INVESTIGATION OF THE COMPOSITION OF ACTIVE PHARMACEUTICAL INGREDIENTS IN “ APISED ” CAPSULES BY THE METHOD OF HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

As high-performance liquid chromatography is the most optimal method for qualitative and quantitative analysis of multicomponent medicines, including phytomedicines, – owing to the possibility to identify and quantify the substances of different volatility degree in the same sample, – the qualitative composition and quantitative content of the complex apiphytomedicine “Apised” developed in the form of capsules for treatment of mental and emotional disorders and depression states in sports medicine has been investigated using HPLC-analyser on the basis of a Milichrome А-02 chromatograph. At the preliminary stage of our research of the active pharmaceutical ingredients of “Apised” capsules and the apiphytomedicine directly developed it has been found that there are 14 compounds in the composition of the standardized substance of natural powdered honey, 19 compounds in the composition of the garden balm herb, 24 compounds in the composition of the hop cones, 20 compounds in the composition of the inflorescences of spike lavender, 27 compounds in the composition of “Apised” capsules. By comparing the retention times and spectral ratios of the peaks obtained with the device database the presence of linalool, rosmarinic and chlorogenic acids in the composition of the garden balm herb, quercetin and myrcene in the composition of the hop cones, linalool in the composition of the inflorescences of spike lavender, linalool and myrcene in the composition of natural powdered honey has been shown. The experimental data concerning the percentage of biologically active substances in the apiphytomedicine “Apised” correlate well with the composition of the medicine developed and the literary information about the chemical composition of the medicinal plant raw material studied. The method offered gives the possibility to carry out standardization of the composition of the medicine developed, namely “Apised” capsules, regardless of the way of obtaining and origin of the medicinal plant raw material.

As high-performance liquid chromatography is the most optimal method for qualitative and quantitative analysis of multicomponent medicines, including phytomedicines, -owing to the possibility to identify and quantify the substances of different volatility degree in the same sample, -the qualitative composition and quantitative content of the complex apiphytomedicine "Apised" developed in the form of capsules for treatment of mental and emotional disorders and depression states in sports medicine has been investigated using HPLC-analyser on the basis of a Milichrome А-02 chromatograph.At the preliminary stage of our research of the active pharmaceutical ingredients of "Apised" capsules and the apiphytomedicine directly developed it has been found that there are 14 compounds in the composition of the standardized substance of natural powdered honey, 19 compounds in the composition of the garden balm herb, 24 compounds in the composition of the hop cones, 20 compounds in the composition of the inflorescences of spike lavender, 27 compounds in the composition of "Apised" capsules.By comparing the retention times and spectral ratios of the peaks obtained with the device database the presence of linalool, rosmarinic and chlorogenic acids in the composition of the garden balm herb, quercetin and myrcene in the composition of the hop cones, linalool in the composition of the inflorescences of spike lavender, linalool and myrcene in the composition of natural powdered honey has been shown.The experimental data concerning the percentage of biologically active substances in the apiphytomedicine "Apised" correlate well with the composition of the medicine developed and the literary information about the chemical composition of the medicinal plant raw material studied.The method offered gives the possibility to carry out standardization of the composition of the medicine developed, namely "Apised" capsules, regardless of the way of obtaining and origin of the medicinal plant raw material.
To treat mental and emotional disorders and depressions states in sports medicine the complex apiphytomedicine "Apised" in the form of capsules [2] has been developed at the Department of Pharmacy Technology of Drugs of the National University of Pharmacy; its main active pharmaceutical ingredients (APhI) are the standardized substance of natural powdered honey (NPH) and the essential oil plant raw material such as the garden balm herb (Herba Melissa officinalis), the hop cones (Strobuli Humuli lupuli) and the inflorescences of spike lavender (Flores Lavandulae angustifoliae) [5].
As high-performance liquid chromatography (HPLC) is considered to be the most optimal method for qualitative and quantitative analysis of multicomponent medicines, including phytomedicines [6,10,12], we have set a goal to carry out the research of the apiphytomedicine developed just by this method.
Previously the qualitative composition and quantitative content of the main components of essential oils of APhI of "Apised" capsules was investigated by us using the method of gas chromatography, however, the method developed allows to determine only volatile compounds included in the composition of the medicine [11].
Unlike other research methods of biologically active compounds the advantage of HPLC method con-sists exactly in the possibility to identify and quantify the substances of different volatility degree in the same sample under the chromatographic conditions carefully selected.The presence of such conditions is provided by using the so-called HPLC-analyser -it is the approach, which essence consists in carrying out the analysis of compounds of a certain list (from 20 to 500 substances) using the same chromatographic system.For this purpose the database for the array of standard substances is compiled, and futher identification of peaks on chromatograms of the test samples is carried out by comparison of their retention times, as well as spectral ratios with this database [7,9,10].One of the variants of such a system was developed on the basis of a Milichrome А-02 chromatograph ("EcoNova" JSC, Novosibirsk, Russian Federation) [1] are among them.
In the work the results of determination of biologically active substances of "Apised" capsules and their individual ingredients using the system of HPLC-analyser mentioned have been given.
The standard samples of compounds such as linalool (batch W263516), myrcene (batch 64643), rosmarinic acid (batch 00390580) produced by Sigma-Aldrich Chemie GmbH company (Germany) and pharmacopeial standard samples of quercetin (batch 050107) and chlorogenic acid (batch 140308) were used in our work.
The chromatographic procedure was carried out using a Milichrome А-02 chromatograph.
The sample preparation was carried out in the following way: 0.2000 g of the corresponding test sample was extracted by 20.00 ml of 96% ethanol; the solutions obtained were filtered and analysed under the conditions mentioned above; not less than 5 chromatograms were developed -the sample volume was 4 mcl.

Results and Discussion
At the preliminary stage of our research of the active pharmaceutical ingredients of "Apised" capsules and the apiphytomedicine directly developed it has been found that there are 14 compounds in the composition of the standardized substance of natural powdered honey, 19 compounds in the composition of the garden balm herb, 24 compounds in the composition of the hop cones, 20 compounds in the composition of the inflorescences of spike lavender, 27 compounds in the composition of "Apised" capsules.
By comparing the retention times and spectral ratios of the peaks obtained with the device database the presence of linalool, rosmarinic and chlorogenic acids in the composition of the garden balm herb, quercetin and myrcene in the composition of the hop cones, linalool in the composition of the inflorescences of spike lavender, linalool and myrcene in the composition of natural powdered honey has been shown.
To increase reliability of identification of the compounds previously determined the standard samples (SS) of linalool, myrcene, quercetin, rosmarinic and chlorogenic acids added to the test samples after the primary identification were used, and the repeated research was conducted.
Increase of the peak area of the corresponding compounds and the absence of peaks split in this case confirm the supposition concerning the structure of the compounds identified made during the primary analysis.
The basic chromatographic parameters of the compounds identified under the conditions of performing the analysis are given in Tab. 1.
The typical chromatogram of biologically active compounds of the apiphytomedicine "Apised" obtained under conditions of the experiment is given in Figure .For quantitative determination of the substances identified in the composition of APhI of the apiphytomedicine studied and namely "Apised" capsules at the first stage of the experimental research the batch of model solutions of linalool, myrcene, quercetin, rosmarinic and chlorogenic acids SS was prepared, they were chromatographed under the conditions mentioned above and possibility of carrying out the quantitative analysis of the given substances within the concentrations range from 0.1 to 1 mg/ml was shown.At the second stage of the research the content of each component was calculated according to the chromatograms of the solutions studied with SS addition and without it.
The quantitative content (%) of the basic compounds identified in the apiphytomedicine developed and its separate constituents is given in Tab. 2.

Table 1
The basic chromatographic parameters of linalool, myrcene, rosmarinic acid, quercetin and chlorogenic acid when determining by the HPLC method It should be noted that the experimental data of Tab. 2 concerning the percentage of biologically active compounds determined in the apiphytomedicine "Apised" correlate well with the composition of the medicine developed and the literary information about the chemical composition of the medicinal plant raw material studied [3,4,8].
Thus, the complex of the experimental research conducted concerning identification and quantitative determination of biologically active substances of APhI of the apiphytomedicine developed allows to develop the reliable and reproducible method, which gives the possibility to carry out standardization of "Apised" capsules developed.CONCLUSIONS 1.The qualitative composition and quantitative content of the complex apiphytomedicine "Apised" developed in the form of capsules for treatment of mental and emotional disorders and depression states in sports medicine has been investigated using HPLC-analyser on the basis of a Milichrome А-02 chromatograph.
2. The presence of 27 compounds has been detected in the composition of the "Apised" apiphytomedicine developed; among them linalool, myrcene, rosmarinic acid, quercetin and chlorogenic acid have been identified.
3. The method offered gives the possibility to carry out standardization of the composition of the medicine developed, namely "Apised"capsules, regardless of the way of obtaining and origin of the medicinal plant raw material with the help of high-performance liquid chromatography.Table 2 The results of the quantitative determination of linalool, myrcene, rosmarinic acid, quercetin and chlorogenic acid by the HPLC method

Fig.
Fig. The typical chromatogram of biologically active compounds of "Apised" capsules obtained by the HPLC method under the conditions mentioned above.