CN108129581B

CN108129581B - Metal complex capable of resisting tumor

Info

Publication number: CN108129581B
Application number: CN201710306212.6A
Authority: CN
Inventors: 陈奕; 聂少平; 谢明勇
Original assignee: Lattice Power Jiangxi Corp
Current assignee: Lattice Power Jiangxi Corp
Priority date: 2017-05-03
Filing date: 2017-05-03
Publication date: 2020-07-14
Anticipated expiration: 2037-05-03
Also published as: CN108129581A

Abstract

The invention provides a novel anti-tumor compound, which is prepared by taking ganoderma atrum as a raw material and performing extraction, refining separation and metal complexation on the ganoderma atrum to obtain a product PSG-Fe or PSG-Ca, has good effect of eliminating DPPH free radicals and strong reducing power, can neutralize more linoleic acid free radicals and other free radicals formed in a system, slows down the color fading of β -carotene, and can be used for anti-tumor.

Description

Metal complex capable of resisting tumor

Technical Field

The invention relates to a novel anti-tumor compound, in particular to an anti-tumor metal complex.

Background

In recent years, with the change of life style and the improvement of medical technology, the number of tumor detection people is also increasing, tumors become one of important diseases which pose serious threats to human health, and because of higher damage and higher fatality rate to the organism of a patient, the application and research of anti-tumor drugs are always the focus of attention of people, and new treatment theory and novel drugs are continuously developed and applied.

Scientists synthesize many anti-tumor molecular drugs with definite molecular targets, but because the development period of chemical synthetic drugs is long, the research and development cost is high, and the clinical toxic and side effects are large, the further research and development and the application of the drugs are restricted. Natural medicinal materials such as Chinese herbal medicines, plant medicines and the like have wide bioactive substances, and researchers can search antitumor medicines and antitumor auxiliary medicines with small toxic and side effects and unique pharmacological effects from the traditional Chinese medicines.

Disclosure of Invention

The invention aims to provide a novel anti-tumor medicament which has obvious activation effect on immune macrophages, kills tumor cells by stimulating the immune function of an organism, and does not directly resist tumors through the cytotoxic effect.

The technical scheme of the invention is as follows:

an anti-tumor metal complex is prepared by the following steps:

(1) soaking 100g of dried ganoderma atrum medicinal material in 95% ethanol at room temperature for 48h, filtering, repeating the steps once, placing the medicinal residues at room temperature for airing, mixing the medicinal residues with distilled water according to the mass-volume (kg/L) ratio of 1: 20, performing reflux extraction twice, sequentially extracting for 2h and 1h each time, combining the extracting solutions, performing centrifugal separation, performing reduced pressure concentration, adding an appropriate amount of 95% ethanol into the obtained sample to ensure that the volume fraction of the ethanol is 80%, performing precipitation, standing at 4 ℃ for 24h, performing centrifugal filtration, combining the precipitates, sequentially washing the precipitates with absolute ethanol, absolute ethyl ether and acetone, performing freeze drying to obtain a sample, taking the obtained dried sample, removing protein by a Sevag method, namely uniformly mixing Sevag reagent (the volume ratio of chloroform to n-butyl alcohol is 5: 1) and the dried sample according to the mass ratio of 1:1, standing, removing a lower protein denaturation layer, concentrating the sample, repeating the protein removal operation for 10 times, concentrating the protein removal sample, washing with 95% ethanol, performing freeze drying again, concentrating to obtain a crude extract, dialyzing the crude extract in a fine bag, and drying the sample to obtain a fine extract, and drying the fine extract in a fine bag, performing dialysis to obtain a fine dry sample, wherein the fine dry;

(2) taking 500mg of the obtained ganoderma atrum fine extract, adding distilled water to prepare a solution with the concentration of 3-5 mg/m L, centrifuging, passing supernate through a gel column chromatography, and using a chromatography system

A Purifier100 biological macromolecule purification system comprises a gel column Hiload 26/60Superdex-200prep grade, wherein a sample and eluent pass through a 0.22 mu m microporous membrane to protect a separation column before sample loading, the eluent is distilled water, the flow rate is 2m L/min, the sample loading volume is 5m L, the sample is collected in 8m L/tube in part, an ultraviolet detector and a differential refraction detector (RID-10A differential detector) are used for detecting the elution condition on line, and a 6 th tube to a 14 th tube are combined, concentrated and freeze-dried to obtain ganoderma atrum extract PSG which is yellow or brown flocculent solid;

(3) and complexing the PSG with metal ions to obtain PSG-Fe or PSG-Ca.

The metal complex product PSG-Fe or PSG-Ca has good effect of eliminating DPPH free radicals and strong reducing power, can neutralize more linoleic acid free radicals and other free radicals formed in a system, slows down the color fading of β -carotene, and can be used for resisting tumors.

Drawings

FIG. 1 is a high performance gel permeation chromatogram of PSG-Fe.

FIG. 2 is an infrared spectrum of PSG and PSG-Fe.

FIG. 3 is an infrared spectrum of PSG and PSG-Ca.

FIG. 4 is a graph showing the ability of PSG, PSG-Fe, PSG-Ca to scavenge DPPH radicals.

Fig. 5 is a graph of a ferrous sulfate standard curve.

FIG. 6 is a graph showing the ability of PSG and PSG-Fe, PSG-Ca to inhibit the oxidation of β -carotene-linoleic acid system.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

Example 1: an anti-tumor metal complex is prepared by the following steps:

Purification of Purifier100 biological macromoleculeThe system comprises a gel column Hiload 26/60Superdex-200prep grade, wherein before sample loading, a sample and an eluent are passed through a 0.22 mu m microporous filter membrane to protect a separation column, the eluent is distilled water, the flow rate is 2m L/min, the sample loading volume is 5m L, the sample is collected in 8m L/tube in part, and an ultraviolet detector and a differential refraction detector (RID-10A differential detector) are used for detecting the elution condition on line;

(3) the preparation of PSG-Fe comprises weighing PSG300mg, adding 0.5g sodium citrate as catalyst, dissolving in 30m L deionized water, stirring continuously in 60 deg.C constant temperature water bath, simultaneously dripping 2 mol/L ferric trichloride solution and 20.0% NaOH solution, adjusting dripping speed to 1 drop (about 0.05 ml)/30 s, controlling pH of reaction solution to 8, and adding FeCl just dripped during reaction₃The solution begins to form a precipitate, then the precipitate is dissolved along with the reaction, when the precipitate generated in the reaction is not dissolved any more, the complexation capability of the reaction system is saturated, namely FeCl dropwise addition is stopped₃And continuously stirring the solution and the NaOH solution for lh in a water bath at 60 ℃, reacting and cooling, and then centrifuging, dialyzing and precipitating with ethanol to obtain the PSG-Fe complex.

Example 2: an anti-tumor metal complex is prepared by the following steps:

(3) PSG-Ca preparation comprises weighing 300mg PSG, and mixing with 20M L0.01.01M CaC1₂The solution (prepared with triple distilled water) was dissolved. The reaction l2h was stirred magnetically at a temperature of 50 ℃. And centrifuging at 4000r/min for 10min to obtain supernatant. And putting the supernatant into triple distilled water for fully dialyzing for 1 d. Concentrating the dialysate at 45 deg.C in rotary evaporator, adding excessive ethanol into the concentrated solution, precipitating, filtering or centrifuging, collecting precipitate, washing with anhydrous ethanol for several times, and detecting no chloride ion in the filtrate (detecting whether precipitate is generated with silver nitrate solution). And (3) putting the precipitate into a vacuum drying oven for drying (50 ℃, the vacuum degree is 0.09 atmospheric pressure) for 2h to obtain the grey white powdery poly PSG-Ca complex.

First, the physical and chemical properties of the product

The PSG-Fe and PSG-Ca obtained in example 1-2 were subjected to infrared spectroscopic analysis while measuring the purity, average molecular weight, and iron and calcium mass fractions of the complex.

(1) Purity determination

The purity was checked by high performance gel permeation chromatography, 3mg of the test sample was dissolved in 1m L distilled water, centrifuged at 1000rpm for 10min, filtered through a microfiltration membrane and automatically loaded to 20 μ L, and the elution profile was recorded for determining the purity of the fraction.

The specific test conditions for HP L C used for the experiment were as follows:

the chromatographic column is Ultrahydrogel^TM500300 mm × 7.8.8 mmid, 0.1M NaCl as mobile phase, 0.6m L/min as flow rate and 35 deg.c as column temperature.

(2) Determination of molecular weight

The liquid chromatographic column is Ultrahydrogel^TM500300 mm × 7.8.8 mmid, mobile phase 0.1MNaCl, flow rate 0.6m L/min, column temperature 35 ℃.

Preparing Glc, T10, T40, T70, T500 and T20002% solutions of each standard, injecting HP L C into each solution to obtain corresponding liquid phase map, and setting the elution volume of T2000 as the empty volume V of the column₀Elution volume of Glc as total volume V of column_tExpressed by the formula Kav ═ V (V)_e-V₀)/(V_t-V₀) Calculation, Standard Curve was plotted as L g Mw-Kav, according to V of the sample_eAnd obtaining the molecular weight of the sample from the standard curve. Because the flow rate of the eluent is constant, a standard curve of the relation between the retention time and the molecular weight can be drawn, and the molecular weight of each product can be obtained according to a regression equation of the standard curve.

(3) Determination of the iron and calcium mass fractions of the complex:

and (3) accurately weighing 0.0100g of the prepared sample, adding 5m of L concentrated nitric acid, digesting by using a microwave digestion instrument, measuring the contents of Ca and Fe metal elements in the obtained digestive juice by using ICP-AES (inductively coupled plasma-atomic emission Spectrometry), and further comparing the complexing capacity and the complexing amount of PSG (particle swarm optimization) on the two beneficial metal elements.

(4) Infrared Spectroscopy (IR)

Taking 1-2mg of sample, using KBr tablet to carry out conventional IR analysis, and the determination parameters of the infrared spectrometer are as follows: number of background scans: 128 times; resolution ratio: 4.0cm^-1(ii) a A detector: and (4) DTGS.

TABLE 1 average molecular weight of the product and elemental contents of Fe, Ca

A large number of active groups such as hydroxyl and carboxyl exist in PSG molecules, so that the PSG has good metal coordination capacity and can form a complex with metal ions. As can be seen from Table 1, PSG-Ca has a high average molecular weight and a low complexing degree, while PSG-Fe has a very low average molecular weight and a relatively high complexing degree. This may be for the following reasons: different complexing processes, different degradation degrees of PSG and different complexing degrees; the degree of complexation is also related to the charge carried by the metal ion. Since the iron complexation is carried out under alkaline conditions, there is a possibility that the PSG molecules will be degraded during the complexation process, resulting in a substantial decrease in molecular weight. PSG-Fe after desalting by dialysis is dissolved in water as K₄[Fe(CN)₆]The aqueous solution was examined and showed no particular effect of Fe (III) ion. Confirming the absence of free Fe in the TPC aqueous solution³⁺. Indicating that a more stable complex was formed.

FIGS. 3 and 4 are infrared spectra of PSG-Fe and PSG-Ca. Compared with the PSG infrared absorption spectrum, the basic skeleton of the spectrum of the PSG-Fe complex is obviously changed, especially at 500-1000cm^-1And the vicinity indicates that the PSG is partially degraded in the basic molecular structure after iron complexation. The basic skeleton of the spectrum of the PSG-Ca complex did not change, but the PSG was 3395cm^-1Nearby hydroxyl (-OH) absorption peaks are obviously enhanced in PSG-Ca and PSG-Fe and move to high wavenumbers because the hydrogen bond association of-OH is weakened after the coordination with the hydroxyl of PSG; PSG at 1413cm^-1Stretching vibration of C-O at carboxyl (-COOH) or changing angle of-C-H at methine group in the high wave number 1419cm^-1In the iron complex, the wave number of the medium-to-high wave is 1429cm^-1The C-O of carboxyl (-COOH) participates in coordination; at 1075cm^-1The secondary hydroxyl absorption peak of (2) in the calcium complex is shifted to 1094cm^-1Moving towards 1070cm in the iron complex^-1(ii) a Located at 1035.03cm^-1The O-H absorption peak at the carboxyl group (-COOH) was still present in both complexes, but the intensity of the absorption peak was significantly reduced.

Second, in vitro antioxidant activity test

1. Scavenging DPPH free radical

Since the product of the invention is insoluble in ethanol solution with high concentration, the method needs to be improved to determine the scavenging effect of the product of the invention on DPPH free radicals, and the improved method is as follows:

preparing 0.2 mmol/L solution of DPPH by 95% ethanol, taking sample solutions with different concentrations of 1m L, putting 2m L of freshly prepared DPPH ethanol solution and 2m L95% ethanol in the same test tube with a plug, fully mixing the solutions, reacting in the dark for 30min, taking out the solution, and measuring the absorbance at 525nm, replacing the DPPH solution with 2m L95% ethanol in a blank group, mixing 2m L of DPPH solution and 3m L95% ethanol in a control group, taking triplicate samples of each group, and taking the average value.

The lower the absorbance of the reaction system is, the stronger the ability of eliminating DPPH free radicals is, and the calculation formula of the DPPH free radical clearance is as follows:

clearance rate of [1- (Ai-Aj)/Ac ] × 100% (9.1)

Wherein Ai is the absorbance value of the sample group, Aj is the absorbance value of the blank group, and Ac is the absorbance value of the control group.

2. Determination of Total reducing Capacity

The total reducing capacity of the sample is determined by using an iron ion reduction capacity method (FRAP).

Preparation of a reagent, namely 10 mmol/L2, 4, 6-tri (2-pyridyl) triazine (TPTZ) solution, weighing 0.0312g TPTZ, and fixing the volume to 10m L by using 40 mmol/L hydrochloric acid solution, wherein FRAP working solution consists of 300 mmol/L acetate buffer solution with pH of 3.6, 10 mmol/L TPTZ solution and 20 mmol/L ferric trichloride solution in a ratio of 10:1: 1.

And (3) taking a ferrous sulfate standard solution 0.2m L with different concentrations of 0.1, 0.2, 0.4, 0.6, 0.8 and 1.0 mmol/L into a reaction tube, respectively adding 6m L FRAP working solution and 0.6m L ultrapure water, uniformly mixing, accurately reacting for 5min, measuring absorbance at 593nm, taking the ultrapure water as a blank, and taking the concentration of the ferrous sulfate solution as a horizontal coordinate and the absorbance as a vertical coordinate to obtain a standard curve.

Samples of a given concentration were assayed under identical conditions in triplicate and the average taken. The reducing power of the sample is expressed in units of mmol/g by FRAP value, and the larger the FRAP value, the stronger the reducing power.

3. Experiment for inhibiting β -carotene-linoleic acid system oxidation

Dissolving 5mg of β -carotene in 50m L chloroform, adding 40mg of linoleic acid and 400mg of Qubi-pass X-100 into the chloroform, performing rotary evaporation concentration at 40 ℃ for 5min to remove the chloroform, adding 100m L of distilled water saturated with oxygen, and fully mixing to obtain an emulsion system, taking 4.5m L of the prepared emulsion, adding sample solutions with different concentrations or ultrapure water 0.5m L, uniformly mixing, measuring an absorbance value at 470nm as the absorbance at zero time, placing the reaction liquid in a 50 ℃ water bath for 2h, measuring the absorbance at 470nm again, taking the reaction liquid without β -carotene as a blank reagent, and taking BHT as a positive control, and calculating the antioxidant rate (%) of the sample according to the fading condition of β -carotene according to the following formula:

wherein, A0 and A0' are the absorbances of the sample and the blank group at the zero moment respectively; at and At' are the absorbance after 2h of reaction, respectively.

4. Results and discussion

(1) DPPH radical scavenging action of Each test substance

As can be seen from FIG. 4, the magnitude of the ability of PSG and its metal complex to scavenge DPPH radicals at the same concentration is in the order: PSG-Ca > PSG-Fe, it can be presumed that: after-OH or-COOH on the sugar ring of PSG is complexed with different metal ions, the spatial structure is changed, and the activities of removing free radicals are different. The PSG-Fe has reduced ability to scavenge DPPH free radicals, and may be a great change in structure and conformation after the PSG is complexed with Fe, so that a part of the active sites of the PSG are hidden, thereby having a certain influence on the antioxidant activity.

(2) Determination of Total reducing Capacity of Each test substance

Many studies have demonstrated that antioxidant activity is related to reducing power. The reducing power is an important index representing the ability of an antioxidant substance to supply electrons, and can be used to stabilize a radical by supplying electrons, thereby interrupting the chain reaction of the radical. Research has also shown that the reducing power is related to the presence of reducing ketones, which can provide hydrogen atoms and stop the chain reaction of free radicals; meanwhile, the antioxidant can also react with the precursor of the peroxide to prevent the generation of the peroxide, thereby playing the role of antioxidation.

The reduction capacity of PSG and the derivative thereof is measured by using an iron ion reduction capacity method. The antioxidant can convert Fe³⁺Reduction to Fe²⁺，Fe²⁺The complex with the tripyridyl triazazine (TPTZ) generates a blue substance, the blue substance has characteristic absorption at 593nm, the absorbance and the concentration are in positive correlation, and the greater the absorbance, the greater the FRAP value, the better the antioxidant effect. The standard curve of ferrous sulfate content is shown in fig. 5, different samples are measured simultaneously, the measurements are performed in parallel for three times, FRAP values are calculated through the standard curve, and the obtained results are shown in table 2.

As can be seen from Table 2, PSG has a strong reducing power even exceeding that of the control BHT. The reduction capability of the complexed product is found to be very low after the PSG and the iron are complexed, and the reason may be related to that the PSG is a ferric ion rather than a ferrous ion, the color is interfered, and the molecular structure is changed after the PSG and the ferrous ion are complexed. The reduction capability of PSG-Ca is slightly lower than that of PSG, but the difference is not obvious, which shows that PSG-Ca not only can supplement calcium, but also has no great influence on the reduction effect.

TABLE 2 Total reducing power (FRAP value) of the respective products

Treatment (Treatment)	FRAP Value(mmol/g)
		PSG	0.2066
PSG-Fe	0.0138
		PSG-Ca	0.2026
BHT	0.1594

(3) Determination of the ability of each test substance to inhibit the oxidation of β -Carotene-linoleic acid System

In β -carotene-linolic acid system, which evaluated the total antioxidant capacity, the discoloration of β -carotene was caused by the oxidation of β -carotene by the free radical generated by the hydroperoxide produced by linoleic acid, in this system linoleic acid abstracts a hydrogen atom from one of the diallyl methylene groups to form the linolic acid radical, attacking the highly unsaturated β -carotene, causing β -carotene to be oxidized, losing the chromophore and its characteristic orange color, the presence of antioxidant scavenges the free radical in the reaction system, protects β -carotene from oxidation, preventing discoloration of β -carotene, the total oxidizing capacity of PSG and PSG-Fe, PSG-Ca is shown in FIG. 6, as seen in FIG. 6, the total antioxidant capacity of PSG-Fe, PSG-Ca is better than PSG, indicating that the presence of PSG-Fe, PSG-Ca neutralizes more linolic acid radicals and other free radicals formed in the system, and slows the discoloration of β -carotene.

Claims

1. An antitumor metal complex characterized by: the preparation method comprises the following steps:

(1) taking 100g of dry ganoderma atrum medicinal material, adding 95% ethanol, soaking and extracting for 48h at room temperature, filtering, repeating the steps once, placing the medicinal residue at room temperature for airing, then mixing the medicinal residue with distilled water according to the mass-volume ratio of 1: 20 kg/L, carrying out reflux extraction twice, wherein the extraction time is 2h and 1h in sequence, combining the extracting solutions, carrying out centrifugal separation and reduced pressure concentration, adding a proper amount of 95% ethanol into the obtained sample to ensure that the volume fraction of the ethanol is 80%, then carrying out precipitation, standing for 24h at 4 ℃, carrying out centrifugal filtration, washing the precipitate with absolute ethyl alcohol, absolute ethyl ether and acetone in sequence, carrying out freeze drying to obtain a sample, taking the obtained dried sample, removing protein by a Sevag method, carrying out concentration on the obtained dried sample, carrying out protein removal operation for 10 times, then carrying out concentration by using 95% ethanol, washing the crude extract, dialyzing the crude extract in a small bag, carrying out dialysis, and carrying out freeze drying on the obtained sample to obtain a black ganoderma lucidum sample, wherein the volume ratio of the black ganoderma lucidum is 1:1 h;

(2) taking 500mg of the obtained ganoderma atrum fine extract, adding distilled water to prepare a solution with the concentration of 3-5 mg/m L, centrifuging, carrying out gel column chromatography on the supernatant, wherein the used chromatography system is a Purifier100 biomacromolecule purification system, a gel column Hiload 26/60Superdex-200prep grade, passing a sample and an eluent through a 0.22 mu m microporous filter membrane to protect a separation column before sample loading, the eluent is distilled water with the flow rate of 2m L/min and the sample loading volume of 5m L, collecting the sample and the eluent in parts with the volume of 8m L/tube, detecting the elution condition by an ultraviolet detector and a differential refraction detector on line, combining 6 th-14 th tubes, concentrating, and freeze-drying to obtain a ganoderma atrum extract PSG which is a yellow or brown flocculent solid;

(3) the preparation of PSG-Ca comprises the steps of weighing 300mg of PSG, dissolving the PSG with 20M L0.01.01M CaC12 solution, electromagnetically stirring and reacting at 50 ℃ for l2h, centrifuging at 4000r/min for 10min after the reaction is finished to obtain supernatant, putting the supernatant into triple distilled water for full dialysis for 1d, then concentrating the dialysate on a rotary evaporator at 45 ℃, adding excessive ethanol into the rotary evaporation concentrated solution, separating out precipitate, filtering or centrifuging, collecting the precipitate, washing the precipitate for multiple times with anhydrous ethanol until no chloride ions exist in the filtrate, putting the precipitate into a vacuum drying oven for drying under the drying condition of 50 ℃, the vacuum degree of 0.09 atmospheric pressure and the drying time of 2h to obtain the off-white powdery multi-PSG-Ca complex.