Application of malus spectabilis tea extract in preparation of medicine for treating benign prostatic hyperplasia

Abstract

The invention provides an application of a malus spectabilis tea extract in preparation of a medicine for treating benign prostatic hyperplasia. The influence of the malus spectabilis tea extract on a benign prostatic hyperplasia rat is discussed on the basis of activity inhibition of 5 alpha-reductase. Results show that the malus spectabilis tea water extract and phlorizin have no obvious toxic and side effects on visceral organs of each group of rats; the PI of the malus spectabilis water extract is obviously reduced, the glandular epithelial cell thickness is obviously reduced, the 5AR activity of the malus spectabilis water extract is obviously reduced, the blood glucose of each dose group and PG group of HT is obviously reduced, the concentration of total cholesterol and low-density lipoprotein is obviously reduced, the high-density lipoprotein level can be obviously increased by HT-M, HT-H and PG, and the content of triglyceride can be reduced by HT-H and PG. The results show that HT and PG have a remarkable inhibiting effect on prostatic hyperplasia. The inhibition of 5AR activity and the reduction of DHT generation and T content are one of the action mechanisms, and the important characteristic is that the prostatic hyperplasia can be inhibited and abnormal blood sugar and blood fat can be regulated at the same time.

Description

Technical Field

[0001] The present invention relates to the application of Malus hupehensis Rehd. extract in the preparation of a medicament for treating benign prostatic hyperplasia, and belongs to the field of medical technology. Background Art

[0002] Benign Prostatic Hyperplasia (BPH) is a common urological disease that mostly occurs in middle-aged and elderly men. Epidemiological reports show that among men aged 50 and above, the prevalence of BPH is 50% - 75%; when the age reaches 70 and above, the prevalence of this disease is as high as 80%. The prevalence of BPH shows a gradually increasing trend with age, and the prevalence of BPH in men under 40 years old is also increasing year by year. Currently, it is generally believed that prostatic hyperplasia is mainly related to sex hormone imbalance. In prostate tissue, testosterone (T) is converted under the catalytic action of 5α-reductase (5AR) to generate dihydrotestosterone (DHT). The binding ability of DHT to androgen receptors is 3 - 10 times higher than that of T. When the activity of 5α-reductase increases, a large amount of DHT is generated, promoting the excessive differentiation and proliferation of prostate stromal cells and epithelial cells, and inducing BPH. 5α-reductase inhibitors are the first-line drugs for treating BPH clinically, but it has been found in clinical use that these drugs can cause adverse reactions such as decreased libido, erectile dysfunction, and reduced ejaculatory volume. Therefore, finding safe and effective drugs has become a research hotspot in the treatment of BPH. Traditional Chinese medicine has advantages such as definite curative effect and few adverse reactions in the treatment of prostatic hyperplasia, and there is a good prospect for finding drugs for treating prostatic hyperplasia from traditional Chinese medicine.

[0003] Malus hupehensis Rehd., also known as wild Chinese flowering crabapple, wild flower red, flower black tea, and tea Chinese flowering crabapple, has a long history of using its young leaves as tea for drinking. It is rich in flavonoids mainly composed of phloridzin, and has functions such as lowering blood sugar and protecting the liver, and is mainly used for treating chronic hepatitis, fatty liver, acute and chronic liver injury, hyperglycemia, hyperlipidemia, etc. However, there is currently no report on the treatment of BPH with Malus hupehensis Rehd. and its active ingredients, and there is no research report on the effect on 5α-reductase. The purpose of this study is to explore the effect of Malus hupehensis Rehd. extract, namely the water extract of Malus hupehensis Rehd. tea and the main active ingredient phloridzin, on 5α-reductase, so as to provide a basis for further using Malus hupehensis Rehd. to treat BPH. Summary of the Invention

[0004] The present invention provides a 5α-reductase inhibitor, and the inhibitor is the water extract of Malus hupehensis Rehd. tea.

[0005] The preparation method of the described Malus spectabilis tea extract comprises the following steps: Malus spectabilis tea leaves are added with water and extracted at 80-90 °C for 15-45 min. The extraction solutions are combined, filtered, concentrated under reduced pressure, and spray-dried to obtain the Malus spectabilis tea water extract.

[0006] Application of the described 5α-reductase inhibitor in the preparation of a medicament for treating prostatic hyperplasia.

[0007] The present invention also provides an application of the Malus spectabilis tea water extract in the preparation of a medicament for treating prostatic hyperplasia.

[0008] The application of the described Malus spectabilis tea water extract in the preparation of a medicament for treating prostatic hyperplasia is achieved by reducing the total volume and wet weight of the prostate and seminal vesicles.

[0009] The application of the described Malus spectabilis tea water extract in the preparation of a medicament for treating prostatic hyperplasia is achieved by improving cell morphology, making the vesicular tubular structure clearly visible, reducing the folds in the acini, and decreasing the thickness of the glandular epithelium.

[0010] The application of the described Malus spectabilis tea water extract in the preparation of a medicament for treating prostatic hyperplasia is achieved by reducing the increase in BPH serum T level caused by exogenous testosterone propionate (TP).

[0011] The application of the described Malus spectabilis tea water extract in the preparation of a medicament for treating prostatic hyperplasia is achieved by reducing the BPH serum DHT level.

[0012] The application of the described Malus spectabilis tea water extract in the preparation of a medicament for treating prostatic hyperplasia is achieved by reducing blood sugar and blood lipids.

[0013] The dosage of the Malus spectabilis tea water extract is 60-240 mg·kg -1 。 Description of the Drawings

[0014] Figure 1 For the effect of HT on the sex organs of BPH rats (n = 6, ).

[0015] Figure 2 For HE staining of the rat prostate (n = 6, ). Detailed Description of the Invention

[0016] Example 1

[0017] The leaves of Malus hupehensis were collected from Langping Town, Changyang Tujia Autonomous County, Hubei Province, and identified by Professor Wang Yubing of China Three Gorges University as the leaves of Malus hupehensis (Rosaceae); Phloridzin: Self-made in the laboratory, with a purity of ≥98%; Olive oil (pharmaceutical grade, LOT: C17295559): Shanghai Macklin Biochemical Co., Ltd.; Finasteride (LOT: RH531198): Shanghai Aladdin Biochemical Technology Co., Ltd.; Sodium carboxymethyl cellulose (LOT: 20141209): Sinopharm Chemical Reagent Co., Ltd.; Sodium penicillin (LOT: 20230901): Shaanxi Lixiang Animal Pharmaceutical Co., Ltd.; DHT ELISA Kit (LO: WV05ZR840158), Rat T ELISA Kit (LOT: WV062T602034): Wuhan Elabscience Biotechnology Co., Ltd.; Rat 5AR ELISA Kit (LOT: 111935006207391119): Shanghai Jianglai Biotechnology Co., Ltd.; Triglyceride test kit (LOT: 20241211), Total cholesterol kit (LOT: 20241217), Glucose kit (LOT: 20241113), Low-density lipoprotein kit (LOT: 20241217), High-density lipoprotein kit (LOT: 20241217): Nanjing Jiancheng Bioengineering Institute.

[0018] Animals

[0019] Fifty-six SPF-grade male SD rats, with a body weight of (210±10) g, were provided by the Animal Experiment Center of China Three Gorges University and were housed in the SPF-grade animal laboratory of the Animal Experiment Center. The feeding conditions were as follows: relative humidity (55±5)%, temperature 20-25°C, and a 12 / 12 h light-dark cycle for free eating and drinking. The production license number of experimental animals was SCXK(E)2022-0012, the license number for the use of experimental animal facilities was SYXK(E)2022-0061, and the approval number for animal ethics review was 2024020V1.

[0020] Preparation of aqueous extract of Malus hupehensis tea

[0021] Aqueous extract of Malus hupehensis tea: Malus hupehensis tea leaves were extracted with 10 times the amount of water at 80°C for 15-45 minutes. The extract was combined, filtered, and concentrated under reduced pressure (negative pressure -0.07 MPa, temperature 80°C) to a solid mass fraction of about 30%. Spray drying (inlet air temperature: 185°C, outlet air temperature 100°C, flow rate 15-20 mL per minute), or hot air drying at about 80°C, was used to control the moisture content of the aqueous extract of Malus hupehensis tea between 5% and 10%. The average yield was 20-30%, and the aqueous extract of Malus hupehensis tea contained 30-40% phloridzin (detected by high performance liquid chromatography).

[0022] Establishment of BPH model and grouped administration

[0023] Forty-two male Sprague-Dawley rats, weighing 200 - 220 g, were anesthetized with 2% sodium pentobarbital. After routine disinfection of the skin, the rats in the sham operation group had their skin incised, and the testes were exposed to the air for several seconds without excision, followed by immediate suture. For the remaining rats, the skin was routinely disinfected, and both testes were removed through the scrotum. The stumps were ligated, and after ensuring hemostasis, the skin was sutured. The postoperative rats were intramuscularly injected with sodium penicillin (200,000 U / kg / d) for 5 consecutive days to prevent infection. After one week of recovery, the castrated rats were grouped using the random number table method based on their weighed body weights. They were divided into 7 groups of 6 rats each, namely the control group (Control group), the model group (Model group), the finasteride group (Fina group, 5 mg·kg -1 )), the low-dose group of aqueous extract of Malus spectabilis tea (HT-L, 60 mg g·kg -1 )), the medium-dose group of aqueous extract of Malus spectabilis tea (HT-M, 120 mg g·kg -1 )), the high-dose group of aqueous extract of Malus spectabilis tea (HT-H, 240 mg g·kg -1 ), the phloridzin group (PG, 120 mg·kg -1 ). The castrated rats were subcutaneously injected with testosterone propionate (TP) 5 mg / kg (dissolved in olive oil) daily, and the gavage volume was 0.1 mL per 100 g. The Control group was subcutaneously injected with an equal dose of olive oil and simultaneously gavaged with drugs, and the gavage volume was 1 mL per 100 g. The Control group and the Model group were gavaged with 0.5% sodium carboxymethylcellulose, and the remaining groups were gavaged with the corresponding concentration of drugs. The model was established and drugs were administered continuously for 28 days.

[0024] Animal treatment and sampling

[0025] After the last drug administration, the rats were fasted but allowed to drink water for 12 h, weighed, anesthetized by intraperitoneal injection of 2% sodium pentobarbital, and blood was collected from the abdominal aorta. After standing at room temperature for 2 h, it was centrifuged at 4000 rpm for 15 minutes at 4°C, and the supernatant was taken to obtain rat serum, which was aliquoted and stored in an -80°C refrigerator for subsequent biochemical index detection.

[0026] After the rat blood was collected, the prostate, seminal vesicle gland tissues, and other visceral tissues of the rats were separated, washed with PBS to remove blood, and blotted dry with filter paper and weighed.

[0027] Index observation and detection

[0028] Changes in rat body weight

[0029] The activity of rats in each group was observed and recorded daily during drug administration, and the body weight was recorded every 3 days after the operation.

[0030] Observation of prostate tissue morphology

[0031] Place the washed prostate and seminal vesicle tissues on graph paper and observe the tissue volume size.

[0032] Calculation of organ coefficient

[0033] Take the intact heart, liver, spleen, lungs and kidneys, dry them with filter paper and weigh them, then calculate the organ coefficient: Organ coefficient = Organ mass (g) / Body mass (g) × 100%

[0034] Determination of prostate wet weight and prostate index

[0035] Isolate the prostate from the tissue, weigh it, and calculate the prostate index: Prostate index (PI) = Prostate wet weight (mg) / Body mass (g).

[0036] Pathological observation of prostate tissue

[0037] Fix the prostate tissue in 4% paraformaldehyde for 24 hours, dehydrate it, embed it in paraffin, section it, and perform HE staining, then observe the pathological changes of rat prostate tissue under an inverted microscope.

[0038] Determination of hormone T and DHT levels in serum

[0039] Both the T kit and the DHT kit use the competitive ELISA method. Operate strictly according to the kit instructions, measure the OD value at a wavelength of 450 nm with an enzyme-labeled instrument, and calculate the concentrations of T and DHT in rat serum by drawing a standard curve.

[0040] Determination of 5AR content in serum

[0041] The 5AR kit uses the double antibody sandwich enzyme-linked immunosorbent assay (ELISA). Measure the absorbance (OD value) at a wavelength of 450 nm with an enzyme-labeled instrument, and calculate the concentration of 5AR in rat serum by drawing a standard curve.

[0042] Determination of blood glucose level in serum

[0043] Use the glucose oxidase method to strictly measure the change in blood glucose content in rat serum according to the kit instructions.

[0044] Determination of blood lipid level in serum

[0045] Total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) are measured strictly according to the kit instructions.

[0046] Statistical analysis

[0047] Statistical analysis was performed using SPSS 25.0, and GraphPad Prism 8.0.1 was used to create tables. All data of each group were expressed as . One-way ANOVA with Dunnett's post hoc test was used for comparison between groups, and P < 0.05 was considered statistically significant.

[0048] Results

[0049] Effect of HT on behavioral activities of rats with benign prostatic hyperplasia

[0050] No animals died during the castration surgery and the period of modeling and drug administration. During the period of modeling and drug administration, the behaviors of rats in each group, such as activity, diet, and defecation, were normal. The body weights of rats were recorded after the surgery, and there was no significant difference in the body weights of rats during the drug administration period compared with the control group.

[0051] Effect of HT on organ coefficients of rats with benign prostatic hyperplasia

[0052] The organ indices of rats in each group were statistically analyzed. The results showed that there were no significant differences in the organ coefficients of the Model group compared with the Control group, and there were also no significant differences in the organ coefficients of the drug intervention groups compared with the Model group and the Control group, indicating that the aqueous extract of Chinese flowering crabapple and phloridzin had no obvious toxic and side effects on the organs of rats after administration at the current dose for 28 days.

[0053] Table 1 Organ coefficients of rats in each group (n = 6, )

[0054]

[0055] Effect of HT on prostate tissue morphology

[0056] Observing the completely dissected prostate, it was found that compared with the Control group, the rats in the Model group showed significant prostate enlargement, and the total volume of the prostate and seminal vesicles increased significantly. By comparison, it was found that compared with the model group, the prostate volumes of the finasteride group, the HT-L, HT-M, HT-H dose groups, and the PG group all decreased to a certain extent (as Figure 1 shown).

[0057] Determination of prostate wet weight and prostate index (PI)

[0058] The prostate index results are shown in Table 2. Compared with the Control group, the wet weight of the prostate in the Model group and the drug intervention groups was significantly increased, and the prostate index was significantly elevated, with significant differences in both cases (P < 0.01), indicating that subcutaneous injection of testosterone propionate for 28 days could successfully induce a BPH model; compared with the Model group, finasteride, high-dose HT, and PG could all significantly reduce the wet weight of the prostate and the prostate index in BPH rats (P < 0.01, P < 0.05), and HT showed a dose-dependence. The above results indicate that both HT and phloridzin, the main active ingredient in Begonia tea, have the potential to treat benign prostatic hyperplasia.

[0059] Table 2 Wet weight and prostate index of rats (n = 6, )

[0060]

[0061] Note: Compared with the Control group, * P < 0.05, ** P < 0.01; compared with the Model group, # P < 0.05, ## P < 0.01 (the same below).

[0062] Effect of HT on histopathological changes of the prostate

[0063] Figure 2 Shown from top to bottom in the middle are HE tissue section images magnified 40 times, 200 times, and 400 times respectively under the same microscope field of view. HE staining showed that the vesicular tubular structure of the prostate tissue in the Control group was clear, the epithelial cells of the glandular lumen were arranged neatly, and there was no hyperplasia in the glands and stroma, and no obvious internal folds protruding into the lumen were seen. Compared with the Control group, the arrangement of epithelial cells in the Model group was disordered, the number of folds in the alveoli increased significantly, the columnar epithelial cells of the prostate showed obvious hyperplasia, and the internal folds increased and protruded into the lumen in a papillary shape.

[0064] The thickness of the glandular epithelial cells in the prostate tissue under 200-fold magnification was measured using Image J analysis software, and the proportion of the area of the hyperplastic glandular epithelial cells in the current field of view was calculated to more clearly reflect the degree of hyperplasia of the prostate tissue. The results are shown in Table 3. Compared with the Model group, the cell morphology in the finasteride group was improved, the vesicular tubular structure was clear, the thickness of the prostatic epithelial cells was significantly reduced (P < 0.01), and the proportion of the area of the glandular epithelial cells in the field of view was also significantly decreased (P < 0.01). A small number of internal folds protruded papillarily into the lumen, and the degree of protrusion was lower than that in the Model group. Compared with the Model group, after HT treatment, the cell morphology was significantly improved, the vesicular tubular structure was clearly visible, similar to that of the control group, the internal folds in the acini were reduced, and the thickness of the glandular epithelium was also significantly decreased (P < 0.05, P < 0.01). Moreover, the proportion of the area of the glandular epithelial cells in the fields of view of the HT-M and HT-H groups was also significantly decreased, showing a highly significant difference (P < 0.01). A small number of internal folds protruded papillarily into the lumen, and the degree of protrusion was significantly lower than that in the Model group. The cell morphology in the PG treatment group was also well improved, the vesicular tubular structure was clearer than that in the Model group, the thickness of the epithelial cells was significantly reduced (P < 0.01), the proportion of the area of the glandular epithelial cells was also significantly decreased compared with the Model group (P < 0.01), and fewer internal folds protruded papillarily into the lumen, and the degree of protrusion was lower than that in the Model group. The above results suggest that HT can significantly alleviate the pathological state of prostate hyperplasia and has a good therapeutic effect on lower urinary tract symptoms.

[0065] Table 3 Thickness and area proportion of rat prostatic epithelium (n = 6, )(n = 6, )

[0066]

[0067] Effect of HT on serum 5AR content in BPH rats

[0068] As shown in Table 4, compared with the Control group, the activity of 5AR in the Model group was significantly increased (P < 0.01). Compared with the Model group, the activities of 5AR in the finasteride group, HT group, and PG group were all significantly decreased (P < 0.01), and the HT group showed a dose-dependence. The above results indicate that HT and PG can improve prostate hyperplasia by inhibiting the activity of 5AR.

[0069] Table 4 Effect of HT on 5AR in BPH rats (n = 6, )

[0070]

[0071] Effect of HT on serum T and DHT contents in BPH rats

[0072] Effect on serum T content

[0073] Compared with the Control group, the content of testosterone (T) in the serum of rats in the Model group increased significantly (P < 0.01); compared with the Model group, the content of T in the serum of rats in the finasteride group, HT group and PG group decreased significantly (P < 0.01). As the dose of HT increased, the content of T in the serum of BPH rats decreased, indicating that HT has a dose-dependent effect in reducing the content of T. The experimental results show that HT and PG can improve the pathological state of prostatic hyperplasia by reducing the testosterone level in the body during benign prostatic hyperplasia.

[0074] Table 5 Effect of HT on the content of T in BPH rats (n = 6, )

[0075]

[0076] Effect on serum DHT content

[0077] As can be seen from Table 6, compared with the Control group, the content of dihydrotestosterone (DHT) in the serum of rats in the Model group increased significantly (P < 0.01); compared with the Model group, the content of DHT in the serum of rats in the finasteride group, HT group and PG group decreased significantly (P < 0.01). As the dose of HT increased, the content of DHT also showed a gradually decreasing trend, indicating that HT has a concentration-dependent effect in reducing the content of DHT; and the effect of PG and high-concentration HT in reducing the content of DHT is better than that of the positive drug finasteride. The change trend of DHT is consistent with the change trend of T concentration, suggesting that HT and PG can also reduce the content of DHT in the body while reducing the content of T in the body, and HT and PG can improve the pathological state of prostatic hyperplasia by simultaneously reducing the levels of DHT and T.

[0078] Table 6 Effect of HT on the content of DHT in BPH rats (n = 6, )

[0079]

[0080] Effect of HT on the serum blood glucose level of BPH rats

[0081] By measuring the blood glucose concentration in the serum of BPH rats, it was found that compared with the Control group, the blood glucose content in the body of BPH rats in the Model group induced by testosterone propionate increased; compared with the Model group, the finasteride group, HT group and PG group could all reduce the blood glucose concentration in the body of BPH rats (P < 0.015, P < 0.01), and the hypoglycemic effect of PG was consistent with that of the positive drug at the same concentration, and it was statistically significant compared with the Model group (P < 0.01). It shows that PG, as the main component of HT, can also treat BPH by reducing blood glucose.

[0082] Table 7 Effects of HT on blood glucose in BPH rats (n = 6, )

[0083]

[0084]

[0085] Effects of HT on serum lipid levels in BPH rats

[0086] As shown in Table 8, compared with the Control group, the total cholesterol content in the Model group increased, indicating an upward trend in serum total cholesterol in rats after modeling. The positive drug finasteride group was also slightly higher than the Control group, suggesting that finasteride might promote the synthesis of total cholesterol. Compared with the Model group, the total cholesterol levels in the high-dose HT group and the PG group were significantly decreased (P < 0.05, P < 0.01), indicating that PG and HT had the effect of reducing total cholesterol at a certain dose.

[0087] Compared with the Control group, the triglyceride level in the Model group increased slightly, and the triglyceride content in the finasteride group was higher, suggesting that finasteride might promote the synthesis of triglyceride. Compared with the Model group, both the HT group and the PG group had the effect of reducing triglyceride, and the effects of PG and high-dose HT on reducing triglyceride were obvious (P < 0.01).

[0088] Compared with the Control group, the low-density lipoprotein contents in both the Model group and the finasteride group were higher (P < 0.01), and the high-density lipoprotein content in the Model group was significantly decreased (P < 0.01); compared with the Model group, the low-density lipoprotein contents in the HT group and the PG group were lower (P < 0.01, P < 0.05), and the high-density lipoprotein content increased, and there were significant differences in the high-dose HT group and the PG group (P < 0.01, P < 0.05), suggesting that HT and PG had the effects of regulating low-density lipoprotein levels and high-density lipoprotein levels.

[0089] Table 8 Effects of HT on blood lipids in BPH rats (n = 6, )

[0090]

[0091]

Claims

1. 5α - reductase inhibitor, characterized in that, The inhibitor is the extract of Chinese flowering crabapple tea.

2. The 5α-reductase inhibitor according to claim 1, wherein The preparation method of the extract of Chinese flowering crabapple tea comprises the following steps: adding water to Chinese flowering crabapple tea leaves, extracting at 80-90 °C for 15-45 min, combining the extracts, filtering, concentrating under reduced pressure, and spray drying to obtain the water extract of Chinese flowering crabapple tea.

3. Use of the 5α-reductase inhibitor according to claim 1 or 2 in the preparation of a medicament for treating benign prostatic hyperplasia.

4. Use of a water extract of Chinese flowering crabapple tea in the preparation of a medicament for treating benign prostatic hyperplasia.

5. The application according to claim 4, characterized in that The use of the water extract of Chinese flowering crabapple tea in the preparation of a medicament for treating benign prostatic hyperplasia is achieved by reducing the total volume and wet weight of the prostate and seminal vesicles.

6. The application according to claim 4, wherein The use of the water extract of Chinese flowering crabapple tea in the preparation of a medicament for treating benign prostatic hyperplasia is achieved by improving cell morphology, making the vesicular tubular structure clearly visible, reducing the folds in the acinus, and decreasing the thickness of the glandular epithelium.

7. The application according to claim 4, wherein The use of the water extract of Chinese flowering crabapple tea in the preparation of a medicament for treating benign prostatic hyperplasia is achieved by reducing the increase in BPH serum T level caused by exogenous testosterone propionate (TP).

8. The application according to claim 4, characterized in that, The use of the water extract of Chinese flowering crabapple tea in the preparation of a medicament for treating benign prostatic hyperplasia is achieved by reducing the BPH serum DHT level.

9. The application according to claim 4, characterized in that, The use of the water extract of Chinese flowering crabapple tea in the preparation of a medicament for treating benign prostatic hyperplasia is achieved by reducing blood sugar and blood lipids.

10. The application according to any one of claims 4-9, characterized in that, The dosage of the aqueous extract of Malus spectabilis is 60 - 240 mg·kg -1 .

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