The expanding importance of mucilage polysaccharides is guiding endeavors to the improvement of fast, proficient and effectively scaleable methods for their purification. An effort has made to distinguish between mucilages and gums on the basis that gums readily dissolve in water, whereas, mucilages form slimy masses. Other investigators have tried to distinguish between them on the basis that mucilages are physiological products and gums are pathological products¹. Another evidence is that mucilages contain protein and pectin². The present research has focussed on the extraction of Basella alba mucilage polysaccharide by using acetone as non-solvent and deproteinization using trichloro acetic acid in comparison with hydrochloric acid for the precipitation of protein by pH adjustment.

Proteins in polysaccharide solutions are acidic and get denatured or can be removed easily when pH changes³.

MATERIALS AND METHODS:

The collection and authentication of plant material, method of extraction, phytochemical characterization and pharmaceutical application of mucilage polysaccharide were already described in our earlier studies⁴. Nucleic acids and proteins absorb in the UV region at wavelength with absorbance maxima at 260 and 280 nm respectively⁵. An UV absorbance ratio at A/260/280 is used to characterize if the polysaccharide contained proteins and nucleic acids. The protein content was assayed by Lowry method using Bovine serum albumin as standard⁶. Polysaccharide was estimated by phenol-sulfuric acid method using glucose as standard⁷.

Extraction and Deproteinization of polysaccharide:

Basella alba leaves (50g) were steeped and extracted in chloroform⁸ (350ml) at 60ºC for 24 hours in a round bottomed flask. The pretreated material was filtered and the leaf residue was air dried. The inactivated raw material was soaked and extracted in distilled water (300 ml) by electrical stirring for 24 hours for the maximum release of mucilage polysaccharide from the residual protein bonds. The material was filtered through a muslin bag to remove the marc from the filtrate. The filtrate was concentrated at 50ºC and filtered. At that point twofold the volume of acetone (4 times the volume of aqueous filtrate) was added to the filtrate for the complete precipitation of crude polysaccharide. Then the aqueous polysaccharide solution was treated with 10% (w/v) trichloroacetic acid⁹ and hydrochloric acid for adjustment of other pH values (3, 4 and 5) separately to remove any adherent protein contaminants and deproteinized.

Polysaccharide quantification and loss ratio:

A 1ml each of polysaccharide solution and 5% phenol were mixed with 5ml of concentrated sulfuric acid in a test tube. Following 10 minutes of standing, the sample was placed in water bath for 15 minutes and afterward absorbance was perused at 520nm. Standard solutions were prepared in same way, except that the 1ml of polysaccharide solution is replaced by distilled water. The total polysaccharide concentration was calculated according to the standard glucose calibration curve. The % loss ratio of polysaccharide was determined¹¹ by Mo-Me/Mo x 100 where, Mo and Me were the polysaccharide proportion before and after partial purification by trichloroacetic acid and hydrochloric acid respectively. The result is given in table 1.

Determination of protein concentration and deproteinization efficiency:

Standard protein solution was pipetted out in the first six test tubes in the volume 0, 0.2, 0.4, 0.6, 0.8 and 1ml respectively and sample solution was taken in the test tubes numbered seven to nine. To these test tubes 0.1 N NaOH was pipetted out in the volume of 0, 0.2, 0.4, 0.6, 0.8 and 1ml respectively. 5ml of freshly prepared copper reagent was pipetted into all the nine test tubes and shaken well. The test tubes are kept in room temperature for 15 minutes. After 15 minutes, 1ml of Folin-Ciocalteau reagent was added and shaken well. The test tubes are kept in room temperature for 30 minutes. The optical density values of all the test tubes were obtained at 660nm. The concentration of protein was calculated according to the standard calibration curve. The percentage of deproteinization efficiency¹¹ was calculated by Co-Ce/Co x 100 where, Co and Ce were the protein concentration before and after deproteinization by trichloroacetic acid and hydrochloric acid respectively.

RESULTS AND DISCUSSION:

The standard procedure was used to extract polysaccharide using water as solvent and acetone as non-solvent. Boiling in the extraction results in denaturation of some proteins and part of protein was removed during filtration. The polysaccharide is off white to brown, odourless and amorphous powder before and after deproteinization. The total yield by acetone precipitation is found to be 12.5% w/w. When dissolved in cold water, it gives a almost neutral pH 6.5, colloidal and viscous solution. It is insoluble in ethyl alcohol, acetone, ethyl acetate and toluene. Nucleic acids and proteins detected as indicated by absorption at 260-280 nm in UV spectrum. The result obtained is presented in Figure 1 which shows the UV spectra of Basella alba polysaccharide.

Figure 1. UV Spectra of polysaccharide solution before deproteinization

In general, a ratio of less than 1.6 indicates less concentration of proteins and nucleic acids in the sample, while ratios above 1.8 are considered to have high concentration. In our study, 280/260 ratio for protein was calculated as 0.68 and A 260/280 ratio for nucleic acids was 1.45. Nucleic acids show absorbance due to presence of purine and pyrimidine bases containing conjugated double bonds and proteins show due to presence of aromatic amino acids. Our values indicates very low concentration of nucleic acids and proteins in the aqueous extract. UV Spectrophotometric measurement of proteins is most common in estimation and also to assess purity. Before deproteinization, the total protein content of the aqueous polysaccharide solution used in the precipitation assay was 120µg/ml using BSA as standard. The total polysaccharide content was found to be 72.05% w/w. Deproteinization is an important step in purification and characterization of polysaccharide from the natural plants.

Two methods of deproteinization were followed for differential precipitation of proteins by pH adjustment. In first method, the initial pH 6.5 was decreased by the addition of HCl to pH 5 and after 2 hours the precipitate was collected by filtration through a membrane of 0.45 µm. The same procedure was followed for the aqueous extract to other pH values (4 and 3). After deproteinization, no much nucleic acids and proteins detected in all the aqueous solutions of different pH as indicated by less absorption at 260-280nm by the effect of hydrochloric acid. Figure 2. shows the UV spectra of Basella alba polysaccharide after deproteinization by HCL at pH 3.

Figure 2. UV Spectra of polysaccharide solution after deproteinization by Hcl at pH 3

The method enabled to reduce the proteins to an average of 40µg/ml of the total protein content. The deproteinization efficiency was 65%. The average total polysaccharide content was found to be 62% w/w. The % polysaccharide loss ratio was found to be 13.94% w/w.

In second method, pH 6.5 was decreased by the addition of 10% w/v trichloroacetic acid to other pH values and after 24 hours the deproteinized polysaccharide was obtained. The same procedure was followed for the aqueous extract with another pH value. Figure 3. shows the UV spectra of Basella alba polysaccharide after deproteinization by TCA at pH 3.

Figure 3. UV Spectra of polysaccharide solution after deproteinization by TCA at pH 3

At 10 % w/v concentration, trichloroacetic acid proved to be efficient in precipitation of protein reducing to an average of 19µg/ml. The result obtained were presented in figures 4-7. The deproteinization efficiency was 83.3%. The total polysaccharide content in an average was found to be 57% w/w. The % polysaccharide loss ratio was found to be 19.5% w/w. The result is given in table 1.

Figure 4. Protein after treatment with Hcl at different pH

Figure 5. Protein after treatment with TCA at different pH

Figure 6. Polysaccharide after treatment with Hcl at different pH

Figure 7. Polysaccharide after treatment with TCA at different pH

Table 1. Comparison of the methods for deproteinization

S. No.	Method	Deproteinization efficiency (%)	Polysaccharide loss (%)
1	HCL	65	13.94
2	TCA	83.3	19.5

The principle involved in trichloroacetic acid method is the binding of protein with trichloroacetic to form an salt which is insoluble and can be precipitated at pH < Pi. In both the methods, the deproteinized polysaccharide obtained by acetone precipitation at different pH adjustments showed slight variation in polysaccharide content. Also further optimization method is required using trichloroacetic acid to deproteinize the polysaccharides to a greater extent. The HCL method excelled over the TCA method in obtaining polysaccharides with little lower percentage of polysaccharide loss.

The solubility of protein also depends on the dielectric constant of the solvent. Acetone is used for extraction of polysaccharides because it not only precipitates polysaccharides but also discourage the dispersion of protein molecules in the aqueous solution because of its low dielectric constant value.

CONCLUSION:

A solitary precipitation may not be adequate to expel different types and concentrations of interfering contaminants in such cases, several precipitations must be performed The Sevage methodis not utilized for the deproteinization, since it contains harmful chloroform which is naturally disadvantageous¹³. The TCA method will offer a room for removing protein from plant polysaccharides in large scale if optimization is studied.

ACKNOWLEDGEMENTS:

The authors would like to thank JSS College of Pharmacy, JSS Academy of Higher Education and Research (JSS AHER), Ooty, Nilgiris, Tamilnadu, India for research grant (REG/DIR(R)/ URG/54/ 2011-12) under the JSS AHER Research Grants / Financial assistance to Faculty sanctioned to Mr. G Ramu, Faculty, Department of Pharmacognosy and also to Department of Pharmacognosy, JSS College of Pharmacy, Ooty, Nilgiris, Tamilnadu, India for providing the necessary research facilities.

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Received on 13.01.2020 Modified on 19.05.2020

Research J. Pharm. and Tech. 2021; 14(6):3093-3096.

DOI: 10.52711/0974-360X.2021.00540