Application of fagopyrum dibotrys or extract thereof in preventing or treating AR-positive prostate cancer or treating benign prostatic hyperplasia

Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of wild buckwheat rhizome or an extract thereof to prevention or treatment of AR-positive prostatic cancer or treatment of benign prostatic hyperplasia. The invention provides application of fagopyrum dibotrys or an extract thereof in preparation of a medicine or a health care product for preventing or treating AR-positive prostatic cancer or treating benign prostatic hyperplasia. Meanwhile, the invention discloses the wild buckwheat rhizome water extract, a preparation method thereof and a pharmaceutical composition or health care product thereof. Researches show that the wild buckwheat rhizome and the extract thereof can significantly reduce expression of AR and PSA, specifically inhibit proliferation of AR-positive prostate cancer cells and induce death of the AR-positive prostate cancer cells, and inhibit proliferation of benign prostatic hyperplasia related cells at the same time. The rhizoma fagopyri dibotryis and the extract thereof can target prostate tissues with high AR expression, a novel intervention scheme with treatment accuracy and clinical safety is provided for prostate cancer prevention and treatment and benign prostatic hyperplasia management, and the rhizoma fagopyri dibotryis and the extract thereof have important clinical application value.

Description

Technical Field

[0001] The present application relates to the field of medical technology, and specifically to the use of golden buckwheat or an extract thereof for preventing or treating AR-positive prostate cancer, or treating benign prostatic hyperplasia. Background Art

[0002] Abnormal activation of the androgen receptor (AR) signaling pathway is the core pathological mechanism of prostate disease. In prostate cancer (PCa), AR overexpression drives tumorigenesis and early progression, while AR gene mutations promote the development of late-stage cancer. In benign prostatic hyperplasia (BPH), AR-mediated cell proliferation leads to glandular enlargement and urethral compression. However, existing interventions for prostate disease have significant limitations:

[0003] 1. Safety issues in prostate cancer prevention and early treatment. Prostate Specific Antigen (PSA) is an internationally recognized core biomarker for prostate cancer, and its dynamic monitoring is of great value in early tumor screening, risk stratification, and treatment response assessment. Epidemiological data show that approximately 38.6% of newly diagnosed patients have low-risk localized prostate cancer (Gleason grade group 1), and guidelines recommend active surveillance (AS) for such patients. This program performs dynamic assessments through periodic PSA testing, multi-parameter magnetic resonance imaging, guided targeted puncture biopsy, and digital rectal examination. However, due to the lack of reliable molecular markers for individualized progression risk prediction, approximately 30% of patients will still progress to more aggressive cancer within 5 years. Existing androgen deprivation therapy (ADT) and androgen receptor signaling pathway inhibitors (such as AR antagonists like enzalutamide) block androgen signaling systemically, but often cause serious side effects, including decreased bone density / osteoporosis (increased risk of pathological fractures, OR ≈ 3.17), increased risk of metabolic syndrome (approximately 2.8 times), and sexual dysfunction (such as a decrease in IIEF-5 score ≥ 60%). These side effects make it difficult to meet the needs of long-term safe intervention for low-risk patients. Similarly, ADT and AR antagonists are not suitable for the prevention of prostatic intraepithelial neoplasia (PIN) or in individuals with a high genetic risk.

[0004] 2. Tolerance issues of drug treatment for benign prostatic hyperplasia.Medical therapy is the core of the management of BPH-related lower urinary tract symptoms (LUTS). 5-α-reductase inhibitors (5-ARIs) and α1-adrenergic receptor blockers are the mainstays of treatment. 5-ARIs inhibit dihydrotestosterone (DHT) synthesis, reducing prostate size and slowing clinical progression (e.g., reducing the risk of acute urinary retention). They are suitable for patients with significantly enlarged prostates, but their sexual side effects (approximately 15-20% of patients report decreased libido, erectile dysfunction, or decreased ejaculation volume) significantly impact treatment willingness. α1-blockers relax the bladder neck and prostate smooth muscle, rapidly improving urinary symptoms. They are suitable for patients with mild to moderate LUTS. Common side effects include dizziness, fatigue, and orthostatic hypotension. Combination therapy (5-ARI + α1-blocker) is a common approach for patients with moderate to severe LUTS or a high risk of progression. While it offers synergistic effects in symptom control and disease progression, the additive side effects of both medications can impact patient tolerance and compliance with long-term treatment.

[0005] 3. Exploration and limitations of plant extract intervention. Plant extracts such as saw palmetto, Pygeum africanum, and Fenugreek have been explored for use in BPH intervention, primarily alleviating symptoms through mechanisms such as inhibition of 5α-reductase activity. However, their effects in modulating AR signaling pathways are limited, and there is a lack of evidence for their selective targeting of AR-positive prostate tissue. Golden buckwheat (Fagopyrum dibotrys), a traditional Chinese medicine / health food ingredient, possesses anti-inflammatory and anti-tumor activities, but limited research has been conducted on its targeted therapy in AR-positive PCa or BPH, and existing public technologies (including patents) have yet to demonstrate its targeted therapeutic effects in AR-positive PCa or BPH.

[0006] In summary, abnormal activation of the AR pathway is a key pathological mechanism of PCa and BPH. However, existing drugs (ADT, AR antagonists, 5-ARI, α-blockers) have significant safety / tolerability issues, and current plant extracts have limited effects and lack precise targeting. Therefore, the development of new intervention strategies that can selectively downregulate AR signaling in prostate tissue while also having high safety is a current technical need for the safe and effective management of prostate cancer (especially prevention and early intervention in low-risk patients, prevention in high-risk populations) and BPH. Summary of the Invention

[0007] In response to the problems existing in the existing technology, this application provides an innovative intervention strategy based on the Polygonaceae plant Fagopyrum dibotrys (hereinafter referred to as Fagopyrum dibotrys) by revealing the role of Fagopyrum dibotrys and its extracts in specifically downregulating the AR signaling pathway. It significantly inhibits the proliferation of AR-positive prostate cancer and BPH cells with low side effects, providing a solution that is both precise and safe for the prevention and treatment of AR-positive prostate cancer and the treatment of benign prostatic hyperplasia, filling the gap in the existing technology.

[0008] Studies have shown that early-stage prostate cancer cells are highly dependent on the androgen-AR signaling pathway to drive proliferation. AR binding to androgen activates the transcription of downstream oncogenes. Therefore, abnormal activation of the androgen-AR signaling pathway is closely associated with the development and progression of prostate cancer. Reducing AR levels or inhibiting the AR signaling pathway is considered a strategy to prevent or delay the development and progression of prostate cancer.

[0009] Prostate-specific antigen (PSA) is an important biomarker for prostate cancer, with elevated levels generally associated with the risk and severity of prostate cancer. PSA expression is directly regulated by the AR signaling pathway; therefore, reducing AR levels leads to decreased PSA levels. Clinically, PSA levels are commonly used for prostate cancer screening and monitoring, and lower PSA levels are associated with a reduced risk of prostate cancer or slower disease progression.

[0010] In benign prostatic hyperplasia (BPH), AR-mediated cell proliferation leads to glandular enlargement and urethral compression. Therefore, reducing AR levels or inhibiting the AR signaling pathway can also inhibit cell proliferation in BPH, delay disease progression, and treat BPH.

[0011] Through research, this application found that golden buckwheat and its extracts can effectively inhibit the proliferation of AR-positive prostate cancer cells by reducing androgen receptor (AR) levels, inducing cell death, and reducing PSA protein expression levels, while having no significant effect on AR-negative cells; it can meet the safe intervention needs of prostate cancer patients, has the effect of preventing or treating AR-positive prostate cancer, and provides a new strategy for the management of early prostate cancer from "passive monitoring" to "active intervention", providing specific targeted drugs for the treatment of prostate cancer patients, filling the gap in chemopreventive drugs and therapeutic drugs with low toxic side effects. Golden buckwheat and its extracts can also inhibit the proliferation of BPH-1 cells, relieve glandular hyperplasia symptoms, and provide safe and effective targeted drugs for BPH patients.

[0012] In a first aspect, the present application provides the use of golden buckwheat or its extract for preparing a medicine or health product for preventing or treating AR-positive prostate cancer, or treating benign prostatic hyperplasia. The present application provides the use of golden buckwheat or its extract for preventing or treating AR-positive prostate cancer, or treating benign prostatic hyperplasia; for preventing or treating AR-positive prostate cancer, or treating benign prostatic hyperplasia.

[0013] In one group of embodiments, the prostate cancer is particularly early-stage prostate cancer.

[0014] In one set of embodiments, the Fagopyrum truncatum is the dried rhizome of Fagopyrum truncatum, and the Fagopyrum truncatum extract is the extract of the dried rhizome of Fagopyrum truncatum.

[0015] In one group of embodiments, the Fagopyrum tiliaceum extract is a water extract, an alcohol extract (such as a C1-4 alcohol extract) or a ketone extract (such as an acetone extract), preferably a water decoction extract, an ethanol extract (such as a 60%-100% ethanol (aqueous solution) extract, which can be 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% ethanol extract).

[0016] In one embodiment, the preparation method of the water extract comprises the following steps: using 5-20 mL (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 mL, such as 8-10 mL) of water per gram of golden buckwheat to decoct, decoct for 20 minutes to 3 hours (e.g., 20, 30, 40, 50 minutes, 1, 1.5, 2, 2.5, 3 hours, preferably 20 minutes to 1 hour), removing the medicinal residue to obtain a water extract; optionally, concentrating and / or drying the aqueous extract (e.g., freeze-drying). Preferably, the decoction temperature is 80-100°C (e.g., 80, 85, 90, 95, 100°C). Preferably, the number of extractions is one or more, such as 2-3 times (e.g., 1, 2, or 3 times). When extracting multiple times, the medicinal residue after the previous extraction is added to water for decoction again, and the multiple extracts are combined to obtain the aqueous extract. When multiple extractions are performed, the amount of water used for each extraction and the decoction temperature are the same as described above. Preferably, the golden buckwheat is crushed or sliced ​​and then decocted with water. Preferably, after decoction, the residue can be removed by filtration or centrifugation.

[0017] In one embodiment, the ethanol extract of Fagopyrum truncatum is an extract directly extracted with ethanol from Fagopyrum truncatum or an extract extracted with ethanol from the residue after water extraction of Fagopyrum truncatum. The ethanol is 60%-100% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%) ethanol. In one embodiment, the method for preparing the ethanol extract comprises the following steps: subjecting Fagopyrum truncatum or the residue after water extraction of Fagopyrum truncatum to ethanol reflux extraction, using 5-20 mL (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 mL, such as 8-10 mL) of ethanol per gram of Fagopyrum truncatum for reflux extraction, removing the residue to obtain an ethanol extract, and optionally, concentrating and / or drying (e.g., freeze-drying) the ethanol extract. Preferably, the extraction is performed once or more, such as 2-3 times (e.g., 1, 2, or 3 times). When multiple extractions are performed, the residue after the previous extraction is further added with ethanol for reflux extraction, and the multiple extracts are combined to obtain an ethanol extract. When multiple extractions are performed, the amount of ethanol used for each extraction is the same as described above. Preferably, the golden buckwheat is crushed or sliced ​​before extraction. Preferably, after reflux extraction, the residue can be removed by filtration or centrifugation.

[0018] In one embodiment, the golden buckwheat extract contains monomeric proanthocyanidins, oligomeric proanthocyanidins, and / or polymeric proanthocyanidins. Preferably, the monomeric proanthocyanidins and oligomeric proanthocyanidins contain one or more of proanthocyanidin B1, proanthocyanidin B2, proanthocyanidin A1 isomer, proanthocyanidin B2 isomer, proanthocyanidin C1 isomer, catechin, or epicatechin. More preferably, the extract contains all seven of the aforementioned components. Preferably, the polymeric proanthocyanidins have a molecular weight of 1,500-20,000 Da, preferably 1,500-15,000 Da, 1,500-13,000 Da, or 3,500-13,000 Da.

[0019] In a second aspect, the present application provides a method for preparing a water extract of golden buckwheat, comprising the following steps: decocting golden buckwheat with water, removing the medicinal residue to obtain a water extract, subjecting the aqueous extract to macroporous adsorption resin column chromatography, and eluting with ethanol to obtain a water extract. After decocting with water, the medicinal residue can be removed by filtration or centrifugation. The ethanol is 5%-100% (5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%) ethanol (aqueous solution), preferably 10%-95% ethanol.

[0020] In one embodiment, 5-20 mL (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 mL, such as 8-10 mL) of water is used for decoction per gram of golden buckwheat. After decoction for 20 min-3 h (e.g., 20, 30, 40, 50 min, 1, 1.5, 2, 2.5, 3 h, preferably 20 min-1 h), the residue is removed to obtain a water extract. Preferably, the decoction temperature is 80-100 ° C (e.g., 80, 85, 90, 95, 100 ° C). Preferably, the number of extractions is 1 or more, such as 2-3 times (e.g., 1, 2 or 3 times). When multiple extractions are performed, the residue after the previous extraction is added to water for decoction, and the multiple extracts are combined to obtain a water extract. When multiple extractions are performed, the amount of water used for each extraction and the decoction temperature are the same as described above. Preferably, the golden buckwheat is crushed or sliced ​​and then boiled in water.

[0021] In one group of embodiments, the water extract is chromatographed on a macroporous adsorption resin column, and eluted first with 5%-50% ethanol to obtain water extract 1; optionally, eluted with 90%-100% (such as 95%) ethanol to obtain water extract 2. Water extract 1 and water extract 2 can be used alone or together. Preferably, the water extract is chromatographed on a macroporous adsorption resin column, and eluted first with 5%-15% (such as 10%) ethanol to obtain water extract 1-1; then eluted with 20%-50% (such as 30%) ethanol to obtain water extract 1-2; optionally, eluted with 90%-100% (such as 95%) ethanol to obtain water extract 2. Water extract 1-1, water extract 1-2 and water extract 2 can be used alone or together. Preferably, water extract 1-1 and water extract 1-2 are used together. The amount of elution solvent used in each gradient is 2-15 column volumes (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 times, preferably 3-10, 5-7 column volumes).

[0022] In one embodiment, the macroporous adsorption resin may be a non-polar macroporous adsorption resin commonly used in the art, such as D101, AB-8, HP20, etc. The amount of the macroporous adsorption resin used is 0.5-2 times the amount of golden buckwheat (by weight), preferably 1-1.5 times the amount.

[0023] In one embodiment, the preparation method further comprises the following steps:

[0024] Before eluting with ethanol, wash with water;

[0025] and / or

[0026] After elution with ethanol, the eluate is concentrated and / or dried (eg, freeze-dried) to obtain an aqueous extract.

[0027] The amount of water used for elution is 2-15 times the column volume (such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 times, preferably 3-10, 5-7 times the column volume).

[0028] In one embodiment, the preparation method further comprises the step of subjecting the aqueous extract obtained by macroporous adsorption resin column chromatography to further separation (e.g., gel chromatography, membrane dialysis) to obtain an aqueous extract containing compounds with a molecular weight greater than or equal to 200 (e.g., molecular weight greater than or equal to 300, molecular weight greater than or equal to 400, molecular weight greater than or equal to 500, molecular weight greater than or equal to 1000, molecular weight greater than or equal to 1500, molecular weight between 500 and 20,000, molecular weight between 500 and 15,000, molecular weight between 500 and 13,000, molecular weight between 1,500 and 20,000, molecular weight between 1,500 and 15,000, molecular weight between 1,500 and 13,000, molecular weight between 3,500 and 13,000). Optionally, after separation, the aqueous extract is concentrated and / or dried (e.g., freeze-dried) to obtain the aqueous extract. The aqueous extract obtained by macroporous adsorption resin column chromatography can be further subjected to membrane dialysis separation, and low molecular weight components can be removed by dialysis using a dialysis bag with a molecular weight cutoff of 200-1000 Da to obtain a high-polymer proanthocyanidin extract of golden buckwheat. Alternatively, the low molecular weight components can be first removed by dialysis using a dialysis bag with a molecular weight cutoff of 200-1000 Da, and then the high molecular weight polymer proanthocyanidin components can be obtained by dialysis using a dialysis bag with a molecular weight cutoff of 1000-50000 Da. Optionally, after separation, the extract can be concentrated and / or dried (e.g., freeze-dried) to obtain an aqueous extract.

[0029] In one set of embodiments, the Fagopyrum truncatum is the dried rhizome of Fagopyrum truncatum.

[0030] In a third aspect, the present application provides a golden buckwheat water extract obtained by the preparation method described in the second aspect. Preferably, it contains monomeric proanthocyanidins, oligomeric proanthocyanidins, and / or polymeric proanthocyanidins. The monomeric proanthocyanidins and oligomeric proanthocyanidins contain one or more of proanthocyanidin B1, proanthocyanidin B2, proanthocyanidin A1 isomer, proanthocyanidin B2 isomer, proanthocyanidin C1 isomer, catechin, or epicatechin, and more preferably contain the above seven components. Preferably, the molecular weight of the polymeric proanthocyanidins is 1500-20000Da, preferably 1500-15000Da, 1500-13000Da, or 3500-13000Da.

[0031] According to the use described in the first aspect, the golden buckwheat extract is the golden buckwheat water extract described in the third aspect.

[0032] In a fourth aspect, the present application provides a pharmaceutical composition or health product containing the golden buckwheat water extract described in the third aspect. Preferably, the pharmaceutical composition further contains a pharmaceutically acceptable excipient.

[0033] In a fifth aspect, the present application provides a method for preventing or treating AR-positive prostate cancer, comprising administering to a subject the golden buckwheat or golden buckwheat extract described in the first aspect, the golden buckwheat water extract described in the third aspect, or the pharmaceutical composition or health product described in the fourth aspect. The present application provides a method for treating benign prostatic hyperplasia, comprising administering to a subject the golden buckwheat or golden buckwheat extract described in the first aspect, the golden buckwheat water extract described in the third aspect, or the pharmaceutical composition or health product described in the fourth aspect.

[0034] After research, it was found that compared with the prior art, this application has at least the following beneficial effects:

[0035] Golden fagopyrum or its extract can be used to prevent AR-positive prostate cancer by downregulating the androgen receptor (AR) signaling pathway. It is suitable for high-risk populations such as prostate intraepithelial neoplasia (PIN), AR gene amplification, or persistently elevated prostate-specific antigen (PSA); treat AR-positive prostate cancer, especially early-stage low-risk patients with a Gleason score ≤6; and treat AR-mediated benign prostatic hyperplasia (BPH) to relieve symptoms of glandular hyperplasia.

[0036] 1. Fagopyrum truncatum and its extracts can specifically downregulate the AR signaling pathway, effectively inhibiting the proliferation of AR-positive prostate cancer cells and inducing cell death, but have no significant inhibitory effect on AR-negative prostate cancer cells; significantly reduce the mRNA and protein levels of AR and PSA; and inhibit the proliferation of BPH-1 cells. The inhibitory effect of Fagopyrum truncatum and its extracts is negatively correlated with the level of cellular AR expression. LNCaP cancer cells with the highest AR expression are the most sensitive, IC 50 The lowest, AR-negative PC-3 cells are the least sensitive, IC 50 The results indicate that the effect of Fagopyrum serrata is AR-dependent and can slow down the progression of prostate cancer, prevent or treat AR-positive prostate cancer, and treat benign prostatic hyperplasia.

[0037] 2. The water extract of golden buckwheat was separated and the activity of different components on prostate cancer LNCaP, C42B and PC-3 cells was tested. The most active components and their composition were determined. The results showed that LF04 and LF05 were the most active components, which contained monomers, oligomers and polymers of proanthocyanidins such as proanthocyanidin B1, proanthocyanidin B2, proanthocyanidin A1 isomer, proanthocyanidin B2 isomer, proanthocyanidin C1 isomer, catechin, epicatechin, etc.

[0038] 3. Fagopyrum serrata and its extracts have an AR-dependent mechanism, which downregulates AR mRNA and protein levels, different from nonspecific antioxidant or apoptosis-inducing mechanisms, providing the potential of plant extracts in AR-targeted therapy.

[0039] 4. Golden buckwheat and its extracts have selective targeting. Through a unique target mechanism, they act on the synthesis and degradation process of androgen receptors and act on prostate tissue with high AR expression, thereby avoiding side effects such as fractures, metabolic syndrome and sexual dysfunction caused by systemic androgen deprivation, avoiding the production of toxic side effects similar to existing anti-androgen drugs, reducing the side effects of drugs on other normal tissues, and meeting the clinical needs for safe intervention for prostate cancer patients, especially early low-risk patients, and patients with benign prostatic hyperplasia.

[0040] 5. This application provides more suitable and effective drugs or health products with low toxic side effects for the prevention or treatment of AR-positive prostate cancer and benign prostatic hyperplasia, and provides patients with a new intervention plan that combines therapeutic precision and clinical safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 This is a statistical analysis chart of the inhibition of golden buckwheat granules on the proliferation of human prostate cancer cells LNCaP and PC-3.

[0042] Figure 2A This is a statistical analysis chart of the inhibition of the proliferation of human prostate cancer cells LNCaP by golden buckwheat granules and golden buckwheat extracts from different origins.

[0043] Figure 2B This is a statistical analysis chart of the inhibition of human prostate cancer cell PC-3 proliferation by golden buckwheat granules and golden buckwheat extracts from different origins.

[0044] Figure 3 This is a flow cytometric analysis of the effect of golden buckwheat granules on the cell cycle distribution of human prostate cancer cells LNCaP.

[0045] Figure 4 This is the immunoblot analysis of the down-regulation of AR and PSA proteins in human prostate cancer cells LNCaP by Fagopyrum truncatum extract.

[0046] Figure 5 This is the separation flow chart of golden buckwheat extract.

[0047] Figure 6A This is a statistical analysis chart of the inhibition of the proliferation of human prostate cancer cells LNCaP and PC-3 by the golden buckwheat component LF01.

[0048] Figure 6B This is a statistical analysis chart of the inhibition of the proliferation of human prostate cancer cells LNCaP and PC-3 by the golden buckwheat component LF02.

[0049] Figure 6C This is a statistical analysis chart of the inhibition of the proliferation of human prostate cancer cells LNCaP and PC-3 by the golden buckwheat component LF03.

[0050] Figure 6D This is a statistical analysis chart of the inhibition of the proliferation of human prostate cancer cells LNCaP and PC-3 by the golden buckwheat component LF04.

[0051] Figure 6E This is a statistical analysis chart of the inhibition of the proliferation of human prostate cancer cells LNCaP and PC-3 by the golden buckwheat component LF05.

[0052] Figure 6F This is a statistical analysis chart of the inhibition of the proliferation of human prostate cancer cells LNCaP and PC-3 by the golden buckwheat component LF06.

[0053] Figure 7A This is the UV chromatogram (275nm) of the golden buckwheat component LF04.

[0054] Figure 7B This is the UV chromatogram (275nm) of the golden buckwheat component LF05.

[0055] Figure 8 This is the immunoblot analysis of the regulation of AR and PSA proteins in human prostate cancer cells LNCaP and C42B by the components extracted from golden buckwheat LF01-LF06.

[0056] Figure 9 LF01-LF06, a component extracted from Fagopyrum truncatum, regulates AR and PSA in human prostate cancer cells LNCaP and C42B

[0057] Graph showing analysis of mRNA levels.

[0058] Figure 10A Figure 1 shows the analysis of the effects of Fagopyrum truncatum extract components LF01-LF06 on PSA levels in the culture medium of human prostate cancer cells LNCaP and C42B.

[0059] Figure 10B Figure 2 shows the analysis of the effects of Fagopyrum truncatum extract components LF01-LF06 on PSA levels in the culture medium of human prostate cancer cells LNCaP and C42B.

[0060] Figure 11A This is a statistical analysis chart of the inhibition of human prostate cancer cell LNCaP proliferation by the components separated from Fagopyrum truncatum LF04 and LF05 after dialysis.

[0061] Figure 11B This is a statistical analysis chart of the inhibition of human prostate cancer cell C42B proliferation by the components separated from Fagopyrum truncatum LF04 and LF05 after dialysis.

[0062] Figure 11C This is a statistical analysis chart of the inhibition of human prostate cancer cell PC-3 proliferation by the components separated from Fagopyrum truncatum LF04 and LF05 after dialysis.

[0063] Figure 12A This is the UV chromatogram, spectrum and mass spectrum of the 500-3500Da component LF04 after dialysis of the golden buckwheat component.

[0064] Figure 12B This is the UV chromatogram, spectrum and mass spectrum of the 3500-13000Da component LF04 after dialysis of the golden buckwheat component.

[0065] Figure 12C This is the UV chromatogram, spectrum and mass spectrum of the 500-3500Da component LF05 after dialysis of the golden buckwheat component.

[0066] Figure 12D This is the UV chromatogram, spectrum and mass spectrum of the 3500-13000Da component LF05 after dialysis.

[0067] Figure 13 This is a statistical analysis chart of the inhibition of the proliferation of human prostate hyperplasia cells BPH-1 by the components of golden buckwheat LF01-LF06. DETAILED DESCRIPTION

[0068] The present application is further described below with reference to the following examples; however, these examples do not limit the scope of the present application. Unless otherwise stated, all reagents used in the examples were obtained from commercial sources; and the instruments and equipment used in the analysis and testing were all commonly used conventional instruments and equipment.

[0069] The terms "include" and "comprising" and any variations thereof in this application are intended to cover non-exclusive inclusions. "Multiple" mentioned in this application refers to two or more.

[0070] Golden buckwheat, scientific name: Fagopyrum dibotrys, is a perennial herb of the genus Fagopyrum in the Polygonaceae family. Its rhizome is used as medicine and is a commonly used Chinese medicine that can be used as health food or medicine in the country. It is often decocted and taken orally and can be used for a long time.

[0071] Golden buckwheat granules are formulated granules made by processing the dried rhizomes of golden buckwheat according to the quality standards of standard decoctions. The preparation method involves boiling golden buckwheat slices with water, filtering, concentrating the filtrate into a clear paste, drying, and adding appropriate auxiliary materials to form granules. Golden buckwheat granules are light brown to brown granules with a faint odor and a slightly astringent taste. The preparation method can be exemplified as follows: 8500g of golden buckwheat slices are boiled with water, filtered, and the filtrate is concentrated into a clear paste (with a paste yield of 5.9-11.8%). The appropriate amount of auxiliary materials is added, dried (or dried and crushed), and then the appropriate amount of auxiliary materials is added, mixed, and granulated to 1000g.

[0072] Fagopyrum truncatum extract generally refers to an extract of the dried rhizome of Fagopyrum truncatum, such as a water extract, an organic solvent extract (such as an alcohol or ketone extract, specifically a lower alcohol (C1-4) extract, a lower ketone extract (C1-4), such as an ethanol extract or an acetone extract).

[0073] Proanthocyanidins are a class of natural polyphenols, flavonoids formed by the condensation of flavan-3-ol monomers with varying degrees of polymerization. Depending on the degree of polymerization, they can be divided into monomeric proanthocyanidins, such as catechin, epicatechin, gallocatechin, and epigallocatechin; oligomeric proanthocyanidins with a degree of polymerization of 2-4, such as proanthocyanidin A1, proanthocyanidin B1, proanthocyanidin B2, and proanthocyanidin C1; and polymeric proanthocyanidins with even higher degrees of polymerization. The molecular weight of monomeric and oligomeric proanthocyanidins ranges from 290 to 1500 Da, while the molecular weight of polymeric proanthocyanidins exceeds 1500 Da.

[0074] The term "pharmaceutical composition" preferably includes "pharmaceutically acceptable excipients", and more preferably includes "pharmaceutically acceptable carriers and excipients". The above-mentioned excipients, carriers, and excipients include, but are not limited to, any adjuvants, carriers, excipients, glidants, sweeteners, diluents, preservatives, dyes / colorants, flavoring agents, surfactants, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, solvents, emulsifiers, and disintegrants approved by relevant government regulatory authorities as acceptable for human or livestock use.

[0075] The pharmaceutical composition and medicine of the present application can be prepared into tablets, pills, capsules, powders, granules, emulsions, suspensions, solutions, injections, inhalants, gels, etc.

[0076] In the present application, the pharmaceutical composition and the drug can be administered orally, transdermally, intramuscularly, subcutaneously, intravenously, or topically.

[0077] The term "subject" refers to any human or non-human organism that can potentially benefit from treatment with Fagopyrum truncatum or an extract thereof as described herein. Exemplary subjects include humans or mammals of any age. Preferably, the subject is a human.

[0078] The term "prophylaxis" includes prophylactic therapy in mammals, particularly humans, intended to reduce the likelihood of developing an infection, disease, or symptom. Patients may be selected for prophylactic therapy based on their increased risk of developing an infection, disease, or symptom compared to the general population. "Prophylaxis" may include treatment of subjects who have not yet developed an infection or clinical condition, and prevention of a second occurrence of the same or similar infection or clinical condition.

[0079] As used herein, the term "treatment" refers to any administration of a therapeutic agent according to a therapeutic regimen that achieves the desired effect, i.e., partially or completely alleviates, improves, alleviates, inhibits, delays the onset of, reduces the severity of, and / or reduces the incidence of one or more symptoms or characteristics of a particular disease, disorder, and / or condition; in some embodiments, the administration of a therapeutic agent according to a therapeutic regimen is associated with the achievement of the desired effect. Such treatment may be directed to subjects who do not exhibit the relevant disease, disorder, and / or condition and / or to subjects who only exhibit early signs of the disease, disorder, and / or condition. Alternatively or in addition, such treatment may be directed to subjects who exhibit one or more determined signs of the relevant disease, disorder, and / or condition. In some embodiments, treatment may be directed to subjects who have been diagnosed with the relevant disease, disorder, and / or condition. In some embodiments, treatment may be directed to subjects who are known to have one or more susceptibility factors that are statistically associated with an increased risk of developing the relevant disease, disorder, and / or condition.

[0080] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any appropriate manner unless there is any contradiction. In order to avoid unnecessary repetition, this application will not further describe various possible combinations.

[0081] In addition, the various implementation methods of the present application may be arbitrarily combined, and as long as they do not violate the concept of the present application, they should also be regarded as the contents disclosed in the present application.

[0082] Experimental Materials:

[0083] Human prostate cancer cells LNCaP and C42B were purchased from the Cell Bank of the Chinese Academy of Sciences, human prostate cancer cells PC-3 were purchased from the ATCC Cell Bank, and human prostate hyperplasia cells BPH-1 were purchased from Wuhan Zishan Biotechnology Co., Ltd. RPMI1640 was purchased from Dalian Meilun Biotechnology Co., Ltd.; fetal bovine serum was purchased from Biosera, France. SYBR Green dye was purchased from Invitrogen, USA. Dimethyl sulfoxide (DMSO) was purchased from Sigma-Aldrich, USA. Golden buckwheat granules were purchased from Jiangyin Tianjiang Pharmaceutical Co., Ltd. Propidium iodide (PI) was purchased from Biosharp, China. RIPA cell lysis buffer, protease inhibitor triple kit, BCA protein concentration kit, and ECL luminescence solution were purchased from Shanghai Weiao Biotechnology Co., Ltd. Skim milk powder was purchased from Shanghai Bright Dairy Co., Ltd. Polyvinylidene fluoride (PVDF) membrane was purchased from Millipore, USA. All other reagents were analytical grade reagents from Shanghai Sinopharm Group.

[0084] Example 1: Investigating the ability of golden buckwheat granules to inhibit the proliferation of human prostate cancer cells LNCaP and PC-3

[0085] 1.1 Experimental methods

[0086] Human prostate cancer cells LNCaP (6×10 3 cells / well) and PC-3 (3×10 3 Cells (cells / well) were seeded in a 96-well plate in RPMI1640 medium containing 10% fetal bovine serum and cultured in a 37°C incubator. Different concentration gradients of drug solutions were prepared using Golden Fagopyrum granules and DMSO: 0 (DMSO control group), 0.0625, 0.125, 0.25, 0.5, and 1 mg / mL. The drug solutions were added to the 96-well plate and incubated in a 37°C incubator for 72 hours. The supernatant was then aspirated and the plate was stored in a -80°C refrigerator. The plate was removed from the refrigerator, and 100 μL of a staining reagent containing 0.01% SYBR GREEN dye and 10% RIPA cell lysis buffer was added to each well.

[0087] Fluorescence values ​​were measured using a microplate reader at an excitation light of 485 / 20 nm and an emission light of 528 / 20 nm. The mean fluorescence value measured in the blank wells was used as the baseline, and the inhibition rate (IC Values) was calculated using Excel; IC Values ​​= [1-(mean value of the treatment group-Baseline) / (mean value of the control group-Baseline)] × 100. The concentration of golden buckwheat when the cell growth inhibition rate reached 50% was the IC 50GraphPad Prism 10.1.2 software was used for graphing and analysis. Two-Way ANOVA was used for analysis of variance, with P < 0.05 as the standard for statistically significant differences. In this experiment, *P < 0.05, **P < 0.01, and ***P < 0.001 indicate significant statistical differences compared with the control group of the same cell line.

[0088] 1.2 Experimental Results

[0089] Figure 1 This is a statistical analysis of the effect of golden buckwheat granules on the proliferation of human prostate cancer cells LNCaP and PC-3. Figure 1 The results showed that when treated with different concentrations of Fagopyrum truncatum granules for 72 hours in LNCaP and PC-3 cells, Fagopyrum truncatum granules showed a strong inhibitory effect on AR-positive LNCaP cells, but had no significant effect on AR-negative PC-3 cells. These results indicate that the anti-prostate cancer effect of Fagopyrum truncatum is closely related to AR activity.

[0090] Example 2: Investigating the ability of Fagopyrum truncatum granules and Fagopyrum truncatum extracts from different origins to inhibit the proliferation of human prostate cancer cells LNCaP and PC-3

[0091] 2.1 Experimental Materials

[0092] The golden buckwheat extract was obtained by decoction of golden buckwheat slices. The three golden buckwheat slices came from Guizhou, Shaanxi and Jiangsu respectively. The obtained extracts were numbered JQM-01, JQM-02 and JQM-03 respectively.

[0093] 2.2 Experimental methods

[0094] Extraction Method: Take 50g of the medicinal material slices, add an appropriate amount of water (covering the material by 2-3cm), soak for 30 minutes, and then boil for 30 minutes. Let cool and filter. Add an appropriate amount of water to the medicinal residue (covering the material by 2-3cm), boil for 30 minutes. Let cool and filter. Combine the two filtrates, concentrate under reduced pressure to an appropriate volume (200-300mL), and freeze-dry to obtain extracts JQM-01: 6.182g, JQM-02: 5.198g, and JQM-03: 5.155g, respectively.

[0095] Different concentrations of DMSO were added to the solution of Fagopyrum truncatum granules or Fagopyrum truncatum extract to prepare the following treatment solutions: 0 (DMSO control), 0.0078125, 0.015625, 0.03125, 0.0625, and 0.125 mg / mL. The growth inhibition rates of Fagopyrum truncatum granules and Fagopyrum truncatum extract on human prostate cancer cells, LNCaP and PC-3, were measured and calculated using the same method as in Example 1. In this experiment, **P<0.01 and ***P<0.001 indicate statistically significant differences compared to the control group.

[0096] 2.3 Experimental Results

[0097] The anti-prostate cancer activities of Fagopyrum truncatum granules and extracts from three batches of Fagopyrum truncatum slices (JQM-01, JQM-02 and JQM-03) from different origins were compared using SYBR Green assay.

[0098] Figures 2A-2B The following is a statistical analysis of the effects of Fagopyrum truncatum granules and Fagopyrum truncatum extracts from different origins on the proliferation of human prostate cancer cells LNCaP and PC-3. Figures 2A-2B The results show that the three batches of golden buckwheat extracts from different origins exhibited similar inhibitory effects on LNCaP cells, while having no significant effect on PC-3 cells at low doses. Statistical comparisons revealed no statistically significant difference in the inhibitory activity of JQM-01, JQM-03, and the granules against prostate cancer cells, while JQM-02 exhibited less activity than JQM-01, JQM-03, and the granules. Subsequent experiments used golden buckwheat extract from Guizhou.

[0099] Example 3: Investigating the ability of golden buckwheat granules to induce cell death in human prostate cancer cells LNCaP

[0100] 3.1 Experimental methods

[0101] Human prostate cancer cells LNCaP (2×10 5 Cells / well) were seeded in 6-well plates in RPMI1640 medium containing 10% fetal bovine serum; the fagopyrum serratum granules were added with DMSO to prepare the drug solution: 0 (DMSO control group), 0.041 mg / mL (IC 50 ) and 0.066 mg / mL (IC 75 ) were added to 6-well plates and incubated in a 37°C incubator for 24 and 48 hours before flow cytometry samples were collected. Upon collection, cells were trypsinized, fixed with 75% ethanol at -20°C, and incubated at 4°C overnight. Cells were then stained with phosphate-buffered saline (PBS) containing RNase A and PI, and analyzed using a flow cytometer and FlowJo software.

[0102] GraphPad Prism 10.1.2 software was used for graphing and analysis. Two-Way ANOVA was used for analysis of variance, with P < 0.05 being considered statistically significant. In this experiment, *P < 0.05 and ***P < 0.001 indicated significant statistical differences compared with the DMSO control group.

[0103] 3.2 Experimental Results

[0104] PI staining was used to detect the expression of Fagopyrum truncatum in IC 50 (0.041 mg / mL) and IC 75 The effects of 1 mg / mL (0.066 mg / mL) on the cell cycle distribution of LNCaP cells at 24 and 48 hours.

[0105] Figure 3 The figure shows the flow cytometry analysis of the effect of golden buckwheat granules on the cell cycle distribution of human prostate cancer cells LNCaP. The results show that under the same action time, the increase in the dose of golden buckwheat granules significantly increased the proportion of subG1 peak cells. 50 At the same dose, the duration of Fagopyrum truncatum action increased, and the proportion of subG1 peak cells increased from 19.6% to 55.5%; IC 75 At the same dose, the proportion of subG1 peak cells increased from 26.8% (24 hours) to 68% (48 hours). The above results show that with the increase of action time and dose, the proportion of LNCaP cell death induced by Golden Fagopyrum Granules also increased significantly.

[0106] Example 4: Investigating the ability of Fagopyrum truncatum extract to downregulate AR and PSA protein levels in human prostate cancer cells LNCaP

[0107] 4.1 Experimental Materials

[0108] Golden buckwheat extract was obtained by decoction from Golden buckwheat slices grown in Guizhou. Trypsin cell digestion solution (0.25% trypsin) and SDS-PAGE loading buffer (5×) were purchased from Shanghai Beyotime Biotechnology Co., Ltd. The BCA assay kit, protease inhibitor cocktail, phosphatase inhibitor cocktail, and PMSF triple pack were all purchased from Shanghai Via Biotechnology Co., Ltd. Prestained color protein markers were purchased from Thermo Fisher Scientific, USA. ECL luminescent solution was purchased from Shanghai Yijia Technology Co., Ltd. Skim milk powder was purchased from Shanghai Sangon Biotechnology Co., Ltd. Methanol was purchased from Shanghai Sinopharm Chemical Reagent Co., Ltd. SDS-PAGE Running Buffer powder and SDS-PAGE Transfer Buffer powder were purchased from Wuhan Saiweier Biotechnology Co., Ltd.

[0109] 4.2 Experimental methods

[0110] The golden buckwheat extract was obtained according to the experimental method in Example 2.

[0111] Human prostate cancer cells LNCaP (2×10 5 Cells / well) were seeded in 6-well plates, cultured in RPMI1640 containing 10% fetal bovine serum, and cultured in a 37°C incubator; the golden buckwheat extract was added to DMSO to prepare the drug solution: 0 (DMSO control group), 0.035 mg / mL (IC 50 ) and 0.059 mg / mL (IC 75 ), the active drug solution was added to a 6-well plate, incubated in a 37°C incubator for 24 hours, and then the cell samples were collected. When collecting the samples, the cells were digested with trypsin, centrifuged and the supernatant was discarded and placed in a 1.5mL centrifuge tube and stored at -80°C. The cells were then pipetted, vortexed, and lysed with RIPA lysis buffer containing 1% protease inhibitors, PMSF (phenylmethylsulfonyl fluoride) and dephosphorylase inhibitors. After centrifugation at 4°C, the supernatant was transferred to a 0.6mL centrifuge tube. The protein concentration was determined by the BCA method. 40μg of protein sample was mixed with loading buffer, the protein was boiled in a 99°C metal bath for 8 minutes, and centrifuged. The proteins were separated by SDS-PAGE one-dimensional electrophoresis technology, and the membrane was transferred using a semi-dry rapid transfer device. After transfer, block the membrane with 5% skim milk powder in TBST for 90 minutes. Wash the membrane three times with TBST, each time for 10 minutes. Dilute the desired primary antibody in TBST containing 3% BSA (refer to the instructions or pre-experimental results for the dilution ratio). Soak the PVDF membrane in the antibody and incubate on a shaker at 4°C overnight. The next day, wash the membrane three times with TBST, each time for 10 minutes. Dilute the secondary antibody in 5% skim milk powder in TBST (refer to the instructions or pre-experimental results for the dilution ratio). Soak the PVDF membrane in the antibody and incubate on a shaker at room temperature for 90 minutes. Wash the membrane three times with TBST, each time for 10 minutes. Finally, mix ECL solutions A and B, apply them dropwise to the membrane, and place it in the machine for exposure.

[0112] 4.3 Experimental Results

[0113] Figure 4 This is an immunoblot analysis of the downregulation of AR and PSA proteins in human prostate cancer cells (LNCaP) by Fagopyrum truncatum extract. The results showed that after 24 hours of treatment with Fagopyrum truncatum extract, the expression levels of AR and prostate-specific antigen (PSA) in LNCaP cells were significantly reduced. This result suggests that Fagopyrum truncatum may inhibit the growth, proliferation, and survival of prostate cancer cells by regulating the AR signaling pathway.

[0114] Because AR plays a crucial role in the early stages of prostate cancer development, reducing AR levels may prevent or delay prostate cancer development before the disease develops. Clinically, PSA levels are commonly used for prostate cancer screening and monitoring, and lower PSA levels are associated with a reduced risk of prostate cancer or slower disease progression. Therefore, golden fagopyrum or golden fagopyrum extracts have the potential to prevent or treat AR-positive prostate cancer and could be used as a health supplement or preventive medication, or as a medication or health supplement after surgical treatment or chemotherapy.

[0115] Example 5: Isolation of Fagopyrum truncatum Extract

[0116] 5.1 Experimental Materials

[0117] The golden buckwheat slices were provided by the production area in Guizhou.

[0118] 5.2 Experimental methods

[0119] Extraction method: Take 19.5 kg of the medicinal material slices, add 200 L of water, soak for 30 minutes, and then decoct for 30 minutes. Let cool and filter. Add 150 L of water to the medicinal residue and decoct for 30 minutes. Let cool and filter. Combine the two filtrates (take 1 / 20 of the total volume, freeze-dry to obtain LF01, and concentrate to obtain 174.2 g of clear paste) to obtain the golden buckwheat aqueous extract. Add 200 L of 80% ethanol aqueous solution to the remaining medicinal residue and reflux extract once. Filter, concentrate the filtrate, and freeze-dry to obtain LF02 (478 g). The aqueous extract of golden buckwheat was loaded onto a 20 kg AB-8 macroporous resin chromatography column. After loading, the column was eluted with water, 10% ethanol (aqueous solution), 30% ethanol (aqueous solution) and 95% ethanol (aqueous solution), with 100 L of elution for each gradient. The loaded effluent and the water washing solution were combined, concentrated and freeze-dried to obtain LF03 (2295 g); the 10% ethanol eluate was concentrated and freeze-dried to obtain LF04 (491 g); the 30% ethanol eluate was concentrated and freeze-dried to obtain LF05 (603 g); the 95% ethanol eluate was concentrated and freeze-dried to obtain LF06 (111 g).

[0120] Figure 5 This is the separation flow chart of Fagopyrum truncatum extract, which yielded 6 different Fagopyrum truncatum components.

[0121] Example 6: Investigating the ability of Fagopyrum truncatum components LF01-LF06 to inhibit the proliferation of human prostate cancer cells LNCaP and PC-3

[0122] 6.1 Experimental methods

[0123] Six different Fagopyrum truncatum fractions, LF01-06, were obtained using the separation process described in Example 5. DMSO was added to prepare therapeutic solutions at varying concentrations: 0 (DMSO control), 0.015625, 0.03125, 0.0625, 0.125, and 0.25 mg / mL. The growth inhibition rates of the Fagopyrum truncatum fractions on human prostate cancer cells, LNCaP and PC-3, were measured and calculated using the same method as in Example 1. In this experiment, *P < 0.05, **P < 0.01, and ***P < 0.001 indicate statistically significant differences compared to the control group for the same cell lines.

[0124] 6.2 Experimental Results

[0125] The SYBR Green assay was used to detect the proliferation inhibitory abilities of these six components in prostate cancer cells with different AR expressions. Figures 6A-6F Figure 2 shows a statistical analysis of the inhibitory effects of Fagopyrum truncatum fractions LF01-LF06 on the proliferation of human prostate cancer cells LNCaP and PC-3. The results showed that, with the exception of LF03, all other fractions exhibited some inhibitory effects on AR-positive LNCaP cells. However, the effects of LF04 and LF05 differed significantly between AR-positive LNCaP cells and AR-negative PC-3 cells. Therefore, components in fractions LF04 and LF05 may underlie Fagopyrum truncatum's AR-dependent inhibition of prostate cancer cell proliferation.

[0126] Example 7: Analysis of components in the golden buckwheat extract fractions LF04 and LF05

[0127] 7.1 Analysis conditions

[0128] 7.1.1 UPLC Chromatographic Conditions

[0129] Chromatograph: Waters H-Class ultra-high performance liquid chromatograph;

[0130] Column: Welch Materials, Ultimate UHPLC AQ-C18, 2.1 × 100 mm, 1.8 μm;

[0131] Column temperature: 30°C; injection volume: 3 μL; flow rate: 0.300 mL / min; detection wavelength: 275 nm;

[0132] Mobile phase gradient setting: see Table 1.

[0133] Table 1 Mobile phase composition and gradient setting (A-0.1% formic acid water, B-methanol)

[0134]

[0135]

[0136] 7.1.2 Mass spectrometry conditions

[0137] Instrument: Sciex Triple TOF 4600LC / MS;

[0138] Detection mode: Positive ion mode / Negative ion mode;

[0139] ESI source parameters: see Table 2;

[0140] Table 2Mass parameters

[0141]

[0142] 7.2 Analysis results

[0143] Figure 7A and Figure 7B This is the UV chromatogram (275nm) of the HPLC-Q-TOF-MS / MS analysis of the components LF04 and LF05 of golden buckwheat. Compared with the catechin, epicatechin, proanthocyanidin B1 and proanthocyanidin B2 reference substances, it was found that the main chromatographic peaks with retention times of 16.18, 17.17, 21.30 and 24.73min in LF04 correspond to proanthocyanidin B1, catechin, proanthocyanidin B2 and epicatechin, respectively; the chromatographic peaks with retention times of 17.26, 21.50 and 24.90min in LF05 correspond to catechin, proanthocyanidin B2 and epicatechin, respectively. In the mass spectrometry detection data of components LF04 and LF05, there are multiple quasi-molecular ion peaks of m / z 577.13, which are detected in their secondary mass spectrometry (MS 2 ) has mass spectrum fragments of 577.13, 451.10, 425.08, 407.07, 289.07, 245.08, 161.02, and 125.02. 2 Data and MS of proanthocyanidin B1 and proanthocyanidin B2 2 The data are basically consistent, so it can be determined that the chromatographic peaks of these quasi-molecular ion peaks m / z 577.13 are proanthocyanidin B1 isomers. The same multiple quasi-molecular ion peaks of m / z 865.20 can also be determined based on their MS 2 The data (865.19, 739.17, 713.15, 695.14, 577.13, 451.10, 425.08, 407.07, 289.07, 287.05, 245.04, 243.03, 161.02, 125.02) were judged to be isomers of proanthocyanidin C1.

[0144] Table 3 LC-MS identification results of Fagopyrum truncatum component LF04

[0145]

[0146]

[0147] Table 4 LC-MS identification results of Fagopyrum truncatum component LF05

[0148]

[0149] Example 8: Investigating the ability of Fagopyrum truncatum extracts to downregulate AR and PSA protein levels in human prostate cancer cells LNCaP and C42B

[0150] 8.1 Experimental Materials

[0151] Golden buckwheat slices were provided by the manufacturer in Guizhou. Trypsin cell digestion solution (0.25% trypsin) and SDS-PAGE loading buffer (5×) were purchased from Shanghai Beyotime Biotechnology Co., Ltd. The BCA assay kit and protease inhibitor cocktail, phosphatase inhibitor cocktail, and PMSF triple pack were all purchased from Shanghai Via Biotechnology Co., Ltd. Prestained protein markers were purchased from Thermo Fisher Scientific, USA. ECL luminescent solution was purchased from Shanghai Yijia Technology Co., Ltd. Skim milk powder was purchased from Shanghai Sangon Biotechnology Co., Ltd. Methanol was purchased from Shanghai Sinopharm Chemical Reagent Co., Ltd. SDS-PAGE Running Buffer Powder and SDS-PAGE Transfer Buffer Powder were purchased from Wuhan Saiweier Biotechnology Co., Ltd.

[0152] 8.2 Experimental methods

[0153] According to the experimental method in Example 5, six kinds of golden buckwheat extract components LF01-06 were obtained.

[0154] Human prostate cancer cells LNCaP (1.5×10 6 cells / well) or C42B (1×10 6 Cells / well) were seeded in a culture dish, and the culture medium was RPMI1640 containing 10% fetal bovine serum, and cultured in a 37°C incubator; the golden buckwheat extract component LF01-06 was extracted according to IC 50The doses (LNCaP: LF01: 0.032 mg / mL; LF02: 0.012 mg / mL; LF04: 0.027 mg / mL; LF05: 0.021 mg / mL; LF06: 0.039 mg / mL; C42B: LF01: 0.14 mg / mL; LF02: 0.057 mg / mL; LF04: 0.049 mg / mL; LF05: 0.032 mg / mL; LF06: 0.043 mg / mL; LF01-06) for C42B were 50 The dosage was determined according to the method of Example 6; since LF03 had no efficacy, the highest dose of IC among LF01-02 and LF04-06 was used. 50 As the addition amount) to prepare the active drug solution. The active drug solution was added to the culture dish respectively, incubated in a 37°C incubator for 24 hours, and then the cell samples were collected. When collecting the samples, the cells were digested with trypsin, centrifuged and the supernatant was discarded and placed in a 1.5mL centrifuge tube and stored at -80°C. The cells were then blown, vortexed, and lysed with RIPA lysis buffer containing 1% protease inhibitors, PMSF (phenylmethylsulfonyl fluoride) and dephosphorylase inhibitors. After centrifugation at 4°C, the supernatant was transferred to a 0.6mL centrifuge tube. The protein concentration was determined by the BCA method. 40μg of protein sample was mixed with the loading buffer, the protein was boiled in a 99°C metal bath for 8 minutes, and centrifuged. The protein was separated by SDS-PAGE one-dimensional electrophoresis technology, and the membrane was transferred using a semi-dry rapid transfer device. After transfer, block the membrane with 5% skim milk powder in TBST for 90 minutes. Wash the membrane three times with TBST, each time for 10 minutes. Dilute the desired primary antibody in TBST containing 3% BSA (refer to the instructions or pre-experimental results for the dilution ratio). Soak the PVDF membrane in the antibody and incubate on a shaker at 4°C overnight. The next day, wash the membrane three times with TBST, each time for 10 minutes. Dilute the secondary antibody in 5% skim milk powder in TBST (refer to the instructions or pre-experimental results for the dilution ratio). Soak the PVDF membrane in the antibody and incubate on a shaker at room temperature for 90 minutes. Wash the membrane three times with TBST, each time for 10 minutes. Finally, mix ECL solutions A and B, apply them dropwise to the membrane, and place it in the machine for exposure.

[0155] 8.3 Experimental Results

[0156] Figure 8This is an immunoblot analysis of the effects of Fagopyrum truncatum extracts on downregulation of AR and PSA proteins in human prostate cancer cells, LNCaP and C42B. C42B cells are also AR-positive prostate cancer cells. In this experiment, C42B cells were added to confirm the regulatory effects of Fagopyrum truncatum extracts on AR and PSA in AR-positive prostate cancer cells. The results showed that after 24 hours of treatment with Fagopyrum truncatum extracts, LF01, LF02, LF04, LF05, and LF06 significantly reduced AR expression in LNCaP and C42B cells; LF04 and LF05 significantly downregulated PSA expression in LNCaP and C42B cells. This result indicates that the active ingredients LF04 and LF05 can also downregulate AR and PSA protein expression levels.

[0157] Example 9: Detection of the effect of Fagopyrum truncatum extract components on regulating AR and PSA mRNA levels in human prostate cancer cells LNCaP and C42B

[0158] 9.1 Experimental Materials

[0159] Golden buckwheat slices were provided by the manufacturer in Guizhou. RNAiso Plus, a total RNA extraction reagent, and PrimeScript RT Reagent Kit were purchased from TAKARA Biotechnology, Japan. SYBR Green Realtime PCR Master Mix was purchased from TOYOBO Life Science, Japan. RNASE AWAY was purchased from Molecular BioProducts, USA. DEPC water (DNase- and RNase-free) was purchased from Shanghai Bio-Technology Co., Ltd. Other reagents, including anhydrous ethanol, chloroform, 75% ethanol, and isopropanol, were purchased from Shanghai Titan Chemical Co., Ltd.

[0160] 9.2 Experimental Methods

[0161] According to the experimental method in Example 5, six kinds of golden buckwheat extract components LF01-06 were obtained.

[0162] Human prostate cancer cells LNCaP (1.5×10 6 cells / well) or C42B (1×10 6 Cells / well) were seeded in a culture dish, and the culture medium was RPMI1640 containing 10% fetal bovine serum, and cultured in a 37°C incubator; the golden buckwheat extract component LF01-06 was extracted according to IC 50The doses (LNCaP: LF01: 0.032 mg / mL; LF02: 0.012 mg / mL; LF04: 0.027 mg / mL; LF05: 0.021 mg / mL; LF06: 0.039 mg / mL; C42B: LF01: 0.14 mg / mL; LF02: 0.057 mg / mL; LF04: 0.049 mg / mL; LF05: 0.032 mg / mL; LF06: 0.043 mg / mL; LF01-06) for C42B were 50 The dosage was determined according to the method of Example 6; since LF03 had no efficacy, the highest dose of IC among LF01-02 and LF04-06 was used. 50 Prepare the active agent solution (using the added amount). Add the active agent solution to each culture dish and incubate in a 37°C incubator for 24 hours. Then, collect the cell samples. To collect the samples, digest the cells with trypsin, centrifuge, discard the supernatant, and place in a 1.5 mL centrifuge tube and store at -80°C. Add RNAiso Plus to the sample to dislodge the cell clumps. Add chloroform and shake upside down to mix thoroughly. Let it stand at room temperature. Centrifuge at 4°C and transfer the top, clear supernatant to a newly labeled EP tube. Add isopropanol and mix thoroughly by inverting until no layers separate. Let it stand at room temperature. Centrifuge at 4°C and discard the supernatant. Add 75% ethanol along the tube wall and gently invert. Centrifuge at 4°C and discard the supernatant. Dissolve the RNA precipitate in DEPC water, as determined by the size of the precipitate. Vortex and centrifuge, then store at -80°C. Determine the RNA concentration using an ultra-micro spectrophotometer. Adjust all samples to the same concentration using DEPC water based on the RNA concentration. Place the prepared RNA reverse transcription system in a reverse transcription machine for reverse transcription. Use the reverse transcribed cDNA to prepare the amplification system, add 10 μL of the amplification system to a 96-well PCR plate, seal the plate with film, and place it in a PCR instrument for amplification.

[0163] 9.3 Experimental Results

[0164] Figure 9 The following is an analysis of the effects of LF01-LF06, a component extracted from golden buckwheat, on the expression of AR and PSA mRNA in human prostate cancer cells LNCaP and C42B. Figure 9 As shown, PC-3 cells are AR-negative and serve as a negative control. After 24 hours of treatment with fractions extracted from Fagopyrum truncatum, LF01, LF02, LF04, LF05, and LF06 all reduced AR and PSA mRNA levels in LNCaP and C42B cells. This result suggests that the active fractions LF04 and LF05 can downregulate AR and PSA mRNA levels.

[0165] Example 10: Detection of the Effects of Fagopyrum truncatum Extract on PSA Levels in Culture Medium of Human Prostate Cancer Cells LNCaP and C42B

[0166] 10.1 Experimental Materials

[0167] Golden buckwheat slices were provided by the Guizhou production area. Human Prostate-Specific Antigen / PSA ELISA Kit (enzyme-linked immunosorbent assay) was purchased from Lianke Biotechnology Co., Ltd.

[0168] 10.2 Experimental Methods

[0169] According to the experimental method in Example 5, six kinds of golden buckwheat extract components LF01-06 were obtained.

[0170] Human prostate cancer cells LNCaP (6×10 3 cells / well) or C42B (44×10 3 Cells (100 cells / well) were seeded in a 96-well plate in RPMI 1640 medium supplemented with 10% fetal bovine serum and incubated at 37°C for 48 hours. The drug component powders were dissolved in DMSO to a stock solution of 400 mg / mL (LF01-03) or 200 mg / mL (LF04-06). Fresh culture medium was used to prepare the drug solution at different concentrations: 0 (DMSO control), 0.015625, 0.03125, 0.0625, 0.125, and 0.25 mg / mL. The old medium in the plate was aspirated and the prepared drug-containing medium was added at 100 μL / well. The plate was incubated at 37°C for 72 hours. The drug-containing medium in the well with 0.03125 mg / mL was aspirated and centrifuged at 300g for 10 minutes at 4°C. The supernatant was stored at -20°C.

[0171] Take the ELISA kit and the drug-containing culture medium stored at -20°C, return them to room temperature, and perform the experiment according to the ELISA kit instructions. Use a microplate reader to detect absorbance at 450nm and 570nm.

[0172] In this experiment, *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001 indicate significant statistical differences compared with the control group of the same cell line.

[0173] 10.3 Experimental Results

[0174] Figures 10A-10BThis is an analysis of the effects of LF01-LF06, a component extracted from golden buckwheat, on PSA levels in culture medium of human prostate cancer cells LNCaP and C42B. Prostate-specific antigen (PSA) is an important marker for prostate cancer. Elevated PSA levels in the blood may indicate prostate cancer or other prostate diseases. Therefore, PSA levels are often used as an indicator to assess AR activity and prostate cancer progression. Figure 10A As shown in the results, the extracts of golden buckwheat were treated in LNCaP and C42B cells for 72 hours, and LF02, LF04, LF05 and LF06 significantly reduced the PSA level in the culture medium. Since there were differences in the number of cells between different groups after 72 hours of culture, Figure 10B The PSA concentration determined by ELISA was divided by the cell absorbance (representing the number of cells) measured using the SYBR Green assay in the same well plate. The results indicate that LF02, LF04, LF05, and LF06 may inhibit the cell's ability to secrete PSA. In summary, the active ingredients LF04 and LF05 significantly reduced PSA levels in the culture medium and may inhibit PSA secretion in AR-positive prostate cancer, suggesting that LF04 and LF05 have the potential to inhibit prostate cancer progression.

[0175] Example 11: Investigating the ability of the dialysis-separated fractions of Fagopyrum truncatum LF04 and LF05 to inhibit the proliferation of human prostate cancer cells LNCaP, C42B, and PC-3

[0176] 11.1 Experimental Methods

[0177] 11.1.1 Dialysis Separation with Dialysis Bags

[0178] Golden buckwheat slices were provided by the Guizhou production area. LF04 was obtained according to the experimental method in Example 5. 2 g of LF04 powder was taken, 150 mL of water was added and ultrasonically heated to dissolve it, and then it was placed in a dialysis bag with a molecular weight cutoff of 500 Da. The dialysis bag was then placed in a beaker, 300 mL of water was added to the beaker, and the dialysis was performed for 24 h. The solution outside the dialysis bag was poured out, and 300 mL of water was added again for dialysis. After dialysis 3 times, the three dialysates were combined to obtain LF04 <500 Da solution, which was concentrated and freeze-dried; then it was passed through dialysis bags with molecular weight cutoffs of 3500 Da and 13000 Da, and dialyzed 3 times each according to the same method to obtain LF04 500-3500 Da, LF04 3500-13000 Da, and LF04>13000 Da.

[0179] LF05 was operated in the same way to obtain LF05<500Da, LF05 500-3500Da, LF05 3.5k-13kDa and LF05>13kDa.

[0180] 11.1.2SYBR Green Experiment

[0181] Eight different golden buckwheat components were obtained through dialysis separation: LF04 < 500Da, LF04 500-3500Da, LF04 3.5k-13kDa, LF04 > 13kDa, LF05 < 500Da, LF05 500-3500Da, LF05 3.5k-13kDa, and LF05 > 13kDa. DMSO was added to prepare different concentration gradients of the active solution: 0 (DMSO control group), 0.015625, 0.03125, 0.0625, 0.125, and 0.25 mg / mL. The test was performed using the same method as in Example 1. In this experiment, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 indicate statistically significant differences compared to the control group of the same cell line.

[0182] 11.2 Experimental Results

[0183] The SYBR Green assay was used to detect the proliferation inhibitory abilities of these eight components in prostate cancer cells with different AR expressions. Figures 11A-11C This is a statistical analysis of the inhibitory effects of dialysis-isolated fractions of Fagopyrum truncatum (LF04 and LF05) on the proliferation of human prostate cancer cells LNCaP, C42B, and PC-3. The results show that fractions with a molecular weight of 500-13,000 Da in LF04 and LF05 have a more potent inhibitory effect on the proliferation of AR-positive prostate cancer cells. Figures 12A-12D These are the UV chromatograms, spectra and mass spectra of the components LF04 and LF05 separated after dialysis. The results indicate that the effective substances of the LF04 and LF05 components may be highly polymerized proanthocyanidins with a molecular weight range of 1500-15000Da.

[0184] Example 12: Detection of the effect of Fagopyrum truncatum component LF01-06 on human prostate hyperplasia cells BPH-1

[0185] 12.1 Experimental Methods

[0186] Fagopyrum truncatum slices were obtained from Guizhou Province. Six different Fagopyrum truncatum fractions, LF01-06, were obtained through the separation process described in Example 5. DMSO was added to prepare therapeutic solutions at varying concentrations: 0 (DMSO control), 0.015625, 0.03125, 0.0625, 0.125, and 0.25 mg / mL. The growth inhibition rates of the Fagopyrum truncatum fractions on human prostate hyperplasia (BPH-1) cells were measured and calculated using the same method as in Example 1. In this experiment, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 indicate statistically significant differences compared to the control group for the same cell line.

[0187] 12.2 Experimental Results

[0188] The SYBR Green assay was used to detect the inhibitory effects of the six components on AR-positive prostate hyperplasia cells. Figure 13 This is a statistical analysis of the inhibition of human prostate hyperplasia (BPH)-1 cell proliferation by Fagopyrum truncatum components LF01-LF06. The results show that Fagopyrum truncatum components LF02, LF04, and LF05 can significantly inhibit the proliferation of human prostate hyperplasia (BPH)-1 cells, indicating that Fagopyrum truncatum also has an inhibitory effect on AR-positive prostate hyperplasia cells and has the potential to treat prostate hyperplasia.

[0189] Through SYBR Green experiments, we obtained IC values ​​for LF04 and LF05 to inhibit the proliferation of BPH-1 cells (experiments have confirmed that the cells express AR positively, and the expression level is lower than that of prostate cancer cells LNCaP and C42B cells). 50 The IC values ​​of LF04 and LF05 for inhibiting the proliferation of prostate cancer LNCaP cells were 0.093 and 0.066 mg / mL, respectively. 50 were 0.027 and 0.021 mg / mL, respectively, which were approximately one-third of that of BPH-1. Compared with C42B cells, the IC 50 The concentrations of LF04 and LF05 in the extracts of Fagopyrum serrata were 0.049 and 0.032 mg / mL, respectively, which were approximately half of those in BPH-1. This indicates that the Fagopyrum serrata components LF04 and LF05 may be more sensitive to prostate cancer cells with higher AR expression and have a certain degree of selectivity.

[0190] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Although the present invention has been disclosed as above in terms of a preferred embodiment, it is not intended to limit the present invention. Any technician familiar with this patent can make slight changes or modifications to equivalent embodiments using the above-mentioned technical contents without departing from the scope of the technical solution of the present invention. However, any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the solution of the present invention.

Claims

1. The use of golden buckwheat or its extract, characterized in that, Used for preparing medicines or health products for preventing or treating AR-positive prostate cancer, or treating benign prostatic hyperplasia; preferably, the prostate cancer is early-stage prostate cancer.

2. The use according to claim 1, characterized in that The extract is a water extract, an alcohol extract or a ketone extract; preferably, the water extract is a water decoction extract, and the alcohol extract is an ethanol extract.

3. The use according to claim 2, characterized in that The ethanol extract is an extract obtained by directly extracting Fagopyrum truncatum with ethanol, or an extract obtained by extracting the residue of Fagopyrum truncatum with water and then subjecting it to ethanol extraction.

4. The use according to any one of claims 1 to 3, characterized in that The extract contains monomeric proanthocyanidins, oligomeric proanthocyanidins and / or polymeric proanthocyanidins; Preferably, the monomeric proanthocyanidins and oligomeric proanthocyanidins contain one or more of proanthocyanidin B1, proanthocyanidin B2, proanthocyanidin A1 isomer, proanthocyanidin B2 isomer, proanthocyanidin C1 isomer, catechin or epicatechin; and the molecular weight of the polymeric proanthocyanidins is 1500-15000 Da.

5. A method for preparing a water extract of Fagopyrum serrata, characterized in that: The method comprises the following steps: decocting golden buckwheat with water, removing the medicinal residue to obtain a water extract, subjecting the water extract to macroporous adsorption resin column chromatography, and eluting with ethanol to obtain a water extract; the ethanol is 5%-100% ethanol; preferably, the ethanol is 10%-95% ethanol.

6. The preparation method according to claim 5, characterized in that The aqueous extract is subjected to macroporous adsorption resin column chromatography, and is first eluted with 5%-50% ethanol to obtain aqueous extract 1; optionally, it is further eluted with 90%-100% ethanol to obtain aqueous extract 2; Preferably, the water extract is chromatographed on a macroporous adsorption resin column, first eluted with 5%-15% ethanol to obtain water extract 1-1; then eluted with 20%-50% ethanol to obtain water extract 1-2; optionally, eluted with 90%-100% ethanol to obtain water extract 2.

7. The preparation method according to claim 5 or 6, characterized in that: It also includes the following steps: Before eluting with ethanol, wash with water; and / or After elution with ethanol, the eluate is concentrated and / or dried to obtain a water extract.

8. The preparation method according to any one of claims 5 to 7, characterized in that The method further comprises the following steps: subjecting the aqueous extract obtained by macroporous adsorption resin column chromatography to further separation to obtain an aqueous extract containing compounds with a molecular weight greater than or equal to 200; optionally, concentrating and / or drying after the further separation; Preferably, a water extract containing compounds with a molecular weight of 500-20,000 is obtained; more preferably, a water extract containing compounds with a molecular weight of 1,500-15,000 is obtained.

9. A golden buckwheat water extract, characterized in that The invention is prepared by the preparation method according to any one of claims 5 to 8.

10. The golden buckwheat water extract according to claim 9, characterized in that Containing monomeric proanthocyanidins, oligomeric proanthocyanidins and / or polymeric proanthocyanidins; Preferably, the monomeric proanthocyanidins and oligomeric proanthocyanidins contain one or more of proanthocyanidin B1, proanthocyanidin B2, proanthocyanidin A1 isomer, proanthocyanidin B2 isomer, proanthocyanidin C1 isomer, catechin or epicatechin; and the molecular weight of the polymeric proanthocyanidins is 1500-15000 Da.

11. The use according to any one of claims 1 to 3, characterized in that The extract is the golden buckwheat water extract according to any one of claims 9-10.

12. A pharmaceutical composition or health product, characterized in that: Contains the golden buckwheat water extract according to any one of claims 9-10.

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