Physico Chemical and Pharmacogenetic Study of Inula Macrophylla and Inula Rhizocephalan Herbs

Taking into account the information revealed by the scientific literature on the rich composition of the BAS target fragment of the metabolome of I. Macrophylla and I. Rhizocephala and their high biological activity, the development of a liquid extract for obtaining an industrial standardized dosage form of the drug for topical use was considered the most appropriate. The use of the entire leafy shoot of a grass plant as a type of raw material with a less laborious stage in the industrial harvesting of LRS was proposed as the initial I. Macrophylla and I. Rhizocephala in the preparation of a liquid extract. The dried leaves and roots of I. Macrophylla and I. Rhizocephala are used by the population in Tajikistan as a means of traditional medicine to obtain aqueous extracts that are used for local wound treatment.

I. Macrophylla and root extract. I Rhizocephala is characterized by the presence of a complex of essential oils that have a beneficial effect on the functional state of the respiratory tract. These compounds promote the active passage of phlegm from the lung tissue, which makes elecampane an effective agent in the treatment of cough. Rhizocephala, has a beneficial effect on the intestinal microflora, contributing to the normalization of digestion and the fight against dysbiosis and constipation. In addition, the plant stimulates the passage of bile, optimizes the functioning of the liver and gallbladder, and also has a beneficial effect on the urinary system. Traditionally, the root of I. macrophylla and I. Rhizocephala, used to treat asthma, bronchitis, cough, diarrhea, nausea and intestinal parasites. The purpose of this work is the physicochemical and pharmacognostic study of the herb inula macrophylla and inula rhizocephala.

The work was carried out on biochemistry and medical biology of the Khatlon State Medical University, Avicenna TSMU, State Institution "Scientific Research Pharmaceutical Center of the Ministry of Health and North-West of the Republic of Tajikistan", Tajik National University and Kursk State Medical University.

Substantiation of the Choice of The Type of Raw Materials I. Macrophylla and I. Rhizocephala

The results of the analysis of the data of traditional medicine in Tajikistan, the assessment of possible chemotherapeutic and pharmacological activities of ALS contained in the metabolome of the plant, the conduct of our own phytochemical studies of raw materials, showed the prospects for the use of the herbs I. Macrophylla and I. Rhizocephala for medical purposes. The use of this raw material to develop a technology for obtaining a liquid extract from it was the basis for the development of methods for establishing the authenticity and quality indicators of the dried herb of the plant. The creation of the layout of the draft regulatory document for the grass I. Macrophylla and I. Rhizocephala was carried out in accordance with the requirements of the State Fund of the Russian Federation XIII ed. to medicinal plant raw materials of a similar morphological group.

Assessment of the Content of Extractives in The Herb I. Macrophylla and I. Rhizocephala

Table 1: Content of extractives extracted by various solvents in samples of the herb I. Macrophylla and I. Rhizocephala.

The results of identifying the most optimal extractant are presented in Table 1. Water and water-alcohol mixtures with a known concentration of alcohol were used as extractants. At the same time, work was carried out with another type of raw material elecampane grass collected during flowering, in a comparative aspect.

From those presented in Table 1. The data show that during extraction with water and 40% alcohol, the maximum extraction of extractives is observed, which is associated with the intensive transition of polar substances. When extracting raw materials with water-ethanol mixtures with a high concentration of alcohol, along with some hydrophilic compounds, less polar representatives of the plant metabolome also pass, which, according to the literature, suggests that they are active in the [1-4]. Study of the content of target ALS in the raw materials of I. macrophylla and I. Rhizocephala. As you know, phenolic compounds are the most significant group of ALS, which is responsible for the antioxidant effect of drugs from medicinal plant raw materials. Among phenolic compounds, flavonoids are responsible for experiencing the biological effects described in the scientific literature. For the express determination of active substances, the content of phenolic compounds in terms of chlorogenic acid was determined by the UV-SPM method in Table 2.

Table 2: Influence of alcohol concentration on the yield of phenolic substances from two types of raw materials I. Macrophylla and I. Rhizocephala.

From the data presented in Table 2, it can be seen that the content of phenolics in the grass collected before flowering is almost twice as high, and at the same time their maximum content is observed when extracted with 70% alcohol.

Table 3: Effect of feedstock: extractant ratio on the recovery of phenolic substances from LRSI I. Macrophylla and I. Rhizocephala.

According to the data presented in Table 3, the content of phenolic compounds is also twice as high in grass harvested before flowering, and is revealed as the maximum possible at a ratio of raw material to extractant from 1:10. With an increase in the ratio, their yield is practically insignificant for the technology of obtaining drugs. In the study of the effect of the grindability of raw materials, I. Macrophylla and I. Rhizocephala used particles of raw materials passing through a sieve with holes of 0.2 size; 0,5; 1; 2 and 3 mm.

Table 4: Influence of the degree of grinding of raw materials on the extraction of phenolic substances.

From the results presented in Tables 4-5, it follows that the content of phenolic substances in the grass I. Macrophylla and I. Rhizocephala, collected before flowering, exceeds the same indicator for the grass collected during the flowering phase by more than 2 times. Since phenolic compounds are one of the target groups of ALS, the expediency of choosing the herb I. Macrophylla and I. Rhizocephala, collected before flowering, as a raw material and object for further research, was confirmed. Thus, the optimal conditions for the extraction of phenolic compounds from I. macrophylla and I. rhizocephala are as follows: 1:10 ratio of raw material to extractant, 70% ethanol extractant, 0.2-3.0 mm grinding degree, 60 min extraction time.

Qualitative Reactions

In order to select the most reliable qualitative reactions in order to establish the authenticity of the herb I. macrophylla and I. rhizocephala, several variants of extractions from raw materials were prepared - with the help of water (aqueous extraction) and different concentrations of water-alcohol mixtures. Qualitative reactions were carried out with the extracts obtained to confirm the literature data on the content of the target groups of ALS in the studied raw materials (flavonoids, tannins, saponins and alkaloids). The results of these tests are presented in Tables 5 and 6.

Table 5: Results of Determination of ALS Groups Using Qualitative Reactions in Various Extracts from Raw Materials.

Note: The amount of "+" indicates the intensity of coloring, sediment and the size of the foam column.

Qualitative reactions to the presence of alkaloids in the raw material of I. Macrophylla and I. Rhizocephala were carried out using general alkaloid reagents. The results of this work are presented in Table 6.

Table 6: Results of Determination of the Presence of Alkaloids in Various Extracts from I. Macrophylla and I. Rhizocephala Raw Materials.

Numerical indicators of the herb I. Macrophylla and I. Rhizocephala

In the production of the extract, crushed raw materials are used, the fractional composition of which is given in Table 7.

Table 7: Fractional composition of particles of chopped grass I. Macrophylla and I. Rhizocephala.

The data in Table 7 show that in the crushed raw materials I. macrophylla and I. Rhizocephala, 90% are particles larger than 2 mm, but not less than 0.5 mm. The fraction of crushed raw materials from fine and dusty particles is at the level of 10%. The results of determining some numerical indicators of the raw materials I. macrophylla and I. Rhizocephala are shown in Table 8. As can be seen from Table 8, the moisture content of I. Macrophylla and I. Rhizocephala grass did not exceed 6%, which corresponds to the level of values of no more than 14% for the dried 83 official raw materials of the morphological group. The total ash content in the grass is about 10%, and ash insoluble in a 10% HCl solution is up to 1%.

Table 8: Some numerical indices of the grass I. Macrophylla and I. Rhizocephala.

As a result of studies on the presence of heavy metals and arsenic in raw materials, as well as pesticide residues, the data presented in Table 9 were obtained.

Table 9: Results of determination of heavy metals, arsenic and residual pesticides in raw materials of I. Macrophylla and I. Rhizocephala.

The study of the herb I. macrophylla and I. Rhizocephala for compliance with safety standards for the indicators "Content of residual pesticides" and "Content of heavy metals and arsenic" showed that the raw material meets the requirements for the purity of medicinal products in the State Branch of the Russian Federation XIII ed. When studying the features of the development of I. Macrophylla and I. Rhizocephala by foreign scientists [5,6,7,8], it was found that elecampane accumulates heavy metals and arsenic in the underground parts, but such elements do not penetrate into the above-ground organs of the plant. Based on this, it is proposed to use elecampane to clean soils from pollution caused by anthropogenic impact.

Development of Methods for The Quantitative Determination of Active Substances in I. Macrophylla and I. Rhizocephala Raw Materials Acceptable for End-To-End Standardization

The data of the literature allow us to consider phenolic compounds, and in particular, flavonoids, as active ingredients of the herbs I. macrophylla and I. Rhizocephala. The results of the study of ALS of the target low-molecular fragment of the metabolome confirm their presence in the studied raw material. When developing a liquid extract from the herb I. Macrophylla and I. Rhizocephala estimated the content of the total amount of phenolic compounds to analyze the efficiency of the head stage of the technological process.

Development of a Method for Determining the Content of The Sum of Phenolic Compounds

Solution of CO chlorogenic acid. A precise load of CO chlorogenic acid (about 0.01 g) is placed in a 50 ml volumetric flask, the volume of the solution is brought to the mark with 70% alcohol and mixed. Place 1 ml of the first solution in a 25 ml volumetric flask and bring the volume of the solution to the mark with 70% alcohol, mix. The solution is used freshly prepared. Tested solution. 1.0 g of dry chopped (no more than 3 mm) herb I. Viscosa is placed in a conical flask of 250 ml with a section. Add 50 ml of 70% alcohol, add to the return refrigerator and heat in a boiling water bath for 1 hour (after boiling). The mixture is cooled, filtered into a 50 ml volumetric flask through a paper (blue ribbon) pleated filter, and the volume is brought to 70% with alcohol. Place 0.5 ml of the tested solution in a 25 ml volumetric flask, bring it to the mark with 70% alcohol and mix. The optical density of the resulting solution is measured on a spectrophotometer at the absorption maximum at 328±2 nm in the thickness of the absorbing layer of 10 mm. 70% alcohol is used as a reference solution. The optical density of the chlorogenic acid solution is measured. 

The content in percentage (X) of phenolic compounds in terms of chlorogenic acid is calculated according to the formula:

where A0 is the optical density of the CO solution of the chlorogenic acid solution;

A is the optical density of the tested solution;

m0 is the mass of chlorogenic acid, g,

m is the mass of the LRS, g,

W is the weight loss on drying, %

P is the content of the main substance in the CO solution of chlorogenic acid.

The UV spectrum of extraction from elecampane raw materials in the region of 280-350 nm coincides with that of chlorogenic acid rice for analytical purposes, the maximum absorption at 328 nm was chosen.

Figure 1: UV spectra of extraction of I. Macrophylla and I. Rhizocephala from grass (1), and a solution of CO chlorogenic acid (2).

In the studied samples of the herb I. Macrophylla and I. Rhizocephala, the content of phenolic compounds in terms of chlorogenic acid ranged from 6.87 to 7.63%. According to GPM.1.1.0012.15, validation was carried out for specificity, linearity, correctness and interlaboratory precision. The validation report is presented in Appendix 5. Validation of the developed method for the quantitative determination of phenolic compounds in terms of chlorogenic acid showed the consistency of the results of the analysis of the sum of phenolic compounds in terms of chlorogenic acid. Since the detailed composition of the sum of phenolic compounds by the UPLC-MS method revealed the presence of a significant number of flavonoids that make a significant contribution to the manifestation of anti-inflammatory activity stated in the literature and predicted by insilico, a method for quantifying the number of flavonoids in terms of Rutin was developed.

Development of A Method for Determining the Content of The Number of Flavonoids in The Raw Materials of The Herb I. Macrophylla and I. Rhizocephala

Flavonoids were determined after a complexation reaction with aluminum chloride, which is selective for this group of phenolic compounds and gives a spectrum shift to the long-wavelength region, which makes it possible to measure the content of mainly flavonoids in the presence of other associated groups of phenolic substances in the extraction from the raw material. When comparing the absorption spectra of flavonoid complexes from LRS I. macrophylla and I. Rhizocephala and RSO Rutin, it was found that in the range of 404-412 nm the absorption maxima coincide, which determined the choice of the analytical wavelength of 410 nm (Figure 2).

Rutin standard sample solution. About 0.2 g (exact weighting) of CO rutin is placed in a 25 ml volumetric flask, the volume of the solution is brought to the mark with 70% alcohol and stirred (solution A CO rutin). The shelf life of the solution is 1 month. Place 2.0 ml of A CO rutin solution in a 25 ml volumetric flask, add 3 ml of 3% alcohol aluminum chloride solution, bring it to the mark with 70% alcohol, stir and stand for 40 minutes (B CO rutin solution). Tested solution. 1.0 g of dry raw materials of the herb I. Macrophylla and I. Rhizocephala (no larger than 3 mm), placed in a conical flask of 250 ml with a section, add 50 ml of 70% alcohol, and heat with a reverse refrigerator in a boiling water bath for 1 hour (from the moment the contents boil). The mixture is cooled, filtered into a 50 ml volumetric flask through a paper filter (blue tape) and the volume is brought to the mark with 70% alcohol (Test solution). 2 ml of the tested solution is placed in a 25 ml volumetric flask, 3 ml of 3% alcohol aluminum chloride solution is added, brought to the mark with 70% alcohol, stirred and kept for 40 minutes. Optical density is measured at a wavelength of 410 nm in a cell with 88 with an absorbing layer thickness of 10 mm. A reference solution is prepared: 2 ml of the tested extract is placed in a 25 ml volumetric flask, 0.5 ml of diluted acetic acid is added, brought to the mark with 70% alcohol, stirred and the optical density is measured. In parallel, they measure the optical density of the CO rutin solution prepared similarly to the test extraction solution.

The content of the sum of flavonoids in % in terms of rutin is calculated according to the formula:

A is the optical density of the solution of the tested extraction;

m0 is the weight of CO rutin in g, 

m is the weight of the raw material in g,

W is the loss in mass during the drying of raw materials,

P is the content of the main substance in CO rutin.

The content of the amount of flavonoids in terms of rutin in the herb I. Macrophylla and I. Rhizocephala is from 1.9 to 2.2 %, i.e., among phenolic compounds, the share of flavonoids is about 30%. Validation of the developed method for quantitative determination of the number of flavonoids in terms of rutin, in the raw material I. Macrophylla and I. Rhizocephala is presented in the form of a report in Appendix 6. The methods proposed for quantitative assessment of the quality of I. Viscosa herb are validated and allow them to be evaluated positively in all parameters. It can be concluded that the use of the developed methods for assessing the content of the sum of phenolic substances and flavonoids gives results that meet the requirements of regulatory documents for LRS and preparations based on it. Substantiation of the biological activity of ALS I. Macrophylla and I. Rhizocephalainsilico. In order to identify the main ALS of the target fragment of the metabolome I. Macrophylla and I. Rhizocephala, their possible potential targets and pharmacological activity, the PASS web service [9-14] was used, the results of which are presented in Table 10.

Table 10: Insilico prediction of pharmacological activity of ALS identified in I. Macrophylla and I. Rhizocephala.

The screening of the biological activity of these compounds, the presence of which in the extract from elecampane raw materials is confirmed by the literature, showed that they may be carriers of pharmacological effects inherent in drugs from I. Macrophylla and I. Rhizocephala, known in traditional medicine and confirmed by modern studies.

Determination of The Authenticity of Raw Materials I. Macrophylla and I. Rhizocephala by UV Spectrophotometry and TLC

In the UV spectrum of extraction (70% alcohol from the LRS I. macrophylla and I. Rhizocephala), two absorption maxima were recorded at wavelengths of 298 and 329 nm (Figure 3). This circumstance suggests the presence of 72 phenolic compounds in the raw material, in particular phenol carboxylic acids and flavonoids. The obtained absorption spectrum can serve as an additional qualitative characteristic for establishing the authenticity of LRS I. macrophylla and I. rhizocephala.

Figure 3: UV Spectrum of Extraction from Raw Materials I. Macrophylla and I. Rhizocephala.

With the help of TLC, the composition of the ALS of the target metabolome fragment in the raw material I. viscosa in the n-butanol-ONION-water system (4:1:1) was studied. Adsorption zones were detected in UV at 365 nm before and after chromatogram treatment with diphenylboryloxyethylamine (1% in methanol) and PEO-400 (5% in methanol) sequentially.

The test extract from LRS was applied to the chromatogram, and the following CO solutions were used as standards: caffeic acid, rutin, gallic and chlorogenic acids in ethanol. Solutions of the standards, on the chromatogram, appear as zones with an Rf of about 0.58 for rutin, with an Rf of about 0.76 gallic acid; with Rf about 0.51 chlorogenic acid and with Rf about 0.75 caffeic acid. Several blue zones (phenolcarboxylic acids) are visible on the chromatogram of the test extraction, the most intense of which have an Rf of about 0.51 and 0.75, which is identical to chlorogenic and caffeic acids, respectively. Zones of brownish glow merging with zones of blue color, one of which corresponds to the rutin standard, the other is close to the zone of the gallic acid standard. The red zone, chlorophyll, is visible closer to the mobile phase front. After spraying the chromatogram with reagent solutions in daylight, three intensely stained flavonoid zones appear on it: with an Rf of about 0.58 (corresponding to rutin) and with an Rf of about 0.69 and with an Rf of about 0.78 (two unidentified substances). On the chromatogram at a wavelength of 365 nm, the blue-greenish adsorption zones belong to phenolcarboxylic acids, and the orange zones correspond to flavonoids (Rf about 0.58, 0.68 and 0.77). The violet fluorescence on chromatogram 74 has a zone of Adsorption of Co Gallich Asid, Booth You Correspondence Zones, Is Wickley Steined on To Chromatogram of You Tested Extraction, To You of Oter Substance on To Sones.

Thus, based on the study of the composition of biomass, extractives extracted by water and 70% alcohol, both types of raw materials are comparable, but the content of phenolic compounds of about 7% in the grass harvested before flowering confirms the expediency of choosing it as LRS. Also, a comparative study of the two possible types of raw materials made it possible to optimize such significant factors for the technology as the degree of dispersion of raw material particles, the concentration of the extractant, the ratio of raw materials to the extractant and the extraction time. Thus, the study of the extraction of I. macrophylla and I. rhizocephala from the raw material by the TLC method made it possible to establish the presence of phenolic substances, the prevailing content (judging by the area of the zones and their intensity of staining and fluorescence) among which are phenolcarboxylic acids, in particular, chlorogenic, caffeic and gallic acids, as well as flavonoids (rutin).

Development of The Composition and Technology of The Application Dosage Form with The Extract of The Herb I. Macrophylla and I. Rhizocephala "Inulan Gel"

Based on the data obtained in the experiments in vivo and in vitro, the presence of pronounced antimicrobial and anti-inflammatory activity of the developed drug "Inulan" was established, which indicates the possibility of developing its application form of gel. Recently, species of the genus Inula (in particular, Inula grandifolia) have been studied as a source of antioxidants and are used to remove free radicals from the body, in the treatment of diseases of the cardiovascular, nervous system, as well as metabolism [15-18].

Development of The Composition of The Application Form

The development of the composition of the application form with I. Macrophylla and I. Rhizocephala extracts involved the choice of the RAP polymer grade, the establishment of the concentration for a specific thickener. Taking into account that the extract contains at least 48 wt% alcohol, it is necessary to ensure the stability of the dispersed 106 system and maintain the ALS in a dissolved form, preventing condensation in the presence of an aqueous gel base of the RAP. The use of alcohols as co-solvents will make it possible to obtain LF "Gel" and should not adversely affect the viscosity characteristics acceptable for application to damaged skin (80-120 Pahs, at 9 rpm). [19-22]. To obtain the gel base of the drug "Inulan", rarely-cross-linked acrylic polymers - RAP - were used. RAP-based gels are the basis of most modern applications used both for transdermal administration of drugs (NSAIDs - Nimulide gel, nice gel, antihistamines - Fenistil, etc.), and for the treatment of the first phase of the wound process (Furagel). The development of the gel composition involves the selection of the polymer grade, the establishment of the working concentration of RAP in the presence of the active substance, as well as the study of the effect of various explosives, in particular, glycerol, PG and PEO-400 alcohols. The presence of alcohols in the composition of various dermatological gels makes it possible to exert a unidirectional effect with the drug on the components of the pathological process and to ensure the physical and chemical stability of the active substances, as well as the microbiological purity of the drug in sufficient quantities Table 11. The results of studying the effect of cosolvents on the viscosity and appearance of gels are presented.

Table 11: Formulations of gel samples with Inulan based on Arespol 1.5%.

Glycerin in dermatological ointments has a moisturizing effect and increases the comfort of use, prevents the drying of the LF. PG is a promoter of absorption and is used in the composition of application drugs in a concentration of up to 60%. PEO-400, as an osmotically active component, is relevant in formulations intended for the treatment of the first phase of the wound process with exudate and the risk of infection. The biopharmaceutical role of solvents was taken into account. The acceptance criteria were considered to be a satisfactory appearance - a transparent homogeneous gel with weak opalescence and dynamic viscosity in the range of 80-120 Pahs, at 9 rpm. The results presented in Table 11 indicate a negative effect of alcohols on the dynamic viscosity of gel samples at an Arespol content of 1.5%. At the same time, the total content of alcohols (PG, PEO-400 and glycerol) from 10 to 30 individually and in combinations does not allow to obtain a transparent, non-opalescent gel. The introduction of ethyl alcohol 70% allows for increased transparency, but leads to a sharp liquefaction (model 14), which excluded it from the samples. Sample 17 was recognized as the most successful according to the combination of the selected criteria. Increasing the content of Arespol to 2.0% increased the viscosity of the gel samples and at the same time the alcohol content to 35-40%, the results are presented in Table 12.

Table 12: Formulations of gel samples with Inulan based on Arespola gel 2.0%.

As can be seen from the results of the experiment presented in Table 12. Sample 2, with an alcohol content of 35%, fully met the viscosity criteria and was more transparent than the other samples. It was also interesting to establish the effect of the extract itself on the appearance and viscosity of the gel form. Table 13 shows the results of the selection of the concentration of Arespol 1.5% or 2.0%, extract 5.0%, 8.0%, 10.0% and alcohols 20-40%.

Table 13: Compositions of samples of gels with Inulin and various polymer and alcohol contents.

The results obtained in Table 13 allow us to consider samples 17 acceptable in appearance. In general, the experiments carried out on the selection of the composition of Inulan gel indicate the wide possibilities for obtaining application LFs based on RAP and require further in-depth substantiation of formulations for various therapeutic tasks. For further work in the course of this study, the composition (sample 1) of the application form based on Arespol 1.5% and the content of Inulan, glycerol, PG and PEO-400 10