JPH066588B2 - Oxyindole, a kind of alkaloid having a stimulating action on the immune system, and a preparation containing the same - Google Patents

Description

Description: TECHNICAL FIELD This invention relates to oxindole alkaloids selected from the group consisting of isopteropodin, pteropodin, isomitrafirin, and mitrafirin having the formula C ₂₁ H ₂₄ O ₄ N ₂ .

The invention further relates to formulations containing such alkaloid oxindole, but which is substantially free of tannin substances and whose fresh endothelium is tan or dark brown. Uncaria tomentosa D
The root extract of C is excluded.

[Background technology]

Despite the significant theoretical development of infectious diseases and tumor diseases caused by antibiotics and cytostatic agents, a large number of therapies against infectious diseases and their infections have increased.

Although a small number of antiviral agents are effective, cytostatic therapy is a disadvantage because it necessarily involves immunosuppression.

Under these circumstances, intensive research has been conducted on alternative therapeutics. This concept of therapy, formerly known as stimulation therapy and today as immunostimulatory activity, has come to the fore again.

The most interesting thing in this context is that the body fluids or cells of any immune stimulant, such as substances or mixtures that either induce specific or non-specific endogenous defense mechanisms or increase the immune response to the co-administered antigen. Is a non-specific increase in the defense mechanism of.

Various experiments have demonstrated that certain compounds can be immunostimulants.

Several compounds, such as algae and fungal polysaccharides, have already been used as formulations in adjuvant therapy of tumor diseases. The disadvantage of these types of compounds is that they are mostly mixtures and are chemically unclear. In this regard, it seems that low molecular weight compounds are satisfied. Most of these compounds are nitrogen-containing compounds along with aristroquine (AS), which is considered to be the prototypical immunostimulatory form. Aristolochia clematitis (Astrachidae) has already been used for all kinds of injuries in ancient Egypt and Greece. Dest model developed (JR Mose and G. Lukas, Phamacolog
ic Research 11,33,1961), thereby demonstrating the stimulatory effect as a possible endogenous defense of the body. An extract of Aristolochia clematisis, prepared according to the Pharmacopeia instructions for a similar therapy, was used as the test extract. In an experiment on the phagocytic activity of rabbit leukocytes, a two-fold increase in the phagocytic activity was observed several days after injection with various dilutions of an extract of Aristolochia. . Diluent D3 was the most effective. No effect was observed with diluted solution D7.

Other known compounds having an immune system stimulating action are shown below, but it is highly possible that an active substance is present in substances of the N-containing compound type, and in phenols, quinones and terpenes, It is remarkable. Almost all compounds are effective in protecting cells.

1. Alkaloids a) Cepharanthin (bisbenzylisoquinoline alkaloid) b) Berbamine (bisbenzylisoquinoline alkaloid) c) Matrine (lupine alkaloid) d) Pilocarpine (imidazole alkaloid) 2. Phenols / quinones a) 2,3-dihydroxybenzoic acid b) ferulic acid c) forfenicin (amino acid derivative) d) anethole e) claistancin (lignan) f) calculigoside (phenol glucocaide) g) sumac All _(C 15 _/ pyrocatechol derivatives having a _{C 17} side chain) h) alpha-tocopherol (vitamin E) i) ubiquinone (mainly Q7, Q8) j) Maesanin _(quinone having _{C 15} side chain) 3. Terpenes a) Zexbrevin A / B (sermaclan-type asquiterpene lactone) b) 12-o-tetradeoanoyl-phorbol-1
3-acetate, TPA (Tetracyclic diterpene) c) Saponin (pentacyclic triterpene) containing non-glycoside oleic acid d) Sinone coside (steroid glycoside) [Disclosure of the Invention] where C ₂₁ H ₂₄ O It has been found that at least one oxindole alkaloid selected from the group consisting of isopteropodin, pteropodin, isomitrafirin and mitrafirin having ₄ N ₂ has an excellent immunostimulatory effect.

As can be seen in WO-A-82 / 1130, the root extract of Uncaria tormentosa DC, which contains mostly no tannin substance and whose fresh endothelium is yellowish brown or dark red, is used as an anti-cancer agent, a contraceptive agent, etc. be able to. Partly very similar to the method of separating plant alkaloids, the method of preparation described therein and the alkaloids (C
According to hemical Abstract, Volvme 82, No. 21, Ref. 135680 K), alkaloids may not be directly related to the above-mentioned uses of root extract, but may be the active substances on which they may be based. Was possible. Based on the idea that various effects of root extract may be caused by non-specific stimulatory effects of the immune system, alkaloids contained in various parts of Uncaria spp. In South America were isolated and their immune effects were analyzed. It was
In this regard, certain effects were observed with crude alkaloid fractions as well as individually isolated purified alkaloids. The immunological properties of similar alkaloids from other Unclea species as well as other alkaloids from various plants were found because of the special nature of the immune system-stimulating action found in the alkaloids tetra- and pentacyclic oxindoles. As a result of the investigation, the results that should be supported were obtained, and the cytotoxicity of the substance having a good immunostimulatory effect became interesting.

The following models of in vitro and in vivo tests have been developed to test various substances for immunostimulatory activity and cytotoxicity.

1. Granulocyte test with improved BRANDT method Neutrophilic leukocyte is the most important phagocytic responder cell together with macrophage and therefore plays an important role against infection. They represent approximately 60-70% of all white blood cells in human blood. The phagocytic effect of all these granulocytes was developed by Brand (Scand.J.
Haematol.Suppl.2,1967), modified by Timper (Munich Med.Weekly 8,251,1978).

Granulocyte fraction is obtained from the blood of healthy probands (P
This abbreviated form of ML-polymorphonuclear leukocytes) is mainly composed of neutral eosinophils and lymphocytes, monocytes, blood cells and very few eosinophilic and basophilic granulocytes. It consists of After adding a fixed amount of yeast cells (Saccharomyces cerevisiae) and the substance to be tested, it was incubated for 10 minutes at 37 ° C., the suspension was spread on a carrier and further incubated for 35 minutes at room temperature. Then
This was dyed and evaluated by a microscope. 100 PM
L-cells were selected per carrier and it was determined how much the yeast particles consisting of individual cells were phagocytosed. According to the index of phagocytosis, the average number of yeast particles that underwent phagocytosis was shown.

Since the test mimics the essential part of the immune action, it is postulated that the substances tested are also active in vivo, provided they act under the same conditions.
As already mentioned, this has already been confirmed following the low molecular weight compound aristroquin. However,
The process of this mechanism is largely unknown.

The test was conducted as follows.

Preparation of granulocyte suspension 15 ml of clinically healthy adult venous blood supplemented with heparin was treated with 1.5% dextran T500 (Pharmacia) 2: 1.
It was diluted to room temperature and left for 30 minutes at room temperature. Inject the supernatant containing white blood cells into a sterilized plastic tube for 500
Centrifuge at rpm for 7 minutes. The pelleting granulocytes were washed twice with 5 ml of RPMI-buffer in each case. The granulocytes were then suspended in about 5 ml of buffer and Neubawell (Neubawell)
uer) Hemocytometer was adjusted to a concentration of 5 × 10 ⁶ cells / ml.

Preparation of Yeast Suspension Yeast suspended in 0.9% NaCl was heated to 100 ° C., passed through gauze 3 times, and adjusted to 3-5 × 10 ⁷ particles / ml.

Preparation of co-injected serum Serum of 5 clinically healthy adults was mixed and frozen in 5 ml units. For testing, it was diluted 1:10 with 0.9% NaCl.

The test was performed as follows.

0.2 ml of the substance dissolved in 0.9% NaCl and 0.2 ml of 0.9% NaCl as a control were mixed with 0.2 ml each of serum, granulocytes and yeast suspension, and the mixture was pipetted and incubated for 10 minutes. did. Then, 5 ml of each of the preparations was applied to a carrier and incubated for another 30 minutes at room temperature under high humidity. Precipitated granulocytes, the supernatant was carefully washed with 0.9% NaCl,
The carrier was dried in a dryer.

Staining 1) 5 drops of May-Grundawald-solution (Merck), excess, undiluted 2) 2 drops of distilled water 3) 15 drops of Giesma-solution (Merck), in 180 ml of water 5
ml 4) 4 drops of distilled water air dried.

Evaluation 100 granulocytes were counted per carrier to determine how phagocytosed the yeast particles of individual cells were. The index of phagocytosis (PJ) indicates the average number of yeast particles per granulocyte. Increase in phagocytic activity at control value (S)
Is calculated according to the following equation.

2. Chemiluminescence test (CL) The granulocyte test according to the BRANDT method is a reliable method for determining the phagocytosis-enhancing properties of substances and extracts, but it is a very time-consuming and labor-intensive method. Therefore,
As one method, chemiluminescence method can be a new method for measuring phagocytosed cells.

ALLEN et al. (Biochem. Biophys. Res. Commun, 47, 129, 1972) first discovered photoemission by neutral eosinophil granulocytes during phagocytosis of latex particles and various bacteria, and NELSON et al. (Infec. Immun.14,129,1975) and WEIDEMANN et al. (FEBS L
This reaction has been applied to experimental and clinical immunology since etters, 89, 136, 1978) demonstrated the L-phenomenon on phagocytic blood macrophages (mononuclear cells).
However, the actual mechanism of the reaction process has not been fully elucidated. In summary, the CL that results in activated phagocytosis is explained as follows: When these cells come into contact with bacteria, viruses or other exogenous substances, such as chemicals, oxygen is first introduced. Increased absorption occurs.
Subsequently, peroxide-anion-radical O ₂ is formed in the cell from molecular oxygen, which then spontaneously or enzymatically reacts with other active oxygen molecules, such as hydrogen peroxide (H ₂ O ₂ ), monomolecular oxygen. ( ¹ O ₂ ), hydroxyl group (OH) and hypochlorite-anion
(OCL ^-) is converted to something like. This process is called a "breathing burst."

Release of reactive oxygen molecules in the immediate vicinity of phagocytosed cells as well as in the phagocytic vacuole itself and activation of the lysozyme enzyme destroys or kills invading microorganisms or cancer cells. To be Other molecules such as so-called cationic agent proteins and lactoferrin are also involved. The cell walls of microorganisms undergo oxidation and reduction and are partially destroyed. CL occurs as a side effect because cell wall material serves as a chemiluminescent material for activated oxygen molecules.

The low light yield of CL-reactions of such phagocytosed cells is increased in vitro by the addition of substances oxidized by activated oxygen, and is a physically measurable wavelength of light. To generate. Suitable substances are luminol (5-amino-2,3-dihydro-1,4-phthalazine diion) and lucigenin (10,10'-).
Dimethyl-9,9'-biagridinium dinitrate)
And these increase the CL-response by 10 ³ fold.

By using a sensitive measuring multiplier whose photons are calculated by a light-multiplier, the CL increased by the substance can be measured.

This study used a 6-channel-CL-device that allowed 6 tests at the same time. Furthermore, the device used is capable of automatically recording CL-responses in a time-dependent manner. Therefore, a time-dependent effect can be added to the dose-effect curve as a third factor.

For in vitro experiments of the phagocytosis-enhancing effect of substances, purified cell populations of granulocytes or macrophages are used or measurements are carried out in directly diluted blood. Activation of phagocytosis is achieved by solid particles, for example bacteria with opsonin,
Zymosan and latex particles or some dissolved material. Known activators are, for example, various ions and fluoride ions.

The test was carried out as follows.

Initial measurements have shown that it is difficult to reproduce the various indications of the literature for performing CL-measurements.

In examining the effect of substances on the formation of phagocytosis-induced reactive oxygen species, the CL-reaction, apart from the different activating capacities of the compounds used, up to a certain extent, CL-enhanced used. The action of phagocytosis as well as the action of compounds depends on the measurement time, temperature, pH value, and composition of the medium.

All tests were performed on a Bellsold 6-channel-Biolmart LB9505 coupled to an Apple computer with a Bellsold CL-evaluation program.

It emits photons with a narrow wavelength distribution of about 510 nm, which is light-
Lucigenin, which is distributed in the maximum range of sensitivity of the multiplier, was selected for light amplification.

The pH value of the measuring solution should be between 7.1 and 7.4, which is achieved by the addition of veronal buffer. The test substances were dissolved in phosphate buffer (PBS-buffer). Since the "breathing out" response is associated with increased glucose metabolism, both buffer systems have 0.1%
Contains glucose.

Zymosan with opsonin was used to activate phagocytosis. The CL of the separated granulocytes was measured by PBS.
Performed with blood consisting of isolated polymorphonuclear leukocytes (PMNL), adjusted to 10 ⁶ cells / μ in buffer.

In CL-measurement with whole blood, the color disappeared due to erythrocytes, so a dilution of 1: 100 was performed with PBS buffer.
Thus, the loss of color was essentially reduced.

The test substances were first incubated with separated granulocytes or whole blood for 10 minutes at room temperature and 10 minutes at 37 ° C. After adding zymosan with opsonin, the measurement was started at 37 ° C. The CL-reaction of each sample was observed for 60 minutes, and the measured values were graphed and displayed in cpm. A curve specific to each substance was obtained. The integrated surface is directly CL
-It is proportional to the strength of the reaction.

A similar procedure for the granulocyte test was used to determine the stimulatory effect of the granulocyte-generated "breathing burst".

Device CL-Measurement: biolumat LB9505, Bellsonade, Wildbad Computer and diskette drive unit: Apple IIe, Apples Program: Bellsold Display: DM5112CX, Sanyo Printer / Plotter: Ipson RX-80 Solution preparation a) Sterilized Ca ²⁺ / PBS buffer without Mg ²⁺ (available as a finished product of biochrome in Berlin) b) Versonal buffer containing Ca ²⁺ , Mg ²⁺ , glucose and albumin Raw solution: 8.3 g NaCl, 1 0.02g of Belsonal, 1
02 mg MgCl ₂ and 22 mg CaCl ₂ were dissolved in 150 ml water. After adding 3.5 ml of 1M HCl, add water and add 200 m.
l

Test solution: 40 ml of stock solution was diluted with 140 ml of test solution, pH was adjusted to 7.0 with 1M-HCl, and water was added to 200 m
l

200 mg of purified bovine blood albumin (Boehringer) and 200
After addition of g glucose, the solution was sterilized by passing.

c) Lucigenin solution Original solution: 25.5 mg of lucigenin was dissolved in 10 ml of water.

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

d) Zymosan with opsonin stock suspension: 25 mg zymosan with 10 ml 0.9% Na
Suspended in Cl and heated in water for 90 minutes. After cooling, the suspension was fractionated into 2 ml units and frozen.

Test solution: 2 ml stock suspension was incubated with 2 ml clinically healthy adult blood sperm for 30 minutes at room temperature, centrifuged,
It was washed with 0.9% NaCl. Zymosan with opsonin was suspended in 2 ml of 0.9% NaCl. Distilled water was used as water in each case.

Preparation of polymorphonuclear leukocytes (PMNL) 20 ml blood was mixed with 30 mg EDTA. Mix 15 ml of this blood with a syringe with 7 ml of dextran and bring to room temperature for 3
It was left for 0 minutes. The supernatant containing white blood cells was transferred to a sterilized centrifuge tube and centrifuged at 1000 rpm for 30 minutes.
The debris consists of white blood cells and the rest of the red blood cells, which can affect the measurement in terms of the loss of color. The remaining red blood cells were removed with a hypotonic alkaline solution. For this, the residue was suspended in 0.25% NaCl solution and mixed in a mixer for 20 minutes. Then mix 20 ml of 1.6% NaCl solution,
Mixed again. After centrifugation at 2000 rpm for 10 minutes, the supernatant was aspirated off and these steps were repeated twice. The residue was then suspended in 2 ml of PBS buffer and diluted 1:10 with Turcks solution. P in Neubauer hemocytometer
It was adjusted to 10 ⁶ cells / ml with BS buffer.

The test was performed as follows.

a) Blood: 460 μ veronal buffer 200 μ diluted blood 100 μ lucigenin solution 200 μ PBS dissolved in PBS and control as PBS b) PMNL: 630 μ veronal buffer 15 μ PMNL 100 μ lucigenin solution 200 μ PBS Dissolved material and PBS as control Preincubation for 10 minutes at room temperature, then 6-channel-bi
After culturing in olumat at 37 ° C. for 10 minutes, 40 μ of opsonin-added zymosan was mixed with stirring to start the measurement. The measurement was stopped after 60 minutes.

Evaluation of test results CL-reactions of the samples were stored in a computer during the measurement period, and after the measurement, displayed as a curve. By surface integration,
The CL-reaction intensity of each sample was obtained in cpm.

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

It is hypothesized from the literature that all extracts and substances which show a positive response in the granulocyte test according to the BRANDT method also give a positive result in the CL-test. It is not known how the two measurements correlate with each other and how the CL-method actually compares to the granulocyte test. Furthermore, it is necessary to compare the CL-measurement with separated granulocytes with the whole blood method.

For the CL-test of the purified substance, we chose the Na salt of aristoloquine for which the phagocytosis-enhancing effect is certain, and we have already obtained concentration-effect data from the granulocyte test.

Accuracy, reproducibility and sensitivity of the granulocyte and whole blood methods As expected, it was confirmed that measurement of isolated granulocytes at a relative rate of change of 7-8% is an accurate method. However, in whole blood, an average value of about 16% was obtained.

In the PMNL method, a significant increase in CL-response was recorded even at ^10-9 % dilution.

In contrast, the whole blood method is much less sensitive. 10 ^-3
There was a measurable increase in the concentration of%. It was a very low value at low concentrations. This is believed to be essentially due to the elimination effect of red blood cells that are still present despite diluting whole blood. The control cpm is generally lower in this test than in the PMML test.

These results limit the extent to which the two CL-methods are applied. Whole blood methods carried out in a short period of time can only be applied to the preselection of substances due to their immunostimulatory effects. However, dose-effect curves can be generated only by CL-measurement with separated granulocytes.

% Increase in CL-reaction by whole blood and PMNL method, test substance is aristoloquine Comparison of CL-method and granulocyte test As in the granulocyte test, a dose-dependent increase in phagocytic activity in the form of a "breathing burst" reaction was observed in the CL-test with isolated granulocytes. Was also found. The CL-reaction at various aristroquine concentrations is shown below. These measurement results correlated very well with the granulocyte test. However, the maximum value was 10 times lower in the CL-test with a concentration of 10 ⁻⁵ % than in the granulocyte test.

The% numbers calculated with the same formula in the granulocyte and CL-tests cannot be compared directly because the two measuring principles are different.
Granulocytes and CL-dose of aristroquin by test-
The effect-curve comparisons were in good agreement with the curved paths of the two test methods.

According to our test, the CL-method using separated granulocytes can be sufficiently replaced by the labor-intensive granulocyte test according to the BRANDT method in terms of accuracy and sensitivity, and further, time-dependent stimulation. The action record can describe in detail the postulated phagocytosis process.

3. Carbon-clearance test (CCT) according to the BIOZZI method. This in vitro test (CR Coc.B) developed by BIOZZI et al.
iol. 148, 431, 1954) (Progr. Liver Dis. 2, 166, 1965) was used to determine the phagocytic activity of tissue macrophages of the reticuloendothelial system (RES). Gelatin-stabilized carbon particles were injected intravenously into test animals, after a period of time, blood was drawn from the retro-orbital venous plexus and carbon release was measured photometrically. Kupfel cells in the liver part had 90% exogenous particle release and 10% macrophages in the spleen part.

The exponential function of carbon emission is shown by the straight line (logE vs t) whose negative slope is the regression coefficient K. If the potentially stimulated substance is for a certain period before the start of the test,
The increase in phagocytosis, given 24 times or 48 hours before, once or several times at a constant concentration, is determined by comparison with the regression coefficient by the blank value. If the relationship is K substance / ^K control <1, the test substance has an inhibitory effect on phagocytic activity. If the relationship is
If> 1, irritation occurs.

Since the regression coefficient K of carbon release differs depending on the weight of liver and spleen, respectively, the true release constant (α-value) is found only after these calculations are combined. In our tests with various substances, the α-value index showed a marked difference from the K-value index.

Test animals: female NMRI-mouse, 25-30g Number of collections: 3 in screening test or 5 in substance
Control and rearing: 2 cages for management and rearing: cage at 20 ° -22 ° C, SFI-standard rearing, water treatment method: A substance dissolved in 0.9% NaCl was administered once or several times. The solution should pass as a purified suspension to avoid cell-stimulatory effects. Intraperitoneal administration of the crude suspension is lethal to the animals. Individual doses were usually 0.1 and 10 mg / kg body weight. The final administration was carried out 24 hours before the start of the test.

The test was performed as follows.

An ink suspension containing 10% gas black (particle size 200-250A) was adjusted to contain 16 mg carbon per ml with 0.9% NaCl containing 1% gelatin, 37
Preheated to ° C. Each mouse contained 0.1 in the tail vein.
ml / 10g was injected. After t = 3,6,9,12,15 minutes,
Twenty-five microliters of blood was drawn from the retro-orbital vein plexus (with heparin-method-cyclopipette method). Blood was hemolyzed in 2 ml of distilled water, and the carbon concentration was determined by photometry to be λ = 650.
It was measured by nm (Hitachi-Spectrometer 181, cell thickness 1 cm).

Evaluation: The regression coefficient K was determined mathematically (logE vs. t) for carbon release as exponential.
An index ^D substance / ^K control could be created to determine the stimulatory or inhibitory effects of RES.

4. Lymphocyte Conversion Assay In a lymphocyte conversion assay, induction of normal lymphocyte embryo-conversion and subsequent mitosis was measured. For this, the substances to be tested were incubated with isolated lymphocytes or diluted whole blood, the preparations were marked with ¹⁴ C- and ³ H-thymidine and incubated again. Marked thymidine bound to cell-DNA was measured with a liquid scintillation spectrometer.

5. Cytotoxicity by the ³ H-thymidine-binding test on ascites cells Since a substance dissolves granulocytes at high concentrations in the granulocyte test, we have modeled the ³ H-thymidine-binding test on ascites cells. Testing for cell-damaging effects,
A relationship between increased phagocytosis and cytotoxicity in activators could be found.

This in vitro test system (Hoppe-Seyher's) according to WEITZEL et al.
Z.physiolg.Chem.348,433,1967), the cytotoxicity of the substance by inhibition of tumor cell DNA-synthesis changes the rate of ³ H-thymidine binding in rat Walter-250-carcinosarcoma ascites cells. Measured numerically by.

The test was conducted as follows.

3 ml Walker-250-carcinosarcoma cell suspension was mixed with 10 ml MEDIUM L-15 (Boehrringer) in a 50 ml centrifuge glass and centrifuged for 10 minutes. The supernatant was decanted and the centrifuge glass was filled up to 1 cm with medium from the lip. From the agitated cell suspension, 1 ml per sample (corresponding to 2 × 10 ⁶ cells / ml) was put in a centrifuge glass, 500 μ of test solution was added, and a control test was performed by adding 500 μ of medium. Each test was performed 4 to 6 times. After preincubation at 37 ° C. for 2 hours, 100 μ of ³ H-thymidine (50 Ci / nM) was added per sample, and the cells were further incubated under stirring. Then 1.5 ml of 1N-perchloric acid was added per sample to stop the binding and the preparation was stored in the freezer overnight. Residues containing unbound precursors are absorbed and removed with paper moistened with perchloric acid
It was washed with trichloroacetic acid and again with ethanol.
The filter was placed in a polyethylene flask and the activity was measured with a toluene scintillator. The results were compared with the control and the inhibitory effect was shown in%.

Comparison of test results In each test, the rate of binding inhibition by the same substance was 30
Since various changes were shown up to%, Methotrexat, which inhibits DNA-synthesis as an antagonist of folic acid, was tested as a reference substance in each test series. Methotrexat
The mean value of the inhibitory effect (43% at 2 mg / ml) was used to determine the relative inhibitory effect H of the substances.

By comparing the obtained results with the results of the granulocyte test, the idea composed of other low molecular weight compounds was confirmed,
According to this, even if the substance showing cytotoxin activity at a high concentration was diluted to 10 ^-2 or 10 ^-3 % again, it had an effect of stimulating the phagocytic effect of separated granulocytes. Even if this can be said generally, it is necessary to further elucidate it by applying it to polymer compounds.

6. Acute toxicity test animals: female or male NMRI-mouse, 30-35g
Management and feeding: 20 ° -22 ° C. cage SFI-standard feeding, water Number of collections: 5 mice The test was carried out as follows: test substance dissolved in water or 0.9% NaCl solution via the throat etc. It was administered to mice.
The behavior of the control group was observed for 14 days.

The prerequisite for all tests is complete solubility of the test material. In the test of lipophilic extracts or substances, compounds such as carboxymethylcellulose or dimethylsulfoxide added as solubilizers can be used as control values at test concentrations because the solubilizers have some self-stimulating effect. used. In CCT, the use of DMSO is discouraged due to high toxins.

Furthermore, the test solution should be prepared as free from bacteria as possible. Gram-positive bacteria contain cell wall polysaccharides that have some of the effects of stimulating the phagocytic effect of granulocytes. The cell wall lipopolysaccharide of Gram-negative bacteria is called so-called endotoxin and affects the test results. Live bacteria and bacterial extracts show a high RES-stimulating effect.
Furthermore, certain endotoxins have side effects such as pyrogenicity, antigenicity and metabolic alterations of bacterial toxins,
These can also be affected in CCT.

It is further advisable to sterilize the test solution and store it in the freezer.

In order to characterize the principle of the effectiveness of the alkaloid oxindole according to the invention,
NH raw water and ethanol extract of Tormen Noosa
_{The three} treated powders were tested, as well as the crude alkaloid mixture, purified alkaloids isolated as well as oxindoles of other alkaloids. The stimulatory effect of NH ₃ alone was previously ruled out.

For the preparation of the extract, the powder treated with 10% NH ₃ alkali and the dried agent were mixed with the eluent in a ratio of 1:10 and extracted with a magnetic stirrer for 2 days at room temperature,
The product was concentrated and dried.

The crude alkaloid mixture was prepared according to the following method. The dried herbs were moistened with 10% NH ₃ , left at room temperature for 1 hour and carefully dried by IR. The drug was then softened by moistening with ethyl acetate for 4 days with a magnetic stirrer. The crude extract thus obtained was concentrated, and the aqueous layer was positive for Vals.
er- until no indicated the reaction was extracted with _2% H 2 SO _4. The aqueous layer was mixed with NaCO ₃ until it became alkaline with pH 8-9. By repeated extraction of the aqueous layer with ethyl acetate, 750 g of drug yielded 6.75 g of light brown crude alkaloid mixture, corresponding to a yield of 0.9%. It was then converted to the salted form of hydrochloric acid.

Individual oxindole, alkaloids were isolated and identified from the crude alkaloid mixture.

They are as follows:

1. Arrow isoprene terrorist positive down the formula _C 21 _H 24 _O 4 _{N 2}
Isomer A with MG: 368 m.p. p. : 204 ° -209 ° Thin layer chromatography: R _f 0.73 (LM I); 0.48 (LM I
I); 0.83 (LM III) HPLC: Rt 8.8min (TS I) [α] ²⁰ _D : -85.1 (c = 0.554 / CHCl ₃ ) UV (MeOH) = λ _max = 208,243,283 (sh) Mass spectrum: m / e (rel.int.): 368 (M ⁺ , 100), 223 (7
0), 208 (35), 180 (14), 146 (11), 145 (9), 144 (5), 130 (25), 6
9 (45) Arrow pterocaryoside positive emissions, isomer B having the formula _{C 21 H 24 O 4 N 2} , MG: 368 m. p. : 214 ° -219 ° C. Tlc: R _f 0.72 (LM I), 0.47 (LM II), 0.81 (LM III) HPLC: Rt 0.8min (TS I) UV (MeOH) = λ _max = 208,243,283 (sh) Mass spectrum : M / e (rel.int.): 368 (M ⁺ , 100), 223 (8
4), 208 (25), 180 (20), 146 (8), 145 (11), 144 (11), 130 (14),
69 (35) 3. N - iso Mitra Villingen formula _C 21 _{H 24} O
Isomer A with ₄ N ₂ ; MG 368 m.p. p. : 96 ° -106 ° C. (Z) Tlc: R _f 0.71 (LM I); 0.47 (LM II), 0.80 (LM III) HPLC: Rt 9.2 min (TS I) [α] ²⁰ _D : + 145 ° (c = 0.758 / CHCl ₃ ) UV (MeOH) = λ _max = 208,243,283 (sh) MS: M / e (rel.int.): 368 (M ⁺ , 70), 223 (100), 208 (1
0), 180 (4), 146 (6), 145 (5), 144 (7), 130 (11), 69 (29) 4. N - Mitorafirin formula _C 21 _H 24 _O 4 N
Isomer B with ₂ ; MG 368 m.p. p. : 264 ° -268 ° C Tlc: R _f 0.55 (LM I), 0.39 (LM II), 0.50 (LM III) HPLC: Rt 10.4min (TS I) [α] ²⁰ _D : -4.3 ° C (c = 0.587 / CHCl ₃ ) UV (MeOH) = λ _max = 208,243,283 (sh) Mass spectrum: m / e (rel.int.): 368 (M ⁺ , 60), 223 (1
00), 208 (10), 180 (4), 146 (6), 145 (7), 144 (10), 130 (10), 6
9 (27) Solvent mixture used (LM) for Tlc LM (I) chloroform-acetone (5: 4) LM (II) chloroform-ethanol (95: 5) LM (III) ethyl acetate-isopropanol-N
H ₃ conc. (100: 2: 1) Uncaria Tormentosa In addition to these oxindole alkaloids isolated from Tormentosa, there is an oxindole alkaloid speciophylline contained in Uncaria spp. , And Gelsemium
Gelsemin, an oxindole alkaloid known as one of the main components of the roots of um semervirens) was used at least as part of the immunological test.

To prepare the water-soluble hydrochloric acid salt, the alkaloid base was dissolved in ether and HCl-gas was introduced from a gas cylinder. The ether was removed, the hydrochloric acid salt was washed again with anhydrous ether and then dried over phosphorus pentoxide in a vacuum desiccator.

The increasing rates of phagocytosis of different extracts and substances were established in the granulocyte test, each at different concentrations.

Cytotoxicity was most often observed at concentrations of 10 ^-1 %.

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

Isoquinoline used for viral eye inflammation
The alkaloid berberine was inactive up to concentrations of ^10-4 %.

Vincristine (aspidospermine type indole
Alkaloids) and chamotesin (quinoline alkaloids) showed no effect even when diluted to 10 ⁻⁶ %. These compounds are used as mitosis-inhibiting agents and carcinostatic agents. In the case of pinklistin, the in vitro-inhibitory effect of macrophages-phagocytosis is ^10-4.
% And ^10-5 % concentration.

Baptisia tinctoria
The main alkaloid of all plants of the family Lycaenidae, the quiniridindin alkaloid cytisine, is used in septic diseases and ulcers, but it is ineffective at concentrations up to 10 ^-4 %.

In the test for the bisbenzylisoquinoline alkaloids talipaverine, taliphabine and fangusanine (tested up to 10 ⁻⁴ %) isolated from Thalictrum faberi (Diptera),
Despite their use as anti-inflammatory agents and folk remedies for the treatment of gastric cancer in China, they have shown no effect.

Among the purified alkaloids separated from the mixture of alkaloids, there is a difference in the effect and the maximum effect is uniformly 10 ^−2.
It was seen despite being in the concentration range of% -10 ^-5 %. The results found in the granulocyte test according to the BRANDT method were confirmed by the chemiluminescence test. The reason for the difference between the results of the two studies (eg due to isolincophilin) has not yet been found. The following table shows the percentage increase in CL-reactivity of the oxindole alkaloids isolated from the crude alkaloid mixture and Uncaria tormentosa.

The stem extract of Uncaria sp., Which contains a small amount of alkaloids, did not show high phagocytic activity for the usual 10% and 20% increase rates.

In contrast, roots containing isopteropodin showed values of 30% and 40% on average.

Leaves with high alkaloid concentrations, such as root, showed a different situation. The 1983 harvested sample did not contain isopteropodin and showed a slightly higher stimulatory value compared to the stem, and the 1981 harvested leaves showed 30% and 40% increase rates. . This leaf contained as much isopteropodin as the root concentration.

Aside from the alkaloid content of the drug, the effect depends on the pattern of alkaloids identified by the combined various phagocytosis enhancing rates of the individual alkaloids.

Examination of the crude alkaloid mixture in the form of a water-soluble hydrochloric acid salt showed an average phagocytosis enhancement of 20%. Since the latter was not higher than in the whole extract with ethanol, water and the substances concomitantly present in the ethanol extract are likely to have an adjuvant role in increasing the effect. This means that the water-soluble catechin extract (pH 6.
10 ml of a 1% aqueous solution of 0) of 0.
It was confirmed by mixing with 10 ml of a 1% aqueous solution, stirring with a magnetic stirrer at room temperature for 24 hours, freeze-drying and finally conducting a carbon-clearance test.

The results of all materials carbon-clearance-tested according to the BIOZZI method are summarized in the table below.

As in the granulocyte test, the efficacy of alkaloids was found in this test as well. Water soaks containing alkaloids stimulate RES to a greater extent than water soaks with low alkaloid content. When the alkaloid mixture was applied together with catechin, which itself had no irritating effect, a similar increase in activity was obtained as in the water-soaked softener.

Lymphocyte conversion test Test whole water extract from Uncaria tormentosa,
A mixture of alkaloids made from Uncaria tormentosa and two mixtures P ₁ and P ₂ were tested. P ₁ is
The alkaloids Isopteropodin, Pteropodin,
And isomitraphyrin, which are active in the granulocyte test, and P ₂ contains the inactive alkaloids mitraphyrin and lincophilin.

Also in also measured by separate lymphocytes in whole blood method, any substances of degree greater than natural conversion ³ tested
No increase in H-thymidine-binding was shown.

Acute Toxicity, Cytotoxicity and Antitumor Efficacy Acute toxicity studies were carried out on mice with whole water extracts from a mixture of Uncaria tormentosa and alkaloids produced by embroidery. The extract was administered orally at a maximum concentration of 5 g / kg body weight and intraperitoneally at 2 g / kg. Alkaloid mixture up to 2 g / kg orally and 1 intraperitoneally
Dosing up to g / kg. When the mice were observed for 14 days or longer, no change in normal behavior was observed.

Cytotoxicity tests were performed on ascites cells by the ³ H-thymidine-binding test. Various extracts and alkaloid concentrations were tested. Proportion of binding inhibition of extract and alkaloid concentrate from Uncaria tormentosa in ³ H-thymidine binding assay a) Cytotoxicity comparison at 0.2% dose b) Proportion of binding inhibition at various concentrations As can be seen, a concentration of 10 ^-1 % alkaloid concentrate has a cytotoxic effect, the more diluting the less toxic. These results show that the alkaloid concentrate P ₁
And P ₂ cytotoxicity test. At concentrations up to 5 μ / ml, the growth of the two cell types is unaffected. Only at the dose of 50 μ / ml (5 × 10 ⁻³ %), the two samples showed a slight cytotoxic effect.

Two cytotoxicity tests revealed a correlation between cytotoxicity and immunostimulatory activity. For example, total alkaloid mixture, to dissolve the granulocytes at concentrations of 10 ^-1%. An immunostimulatory effect was observed with further dilutions with no or little cytotoxic effect. ³ H-
Thymidine - in binding tests, a mixture of Mitorafirin alkaloids which are inert under the granulocytes test P _2, it is interesting that low toxin than a mixture of P ₂ and the active alkaloids.

Claims (6)

[Claims] 1. A formula C ₂₁ H as a component of an immune system stimulant.
An oxindole alkaloid selected from the group consisting of isopteropodin having ₂₄ O ₄ N ₂ , pteropodin, isomitrafirin and mitrafirin. 2. The oxindole alkaloid according to claim 1, wherein at least one is an arrow isomer. 3. The oxindole alkaloid according to claim 1, wherein the immune system stimulant is an aqueous solution of the oxindole alkaloid in the form of a salt with addition of hydrochloric acid at a concentration of 10 ⁻³ % to 10 ⁻⁶ %. . 4. The oxindole alkaloid according to claim 1, wherein the immune system stimulant contains at least one oxindole alkaloid. Arrowisopteropodin, isomer A having the formula: Arrow telopodin, isomer B having the formula: Normal-isomitraphyrin, isomer A having the formula: Normal-mitrafylin, isomer B having the formula 5. The oxindole alkaloid according to claim 1, wherein the immune system stimulant contains at least one alkaloid-catechin complex. 6. The water-soluble catechin extract 1 as the immune system stimulant.
0 parts by volume and water-soluble oxindole alkaloid solution 1
The oxindole alkaloid according to claim 1, which is a mixture with 0 part by volume.