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F I N A L R E P O R T

On the topic: “Evaluation of toxicity and hormone status of combination

of extracts from native medicinal plants in male inbred

mousses strain Balb/c”

Performers: 1. Assoc. Prof. Dr. Petia Genova-Kalou, PhD

Head-Leader of Laboratory of “Rickettsiae and tissue cultures”

Department of Virology, National Centre of Infectious and Parasitic Diseases

44A “Gen. Stoletov” Blvd., 1233 Sofia, Bulgaria

E-mail: petia.d.genova@abv.bg

Cell phone: 00359 889309967

2. Assist. Prof. Stefka Ivanova PhD

Department of Virology, National Centre of Infectious and Parasitic Diseases

44A “Gen. Stoletov” Blvd., 1233 Sofia, Bulgaria

3. Assoc. Prof. Andrey Tchorbanov PhD

Institute of Microbiology, Bulgarian Academy of Sciences

“Geogri Bontchev” Str., bl. 25, Sofia, Bulgaria

4. Adelina Pavlova – biologist

Department of Virology, National Centre of Infectious and Parasitic Diseases

44A “Gen. Stoletov” Blvd., 1233 Sofia, Bulgaria

5. Katia Georgieva-Dimitrova – lab technician

Department of Virology, National Centre of Infectious and Parasitic Diseases

44A “Gen. Stoletov” Blvd., 1233 Sofia, Bulgaria

Introduction

Sexual dysfunction includes erectile dysfunction or impotence, ejaculation

dysfunction, hypogonadism, etc. (Ho C. et al., 2011). It is a serious public health problem

among young as well as old men worldwide, with a prevalence of more than 20% (Laumann

E. et al., 2005). Current sexual dysfunction therapy lack satisfactory success due to adverse

effect, hence patients are seeking complementary and alternative medicine to treat sexual

dysfunction.

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Ayurveda and other Indian literature mention the use of plants in various human

ailments. Researchers conducted in the last few decades on the plants mentioned in ancient

literature or used traditionally for sexual dysfunction. To achieve better sexual desire has led

to the development and use of different substances known as aphrodisiacs (Fugl-Meyer K. &

Fugl-Meyer A, 2000).

Nowadays, herbal medicines are widely used due to various reasons such as

appropriate and sustainable effectiveness, low side effects if used at appropriate doses, low

cost, easy access, etc. (Singh R. et al., 2013).

In our experiments we used four combinations of crude extracts from the following

medical plants: 1) Combination I - Epimedium grandiflorum (10% ikarine) (100 mg); Panax

ginseng extract from root (7 mg); L-arginine (250 mg); Zn (Zinc gluconate) (5 mg); Vitamin

C (32 mg); 2) Combination II – Pygeum africana (100 mg); Panax ginseng extract from root

(5 mg); L-arginine (250 mg); Zn (Zinc gluconate) (7 mg); Niacin (5 mg); Vitamin B6 (3 mg);

Vitamin B12 (10µg); Pantothenic acid (15 mg); 3) Combination III - Pygeum africana (100

mg); Epimedium grandiflorum (10% ikarine) (100 mg); Zn (Zinc gluconate) (7 mg); L-

arginine (180 mg); Vitamine B6 (3 mg); Vitamin B12 (10µg) and 4) Combination IV -

Tribulus terrestris (60% saponines) (320 mg); Niacin (5 mg); Pantothenic acid (20 mg);

Vitamin B6 (3 mg); Vitamin B12 (10µg); Zn (Zinc gluconate) (7 mg).

All of them are well-patronized medicinal herbs by Ayurvedic seers as well as by

modern herbalists. Throughout history, many different cultures have recognized the potential

use of listed above medicinal plants for prevention and treatment of various disorders. Many

indigenous plants have been claimed to have a sex-stimulating effect. These herbs were used

individually as a single therapeutic agent or as a prime or subordinate component with

combination of many vitamins. Some of them are:

• Tribulus terrestris (belonging to family Zygophyllaceae), known as Gokshur or

Gokharu, has been used for a long time for treatment of various kinds of diseases by anti-

inflammatory, anti-diabetic, hypolipidemic, cardiotonic, hepatoprotective, analgesic, anti-

spasmodic, anti-cancer, and anti-bacterial activities (Chhatre S. et al., 2014; Samani N. et al.,

2016). Tribulus terrestris has the potential to increase hormone levels of testosterone and

enhance premature ejaculation, stimulate androgen receptors in the brain, an action that can

help the body respond positively to circulating hormones (Ghosian Moghaddam M. et al.,

2013). It is proven, that Tribulus terrestris can raise serum testosterone levels by stimulating

androgen receptors in the brain, which causes the posterior pituitary gland to secrete

additional luteinizing hormone, thus stimulating the testes to secrete more testosterone

(Saudan C. et al., 2008).

• Panax ginseng is a perennial herb native to Korea and China and has been used as

an herbal remedy in eastern Asia for thousands of years. Modern therapeutic claims refer to

vitality, immune function, cancer, cardiovascular diseases, improvement of cognitive and

physical performance and sexual function. Root of the Korean ginseng has traditionally been

used to treat various diseases, particularly as an adaptogen since it is suggested to normalize

body functions and increase physical strength (Liu C. & Xiao P., 1992). It works as an

antioxidant by enhancing nitric oxide (NO) synthesis in the endothelium of corpora cavernosa

(CC); ginsenosides also cause transmural nerve stimulation-activated relaxation associated

with increased tissue cyclic guanosine monophosphate (Coon J. & Ernst E., 2002; Choi K,

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2008). Panax ginseng enhances the libido possibly through its effects on the CNS and gonadal

tissues, as well as by increasing stamina during intercourse (Jia L. & Zhao Y., 2009).

• Epimedium grandiflorum (“horny goat weed”) have been utilized in Traditional

Chinese Medicine (TCM) for centuries to treat a variety of human illnesses. Recent

investigations into the properties of these plants have suggested that the most metabolically

active extract of Epimedium is icariin (ICA), a flavonol glycoside obtained from the aerial

part of the plant (Xin Z. et al, 2001). ICA has been demonstrated to enhance eNOS expression

and NO production in human endothelial cells as well as decrease caspase-3 expression and

cellular apoptosis in response to hydrogen peroxide (Wang Y. & Huang Z, 2005). It has

hondroprotective, anti-cancer, anti-viral activities, against cardiovascular diseases,

osteoporosis as well as play the role as aphrodisiac. Moreover, it can improve endocrine,

immune, and cognitive functions (Wu B. et al., 2012). In addition to an erectogenic role, it

has been suggested that ICA has testosterone-mimetic properties (Zhang Z. & Yang Q.,

2006). In addition, H. epimedii has been traditionally used in China to treat erectile

dysfunction (Low, W. & Tan, H, 2007).

• Pygeum africana (African cherry), which belongs to the plant family Rosaceae.

This evergreen miraculous plant is only found in sub-Saharan Africa and is highly sought

after owing to its unique anticancer phytochemicals (Ochwang'i, D. et al., 2014). The bark is

used in an attempt to treat fevers, malaria, wound dressing, arrow poison, stomach pain,

purgative, kidney disease, appetite stimulant, gonorrhoea, and insanity (Stewart K., 2003).

• L-arginine (or arginine) is a type of amino acid, and as we know, amino acids are

the “building blocks” of proteins. One of the biggest benefits of taking arginine is its ability to

improve blood flow and circulation. This has multiple benefits, including improving immune

function, fertility, detoxification and brain power. In the body, it is converted into nitric oxide,

which causes blood vessels to open wider. Another important aspect of L-arginine is that it

stimulates the production of certain hormones, especially beneficial growth hormones and

insulin that help usher glucose into cells to be used for growth and energy output (McConell

G., 2007).

• Vitamins (B6, B12, Niacin, Pantothenic acid). The cellular and tissue construction

of thyroid gland needs Vit. B complex. It energies increased hormone flow. B-complex

vitamin guard against impotence, premature ejaculation and premature menopause. Detoxifies

body and maintains healthy skin, tongue, nervous system. Have anti-depressant action

(Bijlwan A. & Kush L., 2013).

• Zinc is a naturally occurring mineral. It is important for growth and for the

development and health of body tissues. Zinc is necessary for the functioning of more than

300 different enzymes and plays a vital role in a large number of biological processes. Zinc is

a cofactor for the antioxidant enzyme superoxide dismutase (SOD) and is in a number of

enzymatic reactions involved in carbohydrate and protein metabolism. Zinc affects different

aspects of mammalian reproduction. Testicular disruption, impaired spermatogenesis and

subsequent poor semen parameters are found in males with zinc deficiency (Haffiez A. et al.,

1984). Zinc is necessary for the maturation of sperm and normal fetal development

(Dissanayake D., et al., 2009).

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• Vitamin C is the most commonly used vitamin. It is a vitamin which takes part in

many biochemical processes in organisms. It is highly soluble in water and functions as an

effective reluctant. Vitamin C has been associated with fertility for many years and may have

evolutionary significance (Millar J., 1992). Vitamin C also contributes to the support of

spermatogenesis at least in part through its capacity to reduce α-tocopherol and maintain this

antioxidant in an active state (Davies M. et al., 1991). It may play a role in mediating

testosterone levels (Fernandes G. et al., 2011).

All experiments were performed on mice model Balb/c. The mice model is an

established model to study effects of agents on humans as they are easy and flexible to handle

and manipulate (Groothuis et al., 2007). Many authors have shown that similarities exist

between mouse and human reproductive organs and cycle thus, allowing careful extrapolation

of findings to human. The reproductive cycles of both mice and humans are under the control

of the endocrine system and are responsible for reproduction. Generally sexual behavior is

enhanced by elevated testosterone levels. Substances I, II, III and IV must induced changes in

neurotransmitter levels or their actions in the cells could also increase sexual behavior

(Yakubu M. et al., 2007).

Materials and methods

Medicinal plant extracts

For the purposes of the experiment were purchased crude medicinal plant extracts in

four combination: 1) Substance I – combination of Epimedium grandiflorum (10% ikarine)

(100 mg); Panax ginseng extract from root (7 mg); L-arginine (250 mg); Zn (Zinc gluconate)

(5 mg); Vitamin C (32 mg); 2) Substance II - combination of Pygeum africana (100 mg);

Panax ginseng extract from root (5 mg); L-arginine (250 mg); Zn (Zinc gluconate) (7 mg);

Niacin (5 mg); Vitamin B6 (3 mg); Vitamin B12 (10µg); Pantothenic acid (15 mg);

3) Substance III – combination of Pygeum africana (100 mg); Epimedium grandiflorum

(10% ikarine) (100 mg); Zn (Zinc gluconate) (7 mg); L-arginine (180 mg); Vitamine B6 (3

mg); Vitamin B12 (10µg) and 4) Substance IV – combination of Tribulus terrestris (60%

saponines) (320 mg); Niacin (5 mg); Pantothenic acid (20 mg); Vitamin B6 (3 mg); Vitamin

B12 (10µg); Zn (Zinc gluconate) (7 mg).

Mice

Male mice Balb/c (20–30 g) aged 8 – 12 months, with proved fertility were used in all

experiments. The animals were bought from a local Animal Center in Sofia (Bulgaria), and

kept in the Animal House of the Department of Virology, National Centre of Infectious and

Parasitic Diseases, Sofia (Bulgaria) for the duration of the experiments. The mice were

handled according to standard guidelines for the use and care of Laboratory experimental

animals as stated by the Ethical committee, National Centre of Infectious and Parasitic

Diseases, Sofia (Bulgaria) as well as the standard guidelines for use of laboratory animals

(National Institute of Health, Bethesda, MD: Public Health Service Policy on Humane Care

and Use of Laboratory animals, 2002) and acclimatized for one weeks before the experiment

proper. Twenty five mousse were housed in a polypropylene cages under controlled

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environmental conditions of humidity and under 12h light and 12h dark conditions. The

temperature was maintained at 28 ± 1oC. They were fed with standard pellet feeds and water.

Mousses were divided in a five groups (one control and four treatment groups), containing

five animals in each group.

Group 1 - Male mouses Balb/c group – control

Group 2 – Male mouses Balb/c group – treated with Substance I

Group 3 - Male mouses Balb/c group – treated with Substance II

Group 4 - Male mouses Balb/c group – treated with Substance III

Group 5 - Male mouses Balb/c group – treated with Substance IV

Group 1 orally received distilled water (1 mL/kg) and served as the control. A single

oral dose of 400 mg/kg of the Substance I, II, III and IV were administered to Groups 2, 3, 4

and 5 respectively, by orally using intragastric tube. All the treatments were carried out for

7 days.

Evaluation of cytotoxicity of herbal combinations (Substances I, II, III and IV)

such as determined maximal non-toxic concentration (MNC) and cytotoxic

concentration 50% (CC50) in cell culture of murine origin

Cell viability was estimated by a modification of the MTT [3-(4,5-dimethylthiazol-2-

yl)-2,5-diphenyltetrazolium bromide] assay (Mosmann T., 1983). The MTT reduction assay

was one of the most frequently used methods for measuring cell proliferation and

cytotoxicity. The intensity of colour (measured spectrophotometrically) of the MTT formazan

produced by living, metabolically active cells by measuring the activity of succinate

dehydrogenase, mostly located in mitochondria was proportional to the number of live cells

present. MTT was a yellow water-soluble tetrazolium dye that was reduced by live, but not

dead, cells to a purple formazan product that was insoluble in aqueous solutions.

In the experiments have been used one murine cell line: L929 (murine fibroblasts) from

European collection of cell cultures (ECACC). Cells were routinely maintained as adherent

cell cultures in DMEM (Dulbecco's Modified Eagle's medium, Sigma-Aldrich) medium and

containing 10% FBS (Fetal bovine serum, Gibco), 2mM L-glutamine (Sigma-Aldrich), 100

U/ml penicillin G sodium (Sigma-Aldrich), 100 µg/ml streptomycin sulphate (Sigma-Aldrich)

at 37oC, in a humidified air incubator containing 5% CO2. Cultivation of the cells was

continued with direct monitoring every two or three days using a phase contrast microscope.

The cells are harvested in 1x trypsin/EDTA solution (Sigma-Aldrich). Cells were passages 1:3

– 1:5 at a density around 5 – 6 x 104 cells/ml, while the passage was resuspended repeatedly.

Cell line was plated at an appropriate density (4,5 x 104 cells/well for L929) in 96-well plates

(Costar Corning) for 24 h. When the adherent cells were stuck to the plastic, the supernatant

was decanted and adding 50 µl of previous prepared dilutions of tested herbal combinations.

The well was completed with culture medium (150 µl) to a final volume of 200 μl. For 48 h

days the plate was incubated at 37oC and 5% content of CO2. Cells grown in medium without

compounds served as a control (negative control). After 48 h incubation, the medium was

replaced with MTT (Sigma-Aldrich) and dissolved at a final concentration of 5 mg/ml in

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serum-free medium, for further 3 h incubation. Then, the MTT-formazan product was

solubilised in ethanol : DMSO (1 : 1), and the optical density was measured at a test wave

length of 540 nm in microplate reader (Bio-Tek Instruments) using wells without sample

containing cells as blanks. Each experiment was in triplicate. Cell viability was reported as

the % of viable cells in the wells treated with different concentrations of the tested compound

compared to the control untreated cells. MNC and CC50 were calculated from the constructed

“dose – cellular survival” curve.

Cytotoxic concentrations 50% (CC50) were defined as the concentrations of

the tested herbal combination at which 50% of the cells die as a result of toxicity of the

herbals.

Maximal non-toxic concentrations (MNC) were defined as the highest

concentrations of the tested herbal combination which do not cause injury or death of the

treated cells.

Acute toxicity study

To determine general short term toxicity, the animals were divided into 5 groups, each

containing 5 mice. Group I animals served as control and received distilled water in an

identical manner. The groups II, III, IV and V were given Substances I, II, III and IV (at the

respective doses - 100 mg/kg p.o., 200 mg/kg p.o. and 400 mg/kg p.o.). The animals were

observed continuously for 1 hr for any gross behavioural changes or death, if any, and

intermittently for the next 6 h and then again at 24 h after substance administrations. The

behavioural parameters like convulsions, hyper activity, sedation, grooming, loss of righting

reflex and increased respiration were observed.

Mounting behavior test

Mount is operationally defined as the male assuming the copulatory position but

failing to achieve intromission. Intromission was defined as the male’s penis entering the

vagina in association with thrusting behavior To quantify mounting behavior, non-estrous

female mice were paired with males treated with single dose of the drugs (400 mg/kg; p.o.).

Animals were observed for 3 h and their behaviors were scored as described (Lawler L.,

1984).

Males were placed individually in a glass cage. After 15 min of acclimatization, a non-

estrous female was introduced into the arena. The number of mounts was recorded during a

15 min observation period at the start of 1 h. The number of mounts was recorded by two

observers, who were uninformed about the drug treatment, during a 15 min observation period

at the start of each hour. All the experiments were performed between 09.00 to 12.00 h a.m. at

room temperature 26 – 27°C.

Hormone assay

On the 3th and 7th days of the experiments, from animals were collected blood samples

by cardiac puncture under anesthesia and isolated sera were analyzed for sexual hormone

secreted (testosterone (T)) and luteinizing hormone (LH). LH and T concentrations were

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determined in duplicate using LH ELISA (IBL, Hamburg, Germany) and a Testosterone

Enzyme Immunoassay kit (Assay Designs, Ann Arbor, MI, USA), respectively, according to

the manufacturer's instructions. The detection limit for LH assay was 0,3 ng/mL. The

detection limit for the T assay was 10,0 pg/mL; cross-reactivity with corticosteroid and other

androgens was minimal (< 1%).

Histopathology and morphology examination

Small pieces of testis and epididymis were fixed in 10% buffered formalin and

processed for embedding in paraffin. Sections of 6 μm thicknesses were cut with a rotary

microtome (LEICA RM2235; Leica Biosystems Inc., Buffalo Grove, IL) and stained with

hematoxylin and eosin (HE) for morphology assessment. Histological sections were examined

using the light microscope (×100).

Statistical analysis

Mean±SD of the sex hormones, body and ovary weights were taken for analysis. The

data was tested for homogeneity of variance and significantly different results were

established by one-way ANOVA using the SPSS software application (version 16). Pair-wise

comparisons were made using the post hoc test. The accepted level of significance was set at

p<0.05.

Results

Evaluation of cytotoxicity of herbal combinations (Substance I and Substance II) such

as determined maximal non-toxic concentration (MNC) and cytotoxic concentration 50%

(CC50) in cell culture of murine origin

Evaluation of cytotoxicity was an important part of the assessment of potential

compounds since the beneficial drugs should be selective for drugs processes with little or no

effects on the metabolisms of normal cells. Both MNC and CC50 values were evaluated

simultaneously by morphological and by cell survival criteria. We used MTT-test,

determining the living and early apoptotic cells (Mosmann T., 1983). When microscopic

observation of the morphology of the monolayers were carried out at 48 h after the treatment

with tested herbal combination in different concentration range a typical cytopathology

characterizing the toxic effect was not registered in tested murine cell line L929.

Dynamics of survival of murine cells treated with Substances I, II, III and IV at 48 h were

presented in Figure 1.

The data presented here showed that the tested herbal combinations (Substances I, II,

III and IV) exhibit low cytotoxicity against murine cell line L929. The most cytotoxic is

Substance I if compared with other substances. These results were dose-dependent. In all

tested substances can not be determined cytotoxic concentration, which reduce cell viability

by 50%(CC50).

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In vitro cytotoxicity data (MNC, mg/ml) for the tested herbal combinations (Substance

I, II, III and IV) were summarized in Table 1.

Table 1. Cytotoxic effect (MNC, mg/ml) of Substance I, II, III and IV on murine cell line

L929 at 48h

Substances MNC, mg/ml

I 0,0001

II 0,001

III 0,005

IV 0,001

Evaluation of acute toxicity study

The Substance I, II, III and IV administered orally in doses 400 mg/kg body weight

did not produce any evident sign of toxicity and any mortality in male mousses Balb/c when

observed up to 14 days after administration.

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Effect of the Substances I, II, III and IV on mounting behavior of male mousses Balb/c

The effect of repeated oral administration (daily for 5 days) with single dose (400

mg/kg; p.o.) of the Substances I, II, III and IV is given in Table 2. All of the treated males

Balb/c mousses displayed excessive mounting behavior as compared to the control. The

number of mounts displayed by the Substances III and IV-treated male animals was more than

three times the values for the untreated controls during the observation period. However, the

Substances I and II-treated mice showed only marginal activity compared to Substances III

and IV-treated ones.

Table 2. Effect of a single dose (400 mg/kg; p.o.) of Substances I, II, III and IV of

mounting behavior of male mousses Balb/c

Group Number of mounts/15 min

1 h 2 h 3 h

Control 2,0 ± 0,9 1,0 ± 0,4 0,5 ± 0,2

Treated with

Substance I 2,5 ± 1,1 1,0 ± 0,4 0 ± 0,3

Treated with

Substance II 3,0 ± 0,9 1,0 ± 0,3 0,8 ± 0,3

Treated with

Substance III 12,0 ± 3,1 5,0 ± 2,4 3,0 ± 0,9

Treated with

Substance IV 11,0 ± 2,5 4,0 ± 1,2 1,0 ± 0,3

Luteinizing hormone (LH) and Testosterone (T) analysis

In the present study, we focused on the influence of botanical substances in relation to

testosterone (T) and luteinizing hormone (LH) concentrations.

Testosterone (T) is a naturally occurring steroid hormone from the androgen group, is

naturally produced in the body and is secreted primarily from the Leydig cells of the testes in

men (> 95%) and to a lesser extent in the theca cells of the ovaries in women, the zona

reticularis of the adrenal cortex, and the skin (Brooks R., 1975; Mooradian A. et al., 1987). It

is the main sex hormone and plays an important role in the development of the male

reproductive system and promotion of secondary sex characteristics such as muscle growth

and strength, bone mass, and growth of body hair (Mooradian A. et al., 1987). Levels of T are

typically more than five times higher in adult male than female (Laughlin G. et al., 2008).

Luteinizing hormone (LH) is produce by the pituitary in a pulsatile fashion, inducing

ovulation and maintenance of the corpus luteum in females. In adult males, LH regulates

spermatogenesis acting on Leydig cells located in the testes, by controlling T production

(Huhtaniemi I., 2015; Saez J., 1994). LH acts upon the Leydig cells of the testis and is

regulated by gonadotropin-releasing hormone (GnRH) (Nomura K. et al., 1989).The Leydig

cells produce testosterone (T) under the control of LH, which regulates the expression of the

enzyme 17β-hydroxysteroid dehydrogenase that is used to convert androstenedione, the

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hormone produced by the testes, to testosterone, an androgen that exerts both endocrine

activity and intratesticular activity on spermatogenesis (Glage S. et al., 2013).

The effect of repeated oral administration with single dose (400 mg/kg; p.o.) of the

Substances I, II, III and IV is given in Table 3. The tested Substances I, II, III and IV acutely

administered at 400 mg/kg to male mosses significantly increased LH and T serum levels in

comparison with non-treated control group.

Table 3. The effect of a single dose (400 mg/kg; p.o.) of Substances I, II, III and IV on

T and LH level in control and treated groups

Groups T level (ng/ml) LH level (mIU/ml)

Control 0,270 ± 0,12 0,0125 ± 0,017

Treated with

Substance I 0,710 ± 0,20 0,079 ± 0,025

Treated with

Substance II 1,05 ± 0,41 0,125 ± 0,058

Treated with

Substance III 4,43 ± 1,04 0,207 ± 0,098

Treated with

Substance IV 6,49 ± 1,56 0,273 ± 0,066

Male mouses Balb/c treated with dose of 400mg/kg body weight with Substances III

and IV have a significant increase (up to 24 times) in level of serum T when compared to the

data of control group and treated mouses with Substances I and II. An increase in testosterone

level in male mouses Balb/c treated with Substances III and IV compared with the control

group has been associated with increase of sexual desire, penile tumescence, and rigidity. The

dose of 400mg/kg of Substances III and IV (up to 22 times) caused a significant increase in

LH (p < 0,01) as compared to control group and treated animals with Substances I and II.

Histological examination of testis of mouses Balb/c treated with Substances I, II, III

and IV in comparison with non-treated control group

In Figure 2 the testis of Substance-treated animals is compared with the testis of an

untreated male mousses Βalb/c. All stages of spermatogenesis were clearly: primary

spermatocytes, secondary spermatocytes, spermatogonia, spermatid and spermatozoa,

basement membrane and leydig`s cells (Figure 2A). No significant changes compared with

the control group could be found in testes morphology of treated with Substance II male

animals (Figure 2B).

The proliferation and increase of leygid’s cells was more pronounced in Substance III

treated mousses as compared to the control group (Figure 2C). The animal treated with

Substance IV showed higher number of spermatozoa in seminiferous tubules as compared to

C. articulata and both control treated groups, which further confirmed the increased

spermatogenesis, which is highly elaborated by an increase in spermatogenic elements as

described Figure 2D.

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Figure 2 Histopathological examination of spermatogenesis in treated with Substances

II, III and IV male mice Balb/c and control group. Shown here are cross-sections

though the testes of male Balb/c mice

.

This study demonstrated the androgenic effects of the both combination Substance III

and Substance IV i.e. increased mounting, intromission, testes weight, spermatogenesis and

Leydig cell proliferation. These effects are explained by the increase in serum testosterone

and LH levels.

A. The light microscopic photograph of mouse

testis in control group staining with

(H & E 40×)

C. The light microscopic photograph of mouse

testis in group treated with Substance III

staining with (H & E)

D. The light microscopic photograph of mouse

testis in group treated with Substance IV

staining with (H & E)

B. The light microscopic photograph of mouse

testis in group treated with Substance II staining

with (H & E)

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8th August 2018 Performers:

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1. Assoc. Prof. Dr. Petia Genova-Kalou, PhD

Head-Leader of Laboratory of

“Rickettsiae and cell cultures”

Department of Virology, National Centre of

Infectious and Parasitic Diseases, 44A “Gen.

Stoletov” Blvd., 1233 Sofia, Bulgaria

2. Assist. Prof. Stefka Ivanova PhD

Department of Virology,

National Centre of Infectious and Parasitic

Diseases, 44A “Gen. Stoletov” Blvd.,

1233 Sofia, Bulgaria

3. Assoc. Prof. Andrey Tchorbanov PhD

Institute of Microbiology,

Bulgarian Academy of Sciences,

“Geogri Bontchev” Str., bl. 25, Sofia,

Bulgaria