Phytochemical analysis and fabrication of silver nanoparticles using Acacia catechu: An efficacious and ecofriendly control tool against selected polyphagous insect pests.

Globally, the farmers are struggling with polyphagous insect pest, and it is the number one enemy of agri-products, which made plenty of economic deterioration. Spodoptera litura and Helicoverpa armigera are the agronomically important polyphagous pests. Most of the farmers are predominately dependent on synthetic chemical insecticides (SCIs) for battle against polyphagous pets. As a result, the broad spectrum usage of SCIs led a lot of detrimental outcomes only inconsequently the researchers search the former-friendly phyto-pesticidal approach. In the present investigation, leaf ethanol extract (LEE) and silver nanoparticles (AgNPs) of A. catechu (Ac) were subjected to various spectral (TLC, CC, UV, FTIR, XRD and SEM) analyses. Larval and pupal toxicity of A. catechu Ac-LEE and Ac-AgNPs were tested against selected polyphagous insect pests. The significant larval and pupal toxicity were experimentally proven, and the highest toxicity noticed in AgNPs than Ac-LEE. The larval and pupal toxicity of Ac-AgNPs tested against S. litura and H. armigera LC50/LC90 values were 71.04/ 74.78, 85.33/ 88.91 µg/mL and 92.57/ 96.21 and 124.43/ 129.95 µg/mL respectively. Ac-AgNPs could be potential phyto-pesticidal effectiveness against selected polyphagous insect pests. In globally, it is significantly sufficient ratification giving towards the prevention of many unauthorized SCPs.

Introduction

Most of the quality and quantity of the agricultural product is directly/ indirectly deteriorated by insect pests in many countries (Elumalai et al., 2010a, Kamaraj et al., 2008, Krishnappa et al., 2010a, Krishnappa and Elumalai, 2012a, Misra, 2014). Spodoptera litura is a predominant polyphagous pest occupied a wide range of hosting around 200 floral species globally, in which 74 floral host species noticed from India (Elumalai et al., 2014, Paulraj et al., 2017). S. litura larvae consumed the different parts of host flora, including rhizome and causing severe damage, which gives above 60% of revenue loss in India (Elumalai et al., 2014, Krishnappa et al., 2010a, Krishnappa et al., 2010b). India is a tropical country, polyphagous pest (S. litura) surviving and high abundance in that particular climate; therefore, recently, agriculture is facing severe economic losses (Paulraj et al., 2017). Helicoverpa armigera is a multivoltine, agronomically predominant polyphagous pest and it consumed a wide range of hosts estimated above 300 floras communities globally (Backiyaraj et al., 2014, Namin et al., 2014). The initial larval stage feeds only soft floral structures then turned to later stages feed on every part of flora (Gokulakrishnan et al., 2012).

Globally, most of the farmers are predominantly depending on synthetic chemical insecticides (SCIs) for battle against polyphagous insect fauna (Elumalai et al., 2010b, Krishnappa and Elumalai, 2012b). As the results of broad-spectrum usage of SCIs led a lot of detrimental outcomes: a drastic conflict between environment and living organisms, insect pest develops resistance against among the SCIs, the broad eradication of non-target fauna and flora, the direct/ indirect toxicity to formers, SCIs has lesser bio-degradable and drastic conventional effects on the natural ecosystem, unpredictable collapse in food chain/ food web, etc., (Govindarajan et al., 2005; Abinaya et al., 2018, Baranitharan et al., 2016, Baranitharan et al., 2020, Benelli and Govindarajan, 2017, Govindarajan, 2011, Govindarajan et al., 2013, Govindarajan et al., 2015, Govindarajan et al., 2016a, Govindarajan et al., 2016b, Govindarajan et al., 2016c, Govindarajan et al., 2016d, Govindarajan et al., 2016e, Govindarajan et al., 2016f, Govindarajan and Benelli, 2016a, Govindarajan and Benelli, 2016b, Govindarajan and Benelli, 2016c, Govindarajan and Benelli, 2016d, Govindarajan and Benelli, 2016e, Govindarajan and Karuppannan, 2011, Govindarajan and Sivakumar, 2012, Krishnappa et al., 2012, Krishnappa et al., 2013, Krishnappa and Elumalai, 2013). Interestingly, the most of the scientist/researchers search for newer alternative SCIs approach usually it belong from naturally available phytoconstituents (PCs) (Govindarajan and Rajeswary, 2015, Govindarajan et al., 2016g, Govindarajan et al., 2016h; Baranitharan et al., 2019, Benelli, 2016, Elumalai et al., 2013, Elumalai et al., 2014, Govindarajan et al., 2016a, Govindarajan and Benelli, 2017, Krishnappa and Elumalai, 2013, Krishnappa and Elumalai, 2014). Because PCs have less/ zero toxic effects to the ecosystem and other related non-targets fauna and flora (Baranitharan et al., 2017, Benelli et al., 2017a, Benelli et al., 2017b, Benelli et al., 2017c, Divya et al., 2018, Karthika et al., 2017, Mathivanan et al., 2010, Muthukumaran et al., 2015, Veerakumar et al., 2014, Zahid et al., 2016). The PCs have multi-special derivatives it has been using in many countries (El-Wakeil, 2013, Sola et al., 2014). Acacia catechu (Leguminoseae) is a highly medicinal value flora, and it distributed in entire Asian and African continents. This plant has a lot of vital PCs; therefore, it has been used as different medicinal properties, and it could cure different human/animal infections. The floral parts like a leaf, branches, bark, fruits, seeds, and rhizomes were used as dental care mouthwash, buccal and alimentary canal infections, gastric ulcers, diarrhea, control of hypertension, dysentery, common cold, cough, asthma, leprosy, antimicrobial, antioxidant activities etc., (Patel et al., 2009, Negi and Dave, 2010, Stohs and Bagchi, 2015, Rahman et al., 2016). Since it is firsthand documentation, existing about the pesticidal activity of A. catechu leaf ethanol extract (Ac-LEE) and green-synthesized nanoparticles (Ac-AgNPs) against selected polyphagous insect pests S. litura and H. armigera. Ac-LEE and Ac-AgNPs of A. catechu were subjected to various spectral and microscopic analyses.

Material and methods

Floral collection and extract preparation

A fresh and cleaned Acacia catechu (Leguminoseae) leaf (Fig. 1A) was collected from parent flora at Namakkal District (11.2189° N, 78.1674° E), Tamilnadu, Southern India. The collected leaves were carefully washed with dechlorinated H2O and immediately kept in the sunshine for 10–15 min. The partially dried and H2O evaporated leaves were carried to the laboratory, which allowed to shade dried on Whatman filter paper (27 ± 2 °C). The dried floral parts were ground as a fine powder (Fig. 1B), which extracted by using the Soxhlet apparatus. Excess of solvent residue in that extract was evaporated naturally, which maintains room temperature on the Petri plate (Fig. 1C) and complete evaporated semi-dried extract weighed, stored in an aseptic glass vial at 0–4 °C in the refrigerator.

Fig. 1

Floral collection and extract preparation. (A) A. catechu plant, (B) Dried leaf powder, (C) A. catechu condensed LEE in a Petri plate.

TLC, CC, and Phyto-chemical analysis

The Ac-LEE was subjected to find potential PCs accountable for different bioassay activities. The Ac-LEE was run on a pre-coated TLC sheet and CC packed with a silica gel column with a height 50 cm and capacity 50 ml. The Ac-LEE diluted with various solvent systems with the composition of ethanol and diethyl ether (Table 1). The phytochemical analysis using for the quality of various PCs found in the competent Ac-LEE described by Nweze et al., 2004, Senthilkumar and Reetha, 2009.

Table 1

Fractions obtained from A. catechu LEE by different ratios of elutants.

Sl. No.	Various solvent systems	Number of fractions obtained
1.	Ethanol:Diethyl ether – 7.5:2.5	2
2.	Ethanol:Diethyl ether – 8.0:2.0	3
3.	Ethanol:Diethyl ether – 8.5:1.5	4
4.	Ethanol:Diethyl ether – 9.0:1.0	5
5.	Ethanol:Diethyl ether – 9.5:0.5	3

Silver nanoparticle (AgNP) synthesis

The Ac-AgNO3 90 ml Mm added with Ac-LEE 10 ml was prepared in 200 ml Erlenmeyer flasks for reduction into Ag + ions. AgNO3 + Ac-LEE mixture was retained 1 h at 27 ± 3° C for complete bio-reduction. The preliminary finding of Ac-AgNPs found the colour change in the AgNO3 + Ac-LEE mixture. The whole reactions allowed in darkness to avoid photoactivation. For the purification process, obtained Ac-AgNPs subjected to ultra-centrifugation above 5,000 rpm for 25 min. After the centrifugation, supernatant discarded and pellet carefully diluted with distilled H2O (Satyavani et al., 2011), the blend was stored in a glass vial, labeled and stored for further analysis.

Characterization of AgNPs

The bio-reduction of Ag + ions solution was keenly monitored by using UV–vis spectroscopy (Rajesh et al., 2009). FTIR spectroscopy evaluated the bio-molecules present in purified Ac-AgNPs (Ashokkumar and Ramaswamy 2014). Ac-AgNPs were allowed to dry at 60° C, and the dried powder was subjected to XRD spectroscopy to identify their exact structure and material.

Polyphagous insect pests rearing

The polyphagous insect pests of S. litura and H. armigera its eggs/ egg masses (Fig. 2A and Fig. 3.A), larvae, pupae, and adults were collected from castor and legume field in Mayiladuthurai District (Latitude: 11° 06′ 12.74″ N; Longitude: 79° 39′ 18.00″ E), Tamilnadu. Before hatching, the collected eggs were sterilized with 0.02% NaClO and neonate larvae were separately kept in the rearing chamber, S. litura larvae allowed on fresh tender castor leaves and bendi fruits for H. armigera. Heat sterilized soil was provided for pupation at maintained 27 ± 2 °C with light 12 h : dark 12 h photoperiod and 70 ± 5% relative humidity in insectariums. Pupae were collected from soil and placed inside the oviposition chamber here cotton soaked with 10–20% (w/v) cane sugar with 1 (or) 2 drops of multivitamins and natural honey was provided for gravid moths to increase the fecundity.

Fig. 2

Bioassay experimental setup of A. catechu LEE and AgNPs against S. litura. (A) S. litura eggs collected from castor plant leaf, (B) Larvicidal activity setup, (C) Pupicidal activity setup.

Fig. 3

Bioassay experimental setup of A. catechu LEE and AgNPs against H. armigera. (A) H. armigera eggs collected from legume plant leaf, (B) Larvicidal activity setup, (C) Pupicidal activity setup.

Insect toxicity

Larval and pupal toxicity of Ac-LEE and Ac-AgNPs were evaluated by the method of Abbott, (1925), and mortality was calculated by probit analysis (Finney, 1971). Five batches of 0–6 h old, 25 number, well active, uniform size, hale and healthy 2nd instar larvae of S. litura and H. armigera were separately introduced in 100 x15 mm petri dish. Larval toxicity of Ac-LEE and Ac-AgNPs of different concentrations (20–150 µg/mL) sprayed on several host plant like castor leaf for S. litura and legume leaf for H. armigera which separately provided to polyphagous pests (Fig. 2B and 3B). Larval death were noted every 4 hrs interval, total % of mortality were calculated and it maintained four replicates.where

%LMT = % larval mortality in the treatment

%LMC = % larval mortality in the control

The same methodology has been applied to pupal toxicity; the hale and healthy of even-sized fifth instars larvae of the selected polyphagous pests were allowed to feed with different concentrations (20–150 ppm) Ac-LEE and Ac-AgNPs sprayed on respective host leaves. All phytochemicals (LEE and AgNPs) treated and control larvae were separately allowed to pupate to evaluate the pupal toxicity in which 15 numbers of pupae for S. litura as well as 12 numbers of pupae for H. armigera. Single experiment setup of pupal toxicity maintained five batches in which a total of 75 and 60 pupae tested against S. litura and for H. armigera, respectively (Fig. 2C and 3C). The appropriate concentration of Ac-LEE/ Ac-AgNPs was mixed with dechlorinated H2O applied on the respective host plant of selected pests. The larval and pupal toxicity were assessed on both narrow and broad range test.where

%PMT = % pupal mortality in the treatment

%PMC = % pupal mortality in the control

Statistical analysis

The % mortality data of polyphagous insect pests S. litura and H. armigera its larvae and pupae were subjected to different statistical tools, LC50/LC90, LCL, UCL, regression, chi-square, etc. All the values were calculated by (IBM) SPSS statistics new version 25.0 version.

Results

TLC, CC analysis and phytochemical screening

The Ac-LEE was subjected to the TLC plate to identify the bio-efficiency of PCs were examined. From TLC experiment provided 5 fractions which get in the solvent system ratio of ethanol 9: diethyl ether 1(Fig. 4A). The same principle was applied on CC; the Ac-LEE capable PCs were assessed by CC tightly packed with silica gel, which runs with the 50 ml solvent ratio of ethanol 9: diethyl ether 1, from the experiment totally we achieved 5 fractions (Fig. 4B). From the A. catechu extracts evaluated to phytochemical screening, the maximum phytochemicals (Alkaloids, flavonoids, saponins, tannins, triterpenes, coumarins, anthraquinones and phenolics) gathered from higher polarity solvent like Ac-LEE and it has been listed in Table 2.

Fig. 4

Various fractionation units. (A) Air shield TLC unit, (B) CC unit.

Table 2

Phytochemical screening of different solvent extracts of A. catechu.

Sl. No.	Phytochemical screening	A. catechu, various leaf extracts
		HNE	DER	DME	EAE	ETL
1.	Carbohydrates	–	+	–	–	–
2.	Alkaloids	–	–	–	–	+
3.	Flavonoids	+	+	–	+	+
4.	Saponins	–	+	–	–	+
5.	Tannins	+	+	+	–	+
6.	Triterpenes	+	+	+	–	+
7.	Resins	+	+	–	–	–
8.	Coumarins	+	+	+	–	+
9.	Anthraquinones	+	+	+	–	+
10.	Phenolics	+	–	+	–	+

HNE: Hexane; DER: Diethyl ether; DME: Dichloromethane; EAE: Ethyl acetate; ETL: Ethanol.

+ = noted for the presence of a phytochemical group.

– = noted for the absence of phytochemical group.

Ac-AgNPs synthesis

The Ac-LEE of Ac-AgNPs (AgNO3 + LEE) composite mixture was clearly indicated and confirming through the colour change (dark brown colour) by adding AgNO3 with Ac-LEE. It is evidently examined in the Fig. 5.

Fig. 5

Colour change observation in A. catechu LEE AgNPs.

UV, FTIR, and XRD analysis

Colour change is the basic observation of Ac-AgNPs synthesis which subjected into UV and FTIR spectral analysis of Ac-LEE of Ac-AgNPs are clearly explained in Fig. 6 and Fig. 7 and FTIR analysis supports our hypothesis that the bioreduction of Ag+ ions to Ag° carried out by C A. catechu leaf borne metabolite. Indeed, the FTIR spectrum showed major peaks at 3422.49, 2922.44, 2853.81, 1608.95, 1588.38, 1489.26, 1444.39, 1383.51, 1268.82, 1237.65, 1110.98, 1036.97, 888.44, 749.31, 724.33, 608.00, 539.81, 487.41, 429.37 cm−1. Above the peak value is strong and broad, they corresponded to functional group like alcohols, phenols (O—H stretch, H-bonded, 3422.49 cm−1). Alkanes (C-H stretch, 2922.44, 2853.81 cm−1), 1* amines is medium (N—H bend, 1608.95 cm−1), aromatics are medium (C—C stretch (in-ring), 1588.38, 1489.26, 1444.39 cm−1), alcohols, carboxylic acids, esters, ethers are strong (C—O stretch, 1268.82, 1237.65, 1110.98, 1036.97 cm−1), alkenes are strong (C—H bend stretch, 888.44, 749.31, 724.33 cm−1), alkynes is broad and strong (—CC—H: C—H bend stretch, 608.00 cm−1), and alkyl halides is medium (C—Br stretch, 539.81 cm−1). The XRD analysis of Ac-LEE of Ac-AgNPs is obviously indicated in (Fig. 8), the appeared peaks were likely matched, and their data were confirming with NIST chemical library.

Fig. 6

Spectrum observation of A. catechu LEE of AgNPs through UV-Vis.

Fig. 7

FTIR spectrum of A. catechu LEE AgNPs.

Fig.8

XRD spectrum of A. catechu LEE AgNPs.

SEM analysis

Green synthesized Ac-LEE of Ac-AgNPs evaluated by SEM analysis and its image has been displayed in Fig. 9. From the image obviously declared, the Ac-AgNPs adhered with the Ac-LEE, and Ac-AgNPs confirmed the particle size range from 23.5 nm and 53.4 nm. SEM image has been magnified into different range, and Ac-AgNPs particles appeared like beads shaped it was indicated the category of NPs.

Fig. 9

SEM image of A. catechu LEE AgNPs. (Magnified at different range (A) 10000X, (B) 20000X, (C) 30000X, (D) 50000X).

Polyphagous insect pests, S. litura and H. armigera larval and pupal toxicity values of Ac-LEE and Ac-AgNPs expressed in Table 3, Table 4. The predominant larval and pupal toxicity were experimentally demonstrated, and the highest toxicity was noticed in Ac-AgNPs than Ac-LEE. The larval toxicity of Ac-LEE and Ac-AgNPs tested against S. litura and H. armigera LC50/LC90 values were 112.48/ 114.20, 71.04/ 74.78 µg/mL and 176.53/ 185.60 and 92.57/ 96.21 µg/mL respectively. The pupal toxicity of Ac-LEE and Ac-AgNPs tested against S. litura and H. armigera LC50/LC90 values were 122.31/ 127.25, 85.33/ 88.91 µg/mL and 208.49/ 212.48 and 124.43/ 129.95 µg/mL respectively. The other statistical values were apparently demonstrated in Table 3, Table 4.

Table 3

Larval toxicity induced by A. catechu LLE and AgNPs on the larvae of selected polyphagous insect pests.

Species tested	LC50(µg/mL)	95% Fiducial limit (µg/mL)		LC90(µg/mL)	95% Fiducial limit (µg/mL)		R-value	χ2
		LCL	UCL		LCL	UCL
LEE of A. catechu
S. litura	65.47	41.73	82.45	129.27	107.66	178.04	y = 15.03 + 1.65x	8.057
H. armigera	77.35	70.00	84.20	149.43	137.84	165.17	y = 4.88 + 1.67x	3.594
AgNPs
S. litura	22.32	14.33	28.11	43.51	36.16	60.47	y = 4.24 + 0.54x	8.466
H. armigera	26.17	23.93	28.30	48.16	44.67	52.79	y = 0.50 + 0.52x	2.785

LC50 = Lethal Concentration brings out 50% mortality; LC90 = Lethal Concentration brings out 90% mortality; LCL = Lower Confidence Limit; UCL = Upper Confidence Limit; R-value = Regrasion value; χ2 = Chi- square.

Table 4

Pupal toxicity induced by A. catechu LLE and AgNPs on the fresh pupae of selected polyphagous insect pests.

Species tested	LC50(µg/mL)	95% Fiducial limit (µg/mL)		LC90(µg/mL)	95% Fiducial limit (µg/mL)			R- value	χ2
		LCL	UCL		LCL	UCL
LEE of A. catechu
S. litura	91.28	59.247	115.01	173.15	143.62	243.33		y = 9.81 + 2.08x	9.257
H. armigera	107.84	98.03	117.16	206.63	190.27	229.05		y = 2.67 + 2.24x	4.857
AgNPs
S. litura	35.90	21.07	46.67	68.24	55.22	105.15		y = 2.99 + 0.80x	11.921
H. armigera	41.14	37.78	44.38	74.85	69.38	82.18		y = 2.34 + 0.79x	3.368

LC50 = Lethal Concentration brings out 50% mortality; LC90 = Lethal Concentration brings out 90% mortality; LCL = Lower Confidence Limit; UCL = Upper Confidence Limit; R-value = Regrasion value; χ2 = Chi- square.

Discussion

Preliminary phytochemical screening

A. catechu different leaf extracts were evaluated for identifying the qualitative abundance of phytochemical screening, and that results were verified in higher number of PCs groups were noticed in high polarity organic solvent like Ac-LEE. In previously, numerous PCs investigations have been reported in various floral sources, and it has efficiently controlled in various stages of insect pests. The PCs could be extracted from various parts of flora as well as species. It is incredibly efficient bio-recourse phyto-insecticidal agents, zero effectiveness on non-target fauna/ flora and PCs are best and supreme alternative recourse against SCPs. (Mkindi et al., 2019, Mungenge et al., 2014, Riaz et al., 2018, Ahmed et al., 2020).

Ag NPs synthesis

The several previous reports obviously synthesized Ac-AgNPs have murky brown/ reddish in colour. A similar propensity has been noticed in the present observation of selected flora Ac-LEE of Ac-AgNPs. Many reports strongly empathize with our present investigation (Pirtarighat et al., 2018, Morejón et al., 2018, Roy et al., 2019, Sutthanont et al., 2019).

Ag NPs characterization

UV–Vis spectral analysis: The confirmation of Ac-LEE green synthesized Ac-AgNPs have been proven by colour change from yellow to brown it can be experimentally observed by UV–vis spectral analysis. In earlier, many kinds of research have been supported to our present investigation. (Ndikau et al., 2017, Elamawi et al., 2018, Femi-Adepoju et al., 2019, Pilaquinga et al., 2019). FTIR spectral analysis: The floral community has several functional groups such as alkaloids, anthraquinones, flavonoids, triterpenes, polyphenols etc., and it could be confirmed and observing through FTIR spectral analysis (Asmathunisha et al., 2010). Therefore, Ac-LEE found various essential function groups and it confirmed their phyto-pesticidal effects while involving with Ac-AgNPs synthesis. Many similar types of works have been observed in previously published experiments (Devaraj et al., 2013, Ajitha et al., 2014, Jyoti et al., 2016, Suresh et al., 2016, Zia et al., 2016). XRD analysis: The periodic steps of lyophilization and acquisition Ac-LEE of Ac-AgNPs dried sample subjected to XRD spectral analysis. Results of XRD spectral investigation of Ac-AgNPs crystal structure strongly agreement with earlier reported values (Ramesh et al., 2016, Kumar et al., 2017, Vizuete et al., 2017, Oves et al., 2018, Shanmuganathan et al., 2018). SEM analysis: SEM reflected image has been magnified into various range it was produced the exact size and shape (morphology) of NPs. Similar propensity were recorded in many earlier outcome (Rautela et al., 2019, Pilaquinga et al., 2019, Erdogan et al., 2019).

Larval and pupal toxicity of Ac-LEE and Ac-AgNPs were tested against polyphagous insect pests S. litura and H. armigera, the predominant toxicity was noticed in Ac-AgNPs than Ac-LEE. The similar examinations were noticed in several previously reported insecticidal activities of different insect pests (agricultural/medical pests). The naturally available photoproducts like F. religiosa and F.benghalensis leaf AgNPs are significantly reduced gut protease activity of H.armigera (Kantrao et al., 2017). The environmental safety and predominant C. cephalonica larval toxicity was noticed on O. sanctum leaf AgNPs (Gogate et al., 2018). The green products of E. hirta leaf AgNPs against fourth instar larvae and pupae of H. armigera, the selected floral AgNPs provided statistically proven significant insecticidal activity (Durga Devi et al., 2014). The Ag NPs synthesized from different flora of O.europaea, F. carica, E. japonica, C. limon, P. vera and M. nigra prepared different concentrations against various life stages of D. melanogaster and their results were given predominant toxicity were recorded from Ag NPs (Araj et al., 2015). The original novel larvicidal agent prepared from C. zedoaria AgNPs, which tested against the larvae of Cx. quinquefasciatus and it produced a prime effect as well as a significant an eco-friendly approach (Sutthanont et al., 2019). S. mammosum AgNPs showed remarkable larval toxicity discovered against vector mosquitoes and it could be acted as a potential alternative tool against disease-spreading human vectors (Pilaquinga et al., 2019). The exploration of leaf extracts of C. aromaticus and W. tinctoria AgNPs against Cx. quinquefasciatus and it could be produced maximum toxicity to selected medical pest (Dass and Mariappan, 2018).

Conclusion

Ac-LEE green synthesized Ac-AgNPs could be potential phyto-pesticidal effectiveness against selected polyphagous insect pests. Around the world above 2000, the floral community has been used as medicinal/ pesticidal properties. In globally, it is significantly sufficient ratification giving towards the prevention of many unauthorized SCPs. Based, on the present research, A. catechu could be a potential multidirectional bioactive agent to combat the polyphagous insect pests.