Food components have biological characteristics such as anticarcinogenic, antimutagenicity, antioxidative activity, and antiageing activities. The antioxidant activity is mainly due to phenolic components such as flavonoids, phenolic acids, and phenolic diterpenes [1,2].
Traditionally, herbs and spices have been added to different types of food to improve the flavor . Currently, there is a growing awareness that spices also improve the oxidative stability of processed products and, as a consequence, spice extracts are being marked as antioxidants for use in the food industry [4–7].
Various herbs and spices contain numerous phytochemicals in addition to phenolic compounds; many of these phytochemicals possess significant antioxidant capacities that are associated with lower incidence and lower mortality rates of cancer in several human cohorts .
Tea has been used as a popular daily beverage classified as black, green, red, yellow, white, and dark compressed tea. Black tea is the most popular tea in the world. Black tea is consumed as a hot or cold beverage across the world, especially in Egypt. Tea possesses antipyretic and diuretic effects, and the pharmacological effects of tea are reviewed, including antioxidative activity  and antimutagenic  and anticancer effects [11,12].
Recently, aromatic plants have received much attention as sources of biologically active substances including antioxidants, antimutagens, and anticarcinogens. It is well known that the antioxidant activity of some plant active ingredients is parallel to their chemopreventive effect .
Therefore, this study aimed to select some aromatic plants namely basil, rosemary, clove, spearmint, and three types of tea (green, red, and black), which are known to possess antioxidant activities to be used as ingredients in blend that gives acceptable flavor and healthy effects after reconstitution in hot water. In addition, the protective effect of the blend infusion was evaluated on carbon tetrachloride (CCl4)-induced acute liver damage in rats.
Materials and methods
Dry leaves of basil (Ocimum basilicum), rosemary (Rosmarinus officinalis), spearmint (Mentha spicata), and clove buds (Syzygium aromaticium) were purchased from the specialized local market. Tea (Camellia sinensis) including Japanese green tea, Chinese red tea, and Chinese black tea were purchased from the local market.
Preparation of blend infusion
The aromatic plants under investigation were separately grounded and blended to prepare blend that contains 20% green tea, 20% black tea, 10% red tea, 10% clove, 20% basil, 10% rosemary, and 10% spearmint. One gram of each grounded aromatic plant was infused with 100 ml freshly boiled water for 5 min followed by filtration.
Animals and diets
Male albino rats with initial weight ranging from 120 to 145 g were used as experimental animals for biochemical and histopathological studies. All experimental animals were provided from the breeding unit of the National Research Center (Cairo, Egypt). The animals were housed individually in stainless steel wire mesh cages. They were maintained for 1 week to allow acclimatization. Throughout the experiment periods, the rats were fed on standard pellets prepared by Cairo Company of Oil and Soap, Egypt. The pellets consisted of protein (approximately 23%), fat (approximately 6.5%), fibers (approximately 4%), ash (approximately 8%), added minerals (approximately 2.5%), and carbohydrates (approximately 56%). Rats were provided with food and water ad libitum. The protocol of this study was approved by the appropriate animal care of the National Research Center.
Thirty male albino rats were used for studying the effects of blend infusion on CCl4-induced liver injury . The animals were divided into three equal groups as follows: control group: rats were provided commercial standard diet and tap water ad libitum. CCl4-intoxicated group: animals were injected intraperitoneally with CCl4 (1.195 ml/kg body weight) three times a week for 2 weeks. Protected group: rats were maintained on a standard diet and blend infusion, instead of water, for 2 weeks followed by intraperitoneal injection of CCl4 (1.195 ml/kg body weight) three times a week for another 2 weeks with continuous supplementation with blend infusion.
At the end of the experiment period, blood samples were collected from each rat by orbital puncture and withdrawn on heparinized tubes (Burlington, North Carolina, USA). Plasma was collected after centrifugation at 3000 rpm equivalent to 200×g for 10 min at 4°C and divided into aliquots to avoid freezing and thawing. Aliquots were then stored at −20°C pending assay. Plasma was used for determination of liver function, proteins, and glucose.
The sediment containing red cells was washed several times with ice-cold saline solution. The packed red blood cells (RBCs) were stored at −20°C for determination of malondialdehyde (MDA) and antioxidant enzymes.
Tissue sampling and processing
Rats were killed by decapitation under ether anesthesia. Liver was excised, rinsed with cold saline, blotted dry, and weighed. A portion of the liver tissue was dropped into 10% formalin for histological and morphometric examinations. The remainder of the liver samples was stored at −20°C for MDA determination.
In this study, assay kits were purchased from Elitek Diagnostic (Spain), Boehringer-Mannheim (Germany), Lincer Chemicals (Italy), Stanbio (Spain), Sigma Diagnostic (USA), and RANDOX (USA). Alanine aminotransferase (ALT) and aspartate aminotransferase (AST), alkaline phosphatase (ALP), γ-glutamyltransferase (γ-GT), and lactate dehydrogenase (LDH) activities were determined by the methods described by Bergmeyer et al. , Rosalki et al. , Szasz , and Anon , respectively. Total and direct bilirubin, total proteins, and albumin levels were determined in plasma samples according to the colorimetric method described by Jendrassik and Gróf , Peters , and Doumas and Biggs , respectively. The activity of catalase (CAT), glutathione peroxidase (GPx), and glucose-6-phosphate dehydrogenase (G-6-PDH) in RBCs were measured by the methods described by Chance , Paglia and Valentine , and Lohar and Wall , respectively.
For histopathological examination, the fixed liver samples in formalin were embedded in paraffin cubes. The cubes were cut into 5 μm thickness and stained with hematoxylin and eosin stain. The sections were examined for pathological changes and photographed.
Karyometric and morphometric studies
After the histopathological study, the hematoxylin and eosin-stained sections were used in karyometric and morphometric analyses. The nuclear areas, nuclear volume, and the ratio of nuclear/cellular volume were estimated. One hundred and fifty cells/animal were measured in the liver of control and treated rats. In addition, damaged areas in liver of control and treated rats were determined using Sigma Scan Image Analysis Program (Jandel Scientific, SPSS Inc., Chicago, USA).
Data were presented as mean±standard error. The difference between two groups was calculated using one-way analysis of variance using MSTAT-C version 4 (Michigan Univ. East Lansing, USA) program according to Snedecor and Cochran .
Effect of blend infusion on body weight
Results inTable 1 indicated significant increases (P<0.001) in body weight of animals of control and blend protected groups after 4 weeks compared with their initial body weight. The percentages of change are 21.64% and 21.85% in the control and protected rats, respectively. In contrast, a significant increase (P<0.001) in body weight of CCl4-intoxicated rats (after 4 weeks) and the percentage of change (13.63%) are much lower than controls.
Biochemical parameters, ALT, AST, ALP, γ-GT, LDH activities, total bilirubin, direct bilirubin, and indirect bilirubin levels exhibited a significant higher level (P<0.001) on CCl4-intoxicated group compared with the control group. The administration of blend for 4 weeks along with CCl4 administration exhibited reduction in the levels of all seven before mentioned biochemical parameters (Tables 2 and 3).
In contrast, total proteins, albumin, globulin levels, and A/G ratio in the CCl4-intoxicated rats showed a significant lower level (P<0.001) than controls. Supplementation with the blend revealed a significant protection against CCl4-induced decrease in total proteins, albumin, globulin levels, and A/G ratio (Table 4).
The mean values±standard error for MDA content in RBCs and liver as well as GPx, CAT, and G-6-PDH activities in the control, CCl4-intoxicated and blend infusion-protected groups are presented inTables 5 and 6.
In CCl4-intoxicated group, a higher significant in MDA level was observed as compared with the control one. Significant lower level in the activities of both GPx and G-6-PDH, while nonsignificant change in CAT activity was recorded in CCl4-intoxicated group as compared with control group.
Analysis of variance indicated significant higher value for RBCs and GPx and G-6-PDH activities between groups. Significant difference was obtained when the mean value of CCl4-intoxicated group was compared with both control and blend infusion-protected groups. Lack of significant difference (P>0.05) was noted when rats were protected with blend infusion before CCl4 injection, compared with the control group. Significant lower level in GPx and G-6-PDH activities in CCl4-intoxicated group compared with control and protected groups was noted. In contrast, supplementation of blend infusion to rats did protect them from the damaging effect of CCl4, as evident from the insignificant difference (P>0.05) between mean values of G-6-PDH and GPx in both control and protected groups.
The liver of control rats seems to be divided into the classical hepatic lobules; each is formed of cords of hepatocytes radiating from the central vein to the periphery of the lobule. The cell cords were separated by narrow blood sinusoids. The hepatocytes were polyhedral cells with acidophilic cytoplasm (Fig. 1a).
The intraperitoneal administration of a dose equivalent to 1.195 ml/kg body weight of CCl4 to rats caused severe damage in the liver, as manifested by fatty change, massive necrosis, lipid droplets, and broad infiltration of lymphocytes around the central vein. Pyknotic or karyolitic nuclei were also seen. In addition, blood sinusoids showed dilated and congested features (Fig. 1b and c).
In the animals supplemented with blend infusion along with CCl4, most characteristic histological alteration in the liver tissue was reduced and the liver seemed more or less similar to the normal architecture. Some nuclei showed pyknosis and others showed hyperchromatic large nuclei (Fig. 1d).
The quantitative data of the nuclear area (in square pixel), nuclear volume (in cubic pixel), and the ratio of nuclear/cellular volume of the liver of control, CCl4-administered and CCl4-administered plus blend rats are summarized in Table 7. CCl4 administration lowered the mean value of nuclear area (48%) compared with those of controls. Statistically, the inhibition was significant (P<0.01). Blend treatment to the CCl4-administered rats restored the nuclear area (35%); the stimulation was statistically significant (P<0.01). Both the nuclear volume and nuclear/cellular volume were lowered with 64% and 32%, respectively, in the CCl4-intoxicated animals versus those of controls. Statistically, the inhibition was significant with a level of P value less than 0.01 and P value less than 0.05 in the nuclear volume and nuclear/cellular volume, respectively. Cotreatment with blend significantly restored (P<0.01) the nuclear volume with 62%, whereas the restoration of the nuclear/cellular volume did not prove to be statistically significant (P<0.05).
Damaged areas of the liver were assessed directly by morphometric analysis as indicated in Table 8. It was found that the percentage of the damaged areas in the liver of CCl4-intoxicated rats were 50.77±2.49, whereas it was 24.32±1.66 in the CCl4-intoxicated rats and supplemented with blend.
More attention has been focused on the protective biochemical function of naturally occurring antioxidants in the biological system and on the mechanisms of their actions [26,27].
In this study, animals intoxicated with CCl4 showed significant reduction in body weights whereas those pretreated with blend infusion and ingested with CCl4 showed insignificant changes in body weights after 4 weeks compared with controls. CCl4-intoxicated rats exhibited less increase in body weight after 4 weeks compared with their initial body weight. Rats protected with blend infusion and intoxicated with CCl4 exhibited higher increase in body weight after 2 and 4 weeks, compared with their initial body weight. Rats protected with blend infusion and intoxicated with CCl4 showed improvement in their final body weight compared with intoxicated rats. Possible effect due to changes in resting metabolic rate and appetite suppression by carbon tetrachloride consumption is suggested [28,29].
Significant changes in liver weights were noted in CCl4-intoxicated rats compared with controls. High significant increase was observed in liver weight/total body weight of CCl4treated rats. Protection with blend infusion before CCl4 injection decreased liver weight, which becomes comparable with controls. These results are in accordance with the findings by Morrison et al.  who reported that advanced liver cirrhosis induced by CCl4 accompanies muscle wasting due to decreased muscle protein synthesis or increased muscle protein breakdown. Gaemers et al.  suggested that malnutrition is a classical feature of liver cirrhosis. They also observed an increase in the liver wet weight and elevated liver weight/whole-body weight ratio in CCl4-intoxicated rats.
Activities of plasma ALT, AST, ALP, γ-GT, and LDH, sensitive indicator of liver function were studied and used as an indirect biochemical index of hepatocellular damage . In this study, CCl4 caused significant elevation in all tested liver enzyme activities compared with the control. El-Demerdash et al.  and Song and Yen  observed the same results.
The metabolism of CCl4 involves the electron oxidase system to yield the trichloromethyl radical CCl3  or possibly the trichloromethyl peroxy radical CCl3OO . Such free radicals can act in two ways. The direct way occurred by covalent binding to membrane proteins and lipids with resulting alkylation reactions and possible enzyme inactivation. The indirect way through interactions with membrane of lipid peroxidation  is an important pathogenic mechanism for liver necrosis.
Galisteo et al.  studied the effects of rosemary as antihepatotoxic activity in rats with acute liver damage by thioacetamide. Pretreatment with rosemary exhibited a significant reduction in the activities of plasma AST, ALT, ALP, and LDH compared with thioacetamide-treated animals.
Effects of restraint stress and its modulation by basil and clove on some biochemical parameters in rats were evaluated by Sen et al. . Basil and clove effectively lowered restraint stress-induced elevations in LDH and ALP activities. The results indicated that rats protected with blend infusion before CCl4 injection improved liver enzyme activities. Our results are in accordance with the findings of Ulicná et al.  who reported that black tea significantly suppressed the increase of ALT and AST levels in rats treated with CCl4.
In this study, CCl4 caused a significant increase in plasma γ-glutamyl transferase activity. These results are in accordance with El-Demerdash et al.  The researcher found a significant increase in liver glutathione level accompanied by a significant increase in hepatic γ-GT activity. Drinking of blend infusion before CCl4-injection improves the level of γ-GT, which becomes near to control.
These data showed that CCl4 induced a significant elevation in LDH activity compared with control. The protection with blend infusion suppressed mainly the toxic elevation in plasma activity of LDH induced by CCl4. This finding is also reported by Naziroğlu et al. , El-Khatib and Mansour , and Joyeux et al.  who stated that an aqueous extract of rosemary reduced the enzymatic release of LDH from the hepatocytes significantly in a tissue culture experiment.
In this study, the animals intoxicated with CCl4 showed very high significant increase in total bilirubin, direct bilirubin, and indirect bilirubin compared with controls. Similar results were obtained by Naziroğlu et al. . Our results showed that pretreated rats with blend infusion suppressed the increase in plasma total bilirubin, direct bilirubin, and indirect bilirubin compared with CCl4-intoxicated animals. Ulicná et al.  reported that tea extract significantly suppresses the increase in plasma total bilirubin. Fahim et al.  reported that rosemary caused a considerable fall in the serum direct bilirubin level compared with rats intoxicated with CCl4.
It is obvious from our data that CCl4-intoxicated rats showed a very high significant reduction in plasma total proteins, albumin levels, and globulin levels. Parallel results were reported by Smuckler and Beneditt  and Agha et al.  who stated that a very important alteration produced by CCl4 in the liver cell causes inhibition of protein synthesis by interaction with the thiol group. The intoxicated animals pretreated with blend infusion showed a noticeable improvement in the toxic depletion of plasma total proteins, albumin levels, and globulin levels induced by CCl4. These results are in accordance with Fahim et al.  who reported that pretreatment of rats with rosemary essential oil followed by intoxication with CCl4 caused significant increase in diminished serum total proteins, albumin levels, and globulin levels, compared with intoxicated animals.
CCl4 yields the reactive metabolite trichloromethyl radical (CCl3−). This free radical can produce lipid hydroperoxides. These hydroperoxides can decompose to alkoxy (RO−) and peroxy (ROO−) free radicals that can oxidize other cell components, resulting in changes in enzyme activity or the generation of mediators [i.e. MDA and reactive oxygen species] .
Results obtained from this study indicated that supplementation of blend infusion to rats before CCl4 intoxication resulted in significant reduction in MDA content of RBCs compared with CCl4-intoxicated rats. These results are in agreement with those of Frei and Higdon , Dorman et al. , Leal et al. , and Sharma et al.  who reported that tea polyphenols mentha basil rosemary extracts act as antioxidants in vitro by scavenging reactive oxygen and nitrogen species and chelating redox-active transition metal ions.
The impact of free radical on liver detoxification enzymes (CAT, superoxide dismutases, and peroxidase) affected enzyme activity, mainly CAT and peroxidase, due to enzyme inactivation during the catalytic cycle. Enzymes such as GPx and CAT also play important roles in protection against free-radical damage, and are considered to be the primary antioxidant enzymes as they are involved in direct elimination of active oxygen species.
Our results revealed that rats protected with blend infusion increased the GPx activity compared with the CCl4-infused group, and with insignificant change compared with controls.
It was documented that the fall in the activity of cytoplasmic antioxidative liver enzymes is not only a result of the immediate inactivating effect of free-radical reactions initiated by CCl4, but also is evidently caused by the covalent binding of its radical metabolites with corresponding macromolecules .
G-6-PDH is the key enzyme of the pentose phosphate pathway of carbohydrate metabolism. G-6-PDH is the principal source of nicotinamide adenine dinucleotide phosphate (NADPH), serves as an antioxidant enzyme, and deficient G-6-PDH activity is associated with increase of endothelial cell oxidant stress.
Petrat et al.  demonstrated that NADPH is a primary target for singlet O2 in hepatocyte mitochondria; thus, NADPH may operate directly as intracellular antioxidant, as it is regenerated.
Our data showed that rats intoxicated with CCl4 exhibit highly significant reduction in the blood level of G-6-PDH activity compared with normal rats. These results are in harmony with those of Ip et al. .
Hepatotoxicity induced by CCl4 in rats is associated with impairment in the formation of hepatic-reduced glutathione. Oxidized glutathione (GSSG) generated from both nonenzymatic and enzymatic scavenging reactions can be converted to reduced glutathione by the glutathione reductase-catalyzed and NADPH-mediated reactions, which are mainly derived from G-6-PDH  The glutathione antioxidant system consists of an array of nonenzymatic and enzymatic reaction pathways that are involved in the neutralization of reactive-free-radical species [53,54]. These results showed that blend infusion improved G-6-PDH activity comparable with intoxicated rats. These results are in parallel to those of Kumaravelu et al.  who reported that CCl4 intoxication imposes deleterious effects on the second-line antioxidation enzymes such as GST, GR, and G-6-PD, as evidenced by the decrease in their activities. Simultaneous administration of eugenol with CCl4 prevents the toxic effects of CCl4 on G-6-PDH. Administration of tea and tea polyphenols has been reported to prevent or attenuate decreases in antioxidant enzyme activities in a number of animal models of oxidative stress .
In this study, the histopathological examination of rats injected with CCl4 showed massive fatty change, focal necrosis, and lymphocytic infiltration.
Recent data have suggested that lipid peroxidative processes also affect collagen synthesis. In fact, it has been reported that lipid peroxidation can stimulate collagen synthesis by fibroblasts . Blend infusion did inhibit this stimulation, presumably by inhibiting lipid peroxidation. Moreover, it is well known that reactive oxygen species produced by inflammatory cells and free-radical-mediated reactions are involved in the inflammatory response and can contribute to liver necrosis . Iron chelators, potent inhibitors of iron-induced lipid peroxidation, as well as free-radical scavengers, can efficiently protect against these phenomena . Tea polyphenols would seem to inhibit Fe absorption . Severe and massive cell injuries, necrosis, inflammatory infiltration, and fatty metamorphosis were observed in the CCl4-induced group. Injury results were initially generated from the metabolism of CCl4 to CCl3, which initiates lipid peroxidation and covalently binds to essential macromolecules due to dysfunction of Ca2+-ATPases and ATP depletion. Subsequently, this causes a rise in intracellular Ca2+ and H2O2 resulting in cell hydropic degeneration and necrosis. Necrosis of hepatocytes as indicated by the presence of pyknotic nuclei occurred frequently around the central vein, compared with the control group. Second, it could be due to liver injury that occurs from inflammatory processes initiated by the activation of kupffer cells and release chemoattractants and activators of neutrophils . The antioxidant activity of blend infusion polyphenols and inhibition of free-radical generation are important in terms of protecting the liver from CCl4-induced damage.
Fahim et al.  reported that rosemary contain potent antioxidant components that lowered the liver inflammatory responses by lowering reactive oxygen species and thus leading to the formation of arachidonic acid metabolites. Ulicná et al.  indicated that tea contains many types of polyhydroxy compounds that can function as a natural antioxidant against radicals CCl3− and CCl3O3− , which cause hepatocellular damage.
These results revealed significant decrease in the mean values of nuclear areas, nuclear volume, and the ratio of nuclear/cellular volume of the liver of carbonate tetrachloride-intoxicated rats versus those of controls. Several studies were dealt with the nuclear volume as an indication of cellular function under different circumstances. It should be stressed that changes in nuclear size are dependent on the functioning of the cell. Enhanced cellular activity is related to an increase of nuclear volume. In contrast, decreased cellular function is associated with the diminution of the volume of the nucleus . Watanabe and Tanaka  claimed that the nuclear size decreased in proportion to cell size in aged human hepatocytes. Vidal et al.  found a significant reduction in the nuclear volume of relay neurons as a result of ageing. The reduction in the liver nuclear areas, nuclear volume, and the ratio of nuclear/cellular volume due to lead toxicity is in agreement with the previous study by El-Sokkary et al.  who reported that decreased nuclear areas, nuclear volume, and the ratio of nuclear/cellular volume in the organs of lead-intoxicated rats and melatonin cotreatment restored the measured morphometric parameters (nuclear areas, nuclear volume, and the ratio of nuclear/cellular volume of the liver).
From the histological results, it can be concluded that natural antioxidant and scavenging agents in blend infusion can be used as a functional acceptable beverage for protection against hepatocellular damage.
This study was supported by the National Research Center Dokki, Cairo, Egypt.
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