“EFFECT OF FLAVANOIDS OF KIGELIA AFRICANA ON ADRENERGIC RECEPTORS OF ISOLATED TISSUES”
M. Pharm. Dissertation Protocol
SUBMITTED TO
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, BANGALORE
KARNATAKA
Bangalore – 560041
In partial fulfillment
Of the requirement for the Degree of
Master of pharmacy In Pharmacology
Under the Guidance Of
Dr. NAGARATHNA P.K.M
ASST. PROFESSOR OF PHARMACOLOGY
BY
VASANTHA KUMAR .C
DEPARTMENT OF PHARMACOLOGY
KARNATAKA COLLEGE OF PHARMACY,
BANGALORE-64
2011-2013
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES
KARNATAKA, BANGALORE.
ANNEXURE-II
PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION
1. / Name of the candidate and address / VASANTHA KUMAR .CS/O CHANDRAIAH M
#451 SUBASHCHANDRA BOSE ROAD
ADARSHANAGAR ARISHINAKUNE
NELAMANGALA TQ
BANGALORE-562123
2. / Name of the Institution / Karnataka College of Pharmacy,
THIRUMENAHALLI,YELAHANKA HOBLI, JAKKUR POST
BENGALURU-560064
KARNATAKA, INDIA
3. /
Course of study and subject
/ M.Pharm-PHARMACOLOGY4. /
Date of the admission
/ 29-08-20115. /
Title of the topic
“EFFECT OF FLAVANOIDS OF KIGELIA AFRICANA ON ADRENERGIC RECEPTORS OF ISOLATED TISSUES”6. /
BRIEF RESUME OF THE INTENDED WORK
7.8.
9.
10.
11. /
6.1 Introduction and Need of the work Enclosure I
6.2 Review of Literature Enclosure II
6.3 Aim and Objective of the study Enclosure III
7.1 Source of data Enclosure IV
7.2 Methods of collection of data Enclosure V
7.3 Does the study require investigation on animals?
a. If yes give details Enclosure VI
7.4 Has ethical clearance been obtained from Applied
your institution in case of 7?
List of references Enclosure VII
Signature of the candidate
VASANTHA KUMAR .C
11.1. Name and Designation
of Guide: / Dr. NAGARATHNA P.K.M
ASST. PROFESSOR OF PHARMACOLOGY
KARNATAKA COLLEGE OF PHARMACY
THIRUMENAHALLI BANGALORE
11.2. Signature of Guide:
11.3. Head of the Department
11.4. Signature of HOD: / [Dr. NAGARATHNA P.K.M.]
Dr.RAJU KONERI
Professor & Dean.
Dept. of Pharmacology,
Karnataka college of pharmacy,
Bengaluru-64.
[Dr.RAJU KONERI]
Remarks of the guide: The topic selected for dissertation is satisfactory. Adequate equipment and chemicals are available to carry out the project work.
12. / Remarks of the Principal: All the required facilities will be provided to carry out dissertation work under the supervision of guide.
Principal: / Dr.K.Ramesh.
Director
Karnataka college of pharmacy,
Thirumenahalli,
Bangalore –64.
Signature of the Principal: /
(Dr.K.RAMESH)
ENCLOSURE – I
6. BRIEF RESUME OF INTENDED WORK:
6.1. INTRODUCTION AND NEED OF THE WORK:
Plant-Kigelia Africana(Bignoniaceae):
Synonyms: Bignonia africana Lam, Crescentia pinnata Jacq., Kigelia aethiopum (Fenzl) Dandy, K. pinnata (Jacq.) DC and others. The species is very variable but it is now generally accepted that there is only one species in the genus and thus all Kigelia spp. are synonymous for Kigelia africana.
Vernacular/common names: sausage tree, cucumber tree8.
Kigelia africana (Bignoniaceae) plant grows commonly in tropical west, east and central African regions especially in low plains along Nigeria and cultivated in many parts of India as an ornamental and roadside tree6. It represents an interesting example of a plant used in traditional medicine for many years, but which is now attracting interest and use far beyond its original geographical range (Kolodziej, 1997). It is commonly called ‘Sausage tree’ or ‘Cucumber tree’ due to its long sausage-like fruit1.
Phytochemicals have been documented with Antidiarrheal, Antileprotic, Antimalarial, Anti-inflammatory, Anti-microbial, Anticancer, anti-implantation, treatment of gynaecological disorders, Central nervous system (CNS) stimulant and anti-oxidative property {which apparently makes it useful in the treatment of diseases especially the liver-borne disease, (Olaive and Rocha, 2007)}.
The ethno medicinal plant bark is used for the treatment of rheumatism, dysentery and veneral diseases. It is also used as ringworm and tapeworm expellant, while other uses include treatment of haemorrhages, diabetes, pneumonia and toothache2. Dried powdered fruit is used as purgative, for the treatment and dressing for ulcers and to increase the flow of milk in lactating women7. Various chemical investigations have been carried out on K. africana and many chemical compounds mainly iridoids, naphthaquinones, monoterpenoid-naphthaquinones, isocoumarins, lignans sterols and flavonoids have been identified2. It is also reported to have ˠ-sitosterol (cytotoxic sterol) and tritriacontane in the root bark5. In South eastern Nigeria, the fruits and flowers are mixed with alcohol or water and used by traditional healers for fertility treatment among women and men of child bearing age (Ogbeche et al., 2002)3. K.africana has strong anti‐oxidative effects against hepatotoxicity induced by paracetamol toxicity. It is speculated that the antioxidant activity is attributed to the caffeic acid derivative and compounds unique to Kigelia. Other notable bioactivities include its antimicrobial action against sexually transmitted diseases4. The ethanolic extract of Kigelia africana has a potential tocolytic effect that can be explored for therapeutic advantage as an alternative treatment for threatened abortion and dysmenorrhoea9.
Adrenergic receptors:
Adrenergic receptors (α1A-, α1B-, α1D-, α2A-, α2B-, α2C-, β1, β2, β3) are members of the G-protein-coupled receptor (GPCR) superfamily of membrane proteins that mediate the actions of the endogenous catecholamine’s, norepinephrine and epinephrine10. In the sympathetic nervous system two families of adrenergic receptors can be distinguished pharmacologically i.e., α and β on the basis of their response to the adrenergic agonists.
1) α- adrenergic receptors –
The α-adrenergic receptors are divided into two sub group’s α1, α2 based on their affinities for α agonists and blocking drugs. The rank order of potency is
Epinephrine ≥ norepinephrine ≥ isoproterenol.
α1 receptors –
These are present on the post-synaptic membrane of the effector organ and mediate many of the classic effects like contraction of smooth muscle. Activation of α1 receptors initiates a series of reactions through G protein activation of phospholipase C, resulting in the generation of inosotol-1,4,5-triphosphate from phosphatidylinositol, causing release of calcium from the endoplasmic reticulum into the cytosol.
α2 receptors –
These receptors primarily located on presynaptic nerve endings and other cells such as the β cell of the pancreas. Circles back norepinephrine reacts with α2receptors on the neuronal membrane. The effects of binding at the alpha 2 receptors are mediated by inhibition of adenylyl cyclase and a fall in the levels of intracellular cAMP.
Tissues used for screening of α1, α2 receptors-
α1- Rat isolated anococcygeus muscle,
Rabbit isolated jejunum,
Guinea pig isolated ileum.
α2 - Guineapig isolated ileum,
Rabbit isolated jejunum.
2) β-adrenergic receptors –
The beta adrenergic receptors can be subdivided into two major subgroups β1, β2 based on their affinities for adrenergic agonists and antagonists.β1receptors have equal affinities for epinephrine and norepinephrine. β2 receptors have higher affinity for epinephrine than for norepinephrine. Binding of neurotransmitter to the beta receptors causes activation of adenylyl cyclase and increased concentration of cAMP within the cell.
The rank order of potency is:
Isoproterenol > epinephrine > norepinephrine.
Tissues used for screening of β 1, β2 receptors-
β1 – Rabbit isolated jejunum.
β2 – Rat isolated uterus,
Guinea pig isolated trachea.
Blockade of vasoconstrictor α1 causes reduce in cardiac output and fall in BP. Hypotension produced by alpha blockers can reduce renal blood flow. β-receptor antagonists decreases force of contraction and cardiac output. α-blockers are used in Pheochrimocytoma, congestive heart failure and Benign hypertrophy of prostate etc. β-blockers are used in Hypertension, Angina pectoris, Cardiac arrhythmias, Myocardial infraction, Congestive heart failure, Migrane and Anxiety etc.
ENCLOSURE – II
6.3- REVIEW OF LITERATURE:
Kigelia africana (Bignoniaceae) commonly found in tropical west, east and central African regions especially in low plains along Nigeria and also in various states of India especially in West Bengal. The plants is used traditionally for treating cancer of the breast, uterus and skin, digestive disorder, genito-urinary tract, veneral diseases, gynaecological disorder, bladder ailments, sickle-cell anaemia, epilepsy, nutritional illness, leg oedemas, internal parasitic infestations (especially tapeworm), leprosy, rheumatism, boil, acne, cysts, whitlows and psoriasis2. Many phytoconstituents of Kigelia africana are isolated through systematic phytochemical approach.
The phytochemicals present in K.africana shows its anti-microbial action by disc diffusion method. The methanol extract presented a higher activity than the aqueous extracts and chloroform extracts against all except E.aerogens, Klebsiella Pneumoniae and Psedomonas aeruginosa which presented less activity. The dichlomethane extracts of the root bark and stem bark exhibited antitrypanosomal activity against Trypanosoma brucei brucei (in vitro). The extract of the tree stem bark was also established to inhibit a number of harmful micro-organisms which include E. coli (responsible for abscesses), P.aeruginosa (which causes skin sepsis and infections), S.aureus (which causes impetigo and skin abscesses) and albican (a fungal organism that causes thrust) in another experiment. The antibacterial and antifungal test carried out on the crude ethanolic stem bark extract revealed exhibited antibacterial and antifungal activities against s. aureus and candida albicans. The aqueous extract exhibited no antibacterial and antifungal activity whereas the activity of the crude ethanolic extract (20 mg/ml) is comparable to amoxicillin drug. Butanolic extract of the stem bark exhibited invitro antiamoebic activity when tested against HK-9 strain of Entamoeba histolytica (micro dilution method) using metronidazole as reference drug. It was found that verminoside (in the extract) has two fold antiamoebic activity as compare to the standard drug while specioside showed comparable activity with metronidazole2.
Ahlquist in 1948 introduced the concentration of excitatory α-receptors and inhibitory β-receptors. Alpha receptors are present pre and post-synaptically11. α1-Adrenergic receptors mediate vasoconstriction in the pulmonary and systemic vasculature12 whereas β1-Adrenergic receptors can activate extracellular signal-regulated kinases (ERKs) via different mechanisms13.
The rank order of potency for α-receptors is
Epinephrine ≥ norepinephrine ≥ isoproterenol.
The rank order of potency for β-receptors is
Isoproterenol > epinephrine > norepinephrine.
ENCLOSURE – III
6.4-OBJECTIVE OF THE STUDY:
Ø To isolate flavonoids from the leaves of Kigelia africana.
Ø To carry out the effect of flavonoids of Kigelia africana on adrenergic receptors α1 by using anococcygeus muscle.
Ø To carry out the effect of flavonoids of Kigelia africana on adrenergic receptors α2 by using guineapig ileum.
Ø To carry out the effect of flavonoids of Kigelia africana on adrenergic receptors β1 by using rat Jejunum.
Ø To carry out the effect of flavonoids of Kigelia africana on adrenergic receptors β2 by using rat uterus and Guinea pig Trachea.
ENCLOSURE – IV
6.5- SOURCE OF DATA:
Whole work is planned to generate data from laboratory studies i.e. experiments are performed as described in references.
Experimental studies in National and International Journals and in text books available with college, IISc and other libraries.
Literature is searched from various web sites in the internet, CD ROM, Chemical abstracts.
ENCLOSURE – V
6.6- MATERIALS AND METHODS
A. Preparation Of Extract:
Powdered leaves were subjected to successive extraction in a Soxhlet extractor with methanol. The extract obtained was concentrated in a rotary shaker and evaporated to dryness to get a constant weight.
B. MATERIALS AND METHODS
Methodology:
1) α1 - Depolarization of anococcygeus muscle(α1) 14:
Tissues will be obtained from male Wistar rats (250-350g).The pair of anococcygeus muscles will be split at the ventral bar. Tissues will be mounted ventrically in an organ bath (70ml) between a horizontal glass rod and an isometric tension transducer (Grass FT03C), under a tension of 1g.Tissues will be incubated with Krebs-Henseleit solution of the following composition:
NaCl-118mM, KCl-4.7mM, CaCl2-1.6mM, MgSO4-1.2mM, NaHCO3-24.9mM, KH2PO4-1.2mM, glucose-11.1mM, Ascorbic acid-0.28mM and Na2EDTA-0.03mM.
The solution will be equilibrated at 380c with a mixture of 95%O2 and 5%CO2 and contained propranolol (4x10-6M) to block neuronal and Extraneuronal amine uptake and β-adrenoceptors respectively.
a. Antagonist drugs employing are: Rauwolscine (selective α2) and Prazosin (selective α1).
b. Agonist drugs employing are: Phenylephrine (selective α1) α-Methylnoradrenaline (mixed) Noradrenaline and Xylazine(α2).
Tissues will be initially incubated for 1hr, with change of bathing fluid every 15min.Two cumulative concentration response curves, separated by 2hr, will be obtained to the agonist. For interaction experiments, the tissue will be exposed to a single concentration of the test antagonist from 1hr before the second concentration response curve to the agonist. Experiments will be carried out using concentrations of antagonist which gives shifts in the agonist concentration response curve of 2-100 times. From the concentration response curves, EC50 values of the agonist will be estimated by probit analysis and will be used to calculate dose ratios and PA2values.
2. EFFECT ON RAT UTERUS (α2)15:
Uterine fragments will be obtained from virgin Wistar rats (180-250 g), 24hr after treatment with stilbestrol 0.1mg/kg. Animal will be sacrificed by a blow on head and carotid bleeding. Pelvic region will be cut opened and both the horns of uterus are exposed. They are separated gently from the surrounding fatty material and transferred them in to a dish containing De Jalon’s solution. When the rat is in oestrus stage generally the uterus is fleshy and pink in colour. Tissue will be mounted in an organ bath containing De Jalon’s solution at 30-320c. Tensions of 0.5g will be applied and the tissue will be allowed to equilibrate for 30min. Then relaxations will be recorded due to different concentrations of noradrenaline using frontal writing lever .Contact time of 30sec and 3min time cycle will used for proper recording of the responses. At least four responses will be recorded due to different concentrations of noradrenaline. Once reproducible curves to a given agonist were obtained, different concentration of yohimbine will be added, which is specific alpha-2 antagonist and causes relaxation, and concentration response curve is recorded. Then add the test compound and see the effect on uterus. From the concentration response curves, EC50 values of the agonist will be estimated and also it will be used to calculate dose ratios and PA2 values.
3) EFFECT ON GUINEAPIG TRACHEA (β2)16 -
Tissue preparations
Male guinea pigs (400-700 gr) will be sacrificed by a blow on the neck and tracheae will be removed. Each trachea will be cut into 10 rings (each containing 2-3 cartilaginous rings). All the rings will be then cut opened opposite the tracheal muscle, and sutured together to form a tracheal chain. Tissue will be then suspended in a 10 mL organ bath (organ bath 61300, Bio Science Palmer-Washington, Sheerness, Kent U.K.) containing Krebs-Henseliet solution of the following composition (mM): NaCl 120, NaHCO3 25, MgSO4 0.5, KH2 PO4 1.2, KCl 4.72, CaCl2 2.5 and dextrose 11.The Krebs solution will be maintained at 370C and also gassed with 95% O2 and 5% CO2 .Tissue will be suspended under isotonic tension of 1g and allowed to equilibrate for at least 1hr while it will be washed with Krebs solution every 15 min. Contractions are then recorded which is due to different concentrations of isoproterenol using frontal writing lever. Contact time of 30sec and 3min time cycle is used for proper recording of the responses. At least four responses will be recorded due to different concentrations of isoproterenol. Once reproducible curves to a given agonist were obtained, the crude extract of Kigelia africana (0.5 to 4mg /ml) will be added to the bath and a last curve is constructed in its presence. Each preparation will be exposed to only one agonist. Propranolol will be used as the standard beta-2 antagonist. The sensitivity to agonist will be evaluated at the EC50 level. The pA2 value for the extract against isoproterenol (as - log g ml') will be calculated by graphical interpolation according to the method of Arunlakshana & Schild (1959). Oxytocin (Syntocinon, Sandoz, Sio Paulo) will be dissolved in aqueous solution.