CN104237151A - Mass detection method for dendrobium officinale medicine - Google Patents
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Abstract
The invention discloses a mass detection method for dendrobium officinale medicine; total bibenzyl content in the medicine is detected by an ultraviolet spectroscopy; the mass detection method comprises the steps of: adopting bibenzyl compound as a reference substance, detecting total bibenzyl content by using an external standard method at 270-290nm. The method is simple, convenient, easy to implement and good in repeatability, can accurately reflect mass of the dendrobium officinale medicine, further completes quality standards of the dendrobium officinale medicine, and covers the shortage that mass evaluation of the dendrobium officinale is lack of the method for detecting fat-soluble characteristic components which are represented by bibenzyl; and therefore, the mass detection technology of the dendrobium officinale medicine is more scientific and reasonable. By adopting the detection method provided by the invention, mass of the dendrobium officinale medicine can be effectively controlled, and thereby safety, stability and effectiveness of clinical application of the dendrobium officinale medicine are ensured.
Description
Technical field
The present invention relates to medical art, specifically a kind of quality determining method of dendrobium candidum medicinal material.
Background technology
Dendrobium candidum is the orchid family Dendrobium per nnial herb, and its main medicinal part is fresh or dry stem, and being mainly distributed in the ground such as Anhui, Zhejiang, Guangxi, Hunan, Yunnan, Guizhou, is traditional rare traditional Chinese medicine.Dendrobium candidum has reinforcing stomach reg fluid, and nourishing Yin and clearing heat effect, hinders body fluid deficiency for the moon, dry polydipsia, deficiency of stomach-Yin, and food is few retches, and abnormal heat is not moved back after being ill, fire excess from yin deficiency, hectic fever due to yin labor heat, and order is secretly failed to understand, muscles and bones impotence is soft waits disease.Shennong's Herbal is classified as top grade, first of it is described as by Taoism regimen classics " Taoist Scriptures " " Chinese nine immortal grass ".
Main containing water miscible polysaccharide, fat-soluble bibenzyl in dendrobium candidum, also containing a small amount of water miscible amino acid, water miscible trace element, water miscible phenolic acid class, and the partially fat-soluble stem of noble dendrobium alkaloid of trace, fat-soluble stem of noble dendrobium sequiterpene, partially fat-soluble Fluorenone compounds, partially fat-soluble lignanoid, water miscible phenols, partially fat-soluble flavones, fat-soluble steroidal etc., wherein bibenzyl has the valuable pharmacological active functions such as antitumor, anti-oxidant.
But current method of quality control still backwardness, 2005 version " Chinese Pharmacopoeia " using proterties and microscopic features as the only resource differentiating the stem of noble dendrobium, 2010 version " Chinese Pharmacopoeia " improve to some extent, although dendrobium candidum is out single-row from stem of noble dendrobium medicinal material, add the item inspection such as moisture, extract, meanwhile, assay is carried out to Dendrobium officinale polysaccharide and mannose.But the existence in plant of polysaccharide and mannose is comparatively general, its distinctive secondary metabolite is not still carried out to the control of science.At present, differentiate dendrobium candidum raw material with proterties and take Thick many candies as the way that leading indicator differentiates product, both be difficult to distinguish and whether contained dendrobium candidum characteristic component, being difficult to again the content measuring dendrobium candidum feature effective constituent, is particularly the fat-soluble characteristic component of representative with bibenzyl.Therefore, in the urgent need to providing the detection method of a kind of science, comprehensively reflection dendrobium candidum quality of medicinal material.
Summary of the invention
The object of the present invention is to provide a kind of quality determining method of dendrobium candidum medicinal material, effectively can control the quality of dendrobium candidum medicinal material, thus guarantee the security of its clinical practice, stability and validity.
For achieving the above object, the invention provides following technical scheme:
A quality determining method for dendrobium candidum medicinal material, adopts ultraviolet spectrophotometry, take Bibenzyl compound as reference substance, carry out assay by external standard method to the total bibenzyl in dendrobium candidum.
The quality determining method of described dendrobium candidum medicinal material, concrete detecting step is as follows:
1) preparation of need testing solution: get dendrobium candidum medicinal material, pulverize, cross 40-60 mesh sieve, precision takes 0.5-1.0g, take weight/volume as the high concentration alcohol water system of 30-70 times amount or Semi-polarity Solvent Extract methods 2-3 time, extract to be transferred in volumetric flask and constant volume, shake up, get the centrifugal 10-20min of 1-5mL, rotating speed is 10000r/min, precision measures supernatant methanol dilution, obtains need testing solution;
2) preparation of reference substance solution: precision takes Bibenzyl compound reference substance, adds methyl alcohol dissolving and makes solution;
3) determination method: with methyl alcohol for blank reference solvent; Adopt ultraviolet spectrophotometry, test the absorbance at 270-290nm wavelength place, blank solution is noiseless; Content is calculated with calibration curve method.
As the further scheme of the present invention: concrete detecting step is as follows:
1) preparation of need testing solution: get dendrobium candidum medicinal material, pulverize, cross 50 mesh sieves, precision takes 1g, put in 100ml round-bottomed flask, add 50% ethanol 70ml water-bath refluxing extraction 3 times, each 3 hours, merge extract, get the centrifugal 15min of 4ml wherein, rotating speed is 10000r/min, and precision measures supernatant 1ml methanol dilution 10 times, obtains need testing solution;
2) preparation of reference substance solution: precision takes EN reference substance 25.0mg, be placed in 25ml volumetric flask, add methyl alcohol to dissolve, constant volume, precision measure EN standard solution 0.5,0.6,0.7,0.8,0.9,1.0,1.1,1.2,1.3,1.4,1.5,1.6ml, be placed in 25ml volumetric flask constant volume respectively, obtain reference substance solution;
3) determination method: with methyl alcohol for blank reference solvent; Survey absorbance according to ultraviolet spectrophotometry at 280nm wavelength place, blank solution is noiseless; Calculating content with calibration curve method, take absorbance as ordinate Y, and EN concentration is that horizontal ordinate X carries out linear regression, trying to achieve regression equation is A=11.002C+0.018, the range of linearity is 0.02mg/ml-0.064mg/ml, and correlation coefficient r=0.9997, linear relationship is good.
As the further scheme of the present invention: described reference substance includes but not limited to EN and dihydro resveratrol.
As the further scheme of the present invention: described Semi-polarity organic solvent includes but not limited to methylene chloride, ethyl acetate, chloroform and acetone, described high concentration alcohol water system includes but not limited to that concentration is the methanol aqueous solution system of 50%-95%, ethanol water system and isopropanol water solution system.
As the further scheme of the present invention: the content of the total bibenzyl in described dendrobium candidum medicinal material is not less than 5%.
Compared with prior art, the invention has the beneficial effects as follows: easy, easy, reproducible, can definitely reflect dendrobium candidum quality of medicinal material, the perfect further quality standard of dendrobium candidum medicinal material, compensate in dendrobium candidum quality assessment the deficiency of the detection method lacked the fat-soluble characteristic component taking bibenzyl as representative, make the quality detection technology of dendrobium candidum medicinal material more scientific, reasonable; Effectively can control the quality of dendrobium candidum medicinal material, thus guarantee the security of its clinical practice, stability and validity.
Accompanying drawing explanation
Fig. 1 is the relation of bibenzyl extraction ratio and concentration of alcohol;
Fig. 2 is the relation of bibenzyl extraction ratio and Extracting temperature;
Fig. 3 is the relation of bibenzyl extraction ratio and extraction time;
Fig. 4 is the relation of bibenzyl extraction ratio and liquid ratio;
Fig. 5 is the relation of bibenzyl extraction ratio and extraction time;
Fig. 6 is EN canonical plotting.
Embodiment
Below in conjunction with the embodiment of the present invention, be clearly and completely described the technical scheme in the embodiment of the present invention, obviously, described embodiment is only the present invention's part embodiment, instead of whole embodiments.Based on the embodiment in the present invention, those of ordinary skill in the art, not making the every other embodiment obtained under creative work prerequisite, belong to the scope of protection of the invention.
Embodiment 1: extracting method is investigated
1. take the dendrobium candidum powder that 1g crosses 50 mesh sieves, totally 4 parts, be designated as A, B, C, D respectively;
A, 50ml ethyl acetate ultrasonic extraction 20min; The ethanol heat of B, 50ml80% carries 2h; C, 40ml acetone ultrasonic extraction 30min; D, 60ml methyl alcohol heat carries 1h.
Respectively get the centrifugal 10min of 4ml extract, rotating speed is 10000r/min, gets supernatant dilution certain multiple, measures its total bibenzyl content, in each extracting method, total bibenzyl extraction ratio is as shown in table 1: Bibenzyl compound can extract by these solvents, the hot formulation of preferred alcohol.
Total bibenzyl extraction ratio of table 1 Different Extraction Method
| Test number | A | B | C | D |
| Extraction ratio (%) | 4.01 | 6.88 | 4.56 | 5.18 |
After above A, B, C, D extract evaporate to dryness, redissolve with methyl alcohol, then detect with Waters 2695-2996Photodiode Array Detector high performance liquid chromatograph, a large amount of containing this compounds with bibenzyl feature ultra-violet absorption spectrum in each extract of result, extract with 80% alcohol reflux the Bibenzyl compound kind proposed many, content is high.
Embodiment 2: bibenzyl reference substance
By measuring the ultra-violet absorption spectrum of reference substance and extract, as shown in table 2, determine near 280nm for reasonably total bibenzyl determined by ultraviolet spectrophotometry is interval.
The ultraviolet absorption maximum of table 2 different material
Embodiment 3: the hot bill of lading Factor Experiment of ethanol
3.1 concentration of alcohol
Take the dendrobium candidum powder that 0.5g crosses 50 mesh sieves, add the ethanol water of 25ml variable concentrations, refluxing extraction 2h, let cool, filter, precision measures filtrate and dilutes 50 times, records absorbance A, then calculate concentration C at 280nm place from regression equation, and then calculate bibenzyl extraction ratio, the relation of the concentration of alcohol obtained and bibenzyl extraction ratio is as shown in Figure 1: when concentration of alcohol is 50%, the extraction ratio of bibenzyl is the highest, is 8.87%.
3.2 Extracting temperature
Take the dry stem powder of dendrobium candidum that 0.5g crosses 50 mesh sieves, add the ethanol of 25ml50%, under different temperatures, extract 2h, let cool, filter, precision measures filtrate and carries out dilution 50 times, records A at 280nm place, C is read again from regression equation, calculate bibenzyl extraction ratio, the relation of the Extracting temperature obtained and bibenzyl extraction ratio as shown in Figure 2: along with the rising of temperature, the extraction ratio of bibenzyl increases gradually, after reaching 90 degree of boilings, the extraction ratio increase of bibenzyl eases up.
3.3 extraction time
Take the dry stem powder of dendrobium candidum that 0.5g crosses 50 mesh sieves, add the ethanol of 25ml50%, refluxing extraction, let cool, filter, precision measures filtrate and carries out dilution 50 times, records A, then read C from regression equation at 280nm place, calculate bibenzyl extraction ratio, the relation of the extraction time obtained and bibenzyl extraction ratio is as shown in Figure 3: time upon extracting for 2h, the extraction ratio of bibenzyl is the highest, is 8.07%.
3.4 liquid ratio
Take the dry stem powder of dendrobium candidum that 0.5g crosses 50 mesh sieves, add the ethanol of 10-35ml50%, refluxing extraction, let cool, filter, precision measures filtrate and carries out dilution 50 or 100 times, records A, then read C from regression equation at 280nm place, calculate bibenzyl extraction ratio, the relation of the liquid ratio obtained and bibenzyl extraction ratio is as shown in Figure 4: when liquid ratio is 1:30, the extraction ratio of bibenzyl is the highest, is 10.9%.
3.5 extraction time
Take the dry stem powder of dendrobium candidum that 0.5g crosses 50 mesh sieves, add the ethanol of 25ml50%, refluxing extraction repeatedly, lets cool, and filter, precision measures filtrate, dilutes 50 times, 20 times, 5 times and do not dilute respectively.Record A at 280nm place, then read C from regression equation, calculate bibenzyl extraction ratio, the relation of the extraction time obtained and bibenzyl extraction ratio as shown in Figure 5: extracting twice can propose bibenzyl composition more than 90%.
Table 3 orthogonal test factor level table
Embodiment 4: alcohol extract orthogonal test
4.1 choose on the larger concentration of alcohol of bibenzyl extraction ratio impact, liquid ratio, extraction time and extraction time four factors carry out orthogonal test, as shown in table 3.
Take the dry stem powder of dendrobium candidum that 0.5g crosses 50 mesh sieves, add the ethanol of the variable concentrations of 15-35ml, refluxing extraction repeatedly, lets cool, and filter, precision measures filtrate, suitably dilutes respectively.Record A at 280nm place, then read C from regression equation, calculate bibenzyl extraction ratio, result is as shown in following table 4-table 5.
The bibenzyl extraction ratio result of table 4 orthogonal test
Orthogonal experiment results shows, the influence degree of each factor is followed successively by C>D>A>B, i.e. extraction time > extraction time > concentration of alcohol > liquid ratio, optimum extraction process is C
3a
1b
3d
3, i.e. the alcohol extract 3 times of 70 times 50%, each 3h.
The variance analysis of table 5 orthogonal test
4.2 demonstration test
According to extraction process A
1b
3c
3d
3verify.
Take the dry stem powder of dendrobium candidum that 0.5g crosses 50 mesh sieves, add the ethanol of 50% of 35ml, refluxing extraction 3 times, lets cool, and merges No. 3 extracts, and filter, precision measures filtrate, dilutes 10 times.Record A at 280nm place, then read C from regression equation, calculate bibenzyl extraction ratio, result is as shown in table 6 below: this extraction process is stablized, and extraction ratio is 6.65%.
Table 6 demonstration test result
Embodiment 5: methodological study
The withinday precision of table 7 instrument
5.1 withinday precision
The EN standard solution of same concentration, in 280nm place duplicate measurements A value 5 times, as shown in table 7, calculate to obtain relative standard deviation=1.58%, show that instrument withinday precision is good.
5.2 day to day precision
The EN standard solution of same concentration, continuous 5 days, measures A value in 280nm place, as shown in table 8, calculates to obtain relative standard deviation=1.43%, shows that instrument day to day precision is good.
The day to day precision of table 8 instrument
5.3 repeated experiment
Table 9 replica test result
Take the dry stem powder of dendrobium candidum that 0.5g crosses 50 mesh sieves, totally 6 parts.Add the ethanol of 25ml50% respectively, refluxing extraction 2h, let cool, filter, precision measures filtrate and carries out dilution 50 times, A is recorded at 280nm place, read C from regression equation again, calculate bibenzyl extraction ratio, result of calculation is as shown in table 9, calculate to obtain relative standard deviation=0.86%, show that the repeatability of the method is good.
5.4 accuracy
Application of sample recovery experiment
Accurately take the dry stem powder of dendrobium candidum that 0.5g crosses 50 mesh sieves, totally 9 parts, add EN standard items respectively in right amount, carry out extraction ratio mensuration by above-mentioned test liquid preparation and total bibenzyl determination method.Result is as shown in table 10 below.
Table 10 application of sample recovery test result
Table 11 stability experiment result
5.5 stability experiment
Same need testing solution, after diluting 50 times, measure A value at 0min, 12h and 24h respectively, as shown in table 11, gained relative standard deviation=0.62%, illustrates that test sample 24h internal stability is good.
Embodiment 6: content assaying method
1) preparation of need testing solution: get dendrobium candidum medicinal material, pulverize, cross 50 mesh sieves, precision takes 1g, put in 100ml round-bottomed flask, add the ethanol water bath refluxing extraction 3 times of 70ml 50%, each 3 hours, merge No. 3 extracts, get the centrifugal 15min of 4ml wherein, rotating speed is 10000r/min, and precision measures supernatant methanol dilution 10 times, obtains need testing solution;
2) preparation of reference substance solution: precision takes EN reference substance 25.0mg, be placed in 25ml volumetric flask, add methyl alcohol to dissolve, constant volume, precision measure EN standard solution 0.5,0.6,0.7,0.8,0.9,1.0,1.1,1.2,1.3,1.4,1.5,1.6ml, be placed in 25ml volumetric flask constant volume respectively, obtain reference substance solution.
3) determination method: with methyl alcohol for blank reference solvent; Survey absorbance according to ultraviolet spectrophotometry at 280nm wavelength place, blank solution is noiseless; Calculating content with calibration curve method, take absorbance as ordinate Y, and EN concentration is that horizontal ordinate X carries out linear regression, trying to achieve regression equation is A=11.002C+0.018, the range of linearity is 0.02mg/ml-0.064mg/ml, and correlation coefficient r=0.9997, linear relationship is good.
In dendrobium candidum, the content of total bibenzyl is not less than 5%.
Embodiment 7: content assaying method
1) preparation of need testing solution: get dendrobium candidum medicinal material, pulverizes, and crosses 60 mesh sieves, precision takes 1g, put in 100ml triangular flask, add the ultrasonic extraction of acetone 2 times, each 30min, extract is transferred to constant volume in volumetric flask, shake up, get the centrifugal 20min of 2ml wherein, rotating speed is 10000r/min, precision measures supernatant methanol dilution 15 times, obtains need testing solution;
2) preparation of reference substance solution: precision takes dihydro resveratrol reference substance 25.0mg, be placed in 25ml volumetric flask, add methyl alcohol to dissolve, constant volume, precision measure dihydro resveratrol standard solution 0.6,0.8,1.0,1.2,1.4,1.6ml, be placed in 25ml volumetric flask constant volume respectively, obtain reference substance solution;
3) determination method: with methyl alcohol for blank reference solvent; Survey absorbance according to ultraviolet spectrophotometry at 285nm wavelength place, blank solution is noiseless; Calculate content with calibration curve method, as shown in Figure 6: take absorbance as ordinate Y, EN concentration is that horizontal ordinate X carries out linear regression, trying to achieve regression equation is A=12.324C+0.012, range of linearity 0.024mg/ml-0.064mg/ml, correlation coefficient r=0.9992, linear relationship is good.
In dendrobium candidum, the content of total bibenzyl is not less than 5%.
Embodiment 8: assay is tested
Take the dry stem powder of dendrobium candidum that 1g crosses 2013 Zhejiang of 50 mesh sieves, 2014 Zhejiang, 2013 Yunnan, 2014 Yunnan, 2014 Hunan respectively, add the ethanol of 50% of 70ml, refluxing extraction 3 times, each 3h, lets cool, and merges No. 3 extracts, filter, precision measures filtrate, dilutes 10 times.Record A at 280nm place, then read C from the regression equation embodiment 1, calculate extraction ratio as shown in table 12 below: in dendrobium candidum, the content of total bibenzyl is not less than 5%.
The bibenzyl extraction ratio of the dendrobium candidum medicinal material of table 12 different batches
The present invention is easy, easy, reproducible, can definitely reflect dendrobium candidum quality of medicinal material, the perfect further quality standard of dendrobium candidum medicinal material, compensate in dendrobium candidum quality assessment the deficiency of the detection method lacked the fat-soluble characteristic component taking bibenzyl as representative, make the quality detection technology of dendrobium candidum medicinal material more scientific, reasonable; Effectively can control the quality of dendrobium candidum medicinal material, thus guarantee the security of its clinical practice, stability and validity.
To those skilled in the art, obviously the invention is not restricted to the details of above-mentioned one exemplary embodiment, and when not deviating from spirit of the present invention or essential characteristic, the present invention can be realized in other specific forms.Therefore, no matter from which point, all should embodiment be regarded as exemplary, and be nonrestrictive, scope of the present invention is limited by claims instead of above-mentioned explanation, and all changes be therefore intended in the implication of the equivalency by dropping on claim and scope are included in the present invention.
In addition, be to be understood that, although this instructions is described according to embodiment, but not each embodiment only comprises an independently technical scheme, this narrating mode of instructions is only for clarity sake, those skilled in the art should by instructions integrally, and the technical scheme in each embodiment also through appropriately combined, can form other embodiments that it will be appreciated by those skilled in the art that.
Claims (6)
1. a quality determining method for dendrobium candidum medicinal material, is characterized in that, adopts ultraviolet spectrophotometry, take Bibenzyl compound as reference substance, carry out assay by external standard method to the total bibenzyl in dendrobium candidum.
2. the quality determining method of dendrobium candidum medicinal material according to claim 1, is characterized in that, concrete detecting step is as follows:
1) preparation of need testing solution: get dendrobium candidum medicinal material, pulverize, cross 40-60 mesh sieve, precision takes 0.5-1.0g, take weight/volume as the high concentration alcohol water system of 30-70 times amount or Semi-polarity Solvent Extract methods 2-3 time, extract to be transferred in volumetric flask and constant volume, shake up, get the centrifugal 10-20min of 1-5mL, rotating speed is 10000r/min, precision measures supernatant methanol dilution, obtains need testing solution;
2) preparation of reference substance solution: precision takes Bibenzyl compound reference substance, adds methyl alcohol dissolving and makes solution;
3) determination method: with methyl alcohol for blank reference solvent; Adopt ultraviolet spectrophotometry, test the absorbance at 270-290nm wavelength place, blank solution is noiseless; Content is calculated with calibration curve method.
3. the quality determining method of dendrobium candidum medicinal material according to claim 2, is characterized in that, concrete detecting step is as follows:
1) preparation of need testing solution: get dendrobium candidum medicinal material, pulverizes, and crosses 50 mesh sieves, precision takes 1g, put in 100ml round-bottomed flask, add the ethanol of 70ml 50%, water-bath refluxing extraction 3 times, each 3 hours, merge extract, get the centrifugal 15min of 4ml wherein, rotating speed is 10000r/min, precision measures supernatant 1ml methanol dilution 10 times, obtains need testing solution;
2) preparation of reference substance solution: precision takes EN reference substance 25.0mg, be placed in 25ml volumetric flask, add methyl alcohol to dissolve, constant volume, precision measure EN standard solution 0.5,0.6,0.7,0.8,0.9,1.0,1.1,1.2,1.3,1.4,1.5,1.6ml, be placed in 25ml volumetric flask constant volume respectively, obtain reference substance solution;
3) determination method: with methyl alcohol for blank reference solvent; Survey absorbance according to ultraviolet spectrophotometry at 280nm wavelength place, blank solution is noiseless; Calculating content with calibration curve method, take absorbance as ordinate Y, and EN concentration is that horizontal ordinate X carries out linear regression, trying to achieve regression equation is A=11.002C+0.018, the range of linearity is 0.02mg/ml-0.064mg/ml, and correlation coefficient r=0.9997, linear relationship is good.
4., according to the quality determining method of the arbitrary described dendrobium candidum medicinal material of claim 1-2, it is characterized in that, described reference substance includes but not limited to EN and dihydro resveratrol.
5. the quality determining method of dendrobium candidum medicinal material according to claim 2, it is characterized in that, described Semi-polarity organic solvent includes but not limited to methylene chloride, ethyl acetate, chloroform and acetone, and described high concentration alcohol water system includes but not limited to that concentration is the methanol aqueous solution system of 50%-95%, ethanol water system and isopropanol water solution system.
6., according to the quality determining method of the arbitrary described dendrobium candidum medicinal material of claim 1-2, it is characterized in that, the content of the total bibenzyl in described dendrobium candidum medicinal material is not less than 5%.
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