Manoj Mandal¹, Sushmita Nandy¹, Bidu Pal¹*, Md Enamul Haque² and Nayan Chandra Howlader³

¹Department of Biochemistry and Molecular Biology, Life Science Faculty, Gopal- ganj Science and Technology University, Gopalganj-8105, Bangladesh
²Department of Biochemistry and Molecular Biology, University of Dhaka, Dhaka 1000, Bangladesh
³Department of Horticulture, Faculty of Agriculture, Bangladesh Agricultural Uni- versity, Mymensingh 2202, Bangladesh

*Corresponding Author: Bidu Pal, Department of Biochemistry and Molecular Biology, Life Science Faculty, Gopalganj Science and Technology University, Gopal- ganj-8105, Bangladesh.

Received: August 03, 2026; Published: August 27, 2026

Abstract

Diospyros blancoi (velvet apple) is a medicinal plant whose stem bark remains relatively underexplored phytochemically. This study aimed to isolate and characterize a major constituent from the chloroform extract of D. blancoi stem bark and to evaluate its antibacterial, antioxidant, and preliminary cytotoxic activities. Fractionation of the chloroform extract by vacuum liquid chromatography followed by preparative thin-layer chromatography yielded compound DB-F7-B1, which was identified as α-linolenic acid based on its ¹H and ¹³C nuclear magnetic resonance spectroscopic characteristics and comparison with reported literature data. The chloroform extract exhibited antibacterial activity against six of nine tested Gram-positive and Gram-negative bacterial strains, with inhibition zones ranging from 8 to 13 mm. The extract contained 11.86 mg gallic acid equivalents (GAE)/g of total phenolics and 4.91 mg quercetin equivalents (QE)/g of total flavonoids. Among the tested fractions, the chloroform-soluble fraction showed the strongest DPPH radical-scavenging activity, with an IC₅₀ value of 41.0 μg/mL, whereas the crude extract exhibited preliminary cytotoxic activity in the brine shrimp lethality assay, with an LC₅₀ value of 3.255 μg/mL. Overall, the findings contribute to the phytochemical characterization of D. blancoi stem bark and demonstrate its preliminary antibacterial, antioxidant, and cytotoxic potential. Further studies involving MIC/MBC determination, bioactivity-guided fractionation, and in vivo evaluation are warranted to establish the therapeutic relevance of its bioactive constituents.

Keywords: Diospyros blancoi; α-linolenic Acid; Phytochemical Isolation; Antibacterial Activity; Antioxidant Activity; Brine Shrimp Lethality Assay

References

1. Nasim N., et al. “Plant-derived natural products for drug discovery: current approaches and prospects”. The Nucleus3 (2022): 399-411.
2. Khan J., et al. “Screening of Medicinal Plants, Herbal Drug Formulation, and Challenges”. In Sustainable Healthcare (Advances in Science, Technology and Innovation) (2026): 35-44.
3. Tamboli F A. “Phytochemical synergy: A forgotten key to therapeutic efficacy”. Journal of Pharmaceutical and Biological Sciences2 (2026): 68-69.
4. Alam M K and Sarwar A K M G. “Ethnobotanical Research in Bangladesh - A Review”. Plant Science Today (2024).
5. Rauf A., et al. “Diospyros, an under-utilized, multi-purpose plant genus: A review”. Biomed Pharmacotherapy 91 (2017): 714-730.
6. Das SC., et al. “Phytochemical and Preliminary Pharmacological Evaluation of Diospyros blancoi Fruits”. Dhaka University Journal of Pharmaceutical Sciences2 (2024): 113-122.
7. Nahar L., et al. “Phytometabolites, Pharmacological Effects, Ethnomedicinal Properties, and Bioeconomic Potential of Velvet Apple (Diospyros discolor Willd.): A Review”. Chemistry and Biodiversity5 (2025): e202402168.
8. Ribeiro A., et al. “The Genus Diospyros: A Review of Novel Insights into the Biological Activity and Species of Mozambican Flora”. Plants15 (2023): 2833.
9. Calder PC. “Functional Roles of Fatty Acids and Their Effects on Human Health”. Journal of Parenteral and Enteral Nutrition1S (2015): 18S-32S.
10. Bertoni C., et al. “Alpha-Linolenic Acid and Cardiovascular Events: A Narrative Review”. International Journal of Molecular Sciences18 (2023): 14319.
11. Ren J and Chung SH. “Anti-inflammatory Effect of α-Linolenic Acid and Its Mode of Action through the Inhibition of Nitric Oxide Production and Inducible Nitric Oxide Synthase Gene Expression via NF-κB and Mitogen-Activated Protein Kinase Pathways”. Journal of Agricultural and Food Chemistry13 (2007): 5073-5080.
12. Yan H., et al. “The Antitumor Effects of α-Linolenic Acid”. Journal of Personalized Medicine3 (2024): 260.
13. Yang Q., et al. “Anti-thrombotic effects of α-Linolenic Acid isolated from Zanthoxylum bungeanum Maxim seeds”. BMC Complementary and Alternative Medicine1 (2014): 348.
14. Sultana H., et al. “An Updated Review on Extraction, Isolation, and Identification of Bioactive Compounds from Plant Extracts”. Scholars Academic Journal of Pharmacy7 (2023): 154-171.
15. Um S., et al. “Extraction, Isolation, and Structural Elucidation of Acylated Triterpene Saponins from Asteraceae Family”. In Y. H. Choi, O. Erol, and S. Irmisch (Eds. Natural Product Isolation and Identification (Methods in Molecular Biology) (2025): 201-225.
16. Debnath S., et al. “Advances in chromatography: contemporary techniques and applications”. Essential Chemistry1 (2025): 1-27.
17. Alexandri E., et al. “High Resolution NMR Spectroscopy as a Structural and Analytical Tool for Unsaturated Lipids in Solution”. Molecules10 (2017): 1663.
18. Kılınc H., et al. “LC-MS and NMR Based Plant Metabolomics: A Comprehensive Phytochemical Investigation of Symphytum anatolicum”. Metabolites10 (2023): 1051.
19. Sari KRP., et al. “Micro-titer plate assay for measurement of total phenolic and total flavonoid contents in medicinal plant extracts”. Arabian Journal of Chemistry 9 (2023): 105003.
20. Universiti Teknologi MARA., et al. “Determination of Antioxidant Activity, Total Phenolic and Flavonoid Contents of Malaysian Stingless Bee Propolis Extracts”. Journal of Sustainability Science and Management12 (2022): 132-143.
21. Kato R., et al. “DPPH Measurement for Phenols and Prediction of Antioxidant Activity of Phenolic Compounds in Food”. Current Issues in Molecular Biology1 (2025): 12.
22. Jalal TK., et al. “Evaluation of Antioxidant, Total Phenol and Flavonoid Content and Antimicrobial Activities of Artocarpus altilis (Breadfruit) of Underutilized Tropical Fruit Extracts”. Applied Biochemistry and Biotechnology7 (2015): 3231-3243.
23. Mabuza MJ., et al. “In vitro antiplasmodial activity and cytotoxicity of three Ziziphus (Rhamnaceae) species from South Africa”. Journal of Ethnopharmacology 349 (2025): 119918.
24. Sukkum C., et al. “nti-cancer activity and brine shrimp lethality assay of the extracts and isolated compounds from Garcinia schomburgkiana Pierre”. Journal of Applied Pharmaceutical Science (2024).
25. Huang W., et al. “Biosynthesis Investigations of Terpenoid, Alkaloid, and Flavonoid Antimicrobial Agents Derived from Medicinal Plants”. Antibiotics10 (2022): 1380.
26. Cushnie T PT., et al. “Alkaloids: an overview of their antibacterial, antibiotic-enhancing and antivirulence activities”. International Journal of Antimicrobial Agents5 (2014): 377-386.
27. Pandey R., et al. “Antibacterial potential and drug discovery of genus Diospyros: A review”. Medicinal Plants - International Journal of Phytomedicines and Related Industries 3 (2024): 410-425.
28. Wagner H. “Synergy research: Approaching a new generation of phytopharmaceuticals”. Fitoterapia 1 (2011): 34-37.
29. Kadeřábková N., et al. “Antibiotic susceptibility testing using minimum inhibitory concentration (MIC) assays”. npj Antimicrobials and Resistance1 (2024): 37.
30. Leopoldini M., et al. “The molecular basis of working mechanism of natural polyphenolic antioxidants”. Food Chemistry2 (2011): 288-306.
31. Pisoschi AM and Pop A. “The role of antioxidants in the chemistry of oxidative stress: A review”. European Journal of Medicinal Chemistry 97 (2015): 55-74.
32. Panche AN., et al. “Flavonoids: an overview”. Journal of Nutritional Science 5 (2016): e47.
33. Hernández-Miranda J., et al. “Impacts of Phenolic Compounds and Their Benefits on Human Health: Germination”. Metabolites 7 (2025): 425.
34. Oluwole O., et al. “Role of phenolic acid, tannins, stilbenes, lignans and flavonoids in human health - a review”. International Journal of Food Science and Technology10 (2022): 6326-6335.
35. Rauf A., et al. “Anticancer therapeutic potential of genus Diospyros: From phytochemistry to clinical applications—A review”. Food Science and Nutrition10 (2024): 7033-7047.
36. Ahsan S M., et al. “Epigenome Editing for Cannabinoid Yield: Targets, Tools, and Priorities in Cannabis sativa”. International Journal of Molecular Sciences14 (2026): 6299.
37. Meshu SS., et al. “Ripening Delay and Prolonging Shelf Life of Postharvest Bananas Through Application of Botanical Coating”. Applied Fruit Science 68 (2026): 221.
38. Howlader NC., et al. “Application of Botanical Extracts for Postharvest Preservation and Quality Enhancement of Horticultural Produce in Developing Countries: A Potential Review”. Journal of Crop Health 78 (2026): 17.
39. MIU Hoque., et al. “Revolutionizing Metabolic Engineering in Cannabis sativa: Harnessing the Power of Hairy Root Culture”. Phyton-International Journal of Experimental Botany12 (2025): 3805-3826.
40. Abdul Fahim M., et al. “Prolonging Shelf Life and Preserving Quality: A Study on Orange Varieties Under Different Storage Conditions”. Acta Horticulturae et Regiotecturae2 (2025): 89-98.
41. Robi R I., et al. “Efficacy of Indole-3-Butyric Acid and Different Stem Portions on the Growth and Rooting Performance of Horitoki (Terminalia chebula)”. Turkish Journal of Agriculture - Food Science and Technology 11 (2025): 3164-3174.
42. Mou A A., et al. “Assessment of Allelopathic Potentiality of Vimraj (Wedelia chinensis) Against Some Stored Grain Insect Pests”. International Journal of Agriculture Environment and Food Sciences3 (2025): 869-877.
43. Fahim M A., et al. “Assessment of Different Storage Methods on Postharvest Quality and Shelf Life of Darjeeling and Chinese Mandarins”. Journal of Plant Stress Physiology 11 (2025): 32-47.
44. Ahsan S M., et al. “Haploid Production in Cannabis sativa: Recent Updates, Prospects, and Perspectives”. Biology 6 (2025): 701.
45. Howlader NC., et al. “Assessing rice genotypes based on agro-morphological characterization and diversity analysis in southern Bangladesh”. Discovery Plants 2 (2025): 189.
46. Karim F., et al. “Biological control of foot and root rot disease of pea by using formulated product of Trichoderma”. Journal of Agricultural Sciences (Belgrade) 69.2 (2024).
47. Sultana A., et al. “Bio-control management of foot and root rot disease of lentil as impacted by different formulations of Trichoderma product”. Journal of Oasis Agriculture and Sustainable Development2 (2025): 1-12.
48. El-Beltagi H S., et al. “GC-MS analysis, antioxidant, antimicrobial and anticancer activities of extracts from Ficus sycomorus fruits and leaves”. Notulae Botanicae Horti Agrobotanici Cluj-Napoca2 (2019): 493-505.
49. Akanda M K M and Hasan AN. “Characterization of pharmacological properties of methanolic seed and stem bark extracts of Ziziphus mauritiana (BAU Kul) using in-vitro and in-vivo animal (Swiss albino male mice) model”. Clinical Phytoscience1 (2021): 8.
50. Yuan Q., et al. “The review of α-linolenic acid: Sources, metabolism, and pharmacology”. Phytotherapy Research 1 (2021): 164-188.
51. Dewi Kusumah., et al. “Linoleic acid, α-linolenic acid, and monolinolenins as antibacterial substances in the heat-processed soybean fermented with Rhizopus oligosporus”. Bioscience, Biotechnology, and Biochemistry (2020): 1285-1290.

Citation

Citation: Bidu Pal., et al. “Phytochemical Isolation of α-Linolenic Acid from Velvet Apple (Diospyros blancoi) Stem Bark and Assessment of Its Antibacte- rial, Antioxidant, and Cytotoxic Activities". Acta Scientific Agriculture 10.6 (2026): 32-45.

Copyright

Copyright: © 2026 Bidu Pal., et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.