دستیابی به فیتواستروژن فروتینین موجود در ریشه گیاه کما (Ferula ovina) در شرایط درون شیشه‌ای با تأکید بر حفظ ذخایر ژرم پلاسم

نوع مقاله : مقالات پژوهشی


1 دانشگاه فردوسی مشهد

2 دانشگاه علوم پزشکی مشهد


ایران تنها رویشگاه طبیعی گیاه کما ‌(Ferula ovina) است. ریشه‌های این گیاه دارای فروتینین، یکی از قوی‌ترین و گران‌قیمت‌ترین فیتواستروژن‌ها، با پتانسیل درمان پوکی استخوان است. با توجه به خواب عمیق بذر کما، عدم امکان تولید فروتینین با روش‌های شیمیایی و نیاز به تخریب منابع طبیعی به دلیل وجود فروتینین در ریشه کما، دستیابی به فروتینین در شرایط آزمایشگاهی ضرورتی اجتناب‌ناپذیر است. لذا پژوهش حاضر با هدف کاهش ریشه‌کنی گیاه در طبیعت و دستیابی به فروتینین در شرایط درون شیشه‌ای انجام شد. به منظور نگهداری گیاه در شرایط درون شیشه‌ای، 24 محیط کشت باززایی غیرمستقیم و 11 محیط کشت ریشه‌زایی با استفاده از طرح کاملاً تصادفی مورد بررسی قرار گرفت و نتایج آزمون TLC نمونه‌های کشت بافتی در جهت دسترسی به فروتینین بدون آسیب رساندن به ذخایر ژرم پلاسم مورد بررسی قرار گرفت. نتایج مقایسه میانگین‌ها با استفاده از آزمون توکی نشان داد، کالوس‌های سبز فشرده بزرگ در محیط کشتMS  دارای 5/1 میلی‌گرم بر لیتر IAA و 1 میلی‌گرم بر لیتر، شاخه‌های بلند و شاداب در محیط کشت  MSبا ترکیب 5/1 میلی‌گرم بر لیتر IAA و 1 میلی‌گرم بر لیتر kin  MSو ریشه در شرایط درون شیشه‌ای در محیط کشت ½MS با ترکیب 1 میلی‌گرم بر لیتر IBA  با اختلاف معنی‌دار نسبت به با سایر محیط‌های‌کشت، تولید می‌شود. با توجه به نتایج پژوهش حاضر، امکان تولید و نگهداری درون شیشه‌ای گیاه کما از طریق کشت بافت وجود دارد. همچنین با تأیید وجود فروتینین در نمونه‌های کشت بافتی، تولید انبوه این ماده از طریق کشت سوسپانسیون سلولی و با استفاده از بیوراکتور امکان‌پذیر است. نگهداری F. ovina در شرایط درون شیشه‌ای، امکان دستیابی به گیاه کما بدون محدودیت‌های فصل رشد و رویشگاه و در نتیجه دسترسی به فروتینین موجود در ریشه آن با حفظ ذخایر ژرم پلاسم گیاه را فراهم آورد.


عنوان مقاله [English]

In Vitro Achievement of Ferutinin from Root of Ferula ovina with Emphasis on Germplasm Resources Conservation

نویسندگان [English]

  • Hoda Zare Mirakabad 1
  • Mohammad Farsi 1
  • Saeed Malekzadeh Shafaroudi 1
  • Mehrdad Iranshahi 2
  • Abdolreza Bagheri 1
  • Nasrin Moshtaghi 1
1 Ferdowsi University of Mashhad
2 Mashhad University of Medical Sciences
چکیده [English]

Introduction: There is a growing body of the literature that recognizes the importance of ferutinin (C22H30O4) as one of the natural phytoestrogens with potency to treat osteoporosis and some kind of cancers. One of the greatest challenges is availability of ferutinin that is found just in root of some plants of the genus Ferula (Apiaceae), which is the reason of high price of it in global markets. Ferula ovina , an endemic plant of Iran, is known as one of the greatest sources of ferutinin. Unfortunately, access to ferutinin requires uprooting Ferula ovina especially older plants with more secondary metabolites content. There is a large volume of published studies describing the importance of tissue culture in propagation of endangered plants including secondary metabolites without possibility of chemical production and with deep dormancy seed exactly like the characters of F. ovina. Up to now, far too little attention has been paid to importance of tissue culture in accessing ferutinin without degrading the germplasm resources of it. The main aim of this study was to find an approach to access ferutinin associated F. ovina germplasm conservation.
Materials and Methods: In this experiment, the aerial parts of F. ovina were collected from Zoshk area (Mashhad, Iran). The plants were recognized as F. ovina by the Institute of Plant Sciences (Ferdowsi University of Mashhad). Tissue culture part was performed with preparation of sterilized media and explants. Therefore, the MS salts and vitamins was applied as basic medium, however MS salt was decreased to half strength for rooting of shoots. The node (root junction) explants were cultured on 24 callus induction/shooting media with different combinations of plant growth regulators (BAP, IAA, Kin, NAA and IBA). The shoots from direct and indirect regeneration were then transferred to media with different combinations of PGRs (BAP, IBA, IAA and NAA) in order to find rooting medium. Following these treatments, unbalanced ANOVA for analysis of data were performed using IBM SPSS 19. The final stage of the study comprised a TLC test for the purpose of finding ferutinin in samples which resulted from tissue culture. For this purpose, air-dried parts of samples were powdered and extraction was done after being in 3-5 times dichloromethane for 24hours. Then after optimizing solvent system was done with selection the ratio that presented bands in middle of TLC paper.
Results and Discussions: The results indicated that from 24 tested media, just 8 of them had potency of callus formation, but just L6 on MS medium containing 1 mg/l BAP and 1.5 mg/l IAA showed significant difference for percentage of callus induction at 5% level with compact green and the heaviest calluses. Although direct and indirect shoot regeneration was observed in this study, L9 on MS medium along with 1.5 mg/l IAA and 3 mg/l kin demonstrated significant difference for percentage of shooting at 5% level. Moreover, R6 on ½MS with 1 mg/l IBA showed significant difference for percentage of rooting of shoots at 5% level. The most surprising observation to emerge from the data comparison was L24 on half strength MS medium containing 0.2 mg/l BAP and 3 mg/l IBA with potency of callus induction, shooting and rooting of shoots. Regarding to the results of TLC test, ferutinin positive bands were observed in some samples. 
Conclusions: The aim of the present research was to examine possibility of achieving ferutinin without need to uproot F. ovina because there are several problems to achieve this valuable sesquiterpene: 1) Chemical production of ferutinin is impossible, 2) It could be accessible just from roots of genus Ferula, 3) Propagation with seeds of F. ovina is limited because of morphophysiological dormancy of them, 4) Natural habitat of this plant in Iran is going to be destructed, 5) Access to natural habitat is difficult, 6) Time of access is limited with short growth season, 7) Maintaining F. ovina in greenhouse condition is impossible. The results of this research support the idea that producing ferutinin in Iran without any harmful effect on germplasm resources of F. ovina is possible. This is the first study of high scale commercial production of ferutinin which examine associations between tissue culture and ferutinin production.

کلیدواژه‌ها [English]

  • Ferula ovina
  • Ferutinin
  • Tissue culture
  • Thin layer chromatography
  • Germplasm conservation
1- Amooaghaie R. 2009. The Effect Mechanism of Moist-Chilling and GA3 on Seed Germination and Subsequent Seedling Growth of Ferula ovina Boiss. The Open Plant Science Journal, 3: 22-28.
2- Ansari N., Fayaz M., and Ghasemi M.H. 2009. Estimate of Iran-Turanian zone rangelands degradation rate by measuring and suggestion Index. Iranian Journal of Range and Desert Research, 16(3): 293-304. (In Persian)
3- Arnoldi L., Ballero M., Fuzzati N., Maxia A., Mercalli E., and Pagni L. 2004. HPLC-DAD-MS identification of bioactive secondary metabolites from Ferula communis roots. Fitoterapia, 75(3): 342-354
4- Azhir F., and Shahmoradi A. 2007. Autecology of Ferula ovina Boiss. In Tehran Province Iranian Journal of Range and Desert Reseach, 14(3): 359-367. (In Persian)
5- Bernard F., Shaker Bazarnov H., Javadi Khatab L., Shafiei Darabi A., and Sheidai M. 2007. Ferula gummosa Boiss, Embryogenic culture and karyological changes. Pakistan Journal of Biological Sciences, 10(12): 1977-1983.
6- Cavani F., Ferretti M., Carnevale G., Bertoni L., Zavatti M., and Palumbo C. 2012. Effects of different doses of ferutinin on bone formation/resorption in ovariectomized rats. Journal of Bone and Mineral Metabolism, 30(6): 619-629.
7- Fasih M., and Tavakkol Afshari R. 2018. The morphophysiological dormancy of Ferula ovina seeds is alleviated by low temperature and hydrogen peroxide. Seed Science Research, 28(1): 52-62.
8- Ferretti M., Bertoni L., Cavani F., Zavatti M., Resca E., Carnevale G., Benelli A., Zanoli P., and Palumbo C. 2010. Influence of ferutinin on bone metabolism in ovariectomized rats. II: Role in recovering osteoporosis. Journal of Anatomy, 217(1): 48-56.
9- Ferretti M., Cavani F., Bertoni L., Zavatti M., Taronna A., Carnevale G., Benelli A., Zanoli P., Marotti G., Palumbo C. 2010. New aspects of Ferutinin effect in preventing osteoporosis. Italian Journal of Anatomy and Embryology, Vol 115(1/2).
10- Ghasemi S., Heidary M., Rezaalizadeh Rooshan F., and Habibi Z. 2013. New Monoterpene Ester Derivative from Aerial Parts of Ferula Ovina Boiss. 2nd National Congress on Medicinal Plants. Tehran- Iran.
11- Hadi N., Omidbaigi R., and Moeinei A. 2011. In vitro conservation of Ferula gummosa germplasm and its callus induction. Iranian Journal of Rangelands and Forests Plant Breeding and Genetic Research 19(1): 38.
12- Iranshahi M., Famili A., Basarlv C., and Pyasnth S. 2009. Purification and determination of compounds in Ferula ovina Boiss. roots. Journal of Medicinal Plants, 9(36): 72-80. (In Persian)
13- Mozaffarian V. 1996. Dictionary of Iranian Plant Names; Farhang Moaser publishers, Tehran, Iran.
14- Murashige T., and Skoog F. 1962. A revised method for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15: 473- 497.
15- Omidi M., and Farzin N. 2012. Biotechnology Approaches for improvement of medicinal plants. Modern Genetics Journal. 7(3): 209-220.
16- Palumbo C., Ferretti M., Bertoni L., Cavani F., Resca E., Casolari B., Carnevale G., Zavatti M., Montanari C., Benelli A., and Zanoli P. 2009. Influence of ferutinin on bone metabolism in ovariectomized rats. I: role in preventing osteoporosis. Journal of Bone and Mineral Metabolism, 27(5): 538-545.
17- Rahnama-Ghahfarokhi A., and Tavakkol-Afshari R. 2007. Methods for Dormancy Breaking and Germination of Galbanum Seeds (Ferula gummosa). Asian Journal of Plant Sciences, 6(4): 611-616.
18- Tabrizi L., and Koocheki A.R. 2014. Medicinal Plants Ecology, Production and Sustainable Utilization. University of Tehran. (In Persian)
19- Zanoli P., Zavatti M., Geminiani E., Corsi L., and Baraldi M. 2009. The phytoestrogen ferutinin affects female sexual behavior modulating ERα expression in the hypothalamus. Behavioural Brain Research, 199(2): 283-287.
20- Zavatti M., Benelli A., Montanari C., and Zanoli P. 2009. The phytoestrogen ferutinin improves sexual behavior in ovariectomized rats. Phytomedicine, 16(6): 547-554.