CN101804082B - Method for identifying cordyceps sinensis powder through microscopic dyeing - Google Patents
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Abstract
The invention belongs to the medicinal material identifying field and in particular relates to a method for identifying cordyceps sinensis powder through microscopic dyeing. The invention aims to solve the technical problem that the sold pure cordyceps sinensis powder in market is difficult to identify. The technical scheme of the invention used for solving the problem is to provide a method for accurately identifying cordyceps sinensis powder. The method comprises the following steps: taking cordyceps sinensis powder to be detected, dying to prepare smears; placing the prepared smears under a microscope for microscopic observation, and if the special structure of cordyceps sinensis can be clearly observed on each smear without obvious blue spots caused by iodine staining and no obvious plant issue can be observed, judging that the sample is pure cordyceps sinensis powder; and if not, judging that the sample is not pure cordyceps sinensis powder. The method of the invention can be used to clearly identify fine cordyceps sinensis powder prepared for decoction and substances not belonging to cordyceps sinensis, the cost is low, the efficiency is high, method can be not easily interfered by various additives, the accuracy is very high, and the method has good application prospects in the medicinal material identifying field.
Description
Technical field
The invention belongs to medicinal material and differentiate the field, be specifically related to the method that a kind of microscopic dyeing is differentiated Chinese caterpillar fungus powder
Background technology
In the traditional medicine of China, Cordyceps sinensis is exactly high tonic always, medicinal status very significantly, with genseng, pilose antler and be called " three big tonics ".Further investigation along with each related discipline field; The report that the new kind of Chinese caterpillar fungus, new distribution are constantly arranged in recent years; Chemical constitution is constantly separated, identify, and relevant many-sided pharmacology, pharmacodynamic study such as immune, antitumor have fully confirmed the drug effect of Cordyceps sinensis.Because it is special growing environment, special geographical environment and the important economic value of Cordyceps sinensis make that people's research interest is extraordinarily strong, especially very active especially to the research of Cordyceps sinensis and substitute thereof.
Existing market circulation and the pure Chinese caterpillar fungus powder of selling owing to be made into certain formulation, are difficult to distinguish the true and false; Though can use the means of testing instruments, prove the existence of some labels of Cordyceps sinensis, like the content of cordycepin and adenosine; But these labels have been bought easily, and also inexpensive, can be used for adjuvant in theory; So physical and chemical inspection can not detect impurity, can not distinguish fakement.Because Chinese caterpillar fungus powder expensive, in order to protect consumer's interests, the method for the pure Chinese caterpillar fungus powder true and false of accurate evaluation need be set up in this area.
Summary of the invention
The present invention will solve is the technical matters that the pure Chinese caterpillar fungus powder of market circulation and sale is difficult to the branch true and false.The technical scheme that the present invention addresses this problem provides a kind of method of accurate discriminating Chinese caterpillar fungus powder.This method may further comprise the steps: get Chinese caterpillar fungus powder to be checked, smear is processed in dyeing; Said dyeing is for carrying out at least a dyeing in hematoxylin-eosin-iodine staining, sarranine-fast green dyeing, the Si Shi dye liquor-iodine staining respectively;
The smear of processing is carried out microscopic observation in microscopically; Like the every clear peculiar structure of observing Cordyceps sinensis of equal ability; And there is not the obvious blue spot under the iodine staining; And do not observe tangible plant tissue, can be judged as pure Chinese caterpillar fungus powder, otherwise be impure Chinese caterpillar fungus powder.
Wherein, the present invention differentiates that the peculiar structure of the Cordyceps sinensis described in the method for pure Chinese caterpillar fungus powder is at least 4 kinds in the stroma epidermal area of stroma mycelium or Cordyceps sinensis of polypide musculature, Cordyceps sinensis of polypide epidermal area, the Cordyceps sinensis of polypide mycelium, the Cordyceps sinensis of Cordyceps sinensis.
Wherein, The present invention differentiates that the colouring method of the Cordyceps sinensis described in the method for pure Chinese caterpillar fungus powder is for dripping bush dye liquor 10min dyeing after washing 5min; Wash with 50%, 75%, 95% ethanol gradient respectively successively then, drip eosin stain dyeing 5min then, drip iodine staining 5min then; Then successively respectively with the washing of 50%, 75%, 95%, 100% ethanol gradient, be placed in the xylene liquid 5～10min to accomplish dyeing.
Wherein, The colouring method that the present invention differentiates the Cordyceps sinensis described in the method for pure Chinese caterpillar fungus powder dyes for smear is dripped sarranine dye liquor 25min after with 50% alcohol immersion; Wash with 50%, 75%, 95% ethanol gradient respectively successively then; Drip fast green dyeing 1min then; Wash with 95%, 95%, 100%, 100% ethanol gradient respectively successively then, be placed on 5～10min in xylene and 100% alcohol mixeding liquid, place pure xylene liquid 5～10min again to accomplish dyeing.
Wherein, when the present invention differentiated the peculiar structure of differentiating Cordyceps sinensis in the method for pure Chinese caterpillar fungus powder, the standard section with the peculiar structure of Cordyceps sinensis that can use mode of the same race to dye was standard.Polypide transverse section and the Cordyceps sinensis stroma transverse section that can also use Cordyceps sinensis be standard as a reference.Produce erroneous judgement to prevent the reviewer owing to lack experience.
Wherein, the present invention differentiates in the method for pure Chinese caterpillar fungus powder 4～10 parts of every batch of pure Cordyceps sinensis powder material samplings to be checked.Generally get 6 parts and detect, to improve accuracy.To every batch to be checked pure winter worm every smear in per 1 on all to have at least 4 kinds of peculiar structures just to be genuine piece.
Particularly, the inventive method can adopt following detailed implementation process:
Get 4～10 parts of Chinese caterpillar fungus powders to be checked, generally get 6 parts.Smear is processed in the appearance dyeing of getting; Said dyeing is at least a dyeing of carrying out respectively in hematoxylin eosin staining, sarranine-fast green dyeing, the dyeing of Si Shi dye liquor, and carries out iodine staining;
Wherein the method for hematoxylin eosin staining is for dripping bush dye liquor 10min dyeing after washing 5min; Wash with 50%, 75%, 95% ethanol gradient respectively successively then; Drip eosin stain dyeing 5min then; Drip iodine staining 5min then,, be placed in the xylene liquid 5～10min to accomplish dyeing then successively respectively with the washing of 50%, 75%, 95%, 100% ethanol gradient.
Wherein the method for sarranine-fast green dyeing is that smear is dripped sarranine dye liquor 25min dyeing after with 50% alcohol immersion; Wash with 50%, 75%, 95% ethanol gradient respectively successively then; Drip fast green dyeing 1min then; Wash with 95%, 95%, 100%, 100% ethanol gradient respectively successively then, be placed on 5～10min in xylene and 100% alcohol mixeding liquid, place pure xylene liquid 5～10min again to accomplish dyeing.
Then the smear of processing is carried out microscopic observation in microscopically; Like the every clear peculiar structure of observing Cordyceps sinensis of equal ability; And there is not the obvious blue spot under the iodine staining; And do not observe tangible plant tissue, can be judged as pure Chinese caterpillar fungus powder, otherwise be impure Chinese caterpillar fungus powder.The peculiar structure of described Cordyceps sinensis is that at least 4 kinds of peculiar structures in the stroma epidermal area of stroma mycelium or Cordyceps sinensis of polypide musculature, Cordyceps sinensis of polypide epidermal area, the Cordyceps sinensis of polypide mycelium, the Cordyceps sinensis of Cordyceps sinensis can both observe at microscopically.
The beneficial effect of the inventive method is: the thinking that the present invention mainly adopts microscopic dyeing to differentiate, to the special circumstances of Cordyceps sinensis, develop the inventive method.The inventive method is through to the dyeing of Cordyceps sinensis microtexture, through biological microscope, under different magnifications, can clear identification tiny medicine materical crude slice powder with tell the material that does not belong to Cordyceps sinensis.And this method cost is low, efficient is high, is not subject to the interference of various adding ingredients, and accuracy is very high, is applicable to the discriminating observation of 1500 orders with interior superfine powder.Can use separately to differentiate pure Cordyceps sinensis superfine powder, the Cordyceps sinensis superfine powder of judgement existing market sale and the confidence level and the pure degree of Cordyceps sinensis preparation are had crucial meaning.The inventive method can be used the true and false of the precious Cordyceps sinensis of true judgement separately, also can combine other Cordyceps sinensis and Cordyceps sinensis superfine powder passing through discrimination method, like the detection of shape, color and luster, smell and flavour, ring grain, worm foot in the Chinese caterpillar fungus proterties; Outward appearance, color and luster, smell and flavour that organoleptic quality requires; Its quality level is confirmed in detection to moisture, adenosine, impurity, heavy metal, microorganism in the physical and chemical index on the basis of the true and false of judging precious Cordyceps sinensis, these interests to the protection consumer have profound significance, in this area good application prospects are arranged.
Description of drawings
Fig. 1 is Cordyceps sinensis longitudinal section (40X)
Fig. 2 is the Cordyceps sinensis transverse section.A left side is a Cordyceps sinensis polypide transverse section (40X), and the right side is a Cordyceps sinensis stroma transverse section (40X).
Fig. 3 is polypide mycelium (100X)
Fig. 4 is Cordyceps sinensis superfine powder-polypide mycelium (100X), eosin stain.
Fig. 5 is polypide epidermal area (100X), the bush dye liquor.
Fig. 6 is Cordyceps sinensis superfine powder-polypide epidermal area (100X), eosin stain.
Fig. 7 is polypide musculature (100X), and a left side is the bush dye liquor; The right side is an eosin stain.
Fig. 8 is Cordyceps sinensis superfine powder-polypide musculature (100X), under the eosin stain.
Fig. 9 is stroma mycelium (100X), and a left side is under the bush dye liquor, and the right side is under the fast green dye liquor.
Figure 10 is Cordyceps sinensis superfine powder-stroma mycelium (100X), under the eosin stain.
Figure 11 is stroma epidermal area (100X), and a left side is under the bush dye liquor, and the right is under the eosin stain.
Figure 12 is Cordyceps sinensis superfine powder-stroma epidermal area (100X), under the eosin stain.
Figure 13 is the starch granules (100X) of iodine staining
Figure 14 is plant tissue-leaf under the bush dye liquor (100X)
Wherein: Fig. 3,5,7,9,11 is meant the part picture (being Fig. 1, the partial enlarged drawing of Fig. 2) of the corresponding typical structure that in the whole section of Cordyceps sinensis, finds.Fig. 4,6,8,10,12 is the result of Cordyceps sinensis superfine powder smear to be measured.
Embodiment
Five, experimental technique and parameter:
1. laboratory sample:
Cordyceps sinensis section, the pure Cordyceps sinensis superfine powder of commercially available extremely careless board, the potpourri of starch superfine powder, starch superfine powder and pure Cordyceps sinensis superfine powder.
2. coloring agent
Fast green dye liquor: Chemical Reagent Co., Ltd., Sinopharm Group; Sarranine dye liquor: Chemical Reagent Co., Ltd., Sinopharm Group; Eosin stain: the new photochemical factory in Shenyang; Haematine dye liquor: the new photochemical factory in Shenyang; Si Shi liquid: Beijing Chemical Plant; Iodine: former chemical company limited is newly changed in Nanjing, is made into dye liquor by conventional method.
3. instrument or other apparatus
Double-tube biologic microscope, photograph interconnecting device, Canon's slr camera.Spirit lamp, microslide, cover glass, staining rack, cedar oil, absolute ethyl alcohol, xylene, canada balsam, lens wiping paper, conventional equipment such as distilled water.
4. flow process
Fixing → transparent → smear (load) → dyeing → envelope Tibetan → drying → microscopy.
5. step
5.1 it is fixing
It is an amount of to get the sample that stirs, and uses distilled water immersion 4h, uses fixedly 4-24h of FAA (surplus is the mixed liquor of distilled water for formaldehyde 10%, ethanol 50%-75%) again.
5.2. it is transparent
The Cordyceps sinensis superfine powder that fixes is washed (each step washing 3-5 minutes) through 30%, 50%, 75%, 95%, 100% ethanol gradient, placed xylene liquid 10 minutes.
5.3. smear (load)
Get clean microslide, the Cordyceps sinensis superfine powder of getting after transparent is an amount of, and mixing and load are unsuitable blocked up.Process six groups with method.
Get clean microslide, it is an amount of to get the starch superfine powder, with Si Shi liquid mixing and load, unsuitable blocked up.Process six groups with method.
5.4. dyeing
(1) hematoxylin eosin staining.
Six groups of slides of every kind of sample are lain against in the double dish; Drip dye liquor (bush dye liquor 10min → washing 5min → 50%, 75%, 95% ethanol washes → washing with alcohol of eosin stain 5min → iodine liquid 5min → 50%, 75%, 95%, 100%, 100%, place xylene liquid 10min.) 1～2 droplet on corresponding smear (dye liquor just covers the smear film and is advisable).About 25-30min dyes.
Coloration result: nucleus is dyed by haematine and is blue look, and cytoplasm (tenuigenin), kernel, muscle fibre, collagen fiber, red blood cell etc. are dyed the different cerise of degree by Yihong.Plasmacytic secretory granules are because it can be shown darkviolet by h and E dyeing event simultaneously.
Then dye blue particle if any starch granules.Used Yihong can be red with epithelial cell, meat fiber and cell pulp.
(2), sarranine-fast green dye liquor dyeing:
Six groups of slides of every kind of sample are lain against in the double dish; With (50% alcohol immersion → sarranine dye liquor 25min → wash → fast green 1min → 95%, 95%, 100%, 100% washing with alcohol with 50%, 75%, 95% ethanol; Place xylene and 100% alcohol mixeding liquid, 5-10min, place pure xylene liquid 5-10min.) 1～2 droplet on corresponding smear (dye liquor just covers the smear film and is advisable).About 25-30min dyes.
Fast green is acid dyes, can water-soluble (solubleness is 4%) and alcohol (solubleness is 9%).Fast green is a kind of coloring agent that contains the cellulose cell tissue of starching matter that dyes, and on transfect cell and plant tissue, uses extremely wide and haematine, sarranine are listed as on the plant histology the most frequently used dyestuff in three.Sarranine is dissolvable in water water and ethanol, is made into the xylem that 1% WS dyes plant usually, also can be made into aniline sarranine liquid according to following prescription and tissue is carried out piece dye: sarranine 5g, 95% ethanol 50mL, aniline oil 20mL, distilled water 450mL.Sarranine can partly dye to lignification, bolt materialization and the gelatinize of plant, and the part after the dyeing is red.Dyeing time is generally 2～24hrs, the dyeing of cooperating of normal and fast green grade.
(3) Si Shi dye liquor-iodine staining:
Six groups of slides of sample of Si Shi liquid mixing are lain against in the double dish, drip the washing with alcohol of iodine liquid 5min → 50%, 75%, 95%, 100%, 100%, place xylene and 100% alcohol mixeding liquid, 5～10min, place xylene liquid 5～10min.
5.5 envelope is hidden
From xylene solution, take out microslide, drip 1-2 in the position that powder is arranged and drip the gum-solution of putting on airs, add the cover glass envelope and hide.
5.6 it is dry
Envelope is hidden good microslide, and air dry, hair dryer dry up or with 40 ℃-50 ℃ oven dry of constant temperature roaster (temperature can not be too high).
5.7 microscopy
Slide is done the back microscopy.Under different multiples, observe every kind of structures of samples (identifying true and false Cordyceps sinensis) with binocular biological microscope or digital biological microscope.As use oily sem observation, then slide must bone dry.
Six, experiment conclusion
Use the above-mentioned staining procedure of the inventive method, can see having or not in the genuine pure Chinese caterpillar fungus powder and mix some other materials: such as: starch, plant tissue; Not by the polypide of fungal infection; The mycelium of commercially available artificial culture etc.
When the judgement of carrying out pure Cordyceps sinensis superfine powder whether, lack experience like the testing staff, can't judge directly that then can use the section of Cordyceps sinensis tissue or smear that pure Cordyceps sinensis superfine powder is made is with reference to the standard of judging.
The spot of visible corresponding coloring agent color is observed in the section of Cordyceps sinensis tissue down through 4X, 10X, 40X times object lens.Through observation fine structure under the 100X oil mirror, except that the iodine staining sheet, for all the other dyeing liquors; Especially pass through eosin stain, haematine dye liquor and fast green dye liquor, in the section of Cordyceps sinensis standard, high-visible polypide mycelium (see figure 3); Polypide epidermal area (see figure 5), polypide musculature (see figure 7), stroma mycelium (see figure 9); The isostructural fragment of stroma epidermal area (seeing Figure 11) is not found the material of different shape.
In the present embodiment, any four of can detect in following five structures of pure Cordyceps sinensis superfine powder to be measured just can be judged as true Chinese caterpillar fungus powder:
Polypide mycelium (see figure 3); Polypide epidermal area (see figure 5); Polypide musculature (see figure 7); Stroma mycelium (see figure 9); Stroma epidermal area (seeing Figure 11).Because detection material is the following Cordyceps sinensis ultra-micro powders of 1500 orders, so under the 100X object lens, observe.
Testing result shows in the commercially available pure Cordyceps sinensis superfine powder of the extremely careless board smear; All high-visible polypide mycelium (see figure 4), polypide epidermal area (see figure 6), polypide musculature (see figure 8); Stroma mycelium (see figure 10); The isostructural fragment of stroma epidermal area (seeing Figure 12), with homemade Cordyceps sinensis section and the contrast of pure Cordyceps sinensis superfine powder smear, each structure is the peculiar structure of Cordyceps sinensis.Contrast Cordyceps sinensis longitudinal section Fig. 1, transverse section Fig. 2, do not find the material of different shape.And not observing does not have tangible plant tissue and tangible starch granules, so decidable is pure Cordyceps sinensis superfine powder for the extremely careless board Cordyceps sinensis of this batch superfine powder.And be added with the Chinese caterpillar fungus powder of starch, owing to can detect the blue particle of represent starch, direct is impure Chinese caterpillar fungus powder with regard to decidable.
Claims (5)
1. differentiate the method for pure Chinese caterpillar fungus powder, it is characterized in that: may further comprise the steps:
Get Chinese caterpillar fungus powder to be checked, smear is processed in dyeing respectively; Said dyeing is for carrying out hematoxylin-eosin-iodine staining;
The smear of processing is carried out microscopic observation in microscopically; Like the every clear peculiar structure of observing Cordyceps sinensis of equal ability; And do not observe tangible plant tissue or do not have the obvious blue spot under the iodine staining, can be judged as pure Chinese caterpillar fungus powder, otherwise be impure Chinese caterpillar fungus powder; The peculiar structure of described Cordyceps sinensis is at least 4 kinds in the stroma epidermal area of stroma mycelium or Cordyceps sinensis of polypide musculature, Cordyceps sinensis of polypide epidermal area, the Cordyceps sinensis of polypide mycelium, the Cordyceps sinensis of Cordyceps sinensis;
Described hematoxylin-eosin-iodine staining method is: drip bush dye liquor 10min dyeing after washing 5min; Wash with 50%, 75%, 95% ethanol gradient respectively successively then; Drip eosin stain dyeing 5min then; Drip iodine staining 5min then then successively respectively with the washing of 50%, 75%, 95%, 100% ethanol gradient, be placed in the xylene liquid 5～10min to accomplish dyeing.
2. differentiate the method for pure Chinese caterpillar fungus powder, it is characterized in that: may further comprise the steps:
Get Chinese caterpillar fungus powder to be checked, smear is processed in dyeing respectively; Said dyeing is for carrying out hematoxylin-eosin-iodine staining and sarranine-fast green dyeing;
The smear of processing is carried out microscopic observation in microscopically; Like the every clear peculiar structure of observing Cordyceps sinensis of equal ability; And do not observe tangible plant tissue or do not have the obvious blue spot under the iodine staining, can be judged as pure Chinese caterpillar fungus powder, otherwise be impure Chinese caterpillar fungus powder; The peculiar structure of described Cordyceps sinensis is at least 4 kinds in the stroma epidermal area of stroma mycelium or Cordyceps sinensis of polypide musculature, Cordyceps sinensis of polypide epidermal area, the Cordyceps sinensis of polypide mycelium, the Cordyceps sinensis of Cordyceps sinensis;
Described hematoxylin-eosin-iodine staining method is: drip bush dye liquor 10min dyeing after washing 5min; Wash with 50%, 75%, 95% ethanol gradient respectively successively then; Drip eosin stain dyeing 5min then; Drip iodine staining 5min then then successively respectively with the washing of 50%, 75%, 95%, 100% ethanol gradient, be placed in the xylene liquid 5～10min to accomplish dyeing;
Described sarranine-fast green colouring method is: smear is dripped sarranine dye liquor 25min dyeing after with 50% alcohol immersion; Wash with 50%, 75%, 95% ethanol gradient respectively successively then; Drip fast green dyeing 1min then; Wash with 95%, 95%, 100%, 100% ethanol gradient respectively successively then, be placed on 5～10min in xylene and 100% alcohol mixeding liquid, place pure xylene liquid 5～10min again to accomplish dyeing.
3. the method for the pure Chinese caterpillar fungus powder of discriminating according to claim 1 and 2 is characterized in that: when differentiating the peculiar structure of Cordyceps sinensis, be normative reference with the standard section with the peculiar structure of Cordyceps sinensis of using mode of the same race to dye.
4. the method for the pure Chinese caterpillar fungus powder of discriminating according to claim 3 is characterized in that: every batch of pure Cordyceps sinensis powder material sampling 4-10 part to be checked is processed smear respectively.
5. the method for the pure Chinese caterpillar fungus powder of discriminating according to claim 4 is characterized in that: on per 1 in 6 smears of every batch of pure Cordyceps sinensis to be checked, all will have at least 4 kinds of peculiar structures just to be genuine piece.
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| 王国栋.粉末鉴定.《冬虫夏草类：生态 培植 应用》.科学技术文献出版社,1995,(第1版),86-87. * |
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