HK1085386B - Pharmaceuticals comprising shikonins as active constituent - Google Patents

Description

Medicine containing shikonins compound as active ingredient

Technical Field

The present invention relates to a medicament containing Shikonin compounds or salts thereof, and more particularly to a medicament containing Shikonin compounds or salts thereof as an active ingredient for preventing and treating microbial infection, inflammation, tumor, hemorrhage, hematologic disorders or immunomodulatory diseases in a human body.

Background

The shikonins are compounds disclosed in literature reports (Linzhen et al, Beijing medical college, 1980, Vol. 12, p. 2, page 101-105), wherein the shikonins have the following general structure:

the shikonins are insoluble in water and soluble in oil, alcohol or ether, and can be extracted from Boraginaceae plants such as radix Arnebiae (Lithospermum erythrorhizon Sieb. et Zucc.), radix Arnebiae (Arnebiaeuthroma (Royle) Johnst.), radix Arnebiae (Arnebia guttata Bunge), etc. It is known that a mixed extract of lithospermum erythrorhizon has an anti-inflammatory action, but it is used in the form of a mixed extract at the time of administration, and there has not been reported about the use of a lithospermum quinone compound isolated from a mixed extract of lithospermum erythrorhizon and an artificially and biologically synthesized lithospermum quinone compound as a single compound or a combination of a small number of compounds for the pharmaceutical preparation, particularly the preparation of a medicament for the prevention and treatment of microbial infection, inflammation, tumor, bleeding, blood diseases, immunoregulatory diseases, and the like of the human body.

Disclosure of Invention

Accordingly, an object of the present invention is to provide a medicament for preventing and treating microbial infection, inflammation, tumor, hemorrhage, hematological disorders or immunoregulatory diseases of a human body, which is prepared from a single compound or a small number of compounds isolated from an extract of lithospermum erythrorhizon.

The invention provides a medicament for preventing and treating microbial infection, inflammation, tumor, hemorrhage, hematopathy or immunoregulatory disease of human body, which comprises shikonins compound represented by the following formula (I) or salts thereof,

wherein R is selected from H (deoxyshikonin), OH (shikonin), (CH)₃)₂C ═ chc (O) O- (. beta.,. beta. -dimethylacryloyl shikonin), CH₃C (O) O- (Acetylshikonin), (CH)₃)₂C＝C(CH₃)CH₂C (O) O- (2, 3-dimethyl pentenoyl shikonin), (CH)₃)₂COHCH₂C (O) O- (. beta. -hydroxyisovalerylshikonin), (CH)₃)₂C[OC(O)CH₃]CH₂C (O) 1-5 of O- (beta-acetoxy isovaleryl shikonin); wherein the extract preferably contains 1-3 of shikonin, beta-dimethylacryloyl shikonin and acetyl shikonin; more preferably contains beta, beta-dimethylacryloyl shikonin and/or acetylshikonin; most preferably contains beta, beta-dimethylacryloyl shikonin. The salt of the shikonins compound comprises alkali metal, alkaline earth metal, ammonium salt and the like.

The medicine of the invention can contain any single compound in the shikonins compounds as the raw material medicine, and also can contain the combination of a few compounds, wherein the purity of each single compound is 80% or more than 80%, and the preferred purity is 90% or more than 90%. When the composition contains a few compounds, the content of the active ingredients in the composition of the few compounds is 70% or more than 70%.

The medicament of the present invention may further contain other drugs as active ingredients, as required. The other drugs are not particularly limited, and those skilled in the art can appropriately select them based on the knowledge of the prior art.

The content range of the shikonins compound contained in the medicine of the invention is 0.0001-75% (weight percentage), and can be properly selected according to different preparations and diseases. When the shikonin is used for human body, the daily dosage of shikonin can be controlled between 10 μ g-20g, preferably 100 μ g-10g, more preferably 1mg-8 g, and most preferably 5 g. Can be selected according to different conditions of patients, such as age, weight, disease conditions, etc. Administration can be single or divided into multiple administrations. The medicament of the present invention can be administered orally, topically, by injection, by inhalation or transdermally.

The medicine containing the shikonins compound can be used for preventing and treating microbial infection of a human body, wherein the microbes comprise pathogenic gram-positive cocci such as staphylococcus aureus, streptococcus pneumoniae, staphylococcus epidermidis, enterococcus faecalis and the like; pathogenic gram-negative cocci such as Klebsiella pneumoniae, Serratia marcescens, and Bacillus maltophilia; anaerobic or microaerophilic pathogenic bacteria such as: helicobacter pylori and the like; deep and superficial fungi such as Candida, Aspergillus fumigatus, Cryptococcus, dermatophytes, Candida krusei, Neurospora tipitata, etc.; various mycoplasma infections such as mycoplasma urealyticum, mycoplasma pneumoniae, etc.; infection by various chlamydiae; viruses such as hepatitis virus, influenza virus, herpes virus, and HIV virus, and the like.

The medicine containing the shikonins compound is sensitive to pathogenic microorganisms, but is not sensitive to microorganisms beneficial to human bodies, such as lactobacillus, bifidobacterium and the like.

The medicine containing the shikonins compound can be used for preventing and treating inflammation of a human body. The inflammation includes, for example, phlebitis, vascular purpura, vaginitis, edema, and the like.

The medicine containing shikonins can also be used for preventing and treating bleeding and blood diseases of human bodies, such as burns and scalds, various dermatitis, septicemia, hemophilia, essential thrombocytosis, leukemia and the like.

The medicine containing the shikonins compound can also be used for preventing and treating tumors of human bodies, particularly malignant tumors, such as ascites tumors: liver cancer, L1210; solid tumors: w256, S180, gastric cancer 823, squamous carcinoma 109, Lewis lung carcinoma, etc.

The medicine containing shikonins can be used for preventing and treating immunoregulatory diseases of human bodies, namely, the nonspecific immunity and the specific cellular immunity of organisms can be promoted by promoting the immune response of T lymphocytes and the like.

Therefore, the drug of the present invention can be used in the respiratory system, digestive system, urinary system, reproductive system, blood system, circulatory system, and skin and mucosal sites of the human body.

Detailed Description

The preparation of the pharmaceutical preparation containing shikonins of the present invention and the pharmacodynamic test will be described in detail below, but the contents of the present invention are not limited thereto.

Preparation example 1.

2 kg of arnebia euchroma (Royle) Johnst is crushed, extracted by petroleum ether until arnebia euchroma (Royle) Johnst residue is colorless, and the solvent is recovered to obtain 80 g of dark red pulp. The obtained slurry is separated by silica gel H column liquid chromatography, namely: the eluents were ethyl acetate-petroleum ether (1: 99) and ethyl acetate-petroleum ether (5: 95) to obtain the 7 gromwell quinone monomers. Namely 2.944 g of deoxidized shikonin (yield: 3.68%), 0.712 g of shikonin (yield: 0.89%), 29.024 g of beta, beta-dimethylacryloyl shikonin (yield: 36.28%), 13.27 g of acetyl shikonin (yield: 16.59%), 6.032 g of 2, 3-dimethyl valeryl shikonin (yield: 7.54%), 0.776 g of beta-hydroxyisovaleryl shikonin (yield: 0.97%), and 0.792 g of beta-acetoxy isovaleryl shikonin (yield: 0.99%). The purity of the product is over 90% by high pressure liquid chromatography.

Preparation example 2.

2 kg of arnebia euchroma is crushed and sieved by a 20-40 mesh sieve, and CO is used₂Supercritical extraction equipment, obtaining 70 g of red paste, prepared by separation by high pressure liquid preparative chromatography (Knauer K1001, germany), preparative column: silica gel H10 μm 50X 300 mm; namely: the eluents were ethyl acetate-petroleum ether (1: 99) and ethyl acetate-petroleum ether (5: 95) to give the red monomers of the 7 shikonins. Namely: 3.486 gThe purity of the product is over 90% by high pressure liquid chromatography analysis, wherein the yield of the product is 4.98% of oxagromwell quinone, 0.707 g of gromwell quinone (the yield is 1.01%), 30.877 g of beta, beta-dimethylacryloyl gromwell quinone (the yield is 44.11%), 15.869 g of acetyl gromwell quinone (the yield is 22.67%), 6.034 g of 2, 3-dimethyl valeryl gromwell quinone (the yield is 8.62%), 0.91 g of beta-hydroxyisovaleryl gromwell quinone (the yield is 1.30%) and 0.77 g of beta-acetoxyisovaleryl gromwell quinone (the yield is 1.10%).

Example 1

The tablets of one or a few of the 7 compounds are prepared according to the method known by the person skilled in the art, wherein the tablets can be prepared to contain 10-70% of the shikonins according to the actual needs.

Under aseptic conditions, 100 g of β, β -dimethylacryloyl shikonin obtained in the above preparation example 1 or 2, 100 g of microcrystalline fiber, 30 g of magnesium stearate, 4 g of hydroxypropylmethyl cellulose were taken. Tablets of 0.5 grams were made according to known tableting techniques and equipment.

Example 2

A tablet of 0.5 g was prepared in the same manner as in example 1 except that 100 g of the composition of shikonin compounds obtained in preparation example 1 or preparation example 2 (the mixing ratio of shikonin, β -dimethylacryloyl shikonin and acetylshikonin was 1: 2).

Example 3

The 7 shikonins were prepared as ointments according to methods known to those skilled in the art. Wherein the ointment can be prepared into the compound containing 0.0001-10% of shikonins according to actual requirements. Under aseptic conditions, 0.5 g of shikonin compounds (deoxyshikonin, shikonin, β -dimethylacryloyl shikonin, acetylshikonin and β -hydroxyisovalerylshikonin in a mixing ratio of 0.7: 1: 2: 0.5) obtained in preparation example 1 or preparation example 2, 80 g of vaseline, 10g of liquid paraffin and 10g of anhydrous lanolin were ground in a mortar and packaged to prepare a product for external use. The ointment can also be used to form a patch for transdermal absorption according to methods known to those skilled in the art.

Example 4

The injection solution of the 7 shikonins is prepared according to the method known by the technical personnel in the field. Under the aseptic operation condition, 0.5 g of beta, beta-dimethylacryloyl alkannin obtained in the preparation example 1 or the preparation example 2, 400 ml of propylene glycol, 100 ml of ethanol, 20 ml of tween-80 and 15 ml of benzyl alcohol are taken, the mixture is fully dissolved, water is added to 1000 ml, and after uniform mixing, the mixture is bottled to prepare an injection product.

The results of the pharmacodynamic test of the drug containing shikonins are described below.

(1) Preparation of drugs

Separately, 5.0 mg of shikonin, β -dimethylacryloyl shikonin and acetyl shikonin obtained in preparation example 1 or preparation example 2 were weighed. The drug was dissolved in 1 ml DMSO. The cells were diluted 50-fold in RPMI-1640 medium, dispensed, and further diluted to concentrations of 100, 50, 25, 12.5, 6.25, 3.125, 1.5625, 0.78125, 0.390625(ug/ml), respectively.

(2) Drug susceptibility testing

Packaging the above medicines into a well plate, and using prepared bacteria with concentration of about 10³-10⁶The respective strains of (a) were inoculated.

The test result shows that: shikonin, beta-dimethylacryloyl shikonin and acetyl shikonin have equal sensitivity to gram-positive staphylococcus aureus, and the sensitivity range (MIC) of the shikonin, the beta, beta-dimethylacryloyl shikonin and the acetyl shikonin is 0.391-12.5 mu g/ml; the sensitive MIC range of the Klebsiella pneumoniae which is common gram-negative pathogenic bacteria is 0.391-6.25 mug/ml, and the few strains are 12.5-50 mug/ml; the sensitivity range of most strains of the sarpsidium marcescens (most clinical isolates) and the maltophilia is 0.391-3.125 mu g/ml. Wherein the berberine hydrochloride is particularly effective to the maltophilia, the sensitivity range is 0.391-0.781 mug/ml, and the berberine is 8-32 mug/ml, namely, the berberine hydrochloride is obviously superior to the berberine hydrochloride; MIC for Bacteroides, especially Bacteroides fragilis is 0.391-6.25 μ g/ml; is quite sensitive to helicobacter pylori, and the MIC of the vaccine is 0.391-0.781 mug/ml.

In addition, the results of the in vitro antifungal test of β, β -dimethylacryloyl shikonin show that: MIC range for Candida and Cryptococcus is between 2.08-33.3 μ g/ml, MIC₉₀33.3. mu.g/ml, while the MIC of fluconazole ranges between 0.125-64. mu.g/ml, the MIC₉₀69 μ g/ml; the MIC range of dermatophytes is 4.16-8.32 μ g/ml, MIC₉₀4.16. mu.g/ml, whereas the MIC of fluconazole for most strains of dermatophytes ranges between 32-64. mu.g/ml, the MIC₉₀The concentration was 64. mu.g/ml, and the difference was significant. In addition, surprisingly, the beta, beta-dimethylacryloyl shikonin has a good inhibition effect on the fluconazole-resistant candida krusei, the MIC is 8.32-16.6 mu g/ml, and the MIC of the fusarium oxysporum, which is insensitive to most antifungal drugs such as fluconazole, is 4.16-8.32 mu g/ml. In addition, the MIC of the acetoshikonin to the cryptococcus neoformans is 3.90625 mu g/ml, and the MIC to the trichophyton rubrum is 0.90625-62.5 mu g/ml; the MIC of the beta, beta-dimethylacryloyl shikonin to aspergillus fumigatus, cryptococcus and trichophyton rubrum is 3.0625-250 mu g/ml. Therefore, the shikonins compound is a broad-spectrum and powerful antifungal drug.

In addition, MIC of the shikonin compound of the present invention to beneficial microorganisms such as Lactobacillus and Bifidobacterium are all above 200 μ g/ml, and thus, it can be seen from the above data that the drug containing shikonin compound of the present invention is sensitive to pathogenic microorganisms but not to beneficial microorganisms.

As can be seen from the comparative tests of the mixed extract of Lithospermum erythrorhizon with 1 to 3 of the shikonins, the MIC (μ g/ml) results of the inventive drug containing 1 to 3 of the shikonins are significantly better than those of the mixed extract of Lithospermum erythrorhizon are shown in Table 1.

TABLE 1

Strain name	A	B	C
				Staphylococcus epidermidis	12.5	0.391	0.7812
Serratia marcescens	25	0.781	3.125
				Bacteroides	＞200	0.391	0.391
Candida albicans	500	3.9062	250

Note: a is the mixed extract of radix Arnebiae;

b is beta, beta-dimethylacryloyl alkannin;

c is a mixture of shikonin compounds (shikonin, beta-dimethylacryloyl shikonin and acetyl shikonin at a ratio of 1: 2)

The results of the test on the bacteriostatic effect of the shikonin, the beta, beta-dimethylacryloyl shikonin and the acetyl shikonin on the mycoplasma pneumoniae show that the MICs of the shikonin, the beta, beta-dimethylacryloyl shikonin and the acetyl shikonin for inhibiting the mycoplasma pneumoniae have the concentrations of 3.751 mug/ml, 2 mug/ml and 7.819 mug/ml respectively, and are equivalent to the inhibiting effect of 0.1925 mug/ml of pure erythromycin for injection.

The results of the test of the partial diseases by the external skin application using the shikonin ointment prepared in example 3 are shown in table 2 below.

TABLE 2

Disorders of the disease	Number of human subjects	High efficiency	Rate of treatment	Days of treatment	Route of administration	Remarks for note
							Burn and scald	300	100％	100％	6-20	Directly administered to affected part	Scald 92, superficial 2 degree 186 persons, deep 2 and 3 degree 114 persons
Hemorrhoid treatment	117	100％	97.4％	15	Directly administered to affected part	Three cases recurred after half a year
							Herpes zoster	98	100％	100％	3-7	Directly administered to affected part	In 12 cases, polyinosinic cells are added
Cervical erosion	80	100％	100％	10-20	Vaginal administration
							Infantile epistaxis	257	99.6％	72.8％	15	Nasal administration
Verruca plana	100	96％	81％	10-30	Directly administered to affected part
							Chronic prostatitis	40	82.5％	57.5％	10-20	Administration to anus
Acne	50	92％	60％	15	Directly administered to affected part
							Bedsore	30	100％	100％	7-21	Directly administered to affected part
Eczema rhagadiforme	98	94.9％	66.3％	10-30	Directly administered to affected part
							Condyloma acuminata	55	100％	100％	5-35	Directly administered to affected part
Dermatitis of baby diaper	208	100％	100％	2-6	Directly administered to affected part

As can be seen from the above table, the external skin application of the shikonins compound is suitable for treating most abscesses, scabies and herpes, has obvious effect on burns and scalds, and does not leave scars after healing.

The results of animal experiments on transplanted tumors by shikonin, β -dimethylacryloyl shikonin and acetylshikonin are shown in table 3 below.

TABLE 3

Tumor type	Beta, beta-dimethylacryloyl alkannin		Acetylshikonin		Lithospermum quinone
								Tumor inhibition rate	Rate of life prolongation	Tumor inhibition rate	Rate of life prolongation	Tumor inhibition rate	Rate of life prolongation
Ascites type liver cancer		113.4％	47.8％	112.6％		130.8％
							Sarcoma 180	9.63％		35.7％
Lewis lung carcinoma	42.8％		52.6％
							L1210				128％
W256			77％

As can be seen from the above table, the beta, beta-dimethylacryloyl alkannin has different degrees of curative effects on liver cancer, sarcoma 180 and Lewis lung cancer; the acetyl alkannin has different degrees of curative effects on liver cancer, sarcoma 180, L1210, Lewis lung cancer and W256; shikonin is effective only on liver cancer.

The test result of the shikonins compound on the virus shows that: the beta, beta-dimethylacryloyl alkannin is orally taken 7 days after duck hepatitis B virus infects the duck, 100mg/kg is taken 2 times a day, the inhibiting effect on the level of DHBV-DNA of the infected duck blood serum is obvious (P is less than 0.05-0.01) in 10 days, and no toxic reaction is caused; the 50mg/kg group has obvious inhibition effect (P is less than 0.05).

The in vitro test of beta, beta-dimethylacryloyl alkannin on hepatitis B virus shows that the concentration is 30 mug/ml, the average inhibition rate on HBsAg is 96.2601%, and the average inhibition rate on HBeAg is 91.6056%.

The results of in vitro tests of shikonin and β, β -dimethylacryloyl shikonin against HIV-1 reverse transcriptase and integrase are shown in table 4 below.

TABLE 4

		IC(μg/ml)
			anti-HIV reverse transcriptase	Positive control PFA	0.097
Gromwell quinone compounds	＞20
		anti-HIV integrase		Positive control ABPS-Y	0.922
Gromwell quinone compounds	12.467

The action of shikonins is researched by using a mouse immune function hypofunction model caused by mitomycin C. The result shows that 6mg/kg of beta, beta-dimethylacryloyl alkannin is intraperitoneally injected every day, the NK cell cytotoxicity of the splenocytes of the mice is improved by about 20 percent (P is less than 0.001) after 5 days of continuous injection, the damage of macrophages in abdominal cavities of the mice can be recovered, the migration capacity of the macrophages in the abdominal cavities can be increased, the activity of T lymphocytes can be increased, the immune response effect of the T lymphocytes can be promoted, and the non-specific immunity and the specific cellular immunity effect of an organism can be promoted.

Claims (10)

1. The application of shikonins compound shown as the following formula (I) in preparing anti-hepatitis B virus drugs,

wherein R is selected from the group consisting of H, OH, (CH)₃)₂C＝CHC(O)O-、CH₃C(O)O-、(CH₃)₂C＝C(CH₃)CH₂C(O)O-、(CH₃)₂COHCH₂C(O)O-、(CH₃)₃C[OC(O)CH₃]CH₂C (O) a substituent of the group consisting of O-.

2. The application of shikonins compound shown as the following formula (I) in preparing anti-HIV virus drugs,

wherein R is selected from the group consisting of H, OH, (CH)₃)₂C＝CHC(O)O-、CH₃C(O)O-、(CH₃)₂C＝C(CH₃)CH₂C(O)O-、(CH₃)₂COHCH₂C(O)O-、(CH₃)₂C[OC(O)CH₃]CH₂C (O) a substituent of the group consisting of O-.

3. The application of shikonins compound shown as the following formula (I) in preparing antiviral pneumonia medicaments,

wherein R is selected from the group consisting of H, OH, (CH)₃)₂C＝CHC(O)O-、CH₃C(O)O-、(CH₃)₂C＝C(CH₃)CH₂C(O)O-、(CH₃)₂COHCH₂C(O)O-、(CH₃)₂C[OC(O)CH₃]CH₂C (O) a substituent of the group consisting of O-.

4. The use according to any one of claims 1 to 3, wherein R is selected from OH, (CH)₃)₂C ═ CHC (O) O-and CH₃C (O) a substituent of O-.

5. The use of claim 4, wherein R is (CH)₃)₂C ═ CHC (O) O-and/or CH₃C(O)O-。

6. The use of claim 5, wherein R is (CH)₃)₂C＝CHC(O)O-。

7. The use as claimed in any one of claims 1 to 3, wherein the purity of each of the shikonins is 80% or more than 80%.

8. The use as claimed in claim 7, wherein the purity of the shikonins is 90% or more than 90%.

9. The use as claimed in any one of claims 1 to 3, wherein, when the medicament contains a combination of 1 to 3 of the shikonins, the total content of the effective ingredients thereof is 70% or more than 70%.

10. Use according to any one of claims 1 to 3, wherein the medicament further comprises a further pharmaceutically active ingredient.