The polypeptide-k was subjected to HPTLC, which showed a single peak after scanning at wavelength of 254 nm.  (Fig. 1). The protein is insoluble in water but completely soluble in 10% formic acid. On hydrolysis it was found to contain 19 different amino acids with 160 residues and a mol. wt. of 18000. Tryptophan and cystine residues could not be determined (Table 1) ⁵. On further analysis 35 amino acids were found arranged in four cycles with interchangeable amino acids at positions 12, 13, 15-19, 25-27 and 31-34. Mixture of sequences homologous to various legumin and Prunes proteins (Table 2) ⁶ were observed Amino acid mass concentrations of one sample was also determined (Table 3).

Electrospray mass spectra of the sample allowed the detections of two different compounds with molecular masses at 11474.32 + 8.77 and 11708.57 + 6.30 with a complex mixture from which it was difficult to distinguish peaks ((Fig. 2). A. B). When MS/MS ion search on trypsin digest of polypeptide-k was carried out after variable modifications as oxidation with propionamide on ESI-QUAD-TOF instrument, two hundred twenty nine peptides were observed after calculating the probability based on more than 51 Mowse auto score hits out of which only two significant hits resembling fragments of Wolinella succinogenes and Pau troglodytes were observed when Mascot search was carried out (Table 4, 1A-9A). The remaining peptides did not match with any of the known peptides as shown in the Database.

However, amino acid Edman sequencing (Fig. 3) results from peak 81 of polypeptide-k trypsin digest sample (run 2 H P 017) showed very strong sequence (Table. 5, 6) which matched very well with 11S globulin subunit beta precursor from Cucurbita maxima (accession #: P 13744) in the NCBI nr database residues 186-196. The predicted mass (1303.45 Da) was in excellent agreement with the mass observed (1303.8 Da) in the MALDI data. There was a tryptic cleavage site in the known sequence prior to the first residue of this peptide, so a trypsin digest of that protein would generate this peptide. Major mass at 1479.91 and 2101.97 in sample 2HP017-113 and 2HP017 - 126 respectively were observed but these were either unsuitable for Edman sequencing  due to a mixture (Table 7) or did not match with any of the known NCBI nr Database    (Table 8).

The polypeptide-k was tested for heavy metals, total aerobic microbial count (E. Coli and Salmonella sp), hexane and acetone (Gas Chromatography) before formulation and manufacturing into Tablets before animal and clinical trials.