Bioseparation of phytochemical constituents from leaf and stem extracts of Mimosa pudica

The isolation procedure of chemical constituents was mainly based on fractionation by solvents of varying polarity. After cold extraction, solvent-solvent partitioning of extract was done with different solvents to yield different extracts of leaf and stem of Mimosa pudica L. Gas Chromatography-Mass Spectrometry (GC-MS) analysis was carried out for petroleum ether, chloroform and ethyl acetate extracts of leaf as well as chloroform and ethyl acetate extracts of stem and 16, 11, 3, 26 and 3 compounds were identified respectively. The major compounds were benzene,1-ethyl-3-methyl-(14.83%) for petroleum ether extract of leaf, fumaric acid, ethyl 2-methylallyl ester (16.96%) for chloroform extract of leaf, glycerin (74.71%) for ethyl acetate extract of leaf, ticlopidine (80.90%) for chloroform extract of stem and hexadecenoic acid, methyl ester (69.95%) for ethyl acetate extract of stem.


Introduction
Around 50% of the modern drugs are of plant origin. But only small fractions of the biologically active medicinal plant molecules have been assayed and much phytochemical investigations of higher plants are going on. After isolation of the phytoconstituents, they are screened for different types of biological activities. Crude plant extracts of particular activities are assayed and then the active fractions are analyzed phytochemically (Harborne, 1998). Plants have been using as food as well as medicine and the medicinal plants are using as raw materials for manufacturing drugs as well as synthesizing phytochemicals that are beneficial for human health which are not synthesized in human body (Martinez et al., 2008). The present study aims to investigate the biochemical activities of different crude extracts of leaf and stem of M. pudica, followed by further fractionation by column chromatography, and analyze the fraction by Gas Chromatography-Mass Spectrometry (GC-MS) coupled system.

Collection of sample plants
M. pudica was collected from the Botanical Garden of Rajshahi University Campus, and the leaf and stem were separated. The petioles were separated from the rachis and the stem was chopped into small pieces. The leaf and stem were dried in normal room temperature keeping them into wooden trays. Drying was carried out under shed to prevent the changes of the constituents in it due to drying. After drying they were grinded into dust Figure 1.

Cold extraction
The extraction procedure was adopted from Alam et al. (2002). The total weight of powdered leaf material was 650 g. That amount was taken in an amber colored extraction bottle and soaked in 100% methanol (2.0L x 3 times). The bottle was kept first time for 7 days with occasional shaking and stirring and also submitted to ultrasonic agitation for an hour daily in a sonicator (Power Sonic 510). The organic phase or the supernatant was then filtered separately through cotton followed by Double Rings Filter paper No. 102 and collected in a beaker. Second time the bottle was kept for 5 days and third time for 3 days with occasional shaking and stirring and also ultrasonic agitation daily in a sonicator and the filtered mixture was collected in the same beaker. Then the extraction was kept open in room temperature, aeration by an aerator for evaporation of the solvent to afford crude extract (32 g).

Figure 5
Schematic representation of solvent-solvent partitioning of crude methanolic extract

Solvent-solvent partitioning of crude extracts
Solvent-solvent partitioning of extracts was done using the protocol designed by Kupchan and modified by Wagenen et al. (1993).

Extraction with petroleum ether
The crude methanolic extract was made slurry with water (100 ml) and taken in a separating funnel of 500 ml. Petroleum ether (100 ml) was added with the aqueous methanolic solution and shaken well. The mixture was kept untouched until the layers were separated. The upper organic layer was then collected and repeated the process for five times. The collected combined petroleum ether extract was filtered and allowed the solvent to evaporate off in room temperature. The dried extract afforded a bluish-black colored oily mass (10.5 g).

Extraction with chloroform
The aqueous fraction was then added with chloroform (100 ml) and shaken well. When the layers were separated, the lower organic layer was collected in a beaker. The process was repeated twice. The combined chloroform extract was filtered and evaporated off in normal room temperature. The dried, concentrated extract obtained a coffee-colored mass (5.2 g).

Figure 6
Filtering of the extractions Figure 7 Solvent-solvent partitioning of extracts

Extraction with ethyl acetate
After extraction of chloroform fraction, the aqueous fraction was extracted with ethyl acetate (100 ml x 3 times) and shaken well. The combined ethyl acetate extract was collected, filtered and evaporated off the solvent. The dried extract obtained a coffee-colored mass (2.1 g).

Extraction with methanol
Finally, the left aqueous fraction was dissolved in methanol and the combined methanol extract was evaporated off, filtered and dried to obtain a blackish mass (14.2g).
The same procedure was followed to yield the extracts of stem of M. pudica. All the output extracts were removed to glass vials and preserved in refrigerator at 4 0 C with proper labeling.

Identification of bioactive compounds by GC-MS
The GC-MS analysis of the plant extract was made in a Shimadzu QP 2020 (Japan) instrument. About 1μL of the methanol extract was injected into the GC-MS using a micro syringe and the scanning was done for 55 minutes.
GC-MS analysis is a common confirmation test. It is best used to make an effective chemical analysis. This analysis provides a representative spectral output of all the compounds that get separated from the sample. The injection of the sample to the injected port of the GC device is the first step of GC-MS method. The GC-MS instrument vaporizes the sample and then separates and analyzes of the various components. Each component ideally produces a specific spectral peak that may be recorded on a paper chart electronically. The time elapsed between elution and injection is called retention time. Differentiation among the compounds is identified using the retention time. The peak is measured from the base to the tip of the peak.
Retention indices (RI) of the compounds are determined by comparing the retention time of a series, and identification of each component is confirmed by comparison of its RI with data in the literature. Interpretation of mass spectrum is carried out by using the database of National Institute of Standards and Technology (NIST) having more than 62,000 patterns. The spectrum of unknown components is compared with the spectrum of known components which is stored in the NIST library.

Gas Chromatography-Mass Spectrometry (GC-MS) analysis of the samples
GC-MS analysis was carried out for crude extracts of leaf and stem of the sample plant M. pudica.

Chloroform extract of stem
GC-MS analysis of chloroform extract of stem showed twenty six compounds. Among them tigloidine (80.902%) was the major compound (Table 4) (Fig 11).