TWI409073B - Treatment of pain, inflammation and liver injury and hepatoprotection with ergosta-7,9(11),22e-trien-3β-ol - Google Patents

Abstract

Disclosed herein is that ergosta-7, 9(11), 22E-trien-3 &bgr; -ol can be used in the treatment of pain, inflammation and liver injury and hepatoprotection.

Description

Use ergoside-7,9(11),22 E-triene-3β-ol to treat pain, inflammation and liver damage, and protect the liver

The present invention relates to the use of ergot-7,9(11),22 E -triene-3β-alcohol for the treatment of pain, inflammation and liver injury, and protection of the liver (hepatoprotection). .

Reactive oxygen species (ROS) are known to occur during the respiration of oxygenated organisms using oxygen (eg, peroxides and hydrogen peroxide). )] and free radicals [eg, hydroxyl radicals and peroxy radicals], and ionizing radiation and exposure to drugs or xenobiotics [eg, Carbon tetrachloride (CCl ₄ )] also leads to the formation of reactive oxygen species and free radicals. Reactive oxygen species and free radicals are very unstable, and they react rapidly with groups or substances in the organism, leading to oxidative damage to cells or tissues, such as DNA damage, polyunsaturated fatty acids. Acid) oxidation [also known as lipid peroxidation] and oxidation of amino acids.

In general, there is an interaction network composed of antioxidant enzymes in the organism to protect cells or tissues from oxidative damage. The most well-known antioxidant enzymes include: superoxide disproportionation. Superoxide dismutase (SOD) (EC 1.15.1.1), catalase (CAT) (EC 1.11.1.6), glutathione peroxidase (GPx) (EC 1.11.1.9) and Glutathione reductase (GSH reductase) (EC 1.8.1.7). However, when the amount of reactive oxygen species and free radicals exceeds the antioxidant capacity of the cells or tissues themselves, oxidative stress is formed. Oxidative stress has now been found to play an important role in the degenerative or pathological processes of various diseases: aging, cancer, inflammation, and liver damage. (liver injury).

Inflammation means an organism's protective response against external stressful stimuli or pathogens. In the early stages of inflammation, damaged cells or tissues release large amounts of chemokines, making polynuclear leukocytes (eg, neutrophil) and mononuclear balls in the immune system (eg, nucleus) Monocytes) accumulate in the affected area [referred to as infiltration]. At the same time, macrophages are activated and promote the hydrolysis of phosphatidylcholine (PC) on the cell membrane by phospholipase A2 to arachidonic acid (AA), followed by arachidonic acid Cyclooxygenase-2 (COX-2) is metabolized into a large number of prostaglandin (PG), prostacyclin and thromboxane A2 to enhance the inflammatory response. Activated macrophages also promote the release of various proinflammatory cytokine (including interleukin) by other macrophages by releasing tumor necrosis factor-α (TNF-α). Interleukin-1 (IL-1), IL-6 and IL-8], and through the action of inducible nitric oxide synthase (iNOS) to produce vasodilating and killing pathogens Nitric oxide. In addition, reactive oxygen species and free radicals are also used by the immune system to kill pathogens and become important members of the inflammatory response.

Although many artificial compounds are currently available for improving oxidative stress and inflammation, respectively, these artificial compounds produce varying degrees of side effects [such as gastric ulcer and gastritis]. If you can find compounds or extracts that can improve oxidative stress and have anti-inflammatory and analgesia activities from natural herbal medicines with mild medicinal properties and low side effects, you should be able to provide one more Safe drug.

Antrodia camphorata (also known as "Bagba") belongs to the genus Aphyllophorales, Polyporaceae, and Antrodia , and is mainly grown on high-altitude eucalyptus ( Cinnamomum kanehirai ) in Taiwan. . In Taiwanese folk medicine, the fruiting body of Antrodia camphorata is used to treat food or drug intoxication, diarrhea, abdominal pain, hypertension, and itchy skin. Skin itching) and cancer (Peng CC et al. (2007), J. Ethnopharmacol ., 109: 93-103). In addition, the fruiting bodies of Antrodia camphorata have also been shown to have immunomodulating, antioxidative, and hepatoprotective effects (Shen YC et al. (2004), Planta. Med ., 70:310-314; Hsiao G. et al. (2003), J. Agricultural And Food Chemistry , 51: 3302-3308).

It has been reported in the literature that fermented culture broth has cytotoxic activity against several tumor cell lines (Peng CC et al . (2007), same as above); fermentation broth of Antrodia camphorata the filtrate (filtrate) effective against carbon tetrachloride -. liver protection utility induced hepatotoxicity (CCl ₄ -induced hepatic toxicity), and antioxidant properties (antioxidant properties) (Song TY et al (2003), J Agricultural and Food Chemistry , 51: 1571-1577); mycelia of Antrodia camphorata has anti-inflammation and vasorelaxation effects, cytotoxicity against various cancer cell lines, and anti-B hepatitis Anti-hepatitis B virus activity (Shen YC et al . (2004), FEMS Microbiology Letter , 231: 137-143; Wang GJ et al . (2003), Life Science , 73: 2769-2783; Yeh CT Et al . (2009), Cancer Letter , 285: 73-79; Lee IH et al . (2002), FEMS Microbiology Letter , 209: 63-67).

In a previous study, the inventors successfully isolated 12 compounds from the submerged broth of Antrodia camphorata, including 11 known compounds [ie, ergot -7,9 (11), 22 E -trien-3β-ol (ergosta-7,9(11),22 E- trien-3β-ol), ergosterol peroxide, methyl (4-hydroxyphenyl)acetate [methyl(4-hydroxyphenyl) acetate], vanillin, 4-hydroxybenzaldehyde, hexadecanoic acid, 5-methoxymethylfuran-2-carbaldehyde (5- Methoxymethylfuran-2-carbaldehyde), 5-hydroxymethylfuran-2-carbaldehyde, 11-hydroxy-γ-dodecalactone, 2-( 2-hydroxyethyl phenol] and 12-hydroxydodecanoic acid methyl ester, and a novel compound [ie 10-hydroxy-γ- 10-hydroxy-γ-dodecalactone] (Shao YY et al. (2008), Natural Product Research , 22: 1151-1157).

In order to understand the biological activity of the compound isolated from the immersion culture solution of Antrodia camphorata, in a subsequent study, the inventors selected ergot 甾-7,9(11),22 having a chemical formula as shown below. E -trien-3β-ol for in vitro anticancer experiments:

It was confirmed by experimental results that ergot 甾-7,9(11), 22 E -triene-3β-alcohol has the effect of inhibiting the growth of liver cancer cells, and its 50% inhibitory concentration on human hepatoma cell line PLC/PRF/5 ( 50% inhibition concentration, IC ₅₀ ) was 20.5 μg/mL (unpublished data).

After research, the inventors unexpectedly discovered that ergosta-7,9(11),22 E -triene-3β-alcohol has analgesic and anti-inflammatory effects in addition to its potency in inhibiting the growth of liver cancer cells. -inflammatory) and the utility of improving ameliorating liver injury and are therefore expected to be useful for the treatment of pain, inflammation and liver damage, and protection of the liver (hepatoprotection).

Summary of invention

Thus, in a first aspect, the present invention provides a pharmaceutical composition for treating pain comprising ergosta-7,9(11),22 E -triene-3β-ol.

In a second aspect, the present invention provides a pharmaceutical composition for treating inflammation comprising ergosta-7,9(11),22 E -triene-3β-ol.

In a third aspect, the present invention provides a pharmaceutical composition for treating liver damage comprising ergosta-7,9(11),22 E -triene-3β-ol.

In a fourth aspect, the present invention provides a composition for protecting a liver comprising ergosta-7,9(11),22 E -triene-3β-ol.

The above and other objects, features and advantages of the present invention will become apparent from

Detailed description of the invention

It is to be understood that if any of the previous publications is quoted here, the prior publication does not constitute an acknowledgement that in Taiwan or any other country, the pre-existing publication forms a common general knowledge in the art. Part of it.

For the purposes of this specification, it will be clearly understood that the term "comprising" means "including but not limited to" and the term "comprises" has a corresponding meaning.

All technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the invention pertains, unless otherwise defined. A person skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which can be used to practice the invention. Of course, the invention is in no way limited by the methods and materials described.

It is known that the mycelium of Antrodia camphorata has anti-inflammatory and vasodilating effects, cytotoxicity against various cancer cell lines, and anti-B hepatitis virus activity, while the filtrate of the fermentation culture of Antrodia camphorata has anti-carbon tetrachloride- Hepatic protective effects and antioxidant properties of induced hepatotoxicity. In order to develop a drug which can be used for the treatment of pain and inflammation, the inventors attempted to find an effective active ingredient having analgesic and anti-inflammatory activity from a soaking culture of Antrodia camphorata. Therefore, the inventors selected the ergosporin-7,9(11),22 E -triene-3β-ol previously isolated from the immersion culture of Antrodia camphorata for in vivo analgesia test. [Including acetic acid-induced writhing test and formalin-induced paw licking test]. The acetic acid-induced writhing test is a method used to assess whether a test substance has a peripheral analgesia effect, and the formalin-induced foot sputum test is a method of injecting fumarin to a mouse. The soles of the feet mimic human clinical pain conditions and are in the 0 to 5 minute period after injection [referred to as early phase, which can reflect neurogenic pain] and 15th A period of 40 minutes [referred to as late phase, which reflects inflammatory pain] to observe the time of the paw of the mouse, thereby confirming a test object for neuropathic pain and inflammatory Whether pain has an analgesic effect. It was confirmed by experimental results that ergot -7,9(11), 22 E -triene-3β-ol has a potency for alleviating inflammatory pain, and thus is expected to have great potential for treating pain. Thus, the present invention discloses the use of ergot-7,9(11),22 E -triene-3β-ol for the preparation of a medicament for the treatment of pain.

Accordingly, the present invention provides a pharmaceutical composition for treating pain comprising ergosta-7,9(11),22 E -triene-3β-ol.

As used herein, "treating" or "treatment" means reducing, alleviating, ameliorating, relieving, or controlling a disease ( One or more clinical signs of disease or disorder, and lowering, stopping, or reversing a condition or symptom that is being treated. The progress of severity.

The pharmaceutical composition according to the present invention can be manufactured into a dosage form suitable for parenterally, topically or orally, using techniques well known to those skilled in the art, including , but not limited to, an injection [eg, a sterile aqueous solution or dispersion], a sterile powder, a tablet, a troche, a pellet, a capsule, and the like. Similar things.

The pharmaceutical composition according to the present invention may be administered by a parenteral route selected from the group consisting of: intraperitoneal injection, subcutaneous injection, intramuscular injection. Intravenous injection, sublingual administration, and transdermal administration.

In a preferred embodiment of the invention, the pharmaceutical composition is formulated for administration by intraperitoneal injection.

In another preferred embodiment of the invention, the pharmaceutical composition is formulated into a dosage form suitable for oral administration.

The pharmaceutical composition according to the present invention may further comprise a pharmaceutically acceptable carrier which is widely used in pharmaceutical manufacturing techniques. For example, the pharmaceutically acceptable carrier can comprise one or more agents selected from the group consisting of solvents, emulsifiers, suspending agents, decomposers, and binding agents. Agent), excipient, stabilizing agent, chelating agent, diluent, gelling agent, preservative, lubricant, Absorption delaying agents, liposomes, and the like.

In a preferred embodiment of the present invention, the pharmaceutical composition comprises a pharmaceutically acceptable solvent, and the solvent is any one of the following: a sugar-containing solution, containing an alcohol [such as ethanol, propanediol, glycerin And an aqueous solution of mannitol, etc., oil [such as peanut oil, olive oil, sesame oil, castor oil, cottonseed oil, and Soybean oil, etc.], glycerin, organic solvents, and liposomes.

The invention also provides a method for treating a subject with pain comprising administering to the individual ergosta-7,9(11),22 E -triene-3β-ol.

In addition, the inventors took ergosin-7,9(11),22 E -triene-3β-ol for carrageenan-induced paw edema test, and evaluated mai Whether or not keratin-7,9(11),22 E -triene-3β-alcohol has anti-inflammatory activity can effectively inhibit acute inflammation caused by carrageenan injection. It was confirmed by experimental results that ergot -7,9(11), 22 E -triene-3β-alcohol has an anti-inflammatory effect. Thus, the present invention discloses the use of a pharmaceutical composition for the preparation of a medicament for the treatment of inflammation.

Accordingly, the present invention provides a pharmaceutical composition for treating inflammation comprising ergosta-7,9(11),22 E -triene-3β-ol.

In accordance with the present invention, the route of administration, dosage form, and pharmaceutically acceptable carrier for which the pharmaceutical composition is acceptable are as described above.

The invention also provides a method for treating an individual with or suspected of having an inflammation comprising administering to the individual ergosta-7,9(11),22 E -triene-3β-ol.

In the carrageenan-induced paw edema test, the inventors observed three antioxidant enzymes [including superoxide dismutase (superoxide dismutase) in the liver of mice after acute inflammation induced by carrageenan injection. The activity of SOD), catalase (CAT) and glutathione peroxidase (GPx) was increased, which showed that ergot-7,9(11),22 E -triene -3β-alcohol has the utility of improving liver damage. Therefore, the inventors selected mice with CCl ₄ -induced chemical liver injury as an animal model to evaluate ergot -7,9(11),22 E -triene. The utility of -3β-alcohol in improving liver damage. It was confirmed by experimental results that ergoside-7,9(11),22 E -triene-3β-alcohol can significantly improve the damage caused by carbon tetrachloride to the liver.

Based on the above, ergosta-7,9(11),22 E -triene-3β-ol is expected to have a high potential for treating liver damage. Accordingly, the present invention discloses ergosterol -7,9 (11), 22 E - trien-ol -3β- supply for therapeutic use for the preparation of a pharmaceutical composition of liver damage.

Accordingly, the present invention provides a pharmaceutical composition for treating liver damage comprising ergosta-7,9(11),22 E -triene-3β-ol.

As used herein, "liver injury" means any structural or functional damage to the liver caused directly or indirectly by one or more external or external factors and combinations thereof. Other factors include, but are not limited to, exposure to hepatotoxic compounds or radiation, mechanical damage, genetic predisposition, viral infections, and autoimmune diseases. (autoimmune diseases).

In a preferred embodiment of the invention, the liver injury is a chemical liver injury, a viral liver injury or an alcoholic liver injury. In a preferred embodiment of the invention, the liver injury is a chemical liver injury. In a more preferred embodiment of the invention, the chemical liver injury is carbon tetrachloride (CCl ₄ ) induced liver damage.

In accordance with the present invention, the route of administration, dosage form, and pharmaceutically acceptable carrier for which the pharmaceutical composition is acceptable are as described above.

The invention also provides a method for treating an individual with or suspected of having a liver injury comprising administering to the individual ergosta-7,9(11),22 E -triene-3β-ol.

Based on ergot 甾-7,9(11), 22 E -triene-3β-alcohol can significantly improve the damage caused by carbon tetrachloride to the liver, and the present invention also contemplates ergot -7,9(11) The 22 E -triene-3β-alcohol is supplied for the preparation of a composition for protecting the liver.

Accordingly, the present invention provides a composition for protecting a liver comprising ergoside-7,9(11),22 E -triene-3β-ol.

As used herein, "hepatoprotection" means protecting or preventing the liver and associated tissues [including, but not limited to, hepatic veins and portal veins] from damage.

According to the invention, the composition is manufactured in the form of, for example, a pharmaceutical or food product.

In accordance with the present invention, the pharmaceutical administration route, dosage form, and pharmaceutically acceptable carrier for use are as described above.

According to the present invention, the composition can be added as a food additive, added by a conventional method at the time of preparation of the raw material, or added during the production of the food, and matched with any edible material. Made into food products for human and non-human animals.

According to the present invention, the types of food products include, but are not limited to, milk powder, beverages, confectionery, candies, fermented foods, bakery products, Animal feeds, health foods, and dietary supplements.

The invention also provides a method for protecting a liver of a body comprising administering to the individual ergosta-7,9(11),22 E -triene-3β-ol.

Detailed description of the preferred embodiment

The invention is further described in the following examples, but it should be understood that these examples are for illustrative purposes only and are not to be construed as limiting.

Example Experimental Materials:

1. The freeze-dried powder of the submerged culture of Antrodia camphorata used in the following examples was developed by the Biotechnology Center of Grape. Provided by King, Inc.) (Taiwan, China), the production batch number is MZ-247.

2. The male imprinting control region (ICR) mice used in the examples below (6 to 8 weeks old, weighing approximately 18 to 25 g) were purchased from Lesco Biotech Co., Ltd. (BioLasco Taiwan Co., Ltd). All experimental animals were housed in plexiglass cages with light and dark for 12 hours, room temperature maintained at 22 ± 1 ° C, and relative humidity maintained at 55 ± 5%, and water and feed were adequately supplied. . Animals were given at least 2 weeks to adjust to the environment prior to the experiment. All experimental procedures for laboratory animals are based on the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals and are in compliance with the International Association for Pain Research (International Association). The guiding principles for the Study of Pain).

General experimental method : l. Preparation of general serum sample

The blood collected from the experimental animals was centrifuged at 3,000 rpm for 10 minutes at 4 ° C, and the obtained serum was used as a general serum sample and stored frozen at -80 ° C until use.

2. Preparation of tissue extract sample

The tissue obtained from the experimental animals was washed with cold normal saline and immediately transferred to an equal volume of cold physiological saline, followed by a homogenizer (Polytron PT-1200, at 4 ° C, Kinematica) was homogenized for 1 minute, and then homogenate formed by centrifugation at 12,000 xg at 4 °C for 5 minutes. Thereafter, the supernatant was collected, and the collected supernatant was added to physiological saline at a final concentration of 10% (v/v) and uniformly mixed, whereby a tissue extract sample was obtained for use.

3. Statistical analysis

In the following examples, the experimental data is expressed as a mean error of the mean (SEM). The difference in experimental data between the groups was first evaluated by one-way ANOVA followed by Scheffe's multiple range test. If the statistical analysis obtained is p < 0.05, representing statistical significance.

Example 1. Ergot 甾-7,9(11),22 derived from a soaking culture of Antrodia camphorata E -trien-3β-alcohol [ergosta-7,9(11),22 E Purification of -trien-3β-ol]

The ergoside-7,9(11),22 E -triene-3β-ol derived from the immersion culture of Antrodia camphora is referred to YI-YUAN SHAO et al . (2008), Natural Product Research , 22:1151 The method described in -1157 is carried out for purification. Briefly, a lyophilized powder of 1.6 kg of immersion culture of Antrodia camphorata was weighed, immersed in 16 L of methanol at room temperature and allowed to stand for 1 day, and then the methanol was evaporated under vacuum. A residue is obtained. This soaking-resting-evaporating step was repeated 3 times to obtain a methanol extract of a immersed culture of brown amaranth. The methanol extract of the obtained immersion culture of Antrodia camphorata was suspended in 1 L of sterilized water, followed by partitioning with water and ethyl acetate (ethyl acetate) for a total of three times. The obtained ethyl acetate fraction (EtOAc fraction) was chromatographed on silica gel using a mixed solution containing hexane (hexane) and ethyl acetate as an eluent, followed by use of 10% acetic acid. The ethyl ester (in hexane) was further purified as a mobile phase in high performance liquid chromatography (HPLC).

The ¹ H-NMR and ¹³ C-NMR spectra of the above purified compounds were detected using a Bruker DMX-400 spectrometer using a solvent of CDCl ₃ and a chemical shift (δ) in ppm. For the unit. The ¹ H-NMR and ¹³ C-NMR spectral data measured by this compound are summarized as follows:

¹ H NMR (CDCl ₃ , 400 MHz) δ: 0.61 (s, 3 H, CH ₃ ), 0.80 (d, J = 6.4 Hz, 3H, CH ₃ ), 0.82 (d, J = 6.4 Hz, 3H, CH ₃ ), 0.89 (d, J = 7.2 Hz, 3H, CH ₃ ), 0.92 (s, 3H, CH ₃ ), 1.01 (d, J = 6.4 Hz, 3H, CH ₃ ), 3.61 (m, 1H, CH) ), 5.14 (dd, J = 15.6, 7.2 Hz, 1H, CH), 5.20 (dd, J = 15.6, 8.0 Hz, 1H, CH), 5.35 (m, 1H, CH), 5.55 (dd, J = 5.6) , 2.0 Hz, CH).

¹³ C NMR (CDCl ₃ , 100 MHz) δ: 12.0, 16.3, 17.6, 19.6, 20.0, 21.1, 23.0, 28.3, 32.0, 33.1, 37.0, 38.4, 39.1, 40.4, 40.8, 42.8, 46.2, 54.6, 55.7, 70.5, 116.3, 119.6, 132.0, 135.6, 139.8, 141.3.

Based on the measured spectral data, the compound was confirmed to be a known compound having the following chemical structural formula [ie, ergosta-7, 9 (11), 22 E -triene-3β-ol]:

Example 2. Ergot 甾-7,9(11),22 E - In vivo analgesic effect of triene-3β-alcohol ( In vivo Analgesia effect)

In order to understand whether ergosta-7,9(11),22 E -triene-3β-alcohol has an effect of inhibiting pain in vivo, according to the ergosin-7,9 (11) obtained in the above Example 1. The 22 E -triene-3β-alcohol was subjected to the following acetic acid-induced writhing test and a formalin-induced paw licking test.

A, acetic acid-induced writhing test

Male ICR mice were randomly divided into 5 groups (n=6 per group), including 1 pathological control group, 1 positive control group, and 3 experimental groups (ie, experiments). Groups 1, 2 and 3). The pathological control group and the positive control group were administered with intraperitoneal injection of physiological saline and indomethacin (purchased from Sigma) (in physiological saline at a dose of 10 mg). /kg), while mice in the experimental group were administered via intraperitoneal injection of ergosta-7,9(11),22 E -triene-3β-ol [with carboxymethyl cellulose (CMC) In the medium], the doses of the experimental groups 1 to 3 were 1, 5, and 10 mg/kg, respectively. At the 25th minute after administration, 1% (v/v) acetic acid solution (Merck, Darmstadt, Germany) (dose of 10 mL/kg) was intraperitoneally injected into each group of mice. The number of writhings in the mice was recorded within 5 minutes from the injection of acetic acid. The experimental results obtained are shown in Figure 1.

As can be seen from Fig. 1, compared with the pathological control group, the number of writhings in the experimental groups 1 to 3 showed a decrease, and along with ergot -7, 9 (11), 22 E - triene - The increase in the dose of 3β-alcohol is more pronounced. The results of this experiment show that ergoside-7,9(11),22 E -triene-3β-alcohol achieves a peripheral analgesia effect in a dose-dependent manner.

B. Formalin-induced paw test

In this experiment, mice were grouped and administered according to the method described in item A above. At the 30th minute after administration, 20 μL of 5% fumarin (purchased from Biosource International, Inc.) was injected to the dorsal surface of the right hind paw of each group of mice, followed by the injection after the injection. The time taken by the mouse to the right hind paw was recorded during the period of 0 to 5 minutes (i.e., early) and during the period of 15 to 40 minutes (i.e., late). The experimental results obtained are shown in Figure 2.

As can be seen from Fig. 2, compared with the pathological control group, during the 0 to 5 minute period (early period) after the formalin injection, there was no significant difference in the sputum time of the experimental group 1 to 3 mice, but in Fuma. During the 15th to 40th minute period after the injection (late stage), the sputum time of the experimental group 1 to 3 mice decreased, and along with ergot -7, 9 (11), 22 E - The dose of triene-3β-alcohol is more pronounced and more pronounced. In particular, the experimental group 3 had less sputum time than the positive control group. The results showed: ergosterol -7,9 (11), 22 E - trien-ol -3β- a dose-dependent manner to inhibit inflammatory pain (inflammatory pain) utility.

Based on the results of the above acetic acid-induced writhing test and the fumarin-induced paw sputum test, the inventors believe that ergosta-7,9(11),22 E -triene-3β-alcohol is transmitted through the periphery. The effect of inhibiting inflammatory pain in vivo is achieved by a peripheral action.

Example 3. Ergot 甾-7,9(11),22 E In vivo anti-inflammatory effects of triene-3β-alcohol ( In vivo Anti-inflammatory effect

In order to investigate whether ergosta-7,9(11),22 E -triene-3β-ol has an anti-inflammatory effect in addition to analgesic effect in vivo, the ergot obtained according to the above Example 1 -7,9(11),22 E -triene-3β-ol was taken for the following carrageenan-induced paw edema test.

experimental method: A, carrageenan-induced foot edema test

Male ICR mice were randomly divided into 6 groups (n=6 per group), including 1 normal control group, 1 pathological control group, 1 positive control group, and 3 experimental groups (ie, Experimental groups 1, 2 and 3). In addition to the pathological control group, the normal control group and the positive control group were administered with physiological saline and indomethacin (dose of 10 mg/kg) via intraperitoneal injection, respectively, while the experimental group of mice passed through the peritoneum. The internal injection was administered with ergosta-7,9(11), 22 E -triene-3β-ol (in CMC), and the doses of the experimental groups 1 to 3 were 1, 5, and 10 mg/kg, respectively. .

At the 30th minute after administration, in addition to the normal control group, 50 μL of 1% carrageenan suspension (carrageenan was purchased from Sigma, in physiological saline) was injected to the right of the other groups of mice. The plantar surface of the hind paw induces acute inflammation, which causes the right hind paw of the mouse to edema. As for the normal control group, the sodium carrageenan suspension was replaced by physiological saline.

The right side of the mouse was measured using a plethysmometer (Cat. No. 7159, Ugo Basile, Varese, Italy) before the carrageenan injection and at 1, 2, 3, 4 and 5 hours after the injection. The volume of the hind foot. The volume of edema in the right hind paw of the mouse is expressed as the difference between the volume measured after the injection of carrageenan in the right hind paw of the mouse minus the difference measured before the carrageenan injection.

Next, blood was collected from the inferior vena cava of each group of mice by puncture and according to the method described in item 1 "Preparation of general serum samples" of "General Experimental Methods" above. General serum samples were prepared and the resulting general serum samples were taken for analysis of items B to C below.

In addition, each group of blood-collected mice was sacrificed and the liver and right hind paw were taken out respectively, wherein the liver was taken for analysis of the following D items, and the right hind paw was taken for analysis of the following E to G items.

B. Determination of serum nitrite concentration

Serum nitric oxide concentrations in each group of mice were determined indirectly by measuring the concentration of nitrite in the serum, and are generally referred to by Recknagel RO et al. (1991), Hepatotoxicology CRC Press, 401 The method described in -436 is performed. Briefly, take an appropriate amount of the general serum sample obtained in item A above and dilute it 5 times with distilled water, then add an appropriate amount of zinc sulfate to a final concentration of 15 g/L and mix well, then Centrifuge at 10,000 xg for 5 minutes at room temperature. The supernatant was collected, and the supernatant was separately added to each well of a microplate in a volume of 100 μL per well. Next, 100 μL of Griess reagent [containing 1% sulfanilamide and 0.1% N-naphthylethylenediamine dihydrochloride) was added to each well. 2.5% in polyphosphoric acid] and the reaction was carried out at room temperature for 10 minutes. Thereafter, the absorbance (OD ₅₄₀ ) of each well at a wavelength of 540 nm was read with a micro reader (Molecular Devices, Orleans Drive, Sunnyvale, CA). The resulting OD ₅₄₀ values were converted to nitrite concentrations (μM) based on a standard curve previously prepared with sodium nitrite (NaNO ₂ ) having different known concentrations relative to their own OD ₅₄₀ values.

C, determination of serum TNF-α concentration

Serum TNF-[alpha] concentrations for each group of mice were determined using a BioSource ^(TM) TNF-[alpha] ELISA kit (Cat. No. KMC3011, Invitrogen). The absorbance values measured for each group were converted to TNF-α concentrations according to a standard curve previously prepared with respect to their own absorbance values of TNF-α standards (Camarillo, CA) having different known concentrations. Pg/mL).

D. Determination of antioxidant enzyme activity in mouse liver tissue

In this experiment, the inventors selected superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) as indicators of antioxidant enzyme activity. , to assess whether ergot -7,9 (11), 22 E -triene-3β-alcohol can prevent the active oxygen species (ROS) and free radicals from being activated by increasing antioxidant enzyme activity during acute inflammation. It is produced to reduce oxidative damage to the liver.

First, the mouse liver obtained in the above item A is subjected to the preparation of the tissue extract sample according to the method described in the second item "Preparation of the tissue extract sample" of the "General Experimental Method" above, and the obtained tissue extract is obtained. The samples were taken for the following enzyme activity assays.

The determination of total superoxide dismutase (SOD) activity is carried out by the method described in Flohe L. and Otting F. (1984), Methods Enzymol. , 105: 93-104. Briefly, take 10 μL of the tissue extract sample and add 3 mL of Tris-HCl buffer (pH 8.2) and 7 μL of pyrogallol. Immediately after mixing, use a spectrophotometer at 25 ° C (Beckman DU530). The absorbance at a wavelength of 325 nm was read at a time interval of 15 seconds for 1 minute. In the present assay, one unit of superoxide dismutase activity is defined as the amount of SOD required to reduce the autooxidation rate of gallicol by up to 50%, and the value obtained is U/mg protein. To represent.

The determination of total catalase (CAT) activity is carried out by the method described in Aebi H. (1984), Methods Enzymol ., 105: 121-126. Briefly, take an appropriate amount of tissue extract sample and dilute it 1000 times with 50 mM potassium phosphate buffer, then take 2 mL of the diluted tissue extract sample and 1 mL of hydrogen peroxide (30 mM) and mix well, immediately after The absorbance at a wavelength of 240 nm was read at 25 ° C for 1 minute using a spectrophotometer at a time interval of 15 seconds. In the present assay, one unit of catalase activity is defined as the number of nanomoles (nmol) of hydrogen peroxide consumed per minute, and the values obtained are expressed in U/mg protein.

The determination of total glutathione peroxidase (GPx) activity is carried out by the method described in Paglia ED and Valentine WN (1967), J. Lab . Clin . Med ., 70: 158-169. Briefly, 200 μL of glutathione reductase (GR) (5 U/mL), 50 μL of GSH (40 mM), 620 μL of potassium phosphate buffer (0.25 M, pH 7.4) and 100 μL of the tissue extract sample diluted 20 times with phosphoric acid solution and mixed well. Next, freshly prepared 10 μL of NADPH (20 mM) and 20 μL of cumene hydroperoxide (15 mM) were added sequentially, followed by a spectrophotometer at 25 ° C for 15 seconds. The interval was taken to read the absorbance at a wavelength of 340 nm for 1 minute. In the present assay, one unit of glutathione peroxidase activity is defined as the number of nanomolecules (nmol) of NADPH oxidized per minute, and the values obtained are expressed in U/mg protein.

E. Determination of malondialdehyde (MDA) concentration in mouse paw tissue

Malondialdehyde (MDA) is the main product of oxidation of polyunsaturated fatty acids, and the concentration of malondialdehyde can be used to assess the extent of oxidative damage. The determination of the concentration of malondialdehyde was carried out in accordance with the method described in Tatum VL and Chow CK (1996), Free Radical Research , 25: 133-139. Briefly, each group of one of the soles obtained from item A above was prepared according to the second item "Preparation of tissue extract samples" of the "General Experimental Method" above, and then 0.4 was taken. mL tissue extract sample was added with 0.4 mL thioarbituric acid reagent (TBA reagent) [containing 0.4% thiobarbituric acid and 0.2% butylated hydroxytoluene (BHT)] and mixed Uniform, then placed in a 90 ° C water bath for 45 minutes. After the reaction was cooled to room temperature, an equal volume of n-butanol was added and mixed well, and the resulting mixture was centrifuged at 3,000 rpm for 10 minutes. Thereafter, a portion of the supernatant was collected and the BCA Protein Assay Kit (Pierce) was used to determine the protein content of the supernatant, while the remaining supernatant was used as a VERSAmax microplate. The absorbance (OD ₅₃₅ ) was measured at a wavelength of 535 nm by a reader (VERSAmax microplate reader, Molecular Devices).

On the other hand, solutions of 1,1,3,3-tetraethoxypropane (TEP) having different known concentrations were taken for the same experiment and based on the concentration and A standard curve is made corresponding to the absorbance value (OD ₅₃₅ ). The absorbance (OD ₅₃₅ ) measured by each group is converted into the malondialdehyde concentration (nmol) by the standard curve, and divided by the corresponding protein content, thereby calculating the C content per mg of protein. Dialdehyde concentration (nmol/mg protein).

F, Western blot analysis of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in mouse paw tissue.

Each group of the soles obtained from item A above is generally prepared according to the second item "Preparation of tissue extract samples" in the "General Experimental Method" above, except that: one is used. Containing 10 mM 3-[(3-cholestyryl)dimethylammonio]-1-propane-sulfonate {3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate}, 1 Replacement of mM methylsulfonyl fluoride, 5 μg/mL aprotinin, 1 μM aprotinin (pepstatin), and 10 μM leupeptin lysis buffer Physiological saline solution.

Tissue extract samples from each group were subjected to SDS-PAGE analysis using a mini vertical electrophoresis tank (Bio-Rad, USA), followed by mouse anti-iNOS monoclonal antibody and mouse anti-COX-2 monoclonal antibody ( Both were purchased from Santa Cruz, CA) as a primary antibody (500-fold diluted with 5% skim milk), and goat anti-mouse IgG conjugated with horseradish catalase (purchased from Sigma) as a secondary antibody (secondary antibody) (to be diluted with 5% skim milk to 2,000-fold), and using ECL reagent and Hyperfilm ^TM (are all purchased from Amersham International plc.) Western blot analysis is performed. Finally, a gel imaging system (gel logic 1500, Kodak) was used for photography.

In addition, β-actin was used as an internal control and an anti-β-actin antibody (sc-47778) (Santa Cruz, CA) was used as a primary control. Antibody (diluted 2000 times with 5% skim milk) and goat anti-mouse IgG antibody conjugated with horseradish catalase as a secondary antibody (1000-fold diluted with 5% skim milk) The same experiment.

The resulting images were analyzed using Kodak Molecular Imaging Software (version 4.0.5) (Eastman Kodak Company, Rochester, NY), whereby the protein band was able to be calculated semi-quantitatively. Corresponding protein expression, protein expression is normalized by the corresponding amount of β-actin protein expression, and then the normalized protein expression of the pathological control group is taken as 100%. The percentage of protein expression of the other groups relative to the pathological control group (% pathological control group) was calculated.

G, histopathological examination (histopathological examination)

The remaining paws of each group were immersed in a fixing solution [containing 1.85% formaldehyde (formaldehyde) and 1% acetic acid] at room temperature and fixed for 1 week. The fixed mouse paws were subjected to ethanol dehydration treatment, then embedded in paraffin (Sherwood Medical), and sectioned to obtain sections having a thickness of 5 μm. The dewaxed sections were stained with hematoxylin-eosin and then visualized using a light microscope (ECLIPSE, TS100, Nikon) at a magnification of 100X and using a digit Take a photo with the camera (NIS-Elements D2.30, SP4, Build 387).

In addition, the counting of the neutrophils was performed at a magnification of 400X. The count for the neutrophil was randomly selected from each group of 3-5 sections, and each section was selected to have 5 fields of view. The experimental data is expressed as the average of the number of neutrophils counted in all fields of view.

result: A, volume change of mouse edema sole

Figure 3 shows that mice were dosed with different doses of ergosta-7,9(11),22 E -triene-3β-ol via an intraperitoneal injection prior to the carrageenan-induced paw edema test, in carrageen A volumetric analyzer was used to measure the change in edema volume of the right hind paw of the mouse before the gel injection (i.e., the 0th hour) and at 1, 2, 3, 4, and 5 hours after the injection.

As can be seen from Fig. 3, compared with the pathological control group, there was no significant difference in the edema volume of the paws of the experimental groups 1 to 3 at 1, 2, and 3 hours after the carrageenan injection, but after the carrageenan injection. At 4 and 5 hours, the volume of the edema of the feet of the experimental groups 2 and 3 decreased, and the dose of ergoside-7,9(11),22 E -triene-3β-alcohol increased. More obvious. The results of this experiment show that ergot -7,9(11), 22 E -triene-3β-alcohol has the effect of improving the edema of the foot.

B. Determination of serum nitrite concentration

Figure 4 shows mice administered different doses of ergosta-7,9(11),22 E -triene-3β-ol via intraperitoneal injection prior to the carrageenan-induced paw edema test, in carrageen Serum nitrite concentration measured at 5 hours after gel injection.

As can be seen from Fig. 4, compared with the pathological control group, the serum nitrite concentrations in the experimental groups 1 to 3 all decreased, and along with the ergot -7, 9 (11), 22 E - three The dose of ene-3β-alcohol is more pronounced and more pronounced. In particular, the serum nitrite concentration of the experimental group 3 was almost the same as that of the positive control group. The results of this experiment show that ergot -7,9(11),22 E -triene-3β-ol inhibits nitric oxide production in a dose-dependent manner.

C, determination of serum TNF-α concentration

Figure 5 shows that mice were dosed with different doses of ergosta-7,9(11),22 E -triene-3β-ol via an intraperitoneal injection prior to the carrageenan-induced paw edema test, in carrageen Serum TNF-α concentration measured at 5 hours after gel injection.

As can be seen from Fig. 5, the serum TNF-α concentrations of the experimental groups 2 and 3 were significantly lowered as compared with the pathological control group. The results of this experiment show that ergosta-7,9(11),22 E -triene-3β-alcohol has the effect of lowering the serum TNF-α concentration.

D. Determination of antioxidant enzyme activity in mouse liver tissue

The results of assays for the activity of superoxide dismutase, catalase, and glutathione peroxidase in mouse liver tissues are summarized in Table 1 below.

As can be seen from Table 1, the superoxide dismutase, catalase and glutathione peroxidase activities of the pathological control group were reduced as compared with the normal control group. Compared with the pathological control group, the activities of superoxide dismutase, catalase and glutathione peroxidase in the positive control group and the experimental group 3 were significantly increased ( p <0.01). The results of this experiment show that ergot-7,9(11),22 E -triene-3β-alcohol has superoxide dismutase, catalase and glutathione peroxidase in the liver of mice. The utility of the activity.

E. Determination of malondialdehyde concentration in mouse paw tissue

Figure 6 shows that mice were administered different doses of ergosta-7,9(11),22 E -triene-3β-ol via intraperitoneal injection prior to the carrageenan-induced paw edema test, in carrageen The malondialdehyde concentration measured on the right hind paw tissue was collected at the 5th hour after the gel injection.

As can be seen from Fig. 6, compared with the pathological control group, the malondialdehyde concentrations in the experimental groups 1 to 3 all decreased, and along with the ergot -7,9(11),22 E -triene The increase in the dose of -3β-alcohol is more pronounced. In particular, the malondialdehyde concentration of the experimental group 3 was lower than that of the positive control group. The results showed: ergosterol -7,9 (11), 22 E - trien-ol -3β- a dose - dependent manner to reduce oxidative damage tissue at the foot edema.

F. Western blot analysis of iNOS and COX-2 expression in mouse paw tissue

Figure 7 shows the carrageenan - induced paw edema test prior via intraperitoneal injection to mice administered ergosterol -7,9 (11), 22 E - triene -3β- alcohol, after carrageenan injection At the 5th hour, the right hind paw tissue was collected for Western blot analysis and the results of iNOS and COX-2 measurements were measured.

As can be seen from Fig. 7, compared with the pathological control group (the protein expression of the pathological control group was regarded as 100%), the iNOS and COX-2 expressions of the positive and the experimental group 3 mice were significantly decreased. In the case, in particular, the iNOS and COX-2 expression amounts of the experimental group 3 were lower than those of the positive control group, respectively. The results of this experiment show that ergot -7,9(11), 22 E -triene-3β-alcohol has the utility of reducing iNOS and COX-2 expression in edema soles.

G, histopathological examination

FIG 8 is a slice staining pattern, which is displayed in carageenan - via intraperitoneal injection to mice administered steroid ergosterol-7,9 (11) prior to foot edema test induced, 22 E - triene -3β- alcohol, And the results of hematoxylin-eosin staining of the right hind paw slice obtained at the 5th hour after the carrageenan injection. Fig. 9 is a result of neutrophil counting by selecting five fields of view based on the result of the staining of Fig. 8.

As can be seen from Fig. 8, compared with the normal control group (A region), the section of the pathological control group (B area) showed moderate extravascular red blood cells and a large number of neutrophils. Infiltrating hemorrhage. Compared with the pathological control group, the sections of the positive control group (C area) and the experimental group 3 (D area) showed a decrease in the inflammatory response. As can be seen from Fig. 9, in the positive control group and the experimental group 3 mice, the average number of neutrophils per field of view was significantly reduced to 40 or less. In particular, in the experimental group 3, the average number of neutrophils per field of view was lower than that of the positive control group. The results of this experiment show that ergot -7,9(11), 22 E -triene-3β-alcohol has the effect of reducing neutrophil infiltration and reducing inflammation.

Based on the above experimental results, the inventors believe that the anti-inflammatory effect of ergosta-7,9(11),22 E -triene-3β-alcohol may be through the enhancement of superoxide dismutase and hydrogen peroxide in the liver. The enzyme and glutathione peroxidase activity reduce the expression of malondialdehyde, iNOS and COX-2 in the edema of the foot by inhibiting the expression of TNF-α and nitric oxide.

In addition, the inventors noted that ergosta-7,9(11),22 E -triene-3β-ol can increase superoxide dismutase and catalase in the carrageenan-induced edema foot test. And the activity of three antioxidant enzymes, glutathione peroxidase. The inventors believe that ergot -7,9(11),22 E -triene-3β-alcohol has an effect of improving liver damage, and is used for preparing a medicament for treating liver damage and a composition for protecting the liver. There should be great potential. Thus, in the following embodiments, the inventors selected with carbon tetrachloride - mice induced chemical liver injury (CCl ₄ -induced chemical liver injury) as an animal model to evaluate ergosterol 7,9 (11) The utility of 22 E -triene-3β-alcohol in the treatment of liver damage and protection of the liver.

Example 4. Ergot 甾-7,9(11),22 E -Triene-3β-alcohol for the evaluation of chemical liver injury A, with CCl ₄ Induced chemical liver injury

Male ICR mice were randomly divided into 6 groups (n=6 per group), including 1 normal control group, 1 pathological control group, 1 positive control group and 3 experimental groups (ie, experimental group 1, 2). And 3). The mice in the normal control group and the pathological control group were administered with physiological saline (dose at a dose of 1.5 mL/kg) via intraperitoneal injection, while the mice in the positive control group and the experimental group were administered via the intraperitoneal injection to the milk thistle. (silymarin) (with a dose of 25 mg/kg in 1% CMC) and ergosta-7,9(11), 22 E -triene-3β-ol (in 1% CMC, the doses are 2.5, 5 and 10 mg/kg), once a day for 7 days.

In the first hour after the last injection, in addition to the normal control group, 20% CCl ₄ (1.5 mL/kg) in olive oil was intraperitoneally injected into mice of other groups. , to induce chemical liver damage. As for the normal control group, physiological saline was used instead of CCl ₄ .

Thereafter, the mice were anesthetized with diethyl ether at 24 hours after the injection of CCl ₄ , and blood was collected from the carotid arteries of the mice by puncture. A part of the blood was centrifuged at 1,700 xg for 30 minutes at 4 ° C, and the obtained serum sample was taken for the analysis of the following item B , and the rest of the blood was in accordance with the above-mentioned "general experimental method" item 1 "general serum Preparation of a general serum sample by the method described in the preparation of the sample, and the obtained general serum sample was taken for analysis of items C to D below.

In addition, each group of blood-collected mice was sacrificed and the liver was taken out, and the obtained liver was divided into two parts, some of which were taken for analysis of the following E to H items, and the other part of the liver was taken for the following. Analysis of item I.

B, serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activity determination

The determination of alanine transaminase and aspartate transaminase activity in the serum samples obtained in item A above was carried out by the Easy-Check Clinical Laboratory. The experimental results obtained are shown in Figures 10 to 11.

As can be seen from Fig. 10 and Fig. 11, the serum alanine transaminase and aspartate transaminase activities of the pathological control group were significantly increased after CCl ₄ injection as compared with the normal control group. Compared with the pathological control group, the serum alanine transaminase and aspartate transaminase activities in the positive control group and the experimental group 1 to 3 were significantly reduced, in particular, the experimental group 1 to 3 would With the increase in the dose of ergosta-7,9(11), 22 E -triene-3β-ol, it became more apparent. The results of this experiment show that ergosta-7,9(11),22 E -triene-3β-ol prevents serum alanine transaminase and aspartate transaminase activity in a dose-dependent manner. The rise.

C, determination of serum nitrite concentration

In this experiment, the serum nitrite concentration was measured in accordance with the method described in "B, Measurement of serum nitrite concentration" in Example 3 above. The experimental results obtained are shown in Fig. 12.

As can be seen from Fig. 12, the serum nitrite concentration of the pathological control group was significantly increased as compared with the normal control group. Compared with the pathological control group, the serum nitrite concentrations in the experimental groups 1 to 3 showed a decrease, and along with the ergot -7,9 (11), 22 E -triene-3β- The increase in alcohol dose is more pronounced. In particular, the serum nitrite concentration of the experimental group 3 was lower than that of the positive control group. The results of this experiment show that ergot -7,9(11),22 E -triene-3β-ol inhibits nitric oxide production in a dose-dependent manner.

D, determination of serum TNF-α concentration

In the present experiment, the serum TNF-α concentration was measured in accordance with the method described in "C, Measurement of serum TNF-α concentration" in Example 3 above. The experimental results obtained are shown in Fig. 13.

As can be seen from Fig. 13, the serum TNF-α concentration of the pathological control group was significantly increased as compared with the normal control group. Compared with the pathological control group, the serum TNF-α concentrations in the experimental groups 1 to 3 showed a decrease, and along with the ergot -7,9 (11), 22 E -triene-3β- The increase in alcohol dose is more pronounced. In particular, the serum TNF-α concentration of the experimental group 3 was lower than that of the positive control group. The results of this experiment show that ergot -7,9(11),22 E -triene-3β-ol reduces serum TNF-α concentration in a dose-dependent manner.

E. Determination of antioxidant enzyme activity in mouse liver tissue

In this experiment, superoxide dismutase, catalase, and glutathione peroxidase were carried out in accordance with the method described in "D, Determination of Antioxidant Enzyme Activity in Mouse Liver Tissue" in Example 3 above. Activity assay. The experimental results obtained are shown in Figures 14 to 16.

As can be seen from Figures 14 to 16, after the injection of CCl ₄ , the superoxide dismutase, catalase and glutathione peroxidase activities of the pathological control group were significantly reduced compared with the normal control group. . Compared with the pathological control group, the activities of superoxide dismutase, catalase and glutathione peroxidase in the experimental group 1 to 3 were significantly improved, and along with the ergot -7 , 9(11), 22 E -triene-3β-alcohol increased the dose more obviously. In particular, the superoxide dismutase, catalase, and glutathione peroxidase activities of the experimental group 3 were higher than those of the positive control group, respectively. The results of this experiment show that ergoside-7,9(11),22 E -triene-3β-alcohol has the utility of increasing superoxide dismutase, catalase and glutathione peroxidase activity.

F, Determination of malondialdehyde concentration in mouse liver tissue

In the present experiment, the concentration of malondialdehyde was measured in accordance with the method described in "E, Measurement of Malondialdehyde Concentration in Mouse Foot Tissue" in Example 3 above. The experimental results obtained are shown in Fig. 17.

As can be seen from Fig. 17, compared with the pathological control group, the malondialdehyde concentrations in the experimental groups 1 to 3 all decreased, and along with the ergot -7, 9 (11), 22 E - triene The increase in the dose of -3β-alcohol is more pronounced. In particular, the malondialdehyde concentration of the experimental group 3 was lower than that of the positive control group. The results of this experiment show that ergoside-7,9(11),22 E -triene-3β-ol reduces the degree of oxidative damage in liver tissue in a dose-dependent manner.

G, Western blots of iNOS and COX-2 expression in mouse liver tissue Point analysis

In the present experiment, the expression of iNOS and COX-2 was analyzed in accordance with the method described in "F, Western blot analysis of iNOS and COX-2 expression in mouse paw tissue" in Example 3 above. The experimental results obtained are shown in Fig. 18.

As can be seen from Fig. 18, the iNOS and COX-2 expression levels of the pathological control group were significantly increased as compared with the normal control group (the protein expression amount of the normal control group was regarded as 100%). Compared with the pathological control group, the iNOS and COX-2 expressions in the positive control group and the experimental group 3 all showed a significant decrease. In particular, the iNOS and COX-2 expressions in the experimental group 3 were respectively higher than those in the experimental group. The positive control group is still low. The results of this experiment show that for mice with chemical liver injury, ergosta-7,9(11),22 E -triene-3β-alcohol has reduced iNOS and COX-2 expression in liver tissue. The utility.

H, Determination of glutathione concentration in mouse liver tissue

First, the mouse paws of each group obtained in the above item A are generally prepared according to the second item "Preparation of tissue extract samples" in the "General Experimental Method" above, and the preparation of the tissue extract samples is different. The point is that 200 mM Tris-HCl (pH 7.2) was used instead of physiological saline.

The measurement of the concentration of glutathione in the liver tissue was carried out in accordance with the method described in Ellman, GL (1959), Archives of Biochemistry and Biophysics , 82: 70-77 with a slight modification. Briefly, 720 μL of Tris-HCl buffer (200 mM, pH 7.2) was added to the tissue extract sample, followed by the addition of 160 μL of 5% trichloroacetic acid (TCA) and mixed well. The resulting mixture was centrifuged at 10,000 xg for 5 minutes at 4 °C. 330 μL of the supernatant was mixed well with 660 μL of Ellman's reagent, and then the absorbance (OD ₄₀₅ ) at a wavelength of 405 nm was read with a VERSA max microplate reader.

The OD ₄₀₅ values measured for each group were converted to glutathione concentrations (nmol) based on correlation curves previously obtained with glutathione values of different known concentrations relative to their own OD ₄₀₅ values, respectively. The glutathione concentration (nmol/mg protein) per mg of protein in each group was calculated by dividing the total amount of protein measured by each group according to item F above. The experimental results obtained are shown in Fig. 19.

As can be seen from Fig. 19, the GSH concentration of the pathological control group was significantly lowered as compared with the normal control group. Compared with the pathological control group, the glutathione concentrations in the experimental group 1 to 3 mice were significantly increased, and along with the ergot -7,9(11),22 E -triene The increase in the dose of -3β-alcohol is more pronounced. In particular, the glutathione concentrations of the experimental groups 2 to 3 were higher than those of the positive control group. The results of this experiment show that ergot -7,9(11),22 E -triene-3β-alcohol increases the glutathione concentration of liver tissue in a dose-dependent manner and reduces oxidative stress.

I, histopathological examination

This experiment was carried out in accordance with the method described in "G, Histopathology Test" of Example 3 above, except that observation and photographing were performed at a magnification of 200X. The results taken are shown in FIG.

As can be seen from Figure 20, in the pathological control group (Z), CCl ₄ can induce histological changes including increased degeneration, necrosis, hepatitis, and portal triaditis. . In addition to the normal control group, all groups exhibited ballooning degeneration and hepatocyte necrosis in the central centrolobular zone. Compared with the pathological control group, the mice in the experimental group 1 to 3 had less degree of ballooning degradation and hepatocyte necrosis (D area ~ F area). The results of this experiment show that ergot -7,9(11), 22 E -triene-3β-alcohol has the effect of improving chemical liver damage.

Based on the above experimental results, the inventors believe that ergosta-7,9(11),22 E -triene-3β-ol can inhibit lipid peroxidation and increase antioxidant enzyme superoxide dismutase in the liver. Hydrogenase and glutathione peroxidase activity, decreased serum concentrations of TNF-α and nitric oxide, and decreased inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) The amount of performance protects the liver from chemical liver damage through anti-inflammatory effects, which in turn has the effect of treating liver damage and protecting the liver.

All of the patents and documents cited in this specification are hereby incorporated by reference in their entirety. In the event of a conflict, the detailed description of the case (including definitions) will prevail.

While the invention has been described with respect to the specific embodiments of the invention, it will be understood that many modifications and changes can be made without departing from the scope and spirit of the invention. It is therefore intended that the invention be limited only by the scope of the appended claims.

1 shows in acetic acid - induced writhing test prior via intraperitoneal injection in mice administered various doses of ergosterol -7,9 (11), 22 E - triene -3β- alcohol, acetic acid and injected starting from Results of the number of writhing in mice recorded within 5 minutes, in which the pathological control group indicated mice induced pain by acetic acid; the positive control group indicated that mice treated with acetic acid-induced pain were treated with 10 mg/kg 吲哚美辛辛Experimental groups 1 to 3 represent mice treated with acetic acid-induced pain at 1, 5 and 10 mg/kg ergosta-7, 9 (11), 22 E -trien-3β-ol, respectively; ",""**" and "***" indicate p < 0.05, p < 0.01, and p < 0.001, respectively, compared with the pathological control group;

Figure 2 shows that mice were dosed with different doses of ergosta-7,9(11),22 E -triene-3β-ol via intraperitoneal injection prior to the formalin-induced paw sputum test, and The time of the right hind paw of the mouse recorded during the 0th to 5th minute period (early period) and the 15th to 40th minute period (late stage) after the formalin injection, wherein the pathological control group was treated with physiological saline solution Mice with pain induced by formalin; positive control group showed that mice treated with formalin-induced pain were treated with 10 mg/kg of indomethacin; groups 1 to 3 showed 1, 5 and 10 mg/kg, respectively. ergosterol -7,9 (11), 22 E - trien-ol -3β- processed formalin-induced pain in mice; and "*", "**" and "***" respectively represents p < 0.05, p <0.01 and p <0.001, compared with the pathological control group;

Figure 3 shows that mice were dosed with different doses of ergosta-7,9(11),22 E -triene-3β-ol via an intraperitoneal injection prior to the carrageenan-induced paw edema test, in carrageen The edema volume change of the right hind paw of the mouse was measured before the gel injection (0 hour) and at 1, 2, 3, 4 and 5 hours after the injection, and the volume of the edema of the mouse foot was small. The volume of the right hind paw of the rat after the injection of the carrageenan suspension was subtracted from the difference measured before the injection of the carrageenan suspension. The pathological control group indicated that the antler was treated with physiological saline. The mice in the control group induced edema of the foot; the positive control group indicated that the mice with edema induced by the carrageenan were treated with 10 mg/kg 吲哚美辛辛; the experimental groups 1 to 3 showed 1, 5 and 10 mg, respectively. /kg ergoside-7,9(11),22 E -triene-3β-ol to treat mice with edema induced by carrageenan; and “*”, “**” and “***” P < 0.05, p < 0.01 and p < 0.001, respectively, compared with the pathological control group;

Figure 4 shows mice administered different doses of ergosta-7,9(11),22 E -triene-3β-ol via intraperitoneal injection prior to the carrageenan-induced paw edema test, in carrageen The serum nitrite concentration measured at the 5th hour after the injection, wherein the normal control group indicated that the male ICR mice were treated with physiological saline; the pathological control group indicated that the sodium edema induced by the carrageenan was treated with physiological saline. The mice in the positive control group indicated that mice treated with carrageenan-induced edema of the foot were treated with 10 mg/kg of saponin; the experimental groups 1 to 3 showed ergot at 1, 5 and 10 mg/kg, respectively. -7,9(11),22 E -triene-3β-ol to treat mice with edema induced by carrageenan; "^###" means p <0.001 compared with normal control group; and "* *" and "***" indicate p < 0.01 and p < 0.001, respectively, compared with the pathological control group;

Figure 5 shows that mice were dosed with different doses of ergosta-7,9(11),22 E -triene-3β-ol via an intraperitoneal injection prior to the carrageenan-induced paw edema test, in carrageen The serum TNF-α concentration measured at the 5th hour after the injection, wherein the normal control group indicated that the male ICR mice were treated with physiological saline; the pathological control group indicated that the sodium edema induced by the carrageenan was treated with physiological saline. The mice in the positive control group indicated that mice treated with carrageenan-induced edema of the foot were treated with 10 mg/kg of saponin; the experimental groups 1 to 3 showed ergot at 1, 5 and 10 mg/kg, respectively. -7,9(11),22 E -triene-3β-ol to treat mice with edema induced by carrageenan; "^###" means p <0.001 compared with normal control group; and "* *" and "***" indicate p < 0.01 and p < 0.001, respectively, compared with the pathological control group;

Figure 6 shows that mice were administered different doses of ergosta-7,9(11),22 E -triene-3β-ol via intraperitoneal injection prior to the carrageenan-induced paw edema test, in carrageen The concentration of malondialdehyde measured in the right hind paw tissue was collected at the 5th hour after the gel injection, wherein the normal control group indicated that the male ICR mice were treated with physiological saline; the pathological control group indicated that the processed carrageen was treated with physiological saline. Mice inducing foot edema in the sac; the positive control group indicated that mice treated with carrageenan induced foot edema were treated with 10 mg/kg 吲哚美辛辛; experimental groups 1 to 3 indicated 1, 5 and 10 mg/ Kg ergoside-7,9(11),22 E -triene-3β-ol to treat mice with edema induced by carrageenan; "^###" means p <0.001 compared with normal control group ; and "*", "**", and "***" indicate p < 0.05, p < 0.01, and p < 0.001, respectively, compared with the pathological control group;

Figure 7 shows the carrageenan - induced paw edema test prior via intraperitoneal injection to mice administered ergosterol -7,9 (11), 22 E - triene -3β- alcohol, after carrageenan injection At the 5th hour, the right hind paw tissue was collected for Western blot analysis and the results of iNOS and COX-2 were measured. The normal control group indicated that the male ICR mice were treated with physiological saline; the pathological control group indicated that the physiological saline was used. Water was used to treat mice with edema induced by carrageenan; positive control group showed that mice treated with carrageenan induced foot edema were treated with 10 mg/kg 吲哚美辛辛; experimental group 3 indicated 10 mg/kg ergosterol -7,9 (11), 22 E - trien -3β- ol treated mice induced by carageenan paw edema; ^"###" represents p <0.001, compared with normal control group; And "**" and "***" respectively indicate p < 0.01 and p < 0.001, compared with the pathological control group;

FIG 8 is a slice staining pattern, which is displayed in carageenan - via intraperitoneal injection to mice administered steroid ergosterol-7,9 (11) prior to foot edema test induced, 22 E - triene -3β- alcohol, And the results of hematoxylin-eosin staining of the right hind paw slice obtained at the 5th hour after the injection of carrageenan, wherein the area A indicates the normal control group of male ICR mice treated with physiological saline; the B area indicates The pathological control group of mice treated with carrageenan induced foot edema was treated with physiological saline; the C area showed positive control group of mice treated with carrageenan-induced foot edema with 10 mg/kg 吲哚美辛辛. And the D region represents the experimental group 3 of mice treated with carrageenan-induced paw edema with 10 mg/kg ergos-7,9(11), 22 E -triene-3β-ol;

Figure 9 is a result of neutrophil counts based on the results of the staining of Figure 8, optionally with five fields of view, wherein the normal control group indicated that male ICR mice were treated with physiological saline; the pathological control group indicated that saline was used. Treatment of mice with edema induced by carrageenan; positive control group indicated that mice treated with carrageenan induced foot edema were treated with 10 mg/kg 吲哚美辛辛; experimental group 3 indicated ergot at 10 mg/kg steroid -7,9 (11), 22 E - trien -3β- ol treated mice induced by carageenan paw edema; ^"###" represents p <0.001, compared with normal control group; and "**" and "***" indicate p < 0.01 and p < 0.001, respectively, compared with the pathological control group;

Figure 10 shows that mice were administered different doses of ergosta-7,9(11),22 E -triene-3β- via intraperitoneal injection prior to induction of chemical liver injury with carbon tetrachloride (CCl ₄ ). Alcohol, serum alanine aminotransferase (ALT) activity measured at 24 hours after CCl ₄ injection, in which the normal control group indicated that male ICR mice were treated with physiological saline; the pathological control group indicated The mice with chemical liver injury induced by CCl ₄ were treated with physiological saline; the positive control group was treated with 25 mg/kg silymarin to treat mice with chemical liver injury induced by CCl ₄ ; the experimental groups 1 to 3 showed Mice with chemically induced liver injury induced by CCl ₄ were treated with 2.5, 5, and 10 mg/kg ergot -7,9(11), 22 E -triene-3β-ol, respectively; “ ^### ” indicates p < 0.001, compared with the normal control group; and "*", "**" and "***" indicate p < 0.05, p < 0.01 and p < 0.001, respectively, compared with the pathological control group;

Figure 11 shows that mice were administered different doses of ergosta-7,9(11),22 E -triene-3β-ol via intraperitoneal injection prior to induction of chemical liver injury with CCl ₄ in CCl ₄ injection. The serum aspartate aminotransferase (AST) activity measured at the 24th hour thereafter, in which the normal control group indicated that male ICR mice were treated with physiological saline; the pathological control group indicated that it was treated with physiological saline. ₄ induced chemical liver injury in mice treated with CCl; positive control represented at 25 mg / kg of silymarin treated mice induced by CCl _4, chemical liver injury; 1-3 represents the experimental group, respectively of 2.5, 5 And 10 mg/kg ergosta-7,9(11),22 E -triene-3β-ol to treat mice induced by CCl ₄ induced chemical liver injury; “ ^### ” means p <0.001, and Comparison with the normal control group; and "**" and "***" indicate p < 0.01 and p < 0.001, respectively, compared with the pathological control group;

Figure 12 shows in CCl ₄ via intraperitoneal injection in mice administered various doses of chemical liver injury induced before ergosterol -7,9 (11), 22 E - triene -3β- alcohol in CCl ₄ injection The serum nitrite concentration measured at the 24th hour thereafter, in which the normal control group indicated that the male ICR mice were treated with physiological saline; the pathological control group indicated that the physiological saline solution was used to treat the chemical liver injury induced by CCl ₄ . Mice; the positive control group indicated that mice treated with CCl ₄ induced chemical liver injury were treated with 25 mg/kg silymarin; the experimental groups 1 to 3 showed 2.5, 5 and 10 mg/kg ergosin-7, respectively. , 9(11), 22 E -triene-3β-alcohol to treat mice with chemical liver injury induced by CCl ₄ ; “ ^### ” means p <0.001 compared with normal control group; and “*” "**" and "***" indicate p < 0.05, p < 0.01 and p < 0.001, respectively, compared with the pathological control group;

Figure 13 shows in CCl ₄ via intraperitoneal injection in mice administered various doses of chemical liver injury induced before ergosterol -7,9 (11), 22 E - triene -3β- alcohol in CCl ₄ injection The serum TNF-α concentration measured at the 24th hour thereafter, in which the normal control group indicated that the male ICR mice were treated with physiological saline; the pathological control group indicated that the physiological saline solution was used to treat the chemical liver injury induced by CCl ₄ . Mice; the positive control group indicated that mice treated with CCl ₄ induced chemical liver injury were treated with 25 mg/kg silymarin; the experimental groups 1 to 3 showed 2.5, 5 and 10 mg/kg ergosin-7, respectively. , 9(11), 22 E -triene-3β-alcohol to treat mice induced by CCl ₄ induced chemical liver injury; “ ^### ” means p <0.001 compared with normal control group; and “** "" and "***" indicate p < 0.01 and p < 0.001, respectively, compared with the pathological control group;

Figure 14 shows that mice were administered different doses of ergosta-7,9(11),22 E -triene-3β-ol via intraperitoneal injection prior to induction of chemical liver injury with CCl ₄ in CCl ₄ injection. The superoxide dismutase activity measured by liver tissue was collected at 24 hours thereafter. The normal control group indicated that male ICR mice were treated with physiological saline; the pathological control group indicated that CCl ₄ induced chemistry was treated with physiological saline. Mice with liver injury; positive control group indicated that mice treated with CCl ₄ induced chemical liver injury were treated with 25 mg/kg silymarin; group 1 to 3 showed 2.5, 5 and 10 mg/kg wheat, respectively. angle steroid -7,9 (11), 22 E - trien -3β- ol induced mice treated with CCl ₄ chemical liver injury; ^"###" represents p <0.001, compared with normal control group; And "*", "**" and "***" respectively indicate p < 0.05, p < 0.01 and p < 0.001, compared with the pathological control group;

Figure 15 shows prior to CCl ₄ induced chemical liver injury in mice via intraperitoneal injection of different doses administered ergosterol -7,9 (11), 22 E - triene -3β- alcohol in CCl ₄ injection At the 24th hour thereafter, the catalase activity measured by liver tissue was collected. The normal control group indicated that the male ICR mice were treated with physiological saline; the pathological control group indicated that the CCl ₄ induced chemical treatment was treated with physiological saline. Liver-injured mice; positive control group indicated that mice treated with CCl ₄ induced chemical liver injury were treated with 25 mg/kg silymarin; experimental groups 1 to 3 showed ergot at 2.5, 5, and 10 mg/kg, respectively.甾-7,9(11),22 E -triene-3β-ol was used to treat mice with chemical liver injury induced by CCl ₄ ; “ ^### ” indicates p <0.001 compared with the normal control group; "*", "**" and "***" indicate p < 0.05, p < 0.01 and p < 0.001, respectively, compared with the pathological control group;

Figure 16 shows that mice were administered different doses of ergosta-7,9(11),22 E -triene-3β-ol via intraperitoneal injection prior to induction of chemical liver injury with CCl ₄ in CCl ₄ injection. The glutathione peroxidase activity measured by liver tissue was collected at 24 hours thereafter. The normal control group indicated that male ICR mice were treated with physiological saline; the pathological control group indicated that CCl _{4 was} treated with physiological saline. Mice that induced chemical liver injury; positive control group indicated that mice treated with CCl ₄ induced chemical liver injury were treated with 25 mg/kg silymarin; and experimental groups 1 to 3 showed 2.5, 5, and 10 mg, respectively. /kg ergoside-7,9(11),22 E -triene-3β-ol to treat mice with chemical liver injury induced by CCl ₄ ; "^###" means p <0.001, compared with normal control group Comparing; and "*", "**", and "***" indicate p < 0.05, p < 0.01, and p < 0.001, respectively, compared with the pathological control group;

Figure 17 shows that mice were administered different doses of ergosta-7,9(11),22 E -triene-3β-ol via intraperitoneal injection prior to induction of chemical liver injury with CCl ₄ in CCl ₄ injection. The concentration of malondialdehyde measured by liver tissue was collected at the 24th hour thereafter. The normal control group indicated that the male ICR mice were treated with physiological saline; the pathological control group indicated that the chemical liver was treated with CCl ₄ by physiological saline. Injured mice; positive control group indicated that mice treated with CCl ₄ induced chemical liver injury were treated with 25 mg/kg silymarin; experimental groups 1 to 3 showed ergot at 2.5, 5 and 10 mg/kg, respectively. -7,9(11),22 E -triene-3β-ol was used to treat mice with chemical liver injury induced by CCl ₄ ; “ ^### ” indicates p <0.001 compared with the normal control group; and “ *", "**" and "***" indicate p < 0.05, p < 0.01 and p < 0.001, respectively, compared with the pathological control group;

Figure 18 shows prior to CCl ₄ induced chemical liver injury in mice via intraperitoneal injection administered ergosterol -7,9 (11), 22 E - triene -3β- alcohol, CCl ₄ after the first injection The results of analysis of iNOS and COX-2 measured by Western blot analysis were collected for 24 hours. The normal control group indicated that male ICR mice were treated with physiological saline; the pathological control group was treated with physiological saline. mice induced by CCl _4, chemical liver injury; positive control represented at 25 mg / kg of silymarin treated mice induced by CCl _4, chemical liver injury; 3 experimental groups expressed at 10 mg / kg ergosterol -7,9(11),22 E -triene-3β-ol was used to treat mice with chemical liver injury induced by CCl ₄ ; “ ^### ” indicates p <0.001 compared with the normal control group; and “ **" and "***" indicate p < 0.01 and p < 0.001, respectively, compared with the pathological control group;

19 shows in CCl ₄ via intraperitoneal injection in mice administered various doses of chemical liver injury induced before ergosterol -7,9 (11), 22 E - triene -3β- alcohol in CCl ₄ injection The glutathione concentration measured by liver tissue was collected at 24 hours thereafter. The normal control group indicated that male ICR mice were treated with physiological saline; the pathological control group indicated that CCl ₄ induced chemical treatment with physiological saline. Liver-injured mice; positive control group indicated that mice treated with CCl ₄ induced chemical liver injury were treated with 25 mg/kg silymarin; experimental groups 1 to 3 showed ergot at 2.5, 5, and 10 mg/kg, respectively.甾-7,9(11),22 E -triene-3β-ol was used to treat mice with chemical liver injury induced by CCl ₄ ; “ ^### ” indicates p <0.001 compared with the normal control group; "*", "**", and "***" indicate p < 0.05, p < 0.01, and p < 0.001, respectively, compared with the pathological control group;

Figure 20 is a section staining diagram showing administration of different doses of ergosta-7,9(11),22 E -triene-3β to mice by intraperitoneal injection prior to induction of chemical liver injury with CCl _{4 .} - alcohol, hematoxylin-eosin staining of the foot slice obtained at the 24th hour after CCl ₄ injection, wherein area A represents the normal control group of male ICR mice treated with physiological saline; Physiological saline was used to treat the pathological control group of mice induced by CCl ₄ induced chemical liver injury; C area showed positive control of mice treated with CCl ₄ induced chemical liver injury with 10 mL/kg 吲哚美辛辛Groups; and D to F regions represent small, chemically induced liver injury induced by CCl ₄ at 2.5, 5, and 10 mg/kg ergosta-7,9(11),22 E -triene-3β-ol, respectively. Rat experimental groups 1 to 3.

Claims (27)

A pharmaceutical composition for treating pain comprising ergoside-7,9(11),22 E -triene-3β-ol. For example, the pharmaceutical composition of claim 1 is in a dosage form for oral administration. A pharmaceutical composition according to claim 1, which is in a form for parenteral administration. A ergot 甾-7,9(11),22 E -triene-3β-ol is used for the preparation of a medicament for the treatment of pain. The use of the fourth aspect of the patent application, wherein the pharmaceutical product is in a dosage form for oral administration. The use of claim 4, wherein the pharmaceutical product is in a form for parenteral administration. A pharmaceutical composition for treating inflammation comprising ergosta-7,9(11),22 E -triene-3β-ol. A pharmaceutical composition according to claim 7 of the patent application, which is in the form of a dosage form for oral administration. A pharmaceutical composition according to claim 7 of the patent application, which is in a form for parenteral administration. A ergot 甾-7,9(11),22 E -triene-3β-ol is supplied for the preparation of a medicament for the treatment of inflammation. The use of claim 10, wherein the pharmaceutical product is in a dosage form for oral administration. The use of claim 10, wherein the pharmaceutical product is in a form for parenteral administration. A pharmaceutical composition for treating liver damage comprising ergoside-7,9(11),22 E -triene-3β-ol. The pharmaceutical composition of claim 13, wherein the liver damage is chemical liver damage. The pharmaceutical composition of claim 14, wherein the chemical liver damage is a carbon tetrachloride-induced chemical liver injury. A pharmaceutical composition according to claim 13 of the patent application, which is in the form of a dosage form for oral administration. A pharmaceutical composition according to claim 13 of the patent application, which is in a form for parenteral administration. A ergot 甾-7,9(11),22 E -triene-3β-ol is used for the preparation of a medicament for treating liver damage. The use of claim 18, wherein the pharmaceutical product is in a dosage form for oral administration. The use of claim 18, wherein the pharmaceutical product is in a form for parenteral administration. A composition for protecting the liver comprising ergoside-7,9(11),22 E -triene-3β-ol. As in the composition of claim 21, it can be manufactured in the form of a pharmaceutical or food product. The composition of claim 22, wherein the pharmaceutical product is in a dosage form for oral administration. The composition of claim 22, wherein the pharmaceutical product is in a form for parenteral administration. An ergot 甾-7,9(11),22 E -triene-3β-ol is used for the preparation of a pharmaceutical or food product for protecting the liver. The use of claim 25, wherein the pharmaceutical product is in a dosage form for oral administration. The use of claim 25, wherein the pharmaceutical product is in a form for parenteral administration.