Morphology and withanolide production of Withania coagulans hairy root cultures

Abstract

Inoculation of leaf sections of Withania coagulans with Agrobacterium tumefaciens strain C58C1 (pRiA4) induced transformed roots with the capacity to produce the most important bioactive compounds of Withania species, withanolide A and withaferin A. The hairy roots obtained showed two morphologies: callus‐like roots (CR) with a high capacity to produce withanolides and typical hairy roots (HR) with faster growth capacity and lower withanolide accumulation. The aux1 gene of pRiA4 was detected by PCR analyses in all roots showing callus‐like morphology. However, this gene was only detected in 12.5% of the roots showing typical hairy root morphology. This fact suggests a significant role of aux genes in the morphology of transformed roots. Time course studies of withanolide production showed that withanolide A accumulated during the first part of the culture whereas the maximum accumulation of withaferin A occurred at the end of the culture period. Some transformed root lines, such as HR112 and CR26, showed considerable potential to produce withanolides in a scaled up bioreactor system, especially the important pharmaceutical compound withanolide A.

Hypoglycemic and Antihyperglycemic Effects of Different Extracts and Combinations of Withania coagulans Dunal and Acacia arabica Lamk in Normal and Alloxan Induced Diabetic Rats

“…Previous phytochemical examination of the whole plant resulted in the isolation of 25 compounds, including 24 withanolides and one dimeric lignan, bispicropodophyllin glucoside. [12] Acacia arabica belongs to family Mimosaceae and is reported for In vitro antibacterial activity [13] Flavonoids, sterols/triterpenoids, alkaloids and phenolics are known to be bioactive antidiabetic principles. [14,15] Flavonoids are known to regenerate the damaged beta cells in the alloxan induced diabetic rats.…”

LC-MS/MS method for the quantification of withaferin-A in plant extracts ofWithaniaspp.

“…Sterilization of seeds, in vitro culturing of roots and extraction of withaferin-A were carried out according to Mirjalili et al [11]. To extract the withaferin-A, we followed the method reported by Chaurasiya et al [5] with some modifications.…”

“…Separations were carried out using a Waters XBridge Shield RP18 (2.1 × 100 mm; 3.5 μm) column. The mobile phase consisted of water (A) and ACN (B) using gradient elution: (time, %B) = (0, 20), (5, 55), (5.5, 80), (5.8, 80), (6,20), (11,20) at a flow rate of 0.4 mL/min. MS/MS analysis was performed on a triple quad-rupole mass spectrometer API 3000 (ABSciex, Ontario, Canada) equipped with a Turbo Ionspray source operating in positive ion mode with the following settings: nebulizer gas (N 2 ) at 10 (arbitrary units), curtain gas (N 2 ) at 12 (arbitrary units), auxiliary gas (N 2 ) at 8000 cc/min and CAD gas (N 2 ) at 6 (arbitrary units).…”

“…However, since the withanolide UV absorption spectrum varies considerably in the number of permutations of chromophoric groups [6] and most withanolides show a low UV maximum, these compounds are in-creasingly being determined by HPLC/MS [7]. Recently, withaferin-A content of the aerial parts and roots of W. somnifera and W. coagulans has been analysed by HPTLC [8]. Quantitative liquid chromatography/mass spectrometry (LC/MS) analyses of withanolides have been reported by several authors, but generally using the less specific single-ion monitoring (SIM) mode or full scan methods [9,10] and not the MS/MS method, which is more sensitive and reliable.…”

Withanolides: Strategies for Enhanced Production and Mechanistic Insights into their Mode of Actio

“…Besides withanolides, there are a number of phytocompounds listed in Table 1, which exhibit similar properties. Biological activity of withanolides is due to C 28 side chain in which C-22 and C-26 are oxidized and are rearranged in a ergostane framework to form a six-membered lactone ring (Mirjalili et al, 2009). Structure-activity corelation of withanolides has been studied which has revealed that unsaturation at α, β position of ketone ring in ring A and epoxy group at position 5β, 6β in ring B are necessary for withanolides to remain biologically active (Chen et al, 2011).…”