Callus Culture of Plumbago indica for Production of Plumbagin

Shyam Baboo Prasad 1*, Rasheeduz Zafar 2, Vidhu Aeri2

Abstracts: Plumbago indica (Plumbaginaceae) is an important indigenous medicinal plant commonly known as Lal Chitra. The roots contain an orange-yellow pigment plumbagin. Chemically, plumbagin is 2-methyl-5-hydroxy-1, 4-napthoquinone. The roots are used as antimalarial, anticancer, antimicrobial, cardiotonic and also posses anti-fertility activity. The present work is an attempt to search for an alternative source of plumbagin. The callus was initiated from leaf and stem explants on MS medium supplemented with 2, 4-D + NAA (1ppm each). The plumbagin content of callus as well as natural root were determined using HPLC and it is found that callus contain more plumbagin as compared to natural root. Key Words: Callus culture, HPLC, Plumbago indica, plumbagin, Lal chitra. The explants were sterelised using mercuric chloride 0.1% for 10 min followed by washing with double distilled sterile water. The surface sterelised explant was inoculated on MS medium supplemented with various growth hormones. The leaf callus was initiated, developed and maintained for 3 month on MS medium supplemented with 2,4-D + NAA (1PPM each) at 252 c under 16 hrs diffused light (1600lux) and 8 hrs dark cycle. Qualitative Phytochemical Analysis10 The alcoholic extract of natural root and leaf callus were prepared and tested for presence and absence of secondary metabolites.

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Calibration Curve of Plumbagin Different conc. (0.25mg/ml, 0.5mg/ml, 0.75mg/ml, 1mg/ml, 2mg/ml, and 4mg/ml) of Plumbagin (purchased from sigma-aldrich) was prepared in HPLC grade methanol and was subjected to HPLC analysis. Quantification of Plumbagin in Natural Root 1gm of finely powdered root was kept overnight with 10 ml of methanol after that it was filtered and volume was made to 10 ml. 20 l of sample was subjected to HPLC analysis.

INTRODUCTION Plumbago indica (Plumbaginaceae) a rare pretty ornamental plant is an important indigenous medicinal plant commonly known as Lal Chitra. The roots contain an orange-yellow pigment, plumbagin. Chemically, plumbagin is 5-hydroxy-2methyl-1, 4-napthoquinone.1 the roots are used as antimalarial2, anticancer3, antimicrobial4, cardio tonic5 and also posses anti-fertility activity6. Plumbagin is mainly obtained from the roots of Plumbago species (P.indica L, P.zeylanica L, P.europea L), however these plants grow quite slowly and it takes long time (3 years) until the roots are suitable for use7. Propagation through seed is difficult due to poor germination and death of young seedlings1. Report on its cultivation and improvements are also limited. Among all species of Plumbago, Plumbago indica is the richest source of Plumbagin8. Pharmaceutical companies are largely dependent upon material procured from natural sources, which are being depleted rapidly, justifying the development of in vitro technique for P. indica. The present investigation was taken to establish in-vitro callus cultures for production of plumbagin. The plumbagin content of natural root as well as callus were determined using HPLC and it was found that callus contain more plumbagin as compared to natural root. MATERIAL AND METHODS Plant Material Leaves of Plumbago indica were collected from the plants growing in Herbal Garden, Jamia Hamdard, Hamdard Nagar, and New Delhi. The plant was identified by the botanist of university and a herbarium specimen is kept in department of Pharmacognosy and Phytochemistry, faculty of Pharmacy Jamia Hamdard, New Delhi. Initiation and Maintainance of Callus9

Quantification of Plumbagin in Callus 1gm of finely powdered callus of 30 days ,60 days, 90 days and 120 days were kept overnight with 10 ml of methanol separately after that it was filtered and volume was made to 10 ml. 20 l of sample was subjected to HPLC analysis.

Estimaion of Plumbagin in Natural Root and Leaf Callus11 Various methods are available for the estimation of plumbagin. The usual methods are spectroscopy method, HPTLC, and HPLC etc. Among these methods HPLC method is much more sensitive and provided superior resolution of Plumbagin.

School of Pharmaceutical Sciences; Lovely Professional University, Phagwara (Punjab), India. E-mail: b.shyam2@gmail.com *Corresponding author
2Jamia

1Lovely

India.

Hamdard, Hamdard Nagar (New Delhi),

Chromatographic Condition11 For estimation of plumbagin HPLC instrument SHIMADZU (LC-10AT VP) with C18 (5, 2504.6mm) coloumn was used. Methanol and NaH2Po4 5mM in 9:1 ratio is used as mobile phase and filtered before use through 0.45 m membrane filter. The flow rate of mobile phase was maintained at 1ml/min. The coloumn temperature was maintained at 40 c. The UV detector was set at 254 nm wavelenth and injection volumewas 20l.

Figure 1: Callus culture of Plumbago indica

CONCLUSION An attempt has been made in present investigation to develop the static from leaf of Plumbago indica. The hormonal combination on which the callus is developed and maintained is new combination which is reported for the first time. The present study is significant for the point of view of the fact that the quantitative estimation of plumbagin has been carried out by HPLC method which is most reliable, less time consuming and reproducible method. The study is also significant because plumbagin content of callus was found to be more than natural root. It is suggested that the culture may be tried in immobilized condition using other advance techniques by which the ability of the calli may be improved for production of useful metabolites. REFERENCES AND NOTES

RESULT AND DISCUSSION The leaf callus of Plumbago indica was successfully initiated developed and maintained on MS medium supplemented with 2, 4-D + NAA (1PPM each) as shown in Fig 1. Prelimnary phytochemical screening of alcoholic extract of natural root and callus culture revealed the presence of napthoquinone, alkaloid, aminoacid, flavonoid, steroid, coumarin, phenol carbohydrate and saponin. The plumbagin content of natural root and callus was determined using HPLC. The retention time of standard plumbagin was found to be 4.635 as shown in Fig 2. Standard plot was plotted using different concentration as shown in tab 1and fig 3, the regression was found to be 0.999. The content of plumbagin in natural root, 30, 60, 90, and 120 days callus was quantified using proposed analytical method and was found to be as shown in Fig 4-8, Table 2.

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Table 1 : Area value of standard plumbagin S. No. Conc.(x) mg/ml 1 0.25 2 0.5 3 0.75 4 1.0 5 2.0 6 4.0

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Table 2: Area value and amount of plumbagin in natural root and leaf callus S. No. Samples AUC Content in g/l 1 Root 45827238 0.89 2 Callus 30 days old 48523475 0.94 3 Callus 60 days old 49881916 0.97 4 Callus 90 days old 49881981 0.97 5 Callus 120 days old 49881871 0.97

Retention time(Rt) 4.625 4.625 4.525 4.625 4.892 4.525 Content in mg/ml 0.89 0.94 0.97 0.97 0.97

AUC 12901678 25603352 38205013 51414356 101215301 205315893 Content in% w/w 0.89 0.94 0.97 0.97 0.97

Figure 2 : HPLC chromatogram of standard plumbagin

Figure 3: Calibration curve of plumbagin

Figure 4: HPLC chromatogram of methenolic extract of root

Figure 5: HPLC chromatogram of methenolic extract of 60 days old callus

Figure 6: HPLC chromatogram of methenolic extract of 60 days old callus

Figure 7: HPLC chromatogram of methenolic extract of 90 days old callus

Figure 8: HPLC chromatogram of methenolic extract of 120 days old callus

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3. R Primula, P Sachdanandam, Molecular and cellular biochemistry, 12, 59-63 (1993) 4. M Didri, Die Pharmazie, 49, 681-683 (1994) 5. S Itoigawa, Planta medica, 57, 317-319 (1999) 6. SK Bhargava, Indian journal of experimental biology, 22, 153-156 (1984)

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7. GM Kitanov, PP Pashankov, Pharmazie,1994; 49, 462 8. SK Nayana, AI Shalini, BS Mamta, Pharmaceutical Biology, 43, 551- 553 (2005) 9. DE Evans, JOD Coleman, Plant cell culture, (BIOS Scientific Publishers Tylor & Francis group, London and New York,2002)

10. CK Kokate, Practical Pharmacognosy, (Vallabh Prakashan, New Delhi, 2005) 11. KP Unnikrishnan, SS Raja, I Balachandran, Indian Journal of Pharmaceutical Sciences, 70, 844-847, (2008).

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