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Detection and identification of two composition components in toad skin extract

A technology of extracts and compositions, applied in the field of traditional Chinese medicine, can solve problems such as difficulties, complex analysis, and unclear basis of effect substances, and achieve the effect of strong persuasion and little interference

Active Publication Date: 2014-09-24
ANHUI CHINA RESOURCES JINCHAN PHARMA CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Based on the lack of comprehensive and in-depth research on the chemical patterns of bufoligin components in toad skin extract, the effector basis of its clinical anti-tumor effect has not been clarified for a long time
On the other hand, the analysis of the composition of multiple polypeptide components is more complicated and difficult

Method used

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  • Detection and identification of two composition components in toad skin extract
  • Detection and identification of two composition components in toad skin extract
  • Detection and identification of two composition components in toad skin extract

Examples

Experimental program
Comparison scheme
Effect test

experiment example 1

[0030] The specific operation method is as follows: after taking the dried toad skin for repair, add 8 times of water (V / m), decoct twice for 30 minutes each time, filter, combine the filtrate, and concentrate. Concentrate to a small volume (relative density is 1.01~1.35, 80°C), cool to 40°C, add 95% ethanol to adjust the alcohol concentration to 40~60%, carry out alcohol precipitation once, then pour out the supernatant; recover the supernatant Ethanol until there is no alcohol smell, add 95% ethanol to adjust the alcohol concentration to 75-90% for secondary alcohol precipitation, after standing still, filter under normal pressure, concentrate the filtrate to a thick paste, refrigerate for 24 hours, and centrifuge to obtain toad skin extract .

[0031] Experimental example 2: Preparation of polypeptide composition in toad skin extract of the present invention

[0032] Sephadex G10, G15, G25, G50, G75 or LH20 was used to separate and purify the toad skin extract described in...

experiment example 2

[0033] Alternatively, use polymethacrylate gel HW40, HW50 or HW65 to separate and purify the intermediate, the ratio of column diameter to height is 1:15~20, the ratio of loading volume to sample is 1:10~40, and the constant flow rate is 3~10ml / min Eluted with deionized water, collected according to 1 / 10 of the column volume, followed by biuret reaction, collected positive fractions, combined and freeze-dried to obtain a polypeptide composition.

[0034] Experimental example 3: Estimation of the molecular weight value of the polypeptide composition

[0035] 3.1 Analysis conditions and results of standard products:

experiment example 3

[0036] 3.1.1 Standard product and its preparation:

[0037] Standard: Somatostatin MW 1638

[0038] Thymosin Alpha1 MW 3108

[0039] Insulin: MW 5808

[0040] RNase MW 13700

[0041] The above standard was dissolved in water and prepared into a solution with a concentration of 1 mg / mL for testing.

[0042] 3.1.2 Analysis conditions: Chromatographic column: G2000SWXL (7.8mm*30cm)

[0043] Column No.: S0042

[0044] Mobile phase: ACN:H2O=2:8 (plus 0.05%TFA)

[0045] Flow rate: 0.7mL / min

[0046] Detector: UV214nm

[0047] 3.1.3 Analysis results: (see Table 1, figure 1)

[0048] Table 1 Standard retention time, retention volume and logarithm of molecular weight

[0049] 3.2 Determination conditions and results of peptide samples

[0050] 3.2.1 Preparation of test samples:

[0051] The polypeptide sample is the polypeptide composition in the toad skin extract described in Example 2 of the present invention.

[0052] Take the polypeptide composition in Example 2, diss...

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Abstract

The invention provides a detection and identification method of two composition components in a toad skin extract. The method comprises the following steps: preparing dry toad skin as a raw material into a middle extract by water extraction and alcohol precipitation methods, and carrying out gel chromatographic separation and ODS purification, so as to obtain a composition formed by one or N polypeptides. A correction curve is prepared by adopting an HPLC-gel chromatography method and combining with a known standard product. The molecular weight distribution range of the composition components is 1000-10000Da. The invention also relates to a fat-soluble composition in the toad skin. The fat-soluble composition is the composition composed by one or N fat-soluble components obtained by taking the dry toad skin as the raw material and adopting a solvent or chromatography method after preparing the middle extract by the water extraction and alcohol precipitation methods. According to the high performance liquid chromatography, octadecylsilane chemically bonded silica is taken as a filler, acetonitrile-water is taken as a mobile phase to carry out gradient elution, and the compound represented by 33 chromatographic peaks in the fat-soluble composition of the toad skin is identified by the combined mass spectrum (HPLC-MS) by deducing fragment information in the mass spectrum and comparing with the known standard substance.

Description

technical field [0001] The invention belongs to the technical field of traditional Chinese medicine, and in particular relates to the detection and identification of two types of composition components in toad skin extract and preparations containing the one or two compositions. Background technique [0002] Toad skin is the dried skin of Bufo Bufo Bufo or Bufo Black Orbitus, which has the effects of clearing away heat and detoxification, diuresis and reducing swelling. A number of drugs developed from the water-soluble parts of toad skin have been marketed, especially cinobufacin injection, which is a water-soluble preparation processed from the skin of Bufo bufo. The overall effective rate is 56%, the tumor shrinkage rates are 10% and 16%, respectively, and it has a synergistic effect on chemotherapy and radiotherapy. Clinical application shows that cinobufacin injection has the effects of detoxification, detumescence and pain relief. It is used to treat middle and advanc...

Claims

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Application Information

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IPC IPC(8): A61K35/56A61K38/00G01N30/88A61P35/00A61P31/20
Inventor 边宝林高波司南王宏洁罗川
Owner ANHUI CHINA RESOURCES JINCHAN PHARMA CO LTD
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