CN117049960A - Preparation method of crocetin and application of crocetin in antioxidation and eye protection - Google Patents

Preparation method of crocetin and application of crocetin in antioxidation and eye protection Download PDF

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CN117049960A
CN117049960A CN202210522534.5A CN202210522534A CN117049960A CN 117049960 A CN117049960 A CN 117049960A CN 202210522534 A CN202210522534 A CN 202210522534A CN 117049960 A CN117049960 A CN 117049960A
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crocus sativus
crocetin
extract
crocin
hydrolysate
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连秀屘
陈家章
曾景瑞
黄朝禄
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Syi Biotechnology Co ltd
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    • A—HUMAN NECESSITIES
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    • A61P27/02—Ophthalmic agents

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Abstract

The invention provides a preparation method of crocin acid and an anti-oxidation eye protection application, wherein the preparation method of crocin extraction hydrolysate containing high-content crocin acid comprises crocin crushing, ethanol water solution extraction and alkali hydrolysis to obtain crocin extract containing crocin, and crocin hydrolysate containing high-content crocin acid can be obtained after hydrolysis, so that the crocin hydrolysate has an eye protection effect and can effectively improve the survival rate of retinal pigment epithelial cells subjected to oxidation pressure.

Description

Preparation method of crocetin and application of crocetin in antioxidation and eye protection
Technical Field
The invention relates to the technical field of crocetin preparation methods, in particular to a crocetin preparation method and an antioxidant eye protection application.
Background
Eye diseases are commonly found in retinopathy, glaucoma and Age-related macular degeneration (Age-related macular degeneration, AMD) caused by diabetes, and the main cause of AMD is maculopathy caused by oxidative damage of retinal pigment epithelial cells due to imbalance of active oxygen in the eye. AMD is a pathogenesis of excessive reactive oxygen species that interfere with the normal function of retinal epithelial cells, and reduce the integrity of the retinal pigment epithelial cell junctions, leading to apoptosis.
Retinal pigment epithelial cells (retinal pigment epithelium, RPE) are a monolayer of cells located between the photoreceptor cells and the choroid of the neural retinal layer. Functionally playing the role of an outer blood-retinal barrier (outer) and the close association (light junction) of endothelial cell formation are critical for transporting fluids or substances across the barrier, so RPE cells have the function of maintaining retinal layer integrity.
Reactive oxygen species are relatively reactive oxygen-containing molecules, generally referred to as oxygen-containing free radicals and non-free radicals. Non-radical molecules are rapidly converted to free radicals when subjected to oxidation. Hydrogen peroxide (hydrogen peroxide, H) 2 O 2 ) Is an active oxygen species, has high polarity, does not have unpaired electrons, but is an intermediate product formed by oxygen free radicals; can flow and spread in cells or among cells, causing oxidative damage to the cells.
Lutein is currently the most common eye care food, but can only bring partial benefit to retina and can not provide complete protection to retina, scientists still continuously search for components with better eye care effect.
Crocin (Crocetin) is carotenoid with small molecule, can be absorbed by human body rapidly, can relieve eye fatigue, and can be obtained by converting Crocin (Crocin) extracted from Crocin with complicated steps. The mechanism of crocetin for ocular protection has not been fully elucidated in the current studies.
There are many improvements in crocetin extraction technology today. Chinese patent CN106905145a discloses a method for extracting crocetin, wherein the gardenia extract uses 4% sodium hydroxide aqueous solution, hydrolyzes for 2.5 hours, the crocetin content in the crude crocetin product is only 10.6%, and the crocetin product needs to be crystallized by using 10% sodium hydroxide aqueous solution and leached by methanol, so that the crocetin with higher purity can be obtained, and the prior art needs not only the step of twice alkali treatment with different concentrations, but also the step of using high concentration alkali; chinese patent CN108203378A discloses a method for extracting crocetin, wherein the gardenia extract is hydrolyzed by potassium hydroxide, neutralized by adding acid, then heated and stirred by adding methanol, filtering, adding a filter cake into acetone for crystallization to obtain high-purity crocetin, and high-concentration alkali, organic solution methanol and acetone are needed in the process; chinese patent CN103695494B discloses a method for extracting crocetin, which uses gardenia methanol extract as raw material, and only crude product with crocetin content of only 10.6% can be obtained through alkali hydrolysis and ultrasonic acceleration; the product of crocetin with high purity can be obtained by secondary alkaline hydrolysis and secondary enzyme catalysis. As can be seen from the foregoing patent, the prior art for preparing crocetin requires complicated steps, besides alkaline hydrolysis, crystallization with strong alkali or organic solvent or enzyme action, and can obtain high purity crocetin, and various organic solvents or high concentration alkali are used in the process, which may remain in the finished product to cause toxicity or may cause long-term harm on the body of the extractor in the extraction process.
Therefore, the invention provides a simple and safe method for extracting crocetin, and confirms that the crocetin has antioxidant eye protection purpose.
Disclosure of Invention
In view of the above, the present inventors have succeeded in developing and completing a simple and safe preparation method of crocetin through improvement and innovation of many years of research, comprising the following steps:
step one: crushing saffron;
step two: adding a first ethanol aqueous solution for extraction, and filtering to obtain a saffron extract, wherein the first ethanol aqueous solution contains 40-60 wt% of ethanol, the extraction temperature is 40-60 ℃, and the extraction time is 5-7 hours;
step three: the crocus sativus extract is prepared in a second ethanol aqueous solution, and sodium hydroxide is added for hydrolysis, wherein the second ethanol aqueous solution contains 12-30wt% of ethanol, the hydrolysis temperature is 65-85 ℃, and the hydrolysis time is 1.5-3 hours;
step four: adding hydrochloric acid for neutralization to obtain a saffron hydrolysate; the crocus sativus hydrolysate contains high crocus sativus acid content, and after drying, the crocus sativus hydrolysate contains 13-20 mg crocus sativus acid per gram.
To achieve the above object, the saffron extract is further concentrated to obtain a saffron concentrated extract or freeze-dried to obtain a saffron extract powder, and then the saffron extract powder is placed in the second ethanol aqueous solution.
To achieve the above object, the weight ratio of the crocus sativus to the first ethanol aqueous solution is (1:40) - (1:60).
To achieve the above object, the weight ratio of the crocus sativus extract to the second aqueous ethanol solution is (1:8) - (1:13).
To achieve the above object, the weight ratio of the crocus sativus concentrated extract to the second ethanol aqueous solution is (1:8) - (1:13); the weight ratio of the crocus sativus extract powder to the second aqueous ethanol solution is (1:8) - (1:13).
To achieve the above object, the weight ratio of the second ethanol aqueous solution to the sodium hydroxide is 35: 1) And (3) the following steps: 1).
To achieve the above object, the crocus sativus hydrolysate is freeze-dried to obtain a crocus sativus hydrolysate powder, wherein each gram of crocus sativus hydrolysate powder contains 15-18 mg of crocus sativus acid.
The invention further provides the use of crocetin for the preparation of a pharmaceutical composition for eye protection.
To achieve the above object, the crocetin is obtained from crocus sativus through the preparation method described above.
To achieve the above object, wherein protecting eyes means effectively improving survival rate of retinal pigment epithelial cells subjected to oxidative stress
To achieve the above object, the effective concentration of crocetin is 6 mug/mL-200 mug/mL.
To achieve the above object, the effective concentration of crocetin is 8 mug/mL-100 mug/mL.
In summary, the present invention provides a simple and safe method for extracting crocetin, which can obtain high crocin and crocetin content without using harmful organic solvents, protect eyes and effectively improve survival rate of retinal pigment epithelial cells subjected to oxidative stress.
Drawings
FIG. 1 is a flow chart of a process for preparing crocus sativus hydrolysate containing high levels of crocus sativus acid.
Fig. 2A is an HPLC profile of crocin standard and crocin acid standard.
Fig. 2B is an HPLC profile of crocus sativus extract and crocus sativus hydrolysate.
FIG. 3A shows crocus sativus hydrolysate pair H 2 O 2 Influence of oxidative damage on retinal pigment epithelial cells (mean P)<0.05)。
FIG. 3B shows crocus sativus extract pair H 2 O 2 Influence of oxidative damage on retinal pigment epithelial cells (mean P)<0.05)。
FIG. 3C is calendula extract pair H 2 O 2 Influence of oxidative damage on retinal pigment epithelial cells (mean P)<0.05)。
Detailed Description
[ definition of terms ]
In the following description, the following definitions are provided for a clear and consistent understanding of the present specification and claims, as well as the scope of the terms that are assigned thereto, as a result of which many technical and scientific terms that are conventional in the biotechnology arts are widely used in this specification. Other terms, not specifically defined below, are to be construed as commonly understood by one of ordinary skill in the art.
The term "individual" refers to any mammal in need of or believed to potentially require the crocetin-containing crocetin hydrolysate of the present invention that contains primates, rodents, pets, laboratory test animals, and wild animals. For example, this may include, but is not limited to: monkey, human, pig, cow, sheep, goat, equine, mouse, rat, guinea pig, hamster, rabbit, cat (felines), dog (cannines). Preferably, the subject is a mouse or a human.
The term "treatment" and the like is used herein to administer an agent in order to achieve a certain effect. The effect is a therapeutic partial or complete effective cure of a disease and/or symptoms of the disease.
The terms "therapeutically effective amount," "therapeutic dose," and the like are used herein to treat, cure, prevent, or ameliorate a disease, disorder, or side effect, or to reduce the rate of progression of a disease or disorder. The term also includes within its scope an amount effective to enhance normal physiological function.
The term "pharmaceutically acceptable" means that the substance or composition must be compatible with the other ingredients of the pharmaceutical formulation thereof and not exacerbate the symptoms of the patient.
The pharmaceutical composition provided by the invention can be prepared into a dosage form suitable for the composition by utilizing the technology known to a person with ordinary skill in the art to which the invention belongs, and the active ingredient or the composition provided by the invention and at least one pharmaceutically acceptable carrier (vehicle). Wherein the dosage form includes, but is not limited to, solutions, emulsions, suspensions, powders, lozenges, troches, tablets, chewing gums, capsules, and other similar or suitable dosage forms for the present invention.
The term "pharmaceutically acceptable carrier" includes one or more types of ingredients selected from the group consisting of: solvents, emulsifiers, suspending agents, disintegrants, binders, excipients, stabilizers, chelating agents, diluents, gelling agents, preservatives, lubricants, surfactants, and other similar or suitable carriers for the invention.
One or more of dissolution aids, buffers, colorants, flavors, and the like which are generally used in the field of preparation may be added to the above composition as needed.
The term "pharmaceutically acceptable excipient" includes, but is not limited to, at least one of polymers, resins, plasticizers, fillers, lubricants, diluents, binders, disintegrants, solvents, co-solvents, surfactants, preservatives, sweeteners, flavoring agents, pharmaceutical grade dyes or pigments, and viscosity agents.
The term "cell culture" and the like are used herein to maintain cells in an artificial in vitro environment. However, it should be understood that the term "cell culture" is a generic term and can be used to encompass not only the cultivation of individual cells, but also the cultivation of tissues or organs.
The methods of the present invention relate to administering crocus hydrolysates containing high levels of crocus acid to an individual (e.g., a human patient) to protect the eyes from oxidative damage. The methods of treatment according to the methods of the invention may also treat, cure, alleviate, alter, remedy, ameliorate, augment or affect the disease, the symptoms or conditions of the disease, the disability caused by the disease, or the progression of the disease.
[ administration of crocus sativus hydrolysate containing high crocus sativus acid ]
The pharmaceutical compositions of the invention may be delivered by any physiologically acceptable route and may be administered topically or systemically, with or without the addition of excipients, such as orally, parenterally (e.g., intramuscularly, intravenously, subcutaneously, and intraperitoneally), transdermally, suppository, ophthalmic, and intranasal.
[ dose ]
In the methods of treatment of the present invention, an effective amount of crocus sativus hydrolysate containing a high level of crocus sativus acid is administered to a subject in need thereof. In particular, the type of crocus sativus hydrolysate containing a high level of crocus sativus acid will vary depending on the purpose of administration, the health and physical condition of the individual to be treated, the age, the taxonomic group of individuals to be treated (e.g., human, non-human primate, etc.), the evaluation of the medical condition by the treating clinician, and other relevant factors. It is expected that this amount will be within a relatively wide range, which can be determined via routine experimentation.
[ pharmaceutical composition ]
Crocus sativus hydrolysates containing high levels of crocus sativus acid can be mixed with pharmaceutically acceptable carriers to prepare pharmaceutical compositions; can also be packaged in a container and prepared into a kit or product together with a package insert which records information related to the crocus sativus extract, the method of use, etc.
The present invention is illustrated by the following examples, which are, however, merely illustrative of the present invention and should not be construed as limiting the scope of the invention in any way, and in addition, all materials used herein are readily available from commercial sources.
Referring to fig. 1, according to an embodiment of the present invention, there is provided a method for producing crocus sativus hydrolysate containing crocus sativus acid in high content, comprising the steps of (S1) pulverizing crocus sativus; (S2) adding 40-60 wt% ethanol water solution for extraction at 40-60 ℃ for 5-7 hours, and filtering to obtain a saffron extract; (S3) preparing the crocus sativus extract into 12-30wt% ethanol water solution, adding sodium hydroxide for hydrolysis, wherein the hydrolysis temperature is 65-85 ℃ and the hydrolysis time is 1.5-3 hours; (S4) adding hydrochloric acid for neutralization to obtain the saffron hydrolysate.
Example one, crocus sativus extraction method
Crushing crocus sativus, adding a first ethanol aqueous solution with the weight being 40-60 times that of the crocus sativus, placing the solution for extraction for 5-7 hours at the extraction temperature of 40-60 ℃, filtering to retain liquid, concentrating the filtrate in vacuum to obtain crocus sativus extract, freeze-drying to obtain crocus sativus extract powder, adding a second ethanol aqueous solution with the weight being 8-13 times that of the crocus sativus extract powder, wherein the second ethanol aqueous solution contains 12-30% of ethanol, adding sodium hydroxide (30-50 g/L), placing the solution in the hydrolysis temperature of 70-80 ℃ for hydrolysis for 1.5-3 hours, adding hydrochloric acid for neutralization to obtain crocus sativus hydrolysate, and freeze-drying to obtain crocus sativus hydrolysate powder.
The amount of the first ethanol aqueous solution is preferably 50 times the weight of the crocus sativus; the first ethanol aqueous solution is preferably 50wt% ethanol; the extraction temperature is optimized at 50 ℃; the extraction time was optimal at 6 hours.
In the example, 30g of crocus sativus is weighed, 1500ml of 50% ethanol is added, extraction is carried out for 6 hours at 50 ℃, and after the filtrate is concentrated in vacuum, the crocus sativus extract powder is obtained after freeze drying.
The dosage of the second ethanol aqueous solution is 10.4 times of the weight of the crocus sativus extract powder; the second ethanol aqueous solution is preferably 20wt% ethanol; the dosage of sodium hydroxide is optimally 40 g/L; the hydrolysis temperature is optimal at 75 ℃, and the hydrolysis time is optimal at 2 hours; hydrochloric acid for neutralization is preferably used at a concentration of 12M.
In the example, 9.6g of crocus sativus extract powder is weighed, 100mL of 20% ethanol is added, naOH (40 g/L) is added, hydrolysis is carried out for 2 hours at 75 ℃, 12M HCl (6 mL) is added for neutralization, and freeze drying is carried out, thus obtaining crocus sativus hydrolysate powder.
Example two, crocus sativus extract and analysis of the content of hydrolysate thereof
HPLC analysis conditions:
chromatographic column: inertsil 250X 4.6 mm i.d.5 microns;
detection wavelength: 440nm;
column temperature: 35 ℃;
mobile phase: a-0.1% formic acid aqueous solution, B-acetonitrile, C-methanol;
flow rate: 0.8 milliliters per minute;
and (3) gradient stripping: as shown in Table 1
TABLE 1
Time (minutes) ％A ％B ％C
0 80 20 0
18 65 35 0
23 25 75 0
36 12 0 88
40 12 0 88
41 0 0 100
Crocin (Crocin) standard formulation: weighing crocin 5 mg, placing in a 50 ml quantitative bottle, adding 50% ethanol to scale, filtering to obtain 0.45 micrometer filter membrane, and injecting into 10 microliters.
Crocetin (Crocetin) standard formulation: weighing crocetin 5 mg, placing into a 50 ml quantitative bottle, adding 50% ethanol to the scale, weighing the 5ml of crocetin to 10 ml with methanol, filtering with 0.45 micrometer filter membrane, and injecting 10 microliters.
Preparing crocus sativus extract: weighing 4 mg of crocus sativus extract powder, placing in a 50 ml quantitative bottle, adding 50% ethanol to the scale, filtering with 0.45 μm filter membrane, and injecting into 10 microliters.
Preparing crocus sativus hydrolysate: weighing crocus sativus hydrolysate powder 25 mg, placing in 50 ml quantitative bottle, adding 50% ethanol to scale, filtering with 0.45 micrometer filter membrane, and injecting into 10 microliters.
Conversion calculation:
C a : total amount of Crocetin (Crocetin) after hydrolysis;
C b : total amount of Crocin (Crocin) before hydrolysis;
MW 2 : crocetin (Crocetin) molecular weight 328.4;
MW 1 : crocin (Crocin) molecular weight 977.0;
FIG. 2A is an HPLC chromatographic chart of Crocin (Crocin) and Crocin (Crocin) standard, 9.6g of unhydrolyzed Crocin extract powder is taken, 7.67g of Crocin hydrolysate powder is obtained after hydrolysis, and according to the HPLC analysis result, the Crocin extract chart in FIG. 2B is marked as Crocin extract (before hydrolysis), the Crocin (Crocin) content in the Crocin extract prepared from the Crocin extract powder is 52.2mg/g, wherein Crocin is not detected; the Crocin hydrolysate pattern in FIG. 2B was designated as Crocin extract (after hydrolysis) and Crocin hydrolysate powder was formulated to have a Crocin acid (Crocin) content of 16.7mg/g, crocin was not detected in Crocin hydrolysate, and the conversion was calculated to be 76.33%.
Example III, crocus sativus extract, crocus sativus hydrolysate protective action on retinal pigment epithelial cells
[H 2 O 2 Oxidative injury cell experiment]Human retinal pigment epithelial cells (ARPE-19) were cultured in 24 well (well) microplates at a cell density of 3X 10 4 cell/well, 5% CO at 37deg.C 2 Culturing in an incubator for 24 hours, and pasting ARPE-19 cells on the dish. Then removing the old culture solution, adding 0.5mL of cell culture solution containing test samples with different concentrations, culturing for 24 hours, removing the culture solution containing the test samples, and then adding the culture solution containing 1mM H 2 O 2 Is cultured for 3 hours to cause oxidative damage, the culture solution is sucked off, and 0.4mL of a medium containing 1mg/mL MTT (light shielding in CO is required) is added to each well 2 The reaction was carried out in an incubator for 2 hours, the medium containing MTT reagent was removed, 0.4mL of DMSO was added thereto, and the crystals were dissolved uniformly by shaking, and the absorbance was measured at 570nm by ELISA reader, and the cell viability was calculated.
The purpose of this experiment was to investigate the H pair of crocus sativus extracts before and after hydrolysis 2 O 2 Protection against oxidative damage of retinal pigment epithelial cells (ARPE-19) was achieved with calendula extract containing 5% lutein as control.
FIG. 3A shows that the crocus sativus hydrolysate sample prepared from crocus sativus hydrolysate powder can reduce oxidation injury of retinal cells, and can significantly reduce H at concentration of more than 10 μg/mL 2 O 2 The resulting oxidative damage was 31% less oxidative damage at 10 μg/mL, 53% less oxidative damage at 25 μg/mL, 79% less oxidative damage at 50 μg/mL, and dose-effect correlation.
FIG. 3B shows that samples of crocus sativus extract formulated from crocus sativus extract powder significantly reduced H at concentrations above 25 μg/mL 2 O 2 The resulting oxidative damage was 41% reduced with 25 μg/mL and 63% reduced with 50 μg/mL, and a dose-effect correlation was established.
FIG. 3C shows that the calendula extract samples used as the control group increased H-channel 2 O 2 Reduced cell viability of oxidative damage, calendula extract at maximum test concentration (50 μg/mL) was significantly reduced visionEffect of oxidative damage of omental pigment epithelial cells 57%.
From the above results, it was found that the crocin hydrolysate (containing crocin), crocin extract (containing crocin), calendula extract (containing 5% lutein) can improve the H-channel 2 O 2 Reduced cell viability with oxidative damage, with the best results with crocus hydrolysates containing high levels of crocetin, the next-second crocus extracts, and the worst calendula extracts.
In view of the above, the present invention provides a simple and safe process for extracting crocetin, which can obtain crocetin hydrolysate containing high crocetin content without using harmful organic solvents; at the same dose, the protection effect of the crocus sativus extract (unhydrolyzed and hydrolyzed) on the oxidative damage of the retina cells is better than that of the calendula extract (containing 5% of lutein), and the crocus sativus hydrolysate containing high crocus sativus acid has the potential of developing health care raw materials for protecting eyes and preventing maculopathy.
The above embodiments are provided to illustrate the technical spirit and features of the present invention, and to enable those skilled in the art to understand the present invention and to implement it according to the present invention, and should not limit the scope of the present invention, i.e. all equivalent changes or modifications that are substantially in accordance with the spirit of the present invention are included in the scope of the present invention.
[ PREPARATION ] S1-S4.

Claims (12)

1. A method for preparing crocetin, comprising the steps of:
step one: crushing saffron;
step two: adding a first ethanol aqueous solution for extraction, and filtering to obtain a saffron extract, wherein the first ethanol aqueous solution contains 40-60 wt% of ethanol, the extraction temperature is 40-60 ℃, and the extraction time is 5-7 hours;
step three: the crocus sativus extract is arranged in a second ethanol aqueous solution, sodium hydroxide is added for hydrolysis, the second ethanol aqueous solution contains 12-30wt% of ethanol, the hydrolysis temperature is 65-85 ℃, and the hydrolysis time is 1.5-3 hours;
step four: adding hydrochloric acid for neutralization to obtain a saffron hydrolysate; the crocus sativus hydrolysate contains crocus sativus acid, and the crocus sativus hydrolysate contains 13-20 mg of crocus sativus acid per gram after being dried.
2. The method of claim 1, wherein the saffron extract is further subjected to liquid concentration to obtain a saffron concentrated extract or freeze-dried to obtain a saffron extract powder, and is further disposed in the second aqueous ethanol solution.
3. The method of claim 1, wherein the weight ratio of crocus sativus to the first aqueous ethanol solution is from (1:40) to (1:60).
4. The method of claim 1, wherein the weight ratio of the crocus sativus extract to the second aqueous ethanol solution is from (1:8) to (1:13).
5. The method of claim 2, wherein the weight ratio of the saffron concentrate extract to the second aqueous ethanol solution is from (1:8) to (1:13); the weight ratio of the crocus sativus extract powder to the second ethanol aqueous solution is (1:8) - (1:13).
6. The method of claim 1, wherein the weight ratio of the second aqueous ethanol solution to the sodium hydroxide is (35:1) - (20:1).
7. The method of claim 1, wherein said crocus sativus hydrolysate is freeze-dried to provide a crocus sativus hydrolysate powder comprising from 15 mg to 18 mg crocus sativus acid per gram of said crocus sativus hydrolysate powder.
8. Use of crocetin for the preparation of a pharmaceutical composition for protecting the eye, wherein protecting the eye is effective to increase the survival rate of retinal pigment epithelial cells subjected to oxidative stress.
9. Use according to claim 8, characterized in that the crocetin is obtained from crocus sativus via the preparation method according to claim 1.
10. The use according to claim 8, wherein the protection of the eye is effective to increase survival of retinal pigment epithelial cells subjected to oxidative stress.
11. The use according to claim 8, wherein the effective concentration of crocetin is between 6 μg/mL and 200 μg/mL.
12. The use according to claim 8, wherein the effective concentration of crocetin is between 8 μg/mL and 100 μg/mL.
CN202210522534.5A 2022-05-04 2022-05-13 Preparation method of crocetin and application of crocetin in antioxidation and eye protection Pending CN117049960A (en)

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