CN113304702B - Preparation method of fructus amomi essential oil microcapsule - Google Patents
Preparation method of fructus amomi essential oil microcapsule Download PDFInfo
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Abstract
The invention provides a preparation method of fructus amomi essential oil microcapsule. The method mainly comprises the following steps: fructus amomi essential oil is used as a core material, one or more of an inulin-lysozyme aqueous solution, an inulin-chitosan aqueous solution, a pectin-lysozyme aqueous solution and a pectin-chitosan aqueous solution are used as wall materials, the mass ratio of the fructus amomi essential oil to the nanoparticle aqueous solution is 2:1-1:2, the pH value of the system is adjusted to 3-10, a Pickering emulsion product is obtained under high-speed stirring at 800-1600 r/min, ultrasonic dispersion is carried out for 30-60 min under the power of 200W, and then the fructus amomi essential oil microcapsule is obtained through spray drying. The fructus amomi essential oil microcapsule prepared by the method can protect the activity of fructus amomi essential oil, realize slow release of the fructus amomi essential oil, have biological activity of inducing apoptosis of human hepatoma cells HepG2, and can realize wider application of the fructus amomi essential oil in the fields of foods, health products and medicines.
Description
Technical Field
The invention belongs to the technical field of microcapsule preparation, and relates to a preparation method of fructus amomi essential oil microcapsules, belonging to the technical field of food processing.
Background
Fructus AmomiAmomum villosumLour.) the perennial herb of the genus cardamom of the family zingiberaceae is a raw material of homology of medicine and food, widely used in the areas of chinese fowls, guangdong and yunnan, etc. Has the effects of resolving dampness and stimulating appetite, warming spleen and relieving diarrhea, regulating qi and preventing miscarriage, is mainly used for clinically treating damp turbidity and middle energizer obstruction, epigastric fullness and allaying hunger, spleen and stomach deficiency-cold, vomiting and diarrhea, vomiting of pregnancy and lochia,the treatment of fetal irritability and other diseases has better effect. In recent years, researches show that fructus amomi has abundant pharmacological activity, and particularly has remarkable advantages in the aspects of gastrointestinal protection, pain relief, diarrhea relieving, bacteriostasis, antioxidation and the like, but less related products are developed.
Fructus Amomi essential oil is volatile oil component extracted from fructus Amomi fruit, and is the main bioactive component of fructus Amomi. Fructus Amomi essential oil has various pharmacological effects, and is mainly used for treating gastrointestinal spasm, ulcer, relieving pain, promoting digestion, eliminating gastrointestinal qi stagnation, promoting gastric secretion, enhancing gastrointestinal motility, relieving diarrhea, promoting bile flow, relieving inflammation, relieving cough, inhibiting bacteria, preventing corrosion, resisting oxidation, etc. Many studies have shown that gastrointestinal health is closely related to the health of the whole body. At present, related researches on fructus amomi essential oil mainly concentrate on aspects of extraction, component measurement, pharmacological activity and the like, and activities are also concentrated on reports of gastrointestinal conditioning, and preparation of fructus amomi essential oil microcapsules and effects of the fructus amomi essential oil microcapsules on the effects are not reported.
Microcapsule technology refers to technology in which individual droplets or particles of solid, gaseous or liquid material are embedded in capsules in the micrometer to millimeter range by a specific process technology, and the core material can be controllably released under specific conditions. The microcapsule technology is adopted to seal the grease in the wall material, and the grease release is controlled to weaken the influence of oxygen, illumination and the like in the processing and storage environment on the quality of the grease, so that the oxidation stability of the grease is enhanced and the application range of the grease is widened. At present, the plant essential oil microcapsule technology is widely applied in the fields of food, chemical industry, medicine and the like. The Pickering emulsion is widely accepted as a universal template for forming a heterogeneous structure of the microcapsule, so that not only is environmental pollution avoided, but also the Pickering emulsion can participate in forming a shell, and the mechanical strength, the thermal stability, the permeation resistance, the magnetic response and the like of a polymer shell can be improved while the uniform and complete spherical morphology of the microcapsule is endowed, so that the Pickering emulsion becomes the future development of the field of the microcapsule.
The fructus amomi essential oil is embedded by using a Pickering emulsion microcapsule technology to prepare the fructus amomi essential oil microcapsule, so that the biological activity of the fructus amomi essential oil can be effectively protected, the fructus amomi essential oil can be slowly released, the biological activity of inducing apoptosis of human liver cancer cells HepG2 can be simultaneously realized, and the fructus amomi essential oil can be widely applied to the fields of foods, health products and even medicines.
Disclosure of Invention
The invention aims to provide the fructus amomi essential oil microcapsule, which can effectively protect the biological activity of the fructus amomi essential oil, slowly release the fructus amomi essential oil, simultaneously has the biological activity of inducing the apoptosis of human hepatoma cell HepG2, can realize the wider application of the fructus amomi essential oil in the fields of food, health care products and medicines, and ensures that the product develops towards nutrition, health care and safety.
In order to achieve the purpose, the invention adopts the technical scheme that.
(1) Respectively dissolving inulin, pectin, chitosan and lysozyme with deionized water to obtain 1mg/ml water solution.
(2) Respectively preparing an aqueous solution of synanthrin-lysozyme nano particles, an aqueous solution of synanthrin-chitosan nano particles, an aqueous solution of pectin-lysozyme nano particles and an aqueous solution of pectin-chitosan nano particles according to the mass ratio of the two components of 1:4-4:1, and performing ultrasonic dispersion for 30-60 min under the power of 200W.
(3) And (3) respectively using 0.5mol/L hydrochloric acid and sodium hydroxide aqueous solution to adjust the pH of the mixed solution in the step (2) to 3-10.
(4) Adding fructus amomi essential oil into the aqueous solution of the nano particles obtained in the step (3), wherein the mass ratio of the fructus amomi essential oil to the aqueous solution of the nano particles is 2:1-1:2, and dispersing the obtained mixed solution at a high speed of 800-1600 r/min for 5-30 min.
(5) Spray drying the mixed solution obtained in the step (4), setting the inlet air temperature to 180 ℃, the outlet air temperature to 85 ℃ and the feeding rate to 5mL/min, and obtaining the fructus amomi essential oil microcapsule.
The preparation method of the fructus amomi essential oil microcapsule is based on Pickering emulsion microcapsule technology, wherein the wall materials used are inulin, chitosan, pectin and lysozyme, and the fructus amomi essential oil microcapsule belongs to natural polymer active ingredients and has the effects of providing embedding with high embedding rate and good slow release property for the fructus amomi essential oil.
According to the preparation method of the fructus amomi essential oil microcapsule, the embedding rate of the fructus amomi essential oil nano microcapsule prepared by the preparation method is more than 85%, the average particle size is between 180 and 300 and nm, the fructus amomi essential oil microcapsule has good protection and slow release properties, and the biological effect of inducing apoptosis of human hepatoma cells HepG2 is achieved.
The beneficial effects of the invention are as follows:
(1) The fructus amomi essential oil microcapsule has the slow-release characteristic, can slowly release fructus amomi essential oil, improves the utilization efficiency of the fructus amomi essential oil, prolongs the acting time of the fructus amomi essential oil and the like.
(2) The fructus amomi essential oil microcapsule prepared by the invention takes the natural high molecular active ingredients of inulin, chitosan, pectin and lysozyme as wall materials, and the fructus amomi essential oil as core material, so that the microcapsule is nontoxic, harmless, safe, reliable, simple to prepare, obvious in antioxidant activity, convenient to use and wide in application.
(3) The fructus amomi essential oil microcapsule prepared by the invention has the biological effect of inducing apoptosis of human hepatoma cell HepG2, and can realize wider application of fructus amomi essential oil in the fields of food, health care products and even medicines.
Drawings
The particle size distribution diagrams of the amomum fruit essential oil microcapsules obtained in examples 1 to 4 of FIG. 1 ((1): example 1, (2): example 2, (3): example 3 and (4): example 4).
FIG. 2 optical photomicrographs of the amomum villosum essential oil microcapsules obtained in examples 1-4 (A: example 1; B: example 2; C: example 3; D: example 4).
FIG. 3 effects of different concentrations of the amomum essential oil microcapsule solution obtained in example 1 on proliferation of HepG2 tumor cells.
FIG. 4 shows the apoptosis of HepG2 tumor cells (. Times.10) (a: 24h-control; b:24 h-60. Mu. Mol/L; c:24 h-120. Mu. Mol/L; d:24 h-180. Mu. Mol/L; e:48h-control; f:48 h-60. Mu. Mol/L; g:48 h-120. Mu. Mol/L; h:48 h-180. Mu. Mol/L; i:72h-control; j:72 h-60. Mu. Mol/L; k:72 h-120. Mu. Mol/L; L:72 h-180. Mu. Mol/L) after the treatment of the microcapsules of the fructus amomi essential oils of different concentrations obtained in example 1 by inverted phase contrast microscopy.
FIG. 5 fluorescence inversion microscopy shows the apoptosis of HepG2 tumor cells (20X) after 72h treatment of the amomum essential oil microcapsules of different concentrations obtained in example 1 (a: control group, inversion; b: control group, fluorescence; c: 30. Mu. Mol/L, inversion; d: 30. Mu. Mol/L, fluorescence; e: 60. Mu. Mol/L, inversion; f: 60. Mu. Mol/L, fluorescence; g: 90. Mu. Mol/L, inversion; h: 90. Mu. Mol/L, fluorescence; i: 120. Mu. Mol/L, inversion; j: 120. Mu. Mol/L, fluorescence; k: 150. Mu. Mol/L, inversion; L: 150. Mu. Mol/L, fluorescence).
The specific embodiment is as follows:
in order that those skilled in the art will better understand the technical solution of the present invention, the present invention will be further described with reference to specific examples, but the examples are not intended to limit the present invention.
The preparation method of the fructus amomi essential oil microcapsule provided by the embodiment of the invention comprises the following steps.
(1) Respectively dissolving inulin, pectin, chitosan and lysozyme with deionized water to obtain 1mg/ml water solution.
(2) Respectively preparing an aqueous solution of synanthrin-lysozyme nano particles, an aqueous solution of synanthrin-chitosan nano particles, an aqueous solution of pectin-lysozyme nano particles and an aqueous solution of pectin-chitosan nano particles according to the mass ratio of the two components of 1:4-4:1, and performing ultrasonic dispersion for 30-60 min under the power of 200W.
(3) And (3) respectively using 0.5mol/L hydrochloric acid and sodium hydroxide aqueous solution to adjust the pH of the mixed solution in the step (2) to 3-10.
(4) Adding fructus amomi essential oil into the aqueous solution of the nano particles obtained in the step (3), wherein the mass ratio of the fructus amomi essential oil to the aqueous solution of the nano particles is 2:1-1:2, and dispersing the obtained mixed solution at a high speed of 800-1600 r/min for 5-30 min.
(5) Spray drying the mixed solution obtained in the step (4), setting the inlet air temperature to 180 ℃, the outlet air temperature to 85 ℃ and the feeding rate to 5mL/min, and obtaining the fructus amomi essential oil microcapsule.
The characterization method comprises the following steps:
(1) Number average particle diameter and morphology observation of microcapsules: the test was performed using a Malvern ZETASIZER 3000 HAS nano laser particle sizer at 25 ℃. The morphology of the microcapsules was observed with a phase contrast microscope of Leka DM 500.
(2) Embedding rate: the embedding rate was calculated according to the following formula:
(3) MTT method (tetramethyl azo salt trace enzyme reaction colorimetric method) for detecting relative activity of cells
The tumor cells are dosed by direct dosing. After culturing 24, 48, 72, h cells, 10 μl of MTT solution was added per well; 37. after continuing to incubate at 4℃ 4h, the cells were removed from the incubator and the cell culture supernatant in the wells was gently removed; dimethyl sulfoxide (dimethyl sulfoxide, DMSO) solution, 150 μl/well, was added to the cell culture flasks, respectively; oscillating the 96-well plate on a decoloring shaking table for 10 min to enable the crystals to be fully dissolved; absorbance (a) at 490 nm wavelength was measured for each well with a microplate reader and cell viability was calculated according to the following formula.
Wherein: a is that 0 Absorbance for the blank control wells; a is that 1 Absorbance for non-dosing wells; a is that 2 Absorbance was measured for the dosing wells.
(4) Observing cell morphology by inverted phase contrast microscope
The density of sensitive cells was adjusted to 1X 10 5 Each mL, inoculated into 6-well plates, 3 mL/well; to each well of the 6-well plate, 0, 22.5, 45.0, 67.5, 90.0, 112.5. Mu.L of 4,000. Mu. Mol/L complex n-3 PUFAs was added to give final concentrations of 0, 30, 60, 90, 120, 150. Mu. Mol/L, respectively. Meanwhile, a culture solution without fructus amomi essential oil microcapsules is used as a control group, and after 24, 48 and 72h are cultured, the cell morphology is observed by an inverted phase contrast microscope.
(5) Observation of sensitive cell apoptosis by fluorescent inverted microscope
The operation was performed according to the Annexin V-FITC apoptosis assay kit method. About 1X 10 5 ～5×10 5 The resuspended sensitive cells were centrifuged at 1,000Xg for 5 min; removing the supernatant, adding 500 mu L Annexin V-FITC binding solution, and lightly suspending the sensitive cells again; adding 5 mu L of Annexin V-FITC solution, uniformly mixing, adding 5 mu L of Propidium Iodide (PI) solution into the reaction system, and gently mixing; at room temperature (20-25 ℃), adopting aluminum foil to incubate for 10 min in dark place; and (3) dripping the stained cell suspension on a glass slide, and lightly covering the cells with a cover slip, so that the detection can be performed by using an inverted fluorescence microscope.
Example 1
(1) Pectin and lysozyme are respectively dissolved by deionized water to prepare 1mg/ml aqueous solution.
(2) And (3) configuring the pectin-lysozyme nanoparticle aqueous solution in the step (1) according to the mass ratio of the two components of 4:1, and performing ultrasonic dispersion for 60min under the power of 200W.
(3) And (3) respectively adjusting the pH value of the mixed solution in the step (2) to 10 by using 0.5mol/L sodium hydroxide aqueous solution.
(4) Adding fructus amomi essential oil into the aqueous solution of the nano particles obtained in the step (3), wherein the mass ratio of the fructus amomi essential oil to the aqueous solution of the nano particles is 1:2, and dispersing the obtained mixed solution at a high speed for 5min at 1600 r/min.
(5) Spray drying the mixed solution obtained in the step (4), setting the inlet air temperature to 180 ℃, the outlet air temperature to 85 ℃ and the feeding rate to 5mL/min, and obtaining the fructus amomi essential oil microcapsule.
The particle size distribution of the fructus amomi essential oil microcapsule is shown in a graph 1 ((1)), the average particle size is 289.45nm, and the embedding rate is 87.6%.
The results of observing the cell morphology under a microscope are shown in FIG. 4. As can be seen from fig. 4, the dose and the duration of action of the different amomum essential oil microcapsules have a significant effect on the number and morphology of HepG2 cells. The control group HepG2 cells have clear outlines, are fusiform, have compact structures among cells and are vigorous in growth; the shape of the HepG2 cells of the treatment group is changed, and the HepG2 cells are in a random state, so that the outline sense of the cells is reduced, the cells float, the distance between the cells is increased, the structure is loose, the number of the cells is reduced, and dead cells are increased.
The result of observing sensitive cells apoptosis by a fluorescence inversion microscope is shown in fig. 5. As shown in fig. 5, the apoptosis phenomenon of HepG2 cells is more severe due to the increase of the additive amount of the amomum essential oil microcapsule, and 92.7% of human liver cancer cells HepG2 of the experimental group can be induced to apoptosis after 72 hours treatment. Meanwhile, fig. 3 can not only observe green fluorescence generated in early apoptosis, red fluorescence generated by necrotic cells or apoptotic bodies losing cell membrane integrity in late apoptosis, and green fluorescence generated in necrotic cell membranes, but also obviously observe the variation of the morphology and quantity of corresponding apoptotic cells.
Example 2
(1) Pectin and chitosan are respectively dissolved by deionized water to prepare 1mg/ml water solution.
(2) And (3) configuring the pectin-chitosan nanoparticle aqueous solution in the step (1) according to the mass ratio of the two components of 2:1, and performing ultrasonic dispersion for 45 min under the power of 200W.
(3) And (3) respectively adjusting the pH value of the mixed solution in the step (2) to 3 by using 0.5mol/L hydrochloric acid aqueous solution.
(4) Adding fructus Amomi essential oil into the aqueous solution of the nano particles obtained in the step (3), wherein the mass ratio of the fructus Amomi essential oil to the aqueous solution of the nano particles is 2:1, and dispersing the obtained mixed solution at a high speed for 30min under 800 r/min.
(5) Spray drying the mixed solution obtained in the step (4), setting the inlet air temperature to 180 ℃, the outlet air temperature to 85 ℃ and the feeding rate to 5mL/min, and obtaining the fructus amomi essential oil microcapsule.
The particle size distribution of the fructus amomi essential oil microcapsule is shown in a graph 1 ((2)), the average particle size is 258.11nm, and the embedding rate is 85.9%.
The results of observing the cell morphology under a microscope are shown in FIG. 4. After being treated by fructus amomi essential oil microcapsule for 72 hours, the extract can induce apoptosis of 90.1% of human liver cancer cells HepG2 of an experimental group.
Example 3
(1) Dissolving inulin and chitosan with deionized water respectively to obtain 1mg/ml water solution.
(2) And (3) preparing an aqueous solution of inulin-chitosan nano particles according to the mass ratio of the two components of 1:2, and performing ultrasonic dispersion for 30min under the power of 200W.
(3) And (3) respectively adjusting the pH value of the mixed solution in the step (2) to 3 by using 0.5mol/L hydrochloric acid aqueous solution.
(4) Adding fructus amomi essential oil into the aqueous solution of the nano particles obtained in the step (3), wherein the mass ratio of the fructus amomi essential oil to the aqueous solution of the nano particles is 1:1, and dispersing the obtained mixed solution at a high speed of 1200 r/min for 15min.
(5) Spray drying the mixed solution obtained in the step (4), setting the inlet air temperature to 180 ℃, the outlet air temperature to 85 ℃ and the feeding rate to 5mL/min, and obtaining the fructus amomi essential oil microcapsule.
The particle size distribution of the fructus amomi essential oil microcapsule is shown in a graph 1 ((3)), the average particle size is 215.62nm, and the embedding rate is 80.2%.
The results of observing the cell morphology under a microscope are shown in FIG. 4. After being treated by fructus amomi essential oil microcapsule for 72 hours, 88.5% of human liver cancer cells HepG2 of the experimental group can be induced to apoptosis.
Example 4
(1) Respectively dissolving inulin and lysozyme with deionized water to obtain 1mg/ml water solution.
(2) And (3) preparing an aqueous solution of inulin-chitosan nano particles according to the mass ratio of the two components of 1:4, and performing ultrasonic dispersion for 45 min under the power of 200W.
(3) And (3) respectively adjusting the pH value of the mixed solution in the step (2) to 10 by using 0.5mol/L sodium hydroxide aqueous solution.
(4) Adding fructus amomi essential oil into the aqueous solution of the nano particles obtained in the step (3), wherein the mass ratio of the fructus amomi essential oil to the aqueous solution of the nano particles is 1:2, and dispersing the obtained mixed solution at a high speed for 15min at 1600 r/min.
(5) And (3) spray-drying the mixed solution obtained in the step (4), setting the air inlet temperature to 180 ℃, setting the outlet temperature to 85 ℃, and carrying out spray-drying at the feeding rate of 5mL/min to obtain amomum essential oil microcapsules, and inducing apoptosis of 60% human liver cancer cells HepG2 of an experimental group after 72 hours.
The particle size distribution of the fructus amomi essential oil microcapsule is shown in a graph (4), the average particle size is 182.73nm, and the embedding rate is 78.6%.
The results of observing the cell morphology under a microscope are shown in FIG. 4. After being treated by fructus amomi essential oil microcapsule for 72 hours, the extract can induce 86.3% of human liver cancer cells HepG2 of an experimental group to apoptosis.
Claims (4)
1. A preparation method of fructus amomi essential oil microcapsule is characterized by comprising the following steps: fructus Amomi essential oil is used as a core material, nano particles are used as wall materials, a Pickering emulsion product is obtained under high-speed stirring, and then the fructus Amomi essential oil microcapsule is obtained through spray drying, wherein the nano particles are one or more of inulin-lysozyme, inulin-chitosan, pectin-lysozyme and pectin-chitosan.
2. The preparation method of the fructus amomi essential oil microcapsule according to claim 1, which is characterized by comprising the following steps:
(1) Adding fructus amomi essential oil into the aqueous solution of the nano particles, and dispersing the obtained mixed solution at a high speed of between 800 and 1600r/min for 5 to 30 minutes;
(2) And (3) spray drying the mixed solution obtained in the step (1), wherein the inlet air temperature is 175-185 ℃, the outlet air temperature is 80-90 ℃, and the feeding rate is 4-6 mL/min, so as to obtain the fructus amomi essential oil microcapsule.
3. The method for preparing fructus amomi essential oil microcapsule according to claim 2, wherein in the step (1), the mass ratio of the fructus amomi essential oil to the nanoparticle aqueous solution is 2:1-1:2.
4. The method for preparing amomum essential oil microcapsule according to claim 2, wherein the method for preparing the nanoparticle aqueous solution in the step (1) comprises the following steps:
(1) Respectively dissolving inulin, pectin, chitosan and lysozyme with deionized water to prepare 1mg/ml water solution;
(2) Respectively preparing an inulin-lysozyme aqueous solution, an inulin-chitosan aqueous solution, a pectin-lysozyme aqueous solution and a pectin-chitosan aqueous solution according to the mass ratio of the two components of 1:4-4:1, and performing ultrasonic dispersion for 30-60 min under the power of 200W;
(3) The pH value of the system is adjusted to 3-10 by 0.5mol/L hydrochloric acid and sodium hydroxide aqueous solution respectively.
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