CN108014152A - A kind of pharmaceutical composition of diastole uterine smooth muscle and preparation method thereof - Google Patents
A kind of pharmaceutical composition of diastole uterine smooth muscle and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a kind of pharmaceutical composition of diastole uterine smooth muscle, it is using Rhizoma Chuanxiong total phenolic acids as active ingredient, is prepared plus pharmaceutically acceptable auxiliary material or complementary component.The invention also discloses a kind of method for extracting Rhizoma Chuanxiong total phenolic acids：(1) Szechwan Lovage Rhizome is added into 65~95% ethanol, ultrasonic extraction 2 times, 30~55min, concentrated extracting solution, obtain Rhizoma Chuanxiong total phenolic acids crude product A every time；Wherein the solid-liquid ratio of Ligusticum wallichii and ethanol is 1:7.5~14g/mL；(2) crude product A is dissolved in ammonium hydroxide, it is 2~3 to adjust pH, and extraction is concentrated to give Rhizoma Chuanxiong total phenolic acids B；Forulic acid yield is 1.87mg/g in Rhizoma Chuanxiong total phenolic acids extract, and chlorogenic acid yield is 0.138mg/g.The purposes of Isolated Rat Uterus smooth muscle contraction inhibitory activity is caused to oxytocin present invention also offers Rhizoma Chuanxiong total phenolic acids, new selection is provided clinically to prevent or treating dysmenorrhoea medicine.Preparation method provided by the invention is reliable and stable, and reappearance is good, and the exploitation for Ligusticum chuanxiong Hort and its preparation provides beneficial foundation and reference.
Description
Technical field
The invention belongs to field of medicaments, and in particular to a kind of pharmaceutical composition of diastole uterine smooth muscle and its preparation side
Method.
Background technology
The dry rhizome of Ligusticum wallichii system Umbelliferae Ligusticum herbaceos perennial Ligusticum wallichii,《Sheng Nong's herbal classic》It is classified as
Product, its gas is aromatic, bitterly, and property pungent-warm, is attributed to liver, courage, pericardium channel, has blood-activating and qi-promoting, wind-expelling pain-stopping, the work(of soothing liver-qi stagnation
Effect, applicating history are long.It is usually used in treating headache, closed dysmenorrhea, the disease such as tumbling and swelling, chest side of body shouting pain.
Ligusticum wallichii mainly contains the components such as volatile oil, phenolic acid class, alkaloid, lactone, polysaccharide and inorganic elements, living at present
Journal of Sex Research more mainly volatile oil and phenolic acid class：
(1) volatile oil：Mainly include alkylbenzene peptide, hydroxy benzenes peptide and benzene peptide dimer etc..Research shows, in Ligusticum wallichii
Alkylbenzene peptide has a light and heat unstability, meets light and is easily polymerized to benzene peptide dimer, heated then be easily converted into hydroxy benzenes peptide.It is modern
Research shows that Ligusticum wallichii benzene peptide constituents have anticonvulsion, antithrombus formation, reduce blood viscosity, adjust cardiac function, prevent
Cerebral ischemia, calmness and hypnosis and other effects.
(2) phenolic acid class：Rhizoma Chuanxiong total phenolic acids are as an effective component group, the weight especially using forulic acid and chlorogenic acid as representative
Active ingredient is wanted, there are anti-platelet aggregation, antithrombotic, anti-oxidant, removing a variety of pharmacology such as free radical and prevention and treatment of coronary heart disease
Activity.
Ligusticum wallichii clinically frequently as the product of promoting blood circulation for regulating menstruation, more with Ligusticum wallichii compound treatment primary dysmenorrhea, mainly passes through suppression
Uterine smooth muscle processed is excited, to alleviate the dysmenorrhoea caused by the strong contraction of uterus.Current research thinks rhizoma chuanxiong volatile oil to mouse
Isolated uterine smooth muscle contraction has certain inhibitory action, effective active composition group of the Rhizoma Chuanxiong total phenolic acids as Ligusticum wallichii, to uterus
The influence of smooth muscle contractile activity there is no report at present.
In addition, on Rhizoma Chuanxiong total phenolic acids extraction process, document《Wang Wenxiang etc., the total phenol extraction of the preferred Ligusticum wallichii of orthogonal experiment
Technique Chinese patent drugs, 2000 (05):The 7-9 pages》Disclose by screening of the orthogonal experiment to Ligusticum wallichii extraction process, obtain compared with
The method of high ferulaic acid content, but the content of its gained forulic acid is only 0.72mg/g, it is still too low.
Therefore, it is badly in need of a kind of side that can extract the Rhizoma Chuanxiong total phenolic acids especially Rhizoma Chuanxiong extract of forulic acid and chlorogenic acid
Method, guidance is provided for industrialization extraction production.
The content of the invention
To solve the above-mentioned problems, the pharmaceutical composition the present invention provides a kind of diastole uterine smooth muscle and its preparation side
Method, the pharmaceutical composition be using Rhizoma Chuanxiong total phenolic acids as active ingredient, the present invention provides Rhizoma Chuanxiong total phenolic acids extracting method and its
The new application of diastole uterine smooth muscle, to provide more medicament selection approach for clinical remission dysmenorrhoea.
The present invention provides a kind of method for extracting Rhizoma Chuanxiong total phenolic acids, step are as follows：
(1) Szechwan Lovage Rhizome is added into 65~95% ethanol, ultrasonic extraction 2 times, 30~55min every time, concentrated extracting solution, obtains
Rhizoma Chuanxiong total phenolic acids crude product A；Wherein the solid-liquid ratio of Ligusticum wallichii and ethanol is 1:7.5~14g/mL；
(2) crude product A is dissolved in ammonium hydroxide, it is 2~3 to adjust pH, and extraction is concentrated to give Rhizoma Chuanxiong total phenolic acids B.
Wherein, in step (1), the concentration of the ethanol is 65%~80%；And/or the ultrasonic extraction time is every
Secondary 45~55min；And/or the solid-liquid ratio of the Ligusticum wallichii and ethanol is 1:9~14g/mL.
Preferably, the concentration of the ethanol is 65~75%, is preferably 65%；And/or the ultrasonic extraction time is every
Secondary 55min；And/or the solid-liquid ratio of the Ligusticum wallichii and ethanol is 1:13g/mL.
Wherein, in step (2), the mass volume ratio of the crude product A and ammonium hydroxide are 1:40~50g/mL；The ammonium hydroxide it is dense
Spend for 80%.
Wherein, in step (2), the extraction is to utilize ethyl acetate, chloroform or dichloromethane extraction.
Wherein, in step (2), the yield of the Rhizoma Chuanxiong total phenolic acids B is 4.63%.
Wherein, forulic acid yield is 1.87mg/g in the Rhizoma Chuanxiong total phenolic acids extract, and chlorogenic acid yield is 0.138mg/
g。
The Rhizoma Chuanxiong total phenolic acids that the above method is prepared.
Present invention also offers above-mentioned Rhizoma Chuanxiong total phenolic acids extract to prepare the medicine with the effect of diastole uterine smooth muscle
In application.
Present invention also offers application of the above-mentioned Rhizoma Chuanxiong total phenolic acids extract in the medicine for preparing treatment dysmenorrhoea.
The extracting method of Rhizoma Chuanxiong total phenolic acids of the present invention, by the cooperation of concentration of alcohol, liquid ratio and ultrasonic time, reaches
The effectively purpose of extraction Rhizoma Chuanxiong total phenolic acids, extract average yield reach 4.63%, and the content of the component such as forulic acid is high, preparation side
Method is reliable and stable, and reappearance is good.Rhizoma Chuanxiong total phenolic acids of the present invention cause Isolated Rat Uterus smooth muscle contraction to have suppression oxytocin
System activity, can be with diastole uterine smooth muscle, and then prevents or treat dysmenorrhoea, and new selection is provided for clinical medicine.
Obviously, the above according to the present invention, according to the ordinary technical knowledge and customary means of this area, is not departing from
Under the premise of the above-mentioned basic fundamental thought of the present invention, the modification, replacement or change of other diversified forms can also be made.
The embodiment of form by the following examples, remakes further specifically the above of the present invention
It is bright.But the scope that this should not be interpreted as to the above-mentioned theme of the present invention is only limitted to following example.It is all to be based on the above of the present invention
The technology realized belongs to the scope of the present invention.
Brief description of the drawings
Fig. 1 is influence of the Rhizoma Chuanxiong total phenolic acids to Isolated Rat Uterus contraction movement power caused by oxytocin in experimental example 2；
Fig. 2 is influence of the Rhizoma Chuanxiong total phenolic acids to Isolated Rat Uterus shrink tension average value caused by oxytocin in experimental example 2；
Inhibitory action of the Fig. 3 for Rhizoma Chuanxiong total phenolic acids in experimental example 2 to Isolated Rat Uterus contraction movement power caused by oxytocin
Figure；
Fig. 4 is suppression of the Rhizoma Chuanxiong total phenolic acids to Isolated Rat Uterus shrink tension average value caused by oxytocin in experimental example 2
Action diagram；
Inhibitory action figures of the Fig. 5 for Rhizoma Chuanxiong total phenolic acids in experimental example 2 to Isolated Rat Uterus contraction movement caused by oxytocin.
Embodiment
The raw material that is used in the specific embodiment of the invention, equipment are known product, are obtained by buying commercial product.
1st, laboratory apparatus
Agilent LC-1100 type high performance liquid chromatographs：Agilent companies；
FR2004b type analysis balances：Shanghai Yue Ping scientific instrument Co., Ltd；
Electronic analytical balance：AUW220D SHIMADZU CORPORATION；
HX-200 type high speed medicinal herb grinders：Zhejiang Province Yongkang City small stream bank hardware medical instrument factory；
SB25-12D ultrasonic washing instruments：Ningbo Xin Yi ultrasonic devices Co., Ltd.
2nd, medicinal material and reagent
Ligusticum wallichii decoction piece：Sichuan New-green Pharmaceutical Technology Development Co., Ltd. provides, and it is perennial to be identified as Umbelliferae
The rhizome of herbaceous plant Ligusticum wallichii Ligusticum chuanxiong Hort.；
Forulic acid：Chengdu Man Site bio tech ltd, lot number：MUST-1710908, content 99.32%；
Chlorogenic acid：Chengdu Man Site bio tech ltd, lot number：MUST-17030620, content 99.39%；
Methanol：Chromatographic grade, Sigma companies；
Other reagents used are that commercially available analysis is pure.
The extraction of 1 Rhizoma Chuanxiong total phenolic acids of the present invention of embodiment
Ligusticum wallichii decoction piece is taken, is crushed, precision weighs Szechwan Lovage Rhizome 10.0g, puts in conical flask with cover, adds 13 times of amounts 65%
The ethanol of concentration, ultrasonic extraction 2 times, each 55min, filtering, filtrate concentration are evaporated.Residue adds ammonium hydroxide fully to dissolve, and filters, filter
Liquid concentrated hydrochloric acid tune pH is 2~3, then 3 times (50,40,30mL) are extracted with ethyl acetate, combined ethyl acetate extract, concentration
It is evaporated, residue is total phenolics.
Illustrate beneficial effects of the present invention with the mode of experimental example below：
The screening of 1 Rhizoma Chuanxiong total phenolic acids extraction process of experimental example
1st, total phenol acid content in HPLC methods measure Ligusticum wallichii
1. chromatographic condition
Chromatographic column：Octadecylsilane chemically bonded silica column Eclipse Plus C18 columns：4.6×100mm× 3.5μm.
Forulic acid mobile phase：The aqueous acetic acid of methanol -1% (30：70, v/v)；Flow velocity：1mL/min；Detection wavelength：
323nm；Column temperature：25℃.
Chlorogenic acid mobile phase：The phosphate aqueous solution of methanol -0.1% (18：82, v/v)；Flow velocity：1mL/min；Detection wavelength：
327nm；Column temperature：30℃.
2. the preparation of reference substance solution
Accurately weighed forulic acid and chlorogenic acid reference substance are appropriate respectively, put in 25mL brown volumetric flasks, and forulic acid is with 70%
Methanol dissolves, and chlorogenic acid is dissolved with 50% methanol and is settled to scale, and each reference substance solution is made；
3. the preparation of standard curve
It is accurate respectively to draw forulic acid, chlorogenic acid reference substance solution 0.1mL, 0.2mL, 0.4mL, 0.6mL, 0.8mL, 1mL
In 6 2mL brown volumetric flasks, add corresponding solvent and be settled to scale, shake up, (0.45 μm) filtering of miillpore filter.In chromatography
Under the conditions of 10 μ L of auto injection for HPLC analyze, using sample size X as abscissa, peak area Y is ordinate, obtains forulic acid recurrence side
Cheng Wei：Y=5490.4X-88.759, correlation coefficient r=0.9998, chlorogenic acid regression equation are：Y=1330.1X-2.93, phase
Relation number r=0.9995.The result shows that forulic acid, in 0.1026~1.026 μ g ranges, chlorogenic acid is 0.0236~0.236
In μ g ranges, in good linear relationship；
4. the preparation of test solution
Ligusticum wallichii decoction piece is taken, is crushed, precision weighs Szechwan Lovage Rhizome 10.0g, puts in conical flask with cover, is added by certain liquid ratio
Certain density ethanol, ultrasonic extraction certain time, filtering, filtrate concentration are evaporated.Residue adds ammonium hydroxide fully to dissolve, and filters, filter
Liquid concentrated hydrochloric acid tune pH is 2~3, then 3 times (50,40,30mL) are extracted with ethyl acetate, combined ethyl acetate extract, concentration
It is evaporated, residue is total phenol acid crude.Precision weighs a certain amount of total phenol acid crude, puts brown volumetric flask, respectively with 70% first
Alcohol, the dissolving of 50% methanol are settled to scale, up to test solution；
5. Precision Experiment
Forulic acid, chlorogenic acid is taken to be measured with a reference substance solution by 1. middle chromatographic condition, continuous sample introduction 5 times, often respectively
Secondary 10 μ L, measure peak area, and forulic acid RSD is 0.22% (n=5), and chlorogenic acid RSD is 0.16% (n=5), the results show instrument
Device precision is good；
6. stability experiment
2 parts of same sample is taken, is settled to scale with 70% methanol, the dissolving of 50% methanol respectively, respectively at 0,2,4,6,8,
10h measures forulic acid and chlorogenic acid content, and 10 μ L of sample introduction, measure peak area, and it is 0.47% (n=6) to obtain forulic acid RSD, green original
Sour RSD is 0.75% (n=6), the results showed that test solution is stablized in 10h；
7. sample recovery rate is tested
Take 5 parts of sample solution for having measured content, respectively with forulic acid, chlorogenic acid reference substance solution mixed in equal amounts, by height
Effect liquid phase chromatogram condition measures to obtain forulic acid average recovery rate 99.04%, and RSD is 1.45% (n=5)；Chlorogenic acid averagely recycles
Rate 100.78%, RSD are 2.57% (n=5)
8. repeated experiment
10 parts of same sample is weighed respectively, wherein 5 parts of 70% methanol of use dissolvings, 5 parts of 50% methanol of use dissolvings are settled to quarter
Degree, contained forulic acid, chlorogenic acid content in test solution are measured by high-efficient liquid phase chromatogram condition, continuous sample introduction 5 times, and every time 10
μ L, measure peak area, it is 2.93% (n=5) to obtain forulic acid RSD, and chlorogenic acid RSD is 2.83% (n=5), the results showed that we
The repeatability of method is preferably.
2nd, in Ligusticum wallichii total phenolics extraction process
Precision weighs Szechwan Lovage Rhizome 10.0g, parallel to be tested, and utilizes HPLC methods measure ferulic acid in Chuanxiong, chlorogenic acid
And the content of total phenolics, as shown in table 1.
1 experimental design of table and result
Analyzed according to table 1, experiment condition is：Concentration of alcohol is 65%~95%, is preferably 65%~85%；Liquid ratio is
7.5:1~14:1, it is preferably 10:1~14:1,30~55min of ultrasonic time, are preferably 40~55 min.
Optimal conditions is：Concentration of alcohol 65%, liquid ratio 13:1, ultrasonic time 55min, the content of total phenolics under the conditions of this
It is expected that reach 43.46mg/g.
3rd, process certification
Select optimised process (concentration of alcohol 65%, liquid ratio 13.05:1, ultrasonic time 55min), calculate extract and obtain
Rate, general formula calculate the rate of transform, are shown in Table 2.
2 recovery rate of table and rate of transform result (n=3)
4th, the measure of Rhizoma Chuanxiong total phenolic acids mean transferred rate：
1) Ligusticum chuanxiong Hort ferulaic acid content measures：Szechwan Lovage Rhizome (crossing No. four sieves) about 0.5g is taken, it is accurately weighed, put tool plug cone
In shape bottle, precision adds 70% methanol 50mL, and close plug, weighed weight, is heated to reflux 30min, lets cool, then weighed weight, uses
70% methanol supplies the weight of less loss, shakes up, and stands, takes supernatant to cross 0.45 μm of miillpore filter, take subsequent filtrate, measured with HPLC
Content, the content for obtaining ferulic acid in Chuanxiong are 2.11mg/g.Meet 2015 editions《Chinese Pharmacopoeia》Content regulation under one item is (no
It must be less than 0.10%).
2) assay of Ligusticum chuanxiong Hort chlorogenic acid：Szechwan Lovage Rhizome (crossing No. four sieves) about 0.5g is taken, it is accurately weighed, put tool plug
In conical flask, precision adds 50% methanol 50mL, and close plug, weighed weight, is ultrasonically treated (power 250W, frequency 35Hz) 30min,
Let cool, then weighed weight, the weight of less loss is supplied with 50% methanol, is shaken up, stands, takes supernatant to cross 0.45 μm of miillpore filter,
Subsequent filtrate is taken, measures content with HPLC, the content for obtaining Ligusticum wallichii Content of Chlorogenic Acid is 1.70mg/g.That is Ligusticum chuanxiong Hort total phenol acid content
For 3.81mg/g.I.e. Ligusticum chuanxiong Hort total phenol acid content is 3.81mg/g.
3) Rhizoma Chuanxiong extract total phenolics assay：70% methanol, the test sample of 50% methanol dissolving are drawn respectively with micro-
(0.45 μm) filtering of hole filter membrane.10 μ L of auto injection are analyzed for HPLC under corresponding chromatographic condition.Calculate turning for Rhizoma Chuanxiong total phenolic acids
Shifting rate, total phenol acid content × 100% in total phenol acid content/Ligusticum chuanxiong Hort in total phenol acid transfer rate=Rhizoma Chuanxiong extract.Extract
Average yield is 4.63%, and forulic acid and chlorogenic acid mean transferred rate are respectively 49.14%, 3.63%, total phenolics mean transferred
Rate is 52.78%, RSD 1.66%, illustrates that the rational technology is feasible, reliable and stable, has preferable reappearance.It is shown in Table 2.
The Rhizoma Chuanxiong total phenolic acids extract diastole uterine smooth muscle experiment of the present invention of experimental example 2
1st, experiment material
1. medicine
Rhizoma Chuanxiong total phenolic acids：Every gram of medicinal extract total phenolics containing 43.46mg (prepared by the method for embodiment 1), is made into DMSO
0.1g/mL, it is spare.
2. animal
SD rats, regular grade, complete female, 220~250g of weight, tests Company of Animals Ltd. up to rich fruit by Chengdu and provides, production
Credit number：SCXK (river) 2015-030, is used after quarantine.
3. reagent
Oxytocin injection, producer：Anhui Hongye Medicine Co., Ltd, lot number：140347, it is made into physiological saline water
1U·mL-1, it is spare；
Estradiol valerate tablet, producer：DELPHARM Lille S.A.S., lot number：309A, 0.8mg/mL is made into pure water,
It is spare；
Verapamil hydrochloride, national drug standards material, by the detection of Food In Sichuan Province drug inspection, institute provides, and is matched somebody with somebody with pure water
Into 1 × 10-3The mother liquor of mol/L, it is spare.
NaCl、KCl、CaCl2、NaHCO3, glucose reagent, by Chengdu Ke Long chemical reagents company produce, for
Put Rockwell liquid (1000mL 9.2g containing NaCl, KC1 0.42g, CaCl2 0.24g、 NaHCO30.15g, glucose 1.0g)；
DMSO, producer：Chengdu Ke Long chemical reagents company, lot number：2016102401.
4. laboratory apparatus
16 physiological record systems：P13516 types, Australian Powerlab companies；
In vitro tissue perfusion system：ML0186 types, Australian Powerlab companies；
Electronic balance：FA2004B types, Shanghai Yue Ping scientific instrument Co., Ltd；
Electric-heated thermostatic water bath：DZKW-S-4 types, bright Medical Instruments Co., Ltd forever of Beijing；
Reverse osmose pure-water instrument：UPT-II-20T types, Chengdu Ultra Pure Science ＆ Technology Co., Ltd；
Adjustable pipette：Thermo Fisher Scinentific types, Shanghai monarch's wing experimental instruments and equipment limited.
2nd, experimental method
Health, the non-pregnant rat of Adult female are taken, the daily dosage by 8mg/kg on the 2nd carries out gavage Estradiol Valerate before experiment
Solution, to cause estrus rat model.Animal is put to death in cervical dislocation on the 3rd, uterus is taken rapidly and is placed in and fill Rockwell liquid
Glass dish in, gently peel off the connective tissue and adipose tissue had on uterus.Then 2~3cm of interlude uterus is taken, one
The bath for filling Rockwell liquid 20mL is fixed at end, and other end connection tonotransducer, heavy burden 1g, keeps the nutrient solution in bath to exist
(37 ± 0.5) DEG C, and pass to 95%O2+ 5%CO2Mixed gas, 1~2 minute bubbles/s of speed, with 16 passage physiology remember
Recording system records uterine contractile curve.After curve stablizes 30min, after adding 1U/mL oxytocins 160 μ L, modeling 10min, give
Rhizoma Chuanxiong total phenolic acids are given, are administered according to basic, normal, high dosage (final concentration of：0.025mg/mL, 0.05mg/mL, 0.1mg/mL,
DMSO is blank control group, and verapamil hydrochloride (Ver) is positive drug, according to isometric administration, positive drug it is final concentration of：
5×10-7mol/L；After administration, the change of isolated uterine smooth muscle contractile activity is observed, records 10min, 3 are rinsed with Rockwell liquid
After secondary, then carry out Germicidal efficacy next time.
Before carrying out paired t-test statistical analysis modeling with SPSS19.0 statistical softwares with after modeling and after modeling with administration
Change afterwards, and the inhibiting rate after administration is calculated, calculation formula is：Inhibiting rate (%)=(parameter after parameter-administration before administration)
Parameter × 100% before ÷ administrations, as a result represents, comparison among groups is analyzed using one-way ANOVA, P with (mean ± SEM)<
0.05 indicates significant difference, P<0.01 indicates pole significant difference.
3rd, experimental result and evaluation
There is strong contraction immediately after oxytocin 160U/mL is given in normal rat isolated uterine, and shows as contraction movement
Power, shrink tension average value are significantly raised, there is significant difference (P compared with before modeling<0.05 or P<0.01), and to shrinking
Frequency has no significant effect.Add various concentrations Rhizoma Chuanxiong total phenolic acids and be respectively acting on uterine smooth muscle 10min, can be different degrees of
The strong contraction of rat uterus smooth muscle caused by ground confrontation oxytocin, as shown in table 3, table 4 and Fig. 1, Fig. 2.
3 Rhizoma Chuanxiong total phenolic acids of table to Isolated Rat Uterus contraction movement power caused by oxytocin influence (N=6)
Note：Compared with before modeling, * P<0.05, * * P<0.01；Compared with after modeling,▲P<0.05,▲▲P<0.01。
4 Rhizoma Chuanxiong total phenolic acids of table to Isolated Rat Uterus shrink tension average value caused by oxytocin influence (N=
6)
Note：Compared with before modeling, * P<0.05, * * P<0.01；Compared with after modeling,▲P<0.05,▲▲P<0.01。
After adding verapamil hydrochloride, contraction movement power, shrink tension average value can be significantly reduced, and there is pole conspicuousness
Difference (P<0.01)；The Rhizoma Chuanxiong total phenolic acids of various dose are separately added into, can substantially reduce uterine contraction activity power and shrink tension
Average value, and it is respectively provided with significant difference (P<0.05 or P<0.01)；It can be seen that by table 5, Fig. 3 and Fig. 4 in test dose
In the range of, Rhizoma Chuanxiong total phenolic acids are inhibited to Isolated Rat Uterus contraction movement caused by oxytocin, and Rhizoma Chuanxiong total phenolic acids
Inhibitory action to uterine smooth muscle is in dose-effect relationship.
As seen in Figure 5, blank group, when not adding oxytocin, uterus is put down for positive group and Rhizoma Chuanxiong total phenolic acids group
Sliding flesh is in Spontaneous Contraction, and after oxytocin is added, the contraction frequency of the uterine muscle strips of 3 groups, contraction movement power
Significantly raising, after relative medicine is separately added into for each group, the uterine muscle strips contraction movement of 3 groups differs,
It is embodied in：Blank control group shows (Fig. 5-A), adds 10min after DMSO, rat uterine contractility frequency, shrink vigor with
And shrink tension is without significant change；And positive group shows (Fig. 5-B), 10min after VER is added, rat uterine contractility vigor, shrinks
Tension force and the equal conspicuousness of contraction frequency reduce, and Rhizoma Chuanxiong total phenolic acids group is shown (Fig. 5-C), adds 10min after Rhizoma Chuanxiong total phenolic acids, greatly
The uterine contractile vigor of mouse, shrink tension and contraction frequency also decrease, but act on not as good as positive group.
Above-mentioned experiment shows：Blank group DMSO has no significant effect uterine smooth muscle, positive drug VER and Rhizoma Chuanxiong total phenolic acids
It is inhibited to Isolated Rat Uterus contraction movement caused by oxytocin, but Rhizoma Chuanxiong total phenolic acids are to the receipts of uterine smooth muscle
The inhibitory action of contracting is weaker than the inhibitory action of contraction of the positive drug to uterine smooth muscle.
5 Rhizoma Chuanxiong total phenolic acids of table to the inhibitory action of Isolated Rat Uterus contraction movement caused by oxytocin (N=6)
| Group | Dosage | Contraction movement power inhibiting rate (%) | Shrink tension average value inhibiting rate (%) |
| Blank group | — | 13.74±11.71 | 4.74±5.98 |
| Verapamil hydrochloride | 5×10-7mol/L | 92.90±2.54## | 75.77±3.61## |
| Rhizoma Chuanxiong total phenolic acids | 0.1mg/mL | 58.26±10.08## | 52.34±6.12## |
| Rhizoma Chuanxiong total phenolic acids | 0.05mg/mL | 45.65±10.14# | 41.20±4.99## |
| Rhizoma Chuanxiong total phenolic acids | 0.025mg/mL | 31.07±10.07 | 22.09±6.52 |
Note：Compared with blank group,#P<0.05,##P<0.01。
Result of the test shows that instant component is acted on preferable diastole uterine smooth muscle, is clinically screening/preparation
Dysmenorrhoea medicine provides a kind of new selection.
The extracting method of Rhizoma Chuanxiong total phenolic acids of the present invention, by the cooperation of concentration of alcohol, liquid ratio and ultrasonic time, reaches
The effectively purpose of extraction Rhizoma Chuanxiong total phenolic acids, extract average yield reach 4.63%, and the content of the component such as forulic acid is high, preparation side
Method is reliable and stable, and reappearance is good.Rhizoma Chuanxiong total phenolic acids of the present invention cause Isolated Rat Uterus smooth muscle contraction to have suppression oxytocin
System activity, can be with diastole uterine smooth muscle, and then prevents or treat dysmenorrhoea, and new selection is provided for clinical medicine.
Claims (10)
- A kind of 1. method for extracting Rhizoma Chuanxiong total phenolic acids, it is characterised in that：Step is as follows：(1) Szechwan Lovage Rhizome is added into 65~95% ethanol, ultrasonic extraction 2 times, 30~55min every time, concentrated extracting solution, obtains Ligusticum wallichii Total phenol acid crude A；Wherein the solid-liquid ratio of Ligusticum wallichii and ethanol is 1: 7.5~14g/mL；(2) crude product A is dissolved in ammonium hydroxide, it is 2~3 to adjust pH, and extraction is concentrated to give Rhizoma Chuanxiong total phenolic acids B.
- 2. extracting method according to claim 1, it is characterised in that：In step (1), the concentration of the ethanol for 65%~ 80%；And/or the ultrasonic extraction time is 45~55min every time；And/or the solid-liquid ratio of the Ligusticum wallichii and ethanol is 1: 9 ~14g/mL.
- 3. extracting method according to claim 2, it is characterised in that：The concentration of the ethanol is 65~75%, is preferably 65%；And/or the ultrasonic extraction time is each 55min；And/or the solid-liquid ratio of the Ligusticum wallichii and ethanol is 1: 13g/ mL。
- 4. extracting method according to claim 1, it is characterised in that：In step (2), the quality of the crude product A and ammonium hydroxide Volume ratio is 1: 40~50g/mL；And/or the concentration of the ammonium hydroxide is 80%.
- 5. extracting method according to claim 1, it is characterised in that：In step (2), the extraction is to utilize acetic acid second Ester, chloroform or dichloromethane extraction.
- 6. extracting method according to claim 1, it is characterised in that：In step (2), the yield of the Rhizoma Chuanxiong total phenolic acids B For 4.63%.
- 7. extracting method according to claim 1, it is characterised in that：Forulic acid yield in the Rhizoma Chuanxiong total phenolic acids extract For 1.87mg/g, chlorogenic acid yield is 0.138mg/g.
- 8. the Rhizoma Chuanxiong total phenolic acids that claim 1~7 any one method is prepared.
- 9. application of the Rhizoma Chuanxiong total phenolic acids extract in the medicine for preparing diastole uterine smooth muscle described in claim 8.
- 10. application of the Rhizoma Chuanxiong total phenolic acids extract in the medicine for preparing treatment dysmenorrhoea described in claim 8.
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| CN113476489A (en) * | 2020-12-31 | 2021-10-08 | 成都中医药大学 | Ligusticum wallichii volatile oil plaster for treating dysmenorrhea as well as preparation method and application thereof |
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| US20090053338A1 (en) * | 2003-08-20 | 2009-02-26 | Eu Leong Yong | Method for the preparation of phytoprogestogenic extracts from rhizoma ligusticum chuanxiong and uses thereof |
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| US20090053338A1 (en) * | 2003-08-20 | 2009-02-26 | Eu Leong Yong | Method for the preparation of phytoprogestogenic extracts from rhizoma ligusticum chuanxiong and uses thereof |
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| 廖律等: "米糠中阿魏酸的提取及荧光分光光度法检测", 《食品科学》 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113476489A (en) * | 2020-12-31 | 2021-10-08 | 成都中医药大学 | Ligusticum wallichii volatile oil plaster for treating dysmenorrhea as well as preparation method and application thereof |
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