CN114920795B - Preparation method of dried toad skin bufogenin lactone component - Google Patents
Preparation method of dried toad skin bufogenin lactone component Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J71/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton is condensed with a heterocyclic ring
- C07J71/0005—Oxygen-containing hetero ring
- C07J71/001—Oxiranes
- C07J71/0021—Oxiranes at position 14(15)
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/56—Materials from animals other than mammals
- A61K35/65—Amphibians, e.g. toads, frogs, salamanders or newts
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J19/00—Normal steroids containing carbon, hydrogen, halogen or oxygen, substituted in position 17 by a lactone ring
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/10—Preparation or pretreatment of starting material
- A61K2236/15—Preparation or pretreatment of starting material involving mechanical treatment, e.g. chopping up, cutting or grinding
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation, decoction
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/53—Liquid-solid separation, e.g. centrifugation, sedimentation or crystallization
Abstract
The invention discloses a preparation method of a cutis Bufonis bufalin lactone component, which comprises the processes of extraction, membrane separation, reversed-phase preparative chromatography refining, concentration, drying and the like. The yield and the content of the components of the cutis Bufonis bufogenin prepared by the method are high; the preparation process has the characteristics of strong specificity to the cutis Bufonis bufalin lactones, high preparation efficiency, no harmful reagent, harmless emission, low preparation cost and the like, and the obtained cutis Bufonis bufalin lactones have clear and definite compositions and better consistency and stability. The invention establishes a new method for preparing the bufogenin components of the cutis Bufonis, and provides technical support for the deep research on the biological activity of the compounds and the research and development of new drugs.
Description
Technical Field
The invention relates to the technical field of traditional Chinese medicine extraction, in particular to a preparation method of a toad skin bufogenin lactone component.
Background
The dried toad skin is the dried whole skin of Bufo Bufo gargarizans Cantor or Bufo melanostictus B.melanostictus of toads, which is the animal of Bufo Bufo gargarizans, and is recorded in the origin of meridian, as a national drug, the dried toad skin is a toad venom-removed and viscera-removed and dried substance, is a common Zhuang and Yao medicinal material in Guangxi, has the efficacies of clearing away heat and toxic materials, inducing diuresis and relieving distension, and is mainly used for treating carbuncle, pyogenic infections, scrofula, tumors and the like.
Modern medical research shows that the chemical components in the dried toad skin are various, wherein toad venom lactones and indole alkaloids are taken as main components, and toad cyclic amides, steroids and other components are also included.
Modern pharmacological studies show that the bufogenin compounds are the main active ingredients in dried toad skin, and documents prove that the bufogenin compounds such as bufalin, bufogenin and the like have better anti-tumor effect.
At present, methods for extracting bufogenin components from dried toad skin generally comprise two methods:
firstly, water extraction and alcohol precipitation are carried out, the extracted main components are alkaloid components containing a very small amount of bufogenin components with extremely low purity, and the cinobufagin extract disclosed by Chinese patent (publication number: CN 101396373A) and the preparation method thereof, wherein the cinobufagin content is more than 0.15 percent and the resibufogenin content is more than 0.10 percent;
the other is organic solvent extraction, passing through macroporous adsorbent resin column, eluting with organic solvent, chinese patent (publication No. CN 101209264A) discloses a preparation method of total bufogenin extract with anti-tumor effect, wherein the resibufogenin content is more than 12%, the cinobufagin content is more than 4%, and the bufalin content is more than 10%, but the used organic solvent is toxic and harmful solvents such as ethyl acetate, diethyl ether, etc., and is not suitable for large-scale production.
Disclosure of Invention
In view of the above situation, the present invention aims to provide a method for preparing a bufogenin component from cutis Bufonis, which can obtain a bufogenin component with high purity through a series of preparation processes on the premise of ensuring safety, environmental protection, no toxicity and no harm.
The technical scheme of the invention is as follows:
a method for preparing cutis Bufonis venenum lactone components comprises the following steps:
1) Preparing a crude extract of the dried toad skin and toad venom lactone components: taking a dried toad skin medicinal material, cutting up, taking water as an extraction solvent, soaking for 0.5-2 hours, then decocting for 0.5-3 hours, and filtering to obtain a liquid medicine a; adding the extraction solvent into the dregs of a decoction again for decocting for 1-3 times, wherein each time of decoction is 0.5-3 hours, filtering to obtain liquid medicine, adding the extraction solvent into the dregs of a decoction for decocting, filtering to obtain liquid medicine for 1-3 times, filtering the liquid medicine after the dregs of a decoction, and mixing the liquid medicine with the liquid medicine a to obtain a crude extract solution of the bufogenin components;
2) Preparing a clarified liquid of the components of the cutis Bufonis venenum lactone: separating the solution of the crude extract of the bufogenin components obtained in the step 1) by at least two or more than two stages of membranes to remove insoluble particulate matters, pigments and exogenous toxic and harmful substances, so as to obtain a bufogenin component clarified liquid;
3) Preparing a refined liquid of the dried toad skin and toad venom lactone components: refining and purifying the clarified liquid of the dried toad skin bufogenin lactone components obtained in the step 2) by high performance preparative liquid chromatography, and collecting column flow-through liquid to obtain refined liquid of the dried toad skin bufogenin lactone components;
4) Preparing powder of dried toad skin and toad venom lactone components: concentrating and drying the refined liquid of the dried toad skin bufogenin components obtained in the step 3) to obtain pure powder of the dried toad skin bufogenin components.
The preparation method of the dried toad skin bufogenin lactone component comprises the step 1), wherein the weight of the extracting solvent added in each time is 2-20 times of the weight of the dried toad skin medicinal material, and the decocting temperature is 40-100 ℃.
In the step 2), the filtering membrane used for membrane separation is an inorganic membrane or an organic membrane, the membrane component is one or at least two of a hollow fiber membrane, a ceramic membrane, a spiral membrane, a tubular membrane or a flat membrane, and when two or more than two stages of membranes are separated, the aperture of the next stage of membrane in the separation process is smaller than that of the previous stage of membrane;
the preparation method of the dry toad skin bufogenin lactone component comprises the step 2), two-stage membrane separation is adopted, the aperture of the first-stage filtration membrane is 100-500 nm, and the aperture of the second-stage filtration membrane is 5-1000 kd.
The preparation method of the dry toad skin bufogenin lactone component comprises the following steps of (1) in the step 2), the membrane filtration flux is 0.05-2.50L/min, the membrane feeding pressure is 0-0.4 MPa, and the membrane passing temperature is 15-35 ℃; after the solution of the crude extract of the bufogenin components of the dry toad skin obtained in the step 1) passes through a membrane, washing and filtering residues on the membrane by using water, wherein the addition of 0.5-10L of washing and filtering water is added for every 50-1000 g of dry toad skin medicinal material.
The preparation method of the cutis Bufonis bufogenin lactone component comprises, in step 3), the high performance preparative liquid chromatography preparation method comprises: the sample concentration is 0.1-100 mg/mL, firstly methanol with 2-9 times of column volume is used for activating to prepare a liquid chromatographic column, then pure water balancing liquid with 1-10 times of column volume is used for washing the chromatographic column, and the sample loading speed is 10-80 mL/min; one or at least two of isocratic elution, linear gradient elution or step gradient elution are adopted.
The preparation method of the dried toad skin bufogenin lactone component comprises the following steps of 1 elution, wherein elution solvent is 0-30% ethanol water solution in volume concentration, elution volume is 1-9 times of column volume, and flow rate is 10-80 mL/min; the solvent for eluting the dried toad skin and toad venom lactone components is 30-95% alcohol water solution, the eluent is collected and decompressed and concentrated until the alcohol is completely removed, and the concentration temperature is 30-70 ℃.
The preparation method of the dried toad skin bufogenin lactone component comprises the following steps of preparing one or two of macroporous resin and silica gel by using a high performance preparative liquid chromatography, wherein the macroporous resin comprises one or at least two of WA30, HPD400 and PS-NVP; the silica gel comprises one or at least two of C18 (ODS), C8, C4, and C18 YE.
The preparation method of the cutis Bufonis bufalin lactone component comprises, in step 4), concentrating by one or at least two of normal pressure evaporation, reduced pressure evaporation, thin film evaporation and multiple effect evaporation; the drying method is one or at least two of reduced pressure drying, freeze drying, microwave drying and spray drying.
The preparation method of the dried toad skin bufogenin lactone component provided by the invention has the following beneficial effects:
1) The invention firstly extracts the dried toad skin with water to obtain a crude extract (containing bufogenin and alkaloid components), removes the alkaloid components without antitumor activity and other impurities through membrane separation and column chromatography, and efficiently enriches the bufogenin components with antitumor activity;
2) The invention adopts the segmented membrane filtration technology, can realize separation and concentration at normal temperature, has no phase change and chemical change in the whole process, has low energy consumption, good selectivity, strong adaptability, simple, economical and practical operation and higher efficiency and stability, and is suitable for industrial production;
3) The method is simple to operate, all used reagents are nontoxic and harmless, the organic solvent can be recycled, the method is environment-friendly and efficient, and the method can be used for large-scale production and has good industrial prospect.
Therefore, the invention establishes a new preparation method of the bufogenin lactone component of the cutis Bufonis, and provides technical support for the deep research on the biological activity of the compound, the research and development of new drugs and the industrial production.
Drawings
FIG. 1 is a schematic flow chart of the preparation method of the bufogenin component of the cutis Bufonis provided by the present invention;
fig. 2 is a UPLC analysis chromatogram of a crude extract, a clarified liquid and a refined liquid of bufogenin components from toad skin in the experimental process of example 1 of the present invention;
FIG. 3 is UPLC analysis chromatogram of pure powder of bufogenin components of Bufo siccus skin in the experimental process of example 1 of the present invention;
FIG. 4 is a graph showing the results of measuring the contents of the crude extract, clarified solution, refined solution of the bufogenin components of Bufo siccus in example 1, and the pure powder of the bufogenin components of Bufo siccus.
Detailed Description
To facilitate an understanding of the invention, the invention will now be described more fully hereinafter with reference to the accompanying drawings. Several embodiments of the invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
Referring to fig. 1, the present invention provides a method for preparing bufogenin components from cutis Bufonis, comprising the following steps:
1) Preparing a crude extract of the dried toad skin and toad venom lactone components: taking a dried toad skin medicinal material, cutting up, taking water as an extraction solvent, soaking for 0.5-2 hours, then decocting for 0.5-3 hours, and filtering to obtain a liquid medicine a; adding the extraction solvent into the dregs of a decoction again for decocting for 1-3 times, wherein each time of decoction is 0.5-3 hours, filtering to obtain liquid medicine, adding the extraction solvent into the dregs of a decoction for decocting, filtering to obtain liquid medicine for 1-3 times, filtering the liquid medicine after the dregs of a decoction, and mixing the liquid medicine with the liquid medicine a to obtain a crude extract solution of the bufogenin components;
2) Preparing a toad skin and toad venom lactone component clear solution: separating the crude extract solution of the dry toad skin bufogenin components obtained in the step 1) by at least two or more than two stages of membranes to remove insoluble particulate matters, pigments and exogenous toxic and harmful substances, so as to obtain a clear solution of the dry toad skin bufogenin components;
3) Preparing a refined liquid of the dried toad skin bufalin lactone component: refining and purifying the clarified liquid of the dried toad skin bufogenin lactone components obtained in the step 2) by high performance preparative liquid chromatography, and collecting column flow-through liquid to obtain refined liquid of the dried toad skin bufogenin lactone components;
4) Preparing powder of dried toad skin and toad venom lactone components: concentrating and drying the refined liquid of the dried toad skin bufogenin components obtained in the step 3) to obtain the pure powder of the dried toad skin bufogenin components.
The preparation method of the dried toad skin bufogenin lactone component comprises the step 1), wherein the weight of the extracting solvent added in each time is 2-20 times of the weight of the dried toad skin medicinal material, and the decocting temperature is 40-100 ℃.
In the step 2), the filtering membrane used for membrane separation is an inorganic membrane or an organic membrane, the membrane component is one or at least two of a hollow fiber membrane, a ceramic membrane, a spiral membrane, a tubular membrane or a flat membrane, and when two or more than two stages of membranes are separated, the aperture of the next stage of membrane in the separation process is smaller than that of the previous stage of membrane;
the preparation method of the cutis Bufonis bufalin lactone component comprises, in step 2), two-stage membrane separation is adopted, the aperture of the first stage filtration membrane is 100-500 nm, and the aperture of the second stage filtration membrane is 5-1000 kd.
The preparation method of the dry toad skin bufogenin lactone component comprises the following steps of (1) in the step 2), the membrane filtration flux is 0.05-2.50L/min, the membrane feeding pressure is 0-0.4 MPa, and the membrane passing temperature is 15-35 ℃; after the crude extract solution of the dry toad skin bufogenin lactone components obtained in the step 1) passes through a membrane, washing and filtering residues on the membrane by using water, wherein the addition amount of the washing and filtering water of each 50-1000 g of dry toad skin medicinal material is 0.5-10L.
The preparation method of the cutis Bufonis bufogenin lactone component comprises, in step 3), the high performance preparative liquid chromatography preparation method comprises: the sample concentration is 0.1-100 mg/mL, firstly methanol with 2-9 times of column volume is used for activation to prepare a liquid chromatographic column, then pure water equilibrium liquid with 1-10 times of column volume is used for washing the chromatographic column, and the sample loading speed is 10-80 mL/min; one or at least two of isocratic elution, linear gradient elution or step gradient elution are adopted.
The preparation method of the cutis Bufonis bufalin lactone component comprises the following steps of eluting for 1 time, wherein the elution solvent is 0-30% ethanol aqueous solution (comprising ethanol, methanol and other organic solvents) in volume concentration, the elution volume is 1-9 times of column volume, and the flow rate is 10-80 mL/min; the solvent for eluting the dried toad skin bufonin lactone components is an alcohol aqueous solution with the volume concentration of 30-95%, the eluent is collected and concentrated under reduced pressure until the alcohol is completely removed, and the concentration temperature is 30-70 ℃.
The preparation method of the dried toad skin bufogenin lactone component comprises the following steps of preparing one or two of macroporous resin and silica gel by using a high performance preparative liquid chromatography, wherein the macroporous resin comprises one or at least two of WA30, HPD400 and PS-NVP; the silica gel comprises one or at least two of C18 (ODS), C8, C4, and C18 YE.
The preparation method of the cutis Bufonis bufogenin lactone component comprises the steps of, wherein the concentration mode in step 4) is one or at least two of normal pressure evaporation, reduced pressure evaporation, thin film evaporation and multiple-effect evaporation; the drying method is one or at least two of reduced pressure drying, freeze drying, microwave drying and spray drying.
The invention is further illustrated below by means of a number of examples. The following examples are only preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the following examples, and any other changes, substitutions, combinations or simplifications which do not depart from the gist of the present invention should be construed as being equivalent replacements within the scope of the present invention.
Example one
A method for preparing cutis Bufonis venenum lactone components comprises the following steps:
step 1: cutting dried toad skin medicinal materials into pieces with scissors, putting 300g of the dried toad skin medicinal materials into a decocting machine, adding 3.0L of pure water, soaking for 30 minutes, decocting for 1 hour at 100 ℃, filtering with a 200-mesh filter screen, collecting filtrate, adding 3L of pure water into dregs of a decoction, putting the dregs of a decoction into the decocting machine again, decocting for 1 hour at 100 ℃, filtering with a 200-mesh filter screen, collecting filtrate, mixing the filtrate obtained after decocting the dregs of a decoction with the filtrate obtained after decocting the medicinal materials to obtain a crude extract of dried toad skin bufonin lactone components, carrying out UPLC analysis on a sample of the crude extract, and obtaining a chromatographic analysis result as shown in figure 2;
step 2: the first stage membrane separation is that 500nm ceramic membrane (membrane aperture 500nm, effective membrane area 0.12m2) is connected to a membrane filtration system (model: ceramem 0100-01), the membrane passing temperature is 25 ℃, the membrane pressure is 0.3MPa, the membrane flux is 1.5L/min, membrane permeation liquid is collected, 3L pure water is taken, 500mL of pure water is added every 5 minutes for washing a filter membrane, membrane permeation washing liquid is collected, substances with the wavelength of more than 500nm are removed, and the membrane permeation liquid and the membrane permeation washing liquid are combined to obtain a primary clarified liquid of the dry bufogenin components; connecting a 10KD roll-type membrane (the membrane aperture is 10KD, and the effective membrane area is 0.36m2) to a membrane filtration system (model: RNF 0460-016) for secondary membrane separation, taking a primary clarified liquid of the dried bufogenin components, performing membrane separation at the membrane passing temperature of 25 ℃ and the membrane pressure of 0.3MPa, wherein the membrane flux is 0.5L/min, collecting membrane permeate, taking 3L of pure water, adding 500mL every 5 minutes for washing and filtering, collecting membrane permeate washing filtrate, removing substances above 10KD, combining the membrane permeate and the membrane permeate washing filtrate to obtain a clarified liquid of the dried bufogenin components, performing UPLC analysis on a sample of the clarified liquid, and obtaining a chromatographic analysis result shown in figure 2;
and step 3: connecting a chromatographic column (the type of a filler is C18YE, the specification is 30 x 240mm, the weight of the filler is 104g, the particle size is 30 mu m, and the column volume is about 170 mL) to a high-pressure liquid phase system, designing the flow rate to be 25mL/min, firstly activating by using 3 times of column volume of methanol to prepare the liquid chromatographic column, then flushing 3 times of column volume of the chromatographic column by using pure water balance liquid, flowing a dried toad skin bufogenin lactone component clear solution (the sample concentration is 8.48 mg/mL) through a system pump to purify the chromatographic column, eluting by using 30% ethanol aqueous solution in volume concentration to remove impurities (3 times of column volume), eluting a target object (3 times of column volume) by using 70% ethanol aqueous solution in volume concentration, collecting 70% ethanol eluent to obtain dried toad skin bufogenin lactone component refined solution, and carrying out UPLC analysis, wherein the chromatographic analysis result is shown in figure 2;
and 4, step 4: the refined liquid is put in a refrigerator with the temperature of minus 80 ℃ for freezing for 6 hours after the ethanol is removed by rotary evaporation at the temperature of 50 ℃, the prefreezed refined liquid is taken and put in a vacuum freeze drier, the vacuum freeze drying is carried out under the condition of 0.2MPa and minus 80 ℃ until the moisture is completely removed, the freeze-dried powder is collected, namely the powder of the dried toad skin bufonin lactone components, and the weight of the obtained sample is weighed. The content of total bufogenin in the toad skin bufogenin lactone component sample is calculated by adopting liquid phase analysis weighing analysis, and the UPLC analysis result of the powder of the toad skin bufogenin lactone component is shown in figure 3, the content of the bufogenin is about 25.38%, and the yield is about 98%.
In this embodiment, a graph of the detection results of the crude extract, the clear solution, the refined solution and the pure powder of the bufogenin component can be seen in fig. 4.
Example two
A method for preparing cutis Bufonis venenum lactone components comprises the following steps:
step 1: cutting dried toad skin medicinal materials into pieces by using scissors, putting 300g of the dried toad skin medicinal materials into a decocting machine, adding 3.0L of pure water, soaking for 30 minutes, decocting for 1 hour at 100 ℃, filtering by using a 200-mesh filter screen, collecting filtrate, adding 3L of pure water into dregs of a decoction, putting the dregs of a decoction into the decocting machine again, decocting for 1 hour at 100 ℃, filtering by using a 200-mesh filter screen, collecting filtrate, filtering out liquid medicine after decocting the dregs of a decoction, combining the filtered liquid medicine after decocting the medicinal materials, and obtaining crude extract of the dried toad skin bufogenin lactone components;
step 2: the first stage of membrane separation is to connect a 500nm ceramic membrane (with the membrane aperture of 500nm and the effective membrane area of 0.12m2) to a membrane filtration system (model: ceramem 0100-01), collect membrane permeate, take 3L pure water, add 500mL of pure water every 5 minutes to wash the filtration membrane, collect membrane permeate washing filtrate to remove substances with the size of more than 500nm, combine the membrane permeate and the membrane permeate washing filtrate to obtain a primary clarified solution of the dried toad skin bufogenin components; connecting a 100KD roll-type membrane (the membrane aperture is 100KD, and the effective membrane area is 0.36m2) to a membrane filtration system (model: RNF 0460-016) by secondary membrane separation, taking a primary clarified liquid of the dried toad skin bufogenin components, carrying out membrane separation under the conditions that the membrane passing temperature is 25 ℃ and the membrane pressure is 0.3MPa, wherein the membrane flux is 1.0L/min, collecting membrane permeate, taking 3L of pure water, adding 500mL of purified water every 5 minutes for washing and filtering, collecting membrane permeation washing filtrate, removing substances with the density of more than 100KD, and combining the membrane permeate and the membrane permeation washing filtrate to obtain the clarified liquid of the dried toad skin bufogenin components;
and step 3: connecting a chromatographic column (the type of a filler is C18YE, the specification is 30 x 240mm, the weight of the filler is 104g, the particle size is 30 mu m, and the column volume is about 170 mL) to a high-pressure liquid phase system, designing the flow rate to be 25mL/min, firstly, activating by using 3 times of column volume of methanol to prepare the liquid chromatographic column, then, flushing 3 times of column volume of the chromatographic column by using pure water balance liquid, flowing a clarified liquid (the sample concentration is 8.48 mg/mL) of a bufogenin lactone component through a system pump, purifying by using a 30% ethanol aqueous solution in volume concentration to remove impurities (3 times of column volume), eluting a target object (3 times of column volume) by using a 70% ethanol aqueous solution in volume concentration, and collecting 70% ethanol eluent, namely the refined liquid of the bufogenin components of the bufogenin the cutis bufogenin the dried bufogenin;
and 4, step 4: removing ethanol from the refined liquid by rotary evaporation at 50 deg.C, freezing in a refrigerator at-80 deg.C for 6 hr, vacuum freeze-drying at-80 deg.C under 0.2MPa in a vacuum freeze-drying machine until water is removed completely, collecting freeze-dried powder, i.e. cutis Bufonis bufogenin lactone component powder, and weighing the obtained sample. The content of the total bufogenin in the dried toad skin bufogenin lactone component sample is calculated by adopting liquid phase analysis weighing analysis, the content of the bufogenin is about 22.44 percent, and the yield is about 97 percent.
EXAMPLE III
A preparation method of a toad skin bufogenin lactone component comprises the following steps:
step 1: cutting dried toad skin medicinal materials into pieces by using scissors, putting 300g of the dried toad skin medicinal materials into a decocting machine, adding 3.0L of pure water, soaking for 30 minutes, decocting for 1 hour at 100 ℃, filtering by using a 200-mesh filter screen, collecting filtrate, adding 3L of pure water into dregs of a decoction, putting the dregs of a decoction into the decocting machine again, decocting for 1 hour at 100 ℃, filtering by using a 200-mesh filter screen, collecting filtrate, filtering out liquid medicine after decocting the dregs of a decoction, combining the filtered liquid medicine after decocting the medicinal materials, and obtaining crude extract of the dried toad skin bufogenin lactone components;
step 2: the first stage membrane separation is that 500nm ceramic membrane (membrane aperture 500nm, effective membrane area 0.12m2) is connected to a membrane filtration system (model: ceramem 0100-01), the membrane passing temperature is 25 ℃, the membrane pressure is 0.2MPa, the membrane flux is 1.5L/min, membrane permeation liquid is collected, 3L pure water is taken, 500mL of pure water is added every 5 minutes for washing a filter membrane, membrane permeation washing liquid is collected, substances with the wavelength of more than 500nm are removed, and the membrane permeation liquid and the membrane permeation washing liquid are combined to obtain a dried toad skin bufogenin component clear liquid;
and 3, step 3: connecting a chromatographic column (the type of a filler is C18YE, the specification is 30 multiplied by 240mm, the weight of the filler is 104g, the particle size is 30 microns, and the column volume is about 170 mL) to a high-pressure liquid phase system, designing the flow rate to be 25mL/min, firstly activating by using methanol with 3 times of the column volume to prepare the liquid-phase chromatographic column, then washing the column with 3 times of the column volume by using pure water balance liquid, flowing clear liquid (the sample concentration is 8.48 mg/mL) of the bufogenin lactone components through a system pump, purifying by using an ethanol aqueous solution with the volume concentration of 30 percent to remove impurities (3 times of the column volume), eluting a target object (3 times of the column volume) by using an ethanol aqueous solution with the volume concentration of 70 percent, and collecting 70 percent ethanol eluent, namely the bufogenin component refined liquid of the bufogenin cutis bufogenin;
and 4, step 4: the refined liquid is put in a refrigerator with the temperature of minus 80 ℃ for freezing for 6 hours after the ethanol is removed by rotary evaporation at the temperature of 50 ℃, the prefreezed refined liquid is taken and put in a vacuum freeze drier, the vacuum freeze drying is carried out under the condition of 0.2MPa and minus 80 ℃ until the moisture is completely removed, the freeze-dried powder is collected, namely the powder of the dried toad skin bufonin lactone components, and the weight of the obtained sample is weighed. The content of the total bufogenin in the dried toad skin bufogenin lactone component sample is calculated by adopting liquid phase analysis weighing analysis, the content of the bufogenin is about 15.38 percent, and the yield is about 96 percent.
In the above examples, the UPLC analysis conditions were as follows:
(1) Preparing a reference solution: taking appropriate amount of bufotalin and resibufogenin reference substances, and mixing with methanol to obtain reference substance solution containing 100 μ g of bufotalin per 1 mL.
(2) Preparing a test solution: taking 5mg of sample component powder to be detected, precisely weighing, adding 5mL of 70% ethanol aqueous solution with volume concentration into a 5mL measuring flask, dissolving to scale, shaking up, performing ultrasonic treatment for 10 minutes, cooling to room temperature, shaking up, centrifuging for 10 minutes at 10000 r.min < -1 >, taking supernatant, filtering, and taking subsequent filtrate to obtain the product.
(3) Respectively injecting the test solution and the reference mixed solution into an ultra-high performance liquid chromatograph for analysis, and determining corresponding peak areas of the bufotalin and other bufogenin components in the reference and the test solution, wherein the chromatographic conditions are as follows:
type of chromatographic column: c18 HD (2.5 μm, 2.1X 100 mm);
mobile phase A: acetonitrile; mobile phase B: 0.1% by volume aqueous formic acid solution;
flow rate: 0.4mL/min;
gradient conditions: 0 min-10 min, from 5-90% by weight, A, 10min-15min 90%;
column temperature: 30 ℃;
detection wavelength: 296nm;
sample introduction volume: 1 μ L.
(4) And (3) calculating the content: external standard method (
Wherein Ax represents the peak area of the component to be measured in the sample to be measured, rf represents a correction factor, and m represents the sample weighing mass of the sample to be measured) (correction factor)
) And calculating the content of mangiferin and isomangiferin in the sample to be detected.
(5) And (3) calculating yield:
(6) The calculation method of the total bufogenin components comprises the following steps: according to the definition result of the major components and the main components, the total content of the bufotalin, the cinobufagin and the lipobufogenin is taken as the content of the bufogenin type components of the cutis Bufonis bufalin lactone.
The sample powder of the components of the toad skin bufogenin obtained by refining the preparation method in each embodiment has uniform color and luster and fine texture; adding purified water into the final pure powder of the cutis Bufonis lactone component, and dissolving rapidly to obtain clear solution;
as shown in fig. 2 and 3, in the first embodiment, the UPLC analysis results of the steps show that the target peak in the analysis spectrogram of the sample before and after purification has not significantly changed, and the calculation according to the total volume, the integrated peak area and the weight shows that the content of the main component of the purified cutis Bufonis lactone component has not significantly decreased, the yield of the bufogenin lactone substances is more than 95%, and the total bufogenin content is more than 10%. The bufogenin lactone component obtained by the method has the advantages of high yield, safety and environmental friendliness.
TABLE 1 comparison of Total bufogenin content obtained in examples of the present invention and the prior art preparation method
As can be seen from table 1, compared with the prior art, the content of the total bufogenin obtained by the method of the present invention is higher than that of the prior art on the premise of ensuring safety, environmental protection, no toxicity and no harm, and obviously, the preparation method of the present invention can obtain better product and better technical effect.
In conclusion, the preparation method of the cutis Bufonis venenum lactone component provided by the invention has the following beneficial effects:
1) The invention firstly extracts the dried toad skin with water to obtain a crude extract (containing bufogenin and alkaloid components), removes the alkaloid components without antitumor activity and other impurities through membrane separation and column chromatography, and efficiently enriches the bufogenin components with antitumor activity;
2) The invention adopts the segmented membrane filtration technology, can realize separation and concentration at normal temperature, has no phase change or chemical change in the whole process, has low energy consumption, good selectivity, strong adaptability, simple, economical and practical operation and higher efficiency and stability, and is suitable for industrial production;
3) The method is simple to operate, all used reagents are nontoxic and harmless, organic solvents can be recycled, the method is environment-friendly and efficient, and the method can be used for large-scale production and has good industrial prospect.
Therefore, the invention establishes a new preparation method of the bufogenin lactone component of the cutis Bufonis, and provides technical support for the deep research on the biological activity of the compound, the research and development of new drugs and the industrial production.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is specific and detailed, but not to be understood as limiting the scope of the present invention. It should be noted that various changes and modifications can be made by those skilled in the art without departing from the spirit of the invention, and these changes and modifications are all within the scope of the invention. Therefore, the protection scope of the present patent should be subject to the appended claims.
Claims (6)
1. A preparation method of a cutis Bufonis venenum lactone component is characterized by comprising the following steps:
1) Preparing a crude extract of the dried toad skin and toad venom lactone components: taking a dried toad skin medicinal material, cutting up, taking water as an extraction solvent, soaking for 0.5-2 hours, then decocting for 0.5-3 hours, and filtering to obtain a liquid medicine a; adding the extraction solvent into the dregs of a decoction again for decocting for 1-3 times, wherein each time of decoction is 0.5-3 hours, filtering to obtain liquid medicine, adding the extraction solvent into the dregs of a decoction for decocting, filtering to obtain liquid medicine for 1-3 times, filtering the liquid medicine after the dregs of a decoction, and mixing the liquid medicine with the liquid medicine a to obtain a crude extract solution of the bufogenin components;
2) Preparing a toad skin and toad venom lactone component clear solution: separating the solution of the crude extract of the bufogenin components obtained in the step 1) by at least two or more than two stages of membranes to remove insoluble particulate matters, pigments and exogenous toxic and harmful substances, so as to obtain a bufogenin component clarified liquid;
3) Preparing a refined liquid of the dried toad skin bufalin lactone component: refining and purifying the toad skin bufogenin component clear solution obtained in the step 2) by high performance liquid chromatography, and collecting column flow-through liquid to obtain a toad skin bufogenin component refined solution;
4) Preparing powder of dried toad skin and toad venom lactone components: concentrating and drying the refined liquid of the dried toad skin bufogenin components obtained in the step 3) to obtain pure powder of the dried toad skin bufogenin components;
wherein, in the step 3), the preparation method of the high performance preparative liquid chromatography comprises the following steps: the sample concentration is 0.1-100 mg/mL, firstly methanol with 2-9 times of column volume is used for activating to prepare a liquid chromatographic column, then pure water balancing liquid with 1-10 times of column volume is used for washing the chromatographic column, and the sample loading speed is 10-80 mL/min; adopting one or at least two of isocratic elution, linear gradient elution or step gradient elution;
the elution mode is 1 time of elution, the elution solvent is an ethanol aqueous solution with the volume concentration of 0-30%, the elution volume is 1-9 times of the column volume, and the flow rate is 10-80 mL/min; the solvent for eluting the dried toad skin and toad venom lactone components is an alcohol aqueous solution with the volume concentration of 30-95 percent, the eluent is collected and concentrated under reduced pressure until the alcohol is completely removed, and the concentration temperature is 30-70 ℃;
the high performance preparative liquid chromatography preparative column packing is one or two of macroporous resin and silica gel, wherein the macroporous resin comprises one or at least two of WA30, HPD400 and PS-NVP; the silica gel comprises one or at least two of C18 (ODS), C8, C4, and C18 YE.
2. The method for preparing bufogenin lactone components of cutis Bufonis as claimed in claim 1, wherein in step 1), the weight of the extraction solvent added each time is 2-20 times of the weight of cutis Bufonis, and the decocting temperature is 40-100 deg.C.
3. The method for preparing bufogenin components according to claim 1, wherein in step 2), the filtering membrane used for membrane separation is an inorganic membrane or an organic membrane, the membrane module is one or at least two of a hollow fiber membrane, a ceramic membrane, a spiral membrane, a tubular membrane or a flat membrane, and when two-stage or more than two-stage membrane separation is performed, the pore diameter of the membrane at the next stage in the separation process is smaller than that of the membrane at the previous stage.
4. The method for preparing bufogenin lactone according to claim 1, wherein in step 2), two-stage membrane separation is adopted, the aperture of the first stage filtration membrane is 100 nm-500 nm, and the aperture of the second stage filtration membrane is 5 kd-1000 kd.
5. The method for preparing the bufogenin component of claim 3 or 4, wherein in the step 2), the membrane filtration flux is 0.05-2.50L/min, the membrane pressure is 0-0.4 MPa, and the membrane passing temperature is 15-35 ℃; after the crude extract solution of the dry toad skin bufogenin lactone components obtained in the step 1) passes through a membrane, washing and filtering residues on the membrane by using water, wherein the addition amount of the washing and filtering water of each 50-1000 g of dry toad skin medicinal material is 0.5-10L.
6. The method for preparing the bufogenin lactone component according to claim 1, wherein the concentration mode in step 4) is one or at least two of normal pressure evaporation, reduced pressure evaporation, thin film evaporation and multiple effect evaporation; the drying method is one or at least two of reduced pressure drying, freeze drying, microwave drying and spray drying.
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