Evaluation of Cardiotonic
Activity of Wedelia chinensis (Osbeck) Merr. Leaves
Swathi Sree Karumuri*, Venkata Naveen Kasagana.
Department of Pharmaceutics ,Sankaralingam Bhuvaneswari College of Pharmacy, Sivakasi,
Tamilnadu, India.
ABSTRACT:
Cardiotonic drugs increases the force and rate of contraction of
the cardiac muscles in conditions like congestive cardiac failure. But at the
same time they produce cardiac arrest even with a slight increase in the dose.
To overcome this problem the present attempt has been made to investigate the Cardiotonic activity of Wedelia chinensis (Osbeck)
Merr, without producing cardiac arrest. The aqueous
leaf extract was studied for preliminary phytochemical
screening and found to have the presence of Cardiac Glycosides. The activity was performed on the isolated frog (Rana hexadactyla.sp) heart that was perfused with digoxin (0.1 ml,
0.2 ml, 0.4 ml) of 0.5 mg/ml and the aqueous extract of Wedelia chinensis (0.1ml, 0.2 ml, 0.4 ml) of
5mg/ml, and were evaluated for force and rate of contraction of cardiac
muscles. From Kymograph record, it was observed that 0.4 ml (2mg) of test extract significantly increased the height
of contraction without any cardiac arrest ,
compared to digoxin 0.4 ml (0.2 mg) which recorded
cardiac arrest. From the above study, it can be
observed that the aqueous extract of Wedelia chinensis leaves been
found to have Cardiotonic activity without producing
the cardiac arrest even at high dose when compared to marketed drug Digoxin. So this test drug can be a better alternative for
the Cardiotonic drugs.
KEYWORDS: Wedelia chinensis, Cardiotonic activity, preliminary phytochemical
screening, pharmacological studies.
INTRODUCTION:
Plants continue
to provide as new chemical entities for the development of drugs against
various pharmacological targets, including AIDS, Cancer, Cardiac Stimulant,
Alzheimer’s disease and pain. According to WHO, 80% of the world population use
plant based remedies as their primary form of health care. Over the past decade, herbal medicine has
become a topic of global importance, making an impact on both world health and
international trade. The literature survey reveals that the plant Wedelia Chinensis (Osbeck) Merr, has not been dealt
in detail. In spite of its use as Cardiotonic, no systematic study has been reported in the
literature. Cardiovascular disease
incurs a greater economical constraint than any other illness especially in the
developing countries. It is the most common cause of death by the year 2020.
Therefore, cardiovascular disease becomes a very common problem in the affluent
societies related to their life style.1 Keeping this in view, it was
considered worthwhile to investigate the Cardiotonic
activity of Wedelia chinensis (Osbeck) Merr. The above
investigations are believed to be worthy addition to the wealth of medicinal
plants.
MATERIALS AND METHODS:
Pharmacognostical
Studies:
Collection and Authentification of Plant:
The plant Wedelia chinensis Osbeck Merr was
collected from the region of thiruninravur, thiruvallur Dist, Chennai, Tamil Nadu, India in the month
of Feb.2010. The collected plant was identified by Prof. P. Jayaraman, Ph.D, Director, National Institute Of
Herbal Science (Retd, Professor, Presidency College, Chennai).
Physio-Chemical
and Phytochemical Investigation:
The preliminary phytochemical
analysis was carried out to detect different chemical components present in
aqueous extract.
Physico
Chemical Studies:
Ash values 2
1.
Total Ash:
The method for determining the total ash involved
weighing out of 5 mg of the sample in to silica crucible which has been
previously ignited and cooled before weighing.
The ignition was repeated until constant weight was obtained.
2.
Water soluble Ash:
The ash was boiled with 25 ml water was filtered
through an ash less filter paper (Whatmann 41). It was followed by washing with hot
water. The filter paper was ignited in
the silica crucible, cooled and the water insoluble ash was weighted. The water soluble ash was calculated by subtracting
the water insoluble ash from the total ash.
3.
Acid insoluble ash:
It was
determined by boiling water insoluble ash with 25 ml dilute hydrochloric acid
(100% w/v) for five minutes and filtering though an ash less paper. The filter
paper was ignited in the silica crucible, cooled and acid insoluble ash was
weighed.
4.
Sulphated
Ash:
About 1 gm of the powdered drug was accurately weighed
in a crucible, ignited gently at first. The drug was moistened with 1 ml of sulphuric acid, heated gently until the white fumes are no
longer evolved and ignited at 800°C ±
25°C until all black particles has disappeared. The crucible was allowed to
cool and weighed. The operation was
repeated until two successive weighing did not differ by more than 0.5 mg. The percentage of Sulphated
ash was calculated with reference to the air dried drug.
Water
Soluble Extractive:
Add
5g to 50 ml water at 800 C in a stoppered
flask. Shake well and allow to stand for 10 minutes, cool, add 2g of keiselghur and filter. Transfer 5ml of the filtrate to a
an evaporating dish, 7.5cm in diameter,
evaporate the solvent on a water bath, continue drying for 30min, finally dry
in a steam oven for 2 hours and weigh the residue. The percentage of water
soluble extractive was calculated with reference to the air-dried drug.
Phytochemical Screening:
The phytochemical investigation
of a plant may thus involve the following Quantitative evaluations.
Qualitative Analysis3
The solvents and reagents used for qualitative and
quantitative analysis are of analytical grade (AR-grade) only.
Extraction Procedure:
The leaves were collected and dried under shade for 14
days and then made in to coarse powder with mechanical mixer, then passed with
sieve no: 40 and stored in air tight container for further use.
Preparation of Aqueous Extract:
The dried powdered plant material pf
the plant (653gm) was first macerated (cold maceration) with Water: Chloroform
solvent system (95:5) for 8 Days and extraction is done. The marc was again
macerated with Water: Chloroform solvent system (95:5) for 3 Days and
extraction is done. Both the extracts (2.950 lit and 2.865 lit respectively)
were concentrated to (130ml) in water bath until a semi solid mass is obtained.
Preliminary Phytochemical
Screening:
Preliminary phytochemical
analysis was done for the aqueous extract of coarsely powdered leaves of Wedelia chinensis. The
following chemical tests were carried out on the aqueous extract to identify
the presence of various Phytochemical constituents.4, 5
Qualitative phytochemical analysis:
Glycosides:
Cardiac glycoside: Keller-Killiani Test-:
To 2 ml extract, add glacial acetic acid, One drop 5 %
ferric chloride and conc. H2SO4. Reddish brown colour appears at junction of the two liquid layers and
upper layer appears bluish green.
Anthraquinone
glycosides: Borntrager’s Test –
To 3 ml extract add dil. H2SO4.
Boil and filter. To cold filtrate add equal volume benzene or chloroform. Shake
well. Separate the organic solvent. Add ammonia. Ammonical
layer turns pink or red.
Saponin
glycosides:
Foam test - Shake the drug extract or dry powder
vigorously with water. Persistent foam observed.
Coumarin
glycosides:
Alcoholic extract when made alkaline, shows blue or
green fluorescence.
In addition to the identification of Cardiac
Glycosides, the tests for the presence of following constituents were performed:
Carbohydrates, Flavonoids, Alkaloids, Phenols,
Tannins, Mucilage, Proteins, Amino acids, Fats and Fixed Oils, Steroids.
Pharmacological studies:
Preparation of
Reference solution
The marketed digoxin ampoules were obtained from local market and labelled as Digoxin – 0.5
mg/ml.
Test Drug: Aqueous Extract of Wedelia
Chinensis;
Chemical: Digoxin;
Adrenaline, Ringer Solution;
Animal: Frog;
Instruments: Sherrington Rotating Drum
Frog Preparation:
Frogs of Rana hexadactyla species (200-260 g) (procured from animal
house of Vel’s College of Pharmacy) housed in cages
at 27 ± 2˚C on a 12 hour light/dark cycle was used. The animal was fed
with water adlibitum. The animals were maintained as per the
norms of CPCSEA and cleared by CPCSEA (290/CPCSEA/ pharm-col-9/ 18810) and institutional
ethics committee (Vel’s College of Pharmacy).
Experimental setup:
The frogs were pithed so as
to destroy the central nervous system but without causing any injury to their
heart and associated blood vessels. The sternum was completely removed and the
pericardium was cut open exposing the heart.
The liver was pushed aside from the inferior vena cava as far as the
hepatic veins. A small cut was made into the venous sinuses; Syme’s cannula was inserted
towards the heart and isolated. A steady flow of the perfusion fluid (Frog
ringer) having the following composition, was perfused
through this cannula. Through the opening of the cannula drugs could be injected by pushing a capillary tube
attached to a syringe through an injection needle. A small hook was attached to
the tip of beating heart which was tied with a thread. The other end of the
thread was attached to the Simple lever so that the movements of the beating
heart could be recorded on a kymograph paper. The force of contraction was
recorded and the rate of contraction was counted and tabulated 6, 7.
Evaluation of Cardiotonic activity8,9
Set up the perfusion of frog heart with normal frog
Ringer solution. After basal recording, 0.5 mg/ml of Adrenaline was
administered to identify the sensitivity of the myocardium on the heart rate
and the force of contraction was studied. Sufficient time was allowed to the
preparation to return to base line after every dose. Replace the perfusion
fluid with modified Ringer containing only 1/2th the calcium chloride.
Note the change in the pattern of recording of the heart. The isolated hearts
were then perfused with digoxin
(0.1 ml, 0.2 ml, 0.4 ml) and the aqueous extract of wedelia
chinensis (0.1ml, 0.2 ml, 0.4 ml) were evaluated for
their Cardiotonic activity. Compare the responses of
these drugs
in hypodynamic heart. The rate and force of heart contraction
were noted.
RESULTS:
Physio - Chemical Studies
The results of physiochemical parameters of are given in the Table – 1.
Table: 1.Physiochemical Constants
of the leaves of Wedelia chinensis
|
S.
No. |
Parameters |
Percentage(%w/w) |
|
ASH
VALUES |
||
|
1 |
Total
ash |
22.5 |
|
2 |
Acid
insoluble ash |
18.5 |
|
3 |
Water
soluble ash |
73.5 |
|
4 |
Sulphated ash |
31 |
|
EXTRACTIVE
VALUE |
||
|
1 |
Water
soluble extractive |
18.40
(w/v) |
Phytochemical Screening:
Table: 2. Qualitative Phytochemical analysis
|
S.NO |
TEST |
INFERENCE |
|
|
POWDERED
DRUG |
EXTRACT |
||
|
1 |
CARBOHYDRATE |
+ |
+ |
|
2 |
PROTEINS |
+ |
+ |
|
3 |
AMINO
ACIDS |
+ |
+ |
|
4 |
STEROIDS |
+ |
+ |
|
5 |
GLYCOSIDES (CARDIAC) |
+ |
+ |
|
6 |
ALKALOIDS |
+ |
+ |
|
7 |
TANNINS
and PHENOLIC
COMPOUNDS |
+ |
+ |
|
8 |
VITAMINS |
- |
- |
|
9 |
FLAVONOIDS |
+ |
+ |
|
10 |
MUCILAGE |
- |
- |
|
11 |
FATS
and OILS |
+ |
+ |
Note:
+ denotes the
presence of the respective phytoconstituents
- denotes the absence of the respective phytoconstituents
Pharmacological activity
Fig. 1. Note: b/m-beats/minute; NHR- Normal heart rate; CA- Cardiac
arrest.
TABLE: 3. EFFECT OF CALCIUM
CONCENTRATION
|
Drug |
Conc. of Ca.
with respect to its concentration in normal ringer |
HR |
HFC in mm |
Change in HFC % |
Cardiac output |
|
(heart rate beats/min) |
(height in force of contraction) |
(HR × HFC) |
|||
|
Ringer |
Normal |
33 |
12 |
100 |
396 |
|
|
1/2 nd |
36 |
16 |
133.33 |
576 |
TABLE: 4. EFFECT ON HYPODYNAMIC
HEART DIGOXIN vs TEST DRUG
|
S No |
Drug |
Dose (in ml) |
Beats/min |
Change in force |
|
1 |
- |
Normal |
36 |
Normal |
|
2 |
Digoxin |
0.1 |
55 |
Rapid Increase |
|
0.2 |
43 |
Slight decrease |
||
|
0.4 |
- |
No Response |
||
|
3 |
Test Drug |
0.1 |
63 |
Rapid Increase |
|
0.2 |
47 |
Slight decrease |
||
|
0.4 |
38 |
Slight decrease |
TABLE: 5. EFFECT ON CARDIOTONIC
ACTIVITY
|
Drug |
Conc. (mg/ml) |
Dose (ml) |
Conc. at diff. Doses (mg) |
HR (beats/min) |
HFC (mm) |
Change in HFC (%) |
Cardiac output (HR × HFC) |
|
Digoxin |
0.5 |
0.1 |
0.05 |
55 |
14 |
127.27 |
770 |
|
|
0.2 |
0.1 |
43 |
18 |
163.63 |
774 |
|
|
|
0.4 |
0.2 |
-- |
Heart Blocked |
-- |
-- |
|
|
Test drug |
5 |
0.1 |
0.5 |
63 |
11 |
100 |
693 |
|
0.2 |
1 |
47 |
15 |
146.38 |
705 |
||
|
0.4 |
2 |
38 |
19 |
172.72 |
792 |
Note : Ca – Calcium; Conc. – Concentration ;
diff. – different; HFC - height in force of contraction; HR – heart rate
Fig.2. Concentration vs Height in Force with Digoxin
Fig.3.
Concentration vs
Height in Force with Test Drug
DISCUSSION:
The documented reports indicate that the
glycosides (which is present in the aqueous extract – Table no. 2. might be
responsible for the positive ionotropic effect, while
the tannins and flavonoids provide free radical
antioxidant activity and vascular strengthening10. Na+ K+ ATPase inhibition by
cardiac glycosides leads ultimately to increase intracellular Ca2+
concentrations through Na+/Ca2+ exchange and an associated increase in slow
inward Ca2+ current as well as in transient Ca2+ current11,12.
From the resultant kymograph, even lower dose of test extract gives a
significant increase in the height of contraction, characterized by positive ionotropic and negative chronotropic
actions. The above results shows the cardio tonic activity of aqueous extract
of Wedelia chinensis
showed a therapeutic
effect in 0.4 ml(2 mg) without any cardiac arrest, where digoxin showed cardiac
arrest at the dose of 0.4 ml (0.2mg). Therefore it serves as a safe alternative to digoxin in
congestive cardiac failure.
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Received on 31.07.2012
Modified on 08.08.2012
Accepted on 18.08.2012
© A&V Publication all right reserved
Research Journal of Pharmacognosy and
Phytochemistry. 4(5): September – October 2012, 262-266