CN106798734A - A kind of soft capsule containing Co-Q10 and its preparation and application - Google Patents

A kind of soft capsule containing Co-Q10 and its preparation and application Download PDF

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CN106798734A
CN106798734A CN201510830305.XA CN201510830305A CN106798734A CN 106798734 A CN106798734 A CN 106798734A CN 201510830305 A CN201510830305 A CN 201510830305A CN 106798734 A CN106798734 A CN 106798734A
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ubiquinone
weight portions
capsule
beeswax
vitamin
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邓金明
吴海英
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AISHENG PHARMACEUTICAL Co Ltd ZHEJIANG
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AISHENG PHARMACEUTICAL Co Ltd ZHEJIANG
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00Medicinal preparations characterised by special physical form
    • A61K9/48Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/4841Filling excipients; Inactive ingredients
    • A61K9/4858Organic compounds
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/12Ketones
    • A61K31/122Ketones having the oxygen directly attached to a ring, e.g. quinones, vitamin K1, anthralin
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18Magnoliophyta (angiosperms)
    • A61K36/88Liliopsida (monocotyledons)
    • A61K36/899Poaceae or Gramineae (Grass family), e.g. bamboo, corn or sugar cane
    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23VINDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00Food compositions, function of food ingredients or processes for food or foodstuffs

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Abstract

The invention discloses a kind of ubiquinone for alleviating physical fatigue, anti-aging10Soft capsule and its preparation method and application.The ubiquinone10Capsule-core composition and the weight portion composition of soft capsule are as follows:Ubiquinone1020 50 weight portions, the weight portion of vitamin E 15 60, the weight portion of corn oil 200 500, the weight portion present invention application ubiquinone of beeswax 5 2010Physiological function effect, be aided with vitamin E, and screen the solvent with synergy, compacting pelletization, improves the stability of product, and the dissolution rate of finished product is good, be conducive to absorption of human body, the effect with good fatigue-relieving, anti-aging has preferable application prospect.

Description

A kind of soft capsule containing Co-Q10 and its preparation and application
Technical field
The present invention relates to a kind of soft capsule health product and its preparation method and application, and in particular to Yi Zhonghan There is ubiquinone10Soft capsule and its preparation and application, belong to health product technical field.
Background technology
Immune refers to that body immune system recognizes itself and dissident's material, and is excluded by immune response anti- Originality foreign matter, with the function of maintaining organism physiology to balance.The immunity of human body has and is divided into congenital immunity With acquired immunity, both acquisition pattern differences.When a variety of reasons cause the immune of body System is lacked of proper care, and the protective effect of immune system is affected, so as to result in the generation of disease.Exempt from What epidemic disease power lowly often showed is exactly liable to illness, One's spirits are drooping, easy fatigue, appetite reduction etc. Symptom.
Fatigue is a kind of physiological phenomenon, is a kind of protective mechanism for people.This is body to me Send the signal that rest, if no matter ignored, body will suffer damage, and finally be overworked for a very long period into Disease.Fatigue generally can be divided into physical fatigue, mental fatigue, mental fatigue and pathology fatigue.Fatigue can Cause big energy to be consumed, kinotoxin is produced in vivo.Kinotoxin stimulates nervous centralis, produces Whole body fatigue is reacted.Because energy is not enough, cell viability declines, and each systemic-function of body goes down, Kinotoxin is built up in vivo, and vivo environment deteriorates, cytotoxic, excitement decay.These factors, It is highly susceptible to induce passive death.
Research finds that, when the hypoimmunity of body, internal free radical, simple gland peptide oxygen will be produced Raw peroxidation.
Ubiquinone in human body10Total content be only 500-1500mg and reduced with older.People's Ubiquinone in organ10Content peaked at 20 years old, it is then rapid to reduce.The coenzyme in heart Q10The reduction of concentration is particularly evident.Ubiquinone in the young Autopsy Cases of the old man than 20 years old of 77 years old10 Reduce 57%.
Ubiquinone10Important physiological function is played in human body as a kind of coenzyme of vitamins.It Participate in the generation process of Mitochondria and ATP, regulating cell redox environment.It is auxiliary Enzyme Q10With metabolic cardiac stimulant function, there is strong effect of scavenging radical, the antioxidase in human body In the presence of, suppress damage of the free radical to biomembrane, it is natural antioxidant and radicals scavenging Agent.Ubiquinone10It is the pith of immune link.When internal ubiquinone10Level raise when, exempt from Epidemic disease system can more be helped from the use of antibiotic and antiviral drugs.But antibiotic Can not help the reconstruction of immune system, and ubiquinone10Can stimulating immune system naturally so that To larger range of benefit and resisting effect.Meanwhile, ubiquinone10Or vitamin E synergist.It is auxiliary Enzyme Q10The work of LDL oxidation is being prevented with redder than vitamin E, carrotene and tomato Element is stronger.It is different in vivo although the main matter for preventing lipid oxidation in vivo is vitamin E Under conditions of, vitamin E can accelerate the oxidation of lipid in turn.This phenomenon can be prevented is exactly auxiliary Enzyme Q10.Ubiquinone10Make vitamin E vigor palingenesis with vitamin c class seemingly, be unique one Plant naturally occurring, the fat-soluble antioxidant that can be regenerated (change into activity form) in human body.Mesh Before, in the market ubiquinone10It is widely used to Medicines and Health Product and cosmetics.
Ubiquinone at room temperature10It is yellow to orange-yellow crystalline powder, fusing point is relatively low (49-52 DEG C), nothing It is smelly tasteless, it is atomic to be dissolved in ethanol in the organic solvent such as dissolvable chloroform, ether or petroleum ether, Water insoluble and methyl alcohol.Ubiquinone10Bioavilability it is very poor, be unsuitable for absorption of human body;Ubiquinone10 To light sensitive, meet light and resolve into red material, temperature and humidity influences smaller to it.
Soft capsule mean by a certain amount of medicine, medicinal substances extract add suitable auxiliary material be sealed in it is spherical, The formulation being made in the soft capsule material of oval or other shapes.
The content of the invention
The purpose of the present invention is exactly the health care needs for meeting current sub-health population, and is tackled above-mentioned existing The technical problem of presence provides a kind of strengthen immunity, the ubiquinone of fatigue-relieving function10Soft capsule and its Preparation method.
The purpose of the present invention can be achieved through the following technical solutions:
A kind of ubiquinone10Soft capsule, including utricule and the capsule-core enclosed in utricule, described capsule-core by with The raw material composition of lower component and weight portion:Ubiquinone1020-50 weight portions, vitamin E 15-60 weight portions, Corn oil 200-500 weight portions, beeswax 5-20 weight portions.
Further, described capsule-core is made up of the raw material of following components and weight portion:Ubiquinone1030-40 weights Amount part, vitamin E 25-55 weight portions, corn oil 300-450 weight portions, beeswax 5.5-18 weight portions.
Further, described capsule-core is made up of the raw material of following components and weight portion:Ubiquinone1035-40 Weight portion, vitamin E 45-50 weight portions, corn oil 380-400 weight portions, beeswax 10-15 weight portions.
Further, described capsule-core is made up of the raw material of following components and weight portion:Ubiquinone1040 weights Amount part, the weight portion of vitamin E 50, the weight portion of corn oil 395, the weight portion of beeswax 15.
Ubiquinone of the present invention10Soft capsule is prepared as follows:
(1) preparation of capsule-core feed liquid:Beeswax, corn oil, heating fusing beeswax are taken by formula rate;By matching somebody with somebody Square ratio takes vitamin E, ubiquinone10, mix with beeswax, corn oil, obtain capsule-core feed liquid;
(2) preparation of capsule hide glue gelatin:Purified water is heated, by gelatin, glycerine, titanium dioxide, burnt sugar coloring, Monascus color is added in purified water, heating, and stirring obtains capsule hide glue gelatin;
(3) soft capsule is prepared:The capsule hide glue gelatin that capsule-core feed liquid obtained in step (1) and step (2) are prepared Mix, by pelleting, sizing, dry, prepare coenzyme Q 10 soft capsule.
Further, the weight portion of gelatin, glycerine and pure water used in step (2) is 1~5:0.5~2:2~8.
Further, the weight portion of selected gelatin, glycerine and pure water is 3:1:4.
The present invention provides a kind of composition and is preparing the health care with strengthen immunity, fatigue-relieving effect Application in product, the raw material of the composition is by as follows into being grouped into:
Ubiquinone1020-50 weight portions, vitamin E 15-60 weight portions, corn oil 200-500 weight portions, Beeswax 5-20 weight portions.
Further, it is made up of the raw material of following components and weight portion:Ubiquinone1030-40 weight portions, dimension life Plain E 25-55 weight portions, corn oil 300-450 weight portions, beeswax 5.5-18 weight portions.
Further, it is made up of the raw material of following components and weight portion:Ubiquinone1035-40 weight portions, dimension Raw element E 45-50 weight portions, corn oil 380-400 weight portions, beeswax 10-15 weight portions.
Further, it is made up of the raw material of following components and weight portion:Ubiquinone1040 weight portions, dimension life The weight portions of plain E 50, the weight portion of corn oil 395, the weight portion of beeswax 15.
The present invention provides a kind of composition in preparing with the health products for dispelling facial pigmentation effect Application, the raw material of the composition is by as follows into being grouped into:
Ubiquinone1020-50 weight portions, vitamin E 15-60 weight portions, corn oil 200-500 weight portions, Beeswax 5-20 weight portions.
Further, it is made up of the raw material of following components and weight portion:Ubiquinone1030-40 weight portions, dimension life Plain E 25-55 weight portions, corn oil 300-450 weight portions, beeswax 5.5-18 weight portions.
Further, it is made up of the raw material of following components and weight portion:Ubiquinone1035-40 weight portions, dimension Raw element E 45-50 weight portions, corn oil 380-400 weight portions, beeswax 10-15 weight portions.
Further, it is made up of the raw material of following components and weight portion:Ubiquinone1040 weight portions, dimension life The weight portions of plain E 50, the weight portion of corn oil 395, the weight portion of beeswax 15.
Primary raw material ubiquinone in inventive formulation10(Coenzyme Q10) also known as Ubidecarenone, are a kind of Quinone cyclics, its structure is similar to vitamin K, the ubiquinone side chain iso-amylene unit of separate sources Number it is different, in the ubiquinone of the mankind and other mammals, isopentene group unit numbers are for 10 Gain the name, have presence in the heart, liver, kidney, the pancreas in human body, the function of determination is compared at present there are two Aspect, one is played an important role during nutriment is converted into energy in mitochondria, is thin The activator that born of the same parents breathe and are metabolized;Two is have obvious lipoid peroxidization resistant, is important anti-oxidant Agent;In recent years, scholar observes, ubiquinone10For the disease of malignant tumour, AIDS and ulcers Shape has some improvement, and then proposes ubiquinone10The also effect with strengthen immunity, in machine The non-specificity of body has the effect of Immune-enhancing effect.
Ubiquinone10Chemical name CoQ10, molecular formula C59H90O4, molecular weight 862, structure belongs to quinone cyclics similar to vitamin K.At room temperature In orange-yellow crystal, odorless, tasteless.Its fusing point is 49 DEG C, is influenceed smaller by temperature and humidity, but illumination Under the conditions of it is unstable, be easily decomposed.Ubiquinone10Benzoquinones has hydroxyl substituent and makes in the quinone ring of structure It tends to polarity, and polyisoprene side chain then makes it in hydrophobic environment with relatively low free energy, This characteristic can spread and be enriched with rapidly in mitochondrial inner membrane.In addition, its quinone ring has typical case Reversible oxidation reduction characteristic.Exist in quinone form under oxidative conditions, under anaerobic, can be also Originally it was hydroquinones, the form with oxidized form, reduced form and free radical semiquinone in cell.More than being based on Reason, ubiquinone10After receiving hydrogen from Mitochondria complex, hydrogen is on the one hand directly transmitted to free radical, Reacted under antioxidase effect, suppress destruction of the free radical to cell membrane, on the other hand by proton In release to mitochondrial matrix, electronics then passes to cytochromes, promotes oxidative phosphorylation and electronics actively Transfer, is consequently formed the main matter ATP of organism ability storage, and carry by reducing AMP losses ATP levels high, reduce calcium ion and are lost in, and maintain calcium channel integrality and stabilizing cell membrane.In reality In the application of border, ubiquinone10It is considered as a kind of natural good antioxidant and free radical scavenger, hair Wave the effect of the multiple efficacies such as alleviation physical fatigue, enhance immunity.Therefore ubiquinone10It is a kind of immune tune Section agent, plays the healthcare function of strengthen immunity.Additionally, ubiquinone10Molecule is big, poorly water-soluble, Gastrointestinal absorption is slow, and the time that valid density is reached after oral administration is more long, about needs (put down within 5~10 hours Equal 6.5 hours), therefore the present invention is from the formulation of soft capsule.
Vitamin E is a kind of liposoluble vitamin being widely present in natural animal-plant, also known as tocopherol, To sour and thermally-stabilised, in the presence of having oxygen, it is heated to 200 DEG C and remains to keep stabilization.Vitamin E has There are different physiological roles, be also that one of vitamin needed by human is one of topmost antioxidant.Dimension life Plain E is positioned on cell membrane, belongs to fat-soluble chain rupture type antioxidant, and it provides hydrogen to fat oxygen radical Ion, makes lipid peroxidation chain interruption, the structure together with superoxide dismutase, glutathione peroxidase Antioxidant system in adult, protects cell membrane and intracellular nucleic acid from the attack of free radical.Additionally, Vitamin E can also melt go out singlet oxygen, improve the oxidation resistance of solvent naphtha.In formula of the invention, Addition vitamin E can effectively protect ubiquinone as auxiliary material10Oxidative inactivation.
Vitamin E is formed by Hydrolysis kinetics in natural plants, compared to synthesising complex E security more Height, vitamin E absorbs high 3.5 times than composite, and half-life period is longer, and physiologically active is approximately composite 3-8 times.Thus the present invention selects vitamin E as the major auxiliary burden of addition.
Ubiquinone10Belong to liposoluble substance, according to the similar principle for mixing, select oily substance as flexible glue Capsule matrix.Corn oil be it is a kind of by maize extract high quality food oil, and its natural anti-oxidation and Stability is fairly good, high temperature resistant frying.Corn oil is rich in unrighted acid, and its Linoleic acid accounts for grease More than the 50% of total amount, rich in vitamin E, phytosterol, phosphatide etc., is in the world referred to as corn oil Nutrition oil, is usually used in medicine and health food production.Linoleic acid contained by corn oil is human body Essential fatty acid, it is not only the necessary constituent of cell membrane, is also the base of body synthesis of prostaglandins Plinth material, it can be combined into ester, promote the transhipment of cholesterol, and promote cholesterol to be changed into cholesterol Cholic acid is excreted, and has good effect for reducing fat, moreover it is possible to is suppressed blood platelet aggegation on vascular wall, is prevented Thrombosis, plays antiatherosclerosis, the effect of pre- angiocardiopathy.The sterol of corn oil plant Content is that other edible oils can not compare, and sterol has the work for directly, effectively, quickly reducing cholesterol With.Modern medicine field of food has developed corn oil for the preventing and treating of cardiovascular and cerebrovascular disease.Meanwhile, it is modern Research is tested mouse, and researcher has found long-term maize of the intake rich in polyunsaturated fatty acid Bud oil, may change the species and level of aliphatic acid in mouse immune cell membrane phospholipid, so as to improve The immunity of mouse, points out the effect with enhancing mouse immunity.The present invention is flexible glue from corn oil The matrix of capsule, can both solve ubiquinone10Indissoluble sex chromosome mosaicism, the Multiple components that wherein corn oil contains With auxiliary lipid-lowering function, corn oil is big compared to the proportioning consumption that other are invented in invention, and it sets out It is corn oil consumption and security reliability in itself to select, and corn oil has good health-care efficacy in itself, Therefore the present invention improves the proportioning consumption of corn oil, it is intended to strengthen ubiquinone10Anti-oxidant and regulation reducing blood lipid Function, the two is mutually applied more is beneficial to immunizing health effect of product.Found in experimentation It is simple to utilize corn oil to ubiquinone10Dissolved not thorough, easily before the encapsulated of soft capsule, encapsulated During and encapsulated after corn oil in produce solids sedimentation, and production and old storage during Sedimentation can slowly be layered, be helped to ensure that suspending liquid has good stability, therefore need to add wherein Suspension, suspending agent can be played to be prevented from settling, is layered, it is ensured that the suspending of product, it is ensured that the standard of loading amount Really.Conventional oiliness suspending agent has ceride class, lecithin, aluminum monostearate etc. at present, it is contemplated that raw material It is economic, practical with it is safe, it is most suitable using beeswax.Beeswax is the wax of honeybee secretion, and honeycomb is through excluding Or obtained a kind of organic mixture after centrifugation removal honey, beeswax can be by influenceing system viscosity and rheology Property is learned, every stability indicator is changed.Concentration of beeswax is higher, and plastic yield is bigger, starts The time for settling gets over extension, and sinking speed is smaller, meets stokes formula.Further, since beeswax Main component be palmitic acid beeswax alcohol ester, simultaneously containing a small amount of free polyols, thus with certain Emulsibility, reduce surface tension, this also causes that the sinking speed of suspending liquid slows down because being obstructed.Beeswax Safety, with low cost, the present invention uses the beeswax of certain concentration to assign product good stream as suspending agent Dynamic property, contributes to the loading amount of soft capsule capsule-core active ingredient and the stabilization of finished dosage forms.
At present using auxiliary material and coenzyme such as dried starch, calcium monohydrogen phosphate, magnesium stearates more than domestic production technology Q10Raw material powder is simply mixed to be dispensed again.Because Co-Q10 easily receives extraneous factor, such as light, oxidation The influence of agent etc., causes content to decline (the maximum range of decrease is more than 20%).It is different from common hard capsule, Formulation of the invention is soft capsule.Oral soft capsule agent and other conventional dosage forms for example tablet, hard capsule, Granule and solution etc. are compared, with bioavilability it is high, medicine stability is good, content is accurate The advantages of.Soft capsule is also referred to as capsule and pill, is by oils or to solution of the gelatin without dissolution, mixed Suspension, even solid and semisolid etc. are closed in a kind of preparation in soft gel coat, are used for some property Matter it is unstable (such as photaesthesia, it is oxidizable or volatilization, meet it is damp and hot unstable) water-insoluble or water-soluble pesticide Thing and have the medicine of poor taste, smell, increase stability can be played, improve bioavilability and The effect of taste masking.Many experiments at present have been compared and drawn, in solid pharmaceutical preparation oral application, soft capsule gram The shortcomings of having taken the easy moisture absorption of hard shell capsules formulation, stability difference, while the bioavilability of preparation can be improved. The present invention is with ubiquinone10Compounding is carried out with the specific ratio such as corn oil, vitamin E, suspending agent to be obtained, The accelerated experiment of product and long-time stability are investigated, steady quality.Product is better than traditional solid dosage forms system Agent, the bioavilability of product is improve with soft capsule, it is ensured that product quality as formulation.
Compared with prior art, the present invention is with ubiquinone10Be main both effectiveness composition, be aided with vitamin E, Corn oil and beeswax are made soft capsule preparation, the healthcare function with strengthen immunity, are mainly used in enhancing The gentle physical fatigue of immunity, anti-oxidant, auxiliary adjustment of blood fat.The product quality that the present invention is provided is steady It is fixed, convenient to take, meet requirement of the State Food and Drug Administration to health food, safely and effectively, Can long-term taking, be the preferable health food of hypoimmunity crowd.
Experimental example 1
Strengthen immunity function to coenzyme Q 10 soft capsule manufactured in the present embodiment is detected.
1st, sample:The soft capsule of DPN diphosphopyridine nucleotide 0 prepared by embodiment 5.
2nd, experimental animal and environment:
SPF grades of healthy Male Kunming strain mice 288, body weight 18g~22g, by Zhejiang Academy of Medical Sciences Experimental Animal Center is provided, Quality of Experimental Animals quality certification number:NO.0051154.Every 48 mouse are I Batch, totally 6 batches.VI VIII batches, carry out the mouse spleen lymphocyte transformation experiment of ConA inductions;II batch, carry out Delayed allergy is tested;III batch, serum hemolysin is determined and antibody-producting cell number is determined;IV~VI Batch, carbonic clearance experiment, peritoneal macrophage phagocytosis chicken red blood cell experiment and NK cytoactives are carried out respectively to be surveyed It is fixed.
22 ± 2 DEG C of experimental situation temperature, relative humidity 50% ± 10%.
3rd, dose design and tested material give mode:Ubiquinone of the invention10Recommendation of the soft capsule to human body Dosage is daily 1.0g/60kg body weight, equivalent to 0.0167g/kg, BW, by equivalent to human body recommended dose 5,10,30 multiple doses determine the low middle Three doses high of mouse, to the dosage point of mouse in the present embodiment 0.083g/kg, BW, 0.167g/kg, BW, 0.50g/kg, BW, three kind are not set to, it is oral daily Gavage gives mouse tested material, and gavage volume presses 10ml/kg, BW.Prepared with corn oil before gavage Tested material, in basic, normal, high dosage group the concentration of tested material be respectively 8.33g/L, 16.667g/L and 50.00g/L, negative control group replaces tested material with isometric distilled water, and solvent control group is with isometric Corn oil replaces tested material, and every immune indexes are tested after 30 days.Mouse is with outbred mice special feed Feeding.
4th, experimental technique
4.1 organ weight ratio values are determined
The de- white execution of cervical vertebra, takes its thymus gland, spleen after mouse weights, removes most manadesma, is blotted with filter paper dirty Device surface blood stains are simultaneously weighed, and calculate thymus gland body weight ratio and spleen weight ratio.The data obtained PEMS Statistical software carries out variance analysis.
The mouse lymphocyte transformation function (mtt assay) of 4.2 Con A inductions
Continuously to sample after 30 days, the de- white execution of cervical vertebra takes spleen to animal, is made splenocyte suspension, adjusts Cell concentration is 3 × 106Individual/ml, is divided into cell suspension holes and adds 24 well culture plates China, per hole 1ml, One hole adds 75 μ LConA liquid (equivalent to 7.Syg/ml), and 5%C02,37 DEG C of trainings are put in another hole as control Support 68h.Culture terminates preceding 4h, per hole gentle aspiration supernatant 0.7ml, adds 0.7ml to be free of calf serum PRMI1640 nutrient solutions, while add MTT (5mg/ml) 50 μ L/ holes, continue cultivate 4h, culture knot Shu Hou, 1ml acid isopropyl alcohol is added per hole, and piping and druming is mixed, and after being completely dissolved purple crystal, then will Liquid is moved into 96 orifice plates, and 3 Duplicate Samples are added per hole, and extinction is measured in 570nm wavelength with ELIASA Angle value (A values), calculates the difference of test hole A values and control wells A values, to represent the competence for added value of lymphocyte. Result is analyzed with variance analysis.If the multiplication capacity of tested material group lymphocyte is higher than control group, and Difference has conspicuousness, can determine that the tested material is improved the mouse spleen lymphocyte conversion capability of ConA inductions Effect.
4.3 delayed allergies (vola pedis thickens method) (DTH)
Sheep blood is taken, with brine 3 times, (v/v is matched somebody with somebody every mouse with physiological saline through intraperitoneal injection 2% Put) hematocrit SRBC (2000r/min, 10min) 0.2ml, 4 days after sensitization, left and right vola pedis thickness is measured, together One position measures 3 times, averages, and then in measuring point hypodermic injection 20%, (v/v uses physiological saline Configuration) hematocrit SRBC20yl, 24 hours measurement left and right vola pedis thickness after injection, to attack front and rear vola pedis thickness Difference represent the degree of DTH.The data obtained is measurement data, and variance analysis is carried out with SPSS statistical softwares, If the difference that tested material attacks front and rear vola pedis thickness is higher than control group, and difference has conspicuousness (P<0.05), Can conclude that the tested material is improved the effect of mouse delayed allergy ability.
4.4 antibody-producting cells determine (Jerne improves slide methods)
Continuously to sample after 30 days, after de- fiber Mianyang erythrocyte immune 5 days, animal cervical vertebra is de- to be located animal in vain Extremely, spleen is taken out, splenocyte suspension is made, adjustment cell concentration is 5 × 106Individual/ml.Top layer is cultivated After base (Ig agaroses add distilled water 100ml) heating for dissolving, 45 DEG C of water-bath insulations are put into, with equivalent PH7.2-7.4, the Hanks liquid of 2 times of concentration mix, and dispense small test tube, often pipe 0.5ml, then add in pipe 50 μ L 10%SRBC, 20 μ L splenocyte suspensions are rapid to mix, and are poured into the 6cm of brush thin agar layer On plate, after continuing to incubate 1.5h, hemolysis plaque number is counted.Result is counted with variance analysis.If The multiplication capacity of tested material group lymphocyte is higher than control group, and difference has conspicuousness, can determine that this is tested Thing plays the role of to strengthen mouse antibodies cell number.
4.5 mice serum hemolysin tests:
Take sheep blood, with brine 3 times, every mouse through intraperitoneal injection 2% (use normal saline, Volume ratio) hematocrit SRBC (2000r/min, 10min) 0.2ml, after being immunized 5 days, extracts eyeball of mouse and takes blood In centrifuge tube, place about 1 hour, solidification blood and tube wall are peeled off, serum is collected in centrifugation, uses physiology Be respectively placed in the solidifying brassboard of micro snow different dilution factor serum to serum doubling dilution, per hole by salt solution 100 μ L, add the μ L of 0.5%SRBC suspensions 100, mix Uniform, put in wet box, in 37 DEG C of incubations 3 Hour, record hemagglutination degree, calculating antibody product (serum two-fold dilution index and aggegation degree product it With).
Serum agglutination degree is divided into 5 grades, clicks standard determination.O grades:SRBC all sinks, and concentrates on hole Bottom forms the round point shape of densification, and surrounding liquid clear level=SRBC major parts are deposited on bottom hole portion into round point shape, Surrounding has the SRBC of a small amount of aggegation;11 grades:The SRBC of aggegation forms thin layer in bottom hole, and center can be with bright It is aobvious to see a loose red point;111 grades:The SRBC of aggegation and paving be dispersed in bottom hole into a thin layer, in The heart mays be seen indistinctly a small red dot;IV grades:Uniformly paving is dispersed in bottom hole into a thin layer to the SRBC of aggegation, coagulates Block is sometimes into convolution shape.
The data obtained carries out variance analysis with PEMS softwares, if tested material group Hemolysin product is high In control group, and difference has conspicuousness, can determine that tested material is improved the work of mice serum hemolysin level With.
4.6 mouse carbonic clearance ability tests
Animal is continuously to sample after 30 days, tail vein injection 1:The india ink of 5 dilutions, treats that prepared Chinese ink injects, and stands That is timing.2,10 minutes after injection prepared Chinese ink, the μ L of blood 20 are taken from angular vein clump respectively, and be added into 2ml In sodium carbonate liquor, absorbance is tested at 600nm wavelength with spectrophotometer, with sodium carbonate liquor Do blank.According to the weight of animals, liver is weighed and spleen re-computation phagocytic index, is as a result entered with variance analysis Row statistics.If tested material group Hemolysin product is higher than control group, and difference has conspicuousness, can Judge that tested material is improved the effect of the carbonic clearance ability of mouse monokaryon-macrophage.
4.7 Turnover of Mouse Peritoneal Macrophages swallow chicken red blood cell (half intracorporal method) experiment
Animal, per the chicken erythrocyte suspension 1ml of mouse intraperitoneal injection 20%, is spaced 30 minutes continuously to sample after 30 days, Cervical dislocation is put to death, and is fixed on mouse plate, cuts off abdominal skin, and injecting normal saline 2ml rotates mouse plate 1 minute, abdominal cavity washing lotion lml is suctioned out, point dripped in 2 sheets, 37 DEG C of incubators are wet to incubate 30 minutes, with life Reason saline rinse, dries, with 1:1 acetone methanol solution is fixed, 4%Giemsa- phosphate buffers dyeing 3 Minute, then dried with distilled water rinsing, oil mirror microscopy is used, calculate percentage phagocytosis and phagocytic index.Its In, number of macrophages × 100 of the number of macrophages/counting of phagocytic rate (%)=phagocytosis chicken red blood cell;
The number of macrophages of the chicken red blood cell sum of phagocytic index=swallowed/count.
The phagocytic rate of acquired results presses X=Sin-1P1/2Converted, P is phagocytic percentage in formula, decimally Represent, if the phagocytic rate or phagocytic index of tested material are apparently higher than control group, and difference has conspicuousness, can Judge that tested material is improved the effect of macrophage phagocytic chicken red blood cell ability.
4.8 NK cells in mice determinations of activity:
Continuously to sample after 30 days, the de- white execution of cervical vertebra takes spleen to animal, and (effect is thin to be made splenocyte suspension Born of the same parents), 24h YAC-1 cells plus 1640 and are cultivated completely after taking passage, and adjustment cell concentration is 4 × 105Individual/ml (target cell), takes target cell and each 100 μ L effect targets ratio (50 of effector cell:1) culture of the hole of U-shaped 96, is added Plate, target cell Spontaneous release hole adds target cell and each 100 μ L of nutrient solution, maximum release aperture add target cell and Each 100 μ L of 1%NP40, above-mentioned items are equipped with three multiple holes, with 37 DEG C, 5% CO2gas incubator Middle culture 4 hours, every hole Aspirate supernatant 100u 1 is placed in flat 96 well culture plate, while adding LDH The μ L of matrix liquid 100, react 3min, add the HCL30 μ L of lmol/L to terminate per hole, are existed with ELIASA A values are determined at 490mm.NK cytoactives are calculated as follows:
NK cytoactives %=(reacting hole A- Spontaneous releases hole A)/(maximum release aperture A- Spontaneous releases hole A) × 100;
NK cytoactives need to be by X=Sin-1P1/2Data conversion is carried out, P is NK cytoactives in formula, decimally table Show, if the phagocytic rate or phagocytic index of tested material are apparently higher than control group, and difference has conspicuousness, can sentence Determine the effect that tested material is improved NK cells in mice activity.
5 experimental results
5.1 experimental results judge
Enhancing immunocompetence function judges:It is thin in cellular immune function, humoral immune function, monokaryon-macrophage Born of the same parents' function, four aspect any two aspect results of NK cytoactives are positive, you can judge that the tested material has There is strengthen immunity function.
Two experimental results wherein in cellular immune function assay project are the positive, or any experiment Two dosage group results are the positive, can determine that cellular immune function assay result is the positive.Humoral immunity work( Two dosage group results that two experimental results in energy measure project are positive or any experiment are sun Property, can determine that humoral immune function measurement result is positive.In monocytes/macrophages functional examination project two Two dosage group results that individual experimental result is positive or any experiment are positive, can determine that monokaryon _ macrophage is thin Born of the same parents' functional examination result is positive.More than one dosage group result of NK cytoactive detections experiment is positive, can Judge that NK cytoactives result is positive.
5.2 ubiquinones10Influence of the soft capsule to Mouse Weight is shown in Table 1
The sample of table 1 is to being immunized one group of influence of the weight of animals (gram)
Different dosage is compared with control group as can be seen from Table 1, and the weight gain of mouse is poor without conspicuousness It is different, therefore ubiquinone10Soft capsule has not significant impact to the body weight of animal.
5.3 ubiquinones10Influence of the soft capsule to mice organs body weight ratio:It is shown in Table 2, table 3.
The ubiquinone of table 210Influence (x ± SD) of the soft capsule to mouse spleen body weight ratio
Group Number of animals (only) Spleen weight ratio (mg/g) P
Water control group 10 5.04±0.41 0.773
Solvent control group 10 4.98±0.62
Low dose group 10 4.93±0.61
Middle dose group 10 4.85±0.80
High dose group 10 4.69±0.68
The ubiquinone of table 310Influence (x ± SD) of the soft capsule to the thymus gland body weight ratio of mouse
Compared with control group, the thymus gland body weight ratio of mouse is without notable for different dosage as can be seen from Table 3 Sex differernce, therefore ubiquinone10Soft capsule has not significant impact to the thymus gland body weight ratio of animal.
5.4 ubiquinones10Influence of the soft capsule to mouse cell immunologic function
A, ubiquinone10Influence of the soft capsule to mouse delayed allergy (DTH) is shown in Table 4.
The ubiquinone of table 410Influence (x ± SD) of the soft capsule to mouse delayed allergy (DTH)
From table 4, water control group, solvent control group and 3 mouse swelling degree of the paw of dosage group Influence is different, and influence of the middle dose group with high dose group to mouse swelling degree of the paw has obvious with control group ratio Difference, it is believed that ubiquinone10Soft capsule is improved the ability of mouse delayed allergy (DTH).
B, ubiquinone10The influence of the mouse spleen lymphocyte transformation experiment that soft capsule is induced ConA, is shown in Table 5.
The influence of the mouse spleen lymphocyte transformation experiment that the coenzyme Q 10 soft capsule of table 5 is induced ConA
From table 5, water control group, solvent control group and 3 dosage groups increase to the lymphocyte of mouse Grow between ability that there was no significant difference, it is believed that ubiquinone10Soft capsule is to improving mouse lymphocyte propagation energy Power has not significant impact.
5.4 ubiquinones10Influence of the soft capsule to mouse humoral immune function
A, ubiquinone10Influence of the soft capsule to mouse antibodies cellulation number, is shown in Table 6.
The ubiquinone of table 610Influence (x ± SD) of the soft capsule to mouse antibodies cellulation number
From table 6, water control group has obvious with the influence of middle dose group and high dose group to plaque number Difference, it is believed that ubiquinone10Soft capsule has the ability for increasing mouse antibodies cellulation number.
B, ubiquinone10Influence of the soft capsule to mice serum hemolysin level, is shown in Table 7.
The ubiquinone of table 710Influence (x ± SD) of the soft capsule to mice serum hemolysin level
From table 7, water control group has obvious with the influence of middle dose group and high dose group antagonist product Difference, it is believed that ubiquinone10Soft capsule has the ability for improving mice serum hemolysin level.
5.5 ubiquinones10Influence of the soft capsule to mouse monokaryon _ macrophage phagocytic function
A, ubiquinone10Influence of the soft capsule to mouse monokaryon _ macrophage carbonic clearance ability, is shown in Table 8.
The ubiquinone of table 810Influence (x ± SD) of the soft capsule to mouse monokaryon macrophage carbonic clearance ability
From table 8, water control group, solvent control group and 3 dosage groups are thin to the monokaryon-macrophage of mouse There was no significant difference between born of the same parents' carbonic clearance ability, it is believed that ubiquinone10Soft capsule does not have to mouse carbonic clearance ability Have a significant effect.
B, ubiquinone10Soft capsule swallows the influence of chicken red blood cell ability to Turnover of Mouse Peritoneal Macrophages, is shown in Table 9th, table 10.
The ubiquinone of table 910Influence (x ± SD) of the soft capsule to Turnover of Mouse Peritoneal Macrophages chicken red blood cell phagocytic rate
Influence (x ± SD) of the coenzyme Q 10 soft capsule of table 10 to Turnover of Mouse Peritoneal Macrophages chicken red blood cell phagocytic index
From table 9 and table 10, water control group, solvent control group and 3 dosage groups are huge to mouse peritoneal There was no significant difference between phagocyte phagocytosis chicken red blood cell ability, it is believed that ubiquinone10Soft capsule is to mouse abdomen Chamber macrophage phagocytic chicken red blood cell ability has not significant impact.
5.6 ubiquinones10Influence of the soft capsule to NK cells in mice activity, is shown in Table 11.
Influence (x ± SD) of the CoQ1 q soft capsules of table 11 to NK cells in mice activity
From table 11, water control group, solvent control group and 3 dosage groups are to NK cells in mice activity Between there was no significant difference, it is believed that ubiquinone10Soft capsule has not significant impact to NK cells in mice activity.
To sum up, the ubiquinone of orally administration mouse various dose group10Soft capsule 30 days, increases to Mouse Weight Length has no adverse effects, on mouse spleen and thymus gland body weight ratio without influence, the humoral immune function of mouse, Cellular immune function assay result is the positive, mononuclear macrophage phagocytic activity, NK cytoactive detections Result is feminine gender, shows ubiquinone10Soft capsule has the effect of strengthen immunity function.
Specific embodiment
Embodiment 1
A kind of ubiquinone10Soft capsule, including capsule-core and capsule skin, capsule-core is by following components and the original of weight portion Material composition:Ubiquinone1035g, vitamin E 35g, corn oil 225g, beeswax 7.5g;
Capsule skin is made up of the raw material of following components and weight portion:Gelatin 140g, glycerine 56g, purified water 133g, titanium dioxide 0.5g, Monascus color 1.2g, burnt sugar coloring 1.0g;
Preparation method:
(1) preparation of capsule-core feed liquid:Ubiquinone10The advance mesh sieve of mistake 80 of raw material, it is standby.Honeybee is taken by formula rate Wax, corn oil, are heated to 70 DEG C of fusing beeswaxs, stir and evenly mix, and obtain mixed material, put to room temperature, standby With.Vitamin E, ubiquinone are taken by formula rate10, it is added in mixed material, stir and evenly mix, cross glue Body barreling wears into the fine and smooth slurry of quality, stands vacuum outgas bubble, standby.
(2) preparation of capsule hide glue gelatin:When purified water is heated into 60 DEG C, by what is weighed up by formula rate in advance Gelatin, glycerine, titanium dioxide, burnt sugar coloring, Monascus color are added in purified water, are sealed, and are started Agitating paddle is stirred, and is heated to 70 DEG C, and stirring makes uniform glue, stops stirring, is stood true Empty de-bubbled 1h, it is standby.
(3) soft capsule is prepared:By the capsule hide glue that capsule-core feed liquid obtained in step (1) is defeated and step (2) is prepared Liquid is mixed in being transported to the storage glue groove of encapsulating machine, standby.Start encapsulating machine, by pelleting, sizing, Wash ball, drying, pick ball and packing step, prepare ubiquinone10Soft capsule.
Embodiment 2
Difference from Example 1 is:Capsule-core is made up of the raw material of following components and weight portion:Ubiquinone10 38g, vitamin E 40g, corn oil 226g, beeswax 5.5g.
Embodiment 3
Difference from Example 1 is:Capsule-core is made up of the raw material of following components and weight portion:Coenzyme Q1040g, vitamin E 37g, corn oil 260g, beeswax 15g.
Embodiment 4
Difference from Example 1 is:Capsule-core is made up of the raw material of following components and weight portion:Coenzyme Q1030g, vitamin E2 5g, corn oil 286g, beeswax 5.5g.
Embodiment 5
Difference from Example 1 is:Capsule-core is made up of the raw material of following components and weight portion:Coenzyme Q1040g, vitamin E 50g, corn oil 395g, beeswax 15g.
Embodiment 6
Difference from Example 1 is:Capsule-core is made up of the raw material of following components and weight portion:Coenzyme Q1038g, vitamin E 27g, corn oil 200g, beeswax 5.5g.
Embodiment 7
Difference from Example 1 is:Capsule-core is made up of the raw material of following components and weight portion:Coenzyme Q1040g, vitamin E 60g, corn oil 250g, beeswax 15g.
Embodiment 8
Difference from Example 1 is:Capsule-core is made up of the raw material of following components and weight portion:Coenzyme Q1040g, vitamin E 37g, corn oil 200g, beeswax 10g.
Comparative examples 1
Capsule-core:Ubiquinone1048g, vitamin E 37g, corn oil 860g, beeswax 55g;
Capsule skin constituent and soft capsule preparation method are with embodiment 1.
Comparative examples 2
Capsule-core:Ubiquinone1010g, vitamin E 30g, corn oil 950g, beeswax 10g;
Capsule skin constituent and soft capsule preparation method are with embodiment 1.
Comparative examples 3
Capsule-core:Ubiquinone1040g, vitamin E 0.2g, corn oil 140g, beeswax 19.8g;
Capsule skin constituent and soft capsule preparation method are with embodiment 1.
Effect experiment:
Trier 660,30-40 Sui, 40-50 Sui (being free of 40 years old), 50-70 Sui (is free of 50 Year) each 220 people, it is divided into 11 groups, every group includes 30-40 Sui respectively, and 40-50 Sui, 50-70 Sui is each 20 people.Three groups of triers use the product of embodiment 1-8 and comparative examples 1-3 respectively.Crowd on probation Skin is intensely dark, there is pigmentation.
Test method:Ubiquinone prepared by embodiment 1-8 and comparative examples 1-310Soft capsule is sent out at random To trier, daily 1,60/bottle try out 2 months.
Standards of grading:
0 point:Observed under natural light, be visible by naked eyes pigmentation;
2 points:Observed under natural light, slight pigmentation, color is light brown, in 50cm or more than 50cm Distance observation not substantially, general cosmetic or beard, chaeta can be covered;
4 points:Observed under natural light, moderate pigmentation, color yellowish-brown, in 50cm or more than 50cm Distance observation not substantially, general cosmetic or beard, chaeta are difficult to cover;
6 points:Observed under natural light, severe pigmentation, color dark brown is observed also very outside 50cm Substantially, general cosmetic or beard, chaeta are difficult to cover.
Therapeutic evaluation:
Therapeutic index=[scoring before (being scored after the treatment of scoring one before treatment)/treatment] * 100%
Clinical epidemic disease heals:Pigmentation all disappears, clinical symptom disappearance, and symptom score value reduces >=95%.
It is effective:Pigmentation is most of to disappear, and clinical symptoms substantially mitigate, and 95% ﹥ symptom score values subtract Few >=70%.
Effectively:Pigmentation partial remission, clinical symptom relief, 70% ﹥ symptom scores value reduces >=30%.
It is invalid:Pigmentation disappears not substantially, and clinical symptoms do not mitigate or deteriorate, and symptom score value is reduced ﹤ 30%.
Efficient=(recovery from illness+effective+effectively)/60*100%
Trial effect is paid a return visit in questionnaire form.Trial effect such as following table:
The trial effect of the embodiment 1-8 of table 12 and comparative examples 1-3 products
The product of embodiment 1 30-40 Sui/people 40-50 Sui/people 50-70 Sui/people It is efficient
Dispel pigmentation 16 16 16 80.00%
The product of embodiment 2 30-40 Sui/people 40-50 Sui/people 50-70 Sui/people It is efficient
Dispel pigmentation 16 18 16 83.30%
The product of embodiment 3 30-40 Sui/people 40-50 Sui/people 50-70 Sui/people It is efficient
Dispel pigmentation 16 16 17 81.60%
The product of embodiment 4 30-40 Sui/people 40-50 Sui/people 50-70 Sui/people It is efficient
Dispel pigmentation 16 16 17 81.60%
Total effective rate 82.75% 84.00% 83.00% 81.50%
The product of embodiment 5 30-40 Sui/people 40-50 Sui/people 50-70 Sui/people It is efficient
Dispel pigmentation 18 18 16 87.30%
The product of embodiment 6 30-40 Sui/people 40-50 Sui/people 50-70 Sui/people It is efficient
Dispel pigmentation 16 16 17 81.60%
The product of embodiment 7 30-40 Sui/people 40-50 Sui/people 50-70 Sui/people It is efficient
Dispel pigmentation 18 18 16 83.30%
The product of embodiment 8 30-40 Sui/people 40-50 Sui/people 50-70 Sui/people It is efficient
Dispel pigmentation 18 16 18 83.30%
The product of comparative examples 1 30-40 Sui/people 40-50 Sui/people 50-70 Sui/people It is efficient
Dispel pigmentation 16 14 15 74.20%
The product of comparative examples 2 30-40 Sui/people 40-50 Sui/people 50-70 Sui/people It is efficient
Dispel pigmentation 14 14 13 71.25%
The product of comparative examples 3 30-40 Sui/people 40-50 Sui/people 50-70 Sui/people It is efficient
Dispel pigmentation 15 15 14 76.15%
Conclusion:From examples detailed above, soft capsule of the present invention can effectively dispel facial pigmentation, effect Fruit is significantly better than the product of comparative examples 1-3, wherein with the effect of product obtained by the formula of embodiment 5 Fruit is optimal, efficient higher compared with other similar proportioning compositions, and therapeutic effect is more preferable.

Claims (10)

1. one kind contains ubiquinone10Soft capsule, including capsule skin and capsule-core, it is characterised in that described Capsule-core is made up of the raw material of following components and weight portion:Ubiquinone1020-50 weight portions, vitamin E 15-60 Weight portion, corn oil 200-500 weight portions, beeswax 5-20 weight portions.
2. a kind of ubiquinone as claimed in claim 110Soft capsule, it is characterised in that described capsule-core It is made up of the raw material of following components and weight portion:Ubiquinone1030-40 weight portions, vitamin E 25-55 weights Amount part, corn oil 300-450 weight portions, beeswax 5.5-18 weight portions.
3. a kind of ubiquinone as claimed in claim 210Soft capsule, it is characterised in that described capsule-core It is made up of the raw material of following components and weight portion:Ubiquinone1035-40 weight portions, vitamin E 45-50 weights Amount part, corn oil 380-400 weight portions, beeswax 10-15 weight portions.
4. a kind of ubiquinone as claimed in claim 310Soft capsule, it is characterised in that described capsule-core It is made up of the raw material of following components and weight portion:Ubiquinone1040 weight portions, the weight portion of vitamin E 50, The weight portion of corn oil 395, the weight portion of beeswax 15.
5. a kind of ubiquinone as described in Claims 1 to 4 is any10The preparation method of soft capsule, its feature It is that the method is comprised the following steps:
(1) preparation of capsule-core feed liquid:Beeswax, corn oil, heating fusing beeswax are taken by formula rate;By matching somebody with somebody Square ratio takes vitamin E, ubiquinone10, mix with beeswax, corn oil, obtain capsule-core feed liquid;
(2) preparation of capsule hide glue gelatin:Purified water is heated, by gelatin, glycerine, titanium dioxide, burnt sugar coloring, Monascus color is added in purified water, is stirred and evenly mixed, and obtains capsule hide glue gelatin;
(3) soft capsule is prepared:The capsule hide glue gelatin that capsule-core feed liquid obtained in step (1) and step (2) are prepared Mix, by pelleting, sizing, dry, prepare ubiquinone10Soft capsule.
6. preparation method as claimed in claim 5, it is characterised in that selected gelatin in step (2), The weight ratio of glycerine and pure water is (1~5):(0.5~2):(2~8).
7. preparation method as claimed in claim 6, it is characterised in that selected gelatin in step (2), The weight ratio of glycerine and pure water is 3:1:4.
8. a kind of composition is preparing answering in having strengthen immunity, the health products of fatigue-relieving effect With, it is characterised in that the raw material of the composition by as follows into being grouped into:
Ubiquinone1020-50 weight portions, vitamin E 15-60 weight portions, corn oil 200-500 weight portions, Beeswax 5-20 weight portions.
9. application as claimed in claim 8, it is characterised in that the raw material of the composition by as follows into It is grouped into:
Ubiquinone1030-40 weight portions, vitamin E 25-55 weight portions, corn oil 300-450 weight portions, Beeswax 5.5-18 weight portions.
10. application of a kind of composition in preparing with the health products for dispelling facial pigmentation effect, Characterized in that, the raw material of the composition is by as follows into being grouped into:
Ubiquinone1020-50 weight portions, vitamin E 15-60 weight portions, corn oil 200-500 weight portions, Beeswax 5-20 weight portions.
CN201510830305.XA 2015-11-25 2015-11-25 A kind of soft capsule containing Co-Q10 and its preparation and application Pending CN106798734A (en)

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CN103083198A (en) * 2011-10-27 2013-05-08 北京天辰空间生物医药研发有限公司 Whitening and freckle removing functions of coenzyme Q10, and application thereof in cosmetics
CN103156193A (en) * 2013-04-03 2013-06-19 上海春芝堂生物制品有限公司 Coenzyme Q10 soft capsules and preparation method thereof
CN104840385A (en) * 2015-04-11 2015-08-19 广东医学院 A group of coenzyme Q10 skin protection creams with effects of skin moisturizing, wrinkle preventing and aging resistance
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CN103156193A (en) * 2013-04-03 2013-06-19 上海春芝堂生物制品有限公司 Coenzyme Q10 soft capsules and preparation method thereof
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