CN105748610A - Solid fermentation method for polygonum hydropiper linn preparations by aid of bacillus subtilis, fermentation products of solid fermentation method and application - Google Patents
Solid fermentation method for polygonum hydropiper linn preparations by aid of bacillus subtilis, fermentation products of solid fermentation method and application Download PDFInfo
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Abstract
The invention discloses a solid fermentation method for polygonum hydropiper linn preparations by the aid of bacillus subtilis, fermentation products of the solid fermentation method and application.The solid fermentation method includes smashing daphniphyllum calycillum benth and polygonum hydropiper; uniformly mixing the daphniphyllum calycillum benth and the polygonum hydropiper with each other to obtain polygonum hydropiper linn powder; sterilizing the polygonum hydropiper linn powder; adding nutrient meat juice culture media and the bacillus subtilis into the polygonum hydropiper linn powder; fermenting and cultivating the polygonum hydropiper linn powder under the conditions of the temperatures of 22-30 DEG C and the humidity of 50%-90%.The solid fermentation method, the fermentation products and the application have the advantages that fermentation tests are carried out on polygonum hydropiper linn compounds by the aid of microbial fermentation technologies, fermentation strains are screened, and the fermentation conditions are optimized, so that the inexpensive, efficient and zero-pollution Chinese herbal compound fermentation finished products can be obtained; as discovered by experiments, obvious inhibition effects can be realized by fermentation extract liquid for writhing response of mice due to acetic acid; certain inhibition effects can be realized for increase of capillary permeability of the mice due to the acetic acid, obvious inhibition effects can be realized for ear swelling due to dimethylbenzene and foot swelling due to egg white, gastric mucosa damage due to the acetic acid can be reduced, exudation of HA in tissues can be obviously inhibited, excellent anti-inflammation and analgesia curative effectives can be realized, and curative effects of the fermentation products are obviously superior to curative effects of unfermented polygonum hydropiper linn extract.
Description
Technical field
The present invention relates to the method for bacillus subtilis bacteria solid fermentation maple knotweed preparation and tunning thereof and application.
Background technology
Herba polygoni hydropiperis is the dry herb of Polygonaceae (Polygonaceae) Polygonum (Polygonum) plant PolygonumhydropiperLinn..Acrid in the mouth, warm in nature；There is the effect such as invigorating the spleen for eliminating dampness, dispeling the wind stagnant, eliminating stasis to stop pain, removing toxic substances and promoting subsidence of swelling, killing parasites for relieving itching；Clinic is used for dysentery, gastroenteritis, diarrhoea, rheumatic arthritis, treating swelling and pain by traumatic injury, dysfunctional uterine hemorrhage；External can control venom, skin eczema etc..Research in recent years shows, water Herba polygoni hydropiperis has antimicrobial acivity, antioxidation, antiinflammatory, antibacterial, antineoplastic action.Water Herba polygoni hydropiperis mainly contains the compounds such as flavonoid, volatile oil, sesquiterpenoids, tannin class, fatty acid, Anthraquinones, Herba polygoni hydropiperis has widely distributed in China, there is abundant plant resources, good DEVELOPMENT PROSPECT is had in medicine, food, botanical pesticide etc., for comprehensively utilizing the plant resources of this preciousness, promote comprehensive exploitation and the utilization of Herba polygoni hydropiperis, carry out this problem.Daphniphyllum calycinum is the dry stem and branch with leaf of Daphniphyllaceae (Daphniphyllaceae) Daphniphyllum (Daphniphyllum) plant DaphniphyllumcalycinumBench..Bitter in the mouth, puckery, property is put down, poisonous.There is antitumor, antiinflammatory, parasite killing, antibacterial function, cure mainly rheumatic ostalgia, sore swollen toxin, injury and bone fracture, venom.Daphniphyllum calycinum is the principal agent of Hainan 'Fengliaochangweikang' ' preparation, mainly contains flavones ingredient and alkaloids composition.
Microorganism has abundant enzyme system, has the ability of powerful decomposition and transformation substance.Fermented altogether by microorganism and Chinese medicine and carry out Chinese medicine processing, it is possible to obtain the medicine that drug effect is higher.The possible mechanism of action has following several: the secondary metabolites that fermentable produces and effective ingredient generation synergism and then enhancing drug effect；Microorganism makes effective ingredient be converted into small-molecule substance or be changed into free state from combined state during the fermentation, is more conducive to the absorption of health；Chemical composition of Chinese materia medica is carried out bioconversion by microorganism, produces new compound or causes the change of some compositions, content in Chinese medicine.Screening for reactive compound is all provided rich in natural resources by these.
Summary of the invention
The present invention adopts microbial fermentation technology that maple knotweed compound recipe carries out fermentation test, screens fermented bacterium, optimization of fermentation conditions.Obtain Chinese medicine compound fermentation finished product cheap, efficient, no pollution.
It is an object of the invention to provide the method for bacillus subtilis bacteria solid fermentation maple knotweed preparation and tunning thereof and application.
The technical solution used in the present invention is:
A kind of method of bacillus subtilis bacteria solid fermentation maple knotweed preparation, comprises the following steps:
1) Daphniphyllum calycinum, Herba polygoni hydropiperis are pulverized, in mass ratio (1.5～2.5): 1 mixing, obtain maple knotweed powder；
2) solid fermentation: by maple knotweed powder sterilizing, add nutrient culture medium and Bacillus subtillis, mixing, in 22～30 DEG C, humidity is that 50%～90% condition bottom fermentation is cultivated 1～7 day,.
Further, step 2) in the mass volume ratio of maple knotweed powder and nutrient culture medium be 1g:(1～3) mL.
Further, step 2) in the consumption of bacillus subtilis be that every gram of maple knotweed powder adds 30～35 μ l bacillus subtilis bacterium solution, bacterial concentration is 106～108cfu/mL。
Further, the pH of above-mentioned nutrient culture medium is 6～8.
Further, step 2) temperature fermented is 28 DEG C.
Further, step 2) humidity fermented is 80%.
Further, step 2) fermented incubation time is 24h.
The extracting method of active substance in said method gained tunning, comprise the following steps: take method described above fermentation products therefrom, dry, grinding to obtain powder, be added to the water by powder, the mass volume ratio of powder and water is 1g:7～9mL, soak 1.8～2.2h, 88～92 DEG C decoct 88～92min, centrifugal, take clear liquid；Precipitation being added in water, the mass volume ratio of precipitation and water is 1g:5～7mL, and 88～92 DEG C decoct 55～65min, centrifugal, take clear liquid, merge the supernatant of twice, and concentrated supernatant, to crude drug concentration 0.8～1.2g/mL, obtains extract.
Further, above-mentioned drying temperature is 40～60 DEG C.
The extract that said method obtains is preparing antiinflammatory or/and application in Pain relief agents.
The invention has the beneficial effects as follows:
The present invention utilizes bacillus subtilis that the fermentation mode of maple knotweed preparation obtains maple knotweed fermented extracted liquid, this fermented extracted liquid is found through experiments acetic acid causes mouse writhing reaction significantly inhibit effect (P < 0.01), but mouse hot-plate is licked metapedes unrestraint effect；Acetic acid is caused the rising of mice capillary permeability and has certain inhibitory action (P>0.05), xylol causes ear swelling, Ovum Gallus domesticus album causes foot swelling and all significantly inhibits effect (P<0.01), there is the reduction ethanol damage (P<0.01) to gastric mucosa, substantially reduce the concentration (P<0.01) of IL-1 α in serum, IL-1 β, significantly inhibit HA in tissue and ooze out (P<0.01), PEG2 is oozed out and has certain suppression, having antiinflammatory very and analgesia effect, its curative effect is substantially better than the extract of unleavened maple knotweed.
Detailed description of the invention
A kind of method of bacillus subtilis bacteria solid fermentation maple knotweed preparation, comprises the following steps:
1) Daphniphyllum calycinum, Herba polygoni hydropiperis are pulverized, in mass ratio (1.5～2.5): 1 mixing, obtain maple knotweed powder；
2) solid fermentation: by maple knotweed powder sterilizing, add nutrient culture medium and Bacillus subtillis, mixing, in 22～30 DEG C, humidity is that 50%～90% condition bottom fermentation is cultivated 1～7 day,.
Preferably, step 1) Daphniphyllum calycinum, Herba polygoni hydropiperis be crushed to 20 orders with top sieve.
Preferably, step 1) Daphniphyllum calycinum, Herba polygoni hydropiperis mass ratio be 2:1.
Preferably, step 2) in the mass volume ratio of maple knotweed powder and nutrient culture medium be 1g:(1～3) mL.
Preferably, maple knotweed powder is 1g:1mL with the mass volume ratio of nutrient culture medium.
Preferably, temperature during described sterilizing is 121 DEG C, and pressure is 14.5～15.5 pounds per inch2, the time is 15～20min.
Preferably, step 2) in the consumption of bacillus subtilis be that every gram of maple knotweed powder adds 30～35 μ l bacillus subtilis bacterium solution, bacterial concentration is 106～108cfu/mL。
Preferably, the pH of described nutrient culture medium is 6～8.
Preferably, the pH of described nutrient culture medium is 7.
Preferably, described nutrient culture medium prescription is: cultivating wherein containing Carnis Bovis seu Bubali cream 3g, peptone 5g, NaCl5g for every liter, surplus is water, standby after sterilizing.
Preferably, step 2) temperature fermented is 28 DEG C.
Preferably, step 3) humidity fermented is 80%.
Preferably, step 3) fermented incubation time is 24h.
The extracting method of active substance in any of the above-described described method gained tunning, comprise the following steps: take method described above fermentation products therefrom, dry, grinding to obtain powder, be added to the water by powder, the mass volume ratio of powder and water is 1g:7～9mL, soak 1.8～2.2h, 88～92 DEG C decoct 88～92min, centrifugal, take clear liquid；Precipitation being added in water, the mass volume ratio of precipitation and water is 1g:5～7mL, and 88～92 DEG C decoct 55～65min, centrifugal, take clear liquid, merge the supernatant of twice, and concentrated supernatant, to crude drug concentration 0.8～1.2g/mL, obtains extract.
Preferably, described drying temperature is 40～60 DEG C.
The extract that method described above obtains is preparing antiinflammatory or/and application in Pain relief agents.
Below in conjunction with specific embodiment, the present invention is further illustrated, but is not limited thereto.
The method of embodiment 1 bacillus subtilis bacteria solid fermentation maple knotweed preparation
1) Daphniphyllum calycinum, Herba polygoni hydropiperis are pulverized, cross 20 orders with top sieve, by Daphniphyllum calycinum: Herba polygoni hydropiperis mass ratio is 2:1 mixing, obtains maple knotweed powder；
2) solid fermentation: weighing the maple knotweed powder 30g of mix homogeneously, 121 DEG C (pressure is 15 pounds per inch2) sterilizing 15min, adding nutrient culture medium 30ml, Bacillus subtillis suspension 1ml, bacterial concentration is 106～108Cfu/mL, mixing；In 28 DEG C, the constant temperature and humidity incubator fermentation culture of humidity 80% 1 day,.
Above-mentioned Bacillus subtillis (GIM1.129) is purchased from Guangdong Province's Culture Collection.
The formula of above-mentioned nutrient culture medium is, Carnis Bovis seu Bubali cream 3g, peptone 5g, NaCl5g, adds distilled water to 1000ml, and adjusting pH is 7；In 121 DEG C, (pressure is 15 pounds per inch2) sterilizing 15min, standby.
In the present invention in research process, by substantial amounts of experimentation data, it is thus achieved that following information:
1. above-mentioned Daphniphyllum calycinum: Herba polygoni hydropiperis mass ratio is in (1.5～2.5): in 1 scope, but Daphniphyllum calycinum: when Herba polygoni hydropiperis mass ratio is 2:1, gained ferment effect is best, and the drug effect of the extract of tunning is best.
2., during fermentation, the mass volume ratio of maple knotweed powder and nutrient culture medium can be 1g:(1～3) mL, when only the mass volume ratio of three is 1g:1mL, the stripping quantity of tunning total flavones reaches maximum, and drug effect is best simultaneously.
3. temperature during fermentation can be 22～30 DEG C, and humidity can be 50～90%, and fermentation time can be 1～7 day, but only when fermentation temperature is 28 DEG C, humidity is 80%, when the time is 1 day, the stripping quantity of tunning total flavones can be only achieved maximum, and drug effect is best simultaneously.
4. the pH value of above-mentioned nutrient culture medium is 6～8, is when 7.0 at pH, and the stripping quantity of tunning total flavones can be only achieved maximum, and drug effect is best simultaneously.
5. the consumption of bacillus subtilis can be 30～35 μ l/g (namely every gram of maple knotweed powder adds 30～35 μ l bacillus subtilis bacterium solution), and the suitableeest bacterium amount is 33 μ l/g, and bacterial concentration is 106～108Cfu/mL, most beneficial for the dissolution of flavonoid compound.
The method of embodiment 2 bacillus subtilis bacteria solid fermentation maple knotweed preparation
1) Daphniphyllum calycinum, Herba polygoni hydropiperis are pulverized, cross 20 orders with top sieve, by Daphniphyllum calycinum: Herba polygoni hydropiperis mass ratio is 1.5:1 mixing, obtains maple knotweed powder；
2) solid fermentation: weighing the maple knotweed powder 30g of mix homogeneously, 121 DEG C (pressure is 15 pounds per inch2) sterilizing 15min, adding nutrient culture medium 3ml, Bacillus subtillis suspension 1ml, bacterial concentration is 106～108Cfu/mL, mixing；In 28 DEG C, the constant temperature and humidity incubator fermentation culture 24h of humidity 80%,.
Above-mentioned Bacillus subtillis (GIM1.129) is purchased from Guangdong Province's Culture Collection.
The formula of above-mentioned nutrient culture medium is, Carnis Bovis seu Bubali cream 3g, peptone 5g, NaCl5g, add distilled water to 1000ml, adjusting pH is 7.0；In 121 DEG C, (pressure is 15 pounds per inch2) sterilizing 15min, standby.
The method of embodiment 3 bacillus subtilis bacteria solid fermentation maple knotweed preparation
1) Daphniphyllum calycinum, Herba polygoni hydropiperis are pulverized, cross 20 orders with top sieve, by Daphniphyllum calycinum: Herba polygoni hydropiperis mass ratio is 2.5:1 mixing, obtains maple knotweed powder；
2) solid fermentation: weighing the maple knotweed powder 30g of mix homogeneously, 121 DEG C (pressure is 15 pounds per inch2) sterilizing 15min, adding nutrient culture medium 40ml, Bacillus subtillis suspension 1ml, bacterial concentration is 106～108Cfu/mL, mixing；In 22 DEG C, the constant temperature and humidity incubator fermentation culture of humidity 50% 7 days,.
Above-mentioned Bacillus subtillis (GIM1.129) is purchased from Guangdong Province's Culture Collection.
The formula of above-mentioned nutrient culture medium is, Carnis Bovis seu Bubali cream 3g, peptone 5g, NaCl5g, adds distilled water to 1000ml, and adjusting pH is 7.0；In 121 DEG C, (pressure is 15 pounds per inch2) sterilizing 15min, standby.
The method of embodiment 4 bacillus subtilis bacteria solid fermentation maple knotweed preparation
1) Daphniphyllum calycinum, Herba polygoni hydropiperis are pulverized, cross 20 mesh sieves, by Daphniphyllum calycinum: Herba polygoni hydropiperis mass ratio is 2:1 mixing, obtains maple knotweed powder；
2) solid fermentation: weighing the maple knotweed powder 30g of mix homogeneously, 121 DEG C (pressure is 15 pounds per inch2) sterilizing 15min, adding nutrient culture medium 30ml, Bacillus subtillis suspension 2ml, bacterial concentration is 106～108Cfu/mL, mixing；In 30 DEG C, the constant temperature and humidity incubator fermentation culture of humidity 80% 1 day.
Bacillus subtillis (GIM1.129) is purchased from Guangdong Province's Culture Collection.
Wherein, the formula of nutrient culture medium is: Carnis Bovis seu Bubali cream 3g, peptone 5g, NaCl5g, agar 20g, adds distilled water to 1000ml, and adjusting pH is 6.0.
Herba polygoni hydropiperis place of production Guangzhou Guangdong, batches 201309；No. 2 samples: Daphniphyllum calycinum place of production Period In Maoming, batches 201306.The herb that No. 1 sample is polygonaceae plant water Herba polygoni hydropiperis (PolygonumhydropiperLinn.) is identified through Guangdong Pharmaceutical University professor Li Shuyuan.
Daphniphyllaceae (Daphniphyllaceae) Daphniphyllum (Daphniphyllum) Daphniphyllum calycinum plant, medical material, after cold drying, is ground into coarse powder with Chinese medicine grinder, crosses 20 mesh sieves, standby.
The extracting method of effective substance in embodiment 5 bacillus subtilis bacteria solid fermentation maple knotweed formulation product
1) tunning of the 1st day after Example 1 fermentation of bacillus subtilis, is placed in 40～60 DEG C of baking oven inner dryings, fermentating drying product is ground to obtain powder；
2) being added to the water by powder, the solid-liquid ratio of powder and aqueous solution is 1g:7～9mL, soaks 2h, then decocts that (temperature of decoction is90DEG C) 0.5h, centrifugal, take clear liquid；Precipitation is added in aqueous solution, soak 2h and decoct (temperature of decoction is 90 DEG C) 0.5h, centrifugal, take clear liquid, merging the supernatant of twice, concentrated supernatant, to crude drug concentration 1g/mL, obtains drug effect extract (hereinafter referred to as maple knotweed fermented extracted liquid group, ferment referred to as maple knotweed), can be directly used for following experiment.
The extracting method of effective substance in the unfermentable maple knotweed preparation of embodiment 6
1) take and be cut into the Daphniphyllum calycinum 400g of 1～2cm, Herba polygoni hydropiperis 200g；
2) add 8 times of water (4.8L) and soak 120min；
3) 90min decocts；
4) 6 times of water (3.6L) decoct 60min；
5) merge twice maple knotweed decoction liquor and be filtered；
6) after rotary evaporator concentrates centrifugal (3000r/min, 20min), supernatant is taken；
7) rotary evaporator is again concentrated to 600mL and crude drug concentration 1g/mL and namely obtains drug effect extract, and 4 DEG C save backup；(hereinafter referred to as maple knotweed extracting solution group of not fermenting, be called for short maple knotweed and do not ferment), can be directly used for following experiment, as the contrast of maple knotweed fermented extracted liquid group.
The application of drug effect extract in embodiment 7 bacillus subtilis bacteria solid fermentation maple knotweed formulation product
1. pharmacodynamic experiment method
1.1 antiinflammatories and analgesic test
Acetic acid causes mice Rat paw edema test and writhing method: mice 60, male and female half and half, divide equally 6 groups, matched group: normal saline at random, aspirin matched group 0.2g/kg, the intestines and stomach health group 10g/kg, maple knotweed fermented extracted liquid group dosage is made a living dose 20.0 and 10.0,5.0g/kg, the extracting solution crude drug amount 20.0 and 10.0 of the maple knotweed that do not ferment, 5.0g/kg (i.e. extracting solution obtained by embodiment 6, do not ferment hereinafter referred to as maple knotweed), it is administered by 0.5ml/20g body weight, every day 1 time, continuous 3d.After last administration 1h, the acetum 0.2ml/ of lumbar injection 0.6%, the writhing number of times in observed and recorded mice 20min, compares between result group.Tail vein injection 5mg/ml azovan blue normal saline solution 0.2mL/ is only subsequently, puts to death after 20min, note normal saline 6ml in disposable celiac, gently rubs abdominal part 1min, draws 4-6ml flushing liquor, centrifuging and taking supernatant.With normal saline as blank, survey absorbance in 610nm, record A value, calculate suppression ratio, compare between organizing.
Suppression ratio (%)=(control group A-administration group A)/control group A × 100%
Ovum Gallus domesticus album causes mice foot swelling method: mice 60, male and female half and half, group technology, medication, approach and dosage are ibid.Picric acid labelling mice, before last is administered, drainage measures every left back sufficient sole of the foot normal volume of mice, after last administration 60min, in left back sufficient plantar aponeurosis hemostasis fresh albumen 0.03ml/ only, measure after administration 1,2,3, the swollen volume of the foot of 4h, calculate paw swelling (mL)=medicine metapedes volume-medicine front foot volume, the difference of relatively more each group.
Dimethylbenzene brings out mice ear method: mice 60, male and female half and half, group technology, medication, approach and dosage are ibid.Infiltrating dimethylbenzene 0.02mL in mouse right ear two sides after last administration 1h, left ear is not painted with normal ear.Put to death after 2h, lay the round auricle of left and right ear by diameter 8mm card punch same area, with scales/electronic balance weighing, calculate ear swelling degree: ear swelling degree (mg)=auris dextra weight-left ear weight.
Hot plate method: screening pain threshold is between 5-30s, female mice 60, and group technology, medication, approach and dosage are ibid.Before first time medicine, each group mice picric acid labelling, surveys pain threshold with hot plate method, and after last administration, 15,30,60,90,120min hot plate methods measure pain threshold.
* note: g/kg all means that every kilogram of mice needs medicine
1.2 animal intestines and stomach inflammation models
Rat 50, male and female half and half, it is divided into 5 groups at random, Normal group, wait appearance normal saline, the intestines and stomach health group 10g/kg, maple knotweed fermented product extract group dosage is crude drug 20.0g and 10.0,5g/kg dosage group.It is administered by 2ml/200g body weight, every day 1 time, continuous 4d, fasting 24h before last 1 medicine, normal water, 95% ethanol 1ml is filled after administration 1h, put to death rat, ligation cardia and pylorus after 1h, inject 5ml normal saline, take out and fix 10min in 10% formalin, cut off flushing observed result along greater gastric curvature.Calculate glandular stomach portion the number of ulcer point occurs and measures ulcer length, gastric mucosal damage index is calculated according to GUTH method, (each petechia counts 1 point, rotten to the corn meter 2 points, ulcer length counts 3 points less than 1mm, counting 4 points more than 1mm less than 2mm, length counts 5 points at more than 2mm) score is added the ulcer index being this animal, compare group difference and calculate ulcer inhibition rate.
The impact that PEG2, HA in IL-1 α, IL-1 β in rat blood serum and tissue are oozed out by 1.3 maple knotweed fermented extracted liquid
Rat 48, male and female half and half, divide equally 6 groups at random, medication, approach and dosage ibid, after last 1 administration after 1h, in right metapedes plantar subcutaneous injection fresh albumen 0.1ml, normal water, plucks after 1h after eyeball takes blood 2ml and puts to death, and on ankle joint, 0.5cm place cuts right metapedes.Peel off the normal saline adding 5ml after skin shreds and soak 1h, take out foot pawl, centrifugal blood and soak, take supernatant, survey the OD value of IL-1 α, IL-1 β and transudate PEG2, HA in serum, compare group difference.
2 statistical procedures
Adopting Excel2007, SPSS17.0 software to carry out statistical analysis, result represents with average ± standard deviation (x ± S).
2.1 antiinflammatories and analgesic test result
Acetic acid is utilized to cause the antalgic and inflammation relieving effect of mice Rat paw edema test and writhing method detection maple knotweed fermented extracted liquid respectively in Table 1: aspirin group, the intestines and stomach health group, maple knotweed fermentation 10g/kg and 20g/kg, the maple knotweed 20g/kg group mouse writhing number of times that do not ferment is below normal saline group and difference extremely significantly (p < 0.01)；Aspirin group antiinflammatory suppression ratio is up to 64.13%, and the absorbance of abdominal cavity penetrating fluid is lower than normal saline group, and difference is extremely notable.Other respectively organize the absorbance of abdominal cavity penetrating fluid lower than normal saline group, have certain antiinflammatory action, but difference is not notable；But data from which, the Daphniphyllum calycinum of equivalent, Herba polygoni hydropiperis powder extracting directly liquid (maple knotweed does not ferment) effect not as good as the extracting solution (fermentation of maple knotweed) of maple knotweed powder by fermentation, illustrating that the drug effect of the extracting solution after by fermentation is better, antiinflammatory is with analgesia effect more preferably.
Table 1 maple knotweed fermented extracted liquid acetic acid causes the impact of mice Rat paw edema test and writhing method
Note: compare * P < 0.05, * * P < 0.01 with normal saline group, lower same.
The result of maple knotweed fermented extracted liquid xylol induced mice ear swelling impact is in Table 2: aspirin group, maple knotweed fermentation 20g/kg group mice auricle swelling degree is below normal saline group and difference extremely notable (p < 0.01)；The intestines and stomach health group, maple knotweed fermentation 5g/kg, 10g/kg group mice auricle swelling degree is below normal saline group, but difference is not significantly (p > 0.05).Data from which, the Daphniphyllum calycinum of equivalent, Herba polygoni hydropiperis powder extracting directly liquid (maple knotweed does not ferment) effect not as good as the extracting solution (fermentation of maple knotweed) of maple knotweed powder by fermentation, illustrate that the drug effect of the extracting solution after by fermentation is better, it is suppressed that the curative effect of swelling is more preferably.
Table 2 maple knotweed fermented extracted liquid on the impact of white mice auricle edema (N=10)
The result of maple knotweed fermented extracted liquid xylol induced mice foot swelling impact is in Table 3: 1h, 2h, 3h, 4h upon administration, the left foot of mice all has certain foot swelling, after medicine, paw swelling is when 1h, compares with normal saline, difference all extremely notable (P < 0.01)；When 2h, aspirin group compares with normal saline, significant difference (P < 0.05), and the fermentation of maple knotweed is compared with normal saline with 5g/kg, 10g/kg, 20g/kg group of not fermenting, difference extremely notable (P < 0.01).
Table 3 maple knotweed fermented extracted liquid on the impact of white mice foot swelling (N=10)
Utilize the analgesic effect of hot plate method detection maple knotweed fermented extracted liquid in Table 4: pain threshold significant difference during aspirin group administration 30min.Respectively group difference is not notable for other.
The impact on the woolfe-Macdonald method threshold of pain of the table 4 maple knotweed fermented extracted liquid
Note: with pain threshold contrast * * P < 0.01, * P < 0.05 before self medicine
2.2 maple knotweed fermented extracted liquid to alcohol-induced rat pipe film injury result in Table 5, each process group ulcer index is below normal saline group and difference extremely notable (p < 0.01), data from which, the Daphniphyllum calycinum of equivalent, Herba polygoni hydropiperis powder extracting directly liquid (maple knotweed does not ferment) effect not as good as the extracting solution (fermentation of maple knotweed) of maple knotweed powder by fermentation, illustrate that the drug effect of the extracting solution after by fermentation is better, it is suppressed that the curative effect of ulcer is more preferably.
The impact on alcohol-induced rat gastric ulcer of the table 5 maple knotweed fermented extracted liquid
Note: contrast * P < 0.05, * * P < 0.01 with normal saline
The result of impact that PEG2, HA in IL-1 α, IL-1 β in rat blood serum and tissue are oozed out by 2.3 maple knotweed preparations is in Table 6, and in each process group serum, IL-1 α is below normal saline group and difference extremely notable (p < 0.01)；In the intestines and stomach health group serum, IL-1 β is lower than normal saline group, significant difference (p < 0.05).In aspirin group, maple knotweed fermentation 5g/kg, 10g/kg, 20g/kg group serum, IL-1 β is below normal saline group and difference extremely notable (p < 0.01).In each process group serum, PEG2 is below normal saline group, but difference is not significantly (p > 0.05)；In aspirin group serum, HA is below normal saline group and significant difference (p < 0.05), and in the intestines and stomach health group, maple knotweed fermentation 5g/kg, 10g/kg, 20g/kg group serum, HA is below normal saline group and difference extremely notable (p < 0.01).Data from which, the Daphniphyllum calycinum of equivalent, Herba polygoni hydropiperis powder extracting directly liquid (maple knotweed does not ferment) effect not as good as the extracting solution (fermentation of maple knotweed) of maple knotweed powder by fermentation, illustrate that the drug effect of the extracting solution after by fermentation is better.
The impact that PEG2, HA in IL-1 α, IL-1 β in rat blood serum and tissue are oozed out by table 6 maple knotweed preparation
Note: contrast * P < 0.05, * * P < 0.01 with normal saline
The optimal conditions of fermentation of fermentation of bacillus subtilis maple knotweed coarse powder of the present invention is medium pH in sum is 7.0, and fermentation temperature is with 28 DEG C, and humidity 80% of fermenting, best solid-liquid ratio is maple knotweed: culture medium is 1g:1mL, and the suitableeest bacterium amount is 33 μ l/g, and bacterial concentration is 106～108cfu/mL。
Animal pharmacodynamics experimental result shows: the foot swelling that maple knotweed fermented extracted liquid has the auricle edema that reduces capillary permeability, xylol and cause, Ovum Gallus domesticus album causes has inhibitory action, and can suppress the release of inflammation part inflammatory factor.Dichlorodiphenyl Acetate causes that writhing response has inhibitory action, but the pain that hot plate is caused is without effect.The drug effect of gained maple knotweed fermented extracted liquid of the present invention is significantly better than the extract of unleavened maple knotweed.
The gastric mucosa injury effect that knotweed fermented extracted liquid of the present invention has an anti-inflammatory and analgesic effect and anti-ethanol causes is shown in conjunction with pharmacodynamic experiment result.Pain is mainly impaired by sensory nerve or inflammatory stimulus causes.Writhing response is a kind of inflammatory pain reaction, due to H after acetic acid lumbar injection+Substantial amounts of assemble stimulate teleneuron cause pain, and acetic acid stimulates and causes Abdominal inflammation generation oxygen-derived free radicals induced pain, substantially reducing of mouse writhing number of times is likely to suppress the tissue injury that oxygen-derived free radicals etc. produces relevant with maple knotweed fermented product extract, and maple knotweed fermented product extract can reduce the writhing response number of times caused by acetic acid significantly.Maple knotweed fermented product extract can not reduce the pain threshold of mouse hot-plate reaction substantially, illustrates there being high-order maincenter participation protective to add foot reagentia more weak.In Mice Auricle caused by dimethylbenzene xylene inflammation is tested, maple knotweed fermented product extract can suppress Mice Auricle tissue to ooze out and swelling night, it was shown that maple knotweed fermented product extract is to oozing out and the inflammatory reaction such as tissue edema has inhibitory action.This present invention records maple knotweed fermented extracted liquid has inhibitory action to inflammatory mediator HA, to PEG2 without effect, demonstrate swelling of the feet caused by Ovum Gallus domesticus album and essentially consist in histamine and 5-HT release, antler glue then essentially consists in PG release, scalding the wounded IL-1 relevant to bradykinin is inflammation Acute phase brute force derivant, having multiple hypotype, wherein IL-1 α, IL-1 β are hypotypes closely-related with inflammation, and IL-1 is the main effect played in inflammatory process: (1) pyrogen effect；(2) inflammatory cell is destroyed；(3) connective tissue is destroyed.Maple knotweed fermentation decocting liquid suppresses the generation of IL-1, is to one of mechanism having antiinflammatory action.
Above-described embodiment is the present invention preferably embodiment; but embodiments of the present invention are also not restricted to the described embodiments; the change made under other any spirit without departing from the present invention and principle, modification, replacement, combination, simplification; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (10)
1. the method for a bacillus subtilis bacteria solid fermentation maple knotweed preparation, it is characterised in that: comprise the following steps:
1) Daphniphyllum calycinum, Herba polygoni hydropiperis are pulverized, in mass ratio (1.5 ~ 2.5): 1 mixing, obtain maple knotweed powder；
2) solid fermentation: by maple knotweed powder sterilizing, add nutrient culture medium and Bacillus subtillis, mixing, in 22～30 DEG C, humidity is that 50%～90% condition bottom fermentation is cultivated 1～7 day,.
2. method according to claim 1, it is characterised in that: step 2) in the mass volume ratio of maple knotweed powder and nutrient culture medium be 1g:(1～3) mL.
3. method according to claim 1, it is characterised in that: step 2) in the consumption of bacillus subtilis be that every gram of maple knotweed powder adds 30～35 μ l bacillus subtilis bacterium solution, bacterial concentration is 106～108cfu/mL。
4. method according to claim 1, it is characterised in that: the pH of described nutrient culture medium is 6 ~ 8.
5. method according to claim 1, it is characterised in that: step 2) temperature fermented is 28 DEG C.
6. method according to claim 1, it is characterised in that: step 2) humidity fermented is 80%.
7. method according to claim 1, it is characterised in that: step 2) fermented incubation time is 24h.
8. the extracting method of active substance in the arbitrary described method gained tunning of claim 1～7, it is characterized in that: comprise the following steps: take the arbitrary described method fermentation products therefrom of claim 1～7, dry, grinding to obtain powder, be added to the water by powder, the mass volume ratio of powder and water is 1g:7～9mL, soak 1.8～2.2h, 88～92 DEG C decoct 88～92min, centrifugal, take clear liquid；Precipitation being added in water, the mass volume ratio of precipitation and water is 1g:5～7mL, and 88～92 DEG C decoct 55～65min, centrifugal, take clear liquid, merge the supernatant of twice, and concentrated supernatant, to crude drug concentration 0.8～1.2g/mL, obtains extract.
9. method according to claim 8, it is characterised in that: described drying temperature is 40～60 DEG C.
10. the extract that the arbitrary described method of claim 8～9 obtains is preparing antiinflammatory or/and application in Pain relief agents.
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| CN111117937A (en) * | 2020-02-16 | 2020-05-08 | 广东省资源综合利用研究所 | Novel acid-resistant salt-resistant composite bacterium for degrading kitchen waste and preparation method and application thereof |
| CN116616065A (en) * | 2023-06-20 | 2023-08-22 | 东北农业大学 | Summer cutting method for verbena |
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| CN1843418A (en) * | 2005-04-07 | 2006-10-11 | 海南医学院 | Traditional Chinese medicine extract for treating gastroenteritis and traditional Chinese medicine composition containing same |
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| CN1843418A (en) * | 2005-04-07 | 2006-10-11 | 海南医学院 | Traditional Chinese medicine extract for treating gastroenteritis and traditional Chinese medicine composition containing same |
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| 贾天柱等: "《中药炮制化学》", 31 October 2015, 上海科学技术出版社 * |
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| CN111117937A (en) * | 2020-02-16 | 2020-05-08 | 广东省资源综合利用研究所 | Novel acid-resistant salt-resistant composite bacterium for degrading kitchen waste and preparation method and application thereof |
| CN116616065A (en) * | 2023-06-20 | 2023-08-22 | 东北农业大学 | Summer cutting method for verbena |
| CN116616065B (en) * | 2023-06-20 | 2024-04-26 | 东北农业大学 | Summer cutting method for verbena |
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