachal Pradesh, India in January 2019. The plant material was authenticated by a taxonomy expert at CSIR-IHBT and submitted to the herbarium of CSIR-IHBT, Palampur with voucher specimen no. PLP16688. Plant material was shade-dried for about a week and then ground into uniform powder using a MAC Willy mill PLT 210 grinder.

Preparation of Extracts, Fractions and Decoctions
Shade-dried 2-kg powdered roots were extracted thrice with ethanol:water (4:1, v/v) using the percolation method at room temperature. The percolate collected from the extraction was evaporated in a rotary evaporator at 50 • C to obtain 239.2 g of hydro-ethanolic crude extract. This dried crude extract was dissolved in 700 mL of distilled water, and after that the dissolved part was further fractionated with organic solvents (600 mL × 3 times), i.e., nhexane, chloroform, and n-butanol to yield fractions of different polarity i.e., n-hexane (26.1 g), chloroform (12.4 g), n-butanol (26.5 g), and water (110.9 g) (Figure 1). Similarly, the stem part (2 kg) was processed by the above procedure to obtain crude extract 219.2 g and fractions as follows: n-hexane (12.6 g), ethyl acetate (7.3 g), n-butanol (23.5 g), and water (104.2 g) (Figure 1). Decoctions were prepared by boiling the plant material with distilled water at a temperature of 85 • C for 30 min. Root decoction was prepared by boiling the crushed roots (100 g) with 800 mL of distilled water. Then the decoction was concentrated on a rotary evaporator at a temperature of 50 • C to obtain 15.1 g of the water extract. The combined decoction of roots (50 g) and stem (50 g) was prepared in a similar way to obtain 10.6 g of the water extract ( Figure 1).

Isolation of Pure Molecules
The chloroform fraction (12.0 g) of roots was chromatographed on silica gel (60-120
The n-butanol fraction (25.0 g) of roots was subjected to chromatography over silica gel (60-120 mesh) and eluted with increasing gradient of CH 3 OH:CHCl 3

Characterization of Molecules Isolated from C. pareira
All the molecules isolated from C. pareira were characterized by 1 H-NMR, 13 C-NMR, UV/Vis, and IR spectroscopy. The melting points of all the molecules were noted on Brønsted Electro thermal 9100. NMR spectral analysis of these molecules was done on Bruker-Avance 600 MHz instrument. UV-Vis analysis was performed on Shimadzu UV-VIS spectrometer-2600. IR analysis was done on Shimadzu IR Prestige-21with ZnSe single reflection ATR accessory.

Preparation of Methyl Esters
The n-hexane fractions of root (50.1 mg) and stem (52.1 mg) were derivatized using methanol and sulfuric acid under nitrogen atmosphere [48]. The derivatized fractions thus obtained were evaluated by gas chromatography (GC-FID and GC-MS).

Quantification of Compounds in Extract, Fractions and Decoctions of C. pareira
The quantification of marker compounds (1, 2, and 3) in different extracts (root and stem) and fractions as well as decoctions of root and stem part of C. pareira was performed by UPLC-DAD method reported earlier by our group [33]. Chromatograms for quantification of compounds in extract, fractions, and decoctions are shown in Supplementary Figure S7a-d.

GC-FID Analysis of n-Hexane Fractions
The n-hexane fractions were subjected to GC-FID analysis using GC Shimadzu 2010 coupled with AOC-20i auto-injector, SH-Rxi-5Sil MS column (30 m × 0.25 mm i.d., 0.25 µm) and FID-detector. Nitrogen was used as a carrier gas with a flow rate of 1.24 mL/min. The initial temperature of oven was 40 • C for 4 min and programmed to 220 • C at 4 min, then held for 15 min at 220 • C. Other parameters for GC analysis were an injector temperature of 250 • C, oven temperature of 250 • C, and the split mode was used. A standard solution of n-alkanes (C 9 -C 23 ) was used to obtain the retention indices. Individual components were identified by matching their retention indices (RI) with those reported in the literature.

Gas Chromatography-Mass Spectrometry Analysis
The GC-MS analysis was carried out on a Shimadzu (GC 2010) GC-MS equipped with an AOC-5000 auto-injector coupled and an SH-Rxi-5Sil MS capillary column (30 m × 0.25 mm i.d., 0.25 µm). The initial temperature of the column was 40 • C held for 4 min and was programmed to 220 • C at 4 min, then held for 21 min at 220 • C; the sample injection volume was 1 µL in the HPLC-grade dichloromethane. Helium was used as carrier gas at a flow rate of 1.28 mL min −1 on the split mode (1:10). Individual components were identified by matching their mass spectra with literature, NIST database, and Adams's libraries [49,50]. GC-MS chromatograms for n-hexane fractions of root and stem are shown in Supplementary Figure S8

Test Insect
Aphis craccivora collected on leguminous plants in the field and reared under controlled conditions (26 ± 2 • C temperature, 60 ± 5% humidity, and photoperiod 16:8 L:D) in the lab on the live host (Phaseolus vulgaris L.) more than 100-120 generations. The uniformly sized nymphs of 3-4 days old aphid were used for bioassay study.

Dose Optimization
Preliminary screening of root/stem extracts and their fractions was carried out at 5000 and 10,000 mg/L) for their bio-efficacy against A. craccivora. Five concentrations were fixed and assessed against aphids in the main bioassay studies based on preliminary efficacy data.

Bioassay of Extracts, Fractions, and Pure Molecules of C. pareira against A. craccivora
Briefly, test samples were dissolved in Triton X 0.05% solution (SD Fine Chemicals Limited, Mumbai, India) in water and then ultrasonicated for complete dissolution. Five concentrations of root extracts/fractions (625 to 10,000 mg/L) and pure compounds (313 to 5000 mg/L) were prepared from stock solutions by serial dilution for dose response bioassay. Fresh bean discs (3 cm diameter) were prepared and pressed over the wateragar medium (1.5%) in Petri plates sprayed with 2 mL of the test solution at different concentrations under Potter's spray tower operated at 1.1 kg/cm 2 pressure and the solvent was evaporated under room temperature for 2 h. For control, leaf disks were sprayed with distilled water containing Triton. In each Petri dish, 10 nymphs were released then sealed with parafilm and kept in the laboratory conditions at 25 ± 2 • C temperature, 60 ± 5% relative humidity, and a photoperiod of 16:8 (L:D) for observations. All the treatments including control were replicated three times. Mortality was determined after 72 and 96 h of treatment. There were five treatments and three replications (5 × 30 = 150 insects, each replication contains 10 insects. The commercially available neem formulation (Neem Baan 0.15 EC, i.e., containing azadirachtin 1500 ppm) available in the market (manufactured by Pest Control India Pvt. Limited, Goa, India) used by the farmers/growers at the recommended dose (5 mL/L of water) for the control of aphid on crop plants was used as a positive control for comparison.

Data Analysis
The mortality data of aphid based on bioassays of extracts/fractions/compounds was compiled. Corrected mortality was not calculated because the test insect nymphs were not died in the untreated control. Lethal concentration to kill 50% test population (LC 50 values) and regression parameters were worked out by Probit analysis [51] using SPSS software version 16. Similarly, the percent mortality data against test insect was also analyzed using analysis of variance (ANOVA), and means were compared by Tukey's post hoc test [52].

Conclusions
The present study concludes that parent extract and fractions of C. pareira possess promising activity against aphids which can serve as potential biopesticide, as currently used chemical pesticides are being prone to resistance by insect pest along with their adverse environmental hazards. Hence lab-scale bioactivity evaluation can serve as a potential footstep for advanced studies in the demand of plants-based bio-pesticides. However, field bio-efficacy studies are necessary to validate the current findings against target pest for the development of botanical formulation.