CN103768087A - Anti-liver-cancer medicinal compound and preparation method thereof - Google Patents

Abstract

The invention provides an anti-liver-cancer medicinal compound. The compound is baicalin metal complex (BMC) with a general molecular formula of Na2M(C21H16O11)2 .xH2O, wherein the molar ratio of baicalin to a metal ion is 2:1; the metal ion in the metal complex is one of Ni<2+>, Co<2+> and Cu<2+>. The anti-liver-cancer medicinal compound is prepared in the following three steps: 1, preparing materials; 2, preparing methanol solution and NaOH solution; 3, synthesizing MBC: preparing a solution A and a solution B, slowly mixing the solution A and the solution B, refluxing, filtering, washing and drying the mixed solution. When the prepared BMC acts on liver cancer cells, proliferation of the cells can be remarkably inhibited, and cell apoptosis is induced. The method is a new medicine development method of a medicinal compound by combining traditional Chinese medicinal effective components and metal ion, and a novel low-cost anti-live-cancer medicinal compound is provided.

Description

Medical compounds of anti-hepatocarcinoma and preparation method thereof

Technical field

The present invention relates to a kind of anti-cancer drug compounds and prepare the method for this compound.

Background technology

Hepatocarcinoma serious threat human health and life, therefore medicines resistant to liver cancer research is subject to extensive concern.At present, people actively find anti-cancer agent, extract on the one hand new drug from natural product, explore on the other hand and improve existing medicine, increase new function; Because new drug development difficulty is large, the cycle is long, and improving existing medicine becomes study hotspot.Chinese medicine is the conventional medicament of China, improves herbal medicine efficacy activity significant, and medicine promotes in active process, Chinese medicine co-ordination complex be prepared as one of main method.

Baicalin ( baicalin, BC) be China's Chinese medicine Radix Scutellariae ( scutellaria baicalensis Georgi, CBG) main component, there is the multiple biological activitys such as antibacterial, antiviral, blood sugar lowering, removing free radical, it also has inhibitory action to tumors such as carcinoma of prostate, cervical cancer, pulmonary carcinoma, malignant lymphoma, myeloma, hepatocarcinoma.In BC molecular structure, contain the group such as hydroxyl or carbonyl and have stronger chelation with metal ion, at present existing bibliographical information baicalin-metal complex ( baicalin metal complexes, BMC) and there is the effects such as the free radical, antioxidation, bacteriostatic activity of removing.Through experiment, using BC as part, select Ni ²⁺, Co ²⁺, Cu ²⁺three metal ion species and BC complexation under alkali condition forms coordination compound, has measured the composition of coordination compound by elementary analysis, spectrographic method, thermogravimetric analysis, resolves the structure of coordination compound; And explored the impact of BMC on people's SMMC-7721 liver cancer cells with reference to pertinent literature, and observe the impact of BMC on Bcl-2 and Bax expression from gene level and protein level, inquire into its mechanism of action.Above-mentioned related work, so far there are no bibliographical information, related results can provide reference for the anticancer machine-processed studies and clinical application of Chinese medicine coordination compound.

Summary of the invention

The technical problem to be solved in the present invention is to pass through series of experiments, detect the impact of BMC on people's hepatocellular carcinoma cell line SMMC-7721 proliferation, cycle and apoptosis, combining form is learned and is observed its active anticancer, also take the DNA of hepatoma carcinoma cell as target spot, observe BMC and the power of DNA binding ability and the relatedness of active anticancer etc., on the basis of above-mentioned test and study, medical compounds of a kind of anti-hepatocarcinoma and preparation method thereof is proposed.

Solve the problems of the technologies described above and adopt following technical scheme: the medical compounds of this anti-hepatocarcinoma is that baicalin metal complex is BMC, and its general formula of molecular structure is:

General molecular formula is: Na ₂m (C ₂₁h ₁₆o ₁₁) ₂. xh ₂o, wherein M is metal ion, xfor water of crystallization number;

Described baicalin metal complex is that to be respectively baicalin-nickel complex be that BC-Ni, baicalin-cobalt complex are that BC-Co or baicalin-copper complex are BC-Cu to BMC; Three's molecular composition is respectively: Na ₂ni (C ₂₁h ₁₆o ₁₁) ₂10H ₂o, Na ₂co (C ₂₁h ₁₆o ₁₁) ₂8H ₂o, Na ₂cu (C ₂₁h ₁₆o ₁₁) ₂8H ₂o; Baicalin is BC and metal ion Ni ²⁺, Co ²⁺, Cu ²⁺the mol ratio coordinating is 2:1; The purity of BC-Ni, BC-Co and BC-Cu is respectively 97.4%, 98.3% and 95.6%;

Three kinds of baicalin metal complexs have following physicochemical property:

	Ultraviolet spectra UV	Mass spectrum MS m/z	Molecular weight
				BC-Ni	λmax=278.5 nm	1172.9 992.6 794.5 596.3 466.9	1173
BC-Co	λmax =279.5 nm	1173.0 1028.1 830.2 632.4 467.6	1173
				BC-Cu	λmax =288.0 nm	1177.7 1033.4 835.6 637.6 467.6	1178

Infrared spectrum IR shows: BC-Ni v: 3391 cm ^-1(O-H), 1624 cm ^-1(C=O), 1188 cm ^-1(C=O);

BC-Co v: 3391 cm ^-1（O-H）、1624 cm ^-1（C=O）、1189 cm ^-1（C=O）；

BC-Cu v:3391 cm ^-1（O-H）、1624 cm ^-1（C=O）、1192 cm ^-1（C=O）；

The preparation method of described anti-liver cancer drug compounds baicalin metal complex BMC is carried out as follows:

(1) get the raw materials ready: the enough mass ratioes of commercial or net purchase are greater than 98% baicalin powder, analytically pure solid four hydration nickel acetates, analytically pure solid four hydration cobaltous acetate, analytically pure solid one hydration Schweinfurt green, analytically pure solid sodium hydroxide, absolute methanol and distilled water;

(2) preparation of methanol solution and sodium hydroxide solution: being mixed with volumetric concentration with 600 mL absolute methanols and 400 mL distilled water is 60% methanol solution; Being mixed with mass concentration with 4 g solid sodium hydroxides and 6 mL distilled water is 40% sodium hydroxide solution;

(3) baicalin metal complex is the synthetic of BMC:

1. the preparation of A solution: get 0.446 g baicalin powder and add in the methanol solution that 70 mL volumetric concentrations are 60%, 65 ℃ of water-soluble stirring and dissolving, then the sodium hydroxide solution that is 40% by mass concentration adjusting pH to 9.0, make A solution;

2. the preparation of B solution: get 0.5 mmo1 solid metal salt and add in the methanol solution that 20 mL volumetric concentrations are 60%, stirring and dissolving, makes B solution;

3. A solution mixes with B solution: under 65 ℃ of water bath condition, B solution is slowly added in A solution, separate out precipitation;

4. reflux, filter, wash and be dried: 2 h that reflux under 65 ℃ of water bath condition, filtered while hot, removes unreacted baicalin and slaine with the methanol solution washing of volumetric concentration 60%, and 50 ℃ of vacuum dryings are to constant weight.

Described (3) 2. a kind of solid metal salt in the preparation of B solution be in solid four hydration nickel acetates, solid four hydration cobaltous acetate or solid-hydration Schweinfurt green any.

The invention has the beneficial effects as follows: the synthetic method that three kinds of anti-liver cancer drug compounds are provided, the accurate structure of this medical compounds is disclosed by analysis and characterization, BMC is acted on to SMMC-7721 cell, show that its active anticancer is apparently higher than BC, can suppress significantly the propagation of SMMC-7721 cell, inducing cell generation apoptosis; Be combined for Effective Component of Chinese Medicine the method for medical compounds made from metal ion, a kind of new anti-liver cancer drug compounds is provided.

Accompanying drawing explanation

Fig. 1 is the ultraviolet spectrogram of BMC;

Fig. 2 is the infrared spectrogram of BMC;

Fig. 3 is the mass spectrum of three kinds of BMC coordinating with Ni, Co, Cu respectively;

Fig. 4 is the thermogravimetric analysis figure of three kinds of BMC coordinating with Ni, Co, Cu respectively;

Fig. 5 is the chemical structural drawing of BMC, and in figure, M is metal ion, and x is water of crystallization number;

Fig. 6 is the titration curve figure of three kinds of BMC coordinating with Ni, Co, Cu respectively;

Fig. 7 is the cellular morphology figure after variable concentrations BMC treatment S MMC-7721 cell 48 h;

Fig. 8 is the affect figure of BMC on SMMC-7721 cell cycle;

Fig. 9 is that BMC is on the apoptotic figure that affects of SMMC-7721;

Figure 10 is the melting curve figure that fluorescence quantitative RT-RCR detects β-actin, Bcl-2 and Bax gene;

Figure 11 is β-actin, Bcl-2, Bax gene fluorescence quantitative RT-PCR amplification efficiency curve chart;

Figure 12 is the affect figure of BMC on SMMC-7721 cell Bcl-2 protein expression;

Figure 13 is the affect figure of BMC on SMMC-7721 cell Bax protein expression;

Figure 14 is DNA, BMC and the cyclic voltammogram of the two mixture on F-CNTs/GCE electrode;

Figure 15 is lg[△ i _p/ (△ i _{p, max}-△ i _p)] and lg c _bMCgraph of a relation.

The specific embodiment

The present invention is below described in further detail in connection with embodiment and with reference to accompanying drawing:

The medical compounds of this anti-hepatocarcinoma is that baicalin metal complex is BMC, and its general formula of molecular structure is:

General molecular formula is: Na ₂m (C ₂₁h ₁₆o ₁₁) ₂. xh ₂o, wherein M is metal ion, xfor water of crystallization number;

Described baicalin metal complex is that to be respectively baicalin-nickel complex be that BC-Ni, baicalin-cobalt complex are that BC-Co or baicalin-copper complex are BC-Cu to BMC; Three's molecular composition is respectively: Na ₂ni (C ₂₁h ₁₆o ₁₁) ₂10H ₂o, Na ₂co (C ₂₁h ₁₆o ₁₁) ₂8H ₂o, Na ₂cu (C ₂₁h ₁₆o ₁₁) ₂8H ₂o; Baicalin is BC and metal ion Ni ²⁺, Co ²⁺, Cu ²⁺the mol ratio coordinating is 2:1; The purity of BC-Ni, BC-Co and BC-Cu is respectively 97.4%, 98.3% and 95.6%;

Three kinds of baicalin metal complexs have following physicochemical property:

	Ultraviolet spectra UV	Mass spectrum MS m/z	Molecular weight
				BC-Ni	λmax=278.5 nm	1172.9 992.6 794.5 596.3 466.9	1173
BC-Co	λmax =279.5 nm	1173.0 1028.1 830.2 632.4 467.6	1173
				BC-Cu	λmax =288.0 nm	1177.7 1033.4 835.6 637.6 467.6	1178

Infrared spectrum IR shows: BC-Ni v: 3391 cm ^-1(O-H), 1624 cm ^-1(C=O), 1188 cm ^-1(C=O);

BC-Co v: 3391 cm ^-1（O—H）、1624 cm ^-1（C=O）、1189 cm ^-1（C=O）；

BC-Cu v:3391 cm ^-1（O—H）、1624 cm ^-1（C=O）、1192 cm ^-1（C=O）；

Described baicalin metal complex is that the preparation method of BMC is carried out as follows:

(1) get the raw materials ready: the enough mass ratioes of commercial or net purchase are greater than 98% baicalin powder, analytically pure solid four hydration nickel acetates, analytically pure solid four hydration cobaltous acetate, analytically pure solid one hydration Schweinfurt green, analytically pure solid sodium hydroxide, absolute methanol and distilled water;

(2) preparation of methanol solution and sodium hydroxide solution: being mixed with volumetric concentration with 600 mL absolute methanols and 400 mL distilled water is 60% methanol solution; Being mixed with mass concentration with 4 g solid sodium hydroxides and 6 mL distilled water is 40% sodium hydroxide solution;

(3) baicalin metal complex is the synthetic of BMC:

1. the preparation of A solution: get 0.446 g baicalin powder and add in the methanol solution that 70 mL volumetric concentrations are 60%, 65 ℃ of water-soluble stirring and dissolving, then the sodium hydroxide solution that is 40% by mass concentration adjusting pH to 9.0, make A solution;

2. the preparation of B solution: get 0.5 mmo1 solid metal salt and add in the methanol solution that 20 mL volumetric concentrations are 60%, stirring and dissolving, makes B solution;

3. A solution mixes with B solution: under 65 ℃ of water bath condition, B solution is slowly added in A solution, separate out precipitation;

4. reflux, filter, wash and be dried: 2 h that reflux under 65 ℃ of water bath condition, filtered while hot, removes unreacted baicalin and slaine with the methanol solution washing of volumetric concentration 60%, and 50 ℃ of vacuum dryings are to constant weight.

Described (3) 2. a kind of solid metal salt in the preparation of B solution be in solid four hydration nickel acetates, solid four hydration cobaltous acetate or solid-hydration Schweinfurt green any.

It should be noted that in BMC preparation method, each raw material is monodrome but not interval value, therefore can regard unique embodiment as most preferred embodiment, enumerate no longer one by one; Raw material is pressed g, mL metering, in like manner, with kg, L metering or with weight fraction metering, is converted and can be realized by routine, does not carefully state.

For checking the present invention, the existing correlation test that the applicant is done is described below:

1, the preparation of raw material:

Human hepatoma cell strain SMMC-7721 is purchased from Chinese Academy of Medical Sciences's tumor cell storehouse; The more than 98% baicalin powder of mass concentration ( baicalin, BC) and be purchased from Jin Sui bio tech ltd, Shanghai; Hyclone (FBS) is purchased from Hangzhou Sijiqing Biological Engineering Material Co., Ltd.; Be purchased from Nanjing KaiJi Biology Science Development Co., Ltd containing 1% dual anti-RPMI-1640 culture medium; Mass concentration is that 0.25% trypsin solution is purchased from Nanjing KaiJi Biology Science Development Co., Ltd; Thiazolyl blue ( methyl thiazolyl tetrazolium, MTT) and be purchased from Nanjing KaiJi Biology Science Development Co., Ltd; AnnexinV-FITC/PI detection kit is purchased from Nanjing KaiJi Biology Science Development Co., Ltd; The total RNA of AxyPrep prepares in a small amount test kit and is purchased from and likes to pursue progress Bioisystech Co., Ltd; PrimeScript ^tMrT reagent Kit (Perfect Real Time) reverse transcription test kit is purchased from Takara company; SYBR Premix Ex Taq II (Tli RNaseH Plus) fluorescence quantitative kit is purchased from Takara company; Mouse-anti people Bcl-2 and Bax monoclonal antibody are purchased from Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge; FITC labelling goat anti-mouse IgG is purchased from Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge; Other reagent is analytical pure.

, the instrument selected:

UV-2550 type ultraviolet spectrophotometer is Japanese Shimadzu company product; IRPrestige-21 type Fourier transformation infrared spectrometer is Japanese Shimadzu company product; Uplc-Q-Tofmicro type mass spectrograph is Waters company of U.S. product; Pyris 1 TGA thermogravimetric analyzer is Perkin-Elmer company of U.S. product; Flash EA-1112 type elemental analyser is Italian CE company product; Inductive coupling plasma emission spectrograph is PE company of U.S. product; HF90 type CO2 gas incubator is the holding company limited of middle national power health biologic medical science and technology product; Thermo Scientific two stage biological safety cabinet is Sai Feishier scientific & technical corporation of U.S. product; CKX31 type inverted microscope is Japanese Olympus company product; Bio-Rad 680 microplate reader are Bio-Rad company of U.S. product; BD FACSCalibur flow cytometer is U.S. company BD product; MLS-3750 autoclave is Japanese Sanyo company product; 3K15 type centrifuge is German Sigma company product; Nucleic acid-protein analyser is German Eppendorf company product; Universal Hood II type gel imaging analysis system is Bio-Rad company of U.S. product; CHI660C electrochemical workstation is Chinese Shanghai Chen Hua Instrument Ltd. product; SK3210HP ultrasonic cleaner is Chinese Shanghai Ke Dao ultrasonic instrument company limited product; Electronic analytical balance is German Sartorius company product; DHG-9123A electric heating constant-temperature blowing drying box is the permanent Science and Technology Ltd. of Chinese Shanghai product.

, solution preparation:

1. three lysates: get 10 g sodium lauryl sulphate solids, 5 mL isobutanol, 1.2 mmol hydrochloric acid, are made into 100 mL with distilled water;

2. PBS solution: take 8.0 g solid sodium chloride, 0.2 g potassium dihydrogen phosphate solid, 0.2 g solid potassium chloride, 3.49g disodium hydrogen phosphate dodecahydrate solid, stirring and dissolving is in three lysates, and adding distil water dilution is settled to 1000 mL, after high temperature sterilize, saves backup in-4 ℃;

3. Thiazolyl blue is MTT solution: claim 50 mg solid-state MTT, be fully dissolved in 10 mL PBS solution, being made into concentration is 5 mgmL ^-1solution, after filtering with 0.22 μ m microfilter ,-4 ℃ keep in Dark Place, effective in two weeks;

4, medical compounds is synthetic:

(1) get the raw materials ready: baicalin powder, solid four hydration nickel acetates, solid four hydration cobaltous acetate, solid one hydration Schweinfurt green that the enough mass concentrations of commercial or net purchase are greater than 98%; The solid sodium hydroxide of the commercial or adjusting pH that net purchase is enough; Commercial or net purchase is enough solvent absolute methanol and distilled water;

(2) preparation of solution:

1. get 600 mL absolute methanols, being mixed with volumetric concentration with 400 mL distilled water is 60% methanol solution;

2. get 4 g solid sodium hydroxides, the sodium hydroxide solution that is 40% by the mass concentration that 6 mL distilled water are mixed with;

(3) baicalin-metal complex (Baicalin metal complexes, BMC) is synthetic:

1. get 0.446 g baicalin powder and be placed in the methanol solution that 70 mL volumetric concentrations are 60%, stirring and dissolving in 65 ℃ of water-baths, then the sodium hydroxide solution that is 40% by mass concentration tune pH to 9.0, be A solution;

2. get 0.5 mmol solid metal salt and be placed in the methanol solution that 20 mL volumetric concentrations are 60%, stirring and dissolving, is B solution;

3. under 65 ℃ of heating in water bath stir, B solution is slowly added in A solution, separate out precipitation;

4. 2 h that reflux in 65 ℃ of water-baths, unreacted baicalin and slaine are removed in the methanol solution washing that filtered while hot is 60% by volumetric concentration, and 50 ℃ of vacuum dryings are to constant weight.

, coordination compound sign:

(1) method:

The UV spectrum absorption difference of UV spectrophotometer measuring BC and BMC 200-600 nm range of wavelengths in 60% alcoholic solution, infers coordination compound complexation reaction, the results are shown in Figure 1; KBr tabletting infrared spectrometry is analyzed the architectural difference of BC and BMC, infers coordination compound coordinating group and coordination bonding position, and sweep limits is 500～4000 cm ^-1, the results are shown in Figure 2; Mass spectrometric data all completes on Uplc-Q-Tofmicro type mass spectrograph, the results are shown in Figure 3; Thermogravimetric analysis BMC sample, N ₂atmosphere, 20 ℃ of min ^-1be warming up to 600 ℃, observe the weightless situation of each BMC, the results are shown in Figure 4; Elemental microanalysis method is determined the content of C in BMC, H, and inductive coupling plasma emission spectrograph is measured the content of metal ion in BMC, further determines the mol ratio of BC and metal ion, and extrapolates the purity of coordination compound; Acid-base titrations detects the stability of BMC, investigates its stability in culture fluid, the results are shown in Figure 6.

Result:

1. from the ultraviolet spectra (UV) of Fig. 1 BMC, UV (BC): λ _max=277 nm; UV (BC-Ni): λ _max=278.5 nm; UV (BC-Co): λ _max=279.5 nm; UV (BC-Cu): λ _max=288 nm.BC forms after coordination compound, and the electron delocalization degree of molecule increases, and coplanarity strengthens, and the energy needing while causing electron transition reduces, λ _maxred shift, illustrates that BC reacts and formed coordination compound with metallic ion coordination.

2. from the infrared spectrum (IR) of Fig. 2 BMC, IR (BC) v: 3391 cm ^-1(O-H), 2924 cm ^-1(Hydrogen bond), 1728,1658 cm ^-1(C=O), 1203 cm ^-1(C-O); IR (BC-Ni) v: 3391 cm ^-1(O-H), 1624 cm ^-1(C=O), 1188 cm ^-1(C-O); IR (BC-Co) v: 3391 cm ^-1(O-H), 1624 cm ^-1(C=O), 1189 cm ^-1(C-O); IR (BC-Cu) v: 3391 cm ^-1(O-H), 1624 cm ^-1(C=O), 1192 cm ^-1(C-O).BC forms after coordination compound, 2924 cm ^-1the intramolecular hydrogen bond peak of 5-OH and 4 C=O disappears substantially, and 3391 cm ^-1 v _o-Hintensity obviously weakens, 1203 cm ^-1 v _c-Ored shift in various degree occurs, illustrate that the oxygen on 5-OH of BC coordinates with metal ion, intramolecular hydrogen bond is destroyed, and because metal ion makes the electron delocalization effect of oxygen in C-O v _c-Othere is red shift.In addition BC 1728,1658 cm, ^-1 v _c=Oforming all disappearances after coordination compound, and at 1624 cm ^-1place occur a new absworption peak, infer this may be carboxylic carbonyl absorption peak and 4 C=O absworption peaks overlapping due to.Reason is in the process of experiment, to have added NaOH solution to adjust pH to 9, glucal acidic group has become D-Glucuronic acid sodium salt, cause carboxylic carbonyl absorption peak that larger displacement occurs, the upper lone pair electrons of oxygen of while 4 C=O and the unoccupied orbital of metal ion have formed coordinate bond, cause the cloud density of C=O to reduce, thereby red shift occurs.By comparison and analysis to BC and BMC infrared spectrum principal character peak, can illustrate that coordination has occurred for 4 C=O of BC and 5-OH and each metal ion.

3. from the mass spectrum (MS) of Fig. 3 BMC, MS (BC-Ni) m/z:1172.9,992.6,794.5,596.3,466.9; MS (BC-Co) m/z:1173.0,1028.1,830.2,632.4,467.6; MS (BC-Cu) m/z:1177.7,1033.4,835.6,637.6,467.6.In BC-Ni second order ms figure, fragment peak 992.6 loses 10 hydrones (180 Da) gained for molecular ion ruptures, 794.5 lose a part D-Glucuronic acid sodium salt (198 Da) gained for fragment peak 992.6 ruptures, 596.3 lose 2 molecule glucose aldehydic acid sodium (396 Da) gained for fragment peak 992.6 ruptures, fragment peak 466.9 is the baicalin sodium salt peak of the last gained of fracture, and visible BC-Ni is by " baicalin sodium salt × 2+Ni+10H ₂o " composition.In like manner can be derived from BC-Co by " baicalin sodium salt × 2+Co+8H ₂o " composition, BC-Cu is by " baicalin sodium salt × 2+Cu+8H ₂o " composition.

4. from the thermogravimetric analysis (TGA) of Fig. 4 BMC, 40 ℃～160 ℃ stages, the weight-loss ratio of BC-Ni, BC-Co and BC-Cu is respectively 15.3%, 12.7% and 12.3%, the weight-loss ratio of BC-Ni with in the structure of supposition, lose 10 mol water of crystallization and conform to, in BC-Co and the weight-loss ratio of BC-Cu and the structure of supposition, lose 8 mol water of crystallization and conform to.

5. elementary analysis and inductively coupled plasma atomic emission (ICP) are measured each constituent content (being theoretical value in bracket).BC-Ni: C 41.84% ( 42.97%)，H 4.4%(4.43%)，Ni 5.08(5.03%)；BC-Co: C 43.58%(44.33%)，H 4.18%(4.22%)，Co 5.53(5.19%)；BC-Cu: C 42.18%(44.13%)，H 4.12%(4.20%)，Cu 5.78(5.60%)。Can determine that according to results of elemental analyses in three kinds of BMC molecules, BC all coordinates with the ratio of 2:1 with metal ion.

Comprehensive UV, IR, MS and TGA result, can determine BC and Ni in BMC molecule ²⁺, Co ²⁺and Cu ²⁺the mol ratio coordinating is 2:1, and molecular formula is respectively Na ₂ni (C ₂₁h ₁₆o ₁₁) ₂10H ₂o, Na ₂co (C ₂₁h ₁₆o ₁₁) ₂8H ₂o, Na ₂cu (C ₂₁h ₁₆o ₁₁) ₂8H ₂o, structure as shown in Figure 5.Elementary analysis and ICP measurement result can further determine that the proportioning of BC and metal ion is 2:1, and are respectively 97.4%, 98.3%, 95.6% according to the purity that the content of carbon can estimate BC-Ni, BC-Co and BC-Cu, and purity all can reach the standard of pharmacological research.

6. with 60 % ethanol waters, BMC being made into concentration is 0.005 mmolL ^-1solution, use 0.01 molL simultaneously ^-1the titration of KOH solution, titration curve is as shown in Figure 6.The pH mutation range of three kinds of coordination compounds all, between 5 ~ 9, illustrates that BMC is more stable in RPMI-1640 culture fluid (pH approximately 7.2).

, the impact of BMC on SMMC-7721 cell proliferation:

(1) method:

By SMMC-7721 cell culture in containing 10% hyclone and penicillin and each 100 UL of streptomycin ^-1rPMI-1640 culture fluid in, 37 ℃, 5% CO ₂in concentration level saturated humidity constant incubator, cultivate.The take the logarithm cell of trophophase, 0.25% trypsinization makes 5 × 10 ⁴individual mL ^-1single-cell suspension liquid, be inoculated in 96 orifice plates with every hole 100 μ L, be positioned over 37 ℃, 5% CO ₂in incubator, cultivate, establish blank group, negative control group and dosing experimental group.After 24 h, inhale and abandon original fluid in hole, experimental group adds respectively pastille culture medium, and BMC final concentration is 6.25,12.5,25,50, l00 μ gmL ^-1with the BC of concentration, as positive controls, blank group and negative control group add equivalent RPMI-1640 culture fluid, and every concentration group and matched group are all established 3 and answered holes, in incubator, cultivate respectively 24 h, 48 h, 72 h, cultivate the front 4 every holes of h of termination and add 20 μ L MTT (5 mgmL ^-1), to continue to cultivate after 4 h, every hole adds 100 μ L tri-lysates, hatches after 6 h, and microplate reader is tested the OD value of each hole at 570 nm places, calculates each group of cell proliferation inhibition rate by formula (1), the results are shown in Table 1.

(1)

(2) result:

Table 1 BC and the BMC impact (mean ± s, n=3) on SMMC-7721 cell proliferation

Note: with matched group comparison, significant difference, * P<0.05; With the comparison of BC processed group, significant difference, ▽ P<0.05.

From table 1, BC and BMC have obvious inhibitory action to the propagation of SMMC-7721 cell, and strengthen with the prolongation of action time and the rising of activity, are regular hour-effect and docs-effect dependency.BC-Ni, BC-Co and BC-Cu coordination compound to the suppression ratio of SMMC-7721 cell apparently higher than BC, illustrate that BMC has not only retained the anti-tumor activity of BC, but also obviously strengthen, its inhibitory action size is: BC-Cu>BC-Co>BC-NiGreatT.Grea T.GTBC.

, the impact of BMC on SMMC-7721 cellular morphology:

(1) method:

The take the logarithm SMMC-7721 cell of trophophase, 0.25% trypsinization makes 1 × 10 ⁵individual mL ^-1single-cell suspension liquid, be inoculated on the coverslip in 6 orifice plates with every hole 100 μ L, in 37 ℃, 5% CO ₂in incubator, cultivate, after cell attachment, add pastille culture medium, make BMC final concentration be respectively 25,50 μ gmL ^-1, establish blank group and add equivalent culture medium, continue carefully to take out the coverslip in hole after cultivation 48 h, PBS washing 3 times, the form of inverted microscope observation of cell and upgrowth situation, refer to Fig. 7, and wherein a is matched group, and b is 25 μ gmL ^-1bC group, c is 50 μ gmL ^-1bC group, d is 25 μ gmL ^-1bC-Ni group, e is 50 μ gmL ^-1bC-Ni group, f is 25 μ gmL ^-1bC-Co group, g is 50 μ gmL ^-1bC-Co group, h is 25 μ gmL ^-1bC-Cu group, i is 50 μ gmL ^-1bC-Cu group.

Result:

As seen from Figure 7, the SMMC-7721 Growth of Cells of matched group is vigorous, and substantially all adherent, cell is full, clear-cut.After drug treating 48 h, cell gradually becomes fusiformis, edge roughness, and there is de-wall phenomenon in cell, cellularity does not have matched group tight.Along with the increase of coordination compound concentration, it is many that suspension cell becomes, and breaking appears in cell, becomes fine debris shape, is necrosis, illustrates that BC and BMC all have inhibitory action to the growth of SMMC-7721 cell, and is certain dose-effect relationship.

, the impact of BMC on SMMC-7721 cell cycle:

(1) method:

With 25,50 μ gmL ^-1bMC treatment S MMC-7721 cell 48 h after, collect 5 × 10 ⁵individual cell is made single-cell suspension liquid, PBS washed twice (2000 rmin ^-1centrifugal 5 min), add 70% ethanol of-20 ℃ of pre-coolings, fixedly spend the night in 4 ℃.After centrifugal collecting cell, with PBS washed cell, to remove ethanol, cell is resuspended in 500 μ L PBS; Add RNase A, making its final concentration is 0. 25 mgmL ^-1, 37 ℃ of shaking tables, 30 min that vibrate; Add 5 μ L PI to mix, room temperature lucifuge 30 min that dye, the variation in cells were tested by flow cytometry cycle, refers to Fig. 8, and wherein first peak is G ₀/ G ₁phase, the presynthetic phase of representing DNA; Second peak is G ₂/ M the phase, represent DNA post-synthetic phase; Two peak-to-peak platforms are the S phase, represent DNA synthesis stage; A is matched group, and b is 25 μ gmL ^-1bC-Ni group, c is 50 μ gmL ^-1bC-Ni group, d is 25 μ gmL ^-1bC-Co group, e is 50 μ gmL ^-1bC-Co group, f is 25 μ gmL ^-1bC-Cu group, g is 50 μ gmL ^-1bC-Cu group.

Result:

Can be found out by Fig. 8, BMC processed group compared with matched group, G ₀/ G ₁phase ratio rises, S phase and G ₂/ M phase ratio declines, and concentration is higher, and this trend is more obvious, illustrates that BMC makes cell block in G ₀/ G ₁phase, entry deterrence S phase and G ₂/ M the phase, S phase and G ₂/ M the phase is mainly that cell carries out DNA replication dna and division stage in later stage, thereby suppresses cell proliferation.In addition, BMC processed group is at G ₀/ G ₁before peak, occur the peak group at hypodiploid cell peak, i.e. typical Apoptotic cell peak, along with this peak value of increase of BMC concentration also increases gradually, therefore can infer, BMC suppresses SMMC-7721 cell proliferation by retardance cell cycle.Each processed group G ₀/ G ₁the obvious rising of phase cell percentage and matched group, S phase and G ₂/ M phase cell percentage obviously reduces, and difference has statistical significance (P<0.05), refers to table 2.

The impact (mean ± s, n=3) of table 2 BMC on SMMC-7721 Cellular cycle and apoptosis

Note: with matched group comparison, significant difference, * P<0.05; With material low concentration processed group of the same race comparison, significant difference, ▽ P<0.05.

, BMC is on the apoptotic impact of SMMC-7721:

(1) method:

With 25,50 μ gmL ^-1bMC treatment S MMC-7721 cell 48 h after, collect 5 × 10 ⁵individual cell is made single-cell suspension liquid, PBS washed twice (2000 rmin ^-1centrifugal 5 min), add 500 μ L Binding Buffer re-suspended cells, after adding again 5 μ L Annexin V-FITC to mix, add 5 μ L Propidium Iodide, after mixing, react 15 min in room temperature lucifuge, in 1 h, detect Apoptosis with flow cytometer, the results detailed in Fig. 9, this is the measured scatterplot of flow cytometer, left lower quadrant is living cells, shows as Annexin-/PI-; Right lower quadrant is viable apoptotic cell, shows as Annexin+/PI-; Right upper quadrant is non-viable apoptotic cell, shows as Annexin+/PI+; Left upper quadrant is dead cell, shows as Annexin-/PI+, apoptosis rate=early stage apoptosis rate in apoptosis rate+late period; A is matched group, and b is 25 μ gmL ^-1bC-Ni group, c is 50 μ gmL ^-1bC-Ni group, d is 25 μ gmL ^-1bC-Co group, e is 50 μ gmL ^-1bC-Co group, f is 25 μ gmL ^-1bC-Cu group, g is 50 μ gmL ^-1bC-Cu group.

Result:

As shown in Figure 9, after BMC treatment S MMC-7721 cell, apoptosis phenomenon is obvious, along with rising early apoptosis and the non-viable apoptotic cell quantity of BMC concentration also increase thereupon.Apoptosis rate is shown in Table 2, it is different that each BMC acts on the apoptosis rate of SMMC-7721 cell, and its size order is: BC-Cu>BC-Co>BC-Ni, and consistent with mtt assay result, each concentration group compared with matched group, difference have statistical significance ( p<0.05), refer to table 2.

, the impact of BMC on SMMC-7721 cell Bcl-2, Bax mrna expression:

(1) method:

With 25,50 μ gmL ^-1bMC treatment S MMC-7721 hepatoma carcinoma cell 48 h after, collect each group of cell.Adopt the total RNA of AxyPrep to prepare in a small amount test kit and extract cell total rna, and carry out purity analysis, quantitatively and 2% agarose gel electrophoresis observe.Utilize PrimeScript ^tMrT reagent Kit (Perfect Real Time) reverse transcription test kit reverse transcription generates cDNA, adopts SYBR premix Ex Taq II (Tli RNaseH Plus) fluorescent quantitation reagent, in the enterprising performing PCR reaction of Bio-rad IQ5 quantitative real time PCR Instrument.Impact and the Mechanism Study design thereof of the oleanolic acid of primer sequence impact research and Li Hongmei etc. on SMMC-7721 apoptosis and Bcl-2/Bax gene mRNA expression with reference to the daphnetin of condition Nan Zhen etc. on cisplatin resistance SGC-7901 cell propagation synthesized, and refers to table 3; Response procedures: 95 ℃ of 10 s, 95 ℃ of 5 s, 60 ℃ of 30 s, totally 40 circulations; Reaction system is 25 μ L:DNA template 1 μ L, forward primer 1 μ L, downstream primer 1 μ L, SYBR premix Ex Taq II (Tli RNaseH Plus) (2 ×) 12.5 μ L, Free H ₂o complements to 25 μ L.Each sample does 3 multiple holes, gets the meansigma methods analysis in 3 holes.Judged the specificity of PCR reaction by melting curve.Adopt 2 according to the situation of amplification efficiency ^{-▽ ▽ Ct}method is carried out relative quantitative assay, the results are shown in Figure 10 ~ 11, and gene expression results is in table 4.

Table 3 β-actin, Bcl-2, Bax mRNA primer sequence

(2) result:

1. can see that from the melting curve of Figure 10 β-actin, Bcl-2 and Bax gene RT-PCR amplified production occur simple spike when 88 ℃, 86.5 ℃ and 89 ℃ respectively, illustrate that PCR product is single specificity product.

2. further verify the variation of genes of interest in cells amount, adopt the RT-PCR product that genes of interest Bc1-2, Bax and reference gene β-actin expression are high ( ct value is less) as standard substance, carry out fluorescence quantitative RT-RCR detection take the RT-PCR product of different extension rates as template, use draws ct value is made amplification efficiency standard curve, result as shown in figure 11, the rectilinear regression correlation coefficient of reference gene β-actin and genes of interest Bcl-2, Bax amplification efficiency standard curve is all greater than 0.98, and amplification efficiency (Eff) is respectively 95.67%, 92.5% and 94.6%, meets 2 ^{-▽ ▽ Ct}the condition of method.

Table 4 fluorescence quantitative RT-RCR detects the expression (n=3, mean ± SD) of Bcl-2 and Bax mRNA

Note: with matched group comparison, significant difference, * P<0.05; With material low concentration processed group of the same race comparison, significant difference, ▽ P<0.05.

3. as shown in Table 4, after each BMC treatment S MMC-7721 cell 48h, the obvious reduction compared with matched group of Bcl-2 mrna expression level, Bax mrna expression level is apparently higher than matched group; Along with the increase Bcl-2 mrna expression amount of concentration reduces, Bax mrna expression amount increases, and has obvious dose-dependence.BC-Ni 50 μ gmL ^-1processed group, the each concentration processed group of BC-Co and the each concentration group of BC-Cu and matched group comparison, difference have statistical significance ( p<0.05); Coordination compound variable concentrations group of the same race comparison, difference have statistical significance ( p<0.05).

, the impact of BMC on SMMC-7721 cell Bcl-2, Bax protein expression:

(1) method:

With 25,50 μ gmL ^-1bMC treatment S MMC-7721 cell 48 h after, collect 5 × 10 ⁵individual cell is made single-cell suspension liquid, PBS washed twice (2000 rmin ^-1centrifugal 5 min), add mouse anti human primary antibodie working solution 50 μ L, room temperature is placed 30 min, 10 mL PBS washing (2000 rmin ^-1centrifugal 5 min), to abandon supernatant, then add 100 μ L goat-anti mice FITC-IgG bis-anti-working solutions, room temperature lucifuge is hatched 30 min, adds 10 mL PBS washing (1000 rmin ^-1centrifugal 5 min), abandon supernatant, then add PBS 1.0 mL, filter through 200 mesh filter screens, flow cytometer detects the relative amount of the interior Bcl-2 of cell or Bax albumen, the results are shown in Figure 12 ~ 13, wherein the equal road of fluorescence value is larger, the content that the interior Bcl-2 of cell or Bax albumen are described is higher, otherwise protein content is lower; A is matched group, and b is 25 μ gmL ^-1bC-Ni group, c is 50 μ gmL ^-1bC-Ni group, d is 25 μ gmL ^-1bC-Co group, e is 50 μ gmL ^-1bC-Co group, f is 25 μ gmL ^-1bC-Cu group, g is 50 μ gmL ^-1bC-Cu group, protein expression the results are shown in Table 5.

Result:

The expression (n=3, mean ± SD) of table 5 BMC treatment S MMC-7721 cell 48h apoptosis-related protein

Note: with matched group comparison, significant difference, * P<0.05; With material low concentration processed group of the same race comparison, significant difference, ▽ P<0.05.

As known from Table 5, after each BMC treatment S MMC-7721 cell 48h, Bcl-2 protein expression is starkly lower than matched group, and Bax protein expression is apparently higher than matched group; Along with the increase of concentration, the expression of Bcl-2 albumen reduces, and the expression of Bax albumen increases, and has obvious dose-dependence.Each concentration group and matched group comparison, difference have statistical significance ( p<0.05); Coordination compound variable concentrations group of the same race comparison, difference have statistical significance ( p<0.05).

, BMC and SMMC-7721 cell DNA binding constant βmensuration:

(1) method:

1. the extraction of cell DNA: SMMC-7721 cell culture is in containing 10% hyclone and penicillin and each 100 UL of streptomycin ^-1rPMI-1640 culture fluid in, in 37 ℃, 5% CO ₂in concentration level saturated humidity constant incubator, cultivate.Collect the cell of exponential phase, press genome DNA extracting reagent kit and extract the total DNA of cell, nucleic acid-protein analyser carries out quantitatively and purity analysis, 1% agarose gel electrophoresis is observed its integrity, double-stranded DNA (the doubleb stranded DNA that finite concentration is extracted, dsDNA) in 100 ℃ of boiling water baths, heat 30 min, be then transferred to rapidly and in ice-water bath, be cooled to room temperature.Herein under reason condition, the dsDNA degeneration of unwinding is single stranded DNA (single stranded DMA, ssDNA), and-20 ℃ save backup.If not particularly point out in experiment, indication DNA is dsDNA;

2. the functionalization of multi-walled carbon nano-tubes (CNTs): get 10 mg CNTs and be dissolved in 50 mL concentrated hydrochloric acid, reflux 7 h under magnetic agitation, to remove metallic catalyst.CNTs after purification be placed in 80 mL acid solutions ( v _{nitric acid}: v _{sulphuric acid}=1:3) in, room temperature ultrasonic reaction 10 h.Distilled water is washed till neutrality, obtains the multi-walled carbon nano-tubes (F-CNTs) of functionalization, is dried to powder.Get 10 mg F-CNTs and be dissolved in DMF, it is for subsequent use that ultrasonic 20 min are uniformly dispersed it;

3. the preparation of F-CNTs modified electrode: glass-carbon electrode carries out polishing with the aluminium sesquioxide powder of 0.3 and 0.05 μ m respectively on polishing cloth, after redistilled water rinses, glass-carbon electrode is moved into and in ultrasonic cleaning instrument, use successively redistilled water, dehydrated alcohol, redistilled water ultrasonic cleaning (5 min/ time), room temperature is dried.Get 5 μ L F-CNTs solution and drip and be applied to the glass-carbon electrode surface that pretreatment is good, natural drying is made F-CNTs/GCE modified electrode;

4. the interactional electro-chemical test of BMC and DNA: on CHI660C electrochemical analyser, application cycle voltammetry detects the interaction of DNA and BMC, the results are shown in Figure 14, and wherein a is BC group, and b is BC-Ni group, and c is BC-Co group, and d is BC-Cu group.Calculate electron gain transfer ratio according to correlation formula αwith electron transfer rate constant k _sand two-way interaction's combination number mand binding constant β, refer to table 6.

(2) result:

1. cyclic voltammetry has been investigated electrolyte solution, pH value of solution, has been swept speed and BMC and the impact of DNA interaction time on peak current, and result shows that three kinds of BMC and BC are at 0.05 mol L ^-1in the B-R buffer solution of pH 8.0, baseline is the most stable, and the symmetry of peak type is best, and electrode reaction reversibility is best, investigates the C-V characteristic of BMC on F-CNTs modified electrode under optimum reaction condition, the results are shown in Figure 14.

2. as shown in Figure 14, in-0.2 ~ 0.6V potential range, DNA does not have peak current to produce (curve on F-CNTs modified electrode a); And BMC can produce a pair of oxidoreduction peak (curve b); BMC and DNA interact (curve c) after, its oxidoreduction peak current all obviously declines, and the negative (curve that moves in various degree occurs spike potential c).According to Bard theory: in the time that coordination compound and DNA have an effect, move if spike potential is negative, coordination compound and DNA generation electrostatic interaction be describeds, if spike potential is shuffled, be illustrated as insertion effect.Judge that with this electrostatic interaction has all occurred for BC and three kinds of BMC and DNA.Curve dfor the voltammogram after BMC and ssDNA effect, known adding after ssDNA, also there is negative moving in spike potential, but peak current declines compared with the lacking of DNA, and illustrates that BMC and DNA also exist insertion effect each other.Adding DNA postpeak current reduction may be because BMC is inserted in the double-spiral structure of DNA, and electronics transmits conductively-closed, and peak current is reduced; And while adding ssDNA, do not have this insertion shielding action, thereby peak current reduces few.Therefore a kind of non-electroactive super molecular compound of pattern formation that BMC mixes by electrostatic interaction and insertion effect with hepatocarcinoma DNA.

3. for the non-electroactive super molecular compound of BMC and DNA interaction formation, can ask the combination number that calculates two-way interaction according to equation (2) mand binding constant β.

(2)

△ in formula i _pfor adding the peak current before and after DNA poor, △ i _{p, max}for adding the maximum peak difference between current before and after DNA.Be lg[△ i _p/ (△ i _{p, max}-△ i _p)] ~ lg c _bMCrelation curve, can obtain respectively in conjunction with number according to slope and intercept mand binding constant β, the results are shown in Figure 15, wherein a is BC group, and b is BC-Ni group, and c is BC-Co group, and d is BC-Cu group; Fit equation is in table 6.

Table 6 lg[△ i _p/ (△ i _{p, max}-△ i _p)] and lg c _bMCthe fit equation of relation curve

5. try to achieve BC and three kinds of BMC and the interactional combination of DNA according to the slope of equation in table 6 and count m and be respectively 1.53,1.61,1.67,1.62, be about 2, show that four kinds of materials and DNA form the super molecular compound of 2:1 type.For verifying this conclusion, suppose mbe respectively 1,2,3, then make corresponding 1/ △ i _p~ 1/ c _bMC ^m relation curve, obtains respectively △ by slope and the intercept of straight line i _{p, max}with β.Result shows that four kinds of materials all exist m=2 o'clock, △ i _{p, max}approaching with the experiment institute numerical value of surveying, therefore four kinds of materials are all correct with the hypothesis of DNA formation 2:1 complex.As shown in Table 6, the size order of four kinds of materials and DNA binding constant is: β _bC-Cu> β _bC-Co> β _bC-Ni> β _bCthe power that is BMC and DNA combination is: BC-Cu>BC-Co>BC-NiGreatT.Grea T.GTBC, in the same size with its anti-tumor activity, binding ability and its anti-tumor activity that BMC and hepatocarcinoma DNA are described have certain relatedness, be that binding ability is stronger, anti-tumor activity is also stronger.

, conclusion:

Three kinds of synthetic BMC herein, have obtained the accurate structure of coordination compound by analysis and characterization.BMC is acted on to SMMC-7721 cell, and the super molecular compound of the pattern formation 2:1 type that BMC mixes by electrostatic interaction and insertion effect with DNA, is obstructed copying of DNA, and cell block is in G ₀/ G ₁phase, entry deterrence S phase and G ₂/ M the phase, and then inhibition cell proliferation, inducing cell generation apoptosis, mechanism may be relevant with the expression of lowering Bcl-2 and rise Bax, anti-tumor activity size is: BC-Cu>BC-Co>BC-NiGreatT.Grea T.GTBC, and present amount-result relation.Effective Component of Chinese Medicine is combined with metal ion and may be brought into play the two synergism, strengthens its biological activity by chemical coordination effect.The coordination compound of Effective Component of Chinese Medicine and metal ion is expected to one of source becoming new Chinese medicine, and relevant experimental result can provide the data refer of definite meaning herein.

Claims (4)

1. a medical compounds for anti-hepatocarcinoma, is characterized in that this medical compounds is that baicalin metal complex is BMC, and its general formula of molecular structure is:

General molecular formula is: Na ₂m (C ₂₁h ₁₆o ₁₁) ₂. xh ₂o, wherein M is metal ion, xfor water of crystallization number.

2. the medical compounds of anti-hepatocarcinoma as claimed in claim 1, is characterized in that described baicalin metal complex is that to be respectively baicalin-nickel complex be that BC-Ni, baicalin-cobalt complex are that BC-Co or baicalin-copper complex are BC-Cu to BMC; Three's molecular composition is respectively: Na ₂ni (C ₂₁h ₁₆o ₁₁) ₂10H ₂o, Na ₂co (C ₂₁h ₁₆o ₁₁) ₂8H ₂o, Na ₂cu (C ₂₁h ₁₆o ₁₁) ₂8H ₂o; Baicalin is BC and metal ion Ni ²⁺, Co ²⁺, Cu ²⁺the mol ratio coordinating is 2:1; The purity of BC-Ni, BC-Co and BC-Cu is respectively 97.4%, 98.3% and 95.6%;

Three kinds of baicalin metal complexs have following physicochemical property:

Ultraviolet spectra UV shows:

BC-Ni λ _max =278.5 nm；BC-Co λ _max =279.5 nm；BC-Cu λ _max =288.0 nm；

Mass spectrum MS shows:

BC-Ni m/z is 1172.9 992.6 794.5 596.3 466.9, molecular weight is 1173;

BC-Co m/z is 1173.0 1028.1 830.2 632.4 467.6, molecular weight is 1173;

BC-Cu m/z is 1177.7 1033.4 835.6 637.6 467.6, molecular weight is 1178;

Infrared spectrum IR shows:

BC- Ni v: 3391 cm ^-1（O-H）、1624 cm ^-1（C=O）、1188 cm ^-1（C=O）；

BC-Co v: 3391 cm ^-1（O-H）、1624 cm ^-1（C=O）、1189 cm ^-1（C=O）；

BC-Cu v: 3391 cm ^-1（O-H）、1624 cm ^-1（C=O）、1192 cm ^-1（C=O）。

3. anti-liver cancer drug compounds as claimed in claim 1 or 2, the baicalin metal complex described in it is characterized in that is that the preparation method of BMC is carried out as follows:

(1) get the raw materials ready: the enough mass ratioes of commercial or net purchase are greater than 98% baicalin powder, analytically pure solid four hydration nickel acetates, analytically pure solid four hydration cobaltous acetate, analytically pure solid one hydration Schweinfurt green, analytically pure solid sodium hydroxide, absolute methanol and distilled water;

(2) preparation of methanol solution and sodium hydroxide solution: being mixed with volumetric concentration with 600 mL absolute methanols and 400 mL distilled water is 60% methanol solution; Being mixed with mass concentration with 4 g solid sodium hydroxides and 6 mL distilled water is 40% sodium hydroxide solution;

(3) baicalin metal complex is the synthetic of BMC:

1. the preparation of A solution: get 0.446 g baicalin powder and add in the methanol solution that 70 mL volumetric concentrations are 60%, 65 ℃ of water-soluble stirring and dissolving, then the sodium hydroxide solution that is 40% by mass concentration adjusting pH to 9.0, make A solution;

2. the preparation of B solution: get 0.5 mmo1 solid metal salt and add in the methanol solution that 20 mL volumetric concentrations are 60%, stirring and dissolving, makes B solution;

3. A solution mixes with B solution: under 65 ℃ of water bath condition, B solution is slowly added in A solution, separate out precipitation;

4. reflux, filter, wash and be dried: 2 h that reflux under 65 ℃ of water bath condition, filtered while hot, removes unreacted baicalin and slaine with the methanol solution washing of volumetric concentration 60%, and 50 ℃ of vacuum dryings are to constant weight.

4. the medical compounds of anti-hepatocarcinoma as claimed in claim 3, it is characterized in that described step (3) 2. a kind of solid metal salt in the preparation of B solution be in solid four hydration nickel acetates, solid four hydration cobaltous acetate or solid-hydration Schweinfurt green any.

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