CN112043731A - Preparation method of whole ganoderma lucidum spore oil nanoemulsion rich in ganoderic acid - Google Patents
Preparation method of whole ganoderma lucidum spore oil nanoemulsion rich in ganoderic acid Download PDFInfo
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Abstract
The invention discloses a preparation method of whole ganoderma lucidum spore oil nanoemulsion rich in ganoderic acid. The method comprises the following steps: (1) using whole ganoderma spore powder as raw material, dry-pressing and granulating, placing in supercritical CO2Extracting in an extraction kettle for supercritical CO2Extracting to obtain light yellow whole ganoderma lucidum spore oil rich in ganoderic acid; (2) the whole ganoderma lucidum spore oil is prepared into the whole ganoderma lucidum spore oil nanoemulsion together with the auxiliary agent. The Ganoderma spore oil is rich in antitumor substancesThe effective component of ganoderic acid, its representative component ganoderic acid B content is more than 3 times of pure ganoderma spore oil. The invention not only expands the application resources of the ganoderma lucidum and the ganoderma lucidum spore powder, but also greatly improves the content of ganoderic acid in the ganoderma lucidum spore oil. The method provided by the invention has simple process flow and convenient operation, and is suitable for industrial production; the product has stable quality and convenient quality control, and can be used in medicine and food.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a preparation method of a whole ganoderma lucidum spore oil nanoemulsion rich in ganoderic acid.
Background
Ganoderma lucidum has medical and edible history for thousands of years in China, is a traditional famous and precious traditional Chinese medicine for strengthening body resistance, consolidating constitution, nourishing and strengthening in China, is called as the top grade by the book of the book, and is regarded as Ling Yao Xian Dan in folk; ganoderma lucidum is a general name of Ganoderma lucidum G.lucidum.karst and Ganoderma japonicum G.japonicum Lloyd of Ganoderma (Polyporaceae) in Polyporaceae of Basidiomycetes, and the main components of Ganoderma lucidum reported in literature include: triterpenes, polysaccharides, nucleosides, sterols, amino acids, vitamins, alkaloids, inorganic ions, etc. Wherein the ganoderic acid is a component with strong biological activity in ganoderma, and ganoderic acid A is a main representative component of ganoderic acid in ganoderma. The ganoderma lucidum triterpene component has poor water solubility, and because the ganoderma lucidum is tough and hard to break and cut, the lipid components such as the ganoderma lucidum triterpene and the like are difficult to extract by a conventional water decoction method, and the effect of the ganoderma lucidum is difficult to fully exert by water decoction extraction.
The Ganoderma spore is germ cell continuously ejected from pileus of Ganoderma in maturation period, is seed for reproducing next generation Ganoderma, and has all genetic active substances of Ganoderma. From the beginning of the eighties of the last century, more research foundations for the anti-tumor aspect of ganoderma lucidum and ganoderma lucidum spore powder exist, in recent years, more artificial cultured ganoderma lucidum spore powder and ganoderma lucidum spore extract are researched and applied, and Chinese scientists find that lipid is the disease-resistant 'main power' of ganoderma lucidum spores. The lipid active substance extracted by supercritical carbon dioxide is Ganoderma spore oil. The main components of the ganoderma lucidum spore comprise: fatty acids, sterols, triterpenes, vitamins, carotenes, lactones, proteins and amino acids, glycopeptides, alkaloids, inorganic ions, etc., are now made using supercritical CO2The invention discloses a plurality of patents for extracting ganoderma lucidum spore oil from ganoderma lucidum spores, but the methods are all to simply obtain the ganoderma lucidum spore oil from the ganoderma lucidum spores.
Although the ganoderma contains basically the same active substances as ganoderma spores, actually, the content of ganoderma triterpenic acid ingredients with strong biological activity in the ganoderma is higher than that in the ganoderma spores, and particularly the content of ganoderic acid B is much higher than that in the ganoderma spores. Therefore, the ganoderma lucidum spore oil is extracted by combining the ganoderma lucidum and the ganoderma lucidum spores, the acquisition resources of the ganoderma lucidum spore oil are expanded, and the ganoderic acid B component in the obtained ganoderma lucidum spore oilThe content of the ganoderma lucidum spore oil is far higher than that of the pure ganoderma lucidum spore oil, and the clinical application effect is better. Chinese patent CN 200410017468.8 discloses supercritical CO of effective component in total ganoderma spore2The extraction method uses Ganoderma encarpium and Ganoderma spore pulverized into granule as raw materials, but at supercritical C02The extraction is carried out by using edible ethanol as an extracting agent, the obtained extract is in the form of ointment, contains lipid components and water-soluble polysaccharide, has a great difference with the components of the ganoderma lucidum spore oil, and the ointment is difficult to prepare into a nano-emulsion preparation. Chinese patent CN 200610035574.8 discloses a method for preparing total ganoderma spore oil, which selects most (50-100%) ganoderma spore powder, less (0-50%) ganoderma powder, some nutrient components and water as initial raw materials, and extracts supercritical CO as final raw material by inoculating ganoderma strain to culture product for up to 60 days2The method has complex process and long time consumption, and the extract is difficult to mix and treat; the invention fully utilizes the characteristic that the components of the ganoderma lucidum and the ganoderma lucidum spore have better complementarity, directly selects the whole ganoderma lucidum which is composed of the ganoderma lucidum with the mass percent accounting for most (51-60%, preferably 55%) and the ganoderma lucidum with the mass percent accounting for less (40-49%, preferably 45%) as the raw materials, respectively carries out superfine grinding and wall breaking treatment, then mixes the raw materials to form the whole ganoderma lucidum spore powder, then adds a small amount of adhesive to mix the whole ganoderma lucidum spore powder evenly, carries out dry pressing granulation, carries out supercritical CO granulation by using the optimized conditions2Extracting to obtain a lipid transparent oily liquid with high content of ganoderic acid B: whole ganoderma spore oil.
The stable whole ganoderma lucidum spore oil nanoemulsion preparation is prepared by the obtained whole ganoderma lucidum spore oil with high content of ganoderic acid B through optimized nanoemulsion preparation conditions for the first time, so that the bioavailability of the whole ganoderma lucidum spore oil can be improved, the effect is fast, the defect of relatively low bioavailability of a common preparation of the whole ganoderma lucidum spore oil is overcome, and the anti-tumor effect of the whole ganoderma lucidum spore oil is obviously enhanced. Meanwhile, the product is used as a new formulation of the whole ganoderma lucidum spore oil, and can provide a new choice for consumers.
Disclosure of Invention
The invention aims to solve the problems, provides whole ganoderma lucidum spore oil with high content of ganoderic acid B, and provides a preparation method of whole ganoderma lucidum spore oil nanoemulsion rich in ganoderic acid, which has high bioavailability, safe and convenient use and can obviously enhance the anti-tumor effect of the whole ganoderma lucidum spore oil.
In order to achieve the purpose, the invention adopts the technical scheme that:
the ganoderma lucidum (ganoderma lucidum mycelium or ganoderma lucidum fruiting body) has rich resources and contains active substances of ganoderma lucidum triterpenic acids (ganoderic acids) with strong physiological activity, but because the water solubility of the ganoderic acids is poor, the actual extraction effect of the traditional water decoction method on the ganoderic acids in the ganoderma lucidum decoction pieces is poor, most of the ganoderic acids cannot be extracted, and the resource waste is caused; even if wine extraction is used, only part of ganoderic acid components in the ganoderma lucidum decoction pieces are extracted due to the compact texture of the ganoderma lucidum, and the resource waste is also caused. Although the ganoderma lucidum spores have all the genetic active substances of the ganoderma lucidum (namely the ganoderma lucidum spores contain basically the same active substances as the ganoderma lucidum), the research finds that the content of ganoderma lucidum acid components with very strong biological activity is much higher than that of the ganoderma lucidum spores.
Because the resource of the ganoderma lucidum spore is limited, in order to expand the resource source of the ganoderma lucidum spore oil and improve the content and the physiological activity of the ganoderic acid components in the ganoderma lucidum spore oil, according to the existence proportion of the ganoderma lucidum and the ganoderma lucidum spore in the nature, the invention fully utilizes the characteristic that the components contained in the ganoderma lucidum and the ganoderma lucidum spore have better complementarity, selects the whole ganoderma lucidum consisting of ganoderma lucidum and ganoderma lucidum spore powder with certain mass percentage as the raw material, respectively carries out superfine grinding and wall breaking treatment, then mixes the whole ganoderma lucidum spore powder, then adds a small amount of binder, carries out dry pressing granulation, and carries out supercritical CO granulation by using the preferred conditions2Extracting to obtain a lipid transparent oily liquid with high content of ganoderic acid B: whole ganoderma spore oil. The whole ganoderma lucidum spore oil is different from the general ganoderma lucidum spore oil, and the content of ganoderic acid B is far higher than that in ganoderma lucidum spores.
A method for preparing whole ganoderma lucidum spore oil nanoemulsion rich in ganoderic acid comprises the following steps:
(1) using whole ganoderma spore powder as raw material, dry-pressing and granulating, placing in supercritical CO2Extracting in an extraction kettle for supercritical CO2Extracting to obtain light yellow whole ganoderma lucidum spore oil rich in ganoderic acid;
(2) the whole ganoderma lucidum spore oil is prepared into the whole ganoderma lucidum spore oil nanoemulsion together with the auxiliary agent. The obtained whole ganoderma spore oil is light yellow, the oil is rich in ganoderma triterpenic acid with anti-tumor effect, and the content of the representative component ganoderma acid B is more than or equal to 75mg/100g and is more than 3 times of the content of pure ganoderma spore oil ganoderma acid B25 mg/100 g.
Preferably, in the above method for preparing ganoderma lucidum spore oil nanoemulsion rich in ganoderic acid, the ganoderma lucidum consists of 51% -60% of ganoderma lucidum fruiting body and 40% -49% of ganoderma lucidum spore powder; the whole ganoderma lucidum spore powder is prepared by the following method: pulverizing Ganoderma fruiting body into superfine powder of 150 meshes or more, and breaking cell wall of Ganoderma spore powder to obtain cell wall-broken Ganoderma spore powder with cell wall breaking rate of 96% or more, and mixing the two powders to obtain the final product.
Preferably, in the above method for preparing the ganoderma lucidum spore oil nanoemulsion rich in ganoderic acid, the step (1) is: firstly, adding 0.5-1.2% of starch or dextrin into the whole ganoderma lucidum spore powder raw material, uniformly mixing, preparing granules with the particle size of 0.1-0.3 cm by adopting a dry pressing method of a granulator in one step, selecting the granulator with the main pressure of 5-9 MPa and the lateral pressure of 0.5-0.9 MPa during granulation, and then placing the granules in a supercritical CO2And (5) extracting in an extraction kettle.
Preferably, in the above method for preparing ganoderma lucidum spore oil nanoemulsion rich in ganoderic acid, the supercritical fluid is CO2The extraction temperature is 23-45 ℃, the extraction pressure is 12-34 MPa, the extraction time is 2-5 hours, and the extraction flow is 32-58 kg/h; two-stage separation is adopted, and the separation process conditions are as follows: the pressure of the primary analyzer is 9-13 MPa, and the separation temperature is 37-43 ℃; the pressure of the secondary analyzer is 4-6 Mpa, and the separation temperature is 27-33 ℃; get shallowYellow whole ganoderma spore oil.
Preferably, in the above method for preparing the ganoderma lucidum spore oil nanoemulsion rich in ganoderic acid, the ganoderma lucidum spore oil and the auxiliary agent are composed of the following components in parts by weight:
5 to 20 percent of whole ganoderma lucidum spore oil,
1 to 12 percent of emulsifier,
1-8% of solubilizer,
0.0 to 2.5 percent of flavoring agent,
0 to 3 percent of isotonic regulator,
0.0 to 0.2 percent of essence,
0.00 to 0.08 percent of preservative,
0.01 to 0.04 percent of antioxidant,
the pH value regulator is a proper amount,
the balance being water.
Preferably, in the above method for preparing the ganoderma lucidum spore oil nanoemulsion rich in ganoderic acid, the ganoderma lucidum spore oil nanoemulsion is in the form of nanoemulsion for injection, and the ganoderma lucidum spore oil and the auxiliary agent are composed of the following components in parts by weight:
6 to 12 percent of whole ganoderma lucidum spore oil,
2 to 10 percent of emulsifier,
1 to 5 percent of solubilizer,
0.2 to 2.5 percent of isoosmotic adjusting agent,
0.01 to 0.02 percent of antioxidant,
the pH value regulator is a proper amount,
the balance of water;
the preparation method comprises the following steps:
(1) taking the whole ganoderma lucidum spore oil, adding part of emulsifier and antioxidant into the whole ganoderma lucidum spore oil in water bath at the temperature of 60-80 ℃ under the protection of nitrogen, stirring to dissolve the emulsifier and the antioxidant, uniformly mixing, and cooling to room temperature to obtain an oil phase;
(2) mixing the rest emulsifier, solubilizer and isotonic regulator in water bath at 60-80 ℃ under the protection of nitrogen, stirring, dissolving in water for injection, shearing and stirring if necessary to dissolve and mix uniformly, and cooling to room temperature to obtain a water phase;
(3) slowly adding the oil phase into the water phase under the stirring condition, simultaneously carrying out shearing stirring for 15-60 minutes under the protection of nitrogen, then adjusting the pH to about 7.0, carrying out primary homogenization, wherein the homogenization pressure is 150-300 bar, the homogenization time is 5-10 minutes, and then adding water for injection to 100% to obtain milky primary emulsion;
(4) and (3) transferring the prepared white colostrum into a high-pressure homogenizer with the high-pressure homogenizing pressure of 800-1300 bar, homogenizing for 10-15 times, filtering by using a microporous filter membrane after homogenization, filling nitrogen, filling, sealing and sterilizing to obtain the uniform and stable nano-emulsion preparation for injection of the ganoderma lucidum spore oil.
Preferably, in the above method for preparing the ganoderma lucidum spore oil nanoemulsion rich in ganoderic acid, the ganoderma lucidum spore oil nanoemulsion is in the form of oral nanoemulsion, and the ganoderma lucidum spore oil and the auxiliary agent are composed of the following components in parts by weight:
8 to 18 percent of whole ganoderma lucidum spore oil,
3 to 8 percent of emulsifier,
1 to 6 percent of solubilizer,
0.02 to 2.5 percent of flavoring agent,
0.0 to 0.2 percent of essence,
0.00 to 0.05 percent of preservative,
the balance of water;
the preparation method comprises the following steps:
(1) taking the whole ganoderma lucidum spore oil, adding part of emulsifier and antioxidant into the whole ganoderma lucidum spore oil in water bath at the temperature of 60-80 ℃ under the protection of nitrogen, stirring to dissolve the emulsifier and the antioxidant, uniformly mixing, and cooling to room temperature to obtain an oil phase;
(2) mixing the rest emulsifier, solubilizer, flavoring agent, preservative and essence in water bath at 60-80 ℃ under the protection of nitrogen, stirring, dissolving in water for injection, shearing and stirring if necessary to dissolve and mix uniformly, and cooling to room temperature to obtain a water phase;
(3) slowly adding the oil phase into the water phase under the stirring condition, simultaneously carrying out shearing stirring for 15-60 minutes under the protection of nitrogen, then carrying out primary homogenization, wherein the homogenization pressure is 150-300 bar, the homogenization time is 5-10 minutes, and then adding water for injection to 100% to obtain milky primary emulsion;
(4) and (3) transferring the prepared white colostrum into a high-pressure homogenizer with the high-pressure homogenizing pressure of 800-1300 bar, homogenizing for 8-12 times, filtering by a microporous filter membrane after homogenization, or filling nitrogen, filling and sealing, and sterilizing to obtain the uniform and stable nanoemulsion for the whole ganoderma spore oil for oral administration.
Preferably, in the preparation method of the ganoderma lucidum spore oil nanoemulsion rich in ganoderic acid, the emulsifier is one or more of soybean lecithin, yolk lecithin phospholipid, liquid lecithin and/or cephalin; the solubilizer is one or two of glycerol and polyethylene glycol 400; the flavoring agent is stevioside, aspartame, glucose or fructose; the isoosmotic adjusting agent is glycerin for injection; the essence is mint essence, lemon essence, rose essence, milk essence, mint essence or a composition of various essences; the antioxidant is vitamin E; the preservative is potassium sorbate; the pH value regulator is NaOH or Na2CO3Or NaHCO3。
The obtained Ganoderma lucidum spore oil nanoemulsion can be used for preparing anti-tumor and rabbit epidemic force improving medicines.
The grain size of the emulsion of the nanoemulsion preparation for whole ganoderma spore oil injection is controlled to be 10-200 nm, and the grain size of the emulsion of the nanoemulsion preparation for whole ganoderma spore oil oral administration is controlled to be 20-500 nm.
The ganoderic acid components are ingredients with strong activity in ganoderma lucidum and ganoderma lucidum spores, triterpenoid ganoderic acid components such as ganoderic acid B, ganoderic acid C, ganoderic acid E and the like are detected in the ganoderma lucidum and the ganoderma lucidum spores, but the content of the triterpenoid ganoderic acid components is greatly different, the content of the ganoderic acid components in the ganoderma lucidum is obviously higher than that of the ganoderma lucidum spores, and the ganoderic acid components in the ganoderma lucidum are difficult to extract by using a traditional water decoction method, so that the resource waste is caused; because the content difference of the ganoderic acid components in the ganoderma lucidum and ganoderma lucidum spore has strong complementarity, according to the existence proportion of ganoderma lucidum and ganoderma lucidum spore in nature, the whole ganoderma lucidum composed of ganoderma lucidum and ganoderma lucidum spore powder with a certain mass percentage is selected as raw material, and the raw material is respectively passed throughMicronizing, breaking cell wall, mixing to obtain Ganoderma spore powder, and supercritical CO pulverizing2The extraction method extracts the whole ganoderma lucidum spore oil, and unexpected beneficial effects are obtained.
Compared with the prior art, the invention has the following beneficial effects:
1. the whole ganoderma lucidum spore is prepared from ganoderma lucidum and ganoderma lucidum spore in a reasonable proportion, so that the waste of ganoderma lucidum resources is reduced, and the application resources of ganoderma lucidum spore are expanded, thereby saving the production cost;
2. the main triterpene ganoderic acid chemical components in the whole ganoderma lucidum spore oil obtained by the invention are clear in composition, and the quality of the product is easy to control by HPLC content determination of ganoderic acid B;
3. the invention adopts supercritical CO2The fluid extraction method for preparing Ganoderma spore oil adopts dry-pressing one-step granulation method to solve supercritical CO2The safety of the material mixing during extraction and incomplete extraction are solved, the granulation method is simple, the heating and drying processes are not needed, and the possibility of component damage caused by heating is avoided;
4. the whole ganoderma lucidum spore oil and the nanoemulsion thereof obtained by the invention not only have the similar effect as the pure ganoderma lucidum spore oil, but also have the content of the ganoderic acid B in the whole ganoderma lucidum spore oil which is more than 3 times of the content in the pure ganoderma lucidum spore oil through the determination of the chemical components of the ganoderic acid and the comparison of the anti-tumor activity, and the anti-tumor action strength of the whole ganoderma lucidum spore oil and the nanoemulsion thereof is obviously higher than that of the pure ganoderma lucidum spore oil, thereby generating unexpected beneficial effect.
The technical solution of the present invention is further illustrated by the following examples.
Detailed Description
Example 1 method for determining content of Ganoderic acid B in Whole or pure Ganoderma spore oil
The Ganoderma spore oil contains multiple chemical components, wherein Ganoderma triterpene acids (Ganoderma acids) compound is one of main effective components, and has various pharmacological effects of resisting tumor, protecting liver, removing toxic substance, and resisting aging; the whole ganoderma lucidum spore oil contains all fat-soluble components contained in ganoderma lucidum and ganoderma lucidum spores, and at least more than twenty ganoderma lucidum acid components are found in the oil at present. At present, a High Performance Liquid Chromatography (HPLC) method is mostly adopted for more accurate determination of ganoderic acid components in ganoderma lucidum spore oil.
Determining the content of ganoderic acid B in the whole ganoderma lucidum spore oil or the pure ganoderma lucidum spore oil: measuring chromatographic conditions and system applicability by high performance liquid chromatography (0512 in the chapter of the first division of the Chinese pharmacopoeia 2015) with octadecylsilane chemically bonded silica as filler; gradient elution was carried out using acetonitrile (A) -as mobile phase A and 0.1% glacial acetic acid solution as mobile phase B as specified in the following table, and the detection wavelength was 254 nm. The number of theoretical plates is not less than 2000 calculated by ganoderic acid B.
| Time (minutes) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 30 | 70 |
| 0～20 min | 30→40 | 70→60 |
Preparation of control solution A proper amount of ganoderic acid B control was precisely weighed, added with methanol to obtain a solution containing 0.08mg of ganoderic acid B per lml, and shaken well to obtain a control solution.
Preparation of test solution Whole Ganoderma spore oil or pure Ganoderma spore oil 1g is precisely weighed, dissolved with petroleum ether 10, added to treated silica gel column (8 g,18mm × 400 mm), and added with 100ml ethyl acetate: eluting with 25:75 mixed solution of petroleum ether (60-90 ℃), discarding eluent, and adding 120ml ethyl acetate to silica gel column: methanol 80: and (3) eluting the mixed solution of 20, collecting eluent, recovering the solvent until the solvent is dry, dissolving the residue with methanol, transferring the residue into a measuring flask, and fixing the volume to obtain the test solution.
The determination method comprises precisely sucking 10 μ l of reference solution and sample solution, injecting into liquid chromatograph, determining, and calculating ganoderic acid B content in whole or pure Ganoderma spore oil.
Example 2 preparation of Whole Ganoderma spore oil and its nanoemulsion
Taking 3.9 kg of lucid ganoderma, and crushing the lucid ganoderma into superfine powder of 200 meshes by a superfine crusher to obtain about 3.6 kg of lucid ganoderma superfine powder; and 3.2kg of ganoderma lucidum spore powder is taken, and is subjected to freezing ultramicro wall breaking treatment to obtain about 3.0kg of wall-broken ganoderma lucidum spore powder with the wall breaking rate of about 98%. And (3) adding 60g of starch into the obtained ganoderma lucidum superfine powder and wall-broken ganoderma lucidum spore powder, uniformly mixing, directly performing dry pressing granulation by using a dry pressing granulator, wherein the main pressure is 5MPa, and the lateral pressure is 0.6MPa, preparing into granules of 0.2-0.3 cm, sieving to remove fine powder of more than 60 meshes, and granulating the fine powder by the same method. Placing the obtained Ganoderma spore powder in supercritical carbon dioxide extraction kettle, and placing at C02Pressurizing by a high-pressure pump, and circularly extracting. The pressure of the extraction kettle is 26MPa, the temperature is 41 ℃, the primary separation is 10MPa and 42 ℃, and the secondary separation is 6MPa and 31 ℃. After continuous extraction for 3 hours, collecting the spore oil in a separator to obtain 765.6g of light yellow ganoderma lucidum spore oil with special smell of ganoderma lucidum spores. The obtained Ganoderma spore oil is determined by the above method (high performance liquid chromatography) for determining content of ganoderic acid B in Ganoderma spore oil or pure Ganoderma spore oil, and the determined content of ganoderic acid B is 92.6mg/100g, which is 3.66 times of content of ganoderic acid B in pure Ganoderma spore oil of 25.3mg/100 g.
The preparation of the nanoemulsion of the whole ganoderma lucidum spore oil for injection comprises the following steps:
(1) taking 100g of the obtained whole ganoderma spore oil under the protection of water bath at the temperature of about 60 ℃ and nitrogen, adding 20g of soybean lecithin and 100mg of vitamin E, stirring until the phospholipid is dissolved to form a uniform mixed phase, and cooling to room temperature to obtain an oil phase;
(2) placing 800ml of water for injection in a container in water bath at about 60 ℃ under the protection of nitrogen, adding 22g of glycerol and 15g of the other part of soybean lecithin, shearing and stirring to dissolve and uniformly mix, and cooling to room temperature to obtain a water phase;
(3) slowly adding the oil phase into the water phase under the stirring condition, simultaneously carrying out shearing stirring for about 20 minutes under the protection of nitrogen, then adjusting the pH to 6.5-7.0 by using a 1% NaOH solution, carrying out primary homogenization, wherein the homogenization pressure is 200bar, the homogenization time is 10 minutes, and then adding water for injection to 1000ml to obtain milky primary emulsion;
(4) transferring the prepared white colostrum into a high-pressure homogenizer with a high-pressure homogenizing pressure of 1100bar, homogenizing at about 60 deg.C for 12 times, checking the particle size of emulsion droplets, homogenizing, filtering with microporous membrane, charging nitrogen, bottling, and capping; the sterilization was performed at 100 ℃ and FO of 20 using a rotary autoclave. After sterilization, gradually cooling. Stored at room temperature. Thus preparing the nano-emulsion for injecting whole ganoderma lucidum spore oil.
Fifthly, measuring stability: the stability determination method adopts freshly prepared nano-emulsion, the nano-emulsion is placed in a centrifuge tube with a plug, and after the nano-emulsion is centrifuged at 4000rpm for 20min, no layering is found, and no precipitation is found. When the emulsion is stored for one year at room temperature in shade, the physical and chemical properties such as the appearance, the grain diameter, the Zeta potential and the like of the emulsion and the content of the emulsion are not obviously changed, and the average grain diameter of the emulsion grains is about 120nm, which indicates that the emulsion is stable.
Example 3 preparation of Whole Ganoderma spore oil and its nanoemulsion
Taking 5.1kg of lucid ganoderma, and crushing the lucid ganoderma into superfine powder of 200 meshes by a superfine crusher to obtain about 4.5kg of lucid ganoderma superfine powder; and 3.2kg of ganoderma lucidum spore powder is taken, and is subjected to freezing ultramicro wall breaking treatment to obtain about 3.0kg of wall-broken ganoderma lucidum spore powder with the wall breaking rate of about 98.7%. And (3) adding 70g of dextrin into the obtained ganoderma lucidum superfine powder and the wall-broken ganoderma lucidum spore powder, uniformly mixing, directly performing dry pressing granulation by using a dry pressing granulator, wherein the main pressure is 5MPa and the lateral pressure is 0.5MPa, preparing into granules of 0.2-0.3 cm, sieving to remove fine powder of more than 60 meshes, and performing granulation by the same method on the fine powder. The prepared whole ganoderma lucidum spore powder particles,placing in supercritical carbon dioxide extraction kettle, C02Pressurizing by a high-pressure pump, and circularly extracting. The pressure of the extraction kettle is 29MPa, the temperature is 39 ℃, the primary separation is 9MPa and 40 ℃, and the secondary separation is 5MPa and 30 ℃. After continuous extraction for 3 hours, collecting spore oil in a separator to obtain 771.4g of light yellow whole ganoderma lucidum spore oil with special smell of ganoderma lucidum spores. The obtained Ganoderma spore oil is determined by the above method (high performance liquid chromatography) for determining content of ganoderic acid B in Ganoderma spore oil or pure Ganoderma spore oil, and the determined content of ganoderic acid B is 107.4mg/100g, which is 4.25 times of content of ganoderic acid B in pure Ganoderma spore oil of 25.3mg/100 g.
Preparing the oral nano-emulsion of the whole ganoderma lucidum spore oil:
(1) taking 100g of the obtained whole ganoderma spore oil under the protection of water bath at the temperature of about 70 ℃ and nitrogen, adding 20g of egg yolk lecithin and 100mg of vitamin E, stirring until the lecithin is dissolved to form a uniform mixed phase, and cooling to room temperature to obtain an oil phase;
(2) placing 800ml of purified water into a container under the protection of water bath at about 70 ℃ and nitrogen, adding 30g of glycerol, 200mg of stevioside, 500mg of potassium sorbate and 15g of the other part of egg yolk lecithin, shearing and stirring to dissolve and uniformly mix, and cooling to room temperature to obtain a water phase;
(3) slowly adding the oil phase into the water phase under stirring, simultaneously shearing and stirring for about 15 minutes under the protection of nitrogen, preliminarily homogenizing under the homogenizing pressure of 180bar for 10 minutes, and adding water for injection to 1000ml to obtain milky primary emulsion;
(4) transferring the prepared white colostrum into a high-pressure homogenizer with a high-pressure homogenizing pressure of 1000bar, homogenizing for 10 times at about 70 deg.C, checking the particle size of emulsion droplets, homogenizing, filtering with microporous membrane, charging nitrogen, bottling, and capping; the sterilization was performed at 100 ℃ and FO of 20 using a rotary autoclave. After sterilization, gradually cooling. Stored at room temperature. Thus preparing the whole ganoderma lucidum spore oil oral nano-emulsion.
Fifthly, measuring stability: the stability determination method adopts the freshly prepared nano emulsion, places the nano emulsion in a centrifuge tube with a plug, and after the nano emulsion is centrifuged at 4000rpm for 15min, no layering is found, and no precipitation is found. When the emulsion is stored for one year at room temperature in shade, the physical and chemical properties such as the appearance, the grain diameter, the Zeta potential and the like of the emulsion and the content of the emulsion are not obviously changed, and the average grain diameter of the emulsion is 250nm, which shows that the emulsion is stable.
Example 4 preparation of Whole Ganoderma spore oil and nanoemulsion thereof
Taking 3.5kg of lucid ganoderma, and crushing the lucid ganoderma into superfine powder of 220 meshes by a superfine crusher to obtain about 3.1kg of lucid ganoderma superfine powder; and 3.2kg of ganoderma lucidum spore powder is taken, and is subjected to freezing ultramicro wall breaking treatment to obtain about 3.0kg of wall-broken ganoderma lucidum spore powder with the wall breaking rate of about 98.3%. And (3) adding 58g of dextrin into the obtained ganoderma lucidum superfine powder and the wall-broken ganoderma lucidum spore powder, uniformly mixing, directly performing dry pressing granulation by using a dry pressing granulator, wherein the main pressure is 6MPa and the lateral pressure is 0.6MPa, preparing into granules of 0.1-0.2 cm, sieving to remove fine powder of more than 60 meshes, and performing granulation by the same method on the fine powder. Placing the obtained Ganoderma spore powder in supercritical carbon dioxide extraction kettle, and placing at C02Pressurizing by a high-pressure pump, and circularly extracting. The pressure of the extraction kettle is 34MPa, the temperature is 30 ℃, the primary separation is 9MPa and 38 ℃, and the secondary separation is 4MPa and 28 ℃. After continuous extraction for 3.0 hours, the spore oil was collected in a separator to obtain 762.7g of light yellow whole ganoderma spore oil with specific smell of ganoderma spores. The obtained Ganoderma spore oil is determined by the above method (high performance liquid chromatography) for determining content of ganoderic acid B in Ganoderma spore oil or pure Ganoderma spore oil, and the determined content of ganoderic acid B is 85.9mg/100g, which is 3.40 times of content of ganoderic acid B in pure Ganoderma spore oil of 25.3mg/100 g.
Preparing the oral nano-emulsion of the whole ganoderma lucidum spore oil:
(1) under the protection of water bath and nitrogen at the temperature of about 80 ℃, 100g of whole ganoderma lucidum spore oil is taken and stirred to be uniform, 15g of soybean phospholipid and 100mg of vitamin C are added and stirred until the phospholipid is dissolved to form a uniform mixed phase, and the mixed phase is cooled to room temperature to obtain an oil phase;
(2) placing 800ml of purified water into a container in water bath at the temperature of about 80 ℃ and under the protection of nitrogen, adding 40030 g of polyethylene glycol, 20g of glucose, 1.6g of rose essence, 500mg of potassium sorbate and 15g of the other part of soybean lecithin, shearing and stirring to dissolve and uniformly mix, and cooling to room temperature to obtain a water phase;
(3) slowly adding the oil phase into the water phase under stirring, simultaneously shearing and stirring for about 20 minutes under the protection of nitrogen, preliminarily homogenizing under the homogenizing pressure of 200bar for 10 minutes, and adding purified water to 1000ml to obtain milky primary emulsion;
(4) transferring the prepared white colostrum into a high-pressure homogenizer with a high-pressure homogenizing pressure of 1150bar, homogenizing at about 60 deg.C for 9 times, checking the particle size of emulsion droplets, homogenizing, filtering with microporous membrane, charging nitrogen, bottling, and capping; the sterilization was performed at 100 ℃ and FO of 20 using a rotary autoclave. After sterilization, gradually cooling. Stored at room temperature. The whole ganoderma lucidum spore oil oral nanoemulsion is prepared.
Fifthly, measuring stability: the stability determination method adopts freshly prepared nano-emulsion, the nano-emulsion is placed in a centrifuge tube with a plug, and after the nano-emulsion is centrifuged at 4000rpm for 15min, no layering is found, and no precipitation is found. When the emulsion is stored for one year at room temperature in shade, the physical and chemical properties such as the appearance, the grain diameter, the Zeta potential and the like of the emulsion and the content of the emulsion are not obviously changed, and the average grain diameter of the emulsion grains is about 300nm, which indicates that the emulsion is stable.
The preparation of the nanoemulsion of the whole ganoderma lucidum spore oil for injection comprises the following steps:
(1) taking 100g of the obtained whole ganoderma spore oil under the protection of water bath at the temperature of about 80 ℃ and nitrogen, adding 20g of egg yolk lecithin and 100mg of vitamin E, stirring until the lecithin is dissolved to form a uniform mixed phase, and cooling to room temperature to obtain an oil phase;
(2) placing 800ml of water for injection in a container in water bath at the temperature of about 80 ℃ under the protection of nitrogen, adding 22g of glycerol and 15g of the other part of egg yolk lecithin, shearing and stirring to dissolve and uniformly mix the components, and cooling to room temperature to obtain a water phase;
(3) slowly adding the oil phase into the water phase under the stirring condition, simultaneously carrying out shearing stirring for about 20 minutes under the protection of nitrogen, then adjusting the pH to 6.5-7.0 by using a 1% NaOH solution, carrying out primary homogenization, wherein the homogenization pressure is 250bar, the homogenization time is 15 minutes, and then adding injection water to 1000ml to obtain milky primary emulsion;
(4) transferring the prepared white colostrum into a high-pressure homogenizer with high-pressure homogenizing pressure of 1200bar, homogenizing at 60 deg.C for 13 times, checking the particle size of emulsion drop, homogenizing, filtering with microporous membrane, charging nitrogen, bottling, and capping; the sterilization was performed at 100 ℃ and FO of 20 using a rotary autoclave. After sterilization, gradually cooling. Stored at room temperature. Thus preparing the nano-emulsion for injecting whole ganoderma lucidum spore oil.
Fifthly, measuring stability: the stability determination method adopts freshly prepared nano-emulsion, the nano-emulsion is placed in a centrifuge tube with a plug, and after the nano-emulsion is centrifuged at 4000rpm for 20min, no layering is found, and no precipitation is found. When the emulsion is stored for one year at room temperature in shade, the physical and chemical properties such as the appearance, the grain diameter, the Zeta potential and the like of the emulsion and the content of the emulsion are not obviously changed, and the average grain diameter of the emulsion grains is 110nm, which shows that the emulsion is stable.
Example 5 Whole ganoderma lucidum spore oil and its nanoemulsion and tumor inhibition rate experiment of pure ganoderma lucidum spore oil and its nanoemulsion
The efficacy of the whole ganoderma lucidum spore oil and the nanoemulsion thereof provided by the invention is compared with the efficacy of the pure ganoderma lucidum spore oil and the nanoemulsion thereof in resisting tumors through a pharmacodynamic test.
1. Test sample
Preparing whole ganoderma spore oil and nanoemulsion thereof: taking the whole ganoderma lucidum spore oil and the whole ganoderma lucidum spore oil nanoemulsion prepared in the embodiment 2 as the samples of the whole ganoderma lucidum spore oil and the whole ganoderma lucidum spore oil nanoemulsion; preparing pure ganoderma lucidum spore oil and nanoemulsion thereof: pure ganoderma lucidum spore oil and pure ganoderma lucidum spore oil nanoemulsion serving as samples of pure ganoderma lucidum spore oil and pure ganoderma lucidum spore oil nanoemulsion are prepared from wall-broken pure ganoderma lucidum spore powder with the wall-broken rate of 98% according to the conditions and the method of the example 2.
2. Grouping of experimental animals and experimental conditions
90 mice of Kunming species with male closed groups, weighing 18-22 g, were prepared into EAC model mice, which were randomly divided into A, B, C groups of 30 mice each. Respectively as group A: a saline control group; group B: pure ganoderma spore oil nanoemulsion injection (prepared according to the conditions and method of example 2) treatment group; c: whole ganoderma spore oil nanoemulsion injection (prepared in example 2) treatment group. The experimental temperature is 23-25 ℃, and the humidity is 58-60%.
Preparation of S180 ascites tumor model mouse
Well-grown S180 ascites tumor was taken under aseptic conditions, diluted with physiological saline to prepare a tumor cell suspension of 1.O X106 cells/mL, and inoculated into the abdominal cavity of 90 mice, each of which was inoculated with 0.2 mL.
4. Experimental methods
4.1 group a saline control group: each mouse was injected intravenously with O.9% normal saline 0.4m1 once a day for 12 consecutive days.
4.2 group B pure ganoderma spore oil nanoemulsion injection treatment group: the pure ganoderma spore oil nanoemulsion injection is injected into the tail of each mouse at a concentration of 0.4m1, and is injected once a day for 12 days continuously.
4.3 group C Whole ganoderma lucidum spore oil nanoemulsion injection treatment group: the whole ganoderma spore oil nanoemulsion injection is injected into the tail of each mouse at a ratio of 0.4m1, and is injected once a day for 12 days continuously.
4.4 animal feeding and handling
The mice were fed regular chow and water was normally drunk during the dosing period. After 12 days, the mice were sacrificed by cutting the cone, ascites tumor bodies were separated, epidermal tissues were removed, and accurate weighing was performed, and the tumor inhibition rate was calculated.
4.5 tumor inhibition Rate calculation
Tumor inhibition rate = (A-B)/A × 100%
(A is the mean tumor weight of the normal saline control group, B is the mean tumor weight of the treatment group)
Comparing whether the tumor inhibition rates of the normal saline control group, the pure ganoderma spore oil nanoemulsion injection treatment group and the whole ganoderma spore oil nanoemulsion injection treatment group and the tumor inhibition rates of the pure ganoderma spore oil nanoemulsion injection treatment group and the whole ganoderma spore oil nanoemulsion injection treatment group have significant differences or not.
5 results of the experiment
After continuous injection for 15 days, the average tumor weight of the normal saline control group is 3.91 g, the average tumor weight of the pure ganoderma lucidum spore oil nanoemulsion injection treatment group is 1.94 g, and the inhibition rate is 50.38%; the average tumor weight of the whole ganoderma lucidum spore oil nanoemulsion injection treatment group is 0.81 g, and the inhibition rate is 79.28%. Compared with the normal saline control group, the tumor inhibition rates of the pure ganoderma lucidum spore oil nanoemulsion injection treatment group and the whole ganoderma lucidum spore oil nanoemulsion injection treatment group are remarkably different (P is less than 0.01); the tumor inhibition rates of the pure ganoderma lucidum spore oil nanoemulsion injection treatment group and the whole ganoderma lucidum spore oil nanoemulsion injection treatment group are also significantly different (P is less than 0.01), and the tumor inhibition rate of the whole ganoderma lucidum spore oil nanoemulsion injection is about 1.6 times of the tumor inhibition rate of the pure ganoderma lucidum spore oil nanoemulsion injection.
6 conclusion and analysis
6.1 comparing the treated group and the control group of the pure ganoderma lucidum spore oil nanoemulsion injection, the tumor inhibition rate has very obvious difference, which shows that the pure ganoderma lucidum spore oil nanoemulsion injection has better tumor inhibition effect. Because the pure ganoderma spore oil is emulsified and exists in the emulsion droplets of the fat emulsion in a nano form, the activity and the efficacy are improved.
6.2 similarly, compared with the control group, the whole ganoderma lucidum spore oil nanoemulsion injection treatment group has very obvious difference in tumor inhibition rate, which shows that the whole ganoderma lucidum spore oil nanoemulsion injection has better tumor inhibition effect. Because the ganoderma lucidum spore oil is emulsified and exists in emulsion droplets of the fat emulsion in a nano form, the activity and the drug effect are greatly improved.
6.3 compared with the pure ganoderma spore oil nanoemulsion injection treatment group, the whole ganoderma spore oil nanoemulsion injection treatment group has very significant difference in tumor inhibition rate, which shows that the whole ganoderma spore oil nanoemulsion has very good tumor inhibition effect. Because the pure ganoderma lucidum spore oil contains the ganoderma lucidum acid component with the traditional anti-tumor effect and the ganoderma lucidum acid component ganoderma lucidum acid B with the anti-tumor effect in the whole ganoderma lucidum spore oil is increased by double, the activity and the drug effect of the whole ganoderma lucidum spore oil are greatly improved.
Claims (9)
1. A method for preparing whole ganoderma lucidum spore oil nanoemulsion rich in ganoderic acid is characterized by comprising the following steps:
(1) using whole ganoderma spore powder as raw material, dry-pressing and granulating, placing in supercritical CO2Extracting in an extraction kettle for supercritical CO2Extracting to obtain light yellow whole ganoderma lucidum spore oil rich in ganoderic acid;
(2) the whole ganoderma lucidum spore oil is prepared into the whole ganoderma lucidum spore oil nanoemulsion together with the auxiliary agent.
2. The method for preparing ganoderma lucidum spore oil nanoemulsion rich in ganoderic acid according to claim 1, wherein the ganoderma lucidum consists of 51% -60% of ganoderma lucidum fruit bodies and 40% -49% of ganoderma lucidum spore powder; the whole ganoderma lucidum spore powder is prepared by the following method: pulverizing Ganoderma fruiting body into superfine powder of 150 meshes or more, and breaking cell wall of Ganoderma spore powder to obtain cell wall-broken Ganoderma spore powder with cell wall breaking rate of 96% or more, and mixing the two powders to obtain the final product.
3. The method for preparing ganoderma lucidum spore oil nanoemulsion rich in ganoderic acid as claimed in claim 1, wherein the step (1) is as follows: firstly, adding 0.5-1.2% of starch or dextrin into the whole ganoderma lucidum spore powder raw material, uniformly mixing, preparing granules with the particle size of 0.1-0.3 cm by adopting a dry pressing method of a granulator in one step, selecting the granulator with the main pressure of 5-9 MPa and the lateral pressure of 0.5-0.9 MPa during granulation, and then placing the granules in a supercritical CO2And (5) extracting in an extraction kettle.
4. The method for preparing ganoderma lucidum spore oil nanoemulsion rich in ganoderic acid as claimed in claim 1 or 3, wherein the supercritical fluid is CO2The extraction temperature is 23-45 ℃, the extraction pressure is 12-34 MPa, the extraction time is 2-5 hours, and the extraction flow is 32-58 kg/h; two-stage separation is adopted, and the separation process conditions are as follows: the pressure of the primary analyzer is 9-13 MPa, and the separation temperature is 37-43 ℃; the pressure of the secondary analyzer is 4-6 Mpa, and the separation temperature is 27-33 ℃; obtaining light yellow whole ganoderma lucidum spore oil.
5. The method for preparing ganoderma lucidum spore oil nanoemulsion rich in ganoderic acid as claimed in claim 1, wherein the ganoderma lucidum spore oil and the auxiliary agent are composed of the following components in parts by weight:
5 to 20 percent of whole ganoderma lucidum spore oil,
1 to 12 percent of emulsifier,
1-8% of solubilizer,
0.0 to 2.5 percent of flavoring agent,
0 to 3 percent of isotonic regulator,
0.0 to 0.2 percent of essence,
0.00 to 0.08 percent of preservative,
0.01 to 0.04 percent of antioxidant,
the pH value regulator is a proper amount,
the balance being water.
6. The method for preparing ganoderma lucidum spore oil nanoemulsion rich in ganoderic acid according to claim 1, wherein the ganoderma lucidum spore oil nanoemulsion is a nanoemulsion for injection, and the ganoderma lucidum spore oil and the auxiliary agent consist of the following components in parts by weight:
6 to 12 percent of whole ganoderma lucidum spore oil,
2 to 10 percent of emulsifier,
1 to 5 percent of solubilizer,
0.2 to 2.5 percent of isoosmotic adjusting agent,
0.01 to 0.02 percent of antioxidant,
the pH value regulator is a proper amount,
the balance of water;
the preparation method comprises the following steps:
(1) taking the whole ganoderma lucidum spore oil, adding part of emulsifier and antioxidant into the whole ganoderma lucidum spore oil in water bath at the temperature of 60-80 ℃ under the protection of nitrogen, stirring to dissolve the emulsifier and the antioxidant, uniformly mixing, and cooling to room temperature to obtain an oil phase;
(2) mixing the rest emulsifier, solubilizer and isotonic regulator in water bath at 60-80 ℃ under the protection of nitrogen, stirring, dissolving in water for injection, shearing and stirring if necessary to dissolve and mix uniformly, and cooling to room temperature to obtain a water phase;
(3) slowly adding the oil phase into the water phase under the stirring condition, simultaneously carrying out shearing stirring for 15-60 minutes under the protection of nitrogen, then adjusting the pH to about 7.0, carrying out primary homogenization, wherein the homogenization pressure is 150-300 bar, the homogenization time is 5-10 minutes, and then adding water for injection to 100% to obtain milky primary emulsion;
(4) and (3) transferring the prepared white colostrum into a high-pressure homogenizer with the high-pressure homogenizing pressure of 800-1300 bar, homogenizing for 10-15 times, filtering by using a microporous filter membrane after homogenization, filling nitrogen, filling, sealing and sterilizing to obtain the uniform and stable nano-emulsion preparation for injection of the ganoderma lucidum spore oil.
7. The method for preparing ganoderma lucidum spore oil nanoemulsion rich in ganoderic acid according to claim 1, wherein the ganoderma lucidum spore oil nanoemulsion is in a nanoemulsion form for oral administration, and the ganoderma lucidum spore oil and the auxiliary agent consist of the following components in parts by weight:
8 to 18 percent of whole ganoderma lucidum spore oil,
3 to 8 percent of emulsifier,
1 to 6 percent of solubilizer,
0.02 to 2.5 percent of flavoring agent,
0.0 to 0.2 percent of essence,
0.00 to 0.05 percent of preservative,
the balance of water;
the preparation method comprises the following steps:
(1) taking the whole ganoderma lucidum spore oil, adding part of emulsifier and antioxidant into the whole ganoderma lucidum spore oil in water bath at the temperature of 60-80 ℃ under the protection of nitrogen, stirring to dissolve the emulsifier and the antioxidant, uniformly mixing, and cooling to room temperature to obtain an oil phase;
(2) mixing the rest emulsifier, solubilizer, flavoring agent, preservative and essence in water bath at 60-80 ℃ under the protection of nitrogen, stirring, dissolving in water for injection, shearing and stirring if necessary to dissolve and mix uniformly, and cooling to room temperature to obtain a water phase;
(3) slowly adding the oil phase into the water phase under the stirring condition, simultaneously carrying out shearing stirring for 15-60 minutes under the protection of nitrogen, then carrying out primary homogenization, wherein the homogenization pressure is 150-300 bar, the homogenization time is 5-10 minutes, and then adding water for injection to 100% to obtain milky primary emulsion;
(4) and (3) transferring the prepared white colostrum into a high-pressure homogenizer with the high-pressure homogenizing pressure of 800-1300 bar, homogenizing for 8-12 times, filtering by a microporous filter membrane after homogenization, or filling nitrogen, filling and sealing, and sterilizing to obtain the uniform and stable nanoemulsion for the whole ganoderma spore oil for oral administration.
8. The method for preparing ganoderma lucidum spore oil nanoemulsion as claimed in claim 5, 6 or 7, wherein the emulsifier is one or more of soybean lecithin, egg yolk lecithin phospholipid, liquid lecithin and/or cephalin; the solubilizer is one or two of glycerol and polyethylene glycol 400; the flavoring agent is stevioside, aspartame, glucose or fructose; the isoosmotic adjusting agent is glycerin for injection; the essence is mint essence, lemon essence, rose essence, milk essence, mint essence or a composition of various essences; the antioxidant is vitamin E; the preservative is potassium sorbate; the pH value regulator is NaOH or Na2CO3Or NaHCO3。
9. The use of the whole ganoderma lucidum spore oil nanoemulsion as defined in any one of claims 1-8 in the preparation of anti-tumor and rabbit epidemic disease improving drugs.
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| CN114672370A (en) * | 2022-03-15 | 2022-06-28 | 苏凤全 | Preparation method of ganoderma lucidum spore oil and skin care product containing ganoderma lucidum spore oil |
| CN116726071A (en) * | 2022-08-03 | 2023-09-12 | 江西鑫康健科技发展有限公司 | Prunella spike seed oil nanoemulsion with anti-tumor effect and preparation method and application thereof |
| CN116210906A (en) * | 2022-12-30 | 2023-06-06 | 广州白云山汉方现代药业有限公司 | Preparation method and application of ganoderma lucidum spore oil oral liquid for improving taste |
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