Determination of Sugar Profile in Viburnum Fruits and its Dosage Forms by HPLC-RID

Syrups are


INTRODUCTION
In Russia, the fruits of guelder-rose (Viburnum opulus L.) and Sargent's guelder-rose (Viburnum sargentii Koehne) of honeysuckle (Caprifoliaceae Juss.) family are approved for medical use (Figure 1).State Pharmacopoeia of the USSR (XI edition, Issue 2) contains a pharmacopoeial monograph for the guelder-rose dried fruits 1 ; the Russian State Pharmacopoeia (XIV edition, Volume IV) -for the fresh fruits of guelder-rose and Sargent's guelder-rose. 2"Viburnum fruits" for the infusion preparation and "Viburnum syrup" are medicines based on medicinal herbal raw materials. 3,4iburnum fruits and products based on it are included in the Russian Register of Products.These substances have passed state registration as raw materials for biologically active additives, food, herbal teas, alcoholic and nonalcoholic balms preparation.
Viburnum fruits contain a large variety of biologically active substances (BAS) [5][6][7][8][9][10] : anthocyanins, proanthocyanidins [11][12][13] , flavonoids, hydroxycinnamic acids, iridoids, polysaccharides, organic acids 14,15 , lipids 16 , aromatic components. 17e raw material contains a unique vitamin complex: ascorbic, nicotinic acid, vitamin A, vitamin K, folic acid, tocopherol; micro-and macro-elements: molybdenum, magnesium, selenium, copper, manganese, zinc, iron, chromium, potassium, cobalt, calcium. 18nti-inflammatory, antispasmodic, hypotensive, antibacterial, K-vitamin, C-vitamin, diuretic, antiradical and other types of pharmacological activity have been established for the viburnum fruits. 3rups are one of the dosage forms that have become widespread.Syrup -a liquid dosage form for internal use; it is a concentrated, thick aqueous solution of various sugars with medicinal substances, extracts, tinctures, fruit juices or without them.Sugar syrup (64% sucrose solution) is used as a consistent additive and flavoring for the preparation of syrups. 19t increases osmotic pressure and completely inhibits the growth and development of microflora in the dosage form.Invert syrup, a mixture of an equal amount of glucose and fructose, is obtained from sugar syrup by inverting (hydrolyzing) sucrose by heating the sugar syrup in the presence of acid (catalyst).Viburnum syrup (Viburni sirupus) has a tonic, diaphoretic and a light diuretic effect.
Viburnum syrup is used in the complex treatment of colds, during the recovery period after diseases.
Current requirements for the standardization of medicinal plant raw materials (MPRM) include indicators characterizing the composition of BAS.The BAS quantitative determination is carried out by acid-base titration according to the pharmacopoeial monograph "Viburnum fruits".It regulates organic acids content in terms of malic acid that should be not less than 6 % in fresh viburnum fruits.Carbohydrate composition and content is also an important indicator since sugars also contribute to the organoleptic properties of drugs together with organic acids.
This study aims to determine the profile and content of carbohydrates in viburnum fruits and drugs based on it.

MATERIALS AND METHODS
Viburnum fruits ("Kamelia-LT' LLC, Technic Specifications 9197-003-81930399-09) are round, flattened on both sides, wrinkled, shiny fruitdrupe with a diameter of 8-12 mm (Figure 2.1).It has a barely visible column and sepals, a deepening at the place of stalk separation.In the pulp is one hard separable flat heart-shaped pit (seed).The color of the fruit is dark red or orange-red; the seed color is light brown.The smell of raw materials is weak, the taste is bitter-sour.
Viburnum infusion is a clear liquid of golden color with a pink tint, has a slight smell, a bitter-sour taste (Figure 2.

2).
Viburnum syrup (registration number: N003534/01, manufacturer of ZAO "VIFITEH") is a thick liquid from orange-red to dark red color with a characteristic odor; a slight gelatinous precipitate may be observed, which dissolves when heated up to 40 °C (Figure 2.3).The smell of syrup is weak, it has a sweet taste, with a slight bitter-sour finish.
Preparation of aqueous extraction from viburnum fruits An analytical sample of the raw material is ground to a particle size passing through a sieve with a hole size of 1 mm.About 2 g (accurately weighed) of the crushed raw material is placed in a 100 ml flask, 40 ml of purified water is added, mixed thoroughly and kept in a boiling water bath for 1 hour.The extract is cooled, filtered through a paper filter into a 100 ml volumetric flask.The raw materials remaining in the flask and on the filter are washed with 20 ml portions of purified water twice, transferred back to the flask and the extraction is repeated for 1 hour.Then the extract is cooled, filtered through a paper filter into a 100 ml volumetric flask, the remaining raw materials in the flask and on the filter are washed by 10 ml of purified water, the volume is adjusted to the mark with purified water and mixed.
Infusion preparation About 10 g (1 tablespoon) of the fruits is placed in an enamel dishware, 200 ml (1 cup) of hot boiled water is poured, covered with a lid and heated in a boiling water bath for 15 minutes,  cooled at room temperature for 45 minutes, filtered, the remaining raw materials are squeezed out.The volume of the resulting infusion is adjusted with boiled water to 200 ml.The resulting infusion is filtered through a paper filter.
Syrup sample preparation About 5 g (accurately weighed) of the syrup is placed in a 100 ml flask, 70 ml of purified water are added, mixed thoroughly, filtered through a paper filter into a 100 ml volumetric flask.
The flask and filter are washed twice with 10 ml portions of purified water.The volume of the resulting infusion is adjusted to the mark with purified water and mixed.
Isocratic elution was performed using purified water (with the addition of Ca-EDTA 0.05 mg/ml) as the mobile phase, at a flow rate of 0.5 mL/min.The column was maintained at 80 °C, the refractive index detector -at 35 °C.The injection volume was 10 μL, analysis time -20 min.Qualitative and quantitative determinations were based on the injection of an external standard solutions prepared as follows: 100 mL water containing 1.5 g of glucose, 1.5 g of fructose and 1.0 g of sucrose.

RESULTS AND DISCUSSION
The main free monomeric carbohydrates of viburnum fruits and preparations based on them are glucose, fructose, and sucrose (Figure 3).The composition of carbohydrates is an important characteristic along with the content of organic acids.The total content of simple sugars in the raw materials of viburnum is 28.393 ± 0.528%, in the infusion -0.499 ± 0.015%, in the syrup -69.736 ± 1.551% (Table 1).
The typical chromatograms are presented in Figure 3.The sugars' degree of passing into infusion is 74.34%.The characteristic ratio of glucose: fructose: sucrose in dried viburnum fruits is 8.1:5.4:1, in infusion -7.0:5.7:1, and in syrup -9.2:8.8:1(Figure 4).This ratio can be used in the control of drugs based on viburnum fruits.According to the study, 5 the ratio of glucose: fructose in samples of fresh viburnum fruits is 1.0-1.5;our findings for dried fruits fall within this range.The profile and content of monomeric sugars, along with other BAS groups, can serve as indicators of identity and good quality when standardizing dried, fresh, freshly frozen viburnum fruits, drugs, extracts, and dietary supplements based on it.

CONCLUSION
As a result of the studies, the technique for determining the concentrations of sucrose, glucose, and fructose was tested in the analysis of the viburnum fruits and drugs based on it using the HPLC-RID method.The advantages of this technique are the lack of time-consuming sample preparation and, as a result, the rapidity of the technique.Using this technique, it is possible to monitor the carbohydrate composition at all stages of the pharmaceutical manufacturing process.Techniques based on the HPLC-RID method should be introduced into the routine pharmacopoeial analysis since it is very informative and makes it easy to identify poor-quality drugs.

Figure 4 :
Figure 4: Monomeric sugar ratio in viburnum fruits and drugs based on it.