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Protective Effect of Hypericum triquetrifolium
Turra. on Cyclophosphamide-Induced
Hepatotoxicity in Rat
Asst.Prof. Songul CETIK
Mardin Artuklu University, TURKEY
İntroduction
Cyclophosphamide (CP) is the most widely used alkylating agent in
chemotherapy with a high therapeutic index and broad spectrum of
activity against a variety of cancers since the late-1950s 1.
The antitumor effect of CP is in proportion to the dose of CP
administered, which often results in hepatotoxicity, immunosuppression
and cytotoxic effects 2 .
Side Effect of CP
Therapeutic effectiveness of anticancer drugs is associated with severe side
effects due to their toxicity.
Liver involvement during CP therapy may represent treatment failure, disease
progression or CP-induced hepatotoxicity.
Thus, strategies to minimize the side effects of chemotherapeutic agents while
preserving their chemotherapeutic efficacy are needed.
CP Metabolits
CP produces two active metabolites such as phosphoramide mustard (PAM) and
acrolein (ACR). Lipid peroxidation (LPO) is one of the principal causes of CPinduced toxicity and is mediated by the production of ACR, a metabolite for much
of its toxicity 3, 4.5.
Acrolein interferes with the tissue antioxidant defense system, produces highly
ROS, and interacts with protein amino acids causing structural and functional
changes in enzymes 6,7.
Experimental evidence suggests that oxidative stress is responsible for
cyclophosphamide hepatotoxicity8,9.
In CP treatment, toxicity is a vital dose limiting factor. Plasma
antioxidant concentration has shown a decrease of patients who had a
high dose chemotheraphy.
As also suggested by previous studies, in order to benefit from CP at
higher doses, there is a need for protective agents that would eliminate
the toxic side effects of CP.
We need new agents to let CP's higher dose useges by preventing the CP's
toxic effects, without tumor preserving and tumor growth stimulating
properties, to protect normal tissues from chemotherapy induced toxicity.
By preventing dose-limiting antineoplastic drugs toxic effects, several methods
have been developed to let their more effective doses in many conducted
experimental and clinical studies, in aspect of importance of role of some the
natural phenolic compounds on body.
Hypericum
Antioxidants that can eliminate toxic side effects of chemotherapy can
provide the use of higher and more effective doses of the anticancer drugs.
The genus Hypericum (family Hypericaceae) have been ethnomedically used in
different parts of the world for its sedative, antiseptic, antioxidant,
antimicrobial and cytotoxic properties.
Hypericum triquetrifolium TURRA.
Hypericum triquetrifolium Turra (HT) belongs to the Hypericaceae family and it is
native to eastern Europe and the Mediterranean area.
The methanolic extract of H.triquetrifolium contain chlorogenic acid, rutin,
hyperoside, quercitrin, quercetin, kaempferol, phenolic - flavonoid compounds
and other water-soluble components that possess a wide array of biological
properties.
HT is a phenolic component with antioxidative, analgesic and anticarcinogenic
properties.
The methanolic extract of H. triquetrifolium aerial parts, possesses a significant
antioxidant activity.
PURPOSE
This study aimed to investigate the possible protective effect of HT on CPinduced hepatotoxicity.
In order to determine the protective effect of HT upon the liver, the levels of
alanine transaminase (ALT), aspartate transaminase (AST),
alkaline
phosphatase (ALP) and lactate dehydrogenase (LDH) as well as the total
levels of total oxidant capacity (TOC) and total antioxidant capacity (TAC)
were determined. Also, oxidative stres index (OSI) was measured.
Furthermore, the liver tissues were analysed histologically.
Plant Extraction
Wild-growing plants (H. triquetrifolium seeds) were harvested at seeding stages
from August to September, 2015 from wild populations located in Zınar, Mardin,
Turkey.
A total of 20 g of each dried material were ground in a grinder with a 2-mmdiameter mesh and then incubated into a glass flask with 200 mL (99%) methanol
for 3 days under magnetic stirrer. After it was filtrated, the methanol was removed
on rotary evaporator.
Approximately, 2 g of the crude methanol extracts of H. triquetrifolium seed was
obtained and kept in dark and airtight glass bottles at 4ºC until it was used.
Then when used for injetions solved with % 0.2 DMSO.
Experimental Groups
Albino rats (Wistar, 3-4 months old, male, weight 220 ± 20 g healthy) were
randomly divided in 9 groups, each including seven animals:
Group 1 (control) treated with saline;
Group 2 treated with 150 mg/kg CP, respectively;
Groups 3, 4 and 5 treated with 25, 50 and 100 mg/kg HT;
Groups 6, 7 and 8 treated with 25, 50 or 100 mg/kg HT+CP,
Group 9 treated with 0.5ml - %0.2 DMSO.
All injections i.p.
CP and HT Administered to Groups
Days
--->
Given Matter
v
1
2
3
CP+HT
Control & DMSO
5
6
+
CP (150mg/kg)
HT(25, 50,100)
4
7
Sacrifice
+
+
+
+
+
+
Sacrifice
HT
HT
HT
CP + HT
HT
HT
Sacrifice
SF
SF
SF
SF
SF
SF
Sacrifice
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
Statistical Analysis
The results were expressed as means ± S.E.M. Statistical analysis was
performed by using One Way Analysis of Variance and Kruskal-Wallis One
Way Analysis of Variance on Ranks Test and p<0.05 accepted as considering
statistical significance.
Each experiment was repeated at least three times.
Histological Analysis
Histological: Liver were fixed with 10% formaldehyde solution. Through
routine histological preperations, samples were embedded in paraffin and 5.0
micron thick serial sections were made, which were stained with HematoxylinEosin and histopathologic features were evaluated.
The results was analyzed by One Way Analysis of Variance and Kruskal-Wallis
One Way Analysis of Variance on Ranks Test.
Results
Our study demonstrated that in line with the rise in the dose of 150 mg/kg
CP, we determined an increase the levels of serum ALT, AST, ALP, LDH,
TOS and OSI, besides of moderate degeneration in the liver tissue.
With 25, 50 and 100 mg/kg HT, there was an important decrease with respect
to CP toxicity.
In the groups given both CP plus HT, there was a rise in serum TAS levels,
while the levels of AST, ALT, ALP, LDH, TOS and OSI showed a remarkable
decrease.
DMSO(Dimethyl Sulfoxide)
When the obtained results are evaluated, the control values close to the %0.2
DMSO, which we used for solving the extracts. This result showed that
DMSO doesn’t affect on the potential of plants and the test results.
ALT (U/L)
ALT
30.00
ALT (U/L)
25.00
20.00
15.00
10.00
5.00
0.00
DOSES (mg/kg)
AST (U/L)
AST
120.00
AST (U/L)
100.00
80.00
60.00
40.00
20.00
0.00
DOSES (mg/kg)
ALP (U/L)
ALP (U/L)
ALP
100.00
90.00
80.00
70.00
60.00
50.00
40.00
30.00
20.00
10.00
0.00
DOSES (mg/kg)
LDH (U/L)
LDH
600.00
LDH (U/L)
500.00
400.00
300.00
200.00
100.00
0.00
DOSES (mg/kg)
TOC ( μmol H2O2 equivalent/L )
TOC (μmol H2O2 equivalent/L)
TOC
3.00
2.50
2.00
1.50
1.00
0.50
0.00
DOSES (mg/kg)
TAC ( μmolTroloxequivalent/L )
(TAC, μmolTroloxequivalent/L)
TAC
1.60
1.40
1.20
1.00
0.80
0.60
0.40
0.20
0.00
DOSES mg/kg
OSI( TOC/TAC X 100 )
OSI
OSI ( TOC/TAC x 100 )
0.30
0.25
0.20
0.15
0.10
0.05
0.00
DOSES (mg/kg)
HISTOLOGICAL RESULTS
Fig. 1- CP grubu A: H-E, B: kaspaz-3, C: Bcl-2, D: Bax.
Fig. 2- 100 HP grubu A: H-E, B: kaspaz-3, C: Bcl-2, D: Bax.
Fig. 3- CP+100 HP group A: H-E, B: kaspaz-3, C: Bcl-2, D: Bax.
Conclusion
Results showed for the first time that the methanolic extract of H.
triquetrifolium seed extract, possesses a significant antioxidant activity.
The signs of recovery in the serum biochemical levels were also
true fort the histological findings of the liver.
Conclusion
CP induced oxidative stress in the liver as evident from the increased lipid
peroxidation (LPO), reactive oxygen species (ROS).
Our data suggest that HT is a highly effective antioxidant substance with a
cell-protecting effect on LPO and ROS.
Therefore, HT could serve as effective agent that could lessen the adverse
effects of anticancer drugs in the course of chemotherapy protocols.
REFERENCES
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THANKS for your attention.