CN117105993A - Method for integrated co-extraction of fritillary alkaloids and polysaccharides - Google Patents
Method for integrated co-extraction of fritillary alkaloids and polysaccharides Download PDFInfo
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- CN117105993A CN117105993A CN202311206472.8A CN202311206472A CN117105993A CN 117105993 A CN117105993 A CN 117105993A CN 202311206472 A CN202311206472 A CN 202311206472A CN 117105993 A CN117105993 A CN 117105993A
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- IUKLSMSEHKDIIP-BZMYINFQSA-N Verticine Chemical compound C([C@@H]1[C@@H](O)C[C@H]2[C@@H]3CC[C@@H]4[C@]5(C)O)[C@@H](O)CC[C@]1(C)[C@H]2C[C@H]3[C@@H]4CN1[C@H]5CC[C@H](C)C1 IUKLSMSEHKDIIP-BZMYINFQSA-N 0.000 description 3
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- 229920002498 Beta-glucan Polymers 0.000 description 1
- 241000605372 Fritillaria Species 0.000 description 1
- 241000234280 Liliaceae Species 0.000 description 1
- 241001629511 Litchi Species 0.000 description 1
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- OQUKIQWCVTZJAF-UHFFFAOYSA-N phenol;sulfuric acid Chemical compound OS(O)(=O)=O.OC1=CC=CC=C1 OQUKIQWCVTZJAF-UHFFFAOYSA-N 0.000 description 1
- IWZKICVEHNUQTL-UHFFFAOYSA-M potassium hydrogen phthalate Chemical compound [K+].OC(=O)C1=CC=CC=C1C([O-])=O IWZKICVEHNUQTL-UHFFFAOYSA-M 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
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- 229930003352 steroid alkaloid Natural products 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07G—COMPOUNDS OF UNKNOWN CONSTITUTION
- C07G5/00—Alkaloids
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/88—Liliopsida (monocotyledons)
- A61K36/896—Liliaceae (Lily family), e.g. daylily, plantain lily, Hyacinth or narcissus
- A61K36/8966—Fritillaria, e.g. checker lily or mission bells
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D11/00—Solvent extraction
- B01D11/02—Solvent extraction of solids
- B01D11/0261—Solvent extraction of solids comprising vibrating mechanisms, e.g. mechanical, acoustical
- B01D11/0265—Applying ultrasound
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D11/00—Solvent extraction
- B01D11/02—Solvent extraction of solids
- B01D11/028—Flow sheets
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D11/00—Solvent extraction
- B01D11/02—Solvent extraction of solids
- B01D11/0288—Applications, solvents
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Organic Chemistry (AREA)
- Mycology (AREA)
- Polymers & Plastics (AREA)
- Acoustics & Sound (AREA)
- Botany (AREA)
- Medicinal Chemistry (AREA)
- Food Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Nutrition Science (AREA)
- General Health & Medical Sciences (AREA)
- Medical Informatics (AREA)
- Epidemiology (AREA)
- Alternative & Traditional Medicine (AREA)
- Materials Engineering (AREA)
- Microbiology (AREA)
- Sustainable Development (AREA)
- Pharmacology & Pharmacy (AREA)
- Biotechnology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Mechanical Engineering (AREA)
- Biochemistry (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention relates to a technology for integrating and extracting alkaloids and polysaccharides from fritillary bulb, which comprises the following steps: and (3) circularly freezing and thawing the fritillary bulb powder, extracting by adopting an ultrasonic-assisted ethanol-ammonium sulfate double-aqueous-phase compound enzyme system, and purifying an upper phase extracting solution and a lower phase extracting solution by dialysis, concentration, alcohol precipitation and D4006 macroporous resin column to obtain various active ingredients of alkaloid and polysaccharide. The invention integrates the damage of freeze thawing to cell walls and organelle membranes into an ultrasonic-assisted double-aqueous-phase compound enzyme extraction method, promotes release of intracellular secondary metabolites to the greatest extent, simplifies the separation step, simultaneously separates multiple components and improves the utilization rate of raw materials. The method has mild conditions, simple process operation, difficult denaturation or structural damage of the fritillary alkaloids and the polysaccharide in the operation process, and easy industrial production and use.
Description
Technical field:
the invention relates to a method for extracting and separating natural products, in particular to a method for integrally co-extracting fritillary alkaloids and polysaccharides.
The background technology is as follows:
fritillary bulb (Fritillaria ussuriensisMaxim) is also called fritillary bulb, which is a perennial herb of fritillary genus of the family liliaceae, the medicinal part is a dried bulb of fritillary bulb, and the fritillary bulb is collected into Chinese pharmacopoeia by virtue of the efficacy of clearing heat, moistening lung, resolving phlegm and relieving cough, and is one of the medicinal materials in northeast district and region of China. The Pinctada comprises various active substances such as steroid alkaloid, polysaccharide, saponin, terpenoid, etc. Wherein the Pinctada polysaccharide has anti-tumor, antioxidant, antifatigue, and blood sugar lowering effects; the fritillary alkaloid has good curative effects in clearing heat and moistening lung, calming, anti-inflammatory, resolving phlegm and relieving cough and the like.
With the emphasis of self health and environmental protection, attention is paid to natural medicines rich in secondary metabolites which have undergone long-term evolution. The fritillary bulb contains secondary metabolites such as alkaloid and polysaccharide with various pharmacological activities, and the secondary metabolites become research hot spots for developing dragon medicines, and the process method for extracting the alkaloid and the polysaccharide with high efficiency and green is a key step for realizing the processing of the fritillary bulb high-added-value products. In the past ten years, extraction of Yu Pingbei alkaloids and polysaccharides by water/organic solvent extraction, microwave-assisted extraction, supercritical extraction and the like have been widely applied, but methods for simultaneously integrating two active ingredients of alkaloids and polysaccharides have been reported. Ultrasonic Assisted Extraction (UAE) utilizes ultrasonic waves to generate high-speed and strong cavitation effect and stirring effect to destroy cells of plant medicinal materials and enable solvents to permeate into the medicinal material cells, so that the extraction time is shortened and the extraction efficiency is improved. Aqueous two-phase extraction (ATPE) has a biphasic extraction capacity and selectivity such that the target component can be extracted into the upper and lower phases, respectively. The enzyme-assisted extraction (EAE) has specificity and can remarkably improve the extraction efficiency of target substances. The ultrasonic-assisted double-aqueous-phase compound enzyme extraction technology (UA-ATPE-EAE) combines the advantages of ultrasonic action and double-aqueous-phase compound enzyme extraction, utilizes the cavitation phenomenon of ultrasonic waves and liquid flow power to effectively crush suspended particles, uniformly mixes samples, accelerates the transmission and distribution between a target substance and two phases, and greatly improves the extraction efficiency of active ingredients. In general, the dissolution of intracellular secondary metabolites such as alkaloids and polysaccharides needs to penetrate through cell walls (membranes), and a circulating freeze thawing method (FTC) can repeatedly freeze and thaw samples, so that tiny ice crystals are formed in plant cells in the process, and cell wall tissues are damaged, so that the extraction rate of the secondary metabolites such as alkaloids and polysaccharides is improved. At the same time, FTC can alter membrane permeability of the organelle, allowing rapid delivery of cytoplasmic active ingredients into the extraction solvent. The invention integrates circulating freeze thawing and ultrasonic assisted double water phase compound enzyme, establishes an FTC-UA-ATPE-EAE integrated co-extraction method for simultaneously extracting alkaloid and polysaccharide from the fritillary, and has the specific operation that firstly, FTC circulating freeze thawing flat fritillary powder is used for destroying cell walls (membranes) of the fritillary, uA-ATPE-EAE is used for simultaneously co-extracting the fritillary alkaloid and the polysaccharide, and the ethanol-ammonium sulfate double water phase compound enzyme system adopted by the invention is nontoxic and harmless and environment-friendly, realizes one-step feeding and simultaneously obtains the alkaloid and the polysaccharide, greatly improves the extraction efficiency of target substances by circulating freeze thawing, has high purity, smaller particle size and higher solubility, and saves the cost for the next production. The method combines the advantages of FTC pretreatment and UA-ATPE-EAE technology, has mild conditions and simple operation, does not cause the inactivation or denaturation of the fritillary alkaloids and the polysaccharide, and is easy for industrial production and application.
At present, no report of applying FTC pretreatment and UA-ATPE-EAE technology to extraction of fritillary alkaloids and polysaccharides is seen. Chinese patent CN104311695a combines FTC treatment with warm water extraction to extract β -glucan from oat, although no enzyme preparation and organic solvent are required, single-phase solvent reduces selectivity of extraction of target substance, warm water extraction is time-consuming, extraction efficiency is low, and subsequent purification steps are complicated. Chinese patent CN110652008A uses UA-ATPE method to extract litchi polyphenol, adopts low-temperature vacuum drying and forced air drying technology to pretreat raw materials, and the pretreatments are relatively energy-consuming and time-consuming, and the invention only extracts and separates one kind of active substances by using UA-ATPE technology, and does not optimize the extraction condition, so that the raw material utilization rate is low. According to the invention, the advantages of the FTC method and the UA-ATPE-EAE are combined, the intracellular secondary metabolite is more easily dissolved out by circulating freeze thawing, the ultrasonic cavitation oscillation effect accelerates the transmission and distribution of two target substances between two phases, and the yields of alkaloid and polysaccharide are obviously improved. Therefore, the invention establishes a high-efficiency, green and energy-saving one-step integrated method for jointly extracting the two active ingredients of the fritillary bulb, and has wide application prospect.
Disclosure of Invention
The invention aims to provide a method for efficiently and integrally extracting the fritillary alkaloid and the polysaccharide in one step to realize comprehensive utilization of the fritillary biological resources.
The invention is realized in the following way:
a method for integrating and co-extracting alkaloid and polysaccharide of Pinctada maxima, which takes Pinctada maxima powder as raw materials, comprises the following steps: (1) washing, drying and crushing the fritillary bulb, mixing the fritillary bulb with deionized water in a ratio of 1:3 to form paste, circularly freezing and thawing for 3 times at low temperature, drying, crushing again, adding the frozen and thawed fritillary bulb powder into a prepared ethanol-ammonium sulfate double-aqueous phase complex enzyme system according to a certain feed-liquid ratio, and extracting under the optimal ultrasonic auxiliary condition; (2) filtering the extracting solution obtained in the step (1) under reduced pressure, centrifuging, standing for phase separation, concentrating the upper and lower phase solutions respectively, dialyzing, and freeze-drying to obtain upper and lower phase crude extracts; (3) respectively dissolving the upper and lower phase crude extracts obtained in the step (2), loading the obtained solution to a D4006 macroporous adsorbent resin column, eluting the resin by using deionized water until polysaccharide is not detected, respectively concentrating the eluent, and freeze-drying to obtain upper and lower phase polysaccharide; (4) eluting the alkaloid adsorbed in the D4006 macroporous resin column in the step (3) by using an ethanol aqueous solution, collecting eluent, concentrating, and freeze-drying to obtain the upper-phase alkaloid.
Compared with the prior art, the invention has the following technical improvements:
1. the invention uses the circulating freeze thawing technology to pretreat the Pinctada martensii powder, thereby effectively improving the yield of target substances; the method combines the advantages of ultrasonic-assisted aqueous two-phase compound enzyme extraction technology, realizes one-step co-extraction and separation to obtain the fritillary alkaloids and the polysaccharide, and has simple operation, high extraction efficiency and easy industrial process amplification.
2. According to the invention, the response surface model is adopted to optimize the UA-ATPE-EAE extraction condition, the obtained optimal process condition can effectively accelerate the dissolution of polysaccharide and alkaloid from the fritillary cells, and the yield is higher.
3. The ethanol-ammonium sulfate double-aqueous phase compound enzyme system adopted by the invention has the advantages that the solvent is nontoxic and low in cost, the requirements of environmental protection and industrial production are well met, three active components can be obtained by one-step feeding, the resource utilization rate is high, and the Yu Pingbei alkaloid and polysaccharide can be used for developing medicines, health products and foods.
4. Compared with the traditional extraction process, the integrated co-extraction technology has higher product purity, smaller average particle size and higher solubility, and is more beneficial to drug release and biological absorption.
Drawings
Fig. 1: morphology contrast under 200 times microscope before and after the cyclic freeze thawing of the Pink Bettner powder: (a) flat powder particles not subjected to freeze thawing treatment; (B) freeze thawing the particles of the flat shellfish powder 1 time; (C) circulating freeze thawing for 2 times to obtain Pinctada powder particles; (D) cyclic freeze thawing of the particles of seashell powder 3 times; (E) circulating freeze thawing for 4 times of the particles of the Pink shell powder; (F) circulating freeze thawing for 5 times to obtain the Pinctada powder particles.
Fig. 2: and (5) obtaining the polysaccharide and alkaloid of the fritillary bulb after the cyclic freeze thawing treatment for different times.
Detailed Description
The present invention will be described in detail with reference to examples below to facilitate further understanding of the present invention, but the scope of the present invention is not limited thereto.
Determination of optimal co-conditions for the alkaloid and polysaccharide of fritillary bulb:
single factor test:
selecting a feed liquid ratio:
the method comprises the steps of weighing fritillary powder, adding the fritillary powder into a double-aqueous phase complex enzyme system consisting of 48.31% -54.29% of ethanol, 18.37% -21.42% of ammonium sulfate and 0.5% -5% (W/v) of hydrogel immobilized complex enzyme (pectase: neutral protease: cellulase=1-3:1-3) according to a certain feed-liquid ratio, extracting for 30min at the condition of ultrasonic power of 480W and temperature of 40 ℃, separating extract liquid, calculating the yields of upper phase polysaccharide, lower phase polysaccharide and upper phase alkaloid, and determining that the feed-liquid ratio is in the range of 1:50-1:90 (g/mL).
Selection of extraction time:
the method comprises the steps of weighing fritillary powder, adding the fritillary powder into a double-aqueous phase complex enzyme system consisting of 48.31% -54.29% of ethanol, 18.37% -21.42% of ammonium sulfate and 0.5% -5% (W/v) of hydrogel immobilized complex enzyme (pectase: neutral protease: cellulase=1-3:1-3) according to a liquid-to-material ratio of 1:50-1:90 (g/mL), extracting at an ultrasonic power of 480W at an extraction temperature of 40 ℃, calculating the yields of upper phase polysaccharide, lower phase polysaccharide and upper phase alkaloid after separating the extract, and determining the extraction time within a range of 20-90 min.
Selection of extraction temperature:
the method comprises the steps of weighing fritillary powder, adding the fritillary powder into a double-aqueous phase complex enzyme system consisting of 48.31% -54.29% of ethanol, 18.37% -21.42% of ammonium sulfate and 0.5% -5% (W/v) of hydrogel immobilized complex enzyme (pectase: neutral protease: cellulase=1-3:1-3) according to a liquid-to-material ratio of 1:50-1:90 (g/mL), extracting for 20-90 min under ultrasonic power of 480W, separating extract, calculating upper and lower phase polysaccharide and upper phase alkaloid yields, and determining the extraction temperature within a range of 20-70 ℃.
Selection of ultrasonic power:
the method comprises the steps of weighing fritillary powder, adding the fritillary powder into a double-aqueous phase complex enzyme system consisting of 48.31% -54.29% of ethanol, 18.37% -21.42% of ammonium sulfate and 0.5% -5% (W/v) of hydrogel immobilized complex enzyme (pectase: neutral protease: cellulase=1-3:1-3) according to a liquid-to-material ratio of 1:50-1:90 (g/mL), extracting for 20-90 min at a temperature of 20-70 ℃, separating extract, calculating the yields of upper phase polysaccharide, lower phase polysaccharide and upper phase alkaloid, and determining the ultrasonic power to be in a range of 360-840W.
Response surface optimization experiment:
based on the single-factor experimental result, a response surface optimization method is adopted to carry out four-factor three-level optimization experiment, and the yield of the polysaccharide in the above phase is Y 1 Yield of polysaccharide in lower phase Y 2 Yield of upper phase alkaloid Y 3 To extract time X as response value 1 (min) extraction temperature X 2 (DEGC), ultrasonic power X 3 (W) and feed liquid ratio X 4 And (g/mL) performing ultrasonic-assisted double-aqueous-phase compound enzyme method extraction of the response surface process optimization of the fritillary alkaloids and the polysaccharide.
TABLE 1 model 1 (upper phase polysaccharide yield, Y 1 ) Analysis of variance results
*p<0.05,**p<0.01,***p<0.001
As shown in Table 1, Y 1 The model analysis of variance shows that the experimental result is obvious (p is less than 0.01) and the response surface model (F= 37.81) is fitted, the mismatching term is not obvious, and the variation coefficient C.V. is 1.81%, so that the model can accurately predict the experimental result. R is R 2 0.9614, the model has 96.14% compliance with experimental data, and the equation model has high reliability. The influence order of the F value known factors on the yield of the upper phase polysaccharide is as follows: the feed liquid ratio is greater than the ultrasonic power, the extraction temperature is greater than the extraction time.
TABLE 2 model 2 (lower polysaccharide yield, Y 2 ) Analysis of variance results
*p<0.05,**p<0.01,***p<0.001
As shown in Table 2, Y 2 The model analysis of variance shows that the experimental result is obvious (p is less than 0.01) and the response surface model (F=43.18) is fitted, the mismatching term is not obvious, and the variation coefficient C.V. is 1.99%, so that the model can accurately predict the experimental result. R is R 2 0.9774, this model showed 97.74% compliance with experimental data and the equation model had high confidence. The influence of the factors of the F value on the yield of the lower phase polysaccharide is as follows: extraction temperature > feed-liquid ratio > extraction time > ultrasonic power.
TABLE 3 model 3 (upper phase alkaloid yield, Y 3 ) Analysis of variance results
*p<0.05,**p<0.01,***p<0.001
As shown in Table 3, Y 3 The model analysis of variance shows that the experimental result is obvious (p is less than 0.01) and the response surface model (F=39.89) is fitted, the mismatching term is not obvious, and the variation coefficient C.V. is 3.01%, so that the model can accurately predict the experimental result. R is R 2 0.9655 shows that the model has 96.55% compliance with experimental data and the equation model has high reliability. The influence of the factors of the F value on the yield of the upper phase alkaloid is as follows: the extraction time is more than the feed liquid ratio is more than the ultrasonic power is more than the extraction temperature.
Example 1:
the method for determining the freeze thawing cycle times of the raw materials comprises the following steps:
adding deionized water into the Pink shell powder 1:3 (g/mL) according to the optimal extraction condition obtained by optimizing the response surface, stirring to paste, freezing at-10 ℃ for 8, thawing at 20 ℃ for 4 hours, taking the mixture as a complete cycle, circulating freeze thawing, drying and crushing, extracting under the optimal UA-ATPE-EAE condition, separating the extract, calculating the total yield of polysaccharides in the upper phase and the lower phase, the yield of alkaloids in the upper phase, and determining the circulation times.
As can be seen from fig. 1A, the sample without freeze thawing treatment has darker color, poor light transmittance, and clear surrounding solution, indicating that no cytoplasm is dissolved in the aqueous phase; as can be seen from fig. 1B to F, after 1 to 5 times of cyclic freeze thawing, the light transmittance of the fritillary powder particles is gradually increased due to the damage of the ice crystals to the fritillary powder, and the color of the surrounding solution is gradually deepened, which indicates that the cell content has flowed out, thereby improving the yields of the fritillary alkaloids and the polysaccharides in the subsequent extraction experiments. As shown in fig. 2, the yields of the polysaccharides and alkaloids in the upper and lower phases are in an increasing trend with the increase of the number of times of circulation, and the increasing trend of the yields is slowed down after 3 times of circulation, so that the Pinctada martensii powder is extracted by 3 times of circulation.
Example 2:
a method for integrating and extracting alkaloid and polysaccharide of Pinctada comprises the following steps:
(1) Adding deionized water into Pinctada powder at a ratio of 1:3, stirring to paste, freezing at-20deg.C for 12 hr, thawing at 20deg.C for 4 hr, oven drying, and pulverizing.
(2) Adding the circularly frozen and thawed Pinctada powder into a double-aqueous-phase compound enzyme system consisting of 53.24% ethanol, 19.33% ammonium sulfate and 1% (W/v) hydrogel immobilized compound enzyme (pectase: neutral protease: cellulase=1:1:1) according to a liquid-to-material ratio of 1:50, and treating for 30min at an extraction temperature of 40 ℃ and an extraction power of 360W. The upper phase and the lower phase of the extract are collected by centrifugal separation, and the volumes are measured respectively.
(3) And (3) taking the upper phase extract, performing rotary evaporation concentration, treating with a 200Da dialysis bag at room temperature for 72 hours, concentrating the dialysate, and performing freeze drying to obtain an upper phase crude extract.
(4) Taking out the lower phase extract, performing rotary evaporation concentration, treating with a 2000Da dialysis bag at room temperature for 48 hours, removing inorganic salt and other small molecular impurities, concentrating the dialysate, and freeze-drying to obtain the lower phase crude extract.
(5) Dissolving 20.0mg of the crude extract of the upper phase in 100.0mL of deionized water, loading the crude extract on a D4006 macroporous resin column, adsorbing alkaloid in the column by macroporous resin, eluting unadsorbed polysaccharide by using deionized water at the eluting flow rate of 1.5mL/min, collecting eluent, concentrating, freeze-drying to obtain the polysaccharide of the upper phase, eluting alkaloid by using 95% ethanol solution as eluent after the polysaccharide is not detected at the flow rate of 1.5mL/min, collecting eluent, concentrating, and freeze-drying to obtain the alkaloid of the upper phase.
(6) Taking 25.0mg of the crude extract of the lower phase, dissolving in 100.0mL of deionized water, loading on a D4006 macroporous resin column, eluting the non-adsorbed polysaccharide with deionized water at the elution flow rate of 1.5mL/min, collecting the eluent, concentrating, and freeze-drying to obtain the polysaccharide of the lower phase.
The determination shows that the yield of the alkaloid in the upper phase is 2.10+/-0.26 mg/g, the yield of the polysaccharide in the upper phase is 3.98+/-0.13%, and the yield of the polysaccharide in the lower phase is 11.72+/-0.21%.
The method comprises the following steps of:
the polysaccharide is measured by adopting a phenol-sulfuric acid method, 1.0mL of glucose standard solution with the concentration of 0.02-0.10 mg/mL is respectively added into a test tube, 1.0mL of deionized water and 1.0mL of phenol solution (5% w/v) are respectively added, the mixture is fully oscillated, and then 5.0mL of concentrated sulfuric acid is added for uniform mixing. And (3) after the reaction system is cooled to normal temperature, measuring the absorbance of the sample solution at 490nm, and replacing the glucose solution with deionized water by using a blank control. And drawing a standard curve by taking the concentration of the glucose solution and the corresponding absorbance as the abscissa and the ordinate respectively to obtain a linear regression equation. And (3) calculating the polysaccharide concentration of the liquid to be detected according to a regression equation, and calculating the polysaccharide yield according to a formula (1).
Wherein: c is the concentration of polysaccharide in the extract, mg/mL;
v is the volume of the solution to be measured, mL;
d is dilution multiple;
m is the mass of the fritillary bulb powder and g.
The alkaloid yield is determined by using a bromothymol blue compounding method, firstly, a standard alkaloid curve is drawn by taking peimine as a standard substance, 0.10mg/mL of a chloroform solution of the peimine is precisely prepared, 2.0-10.0 mL of the chloroform solution is respectively transferred to a 10.0mL volumetric flask, chloroform is used for volume metering to scale, the chloroform is transferred to a beaker, 2.0mL of buffer solution (prepared by 0.2mol/L potassium hydrogen phthalate and 0.2mol/L sodium hydroxide solution in a volume ratio of 100:35, pH=5) and 2.0mL of bromothymol blue alkaline solution (prepared by adding 0.312g of bromothymol blue into 5.0mL of 1mol/L sodium hydroxide solution) are fully mixed and then are stationary overnight, the upper and lower phase solutions are transparent and clarified, the chloroform phase is collected, the absorbance is measured at a wavelength of 410nm by using an ultraviolet spectrophotometer, the pure chloroform is a blank control, the absorbance is plotted as an ordinate, and the standard curve of the alkaloid is drawn by taking the concentration of the peimine as an ordinate, and linear regression is obtained. And (3) calculating the alkaloid concentration of the liquid to be detected according to a regression equation, and calculating the alkaloid yield according to a formula (2).
Wherein: c is the concentration of alkaloid in the extract, mg/mL;
v is the volume of the solution to be measured, mL;
d is dilution multiple;
m is the mass of the raw material, g.
Comparative example 1:
comparison of yields of alkaloid and polysaccharide extracted by different extraction methods
The samples were subjected to a cyclic freeze-thawing pretreatment according to the method of example 1, and then an ethanol-ammonium sulfate aqueous two-phase system determined according to the present invention was used for the integrated co-extraction of fritillary alkaloids and polysaccharides by respectively using an ultrasonic assisted aqueous two-phase extraction method (UA-ATPE), a microwave assisted aqueous two-phase extraction method (MA-ATPE) and a low temperature plasma assisted aqueous two-phase extraction method (CP-aTPE), and compared with the UA-ATPE-EAE of the present invention. The specific operating conditions and the yields of alkaloids and polysaccharides are shown in Table 4.
TABLE 4 results of alkaloid and polysaccharide yields from different extraction methods
As can be seen from Table 4, the same two-aqueous phase system is used as the extraction solvent, and different auxiliary methods have a certain influence on the yields of the three active ingredients, and the extraction rates of the fritillary bulb and the alkaloid extracted by the microwave/low-temperature plasma auxiliary two-aqueous phase method are smaller than those of the ultrasonic auxiliary method, so that the ultrasonic auxiliary method is proved to be more effective. The UA-ATPE-EAE adopted by the invention combines the advantages of ultrasonic wave and compound enzyme, so that the yields of alkaloid and polysaccharide are further improved. Therefore, uA-ATPE-EAE is a process method for efficiently and integrally extracting the alkaloid and the polysaccharide of the fritillary bulb.
Comparative example 2:
comparison of polysaccharide chemical component extracted by different extraction methods
The polysaccharide chemical composition was measured after purifying the polysaccharide obtained from UA-ATPE, mA-ATPE, CP-ATPE and UA-ATPE-EAE used in the present invention by the same method, and the results are shown in Table 5.
TABLE 5 comparison of chemical Components contained in polysaccharides obtained by different extraction methods
Note that: "-1" indicates an upper phase polysaccharide, "-2" indicates a lower phase polysaccharide, and "nd" indicates undetected.
As can be seen from Table 5, after the polysaccharide obtained by adopting the same aqueous two-phase extraction solvent and different auxiliary methods is purified by D4006 macroporous adsorbent resin, the chemical composition difference is obvious, the total sugar content of the upper phase and the lower phase obtained by adopting the UA-ATPE-EAE in the invention is higher than that of the polysaccharide obtained by adopting other methods, and the reducing sugar content is lower than that of the polysaccharide obtained by adopting other methods, so that the purity of the polysaccharide extracted by the UA-ATPE-EAE is higher; and the contents of protein, polyphenol and the like are lower than those of other methods, which shows that the impurity of the polysaccharide extracted from the UA-ATPE-EAE is less. Thus, the UA-ATPE-EAE process can obtain polysaccharides of relatively high purity, reducing subsequent purification steps.
Comparative example 3:
the partial physicochemical property comparison of polysaccharide is obtained by different extraction methods
The polysaccharide average particle size, polymer Dispersion Index (PDI), zeta potential and solubility were determined after purifying the polysaccharide obtained from UA-ATPE, mA-ATPE, CP-ATPE and UA-ATPE-EAE used in the present invention by the same method, and the results are shown in Table 6.
TABLE 6 comparison of partial physicochemical Properties of polysaccharide obtained by extraction by different methods
As can be seen from table 6, the average particle size of the polysaccharide extracted from UA-Ape-EAE is smaller, the particle size distribution is more uniform, and the aqueous system is more stable than the other methods; the absolute value of Zeta potential of the polysaccharide is larger than that of other 6 polysaccharides, which shows that the repulsive force of the particles in the aqueous solution is larger, and the system tends to be in a stable state of dispersion but not aggregation; the polysaccharide extracted by the UA-ATPE-EAE method has higher solubility, so that the polysaccharide is easier to be absorbed and utilized in the body; therefore, the polysaccharide extracted by the UA-ATPE-EAE method has better physicochemical property, and is beneficial to the application in the fields of food, health care products and the like.
Claims (4)
1. A method for integrating and co-extracting alkaloid and polysaccharide of fritillary bulb is characterized in that:
(1) crushing Ping Bei Honggan, mixing with deionized water to form paste, circularly freezing and thawing at low temperature, drying, crushing again, adding the dry powder after freezing and thawing treatment into an ethanol-ammonium sulfate aqueous two-phase complex enzyme system according to a certain feed liquid ratio, and extracting under the optimal ultrasonic condition; (2) filtering the extracting solution obtained in the step (1) under reduced pressure, centrifuging, standing for phase separation, concentrating the upper and lower phase solutions respectively, dialyzing, and freeze-drying to obtain upper and lower phase crude extracts; (3) respectively dissolving the upper and lower phase crude extracts obtained in the step (2), loading the obtained solution to a D4006 macroporous adsorbent resin column, eluting the resin by using deionized water until polysaccharide is not detected, respectively concentrating the eluent, and freeze-drying to obtain upper and lower phase polysaccharide; (4) eluting the alkaloid adsorbed in the D4006 macroporous resin column in the step (3) by using an ethanol aqueous solution, collecting eluent, concentrating, and freeze-drying to obtain the upper-phase alkaloid.
2. The method for integrating and extracting alkaloids and polysaccharides from fritillary bulb according to claim 1, wherein the freezing and thawing treatment conditions in the step (1) are as follows: freezing temperature is-10 to-35 ℃, freezing time is 8-16 h, thawing temperature is 20-30 ℃, and thawing time is 4-8 h.
3. The method for integrated co-extraction of the fritillary alkaloids and polysaccharides according to claim 1, wherein the ethanol-ammonium sulfate aqueous two-phase system in the step (1) comprises 0.5% -5% (w/v) hydrogel immobilized complex enzyme (pectase: neutral protease: cellulase=1-3:1-3); the ethanol-ammonium sulfate aqueous two-phase system comprises the following components: 48.31 to 54.29 percent of ethanol and 18.37 to 21.42 percent of ammonium sulfate; ping Beigan powder and ethanol-ammonium sulfate double water phase system in the feed liquid ratio of 1:50-1:90 (g/mL); the ultrasonic auxiliary extraction conditions are as follows: the extraction temperature is 20-70 ℃, the ultrasonic power is 360-840W, and the extraction time is 20-90 min.
4. The method for integrated co-extraction of the alkaloid and the polysaccharide of the fritillary bulb according to claim 1, wherein the molecular weight cut-off of the upper phase dialysis bag in the step (2) is 200-1000 Da, and the molecular weight cut-off of the lower phase dialysis bag is 1000-10000 Da.
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