WO2018186557A1 - Pharmaceutical composition for preventing or treating andropause syndrome, containing elaeagnus multiflora thunb. extract as active ingredient - Google Patents

Abstract

The present invention provides a pharmaceutical composition for preventing or ameliorating andropause syndrome, comprising an Elaeagnus multiflora Thunb. extract as an active ingredient, wherein the Elaeagnus multiflora Thunb. extract is extracted by using any one selected from among water, a lower alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof. Thus, Elaeagnus multiflora Thunb. which is a natural resource of Korea is used as a resource, and it is possible to safely provide a composition for preventing, ameliorating, or treating andropause syndrome without toxicity or side effects.

Description

A pharmaceutical composition for preventing or treating menopausal syndrome in men, which contains an extract of elderberry as an active ingredient

The present invention relates to a pharmaceutical composition for the prevention or treatment of male menopausal syndrome, characterized in that it contains an extract of Elaeagnus multiflora Thunb. As an active ingredient. More specifically, the present invention relates to a pharmaceutical composition for improving the secretion of testosterone in the body to improve male menopausal disorder or prevent and treat male menopausal disease.

Menopause refers to the period during which several symptoms occur due to a decrease in sex hormones. Since the arrival of menopause, the female hormones are rapidly reduced and various physical changes occur. Therefore, women have been used in general. Recently, however, men's menopausal syndrome has emerged as men report a change in endocrine system due to the gradual decrease of male hormones in the body due to aging. Late-onset hypogonadism (LOH) and age-associated testosterone deficiency syndrome (TDS) at the International Society for the Sutudy of the Aging Male, 2004 Was named.

Testosterone, a testosterone, is produced primarily in men's testicles and controls men's physical health and mental state, and plays a leading role in masculinity and sexual life. However, it has been reported that male hormone decreases by about 1% every year since the age of 30s and about 30-50% of males in their 50s and 70s are lower than normal levels. There are many differences in the decrease in male hormones, mainly drinking, smoking, obesity, poor lifestyle, stress, hypertension, diabetes, chronic diseases, and menopausal menopausal, steroids, cimetidine, diuretics (spinloactone) ), Such as athlete's foot (ketocanazole) may be the cause.

 These menopausal menopausal disorders (Andropause) are clinical and biochemical syndromes associated with an increase in age and are defined as typical symptoms and lack of serum testosterone levels, and can have a negative effect on the function of various body organs. Accompanying symptoms include: lethargy with no known cause, chronic fatigue, decreased concentration, depression, insomnia, loss of confidence, abdominal obesity, hair loss, muscle strength, joint pain, skin aging, hot flashes, palpitations, sweating, osteoporosis, weight And a decrease in muscle mass.

For treatment, the possibility of using male hormone replacement therapy in a similar way to the use of estrogen replacement therapy in postmenopausal women has been claimed. One study reported that significant improvements in treatment of male menopausal patients with testosterone formulations were obtained. However, there is also a risk that the administration of testosterone may adversely affect liver, lipid status, cardiovascular and prostate diseases. In addition, in view of adenomas and adenocarcinomas, about 30% of patients with menopausal symptoms are known to be unable to receive testosterone replacement therapy. Therefore, there is a need for the development of new therapeutic agents useful for the prevention and treatment of male menopausal syndrome caused by aging or caused by chemical or surgical castration that does not provide the disadvantage of hormone replacement therapy.

On the other hand, elderagnus multriflora Thunb.) is known as a gardening tree that is planted in a garden and eats its fruit, but traditionally, herbs , leaves, stems, and roots have been used as medicine. The elder tree is a genus of Elaeagnus native to various parts of Korea. It is called Yaindo or Swolja, and it is known as Mokbanha as the name of the herb. It is also commonly called home tree and true sugar tree, and young branches are covered with reddish brown scale hairs. It looks like a bodhi tree, but because it is large in fruit growing, it is called as a bodhi tree, and when used as a medicine, it is mainly used as a tang.

Leaves are alternate, long oval of 3 ~ 10cm long, narrow at both ends, sharp and without sawtooth at edge. On the front, there are scaly hairs when young, but gradually disappear, and on the back, white scaly hair and brown scaly hair are mixed. Flowers bloom pale yellow in April to May, hanging on the axillas of 1-2 leaves. Flowers have white and brown scaly hairs. The calyx tube is rapidly narrowed at the bottom and surrounds the ovary and has four ends, four stamens, and one pistil. In July, a long oval nucleus, about 1.5cm long, sags down and ripens red. Water pipes are deciduous and grow up to 2-3m in height.

Lime tree ( Elaeagnus) umbellata Although similar in appearance to Thunberg, the tree has many white scaly hairs on its twigs, and its fruit is less than 1cm long, which distinguishes it from the tree. According to the <Ilbosan War>, the berry tree mainly controls circulatory diseases, and is known to have good effects on poisons, bruises, abundance, customs, swelling, and haemostasis. It has also been used to stop coughs, sputum, asthma, bruises, hemorrhoids, improved blood circulation and diarrhea. In particular, the fruit of the hawthorn tree is known to replenish the five intestines, to eliminate heat and calories, to improve indigestion, osteomyelitis, edema, dysmenorrhea, hemorrhoids and swelling, and to cure asthma, no matter how old and poor. It helps to stop the bleeding and to bleed out, to remove the wind, to release the habit, to lower the food and to relieve sore throat. It is also good for coughing, vomiting blood, sputum, hemoptysis, intestinal bleeding, excessive menstruation, rheumatism, and jaundice.

While researching the efficacy of natural products such as horseradish trees traditionally known in recent years, we have been trying to develop materials that have little side effects and are useful for the prevention and recovery of diseases. There is a lack of relevant information on active functionality.

An object of the present invention is to provide a composition for the prevention, improvement or treatment of male menopausal syndrome that can be safely used without toxicity and side effects using the natural bark leaves or fruit extracts.

In order to achieve the above object, the present invention is an elderberry ( Elaeagnus) multiflora Thunb.) leaf or fruit extract as an active ingredient, the berry leaf or fruit extract is characterized in that the extract is available from any one selected from water or lower alcohols having 1 to 4 carbon atoms or mixed solvents thereof. It provides a pharmaceutical composition for preventing or ameliorating male menopausal diseases.

The composition may be prepared by including the amount of 0.01 to 10 g / day of the extract of the camphor tree, or may be prepared by including the extract of 0.01% to 100% by weight.

The present invention utilizes the natural resources of camphor tree as a natural resource through the pharmaceutical composition for the prevention or treatment of male menopausal syndrome containing an extract of Aspenia as an active ingredient, the composition for the prevention, improvement or treatment of male menopausal syndrome It can be safely provided without toxicity or side effects.

1 is a view showing that the leaves of the leaves of the present invention showing the cell viability in TM3 cells.

2 is a view showing that the berry fruit extract of the present invention shows cell viability in TM3 cells.

Figure 3 shows that the leaves of the leaves of the present invention of the present invention shows the effect of increasing the testosterone secretion in TM3 cells.

4 is a view showing that the berry fruit extract of the present invention has an effect of increasing the testosterone secretion in TM3 cells.

5 is a view showing the effect of increasing the testosterone secretion in the body of the elderberry leaf extract of the present invention in old male rats.

6 is a view showing a rotarod exercise equipment used to measure the muscle strength of the rat.

7 is a view showing that the leaves of the leaves of the present invention shows the effect of improving the muscle strength in the elderly male rats.

8 is a view showing a treadmill exercise device used to measure the fatigue of the rat.

9 is a view showing that the leaves of the leaves of the present invention shows the effect of reducing fatigue in the male rats of old age.

10 is a view showing that the leaves of the leaves of the present invention shows the effect of increasing the number of sperm in old male rats.

The present invention relates to a pharmaceutical composition for the prevention or treatment of male menopausal syndrome, characterized in that it contains Elaeagnus multiflora Thunb. Leaf or fruit extract as an active ingredient. It will be described in detail with reference to the following specific examples.

1. Manufacture of Fern Leaf and Fruit Extract

1.1 Preparation of Fern Tree Water Extract

Aspen leaf and fruit extract can be extracted using any solvent selected from water or a lower alcohol having 1 to 4 carbon atoms or a mixed solvent thereof. Specifically, 100 g of the leaves of the deciduous tree was washed with distilled water, and then 1 L of distilled water was added, and heated and extracted at 100 ° C. for 3 hours in a reflux extractor. Filtration was carried out using filter paper (Watman No. 41), and the filtrate was depressurized and concentrated. After filtration, the residue was extracted, filtered and concentrated under reduced pressure once more using the same amount of distilled water. The concentrated hot water extract was freeze-dried at -50 ° C for 48 hours using a freeze dryer. According to the above method, the hot water extract according to the extraction solvent was obtained 16.8 g (16.8%) of the hot water extract of leaf of the barley tree and used as a sample of the following experimental example (hereinafter referred to as "EM").

Aspenella fruit extract was added 1L of distilled water to 100g of dried lime tree fruit, and heated and extracted at 100 ° C. for 3 hours in a reflux extractor. Filter using filter paper (Wattman 41), and depressurize and concentrate. After filtration, the residue was extracted, filtered and concentrated under reduced pressure once more using the same amount of distilled water. The concentrated hot water extract was freeze-dried at -50 ° C for 48 hours using a freeze dryer. According to the above method, the hot water extract according to the extraction solvent was obtained 12.4 g (12.4%) of the lycheeola berry hot water extract and used as a sample of the following experimental example (hereinafter referred to as "EMF").

1.2 Preparation of Polar and Nonpolar Solvent Soluble Fractions

The stepberry hot water extract of the step was fractionated as follows using an organic solvent.

1.2.1 Hexane Soluble Fractionation

After dissolving 12.4 g of the extract of hawthorn tree in 5L of distilled water, the mixture was placed in a fractional filter head and 5L of hexane was added to separate the hexane-insoluble layer (aqueous layer) and the hexane-soluble layer. Again, the same process was repeated three times for the hexane insoluble layer (aqueous layer) to collect the hexane insoluble fraction and the soluble fraction.

1.2.2 Chloroform Soluble Fraction Separation

After adding 5 L of chloroform to the nucleic acid insoluble fraction (aqueous layer), the chloroform soluble fraction and the insoluble fraction were separated, and the same procedure was repeated three times for the chloroform insoluble layer (aqueous layer) to collect the chloroform insoluble fraction and the soluble fraction.

1.2.3 Ethyl Acetate Soluble Fraction Separation

After adding 5 L of ethyl acetate to the chloroform insoluble fraction (aqueous layer), the ethyl acetate soluble fraction and the insoluble fraction were separated, and the same process was repeated three times on the ethyl acetate insoluble layer (aqueous layer) to obtain the ethyl acetate insoluble fraction and the soluble fraction. Collected.

1.2.4. Butanol Soluble Fractionation

After adding 5 L of butanol to the ethyl acetate insoluble fraction (aqueous layer), the butanol soluble fraction and the insoluble fraction were separated, and the same process was repeated three times for the butanol insoluble layer to collect the butanol insoluble fraction and the soluble fraction.

1.2.5 Water Fraction Separation

After dissolving 12.5 g of the extract of Fructus japonica in 5L of distilled water, the mixture was put into a fractional filter, and the hexane soluble layer, the chloroform soluble layer, the ethyl acetate soluble layer, and the butanol soluble layer were separated and concentrated to remove the remaining organic solvent. Fractions were collected.

The fraction obtained by freeze-drying the hexane soluble fraction, the chloroform soluble fraction, the ethyl acetate soluble fraction and the requestol soluble fraction in 12.5 g of the barley tree hot water extract prepared by the process of extracting the hot water from the elder barley and extracting the fractions Used as a sample.

2. Cytotoxicity Measurements of Green Leaf and Fruit Extracts

Prior to confirming the effect of the leaves and fruit extracts of the present invention, cytotoxicity tests were performed to determine suitable concentrations that would be effective without showing cytotoxicity. Cytotoxicity of the extracts of the leaves of Fructus japonica was measured in the TM3 cell line, Leydig cells derived from rat testicular cells. TM3 cells were cultured at 37 ° C. and 5% CO ₂ in DMEM with 10% FBS and 1% penicillin / streptomycin.

TM3 cells were placed in a 96-well plate at a concentration of 1 × 10 ⁴ cells / well and incubated at 37 ° C. and 5% CO ₂ . Aspen leaf or fruit extracts were treated in each well at 10, 50, 100 and 200 μg / mL concentrations and incubated for 24 hours. After 3 hours after the addition of the MTT solution, the supernatant was removed, and 100 μL of DMSO was added per well. The absorbance was measured at 540 nm using an ELISA reader, and the results are shown in FIGS. For reference, all measured results shown in FIGS. 1 to 5, 7, and 9 to 10 are expressed as average and standard error, and statistical significance test is performed using Student's t-test method, and the p value is less than 0.05. ( p <0.05) was considered statistically significant.

Referring to FIG. 1, the cell survival rate was 97%, 96%, 96%, 95% compared to the control (Control) when treated with concentration (10, 50, 100 and 200 μg / mL) It was confirmed that it is not toxic to TM3 cells.

Referring to FIG. 2, the cell viability was 98%, 96%, 93%, 93% compared to the control group when the berry extract was treated by concentration (10, 50, 100 and 200 μg / mL). It was confirmed that it is not toxic to TM3 cells.

3. Measurement of testosterone secretion effect of TM3 cells

TM3 cells were placed in a 24-well plate at a concentration of 2 × 10 ⁵ cells / well and incubated at 37 ° C. and 5% CO ₂ . Aspen leaf and fruit extracts (EM) were mixed to concentrations of 10, 50 and 100 μg / mL and treated in each well and incubated for 24 hours. After taking the supernatant, testosterone content was measured using a Testosterone ELISA kit (ADI-900-065, ENZO Life Sciences, USA), and the results are shown in FIGS. 3 and 4.

Referring to Figure 3, when treated with concentration (10, 50, 100 and 200 μg / mL) of the elder leaves extract 15 pg / mL (130%), 17 pg / mL compared to the control (Control), respectively (148%), 18 pg / mL (150%) and 18 pg / mL (153%) significantly increased testosterone content.

Referring to Figure 4, when treated with concentration (10, 50, 100 and 200 μg / mL) of the elderberry fruit extract 11 pg / mL (133%), 13 pg / mL compared to the control (Control), respectively (166%), 14 pg / mL (181%) and 16 pg / mL (186%) significantly increased testosterone content.

4. Weight gain measurement

After the oral administration of aged male rats for 4 weeks, the results of the comparison with the effect of the leaves of the leaves of the tree (EM) on the body weight are compared.

Groups	Body weight
	Initial	Final	Gains (g / day)
Control	557.4 ± 13.6	589.4 ± 20.4	32.0 ± 6.9
EM 50	556.1 ± 17.2	572.3 ± 20.7	16.3 ± 3.9
EM 100	555.7 ± 14.4	560.0 ± 13.5	4.3 ± 9.8

As shown in Table 1, it was confirmed that the weight gain of the experimental group (50 and 100 mg / mL) administered with the needles and leaves (EM) during the experimental period was reduced to 16g, 4g compared to the control (32g).

5. Long-term weight measurement

After the oral administration of aged male rats for 4 weeks, the effect of the extract of the leaves of the barley tree on organ weight is shown in Table 2.

Groups	Testes (g)	Epididymis (g)	Seminal vesicle (g)	Ventral prostate (g)	Liver (g)	Kidney (g)	Spleen (g)
Control	4.28 ± 0.10	1.42 ± 0.14	1.55 ± 0.19	0.80 ± 0.07	15.82 ± 0.59	3.74 ± 0.08	0.92 ± 0.05
EM 50	4.02 ± 0.17	1.58 ± 0.11	2.13 ± 0.27	1.30 ± 0.26	14.87 ± 1.01	3.56 ± 0.18	0.96 ± 0.06
EM 100	4.23 ± 0.08	1.53 ± 0.02	2.41 ± 0.31	1.55 ± 0.13	14.08 ± 0.91	3.57 ± 0.07	0.94 ± 0.06

As shown in Table 2, as a result of measuring the change in long-term weight of the testicle, epididymis, seminal vesicle, prostate, liver, spleen, kidney, no significant change was observed due to the administration of the leaves of the leaves.

6. Measures the effect of increased testosterone secretion in the body

The effect of increasing testosterone secretion at the in vivo level was confirmed in elderly male rats. SD rats at 6 months of age were adapted for 1 week, and then used in experiments divided into experimental and non-administered control groups. The breeding conditions of the animal laboratory were 23 ± 1 ° C, 45 ± 5% humidity, and the brightness was automatically controlled at 12 hour intervals. Water and diet were ingested freely. The experimental group was dissolved in distilled water at 50, 100 mg / mL concentration of oral administration at the same time every morning, and the control group was orally administered the same amount of distilled water. After oral administration for 4 weeks, blood was collected after the last oral administration, and serum was separated by centrifugation at 4000 rpm for 15 minutes. Testosterone content was measured using the Testosterone ELISA kit (ADI-900-065, ENZO Life Sciences, U.S.A) on the isolated serum and the results are shown in FIG. 5.

Referring to FIG. 5, in the experimental group (50 and 100 mg / mL) administered with the leaves of Fructus japonica, serum testosterone secretion was increased by 211% and 287%, respectively, compared to the control group.

7. Measure the strength strengthening effect

The strength enhancing effect of the extract of the hawthorn tree was measured by assessing the muscle strength state by exercising in the elderly male rats using the rotarod of the rotatable cylindrical shape shown in FIG. The age, breeding conditions, and administration conditions of the leaves of Fructus japonica were the same as the test for measuring the effect of increasing testosterone secretion in the body, and the rats were forced to walk on the rotarod cylinder at a rotational speed of 20 rpm on the last day of the administration. Adapted to the load. After 24 hours, the rats were raised and walked again on the rotating cylinder at the same speed, and the rats were unbalanced and dropped on the cylinder. The measured result is shown in FIG.

Referring to FIG. 7, as a result of evaluating the muscle strength through the rotarod exercise, the rotarod exercise time of the experimental group (50 and 100 mg / mL) administered with the leaves of Fructus japonica was the rotarod exercise time of the control group. Compared with (29 seconds), it can be seen that the strength increase effect is increased to 154 seconds and 270 seconds, respectively.

8. Fatigue improvement effect measurement

The fatigue improvement effect of the extract of the hawthorn tree was measured by applying a treadmill exercise device using the electric aversive stimulation shown in Figure 8 to the elderly male rats. The angle of the treadmill was tilted toward the electrical pole at an angle of 20 ° to the ground. The age, breeding conditions, and administration conditions of the oleander leaf extract (EM) of the used mice were the same as the test for measuring the effect of increasing the testosterone secretion in the body, and trained for 10 minutes on the last day of the administration. After 24 hours, start exercise for 3 minutes at 9 m / min to assess endurance performance, and then increase the intensity of exercise at a rate of 3 m / min every 3 minutes, and continue to exhaustion. Was specified. The measured result is shown in FIG.

Referring to Figure 9, as a result of measuring the exercise duration through the treadmill exercise, the exercise duration of the experimental group (50 and 100 mg / mL) administered with the berry leaf extract compared to the control group (9 minutes), respectively 10 minutes In addition, it increased to 11 minutes and no significant change was observed.

7. Sperm Count

Aged male rats were orally administered for 4 weeks, then the tails of the epididymis were cut and mixed with 1 ml of saline, 10 μL was placed on a hemacytometer, and the total number of sperm present in the epididymis was calculated by multiplying the sperm. The measured result is shown in FIG.

Referring to FIG. 10, the total sperm count of the experimental group (50 and 100 mg / mL) administered with Fructus leaf extract increased 139% and 152%, respectively, compared to the control group.

As can be seen from the above results, the pharmaceutical composition for preventing or treating male menopausal syndrome containing the aspen actives extract of the present invention increases the secretion of testosterone in TM3 cells and old mice, muscle strength in old mice It has been shown to show no cytotoxicity while increasing sperm count, suppressing weight gain, and providing a composition for preventing, improving or treating male menopausal syndrome, which can be safely used without toxicity and side effects. .

The pharmaceutical composition for preventing or treating male menopausal syndrome containing the extract of the camphor tree of the present invention as an active ingredient can be used as a composition for preventing, improving or treating male menopausal syndrome and functional food, which can be safely used without toxicity and side effects. It can be used in human health and disease treatment, so there is industrial availability.

Claims (6)

1. A pharmaceutical composition for preventing or ameliorating male menopausal syndrome, which comprises an extract of Elaeagnus multiflora Thunb. As an active ingredient.
2. The method of claim 1, wherein the extract of the camphor tree is male or menopausal disease prevention or improvement pharmaceutical composition, characterized in that the extract available from any one selected from water or lower alcohol having 1 to 4 carbon atoms or a mixed solvent thereof.
3. A decrease in sperm count and activity, increase visceral fat, decrease muscle mass, decrease muscle strength, decrease bone density, increase arteriosclerosis and increase carotid artery thickness, increase insulin resistance, accompanied by a decrease in testosterone including the extract of claim 1 or 2 as an active ingredient. A pharmaceutical composition for preventing or improving one or more menopausal syndrome, selected from the group consisting of: intellectual activity, cognitive ability, reduction of spatial orientation, fatigue, depression, impatience, mood changes, sleep disorders, hair loss, and skin changes.
4. The pharmaceutical composition for preventing or ameliorating male menopausal syndrome according to claim 2, wherein the pharmaceutical composition is prepared by using an extract of camphor tree in an amount of 0.01 to 10 g / day.
5. The pharmaceutical composition for preventing or ameliorating male menopausal syndrome according to claim 2, wherein the pharmaceutical composition is prepared by using an extract of camphor tree in an amount of 0.01 to 100% by weight.
6. Functional food for the prevention or improvement of male menopausal syndrome, characterized in that it comprises an extract of Elaeagnus multiflora Thunb. As an active ingredient.

Images