CN110746475A - Compound for adjuvant therapy of tumor dyscrasia and application thereof - Google Patents

Abstract

The invention relates to a flavonoid glycoside compound, in particular to a compound which can be used for preparing a medicament for the adjuvant treatment of tumor dyscrasia stage. A compound for adjuvant treatment of tumor dyscrasia with molecular formula C₃₉H₄₄O₁₉Or C₃₈H₄₂O₁₈The invention is used for solving the problems of high content of inflammatory cytokines TNF- α and IL-6 in the malignant tumor stage, anorexia, emaciation and the like caused by poor nutrient absorption.

Description

Compound for adjuvant therapy of tumor dyscrasia and application thereof

Technical Field

The invention relates to a flavonoid glycoside compound, in particular to a compound which can be used for preparing a medicament for the adjuvant treatment of tumor dyscrasia stage.

Background

Cachexia is a critical stage in the development of most malignant tumors to the advanced stage, the probability of cachexia in patients with advanced cancer is as high as 80%, and the main cause of death of patients with advanced malignant tumors is closely related to cachexia. The clinical manifestations are aversion to food, somnolence, emaciation, anemia, metabolic disorders of three nutrients (protein, lipid and carbohydrate), electrolyte disorders and the like. The occurrence mechanism of cachexia is complex, and the exact occurrence mechanism is still uncertain at present, so that a targeted effective scheme in the aspect of cachexia treatment is lacked.

The medical community currently recognizes that systemic chronic inflammation and metabolic disorders play an important role in the development of cachexia. In the state of cachexia, the energy requirement of the organism is increased, the utilization of ineffective energy is increased, and the basal metabolic rate is increased, so that the blood sugar content of the organism is reduced, and muscle adipose tissues are greatly consumed. The energy required for normal body's vital activities is obtained by carbohydrate aerobic respiration, but in the dyscrasia stage, body's energy acquisition almost completely depends on glycolysis, which is a low-efficiency energy production mode, so that tumors consume a large amount of glucose, resulting in hypoglycemia. In general, metabolism in the cachectic state is a decrease in synthesis and an increase in consumption.

Through literature investigation, 2 types of cytokines, namely inflammatory cytokines (such as IL-6, TNF- α -1, INF-y and the like) and inflammation-inhibiting cytokines (such as soluble tumor necrosis factor receptor sTNFR, IL-1 receptor antagonists IL-lra, IL-4, IL-10, IL-13, IL-15 and the like) exist in normal animals, and the imbalance of the two factors plays a key role in cancer cachexia^[1-3]The micro-environment of the tumor cells is a sterile inflammatory state, which often leads to serious imbalance of in-vivo cytokine networks due to abnormal and continuous increase of the content of one or more inflammatory cytokines, so that the body is in a chronic inflammatory state for a long time, and the cachexia is more and more seriousIt has been shown that in cachexia patients and/or tumor-bearing cachexia animals, the expression level of TNF- α is significantly increased, and the cachexia is significantly alleviated after the intervention of a monoclonal antibody against TNF- α, so that serum TNF- α is currently considered to be an important inducing factor for malignant tumor cachexia^[3-5]. It is clear from the current research that the cytokine interleukin-6 (IL-6) is closely related to the malignant dyscrasia of tumors^[5]IL-6 is mainly secreted from Th2 cells, and other cells include fibroblasts and monocytes. Animal experiments show that^[6]Given to rats injected with IL-6, the rapid degradation of skeletal muscle in rats was found, which suggests that IL-6 has a direct effect on enhancing protein degradation, which may explain why tumor patients in the cachexia stage have progressive or even extreme wasting phenomena, and that IL-6 concentration is positively correlated with tumor cachexia. In addition, researchers found that monoclonal antibodies to CNT0328-IL-6 can act well against tumor dyscrasia by blocking the IL-6 function^[7]。

In addition, the cachexia patient is extremely thin and gradually loses weight, except for large energy consumption, the digestive absorption function of the patient is seriously reduced, and the absorption efficiency of nutrient substances is low.

The representative medicine for treating dyscrasia at present is medroxyprogesterone acetate, which has the defects that the medicine is mainly used for solid tumors, and has no advantages for improving body weight, increasing nutrient absorption capacity and reducing overhigh cytokine level.

From the above analysis, although the occurrence mechanism of cachexia is complex, the exact occurrence mechanism is still unclear, and the treatment aspect is a lack of effective scheme, aiming at the emaciation, the malnutrition and the high-level inflammatory cytokine state of the patient, the improvement of the cachexia inflammatory state and the improvement of the nutrient absorption by reducing the levels of TNF- α and IL-6 are important measures for relieving the pain of cancer of the patient and improving the quality of life, and at present, no relevant report on the improvement of the cachexia inflammatory state and the improvement of the nutrient absorption by reducing the levels of TNF- α and IL-6 is presented.

When the inventor conducts drug molecule activity screening, the inventor accidentally finds that two flavonoid glycoside compounds in natural barley seedlings can purposefully reduce the excessive TNF- α and IL-6 levels in the dyscrasia state, and simultaneously improves the problems of poor nutrition absorption, anorexia and emaciation caused by the two flavonoid glycoside compounds, thereby finding that the compounds can be used for preparing a drug for the adjuvant therapy of dyscrasia.

Reference documents:

[1]L Wang,J.G.Qiu,J.He.Suppression of miR-143contributes tooverexpression of IL-6, HIF-1αand NF-κB p65 in Cr(VI)-induced human exposureand tumor growth.Toxicology and Applied Pharmacology.DOI:10.1016/j.taap.2019.114603

[2]NorioIizukaa,Koji Miyamotoa,Shoichi Hazamac,et al.Anticachecticeffects of Coptidis rhizoma,an anti-inflammatory herb,on esophageal cancercells that produce interleukin 6.Cancer Letters 158(2000)35-41

[3]O.Kemik，A.Sumer，A.S.Kemik，et al.The relationship among acute-phaseresponse proteins，cytokines and hormones in cachectic patients with coloncancer[J].World J Surg.Oncol., 8(2010)85-90.

[4]H.J.Patel,B.M.Patel.TNF-α and cancer cachexia:Molecular insightsand clinical implications[J].Life Sciences 170(2017)56–63.

[5]C Paola，C Neus，T Luclana，et al.Tumor necrosis factor-α mediateschanges in tissue protein turn over in a rat cancer cachexia model[J].Clinlnvest,92(1993)2783-2789.

[6]Martin F，Santolaria F,Batista N,et al.Cytokine levels(IL-6and IFN-gamma)，acute phase response and nutritional status as prognostic factors inlung cancer.Cytokine， 11(1999)80-86

[7]Zaki MH，Nemeth JA,Trikha M.CNT0328，a monoclonal antibody to IL-6，inbitits human tumor-induced cachexia in nude mice.Cancer,111(2004)592-595.

disclosure of Invention

The invention aims to solve the problems of high content of inflammatory cytokines TNF- α and IL-6 in the dyscrasia stage of tumors and anorexia and emaciation caused by poor nutrient absorption, and provides a compound for preparing a medicament for adjuvant therapy of the dyscrasia stage of tumors.

The invention relates to a compound for preparing an adjuvant therapy of tumor dyscrasia, which is characterized by having the following structural formula:

wherein R is₁Is OCH₃Or H

When R is₁Is OCH₃When the molecular formula is C₃₉H₄₄O₁₉

When R is₁When it is H, the molecular formula is C₃₈H₄₂O₁₈

A preparation process of a compound for reducing contents of inflammatory cytokines TNF- α and IL-6 in a tumor dyscrasia stage is characterized by comprising the following steps of collecting fresh barley seedlings, freeze-drying, crushing, extracting for 3-6 times by using 50% polar solvent aqueous solution according to the weight ratio of raw materials to an extraction solvent of 1: 4-10 at room temperature, combining extraction solutions, concentrating the extraction solution to a specific gravity of 1.00-1.28, adsorbing the concentrated extraction solution on a macroporous resin column, washing with water, eluting by using 8-50% polar solvent aqueous solution in sequence, collecting 18-20% polar solvent aqueous solution fraction rich in flavonoid components, separating and purifying by using reverse phase preparative liquid chromatography, using 20% polar solvent aqueous solution as a mobile phase, and detecting an outflow component at a position of 254nm to obtain the compound.

Aiming at the problems of excessive secretion levels of inflammatory cytokines TNF- α and IL-6 in the malignant tumor dyscrasia stage and anorexia and emaciation caused by the excessive secretion levels, the compound has the following mechanism for assisting in treating the malignant tumor dyscrasia:

(1) the compound can reduce the levels of TNF- α and IL-6 in a targeted manner, fundamentally improve aseptic inflammation worsening state of a tumor microenvironment caused by overhigh content of inflammatory cytokines in a tumor dyscrasia stage, improve digestive absorption dysfunction caused by a long-term inflammation state, and change anorexia and emaciation in the dyscrasia stage;

(2) the compound has a parent structure of a flavonoid compound, and the flavonoid compound is a recognized antioxidant, has a strong function of removing free radicals, and reduces further damage of various free radicals inside and outside a body to the body, thereby reducing further deterioration of inflammation.

Drawings

FIG. 1: a normal mouse model;

FIG. 2: tumor dyscrasia mouse model;

FIG. 3: cell morphology changes before and after addition of the compounds of the invention;

FIG. 4: liver and kidney tissue section before and after using the compound of the invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.

Example 1

The invention relates to a compound for preparing adjuvant therapy of tumor dyscrasia, which has the following structural formula:

the compound is abbreviated as compound 1

The compound 1 is amorphous powder with molecular formula of C₃₉H₄₄O₁₉

Mass Spectrum (MS): 816.25

333(4.38)

Hydrogen spectrum of nuclear magnetic resonance¹H-NMR(DMSO-d₆，500MHz，δ):3.67(6H,s,sinapoyl OCH₃×2),4.74(1H， d，J＝9Hz，6-C-glc-H-1),5.05(1H，d，J＝7Hz，7-O-glc-H-1),6.37(1H，d,J＝16Hz,sinapoyl H-8), 6.66(1H,s,H-8),6.71(2H,s,sinapoyl H-2,6),6.81(1H,s,H-3),6.85(2H,d,J＝9Hz,H-3',5'), 7.48(1H,d,J＝16Hz,sinapoyl H-7),7.78(2H,d,J＝9Hz,H-2',6'),13.41(1H,s,C-5-OH)

Nuclear magnetic resonance carbon spectrum¹³C-NMR(DMSO-d₆,125MHz):

164.0(C-2),102.7(C-3),181.8(C-4),159.5(C-5),110.7(C-6),161.9(C-7),93.7(C-8),156.1(C-9), 105.0(C-10),120.7(C-1'),127.9(C-2',6'),115.6(C-3',5'),161(C-4'),73.3(C-1”),70.6(C-2”),78.8(C-3” ),70.l(C-4”),80.8(C-5”),60.7(C-6”),101.4(C-1”'),72.6(C-2”'),75.7(C-3”'),69.7(C-4”'),73.8(C-5”'),63 .1(C-6”'),124.2(Sin C-1),106.4(Sin C-2),147.8(Sin C-3),138.5(Sin C-4),145.1(Sin C-5),114.4(Sin C-6),166.1(Sin C-7),55.9(Sin OCH₃)

Example 2

The invention relates to a compound for preparing adjuvant therapy of tumor dyscrasia, which has the following structural formula:

the structural formula is compound 2.

The compound 2 is amorphous powder with a molecular formula of C₃₈H₄₂O₁₈

Mass Spectrum (MS): 786.24

329(4.47)

Hydrogen spectrum of nuclear magnetic resonance¹H-NMR(DMSO-d₆，500MHz，δ):3.69(3H,s,feruloyl-OCH₃),4.73(1H，d，J＝9Hz，6-C-glc-H-1),5.05(1H，d，J＝7Hz，7-O-glc-H-1),6.32(1H，d，J＝16Hz,feruloyl H-8),6.60(1H,d,J＝8Hz,feruloyl H-6),6.68(1H,s,H-8),6.82(1H,s,H-3),6.86(2H,d,J＝9Hz,H-3',5'), 7.03(1H,d,J＝2Hz,feruloyl H-2),7.48(1H,d,J＝16Hz,feruloyl H-7),7.82(2H,d,J＝9Hz,H-2',6'), 13.41(1H,s,C-5-OH)

Nuclear magnetic resonance carbon spectrum¹³C-NMR(DMSO-d₆,125MHz):

164.1(C-2),102.9(C-3),18l.9(C-4),159.5(C-5),110.7(C-6),161.8(C-7),93.7(C-8),156(C-9),10 4.5(C-10),120.8(C-1'),128(C-2',6'),115.8(C-3',5'),161(C-4'),73.3(C-1”),70.6(C-2”),78.8(C-3”),70. l(C-4”),80.7(C-5”),60.5(C-6”),100.9(C-1”'),72.7(C-2”'),75.8(C-3”'),70.1(C-4”'),73.8(C-5”'),63.2(C -6”'),125.4(FerC-1),111.7(FerC-2),149.2(FerC-3),147.7(FerC-4),115.4(FerC-5),122.3(FerC-6),14 4.9(FerC-7),114.5(FerC-8),166(Fer C-9),55.6(Fer OCH₃)

Example 3

The preparation method of the compound of the invention comprises the following steps: the compound is prepared from natural plant barley seedling (obtained by culturing mature fruit of Hordeum Vulgare L. of Gramineae), collecting fresh 20-30 days old barley seedling, lyophilizing at-20 deg.C, pulverizing, weighing 10kg, and adding 50% acetonitrile (CH) at room temperature₃CN) aqueous solution for 3-6 times, combining the extraction solutions, concentrating the extraction solution, adsorbing the concentrated extraction solution on an Amberlite XAD-2 macroporous adsorption resin column, washing with water, and then, sequentially carrying outSecondarily using 8-50 percent of CH₃And (4) eluting with a CN aqueous solution. 18 to 20 weight percent of CH rich in flavonoid component₃The CN aqueous solution fraction was collected, and then separated and purified by preparative liquid chromatography ODS-HPLC (20 mm. times.250 mm) to give 20% by weight of CH₃CN aqueous solution is mobile phase, and outflow components are detected at 254 nm. The obtained fractions were concentrated to dryness in vacuo and stored at-20 ℃. Two pure single-component compounds, namely compound 1 and compound 2, are extracted from the sample. By Mass Spectrometry (MS), nuclear magnetic resonance hydrogen spectroscopy¹H-NMR, nuclear magnetic resonance carbon Spectroscopy¹³The structural formula of the compound of the invention is confirmed by C-NMR, ultraviolet visible spectrum (UV) and infrared spectrum (IR).

Example 4

This example is intended to observe whether the compounds of the invention reduce the levels of TNF- α and IL-6 in the malignant tumor stage and the efficacy in mice with cachexia.

1.1 preparation of Colon cancer mouse cachexia model

1.1.1 animals

SPF (specific pathogen free) healthy BALB/c mice, male, aged 6-8 weeks, weighing 22-24 g, purchased from experimental animals Breeding company Limited, and available from experimental animals production license number SCXK (lu) 2014-. The mice are fed with conventional feed (protein 20% -50%, fat 5% -10%, crude fiber 3% -5%) provided by Jinanpunyue laboratory animal Breeding Co., Ltd.

1.1.2 establishment of BALB/c mouse Colon cancer cachexia model

A mouse colon adenocarcinoma cell strain colon26, which is purchased from the cell center of the institute of basic medicine of Chinese academy of medical sciences; BALB/c mice, 6-8 weeks old, with body mass of about 23g, were subjected to Zhouwei [ Zhouwei, Jiangxiwei, Jiangjun, etc. ] establishment of an animal model with cancer cachexia [ J]J. China Experimental surgery, 2004, 21(4): 490-491]And establishing a colon cancer mouse dyscrasia model. The mice in the malignant tumor group and the drug intervention group were implanted with the mouse Colon adenocarcinoma cell line Colon26 (0.2 mL of the drug was injected subcutaneously into each mouse, and the cell line contained about 1X 10⁶Individual cancer cell), visible 7 days after implantation of the cancer cellThe generation of tumor nodules is observed, the mice appear obvious enlargement of lumps, emaciation, weight reduction, food intake and water intake reduction and activity reduction in 11 days, and when the difference has statistical significance (P is less than 0.05) compared with a healthy control group, the mice enter a dyscrasia state, and the tumor dyscrasia mouse model is successfully constructed. FIG. 1 is a normal mouse, the mouse having a bright and smooth hair color; FIG. 2 shows that the body hair of mice with tumor dyscrasia is sparse and unsmooth, and the mice are cachectic.

1.2 animal groups:

BALE/c mice were acclimatized for 1 week, the weights were individually weighed, and 60 mice were randomly divided into 6 groups, each numbered A, B, C, D, E, F, using a randomization method according to the weight ranking.

1.3 preparation of the medicine:

each of the compounds of the present invention was dissolved in 0.9% physiological saline to prepare 200 mg/kg^-1.d^-1，400mg.kg^-1.d^-1Two dose groups, gavage.

The positive drug is medroxyprogesterone acetate dissolved in 0.9% normal saline, 120mg^-1.d^-1。

1.4 Experimental intervention procedures and specimen Collection

According to the conversion formula of the human common dosage and the mouse dosage,treatment was started after all groups B-F entered cachexia (day 12), as follows:

group A is blank control group. Raising normally without any treatment.

Group B: cachexia model group. The stomach was perfused with 2m1 physiological saline for seven consecutive days

Group C: an anticancer drug group. In an amount of 120mg.kg^-1.d^-1Medroxyprogesterone acetate, and gastric lavage for seven consecutive days

Group D: compound 1 low dose group (200 mg.kg)^-1.d^-1) + 120mg of anticancer drug^-1.d^-1Medroxyprogesterone acetate, and gastric lavage for seven consecutive days

Group E: transformingHigh dose group of compound 1 (400 mg.kg)^-1.d^-1) + 120mg of anticancer drug^-1.d^-1Medroxyprogesterone acetate, and gastric lavage for seven consecutive days

And F group: compound 1 group (400 mg.kg)^-1.d^-1)

And (3) by the day 19, taking blood from eyeballs of the mice, collecting the blood, standing for two hours, centrifuging at 4 ℃ at 4000r/min for ten minutes, sucking supernatant, dividing the supernatant into two parts, detecting nutritional indexes of one part, and freezing and storing the other part at-20 ℃ for later use to detect cytokines. The mice die after a large amount of blood loss, and the tumors of the mice are weighed and stored in liquid nitrogen for later use.

1.5 Observation index

(1) General conditions of

Mice were observed for gross color and activity.

(2) Body weight and food intake

The body weight of the mice was measured with an electronic balance at a fixed time each day. The mouse feed was dosed 50 grams at a fixed time per day, and the amount of mouse feed remaining in the cassette was measured the following day at 11 am, with daily food consumption (the previous day mouse feed weight) of one (the next day mouse feed weight).

(3) Quality of gastrocnemius and epididymis fat in mice and quality of tumor in mice

After the mice were sacrificed, the left intact gastrocnemius muscle was taken and weighed immediately on the electronic balance, the fat of the epididymis on both sides of the mice was taken and weighed immediately on the electronic balance. Stripping off tumor, removing non-tumor tissues such as peripheral muscle and skin, weighing with electronic balance, and storing in liquid nitrogen.

(4) And detection of nutritional indicators

The detection of serum albumin, blood sugar and the like is carried out by adopting a full-automatic biochemical detector.

(5) Serum cytokine detection

Serum cytokines were detected by ELISA with reference to the protocol of each cytokine kit.

1.6 statistical analysis

Data processing is carried out by adopting the sps 16.0 statistical analysis software, and the results are expressed as mean +/-standard deviation

Showing that each group of data is respectively subjected to normality test, two groups of comparison are tested by using independent samples t, and multiple groups of comparison are tested by adopting one-factor analysis of variance, p<0.05 the difference was considered statistically significant, p<0.01 the difference was considered to be of high statistical significance.

1.7 results and analysis

(1) General index

The food intake, activity and hair color of the mice in the group A are kept in normal state all the time, and the mice are slightly fat in body shape; and B-F groups of tumor-bearing mice inoculated with the Colon26 cell suspension subcutaneously can touch subcutaneous nodules on the right upper axilla and grow gradually in 3-5 days of the experiment, subcutaneous raised nodules can be observed by naked eyes after six-seven days, the subcutaneous raised nodules grow to the size of lcm multiplied by lcm within about ten days, the mobility of the tumor-bearing mice is gradually reduced and becomes slow after the tumor cells are inoculated, the hair color is gradually changed from white smooth to dark rough, and the food intake is reduced day by day (see table 3). On the twelfth day, the food intake of mice in the group B model group was significantly reduced, the activity was decreased, the hair color was significantly dull and thin, and the body was weak. After the group C is given the anti-cancer drugs, the food intake of the mice is slightly increased, the activity is increased, and the hair color is gradually good. After the D-E groups are respectively given the compound and the anticancer drug to be combined for dry prediction, the food consumption of the mice is obviously increased, the activity is obviously increased, and the hair color is obviously improved compared with the mice before treatment. After the compound is administered to the group F, the food intake of the mice is obviously increased, the activity is obviously increased, and the hair color is obviously improved compared with the model group. The compound has the obvious effect of improving general physical indexes such as food intake, activity degree, hair color and the like for mice with dyscrasia, and is combined with anticancer drugs to improve more obviously.

(2) Change in body weight

Before treatment, the weights of the mice in each group gradually increased, and the weights of the mice in each group with tumor decreased and the weights of the mice in the A group still increased till the ninth day of tumor inoculation, which is shown in Table 1. The body weight differences among the six groups of mice on day 1 of the experiment were not statistically significant (p >0.05, on day eleven of the vaccinated mice, the body weights of the 5 groups of tumor-vaccinated mice were significantly lower than those of the A group of mice (p <0.05), and the body weight differences among the 5 groups of tumor-vaccinated mice were not statistically significant (p >0.05), indicating that the establishment of a mouse colon cancer cachexia model was successful on day eleven after vaccination from the viewpoint of weight loss.

Table 1 the mice of each group showed changes in body weight at different time points (g,)

compound 1 of the present invention, an anticancer agent and a physiological saline intervention were administered starting on the twelfth day, and the drug intervention was terminated as the first day of treatment, from the experiment to the 19 th day. As can be seen in Table 2, the end body weight was maximal in group A mice; the smallest of the mice in group B has a significant difference P <0.01 compared with the mice in group A; the terminal body weight of the mice in group C, although significantly less than the mice in group a, was also significantly increased compared to the mice in group B (p < 0.05); the terminal weight of mice in groups D and E is obviously higher than that of mice in group B, and both the mice in groups B and E have statistical significance (p is less than 0.05), compared with the mice in group C, the compound low-dose group (group D) is slightly higher than the mice in group C, and the terminal weight of the mice in group E is obviously higher than that of the mice in group C along with the increase of the dose, so that the statistical significance (p is less than 0.05) is achieved, the obvious dose correlation exists along with the increase of the dose of the compound 1, the compound 1 can obviously assist the anticancer drug to improve the terminal weight, and the high dose effect is the best. From group F, it can be seen that when only compound 1 was present, weight gain was significantly better than the anti-cancer drug group and model group (p <0.05) despite not being combined with the anti-cancer drug, indicating that compound 1 itself had a better effect.

Among the tumor masses of 5 groups of tumor-bearing mice, the group B is the largest, compared with the model group, the tumor mass of the group C is obviously reduced, the tumor mass size between the group D and the group F is not statistically different (p is greater than 0.05), but compared with the group C, the tumor mass is obviously reduced (p is less than 0.05); the tumor mass of the group F pure compound group was slightly reduced compared to the model group, but was not statistically different (p > 0.05). See table 9.

The test shows that: the compound 1 can not act on tumor body tissues to reduce the tumor body tissues, but can assist anti-cancer drugs to reduce the tumor body mass.

Table 2 comparison of tumor growth in groups of mice (g,

)

(3) change in food intake of mice

As can be seen from Table 3, the food intake of the group A is stable and does not change obviously, after the group B-F mice are inoculated with the Colon26 tumor cell suspension, the food intake tends to rise in the initial period and decline in the sixth to seventh days of the experiment until the eleventh day of the experiment, and is significantly reduced compared with the food intake of the group A mice, and the food intake of the group A mice is statistically different (p is less than 0.05), but the food intake of the five groups of mice is relatively meaningless (p is more than 0.05).

Seven days after the compound 1, the anticancer drug and the physiological saline are respectively administered for intervention on the twelfth day, the food intake of the group B and the group C is obviously lower than that of the group A (p is less than 0.01), but the food intake of the group D, E, F is obviously increased compared with that of the group B (p is less than 0.05); moreover, after the compound 1 is used, the food consumption of E, F group is obviously higher than that of the anti-cancer drug C group (p <0.05), and the food consumption is positively correlated with the amount of the compound (p < 0.05); the group F using compound 1 alone also significantly increased food intake (p < 0.05).

The results show that the compound 1 can obviously improve the food intake of mice with dyscrasia and improve the anorexia.

Table 3 the mice of each group received food (g,

)

(4) changes in mouse gastrocnemius muscle weight and epididymal fat weight

The weight of epididymal fat of the mice in the group B is obviously lower than that of the mice in the group A (p is less than 0.01), which indicates that the modeling is successful, and the weight of epididymal fat of the mice in the group C is obviously higher than that of the mice in the group B (p is less than 0.05), which indicates that the anticancer drugs can properly improve the absorption of nutrients such as lipid, protein and the like, but is still obviously lower than that of the mice in the group A, which indicates that the improvement effect is not ideal; the left gastrocnemius weight was significantly higher in the D-F group than in the C group (p <0.01), as was the epididymal fat weight (p <0.01, p <0.05, respectively). The experiment shows that the compound 1 effectively promotes nutrient absorption, promotes the increase of the weight of gastrocnemius and epididymis fat of mice, is beneficial to improving the consumption of the muscle and the fat of mice with dyscrasia, and can effectively and synergistically reduce the consumption of the muscle and the fat after the compound 1 is combined with an anticancer drug. See Table 4

Table 4 calf muscle weight and epididymal fat weight (g,

)

(5) change in mouse nutritional index

In each experimental mouse group, the serum albumin content of the group B and the group C is obviously lower than that of the group A (p is less than 0.05, and p is less than 0.01 respectively), but the serum albumin content of the group D-F is obviously higher than that of the group B and the group C (p is less than 0.01); the blood sugar content of the group B-F is obviously lower than that of the group A (P <0.05), but the blood sugar content of the group D-F is obviously higher than that of the group B of the dyscrasia model (P < 0.05); the compound 1 of the invention can obviously improve the content of albumin, blood sugar and other nutrient substances when used alone in the group F, and has more obvious synergistic effect when used together with anticancer drugs. And is shown in table 5.

Table 5 comparison of nutritional indicators for each group of mice (g,

)

(6) the content of the cytokines TNF- α and IL-6 in the serum of mice with dyscrasia is changed

The experimental results in Table 6 show that although the serum TNF- α and IL-6 contents of the mice in the group B of the anticancer drugs are lower than those in the model group B (P <0.05), the contents of the serum TNF- α and the IL-6 in the mice in the group D-F are still high, along with the addition of the compound 1, the serum TNF- α and the IL-6 contents of the mice in the group D-F are rapidly and obviously reduced, the reduction degree is obviously related to the amount of the compound 1, the high-dose effect is more obvious (P <0.05), when the anticancer drugs do not exist, the serum TNF- α and the IL-6 contents of the mice in the group F can also be obviously reduced by the compound 1, and the compound can play an obvious synergistic effect when being used together with.

Table 6 comparison of cytokine indices for each group of mice (pg/ml,

)

example 5

Example 2 efficacy test

Referring to example 4, the pharmacodynamic test using the same method as compound 1 revealed that compound 2 had very similar pharmacological effects to compound 1, compound 2 also significantly reduced the serum TNF- α and IL-6 levels in mice (TNF- α value of 9.21 ± 0.87pg/ml and IL-6 value of 6.72 ± 2.10pg/ml), compound 2 also significantly promoted albumin and blood glucose levels in the blood of mice (albumin value of 9.19 ± 1.02g/L and blood glucose value of 6.00 ± 0.60 umol/L), increased food intake in mice (0.52 ± 0.08g), increased body weight in mice (24.54 ± 2.00g), increased gastrocnemius weight (0.118 ± 0.006g) and epididymal fat mass (0.31 ± 0.007g), the above indices were very different from the positive group with a significant difference P <0.01, and similarly, compound 2 did not have significant effects on solid tumors, and had synergistic effects with epididymis similar to compound 1, although the two basic structural effects were found to be synergistic, and the two are similar in the combined, the two of the two are considered synergistic effects.

Example 6

At the cellular experimental level, experimental study of the compounds of the invention on cell-secreted cytokines

The animal experiments of tumor dyscrasia have proved that the compound can obviously reduce the levels of tumor necrosis factor α (TNF- α) and interleukin-6 (IL-6) in the serum of dyscrasia mice, but the results are obtained from the experiments of the whole animal level, the results of the animal experiments do not make clear the action targets of the compound in consideration of the complex influence factors of the whole animal experiments, the cause and effect relationship is unclear, in order to determine whether the compound improves the nutrient absorption by reducing the levels of TNF- α and IL-6 and whether the compound can influence the levels of TNF- α and IL-6 secreted by cells, the most classical immune cells, namely primary macrophages in the abdominal cavity of the mice, are selected as research objects by adopting a cell experiment method, the cells are cultured in vitro, and then whether the compound influences the inflammatory cytokine secretion of the compound is observed.

Because the inflammatory cytokines related to the tumor dyscrasia mainly comprise tumor necrosis factor α (TNF- α), interleukin 1 β (IL-1 β) and interleukin 6(IL-6), therefore, the three cytokines are selected as observation indexes in the cell experiment, and whether the action mechanism of the compound for promoting nutrient absorption and assisting in treating the tumor dyscrasia is related to the fact that the compound reduces the levels of TNF- α and IL-6 and is also related to the fact that the compound reduces IL-1 β is hoped to be further clarified.

1.1 materials and methods

Kunming mouse, week age 6-8 weeks, weight 18-22g, Jinanpunyue laboratory animal breeding company Limited, laboratory animal production license number SCXK (lu) 2014-0007.

RPMI-1640 cell culture medium, Gibco;

bacterial Lipopolysaccharide (LPS), shanghai hima biotechnology limited;

tetramethyltetrazolium blue (MTT), Sigma;

dimethyl sulfoxide (DMSO), analytically pure, department of tianjin, europe chemical reagent development center;

fetal Bovine Serum (FBS), bio-engineering (shanghai) ltd;

ELISA kit, Shanghai Liaoshu Biotech Co., Ltd;

alcohol, Betai III and chemical reagents, Inc.;

PBS buffer: NaCl 8g, Na₂HPO₄·12H₂O 2.88g，KCl 0.2g，KH₂PO₄0.2g, distilled water to 1000mL, the concentration of 0.01mol/L, and the pH value is adjusted to 7.4.

Cell washing solution: EDTA-Na 0.02g, NaCl 0.8g, KCl 0.04g, distilled water to 100mL, adjusting pH to 7.8-8.0, and autoclaving at 121 ℃ for 20 minutes.

Erythrocyte lysate: tris 0.03735g, NH₄0.13g of Cl and double distilled water to 100mL, and adjusting the pH value to 7.2. Filtering and sterilizing with 0.22 μm microporous filter membrane.

The RPMI-1640 culture solution comprises ① RPMI-1640 bags, sodium pyruvate 0.11g, ampicillin 0.11g, streptomycin 0.125g, dissolved in sterilized 800mL distilled water, magnetically stirred for 1 hr, ② NaHCO₃2.0g was dissolved in 200mL of sterile distilled water ①② and mixed and stirred magnetically for 1 hour.

10% New bovine serum Eagle's MEM culture solution ① MEM 9.4g in 800mL sterile distilled water ② NaHCO₃2.0g of the extract is dissolved in 200mL of sterile distilled water, ① is mixed, the pH value is adjusted to 7.0 to 7.2,filtering and sterilizing with a 0.22 μm microporous filter membrane.

LPS solution: 100. mu.g of LPS was weighed and dissolved in 1mL of RPMI-1640 medium to prepare a 100. mu.g/mL LPS solution, which was then sterilized by filtration through a 0.22 μm microporous membrane.

1.2 instrumentation

A water bath constant temperature oscillator, a gold jar, Hengfeng apparatus factory;

TDL-5-A low-speed large-capacity centrifuge, Shanghai' an pavilion scientific instrument factory;

ultra clean bench, SW-CJ-1CU Sujing group Suzhou Antai air technology, Inc.;

MCO-17A CO₂incubator, SANYO corporation, japan;

LDZX-40BI stereoscopic automatic electric heating pressure steam sterilizer, Shanghai Shenan medical instrument factory;

AL104 electronic balance, mettler-toledo instruments (shanghai) ltd;

a liquid transfer gun (10-100. mu.L, 100-1000. mu.L, 1-5mL), Thermo;

BIO-RAD550 enzyme-labeled quantitative analyzer, BIO-RAD;

XDS-1 type B inverted microscope, COIC;

a microporous filter membrane filter with the pore diameter of 0.22 mu m, available from Jinteng laboratory instruments Co., Ltd., Tianjin;

96-well cell culture plates; 1-10mL of injector and needle; beaker, glass rod, centrifuge tube, scissors, tweezers, etc.

1.3 Experimental methods

1.3.1 preparation of the Compounds of the invention

The extracted and purified single pure compound (purity is more than or equal to 98 percent, HPLC) is respectively prepared into 800ug/L with 0.9 percent normal saline, and is diluted into proper concentration when in use.

1.3.2 extraction and culture of mouse peritoneal macrophages

Taking 10 healthy Kunming mice, dislocation and killing cervical vertebrae of the mice, soaking in 75% alcohol for 5-10min for sterilization, opening abdominal cavity of the mice to expose peritoneum under aseptic condition, and injecting about 4mL serum-free RPMI-1640 culture solution into the abdominal cavity of each mouseGently kneading abdomen with alcohol cotton for 2-3min to make it flow thoroughly, sucking abdominal cavity liquid into centrifuge tube, mixing all abdominal cavity liquids, centrifuging at 1200r/min for 5 min, discarding supernatant, re-suspending cells with RPMI-1640 culture solution containing 10% newborn calf serum, counting total cells, and adjusting cell concentration to 4 × 10 with RPMI-1640 culture solution containing 10% newborn calf serum⁵Per mL, 100. mu.L macrophage suspension was inoculated into 96-well culture plates and placed in 4% CO₂Incubation at 37 ℃ in a constant temperature incubator.

1.3.3 Experimental design grouping and cell culture Process

To examine the effect of the compounds of the present invention on normal macrophages and pathologically altered macrophages, the most commonly used bacterial Lipopolysaccharide (LPS) in immunology was used to stimulate macrophages to create pathological models of macrophages in inflammatory states. Cells cultured at 1.3.2 were grouped as follows.

Blank control group: primary macrophages were cultured in RPMI-1640 medium.

Positive control group: LPS-stimulated macrophages are cultured in LPS and RPMI-1640 medium

Drug group: the effect of each compound of the invention on primary macrophage cells and LPS-stimulated macrophages was observed separately from each single compound solution that had been prepared.

Each group of 3 multiple wells. The RPMI-1640 culture solution group was 100. mu.L of RPMI-1640 culture solution, the positive control group was 15. mu.L of LLPS, and 85. mu.L of RPMI-1640 culture solution.

Experimental groups: 100uL of each single compound solution was added, and 200uL of RPMI-1640 medium was supplemented to each replicate well, which did not reach 200 uL.

After 72 hours of cell culture, the supernatants from each well were taken for cytokine assay.

1.3.4 double antibody sandwich enzyme-linked immunosorbent assay (ELISA) is used for measuring the amount of the cell factors secreted by the macrophages in the abdominal cavity of the mouse.

The analytical procedure was as follows:

(1) and (3) diluting the standard: the original standard substance is sequentially diluted into five required standard concentrations by standard diluent in a small centrifuge tube. Wherein:

five gradient concentrations of TNF- α are 480ng/L, 240ng/L, 120ng/L, 60ng/L and 30 ng/L;

the five gradient concentrations of IL-1 β are 80ng/L, 40ng/L, 20ng/L, 10ng/L and 5 ng/L;

the five gradient concentrations of IL-6 are 1200ng/L, 600ng/L, 300ng/L, 150ng/L and 75 ng/L;

(2) loading, namely respectively arranging a blank hole, a standard hole and a sample hole to be tested, wherein the blank hole is not loaded with a sample, a biotin-labeled TNF- α antibody and streptavidin-HRP, only adding a color reagent A, B and a stop solution, the rest steps are the same, the standard hole is loaded with 50 mu L of the standard, 50 mu L of the streptavidin-HRP (biotin-HRP is not loaded because the biotin is integrated in the standard in advance), the sample hole to be tested is loaded with 40 mu L of the sample to be tested (cell culture supernatant), then 10 mu L of anti-TNF- α (IL-1 β and IL-6) antibody and 50 mu L of the streptavidin-HRP are respectively loaded, the sample is loaded at the bottom of the enzyme-labeled plate hole, the hole wall is not touched as much as possible, a sealing plate film is covered, and the sample hole is lightly shaken and evenly mixed.

(3) And (3) incubation: incubate at 37 ℃ for 60 minutes.

(4) Preparing liquid: and diluting the 30 times of concentrated washing liquid by 30 times of distilled water for later use.

(5) Washing: carefully uncovering the sealing plate film, discarding liquid, spin-drying, filling washing liquid into each hole, standing for 30 seconds, then discarding, repeating the steps for 5 times, and patting dry.

(6) Color development: 50 mu L of color developing agent A is added into each hole, 50 mu L of color developing agent B is added into each hole, the mixture is evenly mixed by gentle shaking, and the mixture is developed for 15 minutes in a dark place at 37 ℃.

(7) And (4) terminating: the reaction was stopped by adding 50. mu.L of stop solution to each well (blue color turned to yellow color).

(8) And (3) determination: the absorbance (OD value) of each well was measured sequentially at a wavelength of 450nm with the blank well being zeroed. The measurement should be performed within 10 minutes after the addition of the stop solution.

(9) And (3) analysis: and calculating a linear regression equation of the standard curve according to the concentration of the standard substance and the corresponding OD value, and calculating the corresponding sample concentration on the regression equation according to the OD value of the sample.

1.3.5 statistical analysis of the results of the experiment

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The statistical method comprises the following steps: one-way anova was performed using SPSS16.0 statistical software and the results were expressed in x ± s.

1.4 results and analysis

(1) Drawing standard curve of cytokine

Through experiments, it can be seen that the working curve equation of each cytokine is obtained by taking the OD value of the solution at 450nm as the ordinate and the concentration of the cytokine as the abscissa.

The standard working curve of TNF- α is y is 0.0046x +0.1488, the correlation coefficient r is 0.9991, and the linear range is 30-480 ng/L;

the IL-6 standard working curve is that y is 0.0032x +0.5496, the correlation coefficient r is 0.9939, and the linear range is 75-800 ng/L;

the standard working curve of IL-1 β is y 0.0471x +0.1275, the correlation coefficient r is 0.9996, and the linear range is 5-80 ng/L;

(2) morphological observation results of mouse macrophage before and after experiment

Under a microscope, as shown in figure 3, most of macrophage morphologies of abdominal cavities of mice are round and uniform in size before the compound is used, and after the compound is added for 12 hours, the macrophage morphologies are obviously changed, some cells form pseudopodia from the round shape, some cells become slender and the cell volume is increased. This phenomenon indicates that the compounds of the present invention exert an effect on macrophages.

(3) Secreted amounts of macrophage inflammatory cytokines

In order to clarify the influence of the compound of the present invention on the secretion of cytokines by macrophages, the influence of the compound of the present invention on the secretion of cytokines by macrophages in the abdominal cavity of mice was observed by further quantifying the content of cytokines in the liquid surrounding the macrophages in the abdominal cavity of mice.

As can be seen from Table 7, the secretion of TNF- α, IL-6 and IL-1 β by various drugs in the compound of the invention is slightly reduced in normal mouse abdominal cavity macrophages without LPS induction, but the difference is not obvious (p is more than 0.05) compared with a blank group, which indicates that the compound of the invention has no obvious influence on the secretion of inflammatory cytokines by macrophages in physiological states.

As can be seen from Table 8, after LPS is added to mouse abdominal macrophages induced by LPS, TNF- α, IL-6 and IL-1 β secreted by the macrophages are obviously increased (p is less than 0.05) compared with normal abdominal macrophages, which indicates that the LPS can stimulate the abdominal macrophages to have obvious effect, and the macrophages stimulated by the LPS can simulate inflammatory pathological states.

TABLE 7 Effect of the Compounds of the present invention on cytokine secretion from normal mouse peritoneal macrophages

TABLE 8 Effect of the Compounds of the present invention on secretion of cytokines by LPS-stimulated mouse peritoneal macrophages

2.5 conclusion of the experiment

The compound can inhibit the secretion of TNF- α and IL-6 to different degrees, and has significance (P is less than 0.05) compared with a positive control group, but has no influence on the mouse abdominal macrophages after being stimulated by bacterial Lipopolysaccharide (LPS). the in vitro cell experimental result is consistent with the experimental result of the mice with dyscrasia, which shows that the compound promotes the nutrition absorption of the mice with dyscrasia, improves the weight and the food intake, assists the anti-cancer drug to treat the tumor dyscrasia and is closely related with the contents of TNF- α and IL-6 under the state of dyscrasia.

Example 7

Toxicity testing of Compounds of the invention

1.1 mice acute oral toxicity test:

1.1.1 Experimental animals

The method comprises the steps of selecting 20 healthy and mature Kunming mice with the weight of 18-22g and half female and half male. 20 mice were divided into two groups of 10 mice each and 5 mice each, and each was used to determine toxicity of the compounds of the present invention. Kunming mouse, week age 6-8 weeks, weight 18-22g, Jinanpunyue laboratory animal breeding company Limited, laboratory animal production license number SCXK (lu) 2014-0007.

Detecting the environmental conditions, wherein the room temperature is 20-25 ℃, and the relative humidity ranges from 40% to 70%.

1.1.2 doses and methods of administration

One dose group was set at 20.0g/kg body weight according to the Maximum Tolerated Dose (MTD) method. Before the experiment, 20g of the sample is weighed, and 40ml of sample solution is prepared by taking 1% sodium carboxymethylcellulose as a solvent. Mice were fasted (without water) for 16 hours before gavage, and gavage was performed twice at a gavage volume of 20ml/kg body weight, with 4 hours intervals. After 2 hours of the last gastric lavage, the animals freely eat and drink water, and the poisoning and death conditions of the animals and the pathological changes of all organs are recorded.

1.1.3 Observation index

After gavage, mice were observed for general status, toxic manifestations and death. The observation period was 15 days, and the initial and final body weights and pathological conditions of the mice were recorded.

1.1.4 observations

Mouse acute oral toxicity test: the death of the mice is shown in table 9. During the experiment, no obvious toxic effect and no death of each mouse were observed. The maximum oral acute toxicity tolerance (MTD) of the compound of the invention to male and female mice is more than 20.0 g/kgBW, belonging to nontoxic grade.

TABLE 9 acute toxic death status in mice of the compounds of the invention

After the mice are subjected to the oral acute toxicity test, the general state is good, the behaviors are not obviously changed, and the mice do not die after 15 days of each dosage group. The pathological results of the organs after 15 days of observation are shown in FIG. 3. Shows that no pathological changes are found in the liver and kidney of important organs after gastric lavage, further indicates that the compound of the invention is non-toxic, and is shown in figure 4.

Example 8

Taking 1g of the compound prepared in the above examples, adding 29 g of excipient medicinal starch, mixing uniformly, granulating, tabletting, and making into 100 tablets, each tablet containing 10 mg of the compound.

Example 9 2g of the compound prepared in the above example was added to 1998 g of distilled water, and an appropriate amount of a corrigent and a preservative was added to prepare an oral liquid containing 10 mg of the compound per ml.

Claims (4)

1. A compound for use in the adjuvant treatment of tumor cachexia, characterized by the structural formula:

wherein R is₁Is OCH₃Or H

When R is₁Is OCH₃When the molecular formula is C₃₉H₄₄O₁₉

When R is₁When it is H, the molecular formula is C₃₈H₄₂O₁₈。

2. The process according to claim 1 for the preparation of a compound for the adjuvant treatment of tumor cachexia, characterized by the following steps:

collecting fresh barley seedlings, freeze-drying, crushing, extracting for 3-6 times by using 50% polar solvent aqueous solution according to the weight ratio of the raw materials to the extraction solvent of 1: 4-10 at room temperature, combining the extraction solutions, concentrating the extraction solution until the specific gravity is 1.00-1.28, adsorbing the concentrated extraction solution onto a macroporous resin column, washing with water, and then sequentially eluting with 8-50% polar solvent aqueous solution. Collecting the polar solvent aqueous solution fraction which is rich in flavonoid components and is 18-20%, further separating and purifying by using a reversed-phase preparative liquid chromatography, and detecting an effluent component at 254nm by using a 20% polar solvent aqueous solution as a mobile phase to obtain the compound.

3. Use of a compound according to claim 1 for the adjunctive treatment of tumor cachexia for the reduction of TNF- α, IL-6 levels at the tumor cachexia stage.

4. Use of a compound according to claim 1 for the adjuvant treatment of tumor cachexia to ameliorate the anorexia and wasting caused by an excessive level of TNF- α, IL-6 in the cachexia state.

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