{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,27]],"date-time":"2026-05-27T21:19:45Z","timestamp":1779916785895,"version":"3.53.1"},"reference-count":49,"publisher":"MDPI AG","issue":"8","license":[{"start":{"date-parts":[[2025,8,14]],"date-time":"2025-08-14T00:00:00Z","timestamp":1755129600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biology"],"abstract":"<jats:p>Anemia remains a widespread public health concern, and the search for interventions demonstrating potent anti-anemic activity is critical for reducing its impact among high-risk populations. Conventional iron therapies are associated with several complications and potential adverse effects. This study explored a polyherbal approach to develop a safer and more effective alternative treatment for anemia. A molecular docking study was initially performed to screen and evaluate alizarin, catechin, kaempferol, recesmol, rubiadin, and rutin, which are known for their antioxidant and hematinic potential. Using AutoDock Vina, these compounds were docked against the target protein (PDB ID: 6MOE) with EPE and ferrous ions as controls. Rutin demonstrated the highest binding affinity of \u22126.4 kcal\/moL, whereas alizarin and rubiadin both followed closely with \u22126.3 kcal\/moL, while kaempferol and ellagic acid exhibited a binding affinity of \u22126.2 kcal\/moL. In comparison, the reference compounds tested ferrous ions, and native ligand EPE (\u22125.0 kcal\/moL) and iron (\u22124.8 kcal\/moL), showed mild affinities. Moreover, the tested compounds demonstrated stable binding, suggesting their potential relevance in modulating anemia-related pathways. Based on the docking results and traditional therapeutic values, a polyherbal formulation (PHF) was developed using methanolic extracts of Trigonella foenum-graecum, Emblica officinalis, Pterocarpus marsupium, Withania somnifera, Asparagus racemosus, Zingiber officinale, Rubia cordifolia, Boerhavia diffusa, and Adhatoda vasica. Phytochemical screening via HPTLC analysis was used to quantify the presence of gallic and ellagic acids. In addition, PHF showed significant antioxidant potential (DPPH IC50: 14.29 \u00b5g\/mL; FRAP IC50: 58.57 \u00b5g\/mL) and iron content (98.47 ppm) values. Furthermore, in vivo evaluation using a phenylhydrazine-induced hemolytic anemia model in Sprague Dawley rats revealed that the PHF achieved complete restoration of RBCs (6.15 \u00b1 0.04), hemoglobin (14.82 \u00b1 0.03 g\/dL), and hematocrit (43.08 \u00b1 0.28%) in anemic rats and improved histopathological features in the liver, spleen, and bone marrow. These results demonstrate that combined molecular and pharmacological evidence support the efficacy of PHF as a promising candidate for the management of anemia by enhancing erythropoiesis, improving iron metabolism, and reducing oxidative stress.<\/jats:p>","DOI":"10.3390\/biology14081052","type":"journal-article","created":{"date-parts":[[2025,8,14]],"date-time":"2025-08-14T14:51:46Z","timestamp":1755183106000},"page":"1052","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Development, Molecular Docking, and Anti-Anemia Potential of Polyherbal Formulation"],"prefix":"10.3390","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-3739-2178","authenticated-orcid":false,"given":"Deepak","family":"Bharati","sequence":"first","affiliation":[{"name":"Department of Pharmacology, AETs St. John Institute of Pharmacy and Research, Palghar 401404, Maharashtra, India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0004-1590-0121","authenticated-orcid":false,"given":"Sakshi","family":"Nirhali","sequence":"additional","affiliation":[{"name":"Department of Pharmacology, AETs St. John Institute of Pharmacy and Research, Palghar 401404, Maharashtra, India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4537-4909","authenticated-orcid":false,"given":"Abhijeet","family":"Puri","sequence":"additional","affiliation":[{"name":"Department of Pharmacognosy, AETs St. John Institute of Pharmacy and Research, Palghar 401404, Maharashtra, India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4536-4444","authenticated-orcid":false,"given":"Popat","family":"Mohite","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical Chemistry, AETs St. John Institute of Pharmacy and Research, Palghar 401404, Maharashtra, India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7929-3322","authenticated-orcid":false,"given":"Sudarshan","family":"Singh","sequence":"additional","affiliation":[{"name":"Office of Research Administration, Chiang Mai University, Chiang Mai 50200, Thailand"},{"name":"Faculty of Pharmacy, Chiang Mai University, Chiang Mai 50200, Thailand"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,8,14]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"S7","DOI":"10.1016\/j.tjnut.2023.07.018","article-title":"Biology of Anemia: A Public Health Perspective","volume":"153","author":"Brittenham","year":"2023","journal-title":"J. Nutr."},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Abdilahi, M.M., Kiruja, J., Farah, B.O., Abdirahman, F.M., Mohamed, A.I., Mohamed, J., and Ahmed, A.M. (2024). Prevalence of anemia and associated factors among pregnant women at Hargeisa Group Hospital, Somaliland. BMC Pregnancy Childbirth, 24.","DOI":"10.1186\/s12884-024-06539-3"},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Addo, O.Y., Yu, E.X., Williams, A.M., Young, M.F., Sharma, A.J., Mei, Z., Kassebaum, N.J., Jefferds, M.E.D., and Suchdev, P.S. (2021). Evaluation of Hemoglobin Cutoff Levels to Define Anemia Among Healthy Individuals. JAMA Netw. Open, 4.","DOI":"10.1001\/jamanetworkopen.2021.19123"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"801","DOI":"10.1182\/blood.2019004017","article-title":"How I treat cancer-associated anemia","volume":"136","author":"Gilreath","year":"2020","journal-title":"Blood"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"49","DOI":"10.1093\/nutrit\/nuw055","article-title":"Dietary strategies for improving iron status: Balancing safety and efficacy","volume":"75","author":"Prentice","year":"2017","journal-title":"Nutr. Rev."},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Cotoraci, C., Ciceu, A., Sasu, A., and Hermenean, A. (2021). Natural Antioxidants in Anemia Treatment. Int. J. Mol. Sci., 22.","DOI":"10.3390\/ijms22041883"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"340","DOI":"10.1016\/j.jaim.2021.02.009","article-title":"Antianemic activity of polyherbal formulation, Raktavardhak Kadha, against phenylhydrazine-induced anemia in rats","volume":"12","author":"Sheth","year":"2021","journal-title":"J. Ayurveda Integr. Med."},{"key":"ref_8","first-page":"41","article-title":"Safety and efficacy of a polyherbal unani formulation (sharbat-e-faulad) in s\u016b\u2019al-qinya (anaemia)\u2014A multi centric study","volume":"6","author":"Khan","year":"2019","journal-title":"Safety"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"2500","DOI":"10.1002\/fsn3.3308","article-title":"Phytobioactive compounds as therapeutic agents for human diseases: A review","volume":"11","author":"Riaz","year":"2023","journal-title":"Food Sci. Nutr."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"359","DOI":"10.1080\/10942912.2022.2157425","article-title":"Antioxidant, phytochemical, and therapeutic properties of medicinal plants: A review","volume":"26","author":"Nwozo","year":"2023","journal-title":"Int. J. Food Prop."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"227","DOI":"10.1007\/s43188-019-00021-5","article-title":"Ameliorative effect of aqueous leaf extract of Solanum aethiopicum on phenylhydrazine-induced anaemia and toxicity in rats","volume":"36","author":"Ekweogu","year":"2020","journal-title":"Toxicol. Res."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"300","DOI":"10.1016\/j.sjbs.2015.09.015","article-title":"Fenugreek a multipurpose crop: Potentialities and improvements","volume":"23","author":"Ahmad","year":"2016","journal-title":"Saudi J. Biol. Sci."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"10","DOI":"10.1016\/j.jnim.2018.10.001","article-title":"Beneficial effects of an aqueous ginger extract on the immune system cells and antibodies, hematology, and thyroid hormones in male smokers and non-smokers","volume":"15","author":"Mahassni","year":"2019","journal-title":"J. Nutr. Intermed. Metab."},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Gaur, P.K., Rastogi, S., and Lata, K. (2022). Correlation between phytocompounds and pharmacological activities of Boerhavia diffusa Linn with traditional-ethnopharmacological insights. Phytomed. Plus, 2.","DOI":"10.1016\/j.phyplu.2022.100260"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"45","DOI":"10.1016\/j.htct.2021.08.015","article-title":"Sickle cell anemia: Hierarchical cluster analysis and clinical profile in a cohort in Brazil","volume":"45","author":"Dutra","year":"2023","journal-title":"Hematol. Transfus. Cell Ther."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"504","DOI":"10.1016\/j.ijbiomac.2019.06.139","article-title":"Immunomodulatory and antioxidant effects of pomegranate peel polysaccharides on immunosuppressed mice","volume":"137","author":"Wu","year":"2019","journal-title":"Int. J. Biol. Macromol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"209","DOI":"10.4103\/0975-9476.104444","article-title":"Effects of eight-week supplementation of Ashwagandha on cardiorespiratory endurance in elite Indian cyclists","volume":"3","author":"Shenoy","year":"2012","journal-title":"J. Ayurveda Integr. Med."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"23238","DOI":"10.1039\/C7RA02138K","article-title":"Effects of naturally occurring flavonoids on ferroportin expression in the spleen in iron deficiency anemia in vivo","volume":"7","author":"Mazhar","year":"2017","journal-title":"RSC Adv."},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Intharuksa, A., Kuljarusnont, S., Sasaki, Y., and Tungmunnithum, D. (2024). Flavonoids and Other Polyphenols: Bioactive Molecules from Traditional Medicine Recipes\/Medicinal Plants and Their Potential for Phytopharmaceutical and Medical Application. Molecules, 29.","DOI":"10.3390\/molecules29235760"},{"key":"ref_20","first-page":"42","article-title":"Phenolics and flavonoids contents of medicinal plants, as natural ingredients for many therapeutic purposes\u2014A review","volume":"10","year":"2020","journal-title":"IOSR J. Pharm."},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Imam, M.U., Zhang, S., Ma, J., Wang, H., and Wang, F. (2017). Antioxidants mediate both iron homeostasis and oxidative stress. Nutrients, 9.","DOI":"10.3390\/nu9070671"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"311","DOI":"10.1146\/annurev-nutr-062320-112625","article-title":"Molecular mechanisms of iron and heme metabolism","volume":"42","author":"Dutt","year":"2022","journal-title":"Annu. Rev. Nutr."},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Chumpolphant, S., Suwatronnakorn, M., Issaravanich, S., Tencomnao, T., and Prasansuklab, A. (2022). Polyherbal formulation exerts wound healing, anti-inflammatory, angiogenic and antimicrobial properties: Potential role in the treatment of diabetic foot ulcers. Saudi J. Biol. Sci., 29.","DOI":"10.1016\/j.sjbs.2022.103330"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"603","DOI":"10.22271\/chemi.2020.v8.i2i.8834","article-title":"Qualitative tests for preliminary phytochemical screening: An overview","volume":"8","author":"Shaikh","year":"2020","journal-title":"Int. J. Chem. Stud."},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Machado, J.C.B., Cristina da Silva, J., Leite, G.V.B., Dos Santos Dantas, T., Daniele-Silva, A., de Freitas Fernandes-Pedrosa, M., de Oliveira, A.M., Dantas da Cruz, R.C., de Souza, I.A., and Weilack, I. (2025). Phytochemical Profile, Acute, and Subacute Toxicity of Spray-Dried Hydroethanolic Extract From Punica granatum Leaves. Chem. Biodivers., 22.","DOI":"10.1002\/cbdv.202402429"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"334","DOI":"10.7897\/2277-4343.05369","article-title":"A study of some anthraquinones of Rubia cordifolia L. incorporated into pinda oil: An ayurvedic medicinal oil used for topical application in dermatological and inflammatory conditions","volume":"5","author":"Abeysekera","year":"2014","journal-title":"Int. J. Res. Ayurveda Pharm."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/S0378-8741(00)00225-7","article-title":"Adhatoda vasica: A critical review of ethnopharmacological and toxicological data","volume":"72","author":"Claeson","year":"2000","journal-title":"J. Ethnopharmacol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"287","DOI":"10.1080\/08927022.2024.2302022","article-title":"Pharmacophore-based computational study on inhibitor of TMPRSS6 as hepcidin modulator in an iron overload of beta-thalassaemia","volume":"50","author":"Yadav","year":"2024","journal-title":"Mol. Simul."},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Wang, Y., Qinqin, H., Wang, H., Zhang, H., Zhang, X., Liu, W., Xiang, Z., and Gu, Y. (2023). Network pharmacology and molecular docking to explore the mechanism of Sheng Xue Bao mixture against iron deficiency anemia. Medicine, 102.","DOI":"10.1097\/MD.0000000000035012"},{"key":"ref_30","first-page":"128","article-title":"Comparative phytochemical, thin layer chromatographic profiling and antioxidant activity of extracts from some Indian herbal drugs","volume":"7","author":"Akhtar","year":"2022","journal-title":"J. Bioresour. Bioprod."},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Gupta, M., Sumaiya, S., Ali, S., Naved, T., Sharma, A., Ahmad, A., Sikander, M., and Sarwat, M. (2023). Pharmacognostical and Phytochemical Evaluation of a Unani Polyherbal Formulation: Dawa ul Kurkum by HPTLC. Separations, 10.","DOI":"10.3390\/separations10020089"},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"Michiu, D., Socaciu, M.-I., Fogarasi, M., Jimborean, A.M., Ranga, F., Mure\u015fan, V., and Semeniuc, C.A. (2022). Implementation of an Analytical Method for Spectrophotometric Evaluation of Total Phenolic Content in Essential Oils. Molecules, 27.","DOI":"10.3390\/molecules27041345"},{"key":"ref_33","doi-asserted-by":"crossref","unstructured":"Shraim, A.M., Ahmed, T.A., Rahman, M.M., and Hijji, Y.M. (2021). Determination of total flavonoid content by aluminum chloride assay: A critical evaluation. LWT, 150.","DOI":"10.1016\/j.lwt.2021.111932"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"4931","DOI":"10.52711\/0974-360X.2022.00828","article-title":"Phytochemical content and antioxidant property of polyherbal formulation, raktavardhak kadha","volume":"15","author":"Sheth","year":"2022","journal-title":"Res. J. Pharm. Technol."},{"key":"ref_35","first-page":"2","article-title":"Exploration and Evaluation of In-vitro Antioxidant Activity of ABANA: A Polyherbal Formulation","volume":"1","author":"Deepak","year":"2023","journal-title":"Curr. Funct. Foods"},{"key":"ref_36","doi-asserted-by":"crossref","unstructured":"Aloke, C., Uche Emelike, C., Ajuka Obasi, N., Nkemjika Ogbu, P., Oswald Edeogu, C., Godwin Uzomba, C., Ekakitie, O., Adewale Iyaniwura, A., Okoro, C.C., and Peter Okey, B. (2021). HPLC profiling and studies on Copaifera salikounda methanol leaf extract on phenylhydrazine-induced hematotoxicity and oxidative stress in rats. Arab. J. Chem., 14.","DOI":"10.1016\/j.arabjc.2021.103428"},{"key":"ref_37","unstructured":"OECD (2022). Test No. 425: Acute Oral Toxicity: Up-and-Down Procedure, OECD Publishing."},{"key":"ref_38","doi-asserted-by":"crossref","unstructured":"Aguilera-Alcala, N., Morales-Reyes, Z., Martin-Lopez, B., Moleon, M., and Sanchez-Zapata, J.A. (2020). Role of scavengers in providing non-material contributions to people. Ecol. Indic., 117.","DOI":"10.1016\/j.ecolind.2020.106643"},{"key":"ref_39","doi-asserted-by":"crossref","unstructured":"Cui, C., Wu, Y., Guo, X., Hong, Z., Xiao, J., Wan, X., Zong, J., and Hou, R. (2024). Accurate and rapid quantification of tea saponins using a thin-layer chromatography (TLC) method based on hemolysis and machine vision. LWT, 199.","DOI":"10.1016\/j.lwt.2024.116139"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"51","DOI":"10.2147\/JEP.S379805","article-title":"Phytochemical Screening and Antimicrobial Activity Evaluation of Selected Medicinal Plants in Ethiopia","volume":"15","author":"Dubale","year":"2023","journal-title":"J. Exp. Pharmacol."},{"key":"ref_41","doi-asserted-by":"crossref","unstructured":"Checkouri, E., Reignier, F., Robert-Da Silva, C., and Meilhac, O. (2020). Evaluation of Polyphenol Content and Antioxidant Capacity of Aqueous Extracts from Eight Medicinal Plants from Reunion Island: Protection against Oxidative Stress in Red Blood Cells and Preadipocytes. Antioxidants, 9.","DOI":"10.3390\/antiox9100959"},{"key":"ref_42","doi-asserted-by":"crossref","unstructured":"Molino, S., Pilar Francino, M., and \u00c1ngel Rufi\u00e1n Henares, J. (2023). Why is it important to understand the nature and chemistry of tannins to exploit their potential as nutraceuticals?. Food Res. Int., 173.","DOI":"10.1016\/j.foodres.2023.113329"},{"key":"ref_43","doi-asserted-by":"crossref","unstructured":"Yap, V.L., Tan, L.F., Rajagopal, M., Wiart, C., Selvaraja, M., Leong, M.Y., and Tan, P.L. (2023). Evaluation of phytochemicals and antioxidant potential of a new polyherbal formulation TC-16: Additive, synergistic or antagonistic?. BMC Complement. Med. Ther., 23.","DOI":"10.1186\/s12906-023-03921-0"},{"key":"ref_44","doi-asserted-by":"crossref","unstructured":"Gulcin, \u0130., and Alwasel, S.H. (2023). DPPH Radical Scavenging Assay. Processes, 11.","DOI":"10.3390\/pr11082248"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"451","DOI":"10.1007\/s13197-015-1994-x","article-title":"Evaluation of antioxidant capacity of 13 plant extracts by three different methods: Cluster analyses applied for selection of the natural extracts with higher antioxidant capacity to replace synthetic antioxidant in lamb burgers","volume":"53","author":"Fernandes","year":"2016","journal-title":"J. Food Sci. Technol."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"93","DOI":"10.1016\/j.jtcme.2024.03.017","article-title":"Therapeutic efficacy of Punarnavadi mandura against phenylhydrazine-induced hemolytic anemia in rats","volume":"15","author":"Rajendran","year":"2025","journal-title":"J. Tradit. Complement. Med."},{"key":"ref_47","doi-asserted-by":"crossref","unstructured":"Ali, F.A.Z., Abdel-Maksoud, F.M., Abd Elaziz, H.O., Al-Brakati, A., and Elmahallawy, E.K. (2021). Descriptive Histopathological and Ultrastructural Study of Hepatocellular Alterations Induced by Aflatoxin B1 in Rats. Animals, 11.","DOI":"10.3390\/ani11020509"},{"key":"ref_48","doi-asserted-by":"crossref","unstructured":"Ousaaid, D., Ghouizi, A.E., Laaroussi, H., Bakour, M., Mechchate, H., Es-Safi, I., Kamaly, O.A., Saleh, A., Conte, R., and Lyoussi, B. (2022). Anti-Anemic Effect of Antioxidant-Rich Apple Vinegar against Phenylhydrazine-Induced Hemolytic Anemia in Rats. Life, 12.","DOI":"10.3390\/life12020239"},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"774","DOI":"10.1089\/jmf.2012.2264","article-title":"Hematopoietic effect of Bacillus subtilis-fermented antler extract on phenylhydrazine-induced hemolytic anemia in Sprague-Dawley rats","volume":"15","author":"Lee","year":"2012","journal-title":"J. Med. Food"}],"container-title":["Biology"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2079-7737\/14\/8\/1052\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,9]],"date-time":"2025-10-09T18:27:45Z","timestamp":1760034465000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2079-7737\/14\/8\/1052"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,8,14]]},"references-count":49,"journal-issue":{"issue":"8","published-online":{"date-parts":[[2025,8]]}},"alternative-id":["biology14081052"],"URL":"https:\/\/doi.org\/10.3390\/biology14081052","relation":{},"ISSN":["2079-7737"],"issn-type":[{"value":"2079-7737","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,8,14]]}}}