invitro anti-inflammatoryand invivo

Research ArticleInVitroAnti-Inflammatory and InVivoAntiarthritic Activities ofAqueous and Ethanolic Extracts of Dissotis thollonii Cogn(Melastomataceae) in Rats

Stephanie Flore Djuichou Nguemnang1 Eric Gonzal Tsafack1 Marius Mbiantcha 1

Ateufack Gilbert 1 Albert Donatien Atsamo 2 William Yousseu Nana1

Vanessa Matah Marthe Mba1 and Carine Flore Adjouzem1

1Laboratory of Animal Physiology and Phytopharmacology Department of Animal Biology Faculty of ScienceUniversity of Dschang PO Box 67 Dschang Cameroon2Laboratory of Animal Physiology Faculty of Science University of Yaounde I PO Box 812 Yaounde Cameroon

Correspondence should be addressed to Marius Mbiantcha mbiantchamariusyahoofr and Ateufack Gilbertateufack2000yahoofr

Received 15 July 2019 Accepted 1 November 2019 Published 15 November 2019

Academic Editor Jae Youl Cho

Copyright copy 2019 Stephanie Flore Djuichou Nguemnang et al -is is an open access article distributed under the CreativeCommons Attribution License which permits unrestricted use distribution and reproduction in any medium provided theoriginal work is properly cited

Dissotis thollonii Cogn (Melastomataceae) is a tropical plant widely used in traditional Cameroonianmedicine to relieve and treatmany pathologies It is widespread in the western region where it is used to treat typhoid fever gastrointestinal disorders andinflammatory diseases-e purpose of this study is to scientifically demonstrate the anti-inflammatory and antiarthritic propertiesof the aqueous and ethanolic extracts of the leaves ofDissotis thollonii-e anti-inflammatory properties were evaluated in vitro byinhibition tests for cyclooxygenase 5-lipoxygenase protein denaturation extracellular ROS production and cell proliferationwhile antiarthritic properties were evaluated in vivo in rats using the zymosan A-induced monoarthritis test and the CFA-inducedpolyarthritis model -is study shows that aqueous and ethanolic extracts at a concentration of 1000 μgml inhibit the activity ofcyclooxygenase (4707 and 6336) and 5-lipoxygenase (6679 and 777) and protein denaturation (4251 and 4444)Similarly both extracts inhibited extracellular ROS production (IC50 574 μgml and 296 μgml for polymorphonuclear leu-kocytes 747 μgml and 328 μg ml for peritoneal macrophages of mouse) and cell proliferation (IC50 1689 μgml and 329 μgml) At a dose of 500mgkg aqueous and ethanolic extracts significantly reduce edema induced by zymosan A (6930 and8180) and CFA (7185 and 7903) At the same dose both extracts decreased sensitivity to mechanical hyperalgesia with6900 and 7035 inhibition respectively Systemic and histological analyzes show that both extracts maintain the studiedparameters very close to normal and greatly restored the normal architecture of the joint in animals Dissotis thollonii wouldtherefore be a very promising source for the treatment of inflammatory diseases

1 Introduction

Rheumatoid arthritis (RA) is a chronic disabling andprogressive autoimmune disease in which chronic pro-liferative synovitis and synovial inflammation arde observedwith significant bone destruction and cartilage destructionresulting in significant joint damage and reduced func-tionality [1ndash3] -is pathology can evolve very quickly in anindividual and affect several parts of the body that become

inflamed or extremely painful particularly affecting the el-derly but also individuals with degenerative bone disorderor immune system dysfunction [4] -is pathology whichcan also occur as a result of the immune system attacking thesynovial membrane is accompanied by swelling stiffnesspain and a reduction or loss of joint function [4] During theestablishment and development of rheumatoid arthritismany inflammatory mediators play a key role in bone de-struction and inflammation of the synovial membrane

HindawiEvidence-Based Complementary and Alternative MedicineVolume 2019 Article ID 3612481 17 pageshttpsdoiorg10115520193612481

including tumor necrosis factor (TNF-α) interleukin-1βinterleukin-6 nitric oxide (NO) prostaglandins reactiveoxygen species (ROS) platelet-activating factor leukotri-enes enzymes (lipoxygenases cyclooxygenases (COX-1 andCOX-2) and phospholipases) [5 6]

Animal models of zymosan A-induced monoarthritis andFreundrsquos complete adjuvant-induced polyarthritis (CFA) arewidely used in research for the activity of many pharmaco-logical substances on rheumatoid arthritis In fact the in-jection of zymosan into the knee joint of the rats causeserosive synovitis with increased vascular permeability neu-trophil infiltration and formation of edemas and exudates inthe acute phase and then in the chronic phase infiltration ofmacrophages and lymphocytes pannus formation and fi-broblastic reaction characteristic of chronic rheumatoid sy-novitis [7 8] CFA is an immunogenic adjuvant consisting ofa suspension of Mycobacterium tuberculosum or Mycobac-terium butyricum killed by heat When injected at the base ofthe animalrsquos tail it causes the development of polyarthritisthat evolves in a two-phase cycle of time the first phaseappears in a few hours and disappears after 3 to 5 days andmanifests itself by an acute local inflammatory reaction andthen the second phase appears after two weeks and corre-sponds to a chronic systemic reaction [9ndash11] -is poly-arthritis is not primarily aimed at the knee joint and it canaffect the general state of the animal body it is a real systemicdisease resulting in inflammation of the distal joints of thelimbs vertebrae lesions of the genitourinary tract gastro-intestinal tract eyes nose ears skin and anorexia accom-panied by significant weight loss [9 11] In addition thepathology will persist and other symptoms will appearnamely joint deformity synovitis synovial hyperplasiacapsular fibrosis angiogenesis pannus formation cartilagedestruction bone erosion inflammation of the bone marrowresorption of bone matrix and ankylosis [12]

-e severity and persistence of rheumatoid arthritisrequire long-term management with anti-inflammatorydrugs Nevertheless these anti-inflammatory drugs have forthe most part risks of toxicity for long-term use whichseriously limits their use Current research in the manage-ment of rheumatoid arthritis is turning to a new generationof substances capable of selectively inhibiting TNF alphaandor cyclooxygenase (COX-2) and having no major sideeffects [13] Recent interest in alternative treatments forarthritis favors the use of traditional medicine althoughscientific evidence of efficacy for most cases is lackingNevertheless several herbs used in a care program and avery effective preventive medicine can act individually andor in synergy to reduce chronic joint inflammation (oste-oarthritis andor rheumatoid arthritis) [14ndash16] To reach thetotal health care coverage of the worldrsquos population tradi-tional medicine is considered by WHO to be the most ef-fective means since about 25 of modern prescription drugsare more or less obtained from plants [17 18]

Comprising about 163 genera the family of Mela-stomataceae which are mainly pantropical plants includemore than 4300 species somany of them are known for theireffectiveness in traditional medicine as antihepatitic anti-hypertensive anti-inflammatory antihyperglycaemic

antioxidant hemostatic and antidiarrheal [19ndash24] Dissotisthollonii (D thollonii) is one of many species of the family ofMelastomataceae used in traditional medicine in Cameroonto treat typhoid fever gastrointestinal disorders in-flammatory diseases and sinusitis [25ndash27] -e leaves arerecommended for the treatment of ulcers and gastrointes-tinal disorders A previous study showed that D tholloniisignificantly inhibited fluid accumulation in intestine in-duced by prostaglandin E2 [28] Based on recent work byAteufack et al [29] this plant has antidiarrheic and anti-bacterial properties and then has several secondary me-tabolites including tannins flavonoids sterolsanthraquinones phenols and polyphenols In addition thework of TadjouaTchoumbou et al [24] showed that thisplant significantly inhibited leukocyte migration in perito-neal fluid intracellular ROS production proliferation ofHela cell lines and TNF-α production Tala et al [27]showed that aqueous and ethanolic extracts were devoid oftoxicity after 28 days of daily treatment Similarly Nonoet al [30] showed the antimicrobial and antioxidantproperties of this plant Several compounds have alreadybeen isolated from this plant among which 33prime-dio-methylellagic acid 4prime-O-β-D-xylopyranoside 3-4prime-O-β-D-arabinopyranoside casuarinine betulinic acid β-sitosterol-3-O-D-glucopyranosyl-6prime-mirystate cellobiosylsterol β-si-tosterol β-sitosterol-3-O-β-D-glucopyranoside arjunolicacid 33prime-diomethylellagic acid ellagic acid and 33prime-dio-methylellagic acid 4prime-O-β-D-glucopyranoside [30] Al-though this plant is traditionally used to relieve manydisorders of the body no information or scientific report toour knowledge has been found in the literature relative to itsantiarthritic properties In our continuous search for bio-active extracts from plants used in traditional Cameroonianmedicine [31 32] and in order to support and improve thetraditional use of D thollonii we undertook to carry out thepresent study on in vitro anti-inflammatory activities and invivo antiarthritic activities of the leave extracts of Dthollonii

2 Materials and Methods

21 Plant Material and Extraction -e plant material ref-erenced to the national herbarium of Cameroon under thenumber Ndeg 133292SRF Cam in the name of D thollonii(Melastomotaceae) was used in this study -e fresh leaveswere harvested in the town of Dschang (western Cameroon)dried in the shade and then crushed into a fine powder Inorder to prepare the aqueous extract 500 g of powder wasmixed into 500ml of distilled water during 72 hours andfiltrated (Whatman paper No 4) the filtrate obtained wasevaporated at 40degC to give the aqueous extract (82 yield)-e same weight of dried powder plant was mixed into500ml of ethanol for 72 hours and then filtered -e filtratewas concentrated with a rotary evaporator set at 96degC to givethe ethanolic extract with 96 yield

22 Phytochemical Assay of D thollonii Extracts -e dif-ferent extracts were subjected to chemical screening in order

2 Evidence-Based Complementary and Alternative Medicine

to detect the presence of the main groups of compoundsfollowing the principles stated by Matos [33]

23 Triterpenes and Steroids LiebermanndashBurchard TestIn a tube containing 3ml of MeOH 01 g of extracts wasdissolved and then 02ml of each of the following reagentswas added chloroform glacial acetic anhydride and con-centrated H2SO4 -e mixture was observed to look for theappearance of the greenish-blue or purple-pink colorcharacteristics of the presence of sterols and triterpenesrespectively

24 Phenols -e tube contained 3ml of ethanol 01 g ofextracts was dissolved and then three drops of 10 iron IIIchloride were added -e solution was then observed toobserve for the appearance of the blue-violet or greenishcoloration which characterizes the presence of the phenols

25 Tannins 5ml of MeOH was introduced in the tube and01 g of extracts was dissolved -e solution was then addedwith 5 drops of 05 sulfuric acid and the mixture wasobserved to detect the green or blue-black color indicatingthe presence of tannins

26 Flavonoids Shinoda Test In a tube containing 3ml ofMeOH 01 g of extracts was dissolved and the mixture wasthen treated with 005 g of magnesium chloride chips and 3drops of concentrated H2SO4 -e flavonoids were high-lighted by the appearance of the following colorings orangefor flavones red for xanthones and pink for flavonols

27 Anthocyanings In a test tube containing 01 g of ex-tracts 5 drops of concentrated hydrochloric acid were in-troduced -e solution was then observed to look for theappearance of red color indicating the presence ofanthocyanins

28 Saponins -e presence of saponins is generally mate-rialized by the formation of a stable foam after stirring asolution -us a solution of 5ml of distilled water and 5mlof each extract was vigorously shaken to check for thepresence or absence of saponins in each extract of our plant

29 Anthraquinone -e presence of free anthraquinonesandor anthraquinone derivatives in a mixture is indicatedby a pink violet or red coloration in the lower phase(ammoniacal phase) of the mixture after stirring -us twomethods made it possible to verify the presence of this classof compound in our various extracts

(i) Stirring a solution prepared in the followingmannerextract (3ml) and benzene (3ml) followed by fil-tration and then 5ml ammonia (10) in the filtrate

(ii) A mixture of extract (3ml) and sulfuric acid (3ml) isboiled and filtered while hot and benzene (3ml) isadded to the filtrate followed by stirring After

separation of the benzene layer ammonia preparedat 10 (3ml) was added

3 In Vitro Anti-Inflammatory Assays

31 Inhibition of Protein Denaturation To evaluate the anti-inflammatory effects of the extracts the protocol describedby Padmanabhan and Jangle [34] and Elias and Rao [35] wasused with small modifications A volume of 1ml of extracts(aqueous and ethanolic) or of diclofenac sodium at differentconcentrations (100 200 500 and 1000 μgml) washomogenised with 1ml of aqueous solution of bovine serumalbumin (5) and incubated at 27degC for 15 minutes -emixture of distilled water and BSA constituted the controltube Denaturation of the proteins was caused by placing themixture in a water bath for 10 minutes at 70degC -e mixturewas cooling inside the ambient room temperature and theactivity each mixture was measured at 660 nm Each test wasdone three times -e following formula was used to cal-culated inhibition percentage

inhibition absorbance of control minus absorbance of sample

absorbance of controltimes 100

(1)

4 Assay of Cyclooxygenase and5-Lipoxygenase Inhibition

41 Lymphocyte Culture Preparation RPMI 1640 (HIME-DIA) added with inactivated fetal calf serum penicillin andstreptomycin was used for culture of humanperipheral lymphocytes and cell proliferation was inducedby phytohemagglutinin (HIMEDIA) After filtration (using02 micron cellulose acetate Sartorios) plasma was added(1times 106 cellsml) and incubated for 72 hours and the culturewas activated by the addition of lipopolysaccharide (1 μl) andincubated again for 24 hours Extracts (aqueous and etha-nolic) and ibuprofen were added at a final concentration of100 200 500 and 1000 μgml incubated for 24 hours andcentrifuged for sedimentation at 6000 rpm for 10min Afterremoval of the supernatant cell lysis buffer was added(50 μl) and the mixture was again centrifuged at 6000 rpmfor 10min -e anti-inflammatory test was performedaccording to the method used by Viji and Helen [36]

42 Assay of Cyclooxygenase A mixture of glutathione tris-HCl buffer enzyme and hemoglobin was used to make theassays After addition of arachidonic acid and TCA (10 in1N HCl 02ml) the mixture was incubated at 37degC (20minutes) -e TBA (02ml) was added to the contents whichwere then heated (for 20 minutes in boiling water) and aftercooling the mixture was centrifuged (1000 rpm 3min) andthe supernatant was used to measure COX activity at 632 nm[36]

43 Assay of 5-Lipoxygenase In 4ml of nonoxygenatedwater linoleic acid (70mg) and an equal weight of in-terpolation were dissolved and pipetted followed by sodium

Evidence-Based Complementary and Alternative Medicine 3

hydroxide (05N) and water without oxygen (25mL) wasadded -e final solution was divided into small portions of05ml each and rinsed with nitrogen and frozen A quartzcuvette (25degC) optical path of 1 cm made it possible to carryout the reaction -e ODmeasured at 234 nm was performedwith a mixture of tris buffer (275ml pH 74) sodium li-noleate (02ml) and enzyme (50ml) [36] -e followingformula was served to determine percent inhibition

inhibition abs control minus abs sample1113864 1113865

abs control1113890 1113891 times 100 (2)

44 Chemiluminescence Assay -e human blood samplesused in this work were received from a donor following theprocedure accepted by the Independent Ethics CommitteeICCBS University of Karachi No ICCBSIEC-008-BC-2015Protocol10 -e blood donors were informed that itshould be used for an experimental study

45 Isolation of Human Polymorphoneutrophils (PMNs)Aseptically a volume of 10ml of venous blood was takenfrom a very healthy 33-year-old volunteer donor and thenintroduced into a tube with anticoagulant (heparin)Neutrophils were isolated following the protocol describedby hypoflat Ficoll density gradient centrifugation [37] Forthis in an empty tube of 45ml the whole blood HBSS andLSM (lymphocyte separation medium) were introduced atequal volume and after 30min latency the supernatant ofthis mixture was then removed and introduced into a 15mltube previously containing 5ml of LSM then the tube wascentrifuged at 400 g for 20 minutes (room temperature)After removal of the supernatant 1ml of distilled water wasintroduced into the tube (for lysis of red blood cells) after aduration of 1 minute the HBSS (2x) (1ml) was also in-troduced (to stop the lysis) After adding 5ml of HBSSagain the tube was centrifuged for 10 minutes at 4degC(300 g) and 1ml of HBSS was added after removal of thesupernatant and the tube was kept in ice -e trypan bluetechnique was used to assess viability while the hemocy-tometer counted the cells For each test the cell concen-tration used was 1times 106 cellsml

46 Peritoneal Macrophage Isolation from Mice One milli-liter of FBS was injected (intraperitoneally) into NMRI miceweighing an average of 22 g and these animals were kept andobserved for 3 days then they underwent cervical dislo-cation for sacrifice -e RPMI medium (10 10ml) wasintroduced into the peritoneal cavity after 2 minutes ofmassage the skin of the abdomen was removed and theperitoneal cavity was exposed Using a syringe the RPMImedium injected into the peritoneum and containing themacrophages was removed introduced into a tubecentrifuged (400 g 20 minutes 4degC) the supernatant wasremoved 5ml incomplete RPMI was supplemented thetube was further centrifuged (300 g 10 minutes 4degC) andthen RPMIHBSS (1ml) was supplemented -e trypan bluetechnique was used to assess viability while the

hemocytometer counted the cells For each test the cellconcentration used was 1times 106 cellsml [38 39]

For the chemiluminescence assay the modified protocolof Mbiantcha et al [40] was used In white plates (96 wells)25 μl of PMNs cells whole blood or macrophages 25 μl ofextracts (aqueous or ethanolic) or ibuprofen were mixedWhile the well without extracts represent the control andreceived only cells and HBSS++ After 20 minutes of in-cubation each well obtained 25 μl of luminol and 25 μl ofPMA which made it possible to obtain a total volume of100 μl in each well After completing this test the results wereexpressed in RLU (relative light units) and the followingformula allowed to calculate the percentage of inhibition

inhibition() (RLUcontrol minus RLUsample)

RLUcontroltimes 100 (3)

47 T-Cell ProliferationAssay [41] 96-well white plates wereused for this test In each well were introduced the extracts(2 10 and 50 μgml) prednisolone (diluted in RPMI (5))50 μl of T lymphocytes at a concentration of 2times106 cellsml50 μl of PHAL at a concentration of 75 μgml (phytohe-magglutinin-L) Wells considered as negative controls re-ceived only 550 μl of cells and 150 μl of RPMI (5) whereasthose considered as positive controls received 50 μl of cells50 μl of PHA and 100 μl of RPMI (5)-e plates were thenincubated (3 days 37degC CO2 (5)) 25 μl of 05 μCiwell(methyl 7 3H) thymidine was used to pulse the culturesfollowed by a second incubation for 18 hours and then thecells were harvested (using a fiberglass filter) -e level ofthymidine integrated in the cells was determined using acounter (LS65000 liquid scintillation) -e following for-mula allowed to calculate the percentage of inhibition usingCPM (counts per minute)

inhibitory activity() (CPMcontrol minus CPMsample)

CPMcontroltimes 100

(4)

5 In Vivo Antiarthritis Assays

51 Animals Female Wistar rats (3 to 4 months old and 150to 200 g) were used for these tests -e animals were breededfrom the faculty of science (laboratory of animal physiologyand phytopharmacology) of the University of Dschang(Cameroon) -ey were raised under normal conditions(19ndash23degC 12 hour light) with water and access without diet

-e experimental procedures have been approved by thelocal Ethics Committee and are in accordance with theguidelines for the study of pain in awake animals publishedby the NIH (publication no 85-23 ldquoPrinciples of AnimalProtectionrdquo Laboratory and Study of Pain Ministry ofScientific Research and Technology which adopted theEuropean Union Guidelines on Animal Care and Experi-mentation (EWC 86609)

52 Treatment Regimen Animals were grouped by theirweight into different cages and in each group they were


identified by the tail with a number using a marker pen Ineach test 42 female rats were divided into 7 groups (6 ratseach) group 1 (healthy control) received no treatment and noinjection of zymosan A (Sigma Chemical Co St LouisMissouri USA) or CFA (Sigma Chemical Co St LouisMissouri USA) group 2 (arthritic control group) received thesolution of DMSO (5)+PBS with injection of zymosan A orCFA group 3 (positive control) received diclofenac (5mgkg)with injection of zymosan A or CFA groups 4 and 5 receivedthe aqueous extract of D thollonii (250 and 500mgkg) withinjection of zymosan A or CFA and groups 6 and 7 receivedthe ethanolic extract of D thollonii (250 and 500mgkg) withinjection of zymosan A or CFA All treatments were ad-ministered orally 1 hour before the induction of zymosan A orCFA (day 0) and then the animals were treated daily for 5days for zymosan A-induced monoarthritis and 35 days forpolyarthritis induced by the CFA

53 Monoarthritis Induced by Zymosan In this test theprotocol of Mbiantcha et al [31] has been used with somemodifications One hour after oral administration of thevarious treatments the thiopental is injected into eachanimal (01ml100 g intraperitoneal route) and then zy-mosan A (03ml 09 vv NaCl) was injected in the kneejoint using a digital caliper (Mitutoyo Japan) before the oraltreatment the thickness of the injected joint was measuredand then 1 2 3 4 5 6 24 48 72 96 and 120 h after theinjection of zymosan A On the fifth day after taking the lastparameters the animals were again anesthetized and theinjected knee joint was incised and preserved in a formalin-PBS mixture (10) -e general scheme of method in his-tology was followed for histological analysis

54 Polyarthritis Induced by CFA -e modified protocol ofMbiantcha et al [40] was used One hour after adminis-tration of each treatment the animals were anesthetized byinhalation of ether vapor and then 100 μl of CFA (10mgml)was injected into the tail vein and then the animals werereturned to well-labeled cages and observed

-e severity of arthritis was assessed by measuring thethickness of the hind leg joint using a digital caliper (days 0 13 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 and 35) thepain sensitivity threshold using an analgesimeter (days 2 4 68 10 12 14 16 18 20 22 24 26 28 30 32 and 34) and thearthritic score every day with certain criteria (score0 normal state score 1 edema on one foot or nodule score2 two feet with edema or paw and nodule tail score 3 twofeet with edema and nodosity of the tail or three feet withedema and score 4 three feet with edema and nodosity oftail or edema of four feet) and body weight every week

At day 36 the animals were anesthetized with thiopental(01ml100 g body weight) the blood was removed bycatheterization of the abdominal artery and introduced intotwo different types of tubes tubes containing EDTA asanticoagulant for analysis of hematological parameters bystandard laboratory method and tubes containing no anti-coagulant were centrifuged (4900 rpm 5 minutes) serumwas collected to evaluate ALT AST ALP MDA creatinine

total protein and stress parameters (NO MAD SODcatalase and glutathione) -e weights of various organssuch as liver kidneys thymus and spleen were weighed andknee joints of all animals were collected and stored in aformalin-PBS mixture (10) for histological analysis

55 Statistical Analysis All in vitro test data indicatemeanplusmn standard deviation in triplicate while for the in vivotest the data are presented as an average of 6 animalsplusmn SEMDifferences between groups were assessed by ANOVA (oneway and two way) followed by Bonferroni posttest Signif-icant differences were considered at plt 005 a b and c or αβ and λ denote significant differences with respect to thehealthy control group andor the arthritic control group

6 Results

61 Chemical Composition Several groups of chemicalcompounds have been demonstrated in extracts of D thol-lonii It can be seen from this table that the ethanolic extractcontains all the test compounds with the exception of an-thocyanins and triterpenes whereas the aqueous extractcontains only flavonoids phenols and polyphenols (Table 1)

7 In Vitro Anti-Inflammatory Activity

71 Inhibition of ProteinDenaturation For the results of thisstudy aqueous and ethanolic extracts effectively inhibitprotein denaturation (albumin) caused by heat Table 2shows significant inhibition (plt 0001) of 4251 and4444 respectively for the aqueous extract and the etha-nolic extract at a concentration of 1000 μgml whereasdiclofenac sodium produced 8919 inhibition

72 Cyclooxygenase Inhibitory Assay Evaluation of cyclo-oxygenase activity determined the effect of both extracts onprostaglandin production -e results show that at 1000 μgml the aqueous extract the ethanolic extract and ibuprofensignificantly (plt 0001) inhibit the activity of cyclo-oxygenase with 4707 6336 and 9788 respectively(Table 2)

73 5-Lipoxygenase Inhibitory Assay -e evaluation of theactivity of 5-lipoxygenase was used to study the effect of thevarious extracts on the production of leukotrienes-is tableshows that the aqueous and ethanolic extracts as well asibuprofen have a significant inhibitory effect (plt 0001) onthe activity of 5-lipoxygenase with 6679 inhibition7748 and 9531 respectively (Table 2)

74 Effect of D thollonii on Production of Intracellular ROSand T-Cell Proliferation Table 3 presents the results forextracellular ROS production and T-cell proliferation PMAis used to activate ROS and luminol as a developer -eethanolic extract of D thollonii significantly inhibited therelease of ROS in whole blood with an IC50 of 498 μgml Inthe presence of polymorphonuclear an inhibition was


observed with an IC50 of 5 74 μgml and an IC50 of 296 μgml with aqueous and ethanolic extracts -ese extracts againinhibited the ROS produced by the macrophages with anIC50 of 747 μgml for the aqueous extract and an IC50 of328 μgml for the ethanolic extract

With respect to T-cell proliferation both extractsshowed significant antiproliferative property Table 3 showsthat the aqueous extract has an antiproliferative activity withan IC50 of 1686 μgml while the ethanolic extract is 329 μgml and that of prednisolone is less than 310 μgml


81 Effect of D thollonii Treatment on Monoarthritis Inducedby Zymosan A in Rats After injection of zymozan A in

articulation of rats knee joint results in the significant growth(plt 0001) in the articular diameter which is maximal fromthe first hour and still from the fifth day after the adminis-tration of zymosan A (Figure 1) Ethanolic and aqueousextracts of D thollonii leaves (250 and 500mgkg) signifi-cantly (plt 005) inhibited zymosan A-induced edema -einhibition of edema caused by the injection of the zymosan Awas significant (plt 001) from the 3rd hour with a percentageinhibition of 5290 for the ethanolic extract (500mgkg) andfrom the 4th hour with the aqueous extract (3330 plt 005500mgkg) and diclofenac (3630 plt 001 5mgkg) -emaximum and significant inhibitory effect (plt 0001) wasobserved at the 5th day for the ethanolic extract (8180500mgkg) at the 5th day for the aqueous extract (6930500mgkg) and at day 3 for diclofenac (5310 5mgkg)

Table 2 Effect of aqueous and ethanolic extracts of Dissotis thollonii on protein denaturation cyclooxygenase inhibition and 5-lip-oxygenase inhibition

Compounds Dose (μgml)Inhibition ()

Protein denaturation Cyclooxygenase 5-lipoxygenase

Diclofenac

100 7441 mdash mdash200 7672 mdash mdash500 8148 mdash mdash1000 8919 mdash mdash

Ibuprofen

100 mdash 8346 8203200 mdash 8990 8863500 mdash 9339 92961000 mdash 9788 9531

Aqueous extract

100 2733 2472 3126200 2926 3414 4560500 3383 4070 58171000 4251 4707 6679

Ethanolic extract

100 3087 2814 3612200 3279 4089 4824500 3730 5355 64111000 4444 6336 7748

-e percentage values were obtained using various concentrations of test compounds and readings are presented as mean of triplicates

Table 1 Phytochemical profile of Dissotis thollonii

ExtractsPhytochemical compounds

1 2 3 4 5 6 7 8 9Aqueous extract minus minus minus + minus minus + + minus

Ethanolic extract minus + + + - + + + minus

minus absent + present 1 triterpenoids 2 sterols 3 tannins 4 flavonoids 5 anthocyanings 6 anthraquinones 7 phenolpolyphenol 8 saponins 9 cardiacglycosides

Table 3 IC50 value of aqueous and ethanolic extracts of Dissotis thollonii on human whole blood evaluated by luminal-amplifiedchemiluminescence

Oxidative burst (IC50 (μgml))T-cell proliferation (IC50 (μgml))

WB PMNs MQAqueous extract gt100 574plusmn 065 747plusmn 268 1689plusmn 255Ethanolic extract 489plusmn 049 296plusmn 012 328plusmn 009 329plusmn 196Ibuprofen 1271plusmn 013 1253plusmn 021 1328plusmn 107 mdashPrednisolone mdash mdash mdash lt310-e IC50 values were obtained using various concentrations of test compounds and readings are presented as meanplusmn SD of triplicates WB whole bloodPMNs polymorphonuclear leukocytes MQ mice peritoneal macrophages


Figure 2 shows the histological analysis of the knee jointstaken after injection of zymosan A e architecture of thejoint has a normal appearance in the nonarthritic controlgroup when in arthritic control there is an erosion of thesynovial membrane a very large joint space and erosion ofbone and articular cartilage However treatment withaqueous and ethanolic extracts of D thollonii prevented thedestruction of the articular architecture of treated animals

82 Eect ofD thollonii Treatment onPolyarthritis Induced byCFA in Rats

821 Eect of D thollonii Treatment on Joint DiameterAfter the injection of CFA into the tail the diameter of thejoint increased signicantly on the 11th day after injection(Figure 3) Aqueous and ethanolic extracts (500mgkg)bring out the signicative (plt 0001) reduction of jointdiameter from day 13 with inhibition percentages of 5434and 6548 respectively compared with the negativecontrol e inhibitory eect of dierent extracts remainedsignicant during the days of experimentation e maxi-mum signicant activity (plt 0001) is observed at day 23 fordiclofenac (6003 5mgkg) at day 23 for the aqueousextract (7185 500mgkg) and at the 31st day for theethanolic extract (7903 500mgkg)

822 Eect of D thollonii on Mechanical Nociceptive Painreshold e induction of arthritis with CFA in ratsgenerates the hypersensitivity threshold to mechanical painwhich increased to day 10 still the end of experimentation(34th day) with negative control e continuous adminis-tration of dierent treatments signicantly protected(plt 005 plt 001 and plt 0001) animals against me-chanical pain which was observed from the 10th day untilthe end of treatment e maximal inhibitory activity isobserved on the 12th day with the aqueous extract (500mgkg plt 0001 6900) and on the 34th day with the etha-nolic extract (500mgkg plt 0001 7035 ) and at day 34with diclofenac (5mgkg plt 0001 4205 ) Howeverthere is little improvement observed with the aqueous ex-tract (250mgkg) with respect to mechanical pain sensitivitythreshold (Figure 4)

823 Eect of D thollonii on Arthritic Score e arthriticscore does not materialize in normal controls but in thegroups that received CFA it is very well materialized Ar-thritic control groups are more aected from 11 until the endof treatment (Figure 5) Nevertheless animals treated withdierent extracts showed a decrease in arthritic score valuesuntil the end of treatment At the dose of 500mgkgethanolic extract has more activity than the aqueous extract

0 1 2 3 4 5 6 24 48 72 96 1205

6

7

8

9

10

αc

λ

λ

λ

βcλc

λcλbλ λbλb

λaλ

λ λ

λ λ λ

λ λ λ λ

λ λ λ

λ λb

λcλcλ λ

λ λ

λb

λ λ

λ

λ λ

λ

λ λ λ λλ λ

λ

λ

αcβcαcβc

λb

λ

λ

λ λ λbλ λ λ

λ λ

ccc

Time (hour)

Join

t dia

met

er (m

m)

AE 250mgkgAE 500mgkg

Healthy controlArthritic controlDiclofenac 5mgkg

EE 250mgkgEE 500mgkg

Figure 1 Eect of aqueous (AE) and ethanolic (EE) extracts ofDissotis thollonii on joint diameter in zymosan A-inducedmonoarthritis Valuesare expressed as meanplusmn SEM for six animals and analyses by two-way ANOVA followed by Bonferroni post hoc test αplt 005 βplt 001 andλplt 0001 when compared with the healthy control and aplt 005 bplt 001 and cplt 0001 when compared with the arthritis control


because it has not only delayed the onset of arthritis ma-terialization but has also reduced the physical value ofmaterialization However animals treated with diclofenacand ethanolic extract presented a signicant (plt 0001)reduction in the physical value of this materialization at theend of treatment

824 Eect of D thollonii on Body Weight and OrganWeight In the arthritic control group body weight decreasedprogressively and became signicant (plt 001) fromweek 2 onpositive group animals In animals treated with the ethanolicextract (500mgkg) the change in body weight was signicant(plt 005) at week 3 and persisted throughout treatmentcompared with arthritic control group animals In animalsfrom dierent groups treated with aqueous extract (500 and

250mgkg) with ethanolic extract (250mgmg) and withdiclofenac (5mgkg) the change in body weight did not occurand was not signicant (plt 005) throughout treatment(Figure 6)

Results showed that weights of the liver spleen andkidney increased signicantly (plt 001) and the weight ofthymus decreased signicantly (plt 001) in all control an-imals arthritic compared with the healthy control groupRecovery of organ weight balance was observed with con-tinuous administration of the dierent extracts in arthriticanimals compared with negative control (Figure 7)

825 Eect of D thollonii Extracts on HematologicalParameters e results of the change in hematologicalparameters are shown in Table 4 In animals in the arthritic

(a)

A

B

C

(b)

(c) (d)

λ

(e)

Figure 2 Histopathological analysis of ankle joints stained with HampE (a) Healthy control showing normal structure with small joint space(b) Arthritic control showing very large joint space (A) erosion of articular cartilage (B) and bone erosion (C) (c) diclofenac 5mgkgtreated (d) aqueous extract (AE) 500mgkg treated and (e) ethanolic extract (EE) 500mgkg treated showing a decrease in joint spaceerosion of the synovial membrane and a reduction of erosion of articular cartilage


control group platelet and WBC levels increased sig-nicantly (plt 0001) in contrast to RBC hemoglobinand of hematocrit which decreased signicantly(plt 0001) compared with the healthy control In treated

animals with dierent doses of extracts (250 and500mgkg) and diclofenac (5 mgkg) the various pa-rameters evaluated are close to those of animals in thehealthy control group

λcλcλcβcλc

λcλcβcλc

λcλc

λcλcλcλcλb

λcλaλc

λb

λaλcλcλc

λcλc

λcλc

λcλcλcλc

λc

α

λλλλ

λλλλ

λλ

λ

λ

λ

λc

αcαc

λcλcλc

λcλc

λcλc

λcλbλc

λc

βcβ βcλc

λc

βcαcβcβcλcβcαcλc

c

95

100

105

110

115

120

125

130

Join

t dia

met

er (

incr

ease

)

3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 351Period (week)




Figure 3 Eect of aqueous (AE) and ethanolic (EE) extracts of Dissotis thollonii on change in joint diameter in CFA-induced arthritis Valuesare expressed as meanplusmn SEM for six animals and analyses by two-way ANOVA followed by Bonferroni post hoc test αplt 005 βplt 001 andλplt 0001 when compared with the healthy control and aplt 005 bplt 001 and cplt 0001 when compared with the arthritic control

1 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 340

10

20

30

40

50

60

70

80

90

100

110

120

λcλc

λcλcλcλcλc

λc

λλcλc

λcλc

λc

λcλcλc

λc

λc

λcλcλcλc

λb

λc

λc

λcλcλc

λcλcλcλc

λcλc

λc

λa

λλλλλλλλ

λλλλ

λ

λcλcλcλc

λcλcλcλc

λcλc

λc

λc

λcλcλcλc

λcλcλcλc

λc

λcλc

λcλb

λcλcλc

λc

Period (week)

Sens

ibili

ty (

)




Figure 4 Eect of aqueous (AE) and methanolic (EE) extracts of Dissotis thollonii on mechanical hyperalgesia in CFA-induced arthritisValues are expressed as meanplusmn SEM for six animals and analyses by two-way ANOVA followed by Bonferroni post hoc test βplt 001 andλplt 0001 when compared with the healthy control and aplt 005 bplt 001 and cplt 0001 when compared with the arthritic control


826 Eect of D thollonii Extracts on BiochemicalParameters e biochemical parameters are shown inTable 5 Serum levels in arthritic animals show a signicant

(plt 0001) increase in AST ALT ALP and creatinine and asignicant decrease (plt 0001) of total protein relative to theserum level of the healthy control ese dierent levels

ccccc

c

c

cccc

c

cccbb

βββλ

λλ

λλλ

λλ

λ

λ

ccccc

cc

ccccc

b

ndash05

00

05

10

15

20

25

Art

hriti

s sco

re

33 3527 29 31211917 23 2513 15117 93 51Period (week)




Figure 5 Eect of aqueous (AE) and ethanolic (EE) extracts of Dissotis thollonii on the arthritis score in CFA-induced arthritis Values areexpressed as meanplusmn SEM for six animals and analyses by two-way ANOVA followed by Bonferroni post hoc test λplt 0001 when comparedwith the healthy control and bplt 001 and cplt 0001 when compared with the arthritic control

0 1 2 3 4 585

90

95

100

105

110

115

λλλ

λλ

λλ

λ

λα

λλ

λ

β

λbλaλa

λ

λλ

Period (week)

Relat

ive b

ody

wei

ght (

)




Figure 6 Eect of aqueous (AE) and ethanolic (EE) extracts of Dissotis thollonii on body weight in CFA-induced arthritis Values areexpressed as meanplusmn SEM for six animals and analyses by two-way ANOVA followed by Bonferroni post hoc test αplt 005 βplt 001 andλplt 0001 when compared with the healthy control and aplt 005 and bplt 001 when compared with the arthritic control


improved considerably in animals treated with differentextracts (aqueous and ethanolic) and diclofenac

827 Effect of D thollonii Extracts on Oxidative StressParameters -e results on the change in oxidative stressparameters shown in Table 6 indicate that levels of NO andMDA increased significantly (plt 001) in arthritic animalswhereas levels of glutathione catalase and SOD decreased

significantly (plt 001) compared with the healthy controlAnimals treated with aqueous and ethanolic extracts such asdiclofenac tend to improve these values

828 Histopathological Study A histopathological study ofthe knee joint showed no evidence of inflammation boneerosion cartilage destruction or cellular infiltration in thehealthy control Animals in the arthritic control group

Healthy controlArthritic controlDiclofenac 5 mgkgAE 250 mgkg

AE 500 mgkgEE 250 mgkgEE 500 mgkg

βbαββaαβ

ba

αβ

ba

Kidney Thymus SpleenLiver0

2

4

6

8

10

12

Org

an w

eigh

t (g)

Figure 7 Effect of aqueous (AE) and ethanolic extracts (EE) of Dissotis thollonii on organ weight in CFA-induced arthritis Values areexpressed as meanplusmn SEM for six animals and analyses by one-way ANOVA followed by Bonferroni post hoc test αplt 005 and βplt 001when compared with the healthy control and aplt 005 and bplt 001 when compared with the arthritic control

Table 4 Influence of the aqueous and ethanolic extracts of Dissotis thollonii on haematological in CFA-induced arthritis in rats

Treatment Dose (mgkg) Haemoglobin (gdl) Hematocrit () Platelet (109L) RBC (millionμl) WBC (109L)Healthy control mdash 1640plusmn 073 3878plusmn 103 36525plusmn 2623 722plusmn 034 785plusmn 044Arthritic control mdash 943plusmn 020λ 2565plusmn 127λ 55475plusmn 2154λ 390plusmn 037λ 1378plusmn 056λDiclofenac 5 1415plusmn 062b 3388plusmn 259a 48175plusmn 1331β 565plusmn 024a 1063plusmn 064αa

Aqueous extract 250 1428plusmn 067b 3115plusmn 123α 47874plusmn 1871β 574plusmn 040a 1185plusmn 080λ500 1555plusmn 059c 3773plusmn 128c 38350plusmn 975c 693plusmn 025c 1025plusmn 041b

Ethanolic extract 250 1433plusmn 102b 3220plusmn 170 42600plusmn 1811c 631plusmn 038c 1115plusmn 048β500 1688plusmn 132c 3838plusmn 099c 37475plusmn 828c 736plusmn 029c 828plusmn 042c

CFA complete Freundrsquos adjuvant RBC red blood cell WBC white blood cell Each value represents the meanplusmnESM of six animals αplt 005 βplt 001λplt 0001 statistically significant compared with the healthy control Each value represents the meanplusmnESM of 6 animals aplt 005 bplt 001 cplt 0001statistically significant compared to arthritic control

Table 5 Effect of aqueous and ethanolic extracts of Dissotis thollonii on serum parameters in CFA-induced arthritis in rats

Treatment Dose (mgkg) ALT (UI) AST (UI) ALP (UI) Creatinine (μmoll) Total protein (gdl)Healthy control mdash 4709plusmn 191 17389plusmn 1161 9027plusmn 336 048plusmn 007 745plusmn 024Arthritic control mdash 7788plusmn 207λ 42309plusmn 2351λ 44438plusmn 1369λ 139plusmn 013λ 590plusmn 017βDiclofenac 5 6346plusmn 150λc 25070plusmn 1676βc 24011plusmn 1628λc 093plusmn 003λc 665plusmn 028

Aqueous extract 250 6397plusmn 172λc 29979plusmn 1176λc 31740plusmn 1720λc 098plusmn 005λc 635plusmn 034500 5514plusmn 116αc 22259plusmn 607c 20733plusmn 989λc 069plusmn 003c 731plusmn 019a

Ethanolic extract 250 5541plusmn 184αc 25330plusmn 277βc 27097plusmn 743λc 086plusmn 03βc 716plusmn 029a500 4612plusmn 142c 17762plusmn 762c 14300plusmn 534c 060plusmn 002c 754plusmn 028b

CFA complete Freundrsquos adjuvant ALP alkaline phosphatase AST aminotransferase ALT alanine aminotransferase Each value represents the meanplusmnESMfor six animals and analyses by one-way ANOVA followed by Bonferroni post hoc test αplt 005 βplt 001 λplt 0001 when compared with the healthycontrol and aplt 005 bplt 001 cplt 0001 when compared with the arthritic control


presented joint architecture exhibiting cartilage destructionbone erosion and cellular infiltration but animals treatedwith different extracts (250 and 500mgkg) and diclofenac(5mgkg) showed a significant protection of the architectureof the joint (Figure 8) by reducing bone erosion cartilagedestruction and cellular infiltration

9 Discussion

In the present study the results show that aqueous andethanolic extracts ofD thollonii have well anti-inflammatoryproperties in vitro in several models such as the inhibition ofdenaturation of proteins 5-LOX COX and ROS In-flammation which is a very complex physiopathologicalresponse involves the production of free radicals derivedfrom neutrophils NO ROS cytokines and prostaglandinsduring its process [42] Protein denaturation is the processby which proteins lose their tertiary structure and secondarystructure Proteins denaturation is a well-documented causeof inflammation [43] -e inflammation mechanism in-volves a series of events in which the metabolism of ara-chidonic acid plays an important role Phenylbutazonesalicylic acid flufenamic acid (anti-inflammatory drugs)etc have shown a dose-dependent ability to thermally in-duced protein denaturation [44] -e pathogenesis of in-flammatory diseases involves the overproduction ofsubstances such as prostaglandin I2 thromboxane A2prostaglandin E2 arachidonic acid and leukotrienesthrough twometabolic pathways the cyclooxygenase (COX)pathway and the 5-lipoxygenase (5-LOX) pathway [45] Invarious inflammatory and allergic disorders COX and 5-LOX are the main enzymes in the synthesis of prostanoidsand eicosanoids from polyunsaturated fatty acids -e ef-fective reduction of chronic inflammatory conditions isimportant by double inhibition of LOX and COX [46]Substances capable of producing double inhibition of COXand 5-LOX with consequent substantial reduction in leu-kotriene and prostaglandin production produce a broadspectrum of anti-inflammatory activity and can be con-sidered to have an excellent profile of pharmacological safetyin clinical practice [47] -e anti-inflammatory activity ofthe aqueous and ethanolic extract of D thollonii was de-termined using two methods which were cyclooxygenase-2(COX-2) and lipoxygenase (LOX) assays All extracts testedshowed significant activities vis-a-vis both tests -e in-hibitory activity of aqueous and ethanolic extracts of Dthollonii on the denaturation of proteins the cyclooxygenase

and 5-lipoxygenase pathways show that these two extractsare capable of significantly inhibiting the production ofprostaglandins and leukotrienes which gives these twoextracts anti-inflammatory properties -ese results arejustified by the fact that compounds such as betulinic acidisolated from D thollonii possess an in vitro inhibitionproperty of cyclooxygenase (COX-1 and COX-2) and leu-kotriene B4 formation mediated by 5-LOX [48 49]

-e injection of zymosan into the knee joint causes aproliferative inflammatory monoarthritis resulting from theonset of an inflammatory reaction accompanied by hyper-nociception an influx of neutrophils and leukocytes andthen production by the infiltrated synovial cells andor manyinflammatory mediators include cytokines free radicalsprostaglandins and leukotrienes which are responsible forthe breakdown of articular cartilage pannus formation andsynovial hypertrophy [50 51] Clinical management of ar-thritis is primarily aimed at reducing the intensity of painjoint swelling and then preventing or significantly reducingbone erosion and the various joint damages observed [52] Inthis study aqueous and ethanolic extracts of D tholloniisignificantly reduced joint swelling up to the fifth hour ofsingle administration and the effect remains significant untilthe fifth day in continuous treatment Similarly the histo-logical analysis of the joints has shown that the extracts ofthis plant as well as diclofenac induce a protective effect onthe alteration of the synovial membrane on bone erosionand considerably reduce cartilage lesions

-e caudal injection-induced polyarthritis of CFA in ratsis a very good experimental model for preclinical evaluationof new antiarthritic agents this model has many similaritieswith human rheumatoid diseases [53ndash55] In addition CFAinjection induces hyperalgesia and allodynia by alteringsensitivity to high-threshold nociceptor transduction [56]In the present study D thollonii extracts showed an anti-arthritic potential on all evaluated inflammatory parameters-ese extracts significantly reduced the diameter of the jointin arthritic animals and they significantly reduced thesusceptibility of arthritic animals to mechanical pain andthey significantly improved the arthritic score On theseimportant parameters the inhibitory effect of the extractswas significantly greater than that of diclofenac In manydisease states such as rheumatoid arthritis decreased bodyweight is an important predictor of health [57]-e results ofthis study show that the extracts improved the body mass ofthe treated animals when compared with the untreatedanimals During the development of rheumatoid arthritis

Table 6 Effect of aqueous and ethanolic extracts ofDissotis thollonii on some parameters of oxidative stress in CFA-induced arthritis in rats

Treatment Dose (mgkg) NO (μM) Glutathion (times103 activity) Catalase (activity) MDA (times103moll) SOD (activity)Healthy control mdash 0048plusmn 0003 028plusmn 003 5299plusmn 067 0087plusmn 0021 193plusmn 002Arthritic control mdash 0089plusmn 0003λ 017plusmn 001β 3689plusmn 072λ 0138plusmn 0027 151plusmn 003λDiclofenac 5 0044plusmn 0001c 021plusmn 001 3772plusmn 074c 0112plusmn 0035 192plusmn 003c

Aqueous extract 250 0073plusmn 0005λa 018plusmn 002β 4533plusmn 097 0127plusmn 0020 193plusmn 002c500 0064plusmn 0003αc 024plusmn 001 5145plusmn 170c 0109plusmn 0038 195plusmn 004c

Ethanolic extract 250 0067plusmn 0001βc 022plusmn 001 4895plusmn 118c 0125plusmn 0005 194plusmn 002c500 0046plusmn 0003c 027plusmn 001b 5239plusmn 409c 0071plusmn 0020 195plusmn 008c

Each value represents the meanplusmnESM for six animals and analyses by one-way ANOVA followed by Bonferroni post hoc test αplt 005 βplt 001 andλplt 0001 when compared with the healthy control and aplt 005 cplt 0001 when compared with the arthritic control


several enzymes are highlighted and represent good indicesaccording to their importance (case of alkaline phosphataseand transaminases) e increase in serum levels of theseenzymes would be implicated in periarticular osteopenia inbone erosion and also indicate severe liver injury resulting inthe production of biologically active substances in the in-iexclammatory process [58ndash61] In the present study arthriticrat serum values ALP AST and ALT signicantly increaseswhereas in animals submited to dierent treatments(aqueous and ethanolic extracts of the leaves of D tholloniior diclofenac) increased levels of these enzymes have beensignicantly reduced

In the pathogenicity of rheumatoid arthritis ROS whichare considered to be enhancers of iniexclammatory proliferationof the synovial membrane play a key role in that their

increased production increases the destruction of cartilageand even bones activates or removes NF-κB transcriptionfactor induces the production of many cytokines and acti-vates enzymes such as COX and 5-lipoxygenase or eveninducible nitrogen monoxide [62ndash65] e NF-κB tran-scription factor is present in the cytosol of many cells where itis bound with the factor I-kB especially those expressingcytokines growth factors chemokines and adhesion mole-cules Activation of the cell causes the phosphorylation of I-kBwhich is degraded followed by the release of the factor NF-κBwhich is introduced into the nucleus of the cell and causes thetranscription of many proiniexclammatory mediators includingiNOS COX-2 and TNF-α and IL-1β IL-6 and IL-8 [66]thus bone erosion and cartilage destruction observed inrheumatoid arthritis are due to overproduction of cytokines

(a)

A

B

C

D

(b)

(c) (d)

(e)

Figure 8 Histopathological analysis of ankle joints stained with HampE (a) Healthy control showing normal structure with small joint space(b) arthritic control showing very large joint space (A) destruction of cartilage (B) bone erosion (C) and cellular inltration (D) (c)diclofenac 5mgkg treated (d) aqueous extract (AE) 250mgkg treated and (e) ethanolic extract (EE) 250mgkg treated showing a decreasein joint space a reduction of cells inltration and reduction of cartilage destruction


and inflammatory mediators -e aqueous and ethanolicextracts of the leaves of D thollonii showed excellent anti-oxidant power and in this study the inhibition of the pro-duction of extracellular ROS in whole blood and in variousphagocytic cells was significative (neutrophils and macro-phages) -is activity is thought to be due to the inhibition ofpro inflammatory cytokine production (TNF alpha) theinhibition of denaturation of protein and the inhibition of theactivity of proinflammatory enzymes such as COX and 5-lipoxygenase

To investigate the effect of aqueous and ethanolicextracts of the leaves of D thollonii on another aspect ofthe cellular immune response the assay on T-cell pro-liferation was used-e results show that these D tholloniiextracts significantly inhibit T-cell proliferation with avery significant result for the extracts compared withprednisolone used as a positive control -e resultssuggest that the compounds present in D thollonii ex-tracts are capable of significantly modulating at differentstages the immune response -e significant decrease inthe diameter of the joints observed macroscopically andhistopathologically followed by a decreased insensitivityto pain clearly reveals the anti-inflammatory anti-hyperalgic and antiarthritic potential of D thollonii ex-tracts It is possible that the effect of our different extractsis associated with an inhibition of the phosphorylation ofthe transcription factor NF-κB -is is justified by the factthat compounds such as betulinic acid ellagic acidβ-sitosterol and ajuronic acid present in the extract of Dthollonii [30] have shown their ability to prevent degra-dation of the inhibitory IκB-α protein inducing inhibitionof NF-κB nuclear transcription factor activation sub-sequent reduction of COX-2 expression iNOS in-flammatory cell quantity the levels of TNF-α IL-6 IL-8and increase the production of IL-10 [67ndash72] Given thatduring the development of the inflammatory reaction thestimulation of the production of TNF-α IL-1β NO andPGE2 would be linked to the activation of the NF-κBIκB-α axis [73] thus the suppression of this axis has a sig-nificant therapeutic effect [72] In addition D tholloniiextracts showed in vitro inhibitory effects on TNF-αproduction intracellular ROS production leukocytemigration and cell-proliferative HeLa cell line [24]

10 Conclusions

At the end of this study the conclusion that we can deduce isthat D thollonii is a plant containing several groups ofchemical compounds with anti-inflammatory and antiar-thritic potential -ese properties have been evaluated by invitro and in vivo studies In vitro studies have shown that Dthollonii has a very strong anti-inflammatory property in-hibition of protein denaturation inhibition of 5-LOX in-hibition of COX and ROS and in vivo studies that haveshown antiarthritis activity of the plant on a zymosan-in-duced monoarthritis model and a model of CFA-inducedpolyarthritis in the rat -ese results confirm the utilizationof this plant in the traditional treatment of chronic in-flammatory diseases and consider it a potential candidate for

the isolation of new anti-inflammatory andor antiarthriticproducts

Data Availability

All data supporting our findings are adequately containedwithin the manuscript

Ethical Approval

-e experimental procedures have been approved by thelocal Ethics Committee and are in accordance with theguidelines for the study of pain in awake animals publishedby the NIH (publication no 85-23 ldquoPrinciples of AnimalProtectionrdquo Laboratory Study of Pain Ministry of ScientificResearch and Technology which adopted the EuropeanUnion Guidelines on Animal Care and Experimentation(EWC 86609)

For the donation of human blood samples all processesof collecting blood are accepted by the Independent EthicsCommittee ICCBS University of Karachi Ndeg ICCBSIEC-008-BC-2015Protocol10 -e blood donors were providedinformed approval for the use of their blood for the purposesof this study

Conflicts of Interest

MM (PhD) is a Senior Lecturer in the Department of AnimalBiology Faculty of Science University of Dschang Came-roon AAD (PhD) is a Senior Lecturer in the Department ofAnimal Biology Faculty of Science University of Yaounde 1Cameroon DNSF TEG YNW MMMV and ACF are PhDstudents in the Department of Animal Biology Faculty ofScience University of Dschang Cameroon AG is an As-sociate Professor in the Department of Animal BiologyFaculty of Science University of Dschang Cameroon -eauthors declare that they have no conflicts of interest

Authorsrsquo Contributions

DNSF MM and AG designed the work DNSF MM TEGAAD MMMV and ACF conducted the work and collectedand analysed the data MM NYW AG and AAD drafted themanuscript and revised it critically All authors agree to beaccountable for all aspects of the work

References

[1] H Jeon W J Yoon Y M Ham S A Yoon and S C KangldquoAnti-arthritis effect through the anti-inflammatory effect ofSargassum muticum extract in collagen-induced arthritic(CIA) micerdquo Molecules vol 24 no 2 p 276 2019

[2] E H Chou and G S Panayi ldquoCytokine pathways and jointinflammation in rheumatoid arthritisrdquo New England Journalof Medicine vol 344 no 12 pp 907ndash916 2001

[3] Y-A Lee J Y Kim S-J Hong et al ldquoSynovial proliferationdifferentially affects hypoxia in the joint cavities of rheu-matoid arthritis and osteoarthritis patientsrdquo Clinical Rheu-matology vol 26 no 12 pp 2023ndash2029 2007

[4] G Murugananthan K G Sudheer C P Sathya andS Mohan ldquoAnti-arthritic and anti-inflammatory constituents


from medicinal plantsrdquo Journal of Applied PharmaceuticalScience vol 3 no 4 pp 161ndash164 2013

[5] M Feldmann F M Brennan and R N Maini ldquoRole ofcytokines in rheumatoid arthritisrdquo Annual Review of Im-munology vol 14 no 1 pp 397ndash440 1996

[6] A B Irem C G Ahmet K Hasan and Y Erdem ldquoEvaluationof the in vitro anti-inflammatory activity of Nerium oleanderL flower extracts and activity-guided isolation of the activeconstituentsrdquo Records of Natural Products vol 12 no 2pp 128ndash141 2018

[7] C Penido F P Conte M S S Chagas C A B RodriguesJ F G Pereira and M G M O Henriques ldquoAntiin-flammatory effects of natural tetranortriterpenoids isolatedfrom Carapa guianensis Aublet on zymosan-induced arthritisin micerdquo Inflammation Research vol 55 no 11 pp 457ndash4642006

[8] J C da S Rocha M E Peixoto S Jancar et al ldquoDual effect ofnitric oxide in articular inflammatory pain in zymosan in-duced arthritis in ratsrdquo British Journal of Pharmacologyvol 136 no 4 pp 588ndash596 2002

[9] A Bendele ldquoAnimal models of rheumatoid arthritisrdquo JournalMusculoskeletal Neuronal Interaction vol 1 no 4 pp 377ndash385 2001

[10] R Holmdahl J C Lorentzen S Lu et al ldquoArthritis induced inrats with non-immunogenic adjuvants as models for rheu-matoid arthritisrdquo Immunological Reviews vol 184 no 1pp 184ndash202 2001

[11] H-G Schaible and B D Grubb ldquoAfferent and spinalmechanisms of joint painrdquo Pain vol 55 no 1 pp 5ndash54 1993

[12] X Cai Y F Wong H Zhou et al ldquo-e comparative study ofsprague-dawley and lewis rats in adjuvant-induced arthritisrdquoNaunyn Schmiedebergs Archives of Pharmacology vol 373no 2 pp 140ndash147 2006

[13] B Zhang X-L He Y Ding and G-H Du ldquoGaultherin anatural salicylate derivative from Gaultheria yunnanensistowards a better non-steroidal anti-inflammatory drugrdquoEuropean Journal of Pharmacology vol 530 no 1-2pp 166ndash171 2006

[14] A R Gaby ldquoAlternative treatments for rheumatoid arthritisrdquoAlternative Medicine Review vol 4 no 6 pp 392ndash402 1999

[15] J W G Jacobs F W Kraaimaat and J W J Bijlsma ldquoWhydo patients with rheumatoid arthritis use alternative treat-mentsrdquo Clinical Rheumatology vol 20 no 3 pp 192ndash1962001

[16] J S Bang D H Oh H M Choi et al ldquoAnti-inflammatoryand antiarthritic effects of piperine in humaninterleukin 1β-stimulated fibroblast-like synoviocytes and in rat arthritismodelsrdquo Arthritis Research amp Cerapy vol 11 no 2 pp 1ndash92009

[17] M Hegen J C Keith M Collins and C L Nickerson-NutterldquoUtility of animal models for identification of potentialtherapeutics for rheumatoid arthritisrdquo Annals of the Rheu-matic Diseases vol 67 no 11 pp 1505ndash1515 2008

[18] A Okunlola and B A Adewoyin O A Odeku Evaluation ofpharmaceutical and microbial qualities of some herbal me-dicinal products in South Western Nigeriardquo Tropical Journalof Pharmaceutical Research vol 6 no 1 pp 661ndash670 2007

[19] D Susanti H M Sirat F Ahmad R M Ali N Aimi andM Kitajima ldquoAntioxidant and cytotoxic flavonoids from theflowers of Melastoma malabathricum Lrdquo Food Chemistryvol 103 no 3 pp 710ndash716 2007

[20] J-T Cheng F-L Hsu and H-F Chen ldquoAntihypertensiveprinciples from the leaves ofMelastoma candidumrdquo PlantaMedica vol 59 no 5 pp 405-406 1993

[21] T Amalraj and S Ignacimuthu ldquoEvaluation of the hypo-glycaemic effect of Memecylon umbellatum in normal andalloxan diabetic micerdquo Journal of Ethnopharmacology vol 62no 3 pp 247ndash250 1998

[22] D S Nicholl H M Daniels M Ira -abrew R J GrayerM S J Simmonds and R D Hughes ldquoIn vitro studies on theimmunomodulatory effects of extracts of Osbeckia asperardquoJournal of Ethnopharmacology vol 78 no 1 pp 39ndash44 2001

[23] T A Abere P E Okoto and F O Agoreyo ldquoAntidiarrhoeaand toxicological evaluation of the leaf extract of Dissotisrotundifoliatriana (Melastomataceae)rdquo BMC Complementaryand Alternative Medicine vol 10 no 1 p 71 2010

[24] H TadjouaTchoumbou G Ateufack W Yousseu Nana et alldquoAntidiarrhoeal anti-inflammatory and cytotoxic effects ofaqueous and methanolic leaves extract of Dissotis tholloniiCogn (Melastomataceae)rdquo European Journal of Pharma-ceutical and Medical Research vol 5 no 6 pp 306ndash314 2018

[25] H A Loigier Descriptive Flora of Puerto Rico and AdjacentIslands Spermaphyta Editorial de la Universidad de PuertoRico San Juan Puerto Rico 1994

[26] IPNI Ce International Plant Names Index InternationalPlant Names Index Cambridge MA USA 2012

[27] D S Tala S P C Fodouop D N Tsafack N KodjioC Fokunang and D Gatsing ldquoToxicological investigations ofethanolic leaves extract of dissothis thollonii (Mela-stomataceae)rdquo Journal of Pharmaceutical Research In-ternational vol 24 no 4 pp 1ndash14 2018

[28] T H Tadjoua G Ateufack U S Shabana et al ldquoAntidiar-rhoeal antibacterial activities and growth stimulatory effecton some probiotic organisms of aqueous and methanol leavesextract of Dissotis tholloniirdquo Journal of Chemical and Phar-maceutical Research vol 9 no 10 pp 95ndash105 2017

[29] G Ateufack T H Tadjoua N W Yousseu S F LeonardJ R Kuate and A Kamanyi ldquoAntidiarrhoeal and antibacterialactivity of aqueous and methanolic leaves extracts of Dissotisthollonii Cogn(Melastomataceae)rdquo Asian Pacific Journal ofTropical Biomedicine vol 4 no 2 pp S672ndashS678 2014

[30] R N Nono L Barboni R B Teponno et al ldquoAntimicrobialantioxidant anti-inflammatory activities and phytocon-stituents of extracts from the roots of Dissotis thollonii Cogn(Melastomataceae)rdquo South African Journal of Botany vol 93pp 19ndash26 2014

[31] M Mbiantcha E G Tsafack G Ateufack et al ldquoAnalgesicanti-inflammatory and anti-arthriticproperties of aqueousand methanolic stem bark extracts from Nauclea pobeguinii(Rubiacee) in ratsrdquo Journal of Complementary and IntegrativeMedicine vol 15 no 4 pp 1ndash9 2018

[32] MMbiantcha J Almas A D Atsamo et al ldquoAnti-inflammatoryand anti-arthritic effects of methanol extract of the stem bark ofboswellia dalzielii hutch (Burseraceae) in ratsrdquo Inflammo-pharmacology vol 26 no 6 pp 1383ndash1398 2018

[33] F J A Matos Introducao a Fitoquacuteımica ExperimentalEdicoes UFC Fortaleza Brazil 2nd edition 1997

[34] P Padmanabhan and S N Jangle ldquoEvaluation of in-vitro anti-inflammatory activity of herbal preparation a combination offour medicinal plantsrdquo International Journal of Basic andApplied Medical Sciences vol 2 no 1 pp 109ndash116 2012

[35] G Elias and M N Rao ldquoInhibition of albumin denaturationand anti-inflammatory activity of dehydrozingerone and itsanalogsrdquo Indian Journal of Experimental Biology vol 26no 10 pp 540ndash542 1988

[36] V Viji and A Helen ldquoInhibition of lipoxygenases andcyclooxygenase-2 enzymes by extracts isolated from Bacopa


monniera (L) wettstrdquo Journal of Ethnopharmacology vol 118no 2 pp 305ndash311 2008

[37] M Zimmerman ldquoEthical guidelines for investigations ofexperimental pain in conscious animalsrdquo Pain vol 16 no 2pp 109-110 1983

[38] M Mesaik Z Zaheerulhaq S Murad et al ldquoBiological andmolecular docking studies on coagulin-H human IL-2 novelnatural inhibitorrdquo Molecular Immunology vol 43 no 11pp 1855ndash1863 2006

[39] M F Mahomoodally A Gurib-Fakim and A H SubrattyldquoEffect of exogenous ATP on Momordica charantia Linn(Cucurbitaceae) induced inhibition of d-glucose l-tyrosineand fluid transport across rat everted intestinal sacs in vitrordquoJournal of Ethnopharmacology vol 110 no 2 pp 257ndash2632007

[40] M Mbiantcha J Almas U S Shabana D Nida and F AishaldquoAnti-arthritic property of crude extracts of Piptadeniastrumafricanum (Mimosaceae) in complete Freundrsquos adjuvant-in-duced arthritis in ratsrdquo BMC Complementary and AlternativeMedicine vol 17 no 1 pp 1ndash16 2017

[41] C Rita V Bruno R Helena et al ldquoPotent anti-inflammatoryand antiproliferative effects of gambogic acid in a rat model ofantigen-induced arthritisrdquo Mediators of Inflammationvol 2014 Article ID 195327 7 pages 2014

[42] M M Mannan M Maridass and B Victor ldquoA review on thepotential uses of fernsrdquo Ethnobotanical Leaflets vol 12no 24 pp 281ndash285 2008

[43] M V Anoop and A R Bindu ldquoIn-vitro anti-inflammatoryactivity studies on Syzygium zeylanicum (L) DC leavesrdquo In-ternational Journal of Pharmacology Researchamp Review vol 4no 8 pp 18ndash27 2015

[44] Y Mizushima and M Kobayashi ldquoInteraction of anti-in-flammatory drugs with serum proteins especially with somebiologically active proteinsrdquo Journal of Pharmacy andPharmacology vol 20 no 3 pp 169ndash173 1968

[45] G Ghosh P Panda M Rath A Pal T Sharma and D DasldquoGC-MS analysis of bioactive compounds in the methanolextract of Clerodendrum viscosum leavesrdquo PharmacognosyResarch vol 7 no 1 pp 110ndash113 2015

[46] G de Gaetano M B Donati and C Cerletti ldquoPrevention ofthrombosis and vascular inflammation benefits and limita-tions of selective or combined COX-1 COX-2 and 5-LOXinhibitorsrdquo Trends in Pharmacological Sciences vol 24 no 5pp 245ndash252 2003

[47] F M Frey and R Meyers ldquoAntibacterial activity of traditionalmedicinal plants used by Haudenosaunee peoples of NewYork Staterdquo BMC Complementary and Alternative Medecinevol 10 no 1 p 64 2010

[48] E M Wenzig U Widowitz O Kunert et al ldquoPhytochemicalcom-position and in vitro pharmacological activity of two rosehip (Rosa caninaL) preparationsrdquo Phytomedicine vol 15no 10 pp 826ndash835 2008

[49] G M Mansour B H A Faujan and S K Alireza ldquoBiologicalactivity of betulinic acid a reviewrdquo Pharmacology amp Phar-macy vol 3 no 2 pp 119ndash123 2012

[50] L A Darren MM Ashley B M Iain and Y L Foo ldquoAnimalmodels of rheumatoid arthritisrdquo European Journal of Im-munology vol 39 no 8 pp 2040ndash2044 2009

[51] F A da Rocha M M Teixeira J C Rocha et al ldquoBlockade ofleukotriene B4 prevents articular incapacitation in rat zy-mosan-induced arthritisrdquo European Journal of Pharmacologyvol 497 no 1 pp 81ndash86 2004

[52] K D Tripathi Essentials of Medical Pharmacology JaypeeBrothers Medical Publishers Ajanta Offset and PackagingsLtd New Delhi India 6th edition 2008

[53] D N Benslay and A M Bendele ldquoDevelopment of a rapidscreen for detecting and differentiating immunomodulatoryvs anti-inflammatory compounds in ratsrdquo Agents and Ac-tions vol 34 no 1-2 pp 254ndash256 1991

[54] F C Colpaert T Meert P DeWitte and P Schmitt ldquoFurtherevidence validating adjuvant arthritis as an experimentalmodel of chronic pain in the ratrdquo Life Sciences vol 31 no 1pp 67ndash75 1982

[55] S Singh and D K Majumdar ldquoEffect of fixed oil of ocimumsanctum against experimentally induced arthritis and jointedema in laboratory animalsrdquo International Journal ofPharmacognosy vol 34 no 3 pp 218ndash222 1996

[56] M Higgins R DrsquoAgostino W Kannel and J Cobb ldquoBenefitsand adverse effects of weight loss observations from theframingham studyrdquo Annals of Internal Medicine vol 119no 7_Part_2 pp 758ndash763 1993

[57] E M Glenn J Gray and W Kooyers ldquoChemical changes inadjuvant induced polyarthritis of ratsrdquo American Journal ofVeterinary Research vol 26 no 114 pp 1195ndash1203 1965

[58] Y Niino-Nanke H Akama M Hara and S KashiwazakildquoAlkaline phasphatase (ALP) activity in rheumatoid arthritis(RA) its clinical significance and synthesis of ALP in RAsynoviumrdquo Ryumachi vol 38 no 4 pp 581ndash588 1998

[59] Q Rehman and N E Lane ldquoBone loss -erapeutic ap-proaches for preventing bone loss in inflammatory arthritisrdquoArthritis Research vol 3 no 4 pp 221ndash227 2001

[60] S Aida ldquoAlkaline phosphatase isoenzyme activities inrheumatoid arthritis hepatobiliary enzyme dissociation andrelation to disease activityrdquo Annals of the Rheumatic Diseasesvol 52 no 7 pp 511ndash516 1993

[61] C A Hitchon and H S El-Gabalawy ldquoOxidation in rheu-matoid arthritisrdquo Arthritis Research amp Cerapy vol 6 no 6pp 265ndash278 2004

[62] J Almas M A Mesaik U S Shabana S B Lubna andF Shaheen ldquoAnti-TNF-α and antiarthritis of patuletin a rareflavonoid from Tagetes patulardquo International Immuno-pharmacology vol 36 pp 232ndash240 2016

[63] J Almas Study of the suppression of inflammatory arthritis atmolecular level by natural and synthetic inhibition of TNFαand IL-1β PhD thesis p 190 2013

[64] L Zhou W Meng C Lvyi X Guangjimg L Xiao andZ Xiuqiao ldquoRole of sinomenine on complete freundrsquosadjuvand-induced arthritis in ratsrdquo International Union ofBiochemistory and Molecular Biology Life vol 68 no 6pp 429ndash435 2016

[65] S Guan B Fang B Song Y Xiong and J Lu ldquoImmuno-suppressive activity of alpinetin on activation and cytokinessecretion of murine T lymphocytesrdquo Immunopharmacologyand Immunotoxicology vol 36 no 4 pp 290ndash296 2014

[66] S Guan Y Zheng X Yu W Li B Han and J Lu ldquoEllagicacid protects against LPS-induced acute lung injury throughinhibition of nuclear factor kappa B proinflammatory cy-tokines and enhancement of interleukin-10rdquo Food and Ag-ricultural Immunology vol 28 no 6 pp 1347ndash1361 2017

[67] M A Rosillo M Sanchez-Hidalgo A Cardeno andC Alarcon de la Lastra ldquoProtective effect of ellagic acid anatural polyphenolic compound in a murine model ofCrohnrsquos diseaserdquo Biochemical Pharmacology vol 82 no 7pp 737ndash745 2011

[68] T Hemalatha S Pulavendran C Balachandran B MManoharand R Puvanakrishan ldquoArjuronic acid a novel phytomedicine


with multifunctional therapeutic applicationsrdquo Indian Journal ofExperimental Biology vol 48 no 3 pp 238ndash247 2010

[69] Y Takada and B B Aggarwal ldquoBetulinic acid suppressescarcinogen-induced NF-κB activation through inhibition ofIκB-α kinase and p65 phosphorylation abrogation of cyclo-oxygenase-2 and matrix metalloprotease-9rdquo Ce Journal ofImmunology vol 171 no 6 pp 3278ndash3286 2003

[70] T Rabi S Shukla and S Gupta ldquoBetulinic acid suppressesconstitutive and TNFα-induced NF-κB activation and inducesapoptosis in human prostate carcinoma PC-3 cellsrdquo Molec-ular Carcinogenesis vol 47 no 12 pp 964ndash973 2008

[71] K-A Kim I-A Lee W Gu S R Hyam and D-H Kimldquoβ-Sitosterol attenuates high-fat diet-induced intestinal in-flammation in mice by inhibiting the binding of lipopoly-saccharide to toll-like receptor 4 in the NF-κB pathwayrdquoMolecular Nutrition amp Food Research vol 58 no 5pp 963ndash972 2014

[72] S G Harris J Padilla L Koumas D Ray and R P PhippsldquoProstaglandins as modulators of immunityrdquo Trends in Im-munology vol 23 no 3 pp 144ndash150 2002

[73] S Dudhgaonkar A -yagarajan and D Sliva ldquoSuppressionof the inflammatory response by triterpenes isolated from themushroom Ganoderma lucidumrdquo International Immuno-pharmacology vol 9 no 11 pp 1272ndash1280 2009


Stem Cells International

Hindawiwwwhindawicom Volume 2018


MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine


Behavioural Neurology

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom

including tumor necrosis factor (TNF-α) interleukin-1βinterleukin-6 nitric oxide (NO) prostaglandins reactiveoxygen species (ROS) platelet-activating factor leukotri-enes enzymes (lipoxygenases cyclooxygenases (COX-1 andCOX-2) and phospholipases) [5 6]

Animal models of zymosan A-induced monoarthritis andFreundrsquos complete adjuvant-induced polyarthritis (CFA) arewidely used in research for the activity of many pharmaco-logical substances on rheumatoid arthritis In fact the in-jection of zymosan into the knee joint of the rats causeserosive synovitis with increased vascular permeability neu-trophil infiltration and formation of edemas and exudates inthe acute phase and then in the chronic phase infiltration ofmacrophages and lymphocytes pannus formation and fi-broblastic reaction characteristic of chronic rheumatoid sy-novitis [7 8] CFA is an immunogenic adjuvant consisting ofa suspension of Mycobacterium tuberculosum or Mycobac-terium butyricum killed by heat When injected at the base ofthe animalrsquos tail it causes the development of polyarthritisthat evolves in a two-phase cycle of time the first phaseappears in a few hours and disappears after 3 to 5 days andmanifests itself by an acute local inflammatory reaction andthen the second phase appears after two weeks and corre-sponds to a chronic systemic reaction [9ndash11] -is poly-arthritis is not primarily aimed at the knee joint and it canaffect the general state of the animal body it is a real systemicdisease resulting in inflammation of the distal joints of thelimbs vertebrae lesions of the genitourinary tract gastro-intestinal tract eyes nose ears skin and anorexia accom-panied by significant weight loss [9 11] In addition thepathology will persist and other symptoms will appearnamely joint deformity synovitis synovial hyperplasiacapsular fibrosis angiogenesis pannus formation cartilagedestruction bone erosion inflammation of the bone marrowresorption of bone matrix and ankylosis [12]

-e severity and persistence of rheumatoid arthritisrequire long-term management with anti-inflammatorydrugs Nevertheless these anti-inflammatory drugs have forthe most part risks of toxicity for long-term use whichseriously limits their use Current research in the manage-ment of rheumatoid arthritis is turning to a new generationof substances capable of selectively inhibiting TNF alphaandor cyclooxygenase (COX-2) and having no major sideeffects [13] Recent interest in alternative treatments forarthritis favors the use of traditional medicine althoughscientific evidence of efficacy for most cases is lackingNevertheless several herbs used in a care program and avery effective preventive medicine can act individually andor in synergy to reduce chronic joint inflammation (oste-oarthritis andor rheumatoid arthritis) [14ndash16] To reach thetotal health care coverage of the worldrsquos population tradi-tional medicine is considered by WHO to be the most ef-fective means since about 25 of modern prescription drugsare more or less obtained from plants [17 18]

Comprising about 163 genera the family of Mela-stomataceae which are mainly pantropical plants includemore than 4300 species somany of them are known for theireffectiveness in traditional medicine as antihepatitic anti-hypertensive anti-inflammatory antihyperglycaemic

antioxidant hemostatic and antidiarrheal [19ndash24] Dissotisthollonii (D thollonii) is one of many species of the family ofMelastomataceae used in traditional medicine in Cameroonto treat typhoid fever gastrointestinal disorders in-flammatory diseases and sinusitis [25ndash27] -e leaves arerecommended for the treatment of ulcers and gastrointes-tinal disorders A previous study showed that D tholloniisignificantly inhibited fluid accumulation in intestine in-duced by prostaglandin E2 [28] Based on recent work byAteufack et al [29] this plant has antidiarrheic and anti-bacterial properties and then has several secondary me-tabolites including tannins flavonoids sterolsanthraquinones phenols and polyphenols In addition thework of TadjouaTchoumbou et al [24] showed that thisplant significantly inhibited leukocyte migration in perito-neal fluid intracellular ROS production proliferation ofHela cell lines and TNF-α production Tala et al [27]showed that aqueous and ethanolic extracts were devoid oftoxicity after 28 days of daily treatment Similarly Nonoet al [30] showed the antimicrobial and antioxidantproperties of this plant Several compounds have alreadybeen isolated from this plant among which 33prime-dio-methylellagic acid 4prime-O-β-D-xylopyranoside 3-4prime-O-β-D-arabinopyranoside casuarinine betulinic acid β-sitosterol-3-O-D-glucopyranosyl-6prime-mirystate cellobiosylsterol β-si-tosterol β-sitosterol-3-O-β-D-glucopyranoside arjunolicacid 33prime-diomethylellagic acid ellagic acid and 33prime-dio-methylellagic acid 4prime-O-β-D-glucopyranoside [30] Al-though this plant is traditionally used to relieve manydisorders of the body no information or scientific report toour knowledge has been found in the literature relative to itsantiarthritic properties In our continuous search for bio-active extracts from plants used in traditional Cameroonianmedicine [31 32] and in order to support and improve thetraditional use of D thollonii we undertook to carry out thepresent study on in vitro anti-inflammatory activities and invivo antiarthritic activities of the leave extracts of Dthollonii

2 Materials and Methods

21 Plant Material and Extraction -e plant material ref-erenced to the national herbarium of Cameroon under thenumber Ndeg 133292SRF Cam in the name of D thollonii(Melastomotaceae) was used in this study -e fresh leaveswere harvested in the town of Dschang (western Cameroon)dried in the shade and then crushed into a fine powder Inorder to prepare the aqueous extract 500 g of powder wasmixed into 500ml of distilled water during 72 hours andfiltrated (Whatman paper No 4) the filtrate obtained wasevaporated at 40degC to give the aqueous extract (82 yield)-e same weight of dried powder plant was mixed into500ml of ethanol for 72 hours and then filtered -e filtratewas concentrated with a rotary evaporator set at 96degC to givethe ethanolic extract with 96 yield

22 Phytochemical Assay of D thollonii Extracts -e dif-ferent extracts were subjected to chemical screening in order

















(1)


















CPMcontroltimes 100

(4)













6 Results
















Diclofenac


Ibuprofen


Aqueous extract

100 2733 2472 3126200 2926 3414 4560500 3383 4070 58171000 4251 4707 6679

Ethanolic extract

100 3087 2814 3612200 3279 4089 4824500 3730 5355 64111000 4444 6336 7748
















0 1 2 3 4 5 6 24 48 72 96 1205

6

7

8

9

10

αc

λ

λ

λ

βcλc

λcλbλ λbλb

λaλ

λ λ

λ λ λ

λ λ λ λ

λ λ λ

λ λb

λcλcλ λ

λ λ

λb

λ λ

λ

λ λ

λ

λ λ λ λλ λ

λ

λ

αcβcαcβc

λb

λ

λ

λ λ λbλ λ λ

λ λ

ccc

Time (hour)

Join

t dia

met

er (m

m)











(a)

A

B

C

(b)

(c) (d)

λ

(e)





λcλcλcβcλc

λcλcβcλc

λcλc

λcλcλcλcλb

λcλaλc

λb

λaλcλcλc

λcλc

λcλc

λcλcλcλc

λc

α

λλλλ

λλλλ

λλ

λ

λ

λ

λc

αcαc

λcλcλc

λcλc

λcλc

λcλbλc

λc

βcβ βcλc

λc


c

95

100

105

110

115

120

125

130

Join

t dia

met

er (

incr

ease

)

3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 351Period (week)





1 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 340

10

20

30

40

50

60

70

80

90

100

110

120

λcλc

λcλcλcλcλc

λc

λλcλc

λcλc

λc

λcλcλc

λc

λc

λcλcλcλc

λb

λc

λc

λcλcλc

λcλcλcλc

λcλc

λc

λa

λλλλλλλλ

λλλλ

λ

λcλcλcλc

λcλcλcλc

λcλc

λc

λc

λcλcλcλc

λcλcλcλc

λc

λcλc

λcλb

λcλcλc

λc

Period (week)

Sens

ibili

ty (

)








ccccc

c

c

cccc

c

cccbb

βββλ

λλ

λλλ

λλ

λ

λ

ccccc

cc

ccccc

b

ndash05

00

05

10

15

20

25

Art

hriti

s sco

re

33 3527 29 31211917 23 2513 15117 93 51Period (week)





0 1 2 3 4 585

90

95

100

105

110

115

λλλ

λλ

λλ

λ

λα

λλ

λ

β

λbλaλa

λ

λλ

Period (week)

Relat

ive b

ody

wei

ght (

)












βbαββaαβ

ba

αβ

ba


2

4

6

8

10

12

Org

an w

eigh

t (g)














9 Discussion














(a)

A

B

C

D

(b)

(c) (d)

(e)





10 Conclusions



Data Availability


Ethical Approval







References





















































































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of


































(1)


















CPMcontroltimes 100

(4)













6 Results
















Diclofenac


Ibuprofen


Aqueous extract

100 2733 2472 3126200 2926 3414 4560500 3383 4070 58171000 4251 4707 6679

Ethanolic extract

100 3087 2814 3612200 3279 4089 4824500 3730 5355 64111000 4444 6336 7748
















0 1 2 3 4 5 6 24 48 72 96 1205

6

7

8

9

10

αc

λ

λ

λ

βcλc

λcλbλ λbλb

λaλ

λ λ

λ λ λ

λ λ λ λ

λ λ λ

λ λb

λcλcλ λ

λ λ

λb

λ λ

λ

λ λ

λ

λ λ λ λλ λ

λ

λ

αcβcαcβc

λb

λ

λ

λ λ λbλ λ λ

λ λ

ccc

Time (hour)

Join

t dia

met

er (m

m)











(a)

A

B

C

(b)

(c) (d)

λ

(e)





λcλcλcβcλc

λcλcβcλc

λcλc

λcλcλcλcλb

λcλaλc

λb

λaλcλcλc

λcλc

λcλc

λcλcλcλc

λc

α

λλλλ

λλλλ

λλ

λ

λ

λ

λc

αcαc

λcλcλc

λcλc

λcλc

λcλbλc

λc

βcβ βcλc

λc


c

95

100

105

110

115

120

125

130

Join

t dia

met

er (

incr

ease

)

3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 351Period (week)





1 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 340

10

20

30

40

50

60

70

80

90

100

110

120

λcλc

λcλcλcλcλc

λc

λλcλc

λcλc

λc

λcλcλc

λc

λc

λcλcλcλc

λb

λc

λc

λcλcλc

λcλcλcλc

λcλc

λc

λa

λλλλλλλλ

λλλλ

λ

λcλcλcλc

λcλcλcλc

λcλc

λc

λc

λcλcλcλc

λcλcλcλc

λc

λcλc

λcλb

λcλcλc

λc

Period (week)

Sens

ibili

ty (

)








ccccc

c

c

cccc

c

cccbb

βββλ

λλ

λλλ

λλ

λ

λ

ccccc

cc

ccccc

b

ndash05

00

05

10

15

20

25

Art

hriti

s sco

re

33 3527 29 31211917 23 2513 15117 93 51Period (week)





0 1 2 3 4 585

90

95

100

105

110

115

λλλ

λλ

λλ

λ

λα

λλ

λ

β

λbλaλa

λ

λλ

Period (week)

Relat

ive b

ody

wei

ght (

)












βbαββaαβ

ba

αβ

ba


2

4

6

8

10

12

Org

an w

eigh

t (g)














9 Discussion














(a)

A

B

C

D

(b)

(c) (d)

(e)





10 Conclusions



Data Availability


Ethical Approval







References





















































































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of































CPMcontroltimes 100

(4)













6 Results
















Diclofenac


Ibuprofen


Aqueous extract

100 2733 2472 3126200 2926 3414 4560500 3383 4070 58171000 4251 4707 6679

Ethanolic extract

100 3087 2814 3612200 3279 4089 4824500 3730 5355 64111000 4444 6336 7748
















0 1 2 3 4 5 6 24 48 72 96 1205

6

7

8

9

10

αc

λ

λ

λ

βcλc

λcλbλ λbλb

λaλ

λ λ

λ λ λ

λ λ λ λ

λ λ λ

λ λb

λcλcλ λ

λ λ

λb

λ λ

λ

λ λ

λ

λ λ λ λλ λ

λ

λ

αcβcαcβc

λb

λ

λ

λ λ λbλ λ λ

λ λ

ccc

Time (hour)

Join

t dia

met

er (m

m)











(a)

A

B

C

(b)

(c) (d)

λ

(e)





λcλcλcβcλc

λcλcβcλc

λcλc

λcλcλcλcλb

λcλaλc

λb

λaλcλcλc

λcλc

λcλc

λcλcλcλc

λc

α

λλλλ

λλλλ

λλ

λ

λ

λ

λc

αcαc

λcλcλc

λcλc

λcλc

λcλbλc

λc

βcβ βcλc

λc


c

95

100

105

110

115

120

125

130

Join

t dia

met

er (

incr

ease

)

3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 351Period (week)





1 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 340

10

20

30

40

50

60

70

80

90

100

110

120

λcλc

λcλcλcλcλc

λc

λλcλc

λcλc

λc

λcλcλc

λc

λc

λcλcλcλc

λb

λc

λc

λcλcλc

λcλcλcλc

λcλc

λc

λa

λλλλλλλλ

λλλλ

λ

λcλcλcλc

λcλcλcλc

λcλc

λc

λc

λcλcλcλc

λcλcλcλc

λc

λcλc

λcλb

λcλcλc

λc

Period (week)

Sens

ibili

ty (

)








ccccc

c

c

cccc

c

cccbb

βββλ

λλ

λλλ

λλ

λ

λ

ccccc

cc

ccccc

b

ndash05

00

05

10

15

20

25

Art

hriti

s sco

re

33 3527 29 31211917 23 2513 15117 93 51Period (week)





0 1 2 3 4 585

90

95

100

105

110

115

λλλ

λλ

λλ

λ

λα

λλ

λ

β

λbλaλa

λ

λλ

Period (week)

Relat

ive b

ody

wei

ght (

)












βbαββaαβ

ba

αβ

ba


2

4

6

8

10

12

Org

an w

eigh

t (g)














9 Discussion














(a)

A

B

C

D

(b)

(c) (d)

(e)





10 Conclusions



Data Availability


Ethical Approval







References





















































































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of


























6 Results
















Diclofenac


Ibuprofen


Aqueous extract

100 2733 2472 3126200 2926 3414 4560500 3383 4070 58171000 4251 4707 6679

Ethanolic extract

100 3087 2814 3612200 3279 4089 4824500 3730 5355 64111000 4444 6336 7748
















0 1 2 3 4 5 6 24 48 72 96 1205

6

7

8

9

10

αc

λ

λ

λ

βcλc

λcλbλ λbλb

λaλ

λ λ

λ λ λ

λ λ λ λ

λ λ λ

λ λb

λcλcλ λ

λ λ

λb

λ λ

λ

λ λ

λ

λ λ λ λλ λ

λ

λ

αcβcαcβc

λb

λ

λ

λ λ λbλ λ λ

λ λ

ccc

Time (hour)

Join

t dia

met

er (m

m)











(a)

A

B

C

(b)

(c) (d)

λ

(e)





λcλcλcβcλc

λcλcβcλc

λcλc

λcλcλcλcλb

λcλaλc

λb

λaλcλcλc

λcλc

λcλc

λcλcλcλc

λc

α

λλλλ

λλλλ

λλ

λ

λ

λ

λc

αcαc

λcλcλc

λcλc

λcλc

λcλbλc

λc

βcβ βcλc

λc


c

95

100

105

110

115

120

125

130

Join

t dia

met

er (

incr

ease

)

3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 351Period (week)





1 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 340

10

20

30

40

50

60

70

80

90

100

110

120

λcλc

λcλcλcλcλc

λc

λλcλc

λcλc

λc

λcλcλc

λc

λc

λcλcλcλc

λb

λc

λc

λcλcλc

λcλcλcλc

λcλc

λc

λa

λλλλλλλλ

λλλλ

λ

λcλcλcλc

λcλcλcλc

λcλc

λc

λc

λcλcλcλc

λcλcλcλc

λc

λcλc

λcλb

λcλcλc

λc

Period (week)

Sens

ibili

ty (

)








ccccc

c

c

cccc

c

cccbb

βββλ

λλ

λλλ

λλ

λ

λ

ccccc

cc

ccccc

b

ndash05

00

05

10

15

20

25

Art

hriti

s sco

re

33 3527 29 31211917 23 2513 15117 93 51Period (week)





0 1 2 3 4 585

90

95

100

105

110

115

λλλ

λλ

λλ

λ

λα

λλ

λ

β

λbλaλa

λ

λλ

Period (week)

Relat

ive b

ody

wei

ght (

)












βbαββaαβ

ba

αβ

ba


2

4

6

8

10

12

Org

an w

eigh

t (g)














9 Discussion














(a)

A

B

C

D

(b)

(c) (d)

(e)





10 Conclusions



Data Availability


Ethical Approval







References





















































































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



























Diclofenac


Ibuprofen


Aqueous extract

100 2733 2472 3126200 2926 3414 4560500 3383 4070 58171000 4251 4707 6679

Ethanolic extract

100 3087 2814 3612200 3279 4089 4824500 3730 5355 64111000 4444 6336 7748
















0 1 2 3 4 5 6 24 48 72 96 1205

6

7

8

9

10

αc

λ

λ

λ

βcλc

λcλbλ λbλb

λaλ

λ λ

λ λ λ

λ λ λ λ

λ λ λ

λ λb

λcλcλ λ

λ λ

λb

λ λ

λ

λ λ

λ

λ λ λ λλ λ

λ

λ

αcβcαcβc

λb

λ

λ

λ λ λbλ λ λ

λ λ

ccc

Time (hour)

Join

t dia

met

er (m

m)











(a)

A

B

C

(b)

(c) (d)

λ

(e)





λcλcλcβcλc

λcλcβcλc

λcλc

λcλcλcλcλb

λcλaλc

λb

λaλcλcλc

λcλc

λcλc

λcλcλcλc

λc

α

λλλλ

λλλλ

λλ

λ

λ

λ

λc

αcαc

λcλcλc

λcλc

λcλc

λcλbλc

λc

βcβ βcλc

λc


c

95

100

105

110

115

120

125

130

Join

t dia

met

er (

incr

ease

)

3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 351Period (week)





1 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 340

10

20

30

40

50

60

70

80

90

100

110

120

λcλc

λcλcλcλcλc

λc

λλcλc

λcλc

λc

λcλcλc

λc

λc

λcλcλcλc

λb

λc

λc

λcλcλc

λcλcλcλc

λcλc

λc

λa

λλλλλλλλ

λλλλ

λ

λcλcλcλc

λcλcλcλc

λcλc

λc

λc

λcλcλcλc

λcλcλcλc

λc

λcλc

λcλb

λcλcλc

λc

Period (week)

Sens

ibili

ty (

)








ccccc

c

c

cccc

c

cccbb

βββλ

λλ

λλλ

λλ

λ

λ

ccccc

cc

ccccc

b

ndash05

00

05

10

15

20

25

Art

hriti

s sco

re

33 3527 29 31211917 23 2513 15117 93 51Period (week)





0 1 2 3 4 585

90

95

100

105

110

115

λλλ

λλ

λλ

λ

λα

λλ

λ

β

λbλaλa

λ

λλ

Period (week)

Relat

ive b

ody

wei

ght (

)












βbαββaαβ

ba

αβ

ba


2

4

6

8

10

12

Org

an w

eigh

t (g)














9 Discussion














(a)

A

B

C

D

(b)

(c) (d)

(e)





10 Conclusions



Data Availability


Ethical Approval







References





















































































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of
























0 1 2 3 4 5 6 24 48 72 96 1205

6

7

8

9

10

αc

λ

λ

λ

βcλc

λcλbλ λbλb

λaλ

λ λ

λ λ λ

λ λ λ λ

λ λ λ

λ λb

λcλcλ λ

λ λ

λb

λ λ

λ

λ λ

λ

λ λ λ λλ λ

λ

λ

αcβcαcβc

λb

λ

λ

λ λ λbλ λ λ

λ λ

ccc

Time (hour)

Join

t dia

met

er (m

m)











(a)

A

B

C

(b)

(c) (d)

λ

(e)





λcλcλcβcλc

λcλcβcλc

λcλc

λcλcλcλcλb

λcλaλc

λb

λaλcλcλc

λcλc

λcλc

λcλcλcλc

λc

α

λλλλ

λλλλ

λλ

λ

λ

λ

λc

αcαc

λcλcλc

λcλc

λcλc

λcλbλc

λc

βcβ βcλc

λc


c

95

100

105

110

115

120

125

130

Join

t dia

met

er (

incr

ease

)

3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 351Period (week)





1 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 340

10

20

30

40

50

60

70

80

90

100

110

120

λcλc

λcλcλcλcλc

λc

λλcλc

λcλc

λc

λcλcλc

λc

λc

λcλcλcλc

λb

λc

λc

λcλcλc

λcλcλcλc

λcλc

λc

λa

λλλλλλλλ

λλλλ

λ

λcλcλcλc

λcλcλcλc

λcλc

λc

λc

λcλcλcλc

λcλcλcλc

λc

λcλc

λcλb

λcλcλc

λc

Period (week)

Sens

ibili

ty (

)








ccccc

c

c

cccc

c

cccbb

βββλ

λλ

λλλ

λλ

λ

λ

ccccc

cc

ccccc

b

ndash05

00

05

10

15

20

25

Art

hriti

s sco

re

33 3527 29 31211917 23 2513 15117 93 51Period (week)





0 1 2 3 4 585

90

95

100

105

110

115

λλλ

λλ

λλ

λ

λα

λλ

λ

β

λbλaλa

λ

λλ

Period (week)

Relat

ive b

ody

wei

ght (

)












βbαββaαβ

ba

αβ

ba


2

4

6

8

10

12

Org

an w

eigh

t (g)














9 Discussion














(a)

A

B

C

D

(b)

(c) (d)

(e)





10 Conclusions



Data Availability


Ethical Approval







References





















































































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of
























(a)

A

B

C

(b)

(c) (d)

λ

(e)





λcλcλcβcλc

λcλcβcλc

λcλc

λcλcλcλcλb

λcλaλc

λb

λaλcλcλc

λcλc

λcλc

λcλcλcλc

λc

α

λλλλ

λλλλ

λλ

λ

λ

λ

λc

αcαc

λcλcλc

λcλc

λcλc

λcλbλc

λc

βcβ βcλc

λc


c

95

100

105

110

115

120

125

130

Join

t dia

met

er (

incr

ease

)

3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 351Period (week)





1 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 340

10

20

30

40

50

60

70

80

90

100

110

120

λcλc

λcλcλcλcλc

λc

λλcλc

λcλc

λc

λcλcλc

λc

λc

λcλcλcλc

λb

λc

λc

λcλcλc

λcλcλcλc

λcλc

λc

λa

λλλλλλλλ

λλλλ

λ

λcλcλcλc

λcλcλcλc

λcλc

λc

λc

λcλcλcλc

λcλcλcλc

λc

λcλc

λcλb

λcλcλc

λc

Period (week)

Sens

ibili

ty (

)








ccccc

c

c

cccc

c

cccbb

βββλ

λλ

λλλ

λλ

λ

λ

ccccc

cc

ccccc

b

ndash05

00

05

10

15

20

25

Art

hriti

s sco

re

33 3527 29 31211917 23 2513 15117 93 51Period (week)





0 1 2 3 4 585

90

95

100

105

110

115

λλλ

λλ

λλ

λ

λα

λλ

λ

β

λbλaλa

λ

λλ

Period (week)

Relat

ive b

ody

wei

ght (

)












βbαββaαβ

ba

αβ

ba


2

4

6

8

10

12

Org

an w

eigh

t (g)














9 Discussion














(a)

A

B

C

D

(b)

(c) (d)

(e)





10 Conclusions



Data Availability


Ethical Approval







References





















































































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of





















λcλcλcβcλc

λcλcβcλc

λcλc

λcλcλcλcλb

λcλaλc

λb

λaλcλcλc

λcλc

λcλc

λcλcλcλc

λc

α

λλλλ

λλλλ

λλ

λ

λ

λ

λc

αcαc

λcλcλc

λcλc

λcλc

λcλbλc

λc

βcβ βcλc

λc


c

95

100

105

110

115

120

125

130

Join

t dia

met

er (

incr

ease

)

3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 351Period (week)





1 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 340

10

20

30

40

50

60

70

80

90

100

110

120

λcλc

λcλcλcλcλc

λc

λλcλc

λcλc

λc

λcλcλc

λc

λc

λcλcλcλc

λb

λc

λc

λcλcλc

λcλcλcλc

λcλc

λc

λa

λλλλλλλλ

λλλλ

λ

λcλcλcλc

λcλcλcλc

λcλc

λc

λc

λcλcλcλc

λcλcλcλc

λc

λcλc

λcλb

λcλcλc

λc

Period (week)

Sens

ibili

ty (

)








ccccc

c

c

cccc

c

cccbb

βββλ

λλ

λλλ

λλ

λ

λ

ccccc

cc

ccccc

b

ndash05

00

05

10

15

20

25

Art

hriti

s sco

re

33 3527 29 31211917 23 2513 15117 93 51Period (week)





0 1 2 3 4 585

90

95

100

105

110

115

λλλ

λλ

λλ

λ

λα

λλ

λ

β

λbλaλa

λ

λλ

Period (week)

Relat

ive b

ody

wei

ght (

)












βbαββaαβ

ba

αβ

ba


2

4

6

8

10

12

Org

an w

eigh

t (g)














9 Discussion














(a)

A

B

C

D

(b)

(c) (d)

(e)





10 Conclusions



Data Availability


Ethical Approval







References





















































































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of





















ccccc

c

c

cccc

c

cccbb

βββλ

λλ

λλλ

λλ

λ

λ

ccccc

cc

ccccc

b

ndash05

00

05

10

15

20

25

Art

hriti

s sco

re

33 3527 29 31211917 23 2513 15117 93 51Period (week)





0 1 2 3 4 585

90

95

100

105

110

115

λλλ

λλ

λλ

λ

λα

λλ

λ

β

λbλaλa

λ

λλ

Period (week)

Relat

ive b

ody

wei

ght (

)












βbαββaαβ

ba

αβ

ba


2

4

6

8

10

12

Org

an w

eigh

t (g)














9 Discussion














(a)

A

B

C

D

(b)

(c) (d)

(e)





10 Conclusions



Data Availability


Ethical Approval







References





















































































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of

























βbαββaαβ

ba

αβ

ba


2

4

6

8

10

12

Org

an w

eigh

t (g)














9 Discussion














(a)

A

B

C

D

(b)

(c) (d)

(e)





10 Conclusions



Data Availability


Ethical Approval







References





















































































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















9 Discussion














(a)

A

B

C

D

(b)

(c) (d)

(e)





10 Conclusions



Data Availability


Ethical Approval







References





















































































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of






















(a)

A

B

C

D

(b)

(c) (d)

(e)





10 Conclusions



Data Availability


Ethical Approval







References





















































































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of





















10 Conclusions



Data Availability


Ethical Approval







References





















































































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of


































































































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



















invitro anti-inflammatoryand invivo

Documents