Plants have been the basis of traditional medicine since the dawn of civilizations. Different plant parts possess various phytochemicals, playing important roles in preventing and curing diseases. Scientists, through extensive experimental studies, are playing an important part in establishing the use of phytochemicals in medicine. However, there are still a large number of medicinal plants which need to be studied for their phytochemical profile. In this study, the objective was to isolate phytochemicals from bark of Bauhinia variegata L. and to study them for their antioxidant and cytotoxic activities. The bark was extracted with methanol, followed by column chromatography and thus isolating kaempferol, stigmasterol, protocatechuic acid-methyl ester (PCA-ME) and protocatechuic acid (PCA). 2,2-azinobis-3-ethyl-benzothiazoline-6-sulfonic acid (ABTS) and 2, 2'-diphenyl-1-picrylhydrazyl radical (DPPH) radical scavenging assays were utilized for assessment of antioxidant activity, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) dye reduction assay was used to determine cytotoxic activity against C-6 glioma rat brain, MCF-7 breast cancer, and HCT-15 colon cancer cell lines. The compounds were found to have significant antioxidant and cytotoxic activity. Since there is a considerable increase in characterizing novel chemical compounds from plant parts, the present study might be helpful for chemotaxonomic determinations, for understanding of medicinal properties as well as for the quality assessment of herbal supplements containing B. variegata bark, thus establishing its use in traditional medicine.

Department of Agriculture Food and Environment University of Pisa 56124 Pisa Italy

Department of Biophysics Panjab University Chandigarh 160014 India

Department of Botanical and Environmental Sciences Guru Nanak Dev University Amritsar 143005 India

Department of Botany and Plant Physiology Faculty of Agrobiology Food and Natural Resources Czech University of Life Sciences 16500 Prague Czech Republic

Department of Molecular Biology and Biochemistry Guru Nanak Dev University Amritsar 143005 India

Department of Plant Physiology Faculty of Agrobiology and Food Resources Slovak University of Agriculture Nitra 94976 Slovakia

Natural Product Chemistry and Process Development Division CSIR Institute of Himalayan Bioresource Technology Palampur Himachal Pradesh 176061 India

State Key Laboratory of Subtropical Silviculture Zhejiang A and F University Hangzhou 311300 China

See more in PubMed

Kawata K., Yokoo H., Shimazaki R., Okabe S. Classification of heavy-metal toxicity by human DNA microarray analysis. Environ. Sci. Technol. 2007;41:3769–3774. doi: 10.1021/es062717d. PubMed DOI

Rehman K., Fatima F., Waheed I., Akash M.S.H. Prevalence of exposure of heavy metals and their impact on health consequences. J. Cell. Biochem. 2018;119:157–184. doi: 10.1002/jcb.26234. PubMed DOI

Sharma A., Kumar V., Thukral A., Bhardwaj R. Responses of Plants to Pesticide Toxicity: An Overview. Planta Daninha. 2019;37:e019184291. doi: 10.1590/s0100-83582019370100065. DOI

De Gruijl F.R. Photocarcinogenesis: UVA vs. UVB radiation. Ski. Pharmacol. Physiol. 2002;15:316–320. doi: 10.1159/000064535. PubMed DOI

Ruiz-Ramos R., Lopez-Carrillo L., Rios-Perez A.D., De Vizcaya-Ruíz A., Cebrian M.E. Sodium arsenite induces ROS generation, DNA oxidative damage, HO-1 and c-Myc proteins, NF-κB activation and cell proliferation in human breast cancer MCF-7 cells. Mutat. Res. Genet. Toxicol. Environ. Mutagenesis. 2009;674:109–115. doi: 10.1016/j.mrgentox.2008.09.021. PubMed DOI

Sharma V., Anderson D., Dhawan A. Zinc oxide nanoparticles induce oxidative DNA damage and ROS-triggered mitochondria mediated apoptosis in human liver cells (HepG2) Apoptosis. 2012;17:852–870. doi: 10.1007/s10495-012-0705-6. PubMed DOI

Barzilai A., Yamamoto K.-I. DNA damage responses to oxidative stress. DNA Repair. 2004;3:1109–1115. doi: 10.1016/j.dnarep.2004.03.002. PubMed DOI

Gupta S.C., Hevia D., Patchva S., Park B., Koh W., Aggarwal B.B. Upsides and downsides of reactive oxygen species for cancer: The roles of reactive oxygen species in tumorigenesis, prevention, and therapy. Antioxid. Redox Signal. 2012;16:1295–1322. doi: 10.1089/ars.2011.4414. PubMed DOI PMC

Dikshit R., Gupta P.C., Ramasundarahettige C., Gajalakshmi V., Aleksandrowicz L., Badwe R., Kumar R., Roy S., Suraweera W., Bray F., et al. Cancer mortality in India: A nationally representative survey. Lancet (Lond. Engl.) 2012;379:1807–1816. doi: 10.1016/S0140-6736(12)60358-4. PubMed DOI

Stewart B.W., Wild C.P. World Cancer Report 2014. World Health Organization; Geneva, Switzerland: 2014.

Ramirez-Mares M.V., Chandra S., de Mejia E.G. In vitro chemopreventive activity of Camellia sinensis, Ilex paraguariensis and Ardisia compressa tea extracts and selected polyphenols. Mutat. Res. 2004;554:53–65. doi: 10.1016/j.mrfmmm.2004.03.002. PubMed DOI

Nemeikaite-Ceniene A., Imbrasaite A., Sergediene E., Cenas N. Quantitative structure-activity relationships in prooxidant cytotoxicity of polyphenols: Role of potential of phenoxyl radical/phenol redox couple. Arch. Biochem. Biophys. 2005;441:182–190. doi: 10.1016/j.abb.2005.07.002. PubMed DOI

Aravindaram K., Yang N.S. Anti-inflammatory plant natural products for cancer therapy. Planta Med. 2010;76:1103–1117. doi: 10.1055/s-0030-1249859. PubMed DOI

Bahadoran Z., Golzarand M., Mirmiran P., Saadati N., Azizi F. The association of dietary phytochemical index and cardiometabolic risk factors in adults: Tehran Lipid and Glucose Study. J. Hum. Nutr. Diet. Off. J. Br. Diet. Assoc. 2013;26(Suppl. 1):145–153. doi: 10.1111/jhn.12048. PubMed DOI

Fresco P., Borges F., Diniz C., Marques M.P. New insights on the anticancer properties of dietary polyphenols. Med. Res. Rev. 2006;26:747–766. doi: 10.1002/med.20060. PubMed DOI

Anand P., Kunnumakkara A.B., Sundaram C., Harikumar K.B., Tharakan S.T., Lai O.S., Sung B., Aggarwal B.B. Cancer is a preventable disease that requires major lifestyle changes. Pharm. Res. 2008;25:2097–2116. doi: 10.1007/s11095-008-9661-9. PubMed DOI PMC

Rivera-Mondragon A., Bijttebier S., Tuenter E., Custers D., Ortiz O.O., Pieters L., Caballero-George C., Apers S., Foubert K. Phytochemical characterization and comparative studies of four Cecropia species collected in Panama using multivariate data analysis. Sci. Rep. 2019;9:1763. doi: 10.1038/s41598-018-38334-4. PubMed DOI PMC

Fent K., Weston A.A., Caminada D. Ecotoxicology of human pharmaceuticals. Aquat. Toxicol. 2006;76:122–159. doi: 10.1016/j.aquatox.2005.09.009. PubMed DOI

Huang D. Dietary Antioxidants and Health Promotion. Antioxidants. 2018;7:9. doi: 10.3390/antiox7010009. PubMed DOI PMC

San Marcos Growers Bauhinia blakeana (Hong Kong Orchid Tree) [(accessed on 7 October 2019)]; Available online: https://www.smgrowers.com/products/plants/plantdisplay.asp?plant_id=234.

Silva K.L.D., Cechinel Filho V. Plants of the genus Bauhinia: Chemical composition and pharmacological potential. Química Nova. 2002;25:449–454. doi: 10.1590/S0100-40422002000300018. DOI

Cavalcanti K., Favoretto R. Bauhinia forficata Link. In: Amaral A., Simões E., Ferreira K., Fiocruz R.D.J., editors. Coletânea Científica de Plantas de Uso Medicinal. Fiocruz; Rio de Janeiro, Brazil: 2005. pp. 1–17.

Modh K.M., Parmar P.T., Panigrahi B., Anand I.S., Patel C.N. Pharmacognostical and Phytochemical Evaluation of Leaves of Bauhinia variegata Linn. Pharmacogn. J. 2011;3:45–49. doi: 10.5530/pj.2011.24.9. DOI

Bodakhe S.H., Alpana R. Hepatoprotective properties of Bauhinia variegata extract. Pharm. J. Jpn. 2007;127:1503–1507. doi: 10.1248/yakushi.127.1503. PubMed DOI

Rajkapoor B., Jayakar B., Murugesh N. Antitumour activity of Bauhinia variegata on Dalton’s ascitic lymphoma. J. Ethnopharmacol. 2003;89:107–109. doi: 10.1016/S0378-8741(03)00264-2. PubMed DOI

Rajkapoor B., Jayakar B., Murugesh N. Antitumour activity of Bauhinia variegata against Ehrlich ascites carcinoma induced mice. Pharm. Biol. 2003;41:604–607. doi: 10.1080/13880200390501947. DOI

Shahana S., Nikalje A.P.G. A Brief Review on Bauhinia variegata: Phytochemistry, Antidiabetic and Antioxidant potential. Am. J. Pharmtech Res. 2017;7:186–197.

Re R., Pellegrini N., Proteggente A., Pannala A., Yang M., Rice-Evans C. Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radic. Biol. Med. 1999;26:1231–1237. doi: 10.1016/S0891-5849(98)00315-3. PubMed DOI

Blois M.S. Antioxidant determinations by the use of a stable free radical. Nature. 1958;181:1199–1200. doi: 10.1038/1811199a0. DOI

Igarashi M., Miyazawa T. The growth inhibitory effect of conjugated linoleic acid on a human hepatoma cell line, HepG2, is induced by a change in fatty acid metabolism, but not the facilitation of lipid peroxidation in the cells. Biochim. Biophys. Acta. 2001;1530:162–171. doi: 10.1016/S1388-1981(00)00180-3. PubMed DOI

Xiao Z., Wu H., Wu T., Shi H., Hang B., Aisa H. Kaempferol and quercetin flavonoids from Rosa rugosa. Chem. Nat. Compd. 2006;42:736–737. doi: 10.1007/s10600-006-0267-3. DOI

Habib M., Nikkon F., Rahman M., Haque Z., Karim M. Isolation of stigmasterol and ß-sitosterol from methanolic extract of root bark of Calotropis gigantean (Linn.) Pak. J. Biol. Sci. 2007;10:4174–4176. PubMed

Moghaddam F.M., Farimani M.M., Salahvarzi S., Amin G. Chemical Constituents of Dichloromethane Extract of Cultivated Satureja khuzistanica. Evid. Based Complement. Altern. Med. eCAM. 2007;4:95–98. doi: 10.1093/ecam/nel065. PubMed DOI PMC

Jamal A., Ahmad W.Y.W., Din L.B. A chemical study on Phyllanthus columnaris. Eur. J. Sci. Res. 2009;28:76–81.

He D., Gu D., Huang Y., Ayupbek A., Yang Y., Aisa H.A., Ito Y. Separation and Purification of Phenolic Acids and Myricetin from Black Currant by High Speed Countercurrent Chromatography. J. Liq. Chromatogr. Relat. Technol. 2009;32:3077–3088. doi: 10.1080/10826070903320756. PubMed DOI PMC

Desai M.C., Chackalamannil S. Rediscovering the role of natural products in drug discovery. Curr. Opin. Drug Discov. Dev. 2008;11:436–437. PubMed

Lam K.S. New aspects of natural products in drug discovery. Trends Microbiol. 2007;15:279–289. doi: 10.1016/j.tim.2007.04.001. PubMed DOI

Mali R.G., Mahajan S.G., Mehta A.A. Rakta Kanchan (Bauhinia variegata): Chemistry, traditional and medicinal uses-a review. Pharmacogn. Rev. 2007;1:314.

Wang J., Fang X., Ge L., Cao F., Zhao L., Wang Z., Xiao W. Antitumor, antioxidant and anti-inflammatory activities of kaempferol and its corresponding glycosides and the enzymatic preparation of kaempferol. PLoS ONE. 2018;13:e0197563. doi: 10.1371/journal.pone.0197563. PubMed DOI PMC

Van Acker S.A., van den Berg D.J., Tromp M.N., Griffioen D.H., van Bennekom W.P., van der Vijgh W.J., Bast A. Structural aspects of antioxidant activity of flavonoids. Free Radic. Biol. Med. 1996;20:331–342. doi: 10.1016/0891-5849(95)02047-0. PubMed DOI

Rice-Evans C. Flavonoid antioxidants. Curr. Med. Chem. 2001;8:797–807. doi: 10.2174/0929867013373011. PubMed DOI

Kampkotter A., Nkwonkam C.G., Zurawski R.F., Timpel C., Chovolou Y., Watjen W., Kahl R. Investigations of protective effects of the flavonoids quercetin and rutin on stress resistance in the model organism Caenorhabditis elegans. Toxicology. 2007;234:113–123. doi: 10.1016/j.tox.2007.02.006. PubMed DOI

M Calderon-Montano J., Burgos-Morón E., Pérez-Guerrero C., López-Lázaro M. A review on the dietary flavonoid kaempferol. Mini Rev. Med. Chem. 2011;11:298–344. doi: 10.2174/138955711795305335. PubMed DOI

Kanimozhi D., Bai V. Evaluation of Phytochemical antioxidant antimicrobial activity determination of bioactive components of ethanolic extract of aerial and underground parts of Cynodon dactylon L. Int. J. Sci. Res. Rev. 2012;1:33–48.

Shanthakumar B., Sathish M., Suresh A.J. In Vitro Anti Oxidant Activity of Extracts and Stigmasterol from Leaves of Clerodendrum inerme Linn. Res. J. Pharm. Biol. Chem. Sci. 2013;4:1411–1418.

Kakkar S., Bais S. A review on protocatechuic acid and its pharmacological potential. ISRN Pharmacol. 2014;2014 doi: 10.1155/2014/952943. PubMed DOI PMC

Saito S., Kawabata J. DPPH (=2,2-diphenyl-1-picrylhydrazyl) radical-scavenging reaction of protocatechuic acid (=3,4-dihydroxybenzoic acid): Difference in reactivity between acids and their esters. Helv. Chim. Acta. 2006;89:1395–1407. doi: 10.1002/hlca.200690139. DOI

Benda P., Lightbody J., Sato G., Levine L., Sweet W. Differentiated rat glial cell strain in tissue culture. Science (New York N. Y.) 1968;161:370–371. doi: 10.1126/science.161.3839.370. PubMed DOI

Soule H.D., Vazguez J., Long A., Albert S., Brennan M. A human cell line from a pleural effusion derived from a breast carcinoma. J. Natl. Cancer Inst. 1973;51:1409–1416. doi: 10.1093/jnci/51.5.1409. PubMed DOI

Diantini A., Subarnas A., Lestari K., Halimah E., Susilawati Y., Supriyatna S., Julaeha E., Achmad T.H., Suradji E.W., Yamazaki C., et al. Kaempferol-3-O-rhamnoside isolated from the leaves of Schima wallichii Korth. inhibits MCF-7 breast cancer cell proliferation through activation of the caspase cascade pathway. Oncol. Lett. 2012;3:1069–1072. doi: 10.3892/ol.2012.596. PubMed DOI PMC

Hong J.T., Yen J.H., Wang L., Lo Y.H., Chen Z.T., Wu M.J. Regulation of heme oxygenase-1 expression and MAPK pathways in response to kaempferol and rhamnocitrin in PC12 cells. Toxicol. Appl. Pharmacol. 2009;237:59–68. doi: 10.1016/j.taap.2009.02.014. PubMed DOI

Filomeni G., Desideri E., Cardaci S., Graziani I., Piccirillo S., Rotilio G., Ciriolo M.R. Carcinoma cells activate AMP-activated protein kinase-dependent autophagy as survival response to kaempferol-mediated energetic impairment. Autophagy. 2010;6:202–216. doi: 10.4161/auto.6.2.10971. PubMed DOI

Bigovic D., Savikin K., Jankovic T., Menkovic N., Zdunic G., Stanojkovic T., Djuric Z. Antiradical and cytotoxic activity of different Helichrysum plicatum flower extracts. Nat. Prod. Commun. 2011;6:819–822. doi: 10.1177/1934578X1100600617. PubMed DOI

Luo H., Rankin G.O., Li Z., Depriest L., Chen Y.C. Kaempferol induces apoptosis in ovarian cancer cells through activating p53 in the intrinsic pathway. Food Chem. 2011;128:513–519. doi: 10.1016/j.foodchem.2011.03.073. PubMed DOI PMC

Yang J.H., Kondratyuk T.P., Jermihov K.C., Marler L.E., Qiu X., Choi Y., Cao H., Yu R., Sturdy M., Huang R., et al. Bioactive compounds from the fern Lepisorus contortus. J. Nat. Prod. 2011;74:129–136. doi: 10.1021/np100373f. PubMed DOI PMC

Kim S.H., Hwang K.A., Choi K.C. Treatment with kaempferol suppresses breast cancer cell growth caused by estrogen and triclosan in cellular and xenograft breast cancer models. J. Nutr. Biochem. 2016;28:70–82. doi: 10.1016/j.jnutbio.2015.09.027. PubMed DOI

Rajendran P., Rengarajan T., Nandakumar N., Palaniswami R., Nishigaki Y., Nishigaki I. Kaempferol, a potential cytostatic and cure for inflammatory disorders. Eur. J. Med. Chem. 2014;86:103–112. doi: 10.1016/j.ejmech.2014.08.011. PubMed DOI

Imran M., Salehi B., Sharifi-Rad J., Aslam Gondal T., Saeed F., Imran A., Shahbaz M., Tsouh Fokou P.V., Umair Arshad M., Khan H., et al. Kaempferol: A Key Emphasis to Its Anticancer Potential. Molecules. 2019;24:2277. doi: 10.3390/molecules24122277. PubMed DOI PMC

Malikova J., Swaczynova J., Kolar Z., Strnad M. Anticancer and antiproliferative activity of natural brassinosteroids. Phytochemistry. 2008;69:418–426. doi: 10.1016/j.phytochem.2007.07.028. PubMed DOI

Ali H., Dixit S., Ali D., Alqahtani S.M., Alkahtani S., Alarifi S. Isolation and evaluation of anticancer efficacy of stigmasterol in a mouse model of DMBA-induced skin carcinoma. Drug Des. Dev. Ther. 2015;9:2793–2800. doi: 10.2147/DDDT.S83514. PubMed DOI PMC

Chao C.-Y., Yin M.-C. Antibacterial effects of roselle calyx extracts and protocatechuic acid in ground beef and apple juice. Foodborne Pathog. Dis. 2009;6:201–206. doi: 10.1089/fpd.2008.0187. PubMed DOI

Lende A.B., Kshirsagar A.D., Deshpande A.D., Muley M.M., Patil R.R., Bafna P.A., Naik S.R. Anti-inflammatory and analgesic activity of protocatechuic acid in rats and mice. Inflammopharmacology. 2011;19:255–263. doi: 10.1007/s10787-011-0086-4. PubMed DOI

Son J.H., Kim S.-Y., Jang H.H., Lee S.N., Ahn K.J. Protective effect of protocatechuic acid against inflammatory stress induced in human dermal fibroblasts. Biomed. Dermatol. 2018;2:9. doi: 10.1186/s41702-017-0018-z. DOI

Bullo S., Buskaran K., Baby R., Dorniani D., Fakurazi S., Hussein M.Z. Dual Drugs Anticancer Nanoformulation using Graphene Oxide-PEG as Nanocarrier for Protocatechuic Acid and Chlorogenic Acid. Pharm. Res. 2019;36:91. doi: 10.1007/s11095-019-2621-8. PubMed DOI

Al-Snafi A.E. The pharmacological importance of Bauhinia variegata—A Review. Int. J. Pharm. Sci. Res. (IJPSR) 2013;4:160–164.

Rajkapoor B., Jayakar B., Murugesh N., Sakthisekaran D. Chemoprevention and cytotoxic effect of Bauhinia variegata against N-nitrosodiethylamine induced liver tumors and human cancer cell lines. J. Ethnopharmacol. 2006;104:407–409. doi: 10.1016/j.jep.2005.08.074. PubMed DOI

Sharma N., Bhardwaj R., Kumar S., Kaur S. Evaluation of Bauhinia variegata L. bark fractions for in vitro antioxidant potential and protective effect against H2O2-induced oxidative damage to pBR322 DNA. Afr. J. Pharm. Pharmacol. 2011;5:1494–1500. doi: 10.5897/AJPP11.457. DOI

Sharma N., Bhardwaj R., Singh B., Kaur S. Essential Oil Composition of Bauhinia variegata L. flowers World J. Pharm. Pharm. Sci. 2013;2:3604–3611.