JPH09241157A - Medicinal composition for protecting liver containing lithospermate b - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a medicinal composition for liver protection which contains lithospermate B obtained from Tannzinn (Salviae miltiorrhizae radix) having liver-protecting action. SOLUTION: This medicinal composition contains lithospermate B of the formula (M<++> is Mg<++> , Ca<++> or the like) as an active ingredient. The lithospermate B is obtained by extracting Tannzinn (root of Salviae miltiorrhiza Bunge) self-growing in mountains and fields of People's Republic of China with water or a polar solvent such as methanol under neutral conditions, concentrating. separating and purifying the extract. A pharmaceutically permissible substrate and an excipient are suitably formulated thereto to prepare preferably powder, syrup, capsules, granules, emulsion, suspension, drops and administered. The daily dose is 10-10,000mg/adult calculated as lithospermate B.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to Danshen's lysospermate B having a hepatoprotective action.

[0002]

2. Description of the Related Art Since the liver has a strong natural healing power and a slight disorder does not cause any manifestation of symptoms, it is also called a "silent organ", which is a substance metabolism, regulation of blood glucose, detoxification, It plays a vital role in maintaining human life, such as regulating bile circulation and storing nutrients. However, in recent years in Japan, the number of patients exhibiting liver function abnormalities has increased with the increase in alcohol consumption. Although the etiology and pathophysiology of liver damage are diverse, the development of therapeutic agents is most sought after for chronic active hepatitis, which has a high medical need. Research and development of various therapeutic agents including antiviral agents and immunomodulators are being actively conducted.

For such liver diseases, folk remedies have been used since ancient times for shosaikoto (saiko, half-summer, carrot, rhubarb,
Licorice, ginger, turmeric), Shiraiyu (Chen, Sat, Huanghe car, Gomiko, Tomei, Daioh) and other herbal medicines are used to "cleanse the liver,""remove liver heat," and "help the liver's energy." However, its active ingredient is not clear. Further, there is only a description (Japanese Patent Publication No. 6-86481) of a polysaccharide obtained by fermenting grass fiber in a specific culture medium for liver diseases and having a therapeutic effect on hepatitis B. is there. Treatment of chronic active hepatitis or the like requires administration of a drug for a long period of time due to its nature, which causes side effects. Therefore, the development of a safe preventive / therapeutic drug for liver disease that has a high therapeutic effect regardless of the cause of liver disease such as virus, drug poisoning, alcohol, etc. .

As a result of repeated studies on various crude drugs, the present inventors have found that lithospermate B contained in the root of Danshen has a remarkable inhibitory effect on liver diseases. Found and completed the present invention.

[0005]

[Means for solving the problem] Danshen (salviae miltiorrh
izae radix) is a perennial of the Lamiaceae that grows naturally in the mountains of China.
It is the root of Salvia miltiorrhiza BUNGE. The components are phenanthrenequinones Tanshinone I, Tanshinone IIA,
Tanshinone IIB, cryptotanshinone, isotanshinone I and the like are known. It is a Japanese-Chinese medicine that has been used as a live blood and a menstrual medicine, and although there are many reports of its effects on the circulatory system, it is also well known that it has antioxidant effects. Since oxidative damage contributes to various liver damages, we examined the hepatoprotective effect of Danshen based on its antioxidant effect. It has not been reported that lysospermate B has a hepatoprotective effect.

CC using primary cultured hepatocytes derived from rat
In in vitro experimental systems to induce hepatocellular damage in l _4, Nisansui extract inhibited significantly elevated GOT concentration in the culture broth. Therefore, separation and purification were carried out while monitoring the activity, and lysopermate B, which is a tetramer of caffeic acid, was isolated as an active ingredient. Then, when the hepatoprotective action was examined in vitro, lyspermate B was found to be CCl ₄ -induced liver injury in rats and D-galactos in mice.
A hepatoprotective effect was confirmed by pharmacological experiments such as amine / LPS-induced liver injury.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION Danshen used in the present invention may be the root of Salvia miltiorrhiza Bunge or a root of its related plant. From Danshen, with water, polar solvent, or their mixed solvent,
Extraction is performed under neutral conditions, and the extract is concentrated, separated, and purified to obtain lysospermate B having a hepatoprotective action having the following formula.

Embedded image (In the formula, M ⁺⁺ is, for example, Mg ⁺⁺ , Ca ⁺⁺ .)

Preparation of Danshen Water Extract Extract First, add water (40 liters) to the dried roots of Danshen (about 8 kg) and
The mixture was heated and refluxed twice for each hour, and the extract obtained by the heating was filtered with a paper filter and freeze-dried to obtain a brown water extract (170 g).
Get.

Dansui water extraction for CCl ₄ induced hepatocellular injury
Effect of Extract The effect of a water extract obtained from Danshen on CCl ₄ -induced liver damage was examined. Since the CCl ₄ liver injury model is simple, it is most widely used as a primary evaluation system for the effect on liver injury. CCl ₄ causes chemical (toxic) liver damage (T.
Yokozawa et al., Biopharmaceutical Journal 47, 229 (199)
3), N. Ishida et al., J. Hepatology 13,200.
(1991)). There are many studies on the mechanism of hepatic injury by CCl _4, but trichloromethyl radical (CCl ₃ ) is generated in the endoplasmic reticulum drug metabolism pathway, which covalently binds to hepatocyte proteins, lipids, etc. to give a functional change. Along with this, it also acts on the cell membrane and
The theory is that LPO) is produced, the membrane is altered, and finally cell necrosis is caused (Paul B McCay et a
l., J. Biological Chem., 259 , 2135 (1984)).

Dissolved in physiological saline or 0.5% C
Sprangue-Dawle was used as a test drug evenly suspended in MC aqueous solution.
After intraperitoneal administration to y rats (6 weeks old) three times at 0, 12, and 23 hours, 6 ml / kg of CCl ₄ / olive oil mixture (1: 1 v / v) was subcutaneously applied to the back at 24 hours. I made an injection. In the control group, physiological saline or 0.5% CMC aqueous solution alone was administered, and then CCl ₄ was similarly injected. Blood was collected 24 hours after the administration of CCl _{4 in} which the degree of liver damage was most pronounced. The GOT value was measured for the collected blood.
The GOT value was measured using a refretron.

[0011]

[Table 1] Effect group of water extract of ginseng on CCl ₄ induced hepatocyte damage Concentration GOT ^a) protection ^b) (μg / ml) (U / L) (%) Normal −24.8 ± 10 Control−294 ± 22− Danshen Treatment 10 239 ± 43 * 20.4 500 115 ± 16 ** 66.5 The result is the average of the measurement results ± standard deviation, n = 4, p <0.
05 and p <0.001. After 24 hours of pre-incubation, the medium was replaced with fresh medium containing 10 mM CCl ₄ and 100 or 500 μg / ml of sample and incubated for 1 hour. ^a) GOT concentration in the medium after 1 hour of CCl ₄ challenge. ^b) Hepatocyte protection rate compared to CCl ₄ control.

As shown in Table 1, the GOT value in the normal group was 24.8 ± 10 U / L, whereas it was 29 in the CCl ₄ induction model.
It was 4 ± 22 U / L. However, the GOT values of the water extract of Danshen (10 μg / ml and 500 μg / ml) added to the culture were 239 ± 25 and 11 respectively.
It was 5 ± 16 U / L. Since we found that Danshen had a significant protective effect against hepatocyte necrosis, we next examined MCl
A gel was used to search for active ingredients.

[0013] Of the hepatoprotective active ingredient in the Danshen water extract
The effect of each of the above-mentioned fractions of the water extract and the methanol extract obtained from Danshen on the CCl ₄ -induced liver injury was examined. Water extract (170.6 g) was MCl-gel CHP-
The mixture was subjected to 20P column chromatography, and the concentration of methanol was gradually increased in water to elute the mixture. Seven fractions (fraction A (110.07 g), fraction B (1.49 g), fraction C (1.44 g), Fraction D (5.1 g), Fraction E (1.01 g), Fraction F (0.72 g), Fraction G (0.43)
g)) is obtained. Lysospermate B (3.2 g) is obtained from Fraction D eluted with 50% water-methanol.

Carbon tetrachloride using primary culture hepatocytes (CC
l ₄₎ by induced hepatopathy model hepatoprotective active ingredient Danshen
Isolation Figure 1 shows the in vitro hepatocyte protection effect of each fraction of Danshen after fractionation by MCI CHP-20 gel column chromatography. SD rat (male 7 weeks old)
Liver parenchymal cells were isolated from collagen liver by collagenase perfusion method and cultured for 24 hours, then 1 μg / ml sample and 10 mMC
The medium was replaced with a medium containing Cl ₄ and after 1 hour, the GOT concentration in the medium was measured. The results show the GPT concentration in the medium 1 hour after replacement with the medium containing CCl ₄ as the average value ± standard difference.
Significantly different from control, * P <0.05. The most active Fr. D is a single compound and is lysospermate B
It turned out. As shown in Figure 1, the hepatoprotective activity of the seven eluted fractions was examined using the assay described above. In the control group induced by CCl ₄ , the GOT value was 324.
± 58 U / L, but fractions C and D are 1μ
In the group added at the concentration of g / ml, it was 154 ± 33 and 138 ± 37 U / L, respectively. The active ingredient of Fraction D, which is the most active of the seven fractions, is ¹
It was identified as lysospermate B by 1 H- and ¹³ C-NMR. ¹ H- and ¹³ C-NMR show acetone-d ₆
It was measured in a solvent (FIGS. 2 and 3).

FIG. 4 shows in vitro using primary culture hepatocytes.
₃ shows the effect of the Danshen active ingredient lysopermate B on CCl ₄ -induced liver injury in the assay system. Liver parenchymal cells were isolated from the livers of SD rats (male 7 weeks old) by the collagenase perfusion method, and after culturing for 24 hours, they were replaced with a medium containing various concentrations of lysospermate B and 10 mM CCl _4, and after 1 hour, the medium was replaced. The GOT concentration inside was measured. The results are shown as the average value ± standard difference. When a bioassay was performed using student's t-test, a significant difference (P <0.05) was observed in all the groups having a concentration of 1 μg / ml or more. As shown in FIG. 4, CCl
_4. Lysospermate B 0.1 to 10 for _4- induced liver injury
At 0 μg / ml, a significant protective activity was shown in a concentration-dependent manner.

FIG. 5 shows the hepatoprotective effect of lysospermate B on CCl ₄ -induced liver injury rats. The results are shown as the average value ± standard error of 7 rats. Significant difference from control, * P <0.05, ** p <0.01, *** p <
0.001. CCl ₄ with various concentrations of lysospermate B
A total of 3 intraperitoneal injections were made 24, 12 and 1 hour before administration of (3 ml / kg). Blood samples were collected 24 hours after CCl ₄ administration and the serum enzyme concentration was measured. Group A: normal group, B
Group: control group, C group: lysopermate B (50 mg / kg)
Administration group, group D: lysopermate B (200 mg / kg) administration group.

As shown in FIG. 5, serum GP of normal group
T, GOT and LDH values are 37 ± 4.9,
74 ± 2.5, 156 ± 24 U / L, GPT, GOT and LDH at 24 hours after intraperitoneal administration of CCl ₄
Values are 493 ± 84, 1360 ± 86, 1509, respectively.
It was ± 208. On the other hand, lysospermate B (50 mg
/ Kg) was administered intraperitoneally three times, serum GPT, GO
T and LDH values are 248 ± 58, 621 ± 97, 598 ±
It was 89 U / L. Also, Lithospermate B (200
(mg / kg), GPT, GOT and LDH levels were 134 ± 39, 364 ± 79 and 354 ± 92 U /, respectively.
L, and lysospermate B showed significant protective activity against CCl ₄ -induced liver injury.

D-galactosamine / LPS-induced liver injury model
Evaluation by Dell D-galactosamine (hereinafter abbreviated as D-gal) / Lip
opolysaccharide (hereinafter abbreviated as LPS) -induced liver injury model (J. Wang et al., Biochem. Pharm. 3)
9, 267 (1990), A.S. Wendel et al., Bioch
em.Pharm. 35, 2115 (1986)), the liver damage is a reaction via autacoids and cytokines such as LTD ₄ and TNF-α, and therefore has a high correlation with clinical results as an immunological liver damage occurrence model. I think that the. D-galactosamine is metabolized in cells and UD
It becomes P-galactosamine and uridine phosphate (UM
Deficiency of P, UDP, UTP) occurs. Therefore RNA
In addition to a decrease in synthetic ability, UDP-glucose and UDP-glucuronic acid decrease, resulting in inhibition of protein and lipid metabolism, resulting in cell necrosis.

Mice are generally resistant to this liver injury. However, when a small amount of Endotoxin (LPS) is administered to a mouse sensitized with galactosamine, a remarkable liver damage model is obtained. This is due to the fact that LPS induces LTD ₄ , the blood vessels are temporarily in an ischemic state, and superoxide is generated when the blood flow starts to flow again as the concentration of LTD ₄ decreases. That is, TNF-α secreted from macrophage activated by superoxide (which is also directly activated by LPS itself) destroys cells sensitized by D-gal. The above is the currently considered mechanism of action of liver injury by D-gal / LPS (Shingo Hirooka et al., Pharmaceutical Research 13, 1046 (198).
2); Sommer BG et al., Transplant Proc. 1
1,578 (1979); G. Tiegs et al. Biochem.
Pharm. 38, 627 (1989); Keppler D. et a.
I., Eur. J. Biochem. 10, 219 (1969)).

FIG. 6 shows the hepatoprotective effect of lysospermate B on D-galactosamine / LPS-induced liver injury mice. Results are expressed as mean ± standard error of 10 mice, significant difference from control * P <0.05. Samples are D-galactosamine (700 mg / kg) and LPS (10 μg)
/ Ml) was administered orally or intraperitoneally at each dose. The GOT concentration in blood was measured 8 hours after the administration of D-galactosamine / LPS. Group A: untreated group, B
Group: control group, group C: lysospermate B, 150 mg / k
g, twice daily for 1 week, group D: lysopermate B, 500 mg / kg, twice daily for one week, group E
Group: Lysopermate B, 50 mg / kg, twice subcutaneous administration group.

2,18 hours after subcutaneous administration of lyspermate B to male ddY mice (6 weeks old),
D-gal (700 mg / kg) and LPS (10 μg / kg)
Was simultaneously administered ip. After only the solvent was administered to the pathological control group, D-gal and LPS were similarly administered. D-g
Blood was collected 8 hours after administration of al and LPS, and G
The PT value was measured. The protective effect of lysospermate B on D-gal / LPS-induced liver injury mice is shown in FIG. GPT level in blood is 66 ± 17 U / L in normal group
2452 ± 5 in the control treated with D-gal / LPS
It was 24 U / L. On the other hand, 2 hours and 8 hours before administration of D-gal / LPS, lysopermate B (50 mg / k)
GPT value of the group which received g) twice subcutaneously was 996 ± 260
It was U / L. In addition, before administration of D-gal / LPS, lysopermate B (150 mg / kg or 50
(0 mg / kg) GPT value when administered orally twice a day for 1 week
2339 ± 503 and 1184 ± 256 U / respectively
L. From the above results, lysospermate B is D
−50 mg subcutaneously for gal / LPS-induced liver injury
/ Kg, and orally administered at 500 mg / kg, a significant hepatoprotective effect was exhibited.

Superoxide of lysospermate B
Nion radical scavenging action Generation of superoxide anion by XOD / xanthine SOD (superoxide) that eliminates active oxygen generated in the body
As a method for measuring the activity of disumutase (superoxide dismutase), a method of generating active oxygen by xanthine / XOD and measuring the degree of reduction of NBT (nitroblue tetrazolium) was used. 50mM Na ₂ CO ₃ (pH 10.2), 0.2mM xanthi
ne, 0.2 mM EDTA, 0.1 mg / ml BSA, 0.15
mM NBT, various concentrations of lysospermate B and 0.1 mg / ml XOD (xanthine oxidase: xanthine)
The oxidase) mixture is incubated at 25 ° C. for 20 minutes. The reaction is stopped by adding 0.1 ml of 6 mM CuCl ₂ and the absorbance at 560 nm is measured.

FIG. 7 shows the superoxide anion radical scavenging action of lysospermate B. Superoxide radical anion was detected by the NBT reduction method. The reaction solution is 50 mM Na ₂ CO ₃ buffer (pH 10.2)
0.1 mM xanthine, 0.1 mM EDTA, 50 μg / mlB
It consisted of SA, 25 μg / ml NBT, 0.1-50 μg / ml lysopermate B and 50 μg / ml XOD, and the absorbance at 560 nm was measured after the reaction was stopped. As a result, it was revealed that lysopermate B had a strong erasing activity with an IC ₅₀ of 2.32 μg / ml.

Macrophage-like cells from J774.1
Of lysospermate B for nitric oxide (NO) production
Effect NO releasing action from macrophage-like cells J774.1 J774.1 cells are seeded in a 24-well culture plate at a concentration of 1 × 10 ⁶ cells / ml. After preculturing for 24 hours,
The medium was replaced with a fresh medium containing dotoxin (10 μg / ml) and various concentrations of lysospermate B. The cells are cultured for 48 hours at 37 ° C. in air containing 5% CO ₂ . J
The production of NO from 774.1 cells was measured by the Griess reaction.

[0025]

[Table 2] Production of nitric oxide (NO) from macrophage-like cells J774.1Effect of lysospermate B on Group concentration Nitrite^a) inhibition^b)  (μg / ml) (μM10⁶cells / 48h) (%) Control 14.6 ± 0.8-Lysospermate B 1 14.4 ± 1.0 1.04 10 12.8 ± 0.8 * 12.5 100 11.4 ± 0.3 * 21.4500 2.3 ± 0.2 ** 84.5

The results are the average of the measurement results ± standard deviation, n =
4, p <0.05 and p <0.001. Endotoxin (derived from E. coli, 10 μg / m
48) with l) and various concentrations of lysospermate B
Cultured for hours. ^{a) The} amount of NO produced was converted to the nitrite ion concentration and measured. ^b) N compared to endotoxin treated control
It is the inhibition rate of O production. As shown in Table 2, lysopermate B significantly inhibited NO production at a concentration of 10 μg / ml or higher.

FIG. 8 shows the effect of lysospermate B on the survival of J774.1 cells. J77
4.1 cells with various concentrations of lysospermate B 4
The cells were cultured for 8 hours. Cell viability was measured by the MTT method. On the other hand, the viability of cells in the presence of lysopermate B was 95% at a concentration of 50 to 250 μg / ml, 500 μg.
It was 83.5% or more in the case of / ml.

When the lysospermate B of the present invention is used as a prophylactic / therapeutic drug for liver damage, it can coexist with a pharmaceutically acceptable carrier or excipient and is suitable for respiration from the gastrointestinal tract. It is desirable to administer in the form. For example, the composition for oral administration may be solid or liquid, and may be powder, syrup, capsule, granule, emulsion, suspension, drop and the like. Carriers or excipients for compositions of this type are well known. Examples of excipients for tablets are lactose, potato and soluble starch, magnesium stearate, etc., Examples of carriers for injection are sterile water, physiological saline, almond oil, etc.
They can be placed in ampoules or added to the active substance before use. If desired, the composition further comprises a binder,
It may contain conventional materials such as stabilizers, emulsifiers, suspending agents, dispersing agents, lubricants, preservatives and extenders.

The lysospermate B of the present invention can be used as it is, or as an aqueous extract from Danshen, and in a state of being appropriately purified, as a food additive in health foods, functional foods and the like.

The daily dose of the preventive or therapeutic agent for liver damage according to the present invention is 10 to 1 in terms of lysospermate B.
0000 mg / adult is preferable. Danshen containing the lysospermate B of the present invention has been generally used as a crude drug for various medical purposes for many years, and toxicity does not cause any problem at least at the above doses.

[0031]

【Example】

Example 1 Tablet According to a conventional method, 100 mg of lysospermate B and lactose 1
g, starch 300 mg, methylcellulose 50 mg,
30 mg of talc is prepared into 10 tablets and sugar-coated with white sugar.

[Brief description of drawings]

FIG. 1 is a bar graph showing the in vitro hepatocyte protection effect of each fraction of Danshen.

FIG. 2 is a ¹ H-NMR chart of lysospermate B.

FIG. 3 is a ¹³ C-NMR chart of lysospermate B.

FIG. 4 is a bar graph showing the effect of lysospermate B on CCl ₄ -induced hepatopathy cells.

FIG. 5 is a bar graph showing the hepatoprotective effect of lysospermate B on CCl ₄ -induced liver injury rats.

FIG. 6 is a bar graph showing the hepatoprotective effect of lysospermate B on D-galactosamine / LPS-induced liver injury mice.

FIG. 7 is a bar graph showing the superoxide anion radical scavenging action of lysospermate B.

FIG. 8 is a line graph showing the effect of lysospermate B on the survival of J774.1 cells.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Shigetoshi Kadota 2556-4 2-402, Gofuku Suehirocho, Toyama City, Toyama Prefecture

Claims (1)

[Claims] 1. A hepatoprotective pharmaceutical composition containing lysospermate B as an active ingredient.