WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES Chandan et al.
World Journal of Pharmacy and Pharmaceutical Sciences
Volume 2, Issue 6, 5134-5142.
Research Article
ISSN 2278 – 4357
IN VITRO REGENERATION AND QUANTIFICATION OF ALIZARIN IN RUBIA CORDIFOLIA *Chandan K Jha, Rachna Singh, Gursatya Singh Algh, Summet Basak and Dr. Khatri
Allele Lifesciences Pvt. Ltd.
Article Received on 22 August 2013,
ABSTRACT
Revised on 30 Sept 2013, Accepted on 04 November 2013
of Rubia cordifolia.Various methods were used to isolate the important
Through this study the Alizarin was qualtify and quantify from the root
secondary metabolites like anthriquinones,
which finds wide
application in the medicinal and dye industry. One of the commercially *Correspondence for
important athriquinone, Alizarin, is widely used as dye in the food and
Author:
cloth industry.The production of alizarin by hairy roots of R. cordifolia
*Chandan K Jha,
was confirmed by HPLC analysis. The results show that the maximum
Allele Lifesciences Pvt. Ltd.,
amount of alizarin production was found in roots grown on MS media
India.
as compared to roots grown on other media.
[email protected]
Key words – Alizarin, Rubia cordifolia, HPLC, Anthriquinones.
INTRODUCTION In the present time focus on plant research has increased all over the globe enormously. They are commonly used as important components of medicines, cosmetics, dyes, beverages etc. Plants are considered chemical laboratories capable of biosynthesizing number of biomolecules of different classes. From roots of Rubia cordifolia many anthraquinones are isolated like 4-dihydroxy 2-methylanthraquinone and 1, 5-dihydroxy 2 methylanthraquinone and 3-prenyl methoxy 1, 4- naphthoquinone, anthraquinone),
alizarin
primeveroside,
ruberythric
anthraquinone,
Mollugin (1-hydroxy-2-methyl-9,10-
(1,3-dihydroxy-2-ethoxymethyl-9,10 acid
anthraquinones,
anthraquinone),
lucidin
2-methyl-1,3,6-trihydroxy-9,10-
2-methyl-1,3,6-trihydroxy-9,10-anthraquinone
3-O-(6'-O-acetyl)-Y-
rhamnosyl-(1_2)-Z-glucoside and 2-methyl-1,3,6-trihydroxy-9,10 anthraquinone 3-O-Yrhamnosyl (1_2)-Z-glucoside, the cytotoxic activity of naphthohydroquinones and
www.wjpps.com
5134
Chandan et al.
World Journal of Pharmacy and Pharmaceutical Sciences
naphthohydroquinones dimmers had been tested. Because it’s wide applications and medicinal properties an attempt has been made to increase the production and extraction of secondary metabolites in this plant. Various methods were used to isolate the important secondary metabolites like anthriquinones, which finds wide application in the medicinal and dye industry. One of he commercially important athriquinone, Alizarin, is widely used as dye in the food and cloth industry. MATERIALS AND METHODS A) Bacterial Strain and Culture Conditions Agrobacterium rhizogenes NCIM 5140 (ATCC 5140)was obtained from NCL, Pune, India and was used for hairy root induction. The bacterial culture was revived and maintained on YEB agar medium. Loop full of bacterial colonies were inoculated in 100ml of liquid YEB medium and the culture was kept on a rotary shaker (100rpm) at 300C overnight till the O.D. at 600 nm was about 0.5. This culture was used for the infection process. B) Infection of explants Bacterial culture of 24 hrs to 30 hrs old were grown to obtain the desired O.D of 0.5-0.8 and centrifuged. 10ml of basal MS medium was added to the pellet. The pellet was dissolved completely and 1ml of this medium added to 9ml of basal MS medium thus achieving 1:9 dilution and the precultutred explants were co-cultivated in a flask at 25oc on rotary shaker in dark. The explants were co-cultivated with A. rhizogenes for 45 minutes and later the explants were blotted dry to remove excess of bacteria. C) Subculturing After 3 days, the explants were transferred to MS medium containing 400 mg/l cefotaxime so as to kill the residual Agrobacterium. The explants were again subcultured on the same medium after a week. Cefotaxime concentration was then halved in subsequent subcultures every week from 400mg/l to 50mg/l and finally cultures free of A. rhizogenes were transferred to MS medium. D)
Analytical studies
Hairy roots of 70 days old were analyzed for the presence of the alizarin, a major anthriquinone present in the roots of Rubia cordifolia. Different secondary metabolites and Alizarin contents in the roots were analyzed by reverse phase-high performance liquid chromatography ( RP-HPLC) (Waters 6785) by the method of Goverdina et. al. (1998). The
www.wjpps.com
5135
Chandan et al.
World Journal of Pharmacy and Pharmaceutical Sciences
hairy roots grown were harvested and the blotted dry to remove the traces of medium. 0.5 gm of fresh weight roots was refluxed with 5ml of methanol, ethanol and D/w. After 18 hrs. the tubes were centrifuged at 5000 rpm for 10 min. and the extract was filtered over a 0.22-um membrane filter. The sample was then analyzed by HPLC. HPLC analysis of alizarin Qualitative determination of alizarin The HPLC apparatus consisted of a water 6785 multi solvent delievery system, equipped with a UV dual detector. Data were processed using Waters Empower software. Chromatography was carried out using an eluent of a 45:55 (v/v) mixture of acetonitrile:20 mM ammonium formate (pH 3.00). The flow-rate was 1 mL min)1. The flow rate of the mobile phase was 1 ml/min. Peaks were detected at 254 nm absorption spectrum. The RT value for alizarin is 7.421 min. Peaks were identified by addition of authentic standards. Quantitative Determination of alizarin Standard solutions of alizarin (25ppm, 50ppm, 100ppm, 200ppm, 300ppm and 400ppm) were prepared in methanol. A 20 uL volume of each standard solution was injected in triplicate onto the HPLC column. The calibration graphs were constructed by plotting the peak areas of alizarin versus their concentrations RESULTS AND DISCUSSION The present work mainly focused on Agrobacterium mediated induction of hairy roots in in vitro grown plants of rubia cordifolia and measurement of anhriquinone content ( alizarin) on different media by using HPLC ( high performance liquid chromatography). Hairy roots In this experiment, in vitro grown stem and leaf discs were used as explants which were incised and pre-cultured for 2 days in order to allow the phenolic compounds to be secreted as wound response which serve as signal for transfer of T-DNA. Duration of co-cultivation was 1hr. the bacterium was eliminated on subsequent transfer from 400 mg/l to 200 mg/l cefatoxime after 25 days of subculture. Hairy roots were observed on 14th to 15th day of infection, stems showing maximum response. The frequency of infection in leaves was found to be low. Hairy roots were induced in two batches and consistent results were observed. The in vitro grown stem segments showed
www.wjpps.com
5136
Chandan et al.
World Journal of Pharmacy and Pharmaceutical Sciences
58.46% induction of hairy roots as compared to the in vitro infected leaves which showed only 34.81% of hairy root induction. The result of amount of hairy roots induced in different explant is shown in table no. 1. Table 1: Induction of hairy roots from different explants isolated from R. cordifolia Days of culture 14 24
Explant Stem Leaf Stem Leaf
% response 56.08 ± 6.43 34.81 ± 12.59 58.46 ± 7.04 34.81 ± 12.59
No. of roots per explant 5.66 ± 0.86 3.00 ± 0.63 7.10 ± 1.26 4.67 ± 0.84
The infected explants were grown on solid MS medium containing 4 % sucrose (plate no. 1). The roots showed red colouring at the root tips on around 25th day indicating production of anthriquinones. Biomass growth was found to be stalled after 20 days. These results are consistent with those observed by Hizozi Suzuki et. al., 1983 which showed that
R.
cordifolia cells in 5 % of sucrose medium reached a maximum dry weight 20 days after the inoculation, while their anthriquinone content decreased at the lag and early exponential phases of growth respectively and then increased till the stationary phase. HPLC analysis of alizarin content in samples: HPLC analysis confirmed the production of alizarin in hairy roots of R. cordifolia. The alizarin content was found maximum in cultures grown in solid MS media around 25.5 ppm/g and least in half MS culture around 3 ppm/g. the alizarin content in various sample is given in table no. 2, while the HPLC results are given in plate no. 2. Similar kind of studies was performed by Banyai et. al. in 2006 on R. tinctorum cultures. They reported alizarin content in hairy root cultures of R. tinctorum around 1.36 mg/g of tissue.
www.wjpps.com
5137
K.Gangabhavani et al.
World Journal of Pharmacy and Pharmaceutical Sciences
Plate no. 1 : Hairy root induction in R. cordifolia.
a)
b)
a) And b) Magnified view of hairy roots obtained in R. cordifolia.
c)
d) c) and d) Hairy roots obtained after subculturing for few weeks.
www.wjpps.com
5138
K.Gangabhavani et al.
World Journal of Pharmacy and Pharmaceutical Sciences
Table no. 2: Alizarin content in R. cordifolia cultures grown in different medium. Sample MS B5 1/2 MS
Alizarin Content (ppm) in 100 Alizarin content (ppm) in 1 gm of mg of fresh hairy root tissue fresh hairy root tissue. 2.55 ± 0.036 25.5 ± 0.42 1.15 ± 0.045 11.5 ± 0.54 0.30 ± 0.012 3.00 ± 0.67
CONCENTRATION (ppm)
ALIZARIN CONTENT IN VARIOUS SAMPLES 30 25 20 15 10 5 0 MS
B5
1/2 MS
MEDIA
Fig 1: Graph showing alizarin content in 1gm samples grown on solid MS, B5 and ½ MS media.
Plate no. 2: HPLC analysis for alizarin content Fig a): HPLC analysis of alizarin standard
www.wjpps.com
5139
K.Gangabhavani et al.
World Journal of Pharmacy and Pharmaceutical Sciences
Fig b): HPLC analysis of hairy roots grown on MS media. CONCLUSION Hairy roots were successfully induced in R. cordifolia. The growth kinetics studies performed on the hairy roots of R. cordifolia showed better growth submerged culture condition compared to those grown on solid media. The production of alizarin by hairy roots of R. cordifolia was confirmed by HPLC analysis. The results show that the maximum amount of alizarin production was found in roots grown on MS media as compared to roots grown on other media. These results show that modification of the culture medium affects the anthraquinone content of Rubia cordifolia hairy roots. The present studies can be utilized for mass production of alizarin dye and other secondary metabolites from hairy roots of R. cordifolia. Further work needs to be done to increase the production of hairy roots using bioreactor and to enhance the production of secondary metabolites. REFERENCES 1. Arreguin., Bonner J., Arch. Biochem., 26, 178 (1950). 2. Babita H., Chhaya G., Goldee P. (2007), Hepatoprotective Activity Of Rubia Cordifolia, Pharmacologyonline3: 73-79. 3. Ba’nyail P., Kuzovkina I.N., Kursinszkil L., Szoke E’. (2006), HPLC analysis of Alizarin and Purpurin Produced by R.tinctorum L. Hairy Root Cultures, Chromatographia, 63:S111-S114. 4. Bauch H.J., Leistner E. (1978)., Planta Medica, 32, 105.
www.wjpps.com
5140
K.Gangabhavani et al.
World Journal of Pharmacy and Pharmaceutical Sciences
5. Catherine M. Schmidt, Karen A. Trentelman M. (2009)., 1064 nm dispersive Raman micro-spectroscopy for the in-situ identification of organic red colorants, e-PS, , 6, 10-21 6. Chang J., Chavez.L, Rubiasins A-C, new anthracene derivatives from the roots and stems of Rubia cordifolia. Tetrahedron Letters. 41(37): 7157-7162. (2000). 7. Derksen G.C.H., Beek T.A, Groota E., Capelle A. (1998), High performance liquid chromatographic method for the analysis of anthriquinone glycosides and aglycones in madder root ( Rubia tinctorum L.), Journal of Chromatography A, 816, 277-281. 8. Deshkar N, Tillo S, Pande V (2008) A Comprehensive Review of Rubia cordifolia Linn. Pharmacognosy Reviews , Vol 2, Issue 3. 9. Dosseh C.H., Tessier A.M., Delaveau P. (1981), Planta Medica, 41, 360. 10. Flores, H.E.; Hoy, M.W.; Pickard, J.J. (1987): Secondary metabolites from root cultures. 11. Trends in Biotechnology (Elsevier Publications, Cambridge)., V015 110.3, pp.64-69. 12. Hill R., Richter D. (1937), Proc. Roy. Soc. (London) Ser. B, 111, 54. 13. Hu ZB and Du M (2006), Hairy Root and Its Application in Plant Genetic Engineering, Journal of Integrative Plant Biology, 48 (2): 121−127 14. Hua H.M., Wang S.X., Wu L.J., Li X., Zhu TR. (1992), Studies on naphthoic acid esters from the roots of Rubia cordifolia., Yao Hsueh Hsueh Pao. 27(4):279-82. 15. Inoue K., Naeshiro H., Inouye H., Zenk M.H. (1981), Phytochemistry, 20, 1683. 16. Itokawa H, Qiao YF, Takeya K, IitakaY. (1989), New triterpenoids from Rubia cordifolia var. pratensis (Rubiaceae). Chemical and Pharmaceutical Bulletin. 37 (6): 1670. 17. Kim Y.J., Weathers P.J., Wyslouzil B.J (2001) Growth of Artemisia annua Hairy Roots in Liquid- and Gas-Phase Reactors, DOI: 10.1002/bit.10389. 18. Leistner E (1973), Phytochemistry, 12, 1699. 19. Leistner E., Zenk M.H. (1969)., Chem. Commun., 210. 20. Liu R, Lu Y, Wu T, Pan Y (2008),Simultaneous Isolation and Purification of Mollugin and Two Anthraquinones from Rubia cordifolia by HSCCC, Online publication. 21. Morita H., Yamamiya T., Takeya K., Itokawa H. (1992), New antitumor bicyclic hexapeptides, RA-XI, -XII, -XIII and -XIV from Rubia cordifolia. Chem Pharm Bull.,40(5):1352-4. 22. Nadkarni, K.M. (1976), Indian material medica pp 1075-1077. Poular Parakshan, Bombay 23. Park S.U., Kim Y.K., Lee S-Y. (2009)., Establishment of hairy root culture of Rubia akane Nakai for alizarin and purpurin production, Scientific Research and Essay Vol.4 (2), pp. 094-097. www.wjpps.com
5141
K.Gangabhavani et al.
World Journal of Pharmacy and Pharmaceutical Sciences
24. Rawal AK, Nath DK, Yadav N, Pande S, Meshram SU, Biswas SK (2009) Rubia cordifolia, Fagonia cretica linn and Tinospora cordifolia exert anti-inflammatory properties bymodulating platelet aggregation and VEGF, COX-2and VCAM gene expressions
in
rat
hippocampalslices
subjected
to
ischemic
reperfusion
injury,International Journal of Applied Research in Natural Products, 25. Rawal A.K., Nath D.K.,Biswas S.K. (2008), Plausible mechanism of antioxidant action of Fagonia cretica linn, Rubia cordifolia and Tinospora cordifolia during ischemic reperfusion injury in rat hippocampusInternational Journal of Applied Research in Natural ProductsVol. 1(2):16-25. 26. Sasson A. (1991), Production of useful biochemicals by higher-plant cell cultures : biotechnological and economic aspects, Options Méditerranéennes - Série Séminaires no 14 -: 59-74. 27. Shin SH (1989) Studies on the Production of Anthraquinone Derivatives by Tissue Culture of Rubia Species, Arch. Pharm. Res. 12(2), 99-102. 28. Singh R., Geetanjali. (2005), Isolation and synthesis of anthraquinones and related compounds of Rubia cordifolia, J. Serb. Chem. Soc. 70 (7) 937–942. 29. Suzuki H., Matsumoto T., Mikami Y. (1984)., Effects of Nutritional Factors on the Formation of Anthraquinones by Rubia cordifolia Plant Cells in Suspension Culture, Agric. Biol. Chem., 48 (3): 603-610 . 30. Suzuki H., Matsumoto T., Obi Y. (1982), Proc. 5th Intl. Cong. Plant Tissue & Cell Culture, , p. 285. 31. Talapatra S.K., Sarkar A.C., Talapatra B. (1981). Two pentacyclic triterpenes from Rubia cordifolia. Phytochemistry. 20 (8):1923-1927. 32. Thomson R.H (1971), Naturally Occurring Quinones, 2nd Ed., Academic Press, London, p. 367.Vol. 2(1):19-26. 33. Wu, L. J., Wang S.X. ((1991)), 6- Methoxygeniposidic acid, an iridoid glycoside from Rubia cordifolia., Phytochemistry. 30(5): 1710-1711. 34. Zenk M.H., Shagi H-E. (1975)., Schulte U., Planta Medica (Suppl.)/ 79.
www.wjpps.com
5142
