Download Cardiospermum halicacabum - Rajiv Gandhi University of Health

Pharmacological study of anti-allergic
activity of Cardiospermum halicacabum
Synopsis for M. Pharm Dissertation
Submitted To
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES
KARNATAKA, BANGALORE
By
Mr. MANJUNATHA. R. 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-BANGALORE
ANNEXURE II
PROFORMA FOR REGISTRATION OF SUBJECTS FOR
DISSERTATION
1.
Name of the Candidate and Address
Residential Address:
Manjunatha. R. M
C/o ATB Hostel, #31, 1st Main, Chamarajpet,
Bangalore-560018
Permanent Address:
Manjunatha. R. M
S/o Chikkamallappa,
K. Raguttahalli, Kotagal(P),
Chintamani(T), Chickaballapur(D)-563125
2.
Name of the Institution
PES College of Pharmacy, Hanumanthanagar,
Bangalore-560050
3.
Course of Study and Subject
Master of Pharmacy - Pharmacology
4.
Date of Admission to Course
29/07/2011
5.
Title of topic:
Pharmacological study of anti-allergic activity of Cardiospermum halicacabum.
6.
Brief resume of the intended work
6.1 General Discussion:
Immediate or type I, hypersensitivity is a rapidly developing immunologic reaction occurring within
minutes after the combination of an antigen with antibody to mast cell in individuals previously
sensitized to the antigen. These reactions are called allergic reactions and antigens eliciting them are
called as allergens. Allergic reaction may occur as two types: Systemic reaction and local reaction.
Systemic reactions usually follow injection of antigen sensitized individual. Sometimes, with in a
minutes the patient goes into a state of shock, is which may be fatal. The nature of local reaction
varies depending on portal entry of allergen and may take the form of localized cutaneous swelling
(skin allergy), nasal and conjunctival discharge (allergic rhinitis & conjunctivitis), hay fever,
bronchial asthma or allergic gastroenteritis (food allergy) in which mast cells are the principal target
cells for the immediate hypersensitivity. As part of allergic response to an antigen, antibodies are
generated and bind to the surface of mast cell via high affinity Fc receptors that are specific for IgE.
Mast cells release histamine during these reactions. Mast cell mediators of inflammatory processes
are histamine, proteases, LTC4, LTB4, PGD2, platelet activating factor. Mast cell degranulation is
believed to be involved in the pathophysiology of CHF, asthma, allergic hypersensitivity reaction
and inflammation1.
Anti-allergy medications are commonly used for preventing and relieving allergy symptoms. There
are countless number of both over-the-counter and prescription. Medications available in the market
to reduce annoying allergic reactions. Anti-allergic drugs include cetirizine, chlorpheniramine
maleate, decongestants, antihistamines, anti-inflammatory agents, anti leukotrienes and other
combination medicines2.
6.2 Need for the study:
The problem with use of modern medicine is potent adverse reactions. The adverse reactions include
drowsiness is the most frequent side effect, and it can interfere with driving ability or adequate
functioning at the work place. Sedative effects can be beneficial in patients who have difficulty
sleeping because of rhinitis symptoms. Other side effects include loss of appetite, nausea, vomiting,
and
epigastric distress. Taking medication with meals or a full glass of water may prevent
gastrointestinal side effects. The modern medicines available as anti-allergic drugs include Sodium
cromoglycate but these drugs are associated with unwanted effects including local irritation,
transient bronchospasm. These drugs are also restricted to use in pregnancy and prolonged use3.
However, there are several plant-derived preparations in the Ancient text of ayurveda and siddha for
the treatment of allergic conditions including asthma. With continuation of new drug discovery,
plants or their preparation scientifically to prove for their clinical applicability is required. In this
view the present study has been selected for evaluation of Cardiospermum halicacabum for its antiallergic property.
6.3 Review of literature:
Cardiospermum halicacabum is a deciduous climber growing to 3 m (9ft 10in) It is hardy to zone 9
and is frost tender. It is in flower from Jul to August, and the seeds ripen from Aug to October. The
flowers are hermaphrodite (have both male and female organs). The plant prefers light (sandy),
medium (loamy) and heavy (clay) soils and requires well-drained Soil. The plant prefers acid,
neutral and basic (alkaline) Soils. It cannot grow in the shade. It requires moist soil.
Plant profile:
Title of plant
: Mudakkatran
Botanical Name: Cardiospermum halicacabum
Family
: Sapindaceae
Genous
: Cardiospermum
Species
: C. halicacabum
Synonyms
: Cardiospermum halicacabum, Balloon vine, Love in a Puff, Heart seed vine
Telugu: Budda teega, Budda kakara, Budduva, Patalitiva, Chinna pottuki theega, Tapakaya,
Budama, Sanduteega.
English: Pigeon’s-knee, Blisters creeper, Heart seed, Ballon vine and Hearts pea4.
Chemical constituents:
The whole plant Cardiospermum halicacabum contains saponins, traces of alkaloids,
flavonoids,
proanthocyanidin, apigenin and phytosterols (e.g, stigmosterol). The seed contains approximately
33% of fatty acids and of these fatty acids about 55% are cyano lipids. The major cyano lipids (49%)
is a diester having two fatty acid moieties esterified with 1-cyano-2-hydroxymethyl-prop-2-ene-1-ol
followed by a diester derived from 1-cyano-2-hydroxymethyl-prop-2-ene-3-ol(6%) of the fatty acids,
11-eicosenic acid is with 42%. The major one (42%) other chief components of the oil are oleic acid
(22%), arachidic acid (10%), linolenic acid (81%), palmitic acid (3%) and stearic acid (2%). In the
leaves larger amount of saponin and alkaloids were found also (+)-pinitol, apigenin, luteolin and
chrysoeriol. The occurrence of esterified fatty acids, pentacyclic triterpenoids various phytosterols
and hydrocyanic acid releasing cyano lipids in the mother tincture was confirmed, although at much
lower concentrations than in the whole fresh plant5.
Seed chemistry studies of Cardiospermum halicacabum L. indicate high protein content. Balloon vine
seed protein content (35.9% by dry weight) and amino acids in g/100g protein (Asp 8.3%, Thr 4.1%, Ser
6.0%, Glu 15.9%, Pro 3.2%, Gly 9.2%, Ala 6.6%, 1/2Cys 1.1%, Val 6.3%, Met 0.9%, Ile 3.9%, Leu
5.7%, Tyr 1.8%, Phe 3.8%, His 2.7%, Lys 3.9%, Arg 5.1%) are equal to or higher than those of many
popular legumes. The high protein content of balloon vine has resulted in its utilization in cattle or
poultry feeds. Some cultures use balloon vine as a high protein food. However, the lack of cystine and
methionine in proteins may cause an imbalance of S-S-amino acids. Hence, the protein from these seeds
will need supplementation with other proteins (i.e., from other plants) rich in these amino acids, if used
as a dietary source6
Medicinal uses : Anti-vatha, analgesic, diuretic, laxative, stomachic, anti-inflammatory
Reported activities:
A case study was done on Cardiospermum halicacabum L. (Modakathon, Balloon Vine) as a
traditional herb for treating rheumatoid arthritis7. Protective role of Cardiospermum halicacabum
was studies in
acetaminophen-induced nephrotoxicity in rats8.
Protective effect of Withania
somnifera and Cardiospermum halicacabum extracts was reported in collagenolytic degradation of
collagen9. Evaluation of indigenous plant extracts was done against the malarial vector, Anopheles
stephensi
(Liston)
(Diptera:
Culicidae)10.
Antioxidant
and
anti-inflammatory
properties
of Cardiospermum halicacabum and its reference compounds ex-vivo and in-vivo11. In vivo
antioxidant and hypolipidemic effect of Cardiospermum halicacabum leaf extract was studied in
streptozotocin-induced diabetic rats12. Cardiospermum halicacabum inhibits cyclophosphamide
induced immunosupression and oxidative stress in mice and also regulates iNOS and COX-2 gene
expression in LPS stimulated macrophages13. Isolation of anxiolytic principle from ethanolic root
extract of Cardiospermum halicacabum14. Cardiospermum halicacabum ethanol extract inhibits LPS
induced COX-2, TNF-alpha and iNOS expression, which is mediated by NF-kappaB regulation, in
RAW264.7 cells15. Effect of Cardiospermum halicacabum on ethanol-induced gastric ulcers was
studied in rats16. In vitro antiparasitic activity of extracts of Cardiospermum halicacabum against
third-stage
larvae
of
strongyloides
stercoralis17.
Effects
of
Cassia
auriculata
Cardospermum halicacabum teas on the steady state blood levels of theophylline in rats18.
and
6.4. OBJECTIVE OF THE STUDY:
To study the anti-allergic effect of aqueous and ethanol extracts of Cardiospermum halicacabum on
clonidine induced mast cell degranulation, milk-induced leukocytosis and eosinophilia and
clonidine-induced catalepsy in experimental animal models.
1.Collection and authentification of Cardiospermum halicacabum
2. Extraction of plant material by water (aqueous) and ethanol using soxhlet apparatus.
3. To establish the pharmacological profile of prepared extracts for its anti-allergic effect in
clonidine induced mast cell degranulation, milk-induced leukocytosis and eosinophilia and
clonidine-induced catalepsy.
Materials and methods:
7.
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: www.sciencedirect.com,
www.pubmed.com,
www.google.com,
www.ijp-online.com
7.2 Materials
:
Plant
: Cardiospermum halicacabum
Chemicals and drugs: Clonidine, sodium cromoglycate, milk, dexamethasone, saline, RPMI-1640
buffer, toluidine blue, aqueous and ethanolic extract of Cardiospermum halicacabum,
Animals
: Albino mice.
Instruments
: Microscope, centrifuge, bar.
7.3 Method:
7.3.1 Preparation of plant extract: Naturally occurring Cardiospermum halicacabum will be
collected and extracted with water and ethanol and these extracts will be used for this study.
7.3.2 Clonidine-Induced mast cell degranulation:
To study the mast cell stabilizing activity aqueous extract Cardiospermum halicacabum against
clonidine –induced degranulation in mice.
Treatment schedule and No mice in each group =6, 4 days
Group
Treatment
Dose and route of administration
I
Control-vehicle treated
Vehicle, i.p,
Duration
in days
4
II
Clonidine
1mg/kg i.p,
4
III
Clonidine + AECH
1mg/kg i.p,+ 100mg/kg p.o,
4
IV
Clonidine + AECH
1mg/kg i.p, + 300mg/kg p.o,
V
Clonidine + Disodium
cromoglycate
1mg/kg + 10mg/kg i.p,
4
4
Method- Mice will be divided into 5 groups of 6 animals each. 4ml of normal saline will be injected
into the peritoneal cavity of mice. After a gentle massage, the peritoneal fluid will be collected and
transferred into the test tubes containing 3-4ml of RPMI-1640 buffer medium (pH 7.2-7.4). The mast
cells will be washed by centrifugation at a low speed (400-500 rpm). Supernatant will be discarded
and the pellets of mast cells will be suspended in the medium. The mast cells will be treated with 1-2
drops clonidine (80µg/ml) incubated at 37oC in a water bath for 10min.These will be stained with
toluidine blue and observed under microscope. 100 mast cells will be observed. The percentage of
protection against degranulation will be computed. The mice will be treated with vehicle (control),
AECH (low and high dose), disodium cromoglycate for 4 days as shown in the table. The last dose
will be administered 30min before collection of mast cells. The results will be expressed as mean +
SEM and the data will be subjected to respective statistical analysis.19
7.3.3 Milk-induced leukocytosis and eosinophilia:
2. To study the effect of aqueous extract Cardiospermum halicacabum on milk induced
leukocytosis and eosinophilia in mice.
No mice in each group = 6
Group
Treatment
Dose and route of administration
I
Control-vehicle
Vehicle, sc
II
Milk
(4ml/kg), sc
III
Milk +AECH
(4ml/kg), sc + 100mg/kg p.o.
IV
Milk +AECH
(4ml/kg), sc + 300mg/kg p.o.
V
Milk + Dexamethasone
(4ml/kg), sc + 1mg/kg sc
Method: Mice will be divided into 5 groups of 6 animals each. Blood samples will be collected from
retro-orbital plexus under light ether anesthesia. The TLC and eosinophil counts will be determined
in each group before AECH administration and 24 h after milk or/and AECH administration. The 5th
group mice will be receiving dexamethasone. It serves as standard. The differences between the TLC
and the eosinophil counts will be recorded.20
7.3.4 Clonidine-induced catalepsy
To study the anti-cataleptic activity of ethanolic extract Cardiospermum halicacabum against
clonidine –induced-catalepsy in mice.
No. of mice in each group=5
Groups
Treatment
I
Control-Vehicle
Dose and route Duration of catalepsy (min)
of administration
0
30 60 90 120
Vehicle, i.p,
II
Clonidine
1mg/kg, i.p,
III
Chlorpheniramine
10mg/kg, I.p,
IV
Clonidine+EECH
V
Clonidine+EECH
1mg/kg+100mg/
kg
1mg/kg+300mg/
kg
Method: Fore paw of the each mouse will be placed on a bar elevated at 3.5cm above the ground
and duration for which the animal maintain the imposed posture will be noted. As the time required
for moving forepaw from the bar. The duration of catalepsy will be measured at 0, 30, 60, 90 and
120 mins. Mean+SEM will be calculated and statistical analysis will be carried out.21
STATISTICAL ANALYSIS
All the values will be expressed as mean± SEM. The data will be analyzed by using one way
ANOVA followed by suitable post-hoc test. Statistical significance will be set at P≤ 0.05.
7.3 Does the study require any investigations or interventions to be conducted on patients or
other humans or animals? If so please describe briefly.
The study requires investigations to be conducted on mice. Mice will be treated with the extracts of
drugs by oral route. Blood samples will be collected by retro orbital route using micro haematocrit
capillaries under light ether anaesthesia to procure serum. Animals will be subjected to different
standard stressor procedures and care will be taken not to cause undue pain.
7.4 Has Ethical Clearance been obtained from your institution in case of 7.3?
Yes, ethical clearance has been obtained.(copy enclosed)
References:
1. Vinay k, Abul k, fausto N, Jon aster c. Robbins and Cortan Pathologic basis of Disease. eight
ed; 2010.
2. Shirwaikar A., Somashekar A.P. Anti-inflammatory activity and free radical scavenging
studies of Aristolochia bracteata Lam. Pharmacol 2003;65(1):67-9.
3. Tripathi M. Essentials of Medical Pharmacology. Third ed: Jaypee brothers medical
publishers (P) Ltd; 1994.
4. http://plants.usda.gov/java/profile?symbol=CAHA13
5. The European Agency for the Evaluation of Medicinal products Veterinary Medicines
Evaluation Unit. EMEA. 1999:664.
6. Ragupathy S, Steven N, G, Gopinadhan P, Candice N, B. Ethnobotany. 2007; 19.
7. Ragupathy S, Steven N, G, Gopinadhan P, Candice N, B. A case study of Cardiospermum
halicacabum L. (Modakathon, Balloon Vine) as a traditional herb for treating rheumatoid
arthritis Ethnobotany,. 2007;19.
8. Parameshappa B, Ali B, MS, Sen S, Chakraborty R, Kumar G, Sagar G, et al.
Acetaminophen-induced nephrotoxicity in rats: Protective role of Cardiospermum
halicacabum. Pharm Biol. 2011;
9. Ganesan K, Sehgal P, Mandal A, S. S. Protective Effect of Withania somnifera and
Cardiospermum halicacabum extracts against collagenolytic degradation of collagen. Appl
Biochem Biotechnol. 2011;3-4(165):1075-91.
10. Govindarajan M. Evaluation of indigenous plant extracts against the malarial vector,
Anopheles stephensi (Liston) (Diptera: Culicidae). Parasitol Res. 2011; 1(109):93-103.
11. Huang M, Huang S, Wang B, Wu C, Sheu M, Hou W, et al. Antioxidant and antiinflammatory properties of Cardiospermum halicacabum and its reference compounds ex
vivo and in vivo. Ethnopharmacol. 2011;2(133):743-50.
12. Veeramani C, Pushpavalli G, Pugalendi K. In vivo antioxidant and hypolipidemic effect of
Cardiospermum halicacabum leaf extract in streptozotocin-induced diabetic rats. Basic Clin
Physiol Pharmacol. 2010;2(21):107-25.
13. Pratheeshkumar P, Kuttan G. Cardiospermum halicacabum inhibits cyclophosphamide
induced immunosupression and oxidative stress in mice and also regulates iNOS and COX-2
gene expression in LPS stimulated macrophages. Asian Pac J Cancer Prev. 2010;5(11):124552.
14. Kumar R, Murugananthan G, Nandakumar K, Talwar S. Isolation of anxiolytic principle
from ethanolic root extract of Cardiospermum halicacabum. Phytomedicine. 2011;23(18):219-23.
15. Sheeba M, Asha V. Cardiospermum halicacabum ethanol extract inhibits LPS induced COX2, TNF-alpha and iNOS expression, which is mediated by NF-kappaB regulation, in
RAW264.7 cells. Ethnopharmacol. 2009;1(124):39-44.
16. Sheeba M, Asha V. Effect of Cardiospermum halicacabum on ethanol-induced gastric ulcers
in rats. Ethnopharmacol. 2006;1(106):105-10.
17. Boonmars T, Khunkitti W, Sithithaworn P, Fujimaki Y. In vitro antiparasitic activity of
extracts of Cardiospermum halicacabum against third-stage larvae of Strongyloides
stercoralis. Parasitol Res. 2005;5(97):417-9.
18. Thabrew M, Munasinghe T, Senarath S, Yapa R. Effects of Cassia auriculata and
Cardospermum halicacabum teas on the steady state blood levels of theophylline in rats.
Drug Metabol Drug Interact. 2004;4(20):263-72.
19. Stanworth DR (1973) , Immediate Hypersensitivity. In: Neuberger A, Tatum EL (Eds). The
molecular basis of the allergic response. North Holland Publishing Company, Amsterdam:p73.
20. Brekhman II, Dardymov IV. New substances of plant origin, which increase nonspecific
resistance. Ann Rev Pharmacol. 1969; 9:419-28.
21. Malaviya S, Nandakumar., Vaghasiya J, Bhalodiya Y, Jivani N, Sheth N, et al. Anxiolytic
activity of root extracts of Cardiospermum halicacabum in mice. The Internet Journal of
Pharmacology. 2009;7.
9.0
10.0
NAME OF CANDIDATE
MANJUNATHA. R. M
SIGNATURE OF THE CNDIDATE
(MANJUNATHA. R. M)
11.0
11.1
REMARKS OF THE GUIDE
NAME
AND
DESIGNATION
OF
THE
GUIDE
Mr. SHIVALINGE GOWDA. K. P
ASST. PROFESSOR,
DEPT.OF PHARMACOLOGY
P.E.S COLLEGE OF PHARMACY
11.2
11.3
SIGNATURE
HEAD OF THE DEPARTMAENT
Mr. SRINATH.R
HOD& ASST.PROFESSOR
DEPT.OF PHARMACOLOGY
P.E.S.COLLEGE OF PHARMACY.
11.4
12.0
12.1
SIGNATURE
REMARKS OF THE PRINCIPAL
FORWARDED FOR APPROVAL
SIGNATURE
Dr. S. Mohan
PRINCIPAL AND DIRECTOR
P.E.S.COLLEGE OF PHARMACY
BENGALORE-560050
PES COLLEGE OF PHARMACY
Institutional Animal Ethics Committee
Ref. No. PESCP/IAEC/04/11
Date: 14/12/11
The Institutional Animal Ethics Committee [IAEC] of PES College of
Pharmacy has held its meeting on 12th December 2011 and has given its consent
to Mr. Manjunatha.R.M, I M Pharm PG student (pharmacology) to carryout
animal experimentation for his dissertation work entitled ‘Pharmacological
study of anti-allergic activity of cardiospermum halicacabum’.
Prof. Dr. S. MOHAN
Mr. R. SRINATH
Members
Dr.Chanda Kulkarni
Prof.B.K.Hanumanthiah
Dr. Nagaraj
Dr. Y.B. Rajeshwari
Prof. J.Saravanan
Dr. C.S.Satish
Mr.Mukund Handral
Dr. K. Lakshaman
50 Feet Road, Hunumanthanagar, Bangalore- 560 050
Phone: 080-26507428, Fax: 080-26506928
E-mail: [email protected] / [email protected]
www.pes.edu