Biological science

Martynov A.M.^*, Dargaeva Т.D.^**, Rozhkova N.Y.^*

^*Irkutsk State Medical Academy for Medical Advanced Studies, the Russian Federation, ^**All-Russia Research Institute of Medicinal and Aromatic Plants (ARRIMAP), Moscow, the Russian Federation

QUANTITATIVE ESTIMATION OF POLYSACCHARIDES IN GRASS VIOLA LANGSDORFFII (VIOLACEAE) BY SPECTROPHOTOMETRY

Plants represent the richest source of biologically active substances with a wide spectrum of pharmacological effect. Within numerous classes of natural compounds polysaccharides make up a large group of high-molecular complexes produced by monosaccharide and their derivatives linked by O–glycoside bonds (homoglycosides), having linear and branchy chains. Polysaccharides of many plant species show a wide spectrum of pharmacological effect, so they are employed as enveloping, anti-inflammatory, emollient and other means. In some violet species the water-soluble polysaccharides implement antineoplastic activity [3, 4]. In the earlier research we identified the monosaccharide structure of carbohydrate complexes of V. langsdorffii [2].

The goal of this investigation was put forward to develop the technique to measure polysaccharide abundance in grass Viola langsdorffii Fischer ex Ging. by spectrophotometry method.

The assigned study object was overground part of V. langsdorffii collected in Sacchalin region in time of its blooming.

Precipitation of polysaccharides from water extracts by 95% ethyl alcohol is a common feature for this group of natural compounds, used both for qualitative and quantitative evaluation of polysaccharide abundance in vegetative raw materials.

The essence of gravimetric method of quantitative measuring polysaccharides in the medicinal vegetative raw materials, described in GF Х1, is their ability to be precipitated by 95 % ethyl alcohol [1]. The method is convenient and simple, however it has some limitations: low accuracy, low specificity and multi-staged analysis.

Therefore, we made up our mind to develop a more exact and reliable method with spectrophotometry involved. This method assumes extraction of polysaccharide complex from raw materials, hydrolysis of the carbohydrates obtained and formation of stained complex of hydrolysis products (monosaccharide) with picrinic acid in the alkaline medium and subsequent measurement of its optical density [5].

While working on the technique the following parameters have been selected: the degree of raw material grinding, duration of extraction, polysaccharide hydrolysis and formation of stained complex. It was eventually found, that it is rational to apply the raw materials crushed to the size of particles passing through a sieve with apertures 1 mm across, with extraction proceeding for 1 hour, raw material/extragent ratio 1:50, hydrolysis lasting 2 hours and reaction time 30 minutes.

The analytical technique is briefly described as follows. The raw material sample was crushed to the size of the particles passing through a sieve with 1 mm aperture. The sample weighing 1 g of crushed raw material was placed into a conic flask 100 ml and then 50 ml of water was added. A flask was connected with a reverse refrigerator and heated on a boiling water bath within 1 hour. After time was over the blend was cooled and filtered through the folded paper filter (a white tape) into a measuring flask 50 ml and the same solvent twas added to the appropriate mark (investigated solution).

Twenty milliliters of solution was poured into a conic flask 100 ml volume and 20 ml of 8 % muriatic acid was added. The flask was connected to the reverse refrigerator and heated on a boiling water bath for 2 hours. Upon termination of hydrolysis the solution was cooled to room temperature then neutralized potentiometrically to pH 6, 5 – 7, 0 with 30 % sodium hydroxide and 10 % solution of hydrochloric acid.

The neutralized hydrolysate was transferred into 50 ml measuring flask, water was added to reach the mark and mixed. The received solution was filtered through folded paper filter (white tape), throwing away upper 15-20 ml of filtrate.

One milliliter of 1 % picrinic acid and 3 ml of 20 % of sodium carbonate solutions were poured into two measuring flasks of 25 ml capacity. Then in one flask we added 1 ml of filtrate (test solution), and 1 ml of water (comparison solution) into another one. Flask contents were stirred and placed on a water bath for 30 minutes then cooled to room temperature and the volume of solutions was increased with water to the mark and mixed. In case of opalescence the test solution was filtered through the folded paper filter (white tape).

The optical density was measured by self-recording spectrophotometer “Lambda 35 UV/VIS” Perkin Elmer instruments (the USA) with wavelength ranging from 400 to 500 nm in a dish with absorbing layer thickness 1 cm.

We simultaneously measured the optical density of glucose with picrinic acid in the alkaline medium.

The study of spectrum of absorbing the complex of monosaccharide V. uniflora has shown that the stained complex has absorption maximum with wavelength 460±2 nm; the similar maximum is available in the complex of glucose with picrinic acid. Thus, it was proposed to use the wavelength 460 nm as the analytical length and to define the sum of monosaccharide after polysaccharide hydrolysis when recalculated for glucose.

The contents of the sum of monosaccharide after polysaccharides hydrolysis in percentage (X) if recalculated for glucose and absolutely dry raw materials were evaluated with the formula:

Where D – optical density of test solution; Dо – optical density of working reference sample (WRS) of glucose; mо – weight of glucose WRS; m – weight of raw material; 100 – W - loss of mass after raw material drying.

Preparation of the reference sample of glucose solution. 0, 1500 g (exact weight) of glucose was placed into measuring flask of 250 ml volume and increased solution volume to the mark and mixed. 1 ml of solution contains 0, 0006 g of glucose.

1 ml of 1% solution of picrinic acid and 3 ml of 20 % of sodium carbonate solution were poured into a measuring flask; after that we added 1 ml of glucose WRS, mixed and heated on a water bath for 30 minutes.

Metrological characteristics are presented in Table 1 to testify satisfactory reproducibility of results.

Table 1

Metrological characteristics of quantitative definition of monosugar sum after hydrolysis of polysaccharides in V. langsdorffii grass

n	f	_ X	S^2	S	Р, %	t(f, Р)	∆Х	Е, %
6	5	5,88	0,0068	0,0823	95	2,57	0,086	1,47

Reliability of the described technique to define monosaccharide contents in raw materials was tested with glucose additives.

Table 2

Results of tests with glucose additives

The sums of monosugars were measured after hydrolysis of polysaccharides in raw materials, grams	Added glucose	Should be, grams	Found, g	Error
				Absolute, g	Relative, %
0,0586	0,0018	0,0604	0,0612	+0,0008	+1,31
0,0586	0,0036	0,0622	0,0617	-0,0005	-0,81
0,0586	0,0048	0,0634	0,0641	+0,0007	+1,09

The relative error of tests with additives occurs within random error of the technique discussed, so it testifies to the absence of regular error.

Conclusions

The technique to quantitatively measure polysaccharides in overground V. langsdorffii by spectrophotometry method has been developed. The quantitative abundance of polysaccharides in the investigated object has been defined. It is proposed that available results can be used in implementing projects on standard documentation.

References

1. The state pharmacopoeia of the USSR: General methods of analysis / МЗ the USSR, 11 edition., supplemented. – М.: Medicine, 1987. V. 1. P. 336.

2. Martynov A.M., Chuparina E.V. Composition of polysaccharide complexes Viola langsdorffii. The Siberian Medical Journal. – 2010. – № 2. – P. 114-116.

3. Pyasytskene, A. Water-soluble polysaccharides of plants, their localization, biological and economic value. – Vilnius, 1994. – P. 74.

4. Vegetative resources of Russia and the adjacent states: Part 1.- Family Lycopodiaceae - Ephedraceae. - Part 2. – Additions to 1-7 volumes. – SPb: the World and a Family, 1996. – P. 157-158.

5. Samylina I.A., Rudakov I.P., Aladysheva Z.I., Kiselyov S.V. Sugar definition by spectrophotometry. Pharmacy. – 2009. – № 4. – P. 3-5.

E-mail: martinov_irk@mail.ru