CN106434533A - Zuogui pill serum containing active ingredients of Zuogui pill serum metabolic substance groups and preparation method and application of Zuogui pill serum - Google Patents
Zuogui pill serum containing active ingredients of Zuogui pill serum metabolic substance groups and preparation method and application of Zuogui pill serum Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0603—Embryonic cells ; Embryoid bodies
- C12N5/0604—Whole embryos; Culture medium therefor
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/14—Blood; Artificial blood
- A61K35/16—Blood plasma; Blood serum
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/70—Undefined extracts
- C12N2500/76—Undefined extracts from plants
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/70—Undefined extracts
- C12N2500/80—Undefined extracts from animals
- C12N2500/84—Undefined extracts from animals from mammals
Abstract
The invention discloses Zuogui pill serum containing active ingredients of Zuogui pill serum metabolic substance groups and a preparation method and application of the Zuogui pill serum. The preparation method includes the steps of (1), mixing medicinal materials for preparing Zuogui pills according to a formulation dosage for preparing the Zuogui pills and performing water extraction so as to obtain water extract; (2), administrating the water extract in animals at a dosage of 10-20 g kg<-1> d<-1> for 7-10 days continuously, once to twice per day, collecting blood after administration, and collecting serum after blood centrifugation so as to obtain the Zuogui pill serum. The Zuogui pill serum is highly rich in glycoside active ingredients and is capable of remarkably increasing an inhibited embryo development rate under intervention of high glucose to promote embryo development.
Description
Technical field
The invention belongs to serum pharmaceutical chemistry technical field is and in particular to the material group of blood serum metabolic containing Bolus as a Kidney-Yin-Tonic is effective
Bolus as a Kidney-Yin-Tonic serum of composition and preparation method and application.
Background technology
With extract oral administration after serum as sample, by traditional medicinal chemistry identical research method, various modern technology
Integrated application, separates from serum, identifies effective constituents, in research serum, effective constituents and the dependency of Traditional Curative Effect, illustrate
Internal direct effect substance metabolism and in vivo dynamic method are referred to as serum pharmaceutical chemistry, study in quality standardization,
The aspect such as scientific of the illustrating of compound compatibility mechanism, the determination of Effective Component of Chinese Medicine and Chinese medicine dosage regimen has important
Research Significance.
At present, research in terms of single medicinal material for the serum pharmaceutical chemistry be related to Herba Artemisiae Scopariae, Radix Angelicae Sinensis, Radix Et Rhizoma Rhei, Folium Isatidiss,
Fructus Psoraleae and Ramulus et folium taxi cuspidatae etc., the research in terms of Chinese medicine compound is related to LIUWEI DIHUANG WAN, Rhizoma Coptidis toxic materials clearing away decoction, compound recipe Herba Artemisiae Scopariae
Soup, Guiling tablets, Lonicerae and Forsythiae Powder and Anti-V capsule etc..
Chinese medicine Bolus as a Kidney-Yin-Tonic comes from《Jing Yue's complete work》Roll up 51 new eight gusts of sides of side, be by Radix Rehmanniae Preparata, Semen Cuscutae, Radix Achyranthis Bidentatae, Testudiniss
Nail gelatin, Colla cornus cervi, Rhizoma Dioscoreae, Fructus Corni, the compound recipe of Fructus Lycii eight taste Chinese medicine composition, have the effect of enriching yin and nourishing kidney, for Kidney-Yin
Deficiency, soreness of waist and knee, night sweat seminal emission, Mental fatigue dry mouth.At present, the research of Bolus as a Kidney-Yin-Tonic is quality evaluation, gene expression, bone marrow mostly
The research of the aspects such as Interstitial cell, gonad cell and Bolus as a Kidney-Yin-Tonic compound recipe.The osteoblast to in-vitro separation, culture for the Bolus as a Kidney-Yin-Tonic serum
Secretion Bone Gla protein has a significant impact, and has facilitation to osteoblastic bone formation, has direct inhibitory action to osteoclast,
And inhibitory action indirectly can be produced to osteoclast by osteoblast, it is one of mechanism of its anti-curing osteoporosis.A left side is returned
Ball serum can promote Bone Marrow Mesenchymal Stem Cells to differentiation alkaline phosphatase expression.And alkali phosphatase is osteoblast
A kind of cell surface marker enzyme, with the increase of Osteoblast Differentiation degree, alkaline phosphatase expression of enzymes strengthens.Bolus as a Kidney-Yin-Tonic serum
Bone marrow stromal cell can be significantly improved to hepatocellular conversion ratio, to hepatocellular differentiation, there is facilitation to medullary cell.
Illustrate that Bolus as a Kidney-Yin-Tonic blood serum metabolic material group has the characteristics that multiple target effect.However, the material group of blood serum metabolic containing Bolus as a Kidney-Yin-Tonic is effective
The Bolus as a Kidney-Yin-Tonic serum of composition does not have been reported that.
Content of the invention
It is an object of the invention to provide the Bolus as a Kidney-Yin-Tonic serum of the material group effective ingredient of blood serum metabolic containing Bolus as a Kidney-Yin-Tonic and its preparation
Method and application, this Bolus as a Kidney-Yin-Tonic serum camber is enriched glycoside effective ingredient, can significantly improve downtrod under high sugar is intervened
Embryo development rate, promotes fetal development.
A kind of preparation method of Bolus as a Kidney-Yin-Tonic serum that the present invention provides, comprises the steps：(1) by the medicine of preparation Bolus as a Kidney-Yin-Tonic
Material carries out water extraction according to after the formula consumption mixing of preparation Bolus as a Kidney-Yin-Tonic, obtains Aqueous extracts；(2) described Aqueous extracts are administered to animal, give
Medicine is taken a blood sample after terminating, and will collect serum, you can obtain described Bolus as a Kidney-Yin-Tonic serum after centrifugal blood；In described dosing step,
Dosage is 10～20g kg-1·d-1, successive administration 7～10 days, daily 1～2 time.
A kind of preparation method of Bolus as a Kidney-Yin-Tonic serum that the present invention provides, comprises the steps：Take isolated blood, by vitro blood
Liquid collected after centrifugation serum, you can obtain described Bolus as a Kidney-Yin-Tonic serum；Described isolated blood is the isolated blood of following animal：(1)
The medical material of preparation Bolus as a Kidney-Yin-Tonic is carried out water extraction according to after the formula consumption mixing of preparation Bolus as a Kidney-Yin-Tonic, obtains Aqueous extracts；(2) animal is given
Aqueous extracts described in medicine, after administration terminates, obtain final product described animal；In described dosing step, dosage is 10～20g kg-1·d-1, successive administration 7～10 days, daily 1～2 time.
In above-mentioned preparation method, in step (1), described medical material be preparation Bolus as a Kidney-Yin-Tonic conventional medical material, specially Radix Rehmanniae Preparata,
Rhizoma Dioscoreae (stir-fry), Fructus Lycii, Fructus Corni, Radix Cyathulae, Colla cornus cervi, Colla Plastri Testudiniss and Semen Cuscutae, described formula consumption is preparation Bolus as a Kidney-Yin-Tonic
Conventional formulation consumption, specially：Radix Rehmanniae Preparata (240g), Rhizoma Dioscoreae (stir-fry) (120g), Fructus Lycii (120g), Fructus Corni (120g), Radix Cyathulae
(90g), Colla cornus cervi (120g), Colla Plastri Testudiniss (120g), Semen Cuscutae (120g).
Above-mentioned preparation method, in step (1), the crude drug amount in described Aqueous extracts can be (0.5～1) g mL-1, specifically
Can be 1g mL-1.
Above-mentioned preparation method, in step (1), the step of described water extraction can be as follows：Heat water to Extracting temperature (such as 50
DEG C) after, the medical material put in addition to Colla cornus cervi and Colla Plastri Testudiniss is decocted, and obtains Aqueous extracts I；Colla Plastri Testudiniss, Colla cornus cervi is taken to add water to add
Heat makes it dissolve, and mixes with described Aqueous extracts I, obtain Aqueous extracts II after all dissolving；Concentrate described Aqueous extracts II, you can obtain
Described Aqueous extracts.
Above-mentioned preparation method, in step (1), in described aqueous extraction step, Extracting temperature can be 50 DEG C～boiling temperature, tool
Body can be 50 DEG C；Extraction time (time extracted every time) can be 1.0～1.5h, concretely 1.5h；The consumption of water (carries every time
The consumption of water when taking) can be 1g medical material：(8～10) mL water, concretely 1g medical material：10mL water；Water extraction number of times can be 2～3 times,
Concretely 3 times.Comprise the following steps that：Take 8 times amount water to be heated to 50 DEG C, put into the medical material heating in addition to Colla cornus cervi and Colla Plastri Testudiniss
Decoct 1.5h；8 times amount decoctings are taken to boil 1.5h for the second time；Third time takes 8 times of decoctings to boil 1.5h；Respectively with eight layers of filtered through gauze, close
And three leachates, obtain Aqueous extracts I.
Above-mentioned preparation method, in step (2), described animal can be male and female rat.
Above-mentioned preparation method, in step (2), described dosage concretely 20g kg-1·d-1；Administration time is concrete
Can be successive administration 7 days, once a day.
Above-mentioned preparation method, in step (2), in described dosing step, administering mode can be gavage.
Above-mentioned preparation method, in described blood sampling step, fasting 12h after the completion of second from the bottom administration, for the last time
After administration start timing, the time control of blood sampling be administered the last time after 30～40min in, specifically can be controlled in last
In the 30min of secondary blood sampling.Blood collection can be eye socket capillary blood sampling tube or ventral aorta blood sampling.
Described isolated blood is the isolated blood of following animal：Fasting 12h after the completion of second from the bottom administration, and for
Animal in 30～40min after single administration afterwards, specifically can be controlled in the 30min of last blood sampling.
Above-mentioned preparation method, in described centrifugation step, centrifugal rotational speed can be 3000～4000r min-1, concretely
4000r·min-1, centrifugation time can be 15～20min, concretely 15min.
Invention further provides a kind of Bolus as a Kidney-Yin-Tonic serum being prepared by above-mentioned preparation method, this Bolus as a Kidney-Yin-Tonic blood
Clear camber is enriched glycoside effective ingredient, such as iridoid glycoside active component.
Present invention also offers above-mentioned Bolus as a Kidney-Yin-Tonic serum has following 1) -2 in preparation) any one of in the medicine of function
Application：
1) improve downtrod embryo development rate under high sugar is intervened；
2) promote the application in fetal development.
The present invention has the advantages that：
1. water is heated to 50 DEG C, and the medical material heating placing in addition to Colla cornus cervi and Colla Plastri Testudiniss decocts, and improves in Aqueous extracts
Glycoside effective ingredient content；
2. laboratory animal dosage reaches 10～20g kg-1·d-1, higher than the dosage of laboratory animal, improve a left side and return
The content of ball blood serum metabolic material group effective ingredient it is achieved that glycoside effective ingredient highly enriched；
3. the Bolus as a Kidney-Yin-Tonic blood serum metabolic material group being obtained by this method, can significantly improve downtrod embryo under high sugar is intervened
Developmental rate, promotes fetal development.
Brief description
Bolus as a Kidney-Yin-Tonic serum of the present invention and the BPI figure of blank serum that Fig. 1 detects for UPLC-Q-TOF/MS, wherein, Fig. 1 (A)
For Bolus as a Kidney-Yin-Tonic blood serum sample of the present invention；Fig. 1 (B) is blank serum samples.
Fig. 2 is the multivariate data analysis figure being carried out based on the chemical composition of UPLC-Q-TOF/HDMS detection, wherein, Fig. 2
(A) it is that administration group (Group 1) is analyzed through OPLS-DA with blank control group (Group 2) serum UPLC-Q/TOP-MS data
Shot chart；Fig. 2 (B) schemes for the S-Plot that OPLS-DA analyzes；Fig. 2 (C) be retention time _ karyoplasmic ratio be 8.31-183.0978 from
The changing trend diagram of son.
The blastaea figure of 4.5dpc period normal blastaea and high sugar culture fluid culture, (note in figure under Fig. 3 light microscopic：A. blank group；
B.20mmol/L glucose group；C.40mmol/L glucose group；D.60mmol/L glucose group；E.80mmol/L glucose group).
The 2- cytological map of 1.5dpc period normal 2- cell and high sugar culture fluid culture, (note in figure under Fig. 4 light microscopic：A. empty
White group；B.20mmol/L glucose group；C.40mmol/L glucose group；D.60mmol/L glucose group；E.80mmol/L Fructus Vitis viniferae
Sugared group).
Specific embodiment
Experimental technique used in following embodiments if no special instructions, is conventional method.
Material used, reagent etc. in following embodiments, if no special instructions, all commercially obtain.
Bolus as a Kidney-Yin-Tonic medical material is purchased from responsible company of Shanxi chain pharmacy company limited of Beijing Tongrentang, Radix Rehmanniae Preparata (Henan, 240g),
Rhizoma Dioscoreae fries (Henan, 120g), Fructus Lycii (Ningxia, 120g), Fructus Corni (Zhejiang, 120g), Radix Cyathulae wine wash cook (Sichuan, 90g),
Colla cornus cervi (120g), Colla Plastri Testudiniss (120g), Semen Cuscutae (Inner Mongol, 120g).
Female sd inbred rats are purchased from National Institute for Food and Drugs Control, credit number：SCXK (capital) 2009-0017.
Embodiment 1, the preparation of Bolus as a Kidney-Yin-Tonic blood serum sample and component analyses
First, the preparation of Bolus as a Kidney-Yin-Tonic blood serum sample
Prepare Bolus as a Kidney-Yin-Tonic blood serum sample in accordance with the following steps：
1st, the preparation of Bolus as a Kidney-Yin-Tonic Aqueous extracts
According to Radix Rehmanniae Preparata：Rhizoma Dioscoreae：Fructus Corni：Fructus Lycii：Semen Cuscutae：Colla cornus cervi：Colla Plastri Testudiniss：Radix Cyathulae=8：4：4：4：4：4：
4：3 mass ratio weighs each medical material.In addition to Colla cornus cervi and Colla Plastri Testudiniss, each medical material is mixed and adds water to decoct three times at 50 DEG C,
Wherein, decoct for the first time as taking 8 times amount (w/v) decocting to boil 1.5h, decoct as taking 8 times amount (w/v) decocting to boil 1.5h for the second time, the
Decoct for three times as taking 8 times of water yields (w/v) to decoct 1.5h, merge three leachates and cross eight layers of filtered through gauze.Take Colla Plastri Testudiniss, Colla cornus cervi
Add suitable quantity of water heating to make dissolving to beaker, be placed in pan-fried well-done medicinal liquid after glue class all dissolves, by Bolus as a Kidney-Yin-Tonic decocting
Liquid is put into and is concentrated into crude drug amount in water-bath is 1g mL-1.
2nd, the preparation of Bolus as a Kidney-Yin-Tonic blood serum sample
Rat is normally raised 5d, is divided into Bolus as a Kidney-Yin-Tonic administration group and blank group.Next day weighs and according to body weight by raw medicinal herbs
Amount 20g kg-1·d-1Dosage gives administration group Bolus as a Kidney-Yin-Tonic Aqueous extracts respectively, blank group distilled water, successive administration 7d, and daily 1
Secondary, weigh before being given daily, strictly control dosage, administering mode is gavage.Beginning fasting 12h after the completion of 6d gavage, the 7th day
Start timing after gavage, taken a blood sample in 30min, Blood collection is taken a blood sample for ventral aorta.Blood is placed 30min at -4 DEG C,
4000r·min-1Centrifugation 15min, it is stand-by that -75 DEG C of ultra cold storage freezers deposited in by collection serum, you can obtains Bolus as a Kidney-Yin-Tonic serum sample
Product.
2nd, Bolus as a Kidney-Yin-Tonic blood serum sample component analyses
1st, sample pretreatment
Take Bolus as a Kidney-Yin-Tonic blood serum sample 2mL, add phosphatase 24 0 μ L, concussion mixes, be loaded to and lived with 3mL methanol, 3mL water in advance
Change on the SPE post having balanced, use 2mL water wash respectively, leacheate discards, then with 2mL methanol-eluted fractions, collect eluent, in 37
Under DEG C water-bath, nitrogen stream dries up, and residue 100 μ L 50% methanol redissolves, ultrasonic 1min, after the concussion 30s that is suspended, 4 DEG C,
13000rpm is centrifuged 10min, takes supernatant to be used for UPLC-Q-TOF/MS analysis.
2nd, UPLC-Q-TOF/MS analysis
Waters AcquityTMUPLC chromatograph of liquid (Waters company, the U.S.)；Waters SynaptTM
HighDefinition MS (HDMS/MS) System mass spectrograph (Waters company, the U.S.)；SPE solid-phase extraction column
WatersOasis HLB 3cc 60mg (Waters company, the U.S.).
2.1 chromatographic condition
Chromatographic column：ACQUTITY UPLCTMHSS T3Column (100mm × 2.1mm i.d., 1.8 μm,
WatersCorp, Milford, USA)；Flow velocity 0.4mL/min；40 DEG C of column temperature；Mobile phase：A：0.1% formic acid acetonitrile, B：0.1%
Formic acid water, elution program is shown in Table 1, the equal 5 μ L of sample size.
Table 1 Bolus as a Kidney-Yin-Tonic enters blood component analysis Ultra Performance Liquid Chromatography gradient
In table 1, A：0.1% formic acid acetonitrile；B：0.1% formic acid water
2.2 Mass Spectrometry Conditions
Electric spray ion source (ESI), sweep parameter：Positive ion mode：Capillary voltage 3.0kV, taper hole voltage 30V, carry
3.5V, 110 DEG C of ion source temperature, 350 DEG C of desolvation temperature are pressed in power taking, and taper hole throughput is 50L/h, desolventizing gas flow
600L/h.Exact mass measures and adopts leucine enkephalin (leucine-enkephalin, [M+H]+=556.2771) solution
For lock mass solution；Negative ion mode：Capillary voltage 2.6kV, taper hole voltage 30V, extract voltage 3.5V, ion source temperature
110 DEG C, 350 DEG C of desolvation temperature, taper hole throughput is 50L/h, desolventizing gas flow 600L/h.Exact mass measures and adopts
Leucine enkephalin (leucine-enkephalin, [M-H]-=554.2615) solution is lock mass solution, mass scanning
Scope：m/z 100-1000Da.
The collection of 2.3 finger printing and the extraction at peak
Collection Bolus as a Kidney-Yin-Tonic blood serum sample and blank serum UPLC-Q/TOF-MS chromatogram under negative ions pattern,
See Fig. 1.The UPLC-Q-TOF/MS chromatogram of collection administration sample and its prescription medicine simultaneously, for entering to constituents absorbed into blood
Row source ownership and the information providing material base for Structural Identification.
Using MarkerLynx V4.1 software (Waters company, the U.S.) to blank serum and Bolus as a Kidney-Yin-Tonic blood serum sample
UPLC-Q/TOF-MS data carries out the extraction at peak.Parameter is as follows：Detection time 0.1-18.0min, mass range 100-
1000Da, retention time disparity range 0.2min, mass number disparity range 0.05Da, noise elimination of level 6, peak intensity threshold value
100.The data extracted is to include retention time tR, the three-dimensional data matrix of ion karyoplasmic ratio m/z and peak intensity, further should
Carry out principal component analysiss (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) with EZinfo 2.0 plug-in unit.OPLS-
DA obtains S-plot, and in " S " figure, upper end and lower end represent the ion of the high expression level in blank and administration group respectively.
Simultaneously according to the corresponding trendgram of each ion (trend plot), thus screening blank group is zero, and the ion of administration group non-zero
Enter blood component ion as Bolus as a Kidney-Yin-Tonic.
The method of application OPLS-DA multivariate pattern recognition compares administration group and blank group, enters blood with find administration group
Composition.In OPLS-DA shot chart, see Fig. 2 (A) it is seen that blank group and administration group significantly divide into two groups, point out to give a left side
After returning ball, in rat serum, composition there occurs obvious change.In order to find its difference composition, in S-plot figure, as Fig. 2 (B) institute
Show, finding blank group content by trend plot is zero, the ion of administration group content non-zero, and such as Fig. 2 (C) illustrates tR-m/z
For the trend plot of 8.31-183.0978 ion, this ion exists only in the serum of administration group, it can thus be appreciated that this ion is
Bolus as a Kidney-Yin-Tonic enter blood component.
The accurate mass that Bolus as a Kidney-Yin-Tonic through OPLS-DA analysis mining is entered with blood component ion carries out elementary composition analysis, meter
Possible molecular formula, and compare with the chemical information of Bolus as a Kidney-Yin-Tonic and its prescription medicine, Preliminary Identification the results are shown in Table 2.
After table 2 Bolus as a Kidney-Yin-Tonic gives female rats, Contained Serum detects in negative ion mode through UPLC-Q/TOF-MS analysis
Enter blood component information
Have detected 27 in the negative ion mode altogether and enter blood component, including 12 prototype compositions and 15 metabolite.Negative
Under ion mode, enter blood component to 13 and identified, wherein have Sweroside from Fructus Corni, loganin, Monot
Glycosides, Loganic acid, table Loganic acid, from the 5 hydroxymethyl 2 furaldehyde glucuronide of Radix Rehmanniae Preparata, dihydro 5- methylol-
2- furfural glucuronide, Radix Rehmanniae hardship aglycon, from the coumaric acid of Fructus Lycii, from the kaempferol glucose of Semen Cuscutae
Aldehydic acid glycosides, cuscutamine.Bolus as a Kidney-Yin-Tonic serum of the present invention is mainly enriched iridoid glycoside active component, is its important medicine
Effect material base.
Embodiment 2, Bolus as a Kidney-Yin-Tonic serum of the present invention ectogenetic impact on mice embryonic
First, the preparation of Bolus as a Kidney-Yin-Tonic blood serum sample
Prepare Bolus as a Kidney-Yin-Tonic blood serum sample according to embodiment 1 identical step.
2nd, experiment agents useful for same and instrument
All chemical drugss are purchased from Sigma company (St.Louis, MO, USA).PMSG and HCG is purchased from Ningbo City's hormone
Products Co., Ltd, hyaluronidase, operate liquid, KSOM culture fluid, DAPI dyeing liquor, superclean bench (Nu-301-330E English
NUVNR company of state), stereomicroscope (261 Japanese Olympus company), fluorescence microscope (Olympus company of BX60 Japan),
CO2Incubator (3110 Forma company of the U.S.).
3rd, experiment content and method
1st, the collection of germ cell and In vitro culture
Only, after 48h, HCG injection 7.5IU/ only carries out superovulation to lumbar injection PMSG 7.5IU/, and same with sexual maturity
The public Mus copulation of system, morning next day (after 12h) checks vaginal suppository.There is bolt raettin to crane one execution in HCG injection 18h, extract fallopian tube kettle
Abdominal part is placed in the culture dish lid filling embryo medium, is expanded transparent for fallopian tube place and tears with syringe needle under microscope
Broken, the zygote group of separate out is removed granular cell in hyaluronidase, washes in culture fluid after being cleaned with operation liquid as early as possible
Operation liquid is washed away with culture fluid in disk.It is respectively put in normal culture fluid and the culture drop containing high concentration glucose, in 37
DEG C, 5%CO2The CO of saturated humidity2Cultivate in incubator.Count 2 cell number after mating 30h, after 102h, count blastaea number.
2nd, experiment content
Experiment one：By concentration of glucose from 20mmol/L (standard K SOM cultivate liquid hold-up) gradually be transferred to 80mmol/L,
It is divided into 5 groups：Normal group, 20mmol/L glucose group, 40mmol/L glucose group, 60mmol/L glucose group, 80mmol/L Portugal
Grape sugar group.The all 1- cell stages shifting ovum pins once being tested collection are randomized into this 5 groups.Observe different glucose
The impact to mice embryonic early development for the culture fluid.Experiment two：Based on experiment one, by screening, 40mmol/L glucose is taken to make
For high Glyco inhabiting fetal development model.The all 1- cell stages shifting ovum pins once being tested collection are randomized into 4 groups：Normally
Group, high sugar group (40mmol/L glucose), Bolus as a Kidney-Yin-Tonic serum group (40mmol/L glucose adds 5% Bolus as a Kidney-Yin-Tonic serum).Observe left
Return the impact to mice embryonic early development for the ball serum.
3rd, embryo quality evaluation
Inverted microscope dynamically observes mice embryonic early development situation in vitro, carries out morphological observation, with blastocyst rate, sight
Examine the ectogenetic impact on mice embryonic of glucose, Serum Containing Zuogui Pill Substances.
4th, statistical analysiss
Data analysiss SAS8.2 software, compares between measurement data group and is checked with t, experimental data is with means standard deviation (x
± s) represent, with p<0.05 is that difference is statistically significant.
3rd, experimental result
1st, embryonic blastula morphological observation
Shown in Fig. 3, it is the blastaea figure of 4.5dpc period normal blastaea and high sugar culture fluid culture under light microscopic.Normal culture fluid
, more than 70%, 20mmol/L glucose group is similar to blank group, is morphologically no clearly distinguished from for middle blastocyst rate；60mmol/L Portugal
The blastocyst rate of grape sugar group is remarkably decreased (p<0.01) and segmentation cavity is less；The nearly no blastaea of 80mmol/L glucose group, embryo
From 2- cell to 4- cell, most of embryos have rested on 2- cell stage forever.
2nd, embryo 2- morphological observation
Fig. 4 is the 2- cytological map of 1.5dpc period normal 2- cell and high sugar culture fluid culture under light microscopic.Under 40 times of light microscopics
Cannot be distinguished from the form of each group 2- cell, under 100 multiples, find that the 2- cellular morphology of 80mmol/L group has a little change, 2- cell
Rate no difference of science of statistics.
3rd, under Bolus as a Kidney-Yin-Tonic serum group high glucose load to 40mmol/L, the impact of Mouse Embryo Development is as shown in table 3.
The impact to Mouse Embryo Development rate under 40mmol/L glucose load for the table 3 Bolus as a Kidney-Yin-Tonic blood serum metabolic material group
Compared with blank group, #p<0.05, ##p<0.01.
This research selects 40mmol/L as glucose inhibition concentration, and from the point of view of blastocyst rate, Bolus as a Kidney-Yin-Tonic serum group can be effective
Ground promotes fetal development, thus showing that Bolus as a Kidney-Yin-Tonic serum of the present invention can improve embryo quality, excites fetal development potential.
Claims (10)
1. a kind of preparation method of Bolus as a Kidney-Yin-Tonic serum, comprises the steps：(1) medical material of preparation Bolus as a Kidney-Yin-Tonic is returned according to a preparation left side
Carry out water extraction after the formula consumption mixing of ball, obtain Aqueous extracts；(2) animal is administered with described Aqueous extracts, administration is adopted after terminating
Blood, will collect serum, you can obtain described Bolus as a Kidney-Yin-Tonic serum after centrifugal blood；In described dosing step, dosage be 10～
20g·kg-1·d-1, successive administration 7～10 days, daily 1～2 time.
2. a kind of preparation method of Bolus as a Kidney-Yin-Tonic serum, comprises the steps：Take isolated blood, by isolated blood collected after centrifugation blood
Clearly, you can obtain described Bolus as a Kidney-Yin-Tonic serum；Described isolated blood is the isolated blood of following animal：(1) by preparation Bolus as a Kidney-Yin-Tonic
Medical material carries out water extraction according to after the formula consumption mixing of preparation Bolus as a Kidney-Yin-Tonic, obtains Aqueous extracts；(2) described Aqueous extracts are administered to animal,
After administration terminates, obtain final product described animal；In described dosing step, dosage is 10～20g kg-1·d-1, successive administration 7～
10 days, daily 1～2 time.
3. preparation method according to claim 1 and 2 it is characterised in that：In step (1), the crude drug amount of described Aqueous extracts
For (0.5～1) g mL-1.
4. the preparation method according to any one of claim 1-3 it is characterised in that：In step (1), the step of described water extraction
Suddenly as follows：After heating water to Extracting temperature, the medical material put in addition to Colla cornus cervi and Colla Plastri Testudiniss is decocted, and obtains Aqueous extracts I；Take
Colla Plastri Testudiniss, Colla cornus cervi add water heating so that it is dissolved, and mix with described Aqueous extracts I, obtain Aqueous extracts II after all dissolving；Concentrate
Described Aqueous extracts II, you can obtain described Aqueous extracts.
5. the preparation method according to any one of claim 1-4 it is characterised in that：In step (1), described aqueous extraction step
In, Extracting temperature is 50～boiling temperature；Extraction time is 1.0～1.5h；The consumption of water is 1g medical material：(8～10) mL water；Carry
Number of times is taken to be 2～3 times.
6. the preparation method according to any one of claim 1-5 it is characterised in that：In step (2), described animal is female
Mus great and mighty or powerful.
7. the preparation method according to any one of claim 1-6 it is characterised in that：In step (2), described dosing step
In, administering mode is gavage.
8. the preparation method according to any one of claim 1-7 it is characterised in that：Described isolated blood is following animal
Isolated blood：Fasting 12h after the completion of second from the bottom administration, and be dynamic in 30～40min after last administration
Thing.
9. the Bolus as a Kidney-Yin-Tonic serum that the preparation method any one of claim 1-8 prepares.
10. the Bolus as a Kidney-Yin-Tonic serum described in claim 9 has following 1) -2 in preparation) any one of in the medicine of function should
With：
1) improve downtrod embryo development rate under high sugar is intervened；
2) promote fetal development.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107748216A (en) * | 2017-12-01 | 2018-03-02 | 海南椰岛酒业发展有限公司 | The detection method of 4 kinds of functional active components in a kind of Rapid Simultaneous Determination YEDAO LUGUI JIU |
| CN110849999A (en) * | 2019-12-05 | 2020-02-28 | 吴海靖 | Liquid chromatography method for separating 8-epiloganin and loganin |
| CN114767781A (en) * | 2022-04-06 | 2022-07-22 | 广州高博士生物医药研究院有限公司 | Traditional Chinese medicine composition for regulating hypothalamus-pituitary growth promoting function and application thereof |
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| CN103479690A (en) * | 2011-08-23 | 2014-01-01 | 成都中医药大学 | New application of traditional Chinese medicine compound medicinal composition |
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2016
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| CN103479690A (en) * | 2011-08-23 | 2014-01-01 | 成都中医药大学 | New application of traditional Chinese medicine compound medicinal composition |
Non-Patent Citations (2)
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| TEMAKA BAI EL AL: "Zuogui Wan rescues the high-glucoseinduced damaging effects on early embryo development", 《BMC COMPLEMENTARY AND ALTERNATIVE MEDICINE》 * |
| 吴笛等: "左归丸含药血清对血管生成体外三维模型的影响", 《中药药理与临床》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107748216A (en) * | 2017-12-01 | 2018-03-02 | 海南椰岛酒业发展有限公司 | The detection method of 4 kinds of functional active components in a kind of Rapid Simultaneous Determination YEDAO LUGUI JIU |
| CN110849999A (en) * | 2019-12-05 | 2020-02-28 | 吴海靖 | Liquid chromatography method for separating 8-epiloganin and loganin |
| CN114767781A (en) * | 2022-04-06 | 2022-07-22 | 广州高博士生物医药研究院有限公司 | Traditional Chinese medicine composition for regulating hypothalamus-pituitary growth promoting function and application thereof |
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Application publication date: 20170222 |