THE INVESTIGATION OF NEUROPROTECTIVE PROPERTY OFADENOCALYMMA ALLIACEUM MIERS LEAVES
IN EXPERIMENTAL ANIMALS
M. Pharm Dissertation Protocol
Submitted to the
Rajiv Gandhi University of Health Sciences, Karnataka, Bengaluru
BY
MR.DIPTENDU BHOWMIK
B.Pharm.
UNDER THE GUIDANCE OF
PROF.ITTAGI SHANMUKHA
M.Pharm., (Ph.D).
P. G. DEPARTMENT OF PHARMACOLOGY
S. C. S. COLLEGE OF PHARMACY,
HARAPANAHALLI-583131
2013-2014
Rajiv Gandhi University of Health Sciences, Karnataka, Bengaluru
Annexure – II
PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION
01 / Name and Address of the Candidate / DIPTENDU BHOWMIKC/O LATE DILIP KUMAR BHOWMIK
vill(post) rajarbag
tripura (state)
udaipur- 799116.
S02 / Name of the Institution / T. M. A. E. Society’s
S. C. S. College of Pharmacy,
Harapanahalli – 583 131
Davangere(dist), Karnataka.
03 / Course of the Study
Branch / M. Pharm.
(Pharmacology)
04 / Date of Admission to course / 30-07-2013
05 / Title of the Topic / THE INVESTIGATION OFNEUROPROTECTIVE PROPERTYOF ADENOCALYMMA ALLIACEUMMIERS LEAVES IN EXPERIMENTAL ANIMALS
06 /
Brief resume of the intended work
6.1. Need for the Study /Enclosure – I
6.2. Review of the Literature / Enclosure – II6.3. Objective of the Study
/ Enclosure – III07 /
Materials and Methods
7.1. Source of data /Enclosure – IV
7.2. Methods of collection of data
/ Enclosure – V7.3. Does the study require any
Investigations on animals?
If yes give details / Enclosure – VI
7.4. Has ethical clearance been
obtained form your institution
in case of 7.3. / Yes, Registration No: 157/PO/C/ 1999/ CPCSEA
(Copies enclosed)
08 /
List of References (About 4 – 6)
/ Enclosure – VII09 /
Signature of the Candidate
/ (diptendu bhowmik)10 /
Remarks of the Guide
/ The present research work is original and not published in any of the journals. This work can be carried out in our Pharmacology laboratory.11 /
Name and Designation of
(in Block Letters)11.1. Guide
11.2.Signature
11.3.Co-Guide (if any)
11.4.Signature
11.5. Head of the Department
11.6. Signature / Prof. ITTAGI SHANMUKHA
M. PHARM.,(PhD)
P.G. Dept. of Pharmacology
S.C.S. College of Pharmacy
Harapanahalli-583 131.
Davangere. (Dist.)
Karnataka
------
Prof. A. VEERANA GOUDA
M. Pharm.
Head of the dept. of pharmacology
S.C.S. College of Pharmacy
Harapanahalli-583 131.
Davangere. (Dist.)
Karnataka
12 /
Remarks of the Principal
12.1. Signature / The present study is permitted to perform in the Pharmacology laboratory of ourinstitution and the study protocol has been approved by IAEC.Dr.R.Nagendra Rao
Principal
ENCLOSURE-I
06. Brief resume of Intended Work
6.1 Need for the study.
Numerous physiological and biochemical processes in the human body may produce oxygen-centered free radicals and other reactive oxygen species as byproducts. Overproduction of such free radicals can cause oxidative damage to biomolecules (e.g. lipids, proteins, DNA), eventually leading to many chronic diseases such as atherosclerosis, cancer, diabetes, aging and other degenerative diseases in humans1. Free radicals are potentially important in a number of ailment states that can have severe effects on the cardiovascular system and nervous systemeither through lipid peroxidation or vasoconstriction.
Plants are exploited as food and medicine since from ancient periods. The therapeutic use of medicinal plants has gained a considerable momentum in the past few decades. The over usage of synthetic drugs resulting in higher incidence of adverse drug reactions have motivated mankind to go back to nature for safer remedies. Continuous exposure to stressful conditions generates free radicals, which may over power the inbuilt protective mechanisms and cause tissue damage. There are many reports that plants possessing free radical scavenging activity are known to have organ protective effect2,3. Carotenoids and non-nutrients like dietary fibers and other phenolic components (falvonoids and tannins) can influence the enzymes involved in the activation and detoxification of xenobiotics including carcinogens4. Similarly, many such herbs are known to protect the organs and organ systems from the environmental, chemical and occupational changes.
Neurodegenerative disorders are a heterogeneous group of diseases of the nervous system including the brain, spinal cord and peripheral nerves that have many different etiologies.5Acute and chronic neurodegenerative diseases are illnesses associated with high morbidity and mortality and few or no effective options are available for their treatment. A characteristic of many neurodegenerative diseases which includes stroke, brain trauma, spinal cord injury, amyotrophic lateral sclerosis, Huntington’s disease, Alzheimer’s disease, and Parkinson’s disease.6Many are hereditary, some are secondary to toxic or metabolic processes and others result from infections. Due to the prevalence of morbidity and mortality of the neurodegenerative diseases, they represent significant medical, social and financial burden on the society. Neuropathologically, these are characterized by abnormalities of relatively specific regions of the brain and specific population of neurons. The degenerating neuron clusters in the different diseases determine the clinical phenotype of that particular illness. Recent investigations in medical genetics have identified specific genes for various neurodegenerative disorders and specially bred animal models have begun to be used to study the etiologicalfactors and underlying pathogenic mechanisms. There are three learning objectives of basic neuroscience: (i) understanding of fundamental concepts of neurodegenerative disorders, (ii) recognition of specific morphological (gross and microscopic) features of each major disease and their general correlation to disease manifestations and (iii) developing and understanding of genetics, treatment options and clinical features of each individual diseases5.
Mechanism responsible for neuronal degeneration are excitotoxicity, oxidative stress, apoptosis, protein misfolding and inflammation, hence controlling the glutamate release, NMDA receptor antagonists, calcium channel blocker, free radical scavengers, anti-inflammatory and oestrogenic drugs may be beneficial in protecting neurons from damage7.
Adenocalymma alliaceum(Ajos sacha) is an evergreen tropical shrubby vine that is native to the Amazon rainforestwhich is reported to contains several of the main sulfur compounds that garlic does.It is these compounds which are responsible for the garlic-like odor and taste of adenocalymma alliaceum.The leaves and/or flowers of this plant contain the known anti-inflammatory and antibacterial plant steroids beta sitosterol, stigmasterol, daucosterol, and fucosterol. Chemicals reported in ajos sacha thus far include: 24-ethyl-cholest-7-en-3-beta-ol, 3-beta-hydroxy-urs-18-en-27-oic acid, alliin, allyl sulfides, alpha 4-hydroxy-9-methoxy-lapachone, alpha 9-methoxylapachone, apigenins, aspartic acid, beta-sitosterol, beta amyrin, beta-peltoboykinolic acid, cosmosiin, cyanidin-3-o-beta-d-rutinoside, daucosterol, diallyl sulfides, 1-2: 3-vinyl-dithi-4-ene, 1-2: 3-vinyl-dithi-5-ene, dithiacyclopentene, dotriacontan-1-ol, fucosterol, glutamic acid, glycyrrhetol, hentriacontanes, hexacosan-1-ol, hexatriacontans, leucine, luteolin, n-nonacosane, oct-1-en-3-ol, octacosan-1-ol, pentatriacont-1-en-17-ol, scutellarein-7-o-beta-d-glucuronide, stigmasterol, triacontan-1-ol, triallyl sulfides, trithiacyclohexene, n-tritriacontane, and ursolic acid8.
There are reports which are already been documented in the literature by research that the plant has uses for the treatment ofarthritis, nervous shock , nervousness, epilepsy, abdominal pain, for headaches,body aches,asthma, cold, coughs, cramps, epilepsy, fatigue, fever, flu, inflammation, malaria, pneumonia, skin problems and uterine disorders. Adenocalymma alliaceae (Garlic creeper as indian traditional name)using also as analgesic, antipyretic, antirheumatic , antitussive, antiviral, antifungal, depurative, purgative andantitussive, purgative and insect repellent8.
The sulfur compounds (the predominate ones being alliin and various allyl sulfides) in both garlicand ajos sacha have been studied by many and reported over the years to be able to lowercholesterol. Extract of ajossacha leaves was reported to have an antioxidant effect which was attributed to the anthocyanincompounds found in the plant. Antioxidants are reported to have organ protective role2. Since the study plant using in lowering cholesterol level, nervous shock and nervousness. Hence the present study has been under taken with an aim to determine the antioxidant and neuroprotective activity of leaf extract of Adenocalymma alliaceaeMiersin experimental animal models.
ENCLOSURE-II
6.2Review of Literature8,9:
The literature survey reveals that the plant Adenocalymma alliaceum Miers belongs to family Bignoniaceae with the synonyms Adenocalymma alliaceum, Adenocalymma pachypus, Adenocalymma sagotii, Bignonia alliacea, Pachyptera alliacea. Pseudocalymma alliaceum, Pseudocalymma pachypus, Pseudocalymma sagotti8,9.
Common Names8,9:
Adenocalymma pachypus, Adenocalymma sagotii, Pachyptera alliacea, seudocalymma alliaceum,Pseudocalymma pachypus and Pseudocalymma sagottiis also known as Garlic vine.
The taxonomic position ofAdenocalymma alliaceum Miers is as follows:
Family : Bignoniaceae Order : Lamiales
Genus : Adenocalymma
Species : A.alliaceum
Description:
Flowering: March-August. Habitat : Waste ground, disturbed sites, roadsides,railroads. Origin : Native to Amazon rain forest of south America.
Leaves :Leaves are simple, 7-15 cm x 4-5 cm in size, ovate tolanceolate, apex
mucronate,margin entire, surfaceglabrous and glaucous, texture
papery, base symmetricand tapering,venation reticulate, color of
upper surfacegreen and lower surface is light green. Petiole is
roundand green in color . On crushing leaves givesgarlic like smell
and flavour.
Stem:Stem is 7-8cm in length and 10-20mm in diameter.Surface is rough
because of smallpits present on stem.Shape is cylindrical,colour of
young stem is green and old stem is light brownish.It has garlic like
smell and taste.
Chemical constituents9,10:
Ajos sacha contains several of the main sulfur compounds that garlic does. These compounds which are responsible for the garlic-like odor and taste of ajos sacha. The wood of the vine was reported to contain two lapachone. The leaves and/or flowers contain steroids like beta sitosterol,stigmasterol, daucosterol, and fucosterol. Chemicals reported in ajos sacha thus far include: 24-ethyl-cholest-7-en-3-beta-ol, 3-beta-hydroxyurs- 18-en-27-oic acid, alliin, allyl sulfides, alpha 4-hydroxy-9-methoxy-lapachone, alpha 9-methoxylapachone, apigenins, aspartic acid, beta-sitosterol, beta amyrin, beta-peltoboykinolic acid, cyanidin-3-o-beta-d-rutinoside, daucosterol, diallyl sulfides, 1-2: 3-vinyl-dithi-4-ene, 1-2: 3-vinyl-dithi-5-ene, dithiacyclopentene, glutamic acid, glycyrrhetol, hentriacontanes, hexatriacontans, leucine, luteolin, n-nonacosane, octacosan-1-ol, pentatriacont-1-en-17-ol, scutellarein-7-o-beta-d-glucuronide, stigmasterol, triacontan-1-ol, triallyl sulfides, trithiacyclohexene and ursolic acid9,10.
Traditional and Medicinal Uses:
Rheumatism; for coughs, colds, flu, pneumonia and upper respiratory conditions; as a general pain-reliever (headaches, muscles, joints, body aches); for fevers (malaria, flu, etc.); for general inflammation (external and internal), antipyretic.The Creoles in Guyana use the leaves in baths for cramps and fatigue and the Tapajosin Brazil use it in baths for body aches and the flu. The bark is typically prepared in a tincture or a decoction forthese types of conditions but the leaves are used similarly for the same conditions as well. The leaves are generally prepared as an infusion or decoction. The root isalso prepared in a tincture or a cold maceration and takenas a general whole-body tonic9,10.
ENCLOSURE –Ш
6.3 Objectives of the study:
The objective of study is to evaluate the antioxidant and neuroprotective effects of leaf
extract on experimentally induced neurotoxicity in rats.
01. To prepare various extracts (petroleum ether extract, chloroform extract, alcoholic
extract and aqueous extract) by successive extraction technique.
02. To identify the phytoconstituents present in the leaf extract.
03. Quantification of phytoconstituents.
04. To assess acute toxicity of the leafextract.
05. To assess the antioxidant property (Super oxide anion scavenging activity, Hydroxyl
radical scavenging activity and DPPH method).
06. To assess the neuro protective activities [mono sodium glutamate, aluminum
fluoride and Carotid artery occlusioninduced neuro toxicity in rats]by observing
following parameters
- Measurement of body weight change.
- Behavioral tests:General behavior, ambulatory behavior, elevated plus maze test, balance beam test and Rota rod test.
- Brain tissue biochemical estimation:Glutathione, catalase (CAT), superoxide dismutase (SOD), lipid peroxidation and protein estimation.
- Brain tissue histopathological changes.
ENCLOSURE – IV
7. Materials & methods:
7.1 Source of data:
Whole work is aimed to generate data from the laboratory i.e., experiments on animals.
Albino rats and mice will be used for this purpose.
Chemicals:
Nitroblue tetrazolium, nicotinamide adenine dinucleotide, phenazine methosulpahate, phenyl hydrazine hydrochloride, deoxyribose, trichloro acetic acid, monosodium glutamate, aluminum fluoride, ethanol, anesthetic ether, tetraethoxy propane, glutathione reductase, glutathione, phenazine methosulphate, bovine serum albumin, nitroblue tetrazolinium etc
ENCLOSURE – V
1 7.2 Materials and methods
The whole study is divided into four phases to generate the data as follows.
Phase 01. Preparation of extract and Identification of phytoconstituents11,12:
The leaf extract will be prepared by successive soxhlation i.e. extracting dried powder with the solvents of increasing order of polarity i.e. Pet. ether (60-80), chloroform (59.5-61.5), 70% ethanol (64.5-65.5) and water. Extracts will be concentrated under reduced pressure.
Phase 02. Acute toxicity studies13:
The toxicity of leaf extract of A.alliaceae mierswill be determined by using albino female rats (200-250 g) maintained under standard husbandry conditions. The animals will be fasted for 3 hr. prior to the experiment. Animals will be administered with single dose of extract and observed for its mortality up to 48 hr. study period (short term toxicity). Based on the short-term toxicity profile, the next dose will be determined as per OECD guidelines No 423.
Phase 03. Experimental design:
Quantification of phytoconstituents:
Quantification of total phenolic content14:
The total phenolic content of the extract ofwill be determined by taking aliquots of the extractin a 10ml glass tube and the volume will bemade up to 3ml with distilled water. Then 0.5ml of Folin ciocalteau reagent (1:1 with distilled water) and 2 ml Sodium carbonate (20%) will be added subsequently in each test tube. A blue colour will be developed in each test tube because the phenols will undergo complex redox reaction with phosphomolibdic acid in Folin ciocalteau reagent in alkaline medium this results in a blue coloured complex, molybdenum blue. The test solutions will be warmed for 1 min, cooled and measured at 650nm using known concentration of catechol. The concentrations of phenols in the test samples will be calculated from the calibration plot and expressed as mg catechol equivalent of phenol per gram of sample.
Quantification of total flavonoid content14:
To determine the total flavonoidal content, stock solutions of theextract will be prepared with ethanol to a suitable concentration for analysis. For determination of total flavonoidal content, aliquots of each extract will be pipetted out in series of test tubes and the volume will be made up to1ml with distilled water; then Sodium nitrite (5%; 0.3ml) will be added to each test tube and incubated for 5min at room temperature; then aluminium chloride solution (10%; 0.06ml) will be added and incubated for 5min. at room temperature; then Sodium hydroxide (1M; 0.25ml) will be added and total volume will be made to 3ml with distilled water. Absorbance will be measured at 510nm against a reagent blank using U.V. spectrometer and concentration of flavonoids in the test sample will be determined and expressed as mg equivalent per gram of sample.
Quantification of tannins15:
The tannins will be identified by using FeCl3 and gelatin tests. For quantification, 0.1g of the extract will be transferred to a 100ml flask; 50ml of water will be added and boiled for 30min. After filtration with cotton filter, filtrate will be transferred to a 500ml flask and the volume will be made up to the mark with distilled water. 0.5 ml aliquots will be transferred to vials, 1ml of 1% K3Fe(CN)6 and 1 ml of 1% FeCl3 will be added and the volume will be made up to 10ml with distilled water. After 5 min the solution will be measured colorimetric ally at 720nm. The total content of tannins present in the plant extract will be obtained from standard calibration curve which will be made by taking the tannic acid as standard.
Antioxidant property:
Super oxide anion scavenging activity16
Hydroxyl radical scavenging activity17
DPPH 18
Neuro protective activity:
Monosodium glutamate induced neurotoxicity model19
The animals will be divided into six groups of six rats each as follows and treated for 7 days as fallows;
Group 1: Receives normal saline (i.p.) + vehicle (p.o.).
Group 2: Receives MSG 2 g/kg (i.p.) + normal saline (p.o.).
Group 3: Receives MSG 2 g/kg (i.p.) + low dose of 70% alcoholic extract (p.o.)
Group 4: Receives MSG 2 g/kg (i.p.) + median dose of 70% alcoholic extract (p.o.)
Group 5: Receives MSG 2 g/kg (i.p.) + high dose of 70% alcoholic extract (p.o.)
Group 6: Receives MSG 2 g/kg (i.p.) + Quercetin 25 mg/kg,(i.p.)(reference standard) During the drug treatment, rats will be observed for the behavioral changes for 50 minutes daily. On 8th day the rats will be evaluated for ambulatory behavior, elevated plus maze test, balance beam test and rota rod test. On 9th day rats will be sacrificed and brain will be isolated for estimation of glutathione, SOD, CAT, lipidperoxidation and histopathological study.
Aluminum fluoride induced neuro toxicity model20
The animals were divided into six groups of six rats each as follows;
Group 1: Receives normal saline (i.p.) + vehicle (p.o.).
Group 2: Receives (AlF4−) 600ppm + normal saline (p.o.).
Group 3: Receives (AlF4−) 600 ppm + low dose of 70% alcoholic extract (p.o.)
Group 4: Receives (AlF4−) 600 ppm + median dose of 70% alcoholic extract (p.o.)
Group 5: Receives (AlF4−) 600 ppm + higher dose of 70% alcoholic extract (p.o.)
Group 6: Receives (AlF4−) 600ppm + Quercetin 25 mg/kg,(i.p.)(reference standard)
Animals from each group were fed with drug or vehicle for 10 days prior to aluminum fluoride treatment through their drinking water (7 days at a dose of 600 ppm).The behavioral changes for 50 minutes daily, rats will be evaluated for ambulatory behavior, elevated plus maze test, balance beam test and rota rod test. Later rats will be sacrificed and brain will be isolated for estimation of glutathione, SOD, CAT, lipidperoxidation and protein.
Carotid artery occlusion produced cerebral ischemia:21
Rats were randomly divided in to six groups of 6 rats each.
Group 1: Negative control (Receives only vehicle)
Group 2: Positive control (Carotid arteries occlusion + vehicle)
Group 3: Standard group (Carotid arteries occlusion + Vitamin E as referance standard)
Group 4:These animals will be subjected to Carotid arteries occlusion + Low dose of 70%
alcoholic extract.
Group 5:These animals will be subjected to Carotid arteries occlusion + Median dose of 70%
alcoholic extract.
Group 6:These animals will be subjected to Carotid arteries occlusion + High dose of 70%
alcoholic extract.
After the Carotid arteries occlusion, the animals will be subjecting for neurological scoring, ambulatory behavior, elevated plus maze test, balance beam test and rota rod test. After the completion of these tests, rats will besacrifice and brain will be isolated for estimation of GSH, CAT, LPO, SOD and protein.