CN104274528B - A kind of cassia bark polyphenol extract with immunosuppressive activity and its preparation method and application - Google Patents
A kind of cassia bark polyphenol extract with immunosuppressive activity and its preparation method and application Download PDFInfo
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Abstract
The invention discloses a kind of cassia bark polyphenol extract with immunosuppressive activity and its preparation method and application.The extract is derived from the extract of at least one of cinnamomum japonicum, bavin osmanthus, oily osmanthus bark, and the Determination of Polyphenols in the extract reaches more than 50wt%.The half cell-lethal concentration of cassia bark polyphenol extract of the present invention>50 μ g/mL, and propagation to the lymphocytes of mitogen ConA inductions and participate in the secretion of important cytokine IL 6, IFN γ of immunological regulation and inflammatory reaction and be respectively provided with the inhibitory action of concentration dependent, it is expected to be used to prepare the pharmaceutical preparation for treating immunity disease as one of active ingredient or unique active ingredient, especially it is expected to be used to prepare the pharmaceutical preparation for treating rheumatoid arthritis as one of active ingredient or unique active ingredient, there is medical value.
Description
Technical field
The present invention relates to a kind of cassia bark polyphenol extract and its preparation method and application, is to be related to one kind to have specifically
Cassia bark polyphenol extract of immunosuppressive activity and its preparation method and application, belongs to technical field of traditional Chinese medicines.
Background technology
Rheumatoid arthritis (RA) is currently to endanger one of most important diseases of human health, is referred to as " not dead cancer
Disease ".RA be it is a kind of be involved with multi-joint, synovial membrane inflammation, bone and cartilage destruction etc. itself are exempted from for the chronic generalized of main feature
Epidemic disease inflammatory disease, its pathogenesis not yet illustrate completely so far, generally believe disorderly with heredity, infection and the factor such as immunological regulation
It is closely related.The illness rate of China RA about 0.3%~0.4%, there are about 4,000,000 patients, and course of disease 5-10 disability rates are 60%, late period
One of the main reason for 90% patient forfeiture social labor power and self care ability are adult's disabilities, therefore cause
Many family problems and huge burden on society.The method of Current treatments rheumatoid arthritis mainly has operative treatment, changes
Learn the treatment of drug therapy and cytokine class biological agent.It is generally acknowledged that surgical operation is only applicable to late deformity case, and
Somewhat expensive, it is larger to body wound；Chemicals such as non-steroidal anti-inflammatory drugs, antirheumatic drug and glucocorticoid etc. for a long time should
With the serious adverse reaction such as gastrointestinal discomfort, bone marrow suppression, hepatic injury, hypertension can be caused；Biological agent is intervening lesion
While cell, certain effect can be similarly produced to normal cell, the inherent immunity and adaptive immunity of interference body, from
And produce larger toxicity and destroy the risk of body defenses effect.Therefore, the medicine for seeking the prevention RA of high-efficiency low-toxicity is still
The challenge and problem that world medical circle faces.
The common name of the barks such as cassia bark is Lauraceae cinnamon Chinese cassia tree, bavin osmanthus, cinnamomum japonicum, Ceylon osmanthus, oily osmanthus, in common
Medicine, and be food flavor or condiment for cooking.The former plant of commodity cassia bark is more complicated, there are about more than ten and plants.Research shows（It is upper marine
Medical magazine, the 5th phase page 82~86 of volume 45 in 2011）：The chemical composition of variety classes cassia bark there are certain otherness,
Such as：Determination of Polyphenols in Chinese cassia tree is 1.31%, and the Determination of Polyphenols in bavin osmanthus is 1.92%.Although Li Yaohua etc. is reported
（Sichuan Journal of Traditional Chinese Medicine, 1989,2：28）：40 cases of atrophic arthritis is treated in right amount with Chinese cassia tree 25g+ mung bean 200g+ slaked limes,
Effective 34, effective 6；But it is also indefinite to its mechanism of action, it plays the material base of drug effect and also not yet understands fully.
The content of the invention
In view of the above-mentioned problems existing in the prior art, the object of the present invention is to provide a kind of osmanthus with immunosuppressive activity
Skin polyphenol extract and its preparation method and application.
Cassia bark polyphenol extract of the present invention with immunosuppressive activity, is derived from cinnamomum japonicum, bavin osmanthus, oily osmanthus
At least one of bark extract, the Determination of Polyphenols in the extract reaches more than 50wt%.
Furtherly, the Determination of Polyphenols in the extract reaches 70wt%~90wt%.
The preparation method of extract of the present invention, includes the following steps：
A) at least one of cinnamomum japonicum, bavin osmanthus, oily osmanthus bark medicinal material are carried out with ethanol water or aqueous acetone solution
Extraction；
B) vacuum distillation removes the ethanol or acetone in extracting solution, and then plus suitable quantity of water dilutes concentrate, obtains crude extract；
C) crude extract is extracted with ethyl acetate 2~6 times, merges the ethyl acetate layer being obtained by extraction, vacuum distillation removes it
In ethyl acetate, obtain ethyl acetate extract；
D) it is diluted with water after ethyl acetate extract is dissolved with a small amount of methanol or ethanol and is splined on DM130 macroreticular resins
Column, successively with 3~5 cylinder ponding, 3~5 column volume 10vol% ethanol waters, 3~5 column volume 35vol% ethanol waters and
3~5 column volume 70vol% ethanol waters elute, and collect the ethanol flow point of 35vol%, are concentrated under reduced pressure, dry, up to described
Extract.
Extraction described in step a) can be cold soaking extraction, temperature extraction takes or refluxing extraction.
Extraction described in step a) can be 1~3 time；Every time during extraction, 1 gram of bark medicinal material uses 5~50 milliliters
Ethanol water or aqueous acetone solution.
Above-mentioned ethanol water and the concentration of volume percent of aqueous acetone solution are both preferably 20%~70%.
Extraction in step c), the volume for the ethyl acetate for being used to extract every time are recommended as the crude extract volume being extracted
0.5~1.5 times.
Drying described in step d) is preferably to be dried in vacuo or be freeze-dried.
Research shows：The half cell-lethal concentration of cassia bark polyphenol extract of the present invention（CC50）>50 μ g/mL,
And propagation to the lymphocytes of mitogen ConA inductions and participate in immunological regulation and inflammatory reaction important cytokine IL-6,
The secretion of IFN-γ is respectively provided with the inhibitory action of concentration dependent, it is expected to is used as one of active ingredient or unique active ingredient
In the pharmaceutical preparation for preparing treatment immunity disease, especially it is expected to be used to make as one of active ingredient or unique active ingredient
The pharmaceutical preparation of standby treatment rheumatoid arthritis.
Heretofore described pharmaceutical dosage form are any pharmaceutically useful peroral dosage forms or injection, including：Tablet, sugar
Garment piece agent, film coated tablet, enteric coated tablet, capsule, hard capsule, soft capsule, oral liquid, mouth containing agent, granule, punching
Agent, pill, powder, parenteral solution, powder-injection etc..
Embodiment
The present invention is made with reference to embodiment further in detail, intactly to illustrate, but is not intended to limit the present invention；This
Some nonessential improvement or replacement that the technical staff in field is made according to the description below belong to protection scope of the present invention.
Cinnamomum japonicum bark medicinal material used originates from Jiangxi Shangrao in embodiment, bavin osmanthus used and oily cassia tree skin in embodiment
Medicinal material originates from Yunnan.Measure in embodiment on the Determination of Polyphenols in extract is that " Shanghai Chinese is miscellaneous for reference literature
Method described in will, the 5th phase page 83 of volume 45 in 2011 ".
Embodiment 1
A) temperature extraction is carried out to cinnamomum japonicum bark medicinal material with the aqueous acetone solution that volumetric concentration is 50% at 40 DEG C to take 1 time, its
In：1 gram of cinnamomum japonicum bark medicinal material, 50 milliliters of aqueous acetone solutions；
B) vacuum distillation removes the acetone in extracting solution, and then plus suitable quantity of water dilutes concentrate, obtains crude extract；
C) crude extract is extracted with ethyl acetate, the volume of extraction ethyl acetate used is the crude extract body being extracted every time
Long-pending 1 times, coextraction 3 times；Merge the ethyl acetate layer being obtained by extraction, vacuum distillation removes ethyl acetate therein, obtains second
Acetoacetic ester position；
D) it is diluted with water after obtained ethyl acetate extract is dissolved with a small amount of ethanol and is splined on DM130 macroreticular resins
Column, successively with 4 cylinder ponding, 4 column volume 10vol% ethanol waters, 4 column volume 35vol% ethanol waters and 4 column volumes
70vol% ethanol waters elute, and collect the ethanol flow point of 35vol%, are concentrated under reduced pressure, and are freeze-dried, up to of the present invention
Extract.
Analyze after measured, in extract made from the present embodiment, the content of total polyphenols is 76wt%.
Embodiment 2
A) at room temperature, extraction 2 is stirred to cinnamomum japonicum bark medicinal material with the aqueous acetone solution that volumetric concentration is 50%
It is secondary, wherein：1 gram of cinnamomum japonicum bark medicinal material, 25 milliliters of aqueous acetone solutions；
B) vacuum distillation removes the acetone in extracting solution, and then plus suitable quantity of water dilutes concentrate, obtains crude extract；
C) crude extract is extracted with ethyl acetate, the volume of extraction ethyl acetate used is the crude extract body being extracted every time
Long-pending 1 times, coextraction 5 times；Merge the ethyl acetate layer being obtained by extraction, vacuum distillation removes ethyl acetate therein, obtains second
Acetoacetic ester position；
D) it is diluted with water after obtained ethyl acetate extract is dissolved with a small amount of ethanol and is splined on DM130 macroreticular resins
Column, successively with 4 cylinder ponding, 4 column volume 10vol% ethanol waters, 4 column volume 35vol% ethanol waters and 4 column volumes
70vol% ethanol waters elute, and collect the ethanol flow point of 35vol%, are concentrated under reduced pressure, and are freeze-dried, up to of the present invention
Extract.
Analyze after measured, in extract made from the present embodiment, the content of total polyphenols is 72wt%.
Embodiment 3
A) temperature extraction is carried out to bavin osmanthus bark medicinal material with the aqueous acetone solution that volumetric concentration is 20% at 40 DEG C to take 2 times, its
In：1 gram of bavin osmanthus bark medicinal material, 25 milliliters of aqueous acetone solutions；
B) vacuum distillation removes the acetone in extracting solution, and then plus suitable quantity of water dilutes concentrate, obtains crude extract；
C) crude extract is extracted with ethyl acetate, the volume of extraction ethyl acetate used is the crude extract body being extracted every time
Long-pending 1.5 times, coextraction 2 times；Merge the ethyl acetate layer being obtained by extraction, vacuum distillation removes ethyl acetate therein, obtains
Ethyl acetate extract；
D) it is diluted with water after obtained ethyl acetate extract is dissolved with a small amount of ethanol and is splined on DM130 macroreticular resins
Column, successively with 4 cylinder ponding, 4 column volume 10vol% ethanol waters, 4 column volume 35vol% ethanol waters and 4 column volumes
70vol% ethanol waters elute, and collect the ethanol flow point of 35vol%, are concentrated under reduced pressure, and are freeze-dried, up to of the present invention
Extract.
Analyze after measured, in extract made from the present embodiment, the content of total polyphenols is 70wt%.
Embodiment 4
A) refluxing extraction is carried out 1 time to bavin osmanthus bark medicinal material with the aqueous acetone solution that volumetric concentration is 20%, wherein：1 gram of bavin
50 milliliters of aqueous acetone solutions of cassia tree skin medicinal material；
B) vacuum distillation removes the acetone in extracting solution, and then plus suitable quantity of water dilutes concentrate, obtains crude extract；
C) crude extract is extracted with ethyl acetate, the volume of extraction ethyl acetate used is the crude extract body being extracted every time
Long-pending 1 times, coextraction 3 times；Merge the ethyl acetate layer being obtained by extraction, vacuum distillation removes ethyl acetate therein, obtains second
Acetoacetic ester position；
D) it is diluted with water after obtained ethyl acetate extract is dissolved with a small amount of ethanol and is splined on DM130 macroreticular resins
Column, successively with 5 cylinder ponding, 5 column volume 10vol% ethanol waters, 5 column volume 35vol% ethanol waters and 5 column volumes
70vol% ethanol waters elute, and collect the ethanol flow point of 35vol%, are concentrated under reduced pressure, and are freeze-dried, up to of the present invention
Extract.
Analyze after measured, in extract made from the present embodiment, the content of total polyphenols is 70wt%.
Embodiment 5
A) at room temperature, extraction 1 time is stirred to bavin osmanthus bark medicinal material with the ethanol water that volumetric concentration is 70%,
Wherein：1 gram of bavin osmanthus bark medicinal material, 50 milliliters of ethanol waters；
B) vacuum distillation removes the ethanol in extracting solution, and then plus suitable quantity of water dilutes concentrate, obtains crude extract；
C) crude extract is extracted with ethyl acetate, the volume of extraction ethyl acetate used is the crude extract body being extracted every time
Long-pending 1 times, coextraction 3 times；Merge the ethyl acetate layer being obtained by extraction, vacuum distillation removes ethyl acetate therein, obtains second
Acetoacetic ester position；
D) it is diluted with water after obtained ethyl acetate extract is dissolved with a small amount of ethanol and is splined on DM130 macroreticular resins
Column, successively with 5 cylinder ponding, 5 column volume 10vol% ethanol waters, 5 column volume 35vol% ethanol waters and 5 column volumes
70vol% ethanol waters elute, and collect the ethanol flow point of 35vol%, are concentrated under reduced pressure, and are freeze-dried, up to of the present invention
Extract.
Analyze after measured, in extract made from the present embodiment, the content of total polyphenols is 78wt%.
Embodiment 6
A) at room temperature, extraction 2 times is stirred to oily cassia tree skin medicinal material with the ethanol water that volumetric concentration is 50%,
Wherein：1 gram of oily cassia tree skin medicinal material, 25 milliliters of ethanol waters；
B) vacuum distillation removes the ethanol in extracting solution, and then plus suitable quantity of water dilutes concentrate, obtains crude extract；
C) crude extract is extracted with ethyl acetate, the volume of extraction ethyl acetate used is the crude extract body being extracted every time
Long-pending 1 times, coextraction 5 times；Merge the ethyl acetate layer being obtained by extraction, vacuum distillation removes ethyl acetate therein, obtains second
Acetoacetic ester position；
D) it is diluted with water after obtained ethyl acetate extract is dissolved with a small amount of ethanol and is splined on DM130 macroreticular resins
Column, successively with 3 cylinder ponding, 3 column volume 10vol% ethanol waters, 3 column volume 35vol% ethanol waters and 3 column volumes
70vol% ethanol waters elute, and collect the ethanol flow point of 35vol%, are concentrated under reduced pressure, and are freeze-dried, up to of the present invention
Extract.
Analyze after measured, in extract made from the present embodiment, the content of total polyphenols is 81wt%.
Embodiment 7
A) refluxing extraction is carried out 3 times to oily cassia tree skin medicinal material with the ethanol water that volumetric concentration is 20%, wherein：1 gram of oil
10 milliliters of ethanol waters of cassia tree skin medicinal material；
B) vacuum distillation removes the ethanol in extracting solution, and then plus suitable quantity of water dilutes concentrate, obtains crude extract；
C) crude extract is extracted with ethyl acetate, the volume of extraction ethyl acetate used is the crude extract body being extracted every time
Long-pending 0.5 times, coextraction 6 times；Merge the ethyl acetate layer being obtained by extraction, vacuum distillation removes ethyl acetate therein, obtains
Ethyl acetate extract；
D) it is diluted with water after obtained ethyl acetate extract is dissolved with a small amount of ethanol and is splined on DM130 macroreticular resins
Column, successively with 3 cylinder ponding, 3 column volume 10vol% ethanol waters, 3 column volume 35vol% ethanol waters and 3 column volumes
70vol% ethanol waters elute, and collect the ethanol flow point of 35vol%, are concentrated under reduced pressure, and are freeze-dried, up to of the present invention
Extract.
Analyze after measured, in extract made from the present embodiment, the content of total polyphenols is 71wt%.
Embodiment 8
A) at 40 DEG C, temperature extraction is carried out to oily cassia tree skin medicinal material with the ethanol water that volumetric concentration is 70% and is taken 2 times,
Wherein：1 gram of oily cassia tree skin medicinal material, 25 milliliters of ethanol waters；
B) vacuum distillation removes the ethanol in extracting solution, and then plus suitable quantity of water dilutes concentrate, obtains crude extract；
C) crude extract is extracted with ethyl acetate, the volume of extraction ethyl acetate used is the crude extract body being extracted every time
Long-pending 1 times, coextraction 4 times；Merge the ethyl acetate layer being obtained by extraction, vacuum distillation removes ethyl acetate therein, obtains second
Acetoacetic ester position；
D) it is diluted with water after obtained ethyl acetate extract is dissolved with a small amount of ethanol and is splined on DM130 macroreticular resins
Column, successively with 3 cylinder ponding, 3 column volume 10vol% ethanol waters, 3 column volume 35vol% ethanol waters and 3 column volumes
70vol% ethanol waters elute, and collect the ethanol flow point of 35vol%, are concentrated under reduced pressure, and are freeze-dried, up to of the present invention
Extract.
Analyze after measured, in extract made from the present embodiment, the content of total polyphenols is 76wt%.
Embodiment 9
A) refluxing extraction is carried out 1 time to oily cassia tree skin medicinal material with the ethanol water that volumetric concentration is 70%, wherein：1 gram of oil
50 milliliters of ethanol waters of cassia tree skin medicinal material；
B) vacuum distillation removes the ethanol in extracting solution, and then plus suitable quantity of water dilutes concentrate, obtains crude extract；
C) crude extract is extracted with ethyl acetate, the volume of extraction ethyl acetate used is the crude extract body being extracted every time
Long-pending 1 times, coextraction 6 times；Merge the ethyl acetate layer being obtained by extraction, vacuum distillation removes ethyl acetate therein, obtains second
Acetoacetic ester position；
D) it is diluted with water after obtained ethyl acetate extract is dissolved with a small amount of ethanol and is splined on DM130 macroreticular resins
Column, successively with 3 cylinder ponding, 3 column volume 10vol% ethanol waters, 4 column volume 35vol% ethanol waters and 4 column volumes
70vol% ethanol waters elute, and collect the ethanol flow point of 35vol%, are concentrated under reduced pressure, and are freeze-dried, up to of the present invention
Extract.
Analyze after measured, in extract made from the present embodiment, the content of total polyphenols is 78wt%.
Embodiment 10
A) at 40 DEG C, temperature extraction is carried out to oily cassia tree skin medicinal material with the aqueous acetone solution that volumetric concentration is 50% and is taken 2 times,
Wherein：1 gram of oily cassia tree skin medicinal material, 30 milliliters of aqueous acetone solutions；
B) vacuum distillation removes the acetone in extracting solution, and then plus suitable quantity of water dilutes concentrate, obtains crude extract；
C) crude extract is extracted with ethyl acetate, the volume of extraction ethyl acetate used is the crude extract body being extracted every time
Long-pending 1 times, coextraction 4 times；Merge the ethyl acetate layer being obtained by extraction, vacuum distillation removes ethyl acetate therein, obtains second
Acetoacetic ester position；
D) it is diluted with water after obtained ethyl acetate extract is dissolved with a small amount of ethanol and is splined on DM130 macroreticular resins
Column, successively with 3 cylinder ponding, 3 column volume 10vol% ethanol waters, 3 column volume 35vol% ethanol waters and 3 column volumes
70vol% ethanol waters elute, and collect the ethanol flow point of 35vol%, are concentrated under reduced pressure, and are freeze-dried, up to of the present invention
Extract.
Analyze after measured, in extract made from the present embodiment, the content of total polyphenols is 73wt%.
Embodiment 11
The cytotoxicity of cassia bark polyphenol extract of the present invention is detected with mtt assay：
Sample sets：Concentration is configured to respectively to the extract prepared by embodiment 2,5,6,10 with RPMI-1640 nutrient solutions
For the solution of 12.5 μ g/mL, 25 μ g/mL and 50 μ g/mL；
Control group：RPMI-1640 nutrient solutions；
By BALB/c mouse splenic lymphocytes（4×105A/hole）48h is co-cultured respectively with each experimental group sample, is being cultivated
4h adds MTT solution before end（5mg/mL）.After culture, 150 μ l of culture supernatant are sucked, then add 150 μ l DMSO dissolving first
Za particle, low speed shaking table 10min, OD values are read with microplate reader at 570nm.
Detect the cytoactive value of each experimental group（OD values）, detailed testing result is shown in Table 1.
The cell OD values that 1 each experimental group of table is surveyed after 48h is acted on
| Experimental group | n | OD values |
| Control group | 5 | 0.161±0.028 |
| 2 extract group of embodiment（12.5μg/mL） | 5 | 0.234±0.023 |
| 2 extract group of embodiment（25μg/mL） | 5 | 0.218±0.022 |
| 2 extract group of embodiment（50μg/mL） | 5 | 0.224±0.027 |
| 5 extract group of embodiment（12.5μg/mL） | 5 | 0.229±0.029 |
| 5 extract group of embodiment（25μg/mL） | 5 | 0.225±0.027 |
| 5 extract group of embodiment（50μg/mL） | 5 | 0.206±0.012 |
| 6 extract group of embodiment（12.5μg/mL） | 5 | 0.251±0.012 |
| 6 extract group of embodiment（25μg/mL） | 5 | 0.231±0.021 |
| 6 extract group of embodiment（50μg/mL） | 5 | 0.210±0.023 |
| 10 extract group of embodiment（12.5μg/mL） | 5 | 0.241±0.026 |
| 10 extract group of embodiment（25μg/mL） | 5 | 0.232±0.023 |
| 10 extract group of embodiment（50μg/mL） | 5 | 0.211±0.027 |
From 1 result of table：Extract of the present invention equal nothing in 12.5 μ g/mL, 25 μ g/mL and 50 μ g/mL concentration
Cytotoxicity.
Embodiment 12
With3It is thin that H-TdR incorporation methods detect the lymph that cassia bark polyphenol extract of the present invention induces mitogen ConA
The proliferation inhibition rate of born of the same parents：
Sample sets：Being configured to concentration respectively to the extract prepared by embodiment 2,5,6 with RPMI-1640 nutrient solutions is
3.125 μ g/mL, 6.25 μ g/mL, 12.5 μ g/mL, the solution of 25 μ g/mL and 50 μ g/mL；
Control group：RPMI-1640 nutrient solutions；
By BALB/c mouse splenic lymphocytes（4×105A/hole）With each experimental group sample, mitogen ConA (2 μ g/mL)
48h is co-cultured, 8h mixes 0.25 μ Ci before culture terminates3H-thymidine.At the end of culture, culture plate is frozen in -20
DEG C refrigerator.The cell of freeze thawing is collected to glass fibre with cell collector during measure, scintillation solution is added and is counted after Beta
Instrument reads incorporation cell DNA3H-thymidine, cell proliferative conditions are represented with cpm values.
The cpm values of each experimental group are detected, detailed testing result is shown in Table 2.
The cpm values of 2 each experimental group of table
| Experimental group | n | Cpm values |
| Blank group | 5 | 497±53 |
| Control group | 5 | 81161±6878 |
| 2 extract group of embodiment（3.125μg/mL） | 5 | 81200±5214 |
| 2 extract group of embodiment（6.25μg/mL） | 5 | 65521±6210 |
| 2 extract group of embodiment（12.5μg/mL） | 5 | 35235±2523 |
| 2 extract group of embodiment（25μg/mL） | 5 | 21135±2258 |
| 2 extract group of embodiment（50μg/mL） | 5 | 10204±956 |
| 5 extract group of embodiment（3.125μg/mL） | 5 | 77290±961 |
| 5 extract group of embodiment（6.25μg/mL） | 5 | 19077±5558 |
| 5 extract group of embodiment（12.5μg/mL） | 5 | 6504±1042 |
| 5 extract group of embodiment（25μg/mL） | 5 | 2159±371 |
| 5 extract group of embodiment（50μg/mL） | 5 | 530±174 |
| 6 extract group of embodiment（3.125μg/mL） | 5 | 80231±6235 |
| 6 extract group of embodiment（6.25μg/mL） | 5 | 68521±6123 |
| 6 extract group of embodiment（12.5μg/mL） | 5 | 32145±2145 |
| 6 extract group of embodiment（25μg/mL） | 5 | 12003±987 |
| 6 extract group of embodiment（50μg/mL） | 5 | 9987±861 |
From 2 result of table：Extract of the present invention can effectively suppress the T cell breeder reaction of ConA inductions, and have
There is concentration dependent.
Embodiment 13
Cell with ELISA method detection cassia bark polyphenol extract of the present invention to participation immunological regulation and inflammatory reaction
The secretion inhibiting rate of factor IL-6 and IFN-γ：
Sample sets：It is 2 μ to be configured to concentration respectively to the extract prepared by embodiment 2,5,6 with RPMI-1640 nutrient solutions
The solution of g/mL, 10 μ g/mL and 50 μ g/mL；
Control group：RPMI-1640 nutrient solutions；
By BALB/c mouse splenic lymphocytes（4×105A/hole）With each experimental group sample, mitogen ConA (2 μ g/mL)
Co-culture 48h.Supernatant is collected by centrifugation（5000rpm, 4 DEG C, 5min）, freeze in -20 DEG C.Use ELISA method（According to phase in kit
Speak on somebody's behalf bright）Detect the content of cell factor (IL-6 and IFN-γ) in supernatant.
Detailed testing result is shown in Table 3.
The cytokine content of 3 each experimental group of table
From 3 result of table：Extract of the present invention can effectively suppress the cell factor IL-6 and IFN- of ConA inductions
The generation of γ, and there is concentration dependent.
Embodiment 14
Select rheumatoid arthritis animal model：Collagen-Induced Arthritis model（collagen-induced
Arthritis, CIA）Examine or check therapeutic effect of the cassia bark polyphenol extract of the present invention to rheumatoid arthritis：
II Collagen Type VI of ox adds 0.1M acetic acid, and it is 10mg/mL solution to be made into concentration, and 4 DEG C of refrigerator overnights dissolve.Experimental day
CFA and the fully emulsified mixing of collagen with Mycobacterium tuberculosis strain H37Rv.With 250g emulsifying agents
Sensitization is carried out in DBA/1 mouse tails base portion, is attacked after 3 weeks with same dose of emulsifying agent in afterbody.After attack 5 days really
Recognize model mice morbidity, proceed by the oral treatment of continuous 2 weeks.It is administered within 5 days after attack, visually observes mouse four limbs
Swelling degree carries out clinical score.Standards of grading are：0 point, normally；1 point, mild redness or toes redness and swelling of joints；2 points, moderate
Redness simultaneously extends to whole foot；3 points, heavier redness simultaneously extends to ankle-joint；4 points, toe, foot, ankle are serious red and swollen, after regression
Arthrocleisis.Experimental result is shown in Table 4.
The clinical score of 4 each experimental group of table
| Experimental group | n | Clinical score |
| Control group | 5 | 7.9 |
| 2 extract group of embodiment（11.1μg/mL） | 5 | 6.9 |
| 2 extract group of embodiment（33.3μg/mL） | 5 | 5.3 |
| 2 extract group of embodiment（100μg/mL） | 5 | 3.4 |
| 5 extract group of embodiment（11.1μg/mL） | 5 | 6.1 |
| 5 extract group of embodiment（33.3μg/mL） | 5 | 5.1 |
| 5 extract group of embodiment（100μg/mL） | 5 | 3.3 |
| 6 extract group of embodiment（11.1μg/mL） | 5 | 7.2 |
| 6 extract group of embodiment（33.3μg/mL） | 5 | 5.8 |
| 6 extract group of embodiment（100μg/mL） | 5 | 3.6 |
From 4 result of table：Extract of the present invention can effectively suppress the mouse arthritis morbidity of II Collagen Type VIs induction,
And there is concentration dependent.
It is visible in summary：The half cell-lethal concentration of cassia bark polyphenol extract of the present invention（CC50）>50μg/
ML, and the propagation and the important cytokine of participation immunological regulation and inflammatory reaction of the lymphocyte to mitogen ConA inductions
The secretion of IL-6, IFN-γ are respectively provided with the inhibitory action of concentration dependent, can effectively suppress the mouse joint of II Collagen Type VIs induction
Inflammation morbidity, it is expected to the pharmaceutical preparation for the treatment of immunity disease is used to prepare as active ingredient, is especially expected as active ingredient
One of or unique active ingredient be used to prepare the pharmaceutical preparation for the treatment of rheumatoid arthritis, there is medical value.
Claims (9)
- A kind of 1. cassia bark polyphenol extract with immunosuppressive activity, it is characterised in that：It is derived from cinnamomum japonicum or bavin osmanthus tree The extract of skin, the Determination of Polyphenols in the extract reach more than 50wt%；And the preparation of the extract includes following step Suddenly：A) cinnamomum japonicum or bavin osmanthus bark medicinal material are extracted with ethanol water or aqueous acetone solution；B) vacuum distillation removes the ethanol or acetone in extracting solution, and then plus suitable quantity of water dilutes concentrate, obtains crude extract；C) crude extract is extracted with ethyl acetate 2~6 times, merges the ethyl acetate layer being obtained by extraction, vacuum distillation removes therein Ethyl acetate, obtains ethyl acetate extract；D) it is diluted with water after ethyl acetate extract is dissolved with a small amount of methanol or ethanol and is splined on DM130 macroporous resin columns, Successively with 3~5 cylinder ponding, 3~5 column volume 10vol% ethanol waters, 3~5 column volume 35vol% ethanol waters and 3 ~5 column volume 70vol% ethanol waters elute, and collect the ethanol flow point of 35vol%, are concentrated under reduced pressure, dry, up to described Extract.
- 2. cassia bark polyphenol extract as claimed in claim 1, it is characterised in that：Determination of Polyphenols in the extract reaches 70wt%~90wt%.
- 3. preparation method as claimed in claim 1, it is characterised in that：Cold soaking extraction, temperature leaching are extracted as described in step a) Extraction or refluxing extraction.
- 4. preparation method as claimed in claim 1, it is characterised in that：It is extracted as 1~3 time described in step a)；Carry every time When taking, 1 gram of bark medicinal material uses 5~50 milliliters of ethanol water or aqueous acetone solution.
- 5. the preparation method as described in claim 1 or 4, it is characterised in that：The ethanol water and aqueous acetone solution Concentration of volume percent is 20%~70%.
- 6. preparation method as claimed in claim 1, it is characterised in that：Extraction in step c), is used for the acetic acid extracted every time The volume of ethyl ester is 0.5~1.5 times of the crude extract volume being extracted.
- 7. preparation method as claimed in claim 1, it is characterised in that：Drying described in step d) is vacuum drying or freezing It is dry.
- A kind of 8. application of the cassia bark polyphenol extract described in claim 1 or 2, it is characterised in that：Using the extract as One of active ingredient or unique active ingredient are used to prepare the pharmaceutical preparation for the treatment of immunity disease.
- 9. application as claimed in claim 8, it is characterised in that：One of active ingredient or unique living is used as using the extract Property component be used to prepare treatment rheumatoid arthritis pharmaceutical preparation.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100998650A (en) * | 2006-12-05 | 2007-07-18 | 高秀丽 | Use of cinnamonum cassia for treating diabetes, its products and preparing method |
| CN102872195A (en) * | 2012-11-05 | 2013-01-16 | 天津市聚星康华医药科技有限公司 | Preparation method of cinnamon total polyphenol extract and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100998650A (en) * | 2006-12-05 | 2007-07-18 | 高秀丽 | Use of cinnamonum cassia for treating diabetes, its products and preparing method |
| CN102872195A (en) * | 2012-11-05 | 2013-01-16 | 天津市聚星康华医药科技有限公司 | Preparation method of cinnamon total polyphenol extract and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| Ameliorative Effects of a Polyphenolic Fraction of Cinnamomum zeylanicum L. Bark in Animal Models of Inflammation and Arthritis;Rathi B,et al.;《SciPharm》;20130225;第81卷(第2期);第573页倒数第2段，第572页表3,第574页第2段，第572页表4,第578页，第3段 * |
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