CN109320570A - A kind of icariside I class compound, derivative, officinal salt and application - Google Patents
A kind of icariside I class compound, derivative, officinal salt and application Download PDFInfo
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Abstract
Present disclose provides a kind of icariside I class compound and derivative and officinal salt, structural formula are shown in formula I:
Description
Technical field
The present invention relates to a kind of icariside I class compounds and derivative and its officinal salt, such compound to exist
Prevent and treat the purposes in terms of tumour.
Background technique
The single compound of the principle active component of natural products is as chemicals itself or the guideization of medicament research and development
Closing object is one of the important research method that medicament research and development personnel is engaged in chemicals research and development for many years, the excessive sheep of flavonoid glycoside compound
Leaves of pulse plants glycosides also has become a hot topic of research.Show that icariin has antitumor efficacy, bone strengthening, adjusts immune function, improves angiocarpy
Function, anti-aging and anti-oxidation function, the antitoxin sick physiological activity such as ability and enhancing sexual function of enhancing.
Herba Epimedii is traditional Chinese medicine simply, is used for being to use excessive sheep under instruction of Chinese Medicine theory in the prior art
The decoction of leaves of pulse plants plant, infusing drugs in wine, cook paste, pill dissipate etc..
Medicament categories currently used for treating liver cancer are various, and either Chinese patent drug class is right there are also Chinese and Western medicine composition of medicine class
The state of an illness of patient can only play the role of alleviation, and dosage is big, and the psychology and physiology to patient all cause serious burden,
And the toxic side effect of the anti-tumor drug listed at present is big, once being discontinued, the state of an illness of patient will be aggravated, be made to patient
At serious financial burden.
Summary of the invention
The mixture extracted from longspur epimedium medicinal herbs can also reduce the risk (U.S.20030170292) of cancer.However, still
Which ingredient in indefinite longspur epimedium medicinal herbs may have this function, the antitumous effect of that ingredient in Epimedium plant
It is more satisfactory, and toxic side effect is small, needs a large amount of innovative works, and difficulty is very big.
Inventor is by the research of the natural constituent to Herba Epimedii, while inventor's long campaigns biosynthesis
Research, discovery icariin/element obtain that synthesising biological effect is more preferable, economic benefit is higher excessive by the method for bio-enzyme engineering
The sheep leaves of pulse plants time I analog derivative of glycosides.It can be used for preparing prevention or treat the drug of tumour.
Technical solution is as follows:
A kind of icariside I class compound and derivative and officinal salt, structural formula are shown in formula I:
Wherein,
1)R1Selected from H, OH, OCH3、OC2H5、OCH(CH3)2、CH3COO、NH2、CH3NH、(CH3)2N、CH3CONH or CN；
2)R2Selected from H, OH, OCH3、OC2H5、OCH(CH3)2、OC3H7、CH3COO、CH3、CF3、C1-C6-NH2、NH2、
CH3NH、(CH3)2N、(CH3CH2)2N、CH3CONH, CN, Br, Cl, F, amino acid acyl, amino acid acylamino-, oligopeptides acyl group or
Oligopeptides acylamino-；Wherein, the C1-C6-NH2C1-C6For with 1-6 C alkyl, naphthenic base, alkylene, cycloalkenyl group,
Clopentylamino, Cyclohexylamino, morpholine base or methyl piperazine base；
3)R3Selected from OH, OCH3、OC2H5、OCH(CH3)2、OC3H7、CH3COO、CH3、CF3、C1-C6-NH2、NH2、CH3NH、
(CH3)2N、(CH3CH2)2N、CH3CONH, CN, Br, Cl, F, amino acid acyl, amino acid acylamino-, oligopeptides acyl group or oligopeptides acyl
Amino；Wherein, the C1-C6-NH2C1-C6For the alkyl with 1-6 C, naphthenic base, alkylene, cycloalkenyl group, cyclopenta
Amino, Cyclohexylamino, morpholine base, methyl piperazine base；
4)R4Selected from H or prenyl；
5)R5Selected from H, OH, OCH3、OC2H5Or OC3H7；
6)R6Selected from H, OH, OCH3、OC2H5Or OC3H7。
Further preferably, R1For OH, OCH3, OCH (CH3)2Or OCH2CH3；R2For OH, OCH3Or OC3H7；R3For OH, OCH3,
OCH(CH3)2Or OCH2CH3；R4ForR5、R6For H.
Preferably, icariside I class compound, derivative, officinal salt structural formula can be as follows
One kind.
In addition, present disclose provides a kind of composition, comprising aforementioned icariside I class compound and derivative and can
Pharmaceutical salts also include pharmaceutically acceptable carrier, excipient, diluent, medium or their combination.
Aforesaid compound, derivative, officinal salt or composition, which are used to prepare drug, can be used for preventing, mitigates or treat swollen
Tumor disease.The tumour include liver cancer, lung cancer, colon cancer, oophoroma, cholangiocarcinoma, malignant lymphatic tumor, liver cancer, bladder cancer and
Prostate cancer.
Icariside I class compound is improving human immunity, is improving human body IFN-γ level, upregulation of apoptosis albumen NOXA
Expression etc. there is significant effect.The present invention by mouse experiment in vivo, (H22 cell and hepg2 are thin for liver cancer cells
Born of the same parents) experiment, it was demonstrated that icariside I can significantly inhibit the growth of tumour.
Detailed description of the invention
Fig. 1 is mouse tumor volume change curve graph described in embodiment 13；
Fig. 2 is icariside I described in embodiment 13 to S180 transplantable tumor inhibitory effect histogram；
Fig. 3 is mouse tumor volume change curve graph described in embodiment 14；
Fig. 4 is mouse tumor volume change curve graph described in embodiment 15；
Fig. 5 is mouse tumor volume change curve graph described in embodiment 16；
Fig. 6 is described in embodiment 17 in 5 times of CD3+In T cell, Icariside I handles 48h pairs of T cell and HepG2 cell
The proliferation inhibiting effect of liver cancer cells；
Fig. 7 is described in embodiment 17 in 5 times of CD3+In T cell, Icariside I handles 48h pairs of T cell and Huh7 cell
The proliferation inhibiting effect of liver cancer cells；
Fig. 8 is described in embodiment 17 in 10 times of CD3+In T cell, Icariside I handles T cell and HepG2 cell 48h
To the proliferation inhibiting effect of liver cancer cells；
Fig. 9 is described in embodiment 17 in 10 times of CD3+In T cell, Icariside I handles 48h pairs of T cell and Huh7 cell
The proliferation inhibiting effect of liver cancer cells.
Specific embodiment
Embodiment 1: taking 100mg icariside I, and the DMSO of 2mL, 30mg iodomethane (CH is added3I), 10mg is added
Silver oxide (Ag2O), stirred 4 hours at 40 DEG C.Reaction solution is poured into distilled water, yellow solid will be precipitated after allowing and filter, dry
It is dry, purified with column chromatography method to get R is arrived1Base is the product of methoxyl group.
Embodiment 2: taking 100mg icariside I, and the DMSO of 2mL, 100mg iodomethane (CH is added3I), 10mg is added
Silver oxide (Ag2O), stirred 12 hours at 25 DEG C.Reaction solution is poured into distilled water, yellow solid will be precipitated after allowing and filter, dry
It is dry, purified with column chromatography method to get R is arrived3And R1Base is the product of methoxyl group.
Embodiment 3: taking 100mg icariside I, and the DMSO of 2ml is added, and 50mg dimethyl suflfate is small in 80 DEG C of stirrings 2
When.Reaction solution to be poured into distilled water, yellow solid will be precipitated after allowing and filter, drying is purified with column chromatography method (silicagel column),
Obtain R3Base is the product of methoxyl group.Required R3Base is the icariside class compound of methoxyl group.
Embodiment 4: taking the icariside I of 100mg, is added 5ml ethyl alcohol (95%) and 30mg dithyl sulfate, then plus
Enter 15mg sodium hydroxide.It is stirred 5 hours at 40 DEG C.After the reaction was completed, 100ml water is added.Solid will be precipitated, crude product is with 95%
After ethyl alcohol dissolution, purified with column chromatography method, it can obtain required R1Base is the icariside I class compound of ethyl.
Embodiment 5: taking the icariside I of 100mg, is added 5ml ethyl alcohol (95%) and 100mg dithyl sulfate, then plus
Enter 15mg sodium hydroxide.It is stirred at room temperature 5 hours.After the reaction was completed, 100mL water is added.Solid will be precipitated, crude product is used
After the dissolution of 95% ethyl alcohol, purified with column chromatography method, it can obtain required R1And R3Base is the icariside I class of ethyl
Compound.
Embodiment 6: taking 100mg icariside I, and the tert-butyl alcohol and 30mg dithyl sulfate containing 5% water of 5ml is added.
It is stirred at room temperature 10 hours.After the reaction was completed, 20ml water is added.Crude product is dissolved with ethyl alcohol, then is purified with column chromatography method,
Obtain R1Base is the modified outcome of ethyl.
Embodiment 7: taking 100mg icariside I, and the isopropanol and 30mg sulfuric acid diisopropyl containing 5% water of 5ml is added
Ester.It is stirred 6 hours at 40 DEG C.After the reaction was completed, 20ml water is added, then uses extracted by ether.Ether layer is dry with anhydrous sodium sulfate.
After removing ether, crude product is dissolved with ethyl alcohol, then is purified with column chromatography method to get R is arrived1Base is that the modification of isopropoxy produces
Object.
Embodiment 8: taking 100mg icariside I, and the isopropanol and 100mg sulfuric acid diisopropyl containing 5% water of 5ml is added
Ester.It is stirred at room temperature 10 hours.After the reaction was completed, 20ml water is added, then uses extracted by ether.Ether layer is dry with anhydrous sodium sulfate
It is dry.After removing ether, crude product is dissolved with ethyl alcohol, then is purified with column chromatography method to get R is arrived1And R3Base is repairing for isopropoxy
Adorn product.
Embodiment 9: icariside I anti-tumor experiment preparation of reagents
1%Tween-80 solution: accurately drawing 1mLTween-80, and appropriate physiological saline is added and is settled to 100mL, mixes,
It needs ready-to-use.
Injection icariside I suspension (zoopery use): general according to the administration concentration of each group mouse, mouse
Weight and the quantity of mouse, determine the amount of icariside I, then accurately weigh the icariside I powder of fixed amount, then
The 1%Tween-80 of fixed volume is added, ultrasonic 30min is at uniform suspension.
Injection A Kela determines suspension (zoopery use): according to the administration concentration of mouse, mouse approximate weight and
The quantity of mouse, determines the fixed amount of A Kela, and the A Kela for then accurately weighing fixed amount determines powder, adds fixed volume
1%Tween-80, ultrasonic 30min is at uniform suspension.
Icariside I 10mM liquid storage (cell experiment use): it accurately weighs 1mg icariside I powder and is managed in brown EP
In, 186.85mL DMSO dissolution is added, after mixing well dissolution, Seal and preservation is in -20 DEG C of refrigerators.
A Kelading 10mM liquid storage (cell experiment use): accurately weighing 1mg A Kelading powder in EP pipe, is added appropriate
DMSO dissolution, after mixing well dissolution, Seal and preservation is in -20 DEG C of refrigerators.
75% ethanol solution (tap the abdomen use): it is accurate to measure 225mL dehydrated alcohol, 75mL deionized water is added, sufficiently
It mixes.
10 × PBS buffer salt solution: a certain amount of KCl, NaCl, Na are accurately weighed2HPO4、KH2PO4It is placed in a beaker, so
After suitable deionized water is added, pH value of solution is adjusted to 7.4 using pH meter.
1 × PBS buffer salt solution liquid (is used) for diluting cells subcutaneous injection: accurately measure suitable 10 × PBS, and plus
Enter deionized water be diluted to 1 ×, then adjust pH to 7.4 spare.
Embodiment 10: upper laxative remedy (UDP) surveys the acute toxicity test of icariside I
Upper laxative remedy (UDP) is the acute poison of a kind of substitution tradition that the Organization of Economy and Cooperation Development revised concurrent cloth in 2001
Property test new method, since upper laxative remedy has significant advantage, people generally have an optimistic view of its development prospect, through by FDA and
EPA is approved as the standard technique of acute toxicity testing and highly recommends to use.The maximum administration that acute toxicity testing uses
Concentration is 2000mg/kg, and the series sigma factor selected is 0.5, and the mouse administration concentration ratio between different groups is determined as 3.2.
The setting of the administration concentration of mouse is 1.75,5.5,17.5,55,175,550,2000mg/ respectively in experimentation
Kg, administration mode is intraperitoneal injection, wherein initial dosage is 175mg/kg.If the mouse under initial administration concentration
Survival, then the 2nd mouse gives 550mg/kg；If the dead mouse or dying under initial administration concentration, the 2nd mouse
Give 55mg/kg.Two mouse administration time intervals are greater than 48h when being administered every time in the process, then conscientiously observe animal
Response situation and record.Main pathology and physiological change are observed, the various actions Novel presentation for recording mouse (can
Compared with the control mice of non-administration).
Dosage and animal survival information input AOT425StatPgm, (the upper laxative remedy that is used for of U.S. EPA exploitation is calculated
LD50Software) after software, the termination of the next mouse dosage that should be administered and experiment is determined according to the prompt of software.Entirely
AOT425StatPgm software calculates the LD of icariside I automatically after acute toxicity testing50And 95% fiducial limit.
Icariside I is configured to suspension by 1% Tween-80 in experiment, and initial dose concentration is 175mg/kg,
Animal current status is observed after administration immediately, and records experimental result, as shown in table 1.After administration record short term toxicity (48h) and
Long term toxicity (14d) calculates the LD50 of icariside I, such as 2 institute of table in record result input AOT425StatPgm software
Show.
Table 1: acute toxicity observes table (dosage mg/kg)
Table 2:LD50 calculated result (dosage mg/kg)
2 result of table is input in AOT425StatPgm software, show that the median lethal dose LD50 of icariside I is big
In 2000mg/kg.
In summary, there may be certain deviations for the LD50 value of the icariside I measured with the method, but can consider
Under test dose of the invention, icariside I has no toxic side effect.
The recovery of embodiment 11:S180 source of mouse sarcoma cell strain and ascites passage
Referring to classical ascites tumor modeling method, source of mouse euphorbia egg decoctum cell is taken out from liquid nitrogen container, is immediately placed in 37 DEG C
Melt in water-bath in one minute, is transferred quickly to have been added to containing 20% by cell in the gnotobasis of superclean bench
In the 15mL centrifuge tube of 1640 complete mediums of fetal calf serum, it is centrifuged 5 minutes under the speed conditions of 1000r/min, gently
It discards supernatant.Then 1640 culture medium of 3mL is added, cell is resuspended, cell is transferred in 10 milliliters of culture dishes, 37 DEG C is placed in and contains
5% CO2It is cultivated 24 hours in cell incubator.It cultivates into the centrifuge tube that cell is transferred to 15mL after 24 hours, with
The revolving speed of 1000r/min is centrifuged 5 minutes, and 1640 culture mediums of appropriate serum-free are added and count to cell.
Cell is configured to 3 × 107The cell suspension of a/mL is injected cell suspension by way of intraperitoneal injection small
In mouse body, the volume of every mouse injection cell suspension is 0.2mL.Observe that mouse has obvious abdominal distension after 7 days, by mouse with neck
Vertebra dislocation method is put to death, the mouse of execution is completely disposed in 75% alcohol after impregnating 10min-15min mouse is placed in it is ultra-clean
Mouse skin is carefully cut off using curved scissors and curved tweezer in platform, exposure mouse peritoneum makes mouse lie on one's side convenient for ascites concentration, uses 10mL
Asepsis injector collocation 1mL syringe needle, extracts ascites (the tapped the abdomen amount of every mouse is about 4mL), ascites is placed in centrifuge tube
In with 1000r/min centrifugation 5 minutes, abandon supernatant, appropriate 1640 culture medium of serum-free is added, blows even cell and counts, be configured to 3
×107The cell suspension of a/mL, every mouse peritoneal inject 0.2mL cell suspension, give mouse interior generation within every 6 to 7 days later
Once, stable passage system is formed.
The foundation of embodiment 12:S180 sarcoma model
The purchase and raising of experimental animal: size is bought 3-4 weeks in Zhongshan University's Experimental Animal Center and weight is in 18-
SPF grade female and male C 57 BL/6 J mouse between 22g, and raised and tested in SPF grades of laboratories.Wherein raise
The relative humidity of environment is 55%, and temperature is (25 ± 1) DEG C, ad lib.
The foundation of S180 sarcoma model: being put to death the ascites tumor mouse for stablizing passage system is formed with cervical dislocation,
10min-15min is impregnated in containing the beaker in 75% alcohol, then mouse is placed in super-clean bench using curved scissors
Mouse skin is carefully cut off with curved tweezer, exposure mouse peritoneum extracts ascites, by abdomen with 10mL asepsis injector collocation 1mL syringe needle
Water is placed in centrifuge tube with 1000r/min centrifugation 5 minutes, abandons supernatant, appropriate 1640 culture medium of serum-free is added, blows even cell simultaneously
It counts, is configured to 3 × 107The cell suspension of a/mL, every mouse peritoneal inject 0.2mL cell suspension, every 3-4 weeks
200 μ L cell suspension of C57BL/6J mouse inoculation is subcutaneous in mouse forelimb right axillary.
It is raised 1-2 weeks after inoculation cancer cell, observes the major diameter that mouse situation utilizes vernier caliper to measure tumour simultaneously daily
With minor axis and perform record, the volume of solid tumor is calculated, until tumour grows to about 100mm3When (general 3~5 days) at random point
Group, and tumour original size is marked and records, carry out anti-tumor experiment administration.
Embodiment 13: icariside I inhibits S180 sarcoma cell growth result
The tumor model made is randomly divided into 8 groups: control group (control), icariside I 1mg/kg group, excessive sheep
Then the leaves of pulse plants time glycosides I 5mg/kg group, cyclophosphamide 25mg/kg (CTX) positive controls, every group of 6 mouse half male and half females divide cage
Label, successive administration 9 days.
Start to be administered after mouse modeling success, tumor volume change situation during record administration daily, as shown in Figure 1,
The strain of S180 sarcoma cell can subcutaneously form tumor mass in C57BL/6 mouse, and control group tumour can be in C57BL/6 Mice Body
Interior continued propagation illustrates being successfully established for mouse S180 lotus knurl model.Cyclophosphamide resists swollen as common positive control drug
Tumor effect is clearly.
Control group (control) tumour keeps continued propagation it can be seen from Fig. 1 mouse tumor volume change data,
(9 days) gross tumor volume is about 700mm at the end of experiment3.And the gross tumor volume of cyclophosphamide group and icariside I group is from the 3rd
Its beginning just has significant difference with control group, and tumor growth rate is slower than control group, and when terminal is administered, gross tumor volume is only
For 300mm3Left and right illustrates that compound icariside I has certain antitumor action in C57BL/6 Mice Body.
It weighs to tumor tissues are stripped after mouse dissection, as a result as shown in Fig. 2 tumor weight.By female mice tumour
Cyclophosphamide group tumour inhibiting rate known to weight chart 2 is 62.8%, and icariside I (5mg/kg) in the case where high dose is shown
Preferable antitumous effect, tumour inhibiting rate 74.9%, icariside I low concentration group (1mg/kg) tumor killing effect is slightly weak, tumor suppression
Rate is 56.5%.The result shows that compound icariside I is small in C57BL/6 to the plastidogenetic xenograft tumor of euphorbia egg decoctum
Apparent inhibiting effect is shown in mouse body.Icariside I under the dosage of 5mg/kg can significant mechanism euphorbia egg decoctum it is thin
The growth of born of the same parents.
Embodiment 14: influence of the icariside I to mouse weight and main organs
Mouse growth state is observed during administration daily and weighs every mouse weight, every group of mouse weight variation such as Fig. 3
It is shown.Since (all female mice average weight 19g or so, male before testing there are larger difference for male and female mouse weight itself
Average mice body weight 24g or so), thus when experimental endpoints male and female mouse weight gap it is larger.From statistical result as can be seen that
Mouse basic activity during administration is acted normally, wherein female tumor-bearing mice the 1st to 3 day weight at administration (5mg/kg)
It will appear decline, icariside I administration group is consistent with control group mice weight trend behind third day, reaches normal level；And
Cyclophosphamide group female mice weight was continued to decline since administration the 3rd day, and mouse is obviously thin, food-intake also compared with control group and
Icariside I group mouse is few.The food-intake for illustrating Effect of cyclophosphamide mouse has obvious toxicity to mouse, and excessive sheep
Toxicity of the leaves of pulse plants time glycosides I at administration initial stage has certain irritation, but the small toxicity of icariside I long term administration, administration front and back
Mouse weight is basically unchanged or is increased slightly.
For male tumor-bearing mice, icariside I administration group is consistent with control group mice weight trend, the excessive sheep of compound
The leaves of pulse plants time glycosides I does not influence mouse weight substantially；And cyclophosphamide group mouse weight was continued to decline since administration the 5th day.It says
Bright cyclophosphamide has certain toxicity to mouse, and compound icariside I is smaller to the toxicity of mouse, has little influence on small
Mouse food-intake and weight.
Embodiment 15: influence of the icariside I to S180 tumor formation rate in mouse
Mouse 18 with the certification of fitness are bought, mouse is divided into 3 groups: control group (control), inoculation before being inoculated with
Icariside I 5mg/kg group before preceding icariside I 1mg/kg group, inoculation, every group of 6 mouse half male and half females, then divides
Cage label successive administration 9 days, situations such as observing mouse appetite, defecation, urine, activity during administration daily, is administered daily preceding title
Amount mouse weight simultaneously records, and it is as shown in Figure 4 to draw the changes of weight curve being administered before mouse inoculation.Stop after being administered to mouse
S180 cell is subcutaneously injected in right axillary, and cell concentration is 3 × 106A/mL, observes tumor presence daily after modeling.
Slightly have from the weight of mouse under the dosage that the changes of weight curve of mouse can be seen that 5mg/kg in experiment periods
Fluctuation, and the other mouse weight of 1mg/kg group is basically unchanged.
Do not grow tumour in experiment after all mouse inoculation S180 cells, female mice and male mouse are under all dosages
Modeling success rate is 0%, the reason is that icariside I can significantly reduce S180 in the intracorporal tumor formation of mouse.The present invention can
To obtain, icariside I has significant potentiality in anti-tumor aspect.
Embodiment 16: influence of the icariside I to mouse H22 liver cancer growth
Start to be administered after mouse modeling success, measures and records volume of tumor mass during experiment daily, as shown in figure 5,
S180 sarcoma cell can subcutaneously form tumor mass in C57BL/6J mouse, and control group (control) tumour keeps continued propagation, and
Cyclophosphamide group, icariside I administration group (CTX, 25mg/kg) and A Kela determine the tumour body of group (ICT, 17.5mg/kg)
Product just has significant difference since administration the 3rd day with control group, and shows to continue inhibiting effect.It is compareed in experimental endpoints
Group (control), cyclophosphamide group, A Kela determine group, icariside I 1mg/kg group, icariside I 2.5mg/kg,
Icariside I 3.75mg/kg group, icariside I 5mg/kg group gross tumor volume average value be respectively as follows: 1431.7mm3,
602.4mm3,618.1mm3,1253.8mm3,1133.4mm3,920.5mm3,771.2mm3.Compound group gross tumor volume with it is right
There is apparent anti-H22 liver cancer effect according to, there are significant difference, compound icariside I is illustrated in vivo between group.
Can be seen that icariside I from tumor volume change data can inhibit Mice Body under all dosages
The speed of growth of interior H22 xenograft tumor, middle and high concentration group (5mg/kg) effect is suitable with positive drug group, illustrates Herba Epimedii
Glycosides I is able to suppress the speed of growth of H22 xenograft tumor in C57BL/6J Mice Body, wherein 3.75mg/kg and 5mg/kg group
Tumor killing effect is significant.
Embodiment 17: the antitumous effect of icariside I on a cellular level
01., which tests common solution, prepares
011.1 × PBS buffer salt solution liquid (for preparing MTT solution): appropriate 10 × PBS is accurately measured, and is added and goes
Ionized water is diluted to 1 ×, then adjust pH to 7.4 spare.
012.2.5mg/mL the MTT powder of 125mg, room MTT solution: are added into the centrifuge tube for filling 1 × PBS of 50mL
It is filtered after its all dissolution within temperature ultrasound 15 minutes, is placed in 4 degree of refrigerators and is protected from light storage no more than 7 days.
013.uptake buffer (intake buffer: 60%ethanol, 0.3M HCl): accurate measurement 360mL is anhydrous
Ethyl alcohol is placed in a beaker, and adds 15mL concentrated hydrochloric acid, and finally plus 225mL deionized water is settled to 600mL.
Recovery, culture and the passage of 02. tumour cell
Tumour cell HepG2 and Huh7 (source of people liver cancer cells) attached cell is cultivated, using containing 10% fetal calf serum
DMEM high glucose medium is cultivated.Cell recovery step is with the method in embodiment 3, when cell covers with, is disappeared with trypsase
Change method carries out secondary culture.
The culture of 03. immunocyte
The CD3 of this experiment+T cell is purchased from Guangzhou Randt Biotechnology Co., Ltd, is derived from fresh blood preparations, adopts
Separation preparation is carried out with immunomagnetic beads method, guarantees the stabilization of cell quality between different batches, while having also set up stringent cell
Identity process, provided primary cell are in good condition.CD3+T cell form is small, belongs to suspension cell, using containing 20%
1640 FBS and 1 percent dual anti-high glucose medium cultures.CD3+The general current existing separation of T cell, which is not reused, not to be passed
Generation, and must assure that blood is fresh, the CD3 of fresh separated+T carries out downstream experiment immediately.
04.CCK8 method detects the hepatoma cell proliferation Inhibition test under the conditions of T cell mixed culture
041. fishplate bar: collecting logarithmic phase liver cancer cells (HepG2 and Huh7), is made under inverted microscope using tally counting
The concentration of cell liquid is 5 × 105/ mL, and zeroing hole, control wells and sample well are set, every group of 6 multiple holes.HepG2 and Huh7
It is seeded in 96 porocyte culture plates respectively, every hole cell volume is 0.1mL, is placed in containing 5%CO2Cell incubator in cultivate
6h keeps cell adherent；
042. plus CD3+T cell, dosing: 6h is after liver cancer cells are adherent for culture, and every hole adds in addition to zeroing hole and control wells
The T cell for entering 50 5 times or 10 times quantity of μ L, adds the compound for the various concentration that 50 μ L are prepared with culture medium, and institute is porose
Final system is all 0.2mL, is put into cell incubator and cultivates 24 hours and 48 hours respectively；
043. plus CCK8: the porose cells and supernatant of institute is collected by centrifugation, is placed in -80 DEG C of refrigerators for IFN-γ content
Detection；Tissue culture plate is cleaned twice with sterile phosphate buffer, the new of 0.1mL is then added in each hole
After the CCK8 of fresh culture medium and 0.01mL, it is put into containing 5%CO2Cell incubator in be incubated for 1~4 hour；
044. detection: after the completion of incubation, the OD value at 450nm is measured.Cell proliferation inhibition rate=1- (ASample-AZeroing)/
(AControl-AZeroing) × 100% or cell survival rate=(ASample-AZeroing)/(AControl-AZeroing) × 100%；
045. analysis result: with cell proliferation inhibition rate or survival rate to drug concentration mapping analysis in CD3+T cell with
Inhibited proliferation of the icariside I to liver cancer cells under conditions of liver cancer cells mixed culture.
Inhibited proliferation of the 05. detection icariside I to liver cancer cells
It is tested using CCK8 in CD3+Detection icariside I is to liver under conditions of T cell and liver cancer cells mixed culture
The inhibited proliferation of cancer cell.The CD3 of 5 times and 10 times quantity is added in HepG2 and Huh7+T cell, then with different dense
After the icariside I of degree is handled cell 48 hours, collects cells and supernatant packing and freeze in -80 DEG C for subsequent ELISA
Detect the content of IFN-γ.96 orifice plates of supernatant are removed, every hole is washed 2 times with the 200 sterile 1 × PBS of μ L, then 100 μ are added in every hole
L fresh culture and 10 μ L CCK8 continue to detect the absorbance value at 450nm by microplate reader after being incubated for 1-4 hours, finally
It is calculated by GraphPad Prism 5 and maps to obtain icariside I in CD3+What T cell and liver cancer cells were mixed
Under the conditions of to the proliferation inhibiting effects of liver cancer cells.
(1) CD3 of 5 times of quantity is added in HepG2 and Huh7+Then T cell uses the icariside I of various concentration
After processing cell 48 hours, icariside I is to the proliferation inhibiting effect of HePG2 liver cancer cells as shown in fig. 6, icariside
The proliferation inhibiting effect of I pair of Huh7 liver cancer cells is as shown in Figure 7.
It is separately added into the icariside I processing cell of various concentration, the light absorption at 450nm is read after culture 48 hours
Value, according to formula cell survival rate=(ASample-AZeroing)/(AControl-AZeroingIcariside I is calculated different dense in) × 100%
For the survival rate of HepG2 cell and Huh7 cell under degree.As shown in Figure 6 and Figure 7, in CD3+T cell and liver cancer cells are with number
Under conditions of amount is than 5:1 mixed culture, icariside I all significantly inhibits the growing multiplication of two kinds of cells,
And show a degree of concentration dependent.Wherein, to the growth inhibition effect of HepG2 cell 8 μM of concentrations above the most
Obviously, average inhibition reaches 50.6%；It is the most obvious in 16 μM of concentrations above to the growth inhibition effect of Huh7 cell, it is average
Inhibiting rate reaches 65.6%.It is close to the inhibiting effect and immune system of tumour cell that this also further illustrates icariside I
Cut phase is closed.
(2) CD3 of 10 times of quantity is added in HepG2 and Huh7+Then T cell uses the icariside I of various concentration
After processing cell 48 hours, icariside I is to the proliferation inhibiting effect of HePG2 liver cancer cells as shown in figure 8, icariside
The proliferation inhibiting effect of I pair of Huh7 liver cancer cells is as shown in Figure 9.
Icariside I is as shown in Figure 8 and Figure 9 for the survival rate of HepG2 cell and Huh7 cell under various concentration,
In CD3+Under conditions of T cell and liver cancer cells are mixed with quantity ratio 5:1, growth of the icariside I to HepG2 cell
Proliferation significantly inhibits, and the most obvious in 16 μM of concentrations above, average inhibition reaches 49.8%；And all dense
It is unobvious to the growth inhibition effect of Huh7 cell under degree, therefore subsequent experimental uses CD3+T cell and liver cancer cells are with quantity ratio
The condition of 5:1 mixed culture carries out, while also illustrating that icariside I is to play antitumor action by immune system.
Claims (6)
1. a kind of icariside I class compound and derivative and officinal salt, which is characterized in that its structural formula such as Formulas I institute
Show:
Wherein,
1)R1Selected from H, OH, OCH3、OC2H5、OCH(CH3)2、CH3COO、NH2、CH3NH、(CH3)2N、CH3CONH or CN；
2)R2Selected from H, OH, OCH3、OC2H5、OCH(CH3)2、OC3H7、CH3COO、CH3、CF3、C1-C6-NH2、NH2、CH3NH、
(CH3)2N、(CH3CH2)2N、CH3CONH, CN, Br, Cl, F, amino acid acyl, amino acid acylamino-, oligopeptides acyl group or oligopeptides acyl
Amino；Wherein, the C1-C6-NH2C1-C6For the alkyl with 1-6 C, naphthenic base, alkylene, cycloalkenyl group, cyclopenta
Amino, Cyclohexylamino, morpholine base or methyl piperazine base；
3)R3Selected from OH, OCH3、OC2H5、OCH(CH3)2、OC3H7、CH3COO、CH3、CF3、C1-C6-NH2、NH2、CH3NH、(CH3)2N、(CH3CH2)2N、CH3CONH, CN, Br, Cl, F, amino acid acyl, amino acid acylamino-, oligopeptides acyl group or oligopeptides acylamino-；
Wherein, the C1-C6-NH2C1-C6For with 1-6 C alkyl, naphthenic base, alkylene, cycloalkenyl group, clopentylamino,
Cyclohexylamino, morpholine base, methyl piperazine base；
4)R4Selected from H or prenyl；
5)R5Selected from H, OH, OCH3、OC2H5Or OC3H7；
6)R6Selected from H, OH, OCH3、OC2H5Or OC3H7。
2. icariside I class compound according to claim 1 and derivative and officinal salt, it is characterised in that: R1
For OH, OCH3, OCH (CH3)2Or OCH2CH3；R2For OH, OCH3Or OC3H7；R3For OH, OCH3, OCH (CH3)2Or OCH2CH3；R4
ForR5、R6For H.
3. icariside I class compound according to claim 1 and derivative and officinal salt, it is characterised in that:
Its structural formula is
One of.
4. a kind of composition, it is characterised in that: comprising the described in any item icariside I class compounds of claims 1 to 3 with
And derivative and officinal salt, it also include pharmaceutically acceptable carrier, excipient, diluent, medium or their combination.
It, should 5. Claims 1 to 4 any one compound, derivative, officinal salt or composition are used to prepare the purposes of drug
Drug is used to preventing, mitigate or treating the purposes in tumor disease.
6. purposes according to claim 5, it is characterised in that: the tumour includes liver cancer, lung cancer, colon cancer, oophoroma, gallbladder
Pipe cancer, malignant lymphatic tumor, liver cancer, bladder cancer and prostate cancer.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111228287A (en) * | 2020-04-15 | 2020-06-05 | 广东金骏康生物技术有限公司 | Application of epimedium flavone glycoside compound in preparing medicine for treating melanoma |
| CN111700900A (en) * | 2020-05-15 | 2020-09-25 | 广东金骏康生物技术有限公司 | Natural immune activator, TIL cell promoter and application thereof |
| CN111870609A (en) * | 2020-07-01 | 2020-11-03 | 广东金骏康生物技术有限公司 | Intestinal flora regulator and application thereof |
| CN114410734A (en) * | 2021-12-07 | 2022-04-29 | 广东工业大学 | Application of icariside I or phloretin in preparation of nucleic acid cytosine deaminase APOBEC3B inhibitor |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101113157A (en) * | 2006-07-28 | 2008-01-30 | 上海特化医药科技有限公司 | Isoamylene radical chromocor derivative, preparation method and uses thereof |
| CN104825479A (en) * | 2015-05-20 | 2015-08-12 | 佛山市金骏康健康科技有限公司 | Icariside derivatives as well as preparation method and application thereof in promoting human cells to generate interferon-gamma and treating disease |
-
2018
- 2018-10-29 CN CN201811269591.7A patent/CN109320570A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101113157A (en) * | 2006-07-28 | 2008-01-30 | 上海特化医药科技有限公司 | Isoamylene radical chromocor derivative, preparation method and uses thereof |
| CN104825479A (en) * | 2015-05-20 | 2015-08-12 | 佛山市金骏康健康科技有限公司 | Icariside derivatives as well as preparation method and application thereof in promoting human cells to generate interferon-gamma and treating disease |
Non-Patent Citations (1)
| Title |
|---|
| 韩惠，等: "箭叶淫羊藿中化学成分及其体外抗肿瘤活性研究", 《现代药物与临床》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111228287A (en) * | 2020-04-15 | 2020-06-05 | 广东金骏康生物技术有限公司 | Application of epimedium flavone glycoside compound in preparing medicine for treating melanoma |
| CN111700900A (en) * | 2020-05-15 | 2020-09-25 | 广东金骏康生物技术有限公司 | Natural immune activator, TIL cell promoter and application thereof |
| CN111870609A (en) * | 2020-07-01 | 2020-11-03 | 广东金骏康生物技术有限公司 | Intestinal flora regulator and application thereof |
| CN114410734A (en) * | 2021-12-07 | 2022-04-29 | 广东工业大学 | Application of icariside I or phloretin in preparation of nucleic acid cytosine deaminase APOBEC3B inhibitor |
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