阅读说明：本技术 一种石榴皮配方颗粒半仿生提取制备方法 (Semi-bionic extraction preparation method of pomegranate peel formula particles ) 是由 王京龙 郑丹丹 于 2020-06-01 设计创作，主要内容包括：本发明属于半仿生提取技术领域,公开了一种石榴皮配方颗粒半仿生提取制备方法,首次采用U<Sub>9</Sub>(9<Sup>1</Sup>×3<Sup>3</Sup>)均匀设计表,以鞣花酸、没食子酸、总酚、干浸膏为测定指标,采用层次分析法(AHP)、指标相关性权重确定法(CRITIC)、AHP-CRITIC混合加权法,并将各指标值进行标准化处理,综合评价,预测石榴皮半仿生最佳工艺参数。本发明优化条件为：三煎用水的pH值依次为6.0、7.4、9.0,煎煮总时间为4h,采用均匀设计优选石榴皮的半仿生提取工艺参数合理,工艺验证可行。利用本发明的半仿生提取法制备得到的石榴皮配方颗粒,有效成分含量得到了大幅度的提高,生物利用度更高,更适合人体吸收。(The invention belongs to the technical field of semi-bionic extraction, and discloses a semi-bionic extraction preparation method of pomegranate peel formula particles, which adopts U for the first time 9 (9 1 ×3 3 ) Uniformly designing table with ellagic acid, gallic acid, total phenols and dry extract as raw materialsAnd measuring indexes, namely adopting an Analytic Hierarchy Process (AHP), an index correlation weight determination method (CRITIC) and an AHP-CRITIC mixed weighting method, standardizing all index values, comprehensively evaluating, and predicting the semi-bionic optimal process parameters of the pomegranate rind. The optimization conditions of the invention are as follows: the pH value of water for three-decoction is 6.0, 7.4 and 9.0 in sequence, the total time for decoction is 4 hours, the semi-bionic extraction process parameters for selecting pomegranate rind by uniform design are reasonable, and the process verification is feasible. The pomegranate rind formula particles prepared by the semi-bionic extraction method have the advantages that the content of effective components is greatly improved, the bioavailability is higher, and the pomegranate rind formula particles are more suitable for being absorbed by human bodies.)

1. The semi-bionic extraction preparation method of the pomegranate bark formula particles is characterized by comprising the following steps of:

step one, adopting U₉(9¹×3³) Uniformly designing a table, taking ellagic acid, gallic acid, total phenol and dry extract as measurement indexes, adopting an analytic hierarchy process AHP, an index correlation weight determination method CRITIC and an AHP-CRITIC mixed weighting method, standardizing all index values, comprehensively evaluating, and optimizing the optimal semi-bionic process parameters of the pomegranate rind;

step two, according to optimized conditions, taking pomegranate rind, crushing, soaking, adding water, adjusting the pH value, heating and extracting, filtering and centrifuging;

adding water into the extracted dregs, adjusting the pH value, heating and extracting, filtering and centrifuging;

adding water into the extracted dregs, adjusting the pH value, heating and extracting, filtering and centrifuging;

step five, combining the extracted filtrates, concentrating to obtain clear paste, adding the available pharmaceutical excipients, and spray drying to obtain extract powder;

and step six, mixing the extract powder with auxiliary materials, and performing dry granulation.

2. The method for preparing the pomegranate rind formula particle through semi-bionic extraction according to claim 1, wherein in the first step, the process conditions are as follows: performing semi-bionic extraction under the conditions of the same medicinal material granularity, decocting water consumption and filtering extraction conditions by using the pH value of water for decocting for three times and the total decocting time, and uniformly designing a table U₉(9¹×3³) And arranging a spotting experiment.

3. The method for semi-biomimetic extraction and preparation of pomegranate rind formula particles as claimed in claim 1, wherein in the first step, the preparation method of the sample solution comprises: weighing 15g of processed pomegranate peel powder, and continuously performing reflux extraction under normal pressure, wherein the amount of the solvent is 10, 8 and 8 times of the weight of the decoction pieces respectively; soaking for 30 min; and (3) sequentially taking the three times of decoction and extraction at a time ratio h of 2:1:1, filtering the three times of extraction solutions by using gauze, centrifuging at 4000r/min for 20min, combining the supernatants, placing the combined supernatants in a 500mL volumetric flask, and adding water to scale marks to obtain No. 1-9 sample solution.

4. The semi-bionic extraction preparation method of the pomegranate rind formula particles of claim 1, wherein in the second step, the chromatographic conditions are set as follows: the chromatographic column is Hypersil ODS C₁₈× 250mm with the thickness of 4.6mm and the thickness of 5 microns, the mobile phase is methanol A, acetonitrile B and 0.4 percent phosphoric acid solution C, the column temperature is 30 ℃, the gradient elution is carried out for 0 to 11min, the ratio of A to B to C is 1.5: 1.5: 97, the detection wavelength is 270nm, the flow rate is 0.8mL/min, the ratio of A to B to C is 15: 15: 70, the detection wavelength is 254nm, and the flow rate is 1 mL/min.

5. The semi-bionic extraction preparation method of the pomegranate rind formula particle of claim 1, wherein in the second step, the preparation method of the mixed reference solution comprises the following steps:

(1) precisely weighing 2mg of gallic acid standard, placing in a 10mL volumetric flask, adding a proper amount of water for dissolving, fixing the volume, and shaking up to obtain 0.2mg/mL of gallic acid reference solution;

(2) precisely weighing 2.62mg of ellagic acid standard substance, placing in a 10mL volumetric flask, adding dimethyl sulfoxide DMSO, dissolving, fixing volume, and shaking to obtain 0.262mg/mL of ellagic acid reference substance solution;

(3) precisely sucking 5mL of each of the gallic acid reference solution and the ellagic acid reference solution, vortexing, shaking, and mixing to obtain a mixed reference solution, wherein the gallic acid content is 0.1mg/mL, and the ellagic acid content is 0.131 mg/mL.

6. The semi-bionic extraction preparation method of the pomegranate rind formula particles as claimed in claim 1, wherein in the step two, the preparation method of the test solution comprises the following steps: precisely measuring 10mL of each sample solution in a 25mL volumetric flask, adding water to a constant volume, and shaking up to obtain a test solution A₁～A₉。

7. The semi-bionic extraction preparation method of the pomegranate rind formula particles as claimed in claim 1, wherein in the second step, the standard curve is drawn by a method comprising the following steps: precisely sucking 2 muL, 5 muL, 10 muL, 15 muL and 20 muL of the mixed reference substance solution of gallic acid and ellagic acid respectively, respectively injecting samples, measuring peak areas at the wavelength of 270nm, respectively taking the content mug of ellagic acid and gallic acid as abscissa and the peak areas of the two components as ordinate, and drawing a standard curve;

according to the optimized conditions, crushing pomegranate rind, soaking, adding water, adjusting the pH value, heating, extracting, filtering and centrifuging;

adding water into the extracted dregs, adjusting the pH value, heating and extracting, filtering and centrifuging;

and step four, adding water into the extracted dregs, adjusting the pH value, heating and extracting, filtering and centrifuging.

8. The semi-bionic extraction preparation method of the pomegranate rind formula particles as claimed in claim 1, wherein in the second step, the precision test is as follows: and continuously feeding sample into the mixed control solution for 6 times, wherein each time is 20 mu L, recording the peak areas of the ellagic acid and the gallic acid, and calculating to obtain the peak areas RSD of the ellagic acid and the gallic acid which are respectively 0.67% and 1.48%.

9. The semi-bionic extraction preparation method of the pomegranate rind formula particles as claimed in claim 1, wherein the stability test is as follows: precisely sucking 20 mu L of each sample solution No. 1, respectively injecting samples in 1, 2, 6, 12, 24 and 48h, recording the peak areas of ellagic acid and gallic acid, calculating to obtain peak areas RSD of ellagic acid and gallic acid of 1.27% and 1.96%, respectively, and stabilizing the sample solution in 48 h;

precisely sucking 20 mu L of sample solution No. 2, continuously feeding samples for 6 times, recording the peak areas of ellagic acid and gallic acid, and calculating to obtain gallic acid and ellagic acid peak areas RSD of 2.31% and 2.49%, respectively;

in the second step, the calculation method of the contents of the gallic acid and the ellagic acidThe method comprises the following steps: get A₁～A₉Respectively injecting sample solution of sample No. 20 μ L, recording peak areas of gallic acid and ellagic acid, and calculating content;

in the third step, the preparation method of the gallic acid standard solution comprises the following steps: accurately weighing 100mg of gallic acid, dissolving with appropriate amount of anhydrous ethanol, and diluting to 100mL to obtain 1mg/mL gallic acid standard solution;

in the third step, the standard curve is drawn by the following method:

1) precisely sucking gallic acid standard solution 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2mL respectively, placing in 25mL volumetric flask, adding ferrous tartrate solution 6mL respectively, shaking, and standing for 10 min;

2) then phosphate buffer solution with pH of 7.5 is added to the constant volume until the scale mark is shaken up, and the mixture is kept stand for 30min for color development;

3) respectively measuring absorbance by using an ultraviolet spectrophotometer under the wavelength of 540nm by using a 0 tube as a blank, and drawing a standard curve by using the absorbance A as a vertical coordinate and the concentration mg/mL as a horizontal coordinate;

in the third step, the method for calculating the total polyphenol content comprises the following steps: respectively and precisely measuring A₁～A₉1.0mL of each sample solution is used for measuring the total polyphenol content;

in the fourth step, the method for measuring the yield of the dry extract comprises the following steps: precisely measuring 20mL of No. 1-9 sample solution, placing in an evaporating dish which is dried to constant weight, evaporating in a water bath, placing in an oven, drying at 105 ℃ for 3h, placing in a dryer, cooling to room temperature for 30min, weighing, and calculating the yield of dry extract.

10. The semi-bionic extraction preparation method of the pomegranate rind formula particles of claim 1, wherein the fifth step comprises; mixing the filtrates, concentrating to obtain fluid extract, adding medicinal adjuvants, and spray drying to obtain extract powder. Semi-bionic pomegranate rind extracting solution is concentrated in vacuum to form clear paste with the relative density of 1.05-1.10, and the clear paste is measured at 65 ℃ and spray-dried to prepare pomegranate rind extract powder;

the sixth step comprises: and (3) mixing the extract powder with auxiliary materials, performing dry granulation, adding 0.03-0.06 times of ethanol, 0.03-0.06 times of sodium carboxymethyl starch and 0.03-0.06 times of povidone k30 into the pomegranate bark extract powder, uniformly mixing, and performing dry granulation to obtain the pomegranate bark formula granules.

Technical Field

The invention belongs to the technical field of semi-bionic extraction, and particularly relates to a semi-bionic extraction preparation method of pomegranate bark formula particles.

Background

Currently, pomegranate rind is the dried pericarp of pomegranate. Punica granatum (Punica grantum L.) belongs to the family Punicaceae, genus Punica, such as deciduous shrub or small tree. The root, leaf, flower, fruit and seed of pomegranate contain various physiological active ingredients, including polyphenol compounds, flavonoid compounds, alkaloid, organic acid and the like, and the polyphenol compounds are mainly concentrated in pomegranate bark.

In 2015, in the Chinese pharmacopoeia, the pomegranate rind is collected as a traditional Chinese medicine, is slightly fragrant, bitter and astringent in taste, is warm in nature after being processed and fried to charcoal, and enters large intestine channels, and has the effects of astringing to stop diarrhea, stopping bleeding, expelling parasites and the like. The pericarpium Granati polyphenol has antioxidant, antibacterial, blood lipid reducing, and anticancer effects. Ellagic acid is a main natural polyphenol component in pomegranate rind, and has biological activity functions of resisting oxidation, canceration, bacteria, mutation and the like.

The semi-bionic extraction method (SBE method) combines the whole medicine research method and the molecular medicine research method from the perspective of biological pharmacy, simulates the absorption and distribution process of oral medicines in the gastrointestinal tract, and is an extraction technology established for the traditional Chinese medicine and the compound thereof which are administrated through the digestive tract. The semi-bionic extraction method adopts acidic water and alkaline water with selected pH value to extract active mixture with high index components. The extraction method does not use organic solvents such as acetone and ethanol, reduces cost in production process, and can extract and retain more effective components. The method controls the internal quality of the Chinese medicinal preparation by using the content of one or more index components, embodies the comprehensive action characteristics of the traditional Chinese medicine clinical medication, and conforms to the absorption and distribution principle of oral medicaments through gastrointestinal tracts. However, no report has been found on the prior art of combining the semi-bionic extraction method with the extraction of polyphenols from pomegranate rind.

The traditional Chinese medicine formula particle is a particle prepared by extracting and concentrating single traditional Chinese medicine decoction pieces and used for a traditional Chinese medicine clinical formula. The traditional Chinese medicine concentrated granule is called as single traditional Chinese medicine concentrated granule in China, and the trade name and folk name of the traditional Chinese medicine concentrated granule are non-decoction traditional Chinese medicine decoction pieces, new decoction pieces, refined decoction pieces, beverage type decoction pieces, scientific traditional Chinese medicine and the like. The novel prescription medicine is prepared by processing traditional Chinese medicine decoction pieces serving as raw materials through production processes of extraction, separation, concentration, drying, granulation, packaging and the like, and has the advantages of unified specification, unified dosage and unified quality standard. The formula granule has the advantages of convenient use, small dosage and quick action, has the advantages of addition and subtraction according to symptoms compared with the finished formula granule, and has wide popularization prospect.

In summary, the problems of the prior art are as follows: the traditional water decoction extraction of the pomegranate rind has low extraction rate of small molecular active ingredients and low extract amount, thereby causing weak drug effect and high cost of the prepared formula particles.

The difficulty of solving the technical problems is as follows: under the condition of ensuring the characteristics of the original traditional Chinese medicine decoction pieces, the extract yield is improved, the content of small molecular active ingredients is improved, and the effect of using the medicine is improved.

The significance of solving the technical problems is as follows: the pomegranate rind is extracted by adopting a semi-bionic extraction method, and the effective substances of the pomegranate rind can be more efficiently extracted by simulating the oral administration process of a human body and taking the aqueous solution with selected pH as an extraction solvent. The extract yield is increased, and the degradation of macromolecular tannins such as punicalagin and the like into micromolecular phenols with strong pharmacological activity such as ellagic acid, gallic acid and the like is facilitated by simulating the acid-base environment of the stomach and intestine, so that the absorption by the organism is facilitated and the function is played. The invention firstly takes the total polyphenol content, the ellagic acid content, the gallic acid content and the dry extract yield as evaluation indexes, optimizes the semi-bionic extraction conditions in the pomegranate rind by adopting a uniform design method, and then prepares the pomegranate rind formula particles.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a semi-bionic extraction preparation method of pomegranate rind formula particles, aiming at optimizing the optimal process conditions of the semi-bionic extraction of pomegranate rind.

The invention is realized in such a way that a semi-bionic extraction preparation method of pomegranate bark formula particles comprises the following steps:

step one, adopting U₉(9¹×3³) Uniformly designing a table, taking ellagic acid, gallic acid, total phenol and dry extract as measurement indexes, adopting an analytic hierarchy process AHP, an index correlation weight determination method CRITIC and an AHP-CRITIC mixed weighting method, standardizing all index values, comprehensively evaluating, and optimizing the optimal semi-bionic process parameters of the pomegranate rind;

step two, according to optimized conditions, taking pomegranate rind, crushing, soaking for 30min, adding 10 times of water, adjusting the pH value to 2.0-6.0, heating, extracting, filtering and centrifuging;

adding 8 times of water into the extracted medicine residues, adjusting the pH value to 6.5-7.5, heating, extracting, filtering and centrifuging;

and step four, adding 8 times of water into the extracted medicine residues, adjusting the pH value to 8.0-9.0, heating and extracting, filtering and centrifuging.

Step five, combining the 3 times of extraction filtrates, concentrating to obtain clear paste, adding the available pharmaceutical excipients, and spray drying to obtain extract powder.

And step six, mixing the extract powder with auxiliary materials, and performing dry granulation.

Further, in the step one, the process conditions are as follows: performing semi-bionic extraction under the conditions of the same medicinal material granularity, decocting water consumption and filtering extraction conditions by using the pH value of water for decocting for three times and the total decocting time, and uniformly designing a table U₉(9¹×3³) And arranging a spotting experiment.

Further, in the first step, the preparation method of the sample solution comprises: weighing 15g of processed pomegranate peel powder, and continuously performing reflux extraction under normal pressure, wherein the amount of the solvent is 10, 8 and 8 times of the weight of the decoction pieces respectively; soaking for 30 min; and (3) sequentially decocting for three times at a ratio of 2:1:1, filtering the three extracting solutions by using gauze, centrifuging for 20min at 4000r/min, combining the supernate, placing the supernate in a 500mL volumetric flask, and adding water until the scale marks, thus obtaining a No. 1-9 sample solution.

Further, in the second step, the chromatographic conditions are set as follows: the chromatographic column is Hypersil ODSC₁₈4.6mm × 250mm, 5 μm, mobile phase of methanol A, acetonitrile B, 0.4% phosphoric acid solution C, columnTemperature 30 ℃, gradient elution: 0-11 min, A: b: c is 1.5: 1.5: 97, the detection wavelength is 270nm, and the flow rate is 0.8 mL/min; 12-25 min, A: b: c is 15: 15: 70, the detection wavelength is 254nm, and the flow rate is 1 mL/min.

Further, in the second step, the preparation method of the mixed reference solution comprises:

(1) precisely weighing 2mg of gallic acid standard, placing in a 10mL volumetric flask, adding a proper amount of water for dissolving, fixing the volume, and shaking up to obtain 0.2mg/mL of gallic acid reference solution;

(2) precisely weighing 2.62mg of ellagic acid standard substance, placing in a 10mL volumetric flask, adding appropriate amount of dimethyl sulfoxide DMSO, dissolving, diluting to constant volume, and shaking to obtain ellagic acid reference substance solution 0.262 mg/mL;

(3) precisely sucking 5mL of each of the gallic acid reference solution and the ellagic acid reference solution, vortexing, shaking, and mixing to obtain a mixed reference solution, wherein the gallic acid content is 0.1mg/mL, and the ellagic acid content is 0.131 mg/mL.

Further, in the second step, the preparation method of the test solution comprises: precisely measuring 10mL of each sample solution in a 25mL volumetric flask, adding water to a constant volume, and shaking up to obtain a test solution A₁～A₉。

Further, in the second step, the standard curve is drawn by the following method: precisely sucking 2 muL, 5 muL, 10 muL, 15 muL and 20 muL of the mixed reference substance solution of gallic acid and ellagic acid respectively, respectively injecting samples, measuring peak areas at the wavelength of 270nm, respectively taking the content of ellagic acid and gallic acid as abscissa and the peak areas of the two components as ordinate, and drawing a standard curve.

Further, in the second step, the precision test is as follows: and continuously feeding sample into the mixed control solution for 6 times, wherein each time is 20 mu L, recording the peak areas of the ellagic acid and the gallic acid, and calculating to obtain the peak areas RSD of the ellagic acid and the gallic acid which are respectively 0.67% and 1.48%.

Further, the stability test is: precisely sucking 20 mu L of sample solution No. 1, respectively injecting samples in 1, 2, 6, 12, 24 and 48h, recording the peak areas of ellagic acid and gallic acid, calculating to obtain peak areas RSD of ellagic acid and gallic acid of 1.27% and 1.96%, respectively, and making the sample solution stable in 48 h.

Further, the repeatability test is as follows: precisely sucking 20 mu L of sample solution No. 2, continuously feeding samples for 6 times, recording the peak areas of ellagic acid and gallic acid, and calculating to obtain gallic acid and ellagic acid peak areas RSD of 2.31% and 2.49%, respectively;

in the second step, the calculation method of the content of the gallic acid and the ellagic acid comprises the following steps: get A₁～A₉Respectively injecting sample solution of sample No. 20 μ L, recording peak areas of gallic acid and ellagic acid, and calculating content;

in the third step, the preparation method of the gallic acid standard solution comprises the following steps: accurately weighing 100mg of gallic acid, dissolving with appropriate amount of anhydrous ethanol, and diluting to 100mL to obtain 1mg/mL gallic acid standard solution;

in the third step, the standard curve is drawn by the following method:

1) precisely sucking gallic acid standard solution 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2mL respectively, placing in 25mL volumetric flask, adding ferrous tartrate solution 6mL respectively, shaking, and standing for 10 min;

2) then phosphate buffer solution with pH of 7.5 is added to the constant volume until the scale mark is shaken up, and the mixture is kept stand for 30min for color development;

3) respectively measuring absorbance by using an ultraviolet spectrophotometer under the wavelength of 540nm by using a 0 tube as a blank, and drawing a standard curve by using the absorbance A as a vertical coordinate and the concentration mg/mL as a horizontal coordinate;

in the third step, the method for calculating the total polyphenol content comprises the following steps: respectively and precisely measuring A₁～A₉1.0mL of each sample solution is used for measuring the total polyphenol content;

in the fourth step, the method for measuring the yield of the dry extract comprises the following steps: precisely measuring 20mL of No. 1-9 sample solution, placing in an evaporating dish which is dried to constant weight, evaporating in a water bath, placing in an oven, drying at 105 ℃ for 3h, placing in a dryer, cooling to room temperature for 30min, weighing, and calculating the yield of dry extract.

Further s, the fifth step comprises; mixing the filtrates, concentrating to obtain fluid extract, adding medicinal adjuvants, and spray drying to obtain extract powder. Semi-bionic pomegranate rind extracting solution is concentrated in vacuum to form clear paste with the relative density of 1.05-1.10, and the clear paste is measured at 65 ℃ and spray-dried to prepare pomegranate rind extract powder;

the sixth step comprises: and (3) mixing the extract powder with auxiliary materials, performing dry granulation, adding 0.03-0.06 times of ethanol, 0.03-0.06 times of sodium carboxymethyl starch and 0.03-0.06 times of povidone k30 into the pomegranate bark extract powder, uniformly mixing, and performing dry granulation to obtain the pomegranate bark formula granules.

In summary, the advantages and positive effects of the invention are: the semi-bionic extraction preparation method of pomegranate rind formula particles provided by the invention takes ellagic acid content, gallic acid content, total polyphenol content and extract yield as evaluation indexes, and adopts uniform design tests to carry out process optimization of a semi-bionic extraction method (SBE method for short). The invention adopts a semi-bionic extraction method to extract the polyphenols in the pomegranate rind, and adopts aqueous solution with selected pH as an extraction solvent by simulating the oral administration process of a human body, so that the use of an organic solvent can be avoided, the solvent residue and the physical injury to operators are reduced, and the polyphenols in the pomegranate rind can be extracted more efficiently. The result shows that the optimal process conditions for the semi-bionic extraction of the pomegranate rind are as follows: the pH value of water for three-decoction is 6.0, 7.4 and 9.0 in sequence, the total time of decoction is 4 hours, the semi-bionic extraction process parameters for selecting pomegranate rind by adopting uniform design are reasonable, the process verification is feasible, and theoretical support can be provided for extraction of the pomegranate rind.

The invention adopts a mobile phase system of methanol-acetonitrile-phosphoric acid water to carry out gradient elution, adopts variable wavelength detection, sets different wavelengths within a certain time range according to the difference of retention time of two components of gallic acid and ellagic acid to measure the contents of the gallic acid and the ellagic acid, and improves the detection sensitivity. The chromatographic peak separation degree of the gallic acid and the ellagic acid is proper, the operation is simple and convenient, and the result is accurate and reliable.

Drawings

Fig. 1 is a flow chart of a semi-bionic extraction preparation method of pomegranate rind formula particles provided by the embodiment of the invention.

FIG. 2 is an HPLC plot of a control solution provided in an embodiment of the invention.

FIG. 3 is an HPLC chart of a sample solution provided in an embodiment of the present invention.

Fig. 4 is a graph of gallic acid standard curve provided by the embodiment of the present invention.

FIG. 5 is a graph of the standard ellagic acid provided by an example of the present invention.

Fig. 6 is a standard graph of total polyphenols provided by an embodiment of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Aiming at the problems in the prior art, the invention provides a semi-bionic extraction preparation method of pomegranate rind formula particles, and the invention is described in detail with reference to the accompanying drawings.

As shown in fig. 1, the semi-bionic extraction preparation method of pomegranate rind formula particles provided by the embodiment of the invention comprises the following steps:

s101: by U₉(9¹×3³) Uniformly designing a table, taking ellagic acid, gallic acid, total phenol and dry extract as measurement indexes, adopting an analytic hierarchy process AHP, an index correlation weight determination method CRITIC and an AHP-CRITIC mixed weighting method, standardizing all index values, comprehensively evaluating, and optimizing the optimal semi-bionic process parameters of the pomegranate rind;

s102: according to optimized conditions, taking pomegranate rind, crushing, soaking for 30min, adding 10 times of water, adjusting the pH value to 2.0-6.0, heating, extracting, filtering and centrifuging;

s103: adding 8 times of water into the extracted medicine residues, adjusting the pH value to 6.5-7.5, heating and extracting, filtering and centrifuging;

s104: adding 8 times of water into the extracted dregs, adjusting the pH value to 8.0-9.0, heating and extracting, filtering and centrifuging.

S105: mixing the filtrates, concentrating to obtain fluid extract, adding medicinal adjuvants, and spray drying to obtain extract powder.

S106: mixing the extract powder with adjuvants, and granulating by dry method.

The semi-bionic extraction preparation method of the pomegranate bark formula particles provided by the embodiment of the invention specifically comprises the following steps:

step one, adopting U₉(9¹×3³) Uniformly designing a table, taking ellagic acid, gallic acid, total phenol and dry extract as measurement indexes, adopting an analytic hierarchy process AHP, an index correlation weight determination method CRITIC and an AHP-CRITIC mixed weighting method, standardizing all index values, comprehensively evaluating, and optimizing the optimal semi-bionic process parameters of the pomegranate rind;

step two, according to optimized conditions, taking pomegranate rind, crushing, soaking for 30min, adding 10 times of water, adjusting the pH value to 2.0-6.0, heating, extracting, filtering and centrifuging;

adding 8 times of water into the extracted medicine residues, adjusting the pH value to 6.5-7.5, heating, extracting, filtering and centrifuging;

and step four, adding 8 times of water into the extracted medicine residues, adjusting the pH value to 8.0-9.0, heating and extracting, filtering and centrifuging.

Step five, combining the 3 times of extraction filtrates, concentrating to obtain clear paste, adding the available pharmaceutical excipients, and spray drying to obtain extract powder. Semi-bionic pomegranate rind extracting solution is concentrated in vacuum to form clear paste with the relative density of 1.05-1.10 (measured at 65 ℃), and spray drying is carried out to prepare pomegranate rind extract powder.

And step six, mixing the extract powder with auxiliary materials, performing dry granulation, adding 0.03-0.06 times of ethanol, 0.03-0.06 times of sodium carboxymethyl starch and 0.03-0.06 times of povidone k30 into the pomegranate bark extract powder, uniformly mixing, and performing dry granulation to obtain the pomegranate bark formula granules.

The technical solution of the present invention will be further described with reference to the following examples.

1. Apparatus and materials

1.1 materials and reagents

Materials and reagents used in the experiment are shown in table 1. The pericarpium Granati is dry peel of Punicaceae Punicandratauml, dried, pulverized, and sieved with 10-20 mesh sieve.

TABLE 1 materials and reagents

1.2 instruments and devices

The instruments and equipment used in the experiment are shown in Table 2.

TABLE 2 instruments and apparatus

2. Experimental methods

2.1 design of the experiment

In order to ensure comparability and repeatability of the experiment, and to determine main factors to be investigated by looking up documents, the technological conditions selected in the experiment are the pH value of water for decocting three times and the total time of decoction, semi-bionic extraction is carried out on the premise that the extraction conditions such as granularity of medicinal materials, water consumption for decocting, filtration and the like are the same, and a uniform design table U is adopted₉(9¹×3³) The spotting experiments were arranged and are shown in table 3.

TABLE 3U₉(9¹×3³) Experimental design table

Note: the extraction time ratio of the three decoctions is 2:1: 1.

2.2 preparation of sample solutions

The experiments were spotted as in Table 3. Weighing 15g of processed pomegranate peel powder, continuously performing reflux extraction under normal pressure (the amount of a solvent is respectively 10, 8 and 8 times of the weight of decoction pieces, soaking for 30min, and the time ratio (h) of three times of decoction is 2:1:1 in sequence), respectively filtering three times of extract with gauze, centrifuging (4000r/min, 20min), combining supernatants, placing in a 500mL volumetric flask, and adding water to scale marks to obtain No. 1-9 sample solution.

2.3 assay of ellagic acid and Gallic acid

2.3.1 chromatographic conditions

The chromatographic column is Hypersil ODSC₁₈(4.6mm × 250mm, 5 μm), the mobile phase is methanol (A), acetonitrile (B) and 0.4% phosphoric acid solution (C), the column temperature is 30 ℃, the gradient elution is 0-11 min, the ratio of A to B to C is 1.5: 1.5: 97, the detection wavelength is 270nm, the flow rate is 0.8mL/min, the ratio of A to B to C is 15: 15: 70, the detection wavelength is 254nm, and the flow rate is 1 mL/min.

2.3.2 preparation of Mixed control solutions

Precisely weighing 2mg of gallic acid standard, placing in a 10mL volumetric flask, adding appropriate amount of water for dissolving, diluting to desired volume, and shaking to obtain gallic acid reference solution (0.2 mg/mL).

Precisely weighing 2.62mg of ellagic acid standard substance, placing in a 10mL volumetric flask, adding appropriate amount of dimethyl sulfoxide (DMSO), dissolving, diluting to desired volume, and shaking to obtain ellagic acid reference substance solution (0.262 mg/mL).

Precisely sucking 5mL of each of the gallic acid control solution and ellagic acid control solution, and vortexing, shaking, and mixing to obtain mixed control solution (gallic acid 0.1mg/mL, ellagic acid 0.131 mg/mL).

2.3.3 preparation of test solutions

Precisely measuring 10mL of each sample solution in a 25mL volumetric flask, adding water to a constant volume, and shaking up to obtain a test solution A₁～A₉。

2.3.4 drawing of Standard Curve

Precisely sucking 2 muL, 5 muL, 10 muL, 15 muL and 20 muL of mixed reference substance solution of gallic acid and ellagic acid respectively, respectively injecting samples, measuring peak areas at a wavelength of 270nm, respectively taking ellagic acid and gallic acid contents (mug) as abscissa and peak areas of two components as ordinate, and drawing a standard curve.

2.3.5 precision test

And continuously feeding sample into the mixed reference substance solution for 6 times, wherein the sample feeding amount is 20 mu L each time, recording the peak areas of the ellagic acid and the gallic acid, and calculating to obtain the peak areas RSD of the ellagic acid and the gallic acid which are respectively 0.67% and 1.48%, so that the precision of the instrument is good.

2.3.6 stability test

Precisely sucking 20 mu L of sample solution No. 1, respectively injecting samples in 1, 2, 6, 12, 24 and 48h, recording the peak areas of ellagic acid and gallic acid, calculating to obtain peak areas RSD of ellagic acid and gallic acid of 1.27% and 1.96%, respectively, and making the sample solution stable in 48 h.

2.3.7 repeatability test

Precisely sucking 20 μ L of sample solution No. 2, continuously sampling for 6 times, recording the peak areas of ellagic acid and gallic acid, and calculating to obtain gallic acid and ellagic acid peak areas RSD of 2.31% and 2.49%, respectively.

2.3.8 gallic acid and ellagic acid content

Get A₁～A₉And (4) feeding 20 mu L of sample solution to be tested, recording peak areas of gallic acid and ellagic acid, and calculating the content.

2.4 Total Polyphenol assay

2.4.1 preparation of Gallic acid Standard solution

Accurately weighing 100mg of gallic acid, dissolving with appropriate amount of anhydrous ethanol, and diluting to 100mL to obtain 1mg/mL gallic acid standard solution.

2.4.2 drawing of Standard Curve

Precisely sucking gallic acid standard solution 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2mL respectively, placing in 25mL volumetric flask, adding ferrous tartrate solution 6mL respectively, shaking, and standing for 10 min; then phosphate buffer solution with pH7.5 is added to the constant volume until the scale mark is shaken up, and the mixture is kept stand for 30min for color development. And (3) taking a 0 tube as a blank, respectively measuring the absorbance at the wavelength of 540nm by using an ultraviolet spectrophotometer, and drawing a standard curve by taking the absorbance A as a vertical coordinate and the concentration mg/mL as a horizontal coordinate.

2.4.3 Total Polyphenol content

Respectively and precisely measuring A under the item of' 2.3.3₁～A₉The total polyphenol content of each 1.0mL of the test sample solution is determined by the method of 2.4.2.

2.5 determination of Dry extract yield

Precisely measuring 20mL of No. 1-9 sample solution, placing in an evaporating dish which is dried to constant weight, evaporating in a water bath, placing in an oven, drying at 105 ℃ for 3h, placing in a dryer, cooling to room temperature for 30min, weighing, and calculating the yield of dry extract.

3. Results and analysis

3.1 Standard Curve

The HPLC chart of the control solution is shown in FIG. 2, and that of the test solution is shown in FIG. 3. In fig. 2 and 3, a reference numeral A is a gallic acid reference substance, and a reference numeral B is an ellagic acid reference substance. The separation of gallic acid, ellagic acid and other components can reach baseline, and the separation effect of gallic acid and ellagic acid is good under the chromatographic condition of the experiment.

The gallic acid standard curve is shown in FIG. 4. From the data, the regression equation between gallic acid content (μ g) and peak area (a): 3635.6X-3.2146, R²0.9996. The content of the gallic acid reference substance has a good linear relation within the range of 0.2-2.0 mu g as can be seen by a regression equation and a standard curve.

The standard curve of ellagic acid is shown in fig. 5. From the data, the regression equation between the ellagic acid content (μ g) and the peak area (a) is obtained: 10380X +750.5, R²0.9997. The regression equation and the standard curve show that the content of the ellagic acid reference substance presents a good linear relation within the range of 0.262-2.620 mu g.

The total polyphenol standard curve is shown in figure 6. From the data, the regression equation between gallic acid concentration (mg/mL) and absorbance (A): 14.336X +0.0009, R²0.9994. The regression equation and the standard curve show that the linear relation of the concentration of the gallic acid reference substance is good in the range of 0.008-0.048 mg/mL.

TABLE 4 index pairwise comparison priority decision matrix

3.2.2CRITIC method for determining weighting coefficients

The CRITIC method is a calculation method for objectively reflecting index weight, reflects the contrast strength and conflict among the investigation indexes by linear interpolation processing, and is more objective and scientific than a subjective scoring method. According to the formula: the measured data of the uniform design test in table 1 were entered as (measured value-minimum)/(maximum value-minimum) index componentLinear interpolation processing, calculating the variability(s) between indices according to the SPSS17.0 software_i) Conflict (b)_i) Composite weight (c)_i) And weight (ω)_i) The results are shown in Table 5.

From the results in table 5, the weight coefficients of total phenol, ellagic acid, gallic acid, and dry extract were 26.07%, 23.47%, 28.67%, and 21.79%, respectively.

TABLE 5 index component CRITIC method treatment results

3.2.3 hybrid weighting method for determining weights

According to the difference of main medicinal components and pharmacological action in pomegranate rind, the AHP method and the CRITIC method respectively conduct empowerment discussion on 4 indexes of total phenol, ellagic acid, gallic acid and dry extract yield from subjective and objective aspects. The AHP method adopts pairwise comparison and judgment matrix processing, so that subjective weighting is more objective, and the difference of contribution degrees of all indexes to the overall extraction effect is reflected. The CRITIC method balances the influence of variability and conflict of index measurement data on the weighting, so that the weighting is more objective. The AHP-CRITIC method combines 2 empowerment methods, has the advantages of the AHP method and the CRITIC method, and enables the evaluation result to be more scientific and reasonable. Calculating the formula: the integrated weight ω_{Synthesis ij}＝ω_AHP-ijω_CRITIC-ij/∑ω_AHP-ijω_CRITIC-ij(where ω is_AHP-ijWeight value, ω, representing AHP process_CRITIC-ijRepresenting the weight value processed by the circic method, i representing the ith factor and j representing the jth sample). The AHP-CRITIC method calculates the comprehensive weight value of each index as follows: the weight coefficients of total phenol, ellagic acid, gallic acid and dry extract are 46.68%, 28.51%, 14.44% and 10.37%, respectively.

Comparison of 3.2.43 weight methods

The measured values of the indices in Table 6 were calculated by using 3 kinds of weighting factors, and the obtained data were subjected to correlation analysis by SPSS17.0 software. As a result, the correlation coefficient between the AHP method and the ACH-CRITIC method is 0.997, the correlation coefficient between the CRITIC method and the ACH-CRITIC method is 0.889, the correlation coefficient between the AHP method and the CRITIC method is 0.908, and the correlation of the AHP method and the CRITIC method is significant (P < 0.05). However, comparing the weight coefficients of the AHP method and the CRITIC method, the correlation coefficient is-0.174, and the correlation between the AHP method and the CRITIC method is not significant (P ═ 0.826 > 0.05), which indicates that the information of the two methods assigned responses has no superposition and is in negative correlation to a smaller extent. The data show that the actual situation of the extraction effect is comprehensively reflected in the aspects of subjectivity and objectivity after comprehensive weighting processing.

3.3 determination of extraction Process

Measuring the data of each index under the items of 2.3 to 2.4 according to a formulaStandardized to x 'in the formula'_i.jIs a normalized value, x_i.jIs the content of the component j in the sample liquid i,

is the average value, s, of the component j in each sample liquid i_jThe value of the overall evaluation index Y was calculated from the overall weight value calculated under the term "2.6", Y ═ total phenol × 46.68% + ellagic acid × 28.51.51% + gallic acid × 14.44% + dry extract × 10.37.37%.

And (3) carrying out quadratic polynomial stepwise regression treatment on the standardized values and the comprehensive evaluation Y values of the index components in the table 6 by using SPSS17.0 software to obtain a regression equation:

Y＝-3.428+0.079A*D+0.034C²and P is 0.006 (< 0.01), the model has statistical significance, the pH value of the first decoction (A), the pH value of the third decoction (C) and the extraction time (D) have obvious influence on the extraction effect, and the pH value of the second decoction has little influence. Planning, solving and predicting the regression equation, and obtaining the optimization conditions as follows: a is 6.0, B is 7.36, C is 9.0, D is 4.0h, and predicted value is Y is 1.2220. And determining the pH values of the water for decoction for 3 times by combining with actual operation: 6.0, 7.4 and 9.0, and the extraction time is as follows in sequence: 2.0h, 1.0h and 1.0 h.

Table 69 set of samples for each index content and standardized processing results

3.4 validation of optimization conditions

According to optimized process conditions, 3 verification tests are carried out according to the method under the item of 2.2, and the standardized values are calculated after the gallic acid content, the ellagic acid content, the total polyphenol content and the dry extract yield are measured. The results are shown in Table 7.

Weighing the pomegranate rind powder, carrying out verification test according to the optimized process conditions, wherein the test result is close to the predicted value, and the optimized conditions are the better extraction process conditions.

Table 7 verifies the test results

4. Results

4.1 conclusion

The invention takes the optimization of the technological conditions of semi-bionic extraction of pomegranate rind as the main research content. The process optimization of the semi-bionic extraction method (SBE) is carried out by adopting a uniform design test by taking the ellagic acid content, the gallic acid content, the total polyphenol content and the dry extract yield as comprehensive evaluation indexes. The optimized process conditions are as follows: the pH values of the water for the three times of decoction and extraction are respectively 6.0, 7.4 and 9.0, and the time duration of the decoction and extraction is 2.0, 1.0 and 1.0h (the total time is 4 h). And (4) verifying that the test result is closer to the predicted value, which indicates that the preferable conditions are feasible. The research process is simple, convenient, reasonable and feasible.

Pomegranate rind belongs to astringent, is rich in active ingredients mainly including tannin, mainly including ellagic acid tannin and gallotannin. However, a large number of researches show that tannin macromolecular substances are hardly absorbed into blood in a prototype mode and are decomposed under the acid-base condition of the intestinal tract, and the blood components are mostly micromolecular phenolic substances such as ellagic acid, gallic acid, urolithin metabolites and the like. Therefore, the total polyphenol, the ellagic acid and the gallic acid which are decomposition products are selected as main investigation indexes in the experiment. Under the optimal optimized SBE condition, the extraction rates of the ellagic acid and the gallic acid respectively reach about 5 times and 4 times of the aqueous extract under the same condition, which indicates that after the semi-bionic extraction, part of tannin components in the pomegranate peel are converted into micromolecule phenolic compounds, thereby being more beneficial to the quick absorption of gastrointestinal tracts. The extraction method can obtain extract, and the preparation of formula granules can undoubtedly increase the clinical curative effect of the granules.

HPLC chromatographic conditions of ellagic acid and gallic acid adopt a variable wavelength gradient elution method, the maximum absorption wavelength of the gallic acid is 270nm, the maximum absorption wavelength of the ellagic acid is 254nm, and due to the fact that the content of the gallic acid is low, the peak shape and the separation degree of the two components are better determined by adopting 270nm in the first 11min and 254nm in the second 13 min.

The extraction process of the traditional Chinese medicine is optimized, so that the holistic concept of the traditional Chinese medicine is reflected, a plurality of indexes are required to be selected for comprehensive evaluation, and how to achieve scientific and reasonable multi-index weight assignment needs to be considered in a key way. In the experiment, an Analytic Hierarchy Process (AHP) and an objective evaluation process (CRITIC) are mixed and weighted, so that the difference of the contribution degree of each index to the traditional Chinese medicine can be reflected, and the influence caused by the conflict and variability of each experimental data can be fully balanced. Compared with a single weighting method, the AHP-CRITIC mixed weighting method has more scientific and reasonable results. In the experiment, a verification experiment shows that the optimized process parameters are reasonable, the extraction process is feasible, and data support can be provided for further research of the pomegranate rind.

The semi-bionic extraction method used in the experiment adopts various evaluation indexes for optimization of process conditions, and most researches take monomer components, total extract and the like as indexes. The preparation not only considers monomer components, but also considers active mixed components, accords with the characteristics of the comprehensive action of clinical medication of the traditional Chinese medicine, and is beneficial to controlling the quality of the preparation by utilizing various evaluation indexes during optimization. The semi-bionic extraction method adopts high-temperature decoction, which is beneficial to extracting effective components, but the long-time high-temperature decoction can destroy the structures of some bioactive components, cause the change of traditional Chinese medicine components and cause the loss of the efficacy to a certain extent.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.