CN111658740A - Medicine and food dual-purpose composition capable of improving age-related macular degeneration as well as preparation method and application thereof - Google Patents
Medicine and food dual-purpose composition capable of improving age-related macular degeneration as well as preparation method and application thereof Download PDFInfo
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- CN111658740A CN111658740A CN202010567201.5A CN202010567201A CN111658740A CN 111658740 A CN111658740 A CN 111658740A CN 202010567201 A CN202010567201 A CN 202010567201A CN 111658740 A CN111658740 A CN 111658740A
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Abstract
The invention discloses a medicine and food dual-purpose composition for improving age-related macular degeneration and a preparation method and application thereof, wherein the medicine and food dual-purpose composition is prepared from the following raw materials in parts by weight: 1-5 parts of wolfberry fruit, 4-8 parts of hawthorn, 4-8 parts of astragalus membranaceus, 1-5 parts of fructus alpiniae oxyphyllae, 1-5 parts of dendrobium officinale, 5-10 parts of oyster, 1-5 parts of chrysanthemum, 0.01-0.1 part of grape seed procyanidine, 0.01-0.1 part of lutein and 0.01-0.1 part of taurine. The medicine and food dual-purpose composition provided by the invention can improve age-related macular degeneration, has good safety, and can be used for preparing common foods, health-care products or medicines for improving age-related macular degeneration.
Description
Technical Field
The invention relates to a medicine and food dual purpose composition, in particular to a medicine and food dual purpose composition for improving age-related macular degeneration and a preparation method and application thereof.
Background
All the data show that China has entered the aging society and is in an accelerated situation. The existing aged population in china has exceeded 1.6 billion and has increased at a rate of nearly 800 million per year. It is expected that by 2050, the population of the aged people in china will reach one third of the total population. The rapid increase of the population of the old, particularly the 100-ten-thousand growth rate of the old with the age of more than 80 years old and the disabled, increasingly remarkable needs for the life care, rehabilitation nursing, medical care, mental culture and the like of the old, and the problem of old care becomes severe day by day.
Among the aging diseases, the disease most affecting vision is age-related macular degeneration (AMD). This is a disease that affects the macula area of the retina, resulting in progressive loss of vision. Clinical symptoms of early AMD include drusen appearing in the retina, abnormal retinitis pigmentosa. Advanced AMD has two clinical manifestations: atrophic (dry) forms are characterized by progressive loss of Retinal Pigment Epithelial (RPE) cells and photoreceptor cells, accounting for 85% -90% of clinical cases. Exudative (wet), the retinal retina of the patient develops with immature choroidal neovascularization and is accompanied by retinal edema or subretinal hemorrhage, accounting for 10% -15% of clinical cases. In the advanced stages of the disease, patients may develop severe and permanent vision impairment or blindness. AMD mainly affects the elderly over 60 years of age and is the leading cause of irreversible visual impairment in the elderly worldwide.
Currently, the global prevalence of AMD is about 8.7% over the age range of 45-85 years, with early prevalence of about 8% and late prevalence of about 0.4%. As the population ages, the number of people affected by AMD is expected to increase to 2.88 billion in 2040 years worldwide. An epidemiological study found that the prevalence of early AMD in developed cities in china is 9.5% and the prevalence of late AMD is 1.0% in people over 50 years of age.
AMD is a complex multifactorial disease. It is currently believed that AMD can be associated with age, ethnicity, family history, smoking, Body Mass Index (BMI), dietary habits (low vitamin C, vitamin E, carotenoids, omega-3 unsaturated fatty acid intake and high saturated fatty acid, high cholesterol intake), and the like. For wet AMD, clinically, the American Academy of Ophthalmology (AAO) clinical guidelines for AMD in 2019 suggest intravitreal injections with anti-Vascular Endothelial Growth Factor (VEGF) drugs (Ranibizumab, Aflibercept, Brolucizumab) as a first-line treatment regimen for treatment; for the treatment of dry AMD, there is no internationally approved clinical drug. With the advancement of aging society, there is an urgent need for new prevention and treatment regimens for AMD, given its significant medical, personal, socioeconomic costs.
Disclosure of Invention
The purpose of the invention is as follows: the invention aims to provide a medicine-food dual-purpose composition with good safety and the effect of improving age-related macular degeneration. Another object of the present invention is to provide a process for the preparation and use of the above composition.
The technical scheme is as follows: in order to achieve the above purpose, the invention adopts the technical scheme that:
a medicine and food dual purpose composition for improving age related macular degeneration is characterized by comprising the following raw materials in parts by weight: 1-5 parts of wolfberry fruit, 4-8 parts of hawthorn, 4-8 parts of astragalus membranaceus, 1-5 parts of fructus alpiniae oxyphyllae, 1-5 parts of dendrobium officinale, 5-10 parts of oyster, 1-5 parts of chrysanthemum, 0.01-0.1 part of grape seed procyanidine, 0.01-0.1 part of lutein and 0.01-0.1 part of taurine.
A preferred formula formed on the basis comprises the following raw materials in parts by weight: 2-4 parts of wolfberry fruit, 5-7 parts of hawthorn, 5-7 parts of astragalus membranaceus, 2-4 parts of fructus alpiniae oxyphyllae, 6-9 parts of oyster, 2-4 parts of dendrobium officinale, 2-4 parts of chrysanthemum, 0.03-0.08 part of grape seed procyanidine, 0.03-0.08 part of lutein and 0.03-0.08 part of taurine.
The optimal formula obtained after further optimization comprises the following raw materials in parts by weight: 3 parts of medlar, 6 parts of hawthorn, 6 parts of astragalus, 3 parts of sharpleaf galangal fruit, 3 parts of dendrobium officinale, 8 parts of oyster, 3 parts of chrysanthemum, 0.05 part of grape seed procyanidin, 0.05 part of lutein and 0.05 part of taurine.
The preparation method of the medical and edible dual-purpose composition for improving age-related macular degeneration and asthenopia comprises the following steps:
(1) taking the wolfberry fruit, the hawthorn fruit, the astragalus root, the sharpleaf galangal fruit, the dendrobium officinale, the oyster and the chrysanthemum according to the parts by weight, adding an extraction solvent, carrying out reflux extraction, and filtering; the filtrate was collected.
(2) Adding an extraction solvent into the medicine residues obtained in the step (1), performing reflux extraction, and filtering; the filtrate was collected.
(3) And (3) combining the filtrates obtained in the steps (1) and (2), concentrating under reduced pressure, and adding grape seed procyanidin, lutein and taurine to obtain the composition.
Preferably, the reflux extraction conditions in step (1) are as follows: firstly, adding 10 times volume of extraction solvent water of the weight of the medicinal materials, and carrying out reflux extraction at 100 ℃ for 120 min. The reflux conditions in the step (2) are as follows: adding 10 times volume of extraction solvent water, and reflux extracting at 100 deg.C for 120 min.
The invention has the advantages that: the present invention is in the absence of effective drugs for AMD, particularly dry AMD. Based on the traditional Chinese medicine theory and the pathological mechanism of AMD, the effective composition for improving AMD is preferably obtained.
AMD is classified into the categories of "bent vision due to direct vision", "blurred vision" and "sudden blindness" in TCM. The book of Lingshu & big Huoxian (treatise on the book of treatises) is characterized by: the essence of the five zang-organs and six fu-organs is the essence by injecting into the eyes; the relationship between eyes and viscera is clarified, and the occurrence and development of AMD are closely related to the aging of viscera. Traditional Chinese medicine considers that the main pathogenesis of AMD is as follows: the aged and weak people with increasingly severe deficiency of liver and kidney and deficiency of yin-fluid can not nourish eyes and produce fundus degeneration. Meanwhile, with the age, the deficiency of the spleen and the stomach, the transportation and transformation and hypodynamia, the water and the grain are not normalized, the water dampness is generated, the dampness is collected into phlegm, the phlegm and the dampness are internally blocked, the eyeground is accumulated, and the drusen and the exudation are generated; deficiency of spleen and stomach, deficiency of qi and blood, malnutrition of eyes, and atrophy of macula of fundus; spleen qi deficiency, the general control of debilitation, fluid and blood overflow outside the vessels, forming eyeground bleeding, exudation, staying in the eyeground, forming blood stasis and phlegm dampness. Therefore, based on AMD, the treatment should be mainly to tonify liver and kidney, invigorate spleen and promote diuresis, nourish qi and blood, and supplement blood and remove blood stasis, activate blood and improve eyesight, soften hardness and dissipate stagnation for eliminating phlegm.
In the medicine and food dual-purpose composition, the medlar has the functions of nourishing liver and kidney, and replenishing vital essence to improve eyesight. Hawthorn fruit has the effects of promoting blood circulation to remove blood stasis and strengthening the spleen and stomach. The two are combined together to promote the circulation of blood and essence without worry, and the eyes can be nourished. The astragalus root has the effects of tonifying spleen and qi, benefiting intelligence, warming spleen and kidney, and can strengthen spleen and transport, help paddy to generate essence and micro, and control blood circulation without paradoxical flow by combining the astragalus root and the hawthorn. The dendrobium officinale can nourish liver and kidney yin, the oyster can calm the liver and subdue yang, and the chrysanthemum can calm the liver and improve eyesight, and the dendrobium officinale, the oyster and the chrysanthemum can prevent eye burn caused by yin deficiency and yang hyperactivity and flaring up of deficient fire. Mu Li can soften hardness and dissipate nodulation, resolve phlegm-dampness stagnation and make eyeground stasis disperse.
From the modern pharmacological research of the medicines in the formula, the zeaxanthin in the medlar can prevent macular hypopigmentation and drusen accumulation of the old; the polysaccharide and carotenoid components effectively protect retinal pigment epithelial cells from oxidation and inflammatory injury; betaine can inhibit retinal neovascularization in diabetic patients. The hawthorn has obvious effects of reducing blood pressure, reducing blood fat and resisting atherosclerosis, improves lipid metabolism of the old, reduces peroxidation of lipid metabolites and protects retina microvasculature. Astragaloside IV and Astragalus polysaccharides not only have obvious protective effect on retinal ischemia reperfusion injury, but also can clear away oxygen free radicals, relieve lipid peroxidation state of retinal ganglion cell membranes, maintain integrity of cell membranes and inhibit apoptosis. The fructus alpiniae oxyphyllae water extract has high functions of eliminating hydrogen peroxide and oxygen free radicals and has an anti-aging effect. The polysaccharide component in Dendrobium officinale Kimura et Migo has effects of reducing retina and overall inflammatory factor level, inhibiting retinal vascular endothelial factor (VEGF) expression, and inhibiting new blood vessel growth. The Concha Ostreae is rich in zinc, magnesium and other microelements, and can reduce oxygen free radical level. The flavonoids such as apigenin and luteolin contained in flos Chrysanthemi can protect retinal pigment epithelial cells from oxidation and inflammation injury, reduce apoptosis caused by the above injury, and protect retinal pigment epithelial cells.
In addition, the added grape seed procyanidin, lutein and taurine are all beneficial to age-related macular degeneration. Grape seed procyanidin can inhibit the generation of lens oxygen free radicals and lipid peroxidation, reduce capillary wall fragility, protect capillary substance transport capacity, and prevent ultraviolet-induced DNA oxidative damage. The lutein can protect retina from damage by filtering short wavelength light causing light damage in visible light, enhance the function of outer segment membrane of cone against oxidation damage, and protect retinal ganglion cells from anoxia and oxidation damage. Taurine can protect retina from light injury by improving membrane permeability, resisting lipid peroxidation, increasing antioxidant enzyme activity, inhibiting apoptosis, etc.
Animal model experimental results of the medicinal and edible composition provided by the invention show that: the medicine and food dual-purpose composition provided by the invention can effectively reduce the most main pathological link of age-related macular degeneration caused by high oxidative stress level, thereby protecting the retina of an animal from damage. Cell model experiment results show that the medicine and food dual-purpose composition provided by the invention can improve the capability of retinal pigment epithelial cells in resisting oxidative damage and increase the expression of neurotrophic factors in retinal Miller cells, thereby achieving the effect of protecting retinal damage.
The invention discloses application of a medicine-food dual-purpose composition for improving age-related macular degeneration in age-related macular degeneration. Particularly, all the medicinal ingredients and components of the medicinal and edible composition are medicinal and edible varieties, so the medicinal and edible composition has good safety, is suitable for long-term use, and can play a role in preventing and improving AMD.
Drawings
FIG. 1 is a bar graph of the effect of a combination of both drugs and food on ROS in mouse serum.
FIG. 2 is a bar graph of the effect of the composition on MDA in mouse serum.
FIG. 3 is a bar graph showing the effect of the composition on SOD activity in mouse serum.
FIG. 4 is a bar graph of the effect of the combination of both drugs and food on the activity of GSH-Px in mouse serum.
FIG. 5 is a bar graph of the effect of the composition on CAT activity in mouse serum.
FIG. 6 is a bar graph of the effect of a combination of both pharmaceutical and edible properties on the expression level of Nrf2 mRNA in mouse retinas.
FIG. 7 is a bar graph of the effect of a combination of both pharmaceutical and edible properties on the expression level of HO-1 mRNA in mouse retina.
FIG. 8 is a bar graph of the effect of a combination of pharmaceutical and edible properties on the expression level of NQO-1 mRNA in mouse retina.
FIG. 9 is a bar graph of the effect of a combination of both drug and food on the expression level of GCL mRNA in mouse retinas.
FIG. 10 is a bar graph of the effect of a combination of both pharmaceutical and edible properties on VEGF expression in mouse retinas.
FIG. 11 is a bar graph of the effect of a combination of both drug and food on oxidative damage to the survival rate of APRE-19 cells.
FIG. 12 is a bar graph of the effect of a combination of drug and food on BBDNF expression in Muller cells.
Detailed Description
The present invention is further illustrated by the following examples, which are intended to be purely exemplary and are not intended to limit the scope of the invention, as various equivalent modifications of the invention will occur to those skilled in the art upon reading the present disclosure and fall within the scope of the appended claims.
Embodiment 1 a method for preparing a composition for both medicine and food with the effect of improving age-related macular degeneration, comprising the following steps:
(1) mixing fructus Lycii 1 part, fructus crataegi 2 parts, radix astragali 2 parts, fructus Alpinae Oxyphyllae 1 part, herba Dendrobii 2 parts, Concha Ostreae 2 parts, and flos Chrysanthemi 1 part. Adding water 8 times the weight of the medicinal materials, reflux extracting for 120min, filtering, and collecting filtrate and residue.
(2) And (2) adding water with the volume amount of 10 times of the weight of the medicinal materials into the medicine residues obtained in the step (1), performing reflux extraction for 120min, filtering, and collecting filtrate.
(3) And (3) combining the filtrates obtained in the step (1) and the step (2), concentrating under reduced pressure to obtain an extract, adding 0.01 part of grape seed procyanidin, 0.01 part of lutein and 0.01 part of taurine, and reducing pressure at low temperature to obtain an extract.
Embodiment 2 a method for preparing a composition for both medicine and food with the effect of improving age-related macular degeneration, comprising the following steps:
(1) mixing 4 parts of wolfberry fruit, 7 parts of hawthorn, 7 parts of astragalus, 4 parts of fructus alpiniae oxyphyllae, 4 parts of dendrobium officinale, 9 parts of oyster and 4 parts of chrysanthemum. Adding water 8 times the weight of the medicinal materials, reflux extracting for 120min, filtering, and collecting filtrate and residue.
(2) And (2) adding water with the volume amount of 10 times of the weight of the medicinal materials into the medicine residues obtained in the step (1), performing reflux extraction for 120min, filtering, and collecting filtrate.
(3) And (3) combining the filtrates obtained in the step (1) and the step (2), concentrating under reduced pressure to obtain an extract, adding 0.08 part of grape seed procyanidin, 0.08 part of lutein and 0.08 part of taurine, and reducing pressure at low temperature to obtain an extract.
Embodiment 3 a method for preparing a composition for both medicine and food with the effect of improving age-related macular degeneration, comprising the following steps:
(1) mixing 3 parts of wolfberry fruit, 6 parts of hawthorn, 6 parts of astragalus root, 3 parts of sharpleaf galangal fruit, 3 parts of dendrobium officinale, 8 parts of oyster and 3 parts of chrysanthemum. Adding water 8 times the weight of the medicinal materials, reflux extracting for 120min, filtering, and collecting filtrate and residue.
(2) And (2) adding water with the volume amount of 10 times of the weight of the medicinal materials into the medicine residues obtained in the step (1), performing reflux extraction for 120min, filtering, and collecting filtrate.
(3) And (3) combining the filtrates obtained in the step (1) and the step (2), concentrating under reduced pressure to obtain an extract, adding 0.05 part of grape seed procyanidin, 0.05 part of lutein and 0.05 part of taurine, and reducing pressure at low temperature to obtain an extract.
Example 4 animal Experimental study on improvement of age-related macular degeneration by medicinal and edible composition
First, experimental material and medicine
1. Laboratory animal
SPF-grade female Kunming mice weighing 18-22 g were purchased from Nanjing university of traditional Chinese medicine laboratory animal center under the certification number SCXK (Su) 2015-0002. The experimental mice are placed in the experimental animal center of Nanjing university of traditional Chinese medicine for feeding, and the facility use license SYXK (Su) 2012-0047 of the experimental animals.
2. Drugs and agents
Hydroquinone (0.8% concentration, Alfa Aesar); high fat diet (Research Diets); catalase (CAT) kit, glutathione peroxidase (GSH-PX) kit, Reactive Oxygen Species (ROS) test kit, superoxide dismutase (SOD) kit and Malondialdehyde (MDA) kit (cargo number: A003-1) are all purchased from Nanjing to build the bioengineering institute. TRIzol RNA extraction kit (15596-026, Invitrogen). RT-PCR reverse transcription kit (PrimeScript)TMRT reagent Kit with gDNA Eraser, RR047A, TAKARA); SYBR fluorescent quantitative PCR reagents (FastStart Universal SYBR Green Master (ROX),04913914001, Roche). Bradford kit (T9310A, TAKARA).
3. Laboratory apparatus
Microplate reader (Perkin-Elmer, USA); BT125 type electronic balance (sydows scientific instruments ltd); KQ-250E ultrasonic cleaner (kunshan grass ultrasonic instrument ltd); AnkeGL-16GII type centrifuge (Shanghai' an pavilion scientific Instrument plant).
Second, Experimental methods
1. Establishment of mouse retina macular degeneration disease model and drug administration of medicine-food dual-purpose composition
After 5d of adaptive feeding of healthy mice, randomly grouping: normal control group (normal diet), model control group (high fat diet decahydroquinone), administration group (high fat diet + hydroquinone + drug). The intervention drugs were administered to each group of 8 animals by gavage. Normal control group: normal diet, normal drinking water. Model control group: adding 0.8% oxide hydroquinone into drinking water, and feeding for 3 months in combination with high fat diet. Administration group: examples 1, 2 and 3 were divided into two groups of high dose (8g/kg) and low dose (4 g/kg). After a high fat diet was given and fed with 0.8% hydroquinone in drinking water for 3 months, examples 1, 2, and 3 were administered for 3 months. Gavage was started at 2 pm each day, mouse body weight was recorded once a week, and mouse status was observed in the morning and evening each day, as well as food intake and water intake.
2. Determination of SOD, GSH-Px and CAT activity, ROS and MDA content in mouse serum
After the observation period, the animals are sacrificed, the eyeballs are picked up, blood is taken from the peripheral veins of the eyes, the blood is centrifuged at 3000rpm and 4 ℃ for 10min, and serum is separated and stored at-20 ℃ for standby. And respectively measuring the SOD, GSH-Px and CAT activities and the contents of ROS and MDA in serum according to SOD, GSH-Px, CAT, ROS and MDA kit specifications.
3. Determination of mRNA expression level of key factor of oxidative stress pathway in mouse retina
Taking out the treated eyeball tissue, grinding uniformly, adding lysis solution to distribute the lysis solution on the tissue surface uniformly, standing at room temperature for 2min, and transferring trizol to an EP tube without RNA enzyme. Adding chloroform 200ul, shaking vigorously for 15s, standing at room temperature for 15min, centrifuging (4 deg.C, 12,000rpm, 15min), collecting the upper layer liquid, and adding into an EP tube. Then, the same volume of isopropyl alcohol was added thereto, the mixture was turned upside down and mixed, and the mixture was allowed to stand at room temperature for 5min and centrifuged (4 ℃, 12,000rpm, 15 min). Removing supernatant, adding 1ml of 75% ethanol into the precipitate, slightly shaking for 15s, and centrifuging at 4 deg.C and 7500rpm for 5 min. The supernatant was removed and the pellet was allowed to dry as much as possible, after which 50uLDEPC water was added.
Preparation of cDNA: RNA concentration and purity were measured by Nanodrop, and the reverse transcription was performed using 1. mu.g of RNA in total according to the instructions of TaKaRa reverse transcription kit.
According to the operation steps of the SYBR fluorescent real-time quantitative kit, a fluorescent real-time quantitative PCR instrument is utilized to measure the gene transcription level expression of the key factors of the oxidative stress pathway in the retina of the mouse. The primer sequences used are shown in table 1 below: nrf2, HO-1, NQO-1, GCL
TABLE 1 primer sequences
4. Determination of vascular growth factor (VEGF) expression levels in mouse retina
And taking out eyeballs of the mice of each group, adding HBSS buffer solution for homogenization, centrifuging to take supernatant, and measuring the VEGF expression in the eyeballs of the mice of each group by using a mouse VEGFELISA kit.
5. Statistical treatment
All experimental data were statistically processed using SPSS18.0 statistical processing software, and the results were obtained
Showing that the difference between groups was measured by t-test, P<A difference of 0.05 is statistically significant.
Third, experimental results
1. Medicine and food dual purpose composition for significantly reducing oxidative stress level in serum of mouse
As shown in fig. 1-2, the serum total Reactive Oxygen Species (ROS) level of the model control group (decahydroquinone after high fat diet) mice was significantly increased (P <0.01) compared to the normal control group, indicating that there was a high level of oxidative stress in the model mice; meanwhile, the content of lipid peroxide Malondialdehyde (MDA) in the model group is also remarkably increased (P <0.01), which indicates that the mice fed with high-fat diet and hydroquinone have high-level oxidative stress in retrobulbar venous blood (P < 0.01).
After the gavage treatment of the three medicine and food dual-purpose compositions with different proportions in examples 1-3, compared with a model control group, the total ROS level in the serum of a mouse in a low-dosage and high-dosage group is obviously reduced, and the MDA content in the serum is also obviously reduced (P is less than 0.01), which shows that the medicine and food dual-purpose composition can reduce the total ROS and MDA content in the serum, improve the oxidative stress state of the retina of the model mouse, and reduce the damage of active oxygen and various oxygen free radicals to the retina of the mouse. In addition, through comparison, the optimal proportion of the medical and edible dual-purpose composition in the example 3 is the best for reducing the oxidative stress level in the three different proportions of the medical and edible dual-purpose compositions in the examples 1 to 3.
2. Medicine and food dual-purpose composition for remarkably improving antioxidant kinase level in mice
As shown in FIGS. 3 to 5, the superoxide dismutase (SOD) activity was significantly reduced (P) in the serum of the model mice fed with high-fat diet and hydroquinone, as compared with the normal control group<0.01), and simultaneously, the activity of glutathione peroxidase (GSH-PX) in serum is also obviously reduced (P)<0.01), suggesting that the level of antioxidant stress factors is reduced in the body of the model mouse due to the existence of higher level of oxidative damage. Meanwhile, the Catalase (CAT) activity in the serum of the model group mice is obviously reduced (P)<0.01) since CAT is a hydroxyl radical (OH)-) The reduction of CAT activity results in a relative increase in the level of oxygen free radicals, indicating the presence of oxidative stress in the model mice. Causing strong oxidative damage to mouse retina tissue.
Compared with a model control group, the medical and edible dual-purpose compositions in the examples 1 to 3 can obviously improve the activities of SOD, GSH-Px and CAT in the serum of the mice with oxidative damage, and the high-dose group of each composition is superior to the low-dose group. And through comparison, the optimal proportion of the composition in the embodiment 3 has the most obvious effect (P <0.01), and the suggestion that the medicine and food dual-purpose composition can improve the activity of antioxidant enzyme in the retinal serum of an oxidative damage mouse, has dose dependence and plays a role in antioxidation, thereby improving the oxidative stress state of retina and resisting the oxidative damage of retina
3. Medicine and food dual-purpose composition for remarkably inhibiting activation of oxidative stress pathway in mouse
Based on the above experiments, we examined the expression level of a key kinase in the oxidative stress pathway in mice. As shown in FIGS. 6-9, compared with the normal control group, the mRNA expression levels of the key kinases Nrf2, HO-1, NQO-1 and GCL in the oxidative stress pathway of the model group are obviously increased (P <0.01), which indicates that the mouse body is in an oxidative stress state due to high fat diet and hydroquinone modeling, so that the Nrf2 pathway is activated, and the phase II enzyme is stimulated to release by itself to play an anti-oxidation role. Compared with a model control group, the Nrf2, HO-1, NQO-1 and GCLmRNA expression levels of the administration group are reduced by the treatment of the medicine and food dual-purpose composition in the embodiments 1 to 3, which indicates that the medicine and food dual-purpose composition can reduce the oxidative stress state of an organism and restore the balance and stable state of cells, thereby protecting retinal cells from oxidative damage. And the effect of the embodiment 3 is the best (P <0.01) in the three medicinal and edible dual-purpose compositions.
4. Medicine and food dual purpose composition for remarkably inhibiting expression of VEGF in mouse eyeball
The expression of VEGF in the mouse eyeball is detected, as shown in figure 10, compared with the normal control group, the expression level of VEGF in the mouse eyeball of the model group is obviously increased (P <0.01), which indicates that the mouse eyeball has a vascular proliferation trend due to high-fat diet and hydroquinone molding. Compared with a model control group, the VEGF expression level of the administration group is reduced by the treatment of the medicine-food dual-purpose composition, which prompts that the medicine-food dual-purpose composition can inhibit vascular proliferation caused by a high oxidative stress state in an animal body, avoid fundus hemorrhage and inhibit the generation of wet AMD. In the three medicinal and edible dual-purpose compositions of examples 1-3, the effect of example 3 is also the best (P < 0.01).
EXAMPLE 5 combination of drug and food for combating oxidative damage to retinal pigment epithelial cells (ARPE-19)
First, experimental material and medicine
1. Cells
Human retinal epithelial pigment cells (ARPE-19) are purchased from ATCC and are stored in the center of collaborative innovation in the industrialization process of traditional Chinese medicine resources in Jiangsu province.
2. Laboratory apparatus
Carbon dioxide incubator (Forma series II water jack CO2 incubator, Thermo Co.), 1300series A2 clean bench (Thermo Co.), AutoVertA1 inverted fluorescence microscope (ZEISS Co.), Nanodrop (DS11spectrophotometer, DeNovix Co.), ABI7500 fluorescent real-time quantitative PCR instrument (Invitrogen Co.), high speed Centrifuge (Allegra X-12R Centricure and Microfuge 22RCentrifuge, Backman Co.), constant temperature water bath (Blueard Co.), constant temperature oscillator (IS-RDV1 incubatorsake, CRYSTAL Co.), cell culture vessel (CORNINING Co.).
3. Reagent
Absolute ethanol (analytically pure), H2O2Solutions (analytically pure) were purchased from Shanghai, national medicine. DMEM/F12 medium, 10% Fetal Bovine Serum (FBS), 1% double antibody (penicillin and streptomycin, P/S) purchased from Biological InCell culture consumables were purchased from Corning, TNF- α from Novel Protein, other reagents not specifically identified, and Sigma-Aldrich.
Second, Experimental methods
1. Cell culture and drug delivery
ARPE-19 cells were human retinal pigment epithelial cells, the medium was DMEM/F12, and 10% FBS and 1% P/S were added thereto. The ARPE-19 cells frozen in a liquid nitrogen tank are revived in a water bath at 37 ℃ and placed in a culture dish of 60mm for culture. Changing the culture solution 1 time every 48h, observing the cell density, and when the cell density reaches 85%, blowing and subculturing to make the number of cells in each 60mm culture dish be 1X105After three passages, the cells were stable and were concentrated at 5 × 103One well, inoculated into a 96-well plate, each well of culture volume 100 u L, dose 3 hours before the replacement of new medium. Examples 1 to 3 the composition for both medicine and food was administered in two doses, high (10. mu.g/mL) and low (3. mu.g/mL), for 48 hours.
The hydrogen peroxide is diluted with culture medium to corresponding dilution times, and is used together with the medicine or is pre-administered and then injured.
MTT assay
ARPE-19 cells were cultured in the composition for both drug and food for 48h, and 10. mu.L of MTT (5mg/mL) was added. Culturing in incubator for 3 hr, removing culture solution with pipette, adding 150 μ L dimethyl sulfoxide into each well, shaking on shaking table at room temperature for 30min to dissolve crystal, and measuring absorbance (A) of each well at OD570nm of ELISA detector570) Cell survival was calculated for each group as (%) ═ a (experimental group)/a (control group) × 100% 100.
3. Statistical treatment
All experimental data were statistically processed using SPSS18.0 statistical processing software, and the results were obtained
Showing that the difference between groups was measured by t-test, P<A difference of 0.05 is statistically significant.
Third, experimental results
The cell survival rate of ARPE-19 cells cultured in vitro is detected by an MTT method after an oxidative damage model is constructed by using hydrogen peroxide and different proportions of medicinal and edible compositions are added for 24 h. As shown in figure 11, the medical and edible dual-purpose compositions of the examples can remarkably protect ARPE-19 cells from being damaged by hydrogen peroxide, wherein the protection effect of the example 3 is the most remarkable.
Example 6 pharmaceutical and edible compositions for promoting expression of neurotrophic factors in retinal Muller cells
First, experimental material and medicine
1. Cells
Miller cells (Muller) were purchased from ATCC and were maintained at the center of collaborative innovation in the industrialization process of Chinese medicinal resources in Jiangsu province.
2. Laboratory apparatus
Carbon dioxide incubator (Forma series II water jack CO2 incubator, Thermo Co.), 1300series A2 clean bench (Thermo Co.), Auto Vert A1 inverted fluorescence microscope (ZEISS Co.), Nanodrop (DS11spectrophotometer, DeNovix Co.), ABI7500 fluorescent real-time quantitative PCR instrument (Invitrogen Co.), high speed Centrifuge (Allegra X-12R Centricure and Microfuge 22 RCentfuge, Backman Co.), constant temperature water bath (Bluepard Co.), constant temperature oscillator (IS-RDV1 incubator, CRYSTAL Co.), cell culture vessel (CORNING Co.).
3. Reagent
TRIzol RNA extraction kit (15596-026, Invitrogen). RT-PCR reverse transcription kit (PrimeScript)TMRT reagent Kit with gDNA Eraser, RR047A, TAKARA); SYBR fluorescent quantitative PCR reagents (FastStart Universal SYBR Green Master (ROX),04913914001, Roche). Bradford kit (T9310A, TAKARA).
Second, Experimental methods
1. Cell culture and drug delivery
M ü ller cell is human retina Miller cell, culture medium is DMEM, 10% FBS and 1% P/S are added, M ü ller cell frozen in liquid nitrogen tank is revived in water bath at 37 deg.C, and cultured in 60mm culture dish, and cell density is observed after changing liquid 1 time every 48hAt 85%, the cells were blown down for passage, so that the number of cells per 60mm dish was 1 × 106Cell density per dish was seeded in culture dishes 60mm in diameter.
When the cells in the culture dish grow to have a confluency of 90%, the cells are administeredExamples 1 to 3The medicine and food dual-purpose composition has two doses of high dose (10 mu g/mL) and low dose (3 mu g/mL) and positive medicine cAMP (50 mu M), and is cultured for 24 h.
2. Real-time quantitative fluorescent PCR instrument for measuring transcription level expression of neurotrophic factor
RNA extraction: the cell culture medium was aspirated, washed with 2mL of PBS, and 500. mu.L of TRIzol was added to each plate of cells, according to the procedure set forth in the TRIzol RNA extraction kit. The extracted RNA was stored at-80 ℃.
Preparation of cDNA: RNA concentration and purity were measured by Nanodrop, and the reverse transcription was performed using 1. mu.g of RNA in total according to the instructions of TaKaRa reverse transcription kit.
And (3) according to the operation steps of the SYBR fluorescent real-time quantitative kit, determining the gene transcription level expression of the neurotrophic factor by using a fluorescent real-time quantitative PCR instrument. The primer sequences used were as follows:
TABLE 2 primer sequences
3. Statistical treatment
All experimental data were statistically processed using SPSS18.0 statistical processing software, and the results were obtained
Showing that the difference between groups was measured by t-test, P<A difference of 0.05 is statistically significant.
Third, experimental results
The retinal miller cells can provide neurotrophic factors and maintain the normal growth of retinal ganglion cells. Adding different proportions of the medicinal and edible dual-purpose composition into human retina muller cells cultured in vitro for 24h, and detecting the expression of the neurotrophic factor BDNF in the cells by utilizing a qPCR technology. As shown in fig. 12, the dual-purpose composition for medicine and food at different concentrations can significantly promote the expression of BDNF in human retinal miller cells, show better neurotrophic activity and present concentration dependence.
Animal model experimental results of the medicinal and edible composition provided by the invention show that: the medicine and food dual-purpose composition provided by the invention can effectively reduce the most main pathological link of age-related macular degeneration caused by high oxidative stress level, thereby protecting the retina of an animal from damage. Cell model experiment results show that the medicine and food dual-purpose composition provided by the invention can improve the capability of retinal pigment epithelial cells in resisting oxidative damage and increase the expression of neurotrophic factors in retinal Miller cells, thereby achieving the effect of protecting retinal damage.
The experimental results show that the medicinal and edible composition provided by the invention has a remarkable function of resisting retinal damage caused by oxidative stress, and the medicinal and edible composition provided by the invention has a good effect of improving age-related macular degeneration caused by high oxidative stress. The composition can improve the capability of retinal pigment epithelial cells in resisting oxidative damage, and can also increase the expression of neurotrophic factors in retinal Miller cells, thereby achieving the effect of protecting retinal damage and having the action characteristic of multiple target points. Particularly, all the medicinal ingredients and components of the medicinal and edible composition are medicinal and edible varieties, so the medicinal and edible composition has good safety, is suitable for long-term use, and can play a role in preventing and improving AMD.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (6)
1. A medicine and food dual purpose composition for improving age related macular degeneration is characterized by comprising the following raw materials in parts by weight: 1-5 parts of wolfberry fruit, 4-8 parts of hawthorn, 4-8 parts of astragalus membranaceus, 5-10 parts of oyster, 1-5 parts of dendrobium officinale, 1-5 parts of chrysanthemum, 1-5 parts of fructus alpiniae oxyphyllae, 0.01-0.1 part of grape seed procyanidine, 0.01-0.1 part of lutein and 0.01-0.1 part of taurine.
2. The composition for use as both medicine and food for improving age-related macular degeneration according to claim 1, which comprises the following raw materials in parts by weight: 2-4 parts of wolfberry fruit, 5-7 parts of hawthorn, 5-7 parts of astragalus membranaceus, 6-9 parts of oyster, 2-4 parts of dendrobium officinale, 2-4 parts of chrysanthemum, 2-4 parts of fructus alpiniae oxyphyllae, 0.03-0.08 part of grape seed procyanidine, 0.03-0.08 part of lutein and 0.03-0.08 part of taurine.
3. The composition for use as both medicine and food for improving age-related macular degeneration according to claim 1, which comprises the following raw materials in parts by weight: 3 parts of medlar, 6 parts of hawthorn, 6 parts of astragalus, 8 parts of oyster, 3 parts of dendrobium officinale, 3 parts of chrysanthemum, 3 parts of fructus alpiniae oxyphyllae, 0.05 part of grape seed procyanidine, 0.05 part of lutein and 0.05 part of taurine.
4. The preparation method of the medicine and food dual-purpose composition for improving asthenopia and age-related macular degeneration as claimed in any one of claims 1 to 3, characterized in that it comprises the following steps:
(1) taking the wolfberry fruit, the hawthorn fruit, the astragalus root, the oyster, the dendrobium officinale, the chrysanthemum and the fructus alpiniae oxyphyllae according to the weight parts, adding 5-15 times of water, performing reflux extraction for 1-2 hours, and filtering; collecting the filtrate;
(2) taking the dregs obtained in the step (1), adding 5-15 times of water, performing reflux extraction for 1-2 hours, and filtering; collecting the filtrate;
(3) and (3) combining the filtrates obtained in the steps (1) and (2), concentrating under reduced pressure, and adding grape seed procyanidin, lutein and taurine to obtain the composition.
5. Use of the dual-purpose composition as medicine and food for improving age-related macular degeneration according to any one of claims 1 to 3 in the preparation of food or health product or medicine for improving age-related macular degeneration.
6. The use of claim 5, wherein the extract of the composition, the food and the pharmaceutically acceptable carrier are formulated into oral liquid, soft candy, tablet, decoction, granule, soft extract or extract.
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| CN116139235B (en) * | 2021-11-19 | 2023-11-21 | 宁夏枸杞创新中心(有限公司) | Medicinal and edible composition with function of relieving asthenopia of teenagers and preparation method and application thereof |
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