CN104013611B - Application of mango aglycone and derivative thereof in preparation of anti-prostate hyperplasia drug - Google Patents

Abstract

The invention belongs to the field of medicines, and discloses novel application of a mango aglycone derivative. An in-vitro test shows that the mango aglycone derivative can obviously inhibit activity of 5 alpha-reductase, can remarkably lower prostate wet weight index of a testosterone propionate replication prostate hyperplasia model mouse, can lower a ratio of E2/T in blood serum, and can lower activity of the 5 alpha-reductase in a liver tissue, so that the mango aglycone derivative is indicated to have effect in inhibiting activity of 5 alpha-reductase, can be used for treating 5 alpha-reductase expression or superhigh-activity-associated diseases, for example testosterone propionate; moreover, the mouse stomach is filled with the drug in maximum administration dosage of 21.6g/kg, and therefore, the drug has very good clinical application prospect.

Description

Application of mangiferin and its derivatives in the preparation of anti-prostatic hyperplasia drugs

technical field

The invention relates to the technical field of medicine, in particular to the application of mangiferin and its derivatives in the preparation of anti-prostatic hyperplasia drugs.

Background technique

Benign prostatic hyperplasia (BPH) is a common disease in middle-aged and elderly men characterized by progressive urinary frequency and dysuria due to the obvious enlargement of the prostate gland. Epidemiological studies have shown that the incidence of benign prostatic hyperplasia is very low before the age of 40, accounts for 40% at the age of 50, and is close to 90% after the age of 80. By the age of 90, almost 100% of prostate hyperplasia will be found if the histological examination of the prostate is performed. With the aging of my country's population, its incidence is on the rise in recent years.

The treatment of BPH is mainly divided into surgery and drug therapy. Surgical resection was once considered to be the first choice for the radical cure of BPH. However, the advanced age of the patients often makes the surgical treatment have certain limitations, and it is easy to cause a variety of complications. According to statistics, surgical complications are about 15%, and the fatality rate is about 1%. . Studies have pointed out that the prostate can produce a variety of immunoglobulins, can synthesize zinc-containing polypeptides with various antibacterial effects, and has local immune functions to protect the reproductive system from bacteria and other pathogenic microorganisms, which should be preserved as much as possible.

The main cause of BPH is related to the increase of dihydrotestosterone content in prostate tissue. In the body, 5α-reductase can catalyze the reduction of testosterone (T) to dihydrotestosterone (DHT), and inhibiting the activity of 5α-reductase can reduce the content of DHT in prostate tissue. Studies have shown that diseases related to excessive expression or activity of 5α-reductase include benign prostatic hyperplasia, prostate cancer, androgenetic alopecia, female hirsutism, acne (Wu Xueyan et al. Steroid 5α-reductase and 5α-reductase type 2 deficiency Progress in Research on Diseases, Basic Medicine and Clinics, 2006,3:225. Li Yongfang et al. Research Progress on Active Components Inhibiting 5A-Reductase in Plants, Chinese Herbal Medicine, 2006,11:1740).

In recent years, drug treatment of BPH has gradually become the main treatment, mainly including 5α-reductase inhibitors, α-adrenoceptor antagonists, herbal medicines and Chinese patent medicines. α-receptor blockers can block the contraction of prostatic smooth muscle and relieve urethral stricture obstruction in patients with benign prostatic hyperplasia; 5α-reductase inhibitors can inhibit the conversion of testosterone to dihydrotestosterone in the prostate, thereby reducing the volume of the prostatic hyperplasia. Whether it is symptomatic, corrective or combined treatment, it can restore the urinary function of patients with benign prostatic hyperplasia to varying degrees and improve their quality of life. However, long-term use of chemical drugs has obvious adverse reactions. The current status of unclear targets, difficult quality control, and difficult to guarantee curative effect.

According to data, about 80% of antibacterial drugs and 60% of anticancer drugs are directly or indirectly derived from natural products, and natural drugs are becoming an important source of human therapeutic drug development. At present, there are no reports on the obvious inhibitory effect of natural medicine on 5α-reductase and its application in anti-prostatic hyperplasia.

Contents of the invention

The purpose of the present invention is to provide new applications of natural medicine mangiferin and its derivatives.

In recent years, studies have found that mangiferin is an active metabolite of mangiferin, which has various pharmacological effects such as anti-oxidation, inhibiting PTP1B activity, lowering blood sugar, anti-tumor, lowering uric acid and anti-gout, but the content of mangiferin in plants is extremely low , it is difficult to meet the needs by simply relying on plant extraction methods. The inventors prepared a series of mangiferin derivatives with a purity >98% through artificial synthesis in the early stage. They are light yellow powders with the structural formula:

Wherein, R ₁ , R ₂ , R ₃ and R ₄ are H or acetyl.

The present invention provides the application of the compound with the following structural formula in the preparation of medicines for the treatment of diseases related to the expression or overactivity of 5α-reductase,

Wherein, R ₁ , R ₂ , R ₃ and R ₄ are H or acetyl.

The present invention conducts in vivo and in vitro pharmacodynamic studies on mangiferin and its derivatives. The results show that: mangiferin and its derivatives have different degrees of inhibitory activity on 5α-reductase in vitro, and its inhibitory activity on 5α-reductase is linearly dependent; continuous intragastric administration for 21 days, mangiferin can Significantly reduce the wet weight and index of the prostate in the prostatic hyperplasia model mice replicated by testosterone propionate, reduce the ratio of E2 and E/T in the serum, and reduce the activity of 5α-reductase in the liver tissue, indicating that it can effectively inhibit the activity of 5α-reductase. It has the effect of treating diseases related to the expression or activity of 5α-reductase, such as benign prostatic hyperplasia.

Preferably, the disease associated with increased expression or activity of 5α-reductase is benign prostatic hyperplasia, prostate cancer, androgenetic alopecia, female hirsutism or acne.

More preferably, the effective dose of the compound is 10-1000 mg/kg/d.

The experiment of the present invention proves that mangiferin does not show lethal toxicity in the acute toxicity test of mice, and its maximum tolerated dose of intragastric administration in one day exceeds 21.6g/kg, which is equivalent to the pharmacodynamically effective dose of the same animal 1440 times of 15mg/kg.

The mangiferin aglycone and its derivatives of the present invention can be used directly alone or in combination with each other; they can be used in a compound form with other medicines including plant extracts, and can also be used in the form of health care products and food in a conventional manner; Tablets, granules, capsules, pills, suspensions or injections prepared by adding the above-mentioned mangiferin derivatives with pharmaceutically acceptable auxiliary materials. Mangiferin and its derivatives have simple structures, are easy to chemically synthesize, and have high clinical application prospects.

Description of drawings

Figure 1 is the relationship between different concentrations of protein and absorbance values;

The linear relationship between different testosterone amounts and peak area of Fig. 2;

The influence of Fig. 3 different enzyme concentrations on enzyme activity;

The influence of Fig. 4 different testosterone concentrations and enzyme activity;

The influence of NADPH of different concentrations of Fig. 5 on enzyme activity;

The inhibitory effect of Fig. 6 finasteride on 5α-reductase;

The inhibitory effect of Fig. 7 mangiferin aglycon on 5α-reductase;

Fig. 8 1-Hydroxy-3,6,7-triacetoxymangiferin inhibits 5a-reductase;

Figure 9 3-Hydroxy-1,6,7-triacetoxymangiferin inhibits 5a-reductase;

Figure 10 1,3-dihydroxy-6,7-diacetoxymangiferin inhibits 5a-reductase;

Figure 11 Inhibitory effect of 6,7-dihydroxy-1,3-diacetoxymangiferin on 5a-reductase.

detailed description

The invention discloses the application of mangiferin and its derivatives in the preparation of anti-prostatic hyperplasia drugs, and those skilled in the art can learn from the content of this article and appropriately improve the process parameters to realize it. In particular, it should be pointed out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The application of the present invention has been described through the preferred embodiments, and the relevant personnel can obviously make changes or appropriate changes and combinations to the methods and applications described herein without departing from the content, spirit and scope of the present invention to realize and apply the present invention. Invent technology.

In order to enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below in conjunction with specific examples.

Example 1: Inhibition of 5α-reductase in vitro

1. Experimental materials

SD female mice, Kunming Medical University; Testosterone, Shanghai Yuanye Biotechnology Co., Ltd. (20130409); NADPH, sigma company (Lot SLBC6718V); Finasteride, Hubei Subang Pharmaceutical Co., Ltd. (20121001); Tris-base, BIOSHARP Company (Amresco0497); Sodium Chloride, Sangon Biological Company (Lot3004B220); DTT, Sigma Company (LotBCBK8875V); Absolute Ethanol, Yunnan Shandian Pharmaceutical Co., Ltd. (Lot LN70N21); Dichloromethane, Tianjin Windboat Chemical Reagent Technology Co., Ltd. (20100926); Chromatographic methanol, Beijing Bailingwei Technology Co., Ltd. (116481); Mangiferin; 1-hydroxy-3,6,7-triacetoxymangiferin; 3-hydroxy-1,6 , 7-triacetoxymangiferin; 1,3-dihydroxy-6,7-diacetoxymangiferin; 6,7-dihydroxy-1,3-diacetoxymangiferin, purity All are greater than 98%, provided by Kunming Pharmaceutical Group Drug Research Institute.

2. Experimental equipment

DHG-9245A electric blast drying oven, Shanghai Yiheng Scientific Instrument Co., Ltd.; Biofuge refrigerated high-speed centrifuge, Thermo Company; e2695 high performance liquid chromatography, Waters Company.

3. Experimental content

3.1 Protein quantification

The liver of female SD rats was fasted overnight to prepare 5α-reductase, and the improved BCA method was used for protein quantification, and the operation steps were strictly followed the instructions of the kit. The protein quantification standard curve is as follows:

3.2 System establishment

This system is optimized on the basis of the original system. Use the high-performance liquid chromatography external standard method to measure the remaining amount of the substrate testosterone, and then subtract the remaining amount of testosterone after the reaction from the total amount of testosterone participating in the reaction to obtain the amount of testosterone consumed in the reaction system, and then calculate the enzyme activity.

Chromatographic conditions: luna C18(2) column (4.6x250mm, 59m); column temperature: 40°C; injection volume: 10uL; flow rate: 1mL/min; mobile phase: 70% methanol; detection wavelength: 242nm.

3.2.1 Testosterone standard curve formulation

As shown in Figure 2, the abscissa is the amount of 10uL testosterone, and the ordinate is the peak area. It can be seen from the figure that the amount of testosterone and the peak area have a good linear relationship.

3.2.25 α-reductase concentration determination

It can be seen from Figure 3 that as the enzyme concentration increases, its activity increases, and when the enzyme concentration reaches 8.675mg/mL, the enzyme activity decreases instead, which may be caused by the excessively high enzyme concentration inhibiting the conversion of testosterone.

3.2.3 Effect of different substrate concentrations on enzyme activity

As shown in Figure 4, the enzyme activity increased with the increase of the substrate concentration, and the enzyme activity was maximum when the substrate concentration was 2 mg/mL.

3.2.4 Effect of different concentrations of NADPH on enzyme activity

As shown in Figure 5, as the concentration of NADPH increases, the enzyme activity also increases. On the premise of not affecting the results, the concentration of NADPH is selected to be 5 mg/mL from an economic point of view.

In summary, it was determined that the enzyme concentration in the reaction system was 5.045 mg/mL, the substrate testosterone concentration was 2 mg/mL, and the NADPH concentration was 5 mg/mL (maximum enzyme activity).

3.3 Determination of the inhibitory activity of finasteride on 5α-reductase

Finasteride is a commonly used drug in the clinical treatment of benign prostatic hyperplasia and is an effective 5α-reductase inhibitor. Under certain reaction conditions, absolute ethanol was replaced by finasteride, and the inhibitory activity of finasteride at various concentrations to 5α-reductase was measured, and then an absolute ethanol reaction well and a blank well (before adding the enzyme) were made. Dichloromethane was added to terminate the reaction). According to the inhibitory activity=(T _finasteride- T _reaction )/(T _blank -T _reaction )×100%, the IC ₅₀ value of finasteride inhibiting 5α-reductase was calculated. The results are shown in Figure 6, calculated from Figure 6, the IC ₅₀ of finasteride inhibiting 5a-reductase is 275.7nM, which is similar to that reported in the literature. It can be confirmed that the enzyme extract derived from female rat liver microsomes has 5α-reductase activity, and the in vitro screening method for anti-prostatic hyperplasia drugs targeting 5α-reductase is feasible and can be used for drug screening.

4. Determination of the inhibitory activity of mangiferin and its derivatives on 5α-reductase

It has been reported in the literature that mangiferin has an inhibitory effect on 5α-reductase. Our previous research results also show that the bioavailability of mangiferin is much higher than that of mangiferin, and the cost of preparing mangiferin by synthesis is also significantly lower than that of mangiferin. .

Prepare different concentrations of mangiferin and its derivatives with 100% DMSO, make a 100% DMSO reaction well and a blank well (add dichloromethane before adding enzyme to terminate the reaction). Inhibitory activity of mangiferin and its derivatives on 5α-reductase=(T _sample -T _reaction )/(T _blank -T _reaction )*100%. The results are shown in Figure 7-11. The calculated IC ₅₀ of mangiferin and its derivatives is between 17.7-43.63uM, and the inhibitory activity of different concentrations of mangiferin and its derivatives on 5α-reductase is linearly dependent sex.

Example 2: Investigation of the In Vivo Pharmacological Effects of Testosterone Propionate Replication Prostatic Hyperplasia Model Mice

1. Test material

1.1 Animals

SPF-grade male ICR mice, weighing 20-22 g, were provided by the Animal Department of Kunming Pharmaceutical Group Co., Ltd., license number SCXK (Dian).

1.2 Test substance

Mangiferin, light yellow powder, purity >98%, provided by Kunming Pharmaceutical Group Research Institute, batch number 20130903; Finasteride Tablets (Guoyao Zhunzi H20070146), 5mg/tablet, Hubei Subang Pharmaceutical Co., Ltd., batch number: 0121001 ; Longbishu Capsules (Z10960007), 0.3g/capsule, Shijiazhuang Kedi Pharmaceutical Co., Ltd., batch number: 111113; use pure water to make suspensions of different concentrations for later use.

1.3 Reagents

Testosterone Propionate Injection (Guoyao Zhunzi H12020531), 10mg/mL, Tianjin Jinyao Amino Acid Co., Ltd., batch number 1202051; Mouse Androgen (T), Estrogen (E2) Kit, R&D Company, batch number 05/2013.

2. Experimental method

2.1 Experimental process

84 20-22g male Kunming mice were randomly divided into 7 groups according to body weight: normal control; model control; finasteride 1mg.kg ^-1 ; Longbishu capsule 0.45g.kg ^-1 ; mangiferin aglycone 60, 30, 15 mg.kg ^-1 ; 12 rats in each group. Except for the normal control group, each model animal group was dosed with 5 mg.kg ^-1 sc testosterone propionate in the morning and intragastrically in the afternoon. The model group was given normal saline with a volume of 20mL.kg ^-1 ; Day, weighed once a week. After the last administration, fasting without food and water for 12 hours, taking blood after weighing, separating the serum and measuring the T and E2 levels in the serum according to the method of ELLISA kit; killing the mice by dislocation of the cervical spine, quickly taking out the prostate and weighing, and calculating the prostate index of the mice in each group (Prostate index=mg prostate wet weight/mouse body weight g×10).

2.2 Data processing

The measurement data are used as mean ± standard deviation ( ) indicates that the t test is used for homogeneous variance, and the t' test is used for uneven variance; non-parametric rank sum test is used for skewed distribution; P<0.05 is statistically significant,

P<0.01 is statistically significant.

3. Experimental results

3.1 Effects on body weight of BPH mice

It can be seen from Table 1 that the weight of the mice in each group continued to increase during the experiment, and there was no significant difference in the weight increase of the mice between the model group and the normal control group, and between the administration groups and the model group.

Table 1 Effects on body weight of BPH mice

Compared with the model group: P>0.05

3.2 Effects on wet weight and index of prostate in BPH mice

It can be seen from Table 2 that the wet weight and index of the prostate in the model group mice were significantly increased, and the difference was very significant compared with the normal control group (P<0.01), indicating that the modeling was successful; In addition to the close statistical difference (P>0.05), the three dose groups of mangiferin aglycone and the positive control finasteride group can significantly reduce the wet weight and index of the prostate in model mice (P<0.05/0.01), in which mango The effects of the three dose groups of aglycon are dose-dependent.

Table 2 Effects on BPH mouse prostate wet weight and index

Compared with the normal group: ^▲▲ P<0.01; compared with the model group:

^*/** P<0.05/0.01

3.3 Effects on 5α-reductase activity in the liver of BPH mice

It can be seen from Table 3 that the activity of 5a-reductase in the liver homogenate of the mice in the model group was significantly higher than that of the normal control group, indicating that a large amount of exogenous androgen induced the increase of the activity of 5a-reductase; 5α-reductase activity in the liver homogenate of the positive control finasteride and mangiferin three dose groups was significantly lower than that of the model group (P<0.05/0.01) , which is consistent with the in vitro test results.

Table 3 Effects on 5a-reductase activity in the liver of BPH mice

Compared with the normal group: ^▲ P<0.05; compared with the model group: **P<0.01

3.4 Effects on Estrogen/Androgen Levels in Serum of BPH Mice

As can be seen from Table 4, the estrogen level and E2/T ratio in the serum of the model group mice were significantly higher than the normal control group (P<0.01/0.05), and the androgen level was only higher than the normal group (P>0.05). It shows that BPH mice are accompanied by endocrine disorders. Except for nasteride, each administration group can reduce the serum E2 level and E2/T ratio of the model animals to varying degrees, and the three doses of mangiferin aglycone are significantly different from those of the control group (P<0.05/0.01); unless that In addition to andramine, each administration group can increase the serum T level of model animals to varying degrees (P>0.05), and the effect of the dose of mangiferin aglycon is close to statistical difference.

The impact of table-4 on hormone levels in serum of BPH mice

Compared with the normal group, ^▲/▲▲ P<0.05/0.01; compared with the model group, ^*/** P<0.05/0.01

4. Summary

Testosterone is the main androgen in the body, it becomes dihydrotestosterone under the action of 5α-reductase, and the increase of DHT concentration in the prostate can lead to glandular hyperplasia. In addition, the theory of estrogen and androgen disorder believes that an increase in the E/T ratio will lead to benign prostatic hyperplasia. Prostate wet weight and prostate index can directly reflect the status of benign prostatic hyperplasia.

The model mice were subcutaneously injected with exogenous testosterone propionate for 21 consecutive days, but the serum testosterone level only increased compared with the normal group, while the estrogen level and the E2/T ratio were significantly increased, suggesting that a large amount of testosterone removed In addition to being transformed into DHT that causes glandular hyperplasia in the prostate, some of it may be converted into E2 through the action of aromatase. The level of E2 in the finasteride group was higher than that in the model group, which may be related to the high selective inhibitory activity of finasteride on type II 5α-reductase. Finasteride reduces the production of dihydrotestosterone by inhibiting 5α-reductase, Due to the negative feedback regulation, T is converted into E2 through the aromatase pathway, which leads to the side effects of long-term use of finasteride (male feminization and other characteristics). The E2 level and E2/T ratio in the mangiferin group were significantly lower than those in the model group, but the T level was increased, which was different from the action characteristics of finasteride, which may be related to the multi-target effect of natural medicines.

With reference to the above experimental method, the in vivo pharmacological effects of mangiferin aglycon derivatives on testosterone propionate replicating benign prostatic hyperplasia model mice were investigated, and the results showed that they had similar pharmacological effects to mangiferin aglycone.

Example 3: Acute Toxicity Test by Oral Gavage in Mice

Preliminary tests showed that the LD ₅₀ of mangiferin aglycone could not be measured by oral administration to mice, so the acute toxicity of the test substance was reflected by the maximum dose administered twice a day. During the test, after the animals were fasted and watered for 8 hours, 40 animals weighing 19.5-21.9 g were selected, half male and half male, and randomly divided into a treatment group and a control group according to sex and body weight, with 10 male and 10 males in each group. The mice in the treatment group were given 0.27g/mL mangiferin paste by oral gavage at the maximum capacity of 40mL/kg, and then administered again after an interval of 6h. The equivalent dose was 21.6g/kg.d, and the control group was given the same volume of pure water. As a result, there were no obvious toxic reactions in the appearance, behavior, mental state, appetite, urine and stool, fur, skin color and breathing of the two groups of animals. After 14 days of continuous observation, all the animals survived in good health, and their body weight increased significantly. There was no statistical difference between the groups. The results are shown in Table 5. Animals were killed and dissected, and visceral tissues such as heart, liver, spleen, lung, and kidney were observed with naked eyes, and no obvious abnormalities were found.

Table 5 Mangiferin mice gavage maximum dosage test body weight observation ( g)

Compared with the control group, p>0.05

The maximum administration dose of mangiferin aglycon in mice was 21.6g/kg in one day, equivalent to 1440 times of the pharmacodynamically effective dose of 15mg/kg for the same animal, and no obvious toxic reaction or death occurred in the animals.

In summary, mangiferin aglycon has obvious inhibitory effect on 5α-reductase both in vivo and in vitro, and can significantly reduce the wet weight and index of the prostate gland of testosterone propionate-replicated benign prostatic hyperplasia model mice, and significantly reduce the serum estrogen level and E/T ratio. No obvious toxic reaction was found in the acute toxicity test of the maximum dosage in mice. The inhibitory effect of mangiferin on benign prostatic hyperplasia is closely related to the inhibition of 5α-reductase, and is also related to the regulation of endocrine. Mangiferin aglycone has potential multi-target therapeutic properties and high safety for benign prostatic hyperplasia, and has a simple structure and is easy to chemically synthesize, so it has high potential for clinical application.

Embodiment 4 : 1,3,6,7-tetraacetoxymangiferin soft capsules

Preparation method: Take mangiferin, add it to soybean oil, stir to dissolve it completely, press into soft capsules, dry, and make 1000 capsules, that is, the product with a specification of 30mg.

Embodiment 5 : mangiferin soft capsule

Preparation method: Take mangiferin, add it to soybean oil, stir to dissolve it completely, press into soft capsules, dry, and make 1000 capsules, that is, the product with a specification of 200mg.

Embodiment 6 : 3-hydroxyl-1,6,7-triacetoxy mangiferin soft capsules

Preparation method: take mangiferin aglycon, add it to peanut oil, stir to dissolve it completely, press soft capsule, dry, and make 1000 capsules, that is, the product with a specification of 500mg.

Embodiment 7 : 1,3-dihydroxy-6,7-diacetoxy mangiferin tablets

Preparation method: pass the materials through 100 mesh sieve respectively, weigh the materials according to the prescription amount, mix evenly, stir to make soft material, granulate with 18-24 mesh sieve, ventilate and dry at 70°C, press into tablets, coat with film, and get the specification It is a 60mg product.

Embodiment 8 : 6,7-dihydroxy-1,3-diacetoxymangiferin tablets

Preparation method: pass the materials through 100 mesh sieve respectively, weigh the materials according to the prescription amount, mix evenly, stir to make soft material, granulate with 18-24 mesh sieve, ventilate and dry at 70°C, press into tablets, coat with film, and get the specification For 100mg product.

Embodiment 9 : 1,3,6,7-tetraacetoxymangiferin capsules

Preparation method: pass the materials through a 100 mesh sieve, weigh the materials according to the prescription, mix them evenly, stir to make soft materials, granulate with a 18-24 mesh sieve, mix evenly, ventilate and dry at 60°C, and fill them in hard capsules That is the product whose specification is 300 mg.

The above is only a preferred embodiment of the present invention, it should be pointed out that, for those of ordinary skill in the art, without departing from the principle of the present invention, some improvements and modifications can also be made, and these improvements and modifications can also be made. It should be regarded as the protection scope of the present invention.

Claims (3)

1. have the application of the compound of following structural formula in the medicine for the preparation treatment of benign prostatic hyperplasia, Wherein, R ₁ , R ₂ , R ₃ and R ₄ are H. 2. The application according to claim 1, characterized in that the effective dose of the compound is 10-1000 mg/kg/d. 3. The application according to claim 1, wherein the medicine is: a tablet, a granule, a capsule, a pill, a suspension or an injection prepared by adding a pharmaceutically acceptable auxiliary material to the compound.