CN101491573B - Plant extract for treating rheumatoid arthritis - Google Patents
Plant extract for treating rheumatoid arthritis Download PDFInfo
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- CN101491573B CN101491573B CN2008100010915A CN200810001091A CN101491573B CN 101491573 B CN101491573 B CN 101491573B CN 2008100010915 A CN2008100010915 A CN 2008100010915A CN 200810001091 A CN200810001091 A CN 200810001091A CN 101491573 B CN101491573 B CN 101491573B
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Abstract
The invention provides a medical composition containing a Plectranthus Amboinicus (Plectranthus Amboinicus(Lour.)Spreng; PA) coarse extract or extract, which is used for treating rheumatoid arthritis. The invention also provides application of the drug using the Plectranthus Amboinicus coarse extract or extract to treat rheumatoid arthritis.
Description
Technical field
The present invention relates to the Chinese herbal medicine extract.Specifically, the present invention relates to utilize Plectlanthus amboinicus (Lour) Spreng (PlectranthusAmboinicus (Lour.) Spreng; PA) slightly come together thing or extract with treatment rheumatoid arthritis (Rheumatoidarthritis; RA) purposes.
Background technology
Because the existing in recent years significant progress of the use of various alleviation property medicines and immunosuppressant, the treatment of rheumatoid arthritis.Most research is presented at the rheumatoid arthritis their early stage and promptly brings into use the property alleviated medicine or immunosuppressant, can slow down the speed that skeleton is damaged effectively.The healing potion of rheumatoid arthritis can be divided into three major types at present:
1. on-steroidal anti-inflammatory medicine (NSAIDs), for example Aspirin, indomethacin (indomethacin) or naproxen (naproxen), it is inflammation-inhibiting effectively, the pain of releiving effect.
2. antirheumatic (anti-rheumatic drugs; ARDs) or claim to change the moist medicine of wind resistance (the diseasemodifying anti-rheumatic drugs of the state of an illness; DMARDs), for example: golden preparation, hydroxychloroquine peaceful (hydroxychloroquine), methotrexate (methotrexate; MTX) or penicillamine (penicillamine), it can suppress disease and improve dysimmunity.
3. steroid dose (steroid) or title adrenal cortex hormone agent (corticosteroid), it has anti-inflammatory effect, can be used as immunosuppressant.
In addition, alleviation property medicine commonly used clinically comprises that still salazosulfamide (sulfasalazine) and immunosuppressant comprise ciclosporin (cyclosporine), azathioprine (azathioprine) or cyclophosphamide (cyclophosphamide).In addition, because some antibiotic such as minocycline (minocycline) have the effect that suppresses ferment, the generation that suppresses skeleton absorption and inflammation material, also be used to treat rheumatoid arthritis.About medicine use and treatment then has multiple treatment pattern to be suggested opportunity, like zigzag therapy, downstairs therapy, classification therapy and targeted therapy.The spirit of these treatment patterns is used multiple alleviation property medicine or immunosuppressant nothing more than separately early stage or merging.
Yet the shortcoming of these medicines is that effective medicine also brings the medicine of very big side effect, particularly steroid simultaneously.Symptoms such as the medicine of anti-inflammatory also often causes the undesired of intestinal aspect, and is for example hemorrhage.
Also make the pharmaceutical grade protein antagonist clinically, to slow down disease fast, still need treat comparatively inconvenience with the invasive mode in the use like TNF-a.In addition, because the Chinese herbal medicine Radix Tripterygii Wilfordii has effects such as antiinflammatory, antibacterial and town's pyrolysis pain, the present known rheumatoid arthritis that is used for treating, but because it has toxic side effects, therefore the consideration of safety is arranged.
Through statistics, the whole world has 1% people to suffer from rheumatoid arthritis approximately.So exploitation is convenient, safe and effective medicine is crucial with the treatment rheumatoid arthritis.
Plectlanthus amboinicus (Lour) Spreng be a kind of originate in Malaysia and India be general family often cultivate view and admire medicinal herbs.General alleged medical material Plectlanthus amboinicus (Lour) Spreng, the ground that is meant the Plectlanthus amboinicus (Lour) Spreng plant partly, its another name is for Lysimachia capillipes Hemsl, foreign Herba Menthae, Radix agastaches, perfume (or spice) in one's hands, yerba buena or receive and distribute perfume (or spice).People from East India used this as cloth aromatic, and the Englishman found the tempting fragrance of Plectlanthus amboinicus (Lour) Spreng when India introduces the tippet cloth in 1820.If the Plectlanthus amboinicus (Lour) Spreng blade is directly placed medicated clothing, not only have the effect that makes that cloth is fragrant, can also prevent medicated clothing to be damaged by worms.The antibacterial of Plectlanthus amboinicus (Lour) Spreng tool, excitement or anthelmintic function are looked in one band area, Southeast Asia.In addition, Plectlanthus amboinicus (Lour) Spreng also can treat poisonous snake or mosquito is bitten, and tool is alleviated headache, flatulence, vomiting, diarrhoea and fever symptom.Plectlanthus amboinicus (Lour) Spreng essential oils area, Asia are especially made the favorite of spice, and in the incense therapy, Plectlanthus amboinicus (Lour) Spreng is used as the usefulness that promotes that epithelial cell regeneration, acne treatment, eczema, tinea pedis and chapped skin are releived.And Plectlanthus amboinicus (Lour) Spreng also is a kind of good tranquilizer and urges excessive dose that tool relieves the effect of anxiety and hypersexuality.
In the present invention, find slightly the come together effect of thing or extract tool treatment rheumatoid arthritis of Plectlanthus amboinicus (Lour) Spreng surprisingly.
Summary of the invention
One of the object of the invention provides a kind of medical composition that is used to treat rheumatoid arthritis, and it comprises the Plectlanthus amboinicus (Lour) Spreng of treating effective dose slightly come together thing or extract.
Another object of the present invention relates to Plectlanthus amboinicus (Lour) Spreng and slightly comes together thing or extract in the purposes of the medicine of preparation treatment rheumatoid arthritis.
Another purpose of the present invention provides the method for preparing fragrant thick collection thing in a left side or extract.
Detailed description of the present invention is set forth in the description.In explanation of the present invention and claims with obvious further feature of the present invention, purpose and advantage.
Description of drawings
Fig. 1 is slightly the come together HPLC collection of illustrative plates of thing of the Plectlanthus amboinicus (Lour) Spreng of embodiment 1.
The animal experiment flow chart of Fig. 2 illustrative embodiment 2.
Fig. 3 explains the feeding PA influence of thing to the CIA rat body weight of slightly coming together.
Fig. 4 explains that feeding PA slightly comes together thing to the exponential influence of CIA rat arthritis.
Fig. 5 explains the feeding PA influence of thing to CIA rat arthritis swelling degree of slightly coming together.
Fig. 6 explains the feeding PA influence of thing to CIA rat blood serum RF of slightly coming together.
Fig. 7 explains the feeding PA influence of thing to CIA rat blood serum CRP of slightly coming together
Fig. 8 explains that the feeding PA thing that slightly comes together is the influence of CIA rat abdominal cavity cytohormone TNF-α and IL-1 β.
Fig. 9 explains the feeding PA influence of thing to CIA rat abdominal cavity cytohormone IL-6 of slightly coming together.
Figure 10 is the HPLC collection of illustrative plates of the Plectlanthus amboinicus (Lour) Spreng extract PA-F2 of embodiment 3.
The animal experiment flow chart of Figure 11 illustrative embodiment 4.
Figure 12 explains the influence of feeding PA extract to the CIA rat body weight.
Figure 13 explains that feeding PA extract is to the exponential influence of CIA rat arthritis.
Figure 14 explains the influence of feeding PA extract to CIA rat arthritis swelling degree.
Figure 15 explains the influence of feeding PA extract to CIA rat blood serum RF.
Figure 16 explains that feeding PA extract is the influence of CIA rat abdominal cavity cytohormone IL-6.
Figure 17 explains the influence of feeding PA extract to CIA rat abdominal cavity cytohormone IL-1 β.
The specific embodiment
Be meant in term used herein " treatment " or " processing " and improve symptom.
" patient " is meant animal, particularly mammal in term used herein.In a preferred specific embodiment, the patient is meant the mankind.
In term used herein " treatment effective dose " be meant separately or and the processing of medical composition component of the present invention with other medicines in symptom on the amount of treating interests can be provided.
Be meant the diluent that can be used for preparing medical composition, excipient, accepting agent or its analog that one of ordinary skill in the art know in term used herein " supporting agent " or " pharmaceutically acceptable supporting agent ".
Be meant that in term used herein " Plectlanthus amboinicus (Lour) Spreng slightly come together thing " or " Plectlanthus amboinicus (Lour) Spreng extract " the Plectlanthus amboinicus (Lour) Spreng plant is on the ground partly through directly squeezing the juice or through extraction process gained person.
Be meant the solvent that has the highest polarity at the solvent that is used for method for preparing in term used herein " high polar solvent ".High polar solvent includes, but are not limited to two kinds or above mixture of water, methanol, ethanol or aforementioned solvents.
Be meant the solvent that has minimum polarity at the solvent that is used for method for preparing in term used herein " low polar solvent ".One or more and one or more solvent that low polar solvent includes, but are not limited to chloroform, isopropyl alcohol, acetone, ethyl acetate, aforementioned solvents two kinds or above mixture or aforementioned solvents has about 70: 30 to about 50: 50 ratio (v: mixture v).
Be meant to have the polarity that is lower than the high polar solvent that is used for method for preparing in term used herein " inferior high polar solvent ", but be higher than the polar solvent of the Semi-polarity solvent that is used for method for preparing.Inferior high polar solvent can be by with about 70: 30 to about 30: 70, and (v: high polar solvent v) gets with the solvent with low polarity to be preferably about 60: 40 to about 40: 60 ratio.
Be meant to have the polarity that is lower than the inferior high polar solvent that is used for method for preparing in term used herein " Semi-polarity solvent ", but be higher than the polar solvent of the low polar solvent that is used for method for preparing.The Semi-polarity solvent can be by with about 30: 70 to about 5: 95, and (v: high polar solvent v) gets with the solvent with low polarity to be preferably about 15: 85 to about 5: 95 ratio.
The present invention is characterised in that and uses slightly come together thing or extract of Plectlanthus amboinicus (Lour) Spreng to treat rheumatic arthritis.Therefore the present invention provides a kind of medical composition that is used to treat rheumatoid arthritis, and it comprises the Plectlanthus amboinicus (Lour) Spreng of treating effective dose slightly come together thing or extract.
The those skilled in the art can be easy to judge and be best suited for pending path and dosage.According to the present invention, the path of preferably offeing medicine is an oral administration medicine supplying, such as but not limited to capsule, lozenge, powder, ointment, liquor or spray etc.Dosage will depend on character and state, pending patient's age and general health, dispensing path and any before enforceable treatment of pending symptom.It will be understood by one of ordinary skill in the art that said dosage can change with individual patients, look individual age, size and health status and correlation factor and decide.In addition, can be if need with its antibacterial, or with any pharmaceutically acceptable supporting agent or mixed with excipients.The preparation of medical composition of the present invention can be carried out by the those skilled in the art according to conventional methods.
In aspect preferred enforcement of the present invention, slightly the come together method for preparing of thing and Plectlanthus amboinicus (Lour) Spreng extract of Plectlanthus amboinicus (Lour) Spreng, as follows:
Slightly the come together preparation of thing of Plectlanthus amboinicus (Lour) Spreng
Get fresh Plectlanthus amboinicus (Lour) Spreng plant and clean, press juice with juice extractor then with clear water.Then Plectlanthus amboinicus (Lour) Spreng juice is given drying with the lyophilization mode, get dried powder; Reuse appropriate solvent, for example chloroform or the methanol extraction Plectlanthus amboinicus (Lour) Spreng thing that slightly comes together.
The preparation of Plectlanthus amboinicus (Lour) Spreng extract
Get quantitative Plectlanthus amboinicus (Lour) Spreng dry product and soak, after the filtration, after an amount of high polar solvent of reuse soaks, the collection fluid of Plectlanthus amboinicus (Lour) Spreng is rotated concentrating instrument with decompression be concentrated to 2～3% of original volume with an amount of high polar solvent; With separating with tubing string behind the solvent dilution.Optionally, can be continuously carry out eluting to the solution of four sections opposed polarities of low polar solvent five equilibrium (divide another name be high polar solvent, inferior high polar solvent, Semi-polarity solvent, low polar solvent) with high polar solvent.High polar solvent, inferior high polar solvent, Semi-polarity solvent and rudimentary property solvent system such as preceding text definition.Preferred person, the tubing string separation method is to use the DIAION tubing string of using methanol treatment, for example; Weigh DIAION with weight such as dry Plectlanthus amboinicus (Lour) Spreng; Soak with methanol, the DIAION that will soak methanol then is filled in the tubing string, and filling finishes the back earlier with 1～2 times of volumes methanol flushing DIAION; 5～6 times of volume secondaries of reuse water flushing DIAION is that filling is accomplished after flushing finishes.
The extract component analysis of Plectlanthus amboinicus (Lour) Spreng medical material
Instrument and equipment
High performance liquid chromatogram chromatographic analysis appearance
Pump: Spectra-Physics P4000
Detecting instrument: UV/VIS Spectra-Physics Spectra System UV600OLP
Automatic sampler: Thermo Separation Pruducts AS3500
Software: Thermo Separation Pruducts Chrom Quest
System's control: Thermo Separation Pruducts SN4000
Liquid phase chromatographic analysis condition
Chromatograph tubing string: COSMOSIL, 4.6 * 250mm, 5C18-MS
Chromatograph flow velocity: 1.0ml/min Pressure Limite:250kgf/cm
2
Sample injection volume: 10 μ l
The PDA condition:
Sampling: 0.64sec
Wave-length coverage: 190-370nm
UV wavelength: 254nm
Elution requirement (one):
| Time (minute) | ||||
| |
0 | 10 | 55 | 60 |
| Water | 90％ | 90％ | 20％ | 80 |
| Second | ||||
| 10％ | 10％ | 80％ | 20％ | |
Elution requirement (two):
| Time (minute) | ||||
| |
0 | 11 | 20 | 30 |
| Water | 95％ | 67％ | 67％ | 60％ |
| Second | 5％ | 33％ | 33％ | 40％ |
With reference to following limiting examples the present invention is detailed.Carry out follow procedure to confirm Plectlanthus amboinicus (Lour) Spreng thing or extract the influence of slightly coming together for treatment rheumatic arthritis.Modification that any those skilled in the art can reach easily and change include in the scope of this case description disclosure and appended claims.
Embodiment
Embodiment 1: use directly squeeze the juice and the Plectlanthus amboinicus (Lour) Spreng thing (PA) that slightly comes together
It is clean with clear water to get 1.25 kilograms of fresh Plectlanthus amboinicus (Lour) Spreng, presses juice with juice extractor then, and Plectlanthus amboinicus (Lour) Spreng juice is used the graduated cylinder measurement volumes; And it is dry with the lyophilization mode to take out 1050 milliliters of Plectlanthus amboinicus (Lour) Spreng juice; Get dried powder 19 grams at last, productive rate is 1.5%, and the HPLC collection of illustrative plates is as shown in Figure 1.
Embodiment 2: with Plectlanthus amboinicus (Lour) Spreng thing (PA) the treatment rheumatoid arthritis animal of slightly coming together
Animal experiment
[experimental animal]
Experimental animal Lewis rat is all available from national animal center; Mus 8 weeks of age; About 155 to 165 grams of body weight, raise in 12 little time secretly circulate, in 23 ± 1 ℃ of the room temperatures, humidity is moderate and air-conditioning is good Animal House, moisture and supply of forage all give freely getting food (ad libitum).In addition, experimental animal all meets the standard of laboratory animal committee standard article during test operation.
[medicine]
1. the collagen protein II type (Sigma C-1188) that obtains by the calf tracheal cartilages
2. formula Freund's complete adjuvant (complete Freund ' s adjuvant, CFA) (BD BBL not
TM231131)
3. formula Freund (incomplete Freund ' s adjuvant, IFA) (BD BBL not
TM263910)
The ferment immunity inspection reagent test kit (ELISA kit) of the tumor necrosis factor of rat (TNF-α), interleukin-6 (IL-6) and interleukin-1 ' beta ' (IL-1 β) (R&D, Duoset)
5. the c reactive protein of rat (CRP) ELISA kit (BD
TMPharmingen 557825)
6. indomethacin (available from strong gloomy chemical pharmacy company, triple cities, Taipei)
7. fresh Chinese herbal medicine PA directly squeezes the juice, and behind concentrating under reduced pressure, forms the PA concentrated solution, is diluted to high concentration (PA-H) of 22.5 gram crude drug amount/kilograms and the low concentration (PA-L) of 4.5 gram crude drug amount/kilograms more respectively, at last according to direct mouthful of food of throwing something and feeding of rat ABW
[equipment]
1. syringe: 1 milliliter, 3 milliliters and 5 milliliters (Terumo)
2. Libra
3. vernier cursor (Mitutoyo Corporation)
4. stop,threeway pipe
5. oscillator (vortex)
6.ELISA interpretoscope (Dynex, Thermo Labsystems)
[test method]
1. antigen preparation and inoculation
The animal experiment flow chart is as shown in Figure 2.The collagen protein II type (Bovine C II) of calf is dissolved in the 0.1M acetum, and stirring is fully dissolved it, and is mixed with the solution of concentration 1.5 and 3 mg/ml, places 4 ℃ to preserve subsequent use down.When carrying out first time inoculation, get the CII solution of 100 microlitres and the CFA emulsifying of equivalent respectively, emulsifying is accomplished the back and is carried out subcutaneous injection (200 microlitres /) in rat tail heel.Rat was write down body weight once in per 3 days after the immunity first time, and whether the observation extremities joint has the swelling situation; After the 15th day, carry out the inoculation second time, get the IFA emulsifying of 100 microlitre CII solution and equivalent respectively, carry out subcutaneous injection (200 microlitres/only) in rat tail heel equally after emulsifying is accomplished.On about the 20th day, begin to observe arthritic symptom (for CIA rat) greatly, and beginning feeding PA and indomethacin to the are till 45 days.
2. animal divides into groups and handles
N=3 rat/group
| Group | Handle | Feeding (gavage) |
| A | Normal rat (blank) | Normal feeding |
| B | CIA rat+mediator (matched group that disease produces) | The feeding primary water |
| C | CIA rat+indomethacin (marketed drug treatment matched group) | 2.5 milligrams/kg/day of feeding indomethacin |
| D | CIA rat+PA-L | Feeding PA75 milligram/kg/day |
| E | CIA rat+PA-H | 375 milligrams/kg/day of feeding PA |
[pilot project and pointer]
1. body weight is observed: per 3 days weighings are observed and are showed
2. rheumatoid arthritis is inspected assessment: with maximum rheumatic arthritis index assessment (maximum arthritic index; MAI)
3. arthroncus rate: measure (Day 15～28Arthritis development) with vernier cursor
4. the rheumatoid joint factor (rheumatoid factor; RF)
5. acute inflammation reactive protein (C-reactive protein; CRP)
6. cytohormone: TNF-α, IL-1 β and IL-6 (inflammatory cell hormone, inflammatory cytokine)
Symptoms of rheumatoid arthritis is inspected assessment
Rat is after immunity, and each week observes 3 times.The redness that writes down its extremities joint swells etc. and to change situation, taking pictures deposit relatively, and inspecting score basis with 5 degree difference conducts:
0: do not present any joint symptom.
1: sole and instep portion (tarsus) take on a red color, and slight swollen.
2: instep portion and ankle moderate are red and swollen.
3: instep portion and ankle are seriously red and swollen.
4: ankylosis, bone distortion.
Maximum rheumatic arthritis index, every group of average MAI calculates as follows:
(0 minute: no CIA took place the summation of every rat extremity record of average MAI=gained MAI; 16 minutes: top score)/4/ every group of number of rats
Arthritis swelling degree measurement
Measure rat sole varied in thickness (2 times/week) with vernier cursor, measurement point be forward foot in a step left and right sides sole central point each one, each three measurement point (ankle joint, sole and toe root) of the left and right sides rear foot.Every rat is totally eight measurement points.
Serum RF analyzes
1. be 40 mcg/ml with coating buffer (coating buffer) preparation collagen concentration, and add 0.1 milliliter respectively in the trace dish of 96 holes, overnightly be statically placed in 4 ℃.
2. after Tris buffer solution for cleaning three times, each hole adds 0.2 milliliter of blocking-up buffer (blocking buffer) that contains 1% bovine serum albumin (BSA), at room temperature reacts reuse Tris buffer solution for cleaning three times 2 hours.
3. with the Tris buffer that contains 0.05%Tween 20 blood serum sample is done suitably dilution back (1/20 or 1/40), each hole adds 0.1 milliliter blood serum sample in the trace dish of 96 holes, at room temperature reacts reuse Tris buffer solution for cleaning three times 2 hours.
4. the anti-rat immune globulin M (IgM) that will coat combines to do suitably dilution back (1/12000) with horseradish peroxidase (HRP), be added in the trace dish of 96 holes, at room temperature reacts reuse Tris buffer solution for cleaning three times 2 hours.
5. each hole directly adds 0.1 milliliter tetramethyl benzidine (TMB) substrate, carries out color reaction, adds the stop bath stopped reaction subsequently.At last, the wavelength with 450 nanometers reads extinction (O.D) value.
Serum C-reactive protein (CRP) is analyzed
1.CRP ELISA kit the is precoating porose disc (pre-coated microplate) that declines can directly add through 0.1 milliliter of the blood serum sample of suitable dilution, room temperature reaction cleaned 4 times with lavation buffer solution after 1 hour.
2. the anti-Mus c reactive protein of rabbit (CRP) is combined with HRP Ab, after 100 times of lavation buffer solution dilutions, each hole adds 0.1 milliliter, and room temperature reaction cleaned 4 times with lavation buffer solution after 1 hour.
3. each hole adds 0.1 milliliter tetramethyl benzidine and carries out color reaction, about 5 to 10 minutes, adds the stop bath stopped reaction subsequently.At last, the wavelength with 450 nanometers reads the O.D value.
(the appended detection rule of the equal reference reagent box of above analytical method is carried out)
Abdominal cavity cell is cultivated and the cytohormone analysis
1. rat is cut off rat abdominal cavity outside fur with shears after carbon dioxide euthanasia, and whole abdominal cavity is exposed.
2. with 10 milliliters of syringes the abdominal cavity is injected in the gradation of HBSS buffer, make cumulative volume be about 20 milliliters/.
3. cut off the abdominal cavity behind the slight massaging rat abdomen, cut off the about 2 centimeters long openings in abdominal cavity with shears, the reuse syringe is drawn peritoneal exudate cells (peritoneal exudation cell; PEC) liquid (can collect 10 to 15 ml cells liquid approximately), and place 50 milliliters of centrifuge tubes.
4. outwell supernatant through 1500rpm after centrifugal 5 minutes, add after 10 milliliters of cleanings of HBSS buffer centrifugally, outwell supernatant.
5. reuse fresh culture (containing antibiotic) is adjusted cell concentration to 2 * 10
6Cells/ml.
6. the cell suspending liquid branch is installed to 48 porose discs (well plate), 0.5 milliliter/hole.
7. add 0.5 milliliter of lipopolysaccharide class (LPS) (20 mcg/ml) in addition more respectively, making final concentration is 10 mcg/ml.
8. place 37 ℃ of incubators to cultivate at last 24 hours, collect supernatant and place-20 ℃ of preservations.Utilize the concentration of ELISAkit analysis of cells hormone TNF-α, IL-6 and IL-1 β.
[result and discussion]
One, Mus body weight change
In animal experiment, no matter be to throw to give medicine or bestow other external force, be the variation that directly or indirectly has influence on body weight, therefore, body weight is observed also just directly becomes most important in appearance index.Result by measured body weight shows; Bringing out arthritic symptom via collagen protein produced after the 20th day; Obviously descend compared to its body weight of normal rat, as shown in Figure 3, however the group of feeding PA-H and indomethacin; Then effectively avoid weight loss, the isometric growth curve is arranged with compared with normal.In addition, feeding PA-L then can't effectively suppress the phenomenon of losing weight.
Two, maximum rheumatic arthritis index
Maximum rheumatic arthritis index (MAI) described in materials and methods, as the foundation of inspecting scoring, is one of appearance index with 5 degree differences.Rat reached peak on the 35th day after collagen protein brings out arthritis, as shown in Figure 4, and the group of feeding PA and indomethacin is arranged, and all can effectively reduce arthritis index, wherein with the effect of PA-H and indomethacin most preferably.
Three, arthroncus degree
Rat after the antigen injection second time on about the 20th day; Begin to produce arthritic symptom successively; And it is actual with light tape measure extremities joint position; Its arthroncus rate (being 8 meansigma methodss that measurement point records) by 20% continue to increase to the 39th day for the highest by 61%, as shown in Figure 5, reached significant difference (P＜0.01) with compared with normal.And aspect feeding PA and indomethacin,, wherein still most preferably, show that PA-H has the drug effect of similar indomethacin anti-inflammatory with the effect of PA-H and indomethacin no matter be that PA-H or PA-L all have remarkable inhibition arthroncus.
Four, feeding PA is to the influence of serum RF
Rheumatoid arthritis tends to supervene several kinds of different autoantibody in pathogenic process; And in clinical diagnosis; Whether autoantibody exists among the patients serum becomes the main foundation that takes a decision as to whether rheumatoid arthritis, wherein has with the autoantibody of RF the most important.Therefore, the zootype of CIA also will be with RF as important biochemical indicator in this test.Past studies have pointed out, utilize the method for ELISA can analyze RF (Vittecoqet al., 2001 in the mankind or the rat blood serum; Jonsson et al., 1986).In this research process, improve according to analytical method in the past, and rebulid the technology platform that is applicable to analyzing rat serum RF.Analysis result shows, after rat is injected via twice antigen, in the time of the 20th day; Serum RF titer all reaches peak, and is as shown in Figure 6, yet the group of feeding PA and indomethacin is arranged; In the time of the 35th day; Significantly reduce by 33% and 47% respectively with relatively playing serum RF value on the 20th day, during by the 45th day, drop to 39% and 51% more respectively.In addition; This research is also found; The serum RF value of negative control group had the trend of successively decreasing gradually later at the 20th day; After showing that rat is brought out arthritic symptom via collagen protein,, natural law can be tending towards relaxing even restoring, so infer that be the 20th day to 45 days gold period of falling ill along with increasing arthritic symptom.
Five, the influence of feeding PA change of serum C RP
Change of serum C RP is mainly produced by liver, for causing the index of general inflammatory response, also is simultaneously that acute inflammation is present in topmost reactant in the serum period.Have document to point out, development has close association (Nakamu rheumatoid arthritis 2000) for arhritis conditions in the generation of CRP content and IL-1 and TNF-α in the rheumatoid arthritis human serum.Result of study shows that rat lures that successfully rat blood serum CRP concentration obviously increases into after the injection of secondary antigen immune, and in the time of the 35th day, arrives summit, and is as shown in Figure 7, and following significant difference (P＜0.01) is arranged with normal group is compared; Find also in the time of the 35th day that equally the CIA rat can effectively suppress change of serum C RP concentration behind feeding PA-H and indomethacin, and still have identical effect during by the 45th day, show that feeding PA-H has the similar indomethacin therapeutic effect of anti-inflammatory medication clinically.The effect of then not having remarkable inhibition inflammation as for feeding PA-L.
Six, the variation of PEC cytohormone
Except whether above-mentioned several important biochemical indicator assessment Plectlanthus amboinicus (Lour) Spreng have the effect of detumescence or anti-inflammatory; The influence of cytohormone secretion to inflammatory response also further inquired in this research; So that more the easy master rat changes situation before and after producing symptoms of rheumatoid arthritis; Wherein to be inflammatory cell hormone (inflammatorycytokine) be most important index for TNF-α, IL-6 and IL-1 β etc., and concentration number and inflammatory response are closely bound up in vivo to that is to say said cytohormone.Result of study confirms that CIA rat feeding PA-H has remarkable inhibition PEC TNF secretion-α and IL-1 β (as shown in Figure 8), and IL-6 (as shown in Figure 9).In addition, but do not have the effect that any inhibition PEC secretes the inflammatory cell hormone in the part of feeding indomethacin, supposition possibly be that the mechanism of action of indomethacin is not at this, also so more highlights the using value of PA in treatment detumescence or anti-inflammatory purposes.
From the above, the medicine of the present invention and control group, indomethacin relatively, slightly the come together drug effect of thing and 2.5 milligrams/kilogram indomethacin of high concentration Plectlanthus amboinicus (Lour) Spreng is suitable.In addition, indomethacin is the COX inhibitor, and from the inhibition phenomenon of cytohormone of performance, its pharmacological action is possible inequality with Plectlanthus amboinicus (Lour) Spreng.
Embodiment 3: use the tubing string separation and purification and Plectlanthus amboinicus (Lour) Spreng extract (PA-EtOH, PA-F1, PA-F2, PA-F3 and PA-F4)
The Plectlanthus amboinicus (Lour) Spreng dry product of getting 2 kilograms soaked 24 hours with 10 times of alcohol in high concentration; After the filtration; 10 times of alcohol in high concentration of reuse soaked after 24 hours, with the collection fluid of Plectlanthus amboinicus (Lour) Spreng with decompression rotation concentrating instrument be concentrated to original volume 2～3% (if be dried to powder is PA-EtOH; Weight 30 grams, productive rate is 1.5%.
With being filled into behind the solvent dilution in the DIAION tubing string of having handled.After the high polar solvent flushing with about 10 times of dried medical material volumes, and collect elution buffer, be designated as PA-F1, weight 8.5g, productive rate are 0.43.
With about 5 to the 10 times of dried medical material volumes flushings of tubing string reuse time high polar solvent and collect elution buffer, be designated as and be PA-F2; Weight 12g, productive rate are 0.6%, and the HPLC collection of illustrative plates is shown in figure 10.
Flushing and collection elution buffer with about 5 to the 10 times of dried medical material volumes of tubing string reuse Semi-polarity solvent are designated as PA-F3; Weight 15g, productive rate are 0.75%.
Flushing and collection elution buffer with about 5 to the 10 times of dried medical material volumes of tubing string reuse low polar solvent are designated as PA-F4; Weight 12g, productive rate are 0.6%.
Embodiment 4: with the animal experiment of Plectlanthus amboinicus (Lour) Spreng extract treatment rheumatoid arthritis animal
In this animal experiment, employed experimental animal and equipment and like 2 announcements of embodiment.
[medicine]
1. the collagen protein II type (Sigma C-1188) that obtains by the calf tracheal cartilages
2. formula Freund's complete adjuvant (complete Freund ' s adjuvant, CFA) (BD BBL not
TM231131)
3. formula Freund (incomplete Freund ' s adjuvant, IFA) (BD BBL not
TM263910)
The ferment immunity inspection reagent test kit (ELISAkit) of interleukin-6 (IL-6) and interleukin-1 ' beta ' (IL-1 β) (R&D, Duoset)
5. Celebrex (Ceelebrex; CBX)
6. the Plectlanthus amboinicus (Lour) Spreng extract of embodiment 3 (PA-EtOH, PA-F1, PA-F, PA-F3 and PA-F4) is according to the direct mouth food of throwing something and feeding of rat ABW
[test method]
1. antigen preparation and inoculation
The animal experiment flow chart is shown in figure 11.The collagen protein II type of calf is dissolved in the 0.1M acetum, and stirring is fully dissolved it, and is mixed with the solution of concentration 1.5 and 3 mg/ml, places 4 ℃ to preserve subsequent use down.When carrying out first time inoculation, get the C II solution of 100 microlitres and the CFA emulsifying of equivalent respectively, emulsifying is accomplished the back and is carried out subcutaneous injection (200 microlitres /) in rat tail heel.Rat was write down body weight once in per 3 days after the immunity first time, and whether the observation extremities joint has the swelling situation; After the 15th day, carry out the inoculation second time, get the IFA emulsifying of 100 microlitre C II solution and equivalent respectively, carry out subcutaneous injection (200 microlitres/only) in rat tail heel equally after emulsifying is accomplished.On about the 18th day, begin to observe arthritic symptom (for the CIA rat) greatly, and began feeding PA and CBX lasting 20 days, till the 38th day in the 19th day.
2. animal divides into groups and handles
N=3 rat/group
| Group | Handle | Feeding (gavage) |
| A | CIA rat+PA-EtOH | 40.5 milligrams/kg/day of feeding PA |
| B | CIA rat+PA-F1 | 3.0 milligrams/kg/day of feeding PA |
| C | CIA rat+PA-F2 | 1.13 milligrams/kg/day of feeding PA |
| D | CIA rat+PA-F3 | 11.3 milligrams/kg/day of feeding PA |
| E | CIA rat+PA-F4 | 11.3 milligrams/kg/day of feeding PA |
| F | CIA rat+H 2O | The feeding primary water |
| G | CIA rat+CBX | 18 milligrams/kg/day of feeding CBX |
| J | Normal rat (blank) | Normal feeding |
[result and discussion]
One, Mus body weight change
Shown in figure 12, in each group, body weight does not have obviously too big gap between normal rat and CIA rat.Compared to the matched group of feeding primary water, the rat of feeding PA-EtOH, PA-F2 and PA-F4 has heavier body weight.
Two, maximum rheumatic arthritis index
Rat reached peak on the 31st day after collagen protein brings out arthritis; Shown in figure 13; And the group of feeding PA-EtOH, PA-F1, PA-F2, PA-F3, PA-F4 and CBX is arranged, all can effectively reduce arthritis index, wherein most preferably with the effect of PA-F1 and CBX.
Three, arthroncus degree
Rat through after the inoculation second time on about the 20th day, begin to produce arthritic symptom successively, and actual with light tape measure extremities joint position, its arthroncus rate continues to increase, arrival is the highest by 64% during by the 27th day.Shown in figure 14, reached significant difference with compared with normal.And the rat of feeding PA-EtOH, PA-F1, PA-F2, PA-F3 and PA-F4 and CBX all represents and suppresses the arthroncus effect, and wherein still with the effect of PA-F1 and CBX most preferably, demonstration PA-F1 has the drug effect of similar CBX anti-inflammatory.
Four, feeding PA is to the influence of serum RF
Analysis result shows, rat via twice inoculation after, in the time of the 18th day, serum RF titer all reaches peak, and is shown in figure 15.Yet, the group of feeding PA-F2 and CBX is arranged, reduce RF value about 34% at the 30th day PA-F2; Reduced RF value about 44% on the 37th day; And CBX treatment reduced the RF value to reduce the RF value in about 52%, the 37 day about 66% at the 30th day, and relatively play the remarkable respectively reduction of serum RF value on the 18th day.
Five, the variation of PEC cytohormone
Result of study confirms that CIA rat feeding PA-F2, PA-F3 and PA-F4 have remarkable inhibition PEC secretion IL-6 (shown in figure 16) and feeding PA-F1, PA-F2 and CBX have remarkable inhibition PEC secretion IL-1 β (shown in figure 17).
[conclusion]
With above-mentioned active animal analysis result tabulation, shown in Table I:
Table I
| CBX | PA-EtOH | PA-F1 | PA-F2 | PA-F3 | PA-F4 | |
| Toe thickness | ++ (49％) | + (16％) | ++ (44％) | + (23％) | + (28％) | + (23％) |
| MAI | ++ (40％) | + (34％) | ++ (46％) | + (24％) | + (37％) | + (21％) |
| RF | ++ (43％) | - (0％) | - (0％) | + (16％) | - (0％) | - (0％) |
| IL-6 | ++ (36％) | + (30％) | + (21％) | +++ (52％) | ++++ (82％) | ++++ (78％) |
| IL-1β | ++ (38％) | - (0％) | +++ (64％) | ++ (45％) | + (30％) | + (14％) |
The inhibitory action of " ++ ++ " expression 75 to 100%
The inhibitory action of " +++" expression 50 to 75%
The inhibitory action of " ++ " expression 25 to 50%
The inhibitory action of "+" expression 0 to 25%
Embodiment 5: the pattern analysis of mouse macrophage cell in vitro
One, the assay method of the concentration of TNF-α and IL-1 β
[purpose]
Measure the concentration of TNF-α and IL-1 β in little phagocyte RAW264.7 cell culture fluid of rat, screen thus and can suppress LPS-and bring out-the synthetic active component of TNF-α.
[equipment and material]
1. instrument
(1) ELISA interpretoscope
(2) centrifuge
Testing reagent test kit DY410: rat TNF-α/TNFSF1A (ELISA kit) (R&D, Duoset)
(1) carrier-free proteinic sheep anti mouse TNF-α or IL-1 β antibody: at PBS (phosphatebuffered saline; PBS) 0.8 mcg/ml in
(2) biotinylated sheep anti mouse TNF-α or IL-1 β antibody: 150 nanograms/milliliter in dilution reagent
(3) recombinant rat TNF-α or the IL-1 β in dilution reagent: 2000 pg/ml
(4) Succ-PEG-DSPE-horseradish peroxidase (Streptavidin-HRP)
3. required solution
(1) PBS (PBS): 137mM sodium chloride, 2.7mM potassium chloride, 8.1mM sodium hydrogen phosphate (Na
2HPO
4), 1.5mM sodium dihydrogen phosphate (KH
2PO
4), pH 7.2～7.4
(2) dcq buffer liquid: 0.05%Tween 20 in PBS
(3) blocking-up buffer: 1% bovine serum albumin (BSA), 5% sucrose, 0.05% sodium azide (NaN in PBS
3)
(4) dilution reagent: 1% bovine serum albumin in PBS
(5) substrate solution: 1: 1 colored reagent A and the mixture of colored reagent B (R&D system#DY999)
(6) stop solution: 2N sulphuric acid
4. medicine
(1) Griess reagent: 1% P-aminobenzene-sulfonamide (sulfanilamide) in 5% phosphoric acid and 0.1%N-(1-naphthyl)-ethylene diamine
(2) standard substance: sodium nitrite (sodium nitrite)
(3) immunologic stimulant: lipopolysaccharide (Lipopolysaccharide; LPS)
(4) activity control agent: nitro-L-arginine methyl ester (L-NAME), indomethacin
(5) culture medium: (the fetal calf serum of 10% hyclone in DMEM; FCS)
(6)PBS
(7) trypsin Trypsin)
5. cell: RAW 264.7
[method]
1. cell culture and branch dish:
(1) sop up old culture medium among the T-75, clean 1 to 2 time with PBS, adds 3 milliliters of trypsin, reaction is 3 minutes in 37 ℃, adds 7 milliliters of culture medium (DMEM adds 10% hyclone) termination trypsin acting.
(2) use centrifuge with 1000rpm, descended centrifugal 10 minutes, contain tryptic culture medium, add 10 milliliters of culture medium, behind the mix homogeneously, calculate cell number to remove at 4 ℃.
(3) with RAW 264.7 cell inoculations in 24 porose discs, cell density is 5 * 10
5Cells/well is cultivated in 37 ℃, 5% carbon dioxide.
2.LPS stimulate and drug treating
(1) L-LAME and indomethacin (activity control agent) and sample are made an addition to respectively in the no phenol culture medium that contains 1 mcg/ml LPS.
(2) take down old culture medium, in generation, done three repetitions to contain LPS and the activity control note or the new culture medium of sample, and cultivation is 18 to 24 hours in 37 ℃, 5% carbon dioxide.
3. prepare sandwich ELISA s
(1) capture antibodies (capture antibody) (being diluted to 0.8 mcg/ml with PBS) of adding 100 λ in each hole of 96 porose discs seals overnight incubation at room temperature.
(2) move free benefit and catch antibody, clean three times, add the blocking-up buffer of 300 λ, at room temperature cultivated at least 1 hour, to reduce non-narrow spectrum combination (binding) with cleaning buffer solution.
(3) collecting cell suspension utilizes centrifuge with centrifugal 10 minutes of 10krpm, 4 ℃, is stored under-20 ℃.
4.ELISAs analyze
(1) removes the blocking-up buffer, clean three times, add cell culture fluid or the standard substance (maximum concentration 2000 pg/ml) of 100 λ, in room temperature, cultivated 2 hours through the suitable multiple dilution with cleaning buffer solution.
(2) remove cell culture fluid or standard substance, clean three times, add the detecting antibody (being diluted to 100 nanograms/milliliter) of 100 λ, in room temperature, cultivated 2 hours with reagent with cleaning buffer solution.
(3) move free detecting antibody, clean three times with cleaning buffer solution, add the work diluent of Succ-PEG-DSPE-horseradish peroxidase of 100 λ, at room temperature lucifuge was cultivated 20 minutes.
(4) move free Succ-PEG-DSPE-horseradish peroxidase, clean three times with cleaning buffer solution, what add 100 λ receives matter solution, and at room temperature lucifuge was cultivated 20 minutes.
(5) add the solution that stops of 100 λ, gentle rocking makes mix homogeneously.
(6) wavelength with 450 nanometers reads the O.D value, and the correction of 540 or 570 nano wave lengths is done in suggestion, or directly 450 the value of reading is deducted the value of reading of 570 (or 540).
Two, the mensuration of NO
[purpose]
Measure the concentration of nitrite (NO) in the little phagocyte RAW264.7 cell culture fluid of rat, screening can suppress LPS-and brings out-the synthetic active component of NO thus.
[equipment and material]
1. instrument
(1) ELISA reading machine
(2) centrifuge
2. medicine
(1) Griess reagent: 1% P-aminobenzene-sulfonamide (sulfanilamide) in 5% phosphoric acid and 0.1%N-(1-naphthyl)-ethylene diamine
(2) standard substance: sodium nitrite (sodium nitrite)
(3) immunologic stimulant: lipopolysaccharide (Lipopolysaccharide; LPS)
(4) activity control agent: nitro-L-arginine methyl ester (L-NAME), indomethacin
(5) culture medium: (the fetal calf serum of 10% hyclone in DMEM; FCS)
(6)PBS
(7) trypsin Trypsin)
3. cell: RAW 264.7
[method]
1. cell culture and branch dish:
(1) sop up old culture medium among the T-75, clean 1 to 2 time with PBS, adds 3 milliliters of trypsin, reaction is 3 minutes in 37 ℃, adds 7 milliliters of culture medium (DMEM adds 10%FCS) termination trypsin acting.
(2) use centrifuge with 1000rpm, 4 centrifugal 10 minutes, contain tryptic culture medium to remove, adds 10 milliliters of culture medium, behind the mix homogeneously, the calculating cell number.
(3) with RAW 264.7 cell inoculations in 24 porose discs, cell density is 5 * 10
5Cells/well is cultivated in 37 ℃, 5% carbon dioxide.
2.LPS stimulate and drug treating
(1) L-NAME and indomethacin (activity control agent) and sample are made an addition to respectively in the no phenol culture medium that contains 1 mcg/ml LPS.
(2) move old culture medium, in generation, done three repetitions to contain the new culture medium of LPS and activity control agent or sample, and cultivation is 18 to 24 hours in 37 ℃, 5% carbon dioxide.
(3) collecting cell suspension uses centrifuge with centrifugal 10 minutes of 10krpm, 4 °, is stored under-20 ℃.
3. measuring N O concentration
(1) preparation standard substance: prepare the sodium nitrite (being dissolved in the culture medium) of 100 μ M/mL, doubling dilution obtains 50,25,12.5,6.25,3.13 and 1.56 μ M/mL totally seven standard substance.
(2) with the supernatant of standard substance or cell culture and the Griess reagent mixed with 1: 1, at room temperature lucifuge was cultivated 15 minutes.
(3) wavelength with 550 interior rice reads the O.D value.
Three, prostaglandin (Prostaglandin E2; PGE
2) mensuration
In 24 porose discs, cell density is 105 cells/well with the RAW264.7 cell inoculation, overnight incubation (16 to 24 hours).Activity control agent and specimen are made an addition to respectively in the no phenol culture medium that contains 1 mcg/ml LPS, remove old culture medium after, add 1 milliliter of fresh culture that contains specimen and LPS, co-cultivation.After 24 hours, use centrifuge centrifugal 10 minutes with 1000rpm is drawn supernatant, places-20 ℃ to preserve or directly use PGE
2PGE in the quantitative supernatant of Correlate-EIA kit (Amersham RPN222)
2Content.
[conclusion]
With above-mentioned cell pattern analysis result tabulation, as shown in Tble II:
Table II
| PAW | PA-F1 | PA-F2 | PA-F3 | PA-F4 | |
| NO | - | IC 50 ?＞ 4.27μg/ml | - | - | - |
| PGE 2 | ED
50 ?
 |
- | ED
50 ?
 |
ED
50  |
ED
50  |
| TNF-α | ED 50 ＞ 50μg/ml | ED
50 ?
 |
- | ED
50  |
ED 50 ＞ 1000μg/ml |
| IL-1β | ED
50  |
- | ED
50 ?
 |
ED
50  |
ED
50  |
The PA extract can suppress the mouse macrophage inflammation that lipopolysaccharide class (LPS) is brought out.PA-F1 has significant inhibitory effect for the TNF-α that inflamed cell produced that is brought out by LPS, and PA-F2 can suppress the effect that IL-1 β produces, and PA-F3 can suppress PGE
2, it is the product of a kind of COX.
In sum, slightly come together thing or extract of Plectlanthus amboinicus (Lour) Spreng used in the present invention is directly to be squeezed the juice or separated and get through tubing string by the Plectlanthus amboinicus (Lour) Spreng plant, and it is the high Chinese herbal medicine of safety ten minutes, but also orally-ingestible of external.In addition; The present invention can effectively suppress the animal symptom and relevant biochemical indicator that collagen protein adds the caused autoimmune disease of immunological adjuvant with slightly come together thing or extract of rheumatoid arthritis rat model validation Plectlanthus amboinicus (Lour) Spreng via oral way; Therefore have the curative effect of potential detumescence and anti-inflammatory, can be effective to treat rheumatoid arthritis.
Claims (6)
1. medical composition of treating rheumatoid arthritis, it comprises Plectlanthus amboinicus (Lour) Spreng (plectlanthus amboinicus (Lour.) Spreng that treats effective dose; PA) slightly come together thing or alcoholic extract and pharmaceutically acceptable supporting agent, wherein said thick collection thing are with directly the squeeze the juice product of gained of Plectlanthus amboinicus (Lour) Spreng, and said alcoholic extract is with alcohol-pickled Plectlanthus amboinicus (Lour) Spreng and concentrates the back with the separating obtained product of tubing string.
2. medical composition according to claim 1, it is capsule, lozenge, powder, ointment, liquor or spray pattern.
3. medical composition according to claim 1, wherein said thick collection thing has the HPLC crest of following holdup time:
Wherein said HPLC carries out in following condition:
Chromatograph flow velocity: 1.0ml/min Pressure Limite:250kgf/cm2
Sample injection volume: 10ul
Sampling: 0.64sec
Wave-length coverage: 190-370nm
UV wavelength: 254nm
The eluting collection of illustrative plates:
4. medical composition according to claim 1, wherein said alcoholic extract have the HPLC crest of following holdup time:
Wherein said HPLC carries out in following condition:
Chromatograph flow velocity: 1.0ml/min Pressure Limite:250 kilogram f/cm2
Sample injection volume: 10ul
The PDA condition:
Sampling: 0.64sec
UV wavelength: 254nm
The eluting collection of illustrative plates
5. Plectlanthus amboinicus (Lour) Spreng (plectlanthus amboinicus (Lour.) Spreng; PA) slightly come together thing or alcoholic extract in the purposes of the medicine of preparation treatment rheumatoid arthritis; Wherein said thick collection thing is with directly the squeeze the juice product of gained of Plectlanthus amboinicus (Lour) Spreng, and said alcoholic extract is with alcohol-pickled Plectlanthus amboinicus (Lour) Spreng and concentrates the back with the separating obtained product of tubing string.
6. purposes according to claim 5, wherein said medicine are capsule, lozenge, powder, ointment, liquor or spray pattern.
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