CN110812401A - Traditional Chinese medicine composition for soft tissue injury and detection method thereof - Google Patents
Traditional Chinese medicine composition for soft tissue injury and detection method thereof Download PDFInfo
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- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
Abstract
The invention provides a traditional Chinese medicine composition for acute soft tissue injury, which is characterized by being prepared from the following raw materials in parts by weight: 1-6 parts of raw gardenia, 1-6 parts of raw rhubarb, 1-3 parts of raw white peony root and 1-3 parts of mirabilite. The results of evaluation of injury syndrome index, pathological morphology and tissue water content of model mice show that the composition provided by the invention has the effects of relieving swelling and pain, activating blood and dissolving stasis on acute sprained rats. In addition, the invention establishes a method for simultaneously measuring the content of 7 effective components in the traditional Chinese medicine composition by optimizing an HPLC detection method, and the method is simple, convenient, feasible, stable and reliable, has relatively short detection time, and can be used for process research, quality control and new drug development of the traditional Chinese medicine composition.
Description
Technical Field
The invention relates to the field of traditional Chinese medicines, and in particular relates to a traditional Chinese medicine composition for soft tissue injury, a preparation method and a detection method thereof.
Background
Acute soft tissue injury refers to the injury of soft tissues (such as muscles, tendons, ligaments and blood vessels) of joints or body parts of four limbs, and the condition of fracture, dislocation, skin and flesh damage and the like does not exist. The clinical manifestations are pain and swelling of injured parts, blood stasis generation under the skin, obvious tenderness and limited joint movement, or red, green and purple colors, which are usually found in waist, ankle, knee, shoulder, wrist, elbow, hip, etc. This is caused by severe exercise, improper posture with heavy load, or accidental falling, dragging, and excessive twisting, which may cause the skin, muscle and tendons in some parts to be damaged, resulting in obstruction of meridians, obstruction of qi flow, and stagnation of blood stasis in local parts. The traditional Chinese medicine theory considers that the acute soft tissue injury belongs to the category of 'muscle injury' in traditional Chinese medicine, and reflects the pathogenic characteristics that 'though the muscle injury is outside, qi and blood channels and collaterals, and viscera are injured to cause the whole body reaction of an organism', and 'bones are dry, the muscles are rigid, the muscles and bones are connected, and all muscles and bones are strong from the bones and the muscles, so the muscle injury can damage the bones, and the bones are injured to hurt the muscles and the muscles, wind-induced spasm and urgency' and the like. The pathogenesis is that local stagnation of qi and blood or blood stasis, bad qi movement and blockage of channels and collaterals cause swelling and pain in the initial stage, the injured muscles, tendons and fascia can be changed by broken tendons, ligaments and fascia, and the serious symptoms are serious because the muscles and bones are damaged; if the blood stays inside or is attacked by exogenous pathogenic factors after the injury, the blood stasis can be transformed into heat, or the blood stasis is transfused and the heat toxin is accumulated, so that the body can be caused to have symptoms such as fever or inflammatory infection; in the later stage, the injured part is susceptible to wind-cold-dampness, obstruction of the meridians or failure of the muscles to nourish, and adhesion degeneration can occur, which leads to chronic injury diseases such as tendon strength, tendon thickness, tendon knots, tendon atrophy or various kinds of arthralgia. The principle of treatment is hemostasis, pain relieving, swelling preventing and swelling subsiding, and the treatment is carried out by adopting external application, taking internal medicines with the functions of clearing heat and cooling blood, promoting qi and activating blood, and relieving pain and swelling, or cold compress, pressure dressing, braking and lifting affected limbs.
With respect to the treatment of acute soft tissue injury, the current internationally accepted early treatment principle is PRICE, namely: protection (protection), rest (rest), ice compress (icing), compression, elevation of the injured limb (elevation). The foreign treatment method is generally that in the acute stage, if discontinuous pain points are detected, quick-freezing agent is sprayed for 30 seconds in a traction state, and anti-inflammatory drugs are externally applied. For example, non-steroidal anti-inflammatory drugs (NSAIDs) 1% procaine and cortisone acetate suspension, fenbide or sitagliptin belong to peripheral analgesics, are suitable for mild and moderate pain, and have the main functions of relieving fever, easing pain and diminishing inflammation. Although the foreign therapy has quick response, the treatment is only symptomatic treatment, and most of the foreign therapy is local pain point injection drugs or oral non-steroidal drugs, which easily cause headache, dizziness, tinnitus, hypopsia, lethargy or insomnia, gastrointestinal reaction and anaphylactic reaction, and some patients also have the side effects of granulocytopenia, aplastic anemia or blood coagulation disorder and the like. Therefore, the development of external drugs for treating acute sprain is significant.
Disclosure of Invention
The invention aims to provide a traditional Chinese medicine composition for treating soft tissue injury, which is simple in formula and low in cost, aiming at the technical problems, and the invention is realized by the following technical scheme:
the traditional Chinese medicine composition for soft tissue injury is characterized by being prepared from the following raw materials in parts by weight: 1-6 parts of raw gardenia, 1-6 parts of raw rhubarb, 1-3 parts of raw white peony root and 1-3 parts of mirabilite. The composition can be directly ground into powder, or made into extract or other forms by conventional method. The soft tissue injury is preferably acute soft tissue injury.
Further, the traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 3-6 parts of raw gardenia, 3-6 parts of raw rhubarb, 1-2 parts of raw white peony root and 1-2 parts of mirabilite.
Preferably, the traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 6 parts of raw gardenia, 6 parts of raw rhubarb, 2 parts of raw white peony root and 1 part of mirabilite.
The invention also provides a preparation method of the traditional Chinese medicine composition, which is characterized by weighing 1-6 parts of raw gardenia, 1-6 parts of raw rhubarb, 1-3 parts of raw white peony root and 1-3 parts of mirabilite, adding 60-90% ethanol solution which is 6-10 times of the total weight of the raw materials for reflux extraction, carrying out reflux extraction for 30-120 min, filtering, adding 4-8 times of 60-90% ethanol solution for reflux extraction of medicine residues, carrying out reflux extraction for 30-120 min, and filtering; mixing the two filtrates, concentrating to obtain soft extract or further drying to obtain fine powder.
The invention also provides application of any one of the compositions in preparing a medicament for treating soft tissue injury.
The invention also provides a medicine for treating soft tissue injury, which is characterized by comprising any one of the compositions and pharmaceutically acceptable auxiliary materials.
Specifically, the auxiliary materials are selected from: binder, humectant, cross-linking agent, penetration enhancer, excipient, preservative, acidity regulator, pH regulator or other pharmaceutically acceptable carrier materials.
The medicine can be prepared into oral administration dosage forms, injection administration dosage forms or external administration preparations by using various pharmaceutically acceptable auxiliary materials. The oral administration dosage forms comprise decoction, granules, capsules, tablets, oral liquid, pills, tinctures, syrups and the like. Preferably, the medicament may also be a cataplasm, a gel patch, an emulsion, a paste or a spray, etc.
The invention also provides a detection method of the traditional Chinese medicine composition for treating soft tissue injury, which comprises the following steps:
preparing the traditional Chinese medicine composition into a test solution, wherein the test conditions of the test solution are as follows: a chromatographic column: kromasil 100-5C18(4.6 mm. times.250 mm,5 μm); mobile phase: acetonitrile (A) -0.1% phosphoric acid water solution (B), and gradient elution (0-11 min, 15% A-85% B;11-43 min, 15% A-85% B → 80% A-20% B; 43-60 min, 15% A-85% B), flow rate of 1.0mL min-1The detection wavelength is 230 nm.
Further, the method further comprises: preparing control solution from paeoniflorin, rhein, emodin, chrysophanol, aloe-emodin, and physcion, and detecting the control solution according to the above detection conditions.
The results of evaluation of injury syndrome index, pathological morphology and tissue water content of model mice show that the composition provided by the invention has the effects of relieving swelling and pain, activating blood and dissolving stasis on acute sprained rats.
Acute (within 48 h) soft tissue injury, aiming at the pathogenesis, the treatment mainly takes the functions of promoting blood circulation to remove blood stasis, and relieving swelling and pain. Raw gardenia in the invention is a monarch drug, has the functions of detumescence and acesodyne for external use, is commonly used for treating sprain and contusion, iridoid glycoside components represented by jasminoidin have a plurality of pharmacological activities of analgesia, anti-inflammation, antibiosis, anticoagulation and the like, and jasminoidin is taken as a quality control index of gardenia medicinal materials in 2015 edition of Chinese pharmacopoeia; raw rhubarb is a ministerial drug which can cool blood, remove food retention, relieve swelling and pain, treat traumatic injury, blood stasis abdominal pain, water-fire scald and the like by external application, and is a common drug for treating blood stasis syndrome, wherein anthraquinone components have pharmacological activities of anti-inflammation, antivirus and the like, and rhein, emodin, chrysophanol, aloe-emodin and physcion are used as quality control indexes of rhubarb medicinal materials in the 2015 part of Chinese pharmacopoeia; raw white peony root is an adjuvant drug, has the efficacy of nourishing blood, relieving pain and the like, and is commonly used for treating various pains. Natrii sulfas is used as adjuvant drug for external use for treating pancreatitis, acute mastitis, traumatic swelling and lymphadenectasis. In conclusion, the medicines of the formula are used together to activate blood to remove blood stasis, dredge channels and collaterals, reduce swelling, promote qi circulation, reduce pain, remove blood stasis and swelling pain, dredge channels and collaterals, and heal sprain. The compatibility of the prescription is characterized in that the prescription is refined and closely attached to the pathogenesis, and the compatibility of the prescription is characterized in that the prescription has the functions of promoting blood circulation to remove blood stasis and relieving swelling and pain, the functions of clearing heat and detoxicating, removing blood stasis and dredging channels are combined, raw gardenia has the main functions of cooling blood and detoxicating and relieving swelling, raw rhubarb and mirabilite help the raw rhubarb to purge fire and detoxify, and raw white peony root helps the raw white peony.
Methods for simultaneously measuring the above-mentioned components by HPLC have not been reported. In recent years, the quality control level of Chinese patent medicines is continuously improved, and the quality control system adopting more single index components is gradually developed into a multi-index quality control system. The method for simultaneously measuring the contents of the plurality of effective components in the traditional Chinese medicine composition is established by optimizing an HPLC (high performance liquid chromatography) detection method, is simple, convenient, feasible, stable and reliable, has relatively short detection time, and can be used for process research, quality control and new medicine development of the traditional Chinese medicine composition.
Drawings
FIG. 1 is a graph showing the evaluation of an injury syndrome index of a model mouse according to the present invention;
FIG. 2 is a graph showing the results of the pathomorphology of a model mouse according to the present invention;
FIG. 3 is a graph showing the evaluation of tissue water content in a model mouse of the present invention;
FIG. 4 is an HPLC chart of a test for the detection specificity of the composition of the present invention; wherein the content of the first and second substances,
A. mixing a reference substance, a test substance B, a gardenia negative sample C, a white peony root negative sample D, a rhubarb negative sample E and a blank F;
1. geniposide, 2. paeoniflorin, 3. aloe-emodin, 4. rhein, 5. emodin, 6. chrysophanol, and 7. physcion.
Detailed Description
The present invention will now be described in more detail with reference to specific embodiments thereof so that the aspects and advantages of the invention may be better understood. However, the contents of the specific embodiments described below are for illustrative purposes only and are not limiting of the present invention.
It should be noted that, if the specific conditions are not indicated, the process is carried out according to the conventional conditions or the conditions suggested by the manufacturer, and the raw materials or auxiliary materials used, and the reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available. All percentages, ratios, proportions, or parts are by weight unless otherwise specified.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention.
EXAMPLE 1 preparation of Whole powder
Taking 6 parts (6g) of raw gardenia, 6 parts (6g) of raw rhubarb, 2 parts (2g) of raw white paeony root and 1 part (1g) of mirabilite, crushing and sieving by a crusher to obtain fine powder.
Example 2 preparation of aqueous extract
Taking 6 parts (6g) of raw gardenia, 6 parts (6g) of raw rhubarb, 2 parts (2g) of raw white peony root and 1 part (1g) of mirabilite, adding 8 times of water for reflux extraction, slightly boiling for 1h, filtering, adding 6 times of water for reflux extraction of medicine dregs, slightly boiling for 1h, and filtering. Mixing the two filtrates, and concentrating under reduced pressure to obtain soft extract.
EXAMPLE 3 preparation of alcohol extract
Taking 6 parts (6g) of raw gardenia, 6 parts (6g) of raw rhubarb, 2 parts (2g) of raw white peony root and 1 part (1g) of mirabilite, adding 8 times of 70% ethanol for reflux extraction, slightly boiling for 1h, filtering, adding 6 times of 70% ethanol for reflux extraction of dregs, slightly boiling for 1h, and filtering. Mixing the two filtrates, and concentrating under reduced pressure to obtain soft extract.
Example 4 the Chinese medicinal composition has repercussive, analgesic, blood circulation promoting, and blood stasis dispelling effects on rats with acute sprain
(I) test materials
Animals: SD rats, 70 male, about 300g, were housed in clean-grade animal laboratories of the animal laboratory center of Shanghai university of medicine.
The tested drugs are: full powder (10%) drug: the cataplasm with the drug loading of 10 percent is prepared by adopting the method of example 1; water extraction (10%) drugs: the cataplasm with the drug loading of 10 percent is prepared by adopting the thick extract as the raw material after the preparation by adopting the method of the embodiment 2; alcohol extraction low dose (10%) drug: the cataplasm with the drug loading of 10 percent is prepared by adopting the method of the embodiment 3; alcohol booster dose (20%) drug: the cataplasm with the drug loading of 20 percent is prepared by adopting the method of example 3.
Positive control drug: compound redbud cataplasm for wound healing, shanghai leiyun pharmaceutical industry ltd, batch number: 170202.
the other reagents are purchased from chemical reagents of national medicine group, Inc., and the purity is analytical purity.
(II) Experimental method
1. Animal grouping: the groups were randomly divided into 7 groups of 10 individuals. Respectively as normal control group, matrix control group, Chinese medicinal composition group (whole powder with drug loading of 10%, water with drug loading of 10%, alcohol with drug loading of 10% and drug loading of 20%), and positive medicinal composition group (compound Cercis chinensis cataplasma) control group. Namely:
(1) 10 Normal control groups (Normal);
(2) 10 of the Vehicle control group (Vehicle);
(3) full powder (10%) drug treatment group (QF 10%) 10;
(4) water extraction (10%) drug treatment group (ST 10%) 10;
(5) alcohol extraction low dose (10%) drug treatment group (CT 10%) 10;
(6) alcohol boost dose (20%) drug treatment group (CT 20%) 10;
(7) 10 positive drugs (compound redbud wound-eliminating cataplasm) control groups (ZJ).
2. Acute soft tissue injury model:
anesthesia: weighing and numbering the rats, and performing respiratory anesthesia by using isoflurane;
depilation: after the anesthesia is effective, the hair of the lower limbs is shaved to the root of the thigh, the hair removal paste removes the hair of the lower limb (to the knee joint), and the residual hair removal paste is removed by wiping with 75% ethanol; and (3) damage: the gastrocnemius muscle was cut and the right lower limb was padded up with a cushion pad (5 cm x 5cm adult diaper) to prevent it from hanging in the air. The right lower limb is slightly rotated outwards to place the posterior muscles of the lower leg on the inner sides of the tibia and the fibula, a fixer (a 50ml centrifuge tube, the bottom of a conical tube is downward, and the tip of the tube is wrapped by a band-aid to prevent skin injury caused by friction) is placed above the gastrocnemius of the lower leg, and the tip is placed at a fixed position. Freely dropping a 500g weight from a height of 45cm along a pipeline, acting on the upper part of the fixer to cause one-time muscle contusion, repeating for 10 times to cause blunt injury to the soft tissue of the right hind limb of the rat; the left hind limb was not treated for any injury as a control.
3. Administration: the cataplasm to be tested is cut into a trapezoidal cataplasm with the top side of 2cm, the bottom side of 4cm and the height of 2cm, the top side corresponds to the distal end of a limb, the part with the damaged gastrocnemius muscle is wrapped, the rubberized fabric is used for fixing the cataplasm on the outer layer, and the dressing change is carried out once a day (24 h).
4. Soft tissue injury scoring:
degree of swelling: no swelling meter is 0 point, mild swelling meter is 1 point, and severe swelling meter is 2 points;
subcutaneous blood stasis: the non-blood stasis meter is 0 point, the small amount of the non-blood stasis meter is 1 point, and the large amount of the non-blood stasis meter is 2 points;
dysfunction: the lameness meter is 0 point, the light limping meter is 1 point, and the heavy limping meter is 2 points;
tenderness response: no tenderness meter for 0 point, mild tenderness meter for 1 point, and obvious tenderness meter for 2 points.
5. Evaluation of pathological morphology: 3 percent sodium pentobarbital is used for intraperitoneal injection and anesthesia at the concentration of 0.1ml/100 g;
right lower limb gastrocnemius muscle (1/3 for histopathological examination, 1/3 for tissue water content determination) was taken and evaluated pathomorphologically, no significant histopathological changes were counted as 0 point, and 1-3 points were counted according to subcutaneous and myofibroedema, blood stasis, capillary hyperplasia and infiltration of inflammatory cells such as neutrophils.
6. Detecting the water content of the tissue: measuring the water content of the tissue by using a wet-weight method, taking the tissue of the fixed part of gastrocnemius, weighing the wet mass by using an electronic balance, baking the wet mass in a constant-temperature dry oven at 110 ℃ for 48h to constant mass, weighing the dry mass by using the electronic balance, and calculating the water content of the muscle tissue according to an Eliliot formula:
Pwater content＝(mWet-mDry matter)/mWet×100％
(III) results of the experiment
1. Evaluation of Damage syndrome index
As shown in fig. 1, each composition group had a therapeutic effect on model mice as compared with the blank matrix group, and from the results of Dysfunction (Dysfunction, (a) group), the 10% water extract was most effective when administered 24 hours after the treatment, the positive drug was most effective after 48 hours, and 10% whole powder was most effective after 72 hours; from the result of Tenderness reaction (Terdenress, (b) group), no obvious treatment effect exists within 24h, 10% of whole powder has obvious curative effect within 48h, and the positive medicine has the best curative effect after 72 h; from the degree of Swelling (Swelling, (c) group), no obvious treatment effect exists within 24h, and the Swelling condition is relieved after 72h of administration; from subcutaneous blood stasis (ecchymosis, (d) group), both 72h and 24h of administration were therapeutically effective, but the difference between groups was not significant.
2. Evaluation of pathomorphology
As shown in fig. 2, from the pathological Morphology (Morphology) results, the 20% alcohol extract showed the best therapeutic effect, and the second group was the positive group, in which the 10% alcohol extract showed a significant therapeutic effect compared to the matrix group.
3. Tissue hydration evaluation
As shown in fig. 3, from the water content results, the 20% alcohol extract has obvious detumescence effect, and the 10% whole powder and 10% alcohol extract have obvious curative effect in all experimental groups compared with the matrix group.
Example 5 detection method of a Chinese medicinal composition for treating acute soft tissue injury
Instrument and reagent
1. Instrument for measuring the position of a moving object
Agilent1260TF-2017016 high performance liquid chromatograph, model ML203T/02 analytical balance (Mettler-Torledo instruments, Inc.); an analytical balance model XS105 (mettler-toledo instruments ltd); SY-360H ultrasonic cleaner (Shanghai Chengxiang instruments, Inc.); model DFY-1000 swing mill (wenzhou zengdian medical devices ltd); Milli-Q type ultrapure water preparation instrument (Mi Rabo China Co., Ltd.); pharmacopeia standard sieve No. 4 (shanghai baocyan laboratory instruments manufacturing ltd); a Centrifuge 5810R type high speed Centrifuge (ibend china limited).
2. Reagent
The control products geniposide (batch No. 110749-201617, content 97.6%), rhein (batch No. 110757-201607, content 99.3%), emodin (batch No. 110756-201512, content 98.7%), chrysophanol (batch No. 110796-201621, content 99.2%), aloe-emodin (batch No. 110795-201609, content 98.1%), physcion (batch No. 110758-201616, content 99.0%), paeoniflorin (batch No. 110736-201640, content 95.2%), all purchased from China food and drug assay research institute. Raw gardenia medicinal decoction pieces (batch No. 170609, Jiangxi, origin, purchased from Shanghai Kangqiao Chinese medicinal decoction pieces Co., Ltd.); crude decoction pieces of radix et rhizoma Rhei (batch No. D1704075, produced in Sichuan, from Sichuan New Lotus herb decoction pieces Co., Ltd.); crude white peony root decoction pieces (batch No. 170328, produced by yao anhui, from shanghai kangqiao decoction pieces ltd); medicinal material extractive solution (batch No. 2018011601, batch No. 2018011602, batch No. 2018011603, batch No. 201801501, batch No. 2018011502, batch No. 2018011503, self-made in laboratory); methanol (batch No. 137467, available from semer feishell science china ltd, chromatographic grade); acetonitrile (batch number JA059930, merck gmbh, chromatographic grade); the rest of the reagents are analytical grade.
(II) methods and results
1. Chromatographic conditions
A chromatographic column: kromasil 100-5C18(4.6 mm. times.250 mm,5 μm); mobile phase: acetonitrile (A) -0.1% phosphoric acid water solution (B), gradient elution (0-11 min, 15% A-85% B, 11-43 min, 15% A-85% B → 80% A-20% B, 43-60 min, 15% A-85% B), flow rate of 1.0mL min-1The detection wavelength is 230nm, the column temperature is 25 ℃, and the sample injection amount is 10 mu L.
2. Preparation of reference stock solutions
Preparing a jasminoidin reference substance stock solution: precisely weighing appropriate amount of geniposide control, and adding methanol to obtain 5.028mg × mL-1(batch No. 2018010501), 1.008 mg/mL-1(batch 2018011801).
Preparation of other control stock solutions: accurately weighing appropriate amount of control substances such as penoniflorin, rhein, emodin, chrysophanol, aloe-emodin, and physcion, and adding methanol to obtain solution with concentration of 1.0060mg mL-1(batch No. 2018010502), 0.0750 mg/mL-1(batch No. 2018010503), 0.0500 mg/mL-1(batch No. 2018010504), 0.1000 mg/mL-1(batch No. 2018010505), 0.0500 mg/mL-1(batch No. 2018010506), 0.0400 mg/mL-1(batch 2018010507).
Preparation of mixed control stock solution: respectively and precisely measuring2.5mL of geniposide stock solution (lot No. 2018010501), 2.5mL of paeoniflorin stock solution (lot No. 2018010502), 5.0mL of rhein stock solution (lot No. 2018010503), 2.5mL of emodin stock solution (lot No. 2018010504), 2.5mL of chrysophanol stock solution (lot No. 2018010505), 2.5mL of aloe-emodin stock solution (lot No. 2018010506), and 5.0mL of physcion stock solution (lot No. 2018010507) in a same 25mL measuring flask, adding methanol to obtain mixed control solution (lot No. 2018010508) containing geniposide, paeoniflorin, rhein, emodin, chrysophanol, aloe-emodin, and physcion, and the concentrations of the mixed control solution are 0.503, 0.101, 0.015, 0.005, 0.010, 0.005, and 0.080mg mL respectively-1。
3. Preparation of test solution
Respectively crushing crude gardenia, crude rhubarb and crude white paeony root medicinal decoction pieces, sieving by a No. 4 standard sieve to obtain powder, respectively weighing 3.0g of crude gardenia powder, 3.0g of crude rhubarb and 1.0g of crude white paeony root powder in the same round-bottom flask, adding 8 times of 70% ethanol, refluxing and extracting for 1 hour, cooling, and filtering; adding 6 times of 70% ethanol, reflux extracting for 1 hr, cooling, and extracting at 4000r min-1Centrifuging at 4 deg.C for 20min, filtering, mixing filtrates, diluting with 70% ethanol to a constant volume of 100mL, mixing, precisely measuring 1mL filtrate, diluting with methanol to a constant volume of 10mL, and collecting the test solution.
4. Specificity test
Preparing negative samples without raw gardenia, raw rhubarb and raw white paeony root according to the prescription proportion and the preparation method respectively, and processing and determining according to the preparation and determination method of the test solution under item 3. The results show that: in the negative sample chromatogram, no chromatographic peak is observed at the corresponding position of retention time of geniposide, paeoniflorin, rhein, emodin, chrysophanol, aloe-emodin and physcion in the sample, which indicates that other medicinal ingredients have no interference to measurement. The peak between 22 and 34min in the chromatogram can be determined to belong to a signal peak caused by solvent phase change by singly feeding the blank solvent methanol, but the determination of the seven components under the liquid phase condition is not influenced by the miscellaneous peaks. See fig. 4.
5. Investigation of linear relationships
The stock solutions of the aforementioned mixed control (lot No. 2018010508) were measured precisely at 8.0, 6.0, 4.0, 2.0, and 0.4mL, and the solutions were quantitatively measured in 10mL measuring flasks with methanol to obtain 6-concentration series of mixed control solutions, and the measurement was performed under the aforementioned chromatographic conditions. Taking the concentration value (X) of the reference substance as an abscissa and the peak area value (Y) as an ordinate to perform linear regression, wherein a regression equation, a linear range and a correlation coefficient are shown in Table 1.
TABLE 17 regression equation, correlation coefficient and Linear Range of the active ingredients
6. Precision test of instrument
Taking the mixed reference solution with the intermediate concentration under the item '5' for continuous sample injection for 6 times, and measuring the peak areas, wherein the RSD of the peak areas of the geniposide, the paeoniflorin, the rhein, the emodin, the chrysophanol, the aloe-emodin and the physcion are respectively 0.8%, 0.9%, 0.5%, 0.2%, 0.4%, 0.3% and 1.4%, and the result shows that the precision of the instrument is good.
7. Stability test
Taking the same sample solution, standing for 1, 2, 4, 6, 8, 10, 12, 24h respectively, and determining according to the above chromatographic conditions, wherein RSD of geniposide, paeoniflorin, rhein, emodin, chrysophanol, aloe-emodin peak areas within 24h are respectively 1%, 0.3%, 0.5%, 0.2%, 2.2%, 0.6%, and RSD of physcion peak areas within 12h is 1.0%.
8. Repeatability test
Preparing 6 parts of test solution in parallel according to the method under item 3, determining the average content of geniposide, paeoniflorin, rhein, emodin, chrysophanol, aloe-emodin, and physcion in the sample according to the chromatographic condition under item 1, respectively 72.86, 31.74, 0.91, 1.88, 0.73, 1.47, and 0.75mg g-1RSD is respectively 3.63%, 3.91%, 2.64%, 3.05%, 3.4%, 3.57% and 3.35%, which shows that the repeatability of the 7 components is better。
9. Sample application recovery test
Taking 9 parts of 0.5mL of the traditional Chinese medicine composition extracting solution, respectively placing the 9 parts into 10mL measuring bottles, respectively and precisely adding a certain amount of reference substances, uniformly mixing, carrying out sample injection measurement, and calculating the recovery rate [ the recovery rate (measured amount-added amount)/the known amount ]. As a result, the average recovery rates of geniposide, paeoniflorin, rhein, emodin, chrysophanol, aloe-emodin and physcion were 100.78%, 97.96%, 100.85%, 104.98%, 101.28%, 100.41% and 101.97%, respectively, and the RSD were 1.8%, 1.5%, 1.6%, 2.1%, 1.4%, 1.8% and 0.8%, respectively. The results are shown in Table 2.
Table 27 sample recovery test results for active ingredients (n ═ 9)
10. Sample assay
Preparing 3 parts of test solution in parallel according to the method under the item 3; preparing 3 parts of water extractive test solution by similar method, respectively, the extractive solution is at 4 deg.C and 4000r min-1And (4) centrifuging for 20 minutes in a centrifuge, sampling and fixing the volume. And (3) according to the chromatographic condition under the item 1, sampling and measuring the peak areas of 7 components, substituting the peak areas into a regression equation, and calculating the contents of geniposide, paeoniflorin, rhein, emodin, chrysophanol, aloe-emodin and physcion in the traditional Chinese medicine composition extracting solution. The results show that 7 components in the alcohol extract of the prescription all show peaks, and no peak is found in the corresponding positions of the chromatogram map of the emodin, chrysophanol, aloe-emodin and physcion in the water extract. See table 3 for details.
TABLE 3 sample determination results of 7 components in water and 70% ethanol extraction test sample solution
The above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.
Claims (10)
1. The traditional Chinese medicine composition for soft tissue injury is characterized by being prepared from the following raw materials in parts by weight: 1-6 parts of raw gardenia, 1-6 parts of raw rhubarb, 1-3 parts of raw white peony root and 1-3 parts of mirabilite.
2. The traditional Chinese medicine composition according to claim 1, wherein the traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 3-6 parts of raw gardenia, 3-6 parts of raw rhubarb, 1-2 parts of raw white peony root and 1-2 parts of mirabilite.
3. The traditional Chinese medicine composition according to claim 1, wherein the traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 6 parts of raw gardenia, 6 parts of raw rhubarb, 2 parts of raw white peony root and 1 part of mirabilite.
4. A preparation method of a traditional Chinese medicine composition is characterized by weighing 1-6 parts of raw gardenia, 1-6 parts of raw rhubarb, 1-3 parts of raw white peony root and 1-3 parts of mirabilite, adding 60-90% ethanol solution which is 6-10 times of the total weight of the raw materials for reflux extraction, carrying out reflux extraction for 30-120 min, filtering, adding 4-8 times of 60-90% ethanol solution for reflux extraction of medicine residues, carrying out reflux extraction for 30-120 min, and filtering; mixing the two filtrates, concentrating to obtain soft extract or further drying to obtain fine powder.
5. Use of a composition according to any one of claims 1 to 3 in the manufacture of a medicament for soft tissue injury.
6. A medicament for soft tissue injury comprising a composition according to any one of claims 1 to 3 and a pharmaceutically acceptable excipient.
7. The medicament of claim 6, wherein the excipient is selected from the group consisting of: binder, humectant, cross-linking agent, penetration enhancer, excipient, preservative, acidity regulator, pH regulator or other pharmaceutically acceptable carrier materials.
8. The medicine of claim 6, which can be prepared into oral administration dosage forms, injection administration dosage forms or external administration preparations by using various pharmaceutically acceptable auxiliary materials, wherein the oral administration dosage forms comprise decoction, granules, capsules, tablets, oral liquid, pills, tinctures and syrups; the external administration preparation is selected from cataplasm, gel plaster, emulsion, paste or spray.
9. A method for detecting a Chinese medicinal composition as claimed in any one of claims 1 to 3, which comprises:
preparing the traditional Chinese medicine composition into a test solution, wherein the test conditions of the test solution are as follows: a chromatographic column: kromasil 100-5C18(4.6 mm. times.250 mm,5 μm); mobile phase: acetonitrile (A) -0.1% phosphoric acid water solution (B), gradient elution (0-11 min, 15% A-85% B, 11-43 min, 15% A-85% B → 80% A-20% B, 43-60 min, 15% A-85% B), flow rate of 1.0mL min-1The detection wavelength is 230 nm.
10. The detection method according to claim 9, characterized in that the method further comprises: preparing paeoniflorin, rhein, emodin, chrysophanol, aloe-emodin and physcion reference substances to obtain reference substance solution, and detecting the reference substance solution according to the detection conditions.
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| CN111429978B (en) * | 2020-03-20 | 2022-12-16 | 广东药科大学 | Method for rapidly discovering effective components of traditional Chinese medicine compound and application |
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