Protein Enrichment From Eurycoma Longifolia Biology Essay

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INTRODUCTION

Eurycoma Longifolia, also locally known as Tongkat Ali, is a popular herbal remedy that is frequently prescribed as a single ingredient or as a mixture with other herbs. The decoction from the Tongkat Ali roots are used as folk medicine for the treatment of aches, persistent fever, malaria, erectile dysfunction (Perry, 1980), dysentery, glandular swelling (Darise et al., 1982; Darise et al., 1983; Lin et al., 2001) and as health supplements. In addition, the crude extracts of this plant were reputed to increase male virility and sexual prowess and gained notoriety as a male aphrodisiac (Ang and Sim, 1998; Ang and Sim, 1997; Ang et al., 2000)

Kuo et al.,2003 found that the protein composition of the water soluble fraction of the methanolic extract of the roots of Eurycoma longifolia comprising of 25 amino acids summing up to 53.39 % of the protein. Moreover, recent research in herbal supplements have revealed that the active ingredients for the effect of the herb may be in the carbohydrate (Leung et al., 2006) or protein fractions (Yu-Haey et al., 2003) rather than the phytochemicals such as in Aloe Vera (Leung et al., 2006) and Ginseng (Sambandan et al., 2004). Furthermore, in 2004, a patent application for the bioactive fraction of Tongkat Ali was filed with the United States Patent and Trademark Office (USPTO) for the peptide fraction with a size of 4300 Da in the water extract with the claim of treating male infertility (Asiah et al., 2007). This peptide fraction has been further isolated in 4 different Malaysian medicinal plants by FRIM using MALDI-TOF mass spectrometry (Bourseana et al., 2009). However, within the patent application, a 3 step chromatographic method for separation was applied which may not be effective on a large scale. Thus there is an economic and technical incentive to explore other more effective separation methods. Membrane separation methods such as ultrafiltration, nanofiltration and reverse osmosis has been used in food processing such as fish protein fractionation (Butylina et al., 2006), whey peptide fractionation (Atra et al., 2005; Yorguna et al., 2009; Cassano et al., 2007), fruit juice clarification and concentration (Laorko et al., 2010; Jain et al., 2010), amino acid and peptide separations (Martin-Orue et al., 1998; Moure et al., 2006), and soy antioxidant enhancement (Kumaresan, 2008). The key principle of membrane separation is the use of a driving force such as pressure through a membrane with specific selectivity by size or affinity to separate a mixture of compounds.

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Tongkat Ali water extract contains various phytochemicals, carbohydrates, glycoproteins and amino acids (Moure et al., 2006). Combining ultrafiltration, which filters compounds below 5000 Da, and nanofiltration, which filters compounds below 500 Da, will separate the desired protein fraction of 4300 Da while concentrating the second stage filtration retentate as done with the fish protein (Butylina et al., 2006) and whey protein (Atra et al., 2005). Similar work on phytochemicals has not been done extensively especially for Malaysian herbal exracts, thus there is a potential to apply a combined membrane separation method to isolate the 4300 Da protein fraction from Tongkat Ali.

OBJECTIVE

The general objective of this study is to produce a protein enriched fraction from Tongkat Ali water extract using two membrane separation methods. Specific objectives include:

1. The preparation of Tongkat Ali water extract fractions with maximum protein content.

2. To develop a protein separation method by combining two membrane separation methods (Ultrafiltration and Nanofiltration) to target the 4300 Da bioactive protein compounds from Tongkat Ali water extracts.

3. To quantify and qualify the separated proteins from the combined membrane system.

METHODOLOGY

Raw Material Preparation

Tongkat Ali extracts will be prepared from ground dry Tongkat Ali Roots from a single geographic source. In addition, extracts will also be prepared from spray dried or freeze dried Tongkat Ali water extracts acquired from the Chemical Engineering Pilot Plant in UTM or from Biotropics Malaysia Berhad. The protein profile from both dry roots and dried extracts will be compared. The optimal extract concentration will be determined for the separation stage.

Analysis Method development and application

The key analysis will be on the overall extract and protein fractions. The overall extract concentration will be measured with a UV-Vis Spectrophotometer to determine the concentration of the overall yield. The protein analysis will be done through Bradford Protein Assay for overall protein yield analysis, size-exclusion chromatography for protein size analysis, and MALDI-TOF Mass Spectrometry for isolated protein identification. Preliminary retentate and permeate samples from membrane separation will be tested on all these analytical methods to determine the protein characteristics as well as the best analytical method settings.

Separation Method setup, optimisation and characterisation

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The Tongkat Ali extract will be separated in two stages, starting with a ultrafiltration stage to separate the compounds below 5000 Da and followed by the nanofiltration stage to remove excess water and compounds below 100 Da. This stage of work will focus on getting the optimal separation parameters such as membrane type, pump pressure, flow rate, the breakthrough curve, flux etc. The protein yield and separation selectivity will be optimised by varying these parameters.

Analysis of Retentate and Permeate

Based on the separation experiments, the retentate and permeate of both ultrafiltration and nanofiltration will be analysed for protein yield and protein size distribution by the aid of Bradford Assay and SDS-PAGE methods respectively . The change in concentration with time will also be modelled.

GRANT’S CHART

Year

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Raw Material

Acquisition and

Literature

Search

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Analytical

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Separation

Method setup

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Separation

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Retentate and

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Experimental

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REQUIRED EQUIPMENTS

UV-Vis Spectrophotometer

UF, NF and RO Membranes

2 high pressure pumps (10 barg cassette)