CN110013499B - Method for preparing lingdan polysaccharide capsule and method for measuring total saponin content thereof - Google Patents
Method for preparing lingdan polysaccharide capsule and method for measuring total saponin content thereof Download PDFInfo
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- 239000002775 capsule Substances 0.000 title claims abstract description 47
- 238000000034 method Methods 0.000 title claims abstract description 39
- 229930182490 saponin Natural products 0.000 title claims abstract description 33
- 150000007949 saponins Chemical class 0.000 title claims abstract description 33
- 229920001282 polysaccharide Polymers 0.000 title claims abstract description 29
- 239000005017 polysaccharide Substances 0.000 title claims abstract description 29
- 150000004676 glycans Chemical class 0.000 title claims abstract description 26
- 239000001397 quillaja saponaria molina bark Substances 0.000 title claims abstract description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 71
- 235000017709 saponins Nutrition 0.000 claims abstract description 32
- 230000008569 process Effects 0.000 claims abstract description 19
- 239000000463 material Substances 0.000 claims abstract description 17
- 238000002360 preparation method Methods 0.000 claims abstract description 13
- 238000003809 water extraction Methods 0.000 claims abstract description 12
- 238000011161 development Methods 0.000 claims abstract description 7
- 239000000203 mixture Substances 0.000 claims abstract description 5
- 241000304195 Salvia miltiorrhiza Species 0.000 claims abstract description 4
- 235000011135 Salvia miltiorrhiza Nutrition 0.000 claims abstract description 4
- 235000006533 astragalus Nutrition 0.000 claims abstract description 4
- 240000008397 Ganoderma lucidum Species 0.000 claims abstract description 3
- 235000001637 Ganoderma lucidum Nutrition 0.000 claims abstract description 3
- 241001061264 Astragalus Species 0.000 claims abstract 2
- 239000004480 active ingredient Substances 0.000 claims abstract 2
- 239000000546 pharmaceutical excipient Substances 0.000 claims abstract 2
- 238000012545 processing Methods 0.000 claims abstract 2
- 210000004233 talus Anatomy 0.000 claims abstract 2
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 46
- 239000000243 solution Substances 0.000 claims description 28
- 238000012360 testing method Methods 0.000 claims description 26
- VLTRZXGMWDSKGL-UHFFFAOYSA-N perchloric acid Chemical compound OCl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-N 0.000 claims description 24
- 229960000583 acetic acid Drugs 0.000 claims description 23
- 239000012362 glacial acetic acid Substances 0.000 claims description 23
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 20
- 238000002835 absorbance Methods 0.000 claims description 19
- 238000000605 extraction Methods 0.000 claims description 18
- PWAOOJDMFUQOKB-WCZZMFLVSA-N ginsenoside Re Chemical compound O[C@@H]1[C@H](O)[C@@H](O)[C@H](C)O[C@H]1O[C@H]1[C@H](O[C@@H]2[C@H]3C(C)(C)[C@@H](O)CC[C@]3(C)[C@@H]3[C@@]([C@@]4(CC[C@@H]([C@H]4[C@H](O)C3)[C@](C)(CCC=C(C)C)O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O3)O)C)(C)C2)O[C@H](CO)[C@@H](O)[C@@H]1O PWAOOJDMFUQOKB-WCZZMFLVSA-N 0.000 claims description 18
- AOGZLQUEBLOQCI-UHFFFAOYSA-N ginsenoside-Re Natural products CC1OC(OCC2OC(OC3CC4(C)C(CC(O)C5C(CCC45C)C(C)(CCC=C(C)C)OC6OC(CO)C(O)C(O)C6O)C7(C)CCC(O)C(C)(C)C37)C(O)C(O)C2O)C(O)C(O)C1O AOGZLQUEBLOQCI-UHFFFAOYSA-N 0.000 claims description 18
- 238000001704 evaporation Methods 0.000 claims description 14
- 239000000523 sample Substances 0.000 claims description 14
- 238000001816 cooling Methods 0.000 claims description 13
- 239000012086 standard solution Substances 0.000 claims description 13
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 12
- 238000001035 drying Methods 0.000 claims description 12
- 239000013558 reference substance Substances 0.000 claims description 12
- MWOOGOJBHIARFG-UHFFFAOYSA-N vanillin Chemical compound COC1=CC(C=O)=CC=C1O MWOOGOJBHIARFG-UHFFFAOYSA-N 0.000 claims description 12
- FGQOOHJZONJGDT-UHFFFAOYSA-N vanillin Natural products COC1=CC(O)=CC(C=O)=C1 FGQOOHJZONJGDT-UHFFFAOYSA-N 0.000 claims description 12
- 235000012141 vanillin Nutrition 0.000 claims description 12
- 238000005303 weighing Methods 0.000 claims description 9
- 239000003480 eluent Substances 0.000 claims description 8
- HQKMJHAJHXVSDF-UHFFFAOYSA-L magnesium stearate Chemical compound [Mg+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O HQKMJHAJHXVSDF-UHFFFAOYSA-L 0.000 claims description 8
- 239000012488 sample solution Substances 0.000 claims description 7
- 239000011347 resin Substances 0.000 claims description 6
- 229920005989 resin Polymers 0.000 claims description 6
- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 claims description 5
- 238000004587 chromatography analysis Methods 0.000 claims description 5
- 238000004440 column chromatography Methods 0.000 claims description 5
- 230000007935 neutral effect Effects 0.000 claims description 5
- 239000006228 supernatant Substances 0.000 claims description 5
- 229920000168 Microcrystalline cellulose Polymers 0.000 claims description 4
- 235000019359 magnesium stearate Nutrition 0.000 claims description 4
- 239000008108 microcrystalline cellulose Substances 0.000 claims description 4
- 235000019813 microcrystalline cellulose Nutrition 0.000 claims description 4
- 229940016286 microcrystalline cellulose Drugs 0.000 claims description 4
- 238000007796 conventional method Methods 0.000 claims description 3
- 239000007779 soft material Substances 0.000 claims description 3
- 238000003556 assay Methods 0.000 claims 2
- 238000010790 dilution Methods 0.000 claims 1
- 239000012895 dilution Substances 0.000 claims 1
- 239000008187 granular material Substances 0.000 claims 1
- 238000005469 granulation Methods 0.000 claims 1
- 230000003179 granulation Effects 0.000 claims 1
- 229940124531 pharmaceutical excipient Drugs 0.000 claims 1
- 239000012088 reference solution Substances 0.000 claims 1
- 238000012546 transfer Methods 0.000 claims 1
- 239000000284 extract Substances 0.000 abstract description 4
- 239000012141 concentrate Substances 0.000 abstract 1
- 229930182494 ginsenoside Natural products 0.000 abstract 1
- 229940089161 ginsenoside Drugs 0.000 abstract 1
- 230000008020 evaporation Effects 0.000 description 10
- 238000002156 mixing Methods 0.000 description 8
- 238000002474 experimental method Methods 0.000 description 7
- 238000005406 washing Methods 0.000 description 7
- 238000007789 sealing Methods 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- 241000222336 Ganoderma Species 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 238000001914 filtration Methods 0.000 description 4
- 239000009636 Huang Qi Substances 0.000 description 3
- HCOLPNRPCMFHOH-UHFFFAOYSA-N Prodigiosin Natural products CCCCCC1C=C(C=C/2N=C(C=C2OC)c3ccc[nH]3)N=C1C HCOLPNRPCMFHOH-UHFFFAOYSA-N 0.000 description 3
- -1 prodigiosin polysaccharide Chemical class 0.000 description 3
- 238000011084 recovery Methods 0.000 description 3
- 238000002791 soaking Methods 0.000 description 3
- 241000045403 Astragalus propinquus Species 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 239000002671 adjuvant Substances 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
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- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000002708 enhancing effect Effects 0.000 description 2
- 235000013402 health food Nutrition 0.000 description 2
- 230000036039 immunity Effects 0.000 description 2
- 238000011835 investigation Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000010298 pulverizing process Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000007873 sieving Methods 0.000 description 2
- 238000009210 therapy by ultrasound Methods 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- 208000018380 Chemical injury Diseases 0.000 description 1
- 102000003712 Complement factor B Human genes 0.000 description 1
- 108090000056 Complement factor B Proteins 0.000 description 1
- 206010067125 Liver injury Diseases 0.000 description 1
- 238000000540 analysis of variance Methods 0.000 description 1
- 238000003287 bathing Methods 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000013400 design of experiment Methods 0.000 description 1
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- 235000013305 food Nutrition 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 231100000753 hepatic injury Toxicity 0.000 description 1
- 238000007654 immersion Methods 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 230000003908 liver function Effects 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 238000000465 moulding Methods 0.000 description 1
- 235000002020 sage Nutrition 0.000 description 1
- 239000012898 sample dilution Substances 0.000 description 1
- 238000013112 stability test Methods 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/06—Fungi, e.g. yeasts
- A61K36/07—Basidiomycota, e.g. Cryptococcus
- A61K36/074—Ganoderma
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
- A61K36/481—Astragalus (milkvetch)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/53—Lamiaceae or Labiatae (Mint family), e.g. thyme, rosemary or lavender
- A61K36/537—Salvia (sage)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/4841—Filling excipients; Inactive ingredients
- A61K9/4866—Organic macromolecular compounds
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation or decoction
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N2030/042—Standards
- G01N2030/047—Standards external
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Abstract
本发明公开了的一种灵丹多糖胶囊的制作方法及其总皂苷含量测定方法，按处方称取灵芝、丹参和黄芪；通过水提提取有效成分，提取2次，每次加药材的8‑10倍量水，每次提取1.8‑3h，混合煎液；浓缩干燥煎液，加入药用辅料按常规工艺制备胶囊。制得灵丹多糖胶囊，其总皂苷含量测定通过制备人参皂苷R、样品处理、显色、测定，计算灵丹多糖胶囊中总皂苷的含量。本发明的水提工艺不但操作简单，且在水提工艺中能很好保留原方用药特色，操作快，实用性好，本发明的总皂苷含量测定方法精密度、稳定性，重复性良好。The invention discloses a preparation method of Lingdan polysaccharide capsules and a method for measuring total saponin content. Ganoderma lucidum, Salvia miltiorrhiza and Astragalus are weighed according to the prescription; the active ingredients are extracted by water extraction, extracted twice, and 8-8% of medicinal materials are added each time. 10 times the amount of water, extract 1.8-3h each time, mix the decoction; concentrate the dry decoction, add medicinal excipients, and prepare capsules according to the conventional process. Preparation of Lingdan Polysaccharide Capsules, Determination of Total Saponins Content of Lingdan Polysaccharide Capsules The content of total saponins in Lingdan polysaccharide capsules is calculated by preparing ginsenoside R, sample processing, color development and determination. The water extraction process of the invention is not only simple to operate, but also can well retain the characteristics of the original prescription in the water extraction process, has fast operation and good practicability, and has good precision, stability and repeatability of the total saponin content determination method of the invention.
Description
Technical Field
The invention belongs to the technical field of health product medicine preparation, and particularly relates to a method for preparing a lingdan polysaccharide capsule and a method for measuring the content of total saponins in the lingdan polysaccharide capsule.
Background
The Lingdan polysaccharide capsule is prepared from Ganoderma, Saviae Miltiorrhizae radix and radix astragali, and has effects in enhancing immunity, and protecting liver from chemical injury. The formula is a development and research project of the Lingdan polysaccharide capsule, namely a science and technology project of provinces in Guangdong provinces. Because of the vacancy of the large brands and the known health food with the double functions of enhancing immunity and assisting in protecting liver function from chemical liver injury in the current health food market, the lingdan polysaccharide capsule can make up the vacancy of the market after realizing industrialization, is beneficial to the expansion of enterprise products, is beneficial to the realization of multidirectional development of enterprises, and realizes better market economic benefit.
Disclosure of Invention
The invention aims to provide a method for preparing a lingdan polysaccharide capsule, which can quickly extract the lingdan polysaccharide capsule.
The invention also aims to establish a method for measuring the content of the total saponins in the lingdan polysaccharide capsule.
The purpose of the invention is realized as follows: a method for preparing a lingdan polysaccharide capsule comprises the following steps: (1) weighing Ganoderma, Saviae Miltiorrhizae radix and radix astragali according to the prescription; (2) extracting the effective components by water extraction: the water extraction process conditions are 2 times of extraction, 8-10 times of water of the medicinal materials is added each time, 1.8-3h of extraction is performed each time, and the decoction of the two times of extraction is mixed; (3) concentrating, drying, adding medicinal adjuvants, and making into capsule by conventional method.
In the step (1), 4-6 parts of lucid ganoderma, 4-6 parts of salvia miltiorrhiza and 5-7 parts of astragalus membranaceus are calculated according to parts by weight.
In the step (2), 10 times of water of the medicinal materials is added for the first time, and 8 times of water of the medicinal materials is added for the second time, and the extraction is carried out for 2 hours each time.
In the step (3), the decoction is filtered, concentrated under reduced pressure and dried under reduced pressure to obtain dry paste, the dry paste is crushed and sieved, microcrystalline cellulose is added, the mixture is mixed, soft materials are prepared, granulated, dried, granulated, added with magnesium stearate, mixed evenly and packaged into capsules.
The method for measuring the content of the total saponins in the lingdan polysaccharide capsule is characterized by comprising the following steps:
(1) preparation of standard solution A proper amount of ginsenoside Re reference substance is precisely weighed in a volumetric flask, and methanol is added to dissolve the ginsenoside Re reference substance into 1ml of standard solution containing ginsenoside Re 2 mg;
(2) precisely weighing 2.0g of capsule content in a sample treatment, placing the capsule content in a 100ml volumetric flask, adding water for ultrasonic treatment, then adding water to a constant volume of 100ml, shaking up, placing, absorbing 1.0ml of supernatant for column chromatography, wherein 3cm of macroporous resin is filled in a chromatographic column, and 1cm of neutral alumina is added; the chromatography process comprises the following steps: sequentially washing the column with ethanol and water, discarding the eluate, precisely adding 1.0ml of sample solution, washing the column with 25ml of water, discarding the eluate, eluting with 25ml of 70% ethanol, collecting the eluate in an evaporation pan, placing in a water bath to evaporate, and collecting the residue for use;
(3) precisely absorbing 80 mu l of a reference substance solution for color development, placing the reference substance solution in a 5ml test tube with a plug, drying the reference substance solution by hot air, precisely adding 0.2ml of a 5% vanillin glacial acetic acid solution, rotating an evaporation dish to fully dissolve residues, precisely adding 0.8ml of perchloric acid, shaking up, sealing the test tube, placing the test tube in a water bath at 60 ℃ for 10min, taking out the test tube, cooling the test tube in the ice bath, accurately adding 5.0ml of glacial acetic acid, shaking up, and measuring the absorbance at the wavelength of 560 nm;
(4) and (3) measuring and taking the residue treated in the step (2), precisely adding 0.2ml of a 5% vanillin-containing glacial acetic acid solution, rotating an evaporation dish to fully dissolve the residue, adding 0.8ml of perchloric acid, uniformly mixing, transferring to a 5ml centrifuge tube with a plug scale, carrying out water bath at 60 ℃ for 10min, taking out, cooling in an ice bath, accurately adding 5.0ml of glacial acetic acid, shaking uniformly, carrying out colorimetric determination on the absorbance with a standard substance at the wavelength of 560nm, and calculating the content of the total saponins in the prodigiosin polysaccharide capsule.
By adopting the technical scheme, compared with the prior art, the water extraction process disclosed by the invention is simple to operate, can well keep the medicinal characteristics of the original prescription in the water extraction process, is quick to operate and good in practicability, and the method for measuring the content of the total saponin has the advantages of high precision, high stability and good repeatability. The research of the invention has no pollution to the environment, and also has important significance for fully, efficiently and comprehensively developing and utilizing traditional Chinese medicine resources and building resource-saving and environment-friendly industries.
Detailed Description
The present invention is described in further detail below, but is not to be construed as being limited thereto.
The invention relates to a method for preparing a lingdan polysaccharide capsule, which comprises the following steps:
(1) weighing Ganoderma, Saviae Miltiorrhizae radix and radix astragali according to the prescription. Preferably, the weight portions of the ganoderma lucidum are 4 to 6 portions, the salvia miltiorrhiza are 4 to 6 portions, and the astragalus membranaceus is 5 to 7 portions. The most preferable weight ratio is 6:5: 5.
(2) Extracting the effective components by water extraction: the water extraction process conditions are 2 times of extraction, 8-10 times of water of the medicinal materials is added each time, 1.8-3h of extraction is performed each time, and the decoction of the two times of extraction is mixed. Most preferably, 10 times of water of the medicinal materials is added for the first time, and 8 times of water of the medicinal materials is added for the second time, and each time of extraction is 2 hours.
(3) Concentrating, drying, adding medicinal adjuvants, and making into capsule by conventional method. The method comprises the following specific steps: filtering the decoction, concentrating under reduced pressure, drying under reduced pressure to obtain dry extract, pulverizing, sieving, adding microcrystalline cellulose, mixing, making soft mass, granulating, drying, grading, adding magnesium stearate, mixing, and making into capsule.
The method for measuring the content of total saponins in the lingdan polysaccharide capsule comprises the following steps:
(1) preparation of standard solution A proper amount of ginsenoside Re reference substance is precisely weighed in a volumetric flask, and methanol is added to dissolve the ginsenoside Re reference substance into 1ml of standard solution containing ginsenoside Re 2 mg;
(2) precisely weighing 2.0g of capsule content in a sample treatment, placing the capsule content in a 100ml volumetric flask, adding water for ultrasonic treatment, then adding water to a constant volume of 100ml, shaking up, placing, absorbing 1.0ml of supernatant for column chromatography, wherein 3cm of macroporous resin is filled in a chromatographic column, and 1cm of neutral alumina is added; the chromatography process comprises the following steps: sequentially washing the column with ethanol and water, discarding the eluate, precisely adding 1.0ml of sample solution, washing the column with 25ml of water, discarding the eluate, eluting with 25ml of 70% ethanol, collecting the eluate in an evaporation pan, placing in a water bath to evaporate, and collecting the residue for use;
(3) precisely absorbing 80 mu l of a reference substance solution for color development, placing the reference substance solution in a 5ml test tube with a plug, drying the reference substance solution by hot air, precisely adding 0.2ml of a 5% vanillin glacial acetic acid solution, rotating an evaporation dish to fully dissolve residues, precisely adding 0.8ml of perchloric acid, shaking up, sealing the test tube, placing the test tube in a water bath at 60 ℃ for 10min, taking out the test tube, cooling the test tube in the ice bath, accurately adding 5.0ml of glacial acetic acid, shaking up, and measuring the absorbance at the wavelength of 560 nm;
(4) and (3) measuring and taking the residue treated in the step (2), precisely adding 0.2ml of a 5% vanillin-containing glacial acetic acid solution, rotating an evaporation dish to fully dissolve the residue, adding 0.8ml of perchloric acid, uniformly mixing, transferring to a 5ml centrifuge tube with a plug scale, carrying out water bath at 60 ℃ for 10min, taking out, cooling in an ice bath, accurately adding 5.0ml of glacial acetic acid, shaking uniformly, carrying out colorimetric determination on the absorbance with a standard substance at the wavelength of 560nm, and calculating the content of the total saponins in the prodigiosin polysaccharide capsule.
The invention applies orthogonal experiment, takes the total saponin extraction amount as an investigation index, takes the water addition amount, the extraction time and the extraction frequency as investigation factors, and takes 3 levels of each factor to carry out the orthogonal experiment.
1 orthogonal design of experiment
1.1 study of Water absorption of medicinal materials
Weighing the medicinal materials (6: 5:5 of radix astragali-Ganoderma-Saviae Miltiorrhizae radix) according to the proportion of the formula, adding 10 times of water, soaking for 2 hr, measuring the water absorption capacity of the medicinal materials to be 2 times, the minimum water immersion capacity of the medicinal materials to be 6 times, and the first water addition capacity is 2 times higher than the second and third times. The design basis of the factor level is taken as the basis.
1.2 contents of the experiment
Weighing the medicinal materials according to the proportion of the formula, adding water for soaking for 2 hours, adopting an orthogonal test method, carrying out water decoction extraction according to a factor level table, filtering, carrying out reduced pressure concentration on a water decoction to about 1000ml, adding water to fix the volume to 1000ml, determining the content of total saponins, and carrying out water decoction process parameters: the decoction time (A), the water addition amount (B) and the decoction times (C) were determined, and 3 levels were selected for each factor as L9 (3)4) Orthogonal experiments, preferably an aqueous extraction process. The factor level table, the orthogonal test table and the variance analysis table are shown in the tables 1, 2 and 3.
TABLE 1 Water decoction Process factor horizon
TABLE 2 Water decoction Process L9(34) Orthogonal test table
TABLE 3 analysis of variance
Carrying out variance analysis according to the total amount of the total saponins, wherein the main action and the secondary action of each factor are A more than C more than B, the decoction time A has significance, and the water addition amount B and the decoction time C have no significance; according to range analysis: factor A is A3＞A2And is obviously > A1(ii) a Factor B though B2＞B1＞B3But the three are not obviously different; factor C of C3＞C2And is obviously > C1。
From the viewpoint of saving man-hour and cost, the optimum process condition is determined as A2B2C2The preparation method comprises decocting in water for 2 times (2 hr each time), wherein the water addition amount is 10 times and 8 times.
1.3 validation test of results of Water decoction Quadrature Process
Weighing the medicinal materials according to the proportion of the prescription, 3 parts, respectively adding water to soak for 2 hours, decocting and extracting for 2 times, wherein the water adding amount is respectively 10 times and 8 times, filtering, concentrating the water decoction under reduced pressure to about 1000ml, fixing the volume to 1000ml, and measuring the content of the total saponin. The results of the water decoction process were verified using the total amount of total saponins as an index, and are shown in table 4.
TABLE 4 verification test of the results of the Water decoction Process
The experimental results show that: the verification result has no obvious difference from the orthogonal extraction result.
1.4 preparation of example 1 sample
Weighing 3 parts of medicinal materials (6: 5:5 of astragalus-ganoderma-red sage root) according to the proportion of the prescription, and respectively preparing the medicinal materials according to the following same processes: soaking in water for 2 hr, decocting for 2 times, each for 2 hr, adding water 10 times and 8 times, filtering, concentrating under reduced pressure, drying under reduced pressure to obtain dry extract, pulverizing, sieving, adding microcrystalline cellulose, mixing for 30min, making soft material, granulating, drying, adding magnesium stearate, mixing, and making into capsule, 0.35g per capsule.
2 Total saponins content determination
2.1 reagents and starting materials
2.1.1 reagents: vanillin, glacial acetic acid, perchloric acid, ethanol, Amberlite-XAD-2 macroporous resin
2.1.2 control: ginsenoside Re; the source is as follows: the Chinese food and drug testing institute; batch number: 110754-; purity: 98.8 percent
2.2 instruments and devices
Uv-visible spectrophotometer: caey 8454 UV-Vis Agilent Technologies; water bath (HHS-12WSZ-133-65 Shanghai dongxing), chromatographic column (diameter 1.5cm)
2.3 analytical procedure
2.3.1 preparation of Standard solution A proper amount of ginsenoside Re reference is precisely weighed in a volumetric flask, and dissolved by adding methanol to prepare 1ml of standard solution containing ginsenoside Re 2 mg.
2.3.2 sample treatment 2.0g of the capsule content prepared in example 1 was precisely weighed, placed in a 100ml volumetric flask, added with water and sonicated for 30min, then water was added to a constant volume of 100ml, shaken up, placed, and 1.0ml of supernatant was taken for column chromatography. The chromatographic column is filled with 3cm Amberlite-XAD-2 macroporous resin, and 1cm neutral alumina is added. Eluting with 25ml 70% ethanol, discarding eluate, eluting with 25ml water, discarding eluate, precisely adding 1.0ml sample solution, eluting with 25ml water, discarding eluate, eluting with 25ml 70% ethanol, collecting eluate, evaporating in evaporating dish, placing in 60 deg.C water bath, and volatilizing to obtain residue.
2.3.3 developing, precisely absorbing 80 μ l of the reference substance solution, placing in a 5ml test tube with a plug, drying by hot air, precisely adding 0.2ml of newly-configured 5% vanillin glacial acetic acid solution, rotating an evaporation dish to fully dissolve residues, precisely adding 0.8ml of perchloric acid, shaking up, sealing the plug, placing in a 60 ℃ water bath for 10min, taking out, cooling in an ice bath, accurately adding 5.0ml of glacial acetic acid, shaking up, and measuring the absorbance at the wavelength of 560 nm.
2.3.4 measuring the residue treated under the item 3.2, precisely adding a newly configured 0.2ml of a 5% vanillin-containing glacial acetic acid solution, rotating an evaporation dish to fully dissolve the residue, adding 0.8ml of perchloric acid, uniformly mixing, transferring to a 5ml centrifugal tube with a plug for 10min in water bath at 60 ℃, taking out, cooling in an ice bath, precisely adding 5.0ml of glacial acetic acid, shaking uniformly, carrying out colorimetric measurement on the absorbance with a standard substance at the wavelength of 560nm, and calculating the content of the total saponins in the prodigiosin polysaccharide capsule.
2.4 calculation and results presentation
In the formula:
x is the content (g/100g) of total saponins (calculated by ginsenoside Re) in the sample;
a1-absorbance value of sample liquid;
a2-absorbance value of standard solution;
c-amount of standard tube ginsenoside Re (μ g);
v-sample dilution volume (ml);
m-sample mass (g);
computing results preserving two significant digits
2.5 Total Saponin detection methodology applicability study
2.5.1 Linear relationship experiment
Precisely absorbing 70, 80, 90, 100 and 110 mu l of the standard solution, respectively placing the solution into 5ml test tubes with plugs, drying the solution by hot air, cooling the solution, precisely adding 0.2ml of newly-configured 5% vanillin glacial acetic acid and 0.8ml of perchloric acid, shaking the solution uniformly, sealing the test tubes, placing the test tubes in a water bath at 60 ℃ for 10min, taking the test tubes out, cooling the test tubes in an ice bath, accurately adding 5.0ml of glacial acetic acid, shaking the test tubes uniformly, and measuring the absorbance at the wavelength of 560 nm. Performing linear regression on the absorbance (A) by using the content m of the ginsenoside Re to obtain a regression equation: a is 3.6999m-0.1512, R2The result shows that the content of the ginsenoside Re is in good linear relation within the range of 0.1-0.22 mg.
2.5.2 precision test
Precisely absorbing 70 mu l of the standard solution, placing the solution in a 5ml test tube with a plug, drying the solution by hot air, cooling the solution, precisely adding 0.2ml of newly-configured 5% vanillin glacial acetic acid and 0.8ml of perchloric acid, shaking the solution uniformly, sealing the plug, placing the solution in a water bath at 60 ℃ for 10min, taking out the solution, cooling the solution in the ice bath, accurately adding 5.0ml of glacial acetic acid, shaking the solution uniformly, measuring the absorbance at the wavelength of 560nm, and continuously measuring the absorbance for 5 times, wherein the RSD is 0.32%. The results show that the precision experiment is good.
2.5.3 repeatability test
2.0g and 6 parts of the content of the capsule prepared in example 1 are precisely weighed, respectively placed in a volumetric flask of 100ml, added with water and ultrasonically treated for 30min, then the volume is determined to be 100ml by water, shaken up, placed, and respectively absorbed with 1.0ml of supernatant for column chromatography. The chromatographic column is filled with 3cm Amberlite-XAD-2 macroporous resin, and 1cm neutral alumina is added. Washing the column with 25ml of 70% ethanol, discarding the eluent, washing the column with 25ml of water, discarding the eluent, precisely adding 1.0ml of each sample solution, washing the column with 25ml of water, discarding the eluent, eluting with 25ml of 70% ethanol, collecting the eluent in an evaporation dish, placing in a 60 ℃ water bath for volatilizing, precisely adding a newly-configured 0.2ml of a 5% vanillin-containing glacial acetic acid solution, rotating the evaporation dish to fully dissolve residues, adding 0.8ml of perchloric acid, uniformly mixing, transferring to a 5ml centrifugal tube with a plug scale, bathing in a 60 ℃ water bath for 10min, taking out, cooling in an ice bath, accurately adding 5.0ml of glacial acetic acid, uniformly shaking, performing colorimetric determination on the absorbance with a standard substance at the wavelength of 560nm, and calculating the content of total saponins. The average content was 9.93mg/g, and the RSD was 0.65%. The results show that the repeatability experiments are good.
2.5.4 stability test
The sample solution No. 6 was sampled, and the absorbance of total saponins was measured at 560nm wavelength of 0, 1, 2, 3, 4, 5 hours, and the RSD was 0.48%. The results show that the total saponins are stable within at least 5 h.
2.5.5 sample recovery test
About 0.5g (total saponin content is 9.93mg/g)6 parts of the capsule content prepared in example 1 are respectively taken, precisely weighed, added into a 100ml volumetric flask, precisely added with 2.5ml of ginsenoside Re standard solution (concentration is 2mg/ml), absorbance is measured according to sample treatment and measurement methods, and the content is calculated. See table 5. The results show that the sample recovery rate is good.
TABLE 5 results of recovery measurement
2.6 determination of the content of three batches of samples
2.0g of the content of 3 batches of capsules prepared in example 1 are precisely weighed respectively, extraction, color development and determination are carried out according to a proposed determination method, and the content is calculated, wherein the content of the total saponins in the 3 batches of capsules is respectively 9.92mg/g, 9.89mg/g and 9.96mg/g, which indicates that the extraction process, the molding process and the content determination method are good.
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