A total salvia miltiorrhiza stem and leaf phenolic acid conversion product with antioxidant activity, protection of vascular endothelial cells and myocardial cells and application thereof

Abstract

The application discloses a total phenolic acid conversion product of Salvia miltiorrhiza stems and leaves with myocardial protection function and a preparation method and application thereof, wherein the conversion product is obtained by adjusting the pH of the total phenolic acid of Salvia miltiorrhiza stems and leaves with hydrochloric acid and then adopting a high-temperature and high-pressure sterilization pot to react. In the conversion product, the content of salvianolic acid A is 7-9%, the content of danshensu is 15-17%, the content of protocatechuic aldehyde is 1-2%, the content of rosmarinic acid is 13-15%, the content of shikonin is 1-2%, and the content of salvianolic acid B is 3-4%. The total phenolic acid conversion product of Salvia miltiorrhiza stems and leaves provided by the application has the activities of antioxidation, myocardial protection and oxidation damage reduction, protection of vascular endothelial cells and myocardial cells, and the function of free radical elimination and oxidation damage reduction, has reliable curative effect, good safety, low adverse reaction, and can be used for preparing a cardiovascular protection medicine.

Description

Technical Field

[0001] This invention relates to a natural product composition, and more particularly to a total phenolic acid conversion product of Salvia miltiorrhiza stems and leaves with antioxidant activity, which protects vascular endothelial cells and cardiomyocytes and reduces oxidative damage, as well as its preparation method and application. Technical Background

[0002] Cardiovascular disease (CVD) is a leading cause of death worldwide, with ischemic heart disease (IHD) being the primary cause. Factors such as hypertension, diabetes, hyperlipidemia, obesity, and smoking can all contribute to atherosclerosis or coronary artery spasm, leading to coronary artery stenosis or occlusion, resulting in insufficient blood supply. Following myocardial ischemia and hypoxia, levels of oxygen free radicals, inflammatory factors, and free fatty acids increase, energy metabolism becomes abnormal, and cardiomyocytes undergo apoptosis, ultimately leading to death. Therefore, in-depth research into the pathogenesis and pathological process of IHD and the development of effective innovative drugs with low side effects are of great significance to global public health.

[0003] Danshen is a plant of the Lamiaceae family called Salvia miltiorrhiza. Salvia miltiorrhiza The dried roots and rhizomes of *B. ge.* have the effects of promoting blood circulation and removing blood stasis, regulating menstruation and relieving pain, clearing the heart and relieving irritability, cooling the blood and reducing swelling. *Salvia miltiorrhiza* is a common bulk medicinal herb. Clinically, it is often used in the treatment of cardiovascular and cerebrovascular diseases with salvia miltiorrhiza injection, compound salvia miltiorrhiza dripping pills, and compound salvia miltiorrhiza tablets. Its above-ground stems and leaves also have certain effects. For example, the Qing Dynasty medical text *Yifang Shouyue* records: "Salvia miltiorrhiza leaves, pounded and mixed with wine lees, applied to the breast, will immediately reduce swelling in its early stages." Furthermore, *Shandong Medicinal Plant Records* also records that "salvia miltiorrhiza stems and leaves have the effects of promoting blood circulation and removing blood stasis, clearing the heart and relieving irritability." Salvia miltiorrhiza stems and leaves have certain medicinal value. Both salvia miltiorrhiza and its stems and leaves contain abundant phenolic acids, which have antioxidant, anti-inflammatory, and antithrombotic pharmacological effects, and are used to treat cardiovascular diseases such as myocardial ischemia and myocardial infarction. Among them, salvia miltiorrhiza acid A has the best activity in protecting against ischemia-reperfusion injury and has clear clinical application value, but its content is low. As traditional non-medicinal parts, the stems and leaves of salvia miltiorrhiza have not been fully developed and utilized, resulting in a great waste of resources and environmental pollution. This invention uses total phenolic acids from the stems and leaves of Salvia miltiorrhiza as a substrate for conversion to increase the content of salvianolic acid A. Based on in vivo and in vitro studies, the antioxidant activity of the composition with relatively increased salvianolic acid A content and its protective effect on vascular endothelial cells and cardiomyocytes are investigated. Summary of the Invention

[0004] Purpose of the invention: The purpose of this invention is to provide a total phenolic acid conversion product from the stems and leaves of *Salvia miltiorrhiza* with cardioprotective function, its preparation method, and its application. A total phenolic acid conversion product from the stems and leaves of *Salvia miltiorrhiza* with antioxidant activity and protective effects on vascular endothelial cells and cardiomyocytes is prepared by the following method: The aerial stems and leaves of Salvia miltiorrhiza were collected, dried with hot air, pulverized in a pulverizer, and sieved to obtain coarse powder of Salvia miltiorrhiza stems and leaves. Ethanol was added, and the mixture was refluxed for extraction. The extracts were combined, and the ethanol was recovered by vacuum concentration. The mixture was then concentrated until no alcohol odor was detected to obtain the Salvia miltiorrhiza stem and leaf extract. The above-mentioned extract of Salvia miltiorrhiza stems and leaves was diluted with water and purified with AB-8 macroporous resin. Before use, the resin was fully swollen with ethanol, and the column was packed by wet method. The column was first washed with pure water until there was no alcohol odor and impurities were removed. The 20% ethanol eluent and the 30% ethanol eluent were used as the total phenolic acid fraction of Salvia miltiorrhiza stems and leaves. The collected eluent was concentrated under reduced pressure to recover ethanol and concentrated until there was no alcohol odor to obtain crude Salvia miltiorrhiza stems and leaves. Dilute the above-mentioned crude product of Salvia miltiorrhiza stems and leaves with water, and adjust with hydrochloric acid. p H, ethyl acetate extraction, combined ethyl acetate fractions, vacuum concentration to recover ethyl acetate and concentrated to dryness to obtain total phenolic acids from tanshinone stems and leaves; The obtained phenolic acids from the stems and leaves of *Salvia miltiorrhiza* were diluted with water, and the conversion solution was adjusted with hydrochloric acid. p H is obtained by reacting in a high-temperature and high-pressure autoclave and then freeze-drying under vacuum.

[0005] As a preferred embodiment, the total phenolic acid conversion product of *Salvia miltiorrhiza* stems and leaves, which has antioxidant activity and protects vascular endothelial cells and cardiomyocytes, is prepared by the following method: The aerial stems and leaves of Salvia miltiorrhiza were collected, dried with hot air at 40-50℃, pulverized, and passed through a standard sieve (No. 3) to obtain coarse powder of Salvia miltiorrhiza stems and leaves. Each extraction was performed by adding 10-15 times the amount of 60% ethanol and refluxing at 80-85℃ for 1-3 times, each time for 1-2 hours. The extracts were combined, and the ethanol was recovered by vacuum concentration until no alcohol odor remained, yielding the Salvia miltiorrhiza stem and leaf extract. The above-mentioned extract of Salvia miltiorrhiza stems and leaves was diluted with water to a concentration of 0.125-1.5g crude drug per mL. It was then purified using AB-8 macroporous resin. Before use, the resin was fully swollen with 2-3 times its volume of 95% ethanol. The column was packed using a wet packing method, with a resin mass to sample loading ratio of 2-3:1 and a column diameter to height ratio of 1:5-1:20. The column was first washed with 3-5 BV of pure water until there was no alcohol odor and impurities were removed. Then, the eluent was eluted with 3-5 BV of 20% ethanol and the eluent was eluted with 1.5-2 BV of 30% ethanol as the total phenolic acid fraction of Salvia miltiorrhiza stems and leaves. The collected eluent was concentrated under reduced pressure to recover the ethanol and concentrated until there was no alcohol odor to obtain the crude product of Salvia miltiorrhiza stems and leaves. Dilute the crude danshen stems and leaves mentioned above with water to a concentration of 0.1-0.3g of raw drug per 1mL, and adjust with 5M hydrochloric acid. p Extract with 2-3 H, extract with twice the amount of ethyl acetate 3-5 times, combine the ethyl acetate fractions, concentrate under vacuum to recover ethyl acetate and concentrate to dryness to obtain total phenolic acids from the stems and leaves of Salvia miltiorrhiza. The obtained tanshinone stem and leaf phenolic acids were diluted with water to a concentration of 10-20 mg of tanshinone B per mL, and the conversion solution was adjusted with 5M hydrochloric acid.p With H=3~5, the reaction is carried out at 105~135℃ for 2~4 hours using a high-temperature and high-pressure sterilizer, followed by vacuum freeze-drying to obtain the conversion product.

[0006] As the optimal solution, the conversion product of the present invention is obtained by the following preparation method: The aerial stems and leaves of Salvia miltiorrhiza were collected, dried with hot air at 40-50℃, pulverized, and passed through a standard sieve (No. 3) to obtain coarse powder of Salvia miltiorrhiza stems and leaves. Ten times the amount of 60% ethanol was added each time, and the mixture was refluxed at 80-85℃ for 1 hour each time, three times. The three extracts were combined, and the ethanol was recovered by vacuum concentration until no alcohol odor remained, yielding the Salvia miltiorrhiza stem and leaf extract.

[0007] The above-mentioned extract of Salvia miltiorrhiza stems and leaves was diluted with water to a concentration of 0.125 g crude drug per mL. It was then purified using AB-8 macroporous resin. Before use, the resin was fully swollen with 2 volumes of 95% ethanol. The column was packed using a wet packing method, with a resin mass to sample loading ratio of 2:1 and a column diameter to height ratio of 1:5 to 1:20. The column was first washed with 5 BV of pure water until no alcohol odor was detected and impurities were removed. The eluent fractions of 5 BV 20% ethanol and the eluent fractions of the first 1.5 BV 30% ethanol were used as the total phenolic acid fraction of Salvia miltiorrhiza stems and leaves. The collected eluent was concentrated under reduced pressure to recover the ethanol and then concentrated until no alcohol odor was detected to obtain the crude product of Salvia miltiorrhiza stems and leaves.

[0008] The crude product of Salvia miltiorrhiza stems and leaves was diluted with water to a concentration of 0.1g of raw drug per mL, the pH was adjusted to 2 with 5M hydrochloric acid, and the product was extracted three times with twice the amount of ethyl acetate. The ethyl acetate fractions were combined, and the ethyl acetate was recovered by vacuum concentration and concentrated to dryness to obtain total phenolic acids from Salvia miltiorrhiza stems and leaves.

[0009] The total phenolic acids obtained from the stems and leaves of *Salvia miltiorrhiza* were diluted with water to a concentration of 15 mg of salvianolic acid B per 1 mL, and the conversion solution was adjusted with 5 M hydrochloric acid. p With H=3, a high-temperature and high-pressure sterilizer was used to react at 135℃ for 2 hours, followed by vacuum freeze-drying to obtain the conversion product.

[0010] The conversion product prepared by this invention includes the following active ingredients in weight percentage: salvianolic acid A 7-9%, tanshinone 15-17%, protocatechuic aldehyde 1-2%, rosmarinic acid 13-15%, shikonin 1-2%, and salvianolic acid B 3-4%.

[0011] This invention provides a method for determining the total phenolic acid conversion products of Salvia miltiorrhiza stems and leaves. Ultra-high performance liquid chromatography (UHPLC) is used to determine the contents of salvianolic acid A, tanshinone, protocatechuic aldehyde, rosmarinic acid, lithospermic acid, and salvianolic acid B. The determination conditions are as follows: Preferably, ultra-high performance liquid chromatography (UHPLC) is used to determine the contents of salvianolic acid A, tanshinone, protocatechuic aldehyde, rosmarinic acid, lithospermic acid, and salvianolic acid B. The determination conditions are as follows: Octadecylsilane-bonded silica gel is used as a filler; Detection wavelength: 280 nm; flow rate: 0.4 mL / min; column temperature: 35 °C; The theoretical plate number, calculated based on salvianolic acid A, should not be less than 10,000; Preparation of reference solution: Accurately weigh appropriate amounts of salvianolic acid A, tanshinone, protocatechuic acid, protocatechuic aldehyde, caffeic acid, rosmarinic acid, lithospermic acid, salvianolic acid B, and salvianolic acid C into a volumetric flask, and add methanol to prepare a mixed reference solution. Preparation of the test solution: Accurately weigh 10 mg of the sample into a 5 mL volumetric flask, add 50% methanol to dissolve and shake well, and dilute to the mark to obtain the solution; Elution was performed using acetonitrile as mobile phase A and 0.1% formic acid aqueous solution as mobile phase B, under the following gradient elution conditions, for 21 minutes.

[0012] During the 0-1 minute period, the proportion of acetonitrile was maintained at 5%, and the proportion of 0.1% formic acid aqueous solution was maintained at 95%. Within 1 to 3 minutes, the proportion of acetonitrile increased from 5% to 10%, and the proportion of 0.1% formic acid aqueous solution decreased from 95% to 90%. Between 3 and 7 minutes, the proportion of acetonitrile increased from 10% to 15%, while the proportion of 0.1% formic acid aqueous solution decreased from 90% to 85%. Between 7 and 11 minutes, the proportion of acetonitrile increased from 15% to 21%, while the proportion of 0.1% formic acid aqueous solution decreased from 85% to 79%. Between 11 and 15 minutes, the proportion of acetonitrile increased from 21% to 33%, while the proportion of 0.1% formic acid aqueous solution decreased from 79% to 66%. Between 15 and 17 minutes, the proportion of acetonitrile increased from 33% to 70%, while the proportion of 0.1% formic acid aqueous solution decreased from 66% to 30%. At 17-18 minutes, the proportion of acetonitrile increased from 70% to 80%, while the proportion of 0.1% formic acid aqueous solution decreased from 30% to 20%. At 18-20 minutes, the acetonitrile ratio is maintained at 80%, and the 0.1% formic acid aqueous solution ratio is maintained at 20%. At 20-21 minutes, the proportion of acetonitrile decreased from 80% to 5%, while the proportion of 0.1% formic acid aqueous solution increased from 20% to 95%. Assay: Accurately pipette 1 mL of the mixed reference solution into each of the test solution into the liquid chromatography vial, and inject automatically. Pipe 2 mL of the mixture into each vial. μ L, determine and calculate the contents of salvianolic acid A, tanshinone, protocatechuic aldehyde, rosmarinic acid, lithospermic acid, and salvianolic acid B.

[0013] This invention obtains the total phenolic acids from the stems and leaves of *Salvia miltiorrhiza* (tanshinone) through extraction, purification, extraction, and high-temperature, high-pressure conversion, with a relatively increased content of salvianolic acid A, i.e., the conversion product. The stems and leaves of *Salvia miltiorrhiza* mainly contain rosmarinic acid and salvianolic acid B. Salvianolic acid B is highly water-soluble but unstable, and easily degrades under high-temperature conditions to form salvianolic acid A and other phenolic acid components. Therefore, 60% ethanol is used for reflux extraction at 80℃~85℃ to ensure that salvianolic acid B is not destroyed.

[0014] The total phenolic acid conversion product of Salvia miltiorrhiza stems and leaves provided by this invention can scavenge DPPH free radicals, scavenge ABTS free radicals, and reduce Fe in vitro. 3+ The results of the Fractional Antioxidant Process (FRAP) assay showed that the antioxidant activity of the transformed product was significantly enhanced compared with that of the total phenolic acids from the stems and leaves of *Salvia miltiorrhiza*. Based on hydrogen peroxide-induced oxidative damage models of human umbilical vein endothelial cells and rat cardiomyocytes, the protective effects of the total phenolic acids from the stems and leaves of *Salvia miltiorrhiza* before and after transformation on cells were investigated. The results showed that after hydrogen peroxide-induced oxidative damage, the SOD level in the model group decreased, while the MDA and LDH levels increased (P<0.0001). The total phenolic acids from the stems and leaves of *Salvia miltiorrhiza* before and after transformation exhibited strong antioxidant activity, comparable to that of vitamin C, and significantly increased SOD levels and decreased MDA levels (P<0.001). Within a certain range, the protective effect was dose-dependent, with higher doses showing significantly greater protective effects than lower doses. The protective effect of the transformed product was superior to that of the total phenolic acids from the stems and leaves of *Salvia miltiorrhiza*. Regarding isoproterenol... The induced acute myocardial ischemia rat model showed good protective effects. After ISO modeling, the heart rate in the model group increased significantly. Prolonged ischemia led to compensatory hypertrophy of the left ventricle and a significant increase in cardiac organ indices (P<0.0001). ST segment depression on electrocardiogram indicated endocardial ischemia-related injury. Myocardial ischemia and hypoxia led to increased oxygen free radicals, increased membrane permeability, and increased secretion of inflammatory factors, reflected in significantly decreased levels of myocardial enzymes AST, LDH, CK, and CK-MB in the model group (P<0.001); significantly decreased activities of antioxidant enzymes SOD, CAT, and GSH-Px (P<0.0001); and increased content of lipid peroxidation end product MDA. The transformed products were more effective than total phenolic acids from tanshinone stems and leaves in downregulating LDH and increasing CAT and SOD activities (P<0.001), with higher doses showing better effects than lower doses. Electrocardiogram results, HE and MASSON pathological section results were consistent with the above findings.

[0015] The total phenolic acid conversion product of Salvia miltiorrhiza stems and leaves provided by this invention has a higher relative content of salvianolic acid A than that of total phenolic acid in Salvia miltiorrhiza stems and leaves, while the contents of rosmarinic acid, shikonin, salvianolic acid B, and salvianolic acid C are reduced. The converted product can significantly improve the ST segment changes in ISO-induced myocardial ischemia rats, increase the activity of antioxidant enzymes, reduce the content of myocardial damaging enzymes, protect myocardial cells, and has a significant anti-myocardial ischemia effect.

[0016] The above studies show that the transformation product provided by the present invention has good antioxidant activity and can play a good protective role against oxidative damage to vascular endothelial cells and cardiomyocytes. It can be used to prepare products that can improve cardiovascular damage caused by coronary artery ischemia and hypoxia.

[0017] The present invention describes a transformation product with antioxidant activity that protects vascular endothelial cells and cardiomyocytes and reduces oxidative damage, as well as its preparation method and application. This transformation product and a pharmaceutically acceptable carrier can be formulated into tablets, pills, powders, decoctions, granules, pastes, or extracts for convenient clinical use. Attached Figure Description

[0018] Figure 1 This is the UPLC spectrum of the mixed reference standard.

[0019] Figure 2 The figure shows the protective effect of the transformation product on the oxidative damage model of HUVEC cells.

[0020] Figure 3 This is a bar chart showing the effect of the transformation products on SOD content in HUVEC cells, a model of oxidative damage.

[0021] Figure 4 This is a bar chart showing the effect of transformation products on MDA content in HUVEC cells, a model of oxidative damage.

[0022] Figure 5 The bar chart shows the effect of the transformation products on the LDH content in the supernatant of the HUVEC cell oxidative damage model.

[0023] Figure 6 The figure shows the protective effect of the transformation product on the oxidative damage model of H9C2 cells.

[0024] Figure 7 This is a bar chart showing the effect of the transformation products on SOD content in H9C2 cell oxidative damage model cells.

[0025] Figure 8 This is a bar chart showing the effect of the transformation products on MDA content in H9C2 cell oxidative damage model cells.

[0026] Figure 9 This is a bar chart showing the effect of the transformation product on the LDH content in the supernatant of the H9C2 cell oxidative damage model.

[0027] Figure 10 A bar chart showing the effect of the transformation product on the cardiac index in a rat model of acute myocardial ischemia.

[0028] Figure 11 This is a bar chart showing the effect of the transformation product on heart rate in a rat model of acute myocardial ischemia.

[0029] Figure 12 This is a bar chart showing the effect of the transformation product on the ST segment on the electrocardiogram of a rat model of acute myocardial ischemia.

[0030] Figure 13 This is a bar chart showing the effect of the transformation product on the electrocardiogram of a rat model of acute myocardial ischemia.

[0031] Figure 14 HE staining results of the transformation product on the myocardium of a rat model of acute myocardial ischemia.

[0032] Figure 15 The results of MASSON staining of the myocardium in a rat model of acute myocardial ischemia by the transformation product.

[0033] Figure 16 This is a bar chart showing the effect of the transformation product on serum CK levels in a rat model of acute myocardial ischemia.

[0034] Figure 17 This is a bar chart showing the effect of the transformation product on the serum CK-MB level in a rat model of acute myocardial ischemia.

[0035] Figure 18 This is a bar chart showing the effect of the transformation product on the serum AST level in a rat model of acute myocardial ischemia.

[0036] Figure 19 This is a bar chart showing the effect of the transformation product on serum LDH levels in a rat model of acute myocardial ischemia.

[0037] Figure 20 This is a bar chart showing the effect of the transformation products on the SOD content in the myocardium of a rat model of acute myocardial ischemia.

[0038] Figure 21 This is a bar chart showing the effect of the transformation products on the MDA content in the myocardium of a rat model of acute myocardial ischemia.

[0039] Figure 22 This is a bar chart showing the effect of the transformation product on the GSH-Px content in the myocardium of a rat model of acute myocardial ischemia.

[0040] Figure 23 This is a bar chart showing the effect of the transformation product on CAT content in the myocardium of a rat model of acute myocardial ischemia. Detailed Implementation

[0041] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. After reading the present invention, any modifications of the present invention in various equivalent forms by those skilled in the art will fall within the scope defined by the appended claims.

[0042] Example 1: A method for preparing a transformation product with antioxidant activity that protects vascular endothelial cells and cardiomyocytes and reduces oxidative damage, comprising the following steps: (1) Take the above-ground stems and leaves of Salvia miltiorrhiza, dry them with hot air at 45℃, pulverize them with a pulverizer and pass them through the No. 3 standard sieve of the pharmacopoeia to obtain coarse powder of Salvia miltiorrhiza stems and leaves. Add 10 times the amount of 60% ethanol each time, and reflux extract at 85℃ for 3 times, 1 hour each time. Combine the 3 extracts, concentrate them under vacuum to recover the ethanol and concentrate them until there is no alcohol taste to obtain the Salvia miltiorrhiza stem and leaf extract.

[0043] (2) The above-mentioned extract of Salvia miltiorrhiza stems and leaves was diluted with water to contain 0.125g of crude drug per 1mL. It was purified with AB-8 macroporous resin. Before use, the resin was fully swollen with 2 times the volume of 95% ethanol. The column was packed by wet method. The ratio of resin mass to sample loading was 2:1 and the ratio of resin column diameter to height was 1:10. The column was first washed with 5BV pure water until there was no alcohol odor and impurities were removed. The elution fraction of 5BV 20% ethanol and the elution fraction of the first 1.5BV 30% ethanol were used as the total phenolic acid fraction of Salvia miltiorrhiza stems and leaves. The collected eluent was concentrated under reduced pressure to recover ethanol and concentrated until there was no alcohol odor to obtain crude Salvia miltiorrhiza stems and leaves.

[0044] (3) Dilute the crude product of Salvia miltiorrhiza stems and leaves with water to a concentration of 0.1g crude drug per 1mL, and adjust with 5M hydrochloric acid. p Extracted three times with twice the amount of ethyl acetate, the ethyl acetate fractions were combined, and the ethyl acetate was recovered by vacuum concentration and concentrated to dryness to obtain total phenolic acids from the stems and leaves of Salvia miltiorrhiza.

[0045] (4) Dilute the obtained tanshinone stem and leaf phenolic acid with water to a concentration of 15 mg of tanshinone B per 1 mL, and adjust the conversion solution with 5 M hydrochloric acid. p With H=3, a high-temperature and high-pressure sterilizer was used to react at 135℃ for 2 hours, followed by vacuum freeze-drying to obtain the transformation product of Salvia miltiorrhiza stems and leaves.

[0046] Example 2 A transformation product with antioxidant activity, which protects vascular endothelial cells and cardiomyocytes and reduces oxidative damage, and its preparation method, comprising the following steps: (1) Take the above-ground stems and leaves of Salvia miltiorrhiza, dry them with hot air at 50℃, pulverize them with a pulverizer and pass them through the No. 3 standard sieve of the pharmacopoeia to obtain coarse powder of Salvia miltiorrhiza stems and leaves. Add 10 times the amount of 60% ethanol each time, and reflux at 80℃ for 3 times, 1 hour each time. Combine the 3 extracts, concentrate them under vacuum to recover the ethanol and concentrate them until there is no alcohol taste to obtain the Salvia miltiorrhiza stem and leaf extract.

[0047] (2) The above-mentioned extract of Salvia miltiorrhiza stems and leaves was diluted with water to contain 0.125g of crude drug per 1mL. It was purified with AB-8 macroporous resin. Before use, the resin was fully swollen with 2 times the volume of 95% ethanol. The column was packed by wet method. The ratio of resin mass to sample loading was 2:1 and the ratio of resin column diameter to height was 1:15. The column was first washed with 5BV pure water until there was no alcohol odor and impurities were removed. The elution fraction of 5BV 20% ethanol and the elution fraction of the first 1.5BV 30% ethanol were used as the total phenolic acid fraction of Salvia miltiorrhiza stems and leaves. The collected eluent was concentrated under reduced pressure to recover ethanol and concentrated until there was no alcohol odor to obtain crude Salvia miltiorrhiza stems and leaves.

[0048] (3) Dilute the crude product of Salvia miltiorrhiza stems and leaves with water to 0.1g of crude drug per 1mL, adjust the pH to 2 with 5M hydrochloric acid, extract with twice the amount of ethyl acetate 3 times, combine the ethyl acetate fraction, concentrate under vacuum to recover the ethyl acetate and concentrate to dryness to obtain total phenolic acids from Salvia miltiorrhiza stems and leaves.

[0049] (4) Dilute the obtained tanshinone stem and leaf phenolic acid with water to 15 mg of tanshinone B per 1 mL, adjust the pH of the conversion solution to 4 with 5M hydrochloric acid, use a high temperature and high pressure sterilizer, react at 135℃ for 2 h, and freeze dry under vacuum to obtain the tanshinone stem and leaf conversion product.

[0050] Example 3: Preparation and Analysis Method of Total Phenolic Acid Conversion Products from Salvia miltiorrhiza Stems and Leaves 1. Instruments and reagents Instruments: Waters Acquity UPLC system (Waters Corporation, USA); Ditect-Q5 pure water preparation system (Millipore Corporation); ML105 0.0001 g electronic balance.

[0051] Column: Waters ACQUITY UPLC BEH C 18 (2.1 mm × 100 mm, 1.7) μ m).

[0052] Reagents: Ultrapure water was prepared in-house using a milli-Q ultrapure water preparation system; 95% ethanol was purchased from Nanjing Wanqing Chemical Glassware Instrument Co., Ltd.; methanol and acetonitrile were purchased from Merck AG, Germany; formic acid (ACS, USA, chromatographic grade).

[0053] Chemical reference standards: salvianolic acid B and protocatechuic acid; tanshinone, lithospermic acid, salvianolic acid A and C, rosmarinic acid and caffeic acid were all purchased from Shanghai Yuanye Biotechnology Co., Ltd.; protocatechuic aldehyde was purchased from Nanjing Chunqiu Bioengineering Co., Ltd., and all had a mass fraction greater than 98%.

[0054] 2. Preparation of reference solution and test solution 2.1 Preparation of reference solutions: Accurately weigh appropriate amounts of each of the following reference standards: tanshinone, protocatechuic acid, protocatechuic aldehyde, caffeic acid, rosmarinic acid, lithospermic acid, salvianolic acid B, salvianolic acid A, and salvianolic acid C, and dilute to the mark with 90% methanol to obtain the mixed reference standard stock solution. Pipette 500 ml of the stock solution... μ Pour L into a new 1mL volumetric flask and dilute to the mark with 90% methanol. The concentrations of tanshinone, protocatechuic acid, protocatechuic aldehyde, caffeic acid, rosmarinic acid, lithospermic acid, salvianolic acid B, salvianolic acid A, and salvianolic acid C are 555, 635, 800, 625, 670, 585, 675, 680, and 595, respectively. μ g / mL, used for subsequent content determination.

[0055] 2.2 Preparation of the test solution: Weigh 5 mg of the conversion product sample powder obtained in Examples 1 and 2, accurately place it in a 1 mL volumetric flask, dissolve completely in 50% methanol, centrifuge at 13000 r / min for 10 min, take the supernatant, and filter through a 0.22 mL filter. μ After filtering with a microporous membrane, it is obtained.

[0056] 3. Chromatographic conditions and system suitability test The column was filled with octadecylsilane-bonded silica gel; the detection wavelength was 280 nm; the flow rate was 0.4 mL / min; the column temperature was 35 °C; and the theoretical plate number, calculated based on salvianolic acid A, should not be less than 10,000. From 0 to 1 minute, the proportion of acetonitrile remained at 5%, and the proportion of 0.1% formic acid aqueous solution remained at 95%; from 1 to 3 minutes, the proportion of acetonitrile increased from 5% to 10%, and the proportion of 0.1% formic acid aqueous solution decreased from 95% to 90%; from 3 to 7 minutes, the proportion of acetonitrile increased from 10% to 15%, and the proportion of 0.1% formic acid aqueous solution decreased from 90% to 85%; from 7 to 11 minutes, the proportion of acetonitrile increased from 15% to 21%, and the proportion of 0.1% formic acid aqueous solution decreased from 85% to 79%; from 11 to 15 minutes, the proportion of acetonitrile increased from 21% to 33%, and the proportion of 0.1% formic acid aqueous solution decreased from 95% to 95%. The proportion of 0.1% formic acid aqueous solution decreased from 79% to 66%; at 15-17 minutes, the proportion of acetonitrile increased from 33% to 70%, and the proportion of 0.1% formic acid aqueous solution decreased from 66% to 30%; at 17-18 minutes, the proportion of acetonitrile increased from 70% to 80%, and the proportion of 0.1% formic acid aqueous solution decreased from 30% to 20%; at 18-20 minutes, the proportion of acetonitrile remained at 80%, and the proportion of 0.1% formic acid aqueous solution remained at 20%; at 20-21 minutes, the proportion of acetonitrile decreased from 80% to 5%, and the proportion of 0.1% formic acid aqueous solution increased from 20% to 95%.

[0057] Under the above conditions, the retention times of the chromatographic peaks of the nine reference standards are shown in Table 1, and the UPLC chromatogram of the mixed reference standards is shown in Table 1. Figure 1 .

[0058] Table 1. Results of chromatographic peak retention times for each reference standard. Danshensu 3.344 Protocatechuic acid 3.486 Protocatechuic aldehyde 4.758 caffeic acid 6.603 Rosemary acid 13.646 Shikonin 13.939 Tanshinone B 14.432 Tanshinone A 15.085 Tanshinone C 16.151 4. Examination of linear relationships Take the mixed reference solution and serially dilute it 10 times (2-fold) to obtain mixed reference solutions of different concentration gradients. Then, dilute the solutions with 0.22... μ Filtered through a microporous membrane, the sample was injected and analyzed sequentially according to the chromatographic conditions under "3. Chromatographic Conditions and System Suitability Test". The peak area integral value was plotted on the ordinate. Y The mass concentration of the reference standard is shown on the x-axis. X The standard curve was plotted, the regression equation was calculated, and the results are shown in Table 2.

[0059] Table 2 Results of linearity study of mixed reference solutions Danshensu 3153.1 5006.6 0.9996 0.5420~555 Protocatechuic acid 7398.2 -10879 0.9996 0.6201~635 Protocatechuic aldehyde 20297 +80237 0.9995 0.7812~800 caffeic acid 15561 -20592 0.9996 0.6104~625 Rosemary acid 7487.8 -6374.7 0.9998 0.6543~670 Shikonin 5217.6 -10474 0.9995 0.5713~585 Tanshinone B 5772.9 -11217 0.9994 0.6592~675 Tanshinone A 14600 -24223 0.9997 0.6641~680 Tanshinone C 10332 -22842 0.9991 0.5811~595 5. Content determination results The conversion product prepared in Example 1 contained 7.34% salvianolic acid A, 15.36% tanshinone, 1.24% protocatechuic aldehyde, 13.70% rosmarinic acid, 1.03% lithospermic acid, and 3.94% salvianolic acid B.

[0060] The conversion product prepared in Example 2 contained 8.16% salvianolic acid A, 16.80% tanshinone, 1.41% protocatechuic aldehyde, 14.91% rosmarinic acid, 1.73% lithospermic acid, and 3.56% salvianolic acid B.

[0061] 6. Precision test Accurately pipette 2 μL of the reference solution μ L was injected six times consecutively according to the conditions under "3. Chromatographic conditions and system suitability test", and the relative standard deviation (RSD) of the peak area of ​​each reference standard was calculated to examine the precision of the instrument. The results showed that the precision was good.

[0062] Table 3 Precision test results Danshensu 178174 179024 178599 181733 186276 184004.5 181301.8 1.82% Protocatechuic acid 364810 367197 366003.5 372729 380051 376390 371196.8 1.67% Protocatechuic aldehyde 1042876 1047480 1045178 1061278 1085023 1073151 1059164 1.62% caffeic acid 747099 754052 750575.5 762056 780825 771440.5 761008 1.72% Rosemary acid 360783 363310 362046.5 367387 377056 372221.5 367134 1.75% Shikonin 191770 191586 191678 193368 198346 195857 193767.5 1.43% Tanshinone B 274415 276792 275603.5 283835 283926 283880.5 279742 1.64% Tanshinone A 594060 600108 597084 609413 633201 621307 609195.5 2.52% Danshensu C 455615 464063 459839 471033 481496 476264.5 468051.8 2.13% 7. Stability Test One portion of the total phenolic acid sample from the stems and leaves of *Salvia miltiorrhiza* was injected at 0, 2, 4, 8, 12, and 24 hours. The chromatographic conditions were determined according to the conditions described in section "3. Chromatographic Conditions and System Suitability Test," and the peak areas were recorded. The RSD values ​​of the peak areas for each component were calculated. The results showed that each compound exhibited good stability within 24 hours.

[0063] Table 4 Stability test results Danshensu 67661.5 67556.5 70701 73652 73084.5 75539 71365.75 4.62% Protocatechuic acid 5378900 5345411 5687672 5763983 5830046 5449741 5575959 3.76% Protocatechuic aldehyde 552520.5 557021 584628 579269 575857 543223 565419.8 2.96% caffeic acid 7933284 8133076 8821640 8165208 8831069 8730559 8435806 4.77% Rosemary acid 351797 360873.5 385169 383719 344792 352786 363189.4 4.75% Shikonin 344376.5 356578.5 378314 370239 368700 338568 359462.7 4.36% Tanshinone B 67661.5 67556.5 70701 73652 73084.5 75539 71365.75 4.62% Tanshinone A 5378900 5345411 5687672 5763983 5830046 5449741 5575959 3.76% Danshensu C 552520.5 557021 584628 579269 575857 543223 565419.8 2.96% 8. Repeatability test The total phenolic acid conversion products from the stems and leaves of *Salvia miltiorrhiza* were taken in parallel and prepared into six test solutions with 50% methanol. The solutions were measured according to the conditions described in section "3. Chromatographic Conditions and System Suitability Test," and the peak areas were recorded. The RSD values ​​of the peak areas of each component were calculated. The results showed that the method had good repeatability.

[0064] Table 5 Repeatability Test Results Danshensu 760750 758152 751371 763949 769673 775653 763258 1.13% Protocatechuic aldehyde 364501 360887 354279 363226 364005 367202 362350 1.23% Rosemary acid 942052 942828 932652 949416 950728 953956 945272 0.82% Tanshinone B 330639 330186 329335 337381 329756 332630 331654.5 0.91% Tanshinone A 723806 697468 696784 712360 686038 689159 700935.8 2.06% 9. Recovery rate test Nine portions of total phenolic acid conversion products from Salvia miltiorrhiza stems and leaves with known content were accurately weighed. Three parallel portions were prepared: 100% sample + 50% standard, 100% sample + 100% standard, and 100% sample + 150% standard. The corresponding standard content was added to each portion. The sample solution was prepared according to the procedure in section "2.2". The determination was performed according to "3. Chromatographic Conditions and System Suitability Test". The peak area was recorded, and the recovery rate and RSD value were calculated. The results indicate a good recovery rate.

[0065] Table 6 Results of Recovery Test 100% Samples + 50% Standards 99.84% 101.44% 103.87% 106.76% 101.94% 99.75% 100% Samples + 100% Standards 98.86% 103.65% 103.62% 97.03% 95.34% 96.67% 100% Samples + 150% Standards 103.03% 104.58% 99.33% 102.55% 97.81% 97.90% average value 100.58% 103.22% 102.28% 102.11% 98.36% 98.11% RSD (%) 2.17% 1.56% 2.50% 4.78% 3.39% 1.58% Example 4: Experimental study on the preparation of total phenolic acid conversion products from Salvia miltiorrhiza stems and leaves 1.1 Evaluation Indicators: Using total phenolic acids from Salvia miltiorrhiza stems and leaves as the transformation substrate, the contents of rosmarinic acid, lithospermic acid, salvianolic acid B, and salvianolic acid C were significantly reduced after transformation. Under the same transformation conditions, reactions targeting these four reduced monomeric components showed that salvianolic acid A was mainly generated from the transformation of salvianolic acid B. Therefore, the conversion rate was calculated as follows: A(Sal A1) and A(Sal A0) are the peak areas of salvianolic acid A before and after transformation, respectively; A(Sal B0) and A(Sal B1) are the peak areas of salvianolic acid B before and after transformation, respectively. Using the conversion rate of salvianolic acid A as the indicator, single-factor analysis was conducted to adjust... p H solvent, p The effects of H value, conversion temperature, conversion time, and substrate concentration on the conversion.

[0066]

[0067] 1.2 Adjustment p Determination of H solvent Take three portions of a total phenolic acid solution (calculated as 10 mg / mL of tanshinone B) from the stems and leaves of *Salvia miltiorrhiza* as test solutions, and adjust the solution with phosphoric acid, hydrochloric acid, phosphate buffer, or sodium hydroxide. p H=5, representing Experiments 1, 2, and 3 respectively. The reaction was carried out in a steam sterilizer at 120℃ for 4 hours. The peak areas of salvianolic acid B and salvianolic acid A before and after conversion were detected by UPLC, and the conversion rates were calculated. The results are shown in Table 7. The results indicate that adjusting with hydrochloric acid... pH is most favorable for the conversion to salvianolic acid A; therefore, hydrochloric acid was used as a regulator in subsequent single-factor experiments. p Solvents with H value.

[0068] Table 7 Adjustment p The effect of solvent type on the conversion rate of salvianolic acid A Experiment 1 hydrochloric acid 104.69 Experiment 2 Phosphoric acid 83.22 Experiment 3 Phosphate buffer 66.1 1.3 p H value Prepare a total phenolic acid solution (calculated as 10 mg / mL of salvianolic acid B) from the stems and leaves of *Salvia miltiorrhiza* as the test solution. Take 5 aliquots of the sample solution, 2 mL each, and adjust with hydrochloric acid or sodium hydroxide respectively. p Experiments with concentrations H1, 3, 5, 7, and 9 were placed in a steam sterilizer for experiments 1-5, reacted at 120℃ for 4 hours, and then removed. The peak areas of salvianolic acid B and salvianolic acid A before and after conversion were measured by UPLC, and the conversion rate was calculated. As shown in Table 8, the conversion rate of salvianolic acid A was high under acidic conditions, and increased with... p The pH value gradually increases, but the conversion rate of salvianolic acid A is extremely low under alkaline conditions, therefore, [the following option is chosen]. p An orthogonal experiment was conducted with H at three levels: 3, 5, and 7.

[0069] Table 8 p Effect of H value on the conversion rate of salvianolic acid A Experiment 1 1 12.65 Experiment 2 3 73.29 Experiment 3 5 104.69 Experiment 4 7 12.33 Experiment 5 9 8.16 1.4 Conversion Temperature Take 5 portions of a total phenolic acid solution from the stems and leaves of *Salvia miltiorrhiza* with a concentration of approximately 10 mg / mL (calculated as tanshinone B), 2 mL for each portion, and adjust with hydrochloric acid and sodium hydroxide. p With H=5, the reactions were carried out at 75℃, 90℃, 105℃, 120℃, and 135℃, respectively, designated as Experiments 1-5. After reacting in a steam sterilizer for 4 hours, the reactions were performed. UPLC was used to detect the peak areas of salvianolic acid B and salvianolic acid A before and after conversion, and the conversion rate was calculated. Reactions above 100℃ were performed in a steam sterilizer, while those below 100℃ were performed in a water bath. As shown in Table 9, temperature has a significant impact on the conversion rate of salvianolic acid A. Higher temperatures above 100℃ are favorable for the conversion of salvianolic acid A, and the conversion rate gradually decreases with increasing temperature. Therefore, orthogonal experiments were conducted at 105℃, 120℃, and 135℃.

[0070] Table 9 Effect of temperature on the conversion rate of salvianolic acid A Experiment 1 75 3.23 Experiment 2 90 13.91 Experiment 3 105 119.07 Experiment 4 120 104.69 Experiment 5 135 31.09 1.5 conversion time Take four 2 mL solutions of total phenolic acid from the stems and leaves of *Salvia miltiorrhiza*, with a concentration of approximately 10 mg / mL (calculated as tanshinone B). Adjust the solutions with hydrochloric acid or sodium hydroxide. pWith H=5, the reaction was carried out at 120℃ for 2, 4, 6, 8, and 10 h respectively, which are experiments 1 to 5. The peak areas of salvianolic acid B and salvianolic acid A before and after conversion were detected by UPLC, and the conversion rate was calculated. See Table 10. Long-term high temperature is not conducive to the stable existence of salvianolic acid A. Therefore, the reaction time was selected as 2, 4, and 6 h for orthogonal experiments.

[0071] Table 10 Effect of conversion time on the conversion rate of salvianolic acid A Experiment 1 2 95.1 Experiment 2 4 104.69 Experiment 3 6 19.07 Experiment 4 8 11.28 Experiment 5 10 46.54 1.6 Effect of Sample Concentration Total phenolic acid solutions from *Salvia miltiorrhiza* stems and leaves were prepared at substrate concentrations of approximately 2, 5, 10, 15, and 20 mg (calculated as salvianolic acid B). Five 2 mL aliquots of each solution were taken and the pH was adjusted to 5 with hydrochloric acid. These were designated as experiments 1-5. The solutions were then sterilized in a steam sterilizer at 120°C for 4 h. The peak areas of salvianolic acid B and salvianolic acid A before and after transformation were detected by UPLC, and the conversion rate was calculated. As shown in Table 11, orthogonal experiments were conducted at three levels of salvianolic acid B concentration (10, 15, and 20 mg / mL).

[0072] Table 11 Effect of substrate salvianolic acid B concentration on salvianolic acid A conversion rate Experiment 1 2 38.01 Experiment 2 5 30.07 Experiment 3 10 104.69 Experiment 4 15 56.61 Experiment 5 20 63.19 2.1 Orthogonal experimental design for optimal conversion process Accurately weigh 9 portions of freeze-dried total phenolic acid powder from Salvia miltiorrhiza stems and leaves. Based on the above experimental results, select L9 (3 4 An orthogonal experiment was conducted using an orthogonal array. The conversion rate of salvianolic acid A was used as the evaluation index. The pH value (A), substrate concentration (B), reaction time (C), and reaction temperature (D) of the sample solution were selected as the factors to be investigated, with three levels selected for each factor. The optimal chemical conversion process was then selected.

[0073] The orthogonal experimental results were analyzed using an ANOVA with the smallest range (C, substrate concentration) as the error term. The influence of each factor on the conversion rate of salvianolic acid A was in the order: A (pH) > B (time) > D (temperature) > C (substrate concentration). The main influencing factor was... p H value ( P <0.05), conversion time ( P <0.05). Based on the range results and the K values ​​of each factor, the optimal chemical conversion process for total phenolic acids in Salvia miltiorrhiza stems and leaves was determined according to the level of the highest K value for each factor. A 1 B 1 C 2 D 3 That is, adjusting with hydrochloric acid pThe H value was 3, the substrate concentration (calculated as salvianolic acid B) was 15 mg / mL, the incubation period was 135℃, and the incubation time was 2 h. The results are shown in Tables 12 and 13.

[0074] Table 12 L9 (3) 4 Orthogonal experimental design and analysis 1 3(1) 2(1) 10(1) 105(1) 46.57 2 3(1) 4(2) 15(2) 120(2) 31.26 3 3(1) 6(3) 20(3) 135(3) 29.11 4 5(2) 2(1) 15(2) 135(3) 35.53 5 5(2) 4(2) 20(3) 105(1) 15.17 6 5(2) 6(3) 10(1) 120(2) 3.87 7 7(3) 2(1) 20(3) 120(2) 16.83 8 7(3) 4(2) 10(1) 135(3) 19.84 9 7(3) 6(3) 15(2) 105(1) 6.39 k1 0.3565 0.3298 0.2343 0.2271 k2 0.1819 0.2209 0.2440 0.1732 k3 0.1435 0.1313 0.2037 0.2816 R 0.2129 0.1985 0.04025 0.1084 Table 13 Analysis of Variance A 0.0773 2 0.0386 29.1797 0.0331 p<0.05 B 0.0593 2 0.0296 22.3829 0.0428 p<0.05 C 0.0026 2 0.0013 1.0000 0.5000 \ D 0.0176 2 0.0088 6.6583 0.1306 p>0.05 Example 5: Experimental Study on the Antioxidant Activity of Total Phenolic Acid Conversion Products from Salvia miltiorrhiza Stems and Leaves I. Experimental Materials and Drugs 1. Experimental Apparatus Ditect-Q5 pure water preparation system (Millipore); TOMY SX-500 steam sterilizer; ML105 0.001 g electronic balance; DLORD D3024R high-speed refrigerated centrifuge; Enspire multi-functional microplate reader (PerkinElmer, USA).

[0075] 2. Drugs and Reagents Ultrapure water was prepared in-house using a milli-Q ultrapure water preparation system; 95% ethanol was purchased from Nanjing Wanqing Chemical Glassware Instrument Co., Ltd.; DPPH was purchased from Solarbio Science & Technology Co., Ltd.; ABTS and TPTZ (tripyridine triazine) test kits were purchased from Nanjing Jiancheng Co., Ltd.; and the post-phenolic acid fraction of Salvia miltiorrhiza stems and leaves before transformation was prepared in-house. 96-well microplates were used.

[0076] II. Experimental Methods 1. Sample extraction and preparation The samples were obtained from the total phenolic acids of Salvia miltiorrhiza stems and leaves before transformation and the transformation products of Salvia miltiorrhiza stems and leaves after transformation in Examples 1 and 2.

[0077] 2. Methods for evaluating in vitro antioxidant activity Accurately weigh the dried samples and redissolve them in 80% ethanol solution to prepare a 1 mg / mL solution. Dilute the solutions sequentially by a factor of 2 to obtain 8 concentration gradients. Analyze the antioxidant capacity using an enzyme-linked immunosorbent assay (ELISA) reader as described below.

[0078] Evaluation of DPPH radical scavenging rate: Add 50g of DPPH radical scavenging agent to the well plate. μ L of test solution and 100 μ L of 0.05 mg / mL DPPH ethanol solution (prepared with anhydrous ethanol) was mixed and reacted at room temperature in the dark for 30 min. The absorbance A was measured at 517 nm. 样品 The absorbance A was measured using the above method, replacing both the DPPH solution and the test solution with 80% ethanol. 对照 and A 空白Calculate the DPPH free radical scavenging rate using the following formula. Vitamin C serves as a positive control.

[0079] DPPH free radical scavenging rate (%) = 1 - (A 样品 -A 对照 ) / A 空白 Calculate IC 50 .

[0080] ABTS scavenging activity evaluation: Following the kit instructions, prepare the ABTS working stock solution by mixing ABTS solution and oxidant solution at a 1:1 ratio. Store at room temperature in the dark for 12-16 hours before use. Dilute the ABTS working stock solution with 80% ethanol to prepare the ABTS working solution before use. Add 200 mg / L of ABTS solution to each well of a 96-well plate. μ L ABTS working solution and 10 μ After mixing the test solution (L), incubate at room temperature for 2–6 min, and then measure the absorbance (A) at a wavelength of 734 nm. 样品 The absorbance A was measured using 80% ethanol instead of the ABTS working solution and the test solution, respectively, according to the method described above. 对照 and A 空白 The ABTS radical scavenging rate is calculated using the following formula.

[0081] ABTS radical scavenging rate (%) = 1 - (A 样品 -A 对照 ) / A 空白 Calculate IC 50 .

[0082] Fe reduction 3+ Proficiency assay (FRAP method): based on the reduction of Fe 3+ Following the instructions of the proficiency assay kit, thoroughly mix the TPTZ diluent and TPTZ solution at a ratio of 10:1, then add an equal volume of detection buffer to the TPTZ solution. Incubate at 37°C to obtain the FRAP working solution. Add 180 μL of the solution to the wells of a 96-well plate. μ L FRAP working fluid and 5 μ Sample A was prepared by mixing the L test solution and measuring it at a wavelength of 593 nm. 27.8 mg of FeSO4·7H2O was weighed and diluted to 1 mL in a volumetric flask to obtain a 100 mmol / mL FeSO4·7H2O solution. The stock solution was diluted to a series of concentrations, and the absorbance was measured using the method described above to obtain a standard curve. The antioxidant activity of the sample is expressed as the amount of FeSO4 (mmol) required to achieve the same absorbance, and its standard equation is Y = 0.2446X + 0.099, R... 2 =0.9937, the higher the measured absorbance A value, the greater the reduction of Fe. 3+ The stronger the ability.

[0083] III. Experimental Results 3.1 Results of DPPH free radical scavenging rate Tables 14 and 16 show that the DPPH free radical scavenging capacity of the transformation products and total phenolic acids from *Salvia miltiorrhiza* stems and leaves under low concentration conditions is in the order of transformation products > total phenolic acids from *Salvia miltiorrhiza* stems and leaves > vitamin C. Furthermore, within the test range, their antioxidant capacity showed a clear dose-dependent relationship with mass concentration, increasing with increasing concentration. The total phenolic acids from *Salvia miltiorrhiza* stems and leaves after transformation (IC50)... 50 3±0.16 mg / mL) compared to total phenolic acids in tanshinone stems and leaves (IC50) 50 The antioxidant capacity was significantly improved (3.4±0.08 mg / mL).

[0084] Table 14 DPPH radical scavenging rate of each sample group (`X±SD, n=3) 31.25 93.88±0.77 95.73±0.25 96.5±0.15 15.63 93.96±0.51 92.98±1.14 93.48±1.88 7.81 80.65±3.95 72.56±4.3 68.47±5.98 3.91 50.61±0.41 45.12±2.35 41.72±2.7 1.95 33.56±1.84 31.84±4.81 28.09±5.05 0.98 25.39±2.53 22.72±2.21 21.4±4.3 3.2 Evaluation of ABTS scavenging activity Tables 15 and 16 show that the ABTS scavenging capacity of the transformed products and total phenolic acids from Salvia miltiorrhiza stems and leaves is comparable to that of vitamin C. Within the measured range, the antioxidant capacity showed a positive dose-dependent relationship with the mass concentration. The results indicate that after the transformation of total phenolic acids (IC50-120%), the ABTS scavenging capacity was significantly increased. 50 The antioxidant capacity of 16.88±0.32 mg / mL was superior to that of total phenolic acids from the stems and leaves of *Salvia miltiorrhiza* (IC50). 50 (20.25±0.48 mg / mL).

[0085] Table 15. ABTS radical scavenging rate of each sample group (`X±SD, n=3) 125.00 98.95±0.46 98.92±0.75 98.66±1.33 62.50 98.35±0.98 97.51±1.85 96.41±4.27 31.25 64.65±2.73 52.15±3.08 49.56±1.95 15.63 33.2±1.06 29.42±4.08 25.46±0.7 7.81 19.28±1.46 15.93±0.64 14.68±1.95 3.91 12.41±1.53 9.57±0.77 10.01±0.63 1.95 8.26±1.28 6.33±1.3 7.89±0.53 0.98 5.9±1.01 5.82±1.26 5.17±1.95 3.3 Reduction of Fe 3+ Proficiency testing (FRAP method) The results are shown in the table. The antioxidant capacity of the total phenolic acid in the stems and leaves of Salvia miltiorrhiza after conversion (1.15±0.03 mmol FeSO4) was improved compared with that of the total phenolic acid in the stems and leaves of Salvia miltiorrhiza (0.98±0.06 mmol FeSO4).

[0086] Table 16 Antioxidant activity of phenolic acid fractions from Salvia miltiorrhiza stems and leaves before and after transformation (X±SD, n=3) Transformation products 3.4±0.08 20.25±0.48 1.154±0.03 Total phenolic acids from the stems and leaves of Salvia miltiorrhiza 3±0.16 16.88±0.32 0.977±0.059 Example 6: Experimental study on the protective effect of the conversion product on the H2O2-induced HUVEC and H9C2 oxidative damage model.

[0087] I. Experimental Materials and Drugs 1. Experimental Apparatus BWS-10 constant temperature water bath (Shanghai Yiheng Scientific Instruments Co., Ltd.), ultrapure water preparation system (MilliQ, Millipore, USA); high-speed centrifuge (Backman); Forma series II water jacket CO2 incubator (Thermo); 1300 series A2 clean bench (Thermo); PrimoStar inverted microscope (ZEISS); TOMY SX-500 autoclave (Nanjing Jitian Biotechnology Co., Ltd.); Enspire multi-functional microplate reader (PerkinEImer, USA); 96-well microplates purchased from Corning, USA; cell culture dishes (ComingIncorporated).

[0088] 2. Reagents High-glucose DMEM medium, antibiotics P / S (penicillin 10000 U / mL, streptomycin 10000 mg / mL), fetal bovine serum FBS, and trypsin-EDTA digestion solution were all obtained from Gibco, USA; dimethyl sulfoxide (DMSO), tetramethylazosulfone blue (MTT), and hydrogen peroxide were purchased from Shanghai Husheng Laboratory Equipment Co., Ltd.; ultrapure water was prepared using a Milli-Q ultrapure water preparation system; the components of total phenolic acids from Salvia miltiorrhiza stems and leaves before and after conversion were prepared in-house (Example 1). Superoxide dismutase (SOD), malondialdehyde (MDA), and lactate dehydrogenase (LDH) kits were purchased from Nanjing Jiancheng Bioengineering Institute.

[0089] 3. Experimental cells Human umbilical vein endothelial cells (HUVEC) were purchased from Nanjing Kaiji Biotechnology Development Co., Ltd., and rat cardiomyocytes (H9C2) were purchased from Wuhan Pronosei Life Science Co., Ltd. In this experiment, cells of passage 3 to 15 were used.

[0090] II. Experimental Methods 1. Cell Culture Both HUVEC and H9C2 cells were cultured in high-glucose DMEM medium containing 10% FBS + 1% P / S, and placed in a cell culture incubator at 37°C, 5% CO2, and saturated humidity. The medium was changed every 2 days. When the cell coverage in the culture dish reached 80%-90%, the cells were passaged using trypsin digestion.

[0091] 2. Cell seeding plate HUVEC and H9C2 cell seeding: When the cell coverage in the culture dish reaches 80%-90%, digest the cells with trypsin for about 2 minutes. Under a light microscope, the cells will become rounded. At this point, use a pipette to mix the cells from the culture dish. Dilute with complete DMEM high-glucose culture medium to prepare a solution with a concentration of 6×10⁻⁶.4 A cell suspension of 100 cells / mL was then seeded into 96-well plates, with 100 cells / mL added to each well. μ The complete culture medium of L was placed in an incubator and cultured under the conditions of 5% CO2 and 37°C.

[0092] 3. Establishment of a hydrogen peroxide cell damage model After changing the cell culture medium, culture for 24 hours, discard the culture medium, wash the cells three times with PBS, and add fresh culture medium (10% FBS) at a depth of 90°C. μ After incubating for 3 hours, add 10 μ Different concentrations of hydrogen peroxide were added. HUVEC cells were incubated with hydrogen peroxide-damaged medium for 2 hours, and H9C2 cells were incubated with hydrogen peroxide-damaged medium for 4 hours, both incubated at 37°C in a 5% CO2 incubator. After the oxidative damage model was established, the old culture medium was discarded, and 90 μL of hydrogen peroxide was added to each well. μ L fresh 10% FBS medium, 10 μ Incubate with L MTT solution (5 mg / mL) in an incubator for 3 hours. After incubation, discard the culture medium from each well and add 150 μL DMSO to each well. μ L, incubated at 37℃ with shaking for 30 min, and the absorbance of each well was measured at 570 nm using a microplate reader. Based on the MTT assay results, the modeling concentration for subsequent experiments was determined using the following calculation formula.

[0093] Cell viability / % = (OD value of model group / OD value of blank group) × 100% 4. Experimental Grouping The experiment was divided into a blank group, a model group, a vitamin C group, and each model plus drug treatment group (total phenolic acid group of tanshinone stems and leaves, and transformation product group). The total phenolic acid of tanshinone stems and leaves and the transformation product were dissolved and diluted with fresh culture medium (10% FBS), and the highest concentration was set at 1 mg / mL. The six concentrations were serially diluted by 2-fold, and the cell viability of each drug treatment group at different concentrations was determined by MTT assay.

[0094] 5. Morphological observation and MTT assay for cell viability in each group. HUVEC and H9C2 cells were cultured at a rate of 6 × 10⁻⁶. 4 Inoculate at a density of 100 cells / mL in 96-well plates, with 100 cells per well. μ L of culture medium was used to incubate 96-well plates in an incubator at 37°C with 5% CO2. Hydrogen peroxide damage group, 2000 μ Cell damage was induced by hydrogen peroxide at mol / L for 2 hours, and drug intervention was administered simultaneously. Cell morphology was observed under an inverted microscope. H9C2 cells in the hydrogen peroxide-damaged group received 800 mol / L hydrogen peroxide. μ Cell damage was induced by mol / L hydrogen peroxide for 4 hours, while drug intervention was administered simultaneously.

[0095] 6. Determination of SOD, MDA, and LDH levels in cell culture medium HUVEC and H9C2 cells (5×10⁻⁶) were used. 5 Cells (100 cells / well) were seeded in 6-well cell culture plates. After modeling and drug administration, the culture supernatant and cells were collected. The levels of superoxide dismutase (SOD), malondialdehyde (MDA), and lactate dehydrogenase (LDH) were measured according to the instructions of the kit.

[0096] 7. Statistical Processing The experimental results were statistically analyzed using SPSS 21.0. Data are expressed as mean ± standard deviation (X ± SD). One-way ANOVA was used for comparisons between groups. P The difference was statistically significant when the value was less than 0.05.

[0097] III. Experimental Results 1. Screening results of modeling concentration Oxidative damage models revealed that the degree of cell damage increased with increasing modeling concentration. After screening modeling concentrations, an optimal concentration was selected when cell viability was approximately 50%. Results showed that hydrogen peroxide 2000... μ The optimal modeling dose for HUVEC cells is 800 mol / L for 2 hours. μ The optimal modeling dose for H9C2 cells is mol / L for 4 hours.

[0098] 2. Protective effect against hydrogen peroxide-damaged HUVEC cells The control group showed good growth of HUVEC cells, exhibiting a cobblestone-like appearance and tight arrangement. In the model group, after oxidative damage, the cells were shrunken, with increased intercellular spaces, uneven size distribution, and disordered arrangement. Compared to the model group, the drug-treated groups showed slight cell shrinkage, slightly larger intercellular spaces, tighter arrangement, and clearer boundaries, with the overall boundary morphology resembling that of the control group. The high-dose and low-dose groups showed cell morphology closer to normal cells. The effects of each drug-treated group on the survival rate of oxidatively damaged HUVEC cells are shown in the figures below. Figure 2 As shown, the MTT results of the model group were significantly lower than those of the blank group ( P <0.0001 indicates successful model establishment. Compared with the model group, each drug-treated group showed significant protective effects against H2O2-damaged HUVECs. P <0.05), showing a positive correlation within a certain concentration range; the higher the dose, the better the protective effect against oxidative damage to cells; the protective effect of the transformation products in each treatment group at the same mass concentration was comparable to that of vitamin C, and superior to the total phenolic acids in the stems and leaves of Salvia miltiorrhiza.

[0099] HUVEC cells after oxidative damage Figure 3 , 4As shown in Figure 5, compared with the control group, the model group showed a significant decrease in SOD secretion (P<0.001) and a significant increase in LDH and MDA levels (P<0.0001). After administration, compared with the model group, the high, medium, and low dose groups showed increased SOD levels in cells and supernatants (P<0.05) and decreased MDA and LDH levels (P<0.01), all showing a dose-response relationship; the higher the dose, the more significant the protective effect. Vitamin C significantly increased SOD levels, and its conversion products significantly decreased MDA and LDH levels (P<0.01).

[0100] In summary, the protective effect of total phenolic acids from tanshinone stems and leaves on oxidatively damaged HUVEC cells at the same mass concentration is comparable to that of vitamin C, and the transformation product is superior to total phenolic acids from tanshinone stems and leaves.

[0101] 3. Protective effect against hydrogen peroxide-damaged H9C2 cells The control group showed good growth of H9C2 cells, which were spindle-shaped or polygonal and tightly packed. In the model group, after oxidative damage, the cells were shrunken, with blurred boundaries, increased gaps, and disordered arrangement. Compared to the model group, the drug-treated groups showed some cell shrinkage, but to a lesser degree, with clearer boundaries, but a tighter arrangement, generally resembling the control group. Among the drug-treated groups, the high-dose and low-dose groups showed cell morphology closer to normal cells. The effects of each drug-treated group on the survival rate of oxidatively damaged H9C2 cells are shown below. Figure 6 The results showed that the MTT in the model group was significantly lower than that in the blank group. P <0.0001), successful model establishment. Compared with the model group, each treatment group showed a protective effect against H2O2-damaged H9C2 cells, exhibiting a positive correlation within a certain concentration range. Higher doses resulted in better protection against oxidative damage, with the vitamin C group showing the best protective effect. High, medium, and low dose groups all significantly protected against oxidative damage to H9C2 cells. At the same mass concentration, the transformation products of the treatment groups showed a stronger protective effect against oxidative damage to H9C2 cells than the total phenolic acids from *Salvia miltiorrhiza* stems and leaves. The experiment found that when the transformation product concentration was below 62.5%, the protective effect against oxidative damage was significantly enhanced. μ At a dosage of g / mL, the total phenolic acids in the stems and leaves of *Salvia miltiorrhiza* are less than 250 g / mL. μ At a concentration of g / mL, almost no significant protective effect was observed against oxidatively damaged H9C2 cells.

[0102] After oxidative damage to H9C2 cells, such as Figure 7 , 8As shown in Figures 9, compared with the control group, the model group showed a significant decrease in SOD secretion (P<0.0001) and a significant increase in MDA and LDH levels (P<0.0001). After administration, compared with the model group, the high- and medium-dose groups showed increased SOD levels in cells and supernatants (P<0.01) and decreased MDA and LDH levels (P<0.001), all exhibiting a dose-response relationship; the higher the dose, the more significant the protective effect. Overall, the transformation product showed the best protective effect, significantly increasing SOD levels and decreasing MDA levels at high doses (P<0.001); Vitamin C significantly decreased LDH levels (P<0.0001); the transformation product, at the same mass concentration, showed better protective effects against oxidative damage to H9C2 cells than the total phenolic acids from Salvia miltiorrhiza stems and leaves.

[0103] Example 7: Experimental Study on Protecting Myocardial Function I. Experimental Materials and Drugs 1. Drugs and reagents Isoproterenol hydrochloride (Ron, CAS 51-30-9); chromatographic grade acetonitrile (Merck, Germany); chromatographic grade formic acid (ACS, USA); ultrapure water (Millipore); medical saline; isoflurane (Reward); lactate dehydrogenase (LDH) assay kit, creatine kinase (CK) assay kit, creatine kinase isoenzyme (CK-MB) assay kit, and aspartate aminotransferase (AST) assay kit were provided by Qiangsheng Biotechnology Co., Ltd. Test drug: Total phenolic acids and transformation products from the stems and leaves of *Salvia miltiorrhiza* prepared in Example 2 of this invention.

[0104] 2. Experimental Apparatus Physiological recorder (AD Instruments PL3508); Microfuge 22R Centrifuge (Beckman Coulter, USA); MX-S adjustable mixer (Dalong Xingchuang Experimental Instruments Co., Ltd.); Fully automated biochemical analyzer (AU480, Beckman Coulter); Small animal anesthetic machine (Jitai).

[0105] 3. Laboratory animals SPF-grade male SD mice, 200±20g, were purchased from the Experimental Animal Center of Nanjing University of Chinese Medicine, certificate number SCXK (Shanghai) 2022-0004.

[0106] II. Experimental Methods 1. Establishment and administration of an acute myocardial ischemia rat model After one week of acclimatization feeding, SPF-grade male SD rats were randomly divided into 8 groups (n=10 per group) using a stratified weight-based method. All rats were administered the drug via gavage for 5 consecutive days. The control group and model group were administered physiological saline via gavage. The propranolol group (positive control group 1): 20 mg / kg / day; the compound Danshen dripping pill group (positive control group 2): 75 mg / kg / day; the low-dose group of total phenolic acids from Danshen stems and leaves (low-dose group 1): 50 mg / kg / day; the high-dose group of total phenolic acids from Danshen stems and leaves (high-dose group 1): 200 mg / kg / day; the low-dose group of total phenolic acid conversion products from Danshen stems and leaves (low-dose group 2): 50 mg / kg / day; and the high-dose group of total phenolic acid conversion products from Danshen stems and leaves (high-dose group 2): 200 mg / kg / day. ISO induction modeling was initiated for 3 consecutive days starting on day 6, with ISO modeling performed half an hour after gavage administration. ISO modeling method: Prepare an ISO concentration of 20 mg / mL with physiological saline, and inject it subcutaneously at multiple points on the neck and back of rats at a modeling dose of 50 mg / Kg to induce an acute myocardial ischemia model. The blank group used physiological saline.

[0107] 2. Measurement of myocardial electrophysiological parameters Two hours after subcutaneous injection of ISO, rats were anesthetized with isoflurane and fixed in a dorsal position on a dissection table. Electrocardiographic parameters were measured using a physiological recorder with subcutaneous lead II, and ECG changes in each group were continuously monitored for 2 minutes. Data recorded by the instrument were analyzed using LabChart.

[0108] 3. Methods for evaluating drug efficacy During gavage administration and model establishment, the rats' mental state, activity, and coat color were observed. On days 5, 6, 7, and 8, electrocardiogram changes were recorded using a physiological recorder before and after model establishment. Three days after model establishment, rats were anesthetized with 10% chloral hydrate via intraperitoneal injection. Whole blood was collected from the abdominal aorta, allowed to stand at room temperature for 2 hours, and then centrifuged in a low-temperature ultracentrifuge (4℃, 3000 rpm / min, 10 min). The separated serum was used to determine LDH, CK, CK-MB, and AST. After blood collection, the hearts were quickly removed, cleaned with physiological saline, blotted dry with filter paper, weighed, placed in sealed plastic bags, and flash-frozen in liquid nitrogen. One heart from each group was randomly selected using a random number table and fixed with paraformaldehyde. HE and MASSON staining were then performed for pathological section analysis.

[0109] 4. Statistical processing The experimental results were statistically analyzed using SPSS 21.0. Data are expressed as mean ± standard deviation (X ± SD). One-way ANOVA was used for comparisons between groups. P The difference was statistically significant when the value was less than 0.05.

[0110] III. Experimental Results After ISO modeling, rats exhibited salivation, frequent chest contractions, huddling together, reduced movement, and sluggish responses. After three consecutive days of modeling, 3 rats died in the model group and W group, 4 in the P group, and 1 in the D group. The cause of death was likely due to excessive vasoconstriction following ISO injection, leading to acute myocardial ischemia and severe pulmonary vasoconstriction, resulting in acute myocardial infarction. Based on mortality data, all treatment groups showed good protective effects against ISO-induced acute myocardial ischemia. Prolonged myocardial ischemia and hypoxia lead to compensatory ventricular hypertrophy in non-necrotic areas to alleviate insufficient myocardial blood supply and enhance myocardial function. Figure 10 There were significant differences in the organ coefficients of the heart between the blank control group and the model group (p<0.01). The group treated with the transformed product showed better cardioprotective effects than the group treated with total phenolic acids from the stems and leaves of Salvia miltiorrhiza, and the lower dose was superior to the higher dose. Figure 11 , 12 Results 13 showed that after rat modeling, heart rate increased significantly and ST segment decreased significantly (P<0.0001), and endocardial ischemia occurred in rats. Compared with the model group, the heart rate and ST segment decrease were improved to varying degrees in each treatment group (P<0.01), with heart rate slowing down and ST segment elevation. The protective effect of the transformation product treatment group was better than that of total phenolic acids from tanshinone stems and leaves.

[0111] After the animal experiments were completed, the heart tissue was quickly removed, the blood was washed away with physiological saline, excess liquid was absorbed with filter paper, and then fixed with paraformaldehyde for HE and MASSON staining. Figure 14 The results showed that the model group's cardiac tissue exhibited significant ischemic damage, disordered arrangement, interstitial congestion, cell swelling, and marked inflammatory cell infiltration. In the drug-treated groups, the low- and high-dose groups of the transformed product treatment group showed significantly reduced myocardial ischemia, significantly improved myocardial fiber rupture and inflammatory cell infiltration, and reduced cell swelling and disordered arrangement. The improvement in myocardial cell damage was superior to that of the group treated with total phenolic acids from *Salvia miltiorrhiza* stems and leaves. Figure 15 The results showed that the cardiac cardiomyocytes in the model group rats were arranged in a disordered manner, with a large number of blue collagen fibers, obvious ischemic damage, and a significant reduction in purplish-red myocardial tissue. All drug administration groups could significantly reduce the blue-stained collagen fibers. Among them, the transformation product drug administration group showed mainly purplish-red cardiomyocytes under the microscope, with a significant reduction in blue-stained collagen fibers, and the effect of improving myocardial fibrosis was better than that of the total phenolic acid group of tanshinone stems and leaves.

[0112] After collecting blood from rats, the levels of CK, CK-MB, AST, and LDH in rat serum were measured according to the kit's instructions. Figure 16 , 17As shown in Figures 18 and 19, the model group rats suffered cardiomyocyte damage, and serum LDH, CK, and AST levels were significantly elevated (P<0.01). All treatment groups effectively downregulated LDH, CK, and AST activities. Both the low-dose and low-dose groups significantly reduced CK-MB levels, demonstrating a protective effect against myocardial injury. The low-dose group was superior to the high-dose group. The transformed product treatment group was more effective than the total phenolic acids from *Salvia miltiorrhiza* stems and leaves in reducing LDH levels. This indicates that the transformed product provided by this invention has excellent cardioprotective function and can effectively protect cardiomyocytes from damage caused by acute myocardial ischemia.

[0113] Example 8 Antioxidant Experimental Study I. Experimental Materials and Drugs 1. Experimental Apparatus Microplate reader (Perkin-Elmer, USA); BT125 electronic balance (Sartorius Scientific Instruments Co., Ltd.); Anke GL-16GII centrifuge (Shanghai Anting Scientific Instruments Factory).

[0114] 2. Drugs and Reagents Isoproterenol hydrochloride (Ron, CAS 51-30-9); medical saline; isoflurane (Reward); superoxide dismutase (SOD) kit, malondialdehyde (MDA) kit, glutathione peroxidase (GSH-Px) kit, and catalase (CAT) kit were purchased from Nanjing Jiancheng Bioengineering Institute.

[0115] Test drug: Total phenolic acids and their transformation products from the stems and leaves of *Salvia miltiorrhiza* prepared in Example 2 of this invention.

[0116] 3. Laboratory animals SPF-grade male SD mice, 200±20g, were purchased from the Experimental Animal Center of Nanjing University of Chinese Medicine, certificate number SCXK (Shanghai) 2022-0004.

[0117] II. Experimental Methods 1. Establishment and administration of an acute myocardial ischemia rat model After one week of acclimatization feeding, SPF-grade male SD rats were randomly divided into groups of 10 rats each using a stratified grouping method based on body weight. All rats were administered the drugs by gavage for 5 consecutive days. The control group and model group were administered physiological saline by gavage; propranolol group (positive control group 1): 20 mg / kg / day; Compound Danshen Dripping Pills group (positive control group 2): 75 mg / kg / day; Low-dose group of total phenolic acids from Danshen stems and leaves (low-dose group 1): 50 mg / kg / day; High-dose group of total phenolic acids from Danshen stems and leaves (high-dose group 1): 200 mg / kg / day; Low-dose group of transformation products (low-dose group 2): 50 mg / kg / day; High-dose group of transformation products (high-dose group 2): 200 mg / kg / day.

[0118] Starting on day 6, ISO induction was performed for three consecutive days. ISO modeling was initiated 30 minutes after gavage administration. ISO modeling method: ISO concentration of 20 mg / mL was prepared with physiological saline and injected subcutaneously at multiple points on the neck and back of rats at a modeling dose of 50 mg / Kg to induce an acute myocardial ischemia model. The blank group used physiological saline.

[0119] 2. Methods for evaluating drug efficacy After the last administration of each group of rats, the rats were anesthetized by intraperitoneal injection of 10% chloral hydrate, and whole blood was collected from the abdominal aorta. After blood collection, the heart was quickly removed, cleaned with physiological saline, dried with filter paper, weighed, placed in a plastic bag, and then quick-frozen in liquid nitrogen.

[0120] Myocardial tissue homogenate was prepared and the SOD, GSH-Px, CAT, and MDA contents were detected according to the kit instructions.

[0121] 3. Statistical processing The experimental results were statistically analyzed using SPSS 21.0. Data are expressed as mean ± standard deviation (X ± SD). One-way ANOVA was used for comparisons between groups. P The difference was statistically significant when the value was less than 0.05.

[0122] III. Experimental Results 1. Results of biochemical index determination After the animal experiments, myocardial tissue homogenate was prepared, and the contents of SOD, MDA, GSH-Px, and CAT in rat myocardium were measured according to the kit's instructions. Figure 20 , 21 Figures 22 and 23 show that after modeling, the activities of GSH-Px, SOD, and CAT decreased, while the content of MDA increased (P<0.0001). All treatment groups effectively downregulated MDA and increased SOD activity (P<0.0001). The high-dose treatment group of the transformation product significantly upregulated GSH-Px (P<0.05), with a protective effect similar to that of the positive control drug compound Danshen dripping pill group. The low-dose treatment group of the transformation product significantly increased CAT activity (P<0.0001). This indicates that the transformation product provided by this invention can effectively scavenge free radicals, has excellent antioxidant effects, and can effectively reduce oxidative damage to myocardial cells caused by acute myocardial ischemia.

Claims

1. A total phenolic acid conversion product from the stems and leaves of *Salvia miltiorrhiza* that possesses antioxidant properties and protects against vascular endothelial cells and cardiomyocytes, characterized in that... The conversion product was prepared using the following method: The above-ground stems and leaves of Salvia miltiorrhiza were dried with hot air at 40-50℃, pulverized and passed through the No. 3 standard sieve of the Pharmacopoeia to obtain coarse powder of Salvia miltiorrhiza stems and leaves; 10-15 times the amount of 60% ethanol was added each time, and the mixture was refluxed at 80℃-85℃ for 1-3 times, each time for 1-2 hours. The extracts were combined, and the ethanol was recovered by vacuum decompression concentration and concentrated until there was no alcohol taste to obtain the Salvia miltiorrhiza stem and leaf extract. The above-mentioned extract of Salvia miltiorrhiza stems and leaves was diluted with water to a concentration of 0.125-1.5g crude drug per mL. It was then purified using AB-8 macroporous resin. Before use, the resin was fully swollen with 2-3 times its volume of 95% ethanol. The column was packed using a wet packing method, with a resin mass to sample loading ratio of 2-3:1 and a column diameter to height ratio of 1:5-1:

20. The column was first washed with 3-5 BV of pure water until there was no alcohol odor and impurities were removed. Then, the eluent was eluted with 3-5 BV of 20% ethanol and the eluent was eluted with 1.5-2 BV of 30% ethanol as the total phenolic acid fraction of Salvia miltiorrhiza stems and leaves. The collected eluent was concentrated under reduced pressure to recover the ethanol and concentrated until there was no alcohol odor to obtain the crude product of Salvia miltiorrhiza stems and leaves. Dilute the crude danshen stems and leaves mentioned above with water to a concentration of 0.1-0.3g of raw drug per 1mL, and adjust with 5M hydrochloric acid. p Extract with 2-3 H, extract with twice the amount of ethyl acetate 3-5 times, combine the ethyl acetate fractions, concentrate under vacuum to recover ethyl acetate and concentrate to dryness to obtain total phenolic acids from the stems and leaves of Salvia miltiorrhiza. The obtained tanshinone stem and leaf phenolic acids were diluted with water to a concentration of 10-20 mg of tanshinone B per mL, and the conversion solution was adjusted with 5M hydrochloric acid. p With H=3, the reaction was carried out at 135℃ for 2 hours using a high-temperature and high-pressure sterilizer, followed by vacuum freeze-drying to obtain the conversion product. The transformation product comprises the following active ingredients in weight percentage: salvianolic acid A 7-9%, tanshinone 15-17%, protocatechuic aldehyde 1-2%, rosmarinic acid 13-15%, shikonin 1-2%, and salvianolic acid B 3-4%.

2. The application of the total phenolic acid conversion product of Salvia miltiorrhiza stems and leaves according to claim 1 in the preparation of drugs that protect vascular endothelial cells and cardiomyocytes.

3. The use of the total phenolic acid conversion product of Salvia miltiorrhiza stems and leaves as described in claim 1 in the preparation of drugs to improve acute myocardial ischemia injury.

4. The application of the total phenolic acid conversion product of Salvia miltiorrhiza stems and leaves as described in claim 1 in the preparation of antioxidant drugs.

5. The application according to any one of claims 2 to 4, characterized in that, Drugs made from the total phenolic acid conversion products of Salvia miltiorrhiza stems and leaves and pharmaceutically acceptable carriers into tablets, pills, powders, decoctions, granules, pastes, or extracts.

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