The assessmenT of The qualiTy parameTers of inulin acTive pharmaceuTical ingredienTs according To physicochemical characTerisTics

Aim. To assess the quality parameters of plant active pharmaceutical ingredients (API) in accordance with the requirements of modern normative documents by such physicochemical characteristics as “Description”, “Solubility”, “Hygroscopy”, “Microscopy”, as well as identification of the structural components of fructan in order to involve the data obtained in development of the national normative documents on the substance of inulin. Results. The study of nine plant API of inulin obtained from chicory, agave and Jerusalem artichoke, as well as the reference standards of fructan from chicory and dahlia has been conducted by such physicochemical characteristics as “Description”, “Solubility”, “Hygroscopy”, “Microscopy”. According to the British Pharmacopoeia 2010 (BP) the chemical reactions of identification confirmed the presence of fructose and glucose after hydrolysis of the substance. According to the United States Pharmacopeia 36 – NF 31 (USP) it has been found by the identification reaction that the substances analyzed (in addition to the standard substances) contain impurities of sugars with the properties of reducing agents. By appearance the samples under study are loose, amorphous powders of practically white and pale yellow color, they are hygroscopic and very hygroscopic, readily soluble and very soluble in hot water. By the microscopic study they are completely or partially fragmented spherical or ovoid fractions with the expressed central micelles and peripheral semi-transparent shells, as well as small parts separated from the main pseudocrystalline formations. Conclusions. The results of studying the substances of inulin by such physicochemical characteristics as “Description”, “Solubility”, “Hygroscopy”, “Microscopy”, as well as identification of the structural components of fructan according to the requirements of the BP and USP, can be used for the input quality control of plant API of inulin to confirm the structure and determine the purity of the substance. The results of studying the physicochemical characteristics of plant API of inulin can be used when developing the national normative documents on the substance of inulin.


STATEMENT OF THE PROBLEM
One of the valuable API having a high biological activity and a specific effect on the organism is the polysaccharide inulin. It has found its application for stabilizing therapeutic proteins, enhancing dissolution of lipophilic drugs, as a filler of tablets, in the form of methacrylate hydrogels for transporting biologically active compounds to the distal segments of the gastrointestinal tract [1]. Inulin is also used as a diagnostic agent in determining the glomerular filtration rate in renal diseases. In addition, this polysaccharide has a prebiotic effect (stimulates the growth and the metabolic activity of bifidobacteria), affects the metabolism of carbohydrates and lipids, participates in the process of elimination of salts of heavy metals, etc., from the body [2,3].
Inulin is a polyfructose chain, in which the molecules of fructose are connected to the β-(2→1)-glycoside bond, usually there is also one molecule of glucose connected with α-D-glucopyranosyl bond ( Fig. 1) [1,[4][5][6]. The properties of inulin depend primarily on the degree of polymerization (DP), i.e. the number of monomeric fructose links in the polysaccharide macromolecule. The length of the fructose chain of the plant inulin varies from 2 to 60. Under the action of acids and specific enzymes fructan can be hydrolyzed to the structural monomers [1,6].
A high-molecular inulin, which number of fructose residues is more than 10, is of interest for use in medical practice since its pharmacological activity is higher [7].
The raw material base for obtaining fructan has been significantly extended; its largest amount is stored in representatives of Asteraceae, Liliaceae and Poaceae families [5,7,8].
However, a significant number of plant sources and variability of production conditions determine the presence of substances of the polysaccharide at the market today; they differ in the number of fructose residues, the degree of purification from impurities, and by their physicochemical properties.

ANALYSIS OF RECENT RESEARCHES AND PUBLICATIONS
The description of physicochemical properties of API of inulin is given in the works of many researchers. Thus, according to the literature sources, inulin is an amorphous, granular, hygroscopic, practically odorless white powder, under the microscope it has the appearance of ribbed irregular particles, does not reduce Fehling's reagent. It is poorly solu-ble in cold water and readily soluble in hot water. Depending on the method of obtaining and polyfructan as the starting material it can be in the form of an amorphous powder and in the form of crystals [2,3,5,9].
Pharmacopoeial requirements for the quality control of inulin are presented in the monographs "Inulin" of the BP and USP (Tab. 1) [10,11]. In the State Pharmacopeia of Ukraine (SPhU) the appropriate normative documents for the substance of inulin are absent.

IDENTIFICATION OF ASPECTS OF THE PROBLEM UNSOLVED PREVIOUSLY
The primary task of quality control of the substances to be analyzed in standardization is to identify a compound and study its critical characteristics. The particle size and morphology, hygroscopicity, and solubility of substances are incomplete list of indicators that determine the physicochemical and technological properties of substances, and this, in turn, is directly related to bioavailability and efficacy of the finished drug, its safety and stability when using [5][6][7][8][9].
Therefore, determination of the physicochemical characteristics of API of inulin and its identification are the initial, but not the less important stage of the input quality control of the object to be analyzed, it allows, if necessary, either to detect adulteration or to prove the quality of the substance.

OBJECTIVE STATEMENT OF THE ARTICLE
The aim of this fragment of complex studies is the quality control of plant API of inulin in accordance with the requirements of modern normative documents by such physicochemical characteristics as "Description", "Solubility", "Hygroscopy", "Microscopy", as well as identification of the structural components of fructan in order to involve the data obtained in development of the national normative documents on the substance of inulin.
The analysis of the objects was carried out in accordance with the requirements of the BP and USP by the following parameters: "Description", "Hygroscopy", "Microscopy", "Solubility in water R, in hot water R, in organic solvents" and by chemical reactions of identification: the reaction with copper tartrate reagent (Fehling's reagent) and with the alcoholic solution of resorcinol (Seliwanoff 's test). The comparative characteristics of requirements regarding the quality of API of inulin according to these pharmacopoeias are given in Tab. 1.
The solubility and the degree of hygroscopicity of the substances were studied according to the re-quirements of the monograph of the State Pharmacopeia of Ukraine (SPhU, 5.11) [12]. The solubility of substances was studied in water R, in hot water R (80-90 ºС) and in organic solvents (chloroform R). Determination of the degree of hygroscopicity was assessed in 24 hours, keeping the substances over the saturated solution of ammonium chloride.
The morphological characteristics (shape and size of the powder) for all API of inulin was studied by the method of microscopy according to the SPhU, 2.9.37, using an "Opton" microscope by "West Germany" company (the magnification range -х 100 -х 200). On a glass slide a small amount of the substance powder uniformly distributing on the surface was placed (approximately 0.01 g) in the mixture with ethanol (96 %) R [12].
Identification of inulin was carried out according to the methods of the BP and USP: Identification В (BP). Dissolve 10 mg of the substance in 2 ml of hot water R, add 3 ml of 0.15 % alcoholic solution of resorcinol, then 3 ml of hydrochloric acid R, mix and heat at 80 °С. A red color must appear (Fig. 2).
Preparation of 0.15 % alcoholic solution of resorcinol: place 0.15 g of resorcinol R in a 100 ml volumetric flask, add a small amount of ethanol R (96 %), dilute to the volume with the same solvent, and mix [11].
Identification С (BP). Boil 5 ml of 10 % aqueous solution of inulin with 0.5 ml of hydrochloric acid R for 2 min, cool and neutralize with sodium hydroxide solution R by litmus paper. Add 0.5 ml of copper tartrate solution R1 prepared according to the requirements of the BP. A red precipitate must appear (Fig. 3).

Preparation of copper tartrate solution R1 (by the requirements of the BP):
Solution А. Dissolve 34.6 g of copper (ІІ) sulfate R in the mixture of 0.5 ml of sulfuric acid R and water R, dilute the solution to the volume of 500 ml. Solution В. Dissolve 176 g of potassium sodium tartrate R and 77 g of sodium hydroxide R in water R, dilute the solution to the volume of 500 ml with the same solvent.
Mix the equal volumes of solutions А and В immediately before use [11].
Identification (USP). Dissolve 10.0 g of inulin in 20 ml of hot water R in a 100 ml volumetric flask, cool, dilute the solution to the volume with water R and mix (solution S). To 2 ml of solution S add 5 ml of copper tartrate solution prepared according to the requirements of the USP: the reaction does not proceed at room temperature, a slight reaction maybe in 1 min of boiling (Fig. 3).

Preparation of copper tartrate solution (by the requirements of the USP):
Solution А. Dissolve 34.6 g of copper (ІІ) sulfate R in water R, dilute the solution to the volume of 500 ml with the same solvent. Solution В. Dissolve 173 g of potassium sodium tartrate R and 50 of sodium hydroxide R in 400 ml of water R. Heat to boiling, cool, dilute the solution obtained to the volume of 500 ml with water R that is free of carbon dioxide.
Mix the equal volumes of solutions А and В immediately before use [10].
The reagents used in the analysis meet the requirements of the SPhU. All tests were performed in several replicates.
According to the BP when carrying out chemical reactions of identification the substances of inulin studied (batches 01-09) and standard substances of inulin with chicory and dahlia (batches 10, 11) had a red color in Seliwanoff 's test (identification В), confirming the presence of fructose, and an orangered / dark-red precipitate in the reaction with Fehling's reagent after hydrolysis (identification С), indicating the presence of glucose.
According to the USP [10] by the reaction with copper tartrate reagent performed without prior hydrolysis of the substances the reaction did not proceed at room temperature, however, when heated for 1 min the appearance of an orange-red precipitate in the compounds analyzed was observed (batches 1-9). This indicates the presence of sugars with the properties of reducing agents in the substances studied and requires further research. In the samples of batches 10, 11 a blue coloration after heating did not change.
In the experiment it has been found that the API of inulin studied (batches 01-09) and the standard substances of inulin with chicory and dahlia (batches 10, 11) are loose, amorphous, powders of white, practically white and pale yellow color by the "Description" [10,11] parameter (Tab. 2).
According to Tab. 3 the test samples are hygroscopic (increase in mass is from 2 % to 15 %) and very hygroscopic (increase in mass is more than 15 %).
By the parameters of "Solubility" [11] in water R (the temperature was 25 ºС) the substances analyzed  were readily soluble, soluble and sparingly soluble in water R, while by the solubility in hot water R (its temperature was 80-90 ºС) all samples were very soluble or readily soluble (Tab. 4). It is known from the literature sources that the less the number of fructose residues in the inulin structure is, the greater is the solubility of the substance [1,2,4]. In this regard, high parameters of solubility in water at the temperature of 25 ºC indicate the potential presence of low molecular weight inulin fractions in the test samples.
The solubility of substances was also checked in chloroform R. It has been experimentally confirmed that in chloroform R all samples are insoluble.
The next step was to study the structure of plant API of inulin by the method of microscopy  (1)(2)(3)(4)(5)(6)(7)(8)(9); standard substance of inulin with chicory (10); standard substance of inulin with dahlia (11); a calibrated stage micrometer (12) in ethanol (96 %) R [11]. The photomicrography is given in Fig. 4. The study of morphology of API of inulin by light microscopy shows that the samples (batches 02-09) look like masses with large spherical or ovoid particles that are completely or partially fragmented. As noted in the works [2], these formations can be described as pseudomorphic or pseudocrystalline with the expressed central micelles looking like black cores and peripheral semi-transparent shells, as well as small parts of inulin (in the form of dark points) separated from the main pseudocrystalline formations.
The samples of batches 01, 10, 11 in this magnification are also completely or partially fragmented spherical or ovoid particles; however, the substances have a smaller size due to the greater degree of fineness.

CONCLUSIONS AND PROSPECTS
FOR FURTHER RESEARCH 1. According to the BP the presence of the structural components -fructose and glucose after hydrolysis of the main substance can be con-firmed by the chemical reactions of identification of plant API of inulin. According to the USP the identification reaction can confirm the presence of inulin as a substance that does not reduce Fehling's reagent, as well as determine the presence of impurities of sugars with the properties of reducing agents. 2. The results of studying the physicochemical characteristics of plant API of inulin according to such parameters as "Appearance", "Solubility", "Hygroscopy", "Microscopy" can be used for the input quality control of API of inulin to confirm the structure and determine the purity of the substance.