International Journal of Life Science and Agriculture Research

Synthetic herbicides generate resistance in weeds and cause environmental toxicity affecting other organisms in the ecosystem. Azadirachta indica (Neem) contains diverse phytochemicals with reported pharmacological and allelopathic potentials which could be employed in controlling growth of weeds. This study examined the bioherbicidal potential of A. indica against Euphorbia heterophylla, and Chromolena odorata. Fresh leaves of A. indica were collected, air-dried and ground into fine powder. The powdered sample was subjected to methanol Soxhlet extraction to obtain Azadirachta indica leaf methanol extract (AILME), followed by rotary evaporation and oven-drying to obtain dry AILME. Fresh leaves of Euphorbia heterophylla and Chromolena odorata weeds were  collected, washed, and macerated (10 g in 30 ml phosphate buffer) and used for biochemical assays. The AIMLE was analyzed using High-performance liquid chromatography (HPLC) and Gas Chromatography-Flame Ionization Detection (GC-FID). In-vitro effects of the extract were tested on the activities of acetolactate synthase (ALS), thioesterase, superoxide dismutase (SOD), catalase and glutathione transferase (GST) in E. heterophylla and C. odorata. The HPLC identified B-Caryophyllene, Trans-B-Farnes, Alpha Ionone, Quercetin and Azadirachtol, while GC-FID showed Azadirachtol and Azadirachnol as major compounds in AIMLE. The AILME reduced ALS and thioesterase activities comparable to the standards, glufosinate and glyphosate in Euphorbiaheterophylla.  The AILME reduced SOD and GST activities in C. odorata weed comparable to standards. This study thus indicates that A. indica leaf extract contains phytochemicals, exerting herbicidal potential involving oxidative stress and disruptions of amino acid and fatty acid biosynthesis in E. heterophylla and C. odorata.

Azadirachta indica, bioherbicide, Chromolena odorata, enzyme activity, Euphorbia heterophylla

1. Nichols, V.; Verhulst, N.; Cox, R.; Govaerts, B. Weed dynamics and conservation agriculture principles: A review. Field Crops Res. 2015, 183, 56–68
2. Oerke, E.C. Crop losses to pests. J. Agric. Sci. 2006, 144, 31–43
3. Zheljazkov V. and Zhalnov I. (1995). Effect of herbicides on yield and quality of Coriandrum sativum L. J. Essent. Oil Res. 7(6): 633-639.
4. Sagarka B., Ramani B., Mathukia K., Khanpara D. (2005). Integrated weed management in coriander. Indian J. Weed Sci. 37(3/4): 231-233.
5. Hasan M., Ahmad-Hamdani M.S., Rosli A.M., Hamdan H. (2021). Bioherbicides: An Eco-Friendly Tool for Sustainable Weed Management. Plants. 10: 1212.
6. Singh, S.; Chhokar, R.S.; Gopal, R.; Ladha, J.K.; Gupta, R.K.; Kumar, V.; Singh, M. IntegratedWeed Management: A key to Success for Direct-seeded Rice in the Indo-Gangetic Plains. In Integrated Crop and Resource Management in the Rice—Wheat System of South Asia; International Rice Research Institute: Los Banos, The Philippines, 2009; pp. 261–278.
7. Tufail, T., U. Ain, H. B., Ijaz, A., Nasir, M. A., Ikram, A., Noreen, S., Arshad, M. T., & Abdullahi, M. A. (2025). Neem ( Azadirachta indica ): A Miracle Herb; Panacea for All Ailments. Food Science & Nutrition, 13(9), e70820.  
8. Nkechinyere, M.K. (2019). The insecticidal activity of neem(Azadirachta indica) against weevils in stored Bambara nuts (Vignasu bterranea) and beans (Phaseolus vulgaris). American Journal of Biomedical and life Sci., 7(2): 31-35.
9. Alzohairy, M. A. (2016). Therapeutics Role of Azadirachta indica (Neem) and Their Active Constituents in Diseases Prevention and Treatment. Evidence-Based Complementary and Alternative Medicine : ECAM, 2016, 7382506. https://doi.org/10.1155/2016/7382506
10. Adeleke G.E.,  Adedosu O.T., AdegbolA P.I., Akintola A.O., Ogunmola I.A., Omidiran O.K. (2021). Chemical screening of Azadirachta indica seed kernel extract and its in-vitro selected enzyme responses in Zonocerus variegatus (Grasshopper). Adv. Biores. 12 (4): 92-103.
11. Wang, Y., Haung, X., Cheng, B.H., Zhang, Z. (2020). Growth performance and enzyme response of the grasshopper, Calliptamus abbreviates orthoptera: Acridae) to six plant-derived compounds. Journal of Insect Science., 20(3): 14; 1-8.
12. Naz S., Khan S. N.,  Khan A, Farooq S. (2013). Herbicidal potential of Azadirachta indica leavesextract against Achyranthes aspera and Senna occidentalis. Mycopath. 11(2): 81-84.
13. Lowry, O.H., Rosbrough, N.J,, Farr, A.L., et al. (1951). Protein measurement with the Folin- phenol reagent. J. Biol. Chem., 193: 265-275.
14. Durigon M. R., Mariani F,, dos Santos F. M., Vargas L., Chavarria G. (2018). Properties of the enzyme acetolactate synthase in herbicide resistant canola. Plant protection. 7.(3): 485-492.
15. Ellman GL (1959) Tissue sulfhydryl groups. Arch Biochem Biophys. 82:70–77
16. Zhuang Z, Song F, Zhao H, Li L, Cao J, Eisenstein E, Herzberg O, Dunaway-Mariano D (2008) Divergence of function in the hot dog fold enzyme superfamily: the bacterial thioesterase YciA. Biochemistry 47:2789–2796
17. Misra, H.P. and Fridovch, J. (1975). The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase. J. Biol. Chem., 1975. 247: 3170-3175.
18. Aebi, H.  (1984). Catalase in vitro. In: Packer L. Editor. Methods in Enzymology. Orlando FL: Academic Press. pp 121-126.
19. Habig W., Pabst M., Jakoby W. (1974). Glutathione S-transferase. The first enzymatic step in mercapturic acid formation. J Biol Chem.; 249: 7130–7139.
20. Parven, A., Meftaul I. M., Venkateswarlu K., Megharaj M. (2024). Pre-emergence herbicides used in urban and agricultural settings: dissipation and ecological implications. Environ. 46(12): 493. 
21. Soltys, D., Krasuska, U., Bogatek, R., & Gniazdowska, A. (2013). Allelochemicals as bioherbicides—present and perspectives. In A. J. Price & J. A. Kelton (Eds.), Herbicides—Current Research and Case Studies in Use (pp. 517-542). InTech Open.  
22. Bashar, H. K., Juraimi, A. S., Ahmad-Hamdani, M. S., Uddin, M. K., Asib, N., Anwar, M. P., & Hossain, A. (2022). Determination and quantification of phytochemicals from the leaf extract of parthenium hysterophorus L. and their physio-biochemical responses to several crop and weed species. Plants, 11(23), 3209. 
23.  Kostina-Bednarz, M., Płonka, J., & Barchanska, H. (2023). Allelopathy as a source of bioherbicides: Challenges and prospects for sustainable agriculture. Reviews in Environmental Science and Bio/Technology, 22, 471-504. 
24.  Triolet, M., Guillemin, J. P., Andre, O., & Steinberg, C. (2020). Fungal-based bioherbicides for weed control: A myth or a reality? Weed Research, 60(1), 60-77. 
25.  Islam, A. K. M. M., Karim, S. M. R., Kheya, S. A., & Yeasmin, S. (2024). Unlocking the potential of bioherbicides for sustainable and environment friendly weed management. Heliyon, 10(16), e35888.
26.  Duke, S. O., Pan, Z., Bajsa-Hirschel, J., & Boyette, C. D. (2022). The potential future roles of natural compounds and microbial bioherbicides in weed management in crops. Advances in Weed Science, 40, e20220039.  
27. Traxler, C., Gaines, T. A., Küpper, A., Luemmen, P., & Dayan, F. E. (2023). The nexus between reactive oxygen species and the mechanism of action of herbicides. The Journal of biological chemistry, 299(11), 105267. 
28. Grene, R. (2002). Oxidative stress and acclimation mechanisms in plants. Arab. Book Am. Soc. Plant Biol., 1, e0036.
29. Quiles, M.J.; López, N.I. (2004) Photoinhibition of photosystems I and II induced by exposure to high light intensity during oat plant growth: Effects on the chloroplast nadh dehydrogenase complex. Plant Sci., 166, 815–823
30. Dietz, K.J.; Mittler, R.; Noctor, G. (2016). Recent progress in understanding the role of reactive oxygen species in plant cell signaling. Plant Physiol., 171, 1535–1539
31. Huang, S.; Van Aken, O.; Schwarzländer, M.; Belt, K.; Millar, A.H. (2016). The roles of mitochondrial reactive oxygen species in cellular signaling and stress response in plants. Plant Physiol. 171, 1551–1559.
32. Khurana, N.; Sharma, N.; Patil, S.; Asmita, G. (2016) Phytopharmacological properties of Sida cordifolia: A review of folklore use and pharmacological activities. Asian J. Pharm. Clin. Res., 2, 52–58.
33. Hasanuzzaman, M., Bhuyan, M. H. M. B., Zulfiqar, F., Raza, A., Mohsin, S. M., Mahmud, J. A., Fujita, M., & Fotopoulos, V. (2020). Reactive oxygen species and antioxidant defense in plants under abiotic stress: Revisiting the crucial role of a universal defense regulator. Antioxidants, 9(8), 681. 
34. Sharma, P., Jha, A. B., Dubey, R. S., & Pessarakli, M. (2012). Reactive oxygen species, oxidative damage, and antioxidative defense mechanism in plants under stressful conditions. Journal of Botany, 2012, 217037.  
35. Gill, S. S., & Tuteja, N. (2010). Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiology and Biochemistry, 48(12), 909-930.  
36. Cummins, I., Dixon, D. P., Freitag-Pohl, S., Skipsey, M., & Edwards, R. (2011). Multiple roles for plant glutathione transferases in xenobiotic detoxification. Drug Metabolism Reviews, 43(2), 266-280.
37.  Edwards, R., Dixon, D. P., & Walbot, V. (2000). Plant glutathione S-transferases: enzymes with multiple functions in sickness and in health. Trends in Plant Science, 5(5), 193-198.  
38. Duggleby, R. G., McCourt, J. A., & Guddat, L. W. (2008). Structure and mechanism of inhibition of plant acetohydroxyacid synthase. Plant Physiology and Biochemistry, 46(3), 309-324.  
39. Zhou, Q., Liu, W., Zhang, Y., & Liu, K. K. (2007). Action mechanisms of acetolactate synthase-inhibiting herbicides. Pesticide Biochemistry and Physiology, 89(2), 89-96.  
40. Caswell B. T., Carvallo C. C., Nguyen H., Roy M., Nguyen T., Cantu D. C. (2022). Thioesterase enzyme families: Functions, structures, and mechanisms. Proteins Sci. 31(3): 652 -676. 
41. Johnen P., Zimmermann S., Betz M., Hendriks J., Zimmermann A., Marnet M., De I., Zimmermann G., Kibat C., Cornaciu I., Mariaule V., Pica A., Clavel D., Marquez J. A., Witschel M. 92022). Inhibition of acyl-ACP thioesterase as site of action of the commercial herbicides cumyluron, oxaziclomefone, brombutide, methyldymron and tebutam.  Pest Management Science. 78(8): 3620 – 3529.