Study of Vasodilating and Regenerative Effect of the Gel with Nettle Juice intended for Telogen Effluvium Treatment

© 2018 Fedorovska Maryana Ivanivna et al. This is an open access article distributed under the terms of the Creative Commons Attribution License -NonCommercial-ShareAlikeUnported License (http://creativecommons.org/licenses/by-nc-sa/3.0/). *Corresponding Author Maryana Fedorovska; Organization and Economics in Pharmacy and Drug Technology Department, Ivano-Frankivsk National Medical University, 124 k, Galytska Str., Ivano-Frankivsk, Ukraine, 76008. E-mail: maryana @ fedorovska.com Study of Vasodilating and Regenerative Effect of the Gel with Nettle Juice intended for Telogen Effluvium Treatment


INTRODUCTION
The prevention and treatment of Telogen effluvium (TE) remains the important task of modern medicine.TE is the most common diffuse hair loss in women of reproductive age, which is due to concomitant somatic pathology (disorders and diseases of the gastrointestinal tract and the endocrine system, infectious diseases, etc.) and/or negative exogenous factors (inappropriate nutrition, drug intoxication, stress, inappropriate hair care, etc.) (Shrivastava, 2009).Hair follicles (HF) affected by harmful factors are characterized with anagen (active growth) termination, which results in the onset of catagen (hair involution) and subsequently 2-3 months telogen (resting phase) finishing with exogen -dead hair shedding (Figure 1) (Harrison and Bergfeld, 2009).
The technological, rheological and microbiological studies were carried out, which resulted in the development of the medical cosmetic gel with nettle juice intended for TE therapy (Fedorovska et al., 2015;Fedorovska and Polovko, 2016).Purpose of the given research was to study the vasodilating effect and follicle-stimulating activity of the developed gel with nettle juice in rats under conditions of wool shedding induced by orally administered boric acid (Belenichev et al., 2008).

Preparation of nettle juice
Nettle fresh upper parts of the stems with 5-6 leaves were collected at the end of May in Ivano-Frankivsk region (Ukraine).Technological process of nettle juice preparation included the following stages: grinding of fresh raw materials, pressing, re-grinding and pressing, inactivation of enzymes in raw juice with 20 min heating in a water bath at 40-60ºС, preservation with 96% ethanol in quantity of 15% by weight, sedimentation at 2-4ºC in a refrigerator for 10 days, filtration, packaging, labeling.Obtained juice was a dark brown transparent liquid with a specific smell; density ranging from 0.994 to 1.106 g/ml; pH from 6.5 to 7.0; dry residue at least 3.5%; identification (thin layer chromatography method) and quantitative determination (spectrophotometric method): hydroxyl cinnamic acids at least 0.01%, carotenoids at least 0.002%, chlorophyll at least 0.003% (Fedorovska et al., 2015).

Preparation of gel with nettle juice
The remedy containing 15% per weight of the nettle juice in a gel base was prepared.Briefly, combine carbopol-so-dium alginate gel base was prepared firstly.Potassium sorbate was used as a carbopol neutralizer as well as a gel preservative, glycerol was used as a sodium alginate solubilizer as well as a moisturizer, purified water was used as a solvent (Fedorovska and Polovko, 2016).After weighing, the nettle juice was added in the gel base by continuous mixing at room temperature.

Experimental animals
White non-linear rats of both sexes with weight range of 200-250 g were obtained from the vivarium of Ivano-Frankivsk State Medical University.All experimental protocols were approved by the Ethical Committee of the University (Minutes No. 69/13, dated 2013.12.03).Animals were kept under standard vivarium conditions, they received food and water ad libitum.

Experimental design
All 28 rats were weighed and tagged; growth destruction of animal wool was induced with oral administration of boric acid (5 mg/kg) during 14 days, then wool was completely shaved on the back section (3 × 7 cm).After that all rats were randomly divided into four groups of seven animals each as followed: 1st group -intact animals; 2 nd group -untreated; 3 rd group -treated with a comparison drug (Alloton spray: complex tincture (1:5) from a mixture of Arctium lappa L. roots -5.6 g, Sophora japonica L. fruit -5.6 g, Acorus calamus L. rhizome -3.2 g, Urtica dioica L. leaves -2.8 g; Humbus lupulus cones -2.8 g); 4 th group -treated with the prepared gel.The developed medical cosmetic remedy and the comparison drug were applied on the shaved back of the rats at the maximum technically spreading dose (app.1.0 g of the developed gel; 3-4 pulverizations of Alloton spray) during next 14 days.

Analytical methods
According to the experiment, the wool length was daily monitored during 14 days.After that period, new wool was completely cut and weighed on scales.The percentage of dystrophic wool (presence of hairs with pointed tip) was observed and determined with help of a microscope "Delta Optical Genetic Pro" with built-in camera: lens 40/0.65 160/0.17;eyepiece WF 10×/18).Additionally, from each group one animal was randomly selected and subjected to euthanasia with compliance of the bioethics; skin biopsy was applied to the chosen animals.The vascular net in different layers of the skin, follicular density (number of follicles per unit area) and hair growth phase (anagen) were estimated.
The study of the vascular net was carried out by the injection method using an ether-chloroform mixture of Paris blue (10 g of substance per 100 ml of a solvent consisting of ether and chloroform in a ratio of 3:1).Fuchseline-picrofuchsine was used for histological examination of the vascular wall, the study of skin structures was carried out with the hematoxylin and eosin staining.The number of hair follicles was determined by morphometric analysis.All histological experiments were performed at the Human Anatomy Department of Ivano-Frankivsk State Medical University according to standard techniques (Bagrij, 2016;Titford, 2009;Kiernan, 2015).The electronic microscopic photographs were made with an electron microscope PEM-125 K at magnifications of 8000-10000 and an accelerating voltage of 75 kV with subsequent photographing; microscopic photo-graphs were made with the microscope "Delta Optical Genetic Pro".

Statistical analysis
The data of external estimations (given in Tables 1-2) were analyzed as the mean ± SEM for seven animals in each group.The data of histological studies (vascular lumen diameter, the number of hair follicles per 1 mm 2 ) were studied as the mean ± SEM for ten skin samples of each selected animal.Statistical analyses were performed using one-way analysis of variance (ANOVA).Differences were considered to be statistically significant when P was < 0.05.All statistical analyses were performed with MS Excel.

RESULTS AND DISCUSSION
The results of the developed gel influence on the growth stimulating activity of the rats' wool are presented in Tables 1-2.
According with the data in Table 1, the developed gel stimulated the recovery of damaged HF growth and slightly exceeded "Alloton" in terms of regenerative action.Thus, wool began to grow on the 3 rd day of the experiment in the 1 st , 3 rd and 4 th groups, but in the 2 nd group (untreated animals) -on the 4 th day.Intensive wool growth in the intact (1 st group) and the treated (3 rd and 4 th group) animals was observed on the 5-8 th days of the research.On the 14 th day of the experiment, the difference in the wool length in the experimental 4 th group was exceeded 3 mm in comparison with the untreated 2 nd group.
On the 15 th day of the experiment (Table 2) the wool weight from the shaved area of 4 th group (experimental animals) was ≈ 480 mg, which significantly exceeded the wool weight of the non-treated 2 nd group (≈371 mg).The wool weight of the healthy animals in the 1st group was slightly higher (≈500 mg), and in the 3 rd group (rats treated with "Alloton") at approximately the same level (≈472).The experimental 4th group also showed a decrease in the percentage of dystrophic wool compared to the untreated animals.So the amount of dystrophic wool in the healthy animals was ≈ 10%, in the experimental 4th group -≈ 17%, and in the untreated animals -≈38% (Table 2).The performed studies confirmed that the external application of the developed gel caused the significant changes in both histological and ultrastructural organization of subcapillary and dermal arterial nets.However, changes in the dermal arterial network were more distinct and accompanied by enlargement of the capillary lumen and by the vascular wall thickening (Figure 2).The widest vascular lumen (diameter = 76.45 ± 0.42 μm) was in the skin samples of the 4 th group after developed gel application (Figure 2 d).The vessels in the skin samples of 1 st group had a diameter of 69.85 ± 0.25 μm (Figure2 a).The skin of animals treated with "Alloton" was characterized by a less distinct vascular pattern (Figure 2 c).
Near the dermal vessels of the 4 th experimental group the number of mast cells significantly increased, which were in a state of strong degranulation (Figure 3).Particularly strongly degranulated mast cells were seen in the reticular layer of the dermis.On the ultrastructure level it was observed a significant decrease of the mast cells size and granules that went beyond their edges (Figure 3 b).High degranulation of mast cells and their functional activity is a factor of changes in blood vessels, which indicate the intensification of blood circulation in the skin.
When applying the gel with nettle juice, a significant increase in the number of new hair follicles was noted (Figure 4).
The number of follicles in the stage of anagen in skin samples of 4 th group was 93% (compared with: 1 st group -87%; 2 nd group -74%; 3 rd group -88%).Also, the intensification of the HF epithelial cells proliferative activity was observed (Figure 5).HF density per unit area (1 mm 2 ) in the skin samples was determined with the morphometric analysis.The HF number in the 1 st group was 36.34 ± 0.14, in the 2 nd group -21.98 ± 0.23, in the 3 rd group -34.56 ± 0.21, in the 4 th group -37.23 ± 0.19.As can be seen from the study results, the application of the developed gel in the experimental animals strongly stimulated HF genesis compared to the untreated rats.The samples of rats' skin tissues from each group are represented in Figure 6 for visual comparison of the HF quantity.

CONCLUSION
Thus, conducted pharmacological and histological studies indicated that the developed gel with nettle juice in comparison with the untreated animals accelerated the wool growth renovation in rats, improved its quality by increasing the weight and reducing the percentage of dystrophic hairs.
It was proved that cutaneous application of the gel had biological effect that significantly dilated the vessels in the reticular layer of the dermis.The intensification of HF blood supply was confirmed by an increase in the number of mast cells and their active degranulation.Strengthening of blood circulation caused the increase of the epithelial cells growth and HF number in the active phase (anagen).

Fig. 2 :
Fig. 2: Dermal arterial net of white rats' skin samples: a -1 st group; b -2 nd group; c -3 rd group; d -4 th group.Vessels injection with Paris blue; an increase in 400.

Fig. 4 :
Fig.4: A significant amount of hair follicles in the skin of the white rat after the application of gel with nettle juice: 1 -epidermis, 2 -dermis, 3 -hair follicles.Coloring with hematoxylin and eosin; microscopic photograph; an increase in 100.

Fig. 5 :
Fig. 5: The structure of the outer and inner epithelial sheath of the white rat HF (1 -root, 2 -internal epithelial sheath, 3 -external epithelial sheath): a -1 st group; b -4 th group.Coloring with hematoxylin and eosin; microscopic photographs; an increase in 400.

Fig. 6 :
Fig. 6: Hair follicles in the skin of white rats: a -1 st group; b -2 nd group; c -3 rd group; d -4 th group.Coloring with hematoxylin and eosin; microscopic photographs; an increase in 200.

Table 1 :
The influence of the developed gel with nettle juice on the experimental animal wool growth (mm).

Table 2 :
Influence of the developed gel on the quality and structure of the rat wool.