CN115093452B - A method for preparing folium Platycladi extract - Google Patents
A method for preparing folium Platycladi extract Download PDFInfo
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Abstract
The invention discloses a preparation method of a cacumen biotae extract, belonging to the technical field of plant extraction. The preparation method comprises the following steps: distilling Chinese arborvitae leaf raw material with microwave-assisted steam, and collecting essential oil and water extract; microwave alcohol extraction of the material dregs, merging water extract and alcohol extract, concentration and solid-liquid separation; passing the solution through macroporous resin, and eluting; concentrating the eluate, passing through polyamide macroporous resin, gradient eluting with ethanol water solution, concentrating and drying the concentrated solution of the target substance to obtain crude extract of folium Platycladi; crystallizing the crude extract with ethanol water solution for multiple times to obtain quercetin extract and myricitrin extract. The invention comprehensively utilizes the effective components in the raw material of the arborvitae leaves and prepares the high-purity extract.
Description
Technical Field
The invention relates to the technical field related to plant extraction, in particular to a preparation method of a cacumen biotae extract.
Background
The cacumen Platycladi is derived from branch tip and leaf of Chinese arborvitae (L.) (Thunb.) of Cupressaceae, is collected in summer and autumn, dried in the shade, has fragrant smell and bitter taste, and has effects of entering lung, liver and spleen channels, cooling blood, stopping bleeding, eliminating phlegm, relieving cough, promoting hair growth and blackening hair, and can be used for treating hematemesis, epistaxis, hemoptysis, hematochezia, metrorrhagia, metrostaxis, lung heat cough, blood heat alopecia, and premature graying of beard and hair. The folium Platycladi chemical components mainly comprise volatile oil, flavonoids, tannin, etc., and the main active components are flavonoids, wherein the total flavonoids have effects of inhibiting alpha-glycosidase, resisting RBC oxidative damage, activating hair mother cell, promoting blood circulation, caring hair, and promoting hair growth. Plant essential oils (cacumen biotae essential oil, etc.) are often used in air fresheners not only for their fragrance effect but also for their bacteriostatic activity. The application of natural plant essential oil in the aspects of disinfection and sterilization, mildew prevention and sterilization, and preservation and fresh keeping is widely accepted. The plant essential oil is used as a natural bacteriostatic agent, and compared with a chemically synthesized bacteriostatic agent, the plant essential oil has the incomparable characteristics of high safety coefficient, no drug resistance, good acceptance by people and the like.
In the related technology, the content of tannin in the cacumen Platycladi is measured to be within 6% by adopting a hide powder method, and in the related technology, the content of total flavone in cacumen Platycladi is measured to be within 1.0% -4.29% by taking the total flavone in the cacumen Platycladi as a research object. The folium Platycladi contains various flavonoids including myricitrin, quercetin, amentoflavone, hinokiflavone, neocedar biflavone, amyrin, rutin, etc., wherein the content of quercetin is highest.
The related art adopts supercritical CO 2 The extraction technology realizes the simultaneous obtaining of volatile oil and total flavone by controlling reaction conditions. However, the purity of the flavonoid compound prepared by the method is not high, only about 46 percent, and the separation of quercetin and myricitrin is not realized. Also disclosed in the related art is an extraction method ofThe method comprises the following steps: extracting with 70% ethanol water solution, directly passing the extractive solution through AB-8 macroporous resin, and collecting the column-passing solution; then eluting with ethanol water solution with the volume fraction of 1BV being 70%; mixing the column-passing solution with 70% ethanol water solution eluate, concentrating under reduced pressure until the volume fraction of ethanol is 40%, passing through polyamide resin, and eluting with 80% ethanol water solution to obtain extract with total flavone content of more than 50%. The method has low extract purity, and does not fully utilize effective components of folium Platycladi, nor realize separation of quercetin and myricitrin.
In summary, it is necessary to develop a method for preparing cacumen Platycladi extract, which can be used to prepare high-purity quercetin and myricitrin.
Disclosure of Invention
The present invention is directed to solving at least one of the problems of the prior art. Therefore, the invention provides a preparation method of the cacumen biotae extract, and high-purity quercetin and myricitrin are prepared by the method.
The method comprises the following specific steps: the invention provides a preparation method of a cacumen biotae extract, which comprises the following steps:
s1, distilling a Chinese arborvitae leaf raw material by microwave-assisted steam, and obtaining an essential oil extract, a water extracting solution and raw material residues after the distillation is finished;
s2, extracting the raw material residues by adopting microwave alcohol to prepare alcohol extract;
s3, mixing the water extract and the alcohol extract, concentrating, and performing solid-liquid separation to obtain a solid and a concentrated solution;
s4, passing the concentrated solution through macroporous resin, and eluting by using water and ethanol water; collecting the ethanol water solution eluent;
s5, concentrating the ethanol water solution eluent, passing through polyamide resin, and sequentially eluting by using water, 30-40% of ethanol water solution in volume fraction and 70-80% of ethanol water solution in volume fraction;
collecting 70-80% ethanol water solution eluate, concentrating to dry to obtain folium Platycladi crude extract;
s6, recrystallizing the crude cacumen biotae extract to obtain a cacumen biotae fine extract;
s7, dissolving the cacumen biotae extract by using 65-75% of ethanol water solution in volume fraction, and then crystallizing for the first time; performing solid-liquid separation, and collecting the solid phase after the first crystallization as a quercitrin extract;
collecting the liquid phase, concentrating, crystallizing for the second time, and performing solid-liquid separation again; and collecting the solid phase after the second crystallization to be the myricitrin extract.
According to one technical scheme of the preparation method provided by the invention, the preparation method at least has the following beneficial effects:
in the invention, microwave assistance is adopted in step S1 and step S2, and the temperature of cells in the micro-tube bundle and glandular cell system in the cacumen biotae rapidly rises (due to absorption of microwave energy) under the action of microwaves; thereby causing the pressure inside the cell to exceed the bearing limit of the expansion of the cell wall and the cell to break; the effective components in the cell can freely flow out.
The steam distillation is pollution-free and low in cost; in the microwave-assisted steam distillation process in the step S1, the cells are cracked by the microwaves, and all volatile oil freely flows out to become free volatile oil, so that the steam distillation process is greatly accelerated, and the distillation time is saved.
Volatile oil can be extracted in a short time by microwave-assisted steam extraction, and the components such as quercetin and myricitrin are degraded under the condition of long-time heating at 100 ℃, so that the microwave-assisted steam extraction method has great advantages compared with direct steam extraction; and microwave-assisted ethanol water extraction is adopted to extract flavonoid components as far as possible and ensure that the effective components are not damaged to the maximum extent. Simultaneously, the water extract and the alcohol extract are combined, so that the yield of the flavonoid compound is further improved.
In the step S4, the myricitrin and the quercitrin are primarily purified through macroporous resin, and most polysaccharides, proteins, tannins and other flavonoid impurities with large polarity difference are removed.
In the process of passing through polyamide resin, water is used for removing impurities with larger polarity, such as polysaccharide, and the like, and low-concentration ethanol water solution (30-40%) is used for removing polyphenol impurities with larger polarity, such as chlorogenic acid, brevifolin carboxylic acid, and the like; thereby realizing that the quercitrin and the myricitrin are enriched in high-concentration ethanol water solution (70 to 80 percent).
And S6, recrystallizing to remove flavonoid impurities with polarity close to that of the myricitrin or the quercitrin, such as myricetin, quercetin, isoquercitrin and the like, and partial myricitrin and quercitrin which are not completely separated.
In the step S7, the solubility difference of the quercetin and the myricitrin in the ethanol water solution with the same concentration is utilized, so that the quercetin and the myricitrin are separated.
The method simultaneously obtains the components of the cacumen platycladi essential oil, the myricitrin, the quercetin and the like through microwave-assisted steam extraction, macroporous resin primary purification, polyamide resin enrichment and repeated crystallization, the purities of the myricitrin and the quercetin are high (both more than 92 percent), other organic reagents are not used except ethanol in the whole process, the industrial production can be realized, and the economic benefit is high.
The cacumen biotae essential oil has the effects of resisting insects, bacteria, oxidation and cancer and the like, and has good application prospect in the aspects of development of natural insecticides, preservatives and health care products.
The main effective component of the cacumen biotae for relieving cough, eliminating phlegm and stopping bleeding is quercetin glycoside in flavonoids compounds, and the content of the quercetin glycoside in the cacumen biotae raw material is 0.2-0.8%.
The quercetin is also called quercetin-3-O-rhamnoside, and the molecular formula is as follows: c 21 H 20 O 11 The CAS number is: 522-12-3, molecular weight: 448.38, acidity index: 6.17. + -. 0.40 (predicted), almost insoluble in cold water, soluble in hot water, ethanol, alkaline solution. The structural formula is shown as the following formula:
the myricitrin component in the arborvitae leaves has the functions of contracting blood vessels, reducing blood sugar, resisting oxidation, protecting liver, benefiting gallbladder, resisting inflammation, resisting mutation, resisting tumor and relieving pain, is one of effective components in arborvitae, and has the content of 0.1-0.5 percent in the raw materials.
The molecular formula of myricitrin is as follows: c 21 H 20 O 12 Molecular weight 464.38, CAS number: 17912-87-7, acidity coefficient: 6.16 +/-0.40 (predicted value), is easy to dissolve in water and can be dissolved in methanol, ethanol and glacial acetic acid. The structural formula of myricitrin is shown as the following formula:
according to some embodiments of the invention, the arborvitae leaf raw material is dried arborvitae shoot and/or dried arborvitae leaf.
According to some embodiments of the invention, the raw material of biota orientalis leaves is crushed to 40-60 mesh.
According to some embodiments of the invention, the mass to volume ratio of the biota orientalis leaf raw material to water in the microwave-assisted steam distillation is 1g to 8ml to 12mL.
According to some embodiments of the invention, the microwave-assisted steam distillation is at a temperature of 95 ℃ to 100 ℃.
According to some embodiments of the invention, the microwave power in the microwave-assisted steam distillation is between 300W and 400W.
According to some embodiments of the invention, the microwave time in the microwave-assisted steam distillation is 12min to 20min.
Microwave energy is too low, resulting in a high ratio of heat dissipation to heat buildup in the extracted system, and a longer period of time for the temperature of the entire system to accumulate to the point where steam is produced in large quantities.
When the microwave power is too high, the heat of the system is too concentrated, so that a large amount of foam is likely to fiercely gush upwards, and certain adverse effect is caused on steam distillation.
The extraction time decreases rapidly with increasing power, and the extraction rate of essential oil decreases slightly at higher power. This is because too high a microwave power causes the evaporation of water to be intensified, resulting in the loss of part of the volatile components with a sudden burst of water vapour into the external environment.
According to some embodiments of the invention, the essential oil extract is an essential oil of cacumen biotae after dehydration treatment.
According to some embodiments of the invention, the dehydration treatment uses anhydrous sodium sulfate or anhydrous magnesium sulfate.
The essential oil extract is efficiently dehydrated by selecting anhydrous sodium sulfate or anhydrous magnesium sulfate, so that the quality of the cacumen biotae essential oil is improved.
According to some embodiments of the invention, the extraction agent in the microwave alcohol extraction is 40% to 80% ethanol aqueous solution.
According to some embodiments of the invention, the extraction agent in the microwave alcohol extraction is 50% to 70% ethanol aqueous solution.
The extraction effect of the quercetin and myricitrin is improved by controlling the concentration of ethanol in the extractant; meanwhile, the dissolution of substances with larger fat solubility is reduced, and the extraction of substances with large polarity is avoided.
According to some embodiments of the present invention, the mass-to-volume ratio of the raw material residue to the extractant in the microwave alcohol extraction is 1g.
According to some embodiments of the invention, the extraction times of the microwave alcohol extraction are 1 to 2 times.
According to some embodiments of the invention, the number of extraction times of the microwave alcohol extraction is 1.
According to some embodiments of the invention, the power of the microwaves in the microwave alcohol extraction is 300W-400W.
Within a certain range, the extraction rate of myricitrin and quercitrin is increased along with the increase of power, but the microwave power is too high, so that heat is too concentrated, and the extraction effect is also influenced.
According to some embodiments of the invention, the time of the microwave in the microwave alcohol extraction is 30min to 40min.
The microwave time is too short, and the extraction rate of myricitrin and quercitrin is lower; too long a microwave time increases energy losses.
According to some embodiments of the invention, the temperature of the microwave alcohol extraction is 40 ℃ to 60 ℃.
The microwave temperature is too low, and the active ingredients are difficult to be completely extracted; when the temperature is too high, myricitrin and quercitrin are all heat-sensitive components, and the degradation loss is large.
According to some embodiments of the invention, the concentration in step S3 is to a volume of 1/18 to 1/15 of the initial volume.
According to some embodiments of the invention, the solid in step S3 is a water-insoluble flavone extract.
According to some embodiments of the invention, the temperature of the concentrate in step S4 is below 60 ℃.
According to some embodiments of the invention, the macroporous resin is one of HPD722, HPD100, LAS-21.
According to some embodiments of the invention, the macroporous resin is eluted with water to colorless and then with an aqueous ethanol solution.
According to some embodiments of the invention, the rate of water elution is from 1BV/h to 2BV/h.
According to some embodiments of the invention, the aqueous ethanol solution is eluted at a rate of 1 to 2BV/h.
According to some embodiments of the invention, the volume fraction of the ethanol aqueous solution in step S4 is 55% to 70%.
The concentration of the elution ethanol is too low, the elution is incomplete, the concentration of the elution ethanol is too high, the elution rate of impurities is increased, the purity is low, and the purification work at the later stage is not facilitated.
According to some embodiments of the invention, the amount of the aqueous ethanol solution used in step S4 is 3.5BV to 4.5BV.
Too little elution solvent, incomplete elution, too much elution, solvent waste and reduced purity of the extract.
According to some embodiments of the invention, the polyamide resin is eluted with water to colorless and then with an aqueous ethanol solution.
According to some embodiments of the invention, the rate of water elution is from 1BV/h to 1.5BV/h.
According to some embodiments of the present invention, the 30 to 40 volume percent aqueous ethanol solution in step S5 is used in an amount of 2 to 2.5BV.
According to some embodiments of the invention, the 30% to 40% aqueous ethanol is eluted at a rate of 1BV/h to 1.5BV/h.
According to some embodiments of the present invention, the 70 to 80 vol% ethanol aqueous solution used in step S5 is used in an amount of 3 to 3.5BV.
According to some embodiments of the invention, the 70% to 80% aqueous ethanol is eluted at a rate of 1 to 1.5BV/h.
According to some embodiments of the invention, the volume fraction of ethanol in the concentrated solution after concentration in step S5 is 30% to 40%.
According to some embodiments of the invention, the recrystallization comprises dissolving by heating, filtering while hot, cooling for the first time, cooling again for crystallization, solid-liquid separation, and collecting the solid phase.
According to some embodiments of the present invention, the solvent used in the recrystallization during the dissolution by heating is 30 to 40% aqueous ethanol.
According to some embodiments of the present invention, the volume-to-mass ratio of the solvent used in the recrystallization during the dissolution by heating to the crude cacumen biotae extract is 8mL to 10mL:1g of the total weight of the composition.
According to some embodiments of the invention, the temperature of the recrystallization during the heated dissolution is 50 ℃ to 60 ℃.
The solubility of myricitrin and quercetin is increased along with the temperature rise, the influence of the water content of an alcoholic solution on the solubility of the quercetin is obviously larger than that of the myricitrin, the myricitrin is dissolved in low-concentration ethanol water in a hot saturation way, after cooling and cooling, the crystallization rate of the quercetin is far larger than that of the myricitrin due to the difference of the solubility change, and the quercetin is separated out before the myricitrin is crystallized, so that the separation effect is achieved. And after the high-concentration ethanol is dissolved in a hydrothermal saturated manner and cooled, the difference of the solubility changes of the two substances is reduced, and the separation effect is poor due to the fact that the quercetin is separated out and the myricitrin is separated out simultaneously.
The temperature is too low, the transfer effect of the effective components is poor, the temperature is too high, the separation effect is slightly poor, and the loss of the active components is large.
According to some embodiments of the invention, the final temperature of the recrystallization at the first cooling is 15 ℃ to 30 ℃.
The natural cooling is carried out before refrigeration, the cooling is too fast, the crystallization rate is too fast, more micro crystals are formed, the solid-liquid separation is difficult, and the collection cannot be carried out, so that the yield and the purity of the quercetin are affected.
According to some embodiments of the invention, the temperature of the recrystallization during the recooling crystallization is from 0 ℃ to 4 ℃.
When the temperature is controlled to be the recrystallization temperature, the quercetin can be crystallized in a short time, the separation effect is good, and the processing time is saved.
According to some embodiments of the invention, the recrystallization time during the recooling crystallization is 4h to 6h.
The refrigeration time is too short, the growth time of the quercetin crystal nucleus is short, the precipitated particles do not have enough time to grow and have no time to agglomerate, the formed particle size is generally small, solid-liquid separation is difficult, and the particles cannot be collected, so that the yield and the purity of the quercetin are affected.
According to some embodiments of the present invention, the mass volume ratio of the cacumen biotae extract and the ethanol aqueous solution with the volume fraction of 65% to 75% in the step S7 is 1g:4mL to 5.5mL.
According to some embodiments of the invention, the temperature of the dissolving in step S7 is 50 ℃ to 60 ℃.
According to some embodiments of the invention, the concentration in step S7 is to 2/5 to 1/3 of the original volume.
According to some embodiments of the invention, the concentration in step S7 is such that just crystals precipitate.
According to some embodiments of the invention, the temperature of the first crystallization is between 0 ℃ and 4 ℃.
According to some embodiments of the invention, the time for the first crystallization is 8h to 16h.
According to some embodiments of the invention, the temperature of the second crystallization is between 0 ℃ and 4 ℃.
According to some embodiments of the invention, the time for the second crystallization is 8h to 16h.
The invention finds that the solubility of myricitrin and quercetin is increased along with the temperature rise, the influence of the water content of an ethanol solution on the solubility of the quercetin is obviously larger than that of the myricitrin, the myricitrin is dissolved in low-concentration ethanol water in a hot saturation manner, after cooling and cooling, the crystallization rate of the quercetin is far higher than that of the myricitrin due to the difference of the solubility change rate, so that the separation effect is achieved, and the natural cooling is transited to refrigeration, so that the over-high crystallization rate of the quercetin can be prevented, and the formation of more micro crystals (which are easily dissolved in the solution again in the separation process, so that the quercetin crystals cannot be completely collected, the purity and the yield of the quercetin are influenced, and the separation effect is poor) is inhibited. The separation of the quercetin and the myricitrin is realized by simultaneously controlling various factors such as the ethanol concentration, the dissolving temperature, the cooling condition, the cold storage time and the like, the solvent crystallization separation method of the quercetin and the myricitrin is successfully developed, a new method is provided for industrial production of the quercetin and the myricitrin to replace a new chromatographic separation process in related industrial production, and the method has important meanings of energy conservation, emission reduction, greenness, high efficiency and the like.
Additional features and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.
Drawings
The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:
FIG. 1 is a liquid chromatogram of quercetin extract prepared in example 1 of the present invention.
FIG. 2 is a liquid chromatogram of the myricitrin extract prepared in example 1 of the present invention.
Detailed Description
The concept and technical effects of the present invention will be clearly and completely described below in conjunction with the embodiments to fully understand the objects, features and effects of the present invention. It is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments, and those skilled in the art can obtain other embodiments without inventive effort based on the embodiments of the present invention, and all embodiments are within the protection scope of the present invention.
In the description of the present invention, reference to the description of the terms "one embodiment," "some embodiments," "an illustrative embodiment," "an example," "a specific example," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
The examples, in which specific conditions are not specified, were carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
Producing area of the cacumen biotae raw material: shandong, essential oil content 3.64%, myricitrin: 0.34%, quercetin 0.59%, total flavonoids: 2.01% (content determined by the following method).
The essential oil is measured according to appendix of China pharmacopoeia 2020 edition-general rule 233 page: 2204 the first method of the volatile oil assay is carried out.
The liquid chromatography conditions of myricitrin and quercetin are as follows:
a chromatographic column: wondasil C18 (250 mm. Times.4.6 mm,5 μm).
Mobile phase: methanol: a 0.01mol/L potassium dihydrogen phosphate-glacial acetic acid solution (the volume fraction of glacial acetic acid in the solution is 1.5%) is 63:37.
flow rate: 1mL/min.
Detection wavelength: 254nm.
Sample introduction amount: 10 μ L.
The conditions of the detection method of the total flavone extract are as follows:
preparation of control solutions: taking a reference substance of quercetin (with a mass content of 99.90%, supplier: dowman Stent Biotechnology Co., ltd.), precisely weighing, and adding anhydrous methanol to obtain a reference substance solution with a concentration of 0.1 mg/mL.
The test solution is prepared by weighing appropriate amount of total flavonoids of cacumen Platycladi, adding into 50mL measuring flask, dissolving with anhydrous methanol, fixing the solution to scale, shaking, and filtering to obtain filtrate.
Drawing a standard curve: precisely sucking 1.0mL, 2.0mL, 4.0mL, 6.0mL, 8.0mL and 10.0mL of the quercetin reference substance solution, respectively placing in 25mL volumetric flasks, adding methanol to 6mL, adding 1mL of 5% sodium nitrite solution, shaking, standing for 6min, adding 1mL of 10% aluminum nitrate solution, shaking, standing for 6min, adding 10mL of 4% sodium hydroxide solution, adding water to scale, shaking, standing for l5min, filtering, taking the filtrate, performing ultraviolet-visible spectrophotometry, measuring absorbance at 428nm wavelength, and drawing a standard curve by using the absorbance as ordinate and the concentration as abscissa.
And (3) sample determination: measuring a proper amount of a test solution, placing the test solution in a 25mL volumetric flask, adding methanol to 6mL, adding 1mL of 5% sodium nitrite solution by mass, shaking up, placing for 6min, adding 1mL of 10% aluminum nitrate solution by mass, shaking up, placing for 6min, adding 10mL of 4% sodium hydroxide solution by mass, then adding methanol to a scale, shaking up, placing for l5min, filtering, taking a filtrate, and measuring the absorbance at a wavelength of 428nm by an ultraviolet-visible spectrophotometry method, and obtaining the content of the total flavonoids from a standard curve.
HPD722 macroporous resins and HPD100B macroporous resins were purchased from cangzhou baien sorbent materials technologies ltd.
LSA-21 macroporous resins were purchased from New scientific materials, inc. of Xian blue, dawn.
The polyamide resin was purchased from Tianjin Jinrilong Fine chemical Co., ltd.
The microwave-assisted steam distillation extraction is realized in a continuous power-adjustable microwave chemical reactor LWMC-205 (Nanjing Ling Jiangjiang science and technology development Limited liability company), the microwave power range is 0W-1000W, the minimum power scale is 20W, and the continuous power adjustment is realized.
Specific examples of the present invention are described in detail below.
Example 1
This example is a method for preparing cacumen biotae extract, comprising the following steps:
s1, crushing 500g of a raw material of oriental arborvitae into 60 meshes, placing the crushed raw material in a round-bottom flask, adding 12 times of water (6000 mL relative to the mass of the raw material), connecting a volatile oil extractor and a reflux condensing device to perform microwave-assisted steam distillation, wherein the microwave power is 300W, the distillation time is 20min, the distillation temperature is 100 ℃, and respectively collecting essential oil and water extract;
dehydrating the essential oil with anhydrous sodium sulfate, and then, carrying out dehydration treatment on 18g of cacumen biotae essential oil;
s2, continuously adding the raw material slag remained in the step S1 into an ethanol aqueous solution with a volume fraction of 50% (mass-to-volume ratio of the raw material slag to the ethanol aqueous solution is 1g, 12mL) for microwave-assisted extraction, wherein the microwave power is 300W, the extraction time is 40min, the extraction is carried out for 1 time, the extraction temperature is 60 ℃, and solid-liquid separation is carried out to prepare an alcohol extract; combining the alcohol extract in the step and the water extract in the step S1 to obtain an extract;
s3, concentrating the extracting solution under reduced pressure to 1/18 of the original volume, carrying out solid-liquid separation while the extracting solution is hot, and collecting a solid and a clear solution;
drying the solid to obtain 15.20g of water insoluble total flavone extract;
s4, passing the hot clear liquid (at the temperature of 58-60 ℃) obtained in the step S3 through LSA-21 macroporous resin, washing with water until the clear liquid is colorless (the elution rate is 1 BV/h), and then eluting with an ethanol water solution with the volume fraction of 3.5BV being 65% (the elution rate is 1 BV/h); collecting the ethanol eluent;
s5, concentrating the ethanol eluent obtained in the step S4 under reduced pressure, and controlling the concentration of the concentrated ethanol to be 30%; passing the concentrated solution through polyamide resin, washing with pure water to colorless (elution rate of 1.5 BV/h), and sequentially eluting with 2.5BV of ethanol aqueous solution with volume fraction of 40% (elution rate of 1.5 BV/h) and 4BV of ethanol aqueous solution with volume fraction of 70% (elution rate of 1.5 BV/h); collecting 70% ethanol water solution eluent (the elution rate is 1.5 BV/h); concentrating under reduced pressure, and drying to obtain crude extract of folium Platycladi;
s6, dissolving the crude cacumen biotae extract obtained in the step S5 in an ethanol aqueous solution with the volume fraction of 30% in a hot saturation manner (the dissolving temperature is 60 ℃, and the volume mass ratio of the ethanol aqueous solution with the volume fraction of 30% to the crude cacumen biotae extract is 10mL);
s7, dissolving the solid obtained in the step S6 by using an ethanol aqueous solution with the volume fraction of 75% (the dissolving temperature is 55 ℃), crystallizing (the volume mass ratio of the ethanol aqueous solution with the volume fraction of 75% to the solid obtained in the step S6 is 4mL, the crystallization temperature is 4 ℃, and the crystallization time is 16h; after the crystallization is finished, carrying out solid-liquid separation, and drying crystals to obtain 2.00g of quercetin extract;
concentrating the mother liquor obtained in step S6 under reduced pressure, stopping concentrating (concentrating to 1/3 of the original volume) when crystal is just precipitated, refrigerating at 4 deg.C for 16 hr, performing solid-liquid separation, and drying the solid to obtain 1.29g myricitrin extract.
HPLC detection shows that the purity of quercetin extract is 95.99% (detection map is shown in FIG. 1, retention time is 24.108 min), and yield is 65.14%;
the myricitrin extract has purity of 94.07% (detection pattern is shown in FIG. 2, retention time is 15.323 min), and yield is 71.16%;
the mass content of the flavonoid compounds in the water-insoluble total flavone extract is 18.36 percent through UV detection.
Example 2
This example is a method for preparing a cacumen biotae extract, comprising the following steps:
s1, crushing 500g of a raw material of oriental arborvitae leaves into 40 meshes, placing the crushed raw material in a round-bottom flask, adding water with the volume being 8 times (4000 mL relative to the mass of the raw material), connecting a volatile oil extractor and a reflux condensing device to perform microwave-assisted steam distillation, wherein the microwave power is 400W, the distillation time is 12min, the distillation temperature is 100 ℃, and respectively collecting essential oil and water extract;
dehydrating the essential oil with anhydrous sodium sulfate, and then 16.2g of cacumen Platycladi essential oil;
s2, continuously adding the residual raw material slag in the step S1 into an ethanol water solution with the volume fraction of 50% (the mass-volume ratio of the raw material slag to the ethanol water solution is 1g, 10mL), performing microwave-assisted extraction with the microwave power of 400W and the extraction time of 30min, extracting for 1 time, and performing solid-liquid separation at the extraction temperature of 40 ℃ to obtain an alcohol extract; combining the alcohol extract in the step and the water extract in the step S1 to obtain an extract;
s3, concentrating the extracting solution under reduced pressure to 1/15 of the original volume, carrying out solid-liquid separation while the extracting solution is hot, and collecting solid and clear liquid;
drying the solid to obtain 16.97g of water insoluble total flavone extract;
s4, passing the clear liquid (at the temperature of 38-40 ℃) obtained in the step S3 through HPD100B macroporous resin while the clear liquid is hot, washing the clear liquid with water until the clear liquid is colorless (the elution rate is 2 BV/h), and then eluting the clear liquid with 4.5BV of ethanol water solution with the volume fraction of 55% (the elution rate is 2 BV/h); collecting the ethanol eluent;
s5, concentrating the ethanol eluent obtained in the step S4 under reduced pressure, and controlling the concentration of the concentrated ethanol to be 40%; passing the concentrated solution through polyamide resin, washing with pure water to colorless (elution rate is 1 BV/h), and sequentially eluting with 2BV of 50% ethanol aqueous solution (elution rate is 1 BV/h) and 3BV of 80% ethanol aqueous solution (elution rate is 1 BV/h); collecting 80% ethanol water eluent; concentrating under reduced pressure and drying to obtain crude extract of folium Platycladi;
s6, dissolving the crude cacumen biotae extract obtained in the step S5 in an ethanol water solution with the volume fraction of 40% in a hot saturation manner (the dissolving temperature is 50 ℃, and the volume mass ratio of the ethanol water solution with the volume fraction of 40% to the crude cacumen biotae extract is 8mL and 1g), carrying out solid-liquid separation while the extract is hot, cooling to 24 ℃, refrigerating for 6 hours at the temperature of 4 ℃ in a refrigerator, and carrying out solid-liquid separation to obtain a solid and a mother solution;
s7, dissolving the solid obtained in the step S6 by using a 65% ethanol aqueous solution in volume fraction (the dissolving temperature is 50 ℃), crystallizing (the volume mass ratio of the 65% ethanol aqueous solution to the solid obtained in the step S6 is 5.5mL and 1g), the crystallizing temperature is 0 ℃, and the crystallizing time is 12h; after the crystallization is finished, carrying out solid-liquid separation, and drying crystals to obtain 2.03g of quercetin extract;
concentrating the mother liquor obtained in step S6 under reduced pressure until crystals precipitate, stopping concentrating (concentrating to 2/5 of the original volume), refrigerating at 0 deg.C for 12 hr, separating solid and liquid, and drying the solid to obtain 1.30g myricitrin extract.
HPLC detection shows that the purity of quercetin extract is 95.46%, and the yield is 65.58%;
the purity of the myricitrin extract is 92.33 percent, and the yield is 70.92 percent;
the mass content of the flavonoid compounds in the water-insoluble total flavone extract is 17.62 percent through UV detection.
Example 3
This example is a method for preparing cacumen biotae extract, comprising the following steps:
s1, crushing 500g of a raw material of oriental arborvitae into 60 meshes, placing the crushed raw material in a round-bottom flask, adding water with the volume being 10 times (5000 mL relative to the mass of the raw material), connecting a volatile oil extractor and a reflux condensing device to perform microwave-assisted steam distillation, wherein the microwave power is 350W, the distillation time is 17min, the distillation temperature is 100 ℃, and respectively collecting essential oil and water extract;
dehydrating the essential oil with anhydrous sodium sulfate, and then 17.28g of cacumen Platycladi essential oil;
s2, continuously adding the residual raw material slag in the step S1 into an ethanol aqueous solution with the volume fraction of 60% (the mass-volume ratio of the raw material slag to the ethanol aqueous solution is 1g, 10mL), performing microwave-assisted extraction, wherein the microwave power is 350W, the extraction time is 30min, extracting for 1 time, the extraction temperature is 55 ℃, and performing solid-liquid separation to obtain an alcohol extract; combining the alcohol extract in the step and the water extract in the step S1 to obtain an extract;
s3, concentrating the extracting solution under reduced pressure to 1/16 of the original volume, carrying out solid-liquid separation while the extracting solution is hot, and collecting solid and clear liquid;
drying the solid to obtain 16.60g of water insoluble total flavone extract;
s4, passing the clear liquid (at the temperature of 53-55 ℃) obtained in the step S3 through HPD722 macroporous resin while the clear liquid is hot, washing the clear liquid with water until the clear liquid is colorless (the elution rate is 1.5 BV/h), and then eluting the clear liquid with 4BV of ethanol aqueous solution with the volume fraction of 60% (the elution rate is 1.5 BV/h); collecting the ethanol eluent;
s5, concentrating the ethanol eluent obtained in the step S4 under reduced pressure, and controlling the concentration of the concentrated ethanol to be 35%; passing the concentrated solution through polyamide resin, washing with pure water to colorless (elution rate is 1 BV/h), and sequentially eluting with 2.5BV of 40% ethanol aqueous solution (elution rate is 1 BV/h) and 4BV of 70% ethanol aqueous solution (elution rate is 1 BV/h); collecting 70% ethanol water eluent; concentrating under reduced pressure and drying to obtain crude extract of folium Platycladi;
s6, dissolving the crude cacumen biotae extract obtained in the step S5 in an ethanol water solution with the volume fraction of 35% in a hot saturation manner (the dissolving temperature is 55 ℃, the volume mass ratio of the ethanol water solution with the volume fraction of 35% to the crude cacumen biotae extract is 9mL and 1 g), carrying out solid-liquid separation while the extract is hot, cooling to 15 ℃, refrigerating for 4 hours at 0 ℃ in a refrigerator, and carrying out solid-liquid separation to obtain a solid and a mother solution;
s7, dissolving the solid obtained in the step S6 by using an ethanol aqueous solution with the volume fraction of 70% (the dissolving temperature is 55 ℃), crystallizing (the volume mass ratio of the ethanol aqueous solution with the volume fraction of 70% to the solid obtained in the step S6 is 1g), wherein the crystallization temperature is 4 ℃, and the crystallization time is 16h; after the crystallization is finished, carrying out solid-liquid separation, and drying crystals to obtain 2.02g of quercetin extract;
concentrating the mother liquor obtained in step S6 under reduced pressure until crystals precipitate, stopping concentrating (concentrating to 2/5 of the original volume), refrigerating at 4 deg.C for 16 hr, separating solid and liquid, and drying the solid to obtain 1.28g myricitrin extract.
HPLC detection shows that the purity of the quercetin extract is 95.49%, and the yield is 65.01%;
the purity of the myricitrin extract is 93.26 percent, and the yield is 70.04 percent;
the mass content of the flavonoid compounds in the water-insoluble total flavone extract is 17.27 percent by UV detection.
Example 4
This comparative example is a method for preparing an extract of cacumen biotae, and is different from example 3 in that: an aqueous solution of ethanol having a volume fraction of 35% (dissolved under heat saturation) of 9 times the volume (relative to the mass of the crude extract of cacumen biotae) in step S6 in example 3 was replaced with an aqueous solution of ethanol having a volume of 60% (dissolved under heat saturation) of 7 times the volume (relative to the mass of the crude extract of cacumen biotae).
17.24g of essential oil, 16.41g of water-insoluble total flavone extract, 1.24g of myricitrin extract and 2.07g of quercetin extract are obtained in the comparative example.
HPLC detection shows that the purity of the quercetin extract is 91.98%, and the yield is 64.43%;
the purity of the myricitrin extract is 84.04%, and the yield is 61.18%;
the mass content of the flavonoid compounds in the water-insoluble total flavone extract is 17.43 percent through UV detection.
Example 5
This comparative example is a method for preparing an extract of cacumen biotae, and is different from example 3 in that: the step of cooling to 15 ℃ in step S6 in example 3 is defaulted; namely, the solid-liquid separation is carried out while the mixture is hot, and the mixture is directly placed in a refrigerator for refrigeration at 0 ℃ for 5 hours.
17.25g of essential oil, 16.38g of water-insoluble total flavone extract, 1.22g of myricitrin extract and 1.90g of quercetin extract are obtained in the comparative example.
HPLC detection shows that the purity of the quercetin extract is 92.62%, and the yield is 59.57%;
the purity of the myricitrin extract is 88.20 percent, and the yield is 63.31 percent;
the mass content of the flavonoid compounds in the water-insoluble total flavone extract is 17.45% by UV detection.
Example 6
This comparative example is a method for preparing an extract of cacumen biotae, and is different from example 3 in that: the cooling time in step S6 in example 3 was extended from 5h to 10h.
17.25g of essential oil, 16.46g of water-insoluble total flavone extract, 1.17g of myricitrin extract and 2.03g of quercetin extract are obtained in the comparative example.
HPLC detection shows that the purity of quercetin extract is 93.93%, and the yield is 64.70%;
the purity of the myricitrin extract is 92.02 percent, and the yield is 63.15 percent;
the mass content of flavonoid compounds in the water-insoluble total flavone extract is 17.49% by UV detection.
Example 7
This comparative example is a method for preparing an extract of cacumen biotae, and is different from example 3 in that: the refrigeration time in step S6 in example 3 was reduced from 5h to 3h.
17.22g of essential oil, 16.61g of water-insoluble total flavone extract, 1.30g of myricitrin extract and 1.89g of quercitrin extract are obtained in the comparative example.
HPLC detection shows that the purity of the quercetin extract is 96.94%, and the yield is 67.58%;
the purity of the myricitrin extract is 88.30 percent, and the yield is 67.58 percent;
the mass content of flavonoid compounds in the water-insoluble total flavone extract is 17.49% by UV detection.
Comparative example 1
This comparative example is a method for preparing an extract of cacumen biotae, and is different from example 3 in that: the microwave-assisted steam distillation in example 3 was replaced by steam distillation; and the distillation time was extended to 1h.
In the comparative example, 18.2g of essential oil, 16.36g of water-insoluble total flavone extract, 1.11g of myricitrin extract and 1.73g of quercetin extract were obtained.
HPLC detection shows that the purity of the quercetin extract is 86.81%, and the yield is 50.84%;
the purity of the myricitrin extract is 81.33 percent, and the yield is 53.11 percent;
the mass content of flavonoid compounds in the water-insoluble total flavone extract is 15.55% by UV detection.
Example 4 differs from example 3 in that: all are dissolved in hot saturation but have poor separation effect: the method has the advantages that the ethanol concentration is too high, the solubilities of the myricitrin and the quercetin are increased, the difference change of the solubilities is reduced, the difference of crystallization rate is also reduced, part of myricitrin is simultaneously separated out, the purity of the quercetin in crystals is reduced, the yield and the purity of recrystallization are further influenced, and meanwhile, the purity of the myricitrin in the mother liquor is obviously reduced, and the yield and the purity of recrystallization are further influenced.
Example 5 differs from example 3 in that: direct cooling crystallization, separation effect is poor: the temperature is reduced too fast, the crystallization rate is too fast, more tiny crystals are easily formed by the quercetin, the solid-liquid separation is difficult, and the quercetin cannot be collected, so that the yield of the quercetin is reduced; the myricitrin is easy to separate out crystals, which not only influences the purity of the quercetin crystals, but also influences the purity and yield of the myricitrin in the solution.
Example 6 differs from example 3 in that: the separation and refrigeration time is prolonged, and the yield of active ingredients is low: the refrigeration time is too long, the crystallization of the quercetin reaches saturation, the precipitation rate of the myricitrin and impurities is increased, the purity of the quercetin is influenced, and the yield and the purity of recrystallization are further influenced; the yield of myricitrin in the solution is reduced, and the purity is not greatly influenced, so that the overall yield is only influenced.
Example 7 differs from example 3 in that: the refrigeration time is too short, and the yield of active ingredients is low: the growth time of the quercetin crystal nucleus is short, the precipitated particles do not have enough time to grow and have no time to agglomerate, the formed particle size is generally small, solid-liquid separation is difficult, and the particles cannot be collected, so that the yield of the quercetin is influenced, and impurities such as myricitrin are less precipitated, so that the purity is slightly high; but the purity of the myricitrin in the solution is low, and the purity and the yield of the recrystallized myricitrin are further influenced.
Comparative example 1 differs from example 3 in that: the yield of essential oil is comparable but the yield of active ingredients is low: the micro tube bundle and glandular cell system inside the material absorbs microwave energy to raise the temperature inside the cell fast, so that the pressure inside the cell exceeds the capacity of the cell wall to expand, and the cell is cracked and the effective components inside the cell flow out freely. The microwave breaks the cells, all volatile oil freely flows out to become the volatile oil in a free state, so the extraction process is greatly accelerated, the extraction time is saved, and the quercetin and the myricitrin are heat-sensitive substances, so the loss of the quercetin and the myricitrin is caused.
In conclusion, the invention provides a preparation method for simultaneously extracting and separating volatile oil, quercetin and myricitrin from cacumen biotae. Therefore, the invention fully excavates the medicinal value of the cacumen biotae product, carries out deeper research and development, develops the cacumen biotae active ingredient product with high added value, and has wide development prospect and practical significance.
While the embodiments of the present invention have been described in detail with reference to the specific embodiments, the present invention is not limited to the embodiments, and various changes can be made without departing from the spirit of the present invention within the knowledge of those skilled in the art. Furthermore, the embodiments of the present invention and the features of the embodiments may be combined with each other without conflict.
Claims (6)
1. A method for extracting an essential oil extract, a quercetin extract and a myricitrin extract from oriental arborvitae leaves is characterized in that: the method comprises the following steps:
s1, distilling a raw material of biota orientalis leaf by microwave-assisted steam, and obtaining an essential oil extract, a water extract and raw material residues after the distillation is finished;
s2, carrying out microwave alcohol extraction on the raw material residues to prepare an alcohol extract;
s3, mixing the water extract and the alcohol extract, concentrating, and performing solid-liquid separation to obtain a solid and a concentrated solution;
s4, passing the concentrated solution through macroporous resin, and eluting by using water and ethanol water; collecting the ethanol water solution eluent;
the macroporous resin is one of HPD722, HPD100 and LAS-21;
the volume fraction of the ethanol aqueous solution in the step S4 is 55-70%, and the using amount of the ethanol aqueous solution in the step S4 is 3.5BV-4.5BV;
s5, concentrating the ethanol water solution eluent, passing through polyamide resin, and sequentially eluting with water, 30-40% by volume of ethanol water solution and 70-80% by volume of ethanol water solution;
collecting the eluent of ethanol water solution with volume fraction of 70% -80%, and concentrating to dryness to obtain a crude extract of cacumen biotae;
s6, recrystallizing the crude cacumen biotae extract to obtain an cacumen biotae extract;
the recrystallization comprises heating for dissolving, filtering while hot, cooling for the first time, cooling again for crystallization to obtain solid and mother liquor, carrying out solid-liquid separation, and collecting solid phase; the solvent used in the heating and dissolving process of the recrystallization is 30-40% of ethanol water solution; the temperature of recrystallization in the heating and dissolving process is 50-60 ℃; the final temperature of recrystallization in the primary cooling process is 15-30 ℃; the temperature of recrystallization in the process of cooling and crystallizing again is 0-4 ℃; the time of recrystallization in the process of cooling crystallization again is 4h to 6h;
s7, dissolving the solid obtained in the step S6 by using 65-75% by volume of ethanol aqueous solution, and crystallizing for the first time; performing solid-liquid separation, and drying the solid to obtain a quercetin extract;
and (4) concentrating the mother liquor obtained in the step (S6) under reduced pressure, precipitating crystals, carrying out solid-liquid separation, and drying the solids to obtain the myricitrin extract.
2. The method of claim 1, wherein: the power of the microwave in the microwave-assisted steam distillation is 300W to 400W, and the time of the microwave in the microwave-assisted steam distillation is 12min to 20min.
3. The method of claim 1, wherein: the extraction agent in the microwave alcohol extraction is 40-80% of ethanol water solution, the power of microwaves in the microwave alcohol extraction is 300W-400W, and the time of the microwaves in the microwave alcohol extraction is 30min-40min.
4. The method of claim 1, wherein: the using amount of the ethanol water solution with the volume fraction of 30-40% in the step S5 is 2BV-2.5BV, and the using amount of the ethanol water solution with the volume fraction of 70-80% in the step S5 is 3BV-3.5BV.
5. The method of claim 1, wherein: and in the step S5, the volume fraction of the ethanol in the concentrated solution is 30-40%.
6. The method according to any one of claims 1 to 5, wherein: the method also comprises the step of dehydrating the essential oil extract to obtain the cacumen biotae essential oil, wherein anhydrous sodium sulfate or anhydrous magnesium sulfate is selected for dehydration.
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