Effect of Bryoniaalba in chemical induced
gouty arthritis in experimental animals
Synopsis for M. Pharm Dissertation
Submitted To
RAJIVGANDHIUNIVERSITY OF HEALTH SCIENCES
KARNATAKA,BANGALORE
By
Mr. YASHWANTH. B. M
Under the guidance of
Mr. K. P. SHIVALINGE GOWDA
Asst. Professor
Department of pharmacology
PES COLLEGE OF PHARMACY,
HANUMANTHANAGAR,KARNATAKA,
BANGALORE-560050
(2011-13)
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES
KARNATAKA-BANGALOR
ANNEXURE II
PROFORMA FOR REGISTRATION OF SUBJECTS FOR
1. /Name of the Candidate and Address
/ Residential Address:Yashwanth B.M
S/o Dr.B.Marimadaiah
#21,2nd main 2nd cross Reshmenagara (Ganigarapalya)Kanakpura road Bangalore-560062
2. /
Name of the Institution
/ PES College Of Pharmacy, Hanumanthanagar, Bangalore-5600503. / Course of Study and Subject / Master Of Pharmacy In Pharmacology
4. / Date of Admission to Course / 25/07/2011
5. /
Title of topic:
Effect of Bryonia alba in chemical induced gouty arthritis in experimental animals.
DISSERTATION
6. /Brief resume of the intended work
6.1 General Discussion:
Uric acid is the end product of purene metabolism in humans. The normal concentration of uric acid in humans is about 3-7 mg/dl. The daily excretion of uric acid is about 500-700mg/day. Hyperuricemia refers to an elevation in the serum uric acid which is sometimes associated with uricosuria (increased uric acid excretion).Gout is a metabolic disorder associated with over production of uric acid. At physiological pH uric acid is in a more soluble form of sodium urate. In severe hyperuricemia crystals of sodium urate get deposited in the soft tissues particularly in the joints. Such deposits are commonly called tophi which causes severe pain and inflammation in the joints called as gouty arthritis. Sodium urate may also precipitate in the kidneys and ureters that result in stone formation and renal damage.
The prevalence of gout is about 3/1000 people mostly affecting males. Post-menopausal women are however as susceptible as men for this disease. Gout is of two types-primary and secondary gout.
1. Primary gout: is an inborn error of metabolism leading to over production of uric acid mostly related to increased production of purene nucleotides as a result of metabolic defects associated with various enzymes of the uric acid pathway.
2. Secondary gout: is due to various other factors causing increased synthesis or decreased excretion of uric acid. Increase degradation of purene nucleotides (hence more uric acid formation) is observed in various cancers, psoriasis, increased tissue breakdown and various other pathological conditions.
The disorders associated with impairment of renal function cause accumulation of uric acid which may lead to gout.
Pseudo gout, the clinical manifestations of pseudo gout are similar to gout and can be easily misunderstood for gout, but this disorder is caused by the deposition of calcium pyrophosphate crystals in the joints. Further the serum uric acid concentration is normal in pseudo gout. [1]
Hyperuricemia does not necessarily lead to gouty arthritis. Many factors contribute to the conversion of asymptomatic hyperuricemia into primary gout, including the following:
•Ageof the individual and duration of the hyperuricemia are factors. Gout rarely appears before 20 to 30 years of hyperuricemia.
•Genetic predispositionis another factor. In addition to the well-defined X-linked abnormalities of HGPRT, primary gout follows multifactorial inheritance and runs in families.
Heavy alcohol consumption predisposes to attacks of gouty arthritis.
• Obesity increases the risk of asymptomatic gout.
• Certain drugs (e.g., thiazides) predispose to the development of gout.
• Lead toxicityincreases the tendency to develop saturnine gout
Central to the pathogenesis of the arthritis is precipitation of monosodium urate crystals into the joints. Synovial fluid is a poorer solvent for monosodium urate than plasma, and so with hyperuricemia the urates in the joint fluid become supersaturated particularly in the peripheral joints (ankle), which may have temperatures as low as 20°C. WithProlonged hyperuricemia, crystals and microtophi of urates develop in the synovium and in the joint cartilage. Some unknown event, possibly trauma, then initiates release of crystalsinto the synovial fluid, which begins a cascade of eventsthat initiates,intensifies,and sustains a powerful inflammatory response that is a hallmark of acute gout attack .[2]
Pathologic changes: the pathologic manifestations of gout include acute gouty arthritis, chronic tophaceous arthritis, tophi in soft tissues and renal lesions as under:
1. Acute gouty arthritis: this stage is characterized by acute synovitis triggered precipitation of sufficient amount of needle-shaped crystals monosodium urate from serum or synovial fluid. There is joint effusion containing numerous polymorphs, macrophages and micro crystals of urates. The mechanism of acute inflammation appears to include phagocytosis of crystals by leucocytes, activation of the kallikrein system, activation of the compliment system and urate-mediated disruption of lysosomes within the leucocytes leading to release of lyososmal products in the joint effusion. Initially there is monoarticular involvement accompanied with intense pain, but later it becomes poly articular along with constitutional symptoms like fever. Acute gouty arthritis is predominantly a disease of the lower extremities, affecting most commonly great toe. Other joints affected in order of decreasing frequency are: the instep, ankles, knees, wrist, fingers and elbows.
2.Chronictophaceous arthritis: recurrent attacks of gouty arthritis lead to progressive evolution into chronic gouty arthritis. The deposits of urate encrust the articular cartilage. There is synovial proliferation, pannus formation and progressive destruction of articular cartilage and subchondrial bone. Deposits of urates in the form of tophi may be found in the periarticular tissues.[3]
A gout attack can be brought on by alcohol, or foods high in Purines such as shellfish, caviar, sardines, anchovies, meats, or organ meats that are commonly used in sausages. A study over a 12-year period of 47,000 adult men revealed that those who ate the most red meat or seafood increased their risk of gout by as much as 50%.[4]
Drugs used in gout:
Allopurinol: allopurinol reduces the synthesis of uric acid by inhibiting xanthene oxidase. It is an analogue of hypoxanthine and inhibits enzyme mainly by substrate competition. Some degree of inhibition of de novo synthesis of purine synthesis also occurs. Allopurinol is converted to
alloxanthine by xanthine oxidase and this metaboloite, which remains in the tissue for a considerable amount of time, is an effective non-competitive inhibitor of the enzyme. Allopurinol reduces the concentration of the relatively insoluble uratesand uric acid in tissues, plasma in urine while increasing the concentration of the more soluble xanthines and hypoxanthines. The deposition of urate crystals in tissues (tophi) is reversed and formation of renal stones is inhibited. Allopurinol is the drug of choice in long term treatment of gout, but it is ineffective in the treatment of acute attack and indeed makes it worse.
Unwanted effects: are only a few. Gastrointestinal disturbances and allergic reactions (mainly skin rashes) can occur but disappear if the drug is stopped. Acute attacks of gout sometimes occur during the early stages of therapy.
Uricosuric agents: ate drugs that increase uric acid excretion by a direct action on the renal tubule. Examples are probenecidandsulfinpyrazole.
Colchicine: has a specific effect in gouty arthritis and can be used both to prevent and to relieve gout acute attacks. It prevents migration of neutrophils into the joint by binding to tubulin, resulting in the depolymerisation of the microtubules and interferes with cell motility. Colchicine treated neutrophils develop a ‘drunken walk’. Colchicines may also prevent production of a putative inflammatory glycoprotein by neutrophils that have phagocytosedurate crystals.
Unwanted effects: The unwanted effects of colchicines are largely gastro intestinal: nausea, vomiting and abdominal pain. Severe diarrhoea may be a problem and with large doses may be associated with gastrointestinal hemorrhage and kidney damage. Rashes sometimes occur, also peripheral neuropathy. Long courses of treatment have occasionally resulted in blood dyscrasias.[5]
6.2 Need for the study:
There are many complications of gout (gouty arthritis) that has major consequence on the way the patient leads his life. In acute gout there is accumulation of sodium urate crystals which triggers an inflammatory response associated with intense pain in the joints which if left untreated may result in chronic gout that further results in many other complications such as kidney stones, cardiac diseases like high blood pressure, congestive heart failure and coronary artery disease, swelling of the joints highly limit the range of motion, cause irreversible bone and joint deformities, and chronic pain. Allopathic medicines even though are specific in their pharmacological and therapeutic actions, they possessevere side effects which are undesirable. So the use of an alternative medicine becomes important here. Alternative medicines like ayurveda, siddha, homeopathy, unanietc, use ‘herbs’ as medicine. In this view the present study has been selected for evaluation of homeopathic medicine Bryoniaalbafor its anti-gouty arthritic property.
6.3 Review of literature:
Plant profile:
WHITE BRYONY (BryoniaalbaL.)Source :
KingdomPlanate Plants
SubkingdomTracheobionta– Vascular plants
Sub divisionSpermatophyta- seed plants
DivisionMagnoliophyta– Flowering plants
ClassMagnoliopsida– Dicotyledons
SubclassDilleniidae
OrderViolales
FamilyCucurbitaceae– Cucumber family
GenusBryoniaL.– bryony
SpeciesBryonia albaL.– white bryony[6]
Origin and Meaning :bryony – climbing plant (Latin), alba – white.[7]
Common name/Synonyms : BryoniaAlbavulg, Vitis Alba, Vitisnigris, White Bryony, Wild Hops.[7]
Habitat/Geographical Distribution: The plant is commonly seen growing in North of Europe, Germany, some parts of France, Spain, and north of Italy.[7]
Description/Morphology: Bryonia alba is a Perennial herbaceous climber with simple tendrils and stalked, alternate leaves. The leaves are caudate, five – lobed, dentate, calloso – scabrous. The Flowers are racemose, corymbose, monoeciousCalyx, in fertile flowers is as long as corolla, Stigma is smooth andBerry is black.[7]
Active Constituents: an alkaloid – bryonicin, and two glycosides – bryonin and bryonidin.[7]
Part used: Fresh roots[7]
Activities (White Bryony): Bryonia alba has been documented for the following activities Abortifacient; Antitumor; Cytotoxic; Diuretic; Emetic; Emmenagogue; Expectorant; Hydragogue; Hypoglycemic; Irritant; Lactagogue; Laxative; Toxic; Vermifuge.[8]
Indications (White Bryony) have been indicated in an array of diseases and disorders such as follows:Abscess; Acne ; Adenopathy; Allergy ; Alopecia; Aposteme ; Arthrosis ; Asthma; Blackhead; Bronchosis ; Cacoethes; an array of Cancers like Cancer of breast; Cancer of colon ; Cancer of face ; Cancer of gland Cancer of joint; Cancer, knee; Cancer, spleen; Cancer, stomach; Cardiopathy; Constipation; Cough; Edema; Enterosis; Epilepsy; Fatigue; Fever; Gastrosis; Gout; Headache; Hepatosis ; High Blood Pressure; hyperglycemia; Hysteria; Indurations; Infection; Inflammation; Insanity ; Insomnia; Leprosy; Lockjaw ; Lumbago; Madness ; Myalgia; Neuralgia; Neuromyososis;Pain; Paralysis; Pertussis; Pleurisy; Polyp; Psoriasis ; Pulmonosis; Radiculomyososis; Sciatica ; Respirosis; Rheumatism; Sclerosis; Splenosis; Tonsilosis;Tuberculosis ; Tumor ; Ulcer ; Virus ; Wart ; Water Retention ;Whitlow ; Worm ; Wound .[8]
Contraindications, Interactions, and Side Effects (White Bryony): Commission E reports the root is a drastic laxative and emetic, while other therapeutic uses are not adequately documented. Contains toxic cucurbitacins. May cause abortion, anuria, blisters, colic, collapse, convulsions, cramps, death, dermatosis, diarrhea, dizziness, emesis, hematochezia, nephrosis, neurosis, paralysis, rash, and/or vomiting (KOM; PH2).[8]
Reported activities:
- Unsaturated Polyhydroxy Acids having Prostaglandin-Like Activity from Bryonia alba II. Major Components[9]
- The effect of homeopathic medicines on yeast growth[10]
- Plant adaptogens. 11.* Bryonia as an adaptogen.[11]
- Effects of heavy physical exercise and adaptogens on nitric oxide content in human saliva[12]
- Flavone C-glycosides from Bryonia alba and B. dioica[13]
- Normalizing effect ofBryonia albaL. on blood phospholipids in alloxan diabetes[14]
7. / 6.4Objectives of the study :
- To investigate the effect of Bryonia alba in MSU (monosodium urate) induced gouty arthritis in mice.
- To investigate the effect of Bryonia alba in potassium oxonate induced gout in rats.
7.1 Source of data:
Whole work is planned to generate data from laboratory i.e., experiments on animals are performed as described in references. The rats and mice will be used for this purpose. Experimental studies in Journals and in text books available with college and various institutions. It is also planned to use the available literature for interpreting the data.
PESCP library, Bangalore
RGUHS digital library (Helinet), Bangalore.
Web site:
7.2 Materials :
Plant : Bryoniaalba.L. The plant is available for use in the form of various
homeopathic formulations for oral use which will be used as test drug for
the following studies.
Chemicals and drugs: MSU,Potassiumoxonate, uric acid kit, acid phosphatase kit.
Animals : Albino Mice, Wistar Rats.
Instruments : Research microscope, Centrifuge, Plethysmometer, Auto analyzer
7.3 Method:
7.3.1 Study of the effect of Bryonia alba in Monosodium urate (MSU) induced Gouty arthritis in rats:
Animals: Adult male albino rats of Wistar strain, weighing about 80±220 g, of either sex will be required.
Drug: the test drug Bryonia alba will be procured from homeopathic pharmacies.
Inducing agent: MSU will be synthesized.
Synthesis of Monosodium urate(MSU):
About 4 g of uric acid was dissolved and heated in 800 ml H2O with NaOH(9 ml/0.5N), adjusted to pH 8.9 at 60 ◦C; cooled overnight in a cold room; washed and dried. Needle-like crystals were recovered which were suspended in sterile saline (20 mg/ml) [15]
Monosodium urate crystal-induced inflammation in rats
Rats were divided into five groups—each comprising six animals. Group I served as controls, In group II, inflammation was induced by intradermal injection of 0.2 ml (4 mg) of monosodium urate crystal suspensioninto the right foot pad, groups III and IV comprised monosodium urate crystal-induced rats treated with Bryonia alba low dose and high dose respectively and group V consisted of monosodium urate crystal-induced rats were treated with standard indomethacin (3 mg/kg body weight).Bryoniaalbaandindomethacin were and orally administered,1 h before the monosodium urate crystal injection and then once daily for 3 days.[15]
Group No / Treatment / Dose and Route / Duration
1. / Control / P.o Vehicle. / All 3 days
2. / MSU control / 0.2 ml (4 mg) i.d Right foot pad. / Only 1st day
3. / MSU / 0.2 ml (4 mg) i.d Right foot pad. / Only 1st day
+ Bryonia alba (test drug) / Low dose, P.o / All 3 days
4. / MSU / 0.2 ml (4 mg) i.d Right foot pad. / Only 1st day
+ Bryonia alba (test drug) / High dose, P.o / All 3 days
5. / MSU / 0.2 ml (4 mg) i.d Right foot pad. / Only 1st day
+ indomethacin (standard) / 3mg/kg , P.o / All 3 days
Parameters to evaluate:
- Inflammation will be quantified by measuring the thickness of the paw with vernier scale /Plethysmometer at different intervals for 3 days.
- At the end of the experimental period (72 h), Blood from each animal will be collected for serum separation and the activity of acid phosphatase was determined by kit method
Animals: Swiss albino mice, 25–30 g, of either sex will be required.
Drug: the test drug Bryonia alba will be procured from homeopathic pharmacies.
Animal model of hyperuricemia in mice
Experimental animal model of hyperuricemia induced by uricase inhibitor potassium oxonate has been used to study drug action. Briefly, mice were injected intraperitoneally (i.p) with potassium oxonate (280 mg/kg) 1 h before the final drug administration to increase the serum urate level. Whole blood samples were collected from mice by tail vein bleeding. The blood was allowed to clot for approximately 1 h at room temperature and then centrifuged at 2500 × g for 10 min to obtain the serum. [16]
Drug administration
Food, but not water, was withdrawn from the animals 1.5 h prior to drug administration. Group I vehicle control andGroup II hyperuricemic control were orally administered with 0.9%saline solution for 1, 3 and 7 days, respectively. Group III will be orally treated with BryoniaalbalowdoseandGroup IV will be orally treated with Bryoniaalbahigh dose along with Potassium oxonatei.p 280mg/kgand Group V will be treated with allopurinol at 10 mg/kg orally and Potassium oxonatei.p 280mg/kg,for 1, 3 and 7 days, respectively. [16]
Group No / Treatment / Dose and Route / Duration
1. / Control / 0.9%saline solution P.o / 1day, 3days, and 7 days.
2. / Potassium oxonateHyperuricemic control / (280 mg/kg) , i.pand 0.9%saline solution P.o / 1day, 3days, and 7 days.
3. / Potassium oxonate / (280 mg/kg) , i.p / 1day, 3days, and 7 days.
+ Bryonia alba (test drug) / Low dose, P.o / 1day, 3days, and 7 days.
4. / Potassium oxonate / (280 mg/kg) , i.p / 1day, 3days, and 7 days.
+ Bryonia alba (test drug) / High dose, P.o / 1day, 3days, and 7 days.
5. / Potassium oxonate / (280 mg/kg) , i.p / 1day, 3days, and 7 days.
+ Allopurinol (standard) / 10 mg/kg, P.o / 1day, 3days, and 7 days.
Parameters to evaluate :
Serum uric acid will be determined by the kit method.
Statistical analysis :
Results were expressed as mean±S.D. and statistical analysis was performed using ANOVA to determine significant differences between groups.
7.4Does the study require any investigation to be conducted on patients or other humans or animals?
Yes the experimental methods require usage of experimental animals.
7.5Has ethical clearance been obtained from your institution in case of 7.4?
Yes, ethical clearance has been obtained [copy of IAEC clearance has been attached].
8.0 / References:
- Satyanarayana U, ChakpaniU. Biochemistry: Books and Allied (P) LTD; 2007.
- VinayK, AbulK, Fausto N, Jon aster C. Robbins and Cortan Pathologic basis of Disease. 8thed.
- Harsh M. Text book of pathology. 5th ed: Jaypee 2005.
- Choi HK, Atkinson K, Karlson EW, Willett W, Curhan G. Purine-rich foods, dairy and protein intake, and the risk of gout in men.N Engl J Med.2004 Mar 11;350(11):1093-103.
- Rang H, Dale M, Ritter J, Flower R. Rang and Dale's Pharmacology. sixth ed2007
- Bryonia alba[online].2011dec12;available from”:URL
- (12/12/2011)
- Duke JA, Bogenschutz-Godwin MJ, DuCellier. J, Duke P-AK. Hand book of Medicinal Herbs. 2nd ed: CRC Press.
- Panossian AG, Avetissian GM, Mnatsakanian VA, Batrakov SG, Vartanian SA, Gabrielian ES, et al. Unsaturated polyhydroxy acids having prostaglandin-like activity from Bryonia alba II. Major components. Planta Med. 1983;47(1):17-25.
- Hagelberg E. The effect of homeopathic medicines on yeast growth. British Homoeopathic journal. 1987;76(3):126-9
- Paossian A, Gabrielian E, Wagner H. Plant adaptogens. 11.Bryoniaas an adaptogen. Phytomedicine. 1997;4(1):85-99.
- Panossian AG, Oganessian AS, Ambartsumian, M, Gabrielian,E.S, Wagner H, Wikman G. Effects of heavy physical exercise and adaptogens on nitric oxide content in human saliva. Phytomedicine. 1999;6(1):17-26
- Krauze-Baranowska M, Cisowski W. Flavone C-glycosides from Bryoniaalba and B. dioica. Phytochemistry. 1995;39( 3 ):727-9.
- Vartanian GS, KaragezianKG. Normalizing effect of Bryoniaalba L. on blood phospholipids in alloxan diabetes. Vopr Med Khim. 1981;27(2):179-81.
- Rasool M, Varalakshmi P. Suppressive effect of Withaniasomnifera root powder on experimental gouty arthritis: An in vivo and in vitro study. Chemico-Biological Interactions 2006 164:174–80.
- Ji Xiao Zhu, Ying Wang, Ling Dong Kong, Cheng Yang, Zhang X. Effects of Biota orientalis extract and its flavonoid constituents, quercetin and rutin on serum uric acid levels in oxonate-induced mice and xanthine dehydrogenase and xanthine oxidase activities in mouse liver. Journal of Ethnopharmacology 2004;93:133–40
9.0 / NAME OF CANDIDATE / YASHWANTH. B. M
10.0 / SIGNATURE OF THE CNDIDATE / (YASHWANTH. B. M)
11.0 / REMARKS OF THE GUIDE
11.1 / NAME AND DESIGNATION OF THE GUIDE / Mr.SHIVALINGE GOWDA.K.P
ASST. PROFESSOR,
DEPT.OF PHARMACOLOGY
P.E.S COLLEGE OF PHARMACY
11.2 / SIGNATURE
11.3 / HEAD OF THE DEPARTMAENT / Mr.SRINATH.R
HOD& ASST.PROFESSOR
DEPT.OF PHARMACOLOGY
P.E.S.COLLEGE OF PHARMACY.
11.4 / SIGNATURE
12.0 / REMARKS OF THE PRINCIPAL / FORWARDED FOR APPROVAL
12.1 / SIGNATURE / Dr.S.Mohan
PRINCIPAL AND DIRECTOR
P.E.S.COLLEGE OF PHARMACY
BENGALORE-560050