Development of Composition and Technology of Anti-microbial Gel with Dracocephalum nutans l. Essential Oil Growing in the Territory of Central Kazakhstan

Sabiyeva Assel¹, Gayane Atazhanova¹, Zholdasbayev Mussa¹, Ashirbekova Bibigul¹,

Aigul Medeshova¹, Kurmantayeva Gulnissa¹, Zhanel Turdiyeva¹, Sholpan Sarsembayeva¹,

Ainur Ibraybekova¹, Mukasheva Gulnar²

¹NCJSC “Karaganda Medical University”, Gogol Str.40., 100000, Karaganda, Kazakhstan.

²NCJSC “Karaganda University named after E.A. Buketov”, 100028, Kazakhstan,

Karaganda, Universitetskaya Str 28, Block 3.

*Corresponding Author E-mail: atazhanova@qmu.kz

ABSTRACT:

As a result of the study on antimicrobial activity it was established that the essential oil of the herb of the drooping dragonhead (Dracocephalum nutans L.) exhibits pronounced antimicrobial activity in relation to strains of gram-positive bacteria Staphylococcus aureus (ATCC 6538), Bacillus subtilis (ATCC 6633) and weak activity in relation to gram-negative bacteria Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853) and weak activity to the yeast fungus Candida albicans (ATCC 10231). Taking into account the above, the purpose of our research is to develop a gel with antimicrobial properties. The essential oil of Dracocephalum nutans L. was chosen as the object of the study. The genus Dracocephalum L. belongs to the family Lamiaceae Lindl. (Labiatae), the plants of which are of interest as sources of medicinal preparations. Studies have shown that some species of Dracocephalum possess antibacterial, antitussive, antidiarrheal, antioxidant, anticancer, anti-inflammatory, antidiabetic and sedative properties. Isolated compounds from the genus Dracocephalum collected from natural ones such as limonene, carvacrol, α-pinene, γ-terpinene, perillaldehyde, caryophyllene oxide, 1,8-cineole, verbenone, perillyl alcohol, neral and geranyl acetate have pronounced antimicrobial properties. Among the plants of this genus, Dracocephalum nutans L. is of interest. Dracocephalum nutans L. (Synonyms: Dracocephalum nutans var. alpinum Kar. & Kir., Dracocephalum nutans subsp. subarctium Kuvaev) contains a complex of biologically active substances and is used in the medicine of the peoples of Southeast Asia in the treatment of kidney inflammation and gastrointestinal diseases such as hepatitis, gastritis, etc. The herb of Dracocephalum nutans L. has a high content of various biologically active compounds that determine the medicinal properties of this species: essential oils, flavonoids, saponins, coumarins, traces of alkaloids. The main natural components of the essential oil of Dracocephalum nutans L. were 1,8-cineole (34%), α-pinene (6.7%), β-pinene (5.2%), β-myrcene (5.3%). The chemical composition of the essential oil was studied by chromatography-mass spectrometry. A unified method for determining the component composition of essential oils, as well as a 7890A Agilent Technologies chromatographic system with a 5975C mass spectrometric detector were used for the analysis. Essential oils have various biological activities: antimicrobial, anti-inflammatory, immunomodulatory, anesthetic. There is data on the use of essential oils as an activator of absorption of pharmacologically active components of the dosage form. One of the main properties of essential oils is a high antimicrobial effect, combined with low toxicity and the absence of side effects in therapeutic use. It is assumed that the mechanism of the antimicrobial action of essential oils lies in the destruction of membrane structures of microbial cells with subsequent disruption of intracellular metabolism and death of microorganisms.

KEYWORDS: Gel, essential oil, Dracocephalum nutans L., antimicrobial activity, bacteria.

INTRODUCTION:

Long-term use of antimicrobial drugs can lead to disruption of reparative processes in the wound, the emergence of antibacterial resistance of the microbial flora. In this regard, it is necessary to expand the range of antimicrobial agents for the treatment of wounds of various etiologies^1,2.

Unlike synthetic drugs, antimicrobial drugs of plant origin are not associated with many side effects and have a huge therapeutic potential for the treatment of many infectious diseases. Recently, there has been a tendency to increase the range of drugs derived from plants. Herbal medicines have high therapeutic efficacy and the lowest toxicity³. Phytochemical studies of domestic medicinal plants provide a valuable material base for the discovery and development of new drugs of natural origin⁴.

Considering the huge potential of plants as a source of antimicrobial drugs, a systematic study of the essential oil of the herb of the drooping dragonhead (Dracocephalum nutans L.), growing in central Kazakhstan, was conducted. According to the literature, the essential oil of the drooping dragonhead has anti-inflammatory and antimicrobial effects. Considering the traditional use of the drooping dragonhead in folk medicine, the development of an antimicrobial dosage form based on it for the treatment of bacterial diseases is of undoubted interest.

The fight against bacterial diseases of the skin and mucous membranes is an important problem of medical practice. Today, clinicians have a significant arsenal of drugs for the treatment of this pathology^5-8.

MATERIALS AND METHODS:

The grass Dracocephalum nutans was harvested in June 2022 near the village of Karkaralinsk (Kazakhstan, Karaganda region, N 49⁰43323, E 75⁰483886) during the full flowering period.

Isolation of the essential oil:

The extraction of essential oil from medicinal plant materials was carried out using the hydrodistillation method on a Clevenger laboratory apparatus in accordance with the State Pharmacopoeia of the Republic of Kazakhstan I, v.1, 2.8.12.

Growth and Maintenance of Test Microorganism for Antimicrobial Studies:

Reference strains of microorganisms from the American Collection of Type Cultures (ATCC): gram-positive bacteria Staphylococcus aureus (ATCC 6538), Bacillus subtilis (ATCC 6633), Gram-negative bacteria Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853) and yeast fungus Candida albicans (ATCC 10231).

Preparation of Inoculum:

The cultures were grown in a liquid medium pH 7.3±0.2 at a temperature of 30 to 35⁰C for 18-20 hours. The cultures were diluted 1:1000 in a sterile 0.9% isotonic sodium chloride solution, 1 ml was added to cups with appropriate elective nutrient media for the studied test strains, and sown according to the “solid lawn” method. After drying, wells 6.0mm in size were formed on the surface of the agar, into which solutions of the test sample, benzylpenicillin sodium salt 10µl each and eucalyptus oil 1µg each were added, 96% ethyl alcohol was used in control in equivolume amounts. Thus, the studied samples were tested in the amount of 1μg, and the reference drug in the amount of 1mg. The inoculations were incubated at 370⁰C, the growing cultures were counted after 24hours.

Preparation of reagents for studying the release of active substances from the ointment:

The study of the release of the active substance from the ointment was carried out by the method of "direct diffusion into agar" in a thermostat for 6 hours. 10ml of 0.1% solution of bromothymol blue indicator was used as a coloring ingredient. Hot agar was immediately poured in equal volumes into 3 Petri dishes and put in the refrigerator to harden until jelly formed, then wells with a diameter of 8.5mm were cut out and ointments based on essential oil with different base compositions weighing 0.25g were placed in them. The studies were carried out in 3 parallels at a temperature of 36.6±0.2° C. The degree of diffusion into agar was assessed visually by a change in the color of the zones in blue.

RESULTS:

The choice of bases for the preparation of the gel is an important problem, since it was necessary to use both lipophilic and hydrophilic bases that do not have allergic, irritating properties and release the drug well from the gel form.

Preliminary studies were conducted to select gelling agents and evaluate the physicochemical and structural-mechanical properties of the bases used. In the process, parameters such as melting point, acid, peroxide and iodine numbers were determined. All selected bases met the requirements of the State Pharmacopoeia of the Republic of Kazakhstan and the Pharmacopoeia of the Eurasian Economic Union for bases for preparing gels. We manufactured 3 gel samples using carbopol and 3 samples with sodium carboxymethylcellulose (Na-CMC).

Table 1: Composition of gel compositions with essential oil of drooping dragonhead (100.0 g)

Name of ingredients	Purpose	Number of components, g
Name of ingredients	Purpose	No. 1	No. 2	No. 3	No. 4	No. 5	No. 6	No. 7
Essential oil	Active ingredient	5,0	5,0	5,0	5,0	5,0	5,0	5,0
Carbopol-940	Gel base	1,0	1,5	2,0	2,5	-	-	-
Sodium CMC	Gel base	-	-	-	-	15,0	18,0	20,0
Twin-80	Stabilizer	1,0	1,5	-	1,0	1,5	-	1,0
10% NaOH	Acidity regulator	pH up to 6,0-7,5				-	-
Glycerol	Plasticizer	10,0	10,0	10,0	10,0	10,0	10,0	10,0
purified water, ml	Solvent up to	100	100	100	100	100	100	100

The gels were prepared taking into account the physicochemical properties of the essential oil and the type of base. When selecting possible ointment bases, their harmlessness, rheological properties, and stability for at least two years were taken into account. The experience of some studies devoted to the development of similar medicinal forms of antimicrobial action was taken into account^9-13.

When analyzing the obtained samples of model gels, organoleptic properties (appearance, color, aroma, uniformity) were established and the pH value was determined. Homogeneity was determined by the absence of visible particles of ingredients, foreign impurities, as well as signs of coagulation, particle accumulation or phase separation. All the studied samples of the basic components that were obtained were distinguished by high transparency and homogeneity.

The therapeutic effectiveness of the gel largely depends on the composition of the basic components. The resulting dosage forms are a homogeneous mass of colorless color. The gels are a viscoplastic flowing mass of colorless color, evenly distributed over the skin, easy to spread, and create a feeling of stickiness on the skin. We used the ointment compositions listed in Table 1.

Samples no. 2,3,4 cause a burning sensation on the skin surface due to the addition of a large amount of sodium hydroxide, which was necessary to control the pH of the gel being developed. Twin -80, also known as polysorbate-80, is often used in cosmetic and pharmaceutical products, including gels. It is added for several reasons: Twin 80 emulsifier helps mix ingredients that are not usually mixed, such as water and oil. This is important for creating a gel with a uniform texture and stable properties. In some cases, Twin -80 can help enhance the penetration of active ingredients into the skin, making the product more effective. So, adding Twin -80 to a gel can provide it with better texture, stability, moisturizing properties and improve its effectiveness. It is observed that samples №5 and №6 have insufficient adhesive properties and do not have enveloping characteristics, as well as unsatisfactory organoleptic characteristics. This is probably characterized by a decrease in glycerin or its complete absence. Glycerin has high moisturizing properties that help soften and moisturize the skin. This is especially important for medicinal gels that can be applied to the skin to treat various conditions such as dryness, irritation, or eczema. Help in the absorption of active ingredients: Glycerin can help the active ingredients penetrate the skin better due to its moisturizing properties. This can improve the effectiveness of the medicinal gel.

With the development of biopharmaceutics, a special place in the assessment of the quality of medicinal preparations is occupied by the determination of the release of active substances in in vitro experiments. The study of release allows for a comparative study of bases and other auxiliary substances that provide an effect on the effectiveness of the medicinal preparation. The criterion for assessing the bases used in gels was the speed and degree of release of active substances. The degree of release of biologically active substances from the bases was judged by the size of the resulting colored zone.

The conducted study of the quality of various gel compositions, manufactured according to samples 1 through 7, showed that samples № 1 and 7 are optimal in terms of texture, ease of use and treatment effectiveness. Based on this, samples № 1 and 7 were selected as the optimal gel composition.

Figure 1: Kinetics of release of essential oil and gel compositions by diffusion into agar

The obtained experimental data indicate that the highest degree of release of biologically active substances occurred from the gel based on NaCMC. As a result of biopharmaceutical studies on the development of the composition and technology of gels based on essential oil, the most promising composition for further research was identified: NaCMC - 20.0g, glycerin 10.0g, Twin -80 -1.0g, purified water to 100ml, essential oil 5.0g. A high degree of release from this sample is observed in the first hours from the moment of application and persists for 24hours.

Rheological properties of the base and gels based on essential oil were studied on a Brookfield NDJ-1F rotational viscometer.

It is known that most gels behave as elastic bodies with reversible deformation under the influence of mechanical forces. At the same time, with a change in conditions, including the deforming force (shear stress), flow rate (shear rate gradient), physicochemical nature of substances, temperature, degree of homogenization and other variable factors, the viscosity of the gels also changes within a fairly wide range. Table 2 shows the dependence of the viscosity of the studied gels on temperature changes.

Table 2: Change in viscosity of the gel base depending on temperature

Speed, rpm	Viscosity, *mPas**	Temperature, °C	Spindle
0,5	138080	20°C	29
1	130160
2	80470
5	52123
10	37766
20	22717
50	12591
100	7620
0,5	137950	30°C
1	97745
2	67016
5	46804
10	33014
20	19389
50	10771
100	6543,3
0,5	134780	40°C
1	86745
2	54860
5	41894
10	24249
20	16621
50	9243,6
100	5589,9

As can be seen from the tables, the viscosity values directly depend on the temperature and the speed of the cylinder. With an increase in temperature in the range from 20°C to 40°C, a significant change in viscosity towards a decrease is observed (sample 1).

To assess the consistency of a soft dosage form, rheograms of its flowability are constructed in the range of shear rates from 0.1 to 25 s–1 at 20^oC (the expected storage temperature of the gel). The specified parameters reflect the ability of the composition to fill tubes during packaging, squeezing out of tubes and other technological properties of the soft dosage form.

The obtained values for the sample of gels based on essential oil were used to plot graphs of the dependence of average values of shear stress and viscosity on the gradient of the applied velocity (Fig. 2, 3), according to which the type of flow of the system and the presence of thixotropic properties were judged.

Figure 2. Rheogram of the viscosity curve of the gel based on Na-CMC with essential oil

Figure 3. Rheograms of the flow of gel based on Na-CMC with essential oil

As can be seen from Fig. 2, there is an inversely proportional dependence of the effective viscosity values on the shear rate values in the entire velocity range. The flow curve indicates that the "ascending" curve characterizing the destruction of the system differs from the "descending" curve characterizing the restoration of the system. This arrangement of the curves is explained by the preservation of residual deformation after strong weakening of the structure under the influence of previously applied stress. To study the thixotropic properties of the studied samples of the base and gel, we plotted curves obtained as a result of deformation of these samples in the coordinates "shear rate - shear stress" (Fig. 3). Such behavior of a thixotropic system is usually called hysteresis, and the rheogram reflecting these processes is called a "hysteresis loop" - a graphical proof of the presence of the thixotropy phenomenon for the objects under study. A hysteresis loop was observed for sample no. 7, and it should be noted that the gel structure was restored with virtually no delay (the descending curve is virtually identical to the ascending curve, but does not overlap it). The obtained curves for sample no. 7 (the so-called flow rheograms) are nonlinear and are described by a line along the "ascending" curve. The "ascending" curve characterizes the destruction and restoration of the system, which retains residual deformation after a strong weakening of the structure under the influence of previously applied stress. The presence of "ascending" curves (Fig. 3) indicates that the studied sample № 7 has thixotropic properties. This means that sample No. 1 is characterized by good spreadability and the ability to be squeezed out of tubes. Similar results were obtained when studying the antimicrobial activity of the obtained ointments using the "disk-diffusion method". The results are presented in Table 3.

Table 3. Results of microbiological study of various gel compositions with essential oil of drooping dragonhead

*Sample code mm*	Staphylococcus aureus АТСС 6538	Bacillus subtilis АТСС 6633	Escheriсhia coli *ATCC 25922*	Pseudomonas aeruginosa АТСС 27853	Сandida albicans АТСС 10231
No. 1	18±0,75	19±0,75	14±1,3	9±0,75	10±0,7
No. 2	14±0,75	12±1,9	11±0,75	-	9±1,9
No. 3	9±1,9	9±1,5	10±1,3	-	10±2,2
No. 4	11±0,7	7±0,7	9±1,3	-	9±0,7
No. 5	9±0,75	7±0,75	8±0,75	-	8±0,3
No. 6	11±0,05	9±0,15	8±0,1	-	8±0,5
No. 7	20 ± 0,2	21 ± 0,3	14± 0,23	10±0,16	10± 0,3
Benzylpenicillin sodium salt	16 ± 0,1	14 ± 0,1	15 ± 0,1	12±1	-
Eucalyptus oil	-	14 ± 0,1	-	-	-
Ethyl alcohol (96%)	10 ± 0,1	10± 0,1	10 ± 0,1	10 ± 0,1	10 ± 0,1

Note: Antimicrobial activity of the samples was assessed by the diameter of the growth inhibition zones of the test strains (mm). The diameter of the growth inhibition zones less than 10 mm continuous growth in the dish was assessed as the absence of antibacterial activity, 10-15 mm - weak activity, 15-20 mm - moderately expressed activity, over 20 mm - expressed ^14-18.

Conducted microbiological studies have shown that gel no. 7 with essential oil based on NaCMC exhibits moderate antimicrobial activity against strains of gram-positive bacteria Staphylococcus aureus, Bacillus subtilis and weak activity against gram-negative bacteria Escherichia coli, Pseudomonas aeruginosa and pronounced antimicrobial activity against the yeast fungus Candida albicans.

DISCUSSION:

The results of the conducted biopharmaceutical and microbiological studies indicate that of the obtained bases, the hydrophilic base with NaCMC, the composition of which is given in Table 1, is optimal for an antimicrobial ointment with essential oil of drooping dragonhead. The results of the conducted studies allowed us to recommend the proposed gel as an antimicrobial agent.

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Received on 21.10.2024 Revised on 14.02.2025

Accepted on 17.04.2025 Published on 02.05.2025

Available online from May 07, 2025

Research J. Pharmacy and Technology. 2025;18(5):2343-2348.

DOI: 10.52711/0974-360X.2025.00335

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