WO2016074203A1 - Cell apoptosis inducer containing chlorine dioxide and use thereof in preparing cosmetics or anti-aging or antineoplastic drugs - Google Patents

Abstract

The present invention relates to a cell apoptosis inducer containing chlorine dioxide. The present invention relates to a cell apoptosis inducer kit containing the following two independent components: the first component: a chlorine dioxide precursor solid, or a solution containing a chlorine dioxide precursor; and the second component: an auqeous solution of an acidity pH adjuster; the two components are stored separately, and can be mixed before use so that in-situ reaction occurs and the cell apoptosis inducer containing chlorine dioxide is prepared; and the amounts and concentrations of the first component and the second component enable the pH of the mixed solution to be 1.5-6.5. The present invention further relates to use of the cell apoptosis inducer containing chlorine dioxide or the cell apoptosis inducer kit containing chlorine dioxide in preparing drugs for treating tumors, or use thereof in preparing anti-aging drugs for target tissues of mammals, or use thereof as cosmetics, or use thereof in preparing chemotherapeutic drugs.

Description

Apoptosis inducing agent comprising chlorine dioxide and use thereof in preparing cosmetic or anti-aging or anti-tumor drugs Technical field

The invention belongs to the field of anti-aging and tumor treatment, and particularly relates to a method for preventing apoptosis and senescence and treating tumor by inducing apoptosis of senescent cells and tumor cells, and chlorine dioxide for preparing cosmetics for mammalian apoptosis inducer or The application of drugs.

Background technique

The aging of the body can not be equated with the aging of the cells, but the former is the cumulative result of the latter, so the body aging can be characterized to a certain extent by the percentage of senescent cells. There are always cells in the body that are constantly aging and dying, and at the same time, cells proliferate to produce new cells to compensate. If the nascent cells produced by cell proliferation not only fully replenish dead cells, but also replace some of the senescent cells, the body will behave younger. Studies have shown that lifetime aging of senescent cells can delay the initial appearance of age-related phenotypes. Moreover, the elimination of later years will delay the development of age-related conditions that have already occurred. This suggests that senescent cells do cause age-related phenotypes, and that their clearance prevents or delays age-related loss of tissue function (Baker DJ, et al. Clearance of p16Ink4a-positive senescent cells delays ageing-associated disorders. Nature, 2011, 479: 232-236). It can be said that by eliminating aging cells and reducing the proportion of aging cells, the body can be younger and can prevent or delay the loss of age-related tissue function. The death of aging cells is essentially apoptosis. (Hui Hongwei et al., "Free Radicals and Apoptosis", Progress in Biochemistry and Biophysics, 1996; 23:12).

Aging does not mean dying. If the culture medium is changed regularly, aging cells can survive for a long time (Seshadri T, Campisi J. Repression of c-fos transcription and an altered genetic program in senescent human fibroblasts. Science, 1990 Jan 12;247 (4939): 205-9). However, the inhibition of proliferation of senescent cells is irreversible, and its growth is arrested in the G1 phase of the cell cycle, and can no longer enter the S phase. This suggests that senescent cells may be anti-apoptotic compared to proliferating young cells, thus maintaining long-term survival (Huang Ying, Zhang Zongyu, Tong Tanjun, "Inducible Apoptosis of Human Aging Fibroblasts", Chinese Journal of Geriatrics, 2000 June, Vol. 19, No. 3).

Therefore, in theory, an anti-aging idea was found, that is, through the induction of apoptosis of senescent cells, the senescent cells are caused to undergo apoptosis and go to death, so that they can be removed from the body. The body will automatically initiate stem cell regeneration, regenerate new cells to replace the cleared cells, and make the body younger.

Consciously inducing apoptosis in senescent cells is the most appropriate way to eliminate senescent cells. In the embryonic development stage, the excess and completed mission cells are cleared by apoptosis, which ensures the normal development of the embryo; in the adult stage, the aging and diseased cells are cleared by apoptosis to ensure the health of the body. For example, in mouse studies, it was found that when old stem cells were placed in a young microenvironment, they were able to rejuvenate (Rescuing replication and osteogenesis of aged mesenchymal stem cells by exposure to a young extracellular matrix Yun Sun, et al. The FASEB Journal, May 2011, vol. 25 no. 51474-1485), that is, younger, also means more regenerative capacity. On the other hand, it is understood that the senescent cells in the tissue are removed, and the stem cells in the tissue are automatically regenerated due to the specific induction space, and the regenerated tissue is younger, so the whole body is younger.

More studies have pointed out that apoptosis helps the body to delay the loss of tissue function associated with aging. For example, DNA fragmentation is also a hallmark of apoptosis. The relationship between DNA double-strand breaks in mouse brain activity indicates that this DNA molecule is broken, even in fully healthy mouse neurons. It happens that in the brains of mice adapted to the new environment, the rate of DNA fragmentation is six times that of mice that are still immobile in the old environment (Elsa Suberbielle, et al. pHysiologic brain activity causes DNA double-strand breaks in neurons , with exacerbation by amyloid-βNature Neuroscience 16, 613–621 (2013)). It is clear that mice adapted to the new environment have stronger memory and thinking ability, which indicates that this apoptotic process of DNA double-strand breaks can promote the brain rejuvenation of mice.

Autophagy promotes the ultimate degradation of apoptotic cells and is the last step in the clean-up of senescent cells. Increased autophagy has an anti-aging effect (Louis R. Lapierre, et al. The TFEB orthologue HLH-30 regulates autopHagy and modulates longevity in Caenorhabditis elegans, Nature Communications. 4, Article number: 2267). Factors that cause cellular oxidation can cause apoptosis. By studying macrophages and monocytes, Albina et al. proposed that NO is also an inducer of apoptosis (Albina JE, Cui S, Mateo RB et al. Nitric oxide-mediated apoptosis in murine peritoneal macropHages.J Immunol, 1993 Jun1 ;150(11):5080-5).

Nitric oxide (NO) has a therapeutic effect on many aging diseases. For example, nitric oxide has a role in preventing neuronal abnormalities that cause Parkinson's disease. Nitric oxide promotes the decomposition of waste proteins and protects them. The role of nerve cells (Kentaro Ozawa, et al. S-nitrosylation regulates mitochondrial quality control via activation of parkin Scientific Reports 3, Article number: 2202 (July 2013). The mechanism of nitric oxide to alleviate aging diseases seems to promote aging The cells undergo apoptosis.

However, in reality, there is no technology that can safely and conveniently target the removal of aging cells and rejuvenate the body.

The Chinese patent "Pearl Extract Extracted from Nitric Oxide" with the application number 201110102598.1 mentions that nitric oxide itself promotes regeneration and anti-aging effects on the skin, and also increases the skin's effective ingredients for traditional cosmetics. absorb.

Patent No. 201210499300.X, "A Method for Initiating Mammalian Stem Cells and Application of Chlorine Dioxide in the Preparation of Drugs for Initiating Mammalian Stem Cells," uses chlorine dioxide to initiate stem cells to promote regeneration, but does not Reference is made to the mechanism of how to rejuvenate tissues by eliminating senescent cells, and there is no anti-aging application that can be used in medicines or cosmetics. Although the patent refers to chlorine dioxide to treat some senile diseases according to the stem cell regeneration mechanism, it does not give more solutions in preventing these diseases.

The roots of cancer and aging are the same (Carlos López-Otín, et al. The Hallmarks of Aging. Cell, 6 June 2013). Induction of tumor cell apoptosis has become an important way to treat tumors. If tumor cells are regarded as senescent cells, the idea of treating tumors by inducing apoptosis of tumor cells and anti-aging by inducing apoptosis of senescent cells are consistent.

Tumor cells usually have a certain mechanism of apoptosis resistance, and the apoptosis mechanism is used to treat tumors, that is, to change the balance between pro-apoptosis and anti-apoptosis at various levels of apoptosis regulation, and induce tumor cell apoptosis.

Although the research on apoptosis mechanism has made great progress in the past ten years, it can explain the apoptosis in detail, especially the mechanism of apoptosis in mammals. There are two commonly accepted classical pathways. Bar: Death receptor pathway and mitochondrial pathway.

In view of the special pH environment of tumor cells, studies have shown that sodium bicarbonate increases tumor pH and inhibits spontaneous tumor metastasis (Ian F. Robey, et al. Bicarbonate Increases Tumor pH and Inhibits Spontaneous Metastases Cancer Res March 15,200969; 2260 ), but it does not form apoptosis induction on tumor cells themselves.

The existing tumor cell apoptosis inducing agents have the following disadvantages: unclear targeting, large side effects, complicated treatment means and high uncertainty, which may cause resistance of tumor cells, and are effective only for a few types of tumor cells.

If the induction of apoptosis in tumor cells is the correct way to treat tumors, it is necessary to find more targeted, lower toxic side effects and more universal apoptosis inducers.

Chlorine dioxide is an internationally recognized new generation of high-efficiency, broad-spectrum, safe sterilization and preservatives. It is an ideal substitute for chlorine preparations and has been widely used in developed countries around the world. Relevant organizations in developed countries such as the United States, Western Europe, Canada, and Japan, such as the US Environmental Protection Agency, the Food and Drug Administration, and the US Department of Agriculture, have approved and recommended the use of chlorine dioxide for disinfection of food, food processing, pharmaceuticals, hospitals, and public environments. Anti-corrosion and preservation of mildew and food. World Health Organization (WHO) and the World Food Organization (FAO) have also listed chlorine dioxide as a Class A1 safe and effective disinfectant. In order to control the production of “three substances” (carcinogenic, teratogenic and mutagenic) in drinking water, chlorine dioxide has been widely used in developed countries in Europe and America to disinfect drinking water. However, chlorine dioxide has not been accepted by the market as a medicine. However, some patents relate to the use of chlorine dioxide for cosmetic or therapeutic use of diseases (eg, CN102137651A, CN101641104A, and CN1199633C, US5750108, CN102441006A), which have not found chlorine dioxide or acidic solutions containing chlorine dioxide. Has the potential to clear aging cells.

Summary of invention

In a first aspect, the invention relates to an apoptosis inducing agent comprising chlorine dioxide.

In a preferred embodiment, the apoptosis inducing agent comprises chlorine dioxide dissolved in water, wherein the concentration of chlorine dioxide is from 500 to 2900 ppm, based on mass.

In a more preferred embodiment, the apoptosis inducing agent of the present invention further comprises an acidic pH adjusting agent such that the pH of the apoptosis inducing agent is 1.5 to 6.5, and the acidic pH adjusting agent is selected from the following collections At least one of:

An organic acid or a salt thereof selected from the group consisting of formic acid, acetic acid, propionic acid, butyric acid, lactic acid, pyruvic acid, citric acid, malic acid, tartaric acid, gluconic acid, glycolic acid, fumaric acid, malonic acid, maleic acid a collection of oxalic acid, succinic acid, acrylic acid, crotonic acid, glutaric acid, and salts thereof;

An inorganic acid or a salt thereof selected from the group consisting of hydrochloric acid, phosphoric acid, boric acid, metaphosphoric acid, pyrophosphoric acid, sulfamic acid, dihydrogen phosphate, and hydrogen phosphate.

Most preferably, wherein the acidic pH adjusting agent is selected from the group consisting of citric acid, acetic acid or dihydrogen phosphate.

In a second aspect, the present invention relates to an apoptosis inducing agent kit comprising chlorine dioxide comprising the following two separate components:

a first component: a chlorine dioxide precursor solid, or a solution comprising a chlorine dioxide precursor;

a second component: an aqueous solution of an acidic pH adjuster;

The two are stored separately and can be mixed before use to react in situ and prepare an apoptosis inducing agent comprising chlorine dioxide; and the amount and concentration of the first component and the second component can enable the mixed solution pH=1.5-6.5;

Wherein the chlorine dioxide precursor is selected from at least one of sodium chlorite, potassium chlorite, lithium chlorite, calcium chlorite, magnesium chlorite or bismuth chlorite;

Wherein the acidic pH adjusting agent is selected from at least one of the following groups:

An organic acid or a salt thereof selected from the group consisting of formic acid, acetic acid, propionic acid, butyric acid, lactic acid, pyruvic acid, citric acid, malic acid, a collection of tartaric acid, gluconic acid, glycolic acid, fumaric acid, malonic acid, maleic acid, oxalic acid, succinic acid, acrylic acid, crotonic acid, glutaric acid, and salts thereof;

An inorganic acid or a salt thereof selected from the group consisting of hydrochloric acid, phosphoric acid, boric acid, metaphosphoric acid, pyrophosphoric acid, sulfamic acid, dihydrogen phosphate, and hydrogen phosphate.

In a third aspect, the present invention relates to the use of the above-described apoptosis inducing agent comprising chlorine dioxide or the above-described apoptosis inducing agent kit comprising chlorine dioxide for preparing a medicament for inducing apoptosis. Preferably, the cell is a mammalian cell.

In a fourth aspect, the present invention relates to the use of the above-described apoptosis inducing agent comprising chlorine dioxide or the above-mentioned apoptosis inducing agent kit comprising chlorine dioxide for preparing a medicament for treating tumor, or for preparing a mammalian target tissue The use of anti-aging drugs, either for cosmetic purposes or for the preparation of chemotherapeutic drugs.

Detailed description of the invention

Based on prior art studies, it is an object of the present invention to provide the use of chlorine dioxide for the preparation of a medicament or cosmetic for use in a mammalian apoptosis inducing agent. According to the application, there is a chlorine dioxide-containing apoptosis inducing agent for anti-aging drugs or cosmetics, which can remove aging cells in target tissues, promote body regeneration, and regenerate young tissues to achieve Anti-aging effect. This application has little side effects or no side effects.

Another object of the present invention is to provide an application of chlorine dioxide in the preparation of a medicament for use in a mammalian apoptosis inducing agent. According to the application, a drug for promoting apoptosis of a tumor cell in a mammal has an apoptosis inducer containing chlorine dioxide, and the drug can specifically induce apoptosis of the tumor cell to achieve better tumor treatment effect. With little side effects or no side effects, the burden on patients is lighter.

In order to achieve the above object, the present invention adopts a technical solution of preparing a chlorine dioxide-containing apoptosis inducing agent and administering the apoptosis inducing agent to a mammalian target tissue, wherein the apoptosis inducing agent is An effective amount of chlorine dioxide is provided when the mammalian target tissue is administered.

Further, the method for preparing an apoptosis inducing agent containing chlorine dioxide is: dissolving chlorine dioxide gas in an acidic solution A having an acidic pH adjuster and having a pH of 1.5 to 6.5 to prepare 500 to 2900 ppm of chlorine dioxide. Solution.

Further, a chlorine dioxide-containing apoptosis inducing agent is prepared by dissolving a chlorine dioxide precursor in water to prepare an aqueous solution of 1% to 40%, and adding an acidic solution B containing an acidic pH adjuster to the aqueous solution. Adjust the pH of the mixed solution to 1.5 to 6.5.

Further, the method for preparing a chlorine dioxide-containing apoptosis inducing agent kit comprises: dissolving a chlorine dioxide precursor in water to prepare an aqueous solution C having a concentration of 1% to 40%; and dissolving the acidic pH adjusting agent in water Prepare acidic solution D; before use Mix the solutions C and D and adjust the pH of the mixed solution to 1.5 to 6.5.

Still further, the chlorine dioxide precursor is sodium chlorite, potassium chlorite, lithium chlorite, calcium chlorite, magnesium chlorite and bismuth chlorite.

Still further, the acidic pH adjusting agent is citric acid, acetic acid or sodium dihydrogen phosphate.

Further, the apoptosis inducing agent is administered by arterial injection, intramuscular injection, subcutaneous injection, intracardiac injection, intrathecal injection, intra-articular injection, puncture injection, rectal administration, nasal administration, transdermal administration or inhalation. Directly to the damaged organ or tissue or to other target tissues.

Further, the apoptosis inducing agent can be prepared as an injection, an ointment, an inhalant, a nasal drop, a lotion, a suppository, a patch, a paste, a tablet, an oral solution, a capsule, a granule, a granule, a pill or Syrup.

The present invention provides an application of chlorine dioxide in the preparation of a medicament for the preparation of a mammalian apoptosis inducer for chlorine dioxide, wherein the medicament or cosmetic is used for anti-aging of a mammalian target tissue, Younger.

Further, this anti-aging includes skin rejuvenation, skin beauty, and rejuvenation of other tissues in the body, such as increased memory, improved sleep therapy, prevention of Alzheimer's disease, Parkinson's syndrome, osteoporosis, diabetes, cardiovascular and cerebrovascular diseases. Diseases and other senile diseases, etc., including but not limited to these.

Further, chlorine dioxide is used for the preparation of a medicament for the mammalian apoptosis inducer, which is used for anti-aging of human tissues and for preventing Alzheimer's disease, Parkinson's syndrome, bone Osteoporosis, diabetes, cardiovascular and cerebrovascular diseases, etc.; the cosmetic is used for skin care, acne or acne removal, and can also be used for oral care mouthwashes, including but not limited to these.

The present invention also provides the use of chlorine dioxide for the preparation of a medicament for inducing apoptosis in a mammal, wherein the medicament is for treating cancer, in particular for inducing tumor cell apoptosis in a mammal.

Further, by inducing apoptosis of tumor cells, the cancers targeted therein include:

Intracranial metastases, meningiomas, skull tumors, brain cancer, pituitary adenomas, auditory nerve sheath tumors, gliomas, brain tumors; maxillary sinus cancer, laryngeal cancer, nasopharyngeal cancer, tongue cancer, thyroid cancer, gingival cancer , lip cancer; thymoma, lung cancer, adenocarcinoma, breast sarcoma, lung metastases, breast fibroma, breast cancer; pancreatic head cancer, stomach cancer, gallbladder cancer, rectal cancer, pancreatic cancer, esophageal cancer, colon cancer, liver cancer; Tumor, penile cancer, urothelial carcinoma, prostate cancer, urethral cancer, testicular cancer, bladder cancer, nephroblastoma, kidney cancer; ovarian cancer, fallopian tube tumor, vulvar cancer, vaginal tumor, uterine cancer, cervical cancer, chorion Cancer, pelvic cancer; skin cancer, liposarcoma, malignant teratoma, fibroids, neurofibromatosis, melanoma, cholangiocarcinoma, squamous cell carcinoma, basal cell carcinoma; chordoma, osteoma, chondroma, osteosarcoma, synovium Sarcoma, giant cell tumor of bone, osteofibrosarcoma; acute leukemia, malignant lymphoma, chronic leukemia; hepatic hemangioma, islet cell carcinoid, blastoma, myxoma, cervical metastases, cardia cancer. These include, but are not limited to, these cancers.

Further, the chlorine dioxide of the present invention is used in the preparation of a medicament for inducing apoptosis in a mammal, the medicament of which may be administered at risk of developing cancer, diagnosed with cancer, during cancer treatment or at The individual of the subject during recovery from cancer treatment, or the drug can be administered as a prophylactic to the individual of the subject to prevent or delay the progression of the cancer.

detailed description

The invention is further described below in conjunction with specific embodiments.

The present invention defines apoptosis as the death of a cell and is eliminated by degradation without strictly distinguishing whether it belongs to apoptosis, necrosis or autophagy. Induction of senescent cells and tumor cell apoptosis in the present invention refers to a method and process for causing death of senescent cells, being degraded by clearing and killing of tumor cells, and being degraded by degradation, without diagnosing death.

Before describing a particular embodiment of the invention, the mechanism of the method provided by the invention will first be described. These descriptions, in conjunction with the specific embodiments, will help those skilled in the art to understand the technical scope and the scope of the invention.

Studies have shown that lifetime aging of senescent cells can delay the initial appearance of age-related phenotypes. Moreover, the elimination of later years will delay the development of age-related conditions that have already occurred. This suggests that senescent cells do cause age-related phenotypes and that their clearance prevents or delays age-related loss of tissue function. (Baker DJ, et al. Clearance of p16 Ink4a-positive senescent cells delays ageing-associated disorders. Nature, 2011, 479: 232-236). It can be said that by eliminating aging cells and reducing the proportion of aging cells, the body can be younger and can prevent or delay the loss of age-related tissue function. The death of aging cells is essentially apoptosis. (Hui Hongwei et al., "Free Radicals and Apoptosis". Progress in Biochemistry and Biophysics, 1996; 23:12).

Autophagy promotes the degradation of apoptotic cells, so it can also play a role in the clearance of senescent cells. The study found that the loss of memory in Drosophila can be reversed by eating foods rich in polyamines. Feeding polyamines such as fruit flies can enhance the lifespan of organisms. It seems to work by reversing the decline in age-related autophagy. Autophagy refers to the mechanism by which cells use to remove their own fragments. Increasing autophagy by genetic technology or limiting calorie intake can also extend the lifespan of fruit flies. (Varun K Gupta, et al. Restoring polyamines protects from age-induced memory impairment in an autopHagy-dependent manner. Nature Neuroscience, 01 September 2013).

High levels of ROS induce apoptosis (Lau AT, Wang Y, Chiu JF. Reactive oxygen species: current knowledge and applications in cancer research and therapeutic. J Cell Biochem. 2008 May 15; 104(2): 657-67) . Studies have shown that lifespan is significantly prolonged when nematodes feed on nitric oxide-producing bacteria (Ivan Gusarov, et al. Bacterial Nitric Oxide Extends the Lifespan Of C.elegans Cell, Volume 152, Issue 4, 818-830, 14 February 2013). This suggests that oxides can promote apoptosis in cells.

Although anti-oxidation has always existed as an effective standard for anti-aging, the actual evidence is rarely supported. If the defective protein cannot be removed in time, it will lead to the loss of protein homeostasis and cause aging-related diseases. For example, in Alzheimer's disease, proteins that cannot be cleared form plaques, leading to neuronal death. Although a large number of free radicals may be harmful, their presence also triggers a protective response. There is no genetic evidence to suggest that enhancing the body's antioxidant defenses can delay aging. (Carlos López-Otín, et al. The Hallmarks of Aging. Cell, 6 June 2013).

In some embodiments, the present invention also demonstrates that a more oxidizing substance (chlorine dioxide) can promote cell apoptosis and contribute to the rejuvenation of the body.

On the other hand, intracellular acidification is prevalent in the growth factor-dependent cell line apoptosis. Intracellular acidification is an intracellular signal change in the process of apoptosis. It promotes apoptosis and has the following facts. Supported: 1 specific Na ⁺ /H ⁺ exchange inhibitor can induce apoptosis by inhibiting Na ⁺ /H ⁺ exchanger by intracellular acidification, in addition to Na ⁺ /H ⁺ exchanger, Na ⁺ -HCO ₃ ^- coordinated transport The change of the device and the Cl ^- /HCO ₃ ^- exchanger is also closely related to apoptosis. There is a dose-effect relationship between the degree of cytoplasmic acidification and the incidence of apoptosis. 2 Simple alkali treatment shows resistance by reducing the degree of intracellular acidification. Inducer-induced apoptosis; there are acidic endonucleases in 3 cells, and intracellular acidification can activate the enzyme to trigger cleavage of nucleosome DNA, which is an important material basis for cell acidification-mediated apoptosis; The activity of many enzymes and proteins involved in apoptosis is enhanced in an acidic environment (Bian Xiaohai et al., "Research progress in intracellular pH in apoptosis", Journal of Changzhi Medical College, 2004, 4).

Aging or apoptotic cells are more likely to die under acidic conditions. Apoptotic cells have enhanced glycolysis and increased acidic metabolites. If externally applied acid pressure (lower pH), apoptotic cells will accelerate toward death. For example, cardiac hypertrophy is a defensive response of myocardial tissue to increased load to temporarily adapt to changes in overall or local myocardial stress. If the need for cardiac physiology exceeds the compensatory capacity of cardiomyocytes, suicide death occurs in cardiomyocytes. That is, apoptosis is one of the main mechanisms for the conversion of cardiac hypertrophy to heart failure. In the absence of oxygen, the rate of glycolysis in hypertrophic cardiomyocytes decreased only slightly, but the rate of glycolysis increased rapidly after reoxygenation, and then reached the peak of the burst and then gradually returned to the level before hypoxia. An increase in the rate of glycolysis inevitably causes intracellular lactic acidosis. The apoptotic rate of hypertrophic cardiomyocytes was significantly higher than that of normal cardiomyocytes during normoxia culture. The apoptosis of both cardiomyocytes was significantly increased after hypoxia. After reoxygenation, the apoptosis of normal cardiomyocytes was gradually reduced, while the hypertrophic cardiomyocytes were reoxygenated. The rate of early apoptosis continued to increase substantially and gradually decreased thereafter. (Feng Bing, Liu Wei, Xu Jing, He Zuoyun, Yang Huibiao, "The relationship between hypertrophic cardiomyocyte apoptosis and energy metabolism pathway transition during hypoxia-reoxygenation", Acta pHysiologica Sinica, October 25, 2005, 57(5): 636-642)

In biochemistry, creatine is a nitrogen-containing organic acid naturally present in vertebrates. Can assist in providing energy to muscles and nerve cells. Studies have found that oral creatine can improve the body performance and prolong life of G93A transgenic mice (muscle atrophic lateral sclerosis), which has a dose-effect relationship and also protects mouse motor neurons and substantia nigra neurons. Klivenyi P, et al. Neuroprotective effects of creatine in a transgenic animal model of amyotrop Hic lateral sclerosis.. Nature Medicine. 1999. mar, 5(3): 347-350). Studies have also found that lipoic acid can slow down the aging process.

It is generally believed that the anti-aging effect of L-ascorbic acid lies in its antioxidant capacity. However, in 2009, important experiments conducted by the Michael Resto laboratory at the University of Jena, Germany, showed that ROS are essential for maintaining the basic functions of the liver. The experimental results show that physical exercise prevents the occurrence of type 2 diabetes by increasing the production of ROS from the electron transport chain system within the mitochondria (the tiny organ of intracellular production, the principle of production through the redox chain). However, if you take the antioxidants vitamin C and vitamin E every day, exercise has no positive effect. A review of Clinical Nutrition published in February 2009 summed up 22 published randomized, double-blind, controlled trials with a total of more than 100,000 participants. The conclusion was that there was no support for "antioxidants to prevent coronary arteriosclerosis." "The saying." A review published in the February 2007 issue of the Journal of the American Medical Association is more resistant to antioxidant health products. A total of 68 studies involving more than 230,000 people were summarized and several antioxidants analyzed had no effect on mortality. If only those low-quality studies were excluded and only 47 high-quality studies involving more than 180,000 people were analyzed, these antioxidants even increased mortality slightly. Therefore, the inventors believe that the anti-aging effect of L-ascorbic acid lies in its acidity.

A variety of acidic substances have a cosmetic effect. For example, with 30% high concentration of salicylic acid as a chemical peeling agent, it can achieve the same effect as lightening pigmentation spots, shrinking pores, removing fine wrinkles and improving aging caused by sun exposure with 70% acid acid peeling. . Skin beauty is to make the skin younger, so it can be concluded that acidic substances can remove aging cells and promote young skin regeneration. The medium-dose and high-dose citric acid treatment groups caused apoptosis in mouse testicular tissue cells (Wang Yanjie: Effect of citric acid on apoptosis of mouse testis tissue [D]; Henan University of Science and Technology: 2012).

The inventors suspect that acidic substances such as L-ascorbic acid, creatine, and lipoic acid can resist aging, and more because their acidic characteristics can accelerate the apoptosis of senescent cells, causing the body to rejuvenate. The inventors have unexpectedly discovered that anti-aging can be achieved by using a chlorine dioxide solution having a higher oxidation potential and supplemented by an acidic environment.

In some embodiments, the present invention demonstrates through experiments that an acidic environment contributes to an increase in the apoptotic effect of a chlorine dioxide formulation on cells.

In various embodiments of the invention, we targeted the administration of the target tissue with an acidic chlorine dioxide solution, demonstrating that the target tissue can be rejuvenated.

Tumor cells have ten characteristics: Self-Sufficiency in Growth Signals; Insensitivity to Antigrowth Signals; Resisting Cell Death); Limitless Replicative Potential; Sustained Angiogenesis; Tissue Invasion and Metastasis; Avoiding Immune Destruction; Promoting Tumor Inflammation (Tumor Promotion) Inflammation); Degegulating Cellular Energetics; Genome Instability and Mutation. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011 Mar 4; 144(5): 646-74). If it inhibits its resistance to cell death, that is, induces tumor cell apoptosis, it greatly inhibits the occurrence of cancer. Logically, since tumor cells are abnormal cells, the body itself should be inhibited by it, that is, these cells should automatically undergo apoptosis, and the reason why apoptosis does not occur is related to its own characteristics. The study found that some cancers may not be completely attributed to genetic damage, but that aging cells bypass the switch that tells them to stop growing. The similarities between behaviors between senescent cells and cancer cells suggest that if aging cells manage to escape death They have the potential to become cancer (Hazel A. Cruickshanks, et al. Senescent cells harbour features of the cancer epigenome, Nature Cell Biology (2013)). The new view is that the root causes of cancer and aging are the same (Carlos López-Otín, et al. The Hallmarks of Aging. Cell, 6 June 2013). The inventors regard tumor cells as senescent cells, which should be directed toward apoptosis. Therefore, the inventors believe that tumor cells are left in the body for a long time because of their special ability to prevent apoptosis.

Two features for tumor cells may be the best way to destroy tumors. These two features are resistance to cell death and abnormal cellular energy. The same applies to the induction of apoptosis in senescent cells. Although tumor cells have their own characteristics, the general cancer patients exist because tumor cells do not automatically die under the action of the body environment, but compared with normal cells, the tumor cells themselves have changed, such as being more afraid of oxides and more afraid of acidic environment. .

Three children with cystic lymphangioma were treated with oral sildenafil, and the tumor volume of the three children was significantly reduced by medication (Glenda L. et al. Swetman, Sildenafil for Severe LympHatic Malformations N Engl J Med 2012;366:384-386 January 26,2012). Sildenafil is a 5-phosphodiesterase inhibitor, and phosphodiesterase is a negative regulator of the NO-cGMP pathway, so sildenafil is able to release nitric oxide, a biologically active substance. Therefore, the inventors believe that nitric oxide, an oxide, acts as a direct inhibitor of tumors.

In experiments with breast and melanoma cancer cells, it was found that nitroglycerin can enhance the action of common chemotherapeutic drugs to kill cancer cells by producing nitric oxide (Barsoum IB, et al. Hypoxia induces escape from innate immunity in cancer cells via increased Expression of ADAM10: role of nitric oxide. Cancer Res. 2011 Dec 15;71(24):7433-41). A prodrug that promotes nitric oxide release, induces death of breast cancer cells while retaining normal mammary epithelial cells (Vanity McMurtry, et al. JS-K, a nitric oxide-releasing pro-drug, induces breast cancer cell death while sparing Normal Mammary epithelial cells, International of journal of oncology, Published online on: Tuesday, January 25, 2011, Pages: 963-971). It is generally believed that high concentrations of nitric oxide synthase can produce toxic effects on tumor cells that inhibit their growth (Xu W, et al. The role of nitric oxide in cancer. Cell Res. 2002 Dec; 12 (5-6 ): 311-20).

Cell proliferation experiments showed that 400μmol/L, 800μmol/L and 1600μmol/L had different degrees of cytotoxicity to Hela cells cultured in vitro, up to 89.0%. After hydrogen peroxide treatment, the cell senescence rate and the level of reactive oxygen species increased with the increase of hydrogen peroxide concentration, showing a dose-effect relationship. Conclusion: Hydrogen peroxide has obvious cytotoxicity to Hela cells, and can induce its senescence and affect the production of reactive oxygen species.

Like aging cells, tumor cells rely more on low-oxygen glycolysis to provide energy. It was discovered more than 80 years ago that the glucose metabolism of tumor cells is stronger than that of normal cells, and even under aerobic conditions, it depends on glycolysis. This phenomenon is called "Warburg effect", and its mechanism is related to the cell membrane of tumor cells. Glucose transporter (Glut) is active and is involved in increased hexokinase activity. The result of the "Warburg effect" is that the cells produce a large amount of glycolysis product, lactic acid. A large amount of H ⁺ removed from sugar cannot be oxidized into water through the respiratory chain like aerobic oxidation, and a large amount of H ⁺ aggregation will threaten the pH (pHi) acidification and apoptosis in the cells, while the tumor cells enhance the acid secretion. The function allows the pHi in the cell to be maintained, but will have an adverse effect on the surrounding normal host cells. The "Warburg effect" of tumor cells causes tumor cells to produce more acid than normal cells, but the pH value of tumor cells is found to be indistinguishable from normal cells, while extracellular pH (pHe) and intracellular acidic vesicles. The pH inside (pHv) is significantly lower than that of normal cells, suggesting that it may be related to the strong acid function of tumor cells. (Izumi H, Torigoe T, Ishiguchi H, et al. Cellular pH regulators: potentially promising molecular targets for cancer chemotherapy. Cancer Treat Rev, 2003, 29(6): 541-549).

In order to avoid the toxicity of the acidic microenvironment, the tumor cells excrete hydrogen ions outward, eventually producing an extracellular acidic environment and an alkaline environment inside the cells. Tumor cells regulate hydrogen ion-associated transporters of the plasma membrane, such as sodium-hydrogen exchange protein (Na ⁺ /H ⁺ ), Na ⁺ /K ⁺ -ATPase, vesicular H ⁺ -ATPases, H ⁺ /CI ^- co-transporters And monocarboxylic acid transporter (MCT), etc., to achieve this mechanism (Harguindey S, et al. The role of pH dynamics and the Na+/H+antiporter in the etiopathogenesis and treatment of cancer. Two faces of the same coin- -one single nature, [J]. Biochim BiopHys Acta, 2005, 1756(1): 1-24).

Therefore, the inventors judged that if the acid secretion function of the tumor cells is lowered by increasing the pressure, the pHi in the tumor cells is lowered, causing acidosis, thereby inducing apoptosis of the tumor cells. While increasing the pressure, even if the pH (pHe) of the tumor's extracellular environment is lowered, the proton pump that transports the H ⁺ ions of the tumor cells increases the pressure and eventually collapses.

There is more literature support, and the acidic environment can induce apoptosis in tumor cells. For example, an acidic environment, through p53 A C Williams, et al. An acidic environment leads to p53 induction of apoptosis in human adenoma and carcinoma cell lines:implications for clonal selection during colorectal carcinogenesis,Oncogene.1999May 27;18(21):3199-204).

Experiments have shown that gastric cancer AGS cells cultured in vitro have an acidic environment with a pH of 6.0. This acidic environment increases the concentration of extracellular H ⁺ ions, possibly by inhibiting the Na ⁺ -H ⁺ exchange function of NHE ^-1 in tumor cells. Excessive H ⁺ ions produced by intracellular glycolysis cannot be discharged to significantly acidify the otherwise alkaline environment, which not only changes the extracellular environment of gastric cancer, but also changes the intracellular environment. The acidification of this internal and external environment of gastric cancer cells changes the pH of the cells inside and outside the environment suitable for its growth and proliferation, so that most gastric cancer cells stop proliferating (Gao Yan et al., "Measurement of pH in gastric cancer AGS cells and acidic environment Effects of Proliferation and Apoptosis, Progress in Modern Biomedicine, 2008, 09).

Rich IN et al. demonstrated that leukocyte lines and peripheral lymphocytes obtained from leukemia patients have a statistically high pH value that is more common than normal hematopoietic tissue cells. This illustrates a direct relationship between intracellular pH and cell cycle regulation of normal hematopoietic and leukemia cells. Using this relationship, they treated leukemia cells with the Na ⁺ /H ⁺ exchanger inhibitor 5-(N,N-hexamethylene)-amiloride (HMA), which reduced intracellular pH and induced apoptosis. (Rich IN, Worthington-White D, Garden OA, Musk P. Apoptosis of leukemic cells accompanies reduction in intracellular pH after targeted inhibition of the Na+/H+exchanger. Blood. 2000, 15, 95(4): 1427~1434) .

In one embodiment of the present invention, we use an acidic chlorine dioxide solution to target tumor cells, cause tumor cells to undergo apoptosis, and with the strengthening of the acidic environment, the tumor cell mortality increases accordingly.

Those skilled in the art understand that strong oxidants and acidic environments have a damaging effect on normal cells, that is, acidic chlorine dioxide solution also accelerates apoptosis in normal cells. However, the inventors believe that normal cell damage stimulates the regeneration of body stem cells, so that general damage can be completely repaired; in addition, senescent cells or cancer cells are dysfunctional cells that resist apoptosis in oxidants or acidic environments. The ability is weaker. Therefore, the technical solution of the present invention is an anti-aging or anti-tumor technique with targeted and less side effects.

In some embodiments, the present invention demonstrates that an acidic chlorine dioxide formulation has a greater apoptosis-inducing effect on senescent cells or tumor cells, but less damage to normal cells, or that the damage is at a level that can be compensated. .

The chlorine dioxide-containing apoptosis inducing agent of the present invention may comprise a single dosage form of chlorine dioxide alone or a combination of several parts comprising a chlorine dioxide precursor. A single dosage form in which chlorine dioxide alone is included can be made as follows:

Method 1: A pH adjuster is added to water to prepare an acidic aqueous solution having a pH of 1.5 to 6.5. By conformity with the regulations, Further, the chlorine dioxide gas is produced by a method of preparing a chlorine dioxide gas having a concentration of 99.9% or more (preferably, the chlorite reacts with an acid). A chlorine dioxide solution of 500 to 2900 ppm was prepared by bubbling and dissolving the chlorine dioxide gas into the above acidic aqueous solution. The solution should be stored in the dark and sealed at a temperature of 4 ° C to 15 ° C before being used in the application of the present invention. The method for preparing a chlorine dioxide-containing preparation is not necessarily an aqueous solution, and only the substance capable of functioning in the target tissue when applied to the target tissue is mainly chlorine dioxide, and in a certain acidic environment.

Method 2: A chlorine dioxide precursor is dissolved in water to prepare a 1% to 40% aqueous solution. An acidic solution (preferably 2% to 50% citric acid solution) containing a pH adjuster is added to the aqueous solution to adjust the pH of the mixed solution to 1.5 to 6.5. The solution should be stored in the dark and sealed at a temperature of 4 ° C to 15 ° C before being used in the application of the present invention. The method for preparing a chlorine dioxide-containing preparation is not necessarily an aqueous solution, and only the substance capable of functioning in the target tissue when applied to the target tissue is mainly chlorine dioxide, and in a certain acidic environment.

The combination of several parts comprising the chlorine dioxide precursor can be prepared by dissolving the chlorine dioxide precursor in water to prepare a 1% to 40% aqueous solution, which is the first solution; preparing an acidic solution with a pH adjuster ( It is preferably a 2% to 50% citric acid solution), which is a second solution. Before use in the application of the present invention, the above solution is mixed in the field, and the pH of the final mixed solution is adjusted to 1.5 to 6.5. After the chlorine dioxide is reacted to generate chlorine dioxide, the solution is applied to the target tissue. The method for preparing a chlorine dioxide-containing preparation is not necessarily an aqueous solution, and only the substance capable of functioning in the target tissue when applied to the target tissue is mainly chlorine dioxide, and in a certain acidic environment.

The chlorine dioxide precursor which can be used in the present invention may, for example, be an alkali metal chlorite or an alkali metal chlorite. Examples of the alkali metal chlorite include sodium chlorite, potassium chlorite, and lithium chlorite. Examples of the alkali metal chlorite include, for example, calcium chlorite, magnesium chlorite, and sub Bismuth chlorate.

Among them, sodium chlorite and potassium chlorite are preferable, and sodium chlorite is more preferable, from the viewpoint of obtaining an easy reason and being excellent in the sustainability of chlorine dioxide activity.

The pH adjuster which can be used in the present invention can be preferably used as long as it is a buffering acid.

Examples of the organic acid or a salt thereof include formic acid, acetic acid, propionic acid, butyric acid, lactic acid, pyruvic acid, citric acid, malic acid, tartaric acid, gluconic acid, glycolic acid, fumaric acid, malonic acid, and maleic acid. , oxalic acid, succinic acid, acrylic acid, crotonic acid, glutaric acid and their salts.

Examples of the inorganic acid include hydrochloric acid, phosphoric acid, boric acid, metaphosphoric acid, pyrophosphoric acid, and sulfamic acid. Examples of the inorganic acid salt include a dihydrogen phosphate salt (sodium salt, potassium salt, the same applies hereinafter), a mixture of a dihydrogen phosphate salt and a hydrogen phosphate salt, and the like. One type of the pH adjuster may be used alone or two or more types may be used in combination.

From the standpoint of use in human safety, the pH adjusting agent is preferably citric acid, acetic acid and sodium dihydrogen phosphate, more preferably citric acid.

Further, the final pH of the chlorine dioxide solution is preferably from 1.5 to 5.5, more preferably from 1.5 to 3.5.

The chlorine dioxide-containing apoptosis inducing agent in the present invention is preferably a liquid preparation.

The dose of the apoptosis inducing agent in the present invention varies depending on the age, body weight, disease properties and condition of the patient. However, in the case of an adult, for example, 1 mg to 5000 mg of chlorine dioxide per day, preferably 1 mg to 1000 mg of chlorine dioxide per day.

The chlorine dioxide-containing apoptosis inducing agent of the present invention can be administered by various means. The chlorine dioxide-containing apoptosis inducing agent can be administered systemically, such as by intravenous, intra-arterial or intraperitoneal administration; or directly administered to a local affected area, the method can be directly administered by transdermal, puncture, etc. The affected area is administered.

The method for administering the chlorine dioxide-containing apoptosis inducing agent in the present invention can be administered by any route which can reach the intended tissue, for example, by intravenous drip, intravenous injection, intraarterial injection, intramuscular injection, subcutaneous Injection, intradermal injection, intracardiac injection, intraperitoneal injection, intrathecal injection, intra-articular injection, puncture injection, rectal administration, sublingual administration, nasal administration, transdermal administration, inhalation or topical administration to the target Organs, tissues, etc., but are not limited to these.

When administered by the above method, the effective amount of the apoptosis inducing agent in the present invention ranges from 0.1 to 500 mg/kg of chlorine dioxide per day due to the size of the target tissue, and sometimes the area of the target tissue is more easily expressed. The effective range of the apoptosis inducing agent in the invention is 0.1 to 500 mg of chlorine dioxide per 100 cm ² per day, and is effective after at least 10 days when administered at the dose.

In any case, the chlorine dioxide-containing apoptosis inducing agent of the present invention can only exert the effect of inducing apoptosis when the chlorine dioxide suitable for the target effective dose enters the target tissue in an appropriate manner, and is not limited thereto. Any form, method and procedure, and the help of other auxiliary substances.

Any of the preparations in the "Chinese Pharmacopoeia General Rules" can be used as a dosage form of the apoptosis inducing agent in the present invention. Examples of the dosage form of the apoptosis inducing agent in the method of the present invention include injections (including suspensions, emulsions) for direct use in the body; ointments (including oily ointments, cream ointments (creams), water-soluble ointments Etc., inhalation, liquid preparation (including eye drops, nasal drops, etc.), suppositories, patches, pastes, lotions and other external preparations; or tablets (including sugar coatings, films, gel coats), liquid preparations , capsules, granules, powders (including fine-grain grade), pills, syrups, lozenges, and the like. These preparations can be prepared according to the general principles of Chinese Pharmacopoeia preparations.

Furthermore, the apoptosis inducing agent in the method of the present invention may also comprise a pharmaceutically acceptable solid or liquid carrier or interventional therapeutic material when administered. As the pharmaceutically acceptable solid or liquid carrier, there may be mentioned solvents, stabilizers, solubilizers, emulsifiers, suspensions, buffers, isotonic agents, colorants, bases, thickeners, excipients, lubricants. And the like, but not limited to, a binder, a disintegrant, a coating agent, a flavoring agent, a conditioning agent, a foaming agent, a superabsorbent resin, a surfactant, a penetration enhancer, and a pH adjuster.

Specific examples include deionized water, lactose, white sugar, fructose, glucose, mannose, sorbitol, etc., or sugar alcohols; Crystalline cellulose, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, low-substituted hydroxypropyl cellulose, hydroxypropyl methyl cellulose, hydroxypropyl methyl cellulose phthalate, Hydroxypropylmethylcellulose acetate succinate, hydroxymethylcellulose, hydroxymethylcellulose calcium, sodium carboxymethylcellulose, crosslinked hydroxymethylcellulose sodium, hydroxymethylethylcellulose, acetic acid Cellulose and its related derivatives such as cellulose phthalate; corn starch, wheat starch, rice starch, potato starch, cyclodextrin, amylopectin and other related derivatives; agar, sodium alginate, arabic Gelatin, gelatin, collagen, shellac, yellow potato gum, xanthan gum and other natural polymers (seaweed, plant mucilage, protein, etc.); polyvinylpyrrolidone, aminoalkyl methacrylic acid copolymer, methacrylic acid copolymerization Synthetic polymer such as hydroxyvinyl copolymer, polyvinyl alcohol, dimethylpolysiloxane; olive oil, cocoa butter, carnauba wax, butter, hardened oil, soybean oil, sesame oil, camellia oil, flaxseed Oil, paraffin, liquid paraffin, yellow beeswax White petroleum jelly, coconut oil, microcrystalline wax and other oils; stearic acid, aluminum stearate, calcium stearate, magnesium stearate, triethyl citrate, triethyl glyceride, medium chain fatty acid triglyceride Fatty acids such as stearin and isopropyl myristate and their derivatives; alcohols and polyols such as ethanol, glycerin, stearyl alcohol, cetyl alcohol, propylene glycol, polyethylene glycol; zinc oxide, calcium hydrogen phosphate, and precipitated calcium carbonate , synthetic aluminum silicate, silicic anhydride, kaolin, dry aluminum hydroxide gel, synthetic hydrotalcite, titanium oxide, talc, bentonite, magnesium aluminum silicate, potassium aluminum sulfate, bismuth sub-gallate, bismuth subsalicylate, lactic acid Inorganic substances and metal salt compounds such as calcium, sodium citrate, sodium chloride, sodium hydrogencarbonate; sucrose fatty acid esters, polyoxyl stearates, hydrogenated castor oil polyoxyethylene ethers, polyoxyethylene polyoxypropylene diols , sorbitan sorbitan ester, sorbitan trioleate, sorbitan monostearate, but sorbitan palmitate, sorbitan monolaurate, polysorbate, single hard Glyceryl glyceride, sodium lauryl sulfate, poly gui Surfactants such as alcohol; osmotic accelerators such as dimethyl sulfoxide and its analogues, azone compounds, pyrrolidone derivatives, alcohol compounds and fatty acid compounds; pigments; But it is not limited to these.

Examples of the interventional treatment material include, but are not limited to, a syringe, a stent, an artificial blood vessel, a puncture syringe, a catheter, a balloon, and the like.

The chlorine dioxide-containing apoptosis inducing agent of the present invention may be administered an anesthetic according to the administration mode before administration, such as an injection type anesthetic such as barbiturate or an inhaled anesthetic such as nitrous oxide, Lidoca. Because of the surface anesthetic, etc., but not limited to these.

The apoptosis inducing agent in the present invention is applied to mammals, and specific examples of mammals include humans, monkeys, dogs, pigs, cats, rabbits, rats, and mice. Among them, people are preferred.

Example

The following examples are provided to illustrate the invention and are not intended to limit the invention in any way.

Example 1: Effect of chlorine dioxide-containing preparation on apoptosis of proliferative scar skin fibroblasts

As a kind of disease that affects organ function and appearance, hypertrophic scar has many causes, but any damage that affects deep layers of the dermis may cause hypertrophic scars. It is generally believed that insufficient apoptosis of fibroblasts is the cause of hypertrophic scars (Ogawa, R. The most current algorithms for the treatment and prevention of hypertrop Hic scars and keloids [J]. Plast Reconstr Surg, 2010, 125: 557-568) . Common sense shows that wounds of the same size are relatively slower to heal and tend to leave scars than younger people. Therefore, the formation of hypertrophic scar can be understood as a phenomenon of skin aging, which may be a good way to cure scarred skin by inducing apoptosis of fibroblasts.

1) Apoptosis test

There were 6 patients with hypertrophic scars, including 5 males and 1 female, with an average age of 42 years. The experiment was divided into 4 groups: the central part of the scar (within the central 2/3 radius), the edge of the scar (within the radius of 1/3 of the periphery), the skin around the scar (within 0.5 cm from the edge of the keloid), and other parts of the scar patient. Normal skin.

Culture of fibroblasts: The specimens were grouped according to the above method, and the epidermis and subcutaneous tissues were excised under aseptic conditions. The specimens were cut into tissue pieces of about 1 mm ³ in a small amount of fetal bovine serum at 37 ° C, 5% CO. ₂ Incubate for 4-6 hours under saturated humidity conditions, so that the tissue block adheres to the bottle wall, then add appropriate amount of DMEM (dulbecco's modified eagle medium) containing 15% fetal bovine serum, and change the solution once every 3-4 days, 2 - After 3 weeks, the primary cells were overgrown and pooled into pellets, passaged once every 3-5 days, and cells 6-8 were used for the experiment.

The first solution was prepared by dissolving a mixed solution of 7.47% sodium chlorite and 1.59% sodium chloride in deionized water; a citric acid solution having a concentration of 16.7% was prepared with deionized water to prepare a second solution. . Take the same volume of solution from the different parts of the solution, mix, wait for the solution to mix and stand for 3 to 5 minutes, then filter with 0.22μm double-layer filter, dilute with deionized water to prepare different concentrations of chlorine dioxide. Solution.

Cells were seeded and different treatment factors applied to induce apoptosis. Each group was taken from a cell in the logarithmic growth phase and inoculated into a 6-well cell culture plate. Each group of cells was seeded with 1 well (1 × 10 ⁵ cells/well) per plate, and a total of 6 plates were inoculated. 24 wells, continue to culture at 37 ° C, 5% CO ₂ saturated humidity for 48-72h, until the cells are full of the bottom of the well, take one of the plates, aspirate the medium, wash with Hank liquid (a balanced salt solution) After adding DMEM medium containing no fetal bovine serum, another three plates were added to the chlorine dioxide-containing preparation to achieve a final concentration of 100 ppm, 1000 ppm, and 2900 ppm, respectively, and one plate was added to FasMcAb (Fas mAb). The final concentration reached 1 μg/ml, and the other plate did not apply any treatment factor, and the apoptosis rate was detected after 24 hours of culture.

PI staining and flow cytometry were used to compare the apoptosis rate of each component fiber cell. The treated cells were digested and collected in a 10 ml centrifuge tube, centrifuged at 1000 r/min for 10 min, washed with pre-cooled PBS (phosphate buffer solution), added with 70% ethanol at 4 ° C overnight, then centrifuged at low speed for 10 min, washed with PBS. Resuspend the cells and add in the remaining 0.5 ml of cell suspension Into RNase A 2μl (20mg/ml), incubate at 37 °C for 30min, immediately put into the ice bath to stop the action of RNase A, then add 500μl PI staining solution (100μg/ml) and incubate for 30min in the dark, blow off the cells, filter through 300 mesh filter Cellular DNA analysis was performed on a flow cytometer to detect apoptosis rate.

The apoptosis rate of all the cells was increased after 24 hours of serum-free culture. However, after comparing the growth rate of apoptosis in each group, the normal skin was found to be significantly higher than the edge of keloid. <0.01), and the skin around the keloid and the central part of the keloid were between the two, and there was no significant difference between the two groups (P>0.05, Table 1). This indicates that the scar that is regarded as an aging tissue has obvious anti-apoptotic ability, so the scar tissue can not only be automatically removed, but will proliferate.

Table 1 Apoptosis rate of scar and its surrounding skin fibroblasts after 24 hours of serum-free culture

Note: *Compared with the edge of the scar, P<0.01

The apoptosis rate of fibroblasts in normal skin under FasMcAb was significantly higher than that in scar and surrounding skin fibroblasts (P<0.01, Table 2). This also indicates that the tissue at the scar has anti-apoptotic ability.

Table 2 Apoptosis rate of fibroblasts in scar and its surrounding skin under FasMcAb

Drawing part	FasMcAb action (1μg/ml)
		Scar edge	7.32±2.18
Central scar	6.52±1.90
		Scar around the skin	13.08±1.97
Normal skin	63.04±6.59*

Note: *Compared with the edge of the scar, P<0.01

Under the action of chlorine dioxide preparation, the apoptosis rate of the four-component fiber cells gradually increased with the increase of the chlorine dioxide concentration gradient, and the increase was significant (P<0.01, Table 3). This shows the chlorine dioxide preparation in acidic environment for hypertrophic scar tissue Has a significant pro-apoptotic effect.

Table 3 Apoptosis rate of fibroblasts in scars and their surrounding skin under chlorine dioxide

2) Treatment of scars

The first solution was prepared by dissolving a mixed solution of 7.47% sodium chlorite and 1.59% sodium chloride in deionized water; a citric acid solution having a concentration of 16.7% was prepared with deionized water to prepare a second solution. . The same volume of solution was taken from the containers of different solutions, mixed, and the solution was allowed to stand still for 3 to 5 minutes, and then filtered through a 0.22 μm double-layer filter. A chlorine dioxide preparation was prepared.

The hypertrophic scar was treated twice with the above chlorine dioxide preparation, the chlorine dioxide preparation was directly used once, and the same amount of dimethyl sulfoxide was added for the second time. That is, the scar is directly applied to the two times, with an interval of 30 minutes in between, and continuous application for 15 days.

Immediately after treatment, the hypertrophic scar was dark red, and the surface of the scar was uniformly distributed with gray to yellowish punctate gasification dander, and there was no obvious bleeding or exudation. On the first day after treatment, the wound surface was dry and red punctate suede was formed. From then on to the fifth day after treatment, the treated wound surface was bright red, and the wound surface gradually fell off. On the twentieth day after the treatment, the treated wounds completely healed, and the suede all fell off. At the same time, the height of the papillary lobes in the center of the scar was observed to be low, and the skin at the scar was smooth and pigmented. About 25 days after the treatment, smooth fresh skin was formed, and the original irregular shape of the scar was completely flat at about 40±20 days.

The scar was taken before treatment, 1 hour after treatment, 2 days, 7 days, 14 days, and 28 days after treatment. The whole layer of the scar was cut, and the tissue was removed by 4% paraformaldehyde, then dehydrated and embedded in paraffin. HE staining was used to observe the morphology, density, collagen fiber arrangement and capillary proliferation of microfibrous cells in each group. TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) method was used to detect the apoptosis rate of fibroblasts.

Using the TUNEL method, the stained area is located in the nucleus. Under normal light microscope, the nuclei of normal fibroblasts were blue, which was a negative reaction. The nucleus of apoptotic cells showed a dark brown color, which was a positive reaction, namely apoptotic cells. Under the observation of 40 times light microscope, each slice was randomly selected to have the highest number of positive cells with the highest number of fibroblast positive cells, and the positive cells were reversed. Rate (R) = number of positive cells / total number of cells, and the results were averaged.

Apoptotic bodies, ie apoptotic cells, are found in fibroblasts, keratinocytes, and vascular endothelial cells. Immediately after treatment, there was an obvious increase in fibroblast apoptosis. The apoptosis rate of fibroblasts was gradually increased at 1h, 2 days, 7 days, 14 days, and 28 days after treatment, and the apoptosis rate was higher than that before treatment. P value <0.01, indicating that the difference is statistically significant.

Table 4 Changes of apoptotic rate of fibroblasts detected by TUNEL in scar tissue

* represents P < 0.01 compared with before treatment.

Therefore, whether in vitro culture or direct treatment, it is shown that the chlorine dioxide preparation in the acidic environment of the present invention can induce apoptosis of the cells in which the cells are accelerated, thereby making the skin healthy and young.

Example 2: Cosmetic test for anti-aging of skin containing chlorine dioxide preparation

Cosmetic preparation for chlorine-containing preparations for skin cosmetic: a mixed solution of 7.47% sodium chlorite and 1.59% sodium chloride is prepared with deionized water to prepare a first solution; concentration is set with deionized water A second solution was prepared as a 16.7% citric acid solution. The same volume of solution was taken from the containers of different solutions, mixed, and the solution was allowed to stand still for 3 to 5 minutes, and then filtered through a 0.22 μm double-layer filter. An equal volume of 90% dimethyl sulfoxide is added, and then sealed in a glass bottle to prepare an anti-aging or youthful cosmetic for the skin.

The female observer (100) was actually applied to the skin and evaluated according to the following evaluations. The evaluation of "the presence or absence of sticky feeling", "the presence or absence of the feeling of hairiness", and "the presence or absence of movement on the skin" And the presence or absence of the effect of wrinkles and slacks (skinning of the skin).

From the results (Table 5), the chlorine dioxide preparation of the present invention has a very excellent cosmetic and skin care function as a cosmetic.

Table 5: Observation of skin cosmetic use effect

The cosmetic prepared by the above invention is taken as an example for observation of the therapeutic effect, and the efficacy test is carried out in the human population, and the improvement of the skin moisture content, elasticity, fine lines and roughness during the use of the user is observed. 30 healthy women and 10 healthy men were selected and applied freely to the skin for 15 days. Before use, 1h, 7 days (after 1h use), 14th day (after 1h use), and 28th day The tester performed an assessment of the condition of the skin, including clinical judgment and non-invasive instrument evaluation. Clinical evaluation included the following parameters: skin moisture content, skin elasticity, skin gloss, skin roughness/smoothness, skin tone uniformity, and fine lines. Non-invasive instrument evaluation was performed by measuring the skin moisture content using a skin moisture meter Corneometer CM820, and skin elasticity was measured using a skin elasticity meter (Cutometer SEM 575). The experimental data is shown in Table 6.

It can be seen from Table 6 that after using this cosmetic, the moisture content of the skin increases sharply, indicating that the product is very effective in improving the skin drying effect, and the water content of the skin continues to be improved continuously as time advances to the 28th day.

The greater the skin elasticity value, the better, indicating that the skin elasticity is better. It can be seen from Table 6 that after 7 days, the elasticity of the skin is obviously improved, the elasticity becomes better and better, and there is a significant difference from the basic value.

Four indicators such as glossiness, the smaller the data value, the better the improvement. From the above data, it can be seen that compared with the basic values, the parameters are improved on the 7th, 14th and 28th days, and there is a significant difference from the basic values (expressed in the downward trend of each value over time). ).

Table 6 Skin improvement after use of chlorine dioxide cosmetics

Note: *P<0.01 compared to the base value.

Therefore, the chlorine dioxide-containing cosmetic of the present invention has a cosmetic effect of skin rejuvenation such as skin care, hydration, freckle, whitening, and wrinkles.

Example 3 Test for the treatment of acne with chlorine dioxide preparation

Acne is a frequent skin problem in adolescents. The main reason is that the aging cuticle cannot be removed in time, clogged pores, causing oil to accumulate and form swelling (acne). After infection by acne bacteria, redness forms pustules. Since chlorine dioxide itself is a highly effective fungicide, it is only used to ensure that aged keratinocytes are removed, so chlorine dioxide can treat acne.

Cosmetic preparation of chlorine dioxide-containing preparation for acne (treatment of acne): Using the preparation method of Example 2, an acne solution was prepared for acne skin application.

Thirty young people with acne problems (male 20, female 10) were selected, with an average age of 22 years. The acne affected area was applied once daily for 5 days with the above-mentioned chlorine dioxide-containing preparation. The specific effects are shown in the table below.

Table 7 Acne Treatment Test

30 people with acne, for 5 days in a row, 90% of acne recovered. It is indicated that the chlorine dioxide-containing preparation of the present invention is very effective for treating acne.

Example 4 Test of the effect of intravenous injection of chlorine dioxide preparation on the lifespan of mice

Preparation of chlorine dioxide preparation: a mixed solution of 2.49% sodium chlorite and 0.53% sodium chloride was prepared with deionized water to prepare a first solution; a citric acid solution having a concentration of 5.57% was prepared with deionized water. A second solution was prepared. Take the same volume of solution from the different parts of the solution, mix, wait for the solution to mix and stand for 5 to 10 minutes, add an equal amount of 50% dimethyl sulfoxide, then filter with 0.22 μm double-layer filter, load immediately syringe. Prepare once and use once.

C57BL/6 male mice, 12 months old, each cage was fed in a single cage. 100 mice were randomly divided into two groups: experimental group (50) and control group (50). Both groups had sufficient feed and drinking water. The experimental group was intravenously injected with the chlorine dioxide preparation prepared by the above method at a dose of 10 ml/kg once a week after 15 months of age. The dates of natural death of each mouse in each group were recorded in detail, and the average lifespan and maximum lifespan of each group of mice were calculated.

The results showed that the average lifespan of the control mice was 696.5 days, and the average lifespan of the mice in the test group was 778.3 days. The weekly chlorine dioxide preparation extended the average lifespan of the mice by 11%. This indicates that the injection of chlorine dioxide preparation significantly prolonged the average lifespan of mice (see Table 8).

According to the currently used maximum life calculation method, the average life expectancy of 10% of the mice that died naturally in the group is the maximum life expectancy. The results showed that the maximum lifespan of the control mice was 932 days, and the maximum lifespan of the mice in the test group was 987.5 days. The injection of chlorine dioxide preparation extended the maximum lifespan of the mice by 5.9%, indicating that the injection of chlorine dioxide was significantly prolonged. The role of the maximum lifespan of mice.

Table 8 Effect of injection of chlorine dioxide on the lifespan of mice

Note: *P<0.05 compared with the control group

At the time of the mouse's age of 19 months, 10 mice were randomly selected from the experimental group and the control group, respectively, and subjected to a water maze and a balanced endurance test. The two test intervals are 2 weeks.

Water maze test: continuous training for 3 days, 2 times / day before the experiment. The square water labyrinth device is a black wooden trough 50cm×30cm×15cm, a starting zone, a 4-blind end meandering loop, and a safety platform. The water depth is controlled at 12 cm and the water temperature is (25 ± 2) °C. The mouse is placed from the entrance end to start timing, and the number of times the blind end is touched is automatically recorded. The mouse is climbed up to the safety station as the end time, which is called the incubation period. The incubation period is set to a maximum of 120s, and animals that cannot be exported can be found after screening for more than 120s.

Balanced endurance test: Continuous training for 3 days, 2 times/day before the experiment. A wire with a diameter of 2 mm is pulled between the two fixed columns at a distance of 2 m and a height of 1 m. Bundle the lower limbs with a rope, hold the mouse tightly with the upper limbs, and use a stopwatch to calculate the time to start gripping to the fall. The observation time was set to 30 s, and the animals that fell within 30 s were screened out.

It can be seen from Table 9 that there is no difference in memory on the first day of each group of animals; on the 3rd, 6th and 9th day of the experiment, the number of errors and latency of the test group were significantly less than that of the control group. It can be seen that the chlorine dioxide preparation significantly improves the learning ability and memory ability of the mouse.

Table 9 Comparison of memory in different groups of mice at different times

Note: *P<0.05 compared with the control group

As can be seen from Table 10, the balance endurance of the test group mice at 1 day, 3 days, 6 days, and 9 days was significantly higher than that of the control group. It is indicated that the chlorine dioxide preparation significantly improves the body capacity of the mouse.

Table 10 Comparison of balanced endurance in different groups of mice at different times

Group	1 day	3 days	6 days	9 days
					Control group	88±48.39	93±45.63	102±56.34	112±60.50
test group	132.90±46.53*	140.30±56.36*	149.35±42.64*	153±60.35*

Note: *P<0.05 compared with the control group

From the experimental data of the above various examples, we can conclude that the chlorine dioxide preparation in an acidic environment can significantly improve the lifespan of the animal, accompanied by the improvement of memory and body capacity, the mechanism should be that the chlorine dioxide preparation can induce senescent cells. Apoptosis occurs and aging cells are cleared, and the body's own ability to regenerate will rejuvenate. Therefore, the chlorine dioxide preparation of the present invention can be used as a cosmetic for the skin rejuvenation, or can be made into a medicine for rejuvenating the body, thereby preventing the occurrence of senile diseases.

Example 5 The killing effect of chlorine dioxide preparation on human non-small cell lung cancer cell A549.

The first solution was prepared by dissolving a mixed solution of 7.47% sodium chlorite and 1.59% sodium chloride in deionized water; a citric acid solution having a concentration of 16.7% was prepared with deionized water to prepare a second solution. . Remove the same volume of solution from the different parts of the container, mix, wait for the solution to mix and stand for 3 to 5 minutes, then filter with 0.22μm double-layer filter, dilute with deionized water to prepare different concentrations of chlorine dioxide solution. .

1) Experimental method

The human non-small cell lung cancer cell A549 cells were cultured in a culture medium containing 10% fetal bovine serum with PRMI1640 and placed in a 37 ° C, 5% CO 2 incubator. The cells were digested with 0.25% trypsin solution every 3 days, and replaced. liquid. Inoculate 96-well plates at a concentration of 0.7×10 ⁴ /well, and incubate at 37 ° C, 5% CO 2 incubator for 24 h, then add chlorine dioxide solution, each chlorine dioxide solution according to the final concentration of 100ppm, 200ppm, 400ppm , 700ppm, 1000ppm, 1500ppm, 2000ppm, 2900ppm were added, the negative control cells were added with 0.1% (v/v) DMSO (dimethyl sulfoxide), and the other group was adjusted to zero (ie, there were no cells in the cell culture well, only added Cell culture medium). Four replicate wells were set in each group, and then 96-well plates were placed in a 37 ° C, 5% CO 2 incubator for another 24 h, and then the killing effect of the compounds on tumor cells was examined as follows.

2) Detection method:

The lactate dehydrogenase (LDH) release test is used to detect tumor cell death rate using a lactate dehydrogenase detection kit, and the specific method is carried out according to the instructions of the lactate dehydrogenase detection kit. Resuspend the reaction substrate (Substrate Mix) with Assay Buffer. 50 μl of the culture supernatant of 3 replicate wells of each group in a 96-well cell culture plate was plated into a new 96-well plate, and the remaining one of each group was added to a Triton with a final concentration of 0.9% (v/v). -x100 (provided in the kit) and placed in a 37 ° C incubator for 50 minutes to lyse the cells, 50 μl of the supernatant was added to the new 96-well plate, and then an equal volume was added to each well in the new 96-well plate. Resuspend the reaction substrate solution, incubate for 30 minutes at room temperature, add 50 μl of stop solution per well, ie 1 M acetic acid (provided in the kit) to stop the reaction, and measure the OD value of each well with a microplate reader (490 nm wavelength). The OD value detected by lysing the cell well was recorded as "the maximum release OD value of the cells", and the OD value of the negative control group was recorded as "the OD value of the natural release control group".

Based on the detected OD490 value, the cell death rate was calculated according to the following formula, and the results were expressed as mean ± standard deviation, and the half-cell killing concentration, i.e., IC ₅₀ , was calculated using the SPSS software Probit module.

Tumor cell mortality (%) = (experimental group OD value - natural release control OD value) / (cell maximum release OD value - natural release control OD value)

3) Test results:

As shown in Table 10, the chlorine dioxide preparation can effectively kill A549 cells, and their killing effect on A549 increases dose-dependently as the concentration increases, and the chlorine dioxide preparation results in half of the calculation by the SPSS software Probit module. effective dose (IC ₅₀₎ A549 cell death is 495ppm.

Table 10 In vitro killing of A549 cells

This example demonstrates that an acidic chlorine dioxide formulation is capable of significantly killing A549 tumor cells.

Example 6 killing effect of chlorine dioxide preparation on human cervical cancer cell line HeLa

A chlorine dioxide preparation was prepared according to the method of Example 5, and human cervical cancer HeLa cells were cultured in a cell culture method according to Example 5 and seeded in a 96-well culture plate. The experimental group, the control group and the zeroing group were set according to the method in Example 5, and the death rate of HeLa cells caused by the chlorine dioxide solvent was detected by the lactate dehydrogenase (LDH) release test, and the death of half of HeLa cells was calculated. Dosage (IC ₅₀ ).

Test results: As shown in Table 11, the chlorine dioxide preparation can effectively kill HeLa cells, and with the concentration of the compound Increasingly, their killing effect on HeLa was dose-dependently increased, and the effective dose of chlorine dioxide preparation resulting in the death of half of HeLa cells was 405 ppm as calculated by the SPSS software Probit module.

Table 11: In vitro killing of HeLa cells by chlorine dioxide preparation

Example 7: Using a lactate dehydrogenase release assay to detect chlorine dioxide preparations against a variety of human tumor cells, including breast, ovarian, liver, nasopharyngeal, gastric, laryngeal, pancreatic, melanoma, bladder cancer And the killing effect of leukemia cells.

A chlorine dioxide preparation was prepared in accordance with the method of Example 5. Breast cancer cells (MCF-7), ovarian cancer cells (SKOV3), liver cancer cells (Bel-7402), nasopharyngeal carcinoma cells (HNE), gastric cancer cells (MKN-45) were preserved as in Example 5. Laryngeal carcinoma cells (Hep-2), pancreatic cancer cells (Pan-1), melanoma cells (A375), bladder cancer cells (Biu-87), and leukemia cells (Jurkat) are also treated with 10% fetal bovine serum PRMI1640. The cells were cultured in a 37 ° C, 5% CO 2 incubator and passaged in a conventional suspension cell culture mode, that is, centrifugation was performed to remove the old culture solution, and then a new culture solution was added. Then, the experimental group, the control group, and the zeroing group were set according to the method of Example 5, and the tumor cell death rate was measured by a lactate dehydrogenase (LDH) release test, and the dose (IC ₅₀ ) which caused half of the tumor cell death was calculated.

As a result, as shown in Table 12, the chlorine dioxide preparation was effective in killing various tumor cells, and as the concentration of chlorine dioxide increased, their killing effect on each tumor also increased in a dose-dependent manner.

It is indicated that the acidic chlorine dioxide preparation provided by the present invention has a broad spectrum of killing effect on tumor cells.

Table 12 Mortality of various human tumor cells at different concentrations of chlorine dioxide (%)

Example 8 Test for Inducing Apoptosis of Tumor Cells by Chlorine Dioxide Formulation

The death and cell cycle changes of the colon cancer cell line LS174T and the breast cancer cell line Cama-1 were analyzed. The cells were cultured for 48 hours in the absence of chlorine dioxide or in the presence of 100 ppm chlorine dioxide (prepared by the method of Example 5). After 30 minutes of pulsed with 1 μg/ml bromodeoxyuridine (BrdU), the cells were fixed overnight in 70% ethanol at 4 ° C, and then stained with FITC-conjugated anti-BrdU monoclonal antibody and 3 μg/ml propidium iodide. Cell death and cell cycle were analyzed by flow cytometry (FACS). Incorporation of BrdU is a method of proliferation measurement, while propidium iodide staining determines the DNA content, particularly the population of near diploid cells that are undergoing apoptosis.

The data showed that the percentage of BrdU-incorporated LS174T cells was changed from 27% before treatment to 6% after 48 hours of culture in the presence of 100 ppm chlorine dioxide preparation. In contrast, the percentage of LS174T cells with near diploid DNA content was changed from 4% before treatment to 23% after 48 hours of culture in the presence of 100 ppm chlorine dioxide preparation, indicating the ability of apoptosis promotion of 100 ppm chlorine dioxide preparation. Strong.

The data also showed that the percentage of Cmad-1 cells spiked with BrdU was changed from 15% before treatment to 2% after 48 hours of incubation in the presence of 100 ppm chlorine dioxide preparation. In contrast, the percentage of Cama-1 cells with near diploid DNA content was changed from 4% before treatment to 17% after 48 hours of culture in the presence of 100 ppm chlorine dioxide preparation, indicating that 100 ppm chlorine dioxide preparation triggered apoptosis. .

These data indicate that the LS 174T and Cama-1 cell lines stopped dividing and were subjected to apoptosis after treatment with a 100 ppm chlorine dioxide formulation for 48 hours.

Example 9 Effect of Chlorine Dioxide Formulation on Apoptosis of Tumor Cells at Different pH Values

To further investigate the effects of chlorine dioxide preparation on breast tumor cell lines, in Cama-1 cells, Cell death was analyzed by annexin V staining. Cells were cultured for 24 hours with or without the presence of a 400 ppm chlorine dioxide formulation (prepared by the method of Example 5). Apoptosis was detected by annexin V staining and flow cytometry. The data showed that more than 70% of Cama-1 cells were stained with annexin V, further demonstrating that 400 ppm chlorine dioxide preparation induced apoptosis. Other conditions remain the same, but the 400 ppm chlorine dioxide preparation is passed through a pH adjuster to increase the pH from 3.5 to 5.5, and only over 50% of Cama-1 cells are stained with annexin V, indicating that the acidic environment contributes Increase the rate of cancer cell apoptosis.

We also attempted to determine the kinetics of chlorine dioxide-induced apoptosis. Cama-1 cells were cultured in the presence or absence of a 500 ppm chlorine dioxide preparation (prepared by the method of Example 5) at a pH of 3.5. After 30 hours, the percentage of apoptotic cells (Annexin positive cells) in the culture was examined. The data showed that after 30 hours, untreated cells showed 15% spontaneous apoptosis. However, 80% of cells treated with the chlorine dioxide formulation showed cell death. Specifically, in Cama-1 cells, chlorine dioxide-induced apoptosis was started 9 hours after the addition of 500 ppm of chlorine dioxide preparation, and after 30 hours of treatment, the apoptotic cells reached 80%. Other conditions were the same, but the 500 ppm chlorine dioxide preparation was adjusted to a pH of 3.0 by a pH adjuster, and only over 45% of the cells showed death, indicating that the acidic environment contributes to an increase in cancer cell apoptosis.

We then attempted to determine the effect of chlorine dioxide preparations on human primary breast tumor cells. The newly recovered tumor single cell suspension was incubated with PBS or chlorine dioxide (100 ppm) formulation for 48 hours. Apoptosis was determined by PI staining. The percentage indicates the proportion of cells (subG0/G1 cells) containing a low amount of DNA, that is, the proportion of apoptotic cells. The data shows that 19.5% of cells treated with PBS have low levels of DNA, while 38.6% of cells treated with chlorine dioxide preparations have low levels of DNA. Thus, a similar 100 ppm chlorine dioxide formulation was observed to have an apoptotic effect on human breast primary tumor cells. Other conditions were unchanged, but the 100 ppm chlorine dioxide formulation was passed through a pH adjuster to increase the pH from 4.5 to 6, and only 25% of the cells treated with the chlorine dioxide formulation had low levels of DNA. This indicates that the acidic environment helps to increase the apoptosis rate of cancer cells.

Combined with the above tumor in vitro killing test, those skilled in the art can judge that the acidic chlorine dioxide preparation has obvious killing effect on tumor cells, and the mechanism of action is that the acidic chlorine dioxide preparation can significantly induce apoptosis of tumor cells. .

Example 10 Effect of Chlorine Dioxide Directly on Tumor Suppressive Effect and Immune Organ Index in S180 Tumor-bearing Mice

The first solution was prepared by dissolving a mixed solution of 7.47% sodium chlorite and 1.59% sodium chloride in deionized water; a citric acid solution having a concentration of 16.7% was prepared with deionized water to prepare a second solution. . The same volume of solution was taken from the containers of different solutions, mixed, and the solution was allowed to stand still for 5 to 10 minutes, then an equal volume of 90% dimethyl sulfoxide was added, and then filtered through a 0.22 μm double-layer filter. 100 ppm, 500 ppm, and 2000 ppm chlorine dioxide formulations of pH = 5 and pH = 3.5 were prepared separately.

Kunming mice were randomly divided into 8 groups, 15 in each group, male and female, respectively: blank control group, cyclophosphamide group, Low, medium and high doses of chlorine dioxide (pH is constant at 5), low dose chlorine dioxide (pH = 3.5), medium dose (pH = 3.5), high dose (pH = 3.5). The dose of Cyclophosphamide ip was 20 mg/kg, and the low, medium and high doses of chloramphenicol injection were 100 ppm, 500 ppm and 2000 ppm, respectively. The blank control group was given normal saline. Each group of mice was inoculated with S180 solid tumors for 24 hours, and the volume was 0.1 ml/10 g once daily for 14 days. The mice were sacrificed on the 5th day of withdrawal, the tumor mass and the spleen and thymus were removed and weighed, and the tumor inhibition rate and spleen and thymus index were calculated.

As can be seen from Table 1, compared with the blank control group, the chlorine dioxide preparation (high pH) low, medium and high dose groups, cyclophosphamide chemotherapy group, and chlorine dioxide preparation (low pH) are low, medium High-dose group significantly inhibited the growth of S180 tumors (P<0.05, P<0.01). Compared with the high pH group, the low pH group of chlorine dioxide preparation can significantly increase the inhibitory effect of chlorine dioxide preparation on S180 tumor growth (P<0.05).

Table 13 Anti-tumor effect of chlorine dioxide preparation on S180 tumor-bearing mice

Note: *P<0.05, **P<0.01 vs. blank control group; #P<0.05, ##P<0.01 vs. high pH group

As can be seen from Table 14, compared with the blank control group, the thymus and spleen index of the mice in the cyclophosphamide chemotherapy group were significantly decreased (P<0.01), indicating that the side effects of chemotherapy were great. The thymus and spleen index of the mice in the high-dose group of chlorine dioxide were significantly increased (P<0.05). For high and low pH values, the effect of chlorine dioxide is not significant. It shows that the side effects of immune dioxide on the process of apoptosis induced by chlorine dioxide are minimal.

Table 14 Effect of chlorine dioxide preparation on immune organ index

Note: *P<0.05, **P<0.01 compared with the blank control group.

It is to be noted that the acidic chlorine dioxide preparation provided by the present invention has obvious apoptosis-inducing effect on tumor cells, and has almost no side effects on the body.

Claims (8)

1. An apoptosis inducing agent comprising chlorine dioxide.
2. The apoptosis inducing agent according to claim 1, which comprises chlorine dioxide dissolved in water, wherein the concentration of chlorine dioxide is from 500 to 2900 ppm, based on the mass.
3. The apoptosis inducing agent according to claim 2, which further comprises an acidic pH adjusting agent such that the pH of the apoptosis inducing agent is 1.5 to 6.5, and the acidic pH adjusting agent is selected from at least one of the following groups:

   An organic acid or a salt thereof selected from the group consisting of formic acid, acetic acid, propionic acid, butyric acid, lactic acid, pyruvic acid, citric acid, malic acid, tartaric acid, gluconic acid, glycolic acid, fumaric acid, malonic acid, maleic acid a collection of oxalic acid, succinic acid, acrylic acid, crotonic acid, glutaric acid, and salts thereof;

   An inorganic acid or a salt thereof selected from the group consisting of hydrochloric acid, phosphoric acid, boric acid, metaphosphoric acid, pyrophosphoric acid, sulfamic acid, dihydrogen phosphate, and hydrogen phosphate.
4. The apoptosis inducing agent according to claim 3, wherein the acidic pH adjusting agent is selected from the group consisting of citric acid, acetic acid or dihydrogen phosphate.
5. An apoptosis inducing agent kit comprising chlorine dioxide comprising the following two independent components:

   a first component: a chlorine dioxide precursor solid, or a solution comprising a chlorine dioxide precursor;

   a second component: an aqueous solution of an acidic pH adjuster;

   The two are stored separately and can be mixed before use to react in situ and prepare an apoptosis inducing agent comprising chlorine dioxide; and the amount and concentration of the first component and the second component can enable the mixed solution pH=1.5-6.5;

   Wherein the chlorine dioxide precursor is selected from at least one of sodium chlorite, potassium chlorite, lithium chlorite, calcium chlorite, magnesium chlorite or bismuth chlorite;

   Wherein the acidic pH adjusting agent is selected from at least one of the following groups:

   An organic acid or a salt thereof selected from the group consisting of formic acid, acetic acid, propionic acid, butyric acid, lactic acid, pyruvic acid, citric acid, malic acid, tartaric acid, gluconic acid, glycolic acid, fumaric acid, malonic acid, maleic acid a collection of oxalic acid, succinic acid, acrylic acid, crotonic acid, glutaric acid, and salts thereof;

   An inorganic acid or a salt thereof selected from the group consisting of hydrochloric acid, phosphoric acid, boric acid, metaphosphoric acid, pyrophosphoric acid, sulfamic acid, A collection of dihydrogen phosphate and hydrogen phosphate.
6. Use of the apoptosis inducing agent according to any one of claims 1 to 4 or the chlorine dioxide-containing apoptosis inducing agent kit according to claim 5 for the preparation of a drug for inducing apoptosis.
7. Use of the chlorine dioxide-containing apoptosis inducing agent according to any one of claims 1 to 4 or the chlorine dioxide-containing apoptosis inducing agent kit of claim 5 for the preparation of a medicament for treating a tumor, or for The use of a mammalian target tissue anti-aging drug, or as a cosmetic, or for the preparation of a chemotherapeutic drug.
8. The use according to claim 7, wherein the tumor comprises: intracranial metastases, meningiomas, skull tumors, brain cancer, pituitary adenomas, acoustic schwannomas, gliomas, brain tumors; maxillary sinus cancer, laryngeal cancer, Nasopharyngeal cancer, tongue cancer, thyroid cancer, gingival cancer, lip cancer; thymoma, lung cancer, adenocarcinoma, breast sarcoma, lung metastasis, breast fibroma, breast cancer; pancreatic head cancer, stomach cancer, gallbladder cancer, rectal cancer, Pancreatic cancer, esophageal cancer, colon cancer, liver cancer; renal pelvic tumor, penile cancer, urothelial cancer, prostate cancer, urethral cancer, testicular cancer, bladder cancer, nephroblastoma, renal cancer; ovarian cancer, fallopian tube tumor, vulvar cancer , vaginal tumor, uterine cancer, cervical cancer, choriocarcinoma, pelvic cancer; skin cancer, liposarcoma, malignant teratoma, fibroids, neurofibromatosis, melanoma, cholangiocarcinoma, squamous cell carcinoma, basal cell carcinoma; chordoma , osteoma, chondroma, osteosarcoma, synovial sarcoma, giant cell tumor of bone, osteofibrosarcoma; acute leukemia, malignant lymphoma, chronic leukemia; hepatic hemangioma, islet cell carcinoid, blastoma, mucus Tumor, neck metastasis, cardia cancer; independently,

   The anti-aging drug includes a drug for preventing memory loss, insomnia, Alzheimer's disease, Parkinson's syndrome, osteoporosis, diabetes or cardiovascular and cerebrovascular diseases: independently,

   The cosmetic comprises a cosmetic which whitens the skin, protects the skin, reduces or eliminates wrinkles, increases elasticity, acne, ecchymoses, scars or rejuvenates the skin.