Detection and quantification of piperine from in vitro cultures of black pepper (Piper nigrum L.)
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Date
2011
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Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara
Abstract
The present study on “Detection and quantification of piperine from in vitro cultures of black pepper (Piper nigrum L.)” was carried out at the Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, during the period 2009-2011. The objective of the study was to standardize the protocol for in vitro synthesis of piperine in black pepper and to detect and quantify the in vitro synthesized piperine.
Callus cultures from tender, pale green leaf from the variety Panniyur 5 and immature berry and epicarp of berry from the variety Panniyur 2 were established successfully. Surface sterilization with mercuric chloride (0.1 %) for 5 min was effective in initiating healthy, contamination free callus from leaf and 10 min were effective in initiating healthy callus from berry explants. It was observed that half strength MS (Murashige and Skoog, 1962) medium supplemented with IAA (1.0 mg l-1) along with BA (1.0 mg l-1) was suitable for initiation and proliferation of calli. Leaf derived cultures were superior with respect to callus initiation and proliferation.
Leaf calli were sub cultured into five different modifications of the culture initiation medium viz., (I) increased sucrose concentration (5 different concentrations), (II) 1/4th nitrate and phosphate concentration in the MS stock solution, (III) different hormone combinations (2, 4-D and BA in four combinations), (IV) stress inducing agent (mannitol) and (V) precursor of piperine (L-lysine) in three different concentrations. The effect of these modified media on piperine production was studied by detecting the presence of piperine and estimating the quantity by High Performance Liquid Chromatography in the respective calli, callus growing in the culture initiation medium (as control) and the calli growing in these modified media.
Piperine was extracted from 1.0 g callus by grinding in 5 ml methanol and the centrifuged crude extract was used for High Performance Liquid Chromatography analysis (Shimadzu, Japan). Gradient elution system using 0.1% ortho-phosphoric acid and 90 % acetonitrile in 0.1% ortho-phosphoric acid was employed for detection of piperine.
The study revealed that increasing the concentration of sucrose up to 7 per cent in the callus initiation medium increased the piperine content from 0.018 % to 0.135% in leaf derived calli whereas, reducing the nitrate nitrogen and phosphorus content of the basal medium to 1/4th of its original strength exerted an inhibitory effect on piperine production (0.003%). Similarly, supplementing the callus initiation medium with mannitol at 4 per cent along with 3 per cent sucrose, resulted in enhanced in vitro synthesis of the target compound (0.107 %). Similarly the medium with 2, 4-D (3.0 mgl-1) enhanced the in vitro synthesis of piperine to 0.042 per cent in berry calli. Precursor feeding with L-lysine at higher concentration (100 mgl-1) also exerted a favorable influence on piperine (0.066 %) synthesis in comparison to other concentration (5 and50 mgl-1).
Among the various explants studied, young leaves were found to be the most suitable for callus induction in black pepper. In vitro piperine production from leaf calli (0.135 %) was higher than that in the leaf used as explant (0.01%). Berry calli produced very less amount of piperine (0.042 %) compared to that of fresh berry used as explant (2.7%). The results indicated that in vitro synthesis of piperine may be exploited by further modifications of the culture media.
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