CN1772033A - Use of cajan seed and its extract in preparing medicine for treating osteal arthritis - Google Patents
Use of cajan seed and its extract in preparing medicine for treating osteal arthritis Download PDFInfo
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Abstract
The present invention is the use of cajan leaf and its extract in preparing medicine for treating osteal arthritis. Cajan leaf and twig after being decocted or crushed and tabletted may be used in treating osteal arthritis. Preferably, cajan leaf is water or alcohol extract to obtain extract, and animal experiment shows that cajan leaf extract has obvious intracorporeal curative effect on osteal arthritis and extracorporeal effect of promoting the proliiferation of cartilage cell and the synthesis of protein inside cartilage and total protein, so that it may be used in preparing medicine for treating osteal arthritis.
Description
Technical field
The present invention relates to field of traditional Chinese medicine pharmacy, particularly, the present invention relates to Chinese medicinal plant Folium Cajani and extract thereof and be used to prepare the purposes for the treatment of medicine for treating arthritis.
Background technology
Osteoarthritis is modal in the world arthrosis, and with the age increase, prevalence rises rapidly; Greater than the X-ray film evidence that osteoarthritis was arranged more than 50% among the crowd in 65 years old, but have 25% to have symptom.Symptom can appear in crowd 80% more than 75 years old.More than 50 years old among the male, osteoarthritis is to be only second to second reason that ischemic heart desease causes the ability to work forfeiture, the labour force is lost reach 53% in the U.S..Osteoarthritis is old people's pain and major cause of morbidity.
The World Health Organization's statistics, osteoarthritis accounts for the 4th in women's prevalence, account for the 8th in the male sicken rate.In China, aging population have 8,000 ten thousand populations to have the X line performance of osteoarthritis about more than 100,000,000 approximately, have 4,000 ten thousand people that symptom is arranged approximately.Along with the aging of population, China has also entered aging country, and this disease can constantly increase.Treat this disease and also will expend huge health care resource.
Folium Cajani is dried leaves and the twig of leguminous plant Semen Cajani (Cajanus canjan (L.) Millsp.), is distributed in the south, Asia, and there is cultivation provinces and regions such as China Hainan, Yunnan, Sichuan, Jiangsu, Guangdong and Guangxi, aboundresources." Chinese medicine voluminous dictionary " and " the herbal illustrated handbook of primary colors China " carries: " property is flat, and is lightly seasoned, separates the pox mild toxicity, and antiinflammatory is swollen for Folium Cajani.Control children's's chickenpox, carbuncle; " the Semen Cajani seed " sweet little acid, warm in nature.Heat-clearing and toxic substances removing, invigorating the spleen and replenishing QI, diuretic helps digestion, hemostasis and dysentery-stopping." the Semen Cajani tender leaf chews the mashed aphtha that is used for the treatment of, and presses the juice jaundice that can disappear for oral administration, the juice of mashing has the putrefaction-removing granulation-promoting effect to wound and sore, and the decoct of leaf is to cough, diarrhoea etc. effectively.Yuan Hao has obtained the national patent mandate (patent No.: ZL97108820.9), find also that in addition the Folium Cajani extract can be used for treating diseases such as fracture, osteoporosis, cerebral infarction with Folium Cajani extract for treating femur head necrosis.But the research of utilization Folium Cajani treatment osteoarthritis is not arranged as yet at present.
Studies show that osteoarthritis is different with femur head necrosis, femur head necrosis system is limited by femoral head blood fortune, causes femoral head sclerotin ischemia, forms the death of the great-hearted composition (comprising osteocyte, bone marrow, hematopoietic cell and adipose cell) of bone.The pathogenic factor of femur head necrosis is a lot, and some is not investigated thoroughly as yet, the known two big classes that are divided into.Damaging femur head necrosis main diseases is because of having: fracture of femoral neck, dislocation of hip joint, hip joint damage etc.Wherein fracture of femoral neck causes femur head necrosis maximum.But secondary femur head necrosis still after the fracture of femoral neck healing.Non-damage femur head necrosis main diseases is because of having: take hormone, chronic alcoholism, hemoglobinopathy, decompression sickness, hemophilia, hyperlipidemia, arteriosclerosis, diabetes and coxa plana etc. for a long time.Wherein hormone, alcoholism cause femur head necrosis more.As hormone capillary permeability being raise causes oozing of blood, vein obstruction to cause backflow obstacle or the like to cause that all intra-osteal pressure increases.The intra-osteal pressure that raises has increased the resistance of blood flow again, thereby further causes ischemia, forms vicious cycle, especially suffers from limb and continues to bear a heavy burden.After the femur head necrosis, the femoral head pressurized of ischemia can be accelerated capital wearing and tearing, subside.The clinical manifestation of femur head necrosis is: (1) pain: Avascular Necrosis Of Femur Head can not have clinical symptoms in early days, but when taking x line sheet, find.The symptom of Chu Xianing is the hip joint or the gonalgia the earliest.Pain can be persistence or intermittence.Hip or knee pain, dull pain or the bloated discomfort of acid etc. appear gradually or suddenly, often to inguinal region or the buttocks rear side or the outside, or the inboard radiation of knee joint, there is feeling of numbness in this district.How not serious pain character is in early days, but increase the weight of gradually, also can be subjected to increasing the weight of suddenly after the wound.Through can respite behind the expectant treatment, but through showing effect once again after a while.Primary disease is very big apart from the time phase difference that pain occurs; (2) ankylosis and limitation of activity: the movable normal or slight forfeiture of early stage patient hip joint shows as to a certain direction moving obstacle, particularly inward turning.Should stretch hip and the hip 90 of going down on one's knees ' position is bent, stretched, interior receipts, abduction and inward turning inspection at horizontal position, the bilateral contrast could be found.Dwindle gradually with the PD range of activity, late period, hip joint was seriously limited to all directions activity owing to the plump contracture of joint capsule, and hip joint merges, and it is stiff hip joint to occur; (3) walk lamely: early stage patient increases owing to femoral head is intrinsic pressure, and intermittent claudication can be arranged, and the back of having a rest takes a turn for the better, and patients with terminal is because collapse of the femoral head and hip joint subluxation can have the persistence limping.The main Therapeutic Method of femur head necrosis is operative treatment and Drug therapy.Operative treatment comprises: decompression and Drilling, blood vessel grafting, free bone grafting operation, allosome cartilage grafting, artificial joint replacement in the bone marrow; Drug therapy comprises: Western medicine does not have a kind of medicine can treat femur head necrosis at present as yet.Chinese patent medicine mainly contains TONGLUO SHENGGU JIAONANG, strong bone life, XIANLING GUBAO JIAONANG and Ma Shi osteocomma etc.Mainly be to reach the effect of repairing osteonecrosis by blood circulation promoting and blood stasis dispelling, reinforcing the kidney to strengthen the bone.
And osteoarthritis is a kind of chronic joint disease, and its main change is the degeneration and the insecondary hyperosteogeny of articular cartilage face.Pilosity was born in after the middle age, and site of pathological change is many in the heavy burden joint.Also can be secondary to after joint deformity, damage and the inflammation, so claim proliferative arthritis, hypertrophiarthritis, degenerative osteoarthritis, senile arthritis, arthritis deformans, traumatic arthritis or the like again.The etiology and pathology of osteoarthritis is mainly followed deficiency, inflammation and constitutional factor and is caused by damage, asymmetric, the blood of articular surface.Main change be articular cartilage and below sclerotin, cartilage weares and teares gradually, the maximum position of pressure at first cartilage disappears, the sclerosis of subchondral bone matter, the edge, joint increases, and forms bony spur.Early stage cartilage flavescence, coarse, tarnish, crack, softening or strip off takes place in the cartilage deep layer that continues, sclerotin exposes.Cartilage surrounding tissue hypertrophy forms hyperosteogeny, promptly general so-called bony spur later on.Sometimes hyperosteogeny may be very big, so that influence the activity in joint.Meeting with stresses and the maximum central part that weares and teares, subchondral bone matter dense sclerosis, and the peripheral position minimum that meets with stresses, subchondral bone generation atrophy shows as osteoporosis.In the heavy burden joint, the blister cavities pathological changes can occur as hip joint, and communicate with the joint, contain synovial fluid.In joint capsule and synovial membrane, there is no the constitutional pathological changes; but owing to strain repeatedly to these tissues; impel it slightly to thicken and fibrosis; joint motion is restricted owing to muscle around the joint produces the protectiveness spasm because of pain; the musculus flexor contracture makes the joint that deformity progressively take place, and this will increase the degeneration at local compression position; the fibrous ankylosis in joint consequently, but rare bony ankylosis.The clinical manifestation of osteoarthritis mainly is an arthralgia and movable dumb, morbidity slowly, patient often feels pain when static, promptly the joint is in certain position and crosses for a long time or early morning, patient feels arthralgia, movable a little back pain can be alleviated.If but hyperactivity produces pain again, the pain increased when it was overcast and rainy.
The medicine of osteoarthritis is divided into the two class medicines that improve symptom and change the state of an illness.Common drug comprises following several: (1) anti-inflammatory analgesic medicine: as acetyl aminophenol; (2) nonsteroidal antiinflammatory drug (NSAIDs): having antiinflammatory, pain relieving and refrigeration function, is the most frequently used medicine of treatment osteoarthritis.As aspirin, salicylic acid, Phenylbutazone, indomethacin and naproxen, diclofenac, meloxicam, nabumetone, etodolac, sulindac and acemetacin etc.; (3) opiates: be last selection as treatment; (4) glucosamine: be also referred to as Chondroprotective agents, can stop the development of knee joint osseous arthritis; (5) diacerein: can suppress MMP activities and stablize lysosome membrane and bring into play antiinflammatory and reach protective effect, thereby improve the course of disease of osteoarthritis to articular cartilage; (6) joint cavity injection medicine: a kind of is hormone medicine, can temporarily ease the pain.Another kind of is transparent hyaluronic acid preparation.
The Chinese patent medicine kind that is specifically designed to the treatment osteoarthritis of present domestic listing is few, and usually all is to derive from treatment lumbago and skelalgia class medicine.Can be divided into external and two big classes for oral administration, external comprises: agent of wine spit of fland such as rheumatalgia medicated wine, shiguogong wine, bone strengthening medicated wine or the like; Unguentum such as SHANGSHI ZHITONG GAO, DONGFANG HUOXUE GAO, sky and ZHUIFENG GAO, Daphne giraldii Nitsche pain-relieving plaster for arthritis or the like, this class medicine for external use mainly plays reducing swelling and alleviating pain, the effect of respite local symptom; Oral medicine principal item has: ZHUANGGU GUANJIE WAN, XIANLING GUBAO JIAONANG, WANGBI CHONGJI, Biqi capsules or the like, this class medicine mainly by the kidney invigorating, invigorate blood circulation, collateral dredging, circulation of qi promoting, make that local QI and blood passages through which vital energy circulates operation is unimpeded, reach the analgesic effect.From modern pharmacological research, the report that at present relevant these oral medicine are repaired articular cartilage damage is actually rare, and from the strict sense, these medicines still can not make the course of disease of osteoarthritis reverse or stop.
We further investigate Folium Cajani, on the osteoarthritis animal model of rabbit, find that this medicine can improve the cartilage moisture content, improve the cartilage glucuronic acid content, improve acid phosphatase, reduce alkali phosphatase, prevent that chondrocyte from destroying, stop the degraded of chondrocyte substrate, promote the synthetic of chondrocyte substrate, promote the reparation of articular cartilage, eliminate the congested swelling of synovial membrane, promote the absorption of arthritis, the osteoarthritis animal model of rabbit is had therapeutical effect preferably.In addition, adopt the chondrocyte In vitro culture, be used for observing influence chondrocyte proliferation and protein synthesis by Folium Cajani extract pastille serum.The result confirms: the pastille serum of Folium Cajani extract can (1) promotes the propagation of chondrocyte; (2) promote the synthetic of interior albumen of chondrocyte and total protein of cell.Thus, finished the Folium Cajani extract in the invention that is used for the treatment of the osteoarthritis purposes.
Summary of the invention
The purpose of this invention is to provide a kind of new purposes of Folium Cajani, promptly Folium Cajani is used to prepare the purposes of the medicine for the treatment of osteoarthritis;
Another object of the present invention provides the purposes that the Folium Cajani extract is used to prepare the medicine for the treatment of osteoarthritis.
Purpose of the present invention realizes by following embodiment.
Folium Cajani of the present invention is meant Folium Cajani dried leaves and twig thereof.Purpose of the present invention can be used for the treatment of osteoarthritis by Folium Cajani is used realization through traditional decoction or pulverizing tabletting.What purpose of the present invention was better is to realize by preparation Folium Cajani extract.
The Folium Cajani extract can prepare by following method:
(1) Folium Cajani water extract
Get Folium Cajani, 10 times of water loggings that add medical material weight were steeped about 1 hour, and heating decocts, and extracts three times, each 1.5 hours, filter, merging filtrate, being evaporated to relative density is 1.05-1.10, filter, the filtrate drying, the dry extract that obtains is " Folium Cajani water extract ".
(2) Folium Cajani alcohol extract
Get Folium Cajani, with the 20%-80% of 4 times of weight (v/v: volume ratio) alcohol reflux, extract each 1 hour 3 times, filter, merging filtrate is behind the decompression filtrate recycling ethanol, regulating relative density is 1.10, drying, and the dry powder that obtains is " Folium Cajani alcohol extract ".
Particularly, Folium Cajani water extract of the present invention and ethanol extract can prepare by following method.
(1) Folium Cajani water extract
Get Folium Cajani, clean the removal of impurity, put in the multi-function extractor, 10 times of water loggings that add the medical material amount were steeped about 1 hour, heating decocts, and extracts each 1.5 hours three times, filter (with 80 order nylon cloth), merging filtrate is put in the vacuum concentrator (vacuum is-0.08Mpa 60 ℃ of temperature), being concentrated into relative density is that 1.05-1.10 (60 ℃ of heat are surveyed) filters (with 80 order nylon cloth), filtrate adopts continuous centrifugal to filter (rotating speed 15000r.p.m, flow are 50L/ hour), the spray-dried device of filtrate (air inlet 180 ℃ * 4 seconds, air outlet is 80 ℃) drying, the dry extract of acquisition is " Folium Cajani water extract ".
(2) Folium Cajani alcohol extract
Get Folium Cajani, clean the removal of impurity, use the Different concentrations of alcohol (20%, 40%, 60%, 80%) of 4 times of amounts to reflux respectively, extract 3 times, each 1 hour, filter merging filtrate, (80 ℃ of decompression filtrate recycling ethanols, 0.08MPa) after, regulating relative density is 1.10, carries out spray drying, obtains dry powder and is " Folium Cajani alcohol extract ".
The present invention studies have shown that: adopt the chondrocyte extracorporeal culturing method, be used for observing the influence of this medicine to chondrocyte proliferation and protein synthesis by pastille serum.Evidence: the pastille serum of Folium Cajani water extract and ethanol extract all can (1) promotes the propagation of chondrocyte; (2) promote the synthetic of interior albumen of chondrocyte and total protein of cell.In addition, the Folium Cajani water extract is used for the treatment of Os Leporis seu Oryctolagi arthritis animal model, has excellent curative.We have carried out the main pharmacodynamics research of Folium Cajani treatment osteoarthritis, set up the animal model that papain causes osteoarthritis, and establish Folium Cajani water extraction object height, in, low dose group, contrast medicine (ZHUANGGU GUANJIE WAN) group, model group and normal control group, check substantially after 4 weeks through Drug therapy, the cartilage pathological section, cartilage biochemical measurement (moisture content, alduronic acid, hydroxyproline) and blood biochemical measure (acid phosphatase, alkali phosphatase, calcium, phosphorus), the result shows: Folium Cajani water extract group can be improved the cartilage moisture content, improve the cartilage glucuronic acid content, improve acid phosphatase, reduce alkali phosphatase, prevent the destruction of chondrocyte, stop the degraded of chondrocyte substrate, promote the synthetic of chondrocyte substrate, promote the reparation of articular cartilage, eliminate the congested swelling of synovial membrane, promote the absorption of arthritis, Os Leporis seu Oryctolagi arthritis animal model is had therapeutical effect preferably.By main pharmacodynamics research, proved that the Folium Cajani water extract can promote the repair of cartilage of the osteoarthritis animal model of rabbit, the propagation of promotion cartilage.Therefore can be used for treating the preparation of medicine for treating arthritis.
The Folium Cajani extract of invention can use separately also can with the other medicines compatibility, be made into pharmaceutical composition, be used for the treatment of osteoarthritis.This pharmaceutical composition can be forms such as tablet, capsule, granule, drop pill, injection, sustained-release preparation.
The specific embodiment
Further specify the present invention below by embodiment.It should be understood that embodiments of the invention are to be used to illustrate the present invention rather than limitation of the present invention.Essence according to the present invention all belongs to the scope of protection of present invention to the simple modifications that the present invention carries out.
Embodiment 1: the preparation of Folium Cajani water extract of the present invention
The preparation of Folium Cajani water extract: get Folium Cajani, clean the removal of impurity, put in the multi-function extractor, 10 times of water loggings that add the medical material amount were steeped about 1 hour, heating decocts, and extracts each 1.5 hours three times, filter (with 80 order nylon cloth), merging filtrate is put in the vacuum concentrator (vacuum is about 0.08Mpa, 60 ℃ of temperature), being concentrated into relative density is 1.05-1.10 (60 ℃ of heat are surveyed), filter (with 80 order nylon cloth), filtrate adopts continuous centrifugal to filter (rotating speed 15000r.p.m, flow are 50L/ hour), the spray-dried device of filtrate (air inlet 180 ℃ * 4 seconds, air outlet is 80 ℃) drying for standby.
Embodiment 2: the preparation of Folium Cajani alcohol extract of the present invention
The preparation of Folium Cajani alcohol extract: get Folium Cajani, clean the removal of impurity, use the Different concentrations of alcohol (20%, 40%, 60%, 80%) of 4 times of amounts to reflux respectively, extract 3 times, each 1 hour, filter merging filtrate, (80 ℃ of decompression filtrate recycling ethanols, 0.08MPa) after, regulating relative density is 1.10, carries out spray drying, standby.
Embodiment 3: Folium Cajani water extract of the present invention is to the therapeutical effect of Os Leporis seu Oryctolagi arthritis animal model
1. material
1.1 medicine
The Folium Cajani water extract: the method according to the embodiment of the invention 1 prepares;
ZHUANGGU GUANJIE WAN is produced by Sanjiu Pharmaceutical Co., Ltd, lot number 0404281, specification 60g/ bottle;
Papain (Sigma) is given birth to worker's biological engineering company limited by Shanghai and is provided;
Glucuronic acid (Sigma), hydroxyproline (Fluka) are provided by Beijing Shu Baier company; All the other reagent are the above product of AR level.
1.2 animal
New zealand white rabbit, regular grade, 10～12 monthly ages, body weight 2.0～2.5kg, male and female half and half are provided by Guangzhou TCM Universities Experimental Animal Center, laboratory animal certification of fitness SCXK (Guangdong) 2003-0001.Animal feed Traditional Chinese Medicine University Of Guangzhou Experimental Animal Center provides standard feed.
1.3 instrument
The UV-754 spectrophotometer, Traditional Chinese Medicine University Of Guangzhou's internal medicine laboratory provides; Electronics full automatic weighing instrument, Traditional Chinese Medicine University Of Guangzhou's orthopaedics laboratory provides; Hitachi's 7060 automatic clinical chemistry analyzers, HIT produces, and Traditional Chinese Medicine University Of Guangzhou's Experimental Animal Center provides; Leica SM2000R type horizontal sliding microtome, German Lycra Instr Ltd. produces, and the Traditional Chinese Medicine University Of Guangzhou Technology Park provides.
2. method
2.1 set up the animal model that papain causes osteoarthritis:
60 of healthy new zealand rabbits, left knee joint peripheral is shaved hair, and alcohol disinfecting carries out the auricular vein injecting anesthetic with the pentobarbital sodium of 30mg/kg.Anesthesia, is injected respectively at the 1st, 4,7 day every 3 days 1 time after knee joint center inserting needle is injected 4% papain distilled water 0.3ml by the kneecap arnold ligament and gone into articular cavity, finishes modeling after the week.
2.2 Drug therapy
After the modeling, carry out corresponding Drug therapy, the high, medium and low dosage group of Folium Cajani water extract is established in this test altogether, the ZHUANGGU GUANJIE WAN group, and model group and normal group see the following form 1.
Each drug dose of table 1 is provided with
| Each dosage group | Dosage | Be equivalent to clinical consumption by weighing machine | By the suitable human body consumption of body surface area | The administration volume |
| Dosage Folium Cajani water extract low dosage ZHUANGGU GUANJIE WAN group model group normal control group in the Folium Cajani water extract high dose Folium Cajani water extract | 0.8g/kg/ days 0.8g/kg/ days distilled water distilled water of it 0.4g/kg/ days 0.2g/kg/ | 8 times 4 times 2 times 4 times-- | 2.8 doubly 1.4 times 0.7 times 1.4 times-- | 10ml 10ml 10ml 10ml 10ml 10ml |
Be provided with by last table various dose, with the drug suspension of distilled water preparation respective concentration, by gastric infusion, every rabbit administration volume is 10ml, and normal group and model group give the distilled water of equivalent respectively, once a day, and 4 weeks of continuous irrigation stomach.
2.3 curative effect detects
The quiet air injection of the whole ear edge in 4 week of administration back is put to death animal, does following inspection respectively.
(1) checks substantially
Dialysis left side knee joint, naked eyes carry out gross examination of skeletal muscle to cartilage, synovial membrane.
(2) articular cartilage check pathological section
With freezing distilled water flushing articular cavity, cut medial tibial platform centre strip cylindrarthrosis face (comprising subchondral bone) and inboard and do the pathology section, articular cartilage specimen 10% formaldehyde fixed is used om observation after paraffin embedding, section, the HE dyeing after the decalcification.
(3) mensuration of articular cartilage biochemical indicator
Measurement of water-content coefficient: after getting articular cartilage, blot cartilage surface moisture with filter paper, accurately take by weighing weight in wet base and be placed in the acetone dehydration 24 hours, vacuum drying 1 thoughtful permanent must the weight at room temperature accurately takes by weighing dry weight then, calculates moisture content.Moisture content=(weight in wet base-dry weight)/weight in wet base * 100%
The mensuration of alduronic acid: accurately take by weighing cartilage samples, put in the 5ml ampoule, add 1ml2N HCL, 100 ℃ of hydrolysis 2 hours.Pour out alduronic acid is measured in cooling back by the kappa azoles method of Bitter etc. content.
Determination of Hydroxyproline: dried cartilage, every duplicate samples takes by weighing 30mg, puts in the 10ml test tube with cover, add 0.3mol/L trichloroacetic acid solution 3ml, put 90 ℃ of calorstats interior 3 hours, cooling back centrifugal (3500r/min) 5 minutes, supernatant is put in the 20ml clean tube; Add 0.3mol/L trichloroacetic acid solution 2ml in the precipitation again, extract again 1 time, merge supernatant according to last method.The test tube that fills extracting solution is put 120 ℃ of evaporates to dryness in the baking oven.Add 6mol/L HCL solution 2ml in the test tube behind evaporate to dryness, dissolve whole residues after, move into the clean peace bottle of 5ml, sealing, 120 ℃ of hydrolysis 10 hours.Get hydrolyzed solution 20 μ l, adding distil water 6000 μ l are made into testing sample after the vibration.Get testing sample 1ml and add 0.05mol/L CuSO
4And each 0.50ML of 10%NaOH solution, add 6%H again
2O
2Solution 0.50ml puts 30 ℃ of water-baths 10 minutes.Vibration is 3 times between resting period.Add dehydrated alcohol 0.50ml, placed again 20 minutes, vibrate therebetween 3 times; Add 6mol/L HCl 1ml, add 5% paradime thylaminobenzaldehyde 1ml again and shake up,, behind the natural cooling, survey the light absorption value of 540nm in 60 degree water-baths 15 minutes.Other gets 2mg hydroxyproline standard substance, is dissolved in the 50ml volumetric flask, is made into storing solution, during experiment, be diluted to variable concentrations by 0,2,4,6,8,10,12,14,16 μ g/ml, measure light absorption value synchronously, the content of hydroxyproline in the calculation sample with testing sample.
(4) blood parameters
Get 3ml blood in rabbit ear medium-sized artery, 2500 rev/mins centrifugal 10-15 minute, get determination of serum phosphatase acid serum, alkali phosphatase, calcium and phosphorus.
(5) date processing and statistical analysis
All measurement datas are all with mean ± standard deviation (x ± s) expression.Adopt the SPSS11.0 statistical package to carry out variance analysis (ANOVA).The significance test level is got α=0.05, and P＜0.05 is item for there being significant difference.
3 results
3.1 specimen is observed substantially
Normal group sees that articular cartilage surface is smooth, and synovial membrane is not seen congestion and edema, and the joint liquid measure is moderate; Model group is seen the articular cartilage flavescence, and the surface is seen the crack, and synovium of joint has congestion and edema, and the joint liquid measure increases; Folium Cajani water extract high dose group articular cartilage surface is smooth does not see the crack, and synovium of joint is not seen obvious inflammatory reaction, and the joint liquid measure is moderate; In the Folium Cajani water extract, as seen low dose group and ZHUANGGU GUANJIE WAN group have the flavescence of part cartilage, the part cartilage surface has rimala, synovial membrane is not seen obvious congestion and edema, joint fluid does not have showed increased.
3.2 articular cartilage biochemistry detection
3.2.1 the mensuration of cartilage moisture content
The Folium Cajani water extract sees Table 2 to the influence of osteoarthritis animal model cartilage moisture content.
Table 2 Folium Cajani water extract is to the influence of cartilage moisture content (x ± s)
| Group | Number of animals (only) | Dosage (g/kg) | Moisture content (%) |
| Dosage group cajan leaf water extract low dose group in the normal group model group zhuanggu guanjie pill group cajan leaf water extract high dose group cajan leaf water extract | 10 10 10 10 10 10 | Distilled water distilled water 0.8 0.8 0.4 0.2 | 60.02±3.95 ## 66.09±3.44 ** 58.59±2.94 ## 59.76±3.97 ## 57.35±3.60 ## 58.42±2.21 ## |
Annotate: compare with normal group:
*P＜0.05
*P＜0.01; Compare with model group: #P＜0.05 ##P＜0.01
The moisture content of model group is apparently higher than normal group (P＜0.01), the moisture content of high, medium and low dosage group of Folium Cajani water extract and ZHUANGGU GUANJIE WAN group does not have the significance meaning with normal group than difference, with the model group ratio obvious decline (P＜0.01) is arranged, the prompting moisture content has the trend of improvement.
3.2.2 the cartilage glucuronic acid content is measured
The Folium Cajani water extract sees Table 3 to the influence of osteoarthritis model cartilage glucuronic acid content.
Table 3 Folium Cajani water extract is to the influence of cartilage glucuronic acid content (x ± s)
| Group | Number of animals (only) | Dosage (g/kg) | Glucuronic acid content (μ g/mg) (dry weight) |
| Dosage group Folium Cajani water extract low dose group in the normal group model group ZHUANGGU GUANJIE WAN group Folium Cajani water extract high dose group Folium Cajani water extract | 10 10 10 10 10 10 | Distilled water distilled water 0.8 0.8 0.4 0.2 | 77.23±2.73 ## 60.06±2.58 ** 67.13±3.51 **## 70.83±2.29 **## 64.84±2.99 **## 64.84±2.99 **## |
Annotate: compare with normal group:
*P＜0.05
*P＜0.01; Compare with model group: #P＜0.05 ##P＜0.01
Model group and each administration group glucuronic acid content are compared with normal group, and content obviously descends, and difference has significance meaning (P＜0.01); High, medium and low dosage group of Folium Cajani water extract and ZHUANGGU GUANJIE WAN group are compared with model group, and glucuronic acid content raises, and difference has significance meaning (p＜0.01).Illustrate that the Folium Cajani water extract can effectively improve osteoarthritis animal model cartilage glucuronic acid content, pointing out this medicine is by improving the content of cartilage matrix protein polysaccharide, thereby promotes the synthetic of cartilage matrix.
3.2.3 cartilage hydroxyproline content
The Folium Cajani water extract sees Table 4 to the influence of osteoarthritis animal model cartilage hydroxyproline content.
Table 4 Folium Cajani water extract is to the influence of cartilage hydroxyproline content (x ± s)
| Group | Number of animals (only) | Hydroxyproline content (μ g/mg) (dry weight) |
| Dosage group Folium Cajani water extract low dose group in the normal group model group ZHUANGGU GUANJIE WAN group Folium Cajani water extract high dose group Folium Cajani water extract | 10 10 10 10 10 10 | 81.62±2.23 79.96±1.54 80.04±1.59 80.86±2.04 81.64±2.49 80.04±1.59 |
Annotate: no significance difference (p＞0.05) on the statistics between each group
Cartilage hydroxyproline content difference does not have significance meaning (P＞0.05) between the high, medium and low dosage group of Folium Cajani water extract, ZHUANGGU GUANJIE WAN group, model group and the normal group, illustrate that the Folium Cajani water extract does not have influence to osteoarthritis animal model cartilage hydroxyproline content, point out this medicine the obviously effect of the synthetic nothing of the collagen of articular cartilage.
3.3 blood biochemical analysis
Table 5 Folium Cajani water extract is to the influence of serum acid, alkali phosphatase, calcium, phosphorus content (x ± s)
| Group | Animal (only) | Acid phosphatase (u/L) | Alkali phosphatase (u/L) | Calcium (mmol/L) | Phosphorus (mmol/L) |
| Dosage Folium Cajani water extract low dosage in the normal group model group ZHUANGGU GUANJIE WAN group Folium Cajani water extract high dose Folium Cajani water extract | 10 10 10 10 10 10 | 13.23±2.40 ## 7.65±1.76 ** 12.55±1.70 ## 13.90±2.25 ## 13.70±2.29 ## 12.31±1.66 ## | 113.36±3.98 ## 144.96±15.43 ** 114.17±3.05 ## 113.65±5.09 ## 114.80±3.30 ## 116.70±5.01 ## | 2.52±0.14 2.54±0.32 2.44±0.31 2.31±0.31 2.44±0.23 2.48±0.36 | 1.93±0.18 1.93±0.21 1.86±0.22 1.85±0.19 1.87±0.17 1.84±0.25 |
Notes compare with normal group:
*P＜0.05
*P＜0.01; Compare with model group: #P＜0.05 ##P＜0.01
Model group and normal group compare, and phosphatase acid serum obviously descends, and difference has significance meaning (P＜0.01); The high, medium and low dosage group of Folium Cajani water extract has been compared obvious rising with ZHUANGGU GUANJIE WAN group phosphatase acid serum with model group, difference has significance meaning (P＜0.01).
Model group and normal group compare, and serum alkaline phosphatase obviously raises, and difference has significance meaning (P＜0.01); The high, medium and low dosage group of Folium Cajani water extract has been compared obvious decline with ZHUANGGU GUANJIE WAN group serum alkaline phosphatase with model group, difference has significance meaning (P＜0.01).
No significant difference between each group of the content of serum calcium, phosphorus.
Show that the Folium Cajani water extract can effectively improve the activity of osteoarthritis animal model phosphatase acid serum and serum alkaline phosphatase.But serum calcium, phosphorus content there is not influence.
3.4 pathological examination
Normal group: cartilage surface is smooth, from the superficial to the deep can be divided into the table shallow-layer, divide a word with a hyphen at the end of a line layer, emitting layer and calcification layer.The table shallow-layer is thinner, and chondrocyte is flat, and its major axis is parallel with articular surface; Layer chondrocyte of dividing a word with a hyphen at the end of a line is less, and nuclear is not obviously seen chondrocyte bunch; The stringer of emitting layer chondrocyte is arranged in column; It for the calcification layer, links to each other with subchondral bone down, and the tide mark line is complete.
Model group: cartilage surface is unsmooth, many cracks occur, and the part cartilage strips off, form defective region, cells of superficial layer disappears, and the confluent monolayer cells of dividing a word with a hyphen at the end of a line is most of to disappear, have individual cells to have visible chondrocyte bunch, the emitting layer cell is columnar arrangement, and cell cluster collection phenomenon is arranged; Tide mark is imperfect.
The ZHUANGGU GUANJIE WAN group: cartilage surface has the shallow crack of table, and table shallow-layer cell partly disappears; The layer of dividing a word with a hyphen at the end of a line sees that chondrocyte bunch is arranged; The emitting layer cell is arranged and is column, and the cell cluster collection is arranged, and tide mark is complete.
Folium Cajani water extract low dose group: cartilage surface has the shallow crack of table, and table shallow-layer cell partly disappears; The layer of dividing a word with a hyphen at the end of a line is seen chondrocyte bunch; The emitting layer cell is arranged and is column, and cell cluster is arranged, and tide mark is imperfect.
Dosage group in the Folium Cajani water extract: cartilage surface has the shallow crack of table, and table shallow-layer cell partly disappears; The layer of dividing a word with a hyphen at the end of a line is seen chondrocyte bunch; The emitting layer cell is arranged and is column, the cell marshalling, and tide mark is complete.
Folium Cajani water extract high dose group: cartilage surface is smooth, and table shallow-layer cell partly disappears; The layer of dividing a word with a hyphen at the end of a line sees that chondrocyte bunch is arranged; The emitting layer cell is arranged and is column, marshalling, and tide mark is complete.
The destruction that pathological section prompting Folium Cajani water extract can effectively prevent articular cartilage promotes the reparation of articular cartilage, suppresses the absorption of arthritis, promotion inflammation.The visible articular surface of high dose group is smooth, the chondrocyte marshalling, and tide mark is complete, and articular cartilage is broken less, near normal group, obviously is better than model group; In, as seen low dose group and ZHUANGGU GUANJIE WAN group still have the destruction of part articular cartilage, still has tangible improvement but compare with model group.
This experimentation shows that the Folium Cajani water extract can prevent the destruction of chondrocyte, stop the degraded of chondrocyte substrate, promote the synthetic of chondrocyte substrate, obviously improve the content of cartilage proteoglycan and water, promote the reparation of articular cartilage, eliminate the congested swelling of synovial membrane, promote the absorption of arthritis, significantly improve phosphatase acid serum and alkaline phosphatase activity.But the content to calcium, phosphorus in chondrigen and the serum does not have tangible influence.
Embodiment 4: Folium Cajani water extract of the present invention and ethanol extract are to the effect of In vitro culture chondrocyte proliferation and protein synthesis
1 material
1.1 medicine is with " embodiment 3 ", in addition, the Folium Cajani alcohol extract prepares according to the method for embodiment 2.
1.2 animal is with " embodiment 3 ".
1.3 main agents and instrument
Cultivation is with liquid and main agents: (every liter of distilled water contains NaCl8g to the D-Hanks balance liquid, KCl0.4g, Na
2HPO
412H
2O1.34g, KH
2PO
40.06g, NaHCO
30.35g, 5 of 6% phenol red liquid, the malleation filtration sterilization divides the bottle encapsulation, puts-4 ℃ of refrigerators and preserves standby); (every liter of distilled water contains NaCl8g to PBS liquid, KCl0.2g, Na
2HPO
412H
2O3.14g, KH
2PO
40.2g, autoclave sterilization); Hyclone (purchase in Yongxing company, Hangzhou Ilex purpurea Hassk.[I.chinensis Sims company limited is made, and uses preceding with 56 ℃ of water-baths 30 minutes); The DMEM culture fluid (produced by GIBCO company, and HEPES is produced by SIGMA company, 13.4gDMEM dry powder, NaHCO by DMEM dry powder
33.7g HEPES2.38g, Penicillin0.0625g, Streptomycin0.1g are dissolved in the 900ml distilled water altogether, the malleation filtration sterilization, and it is standby to divide bottle seal apparatus-4 ℃ refrigerator to preserve, and adds 10% hyclone before the use, adjusting pH value is 7.4); Trypsin is produced by SIGMA company, is mixed with 0.25% concentration before using with PBS liquid, the filtration sterilization packing, and-20 ℃ of refrigerator cold-storages are standby; Collagenase II is produced by SIGMA company, is mixed with 0.2% concentration before using with PBS liquid, the filtration sterilization packing, and-20 ℃ of refrigerator cold-storages are standby; 10% neutral formalin buffer (contain 0.4% cetyl trimethyl ammonia bromide, adjusting pH value with NaOH is 7.0); 4% trypan blue mother solution (takes by weighing the 0.4g trypan blue, adds a small amount of distilled water and grind, add distilled water to 10ml, use filter paper filtering, 4 ℃ of preservations.During use, be diluted to 0.4%) with PBS; 0.04% Toluidine blue staining liquid (is the preparations of 4.0 acetate buffer solutions with the 0.01M pH value); Coomassie brilliant blue liquid (100mg Coomassie brilliant blue G-250 is dissolved in the ethanol of 50ml 95%, adds the phosphoric acid of 100ml 85%, replenishes distilled water to 1000m1); Bovine serum albumin (Wei Jia company).
Key instrument and material: incubator (U.S. SL), superclean bench (Shanghai cleaning equipment factory), inverted microscope (Leica), constant temperature water bath agitator (Jintan City, Jiangsu Province Medical Instruments factory), constant temperature blender with magnetic force (Hangzhou motor for instrument factory), high-speed refrigerated centrifuge (Beijing Medical Centrifugal Machine Factory),, microplate reader (Barad3300), ophthalmic operating set, culture dish, culture bottle, culture plate (Denmark Nunc), enumerator, No. 18 and No. 20 remaining needles.
2 methods
2.1 the preparation of pastille serum
With Folium Cajani water carry, the ethanol extract dose,equivalent (g/kg/ days) of body weight for humans (60kg) conversion animal proportionately, give 1 times of animal, 5 times and 10 multiple doses respectively, be made into 10ml/ solution with distilled water and irritate stomach, normal rabbit serum is with 10ml/ filling of distilled water stomach.Double filling stomach, midfeather 4 hours, irritate stomach ventral aorta blood sampling under the pentobarbital sodium intraperitoneal anesthesia after 1 hour in last, 4 ℃ of refrigerator overnight, centrifugal (2500rpm) 25min extracts serum (hemolytic rejecting is arranged), 56 ℃, 30min deactivation, through 0.22 μ m filter membrane sucking filtration degerming, packing ,-20 ℃ of preservations are standby.
2.2 preparation growth curve
With chondrocyte with every hole 2 * 10
4Individual cell/cm
2Inoculate 5 96 orifice plates, 10% rabbit anteserum is cultivated, and observes continuously 11 days, measures cell proliferation at 5 time points (being respectively 3,5,7,9,11 days) with mtt assay, draws growth curve, determines point of observation.
2.3 pastille serum influences external chondrocyte proliferation
Passage cell is seeded in 96 orifice plates, and inoculum density is 2 * 10
4Individual cell/cm
2Cultivate with 5%, 10%, 15% normal rabbit serum, Folium Cajani extract serum, ZHUANGGU GUANJIE WAN serum and hyclone respectively, change liquid every other day, inhaled the MTT20 μ l/ hole remove to add behind the culture fluid pure DMEM of 100 μ l and 5mg/ml on the 5th day, suction went liquid to add 150 μ l dimethyl sulfoxides (DMSO) after suction went liquid to add PBS100 μ l rinsing after 4 hours in the placement incubator, survey the OD value behind the 10min on microplate reader, wavelength is 490nm.
With the different influences of irritating stomach amount pastille serum of method contrast to chondrocyte proliferation.
2.4 pastille serum is to the influence of external chondrocyte protein synthesis
Get and pass monobasic chondrocyte, be diluted to 2 * 10 with DMEM culture fluid (containing 10% hyclone)
5/ ml, every hole is 1 milliliter in 24 well culture plates, cultivate 24 hour cells adherent after, suction removes supernatant to be replaced by respectively to contain the DMEM culture fluid of blank rabbit anteserum, Folium Cajani water extract pastille serum, Folium Cajani ethanol extract pastille serum, cultivates and adopts the Coomassie brilliant blue method to measure intracellular protein and total protein after five days.
Protein determination method: trypsin is added respectively in the 24 well culture plate holes (measure that intracellular protein then incline supernatant operate), collect each porocyte in corresponding test tube, respectively add 0.3mol/L NaOH0.5ml, put 100 ℃ of water-baths 30 minutes, make lysis.The Coomassie brilliant blue liquid of getting cell pyrolysis liquid 100 μ I and 1ml mixes, place and after 10 minutes liquid is placed 96 orifice plates, on the enzyme-linked immunosorbent assay instrument with bovine serum albumin (1-50 μ g) as standard substance, the 595nm wavelength is surveyed the A value, extrapolates protein content in the sample according to standard curve.
2.5 date processing and statistical analysis
All measurement datas are all with mean ± standard deviation (x ± s) expression.Adopt the SPSS11.0 statistical package to carry out variance analysis (ANOVA).The significance test level is got α=0.05, and P＜0.05 is item for there being significant difference.
3 results
3.1 growth curve
Chondrocyte is at In vitro culture, 3～5 days cell fast breedings, and cell proliferation peaked in 5～7 days, and is mild gradually later on, slightly has a declining tendency; Cell proliferation was active in 3～5 days, doubled 3.5 times altogether, later cell multiplication ability drop.Therefore point of observation determines at the 5th～7 day relatively to be fit to (the chondrocyte point of observation of this experiment employing is the 5th day), sees accompanying drawing 1 (water extract).
Accompanying drawing 1: In vitro culture chondrocyte growth curve: abscissa: day, vertical coordinate: OD value.
3.2 pastille serum influences external chondrocyte proliferation
3.2.1 variable concentrations pastille serum is to the influence of chondrocyte proliferation
By of the trial test of variable concentrations pastille serum, seek more satisfactory, a rational serum-concentration as efficacy of medicine observing concentration, to get rid of in the serum other cytokine as far as possible to the interference of the test of pesticide effectiveness to chondrocyte proliferation.Result such as following table 6.
Table 6 variable concentrations pastille serum is to the influence of chondrocyte proliferation (x ± s)
| Group | Sample number (n) | The OD value | ||
| 5% serum | 10% serum | 15% serum | ||
| 1 times of amount of rabbit anteserum hyclone Folium Cajani ethanol extract 1 times of amount Folium Cajani extract | 16 16 16 16 | 0.381±0.025 0.382±0.035 0.482±0.044 ** 0.663±0.036 ** | 0.464±0.031 0.498±0.033 0.623±0.050 ** 0.836±0.122 ** | 0.569±0.034 0.516±0.026 0.636±0.028 * 0.774±0.085 ** |
Annotate: each group is compared with the rabbit anteserum group:
*P＜0.05;
*P＜0.01.
Folium Cajani water extract group and ethanol extract group all are better than blank rabbit anteserum group and hyclone group as can be seen from Table 6, and be more obvious with Folium Cajani water extract group especially; And zero difference between rabbit anteserum and hyclone group.When 5%, 10% serum-concentration, pastille serum all shows the effect that promotes chondrocyte proliferation; And 15% high concentration serum group does not show the effect that promotes chondrocyte proliferation significantly, and this may be the serum proper constituent too high levels such as cytokine in the high concentration serum, and the phase mutual interference influences or covered due to the drug action.
From above trial test as can be known: (1) rabbit anteserum can substitute hyclone fully and carry out the external test of pesticide effectiveness; (2) serum-concentration of Cai Yonging is comparatively reasonable with 10%.
3.2.2 different influences of irritating 10% serum of stomach amount to chondrocyte proliferation
Table 7 is different irritates 10% serum of stomach amount to the influence of chondrocyte proliferation (x ± s)
| Group | Sample number (n) | The OD value | ||
| 1 times of equivalent | 5 times of equivalents | 10 times of equivalents | ||
| Rabbit anteserum Folium Cajani ethanol extract Folium Cajani water extract | 16 16 16 | 0.464±0.031 0.623±0.050 ** 0.836±0.122 **## | 0.464±0.031 0.685±0.017 ** 0.871±0.138 **## | 0.464±0.031 0.799±0.087 ** 0.966±0.082 **## |
Annotate: compare with the rabbit anteserum group:
*P＜0.01; Compare with the ethanol extract group: ##P＜0.01.
As can be seen from Table 7:
(1) 10% serum of the different filling with ethanol extract of Folium Cajani water extract stomach amount all can promote the propagation of chondrocyte, compares P＜0.01 with rabbit anteserum, and difference has the significance meaning;
(2) different dose forms reveal certain dose-effect relationship;
(3) Folium Cajani water get each dosage group of thing the OD value all than ethanol extract group height (P＜0.01), difference has the significance meaning, illustrates that the Folium Cajani water extract is better than ethanol extract to the influence of chondrocyte proliferation.
3.3 pastille serum is to the synthetic influence of albumen in the external chondrocyte
10% serum that table 8 is different irritates the stomach amount is to the synthetic influence of albumen in the chondrocyte (x ± s)
| Group | Sample number (n) | Intracellular protein (μ g/100 μ l) | ||
| 1 times of equivalent | 5 times of equivalents | 10 times of equivalents | ||
| Rabbit anteserum Folium Cajani ethanol extract Folium Cajani extract | 16 16 16 | 3.640±0.567 4.832±0.436 ** 6.015±0.582 **## | 3.640±0.567 5.370±0.613 ** 6.613±0.607 **## | 3.640±0.567 5.665±0.646 ** 6.711±0.580 **## |
Annotate: compare with the rabbit anteserum group:
*P＜0.01; Compare with the ethanol extract group: ##P＜0.01.
As can be seen from Table 8:
(1) 10% serum of the different filling with ethanol extract of Folium Cajani water extract stomach amount all can promote proteic synthesizing in the chondrocyte, compares P＜0.01 with rabbit anteserum, and difference has the significance meaning;
(2) different dose forms reveal certain dose-effect relationship;
(3) the albumen synthetic quantity all is higher than ethanol extract group (P＜0.01) in each dosage group chondrocyte of Folium Cajani water extract, and difference has the significance meaning.
3.4 pastille serum is to the synthetic influence of external chondrocyte total protein
10% serum that table 9 is different irritates the stomach amount is to the synthetic influence of chondrocyte total protein (x ± s)
| Group | Sample number (n) | Total protein of cell (μ g/100 μ l) | ||
| 1 times of equivalent | 5 times of equivalents | 10 times of equivalents | ||
| Rabbit anteserum Folium Cajani ethanol extract Folium Cajani water extract | 16 16 16 | 6.119±0.829 7.809±0.731 ** 7.811±0.912 ** | 6.119±0.829 7.833±0.812 ** 9.019±0.681 **## | 6.119±0.829 8.143±0.569 ** 9.410±0.760 **## |
Annotate: compare with the rabbit anteserum group:
*P＜0.01; Compare with the ethanol extract group: ##P＜0.01.
As can be seen from Table 9:
(1) 10% serum of the different filling with ethanol extract of Folium Cajani water extract stomach amount all can promote the synthetic of chondrocyte total protein, compares P＜0.01 with rabbit anteserum, and difference has the significance meaning;
(2) different dose forms reveal certain dose-effect relationship;
(3) the chondrocyte total protein synthetic quantity of 5 times and 10 times equivalent 10% serum of Folium Cajani water extract all is higher than ethanol extract group (P＜0.01), and difference has the significance meaning.
Osteoarthritis is a kind of chronic degenerative diseases of Movable joint, is feature with the fracture of articular cartilage collagen fiber, the consume of carrying out property of proteoglycan.And the pathological changes of chondrocyte is the root that this disease produces.We use pastille serum to cultivate chondrocyte, make Chinese medicine approach in vivo effect link more, more can reflect the effect of Chinese medicine to chondrocyte.
Result of the test proves: (1) Folium Cajani water extract and ethanol extract pastille serum all can promote the propagation of In vitro culture chondrocyte; (2) no matter Folium Cajani water extract and ethanol extract pastille serum still are that total protein synthetic all has obvious facilitation to albumen in the chondrocyte.
Claims (9)
1. Folium Cajani is used to prepare the medicinal usage for the treatment of osteoarthritis.
2. according to the purposes of claim 1, use therein Folium Cajani is the Folium Cajani extract.
3. according to the purposes of claim 2, use therein Folium Cajani extract is the Folium Cajani water extract.
4. according to the purposes of claim 2, use therein Folium Cajani extract is the Folium Cajani ethanol extract.
5. according to the purposes of claim 3, use therein Folium Cajani water extract prepares by following method: get Folium Cajani, 10 times of water loggings that add medical material weight were steeped about 1 hour, and heating decocts, extract three times, each 1.5 hours, filter merging filtrate, being evaporated to relative density is 1.05-1.10, filter, the filtrate drying obtains Folium Cajani water extract dry extract.
6. according to the purposes of claim 4, use therein Folium Cajani ethanol extract prepares by following method: get Folium Cajani, 20%-80% (v/v) alcohol reflux with 4 times of weight extracts each 1 hour 3 times, filter, merging filtrate, behind the decompression filtrate recycling ethanol, regulating relative density is 1.10, drying obtains the Folium Cajani alcohol extract.
7. according to the purposes of one of claim 1-6, wherein Folium Cajani or Folium Cajani extract can use separately or use with other compatibility of drugs for the treatment of osteoarthritis.
8. according to the purposes of one of claim 1-6, wherein Folium Cajani or Folium Cajani extract are made the pharmaceutical composition use.
9. purposes according to Claim 8, wherein said pharmaceutical composition is tablet, capsule, granule, drop pill, injection or sustained-release preparation.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010094760A1 (en) * | 2009-02-23 | 2010-08-26 | Dsm Ip Assets B.V. | Cajanus extracts and glucosamine for inflammatory disorders |
| US20110180431A1 (en) * | 2010-01-28 | 2011-07-28 | George Lowe | Sparkle essence system |
| CN104605429A (en) * | 2015-01-27 | 2015-05-13 | 福建省农业科学院作物研究所 | Method for preparing pigeon pea pod health drink |
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| WO2010094760A1 (en) * | 2009-02-23 | 2010-08-26 | Dsm Ip Assets B.V. | Cajanus extracts and glucosamine for inflammatory disorders |
| US20110180431A1 (en) * | 2010-01-28 | 2011-07-28 | George Lowe | Sparkle essence system |
| US8535739B2 (en) * | 2010-01-28 | 2013-09-17 | George Lowe | Sparkle essence system |
| CN104605429A (en) * | 2015-01-27 | 2015-05-13 | 福建省农业科学院作物研究所 | Method for preparing pigeon pea pod health drink |
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