CN110917228B - Method for simultaneously preparing total saponins, total flavonoids and oil unsaponifiable matter of ginseng stem leaves - Google Patents

Method for simultaneously preparing total saponins, total flavonoids and oil unsaponifiable matter of ginseng stem leaves Download PDF

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CN110917228B
CN110917228B CN201911173325.9A CN201911173325A CN110917228B CN 110917228 B CN110917228 B CN 110917228B CN 201911173325 A CN201911173325 A CN 201911173325A CN 110917228 B CN110917228 B CN 110917228B
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刘春明
李赛男
许晶盈
张语迟
时东方
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Beijing Tianyi Biotechnology Co ltd
Dragon Totem Technology Hefei Co ltd
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    • 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
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Abstract

The invention provides a method for simultaneously preparing total saponins, total flavonoids and oil unsaponifiable matters of ginseng stems and leaves, belonging to the field of preparation methods of ginseng extracts. The method comprises processing dried stems and leaves of Ginseng radix into powder, adding petroleum ether, sodium hydroxide, n-hexane, ethyl acetate, acetonitrile and water, and extracting by magnetic stirring; standing after extraction, collecting the upper layer solution, and concentrating under reduced pressure or normal pressure to obtain unsaponifiable matter of ginseng stem and leaf oil; respectively taking the middle layer solution and the lower layer solution, concentrating, loading onto macroporous resin column, eluting with water until the water eluate is colorless, clear and transparent, eluting with ethanol solution until the ethanol eluate is colorless, clear and transparent, collecting the ethanol eluate, recovering ethanol, and drying to obtain dry extract-like total flavonoids of ginseng stem and leaf and total saponins of ginseng stem and leaf. The method is convenient, efficient, easy to operate and low in cost, improves the utilization rate of the ginseng stems and leaves, can meet the development and utilization in the fields of medicine, food and chemical industry, and has great application and development values.

Description

Method for simultaneously preparing total saponins, total flavonoids and oil unsaponifiable matter of ginseng stem leaves
Technical Field
The invention belongs to the field of preparation methods of ginseng extracts, and particularly relates to a method for simultaneously preparing total saponins, total flavonoids and oil unsaponifiable matters of ginseng stems and leaves.
Background
The caulis Et folium Ginseng is selected from Panax ginseng C.A.Meyer of AraliaceaePanax ginseng C.A. Mey). Bitter, sweet and cold in nature. Has the functions of invigorating qi, benefiting lung, dispelling summer heat, promoting the production of body fluid and the like, and is mainly used for qi deficiency cough, fever dysphoria, body fluid injury thirst, unclear head and eyes, tiredness of limbs and the like. Modern pharmacology indicates that the ginseng stem and leaf extract has the effects of resisting fatigue, resisting stress, enhancing immunologic function, regulating organism functions, resisting tumors, aging, oxidation, bacteria, inflammation and viruses, protecting heart and liver, reducing blood fat, resisting arteriosclerosis, resisting coagulation, promoting urination, resisting endotoxin, hemolysis and the like. In addition, the ginseng leaves also have protective effects on the central nervous system and the respiratory system, and can participate in the effects of regulating substance metabolism, promoting cell proliferation, improving sleep quality and the like. The main chemical components comprise chlorophyll, saponin, grease, flavone, amino acid, polysaccharide, vitamin, trace elements and the like. The oil and saponin are important chemical components in ginseng stems and leaves, the oil can be saponified under the alkaline condition, the saponifiable matter is called saponified matter, and the saponified matter can be dissolved in ethanol and water solution, and has low solubility in ether liquid such as petroleum ether. The unsaponifiable substances include partially sterols, high molecular alcohols, hydrocarbons, etc., which are called unsaponifiable substances, which are easily soluble in ether solutions such as petroleum ether, and have low solubility in ethanol and aqueous solutions. Recent studies have shown that unsaponifiable substances of fats and oils have a very strong biological activity, and that unsaponifiable substances in the preparation of fats and oils or the extraction of unsaponifiable substances in fats and oils can be used for the development of foods, health foods, medicines, and the like. In the traditional method, when the grease is extracted from the plant raw materials and the unsaponifiable matters in the grease are prepared, the grease is usually extracted from the raw materials firstly, the extracting solution is concentrated to obtain the grease, potassium hydroxide and ethanol solution are added into the grease for heating and saponification, after the saponification is finished, the petroleum ether, ether and other solutions are used for extraction to obtain extracts of the petroleum ether and ether layers, then the extracts are respectively washed by alkali-containing ethanol and ethanol, and the petroleum ether and the ether are volatilized to dry, so that the unsaponifiable matters in the grease are obtained. The process needs 6 steps of extracting and concentrating the grease from the raw materials, preparing the grease, saponifying, extracting, washing and the like,the steps are relatively independent, the operation is complicated, and manpower and material resources are wasted.
The methods for preparing the grease unsaponifiable matters which are commonly adopted at present are mostly general methods or more complicated methods. The method for extracting unsaponifiable matters from the liquid shea butter, which is proposed by Huangxiancai and other Chinese patents (application number 201210181078.9), comprises the steps of ester exchange, molecular distillation, crystallization and the like, so that a high-purity unsaponifiable matter composition can be obtained, and the composition contains high content of triterpenoid alcohols and sterols; the antotun-bigri et al patent in china (application No. 201080047939.3) proposed a method for extracting unsaponifiable matter from renewable raw materials selected from the group consisting of oil-bearing fruits, oil-bearing seeds, etc., characterized in that it comprises the following steps: dehydrating, adding alcohol and catalyst, extracting, concentrating, saponifying, and extracting saponified material; j.le Ge Lang et al in Chinese patent (application No. 201280006964.6) propose the preparation method of total unsaponifiable matter of vegetable oil, which comprises the steps of firstly obtaining vegetable oil, saponifying with potassium carbonate ethanol solution, extracting with organic solution, recovering solvent, and stripping unsaponifiable matter with carrier gas. Zhandei et al, in Chinese patent (application No. 93118734.6), have proposed a preparation method of solanesol unsaponifiable matter, its main process is to regard mildew tobacco or broken miscellaneous tobacco powder as raw materials, add solvent and catalyst to saponify, then carry on the process such as filtering, mixing, separating, hyperfiltration, concentration, etc., make solanesol unsaponifiable matter; in chinese patent application No. 201310739702.7, gazania zeoho et al, a two-stage column chromatography method for extracting squalene from deodorized distillate of vegetable oil was proposed, which was first saponified and comprised the following process steps: saponifying vegetable oil by common method, extracting and separating to obtain saponified substance, and separating squalene from unsaponifiable substance by column chromatography. Chinese patent (201410427299.9) to Shikei et al proposes a method for extracting squalene from the leftover after natural vitamin E extraction, which comprises the steps of firstly carrying out saponification reaction on the leftover, then extracting, purifying the extract by a 2-stage rectifying tower, and then separating by chromatography. The method is to saponify the vegetable oil by common methods, or to saponify by ester exchange and catalyst addition, or to further purify and separate the saponified substance, so as to separate the pure unsaponifiable substance. In the method, the processes of extracting, saponifying and unsaponifiable matters of the vegetable oil are relatively independent, and the operation is complicated.
In the Chinese patent (application number: 201711140841) of Zhuyixin and the like, a preparation method of a rubber tree seed unsaponifiable matter with immunity improvement is provided, wherein a resin-soluble matter of rubber tree seeds is used as a raw material, excessive potassium hydroxide and ethanol solution are added for saponification reaction, then, the ethanol is recovered by decompression concentration, and then, ether, ethyl acetate or normal hexane and the like are added for extraction to prepare the rubber tree seed oil unsaponifiable matter. A similar saponification method is also adopted in Chinese patent application No. 201711140838.0 of Yangyumei et al to prepare unsaponifiable matters of oil in rubber tree seeds for preventing and treating atherosclerosis. Similar saponification methods are also adopted in China patent application No. 201711137736.3 of Chenwang et al and China patent application No. 201711139152.X of Xushuang et al for preparing unsaponifiable matter in rubber tree seeds. The methods for producing the oil-and-fat unsaponifiable matter in the other reports are substantially the same as the above-mentioned methods. The preparation method of the oil unsaponifiable matter is a common preparation method, namely adding an alkaline ethanol solution into oil for saponification, and then extracting by using ether liquid and the like. The saponification and extraction impurity removal are not finished synchronously, and in addition, the method can be used for preparing stem leaf total saponins and total flavonoids besides preparing unsaponifiable oil. At present, reports of simultaneously preparing stem and leaf total saponins, total flavonoids and grease unsaponifiables in one step are not seen.
In the aspect of preparing total saponins of ginseng stem and leaf, Xihaifeng and the like extract and prepare saponins in ginseng leaf by a reflux method in Chinese patent (application number: 201610014885. X), and then separate and purify the saponins by column chromatography and the like to obtain ginsenosides Re and Rg 1; zhookai et al, in Chinese patent (application No. 201711470015.4), utilize the reflux valve to extract the saponin of the ginseng stem and leaf, after removing impurities by centrifugation, utilize gel column chromatography to purify and separate, prepare the total saponin of the ginseng stem and leaf containing ginsenoside Re, Rg1, Rd with higher purity; von waves in Chinese patent (application number: 201810415236. X) propose a preparation method of total saponins of ginseng stems and leaves with low pesticide residue. Firstly, using low-carbon alcohol to remove pesticide residues from the raw materials of ginseng stems and leaves, then using ethanol to reflux and extract saponin, and concentrating and drying to obtain the total saponin of ginseng stems and leaves. The above reports show that the ginsenoside is extracted by reflux method, or purified by column chromatography or other purification methods. Fish-Red flash et al in Chinese patent (application No. 201510276892.2) propose a method for preparing rare saponins of Ginseng radix by converting ginsenoside stem and leaf with microbial enzyme, which comprises extracting total saponin from ginseng stem and leaf, adding microbial enzyme, converting ginsenoside stem and leaf with microbial enzyme, and converting common ginsenoside Rb3, Rc, Rb2, Rd, etc. into ginsenoside C-Mx, C-Mc, C-Y, C-K.
Disclosure of Invention
The invention aims to provide a method for simultaneously preparing total saponins, total flavonoids and unsaponifiable matters of ginseng stems and leaves, which is convenient, efficient, easy to operate and low in cost.
The invention provides a method for simultaneously preparing total saponins, total flavonoids and oil unsaponifiable matters of ginseng stems and leaves, which comprises the following steps and conditions:
the method comprises the following steps: processing dried stems and leaves of Ginseng radix into powder, adding petroleum ether, sodium hydroxide, n-hexane, ethyl acetate, acetonitrile and water, and extracting by magnetic stirring;
step two: standing after extraction, collecting the upper layer solution, and concentrating under reduced pressure or normal pressure to obtain unsaponifiable matter of ginseng stem and leaf oil;
step three: concentrating the intermediate layer solution to 1/4-1/10 of the original volume, loading onto macroporous resin column, eluting with water until the water eluate is colorless, clear and transparent, and has pH of 7, eluting with ethanol solution until the ethanol eluate is colorless, clear and transparent, collecting ethanol eluate, recovering ethanol, and drying to obtain dry extract-like ginseng stem leaf total flavone;
step four: collecting the lower layer solution, concentrating to 1/4-1/10, separating with macroporous resin column, eluting with water until the water eluate is colorless, clear and transparent, and has pH of 7, eluting with ethanol until the ethanol eluate is colorless, clear and transparent, collecting ethanol eluate, recovering ethanol, and drying to obtain dry extract of total saponins of caulis Et folium Ginseng.
Preferably, the mass M of the ginseng stem and leaf powder in the first step1(g): volume V of Petroleum Ether1(ml): mass M of sodium hydroxide2(g): volume V of n-hexane2(ml): volume V of Ethyl acetate3(ml): volume V of acetonitrile4(ml): volume V of water5(ml) 1: (10-50): (0.2-0.5): (10-50): (10-50): (20-70): (10-50).
Preferably, the extraction temperature in the first step is 100-.
Preferably, the unsaponifiable matter of the ginseng stem and leaf fat in the second step is 3,7,11,15-Tetramethyl-2-hexadecen-1-ol (phytol), gamma-Sitosterol (gamma-Sitosterol), Stigmasta-5,24(28) -dien-3-ol, (3 beta, 24Z) -.
Preferably, the macroporous resin models of the third step and the fourth step are D101, D130, AB8 or NKA.
Preferably, the mass g of the macroporous resin in the third step and the fourth step is as follows: the mass g of the ginseng stem and leaf is 1: (0.5-2).
Preferably, the volume ratio of the ethanol solution in the third step and the fourth step is 40-60%.
Preferably, the total flavonoids of the stem and leaf of ginseng in the third step are rutin, ginseng flavonoid glycoside, quercetin and kaempferol.
Preferably, the total saponins of the ginseng stems and leaves in the fourth step are ginsenoside 20R-Rg3 and 20S-Rg 3.
Principle of the invention
The principle of the method for simultaneously preparing the total saponins, the total flavonoids and the oil unsaponifiable matters of the ginseng stems and leaves is as follows: the oil and its unsaponifiable matter are dissolved in petroleum ether, the oil saponified matter is easily dissolved in acetonitrile and water, the flavone is easily dissolved in ethyl acetate, and the saponin is not dissolved in ethyl acetate and is easily dissolved in acetonitrile and water. Extracting ginseng stem leaves by using petroleum ether, sodium hydroxide, ethyl acetate, normal hexane, acetonitrile and water mixed solution, wherein the mixed solution can be divided into an upper layer solution, a middle layer solution and a lower layer solution under the condition of not stirring, namely the upper layer solution is mainly petroleum ether, the middle layer solution is mainly ethyl acetate, the lower layer solution is mainly acetonitrile and water, the normal hexane is simultaneously dissolved in the upper layer solution and the middle layer solution, the acetonitrile is mainly dissolved in the middle layer solution and the lower layer solution, and the sodium hydroxide is mainly dissolved in the lower layer solution. Under agitation, an emulsion can be formed temporarily, i.e., the solutions are mixed together. Under the action of the emulsion, the oil is extracted by the petroleum ether part in the emulsion, the flavone is extracted by the ethyl acetate and the acetonitrile in the emulsion, the saponin is extracted by the acetonitrile and the aqueous solution part in the emulsion, simultaneously, the oil is saponified under the action of the sodium hydroxide in the emulsion, the saponin is hydrolyzed under the action of the sodium hydroxide in the emulsion to generate the ginsenoside 20R-Rg3 and 20S-Rg3, the mixture of the petroleum ether-sodium hydroxide-n-hexane-ethyl acetate-acetonitrile-aqueous solution is re-divided into an upper layer, a middle layer and a lower layer after the stirring and the extraction are finished, the unsaponifiable matter is dissolved in the petroleum ether solution at the upper layer, and the rest substances are dissolved in the alcoholic solutions at the middle layer and the lower layer. And (4) taking the upper layer petroleum ether solution, and concentrating under reduced pressure until the upper layer petroleum ether solution is dried. The unsaponifiable matter mainly contains 3,7,11,15-Tetramethyl-2-hexadecen-1-ol (phytol), gamma-Sitosterol (gamma-Sitosterol), Stigmasta-5,24(28) -dien-3-ol, (3 beta, 24Z) -, which is detected by using gas chromatography and mass spectrometry. The three substances account for more than 60% of the total unsaponifiable matter calculated by a normalization method. Respectively concentrating the middle layer solution and the lower layer solution, respectively loading onto macroporous resin column, eluting with water, washing off impurities such as sodium hydroxide, oil and chlorophyll saponificate, eluting with ethanol solution, respectively collecting ethanol eluates, concentrating to obtain dry extract containing total flavonoids of stem and leaf of Ginseng radix and total saponins, wherein the total flavonoids mainly contain rutin, ginsengenin glycoside, quercetin and kaempferol. The total saponins mainly contain ginsenoside 20R-Rg3 and 20S-Rg 3.
The invention has the advantages of
The invention provides a method for simultaneously preparing total saponins, total flavonoids and oil unsaponifiable matters of ginseng stem leaves, which takes ginseng stem leaf powder as a starting raw material, uses a composite solution (petroleum ether, sodium hydroxide, normal hexane, ethyl acetate, acetonitrile and water) for extraction, completes saponification and extraction treatment while extracting oil, obtains the oil unsaponifiable matters of ginseng stem leaves in one step, and synchronously completes the extraction, saponification and extraction of oil. The degreasing, the alkaline hydrolysis and the extraction are completed while the ginsenoside is extracted, so that the degreasing, the alkaline hydrolysis and the extraction of the saponin are completed synchronously. The method is convenient and rapid, and can simultaneously obtain oil unsaponifiable matter and total saponin of caulis Et folium Ginseng containing ginsenoside 20R-Rg3 and 20S-Rg 3. The method realizes the rapid preparation from 'plant materials' to 'unsaponifiable matters' and 'ginseng stem and leaf total saponins containing the ginsenosides 20R-Rg3 and 20S-Rg 3', simultaneously extracts the ginseng stem and leaf total flavonoids, stands the extracting solution, is divided into three layers, and the grease unsaponifiable matters, the flavonoids and the saponins are respectively distributed in the three layers of solution, thereby providing convenience for subsequent treatment, simplifying the operation link, saving the time and having advantages on mass production and preparation. The method is convenient, efficient, easy to operate, low in cost, capable of meeting the requirements of development and utilization in the fields of medicine and chemical industry, and high in application and development values.
Drawings
Fig. 1 is a gas chromatography-mass spectrometry ion-flow diagram of an oil-unsaponifiable matter of ginseng stem leaves prepared in example 1.
FIG. 2 is a liquid chromatography-mass spectrometry ion-flow diagram of total saponins in ginseng stems and leaves prepared in example 1.
FIG. 3 is a LC-MS ion flow diagram of total flavonoids in ginseng stem leaves prepared in example 1.
Detailed Description
The invention provides a method for simultaneously preparing total saponins, total flavonoids and oil unsaponifiable matters of ginseng stems and leaves, which comprises the following steps and conditions:
the method comprises the following steps: processing dried stems and leaves of Ginseng radix into powder, adding petroleum ether, sodium hydroxide, n-hexane, ethyl acetate, acetonitrile and water, and extracting by magnetic stirring; the extraction temperature is preferably 100-200 DEG CThe time is preferably 0.5-2 hours, and the stirring speed is preferably 100-300 rpm; the mass M of the ginseng stem and leaf powder1(g): volume V of Petroleum Ether1(ml): mass M of sodium hydroxide2(g): volume V of n-hexane2(ml): volume V of Ethyl acetate3(ml): volume V of acetonitrile4(ml): volume V of water5(ml) is preferably 1: (10-50): (0.2-0.5): (10-50): (10-50): (20-70): (10-50);
step two: standing after extraction, wherein the standing time is preferably 60-300 min, collecting the upper layer solution, and concentrating under reduced pressure or normal pressure to obtain unsaponifiable matter of ginseng stem and leaf oil; the unsaponifiable matter of the ginseng stem and leaf grease is 3,7,11,15-Tetramethyl-2-hexadecen-1-ol (phytol), gamma-Sitosterol (gamma-Sitosterol), Stigmasta-5,24(28) -dien-3-ol (3 beta, 24Z) -;
step three: taking the intermediate layer solution, concentrating to 1/4-1/10 of the original volume, and loading on a macroporous resin column, wherein the type of the macroporous resin is preferably D101, D130, AB8 or NKA, and the mass g of the macroporous resin is as follows: the preferable weight g of the ginseng stem and leaf is 1: (0.5 to 2); eluting with water until the water eluent is colorless, clear and transparent and has a pH value of 7, and then eluting with an ethanol solution, wherein the volume ratio of the ethanol solution is preferably 40-60%; eluting until the ethanol eluate is colorless, clear and transparent, collecting the ethanol eluate, recovering ethanol, and drying to obtain dry extract-like ginseng stem and leaf total flavonoids; the total flavonoids of the ginseng stem leaves are rutin, ginseng flavonoid glycoside, quercetin and kaempferol;
step four: taking the lower layer solution, concentrating to 1/4-1/10 of the original volume, and loading on a macroporous resin column, wherein the type of the macroporous resin is preferably D101, D130, AB8 or NKA, and the mass g of the macroporous resin is as follows: the preferable weight g of the ginseng stem and leaf is 1: (0.5 to 2); eluting with water until the water eluent is colorless, clear and transparent and has a pH value of 7, and then eluting with an ethanol solution, wherein the volume ratio of the ethanol solution is preferably 40-60%; eluting until the ethanol eluate is colorless, clear and transparent, collecting the ethanol eluate, recovering ethanol, and drying to obtain dry extract of total saponins of caulis Et folium Ginseng, wherein the total saponins of caulis Et folium Ginseng are ginsenoside 20R-Rg3 and 20S-Rg 3.
The present invention is further illustrated by reference to the following specific examples, in which the starting materials are all commercially available.
Example 1
Processing 5g of dried stems and leaves of ginseng into powder, adding 50mL of petroleum ether, 1 g of sodium hydroxide, 60 mL of n-hexane, 60 mL of ethyl acetate, 100 mL of acetonitrile and 80mL of water, and extracting by a magnetic stirring method at the extraction temperature of: 100oAnd C, heating for 0.5 hour, stirring at 100 rpm, standing for 60min after heating, collecting the upper solution, and concentrating under reduced pressure or normal pressure to obtain unsaponifiable matter of ginseng stem leaf oil. Concentrating the intermediate layer solution to 1/4 of the original volume, loading into a macroporous resin column of 2.5g, wherein the model of the macroporous resin is D101, eluting with water until the water eluent is colorless, clear and transparent, and the pH value is 7, eluting with 40% ethanol solution by volume until the ethanol eluent is colorless, clear and transparent, collecting the ethanol eluent, recovering ethanol, and drying to obtain dry extract-like ginseng stem leaf total flavonoids; and (3) concentrating the lower layer solution to 1/4 of the original volume, loading the lower layer solution onto a macroporous resin column of 2.5g, wherein the model of the macroporous resin is D101, eluting with water until the water eluent is colorless, clear and transparent and the pH value is 7, eluting with an ethanol solution of which the volume ratio is 40% until the ethanol eluent is colorless, clear and transparent, collecting the ethanol eluent, recovering ethanol, and drying to obtain the dry extract of the ginseng stem leaf total saponins.
The unsaponifiable matter of the ginseng stem and leaf fat prepared in example 1 was analyzed by a combination of gas chromatography and mass spectrometry. The gas chromatograph-mass spectrometer comprises: U.S. Thermoscientific-ITQ 900.
Mass spectrum parameters: an EI source; emission current: 150 muA; electron energy: 70 eV; interface temperature: 280 ℃; ion source temperature: 250 ℃ to obtain a mixture; detecting voltage: 1000V.
Chromatographic parameters: initial temperature of chromatography: 60 ℃; initialization time: 3.0 min; the heating rate is as follows: 9 ℃ per min; end temperature of chromatography: 290 ℃ C; end temperature residence time: and 2 min.
Sample introduction parameters: sample injector temperature: 280 ℃; sample introduction mode: constant current; sample introduction volume: 1 mu L of the solution; a chromatographic column TR-5ms, 30m 0.25mm 0.25 μm; carrier gas: and (e) He.
Fig. 1 is a gas chromatography-mass spectrometry ion-flow diagram of an oil-unsaponifiable matter of ginseng stem leaves prepared in example 1. Fig. 1 shows that all the chromatographic peaks are almost completely eluted within 30 minutes, the obtained chromatographic peaks are completely separated, the sample chromatogram comprises 8 main chromatographic peaks, wherein the peak area of 3 chromatographic peaks is the largest, which shows that the content is higher, and the peak area is respectively 3,7,11,15-Tetramethyl-2-hexadecen-1-ol (phytol), gamma-Sitosterol (gamma-Sitosterol), Stigmasta-5,24(28) -dien-3-ol, (3 beta, 24Z) -, and the content of the 3 compounds accounts for more than 60% of the total unsaponifiable matter by using a normalization method.
Qualitative identification is carried out on chromatographic peaks in a general ion flow diagram of unsaponifiable matters of ginseng stem leaves by applying mass spectrometry, and the results are as follows: chromatographic peak 1: phenol, 2,5-bis (1, 1-dimethyllethyl) - (degree of matching 99%), chromatogram peak 2: 1,1,6, 6-tetramethylpiro [4.4] none (degree of matching 99%), chromatogram peak 3: 3,7,11, 15-tetramethylol-2-hexadecen-1-ol (matching degree 91%); chromatographic peak 4: gamma-Sitosterol (degree of matching 93%), chromatographic peak 5: stigmasta-5,24(28) -dien-3-ol, (3 β,24Z) - (degree of match 95%), peak 6: unknow, chromatographic peak 7: betulin (degree of matching 99%).
Table 1 shows the peak area data of unsaponifiable matter of ginseng stem leaves by gas chromatography and mass spectrometry.
TABLE 1 gas chromatography and mass spectrometry detection data of unsaponifiable matter chemical components of ginseng stem and leaf oil
Chromatographic peak Retention time (min) Molecular formula Molecular weight Name of Compound
1 15.33 C14H22O 206 Phenol, 2,5-bis(1,1-dimethylethyl)-
2 19.03 C13H24 180 1,1,6,6-Tetramethylspiro[4.4]nonane
3 21.71 C20H40O 296 3,7,11,15-Tetramethyl-2-hexadecen-1-ol
4 26.22 C29H50O 414 γ-Sitosterol
5 26.57 C29H48O 412 Stigmasta-5,24(28)-dien-3-ol, (3β,24Z)-
6 27.56 — — Unknown (Unknown)
7 28.26 C30H50O2 442 Betulin
The ginsenosides obtained in example 1 were detected by a combination of liquid chromatography and mass spectrometry. The liquid chromatogram and mass spectrum combined instrument comprises: Thermoscientific-LCQ FLUET type, USA.
Chromatographic conditions are as follows: the chromatographic column was Waters (4.6 mm. times.250 mm, 5 μm), mobile phase A was acetonitrile, and mobile phase B was 0.2% acetic acid aqueous solution. The elution condition is 0-20 min: 25-50% of A and 75-50% of B; 20-60 min: 50-90% of A and 50-10% of B; 60-70 min: 90-100% of A and 10-0% of B. Flow rate: 0.5 mL/min; sample introduction amount: 10 mu L of the solution; detection wavelength: 203 nm; column temperature: at 30 ℃.
Mass spectrum conditions: spraying voltage: 4.5kV, metal capillary temperature: 250V and the sheath gas is N2Pressure of 6.8X 102kV, auxiliary gas He, pressure: 1.0X 102kpa, negative ion mode, scan range: m/z: 150 to 2000.
FIG. 2 is a liquid chromatography-mass spectrometry ion-flow diagram of total saponins in ginseng stems and leaves prepared in example 1. Fig. 2 shows that all chromatographic peaks are eluted substantially completely within 60 minutes and the resulting chromatographic peaks are completely separated. The total saponins of caulis Et folium Ginseng prepared by this method contain ginsenoside 20R-Rg3 and 20S-Rg 3. Wherein (1) Re + Rg1, (2) Rf, (3) Rg2, (4) Rb1, (5) esterified-Rb 1, (6) Rc, (7) Rb2, (8) Rd, (9) Rb3, (10) Ro, (11) Rg6, (12) F4, (13) Rk3, (14) Rh4, (15) 20R-Rg3, and (16) 20S-Rg 3. The sample chromatogram comprises 16 main chromatographic peaks, qualitative identification is carried out on the chromatographic peaks in the total ion flow diagram of the ginsenoside by applying mass spectrometry, and the result is shown in table 2.
TABLE 2 liquid chromatography and mass spectrometry detection data of ginseng stem leaf saponin chemical components
Figure DEST_PATH_IMAGE002
The total saponins of the ginseng stem and leaf prepared by the method mainly comprise the ginsenosides Re + Rg1, Rf, Rg2, Rb1, esterified-Rb 1, Rc, Rb2, Rd, Rb3, Ro, Rg6, F4, Rk3, Rh4, 20R-Rg3 and 20S-Rg 3.
The ginsenosides obtained in example 1 were detected by a combination of liquid chromatography and mass spectrometry. The liquid chromatogram and mass spectrum combined instrument comprises: Thermoscientific-LCQ FLUET type, USA.
Chromatographic conditions are as follows: the chromatographic column is Waters SunfireTMC18 chromatographic column (250 mm × 4.6mm, 5 μm) with a column temperature of 30%oC; mobile phase composition and elution process: mobile phase (A): 0.05% aqueous acetic acid; (B) the method comprises the following steps Acetonitrile, 13% of B, 0-20 min; 13-40% of B for 20-60 min; 40-100% B for 60-90 min; the flow rate of the mobile phase is 0.5 mL/min; the wavelength is 250 nm; the amount of the sample was 10. mu.L.
Mass spectrum conditions: spraying voltage: 4.5kV, metal capillary temperature: 250V and the sheath gas is N2Pressure of 6.5X 102kV, auxiliary gas He, pressure: 1.0X 102kpa, negative ion mode, scan range: m/z: 150 to 2000.
The resulting sample solution was analyzed. FIG. 3 is a LC-MS ion flow diagram of total flavonoids in ginseng stem leaves prepared in example 1. The results show that all chromatographic peaks are eluted substantially completely within 90 minutes and the resulting chromatographic peaks are completely separated. The sample chromatogram comprises 5 main chromatographic peaks, and the total flavone of Ginseng radix stem and leaf prepared by the methodContains (1) Rutin and (2) Kaempferol-3-O-galactoside-O-glucoside (kaempferol-3-)OGalactose-OGlucoside (3) Kaempferol-3-O-glucoside (astragalin), (4) Quercetin (Quercetin), (5) Kaempferol (Kaempferol). The mass spectrometry is applied to qualitatively identify chromatographic peaks in the general ion flow graph of ginseng stem leaf flavone, and the results are shown in table 3.
TABLE 3 liquid chromatography and mass spectrometry detection data for flavonoids in ginseng stem and leaf
Chromatographic peak Name of Compound Retention time (min) Peak material of quasi-molecular ion Spectral data (m/z) Molecular weight (Da) Secondary mass spectral data (m/z)
1 Rutin (Rutin) 5.43 609 [M-H]- 610 447 [M-glu-H]-, 301 [M-glu-rha-H]-
2 Kaempferol-3-O-galactoside-O-glucoside (Kaempferol-3-OGalactose-O-glucoside) 29.56 609 [M-H]- 610 447 [M-glu-H]-, 285 [M-2Glc-H]-
3 Kaempferol-3-O-glucoside (astragalin) 31.77 447 [M-H]- 448 284 [M-C6H10O5-H]- , 151[1,3A]–, 179 [1,2A-H]–
4 Quercetin (Quercetin) 45.39 301 [M-H]- 302 271 [M-CH2O]–, 255 [M-H2O-CO-H]–, 151[1,3A]–, 179 [1,2A-H]–
5 Kaempferol (Kaempferol) 49.19 284 [M-H]- 286 255 ([M -H2O-CO-H]–), 151(1,3A–), 179([1,2A-H]–)
Shows that the total flavone of the stem and leaf of the ginseng prepared by the method mainly comprises Rutin and Kaempferol-3-O-galactoside-O-glucoside (kaempferol-3-)OGalactose-OGlucoside), Kaempferol-3-O-glucoside (astragalin), Quercetin (Quercetin), Kaempferol (Kaempferol).
Example 2
Processing 5g of dried stems and leaves of ginseng into powder, adding 80mL of petroleum ether, 1.5g of sodium hydroxide, 100 mL of n-hexane, 100 mL of ethyl acetate, 130 mL of acetonitrile and 140 mL of water, and extracting by a magnetic stirring method at the extraction temperature of: 150 oCHeating for 1.0 hr, stirring at 150 rpm, standing for 90 min after heating, collecting the upper layer solution, and concentrating under reduced pressure or normal pressure to obtain unsaponifiable matter of ginseng stem and leaf oil. Concentrating the intermediate layer solution to 1/5 of the original volume, loading into a macroporous resin column of 3.5 g, wherein the model of the macroporous resin is D130, eluting with water until the water eluent is colorless, clear and transparent, and the pH value is 7, eluting with 50% ethanol solution by volume until the ethanol eluent is colorless, clear and transparent, collecting the ethanol eluent, recovering ethanol, and drying to obtain dry extract-like ginseng stem leaf total flavonoids; and (3) concentrating the lower layer solution to 1/5 of the original volume, loading the lower layer solution onto a 4.5 g macroporous resin column with the model of D101, eluting with water until the water eluent is colorless, clear and transparent and the pH value is 7, eluting with 50% ethanol solution by volume until the ethanol eluent is colorless, clear and transparent, collecting the ethanol eluent, recovering ethanol, and drying to obtain the dry extract of the ginseng stem leaf total saponins.
Example 3
Processing 5g of dried stems and leaves of ginseng into powder, adding 110 mL of petroleum ether, 1.8 g of sodium hydroxide, 160 mL of n-hexane, 120 mL of ethyl acetate, 190 mL of acetonitrile and 200mL of water, and extracting by a magnetic stirring method at the extraction temperature of: 180 oCHeating for 1.8 hours at a stirring speed of 280 rpm, heatingStanding for 120 min, collecting the upper layer solution, and concentrating under reduced pressure or normal pressure to obtain unsaponifiable matter of caulis Et folium Ginseng oil. Concentrating the intermediate layer solution to 1/6 of the original volume, loading into a 5.0 g macroporous resin column of which the model is AB8, eluting with water until the water eluent is colorless, clear and transparent and the pH value is 7, then eluting with 60% ethanol solution by volume until the ethanol eluent is colorless, clear and transparent, collecting the ethanol eluent, recovering ethanol, and drying to obtain dry extract-like ginseng stem leaf total flavonoids; collecting the lower layer solution, concentrating to 1/5 of the original volume, loading onto 6.0g macroporous resin column with model D130, eluting with water until the water eluate is colorless, clear and transparent, and has pH of 7, eluting with 60% ethanol solution until the ethanol eluate is colorless, clear and transparent, collecting ethanol eluate, recovering ethanol, and drying to obtain dry extract of total saponins of caulis Et folium Ginseng.
Example 4
Processing 5g of dried stems and leaves of ginseng into powder, adding 250mL of petroleum ether, 2.5g of sodium hydroxide, 200mL of n-hexane, 190 mL of ethyl acetate, 300mL of acetonitrile and 200mL of water, and extracting by a magnetic stirring method at the extraction temperature of: 200 oCHeating for 2 hr at stirring speed of 300 rpm, standing for 180 min after heating, collecting the upper layer solution, and concentrating under reduced pressure or normal pressure to obtain unsaponifiable matter of ginseng stem and leaf oil. Concentrating the intermediate layer solution to 1/8 of the original volume, loading into a macroporous resin column of 8.0g, wherein the type of the macroporous resin is NKA, eluting with water until the water eluate is colorless, clear and transparent, and has a pH value of 7, eluting with 50% ethanol solution by volume until the ethanol eluate is colorless, clear and transparent, collecting the ethanol eluate, recovering ethanol, and drying to obtain dry extract-like ginseng stem-leaf total flavonoids; collecting the lower layer solution, concentrating to 1/5 of original volume, loading onto 6.0g macroporous resin column (type AB 8), eluting with water until the water eluate is colorless, clear and transparent, and has pH of 7, eluting with 60% ethanol solution until the ethanol eluate is colorless, clear and transparent, collecting ethanol eluate, recovering ethanol, and drying to obtain dry extract of total saponins of caulis Et folium Ginseng.
Example 5
Processing 5g of dried stems and leaves of ginseng into powder, adding 250mL of petroleum ether, 2.5g of sodium hydroxide, 250mL of n-hexane, 250mL of ethyl acetate, 350 mL of acetonitrile and 250mL of water, and extracting by a magnetic stirring method at the extraction temperature of: heating at 200 deg.C for 2 hr, stirring at 300 rpm, standing for 240 min, collecting the upper layer solution, and concentrating under reduced pressure or normal pressure to obtain unsaponifiable matter of ginseng stem and leaf oil. Concentrating the intermediate layer solution to 1/10 of the original volume, loading into a 10g macroporous resin column with the model of D101, eluting with water until the water eluate is colorless, clear and transparent and has a pH value of 7, eluting with 60% ethanol solution, eluting until the ethanol eluate is colorless, clear and transparent, collecting the ethanol eluate, recovering ethanol, and drying to obtain dry extract-like ginseng stem and leaf total flavonoids; and (3) concentrating the lower layer solution to 1/10 of the original volume, loading the lower layer solution onto a 10g macroporous resin column with the type of NKA, eluting with water until the water eluate is colorless, clear and transparent and the pH value is 7, eluting with 60% ethanol solution by volume until the ethanol eluate is colorless, clear and transparent, collecting the ethanol eluate, recovering ethanol, and drying to obtain the dry extract of the ginseng stem leaf total saponins.
Example 6
Processing 5g of dried stems and leaves of ginseng into powder, adding 100 mL of petroleum ether, 2.0 g of sodium hydroxide, 180 mL of n-hexane, 150 mL of ethyl acetate, 220 mL of acetonitrile and 250mL of water, and extracting by a magnetic stirring method at the extraction temperature of: 180 oCHeating for 1.8 hr at stirring speed of 250 rpm, standing for 300 min after heating, collecting the upper layer solution, and concentrating under reduced pressure or normal pressure to obtain unsaponifiable matter of ginseng stem and leaf oil. Concentrating the intermediate layer solution to 1/8 of the original volume, loading into a macroporous resin column of 8.0g, wherein the model of the macroporous resin is AB8, eluting with water until the water eluent is colorless, clear and transparent, and the pH value is 7, eluting with 55% ethanol solution by volume until the ethanol eluent is colorless, clear and transparent, collecting the ethanol eluent, recovering ethanol, and drying to obtain dry extract-like ginseng stem leaf total flavonoids; collecting the lower layer solution, concentrating to 1/5, and purifying with 5.5 g macroporous resin column (NKA type)Eluting with water until the water eluate is colorless, clear and transparent, and has pH value of 7, eluting with 55% ethanol solution until the ethanol eluate is colorless, clear and transparent, collecting the ethanol eluate, recovering ethanol, and drying to obtain dry extract of total saponins of caulis Et folium Ginseng.

Claims (7)

1. A method for simultaneously preparing total saponins, total flavonoids and unsaponifiable matters of oil and stems of ginseng is characterized by comprising the following steps and conditions:
the method comprises the following steps: processing dried stems and leaves of Ginseng radix into powder, adding petroleum ether, sodium hydroxide, n-hexane, ethyl acetate, acetonitrile and water, and extracting by magnetic stirring;
step two: standing after extraction, collecting the upper layer solution, and concentrating under reduced pressure or normal pressure to obtain unsaponifiable matter of ginseng stem and leaf oil;
step three: concentrating the intermediate layer solution to 1/4-1/10 of the original volume, loading onto macroporous resin column, eluting with water until the water eluate is colorless, clear and transparent, and has pH of 7, eluting with ethanol solution until the ethanol eluate is colorless, clear and transparent, collecting ethanol eluate, recovering ethanol, and drying to obtain dry extract-like ginseng stem leaf total flavone;
step four: collecting the lower layer solution, concentrating to 1/4-1/10 of original volume, loading onto macroporous resin column, eluting with water until the water eluate is colorless, clear and transparent, and has pH of 7, eluting with ethanol until the ethanol eluate is colorless, clear and transparent, collecting ethanol eluate, recovering ethanol, and drying to obtain dry extract of total saponins of caulis Et folium Ginseng;
the mass M of the ginseng stem leaf powder in the step one1: volume V of Petroleum Ether1: mass M of sodium hydroxide2: volume V of n-hexane2: volume V of Ethyl acetate3: volume V of acetonitrile4: volume V of water51 g: (10-50) ml: (0.2-0.5) g: (10-50) ml: (10-50) ml: (20-70) ml: (10-50) ml;
the first step is extracting at 100-200 deg.C for 0.5-2 hr at a stirring speed of 100-300 rpm.
2. The method for simultaneously preparing total saponins, total flavonoids and unsaponifiable matter of ginseng stems and leaves according to claim 1, wherein the unsaponifiable matter of ginseng stems and leaves in the second step is 3,7,11,15-Tetramethyl-2-hexadecen-1-ol, γ -Sitosterol, Stigmasta-5,24(28) -dien-3-ol, (3 β,24Z) -.
3. The method for simultaneously preparing the total saponins, the total flavonoids and the oil unsaponifiable matters of the ginseng stems and leaves according to claim 1, wherein the macroporous resin of the third step and the fourth step is D101, D130, AB8 or NKA.
4. The method for simultaneously preparing the total saponins, the total flavonoids and the oil unsaponifiable matters of the ginseng stems and leaves according to claim 1, wherein the mass g of the macroporous resin in the third step and the fourth step is as follows: the mass g of the ginseng stem and leaf is 1: (0.5-2).
5. The method for simultaneously preparing the total saponins, the total flavonoids and the unsaponifiable oil of the ginseng stems and leaves according to claim 1, wherein the volume ratio of the ethanol solution in the third step to the fourth step is 40-60%.
6. The method for simultaneously preparing total saponins, total flavonoids and oil unsaponifiable matters of ginseng stems and leaves according to claim 1, wherein the total flavonoids of ginseng stems and leaves in the third step are rutin, ginsenosides, quercetin and kaempferol.
7. The method for simultaneously preparing the total saponins, the total flavonoids and the oil unsaponifiable matters of the ginseng stems and leaves according to claim 1, wherein the total saponins of the ginseng stems and leaves in the fourth step are ginsenoside 20R-Rg3 and 20S-Rg 3.
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