WO2020125091A1 - 南瓜籽油中甾醇组合物及其应用和治疗前列腺增生药物 - Google Patents

南瓜籽油中甾醇组合物及其应用和治疗前列腺增生药物 Download PDF

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WO2020125091A1
WO2020125091A1 PCT/CN2019/105930 CN2019105930W WO2020125091A1 WO 2020125091 A1 WO2020125091 A1 WO 2020125091A1 CN 2019105930 W CN2019105930 W CN 2019105930W WO 2020125091 A1 WO2020125091 A1 WO 2020125091A1
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parts
seed oil
pumpkin seed
sterol composition
sterol
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PCT/CN2019/105930
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French (fr)
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刘东波
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湖南中科文泰生物科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/575—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of three or more carbon atoms, e.g. cholane, cholestane, ergosterol, sitosterol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/42—Cucurbitaceae (Cucumber family)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/08—Drugs for disorders of the urinary system of the prostate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine

Definitions

  • the invention belongs to the technical field of medicine, and specifically relates to a sterol composition in pumpkin seed oil, its application and a medicine for treating prostate hyperplasia.
  • Prostatic hyperplasia (benignprostatic hyperplasia, BPH) is a common and frequent disease in middle-aged and elderly men.
  • BPH benignprostatic hyperplasia
  • the treatment of benign prostatic hyperplasia is mainly through Western medicine and non-drug treatment.
  • Commonly used Western medicine 5 ⁇ -reductase inhibitors such as finasteride, ⁇ 1-adrenoceptor blockers such as phenoxybenzamine or mazanib, such drugs
  • the treatment has a certain therapeutic effect, but the effect is slow, usually requires more than 6 months of medication, and the side effects are large, such as body side hypotension, fatigue, blurred vision, ejaculation disorders and other side effects.
  • bee pollen extract namely Qianliekang, saw palmetto extract, which is commercially available Bourson, Bonot capsules, and African hipberry extract, which is Tonguria
  • bee pollen extract namely Qianliekang, saw palmetto extract, which is commercially available Bourson, Bonot capsules, and African hipberry extract, which is Tonguria
  • African hipberry extract which is Tonguria
  • the present invention provides a sterol composition in pumpkin seed oil and its application and medicine for treating prostate hyperplasia, which can quickly and safely treat prostate hyperplasia without side effects.
  • the present invention adopts the following technical solutions:
  • the present invention provides a sterol composition in pumpkin seed oil, which includes the following components in parts by weight: 0.2-2 parts of cholesterol, 0.5-5 parts of campesterol, 1.5-5 parts of ⁇ -sitosterol, and stigmasterol 15 ⁇ 40 parts, ergoster-7,22-diene-3 ⁇ -alcohol 20-40 parts, lanosterol 5-10 parts, cholester-7-ene-3 ⁇ , 5 ⁇ -diol 15-30 parts.
  • the sterol composition in pumpkin seed oil includes the following components in parts by weight: cholesterol 0.5-1 parts, campesterol 1-3 parts, ⁇ -sitosterol 2-3 parts, stigmasterol 20- 35 parts, ergosterol-7,22-diene-3 ⁇ -alcohol 25-35 parts, lanosterol 6-8 parts, cholester-7-ene-3 ⁇ , 5 ⁇ -diol 20-35 parts.
  • the sterol composition in pumpkin seed oil includes the following parts by weight of components: cholesterol 0.99 parts, campesterol sterol 1.35 parts, ⁇ -sitosterol 2.42 parts, stigmasterol 30.06 parts, ergosterol-7, 32.01 parts of 22-diene-3 ⁇ -alcohol, 6.09 parts of lanosterol, 24.12 parts of 5 ⁇ -diol.
  • the sterol composition in pumpkin seed oil and the sterol composition in pumpkin seed oil are prepared by the following steps: pumpkin seed oil is sequentially subjected to saponification reaction and organic solvent extraction, and the upper phase of the extraction is taken as the extract , Washing the extract and drying the crystals to obtain the sterol composition in pumpkin seed oil.
  • the sterol composition in the pumpkin seed oil is more than 90%.
  • the step of washing the extract is specifically: washing the extract in sequence with water, ethanol aqueous solution, water, potassium hydroxide solution, and water, discarding the lower aqueous phase, and retaining the extraction The upper organic phase of the liquid.
  • the step of washing the extract with the potassium hydroxide solution and water in the sterol composition in pumpkin seed oil is repeated at least once, until the lower aqueous phase is neutral.
  • the present invention provides the use of the sterol composition in pumpkin seed oil in the treatment of prostatic hyperplasia.
  • the present invention provides a medicament for treating benign prostatic hyperplasia, including pharmaceutically acceptable excipients, and further comprising an effective dose of the sterol composition in pumpkin seed oil described above.
  • the dosage form of the drug for treating benign prostatic hyperplasia is any one of solution, pill, tablet, capsule, powder, paste and aerosol.
  • the present invention has the following beneficial effects:
  • the sterol composition in pumpkin seed oil according to the present invention is a pumpkin plant sterol composition extracted from pumpkin seed oil, in which cholesterol, campesterol, ⁇ -sitosterol, stigmasterol, ergosterol-7,22-diene -3 ⁇ -alcohol, lanosterol, cholesterol-7-ene-3 ⁇ , 5 ⁇ -diol are all phytosterols, through the compatibility of each phytosterol component, the ratio between each component is controlled, with relief and The effect of treating prostate diseases, especially prostatic hyperplasia.
  • the phytosterols in the sterol composition in the pumpkin seed oil of the present invention have a synergistic effect and have anti-inflammatory and immunomodulatory effects, and the phytosterols in the sterol composition in the pumpkin seed oil show a high degree of prostate tissue Affinity, showing the activity of hormones in the body of animals or humans, can regulate the gene expression of growth factors related to benign prostatic hyperplasia, but without hormone side effects.
  • the sterol composition in the pumpkin seed oil is mainly ⁇ -7 phytosterol, which is the main substance for inhibiting prostate hyperplasia.
  • ⁇ -7 phytosterol has the effect of inhibiting 5 ⁇ -reductase, thereby inhibiting the conversion of testosterone to dihydrotestosterone , Inhibit prostate hyperplasia.
  • the sterol composition in the pumpkin seed oil not only inhibits the expression of 5 ⁇ -reductase, but also inhibits the gene expression of androgen receptor, bFGF and TGF- ⁇ 1, etc., which can significantly reduce the weight and prostate index of prostate organs, making Prostatic hyperplasia is recovered.
  • Example 1 is a GC-MS chart of the sterol composition in pumpkin seed oil according to Example 1 of the present invention
  • FIG. 2 is a diagram of prostate tissue in an embodiment of the present invention.
  • Fig. 3 is a pathological slice of H&E staining in an embodiment of the present invention.
  • FIG. 4 is a graph showing the effect of the sterol composition in pumpkin seed oil described in Example 1 on the expression of androgen receptor and steroid receptor coactivator in prostate hyperplasia rats;
  • FIG. 5 is a graph showing the expression change of 5 ⁇ -reductase gene detected by RT-qPCR in an embodiment of the present invention
  • FIG. 6 is a graph showing the change of bFGF gene expression detected by RT-qPCR in an embodiment of the present invention.
  • FIG. 7 is a graph showing the change of TGF- ⁇ 1 gene expression detected by RT-qPCR in an embodiment of the present invention.
  • the embodiment of the present invention provides a sterol composition in pumpkin seed oil, which includes the following components in parts by weight: cholesterol 0.2-2 parts, campesterol sterol 0.5-5 parts, beta-sitosterol 1.5-5 parts, beans 15-40 parts of sterol, 20-40 parts of ergosterol-7,22-diene-3 ⁇ -alcohol, 5-10 parts of lanosterol, 15-30 parts of cholester-7-ene-3 ⁇ , 5 ⁇ -diol.
  • the molecular formula of the campesterol is C 28 H 48 O, the relative molecular mass is 400.69, and the chemical structural formula is shown in Formula II:
  • the molecular formula of stigmasterol is C 29 H 52 O, the relative molecular mass is 416.73, and the chemical structural formula is as shown in Formula IV:
  • the molecular formula of the lanosterol is C 30 H 50 O, the relative molecular mass is 426.73, and the chemical structural formula is shown in Formula VI:
  • the sterol composition in pumpkin seed oil is a pumpkin plant sterol composition extracted from pumpkin seed oil, in which cholesterol, campesterol, ⁇ -sitosterol, stigmasterol, ergosterol-7,22- Diene-3 ⁇ -alcohol, lanosterol, cholesterol-7-ene-3 ⁇ , 5 ⁇ -diol are all phytosterols, and through the compatibility of each phytosterol component, the ratio between each component is controlled, with The effect of relieving and treating prostate diseases, especially prostatic hyperplasia.
  • the phytosterols in the sterol composition in pumpkin seed oil according to the embodiment of the present invention have a synergistic effect and have anti-inflammatory and immunomodulatory effects, and the phytosterols in the sterol composition in pumpkin seed oil have a performance on prostate tissue It has a high affinity and shows hormone activity in the body of animals or humans, which can regulate the gene expression of growth factors related to benign prostatic hyperplasia, but without hormone side effects.
  • the sterol composition in the pumpkin seed oil is mainly ⁇ -7 phytosterol, which is the main substance for inhibiting prostate hyperplasia.
  • ⁇ -7 phytosterol has the effect of inhibiting 5 ⁇ -reductase, thereby inhibiting the conversion of testosterone to dihydrotestosterone , Inhibit prostate hyperplasia.
  • the sterol composition in pumpkin seed oil not only inhibits the expression of 5 ⁇ -reductase gene, but also inhibits the gene expression of androgen receptor, bFGF and TGF- ⁇ 1, etc., which can significantly reduce the weight and prostate index of prostate organs, Make the prostate hyperplasia recover.
  • the sterol composition in the pumpkin seed oil includes the following components in parts by weight: 0.5 to 1 part of cholesterol, 1 to 3 parts of campesterol, 2 to 3 parts of ⁇ -sitosterol, and 20 to 35 parts of stigmasterol , Ergoster-7,22-diene-3 ⁇ -alcohol 25-35 parts, lanosterol 6-8 parts, cholester-7-ene-3 ⁇ , 5 ⁇ -diol 20-35 parts.
  • the sterol composition in the pumpkin seed oil includes the following parts by weight: cholesterol 0.6-1 parts, campesterol 1.1-2 parts, ⁇ -sitosterol 2.2-2.5 parts, stigmasterol 25-32 parts , Ergoster-7,22-diene-3 ⁇ -alcohol 30-33 parts, lanosterol 6-7 parts, cholester-7-ene-3 ⁇ , 5 ⁇ -diol 22-30 parts.
  • the sterol composition in pumpkin seed oil includes the following components in parts by weight: cholesterol 0.99 parts, campesterol sterol 1.35 parts, ⁇ -sitosterol 2.42 parts, stigmasterol 30.06 parts, ergosterol-7 , 32.01 parts of 22-diene-3 ⁇ -alcohol, 6.09 parts of lanosterol, 24.12 parts of cholester-7-ene-3 ⁇ , 5 ⁇ -diol.
  • the sterol composition in pumpkin seed oil includes the following components by weight: 0.8 parts of cholesterol, 1.5 parts of campesterol, 2.3 parts of ⁇ -sitosterol, 28 parts of stigmasterol, and ergot Sterol-7,22-diene-3 ⁇ -alcohol 31 parts, lanosterol 6.5 parts, cholester-7-ene-3 ⁇ , 5 ⁇ -diol 23 parts.
  • the sterol composition in pumpkin seed oil includes the following parts by weight of components: 0.2 parts of cholesterol, 0.5 parts of campesterol, 1.5 parts of ⁇ -sitosterol, 15 parts of stigmasterol, and ergot Steroid-7,22-diene-3 ⁇ -alcohol 20 parts, lanosterol 5 parts, cholester-7-ene-3 ⁇ , 5 ⁇ -diol 30 parts.
  • the sterol composition in pumpkin seed oil includes the following components in parts by weight: 1.5 parts of cholesterol, 3 parts of campesterol, 3.5 parts of ⁇ -sitosterol, 35 parts of stigmasterol, and ergot Steroid-7,22-diene-3 ⁇ -alcohol 30 parts, lanosterol 7.5 parts, cholester-7-ene-3 ⁇ , 5 ⁇ -diol 22.5 parts.
  • the sterol composition in pumpkin seed oil is a composition obtained from pumpkin seed oil, and the sterol composition in pumpkin seed oil is specifically prepared by the following steps: pumpkin seed oil is sequentially subjected to saponification reaction, Organic solvent extraction, taking the extracted upper phase as the extract, washing the extract and drying the crystals to obtain the sterol composition in pumpkin seed oil.
  • the mass content of phytosterol in the sterol composition in the pumpkin seed oil prepared in the above examples is more than 90%, and compared with the prior art, the purity of the phytosterol prepared in the examples of the present invention is higher.
  • the step of saponification reaction is specifically to prepare a 0.5 mol/L to 1.5 mol/L potassium hydroxide-ethanol solution and mix the pumpkin seed oil with potassium hydroxide-ethanol at a feed-liquid ratio of 1:10 (w:v)
  • the solution is mixed, saponified and refluxed at 65 to 100°C for 0.5 to 2.5 hours. Saponification is completed to obtain a mixed solution.
  • Water with a volume fraction of 12.5% to 25% of the mixed solution is added to the saponified solution to dissolve the fatty acid salt generated by the saponification reaction.
  • the reaction system is cooled to room temperature to obtain a saponified liquid; the room temperature is the ambient temperature where the reaction system is located.
  • the step of organic solvent extraction is that the organic solvent can be extracted with n-hexane, specifically, the saponified liquid is extracted with n-hexane 1 to 3 times, the extraction upper phase is taken, and the extraction upper phase is combined to obtain the extraction liquid; each extraction process In the volume ratio of n-hexane and saponification liquid is 1:1.
  • the steps of washing the extract are specifically: washing the extract in sequence with water, ethanol aqueous solution, water, potassium hydroxide solution, and water, discarding the lower aqueous phase, and retaining the upper organic phase of the extract. Further, the steps of washing the extract with potassium hydroxide solution and water are repeated at least once, until the lower aqueous phase is neutral.
  • the washing solution is water and the volume fraction is 8%-25% ethanol aqueous solution, 0.2 ⁇ 0.5mol/L potassium hydroxide solution; the volume of each washing solution is 10%-30% of the volume of saponification solution, repeat the steps of potassium hydroxide washing and water washing until The lower aqueous phase is neutral, the aqueous layer is discarded, and the upper organic phase of the extract is retained.
  • the steps of drying and crystallization are specifically: combining the upper organic phase, adding anhydrous sodium sulfate to the upper organic phase, and standing for 10-30 minutes to remove water, and then concentrating the dehydrated extract with a reduced-pressure rotary evaporator. Let stand overnight in a refrigerator at 4°C to obtain white needle-like crystals; filter with suction, wash with n-hexane, and dry to obtain a sterol composition in pumpkin seed oil.
  • the saponification liquid obtained by the current alkali saponification is washed with water and alkali during the washing process after extraction. Since the fatty acid salt generated in the saponification process encounters water, a large amount of foam will be generated, emulsifying the upper organic phase in the saponification liquid. Even if the ratio of the lower water phase to the upper organic phase is adjusted, a large amount of emulsified layer will inevitably be formed between the two phases, the time from washing to neutrality will increase, and the sterol composition in pumpkin seed oil will be lost with washing.
  • an aqueous ethanol solution is added for washing, which effectively eliminates the phenomenon that fatty acid salts encounter a large amount of foam when it meets water, reduces the emulsified layer between the two phases, shortens the entire washing process step, and reduces emulsification
  • the loss of the layer also improves the final product yield; the product yield of the above preparation method can reach 1.6-2 ⁇ .
  • the embodiments of the present invention provide the use of the sterol composition in pumpkin seed oil in the treatment of prostatic hyperplasia.
  • the phytosterols in the sterol composition in pumpkin seed oil according to the embodiment of the present invention have a synergistic effect and have anti-inflammatory and immunomodulatory effects, and the phytosterols in the sterol composition in pumpkin seed oil have a performance on prostate tissue It has a high affinity and shows hormone activity in the body of animals or humans, which can regulate the gene expression of growth factors related to benign prostatic hyperplasia, but without hormone side effects.
  • the sterol composition in the pumpkin seed oil is mainly ⁇ -7 phytosterol, which is the main substance for inhibiting prostate hyperplasia.
  • ⁇ -7 phytosterol has the effect of inhibiting 5 ⁇ -reductase, thereby inhibiting the conversion of testosterone to dihydrotestosterone , Inhibit prostate hyperplasia.
  • the sterol composition in pumpkin seed oil not only inhibits the expression of 5 ⁇ -reductase gene, but also inhibits the gene expression of androgen receptor, bFGF and TGF- ⁇ 1, etc., which can significantly reduce the weight and prostate index of prostate organs, Make the prostate hyperplasia recover.
  • the embodiments of the present invention also provide a medicine for treating prostate hyperplasia, including pharmaceutically acceptable
  • the auxiliary material also includes an effective dose of the sterol composition in pumpkin seed oil described in the above embodiments.
  • the effective dose refers to the therapeutically effective amount, and refers to the amount of the sterol composition in the pumpkin seed oil of the present invention sufficient to show benefit or clinical significance to the individual; those skilled in the art will understand that the actual amount or dose administered And the administration schedule will depend on the nature and severity of the disease being treated, the age and general condition of the subject being treated, and the mode of administration.
  • the sterol composition in pumpkin seed oil is the above-mentioned components including the following components and their parts by weight: 0.2-2 parts of cholesterol, 0.5-5 parts of rapeseed sterol, and 1.5- ⁇ -sitosterol 5 parts, stigmasterol 15-40 parts, ergosterol-7,22-diene-3 ⁇ -alcohol 20-40 parts, lanosterol 5-10 parts, cholester-7-ene-3 ⁇ , 5 ⁇ -diol 15 ⁇ 30 parts; in a specific embodiment, the sterol composition in pumpkin seed oil includes the following parts by weight of components: cholesterol 0.99 parts, campesterol sterol 1.35 parts, ⁇ -sitosterol 2.42 parts, stigmasterol 30.06 parts, Ergoster-7,22-diene-3 ⁇ -alcohol 32.01 parts, lanosterol 6.09 parts, cholester-7-ene-3 ⁇ , 5 ⁇ -diol 24.12 parts; compatibility between each component and control of each component The dosage ratio can effectively improve the efficacy
  • the excipients should be non-toxic, do not interfere with or impair the effectiveness of the sterol composition in the pumpkin seed oil described above in the embodiments of the present invention, and the selection of the excipients can be based on the above-mentioned embodiments to treat the presence of prostatic hyperplasia And choose flexibly.
  • the auxiliary material may be at least one of sugars such as lactose, glucose or sucrose, starches such as corn starch or potato starch, malt, gelatin, talc and the like.
  • excipients can also include lubricants such as sodium lauryl sulfate and magnesium stearate, colorants, release agents, coating agents, sweeteners, flavoring agents and fragrances according to the pharmaceutical dosage form or administration method, or according to According to the judgment of those skilled in the art, preservatives and antioxidants are added.
  • the administration method of the above-mentioned prostatic hyperplasia drugs may be animals, including humans. Modes of administration include topical, parenteral, intravenous, intraarterial, intramuscular, subcutaneous, etc., delivery by aerosol, suppository, or oral administration, etc.
  • the above-mentioned drugs for treating benign prostatic hyperplasia can be administered alone or in combination with other compositions if necessary.
  • the adjuvants acceptable to the above-mentioned drugs it is possible to flexibly select the adjuvants acceptable to the above-mentioned drugs to be combined with an effective dose of sterol composition in pumpkin seed oil to prepare different dosage forms, as the drug for treating benign prostatic hyperplasia is a solution Any of the dosage forms, pills, tablets, capsules, powders, pastes and aerosols.
  • the embodiment of the present invention also provides the preparation method of the drug for treating prostatic hyperplasia described above.
  • the preparation method further includes the supplementary excipients of the drug for treating prostatic hyperplasia described above
  • the sterol composition in the pumpkin seed oil with the effective dose is treated according to methods and formulation acceptable processes by the pharmacist. Through this treatment, a prostatic hyperplasia drug required for the administration mode is prepared. Therefore, the preparation method of the medicine for treating benign prostatic hyperplasia according to the embodiments of the present invention can flexibly select the auxiliary materials according to the requirements of the dosage form, and the effective pharmaceutical ingredient sterol composition in pumpkin seed oil can be combined with the auxiliary materials according to the methods and preparations that can be understood by the pharmaceutical personnel.
  • the accepted process treats the finished medicine, and its preparation process is stable, which effectively guarantees the active stability of the medicine and reduces the production cost.
  • SD rats in the normal group are anesthetized, and converted according to the body surface area of humans and animals
  • the equivalent dose method is used to calculate the anesthesia dose. Inject 10% chloral hydrate intraperitoneally according to 0.3ml/100g to anesthetize the SD rats. After disinfecting the skin of the SD rats, remove the bilateral testes and sew them; then in the thighs of the SD rats At the medial muscle, 200,000 U/kg/d penicillin was injected intramuscularly for one week to observe the recovery of SD rats;
  • each SD rat is injected subcutaneously with testosterone propionate injection at a dose of 4 mg/kg for 30 consecutive days.
  • SD rats in the normal group are injected with an equal volume
  • the saline solution is completed.
  • the high-dose group, the middle-dose group, the low-dose group and the finasteride group according to the method of calculating the equivalent dose converted from the body surface area between humans and animals, administer the corresponding drugs respectively, the model group and the normal group
  • the high-dose group was administered with 10 mg/kg (the dose was calculated according to the equivalent dose converted from the body surface area between humans and animals.
  • the sterol in pumpkin seed oil prepared in Example 1 was administered 10 mg), 3.3 mg/kg in the middle-dose group, and 1.1 mg/kg in the low-dose group, which are equivalent to 20 times, 6.66 times, and 2.22 times the clinical dose, respectively, and were formulated into 4.5 mg/mL with corn oil before use.
  • 1.5mg/mL, 0.5mg/mL suspension, the finasteride group was given 0.28mg/kg, once a day by intragastric administration, and the drug was stopped for 1 day after continuous administration for 6 days, with a cycle of 4 weeks.
  • the SD rats were killed, the prostate organs of the SD rats were peeled off with tweezers, and the prostate was weighed;
  • a part of prostate organs was fixed in 10% formalin solution, the above prostate organs were embedded with paraffin, sectioned, hematoxylin-eosin staining (HE&staining, H&E) staining, using optical Observe the morphological changes of the prostate of SD rats with a microscope, and determine the androgen receptor (AR) and steroid receptor coactivator-1 (SRC-1) in prostate tissue by immunohistochemistry; Immunohistochemical method is based on the detection index such as protein and antibody specific binding reaction, positioning, qualitative and relative quantitative research on the index to show the expression level and expression position of the protein.
  • RT-qPCR Reverse transcription-qPCR
  • AR 5 ⁇ -reductase, androgen receptor
  • basic fibroblast growth factor basic fibroblast growth factor
  • bFGF basic fibroblast growth factor
  • Messenger RNA essential RNA
  • transforming growth factor transforming growth factor- ⁇ 1, TGF- ⁇ 1 mRNA
  • B-cell lymphoma-2 gene B-cell lymphoma-2, Bcl-2 expression.
  • prostate index prostate weight (mg) / rat body weight (100g);
  • the body weight of each group of rats did not change significantly.
  • the prostate weight and prostate index of the model group increased significantly, by 28.4% and 39.4% respectively; compared with the model group, the finasteride group
  • the prostate weight and prostate index decreased by 19.1% and 23.5%, respectively; compared with the model group, the prostate index of the middle-dose group and the high-dose group decreased significantly, by 18.4% and 20.5%, respectively.
  • the high-dose group basically achieved the effect of finasteride group.
  • Figure 2 is a prostate tissue diagram.
  • the prostate tissue in the normal group was smooth and pink in color; the prostate tissue in the model group was larger in volume than the normal group, darker in color and dark red in color, with proliferative nodules raised on its surface.
  • the volume of prostate tissue in the finasteride group, high-dose and medium-dose groups was significantly reduced, and the surface of the prostate tissue in the high-dose group returned to a smooth state without obvious hyperplastic nodules, and basically returned to the normal group. .
  • Fig. 3 is a H&E stained pathological slice.
  • the left side of Fig. 3 is magnification ⁇ 200
  • the right side is magnification ⁇ 400
  • prostate tissue was evaluated by H&E staining.
  • the prostate epithelial cells of the model group proliferated significantly, from a single layer to a multi-layer, the cavity area was significantly reduced, and the cavity gap was tight.
  • part of the stratified epithelial cells of prostate tissue in the finasteride group, low-dose group, medium-dose group, and high-dose group returned to single Layer, cavity area increases, cavity gap becomes loose.
  • FIG. 4 is a graph showing the effect of the sterol composition in pumpkin seed oil on the expression of androgen receptor and steroid receptor co-activator of prostate hyperplasia in Example 1; wherein, the left side of FIG. 4 is the magnification ⁇ 200 , The right side is the magnification ⁇ 400.
  • prostatic hyperplasia related factors such as androgen receptor (AR) and steroid receptor co-activator (SRC-1) was evaluated by immunohistochemistry.
  • AR androgen receptor
  • SRC-1 steroid receptor co-activator
  • testosterone is converted to dihydrotestosterone.
  • Testosterone can be combined with AR and steroid receptor coactivator.
  • Dihydrotestosterone can also be combined with AR and steroid receptor coactivator.
  • the binding capacity of AR is 5 times that of testosterone and AR, and dihydrotestosterone is the main reason for stimulating prostate hyperplasia; therefore, by inhibiting the gene expression of 5 ⁇ -reductase, prostate hyperplasia can be effectively controlled. Finasteride can effectively inhibit the gene expression of 5 ⁇ -reductase.
  • the gene expression levels of AR and SRC-1 in the model group were significantly higher than those in the normal group.
  • the finasteride group and the high-dose group, the medium-dose group, and the low-dose group of the sterol composition in pumpkin seed oil described in Example 1 were all Significantly reduced the gene expression of AR and SRC-1.
  • RT-qPCR detects gene expression changes of growth factors and 5 ⁇ -reductase associated with benign prostatic hyperplasia
  • Growth factors are a class of small molecule peptides. There are many kinds of growth factors such as bFGF and TGF- ⁇ 1 in the prostate. The gene expression of these growth factors can regulate the growth, differentiation, proliferation and apoptosis of prostate cells.
  • 5 ⁇ -reductase is a key enzyme that converts testosterone into dihydrotestosterone.
  • the gene expression of 5 ⁇ -reductase in the model group is significantly higher than that in the normal group.
  • the 5 ⁇ -reductase gene in the finasteride group The expression level was significantly reduced, while the gene expression level of 5 ⁇ -reductase in the high-dose group, the middle-dose group and the low-dose group acting on the pumpkin seed oil described in Example 1 was significantly reduced.
  • Basic fibroblast growth factor is an important factor that promotes mitosis in mesoderm and neuroectodermal cells.
  • bFGF mainly stimulates the proliferation of stromal cells, and the overexpression of bFGF may be the mechanism of the pathogenesis of prostate hyperplasia one.
  • the relative gene expression of bFGF in the model group was higher than that in the normal group, while the high-dose group and the middle-dose group significantly reduced the gene expression of bFGF in the prostate tissue, and it was related to the administration concentration. With the increase of the administration concentration, the expression of bFGF gene was controlled. The better the effect.
  • TGF- ⁇ 1 Metastatic growth factor
  • TGF- ⁇ 1 is a kind of polypeptide with wide biological activity, which can regulate the proliferation, development, transformation and differentiation of cells in prostate tissue.
  • TGF- ⁇ 1 has the dual effect of stimulating and inhibiting mitosis.
  • TGF- ⁇ 1 can inhibit the growth of interstitial cells and induce the expression of smooth muscle cell phenotype, and promote the differentiation of interstitial cells into smooth muscle.
  • the enhanced negative regulation of TGF- ⁇ 1 in prostate matrix and epithelial cells, and the decreased positive regulation of bFGF, can accelerate the apoptosis of prostate cells and the atrophy of prostate tissue.
  • Each phytosterol in the sterol composition in the pumpkin seed oil synergistically has an anti-inflammatory effect and an immunomodulatory effect
  • each phytosterol in the sterol composition in the pumpkin seed oil shows a high affinity for prostate tissue, It shows the activity of hormones in SD rats, which can regulate the gene expression of growth factors related to benign prostatic hyperplasia, but without the side effects of hormones.
  • the sterol composition in the pumpkin seed oil is mainly ⁇ -7 phytosterol, which is the main substance for inhibiting prostate hyperplasia.
  • ⁇ -7 phytosterol has the effect of inhibiting 5 ⁇ -reductase, thereby inhibiting the conversion of testosterone to dihydrotestosterone , Inhibit prostate hyperplasia.
  • the sterol composition in the pumpkin seed oil not only inhibits the expression of 5 ⁇ -reductase, but also inhibits the gene expression of androgen receptor, bFGF and TGF- ⁇ 1, etc., which can significantly reduce the weight and prostate index of prostate organs, so that Prostatic hyperplasia is recovered.

Abstract

本发明属于医药技术领域，具体涉及南瓜籽油中甾醇组合物及其应用和治疗前列腺增生药物。公开了南瓜籽油中甾醇组合物，包括以下重量份数的组分：胆甾醇0.2～2份、菜油甾醇0.5～5份、β-谷甾醇1.5～5份、豆甾醇15～40份、麦角甾-7，22-二烯-3β-醇20～40份、羊毛甾醇5～10份、胆甾-7-烯-3β，5α-二醇15～30份。本发明所述南瓜籽油中甾醇组合物是从南瓜籽油中提取获得的南瓜植物甾醇组合物，通过各植物甾醇组分之间的配伍，控制各组分之间的配比，具有缓解和治疗前列腺疾病的效果，特别是前列腺增生疾病。

Description

南瓜籽油中甾醇组合物及其应用和治疗前列腺增生药物 技术领域
本发明属于医药技术领域，具体涉及南瓜籽油中甾醇组合物及其应用和治疗前列腺增生药物。
背景技术
前列腺增生(benignprostatic hyperplasia,BPH)是男性中老年人的常见病、多发病。目前治疗前列腺增生主要是通过西药治疗和非药物治疗，常用的西药5α-还原酶抑制剂如非那雄胺、α1-肾上腺素受体阻滞剂如酚苄明或马沙尼，此类药物治疗具有一定的治疗效果，但是见效慢，通常需要服药6个月以上，且副作用大，如机体出现体位性低血压、乏力、视物模糊、射精障碍等副作用。
对于轻度和中度前列腺增生的患者，通过西药保守治疗有一定疗效，但对于重度增生患者，特别是残余尿较多的患者仍需手术治疗才能解除梗阻。但是手术会伤害机体，同时相关的并发症也给BPH患者带来了明显痛苦。上述药物及手术治疗均存在局限性。
而目前常用治疗前列腺增生的植物药包括：蜂花粉提取物即前列康，锯叶棕提取物即市售伯泌松、柏诺特胶囊，非洲臀果木提取物即通尿灵，但是上述植物药均存在成分复杂，机理不清楚，见效慢等问题，不能很好地满足患者的需要。
因此，迫切需要研发一种可快速、安全有效的治疗前列腺增生疾病的组合物。
发明内容
为解决上述技术问题，本发明提供了南瓜籽油中甾醇组合物及其应用和治疗前列腺增生药物，可以快速、安全的治疗前列腺增生，且无副作用。
为实现上述目的，本发明采用以下技术方案：
一方面，本发明提供了南瓜籽油中甾醇组合物，包括以下重量份数的组分：胆甾醇0.2～2份、菜油甾醇0.5～5份、β-谷甾醇1.5～5份、豆甾醇15～40份、麦角甾-7，22-二烯-3β-醇20～40份、羊毛甾醇5～10份、胆甾-7-烯-3β，5α-二醇15～30份。
优选地，所述的南瓜籽油中甾醇组合物，包括以下重量份数的组分：胆甾醇0.5～1份、菜油甾醇1～3份、β-谷甾醇2～3份、豆甾醇20～35份、麦角甾-7，22-二烯-3β-醇25～35份、羊毛甾醇6～8份、胆甾-7-烯-3β，5α-二醇20～35份。
优选地，所述的南瓜籽油中甾醇组合物，包括以下重量份数的组分：胆甾醇0.99份、菜油甾醇1.35份、β-谷甾醇2.42份、豆甾醇30.06份、麦角甾-7，22-二烯-3β-醇32.01份、羊毛甾醇6.09份、胆甾-7-烯-3β，5α-二醇24.12份。
优选地，所述的南瓜籽油中甾醇组合物，所述的南瓜籽油中甾醇组合物，由以下步骤制备得到：南瓜籽油依次经皂化反应、有机溶剂萃取，取萃取上相为萃取液，洗涤萃取液、干燥结晶，得到所述的南瓜籽油中甾醇组合物。
优选地，所述的南瓜籽油中甾醇组合物，所述南瓜籽油中甾醇组合物的植物甾醇质量含量为90％以上。
优选地，所述的南瓜籽油中甾醇组合物，所述洗涤萃取液的步骤具体为：依次用水、乙醇水溶液、水、氢氧化钾溶液、水洗涤萃取液，弃去下层水相，保留萃取液的上层有机相。
优选地，所述的南瓜籽油中甾醇组合物，用氢氧化钾溶液、水洗涤萃取液 的步骤至少重复一次，洗涤至下层水相为中性。
另一方面，本发明提供了所述的南瓜籽油中甾醇组合物在治疗前列腺增生药物中的应用。
又一方面，本发明提供了一种治疗前列腺增生药物，包括药物可接受的辅料，还包括有效剂量的上文所述的南瓜籽油中甾醇组合物。
优选地，所述的治疗前列腺增生药物，所述治疗前列腺增生药物的剂型为溶液剂、丸剂、片剂、胶囊剂、散剂、糊剂和气溶胶中的任一剂型。
本发明与现有技术相比，具有以下有益效果：
本发明所述南瓜籽油中甾醇组合物是从南瓜籽油中提取获得的南瓜植物甾醇组合物，其中胆甾醇、菜油甾醇、β-谷甾醇、豆甾醇、麦角甾-7，22-二烯-3β-醇、羊毛甾醇、胆甾-7-烯-3β，5α-二醇均为植物甾醇，通过各植物甾醇组分之间的配伍，控制各组分之间的配比，具有缓解和治疗前列腺疾病的效果，特别是前列腺增生疾病。
本发明所述南瓜籽油中甾醇组合物中的各植物甾醇之间协同作用，具有抗炎功效和免疫调节功效，且所述南瓜籽油中甾醇组合物中各植物甾醇对前列腺组织表现出高度的亲和性，在动物或人体的体内表现出激素的活性，可以调节与前列腺增生相关的生长因子的基因表达，但是无激素的副作用。且所述南瓜籽油中甾醇组合物主要为△-7植物甾醇，其为抑制前列腺增生的主要物质，△-7植物甾醇具有抑制5α-还原酶的作用，从而抑制了睾酮转化为双氢睾酮，抑制前列腺增生。所述南瓜籽油中甾醇组合物不仅抑制5α-还原酶的表达，还抑制雄激素受体、bFGF和TGF-β1等的基因表达，可显著减小前列腺组脏器的重量和前列腺指数，使得前列腺增生疾病得以恢复。
附图说明
为了更清楚地说明本发明实施例中的技术方案，下面将对实施例或现有技 术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动性的前提下，还可以根据这些附图获得其他的附图。
图1为本发明实施例1所述南瓜籽油中甾醇组合物的GC-MS图谱；
图2为本发明实施例中前列腺组织图；
图3为本发明实施例中H&E染色病理切片图；
图4为实施例1所述南瓜籽油中甾醇组合物对前列腺增生鼠雄激素受体、类固醇受体辅活化因子表达的影响图；
图5为本发明实施例中RT-qPCR检测5α-还原酶基因表达变化图；
图6为本发明实施例中RT-qPCR检测bFGF基因表达变化图；
图7为本发明实施例中RT-qPCR检测TGF-β1基因表达变化图；
其中，图中各附图标记：
1—胆甾醇；2—菜油甾醇；3—β-谷甾醇；4-豆甾醇；
5—麦角甾-7，22-二烯-3β-醇；6—羊毛甾醇；7—胆甾-7-烯-3β，5α-二醇
具体实施方式
为了使本发明所要解决的技术问题、技术方案及有益效果更加清楚明白，以下结合附图及实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
一方面，本发明实施例提供了南瓜籽油中甾醇组合物，包括以下重量份数的组分：胆甾醇0.2～2份、菜油甾醇0.5～5份、β-谷甾醇1.5～5份、豆甾醇15～40份、麦角甾-7，22-二烯-3β-醇20～40份、羊毛甾醇5～10份、胆甾-7-烯-3β，5α-二醇15～30份。
其中，所述胆甾醇的分子式为C 27H 46O，相对分子质量为386.35，化学结 构式为式I所示：
Figure PCTCN2019105930-appb-000001
所述菜油甾醇的分子式为C 28H 48O，相对分子质量为400.69，化学结构式为式II所示：
Figure PCTCN2019105930-appb-000002
所述β-谷甾醇的分子式为C 29H 50O，相对分子质量为414.72，化学结构式为式III所示：
Figure PCTCN2019105930-appb-000003
所述豆甾醇的分子式为C 29H 52O，相对分子质量为416.73，化学结构式为式IV所示：
Figure PCTCN2019105930-appb-000004
所述麦角甾-7，22-二烯-3β-醇的分子式为C 28H 46O，相对分子质量为398.68，化学结构式为式V所示：
Figure PCTCN2019105930-appb-000005
所述羊毛甾醇的分子式为C 30H 50O，相对分子质量为426.73，化学结构式为式VI所示：
Figure PCTCN2019105930-appb-000006
所述胆甾-7-烯-3β，5α-二醇的分子式为C 27H 46O，相对分子质量为386.35，化学结构式为式VII所示：
Figure PCTCN2019105930-appb-000007
本发明实施例所述南瓜籽油中甾醇组合物是从南瓜籽油中提取获得的南瓜植物甾醇组合物，其中胆甾醇、菜油甾醇、β-谷甾醇、豆甾醇、麦角甾-7，22-二烯-3β-醇、羊毛甾醇、胆甾-7-烯-3β，5α-二醇均为植物甾醇，通过各植物甾醇组分之间的配伍，控制各组分之间的配比，具有缓解和治疗前列腺疾病的效果，特别是前列腺增生疾病。
本发明实施例所述南瓜籽油中甾醇组合物中的各植物甾醇之间协同作用，具有抗炎功效和免疫调节功效，且所述南瓜籽油中甾醇组合物中各植物甾醇对前列腺组织表现出高度的亲和性，在动物或人体的体内表现出激素的活性，可以调节与前列腺增生相关的生长因子的基因表达，但是无激素的副作用。且所述南瓜籽油中甾醇组合物主要为△-7植物甾醇，其为抑制前列腺增生的主要物质，△-7植物甾醇具有抑制5α-还原酶的作用，从而抑制了睾酮转化为双氢睾酮，抑制前列腺增生。所述南瓜籽油中甾醇组合物不仅抑制5α-还原酶基因的表达，还抑制雄激素受体、bFGF和TGF-β1等的基因表达，可显著减小前列腺组脏器的重量和前列腺指数，使得前列腺增生疾病得以恢复。
进一步地，所述南瓜籽油中甾醇组合物包括以下重量份数的组分：胆甾醇0.5～1份、菜油甾醇1～3份、β-谷甾醇2～3份、豆甾醇20～35份、麦角甾-7，22-二烯-3β-醇25～35份、羊毛甾醇6～8份、胆甾-7-烯-3β，5α-二醇20～35份。
进一步地，所述南瓜籽油中甾醇组合物包括以下重量份数的组分：胆甾醇 0.6～1份、菜油甾醇1.1～2份、β-谷甾醇2.2～2.5份、豆甾醇25～32份、麦角甾-7，22-二烯-3β-醇30～33份、羊毛甾醇6～7份、胆甾-7-烯-3β，5α-二醇22～30份。
作为一具体实施例，所述南瓜籽油中甾醇组合物包括以下重量份数的组分：胆甾醇0.99份、菜油甾醇1.35份、β-谷甾醇2.42份、豆甾醇30.06份、麦角甾-7，22-二烯-3β-醇32.01份、羊毛甾醇6.09份、胆甾-7-烯-3β，5α-二醇24.12份。
作为本发明另一具体实施例，所述南瓜籽油中甾醇组合物包括以下重量份数的组分：胆甾醇0.8份、菜油甾醇1.5份、β-谷甾醇2.3份、豆甾醇28份、麦角甾-7，22-二烯-3β-醇31份、羊毛甾醇6.5份、胆甾-7-烯-3β，5α-二醇23份。
作为本发明又一具体实施例，所述胆甾醇2份、菜油甾醇5份、β-谷甾醇5份、豆甾醇40份、麦角甾-7，22-二烯-3β-醇40份、羊毛甾醇10份、胆甾-7-烯-3β，5α-二醇30份。
作为本发明另一具体实施例，所述南瓜籽油中甾醇组合物包括以下重量份数的组分：胆甾醇0.2份、菜油甾醇0.5份、β-谷甾醇1.5份、豆甾醇15份、麦角甾-7，22-二烯-3β-醇20份、羊毛甾醇5份、胆甾-7-烯-3β，5α-二醇30份。
作为本发明另一具体实施例，所述南瓜籽油中甾醇组合物包括以下重量份数的组分：胆甾醇1.5份、菜油甾醇3份、β-谷甾醇3.5份、豆甾醇35份、麦角甾-7，22-二烯-3β-醇30份、羊毛甾醇7.5份、胆甾-7-烯-3β，5α-二醇22.5份。
上述各实施例中，所述南瓜籽油中甾醇组合物是从南瓜仔油中获取的组合物，所述南瓜籽油中甾醇组合物具体由以下步骤制备得到：南瓜籽油依次经皂化反应、有机溶剂萃取，取萃取上相为萃取液，洗涤萃取液、干燥结晶，得到 所述的南瓜籽油中甾醇组合物。
其中，由上述实施例制备得到的南瓜籽油中甾醇组合物中植物甾醇的质量含量为90％以上，相比于现有技术，本发明实施例制备得到的植物甾醇的纯度更高。
其中，皂化反应的步骤具体为，配制0.5mol/L～1.5mol/L的氢氧化钾-乙醇溶液，以料液比为1:10(w：v)将南瓜籽油与氢氧化钾-乙醇溶液混合，于65～100℃皂化回流0.5～2.5h，皂化完成，得到混合液，将体积分数为混合液12.5％～25％的水加入皂化液中，溶解皂化反应产生的脂肪酸盐，将反应体系冷却至室温，得到皂化液；所述室温为反应体系所处的环境温度。
其中，有机溶剂萃取的步骤具体为，有机溶剂可以用正己烷，具体地，正己烷萃取皂化液1～3次，取萃取上相，合并萃取上相，即得萃取液；每次萃取的过程中，正己烷和皂化液的体积比为1：1。
其中，洗涤萃取液的步骤具体为：依次用水、乙醇水溶液、水、氢氧化钾溶液、水洗涤萃取液，弃去下层水相，保留萃取液的上层有机相。进一步地，用氢氧化钾溶液、水洗涤萃取液的步骤至少重复一次，洗涤至下层水相为中性。
具体地，依次以水、体积分数为8％-25％乙醇水溶液、水、0.2～0.5mol/L的氢氧化钾溶液、水洗涤，每次洗涤过程中，洗涤液分别为水、体积分数为8％-25％乙醇水溶液、0.2～0.5mol/L的氢氧化钾溶液；各洗涤液的体积为皂化液体积的10％-30％，重复氢氧化钾洗涤和水洗涤的步骤，直至洗至下层水相为中性，弃去水层，保留萃取液的上层有机相。
其中，干燥结晶的步骤具体为：合并上层有机相，向上层有机相中加入无水硫酸钠后静置10-30分钟除水，后用减压旋转蒸发仪将除水的萃取液浓缩，于4℃冰箱中静置过夜，获得白色针状晶体；抽滤，用正己烷洗涤，烘干，获得南瓜籽油中甾醇组合物。
现行碱法皂化所得皂化液，萃取后洗涤的过程中，采用水和碱洗涤，由于 皂化过程中产生的脂肪酸盐遇水会产生大量的泡沫，使皂化液中的上层有机相乳化。即使调节下层水相与上层有机相的比例，仍不可避免会在两相中间形成大量的乳化层，洗涤至中性的时间加长，以及南瓜籽油中甾醇组合物会随洗涤而损失。
本发明实施例在将洗涤萃取液的过程中，加入了乙醇水溶液洗涤，有效消除了脂肪酸盐遇水产生大量泡沫的现象，减少两相间的乳化层，使整个洗涤过程步骤明显缩短，减少乳化层的损耗，也使最终的产物得率提高；上述制备方法的产物得率可达1.6-2‰。
另一方面，本发明实施例提供了所述的南瓜籽油中甾醇组合物在治疗前列腺增生药物中的应用。
本发明实施例所述南瓜籽油中甾醇组合物中的各植物甾醇之间协同作用，具有抗炎功效和免疫调节功效，且所述南瓜籽油中甾醇组合物中各植物甾醇对前列腺组织表现出高度的亲和性，在动物或人体的体内表现出激素的活性，可以调节与前列腺增生相关的生长因子的基因表达，但是无激素的副作用。且所述南瓜籽油中甾醇组合物主要为△-7植物甾醇，其为抑制前列腺增生的主要物质，△-7植物甾醇具有抑制5α-还原酶的作用，从而抑制了睾酮转化为双氢睾酮，抑制前列腺增生。所述南瓜籽油中甾醇组合物不仅抑制5α-还原酶基因的表达，还抑制雄激素受体、bFGF和TGF-β1等的基因表达，可显著减小前列腺组脏器的重量和前列腺指数，使得前列腺增生疾病得以恢复。
又一方面，在上文所述的南瓜籽油中甾醇组合物在治疗前列腺增生药物中的应用的实施例基础上，本发明实施例还提供了一种治疗前列腺增生药物，包括药物可接受的辅料，还包括有效剂量上述各实施例所述的南瓜籽油中甾醇组合物。
其中，有效剂量是指治疗有效量，是指足以对个体显示益处或临床意义的本发明所述南瓜籽油中甾醇组合物的量；本领域技术人员将会理解，给药的实际量或剂量以及给药时程将取决于被治疗的疾病的性质和严重性、被治疗的受 试者的年龄和一般状况以及给药方式等。
具体地，所述南瓜籽油中甾醇组合物为上文所述的包括以下各组分及其重量份数为：胆甾醇0.2～2份、菜油甾醇0.5～5份、β-谷甾醇1.5～5份、豆甾醇15～40份、麦角甾-7，22-二烯-3β-醇20～40份、羊毛甾醇5～10份、胆甾-7-烯-3β，5α-二醇15～30份；在一具体实施例中，所述南瓜籽油中甾醇组合物包括以下重量份数的组分：胆甾醇0.99份、菜油甾醇1.35份、β-谷甾醇2.42份、豆甾醇30.06份、麦角甾-7，22-二烯-3β-醇32.01份、羊毛甾醇6.09份、胆甾-7-烯-3β，5α-二醇24.12份；各组分之间进行配伍，并控制各组分的用量比例，能够有效提高本发明实施例治疗前列腺增生药物对前列腺疾病的药效，特别是对前列腺增生的药效。
其中，所述辅料应当是无毒的、不干扰或不损害本发明实施例上述南瓜籽油中甾醇组合物的效力，另外，辅料的选用可以根据上述各实施例治疗前列腺增生药物的存在的剂型而灵活选用。在一具体实施例中，辅料可以选用糖类如乳糖、葡萄糖或蔗糖，淀粉如玉米淀粉或马铃薯淀粉，麦芽、明胶、滑石等中的至少一种。上述辅料还可以根据药物剂型或给药方式还包括润滑剂如十二烷基硫酸钠和硬脂酸镁、着色剂、释放剂、包衣剂、增甜剂、调味剂和芳香剂，或根据本领域技术人员的判断，增加防腐剂和抗氧化剂。
另外，上述治疗前列腺增生药物的给药方式可以是动物，包括人。给药模式包括局部、胃肠外、静脉内、动脉内、肌内、皮下等，通过气溶胶、栓剂或口服等递送。上述治疗前列腺增生药物可以单独给药，也可以与如果必要的其他组合物联合给药。
本发明实施例，根据治疗前列腺增生药物的给药模式，可以灵活选用上述药物可接收的辅料与有效剂量的南瓜籽油中甾醇组合物配伍制备成不同剂型，如所述治疗前列腺增生药物为溶液剂、丸剂、片剂、胶囊剂、散剂、糊剂和气溶胶中的任一剂型。
相应地，本发明实施例还提供了上文所述的治疗前列腺增生药物的制备方 法，在一实施例中，该制备方法还包括将上文所述的治疗前列腺增生药物中药物可接收的辅料与所述有效剂量的南瓜籽油中甾醇组合物，按照药学人员能够理解的方法和制剂上能够接受的工艺处理。通过该处理，制备成给药模式所需要的治疗前列腺增生药物。因此，本发明实施例治疗前列腺增生药物制备方法，能够根据剂型的要求而灵活选用辅料，而且该有效药物成分南瓜籽油中甾醇组合物能够与辅料，按照药学人员能够理解的方法和制剂上能够接受的工艺处理成药，其制备工艺稳定，有效保证了药物的活性稳定性，而且还降低了生产成本。
以下通过具体实施例对本发明做进一步说明：
实施例1
S101、配制1.5mol/L的氢氧化钾-乙醇溶液，将南瓜籽油180g与氢氧化钾-乙醇溶液1800mL混合，于80℃皂化回流2h，皂化完成，得到混合液，将体积分数为混合液16％的水加入皂化液中，溶解皂化反应产生的脂肪酸盐，将反应体系冷却至室温，得到皂化液；所述室温为反应体系所处的环境温度。
S102、正己烷萃取皂化液2次，取萃取上相，合并萃取上相，即得萃取液；每次萃取的过程中，正己烷和皂化液的体积比为1：1。
S103、依次用水、体积分数为20％乙醇水溶液、水、0.5mol/L氢氧化钾溶液、水洗涤萃取液，弃去下层水相，洗涤至下层水相为中性，保留萃取液的上层有机相；各洗涤液的体积为皂化液体积的30％。
S104、合并上层有机相，向上层有机相中加入无水硫酸钠后静置10分钟除水，后用减压旋转蒸发仪将除水的萃取液浓缩，于4℃冰箱中静置过夜，获得白色针状晶体；抽滤，用正己烷洗涤，烘干，获得南瓜籽油中甾醇组合物。
结果分析
采用气相色谱-质谱(Gas Chromatography-Mass Spectrometer，GC-MS)联用仪对实施例1所制备的南瓜籽油中甾醇组合物进行分析测试，得到的GC-MS 图谱；结合图1，实施例1中的各组分及组分的质量百分比如表1中所示，且实施例1所制备的南瓜籽油中甾醇组合物的植物甾醇得率为1.76‰。
表1
Figure PCTCN2019105930-appb-000008
实施例2
S201、配制1mol/L的氢氧化钾-乙醇溶液，将南瓜籽油180g与氢氧化钾-乙醇溶液1800mL混合，于70℃皂化回流2h，皂化完成，得到混合液，将体积分数为混合液16％的水加入皂化液中，溶解皂化反应产生的脂肪酸盐，将反应体系冷却至室温，得到皂化液。
S202、正己烷萃取皂化液2次，取萃取上相，合并萃取上相，即得萃取液；每次萃取的过程中，正己烷和皂化液的体积比为1：1。
S203、依次用水、体积分数为20％乙醇水溶液、水、0.5mol/L氢氧化钾溶液、水洗涤萃取液，弃去下层水相，洗涤至下层水相为中性，保留萃取液的上层有机相；各洗涤液的体积为皂化液体积的30％。
S204、合并上层有机相，向上层有机相中加入无水硫酸钠后静置10分钟除水，后用减压旋转蒸发仪将除水的萃取液浓缩，于4℃冰箱中静置过夜，获得白色针状晶体；抽滤，用正己烷洗涤，烘干，获得南瓜籽油中甾醇组合物。所 制备的南瓜籽油中甾醇组合物的植物甾醇得率为1.69‰。
实施例3
对实施例1所制备的南瓜籽油中甾醇组合物进行动物试验：
1、试验分组：
S301、分组：
S3011、选取清洁级SD大鼠80只，4周龄，雄性，体重为200-220g；
S3012、饲养SD大鼠一周后，取其中10只健康的SD大鼠作为正常组，正常组作为空白对照，取其中50只健康的SD大鼠进行造模手术；
S3013、造模手术后观察一周，对50只SD大鼠进行分组，共分5组，每组10只，分别为：模型组，非那雄胺组，低剂量组，中剂量组，高剂量组；其中非那雄胺组选用临床用药如非那雄胺片作为阳性对照，模型组作为造模手术SD大鼠中的空白对照。
2、造模与给药：
S302、造模：
S3021、6组SD大鼠分别称重；
S3022、除正常组的SD大鼠，对其他5组(模型组，非那雄胺组，低剂量组，中剂量组，高剂量组)SD大鼠进行麻醉，按照人与动物的体表面积折算的等效剂量的方法计算麻醉剂量，按照0.3ml/100g腹腔注射10％水合氯醛，麻醉SD大鼠，对SD大鼠皮肤消毒后，摘除双侧睾丸，缝合；然后在SD大鼠的大腿内侧肌处，肌内注射青霉素20万U/kg/d，连续注射一周，观察SD大鼠的恢复状况；
S2023、按照人与动物间体表面积折算的等效剂量的方法，按照4mg/kg的剂量，每只SD大鼠皮下注射丙酸睾酮注射液，连续30天，正常组的SD大鼠注射等体积生理盐水，造模完成。
S303、给药：
高剂量组、中剂量组、低剂量组和非那雄胺组，按照人与动物间体表面积折算的等效剂量的方法计算给药剂量，分别灌服相应药物，模型组和正常组灌服同体积玉米油；其中，高剂量组、中剂量组、低剂量组均采用实施例1所制备的南瓜籽油中甾醇组合物，分别对SD大鼠进行给药。高剂量组给药10mg/kg(按照人与动物间体表面积折算的等效剂量的方法计算给药剂量，SD大鼠的体重为1Kg时，给药实施例1所制备的南瓜籽油中甾醇组合物10mg)、中剂量组3.3mg/kg、低剂量组1.1mg/kg，分别相当于临床剂量的20倍、6.66倍、2.22倍，临用前分别用玉米油分别配成4.5mg/mL、1.5mg/mL、0.5mg/mL的悬液，非那雄胺组给0.28mg/kg，每日灌胃给药1次，连续给药6天后停药1天，周期为4周。
3、指标的测定
末次给药后2h，且已禁食不禁水12h，分别将6组SD大鼠称重；
处死SD大鼠，用镊子剥离SD大鼠的前列腺脏器，将前列腺称重；
将一部分前列腺脏器于10％福尔马林溶液中固定，用石蜡将上述前列腺脏器进行包埋，切片，苏木精-伊红(hematoxylin-eosin staining，HE stain，H&E)染色，采用光学显微镜，观察SD大鼠前列腺的形态变化，采用免疫组化法测定前列腺组织中雄激素受体(androgen receptor，AR)，类固醇受体辅活化因子(steroid receptor coactivator-1，SRC-1)；其中免疫组化法是根据检测指标如蛋白与抗体特异性结合反应，对指标进行定位、定性及相对定量的研究显示蛋白的表达量和表达位置。
将另一部分前列腺脏器迅速放于液氮中，采用RT-qPCR(Reverse transcription-qPCR)技术，检测5α-还原酶、雄激素受体(AR)、碱性成纤维细胞生长因子(basic fibroblast growth factor，bFGF)mRNA(Messenger RNA)、转移生长因子(transforming growth factor-β1，TGF-β1)mRNA和B淋巴细 胞瘤-2基因(B-cell lymphoma-2，Bcl-2)的表达。
4、结果分析：
实施例1所述的南瓜籽油中的甾醇组合物对丙酸睾酮处理的前列腺增生鼠的影响的结果，如表1所示：
表1实施例1所制备的南瓜籽油中甾醇组合物对前列腺指数的影响
Figure PCTCN2019105930-appb-000009
其中，前列腺指数＝前列腺重量(mg)/大鼠体重(100g)；
*表示与正常组相比有明显差异，P<0.05；**表示与正常组相比有非常明显差异，P<0.01。
#表示与模型组相比有明显差异，P<0.05；##表示与模型组相比有非常明显差异，P<0.01。
由表1可知，每组大鼠体重没有明显变化，与正常组相比，模型组的前列腺重量及前列腺指数明显增高，分别增高28.4％及39.4％；与模型组相比，非那雄胺组的前列腺重量和前列腺指数分别降低19.1％和23.5％；与模型组相比，中剂量组和高剂量组前列腺指数明显降低，分别降低18.4％、20.5％。高中剂量组基本达到非那雄胺组效果。
结合图2，图2为前列腺组织图。正常组的前列腺组织表面光滑，颜色为 粉红色；模型组的前列腺组织体积比正常组大，颜色偏深，为暗红色，其表面有增生结节凸起。与模型组相比，非那雄胺组、高剂量和中剂量的前列腺组织体积明显减小，高剂量组的前列腺组织表面恢复到光滑状态，没有明显增生结节，基本恢复到正常组的状态。
结合图3，图3为H&E染色病理切片图。其中，图3左侧为放大倍数×200，右侧为放大倍数×400；前列腺组织通过H&E染色评估。与正常组相比，模型组的前列腺上皮细胞明显增殖，由单层增殖为复层，腔面积明显减小，腔间隙紧密。通过非那雄胺处理、实施例1所述南瓜籽油中的甾醇组合物处理，非那雄胺组、低剂量组、中剂量组和高剂量组的前列腺组织部分复层上皮细胞恢复成单层，腔面积增加，腔间隙变疏松。
结合图4，图4为实施例1所述南瓜籽油中甾醇组合物对前列腺增生鼠雄激素受体、类固醇受体辅活化因子表达的影响图；其中，图4左侧为放大倍数×200，右侧为放大倍数×400。
通过免疫组化评价前列腺增生相关的因子如雄激素受体(AR)和类固醇受体辅活化因子(SRC-1)的基因表达。在5α-还原酶的作用下，睾酮转化为双氢睾酮，睾酮可以与AR及类固醇受体辅活化因子结合，双氢睾酮也可以与AR及类固醇受体辅活化因子结合，而双氢睾酮与AR的结合能力为睾酮与AR结合能力的5倍，而双氢睾酮为刺激前列腺增生的主要原因；因此通过抑制5α-还原酶的基因表达，可以有效的控制前列腺增生。非那雄胺可以有效抑制5α-还原酶的基因表达。
模型组中AR和SRC-1的基因表达量明显高于正常组，非那雄胺组和实施例1所述南瓜籽油中甾醇组合物作用的高剂量组、中剂量组和低剂量组均明显降低了AR和SRC-1的基因表达。
结合图5～图7，RT-qPCR检测前列腺增生相关生长因子和5α-还原酶的基因表达变化图；
生长因子是一类小分子肽,在前列腺内有很多种类的生长因子如bFGF、TGF-β1，这些生长因子的基因表达对前列腺细胞的生长、分化、增殖、凋亡均有调控作用。
5α-还原酶是让睾酮转化为双氢睾酮的关键酶，模型组的5α-还原酶的基因表达量明显高于正常组，和模型组相比，非那雄胺组的5α-还原酶基因表达量显著降低，而实施例1所述南瓜籽油中甾醇组合物作用的高剂量组、中剂量组和低剂量组的5α-还原酶的基因表达量显著降低。
碱性成纤维细胞生长因子(bFGF)是中胚层和神经外胚层细胞的促进有丝分裂的重要因子，在前列腺组织中，bFGF主要是刺激基质细胞增殖，而bFGF的过表达可能是前列腺增生发病的机制之一。模型组bFGF相对基因表达量高于正常组，而高剂量组和中剂量组显著降低前列腺组织中bFGF的基因表达，且与给药浓度相关，随着给药浓度的增加，控制bFGF基因表达的效果越好。
转移生长因子(TGF-β1)是一类具有广泛生物学活性的多肽,对前列腺组织中细胞增殖、发育、转化和分化起调节作用。TGF-β1具有刺激和抑制有丝分裂的双重效应，TGF-β1能抑制间质细胞的生长并诱导平滑肌细胞表型的表达，促进间质细胞向平滑肌分化。TGF-β1在前列腺基质和上皮细胞中负向调节作用的增强，以及bFGF正向调节作用的减弱，可共同加速前列腺组织中细胞的调亡和前列腺组织的萎缩。
所述南瓜籽油中甾醇组合物中各植物甾醇协同作用，具有抗炎功效和免疫调节功效，且所述南瓜籽油中甾醇组合物中各植物甾醇对前列腺组织表现出高度的亲和性，在SD大鼠体内表现出激素的活性，可以调节与前列腺增生相关的生长因子的基因表达，但是无激素的副作用。且所述南瓜籽油中甾醇组合物主要为△-7植物甾醇，其为抑制前列腺增生的主要物质，△-7植物甾醇具有抑制5α-还原酶的作用，从而抑制了睾酮转化为双氢睾酮，抑制前列腺增生。所述南瓜籽油中甾醇组合物不仅抑制5α-还原酶的表达，还抑制雄激素受体、bFGF和TGF-β1等的基因表达，可显著减小前列腺组脏器的重量和前列腺指 数，使前列腺增生疾病得以恢复。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。

Claims (10)

  1. 南瓜籽油中甾醇组合物，其特征在于，包括以下重量份数的组分：胆甾醇0.2～2份、菜油甾醇0.5～5份、β-谷甾醇1.5～5份、豆甾醇15～40份、麦角甾-7，22-二烯-3β-醇20～40份、羊毛甾醇5～10份、胆甾-7-烯-3β，5α-二醇15～30份。
  2. 根据权利要求1所述的南瓜籽油中甾醇组合物，其特征在于，包括以下重量份数的组分：胆甾醇0.5～1份、菜油甾醇1～3份、β-谷甾醇2～3份、豆甾醇20～35份、麦角甾-7，22-二烯-3β-醇25～35份、羊毛甾醇6～8份、胆甾-7-烯-3β，5α-二醇20～35份。
  3. 根据权利要求1所述的南瓜籽油中甾醇组合物，其特征在于，包括以下重量份数的组分：胆甾醇0.99份、菜油甾醇1.35份、β-谷甾醇2.42份、豆甾醇30.06份、麦角甾-7，22-二烯-3β-醇32.01份、羊毛甾醇6.09份、胆甾-7-烯-3β，5α-二醇24.12份。
  4. 根据权利要求1～3任一权利要求所述的南瓜籽油中甾醇组合物，其特征在于，所述的南瓜籽油中甾醇组合物，由以下步骤制备得到：南瓜籽油依次经皂化反应、有机溶剂萃取，取萃取上相为萃取液，洗涤萃取液、干燥结晶，得到所述的南瓜籽油中甾醇组合物。
  5. 根据权利要求4所述的南瓜籽油中甾醇组合物，其特征在于，所述南瓜籽油中甾醇组合物的植物甾醇质量含量为90％以上。
  6. 根据权利要求4所述的南瓜籽油中甾醇组合物，其特征在于，所述洗涤萃取液的步骤具体为：依次用水、乙醇水溶液、水、氢氧化钾溶液、水洗涤萃取液，弃去下层水相，保留萃取液的上层有机相。
  7. 根据权利要求6所述的南瓜籽油中甾醇组合物，其特征在于，用氢氧化钾溶液、水洗涤萃取液的步骤至少重复一次，洗涤至下层水相为中性。
  8. 权利要求1～7任一权利要求所述的南瓜籽油中甾醇组合物在治疗前列腺增 生药物中的应用。
  9. 一种治疗前列腺增生药物，包括药物可接受的辅料，其特征在于：还包括有效剂量的权利要求1～7任一权利要求所述的南瓜籽油中甾醇组合物。
  10. 根据权利要求9所述的治疗前列腺增生药物，其特征在于，所述治疗前列腺增生药物的剂型为溶液剂、丸剂、片剂、胶囊剂、散剂、糊剂和气溶胶中的任一剂型。
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