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Khuseynov Arslonnazar, candidate of chemical sciences, Senior researcher Tashkent Chemical Technological Institute Rakhmonberdiev Gappar, doctor of chemical sciences, professor, Tashkent Chemical Technological Institute
Malikova Mavjuda, candidate of chemical sciences, Ibragimova Kamola, Senior researcher, Tashkent Chemical Technological Institute E-mail: [email protected]
INFLUENCE OF OBTAINING CONDITIONS OF ACETATEINULIN ON THE STRUCTURE AND ITS MICROBIOLOGICAL ACTIVITY
Abstract: Results of obtaining inulin acetates have been presented in two ways. The quantity of acetyls groups has been defined. The structure inulin acetate was investigated by IR-spectroscopy, X-ray crystal analysis was conducted. The sensitivity characteristic of microbes to acetates of inulin was studied.
Keywords: topinambour tubers, inulin, acetateinulin, IR-spectroscopy, diffraction pattern, sensitivity to microbes.
Inulin - plant polysaccharide-polyfructosan -consists of D-fruktofuranose residue bound ^-1,2-glucoside ties, the end residue is presented by sucrose. The key source of inulin is topinambour-Helianthus tuberosus. Topinambour tubers, thanks to the unique biochemical composition, manifest powerful medical and preventive effect.
Topinambour serves as raw materials for obtaining the products widely applied in medicine and the food-processing industry which are based on inulin. As it is known, inulin has rather wide spectrum of biological activity and its modification can lead to obtaining of the compounds possessing new properties which are not characteristic for most inulin. The hydroxyl groups of this polysacharide are sufficiently reactive and define its basic chemical properties.
From the above-stated follows that inulin ethers can be used as biologically active compounds.
Experimental part. The work is devoted to the obtaining of acetateinulin (AI) in various conditions.
1. 1g of inulin was dissolved in 10 ml formamide, then 5 ml of pyridine and 10 ml of acetic anhydride were added. Heated up at 80-90 °C within 2.5 hours. During the reaction colour of a mixture changed from yellow to dark brown colour. Then the mix was cooled and added in the capacity with 200 ml of distilled water. It was left for 24 o'clock for precipitate formation, then the precipitate was washed with water until all reagent residues were completely removed, exsiccated at room temperature. Output of 1.15 - AI-1. The high yield of AI-1 is due to the fact that full acetylation of inulin occurs, as shown by IR-spectroscopic data - the absence of the absorption band of the OH groups (3200-3600 cm-1).
2. 1g of inulin was acetylated as described above, but at room temperature for 24 hours, precipitated
with water, the precipitate was washed with water until complete removal of the reagents, dried at room temperature. Output of 0.44 g.- AI-2. In this case, there is a partial acetylation of inulin.
Quantitative determination of acetyl groups. 0.1 g of acetateinulin was suspended in 10 ml of water, then added 10 ml of 0.5H NaOH, left for saponification for 20-22 hours. Then titrated with 0.5H HCl, indicator: phenolphthalein. At the same time a control experiment was conducted [1]. Consumption of 0.5N HCI was: - control experiment - 10 ml(v1)
Table 1.- Physical-chemical
- worker - 1-7.1 ml (v2)
- worker - 2-6.5 ml (v2) ( -y2 )' F ■ 0.0295 -100
%OAc = -
g
V - the volume of a 0.5 n solution of HCI consumed for the titration of a control sample, ml.
V2 - the volume of a 0.5n solution of HCI consumed for the titration of a working sample, ml. F - correction factor 0.5n solution of HCl. 0.0295 - the number of acetate groups corresponding to 1 ml of exactly 0.5n solution of HCl, g g - a portion of inulin acetate, g
characteristic of acetateinulins
Experimental Conditions Yield,% Quantity of acetyl groups,% IR, sm-1
1 g of inulin, 10 ml of formamide, 5 ml of pyridine, 10 ml of acetic anhydride 24 hours at 20 °C 74 39 3600-3200, 1750, 1427, 1375, 1332, 1240, 1134, 915, 874, 817.
1 g of inulin, 10 ml of formamide, 10 ml of acetic anhydride, 5 ml of pyridine, 2.5 hours at 80-90 °C 115 59-60 1750, 1427, 1375, 1332, 1240, 1134, 915, 874, 817.
It should be noted that in the first case acetateinulin is an amorphous powder readily soluble in acetone. According to the analysis, the quantity of acetyl groups is 85.5%.
Acetateinulin, obtained at room temperature with a yield of 44%, is a white crystalline powder,
also well-soluble in acetone. The number of acetyl groups is 59%.
The synthesized inulin esters were characterized using IR-spectroscopy (Figure 1).
4000 3000 2000 1500 1000 400
v, cm1
Figure 1. Infra-red spectra of inulin (1), AI-1 (2) and AI-2 (3)
The IR-spectra of the samples were taken on a Fourier transform infrared spectrometer System 2000, Perkin-Elmer (USA production), in tablets with KBr infrared range of4000-400 cm-1.
The figure shows the IR spectra of inulin, AI-1 and AI-2. As it can be seen from the figure, in the IR spectrum of inulin there is a wide absorption band of the hydroxyl group in the region of3600-3200 cm-1 and other absorption bands in the regions of 1427, 1332, 1250, 1134, 915, 874, 817 cm-1, characteristic of fructans type of inulin [2; 3].
As a result of esterification with acetic anhydride, the band of the hydroxyl group in the region of 3600-3200 cm-1 AI-1 practically disappears, which indicates the acetylation reaction, while new intense absorption bands appear in the regions of 1750 cm-1 (C = O), 1375 cm-1 (C-CH3) and 1240 cm - 1(C-O) related to bond vibrations in acetate groups.
In the IR spectrum ofAI-1, there is no absorption band of the hydroxyl group, since acetylation is complete; in the case ofAI-2, acetylation occurs partially.
Absorption bands are present in AI-1 and AI-2, which characterize the presence of acetyl groups: 1748, 1241 cm-1.
X-ray phase analysis was performed using a DRON-3M diffractometer (Russia production) [4]. CuKa radiation extracted by nickel filter with a wavelength A = 1.542 A was used. The operating voltage was 22 kV, the anode current strength was 12 mA.
As it can be seen from the experimental data, samples of inulin obtained from tapinambour have a crystal structure, which is visible from X-ray diffraction patterns which is in accordance with the literature data. X-ray phase analysis showed that the samples of inulin AI-1 and AI-2 are observed, the crystalline peak at 20 = 22.5 while at AI-1 the crystal reflexes have a wider shape(Fig. 2). This is due to the fact that in powder samples the distribution of crystallites in various modifications leads to line broadening.
Figure 2. Diffractogram of inulin samples and inulin acetates obtained under different conditions AI-1, AI-2, 3-inulin
The received X-ray diffraction patterns show that and are characterized by different packing of the el-the samples of acetateinulins differ in their crystal ementary units in the crystal cell. structure depending on the preparation conditions
As it can be seen from X-ray phase analysis, inu-lin is observed crystal reflections at 20 = 22.1 °, 29.5 °, 31.9 °, 43 ° and 48.5 °, associated with X-ray diffractions from different planes, which have an interpla-nar distance d = 3.995 A, 3.028 A, 2.818 A, 2.103 A and 1.877 A.
In the samples of inulin acetates, a more amorphous structure and an amorphous halo are observed with a broadening X-ray diffraction angle over the whole region, which have a maximum at 20=22.1 °, associated with changes in the interplanar spacing of inulin crystals.
Currently, the creation of medicinal agents with antibacterial action is a very promising direction, since it allows not only to prolong the effect of the antibacterial drug due to its long-term deposition, but also to change the effect on cellular inflammatory factors.
According to the latest literature data [5; 6; 7], the sensitivity of microorganisms to chemical preparations is determined in two ways:
Table 2. - Characteristics of the sensitivity of
1. Disk-diffusion method is a method of diffusion in agar using paper disks saturated with chemical preparations.
2. The method of serial dilutions of chemicals in dense or liquid nutrient media with the introduction of microbes into them.
Among these methods for determining the sensitivity of microbes to chemicals, the disco-diffusion method is the most common. The frequency of using this method can be explained by its advantages such as technological accessibility of testing, low cost, flexibility - that is, the ability to determine the sensitivity to the drugs that are required in this clinical situation, high reproducibility of the results while observing the conditions of testing and preparation of consumables.
Considering the above, we carried out studies on the sensitivity of some microorganisms that are most commonly found in various biotopes of the human body to acetateinulin according to the first method (Table 2.).
microbes to the acetateinulin preparation(AI-l)
№ Groups of microbes Acetateinulin /M + m/
10 mg 50 mg 100 mg
1 Staphylococcus aureus 13.0 ± 0.2 17.0 ± 0.3 20.0 ± 0.4
2 Staphylococcus epidermidis 10.0 ± 0.1 12.0 ± 0.2 15.0 ± 0.3
3 Staphylococcus saprofiticus 10.0 ± 0.1 12.0 ± 0.2 15.0 ± 0.3
4 Streptococcus pyogenes 20.0 ± 0.4 17.0 ± 0.3 20.0 ± 0.4
5 Escherichia coli 17.0 ± 0.3 12.0 ± 0.2 10.0 ± 0.2
6 Proteus vulgaris 10.0 ± 0.1 12.0 ± 0.2 12.0 ± 0.2
7 Klebsiella 17.0 ± 0.3 25.0 ± 0.5 25.0 ± 0.5
8 Pseudomonas aeruginosa 20.0 ± 0.4 10.0 ± 0.1 10.0 ± 0.2
9 Candida albicans 0 0 0
10 Actinomycetae 12.0 ± 0.2 20.0 ± 0.4 22.0 ± 0.4
Note: units are given in mm of microbes growth inhibition zones
From (table 2) it can be seen that acetateinulin in low concentrations of 10 mg, had an antibacterial effect on the strains of such microbes as: streptococci, Escherichia, Klebsiella and Pseudomonas, all other microbial strains taken in the experiment were poorly-sensitive.
The same drug, only in medium concentrations of 50 mg, had an antibacterial effect on staphylococci, streptococci, Klebsiella and actinomycetes. At the same time, all other microbes were poorly sensitive.
AI-1 at an enhanced concentration of 100 mg, had a significant antibacterial effect on most mi-
crobes and only gram-negative flora proteus and pseudo-manas turned out to be weakly sensitive.
It is interesting to note that the culture of Candida albicans showed insensitivity to acetateinulin in all tested concentrations. Apparently, this is due to the fact that the fungi of the Candida genus in their structure belong to eukaryotes and their cell wall does not contain pectidoglycane.
Thus, based on the conducted microbiological studies, it can be concluded that the acetateinulin preparation, especially in high concentrations of 50 mg, can be used for the treatment of purulent-inflammatory diseases of coccine aetiology, as well
as for diseases in which Klebsiella and actinomy-cetes prevail.
Conclusion:
Acetateinulins were obtained and their physico-chemical properties were investigated. It is shown that the temperature regime influences on the yield of acetateinulin and the amount of bound acetyl groups. The resulting product was investigated by IR-spectroscopy and the presence of absorption bands of 1241, 1748 cm-1 indicates the presence of acetyl groups. And also shows the sensitivity of some microbes to acetateinulin.
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