Preparation method and application of herba epimedii sagittatum active extract

Abstract

The invention relates to the technical field of important extraction, and particularly provides a preparation method and application of an epimedium sagittatum active extract.The preparation method comprises the steps that leaves of epimedium sagittatum and dried rhizomes of bistort rhizome are taken according to the mass ratio of (1-4): 1, and sheep fat is adopted for processing; and extracting active components in the processed medicinal materials by adopting one or a combination of several of a solvent extraction method, a resin adsorption method, a normal-phase column chromatography and a reversed-phase column chromatography. The active extract is simple and practical in process, good in anti-ED curative effect and free of toxic and side effects, and a wide way is provided for application of herba epimedii.

Description

Technical Field

[0001] The invention relates to the technical field of traditional Chinese medicine extraction, and in particular to a preparation method of an active extract of Epimedium sagittatum and application thereof. Background Art

[0002] Sagittatum Epimedium has the effects of nourishing kidney yang and strengthening tendons and bones. It is worth noting that Epimedium has different uses when raw or roasted. The raw product is mainly used to strengthen tendons and bones, while its sheep oil fried product is used to "warm the kidney and assist yang". Traditional theory believes that the kidney is the innate foundation, and "vital energy" originates from the kidney. The "warming kidney" of Epimedium refers to "warming kidney yang", and by "warming kidney yang" to replenish the original yang, so as to achieve the effect of "assisting yang". Modern research also believes that "kidney yang" is related to the basic functions of human reproduction, immunity, metabolism, etc., and is the physiological and pathological basis of the human body.

[0003] At present, a rat model of "kidney yang deficiency" can be constructed by injecting corticosterone. After feeding the model rats with relevant Chinese medicine, the regulatory activity of the model rats' "hypothalamus-pituitary-adrenal axis" (HPA) and the key target PDE5A1 enzyme in the sexual organs is observed, so as to screen out the active components of Chinese medicine with the effect of "warming the kidney and strengthening yang". Although Epimedium sagittatum has been found to have the effect of warming the kidney and strengthening yang for a long time, the effect of directly consuming its leaves or its decoction is not obvious. If a more obvious effect is to be produced, a large amount of Epimedium leaves need to be consumed for a long time, which has certain side effects. Although the processing method can increase the efficacy of the drug, the dosage is still relatively large.

[0004] Bistorta is the dried rhizome of Polygonum bistorta L., a plant of the Polygonaceae family. It has the effects of clearing away heat and detoxifying, reducing swelling and stopping bleeding. It is mainly used to treat dysentery, hot diarrhea, lung heat cough, carbuncle, scrofula, oral sores, blood heat vomiting and bleeding, hemorrhoid bleeding, and snake and insect bites. At present, there is no report on the combined use of Bistorta and Epimedium sagittatum. Summary of the invention

[0005] In view of this, the present invention provides a preparation method of an active extract of Epimedium sagittatum and application thereof.

[0006] The technical solution of the present invention is achieved as follows: The present invention provides a method for preparing an active extract of Epimedium sagittatum, comprising the following steps:

[0007] Step 1: Take the leaves of Epimedium sagittatum and the dried rhizome of Polygonum multiflorum, and prepare them with sheep fat;

[0008] Step 2: Use one or a combination of solvent extraction, solvent extraction, resin adsorption, normal phase column chromatography, and reverse phase column chromatography to extract the processed Epimedium sagittatum and Polygonum multiflorum.

[0009] In some embodiments, the preparation method includes: mixing and stirring equal masses of mutton fat and boiling water, and pouring the mixture on the herbal medicines of epimedium and ginseng after stirring; heating and refining the mutton fat in an oil refining pot, and then evenly stirring the herbal medicines of epimedium and ginseng after pouring with the mutton fat, and keeping them warm and moist; putting the moistened herbal medicines of epimedium and ginseng into a medicine frying machine for frying.

[0010] In some embodiments, the oil-water mixture is 10-30% of the total weight of the Epimedium and Bistoria medicinal materials.

[0011] In some embodiments, the mass ratio of mutton fat to Epimedium and Polygonum multiflorum is 30:(90-140).

[0012] In some embodiments, the wetting temperature is 70-90° C. and the time is 2-4 h.

[0013] In some embodiments, the temperature of the stir-frying machine is 240-280° C., and the stir-frying time is 2-4 min.

[0014] In some embodiments, the refining temperature is 70-90°C.

[0015] In some embodiments, the mass ratio of Epimedium leaf blade to Bistorta parviflora dry rhizome is (1-4):1.

[0016] In some embodiments, the solvent used in the solvent extraction method includes at least one of water, methanol, ethanol, isopropanol and n-butanol, and the extraction method includes: infiltration extraction at room temperature or ultrasonic oscillation extraction using an ultrasonic oscillator; or infiltration extraction or reflux extraction under heating for 1-6 times.

[0017] In some embodiments, the solvent extraction method comprises: extracting the processed medicinal material after degreasing with petroleum ether, dichloromethane or chloroform with n-butanol or aqueous n-butanol for 1-6 times.

[0018] In some embodiments, the resin adsorption method uses LSA-12S adsorption resin, XAD-2 adsorption resin or CHP-20P adsorption resin, and the elution solvent is one or more of water, methanol, ethanol, acetone, aqueous methanol, aqueous ethanol or aqueous acetone, and the elution is performed in an isocratic or gradient manner.

[0019] In some embodiments, the normal phase column chromatography method uses normal pressure or pressurized silica gel column chromatography or alumina column chromatography, the column filler used can be silica gel or alumina for column chromatography, or silica gel or alumina for thin layer chromatography, and the elution is selected from one or more of benzene / acetone, dichloromethane / acetone, benzene / methanol, dichloromethane / methanol, chloroform / methanol.ethyl acetate / methanol, benzene / acetone / water, dichloromethane / acetone / water, chloroform / acetone / water, dichloromethane / methanol / water, chloroform / methanol / water or ethyl acetate / methanol / water, and the elution is performed in an isocratic or gradient manner.

[0020] In some embodiments, the reverse phase column chromatography method uses normal pressure or pressurized reverse phase column chromatography, the column filler used is octadecyl bonded phase (ODS) or octadecyl bonded phase, the eluent is aqueous methanol or aqueous acetonitrile, and the elution is performed in an isocratic or gradient manner.

[0021] In a second aspect, the present invention also provides an active extract of Epimedium sagittatum prepared by the above-mentioned preparation method.

[0022] In a third aspect, the present invention also provides an application of the active extract of Epimedium sagittatum obtained above, for preparing a drug for treating male sexual dysfunction.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The invention uses plants Epimedium sagittatum and Polygonum multiflorum as raw materials, adopts one or more processes of processing method, solvent extraction method, solvent extraction method, resin adsorption method, normal phase column chromatography method, reverse phase column chromatography method, gel chromatography method and the like, and combines with common drying methods such as concentration drying, freeze drying, spray drying and the like to prepare active extracts. The active extract process is simple and practical, has good anti-ED efficacy, and has no toxic side effects, thereby providing a wide range of ways for the application of Epimedium. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0026] Figure 1 This is a comparison chart of the CRH mRNA expression levels of mice in each group in the experimental example of the present invention;

[0027] Figure 2 This is a comparison chart of the ACTH mRNA expression levels of mice in each group in the experimental example of the present invention;

[0028] Figure 3 This is a comparison chart of ATCH concentrations in the serum of mice in each group in the experimental example of the present invention;

[0029] Figure 4 This is a comparison chart of PDE5A1 levels in the corpus cavernosum of each group of mice in the experimental examples of the present invention. DETAILED DESCRIPTION

[0030] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the embodiments of the present invention belong. If the definitions set forth in this section are contrary to or otherwise inconsistent with the definitions set forth in the patents, patent applications, published patent applications, and other publications incorporated herein by reference, the definitions listed in this section take precedence over the definitions incorporated herein by reference.

[0032] Unless otherwise specified, the methods used in the following examples are all conventional methods. Unless otherwise specified, the materials, reagents and instruments used are all conventional materials, reagents and instruments in the art, and can be obtained by those skilled in the art through commercial channels.

[0033] When an amount, concentration or other value or parameter is expressed as a range, a preferred range or a range defined by a series of upper preferred values ​​and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pairing of any range upper limit or preferred value with any range lower limit or preferred value, regardless of whether the range is disclosed separately. For example, when a range of "1 to 5" is disclosed, the described range should be interpreted as including the range "1 to 4", "1 to 3", "1 to 2", "1 to 2 and 4 to 5", "1 to 3 and 5", etc. When a numerical range is described herein, unless otherwise stated, the range is intended to include its end values ​​and all integers and fractions within the range. In the present specification and claims, range definitions can be combined and / or interchanged, and if not otherwise stated, these ranges include all subranges contained therein.

[0034] Example 1

[0035] This embodiment provides a method for preparing an active extract of Epimedium sagittatum, which is carried out according to the following steps:

[0036] Processing: Take 3kg of sagittaria leaves and 1kg of dried rhizomes of bistortia, mix and stir equal amounts of mutton fat and hot water (0.4kg each), and pour on the bistortia and epimedium after stirring; take 1.0kg of mutton fat in an oil refining pot and heat it to 80℃ for refining, then mix the bistortia and epimedium with the mutton fat evenly, and keep it at 80℃ for 3h; turn the temperature of the medicine frying machine to 260℃, put the moistened epimedium and bistort into the medicine frying machine, and stir for 3min;

[0037] Extraction: Take the processed medicinal materials and reflux extract them twice in 50% ethanol at 83°C, each time for 1.5 h. The mass of 50% ethanol used twice is 15 times and 10 times the mass of the medicinal materials, respectively. The extracts are combined and concentrated to obtain 1.68 kg of ethanol extract. The ethanol extract is then dried to obtain 0.83 kg of active extract of Epimedium sagittatum.

[0038] Example 2

[0039] This embodiment provides a method for preparing an active extract of Epimedium sagittatum, which is carried out according to the following steps:

[0040] Processing: Take 3kg of sagittaria leaves and 1kg of dried rhizomes of bistortia, mix and stir equal amounts of mutton fat and hot water (0.4kg each), and pour on the bistortia and epimedium after stirring; take 1.0kg of mutton fat in an oil refining pot and heat it to 80℃ for refining, then mix the bistortia and epimedium with the mutton fat evenly, and keep it at 80℃ for 3h; turn the temperature of the medicine frying machine to 260℃, put the moistened epimedium and bistort into the medicine frying machine, and stir for 3min;

[0041] Extraction: Take the processed medicinal materials and reflux extract them with 50% ethanol at 90°C for two times, each time for 1.5 hours. The mass of 50% ethanol used twice is 15 times and 10 times the mass of the medicinal materials respectively. The extracts are combined and concentrated to obtain an ethanol extract. The ethanol extract is applied to an LSA-12S adsorption resin column, first eluted with water and 5% ethanol / water to remove pigments and impurities, and then eluted with 60% ethanol / water. The 60% ethanol / water elution portion is concentrated under reduced pressure to remove ethanol, and then freeze-dried to obtain the epimedium extract with a yield of 1.3%.

[0042] Comparative Example 1

[0043] This comparative example provides a method for preparing an active extract of Epimedium sagittatum, which is carried out according to the following steps:

[0044] Take 3kg of Epimedium sagittatum leaves and beat them, and 1kg of dried rhizome of Polygonum multiflorum, grind them into powder, and reflux extract them with 50% ethanol at 90°C for 2 times, each time for 1.5h. The mass of 50% ethanol used twice is 15 times and 10 times the mass of the medicinal material, respectively. The extracts are combined and concentrated to obtain an ethanol extract. The ethanol extract is applied to an LSA-12S adsorption resin column, first eluted with water and 5% ethanol / water to remove pigments and impurities, and then eluted with 60% ethanol / water. The 60% ethanol / water elution portion is concentrated under reduced pressure to remove ethanol, and then freeze-dried to obtain the Epimedium extract with a yield of 1.6%.

[0045] Comparative Example 2

[0046] This comparative example provides a method for preparing an active extract of Epimedium sagittatum, which is carried out according to the following steps:

[0047] Processing: Take 4kg of Epimedium sagittatum leaves, mix and stir the same mass of mutton fat and hot water (0.4kg each), and then pour it on the Epimedium and Bistoria medicinal materials after stirring; Take 1.0kg of mutton fat and heat it to 80℃ in an oil refining pot for refining, then mix the Epimedium and Bistoria medicinal materials with the mutton fat evenly, keep it at 80℃ and let it soak for 3h; Set the temperature of the medicine frying machine to 260℃, put the soaked Epimedium and Bistoria medicinal materials into the medicine frying machine, and stir for 3min;

[0048] Extraction: Take the processed product and reflux extract it with 50% ethanol at 90°C for two times, each time for 1.5 hours. The mass of 50% ethanol used for the two times is 15 times and 10 times the mass of the medicinal material respectively. The extracts are combined and concentrated to obtain an ethanol extract. The ethanol extract is loaded on an LSA-12S adsorption resin column, first eluted with water and 5% ethanol / water to remove pigments and impurities, and then eluted with 60% ethanol / water. The 60% ethanol / water elution portion is concentrated under reduced pressure to remove ethanol, and then freeze-dried to obtain the Epimedium extract with a yield of 1.4%.

[0049] Comparative Example 3

[0050] This comparative example provides a method for preparing an active extract of Epimedium sagittatum, which is carried out according to the following steps:

[0051] Processing: Take 4kg of dried rhizome of Bistorta ginseng, mix and stir the same mass of mutton fat and hot water (0.4kg each), and pour on the Bistorta ginseng medicinal materials after stirring; Take 1.0kg of mutton fat and heat it to 80℃ in an oil refining pot for refining, then stir the Bistorta ginseng medicinal materials and mutton fat evenly, keep it at 80℃ and let it soak for 3h; Set the temperature of the stir-frying machine to 260℃, put the moistened Bistorta ginseng medicinal materials into the stir-frying machine, and stir-fry for 3min;

[0052] Extraction: Take the processed product and reflux extract it with 50% ethanol at 90°C for two times, each time for 1.5 hours. The mass of 50% ethanol used twice is 15 times and 10 times the mass of the medicinal material respectively. The extracts are combined and concentrated to obtain an ethanol extract. The ethanol extract is loaded on an LSA-12S adsorption resin column, first eluted with water and 5% ethanol / water to remove pigments and impurities, and then eluted with 60% ethanol / water. The 60% ethanol / water elution portion is concentrated under reduced pressure to remove ethanol, and then freeze-dried to obtain the Polygonum multiflorum extract with a yield of 1.3%.

[0053] Experimental example:

[0054] A total of 60 C57BL / 6 mice were divided into 6 groups, namely 10 normal groups, 10 kidney yang deficiency groups, and 10 mice in each group of the embodiment and comparative examples. Except for the normal group, which was subcutaneously injected with normal saline, the other 5 groups were subcutaneously injected with 20 mg / kg corticosterone once a day for 21 days. The embodiment and comparative example groups were intragastrically administered with 2 times the human dose of the extract, and the normal group and kidney yang deficiency group were intragastrically administered with an equal volume of normal saline for 21 days.

[0055] The hypothalamus and pituitary of each group of mice were removed and stored at -80℃. Fluorescence quantitative PCR was used to detect the mRNA expression levels of corticotropin-releasing hormone (CRH) in the hypothalamus and adrenocorticotropic hormone (ACTH) in the pituitary.

[0056] The results are as follows Figure 1 and Figure 2 As shown, compared with the CRH and ACTH mRNA expression levels of the normal group, the CRH and ACTH mRNA in the kidney yang deficiency group were significantly reduced; compared with the CRH and ACTH mRNA levels in the kidney yang deficiency group, the Example 2 and the Comparative Examples 1-3 groups were significantly increased. The Comparative Examples 1-3 groups were significantly lower than the Example 2 group. The active extracts of Example 2 and Comparative Examples 1-3 have the effect of improving the disordered HPA axis, and the combined extraction of Epimedium and Polygonum multiflorum has a synergistic effect.

[0057] Blood was collected from the subretinal area of ​​each group of mice, and after being kept at 4°C overnight, the blood was centrifuged at 1500 rpm for 5 minutes to obtain serum. The ACTH ELISA kit from CUSABIO TECHNOLOGY was used to detect the level of ACTH in the serum.

[0058] Figure 3 The results showed that compared with the ACTH expression level in the serum of the normal group, the ACTH concentration in the kidney yang deficiency group was significantly reduced; compared with the ACTH level in the kidney yang deficiency group, the ACTH levels in Example 2 and Comparative Example 2 groups increased significantly, and there was no significant difference in Comparative Example 3. Comparative Example 1-3 groups were significantly lower than Example 2 group.

[0059] Regulatory effects of each extract on PDE5A1 in the corpus cavernosum of model mice: The mice were sacrificed, the penises of each group of mice were peeled off, the corpus cavernosum was taken, and after tissue grinding, it was frozen and stored at -80°C. The mRNA expression levels of PDE5A1 were detected by fluorescent quantitative PCR.

[0060] Figure 4 The results showed that compared with the PDE5A1 mRNA expression level of the normal group, the PDE5A1 mRNA in the kidney-yang deficiency group was significantly increased; compared with the PDE5A1 mRNA level in the kidney-yang deficiency group, the levels in Example 2 and Comparative Examples 1-3 were significantly decreased, and both were able to improve the disorder of PDE5A1 in the corpus cavernosum of mice with kidney-yang deficiency. The PDE5A1 mRNA expression levels in Comparative Examples 1-3 were significantly higher than those in Example 2, and the combined extraction of Epimedium and Polygonum multiflorum had a synergistic effect.

[0061] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing an active extract of Epimedium sagittatum, characterized in that: The steps include: Step 1: Take the leaves of Epimedium sagittatum and the dried rhizome of Polygonum multiflorum, and prepare them with sheep fat; Step 2: Use one or a combination of solvent extraction, solvent extraction, resin adsorption, normal phase column chromatography, and reverse phase column chromatography to extract the processed Epimedium sagittatum and Polygonum multiflorum.

2. The method for preparing the active extract of Epimedium sagittatum according to claim 1, characterized in that: The preparation method includes: mixing and stirring equal masses of mutton fat and boiling water, and then pouring the mixture on the herbal medicines of epimedium and ginseng after stirring; heating and refining the mutton fat in an oil refining pot, and then evenly stirring the herbal medicines of epimedium and ginseng after pouring with the mutton fat, and keeping them warm and moist; putting the moistened herbal medicines of epimedium and ginseng into a medicine frying machine for frying.

3. The method for preparing the active extract of Epimedium sagittatum according to claim 2, characterized in that: The oil-water mixture accounts for 10-30% of the total weight of the Epimedium and Bistoria medicinal materials.

4. The method for preparing the active extract of Epimedium sagittatum according to claim 2, characterized in that: The mass ratio of mutton fat to Epimedium and Polygonum multiflorum is 30:(90-140).

5. The method for preparing the active extract of Epimedium sagittatum according to claim 2, characterized in that: The lubrication temperature is 70-90℃ and the time is 2-4h.

6. The method for preparing the active extract of Epimedium sagittatum according to claim 2, characterized in that: The temperature of the medicine frying machine is 240-280℃, and the frying time is 2-4min.

7. The method for preparing the active extract of Epimedium sagittatum according to claim 2, characterized in that: The refining temperature is 70-90°C.

8. The method for preparing the active extract of Epimedium sagittatum according to claim 1, characterized in that: The mass ratio of Epimedium leaves to dried rhizomes of Bistorta ternata is (1-4):

1.

9. An active extract of Epimedium sagittatum, characterized in that: The preparation method is described in any one of claims 1 to 8.

10. The use of the active extract of Epimedium sagittatum according to claim 9, characterized in that: Application in the preparation of drugs for treating male sexual dysfunction.

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