Rapid screening of antioxidant activity, fracture rate and scavenging of free radicals by hairy root of Periwinkle (Catharanthus roseus L. G. Don)

Document Type: Original article

Author

Department of Science and Engineer, Mobarakeh Branch, Islamic Azad University, Mobarakeh, Isfahan, Iran

Abstract

Background & Aim:Oxidative stresses caused by free radicals are known to promote many diseases such as inflammation and cancer; therefore, any plant product that can inhibit free radical production can play an important role in preventing diseases. We aimed to compare the fracture rate and the scavenging activity of free radicals, and the antioxidant composition of rolB-transformed hairy root of Catharanthus roseus with those of the plantʼs natural root and leaf.
Experimental: Hairy root of the Madagascar periwinkle (C. roseus) were induced using Agrobacterium rhizogenes strain ATCC 15834 to transfer the rolB gene. PCR analysis was used to identify the geneʼs presence in the transformed hairy root. Folin-Ciocalteu reagent and HPLC were used to determine total phenolics and gallic acid. This study also entailed rapid screening of antioxidant by dot-blot DPPH staining on thin-layer chromatography (TLC), radicals fracture rate, free radical scavenging by 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay, and nitric oxide-scavenging activities by Griess reagent.
Results: Our results showed that phenolic and gallic acid content in the ethanol extract of hairy root was significantly higher (p ≤ 0.05) than that naturally found in the root and leaf of C. roseus. The hairy root extract had the highest dot-blot staining and fracture rate of radicals and the lowest IC50 for inhibition of DPPH and NO. The transgenic hairy root of C. roseus showed high number of scavenging radicals, whereas the most active ethanol extract of hairy root registered by the highest level of gallic acid and total phenolic content.
Recommended applications/industries: The transformed hairy roots can be considered as a rich natural source of antioxidants.

Article Title [Persian]

بررسی سریع فعالیت آنتی اکسیدانی و سرعت شکست رادیکال های آزاد ریشه های مویین پروانش (کاتارانتوس رزئوس)

Author [Persian]

  • شاهین مردانی نژاد
گروه علوم و مهندسی، واحد مبارکه، دانشگاه ازاد اسلامی، مبارکه، اصفهان، ایران
Abstract [Persian]

Background & Aim:Oxidative stresses caused by free radicals are known to promote many diseases such as inflammation and cancer; therefore, any plant product that can inhibit free radical production can play an important role in preventing diseases. We aimed to compare the fracture rate and the scavenging activity of free radicals, and the antioxidant composition of rolB-transformed hairy root of Catharanthus roseus with those of the plantʼs natural root and leaf.
Experimental: Hairy root of the Madagascar periwinkle (C. roseus) were induced using Agrobacterium rhizogenes strain ATCC 15834 to transfer the rolB gene. PCR analysis was used to identify the geneʼs presence in the transformed hairy root. Folin-Ciocalteu reagent and HPLC were used to determine total phenolics and gallic acid. This study also entailed rapid screening of antioxidant by dot-blot DPPH staining on thin-layer chromatography (TLC), radicals fracture rate, free radical scavenging by 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay, and nitric oxide-scavenging activities by Griess reagent.
Results: Our results showed that phenolic and gallic acid content in the ethanol extract of hairy root was significantly higher (p ≤ 0.05) than that naturally found in the root and leaf of C. roseus. The hairy root extract had the highest dot-blot staining and fracture rate of radicals and the lowest IC50 for inhibition of DPPH and NO. The transgenic hairy root of C. roseus showed high number of scavenging radicals, whereas the most active ethanol extract of hairy root registered by the highest level of gallic acid and total phenolic content.
Recommended applications/industries: The transformed hairy roots can be considered as a rich natural source of antioxidants.

Ahmadi Moghadam, Y., Piri K.H., Bahramnejad, B., Ghiasvand, T. 2014. Dopamine production in hairy root cultures of portulaca oleracea (Purslane) Using Agrobacterium rhizogenes. Journal of Agriculture Science Techology, 16:409-420.

Aslam, J., Khan S.H., Siddiqui, Z.H., Fatima, Z., Maqsood, M., Bhat, M.B., Nasim, S.A., Ilah, A., Ahmad, I.Z., Khan, S.A., Mujib, A. and Sharma, M.P. 2010. Catharanthus roseus (L.) G. Don. an important drug: It’s applications and production. Pharmacie Globale (IJCP), 4: 1–16.

Atta, A.H., Hashim, M.M., Arbid, M.M.S., Nada, S.A., Morgan A, Asaad, G.F. 2007. Antioxidant, hepatorenal and mutagenic effects of butylated hydroxytoluene.Veterinary Medical JournalGiza, 55:101-113.

Baldi, A., Dixi, V.K. 2008. Yield enhancement strategies for artemisinin production bysuspension cultures of Artemisia annua. Bioresour Technology, 99:4609-46.

Broothaerts, W., Mitchell,H.J., Weir, B., Kaines, L.M.A., Yang, W., Mayer, J.E., Roa-Rodriguez, C., Jefferson, R.A. 2005. Gene transfer to plants by diverse species of bacteria. Nature, 433:629-633.

Brand-Williams, W., Cuvelier, M. and Bersel, C. 1995. Use of a free radical method to evaluate antioxidant activity. Food Science and Technology, 28: 25-30.

Chang, H.Y., Sheu, Y.L., Lin, M.J., Lin, Y.H., Tseng, M.C., Wu, S.J., Huang, G.J., Chang, Y.S. 2007. Antioxidant and free radical scavenging activities of Phellinus merrillii extracts,Biochemistry, 48:407-417.

Claudio,  A.F.M., Ferreira,  A.M., Freire,  C.S.R., Silvestre, A.J.D., Freire, M.G., Coutinho, J.A.P. 2012. Optimization of the gallic acid extraction using ionic-liquid-based aqueous two-phase systems. Separation and Purification Technology, 97:142-149.

Dehpour, A.A., Ebrahimzadeh, M.A., Nabavi, S.F., Nabavi, S.M. 2009. Antioxidant activity of methanol extract of Ferula assafoetida and its essential oil composition. Grasas Y Aceites, 60:405-412.

European Food Safety Agency. 2012. Scientific Opinion on the re-evaluation of butylated hydroxytoluene BHT (E 321) as a food additive. EFSA Journal, 10:2588.

Ferreres, F., Pereira, D.M., Valentao, P., Andrade, P.B., Seabra, R.M. and Sottomayor, M. 2008. New phenolic compounds and antioxidant potential of Chataranthus roseus. Journal of Agricultural and Food Chemistry, 56: 9967-9974.

Gangopadhyay, M., Dewanjee, S., Bhattacharyya, S., Bhattacharya, S. 2010.Effect of different strains of Agrobacterium rhizogenes and nature of explants on plumbago indica hairy root culture with special emphasis on root biomass and plumbagin production. Natural Product Communications, 5: 1913-1916.

Gokany, S., Loring, R.H., Glick, J., Lee-Parsons, C.W. 2009. Assessing the limitations to terpenoid indole alkaloid biosynthesis in Catharanthus roseus hairy root cultures through gene expression profiling and precursor feeding. Biotechnology Progress, 25:1989-96.

Govindarajan, R., Rastogi, S., Vijaykumar, M., Shirwaikar, A., Rawat, A.K.,  Mehrotra, S.2003. Studies on antioxidant activities of Desmodium gangeticum. Biological and Pharmaceutical Bulletin, 26:1424-1427.

Huang,D.J., Lin, C.D., Chen, H.J., Hou, W.C., Lin,Y.H. 2004. Active recombinant thioredoxin h protein with antioxidant activities from sweet potato (Ipomoea  batatas) storage roots. Journal of Agriculture Science Techology, 52:4720-4724.

Khan, S., Qureshi, I. M., Alam, T., Abdin, M. Z. 2007. Protocol for isolation of genomic DNA from dry and fresh roots of medicinal plants suitable for RAPD and restriction digestion. African Journal of Biotechnology,6:175-178.

Karthikeyan, B., Joe, M.M., Jaleel,C.A., Deiveekasundaram, M. 2010. Effect of root inoculation with plant growth promoting rhizobacteria (PGPR) on plant growth, alkaloid content and nutrient control of catharanthus roseus (L.) G. Don.Natura. Croatica, 19:205-212.

Lata, B. 2007. Cultivation, mineral nutrition and seed production of Catharanthus roseus(L.) G. Don in the temperate climate zone. Phytochem Rev, 6:403-411.

Lobo, V., Patil, A., Phatak, A., Chandara, N. 2010. Free radicals, antioxidants and functional foods: Impact on human health. Pharmacognosy Reviews, 4:118–126.

Mohammadpour, M., Sadeghi, A., Fassihi, A., Saghaei, L., Movahedian, A., Rostami, M. 2012. Synthesis and antioxidant evaluation of some novel orthohydroxypyridine-4-one iron chelators.Research in Pharmaceutical Sciences,7:171–179.

Molyneux, P. 2004. The use of stable free radical diphenylpicryl hydrazyl for estimating antioxidant activity.Songklanakarin Journal of Science and Technology,26:211-219.

Murashige, T., Skoog, F.A. 1962. Revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum,15:473-497.

Orcic, D.Z., Mimica-Dukic, N.M., Franciskovic, M.M., Petrovics, S.S., Jovin, E.D. 2011. Antioxidant activity relationship of phenolic compounds in Hypericum perforatum L. Chemistry Central Journal, 5:5-34.

Park, N.I., xiaohua, li., Uddin, M.R., Park, S.U. 2011. Phenolic compound production by different morphological phenotypes in hairy root cultures of fagopyrum tataricumGaertn. Archives of Biological Sciences, 63:193-198.

Panicker, V.P., George, S., Krishna, B.D. 2014. Toxicity study of butylated hydroxyl toluene (BHT) in rats. World Journal of Pharmacy and Pharmaceutical Sciences, 3:758-763.

Pereira, D.M., Faria, J., Gasparn, L., Ferreres, F., Valentao, P., Sottomayor, M., Andrade, P.B. 2010. Exploiting Catharanthus roseus roots: source ofantioxidants. Food chemistry,121:56-61.

Pham-Huy, L.A., He, H., Pham-Huy, C. 2008. Free radicals, antioxidants in disease and health. Journal of Biomedical Science, 4:89-96.

Pirian, K.,  Piri,  K.h., Ghiyasvand, T. 2012. Hairy roots Induction from Portulacaoleracea Using  Agrobacterium  rhizogenes to  Noradrenaline’s  Production.  Intl. Res International Journal of Basic Sciences and AppliedResearch, 3: 642-649.

Pistelli, L., Giovanni, A., Ruffoni, B., Bertoli, A. 2010. Hairy root cultures for secondary metabolites production. Landes Bioscience, Springer: 167-184.

Pavlov, A., Kovatcheva, P., Georgiev, V., Koleva, I., Ilieva, M. 2002. Biosynthesis and radical scavenging activity of betalains during the cultivation of Red beet (Beta vulgaris) hairy root cultures. Zeitschrift Fur Naturforschung C, 57:640–644.

Pratap Chandran, R., Potty, V.P. 2008. Induction of hairy roots through the mediation of four strains ofAgrobacterium rhizogenes on five host plants. Indian Journal of Biotechnology, 7:122-128.

Sawant, N.R., Abhijit, R., Chavan, A.R. 2013. Determination of gallic acid from their methanolic extract of punica granatum by hplc method. International Journal of Chemistry Technology Research, 5:2598-2602.

Setamam, N.M., Sidik, N.J., Rahman, Z.A., Zain, C. R.C.M. 2014. Induction of hairy roots by various strains of Agrobacterium rhizogenes in different types of Capsicum species explants. BMC Research Notes, 7:414-422.

Singleton, V.L., Orthofer, R., Lamuela-Raventos, R.M. 1999. Analysis of total phenols and other oxidation substrates and antioxidants by Folin Ciocalteu reagent. Methods Enzymology. 299:152-178.

Shaw, C.A., Taylor, E.L., Megson, I.L., Rossi, A.G. 2005. Nitric oxide and the resolution of inflammation: implications for atherosclerosis. Publication ofInstituto Oswaldo Cruz, 1:67-71.

Soleimani, T., Keyhanfar, M., Piri, K., Hasanloo, T. 2012. Morphological evaluation of hairy roots induced in Artemisia annuaL. and investigating elicitation effects on the hairy roots biomass production.  International Journal of Agricultural Research, 2:1005-1013.

Stoilova, I., Krastano, A., Stoyanova, A., Denev, P., Gargova, S. 2007. Antioxidant activity of ginger extract Zingiber officinale. Food Chemistry, 102:764-770.

Rahnama, H., Hasanloo, T., Shams, M.R., Sepehrifar, R. 2008. Silymarin production by hairy root culture of Silybummarianum L. Gaertn. Iran Journal Biotechnology, 6:113-118.

Tzfira, T., Citovsky, V. 2008. Agrobacterium: from biology to biotechnology. Springer Science, Business Media LLC, NewYork,523–563. 

Verpoorte, R.2002.Contain a and memelink journal of biotechnology for production of plant secondary metabolites. Phytochemistry Reviews, 1:13-25.