CN114805466B - Preparation method of hedera helix extract - Google Patents
Preparation method of hedera helix extract Download PDFInfo
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- CN114805466B CN114805466B CN202210392864.7A CN202210392864A CN114805466B CN 114805466 B CN114805466 B CN 114805466B CN 202210392864 A CN202210392864 A CN 202210392864A CN 114805466 B CN114805466 B CN 114805466B
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Abstract
The invention relates to a preparation method of a hedera helix extract, belonging to the technical field of plant extraction processing. The preparation method comprises the following steps: s1, mixing a hedera helix raw material with an auxiliary agent solution, and performing ultrasonic extraction to obtain an extracting solution; s2, concentrating the extracting solution, mixing with ethanol, and collecting a concentrated solution; s3, eluting the concentrated solution with an aluminum peroxide chromatographic column by adopting water, an ethanol aqueous solution and an acidic ethanol aqueous solution in sequence; collecting the eluent of the ethanol water solution to prepare hederagenin C eluent; collecting the eluent of the acidic ethanol aqueous solution to prepare an alpha-hederagenin eluent; s4, crystallizing the hederagenin C eluent to obtain a hederagenin C extract; crystallizing the alpha-hederacoside eluent to obtain an alpha-hederacoside extract; the extraction aid solution comprises the following preparation raw materials: surfactants and flocculants. The method can obtain hederagenin extract with high purity.
Description
Technical Field
The invention belongs to the technical field of plant extraction and processing, and particularly relates to a preparation method of a hedera helix extract.
Background
Hedera helix (Latin school name: hedera nepalensis var. Sinensis (Tobl.)) is the dried stem and leaf of Hedera helix of Araliaceae, and is called as caulis et folium Hederae sinensis, caulis et folium Hedyotideae Divaricatae, radix seu caulis Kadsurae Longipedunculatae, and radix Trigonellae, which are from Ben Cao gang mu Shiyi, and has effects of dispelling pathogenic wind, removing dampness, promoting blood circulation, relieving swelling, suppressing hyperactive liver, and removing toxic substances. The main components of Hedera helix include triterpenoid saponin, carotene, tannin, and saccharide, wherein the stem contains Hedera helix glycoside, tannin (about 12.01% by mass), resin, etc., and the leaf contains Hedera helix glycoside, inositol, carotene, and saccharide, and contains tannin about 29.4%. The saponin compounds (such as triterpenoid saponins) are components which are found in the Hedera helix plants in a plurality of types and have the best activity, and have a plurality of pharmacological activities such as anti-inflammatory, anti-tumor, antifungal, insecticidal and the like.
Modern pharmacological studies have shown that: the main active ingredient of hedera helix is saponin. Alpha-hederagenin extracted from Hedera helix, hedera helix saponin C, hedera helix saponin E and Hedera helix saponin F have good antiinflammatory effect. Hederagenin C (hederacoside C) and alpha-hederagenin, which are disaccharide chains, are considered as main active ingredients in hedera helix, wherein alpha-hederagenin can be formed from hederagenin C by ester hydrolysis, and the european pharmacopoeia prescribes that the content of hederagenin C in medicinal hederagenin is not lower than 3%. The related art describes that the content of hederagenin C in stems and leaves of hederacea is 1.30% -13.65%, the content of alpha-hederagenin is 0.56% -4.42%, and the content of hederagenin in leaves is 3-4 times of stems.
In the related art, a crude extract (alcohol extract) of hedera helix is adopted to prepare a preparation, and the preparation is used for treating cough, dyspnea and other diseases, and the main medicinal component is hederagenin. It has also been found in the related art that α -hederagenin is particularly helpful in bronchospasmolysis by inhibiting receptor/ligand cellular internalization, constantly activating the adenylate cyclase system, relaxing smooth muscle (spasmolysis), whereas hederagenin C and hederagenin are unable to inhibit the internalization process; however, hederagenin C has the functions of dispelling wind, promoting diuresis, calming liver, detoxifying and the like.
The related technology also provides a method for extracting and separating the total saponins of the Hedera sinensis, which is compared with extraction methods such as solvent extraction, microwave extraction, biological enzyme and the like, and the hederagenin C with the purity of 87 percent is obtained by means of purification by different macroporous resins, preparation type reversed-phase high-performance liquid chromatography separation and the like. The process uses the preparative chromatographic purification technology to separate hederacoside C, has high cost and complex technology, and is not beneficial to industrial production.
The related art also provides a method for preparing the hederagenin leaf extract containing the active ingredients hederagenin C and alpha-hederagenin, and the method is mainly used for preparing the high-efficiency extract which is rich in 4% of alpha-hederagenin and 6.5% of hederagenin C in a mixing way. The method does not disclose how to separate hederacoside C and alpha-hederagenin, and the total content of the separated hederacoside C and alpha-hederagenin is low.
The related art also provides a method for preparing hederacoside C from hedera helix, which comprises the steps of drying and crushing hedera helix stems and leaves, extracting with ethanol and water, extracting with petroleum ether, ethyl acetate and n-butanol sequentially, dissolving n-butanol phase with ethanol, precipitating with ethyl acetate, and separating and purifying with a silica gel column to obtain hederacoside C monomer with the content of more than 98%. The invention can obtain the hederagenin C monomer with high purity, but has complex process and high cost, and the invention uses high-risk reagents such as petroleum ether, chloroform and the like, has low production safety, and does not fully utilize other components in the raw materials, thereby causing resource waste.
The related art also provides a preparation method of the hedera plant extract, which comprises the steps of soaking hedera raw materials, extracting by using an ethanol aqueous solution, enriching by using macroporous resin (elution of ethanol aqueous solution with concentration of more than or equal to 40 percent), and drying the enriched solution under reduced pressure to obtain the extract, wherein the total glycoside content of the extract in the embodiment is not more than 60 percent (colorimetric method), and the monomer content (hederagenin C or alpha-hedera extract) is not more than 25 percent. I.e. the extract obtained by the above method has low purity.
Therefore, there is a need to develop a preparation method of hederagenin extract, which can simultaneously produce hederagenin C extract and α -hederagenin extract in high purity.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a preparation method of a hedera helix extract, which can simultaneously prepare a hedera helix glycoside C extract and an alpha-hedera helix glycoside extract with high purity.
In order to solve the technical problems, the invention provides a preparation method of a hedera helix extract, which comprises the following steps:
s1, mixing a hedera helix raw material with an auxiliary agent solution, and performing ultrasonic extraction to obtain an extracting solution;
s2, concentrating the extracting solution, mixing with ethanol, and collecting a concentrated solution;
s3, eluting the concentrated solution with an aluminum peroxide chromatographic column by adopting water, an ethanol aqueous solution and an acidic ethanol aqueous solution in sequence;
collecting the eluent of the ethanol water solution to prepare hederagenin C eluent;
collecting the eluent of the acidic ethanol aqueous solution to prepare an alpha-hederagenin eluent;
s4, crystallizing the hederagenin C eluent to obtain a hederagenin C extract;
crystallizing the alpha-hederacoside eluent to obtain an alpha-hederacoside extract;
the extraction aid solution comprises the following preparation raw materials:
surfactants and flocculants.
According to at least one of the technical schemes of the preparation method, the preparation method at least has the following beneficial effects:
according to the invention, through the synergistic effect of the extraction aid and ultrasonic extraction, the thickness of a liquid film between the hederacoside compound (alpha-hederacoside and hederacoside C) and the extraction aid solution in the hederacoside raw material is sufficiently thin, the resistance of the hederacoside in the process of diffusing from the surface of the raw material to the extraction aid solution is reduced, and the extraction rate of active ingredients is greatly improved (the total glycoside content in the extraction solution is more than 50%).
The extraction aid is prepared by the interaction of a surfactant (reducing the surface tension of an extraction aid solution, increasing the permeability of cell tissues, the desorption, dissolution, solubilization and the like of active ingredients) and a flocculating agent (flocculating impurities such as tannins and the like in an extracting solution to achieve the impurity removal effect, (2) improving the dissolution assisting effect of the surfactant on the active ingredients), so that the extraction rate of the active ingredients (alpha-hederacoside and hederacoside C) is obviously enhanced, the extraction time is shortened, and the impurity removal effect is achieved.
The hederagenin compound contains hydroxyl and has good solubility in ethanol; ethanol is added into the extracting solution to prevent the hederagenin compound from precipitating due to the reduction of the solvent in the concentration process; so that the hederagenin compound is fully dissolved in the concentrated solution.
The separation of alpha-hederacoside and hederacoside C is achieved by eluting with water, an aqueous ethanol solution and an aqueous acidic ethanol solution.
The invention realizes the simultaneous preparation of high-purity hederacoside C and alpha-hederacoside by the aid of ultrasonic extraction, alumina column chromatographic separation and crystallization, and has high yield; the whole process does not use other organic solvents except ethanol, is safe and environment-friendly, and is economical and efficient.
Hederaoside C has formula of C 59 H 96 O 26 The molecular weight is 1221, and the polymer is soluble in cold water, soluble in solvents with relatively high polarity such as hot water, methanol, ethanol and acetonitrile, and insoluble in organic solvents with low polarity such as petroleum ether. The structural formula is shown as follows:
alpha-hederagenin has molecular formula of C 41 H 66 O 12 Molecular weight of 750, whichPoor water solubility, and easy dissolution in hot methanol, hot ethanol, etc. The structural formula is shown as follows:
according to some embodiments of the invention, the hedera helix raw material is at least one of hedera helix leaf or hedera helix stalk.
According to some embodiments of the invention, the ivy leaf is a ivy dry leaf.
According to some embodiments of the invention, the hedera helix is a hedera helix.
According to some embodiments of the invention, the ivy raw material is crushed to obtain ivy raw material powder.
According to some embodiments of the invention, the hedera helix raw material powder has a particle size of 40 mesh to 60 mesh.
By crushing the raw materials to a proper mesh number, the contact area of the raw materials and the extraction aid solution is increased, so that the extraction efficiency is accelerated.
According to some embodiments of the invention, the surfactant is selected from at least one of tween 20, tween 40 and tween 80.
According to some embodiments of the invention, the flocculant is hydroxypropyl chitosan.
According to some embodiments of the invention, the degree of substitution of the hydroxypropyl chitosan is above 80%.
The hederagenin compound has a plurality of hydroxyl groups, the electron cloud density is high, the compound is electronegative, and an ion pair is formed by the compound and hydroxypropyl chitosan of a cationic polymer, compared with the polar form of a target compound (hederagenin compound), the ion pair form is more easily captured by a non-ionic surfactant Tween (Tween 20, tween 40 and Tween 80), so that the extraction rate is remarkably improved; the extraction rate of the active ingredients is improved, and simultaneously, dissolved impurities such as tannins and the like collide with the hydroxypropyl chitosan at high speed under the assistance of ultrasonic waves, so that a good flocculation effect is achieved.
The hydroxyl in the hydroxypropyl chitosan is introduced to enable a large number of hydroxyl to exist on the whole flocculant molecular chain, so that the fusion of the chitosan molecular structure and the tannic acid molecular structure is stronger, and the hydroxyl in the tannic acid colloidal particle molecule can not only form hydrogen bonds to generate characteristic adsorption effect; ionization thereof generates ions, and electric neutralization thereof can promote flocculation.
According to some embodiments of the invention, the mass ratio of the surfactant to the flocculant is 2.5-6:1.
Under the condition of fixed surfactant, if the flocculant is too much, the excessive positive charge is neutralized with the negative charge of hederacoside, and more loss is caused by flocculation; too little, the impurity removal rate is low, the purity of the effective components is influenced, the subsequent purification effect is further influenced, and the synergistic effect of the solubilizing aid of the surfactant is reduced.
According to some embodiments of the invention, the weight fraction of the auxiliary agent in the auxiliary agent solution is 0.3-0.6%.
When the concentration of the extraction aid is too high, a large amount of foam can be generated, the operation difficulty is increased, and meanwhile, the dissolution of impurities is increased, so that the later separation is not facilitated. When the concentration is too low, the concentration of the formed micelle is insufficient, and the compatibilization effect is not obvious.
According to some embodiments of the invention, the flocculant in the booster solution has a mass fraction of 0.08-0.1%.
According to some embodiments of the invention, the pH of the booster solution is 7.5 to 8.5.
Because the water solubility of the alpha-hederacoside is poor, the extraction rate of the alpha-hederacoside can be effectively improved by adjusting the pH value; the pH is too large, the flocculation effect of the hydroxypropyl chitosan is affected, and the synergistic effect results in low extraction rate and low purity of alpha-hederacoside.
And due to the weak alkaline environment in the extraction process, the partially ionized hederagenin is more easily adsorbed by the alumina column chromatography, and then the gradient elution is adopted to achieve a good separation effect through polarity difference and the difficulty of the adsorption of the target component and the alumina column chromatography.
According to some embodiments of the invention, the pH of the booster solution is adjusted by the selection of alkaline substances.
According to some embodiments of the invention, the alkaline substance comprises an inorganic base, carbonate and bicarbonate.
According to some embodiments of the invention, the inorganic base is at least one of sodium hydroxide, potassium hydroxide, and cesium hydroxide.
According to some embodiments of the invention, the carbonate is at least one of sodium carbonate, potassium carbonate, and cesium carbonate.
According to some embodiments of the invention, the bicarbonate is at least one of sodium bicarbonate, potassium bicarbonate, and cesium bicarbonate.
According to some embodiments of the invention, the starting material for the preparation of the extractant solution further comprises water.
According to some embodiments of the invention, the mass ratio of the booster solution to the hedera helix raw material is 1:12-25.
According to some embodiments of the invention, the mass ratio of the booster solution to the hedera helix raw material is 1:15-20.
According to some embodiments of the invention, the parameters of the ultrasound extraction are as follows: the extraction time is 30-45 min, the extraction temperature is 45-60 ℃, and the ultrasonic frequency is 20-25 kHz.
The structure of the surfactant can be damaged by the excessively high temperature, the extraction efficiency is affected, the hederagenin C is degraded, and in a proper temperature range, the movement speed of the effective component molecules can be accelerated by increasing the temperature, the diffusion can be accelerated, and the dissolution amount can be increased.
The structure of flocculating agent and impurity flocculation can be broken due to overlong ultrasonic time, so that the flocculation effect is poor, and the proper ultrasonic assistance can improve the collision of molecules, promote the formation of linkage acting force between the flocculating agent and the impurity and improve the flocculation effect; too high ultrasonic frequency can destroy the molecular structure formed by the flocculant and impurity molecules, and reduce flocculation effect.
According to some embodiments of the invention, the ultrasonic extraction is intermittent ultrasonic extraction.
According to some embodiments of the invention, the total duration of the intermittent ultrasound extraction consists of several adjacent ultrasound times and interval times.
According to some embodiments of the invention, each batch time of the intermittent ultrasonic extraction is between 5s and 10s.
According to some embodiments of the invention, each time of the intermittent ultrasonic extraction is 5min to 6min.
According to some embodiments of the invention, the concentrating is reduced pressure concentrating.
According to some embodiments of the invention, the concentration process is concentrated to a solids content of 9% to 15%.
According to some embodiments of the invention, the volume fraction of ethanol in the concentrate is 30% to 40%.
The volume fraction of the ethanol in the concentrated solution is controlled within the range, so that the complete dissolution of the alpha-hederacoside is ensured, and the aim of removing part of impurities with larger polarity is fulfilled.
According to some embodiments of the invention, the water is used in step S3 in an amount of 0.25BV to 0.5BV.
A small amount of water is used for removing a small amount of impurities which are relatively large in polarity and not easy to adsorb, and excessive water has no influence on resin adsorbates; but can affect the extraction time and also cause the waste of water resources.
According to some embodiments of the invention, the water flow rate in step S3 is 1BV/h to 1.5BV/h.
According to some embodiments of the invention, the volume fraction of ethanol in the aqueous ethanol solution in step S3 is 50% to 60%.
According to some embodiments of the invention, the aqueous ethanol solution in step S3 is used in an amount of 2BV to 2.5BV.
The dosage is less than the limit range, the elution is incomplete, and above the limit range, a small amount of alpha-hederacoside can be washed off, and the solvent is wasted.
According to some embodiments of the invention, the flow rate of the aqueous ethanol solution in the step S3 is 1BV/h to 1.5BV/h.
According to some embodiments of the invention, the volume fraction of ethanol in the acidic aqueous ethanol solution in step S3 is 70% to 80%.
According to some embodiments of the invention, the amount of the acidic ethanol aqueous solution used in step S3 is 2BV to 3BV.
The dosage is less than the limit range, the elution is incomplete, and a small amount of impurities can be eluted above the limit range, and the solvent is wasted.
According to some embodiments of the invention, the flow rate of the acidic ethanol aqueous solution in the step S3 is 1BV/h to 1.5BV/h.
According to some embodiments of the invention, the pH of the aqueous acidic ethanol solution in step S3 is between 5 and 6.
The pH is not in the above range, the separation effect is poor, the peracid or the alkalinity is high, and impurities are easy to elute, so that the purity of the alpha-hederacoside is affected. And too high a pH may result in incomplete elution of alpha-hederacoside.
According to some embodiments of the invention, the pH of the aqueous acidic ethanol solution is adjusted with an acid.
According to some embodiments of the invention, the acid is at least one of hydrogen chloride or sulfuric acid.
According to some embodiments of the invention, the alumina chromatographic column is a neutral alumina chromatographic column or an acidic alumina chromatographic column.
The hederacoside C and the alpha hederacoside are effectively separated by adopting neutral alumina and acidic alumina column chromatography, and the adsorption capacity and the resolution ratio are relatively high.
According to some embodiments of the invention, the hederagenin C eluent is crystallized at a temperature of 4-10 ℃.
According to some embodiments of the invention, the hederagenin C eluent is crystallized for a time of 8-24 h.
By controlling the crystallization temperature and time, hederagenin C is fully crystallized, and the yield is improved.
According to some embodiments of the invention, the hederagenin C eluate is crystallized, comprising the steps of:
concentrating the hederagenin C eluent under reduced pressure to saturation, crystallizing at 4-10 ℃ for 8-24 h, collecting solid phase, and drying to obtain hederagenin C extract.
According to some embodiments of the invention, the crystallization temperature of the α -hederacoside eluate is between 4 ℃ and 10 ℃.
According to some embodiments of the invention, the time for crystallization of the α -hederacoside eluate is between 8h and 24h.
By controlling the crystallization temperature and time, the alpha-hederacoside is fully crystallized, and the yield is improved.
According to some embodiments of the invention, the crystallization of the α -hederacoside eluate comprises the steps of:
concentrating the alpha-hederacoside eluent under reduced pressure to saturation, crystallizing at 4-10 ℃ for 8-24 h, collecting solid phase, washing and drying to obtain the alpha-hederacoside extract.
According to some embodiments of the invention, the washed detergent is water.
According to some embodiments of the invention, the number of washes is 1-2.
The saturation referred to in the present invention is a state in which crystals are just precipitated.
Drawings
FIG. 1 is a high performance liquid chromatography detection chart of the hederagenin C extract prepared in example 3 of the invention.
FIG. 2 is a high performance liquid chromatography detection chart of the α -hederagenin extract prepared in example 3 of the invention.
Detailed Description
The conception and the technical effects produced by the present invention will be clearly and completely described in conjunction with the embodiments below to fully understand the objects, features and effects of the present invention. It is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments, and that other embodiments obtained by those skilled in the art without inventive effort are within the scope of the present invention based on the embodiments of the present invention.
In the description of the present invention, the descriptions of the terms "one embodiment," "some embodiments," "illustrative embodiments," "examples," "specific examples," or "some examples," etc., mean 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, schematic representations of the above terms do not necessarily refer to the same embodiments or examples. 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 specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The mass content of hederacoside C in the dry hederacoside leaf raw material selected in the embodiment of the invention is 5.34%, and the mass content of alpha-hederacoside is 2.16%.
In the embodiment of the invention, the content of hederacoside C and alpha-hederacoside is detected by high performance liquid chromatography.
The liquid chromatography detection conditions of hederagenin C are as follows:
chromatographic column: wondasiltmc18 column (250 mm (length) ×4.6mm (inner diameter), 5 μm (packing particle size));
flow rate: 1mL/min;
detection wavelength: 203nm;
sample injection amount: 10. Mu.L;
sensitivity: 2.000AUFS;
mobile phase: acetonitrile and water ratio of 28:72;
column temperature: 25 ℃.
The liquid chromatography detection conditions of the alpha-hederagenin are as follows:
chromatographic column: wondasilTMC18 column (250 mm (length). Times.4.6 mm (inner diameter), 5 μm (packing particle size)).
Flow rate: 0.8mL/min;
detection wavelength: 203nm;
sample injection amount: 10. Mu.L;
sensitivity: 2.000AUFS;
mobile phase: the volume ratio of acetonitrile to the phosphoric acid aqueous solution with the mass fraction of 0.1% is 45:55;
column temperature: 25 ℃.
Example 1
The embodiment is a preparation method of a hedera helix extract, which comprises the following steps:
s1, ultrasonic auxiliary extraction:
crushing dried ivy leaf raw materials to 40 meshes, adding 15 times of a lifting aid solution, and carrying out ultrasonic-assisted continuous countercurrent extraction for 30min at 45 ℃; solid-liquid separation; collecting liquid phase to obtain ultrasonic extractive solution (the sum of hederacoside C and alpha-hederacoside content is 53.97%).
The mass fraction of the extraction aid in the extraction aid solution is 0.3 per mill.
The extraction aid consists of Tween 20 (CAS number: 9005-64-5) and hydroxypropyl chitosan (Shanghai ShiFengshi biotechnology Co., ltd., substitution degree is not less than 80%, CAS number: 104673-29-2), wherein the mass ratio of Tween 20 to hydroxypropyl chitosan is 2.5:1.
the pH of the booster solution was 8.5 (pH at 25 ℃ C.; adjusted by the addition of sodium hydroxide).
The ultrasound-assisted conditions were as follows: intermittent ultrasonic with low frequency, the ultrasonic time is 5s/5min (i.e. 5s for each ultrasonic every 5min, the ultrasonic is stopped), and the ultrasonic frequency is 20kHz.
S2, column chromatography:
concentrating the ultrasonic extraction liquid obtained in the step S1 under reduced pressure (the solid mass concentration is 12% +/-3%), adding ethanol, controlling the volume concentration of the ethanol to be 30%, and carrying out solid-liquid separation; collecting liquid phase to obtain reduced pressure concentrated solution;
subjecting the reduced pressure concentrated solution to neutral alumina column chromatography, eluting with 0.25BV water, 2BV 50% ethanol water solution, and 2BV 70% acidic ethanol water solution (pH 5, and adjusted with 35% hydrochloric acid) in sequence at an elution flow rate of 1BV/h;
collecting 50% ethanol water solution eluent to obtain hederagenin C crude extract;
collecting 70% acidic ethanol water solution eluent to obtain alpha-hederagenin crude extract;
s3, crystallizing:
concentrating the hederagenin C crude extract obtained in the step S2 under reduced pressure until just precipitation, refrigerating at 4 ℃ and standing for 8 hours, precipitating crystals, carrying out solid-liquid separation, and drying the crystals to obtain hederagenin C extract;
concentrating the crude alpha-hederagenin extract obtained in the step S2 under reduced pressure until the crude alpha-hederagenin extract is just separated out, refrigerating at 4 ℃ for 8 hours, separating out crystals, carrying out solid-liquid separation, leaching the crystals with water for 2 times, and drying to obtain the alpha-hederagenin extract.
The mass fraction of hederagenin C in the hederagenin C extract is 98.71%, and the yield is 84.17%.
The mass fraction of the alpha-hederacoside in the alpha-hederacoside extract is 98.41%, and the yield is 82.00%.
Example 2
The embodiment is a preparation method of a hedera helix extract, which comprises the following steps:
s1, ultrasonic auxiliary extraction:
crushing dried ivy leaf raw materials to 60 meshes, adding 20 times of extraction aid solution, and carrying out ultrasonic-assisted continuous countercurrent extraction at 60 ℃ for 45min; solid-liquid separation; collecting liquid phase to obtain ultrasonic extractive solution (the sum of hederacoside C and alpha-hederacoside content is 52.79%).
The weight fraction of the extraction aid in the extraction aid solution is 0.6 per mill.
The extraction aid consists of Tween 20 (CAS number: 9005-64-5) and hydroxypropyl chitosan (Shanghai ShiFengshi biotechnology Co., ltd., substitution degree is not less than 80%, CAS number: 104673-29-2), wherein the mass ratio of Tween 20 to hydroxypropyl chitosan is 6:1.
the pH of the booster solution was 7.5 (pH at 25 ℃ C.; adjusted by the addition of sodium bicarbonate).
The ultrasound-assisted conditions were as follows: intermittent ultrasonic with low frequency is carried out for 10s/5min (i.e. every ultrasonic for 5min, ultrasonic is stopped for 10 s), and ultrasonic frequency is 25kHz.
S2, column chromatography:
concentrating the ultrasonic extraction liquid obtained in the step S1 under reduced pressure (the solid mass concentration is 12% +/-3%), adding ethanol, controlling the volume concentration of the ethanol to be 40%, and carrying out solid-liquid separation; collecting liquid phase to obtain reduced pressure concentrated solution;
subjecting the reduced pressure concentrated solution to acidic alumina column chromatography, eluting with 0.5BV water, 2.5BV 60% ethanol water solution, and 3BV 80% acidic ethanol water solution (pH 6, and adjusted with 35% hydrochloric acid) in sequence, with an elution flow rate of 1.5BV/h;
collecting 60% ethanol water solution eluent to obtain hederagenin C crude extract;
collecting the eluent of 80% acidic ethanol aqueous solution to obtain a crude alpha-hederagenin extract;
s3, crystallizing:
concentrating the hederagenin C crude extract obtained in the step S2 under reduced pressure until just precipitation, refrigerating at 10 ℃ and standing for 24 hours, precipitating crystals, carrying out solid-liquid separation, and drying the crystals to obtain hederagenin C extract;
concentrating the crude alpha-hederagenin extract obtained in the step S2 under reduced pressure until the crude alpha-hederagenin extract is just separated out, refrigerating at 10 ℃ for 24 hours, separating out crystals, carrying out solid-liquid separation, leaching the crystals with water for 1 time, and drying to obtain the alpha-hederagenin extract.
The mass fraction of hederagenin C in the hederagenin C extract is 98.59%, and the yield is 84.80%.
The mass fraction of the alpha-hederacoside in the alpha-hederacoside extract is 98.20%, and the yield is 82.82%.
Example 3
The embodiment is a preparation method of a hedera helix extract, which comprises the following steps:
s1, ultrasonic auxiliary extraction:
crushing dry ivy leaf raw materials to 60 meshes, adding an extracting aid solution with the mass of 18 times, and carrying out ultrasonic-assisted continuous countercurrent extraction for 40min at 55 ℃; solid-liquid separation; collecting liquid phase to obtain ultrasonic extractive solution (the sum of hederacoside C and alpha-hederacoside content is 53.07%).
The weight fraction of the extraction aid in the extraction aid solution is 0.4 per mill.
The extraction aid consists of Tween 20 (CAS number: 9005-64-5) and hydroxypropyl chitosan (Shanghai ShiFengshi biotechnology Co., ltd., substitution degree is not less than 80%, CAS number: 104673-29-2), wherein the mass ratio of Tween 20 to hydroxypropyl chitosan is 4:1.
the pH of the booster solution was 8.0 (pH at 25 ℃ C.; adjusted by the addition of potassium hydroxide).
The ultrasound-assisted conditions were as follows: intermittent ultrasonic with low frequency is carried out for 8s/5min (i.e. ultrasonic is stopped for 8s every 5 min), and ultrasonic frequency is 20kHz.
S2, column chromatography:
concentrating the ultrasonic extraction liquid obtained in the step S1 under reduced pressure (the solid mass concentration is 12% +/-3%), adding ethanol, controlling the volume concentration of the ethanol to be 40%, and carrying out solid-liquid separation; collecting liquid phase to obtain reduced pressure concentrated solution;
subjecting the reduced pressure concentrated solution to neutral alumina column chromatography, eluting with 0.25BV water, 2BV 60% ethanol water solution, and 2BV 80% acidic ethanol water solution (pH 5.5, and 50% sulfuric acid solution) in sequence, with an elution flow rate of 1BV/h;
collecting 60% ethanol water solution eluent to obtain hederagenin C crude extract;
collecting the eluent of 80% acidic ethanol aqueous solution to obtain a crude alpha-hederagenin extract;
s3, crystallizing:
concentrating the hederagenin C crude extract obtained in the step S2 under reduced pressure until just precipitation, refrigerating at 6 ℃ and standing for 14 hours, precipitating crystals, carrying out solid-liquid separation, and drying the crystals to obtain hederagenin C extract;
concentrating the crude alpha-hederagenin extract obtained in the step S2 under reduced pressure until the crude alpha-hederagenin extract is just separated out, refrigerating and standing at 6 ℃ for 14h, separating out crystals, carrying out solid-liquid separation, leaching the crystals with water for 1 time, and drying to obtain the alpha-hederagenin extract.
The mass fraction of hederagenin C in the hederagenin C extract is 98.46% and the yield is 83.45% through high performance liquid chromatography detection; the detection pattern is shown in FIG. 1 (retention time is 15.233 min).
The mass fraction of the alpha-hederacoside in the alpha-hederacoside extract is 98.39 percent, and the yield is 81.90 percent; the detection pattern is shown in FIG. 2 (retention time 25.178 min).
Comparative example 1
The comparative example is a preparation method of hedera helix extract, and the difference from example 3 is that:
the extractant solution of example 3 was replaced equally with potassium hydroxide solution (pH 8.0).
The mass fraction of hederacoside C in the hederacoside C extract is 86.76% and the yield is 69.17% through high performance liquid chromatography detection.
The mass fraction of the alpha-hederacoside in the alpha-hederacoside extract is 81.16%, and the yield is 60.04%.
Comparative example 2
The comparative example is a preparation method of hedera helix extract, and the difference from example 3 is that:
the extraction aid solution in example 3 was replaced with tween 20 solution (mass fraction 0.4%o; pH 8.0, adjusted by adding potassium hydroxide).
The mass fraction of hederagenin C in the hederagenin C extract is 86.93%, and the yield is 74.44%.
The mass fraction of the alpha-hederacoside in the alpha-hederacoside extract is 81.42%, and the yield is 69.89%.
Comparative example 3
The comparative example is a preparation method of hedera helix extract, and the difference from example 3 is that:
the ultrasound-assisted extraction in example 3 was replaced with the following:
continuous countercurrent extraction at 55deg.C for 40min.
The mass fraction of hederacoside C in the hederacoside C extract is 89.73% and the yield is 74.64% through high performance liquid chromatography detection.
The mass fraction of the alpha-hederacoside in the alpha-hederacoside extract is 83.94%, and the yield is 71.17%.
Comparative example 4
The comparative example is a preparation method of hedera helix extract, and the difference from example 3 is that:
the neutral alumina column of example 3 was replaced with macroporous resin HP 20.
The mass fraction of hederacoside C in the hederacoside C extract is 85.49% and the yield is 68.01% through high performance liquid chromatography detection.
The mass fraction of the alpha-hederacoside in the alpha-hederacoside extract is 81.61%, and the yield is 66.62%.
Comparative example 5
The comparative example is a preparation method of hedera helix extract, and the difference from example 3 is that:
the mass fraction of the builder in the builder solution in example 3 was adjusted to 0.8% by weight.
The mass fraction of hederagenin C in the hederagenin C extract is 88.89%, and the yield is 74.03%.
The mass fraction of the alpha-hederacoside in the alpha-hederacoside extract is 83.11%, and the yield is 71.25%.
Comparative example 6
The comparative example is a preparation method of hedera helix extract, and the difference from example 3 is that:
the pH of the booster solution in example 3 was not adjusted (pH of the booster solution was 6.8).
The mass fraction of hederagenin C in the hederagenin C extract is 98.18%, and the yield is 80.68%.
The mass fraction of the alpha-hederacoside in the alpha-hederacoside extract is 98.02%, and the yield is 73.36%.
Comparative example 7
The comparative example is a preparation method of hedera helix extract, and the difference from example 3 is that:
the pH of the extractant solution in example 3 was adjusted to 9.
The mass fraction of hederacoside C in the hederacoside C extract is 89.35% and the yield is 74.57% through high performance liquid chromatography detection.
The mass fraction of the alpha-hederacoside in the alpha-hederacoside extract is 84.50%, and the yield is 73.04%.
Comparative example 8
The comparative example is a preparation method of hedera helix extract, and the difference from example 3 is that:
the ultrasound-assisted extraction in example 3 was replaced by intermittent ultrasound with continuous ultrasound.
The mass fraction of hederacoside C in the hederacoside C extract is 91.61% and the yield is 72.99% through high performance liquid chromatography detection.
The mass fraction of the alpha-hederacoside in the alpha-hederacoside extract is 86.05%, and the yield is 70.26%.
Comparative example 9
The comparative example is a preparation method of hedera helix extract, and the difference from example 3 is that:
the aqueous acidic ethanol solution (pH 5.5) with a volume fraction of 80% during the column chromatography elution of example 3 was replaced with an aqueous alkaline ethanol solution (ph=9, sodium hydroxide adjustment) with a volume fraction of 80%.
The mass fraction of hederagenin C in the hederagenin C extract is 98.17%, and the yield is 83.20%.
The mass fraction of the alpha-hederacoside in the alpha-hederacoside extract is 83.59%, and the yield is 75.24%.
Comparative example 10
The comparative example is a preparation method of hedera helix extract, and the difference from example 3 is that:
the 80% volume fraction of the acidic aqueous ethanol solution (pH 5.5) during the column chromatography elution of example 3 was replaced with 80% volume fraction aqueous ethanol solution.
The mass fraction of hederagenin C in the hederagenin C extract is 98.63% and the yield is 83.30% through high performance liquid chromatography detection.
The mass fraction of the alpha-hederacoside in the alpha-hederacoside extract is 93.29%, and the yield is 65.59%.
The difference between comparative example 1 and example 3 is that: no surfactant or flocculant is added; the alpha-hederagenin with poor water solubility has low extraction rate, and the extracted tannins and other impurities cannot be effectively removed, so that the purity of active ingredients in the extracting solution is low, the separation and purification effects and the yield of the alumina resin are affected, and the purity and the yield of a target product after crystallization are further affected.
The difference between comparative example 2 and example 3 is that: no flocculant is added; the method has the advantages that the method has no synergistic extraction of flocculating agent, the extraction rate of alpha-hederagenin is low, and impurities such as extracted tannins cannot be removed, so that the purity of active ingredients in the extracting solution is low, the purification effect and the yield of aluminum oxide resin are affected, and the purity and the yield of a target product after crystallization are further affected.
The difference between comparative example 3 and example 3 is that: ultrasound-assisted extraction was not employed: the extraction rate of the effective components is low and the flocculation effect is slightly poor, so that the purity of the effective components in the extracting solution is low, thereby influencing the separation and purification effect and yield of the alumina resin and further influencing the purity and yield of the target product after crystallization.
The difference between comparative example 4 and example 3 is that: replacing the alumina column with macroporous resin; the difference between macroporous resin and alumina column chromatographic separation is mainly reflected in the separation effect, and the macroporous resin is sent down to separate hederacoside and alpha-hederacoside better in the test, and secondly, the recovery rate is lower, so that the purity of the extract obtained by separation is lower, and the crystallization yield is further influenced.
The difference between comparative example 5 and example 3 is that: the quality fraction of the auxiliary agent becomes high; the dissolution of the effective components reaches saturation, the dissolution of impurities is increased, the purity of the effective components in the extracting solution is influenced, the foaming is serious, the production operation difficulty is increased, the loss is caused, the yield is influenced, and the purity is low, so that the separation and purification effect and the yield of the alumina resin are influenced, and the purity and the yield of a target product after crystallization are further influenced.
The difference between comparative example 6 and example 3 is that: the pH of the booster solution is not adjusted; the water solubility of the alpha-hederagenin is poor, the extraction rate of the alpha-hederagenin can be effectively improved by adjusting the pH value, the extraction rate of active ingredients is influenced by not adjusting the pH value, and the influence on the active ingredients in the extracting solution is small, so that the purity of the target product is higher, and the yield is lower.
The difference between comparative example 7 and example 3 is that: the pH of the booster solution is too high; the flocculation effect of the flocculant is affected, so that the purity of the active ingredients in the extracting solution is lower, the separation and purification effect and the yield of the alumina resin are affected, and the purity and the yield of the target product after crystallization are further affected.
The difference between comparative example 8 and example 3 is that: adopting continuous ultrasonic treatment; intermittent ultrasonic assistance can improve intermolecular collision, not only promote the formation of linkage acting force between flocculant and impurity and improve flocculation effect, but also promote dissolution of effective components; the ultrasonic treatment is too long, so that the flocculating agent and impurity flocculating structure can be broken, the flocculating effect is poor, and the purity of active ingredients in the extracting solution is low, thereby influencing the separation and purification effect and yield of the alumina resin, and further influencing the purity and yield of the crystallized target product.
The difference between comparative example 9 and example 3 is that: replacing the acidic ethanol aqueous solution with an alkaline ethanol aqueous solution; impurities are easy to elute, which affects the purity of alpha-hederacoside, and further results in lower purity and yield of crystals.
The difference between comparative example 10 and example 3 is that: the acidic aqueous ethanol solution is replaced with an aqueous ethanol solution. The alpha-hederacoside cannot be eluted completely, and the purity and the yield of the alpha-hederacoside are affected, so that the purity and the yield of crystals are lower.
The structure of the surfactant can be damaged due to the fact that the temperature is too high in the extraction process, the extraction efficiency is affected, the movement speed of effective component molecules can be accelerated by increasing the temperature in a proper temperature range, diffusion is accelerated, and the dissolution amount is increased. And the temperature is too high, and the hederagenin C is degraded.
The structure of flocculating agent and impurity flocculation can be broken due to overlong ultrasonic time, so that the flocculation effect is poor, and the proper ultrasonic assistance can improve the collision of molecules, promote the formation of linkage acting force between the flocculating agent and the impurity and improve the flocculation effect; too high ultrasonic frequency can destroy the molecular structure formed by the flocculant and impurity molecules, and reduce flocculation effect.
In conclusion, the preparation method of the invention comprises the steps of crushing the dried raw materials of the ivy leaves, carrying out ultrasonic-assisted continuous countercurrent extraction as an extraction aid, and carrying out solid-liquid separation; concentrating the extractive solution under reduced pressure, separating solid from liquid, subjecting the concentrated solution to alumina column chromatography, and gradient eluting; concentrating and crystallizing the target section eluent under reduced pressure to obtain hederacoside C and alpha-hederacoside extract. The invention comprehensively utilizes the active ingredients in the hedera helix raw material, prepares the hedera helix glycoside C and alpha-hedera helix glycoside extract with high content (more than or equal to 98 percent), and can realize industrial production, the process is safe and reliable, and the invention is environment-friendly.
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 above 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, embodiments of the invention and features of the embodiments may be combined with each other without conflict.
Claims (3)
1. A preparation method of a hedera helix extract is characterized by comprising the following steps:
s1, mixing a hedera helix raw material with an auxiliary agent solution, and performing ultrasonic-assisted continuous countercurrent extraction to obtain an extracting solution;
wherein, the extracting aid solution comprises the following preparation raw materials: the extraction assisting agent comprises a surfactant, hydroxypropyl chitosan and water, wherein the surfactant is at least one of Tween 20, tween 40 and Tween 80, the mass fraction of the extraction assisting agent in the extraction assisting agent solution is 0.3 per mill-0.6 per mill, and the pH value of the extraction assisting agent solution is 7.5-8.5; the parameters of the ultrasonic-assisted continuous countercurrent extraction are as follows: extracting for 30-45 min at 45-60 deg.c and ultrasonic frequency of 20-25 kHz;
s2, concentrating the extracting solution, mixing with ethanol, and collecting a concentrated solution;
s3, eluting the concentrated solution with an aluminum peroxide chromatographic column by sequentially adopting water, an ethanol aqueous solution and an acidic ethanol aqueous solution, collecting the eluent of the ethanol aqueous solution to prepare a hederagenin C eluent, and collecting the eluent of the acidic ethanol aqueous solution to prepare an alpha-hederagenin eluent;
wherein the alumina chromatographic column is a neutral alumina chromatographic column or an acidic alumina chromatographic column, the volume fraction of ethanol in the ethanol aqueous solution is 50% -60%, and the dosage of the ethanol aqueous solution is 2 BV-2.5 BV; the volume fraction of the ethanol in the acidic ethanol aqueous solution is 70% -80%, the dosage of the acidic ethanol aqueous solution is 2 BV-3 BV, and the pH value of the acidic ethanol aqueous solution is 5-6;
s4, crystallizing the hederagenin C eluent to obtain a hederagenin C extract; crystallizing the alpha-hederacoside eluent to obtain an alpha-hederacoside extract.
2. The method for preparing a hedera helix extract according to claim 1, characterized in that: the crystallization temperature of the hederagenin C eluent is 4-10 ℃.
3. The method for preparing a hedera helix extract according to claim 1, characterized in that: the crystallization temperature of the alpha-hederagenin eluent is 4-10 ℃.
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Family Cites Families (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2460136B1 (en) * | 1979-07-05 | 1986-01-10 | Synthelabo | PHARMACEUTICAL COMPOSITIONS CONTAINING CLIMBING IVY EXTRACTS |
| CN1384110A (en) * | 2002-05-20 | 2002-12-11 | 吉林天药科技股份有限公司 | Prepn of hederagenin |
| DE10315931A1 (en) * | 2003-04-02 | 2004-11-04 | Engelhard Arzneimittel Gmbh & Co. Kg | Process for the preparation of an extract of ivy leaves |
| DE10345343A1 (en) * | 2003-09-19 | 2005-04-14 | Engelhard Arzneimittel Gmbh & Co. Kg | Production of an ivy leaf extract with both rapid and prolonged action in treating respiratory diseases, comprises mixing a first ivy extract rich in hederacoside C with a second ivy extract rich in alpha-hederin |
| DE10345342A1 (en) * | 2003-09-19 | 2005-04-28 | Engelhard Arzneimittel Gmbh | Producing an ivy leaf extract containing hederacoside C and alpha-hederin, useful for treating respiratory diseases comprises steaming comminuted ivy leaves before extraction |
| CN101235068B (en) * | 2007-01-29 | 2010-08-25 | 中南大学 | Method for preparing hederacoside C from bindwood |
| CN102188465A (en) * | 2011-04-18 | 2011-09-21 | 中国中医科学院中药研究所 | Preparation method of hedera plant extractives and purposes thereof |
| CN103588832B (en) * | 2013-11-26 | 2017-01-04 | 陕西嘉禾生物科技股份有限公司 | Hederacoside C and the method for aglycon is separated from Rhizoma Calystegiae Hederaceae |
| CN105237608B (en) * | 2015-09-30 | 2017-03-22 | 桂林益元素生物科技有限公司 | Method for extracting hederacoside from ivy |
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