Rhizoma corydalis extract and application thereof

Abstract

The invention relates to the field of medicines, and particularly provides a rhizoma corydalis extract and application thereof in medicines and health-care products for preventing and / or treating benign prostatic hyperplasia. The invention also provides a preparation method of the rhizoma corydalis extract, which comprises the following steps: adding water into medicinal materials at 60 DEG C, extracting for 1-3 times, then adding 60-75vt% of alcohol-water solution, extracting for 1-3 times, combining extracting solutions, purifying by a chromatographic column, collecting 60-80% of ethanol eluent, and drying by distillation under reduced pressure to obtain the extract.

Description

Technical Field

[0001] This invention relates to the pharmaceutical field, specifically to a Corydalis extract and its use in the preparation of drugs for the prevention and / or treatment of benign prostatic hyperplasia. Background Technology

[0002] Benign prostatic hyperplasia (BPH), also known as enlarged prostate or benign prostatic hyperplasia, is a common benign condition in older men. It is primarily caused by the proliferation of prostatic glandular and stromal tissues, leading to an enlarged prostate. This enlargement can compress the urethra, causing symptoms such as difficulty urinating, urinary frequency, urgency, and nocturia. The incidence of BPH increases with age. In men aged 41-50, the incidence is approximately 20%. In men aged 51-60, the incidence is approximately 50%. In men over 80 years of age, the incidence can reach as high as 90%. Clinically, the main treatments for benign prostatic hyperplasia (BPH) fall into two categories: surgical treatment and drug therapy. Because BPH patients are generally older and have weaker physiological functions, many are not candidates for surgery, and drug therapy is usually the primary treatment. Commonly used drugs include alpha-receptor blockers (such as doxazosin and terazosin) and 5α-reductase inhibitors (such as finasteride). These drugs can relax the smooth muscle of the urethra and improve symptoms such as difficulty urinating. However, drug therapy requires long-term use and may be accompanied by some side effects, such as dizziness and low blood pressure, and may have a certain impact on the immune system. Searching for novel active substances with anti-prostate hyperplasia properties from extracts of traditional Chinese medicine has become a hot topic in pharmaceutical research.

[0003] Corydalis rhizome, the dried tuber of Corydalis yanhusuo (a plant belonging to the genus Corydalis in the family Papaveraceae), is also known as Yuanhu, Xuanhu, and Xuanhusuo. It is pungent, bitter, and warm in nature, and enters the liver and spleen meridians. The main components of Corydalis rhizome are starch, polysaccharides, alkaloids, mucilage, resin, volatile oil, and inorganic trace elements. To date, more than 60 alkaloids and more than 20 non-alkaloid compounds have been isolated and identified. Traditional Chinese medicine believes that Corydalis rhizome has the effects of promoting blood circulation, removing blood stasis, regulating qi, and relieving pain. The main pharmacodynamic material basis is the total alkaloids of Corydalis rhizome. Pharmacological activity studies have shown that related components of Corydalis rhizome have good sedative, analgesic, anti-myocardial ischemia, anti-gastric ulcer, anti-tumor, and immunomodulatory effects. However, there are no reports on the use of Corydalis rhizome extract in the preparation of drugs for the prevention and / or treatment of benign prostatic hyperplasia. Summary of the Invention

[0004] Therefore, the purpose of this invention is to find effective active components in traditional Chinese medicine for the prevention and / or treatment of benign prostatic hyperplasia, namely, to provide the application of Corydalis extract in the preparation of drugs for the prevention and / or treatment of benign prostatic hyperplasia.

[0005] This invention provides a method for preparing Corydalis extract, characterized in that the method includes the following steps: Step a: Extraction: Take Corydalis rhizome, heat and extract with pure water and / or 60-95% ethanol solution, collect the extract, and concentrate under reduced pressure; Step b: Chromatographic column purification: The extract concentrate is loaded onto macroporous resin and eluted sequentially with 0-30%, 60-80%, and 95% ethanol. The 60-80% ethanol eluent is collected and evaporated to dryness under reduced pressure to obtain the Corydalis extract.

[0006] The preferred step a involves pulverizing Corydalis rhizome, placing it in an extraction tank, adding 8 times the amount of pure water, extracting at 60°C for 0.5 hours, discarding the aqueous extract, and extracting the residue with 5-7 times the amount of 60-75% ethanol (w / v) for 2-3 times, each time for 1-2 hours, at an extraction temperature of 35-45°C, and combining the ethanol extracts.

[0007] For the reverse column chromatography step, a conventional reverse column, such as a C18 column, is used; for the macroporous resin chromatography step, a conventional macroporous resin column, such as an AB-8 column or a D101 column, is used.

[0008] The present invention also provides the use of Corydalis extract, which has the effect of preventing and / or treating benign prostatic hyperplasia, in pharmaceuticals and health products.

[0009] The Corydalis extract is an extract obtained by extracting Corydalis using conventional extraction methods or by extracting it separately using conventional extraction methods; or the effective portion of the extract obtained through a purification process. Preferably, the conventional extraction method includes one or more of maceration extraction, decoction extraction, reflux extraction, percolation extraction, ultrasonic extraction, and steam distillation; the extraction solvent includes water or ethanol solution; the purification process includes one or more of water extraction and alcohol precipitation, extraction, silica gel column separation, and macroporous resin column separation; the drug uses Corydalis extract as the active ingredient and also includes a pharmaceutically acceptable carrier.

[0010] More preferably, the preparation method of the Corydalis extract includes the following steps: taking the pulverized Corydalis herb and extracting it with water at 60°C 1-3 times, then adding 60-75 vt% alcohol aqueous solution and extracting it at 35-45°C 1-3 times, combining the extracts, concentrating, and obtaining the extract.

[0011] The technical solution of this invention has the following advantages: 1. The extraction process provided by this invention does not require the use of organic solvents such as acetone and n-hexane, but only ethanol and water, thereby improving the safety of the production process and the safety of Corydalis extract. 2. The extraction process provided by the present invention first uses pure water for heating and extraction to remove most of the water-soluble impurities such as pigments, starch, and sugars. At the same time, it can fully expand the plant cell walls, which is beneficial to the dissolution of alcohol-soluble active ingredients such as alkaloids during the subsequent ethanol extraction. 3. In the extraction process provided by the present invention, in order to prevent the effective components from being damaged by high temperature during the water extraction process, it was found through experiments that adding 8 times the amount of pure water and extracting at 60°C for 0.5 hours can both remove water-soluble impurities and retain the effective components. 4. The use of the Corydalis extract provided by this invention in the preparation of drugs for the prevention or treatment of benign prostatic hyperplasia (BPH). Studies have found that the Corydalis extract has an inhibitory effect on the increase in prostate volume in rats induced by testosterone propionate, reduces the index of each lobe of the prostate, especially the index of the ventral lobe of the prostate, and increases SOD activity and reduces MDA content. HE staining clearly shows that, compared with the model group, the number of glandular hyperplasia in rats given the Corydalis extract is reduced. In summary, the Corydalis extract of this invention has a good inhibitory effect on BPH. Attached Figure Description

[0012] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0013] Figure 1 This is an HE staining image of the ventral lobe of the prostate gland in the blank group rats in Experiment Example 1 of this invention; Figure 2 This is an HE staining image of the ventral lobe of the prostate gland in the model group rats in Experiment Example 1 of this invention; Figure 3 This is an HE staining image of the ventral lobe of the prostate gland of rats in the drug administration group 1 in Experiment Example 1 of this invention; Figure 4 This is an HE staining image of the ventral lobe of the prostate gland of rats in the drug administration group 3 in Experiment Example 1 of this invention; Detailed Implementation

[0014] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0015] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0016] 50% ethanol aqueous solution refers to the volume percentage of ethanol in a mixture of ethanol and water. 5% alcohol aqueous solution or 50% alcohol aqueous solution refers to the volume percentage of alcohol in a mixture of alcohol and water. Example 1

[0017] This embodiment provides an extraction process for Corydalis extract, including the following steps: Take 1 kg of pulverized Corydalis rhizome, add 8 times its weight of water, heat to 60℃ and extract for 0.5 h, filter, add 5 times its weight of 75% ethanol aqueous solution to the residue and extract twice at 35℃, 1 h each time, combine the extracts, and concentrate under reduced pressure at 40℃ to a solution volume of 1 L to obtain ethanol extract A. Pass half of extract A through an AB-8 chromatography column, loading at a flow rate of 2 BV / h, eluting with 30%, 80%, and 95% ethanol, 3 BV for each gradient, collect 80% of the eluent, evaporate to dryness under reduced pressure at 40℃ to obtain Corydalis extract 1. Example 2

[0018] This embodiment provides an extraction process for Corydalis extract, including the following steps: Take 1 kg of pulverized Corydalis rhizome, add 8 times its weight of water, heat to 60℃ and extract for 0.5 h, filter, add 6 times its weight of 65% ethanol aqueous solution to the residue and extract twice at 40℃, 1.5 h each time, combine the extracts, and concentrate under reduced pressure at 40℃ to a solution volume of 1 L to obtain the ethanol extract. Pass the ethanol extract through an LX20SS chromatography column, with a flow rate of 2 BV / h, and elute with 20%, 60%, and 95% ethanol, 3 BV for each gradient, collect 60% of the eluent, and evaporate to dryness under reduced pressure at 40℃ to obtain Corydalis rhizome extract 2. Example 3

[0019] This embodiment provides an extraction process for Corydalis extract, including the following steps: Take 1 kg of pulverized Corydalis rhizome, add 8 times its weight of water, heat to 60℃ and extract for 0.5 h, filter, add 7 times its weight of 60% ethanol aqueous solution to the residue and extract 3 times at 45℃, 2 h each time, combine the extracts, and concentrate under reduced pressure at 40℃ to a solution volume of 1 L to obtain the ethanol extract. Pass the ethanol extract through an XAD chromatography column at a flow rate of 2 BV / h, elute with pure water, 75%, and 95% ethanol, 3 BV for each gradient, collect 75% of the eluted fraction, concentrate under reduced pressure at 40℃ to 1 L, and pass the concentrate through an NKA-9 chromatography column at a flow rate of 2 BV / h, elute with 30%, 75%, and 95% ethanol, 3 BV for each gradient, collect 75% of the eluted fraction, evaporate to dryness under reduced pressure at 40℃ to obtain Corydalis rhizome extract 3. Experimental Example 1

[0020] 1. Laboratory animals Male healthy SD (Sprague-Dawley) rats, SPF grade, 6-8 weeks old, weighing 200 g ± 20 g.

[0021] 2. Reagents and Medicines Test samples: alcohol extract A prepared according to the process of Example 1, Corydalis extract 2 prepared according to the process of Example 2, and Corydalis extract 3 prepared according to the process of Example 3; Testosterone propionate: purchased from Aladdin Company, specification: 5g / bottle; Positive drug: Finasteride (specification: 5mg / tablet, batch number: 294051, Hangzhou Merck Pharmaceutical Co., Ltd.).

[0022] 3. Experiment Content SD rats were acclimatized for 5 days. Six rats were randomly selected for sham surgery as the control group. The remaining rats were anesthetized with isoflurane and bilateral testicles were removed via the scrotum under sterile conditions. The surgical sites were left open and disinfected with povidone-iodine. The rats were allowed free access to food and were housed individually for one week to recover. On day 8 after castration, the castrated rats were randomly divided into 5 groups of 6 rats each according to their body weight: the model group, the positive control group, and the drug treatment groups 1-3. The grouping and drug administration details are shown in Table 1 below. The rats were administered the drug once by gavage starting on the morning of day 9 after castration, with a treatment period of 21 days. During the treatment period, the model group and the rats receiving drug treatment 1-3 were simultaneously injected subcutaneously with the modeling agent testosterone propionate at a dose of 2.0 mg / kg / day, using commercially available olive oil as the solvent, with an injection volume of 1 mL / kg. The modeling period was 21 days.

[0023] Table 1 Grouping and Dosage

[0024] Twenty-four hours after the last administration, rats were euthanized by cervical dislocation. The prostate tissue (ventral and dorsal lobes) was quickly removed, surface fluid was blotted off with filter paper, adipose tissue was removed, and the wet weight was measured using an electronic balance. The prostate index was calculated using the formula: Prostate Index = Mass of each prostate lobe (g) / Body weight (g). The volume was then measured in a measuring tube containing physiological saline. Equal masses of tissue from the same location were homogenized using a tissue homogenizer. The MDA and SOD contents in the tissues were determined using the MDA and SOD kits from Nanjing Biotechnology Co., Ltd., following the kit instructions.

[0025] The ventral lobe of the prostate tissue of rats in the blank group, model group, drug treatment group 1 and drug treatment group 3 was fixed in 10% formalin solution for routine paraffin sectioning, HE staining, and observation of rat prostate tissue pathology sections under a light microscope. The specific steps for paraffin sectioning are as follows: (1) Take the abdominal lobe of the prostate and place it in fixative (10% formalin) for more than 24 hours. Remove the tissue block from the fixative and place it on clean filter paper to absorb the surface liquid. (2) Dehydrate the tissue block with ethanol: Immerse it in 75% alcohol (5 hours), 85% alcohol (2 hours), 95% alcohol (2 hours), 100% alcohol I (40 minutes), and 100% alcohol II (35 minutes) in sequence. (3) Fix the tissue block for transparency: Immerse it in xylene I (2-10 minutes, observe the tissue at any time, and make sure the edges are transparent to prevent seepage) and xylene II (10 minutes) in sequence. (4) Immerse the tissue block in paraffin I (60 minutes) and paraffin II (60 minutes) in sequence. (5) Embed (including embedding the paraffin, which should be the same as the paraffin used for the last embedding).

[0026] 4. Data statistics and analysis Excel was used for data entry and statistical analysis. Quantitative data in the experimental results are expressed as mean ± standard deviation (mean ± SD). One-way ANOVA was performed using SPSS 13.0 for comparisons between groups; p < 0.05 was considered statistically significant.

[0027] 5. Experimental Results (1) Effects of drugs on prostate volume and prostate index in rats with prostatic hyperplasia induced by testosterone propionate Table 2. Effects of drugs on prostate index and prostate volume in rats with benign prostatic hyperplasia (n=6, mean ± SD)

[0028] Note: * indicates P < 0.05 compared to the model control group (t-test); ** indicates P < 0.01 compared to the model control group (t-test); *** indicates P < 0.001 compared to the model control group (t-test); # indicates P < 0.001 compared to the model control group (t-test); As shown in the table above, 21 days after modeling, compared with the control group, the prostate volume and prostate index of the castration model group were significantly increased (p<0.001), indicating that the modeling was successful.

[0029] In the prostate volume measurement, compared with the model group, the drug administration groups 1 and 3 all inhibited the increase in prostate volume induced by testosterone propionate in rats, and the differences were statistically significant (P<0.05); the drug administration group 2 had a certain inhibitory effect on the increase in prostate volume induced by testosterone propionate in rats, but it was not statistically significant, indicating that the drug of the present invention has a certain inhibitory effect on the increase in prostate volume induced by testosterone propionate in rats.

[0030] In the prostate index determination, compared with the model group, the positive control group and each drug administration group showed varying degrees of reduction in the index of each lobe of the prostate, especially the index of the ventral lobe of the prostate. Among them, the ventral lobe index of drug administration group 3 was significantly reduced (P<0.01), and the ventral lobe index of drug administration group 2 was significantly reduced (P<0.05). It can be seen from both the prostate index and the ventral lobe index that the drug of the present invention has an inhibitory effect on prostatic hyperplasia in rats.

[0031] (2) Effects of drugs on the content of MDA and SOD in prostate tissue Table 3. Effects of drugs on the content of MDA and SOD in prostate tissue (n=6, mean ± SD) Grouping SOD (U / mg) MDA (mmol / mg) Blank group 60.5±1.52 7.07±0.66 Model group 42.27±2.01### 10.4±0.45### Group 1 44.65±1.47 9.06±0.45* Group 2 47.7±3.41 10.23±0.34 Group 3 49.01±0.96** 8.52±0.66** Positive control group 52.13±1.97*** 8.01±0.62*** Note: * indicates P < 0.05 compared to the model control group (t-test); ** indicates P < 0.01 compared to the model control group (t-test); *** indicates P < 0.001 compared to the model control group (t-test); # indicates P < 0.001 compared to the model control group (t-test); Table 3 shows that the SOD activity in the prostate tissue of benign prostatic hyperplasia mice (model group) treated with testosterone propionate was significantly reduced (p<0.001) and the MDA content was significantly increased (p<0.001), indicating successful model establishment. The increased SOD activity and decreased MDA content in the prostate tissue of mice after drug administration indicate that the drug of this invention can effectively improve the reduction in SOD activity and increase in MDA content caused by testosterone propionate-induced benign prostatic hyperplasia.

[0032] (3) Histopathological sections of the ventral lobe of the prostate in rats with prostatic hyperplasia induced by testosterone propionate like Figure 1 As shown in the blank group, most of the prostate glands are regular in shape, with glandular epithelial cells in a single layer and arranged normally. A small number of them have small papillary endothelium. The glandular lumen contains a small amount of secretions, and there is no dilation of the glandular lumen or fibrous tissue hyperplasia in the stroma.

[0033] like Figure 2 As shown, in the model group, the number of prostate glands in rats increased and became denser, with new glands proliferating, some glands dilating, the epithelium being tall columnar, some forming a pseudo-stratified structure, many papillary processes protruding into the lumen, exudate accumulating in the stroma, and a large amount of fibrous tissue proliferating.

[0034] like Figure 3 As shown, in group 1, some glandular lumens were enlarged, with fewer glands than in the model group, and the number of glandular hyperplasia was reduced. A small portion of these glands protruded into the lumen in a papillary manner, a decrease compared to the model group. Half of the prostate gland lumens were regularly shaped, and the epithelial cells were mostly cuboidal columnar epithelium. Exudate was present in the stroma and some glandular lumens, with a small amount of fibrous tissue hyperplasia in the stroma. This indicates that after 21 days of modeling, test sample 1 did not show an inhibitory effect on prostatic hyperplasia in terms of the prostate ventral lobe index and prostate index; however, it did show a certain inhibitory effect on prostatic hyperplasia in paraffin sections.

[0035] like Figure 4 As shown, in drug administration group 3, the number of glandular hyperplasia was reduced, the number of papillary protrusions was significantly reduced, half of the prostate gland cavities were regularly shaped, the epithelial cells were mostly cuboidal columnar epithelium, and there was exudate in the stroma and some gland cavities. Although the glands varied in size, some were larger, but there was a significant improvement compared to the model group.

[0036] The above indicates that the Corydalis extract of the present invention has a good effect on inhibiting benign prostatic hyperplasia.

[0037] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for preparing Corydalis extract, characterized in that, The method includes the following steps: Step a: Extraction: Take Corydalis rhizome, heat and extract with pure water and / or 60-95% ethanol solution, collect the extract, and concentrate under reduced pressure; Step b: Chromatographic column purification: The extract concentrate is loaded onto macroporous resin and eluted sequentially with 0-30%, 60-80%, and 95% ethanol. The 60-80% ethanol eluent is collected and evaporated to dryness under reduced pressure to obtain the Corydalis extract.

2. The method for preparing Corydalis extract according to claim 1, characterized in that, The extraction described in step a involves crushing the Corydalis rhizome into powder, placing it in an extraction tank, adding 8 times the amount of pure water, extracting at 60°C for 0.5 hours, discarding the aqueous extract, and extracting the residue with 5-7 times the amount of the medicinal material using 60-75% ethanol (w / v) 2-3 times, each time for 1-2 hours, at an extraction temperature of 35-45°C. The ethanol extracts are then combined and concentrated under reduced pressure.

3. The method for preparing Corydalis extract according to claim 1, characterized in that, The macroporous resin mentioned in step b is one or a mixture of several of the following: AB-8, XAD, LX60, LX20SS, NKA-9, D-101, or reverse column C18.

4. A Corydalis extract obtained by the extraction method according to any one of claims 1 to 3, which has the effect of preventing and / or treating benign prostatic hyperplasia.

5. The use of a Corydalis extract as described in claim 4, which has the effect of preventing and / or treating benign prostatic hyperplasia, in pharmaceuticals and health products.

6. Use of Corydalis extract in the preparation of drugs for the prevention or treatment of benign prostatic hyperplasia.

7. The use according to claim 6, characterized in that, The preparation method of the Corydalis extract includes the following steps: take the pulverized Corydalis herb and extract it with water at 60℃ 1-3 times, then add 60-75 vt% alcohol aqueous solution and extract it at 35-45℃ 1-3 times, combine the extracts, concentrate and obtain the extract.

8. The use according to claim 6 or 7, characterized in that, The drug uses Corydalis extract as its active ingredient and also includes a pharmaceutically acceptable carrier.

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