CONCLUSION

Based on results of the present study, it can be concluded that garlic is a promising hepatoprotective agent and this protective activity of black garlic may be due to its antioxidant

THE PROTECTIVE EFFECT OF WHITE AND BLACK GARLIC POWDER ON HEPATOTOXICITY IN RATS

¹Doaa A.E. Hussein ; ¹Maysa M. El-Mallah ; ¹Hany G. EL-Masry

and ²Magda S. Mohamed

¹Nutrition and Food Science Department, Faculty of Home Economics, Helwan University, Cairo, Egypt

²Nutrition and Food Science Department, Division of Food industry and nutrition, National Research Center

Key Words: Garlic(Allium sativum), Black Garlic, liver damage, Rats

ABSTRACT

            Garlic (Allium sativum, Family, Alliaceae) is an important component of the Mediterranean diet and it is commonly used for both culinary and medicinal purposes. Black garlic (BG)is a type of fermented fresh garlic (heads) in a temperature (60-70 Ċ) and humidity (85-95%) controlled room for 40 days without any additional treatment and additives. Aged black garlic (ABG) contains high levels of bioactive organic sulfur compounds such as water soluble S-allylcysteine and polyphones and stronger an toxicant compounds. The aim of the present study was to investigate the protective effect of white and black garlic powder on hepatotoxicity in rats. Thirty six adult male albino rats (Sprague-Dawley strain), weighing about (170±5g) were divided randomly into 6 equal groups of 6 rats each: the first group (-ve control= 6 rats) was fed on basal diet. The second group (6 rats) were fed on basal diet and injected with CCl4 1 ml/kg, (1:1) mixture with paraffin oil (2 injections /week) to induce acute liver damage. The other 4 protected groups(3,4,5and 6) were pretreated by  feeding  on diets supplemented with white garlic powder (WGP) and black garlic powder (BGP) at low (1%) and high (2%) concentrations, respectively for 4 weeks and intoxicated by CCl4 as in group 2 . At the end of the experimental period (4 weeks), rats were scarified and serum was collected for biochemical analyses. Results indicated that serum concentrations of aspartate aminotransferase (AST), alanine aminotransferase  (ALT), 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, and significantly (P<0.05) decreased in glutathione peroxidase (GPX). It also indicated that supplemented diet with white and black garlic powder reversed these changes that caused by CCl4 administration. It could be recommended that black garlic powder is worthy treating on liver damage. 

INTRODUCTION

The liver is the most important organ in the body , it plays a pivotal role in regulating various physiological processes.  It is also involved in several vital functions such as metabolism, secretion and storage.  It has great capacity to detoxicate toxic substances and synthesize useful principles (Adewusi and Afolayan 2010).The liver is a vital organ that plays a central role in transforming and cleaning chemicals and is susceptible to the toxicity from these agents (Al-Harbiet al., 2014). A total loss of liver function could leads to death within minutes, demonstrating the liver‘s great importance. At present approximately 2 to 3% of adults (about 5.5 million of people) have chronic liver diseases or cirrhosis (Udompap et al., 2015).

Garlic is one of the most important vegetables throughout the word (Bhandari, 2014). And most important medicinal plants that are intensively used in traditional medicine (Hammami et al., 2012).Despite the numerous health benefits of garlic, global consumption of garlic is declining. Some people are on reluctant to eat raw garlic because of its pungent taste and smell (Tanamai et al., 2004), and raw garlic can cause gastrointestinal discomfort in some people. Food scientists have developed aged garlic preparations to reduce these discomforts.

Black garlic (BG) was obtained by processing fresh garlic at high temperature for 60 to 90 day, without any additional treatments (Bae et al., 2012). During the aging process, the odorous, harsh, and irritating compounds in fresh raw garlic (FRG) are converted naturally into stable and safe compounds (Amagase et al., 2001). As a result, aged black garlic (ABG) has a sweet and sour taste and jelly-like texture. The heating process leads to a Maillard reaction, creating the typical dark brown color, and produces antioxidant compounds (Manzocco et al., 2000 and Kang, 2016). ABG contains bioactive compounds, such as phenols, flavonoids, pyruvate, thiosulfate, S-allylcysteine (SAC), and S-allylmercaptocysteine (SAMC) (Shin et al., 2014 and Choi et al., 2008).

Therefore, this study was conducted to evaluate the effect of white and black garlic powder on hepatotoxicity of carbon tetrachloride (CCl4) in rats, also to evaluate on body weight, feed intake, lipid profile and liver functions that may occur on hepatotoxic rats.

MATERIALS AND METHODS

Materials:      

White garlic heads were purchased from the local market, Cairo, Egypt. And some garlic cloves were freeze drying for preparing (aged) black garlic in National Research Center, Dokki, Egypt. 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. Thirtysix adult male albino rats (Sprague-Dawley strain), weighing about (170±5g) were obtained from the Animal House, Agriculture Research Center, Giza, Egypt.

Methods:

Preparation of Black garlic:

Black garlic created by maintaining fresh garlic (heads) in a temperature (60-70 ^oC) and humidity (85-95%) were controlled for 40 days without any additional treatment and additives, in Incubator. Fresh white garlic were changed its color from white to brown and eventually became black a month later by the Millard and Browning Reaction (Sato et al., 2006) National Research Center, Dokki, Egypt.

Preparation of garlic fine powders:

After drying of white garlic according by AOAC, 2000 and freeze drying to the other the outer peels of them were removed and garlic cloves were grinded using a coffee grinder into fine powders.  Powders were kept in refrigerator at 4 Ċ till used for both chemical analysis and basal diet supplementation.

Induction of Hepatotoxicity:

     Subcutaneous injection of male albino rats with CCl4 (2 injections/week) at 1ml/kg b.wt diluted with liquid paraffin (1:1, V: V)   caused hepatotoxicity for rats (karthikeyan and deepa 2010). 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).

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 Rats were randomly divided into 6 equal groups of 6 rats each.

Group 1:- was kept as a normal (negative) control group, received a single intraperitoneal (i.p.) injection of normal saline (2.5ml/kg).

Group 2:- served as (positive) control group, injected subcutaneously (2 injections/week) by CCl4 at 1ml/kg b.wt. Diluted with liquid paraffin (1:1, V: V) at the last week of the experiment for induction of hepatotoxicity.

The other 4 protected groups(3,4,5and 6) were pretreated by  feeding  on diets supplemented with white garlic powder (WGP) and black garlic powder (BGP) at low (1%) and high (2%) concentrations, respectively for 4 weeks and intoxicated by CCl4 as in group 2 .

   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 8000 rpm for 15 min to obtain the serum for biochemical analysis.

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 % = 

Feed Efficiency Ratio = Gain weight (gm)/food consumed (mg).

Relative organ weight % =.

Biochemical Analysis of Serum:

Aspartate aminotransaminases (AST) and Alanine aminotransaminases (ALT) were determined according to the method described by Young, (1991), alkaline phosphates (ALP) was determined according to Roy, (1970). Total bilirubinwas determined according to the method described byTietz (1995).Malondialdehyde (MDA)was determined according to Pompella et al., (2003) and Glutathione peroxidase (GPx)was determined according to Shin et al., (1972). Serum total cholesterol (TC) and triglyceride (TG)were determined according to the method described byTrinder and Ann, (1969). High density lipoprotein cholesterol (HDL-C), serum low density lipoprotein cholesterol (LDL-C) and very low density lipoprotein cholesterol (VLDL-C) were determined according to Friedewald et al., (1972).

Histopathological Examination:-

Specimens from the liver were placed in 10% neutral buffered formalin for histopathological examination according to (Bancroft and Stevens, 1996). Histopathological examination was done in Veterinary medicine, Cairo University.     

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 statisticalsoftware, 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 White and black garlic powder on feed intake (FI), body weight gain (BWG) and feed efficiency ratio (FER) of  hepatotoxic rats. Feed intake was increased in positive control group compared to negative control group, while treated groups with 1% and 2% white and black garlic powder were close to positive control group. There were significantly decreased (P< 0.05) in BWG for the positive control group compared to the negative control group, it was also showed that all treatment groups with white and black garlic powder significantly increased (P< 0.05) in BWG compared with positive control group. It was also showed that all treatment groups with white and black garlic powder No significant difference (P< 0.05) in FER compared with positive control group.

Result of Feed efficiency ratio was in the same line with Hyun-Sook et al., (2011) who found significant difference in food efficiency compared with positive control group. Aoet al., (2011), confirmed No effects were observed (P > 0.05) in feed intake (FI) throughout the whole experiment. improved the body weight in rats that affected by hepatotoxicity, due to low triglycerides, low density lipoprotein cholesterol (LDL-c), insulin and leptin serum concentrations and higher high density lipoprotein cholesterol (HDL-c) (Amor, 2018).

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 white and black garlic powder compared to positive control group. The highest improvement for liver functions was observed at the group that fed on 2% of black garlic.

The enhancement of results on liver functions may be due to that white and black garlic contains antioxidant, flavonoids and phenolic compounds(Sun and Wang 2017)but some people are reluctant to ingest raw garlic due to its unpleasant odor and taste. the hepatoprotective of black garlic powder may also be because it other properties like anti-inflammatory, antibacterial , anti-cancer, anti-obesity, anti-diabetic, anti-allergic, cardioprotective, hepatoprotective effects,  anti-inflammatory, anti-coagulation, immunomodulatory, and anti-allergic effects compared to raw garlic.as well as antioxidants two times higher than regular garlic due to it consists of S-allycysteine. Dewi, Mustika, (2017). Results of serum ALT, AST, ALP in the current study also in agreement with Tasi et al.,  (2019)  and  Tran et al., (2018).

Hepatocyte cytoplasm is very rich in bilirubin and in cell damage bilirubin is abundantly leaked in to blood (Nkosi et al., 2005). So, a reduction of serum bilirubin in the present study confirms the hepatoprotective activity of white and black garlic powder. Result of serum total bilirubin in the current study was in the same line with Naji et al., (2017).

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 white and black garlic powder 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 white and black garlic powder significantly decreased (P< 0.05) compared to the positive control group. It was also observed that rats were fed on 2% black garlic powder considered the best group for increasing the serum GPX and reducing serum MDA levels.

The increase in MDA implies increased lipid peroxidation as a result of hepatotoxicity which could have been induced by CCl₄ as shown in the results of the present study, MDA level was increased in positive control rats while the antioxidant enzymes GP_X was depleted. The increased lipid peroxidation in the liver indicated tissue injury and a failure of the antioxidant defense mechanism, which prevents the formation of excess of free radicals. This finding is consistent with the previous works reported by Yanpallewar (2003). Also, GP_X result was in agreement withDkhilet al., (2013), and MDA result in agreement with Nwobodo (2017).

Chemical black garlic powder investigation resulted in the isa S-allyl cysteine, S-allyl mercaptocysteine, allicin, and selenium Antioxidant compounds (Wang et al., 2010). SAC as an antioxidant can prevent lipid peroxidation. Giving black garlic to healthy people can increase the activity of SOD and CAT antioxidant enzymes and capture superoxide free radicals thereby reducing myocardial damage by free radicals. These findings, when combined with the present results, indicate that black garlic powder most likely exerts its hepatoprotective effect by acting as an antioxidant inhibiting lipid peroxidation  Saryono and Proverawati (2019).

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 white and black garlic powder decreased significantly (P< 0.05) in serum TC, TG and VLDL-C levels compared to the positive control group, results showed a significant (P< 0.05) decrease in serum LDL-C level of the  group fed on  2% black garlic 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 white and black garlic compared to negative control group. The highest improvement for lipid profile was observed at the group that fed on 2% of black garlic powder.

Results of lipid profile were in the same line with Saryono and  Proverawati, (2019), who showed a significant increase in TC, TG, LDL-c and decrease in HDL-c in hepatotoxic rats (positive control groups) compared with their respective levels in the negative control, while the rats treated with varied doses of white and  black garlic powder, the significant decrease in TC, TG, LDL-c, and increase in HDL-c compared with positive control group indicate the hypolipidaemic effect which might have resulted from antilipidaemic role of black garlic powder  Prihanti et al., (2019). The high content of organosulfur compounds can act as antioxidants, Flavonoids and polyphenols decrease intracellular lipids, which would contribute to lowering the risks of hyperlipidemia and atherosclerosis Ha et al., 2015).

Results of histopathological examinations of liver are shown in Photos (1-8). Photo (1) showed normal histological structure of hepatic(fed on basal diet) revealed that no marked histopathological changes. While, the positive control group (rats with hepatotoxicity) in Photo (2),(3) showed revealed focal hepatic necrosis associated with mononuclear cells infiltration, Kupffer cells activation, sinusoidal leukocytosis and portal infiltration with mononuclear inflammatory cells. Rats fed on 1%  white garlic powder showing slight activation of Kupffer cells activation, portal infiltration with mononuclear inflammatory cells seen in photo (4),(5). Photo (6) indicated a showing hydropic degeneration of hepatocytes and portal infiltration with mononuclear inflammatory.  Rats fed on 2% white garlic powder as shown in photo (7) and Rats fed on 2% black garlic powder as seen in photo (8) showing no histopathological alterations in liver

Results of histopathological examinations of liver were supported by Baligar et al., (2014) who indicated that CCl4-treated rats caused a severe hepatocellular degeneration, necrosis, and congestion of the sinusoids, along with periportal mononuclear cell infiltration due to toxicity. Also, results of the present study were in the line with Alshehri et al., (2014)found that administered Aged Black Garlic (ABG) showed alleviation of histological degenerative changes caused by ccl4. This result was also in agreement with Nakagawat et al., (1989). who found that the protective effect of Sallyhercapto cysteine (SAMC) upon the liver cells were far superior than that of S-ally1 cysteine (SAC) The reversal of all the biochemical enzymatic parameters and histological alterations after treating with garlic powder might be due to antioxidant property of black garlic which preserved the hepatic tissue from possible CCl4–induced oxidative stress which could lead to hepatic damage Masjedia et al., (2011).

Based on results of the present study, it can be concluded that white and black garlic powder is a promising hepatoprotective agent and this protective activity of black garlic powder may be due to its antioxidant content. This was manifested by improvement in the biochemistry and histology of liver of the studied animals injected with CCl₄ induced liver adverse effect.

Table (1): Effect of White and black garlic on 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.

*WGP = White Garlic Powder, BGP= Black Garlic Powder.

Table (2): Effect of White and Black Garlic Powder on Serum Aspartate Aminotransferase (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.

*WGP = White Garlic Powder, BGP= Black Garlic Powder.

Table (3): Effect of White and Black Garlic on Serum Glutathione Peroxidase (GP_X)and Malondialdehyde (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.

*WGP = White Garlic Powder, BGP= Black Garlic Powder.

Table (4): Effect of white and black garlic on Serum Total Cholesterol (TC), Triglyceride (TG), High Density Lipoprotein Cholesterol (HDL-C), Low Density Lipoprotein Cholesterol(LDL-C) and Very Low Density Lipoprotein Cholesterol (VLDL-C) ofHepatotoxic 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.

*WGP = White Garlic Powder, BGP= Black Garlic Powder

Photo (1):Liver of negative control rats fed on basal diet

Photo (2): Liver of rat from group positive control showing focal hepatic necrosis associated with mononuclear cells infiltration (H & E X 400).

Photo (3): Liver of rat from group positive control showing Kupffer cells activation and sinusoidal leukocytosis (H & E X 400).

Photo(4): Liver of rat from group on fed 1% white garlic showing slight activation of Kupffer cells (H & E X 400).

Photo (5): Liver of rat from group on fed 1% white garlic showing portal infiltration with mononuclear inflammatory cells (H & E X 400).

Photo (6): Liver of rat from group on fed 1% black garlic powder showing hydropic degeneration of hepatocytes and portal infiltration with mononuclear inflammatory cells (H & E X 400).

Photo (7): Liver of rat from group on fed 2% white garlic powder showing no histopathological alterations (H & E X 400).

Photo (8): Liver of rat from group on fed 2% black garlic powder showing no histopathological alterations (H & E X 400).

CONCLUSION

Based on results of the present study, it can be concluded that garlic is a promising hepatoprotective agent and this protective activity of black garlic may be due to its antioxidant.

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التأثیر الوقائیلمسحوق الثوم الأبیض والأسود على التسمم الکبدی فی الفئران

دعاء عبد الرحیم حسین، هانی جابر المصری، ما یسه محمد الملاح,ماجده سلیمان محمد

قسم التغذیة وعلوم الأطعمة  - کلیة الاقتصاد المنزلی - جامعة حلوان

الثوم یعتبر مکونًا هامًا من مکونات الأطعمة فى حوض البحر المتوسط حیث یستخدم عادةً لکل من الطهی (کمحسن للنکهة) ولأغراض طبیة. تم الحصول علی الثوم الأسود عن طریق الحفاظ على الثوم الطازج (الرؤوس) فی درجة حرارة (65-80 درجة مئویة) والرطوبة (70-80 ٪) بغرفة محکمة لمدة 30-40 یوما دون أی معالجة إضافیة أو مواد مضافة المعالجة الحراریةالثوم الأسود یحتوی على مستویات عالیة من مرکبات الکبریت العضویة النشطة حیویاً مثل کبرتید الأیلسیستین ​​القابل للذوبان فی الماء والبولیفینول ومرکبات مضادة للأکسدة أقوى. کان الهدف من هذه الدراسة هو التأثیر الوقائی لمسحوق الثوم الأبیض والأسود على سمیة الکبد لدى الفئران.حیث أجریت الدراسة علی ستة وثلاثون فأرا من نوع الالبینو، تتراوح أوزانهم من (170 ± 5 جم) . تم تقسیم الفئران 6 مجموعات متساویة من 6 فئران لکل منهما. المجموعة الأولی (6 فئران ) تم تغذیتهم علی الغذاء الأساسی طوال فترة التجربة  وتعتبر المجموعة الضابطة السالبة . المجموعة الثانیة (6 فأرا) تم تغذیتهم علی الغذاء الأساسی و حقنهم بمادة رابع کلورید الکربون ( ا مل /کجم من وزن الجسم ( بنسبة 1:1 مع خلیط من زیت البارافین (2 حقنه / أسبوع)لإحداث تلف الکبد ،تم معالجة المجموعات الأربع المحمیة الأخرى (3،4،5 و 6) بالتغذیة على الوجبات الغذائیة المدعمة بمسحوق الثوم الأبیض (WGP) ومسحوق الثوم الأسود (BGP) بترکیزات منخفضة (1٪) وعالیة (2٪)  على التوالی.استمرت التجربة لمدة أربعة أسابیع وفی نهایة فترة التجربة تم تشریح الفئران والحصول علی السیرم لإجراء التحالیل البیوکیمیائیة  . حیث أشارات النتائج أن مستویات , MDA, ALT,ALP ,AST total bilirubin، کانت مرتفعة بشکل ملحوظ فی المجموعة الضابطة الموجبة مقارنة بالمجموعة الضابطة السالبة، علاوة علی ذلک  حدوث انخفاض ملحوظ فی مستوی GPx.کما أشارت النتائج  إلى أن إضافة مسحوق الثوم الأبیض والأسود عکس هذه التغییرات التی أحدثتها رابع کلورید الکربون. وبالتالی  یمکن التوصیة بأن مسحوق الثوم وخصوصا مسحوق الثوم الأسود ذو قیمة علاجیة  لتلف الکبد الناتج عن رابع کلورید الکربون

الکلمات المفتاحیة : الثوم ، الثوم الاسود, التلف الکبدی ، الفئران