Egypt. J. Prot. Res. Inst. (2020), 3 (2): 777 - 785

Egyptian Journal of Plant Protection Research Institute www.ejppri.eg.net

Phytochemical and insecticidal evaluation of agro-waste of Lagenaria siceraria () plant against the cotton leafworm Spodoptera littoralis (Lepidoptera: Noctuidae)

Anwaar, M. Abaza Plant Protection Research Institute, Agricultural Research Center, Dokki, Giza, Egypt. ARTICLE INFO Abstract: Article History Lagenaria siceraria (Cucurbitaceae) commonly known as Received: 19/4/2020 bottle gourd cultivated in Africa, Asia and now cultivated in Egypt. Accepted: 28/6/ 2020 After harvesting fruits of L. siceraria, the leaves of the are ______remained as agro-waste in the field. Preliminary phytochemical Keywords screening of L. siceraria leaves revealed the presence of steroids, Agro-waste, Lagenaria alkaloids, flavonoids, terpenoids, tannins and saponnins. The leaves siceraria, cotton extracted by ethanol and fractionated via solvents of different leafworm, Spodoptera polarities; petroleum ether, ethyl acetate and n-butanol. The toxicity of littoralis, Gas extracts was tested against the cotton leafworm, Spodoptera littoralis Chromatography-Mass (Boisduval) (Lepidoptera: Noctuidae) under laboratory conditions. Spectrometry and The petroleum ether extract exhibited more activity followed by control. ethanol, ethyl acetate and n-butanol. The LC were 0.201, 0.352, 50 0.539 and 0.735%, respectively. The petroleum ether extract was

subjected to saponification reaction and its chemical constituents were

investigated by Gas Chromatography-Mass Spectrometry. This

indicated that the agro-waste of L. siceraria plant can be applied as

green insecticide to control the cotton leafworm, S. littoralis. Introduction The Plant kingdom produce a great warmer region of world (Rahman, 2003). variety of phytochemicals (secondary Among all the plants of Cucurbitaceae metabolites) including flavonoids, saponnins, family Lagenaria species is the most popular. tannins, steroids, terpenoids and alkaloids are Lagenaria siceraria (Molina) member of often important for mediating interactions genus Lagenaria that is derived from the between plants and their biotic environment. word lagena, meaning the bottle. L. siceraria They can be models of active defense against known as bottle gourd is a common fruit phytophagous insects and pathogens. vegetable used throughout the India. This Cucurbitaceae family is commonly known as species follows the plant Kingdom : Plantae, gourd, melon and pumpkin family. This Division: Magnoliophyta, Class: family is composed of 118 genera and 825 Magnoliopsida, Order: , Family: species, which are widely distributed in the Cucurbitaceae, Genus: Lagenaria and Species

777

Abaza, 2020

: L. siceraria L. siceraria large pubescent, compressed with two flat facial ridges annual, prostrate or climbing herb cultivated (Figure, 1) . in Africa, Asia and now cultivated in Egypt, its fruit is a source of vitamin-B complex and choline along with fair source of vitamin-C and β-carotene, amongst any other vegetable known to man till date, it is also good source of minerals and amino acids (Kirtikar and Basu, 2001). The fruits have antimicrobial, cytotoxicity, anticancer and

antihepatotoxicity properties (Answar et al., 1984; Desta, 1993; Furukawa et al., 1995 and Shiwaikar and Sreenivasan, 1996). Lagenin, a ribosome inactivating protein (RIP) isolated from the seeds of L. siceraria possesses immune protective, antitumor, anti HIV and anti proliferative properties (Wang and Ng, 2000). After harvesting fruits of L. siceraria, the leaves of the plants are remained as agro- waste in the field. Rahman (2003) described Figure (1): Lagenaria siceraria plant the morphology of this plant species as This study was planned to evaluate follows: Stem: The stem is prostrate or the insecticidal activity of ethanolic extract of climbing in nature with angular, ribbed thick, L. siceraria agro-waste (leaves) and its brittle, softly hairy. Leaves: Leaves are fractions in solvents of different polarities simple with long petiole from 25-30 mm long against the cotton leafworm Spodoptera thick, hallow, densely hairy with two small littoralis (Boisduval) (Lepidoptera: Noctuidae) under laboratory conditions. . lateral glands located at the leaf base. Leaf Depending on the insecticidal evaluation, lamina: The lamina of leaf is usually five identify the chemical constituents in the lobed, broad cordate, pubescent with soft bioactive fraction using chromatographic hairs and the tendrils are branched. The techniques. flowers are solitary axillary, pedicellate, Materials and methods unisexual and monoecious. Petals: Petals are 1. Chemicals: mainly five, white or cream colored which The organic solvents of different polarities (petroleum ether, ethyl acetate, opens in the evening. Fruits: Fruits are green ethanol and n-butanol) used in the present in color which turns to yellow on maturity. work were obtained from Edwic Company. Fruits are large densely hairy often 2. Gas Chromatography-Mass cylindrical or flask shaped or globose. They Spectroscopy analysis (GC/MS): are constricted at the middle. Pulp: Pulp is GC-MS analysis was performed on a Varian pale brown in color and the dried fruit has GC interfaced to Finnegan SSQ 7000 Mass selective Detector (SMD) with ICIS V2.0 thick hard hollow structure. Seeds: Seeds are data system for MS identification of the GC embedded in the spongy pulp and

778

Egypt. J. Plant Prot. Res. Inst. (2020), 3 (2): 777 - 785 components. The column used was DB-5 layer was separated in a flask, and the (J&W Scientific, Folosm, CA) cross-linked process was repeated three times to ensure fused silica capillary column (30 m. long, maximum extraction. In the same manner, 0.25mm. internal diameter) coated with poloy ethyl acetate and n-butanol fractions were dimethyl-siloxane (0.5μm. film thickness). obtained, leaving behind a residual aqueous The oven temperature was programmed from fraction. The solvents were evaporated in 50 °С for 3 min., at isothermal, then heating rotary evaporator and the dried fractions were by 7 °С /min. to 250 °С and isothermally for collected. 10 min., at 250 °С. Injector temperature was 6. Preliminary phytochemical screening; 200 °С and the volume injected was 0.5μl. Preliminary phytochemical screening of Transition-line and ion source temperature L. sicerariaA leaves was carried out as the were 250 °С and 150 °С, respectively. The standard prodedures (Sofowara, 1993 and mass spectrometer had a delay of 3 min. to Harborne, 1998). avoid the solvent plead and then scanned 6.1. Alkaloids: The alcoholic extract from m/z 50 to m/z 300. Ionization energy (corresponding to 2.5 g leaves powder) was set at 70 eV. (Agricultural Research was evaporated to dryness and the residue Center, Dokki, Giza). was heated on a boiling water bath with 3. Experimental insect: 2N HCl (2 ml). After cooling the mixture Newly molted 4th instar larvae of the was filtered and the filtrate was treated cotton leafworm S. littoralis were selected for with few drops of Mayer's reagent. this study. The culture of laboratory Mayer's reagent: dissolve 136 g susceptible stain was reared under optimum HgCl2 in 60 ml water and 5 g KI in 10 ml conditions 25±2°C, 75±5% RH. and (16L:8D) water. Combine both solutions and make up light: dark photoperiod in the plant protection with water to 100 ml. add a few drops to an research institute, Dokki, Giza, Egypt. The acidified solution (HCl or diluted H2SO4), a larvae were reared on castor bean leaves yellow to orange precipitate will appear 4. Plant material: indicated a positive test. The agro-waste leaves ofA L. sicerariaA 6.2. Terpenoid: Alcoholic extract was plant were remained after harvesting fruits. L. evaporated and the residue was treated sicerariaAleaves were collected from field of with anhydrous chloroform and filtered. experimental farm in Zagazig Governorate, Conc. sulphuric acid was added to the Egypt and identified by Dr. Reda Mohamed filtrate and shaken. Yellow color was El-Sayed, Faculty of Agriculture, and produced, changed to orange then red. Zagazig University. The collected leaves 6.3. Steroids: Alcoholic extract was were cleaned, left to dry under shade and evaporated and the residue was treated homogenized to fine powder using electric with anhydrous chloroform and filtered. mill. The filtrate was treated with acetic acid 5. Extraction and fractionation and drops of Conc. sulphuric acid were A weighed amount of the powder (250 added carefully on the side of test tube. gm) was soaked in ethanol, the obtained Greenish color appeared (lower layer). extract from filtration was evaporated under 6.4. Flavonoids: A small quantity of the vacuum at 40°C on rotary evaporator to yield extract was dissolved in diluted sodium (45.32 gm). Part of the crude extract (30 gm) hydroxide (5%) and hydrochloric acid was was suspended in distilled water in a added to the mixture. A yellow solution separating funnel, petroleum ether was added that turns colorless on addition of to the funnel which was shaken gently and hydrochloric acid indicated the presence allowed to stand for about 2 hrs, the pet. ether of flavonoids.

779

Abaza, 2020

6.5. Saponins: one gram of the leaves was corrected mortality percentages, the boiled with 10 ml water for few minutes corresponding toxicity lines (LC-P lines) and filtered. The filtrate was vigorously were estimated in addition to determine LC25 shaken. The persistent froth (1 cm height) and LC50 values and their confidence limits, was observed for 1 hr. indicates the slope values of tested extract were also presence of saponins. estimated. 6.6. Anthraquinones: The alcoholic extract 9. Chromatography and identification of (corresponding to 5g plant material) was chemical constituents: evaporated to dryness and the residue was Petroleum ether fraction, which showed heated on a boiling water bath with 2N the highest toxicity activity against the 4th HCl (10 ml). After cooling the mixture instar larvae, was selected for phytochemical was filtered and the filtrate was extracted investigation through GC/MS technique. with benzene. The benzene extract was Pet. ether extract was subjected to shaken with NH4OH (5 ml) and a positive Saponification reaction; hydrolysis with 10% reaction was evidenced by the formation alcoholic NaOH over water bath under reflux of a red color in the alkaline layer. for 30 min., cooling, then diluted with water 6.7. Tannins: About one gram of the and extracted wit diethyl ether afforded un- powdered sample was boiled with 10 mL saponifiable fraction, while the saponifiable of distilled water for five minutes, filtered fraction was acidified with diluted HCl then while hot and a few drops of ferric extracted with diethyl ether. The saponifiable chloride reagent was added to the filtrate. fraction was analyzed by GC/MS technique A red color indicates a positive test. for characterization the petroleum ether 7. Preparation of tested concentrations: constituents. The prepared fractions of L. siceraria Results and discussion were diluted in distilled water containing one L. siceraria plant was cultivated in drop of an emulsifier Tween®20 (Tween®20; Africa, Asia and now cultivated in Egypt. Sigma-Aldrich), to ensure complete solubility After harvesting fruits of the plant, the leaves of constituents. The applied concentrations are remained as agro-waste. Phytochemical were selected after preliminary bioassays screening of L. sicerariaA leaves showed the with a wide range of concentrations to presence of flavonoids, saponins, tannins, determine the proper range. Two negative steroids, terpenoids and alkaloids active control treatments were used in each phytoconstituents as shown in Table (1). experiment. One of them was for one drop of L. siceraria leaves were extracted by emulsifier (Tween®20) in water, where the ethanol and fractionated via solvents of Tween®20 solutions showed no significant different polarities; petroleum ether, ethyl mortality effect against S. littoralis larvae acetate and n-butanol, and examined as (data not shown) compared with water. natural insecticides against newly molted 4th 8. Toxicological evaluation: instar larvae of Spodoptera littoralis under Leaf dipping technique was applied; laboratory conditions. , the tested extracts of Castor bean leaves were dipped for 30 L. siceraria leaves exhibited a high degree of seconds in each concentration of tested efficiency as insecticide against S. littoralis extracts then left to dry. The treated leaves larvae. were offered to newly molted 4th instar larvae The petroleum ether extract exhibited a of S. littoralis for 48 hrs. then replaced by high degree of efficiency as insecticide untreated ones. Accumulative mortality followed by ethanol, ethyl acetate and finally percentages were recorded, then corrected n-butanol extracts. The activity was according Abbott's formula (1925). From the concentration dependent of the tested

780

Egypt. J. Plant Prot. Res. Inst. (2020), 3 (2): 777 - 785 extracts. LC25 and LC50 values and their suggested that the crude methanol extract of confidence limits obtained from probit L. siceraria leaves produced anthelmintic analysis for mortality values are showed in activity against Indian earthworm, Pheretima Table (2). posthuma when compared with the The obtained results agree with those conventionally used drug (Albendazole). obtained by Amit and Sangh (2012) who

Table (1): Preliminary phytochemical screening of Lagenaria siceraria leaves. Test Observation Result Test for Flavonoids Yellow color was observed in Presence of Flavonoids Filtrate + NaOH (5%) test tube, then turned to colorless on addition of HCl Test for Tannins Brownish green to blue black Presence of Tannins 5g of leaf extract + 10mL of precipitate was observed in test distilled water. Filter + drops of tube. ferric chloride (0.1%)

Test for Saponins Froth was observed in the test Presence of Saponnins (Frothing test) tube, while disappeared after 1g of leaves + distilled water + about one hour. gentle warming and shaking vigorously

Test for Alkaloids An yellowish precipitate was Presence of Alkaloids alcoholic extract + 5mL observed in test tube Aqueous HCl (2N) + drops of Mayer’s solution

Test for Terpenoids yellow layer then turned to Presence of Terpenoids alcoholic extract + 2 ml CHCl3 orange was observed in test tube + 3 ml Conc. H2SO4

Test of Steroids Greenish color layer was Presence of Steroids alcoholic extract + 2 ml CHCl3 observed on test tube + 3 ml Conc. H2SO4 + acetic acid

Test for Anthraquinones No color change was observed. Absence of 5g of leaves + 10mL Benzene. Anthraquinones Filtrate +5mL 10% NH4OH solution

781

Abaza, 2020

Table (2): Insecticidal activity of Lagenaria siceraria leaves against the 4th instar larvae of Spodoptera littoralis.

Tested extract LC25 (%) LC50 (%) Slope ± SE Toxicity index Confidence limits at Confidence limits at 95% 95% Petroleum 0.083 0.201 1.754 ± 0.177 100 Ether (0.057 – 0.108) (0.164 – 0.238) Ethanol 0.159 0.352 1.947 ± 0.179 56.94 (0.125 – 0.191) (0.305 – 0.407)

Ethyl acetate 0.213 0.539 1.669 ± 0.179 37.22 (0.166 – 0.257) (0.457 – 0.654) n-butanol 0.310 0.735 1.801 ± 0.196 27.34 (0.254 – 0.365) (0.618 – 0.915) The obtained insecticidal evaluation reaction, and its chemical constituents were data indicated that pet. Ether fraction is the chemically investigated using GC-MS most toxic extract. For this reason, this technique (Table ,3). extract was subjected to saponification Table (3): Chemical constituents of petroleum ether extract. Component name R.T Area% Molecular formula

Dodecane 10.42 0.41 C12H26

Hexadecene 16.81 0.43 C16H23

Octadecane 20.96 0.20 C18H36

Neophytadiene 24.21 0.35 C20H38 6,10,14-trimethyl-1,2- 25.05 1.16 C H O pentadecanone 18 36 2

3,7,11,15-tetramethyl-1,2- 28.51 53.01 C H O hexadecen-1-ol (Phytol) 20 40

9,12,15-octadecatrienal 32.82 2.05 C18H30O

Eicosene 34.06 1.09 C20H40

Docosene 36.49 2.38 C22H44

Hexacosene 40.11 2.24 C26H52

Squalene 42.33 3.02 C30H50

Cholesterol 44.38 0.32 C27H46O

Campesterol 46.48 1.04 C28H48O

Spinasterol 47.11 7.89 C29H48O

β-Sitosterol 47.76 1.38 C29H50O

Fucosterol 48.01 1.65 C29H48O

22-dihydro-spinasterol 48.78 5.78 C29H50O

782

Egypt. J. Plant Prot. Res. Inst. (2020), 3 (2): 777 - 785

The qualitative and quantitative constituents were phytol (53.1%), spinasterol compositions of petroleum ether extract were (7.89%) and 22-Dihydrospinasterol (5.78%). analyzed GC/MS where, the most abundant

Phytol

Spinasterol 22-Dihydrospinasterol The insecticidal activity of petroleum to potent antimicrobial activity against the ether extract of L. siceraria leaves against the bacterial strains: Escherichia coli, 4th instar larvae of S. littoralis may due to the Enterococcus faecalis, Klebsiella pneumonia, presence of natural phytosterols as Salmonella typhi, Staphylococcus aureus and spinasterol, 22-dihydrospinasterol, antifungal strains: such as Aspergillus flavus, Fucosterol, β-Sitosterol and Campesterol., Aspergillus oryzae and Trichoderma Where spinasterol exhibited different harzianum (Nagaraja et al., 2012). biological activities including anti- Cucurbitaceae family is major source of ulcerogenic (Klein et al., 2010), anti- medicinal agents since ancient time. Various inflammatory (Borges et al., 2014), and plants parts of this family have been antiproliferative activities (Ntie-Kang and established for their pharmacological Yong, 2014). Spinasterol, 22,23- potential. The use of L. siceraria, Cucumis dihydrospinasterol possess pharmacological sativus and maxima leaves as and cytotoxic exertions likewise, it was useful anti-oxidant and cytotoxic agents isolated from Bougainvillea spectabillis and (Alamgir et al., 2016). The potency of exhibited strong inhibition of xanthine methanol, acetone, chloroform and petroleum oxidase being IC50 of 39.21 µM, (Chang et ether extracts of Lagenaria siceraria leaves al., 1994). against the 3rd instar larvae of the housefly, Meneses-Sagrero et al. (2017), Musca domestica (Diptera: Muscidae) which identified spinasterol from the methanol consider as a diseases vector was evaluated extract of Stegnosperma halimifolium and by Mostafa et al., (2018), where all plant evaluated against cancer cell line, they found extracts showed a larvicidal activity against that spinasterol exhibited potential activity as the 3rd instar larvae of M. domestica larvae; antiproliferative against two cell lines of however, the petroleum ether extract was cervical cancer such as HeLa and RAW found that to be the more effective than 264.7. The petroleum ether, chloroform, chloroform, acetone and methanol extracts. methanol, absolute alcohol and water of The LC50 values of methanol, acetone, Lagenaria siceraria plant showed moderate chloroform and petroleum ether extracts

783

Abaza, 2020 recorded 468.5, 432.1, 433. 8 and 101.4 ppm; H.C. (1994): Inhibitory effects of respectively. As well, at the LC50 values of phenolics on xanthine oxidase. the tested extracts exhibited repellent activity Anticancer Res., 14: 501–506. against M. domestica adults. The effective Desta, B. (1993): Ethiopian traditional herbal plant extract that exhibited high antifeedant drugs. Part II: antimicrobial activity of or repellency action was petroleum ether 63 medicinal plants. J. extract as compared to chloroform, acetone Ethnopharmacol., 39: 129 – 139. and methanol extracts. From all mentioned Furukawa, K.; Yamamoto, I.; Tanida, N. ; results, it can be concluded that the petroleum Tsujiai, T.; Nishikawa, M. ; ether extract of L.siceraria leaves (Agro- Nariswa, T. and Shimoyama, T. waste) can applied as natural insecticide to (1995): The effects of dietary fiber control the cotton leafworm, S. littoralis. from Lagenaria siceraria (Yugao References melon) on colonic carcinogenesis in Abbott, W.S. (1925): A method of Mice. Cancer, 75: 1508-1515. computing the effectiveness of an Harborne, J.B. (1998): Phytochemical insecticide. J. Econ. Entomol., 18: methods.A guide to modern 265-267. techniques of plant analysis. 3rd Alamgir, H.M.; Sheikh Arman, M.; edition. Chapman and Hall, London. Muniruddin, A. and Kayser, M. d. Kirtikar, K.R. and Basu, B.D. (2001): S. (2016): Phytochemical and Indian Medicinal Plants. Ed 2nd, pharmacological investigation of Oriental Enterprises, Dehradun, 1116- Lagenaria siceraria, Cucumis sativus 1119. and Cucurbita maxima. European Klein, L.C.; Gandolfi, R.B.; Santin, J.R.; Journal of Medicinal Plants, 12(3): 1- Lemos, M. ; Cechinel Filho, V. and 13. de Andrade, S.F. (2010): Amit, K. and Sangh, P. (2012): In vitro Antiulcerogenic activity of extract, anthelmintic activity of Lagenaria fractions, and some compounds siceraria leaves in Indian adult obtained from Polygala cyparissias earthworm. Journal of St. Hillaire and Moquin Pharmacognosy and Phytochemistry, (Polygalaceae). Naunyn- 1(4): 39-42. Schmiedeberg’s Arch. Pharmacol., Answar, B.S.; Bhakuni, D.S.; Goel, A.K. ; 381: 121-126. Kar, K.; Mehrotra, B.N. and Meneses-Sagrero, S.E.; Navarro-Navarro, Mukherjee, K.C. (1984): Screening M.; Ruiz-Bustos, E.; Del-Toro- of Indian plants for biological Sánche, C.L.; Jiménez-Estrada, M. activity: Part X. Indian. J. Exp. Biol., and Robles-Zepeda, R.E. (2017): 22: 312-332. Antiproliferative activity of Borges, F.R.; Silva, M.D.; Córdova, M.M.; spinasterol isolated of Stegnosperma Schambach, T.R.; Pizzolatti, M.G. halimifolium (Benth, 1844). Saudi and Santos, A.R. (2014): Anti- Pharm. J., 25: 1137-1143. inflammatory action of Mostafa, I.H.; Ahmed, S.B.; Ahmed, Z.I.S. hydroalcoholic extract, and Hossam, A.A. (2018): dichloromethane fraction and steroid Larvicidal and repellent activity of α-spinasterol from Polygala sabulosa Lagenaria siceraria (Cucurbitaceae) in LPS-induced peritonitis in mice. J. extracts against the house fly: Musca Ethnopharmacol., 151: 144-150. Domestica (Diptera: Muscidae). Chang,W.; Chang, Y. ; Lu, F. and Chiang, Journal of the Egyptian Society of

784

Egypt. J. Plant Prot. Res. Inst. (2020), 3 (2): 777 - 785

Parasitology, 48(1): 45 – 53. Nagaraja, Y. P.; Geetha, K. N. and Vinay, M. S. (2012): Antimicrobial effect of Lagenaria siceraria (Mol.) Standley, against certain bacteria and fungal strains. Journal of Applied and Natural Science, 3 (1): 124-127. Ntie-Kang, F. and Yong, J.N. (2014): The chemistry and biological activities of natural products from Northern African plant families: From Aloaceae to Cupressaceae. RSC Adv.,4: 61975– 61991. Rahman, A.S.H. (2003): Bottle gourd (Lagenaria siceraria) A vegetable for good health. Natural Product Radiance., 2(5): 249-250. Shiwaikar, A. and Sreenivasan, K.K. (1996): Chemical investigation and antihepatotoxicity activity of the fruits of Lagenaria siceraria. Indian J. Pharm.Sc., 58: 197-202. Sofowara, A. (1993): Screening plants for bioactive agents. In: Medicinal Plants and Traditional Medicinal in Africa, second ed., Spectrum Books Ltd., Sunshine House, Ibadan, Nigeria, pp. 134-156. Wang, H.X. and Ng, T.B. (2000): Lagenin, a novel ribosome inactivating protein with ribonucleatic activity from bottle gourd (Lagenaria siceraria) seeds. Life Sci., 67: 2631-2638.

785