Pharmacological Study of Anti-Allergic

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

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.

DISSERTATION

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 anti-allergic 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 Cardiospermumhalicacabum was studies in acetaminophen-induced nephrotoxicity in rats8. Protective effect of Withania somnifera andCardiospermum 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 ofCardiospermumhalicacabumand its reference compounds ex-vivo and in-vivo11. In vivo antioxidant and hypolipidemic effect ofCardiospermumhalicacabumleaf extract was studied in streptozotocin-induced diabetic rats12. Cardiospermumhalicacabuminhibits 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 ofCardiospermumhalicacabum14. Cardiospermumhalicacabumethanol extract inhibits LPS induced COX-2, TNF-alpha and iNOS expression, which is mediated by NF-kappaB regulation, in RAW264.7 cells15. Effect ofCardiospermumhalicacabumon ethanol-induced gastric ulcers was studied in rats16. In vitro antiparasitic activity of extracts ofCardiospermumhalicacabumagainst third-stage larvae of strongyloides stercoralis17. Effects of Cassia auriculata and Cardospermumhalicacabumteas on the steady state blood levels of theophylline in rats18.
7. / 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:
7.1 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 / Duration in days
I / Control-vehicle treated / Vehicle, i.p, / 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, / 4
V / Clonidine + Disodium cromoglycate / 1mg/kg + 10mg/kg i.p, / 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 / Dose and route of administration / Duration of catalepsy (min)
0 / 30 / 60 / 90 / 120
I / Control-Vehicle / Vehicle, i.p,
II / Clonidine / 1mg/kg, i.p,
III / Chlorpheniramine / 10mg/kg, I.p,
IV / Clonidine+EECH / 1mg/kg+100mg/kg
V / Clonidine+EECH / 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 anti-inflammatory 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):1245-52.
14.  Kumar R, Murugananthan G, Nandakumar K, Talwar S. Isolation of anxiolytic principle from ethanolic root extract of Cardiospermum halicacabum. Phytomedicine. 2011;2-3(18):219-23.
15.  Sheeba M, Asha V. Cardiospermum halicacabum ethanol extract inhibits LPS induced COX-2, 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:p-73.
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 / NAME OF CANDIDATE / MANJUNATHA. R. M
10.0 / SIGNATURE OF THE CNDIDATE / (MANJUNATHA. R. 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