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Isolation and Identification of Medicinal Compounds from Kalanchoe Pinnata of Crassulaceae Family by 1h Nmr

Sabrina Sharmin
Pharmaceutical Chemistry Department, Faculty of Pharmacy, University of Dhaka, Dhaka 1000, Bangladesh

Md. Zahidul Islam
Biotechnology and Genetic Engineering Discipline, Khulna University, Khulna-9208, Bangladesh

Md. Abdul Jabber
Pharmaceutical Chemistry Department, Faculty of Pharmacy, University of Dhaka, Dhaka 1000, Bangladesh

Abstract/ Executive Summary:

The details of phytochemical and biological investigations of Kalanchoe pinnata, belong to the Crassulaceae family grown in Bangladesh has not been studied extensively. Here, Kalanchoe pinnata has been investigated for the isolation of its secondary metabolites by vacuum liquid chromatography. The powdered leaf of Kalanchoe pinnatawas extracted with ethanol. The concentrated extract was partitioned with n-hexane, carbon tetrachloride and Dichloromethane. Chromatographic fractionation and purification of n-hexane partitionate provided two compounds. These two isolated compounds were identified by extensive analyses of their high resolution 1H NMR spectral data. The purified compounds were campesterol and 5,6,7,8,4' pentahydroxy flavanone. This is the first report of their occurrence in Kalanchoe pinnata.

Keywords: Kalanchoe pinnata, 1H NMR, Campesterol and 5,6,7,8,4' pentahydroxy flavanone, n-Hexane, Carbontetrachloride, Dichloromethane and Ethanol.

Location: Pharmaceutical Chemistry Department, Faculty of Pharmacy, University of Dhaka, Dhaka 1000, Bangladesh

Start Date:

End Date:

Program Area: Development of Host and Medicinal Plants

Commodity: Medicinal Plants

Objectives:

The objective is to explore the possibility of developing new drug candidates from these plants for the treatment of various diseases.

Methodology:

Collection and Preparation of the plant material Plant sample of Kalanchoe pinnata was collected. One voucher specimen has been deposited in Dhaka University Herbarium. The leaves (after cutting into small pieces) were sun dried for several days. The plant materials were then oven dried for 24 hours at considerably low temperature for better grinding. The dried plant was then ground in coarse powder using high capacity grinding machine in the Phytochemical Research Laboratory, Faculty of Pharmacy, University of Dhaka. Crude extract preparation About 200 gm of leaf powdered material was taken in clean, round bottomed flask (5 liters) and add 2.5 liters of ethanol. The container was sealed by cotton plug and aluminum foil and kept for 15 days accompanying occasional shaking and stirring. The whole mixture was then filtered and concentrated by a rotary evaporator at low temperature (40-50°C) under reduced pressure. The concentrated extract thus obtained is termed as crude extract. The concentrated extract was air dried to solid residue. Partitioning with n-Hexane, Carbon tetrachloride, and Dichloromethane Here we used modified Kupchan Partition (Beckett and Stenlake1986) method for the solvent-solvent partition of crude extract. Total 5 gm of ethanol crude extract was triturated with 90 ml of methanol and 10 ml of distilled water. The crude extract was dissolved completely. This is the mother solution, which was partitioned successively by three solvents of different polarity (n-hexane, carbon tetrachloride (CCl4), and dichloromethane (DCM)). The mother solution was taken in a separating funnel. The n-hexane (100 ml) was added to it and the funnel was shaken and then kept in a plane surface. The organic as well as the aqueous phase were collected separately. This process was repeated three times and n-hexane fractions were collected together. Distilled water (12.5 ml) added to the mother solution left after washing with n-hexane and mixed well. This solution was taken in a separating funnel and extracted with CCl4 (100 ml × 3). The CCl4 fractions were collected together. The aqueous fraction was preserved for the next step. To the mother solution that left after washing CCl4, 16 ml of distilled water was added and mixed uniformly. The mother solution was then taken in a separating funnel and extracted with DCM (100 ml × 3). The DCM soluble fractions were collected together. The aqueous methanolic fraction was preserved as aqueous fraction. Finally, all the fractions (organic phases as well as the aqueous phase) were collected separately and evaporated by Rota evaporator to dryness. Each of the fraction was analyzed separately for the detection and identification of pure compounds. Isolation of compounds The crude extract and different partitioned fractions were subjected to Thin Layer Chromatography (TLC) for screening the different types of compounds. The n-hexane fraction (400 mg) was subjected to Vacuum Liquid Chromatography (VLC) for separation. VLC is a relatively recent separation technique which involves short column chromatography under reduced pressure. This technique is used for the initial rapid fractionation of crude extracts. The column was packed with fine TLC grade silica (Kiesel gel 60H). Details of the method have been published by Pelletier et al (Pelletier et al.1986) and by Coll and Bowden (Coll and Bowden1986). The column is packed with silica gel (kiesel gel 60H) under vacuum. The size of the column and the height of the adsorbent layer are dependent upon the amount of extract to be analyzed. A column having 50 cm length & 5 cm in diameter was packed with silica gel up to a height of 30 cm under reduced pressure. The column was initially washed with a non-polar solvent (petroleum ether) to facilitate compact packing. The sample to be separated was adsorbed onto silica gel (kiesel gel 60, mesh 70-230), allowed to dry. The column was eluted with a number of organic solvents of increasing polarity and the fractions were collected in 50 & 100 ml beaker. The VLC fractions were analyzed by TLC. The fractions with satisfactory resolution of components were subjected to Preparative Thin Layer Chromatography (PTLC) to obtain the pure compound. Identification by 1H NMR Spectral data was taken for chemical characterization of compounds. The 1H NMR spectra were recorded using a Broker AMX-400 (400 MHz) instruments. For NMR studies deuterated chloroform was used as solvent and the NMR instrument was locked at the solvent peak and the t5 values for 1H were referenced relative to the residual undeuterated solvent signal.

Source of Information: Biosci Bioeng Commun 2016; 2(2): 123-129

Article Link (Online Journal): https://www.journalbinet.com/

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

These investigations provide two interesting compounds, which may be pharmacologically active. The ethanolic crude extract, n-hexane, carbon tetrachloride, dichloromethane and aqueous soluble fractions will be used as a medicine for various diseases. After successive VLC separation of n-hexane soluble fraction of the ethanolic extract of Kalanchoe pinnatayielded two compounds (1) Campesterol and (2) 5,6,7,8,4' pentahydroxy flavanone. Isolated compounds from Kalanchoe pinnataplant play a key role in the healthcare of the people in near future. These new compounds in this report will be used to reveal antioxidant, antimicrobial and cytotoxic activities and to design new kinds of drugs for different kinds of diseases.

Knowledge Management: Journal