High-purity magnesium lithospermate B and preparation method therefor
10259798 ยท 2019-04-16
Assignee
- Shanghai Institute Of Materia Medica Chinese Academy Of Sciences (Shanghai, CN)
- SHANGHAI GREEN VALLEY PHARMACEUTICAL CO., LTD. (Shanghai, CN)
Inventors
- Lijiang Xuan (Shanghai, CN)
- Yiping Wang (Shanghai, CN)
- Weibin Song (Shanghai, CN)
- Chunguang Chen (Shanghai, CN)
- Dingxiang Li (Shanghai, CN)
- Xiaoyu Zhou (Shanghai, CN)
- Yunlong Gu (Shanghai, CN)
- Liang HU (Shanghai, CN)
- Jing Zhao (Shanghai, CN)
- Wenwei Xu (Shanghai, CN)
- Shumei Wang (Shanghai, CN)
Cpc classification
A61K31/343
HUMAN NECESSITIES
A61P9/10
HUMAN NECESSITIES
A61P7/02
HUMAN NECESSITIES
International classification
A61K36/00
HUMAN NECESSITIES
C07D307/86
CHEMISTRY; METALLURGY
Abstract
The present invention relates to a method for preparing magnesium lithospermate B. The method is characterized in that: magnesium lithospermate B is extracted or purified from a Salvia miltiorrhiza plant material or a Salvia miltiorrhiza extract in the presence of an added magnesium salt. The present invention also relates to a high-purity magnesium lithospermate B product prepared by the method of the present invention, wherein the content of magnesium lithospermate B is more than 95% by weight.
Claims
1. A process for preparing magnesium lithospermate B, comprising: a) obtaining a Salvia miltiorrhiza liquid extract by extracting a Salvia miltiorrhiza plant material with a first alcohol-water solution, and b) separating the Salvia miltiorrhiza liquid extract via chromatography on macroporous absorption resin, wherein a second alcohol-water solution and a third alcohol-water solution is used for elution, collecting an eluate containing magnesium lithospermate B; concentrating the eluate to give a concentrated eluate, adjusting the pH value of the concentrated eluate to 3-7, and then extracting with an organic solvent to give a raffinate comprising magnesium lithospermate B; wherein a magnesium salt is added into at least one of the following: the first alcohol-water solution in a), the Salvia miltiorrhiza liquid extract in b), the second alcohol-water solution in b), or the third alcohol-water solution in b).
2. The process according to claim 1, wherein the magnesium salt is one or more of the following compounds: MgSO.sub.4, Mg(Ac).sub.2, MgCl.sub.2, MgBr.sub.2, MgCO.sub.3, and Mg(HCO.sub.3).sub.2.
3. The process according to claim 1 or 2, wherein the amount of the added magnesium salt is 5-200 mg per 100 g of the Salvia miltiorrhiza plant material or the Salvia miltiorrhiza extract.
4. The process according to claim 3, wherein the organic solvent is selected from C.sub.3-C.sub.6 alcohols, C.sub.1-C.sub.6 alkyl C.sub.1-C.sub.3 carboxylates, and di(C.sub.1-C.sub.5 alkyl)ethers.
5. The process according to claim 4, wherein the organic solvent is one or more selected from the following solvents: n-butyl alcohol, methyl acetate, ethyl acetate, propyl acetate, butyl acetate, methyl t-butyl ether, and diethyl ether.
6. The process according to claim 4 or 5, further comprising concentrating the raffinate comprising magnesium lithospermate B, follow by alcohol-precipitating, filtrating, and drying to give a magnesium lithospermate B solid.
7. The process according to claim 6, wherein the solvent used for alcohol-precipitating is ethanol or an aqueous ethanol solution.
8. The process according to claim 1, wherein the first alcohol-water solution and the second alcohol-water solution are the same or different, and are independently selected from aqueous solutions of C.sub.1-C.sub.4 alcohol with a concentration of 0-80%.
9. The process according to claim 1, wherein the third alcohol-water solution is selected from aqueous solutions of C.sub.1-C.sub.4 alcohol with a concentration of 0-80%.
10. The process according to claim 1, further comprising concentrating the Salvia miltiorrhiza liquid extract to give a concentrated Salvia miltiorrhiza liquid extract.
11. The process according to any one of claims 1, 2, 3, 4-9, and 10, wherein the macroporous resin comprises one or more of the following resins: HP20, HPD-80, HPD-100, HPD-100B, HPD-200A, HPD-300, HPD-450, HPD-722, HPD-826, ADS-5, ADS-8, ADS-21, D101, AB-8, and 1300-I.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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EXAMPLES
(7) The following examples are used to exemplarily illustrate the present invention, but should not be understood as limiting the scope of the present invention in any way.
(8) I. Preparation of Magnesium Lithospermate B
Example 1
(9) 1 kg Salvia miltiorrhiza plant material (purchased from Shanghai Traditional Chinese Medicine Co., Ltd.; lot number: 131001) was taken and smashed. The smashed Salvia miltiorrhiza plant material was sequentially extracted with 7 L, 5 L, and 3 L of 50% ethanol-water solution for three times (2 hours for each time) under reflux condition. The extract liquids were combined and concentrated under reduced pressure until the alcohol was completely removed. The obtained concentrated liquid was filtered to give 6 L filtrate (relative density is 1.02), which was then subjected to adsorption chromatography with 3 kg macroporous resin D101 (Tianjin Haiguang Chemical Engineering Co. Ltd.). Elution was carried out with 6 column volumes of 0% and 6% aqueous ethanol-MgCl.sub.2 in sequence (the eluant 6% aqueous ethanol/magnesium chloride solution comprises 500 mg magnesium chloride in total), then 2 column volumes of 20% ethanol-water eluant, followed with 50% ethanol-water eluant until completion. The eluate comprising magnesium lithospermate B was collected and concentrated under reduced pressure until the alcohol was completely removed. Then, the pH of the concentrated magnesium lithospermate B liquid was adjusted to weak acidity. The concentrated liquid was continuously counter-flow extracted with ethyl acetate. The aqueous solution fraction was collected and concentrated until the concentration of magnesium lithospermate B was about 100 mg/ml, followed by alcohol-precipitating with 95% ethanol and filtration. The filtrate was concentrated and dried under reduced pressure to give 30.8 g magnesium lithospermate B solid, the content of magnesium lithospermate B was 96.70%, and the yield was 2.98% based on the plant material. The primary impurities were 0.76% lithospermic acid and salt(s) thereof, 0.62% rosmarinic acid and salt(s) thereof, and 0.12% danshensu and salt(s) thereof.
Example 2
(10) 1 kg Salvia miltiorrhiza plant material (purchased from Shanghai Traditional Chinese Medicine Co., Ltd.; lot number: 131001) was taken and smashed. The smashed Salvia miltiorrhiza plant material was sequentially extracted with 6 L, 5 L, and 3 L of 70% ethanol-water solution for three times (2 hours for each time) under reflux condition. The extract liquids were combined and concentrated under reduced pressure until the alcohol was completely removed. The concentrated liquid was added with 200 mg magnesium chloride under stirring condition, the liquid was then filtered and subjected to chromatography with macroporous resin HPD-100 (Bon Chemical Engineering Co. Ltd.). Elution was carried out with 6 column volumes of 0% and 6% ethanol-water solutions in sequence, and then 2 column volumes of 20% ethanol-water solution, followed by 45% ethanol-water solution until completion. The eluate comprising magnesium lithospermate B was collected and concentrated under reduced pressure until the alcohol was completely removed. The pH of the resulting concentrated magnesium lithospermate B liquid was adjusted to weak acidity. The concentrated liquid was extracted with 1-fold volume of ethyl acetate for three times. The aqueous solution fraction was collected and concentrated until the concentration of magnesium lithospermate B was about 50 mg/ml, followed by alcohol-precipitating with 90% ethanol and filtration. The filtrate was concentrated and dried under reduced pressure to give 30.6 g magnesium lithospermate B solid, and the content of magnesium lithospermate B was 96.30%, the yield was 2.95% based on the plant material. The primary impurities were 0.80% lithospermic acid and salt(s) thereof, 0.71% rosmarinic acid and salt(s) thereof, and 0.13% danshensu and salt(s) thereof.
(11) II. Detection and Analysis of Magnesium Lithospermate B and Primary Impurities
(12) 1. Chromatographic Analysis and Detection of Magnesium Lithospermate B
(13) Equipment: Agilent 1260 high performance liquid chromatograph, Empower2 chromatography workstation, full wavelength diode array detector; Sartorius cp225D hundred thousandth electronic balance.
(14) Chromatographic column: YMC C18 chromatographic column (2504.6 mm, 5 m);
(15) Reagents: methanol was chromatographically pure; water was ultrapure water prepared by Millipore; all the other reagents were analytically pure.
(16) The lithospermic acid B control was purchased from National Institutes For Food And Drug Control, lot number: 111562-201212, for content determination.
(17) Chromatographic conditions and system suitability testing: octadecylsilane bonded silica gel was used as filler; flow rate: 1.0 ml.Math.min.sup.1; column temperature: 30 C.; detection wavelength: 288 nm. Theoretical plate number calculated on lithospermic acid B was no less than 5000; using methanol and 0.05% formic acid aqueous solutions as mobile phase, isocratic elution (the ratio of methanol was 45%) was performed for 30 mins.
(18) Preparation of lithospermic acid B control solution: lithospermic acid B control was accurately weighed, moved to a volumetric flask, dissolved with water and well shaken at 25 C. ambient temperature, and diluted to scale mark.
(19) Preparation of sample solution: a sample was accurately weighed, dissolved with water and shaken well at 25 C. ambient temperature, and diluted to scale mark.
(20) Determination method: the control solution was accurately taken and injected into the liquid chromatograph, and the chromatogram was generated and recorded; the sample was accurately taken and injected into the liquid chromatograph, the ratio of peak areas was calculated.
(21) The HPLC chromatogram of lithospermic acid B control is shown in
(22) The HPLC chromatogram of the magnesium lithospermate B solid prepared according to Example 1 of the present invention is shown in
(23) 2. Detection of Metal Cations in Magnesium Lithospermate B
(24) Equipment: DIONEX ICS900 ion chromatograph, Thermo DS5 electric conductivity detector; Inhibitor Thermo CSRS 300 4 mm; Workstation Chromeleon 7; SPE-C18 small column; Sartorius cp225D hundred thousandth electronic balance.
(25) Chromatographic column: Dionex IonPac CS12A; Guard column: Dionex IonPac CG12A; eluted with 20 mM aqueous methylsulfonic acid solution.
(26) Six Cation-II Standard was purchased from DIONEX Company for content determination and analysis.
(27) Chromatographic conditions and determination method: flow rate: 1.0 ml.Math.min.sup.1; injection volume: 10 l; the sample of the magnesium lithospermate B was formulated to 1000 ppm mother liquor with deionized water, which was diluted to 100 ppm immediately before experiment; after addition of hydrochloric acid (final concentration of 20 mM), the sample was passed through SPE-C18 adsorption column at a rate of one drop per second, then passed through microporous filter membrane, and injected directly.
(28) Six Cation-II Standard curve: Six Cation-II Standard was accurately weighed, moved to a volumetric flask, dissolved with water and well shaken at 25 C., and diluted to scale mark.
(29) Determination method: The Six Cation-II Standard solution was accurately taken and injected into the ion chromatograph, and the chromatogram was recorded; the sample was accurately taken and injected into the ion chromatograph; the ratio of peak areas was calculated.
(30) The chromatogram of six-cation standard solution control was shown in
(31) The ion chromatogram of magnesium lithospermate B prepared according to Example 6 of the present invention was shown in
(32) The experimental results showed that magnesium lithospermate B was obtained from the present invention.
(33) 3. Structure Confirmation of Magnesium Lithospermate B and Primary Impurities
(34) Impurities were separated from the magnesium lithospermate B product of Example 1 of the present invention by gel column chromatography and C18 column, and structurally confirmed by electrospray ionization mass spectrometry (ESI-MS), nuclear magnetic resonance (NMR), and Thermo ion chromatography. The results were as follows:
(35) 3.1 Sodium Salt of Danshensu
(36) ##STR00002##
(37) .sup.1H NMR (D.sub.2O, 400 MHz) : 6.79 (d, J=2.0 Hz, 1H), 6.67 (d, J=8.2 Hz, 1H), 6.59 (dd, J=8.2, 2.0 Hz, 1H), 5.18 (dd, J=8.4, 4.0 Hz, 1H), 3.42 (dd, J=14.3, 4.0 Hz, 1H), 3.25 (dd, J=14.3, 8.4 Hz, 1H); .sup.13C NMR (D.sub.2O, 100 MHz) : 174.1, 145.3, 144.1, 130.1, 121.7, 117.4, 116.3, 78.6, 39.4; ESI-MS (m/z) 221.2 [M+Na].sup.+; IC: Na.sup.+.
(38) 3.2 Magnesium Salt of Lithospermic Acid
(39) ##STR00003##
(40) .sup.1H NMR (D.sub.2O, 400 MHz) : 7.56 (d, J=16.0 Hz, 1H), 7.03 (d, J=1.5 Hz, 1H), 6.94 (d, J=1.0 Hz, 1H), 6.93 (d, J=8.5 Hz, 1H), 6.88 (dd, J=8.5 Hz, 1H), 6.86 (d, J=8.5 Hz, 1H), 6.83 (dd, J=8.0, 1.5 Hz, 1H), 6.81 (d, J=8.5 Hz, 1H), 6.76 (dd, J=8.5, 1.0 Hz, 1H), 6.20 (d, J=16.0 Hz, 1H), 5.81 (d, J=6.0 Hz, 1H), 4.98 (dd, J=9.5, 3.5 Hz, 1H), 4.23 (d, J=5.5 Hz, 1H), 3.13 (dd, J=14.1, 3.5 Hz), 3.05 (dd, J=14.1, 9.5 Hz, 1H); .sup.13C NMR (D.sub.2O, 100 MHz) : 182.3, 180.1, 171.4, 149.7, 147.3, 147.0, 146.8, 145.6, 145.3, 145.1, 136.3, 133.4, 131.6, 126.5, 124.6, 124.2, 121.3, 120.4, 119.8, 119.2, 119.1, 118.7, 116.2, 91.9, 79.7, 62.3, 40.0; ESI-MS (m/z) 561.1 [M+Na].sup.+; IC: Mg.sup.2+.
(41) 3.3 Rosmarinic Acid
(42) ##STR00004##
(43) .sup.1H NMR ((CD.sub.3).sub.2CO, 400 MHz) : 7.55 (d, J=16.0 Hz, 1H), 7.14 (d, J=2.0 Hz, 1H), 7.01 (dd, J=8.2, 2.0 Hz, 1H), 6.84 (d, J=8.2 Hz, 1H), 6.83 (dd, J=2.0 Hz, 1H), 6.68 (d, J=8.3 Hz, 1H), 6.59 (dd, J=8.3, 2.0 Hz, 1H), 6.23 (d, J=16.0 Hz, 1H), 5.17 (br.d, J=8.0 Hz, 1H), 3.07 (br.d, J=14.3 Hz, 1H), 2.93 (dd, J=14.3, 8.0 Hz, 1H); .sup.13C NMR ((CD.sub.3).sub.2CO, 100 MHz) : 171.1, 166.3, 148.5, 145.6, 145.4, 144.7, 143.3, 128.6, 126.7, 121.6, 120.7, 117.1, 115.3, 115.0, 114.6, 114.2, 72.6, 37.5; ESI-MS (m/z) 361.2 [M+H].sup.+.
(44) 3.4 Magnesium Lithospermate B
(45) ##STR00005##
(46) .sup.1H NMR (CD.sub.3OD, 500 MHz) : 7.52 (d, J=15.9 Hz, 1H), 7.15 (d, J=8.5 Hz, 1H), 6.83 (d, J=8.5 Hz, 1H), 6.76 (d, J=1.9 Hz, 1H), 6.74 (d, J=4.3 Hz, 1H), 6.73 (d, J=1.5 Hz, 1H), 6.69 (d, J=8.0 Hz, 1H), 6.65 (dd, J=8.2, 2.0 Hz, 1H), 6.61 (dd, J=8.2, 1.9 Hz, 1H), 6.54 (d, J=8.0 Hz, 1H), 6.51 (d, J=1.9 Hz, 1H), 6.30 (dd, J=8.1, 1.9 Hz, 1H), 6.20 (d, J=15.9 Hz, 1H), 5.85 (d, J=4.8 Hz, 1H), 5.20-5.15 (m, 2H), 4.35 (d, J=4.8 Hz, 1H), 3.06 (dd, J=14.3, 4.3 Hz, 1H), 3.03-2.96 (m, 2H), 2.83 (dd, J=14.3, 9.6 Hz, 1H); .sup.13C NMR (CD.sub.3OD, 125 MHz) : 171.8, 170.7, 170.4, 166.2, 147.2, 144.9, 144.7, 144.2, 144.1, 143.4, 143.2, 141.7 131.8, 127.4, 127.1, 124.5, 122.8, 120.4, 120.2, 119.9, 116.5, 116.5, 115.7, 115.4, 114.7, 114.6, 114.5, 114.5, 111.5, 86.4, 73.7, 72.8, 56.1, 36.0, 35.6; ESI-MS (m/z) 741.2 [M+Na].sup.+; IC: Mg.sup.2+.
(47) III. Research on Extraction or Purification Process of Magnesium Lithospermate B
(48) 1. Effect of a Magnesium Salt on the Yield of Magnesium Lithospermate B
(49) In order to investigate the effect of the added magnesium salts on the yield of magnesium lithospermate B during extraction and separation process, the following verification experiments were conducted.
Comparative Example 1 (Without Addition of Any Magnesium Salt)
(50) 100 g Salvia miltiorrhiza plant material (purchased from Shanghai Traditional Chinese Medicine Co., Ltd.; lot number: 131001) was taken, and divided into 50 g as one extraction unit. The extraction steps for each unit were as follows:
(51) (1) 50 g Salvia miltiorrhiza plant material was extracted with 300 ml 70% ethanol under reflux for 2 hours, and filtrated, and 180 ml filtrate was collected. The residue was further added with 250 ml 70% ethanol for extraction under reflux for 2 hours and filtrated, and 235 ml filtrate was collected. The residue was further added with 250 ml 70% ethanol for extraction under reflux for 2 hours and filtrated, and 225 ml filtrate was collected. The filtrates were combined (640 ml). The ethanol was recovered, and 82 g liquid extract was obtained with a relative density of 1.091 (60 C.), which was diluted to a relative density of 1.02 with water, and filtrated to obtain 305 ml filtrate. The resulting filtrate was slowly charged into 150 g 1300-I (Yangzhou Pharmaceutical Co., Ltd.) macroporous resin adsorption column. After completion of the adsorption, 300 ml water was used to wash away sugars, proteins, and inorganic salts. Further, 300 ml 6% ethanol was used to remove other salvianolates except magnesium lithospermate B. Finally, 150 ml 20% ethanol was used for elution. Each 10 ml eluate was collected as one fraction, 120 ml volume of eluate was obtained after confirmed by HPLC detection. The eluate was concentrated under reduced pressure at vacuum degree of 0.07 to 0.1 MPa and temperature of 80 C. to obtain 5.7 g extract with a relative density of 1.15 (50 C.).
(52) (2) 50 g of the rest Salvia miltiorrhiza plant material was extracted with 300 ml 70% ethanol under reflux for 2 hours, and filtrated, and 190 ml filtrate was collected. The residue was further added with 250 ml 70% ethanol for extraction under reflux for 2 hours and filtrated, and 225 ml filtrate was collected. The residue was further added with 250 ml 70% ethanol for extraction under reflux for 2 hours and filtrated, and 220 ml filtrate was collected. The filtrates were combined (635 ml). The ethanol was recovered, and 95 g extract paste was obtained with a relative density of 1.090 (60 C.), which was diluted to a relative density of 1.02 with water, and filtrated to obtain 305 ml filtrate. The resulting filtrate was slowly charged into 150 g 1300-I (Yangzhou Pharmaceutical Co., Ltd.) macroporous resin adsorption column. After completion of adsorption, 300 ml water was used to wash away sugars, proteins, and inorganic salts. Further, 300 ml 6% ethanol was used to remove other salvianolates except magnesium lithospermate B. Finally, 150 ml 20% ethanol was used for elution. Each 10 ml eluate was collected for one fraction, 110 ml volume of eluate was obtained after confirmed by HPLC detection. The eluate was concentrated under reduced pressure at vacuum degree of 0.07 to 0.1 MPa and temperature of 80 C. to obtain 5.5 g extract with a relative density of 1.15 (50 C.).
(53) 11.2 g liquid extract was obtained by combining the extracts from above units (1) and (2). After addition of 300 ml 95% ethanol, the mixture was left standing for 2 hours for alcohol precipitation, and then filtrated to give 290 ml clear liquid. The filtrate was subjected to ethanol recovery at vacuum degree of 0.07 to 0.1 MPa with temperature of 60 C., and then dried at vacuum degree of 0.08 MPa and temperature of 60 C. to give 3.27 g magnesium lithospermate B with a content of 85.10% in 2.78% yield (based on plant material).
Comparative Example 2 (With Addition of Potassium Chloride)
(54) The process steps were the same as those of Comparative Example 1 except that potassium chloride (16 mg, 0.21 mmol) was added during the extraction process. 3.17 g of magnesium lithospermate B was obtained with a content of 84.23% in 2.67% yield (based on plant material).
Example 3
(55) The process steps were the same as those of Comparative Example 1 except that magnesium chloride (20 mg, 0.21 mmol) was added during the extraction process. 3.86 g of magnesium lithospermate B was obtained with a content of 86.36% in 3.33% yield (based on plant material).
Example 4
(56) The process steps were the same as those of Comparative Example 1 except that magnesium chloride (20 mg, 0.21 mmol) was added into the concentrated extract liquid. 4.07 g of magnesium lithospermate B was obtained with a content of 84.21% in 3.42% yield (based on plant material).
Example 5
(57) The process steps were the same as those of Comparative Example 1 except that magnesium chloride (20 mg, 0.21 mmol) was added into the water-elution phase during column separation. 3.95 g of magnesium lithospermate B was obtained with a content of 85.31% in 3.37% yield (based on plant material).
(58) Table 1 shows the purities, solid amounts, yields, and relative increasing rates of the yields of magnesium lithospermate B obtained in Comparative Examples 1-2 and Examples 3-5, wherein the relative increasing rate of the yields was calculated based on the yield of Comparative Example 1: relative increasing rate=(yieldyield of Comparative Example 1)/yield100%.
(59) TABLE-US-00001 TABLE 1 Effect of the magnesium salt (magnesium chloride) on the yield of magnesium lithospermate B Purity of Yield of magnesium Amount magnesium Relative litho- of litho- in- Added spermate obtained spermate creasing salt B (%) solid (g) B (%) rate (%) Comparative 85.10 3.27 2.78 0 Example 1 Comparative KCl 84.23 3.17 2.67 3.96 Example 2 Example 3 MgCl.sub.2 86.36 3.86 3.33 19.78 Example 4 MgCl.sub.2 84.21 4.07 3.42 23.02 Example 5 MgCl.sub.2 85.31 3.95 3.37 21.22
(60) The comparison between the results of Comparative Examples 1-2 and Examples 3-5 indicated that the addition of a magnesium salt during extraction and separation process according to the present invention resulted in significantly higher magnesium lithospermate B yields (3.33%, 3.42%, and 3.37%) than the magnesium lithospermate B yields with no magnesium salt added or when a potassium salt was added (2.78% and 2.67%).
(61) 2. Effect of Extraction on the Purity of Magnesium Lithospermate B
Comparative Example 3
(62) 100 g Salvia miltiorrhiza plant material (purchased from Shanghai Traditional Chinese Medicine Co., Ltd.; lot number: 131001) was smashed, decocted and sequentially extracted three times with 4-fold volume, 2-fold volume, and 2-fold volume of water, 2 hours for each time. The extract liquids were combined, cooled to room temperature, and filtrated by centrifugation. The filtrate was slowly passed through an adsorption column packed with 3-fold volume of 1300-I macroporous resin (Yangzhou Pharmaceutical Co., Ltd.). After completion of the adsorption, 6-fold volume of water was used to wash away sugars, proteins, and inorganic salts. Further, 6-fold volume of 20% ethanol was used to remove other salvianolates except magnesium lithospermate B. Finally, 3-fold volume of 50% ethanol was used to elute salvianolates of Salvia miltiorrhiza. Based on TLC detection, the eluate by 50% ethanol contained rich magnesium lithospermate B. It was collected and concentrated under reduced pressure to an amount of 1/10 weight of the plant material; subsequently, anhydrous ethanol was slowly added in an amount of 9/10 weight of the plant material under stirring condition. After standing for 2 hours, the precipitate was discarded by centrifugation. The filtrate was concentrated to an amount of 1/20 weight of the plant material, and then added with anhydrous ethanol in an amount of 19/20 weight of the plant material, left standing for 2 hours, and filtrated by centrifugation. The filtrate was concentrated under reduced pressure till to dry. After pulverization, 2.57 g of magnesium lithospermate B was obtained with a content of 91.16% in 2.37% yield (based on plant material). The primary impurities were 0.78% lithospermic acid and salt(s) thereof, 6.57% rosmarinic acid and salt(s) thereof, and 0.10% danshensu and salt(s) thereof.
Comparative Example 4
(63) 100 g Salvia miltiorrhiza plant material (purchased from Shanghai Traditional Chinese Medicine Co., Ltd.; lot number: 131001) was smashed, decocted and sequentially extracted three times at 90-100 C. with 600, 300, and 250 ml water, 2 hours for each time. The extract liquids were combined and filtrated by centrifugation. The filtrate was concentrated under reduced pressure. The concentrated liquid (250 ml) was slowly passed through an adsorption column charged with 300 ml 1300-I macroporous resin (Yangzhou Pharmaceutical Co., Ltd.). After 800 ml water was used to wash the adsorption column for removing impurities, 600 ml 50% ethanol was further used to elute salvianolates of Salvia miltiorrhiza. The eluate from the 50% ethanol elution was collected and concentrated under reduced pressure to 10 ml, and slowly added with 90 ml anhydrous ethanol with stirring. After standing for 2 hours, it was then centrifuged and the precipitate was discarded. The supernatant was concentrated, dried and smashed to give salvianolates with 80.56% magnesium lithospermate B. The primary impurities were 5.21% lithospermic acid and salt(s) thereof, 8.64% rosmarinic acid and salt(s) thereof, and 0.62% danshensu and salt(s) thereof.
(64) The resulting salvianolates of Salvia miltiorrhiza were dissolved with water, separated via HP20 macroporous resin column. Elution was carried out with 6 column volumes of 0% and 6% aqueous ethanol solutions in sequence, 2 column volumes of 20% aqueous ethanol solution, and then 50% aqueous ethanol solution until completion. By HPLC detection, the 50% ethanol eluate comprising magnesium lithospermate B was collected and concentrated under reduced pressure until the alcohol was completely removed. The pH of the concentrated liquid was adjusted to 5, which was subjected to reverse extracted with ethyl acetate for 3 times. The magnesium lithospermate B fraction was collected and concentrated under reduced pressure until dry. 2.16 g of magnesium lithospermate B was obtained with a content of 98.67% in 2.13% yield (based on plant material). The primary impurities were 0.52% lithospermic acid and salt(s) thereof and 0.32% rosmarinic acid and salt(s) thereof.
Comparative Example 5
(65) 100 g Salvia miltiorrhiza plant material (purchased from Shanghai Traditional Chinese Medicine Co., Ltd.; lot number: 131001) was extracted with 6-fold volume of 60% ethanol under reflux for 2 hours. The extraction was performed for three times in total. The extract liquids were combined and concentrated under reduced pressure until the alcohol was completely removed. The concentrated liquid was separated via HP20 macroporous resin column after filtration. Elution was carried out with 6 column volumes of 0% and 6% aqueous ethanol solutions in sequence, followed with 2 column volumes of 20% ethanol aqueous solution, and then 50% ethanol aqueous solution until completion. By HPLC detection, the 50% ethanol eluate comprising magnesium lithospermate B was collected and concentrated under reduced pressure until no alcohol smell was detected. The pH of the concentrated liquid was adjusted to 5, which was subjected to reverse extracted with ethyl acetate for 3 times. The magnesium lithospermate B fraction was collected and concentrated under reduced pressure until dry. 2.41 g of magnesium lithospermate B was obtained with a content of 96.21% in 2.32% yield (based on plant material). The primary impurities were 1.08% lithospermic acid and salt(s) thereof and 0.96% rosmarinic acid and salt(s) thereof.
Comparative Example 6
(66) 100 g Salvia miltiorrhiza plant material (purchased from Shanghai Traditional Chinese Medicine Co., Ltd.; lot number: 131001) was extracted with 6-fold volume of 60% ethanol under reflux for 2 hours. The extraction was performed three times in total. The extract liquids were combined and concentrated under reduced pressure until no alcohol smell was detected. The concentrated liquid was added with magnesium chloride (16 mg, 0.21 mmol) and stirred for half an hour, then separated via HP20 macroporous resin column after filtration. Elution was carried out with 6 column volumes of 0% and 6% aqueous ethanol solutions in sequence, followed with 2 column volumes of 20% ethanol aqueous solution, and then 50% ethanol aqueous solution until completion. By HPLC detection, the 50% ethanol eluate containing magnesium lithospermate B was collected and concentrated under reduced pressure until no alcohol smell was detected. The pH of the concentrated liquid was adjusted to 5, which was subjected to reverse extracted with ethyl acetate for 3 times. The magnesium lithospermate B fraction was collected and concentrated under reduced pressure until to dry. 2.97 g of magnesium lithospermate B was obtained with a content of 96.92% in 2.88% yield (based on plant material). The primary impurities were 0.92% lithospermic acid and salt(s) thereof and 0.81% rosmarinic acid and salt(s) thereof.
(67) Table 2 shows the purities, solid amounts, yields, and relative increasing rates of the yields of magnesium lithospermate B obtained in Comparative Examples 3-5 and Example 6, wherein the relative increasing rate of the yields was calculated based on the yield of Comparative Example 3: relative increasing rate=(yieldyield of Comparative Example 3)/yield100%.
(68) TABLE-US-00002 TABLE 2 Effect of extraction process on the purity of magnesium lithospermate B Purity of Yield of magnesium Amount of magnesium Relative Added lithospermate obtained lithospermate increasing Extraction salt B (%) solid (g) B (%) rate (%) Comparative No 91.16 2.60 2.37 0 Example 3 Comparative Yes 98.67 2.16 2.13 10.12 Example 4 Comparative Yes 96.21 2.41 2.32 2.11 Example 5 Example 6 Yes MgCl.sub.2 96.92 2.97 2.88 21.52
(69) By comparing the results of Comparative Examples 3-5 and Example 6, it was found that the magnesium lithospermate B contents (reaching above 95%) could be significantly improved by reverse extraction. In addition, by comparing the results of Comparative Examples 4-5 and Example 6, it was further found that the yield of high purity magnesium lithospermate B could be significantly improved by the added magnesium salts. Meanwhile, the result of Comparative Example 6 showed that the content of magnesium lithospermate B could reach 98% when using despite salts of Salvia miltiorrhiza with purity of about 80% as raw materials.
(70) 3. Effect of the Addition Amount of a Magnesium Salt on the Yield and Purity of Magnesium Lithospermate B
Example 7
(71) 100 g Salvia miltiorrhiza plant material (purchased from Shanghai Traditional Chinese Medicine Co., Ltd.; lot number: 131001) was extracted with 6-fold volume of 60% ethanol under reflux condition for 2 hours. The extraction was performed for three times in total. The extract liquids were combined and concentrated under reduced pressure until no alcohol smell was detected. The concentrated liquid was added with 5 mg magnesium chloride and stirred for half an hour, and then separated via HP20 macroporous resin (Mitsubishi Chemical, Japan) column after filtration, and eluted with 6 column volumes of water, 20% ethanol, and 40% ethanol in sequence. By HPLC detection, the 40% eluate containing magnesium lithospermate B was collected and concentrated under reduced pressure until no alcohol smell was detected. The pH of the concentrated liquid was adjusted to 5, which was subjected to reverse extraction with ethyl acetate for 3 times. The magnesium lithospermate B fraction was collected and concentrated under reduced pressure until dry. 2.62 g of magnesium lithospermate B was obtained with a content of 96.63% in 2.52% yield (based on plant material).
Example 8
(72) The process steps were the same as those of Example 5 except that 10 mg magnesium chloride was added into the concentrated extract liquid. 2.91 g of magnesium lithospermate B was obtained with a content of 95.35% in 2.77% yield (based on plant material).
Example 9
(73) The process steps were the same as those of Example 5 except that 20 mg magnesium chloride was added into the concentrated extract liquid. 3.05 g of magnesium lithospermate B was obtained with a content of 96.81% in 2.95% yield (based on plant material).
Example 10
(74) The process steps were the same as those of Example 5 except that 50 mg magnesium chloride was added into the concentrated extract liquid. 3.25 g of magnesium lithospermate B was obtained with a content of 94.11% in 3.06% yield (based on plant material).
Example 11
(75) The process steps were the same as those of Example 5 except that 100 mg magnesium chloride was added into the concentrated extract liquid. 3.52 g of magnesium lithospermate B was obtained with a content of 92.56% in 3.26% yield (based on plant material).
Example 12
(76) The process steps were the same as those of Example 5 except that 200 mg magnesium chloride was added into the concentrated extract liquid. 3.48 g of magnesium lithospermate B was obtained with a content of 93.17% in 3.24% yield (based on plant material).
(77) Table 3 showed the purity, solid amount, and yield of magnesium lithospermate B obtained in each of Examples 7-12.
(78) TABLE-US-00003 TABLE 3 Effect of the addition amount of the magnesium salt (magnesium chloride) on the yield and purity of magnesium lithospermate B Purity of Yield of Amount magnesium magnesium Addition of litho- litho- amount obtained spermate spermate of MgCl.sub.2 (mg) solid (g) B (%) B (%) Comparative 5 0 2.41 96.21 2.32 Example 7 5 2.62 96.63 2.53 Example 8 10 2.91 95.35 2.77 Example 9 20 3.05 96.81 2.95 Example 10 50 3.25 94.11 3.06 Example 11 100 3.52 92.56 3.26 Example 12 200 3.48 93.17 3.24
(79) As shown in Table 3, when the added magnesium chloride at the range of 5-100 mg per 100 g Salvia miltiorrhiza, the more the amount of the added magnesium chloride was, the higher the magnesium lithospermate B yield was. But the magnesium lithospermate B yield was no more significantly increased when the amount of magnesium chloride was increased to 200 mg.
(80) 4. Research on Stability of Magnesium Lithospermate B
(81) The chemical stability of magnesium lithospermate B was investigated by detecting the variation of peak areas (HPLC). Specific experimental method: lithospermic acid B (lab-made; lot number: 20131017) and magnesium lithospermate B prepared in Example 1 of the present invention were respectively formulated to 1 mg/mL aqueous solution at 25 C., which was then diluted 10 times. The variation of peak areas of lithospermic acid B at 288 nm for durations of 0, 2, 4, 6, 8, 12, and 24 hours were detected by HPLC, respectively. 10 microliters was injected for each time. The results are shown in Table 4.
(82) TABLE-US-00004 TABLE 4 Chemical stability of magnesium lithospermate B Time 0 h 2 h 4 h 6 h 8 h 12 h 24 h Average peak peak peak peak peak peak peak peak RSD Component area area area area area area area area (%) Lithospermic 3957 3991 3992 3983 4002 3990 4000 3987.8 0.26 acid B Magnesium 3611 3632 3614 3636 3629 3628 3599 3621.3 0.31 lithospermate B
(83) The experimental results showed that the peak areas did not significantly vary within 24 hours for each component. This illustrated the magnesium lithospermate B prepared according to Example 1 of the present invention had good chemical stability.
(84) IV. Research of Pharmacological Activity of Magnesium Lithospermate B
Experimental Example 1. Research on Cytotoxicity and Antioxidant Activity of Lithospermic Acid B and the Magnesium Lithospermate B
(85) 1.1 Research Material and Method of Cytotoxicity Experiment
(86) 1.1.1. Materials for Cytotoxicity Experiment
(87) Sample and control: magnesium lithospermate B (prepared according to Example 6 of the present application), lithospermic acid B (lab made; lot number: 20131017), formulated with normal saline prior to use; normal saline as solvent control was derived from Jiangsu Yabang Pharmaceutical Co., Ltd.; the other reagents were all commercially available analytically pure products.
(88) Cell lines and reagents: cardiac myoblast (H9C2); Microvascular Endothelial Cell (HMEC-1); Lactic Dehydrogenase Cytotoxicity Detection Kit, Beyotime Biotechnology Institute.
(89) Primary equipment: multifunctional ELISA reader (Spectra MAX M2e), a product from Molecular Devices, USA; microscope (Optiphot-2), Nikon, Japan; CO.sub.2 incubator, Thermo, USA; biosafety cabinet, Boxun, Shanghai.
(90) 1.1.2 Method of Cytotoxicity Experiment
(91) Dosage setting: the dosages of lithospermic acid B and magnesium lithospermate B were set at 10, 50, 100, 200, 400, 800, 1200, 1600 M and the incubation time was set as 24 hours in the present experiment.
(92) Experimental method: H9C2 cells and HMEC-1 cells were respectively incubated in 10% FBS containing low saccharide DMEM medium and MCDB-131 medium, at 37 C. and 5% CO.sub.2. Cells were seeded in a 96-well culture plate, which was added with different concentrations of lithospermic acid B solutions and magnesium lithospermate B solutions on the next day (about 80% fusion). After incubation for 24 hours in total, the detection was conducted according to instructions of the Lactic Dehydrogenase Cytotoxicity Detection Kit.
(93) Observation indicator and observation time: the observation indicator is the level of lactic dehydrogenase in cells; cell survival rate was represented by intracellular LDH content ratio, i.e. (absorbance of treated sample wellabsorbance of sample control well)/(absorbance of cells with maximum enzymatic activityabsorbance of sample control well)100.
(94) Statistical method: the data was presented with averagestandard derivation (meanSD); the comparison of two groups of data was statistically analyzed by student-t test method, P<0.05 indicating statistical difference.
(95) 1.1.3 Results of Cytotoxicity Experiment
(96) Different concentrations of lithospermic acid B and a magnesium lithospermate B were incubated with the cells for 24 hours. The results of intracellular LDH content detection showed that for cardiac myoblast cells H9C2, 400, 800, 1200, 1600 M of lithospermic acid B and 800, 1200, 1600 M of magnesium lithospermate B significantly decreased the intracellular LDH content as compared with normal control wells (P<0.05 or P<0.01); for endothelial cells HMEC-1, 1200 and 1600 M of lithospermic acid B and 1200 and 1600 M of magnesium lithospermate B significantly decreased the intracellular LDH content as compared with normal control wells (P<0.05 or P<0.01). See Table 5,
(97) TABLE-US-00005 TABLE 5 Effect of lithospermic acid B and magnesium lithospermate B on intracellular LDH content (triplicate wells, n = 3) Intracellular LDH content percent (%) Drug name Dosage (M) H9C2 cell HMEC-1 cell Lithospermic 10 99.62 1.67 99.89 2.66 acid B 50 99.46 0.96 98.91 1.64 100 99.29 2.47 98.21 5.59 200 96.18 2.88 98.34 4.26 400 94.27 3.31* 100.94 6.55 800 16.79 5.74** 98.33 6.35 1200 12.47 5.72** 80.65 7.61* 1600 10.1 5.88** 40.93 5.21** Magnesium 10 101.42 2.00 99.70 3.88 lithospermate 50 99.97 1.43 101.05 4.77 B 100 101.25 2.79 100.96 4.01 200 102.39 1.37 98.58 0.24 400 102.75 3.07 97.8 1.03 800 37.72 7.52** 96.93 1.37 1200 21.59 2.86** 96.17 1.00* 1600 14.59 3.05** 84.28 3.28* *P < 0.05, **P < 0.01 vs. normal control group
(98) Conclusion: high dosages of lithospermic acid B and magnesium lithospermate B had toxic effect on both cardiac myoblasts and endothelial cells but magnesium lithospermate B showed lower toxicity than lithospermic acid B, especially on endothelial cell line.
(99) 1.2 Research Materials and Method of in Vitro Anti-Lipid Peroxidation Experiment
(100) 1.2.1 Materials for in Vitro Anti-Lipid Peroxidation Experiment
(101) Sample and control: magnesium lithospermate B (prepared according to Example 6 of the present application), lithospermic acid B (lab-made; lot number: 20131017), formulated with normal saline prior to use; normal saline as solvent control was from Jiangsu Yabang Pharmaceutical Co., Ltd.; the other reagents were all commercially available analytically pure products. Tris base, potassium chloride, dipotassium phosphate, glycerol, etc. (Sinopharm Chemical Reagent Co., Ltd.); Coomassie brilliant blue G-250, bovine serum albumin (Shanghai Lanji Technology Development Co., Ltd.).
(102) Experimental animal: species and strain: SD rat; source: Shanghai SLAC Laboratory Animal Co., Ltd., Laboratory Animal Production License: SCXK (Shanghai) 2012-0002; number and gender: 2, male; animal weight: 200-250 g.
(103) 1.2.2 Method of in Vitro Anti-Lipid Peroxidation Experiment
(104) Steps for preparing liver microsome: liver tissue was taken out, cut into pieces, washed with Buffer A, wiped up, and weighed for 8 g. By adding 10 ml Buffer A per gram of tissue, 80 ml was added in total, followed by automatic homogenization and further homogenization with manual homogenizer. The homogenate was centrifuged at 10,000 g and 4 C. for 30 mins, and the supernatant was taken out. The supernatant was centrifuged at 105,000 g and 4 C. for 60 mins. The resulting precipitate is the microsome, which was then added with Buffer B to obtain microsome suspension, aliquoted into 2 ml small tubes, and stored in 70 C. refrigerator for further use. Determination of protein content: using Bradford method (Coomassie brilliant blue G-250 method).
(105) Determination of lipid oxidation in rat liver microsome: 1 ml liver microsome was added into each tube, followed by addition of different concentrations of reagent solutions. The mixtures were well shaken and incubated in 37 C. water bath for 90 mins. After the tubes were taken out, 1 ml 10% trichloroacetic acid (TCA) and 1 ml 0.67% thiobarbituric acid (TBA) were added into each tube. The tubes were placed in boiling water bath for 15 mins, then cooled with running water and centrifuged at 3000 rpm for 10 mins. The supernatants were taken for colorimetric determination at 532 nm. The contents were calculated with standard tetraethoxypropane and represented with content per mg protein.
(106) Calculation formula:
MDA content (nmol/mg prot)={[(A.sub.sampleA.sub.blank)(A.sub.standardA.sub.blank)]10 nmol/ml}protein content
Inhibition rate %=(MDA.sub.blankMDA.sub.sample)/MDA.sub.blank100%
(107) 1.2.2 Experimental Results
(108) The results are shown in Table 1. At a drug concentration of 100 M, lithospermic acid B and magnesium lithospermate B exhibited considerable anti-lipid peroxidation effects, where the inhibition rate reached around 85%, indicating that the anti-oxidation of these compounds depended on phenolic hydroxy in the chemical structures, while the salt form had no impact in this regard.
(109) TABLE-US-00006 TABLE 6 In vitro antioxidant activity of lithospermic acid B and magnesium lithospermate B (n = 3) Compound Inhibition rate (%) Lithospermic acid B 86.42 100 M Magnesium lithospermate B 84.41 100 M
Experimental Example 2. Effects of Lithospermic Acid B, Dipotassium Lithospermate B, Depside Salts of Salvia Miltiorrhiza, and Magnesium Lithospermate B on Bleeding of Severed Tails of Rats
(110) 2.1 Materials and Method
(111) 2.1.1 Experimental Material
(112) Sample and control: lithospermic acid B, dipotassium lithospermate B, depside salts of Salvia miltiorrhiza (comprising 85% magnesium lithospermate B), magnesium lithospermate B, formulated with normal saline prior to use; normal saline as solvent control was from Jiangsu Yabang Pharmaceutical Co., Ltd.; the other reagents were all commercially available analytically pure products.
(113) Experimental animal: species and strain: SD rat; source: Shanghai SLAC Laboratory Animal Co., Ltd., Laboratory Animal Production License: SCXK (Shanghai) 2012-0002; number and gender: 50, male; animal weight: 120-200 g.
(114) Primary devices: water bath, scalpel, timer
(115) 2.1.2 Experimental Method
(116) Dosage setting: the dosages of lithospermic acid type reagents were set as 50 and 100 mg/kg in the present experiment; normal saline was given as additional solvent control. Tail severing time was 2 mins after administration.
(117) Administration route and administration dosage of sample and control: administration by injection through femoral vein, 2 ml/kg.
(118) Experimental method: the animals were water fasted overnight. 50 mg/kg pentobarbital sodium was intraperitoneally injected for anesthesia prior to the experiment. After anesthesia of the animals, markers were made at 4 mm from tips of rat tails with a marker pen and ruler. Drugs were injected through femoral veins of the animals. 2 mins later, the tails were swiftly severed at marked positions of the animal tails with a scalpel. The remained animal tails were rapidly inserted into prepared normal saline tubes in 37 C. water bath, and timing was started. The bleeding of the animal tails was observed. If no more bleeding was observed within 30 seconds, the time was recorded as coagulation time (unit: second) of the animals. The experiment was stopped when no bleeding was observed in 30 mins, which was recorded as 1800 seconds.
(119) Observation indicator and observation time: the indicator was the bleeding time.
(120) Statistical method: the experimental data was represented with xSD; GraphPad Prism 5 1 way ANOVA was applied for significance test.
(121) 2.2 Experimental Results
(122) As shown in Table 4-1, by administrating 50 and 100 mg/kg of lithospermic acid type compounds to SD rats via intravenous injection, the bleeding time of severed tails of rats can be significantly extended; by administrating the same dosage of lithospermic acid B or the magnesium lithospermate B to SD rats via intravenous injection, the bleeding time of severed tails of rats in magnesium lithospermate B administration group was remarkably longer than lithospermic acid B administration group. In addition, at the same dosage, the bleeding time of severed tails of rats in magnesium lithospermate B administration group was significantly longer than dipotassium lithospermate B and depside salts of Salvia miltiorrhiza (obtained according to the description in paragraph 1 of Comparative Example 4; comprising 80.56% magnesium lithospermate B).
(123) TABLE-US-00007 TABLE 7 Effect of administration of lithospermic acid B type compounds via intravenous injection on the bleeding time of severed tails of rats Animal Bleeding time Drug name Dosage number (second) Solvent control 2 ml/kg 21 360 24 normal saline Lithospermic acid B 50 mg/kg 10 869 71**.sup.## 100 mg/kg 10 1078 137**.sup.## Magnesium 50 mg/kg 26 1098 84** lithospermate B 100 mg/kg 10 1800 0** Dipotassium 50 mg/kg 10 827 70**.sup.$$ lithospermate B 100 mg/kg 10 1301 189**.sup.$$ Depside salts of 50 mg/kg 10 1001 151**.sup.$$ Salvia miltiorrhiza *P < 0.05, **P < 0.01 vs. solvent control; #P < 0.05, .sup.##P < 0.01 lithospermic acid B vs. magnesium lithospermate B in the groups of the same dosage; .sup.$P < 0.05, .sup.$$P < 0.01 dipotassium lithospermate B or depside salts of Salvia miltiorrhiza vs. magnesium lithospermate B in the groups of the same dosage
(124) The above pharmacological experiment results indicated that magnesium lithospermate B exhibited lower toxicity than lithospermic acid B in the cytotoxicity experiments of cardiac myoblast cell H9C2 and endothelial cell HMEC-1; in addition, magnesium lithospermate B exhibited an activity superior to the free acid (lithospermic acid B), other salts (dipotassium lithospermate B), and low purity mixtures (salvianolates of Salvia miltiorrhiza) in the experiment for extending the bleeding time of rat tails.