INTRODUCTION:

Sapropels are natural organic and mineral formations, deposits of freshwater reservoirs, which are formed from dead plant and animal organisms, mineral substances of biochemical and chemical origin and mineral components [1].

Sapropels are used to treat chronic diseases of the musculoskeletal system, the peripheral and central nervous system, diseases of the genital organs, the gastrointestinal tract, ophthalmic, dermatological and other diseases [2,3].

According the literature and own researchers, sapropels contain macro- and micro elements, vitamins, amino acids [4], fatty acids, organic and carboxylic acids [5], humic substances [6], enzymes, sterols, hormonal substances, carotene, chlorophyl and other biologically active substances [7,8].

The composition characteristics and bactericidal properties are due to the sapropel formation, the absence of pathogenic microflora, toxic substances and agents of carcinogenic activity [9], which ensures a rapid discontinuation of inflammatory processes and a good treatment of eczema, dermatitis, burns, etc. Sapropels are an important source of products with a wide range of therapeutic effects.

A wide spectrum of pharmacological activity of sapropel is related to humic substances (HS) [1].

Humic substances (HS) are a group of macromolecular compounds that are non-living organisms, but are products of plant transformation, animal and microbial residues in natural conditions [10].

It was set out that products with HS possess a wide range of biological properties that are widely used in veterinary medicine and human medicine. In particular, preparations containing HS affect the nonspecific and specific resistance of organism, have antioxidant, anti-inflammatory, antiviral, antibacterial, antifungal, membrane-protective and hepatoprotective properties, have the ability to enhance the activity of metabolic processes in the body and normalize the leukocyte blood formula in leukemia [3,11-13].

Nevertheless, our understanding of the biologic effects of HS with regard to their antiviral activity, interactions with environmentally harmful substances has been considerably extended during the last decades [3].

Amosova et al., in evaluating the biologic activity of humic acids (HA) from Tambukan therapeutic mud in animals, found HA (10mg/kg) to suppress both phases of the inflammatory process: the exudation (by 44%) and the proliferation process (by 50-55%) [14].

The inflammatory effect of HS has been supported by a plausible biochemical explanation.

As demonstrated by Schewe et al., naturally occurring HA, and even more synthetic HA-like polymers, inhibit the lipoxygenase pathway of the arachidonic acid cascade [15].

The findings of studies of Dunkelberg et al., Klocking et.al., may be indicating for membrane-protective activities of HA type substances [16,17].

In referring to the function of HS as electron donor-acceptor system, Jurcsik I. discussed the behavior of HS as the consequence of a “buffering effect”: this means that HA are able to produce as well as to bind activated oxygen species. This regulatory system is assumed to be important for the favorable influence of HS on wound healing and killing of cancer cells [18].

The experimental research has shown the presence of significant amounts of carboxylic acids, amino acids, micro- and macroelements, humic acids in the sapropel extracted from Prybych Lake [4-6,19].

Anti-inflammatory and reparative activity of oil and alcohol extracts obtained from the sapropel of Prybych deposit, located at Volyn region, were verified [20,21].

The technology of the aqueous sapropel extract production using cavitation method provides better extraction of HS, since their content exceeds 20% [6].

The composition of the gel containing aqueous extract of sapropel, carbopol Ultrez 10, potassium sorbate and purified water has been developed.

An important phase in the development of pharmaceutical products is the study of the pharmacological activity of the product and the substantiation of the active substance concentration in its composition, therefore the goal of the study was to determine the presence and expressiveness of photoprotective activity of products with different content of sapropel extract in the model of acute photodynamic skin inflammation in rats.

MATERIALS AND METHODS:

Materials:

The objects of the study were the gels samples, based on carbopol "Ultrez 10", with the 10 and 15% concentration aqueous sapropel extract and the “Panthenol” ointment, “Hemofarm AD”, Serbia, which was used as a medicinal product of comparison.

The sapropel extract was obtained from the sapropel of Prybych deposit, located at Volyn region, Ukraine [22]. The sapropel was treated with 0.1 N alkali solution. Cavitation was used at a temperature of (50 – 60)° C and a speed of 3000 rot/min for 60 minutes for obtaining a homogeneous mixture. The obtained extract was evaporated to 1:10 of basal volume [6,23,24].

The gels with aqueous sapropel extract were prepared according the following technology: potassium sorbate (preservative) was dissolved in purified water, adding the pre-weighed carbomer of the Ultrez 10 brand and left to swell for 30-60 minutes. During the swelling periodically the mixer was switched on and mixed at a speed of 60-90 rot/min. Gradually the sapropel extract was added and homogenized at a speed of 60-90 rot/min during 5-10 minutes until obtaining a homogeneous gel.

The product of comparison, "Pantenol" ointment produced by “Hemofarm AD” in Serbia, contains such an active pharmaceutical ingredient as dexpanthenol in 50mg/g concentration. According the manufacturer's annotation, it is used for a rapid healing of skin and mucous membranes of various origin lesions: scratches, thermal and sun burns, aseptic postoperative wounds, bullous and blister dermatitis, skin grafts.

Methods:

Animals studies:

Researches on animals were carried out taking into account the "General Ethical Principles of Animal Experiments" (Ukraine, 2001), Law of Ukraine "On the Protection of Animals from Cruel Treatment" from February 21, 2006, which was confirmed by the protocol of the Bioethical Examination Commission (No. 65 dated 8.11. 2017), the Order of the Ministry of Education and Science, Youth and Sports of Ukraine No 249 dated March 1, 2012 on the "The order of conducting animal investigations, animal experiments by scientific institutions" and in accordance with the provisions of the Directive of the European Parliament and of the Council from 22 September 2010 on the protection of animals used for scientific purposes 2010/63/EU [25-28]. The Commission on Bioethics of the National Pharmaceutical University (NPhU) (protocol No.6 from 20.06.2019) violations of moral and ethical norms during the research work were not found out.

In preclinical studies were used experimental animals reared in the vivarium of the central research laboratory of the NPhU, Kharkiv (certified by the Ministry of Health of Ukraine (MHU), the certificate No. 58/15 dated 12.08.2015, valid until 07.12.2019), which was equipped in accordance with current sanitary and hygienic standards. Animal researches were conducted on the basis of the problem laboratory of morphofunctional researches at the NPhU, Kharkiv (Accreditation Certificate of the National Accreditation Agency of Ukraine No. 2Н1422 dated 07.09.2017, valid until 06.09.2022).

Experimental animals were kept in standard sanitary conditions: during the experiment the animals were in the vivarium at a temperature of (19-24)°С, at humidity no more than 50%, in a natural light regime “day-night”, in plastic cages, and in a balanced diet [29]. Before the experiment, the animals were acclimatized in the room for testing within 7 days.

The studies were conducted on clinically healthy white, outbred rats aged 3-4 months (females) body weight (b.w.) 180-220 g, which were kept in standard vivarium conditions, in a standard diet. [29,30].

Experimental animals were divided into four groups, 6 animals in each (n = 6):

1. Control pathology (CP) - untreated animals with ultraviolet erythema;

2. Comparison group - animals with ultraviolet erythema treated with the comparison product - ointment "Panthenol" ("Hemofarm AD", Serbia).

3. Animals with ultraviolet erythema treated with the medicinal product - sample № 1 (10% gel with sapropel extract;

4. Animals with ultraviolet erythema treated with the medicinal product - sample № 2 (15% gel with sapropel extract).

Method of ultraviolet (UV) erythema of skin inflammation in rats:

Investigation of anti-inflammatory activity of sapropel extract gel samples was conducted on the model of UV erythema in rats. [30].

The acute photodynamic skin injury in rats was caused by the irradiator "Promin" ZEMI" with a mercury-quartz lamp of the DRT 125-1 type (the range of ultraviolet radiation was 230-400nm).

The day before the experiment, the animal wool was removed on the left side. Before irradiation, the animals were narcoded with barbamyl (0.8ml of an 1% aqueous solution of barbamyl per 100g of animal mass), the depilated skin area was covered with a wide plaster with an opening of 1 сm². The irradiator was located at a distance of 10cm from the animal, the exposure time was 60 seconds.

Sapropel extract gels were applied to the skin surface at a conditional therapeutic dose of 25 mg/сm²immediately after irradiation, and then once daily from day the 1^st to the 7^th day of the experiment.

The anti-inflammatory activity of the studied products was evaluated for 7 days by the skinfold thickness, which was measured with calipers in mm, and the condition of the skin.

The degree of the experimental animals skin inflammation was evaluated in points: 0 - the absence of erythema; 1 - weak erythema (pink tone), 2 - moderately expressed erythema (pinkish-red tone), fine skin peeling; 3 - expressed erythema (red tone), large-scale skin peeling, spot hemorrhage; 4 - dramatically expressed erythema (bright red tone), focal necrosis and hemorrhages [31].

Anti-inflammatory activity of medicinal products (AIA, %) was calculated by the formula:

where

ІI_EG - the intensity of inflammatory reaction on the animal skin of the experimental groups (II, III IV), points.

ІI_CP – the intensity of inflammatory reaction on the animal skin of the control pathology group (CP), points.

All experimental animals were cured for a different time period.

Statistical analysis:

The statistical analysis of the obtained results was carried out by the variation statistics method using Student's t-criterion and using the personal computer and Microsoft Excel software with the definition of indices of the arithmetic means (M), the arithmetic means errors(m); the probable difference between the comparison groups. The difference between the arithmetic mean and arithmetic means error was considered statistically significant at: * p <0.05 [32].

RESULTS AND DISCUSSION:

Inflammatory processes are one of the most important problems in general pathology and the clinic [8,9], as they accompany the development of most pathologies of various etiologies, including wounds and burns. Since the evaluation of anti-inflammatory activity of new medicinal products is one of the main objectives of pharmacological research, we investigated the anti-inflammatory effect of sapropel extract gel using the model of acute photodynamic skin inflammation in rats (UV erythema). The ultraviolet (UV) erythema test is one of the most frequently used methods to investigate the anti-inflammatory potency of topical dermatological preparations in vivo [33].

The results of the inflammatory process intensity on the skin, the anti-inflammatory activity of the research products, the skinfold thickness of rats are presented in Tables 1-3 and Figure 1.

А – UV erythema of the I group’s rat (CP), B – UV erythema of the IV group’s rat

Fig. 1. Photodynamic skin inflammation in rats on the 4^thday after UV-irradiation

According the obtained data, the macroscopic signs of photodynamic skin inflammation developed gradually and in the control pathology group (CP) reached the maximum intensity on the 2^nd-3^rd day after irradiation (Table 1). Inflammation of the skin was characterized by swelling tissues, hyperemia, hemorrhages, and the development of hemorrhagic crusts with ulcers. Complete restoration of the skin, in accordance with the indicators of the inflammatory reaction intensity and the skinfold thickness, in the CP group was observed on the 14^th day after irradiation.

On the background of sapropel extract gel treatment on the 2-7^th days of the experiment, there was a significant decrease in the intensity of photodynamic skin inflammation in experimental animals, which was characterized by a decrease in edema and hyperemia, and a decrease in the skinfold thickness in the relation to the CP group (Table 2).

Table 1: Intensity of inflammatory reaction on the skin of rats on the model of acute photodynamic inflammation, points ( M±m, n=6)

Group	Observation time, days
Group	1	2	3	4	6	7
I	1.00±0.00	3.50±0.22	3.50±0.22	3.33±0.21	3.00±0.00	0.83±0.17
II	0.50±0.22*	2.67±0.21*	2.50±0.22*	1.83±0.17*	1.50±0.22*	1.33±0.21*
III	0.67±0.21	1.50±0.22*/#	1.17±0.17*/#	1.00±0.00*/#	0.33±0.21*/#	0.00±0.00*/#
IV	0.33±0.21*	1.00±0.00*/#	0.83±0.17*/#	0.67±0.21*/#	0.00±0.00*/#	0.00±0.00*/#

Notes:

* – the difference is statistically significant with respect to the values of group I (control pathology), р<0.05;

# – the difference is statistically significant with respect to the values of group II (comparison group), p<0.05.

Таble 2: The thickness of the skin fold in rats on the model of acute photodynamic skin inflammation, mm, (M ± m, n=6)

Group	Observation time, days
Group	Initial data	1	2	3	7	14
I	2.07±0.06	2.27±0.06	2.75±0.09	2.79±0.08	2.57±0.09	2.20±0.06
II	2.05±0.06	2.15±0.05	2.47±0.06*	2.43±0.09*	2.23±0.06*	2.12±0.06
III	2.03±0.06	2.17±0.04	2.37±0.08*	2.28±0.07*	2.06±0.04*#	2.08±0.05
IV	2.02±0.07	2.08±0.10	2.22±0.07*#	2.12±0.07*#	2.03±0.05*#	2.04±0.05

Notes:

* – the difference is statistically significant with respect to the values of group I (control pathology), р<0.05;

# – the difference is statistically significant with respect to the values of group II (comparison group), p<0.05.

Table 3: Anti-inflammatory activity of the studied products on the model of acute photodynamic inflammation of the skin in rats, % (n=6)

Group	Observation time, days
Group	1	2	3	4	6	7
II	50.00*	23.81*	28.57*	45.00*	50.00*	52.94*
III	33.33	57.14*	66.67*	70.00*	88.89*	100.00*
IV	66.67*	71.43*	76.19*	80.00*	100.00*	100.00*

Note:

* – the difference is statistically significant with respect to the values of group I (CP), р<0.05.

So, on the 2^nd day after ultraviolet irradiation in groups III (10% sapropel extract gel) and IV (15% sapropel extract gel), the decrease in the intensity of skin injury was observed in comparison with CP to 57.14% and 71.43%, respectively. In this case, the anti-inflammatory activity of gels in groups III and IV was by 47.62% and 33.33% (p <0.05) higher than in the comparison product - “Panthenol” ointment (Gr. II) (Table 3).

The decrease in the intensity of the inflammatory reaction, which we observed in groups III and IV, was accompanied by a significant decrease in the skinfold thickness in the relation to the CP group by 13.8% and 19.3% respectively (Table 2).

On the 3^rd day after ultraviolet irradiation, the anti-inflammatory activity of sapropel extract medicinal product in groups III and IV was 66.67% and 76.19% respectively, which is by 38.10% and 47.62% (p <0.05) respectively higher than in the comparison product (Table 3). The decrease in the intensity of the inflammatory response in groups III and IV was accompanied by a further decrease in the skinfold thickness in relation to the CP group by 18.0% and 24.0%, respectively (Table 2).

On the 6^th day after UV irradiation, complete healing of erythema UV in group IV (Table 1) was observed. In group III the anti-inflammatory activity of sapropel extract medicinal products was 88.89% (Table 3). On the 7^th day after UV irradiation, complete recovery of the skin in group III (Table 1) was recorded.

It should be noted that on the 6^thand 7^th days of the experiment, according to the indices of anti-inflammatory activity and thickness of the skin, the reference product (group II) was significantly weaker compared to the groups III and IV by 38.9 - 50.0% (Table 3).

The choice of dosage form affects the effectiveness of their anti-inflammatory action. However, sapropel extract gels have expressed major anti-inflammatory activity than creams [34].

CONCLUSIONS:

Medicinal products with the sapropel extract have expressed anti-inflammatory properties, allow to reduce the intensity of acute photodynamic inflammation and shorten the duration of its treatment.

Comparative analysis of medicinal products with sapropel extract and the referent product - "Panthenol" ointment has shown that sapropel extract gels, much faster increased the reduction of skin signs with photodynamic inflammation: the intensity of skin lesions on the 1^st-7^th day of treatment was less expressed in the group of animals treated with the "Panthenol" ointment (in 1.3-3.0 times, p <0.05).

ACKNOWLEDGMENTS:

We are thankful to “Zander–Ukraine” LTD for providing free samples of sapropel.

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Received on 03.09.2019 Modified on 10.10.2019

Research J. Pharm. and Tech 2020; 13(9):4381-4386.

DOI: 10.5958/0974-360X.2020.00774.X