JPS61502604A - Oxindole, a type of alkaloid that has an immune system stimulating effect, and preparations containing it - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Oxindole, a type of alkaloid that has a stimulating effect on the immune system, and contains oxindole. formulation with 〔Technical field〕 This invention is based on the general formula (However, in the formula, R1 is a hydrogen atom or a hydroxyl group, and R2 is an oxygen-containing C3 side chain. , R3 is an ethyl or vinyl group, or R2 and R3 together form a pyran ring. ) It relates to the alkaloid oxindole represented by

This invention also provides for formulations containing such alkaloid oxindoles. The fresh endothelium is yellowish-brown or dark red and contains virtually no tannic substances. Uncaria tomentoaa 　D　Extract of the root part of C is excluded.

[Background technology]

Major developments in the theory of infectious and tumor diseases caused by antibiotics and anti-cytostatic agents have also taken place. However, there has been an increase in infectious diseases and the number of therapies to combat their infections.

Although a few antiviral agents are effective, cytostatic therapy inevitably results in immunosuppression. This is a disadvantageous condition.

Under these circumstances, intensive research has been conducted on alternative therapies using adjuvants. Ta. The concept of this therapy was previously used as a stimulatory therapy, and today it is used as an immunostimulatory agent. However, it has once again sparked interest.

The most interesting in this relationship is that $ induces specific or non-specific endogenous defense mechanisms. or a substance or mixture of substances that increases the immune response to antigens administered at the same time. Nonspecific increases in body fluid or cellular defense mechanisms caused by so-called immunostimulants It is about action.

Various experiments have shown that certain compounds can be immunostimulants. There is.

Some compounds, such as polysaccharides from algae and fungi, have shown promise in adjuvant treatment of tumor diseases. It is already used as a preparation in the law.

Most of these compounds are mixtures and chemically unclear. This is a drawback. This point seems to be satisfied by low-molecular compounds. . Most of these compounds are considered to be the prototype with immunostimulatory properties. Along with aristroquine (As), it is a nitrogen-containing compound. Aristolochia Aristolochia clematitis ( The family Amanophyllidae) was already cultivated in ancient Egypt and Greece. It was used for injuries. Test models were developed (T, R, Mo5s and G. Lukas, Pharmacological Research, 11 +33t 1961), thereby stimulating the body as a possible endogenous defense. Its use has been proven. Aristolochia cubes prepared according to pharmacopoeial instructions for similar treatments. Lematitis extract was used as the test extract. rabbit white blood cells In an experiment on phagocytic activity, if the phagocytic activity increases to twice the breaking value, was observed several days after injection of various dilutions of Aristolochia extract. . Dilution D3 was the most effective. No effect was observed for diluent D7. There wasn't.

Other compounds known to have immune system stimulating effects are listed below, including N-containing compounds. The presence of active substances in these types of substances is more likely, and phenol This is particularly noticeable in genus, dinones, and terpenes. Almost all compounds protect cells It is effective for

1. Alkaloids α) Cefplantin (bisbenzylisoquinoline alkaloid) b) Berbamine (bisbenzylisoquinoline alkaloid C) matrine (ru Pin alkaloid) d) Hylocarpine (imidazole alkaloid 9) 2 Phenols/quinones α)′2,3-dihydroxybenzoic acid b) 7 erulic acid C) Forfuenisin (amino acid derivative) d) Anethole g) Claistansine (lignan) f) Calcriboside (phenol gel cocide) f) Urushiol (C□5/ Pyrocatechin derivative with C□7 side chain) h) α-tocopherol (vitamin E) L) ubiquinone (mainly Q7.Q8) g) Maesanin (quinone with C15 side chain) 3 Chiluins α) Zexprevin A/EC selmacran type asquiterbenlactone) b) 12-0-tetrazooanoyl-7orbol-13-acetate, TPA (Tetracyclic dithyl) C) Saponin containing non-steroidal oleonic acid (Pentacyclic trityl) d) Dynoncoside (steroid glycoside) [Disclosure of the invention] And here is the general formula, R1 is a hydrogen atom or a hydroxyl group, R2 is an oxygen-containing C3 side chain, R3 is an ethyl or vinyl group, or R2 and R3 together form a pyran ring. ), The alkaloid oxindole shown in is another group of substances that have immunostimulatory effects. was found to form.

As can be seen in WO-A-82/1130, most do not contain tannin substances. Uncaria tormentosa DC root whose fresh inner bark is yellowish brown or dark red The extract can be used as anticancer agent, contraceptive, etc. plant alkaloids It is partially very similar to the method of isolation of The alkaloids contained in Nkaria species pointed out by Hemingway et al. Chemica'l Abstract, Volvme82, A21. Ref , 135680 K), 7/l/caroids are used in the above-mentioned uses of root extracts. It is possible that it is an active substance that is not directly related to the Obtained. The various effects of root extracts may be caused by non-specific stimulation of the immune system. Based on the idea that the alkaloids contained in various parts of the South American Uncaria species were isolated and their immunological effects were analyzed.

In this regard, certain effects may be observed in crude as well as individually isolated purified alkaloids. The alkaloid fraction was also observed. The special properties of immune system stimulation Found in the caloids tet-2- and pentacyclic oxindole. and the sarcoid alicaloids from rice plants as well as the same from other Lanclair species. As a result of investigating the immunological properties of alkaloids, favorable results were obtained. Then, the cytotoxicity of substances with good immunostimulatory effects became interesting. .

To test a wide range of substances for immunostimulatory and cytotoxic properties, the following in vitro and an in vivo test model was created.

1. Granulocyte test using an improved BRANDT method Neutrophic granulocytes are tested using Macropher It is the most important phagocytic effect cell along with the phagocytosis cells, and therefore plays an important role in fighting infection. It's worth it. They represent about 60-70% of all white blood cells in human blood. do. The phagocytic effect of all these granulocytes has been developed by the brand (5ca nd, J. Haematol, 8upp1゜2.1967), Timper Improved by (MunichMed, Week178.251. (1978) can be measured in vitro.

The granulocyte fraction is obtained from the blood of a healthy proband (PML--preparation of polymorphonuclear leukocytes). (abbreviation) This PML-minute 11211Vs is mainly composed of neutral granulocytes and lymphocytes, monocytogenes, Consists of very few eosinophilic and basophilic granulocytes, similar to the globules and lamina. . A certain amount of yeast cells (Saccharom-7Ce8 CQreViθ1ae ) and the substance to be tested, incubate for 10 minutes at 37°C and carry the suspension. It was spread on the body and incubated for an additional 35 minutes at room temperature. This is then stained and placed under a microscope. I evaluated it more. 100 PML-tubes were selected on the carrier and isolated from individual cells. The extent to which the yeast particles were phagocytosed was measured.

According to the phagocytosis index, the number of phagocytized yeast particles was on average.

Since the test simulates an essential part of immune function, the tested substance is If they act under the same conditions, it is assumed that they also exhibit activity in vivo. It will be done. As already mentioned, this is about the low-molecular-weight compound aristoloquine. Already confirmed. However, the mechanism of this mechanism is largely unknown. stomach.

The test was conducted as follows.

Preparation of granulocyte suspension Heparinized venous blood from clinically healthy adults was mixed with 1.5% dextrough. Translate 2=1 with T500 (Pharmacia) and leave at room temperature for 30 minutes. I left it alone. The supernatant containing white blood cells was press-fitted into a sterilized plastic tube for 50 minutes. Centrifugation was performed at 0 r, pm for 7 minutes. The granulocytes in the precipitate were in each case 51 Washed twice with RPMI-buffer. The granulocytes were then suspended in approximately 5 ml of buffer. 5 x 10 cells/d in a Neubauer hemocytometer. The concentration was adjusted.

Preparation of yeast suspension Yeast suspended in 0.9% NaCJ was heated to 100°C and filtered three times through gauze. The particle size was adjusted to 3-5×10 particles/−.

Preparation of mixed serum Sera from five clinically 1f11i adults were mixed and frozen in 51 units. For the exam diluted 1:10 with 7l of 0.9% NhC.

The test was conducted as follows.

0,2− material dissolved in 0.9% NaC/09% N as a control. a (Pipulate 2 ml of Jα into 0.2 ml of each of the mixed serum, granulocyte, and yeast suspensions. Inject it into a pet and incubate for 10 minutes first.

Next, each sample of 51Lt was applied to the carrier and incubated for an additional 30 minutes at room temperature under high humidity. fed. The precipitated granulocytes and supernatant were carefully washed with 0.9% NaCJ, and the carrier was removed. Dry in a dryer.

staining 1) 5 drops of llllay-Grundwald solution (Merck), filtered; undiluted 2) 2 drops of distilled water 3) 15 drops of G1θsma-solution (Merck), 5TKt4 in water 180°C) 4 drops distilled water Air drying.

evaluation Count 100 granulocytes per carrier to determine how many yeast particles consisting of individual cells have eaten. Determined whether it was affected. The index of phagocytosis (PJ) is the yeast granule per granulocyte. Shows the average number of children. The increase in phagocytic activity (S) in the control value is calculated according to the following formula: calculated.

The granulocyte test according to the BRANDT method determines the phagocytosis-enhancing properties of substances and extracts. Although this method is highly reliable as a determination method, it is very time-consuming and labor-intensive. Ru. Therefore, one method using chemiluminescence is to This could be a new method for measuring cells.

ALIJN et al. (Biochem, Biophys, Rea, Commun , 47. I511972) was the first to phagocytose latex particles and various bacteria. discovered the light emission by neutral granulocytes during the use of neutrophils, and NELSON et al. fec, engineering mmun, 14129? (1975) and WEIDEMANN et al. EBS Letters, 89.136.1978) blood macro that is phagocytized Since the demonstration of the OL-phenomenon for phages (mononuclear cells), this reaction has been applied in clinical and clinical immunology. However, the actual mechanism of the reaction process is It has not yet been fully elucidated. In summary, what occurs during activated phagocytosis CL is explained as follows: these cells are infected with bacteria, viruses or other foreign substances. Upon contact with a causative agent, such as a chemical, first of all an increase in oxygen absorption occurs.

Subsequently, peroxide-anion-radical 02 is formed from oxygen molecules in the cell, Then, naturally or enzymatically, other active oxygen molecules, e.g. Convert to something like ON (OCL). This process is called a “breathing burst.” It is.

Active tR$ molecules are transferred to the phagocytic vacuole itself in the immediate vicinity of the phagocytosed cell. Invading microorganisms are released in the same way as or cancer cells are destroyed or killed. The so-called cationic agents proteins and Other molecules such as cutoferrin are also involved. Microbial cell walls undergo oxidation and oxidation. received and partially destroyed. Chemiluminescence of cell wall substances due to activated oxygen molecules Since it serves as a nessence substance, CL occurs as a side effect.

The low light yield of the OL-reaction in such phagocytosed cells is due to activated oxygen. is increased in in vitro experiments by the addition of oxidized substances, and the physical It produces light of a certain wavelength that can be measured. A preferred material is luminol (5-amino no-2,3-dihydro-L4-7 tarazine diion) and lucigenin (10 , 10'-dimethyl-9,9'-biagridinium dinitrate), which is increase the CL-response by 10-fold.

By using a sensitive measuring multiplier, the photons are calculated by a light-multiplier. Increased CL can be measured by quality.

In this study, we used a 6-channel CL-device that allows six tests at the same time. used. Furthermore, the equipment used allows automatic time-dependent recording of CL-reactions. I can do it. Therefore, time-dependent effects can be added to the dose-effect curve as a third component. can be added.

For in vitro experiments of the phagocytosis-enhancing effect of substances, a cell group produced by f# of granulocytes or macrophages are used or measurements are performed directly in diluted blood. . Activation of phagocytosis can be achieved using solid particles such as opsonized bacteria, zymosan and and latex particles or some kind of dissolved substance. not known Examples of active agents include various ions and fluoride ions.

The test was conducted as follows.

For very first measurements, it is possible to reproduce various instructions in the literature for performing CL-measurements. has been shown to be difficult.

Phagocytosis - When testing the influence of substances on the formation of oxidized active oxygen, CL-reaction Apart from the moderate activating potential of the compounds used, to a certain extent the As well as the effect of the OL-enhancer, the effect of phagocytosis, measurement time, temperature, pa value, Depends on the composition of the medium.

All tests were performed on an Apple computer with Bernrud's CL-evaluation program. Connected to the motor, Berthold's 6-channel pipe Olmat LE9505 It was carried out by

It emits photons with a narrow wavelength distribution of approximately 510 nm, which is the maximum sensitivity of the optical multiplier. Lucigenin, which is distributed in the region, was selected for photoamplification.

The pa value of the measuring solution should be between 7.1 and 7.4 and it should be veronal buffered. This is accomplished by adding liquid. The test substance was dissolved in phosphate buffer (PB S-buffer). The “respiratory burst” response is associated with increased glucose metabolism. Both buffer systems contained 0.1% glucose.

Zysosan with opsonin added was used to activate phagocytosis. separated Measurement of OL of granulocytes was carried out using isolated multipliers adjusted to 10 cells/μl with PBS-buffer. It was performed with blood consisting of morphology nuclear leukocytes (PMNL).

CL- measurements using whole blood were performed at a dilution of 1:100 because the color was lost due to red blood cells. Dilution was performed with PBS buffer. Thus, color loss was substantially reduced.

The test substance was first incubated with isolated granulocytes or whole blood for 10 minutes at room temperature and 37 minutes. The cells were incubated at ℃ for 10 minutes. After adding zymosan with opunin, the measurement was 3. Started at 7°C. The CL-reaction of each sample was incubated for 60 minutes, and the measured values were It was roughened and expressed in cpm. A unique curve for each substance was obtained. integrated surface is directly proportional to the strength of the OL-reaction.

In order to determine the stimulatory effect of a single respiratory burst caused by granulocytes, a granulocyte test was performed. A similar method was used for

Device CL-measurement: biolumat LB9505+Berthornet, Wildpad Computer and diskette drive unit: Apple AE.

apples Program: Bell Shield Display: DM5112CX, Sanyo printer/plotter 12 Epso Preparation of RX-8Q solution α) Sterilized PBS buffer without 7Mg of Ca (Biochrome, Berlin) available as a finished product) b) Base containing Ca, Mg, glucose and albumin lonar buffer Stock solution: 33g Na(J, 1.029Bersonal, 102mFCIMg(J Dissolve 2 and 221Ni of CaC12 in 150- of water.

l　M　-HCI! After adding 3,5-, water was added to make 200 d.

Test solution: Dilute the raw solution of 40 cod with the test solution of 140- The pH was adjusted to 7.0, and water was added to make 200 ILt.

200μ of bovine blood albumin (Boshringer) and 200I of protein. After adding lucose, the solution was filtered and sterilized.

C) Lucigenin solution In the stock solution, 22a5m9 of lucigenin was dissolved in 10-hydrohydraulic solution.

Test solution: The stock solution was diluted with water in a ratio of 1:5.

d) Zymosan with opsonin Storage suspension g: 25 μg of Zymosa was suspended in 7 l of 0.9% NctC of 10-g water. Heated in a bath for 90 minutes. After cooling, the suspension was fractionated into two rainbow units and frozen.

Test solution = 2- stock suspension at room temperature with 2*t clinically healthy adult faces. The cells were incubated for 30 minutes, centrifuged, and washed with 0.9% ΔσCJ. Add openin Zymosan was suspended in 21 Lt of 0.9% NetC1. In each case , distilled water was used as water.

Preparation of polymorphonuclear leukocytes (PMNL) 20 TILt of blood was mixed with 30 μ of WDTA. 15- Inject this blood The mixture was mixed with 7-Rainbow dextran in a container and left at room temperature for 30 minutes. Supernatant containing white blood cells Transfer to a sterilized centrifuge tube and centrifuge at 1.00 Or-pm for 30 minutes. I let go. The residue consists of the remaining parts of white blood cells and red blood cells, which are discolored and disappear. This may affect the measurement in terms of Remaining red blood cells were removed with hypotonic alkaline solution. others For this purpose, the residual 0.25% Nac4 solution was suspended and mixed in a mixer for 20 min. . Then, 201 Lt of 1.6% NaCl solution was mixed and combined again. zo After centrifugation for 10 minutes at oor, p, m, the supernatant was removed by suction and the Repeated many times. The residue was then suspended in 2-PBS buffer and Diluted 1:10 with solution. 1 with PBS buffer in a Fibawell hemocytometer. It was adjusted to 0 cells/gLt.

The test was conducted as follows.

α) Blood: 460 μl Veronal buffer 200 μj diluted blood 100μl lucigenin solution Substances dissolved in 200 μl PBS and PBS as control h) PliNL: 630 μl veronal buffer 15 μl PMNL 100μl lucogenin solution Substance dissolved in PBS at 200 μE and PBS as near/trol Preincubate for 10 min at room temperature and then incubate for 37 min in a 6-channel-biolumat. After incubation for 10 minutes at ℃, 40μ! Zymosan with added opsonin? ll - Mix under stirring and start measurement. The measurement was stopped after 60 minutes.

Evaluation of test results The CL-reaction of the sample is stored in the computer during the measurement period and displayed as a curve after the measurement. It was. The intensity of the CL-reaction in cpm for each sample was obtained by surface integration.

The increase in S of the CL-reaction by the test substance was calculated according to the following formula.

According to the literature, all extracts that showed a positive reaction in the granulocyte test according to the BRANDT method. and Substance B, is assumed to give a positive 0 result in the OL-test as well. two How the two measurement results relate to each other and how the CL method works with the granulocyte test. It is unknown how they will actually be compared. Furthermore, the next granulocytes are separated. It is necessary to compare OL-measurements with whole blood methods.

For CL-testing of purified substances, we use Aris, whose phagocytosis-enhancing effect is certain. We chose the Nα salt of Troquine and already had concentration-effect data from the granulocyte assay. ing.

Granulocyte and Whole Blood Methods Accuracy, Reproducibility and Sensitivity As expected, isolated granules The measurement with a relative change rate of 7-8% of the granulocytes was confirmed to be an accurate method. However, an average value of ri of about 16% was obtained for whole blood.

In the PMNL method, a significant increase in CL-reactivity was recorded even at 10% dilution. .

In contrast, the whole blood method is much less sensitive. Measurable increase at a concentration of 10 It shows a very low value at low t6 concentrations. This essentially means that whole blood This is thought to be due to the elimination effect of red blood cells that still exist despite dilution. It will be done.

Control cpm was generally lower in this study than in the PMML study. There is.

These results limit the scope of application of the two OL methods. short term The whole blood method carried out in can only be applied to the preliminary selection of substances by immunostimulatory effect . However, only dose-effects can be determined by CL-measurement with isolated granulocytes. Curves can be created.

Increase in CL-reaction by whole blood and PMNL method, test substance whole blood -51, 919,221,814,055---PMNL　---85,191568,3 Comparison of 61,95,8,731,3-0L-method and granulocyte test As with the granulocyte test, phagocytic activity is dose-dependent in the form of a 'respiratory burst' response. An increased survival was also found in the OL-test with isolated granulocytes. seed The OL-responses of various aristroquine concentrations are shown below. These measurement results are based on the granule It correlated extremely well with the case of the ball test. However, the maximum value depends on the concentration of 1o-5% In the OL- test it was 10 times lower than in the granulocyte test.

1 control 2.12 50.0 -2 10 4.80 30.0 126.43 10"" 5.52:16 160.3 4 10 3.40 34.6 69.85 10 3.24 32.0 52 ．． 84.10 29.3 93.4 The coefficients calculated using the same formula for the Granule Taku and CL- tests are different because the two measurement principles are different. Direct comparison is not possible. Aristroquine in Granulocytes and CL-Test A comparison of the dose-effect curves showed good agreement with the curve paths of the two test methods.

According to our tests, the CL-method using isolated granulocytes is sufficient in terms of accuracy and sensitivity. The BRANDT method (which can replace the more time-consuming granulocyte test 1) By recording the time-dependent stimulatory effects, the hypothesized phagocytic process can be described in detail. Ru.

3. Nine-carbon clearance test (OCT) according to the B engineering method Nine in vitro tests (C, R, Soc.

Biol, 148.431.1954) (Progr, Liver Di g-2, 166゜1965) K Therefore, the tissue macrophage of the reticuloendothelial system (RES) The phagocytic activity of the medium was determined. Carbon particles stabilized with gelatin? to test animals Intravenously injected, after a certain period of time, the blood is collected from the retroorbital plexus, and the carbohydrate The emission of light was measured photometrically. Kupp 7L cells in the liver part contain exogenous particles. The release of macrophages was 90%, and the macrophages in the pancreatic region were 10%.

The exponential function of carbon prevention is a straight line with a negative slope on the regression coefficient (Io for t gE). If a potentially irritating substance is given once or several times at a fixed concentration, e.g. 24 or 48 hours before The increase in phagocytosis is then determined by comparing the regression coefficient with the blank value. be done. If the substance/control factor involved, the test substance Has active activity (suppressive effect). If the relationship is 〉l, stimulation occurs.

The regression coefficient for carbon release differs depending on the weight of the liver and pancreas, respectively. The true emission constant (α-value) is found only after combining these calculations. we In tests with various substances, the α-value index shows a significant difference from the K-value index. Ta.

Test animal: Soot NMR knee mouse, 25-30g collection collection cleaning test 3 animals for the substance or 5 animals for the substance and 2 animals for the control. Management and feeding: 20 cm cage at 22°C, SF Any semi-housing, water Treatment method 20. The substance dissolved in 9% NaCl was administered once or several times. The solution is N suspensions should be used to avoid cell-stimulating effects. crude Its! Intraperitoneal administration of the suspension results in animal death. The individual dosage is usually The weight was 0.1 and 10~/kg. The final dose was given 24 hours before the start of the study. Performed before.

The test was conducted as follows.

An ink suspension containing 10% gas black (particle size 200-25OA) was 0.9% NaCJ containing 1% gelatin contains 167η of carbon per cod. The mixture was adjusted so that it was warm and preheated to 37°C. Each mouse received 0.1 d in the tail vein. /10g was injected.

t=3. fi9, 12. After 15 minutes, 25 μj of blood was collected from the retroorbital plexus. (one method with the addition of heparin, one cyclopi was one method).

The blood was hemolyzed in 2WLt distilled water, and the carbon concentration was determined by photometry at λ = 559 nm. (Hitachi has a 181° cell thickness meter).

Evaluation: Regression factor for carbon release shown as an exponential function The number K was determined mathematically (logE versus t). Control on index substance/ The stimulatory or inhibitory effects of the RES could be determined.

4. Lymphocyte conversion test In the lymphocyte conversion test, the induction and control of normal lymphocyte embryonic conversion Mitosis was measured. For this purpose, the substance to be tested may be separated or Incubate with diluted whole blood, mark with standard t, 6G- and H-thymidine and incubate again. did. Liquid scintillation of thymidine bound to cell-DNA A friend who measures with an ictrometer.

5.D. Cytotoxicity by H-thymidine-binding test on water cells In the granulocyte test, because certain substances lyse granulocytes at high concentrations, we Testing the cell-damaging effect in a model of H-y-midine binding test in ascites cells. Experiments found a relationship between increased phagocytosis and cytotoxicity in activated substances. I was able to do that.

This in vitro test system (Hoppe-8syher's Z , physiolg, Chem, 348.433.1967), The cytotoxicity of the substance due to inhibition of DNA synthesis in tumor cells has been demonstrated in Waite in rats. Changes in the binding ratio of H-thymidine in ascites cells of r-250 monolayer endoma was measured numerically.

The test was conducted as follows.

3- Walker-250 monolayer intracellular suspension? In a 50-liter centrifuge gas Mix with MED engineering σM L-15 (Boehringer) 10Mt and leave for 10 minutes. Centrifuged for a while. Rinse the supernatant and fill the centrifuge glass to 1 cup from the rim with the medium. did. Sample charge of 1 mA from stirred cell suspension (equivalent to 2 x 10 cells/-) into a centrifuge glass, add 500 μg of test solution, and add 500 μg of test solution for control test. 00 μl of medium was added. Each test was performed 4-6 times.

After pre-incubation for 2 hours at 37°C, 100 μl of h-thymidine (50C110M) was added. It was added per sample and further incubated under stirring. Then 1,5 WLt IN-hypersalt Binding was stopped by adding sample hydroxide to the sample, and the preparation was stored in the freezer overnight. join Absorb and remove the residue containing the precursors with perchloric acid-moistened tear paper. Washed with chloroacetic acid and again with ethanol. Insert the filter into a polyethylene film. The mixture was placed in a flask and its activity was measured using a toluene scintillator. control your results In comparison, the inhibitory effect is indicated by .

Comparison of test results In each test, the percentage of binding inhibition by the same substance varied up to 30%. Therefore, Methotre, which inhibits DNA synthesis as an antagonist of folic acid, xat■ was tested as a reference material in each test series. Methotre The average value of the xat■-inhibitory effect (43% for 2+Y/+++J) is the relative inhibition of the substance. It was used to determine the effect H.

By comparing the obtained results with the results of the granulocyte test, it was possible to determine whether other low-molecular compounds This idea has been confirmed, and according to this, substances that exhibit cytotoxic activity at high concentrations are , re-KIO or diluted to 10% also stimulates the phagocytic effect of isolated granulocytes It had an impact. Although this can be said generally, it also applies to polymer compounds. Further testing is needed to clarify this.

6. Acute toxicity Test animal: female or male NMR knee mouse, 30-35g Management and feeding: 20 'Cage 5FI at -22°C - standard feeding, water Number of collected: 5 mice The test was carried out as follows: fc: test article dissolved in water or 0.9% Me-C1 solution The substance was administered to mice via the throat. 14 days of behavior regarding control group We had a parent's festival.

A prerequisite for all tests is complete solubility of the test material. lipophilic extract or carboxymethylcellulose added as a solubilizer in testing substances. Solubilizers have some self-stimulating properties, such as compounds such as carbonaceous acid or dimethylsulfoxide. It is used as a control value at the test concentration. In OCT , the use of DMSO is not recommended due to high toxins.

Additionally, test solutions should be prepared to remove as much bacteria as possible. gram positive bacteria contains cell wall polysaccharides, which in part have the effect of stimulating the phagocytic effect of granulocytes. Ru. Gram-resistant bacterial cell jffil, I popolysaccharide is a so-called endotoxin. This is said to affect test results. Live bacteria and bacterial extracts have a high R ES-indicates stimulatory effect. Furthermore, some endotoxins are pyrogenic, antigenic and It has secondary effects such as metabolic changes in bacterial toxins, and these are can also be affected.

It also serves to filter and sterilize the test solution and store it in a freezer. It is the right thing to do.

In accordance with this publication, we have specified the principles for the effectiveness of the alkaloid oxindole. Untreated water and ethanol extraction of Uncaria tomenosa to extract and NH3-treated powder, as well as crude alkaloid mixtures and other alkaloids. Similar to the oxindoles, isolated nt-kama alkaloids were tested.

Note that the stimulatory effect of NH3 alone was previously excluded.

To prepare the extract, powder treated with alkali with 10% NH3 and dried agent were used. Mix with eluent at a ratio of 1:10 and extract with a magnetic stirrer at room temperature for 2 days. The solution was taken out, filtered, and product F was concentrated and dried.

The crude alkaloid mixture was prepared according to the following method.

Dried medicinal herbs were moistened with 10% NH3, left at room temperature for 1 hour, and carefully dried with It was dry. The drug was then moistened with ethyl acetate for 4 days using a magnetic thickener to soften it. I let it happen. The crude extract thus obtained was concentrated, and the aqueous layer had a positive 'l/axser-reaction. It was extracted with 2%) 12So4 until it no longer showed any. The aqueous layer is mixed with an alkaline solution with a pH of 8-9. Mix NαCO3 and peel until it becomes potassium. Repeated extraction of the aqueous layer with ethyl acetate Thus, 750 g of drug contains 6.75 g of light brown crude alkaloid mixture. The yield was equivalent to 0.9%. The mixture is then converted into a salt form with addition of hydrochloric acid.

Separation of individual oxindole alkaloids from the alkaloid mixture made by S and identified it.

They are as follows.

1. Arrowisopteropodine 9 Formula C2, isomer A, M with H2404N2 G:368 m, p,: 204c -209℃ Thin layer chromatography: R, 0,73 (LMI); 0.48 (LMll); 0.83 (LM 1ft) HPLC: Rt 8.8m1n (TS I) [α] -85,1 (C=0 ．． 554/CHCl3) UV (MeOH) = λm,, = 201L 243. 283(ah)-r　X　Sdctor:　m/e(rel, int,): 368 (M +100) +223 (70), 208 (35), 180 (14) , 146(11).

145 (9), 144 (5), 130 (25), 69 (45) λ arrow Isomer B with terobodine 2 formula C21H2404N2.

MG:368 to, p・: 214-219℃ Txc: Rfo, 72 (LM l), 0.47 (LM n) - 0, 81 (” fil) HPLC: Rt O, 8 miΩ (TS l) UV (MeO H) :λ. ax=208.243.283(ah)-r xd vector: r rve (rel, int,): 368 (M, 100).

223 (84), 208 (25), 180 (20), 146 (8), 145 (1 1), 144 (11), 130 (14), 69 (35) 3, normal-isomer Isomer A with formula C21H2404N2; MG368 m, p.: 96'-106℃ (2) Tlc: RfO, 71 (LM l) - 0,47 (LMII), 0.80 (LMIn) HPLC: Rt 9.2 m1n (TS l) ((ri, + 145' (c=0.758/CHCl3)tJV(MeOH):λmax= 208.2″43.283(ah)MS:M/e (ral, int,): 368 (M p70), 223 (100), 208 (10), 180 (4), 146(6), 145(5), 144(7).

130(11), 69(29) 4. Isomer with Normal Rouinli/Cofilin 1 formula C2□H2804N2 A, MG: 384 m, p-: 138°-141°C Tlc", RfO, 71 (LMI) - 0,42 (LM [1), 0.70 (LM, n [) HPLC: Rt 13.9 m1n (TS I,) (a) D :+7.8' (c=o, 4zo/cHc13)UV7MeOH) :λ,, =208.243,283 ([1lh) mass is kutor: TIv/e(re’ 1.1nt-): 384 (Mp 100) + 239 (80), 224 (23), 210 (14), 208 (25), 146 (6) 145 (7) , 144(10), 130(14), 69(75)5, normal-mid 2filin, isomer B with formula C21H2404N2, MG:368 m, p,: 264C-268℃ Txc: RfO, 55 (LM I) - 0,39 (LM l), 0.50 (LMl [I) HPLC: Rt 10.4 min ('L'Sl) [α ), -4.3℃ (C=0.5877CHCI3)Ll'/(MeOH):λ max=208. 243. 283 (Elh)-q;x, dctor: m/e (raLint): 368 (M + 60) * 223144 (10), 130 (10) # 69 (27) 6. Normal-Rinkotsu 49fu 2 type C2□H Isomer B with 2804N2, MG:334 m-p: 214'-216℃ T'le: R, 0, 36 (LM 1)) 0.25 (LM 11), 0.31 ( LM In) HPLC: Rt 17.3 m1n ('I'S 1) ((1), - 15,1c (c=o, sst/cuc13) UV (MeOH): λrnax= 208. 243. 283 (θh) mass spectrum #: m/e (rel, int, ): 384 (M + 100) + 293 (80), 224 (30 ), 210(20), 208(24).

146(8), 145e12), 144(13), 130(10).

Solvent mixture used (LM) far TICLMI chloroform-acetate (5:4) LM ■ Chloroform-ethanol (95:5) LM ■ Tylacetate-isopropanol-NH3cone, (100:2:1) These oxindole alkaro isolated from Uncaria tormentosa In addition to ido, oxindole alkaloids contained in Uncaria species There is SR geophyllin, and its structural formula is: and GelsemiumBθmer It is known as one of the main components of the roots of virens) (7. Virens). Oxindole: The alkaloid gelcemin should be at least part of immunological tests. used.

Dissolving the alkaloid base in ether to prepare a water-soluble hydrochloric acid salt Then, HCl-gas was introduced from a gas cylinder to remove t0 ether, and then salted with hydrochloric acid. was washed again with anhydrous ether and then dried with phosphorus pentoxide in a vacuum desiccator. It was dry.

The rate of increase in phagocytosis of various extracts and substances, each at various concentrations, was determined in the granulocyte test. was established.

1. Extract from Uncarla tomentosa H2O-maceration 7.3 21.8 24.1 5.7--H2C-maceration (N H3) 15,6 12,9 29.6 28.4 - -f2tOH-maceration -14,412,026,3--gtOH-maceration (NH3) 7,6 35,6 36,8 25.5--2, Unc, tom, to O Mixture 20.1 21.0 15.7 10.3 - -3, Unc, tom , minutes from Distant friend oxindole alkaloid Isopteropodin 23.4 4&7 55.9 38.2 28,2 13. 6 Buteropodine 11,2 21.7 26.1 23.0--Inmitraphy Lin 16.5 27.0 27.0 19.4 5.7 - Inlin Kafili 10.8 25.2 27.3 16.1 8.8 -4, other oxiins Doll alkaloid This is geophyllin 10,5 18,5 26,0 35.3 - -Gelsemi 2Z1 40,4 33.7 8.2 - - Cytotoxicity is It was observed at a concentration of 10%.

For comparison, granulocyte tests were performed on non-oxindole alkaloids. It was done.

Inquinoline used for viral eye inflammation; Berbery alkaloid was inactive up to a concentration of 10%.

Vincristine (aspidos is a lumin-type indole alkaloid) and duck Totisine (quinoline alkaloid) is effective even when diluted to 10%. I can't see it.9. These compounds are used as mitosis-inhibitors and anticancer agents. be done.

In the case of vincristine, the in vitro inhibition of macrophage phagocytosis is 10 % and 10% concentrations.

Baptisia tinctoria (glue) It is the main alkaloid of all plants of the family Butterflyaceae. Ticine is used for septic diseases and ulcers, but it is It doesn't show any effect.

Thalictrum faberi (Thalictrum faberi) (Thalictrum faberi) Bisbenzylisoquinoline alkaloid talifaverine isolated from In studies with rifabin and fungusanin (tested up to 10%), this They are used as anti-inflammatory agents and folk remedies in the treatment of stomach cancer in China. However, it did not show any effect.

Among purified alkaloids separated from alkaloid mixtures, there are differences in effectiveness. However, although the maximum effect uniformly falls in the 10%-10% concentration range, no It was. The results found in the granulocyte test according to the BRANDT method are Confirmed by sense test. The results of these two tests (e.g. Isolinkov The reason for the difference (due to Ilin) has not yet been found. The following table shows the crude Oxindo isolated from alkaloid mixture and Uncaria tormentosa Figure 2 shows the rate of increase in the CL-reaction of alcohol alkaloids.

Concentration 10・10% 10% 10% 10% 10% 10% Mixture -31°5 3 5.5 75.2 33.2 - -2 Separated Al kaloid Infterobodin-67,266,055,768,155,331,2 Pte Lopodin-10,610,023,715,4--Ingodraphilin-1 5,415,736゜1　46.7　18.6　-Contains a small amount of alkaloids , the stem extract of Uncaria species has a high It did not show phagocytic activity.

In contrast, roots containing imbutylobodein averaged values of 30% and 40%. showed that.

Leaves showing high alkaloid concentrations, like roots, showed a different picture. 1983 The harvested crop does not contain imbutylobodein and has a slightly higher stimulation crop compared to the stem. The leaves harvested in 1981 showed an increase rate of 30% and 40%. Indicated. The leaves contained isobutylobodein at similar concentrations to the roots.

Aside from the alkaloid content of the drug, the effect of each alkaloid of H1+161° It depends on the pattern of alkaloids that is confirmed by integrating various phagocytosis enhancement rates. Ru.

Testing of crude alkaloid mixtures in the form of water-soluble hydrochloric acid salts has shown that an average of 20% It showed increased phagocytosis. The latter was not higher than that of total extract with ethanol. Therefore, substances present concomitantly in the water and ethanol extracts may compensate for the increased efficacy. It seems to have a role as an auxiliary agent. This indicates that water-soluble catechin extract ( 1% aqueous solution (pH 6.0) 10 - 0.1% aqueous solution of the total alkaloid mixture 10 - m1, stirred with a magnetic stirrer to room temperature for 24 hours, lyophilized, This was finally confirmed by conducting a carbon clearance test.

Results of all samples subjected to carbon clearance test according to J3IOZZ construction method The results are summarized in the table below.

As in the granulocyte test, the efficacy of alkaloids was found in this test. Served. Water-soaked drinks containing alkaloids are water-soaked drinks with low alkaloid content. Stimulates REuS to a higher extent than drinks. The alkaloid mixture itself is When applied with non-stimulating catechins, high stone activity similar to that of water-soaked drinks An increase was obtained.

Cell transformation test at Linno A whole water extract from Uncaria tormentosa was tested and found that Uncaria tormentosa An inhibited alkaloid mixture from Sa. and two mixtures P and P2 were tested. Pl are the alkaloids isobutylobodein, butylobodein, and inmitraphyllin. and inlin crophilin, which are active in the granulocyte test and P 2 contains the inactive alkaloids mitraphyllin and lincophyllin.

Both in the whole blood method and in measurements using isolated lymphocytes, no The material also did not show an increase in 3H-thymidine-bonds over the natural conversion.

Acute toxicity, zero cytotoxicity and antitumor effect Acute toxicity tests were carried out on total toxicity from Uncaria tormentosa and the inhibitory alkaloid mixture. carried out on mice by water extract.

The extract is administered orally at a maximum concentration of 5117 to 9 and intraperitoneally at a maximum concentration of 2g to 9. It was administered intracavitally. The alkaloid mixture can be administered orally until 2fllk & intraperitoneally. up to 1 g/kg. When observed for over 14 days, the mice showed normal behavior. No change in behavior was observed.

A cytotoxicity test was carried out on ascites cells by H-thymidine-binding test. Next. Various extracts and alkaloid concentrations were tested.

Extract from Uncaria tormentosa in 3H-thymidine binding test and Rate of binding inhibition effect of alkaloid concentrate α) Cytotoxin at a dose of 0.2% Comparative sample of sex Relative binding inhibition effect (%) H2C-extract 42 H20-Alkaline drug extract 65 MeOH-extract 52 MeOH - Extract of alkaline drugs 64 Total alkaloid mixture 70 PI-1 (Cl 100 P2-1((J69 Percentage of binding inhibition effect at various concentrations Alkaloid 10% 10% 10% 10% 10% total alkaloids Mixture 67 36 38 12- pl　93　86　45　15　15 P2 64 58 20 - - As is clear, alkaloid concentrates at a concentration of 10% have cytotoxic effects. The more diluted it is, the less toxic it becomes.

These results correlate with cytotoxicity testing of alkaloid concentrates P1 and P2 . At concentrations up to 5 μg/-, the growth of the two cell types is unaffected. . Only at a dose of 50μfi7rtt (5X10%) two samples showed a slight cytotoxic effect.

Cytotoxic alkaloids of Po and P2 for KB and P388 cells 0.5 5.0 50 (μ9/1tt) mixture KB p3ss KB p3ss KB P388PI-)IC/100 100 100 Zoo 77 76 P2-) Check the survival rate of 1c1100 100 100 100 77 64 cells. Indicated by

Two cytotoxin tests demonstrate a correlation between cytotoxicity and immunostimulatory effects. found. For example, a total alkaloid mixture inhibits granulocytes at a concentration of 10%. dissolve. Dilute further and cell l! Even if it has no or almost no elementary effect, An immunostimulatory effect was observed. In the d-thymidine-binding test, P2 granulocytes Tests have shown that mixtures with the inactive alkaloids mitraphyllin and lincophyllin It is interesting that the compound is less toxic than P2 mixed with active alkaloids. deep.

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Claims (11)

[Claims] 1. There are general formulas, ▲mathematical formulas, chemical formulas, tables, etc. as components of immune system stimulants▼ (However, during the ceremony, R1 is a hydrogen atom or a hydroxyl group, R2 is an oxygen-containing C3 side chain, R3 is an ethyl or vinyl group, or R2 and R3 together form a pyran ring. ) Oxindole alkaloid represented by. 2. R1 is a hydrogen atom, R2 is methoxyisopropenoic acid methyl ester The oxindole alkaloid of claim 1. 3. The oxy of claim 1, wherein R2 and R3 are taken together to form a dihydropyran ring. Indole alkaloid. 4. The oxindole alkaloid of claim 3 having the formula C21H24O4N2 Do. 5. The compound according to any one of claims 1 to 4, characterized in that it is selected from the following compounds: xiindole alkaloid. Isopteropodin Pteropotein Isomitraphyllin mitraphyllin isorhynchophyllin Lincofilin speciofilin 6. The oxyisomer according to claim 5, characterized in that at least one is an arrow isomer. ndole alkaloid. 7. At least the general formula, ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ (However, during the ceremony, R1 is a hydrogen atom or a hydroxyl group, R2 is an oxygen-containing C3 side chain, R3 is an ethyl or vinyl group, or R2 and R3 together form a pyran ring. ), Preparations containing oxindole alkaloids shown in Uncaria tomentosa The fresh endothelium, which is free from tannic substances, produces a tan or dark red extract. except). 8. in the form of a salt of hydrochloric acid, the water bath solution of which has a concentration of 10-3% to 10-6%. The formulation according to claim 7, characterized in that: 9. Contains at least one of the following oxindole alkaloids. 9. The formulation according to claim 7 or 8, wherein: Arrowisopteropodine, isomer A with the following formula ▲ Numerical formula, chemical formula, table, etc. I'm here▼ Alopetelopodin, isomer B with the following formula ▲ There are mathematical formulas, chemical formulas, tables, etc. S▼ Normal-isomithraphyllin, isomer A with the following formula ▲ Mathematical formula, chemical formula, table There are etc.▼ Normal-lumitraphyllin, isomer B with the following formula ▲ Numerical formula, chemical formula, table, etc. There is▼ Normal-isorhinchofilin, isomer A with the following formula ▲ Mathematical formula, chemical formula, table There are etc.▼ Normal rhynchofilin, isomer B with the following formula ▲ Numerical formula, chemical formula, table, etc. There is▼ 10. At least one oxindole alkaloid is an alkaloid categorical A kind of preparation selected from claims 7 to 9, characterized in that it is a complex. 11. 10 parts of water-soluble catechin extract was dissolved in water-soluble oxindole alkaloid. 11. A formulation according to claim 10, characterized in that it is mixed with one part (by volume) of a liquid.