CN108452010B

CN108452010B - Salvianolic acid, pharmaceutical preparation, determination method and medical application

Info

Publication number: CN108452010B
Application number: CN201710102437.XA
Authority: CN
Inventors: 邢为藩; 吴祖栋; 方传祥; 孙南京
Original assignee: Nanjing Chenxiang Medical Research Co ltd
Current assignee: Nanjing Chenxiang Medical Research Co ltd
Priority date: 2017-02-20
Filing date: 2017-02-20
Publication date: 2022-03-01
Anticipated expiration: 2037-02-20
Also published as: CN108452010A

Abstract

The invention discloses a danol, a pharmaceutical preparation, a determination method and a medical application, wherein the danol is prepared by the following production process: rapidly stirring and extracting for 3 times (10 min, 5min and 5min) with water of which the amount is 5 times of 25 ℃/80-85 ℃, combining extracting solutions, finely filtering, adjusting the pH of filtrate to 3.0-4.0 by using dilute hydrochloric acid, standing for 2-4 h, finely filtering, adjusting the pH of filtrate to 5.0-6.0 by using sodium hydroxide solution, passing through No. 2 macroporous resin, separating, washing sequentially by using water, purified water, injection water and low-concentration ethanol, eluting by using dilute ethanol, adjusting the pH of eluent to 2.0-2.5, passing through No. 3 macroporous resin of chromatography, eluting by using 50% ethanol after washing, and spray-drying to obtain white-like to light yellow salvianolic acid; discloses a series of new formulations of injection danphenol; calculating the content of 6 reference substances by external standard method, calculating the content of the rest peak areas by using salvianolic acid B as reference substance, and calculating the sum of the two reference substances as the content of salvianolic acid.

Description

Salvianolic acid, pharmaceutical preparation, determination method and medical application

Technical Field

The invention relates to the technical field of medicines, in particular to a preparation method of a medicine danshenol for resisting cerebral ischemia-reperfusion injury, a medicine composition, a content determination method and clinical application thereof.

Background

In recent years, the incidence of stroke is rising year by year, is one of three current fatal diseases, and has the characteristics of high incidence, high morbidity, high mortality, high disability rate and high recurrence rate. Cerebral apoplexy is divided into cerebral arterial thrombosis and hemorrhagic cerebral apoplexy, the incidence of the cerebral arterial thrombosis is far higher than that of the hemorrhagic apoplexy and accounts for more than 70 percent of the cerebral arterial thrombosis, ischemic brain tissues recover blood flow under certain conditions, and more serious cerebral dysfunction and structural damage occur, which is called cerebral Ischemia-Reperfusion Injury (IRI). The main approach for treating ischemic stroke is to dredge the cerebral vessels and recover the blood supply, and the alleviation of ischemia-reperfusion injury becomes an indispensable important part in the treatment link.

The Saviae Miltiorrhizae radix is dried root and rhizome of Salvia miltiorrhiza Bunge (Salvia millirrhiza Bunge) belonging to Labiatae. Bitter taste, slightly cold nature, heart and liver meridians entered. Has the effects of activating blood circulation to dissipate blood stasis, stimulating the menstrual flow to relieve pain, clearing away the heart-fire and relieving restlessness, cooling blood and eliminating carbuncle. Can be used for treating thoracic obstruction, heart pain, abdominal pain, hypochondriac pain, abdominal mass, pain due to pyretic arthralgia, vexation, insomnia, menoxenia, dysmenorrhea, amenorrhea, and pyocutaneous disease with swelling and pain. It has effects of preventing and/or treating cerebral ischemia and myocardial ischemia, and inhibiting cataract formation.

The inventor has obtained a patent of 'preparation of salvia miltiorrhiza extract, pharmaceutical preparation and content determination method' (patent number: ZL200510037786.5), and the patent technology continues to use 'water boiling' in the 'water boiling alcohol precipitation' classic technology, and 'alcohol precipitation' is abolished. Therefore, the quality, the quantity, the production process and other aspects of the product are greatly different from those of the salvia miltiorrhiza preparation, the patent extraction process is saved, the purity is good, the yield is high, the cost is low, the file release requirement of the drug evaluation center is renamed, the upper limit and the lower limit of the content of the salvianolic acid are required to be established, and the name of the product is approved by the national food and drug administration to be named as the salvianolic acid so as to show the difference from the existing salvia miltiorrhiza preparation. However, the long-time water boiling in the process not only consumes a large amount of energy, but also causes the degradation of main components. In contrast, the inventor has made a breakthrough progress through years of continuous research and innovation: the mixture is quickly stirred and extracted at room temperature/80-85 ℃ in an ultrashort time, 10min is firstly extracted, 5min is extracted for 2 and 3 times respectively, the degradation of main components is reduced by 10-15%, the yield is improved by more than 10%, the water consumption for extraction is optimized, and the extraction efficiency is improved by 2.4 times; the resin model is optimized, and the purity is improved by more than 20%; the fine filtration mode is changed, and the filtration efficiency is improved by 40 times; the vacuum thermal concentration is changed into dilute purified liquid for room temperature column enrichment, the energy is further saved, the whole processes of extraction, separation and purification and concentration are not heated and/or are rarely heated, according to incomplete statistics, calculated according to the annual salvia miltiorrhiza injection produced in China, 58.368 million tons of standard coal can be saved, 63.037 million yuan RMB is saved, 152.92416 million tons of discharged carbon dioxide, 4962.816 tons of sulfur dioxide and 4319.232 tons of nitrogen oxide are reduced, the pollution of the traditional Chinese medicine pharmaceutical industry to the environment is greatly reduced, the yield is nearly 3 times of that of the original patent, and the green extraction process of the salvianolic acid is realized. This patent is a continuation and addition to the previous patent. Compared with the original patent, the main characteristics of the new patent are shown in the table 1.

TABLE 1 main differences between the new patent and the original patent

The production process of the salvianolic acid is proved by trial production and enlargement, the repeatability of each key technical parameter is good, the feasibility is high, the new patented process makes great progress in the aspects of energy conservation and emission reduction, product purity, production period, cost, quality standard and the like, and a solid step is brought for the modernization of the traditional Chinese medicine.

Disclosure of Invention

The invention finds out that the proportions and the contents of various components of the salvianolic acid in the salvia miltiorrhiza, which are generated and converted by the salvianolic acid and the main component thereof, are different or even greatly different in salvia miltiorrhiza produced in different regions, similar regions, different cultivation conditions, different harvesting seasons, different drying modes or different processing methods. The main active component of salvianolic acid B and most related components in the salvia miltiorrhiza are collected after and/or only after the photosynthesis of the salvia miltiorrhiza is stopped, certain small molecular substrates in the salvia miltiorrhiza generate macromolecular salvianolic acid B under the action of certain enzymes, or the salvianolic acid B is degraded to generate danshensu, protocatechuic aldehyde, caffeic acid, alkannic acid, salvianolic acid A and the like. The synthesis conditions and the method thereof include that the target components can meet the requirements by using sunlight intensity for drying, heating for drying, intensive processing or adjusting an extraction process for cold annealing. Adjusting the extraction temperature among the processes is the most effective, most convenient and most economical process. The operation should be moderate, not excessive, and excessive degradation of the relevant components. Therefore, it is necessary to perform a preliminary test before producing salvianolic acid, that is, collecting a proper amount of representative small sample, pulverizing into fine powder, weighing 20g of radix Salviae Miltiorrhizae powder, and adding water

Extracting with 100g hot water under rapid stirring at room temperature/low temperature for 10min, filtering, collecting residue 1, and adding water

Extracting with 100g hot water under stirring at room temperature/low temperature for 5min, filtering, and adding water into the residue 2

80g of hot waterExtracting at room temperature/low temperature under rapid stirring for 5min, filtering, mixing the three filtrates, and standing

Cooling to room temperature, fine filtering, adjusting pH of the fine filtrate with 18% hydrochloric acid

Placing

And h, performing fine filtration, injecting 20 mu l of subsequent filtrate into an HPLC chromatograph, recording a chromatogram, calculating that the content of the salvianolic acid in the salvia miltiorrhiza is not lower than 3.0%, comparing with a control salvia miltiorrhiza HPLC fingerprint, and ensuring that the similarity is not lower than 90.0%, thereby meeting the enterprise internal control quality standard of the salvia miltiorrhiza.

If the root of red-rooted salvia is powdered

The hot stirring extraction result is obviously improved, but still does not meet the internal control standard of an enterprise, and the hot extraction temperature can be properly increased or the hot extraction time can be properly prolonged so as to meet the requirement. Even if the quality standard of the enterprise internal control can not be achieved, if the treatment is continued, the qualified salvianolic acid can be obtained, but the yield is obviously reduced, and the salvia miltiorrhiza is not suitable to be used as a raw material for preparing the salvianolic acid from economic considerations and even can not be used for medicine.

Whether a qualified extract can be obtained is an important step in the preparation of the salvianolic acid, and further work is purification, concentration and drying.

Purifying the salvia miltiorrhiza extract by a primary column chromatography, passing through a No. 2 macroporous resin column (diameter-height ratio is 325: 1200), washing by 5BV purified water, 3BV 3% ethanol and 3BV 10% ethanol in sequence, eluting by 3-5 BV 20% ethanol at the flow rate of 0.3-0.5 BV/h, collecting the eluent containing the salvianolic acid, adjusting the pH to 2.00-2.50 by 18% hydrochloric acid, and finely filtering if necessary for enrichment by a secondary column chromatography;

performing secondary column chromatography enrichment, passing the purified solution with the pH of 2.00-2.50 through a No. 3 resin column (the diameter-height ratio is 325: 300) for enrichment (concentration), washing with 5BV of water for injection, draining off water in the column, washing with 0.8BV of 30% alcohol, draining off, eluting with 2BV of 50% alcohol, collecting the eluate until the eluate is finished, slightly concentrating to ensure that the concentration of the salvianolic acid is 150-200 mg/ml, performing precise filtration, and performing spray drying;

spray drying the concentrated and purified solution by negative pressure spray drying at air inlet temperature of

The air outlet temperature is 75 +/-5 ℃, the rotating speed of a spray head is 250-300 HZ, the feeding speed is 15-30 rp/min, yellowish-yellow powdery danol is obtained, the weight is weighed, and the yield is calculated.

The invention also discloses a group of salvia miltiorrhiza total water-soluble components 'danshinol' instead of a few components with little content in salvia miltiorrhiza.

The invention also discloses the ultra-short time room temperature/low temperature stirring extraction, high-efficiency separation and purification, enrichment without heating a resin column, reduction of degradation of thermosensitive drugs, or possibility of preparing thermosensitive pure products, and greening of the salvianolic acid process to the maximum extent.

The invention also discloses a novel method for preparing the salvianolic acid with similarity of HPLC fingerprint and contrast HPLC fingerprint of the salvia miltiorrhiza more than 90.0% from salvia miltiorrhiza with certain different qualities.

The preparation method of the salvianolic acid disclosed by the invention has good reproducibility, and the similarity of the fingerprint of the salvianolic acid and the fingerprint of the salvia miltiorrhiza is more than 90.0% instead of the retention time or the relative retention time of a plurality of marked components being similar by calculating the product produced in a test by using the similarity judgment software of the chromatographic fingerprint of traditional Chinese medicines.

The invention also discloses that the additive adopts not less than

A method for measuring the content of salvianolic acid in a reference substance,

the sum of the contents of the components is not less than 95 percent.

The invention also discloses a novel pharmaceutical composition of the danphenol, a novel preparation process and a novel technology thereof.

The invention discloses clinical application of a novel pharmaceutical composition of danphenol in the aspects of preventing and treating ischemic cardiovascular diseases, cerebrovascular diseases, cerebral apoplexy convalescence and the like.

Drawings

FIG. 1 shows HPLC chromatogram of fresh Saviae Miltiorrhizae radix extract (compared with control chromatogram, apparently dissimilar, not included in calculation similarity).

FIG. 2, fresh Danshen root plus

HPLC profile of the extract was stirred rapidly with hot water.

FIG. 3 shows HPLC chromatogram of the boiling and rapid stirring extract of Salvia miltiorrhiza.

FIG. 4, HPLC chromatogram of "danol" produced in trial, is to be regarded as the attached drawing of the abstract of the description.

FIG. 5 shows HPLC chromatogram of "Danphenol dripping pill" produced by trial.

FIG. 6 shows HPLC chromatogram of "injectable salvianolic" produced in test.

FIG. 7 shows the aqueous extract of Salvia miltiorrhiza,

HPLC (high performance liquid chromatography) spectrum similarity analysis reports of the extracting solution which is quickly stirred by hot water, the extracting solution which is quickly stirred by boiling the salvia miltiorrhiza, the danol dropping pill and the danol for injection.

FIG. 8 shows the water decoction of Salvia miltiorrhiza,

And (3) rapidly stirring the extracting solution, the danol for injection and the HPLC map similarity calculation result of the danol dropping pill by hot water.

Note: the standard control HPLC profile was synthesized from 9 profiles, which could not be printed and provided.

Detailed Description

The invention is further described below by way of examples, it being understood that these examples are for illustrative purposes only and in no way limit the scope of the invention.

EXAMPLE 1 preparation of Salvianolic acid

Pre-testing: collecting 20g of radix Salviae Miltiorrhizae (collected in spring, subjected to heating, muggy drying, standing, and pulverizing) powder, adding 100g of water, rapidly stirring at room temperature for 10min, filtering, adding 100g of water into residue 1, rapidly stirring at room temperature for 5min, filtering, adding 80g of water into residue 2, rapidly stirring at room temperature for 5min, filtering, mixing the three filtrates, precisely filtering, collecting 20 μ l of the filtrate, injecting into HPLC chromatograph, recording chromatogram, calculating salvianolic acid content of 3.35% by external standard method, and comparing with HPLC fingerprint chromatogram of control radix Salviae Miltiorrhizae to obtain 98.6% similarity. Meets the internal control quality standard of the salvia miltiorrhiza.

Extracting Saviae Miltiorrhizae radix powder 50kg of the same lot of the above preliminary test, adding water 250kg, rapidly stirring for 10min, centrifuging, filtering, adding water 250kg to residue 1, rapidly stirring for 5min, centrifuging, filtering, adding water 200kg to residue 2, rapidly stirring for 5min, centrifuging, filtering, mixing the three filtrates, standing for 4 hr, cooling to room temperature, fine filtering, adjusting pH of the fine filtrate to 3.65 with 18% hydrochloric acid, standing for 15 hr, fine filtering, adjusting pH of the filtrate to 5.61 with 10% sodium hydroxide solution, and purifying with column.

Purifying the above extractive solution with first column chromatography, passing through No. 2 macroporous resin column (diameter-height ratio 325: 1200), sequentially washing with 5BV purified water, 3BV 3% alcohol, and 3BV 10% alcohol, eluting with 2BV 18% alcohol at flow rate of 500 + -100 ml/min, collecting diluted alcohol eluate 160L, adjusting pH to 2.2 with 18% hydrochloric acid, and enriching with second column chromatography

And (3) enriching the purified solution by passing through a No. 3 resin column (the diameter-height ratio is 325: 300), washing with 5BV of water for injection, draining off water in the column, washing with 0.8BV 30% alcohol, draining, eluting with 1BV 50% alcohol, collecting 20L of eluent, slightly concentrating to obtain the concentration of the salvianolic acid 188mg/ml, precisely filtering, and spray-drying.

Spray drying the purified solution, wherein the purified solution is subjected to negative pressure spray drying, and the spray drying parameters are designed as follows: the air inlet temperature is 105-110 ℃, the air outlet temperature is 70 +/-5 ℃, the rotating speed of a spray head is 250-300 HZ, the feeding speed is 15-30 rp/min, yellow powder is obtained, the weight is 1.265kg, and the yield is 2.53%.

EXAMPLE 2 preparation of Salvianolic acid

Pre-testing: taking 20g of powder of salvia miltiorrhiza (dug, dried and crushed in autumn) produced in Qingdao, adding 100g of water, quickly stirring at room temperature for 10min, filtering, adding 100g of water into filter residue 1, quickly stirring at room temperature for 5min, filtering, adding 80g of water into filter residue 2, quickly stirring at room temperature for 5min, filtering, combining three filtrates, carrying out precision filtration, taking 20 mu l of subsequent filtrate, injecting into an HPLC chromatograph, recording a chromatogram, calculating the content of salvianolic acid to be 2.53% by an external standard method, and comparing with a fingerprint chromatogram of a reference salvia miltiorrhiza, wherein the content is obviously dissimilar. The content of the salvianolic acid B is also obviously lower, which does not meet the internal control quality standard of the salvia miltiorrhiza.

And adding 80-85 ℃ hot water into 20g of the salvia miltiorrhiza powder produced in the same batch of Qingdao in the preliminary test for 100g, quickly stirring for 10min, filtering, adding 80-85 ℃ hot water into 100g of filter residue 1, quickly stirring for 5min, filtering, adding 80-85 ℃ hot water into 80g of filter residue 2, quickly stirring for 15min, filtering, combining three filtrates, cooling for 3-4 h, cooling to room temperature, precisely filtering, adding 20 mu l of subsequent filtrate into an HPLC chromatograph, recording a chromatogram, calculating the content of salvianolic acid in the salvia miltiorrhiza by using an external standard method to be 3.49%, comparing the content of salvianolic acid with a standard control salvia miltiorrhiza HPLC fingerprint chromatogram with the similarity of 99.83%, and meeting the internal control quality standard of the salvia miltiorrhiza.

Extracting 50kg of the same batch of the red sage root powder in the preliminary test, adding 250kg of hot water at the temperature of 80-85 ℃, quickly stirring for 10min, centrifugally throwing and filtering, adding 250kg of hot water at the temperature of 80-85 ℃ into the filter residue 1, quickly stirring for 5min, centrifugally throwing and filtering, adding 200kg of hot water at the temperature of 80-85 ℃ into the filter residue 2, quickly stirring for 5min, centrifugally throwing and filtering, combining the three extraction filtrates, standing for 4h, cooling to room temperature, precisely filtering, adjusting the pH of the fine filtrate to 3.65 by using 18% hydrochloric acid, standing for 15h, precisely filtering, adjusting the pH of the filtrate to 5.62 by using 10% sodium hydroxide solution, and performing column chromatography purification once.

And (3) enriching the purified solution by passing through a No. 3 resin column (the diameter-height ratio is 325: 300), washing with 5BV of water for injection, draining off water in the column, washing with 0.8BV of 30% alcohol, draining, eluting with 1BV of 50% alcohol, collecting 20L of eluent, slightly concentrating to obtain the concentration of the salvianolic acid 188mg/ml, precisely filtering if necessary, and spray-drying.

Spray drying the concentrated and purified solution by negative pressure spray drying, wherein the spray drying parameters are designed as follows: the air inlet temperature is 105-110 ℃, the air outlet temperature is 70 +/-5 ℃, the rotating speed of a spray head is 250-300 HZ, the feeding speed is 15-30 rp/min, yellow powder is obtained, the weight is 1.275kg, and the yield is 2.55%.

EXAMPLE 3 preparation of Salvianolic acid

Extracting 50.1kg of the salvia miltiorrhiza powder in the embodiment 2, adding 250kg of 80-85 ℃ hot water, quickly stirring for 10min, centrifugally throwing, adding 250kg of 80-85 ℃ hot water into the filter residue 1, quickly stirring for 5min, centrifugally throwing, adding 200kg of 80-85 ℃ hot water into the filter residue 2, quickly stirring for 5min, centrifugally throwing, combining three filtrates, standing for 4h to cool to room temperature, precisely filtering, adjusting the pH of the fine filtrate to 3.61 by using 18% hydrochloric acid, standing for 15h, precisely filtering, adjusting the pH of the filtrate to 5.63 by using 10% sodium hydroxide solution, and performing column purification for one time.

And (3) enriching the purified solution by passing through a No. 3 resin column (the diameter-height ratio is 325: 300), washing with 5BV of water for injection, draining off water in the column, washing with 0.8BV of 30% alcohol, draining off, eluting with 1BV of 50% alcohol, collecting 20L of eluent, slightly concentrating to ensure that the concentration of the salvianolic acid is 191mg/ml, finely filtering if necessary, and spray-drying.

Spray drying the concentrated and purified solution by negative pressure spray drying, wherein the spray drying parameters are designed as follows: the air inlet temperature is 105-110 ℃, the air outlet temperature is 70 +/-5 ℃, the rotating speed of a spray head is 250-300 HZ, the feeding speed is 15-30 rp/min, yellow powder is obtained, the weight is 1.287kg, and the yield is 2.574%.

EXAMPLE 4 preparation of Salvianolic acid

Extracting 50.2kg of the salvia miltiorrhiza powder in the embodiment 2, adding 250kg of 80-85 ℃ hot water, quickly stirring for 10min, centrifugally throwing, adding 250kg of 80-85 ℃ hot water into the filter residue 1, quickly stirring for 5min, centrifugally throwing, adding 200kg of 80-85 ℃ hot water into the filter residue 2, quickly stirring for 5min, centrifugally throwing, combining three filtrates, standing for 4h to cool to room temperature, precisely filtering, adjusting the pH of the fine filtrate to 3.61 by using 18% hydrochloric acid, standing for 11 h, precisely filtering, adjusting the pH of the filtrate to 5.7 by using 10% sodium hydroxide solution, and performing column purification for one time.

Purifying the above extractive solution with first column chromatography, passing through No. 2 macroporous resin column (diameter-height ratio 325: 1200), sequentially washing with 5BV purified water, 3BV 3% alcohol, and 3BV 10% alcohol, eluting with 2BV 18% alcohol at flow rate of 500 + -100 ml/min, collecting diluted alcohol eluate 160L, adjusting pH to 2.3 with 18% hydrochloric acid, and enriching with second column chromatography

And (3) enriching the purified solution by passing through a No. 3 resin column (the diameter-height ratio is 325: 300), washing with 5BV of water for injection, draining off water in the column, washing with 0.8BV of 30% alcohol, draining, eluting with 1BV of 50% alcohol, collecting 20L of eluent, slightly concentrating to make the concentration of the salvianolic acid 177mg/ml, finely filtering if necessary, and spray-drying.

Spray drying the purified solution by using negative pressure spray drying, wherein the spray drying parameters are designed as follows: the air inlet temperature is 105-110 ℃, the air outlet temperature is 70 +/-5 ℃, the rotating speed of a spray head is 250-300 HZ, the feeding speed is 15-30 rp/min, yellow powder is obtained, the weight is 1.259kg, and the yield is 2.518%.

The results of examples 2-4 show that the relative deviation between the purity and the yield is small

EXAMPLE 5 preparation of Salvianolic acid

Pre-testing: taking 20g of radix salviae miltiorrhizae (produced in Qingdao, collected in spring, dried and crushed) powder, adding 100g of water, quickly stirring at room temperature for 10min, filtering, adding 100g of water into filter residue 1, quickly stirring at room temperature for 5min, filtering, adding 80g of water into filter residue 2, quickly stirring at room temperature for 5min, filtering, combining three filtrates, standing for 4h, carrying out precision filtration, taking 20 mu l of subsequent filtrate, injecting into an HPLC chromatograph, recording a chromatogram, calculating the content of salvianolic acid to be 2.89% according to an external standard method, and comparing with a standard control radix salviae miltiorrhizae HPLC fingerprint chromatogram to obtain the radix salviae miltiorrhizae extract which is obviously dissimilar. The content of the salvianolic acid B is also obviously lower, which does not meet the internal control quality standard of the salvia miltiorrhiza.

And adding 80-85 ℃ hot water into 20g of the salvia miltiorrhiza powder produced in the same batch of Qingdao in the preliminary test for 100g, quickly stirring for 10min, filtering, adding 80-85 ℃ hot water into 100g of filter residue 1, quickly stirring for 5min, filtering, adding 80-85 ℃ hot water into 80g of filter residue 2, quickly stirring for 5min, filtering, combining three filtrates, standing for 4h, cooling to room temperature, precisely filtering, injecting 20 mu l of subsequent filtrate into an HPLC chromatograph, recording a chromatogram, calculating the content of salvianolic acid in the salvia miltiorrhiza by using an external standard method, comparing with a control salvia miltiorrhiza HPLC fingerprint, wherein the similarity is 99.88%, and the quality standard of internal control of the salvia miltiorrhiza is met.

Extracting 50kg of radix salviae miltiorrhizae powder produced in Qingdao of the same batch as a pre-test, adding 250kg of hot water with the temperature of 80-85 ℃, rapidly stirring for 10min, centrifugally filtering, adding 250kg of hot water with the temperature of 80-85 ℃ into filter residue 1, rapidly stirring for 5min, centrifugally filtering, adding 200kg of hot water with the temperature of 80-85 ℃ into filter residue 2, rapidly stirring for 5min, centrifugally filtering, combining three filtrates, standing for 4h, cooling to room temperature, precisely filtering, adjusting the pH of the fine filtrate to 3.65 by using 18% hydrochloric acid, standing for 15h, precisely filtering, adjusting the pH of the filtrate to 5.62 by using 10% sodium hydroxide solution, and performing column chromatography purification once.

Purifying the extractive solution with primary column chromatography, passing through No. 2 macroporous resin column (diameter-height ratio 325: 1200), sequentially eluting with 5BV purified water, 3BV 3% alcohol, 3BV 10% alcohol, and 2BV 18% alcohol at flow rate of 500 + -100 ml/min, collecting diluted alcohol eluate 160L, adjusting pH to 2.2 with 18% hydrochloric acid, and enriching with secondary column chromatography

And (3) enriching the purified solution by passing through a No. 3 resin column (the diameter-height ratio is 325: 300), washing with 5BV of water for injection, draining off water in the column, washing with 0.8BV of 30% alcohol, draining, eluting with 1BV of 50% alcohol, collecting 20L of eluent, slightly concentrating to obtain the concentration of the salvianolic acid 188mg/ml, performing precision filtration, and performing spray drying.

Spray drying the purified solution by using negative pressure spray drying, wherein the spray drying parameters are designed as follows: the air inlet temperature is 105-110 ℃, the air outlet temperature is 70 +/-5 ℃, the rotating speed of a spray head is 250-300 HZ, the feeding speed is 15-30 rp/min, yellow powder is obtained, the weight is 1.275kg, and the yield is 2.55%.

EXAMPLE 6 preparation of Salvianolic acid

Extracting 50kg of the salvia miltiorrhiza powder in the embodiment 5, adding 250kg of hot water with the temperature of 80-85 ℃, quickly stirring for 10min, and centrifuging and filtering; adding 250kg of hot water with the temperature of 80-85 ℃ into the filter residue 1, quickly stirring for 5min, centrifugally filtering, adding 200kg of hot water with the temperature of 80-85 ℃ into the filter residue 2, stirring for 5min, centrifugally filtering, combining the three filtrates, standing for 4h, cooling to room temperature, precisely filtering, adjusting the pH of the fine filtrate to 3.65 with 18% hydrochloric acid, standing for 16h (standing overnight), precisely filtering, adjusting the pH of the filtrate to 5.6 with 10% sodium hydroxide solution, and allowing the filtrate to pass through a column for purification.

Purifying the extract by a first column chromatography, passing through a No. 2 macroporous resin column (diameter-height ratio is 325: 1200), washing by 5BV purified water, 3BV 3% ethanol and 3BV 10% ethanol in sequence, eluting by 4BV 20% ethanol at the flow rate of 0.3-0.5 BV/h, collecting 160L of eluent, adjusting the pH value to 2.35 by 18% hydrochloric acid, and obtaining a purified solution for enrichment by a second column chromatography.

Performing secondary column chromatography enrichment, namely passing the purified solution through a No. 3 resin column (the diameter-height ratio is 325: 300), washing with 5BV of purified water/water for injection, draining, washing with 0.8BV of 30% alcohol, draining, eluting with 2BV of 50% alcohol, collecting 20L of eluent, slightly concentrating to ensure that the concentration of the salvianolic acid is 150-200 mg/ml, cooling to room temperature, performing precise filtration if necessary, and performing spray drying;

spray drying concentrated purified solution by negative pressure spray drying with inlet air temperature of

The air outlet temperature is 70 +/-5 ℃, the rotating speed of a spray head is 250-300 HZ, the feeding speed is 15-30 rp/min, light yellow powder is obtained, the weight of 1.205kg is the yield of 2.41 percent

EXAMPLE 7 preparation of Salvianolic acid

Extracting Saviae Miltiorrhizae radix powder 50kg of example 5, adding 250kg, rapidly stirring at room temperature for 10min, centrifuging, filtering, adding 250kg of water into residue 1, rapidly stirring at room temperature for 5min, centrifuging, filtering, adding 250kg of water into residue 2, rapidly stirring at room temperature for 5min, centrifuging, filtering, mixing the three filtrates, fine filtering, adjusting pH of the fine filtrate with 18% hydrochloric acid to obtain final product

Standing for 6-11 hours, performing precise filtration, and adjusting the pH of filtrate to be 10% sodium hydroxide solution

And (5) carrying out column purification for one time.

And (3) purifying the extracting solution by a first column chromatography, passing the extracting solution through a No. 2 macroporous resin column (the diameter-height ratio is 325: 1200), washing with 5BV of purified water, 3BV of 3% alcohol, 3BV of 10% alcohol and 2BV of 18% alcohol in sequence at a flow rate of 500 +/-100 ml/min, collecting about 160L of dilute alcohol washing solution, adjusting the pH to be 2.00-2.50 by 18% hydrochloric acid, and waiting for secondary column chromatography.

And (3) enriching the purified solution by passing through a No. 3 resin column (the diameter-height ratio is 325: 300), washing with 5BV of water for injection, draining the water in the column, washing with 0.8BV of 30% alcohol, draining, washing with 1BV of 50% alcohol, discarding 10L of the first eluate, receiving 20L of the eluate, slightly concentrating to make the concentration of the salvianolic acid 175mg/ml, cooling to room temperature, and spray-drying.

Spray drying the concentrated and purified solution by negative pressure spray drying, wherein the spray drying parameters are designed as follows: the air inlet temperature is 105-110 ℃, the air outlet temperature is 70 +/-5 ℃, the rotating speed of a spray head is 250-300 HZ, the feeding speed is 15-30 rp/min, light yellow powder is obtained, and the yield is 2.462% after the weight of 1.231 kg.

The results of examples 5-7 show that the relative deviation between the purity and the yield is small; 2-7 show that the relative deviation of the purity and the yield of the obtained product is small in the same production area and different harvesting seasons.

EXAMPLE 8 preparation of Salvianolic acid for injection

Prescription

Note: the salvianolic acid used in the following examples is prepared by fully and uniformly mixing the salvianolic acid obtained in the above examples 2-7, sieving the mixture with a 80-mesh sieve, and detecting that the content of the salvianolic acid is 98.55% and the water content is 1.43%, thus weighing the salvianolic acid by pure conversion and feeding.

The process comprises the following steps:

placing the danshinol and mannitol in a beaker.

Freshly prepared water for injection was added to dissolve and dilute to 1500 ml.

Adding 0.05% of active carbon for injection, stirring for 10min, and filtering with 0.2 μ l microporous membrane.

Subpackaging in 10ml penicillin bottles, each bottle is 1.5ml, and plugging the rubber plug.

Pre-freezing at-50 deg.C for 3 hr

And (3) placing the precooled sample in a freeze drying box for freeze drying for 16h, then gradually heating to 25 ℃, and continuing to dry for 3 h.

And (3) compacting the rubber plug under vacuum, taking out, capping, labeling, packaging, boxing and warehousing.

Sampling and full inspection.

EXAMPLE 9 preparation of Salvianolic acid drop pills

Pill core prescription

Prescription of coating liquid

Pill core 5000 pill

Opadry 15.0g

Purification of 150ml

Preparation process

Pretreating raw materials and auxiliary materials: sieving salvianolic acid with 100 mesh sieve, oven drying, grinding PEG6000 and PEG4000 respectively, sieving with 100 mesh sieve, stirring Opadry with purified water for more than 45 min, and making into 10% suspension.

Melting: weighing the danol, the PEG6000 and the PEG4000 according to the proportion of the prescription, uniformly mixing, melting in a water bath at 72 +/-2 ℃, and uniformly mixing the liquid medicine by electric stirring.

Transferring liquid medicine: the uniform liquid medicine is quickly transferred to a liquid medicine tank preheated at 70 ℃.

Dripping: regulating and controlling the dripping speed, and dripping the liquid medicine into 200cs methyl silicone oil at a constant speed and collecting pills.

Oil removal: and wiping to remove oil.

Coating: and (3) putting the pill cores into a coating pot, uniformly spraying the Opadry solution on the surfaces of the pill cores by using a spray gun at the rotation speed of 15-20r/min, heating by using warm air to dry the pill cores (the temperature of the pill cores is 30-35 ℃), and increasing the weight of the coating film of the dropping pills by 1-3%.

Packaging: packaging the coated salvianolic acid drop pills in high-density polyethylene bottles, packaging 150 pills in each bottle together with an instruction into a small box, packaging according to the proposed requirements, and warehousing.

And (4) full inspection: sampling and fully inspecting the quality standard of the prepared danol dripping pills

EXAMPLE 10 preparation of Compound Danol dropping pills

Pill core prescription

Coating formula

10000 pellets with pellet core

Opadry 32g

Preparation process

Pretreating raw materials and auxiliary materials: grinding polyethylene glycol 6000, and sieving with 100 mesh sieve; grinding Notoginseng radix total saponin and natural Borneolum, and sieving with 100 mesh sieve to obtain raw materials. Opadry is stirred with purified water for more than 45 minutes to prepare a 10% suspension for later use.

Melting: weighing the danol, the panax notoginseng saponins, the natural borneol and the PEG6000 according to the proportion of the prescription, placing the mixture in a melting tank at the temperature of 75 ℃, carrying out water bath at the temperature of 72 +/-2 ℃, and melting and electrically stirring the mixture to uniformly mix the liquid medicine.

Pelleting: the dripping pill machine is provided with 12-hole dripping disc, the inner diameter of the dripping head is 1.5mm, the temperature of the dripping disc is 75 ℃, the temperature is 75 ℃, the dripping distance is 4cm, the condensate is 200cs dimethyl silicon oil, the temperature of the condensate is 0 ℃, and the temperature of the pipe orifice is

And (5) normally dripping.

Oil removal: wrapping the dripping pill in oil removing cloth to remove oil.

Sieving pills: and (4) screening out the undesirable dripping pills.

Coating: placing pills with round appearance and qualified weight in coating pan at rotation speed of 5-15r/min, uniformly spraying Opadry solution onto the surface of the pill core with spray gun, heating with hot air to dry the pill core (keeping the temperature of the pill core at

) And (5) obtaining the finished product. Coating weight gain of dripping pill

Subpackaging, packaging and warehousing: packaging 10 bottles in each box, 100 boxes in each box, labeling and warehousing according to 70 bottles.

And (4) full inspection: sampling and fully inspecting according to the quality standard of the formulated compound danol dropping pill.

EXAMPLE 11 preparation of Salvianolic acid Soft capsules

Prescription of capsule shell (weight ratio, capsule shell wall thickness)

)：

The prescription of the contents:

preparation process

Setting of main parameters

Sol temperature: 70 deg.C

The temperature of the glue box is as follows: 58 deg.C

Rotating speed of the rolling die: 1r/min

Eductor 33 deg.C

The condensation temperature of the glue solution is as follows: 10 deg.C

The drying temperature of the dripping pill is 30 DEG C

Process for the preparation of a catalyst

Pretreatment of raw and auxiliary materials

Preparing related auxiliary materials according to the prescription of the capsule shell. Preparing related raw and auxiliary materials according to the prescription of the content, crushing solid raw and auxiliary materials, and sieving with a 120-mesh sieve.

Preparation of capsule shell

Swelling, putting gelatin in a melten gel tank, adding water and glycerol, and swelling at room temperature for 2 h.

Melting glue, namely introducing hot water of 80 ℃ into the interlayer of the glue melting tank, and maintaining for 30 minutes when the content in the glue melting tank reaches 70 ℃.

Adding fumaric acid and ethylparaben, and stirring

And (3) decompressing and degassing for 15 minutes by using a water circulation type air pump so as to ensure that the prepared capsule shell has no bubbles.

Starting the soft capsule machine, adjusting relevant parameters of the capsule shell, starting the air compressor to enable the glue solution to enter a rubber sheet of the soft capsule machine, and finely adjusting relevant technical parameters to enable the thickness of the prepared rubber sheet to be 0.80 +/-0.05 mm and enable the thicknesses of the rubber sheets on the left side and the right side to be consistent.

The relative position of the two pressing rollers is adjusted so as to press the capsules.

The contents are prepared according to the proportion of the prescription, the danshinol, the sodium caprate and the sodium metabisulfite are taken and mixed evenly, the mixture of the 1, 2-propylene glycol of the Tween 80 is added and stirred evenly, the PEG400 is added, and after stirring evenly, the mixture is added into a storage tank for the contents for standby.

And pressing the soft capsule to start a body spraying switch, and adjusting the flow to meet the set requirement, thus preparing the salvianolic acid soft capsule.

Screening and removing the unqualified soft capsules with irregular appearance, insufficient filling and the like, and wrapping the soft capsules in an oil removing cloth to remove oil.

Subpackaging and packaging the screened and deoiled soft capsules, subpackaging, labeling, packaging and warehousing.

The total inspection sampling is carried out according to the drawn quality standard of the salvianolic acid soft capsule.

EXAMPLE 12 preparation of Salvianolic acid sustained-Release pellets

Prescription 1 pellet with normal dissolution

Process for the preparation of a catalyst

Pretreating to obtain salvianolic acid, and sieving with 100 mesh sieve.

Mixing with salvianolic acid (100 mesh sieve), starch, pregelatinized starch, carboxymethyl starch sodium (added with 2.5g), and microcrystalline cellulose, and mixing uniformly according to the principle of equivalent increment.

The soft material is wetted by 70% ethanol to obtain soft material with moderate hardness.

Extruding and rounding to obtain soft material, extruding into strips, cutting and rounding to obtain the final product.

Drying the pellets in a vacuum drying oven at 35 deg.C.

Coating with green gastric-soluble film to obtain the final product.

The sample is inspected according to the quality standard for clinical research, and all the inspection indexes are in accordance with the regulations.

Prescription 2 sustained-release pellet for 6h

Process for the preparation of a catalyst

Pretreating the raw materials and adjuvants, sieving danol, lactose, sodium cyclamate, hypromellose, and carbomer with 80 mesh sieve, and sieving with polyvidone K ₃₀5% (w/v) solution was prepared with 75% ethanol.

Weighing danol, lactose, hypromellose and carbomer, mixing, and adding polyvidone K₃₀Making into soft material.

Extruding and rounding the soft material to form strips, cutting and rounding.

Drying, vacuum drying the pellet at 35 deg.C in a vacuum drying oven, and removing fine powder to obtain pellet.

Coating with yellow acrylic resin IV film to obtain the final product.

Prescription 3 pellet capable of slowly releasing for 12h

*: ethyl cellulose was made into a 6% solution with 80% ethanol as a binder. The amount of 80% ethanol may be suitably adjusted as required.

Process for the preparation of a catalyst

Sieving the raw and auxiliary materials pretreated salvianolic acid with a 80-mesh sieve, and sieving hydroxypropyl methylcellulose K100M CR (HPMC K100M CR) with the 80-mesh sieve; soaking Ethyl Cellulose (EC) in 80% ethanol to dissolve to obtain 6% (w/v) solution for use.

Weighing salvianolic acid and hydroxypropyl methylcellulose K100M according to the prescription, mixing, adding 6% EC solution, and kneading repeatedly to obtain uniform soft material

Extruding and rounding, extruding into strips by an extruding and rounding machine, cutting, and rounding to obtain the wet pellets.

Drying the pellets, and drying the wet pellets in a drying box.

Coating with red acrylic resin I film.

Salvianolic acid sustained and controlled release pellet capsule/tablet

The mixed pill is prepared by weighing 100g, 90g and 120g of the pellets in the

formulas

1, 2 and 3 respectively, and fully and uniformly mixing.

And (4) filling the mixed micro-pills into a proper capsule shell, wherein the content of each micro-pill weighs 310mg, and the labeled amount is 150 mg.

Subpackaging, packaging, labeling, packaging and warehousing.

The sample is inspected according to the quality standard for clinical research, and all indexes are in accordance with the regulations.

Content determination method

The invention relates to a method for measuring the content of danol, which uses UHPLC and high-precision mass spectrum together, positions each component of the danol according to molecular weight, and selects the component with relatively high content and activity as the basis for selecting a reference substance. 6 reference substances of danshensu, protocatechualdehyde, rosmarinic acid, alkannic acid, salvianolic acid B and salvianolic acid A are used for calculating the content of each component according to an external standard method, wherein the content of 6 components accounts for 93-97% of the labeled amount. The sum of the other small peak areas takes salvianolic acid B as a reference substance, and the content is calculated according to an external standard method and accounts for about 2-6% of the marked amount. The sum of the contents of the two parts is the content of the salvianolic acid, and the content of the salvianolic acid is more than 98 percent of the marked amount.

Clinical application of danphenol

The invention also relates to a new application of the danol preparation in preparing the medicine for resisting cerebral ischemia reperfusion injury: an animal model of cerebral ischemia reperfusion is established by a rat arterial embolism Model (MCAO), the behaviours, the cerebral infarction area, the brain water content, the levels of Lactate Dehydrogenase (LDH), Creatine Kinase (CK), superoxide dismutase (SOD) and Malondialdehyde (MDA), IL-6, IL-1 beta and TNF-alpha in brain tissues are observed, and the conditions of MCAO molding and rat cortex damage after administration are observed by HE staining. The results all prove that the danol ip 15mg/kg for rats has obvious effect of resisting cerebral ischemia reperfusion injury, is obviously superior to the curative effect of nimodipine ip 20mg/kg for first-line treatment of ischemic stroke, and can be used for preparing the medicine for preventing and/or treating cerebral ischemia reperfusion injury.

Claims

1. The salvianolic acid is characterized by being prepared by the following production process:

extraction: taking red sage root powder, adding 5 times of water, quickly stirring and extracting for 3 times, wherein the extraction temperature is 80-85 ℃, extracting for 10min for the first time, extracting for 5min for the second time and the third time respectively, filtering, combining filtrate obtained in the third time, standing for 3-4 hours, cooling to room temperature, precisely filtering, adjusting the pH of the fine filtrate to 3.5-4.0 by using 18% hydrochloric acid, standing for 4-16 hours, precisely filtering, adjusting the pH of the filtrate to 5.5-6.0, and performing column chromatography purification for one time;

and (3) primary column chromatography purification: loading the filtrate to be subjected to primary column chromatography purification on a No. 2 macroporous resin column, sequentially washing with water, 3% ethanol and 10% ethanol, eluting with 18% ethanol, collecting 18% ethanol eluate, adjusting the pH to 2.0-2.5 with 18% hydrochloric acid, and allowing secondary column chromatography enrichment to be performed;

and (3) secondary column passing enrichment: loading the eluate to be subjected to secondary column chromatography enrichment into a No. 3 resin column, sequentially washing with water and 30% ethanol, eluting with 50% ethanol, collecting 50% ethanol eluate, precisely filtering, and spray drying;

spray drying: and (3) performing negative pressure spray drying on the filtrate to be subjected to spray drying, wherein the air inlet temperature is 105-110 ℃, the air outlet temperature is 75 +/-5 ℃, the rotating speed of a spray head is 250-300 HZ, and the feeding speed is 15-30 rp/min, so that yellowish-yellow powdery danol is obtained.

2. A pharmaceutical preparation comprising the danol of claim 1, wherein the danol of claim 1 is formulated into pharmaceutically acceptable dosage forms with pharmaceutically acceptable carriers, wherein the dosage forms include sustained release pellets, soft capsules, dripping pills, injections and microemulsions.

3. A content measurement method for measuring the content of the salvianolic acid according to claim 1 or the pharmaceutical preparation according to claim 2, which is characterized by comprising the steps of: using liquid chromatography, calculating corresponding content of chromatographic peak with reference substance according to external standard method, calculating content of the rest chromatographic peaks with salvianolic acid B as reference substance according to external standard method, wherein the sum of the two parts is salvianolic acid content, and the salvianolic acid content should be not less than 98.5%.

4. The medical application of the danol in the preparation of the medicine for resisting ischemic cardiovascular and cerebrovascular diseases according to claim 1.