CN102776129B - Isaria cretacea and application thereof - Google Patents
Isaria cretacea and application thereof Download PDFInfo
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Abstract
The invention discloses isaria cretacea and application thereof. The isaria cretacea strain is isaria cretacea A-01of CGMCC (China General Microbiological Culture Collection) No. 6204 registered at a general microbiology center of a China microbial culture collection management committee. The isaria cretacea A-01 and a bacterium agent taking the same as an active ingredient can be used for preparing a product with at least one function: 1) a protective effect on animal bone marrow and small intestine which are irradiated by X-ray; 2) a protective effect on animal chromosomal damage; 3) enhancing of killing role of rays on tumors; 4) cell immune regulation effect; 5) humoral immune regulation; 6) treatment of palpitation; 7) treatment of shortness of breath; 8) treatment of lacking in strength; 9) treatment of chest tightness; 10) treatment of anorexia; 11) treatment of constipation; 12) treatment of insomnia; and 13) treatment of dreaminess.
Description
Technical field
The present invention relates to a strain Isaria mould and application.
Background technology
The world performs the operation exactly in effective method aspect treatment tumour at present, also has operation chemotherapy and radiotherapy afterwards, if but only with operative treatment tumour, the tumour of curing like this, tumor recurrence rate is quite high afterwards, must need to carry out dietotherapy assists, and chemotherapy radiotherapy is very high to the side effect of human body, to patient, bring very large misery, so actual some time after chemicotherapy the time of survival of patients longer than the time of surviving before not than chemotherapy radiotherapy, all modern medicines start to carry out comprehensive therapeutic plan, what is that the so-called complex therapy of complex therapy is exactly at perioperatively, when carrying out chemicotherapy operation, take to have and can alleviate the injury to health afterwards of opposing chemicotherapy and the protective foods of pain.So exploitation can be alleviated the injury to health afterwards of opposing chemicotherapy and the protective foods of pain has great importance to the treatment of tumour.
Summary of the invention
An object of the present invention is to provide a strain Isaria mould and application.
Provided by the present invention javanicanicus mould strain is an Isaria mould (Isaria cretacea) A-01, and it is numbered CGMCC No.6204 registering on the books of China Committee for Culture Collection of Microorganisms's common micro-organisms center.This bacterial classification has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (being called for short CGMCC) on 06 07th, 2012.
Isaria mould (Isaria cretacea) A-01 is that (its cultural characteristic and morphological specificity are as follows: in 25 ℃ of cultivations of peptone dextrose culture-medium for the bacterial classification that separates of Hepialus larva (Hepialius armoricanus) that Cai Chun gathered in August, 1987 from Sichuan Province China Aba area, bacterium colony is white in color, fine hair shape, there is shallow wrinkle, cultivate three or four days mycelia and occur pencil, rear woollen yarn knitting is obvious, two to three weeks, white coremium growth scattered and that grow thickly is vigorous, handle is arranged at bottom, upper end is slightly flat, be branched, coremium reaches 2.5-4.5 centimetre, diameter is 0.6-2.0 millimeter.Coremium is comprised of many parallel and staggered mycelium woollen yarn knittings, is Powdered around, and coremium periphery is covered with a large amount of conidiums and scattered mycelia.This bacterium mycelia is transparent, have every, branch, diameter 2.2-2.8 micron, and bottle stalk grows from mycelia or from a short stalk, water white transparency, Dan Sheng, alternate or to life, slightly expand middle and upper part, shrink on top.It is avette or oval that conidium is, and size is 2.5-3.5 × 2.2-2.8 micron, and colourless smooth transparent, consor obstructs top in bottle.
Another object of the present invention is to provide a kind of microbiobacterial agent.
Microbiobacterial agent provided by the present invention, its activeconstituents is an Isaria mould (Isaria cretacea) A-01, specifically can be conidium and/or the mycelium of an Isaria mould (Isaria cretacea) A-01.
This microbial inoculum has following at least one effect:
1) marrow to x-ray bombardment and small intestine have provide protection;
2) animal chromosome damage is had to provide protection;
3) strengthen the effect of ray to tumor-killing;
4) cellular immunization regulating effect;
5) Humoral Immunological Regulation Roles;
6) treatment palpitaition;
7) treatment is breathed hard;
8) treat weak;
9) treat uncomfortable in chest;
10) treatment receive poor;
11) treatment constipation;
12) Cure for insomnia;
13) treatment dreaminess.
This microbial inoculum can be prepared according to the method comprising the following steps: by mould an Isaria (Isaria cretacea) A-01 access PDA substratum, at 16-20 ℃, cultivate 14 day under dark condition 24 hours every days, and all culture drying and crushing in culture vessel are obtained to described microbial inoculum.
Experiment showed, mould (Isaria cretacea) A-01 of an Isaria and the microbial inoculum take it as activeconstituents of embodiment 2-6 can be used for preparing the product with following at least one function:
1) marrow to x-ray bombardment and small intestine have provide protection;
2) animal chromosome damage is had to provide protection;
3) strengthen the effect of ray to tumor-killing;
4) cellular immunization regulating effect;
5) Humoral Immunological Regulation Roles;
6) treatment palpitaition;
7) treatment is breathed hard;
8) treat weak;
9) treat uncomfortable in chest;
10) treatment receive poor;
11) treatment constipation;
12) Cure for insomnia;
13) treatment dreaminess.
Described product specifically can be medicine or healthcare products.
Strain name a: Isaria is mould
Latin name: Isaria cretacea
Strain number: A-01
Preservation mechanism: China Committee for Culture Collection of Microorganisms's common micro-organisms center
Preservation mechanism is called for short: CGMCC
Address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City
Preservation date: on 06 07th, 2012
The preservation center numbering of registering on the books: CGMCC No.6204
Below in conjunction with specific embodiment, describe the present invention in detail, these embodiment are for understanding rather than restriction the present invention.
Accompanying drawing explanation
Fig. 1 is the strain bag culture photo of an Isaria mould (Isaria Cretacea) A-01CGMCC No.6204.
Embodiment
The experimental technique using in following embodiment if no special instructions, is ordinary method.
In following embodiment, material used, reagent etc., if no special instructions, all can obtain from commercial channels.
Substratum in following embodiment:
Every 1L peptone dextrose culture-medium is made by following material: peptone 10g, glucose 10g, agar 20g and water.Wherein, water is settled to 1000ml by substratum.
Every 1L PD substratum is made by following material: 200g potato, 20.0g glucose and water.Wherein, water is settled to 1000ml by substratum.
Every 1L PDA substratum is made by following material: 200g potato, 20.0g glucose, 20g agar and water.Wherein, water is settled to 1000ml by substratum.
The compound method of PD substratum is as follows: the peeled potatoes being cut into small pieces is boiled to half an hour, then use filtered through gauze, in filtrate, add glucose, water is settled to 1000mL, obtain PD nutrient solution, then PD nutrient solution 150ml is packed in 300ml triangular flask, 121 ℃ of moist heat sterilization 30min obtain PD substratum.
The compound method of long-grained nonglutinous rice substratum is as follows: first 150g long-grained nonglutinous rice is loaded in ready strain bag, adds the unpasteurized PD nutrient solution of 75ml, with tampon sealing, then bandage high-temperature sterilization preparation with kraft paper.Wherein high-temperature sterilization condition: 1.1kPa, under 121 ℃ of conditions, moist heat sterilization half hour.
The compound method of PDA substratum is as follows: the peeled potatoes being cut into small pieces is boiled to half an hour, then use filtered through gauze, in filtrate, add glucose and agar, water is settled to 1000mL, then 121 ℃ of moist heat sterilization 30min obtain PDA substratum.
The compound method of peptone dextrose culture-medium is as follows: agar is put into water and boil molten, add again stirring and dissolving after peptone and glucose, adjust pH to 6.5～7, water is settled to 1000mL, then 121 ℃ of moist heat sterilization 30min obtain peptone dextrose culture-medium.
The preparation of embodiment 1, an Isaria mould (Isaria Cretacea) A-01CGMCC No.6204 bacterial classification
Mould an Isaria (Isaria Cretacea) A-01CGMCC No.6204 is inoculated on the inclined-plane of PDA substratum, at 18 ℃, under dark condition, cultivate 14 days, obtain Isaria mould (Isaria Cretacea) A-01CGMCC No.6204 bacterial classification to the end.
The preparation of embodiment 2, an Isaria mould (Isaria Cretacea) A-01CGMCC No.6204 hypha powder
1, the preparation of strain liquid is inoculated in mould an Isaria (Isaria Cretacea) A-01CGMCC No.6204 bacterial classification in PD substratum, and at 22 ℃, rotating speed 150rpm, carries out shaking culture and within 72 hours, obtain strain liquid under dark condition.
2, prepare hypha powder
By long-grained nonglutinous rice substratum good sterilizing, across strain bag, rub brokenly, under aseptic condition, inoculate into strain liquid 15ml for every bag, continue aseptic technique, seal mouth with tampon, after strain bag kneads, put into culturing room, on culturing rack, not contact tampon, as standard, shakeout as far as possible and put cultivation.Under dark condition, in 18 ℃ of environment, cultivate 21 days.After 21 days, by all cultures in strain bag (as Fig. 1, comprise substratum, conidium and mycelia) under 80 ℃ of conditions, dry 72 hours, after crushed after being dried, cross 120 mesh sieves, obtain Isaria mould (Isaria Cretacea) A-01CGMCC No.6204 hypha powder to the end.
Embodiment 3, marrow and the small intestine of an Isaria mould (Isaria Cretacea) A-01CGMCC No.6204 to x-ray bombardment mouse have provide protection
The present embodiment adopts spleen nodiform established law to observe to be subject to after an Isaria mould (Isaria Cretacea) the A-01CGMCC No.6204 hypha powder that BALB/c mouse takes embodiment 2 survival condition of bone marrow stem cell after various dose roentgen radiation x, take simple exposure and normal mouse as contrast; With crypts of small intestine cell regeneration method observation BALB/c and KM mouse, at an Isaria of taking embodiment 2 mould (Isaria Cretacea) A-01CGMCC No.6204 hypha powder different time, compared with simple irradiation group by the crypts of small intestine cell regeneration situation after various dose roentgen radiation x.
Materials and methods:
1, spleen clone (spleen tubercle) forms experiment:
Mouse: donor mice and acceptor mouse are BALB/c mouse inbred lines, female, hero half and half, body weight 18-22 gram.
Illuminate condition: experiment adopts SL75-20 linear accelerator, 8Mev-X line, SSD:80cm, dose rate: 468cGy/min. pre-irradiation carries out Dose Calibration, during irradiation is placed in mouse in synthetic glass box, and performance covers cured of 2cm, does full-body exposure.
Experimental technique:
Donor mouse: 120 of BALB/c mouse inbred liness (female, hero half and half), divide equally two large groups: irradiation group merely (control group) and dosing group.Dosing group is fed containing spice with following: (Isaria Cretacea) A-01CGMCC No.6204 hypha powder is made containing spice in normal diet, to add an Isaria of embodiment 2 mould, should make the dosage of every mouse reach 3g hypha powder/kg body weight containing the content of an Isaria mould (Isaria Cretacea) the A-01CGMCC No.6204 hypha powder in spice.Simple irradiation group is fed with normal diet.Feed after 10 days, each 60 mouse of dosing group and simple irradiation group are divided into 6 groups, point six dose points (0Gy, 1Gy, 2Gy, 3Gy, 4.5Gy and 6Gy) carry out respectively full-body exposure under waking state.Irradiate disconnected neck in latter 2 hours and put to death mouse.Under aseptic condition, peel off femur, be filled in medullary space with 0.9% physiological saline, repeatedly rinse three times, the marrow of same dosage group is mixed and made into single cell suspension.Under microscope, count number of nucleated cells and according to different irradiation doses, cell dilution is arrived to desired concn, cryopreservation is standby.
Acceptor mouse: 120 of BALB/c mouse inbred liness (female, hero half and half), after full-body exposure 8.5Gy, are divided into 12 groups corresponding with donor mouse experiment condition, every group of 10 mouse.After irradiating, 2 hours above-mentioned stand-by donor mice of expert's tail vein injection (cell quantity of injection is determined according to irradiation dose).After irradiation, with normal diet, feed 9 days, then disconnected neck is put to death mouse, gets spleen, and liquid-solid fixed three days of Bouin ' s, carries out spleen clonogenic unit (colonyforming unit of spleen, CFU) counting under anatomical lens.
Result, as shown in table 1-3, shows the mouse of an Isaria mould (Isaria Cretacea) the A-01CGMCC No.6204 hypha powder of taking embodiment 2 at pre-irradiation, and after irradiated by 1-3Gy, bone marrow stem cell survival number is all higher than simple irradiation group.In non-irradiated mouse, an Isaria mould (Isaria Cretacea) the A-01CGMCC No.6204 hypha powder of taking embodiment 2 also can make the activity of bone marrow stem cell improve.As represented the survival condition of bone marrow stem cell with the form of cell survival curve, when visible 1-3Gy irradiates the bone marrow stem cell survival condition of dosing group all more simple irradiation group be height.Take the provide protection (protecting factor) of Dq ratio as 1.677.An Isaria mould (Isaria Cretacea) the A-01CGMCC No.6204 hypha powder of explanation (during radiotherapy within the scope of every gradation irradiation dose) embodiment 2 when compared with low dosage has significant provide protection to bone marrow stem cell.
Table 1. mice spleen clone counting
The mice spleen clone dosage effect of the simple irradiation group of table 2.
Dose effect curve parameter: Do=0.95, N=2.14, Dq=0.31
The mice spleen clone dosage effect of table 3. dosing group
Dose effect curve parameter: Do=1.02, N=3.24, Dq=0.52
Protecting factor=1.677(Dq ratio)
2, crypts of small intestine cell regeneration experiment:
(1) mouse: experiment adopts respectively KM mouse (27-30 gram of body weight, female, male half and half) and BALB/c mouse (22-24 gram of body weight, female, male half and half).
(2) illuminate condition: with spleen colony formation.But make TAR.
(3) administration and illuminating method: comprise three methods.
Pre-irradiation administration (use BALB/c mouse): 80 of BALB/c mouse inbred liness (female, hero half and half), divide equally two large groups: irradiation group merely (control group) and the front dosing group of photograph.Dosing group is fed containing spice with following: (Isaria Cretacea) A-01CGMCC No.6204 hypha powder is made containing spice in normal diet, to add an Isaria of embodiment 2 mould, should make the dosage of every mouse reach 3g hypha powder/kg body weight containing the content of an Isaria mould (Isaria Cretacea) the A-01CGMCC No.6204 hypha powder in spice.Simple irradiation group is fed with normal diet.Feed after 38 days, will be divided into 4 groups according to each 40 mouse of front dosing group and simple irradiation group, point 4 dose points (10Gy, 12Gy, 14Gy and 16Gy) carry out respectively TAR under waking state.After irradiating, all with normal diet, feed for two groups.
Irradiate forward and backward successive administration (using KM mouse): 80 of KM mouse (female, hero half and half), divide equally two large groups: irradiation group merely (control group) and the rear continuous dosing group of the front photograph of photograph.According to the rear continuous dosing group of front photograph, with following, containing spice, feed: (Isaria Cretacea) A-01CGMCCNo.6204 hypha powder is made containing spice in normal diet, to add an Isaria of embodiment 2 mould, should make the dosage of every mouse reach 3g hypha powder/kg body weight containing the content of an Isaria mould (Isaria Cretacea) the A-01CGMCC No.6204 hypha powder in spice.Simple irradiation group is fed with normal diet.Feed after 7 days, will be divided into 4 groups according to each 40 mouse of the rear continuous dosing group of front photograph and simple irradiation group, point 4 dose points (10Gy, 12Gy, 14Gy and 16Gy) carry out respectively TAR under waking state.After irradiation, according to the rear continuous dosing group of front photograph, still with above-mentioned pastille material, feed, simple irradiation group is still fed with above-mentioned normal diet.
Administration (BALB/c mouse) after irradiating: 10 of BALB/c mouse inbred liness (female, hero half and half), as shining rear dosing group.Pre-irradiation, feeds with above-mentioned normal diet according to the mouse of rear dosing group.10 mouse according to rear dosing group are carried out respectively to TAR with the dosage of 14Gy under waking state.After irradiation, according to rear dosing group, with following, containing spice, feed: (Isaria Cretacea) A-01CGMCCNo.6204 hypha powder is made containing spice in normal diet, to add an Isaria of embodiment 2 mould, should make the dosage of every mouse reach 3g hypha powder/kg body weight containing the content of an Isaria mould (Isaria Cretacea) the A-01CGMCC No.6204 hypha powder in spice.
(4) draw materials and data analysis:
All mouse are all put to death at the disconnected neck of latter 3.5 days of irradiation, according to Withers method, carry out that small intestine is drawn materials and film-making, and counting regeneration crypts of small intestine number under the microscope, all experiments were data learn by statistics and process after computer fitting mapping.
Result is as shown in table 4-7, show be no matter simple pre-irradiation take an Isaria mould (Isaria Cretacea) the A-01CGMCC No.6204 hypha powder one month of embodiment 2 or irradiate after to living, kill three and half in take, small intestine stem cell is had to similar remarkable provide protection (protecting factor=1.95); Before exposure, take 7 days and also take after irradiation, showing equally obvious provide protection (protecting factor=1.32).An Isaria mould (Isaria Cretacea) the A-01CGMCC No.6204 hypha powder that embodiment 2 is described has provide protection to irradiated marrow, especially with more remarkable when compared with low dosage.The dosage range (1-3Gy) that its effect is the strongest; radiotherapy fractionated irradiation each dosage range used just, an Isaria mould (Isaria Cretacea) the A-01CGMCCNo.6204 hypha powder of embodiment 2 has the aobvious provide protection of work to accepting the hemopoietic function of radiation therapy patient.An Isaria mould (Isaria Cretacea) the A-01CGMCC No.6204 hypha powder of embodiment 2 also can have significant provide protection to injury of small intestine common in tumour radiotherapy.
The each group of table 4. is according to average every ring crypts of small intestine number (KM mouse) of the rear successive administration various dose point of front photograph
The dosage effect (S.E.) (KM mouse) of the small intestine regenerative crypt of the each group of table 5.
Average every ring crypts of small intestine number (BALB/c mouse) of the each group of table 6. administration various dose point
The dosage effect (S.E.) (BALB/c mouse) of the each group of table 7. small intestine regenerative crypt
Embodiment 4, an Isaria mould (Isaria Cretacea) A-01CGMCC No.6204 have provide protection to chromosome damage
Choose 70 of NIH mouse (body weight 18-22g), male and female half and half, are divided into 7 groups at random, 10 every group.Negative control group (physiological saline), positive controls (endoxan, 40mg/kg), medicine control group, medicine add endoxan 1,2,3 and 4 dosage groups.The administering mode of each group laboratory animal is as follows: the disposable injection 0.5ml of every mouse of negative control group physiological saline; The normal saline solution 0.5ml of the disposable injection endoxan of every mouse of positive controls makes dosage reach 40mg/kg body weight; The disposable per os of every mouse of medicine control group gives an Isaria mould (Isaria Cretacea) the A-01CGMCC No.6204 hypha powder of embodiment 2, and dosage is 5g hypha powder/kg body weight; Medicine adds the disposable per os of endoxan every mouse of 1 dosage group and gives an Isaria mould (Isaria Cretacea) the A-01CGMCC No.6204 hypha powder of embodiment 2, dosage is 5g hypha powder/kg body weight, after half an hour, the normal saline solution 0.5ml of every disposable injection endoxan of mouse makes dosage reach 40mg/kg body weight; Medicine adds the disposable per os of endoxan every mouse of 2 dosage groups and gives an Isaria mould (Isaria Cretacea) the A-01CGMCC No.6204 hypha powder of embodiment 2, dosage is 2.5g hypha powder/kg body weight, after half an hour, the normal saline solution 0.5ml of every disposable injection endoxan of mouse makes dosage reach 40mg/kg body weight; Medicine adds the disposable per os of endoxan every mouse of 3 dosage groups and gives an Isaria mould (Isaria Cretacea) the A-01CGMCC No.6204 hypha powder of embodiment 2, dosage is 1.25g hypha powder/kg body weight, after half an hour, the normal saline solution 0.5ml of every disposable injection endoxan of mouse makes dosage reach 40mg/kg body weight; Medicine adds the disposable per os of endoxan every mouse of 4 dosage groups and gives an Isaria mould (Isaria Cretacea) the A-01CGMCC No.6204 hypha powder of embodiment 2, dosage is 1g hypha powder/kg body weight, after half an hour, the normal saline solution 0.5ml of every disposable injection endoxan of mouse makes dosage reach 40mg/kg body weight.Laboratory animal is put to death for 24 hours after administration, get femur bone marrow sectioning cells, methyl alcohol is fixed, Giemsa dyeing, 10000 polychromatic erythrocytes of every animal counting, and record occurs with permillage, representing Rate of Appearance Micronuclei (micronuclear rates) by micronucleated cell number, adopt sided t to detect and carry out two sample average comparisons, calculate inhibiting rate.Inhibiting rate=［ (positive group Rate of Appearance Micronuclei one dosage group Rate of Appearance Micronuclei)/positive group Rate of Appearance Micronuclei ］ × 100%.
Result is as shown in table 8; an Isaria mould (Isaria Cretacea) the A-01CGMCCNo.6204 hypha powder that shows embodiment 2 has stronger restraining effect to the erythrocytic micronucleus number of mouse bone marrow cells polychromatophilia; statistics shows there is significant difference; under the condition of experiment; in set dosage range, an Isaria of embodiment 2 mould (Isaria Cretacea) A-01CGMCC No.6204 hypha powder has stronger restraining effect also chromosome damage to be had to provide protection to micronucleus.
There is situation in the micronucleus of the each group of table 8.
Note: NS represents that compared with negative control group, without significant difference, * represents that, compared with positive controls, # represents compared with negative control group.
Embodiment 5, mould (Isaria Cretacea) the A-01CGMCC No.6204 of an Isaria and chemotherapy combined radiotherapy are applied the enhancement to tumor-killing
Materials and methods:
Medication: the feed containing (DCXC) for dosing group (converted and be prepared into pastille piece material by administration problem 3g-kg every day by medical courses in general institute animal center) is fed all experiments were process.Simple irradiation group is fed with normal diet.After 15 days, irradiate local tumor.
Illuminate condition: experiment SL75-20 linear accelerator, 8Mev-X line, SSD=100cm, dose rate=3.8G/min.Pre-irradiation carries out Dose Calibration by the court's radiotherapy material physical cell.During irradiation, conscious mouse is placed in special organic next fixer to shielding other position of health, the band knurl hind leg that point six dose points (2,3.5,7.0,10.5,14,17.5Gy) partial irradiation surface coverage 2cm is cured.
Test method: 120 of T-739 mouse (female, hero half and half), divide equally two large groups: irradiation group merely (control group) and administration and chemotherapy combined radiotherapy set of applications (dosing group).By LA-795(mouse lung adenocarcinoma cell) be seeded in every left back leg muscle of mouse, after inoculation, dosing group is fed containing spice with following: (Isaria Cretacea) A-01CGMCC No.6204 hypha powder is made containing spice in normal diet, to add an Isaria of embodiment 2 mould, should make the dosage of every mouse reach 3g Isaria mould (Isaria Cretacea) A-01CGMCC No.6204 hypha powder/kg body weight containing the content of an Isaria mould (Isaria Cretacea) the A-01CGMCC No.6204 hypha powder in spice.Simple irradiation group is fed with normal diet.Feed after 15 days, each 60 mouse of dosing group and simple irradiation group are divided into 6 groups, points of six dose points (0,3.5,7.0,10.5,14.0 and 17.5Gy) irradiate.Illuminating method is as follows: adopt SL75-20 linear accelerator, 8Mev-X line, SSD=100cm, dose rate=3.8Gy/min.Pre-irradiation carries out Dose Calibration.During irradiation, conscious mouse is placed in synthetic glass fixer to shielding other position of health, the band knurl hind leg that partial irradiation surface coverage 2cm is cured.After irradiation, dosing group is still fed with above-mentioned pastille material, and simple irradiation group is still fed with above-mentioned normal diet.Observe change and the difference of two groups of tumor growth rates.The number of days that tumour reaches a certain size as shown in Table 9 and Table 10, the ratio of the dose,equivalent of dosing group and simple irradiation group is 1.24, and A-01CGMCC No.6204 hypha powder can strengthen the lethal effect of ray to tumour to illustrate that an Isaria of embodiment 2 is mould (Isaria Cretacea).
The tumour of the simple irradiation group of table 9. reach a certain size number of days (my god)
The tumour of table 10. administration and chemotherapy combined radiotherapy set of applications reach a certain size number of days (my god)
Embodiment 6, an Isaria mould (Isaria Cretacea) A-01CGMCC No.6204 have immunoregulation effect
One, cellular immunization regulating effect (delayed type hypersensitivity)
Supply reagent thing: an Isaria mould (Isaria Cretacea) the A-01CGMCC No.6204 hypha powder of embodiment 2, with the positive contrast medicine of LEVAMISOLE HCL (guilin pharmacy factory, No. 1982:11033, the accurate word of medicine defended in osmanthus).
Laboratory animal and medication: 50 male ICR mouses, are divided into 5 groups: dosage group, hypha powder low dose group and positive controls in negative control group, hypha powder high dose group, hypha powder.Administering mode is as follows: every mouse of negative control group is filled with and feeds equal-volume physiological saline; Every mouse of hypha powder high dose group is filled with an Isaria mould (Isaria Cretacea) the A-01CGMCC No.6204 hypha powder of feeding embodiment 2, and dosage is 1000mg hypha powder/kg body weight; In hypha powder, every mouse of dosage group is filled with an Isaria mould (Isaria Cretacea) the A-01CGMCC No.6204 hypha powder of feeding embodiment 2, and dosage is 500mg hypha powder/kg body weight; Every mouse of hypha powder low dose group is filled with an Isaria mould (Isaria Cretacea) the A-01CGMCC No.6204 hypha powder of feeding embodiment 2, and dosage is 250mg hypha powder/kg body weight; Every mouse of positive controls is filled with and feeds LEVAMISOLE HCL, and dosage is 25mg/kg body weight (people is with 10 times of dosage).
According to above-mentioned medication continuous irrigation, feed after 10 days, every mouse web portion unhairing, the about 3*3cm of scope, next day is by 1% DNFB(dinitrofluorobenzene) acetone sesame oil liquid 50ul(acetone: sesame oil=1:1) be evenly applied in to lose hair or feathers and sentence sensitization.Second day repeats sensitization once.After 5 days, be evenly applied to auris dextra two sides attack with 1%DNFB10ul, after attacking, 24h puts to death mouse, takes off the auricle that diameter is 8mm with punch tool, weighs, even if claim thymus gland and spleen weight organ index simultaneously.Organ index=organ weights (g)/the weight of animals (g).
Result is as shown in table 11, in hypha powder, dosage group and hypha powder high dose group can be strengthened mice ear degree, hypha powder high dose group can increase mouse thymus organ coefficient, and A-01CGMCC No.6204 hypha powder can strengthen the cellular immune function of mouse to illustrate that an Isaria of embodiment 2 is mould (Isaria Cretacea).
The cellular immunization regulating effect of table 11. hypha powder
Note: data are the mean+SD of 10 mouse.
Two, humoral immunization experiment (serum hemolysin assay method):
Supply reagent thing: an Isaria mould (Isaria Cretacea) the A-01CGMCC No.6204 hypha powder of embodiment 2, with the positive contrast medicine of LEVAMISOLE HCL (guilin pharmacy factory, No. 1982:11033, the accurate word of medicine defended in osmanthus).
Laboratory animal and medication: 50 male ICR mouses, are divided into 5 groups: dosage group, hypha powder low dose group and positive controls in negative control group, hypha powder high dose group, hypha powder.Administering mode is as follows: every mouse of negative control group is filled with and feeds equal-volume physiological saline; Every mouse of hypha powder high dose group is filled with an Isaria mould (Isaria Cretacea) the A-01CGMCC No.6204 hypha powder of feeding embodiment 2, and dosage is 1000mg hypha powder/kg body weight; In hypha powder, every mouse of dosage group is filled with an Isaria mould (Isaria Cretacea) the A-01CGMCC No.6204 hypha powder of feeding embodiment 2, and dosage is 500mg hypha powder/kg body weight; Every mouse of hypha powder low dose group is filled with an Isaria mould (Isaria Cretacea) the A-01CGMCC No.6204 hypha powder of feeding embodiment 2, and dosage is 250mg hypha powder/kg body weight; Every mouse of positive controls is filled with and feeds LEVAMISOLE HCL, and dosage is 25mg/kg body weight (people is with 10 times of dosage).
According to above-mentioned medication continuous irrigation, feed after 10 days, every mouse abdominal injection 3:5(V/V) dilution sheep red blood cell 0.2ml(approximately 500,000,000 red corpuscle) with sensitization, after 4 days, from mouse heart blood sampling 1ml, after separation of serum with physiological saline two-fold dilution, getting different dilution serum 1ml puts and in vitro puts into ice bath, add successively 10% sheep red blood cell (SRBC) suspension, guinea pig serum (in advance with the sheep red blood cell (SRBC) absorption) 1ml of 1:10 dilution, 37 ℃ of insulation 10min, put after ice bath termination reaction with the centrifugal 10min of 2000rpm, getting supernatant liquor 1ml and 3ml Dou Shi reagent mixes and places after 10min in 540nm colorimetric.
Get red cell suspension 0.25ml, be diluted to 4ml with Dou Shi reagent, mix and place after 10min in 540nm colorimetric, absorbance while obtaining red corpuscle HD50, with the half hemolysis value (HC of following formula calculation sample
50):
Sample HC
50absorbance during absorbance/red corpuscle HD50 of=extension rate × sample
Result is as shown in table 12, and in hypha powder, dosage group can improve hemolytic action, illustrates that in hypha powder, dosage group can improve the humoral immune function of animal.
The HC of table 12. sample
50(mean+SD)
Note:
*expression compared with negative control group, P < 0.01
*expression compared with negative control group, P < 0.001
Data are the mean+SD of 10 mouse.
The clinical efficacy of embodiment 7, an Isaria mould (Isaria Cretacea) A-01CGMCC No.6204
Supply reagent thing: an Isaria mould (Isaria Cretacea) the A-01CGMCC No.6204 hypha powder of embodiment 2
Case: be selected from each sick 50 examples of planting in outpatient service and ward, the wherein male sex's 21 examples, women's 29 examples, maximum 78 years old age, minimal ages 21 years old, 55.7 ± 12.4 years old mean age, each case distributes: coronary heart disease and angina pectoris 17 examples, the outmoded heart obstruct 1 example, irregular pulse 5 examples, rheumatic heart disease 1 example, pulmonary heart disease 1 example, myocardosis 1 example, hypertension 7 examples, hyperlipidemia 2 examples, cerebral infarction 2 examples, pharyngitis 1 example, diabetes 1 example, postoperative 1 example of breast cancer, postoperative 1 example of prostate cancer, lymphosarcoma 1 example, lupus erythematosus 1 example, neurasthenia 6 examples.
Case definition:
With reference to < < new Chinese medicine guideline of clinical investigations > > and < < whole nation higher medical institution internal medicine (doctor trained in Western medicine, the traditional Chinese medical science) teaching material > >, diagnose and differential diagnosis in tcm.
Observe case standard:
Allly conform to above traditional Chinese and western medicine Case definition person, select with the common syndrome of deficient card: the diseases such as fatigue and weak, poor appetite, insomnia and dreamful sleep, palpitation and amnesia, sensation of oppression over the chest with shortness of breath are carried out clinical observation.The focal selection obstruction of qi in the chest, deficiency of both qi and yin (coronary heart diseases and angina pectoris), severe palpitation (irregular pulse), heart spleen or asthenia of both the spleen and kidney (neurasthenia), spleen-deficiency, wet turbid resistance network card (hyperlipidaemia), quench one's thirst, the disease kind syndrome such as syndrome of deficiency of both qi and yin (diabetes) qi and blood or syndrome of deficiency of both qi and yin (after cancer, chemicotherapy) carries out clinical observation.
Medication:
An Isaria mould (Isaria Cretacea) the A-01CGMCC No.6204 hypha powder of oral meal embodiment 2, every day 3 times, an Isaria mould (Isaria Cretacea) the A-01CGMCC No.6204 hypha powder of each 0.33g embodiment 24 weeks is a course for the treatment of.After the oral course for the treatment of, carry out efficacy evaluation.
Efficacy assessment standard:
Adopt syndrome point-score, standard rating: 3 points, symptom is obvious, endurable affects life and work.2 points, heavy when light during symptom, can stand.1 point, mild symptoms or once in a while appearance.0 point, asymptomatic.
Other criterion of therapeutical effect as: electrocardiogram(ECG, blood fat, blood sugar, immunoglobulin (Ig), hepatic and renal function etc. are all evaluated with reference to the standard of < < new Chinese medicine guideline of clinical investigations > > defined with corresponding name of disease.Result as shown in Tables 13 and 14.
Table 13. cardinal symptom efficacy result (example (%))
| Symptom | Effective | Effectively | Invalid | Total effective rate |
| Palpitaition | 8（30.7） | 17（65.5） | 1（3.8） | 25（96.2） |
| Breathe hard | 3（11.2） | 23（85.2） | 1（3.6） | 26（96.2） |
| Weak | 17（45.9） | 17（45.9） | 3（8.2） | 34（91.2） |
| Uncomfortable in chest | 7（25.9） | 17（62.9） | 3（11.2） | 24（88.9） |
| It is poor to receive | 11（42.3） | 13（50.0） | 2（7.7） | 24（92.3） |
| Constipation | 1（14.3） | 4（57.1） | 2（28.6） | 5（71.4） |
| Insomnia | 4（13.8） | 21（72.4） | 4（13.8） | 25（86.2） |
| Dreaminess | 2（9.1） | 16（72.7） | 4（18.2） | 22（81.8） |
| Forgetful | 1（4.8） | 8（38.1） | 12（57.1） | 9（42.9） |
Table 14. objective indicator is improved interpretation of result
| Project | Number of cases | Efficient |
| Heart rate fluctuates in normal range | 50/50 | - |
| Blood pressure drops is to normal | 6/7 | 85.7 |
| Oxyphorase ascensionist | 16/37 | 43.0 |
| Oxyphorase rises to normal person | 6/6 | 100.00 |
| Total cholesterol descender | 4/7 | 57.1 |
| Total cholesterol drops to normal person | 2/7 | 28.5 |
| Triglyceride level descender | 7/8 | 87.5 |
| Triglyceride level drops to normal person | 1/8 | 12.5 |
| IgA ascensionist | 11/27 | 40.7 |
| IgA rises to normal person | 5/5 | 100.00 |
| IgG ascensionist | 10/27 | 37.0 |
| IgG rises to normal person | 2/2 | 100.00 |
| IgM rising person | 5/27 | 18.5 |
Note: in number of cases row, molecule is effective number of cases, and denominator is total number of cases.
Security one, the Salmonella typhimurium/microsomal enzyme mutagenic test (Ames Test) of embodiment 8, an Isaria mould (Isaria Cretacea) A-01CGMCC No.6204
Supply reagent thing: an Isaria mould (Isaria Cretacea) the A-01CGMCC No.6204 hypha powder of embodiment 2
Test strain: Salmonella typhimurium TA97a and TA98 are frame shift type mutant strain.Salmonella typhimurium TA100 and TA102 are base substitution type mutant strain.
Before test, the bacterium liquid of getting 20ul stored frozen is inoculated in 5ml nutrient broth medium, and 37 ℃ of 14 hours water-bath shaking culture, obtain 2 × 10
8cFU bacteria suspension.
Using method: give first warm bath method:
Tested concentration: determine tested material maximal dose standard by pharmaceutical control and administration rules, proof load is: 5mg/ ware, 2.5mg/ ware, 1.25mg/ ware, 0.625mg/ ware, 0.313mg/ ware, five tested concentration.
Positive control: 2.7-=AF 10ug/ ware, 2-AF 10ug/ ware, MMS2ul/ ware, VP-16 25ul/ ware, zhengdingmeisu 20ug/ ware, 9-is ketone 0.2ug/ ware not,
Experiment parameter: (1) is via the rat liver homogenate microsomal enzyme system (S-9) of polychlorobiphenyl (Aroclor-1254) induction, the protein content 34mg/ml of measurement
(2) according to Ames in 19883, revise method, S-9 add-on is 20ul/ ware.
(3) data of test-results are twice experimental result mean number.
Experimental result: give in first temperature bath test and selected five test concentrations, when analyte concentration is increased to 5mg/ ware, four bacterial strains do not occur that significantly antibacterial and jumping phenomenon does not all appear in antibacterial and other four tested concentration of jumping phenomenon.
Conclusion: under the condition of this experiment, in selected dosage range, an Isaria of embodiment 2 mould (Isaria Cretacea) A-01CGMCC No.6204 hypha powder detects bacterial strain to four does not have reverse mutation effect.
Two, to dog long term toxicity test
Supply reagent thing: an Isaria mould (Isaria Cretacea) the A-01CGMCC No.6204 hypha powder of embodiment 2
Laboratory animal: domesticated dog 3-5 monthly age male and female dual-purpose
Main agents and instrument: hepatic and renal function is measured test kit: Shanghai Long March medical science company limited product
Hepatic and renal function bioassay standard liquid: Beijing Chemical Plant produces
Physiological saline etc.: BJ Pharmaceutical Co., Ltd. produces
Dimethylbenzene, formaldehyde, ethanol, two ketone etc.: Beijing Chemical Plant produces
Baker Hematocyte Counter 150 types
Encore I type biochemical measurement instrument
Electrocardiograph XDH-3 type
Olympus BH-2 type microscope
Whizzer, analytical balance, slicing machine etc.
1, method
1) experimental dog is bought rear quarantine three weeks, clean body surface therebetween, expulsion Enterozoa, injection rabies vaccine etc.Except raising the dog material of producing with the feed factory of Jiujiang, Beijing mouth, add weekly and feed meat twice, raising and the sanitation and hygiene of animal are responsible for by special messenger.
2) quarantine is after three weeks, before administration, check outward appearance, body weight, blood phase, the liver function of standby dog, comprising glutamate pyruvate transaminase (SGPT), glutamic-oxal(o)acetic transaminase (SGOT), alkaline phosphatase (ALP), albumin (ALB), sphaeroprotein (GLU) and albumins/globulins (A/G), renal function selects urea ammonia (BUN), routine urinalysis and electrocardiogram(ECG, select indices check result and all belong to each 32 of normal male and female dog, be divided into 4 groups, i.e. control group, low dose group, middle dosage group and high dose group, 4 every group, each 2 of male and female dog.
3) dosage:
Every dog of high dose group gives an Isaria mould (Isaria Cretacea) the A-01CGMCC No.6204 hypha powder of embodiment 2, and dosage is 2.0g hypha powder/kg body weight; Every dog of middle dosage group gives an Isaria mould (Isaria Cretacea) the A-01CGMCC No.6204 hypha powder of embodiment 2, and dosage is 1.0g hypha powder/kg body weight; Every dog of low dose group gives an Isaria mould (Isaria Cretacea) the A-01CGMCC No.6204 hypha powder of embodiment 2, dosage is 0.4g hypha powder/kg body weight, oral by way of administration, control group also imposes the processing of corresponding administration process, but not containing medicine.Offer medicine once every day, continuous 180 days.
4) after administration, aforementioned index, after 30 days (decubation), is checked in 90 days, 180 days, drug withdrawal.Drug withdrawal next day, after the inspection that completes aforementioned index, put to death low, middle dosage group all experiments were dog, control group and high dose group are respectively put to death half animal (each 1 of male and female), and another half dog gives over to decubation and observes, and within 30 days after drug withdrawal, all puts to death.
Animal after execution, cuts open inspection immediately, observes general pathology diseaseization.Leave and take all with the histoorgan such as the part heart, liver, spleen, lung, kidney, stomach, small intestine, pancreas, brain, suprarenal gland, testis or ovary, lymphoglandula and make section microscopy.To the heart, liver,spleen,kidney, suprarenal gland, testis, all organs of ovary, after first claiming it heavy, then leave and take the sample of manufacturing section.
2, result
In experiment, the medication dose of administration group dog is respectively 4 times (low dose group) of quantity, 10 times (middle dosage groups) and 20 times (high dose group).Administration time is clinical 4-6 times.Four treated animals have all completed the whole process of experiment.
2.1 general status:
In medication whole process, before the mouth and nose of animal, eyes, hearing etc. and administration, be as good as, but high dose group dog is in the experiment later stage, hair is aobvious disorderly, and glossiness reduces, but depilation phenomenon does not occur.
In administration first month, the drug reaction of middle and high dosage group dog is take Digestive tract as main, and generally performance has loose stool, receives poorly, and high dose group is more showed the even food refusal that has, or vomiting reaction (incidence 50%) after food
After medication one month, dog adapts to gradually to medicine, and the feed of middle dosage group dog is normal, the defecation frequency rule that also becomes, and high dose group dog recovers normal feed substantially, does not vomit again.
2.2 body weight change:
The experiment initial stage, though middle high dose group dog is more obvious to the digestive tract reaction of medicine, but adapt to very soon, feed takes a turn for the better, so the each treated animal body weight of experimental session has growth in various degree, even the increment of low dose group dog is more than control group, and high dose group dog body weight also has to be increased but too late first three groups.Administration within 180 days, respectively organize afterwards dog than administration before average increasing amount be followed successively by 3.50kg, 5.25kg, 3.75kg and 2.30kg.Drug withdrawal after 30 days control group than administration before the average 4.37kg that increases, high dose group increases 2.75kg.
2.3 blood phases:
2.3.1 leukocyte counts: experiment whole process is made count check four times, before administration, 30 days each detections once after administration 90 days, 180 days and drug withdrawal.In four times no matter check results relatively or are not organized between group with the same period same period, self compare, all no significant differences, all count values are all in normal range.
2.3.2 peripheral blood leucocyte differential count: every animal makes one of blood smear, four countings of every counting 100 cells (neutrophilia, acidophilia, basophilic stippling cell, lymphocyte and monocyte) first three groups animal are made relevant more equal no difference of science of statistics.In high dose group administration, after 180 days, have a blood test, neutrophilic granulocyte counting is than obviously reducing (70.25 ± 0.83 → 64.00 ± 3.56 before administration, P < 0.02), and lymphocyte count compared with administration before raise (22.50 ± 1.66 → 28.00 ± 3.74, P < 0.05).But the neutrophil of dog counting range of normal value is 51-72%, and lymphocyte is 22-49%, and this species diversity is only the fluctuation in range of normal value.
2.3.3 hemochrome content: take a blood sample with leukocyte counts simultaneously, measure.Check for four times hemochrome content without between group or in group, the same period or the difference of the comparison same period not, all in range of normal value.
2.3.4 peripheral blood platelet count: take a blood sample with leukocyte counts, whole process checks for four times totally simultaneously, and result shows each group of dog platelet count, all in range of normal value, and without the significant difference of various comparisons.
2.4 liver function
2.4.1SGPT: omnidistance check for four times except high dose group decubation two dogs than the slight rising of the administration observed value of 180 days (P > 0.05), all the other are all normal.High dose group decubation, the SGPT value of two dogs all raise (P < 0.01) than concurrent control group dog measured value, and A-01CGMCCNo.6204 hypha powder may have minor injury to liver in high dosage medication after 180 days to illustrate that an Isaria of embodiment 2 is mould (Isaria Cretacea).As everyone knows, SGTP is very sensitive liver function indexes, this experiment is determined as 71.00 ± 1.41iu/L for 30 days after drug withdrawal, in illustrating that this dog may the recovery process after high assay value, or the slight rising that causes of an Isaria of embodiment 2 mould (Isaria Cretacea) A-01CGMCC No.6204 hypha powder effect.
2.4.2SGOT: omnidistance four inspections of four treated animals, measured value is all in normal range.
2.4.3ALP: except low dose group administration half way value has rising, all the other are all normal.This time measured value is 143.25 ± 57.68, and higher than normal value, but administration finishes rear mensuration for 180 days, has reduced to normal value (42.50 ± 32.76).It is because two sample hemolysis cause that this measured value is grown tall, irrelevant with an Isaria of embodiment 2 mould (Isaria Cretacea) A-01CGMCC No.6204 hypha powder.
2.4.4 blood plasma total protein: measurement result is all in range of normal value.
2.4.5ALB, GLU and A/G: as shown in table 8, ALB or GLU be all in normal value level, but be greater than 1 except the A/G value of administration after 180 days, and it is all less than or equal to 1 complete three times, and control group also shows equifinality.
2.5 renal function
Each group is omnidistance checks that BUN value is 6.18 ± 1.22-14.50 ± 2.67, all normal values.
2.6 routine urinalysis
Each group is omnidistance to be checked, white corpuscle, red corpuscle, cast epithelial and four observation index of albumen are all without abnormal.
The comparison of 2.7 some organ weight and body weight
The ratio four treated animal result indifferences of the weight of the heart, liver, spleen, lung, kidney and body weight, be normal value, and the ratio of suprarenal gland, testis and body weight, high dose group result is all lower than other three groups (P < 0.05), and ovary result is not obvious.
2.8 electrocardiogram(ECG
Each treated animal electrocardiogram(ECG before, during and after administration and decubation detect all normally, the rhythm of the heart is neat, the each wave band figure of electrocardiogram(ECG is without extremely, do not seen premature beat or the chamber fibre image such as quiver.
2.9 pathological examination
Substantially visual: the animal of twice execution except the testis of high dose group and suprarenal gland less than another three treated animals, abnormal without other.
Microscopic observation: high dose group dog suprarenal gland section, there is no change or other pathological changes of weave construction, cortex, medullary substance are normal, and the ball of cortex, bundle, reticular zone are arranged normal.The pathological change of testis tissue is more obvious, in the dog that administration is put to death for 180 days afterwards, can see Qu Jingguan epithelium arrangement disorder, pyknosis, and in chamber, without mature sperm, performance has serious dyszoospermia.After drug withdrawal, through 30 days, recover, the normal configuration post-equalization of high dose group testis, has spermatogenesis in Qu Jingguan, and spermatogenesis recovers substantially.Ovary tissue, without obvious pathological change, can be seen folliculus growths at different levels and lean type and generate.Organ weight's cardinal principle is basically identical with the observations under mirror.Other histoorgans after one's own heart, liver, spleen, lung, kidney, stomach, pancreas, small intestine, brain, lymphoglandula etc., substantially, be showed no pathology under mirror.
3, sum up
Low dosage is 4 times of quantity, does not occur any toxic side effects, and drug reaction has appearred in middle and high dosage.High dose group is significantly better than middle dosage group, between three dosage, shows clear and definite amount-effect relationship.Toxicity target organ is testis, adrenal tissue and Digestive tract.
The experimental dog initial stage of taking medicine has just rare, and it is poor to receive, even food refusal, especially with high dose group for very.But the highly sensitive bromine of food refusal and dog feels there is certain relation.Until its be hard set and eat and gradually adapt to after body weight have growth in various degree.Even if illustrate under high dosage, for a long time with also adapting to.Four groups of laboratory animal all complete experiment, none death.
High dose group dog decubation SGPI slightly raises, but do not see hepatic tissue structure and cell in pathological section, has any extremely, does not see the changes such as hepatic necrosis, sex change, illustrate that the slight rising of SGPT does not cause tissue morphology variation.
42.20 ± 2.90%, three kinds of sphaeroprotein content that the albumin normal value content of dog accounts for blood plasma total protein account for 57.80 ± 1.53%, so the A/G value of dog is in most cases less than 1, are normal.
Under cardinal principle and mirror, pathological change is the most obvious with testis.Middle and high dosage group testicular spermatogenic function is suppressed, and especially with the latter, attaches most importance to.The spermatogenesis of testis is subject to drug influence most, but because it flushes, so substantially all can recover normal in drug withdrawal in 30 days.
An Isaria mould (Isaria Cretacea) the A-01CGMCC No.6204 hypha powder of embodiment 2 is with 0.4g/kg, and 1.0g/kg and 2.0g/kg take 180 days continuously to dog, and medicine presents dose-effect relationship to experimental dog.Experimental result shows, 2.0g/kg is the tissues such as the testis of reproductive system, stomach, intestines, liver and the suprarenal gland of Digestive tract to the toxicity target organ of dog.0.4g/kg does not produce toxic reaction to dog substantially.Functional or the organic lesion that an Isaria mould (Isaria Cretacea) the A-01CGMCC No.6204 hypha powder of embodiment 2 causes histoorgan is all reversible, after drug withdrawal, in 30 days, substantially can recover.
Three, to rat long term toxicity test
Supply reagent thing: an Isaria mould (Isaria Cretacea) the A-01CGMCC No.6204 hypha powder of embodiment 2
Laboratory animal: Wistar rat is purchased from Chinese Academy of Medical Sciences's Experimental Animal Center, conformity certification number: 5707R08
Main agents and instrument: hepatic and renal function is measured test kit: Shanghai Long March medical science company limited product
Hepatic and renal function bioassay standard liquid: Beijing Chemical Plant produces
Physiological saline etc.: BJ Pharmaceutical Co., Ltd. produces
Dimethylbenzene formaldehyde ethanol two ketone Giemsa ' s staining agents etc.: Beijing Chemical Plant produces
Baker Hematocyte Counter 150 types
Encore I type biochemical measurement instrument
Olympus BM-2 type microscope etc.
1. method
160 of Wistar rats, are divided into 4 groups at random, and 40 every group, male and female half and half, body weight 60-80g, raises the egg milk material of producing with Jiujiang, Chaoyang District, Beijing City mouth feed factory, arbitrarily drinking public water supply.
Control group, basic, normal, high dosage group are established in experiment.Every rat of high dose group gives an Isaria mould (Isaria Cretacea) the A-01CGMCC No.6204 hypha powder of embodiment 2, and dosage is 10.0g hypha powder/kg body weight; Every rat of middle dosage group gives an Isaria mould (Isaria Cretacea) the A-01CGMCCNo.6204 hypha powder of embodiment 2, and dosage is 5.0g hypha powder/kg body weight; Every rat of low dose group gives an Isaria mould (Isaria Cretacea) the A-01CGMCC No.6204 hypha powder of embodiment 2, dosage is 1.0g hypha powder/kg body weight, oral by way of administration, mould an Isaria of embodiment 2 (Isaria Cretacea) A-01CGMCCNo.6204 hypha powder is made to suspension with 20ml/kg body weight oral administration with tap water, once a day, be used in conjunction 180 days; Control group gavages tap water 20ml/kg body weight the every day same period.
Before administration and after administration 90 days, 180 days, after drug withdrawal, 30 days (decubation) checked peripheral blood leucocyte sum, Arneth's count, hemochrome and routine urinalysis.Within after administration 180 days and drug withdrawal 30 days, check liver function (glutamate pyruvate transaminase, alkaline phosphatase, i.e. SGPT and ALP), renal function (blood urea nitrogen is BUN).Whole process is weighed 8 times.After drug withdrawal, put to death the whole rats of low, middle dosage group and control group, high dose group half animal (female male rat each 1/2), another half animal gives over to decubation observation, within 30 days after drug withdrawal, puts to death.The rat of putting to death is except dissecting and carry out gross examination of skeletal muscle immediately, and every mouse is left and taken the heart, liver, spleen, lung, kidney, stomach, small intestine, pancreas, suprarenal gland, lymphoglandula, testis or ovary and makes section microscopy.
2. result
Omnidistance administration 180 days, therebetween, the low dose group rat total amount of taking medicine is 180g/kg, middle dosage group 900g/kg, high dose group 1800g/kg.Each group dosage is equivalent to 100 times of quantity, 50 times and 10 times.Administration time is 4-6 times of the clinical conventional course for the treatment of.Test omnidistance control group and each administration animal without death.
2.1 generalized case
In initial administration one month, animal defecation is half congealed rare, and high dose group is the most obvious, in, low dose group takes second place, and control group is still normally discharged ellipse moulding just, show that loose stool system takes medicine due to.After administration one month, adapt to gradually, low, middle dosage group defecation is gradually normal, though and high dose group is just no longer half congealed, but still not moulding.
In experimentation, the behavior of animal, movable without abnormal, eyes, mouth and nose are without secretory product.High dose group Hair of Rats glossiness is slightly poorer than other three groups in the experiment later stage, slightly dislikes disorderly, but without depilation phenomenon.
Before administration, the average food-intake of each treated animal is close, first 90 days of administration, middle and high dosage group rats eating amount is slightly lower than another two groups (P > 0.5), to administration 180 days, middle dosage group rat appetite increases and control group is as good as, and high dose group is still slightly lower than the appetite (P > 0.7) of another three treated animals, drug withdrawal is after 30 days, high dose group rat appetite and other three treated animals indifference.Although it is slightly poor that middle and high dosage group rat shows appetite at different time, with control rats food-intake not statistically significant.
2.2 body weight change
The front four treated animal mean body weights of administration differ and are less than 1.0 grams.During administration half way, the mean body weight of control group and basic, normal, high three administration groups is respectively 330.74 ± 69.27,299.58 ± 59.63,307.53 ± 55.53 and 291.11 ± 48.43g.No significant difference between low, middle dosage group and control group (P > 0.05), and significantly (P < 0.01) of high dose group and control group comparing difference.Body weight during administration 180 days is followed successively by 353.66 ± 60.49, and 317.50 ± 45.83,346.69 ± 72.58 and 330.87 ± 46.72g, can find out and between group, not have notable difference.After drug withdrawal, 30 days weighing results are 323.68 ± 34.88 and 333.29 ± 71.39g, and after decubation, high dose group body weight and control group are basic identical.
2.3 blood phases
1. leukocyte counts: before administration, have a blood test for 30 days four times after administration 90 days, 180 days and drug withdrawal, compare between group the same period, or compare the interior front and back of the group same period, equal no significant difference, the lifting of numerical value all belongs to the fluctuation in range of normal value, without Special Significance.
2. peripheral blood leucocyte differential count: 100 cells of every blood smear counting, observe neutrophilia, acidophilia, basophilic stippling white corpuscle, the variation of lymphocyte and monocyte percentage.Result shows, in no matter organizing, between group, each phase comparison, does not all have significant difference.
3. hemochrome content: between group, in the same period or group, do not compare the same period self, omnidistance 4 measurement results show, an Isaria mould (Isaria Cretacea) the A-01CGMCC No.6204 hypha powder of embodiment 2 has no significant effect hemochrome content, and its content is all in range of normal value.
2.4 liver function
Owing to only having the eyeball of excision to obtain, measure hepatic and renal function required blood volume, so the rat that the mensuration of hepatic and renal function is only limited to before being about to put to death carries out, extract after eyeball, get blood and do between (after administration 180 days, drug withdrawal 30 days) the group same period relatively; Control group and high dose group are done the decubation of drug withdrawal after 30 days because of need and are observed, thus can with the heaven-made not same period of the comparison of administration 180.
Experimental result shows, SGPT and the ALP value after administration 180 days and drug withdrawal, within 30 days, measured, and each automatic self synchronizing is no significant difference relatively.The SGPT average value ranges of four groups, at 33.58 ± 0.84-52.08 ± 6.25iu/L, all belongs to range of normal value (30-52iu/L).Not also indifference of the measurement result same period.Four groups of the ALP administration measured values of 180 days are close, average value ranges at 221.63 ± 30.58-254.84 ± 27.12iu/L without group difference.Drug withdrawal is two groups of male mouses and control group Female Rats ALP value and 180 days measured results close (P > 0.2) after 30 days, and the Female Rats ALP value of high dose group and the administration corresponding measured value comparison of 180 days drops to 136.30 ± 33.10(P < 0.05 by 235.70 ± 112.15).
2.5 renal functions: based on the cause same with liver functional testing, BUN also can only do twice detection.Result shows, no matter is administration 180 days, or the measured value of 30 days after drug withdrawal, no difference of science of statistics more all in the group same period relatively or not between its mean value group.The mean value that four groups of administrations record for 180 days is followed successively by 18.74 ± 0.06, and 18.81 ± 0.06,18.02 ± 0.01 and 17.98 ± 0.01mg%, the mean value of drug withdrawal after 30 days is 19.89 ± 3.73 and 21.90 ± 7.16mg%, entirely belongs to range of normal value (6-22mg%).
The Female Rats BUN value of 30 days latter two groups of drug withdrawal raises than the administration measured value of 180 days, and P < 0.01 and 0.001(explain according to biochemical chamber mensuration personnel, are because serum amount is few, when machine automatic sampling, have sucked due to a small amount of underlying liquid).The female mouse value of control group and high dose group all has rising, and therefore not an Isaria mould (Isaria Cretacea) the A-01CGMCC No.6204 hypha powder of embodiment 2 causes.
2.6 routine urinalysis: before administration and after administration 90 days, 180 days, within after drug withdrawal 30 days, make altogether four times routine urianlysis, the four indices (red, white corpuscle, cast epithelial, albumen) of observation is all without extremely.
2.7 pathological examinations:
After dissecting, do visual inspection substantially, see control group (2), low dose group (4), there is slight inflammatory lesion, the pathological change that other histoorgans can be seen without naked eyes in middle dosage group (5) and high dose group (5) rat lung.
Under mirror, check and have no the heart, liver,spleen,kidney, suprarenal gland, stomach, small intestine, pancreas, ovary and lymphoglandula generation pathological change.Above-mentioned visual being seen lung changes, and Microscopic observation lesion nature is bronchitis and focal pneumonia, belongs to slight pathological change, consistent with cardinal principle finding.
What administration group pathology incidence was the highest is the testis tissue of reproductive system.Control group and low dose group are without pathological change.In 20 male mouse of middle dosage group, 3 there is slight dyszoospermia; In 10 male mouse that high dose group administration is put to death for 180 days, 3 of slight pathologies, 5 of moderate pathologies, 2 of severe pathologies, i.e. high dose group administration is entirely organized afterwards male mouse testicular spermatogenic function for 180 days and is all subject to restraining effect in various degree.Show as the regression of Qu Jingguan epithelium, cause dyszoospermia.
In another 10 the male mouse of high dose group that 30 days (decubation) puts to death after drug withdrawal; pathology is or start to recover; 6/10 rat testicle spermatogenesis are quite active; 4/10 have started to recover spermatogenesis; though in can seeing or a small amount of sperm be present in Qu Jingguan and pay in testis pipe; do not reach yet the level of aforementioned 6, not yet quite restored be described.
3 sum up
3.1 press 1.0g/kg to rat, the dosage of 5.0g/kg and 10.0g/kg, an Isaria mould (Isaria Cretacea) the A-01CGMCC No.6204 hypha powder of oral embodiment 2 is after 180 days, its toxicity target organ is clear and definite, is mainly the testis tissue of reproductive system and the gi tract of Digestive tract, shows as spermatogenesis obstacle and includes minimizing in; be just rare; body weight gain is slow etc., and toxic side effect increases and increases the weight of with dosage, demonstrates dose-effect relationship.
The testis tissue of 3.2 rats is the primary toxicity target organ of an Isaria of embodiment 2 mould (Isaria Cretacea) A-01CGMCCNo.6204 hypha powder.Low dose group rat is uninfluenced.Middle and high dosage all can cause the obvious regression of rat Qu Jingguan epithelium and dyszoospermia in various degree.Middle dosage treated animal is impaired lighter, only has 3/20 animals to occur that slight production of sperm suppresses when administration finishes, and the high dose group rat situation of getting involved is just more serious.But the damage that this toxic action causes can recover, after drug withdrawal, the animal of 6/10 of 30 days high dose group has recovered the spermatogenesis enlivening very much, and 4/10 have started the spermatogenesis of middle minuent.
The above results shows that damage is reversible, substantially in drug withdrawal 30 days recovers normal.
Though 3.3 Digestive tract have receiving of long period poor high dose group is aobvious; rare just with body weight gain lower than the performances such as other three groups; the digestion organs such as stomach, intestines, liver, pancreas also find no obvious pathology; illustrate, under dosage used, Digestive tract is only caused to some symptoms, do not cause the organic change that can see.
3.4 4 groups of rats have Some Animals to occur the pathologies such as pneumonia, and this is relevant on the occasion of severe winter with the experiment later stage, and not drug toxicity causes.
3.5 Isaria at embodiment 2 mould (Isaria Cretacea) A-01CGMCC No.6204 hypha powder dosage is higher than 100 times of quantities, through long-term prescription, all animals has all completed drug dosage schedule smoothly, and A-01CGMCC No.6204 hypha powder is a medicine that toxicity is very low to illustrate that an Isaria of embodiment 2 is mould (Isaria Cretacea).
An Isaria mould (Isaria Cretacea) the A-01CGMCC No.6204 hypha powder toxicity test result of continuous 180 days of Oral Administration in Rats embodiment 2 shows, 1.0g/kg does not produce poison to animal and pays effect, it is safe dose, 5.0g/kg can cause that minority Spermatogenesis in Rats is slightly suppressed, 10.0g/kg can cause full treated animal testis tissue dyszoospermia and body weight gain Speed Reduction, but these poison effects of paying are reversible, after drug withdrawal, in 30 days, can recover.The above results shows, an Isaria mould (Isaria Cretacea) the A-01CGMCC No.6204 hypha powder of embodiment 2 be a toxicity very generation low medicine.
Result according to an Isaria of embodiment 2 mould (Isaria Cretacea) A-01CGMCC No.6204 hypha powder to rat and the observation of dog long term toxicity test, from toxicology angle, weigh, mould an Isaria of embodiment 2 (Isaria Cretacea) A-01CGMCC No.6204 hypha powder is offered to clinic trial, should be feasible.
Claims (3)
- An Isaria mould ( isaria cretacea), it is characterized in that: described Isaria mould ( isaria cretacea) bacterial strain number be A-01, it is numbered CGMCC No.6204 registering on the books of China Committee for Culture Collection of Microorganisms's common micro-organisms center.
- 2. microbiobacterial agent, its activeconstituents be claimed in claim 1 Isaria mould ( isaria cretacea).
- 3. microbial inoculum according to claim 2, is characterized in that: described Isaria mould ( isaria cretacea) be conidium and/or mycelium.
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