EFFECT OF MILK THISTLE(Silybummarianum)ON HEPATOTOXIC RATS

تأثیر الخرشوف البری على الفئران المصابة بتسمم الکبد

منى محمود علی، هانی جابر المصری، محمد حمدی حجاج

قسم التغذیة وعلوم الأطعمة  - کلیة الاقتصاد المنزلی - جامعة حلوان

ینتمی الخرشوف البری إلى العائلة الأستیراسیة، تم استخدامه منذ 2000 سنة کعشبة علاجیة. یطلق على مستخلص بذور الخرشوف البری اسم السیلیمارین، الذی یحتوی على العدید من الأنشطة العلاجیة کمضاد للسکری، مضاد للإلتهابات ووقایة الأعصاب. کان هدف هذه الدراسة معرفة تأثیر استخدام بذور الخرشوف البری على الفءران المصابة بتسمم الکبد. حیث أجریت الدراسة علی ثلاثون فأرا من نوع الالبینو، تتراوح أوزانهم من (150 ± 10 جم) . تم تقسیم الفئران الی مجموعتین أساسیتین. المجموعة الاولی (5 فئران )تم تغذیتهم علی الغذاء الأساسی طوال فترة التجربة  وتعتبر المجموعة الضابطة السالبة . المجموعة الثانیة (25 فأرا) تم تغذیتهم علی الغذاء الآساسی و حقنهم بمادة رابع کلورید الکربون ( ا مل /کجم من وزن الجسم ( بنسبة 1:1 مع خلیط من زیت البارافین لمدة 3 أیام لإحداث تلف الکبد ، وبعد ذلک تم تقسیم الفئران الی 5 مجامیع فرعیةمن المجموعة (2) إلی المجموعة (6). المجموعة الفرعیة (2):  تم تغذیتهم علی الغذاء الأساسی  فقط وتعتبر مجموعة ضابطة موجبة. المجموعة الفرعیة (3) و(4) : تم تغذیتهم علی الغذاء الأساسی المحتوی علی مسحوقبذورالخرشوف البری بنسبة 5% و10% لکل کجم من الغذاء الأساسی، علی التوالی .  المجموعة الفرعیة (5) و(6) : تم تغذیتهم علی الغذاء الأساسی المحتوی علی المستخلص المائی لبذور الخرشوف البری بترکیز 5% و 10% لکل کجم من الغذاء الأساسی.استمرت التجربة لمدة أربعة أسابیع وفی نهایة فترة التجربة تم تشریح الفئران والحصول علی السیرم لاجراء التحالیل البیوکیمیائیة. أظهرت النتائج أن مستویات ALT, AST, ALP, total bilirubin, MDA ارتفعت بشکل ملحوظ فی المجموعة الضابطة الموجبة مقارنة بالمجموعة الضابطة السالبة، بینما کان هناک انخفاض ملحوظ فی مستوى GPx. کما أشارت النتائج إلى أن إضافة مسحوق والمستخلص المائی لبذور الخرشوف البری عکس هذه التغیرات التی حدثت بالتسمم برابع کلورید الکربون. لذلک توصی هذه الدراسة بأنتناولنبات الخرشوف البریقدیکونمفیدًاللمرضىالذینیعانونمنأمراضالکبدالمرتبطةبالإجهادالتأکسدی.

الکلمات المفتاحیة: الخرشوف البری، السیلیمارین، تسمم الکبد، الفئران.

EFFECT OF MILK THISTLE(Silybummarianum)ON HEPATOTOXIC RATS

Mona M. Ali; H. G. EL-Masry and M. H. Haggag

Nutrition and Food Science Department, Faculty of Home Economics,

Helwan University, Cairo, Egypt

Keywords:Silybummarianum, silymarin, hepatotoxicity, rats.

ABSTRACT

Silybummarianum (SM)(milk thistle, family: Asteraceae)is a therapeuticherb with a 2,000-year history of use. SM seeds extract has been called silymarin which has manytherapeuticactivities asantidiabetic, anti-inflammatory andneuroprotective.The aim of the present study was to investigate the effect of Silybummarianumseeds on hepatotoxicity in rats. Thirty adult male albino rats (Sprague-Dawley strain), weighing about (150±10g)were divided randomly into two main groups as follow: the first group (-ve control= 5 rats) was fed on basal diet. The second group (25 rats) were fed on basal diet and injected with CCl₄ 1ml/kg, (1:1) mixture with paraffin oil for 3 days to induce acute liver damage, then divided into 5 groups from group 2 to group 6. Group 2 (+ve control) fed on basal diet. Group 3 and 4 fed on basal diet supplemented with 5% and 10% SM seeds powder, respectively. Also, group 5 and 6 fed on basal diet supplemented with 5% and 10% SM seeds aqueous extract, respectively. At the end of the experimental period (4 weeks), rats were scarified and serum was collected to determine liver functions, lipid profile and antioxidant enzyme. The results showed that serum levels of alanine aminotransferase (ALT),aspartate aminotransferase (AST), alkaline phosphatase (ALP), total bilirubin and malondialdehyide (MDA) were significantly elevated (P<0.05) by CCl₄ administration (positive control group) compared with negative control group, while glutathione peroxidase (GP_X)significantly decreased (P<0.05). It also found that supplemented diet with Silybummarianumseeds and its extract reversed these changes that caused by CCl₄ administration. The study recommends that intake of Silybiummarianumplant may be beneficial for patients who suffer from liver diseases associated with oxidative stress.

INTRODUCTION

Liver is the largest solid organ in the body, weighing about 1.5 kg in an adult. Liver plays a vital role in various functions of the body such as metabolism, secretion, storage, and detoxification of endogenous and exogenous substances (Raoet al., 2013). During the past 30 years and even after the major progress in the liver disease management, millions of people still suffer from an acute or chronic liver condition worldwide. Liver diseases affect more than 10% of the world population and its mortal end-stage generally follows cirrhosis and liver cancer (Muriel, 2017).Méndez-Sánchezet al., (2005) have estimated approximately two million cases of chronic liver disease by the year 2050.

Medicinal plants have played an important role in world health and are circulated worldwide, found mostly in tropical countries. It is noted that about 25% of all modern medicines are indirectly or directly obtained from medicinal plants (Lesney, 2004). Silybummarianum (SM), commonly known as milk thistle (MT), is an annual/biennial plant of the Asteraceae family, native of Mediterranean area and now growing and cultivated worldwide (Abenavoliet al., 2010 and Bijak, 2017).All parts of the plant can be used, but the milk thistle seeds are considered to be the most medicinally potent for therapeutic use. The seeds and extracts of the milk thistle plant are a well-established herbal food for protecting, detoxifying and regenerating the liver, one of the most important organs of the human body (Apostolet al., 2017).Silybummarianumis a therapeuticherb with a 2,000-year history of use. Silybummarianumseed extract has been called silymarin and consist of silybin, silychristin, silydianin and isosilybin(Surai, 2015).

The aim of the present study was to investigate the effect of Silybummarianumseeds powder and its aqueous extract against the toxicity of carbon tetrachloride (CCl₄), also to evaluate on body weight, feed intake, lipid profile and liver functions that may occur on hepatotoxic rats.

MATERIALS AND METHODS

Materials:      

Dried seeds of Silybummarianum were obtained from local herbal market from Cairo, Egypt. Taxonomic identification of these seeds was established by the staff members of the Department of Botany, Faculty of Agriculture, CairoUniversity.Carbon tetrachloride (CCl₄)_, chemical kits, casein, cellulose, choline chloride, D-L methionine, vitamins and minerals were obtained from El-Gomhoriya Pharm. Starch, corn oil, and sucrose were obtained from the Egyptian local market. Thirty adult male albino rats (Sprague-Dawley strain), weighing about (150±10g) were obtained from the Farm of Experimental Animals, Giza, Egypt.

Methods:

1. Preparation of SilybummarianumSeeds AqueousExtract:

Dried Silybummarianumground seeds will be submerged in (distilled) water and allowed to soak (overnight), then the water extract will be mixed with different levels to basal diet according to Raskovicet al., (2002).

1. Induction of Hepatotoxicity:

Carbon tetrachloride (CCl₄)-induced acute hepatotoxicity in rats. Intraperitoneal injection of male albino rats with CCl₄ 1ml/kg, (1:1) mixture with paraffin oil for 3 days increased serum alanine  transaminase, aspartate transaminase, and alkaline phosphatase activities as well as total bilirubin, triglycerides and total cholesterol levels. This is in addition to the disrupted histology (Karthikeyan and Deepa, 2010).

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   Diet Composition and Experimental Animal Design:

The basal diet was formulated according to AIN-93M diet (Reeves et al., 1993).Animals (30 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 group (5 rats) was fed on basal diet and served as a negative control group (-ve), the second group (25 rats) was injected with 1 ml CCl4/kg b.w.After liver injury rats were divided as follow:-

At the end of the experimental period (4 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.

1. Biological Evaluation:feed intake (FI), body weight gain percent (BWG%)and feed efficiency ratio (FER) were determined according to Chapman et al., (1959) using the following equation:

BWG%=  Final body weight – Initial body weight / Initial body weight × 100

FER     =  Weight gain / Feed intake.

1. Biochemical Analysis of Serum:

Aspartate amino transaminase (AST) and Alanine amino transaminases (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 Fridewaldet al., (1972).

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   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 Silybummarianumon feed intake (FI), body weight gain (BWG) and feed efficiency ratio (FER) of hepatotoxic rats. FI was decreased in positive control group compared to negative control group, while all treated groups with Silybummarianum decreased in FI except group (6) increased in FI compared with the 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. BWG result also showed the best result was group (6) fed on 10% of Silybummarianum seeds extract, while FER result in group 4 and 6 showed the best result compared to negative control group.

Results of BWG and FER were in agreement with Shaker et al.,(2010), who reported that BWG and FER were significantly decreased for positive control (CCl₄) comparing to normal control.  While group treated with ethanolic extract of Silybummarianum seeds increased in BWG and FER. Also, Bouhalit and Kechrid, (2018) confirmed that the administration of SM seeds methanolic extract increased body weight. The last author investigated that the altered body weight was retrieved to near normal levels because of the antioxidant effects of silymarin.

As seen in Table (2), serum concentrations of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP) and total bilirubin were elevated significantly (P< 0.05) by CCl₄ administration (positive control group) compared with negative control group. It was observed significantly (P< 0.05) reduce in serum AST, ALT, ALP and total bilirubin levels for all groups treated with Silybummarianum seeds powder and its extract. The highest improvement for liver functions were observed at the group that fed on 10% of Silybummarianum seeds extract.

One of the most sensitive and dramatic indicators of hepatocyte injury is the release of intracellular enzymes, such as alanine aminotransferase, aspartate aminotransferase and serum alkaline phosphatase in the circulation after CCl₄ administration. Rajesh and Latha (2004) stated that elevated activities of these enzymes are indicative of cellular leakage and loss of the functional integrity of liver cell membranes.

Higher total phenolic content hasbeen known to contribute to the antioxidant activity ofextracts(Yuan et al., 2014), while antioxidant activity has also beenlinked to the hepatoprotective effect of some extracts(Guet al., 2014).Silymarin has hepatoprotective properties and is used intreatment of various liver diseases(Elmowafyet al., 2013). Various studies indicate that silymarin exhibits strong antioxidant tactivity(Simeonovaet al., 2013) and shows protective effects against hepatic toxicity induced by a wide variety of agents by inhibiting lipid peroxidation(Bosisioet al., 1992).

The present results of ALT, AST and ALP were in the same line with Ozturket al., (2012) and Behroujet al., (2015), they found that the treatment with aqueous SM seeds extract significantly decreased the ALT, AST and ALP activities that affected by hepatotoxicity. Regarding to result of serum total bilirubin was in agreement with Masoud, (2018), who indicated that treatment with silymarin significantly decreased in bilirubin that elevated paracetamol administration. Also, results of serum ALT, AST, ALP and total bilirubin in the present study confirmed by Madaniet al., (2008) and Bouhalit and Kechrid, (2018).These findings corroborate with current results on the ability of SM to exert a hepatoprotective activity.

Results in Table (3) showed a significant decrease (P< 0.05) in serum glutathione peroxidase (GP_X)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 GP_X activity for group 4, 5 and 6 compared to the positive control group. Concerning serum malondialdehyide (MDA)level, results showed that serum MDAlevel was increased significantly (P< 0.05) in the positive control group compared with the negative control group, while all treated groups withSilybummarianum showed a significant reduce (P< 0.05) compared with positive control group. It was also observed that rats were fed on high level of Silybummarianum seeds powder and its extract (group 4, 6) considered the best result for increasing the serum GP_X and reducing serum MDA levels.

Malondialdehyde (MDA) level in liver tissue was assessed as an indicator of lipid peroxidation in oxidative liver damage (Nielsen et al., 1997). In addition, the increase of MDA has been considered a key feature in liver injury (Mateoset al., 2005). The antioxidant activity of Silybummarianum extract was proved in the present study by significant increasesin the levels of antioxidant enzyme (GPx) in rats. In this concern,Muriel and Mourelle (1990) and Lawrence et al.,(2000) reported that the flavonoids of SMhad a potent antioxidant effect as indicatedby significant decreases of superoxide anions and freelipid and oxygen radicals due to inhibition of lipidperoxidation. Also, results of MDA and GPx results in agreement with Mansour et al., (2006) and Mesallamyet al., (2011). Moreover, many previous investigations proved the potent invitroantioxidant activity of Silybummarianumplant extract such as those reported by Vargaet al. (2004); Katiyar (2005) and Salleret al., (2007).

Data revealed in Table (4) that serum TC, TG, LDL-C and VLDL-C were increased significantly (P< 0.05) in the positive control group compared with the negative control group. Regarding serum HDL-C level, results a significant (P< 0.05) decrease in serum HDL-C level of the positive control group compared to the negative control group. The highest improvement for lipid profile was observed at the group that fed on 10% of Silybummarianum seeds extract which is close to the negative control.

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 and VLDL, while decreasing in HDL level in CCl₄ inducted rats. Treatment of SM seeds also attenuated these changes of lipid profile of rats treated previously with CCl₄. These results were in the same line with (Sobolovaet al., 2006); Shaker et al.,(2010) and Lien et al., (2016).

It could be concluded from results of the current study that the treatment with Silybummarianumcould significantly attenuate the carbon tetrachloride induced oxidative hepatotoxicity through its antioxidant properties.

Table (1): Effect of Silybummarianumon Feed Intake (FI), Body Weight Gain (BWG) and Feed Efficiency Ratio (FER) of Hepatotoxic Rats

*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.

* SMSP = Silybummarianum Seeds Powder, SMSE = Silybummarianum Seeds Extract.

Table (2): Effect of Silybummarianum on Serum AspartaAminotransferase (AST), Alanine Aminotransferase (ALT) and Alkaline phosphatase (ALP) of Hepatotoxic Rats   

*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.

*SMSP = Silybummarianum Seeds Powder, SMSE = Silybummarianum Seeds Extract.

Table (3): Effect of Silybummarianum on Serum GlutathionPeroxidase(GP_X) and Malondialdehyide (MDA) of Hepatotoxic Rats

*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.

*SMSP = Silybummarianum Seeds Powder, SMSE = SilybummarianumSeeds Extract.

Table (4): Effect of Silybummarianumon Serum Total Cholesterol (TC), Triglyceride (TG), High Density Lipoprotein Cholesterol (HDL-C), Low Density Lipoprotein Cholesterol (LDL-C) and Very Low Density Lipo- protein Cholesterol (VLDL-C) of Hepatotoxic Rats

*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.

* SMSP = Silybummarianum Seeds Powder, SMSE = Silybummarianum Seeds Extract.

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تأثیر الخرشوف البری على الفئران المصابة بتسمم الکبد

منى محمود علی، هانی جابر المصری، محمد حمدی حجاج

قسم التغذیة وعلوم الأطعمة  - کلیة الاقتصاد المنزلی - جامعة حلوان

ینتمی الخرشوف البری إلى العائلة الأستیراسیة، تم استخدامه منذ 2000 سنة کعشبة علاجیة. یطلق على مستخلص بذور الخرشوف البری اسم السیلیمارین، الذی یحتوی على العدید من الأنشطة العلاجیة کمضاد للسکری، مضاد للإلتهابات ووقایة الأعصاب. کان هدف هذه الدراسة معرفة تأثیر استخدام بذور الخرشوف البری على الفءران المصابة بتسمم الکبد. حیث أجریت الدراسة علی ثلاثون فأرا من نوع الالبینو، تتراوح أوزانهم من (150 ± 10 جم) . تم تقسیم الفئران الی مجموعتین أساسیتین. المجموعة الاولی (5 فئران )تم تغذیتهم علی الغذاء الأساسی طوال فترة التجربة  وتعتبر المجموعة الضابطة السالبة . المجموعة الثانیة (25 فأرا) تم تغذیتهم علی الغذاء الآساسی و حقنهم بمادة رابع کلورید الکربون ( ا مل /کجم من وزن الجسم ( بنسبة 1:1 مع خلیط من زیت البارافین لمدة 3 أیام لإحداث تلف الکبد ، وبعد ذلک تم تقسیم الفئران الی 5 مجامیع فرعیةمن المجموعة (2) إلی المجموعة (6). المجموعة الفرعیة (2):  تم تغذیتهم علی الغذاء الأساسی  فقط وتعتبر مجموعة ضابطة موجبة. المجموعة الفرعیة (3) و(4) : تم تغذیتهم علی الغذاء الأساسی المحتوی علی مسحوقبذورالخرشوف البری بنسبة 5% و10% لکل کجم من الغذاء الأساسی، علی التوالی .  المجموعة الفرعیة (5) و(6) : تم تغذیتهم علی الغذاء الأساسی المحتوی علی المستخلص المائی لبذور الخرشوف البری بترکیز 5% و 10% لکل کجم من الغذاء الأساسی.استمرت التجربة لمدة أربعة أسابیع وفی نهایة فترة التجربة تم تشریح الفئران والحصول علی السیرم لاجراء التحالیل البیوکیمیائیة. أظهرت النتائج أن مستویات ALT, AST, ALP, total bilirubin, MDA ارتفعت بشکل ملحوظ فی المجموعة الضابطة الموجبة مقارنة بالمجموعة الضابطة السالبة، بینما کان هناک انخفاض ملحوظ فی مستوى GPx. کما أشارت النتائج إلى أن إضافة مسحوق والمستخلص المائی لبذور الخرشوف البری عکس هذه التغیرات التی حدثت بالتسمم برابع کلورید الکربون. لذلک توصی هذه الدراسة بأنتناولنبات الخرشوف البریقدیکونمفیدًاللمرضىالذینیعانونمنأمراضالکبدالمرتبطةبالإجهادالتأکسدی.

الکلمات المفتاحیة: الخرشوف البری، السیلیمارین، تسمم الکبد، الفئران.