CN109806247A

CN109806247A - The new application of Paeonol and its interior metabolism product

Info

Publication number: CN109806247A
Application number: CN201910055539.XA
Authority: CN
Inventors: 丁丽琴; 邱峰; 曹世杰; 李巍; 胡欣彤; 姜本科; 程丽娜
Original assignee: Tianjin University of Traditional Chinese Medicine
Current assignee: Tianjin University of Traditional Chinese Medicine
Priority date: 2019-01-22
Filing date: 2019-01-22
Publication date: 2019-05-28
Also published as: CN111759829A

Abstract

The invention discloses Paeonol and its new applications of interior metabolism product, more particularly to Paeonol metabolite 2-hydroxy-4-methoxyacetophenone-5-O-sulfate (M2) and 2,5-dihyfroxy-4-methoxyacetophenone (M5) is in preparation for the application in the new application in anti-tumor drug, especially treatment liver cancer, malignant mela noma.Metabolite M2 and M5 can inhibit human liver cancer cell and melanoma cell growth, especially obvious to A375 cell growth inhibition, also have apparent inhibiting effect to the growth of Hep-G2 cell.On the other hand it is preparing more particularly to Paeonol and its metabolite M1-M6 for treating the new application in diabetes medicament.10 μM of Paeonol and its metabolite M1-M6 be not toxic for 3T3-L1 cell.At this dose, Paeonol and its metabolite M1-M6 can obviously increase glucose uptake, and act on similar with positive drug.The metabolite M1-M6 of Paeonol can be used for preparing the drug for the treatment of diabetes.

Description

The new application of Paeonol and its interior metabolism product

Technical field

The present invention relates to field of medicaments, specifically the two of Paeonol and its metabolite kind new application.

Background technique

Traditional Chinese medicine is based on oral medication, and drug is absorbed after entering in vivo, is distributed, is metabolized, draining, metabolic conversion Through entire physiological disposition.In recent years, more and more researches show that, some chemical components of Chinese medicine pass through biotransformationin vivo Afterwards, the metabolite of formation also has certain bioactivity.Active metabolism is obtained by the means of enrichment, separation and synthesis Object studies metabolite, can not only mention for the rule and mechanism of action that simple or even compound are metabolized in vivo For foundation, it has also been found that lead compound, is one of the important channel for researching and developing new drug.

Liver cancer is one of global six big malignant tumours, seriously threatens human health, 85%~95% liver cirrhosis patient can Development is liver cancer.Liver cancer can be divided into primary and secondary two major classes, belong to vascular tumor, can often invade vein and turn to inside and outside liver Move, liver cancer cells to transfer inside and outside liver be the main reason for liver cancer patient is dead.Hepatocyte growth adjusts out of control and Apoptosis Mechanism inactivation, leading tumour indeterminate growth is the principal element that canceration occurs, so inhibiting liver cancer cell growth, inducing cell apoptosis It is one of effective therapeutic strategy of liver cancer.

Malignant mela noma abbreviation melanoma (malignant melanoma, MM) is increased extremely by melanoma cells Production life, majority is developed by normal mole and pigmented spots to be formed, and is mainly in skin.Although melanoma only accounts for skin neoplasin 4%, but having 80% skin neoplasin Died Patients is due to melanoma.Compared with other malignant tumours, melanin Tumor increases faster, and the death rate is higher and life span is shorter.It has now been found that light skin, hair, eyes are quick to ultraviolet light Sense, freckle is more, there is melanoma tumors family history, there are dysplastic nevus, big congenital nevus and to immunosuppressor It uses, each contributes to the formation of melanoma.

The common pathology of aggressive malignant mela noma is divided into: superficial diffusivity (accounting for about 70%), nodositas (account for about 15%), malignant freckle template and acra freckle sample melanoma type (accounting for about 15%).Most melanoma is by the shallow of epidermis Table tumour, which develops, to be formed, most of to maintain as long as the several years in horizontal growth or radial growth stage, this melanoma for being Invasive growth is had not occurred, patient can be made to reach recovery from illness by excision of performing the operation substantially.Once melanoma penetrates into very Skin tissue will be considered as " vertical " growth phase, and have the potential of transfer.

Currently, radiation and chemotherapy is still the critical treatment means of malignant tumour, but effect is treated in some malignant tumours Fruit is unsatisfactory, and side effect is obvious.Chinese traditional treatment is the characteristic treatment method in China, is had in antitumor field unique excellent Gesture, the drug that treatment malignant tumour is found from Chinese medicine should have broad application prospects.

It is also changed correspondingly with living-pattern preservation, dietary structure and eating habit with the improvement of people ' s living standards, And then causing more and more people with diabetes, diabetes have become the third-largest threat people after tumour, cardiovascular disease The great non-infective disease of class health.Having more than 3.5 hundred million people in worldwide at present undergos the torment of diabetes, disease incidence And illness rate is at increased trend year by year.Therefore, the research for treating diabetes has become one of the hot spot of whole world research.Diabetes It is the metabolic disorder as caused by the Different types of etiopathogenises such as autoimmune, heredity, metabolic organ's damage characterized by chronic hyperglycemia Disease.There are four types of be respectively as follows: (1) type 1 diabetes to diabetes at present；(2) diabetes B；(3) gestational diabetes mellitus；(4) special Different class patients with type Ⅰ DM.Wherein diabetes B (T2DM) is that the most common diabetes type and number of patients are at most most important It is a kind of.

During clinical treatment diabetes, long-term use Western medicine side effect is larger, serious to liver and kidney dysfunction.Closely In the coming year, with Traditional Chinese Medicine Chinese medicine increasing in clinical application, advantage of the traditional Chinese medicine in treating diabetes is also gradually highlighted, More and more clinical research reports point out Chinese medicine in the treatment of diabetes with the advantage function of highly significant.Therefore, from The drug that treatment diabetes are found in Chinese medicine should have broad application prospects.

Paeonol (Paeonol) is mainly from Asclepiadaceae plant paniculate swallowwort Cynanchum paniculatum (Bunge) In the root skin of Kitagawa dry root or herb and Ranunculaceae Chinese herbaceous peony platymiscium tree peony Paeonia suff ruticosa Andr. Extract a kind of active constituent separated.Paeonol is one of main active of cortex moutan, and States Pharmacopoeia specifications its contents is not It is bitter, pungent less than 1.2%, it is slightly soluble in water, there are various pharmacology works such as anti-inflammatory, antipyretic, analgesia, antibacterial, calmness, hypnosis With clinic is chiefly used in treating fever, headache, courbature, neuralgia and rheumatoid arthritis etc..

Largely about in the document report being metabolized in Paeonol body, having no the generation in vivo of Paeonol involved in the present invention The metabolite 2-hydroxy-4-methoxyacetophenone-5-O-sulfate (M2) generated after thanking, 2,5- Dihyfroxy-4-methoxyacetophenone (M5) is to human liver cancer cell Hep-G2 and malignant melanoma cell A-375 The relevant report of cell growth inhibition.Meanwhile also having no Paeonol and its interior metabolism product involved in the present invention Resacetophenone (M1), 2-hydroxy-4-methoxyacetophenone-5-O-sulfate (M2), 2- Hydroxy-4-methoxyacetophenone-5-O-glucopyranuronosid (M3), 2- Hydroxyacetophenone-4-O-glucopyranuronoside (M4), 2,5-dihyfroxy-4- The life of methoxyacetophenone (M5), 2,4,5-trihydroxyacetophenone (M6) to 3T3-L1 fat cell Length can obviously increase the relevant report of the glucose consumption of 3T3-L1 fat cell without obvious inhibiting effect.

Summary of the invention

The purpose of the present invention is to provide Paeonol and its two kinds of novel medical uses of metabolite.

The technical solution of the present invention is as follows:

Application of the Paeonol metabolite in preparation treating cancer disease medicament, the Paeonol metabolite are 2- Hydroxy-4-methoxyacetophenone-5-O-sulfate (M2) and 2,5-dihyfroxy-4- Methoxyacetophenone (M5), structural formula is as follows:

The treating cancer disease refers to inhibition growth of tumour cell.The tumour cell be human liver cancer cell Hep-G2 and People's malignant melanoma cell A-375.

The metabolite is the metabolite generated after people and big metabolism in mice.

The application of Paeonol and its six kinds of metabolites in preparation treatment diabetes medicament, the Paeonol metabolite For resacetophenone (M1), 2-hydroxy-4-methoxyacetophenone-5-O-sulfate (M2), 2- Hydroxy-4-methoxyacetophenone-5-O-glucopyranuronosid (M3), 2- Hydroxyacetophenone-4-O-glucopyranuronoside (M4), 2,5-dihyfroxy-4- Methoxyacetophenone (M5), 2,4,5-trihydroxyacetophenone (M6), structural formula is as follows:

The treatment diabetes refer to the glucose consumption for increasing 3T3-L1 fat cell.

The preparation method of the metabolite, including following operating procedure: the collection of biological sample is metabolized in biological sample The separation and purifying of product.

The biological sample is the urine of rat；The separation is separated using chromatography.

The utility model has the advantages that present invention firstly discovers that Paeonol (paeonol) and its metabolite are to 3T3-L1 fat cell Growth without apparent inhibiting effect, the glucose consumption of 3T3-L1 fat cell can be obviously increased, can be prepared for treating The drug of hypoglycemic.Paeonol metabolite M2 and M5, are tested by antitumor activity, are shown a degree of antitumor The activity of cell Hep-G2, A375 proliferation, shows stronger depression effect, can be used for preparing antineoplastic.

Detailed description of the invention

Fig. 1 is influence of the M2 to Hep-G2 cell growth inhibition；

Fig. 2 is influence of the M5 to Hep-G2 cell growth inhibition；

Fig. 3 is influence of the 5-FU to Hep-G2 cell growth inhibition；

Fig. 4 is influence of the M2 to A375 cell growth inhibition；

Fig. 5 is influence of the M5 to A375 cell growth inhibition；

Fig. 6 is influence of the 5-FU to A375 cell growth inhibition；

Fig. 7 is Paeonol and its metabolite M1-M6 to 3T3-L1 fat cell toxicity (A) and glucose consumption (B) It influences.

Specific embodiment

Essentiality content of the invention is further illustrated below with reference to embodiment.

The separation preparation of embodiment 1:M1-M6 and structural identification

The preparation method of M1-M6 is the same as preparation method reported in the literature (Isolation and identification of The metabolites of paeonol in human urine, Xenobiotica, 2012,42 (12): 1206-1212). Report is consistent in M1-M6 compound structure and document.

Proliferation inhibition test of the embodiment 2:M2 and M5 for Hep-G2

1) for trying cell line: human liver cancer cell Hep-G2, American type culture collection is derived from (ATCC,Manassas,VA,USA)

2) experimental material

DMEM culture medium is purchased from Gibco company, and fetal calf serum is purchased from Biological Industries company, tryptose Enzyme is purchased from Biosharp company, and penicillin is purchased from Northeast Pharmaceutical Factory group company Shenyang No. 1 Pharmaceutical Factory, streptomysin, and EDTA is purchased from Sigma Co., USA, DMSO (dimethyl sulfoxide), MTT (3- (4,5- dimethylthiazole -2) -2,5- diphenyltetrazolium bromide bromide: Tetramethyl azo Zo is blue).

3) instrument and equipment

Carbon dioxide constant incubator (Shanghai Li Shen Co., Ltd), Biohazard Safety Equipment (Co., Ltd, China's Haier), Set microscope (Japanese Nikon company), Milli-Q ultrapure water all-in-one machine (Merck Millipore Corp.), multi-function microplate reader (Shanghai gene Co., Ltd), micro oscillator (Fuhua Instrument Ltd., Jintan City)

4) cell culture

Human hepatoma HepG2 cell in the DMEM high glucose medium containing 10% fetal calf serum (penicillin containing 100U/ml, 10 μ g/ml streptomysins).When attached cell HepG2 grows to fine and close single layer, secondary culture is carried out.Former culture medium is discarded, is added Pancreatin, in 37 DEG C of incubation 5min.The cell suspension digested is poured into concentrator bowl, appropriate culture solution is added, with 1000r/ Min is centrifuged 5min.It discards supernatant, fresh culture medium is added later and blows and beats cell uniformly, is transferred to new Tissue Culture Flask In, it is put into 5%CO₂Constant temperature incubation under the conditions of 37 DEG C in cell incubator.

5) cell inhibitory rate detects

By the HepG2 cell in logarithmic growth phase with 7*10³The density kind of cells/well is in 96 porocyte culture plates In, after culture for 24 hours, be added various concentration metabolite M2 and M5, concentration gradient 3.125,6.25,12.5,25,50,100, 150μM.3 multiple holes of every group of setting, while negative control is set.It is placed in 5%CO₂In constant incubator, continue under the conditions of 37 DEG C After cultivating 48h, cell mortality is detected with mtt assay.

6) experimental result and conclusion

This experiment has detected the influence of M2 and M5 for hepatoma cell proliferation, and as depicted in figs. 1 and 2, MTT analyzes result card Bright M2 and M5 can significantly inhibit liver cancer cell growth, and dose dependent is presented in inhibiting rate and drug concentration.The knot of positive drug Fruit is as shown in figure 3, HepG2 inhibitory rate of cell growth is as follows: with drug concentration relationship

Different pharmaceutical is on the growth inhibiting influence of human liver cancer cell HepG2

HepG2 cell experiment is as the result is shown: the IC50 of M2 is 30.4 ± 5.49 μm of ol/L, the IC50 of M5 is 41.71 ± 6.84μmol/L。

Proliferation inhibition test of the embodiment 3:M2 and M5 for A375

1) for trying cell line: people malignant melanoma cell A-375, American type culture is derived from collection(ATCC,Manassas,VA,USA)

2) experimental material

3) instrument and equipment

4) cell culture

5) cell inhibitory rate detects

By the A375 cell in logarithmic growth phase with 3*10³The density kind of cells/well is in 96 porocyte culture plates In, culture for 24 hours after, be added various concentration metabolite M2 and M5,3.125,6.25,12.5,25,50 μM of concentration gradient.Often Group 3 multiple holes of setting, while negative control is set.It is placed in 5%CO₂In constant incubator, continue to cultivate 48h under the conditions of 37 DEG C Afterwards, cell mortality is detected with mtt assay.

6) experimental result and conclusion

This experiment has detected the influence that M2 and M5 are proliferated melanoma cells, as shown in Figure 4 and Figure 5, MTT analysis knot Fruit proves that M2 and M5 can significantly inhibit melanoma cell growth, and dose dependent is presented in inhibiting rate and drug concentration.Sun The result of property medicine is as shown in fig. 6, A375 inhibitory rate of cell growth is as follows: with drug concentration relationship

Different pharmaceutical is on the growth inhibiting influence of melanoma cells A375

A375 cell experiment is as the result is shown: the IC of M5₅₀For 24.90 ± 6.86 μm of ol/L, with negative medicine IC₅₀Value 24.03 ± 7.54 μm of ol/L are close；The IC of M2₅₀Inhibit the effect of A375 cell growth than positive drug 5- for 17.45 ± 4.96 μm of ol/L, M2 FU effect is stronger.

Embodiment 4: the influence of Paeonol and its metabolite M1-M6 to 3T3-L1 fat cell insulin resistance

1) for trying cell line: murine preadipocyte cell 3T3-L1, it is purchased from Beijing Academy of Medical Sciences Institute of Basic Medical Sciences

2) experimental material

DMEM culture medium is purchased from Gibco company, and fetal calf serum is purchased from Biological Industries company, tryptose Enzyme is purchased from Biosharp company, and penicillin is purchased from Northeast Pharmaceutical Factory group company Shenyang No. 1 Pharmaceutical Factory, and streptomysin is purchased from Yushan Hill Eastern Shandong medicine limited liability company, EDTA are purchased from Sigma Co., USA, DMSO (dimethyl sulfoxide), MTT (3- (4,5- diformazans Base thiazole -2) -2,5- diphenyltetrazolium bromide bromide: tetramethyl azo Zo is blue) it is purchased from Sigma Co., USA, Pioglitazone (PI) Purchased from Sigma company, 3-isobutyl-1-methylxanthine (IBMX) is purchased from Sigma company, and dexamethasone (Dex) is purchased from Sigma company, bovine serum albumin (BSA) are purchased from MPBIO company, and glucose oxidase method kit comes purchased from Beijing Puli Company, glucose are purchased from Tianjin great Mao chemical reagent factory, and TNF-α is purchased from Peprotech company.

3) instrument and equipment

4) 3T3-L1 PECTORAL LIMB SKELETON induction differentiation

When 3T3-L1 PECTORAL LIMB SKELETON adherent growth to confluent cultures bottle bottom of bottle, make cell contact inhibition 2 days, then into Row induction differentiation, cell culture fluid change into containing 0.5mM IBMX, 1 μM of DEX, 10 μ g/mL insulin, 3 μM of Pioglitazones (PI) With the DMEM high glucose medium of 10% fetal calf serum, it is incubated for 48h；Culture solution is changed into containing 10 μ g/mL insulin, 3 μM of ROS It is incubated for 48h again with the DMEM high glucose medium of 10% fetal calf serum；Change the DMEM high glucose medium containing 10% fetal calf serum into, It changes the liquid once within every 2 days, until 80% or more cell differentiation is mature.

5) TNF-α induction 3T3-L1 cell establishes insulin resistant model

The 3T3-L1 cell inoculation of logarithmic growth phase sets up 4 groups, is respectively: blank pair in 96 well culture plates According to group, blank control adds insulin group, and model group and model add insulin group.Cell induce differentiation into it is ripe after, culture solution is changed For the plasma-free DMEM medium containing 0.5%BSA, 5%CO₂37 ° of incubation 8h of incubator；The culture solution of blank control group is changed For the DMEM high glucose medium containing 10% fetal calf serum, model group, which is changed to, to be added 10ng/mL TNF-α and contains 10% tire ox The culture medium of the DMEM high glucose medium of serum, blank control group and model group is primary per replacement for 24 hours, after 96h, discards original training Support base；Blank control adds insulin group and model that insulin group is added to be changed to addition containing 100nM insulin, and 10% fetal calf serum is low Sugared DMEM culture medium.Blank control group and model group are changed to 10% fetal calf serum low sugar DMEM culture medium, draw supernatant after 15min Liquid, with glucose content in glucose oxidase method kit detection supernatant.

6) drug hypoglycemic activity screens

The 3T3-L1 cell for having had built up insulin resistant model is taken, the drug that concentration is 10 μM is acted in TNF- ɑ It is last be added in culture medium for 24 hours, draw supernatant as sample glucose oxidase method kit detection.After drawing supernatant The MTT solution of 100 μ L 5mg/mL, 5%CO is added in cell per well₂37 DEG C of incubation 2.5h, 150 μ L DMSO dissolutions of incubator, shake It swings 10 minutes, light absorption value is measured at 490nm.Survival rate is calculated by this formula:

Cell viability (%)=[(A490, sample-A490, Blank)]/[(A490, control-A490, Blank)]×100

7) statistical analysis

It tests independent repetitive operation at least 3 times, experimental result is expressed as average value ± SD.Experimental result is counted using SPSS It learns software and carries out one-way analysis of variance (ANOVA).P < 0.05 is considered to have statistical significance, * P < 0.05, * * P < 0.01, compared with model group (M).

8) experimental result and discussion

Shown in experimental result such as Fig. 7 (A), 10 μM of Paeonol and its metabolite M1-M6 for 3T3-L1 cell not It is toxic.At this dose, as shown in Fig. 7 (B), Paeonol and its metabolite M1-M6 can obviously increase glucose and take the photograph It takes, and acts on similar with positive drug.

Claims

1. application of the Paeonol metabolite in preparation treating cancer disease medicament, which is characterized in that the root bark of tree peony phenol metabolism Product is 2-hydroxy-4-methoxyacetophenone-5-O-sulfate, is denoted as M2 and 2,5-dihyfroxy-4- Methoxyacetophenone is denoted as M5, and structural formula difference is as follows:

。

2. application of the Paeonol metabolite in preparation treating cancer disease medicament, feature exist according to claim 1 In the treating cancer disease refers to inhibition growth of tumour cell.

3. application of the Paeonol metabolite in preparation treating cancer disease medicament, feature exist according to claim 2 In the tumour cell is human liver cancer cell Hep-G2 and people's malignant melanoma cell A-375.

4. Paeonol metabolite described in -3 any one claims is in preparation treating cancer disease medicament according to claim 1 In application, which is characterized in that the metabolite is the metabolite generated after people and big metabolism in mice.

5. the application of Paeonol and its six kinds of metabolites in preparation treatment diabetes medicament, which is characterized in that the root bark of tree peony Phenol metabolism product is resacetophenone, is denoted as M1,2-hydroxy-4-methoxyacetophenone-5-O- Sulfate is denoted as M2, and 2-hydroxy-4-methoxyacetophenone-5-O-glucopyranuronosid is denoted as M3, 2-hydroxyacetophenone-4-O-glucopyranuronoside is denoted as M4, and 2,5-dihyfroxy-4- Methoxyacetophenone is denoted as M5, and 2,4,5-trihydroxyacetophenone are denoted as M6, and Paeonol and its metabolism produce Object structural formula difference is as follows:

。

6. the application of Paeonol and its six kinds of metabolites in preparation treatment diabetes medicament according to claim 5, It is characterized in that, the treatment diabetes refer to the glucose consumption for increasing 3T3-L1 fat cell.

7. treating glycosuria in preparation according to Paeonol described in claim 5-6 any one claim and its six kinds of metabolites Application in medicine, which is characterized in that the metabolite is the metabolite generated after people and big metabolism in mice.