Applied Biochemistry and Biotechnology

, Volume 179, Issue 8, pp 1456–1468 | Cite as

Effect of Rol Genes on Polyphenols Biosynthesis in Artemisia annua and Their Effect on Antioxidant and Cytotoxic Potential of the Plant

  • Erum Dilshad
  • Sara Zafar
  • Hammad Ismail
  • Mohammad Tahir Waheed
  • Rosa Maria Cusido
  • Javier Palazon
  • Bushra MirzaEmail author


Flavonoids are famous for their antioxidant capacity and redox potential. They can combat with cell aging, lipid peroxidation, and cancer. In the present study, Artemisia annua hybrid (Hyb8001r) was subjected to qualitative and quantitative analysis of flavonoids through HPLC. Rol genes transgenics of A. annua were also evaluated for an increase in their flavonoid content along with an increase in antioxidant and cytotoxic potential. This was also correlated with the expression level of flavonoids biosynthetic pathway genes as determined by real-time qPCR. Phenylalanine ammonia-lyase and chalcone synthase genes were found to be significantly more highly expressed in rol B (four to sixfold) and rol C transgenics (3.8–5.5-fold) than the wild-type plant. Flavonoids detected in the wild-type A. annua through HPLC include rutin (0.31 mg/g DW), quercetin (0.01 mg/g DW), isoquercetin (0.107 mg/g DW) and caffeic acid (0.03 mg/g DW). Transgenics of the rol B gene showed up to threefold increase in rutin and caffeic acid, sixfold increase in isoquercetin, and fourfold increase in quercetin. Whereas, in the case of transgenics of rol C gene, threefold increase in rutin and quercetin, 5 fold increase in isoquercetin, and 2.6-fold increase in caffeic acid was followed. Total phenolics and flavonoids content was also found to be increased in rol B (1.5-fold) and rol C (1.4-fold) transgenics as compared to the wild-type plant along with increased free radical scavenging activity. Similarly, the cytotoxic potential of rol gene transgenics against MCF7, HeLA, and HePG2 cancer cell lines was found to be significantly enhanced than the wild-type plant of A. annua. Current findings support the fact that rol genes can alter the secondary metabolism and phytochemical level of the plant. They increased the flavonoids content of A. annua by altering the expression level of flavonoids biosynthetic pathway genes. Increased flavonoid content also enhanced the antioxidant and cytotoxic potential of the plant.


Antioxidants Artemisia annua Cytotoxic potential Flavonoids Hyb8001r 



chalcone synthase


dimethyl sulfoxide




dry weight


phenylalanine ammonia-lyase


tris buffer EDTA


reactive oxygen species



We are appreciative to the Higher Education Commission (HEC) of Pakistan for providing the scholarship to author Erum Dilshad during her Ph.D. Part of this work was financially supported by the Spanish MEC (BIO2014-51861-R) and the Generalitat de Catalunya (2014SGR215).

Authors’ contributions

ED carried out the practical work regarding experiments, i.e., real-time qPCR and MTT assay besides manuscript write-up. SZ and HI carried out HPLC analysis and antioxidant assays. RMC and JP supervised all the work done in the Department of Pharmacy, University of Barcelona, Spain. They also went over the manuscript intellectually and critically to give it final shape for publishing. BM and MTW supervised all the work performed in plant molecular Biology laboratory of QAU Islamabad Pakistan. They too assisted in writing the manuscript and its proofreading. All authors have read and approved the final version of the paper.

Compliance with Ethical Standards

Competing Interests

The authors declare that the have no competing interests.


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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Erum Dilshad
    • 1
  • Sara Zafar
    • 1
  • Hammad Ismail
    • 1
  • Mohammad Tahir Waheed
    • 1
  • Rosa Maria Cusido
    • 2
  • Javier Palazon
    • 2
  • Bushra Mirza
    • 1
    Email author
  1. 1.Department of BiochemistryQuaid-i-Azam University IslamabadIslamabadPakistan
  2. 2.Laboratorio de Fisiologia Vegetal, Facultad de FarmaciaUniversidad de BarcelonaBarcelonaSpain

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