CN115154506A - Composition for improving hypoxia stress state and preparation method and application thereof - Google Patents
Composition for improving hypoxia stress state and preparation method and application thereof Download PDFInfo
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- CN115154506A CN115154506A CN202110358289.4A CN202110358289A CN115154506A CN 115154506 A CN115154506 A CN 115154506A CN 202110358289 A CN202110358289 A CN 202110358289A CN 115154506 A CN115154506 A CN 115154506A
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Abstract
The invention provides a composition for improving an anaerobic stress state, and a preparation method and application thereof. The composition for improving the hypoxia stress state has obvious anti-hypoxia activity, can obviously inhibit the hypoxia apoptosis of brain microglia, obviously improve the survival time of a hypoxic mouse, relieve the brain tissue damage caused by hypoxia, reduce the expression of hypoxia induction factors in animal brain tissues and relieve the oxidative stress state of the brain caused by the hypoxia, and has obvious synergistic effect on the tissue damage caused by the hypoxia.
Description
Technical Field
The invention relates to a composition for improving an anoxic stress state, a preparation method and application thereof, belonging to the fields of food and medical health care.
Background
Hypoxia is a pathological process in which the metabolism, function and morphological structure of a tissue are abnormally changed due to insufficient oxygen supply or oxygen deficiency of the tissue. Hypoxia is the most common stress factor in special environments such as plateau, aviation, diving and the like. Research shows that the continuous and stable anoxic environment can enable the organism to establish anoxic adaptation, which is beneficial to the organism to maintain self balance and internal environment stability, but the over-strong or long-term anoxic stress can bring serious harm to the organism, and finally, important organs of the organism, such as heart, brain, and the like, can die due to insufficient energy supply.
Therefore, finding an effective intervention is crucial to prevent hypoxia-induced pulmonary and cardiovascular diseases. The plant extract has protective effect in preventing anoxia injury, such as folium Ginkgo extract can protect myocardial cell from anoxia injury by antioxidant, antiinflammatory and anti-apoptosis mechanisms; the rhodiola rosea extract improves the plateau pulmonary edema induced by hypoxic mice and lightens ischemic brain injury. Therefore, screening of extracts or monomeric compounds having anti-hypoxic activity from plants is an effective method for developing anti-hypoxic drugs and health products.
Spearmint is a plant of the genus Mentha (Herba Menthae Spicatae) of the family Labiatae, and is described in compendium of materia Medica as pungent, cool, nontoxic, and has effects of inducing perspiration, relieving fever, dispelling pathogenic wind, and relieving itching. When eaten raw or cooked for a long time, can cool kidney qi, eliminate pathogenic toxin, remove fatigue and relieve fatigue, so that the human body has fragrant and clean smell. It can be used for knee sore by decocting and washing. It can also be used for treating excessive phlegm and various kinds of common cold with headache. In addition, the juice can remove heart heat and diseases of mouth and teeth; mashing into juice, gargling tongue and removing astringent; using She Saibi, stopping epistaxis; it can also be used for treating bee sting and snake wound. The book Lelan Xiang fried fermented soya beans soup, warm wine, tea, raw food can be written in the food medicine cardioscope, which is more beneficial than dish. Mainly distributed in Hebei, jiangsu, zhejiang, guangdong, guangxi, sichuan, guizhou, yunnan and other places in China, and can be harvested all the year round, and the sources are rich. The spearmint extract has been reported to have antiviral, antibacterial, antitumor, anti-inflammatory, antioxidant, etc. effects. However, there is no report on the anti-hypoxia property of the spearmint extract.
The wild cherry berry is a deciduous shrub of Rosaceae, is native to wet forest and marsh in east of North America, is a precious tree species integrating edible, medicinal, garden and ecological values, and can be used for processing food and beverage such as fruit juice, fruit wine, jam, can, preserved fruit, etc. The Prunus serrulata Sieb fruit contains components beneficial to human body, such as VB, VE, and VC, and folic acid, quinic acid, polyphenolic acid, tannin, catechin, quercetin, rutin, hesperidin, resveratrol and anthocyanidin etc. The aronia serrulata fruit extract has been reported to have antioxidant, antibacterial, anti-inflammatory, anti-liver injury, anti-obesity, anti-hypertension and other effects. However, the current report of the cherokee rose fruit extract on the aspect of resisting anoxia is not found temporarily.
Therefore, there is a need for a new composition for improving hypoxic stress status, a preparation method thereof and an application thereof, so as to overcome the above problems.
Disclosure of Invention
The composition for improving the hypoxia stress state has obvious anti-hypoxia activity, can obviously inhibit the hypoxia apoptosis of brain microglia, obviously improves the survival time of a hypoxic mouse, relieves the brain tissue damage caused by hypoxia, reduces the expression of hypoxia induction factors in animal brain tissues and the oxidative stress state of the brain caused by hypoxia, and has obvious synergistic effect on the tissue damage caused by hypoxia.
The invention is realized by the following steps:
the invention provides a composition for improving an anaerobic stress state, which comprises a spearmint extract and a prunus maritima fruit extract.
Further, the mass ratio of the spearmint extract to the prunus mume fruit extract is (1-10): (0.5-5).
Further, the mass ratio of the spearmint extract to the prunus mume fruit extract is (2.5-7): (1-3.5).
The invention also provides a preparation method of the composition for improving the hypoxic stress state, which comprises the following steps:
the method comprises the following steps: pulverizing herba Menthae Rotundifoliae, adding ethanol, reflux extracting for 1-3 times, filtering, mixing filtrates, recovering ethanol, concentrating under reduced pressure to obtain extract, spray drying, pulverizing the extract, and sieving to obtain herba Menthae Rotundifoliae extract;
step two: pulverizing fructus Rosae Davuricae, adding water, extracting for 1-3 times, mixing extractive solutions, filtering, concentrating to obtain extract, vacuum drying, pulverizing, and sieving to obtain fructus Rosae Davuricae extract;
step three: mixing the spearmint extract in the step one and the aronia melanocarpa extract in the step two according to the mass ratio of (1-10): (0.5-5) mixing to obtain the composition for improving the anoxic stress state.
Further, in the second step, the vacuum drying treatment is carried out under the conditions that the temperature is kept between 40 and 50 ℃ and the pressure is kept between 0.06 and-0.08 MPa.
The invention also provides application of the composition for improving the hypoxic stress state, and the composition for improving the hypoxic stress state is applied to preparing oral preparations.
Furthermore, the oral preparation can be tablets, capsules, pills, granules, powder, extract or oral liquid.
Further, the composition for improving the hypoxic stress state is applied to preparation of anti-hypoxic drugs, health products or foods.
Furthermore, the anti-hypoxia drug, health product or food refers to a drug, health product or food for preventing or treating injury caused by acute and chronic hypoxia.
Furthermore, the anti-hypoxic drug, health product or food refers to a drug, health product or food for preventing or treating pulmonary and cardiovascular and cerebrovascular diseases caused by the decrease of blood oxygen concentration in vivo.
The invention has the following beneficial effects:
the invention provides a composition for improving an anoxic stress state, namely, the spearmint extract and the aronia melanocarpa fruit extract are compounded for use, so that the composition has obvious anti-anoxic activity, can obviously inhibit the anoxic apoptosis of brain microglia, obviously improve the survival time of an anoxic mouse, relieve brain tissue damage caused by the anoxic, reduce the expression of anoxic induction factors in animal brain tissues and relieve the oxidative stress state of the brain caused by the anoxic, and has obvious synergistic effect on the tissue damage caused by the anoxic.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without inventive exercise.
FIG. 1 is a BV2 cell viability map provided in an example of the present invention;
FIG. 2 is a graph of the survival time of mice induced by sodium nitrite for hypoxia provided by the present invention.
FIG. 3 is a schematic diagram of the expression of HIF-1 alpha (hypoxia inducible factor alpha), SOD (superoxide dismutase) and ROS (reactive oxygen species) in blood of an animal under a hypoxia model.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention provides a composition for improving an anaerobic stress state, which comprises spearmint extract and cherokee rose fruit extract.
Further, the mass ratio of the spearmint extract to the prunus mume fruit extract is (1-10): (0.5-5).
In the preferred embodiment, the mass ratio of the spearmint extract to the prunus mume fruit extract is (2.5-7): (1-3.5).
The invention also provides application of the composition for improving the hypoxic stress state, and the composition for improving the hypoxic stress state is applied to preparing oral preparations. The oral preparation is in the form of tablets, capsules, pills, granules, powder, extract or oral liquid or other dosage forms suitable for oral administration. The oral preparation of the composition can be prepared by adopting a conventional method in the prior pharmaceutical field, and can be added with various pharmaceutically acceptable auxiliary materials including diluents, excipients, fillers, binders, wetting agents, disintegrating agents and the like which are conventional in the pharmaceutical field when necessary.
Further, the composition for improving the hypoxic stress state is applied to preparation of anti-hypoxic drugs, health products or foods. The anti-hypoxia medicine, health-care product or food is a medicine, health-care product or food for preventing or treating injury caused by acute and chronic hypoxia. In addition, the anti-hypoxic drugs, health care products or foods can also be drugs, health care products or foods for preventing or treating pulmonary and cardiovascular and cerebrovascular diseases caused by the decrease of blood oxygen concentration in vivo.
The invention also provides a preparation method of the composition for improving the hypoxia stress state, which comprises the following steps:
the method comprises the following steps: pulverizing herba Menthae Rotundifoliae, adding 3-8 times of 70% ethanol, reflux extracting for 1-3 times, filtering, mixing filtrates, recovering ethanol, concentrating under reduced pressure to obtain extract, spray drying, pulverizing the dry extract, and sieving with 80 mesh sieve to obtain herba Menthae Rotundifoliae extract.
Step two: pulverizing fructus Rosae Davuricae, adding 2-8 times of water, extracting for 1-3 times, the first time for 0.5-2 hr and the second time for 0.5-2 hr, mixing extractive solutions, filtering, concentrating to obtain extract, vacuum drying, pulverizing, and sieving with 80 mesh sieve to obtain fructus Rosae Davuricae extract; wherein the vacuum drying treatment is carried out under the conditions that the temperature is kept between 40 and 50 ℃ and the pressure is kept between 0.06 and-0.08 MPa.
Step three: mixing the spearmint extract in the step one and the aronia melanocarpa extract in the step two according to the mass ratio of (1-10): (0.5-5) mixing to obtain the composition for improving the anoxic stress state.
The spearmint extract and the aronia melanocarpa fruit extract in the traditional Chinese medicine composition can be respectively extracted and processed by proper solvents and methods to obtain extracts, and pharmaceutically acceptable auxiliary materials are added to prepare pharmaceutically acceptable oral preparations. The independent extraction refers to that the extracts of the spearmint and the wild cherry fruits are respectively and independently extracted by different processes.
In the above extraction preparation method of spearmint extract and wild cherry fruit extract, the solvent is pharmaceutically common solvent, such as water or ethanol; the method refers to the general method for extracting Chinese medicinal materials, and can be decoction, reflux extraction, soaking, percolation or continuous extraction. For example, spearmint can be prepared by ethanol reflux extraction or water extraction and alcohol precipitation, and wild cherry berry can be prepared by ethanol reflux extraction to obtain wild cherry berry extract. The invention provides a preferable extraction preparation process of spearmint extract and prunus laevigata fruit extract, which is illustrated by the following specific examples:
the preferable extraction and preparation process of the spearmint extract is as follows: pulverizing the spearmint in parts by weight, adding 6 times of 70% ethanol, reflux-extracting for 3 times, filtering, mixing filtrates, recovering ethanol, concentrating the extract under reduced pressure, spray-drying, pulverizing the dry extract, and sieving with a 80-mesh sieve to obtain the spearmint extract.
The preferred extraction preparation process of the aronia melanocarpa fruit extract is as follows: taking the dried aronia melanocarpa fruit in parts by weight, crushing, adding 8 times of water, extracting for 2 times, 2 hours for the first time and 1.5 hours for the second time, combining extracting solutions, filtering, concentrating to obtain an extract, drying in vacuum, crushing, and sieving with a 80-mesh sieve to obtain the aronia melanocarpa fruit extract. Wherein the low-temperature vacuum drying is carried out at 40-50 ℃ and 0.06-0.08 Mpa.
Mixing the spearmint extract and the aronia melanocarpa fruit extract according to the mass ratio of (1-10): (0.5-5) mixing to obtain the composition for improving the anoxic stress state.
In the case of the example 1, the following examples are given,
in vitro studies of the anti-hypoxic activity of compositions that improve hypoxic stress status:
the experiment adopts MTT method to determine the cell activity inhibition effect of the medicine on the prostate epithelial cells and the stroma cells with abnormal hyperplasia, and uses in vitro experiment to preliminarily verify the antiproliferative activity of the medicine on the prostate hyperplasia cells.
Experimental materials and methods:
(1) Experimental materials: mouse microglia BV2 cells were used as experimental cells.
(2) Grouping experiments: the cell experiment is divided into a blank control group and a medicine group, wherein the medicine group comprises a spearmint extract group (L), a cherokee rose fruit extract group (Y) and a composition group (L/Y), the dosage concentration of each group is respectively 50 mug/ml, 100 mug/ml and 40 mug/ml, and the mass ratio of the spearmint extract group to the cherokee rose fruit extract group in the composition group is 5:3.
BV2 cells were cultured in DMEM medium supplemented with 10% fetal bovine serum and 10 μ l/ml streptomycin/penicillin; culture was maintained at 37 ℃ and 5% CO 2 The culture medium is replaced every 2 to 3 days in the incubator; then the BV2 cell is inoculated on a 96-well plate, after 24 hours of culture, the medicines are added into the 96-well plate individually and in combination according to the medicine concentration of different groups, and the final concentration is 1mMCoCl 2 Cells were treated for 24h.
(3) And (3) detecting the cell viability: cell viability was assessed by MTT colorimetric assay, cell viability after treatment was measured by tetrazolium salt colorimetric assay, and the ability of cells to reduce 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) by mitochondrial succinate dehydrogenase was assessed. MTT enters cells and mitochondria where it is reduced to the colored insoluble product Formazan (Formazan). To make the color visible, the colored formazan particles are dissolved by the addition of DMSO. The MTT lysis reaction needs to be performed in intact cells and is proportional to the degree of metabolic activity of the cells.
At 37 ℃ C, 5% CO 2 After incubating the cells with 100. Mu.l MTT solution (500. Mu.g/ml) in culture medium for 4h under conditions, the supernatant was removed and 150. Mu.l DMSO was added. Then, the optical density (o.d.) was measured by a microplate reader at a wavelength of 570 nm.
Cell viability was expressed as a percentage of optical density relative to untreated blank, and three replicates were performed for each sample.
The experimental results are as follows: the results of the BV2 cell proliferation assay of the different treatment groups are shown in fig. 1, fig. 1 is a BV2 cell activity graph showing the percentage of cell viability of BV2 cells obtained after 24h administration of the spearmint extract group (L), the prunus mume fruit extract group (Y) and the composition group (L/Y) in the presence of 1mm of cocl2 to compare the inhibitory effect of the drug on the hypoxic apoptosis of microglia;
the data obtained from the MTT assay performed show that when the spearmint extract group (L), the prunus mume fruit extract group (Y) were used alone, they both increased the cell viability of BV2 cells and reduced the hypoxic apoptosis of cells due to CoCl 2. The cell viability is obviously increased by treating the same cell line by using the composition group (L/Y), the cell inhibition rate of the drug combination group is 20 percent higher than that of the drug combination group alone, and the drug combination group has obvious synergistic antiproliferative effect.
Judgment standard of drug synergy: the synergistic effect of the three drugs is judged by using a multi-drug effect equation. The multi-drug effect equation is:
wherein (Dx) 1 is the concentration at which the cell survival rate is X% when the drug D1 is administered alone, and (DX) 2 is the same. The drug combination (D) 1 and (D) 2 are concentrations when the cell survival rate is X% when the drugs are used in combination; CI <1, =1, >1 correspond to synergy, addition and antagonism, respectively.
As can be seen from fig. 1, the cell proliferation rates of DX1 and DX2 are about 50%, DX1=50 μ g/ml, and DX2=100 μ g/ml, and if the proliferation rate of the composition is 70%, DX1=70 μ g/ml, DX2=140 μ g/ml, D1=25, and D2=15, CI <1, and the three drugs are synergistically effective in combination.
In the case of the example 2, the following examples are given,
in vivo evaluation of anti-hypoxic activity of composition for improving hypoxic stress status:
(1) Experimental materials: c57 mice were used as experimental animals.
(2) And (3) checking and grouping: the animal experiment is divided into a blank group, a model group and a medicine group, wherein the medicine group comprises a spearmint extract group (L), a aronia serrulata fruit extract group (Y) and a composition group (L/Y), the dosage concentrations are respectively 50mg/kg, 40mg/kg and 30mg/kg, and the mass ratio of the spearmint extract to the aronia serrulata fruit extract in the composition group is 4:1.
(3) The experimental scheme is as follows: the dosing period was 14 days during which the mouse body weight was recorded daily and the dosing schedule was: the mice are dosed according to the gavage proportion of 10g/0.2ml, the blank group and the model group are filled with the gavage physiological saline, and the drug group is filled with the corresponding drug. After the completion of the gavage, naNO2 was prepared into a 10mg/mL solution with physiological saline and injected intraperitoneally (0.2 mL/10 g) into the mice (final dose of 200 mg/kg). The time from injection to mouse death was recorded. Immediately after the death of the mice, blood of the mice is taken to detect the contents of HIF-1 alpha, SOD and ROS.
(4) The experimental results are as follows: as shown in fig. 2, fig. 2 is a graph of survival time of mice induced by hypoxia with sodium nitrite, showing survival time of mice induced by hypoxia death with acute sodium nitrite 14 days after administration of the group of spearmint extract (L), the group of prunus cerasifera fruit extract (Y) and the group of composition (L/Y), to compare the inhibitory effect of the drug on the animal model of acute hypoxia; when the spearmint extract group (L) and the prunus mume fruit extract group (Y) are independently administered, the death time of hypoxic mice can be prolonged, and the effect of the spearmint extract is better than that of the prunus mume fruit extract. After the composition group (L/Y) is administrated, the death time of the hypoxic mice is also remarkably improved, and compared with a single medicine group, the death time is prolonged by nearly 30%. Has obvious synergistic anti-anoxia effect.
As shown in fig. 3, the expression levels of HIF-1 α (hypoxia inducible factor α), SOD (superoxide dismutase), and ROS (reactive oxygen species) in the blood of mice were measured, and the expression levels of HIF-1 α were reduced in the spearmint extract group (L), the prunus mume fructus extract group (Y), and the composition (L/Y), indicating that all of them had a certain anti-hypoxia activity, wherein the composition group (L/Y) had a greater degree of down-regulation of HIF-1 α than the individual administration of the spearmint extract group (L) and the prunus mume fructus extract group. SOD and ROS are classical indexes which can accurately reflect the oxidative stress state in vivo, the more serious the oxidative stress state is, the expression of SOD can be reduced, and the expression of ROS can be increased. Compared with the group (L/Y) which is independently administrated with the spearmint extract and the aronia melanocarpa extract, the composition group (L/Y) obviously increases the expression of SOD and simultaneously reduces the ROS level in vivo, and the data reflect that the composition group (L/Y) has obvious synergistic anti-hypoxia effect.
In the case of the example 3, the following examples are given,
preparing the composition for improving the hypoxia stress state into microcapsules for resisting hypoxia:
1kg of spearmint extract, 0.4kg of aronia serrulata extract, 2kg of lecithin, 0.5kg of emulsifier (mixture of monoglyceride, diglyceride fatty acid ester by weight 1:1), 10kg of oily component (mixture of linseed oil, pumpkin seed oil and coconut oil by weight 1.
The preparation method comprises the following steps:
(1) Mixing lecithin and oily components uniformly to obtain an oil phase, adding the spearmint extract into the oil phase, heating at 80 ℃, and uniformly stirring for 9 hours to uniformly disperse the spearmint extract to form spearmint extract oil phase dispersion liquid;
(2) 1:4 water and emulsifier, adding fructus Rosae Davuricae extract, heating to 70 deg.C, homogenizing and stirring for 1h to form water dispersible emulsion;
(3) Adding the water dispersion emulsion into oil phase dispersion of spearmint extract, heating to 70 deg.C, homogenizing and stirring for 1h;
(4) Adding the rest raw materials, mixing, spray drying with spray tower at inlet air temperature of 120 deg.C, tower internal temperature of 60 deg.C and outlet air temperature of 65 deg.C, cooling with fluidized bed, and cooling to 30 deg.C to obtain brown powder, and obtain microcapsule for resisting anoxia.
In conclusion, the composition for improving the hypoxia stress state, provided by the invention, is prepared by compounding the spearmint extract and the aronia melanocarpa extract, has obvious anti-hypoxia activity, can obviously inhibit the hypoxia apoptosis of brain microglia, obviously improve the survival time of a hypoxic mouse, relieve the brain tissue damage caused by hypoxia, reduce the expression of hypoxia-induced factors in animal brain tissues and relieve the oxidative stress state of the brain caused by hypoxia, and has obvious synergistic effect on the tissue damage caused by hypoxia.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (10)
1. A composition for ameliorating a hypoxic stress state, comprising: the composition for improving the hypoxic stress state comprises spearmint extract and cherokee rose fruit extract.
2. The composition for ameliorating a hypoxic stress state according to claim 1, wherein: the mass ratio of the spearmint extract to the wild cherry fruit extract is (1-10): (0.5-5).
3. The composition for improving hypoxic stress status as claimed in claim 2, wherein: the mass ratio of the spearmint extract to the aronia melanocarpa fruit extract is (2.5-7): (1-3.5).
4. A method for preparing a composition for improving a hypoxic stress state, comprising the steps of:
the method comprises the following steps: pulverizing herba Menthae Rotundifoliae, adding ethanol, reflux extracting for 1-3 times, filtering, mixing filtrates, recovering ethanol, concentrating under reduced pressure to obtain extract, spray drying, pulverizing the extract, and sieving to obtain herba Menthae Rotundifoliae extract;
step two: pulverizing fructus Rosae Davuricae, adding water, extracting for 1-3 times, mixing extractive solutions, filtering, concentrating to obtain extract, vacuum drying, pulverizing, and sieving to obtain fructus Rosae Davuricae extract;
step three: mixing the spearmint extract in the step one and the wild cherry fruit extract in the step two according to the mass ratio of (1-10): (0.5-5) mixing to obtain the composition for improving the anoxic stress state.
5. The method for preparing the composition for improving hypoxic stress state according to claim 4, wherein: in the second step, the vacuum drying treatment is carried out under the conditions that the temperature is kept between 40 and 50 ℃ and the pressure is kept between 0.06 and-0.08 MPa.
6. Use of a composition for improving hypoxic stress status, characterized in that: the composition for improving the hypoxic stress state is applied to preparing oral preparations.
7. Use of a composition for ameliorating a hypoxic stress state according to claim 6, wherein: the oral preparation can be tablets, capsules, pills, granules, powder, extract or oral liquid.
8. Use of a composition for ameliorating a hypoxic stress state according to claim 6 or 7, wherein: the composition for improving the hypoxic stress state is applied to preparing anti-hypoxic drugs, health care products or foods.
9. Use of a composition for ameliorating a hypoxic stress status according to claim 8, wherein: the anti-hypoxia medicine, health-care product or food is a medicine, health-care product or food for preventing or treating injury caused by acute and chronic hypoxia.
10. Use of a composition for ameliorating a hypoxic stress state according to claim 8, wherein: the anti-hypoxic drug, health product or food refers to a drug, health product or food for preventing or treating lung and cardiovascular and cerebrovascular diseases caused by blood oxygen concentration reduction in vivo.
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| CN107404927A (en) * | 2015-01-02 | 2017-11-28 | 麦莱琉卡有限公司 | Multiple complementary goods composition |
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