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Medium Optimization and Investigation of Phenolic Contents in Cell Suspension Culture of Hypericum retusum Auche

Year 2017, Volume: 7 Issue: 2, 97 - 105, 30.06.2017

Abstract

Hypericum retusum has a potential value as a medicinal plant. In this study, we investigated the
effects of different combination of 6-benzyladenine (BAP) and kinetin (KIN) with 2,4-dichlorophenoxyacetic acid
(2,4-D), different sucrose concentrations (15, 30 and 50 g L
-1) and different light conditions (continuous dark,
continuous light and control) and initial pH values (pH 4.5, 5.8 ve 6.5) on the accumulation of biomass and the
production of fve pharmacologically important phenolic constituents (hypericin, hyperoside, chlorogenic acid,
quercetin and pseudohypericin) in cell suspension culture of
H. retusum. Our data suggested that the best biomass
accumulations obtained from on MS medium supplemented with 0.5 mg L
-1 KİN+1.0 mg L-1 2,4-D, 30 g L-1
sucrose, 5.8 pH and under the darkness conditions in cell suspension culture of H. retusum. The highest phenolic
compound contents were obtained from on MS medium containing 0.1 mg L
-1 BAP+ 0.5 mg L-1 2,4-D at and 15
g L
-1 sucrose. The phenolic compound contents of suspension culture under the different light conditions varied
among the treatments. With this study, we have developed a novel cell suspension culture system for
H. retusum,
suggesting a possible technology for large-scale production of phenolics.



References

  • Agostinis P, Vantieghem A, Merlevede W, De WPAM, 2002. Hypericin in cancer treatment: More light on the way. International Journal of Biochemistry & Cell Biol0gy, 34: 221–41.
  • Akgoz Y, Toker Z, 2013. Antioxidant And Antimicrobial Effects Of Hypericum Retusum Aucher Plant Extracts Prepared In Various Solvents. Fresenıus Envıronmental Bulletin, 22: 493 – 499.
  • Bais HP, Walker TS, McGrew JJ, Vivanco JM, 2002. Factors affecting growth of suspension culture of Hypericum perforatum and production of hypericin. In vitro Cellular and Developmental Biology-Plant, 38: 58-65.
  • Bais HP, Walker TS, McGrew JJ, Vivanco JM, 2002. Factors affecting growth of suspension culture of Hypericum perforatum and production of hypericin. In vitro Cellular and Developmental Biology-Plant, 38: 58-65.
  • Barnes J, Anderson LA, Phillipson JD, 2001. St John’s wort (Hypericum perforatum L.): A review of its chemistry, pharmacology and clinical properties. Journal of Pharm Pharmacology, 53: 583–600.
  • Bertoli A, Cirak C, Teixeira da Silva J.A, 2011. Hypericum species as source of valuable essential oils. Medicinal and Aromatic Plant Science and Biotechnology, 5: 29–47.
  • Butterweck V, Bockers T, Korte B, 2002. Long-term effects of St. John’s wort and hypericin on monoamine levels in rat hypothalamus and hippocampus. Brain Research, 930: 21–9.
  • Coste A, Vlase L, Halmagyi A, Deliu C, Coldea G, 2011. Effects of plant growth regulators and elicitors on production of secondary metabolites in shoot cultures of Hypericum hirsutum and Hypericum maculatum. Plant Cell Tissue Organ Culture, 106: 279–288.
  • Crockett SL, 2010. Essential oil and volatile components of the genus Hypericum (Hypericaceae). Natural Product Communucations, 5: 1493–506.
  • Cui X H, Murthy HN, Wu CH, Paek KY, 2010. Sucrose-induced osmotic stress affects biomass, metabolite, and antioxidant levels in root suspension cultures of Hypericum perforatum. Plant Cell Tissue Organ Culture, 103:7–14.
  • Çakır Ö, 2004. Kimyasal kültürleme koşullarının Astragalus chrysochlorus hücre süspansiyon kültürleri üzerindeki çeşitli etkilerinin incelenmesi. İstanbul Üniversitesi Fen Bilimleri Enstitüsü, Yüksek Lisans Tezi, 57s.
  • Çetin ES, 2010. Asmada hücre süspansiyon kültürleri ile sekonder metabolit üretimi üzerine Araştırmalar. Süleyman Demirel Üniversitesi Fen Bilimleri Enstitüsü, Doktora Tezi, 128s.
  • Dall’Agnol R, Ferraz A, Bernardi AP, 2003. Antimicrobial activity of some Hypericum species. Phytomedicine, 10: 511–16.
  • Davis PH, 1988. Flora of Turkey and the East Aegean Islands. Edinburgh: Edinburgh University Press
  • Dias ACP, Seabra RM, Andrade PB, Ferreres F, Fernandes M, 2000. Xanthone biosynthesis and accumulation in calli and suspended cells of Hypericum androsaemum. Plant Science, 150: 93–101.
  • Ellialtıoglu Ş, Üstün A S, Mehmetoğlu Ü, 1998. Hücre süspansiyon kültüründe fitoaleksin üretimi. Bitkilerde Stres Fizyolojisinin Moleküler Temelleri. E.Ü.Bilim-Teknoloji Uygulama ve Araştırma Merkezi, 22-26 Haziran 1998, Bornova-İZMİR, S: 82- 96.
  • Ertas A, Boga M, Yılmaz MA, Yesil Y, Hasimi N, Kaya MS, Kolak U, 2014. Chemical compositions by using LC-MS/MS and GC-MS and biological activities of Sedum sediforme, Jacq. Pau. Journal of Agricultural And Food Chemistry, 62: 4601-4609.
  • Gaspar T, Penel C, Greppin H, 1982. Peroxidases 1970–1980. A Survey of Their Biochemical and Physiological Roles in Higher Plants. University of Geneva, Geneva
  • Glıck BR, Pasternak JJ, 2003. Molecular Biotechnology, Principles and Applications of Recombinant DNA 3rd Edition, Large Scale Production of Proteins from Recombinant Microorganisms, ASM Press, Washington, 481-509. ISBN: 1-55581-269-4.
  • Hahlbrock K, Scheel D, 1989. Physiology and molecular biology ofphenylpropanoid metabolism. Ann. Rev. Plant Physiol. Molecular Biology, 40: 347–369.
  • Iranbakhsh AR, Oshagi MA, Ebadi M, 2007. Growth and production optimization of tropane alkaloids in Datura stramonium cell suspension culture. Pakistan journal of Biological Sciences, 10: 1236-1242.
  • Isikalan C, Karakus P, Kuru IS, Celik KS, 2013. The Effect Of Uv-B On Fatty Acıd Content And Radıcal Scavengıng Actıvıty Of Methanolıc Extracts From Hypericum Retusum Aucher Grown Under In Vitro Conditions. Fresenius Environmental Bulletin, 22: 1184 – 1188.
  • İlker R, 1987. In vitro pigment production: an alternative to color synthesis. Food Technology, 41: 70-72.
  • Kızıl G, Kızıl M, Çeken B, Yavuz M, Demir H, 2011. Protective Ability of Ethanol Extracts of Hypericum Scabrum L. and Hypericum Retusum Aucher Against the Protein Oxidation and DNA Damage. International Journal of Food Properties, 14: 926-940.
  • Kitanov GM, 2001. Hypericin and pseudohypericin in some Hypericum species. Biochemical Systematics And Ecology, 29: 171–178.
  • Liu XN, Zhang Q, Zhang SX, Sun JS, 2007. Effects of cytokinins and elicitors on the production of hypericins and hyperforin metabolites in Hypericum sampsonii and Hypericum perforatum. Plant Cell Tissue and Organ Culture, 9: 1–7.
  • Meruelo D, Lavie G, Lavie D, 1988. Therapeutic agents with dramatic antiretroviral activity and little toxicity at effective doses: Aromatic polycyclic diones hypericin and pseudohypericin. Proceeding of The National Academy of Science of USA, 85: 5230–4.
  • Namlı S, Akbaş F, Işıkalan Ç, Ayaz Tilkat E, Başaran D, 2010. The effect of different plant hormones (PGRs) on multiple shoots of Hypericum retusum Aucher. Plant Omics Journal, 3: 12-17.
  • Namlı S, Işıkalan C, Akbaş F, Toker Z, Ayaz Tilkat E, 2014. Effects of UV-B radiation on total phenolic, flavonoid and hypericin contents in Hypericum retusum Aucher grown under in vitro conditions. Natural Product Research, 28: 2286-2292.
  • Poutaraud A, Gregorio FD, Fook VC, Girardin P, 2001. Effectof light on hypericin content in fresh flowering top parts and inextract of St. John’s wort (Hypericum perforatum L.). Planta Medica, 67: 254–259.
  • Praveen N, Murthy HN, 2010. Establishment of cell suspension cultures of Withania somnifera for the production of withanolide. Bioresource Technology, 101: 6735–6739.
  • Romulo MLZ, Studart-Guimarães C, Landsmann J, Campos FAP, 1999. Establishment of callus and cell suspension cultures of Opuntia ficus-indica. Plant Cell Tissue and Organ Culture, 58: 155-157.
  • Sakuta M, Takagi T, Komamine A, 1986. Growth related accumulation of betacyanin in suspension cultures of Phytolacca americana L. Journal of Plant Physiology, 125: 337-343.
  • Sökmen A, Gürel E, 2001. Sekonder Metabolit Üretimi, 211-261, Bitki Biyoteknolojisi, Doku Kültürü ve Uygulamaları, Selçuk Üniversitesi Basımevi, Konya, ISBN: 975-6652-04-7.
  • Verpoorte R, Contin A, Memelink J, 2002. Biotechnology for the production of plant secondary metabolites. Phytochemistry Reviews, 1: 13–25.
  • Walker TS, Bais HP, Vivanco JM, 2002. Jasmonic acid-induced hypericin production in cell suspension cultures of Hypericum perforatum L. (St. John's wort).Phytochemistry, 60: 289-293.
  • Westwood MN, 1993. Hormones and growth regulators, temperate zone pomology:physiology and culture. Timber Press, Inc. S.W. Wilshire, 124, Portland, Oregon.
  • Yeoman MM, Holden MA, Corchet P, Holden PR, Goy JG, Hobbs MC, 1990. Exploitation of Disorganized Plant Cultures for the Production of Secondary Metabolites, 139-166, Secondary Products from Plant Tissue Culture, Oxford University Press, New York, ISBN: 0-19-857717-6.
  • Zhang W, Furusaki S, 1997. Regulation of Anthocyanin Synthesis in Suspension Cultures of Strawberry Cell by pH. Biotechnology Letters, 11: 1057-1061.
  • Zhang YH, Zhong JJ, Yu JT, 1996. Enhancement of ginseng saponin production in suspension cultures of Panax notoginseng:manipulation of medium sucrose. J Biotechnol, 51: 49–56.Zhong JJ, Yoshida T, 1995. High-density cultivation of Perilla frutescens cell suspensions for anthocyanin production: effects of sucrose concentration and inoculum size. Enzyme Microbiology Technology, 17: 1073–1079.

Hypericum retusum Aucher’in Hücre Süspansiyon Kültürlerinin Optimizasyonu ve Fenolik Bileşen İçeriğinin İncelenmesi

Year 2017, Volume: 7 Issue: 2, 97 - 105, 30.06.2017

Abstract

Hypericum retusum tıbbi bitki olarak potansiyel bir öneme sahiptir. Bu çalışmada, 6-benziladenin (BAP) ve
kinetin (KİN)’in 2,4-diklorofenoksiasetik asit (2,4-D) ile kombinasyonlarının, farklı sukroz konsantrasyonlarının
(15, 30 ve 50 g L
-1), farklı ışık uygulamalarının (Sürekli karanlık, Sürekli aydınlık ve kontrol) ve farklı başlangıç pH
değerlerinin (pH 4.5, 5.8 ve 6.5),
H. retusum’ un hücre süspansiyon kültürlerinde biyokütle birikimi ve farmakolojik
olarak önemli beş adet fenolik bileşiğin (hiperisin, hiperosid, klorogenik asit, kuersetin ve psödohiperisin) miktarları
üzerine etkisini araştırdık. Verilerimiz,
H. retusum’ un hücre süspansiyon kültürlerinde en iyi biyokütle birikiminin,
0.5 mg L
-1 KİN+ 1.0 mg L-1 2,4-D ve 30 g L-1 ilaveli MS besi ortamında, pH’sı 5.8 ve sürekli karanlık şartlar altında
olan ortamdan elde edildiğini ortaya koymuştur. En yüksek fenolik bileşik içeriği, 0.1 mg L
-1 BAP+ 0.5 mg L-1
2,4-D ve 15 g L-1 sukroz ilaveli ve pH’sı 4.5 olan ortamdan elde edilmiştir. Farklı ışık uygulamalarına tabi tutulan
kültürlerdeki fenolik bileşen içeriği ise uygulamalar arasında farklılık göstermiştir. Bu çalışmayla,
H. retusum
için, fenolik bileşiklerin geniş ölçekli üretimi için muhtemel bir teknoloji sunan yeni bir hücre süspansiyon kültür
sistemi geliştirdik.



References

  • Agostinis P, Vantieghem A, Merlevede W, De WPAM, 2002. Hypericin in cancer treatment: More light on the way. International Journal of Biochemistry & Cell Biol0gy, 34: 221–41.
  • Akgoz Y, Toker Z, 2013. Antioxidant And Antimicrobial Effects Of Hypericum Retusum Aucher Plant Extracts Prepared In Various Solvents. Fresenıus Envıronmental Bulletin, 22: 493 – 499.
  • Bais HP, Walker TS, McGrew JJ, Vivanco JM, 2002. Factors affecting growth of suspension culture of Hypericum perforatum and production of hypericin. In vitro Cellular and Developmental Biology-Plant, 38: 58-65.
  • Bais HP, Walker TS, McGrew JJ, Vivanco JM, 2002. Factors affecting growth of suspension culture of Hypericum perforatum and production of hypericin. In vitro Cellular and Developmental Biology-Plant, 38: 58-65.
  • Barnes J, Anderson LA, Phillipson JD, 2001. St John’s wort (Hypericum perforatum L.): A review of its chemistry, pharmacology and clinical properties. Journal of Pharm Pharmacology, 53: 583–600.
  • Bertoli A, Cirak C, Teixeira da Silva J.A, 2011. Hypericum species as source of valuable essential oils. Medicinal and Aromatic Plant Science and Biotechnology, 5: 29–47.
  • Butterweck V, Bockers T, Korte B, 2002. Long-term effects of St. John’s wort and hypericin on monoamine levels in rat hypothalamus and hippocampus. Brain Research, 930: 21–9.
  • Coste A, Vlase L, Halmagyi A, Deliu C, Coldea G, 2011. Effects of plant growth regulators and elicitors on production of secondary metabolites in shoot cultures of Hypericum hirsutum and Hypericum maculatum. Plant Cell Tissue Organ Culture, 106: 279–288.
  • Crockett SL, 2010. Essential oil and volatile components of the genus Hypericum (Hypericaceae). Natural Product Communucations, 5: 1493–506.
  • Cui X H, Murthy HN, Wu CH, Paek KY, 2010. Sucrose-induced osmotic stress affects biomass, metabolite, and antioxidant levels in root suspension cultures of Hypericum perforatum. Plant Cell Tissue Organ Culture, 103:7–14.
  • Çakır Ö, 2004. Kimyasal kültürleme koşullarının Astragalus chrysochlorus hücre süspansiyon kültürleri üzerindeki çeşitli etkilerinin incelenmesi. İstanbul Üniversitesi Fen Bilimleri Enstitüsü, Yüksek Lisans Tezi, 57s.
  • Çetin ES, 2010. Asmada hücre süspansiyon kültürleri ile sekonder metabolit üretimi üzerine Araştırmalar. Süleyman Demirel Üniversitesi Fen Bilimleri Enstitüsü, Doktora Tezi, 128s.
  • Dall’Agnol R, Ferraz A, Bernardi AP, 2003. Antimicrobial activity of some Hypericum species. Phytomedicine, 10: 511–16.
  • Davis PH, 1988. Flora of Turkey and the East Aegean Islands. Edinburgh: Edinburgh University Press
  • Dias ACP, Seabra RM, Andrade PB, Ferreres F, Fernandes M, 2000. Xanthone biosynthesis and accumulation in calli and suspended cells of Hypericum androsaemum. Plant Science, 150: 93–101.
  • Ellialtıoglu Ş, Üstün A S, Mehmetoğlu Ü, 1998. Hücre süspansiyon kültüründe fitoaleksin üretimi. Bitkilerde Stres Fizyolojisinin Moleküler Temelleri. E.Ü.Bilim-Teknoloji Uygulama ve Araştırma Merkezi, 22-26 Haziran 1998, Bornova-İZMİR, S: 82- 96.
  • Ertas A, Boga M, Yılmaz MA, Yesil Y, Hasimi N, Kaya MS, Kolak U, 2014. Chemical compositions by using LC-MS/MS and GC-MS and biological activities of Sedum sediforme, Jacq. Pau. Journal of Agricultural And Food Chemistry, 62: 4601-4609.
  • Gaspar T, Penel C, Greppin H, 1982. Peroxidases 1970–1980. A Survey of Their Biochemical and Physiological Roles in Higher Plants. University of Geneva, Geneva
  • Glıck BR, Pasternak JJ, 2003. Molecular Biotechnology, Principles and Applications of Recombinant DNA 3rd Edition, Large Scale Production of Proteins from Recombinant Microorganisms, ASM Press, Washington, 481-509. ISBN: 1-55581-269-4.
  • Hahlbrock K, Scheel D, 1989. Physiology and molecular biology ofphenylpropanoid metabolism. Ann. Rev. Plant Physiol. Molecular Biology, 40: 347–369.
  • Iranbakhsh AR, Oshagi MA, Ebadi M, 2007. Growth and production optimization of tropane alkaloids in Datura stramonium cell suspension culture. Pakistan journal of Biological Sciences, 10: 1236-1242.
  • Isikalan C, Karakus P, Kuru IS, Celik KS, 2013. The Effect Of Uv-B On Fatty Acıd Content And Radıcal Scavengıng Actıvıty Of Methanolıc Extracts From Hypericum Retusum Aucher Grown Under In Vitro Conditions. Fresenius Environmental Bulletin, 22: 1184 – 1188.
  • İlker R, 1987. In vitro pigment production: an alternative to color synthesis. Food Technology, 41: 70-72.
  • Kızıl G, Kızıl M, Çeken B, Yavuz M, Demir H, 2011. Protective Ability of Ethanol Extracts of Hypericum Scabrum L. and Hypericum Retusum Aucher Against the Protein Oxidation and DNA Damage. International Journal of Food Properties, 14: 926-940.
  • Kitanov GM, 2001. Hypericin and pseudohypericin in some Hypericum species. Biochemical Systematics And Ecology, 29: 171–178.
  • Liu XN, Zhang Q, Zhang SX, Sun JS, 2007. Effects of cytokinins and elicitors on the production of hypericins and hyperforin metabolites in Hypericum sampsonii and Hypericum perforatum. Plant Cell Tissue and Organ Culture, 9: 1–7.
  • Meruelo D, Lavie G, Lavie D, 1988. Therapeutic agents with dramatic antiretroviral activity and little toxicity at effective doses: Aromatic polycyclic diones hypericin and pseudohypericin. Proceeding of The National Academy of Science of USA, 85: 5230–4.
  • Namlı S, Akbaş F, Işıkalan Ç, Ayaz Tilkat E, Başaran D, 2010. The effect of different plant hormones (PGRs) on multiple shoots of Hypericum retusum Aucher. Plant Omics Journal, 3: 12-17.
  • Namlı S, Işıkalan C, Akbaş F, Toker Z, Ayaz Tilkat E, 2014. Effects of UV-B radiation on total phenolic, flavonoid and hypericin contents in Hypericum retusum Aucher grown under in vitro conditions. Natural Product Research, 28: 2286-2292.
  • Poutaraud A, Gregorio FD, Fook VC, Girardin P, 2001. Effectof light on hypericin content in fresh flowering top parts and inextract of St. John’s wort (Hypericum perforatum L.). Planta Medica, 67: 254–259.
  • Praveen N, Murthy HN, 2010. Establishment of cell suspension cultures of Withania somnifera for the production of withanolide. Bioresource Technology, 101: 6735–6739.
  • Romulo MLZ, Studart-Guimarães C, Landsmann J, Campos FAP, 1999. Establishment of callus and cell suspension cultures of Opuntia ficus-indica. Plant Cell Tissue and Organ Culture, 58: 155-157.
  • Sakuta M, Takagi T, Komamine A, 1986. Growth related accumulation of betacyanin in suspension cultures of Phytolacca americana L. Journal of Plant Physiology, 125: 337-343.
  • Sökmen A, Gürel E, 2001. Sekonder Metabolit Üretimi, 211-261, Bitki Biyoteknolojisi, Doku Kültürü ve Uygulamaları, Selçuk Üniversitesi Basımevi, Konya, ISBN: 975-6652-04-7.
  • Verpoorte R, Contin A, Memelink J, 2002. Biotechnology for the production of plant secondary metabolites. Phytochemistry Reviews, 1: 13–25.
  • Walker TS, Bais HP, Vivanco JM, 2002. Jasmonic acid-induced hypericin production in cell suspension cultures of Hypericum perforatum L. (St. John's wort).Phytochemistry, 60: 289-293.
  • Westwood MN, 1993. Hormones and growth regulators, temperate zone pomology:physiology and culture. Timber Press, Inc. S.W. Wilshire, 124, Portland, Oregon.
  • Yeoman MM, Holden MA, Corchet P, Holden PR, Goy JG, Hobbs MC, 1990. Exploitation of Disorganized Plant Cultures for the Production of Secondary Metabolites, 139-166, Secondary Products from Plant Tissue Culture, Oxford University Press, New York, ISBN: 0-19-857717-6.
  • Zhang W, Furusaki S, 1997. Regulation of Anthocyanin Synthesis in Suspension Cultures of Strawberry Cell by pH. Biotechnology Letters, 11: 1057-1061.
  • Zhang YH, Zhong JJ, Yu JT, 1996. Enhancement of ginseng saponin production in suspension cultures of Panax notoginseng:manipulation of medium sucrose. J Biotechnol, 51: 49–56.Zhong JJ, Yoshida T, 1995. High-density cultivation of Perilla frutescens cell suspensions for anthocyanin production: effects of sucrose concentration and inoculum size. Enzyme Microbiology Technology, 17: 1073–1079.
There are 40 citations in total.

Details

Primary Language Turkish
Journal Section Biyoloji / Biology
Authors

Hilal Surmuş Asan

Özen Hasan Çetin This is me

Ahmet Onay This is me

Publication Date June 30, 2017
Submission Date June 26, 2016
Acceptance Date October 18, 2016
Published in Issue Year 2017 Volume: 7 Issue: 2

Cite

APA Surmuş Asan, H., Hasan Çetin, Ö., & Onay, A. (2017). Hypericum retusum Aucher’in Hücre Süspansiyon Kültürlerinin Optimizasyonu ve Fenolik Bileşen İçeriğinin İncelenmesi. Journal of the Institute of Science and Technology, 7(2), 97-105.
AMA Surmuş Asan H, Hasan Çetin Ö, Onay A. Hypericum retusum Aucher’in Hücre Süspansiyon Kültürlerinin Optimizasyonu ve Fenolik Bileşen İçeriğinin İncelenmesi. J. Inst. Sci. and Tech. June 2017;7(2):97-105.
Chicago Surmuş Asan, Hilal, Özen Hasan Çetin, and Ahmet Onay. “Hypericum Retusum Aucher’in Hücre Süspansiyon Kültürlerinin Optimizasyonu Ve Fenolik Bileşen İçeriğinin İncelenmesi”. Journal of the Institute of Science and Technology 7, no. 2 (June 2017): 97-105.
EndNote Surmuş Asan H, Hasan Çetin Ö, Onay A (June 1, 2017) Hypericum retusum Aucher’in Hücre Süspansiyon Kültürlerinin Optimizasyonu ve Fenolik Bileşen İçeriğinin İncelenmesi. Journal of the Institute of Science and Technology 7 2 97–105.
IEEE H. Surmuş Asan, Ö. Hasan Çetin, and A. Onay, “Hypericum retusum Aucher’in Hücre Süspansiyon Kültürlerinin Optimizasyonu ve Fenolik Bileşen İçeriğinin İncelenmesi”, J. Inst. Sci. and Tech., vol. 7, no. 2, pp. 97–105, 2017.
ISNAD Surmuş Asan, Hilal et al. “Hypericum Retusum Aucher’in Hücre Süspansiyon Kültürlerinin Optimizasyonu Ve Fenolik Bileşen İçeriğinin İncelenmesi”. Journal of the Institute of Science and Technology 7/2 (June 2017), 97-105.
JAMA Surmuş Asan H, Hasan Çetin Ö, Onay A. Hypericum retusum Aucher’in Hücre Süspansiyon Kültürlerinin Optimizasyonu ve Fenolik Bileşen İçeriğinin İncelenmesi. J. Inst. Sci. and Tech. 2017;7:97–105.
MLA Surmuş Asan, Hilal et al. “Hypericum Retusum Aucher’in Hücre Süspansiyon Kültürlerinin Optimizasyonu Ve Fenolik Bileşen İçeriğinin İncelenmesi”. Journal of the Institute of Science and Technology, vol. 7, no. 2, 2017, pp. 97-105.
Vancouver Surmuş Asan H, Hasan Çetin Ö, Onay A. Hypericum retusum Aucher’in Hücre Süspansiyon Kültürlerinin Optimizasyonu ve Fenolik Bileşen İçeriğinin İncelenmesi. J. Inst. Sci. and Tech. 2017;7(2):97-105.