CN1245623C - Lentinan molecular weight and molecular weight distribution measuring method - Google Patents

Abstract

The present invention relates to a method for measuring the molecular weight and the distribution of natural macromolecular high polymers of lentinan by a universal correction method. The method is characterized in that gel permeation chromatography-HPGPC with high efficiency is adopted; a moving phase of the HPGPC can be ultrapure water, sodium nitrate with 0.1 to 0.5M, sodium acetate trihydrate with 0.1 to 0.5M and a phosphate buffer solution; analytical columns of the HPGPC can be chromatographic columns suitable for polysaccharide, the number of the analytical columns can be from 1 to 4, and a separation range can be from 2, 000, 000 to 1000 daltons; a column temperature of the analytical columns of the HPGPC can be kept between 30 to 55 DEG C; a flow speed of the HPGPC can be from 0.1 ml/min to 1.0 ml/min; standard materials for drawing standard curves relative to the HPGPC can be Dextran or Pullulan; standard materials for drawing correction curves universally suitable for the HPGPC are graded separation products of lentinan.

Description

Lentinan molecular weight and molecular weight distribution determination method

Technical field

The present invention relates to a kind of molecular weight and molecular weight distribution determination method, specifically a kind of lentinan molecular weight and molecular weight distribution determination method.

Background technology

Lentinan is a kind of biological response modifier (Biological Response Modifier BRM) that is widely used in anti-tumor chemotherapeutic and radiotherapy at present.Particularly be applied to the toxic and side effect that cancer of the stomach, lung cancer, intestinal cancer, breast cancer have tangible chemotherapeutic sensitivity and reduces chemotherapy and radiotherapy, the chemotherapy of other tumours is all had synergy in various degree and reduces toxic and side effect.Lentinan is to be main chain with β-D-(1 → 3) glucose, and β-D-(1 → 6) glucose is the glucosan of side chain, is the superpolymer of polydispersity.Discover that lentinan molecular weight and animal and even clinical antitumor activity have correlationship, therefore, the assay method of research lentinan molecular weight is significant to control lentinan inherent quality.Yet, the method institute accepted standard material of measuring the macromolecular polysaccharide molecular weight at present is often inequality with the chemical constitution of measured matter, what set up thus is relative standard's curve, and measured result is a relative molecular weight, with the actual molecular weight of survey material bigger error is arranged.Therefore, need set up a kind of industrial lentinan molecular weight and Determination of distribution method thereof of being fit to.

Summary of the invention

The objective of the invention is to set up the method that a kind of universal calibration method is measured lentinan natural macromolecular molecular weight of high polymer and distribution thereof.

Principle of the present invention is according to Mark-Houwink experimental formula [η=KM^α] carry out, η is an intrinsic viscosity in the formula, and M is a molecular weight, and K, α are coefficient.

One, the molecular weight of product lentinan of the present invention, molecular formula, chemical structural formula

1, molecular weight determination

Through the efficient gel permeation chromatography, adopt pervasive calibration curve method determining molecular weight mostly between 40～800,000.

2, nuclear magnetic resonance of carbon spectrum (seeing Table 1)

Table 1

C	Lentinan (Lentinan)	Japan produces lentinan	Resolve
				1	104.6	104.6	β-D-(1 → 3) glucose
2	74.5	74.3	β-D-(1 → 3) glucose
				3	87.7	87.7	β-D-(1 → 3) glucose
4	70.2	70.2	β-D-(1 → 3) glucose
				5	77.8	77.9	β-D-(1 → 3) glucose
6	62.5	62.5	β-D-(1 → 3) glucose
				1′	104.9	104.6	β-D-(1 → 6) glucose
6′	71.5	71.5	β-D-(1 → 6) glucose

3, infrared spectrogram (see figure 1)

4, molecular formula

The result shows through trace element analysis, and molecular formula is (C₆H₁₀O₅) n

5, chemical structural formula

In conjunction with spectrographic method, the primary structure formula of lentinan is as follows through chemical reactions such as periodate oxidation and Smith degradeds:

Purpose of the present invention can reach by following measure:

A kind of lentinan molecular weight and molecular weight distribution determination method is characterized in that:

Adopt efficient gel permeation chromatography-HPGPC;

The moving phase of HPGPC is selected ultrapure water, 0.1～0.5M sodium nitrate, 0.1～0.5M sodium acetate and phosphate buffered solution for use;

The analytical column of HPGPC is selected the chromatographic column that is fit to polysaccharide for use, and with one～four, separating ranges is that 2,000,000～1000 daltonian chromatographic column is formed;

The column temperature of HPGPC analytical column remains on 30 ℃～55 ℃;

The flow velocity of HPGPC is 0.1ml/min～1.0ml/min;

The standard substance of drawing HPGPC relative standard curve is with Dextran or Pullulan;

The standard substance of drawing the pervasive calibration curve of HPGPC is the fractionated product of lentinan.

Purpose of the present invention can also reach by following measure:

Adopt the typical curve of pervasive correction to measure the molecular weight and the distribution thereof of lentinan;

Adopt alkali lye dissolving lentinan;

Used alkali lye is NaOH, potassium hydroxide, sodium carbonate or sodium bicarbonate;

The concentration of used alkali lye is 0.1～1MNaOH, 0.1～1MKOH, 0.5～1MNa₂CO₃Or 0.1～1MNaHCO₃

The moving phase of HPGPC is selected sodium acetate and the phosphate buffer of 0.1mol/L for use;

The pH value of described sodium acetate and phosphate buffer is 6.0～8.0;

The array mode of the analytical column of HPGPC is three stainless steel column series connection, and separating ranges is 2,000,000～1000 dalton;

The column temperature of HPGPC analytical column is 30 ℃;

The flow velocity of HPGPC keeps 0.4ml/min;

The weight-average molecular weight dalton of lentinan fractionated product is respectively:

1、62.4×10⁴，2、53.4×10⁴，3、18.4×10⁴，4、10.6×10⁴，5、7.72×10⁴；

Recording lentinan K value is 0.0535～0.0543, and the α value is 0.6～0.8;

The lentinan K value of optimizing is 0.0539, and the α value is 0.607.

The present invention adopts pervasive calibration curve that the lentinan molecular weight is proofreaied and correct the molecular weight that obtained and molecular weight distribution to similar with the light scattering method measured result, and error is less, and degree of accuracy improves.

Description of drawings

Fig. 1 is the infrared spectrogram of lentinan of the present invention;

Fig. 2 is a HPGPC detection system reappearance displayed map of the present invention.

Embodiment

Implementation step of the present invention is to adopt standard substance Pullulan series or dextran (Dextran) series that can buy on the market to measure relative standard's curve respectively, and calculating K, α value.

The present invention adopts its intrinsic viscosity of lentinan fractionated products measure further, and with the molecular weight of its fractionated product of light scattering determining, set up pervasive calibration curve, setting up with Dextran or Pullulan is general efficient gel permeation chromatography (HPGPC) the mensuration lentinan molecular weight of standard and the method for distribution thereof.

The present invention be based upon 25 batches of lentinans (trade name: Lentinan, down with) on the basis of molecular weight and molecular weight distribution (be called for short distribute, down with) experimental study gained data.Data statistic referring to table 2 K, α value.

The mensuration of lentinan K, α value:

In order to measure lentinan K, α value, choose the lentinan fractionated sample of five different molecular weights, use the light scattering determining molecular weight: 62.4 * 10⁴, 53.4 * 10⁴, 18.4 * 10⁴, 10.6 * 10⁴, 7.72 * 10⁴According to the regulation of Chinese Pharmacopoeia (version in 2000) appendix, under 0.1mol/L sodium acetate and phosphate buffer (pH7.8～8.0), 30 ℃ of conditions, measure intrinsic viscosity, use η=KM^αFormula calculating K, α value have been measured 25 groups of K, α value (newly join phosphate buffer at every turn and measure the pH value, detailed numerical value sees the following form) altogether, and arithmetic mean and the standard deviation of calculating K, α are judged the choice of numerical value with the Grubbs method of inspection respectively.

The data statistic of table 2 K, α value

Number of times	Phosphate buffer pH value	K	α
				1	7.89	0.0540	0.605
2	7.91	0.0538	0.607
				3	7.93	0.0540	0.606
4	7.86	0.0538	0.607
				5	7.88	0.0541	0.609
6	7.90	0.0536	0.604
				7	7.84	0.0535	0.609
8	7.92	0.0537	0.610
				9	7.87	0.0539	0.609
10	7.90	0.0537	0.609
				11	7.91	0.0540	0.608
12	7.89	0.0537	0.609
				13	7.90	0.0536	0.610
14	7.86	0.0536	0.608
				15	7.94	0.0540	0.608
16	7.97	0.0542	0.607
				17	7.84	0.0538	0.605
18	7.92	0.0540	0.605
				19	7.90	0.0538	0.602
20	7.95	0.0540	0.608
				21	7.81	0.0543	0.608
22	7.97	0.0538	0.607
				23	7.87	0.0538	0.610
24	7.95	0.0537	0.606
				25	7.89	0.0540	0.607
Mean value	7.90	0.0539	0.607
				Standard deviation	0.0404	0.000200	0.00201

The present invention also adopts laser light scattering method directly to measure 6 lot number lentinan weight-average molecular weight and checks.The result shows that the data that two kinds of methods are surveyed are similar to, and error is less.

Embodiment 1 instrument: the efficient gel permeation chromatograph is U.S. Waters company workstation system, 510 pumps, 410 differential refraction detectors, Millennium2010 system software and a GPC special software.Other high performance liquid chromatograph devices with above-mentioned accessory and function also can.

Can select separating ranges when (1) chromatographic column is selected gel chromatographic columns for use for use is 2,000,000～1000 daltonian chromatographic column, can adopt three stainless steel column series connection.For example: Ultrahydrogel1000,500 and 250.

(2) moving phase is successively with ultrapure water (resistance is 18.2M), 0.1mol/L sodium acetate and phosphate buffered solution (pH=7.8, according to version Chinese Pharmacopoeia appendix preparation in 2000) be moving phase, factors such as solubleness per sample, index of refraction, appearance time and peak shape are selected phosphate buffered solution pH=7.8 for use.

HPGPC collection of illustrative plates when (3) column temperature has successively compared 30 ℃, 35 ℃, 45 ℃, 55 ℃, wherein better, better with 30 ℃ collection of illustrative plates especially with 35 ℃, 30 ℃.

(4) velocity ratio the HPGPC collection of illustrative plates when 0.2ml/min, 0.4ml/min, 0.6ml/min, 0.8ml/min, 1.0ml/min all can, but be excellent with 0.4ml/min.

(5) it is better that sample size has compared the collection of illustrative plates of HPGPC of 100 μ l, 200 μ l, 300 μ l, but with 200 μ l be excellent.

(6) the drafting selection standard material Dextran of relative standard's curve series is five, and its molecular weight is 17.5 * 10⁵, 74.95 * 10⁴, 41.00 * 10⁴, 27.3 * 10⁴, 4.86 * 10⁴Or Pullulan series: 74.9 * 10⁴, 38 * 10⁴, 21.2 * 10⁴, 10 * 10⁴, 4.8 * 10⁴, 2.37 * 10⁴Also can.Its intrinsic viscosity at 30 ℃ of above-mentioned standard substance Dextran is respectively 74.8,57.1,48.6,35.5,23.0.According to formula η=KM^α, K, the α value of calculating standard specimen Dextran.K, α value according to Dextran are drawn relative standard's curve.

(7) drafting of pervasive calibration curve is made into the solution of 1mg/ml concentration with the Dextran standard specimen of 5 known molecular amounts and intrinsic viscosity, injects the high performance liquid chromatogram instrument, and by formula 1g η=1gK+ α 1gM calculates K and α value.The input computing machine is drawn 1g[η by GPC software] M-V₀The typical curve of (drenching volume), K value, α value input computing machine with lentinan obtain pervasive calibration curve, and the K value of lentinan and α value are narrated by following (8).(8) mensuration 1 of lentinan fractionated product and the molecular weight thereof) separation of lentinan graded product, purifying are referring to Nature 1,965 222 687-8 pages or leaves.The graded product of gained purifying is respectively with the molecular weight that light scattering method (being measured by State Bureau of Technical Supervision standard substance research centre) records five products: 62.4 * 10⁴, 53.4 * 10⁴, 18.4 * 10⁴, 10.6 * 10⁴, 7.72 * 10⁴Above-mentioned fractionated product is measured its intrinsic viscosity by 38 pages of VIG three therapeutic methods of traditional Chinese medicine of Chinese Pharmacopoeia two appendix of version in 2000 respectively.2) mensuration of lentinan K, α value.According to formula η=KM^α, recording the K value of lentinan under above-mentioned moving phase system is 0.0539, the α value is 0.607.Lentinan K, α value are determined according to the data of 25 groups of measurings.Referring to table 2.

(9) lentinan HPGPC method determining molecular weight and distribution lentinan test sample 5mg thereof add the sodium hydroxide solution 0.5ml swelling of 0.5mol/L, transfer PH to 7.0～8.0 with the hydrochloric acid of 0.5mol/L again, water is settled to 5ml and shakes up, with 0.45 μ membrane filtration promptly.Draw above-mentioned lentinan need testing solution 200 μ l, inject gel permeation chrommatograph, flow velocity 0.4ml/min, record chromatogram.Promptly get lentinan molecular weight and distribution collection of illustrative plates thereof after pervasive calibration curve is proofreaied and correct.See Table 3,4.

The a collection of molecular weight cumulative weight distribution curve of table 3 lentinan (Lentinan) burst statistical form

Slice Table showing 40 of 3877 slices

#	Cumulative％ (％)	Mol Wt (Daltons)	Retention Time (min)
				1	0.061	1707673	33.333
2	0.449	1496554	34.140
				3	3.015	1311536	34.948
4	9.061	1149391	35.755
				5	15.813	1007293	36.562
6	21.926	882762	37.370
				7	27.394	773626	38.178
8	32.402	677983	38.985
				9	37.043	594164	39.792
10	41.387	520708	40.600
				11	45.514	456333	41.407
12	49.470	399917	42.215
				13	53.210	350476	43.022
14	56.786	307146	43.830
				15	60.219	269174	44.637
16	63.554	235896	45.445
				17	66.759	206732	46.252
18	69.813	181174	47.060
				19	72.661	158776	47.867
20	75.277	139146	48.675
				21	77.721	121944	49.482
22	79.946	106868	50.290
				23	82.033	93656	51.097
24	83.981	82077	51.905
				25	85.793	71930	52.712
26	87.524	63037	53.520
				27	89.191	55244	54.327
28	90.757	48414	55.135
				29	92.273	42429	55.942
30	93.713	37183	56.750
				31	94.999	32586	57.557
32	96.067	28558	58.365
				33	96.971	25027	59.172
34	97.721	21933	59.980
				35	98.338	19222	60.787
36	98.839	16845	61.595
				37	99.277	14763	62.402
38	99.624	12938	63.210

39	99.875	11338	64.017
				40	100.000	9936	64.825

(10) reliability of pervasive calibration curve and evaluation of the accuracy see Table 4.

Table 4 HPGPC method and laser light scattering method are measured lentinan molecular weight comparison sheet as a result

The raw material lot number	Weight-average molecular weight (Mw) (unit: ten thousand)		Relative deviation (%)
				The HPGPC method	Light scattering method
	1	60.0				57.5	2.1
2			50.2	53.0	2.7
	3	70.4				71.8	1.0
4			56.6	61.0	3.7
	5	57.7				59.7	1.7
6			54.9	52.2	2.5

(11) HPGPC detection system reappearance is investigated to investigating the reappearance of HPGPC system, and a collection of lentinan for injection (Lentinan) is repeated sample introduction 5 times, and it the results are shown in Figure 2 and table 5.

The HPGPC collection of illustrative plates parameter that the repetition of certain batch of table 5 lentinan for injection is 5 times relatively

(12) data processing adopts Millennium2010 system software and the GPC special software that Waters company provides, and above software all obtains the U.S. FDA approval.

Claims (13)

1, a kind of lentinan molecular weight and molecular weight distribution determination method is characterized in that:

Adopt efficient gel permeation chromatography-HPGPC;

The moving phase of HPGPC is selected ultrapure water, 0.1～0.5M sodium nitrate, 0.1～0.5M sodium acetate and phosphate buffered solution for use;

The analytical column of HPGPC is selected the chromatographic column that is fit to polysaccharide for use, and with one～four, separating ranges is that 2,000,000～1000 daltonian chromatographic column is formed;

The column temperature of HPGPC analytical column remains on 30 ℃～55 ℃;

The flow velocity of HPGPC is 0.1ml/min～1.0ml/min;

The standard substance of drawing HPGPC relative standard curve is with Dextran or Pullulan;

The standard substance of drawing the pervasive calibration curve of HPGPC is the fractionated product of lentinan.

2, lentinan molecular weight according to claim 1 and molecular weight distribution determination method is characterized in that adopting the typical curve of pervasive correction to measure the molecular weight and the distribution thereof of lentinan.

3, lentinan molecular weight according to claim 2 and molecular weight distribution determination method is characterized in that adopting alkali lye dissolving lentinan.

4, lentinan molecular weight according to claim 3 and molecular weight distribution determination method is characterized in that used alkali lye is NaOH, potassium hydroxide, sodium carbonate or sodium bicarbonate.

5, lentinan molecular weight according to claim 4 and molecular weight distribution determination method, the concentration that it is characterized in that used alkali lye is 0.1～1MNaOH, 0.1～1MKOH, 0.5MNa₂CO₃Or 0.1～1MNaHCO₃

6, lentinan molecular weight according to claim 1 and molecular weight distribution determination method, the moving phase that it is characterized in that HPGPC is selected sodium acetate and the phosphate buffer of 0.1mol/L for use.

7, lentinan molecular weight according to claim 6 and molecular weight distribution determination method, the pH that it is characterized in that described sodium acetate and phosphate buffer is 6.0～8.0.

8, lentinan molecular weight according to claim 1 and molecular weight distribution determination method, the array mode that it is characterized in that the analytical column of HPGPC are three stainless steel column series connection, and separating ranges is 2,000,000～1000 dalton.

9, lentinan molecular weight according to claim 1 and molecular weight distribution determination method, the column temperature that it is characterized in that the HPGPC analytical column is 30 ℃.

10, lentinan molecular weight according to claim 1 and molecular weight distribution determination method is characterized in that the flow velocity of HPGPC keeps 0.4ml/min.

11, lentinan molecular weight according to claim 1 and molecular weight distribution determination method, its principle system is according to Mark-Houwink experimental formula [η=KM^α] carry out, η is an intrinsic viscosity in the formula, and M is a molecular weight, and K, α are coefficient, it is characterized in that the weight-average molecular weight dalton of the fractionated product of lentinan is respectively:

1、62.4×10⁴，2、53.4×10⁴，3、18.4×10⁴，4、10.6×10⁴，5、7.72×10⁴。

12, lentinan molecular weight according to claim 11 and molecular weight distribution determination method, it is characterized in that adopting lentinan fractionated product to record lentinan K value is 0.0535～0.0543, the α value is 0.6～0.8.

13, lentinan molecular weight according to claim 11 and molecular weight distribution determination method is characterized in that lentinan K value is 0.0539, and the α value is 0.607.

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