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УДК: 615

УДК: 577.114.4:582.736

M.А. Duchenko

Vinnytsia National Medical University named after M.I. Pyrohov
Research of polysaccharides of Gleditsia trіacanthos leaves

The results of the study of polysaccharides of prickly leaves of honey locust are presented. The quantitative content of water-soluble polysaccharides, pectin, hemicellulose A and B was determined gravimetrically. By the method of paper chromatography monosaccharides composition of polysaccharides was established. In the extracted polysaccharide fractions the quantitative content of regenerative and acidic sugars was found.

Keywords: Gleditsia trіacanthos, polysaccharides, pectin, hemicellulose, renewable and acidic sugars.

Introduction. In recent years the number of research related to the study of the structure of polysaccharides and study their pharmacological action has significantly increased. Previously polysaccharides were used mainly as excipients in the manufacture of various dosage forms, in recent years, they are mainly considered to be biologically active compounds with a broad spectrum of pharmacological action which is not limited to mitigating and enveloping properties. In particular, it was found that some water-soluble polysaccharides exhibit antiexudative, anti-inflammatory, anti-proliferative activities [2].

������������� All polysaccharides are adsorbents; pectins are the most active of them. Antidiabetic effect is directly correlated with the use of plants in pancreatitis; inulin manifests itself best of all here. As for adaptogenic activity, it was studied in mucus and inulin, but it can be found in pectins as well. Polysaccharides are characterized by two neglected effects: positive impact on the course of pathological processes in the kidney, the second is antifunhal especially manifested in combination with other BAS. [3].

There is no data concerning the carbohydrate composition of prickly leaves of honey locust. That’s why the article is the continuation of research of bioactive substances of prickly leaves of honey locust (Gleditsia triacanthos L.), legume family (Fabaceae). This wood comes from North America, which was brought to us in the last century. In Ukraine, the honey locust is widely cultivated as an ornamental plant. [4,7].
The aim of our work was the extraction and study of polysaccharides of Gleditsia triacanthos L.

Materials and methods. The objects of the study were prickly leaves of honey locust harvested in the botanical garden of Kharkov National University named after V.N. Karazin in summer 2010. The extraction of polysaccharide fractions out of the material was carried out in accordance with N.K. Kochetkov’s methodology [5]. To determine the content of polysaccharide complexes gravimetric method was used [1].

In order to receive water-soluble polysaccharide complex (WSPC) air-dry raw material was crushed into particles of 1 mm, purified by lipophilic impurities by exhaustive extraction with chloroform in a Soxhlet apparatus . Accurate weight of the low fat raw extracted three times with hot water in a boiling water bath, under reflux, with a ratio of the raw material and extractant 1:20, 1:10, 1:10 within 1 , 0.5 , 0.5 hours respectively , stirring occasionally. Extracts were mixed, filtered through the filter paper on the Buchner funnel under vacuum, evaporated to 1/5 of the original volume. PS was precipitated by fivefold (relative to the extract) in volume of 96% ethanol. The precipitate was filtered under vacuum, washed repeatedly by 96 % ethanol, demoisted by acetone, dried in air and weighed.

The mixture after receiving WSPC was used to produce pectin (PR). This mixture was extracted three times with the mixture of 0.5 % solution of oxalic acid and ammonium oxalate (1:1) at the ratio of the raw material and extractant 1:20 for 2 hours. Combined, filtered extracts were evaporated to 1/5 of the original volume and precipitated by fivefold volume of 96% ethanol. The precipitate was filtered, washed with 96% ethanol, dried in air and weighed.The remaining mixture after getting PR, was extracted with the solution of 10 % sodium hydroxide at a ratio of 1:5 of the raw material and extractant for 12 hours at room temperature. By adding glacial acetic acid precipitate hemicellulose A (GC A) was formed, which was filtered, dried and weighed. Twice the volume of 96% ethanol was added to the filtrate – precipitate of hemicellulose B (GC B) was formed, which was washed with alcohol, dried and weighed.

Determination of monosaccharyd composition of WSPC, PR, GC A and GC B was performed after hydrolysis of 10% sulfuric acid at a ratio of 1:50 for 2 hours [5 ]. Hydrolyzate was neutralized with barium carbonate by universal indicator to a neutral reaction. The solution was filtered by washing the precipitate with water to the volume of 20 ml of the filtrate. Five times the volume of 96% ethanol was added to the filtrate and it was left for 24 hours.

The precipitate which was formed was filtered off and the filtrate was evaporated to dryness. The dry residue (neutral monosaccharides) was dissolved in ethanol and chromatographed by descending method on paper Filtrak FN № 4 in a solvent system n-butanol - pyridine - water (6:4:3 ) parallel with credible examples of monosaccharides. The precipitate barium salts of uronic acids were dissolved in water, neutralized with cation exchanger KU- 2 (H +), the filtrate was evaporated. The residue ( acidic monosaccharides ) was dissolved in ethanol and chromatographed in the system - acetic ethyl ether - acetic acid - formic acid - water ( 18:3:1:4 ) as compared to samples of credible uronic acids. Air-dried chromatograms were treated with acidic aniline phthalate and heated in the oven at the temperature of 100-105 ° C; aldoheksozes were shown as brown spots, aldopentozes - as red-brown.

The qualitative content of the amount of renewable sugars in terms of glucose was determined by reaction with picric acid and acid content of sugars in terms of glucuronic acid was determoned by reaction with carbazole spectrophotometric method [6 ].

Results and discussion. The resulting water-soluble polysaccharide complex is amorphous powder of brown color, soluble in water to form light rejecting solutions (pH of 1% water solution is in the range 5.0 - 6.0), insoluble in organic solvents. The resulting WSPC gives positive reaction with ninhydrin solution (free amino acids), biuret reagent (protein), solution of ferriammonium sulphate (tannins ).

Pectin substances, extracted from raw materials represent amorphous powder, light gray, well soluble in water which form viscous solutions (pH of 1% water solution varies between 4.0-5.0 ). Hemicellulose A and B are amorphous powders of yellowish- brown color.

By paper chromatography in comparison with authentic samples of sugars in the hydrolyzate investigated polysaccharide complex glucose, arabinose, rhamnose, galactose, glucuronic and galacturonic acid were identified. By the magnitude and intensity of color spots on the chromatograph it was determined that the constituent monomer units of WSPC of Gleditsia trіacanthos L. is mostly glucose, arabinose, rhamnose, galactose, in composition of PR - glucuronic and galacturonic acids.

By the gravimetric method quantitative content of received was defined, it included water soluble polysaccharides - 4.11 % pectin - 8.22 % hemicellulose A - 3.70 % and hemicellulose B - 7.95% .

It is well known that acidic sugars exhibit cancerstatic and antiviral effect. Taking it into consideration, we have identified the content of renewable and acidic sugars in derived polysaccharide complexes. The results are presented in Table 1. As the table shows, a rather high content of acidic sugars, especially in leaves - 20.83 % the isolated polysaccharide fractions are observed. This gives the reason to anticipate a high pharmacological activity of Gleditsia trіacanthos L.

Table

Content of renewable and acidic sugars in the polysaccharide complex of leaves

of Gleditsia triacanthos L.

Name of fraction

Content of sugar in fractions, %

WSPC

Restoring

55,25

Acidic

20,83

PR

Restoring

27,97

Acidic

10,02

Conclusions.
1. For the first time polysaccharide fractions of Gleditsia trіacanthos leaves such as soluble polysaccharides, pectin, hemicellulose A and B were identified and investigated.
2. Quantitative content of water-soluble polysaccharides, pectin, hemicellulose A and B were identified in the studied raw material by the gravimetric method, it was 4.11 %, 8.22%, 3.70%, 7.95% respectively.
3. By the method of paper chromatography carbohydrate monomer composition of extracated fractions was found. The structure of the polysaccharides include: glucose, arabinose, rhamnose, galactose, glucuronic and galacturonic acid.
4. In the extracted polysaccharide fractions quantitative content of renewable sugars calculated as glucose and acidic sugars calculated as glucuronic acid were found. Sufficiently high content of acidic sugars was noticed - 20.83 %.

Reference
1. Ukrainian State Pharmacopoeia /State. n -in "Scientific – Expert Pharmacopoeial Centre." - 1st ed . - H.: RIREH , 2001. - Appendix 2. - 2008. - 620 p.
2. Rybak L.M., Konovalova O.U., Kovalchuk T.V. Investigation of quantitative content of polysaccharide fractions of different species of  Geranium L / / Topical Issues ofpharmaceutical and medical science and practice. - 2011. - Issue XXIV, № 2. - P.110-112.
3. Pharmacognosy in medicine: Studying guide / AJ Kobzar - K. : Medicine , 2007. -544p.
4. Hrysyuk N.M. Bean Plants in protectional foresting / N.M. Grysyuk , O. Tsarenko . - K.: Vintage, 1991. - 168 p.
5. Stepanenko B.N. Chemistry and biochemistry of carbohydrates: Polysaccharides. - M., 1978. - 256 p.
6. Minin S.A. Chemistry & Technologies of phytopreparations / S. Minin, E. I. Kauhova . - Moscow: Heotar - Honey, 2004 - 516 p.

7. Guryev A.M., Belousov M.V., Yusubov M.S. and others. Study of acute toxicity of complex WSPC of ayra marsh rhizomes / / Bull. Siberian medicine. , 2010. Appendix, № 1. P. 36 - 40.

8. Baertels A. Gleditsia: natural distribution, characteristics / A. Baertels / / Gartenpraxis. - 1995. - Vol. 21 (6). - P. 22-25.

9. Furusawa E., Hirasumi A., Story S. et al. // Phytother. Res. 2003. V. 17, № 10. P. 1158.



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