Histidine acid phytases (HAPhy) are widely distributed enzymes among bacteria, fungi, plants, and some animal tissues. They have a significant role as an animal feed enzyme and in the solubilization of insoluble phosphates and minerals present in the form of phytic acid complex. A set of 50 reference protein sequences representing HAPhy were retrieved from NCBI protein database and characterized for various biochemical properties, multiple sequence alignment (MSA), homology search, phylogenetic analysis, motifs, and superfamily search. MSA using MEGA5 revealed the presence of conserved sequences at N-terminal “RHGXRXP” and C-terminal “HD.” Phylogenetic tree analysis indicates the presence of three clusters representing different HAPhy, that is, PhyA, PhyB, and AppA. Analysis of 10 commonly distributed motifs in the sequences indicates the presence of signature sequence for each class. Motif 1 “SPFCDLFTHEEWIQYDYLQSLGKYYGYGAGNPLGPAQGIGF” was present in 38 protein sequences representing clusters 1 (PhyA) and 2 (PhyB). Cluster 3 (AppA) contains motif 9 “KKGCPQSGQVAIIADVDERTRKTGEAFAAGLAPDCAITVHTQADTSSPDP” as a signature sequence. All sequences belong to histidine acid phosphatase family as resulted from superfamily search. No conserved sequence representing 3- or 6-phytase could be identified using multiple sequence alignment. Thisin silicoanalysis might contribute in the classification and future genetic engineering of this most diverse class of phytase.

This study aimed at optimizing conditions for increased withanolide production in Withania somnifera. The elicitors used for the foliar spray on the aerial parts of the plant were salicylic acid, jasmonic acid, and chitosan for the enhancement of withanolides in Withania somnifera under different environmental regimes. Three different elicitors, i.e., chitosan, jasmonic acid and salicylic acid, were applied on the plants through foliar route every 15th day for 6 months, and later plants were used for sample preparation. Further, the elicitors were used in different concentration, i.e., jasmonic acid (50, 200 and 400 ppm), chitosan (10, 50 and 100 ppm) and salicylic acid (0.5, 1 and 2 ppm). The elicitors were sprayed on the foliar parts of the plant between 10:00–11:00 a.m. on application days. For elicitor spray, a calibrated sprayer was used. The withanolide A/withaferin A was quantified through HPLC. It was found that in an open environment, maximum withaferin A content, i.e., 0.570 mg/g (DW), was recorded with jasmonic acid (50 ppm) treatment in comparison to control (0.067 mg/g DW). Thus, there was an 8.5-fold increase in the withaferin A content. Maximum withanolide A content of 0.352 mg/g (DW) was recorded when chitosan (50 ppm) was sprayed, while in the control, withanolide A content was recorded to be 0.031 mg/g (DW); thus, chitosan application increased the production of withanolide A by 11.3-fold. Under controlled conditions, maximum withaferin A content of 1.659 mg/g (DW) was recorded when plants were sprayed with chitosan (100 ppm), which was 8.1 times greater than the control content of 0.203 mg/g (DW). Maximum withanolide A content of 0.460 mg/g (DW) was recorded when chitosan (100 ppm) was applied, whereas in the control, withanolide A content was found to be 0.061 mg/g (DW). Thus, foliar spraying of elicitors in very low concentrations can serve as a low-cost, eco-friendly, labor-intensive and elegant alternative approach that can be practiced by farmers for the enhancement, consistent production and improved yield of withanolide A/withaferin A. This can be a suitable way to enhance plant productivity, thus increasing the availability of withanolide A and withaferin A for the health and pharma industry.

Fenugreek seeds were evaluated for diosgenin content and productivity which can be useful for pharmaceutical industries and farmers. Statistical significance was tested by ANOVA (n=5) followed by comparisons of means by DMRT (p≤0.05). Fenugreek seeds were grown in the experimental fields of Vegetable Research Center, Pantnagar, Uttarakhand, India for cropping year 2014. Two local varieties Pant Ragini (PR), Pusa Early Bunching (PEB) and eight accessions PM(C)1, PM 2, PM 3, PM 4, PM 5, PM 6, PM 7 and PM 8 were selected to evaluate high diosgenin content and productivity. Results indicate that fenugreek is a good source of diosgenin with high productivity, ranging from 0.40 to 0.60 g/100 g and 7.78 to 14.95 Kg ha^-1, respectively. Significant differences (p≤0.05) were observed among the accessions and local varieties with respect to diosgenin content and productivity. The higher diosgenin content was recorded in local variety, PEB (0.60%) and PR (0.59%), followed by accession PM 7 (0.59%) and PM2 (0.50%) respectively. The highest diosgenin productivity was recorded in PR (14.95 Kg ha^-1) followed by PEB (14.54 Kg ha^-1), PM 7 (14.46 Kg ha^-1) and PM (C)1 (13.90 Kg ha^-1). The maximum seed yield was recorded in accession PM(C)1 (2.98 T ha^-1) followed by PR (2.52 T ha^-1). Fenugreek seeds, used as functional foods or pharmacology industry due to bioactive substance diosgenin, contribute nutraceutical and pharmacological attributes. Fenugreek may be a feasible alternative for diosgenin production because of lower production costs in a short growing period, ease of cultivation and consistent seed yield. Variation in genotype as well as plant species bioactive compound composition has been contradictory for their availability and commercial use. This study may be useful in selecting superior fenugreek genotypes for targeted pharmaceutical purposes and also for a breeding program, further facilitate the development of more reliable genotype for industrial use.