Effect of sample extraction, preparation methods on HPLC quantification of plumbagin in in vivo and in vitro plant parts of Plumbago zeylanica L.

Plumbagin is an important therapeutic compound of Plumbago zeylanica L., the commercial demand of this compound warrants optimizing a suitable method to isolate plumbagin in large scale. The present study was undertaken to obtain the maximum recovery of plumbagin content by employing different extraction methods viz., ultra-assisted extraction (UAE), maceration extraction (ME), soxhlet extraction (SE), serial soxhlet extraction (SSE), and serial maceration extraction (SME) from plant parts of P. zeylanica. Plumbagin content of two different sources such as field grown and hardened in vitro regenerated plants were quantified using reversed-phase high-performance liquid chromatography (RP-HPLC). For in vitro cultures, 6-benzylaminopurine (BAP) and Kinetin (KIN) were used for multiple shoot induction from nodal segments of P. zeylanica. Maximum percentage of shoot induction was obtained on MS medium fortified with BAP (6.66 µM) from nodal segments exposed for 6 weeks. Further multiple shoot proliferation and elongation was achieved in MS medium with a combination of BAP (6.66 µM) and KIN (4.44 µM), with the maximum number of shoots (47.3±0.06) and shoot length (2.0±0.06 cm) per explant after 6 weeks of culture. The optimum root induction was observed on MS medium supplemented with 1.23-µM indole-3 butyric acid (IBA) which produced 10.02±0.2 mean roots with 6.2±0. 8 cm root length. Among the extraction methods, the SME method yielded maximum recovery (99.5%) of plumbagin as compared to others. In vitro leaf extract yielded high content of plumbagin (152.02 mg/g-1 DW) as compared to other plant parts (root 115.41 mg/g-1 dry weight; stem 98.02 mg/g-1 dry weight) whereas in vivo leaf, stem and root samples yielded 96.7, 38.59, and 86.35 mg/g DW of plumbagin, respectively. The present observation suggests that the SME was more efficient for obtaining the maximum recovery of plumbagin and it was confirmed with HPLC quantification. Among the field grown and in vitro regenerated plants, the in vitro culture shows more accumulation of plumbagin and is found suitable for commercial extraction.   Key words: Micropropagation, cytokinin, serial maceration extraction (SME), optimization, methanol, quantification.