PHARMACEUTICAL COMPOSITION FOR CURE AND PREVENTION OF CARDIOVASCULAR DISEASE AND METHOD OF PREPARING THE SAME

Abstract: A pharmaceutical composition for treatment and pre­ vention of cardiovascular disease, and increasing iummune function contains Ginsenoside, Ophiopogonin, Sesquicarene and Chamigrene. Processes for preparing these compounds and the composition, referred to as Pure San-Mai-Sen (PSMS), are desired.

Description: The invention relates to a pharmaceutical composition, especially for treating and preventing cardiovascular disease, and increasing the immune function, and a method of preparing the same. Ginseng roots have long been used in Asia to prepare drugs and medicines. Many methods of extracting the active ingredients of ginseng roots have been used over the years including stewing or extraction with solvents such as ethanol. There is a need to provide a composition comprising several active ingredients which, in combination, are useful in treating and preventing cardiovascular disease in human beings and in increasing resistance to infection (immune response). This object is solved by the pharmaceutical composition according to independent claim 1. Further advantageous features of this composition are evident from dependent claims 2 to 5. Independent claim 6 claims the compositions according to one of claims 1 to 5 for preventing and curing cardiovascular disease and increasing the immune function. Specifically, this invention provides a new composition of four major active ingredients: Ginsenoside, Ophiopogonin, Sesquicarene and Chamigrene. This invention provides a pharmaceutical composition referred to as Pure San-Mai-Sen (PSMS). PSMS comprises Ginsenoside (I), Ophiopogonin (II), Sesquicarene (III) and Chamigrene (IV), which are all derived from plant materials. The approximate composition of PSMS and the sources of its components are listed below in Table 1. The process for producing PSMS comprises extracting powders of the above natural materials with appropriate solvents such as alcohol or water, removing lipids by extraction with ether in case of Ginsenoside and Ophiopogonin, vacuum-distilling the extracts with and/or without addition of butanol, and preparing PSMS by mixing its components in the desired proportion. The chemical structures of the components of PSMS are shown in the following. Ginsenoside (I) includes Ginsenoside g₁ and b₁ as illustrated in the following. Chemical structure of Ginsenoside (I) When R₁ = Glucose 6 1 Glucose R₂ = Glucose 2 1 Glucose , and R₃ = H, then the compound is Ginsenoside b₁ with a melting point of 198-202° C. When R₁ = Glucose R₂ = H , and R₃ = -0- Glucose , then the compound is Ginsenoside g₁ , with a melting point of 192-194°C. Chemical Structure of Substituents of Ginsenoside Ophiopogonin (II) is a mixture of Ophiopogonin B and D as illustrated in the following. Chemical Structure of Ophiopogonin (II) When R = H, the component is Ophiopogonin B, which has a melting point of 269-271 o C. When R has following chemical structure then the component is Ophiopogonin D, which has a melting point of 263-265 o C. Sesquicarene (III) and Chamigrene (IV) are isomers occurring in a mixture. Their structures are illustrated in the following. Chemical Structure of Sesquicarene (III) Chemical Structure of Chamigrene (IV) PSMS is produced by mixing the above components in the proportions given in Table 1 above. Detailed Description The processes for producing the above-mentioned active ingredients of PSMS are described by the following examples: Example 1: Extraction and Purification of Ginsenoside (I). 1000 g of dried ginseng powder are extracted with 2000 ml of 95% ethanol at room temperature for 24 hours. The powder is recovered by filtration. Filtrate A is saved and the powder filtercake is refluxed with additional 2000 ml of 95% ethanol on a steam bath. The mixture is filtered again. Filtrate B is saved and the powder filtercake is refluxed two more times for 6 hours with additional 2000 ml batches of 95% ethanol and filtered, providing filtrates C and D. Filtrates A, B, C, and D are combined and distilled at reduced pressure, preferably 2266.47 Pa (17 mm Hg), whereby ethanol is recovered and a still residue is obtained. This still residue is dissolved in 500 ml of distilled water. This aqueous solution is extracted five times with 500 ml of a lipophilic solvent, e. g. diethyl ether or petroleum ether, whereby lipids are removed from the solution. To this aqueous raffinate is added 500 ml of water-saturated n-butanol and the mixture is distilled at reduced pressure, preferably 2266.47 Pa (17 mm Hg), to dryness, whereby a powder residue is obtained. This powder residue is dissolved in 500 ml of anhydrous ethanol, and 2000 ml of acetone are. added with agitation while a precipitate forms. The precipitate is recovered by filtration and washed twice with acetone and twice with diethyl ether and dried. About 60 g of a white to light yellow powder are recovered. This is Ginsenoside (I). Example 2: Extraction and Purification of Ophiopogonin (II). 1000 g of Ophiopogon japonica Ker-Gawl powder are soaked in 2000 ml of 75% ethanol at room temperature for 24 hours. The mixture is filtered and the filtercake powder is refluxed twice for two hours with 2000 ml of 75% ethanol and filtered. The filtrates are combined and distilled on a steam bath at reduced pressure, preferably 2266.47 Pa (17 mm Hg), whereby ethanol is vaporized and thereby removed. An aqueous still residue is obtained. This still residue is extracted with 500 ml of diethyl ether four times to remove lipids. Other solvents such as petroleum ether may be used. To this aqueous raffinate 500 ml of n-butanol are added and the mixture is evaporated to dryness at reduced pressure, preferably 2266.47 Pa (17 mm Hg) whereby about 30 g of powder residue are obtained. This is Ophiopogonin (II). Example 3: Recovery of sesquicarene/Chamigrene (III)/(IV). 1000 g of Schisandra chinensis Baill is ground into a powder and added to 2000 ml of distilled water. The mixture is vacuum-distilled and the distillate fraction recovered at reduced pressure, preferably 1333.22 Pa (10 mm Hg), between 80 and 130°C is separated. About 14 g of this distillate are obtained. It contains the isomers Sesquicarene (III) and Chamigrene (IV). Example 4: Preparation of PSMS Liquid 120 g of Ginsenoside, 90 g of Ophiopogonin, and 28 g of Sesquicarene/Chamigrene are dispersed and dissolved in 500 ml of distilled water. The pH of this solution is adjusted to 7 by addition of dilute hydrochloric acid. This is PSMS Liquid. On a dry basis, the composition of PSMS may vary as follows: Weight percent Preferred composition weight percent Ginsenoside 25-60 25 Ophiopogonin 25-60 50 Sesquicarene/Chamigrene 5-50 25 The dry ingredients of PSMS, prepared in accordance with the present invention, may be incorporated in tablets, capsules and syrups by conventional methods. The invention will now be described with reference to its beneficial effects, as illustrated by the following tests: Example 5: The influence of PSMS on the survival rate of myocardial cells in a low nutrition medium. Culture of chick heart cell. Hearts were removed from embryos and dissociated at 37 o C for 45 minutes with 0.25% trypsin obtained from (sigma, type III) 0.025% collagenase (obtained from Sigma, type I), and 0.005% pancreatin (obtained from NBCo) prepared in calcium and magnesium-free G (trademark of Surum) containing 4% chicken serum. Then the tissue is dispersed into a single cell suspension in culture medium containing 5mg/ml DNAse I (sigma). Viable cells were counted using a hemocytometer. For the counting, the cells were dispersed into 60 culture dishes (surface area 2000 mm²) at densities of 200 cells/mm². Cultures were maintained in Ham's F-12K cln, a medium for myocardial cells as described in Kaighn, ME, "Tissue culture methods and applications" (Kruse and Potterson, eds. pg. 54-58, Academic Press. 1973) and supplemented with 5% fetal bovine serum, gentamycin (5mg/100ml). Tissue culture plates were incubated under 5% CO₂ and 95% air at 37 o C. All cells were counted in 20 randomly selected field across the entire dish. A Zeiss microscope 25X objective having a field of view of 0.32 mm² was used for cell counting. In a low nutrition culture medium, chick myocardial cells take longer, for example 7 days, for some of the cells to die. Therefore the number of cells decreases. Under similar condition PSMS at 150 mg/ml increases the number of chick myocardial cells (table 2). Chick myocardial cells were divided into 3 groups after being put into culture for 1 day under normal condition. 100% nutrition group is the normal medium, twice a week the medium was changed. The 10% nutrition group uses a 10% medium and 90% physiological saline solution. The medium and physiological saline solution was not changed for a week. The PSMS group used 10% medium , 90% of a physiological saline solution and 150 µg PSMS. The media was not changed for a week. On the 7th day the cell counts were as given in Table 2. The influence of PSMS on chick myocardial cells under low nutrition culture medium conditions. Example 6: The influence of PSMS on the Autoradiogram of myocardial cells in a low nutrition medium. Autoradiograms - cellular labelling was conducted in medium prepared without unlabelled thymidine and containing 5µ Ci /ml methyl-³H-thymidine (New England Nuclear) at a specific activity of 50.8 Ci/mole. Cultures were labelled in medium containing ³H-thymidine labelling agent. After the labelling procedure the plates were rinsed in saline, fixed in formal: ethanol(1:9), stained with periodic acid-, schiff (PAS) and coated with Kodak NTB-3 emulsion, a nuclear emulsion diluted 1:1 with water. Autoradiograms were exposed for 7 days then developed in Dektol counterstained with 1% aqueous fast green and air dried. At least 1000 nuclei per culture were counted at random, cells having more than 50 silver grains over the nucleus were scored as positive labelling, background was usually less than 5 grains. The cellular density of each culture was also determined by recording the number of microscopic fields counted to assess the total area scored. A Zeiss 25X objective having a field of view of 0.32 mm² was used for cell counting. Under similar condition described in table 2, experiment of Autoradiograms was performed. From table 3 we can see PSMS at 150 µg/ml can increase percent of myocardial cell nuclei labelled. That is PSMS increases DNA synthesis of chick myocardial cells. Table 3. The influence of PSMS on percent of myocardial cell nuclei labelled. On the seventh (7th) day, ³H-TdR was added (5µ Ci/ml medium) Example 7. The influence of PSMS on the cardiovascular experiment in animals. The above Four components in animal experiments reflect the following results: A. PSMS can significantly increase myocardial DNA synthesis; treated group/control group = 365.7%, P<0.001. B. PSMS can prolong significantly the survival time of mice under hypoxia. Living minute of the control group is 10 ± 5 min , PSMS group is 56 ± 3 min. , P<0.001 (O₂ about 5%). C. PSMS can enhance markedly the coronary flow on isolated perfused hearts. Drug group/control group = 183.69%. Myocardia uptake of ⁸⁶Pb was raised in rats. Example 8: The influence of PSMS on the immune function in animals. A. PSMS caused markedly increase lymphocyto-blastogenesis. Mice were injected with acetoprednisolone making up a model of inhibited immunity, then establishing the index of stimulation of lymphocyto-blastogenesis. Control group: 16.87 , PSMS group: 30.12 (P<0.001) B. PSMS caused increase of serum complement content in guinea pigs. Control group: 301 µg/ml PSMS group: 359 µg/ml (P<0.05) C. PSMS was found to markedly increase the formation of roset in guinea pigs. Control group: 42.7% PSMS group: 65.3% (P<0.01) D. PSMS could increase the content of serum lysozyme in mice. Control group: 9.0 µg/ml, PSMS group: 11.7 µg/ml (P<0.01). Example 9: Toxicity of PSMS A. The toxicity of PSMS in the human body and animals was found to be very low. When PSMS was administered orally to mice at a dose of 48g/kg neither toxic effect nor death was observed other than a sedative appearance. B. LD₅₀: 1295mg/kg injection in abdominal cavity in mice. C. Each dose for an adult is 20mg. Using 50 kg as the average weight of an adult the dosage is 0.4mg/kg, therefore PSMS is very safe.

Claims: A pharmaceutical composition, comprising, in weight percent, 25-60% Ginsenoside, 25-60% Ophiopogonin, and 5-50% Sesquicarene/Chamigrene, obtainable by preparing Ginsenoside according to the following steps: a) extracting a powder of the roots of Panax quinquefolium L. or Panax ginseng C.A. Meyer with 95% ethanol at room temperature for 24 hours and recovering a filtrate A and a filter cake by filtration, b) refluxing the filter cake of step a) with 95% ethanol on a steam bath and recovering a further filter cake and a filtrate B by filtration, c) repeating step b) two more times for 6 hours to obtain filtrates C and D, d) combining filtrates A, B, C and D and distilling same at reduced pressure, preferably 2266.47 Pa (17 mm Hg), to obtain a still residue, e) dissolving the still residue in distilled water and extracting the aqueous solution five times with a lipophilic solvent, preferably diethyl ether or petroleum ether, f) adding water-saturated n-butanol to the aqueous raffinate of step e) and distilling the mixture at reduced pressure, preferably 2266.47 Pa (17 mm Hg), to dryness to obtain a powder residue, g) dissolving the powder residue in anhydrous ethanol and adding acetone under agitation, h) recovering the precipitate obtained in step g) by filtration and washing the filtrate twice with acetone and twice with diethyl ether, and i) drying the washed precipitate to obtain Ginsenoide powder, Ophiopoganin according to the following steps: a') soaking ground Ophiopogon japonica Ker-Gawl powder in 75% ethanol at room temperature for 24 hours, b') separating the powder from the ethanol extract, c') refluxing the powder from step b') with additional 75% ethanol, and separating it therefrom, d') again separating the powder from the ethanol extract, e') repeating steps c') and d') 1-3 times, f') distilling the ethanol extracts at reduced pressure, preferably 2266.47 Pa (17 mm Hg), thereby removing ethanol, and obtaining an aqueous still residue, g') extracting said aqueous still residue 1-5 times with a lipophilic solvent of low mutual miscibility with water, preferably diethyl ether or petroleum ether, recovering an aqueous raffinate, and adding n-butanol to the aqueous raffinate, and h') evaporating said aqueous raffinate to dryness at reduced pressure, preferably 2266.47 Pa (17 mm Hg) to produce Ophiopogonin powder, and Sesquicarene/Chamigrene according to the following steps: a'') distilling a mixture of powdered Schisandra chinensis Baill and water at a pressure between 933.25 and 1999.83 Pa (7 to 15 mm Hg), and b'') recovering a distillate fraction between 75 and 150°C, to obtain Sesquicarene/Chamigrene, and mixing the components in the above-mentioned ranges. The pharmaceutical composition of claim 1 wherein the refluxing step c') is carried out on a steam bath. The pharmaceutical composition of claim 1 or 2 wherein the evaporating step h') is carried out upon addition of n-butanol. The pharmaceutical composition of one of claims 1 to 3 comprising, in weight percent, 25% Ginsenoide, 50% Ophiopogonin, and 25% Sesquicarene/Chamigrene. The pharmaceutical composition of one of claims 1 to 4 being incorporated in tablets, capsules or syrups. Pharmaceutical composition according to one of claims 1 to 5 for preventing and curing cardiovascular disease and increasing the immune function.

Inventors: LIU, YAGUANG
Applicants: LIU, YAGUANG
Application year: 1986
Main classification: A61K35
IPC codes: A61K31, A61K45, A61K36