6 Discussion(72 73)
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DISCUSSION
DISCUSSION Hemidesmus indicus (L.) R.Br. is an important drug of Indian System of Medicine and used in medicines since time immemorial. A number of phytochemical studies have been carried out and many of the compounds reported are mainly triterpenoids, glycosides, organic acids, aldehydes etc
19-27
. Various pharmacological
activities have been reported such as anticancer, hepatoprotective, anti snake-venom activity, anti-ulcerogenic, antiatherogenic, anti-inflammatory, analgesic, antileprotic and antimicrobial etc 28-64. Although many works have been carried out on this plant but there are scanty reports on specific markers for this plant, HPLC profile of the compounds and standardization. Hence in the present study an attempt had been made to isolate and standardize the constituents which are unique to this plant and characterize them as markers which are chemically defined constituents and can serve as a powerful tool for standardization. Isolation of the phytoconstituents The methanolic root extract of Hemidesmus indicus was refluxed with acetone. The acetone extract was then fractionated using silica gel column chromatography (60200 mesh). Review of literature indicates that column chromatography using silica gel is the most popular method for isolation 19-27. Totally four different column chromatography has been done with different solvent systems which resulted in the isolation of four different compounds. These were given codes as HI-01, HI-02, HI-03 and HI-004.
Department of Pharmacognosy - PESCP
72
DISCUSSION
Analysis of isolated compounds Determination of Melting point and solubility profile were done for the isolated compounds. New HPLC methods were developed to analyze the purity of the compounds. The HPLC method indicated that the compound HI-01 was 99.41 % pure, HI-02 was 32.88 % pure, HI-03 was 46.82% pure and HI-004 was 99.84 % pure. Hence depending upon the purity only HI-01 and HI-004 were taken for the further characterization. Characterization of the isolated compounds The integral values of NMR spectroscopy, the values where the bands obtained in the IR spectroscopy, the CHN analysis and the Mass spectroscopic analysis clearly suggested that the compound HI-01 was found to be a pentacyclic triterpene acetate. The chemical name suggested for this was 20(30)-Ursen-3-acetate. The other compound that is HI-004 was found to be an aromatic aldehyde and the chemical name suggested for this compound was 2-Hydroxy-4-Methoxy benzaldehyde 72, 73. Estimation of isolated compounds In order to check the suitability of the isolated compounds as markers, HPLC analytical methods were developed. The distinct peaks at the retention time of 1.694 min. and 26.021 min. (corresponding to the same value of HI-01 and HI-004 respectively) in HPLC chromatograms of acetone fraction was observed indicating that the method was suitable and sensitive. The acetone fraction of the MeOH extract was found to contain 14.34 % of HI-01 and 12.94 % of HI-004. Hence these compounds can be termed as markers and the extract is said to be standardized.
Department of Pharmacognosy - PESCP
73
DISCUSSION Hemidesmus indicus (L.) R.Br. is an important drug of Indian System of Medicine and used in medicines since time immemorial. A number of phytochemical studies have been carried out and many of the compounds reported are mainly triterpenoids, glycosides, organic acids, aldehydes etc
19-27
. Various pharmacological
activities have been reported such as anticancer, hepatoprotective, anti snake-venom activity, anti-ulcerogenic, antiatherogenic, anti-inflammatory, analgesic, antileprotic and antimicrobial etc 28-64. Although many works have been carried out on this plant but there are scanty reports on specific markers for this plant, HPLC profile of the compounds and standardization. Hence in the present study an attempt had been made to isolate and standardize the constituents which are unique to this plant and characterize them as markers which are chemically defined constituents and can serve as a powerful tool for standardization. Isolation of the phytoconstituents The methanolic root extract of Hemidesmus indicus was refluxed with acetone. The acetone extract was then fractionated using silica gel column chromatography (60200 mesh). Review of literature indicates that column chromatography using silica gel is the most popular method for isolation 19-27. Totally four different column chromatography has been done with different solvent systems which resulted in the isolation of four different compounds. These were given codes as HI-01, HI-02, HI-03 and HI-004.
Department of Pharmacognosy - PESCP
72
DISCUSSION
Analysis of isolated compounds Determination of Melting point and solubility profile were done for the isolated compounds. New HPLC methods were developed to analyze the purity of the compounds. The HPLC method indicated that the compound HI-01 was 99.41 % pure, HI-02 was 32.88 % pure, HI-03 was 46.82% pure and HI-004 was 99.84 % pure. Hence depending upon the purity only HI-01 and HI-004 were taken for the further characterization. Characterization of the isolated compounds The integral values of NMR spectroscopy, the values where the bands obtained in the IR spectroscopy, the CHN analysis and the Mass spectroscopic analysis clearly suggested that the compound HI-01 was found to be a pentacyclic triterpene acetate. The chemical name suggested for this was 20(30)-Ursen-3-acetate. The other compound that is HI-004 was found to be an aromatic aldehyde and the chemical name suggested for this compound was 2-Hydroxy-4-Methoxy benzaldehyde 72, 73. Estimation of isolated compounds In order to check the suitability of the isolated compounds as markers, HPLC analytical methods were developed. The distinct peaks at the retention time of 1.694 min. and 26.021 min. (corresponding to the same value of HI-01 and HI-004 respectively) in HPLC chromatograms of acetone fraction was observed indicating that the method was suitable and sensitive. The acetone fraction of the MeOH extract was found to contain 14.34 % of HI-01 and 12.94 % of HI-004. Hence these compounds can be termed as markers and the extract is said to be standardized.
Department of Pharmacognosy - PESCP
73
