Резюме
Цулун О.В., Деримедвідь Л.В., Зеленін Ю.В. Дослідження гострої токсичності комбінованих мазей природного походження.
Проведено дослідження гострої токсичності на щурах нових мазей природного походження: «СКД» (містить екстракт солодки, коензим Q10, дигідрокверцетин) та «Дерматроп» (містить календулу та ромашку), які було розроблено в НФаУ. Встановлено, що при нашкірному, пероральному та ректальному застосуванні мазей «СКД» та «Дерматроп» у максимальних дозах були відсутні будь-які негативні прояви їх токсичної та подразнюючої дії. За класифікацією К.К. Сидорова мазь СКД та мазь «Дерматроп» відносяться до VI класу токсичності – «відносно нешкідливі» препарати. Отримані результати свідчать про доцільність подальших поглиблених досліджень мазей СКД та «Дерматроп» у зв’язку з їх нешкідливістю та відсутністю місцевоподразнюючої та гематотоксичної дії.
Ключові слова: гостра токсичність, мазь, дерматотоксичність, щури.

Резюме
Цулун А.В., Деримедведь Л.В., Зеленин Ю.В. Исследование острой токсичности комбинированных мазей природного происхождения.
Проведено исследование острой токсичности на крысах новых мазей природного происхождения: «СКД» (содержит экстракт солодки, коэнзим Q10, дигидрокверцетин) и « Дерматроп » (содержит календулу и ромашку ), которые были разработаны в НФаУ. Установлено, что при наружном, пероральном и ректальном применении в максимальных дозах мазей «СКД» и «Дерматроп» отсутствовали какие-либо негативные проявления их токсического и раздражающего действия. По классификации К.К. Сидорова мазь «СКД» и мазь «Дерматроп» относятся к VI классу токсичности – «относительно безвредные» препараты. Полученные результаты свидетельствуют о целесообразности дальнейших углубленных исследований мазей «СКД» и « Дерматроп » в связи с их безвредностью и отсутствием местнораздражающего и гематотоксического действия.
Ключевые слова: острая токсичность, мазь, дерматотоксичность, крысы.

Summary
tsulun o.v., Derymedvid l.v., zelenin y.v. Acute toxicity study of natural origin combined.
The aim of the study was to investigate the acute toxicity of new natural origin ointments “LCD” (contains licorice extract , coenzyme Q10, Dihydroquercetin ) and «Dermatrop»(contains calendula and chamomile ),, that were created at the NUPh. After cutaneous, oral and rectal administration of ointments “LCD” and “Dermatrop” at maximum dose there were no signs of their toxic or irritative effect. Ointments “LCD” and “Dermatrop” according to the classification of Sidorov K.K. belong to toxicity class VI – “relatively safe” drugs. Received data suggest expediency of further advanced study of ointments LCD and “Dermatrop” due to their safety. Therefore, received data suggest expediency of further advanced study of ointments “LCD” and “Dermatrop” due to their safety and absence of local irritative and hematotoxic effects.
Key words : acute toxicity, ointment, dermatotoxicity, rats.

Рецензент: д.мед.н., проф. С.Ю. Штриголь

УДК 615.322:615.262.1:615.454.1:577.127.4

Національний фармацевтичний університет (Харків)

Национальный фармацевтический университет (Харьков)

National University of Pharmacy (Kharkov)

elena_culun@mail.ru, derimedved@mail.ru

Харківська медична академія післядипломної освіти

Харьковская медицинская академия последипломного образования

Kharkov medical academy of Postgraduate Education

Skin is one of the most important organs, which participates in maintenance of persons’ homeostasis in living environment. This organ is a unique multifunctional formation and with the help of sensor and protective mechanisms provides contact between person and external environment, reflects internal organs, endocrine and nervous system functional stage. Skin also has such important functions as barrier, thermoregulatory, secretory and receptor. At least one of these functions violation can lead to different diseases of skin, including dermatitis and dermatosis [1].

According to statistical data, more than 50% of skin diseases in Ukraine are chronical dermatitis (eczema, neurodermatitis, atopic dermatitis). Quantity of inflammatory skin disorders has also increased, especially among people of working age, which emphasizes important medical and social value of the need to prevent and treat this group of diseases [1].

For local treatment of dermatological diseases soft medical forms such as ointments are widely used. This medical form allows to include different in origin and properties hydrophilic and hydrophobic biologically active substances, and due ointment bases regulate the release of biologically active substances, which ultimately may improve the efficacy and safety of drugs.

Today, the pharmaceutical market represents a wide range of drugs for local treatment of dermatitis, wounds, ulcers, etc., 56% of these drugs are imported and only 43% are produced by domestic companies [1]. And yet, despite the significant amount of drugs used for treatment of these diseases, their efficacy and pharmaceutical properties do not fully meet modern requirements. To a large extent it is caused by the fact that most of these ointments incorporate one active substance that can not positively affect all or most of the links of the pathological process.

            Lately, great attention is paid to products containing active substances of plant origin, which are the carriers of complex biologically active compounds have low toxicity, allowing their continued use without severe adverse changes and exert anti-inflammatory and anti-allergic effect.

            The aim of the study was to investigate the acute toxicity of new natural origin ointments "LCD" and "Dermatrop", that were created at the NUPh. First ointment so called "LCD", which was created at the Department of Industrial Technology of Drugs under the supervision of prof. Ruban O.A., contains as active ingredients Coenzyme Q10, Dihydroquercetin (DHA) and licorice extract (Glycyrrhiza glabra L.). The formula and technology were developed and ointment "Dermatrop" containing calendula (Calendula officinalis) and chamomile (Matricaria chamomilla) was prepared at the Department of Drug Technology and Clinical Pharmacy with Pharmaceutical Care of Medical Academy of Postgraduate Education NUPh under the supervision of prof. Piminov O.F.

Materials and methods.

Experiments were performed on animals grown in vivarium CSRL of NUPh in accordance with relevant requirements of sanitary standards on a standard diet and received food and water ad libitum [4, 5, 7, 8]. All experiments were performed according to the procedures and requirements of the Ukrainian State Pharmacological Center of MoH and the Committee on Bioethics [4]. Animals were kept in standard sanitary conditions. During the experiment, animals were kept in the room for testing at t˚ = 20-25⁰С, humidity less than 55%, with natural "day-night" light in standard cages on a diet from granulated feeds [2]. Animals were treated according to the rules of the “European Convention for protection of vertebrate animals used for experimental and scientific purposes” (Strasbourg, 1986).

In the study of drugs acute toxicity doses were used according to the higher doses for toxicity class VI according to the classification of substances for toxicity, recommended by Ukrainian State Pharmacological Center of MoH [4]. Before the introduction of experimental drugs rats starved for 12 hours. Access of animals to water was free, to food was allowed only 6 hours after applying of ointment (first administration). To identify possible toxic effects of ointments comparison with indexes of intact animals group that were in similar conditions of observation was conducted (intact control).

Acute toxicity of ointment was studied on white nonlinear rats weighing 180-200 g. All experimental groups consisted of 12 animals - 6 of each sex.

To study the acute toxicity after cutaneous application, ointments were applied to the prepared (shaved 24 hours before applying preparations) area of skin at the back and sides of the rat size 8´6 cm, which is more than 15% of the total area of animals skin. Studied dose of ointments was 22590 mg/kg (~85 mg/cm²). Taking into account impossibility of a single application of ointments in the specified dose, application was conducted three times at intervals of 1 hour in equal doses - 28 mg/cm² per application (dose calculated based on the average body weight [4, 9] and the application area of drug).

Also conducted a study of acute toxicity of these ointments with oral administration (at a rate of 5 g/kg) and rectal administration [4, 7, 10].

At rectal administration experimental animals were treated with the single 3000 mg/kg dose of ointment. Administration was conducted in the morning on an empty stomach after reflex defecation. The test sample was injected into the rectum very slowly (60-90 s) with a special applicator. Then the rectal hole of the rat was screened for 30 min. with a special tap.

Statistical analysis of obtained results was conducted by methods of variation statistics using standard software packages Excel on a PC Pentium III. When comparing the results between the two groups used a modified parametric t- - criterion of Student for samples with unequal variances [3, 6, 8].

The results of acute toxicity study of ointments "LCD" and "Dermatrop" for two routes of administration are presented in Tables.

Table 1

Survival rate of rats when administered "LCD" and "Dermatrop" at high doses

After cutaneous, rectal and oral administration of ointments "LCD" and "Dermatrop" at maximum dose (22590 mg/kg and 3000 mg/kg and 5000 mg/kg, respectively) signs of toxicity in animals were not observed: animals were tidy, active, had normal appetite , respond to sound and light stimuli, processes of urination and defecation were normal, respiratory failure and seizures were not observed. Reflex excitability in all animals was kept.

According to the methodology of acute toxicity studying for the evaluation of toxic effects of ointments on animals the study of body weight dynamics (figure) was conducted.

The next stage was to investigate the local irritative effect of ointments “LCD” and “Dermatrop”. Local irritative effect on skin was examined in rats after cutaneous application during one month. 0.5 g of ointment per animal was applied on shaved lateral area of skin 2×2 cm, which is 10% of the animals’ skin. Control animals were treated similarly with ointment basis. During experiment state of the skin and behavior of the animals were observed. Table 2 shows results of potential dermatotoxicity evaluation of ointments “LCD” and “Dermatrop”.

According to Table 2, after prolonged usage of preparations the skinfold thickness changes remained within the control group of animals. Results have showen that during the whole period of experimental groups observation there were no signs of irritation, redness, peeling, rash, depigmentation. Regenerating hair coat had a thick undercoat, evenly distributed throughout the whole area. Behavior of the animals during the whole experiment was as usual.  

Data characterizing the state of rats peripheral blood indicate that one month application of experimental preparations had no effect on studied hematological parameters in rats and did not change the leukocyte formula (Table 3).

Table 2

Effect of ointments “LCD” and “Dermatrop” on skinfold thickness in rats

 (M ±m, n=6)

Note: n – number of animals in group.

Table 3

Hemogram profile in rats after 1 month application of ointments “LCD” and “Dermatrop” (M ±m, n=6)

Notes: ¹ – р≤0.05 compared to baseline; n– number of animals in group.

Insignificant changes in hemoglobin concentration, erythrocytes and platelets counts, which are observed during the experiment (Table 3) were within the physiological norm for this type of animal.  Therefore, received data suggest expediency of further advanced study of ointments “LCD” and “Dermatrop” due to their safety and absence of local irritative and hematotoxic effects.

It has been established, that at cutaneous, oral and rectal application of ointments "LCD" and "Dermatrop" and in the negative control group the body weight of animals increased evenly. Overall, results indicate that ointments "LCD" and "Dermatrop" have no toxic properties, which could dramatically affect the overall trophic processes of mammal organism.

The absence of lethal toxicity effects of ointments "LCD" and "Dermatrop" when applied in these doses allows to classify according to the classification of Sidorov K.K. as recommended by Ukrainian State Pharmacological Center of MoH as "relatively safe" drugs at cutaneous application, after oral and rectal administration [4].

Therefore, received data suggest expediency of further advanced study of ointments LCD and “Dermatrop” due to their safety.

The next stage was to investigate the local irritative effect of ointments “LCD” and “Dermatrop”. Local irritative effect on skin was examined in rats after cutaneous application during one month. 0.5 g of ointment per animal was applied on shaved lateral area of skin 2×2 cm, which is 10% of the animals’ skin. Control animals were treated similarly with ointment basis. During experiment state of the skin and behavior of the animals were observed. Table 2 shows results of potential dermatotoxicity evaluation of ointments “LCD” and “Dermatrop”.

According to Table 2, after prolonged usage of preparations the skinfold thickness changes remained within the control group of animals. Results have showen that during the whole period of experimental groups observation there were no signs of irritation, redness, peeling, rash, depigmentation. Regenerating hair coat had a thick undercoat, evenly distributed throughout the whole area. Behavior of the animals during the whole experiment was as usual.

Data characterizing the state of rats peripheral blood indicate that one month application of experimental preparations had no effect on studied hematological parameters in rats and did not change the leukocyte formula (Table 3).

Table 2

Effect of ointments “LCD” and “Dermatrop” on skinfold thickness in rats

 (M ±m, n=6)

Note: n – number of animals in group.

Table 3

Hemogram profile in rats after 1 month application of ointments “LCD” and “Dermatrop” (M ±m, n=6)

Notes: ¹ – р≤0.05 compared to baseline; n– number of animals in group.

Insignificant changes in hemoglobin concentration, erythrocytes and platelets counts, which are observed during the experiment (Table 3) were within the physiological norm for this type of animal.

Therefore, received data suggest expediency of further advanced study of ointments “LCD” and “Dermatrop” due to their safety and absence of local irritative and hematotoxic effects.

Conclusions:

1. After cutaneous, oral and rectal administration of ointments "LCD" and "Dermatrop" at maximum dosethere were no signs of their toxic or irritative effect.

2. Ointments "LCD" and "Dermatrop" according to the classification of Sidorov K.K. belong to toxicity class VI – "relatively safe" drugs.

3. Received data suggest expediency of further advanced study of ointments LCD and “Dermatrop” due to their safety.

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