PROTECTIVE EFFECT OF DIFFERENT TYPES OF COFFEE EXTRACT ON RATS WITH INDUCED ACUTE LIVER TOXICITY

1
PROTECTIVE EFFECT OF DIFFERENT TYPES OF
COFFEE EXTRACT ON RATS WITH INDUCED
ACUTE LIVER TOXICITY
Esraa T. Moawed ; Naeem M. Rabeh and Hany G. EL-Masry
Nutrition and Food Science Department, Faculty of Home Economics,
Helwan University, Cairo, Egypt
Key Words: green coffee, Arabic coffee, caffeine, liver toxicity, CCl4, rats.
Abstract
Coffee (Coffea L.) is the world’s beloved drink that is the most
regularly consumed caffeine-containing beverage. It contains a complex
mixture of chemicals that provide important amounts of chlorogenic acid
and caffeine, which associated with many health benefits. The aim of the
present study was to investigate the protective effects of conventional,
decaffeinated coffee, green coffee and Arabic coffee extract on rats with
induced acute liver toxicity. Thirty six adult male albino rats (Sprague-
Dawley strain), weighing about (180±10 g) were divided randomly into
two main groups as follow: the first group (-ve control= 6 rats) was fed
on basal diet. The second group (30 rats) were divided into 5 subgroups
from subgroup 1 to subgroup 5. Subgroup 2 (+ve control) fed on basal
diet. Subgroups 2 and 5 were given the same dose of extracts 2ml/rat of
Conventional, Decaffeinated, Arabic and Green coffee, respectively.
After 6 weeks, subgroups from (1-5) were injected with one dose of
CCl4. At the end of the experimental period (6 weeks), rats were scarified
and serum was collected to determine biochemical analysis. The results
showed that the administration of CCl4 resulted in significant elevation in
serum activities of aspartate aminotransferase (AST) alanine
aminotransferase (ALT), alkaline phosphatase (ALP) and total bilirubin
whereas serum total proteins albumin and globulin were significantly
reduced, along with significant elevation of malondialdehyde level
(MDA) and significant depletion in the level of reduced glutathione
peroxidase (GPX) compared with negative control. Treatment with
extracts of coffee prior to CCl4 produced protective effects and
attenuated these biochemical changes. The protective effects of extracts
of coffee were more pronounced for the Arabic coffee. It be concluded
that coffee beans exhibited hepatoprotective actions along with the
restoration of lipid profile in acute liver injury animal models.
INTRODUCTION
Liver is one of the largest organs in human body and the chief site
for intense metabolism and excretion. So it has a surprising role in the
maintenance, performance and regulating homeostasis of the body. It is
Egypt. J. of Appl. Sci., 35 (11) 2020 172-185
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involved with almost all the biochemical pathways of growth, fight
against disease, nutrient supply, energy provision and reproduction
(Mroueh et al., 2004). Liver damage is further characterized into
hepatocellular predominantly (initial Alanine transferase elevation -
initial alkaline phosphatase rise) and cholestatic type. Chemicals and
drugs such as Carbon tetrachloride (CCl4) catabolized radicals induced
lipid peroxidation, damage the membranes of liver cells and organelles,
causing the swelling and necrosis of hepatocytes and result to the release
of cytosolic enzymes in to the blood (Mumoli et al., 2006).
Coffee (Coffea L.) is the world’s beloved drink that is the most
regularly consumed caffeine-containing beverage. It contains a complex
mixture of chemicals that provide important amounts of chlorogenic acid
and caffeine, which associated with many health benefits (Higdon and
Frei, 2006). Coffee is appreciated for its aroma and flavour, and is a
mixture of thousands of different compounds, such as carbohydrates,
lipids, alkaloids, vitamins, nitrogenous molecules and phenolic
compounds (Spiller, 1998). The roasting process causes a loss of water
from the green bean and degradation of many of the compounds
including the antioxidant polyphenols; however, there is very little
difference in total antioxidants between the different roasts of a bean
(Daglia et al., 2000).
Green coffee beans contain 5–14% of the reported major
components, chlorogenic acids (CGAs) are a family of esters formed
between certain phenolic acids (trans-cinnamic acids) and quinic acid
(Farah and Donangelo, 2006). The roasting process destroys CGAs;
therefore, the unroasted beans like green coffee and Arabic coffee are
used as the source for this nutritional supplement (Moon and
Shibamoto, 2009).
The aim of the present study was to investigate the protective
effects of conventional, decaffeinated coffee, green coffee and Arabic
coffee extract on rats with CCl4-induced acute liver toxicity, also to
evaluate on body weight, feed intake, lipid profile and liver functions that
may occur on hepatotoxic rats.
MATERIALS AND METHODS
Materials:
Beans of (Conventional coffee and green coffee), and
decaffeinated coffee were obtained from Agriculture Research Center,
Giza, Egypt. Beans were convert to powder and Arabic coffee were
prepared from green coffee with add some flavors with common doses.
Carbon tetrachloride and chemical kits were obtained from Sigma-
Aldrich Group., Cairo, Egypt. Casein, cellulose, sucrose, choline
chloride, D-L methionine, vitamins and minerals constituents were
purchased from El- Gomhoriya Company Cairo, Egypt. Thirty six adult
173 Egypt. J. of Appl. Sci., 35 (11) 2020
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male albino rats (Sprague-Dawley strain), weighing about (180±10 g)
were purchased from the Animal House of the National Research Center,
Dokki, Egypt.
Methods:
1. Preparation of Water Extract of Conventional Coffee,
Decaffeinated Coffee, Green Coffee and Arabic Coffee:
Twenty gram of conventional coffee, decaffeinated coffee, green
coffee and Arabic coffee powder were submerged in 100 ml of distilled
water and were boiled before being given to the rats, then filtered to
obtain a liquid extract. A dose of 2 ml /rat of conventional coffee,
decaffeinated coffee, green coffee, caffeine and Arabic coffee extract
were given orally respectively by stomach tube.
2. Induction of Acute Liver Toxicity:
Carbon tetrachloride (CCl4)-induced acute hepatotoxity in rats at
the end of the experiment. Intraperitoneal injection of male albino rats
with CCl4 1 mL/kg/BW, (1:1) mixture with paraffin oil according to
(karthikeyan and deepa, 2010).
3. Diet Composition and Experimental Animal Design:
The basal diet was formulated according to AIN-93M diet
(Reeves et al., 1993). Animals (36 rats) were housed in well conditions
in biological studies lab of Faculty of Home Economics. They were left
for seven days as adaptation period and they were allowed to feed
standard laboratory food and water. After the period of adaptation,
animals were divided into two main groups, as follows: - the first main
group (6 rats) was fed on basal diet and served as a negative control
group (-ve), the second main group (30 rats) were divided as follow:-
Subgroup (1): Rats were fed on basal diet and given only distilled
water daily as were kept as positive control group.
Subgroup (2): Rats were given 2 ml/ rat of Conventional Coffee
extract.
Subgroup (3): Rats were given 2 ml/ rat of Decaffeinated Coffee
extract.
Subgroup (4): Rats were given 2 ml/ rat of Arabic Coffee extract.
Subgroup (5): Rats were given 2 ml/ rat of Green Coffee extract.
After 6 weeks, subgroups from (1-5) were injected with one dose
of CCl4 (Karthikeyan and Deepa, 2010).
4. Biological Evaluation:
Feed intake will be recorded daily and animals will be weighed at
the beginning and twice a week throughout the experimental period.
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Body weight gain% and feed efficiency ratio will be calculated at the end
of the experiment according to the method of Chapman et al., (1959).
5. Blood Collection And Serum Separation:
At the end of the experimental period (6 weeks), rats were fasted
overnight before scarifying and blood samples were collected from each
rat and were centrifuged at 3000 rpm for 15 min to obtain the serum for
biochemical analysis.
6. Biochemical Analysis of Serum:
Aspartate aminotransaminase (AST) and Alanine
aminotransaminases (ALT) were determined according to the method
described by Young, (2001), and Alkaline phosphates (ALP) was
determined according to Roy, (1970). Total bilirubin, Malondialdehyde
(MDA) and Glutathione peroxidase (GPx) were determined according to
Young, (2001); Draper and Hadley, (1990) and Hissin and Hilf,
(1970), respectively. Serum total cholesterol (TC), triglyceride (TG),
high density lipoprotein cholesterol (HDL-C) were determined according
to Richmond, (1973); Wahlefeld, (1974) and Albers et al., (1983),
respectively. Regarding to serum low density lipoprotein cholesterol
(LDL-C) and very low density lipoprotein cholesterol (VLDL-C) were
calculated according to Fridewald et al., (1972). Total protein, albumin
and globulin were determined in the serum according to method describe
by Burtis and Ashwood, (1999); Young, (2001) and Goldenberg and
Drewes, (1971), respectively. The biochemical determinations were
conducted at the National Research Center, Giza, Egypt.
7. Statistical Analysis:
Results were expressed as the mean standard error ± SE. Data
were statistically analyzed for variance “ANOVA” test at P ≤ (0.05)
using SPSS statistical software, version 20 was used for these
calculations (Armitage and Berry, 1987).
RESULTS AND DISCUSSION
Results recorded in Table 1 showed the effect of diet supplemented
with extracts of Conventional, Decaffeinated, Arabic and Green Coffee
on FI, BWG % and FER of Hepatotoxic Rats. Feed intake was decreased
in positive control group compared to negative control group, while
groups treated with extracts of Conventional, Decaffeinated, Arabic and
Green Coffee were reduced in feed intake compared to negative control
group. There were significantly decreased (P< 0.05) in BWG and FER
for the positive control group compared to the negative control group, it
was also showed that all treatment groups with extracts of Conventional,
Decaffeinated, Arabic and Green Coffee significantly increased (P< 0.05)
in BWG and FER compared with positive control group expect BWG in
175 Egypt. J. of Appl. Sci., 35 (11) 2020
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group treated with Conventional Coffee non significantly compared to
positive control group.
Table (1): Effect of Conventional, Decaffeinated, Arabic and Green
Coffee Extract on Feed Intake (FI), Body Weight Gain%
(BWG %) and Feed Efficiency Ratio (FER) in Rats with
CCl4-Induced Acute Liver Toxicity
Parameters
Groups
FI (g/d)
BWG%
FER
Negative Control 15.00 34.05±0.05a 0.136±0.004a
Positive Control 14.00 17.16±0.84e 0.070±0.001c
Conventional Coffee 13.50 23.53±0.83cd 0.122±0.002b
Decaffeinated Coffee 12.00 21.53±0.53de 0.124±0.024b
Arabic Coffee 13.00 31.94±0.78ab 0.138±0.037a
Green Coffee 14.00 27.51±0.83bc 0.130±0.001ab
*Mean values are expressed as means ± SE.
*Mean values at the same column with the same superscript letters are not
statistically significant at P<0.05.
Results in Table 1 indicated that CCl4 administration significantly
lowered FI, BWG and FER compared to negative control group, these
results were in the same line with Shaker et al., (2010) and Wang et al.,
(2018). Mohmoud et al., (2013) found that the feeding on different doses
of Arabic coffee for 30 days lowered body weight in rats fed on basal
diet. The possible mechanism by which coffee prevented higher body
weight gain in the present study could be by increasing lipolysis via
catecholamines (Kobayashi-Hattori et al., 2005 and Lopez-Garcia et
al., 2006). Caffeine might also have caused body weight loss by
increasing physical activity. It is known that green coffee effects the
body weight, green coffee extract has a hypotensive effect in mice,
decreases visceral fat and body weight (Jeszka-Skowron et al. 2016).
As seen in Table 2, serum concentrations of aspartate
aminotransferase (AST), alanine aminotransferase (ALT), alkaline
phosphatase (ALP) and total bilirubin were significantly (P< 0.05)
elevated by CCl4 administration (positive control group) compared with
negative control group. It was observed significant (P< 0.05) reduce in
serum AST, ALT, ALP and total bilirubin levels for all groups treated
with extracts of Conventional, Decaffeinated, Arabic and Green Coffee
compared to positive control group, group treated with Arabic Coffee
was close to the negative control group.
Mahmoud et al., (2013) evaluated the effect of consumption of
different levels of Arabic coffee on blood parameters concentration.
Blood samples were collected and used for determination of ALT, and
AST. These parameters were found to be significantly lower in group
treated with coffee than control group. These results are in accordance to
other studies in which coffee consumption displayed reduction of liver
Egypt. J. of Appl. Sci., 35 (11) 2020 176
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functioning enzymes such as, AST, ALT and ALP as prevention from
cirrhosis and fibrosis (Sadiq et al., 2019).
Table (2): Effect of Conventional, Decaffeinated, Arabic and Green
Coffee Extract on Serum AST, ALT, ALP and Total
Bilirubin in Rats with CCl4-Induced Acute Liver Toxicity
Parameters
Groups
AST
ALT ALP
Total
Bilirubin
U/L mg/dL
Negative Control 91.96±0.83f 29.64±0.54d 88.84±0.48e 0.39±0.014d
Positive Control 140.79±0.42a 48.01±0.52a 150.26±0.32a 0.91±0.010a
Conventional coffee 129.65±0.30b 34.62±0.98c 108.97±0.58bc 0.58±0.016c
Decaffeinated coffee 124.33±0.89c 38.45±0.90b 114.35±0.98b 0.69±0.017b
Arabic coffee 97.09±0.98e 30.42±0.90d 98.09±0.99d 0.40±0.006d
Green coffee 101.36±0.71d 32.44±0.44cd 103.69±0.95cd 0.43±0.007d
*Mean values are expressed as means ± SE.
*Mean values at the same column with the same superscript letters are not
statistically significant at P<0.05.
Green coffee bean extract contains chlorogenic acid (CGA) as the
principal constituent, and most of the health benefits of decaffeinated
coffee and its by-product have been attributed to chlorogenic acid (Stohs
and Badmaev, 2016). The effects induced by repeated CCl4
administration were attenuated by conventional and decaffeinated,
arabica and green coffee intake. These findings confirm the beneficial
effects of coffee and against liver injury induced by other hepatotoxicants
(Ozercan et al., 2006; Lee et al., 2007; Shi et al., 2010 and Moreno et
al., 2011). However, a suitable evaluation of the protective efficacy of
coffee beverages and caffeine against different stages of carcinogenesis
might be better warranted in other models of rat liver carcinogenesis
(Okano et al., 2008 and Silva‐ Oliveira et al., 2010).
Results in Table 3 showed a significant decrease (P< 0.05) in
serum glutathione peroxidase (GPX) activity of the positive control group
compared with the negative control group. It was clear that, there was
significant (P< 0.05) increase in serum GPX activity for all treated groups
with Coffee compared to the positive control group. Concerning to serum
malondialdehyide (MDA) level, results showed that serum MDA level
was significantly increased (P< 0.05) in the positive control group
compared with the negative control group whereas all treated groups with
Coffee significantly decreased (P< 0.05) compared to the positive control
group. It was also observed that rats were fed on Arabic coffee rextract
177 Egypt. J. of Appl. Sci., 35 (11) 2020
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considered the best group for enhancing the serum GPX and reducing
serum MDA levels.
Table (3): Effect of Conventional, Decaffeinated, Arabic and Green
Coffee Extract on Serum GPX and MDA in Rats with
CCl4-Induced Acute Liver Toxicity
Parameters
Groups
GPX
(U/mg)
MDA
(μmol/dL)
Negative Control 64.03±0.61a 9.80±0.19e
Positive Control 29.10±0.98e 22.11±0.51a
Conventional coffee 46.71±0.93c 15.02±0.40bc
Decaffeinated coffee 40.72±0.29d 17.02±0.34b
Arabic coffee 63.62±0.91a 12.74±0.41d
Green coffee 54.62±0.72b 13.28±0.73cd
*Mean values are expressed as means ± SE.
*Mean values at the same column with the same superscript letters are not
statistically significant at P<0.05.
Demirtaş et al., (2012) reported that caffeine doses decreased lipid
peroxidation (MDA) in liver. Antioxidant enzyme activities in the rat
liver, like GPx, showed a statistically significant increase with caffeine
intake. Chlorogenic acid, a phenolic compound found in green coffee,
has antioxidant activity and has the ability to trap superoxide anions or
hydroxyl radicals (Castro et al., 2018). Cholorogenic acids are strong
reactive oxygen species (ROS) scavengers. ROS are produced
physiologically during various cellular processes such as aerobic
metabolism and should be harmful when the amount is high. Even
though ROS are known to be harmful, in order to sustain cellular
homeostasis through redox cell signalling, they must be at a certain level
(Priftis et al., 2018).
Data revealed in Table 4 that serum TC, TG, LDL-C and
VLDL-C were significantly (P< 0.05) increased in the positive control
group compared with the negative control group. Results also illustrated
that all groups that were treated with coffee decreased significantly (P<
0.05) in serum TC, TG and VLDL-C levels compared to the positive
control group. Regarding serum HDL-C level, results showed a
significant (P< 0.05) decrease in serum HDL-C level of the positive
control group compared to the negative control group. There was a
significant change in serum HDL-C level for all treated groups with
coffee compared to negative control group. The highest improvement for
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lipid profile was observed at the groups treated with by the extracts of
Arabic and Green coffee.
Table (4): Effect of Conventional, Decaffeinated, Arabic and Green
Coffee Extract on Serum TG, TC, HDL-C, LDL-C and
VLDL-C in Rats with CCl4-Induced Acute Liver Toxicity
Parameters
Groups
TG TC HDL
LDL VLDL
mg/dl
Negative Control 45.03±0.38f 89.19±0.38d 44.37±0.63a 35.46±0.48d 9.00±0.76f
Positive Control 112.62±0.83a 146.64±0.65a 25.03±0.24f 99.09±0.57a 22.52±0.16a
Conventional coffee 98.08±0.98c 124.47±0.98b 31.85±0.72d 73.00±0.89b 19.61±0.43c
Decaffeinated coffee 103.00±0.92b 130.47±0.36b 27.68±0.59e 82.02±0.65b 20.75±0.18b
Arabic coffee 67.38±0.73e 93.83±0.99d 36.04±0.43c 44.31±0.89c 13.70±0.14e
Green coffee 73.53±0.60d 105.43±0.97c 39.44±0.35b 51.28±0.97c 14.70±0.12d
*Mean values are expressed as means ± SE.
*Mean values at the same column with the same superscript letters are not
statistically significant at P<0.05.
Liver is the major site for the synthesis and metabolism of
cholesterol (Yang et al., 2011). Distinct alterations in lipid metabolism
have been reported in CCl4 induced hepatotoxicity in rats (Singhal and
Gupta, 2012). The present results expressed the significant increasing in
levels of serum TG, TC, LDL-C and VLDL-C, while decreasing in HDLC
level in CCl4 inducted rats. Treatment of coffee also attenuated these
changes of lipid profile of rats treated previously with CCl4. These results
were in the same line with Sadiq et al., (2019) who reported that Arabica
coffee decreased the levels of TG, TC and LDL in co-treated group,
while an elevation in HDL levels is also there. This could also be
attributed to the various bioactive molecules identified, along with
various fat-soluble compounds (Lee, 2000). It is suggested that, both, the
type and mode of coffee preparation strongly affect the lipid profile
modulation (Corrêa et al., 2013). The bioactive molecules, cafestol and
kahewol are found in considerable amounts in unfiltered boiled coffee,
that enhance TG, TC and LDL (Heckers et al., 1994; Weusten-Van der
Wouw et al., 1994; Urgert et al., 1997).
Green coffee directly affects all the parameters of the lipid
profile (Shimoda et al., 2006). The phytoconstituent of green coffee
beans was reported to significantly improve the levels of HDL and
VLDL (Meng et al., 2013). Additionally, green coffee was reported to
decrease the levels of cholesterol, triglyceride, and LDL (Yukawa et al.,
2004).
179 Egypt. J. of Appl. Sci., 35 (11) 2020
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In Table 5, CCl4 treatment significantly (p< 0.05) reduced serum
total proteins, albumin and globulin as compared to negative control.
Groups treated with extracts of Conventional, Decaffeinated, Arabic and
Green Coffee have a significant increase compared to positive control
group. The best improvement of serum total proteins, albumin and
globulin was observed in the group treated with Arabic coffee.
Results in Table 5 were in agreement with Hussein et al., (2020),
who reported that the treatment with green coffee significantly increased
levels of total protein, albumin and globulin compared to positive control
group. On the other hand, Birkner et al., (2006) showed that the level of
total protein increased after treatment with caffeine.
Table (5): Effect of Conventional, Decaffeinated, Arabic and Green
Coffee Extract on Serum Total Protein, Albumin and
Globulin in Rats with CCl4-Induced Acute Liver Toxicity
Parameters
Groups
Total Protein Albumin Globulin
g/dL
Negative Control 5.02±0.06a 3.65±0.13a 2.18±0.88a
Positive Control 2.43±0.15c 0.98±0.13d 1.17±0.27d
Conventional coffee 2.63±0.13c 2.29±0.11c 1.24±0.24d
Decaffeinated coffee 3.76±0.09b 1.97±0.58c 1.40±0.10cd
Arabic coffee 4.75±0.11a 2.89±0.40b 1.99±0.11ab
Green coffee 4.00±0.10b 3.31±0.97a 1.73±0.14bc
*Mean values are expressed as means ± SE.
*Mean values at the same column with the same superscript letters are not
statistically significant at P<0.05.
CONCLUSION:
Coffee is beneficial for health in general and particularly for
patients with liver disease. Consumption of coffee protects against
progression of almost all forms of liver disease. Usual mechanisms
involved are prevention of fibrosis, carcinogenesis, and antioxidant
effect. The incidence of advanced fibrosis and cirrhosis is lower among
coffee drinkers. The risk of hepatocellular carcinoma also is lower in
coffee drinkers compared to the non-coffee consuming population.
However, in view of retrospective nature of data, more interventional
trials are required before coffee finds its way in the regular prescription
in liver disease patients.
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التأثير الوقائي لمستخمص أنواع مختمفة من القهوة في الفئ ا رن المحدث لها
التسمم الکبدي الحاد
إس ا رء طارق معوض، نعيم محمد ا ربح، هاني جابر المصري
قسم التغذية وعموم الأطعمة - کمية الاقتصاد المنزلي - جامعة حموان
القهوة هي المشروب المحبب في العالم وهى أکثر المشروبات التي تحتوي عمى الکافيين
استهلاکًا بانتظام. يحتوي عمى مزيج معقد من المواد الکيميائية التي توفر کميات مهمة من
حمض الکموروجينيک والکافيين، والتي ترتبط بالعديد من الفوائد الصحية. کان الهدف من هذه
الد ا رسة هو د ا رسة التأثي ا رت الوقائية لمقهوة التقميدية ومنزوعة الکافيين والقهوة الخض ا رء
ومستخمص البن العربي عمى الفئ ا رن المصابة بسمية کبدية حادة. تم تقسيم ستة وثلاثين من
10 ± التي تزن حوالي ) 180 ، )Sprague-Dawley ذکور الفئ ا رن البيضاء البالغة )سلالة
جم( بشکل عشوائي إلى مجموعتين رئيسيتين عمى النحو التالي: المجموعة الأولى )المجموعة
الضابطة = 6 فئ ا رن( تم تغذيتها عمى النظام الغذائي الأساسي. تم تقسيم المجموعة الثانية ) 30
فئ ا رن( إلى 5 مجموعات فرعية من المجموعة الفرعية 1 إلى المجموعة الفرعية 5. المجموعة
الفرعية 2 )مجموعة ضابطة موجبة( تم تغذيتها عمى النظام الغذائي الأساسي. تم إعطاء
المجموعات 2 إلى 5 نفس جرعة المستخمصات 2 مل / فأر من القهوة التقميدية ، منزوعة
الکافيين ، العربية والقهوة الخض ا رء ، عمى التوالي. بعد 6 أسابيع تم حقن مجموعات فرعية من
في نهاية فترة التجربة ) 6 أسابيع( تم تشريح الفئ ا رن وجمع .CCl 5-1 ( بجرعة واحدة من 4 (
السيرم لإج ا رء التحاليل الکيميائية. أظهرت النتائج أن الحقن ب ا ربع کموريد الکربون أدى إلى ارتفاع
والبميروبين الکمي ، بينما انخفض بشکل کبير البروتين ALP ،ALT ،AST کبير في سيرم
وانخفاض کبير في MDA الکمي في الدم ، الألبومين والجموبيولين، مع ارتفاع من مستوى
CCl مقارنة بالمجموعة الضابطة السالبة. أدى العلاج بمستخمصات القهوة قبل 4 GPX مستوى
إلى تأثي ا رت وقائية وقمل من هذه التغي ا رت البيوکيميائية. کانت التأثي ا رت الوقائية لمستخمصات
القهوة أکثر وضوحًا لمقهوة العربية. نستنتج أن حبوب البن أظهرت تأثي ا رت وقائية لمکبد مع
استعادة المظهر الدهني في نماذج حيوانية المصابة بالتسمم الکبدي الحاد.
185 Egypt. J. of Appl. Sci., 35 (11) 2020