International Journal of Agriculture and Crop Sciences. Available online at www.ijagcs.com IJACS/2013/5-0/00-00. ISSN 2227-670X ©2013 IJACS Journal
Increase alpha-tocopherol in cell suspension cultures Elaeagnus angustifolia L Azam Badrhadad*1,Khosro Piri2, Tayebe Ghiasvand3 1. MS ,Biotechnology Department of Bu Ali Sina university,Hamadan,Iran 2. Associate Professor Biotechnology Department of Bu Ali Sina university,,Hamadan,Iran. 3. Research center ofmedical faculty,Bu-Ali Sina university, Hamadan,Iran. Corresponding author email:
[email protected] ABSTRACT: Plants are a tremendous source for the discovery of new medicinal valuable products for drug development. The evolving commercial importance of secondary metabolites has in recent years resulted in a greatinterest in secondary metabolism, particularly in the possibility of altering the production of bioactive plant metabolites by using of tissue culture technology. The presentinvestigation assaytoincrease accumulation of alpha-tocopherol in cell suspension culture of Elaeagnus angustifolia. callus producted was transfered to suspension culture medium ,then Jasmonic acid ( with 5, 25,50 µm)and salicylic acid (with 0.1, 0.5, 1 mM concentrations )elicitors added to samples. HPLC instrument was used to evaluated the rate of alpha tocopherolproduction. Our results showed both of elicitors effective in increasingalpha tocopherol production.the 1mM concentration of SA was most effective in alpha tocopherol production in the cell cultures. The increased concentration of SA was positively corrected with alpha tocopherol accumulation. Key words:Alpha tocopherol, Suspension culture, Elaeagnus angostifolia, Salicylic acid, Jasmonic acid INTRODUCTION Plant secondary products are of immense use as potential drugs, nutraceuticals, and food additives. Due to theirlimited availability and complexity for chemical synthesis an alternative route such as plant cell culture is gainingimportance for large-scale production of desired compounds (Savitha et al., 2006). Usually the production of secondary metabolites remained low in cell cultures and, therefore, various techniques has been employed to improve upon the production including abiotic and biotic elicitors.A wide variety of elicitors have been employed to alter cell metabolism in order to enhance the desired secondary metabolite. Jasmonic acid and salicylic acid are recognized as two important signals in plant defense response and are widely used as chemical inducers for secondary metabolite production in plant cells. Someplants synthesize eight different molecules with vitamin E. Vitamin E, a strong anti-sterility factor belongs to afamily of tocopherols findingin oils and fats. In the presence of oxidizing agents or UV light, this vitamin undergoes degradation and isomerization and yields four major vitamers (i.e., α, β, T, and σtocopherols). Three of these vitamers lower biological activities i.e., 30, 15, and 5% for β , T, and σtocopherols, respectively, while α -tocopherol has 100% biological activity.(Mclaughtin, 1979).Tocopherols, substances physiologically active as vitamin E, are naturalpotential antioxidants and find extensive applications in food, cosmetic and pharmaceutical industries. They play significant roles in food preservation and disease prevention: tocopherols inhibit acylglycerol peroxidation ,suppree cholesterol production in liver, give aeffectiveprotection against certain types of cancers, enhance the body’s immune system and reduce cellular aging.(Ruperez et al., 2001; Chu et al., 2002).The Elaeagnus genus belong to the Elaeagnaceae family , which comprises some important species that growing in Iran, with the common local name Senjed thathas a great interest in Iranian traditional medicine, especially in Hamedan province (Mozaffarian, 1996). Elaeagnus angustifolia is reported to have a wide range of biological activities, suchas Antinociceptive, antiinflammatory ( Ahmadiani, 1999) and antioxidant(Bucur, 2007).The main cosstituents in Elaeagnus angustifolia indicateEthyl cinnamate, 2-phenyl-ethyl benzoate, 2-phenyl-ethyl isovalerate, nerolidole, squalene and acetophenone (Bucur, 2007).
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MATERIALS AND METHODS Callus induction from leaf explants Young leaves oleaster tree (Elaeagnus angustifolia L.) werecollected in June from Hamedan province. Samples were surface sterilized by 70% ethanolfor 30 sec and 2% sodium hypochlorite solution for 15 min followedby three rinses with sterilized distilled water. Medium for callus culture were Morashige and Skoog(MS) containing 1mg/l TDZ(N-phenyl-N0 (1,2,3-thidiazol-5-yl) urea) with 3% sucrose.pH of the culture media were adjusted to 5.8 before adding gelling agent (Agar-Agar, Merck). All culturemedia were sterilized by wet autoclave at 121° C for 15 min. All cultures wereincubated in growth room.( Karami & Piri, 2009) Cell suspention culture Cell suspention culture was initiated by transferring 1g of fresh homogenous callus into a 250 ml Erlenmeyer flasks containing 100 ml medium MS without agar. These cultures were incubated on a rotary shaker at 100 rmp and 25±0.2ºC in dark. The cell cultures were subcultured every 2th wk.Cell cultures were treated with jasmonate acid(JA), salicylic acid(SA).The elicitors were prepared as concentrated stock solutions and added to cultures after filter sterilization at the appropriate concentrations at 72 h of culture ,and stay in before condition for 24h.The cell cultures were harvested, washed with distilled water and filtered with filterpaper. Then fresh weighed(FW) and after that 24 h dry wt(DW) was determinate. Samples storedin freezer until extraction. Extraction of tocopherols Extraction of tocopherols from callus cultures of Elaeagnus angustifolia carried out taking care to protect the operations from light and oxidising conditions. A total of 5 g freeze-dried material were ground to a powder and stirred with 100 ml methanol(Merck) for 3 h in the dark at 25 °C, then sonicated in ultrasound(Banoelin sonorex) bath for 40 min. After filtering and centrifuging at 3000 g for 20 min, the supernatant was dried in a rotary evaporator and weighed. The dried extract was resuspended in 100 ml nhexane(Merck), sonicated again for 40 min, and then centrifuged at 3000 g for 20 min; the supernatant was dried and weighed, and stored at −20 °C until further use.( Antognoni et al., 2008). HPLC conditions The following HPLC conditions were used. HPLC system: Waters Method:Isocratic Column: RP-C18(1250*3.9mm, particle size size 5um) Detection: by fluorimetry at 292nm Fluent composition: Methanol:Acetonitril:Thf: Water(50:41:5:4)% v/v Column temperature: 25°C Injection volume: 20 µL Flow rate: 1.5 µL/min Aufs: 0.05 Pressure:1350 Run time: 12 min Retention Time: 7 min Detector: Uv-vis (Aliver et al., 1992) RESULTS Elicitors are defined as molecules that stimulate defense or stress-induced responses in plants (VanEtten et al., 1994).The exogenous application of elicitors to in vitro cultures is useful for studying plant responses to potential microbe/insect attack as well as for enhanced biotechnological production of valueadded secondary metabolites in fermentation systems. Elicitors including JA and its derivatives are known to stimulate production of secondary metabolites in plants (Mizukami et al., 1993; Sanz et al,. 2000). Therefore,we examined the effects of JA, SA elicitors from on production of alpha tocopherol in cell suspension cultures of E.angostifolia , According to the table1 and fig 1 the amount of tocopherol in three concentrations of acid Jasmonic in comparision of control group, increased. We observed that treatment with 5, 25µm JA under dark conditions amount of alpha tocopherol increased as compared to control. In the
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samples treated with salicylic acid stimulating production of alpha-tocopherol in total concentrations increased. Our results suggest that SA is an effective elicitor for the production of alpha tocopherol in cell suspension cultures of E.angostifolia.Specially in highest concentration of salicylic acid (1 mM) was increased significantly produced intocopherol, This was a 9-fold increase over the control group (Fig2).Therefore, cell suspension cultures of E.angostifolia should be considered as an entirely different experimental system for elicitation studies. Recent studies have shown that addition of JA and specially SA to suspension cultures increased the production of alpha tocopherol . Enhancement of secondary metabolites by elicitation is one of the few useful strategies recently finding commercial application (Savitha et al.2006). Table1. Effect of different concentration of jasmonic acid and salicylic acid on alpha tocopherol content(ppm) in cell culture of Elaeagnus angustifolia grown in MS medium Treatment
concentration
amount of alpha tocopherol(ppm)
control _
1.3
Jasmonic acid
5µm 25µm 50µm
2.12 3.2 2.4
Salicylic acid
0.1mM 0.5mM 1mM
1.15 2.27 9.82
Figure 1. Effect of jasmonic acid to production alpha tocopherol
Figure 2. Effect of salicylic acid to production alpha tocopherol
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CONCLUSION In order to improve the nutraceutical value of plants, efforts have been made to enhance the amount of tocopherols through metabolic engineering (Shintani & DellaPenna 1998). For example, in Arabidopsis transformants, increased leaf and seed tocopherol levels relative to control plants (Tsegaye et al. 2002). Elicitation offers an attractive alternative to understand the regulation of enzymes involved in plant secondary metabolism and effective in the biosynthetic pathway. The results presented demonstrate that Elaeagnus angustifolia represent suitable species for the establishment of a cell culture system able to produce αtocopherol. Indeed, callus cultures can be easily obtained from these plants by culturing explants on a MS medium. Accumulation of secondary metabolites is one of the common responses of plants to pathogen infection and elicitor treatment (Hahlbrock et al,. 2003). Our results show that the elicitor-induced alpha tocopherol production can be suppressed by both JA and SA elicitors , In the case of tocopherols, the interactions between jasmonates and phytohormones may be considered of particular relevance. Considering the well demonstrated beneficial effects of α-tocopherol on human health, and its impact in the prevention of chronic diseases (Brigelius-Flohé & Traber 1999), ), Elaeagnus angustifolia suspension cultures will be established with the aim of further increasing the productivity of this in vitro culture system.In conclusion, callus cultures of Elaeagnus angustifoliarespond to elicitation by JA ,SA in α-tocopherol production, therefore represent a promising system for studies on this metabolic pathway. REFRENCES Ahmadiani A, Hosseiny J, Semnanian S, Javan M, Saeedi F, Kamalinejad M, Saremi S.2000. Antinociceptive and anti-inflammatory effects of Elaeagnus angustifolia fruit extract . J Ethnopharmacology.72: 287 – 292. Aliver RWA, Kafwemve EM. 1992. E new spectrophotometric assay for intermination of vitamin A and related compound in serum. International Journal Vit.nutri.rea. 62:221-227 Antognoni F, Faudale M, Poli F, Biondi S. 2008. Methyl jasmonate differentially affects tocopherol content and tyrosine amino transferase activity in cultured cells of Amaranthus caudatus and Chenopodium quinoa“ . Plant Biology. 11:161-169. Brigelius-Flohé R, Traber MG. 1999. Vitamin E: function and metabolism. FASEB Journal. 13: 1145–1155. Bucur G, Stanciu G, Istudor V. 2007. The GC-MS Analysis of Elaeagnus angustifolia L. Flowers Essential Oil . REV. CHIM. 58 (11): 10271029. Chu BS, Baharin BS, Quek SY. 2002. Factors affecting pre-concentration of tocopherols and tocotrienols from palm fatty acid distillate by lipase-catalysed hydrolisys Food Chem. 79: 1- 55. Hahlbrock K, Bednarek P, Ciolkowski I. 2003. Non-self recognition, transcriptional reprogramming, and second metabolite accumulation during plant/pathogen interactions. Proceedings of the National Academy of Sciences of the United States of America. 10 :14569– 14576. Karami O, Piri Kh. 2009. Shoot organogenesis in oleaster (Elaeagnus angustifolia L.) African Journal of Biotechnology. 8 (3):438-440. McLaughtin PJ, Weihrauch JL. 1979. Vitamin E content of foods. J Am Diet Assoc. 75:847-850. Mizukami H, Tabira Y, Ellis BE. 1993. Methyl jasmonate- induced rosmarinic acidbiosynthesis in Lithospermum erythrorhizon cell suspension cultures. Plant Cell Reports. 12:706-709. Mozaffarian VA. 1996. Dictionary of Iranian Plant, Names. Farhang Moaser, Tehran, Iran Ruperez FJ, Martín D, Herrera E, Barbas C. 2001. Chromatographic analysis of α-tocoferol and related compounds in various matrices . Journal of chromatography A. 9 :35- 45. Sanz MK, Hernandez XE, Tonn CE, Guerreio E. 2000. Enhancement of tessaric acid production in Tessaria absinthioides cell susp ension cultures. Plant Cell Rep. 19:821–824. Savitha BC, Thimmaraju R, Bhagyalakshmi N, Ravinshankar AG. 2006. Different biotic and abiotic elicitors influence betalain production in hairy root cultures of Beta vulgaris in shake-flask and bioreactor. Process Biochemistry.41(1): 50-60. Shintani D, DellaPenna D. 1998 . Elevating the vitamin E content of plants through metabolic engineering. Science. 282: 2098–2100. Tsegaye Y, Shintani DK, DellaPenna D. 2002. Overexpression of the enzyme p-hydroxyphenolpyruvate dioxygenase in Arabidopsis and its relation to tocopherol biosynthesis. Plant Physiology and Biochemistry. 40: 913–920. Van Etten HD, Mansfield JW, Bailey JA, Farmer EE. 1994. Two classes of plant antibiotics: phytoalexins versus ‘‘phytoanticipi ns. Plant Cell. 6: 1191–1192.
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