CN112107577A - Quinolizidine Cu+And or Fe2+Use of chelating agents and pharmaceutically acceptable salts thereof - Google Patents

Abstract

The invention discloses a Cu-containing alloy for the first time⁺And or Fe²⁺The application of the quinolizidine chelating agent with the ion chelation effect and the medicinal salt thereof in copper overload and iron overload diseases creatively provides important guidance for accurate and reasonable clinical medication of quinolizidine medicaments.

Description

Quinolizidine Cu+And or Fe2+Use of chelating agents and pharmaceutically acceptable salts thereof

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a Cu-containing composite material⁺And/or Fe²⁺Use of an ion-chelating alkaloid chelator and pharmaceutically acceptable salts thereof in human or other mammal in a disease associated with copper overload and/or iron overload.

Background

Metal elements are vital substances essential for microorganisms, animals and human beings to maintain vital activities, and affect the vital activities of the body by interacting with biological macromolecules, such as acting as the structure or active center of metalloenzymes, causing changes in specific conformations of proteins and nucleic acids, and the like. According to statistics, nearly 40% of known enzyme systems need metal ions to act as cofactors of enzyme activity parts, so that the research on the metabolic process of a metal ion organism is not only helpful for people to know the life activity process, but also greatly helpful for the prevention and treatment of animal and human diseases and the treatment of environmental pollution.

Copper is one of essential trace metal elements in the body, and in a normal adult, the copper is about 100-200 mg, 50-70% is distributed in muscles and bones, 20% is in the liver, and 5-10% is in the blood. The daily intake of copper in humans is generally 2 mg, with a maximum tolerance of about 8 mg, with Cu being obtained from food primarily through the small intestine²⁺The blood is absorbed in a form, and then the blood is distributed to various tissues of the organism through protein combination (for example, ceruloplasmin combines 90-95% of copper ions in the blood), and the blood participates in the life activities such as oxidation reduction, bone growth, inflammatory immunity, nerve, hematopoiesis, angiogenesis and the like mediated by cytochrome C oxidase, lysyl oxidase, dopamine beta hydroxylase, superoxide dismutase, tyrosinase and the like. However, when the free copper in the blood is too much (containing cuprous ions Cu)⁺And copper ion Cu²⁺, 2Cu²⁺ + 2GSH ⇋ 2Cu⁺ + GSSG + 2H⁺) That is, excessive free copper not bound to ceruloplasmin is distributed and deposited in relevant organs and tissues of the body such as liver, brain, heart, blood vessels and kidney, and causes the generation of hydroxyl radicals and lipid peroxidation in cells, resulting in oxidative stress damage, and thus leading to hereditary and non-hereditary copper overload diseases (discussion of copper uptake and transport and related gene diseases).Chemical industry of Guangdong province. 2016, 4397) such as hereditary Wilson's disease (clinically manifested primarily as liver damage, extrapyramidal symptoms, corneal chroman rings, kidney damage, damage to the blood system, etc.) and non-hereditary Copper-overload diabetes, scleroderma (Copper and the synthesis of elastins and collagen. In: Biological roles of Copper. Ciba Foundation Symposium 79.Amsterdam: Excerpta Medica1980, 163), oral submucosal fibrosis (rased tissue copolymers in oral submucous fibrosis.J Oral Pathol Med. 2000,29241), xeroderma pigmentosum, pellagra, sjogren's syndrome, primary pulmonary hypertension, cardiovascular Disease, tumor invasion and metastasis, amyotrophic lateral sclerosis, parkinson and alzheimer's Disease, etc. (New rolls for cope Metabolism in Cell promotion, Signaling, and Disease.J Bio Chem. 2009, 284, 717；Targeting copper to treat breast cancer. Science. 2015, 349128; copper metabolism and its role in neurological diseasesChina A journal for the elderly.2016, 36, 5456). Relative to Cu²⁺Free Cu⁺More easily leading directly to oxidative stress damage (Cu)⁺ + H₂O₂ ⇋ Cu²⁺ + OH˙ +OH^-) Thus, it was found to have Cu⁺Chelated copper chelators are safe and effective in reducing excess free Cu in the human body⁺And Cu²⁺And lowering OH^·The generation of the above-mentioned substances and the improvement of the oxidative stress damage caused by the above-mentioned substances are potential therapeutic means for the above-mentioned diseases caused by copper overload (chemical therapy in Wilson's disease: from D-penicillamine to the design of selective biochemical intercellular Cu (I) molecules).Dalton Trans. 2012, 41, 6359）。

The iron is one of essential trace metal elements in the organism, the normal adult contains about 4-5 g of iron, the daily intake of the iron is generally 10-15 mg, and the highest tolerance is about 50 mg. The iron in human body can be divided into two categories, functional iron and storage iron according to functions. Functional iron accounts for about 70% of the total iron in the human body, including hemoglobin (about 65%), myoglobin (about 3%), cerebroglobin (about 0.5%), and other iron-containing enzymes (about 1%); stored iron, which accounts for approximately 30% of the total iron content of the human body, is present mainly in the form of ferritin (about 25%) and ferrihemoxanthin in the liver, spleen, bone marrow, nuclei of the extrapyramidal tract of the brain, intestines and placenta. In the human body, iron is mainly extracted from food through the duodenum and upper jejunum as Fe²⁺Form absorption (The type IV mucositis-associated protein TRPML1 is an endolysomic screw release channel.Nature2008, 455, 992), absorbed Fe²⁺A part of the Fe is oxidized into Fe in epithelial cells of small intestinal mucosa³⁺Then storing in soluble ferritin for organism recycling (Fe)²⁺Absorption → Fe³⁺Storage → Fe²⁺Release utilization); the other part absorbs the Fe absorbed into the blood²⁺Oxidation of ceruloplasmin to Fe³⁺And is combined with transferrin in plasma to be distributed to various tissues of the body, and then the combination with transferrin receptor starts the endocytosis of cells to release Fe²⁺And released into the cytoplasm via transport of divalent metal transporters (Fe)²⁺Absorption → Fe³⁺Transport → Fe²⁺Liberation utilization), participate in oxygen transport and electron transport mediated by hemoglobin, oxido/reductases, nitrogenase, peroxidase and the likeTransmission, oxidative phosphorylation, DNA biosynthesis, neurotransmitter synthesis, NO metabolism, hematopoiesis, and immune defense. However, when free Fe is present in blood²⁺And or Fe³⁺Too much, i.e. not effectively bound to ferritin, Fe²⁺、Fe³⁺Excessive Fe will be distributed and deposited in the relevant organs and tissues of the body, such as liver, spleen, pancreas, bone marrow, brain, lung, intestinal tract, kidney and heart²⁺Can be directly connected with H₂O₂Generating more toxic hydroxyl radical OH through Fenton reaction^·And Fe³⁺And O is^2·-With Fe³⁺Fe can be produced again by Haber-Weiss reaction²⁺Thus, the redox cycling reaction can generate a large amount of active oxygen radicals, damage cellular substructure, and induce apoptosis (Muhoberac BB, Vidal R).Front Aging Neurosci. 2013, 532), which in turn causes genetic and non-genetic iron overload diseases (Body iron metabolism and pathobiology of iron overload).Int J Hematol. 2008; 88, 7；Iron Overload in Human Disease. N Engl J Med2012, 366, 348), such as hereditary hemochromatosis (clinically manifested primarily as skin pigmentation, liver cirrhosis, secondary heart disease, arthritis, diabetes), thalassemia (clinically manifested primarily as anemia, progressive exacerbation of hepatosplenomegaly, jaundice, etc.), and myelofibrosis caused by non-hereditary Iron overload, myelodysplastic syndrome, osteoporosis, chronic kidney disease, myocardial infarction, cerebral hemorrhage (Iron and Iron-handling proteins in the brain after membrane arterial hemorrhage.Stroke. 2003, 342964), neurodegenerative diseases, hepatic fibrosis, nonalcoholic fatty liver, pancreatitis, secondary hemochromatosis, delayed-onset porphyria cutanea, Mitochondrial iron overload disease (mitochondrion iron overload: consumers and sequences).Curr Opin Genet Dev. 2016, 3831), liver cancer, lung cancer, colon cancer, esophageal cancer, gastrointestinal tumors, pancreatic cancer, and breast cancer (application of ferritin detection in clinics).Marker immunoassay and clinical. 2012, 19, 378). Thus, Fe was developed²⁺And or Fe³⁺Ion chelators, especially developed with Fe²⁺Chelating chelators are safe and effective in reducing excess iron ions and hydroxyl radical OH in humans^·The generation and the improvement of oxidative stress injury brought by the iron overload are potential treatment means for the diseases caused by the iron overload.

Notably, the copper and iron metabolism are not independent of each other; rather, they have a close relationship and influence (location of a glutathione and a negative cell death by the transformation parameters iron and copper: indications for the use of a related neuro-influence series).Free Radical Bio Med. 2018, 115, 92). For example, when ceruloplasmin is deficient or binding is overloaded, it not only easily leads to free Cu in blood⁺Andor Cu²⁺Excessive, copper-overload disease and easy to cause Fe in blood plasma²⁺Can not be converted into Fe in time³⁺And binds to transferrin, resulting in excess plasma free Iron ions causing Iron overload Disease (Regulation of coater and Iron Homeostatis by Metal Chemists: A polymeric Chemotherapy for Alzheimer's Disease. Acc. Chem. Res. 2018, 48, 1332). As another example, in Parkinson's disease, an increase in iron concentration in The substantia nigra is accompanied by a decrease in The ceruloplasmin ferrous oxidase activity (The neuron and manganese in Parkinson's and Wilson's diseases.J Trace Elem Med Bio. 2015, 31193), which in turn causes plasma copper ion overload. As another example, clinical studies have found that diabetic patients have significantly higher Serum iron (11%) and copper (8%) levels relative to the normal group, and are primarily characterized by increased levels of free iron and free copper (Serum coppers, zinc, and iron levels, and markers of carbohydrate metabolism in a postmetabolic tissue with precursors and types 2 diabetes mellitis.J Trace Elem Med Bio. 2017, 43, 46). Clearly, in the above cases, single chelating metal chelators tend to be difficult to effectively ameliorate the associated disease caused by copper overload and/or iron overload. Thus, develop and becomeThe chelating agent which acts on iron ions and copper ions simultaneously can safely and effectively regulate the excessive free iron and free copper levels in human bodies, and is a more effective potential treatment means for related diseases caused by copper and/or iron overload.

Disclosure of Invention

Although chelating agents against copper overload such as penicillamine, triethylene tetramine (TETA), dimercaptosuccinic acid (DMSA), sodium Dimercaptopropanesulfonate (DMPS), dimercaptopropanol, and desferrioxamine, deferiprone and deferasirox against iron overload have been successively marketed clinically, there are still many problems with this class of drugs in clinical application. For example, the copper overload chelator penicillamine can cause more side effects of allergy and digestive system, and even cause serious side effects such as bone marrow suppression and kidney damage; however, metal chelating agents containing mercapto groups such as dimercaptosuccinic acid often have unpleasant odor, and can easily cause kidney damage after long-term administration; although iron chelators such as deferasirox have good iron chelation selectivity, the problem of effectively improving the metabolic disorder of copper ions caused by iron overload is not reported. More importantly, the metal chelating agent is used for Cu⁺And or Fe²⁺Induced hydroxyl radical OH^·There is no significant improvement in the formation of (i) and the oxidative stress injury it brings. Therefore, there is an urgent need in the art to develop a novel Cu-containing alloy⁺And/or Fe²⁺Metal chelators that chelate and reduce oxidative stress damage they cause.

Based on the above analysis, the present invention provides a Cu-containing alloy⁺And/or Fe²⁺Ion-chelating quinolizidine alkaloid chelating agents and pharmaceutically acceptable salts thereof, are useful for treating diseases caused by copper and/or iron overload.

Preferably, the treatment of diseases caused by copper overload is mainly Wilson's disease, scleroderma, oral submucosal fibrosis, xeroderma pigmentosum, pellagra, Sjogren's syndrome, amyotrophic lateral sclerosis.

Preferably, the treatment of diseases caused by iron overload is mainly hereditary hemochromatosis, secondary hemochromatosis, delayed porphyria cutanea dermalis, mitochondrial iron overload disease, thalassemia, myelofibrosis, myelodysplastic syndrome.

The quinolizidine alkaloid Cu⁺And/or Fe²⁺The ion chelating agent and its pharmaceutically acceptable salt are used for the treatment of hereditary Wilson's disease, more preferably, for improving liver damage, extrapyramidal symptoms, corneal tryptophane ring, kidney damage, and blood system damage caused by Wilson's disease.

The quinolizidine alkaloid Cu⁺And/or Fe²⁺The ion chelating agent and its pharmaceutically acceptable salt are used for treating thalassemia, and more preferably, for improving hereditary hemolytic anemia, progressive exacerbation of hepatosplenomegaly and jaundice symptoms caused by thalassemia.

The quinolizidine alkaloid Cu⁺And/or Fe²⁺Ion chelating agent and its pharmaceutical salt, preferably, the quinolizidine alkaloid is matrine, oxymatrine, sophocarpine, oxysophocarpine, sophoridine, oxysophoridine, the main structure is as follows:

。

the invention also provides pharmaceutically acceptable salts of the quinolizidine alkaloid metal chelating agents.

As used herein, the term "pharmaceutically acceptable salt" refers to a non-toxic acid salt of a compound of the main structural formula. These salts can be prepared in situ during the final isolation and purification of the compounds of the main formula or separately by reacting a suitable organic or inorganic acid with a basic functional group. Representative acids include, but are not limited to, hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, acetic acid, citric acid, phenylbutyric acid, fumaric acid, monomethyl fumarate、Oxalic acid, malonic acid, valproic acid, salicylic acid, malic acid, glucoheptonic acid, digluconic acid, aspartic acid, alginic acid, lactic acid, picric acid, succinic acid, glycerophosphoric acid, heptanoic acid, nicotinic acid, oxalic acid, hexanoic acid, succinic acid, mandelic acid, ascorbic acid, maleic acid, tartaric acid, benzenesulfonic acid, methanesulfonic acid, taurine or isethionic acid (see: salt formation: tartaric acid, benzenesulfonic acid, methanesulfonic acid, taurine or isethionic acid)Research and development of drugs.Pharmaceutical advances. 2012, 36, 151). In addition, the nitrogen-containing basic groups may be quaternized with the following agents: alkyl halides such as methyl, ethyl, propyl, butyl chlorides, bromides and iodides; dialkyl sulfates such as dimethyl, diethyl, dibutyl and diamyl sulfates; long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides; aralkyl halides such as benzyl and phenethyl bromide, and the like. Thus obtaining a water-soluble or oil-soluble or dispersible product.

According to a second aspect of the invention, there is provided a method of treating Cu⁺And or Fe²⁺The medicine composition for treating overload caused relevant diseases contains at least one kind of orally taken medicine prepared with compound in the main structure or its medicinal salt, or nanometer medicine injection preparation prepared with liposome, albumin, polymer and inorganic nanometer particle, or application preparation, spray preparation or composition combined with other medicine.

Advantageous effects

1. The quinolizidine alkaloid chelating agents of the present invention not only have the effect of chelating Cu, as opposed to the existing copper chelating agents such as triethylene tetramine⁺And can remarkably suppress Cu⁺Catalysis H₂O₂The generated hydroxyl free radical reduces the oxidative stress injury of the organism.

2. The quinolizidine alkaloid chelating agents of the invention not only have the function of chelating Fe, as opposed to the existing iron chelating agents such as deferasirox²⁺And can remarkably inhibit Fe²⁺Catalysis H₂O₂The generated hydroxyl free radical reduces the oxidative stress injury of the organism.

3. The invention reports that quinolizidine alkaloids have obvious Cu chelate for the first time⁺And or Fe²⁺The method creatively provides important guidance for accurate and reasonable clinical medication of quinolizidine alkaloid-related medicaments.

Drawings

FIG. 1 is a bone marrow iron staining chart of rat model with iron overload after matrine administration.

FIG. 2 is a graph showing the effect of matrine administration of the present invention on the relative thickness of the epidermis in xeroderma pigmentosum mice.

Detailed Description

For the sake of understanding, the present invention will be described in detail below by way of specific examples. It is to be expressly understood that the description is illustrative only and is not intended as a definition of the limits of the invention. Many variations and modifications of the present invention will be apparent to those skilled in the art in light of the teachings of this specification.

Example 1 quinolizidine alkaloid vs. Cu⁺Chelation Studies

The experimental principle is as follows: 2, 9-Dimethylphenantholine (Dmphen) and Cu⁺Formation of a stable yellow complex Cu (dmphen) in weak acid and neutral solution₂ ⁺The relationship between the absorbance and the concentration of the compound accords with the Lambert-beer law. And the added cuprous chelating agent can competitively chelate Cu with 2, 9-dimethyl phenanthroline⁺Thereby causing a decreasing change in absorbance.

And (3) test solution and preparation: (1) 0.1 g/L copper standard solution: 0.025 g of CuCl was weighed, dissolved in 7 mL of 2N HCl solution and transferred to a 1L volumetric flask, which was then diluted to 0.1 g/L with distilled water. (2) 1 mol/L NaAc solution. (3)1.5 g/L2, 9-dimethyl phenanthroline water solution: is prepared at the time of clinical application. (4) 10% hydroxylamine hydrochloride solution. (5) Matrine samples to be tested: the preparation concentrations are respectively 0 and 10^-5、10^-4、10^-3、10^-2A matrine aqueous solution of mol/L.

The experimental method comprises the following steps: taking 3 test tubes, respectively defining test tube 1 as 2, 9-dimethyl phenanthroline (A)_dmphen) Test tube 2 is blank reference (A)_{Ginseng radix (Panax ginseng C.A. Meyer)}) Test tube 3 is the sample (A)_{Sample (A)}) According to the requirements of table 1, 2 mL of 0.1 g/L CuCl standard solution, 1 mL of hydroxylamine hydrochloride with volume fraction of 10%, 2 mL of matrine sample solution to be tested with different concentrations, 2 mL of 1.5 g/L2, 9-dimethyl phenanthroline and 1 mol/L NaAc 5 mL are respectively added, and each reagent is initially and uniformly mixed. Then, the volume is adjusted to 50mL by deionized water, and the mixture is fully shaken up. Finally, the solution is subjected to colorimetry (with the wavelength of 457 nm) by using an automatic enzyme-labeled reading instrument, and the value of absorbance (A) is measured, wherein the smaller the value of A, the stronger the copper ion chelating capacity is. Each sample was repeated 3 times and averaged.

TABLE 1 chelating agent Cu for quinolizidine alkaloids⁺Sample adding table for chelating effect experiment research

	dmphen (2 mL)	Copper standard solution (2 mL)	Sample (I)	Other reagents
					Test tube 1 (A)phen）	+	+	-	+
Test tube 2 (A)Ginseng radix (Panax ginseng C.A. Meyer)）	+	-	-	+
					Test tube 3 (A)Sample (A)）	+	+	+	+

^*Sample pair Cu⁺The chelation of (a) can be formulated as: c = [ 2 ] _{phen Sample (A) phen}A-A]/[A-A_Ginseng)]×100%。

Detection indexes are as follows: drawing a concentration-chelation curve, and obtaining the corresponding concentration when the chelation is 50 percent from the curve, wherein the corresponding concentration is defined as half of the chelation concentration EC₅₀The results are shown in Table 2.

TABLE 2 chelating agent Cu for quinolizidine alkaloids⁺Chelation

Compound (I)	Cu + chelate EC50	Compound (I)	Cu + chelate EC50
				Matrine	B	Matrine monomethyl fumarate	A
Oxymatrine	A	Matrine fumarate	A
				Sophocarpine	C	Matrine valproate	B
Oxyphylline	B	Sophocarpine monomethyl fumarate salt	B
				Matrine hydrochloride	B	Sophocarpine maleate	B
Matrine methanesulfonate	B	Sophocarpine valproate	B
				Matrine maleate	A	Triethylene tetramine	C

Note: a < 100. mu.M; b, 100-500 mu M; c > 500. mu.M.

Example 2 quinolizidine alkaloid pairs Fe²⁺Chelation Studies

The experimental principle is as follows: phenanthroline (Phen) and Fe²⁺Generating a stable orange-red complex Fe (phen) in a solution with pH = 3-9₃ ²⁺The relationship between the absorbance and the concentration of the compound accords with the Lambert-beer law. While the ferrous chelator can competitively chelate Fe with phenanthroline²⁺Thereby causing a decreasing change in absorbance.

And (3) test solution and preparation: (1) 0.1 g/L iron standard solution: weighing 0.1 g(NH₄)₂Fe(SO₄)₂·6H₂O, dissolved in 7 mL of 2N HCl solution and transferred to a 1L volumetric flask, which is then diluted to 0.1 g/L with distilled water. (2) 1 mol/L NaAc solution. (3)1.5 g/L of an o-diazaphenanthrene aqueous solution: is prepared at the time of clinical application. (4) 10% hydroxylamine hydrochloride solution. (5) Matrine samples to be tested: the preparation concentrations are respectively 0 and 10^-5、10^-4、10^-3、10^-2A matrine aqueous solution of mol/L.

The experimental method comprises the following steps: taking 3 test tubes, respectively defining test tube 1 as phenanthroline (A)_phen) Test tube 2 is blank reference (A)_{Ginseng radix (Panax ginseng C.A. Meyer)}) Test tube 3 is the sample (A)_{Sample (A)}) The standard solution (NH) was added according to the requirements of Table 3₄)₂Fe(SO₄)₂·12H₂O0.1 g/L) 2 mL, 10% hydroxylamine hydrochloride 1 mL, matrine sample solutions to be tested with different concentrations 2 mL, 1.5 g/L phenanthroline 2 mL, 1 mol/L NaAc 5 mL, and each reagent is added and mixed uniformly. Then, the volume was adjusted to 50mL with deionized water and shaken well. Finally, the solution is subjected to colorimetry (with the wavelength of 510 nm) by an automatic enzyme-labeled reading instrument, and the absorbance (A) value is measured, wherein the smaller the A value is, the stronger the iron ion chelating capacity is. Each sample was repeated 3 times and averaged.

TABLE 3 matrine chelating agent Fe²⁺Sample adding table for chelating effect experiment research

	Phen (2 mL)	Iron standard solution (2 mL)	Sample (I)	Other reagents
					Test tube 1 (A)phen）	+	+	-	+
Test tube 2 (A)Ginseng radix (Panax ginseng C.A. Meyer)）	+	-	-	+
					Test tube 3 (A)Sample (A)）	+	+	+	+

^*Sample pair Fe²⁺The chelation of (a) can be formulated as: c = [ 2 ] _{phen Sample (A) phen}A-A]/[A-A_Ginseng)]×100%。

Detection indexes are as follows: drawing a concentration-chelation curve, and obtaining the corresponding concentration when the chelation is 50 percent from the curve, wherein the corresponding concentration is defined as half of the chelation concentration EC₅₀The results are shown in Table 4.

TABLE 4 matrine chelating agent Fe²⁺Chelation

Compound (I)	Fe2+Chelated EC50	Compound (I)	Fe2+Chelated EC50
				Matrine	B	Matrine monomethyl fumarate	A
Oxymatrine	A	Matrine fumarate	A
				Sophocarpine	C	Matrine valproate	B
Oxyphylline	B	Sophocarpine monomethyl fumarate salt	B
				Sophoridine	B	Sophoridine maleate	B
Matrine methanesulfonate	B	Sophocarpine valproate	B
				Matrine maleate	A	Deferiprone	A

Note: a is less than 50 mu M; b, 50-100 mu M; c, 100-500 mu M; d > 500. mu.M.

Example 3 chelating agent of quinolizidine alkaloids vs. Cu⁺Induced hydroxyl radical scavenging experiments

The experimental principle is as follows: the hydroxyl radical may be formed from Cu⁺-H₂O₂The system is generated through a quasi-Fenton reaction, and the absorbance of rhodamine B is obviously reduced after the rhodamine B reacts with the rhodamine B. The antioxidant may be with OH^·The reaction decreases the degree of decrease in absorbance.

Preparing a test solution: (1) rhodamine B solution: accurately weighing 24 mg of rhodamine B, and dissolving the rhodamine B in a 250mL volumetric flask by using distilled water to obtain the rhodamine B with the concentration of 2 multiplied by 10^-4mol/L solution. (2) CuCl solution: 5 mmol/L, accurately weighing 405mg of CuCl, dissolving with water, and fixing the volume in a 100 mL volumetric flask. (3) Hydrogen peroxide solution: accurately measuring 30% of H₂O₂0.55 mL of the solution was taken in a 250mL volumetric flask with distilled water to give a solution having a concentration of 20 mmol/L. (4) Sulfuric acid solution: 0.5 g of 98% concentrated sulfuric acid was measured out accurately, and the solution was taken out in a 250mL volumetric flask with distilled water to obtain a solution having a concentration of 0.02 mmol/L.

The experimental method comprises the following steps: three tubes were used to define tube 1 as a blank reference (A)_{Ginseng radix (Panax ginseng C.A. Meyer)}) Test tube 2 is hydroxyl radical (A)_{Hydroxyl radical}) Test tube 3 is the sample (A)_{Sample (A)}) The reagents were added in the order of Table 5, the reaction was started, and then diluted to 20 mL with distilled water, and the absorbance was measured at 550 nm after 5 min. Each sample was repeated 3 times and averaged.

TABLE 5 matrine chelating agent hydroxyl radical scavenging sample application table

	Rhodamine (0.7 mL)	Sulfuric acid (2 mL)	CuCl (0.3 mL)	Sample (I)	H2O2 (0.2 mL)
						Test tube 1 (A)Ginseng radix (Panax ginseng C.A. Meyer)）	+	+	-	-	-
Test tube 2 (A)Hydroxyl radical）	+	+	+	-	+
						Test tube 3 (A)Sample (A)）	+	+	+	+	+

^*The clearance rate S of hydroxyl radicals of the sample can be expressed by the following formula: s = [ 2 ] _{Sample (A) Hydroxyl radical}A-A]/[A _{Hydroxyl radical}-A_Ginseng)]×100%

Detection indexes are as follows: plotting concentration-clearance curve, obtaining the concentration corresponding to 50% clearance from the curve, and defining the concentration as half inhibition concentration IC₅₀The results are shown in Table 6.

TABLE 6 Renolizidine alkaloids for Cu scavenging⁺Induced hydroxyl radical scavenging action

Compound (I)	IC50	Compound (I)	IC50
				Matrine	A	Matrine monomethyl fumarate	A
Oxymatrine	A	Matrine fumarate	A
				Sophoridine	B	Matrine valproate	B
Oxyphylline	B	Matrine maleate	A
				Triethylene tetramine	D	Sophocarpine valproate	B

Note: a is less than 50 mu M; b, 50-100 mu M; c, 100-500 mu M; d > 500. mu.M.

Example 4 chelating agent pairs of quinolizidine alkaloids to Fe²⁺Induced hydroxyl radical scavenging experiments:

the experimental principle is as follows: the hydroxyl radical may be formed from Fe²⁺-H₂O₂The system is generated through a Fenton reaction, and the absorbance of rhodamine B is obviously reduced after the rhodamine B reacts with the rhodamine B. The antioxidant may be with OH^·The reaction decreases the degree of decrease in absorbance.

Preparing a test solution: (1) rhodamine B solution: accurately weighing 24 mg of rhodamine B, and dissolving the rhodamine B in a 250mL volumetric flask by using distilled water to obtain the rhodamine B with the concentration of 2 multiplied by 10^-4mol/L solution. (2) Ferrous sulfate solution: accurately weighing FeSO₄0.19 g was taken in a 250mL volumetric flask with distilled water to obtain a solution having a concentration of 5 mmol/L. (3) Hydrogen peroxide solution: accurately measuring 30% of H₂O₂0.55 mL of the solution was taken in a 250mL volumetric flask with distilled water to give a solution having a concentration of 20 mmol/L. (4) Sulfuric acid solution: 0.5 g of 98% concentrated sulfuric acid was measured out accurately, and the solution was taken out in a 250mL volumetric flask with distilled water to obtain a solution having a concentration of 0.02 mmol/L.

The experimental method comprises the following steps: three tubes were used to define tube 1 as a blank reference (A)_{Ginseng radix (Panax ginseng C.A. Meyer)}) Test tube 2 is hydroxyl radical (A)_{Hydroxyl radical}) Test tube 3 is the sample (A)_{Sample (A)}) The reagents were added in the order of Table 7, the reaction was started, and then diluted to 20 mL with distilled water, and the absorbance was measured at 550 nm after 5 min. Each sample was repeated 3 times and averaged.

TABLE 7 application of quinolizidine alkaloid chelating agents to scavenge hydroxyl radicals

	Rhodamine (0.7 mL)	Sulfuric acid (2 mL)	FeSO4 (0.3 mL)	Sample (I)	H2O2 (0.2 mL)
						Test tube 1 (A)Ginseng radix (Panax ginseng C.A. Meyer)）	+	+	-	-	-
Test tube 2 (A)Hydroxyl radical）	+	+	+	-	+
						Test tube 3 (A)Sample (A)）	+	+	+	+	+

^*The clearance rate S of hydroxyl radicals of the sample can be expressed by the following formula: s = [ 2 ] _{Sample (A) Hydroxyl radical}A-A]/[A _{Hydroxyl radical}-A_Ginseng)]×100%

Detection indexes are as follows: plotting concentration-clearance curve, obtaining the concentration corresponding to 50% clearance from the curve, and defining the concentration as half inhibition concentration IC₅₀The results are shown in Table 8.

TABLE 8 removal of Fe by quinolizidine alkaloids²⁺Induced hydroxyl radical scavenging action

Compound (I)	IC50Value of	Compound (I)	IC50Value of
				Matrine	A	Matrine monomethyl fumarate	A
Oxymatrine	A	Matrine fumarate	A
				Sophoridine	B	Matrine valproate	B
Oxyphylline	B	Matrine maleate	A
				Triethylene tetramine	D	Sophocarpine valproate	B

Note: a is less than 50 mu M; b, 50-100 mu M; c, 100-500 mu M; d > 500. mu.M.

Example 5 effect of quinolizidine alkaloid chelators on copper levels in a hepatolenticular degeneration model mouse:

grouping experimental animals: animals and grouping pure line healthy male Wistar rats 60, quality 220- & ltSUB & gt 280 g. The test is carried out by adaptive feeding for 1 week before the test, and the test is randomly divided into a normal control group, a model group, a matrine administration group and a penicillamine positive drug control group, wherein each group comprises 10 animals.

Making an experimental animal model and intervening medicines: rats were raised in cages. Normal group (10) rats were fed copper-free basal diet; the experimental group (50) rats were fed a copper-loaded diet (containing l g/kg copper sulfate in powdered feed and 0.185% copper sulfate in deionized water) for 8 weeks; starting at week 5, the surviving 60 model rats were randomized into 6 groups, the normal group: 10 pieces of the Chinese herbal medicine are added; model control group: 10 pieces of the Chinese herbal medicine are added; matrine group: performing intragastric administration for 1 time per day at a dose of 100 mg/kg for 10 patients; oxymatrine group: 10 patients are administered with the gavage of 100 mg/kg 1 time per day; matrine fumarate group: 10 patients are administered with the gavage of 100 mg/kg 1 time per day; positive control group: 10 patients were gavaged with 100 mg/kg penicillamine 1 time a day for 4 weeks.

The experimental method and detection: after 8 weeks of copper-loaded diet, fasting for 18 h and anaesthetizing the animals, taking 2 mL of blood from the abdominal aorta, placing the blood in an anticoagulation tube, standing for half an hour, centrifuging at 4 ℃ for 10 min at 3000 r/min, separating serum, and measuring the content of serum copper. The liver tissue is collected by laparotomy, the sample is repeatedly cleaned by 0.9% NaCl, 500 mg of wet weight is absorbed and weighed by clean filter paper, the digestion is carried out by heating at low temperature by using 10 mL of concentrated nitric acid, and after the tissue is completely dissolved to be yellow, clear and transparent, the copper ion content is measured by adopting an atomic absorption spectrometry. And (3) 24 h urine copper ion determination: placing one 5 d urine sample in metabolism cage for 3 d (72H), shaking, measuring urine volume accurately, and measuring by adding 0.1 mL H into 2 mL urine₂SO₄. Mixing, and measuring by atomic absorption spectrophotometry. Statistical methods the data were expressed as means ± standard deviation (x ± s), statistical methods using one-way anova and LSD-t test.

TABLE 9 Effect of matrine-based chelators on copper levels in Sophragmoid degeneration model mice

Group of	Liver copper (mu g/g)	Urine copper (mu g/24 h)	Serum copper (mu mol/L)
				Normal control group	5.90±0.59	2037.27±868.25	23.98±2.47
Model set	78.70±10.67△△	3656.20±1688.43△△	60.34±5.73△△
				Matrine	28.95±3.85##	7335.26±1038.10##	34.76±3.56##
Oxymatrine	34.90±4.26##	7037.27±1172.25##	36.21±3.42##
				Matrine fumarate	27.24±3.28##	7215.29±1232.13##	32.32±3.16##
Penicillin amines	29.87±4.56##	7235.60±1156.59##	35.28±4.42##

Note: in comparison with the normal group,^△△P<0.01; in comparison with the set of models,^#P<0.05，^##P<0.01。

example 6 Effect of matrine-based chelators on iron-overloaded rat bone marrow, liver, spleen and serum iron

Grouping experimental animals: 60 male Wistar rats (SPF grade) with the weight of 140-: a normal control group, a model group, a matrine administration group and a deferiprone positive drug control group, wherein each group contains 10 animals.

Making an experimental animal model and intervening medicines: rats were raised in cages and iron-free basal diet was prepared according to the AIN-93G formula. The normal group (10) was fed with iron-free basal diet; the experimental group (50) was fed iron-free basal diet and administered 12 mg of iron dextran by intraperitoneal single dose injection every other day for 12 weeks. After 6 weeks, the experimental group randomized model rats were randomized into 5 groups, model control group: 10 pieces of the Chinese herbal medicine are added; matrine group: performing intragastric administration for 1 time per day at a dose of 100 mg/kg for 10 patients; oxymatrine group: 10 patients are administered with the gavage of 100 mg/kg 1 time per day; matrine fumarate group: 10 patients are administered with the gavage of 100 mg/kg 1 time per day; positive control group: 10 patients were gavaged with 100 mg/kg deferiprone 1 time a day for 6 weeks.

The experimental method and detection: after the experiment is finished, the rat is fasted for 8 hours, 10% chloral hydrate is used for abdominal anesthesia, the abdominal aorta is used for blood collection, and organs are taken for testing. Bone marrow iron staining: after the abdominal aorta is bled, the femoral muscle and fascia of the right leg of the rat are removed immediately, the marrow cavity is flushed with 10 mul fetal calf serum, and the tablet is pushed rapidly. Adopting a Prussian blue staining method to semi-quantitatively and qualitatively count the bone marrow extracellular iron: observing the whole marrow membrane under a low power microscope, finding blue-green particles, beads or small blocks in the small marrow particles, and then changing an oil microscope to identify the positive degree (see figure 2). Liver, spleen tissue iron, serum iron: shearing appropriate amount of liver and spleen tissue, adding normal saline to prepare into 10% tissue homogenate, and measuring iron content of liver and spleen tissue by colorimetry; standing fresh anticoagulated blood for half an hour, centrifuging at 4 ℃ for 10 min at 3000 r/min, separating serum, and measuring serum iron content (strictly according to the operation of a tissue iron and serum iron kit instruction). Statistical methods statistical analysis of the results was performed using SPSS 19.0 software. The measured data are expressed as mean + -standard deviation (x + -s), and the statistical method adopts one-way anova and LSD-t test.

TABLE 10 Effect of matrine chelators on iron levels in iron-overloaded model mice

Group of	Liver iron (mug/g)	Spleen iron (mu g/g)	Serum ferrum (mu mol/L)
				Normal control group	44.25±9.34	31.27±5.28	75.57±10.55
Model set	128.70±10.67△△	46.20±8.43△△	120.34±9.74△△
				Matrine administration group	62.92±4.83##	35.12±5.10##	85.75±9.66##
Oxymatrine administration group	74.31±5.25##	37.27±4.95##	96.23±10.42##
				Matrine fumarate	57.86±4.58##	31.23±4.13##	82.38±8.16##
Deferiprone administration group	69.67±7.10##	35.62±5.49##	95.28±10.07##

Note: in comparison with the normal group,^△△P<0.01; in comparison with the set of models,^#P<0.05，^##P<0.01。

example 7 intervention of matrine chelators on xeroderma pigmentosum

Grouping experimental animals: 12C 57BL/6 mice (male, 8-10 weeks, 20-25g, SPF grade, provided by the university of yangzhou comparative medical center), which were randomly divided into a blank control group (control, n = 4), a model group (model, n = 4) and a dosing group (anti-incision scientific oil, n = 4). Mice were acclimatized in advance, and the experimental area on the back of the neck of the mice was shaved 48 hours before the start of the experiment with an electric depilator, followed by weighing, and mice within the same group were numbered at the tail.

Making an experimental animal model and intervening medicines: acetone-ethyl ether-0.1% CuCl aqueous solution is adopted to establish a colorable xeroderma model. Slightly squeezing off excessive liquid from a cotton pad soaked in a mixed solution (a mixture prepared by acetone and diethyl ether at a ratio of 1: 1), applying the cotton pad on the skin of a shaved part of the back and neck of a mouse for 15s, then applying the cotton pad soaked by pure water to the same shaved part of the back and neck of the mouse for 30s, ensuring that the cotton pad is not too wet or too dry, and performing mixed solution stimulation twice a day in the morning and at night at a fixed time, wherein reagents of a blank group (control) are replaced by water. The administration group was administered continuously (0.1M aqueous matrine solution) on days 6-9. On day 9 of molding, the mice were sacrificed, and skin tissues (1 cm. times.1 cm) on the back of the neck of the mice were taken, fixed with 4% paraformaldehyde at 4 ℃ for 48 hours, and then placed in a 20% sucrose solution for 24 hours for sugar precipitation. After the precipitation, the embedded tissues (trimmed to 0.5 × 1cm size rectangular to the skin before embedding) were snap frozen at-80 ℃ with OTC embedding medium, and then sectioned with a cryomicrotome at 16 μm thickness, and the cut sections were stored in a freezer at-20 ℃. The pieces were then HE stained, mounted on neutral gum, photographed (brightfield) and analyzed for skin thickness. The experimental results are shown in figure 2.

Claims (8)

1. Has Cu⁺And or Fe²⁺Use of ion-chelating quinolizidine alkaloids and pharmaceutically acceptable salts thereof for the treatment of diseases caused by copper and/or iron overload.

2. A catalyst having Cu according to claim 1⁺And or Fe²⁺Use of an ion-chelating quinolizidine alkaloid and pharmaceutically acceptable salts thereof, characterized in that: the diseases caused by copper overload are mainly Wilson disease, scleroderma, oral submucosa fibrosis, xeroderma pigmentosum, pellagra, sicca syndrome and amyotrophic lateral sclerosis.

3. A catalyst having Cu according to claim 1⁺And or Fe²⁺Use of an ion-chelating quinolizidine alkaloid and pharmaceutically acceptable salts thereof, characterized in that: the diseases caused by iron overload are mainly hereditary hemochromatosis, secondary hemochromatosis, delayed porphyria cutanea, mitochondrial iron overload disease, thalassemia, myelofibrosis and myelodysplastic syndrome.

4. A method as claimed in claim 2, havingCu⁺And or Fe²⁺The ion-chelating quinolizidine alkaloid and its pharmaceutically acceptable salt are used for treating hereditary Wilson disease, and are characterized by improving liver damage, extrapyramidal symptoms, corneal tryptophane ring, kidney damage, and blood system damage caused by Wilson disease.

5. A catalyst having Cu according to claim 3⁺And or Fe²⁺The quinolizidine alkaloid with ion chelation effect and the medicinal salt thereof are used for treating thalassemia, and are characterized in that hereditary hemolytic anemia, progressive exacerbation of hepatosplenomegaly and jaundice symptoms caused by thalassemia are improved.

6. A catalyst having Cu according to claim 1⁺And or Fe²⁺The quinolizidine alkaloid with the ion chelation effect and the medicinal salt thereof are characterized in that the main structure of the quinolizidine biological chelating agent and the medicinal salt thereof is as follows:

。

7. quinolizidine alkaloid Cu according to claim 1⁺And or Fe²⁺A pharmaceutically acceptable salt of an ion chelating agent, wherein the pharmaceutically acceptable salt is a salt obtained by adding the matrine compound and an acid, and the acid is hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, acetic acid, citric acid, phenylbutyric acid, fumaric acid, and monomethyl fumarate、Oxalic acid, malonic acid, valproic acid, salicylic acid, malic acid, glucoheptanoic acid, digluconic acid, aspartic acid, alginic acid, lactic acid, picric acid, succinic acid, glycerophosphoric acid, heptanoic acid, nicotinic acid, oxalic acid, hexanoic acid, succinic acid, mandelic acid, ascorbic acid, maleic acid, tartaric acid, benzenesulfonic acid, methanesulfonic acid, taurine or isethionic acid.

8. Use according to claim 1, characterized in that it comprises one or more of any of the aboveQuinolizidine alkaloid Cu⁺And or Fe²⁺The chelating agent and the medicinal salt are used as therapeutic components to prepare oral preparations, or nano-drug injection preparations prepared by the assistance of liposome, albumin, high molecular polymer and inorganic nano-particles, or application preparations, eye drops, or spray preparations, or compositions combined with other drugs.

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