CN1216701A - New use of sinomenine - Google Patents

Abstract

The present invention discloses a new pharmaceutical application of sinomenine and its medicinal salt, i. e. application of sinomenine in the preparation of medicine with the functions of scavenging free radical, resisting lipid peroxidation and protecting activity of antioxidase.

Description

New application of sinomenine

The invention relates to a new pharmaceutical application of sinomenine, in particular to an application of sinomenine in preparing a medicament with the effects of scavenging free radicals and resisting lipid peroxidation, belonging to the field of medicaments.

Sinomenine (Sinomenine) is one of alkaloids extracted from stems and roots of Sinomenium acutum (Thunb.) r.etwils, a plant of the tetrandra family, and is a known medicinal substance. Modern pharmacological research shows that sinomenine has various pharmacological effects. In summary, the main pharmacological actions of sinomenine known so far are as follows: antiinflammatory, blood pressure lowering, central nervous system inhibiting, and arrhythmia resisting effects; has inhibitory action on non-specific immunity, cellular immunity and humoral immunity of organism, especially has stronger inhibitory action on the function of T lymphocyte [ Penhuimin, etc., the immunosuppressive action of sinomenine, Chinese pharmacology declaration 1988,9(4)377-380]; sinomenine is also a histamine releasing agent (von Jing Yi, et al, pharmacological action VII of Sinomenine. Effect on gastrointestinal activity and its mechanism, pharmaceutical Proc 1965; 12: 492); clinically, the traditional Chinese medicine composition has obvious curative effect on treating rheumatoid arthritis (Zhang Xin, et al, Shanxi traditional Chinese medicine, 1980,5: 13; scientific research group of traditional Chinese medicine institute in Shupu county, Hunan province, J Chizhi doctor, 1975,12: 612); the mechanism of action of sinomenine in the treatment of rheumatoid arthritis has also been studied in some cases [ Li Xinjiang et al, influence of sinomenine on immune function of mice, Chinese herbal medicine, 1992,23(2):81-83]. To date, there is no report that sinomenine has the effects of scavenging free radicals and resisting lipid peroxidation.

The present inventors have confirmed the above pharmacological actions through experimental studies, and have completed the present invention.

Therefore, the invention aims to provide the application of sinomenine in preparing medicaments for eliminating free radicals and resisting lipid peroxidation.

The experimental research shows that the sinomenine, especially sinomenine hydrochloride, has the functions of inhibiting lipid peroxidation, protecting the activity of antioxidant enzyme and eliminating free radicals.

Sinomenine is sold as a therapeutic drug, for example, Sinomenine (SIN) produced by pharmaceutical factories in the area of Qian Yang in Hunan province, sinomenine hydrochloride powder produced by pharmaceutical factories linked in Wuhan City, etc.

Therefore, sinomenine as a medicine for eliminating free radicals and resisting lipid peroxidation can be prepared into any pharmaceutical dosage form suitable for clinical use according to the conventional preparation method, such as tablets, pills, capsules, granules, oral liquid, emulsion, suspension, injection, suppository, paste and the like.

Besides the sinomenine, the pharmaceutically acceptable salts of sinomenine, such as sinomenine hydrochloride, also have the effects of scavenging free radicals and resisting lipid peroxidation, so that the sinomenine and the pharmaceutically acceptable salts thereof are used for preparing medicaments for scavenging free radicals and resisting lipid peroxidation and belong to the protection scope of the invention.

When the sinomenine and the pharmaceutically acceptable salt thereof are used for preparing medicaments for scavenging free radicals and resisting lipid peroxidation, pharmaceutically common excipients, such as a binding agent, a disintegrating agent, a filling agent, a lubricating agent, a solubilizer, a preservative, a flavoring agent, a solvent, an emulsifier and the like can be added.

The sinomenine and the pharmaceutically acceptable salts thereof can be prepared into medicaments together with other compatible therapeutic medicaments when being used for preparing the medicaments with the effects of scavenging free radicals and resisting lipid peroxidation.

Example 1

The sinomenine hydrochloride powder is 2500mg, purchased from Union pharmaceutical factories in Wuhan City, 250g of medicinal starch and a proper amount of alcohol are granulated, dried and tabletted by a tabletting machine to prepare 1000 tablets, wherein each tablet is 275mg in weight and contains 25mg of sinomenine hydrochloride.

Example 2

20000mg of sinomenine and 180g of starch are fully mixed and encapsulated to prepare 2000 capsules, each capsule is 0.1g in weight and contains 10mg of sinomenine hydrochloride.

Experimental example pharmacological experimental study on scavenging free radicals and resisting lipid peroxidation of sinomenine

Materials and methods

1. Reagent and animal

Tetraethoxypropane (1,1,3, 3-tetraethoxypropane TEP) was purchased from FIuka corporation; o-dianisidine (O-dianisidine) was purchased from Huamei corporation; reduced glutathione (GSH glutathione), reduced coenzyme II (NADPH), glutathione reductase (glutathione reductase) and sodium azide (N)_aN_s) All purchased from Sigma. The rest reagents are all domestic analytical purifiers. Sinomenine hydrochloride is provided for the purification of the national key laboratory of Bei Yi Dabionic and natural medicine.

The experimental animal is a Wistar pure line rat provided by the department of animals in the institute, and the male animal is healthy and has no eye diseases, and the weight of the male animal is about 150 g. Inducing animal autoimmune retinitis pigmentosa (EAU) membrane type rat with retina-free S antigen at different observation time points, immediately taking out eyeball after neck-breaking death, stripping off whole retina tissue under ice bath, and preparing tissue homogenate or storing in-80 deg.C refrigerator for use.

(ii) determination of retinal tissue conjugated diene

Reference is made to Buege et al for a slight modification. Retinal tissue was fractionated under a microscope, and prepared into 10% (W/V) homogenate using an ice-cold 0.05mol/L phosphate buffer pH7.2 (containing 0.5mmol/L EDTA) using a glass homogenizer and stored. To 3ml of a chloroform-methanol (1: 2V/V) mixture was added 0.2ml of the homogenate and the mixture was shaken for 2 minutes, and then to 1ml of the chloroform-methanol mixture was added and the mixture was shaken for 1 minute. Adding 1ml of acidified water (adjusting pH to 2.5 with 0.1N hydrochloric acid), shaking for 1 min, centrifuging at 2000 rpm for 10 min; taking out 1ml of subnatant, introducing nitrogen gas for drying, adding 1ml of ethanol, shaking uniformly, measuring OD value at 233nm, and using phosphate buffer as blank control.

(II) determination of malondialdehyde content

Taking 0.2ml of the homogenate, adding 0.2ml of 8.1% SDS solution, 1.5ml of 20% acetic acid solution and 1.5ml of 0.8% thiobarbituric acid solution, shaking uniformly for 5 minutes, placing in a water bath at 95 ℃, taking out after 1 hour, and placing in an ice bath to terminate the reaction; cooling, centrifuging at 2000 r/m for 5 min, adding 5ml of n-butyl pyridine, performing oscillation extraction for 2 min, centrifuging at 2000 r/m, and layering for 15 min; the n-butanol pyridine layer was aspirated, and the fluorescence intensity was measured on a fluorescence photometer, with an excitation light wavelength (Ex) of 515nm and an emission light wavelength (Em) of 553 nm. Simultaneously, the tetraethoxy propane solution of 1nmoL/L is taken as a standard. And calculating the content of malondialdehyde. Protein content was determined by the Bradford method (the same below).

(III) measurement of fluorescent chromolipid (fluorogenic lipid) content.

0.2ml of the homogenate was added to 3.5ml of a methanol-chloroform mixture (1: 2V/V), shaken for 5 minutes, and then added with 2ml of acidified water (as before). Shaking for 5 minutes; centrifuge at 2000 rpm for 5 minutes. Taking out the methanol aqueous phase layer after layering. The fluorescence intensity was measured, and the excitation light wavelength (Ex) was 360nm and the emission light wavelength (Ex) was 430 nm. Simultaneously, quinine sulfate solution (1ug/ml dissolved in 0.1N H)₂SO₄Middle) was used as a standard to calculate the color lipid content.

(IV) determination of lipid hydrogen perchlorides (lipid hydroperoxides) by the method of peter A. ward et al.

Taking 0.5ml of homogenate, adding 3.5ml of methanol-chloroform mixed solution (1: 2V/V), oscillating for 2 minutes, centrifuging for 2000 r/min, and centrifuging for 5 minutes; after layering, sucking 2ml of subnatant, introducing nitrogen to blow dry, then adding 1.0ml of acetic acid chloroform mixed solution (3: 2/V/V), adding 0.05ml of 12.8% potassium iodide solution under the condition of keeping out of the sun, oscillating for 5 minutes, adding 3.0ml of 0.5% cadmium acetate, and oscillating for 1 minute; centrifuging at 1000 rpm for 5 min, collecting supernatant, and measuring absorbance at 353 nm.

(V) measurement of myeloperoxidase Activity

Retinal tissue was homogenized at 10% (W/V) with 50mmol/L, pH6.0 potassium phosphate buffer (containing 0.5% cetyltrimethylammonium bromide) in a glass homogenizer and sonicated for 10 seconds in an ice bath. After repeated ice-melting 3 times, the cells were again sonicated for 10 seconds. Centrifuge at 4000g, 4 ℃ for 15 min. 0.2ml of the supernatant was added to 2.8ml of phosphate buffer (containing 0.167mg/ml o-dianisidine dihydrochloride and 0.005% hydrogen peroxide and shaken up, followed by continuous monitoring at 460nm, myeloperoxidase activity: 1. mu. mol of hydrogen peroxide consumed per minute at 25 ℃.

(VI) determination of superoxide dismutase Activity

Reference is made to dungbuy et al. Centrifuging the homogenate at 4000 rpm for 20 min, collecting supernatant as extractive solution, adding 4.5ml of 50mmol/L PH8.30K₂HPO₄Buffer, 10ul of 50mmol/L pyrogallol solution (freshly prepared with 10mmol/L HCI) was mixed rapidly and the change in absorbance at 325nm was measured continuously. The reaction was started by adding pyrogallol solution at a reaction temperature of 25 ℃ and a total reaction volume of 4.6 ml. 1 unit of enzyme activity is defined as the amount of enzyme that inhibits pyrogallol autoxidation by 50% per minute per ml of reaction solution at 25℃.

(VII) peroxidase Activity measurement

The principle is as follows: catalase can catalyze the peroxidation and decomposition, so that the content of hydrogen peroxide in the system is continuously reduced, the light absorption value of the hydrogen peroxide at 240nm is gradually reduced, and the activity of the catalase can be measured by utilizing the principle. The sample analysis solution contained 0.05mol/L Tris-HCI buffer solution, pH7.4, a certain amount of hydrogen peroxide and a proper amount of enzyme extract were added, the reaction volume was 1ml, and the change of absorbance at 240nm was continuously observed with the addition of the extract as the reaction start timing. 1 unit of enzyme activity is defined as the amount of enzyme required to catalyze 1u mol of substrate per minute at 37 ℃.

(VIII) measurement of glutathione peroxidase Activity

Reference is made to the method of conjugation of glutathione reductase by paglia et al. The sample analysis solution contains 0.05mol/L phosphate buffer solution (containing 3mmol/L EDTA), 10mmol/L reduced glutathione with pH7.2, 0.3mmol/L reduced coenzyme II, 1mmol/L sodium azide, 1 unit of glutathione reductase and a proper amount of enzyme extract. After mixing evenly, the temperature is kept at 30 ℃, peroxide butane is added until the final concentration is 1mmol/L, and the total volume of the reaction is 1 ml. The reaction was started with the addition of tert-butane peroxide and the change in absorbance at 340nm was measured. 1 unit of enzyme activity is defined as the amount of enzyme required to oxidize 1umol of reduced coenzyme II per minute at 30 ℃.

Second, result in

(ii) effects of sinomenine treatment on retinal lipid peroxidation product levels in EAU rats.

1. Conjugated diene: as shown in Table1-1, the conjugated diene level in the retina was significantly increased by about 4.2 times that of normal rats on the ninth day after S antigen immunization, about 4.8 times that of normal rats on the fourteenth day, and increased to 6.8 times that of normal rats by twenty-one day. Therefore, the level of the compound is in an ascending trend in the observation period, and has very significant difference compared with a normal group, wherein P is less than 0.01; after the sinomenine treatment, the level of the conjugated diene of the retina is reduced by about 8.9 percent on the ninth day and 12.8 percent on the fourteenth day, but compared with the control group, the level of the conjugated diene is not statistically significant, the level of the conjugated diene is reduced more obviously on the twenty-first day after the treatment, about 19.8 percent, statistically significant difference is achieved, and P is less than 0.05. The results show that in the EAU pathological changes of rats, the retinal conjugated diene level is increased, and the sinomenine treatment can inhibit the increase of the retinal conjugated diene level of the EAU rats.

2. Malondialdehyde: as can be seen from tables 1-2, changes in retinal Malondialdehyde (MDA) levels in EAU rats were in synchrony with conjugated dienes, all in an increasing trend. On day 9, 2.27 times that of the normal group, on day 14, 3.28 times that of the normal group, and by day 21, it increased to 3.93 times that of the normal group. Compared with the normal group, the gene has very statistically significant difference, and P is less than 0.01. After the sinomenine treatment, the MDA level of the retina is reduced to different degrees compared with that of a control group. About 8.4% at day 9, about 41.2% at day 14, and about 45.8% at day 21. The results of the comparison between the treated group and the control group were statistically very significant in all groups except the group on day 9, and P was<0.01. The above results indicate that retinal malondialdehyde levels are elevated in the EAU lesions in rats, and that sinomenine treatment can inhibit the extent of elevation of retinal malondialdehyde levels in EAU rats.

3. Fluorescent color lipid: the changes in fluorescent lipid content are shown in tables 1-3, from which it can be seen that the increase in fluorescent lipid content in the retina of EAU rats lags in time with the changes in conjugated diene and MDA, the control group was increased from the normal group on days 9 and 14, but there was no statistically significant difference, and P was>0.05. At day 21 of EAU, the content of EAU was significantly increased, about 7.3 times that of the normal group, and the comparison results of the two groups were statistically very significant, with p<0.01. After sinomenine treatment, the fluorescent lipids of the retina of EAU rats were reduced to different degrees compared with the control group, respectively 12% (day 9), 6.3% (day 14) and 51.0% (day 21). The results of the comparison between the two groups showed that the groups on day 9 and 14 had no statistically significant difference, P was greater than 0.05, and the group on day 21 had a statistically significant difference, P was less than 0.01. The results show that in the EAU pathological changes of rats, the retinal fluorescent color lipid is not obviously changed for 2 weeks, and then the retinal fluorescent color lipid is obviously increased, and the sinomenine treatment can inhibit the increase of the retinal fluorescent color lipid content of the EAU rats.

4. Lipid hydroperoxides: the changes in lipid hydroperoxide levels, unlike the first three products, are shown in tables 1-4 as a significant increase at day 9, approximately 2.49 times normal, and at days 14 and 21, 2.03 and 1.84 times normal, respectively, with statistically significant differences between the two, P>0.05. After sinomenine treatment, the lipid hydroperoxide levels in each time group were reduced to some extent, 19.7% (day 9), 25.2% (day 14) and 5.3% (day 21), respectively, but there were no statistically significant differences, with P<0.05. The results show that the retinal lipid hydroperoxide level is increased in the EAU pathological changes of rats, and the increase degree of the retinal lipid hydroperoxide level of EAU rats after the sinomenine treatment is reduced, but no significant difference exists in statistics.

(II) Effect of Sinomenine treatment on the levels of retinal-associated enzymes in EAU rats

1. Myeloperoxidase: as can be seen from tables 1-5, the retinal myeloperoxidase activity of EAU rats increased continuously within 14 days after immunization, with the increase being most pronounced within the second week and then gradually decreased. After the sinomenine treatment, the activity of myeloperoxidase is reduced to different degrees compared with that of a control group, wherein the activity is reduced by 9.7 percent on the 9 th day, 36.5 percent on the 14 th day and 17.1 percent on the 21 st day. The results of the comparison between the two groups showed that the group on day 14 had statistically significant differences, P was less than 0.01, and the other two groups did not reach the degree of statistically significant differences, P was greater than 0.05. The results show that the activity of retinal myeloperoxidase is increased in the course of EAU lesion of rats, and the peak is at 14 days, and the increase degree of the activity of the retinal myeloperoxidase can be reduced by using the sinomenine treatment.

2. Superoxide dismutase: changes in the activity of EAU rat retinal superoxide dismutase SDD are shown in tables 1-6. On day 9 after immunization, superoxide dismutase activity increased approximately 1.29-fold as compared to normal. The decrease was then most pronounced at day 14, with an activity of only 46.2% of normal. On day 21, the activity was slightly higher than that of the previous day, and was 69.5% of the normal value. After sinomenine treatment, there was no significant change at day 9 compared to the control group. The ratio of the ratio. The results show that the activity of the retinal superoxide dismutase is increased temporarily and then decreased in the process of EAU lesion of rats, and the decrease degree of the activity of the retinal superoxide dismutase is inhibited 2 to 3 weeks after the sinomenine treatment.

3. Catalase: the results are shown in tables 1 to 7. It was found that the catalase activity of the retina of the EAU rat was decreased with time by 22.4% (day 9), 45.6% (day 14) and 52.0% (day 21), respectively, as compared with the normal group. The catalase activity of the latter was higher than that of the treated group at each time point, and the comparison results were statistically significant at day 14 and day 21, with p<0.01 and p<0.05. The above results indicate that the retinal catalase activity decreases during the course of EAU lesions in rats. Sinomenine treatment can reduce the degree of decrease in enzyme activity.

4. Glutathione peroxidase.

Changes in retinal glutathione peroxidase activity in EAU rats are shown in tables 1-8. Unlike the change in catalase activity, the activity did not change much in the early stage of observation (days 9 and 14) compared with the normal control group, but changed significantly by day 21, and decreased by about 20.9% compared with the normal control group, but the statistical difference was not significant, and p was greater than 0.05. The trend of change in glutathione peroxidase activity was similar in the sinomenine-treated group to that in the untreated group. Although the enzyme activity was slightly higher at each time point than in the untreated group, there was no statistically significant difference, p>0.05. The experimental result shows that the activity of the retinal glutathione peroxidase of the EAU rat is not obviously changed in the early observation period and is reduced in the third week. The sinomenine treatment group has no obvious difference from the non-treatment group.

TABLE 1-1 Change in conjugated diene in retinas of EAU rats in the Sinomenine-treated and untreated groups

CD X + -SD (nmol/mg protein) group n untreated group 86.19 + -0.525.63 + -0.95 day 9, 87.10 + -0.406.19 + -1.25 day 21, 810.04 + -3.108.06 + -1.15 normal group (4):1.47 + -0.36^*P<0.05 TABLE 1-2 changes in MDA in retinas of sinomenine-treated and untreated EAU rats

MDA X + -SD (nmol/mg protein) group n untreated group treatment group 80.427 + -0.0350.391 + -0.042 day 9, 80.617 + -0.1020.361 + -0.033 day 21, 80.738 + -0.1050.400 + -0.065 day normal group (4):0.188 + -0.013^**:p＜0.01TABLE 1-3 Fluorochrome in retinas of EAU rats in the Sinomenine-treated and untreated groupsChanges in lipids

FL X + -SD (ug/mg protein) group n untreated group treatment group 80.025 + -0.0070.022 + -0.006 day 9, 80.016 + -0.0050.015 + -0.005 day 21, 80.102 + -0.0350.050 + -0.013 + -normal group (4):0.014 + -0.004^**P<0.01 Table 1-4 changes in lipid hydroperoxides in retinas of sinomenine-treated and untreated groups of EAU rats

LH X + -SD (nmol/mg protein) group n untreated group n treated group 40.386 + -0.1000.310 + -0.120 day 9, 40.314 + -0.1400.235 + -0.085 day 21, 40.285 + -0.0840.270 + -0.064 Normal group (4) 0.155 + -0.046 myeloperoxidase activity in retina of EAU rat in Table 1-5 sinomenine treated group

Myeloperoxidase activity (u/mg protein) group n untreated group treatment group 60.031 ± 0.0060.028 ± 0.005 day 9 60.115 ± 0.0150.073 ± 0.007 day 21 60.041 ± 0.0040.034 ± 0.004^**P<0.01 Table 1-6 SOD activity in retinas of rats of sinomenine-treated group and untreated group EAU

SOD activity (u/mg protein) (X + -SD) group n untreated group 79.92 + -0.829.80 + -0.74 at day 9, 73.56 + -0.934.83 + -0.77 at day 14, 75.36 + -1.696.27 + -1.54 at day 21, normal group (4) 7.71 + -0.25

^*P<0.05 Table 1-7 Catharase Activity in retina of Sinomenine-treated and untreated groups of EAU rats

Catalase (u/mg protein)

(X + -SD) group n untreated group on day 9 80.097 + -0.0270.109 + -0.014 on day 14 80.068 + -0.0060.099 + -0.016 on day 21 80.060 + -0.0060.076 + -0.006 normal group (4) 0.125 + -0.016^*:p＜0.05^**P<0.01 Table 1-8 glutathione peroxidase Activity in retinas of Sinomenine-treated and untreated groups of EAU rats

Glutathione peroxidase Activity (u/mg protein)

(X + -SD) group n untreated group on day 9 40.315 + -0.0310.324 + -0.030 on day 14 40.307 + -0.0140.314 + -0.050 on day 21 40.261 + -0.0410.274 + -0.039 Normal group (4):0.330 + -0.041, discussion

In this experiment, the conjugated dienes, malondialdehyde, lipid hydroperoxides and fluorescent lipids observed were considered to be products of different stages of the lipid peroxidation process.

During the initial phase of lipid peroxidation, free radicals abstract hydrogen from unsaturated fatty acids, causing the formation of conjugated dienes (which have a characteristic absorption peak at 233 nm), which in turn react with oxygen to form lipid peroxides which are further converted to endoperoxides. Lipid endoperoxides can react with a variety of compounds, including hydroxyls, to form alkyl radicals and lipid hydroperoxides, or to form propionaldehyde and malondialdehyde, which, in combination with the amino groups of phospholipids and aromatic anilines, and protein, can form conjugated schiff bases, which have exceptional fluorescence. In addition, hydroperoxides may react to form malondialdehyde.

Our experimental results show that various lipid peroxidation products are increased in different degrees in the retina of the S antigen-induced EAU rat, reflecting that the process of free radical-mediated lipid peroxidation is increased in the course of EAU, and the resulting injury effect is also increased.

Studies on the pathological damage mechanism of EAU have shown that, although T cells are a mediator of pathology, oxidative damage to the retina is polymorphonuclear leukocytes (PMNs)₈) Thereby, the effect is achieved. The content of polyunsaturated fatty acid (PUFA) in the retina photoreceptor cells is higher than that in any other organ tissue₈) And thus are most vulnerable to free radical mediated lipid peroxidation. In the acute phase of EAU, PMN₈Accumulated to the site of inflammation and activated, with a consequent respiratory burst (respiratory burst) process characterized by increased oxygen consumption, pentose bypass activation and release of reactive oxygen species. Wherein the active oxygen product is mainly superoxide anion free radical which can generate H through disproportionation process₂O₂Followed by a Haber-Weiss reaction: and a Featon reaction: generating a more toxic hydroxyl radical (. OH). These active oxygen species are increased in tissues, and may cause lipid peroxidation, and damage to proteins, DNA, and the like.

In recent years, when an EAU animal model is induced, BCG and Bordetella pertussis or Bordetella pertussis toxin are generally used as double adjuvants to immunize animals together with antigens. Experiments show that the infiltration degree of the P MNs of the animal model after double adjuvant immunization is increased, and the retinal lipid peroxidation damage is aggravated. The peak period superoxide anion free radical and hydroxyl free radical of the EAU are obviously increased by utilizing a chemical reflection method. It is thus believed that oxygen radicals play a major role in retinal oxidative damage, whether in the initial phase, or sustained period of EAU, and ultimately lead to retinal degeneration.

The results of this experiment show that the increase in retinal conjugated diene and malondialdehyde levels in EAU rats are essentially synchronized in time, whereas the increase in fluorescent lipid levels lags the first two and increases significantly after the second week. The increase in lipid hydroperoxide levels peaked shortly before the first week and then, although significantly higher than the normal control, tended to decline.

It is presumed from the process of lipid peroxidation that the fluorescent lipid is a product produced by the reaction of malondialdehyde with phospholipids, aromatic aniline and protein amino groups, and the reaction rate may be relatively slow, and the active metabolism of aldehydes in tissues may cause the delay of the fluorescent lipid.

The lipid hydroperoxides (LHOOH) produced during the initiation phase of lipid peroxidation can undergo fragmentation to produce alkoxy radicals (. LHO) and hydroxyl radicals (. OH): . In the extended stage of lipid peroxidation, ferrous ions (Ee)⁺⁺) Can catalyze LHOOH reaction to generate alkoxy free radical: . We therefore speculate that at some stage of EAU pathology the breakdown of LHOOH is greater than production, possibly resulting in a gradual decrease in its total content after the first week.

After the sinomenine treatment, the generation of lipid peroxides of the retina of EAU rats is inhibited to different degrees, and the infiltration degree of polymorphonuclear leukocytes is also reduced by pathological examination. From our previous in vitro experiments, it was found that sinomenine itself can scavenge superoxide anion radical and hydroxyl radical, and has anti-lipid peroxidation effect. Previous experiments have shown that oxidative damage to cell membranes, which may lead to calcium influx, activates phospholipases, leading to increased production of free arachidonic acid, which may be further converted to prostaglandins, and that lipid hydroperoxides can also lead to oxidation of arachidonic acid, both of which contribute to inflammatory cell chemokine production and exacerbate the degree of local inflammatory infiltration. Therefore, we believe that the action of sinomenine in alleviating the inflammatory response of EAU is related to its scavenging of free radicals against lipid peroxidation, thereby inhibiting inflammatory chemokine production.

Myeloperoxidase (MPO) has been used as a marker enzyme for neutrophils at a very high concentration in the neutrophil granules. In observations in this experiment, retinal MPO activity in EAU rats was already elevated at day 9 post immunization, peaked at day 14, and then declined, still higher at day 21 than at day 9. This result is similar to the experimental observations of goto.h et al.

It is well known that there are three mechanisms of protection against oxidative damage in normal ocular tissues: the first is an antioxidase system which can prevent the generation of active oxygen; second, a free radical scavenger; thirdly, the repair mechanism of oxidative damage. Among the three mechanisms, the first one is the antioxidase system, which mainly comprises: superoxide dismutase (SOD), catalase and peroxidase system.

SOD is considered to be the primary component of antioxidant enzyme systems, which is capable of specifically scavenging superoxide anion radicals (.O)₂). Catalase not only scavenges hydrogen peroxide (H)₂O₂) And can reduce the generation of hydroxyl radicals, glutathione peroxidase is a seleno-enzyme which can catalyze the reaction of glutathione with hydrogen peroxide and peroxide to eliminate superoxideToxic effects of the oxides.

In this experiment, we observed changes in SOD, catalase and glutathione peroxidase activities. The experimental results show that the activity of SOD firstly increases at the 9 th day after animal immunization, then rapidly decreases along the course of disease, the activity is reduced to the lowest point at the peak period (14 th day) of inflammation, and then increases. Experiments prove that the retinal SOD is mainly positioned in the inner segment of photoreceptor cells and retinal pigment epithelium, the parts are just the eye tissues mainly invaded by EAU inflammation, and the activity of the retinal SOD is far greater than that of crystalline lens. We therefore speculate that the reactivity of retinal SOD activity is increased in the early stages of EAU disease to eliminate the increasing amount of O₂When O is present₂When the production exceeds the SOD scavenging ability, the SOD activity is significantly reduced. In addition, it has been found that excessive hydrogen peroxide production in the tissue can cause Cu, an important prosthetic group of Cu-Zn SOD⁺⁺Thereby inhibiting the activity of SOD.

We performed on EAU rat retina catalase and cerealsThe result of the activity measurement of the caspase shows that the activity change of the caspase and the caspase is different from that of SOD, and the activity change of the caspase and the caspase always shows a descending trend in anobservation period, and the activity reduction of the catalase is more obvious. The decrease of the activity of the two will lead to excessive H in the tissue₂O₂And peroxides are not effectively scavenged. This not only directly leads to tissue damage, but more importantly to excessive H₂O₂Can react to generate more toxic hydroxyl free radical, thereby activating lipid peroxidation process and aggravating tissue injury.

After the sinomenine is used for treatment, the increase of myeloperoxidase activity reflecting the infiltration degree of neutrophils in tissues is inhibited, and the decrease degrees of SOD and catalase activity are obviously reduced. It is well known that the mechanisms of oxidation and antioxidation in the normal body maintain a dynamic balance. In the antioxidant system, the antioxidant enzyme system has the characteristics of rapid reaction and high activity, so the antioxidant enzyme system becomes a main component in an antioxidant mechanism. As can be seen from our previous observations of retinal lipid peroxidation products from EAU rats, the retinal lipid peroxidation process is exacerbated during EAU. The free radical mediated lipid peroxidation is a chain reaction, has a continuously enhanced tendency, the damage effect is continuously enhanced, and the damage can be stopped or reduced only by breaking the reaction chain through an antioxidant mechanism. Thus, it is believed that protecting the activity of antioxidant enzymes in the EAU and administering antioxidants or antioxidant enzymes from outside the body is an effective method of reducing lipid peroxidation damage in EAU.

In summary, the retinal lipid peroxidation is enhanced during the course of EAU, while the activity of the antioxidant enzyme system is decreased. Sinomenine treatment effective in inhibiting lipid peroxidation, reducing inflammatory infiltration, and protecting antioxidase activity

Example 1

25000mg of sinomenine hydrochloride powder is purchased from Union pharmaceutical factories in Wuhan City, 250g of medicinal starch and a proper amount of alcohol are granulated, dried and tabletted in a tabletting machine to prepare 1000 tablets, each tablet is 275mg in weight and contains 25mg of sinomenine hydrochloride.

Example 2

20000mg of sinomenine and 180g of starch are fully mixed and encapsulated to prepare 2000 capsules, each capsule is 0.1g in weight and contains 10mg of sinomenine hydrochloride.

Claims (3)

1. A new medicinal application of sinomenine and its medicinal salt is characterized by the application of sinomenine and its medicinal salt in preparing medicine with the function of eliminating free radicals.

2. A new application of sinomenine and its medicinal salt in preparing the medicines for preventing lipid peroxidation is disclosed.

3. A new application of sinomenine and its medicinal salt in preparing the medicines for protecting antioxidase activity is disclosed.