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

Abstract

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.

Plumbagin Extraction: A Green Study

“…This procedure offered better plumbagin extraction, in comparison to the previously reported methods 19‐21 (Figures 3, and S3–S8); however, it was not for this study. At this point, the synergy between the cavitation effect and the direct heating by a typical reflux process increased the yield by a higher disruption of the root cell walls and solvent/solute interactions, facilitating the release of the target molecule due to better mass transfer of the metabolite from the sample matrix.…”

“…These samples were mixed with 10, 15, 30, 60, 90, or 120 mL of n -hexane, ethyl acetate, acetone, or methanol and macerating for 2.5, 5, 7.5, or 12 h at room temperature. 21 Each experiment was systematically monitored by TLC ( n -hexane/ethyl acetate 85:15; in silica) employing an authentic commercial sample of plumbagin; after this time, the extracts were filtered and dried under vacuum.…”

“…Independent experiments were conducted with either 1 g or 50 g of dried and milled roots of Plumbago pulchella Boiss with either 10 or 120 mL of ethyl acetate, n -hexane, MeOH, or acetone, respectively, and refluxed for 2.5, 5, or 7.5 h at 63–65 °C. 21 Each extract was systematically monitored by TLC ( n -hexane/ethyl acetate 85:15; on silica gel) employing an authentic commercial sample of plumbagin; after this time, the extracts were filtered and dried under vacuum.…”

“…6 As a consequence of the last comments, some research groups have focused their efforts on improving the extraction of plumbagin, employing several extraction methods: maceration, [19][20][21] serial maceration, 21 dynamic maceration, 20 mantle heating, 20,21 and ultrasound. 20,21 However, some of them have shown disadvantages: the use of chloroform 20 or its mixture with dichloromethane, 19 which are considered toxic solvents, 22 and the requirement of long extraction times, even days, employing as solvents, n-hexane, methanol, water, chloroform, or ethyl acetate. [19][20][21] After an exhaustive search of the literature, and consequently, to our knowledge, there is minimal information related to the chemistry and extraction of Plumbago pulchella Boiss: only three studies were found: cytotoxic activity, 23 skincare formulation, 24 and the hyperaccumulation occurring in ultramafic vegetation.…”

“…20,21 However, some of them have shown disadvantages: the use of chloroform 20 or its mixture with dichloromethane, 19 which are considered toxic solvents, 22 and the requirement of long extraction times, even days, employing as solvents, n -hexane, methanol, water, chloroform, or ethyl acetate. 19‐21…”