CN113248514B

CN113248514B - Dimethyldeuterated carnosol, preparation method thereof and application thereof in preparation of drugs for treating cachexia

Info

Publication number: CN113248514B
Application number: CN202010088747.2A
Authority: CN
Inventors: 章雄文; 李奕伟; 潘俊芳; 刘璇
Original assignee: East China Normal University
Current assignee: East China Normal University
Priority date: 2020-02-12
Filing date: 2020-02-12
Publication date: 2023-03-31
Anticipated expiration: 2040-02-12
Also published as: CN113248514A

Abstract

The invention innovatively provides the application of carnosol compounds in preparing the medicine for treating cachexia for the first time, wherein the carnosol compounds comprise carnosol compounds shown as a formula X, dimethyl deuterated carnosol shown as a formula 1, dimethyl carnosol shown as a formula 2 and the like. The invention also provides a new compound, namely the dimethyl deuterated carnosol shown in the formula 1 and a preparation method thereof. The invention also provides a pharmaceutical composition for treating cachexia. The invention also provides application of the compound in preparing a medicament for treating tumor cachexia, wherein the compound comprises muscular atrophy caused by tumor tissues, fat reduction caused by the tumor tissues, appetite reduction caused by the tumor tissues, inflammatory reaction caused by the tumor tissues and the tumor cachexia caused by cancers related to digestive tracts, liver cancer, lung cancer and colon cancer. The invention also provides application of the compound in preparing a medicament or an inhibitor for treating or inhibiting muscle atrophy, a medicament or an inhibitor for inhibiting or relieving lipolysis of fat cells and a medicament or an inhibitor for inhibiting or relieving weight loss or reduction. The invention has wide application prospect.

Description

Dimethyldeuterated carnosol, preparation method thereof and application thereof in preparation of drugs for treating cachexia

Technical Field

The invention relates to the field of drug synthesis, in particular to a dimethyldeuterated carnosol shown as a formula (1) and a preparation method and application thereof.

Background

Cachexia is a progressive wasting syndrome, accompanied by general failure such as emaciation, anemia, and mental fatigue, and is often caused by cancer and other serious chronic diseases (such as chronic pulmonary obstruction, chronic heart failure, and aids), and cachexia caused by tumors is called tumor cachexia. Tumor cachexia (Cancer cachexia) is a major complication in patients with various malignancies and is the leading cause of death in many patients with tumors. Prevention and treatment of tumor cachexia has become an important component of multidisciplinary comprehensive treatment of malignant tumors, and is receiving more and more attention. Tumor cachexia is a wasting syndrome characterized by general metabolic disturbance, progressive muscle and fat consumption, weight loss, and progressive failure of the organs of the body, which is caused by the combination of tumor cell products and cytokines released by the body, and is mainly characterized by the marked decrease in the body weight of a patient caused by skeletal muscle atrophy. In patients with advanced tumors, the incidence of tumor cachexia is as high as 50% -80%, wherein the incidence of patients with breast cancer and leukemia is 40%, the incidence of patients with lung cancer, colon cancer and prostate cancer is 50%, and the incidence of patients with gastric cancer and pancreatic cancer is as high as 80%, about 20% of patients with tumors directly die of heart and lung failure caused by cachexia, so that the tumor cachexia is the main cause of death of a plurality of tumor patients. The occurrence of tumor cachexia not only weakens the effects of chemotherapy and radiotherapy and shortens the life cycle, but also seriously affects the life quality of patients.

In 2011, the Lancet Oncol journal published a joint consensus of 8 experts on cancer cachexia diagnosis and staging standards, and the consensus determined the screening and diagnosis standards of tumor cachexia: body weight loss of 5% or body mass index BMI <20kg/m2 or body weight loss of 2% in those who have experienced a reduction in skeletal muscle mass. The method is subdivided into the following three stages, namely, the tumor cachexia prophase: the weight loss is less than 5%, and anorexia and metabolic change are accompanied; cachexia stage: means a weight loss of >5% in 6 months or a weight loss of >2% in BMI <20kg/m2, or a weight loss of >2% in limb skeletal muscle index consistent with sarcopenia (< 7.26kg/m2 in men and <5.45kg/m2 in women); refractory cachexia stage: advanced cancer patients show active catabolism, no response to anticancer therapy, low WHO physical status score (3 or 4 points), and less than 3 months of survival. The pathogenesis of tumor cachexia: clinically, the malignant tumor is mostly manifested by weight loss, inappetence, anemia and weakness of patients and is a systemic multi-organ metabolic disorder syndrome, and skeletal muscle and adipose tissue loss caused by the malignant tumor is a main reason of weight loss of the patients. Currently, tumor cachexia is becoming the main cause of death of clinical tumor patients, and the importance of preventing and treating tumor cachexia in tumor treatment is increasingly prominent. The mechanisms underlying tumor cachexia are primarily related to systemic inflammation, skeletal muscle consumption and atrophy, fat consumption and degradation, and disorders of energy metabolism. Systemic inflammation: during the process of tumorigenesis and development, immune cells are induced to produce various inflammatory factors, such as TNF-alpha, IL-6, IL-1, LMF, ZAG, PIF and the like, and in addition, ectopic dysbacteriosis of intestinal tracts and release of lipopolysaccharides and bacterial toxins can activate immune cells to release the inflammatory factors. The inflammatory factors activate corresponding signal pathways to regulate synthesis and decomposition of skeletal muscle and adipose tissue, for example, the Isulin/IGF1-PI3K-Akt pathway in the skeletal muscle can regulate muscle protein synthesis, and the NF-kB pathway is related to muscle protein degradation; the NF-kB pathway in adipose tissue influences HSL phosphorylation and ATGL protein activity, and regulates the process of fat degradation.

Skeletal muscle supports a great deal of body weight, whether skeletal muscle is healthy or not depends on whether skeletal muscle protein synthesis and degradation are in dynamic balance, and active skeletal muscle protein degradation is considered to be an important factor of cachexia such as weight loss and fatigue weakness. Studies have shown that under the combined action of the body's immune system with tumor cells themselves and cytokines and inflammatory factors produced by systemic inflammation, skeletal muscle protein depletion is produced and exacerbated, manifested by decreased skeletal muscle protein synthesis, increased levels of degradation, increased muscle cell apoptosis, and decreased regeneration. Inflammatory factors TNF alpha, IL-1 and the like can activate FOXO transcription factors through an NF-kB signal channel, up-regulate the expression level of E3 ubiquitin ligase MuRF-1, promote the ubiquitination degradation of myogenic protein, also can activate a muscle cell p38/MAPK signal channel, induce caspase activation and promote the apoptosis of skeletal muscle cells; the binding of the cytokines Myostatin and Activin to the receptor ActRIIA/B activates Smad2, which reduces muscle protein synthesis by inhibiting the AKT/mTOR signaling pathway, activates FOXO transcription factors to increase muscle protein degradation, and also activates the P38/MAPK signaling pathway leading to muscle cell apoptosis.

Tumor cachexia is a serious systemic wasting syndrome, and fat is the most important energy storage tissue of the body, so in the process of tumor cachexia development, fat tissue is often consumed before skeletal muscle, wherein the increase of lipolysis due to the reduction of white fat tissue synthesis, the increase of white fat browning, and the reduction of brown fat into white fat are the main causes of fat tissue consumption and degradation. Lipoprotein esterase (LPL) also induces hydrolysis of endogenous and exogenous triglycerides, and its activity decreases slowing down the uptake of fatty acids, affecting the fat synthesis process; hormone sensitive esterase (HSL) and triglyceride esterase (ATGL) mainly regulate the degradation of white adipose tissue, and the expression of both promotes the hydrolysis of triglyceride under the induction of inflammatory factors; in addition, inflammatory factors such as TNF alpha and IL-1 promote the browning of white adipose tissues by increasing the expression of UCP1 in mitochondria, and aggravate the consumption of the adipose tissues. Disorders of energy metabolism: because tumor growth requires competition for a large amount of nutrients with the host, it shows an imbalance in energy intake and consumption for the host organism, increases in energy consumption of the organism and cannot be reversed by a simple nutritional support means, and shows partial sensitivity or even insensitivity to nutritional support. The tumor tissue consumes a large amount of glucose and glutamine and produces a large amount of lactic acid, the lactic acid is regenerated into glucose through gluconeogenesis consumed energy, and the energy is utilized inefficiently due to the large increase of ineffective lactic acid circulation; abnormal fat metabolism, increased catabolism but low effective availability; the breakdown of muscle key proteins also worsens the supply-demand balance of skeletal muscle proteins.

The pathogenesis of tumor cachexia is very complex, an effective treatment means is lacked, and only megestrol is approved by the U.S. drug administration to improve the appetite of patients with tumor cachexia at present. The palliative treatment of stimulating appetite in combination with enhancing the nutritional intake of patients has a certain symptom-improving effect clinically on patients with tumor cachexia, but it is still difficult to achieve a very effective treatment. A variety of antibodies to inflammatory cytokines have also been tried in clinical trials, including antibodies specific for IL-1 α, IL-6, TNF- α, myostatin, etc., but it was found that inhibition of one of the factors did not completely prevent cachexia development. Infliximab (monoclonal antibody of TNF alpha) and Clazakizumab (monoclonal antibody of IL-6) are respectively applied to clinical treatment research of pancreatic tumor cachexia and non-small cell lung cancer tumor cachexia, and the weight loss, skeletal muscle atrophy and life quality of a patient are not obviously improved. In addition, ghrelin receptor agonists, selective Androgen Receptor (AR) agonists, adrenergic beta blockers, and anti-myostatin polypeptides, among others, are of interest. The new drugs that were originally most promising in the clinical research phase in the near future are Anamorelin (Anamorelin, a ghrelin receptor agonist) and Enobosarm (a selective AR agonist), but the phase III clinical studies of these 2 drugs have all ended up with failure.

In conclusion, because the tumor cachexia is a main complication of various malignant tumor patients, the effects of chemotherapy and radiotherapy are weakened, the life cycle is shortened, and the life quality of the patients is also seriously affected. Although the importance and necessity of the treatment of the tumor cachexia are well known, the pathogenesis of the tumor cachexia is complex, the current research is only in Bingshan mountain corner, the treatment method of the tumor cachexia and the research and development of new drugs have no breakthrough progress, the clinical treatment effect is limited, and no drug is approved to be used as a specific drug for the tumor cachexia on the market, so the research on the tumor cachexia and the research and development of new drugs are needed to be further carried out.

Disclosure of Invention

Aiming at the defects in the prior treatment technical means, the invention provides the effect of the traditional Chinese medicine monomer components and the derivatives thereof in the relevant field of anti-tumor cachexia, relates to the dimethyl deuterated carnosol compound, the preparation method and the new medical application thereof, and provides the new application of the carnosol compound in anti-tumor cachexia. The invention provides application of carnosol compounds in resisting cachexia including tumor cachexia, in particular application in treating muscle atrophy and fat degradation caused by cachexia. In particular embodiments, the invention provides the use of carnosol compounds in cell cachexia models and animal cachexia models.

The invention provides application of carnosol compounds in formula (X) in preparing medicines for treating cachexia. Wherein, the structure of formula (X) is as follows:

in the formula (X), R ₁ ＝H、CH ₃ Methyl deuteration; r ₂ ＝H、CH ₃ And methyl deuteration.

Preferably, in the formula (X), R ₁ ＝H、CH ₃ 、R ₁ ＝H、CH ₃ The structural formula is shown as follows:

wherein carnosol (R in structural formula) ₁ ＝R ₂ = H) monomeric compounds have been commercially available, derived from plant extraction, isolation and purification of monomeric compounds, or semi-synthetic or fully synthetic monomeric compounds.

Wherein the carnosol is subjected to hydroxyl methylation to obtain dimethyl carnosol (R in the structural formula) ₁ ＝R ₂ ＝CH ₃ ) Namely, the bismethylcarnosol (compound 2) represented by formula 2 is as follows:

the invention also provides a structural formula of the dimethyl deuterated carnosol (compound 1) with a brand-new structure, which is as follows:

the invention also provides application of the dimethyl deuterated carnosol (compound 1) with a brand-new structure in preparation of a medicine for treating cachexia, and the dimethyl deuterated carnosol has a good anti-cachexia effect.

The invention also provides a preparation method of the dimethyl deuterated carnosol (compound 1) shown in the formula (1), which is characterized in that carnosol and deuterated iodomethane are used as raw materials, potassium carbonate is used as a catalyst, acetone is used as a solvent, and the dimethyl deuterated carnosol (compound 1) shown in the formula (1) is prepared through one-step reaction. The reaction process and the reaction formula of the preparation method are as follows:

the dimethy deuterated carnosol (compound 1) in the formula (1) can be used for preparing anti-cachexia medicines. Wherein, the cachexia includes but is not limited to progressive wasting syndrome induced by tumor, chronic kidney disease, chronic lung obstruction, chronic heart failure, AIDS, etc. The medicament is a medicament for humans and/or animals.

The invention also provides a pharmaceutical composition for treating cachexia, which contains the dimethydeuterohemin shown in the formula (1) and/or other pharmaceutically acceptable components. The product dosage form of the pharmaceutical composition includes, but is not limited to, capsules, tablets, oral preparations, microcapsule preparations, injections, ointments, sprays, suppositories and the like. The product administration mode of the pharmaceutical composition includes but is not limited to injection, oral administration, parenteral administration, inhalation spray or transdermal administration and the like. The dimethy deuterated carnosol shown in the formula (1) accounts for 0.001-100 wt% of the total dry weight of the composition.

In the application of the invention, the cachexia disease comprises tumor cachexia; the tumor is a solid tumor. The tumor cachexia includes but is not limited to muscle atrophy caused by tumor tissue, fat reduction caused by tumor tissue, appetite reduction caused by tumor tissue, inflammatory reaction caused by tumor tissue, tumor cachexia caused by digestive tract related cancer, liver cancer, lung cancer, colon cancer and the like.

The invention also provides a method for treating cachexia, wherein the dosage of the dimethy deuterated carnosol shown in the formula (1) is 0.01 mu g-100 mg/kg of body weight/day.

The invention also provides a preparation method of the bismethyl carnosol (compound 2) shown in the formula (2), which is characterized in that carnosol and methyl iodide are used as raw materials, potassium carbonate is used as a catalyst, acetone is used as a solvent, and the bismethyl carnosol (compound 2) shown in the formula (2) is prepared through one-step reaction and is shown in the following reaction formula;

the invention also provides application of the bismethyl carnosol shown in the formula (2) in preparing a medicament for treating cachexia. The cachexia includes, but is not limited to, progressive wasting syndrome induced by tumor, chronic kidney disease, chronic lung obstruction, chronic heart failure, aids, and the like. The medicament is a medicament for humans and/or animals.

The invention also provides a pharmaceutical composition for treating cachexia, which contains the bismethyl carnosol shown in the formula (2) and/or other pharmaceutically acceptable components. The product dosage form of the pharmaceutical composition includes, but is not limited to, capsules, tablets, oral preparations, microcapsules, injections, ointments, sprays, suppositories, and the like. The product administration mode of the pharmaceutical composition includes but is not limited to injection, oral administration, parenteral administration, inhalation spray or transdermal administration and the like. The content of the bismethylcarnosol shown in the formula (2) is 0.001-100 wt% of the total dry weight of the composition.

In the present invention, the cachexia disease includes tumor cachexia; the tumor is a solid tumor. The tumor cachexia comprises muscle atrophy caused by tumor tissues, fat reduction caused by the tumor tissues, appetite reduction caused by the tumor tissues, inflammatory reaction caused by the tumor tissues and tumor cachexia caused by digestive tract related cancers, liver cancer, lung cancer and colon cancer.

The invention also provides a method for treating cachexia, wherein the dosage of the bismethyl carnosol in the formula (2) is 0.01 mu g-100 mg/kg body weight/day.

The terms "carnosol compound", "dimethy-deuterated carnosol", "dimethy-carnosol" as used in the present invention refer to a single ingredient or to a combination with other pharmaceutically acceptable ingredients.

The term "other pharmaceutically acceptable ingredients" as used in the present invention refers to drugs which do not antagonize "carnosol", "dimethy-deuterol (compound 1)" or "dimethy-carnosol (compound 2)" and may be any one or more pharmaceutically acceptable excipients.

The term "cachexia" as used in the present invention refers to cachexia induced by a solid tumor.

The term "solid tumor" as used in the present invention refers to a solid tumor that is primary or secondary, such as digestive tract-related cancer, liver cancer and lung cancer.

The muscle atrophy of the invention is the muscle atrophy induced by tumor cachexia.

The fat degradation of the invention is the fat degradation caused by tumor cachexia.

The administration modes of the carnosol compound shown in the formula (X), the dimethyl deuterated carnosol (compound 1) and the dimethyl carnosol (compound 2) or the composition formed by the compound and other pharmaceutically acceptable components are injection, oral administration, parenteral administration, inhalation type spray or transdermal administration.

According to the invention, the application of the carnosol compound shown in the formula (X), the dimethy deuterated carnosol (compound 1) shown in the formula (1) and/or the dimethy carnosol (compound 2) in the preparation of the medicine for treating cachexia is disclosed, and the medicine can be used for human and/or animals.

Wherein the amount of the carnosol compound of formula (X), the dimethy deuterated carnosol (compound 1) of formula (1) and/or the dimethy carnosol (compound 2) is 0.01 mu g-100 mg/kg body weight/day.

Wherein the medicament comprises a medicament or inhibitor for treating or inhibiting muscle atrophy, a medicament or inhibitor for inhibiting or relieving lipolysis of fat cells, and a medicament or inhibitor for inhibiting or relieving weight loss or reduction.

The invention also provides a pharmaceutical composition for treating cachexia, which contains the carnosol compound shown in the formula (X) and/or other pharmaceutically acceptable components; whereinIn the formula (X), R ₁ ＝H、CH ₃ Methyl deuteration; r ₂ ＝H、CH ₃ And methyl deuteration. Wherein, the product dosage form of the composition comprises capsules, tablets, oral preparations, microcapsule preparations, injections, ointments, sprays, suppositories and the like. Wherein, the administration mode of the composition includes but is not limited to injection, oral administration, parenteral administration, inhalation spray or transdermal administration, etc. Wherein the carnosol compound shown in the formula (X) accounts for 0.001-100 wt% of the total dry weight of the composition.

In the application of the invention, the cachexia disease comprises tumor cachexia; the tumor is a solid tumor. Wherein, the tumor cachexia includes but is not limited to muscle atrophy caused by tumor tissue, fat reduction caused by tumor tissue, appetite reduction caused by tumor tissue, inflammatory reaction caused by tumor tissue, and tumor cachexia caused by digestive tract related cancer, liver cancer, lung cancer, colon cancer, etc.

The invention also provides a method for treating cachexia, wherein the dosage of the carnosol compound shown in the formula (X), the dimethyl deuterated carnosol (compound 1) shown in the formula (1) and/or the dimethyl carnosol (compound 2) shown in the formula (2) is 0.01 mu g-100 mg/kg of body weight/day. Such administration includes, but is not limited to, injection, oral, parenteral, inhalation spray, transdermal, and the like.

The invention also provides a pharmaceutical composition containing therapeutically effective amount of the carnosol compound shown in the formula (X), the dimethy deuterated carnosol (compound 1) in the formula (1) and/or the dimethy carnosol (compound 2) in the formula (2) and medicinal salt thereof.

The invention also provides that the composition product further comprises a protein source, a fat source and/or a carbohydrate source, and may be in a form selected from nutritionally balanced food, a nutritionally complete formula, a dairy product, a frozen ambient stable beverage, a soup, a nutritional bar, a dessert, a pet food, a pharmaceutical composition, combinations thereof, and the like. The product contains carnosol compound shown in formula (X), dimethy deuterated carnosol (compound 1) shown in formula (1) and/or dimethy carnosol (compound 2) shown in formula (2).

The invention also provides the carnosol compound shown in the formula (X), the dimethy deuterated carnosol (compound 1) shown in the formula (1) and/or the dimethy carnosol (compound 2) shown in the formula (2) which comprise medicines, foods, nutritional supplements and/or health products which are not limited to human and/or pets and are used for human and/or pets.

The invention also provides a carnosol compound shown in the formula (X), the dimethyl deuterated carnosol (compound 1) shown in the formula (1) and/or the dimethyl carnosol (compound 2) shown in the formula (2) as a single component or a composition formed by the compound 2 and other pharmaceutically acceptable components to prepare a product for treating cachexia. The other pharmaceutically acceptable ingredients contained in the composition can be medicines which have no antagonistic action with the compound, and can also be any one or more pharmaceutically allowable auxiliary materials.

The meanings of English and abbreviations in the present invention are as follows:

carnsol: carnosol

FBS: fetal bovine serum, HS: horse serum, DMEM medium: amino acid and glucose containing broth, PBS buffer: phosphate buffered saline, DMSO: dimethyl sulfoxide (DMSO).

The pharmacodynamic test method employed in the present invention is a method well known to those skilled in the art.

In the present invention, C2C12 cells, 3T3-L1 cells, C26 cells and Balb/C mice are commercially available to those skilled in the art.

Carnosol is a plant polyphenol compound derived from various labiatae plants such as sage (Salvia officinalis L.) and rosemary (Rosmarinus officinalis L.) which are industrially used as raw materials, and has a molecular formula of C ₂₀ H ₂₆ O ₄ And the molecular weight is 330.42. Carnosol has a variety of pharmacological activities. Researches show that the carnosol has obvious anti-inflammatory and anti-oxidation effects, can inhibit the expression of COX1 and COX2, and reduce the levels of factors IL4 and IL 10; can reduce damage caused by ischemia reperfusion and inhibit the expression of NF-kB and ICAM-1; in addition, carnosol has no significant anti-tumor activity, and only reports that the carnosol hasCan inhibit the growth of WEHI-164 fibroid. Chinese patent document CN102014893A discloses the use of polyphenols such as carnosol extracted from rosemary in the preparation of products for improving cartilage diseases, especially for preventing and treating musculoskeletal diseases. However, no relevant patent reports exist about the effect of carnosol on treating tumor cachexia. According to the research, the salviol compounds can relieve muscle atrophy in a concentration-dependent manner, and the newly found mechanism of the invention mainly can be used for up-regulating the phosphorylation level of AKT (alkyl ketene dimer) and promoting the synthesis of muscle cell protein; the expression of E3 ubiquitination ligase MuRF-1 is reduced, and the degradation of muscle cell protein is relieved; up-regulates the expression level of myogenic determinant MyoD, and promotes the differentiation and growth of muscle cells. The carnosol compound can relieve fat degradation in a concentration-dependent manner, and a newly-discovered mechanism of the patent mainly has the effects of up-regulating the phosphorylation level of AKT (alkyl ketene dimer) and promoting the survival of fat cells; down-regulating the phosphorylation level of hormone sensitive triglyceride lipase HSL, reducing fat mobilization and reducing the decomposition of triglyceride; the phosphorylation activation of AMPK alpha of AMP-dependent protein kinase is reduced, on one hand, the phosphorylation level of HSL caused by the phosphorylation activation of AMPK alpha is reduced, lipolysis is relieved, on the other hand, excessive energy metabolism caused by the phosphorylation activation of AMPK alpha is relieved, and the lipid loss is relieved. In a word, the carnosol compound relieves the protein degradation in muscle cells, promotes the differentiation and growth of muscle cells, reduces the fat degradation, relieves the excessive energy consumption, can reverse the myotube cell atrophy and the fat cell degradation caused by tumors in vitro, can reverse the muscle tissue atrophy caused by tumors in vivo, relieves the fat tissue degradation, inhibits the inflammatory reaction and inhibits the weight reduction, has a remarkable anti-tumor cachexia effect, and is beneficial to the improvement of cachexia through a brand new molecular action mechanism.

Carnosol is a polyphenol antioxidant and is used for research on anti-inflammatory and antioxidant aspects. The research of the invention finds that the carnosol and the derivatives thereof can relieve the protein degradation in muscle cells, promote the differentiation and growth of muscle cells, reduce the fat degradation and relieve the energy excessive consumption through a brand-new molecular action mechanism, can reverse the myotube cell atrophy and the fat cell degradation caused by tumors in vitro, can reverse the muscle tissue atrophy caused by tumors in vivo, relieve the fat tissue degradation, inhibit the weight reduction, simultaneously can enhance the appetite, have a remarkable anti-tumor cachexia effect, and are beneficial to the improvement of cachexia (the applied application is special). Based on the important function of carnosol and derivatives thereof in relieving tumor cachexia muscle atrophy and fat degradation, the patent research on the aspect of treating tumor cachexia by synthesizing dimethyl deuterated carnosol through phenolic hydroxyl deuterated dimethyl is designed and synthesized. The research finds that the dimethy deuterated carnosol can relieve the muscular atrophy in a concentration gradient manner, and the molecular action mechanism of myotube cells mainly up-regulates the phosphorylation level of AKT (alkyl ketene dimer) to promote the survival of the myotube cells; the expression of E3 ubiquitination ligase Atrogin-1 is reduced, and the degradation of muscle cell protein is relieved; up-regulating the expression level of myogenic determinant MyoD, promoting hypertrophy and fusion of muscle fibers; inhibiting p65 phosphorylation activation and blocking NF-kB signal transduction, thereby inhibiting myotube cell inflammatory response. The deuterated dicarbamic carnosol can relieve fat degradation in a concentration gradient manner, and mainly has the function of up-regulating the phosphorylation level of AKT (alkyl ketene dimer) on the molecular action mechanism of fat cells to promote the survival of the fat cells; down-regulating the phosphorylation level of hormone sensitive triglyceride lipase HSL, reducing fat mobilization and reducing the decomposition of triglyceride; the phosphorylation activation of AMPK alpha of AMP-dependent protein kinase is reduced, on one hand, the phosphorylation level of HSL caused by the phosphorylation activation of AMPK-alpha is reduced, lipolysis is relieved, on the other hand, excessive energy metabolism caused by the phosphorylation activation of AMPK-alpha is relieved, and lipid loss is relieved; inhibiting p65 phosphorylation activation and blocking NF-kB signal transduction, thereby inhibiting adipocyte inflammatory reaction.

In a word, the dimethy deuterated carnosol relieves the protein degradation in muscle cells, promotes the differentiation and growth of muscle cells, reduces the fat degradation, relieves the energy excessive consumption, can reverse the myotube cell atrophy and the fat cell degradation caused by tumors in vitro, can reverse the muscle tissue atrophy caused by tumors in vivo, relieves the fat tissue degradation, inhibits the inflammatory reaction, inhibits the weight reduction, has a remarkable anti-tumor cachexia effect, and is beneficial to the improvement of cachexia through a brand new molecular action mechanism.

The compound provided by the invention can relieve the processes of tumor cachexia, skeletal muscle atrophy and fat degradation by influencing related signal pathways in different tissues, and animal experiments show that the compound can obviously relieve the weight and food intake reduction caused by tumor cachexia, and has a remarkable effect of resisting tumor cachexia. The experimental result shows that the compound has good anti-cachexia activity, can be applied to the treatment of tumor cachexia and related diseases thereof, and can be further used for preparing novel anti-cachexia medicaments.

The invention discloses a new application of carnosol compounds in treating tumor cachexia. The invention provides carnosol and derivatives thereof, and finds that the carnosol and the derivatives thereof can relieve protein degradation in muscle cells, promote muscle cell differentiation and growth, reduce fat degradation and relieve energy excessive consumption through a brand-new molecular action mechanism, can reverse myotube cell atrophy and fat cell degradation caused by tumors in vitro, can reverse muscle tissue atrophy caused by tumors in vivo, relieve fat tissue degradation, inhibit weight loss, enhance appetite, have a remarkable anti-tumor cachexia effect and are beneficial to improving cachexia.

The application and mechanism innovatively provided by the invention comprise that the phosphorylation level of AKT is mainly up-regulated, and the synthesis of myocyte protein is promoted; the expression of E3 ubiquitination ligase MuRF-1 and Atrogin-1 is reduced, and the degradation of muscle cell protein is relieved; up-regulates the expression level of myogenic determinant MyoD, and promotes the differentiation and growth of muscle cells. The carnosol compound is found to inhibit fat degradation caused by tumor cachexia for the first time, and the newly found mechanism of the invention mainly up-regulates the phosphorylation level of AKT and promotes the survival of fat cells; down-regulating the phosphorylation level of hormone sensitive triglyceride lipase HSL, reducing fat mobilization, and reducing triglyceride decomposition; the phosphorylation activation of AMPK alpha is reduced, so that the phosphorylation activation of AMPK alpha is reduced, the phosphorylation level of HSL (high-speed lipoprotein) caused by the phosphorylation activation of AMPK alpha is reduced, lipolysis is relieved, excessive energy metabolism caused by the phosphorylation activation of AMPK alpha is relieved, and lipid loss is relieved. The carnosol compound is found to be capable of effectively inhibiting the secretion of tumor inflammatory factors TNF-alpha for the first time, so that the carnosol and the derivatives thereof have the function of resisting tumor cachexia, can be applied to the treatment of the tumor cachexia and related diseases, and are ideal tumor cachexia treatment medicines.

The invention has the advantages that: the invention provides the novel application of the carnosol and the derivatives thereof for the first time, the carnosol compound is found to inhibit the muscular atrophy caused by tumor cachexia for the first time through in vitro and in vivo experiments, the carnosol compound shown in the formula (X), the dimethy-deuterol (compound 1) shown in the formula (1) and the dimethy-carnosol (compound 2) shown in the formula (2) are subjected to in vitro and in vivo experiments to find that the muscular atrophy and lipolysis caused by the tumor cachexia can be relieved, and the results show that the carnosol shown in the formula (X), the dimethy-deuterol (compound 1) and the dimethy-carnosol (compound 2) shown in the formula (2) have the function of resisting the tumor cachexia, can be applied to the treatment of the tumor cachexia and related diseases, and are ideal tumor cachexia treatment medicines.

Drawings

FIG. 1 shows the results of experiments on the effect of carnosol on the activity of C2C12 muscle cells in example 3.

FIG. 2 is the results of the experiment on the effect of bismethylcarnosol on the activity of C2C12 muscle cells in example 3.

FIG. 3 shows the results of experiments on the effect of dimethy deuterated carnosol on the activity of C2C12 muscle cells in example 3.

FIG. 4 is a graph of HE staining of the control group of Table 2 in example 4.

FIG. 5 is a graph of the HE staining pattern of the model group of Table 2 in example 4.

FIG. 6 is the HE staining pattern of experimental group 1 of Table 2 in example 4.

FIG. 7 is a HE staining pattern of experimental group 2 of Table 2 in example 4.

FIG. 8 is a HE staining pattern of experiment group 3 of Table 2 in example 4.

FIG. 9 is a HE staining pattern of experiment group 4 of Table 2 in example 4.

FIG. 10 is a graph of HE staining of the control group of Table 3 in example 4.

FIG. 11 is a graph of HE staining of the table 3 model group in example 4.

FIG. 12 is the HE staining pattern of experimental group 1 of Table 3 in example 4.

FIG. 13 is a HE staining pattern of experiment group 2 of Table 3 in example 4.

FIG. 14 is the HE staining pattern of experimental group 3 of Table 3 in example 4.

FIG. 15 is HE staining pattern of experiment group 4 in Table 3 in example 4

FIG. 16 is the HE staining pattern of Table 3, experimental group 5, in example 4

FIG. 17 is a HE staining pattern of experiment group 6 of Table 3 in example 4.

Figure 18 is a graph of the statistical results of carnosol alleviating C2C12 muscle cell atrophy in example 4.

Figure 19 is a graph of the statistical results of the C2C12 muscle cell atrophy alleviation by deuterated dimethylcarnosol and dimethylcarnosol of example 4.

FIG. 20 shows the results of Western blotting of carnosol in the C2C12 cell muscle atrophy model in example 4.

FIG. 21 shows the results of Western blotting of bismethylcarnosol in the C2C12 cell muscle atrophy model in example 4.

FIG. 22 shows the result of Western blotting of the dimethy deuterated carnosol in the muscle atrophy model of C2C12 cells in example 4.

FIG. 23 shows the results of experiments on the effect of carnosol on the activity of 3T3-L1 adipocytes in example 5.

FIG. 24 shows the results of experiments on the effect of carnosol on the activity of 3T3-L1 adipocytes in example 5.

FIG. 25 shows the experimental results of the effect of dimethyyldeuterol on the activity of 3T3-L1 adipocytes in example 5.

FIG. 26 is a graph of the oil red O staining of the control group of Table 6 in example 6.

FIG. 27 is a graph of oil red O staining for the C26 model group of Table 6 in example 6.

FIG. 28 is a graph of oil red O staining of experimental group 1 of Table 6 in example 6.

FIG. 29 is a graph showing the oil red O staining pattern of experimental group 2 of Table 6 in example 6.

FIG. 30 is a graph of oil red O staining of experimental group 3 of Table 6 in example 6.

FIG. 31 is a graph of oil red O staining of experimental group 4 of Table 6 in example 6.

FIG. 32 is a graph of the oil red O staining of the control group of Table 7 in example 6.

FIG. 33 is a graph of the oil red O staining of the C26 model group of Table 7 in example 6.

FIG. 34 is a graph showing the oil red O staining pattern of the experimental group 1 of Table 7 in example 6.

FIG. 35 is a graph showing the oil red O staining pattern of experimental group 2 of Table 7 in example 6.

FIG. 36 is a graph showing the oil red O staining pattern of experimental group 3 of Table 7 in example 6.

FIG. 37 is a graph showing the oil red O staining pattern of experimental group 4 of Table 7 in example 6.

FIG. 38 is a graph of oil red O staining of experimental group 5 of Table 7 in example 6.

FIG. 39 is a graph of oil red O staining of experimental group 6 of Table 7 in example 6.

FIG. 40 is a graph of the semi-quantitative results of the oil red O staining experiments in example 6 in which carnosol mitigates lipolysis of 3T3-L1 adipocytes.

FIG. 41 is a semi-quantitative graph of oil red O staining experiments in example 6 in which dimethydeuterium carnosol and dimethycarnosol alleviated lipolysis of 3T3-L1 adipocytes.

FIG. 42 is the results of the glycerol assay in example 6 in which carnosol relieves lipolysis in 3T3-L1 adipocytes.

FIG. 43 is the results of the glycerol assay experiments in example 6 in which dimethy-deuterocol and dimethy-carnosol alleviate lipolysis in 3T3-L1 adipocytes.

FIG. 44 is the results of the triglyceride assay experiment in example 6 in which carnosol ameliorates lipolysis of 3T3-L1 adipocytes.

FIG. 45 shows the results of experiments on triglyceride detection in example 6 in which dimethy-deuterocol and dimethy-carnosol alleviate lipolysis in 3T3-L1 adipocytes.

FIG. 46 shows the results of Western blotting of carnosol in the 3T3-L1 cell lipolysis model in example 6.

FIG. 47 is the result of Western blotting of bismethylcarnosol in the model of 3T3-L1 cell lipolysis in example 6.

FIG. 48 shows the results of Western blotting of dimethy deuterated carnosol in 3T3-L1 cell lipolysis model in example 6.

FIGS. 49 and 50 are graphs of tumor-bearing body weights of mice in example 7.

FIGS. 51 and 52 are graphs of the tumor-free weights of the mice in example 7

FIGS. 53 and 54 are graphs showing the cumulative food intake of the mice in example 7.

FIGS. 55 and 56 are graphs showing the change in the average daily food intake of the mice in example 7.

FIGS. 57 and 58 are graphs showing the body temperature changes of mice in example 7.

FIGS. 59 and 60 are graphs showing the change in tumor volume of the mice in example 7.

FIGS. 61 and 62 are the tumor anatomies of the mice in example 7.

FIG. 63 is a graph of calf muscle mass of the mice from the phenol test of Salvia miltiorrhiza in example 7.

FIG. 64 is a diagram showing the dissection of gastrocnemius muscle of the experimental mouse of carnosol in example 7.

FIG. 65 is a graph showing the staining of the sections of gastrocnemius muscle HE of mice in the sub-experimental healthy group of carnosol in example 7.

FIG. 66 is a graph showing HE staining of a section of gastrocnemius muscle of mice in the C26 tumor model group of the carnosol experiment in example 7.

FIG. 67 is a graph showing the HE staining of a section of gastrocnemius muscle of a mouse (10 mg/kg) in example 7 using carnosol test.

FIG. 68 is a statistical view of the cross-sectional area of gastrocnemius muscle of the mouse used in the phenolic test of sage in example 7.

FIG. 69 is the result of Western blotting of gastrocnemius muscle of mice tested for carnosol in example 7.

FIG. 70 is a graph showing calf muscle quality of the experimental mice with dimethy deuterated salvia miltiorrhiza phenolic time in example 7.

FIG. 71 is a diagram showing the gastrocnemius muscle of the experimental mice in example 7, which shows the effect of the dimethy deuterated salvia miltiorrhiza.

FIG. 72 is a graph showing staining of the sections of gastrocnemius muscle HE of mice in the healthy group in example 7 in the dimethy deuterated salvia phenol test.

FIG. 73 is the HE staining pattern of gastrocnemius section of mice in the C26 tumor model group of the dimethy deuterated salvia phenolic experiment in example 7.

FIG. 74 is the staining pattern of HE section of calf muscle of mice with dimethy deuterated carnosol (20 mg/kg) in example 7.

FIG. 75 is a statistical chart of the cross-sectional area of gastrocnemius muscle of the mouse tested by the dimethy deuterated salvia miltiorrhiza phenolic time in example 7.

FIG. 76 is a graph showing the epididymal fat mass of the mouse obtained by the secondary experiment with carnosol in example 7.

FIG. 77 is the fat profile of epididymal in the experimental mouse with carnosol in example 7.

FIG. 78 is a HE staining pattern of epididymal fat sections of mice in the experimental healthy group of carnosol in example 7.

FIG. 79 is the HE staining pattern of epididymal fat section of mice in the C26 tumor model group of the carnosol experiment in example 7.

FIG. 80 is a graph showing the HE staining of epididymal fat sections of mice in which carnosol was tested in a sub-experiment (10 mg/kg) in example 7.

FIG. 81 is a statistical chart of the cross-sectional area of epididymal fat of the mice tested for carnosol in example 7.

FIG. 82 shows the results of Western blotting of epididymal fat in mice tested for carnosol in example 7.

FIG. 83 is a graph showing the epididymal fat mass of the mouse in the example 7, which is a result of the experiments on the hypo-phenolic value of the dimethy deuterated sage.

FIG. 84 is the fat anatomical map of epididymis of experimental mice with dimethybird sage phenolic acid in example 7.

FIG. 85 is the HE staining pattern of fat section of epididymis of the rat in the experiment healthy group of dimethyl-deuterated carnosol in example 7.

FIG. 86 is the HE staining pattern of fat section of epididymis of mice in C26 tumor model group in example 7 of the bis-methyl deuterated carnosol test.

FIG. 87 is the staining pattern of fat section HE of epididymis fat section of mouse with dimethydeuterocol (20 mg/kg) obtained in example 7.

FIG. 88 is a statistical chart of the cross-sectional area of epididymis fat of the mouse tested by the bis-methyl deuterated carnosol in example 7.

FIG. 89 is a graph of the serum glycerol content of the carnosol test mice of example 7.

FIG. 90 is a graph of serum triglyceride levels of the carnosol test mice of example 7.

FIG. 91 is a graph of the serum TNF-. Alpha.content of the mice from the phenolic trials of sage in example 7.

FIG. 92 is a graph of the serum IL-6 level of the mice from the phenolic trials of sage in example 7.

Detailed Description

The present invention will be described in further detail with reference to the following specific examples and the accompanying drawings. The procedures, conditions, experimental methods and the like for carrying out the present invention are general knowledge and common general knowledge in the art except for the contents specifically mentioned below, and the present invention is not particularly limited.

The invention provides new application of carnosol, dimethyl carnosol and dimethyl deuterated carnosol in treating tumor cachexia and related diseases.

In the present invention, the C2C12 cells (mouse myoblasts), 3T3-L1 cells (mouse adipocytes) and C26 cells (mouse colon cancer cells) used were purchased from cell banks of the national academy of sciences type culture Collection. Bablb/c mice were purchased from Shanghai Ling Biotech, inc.

Carnosol, available from Shanghai Standard Technology co., ltd.

Methyl iodide was purchased from pharmaceutical industry, inc., of the national drug group.

Deuterated iodomethane was purchased from pharmaceutical industry, inc., of the national drug group.

FBS (fetal bovine serum), purchased from Biological Industries.

Horse serum (Horse serum), purchased from Gibco.

High-glucose DMEM medium, purchased from Hyclone.

RPMI-1640 medium, purchased from Hyclone.

Phenol red-free high-glucose DMEM medium purchased from Hyclone.

The P/S double antibody (penicillin-streptomycin mixture) was purchased from Hyclone.

Dexamethasone, purchased from Sigma-Aldrich.

IBMX (3-isobutyl-1-methylxanthine) broad-spectrum phosphodiesterase inhibitor available from Sigma-Aldrich.

Human recombinant insulin, purchased from Shanghai and Jinmai organisms.

Glycerol detection kit, purchased from Beijing prilley Gene technology, inc.

Example 1 Synthesis of Bimetyl carnosol into a 50mL reaction flask were added 300mg carnosol, 250mg potassium carbonate and 20mL anhydrous acetone, 1mL methyl iodide was added under nitrogen protection, reaction was carried out at room temperature in the dark for 48 hours, the reaction solution changed from colorless to brown, the solid was removed by suction filtration, the filtrate was concentrated to dryness and then separated by column chromatography to obtain a white solid 70mg, yield 21.5%. LC-MS (ESI) m/z C ₂₂ H ₃₁ O ₄ [M+H] ⁺ calcd for 359.2, found 359.4。

Example 2 Synthesis of Dimethyldeuterated carnosol

Adding 750mg carnosol, 600mg potassium carbonate and 50mL anhydrous acetone into a 100mL reaction bottle, adding 3mL deuterated iodomethane under the protection of nitrogen, reacting at room temperature in the dark for 48 hours, changing the reaction liquid from colorless to brown, filtering to remove solids, concentrating the filtrate to dryness, and performing column chromatography to obtain 135mg white solid with the yield of 16.3%. LC-MS (ESI) m/z C ₂₂ H ₂₅ D ₆ O ₄ [M+H]+ calcd for 364.3,found 365.5。

Example 3 Effect of carnosol, dimethy carnosol and dimethy deuterated carnosol on the Activity of mouse myoblasts (C2C 12)

The survival rate of the C2C12 cells is detected by adopting an MTT method. The detection principle is that succinate dehydrogenase in mitochondria of living cells can enable exogenous MTT to be reduced into water-insoluble blue-violet crystal Formazan (Formazan) and deposited in the cells, and dead cells do not have the function. The triple liquid (SDS +5% isobutanol +0.01% concentrated hydrochloric acid aqueous solution by 10%) can dissolve formazan in cells, and the light absorption value of the triple liquid is measured by an enzyme-linked immunosorbent assay detector at the wavelength of 570nm, so that the quantity of living cells can be indirectly reflected. Within a certain range of cell number, MTT crystals are formed in an amount proportional to the cell number.

The effect of the drug on the viability of mouse myoblasts (C2C 12) can be assessed by the above method. The specific method comprises the following steps:

inoculating C2C12 cells into 24-well plate, culturing in high-glucose DMEM medium containing 10% FBS, adding 1% diabody, and adding 5% CO ₂ And in an environment of 37 ℃. When the cell growth density reached 50-60%, the culture system was changed to high-glucose DMEM medium containing 2% hs, and 1% diabody was added, and the medium was changed every 48 hours, and differentiated and matured on

day

5 or 6. Carnosol or 6.25 μ M,12.5 μ M,25 μ M,50 μ M bismethylcarnosol or 6.25 μ M,12.5 μ M,25 μ M,50 μ M,100 μ M bismethyldeuterated carnosol was formulated in 2% HS differentiation solution at the following concentrations of 3.125 μ M,6.25 μ M,12.5 μ M,25 μ M,50 μ M,100 μ M and allowed to act on mature C2C12 myotubular cells for 48h. The culture solution was changed to MTT (5 mg/ml): 2% hs =1, 500 ul/well, 3h later 250ul of the triple solution was added per well and after overnight the light absorption was determined with an enzyme linked immunosorbent assay at 570nm wavelength.

Cell viability (%) = (OD) _drug -OD _blank )/(OD _control -OD _blank )×100％

Results and conclusions: referring to FIGS. 1-3, FIGS. 1-3 show the survival rates of C2C12 myotube cells under the action of carnosol, bismethylcarnosol and dimethy-deuterocol at different concentrations. As shown in table 1, the survival rate was 97.78% when the concentration of carnosol was 3.125 μ M; when the concentration of the carnosol is 6.25 mu M, the survival rate is 96.73 percent; the survival rate is 93.00 percent when the concentration is 12.5 mu M; the survival rate is 92.56% when the concentration is 25 muM; at a concentration of 50. Mu.M, the survival rate was 70.67%. When the concentration of the bismethylcarnosol is 6.25 mu M, the survival rate is 97.69 percent; the survival rate is 99.63 percent when the concentration is 12.5 mu M; the survival rate is 95.10 percent when the concentration is 25 mu M; the survival rate is 92.11% when the concentration is 50 muM, and the survival rate is 73.74% when the concentration is 100 muM; when the concentration of the dimethy deuterated carnosol is 6.25 mu M, the survival rate is 101.72 percent; the survival rate is 99.49 percent when the concentration is 12.5 mu M; the survival rate is 103.47% when the concentration is 25 muM; the survival rate was 103.32% at a concentration of 50. Mu.M, and 70.43% at a concentration of 100. Mu.M.

The above results demonstrate that carnosol is at a maximum safe concentration for use in C2C12 muscle cells of 25 μ M; the maximum safe concentration of the bismethylcarnosol for C2C12 muscle cells is 50 μ M; the maximum safe concentration of dimethydinol for use in C2C12 muscle cells was 50 μ M.

Table 1 shows the statistical results of the effect of carnosol on the activity of C2C12 muscle cells in example 3, corresponding to FIGS. 1-3.

TABLE 1 Effect of carnosol, bismethyl carnosol and bismethyl deuterated carnosol on C2C12 cell viability

Compound (concentration)	Survival rate (%)
		Carnosol-3.125. Mu.M	97.78
Carnosol-6.25. Mu.M	96.73
		Carnosol-12.5. Mu.M	93.00
Carnosol-25. Mu.M	92.56
		Carnosol-50. Mu.M	70.76
6.25 mu M of bismethylcarnosol	97.69％
		Bimetyl carnosol-12.5 mu M	99.63％
Bimetyl carnosol-25 mu M	95.10％
		Dichlorocarnosol-50 mu M	92.11％
Dichlorocarnosol-100 mu M	73.74％
		6.25 mu M of dimethy deuterated carnosol	101.72％
Dimethyldeuterated carnosol-12.5 mu M	99.49％
		Dimethyldeuterated carnosol-25 mu M	103.47％
Dimethyldeuterated carnosol-50 mu M	103.32％
		Dimethyldeuterated carnosol-100 mu M	70.43％

Example 4 carnosol, dimethy carnosol and dimethy deuterated carnosol alleviate atrophy of mouse myoblast (C2C 12) induced by supernatant of mouse colon cancer cell (C26) culture solution

Myotube diameter after differentiation of mouse myoblasts (C2C 12) was evaluated by diameter measurement. The staining method used in the experiment is hematoxylin-eosin staining method, which is called HE staining method for short. The hematoxylin staining solution is alkaline and positively charged, and can be easily bonded with negatively charged and acidic deoxyribonucleic acid (DNA) in cell nucleus by ionic bond to stain blue; eosin is an acid dye that dissociates in water into negatively charged anions that readily bind to the amino positive charges of proteins in the cytoplasm and stain red. The stained cells were placed under a high power microscope for pattern collection, and myotube cell diameter was counted using image J software.

Changes in the levels of the relevant proteins in mouse myoblasts (C2C 12) were evaluated using western blot experiments. A protein sample separated by PAGE (polyacrylamide gel electrophoresis) is transferred to a solid phase carrier (such as a nitrocellulose membrane), and the protein is adsorbed by the solid phase carrier in a non-covalent bond form, and the type of the polypeptide separated by the electrophoresis and the biological activity of the polypeptide can be kept unchanged. Taking protein or polypeptide on a solid phase carrier as an antigen, carrying out immunoreaction with a corresponding antibody, then carrying out reaction with a second antibody marked by enzyme or isotope, and carrying out substrate color development or autoradiography to detect protein components expressed by specific target genes separated by electrophoresis.

The effect of the drug on the C2C12 cell muscle atrophy model can be evaluated by the above method. The specific method comprises the following steps: the C2C12 cells were seeded in 24-well plates and differentiated into mature myotube cells using high-glucose DMEM medium containing 2% HS. In addition, the C26 cells were inoculated into T75 bottles and cultured for 48 hours, and then the supernatant was collected. The C26 supernatant and 2% hs differentiation solution 1 were mixed together to prepare a muscular atrophy-inducing solution. Except for the control group to which the 2-vol% hs differentiation solution was added, the same amount of the muscular atrophy-inducing solution was added to each of the other groups, one group was used as a model group, and the other groups were used as administration experiment groups. At the same time, carnosol stock solution was added to the cells at the following concentrations of 3.125. Mu.M, 6.25. Mu.M, 12.5. Mu.M, 25. Mu.M, see Table 2; adding 6.25 μ M,12.5 μ M,25 μ M, and 50 μ M into the obtained solution; the bis-methyl deuterated carnosol stock solution was added at 6.25. Mu.M, 12.5. Mu.M, 25. Mu.M, 50. Mu.M, as shown in Table 3. Table 2 is the dosing regimen of carnosol to C2C12 muscle cell muscle atrophy reversal experiments in example 4.

TABLE 2 administration samples for myoblast (C2C 12) atrophy experiments in example 4

Table 3 is the dosing regimen of the experiment of reversal of muscular atrophy of C2C12 muscle cells with dimethy carnosol and dimethy deuterated carnosol in example 4.

TABLE 3 administration samples for experiments on myoblast (C2C 12) atrophy in example 4

Wherein, μ M means μmol/L.

The myotube diameter measurement method is as follows:

after 48h of drug action, the cells were fixed with a fixative (absolute ethanol: formaldehyde: glacial acetic acid =20 = 2. The muscle atrophy reversal rate was calculated as follows:

muscular atrophy reversal rate = (mean value of myotubes in administration group-mean value of myotubes in model group)/(mean value of myotubes in control group-mean value of myotubes in model group) X100%

The western blot method was as follows:

after 48h of action, the cells were collected and the RIPA lysate containing 1% phosphoprotease inhibitor was added in proportion. After the lysis is finished, the lysate is placed at 13000rpm at 4 ℃ for centrifugation for 30min, supernatant is taken out and diluted for BCA protein quantification, and the protein concentration of the sample is calculated. The loading amount is 20 μ g, the loading system is 20 μ l, protein is subpackaged according to protein concentration and dilution times, heat denaturation is carried out for 10min in a metal bath at 100 ℃, ice cooling is carried out, centrifugation is carried out, and then storage at minus 80 ℃ or direct loading electrophoresis, membrane transfer and immunoreaction are carried out.

Results and conclusions: please refer to fig. 4-19. Fig. 4-17 are HE staining representative pictures of carnosol, bismethylcarnosol, and bismethyldeuterated carnosol for respectively alleviating C2C12 mature myotube cell atrophy induced by C26 cell culture fluid, fig. 18 and 19 are myotube statistical result graphs, and the attached table 4 is a myotube statistical result table. The carnosol, the dimethy carnosol and the dimethy deuterated carnosol have obvious reversal effect on the muscle cell atrophy and are in a concentration dependency relationship. When the concentration of the carnosol is 3.125 mu M, the reversion rate is 23.21 percent; when the concentration of the carnosol is 6.25 mu M, the reversion rate is 36.43 percent; when the concentration is 12.5 mu M, the reversion rate is 50.02 percent; when the concentration is 25 mu M, the reversion rate is 66.39 percent; when the concentration of the bismethylcarnosol is 12.5 mu M, the reversion rate is 28.40 percent; when the concentration is 25 mu M, the reversion rate is 36.24 percent; when the concentration is 50 mu M, the reversion rate is 69.73%; when the concentration of the dimethy deuterated carnosol is 12.5 mu M, the reversion rate is 25.82 percent; when the concentration is 25 mu M, the reversion rate is 33.87 percent; at a concentration of 50. Mu.M, the reversion was 75.86%.

Table 4 is a statistical result of the C2C12 muscle cell atrophy relief by carnosol, bismethyl carnosol and bismethyl deuterated carnosol, corresponding to fig. 4-17.

TABLE 4 reversal rates of carnosol, bismethylcarnosol and bismethyldeuterated carnosol on C2C12 myotubular cell muscular atrophy

Compound (concentration)	Reverse conversion rate of muscular atrophy (%)
		Carnosol-3.125. Mu.M	23.21％
Carnosol-6.25. Mu.M	36.43％
		Carnosol-12.5. Mu.M	50.02％
Carnosol-25 μ M	66.36％
		Bimetyl carnosol-12.5 mu M	28.40％
Bimetyl carnosol-25 mu M	36.24％
		50 mu M of bismethylsulfop-l	69.73％
Dimethyldeuterated carnosol-12.5 mu M	25.82％
		Dimethyldeuterated carnosol-25 mu M	33.87％
Dimethyldeuterated carnosol-50 mu M	75.86％

FIGS. 20-22 show the results of western blotting of carnosol, bismethylcarnosol and bismethyldeuterated carnosol in C2C12 cell muscle atrophy models. The result shows that p-p65 in the C2C12 myotube cells is activated under the stimulation of the C26 cell culture solution, the expression level is up-regulated, and the p-p65 expression can be reduced in a concentration gradient manner by using carnosol, dimethyl carnosol and dimethyl deuterated carnosol; meanwhile, the up-regulation of p-p65 activates the expression of E3 ubiquitination ligase MuRF-1 and Atrogin-1, but the up-regulation of the expression is also inhibited by carnosol, dimethyl carnosol and dimethyl deuterated carnosol; the expression levels of MHC and MyoD are significantly reduced due to the up-regulation of p-p65, while carnosol, bismethylcarnosol and bismethyldeuterated carnosol alleviate the decrease of MHC and MyoD, and this alleviation is concentration-dependent. In addition, carnosol, bismethylcarnosol and bismethyldeuterated carnosol can reverse the reduction of p-AKT caused by C26 cell culture solution. However, carnosol had no alleviating effect on p-p38 activation.

The results show that the carnosol, the dimethy carnosol and the dimethy deuterated carnosol relieve the protein degradation in muscle cells through a brand-new molecular action mechanism, promote the differentiation and growth of the muscle cells, have obvious relieving effect on the muscular atrophy of the muscle cells and are in a concentration-dependent relationship.

Example 5 Effect of carnosol, bimethylcarnosol and Dimethyldeuterated carnosol on Activity of mouse Pre-adipocytes (3T 3-L1)

The survival rate of 3T3-L1 cells is detected by adopting an MTT method. The detection principle is that succinate dehydrogenase in mitochondria of living cells can enable exogenous MTT to be reduced into water-insoluble blue-violet crystal Formazan (Formazan) and deposited in the cells, and dead cells do not have the function. The triple liquid (SDS +5% isobutanol +0.01% concentrated hydrochloric acid aqueous solution by 10%) can dissolve formazan in cells, and the light absorption value of the triple liquid is measured by an enzyme-linked immunosorbent assay detector at the wavelength of 570nm, so that the quantity of living cells can be indirectly reflected. Within a certain range of cell number, MTT crystals are formed in an amount proportional to the cell number.

The effect of the drug on the viability of mouse preadipocytes (3T 3-L1) can be evaluated by the above method. The specific method comprises the following steps:

the 3T3-L1 cells were seeded in a 96-well plate, and the culture system was high-sugar DMEM medium containing 10% fbs, and 1% diabody was added, and placed in an environment of 5% co2, 37 ℃. After the cells are confluent, the cells are differentiated into mature fat cells by using high-sugar DMEM culture solution containing 0.5mM IBMX, 5mg/ml insulin, 1 mu M dexamethasone and 10% FBS, and after the cells are successfully differentiated, the cells are obviously seen to contain a large amount of oil drops. Salvianolic acid was added at the following concentration of 6.25. Mu.M, 12.5. Mu.M, 25. Mu.M, 50. Mu.M, 100. Mu.M or 6.25. Mu.M, 12.5. Mu.M, 25. Mu.M, 50. Mu.M, 100. Mu.M, and bismethylcarnosol was added to prepare a 10-vol% FBS-containing high-glucose DMEM culture solution, which was allowed to act on the differentiated mature 3T3-L1 adipocytes for 48 hours. The culture solution was changed to MTT (5 mg/ml): the culture solution =1, 100 ul/well, 50ul of the triple solution was added to each well after 3h, and the light absorption value was measured at a wavelength of 570nm by an enzyme linked immunosorbent assay after overnight.

Cell viability (%) = (OD) _drug -OD _blank )/(OD _control -OD _blank )×100％

Results and conclusions: please refer to fig. 23-25. FIGS. 23-25 show the survival rates of 3T3-L1 cells with different concentrations of carnosol, bismethyl carnosol and bismethyl deuterated carnosol. As shown in table 5, the survival rate was 99.82% when the concentration of carnosol was 3.125 μ M; when the concentration of the carnosol is 6.25 mu M, the survival rate is 96.19 percent; the survival rate is 96.23 percent when the concentration is 12.5 mu M; the survival rate is 97.18 percent when the concentration is 25 mu M; the survival rate is 90.04% when the concentration is 50 muM; the survival rate is 51.47% when the concentration is 100 mu M; when the concentration of the bismethylcarnosol is 6.25 mu M, the survival rate is 99.03 percent; the survival rate is 95.76 percent when the concentration is 12.5 mu M; the survival rate is 93.60 percent when the concentration is 25 mu M; the survival rate is 91.84% when the concentration is 50 muM; the survival rate is 90.73 percent when the concentration is 100 mu M; when the concentration of the dimethy deuterated carnosol is 25 mu M, the survival rate is 97.19 percent; the survival rate is 92.80 percent when the concentration is 50 mu M; at a concentration of 100. Mu.M, the survival rate was 90.54%.

The above results demonstrate that the maximum safe concentration of carnosol for 3T3-L1 adipocytes is 50 μ M; the maximum safe concentration of the dimethomorph caudal pedunculal phenol on the 3T3-L1 fat cells is 100 mu M; the maximum safe concentration of dimethy deuterated carnosol on 3T3-L1 fat cells is 100 mu M.

Table 5 shows the statistical results of the toxicity tests of carnosol, bismethylcarnosol and bismethyldeuterated carnosol on 3T3-L1 adipocytes in example 5, corresponding to FIGS. 23-25.

TABLE 5 Table of the results of the effects of carnosol, bismethylcarnosol and bismethyldeuterated carnosol on the viability of 3T3-L1 cells

Compound (concentration)	Survival rate (%)
		Carnosol-3.125. Mu.M	99.82％
Carnosol-6.25. Mu.M	96.19％
		Carnosol-12.5. Mu.M	96.23％
Carnosol-25. Mu.M	97.18％
		Carnosol-50 μ M	90.04％
Carnosol-100. Mu.M	51.47％
		6.25 mu M of bismethylcarnosol	99.03％
Bimetyl carnosol-12.5 mu M	95.76％
		Bimetyl carnosol-25 mu M	93.60％
50 mu M of bismethylsulfop-l	91.84％
		Dictamni carnosol-100. Mu.M	90.73％
6.25 mu M of dimethy deuterated carnosol	99.54％
		Dimethyldeuterated carnosol-12.5 mu M	95.92％
Dimethyldeuterated carnosol-25 mu M	97.19％
		Dimethyldeuterated carnosol-50 mu M	92.80％
Dimethyl deuteriumCarnosol-100. Mu.M	90.54％

Example 6 Experimental results of carnosol, bismethylcarnosol and bismethyldeuterated carnosol to alleviate lipolysis in 3T3-L1 adipocytes

The intracellular fat content was assessed by oil red O fat staining. The oil red O is liposoluble dye, can be highly dissolved in fat, and can specifically color neutral fat such as triglyceride in tissue.

Glycerol assay was used to assess intracellular fat content. Glycerokinase phosphorylates glycerol to glycerol-3-phosphate; oxidizing 3-phosphoglycerol by glycerophosphate oxidase to produce hydrogen peroxide; the chromogenic substrate is converted into benzoquinone imine under the action of catalase, and the optical density value is in direct proportion to the concentration of glycerol.

The intracellular fat content was assessed using the triglyceride assay. Decomposing triglyceride in serum into glycerol by lipase; glycerol kinase phosphorylates glycerol to glycerol-3-phosphate; oxidizing 3-phosphoglycerol by glycerophosphate oxidase to produce hydrogen peroxide; the chromogenic substrate is converted into benzoquinone imine under the action of catalase, and the optical density value of the quinone imine is in direct proportion to the concentration of the glycerol and further in direct proportion to the content of the triglyceride.

Changes in the levels of the relevant proteins in mouse adipocytes (3T 3-L1) were evaluated by Western blotting experiments.

The effect of the drug on a model of cellular lipolysis can be assessed by the above method. The specific method comprises the following steps: 3T3-L1 cells were seeded in 6-well plates and cultured in high-sugar DMEM medium containing 10% FBS, 1% diabody was added, and the cells were subjected to 5% CO2 at 37 ℃ in the environment. After the cells are confluent, the cells are differentiated into mature fat cells by using high-sugar DMEM culture solution containing 0.5mM IBMX, 5mg/ml insulin, 1 mu M dexamethasone and 10% FBS, and after the cells are successfully differentiated, the cells are obviously seen to contain a large amount of oil drops. In addition, C26 cells were inoculated in a T75 bottle, 48h of phenol red-free high-sugar DMEM culture solution was collected after full growth, centrifuged at 1000rpm for 3min, the supernatant was taken, centrifuged at 4000rpm for 10min, and the supernatant was taken. The C26 supernatant was mixed with phenol red-free high-glucose DMEM medium 1 as a lipolysis-inducing solution. Except for the control group added with phenol red-free high-sugar DMEM culture solution, the other groups are added with the same amount of lipolysis inducing solution, one group is used as a model group, and the other groups are used as administration experiment groups. Simultaneously, carnosol stock solutions were added to the cells at the following concentrations 6.25. Mu.M, 12.5. Mu.M, 25. Mu.M, 50. Mu.M, see Table 6; the bis-methyl carnosol stock was added at 25. Mu.M, 50. Mu.M, 100. Mu.M, and the bis-methyl deuterated carnosol stock was added at 25. Mu.M, 50. Mu.M, 100. Mu.M, as shown in Table 7.

Table 6 is a dosing regimen for 3T3-L1 adipocyte lipolysis reversal experiments with carnosol as in example 6.

TABLE 6 administration samples of mouse preadipocyte (3T 3-L1) lipolysis assay in example 6

Table 7 shows the dosing schedule of the experiment of the reversion of 3T3-L1 adipocyte lipolysis by dimethy carnosol and dimethy-deuterol in example 6.

TABLE 7 administration samples of mouse preadipocyte (3T 3-L1) lipolysis assay in example 6

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The oil red O staining and semi-quantitative method is as follows: after 48h of action, 0.5% oil red O dye (prepared in isopropanol) is diluted with distilled water 3. Discarding cell culture solution, fixing cells with 4% neutral formaldehyde for more than 1h, sucking the fixing solution with a suction pump, washing with PBS for 3 times, placing in a room temperature ventilation place, drying in the air for 20min, adding oil red O staining solution for staining for 30min, removing, washing with PBS for 2 times, washing with 60% isopropanol for removing floating color, washing with distilled water for 2 times, placing under an inverted high power microscope for observation and taking photos. Bound oil red O in the cells was then dissolved out with isopropanol and run in parallel with an undifferentiated adipocyte staining set as a blank zero set and OD values were measured at 510 nm. Performing oil red O semi-quantification according to the OD value result according to the following formula:

fat content (%) = (OD) _drug -OD _blank )/(OD _control -OD _blank )×100％

The glycerol and triglyceride detection method is as follows: after 48 hours of action, the content of the triglyceride in the supernatant and the content of the triglyceride in the cells are respectively detected by using a glycerol detection kit and a triglyceride detection kit of Beijing prilley gene technology Co.

The western blotting method was the same as that described in example 4.

Results and conclusions: please refer to fig. 26-41. FIGS. 26-39 are representative images of oil red O staining of carnosol, bismethyl carnosol, and bismethyl deuterated carnosol to alleviate lipolysis in 3T3-L1 mature adipocytes induced by C26 cell culture fluid, with a large number of lipid droplets accumulated in normal control cells, larger lipid droplets, and dark and bright staining; after the C26 cell culture solution acts independently, intracellular lipid drops are obviously reduced and the content is reduced sharply; and when carnosol, dimethyl carnosol or dimethyl deuterated carnosol is incubated under the action of the C26 cell culture solution, the content of fat drops stained by oil red O in cells is gradually increased along with the increase of the concentration of the carnosol, the dimethyl carnosol or the dimethyl deuterated carnosol.

FIG. 40, 41 shows the semi-quantitative results of oil red O staining, as shown in FIGS. 40, 41, with carnosol, bismethylcarnosol and bismethyldeuterated carnosol concentration-dependently increasing intracellular oil red O content, indicating an increase in fat content.

FIGS. 42-45 show the results of glycerol and triglyceride assays, respectively, with carnosol, bismethylcarnosol and bismethyldeuterated carnosol concentration-dependently reducing 3T3-L1 mature adipocyte glycerol release by C26 cell culture medium and reducing intracellular triglyceride breakdown.

FIGS. 46-48 show western blot experiments of carnosol, bismethyl carnosol and bismethyl deuterated carnosol in a 3T3-L1 cell lipolysis model. The result shows that p-p65 in the 3T3-L1 fat cell is activated under the stimulation of the C26 cell culture solution, the expression level is up-regulated, and the expression of p-p65 can be reduced in a concentration gradient manner by the carnosol, the dimethyl carnosol and the dimethyl deuterated carnosol; c26 cell culture solution activates phosphorylation of HSL (hormone sensitive esterase), p-HSL expression level is up-regulated, and participates in lipolysis process, and p-HSL expression is reduced along with the increase of concentration of carnosol, dimethyl carnosol and dimethyl deuterated carnosol; the phosphorylation of AMPK alpha (AMP-dependent protein kinase alpha subtype) is activated by the C26 cell culture solution, the expression quantity of p-AMPK alpha is up-regulated, the energy metabolism is intensified, and the expression of p-AMPK alpha is reduced along with the increase of the concentration of carnosol, dimethyl carnosol and dimethyl deuterated carnosol. However, carnosol or bismethylcarnosol has no alleviating effect on p-p38 activation.

The results show that the carnosol, the dimethy carnosol and the dimethy deuterated carnosol reduce the fat degradation and relieve the energy excessive consumption through a brand-new molecular action mechanism, have obvious relieving effect on fat cell lipolysis, and are in a concentration dependence relationship.

Example 7 animal model test results of carnosol and bis-methyl deuterated carnosol for treating tumor cachexia

The C26 cell suspension is inoculated to 100 million of axilla on the left and right sides of a Balb/C mouse. Increasing the tumor volume to 800cm ³ Then, the tumor was taken out and homogenized with 3.5ml of ice physiological saline/g to obtain a tumor tissue suspension. The mice to be inoculated are divided into groups according to the body weight, and the cell suspension is inoculated in the left underarm of Balb/c mice, with the inoculation amount of 100 ul/mouse. Administration was started the following day after inoculation. The carnosol was dissolved in DMSO and a pre-warmed 0.5% CMCNa solution to a uniform and stable solution, the final concentration was 1mg/ml containing 3% DMSO, the dose was 10mg/kg, and the route of administration was intraperitoneal injection. The dimethyl deuterated carnosol is dissolved in 3% DMSO (dimethylsulfoxide), 2% absolute ethyl alcohol, 2% polyoxyethylene castor oil and 93% normal saline solution in a mixed manner to form a uniform and stable solution, the final concentration is 2mg/ml, the dosage is 20mg/kg, and the administration route is intraperitoneal injection. Mice were monitored daily for body weight, body temperature, tumor size and food intake. After 15 days, about 10% of the body weight of the model group mice is considered to enter the advanced stage of cachexia. Biochemical samples such as tibialis anterior muscle, epididymal fat, tumor, and serum were obtained after cervical dislocation of the mice.

Results and conclusions: please refer to fig. 49-92.

FIGS. 49-62 are graphs of tumor bearing body weight, tumor free body weight, mean cumulative food intake, average daily food intake, body temperature change, tumor volume and tumor anatomy during mouse survival. As shown, healthy mice continued to gain weight; the tumor-bearing body weight of the mice in the C26 tumor model group is reduced and then increased from the beginning of the experiment to the 10 th day, the whole body is maintained stable, the tumor-bearing body weight is continuously reduced from the 10 th day to the end of the experiment, and the same is true for the tumor-removing body weight; the tumor-bearing weights of the carnosol group and the dimethy deuterated carnosol group continuously increase, the weight begins to obviously decrease at 14 days, and the tumor-bearing weight and the tumor-removing weight are higher than those of the C26 Model group until the experiment is finished, and the difference has statistical significance (p is less than 0.01). The food intake of mice in the C26 tumor model group is obviously reduced compared with that of healthy groups, and the carnosol and the dimethy deuterated carnosol can slightly improve the appetite of the mice. In addition, the body temperature of the mice in the C26 tumor model group is obviously reduced compared with that in the healthy group, and the body temperature of the carnosol group and the dimethyl deuterated carnosol group is higher than that of the C26 tumor model group, and the difference is larger. However, carnosol and dimethy deuterated carnosol have no obvious inhibiting effect on C26 tumors.

Fig. 63-68, 70-75 are graphs of gastrocnemius mass, gastrocnemius compaction, results of HE staining of gastrocnemius sections, statistical graphs of fascial diameter of gastrocnemius sections, as shown, the gastrocnemius mass of mice in the C26 tumor model group was significantly smaller than that in the healthy group, and the difference was statistically significant (p < 0.001), the gastrocnemius mass was slightly larger than that in the C26 tumor model group in the carnosol group and the dimethy-deuterol group, although there was no statistical significance in mass, the slicing results showed that the diameter of the fascial diameter of the carnosol group in the carnosol group was larger than that in the C26 tumor model group, and the diameter of the gastrocnemius fascial diameter of the dimethy-deuterol group was also larger than that in the C26 tumor model group (p = 0.06).

FIG. 69 is the results of western blotting experiments in mouse gastrocnemius tissues. The result shows that the C26 tumor activates p-p65, the expression level of the p-p65 is up-regulated, and the p-p65 expression can be reduced by the carnosol; meanwhile, the up-regulation of p-p65 also activates the expression of E3 ubiquitination ligase MuRF-1, but the up-regulation of the expression is also inhibited by carnosol; due to the up-regulation of p-p65, the expression levels of MHC and MyoD are significantly reduced, while carnosol can alleviate the reduction of MHC and MyoD. In addition, carnosol can reverse the reduction in p-AKT induced by C26 tumors. However, carnosol had no alleviating effect on p-p38 activation.

Fig. 76-81, 83-88 show the epididymal fat mass, epididymal fat tap, epididymal fat slice HE staining results, epididymal fat slice fat diameter statistical plots, as shown in the figure, the epididymal fat mass of the C26 tumor model group was significantly less than that of the healthy group, and the difference was statistically significant (p < 0.001), and the epididymal fat mass of the carnosol group and the bis-methyl deuterated carnosol group was significantly greater than that of the C26 tumor model group, and the difference was statistically significant (p < 0.01).

FIG. 82 shows the results of western blotting of epididymal adipose tissues in mice. The result shows that the C26 tumor activates the expression of p-p65 in the epididymal fat of the mouse, and the expression of the p-p65 can be reduced by the carnosol; the C26 tumor activates phosphorylation of HSL (hormone sensitive esterase) in epididymis fat of a mouse, the expression quantity of p-HSL is up-regulated, the p-HSL participates in lipolysis process, and the p-HSL is hardly activated under the action of carnosol; the C26 tumor activates phosphorylation of AMPK alpha (AMP-dependent protein kinase alpha subtype) in epididymis fat of mice, expression level of p-AMPK alpha is up-regulated, energy metabolism is aggravated, and the expression level of p-AMPK alpha is obviously reduced by carnosol. But likewise carnosol has no alleviating effect on p-p38 activation.

FIG. 89 shows the measurement of the glycerol content in the mouse serum. The results show that the serum free glycerol content of mice in the C26 tumor model group is obviously higher than that of mice in the healthy group, the difference is statistically significant (p is less than 0.001), the serum free glycerol content of the carnosol group is obviously lower than that of the mice in the C26 tumor model group, and the difference is statistically significant (p is less than 0.01).

FIG. 90 shows measurement of the triglyceride level in mouse serum. The results show that the content of free triglyceride in the serum of mice in the C26 tumor model group is obviously lower than that in the healthy group, the difference is statistically significant (p is less than 0.01), the content of free triglyceride in the serum of carnosol group is obviously higher than that in the C26 tumor model group, and the difference is statistically significant (p is less than 0.05).

FIG. 91 shows the measurement of the serum TNF- α level in mice. The results show that the mouse serum TNF-alpha content in the C26 tumor model group is obviously higher than that in the healthy group, the difference is statistically significant (p is less than 0.05), the TNF-alpha content in the carnosol group is obviously lower than that in the C26 tumor model group, and the difference is statistically significant (p is less than 0.05).

FIG. 92 shows the measurement of IL-6 content in mouse serum. The result shows that the content of IL-6 in the serum of mice in the C26 tumor model group is obviously higher than that in the healthy group, the difference is statistically significant (p is less than 0.01), and the content of IL-6 in the carnosol group is not obviously different from that in the C26 tumor model group.

The above results demonstrate that carnosol and dimethy-deuterated carnosol can relieve weight loss, muscle atrophy, fat degradation and body temperature reduction caused by tumor cachexia without affecting tumor size, and improve appetite.

Claims

1. The dimethyl deuterated carnosol is shown as a formula (1), and is characterized in that the structure is shown as the following formula (1):

2. a preparation method of dimethy deuterated carnosol is characterized in that 750mg of carnosol and 3mL of deuterated iodomethane are used as raw materials, 600mg of potassium carbonate is used as a catalyst, 50mL of acetone is used as a solvent, and the raw materials are reacted at room temperature in a dark place for 48 hours to prepare the dimethy deuterated carnosol shown in the formula (1);

the preparation method is shown as the following reaction formula (A):

3. an application of dimethy deuterated carnosol shown in a formula (1) in preparing a medicament for treating muscular atrophy and fat reduction caused by solid tumor cachexia;

4. use according to claim 3, wherein the medicament is a medicament for humans and/or animals.

5. A pharmaceutical composition for treating muscle atrophy and fat loss caused by solid tumor cachexia, which comprises the dimethyl-deuterated carnosol of formula (1) according to claim 1.

6. The pharmaceutical composition of claim 5, wherein the pharmaceutical composition is in the form of a capsule, tablet, oral preparation, microcapsule, injection, ointment, spray or suppository.

7. The pharmaceutical composition of claim 5, wherein the product is administered by injection, orally, parenterally, by inhalation spray, or transdermally.

8. The pharmaceutical composition of claim 5, wherein the dimethy deuterated carnosol represented by formula (1) accounts for 0.001-100wt.% of the total dry weight of the composition.