Studies
on the Hematological Effect of the Extracts of Cordia dichotoma Forst. F. Fruits
IJ
Kuppast1, P Vasudeva Nayak2, MC Ravi1 and SS Biradar3.
1Department of Pharmacology, National College of
Pharmacy, Balraj Urs Road, Shimoga-577 202 Karnataka, India.
2Department of Studies in Chemistry,
3Department of Pharmaceutics,
ABSTRACT
In the present study anti-anaemic effect of
the petroleum ether, solvent ether, ethyl acetate, butanol and butanon extracts
of Cordia dichotoma Forst.f. fruits was evaluated in albino rats. The rats
were made anaemic as per standard procedure and were treated daily by extracts
in the dose of 100, 200 and 300 mg/kg body weight for 15 days p.o. After 15 days of daily treatment
with the extracts of fruits, the haematological parameters measured are
clotting time, bleeding time, hemoglobin content, total count of red blood
corpuscles, white blood cells and differential count of leucocytes. From the
statistical analysis of the study it has been found that ethyl acetate, butanol
and butanon extract of C. dichotoma
fruits causes significant dose dependent rise in hemoglobin content and RBC
count. However, reduction in clotting and bleeding time was not found to be
dose dependent. The WBC count was found to rise only in higher doses of
extracts (300mg). In case of higher doses (200 and 300mg/kg body weight) the
extracts of fruits reduced the clotting and bleeding time, where as lower dose
(100 mg/kg body weight) there was no significant change in bleeding and
clotting time. The results suggest that the extracts of C. dichotoma
fruits have significant ant-anaemic effect and are haematologically non toxic.
KEY
WORDS: Hematological Effect
Extracts C. dichotoma forst.f. Fruits
1. INTRODUCTION
C.
dichotoma forst.f. a plant
belonging to family Boraginaceae is medium sized tree with a short, usually
crooked trunk 3-4 ft. in girth1. The fruits are globose,
yellowish-brown, pink or black and pulpy. The plant grows in
2. MATERIALS AND METHODS:
Preparation
of plant extracts:
The fruits of C. dichotoma were purchased from local market of Hubli and were
authenticated by Prof V. S Huddar, Department of Botany, H. S. K Science and S.
K Arts College, Hubli. The collected fruits were shade dried, reduced to coarse
powder (2 kg) and subjected to repeated exhaustive extraction in batches with
ethanol in a soxhlet extractor.
TABLE 1: Qualitative chemical examination of various
extracts obtained by successive solvent fractionation of the Cordia dichotma
Forst.f. fruits.
|
Sl. No |
Phytoconstituents |
Pet.
ether fraction |
Solvent
ether fraction |
Ethyl
acetate fraction |
Butanone
fraction |
Butanol
fraction |
|
1. |
Alkaloids |
- |
+ |
- |
- |
- |
|
2. |
Carbohydrates |
+ |
+ |
+ |
+ |
+ |
|
3. |
Phytosterols |
- |
- |
- |
- |
- |
|
4. |
Flavanoids |
- |
- |
+ |
+ |
+ |
|
5. |
Tannins |
- |
- |
- |
- |
- |
|
6. |
Proteins&
Amino acids |
+ |
+ |
+ |
+ |
+ |
|
7. |
Saponins |
+ |
+ |
- |
- |
- |
Table-2: Studies on the Haematological Effect of the
Petroleum ether Extract of C. dichotoma Fruits (Values are in mean ±
S. E.).
|
Parameters Studied |
Control |
300mg/kg b.w. |
200mg/kg b.w. |
100mg/kg b.w. |
|
Clotting time (In
min.) |
3.5 ±
0.02 |
3.4 ±
0.031 |
3. 6 ± 0.042 |
3.3 ± 0.021 |
|
Bleeding time (In
min.) |
3.2 ±
0.11 |
3.4 ±
0.022 |
3.8 ±
0.65 |
3.3 ±
0.14 |
|
Haemoglobin
content (Gm %) |
14.8±
0.2 |
14.8 ±
0.023 |
14.5 ±
0.023 |
14.2 ±
0.056 |
|
Total RBC count (In
millions/cu mm) |
4.4 ±
0.21 |
4.6 ±
0.036 |
4.5 ±
0.54 |
4.7 ±
0.021 |
|
Total WBC count (In
thousand/ cu mm) |
6.2±
0.025 |
6.3 ±
0.023 |
6.4 ±
0.025 |
6.1 ±
0.014 |
* P <0.001 is considered
as significant.
Table-3: Studies on the Haematological Effect of the
Solvent ether Extract of C. dichotoma Fruits (Values are in mean ± S. E.).
|
Parameters Studied |
Control |
300mg/kg b.w. |
200mg/kg b.w. |
100mg/kg b.w. |
|
Clotting time
(In min.) |
3.5 ±
0.12 |
3.4 ±
0.011 |
3.6 ± 0.028 |
3.4 ± 0.011 |
|
Bleeding time
(In min.) |
3.2 ±
0.11 |
3.4 ±
0.028 |
3.3 ±
0.42 |
3.4 ±
0.012 |
|
Haemoglobin
content (Gm %) |
14.8±
0.2 |
14.8 ±
0.041 |
14.5 ±
0.018 |
14.7 ±
0.025 |
|
Total RBC count
(In millions/cu mm) |
4.4 ±
0.21 |
4.5 ±
0.021 |
4.5 ± 0.021 |
4.6 ±
0.051 |
|
Total WBC count
(In thousand/ cu mm) |
6.2±
0.041 |
6.1 ±
0.019 |
6.3 ±
0.024 |
6. 4 ±
0.019 |
* P <0.001 is considered
as significant.
Table-4: Studies on the Haematological Effect of the
Ethyl acetate Extract of C. dichotoma Fruits (Values are in mean ± S. E.).
|
Parameters Studied |
Control |
300mg/kg b.w. |
200mg/kg b.w. |
100mg/kg b.w. |
|
Clotting time
(In min.) |
3.4 ±
0.02 |
2.1 ±
0.024* |
2.6 ± 0.02* |
2.4 ± 0.016 |
|
Bleeding time
(In min.) |
3.1 ±
0.16 |
2.4 ±
0.017* |
2.8 ±
0.012* |
2.6±
0.022 |
|
Haemoglobin
content (Gm %) |
14.9±
003 |
15.8 ±
0.054* |
15.5 ±
0.048* |
15.0 ±
0.021 |
|
Total RBC count
(In millions/cu mm) |
4.3 ±
0.19 |
5.9 ±
0.02* |
5.4 ±
0.041* |
4.15 ±
0.032 |
|
Total WBC count
(In thousand/ cu mm) |
6.1±
0.041 |
7.8 ±
0.063* |
6.4 ±
0.029 |
6.2 ±
0.054 |
* P <0.001 is considered
as significant.
Table-5: Studies on the Haematological Effect of the
butanol Extract of C. dichotoma Fruits (Values are in mean ± S. E.).
|
Parameters Studied |
Control |
300mg/kg b.w. |
200mg/kg b.w. |
100mg/kg b.w. |
|
Clotting time
(In min.) |
3.4 ±
0.02 |
2.3 ±
0.056* |
2.6 ± 0.042* |
3.4 ± 0.021 |
|
Bleeding time
(In min.) |
3.4 ±
0.11 |
2.4 ±
0.021* |
2.6 ±
0.5* |
3.3 ±
0.14 |
|
Haemoglobin
content (Gm %) |
14.7±
0.2 |
15.9 ±
0.054* |
15.5 ±
0.054* |
14.9 ±
0.047 |
|
Total RBC count
(In millions/cu mm) |
4.2 ±
0.29 |
6.1 ±
0.016* |
5.9 ±
0.14 |
4.4 ±
0.035 |
|
Total WBC count
(In thousand/ cu mm) |
6.0±
0.022 |
7.9 ±
0.012* |
6.4 ±
0.029 |
6.2 ±
0.042 |
* P <0.001 is considered
as significant.
Table-6: Studies on the Haematological Effect of the
butanon Extract of C. dichotoma Fruits (Values are in mean ± S. E.).
|
Parameters Studied |
Control |
300mg/kg b.w. |
200mg/kg b.w. |
100mg/kg b.w. |
|
Clotting time
(In min.) |
3.8 ±
0.01 |
2.6 ±
0.041* |
2.3 ± 0.033* |
3..9 ± 0.036 |
|
Bleeding time
(In min.) |
3.4 ±
0.22 |
2.6 ±
0.02* |
2.8 ±
0.61* |
3.8 ±
0.23 |
|
Haemoglobin
content (Gm %) |
14.5±
0.12 |
15.8 ±
0.02* |
15.6 ±
0.011* |
14.7 ±
0.021 |
|
Total RBC count
(In millions/cu mm) |
4.6 ±
0.29 |
6.2 ±
0.033* |
5.8 ±
0.021* |
4.8 ±
0.011 |
|
Total WBC count
(In thousand/ cu mm) |
6.4±
0.033 |
7.7 ±
0.055* |
6.5 ±
0.012* |
6.5 ±
0.013 |
* P <0.001 is considered
as significant.
After complete extraction the alcoholic
extract (68 gm) was then suspended in water and further fractionated using
petroleum ether (8 gm), solvent ether (2 gm), ethyl acetate (4 gm) butanol (5.5
gm) and butanon (2.5) in succession. These extracts were vacuum dried and used
for anti-anaemic activity. Tween 80 (1%) was used as vehicle to suspend the
extracts.
Phytochemical
Screening:
All the extracts were subjected to
phytochemical analysis as per standard procedures6 to know the
nature of phytoconstituents present (TABLE 1). The major active constituents
were found in extracts includes flavonoids, alkaloids and saponins. However among
all active constituents flavonoids were found to be in higher concentrations.
Acute
toxicity studies:
The acute toxicity studies were
carried out as per stair case or “Up and down” method7. Accordingly
the LD50 of all extracts were found to be 3000 mg/kg bodyweight. Three doses within the therapeutic dose i.e.
100, 200 and 300 mg/kg body weight were taken for study.
Experimental
design for Haematological Effect:
Tween-80 (1%) was used as
vehicle to suspend the extracts and was administered per orally. The animals
were divided into six groups of six each. The animals of all groups were made
anaemic by repeated bleeding at the rate of 12 to 15 ml / kg body weight for
consecutive 5 days8. Group one was considered as control and
remaining groups were treated with extracts daily in the dose of 100, 200 and
300 mg/kg body weight for about 15 days p.o.
After 15 days treatment with the extracts of fruits, the haematological
parameters measured are clotting time, bleeding time, haemoglobin content,
total count of red blood corpuscles and white blood cells by standard
procedures.
3. RESULT:
The LD50 of the extracts were found to be 3000mg/kg
body weight by Up and Down method and 1/10th of the LD50
i.e. 300mg/kg body weight was considered as therapeutic dose. Three doses
within the therapeutic dose i.e. 100, 200 and 300 mg/kg body weight were taken
for study. All the results were analysed by student “t” test and level of
significance was P<0.001. The
results of the present study indicates that the ethyl acetate butanol and
butanone extracts of C. dichotoma
fruits causes significant dose dependent rise in haemoglobin content and RBC
count. However the, reduction in clotting and bleeding time was not found to be
dose dependent. The WBC count was found to rise only in higher doses of
extracts (300mg). In case of higher doses (300 and 200mg/kg body weight) the
extracts of fruits reduced the clotting and bleeding time, where as lower dose
(100 mg/kg body weight) there was no significant change in bleeding and
clotting time. The results are summarized in Table 2-6. The present study
confirms our preliminary observations that fruit of C. dichotoma Forst.f.
have anti-anaemic activity.
4. DISCUSSION:
The drug reported to be anti-anaemic effect in
traditional system of medicine and used for that purpose has been shown in rat
significantly. The preliminary
phytochemical studies reveal that the presence of flavonoids in ethyl acetate,
butanol and butanone extracts of C.
dichotoma; various flavonoids have been reported for its anti-anaemic
activity with good effect. So the possible mechanism of anti-anaemic action of C. dichotoma fruits may be due to its
flavonoids content. The possible mechanism of action of flavonoids may be as
fallows. Hemoglobin (Hb)
oxidation leads to the formation of hemichrome, which binds to the membrane and
causes red blood cell removal by the reticuloendothelial system. The effect of
flavonoids on Hb oxidation and their binding to red blood cell (RBC) membranes
were studied. The intrinsic antioxidant activity of RBC was able to prevent the
binding of Hb to the membrane.
Oxidations performed in membrane-free Hb solutions with
an identical oxidative system showed less Hb oxidation. These observations
suggest that erythrocyte membrane lipid peroxidation enhances the oxidative
damage of Hb, increasing its binding to membranes. Flavonoids partially
protected Hb against oxidation. and reduced the levels of the membrane bound
hemichrome. Lipid peroxidation was also significantly suppressed by flavonoids.
Some selcted flavonoids like Rutin and morin had little effect in preventing Hb
binding to RBC membranes, indicating the importance of structure in the
antioxidant properties of flavonoids. In the absence of oxidant, the
peroxidation of erythrocyte membrane and isotonic hemolysis were protected by
flavonoids like quercetin. These results suggest that flavonoids displays a
beneficial role on aging of RBC9. Hence it can be claimed that C. dichotoma provides significant
anti-anaemic effect. Further
study regarding isolation and characterization of active principal responsible
for anti-anaemic activity is currently under progress.
5. REFERENCES:
1. The Wealth of India, Vol. II, A Dictionary of Indian Raw materials and Industrial
product; 1950; Vol. II: CSIR New Delhi: pp.346.
2. Yoganarsimhan SN., Medicinal plants of
3. Rapisarda A, Ficarra R, Tommasin S, Caldbro
ML and Hungsa, Plant Medica, 1992;
42: 643.
4. Wassel G, El-Menshaw B, Saud A, Meharuna G
and El-Merzabani M, Pharmazie, 1987; 42: 709.
5. Rajesh MG, Paul B and Latha M S, Antiseptic, 2000; 7: 320.
6. Evans WC, Trease GE, Text book of
Pharmacognosy, 1984; 4th Editon: ELBS London: pp. 152-158.
7.
8. Pawar DS and Somkumar AP, Anti-anaemic
effect of Embilica Officinalis in rats, 2000;
XXXIII Annuala Conference of the Indian Pharmacological Society,
Gandhinagar: December 28-3: Abstract part IV: 194.
9. Cesquini M, Tenor AC, Torsoni MA, Stoppa,
GR,
Received on 14.05.2009
Accepted on 20.06.2009
© A&V Publication all right reserved
Research J. Pharmacology and
Pharmacodynamics 1(3) Nov - Dec. 2009: 117-119