CN103948648A - Application of freezing technology in drying medicinal fungi - Google Patents

Abstract

The invention relates to application of a freezing technology in drying medicinal fungi. The medicinal fungi are prepared by selecting newly harvested medicinal fungi, freezing, piling in a dank place for 2-5 days and drying. According to the medicinal fungi, the main effective components are high in content and strong in pharmacological activity.

Description

The application of Refrigeration Technique in medicinal fungi is dry

Technical field

The present invention relates to medicine method for making, the particularly application of Refrigeration Technique in medicinal fungi is dry, belongs to medical technical field.

Background technology

Fungal drug is extensive, and usually said medicinal fungi is limited to the higher fungus that can form sporophore more.Conventional medicinal fungi has Ganoderma, Phellinus igniarius (L. ex Fr.) Quel., Ganoderma applanatum (Pers. Ex Wallr) Pat., Inonqqus obliquus, Jin Zhi, Cordyceps, Hericium erinaceus (Bull. Ex Fr.) Pers., Poria, Polyporus etc.

The traditional Chinese medical science is treated human diseases using fungus as medical material have long history.But traditional medicinal fungi drying means, active constituent content is low, and a little less than pharmacologically active, the active constituent content and the pharmacologically active that improve medicinal fungi are medical worker's vital tasks.

Summary of the invention

The object of this invention is to provide the dry new method of a kind of medicinal fungi, the medicinal fungi that the method is dry, mainly contains effective component content high, and pharmacologically active is strong.

The present invention is achieved in that

The application of Refrigeration Technique in medicinal fungi is dry, is characterized in that: get the medicinal fungi of newly gathering, and freezing, the 2-5 days that banks up at dark and damp place is dry.

The application of Refrigeration Technique of the present invention in medicinal fungi is dry, is characterized in that: freezing is to be refrigerated to freeze.

The application of Refrigeration Technique of the present invention in medicinal fungi is dry, is characterized in that: freezing is to be refrigerated to-5 DEG C to freeze below.

The application of Refrigeration Technique of the present invention in medicinal fungi is dry, is characterized in that: dry is that heap postpone is dried or dries or dries.

The application of Refrigeration Technique of the present invention in medicinal fungi is dry, is characterized in that: medicinal fungi is Ganoderma, Phellinus igniarius (L. ex Fr.) Quel., Ganoderma applanatum (Pers. Ex Wallr) Pat., Inonqqus obliquus, Jin Zhi, Hericium erinaceus (Bull. Ex Fr.) Pers..

Ganoderma is On Polyporaceae Ganoderma lucidum (Leyss. Ex Fr.) Karst. Ganodermalucidum (Leyss.ExFr.) Karst. or Ganoderma GanodermasinenseZhao, the dry sporophore of XuetZhang.Phellinus igniarius (L. ex Fr.) Quel. is the dry sporophore of Basidiomycotina (Basidiomycotina) Hymenomycetes (Hymenomycetes) Aphyllophorales (Polyporales) perforated Cordycepps (Hymenochaetacae) Phellinus (Phellinus).Ganoderma applanatum (Pers. Ex Wallr) Pat. is the dry sporophore that basidiomycetes polypor detailed outline Ganoderma applanatum (Pers. Ex Wallr) Pat. section Ganoderma applanatum (Pers. Ex Wallr) Pat. belongs to fungus (Ganodermaapplanatum (Pers.) Pat).Inonqqus obliquus belongs to the dry sporophore of Eumycota, Basidiomycotina, Hymenomycetes, non-brown Zoopagales, Polyporaceae, brown transverse hole fungus genus (Fuscoporia obliqua (Pers.Fr.) Aosh or Inonotus obliquus (Fr.) Pilat).Jin Zhi is the dry sporophore of the Hymenochaetaceae of caprifoliaceae plant Radix Ophiopogonis Lonicera japonica Thunb basal part of stem parasitism lobate layer Pseudomonas fungus currant leaf pore fungi [Phylloporia ribis (Schumach.:Fr.) Ryvarden].Hericium erinaceus (Bull. Ex Fr.) Pers. cries again Hericium erinaceus (Bull. Ex Fr.) Pers., monkey mushroom, and hedgehog hydnum, monkey mushroom is the dry sporophore of Hericium erinaceus.

Of the present inventionly being refrigerated to subzero certain DEG C, is to be refrigerated to and to freeze at this subzero certain DEG C.

So far completed the present invention, the medicinal fungi of preparing by drying means of the present invention, mainly contains effective component content high, and pharmacologically active is strong.

Further illustrate usefulness of the present invention below by test example, test example is intended to further illustrate usefulness of the present invention, but not limitation of the present invention.

One, by with a collection of Ganoderma of newly gathering, Phellinus igniarius (L. ex Fr.) Quel., Ganoderma applanatum (Pers. Ex Wallr) Pat., Inonqqus obliquus, Hericium erinaceus (Bull. Ex Fr.) Pers., Jin Zhi, remove impurity, process by the following method respectively.

1) dry: shady and cool ventilation place, spread out and dry;

2) dry: dry;

3) dry: 50 DEG C of following oven dry;

4) be refrigerated to-5 DEG C to freezing, bank up 3 days at dark and damp place, 50 DEG C of following oven dry.

(1) the dry medicinal fungi polyoses content comparison of distinct methods

The preparation of reference substance solution: get glucose reference substance appropriate, accurately weighed, add water and make the solution of every 1ml containing 0.1mg, to obtain final product.

The preparation of standard curve: precision measures reference substance solution 0.2ml respectively, 0.4m1, 0.6ml, 0.8ml, 1.0ml, 1.2ml, put in 10ml tool plug test tube, add water to 2.0ml, precision adds sulphuric acid anthrone solution, and (precision takes anthrone 0.1g, adding 80% sulfuric acid solution 100ml makes to dissolve, shake up) 6ml, shake up, put in water-bath and heat 15 minutes, take out, put in people's ice bath cooling 15 minutes, taking corresponding reagent as blank, according to " Chinese Pharmacopoeia " ultraviolet visible spectrophotometry, measure absorbance at 625nm wavelength place, taking absorbance as vertical coordinate, concentration is abscissa, drawing standard curve.

The preparation of need testing solution: get the about 2g of each sample powder, accurately weighed, put in apparatus,Soxhlet's, the 90ml that adds water, heating and refluxing extraction is to extracting liquid colourless, and extracting solution is transferred in 100ml measuring bottle, adds water to scale, shakes up.Precision measures 10ml, adds ethanol 150ml, shakes up, and places 12 hours for 4 DEG C, takes out, centrifugal, the supernatant that inclines, and precipitation is dissolved in water, and is transferred in 50ml measuring bottle, adds water to scale, shakes up, and to obtain final product.

Algoscopy: precision measures need testing solution 2ml, put in 10ml tool plug test tube, method under the preparation of sighting target directrix curve, from " precision adds sulphuric acid anthrone solution 6ml ", measure absorbance in accordance with the law, read the weight (mg) containing glucose in need testing solution from standard curve, calculate, to obtain final product.

Press dry product and calculate, in anhydrous glucose (C6H12O6), the results are shown in Table 1,2,3,4,5,6 containing polysaccharide.

Table 1 distinct methods is dried ganoderma polyoses content (%)

Drying means	Dry	Dry	Dry	The freezing oven dry of banking up
					Content (%)	1.76	1.74	1.74	3.26

Table 2 distinct methods is dried phellin polysaccharides content (%)

Drying means	Dry	Dry	Dry	The freezing oven dry of banking up
					Content (%)	3.75	3.81	3.76	5.42

Table 3 distinct methods is dried Ganoderma Applanatum Polysaccharides content (%)

Drying means	Dry	Dry	Dry	The freezing oven dry of banking up
					Content (%)	4.26	4.25	4.25	6.73

Table 4 distinct methods is dried Fuscoporia obliqua polysaccharide content (%)

Drying means	Dry	Dry	Dry	The freezing oven dry of banking up
					Content (%)	5.31	5.24	5.26	9.37

Table 5 distinct methods is dried Hericium Erinaceus Polysaccharide content (%)

Drying means	Dry	Dry	Dry	The freezing oven dry of banking up
					Content (%)	4.38	4.36	4.37	7.04

Table 6 distinct methods is dried golden sesame polyoses content (%)

Drying means	Dry	Dry	The freezing oven dry of banking up
				Content (%)	4.35	4.34	8.38

Found out by table, Ganoderma, Phellinus igniarius (L. ex Fr.) Quel., Ganoderma applanatum (Pers. Ex Wallr) Pat., Inonqqus obliquus, Hericium erinaceus (Bull. Ex Fr.) Pers., Jin Zhi " freezing heap postpone is dry ", polyoses content is apparently higher than " drying ", " drying " and " oven dry ".

(2) the dry anti-human lung of medicinal fungi source adenocarcinoma cell SPCA-1, human hepatoma cell line HepG2 and the people's chronic myelogenous leukemia cell K562 test of distinct methods.

Prepare medicinal fungi extract: get respectively the dry medicinal fungi of distinct methods and pulverize, respectively decoct with water twice, add water for the first time 8 times and measure, decoct 2 hours, add water for the second time 8 times and measure, decoct 1 hour, merge medicinal liquid, medicinal liquid filters, and gets filtrate, and being concentrated into relative density is 1.10(60 DEG C) time, add ethanol to make to reach 75% containing alcohol amount, precipitation, leaves standstill 36 hours, draw supernatant, reclaim ethanol, concentrated, dry, pulverize, obtain the dry Ganoderma applanatum (Pers. Ex Wallr) Pat. extract of distinct methods.

Tumor cell line: people's lung source adenocarcinoma cell SPCA-1, human hepatoma cell line HepG2 and people's chronic myelogenous leukemia cell K562, use RPMI-1649 culture fluid, 37 DEG C, 5%CO2, relative humidity 100% is cultivated, and attached cell 0.25% trypsinization goes down to posterity.

Method: the inhibitory action of MTT colorimetric method for determining extract to growth of tumour cell.The trophophase cell of taking the logarithm, is mixed with cell suspension, 0.8 × 105/ml of 1 × 105/ml of suspension cell attached cell with fresh RPMI-1640 culture fluid.Suspension cell is inoculated in 96 well culture plates after adding respectively variable concentrations Experimental agents; Attached cell is first inoculated in 96 well culture plates, after every hole 90 μ l24h, adds respectively variable concentrations Experimental agents.Final volume is that every hole 100 μ l and every group are established 3 parallel holes, establishes altogether 4 groups: be subject to reagent group T (culture fluid+cell suspension+variable concentrations is subject to reagent), negative control group N (culture fluid+cell suspension), medicine color matched group TC (culture fluid+variable concentrations is subject to reagent), blank group B (culture fluid+normal saline).Tested concentration is followed successively by 10 μ g/ml, 30 μ g/ml, 50 μ g/ml.Put 37 DEG C, in 5%CO2 incubator, cultivate after 72h, after adding the 10 μ l concussions of MTT solution to mix to every hole, continue to cultivate 4h, add SDS90 μ l to stop cultivating, 37 DEG C are spent the night, and then under room temperature, on micro oscillator, shake 10min, microplate reader is measured the absorbance (OD value) at 570nm wavelength place, and experiment repeats 3 times.

Calculate as follows growth inhibition ratio:

Growth inhibition ratio (%)=(N group OD average-B group OD average)-(T group OD average-TC group OD average)/N group OD average-B group OD average × 100%.

Result: distinct methods is dry, and different medicinal fungi extracts the results are shown in following table to the suppression ratio of people's lung source adenocarcinoma cell SPCA-1, human hepatoma cell line HepG2 and people's chronic myelogenous leukemia cell K562.

1, the dry Ganoderma extract of distinct methods the results are shown in Table 7,8,9 to the suppression ratio of tumor cell.

The suppression ratio result of the dry Ganoderma of table 7 distinct methods to SPCA-1

Suppression ratio (%)	10（μg/ml）	30（μg/ml）	50（μg/ml）
				Dry	46.52	53.42	66.51
Dry	46.50	53.42	66.50
				Dry	46.47	53.50	66.53
The freezing oven dry of banking up	57.68	71.38	79.64

The suppression ratio result of the dry Ganoderma of table 8 distinct methods to HepG2

Suppression ratio (%)	10（μg/ml）	30（μg/ml）	50（μg/ml）
				Dry	43.62	54.74	58.67
Dry	43.59	54.74	58.66
				Dry	43.61	54.71	58.64
The freezing oven dry of banking up	56.48	67.49	83.16

The suppression ratio result of the dry Ganoderma of table 9 distinct methods to K562

Suppression ratio (%)	10（μg/ml）	30（μg/ml）	50（μg/ml）
				Dry	57.29	68.44	74.65
Dry	57.28	68.45	74.65
				Dry	57.31	68.42	74.62
The freezing oven dry of banking up	64.38	73.75	83.02

2, the dry Ganoderma applanatum (Pers. Ex Wallr) Pat. extract of distinct methods the results are shown in Table 10,11,12 to the suppression ratio of tumor cell.

The suppression ratio result of the dry Ganoderma applanatum (Pers. Ex Wallr) Pat. of table 10 distinct methods to SPCA-1

Suppression ratio (%)	10（μg/ml）	30（μg/ml）	50（μg/ml）
				Dry	47.43	58.36	67.11
Dry	47.42	58.37	67.12
				Dry	47.42	58.35	67.12
The freezing oven dry of banking up	53.27	73.42	81.44

The suppression ratio result of the dry Ganoderma applanatum (Pers. Ex Wallr) Pat. of table 11 distinct methods to HepG2

Suppression ratio (%)	10（μg/ml）	30（μg/ml）	50（μg/ml）
				Dry	46.72	56.54	65.57
Dry	46.41	56.51	65.58
				Dry	46.34	56.53	65.54
The freezing oven dry of banking up	57.61	73.37	86.18

The suppression ratio result of the dry Ganoderma applanatum (Pers. Ex Wallr) Pat. of table 12 distinct methods to K562

Suppression ratio (%)	10（μg/ml）	30（μg/ml）	50（μg/ml）
				Dry	53.28	66.51	71.62
Dry	53.27	66.52	71.60
				Dry	53.26	66.50	71.56
The freezing oven dry of banking up	61.73	74.35	80.47

3, the dry Inonqqus obliquus extract of distinct methods the results are shown in Table 13,14,15 to the suppression ratio of tumor cell.

The suppression ratio result of the dry Inonqqus obliquus of table 13 distinct methods to SPCA-1

Suppression ratio (%)	10（μg/ml）	30（μg/ml）	50（μg/ml）
				Dry	53.21	61.15	75.33
Dry	53.34	61.21	75.30
				Dry	53.21	61.17	75.34
The freezing oven dry of banking up	64.36	75.32	84.86

The suppression ratio result of the dry Inonqqus obliquus of table 14 distinct methods to HepG2

Suppression ratio (%)	10（μg/ml）	30（μg/ml）	50（μg/ml）
				Dry	43.85	54.20	60.27
Dry	43.82	54.21	60.27
				Dry	43.84	54.21	60.25
The freezing oven dry of banking up	55.74	63.48	86.51

The suppression ratio result of the dry Inonqqus obliquus of table 15 distinct methods to K562

Suppression ratio (%)	10（μg/ml）	30（μg/ml）	50（μg/ml）
				Dry	55.52	70.44	75.78
Dry	55.51	70.43	75.86
				Dry	55.53	70.43	75.85
The freezing oven dry of banking up	71.43	76.82	81.27

Below respectively show result and show, the dry medicinal fungi of distinct methods all has inhibitory action to the propagation of SPCA-1, HepG2, K562 cell, and wherein, the freezing oven dry curative effect of banking up is best.

(3) the dry flavonoids from phellinus content comparison of distinct methods

Take respectively each sample powder (60 order) 5g, add volume fraction 70% alcoholic solution 70mL, reflux, extract, 4 times, merges supernatant, filter, and filtrate recycling ethanol, vacuum drying, obtains flavone extract.

Get a certain amount of dried flavone extract, with dissolve with methanol and be settled to certain volume, adopt NaNO

The content of 2-Al (NO3) colorimetric method for determining flavone, taking rutin as standard substance drawing standard curve, in dry product, the results are shown in Table 16.

Table 16 distinct methods is dried flavonoids from phellinus content (%)

Drying means	Dry	Dry	Dry	The freezing oven dry of banking up
					Content (%)	2.12	2.53	2.57	4.31

Found out by table 16, Phellinus igniarius (L. ex Fr.) Quel. " freezing heap postpone is dry ", flavones content is apparently higher than " drying ", " drying " and " oven dry ".

(4) the dry golden sesame sterols content comparison of distinct methods

1, instrument and material

Instrument: Agilentl 100 high performance liquid chromatographs, G1315A/B (DAD UV-detector); Medicine: ergosterol reference substance, methanol, ultra-pure water, other reagent are analytical pure.

2, method

Chromatographic condition chromatographic column: Zorbax eclips XDB C8 (150mm × 4.6mm); Mobile phase: methanol; Flow velocity: 1.0 mL/ min; Detect wavelength: 282nm; Column temperature: 30 DEG C; Sample size: 20 μ L.Under these conditions, inject sample solution, theoretical cam curve is greater than 10000 in ergosterol.

The preparation of reference substance solution: it is appropriate that precision takes ergosterol reference substance, is mixed with the solution of 0.3mg/mL with methanol, 4 DEG C of cold preservations are for subsequent use.

The preparation of need testing solution: get the about 0.5g of each sample powder (crossing sieve No. three), accurately weighed, be placed in tool plug triangular flask, add methanol 40mL, weighed weight, ultrasonic (400W, 40kHz) 1h, place room temperature, weigh, add methanol to former weight, get supernatant and cross 0.45 μ m filter membrane, obtain need testing solution, 4 DEG C of cold preservations are for subsequent use.

Sample determination: the accurate need testing solution 20 μ m that draw, injection liquid chromatography, records peak area value, calculates content by dry product.

3, result the results are shown in Table 17.

Table 17 distinct methods is dried golden sesame sterols content (%)

Drying means	Dry	Dry	The freezing oven dry of banking up
				Content (%)	0.11	0.12	0.42

Found out by table 17, Jin Zhi " freezing heap postpone is dry ", sterols content is apparently higher than " drying " and " oven dry ", and known the inventive method has advance.

(5) the dry Hericium erinaceus (Bull. Ex Fr.) Pers. of distinct methods causes the protective effect comparison of gastric mucosa damage to ethanol.

Choose healthy SD rat, male and female half and half, weight 180-220g, is divided into that matched group, Hericium erinaceus (Bull. Ex Fr.) Pers. dry group, dry group, oven dry group, the freezing oven dry group of banking up at random, totally 5 groups, 10 every group, every day gastric infusion 1 time, 1 2mg/kg, continuous 7 days, matched group gave equivalent distilled water.Before experiment, first fasting 48 h of rat, freely drink water, after last administration, every rat oral gavage dehydrated alcohol 1mL/ of 1h only, put to death animal after the de-cervical vertebra of 1h, close cardia by clip, inject 1% formaldehyde 5mL from pylorus, folder closes pylorus again, put into the fixing 10min of 1% formalin, cut off coat of the stomach along greater gastric curvature, measure damaged length, using damaged length (width is greater than 1 mm to be doubled) summation as gastric mucosal damage index, and calculate suppression ratio.The results are shown in Table 18.

Suppression ratio (%)=(matched group ulcer index one administration group ulcer index) ÷ matched group ulcer index × 100%

The protection result of the dry Hericium erinaceus (Bull. Ex Fr.) Pers. of table 18 distinct methods to mucosal lesion

Group	Dosage (mg/kg)	Gastric mucosal damage index (mm)	Suppression ratio (%)
				Matched group	?	97.6±34.2	?
Dry group	2	58.3±26.7	40.26
				Dry group	2	58.9±26.6	39.65
Oven dry group	2	58.3±25.9	40.27
				The freezing oven dry group of banking up	2	41.2±22.4	57.79

Result shows, the dry Hericium erinaceus (Bull. Ex Fr.) Pers. of distinct methods causes gastric mucosa damage to ethanol all protective effect, wherein, freezingly banks up that to dry protective effect best.

Detailed description of the invention

embodiment 1

Get respectively the Ganoderma, Phellinus igniarius (L. ex Fr.) Quel., Ganoderma applanatum (Pers. Ex Wallr) Pat., Inonqqus obliquus, Hericium erinaceus (Bull. Ex Fr.) Pers., the Jin Zhi that newly gather, put in freeze dryer, be refrigerated to-5 DEG C to freezing, banking up 2 days in dark and damp place, dries.

embodiment 2

Get respectively the Ganoderma, Phellinus igniarius (L. ex Fr.) Quel., Ganoderma applanatum (Pers. Ex Wallr) Pat., Inonqqus obliquus, Hericium erinaceus (Bull. Ex Fr.) Pers., the Jin Zhi that newly gather, put in freeze dryer, be refrigerated to-10 DEG C, bank up 3 days at dark and damp place, dry.

embodiment 3

Get respectively the Ganoderma, Phellinus igniarius (L. ex Fr.) Quel., Ganoderma applanatum (Pers. Ex Wallr) Pat., Inonqqus obliquus, Hericium erinaceus (Bull. Ex Fr.) Pers., the Jin Zhi that newly gather, put in freeze dryer, be refrigerated to-20 DEG C, bank up 4 days at dark and damp place, dry.

embodiment 4

Get respectively the Ganoderma, Phellinus igniarius (L. ex Fr.) Quel., Ganoderma applanatum (Pers. Ex Wallr) Pat., Inonqqus obliquus, Hericium erinaceus (Bull. Ex Fr.) Pers., the Jin Zhi that newly gather, put in freeze dryer, be refrigerated to-32 DEG C to freezing, bank up 3 days at dark and damp place, dry.

embodiment 6

Get respectively the Ganoderma, Phellinus igniarius (L. ex Fr.) Quel., Ganoderma applanatum (Pers. Ex Wallr) Pat., Inonqqus obliquus, Hericium erinaceus (Bull. Ex Fr.) Pers., the Jin Zhi that newly gather, put in freeze dryer, be refrigerated to-15 DEG C, bank up 5 days at dark and damp place, dry.

Claims (5)

1. the application of Refrigeration Technique in medicinal fungi is dry, is characterized in that: get the medicinal fungi of newly gathering, and freezing, the 2-5 days that banks up at dark and damp place is dry.

2. the application in medicinal fungi is dry according to the Refrigeration Technique of claim 1, is characterized in that: freezing is to be refrigerated to freeze.

3. the application in medicinal fungi is dry according to the Refrigeration Technique of claim 1, is characterized in that: freezing is to be refrigerated to-5 DEG C to freeze below.

4. the application in medicinal fungi is dry according to the Refrigeration Technique of claim 1, is characterized in that: dry is that heap postpone is dried or dries or dries.

5. the application in medicinal fungi is dry according to the Refrigeration Technique of claim 1, is characterized in that: medicinal fungi is Ganoderma, Phellinus igniarius (L. ex Fr.) Quel., Ganoderma applanatum (Pers. Ex Wallr) Pat., Inonqqus obliquus, Jin Zhi, Hericium erinaceus (Bull. Ex Fr.) Pers..