1. Abstract Reference Number : OPP 030
Effect of total extract of Buchanania lanzan leaves against
Hepatocellular Carcinoma(HCC) in Diethyl nitrosamine
induced tumor model
Sumithra M*, Anbu J, Srota Dawn, Ashwini Anjana, Nazeer Ahmed, Ravichandiran V.
Department of Pharmacology, Vels University
Pallavaram, Chennai-600117
Presenting Author
Srota Dawn
M . Pharm (Pharmacology)
2. Introduction:
HCC is the most common type of liver malignancy and 4th most common cause of
cancer mortality.
Worldwide incidence >600,000 cases per year
Liver cancer is the most rapidly increasing cancer in the U.S., Asia and Africa.
19,160 new cases and 16,780 deaths in 2007
More common in men than women (4:1)
For resection, rate of recurrence can be as high as 50% at 2 years
Only 12% are eligible for resection or for transplant
80%-90% of HCC cases occur in cirrhotic livers
3. Factors causing HCC :
Environmental factors
Hepatitis viral infection
Food additives
Excessive alcohol consumption and smoking
Alfatoxins
Exposure with chemical carcinogens
Air and water pollutants
7. About the Plant:
Buchanania lanzan (Anacardiaceae family) is an evergreen deciduous tree, growing 50
ft tall.
popularly known as “Chiranji” in Hindi
Uses:
Buchanania lanzan has folklore evidence for its effective role in curing
asthma, cough, skin diseases, antioxidants and antitumor effect.
All parts of the plant are used for the treatment of various diseases. The oils extracted
from the seed are used for reduction of granular swelling of neck
The kernel is used to treat skin diseases like itching and as prickly heat also
The gum obtained from the bark of Buchanania lanzan used for treating diarrhoea
and intercostals pains.
Leaves of Buchanania lanzan tree are used for promoting wound healing
8. Work plan and procedure
Collection and authentication of plant material
Extraction procedure (soxhlet extractor, 70% ethanol, temperature 3540ºC for 72 hrs.); preliminary phytochemical studies
Selection of animals(30 adult male wistar rats, 100-150 g);
acclimatized for 2 weeks, 12 hrs. day/night cycle, pallet food, water ad libitum
;
experimental procedure and protocol used in this study was reviewed and
approved by Institutional Animal Ethics Committee (IAEC)
The doses are fixed after performing the acute toxicity studies according to
the OECD guideline 423
Continued….
9. Grouping of the selected animals
Experimental animals are randomly divided into 5 groups
(n=6)
GROUP - І
(control)
GROUP – ІІ (DEN
alone)
GROUP - ІІІ
(DEN+TEST 1 ;
200 mg/kg)
GROUP – ІV
(DEN+TEST 2 ;
400mg/kg)
GROUP - V
(DEN+ 5
Flurouracil)
DEN (in DMSO) is given intraperitonially at a dose of 50 mg/kg once in a week for a
period of three weeks to GROUP – ІІ, GROUP – ІІІ, GROUP - ІV and GROUP - V ;
The test drug is given for 15 days;
Continued.........
10. After 21 days of observation the overnight fasted animals were
sacrificed by chemically induced euthanasia using chloroform.
Blood samples were collected by
cardiac puncture
serum was obtained from the blood by
centrifugation at 4,000 rpm for 20
minutes. The supernatant is used for
estimation of
Serum enzymes like
SGPT, SGOT, ALP, total protein
and bilirubin values were
evaluated.
determination of antioxidant
enzymes, liver was dissected
Using ice cold saline and homogenate was
prepared (0.1M Tris–HCL buffer, pH 7.4)
, centrifuged and the supernatant was used for
the estimation of
Tissue LPO , SOD and CAT
level were estimated.
All data obtained from the experiment were represented as mean ± SEM and
analyzed by One way ANOVA and for multiple comparisons of groups with post hoc
Bonferroni’s test was used.
11. Result and graphs:
Table no. 1. Data showing preliminary Phytochemical screening of the ethanolic extract of
Buchanania lanzan leaves extract.
Sl. No.
Phytochemical test
Ethanolic extract of
Buchanania lanzan leaves
extract
1.
Test for Alkaloids
+ve
2.
Test for Steroids
-ve
3.
Test for Tannins
-ve
4.
Test for Saponin
-ve
5.
Test for Gallic acid
+ve
6.
Test for Flavonoids
+ve
7.
Test for Carbohydrates
-ve
8.
Test for Gum and Mucilage’s
-ve
9.
Test for Glycoside
+ve
12. Table no. 2.
Effect of ethanolic extract of Buchanania lanzan leaves on
Biochemical parameters in DEN induced Hepatocarcinoma in rats.
Bilirubin
(mg/dL)
Groups (n=6)
SGOT (IUL-1)
SGPT (IUL-1)
ALP (IUL-1)
TP (mg/100g)
Normal control
51.47±0.33
98.25±0.27
220.25±0.25
767.90±0.23
2.
DEN alone
103.13±0.45
255.51±0.15
295.33±0.62
832.16±0.52
1.517±0.093
3.
DEN+200mg/kg
extract(T1)
91.21±0.23
242.98±0.34
288.12±0.33
818.32±0.43
0.510±0.06
4.
DEN+400mg
extract(T2)
84.45±0.65
180.43±0.33
252.65±0.22
792.21±0.21
0.536±0.12
5.
DEN+ 5FU
81.11±0.54
167.55±0.56
247.55±0.93
788.34±0.99
0.510±0.05
Sl. no.
1.
0.460±0.022
All values are presented as mean ± SEM. Group II was compared with group I and all values were significant
(p< 0.001); group III, group IV and group V were compared with group II and all values were significant. (p<
0.001). P values were calculated by one way ANOVA analysis with post hoc Bonferroni’s test. (N=6)
13. Graphical representation
SGOT & SGPT level (IU/L)
300
Effect of Buchanania lanzan on SGOT and SGPT level in DEN induced Hepatocarcinoma in rats
250
200
150
255.51
242.98
100
180.43
50
98.25
103.13
91.21
84.45
167.55
81.11
51.47
0
Normal control
DEN alone
DEN+200 mg/kg
extract(T1)
DEN+400 mg
extract(T2)
DEN+ 5FU
Groups
SGOT (IUL-1)
SGPT (IUL-1)
* Reitman et.al 1957
14. Effect of Buchanania Lanzan on Biochemical parameters in DEN
induced Hepatocarcinoma in rats.
1000
900
ALP and TP level
800
700
600
500
400
832.16
767.9
818.32
792.21
788.34
300
200
100
220.25
295.33
288.12
252.62
247.55
0
Normal control
DEN alone
DEN+200 mg/kg
extract(T1)
Groups
DEN+400 mg/kg
extract(T2)
DEN+ 5FU
ALP (IUL-1)
TP (mg/100g)
15. 1.8
Effect of Buchanania lanzan on bilirubin level in DEN induced Hepatocarcinoma
in rats.
1.6
Bilirubin level (mg/dl)
1.4
1.2
1
0.8
1.517
0.6
0.4
0.2
0.51
0.46
0.536
0.51
DEN+200mg/kg
extract(T1)
DEN+400mg/kg
extract(T2)
DEN+ 5FU
0
Normal control
DEN alone
Groups
16. Table no. 3. Effect of Buchanania Lanzan on Antioxidant level in DEN induced
Hepatocarcinoma in rats.
Sl. no.
Groups
(n=6)
SOD
CAT
LPO
1.
Normal control
8.89±0.12
63.21±1.45
0.095±.003
2.
DEN alone
4.44±0.10
41.85±1.33
0.219±0.004
3.
DEN+200mg extract(T1)
5.11±0.21**
48.55±1.09**
0.115±0.013
4.
DEN+400mg extract(T2)
7.65±0.33
56.09±0.99
0.178±0.02**
5.
DEN+ 5FU
7.99±0.55
57.99±1.93
0.099±0.003
All values are presented as mean ± SEM. Group II was compared with group I and all values were
significant (p<0.001)
Group III (p<0.01) vs. Group II
Group IV (p<0.01) vs. Group II
Group V (p<0.01) vs. Group II, N=6
SOD = µ moles of MDA/min/mg protein
CAT = µ moles of H2O2 consumed/min/mg protein
LPO = µ moles of MDA/min/mg protein
17. Effect of Buchanania Lanzan on SOD and CAT level in DEN induced Hepatocarcinoma in
rats.
70
60
SOD and CAT level
50
40
SOD
63.21
30
57.99
56.09
48.55
41.85
20
10
8.89
7.65
4.44
7.99
5.11
0
Normal control
DEN alone
DEN+200mg/kg
extract(T1)
Groups
DEN+400mg/kg
extract(T2)
DEN+ 5FU
CAT
18. Effect of Buchanania Lanzan on LPO level in DEN induced Hepatocarcinoma in rats.
0.25
LPO level
0.2
0.15
0.219
0.1
0.05
0.178
0.115
0.095
0.099
0
Normal control
DEN alone
DEN+200mg/kg
extract(T1)
Groups
DEN+400mg/kg
extract(T2)
DEN+ 5FU
19. Discussion:
Elevation of the plasma levels of cytoplasmic and mitochondrial enzymes are
sensitive indicators of liver damage. Elevation of serum SGPT, SGOT, ALP, TP, LPO and
bilirubin is known effect of DEN toxicity which specially affects the liver and activities of
ALP, SGPT, SGOT, and LPO are most commonly used biochemical markers of liver
damage. Lipid peroxidation plays an important role in carcinogenesis
There is a significant dose dependent reduction in SGOT , SGPT, ALP, TP level and
SOD, CAT levels are increased in dose dependent manner in the B. lanzan treated groups.
The low dose of extract shows a better effect in reduction of bilirubin and LPO level
than the high dose.
20. Conclusion:
The preliminary phytochemical studies reveal the presence of flavonoids, Alkaloids, Gallic
acid and Glycoside in total extract of Buchanania lanzan.
After studying many journals we can say that Flavonoid and alkaloids has a chemo
preventive role in cancer. Therefore the possible mechanism of anticancer of Buchanania
lanzan may be due to flavonoid or alkaloid content and further study has to be carried out to
study the exact mechanism behind it and isolation of the active constituents present in the
extract.
21. Acknowledgments
The authors are thankful to Vels University, Pallavaram, Chennai – 600117, India,
for providing the necessary laboratory facilities for this study.
I want to thank my teacher Sumithra M. for giving me the opportunity to present this
paper and I also want to thank the authority of NATIONAL CONFERENCE ON
HERBAL MEDICINE (NCHM’13) for encouraging the students and research scholars
and for giving a platform to present their views.
