Physico chemical parameters-
Physico chemical parameters for both the stem and root are almost the same except for the alcohol soluble extractive. Root gives 18% and for stem 10%.
Isolation of various constituents
Phenols and alkaloids were isolated using general method for isolation given in materials and methods. Organic layer on testing for terpenoids, phenols and glycosides showed the presence of only phenols (blue colour with neutral ferric chloride). Both the stem and root gave the test for phenols. Tannins were detected in the hot aqueous extract of both the stem and root.
TLC study of different extracts of stem and root were carried out with a view to distinguish between them.
TLC study of different fractions separated from stem and root
Different fractions separated (1) Lipids, fats and waxes. (2) Glycosides, Terpenoids and Phenols. (3)Alkaloids
(1) Lipids, fats and waxes.
Solvent system-Benzene-Acetone 9:1
Stem gave one spot Rf 0.84 in UV, whereas root did not give any spot in UV. In Iodine stem gave 2 spots Rf 0.52 and 0.68 and root gave one spot of Rf 0.84. i.e., the three compounds were separated from stem lipids, fats and waxes, and only one compound from root. Also the compounds isolated are different.
Toluene-Ethyl acetate 7:3
In this solvent system, three compounds were separated from root, one spot of Rf 0.85 in UV, and 2 spots of Rf 0.7, 0.6 in Iodine.
(2) TLC study of phenols separated
Solvent system--Benzene-Acetone 9:1
Phenolic compounds of stem could not be detected in this solvent system where as that of root was detected. Three compounds were separated with Rf 0.71 in UV, and 0.21 and 0.84 in Iodine indicating that the phenols present in the root are not present in stem.
Toluene-Ethyl acetate 7:3
In this solvent system, two phenolic compounds could be separated with Rf 0.32, in Iodine and 0.84 in UV and one compound with Rf 0.9 in root also indicating that phenolic compounds in root and stem are different.
(3)TLC study of alkaloids
Solvent system--Dioxan-Ammonia 9:1
Alkaloids separated were subjected to TLC in solvent system Dioxan-Ammonia 9:1. Two different alkaloids were separated from the stem and root, as evidenced by different Rf values. Stem gave one brown fluorescent spot of Rf 0.9, whereas root gave one spot of Rf 0.27.
From the above results, it can be seen that TLC studies of different fractions can be used to distinguish stem from root.
Alkaloids separated by general procedure was used for HPTLC study. Solvent system used was Dioxan-Ammonia 9:1. Linear ascending development was carried out and densitometric scanning was performed on CAMAG′s TLC Scanner in absorbance mode at 254 nm.
Root gives 4 peaks of Rf values 0.09, 0.47, 0.57, 0.82. Stem gives 3 peaks of Rf values 0.12, 0.41, 0.83. One additional peak of Rf 0.57 is present in root which is a quick and easy way of distinguishing stem and root.