issn 1110-7537 protective effect of sage ( salvia officinalis l.) on...

Egypt. J. Comp. Path. & Clinic. Path. Vol. 23 No. 2 (May) 2010; 121 - 137

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Referred byReferred by

Prof. Dr. Alaa Rafat Professor of Clinical Pathology, Fac. Vet. Med., Cairo University

Prof. Dr. Iman Bakr Mohamed Professor of Pathology, Fac. Vet. Med., Cairo University

Protective Effect of Sage (Salvia officinalis L.) on Cyclophosphamide toxicity evaluated By Cytogenetic, Biochemical and Histopathological

Assays in Male Albino Mice By

Ekram S. Ahmed (1), Thanaa M. Shoman. (1) Adel Ezz-Eldin, (1) and Kawkab A. Ahmed(2)

1. Department of Cell Biology National Research Center Egypt

2. Department of Pathology, Faculty of Veterinary Medicine, Cairo Univ.

SUMMARY

T he induction of chromosomal aberration, biochemical and patho-logical alterations by Cyclophosphamide (CP) and their reduction

by two concentrations of Sage (Salvia officinalis L.) were studied in male albino mice. Six groups of mice were used included the control group, cyclophosphamide (CP) treated group and the other groups treated with the extract alone or in combination with CP. The results in-dicated that treatment with CP resulted in significant clastogenetic ef-fects. Also, biochemical results revealed that CP increased Aspartate amino transferase (AST), Alanine amino transferase (ALT) and Glucose levels and decreased testosterone level. Moreover, the results showed different histopathological lesions in kidneys, liver and testes of CP treated mice. However, Salvia did not have toxic effects of its own and the two concentrations of sage extracts exhibited a protection against CP-induced cytogenetic, serum biochemical and pathological changes in mice. The results provide some support for antimutagenic potency of sage in vivo.

Keywords: Chromosomal aberration, Salvia officinalis, Albino mice, Cyclophosphamide, Histopathology.

INTRODUCTION

C hemoprevention of mutation-related disease and cancer is

an important research field and the

dietary use of antimutagens and anticarcingenes has been proposed as the most promising approach to protection human health (Fe-

ISSN 1110-7537


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rguson, 1994 and Vukcevic-Knezevic et al., 2005) Medicinal plant are a valuable natural re-source and regarded as potentially safe drugs (Khalil et al., 2005).

Many species of salvia have been used as medicinal plants and show several activities. There are four main categories of chemical compounds in plants of genus sal-via including monoterpenes, diter-penes, phenolic acids and flavon-oids. Regarding phenolic acids, the majority of these compounds in salvia species and possesses a vari-ety of biological activities includ-ing antioxidant, antiplatelet, antitu-mor and antiviral activities (Weiss and Fintebmann 2000; Lu and Foo 2002 and Hosseinzadeh et al., 2009).

Salvia officinalis L. are well

known for their anti-oxidative properties (Baricevic et al., 1996). Moreover, the antimutagenic po-tential of sage extracts was demon-strated on Escherichia coli repair proficient strains (Filipic and Baricevic 1997 and 1998).

On the other hand it should be

considered that some of the S. offici-nalis that have antimutagenic effect in certain concentrations are cyto-toxic in higher concentrations (Vujosevic and Blagojevic 2004). Also, Abd El-Aziem et al. (2004) reported that sage have a protective

role against acrylamide induced genotoxicity in rats. Therefore, it is of interest to investigate whether sage reveals its antimutagenic activ-ity in mammalian cells in vivo. In this study, the mice bone marrow chromosomal aberrations test was applied to evaluate the role of sage in modulating the cytogenetic damage induced by indirect mutagen cyclo-phosphamide. The activities of ALT, AST as well as levels of Glucose and testosterone were measured. Also, histopathological examination of mice tissues (kidneys, liver and testes) was done.

MATERIALS AND METHODS

1- Cyclophosphamide (CP; CAS No. 6055-19-2) was obtained from Sigma chemical Company (St. Louis, MO, USA).

2- Dried Sage leaves (Salvia offici-nalis) was supplied by the de-partment of crop production, faculty of Agriculture, Ain Shams University.

3- Kits: Transaminase(ALT and AST) were purchased from Randex Labs(San Francisco, CA,USA).Glucose and testos-terone were obtained from Biomerieux Lab of reagents and products (Marcy Letoile, France).

Experimental animals:

Forty eight (six to eight weeks old) Swiss albino male mice (20-


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25g), purchased from animal house colony, Giza, Egypt were main-tained on standard lab diet (Protein: 160, 4; Fat: 36.3; fibre: 41g/kg and metabolizable energy 12.09 MJ), and housed in a room free from any source of chemical contamination, artificially illuminated and ther-mally controlled, at the Animal House Lab., National Research Centre, (N.R.C.) Cairo, Egypt. After an acclimatization period of one week, the animals were divided into six groups (8 mice/group) and housed in filter-top poly carbonate cages. All animals received humane care in compliance with the guide-lines of the Animal Care and Use Committee of the N.R.C. Cairo, Egypt.

Experimental design: Animals within different groups

were orally treated for 2 weeks as fol-low: Group 1: served as negative control Group 2: treated with sage (0.3 g/kg. b.w.). Group 3: treated with sage (0.6 g/kg. b.w.). Group 4: treated with intraperitoneal injection with CP (25 mg/kg b.w.). Group 5: treated with sage (0.3 g/kg, b.w.) for two weeks then injected with CP (25 mg/kg b.w.). Group 6: treated with sage (0.6 g/kg b.w.) for two weeks then injected with CP (25 mg/kg b.w.) . Cytogenetic analysis: for chromo-somal analysis both treated and

control animals were sacrified by cervical dislocation. Two hours be-fore sacrifice, mice were injected with 5mg colchicines/kg b.w. fe-murs were removed and the bone marrow cells were aspirated using saline solution. Metaphase spreads were prepared using the method of Preston et al., 1987). Fifty meta-phase spreads per animals were analyzed for scoring the different types of chromosomal aberrations. Biochemical analysis: At the end of experimental period, blood samples were collected from all animals from the retro-orbital ve-nous plexus for biochemical analy-ses. These analysis included, se-rum ALT, AST (Young, 1995), Glucose (Trinder, 1995), and Tes-tosterone (Hall, 1988).

Histopathological examination: Tissue specimens from kidneys,

liver and testes were collected from all experimental groups at the end of experiment and fixed in neural buff-ered formalin 10%, dehydrated in ascending concentration of ethanol, cleared in xylene and embedded in paraffin. Sections 4-5m thick were prepared and stained with Hema-toxylin and Eosin (Bancroft et al, 1996).

Statistical analysis: The obtained results of Cyto-

genetic examinations or biochemi-cal analysis were statistically ana-lyzed by ANOVA (one way) using Excel 2003 Microsoft Crop


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(11.5612. 5606). Redmond, WA software package.

RESULTS Cytogenetic analysis:

The data on chromosomal ab-errations were shown in Table (1) Cytogenetic results showed that the frequencies of total structural chromosome aberrations were low in the two groups of mice which treated with single treatments of (0.3 or 0.6g/kg b.w.) sage com-pared to those found of the control group of mice.

The established mutagen CP

significantly induced chromosomal aberration in male mice as com-pared to the control and salvia treatment.

The third and fourth groups

which treated with (0.3 or 0.6 of salvia) respectively, resulted in a significant decrease in the total chromosomal aberrations when compared to the control or the positive control (CP). But there in no statistically significant differ-ence between the two concentra-tions of salvia was found on the total chromosomal aberrations in-duced by 0.3 or 0.6 g/kg b.w. (Table 1).

The combined treatments of

salvia (0.3 g/kg b.w.) or (0.6 g/kg b.w.) resulted in a significant de-crease in the frequency of total

chromosomal aberrations in bone marrow of male mice induced by cyclophosphamide (25 mg/kg b.w.), and significant increase when compared to control group and salvia alone. Also, when com-pared slavia (0.6 g/kg b.w. + CP) with slavia (0.3 g/kg b.w. + CP) there was significant decrease in total chromosomal aberration (Table 1).

Biochemical results:

The data of serum biochemi-cal studies on different parameters in all experimental treatments are shown in Table 2. The data show that cyclophosphamide affected the liver enzymes (AST and ALT), Glucose and testosterone. CP caused a statistically significant increase in all measured biochemi-cal parameters of males mice when compared with the control except the plasma testosterone levels it was significally decreased. How-ever, the (AST and ALT) and Glu-cose were significantly decreased at (P < 0.05) when the two concen-trations of salvia singly was ad-ministrated when compared to the control or other treatments.

Meanwhile, a statistically sig-

nificant increase in testosterone level was observed in combined treatments of (Salvia and CP) when compared with the positive control (CP) alone.


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Table (2): Serum levels of AST, ALT, glucose and testosterone in control, Saliva extract and or cyclophosphamide treated mice.

Treatment AST

U/l

ALT

U/l

Glucose

mg/dl

Testoster-

one

ng/ml

Control 120.20 + 1.66d 45.80 + 1.77c 68.40 + 1.89c 9.40 + 0.75c

Salvia (0.3 g/kg) 102.00 + 1.70b 27.00 + 2.55b 42.00 + 2.55b 6.00 + 0.55b

Salvia (0.6 g/kg) 72.00 + 1.70a 17.00 + 2.55a 23.80 + 1.74a 7.20 + 0.58b

Cyclophosmide (25 mg/kg)

194.20 + 1.66f 81.20 + 4.65 181.80 + 0.92f 3.00 + 0.89d

Salvia 0.3 + Cyclophos-

phamide 144.60 + 1.60e 55.80 + 1.66d 133.80 + 1.85e 5.40 + 0.75b

Salvia 0.6 + Cyclophos-

phamide 122.60 + 1.08c 33.60 + 1.86b 99.6 + 3.33d 8.20 + 0.58c

The different letters (a,b,c,d,e,f) in the same column are significantly differ-ent at level (P < 0.05)

Histopathological Results: Kidneys of mice from control

group (group 1) or from mice treated with Salvia either with (0.3g/kg b.w.)or (0.6g/kg b.w.) (groups 2 & 3) revealed normal histological structure of renal parenchyma from cortex and medulla (Figure 1).

Meanwhile, kidneys of mice

treated with CP showed congestion of renal blood vessels and glomeru-lar tufts. There were granular and vacuolar degeneration of epithelial lining renal tubules (Figures. 2&3)

as well as pyknosis of some nuclei of the epithelial lining and focal inter-stitial leucocytic cells infiltration (Figure 3). However, kidneys of mice treated with CP plus salvia 0.3 g/kg b.w revealed no pathological changes except congestion of glomerular tufts (Figure 4). More-over, apparent normal renal paren-chyma was noticed in examined sec-tions from mice treated with CP plus salvia 0.6 g/kg b.w (Figure 5).

Regarding liver, examined

sections from control mice (group 1), or from mice treated with 0.3 or


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0.6 g/kg b.w. of Salvia (groups 2 & 3) revealed no changes as the liver showed the normal histological structure of hepatic lobule from cen-tral vein and concentrically arranged hepatocytes. On the other hand, liver of mice treated with CP (group 4) revealed different histopathologi-cal changes which summarized as vacuolations of hepatocytes and pyknosis of some nuclei of hepato-cytes (Figure 6). Focal hepatic hem-orrhages associated with necrosis of hepatocytes were also observed in all examined sections (Figure 7). Additionally, some examined sec-tions from this group (group 4) showed marked dilatation of bile duct as well as cholangitis described by inflammatory cells infiltration in the wall of bile duct (Figure 8). However, examined liver sections from mice treated with CP com-bined with 0.3k/kg b.w. of Salvia revealed slight improvement in the histopathological picture as exam-ined sections showed kupffer cells activation (Figure 9), vacuolar de-generation of some hepatocytes and cholangitis (Figure 10). On the other hand, liver of mice treated with CP combined with 0.6g/kg b.w. of Sal-via showed marked improvement in the histopathological picture as the liver showed no histopathological changes except Kupffer cells activa-tion (Figure 11).

Concerning testes, examined sections from control mice or from

mice treated with 0.3 or 0.6 g/kg b.w. Salvia revealed the normal his-tology of semineferous tubules which lined by different sper-matogoneal cells (Figure 12).

Microscopically, testes of mice treated with CP revealed vacuolations of spermatogoneal cells lining the semineferous tu-bules (Figure 13), interstitial oe-dema dispersed the semineferous tubules far away from each other, degeneration of spermatogoneal cells lining semineferous tubules (Figure 14) with slight thickening of basement membrane. On the other hand, testes of mice treated with CP plus 0.3g/kg b.w. of Salvia showed improvement in the histopathologi-cal picture, examined sections re-vealed slight degeneration of sper-matogoneal cells lining some semineferous tubules associated with slight interstitial oedema (Figure 15). Whereas, testes sec-tions of mice treated with CP plus 0.6g/kg b.w. of Salvia revealed no histopathological alterations except slight degeneration of sper-matogoneal cells lining of some semineferous tubules associated with incomplete spermatogenesis (Figure 16).


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Fig. (1): Kidney of Control, untreated mice showing the normal histologi-cal structure of renal parenchyma (H and E x 200).

Fig. (2): Kidney of mice treated with CP showing congestion of glome-rular tuft, granular and vacuolar degeneration of epithelial lining renal tubules (arrow) (H and E x 200).

1 2

Fig. (3): Kidney of mice treated with CP showing vacuolar degeneration of epithelial lining renal tubules and focal interstitial leucocytic cells in-filtration (arrow) (H and E x 200)

Fig. (4): Kidney of mice treated with CP and 0.3 Salvia showing con-gestion of glomerular tuft (arrows) (H and E x 200)

3 4


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5 6

Fig. (5): Kidney of mice treated with CP and 0.6 Salvia showing appar-ent normal renal parenchyma (H and E x 200).

Fig. (6): Liver of mice treated with CP showing clearance of the cy-toplasm and pyknosis of some nuclei of hepatocytes (arrow) (H and E x 200) .

7

Fig. (7): Liver of mice treated with CP showing focal hepatic hemorrhage (arrow) assoicated with necrosis of hepatocytes and pyknosis of their nu-clei (H and E x 200)

Fig. (8): Liver of mice treated with CP showing marked dilatation of bile duct (small arrow) associ-ated with cholangitis (large ar-row) (H and E x 200)

8


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9 Fig. (9): Liver of mice treated with

CP and 0.3 Salvia showing kupffer cells activation (arrow) (H and E x 200)

Fig. (10): Liver of mice treated with CP and 0.3 Salvia showing cholangitis (H and E x 200).

10

11

Fig. (11): Liver of mice treated with CP and 0.6 Salvia showing kupffer cells activation (arrow) (H and E x 200).

Fig. (12): Testis of control, untreated mice showing the normal histology of semineferous tubules (arrows) (H and E x 200).

12


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13

Fig. (13). Testis of mice treated with CP showing degeneration of sper-matogoneal cells lining seminefer-ous tubules (arrow) (H and E x 200).

Fig. (14): Testis of mice treated with CP showing vacuolation of sper-matoganeal cells lining the seminiferous tubulesas well as inerstial edema (arrow) (H and E x 200).

14

15

Fig. (15): Testis of mice treated with CP and 0.3 Salvia showing degeneration of spermatogoneal cells lining semine-ferous tubules and interstitial edema (arrow) (H and E x 200).

Fig. (16): Testis of mice treated with CP and 0.6 Salvia showing slight degeneration of spermatogoneal cells (arrow) (H and E x 200) .

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DISCUSSION

T he results of the present study show that Salvia officinalis

(Sage) has some antimutagenic properties against CP induced chromosomal aberrations and im-prove the changes in the biochemi-cal parameters (AST, ALT, Glu-cose and testosterone). From histo-pathological point of view, the pre-sent work indicated that CP was able to induce many of alterations in kidneys as well as in liver and testes tissues. Those alterations could be attributed to the oxidative stress induced by increasing lipid peroxidation production by cyclo-phosphamide as well as to the abil-ity of the activated metabolites of cyclophosphamide (which are al-kylating agents) to cause cross-linking of DNA strands, interfering with normal cell division in all rapidly proliferating tissues. In ad-dition, cyclophosphamide can dis-rupt the redox balance of tissues suggesting that biochemical and physiological disturbances may re-sult from oxidative stress caused by the generation of free radicals and ROS.(Sabik and Abd El-Rahman (2009).

Possible application of plant antimutagens as a dietary preven-tion of cancer and other mutation related disease makes the study of plant antimutagens an important in research field (Craig, 1999). Sal-via sp. is an important genus con-sisting of about 900 species in the

family Lamiaceae. Salvia officinalis is an aro-

matic and medicinal plant of Medi-terranean origin and well known for its antioxidant properties due to its composition in phenolic com-pounds. The antioxidant activity mainly based on results from sev-eral subcellular and non cellular in vitro (Baricevic and Barto 2000). Also, Farhat et al., (2001) re-ported that the antioxidant activity of sage may be due to 1,8-cineole, the main constituent, although other components included ketones such as limonene and alpha, beta-pinenne and alcohols such as bor-neol and linalool. Moreover, sage tea drinking has shown the ability to improve liver antioxidant status in mice and rats (Lima et al., 2005).

Consequently, Amin and Hamza (2005) have shown that the treatment of rats with a water extract of sage protected against azathioprine. In agreement with these findings, we found in the present study that salvia could in-hibit the toxic effects of CP in males mice and the high dose (0.6 g/kg b.w.) of saliva more effective to reduce the mutagenic effects and pathological alterations in all examined tissues. The results of another investigation showed clearly the antioxidant effects at cellular level of sage namely pre-venting cell death, lipid peroxida-


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tion and GSH depletion induced by tert-butylhydroperoxide in HePO2 cells (Lima et al., 2007). Also, Abd - ElAziem et al., (2004) re-ported that sage supplemented diet decreased acrylamide mutagenicity in rats.

On the other hand, it should be considered that some of the Sal-via species (such as S. officinalis) that have antimutagenic effect in certain concentrations, are cyto-toxic in higher concentrations (Vujosevic and Blagojevic 2004). The protective effect of sage from CP may be caused by a change in the activity of biotransformation enzymes needs to produce its ac-tive mutagenic metabolite phos-phoramide mustard (Rompelberg et al., 1995).

The data of the biochemical study in the present work show that oral administration of sage caused a significant decrease in the liver en-zyme (AST and ALT) and Glucose in the two concentrations (0.3 and 0.6 g/b.w.). These biochemical data were supported with the histopa-thological results, as the examined tissues from those groups revealed marked improvement in the histopa-thological picture. Furthermore, sage has a protective role against CP to reduce these biochemical pa-rameters, increase testosterone lev-els and improve the histopathologi-cal picture. These results were in agreement with Hosseinzadeh and Eghbal (2002) who investigated the

possible hepatoprotective effects of the alcoholic leaf extract of Salvia leriifolia in mice. At present, it is known that Salvia has relatively unambiguous antiendoxin effects and can directly neutralize and de-stroy endoxin. It has been pointed out in some studies that Salvia can protect hepatic cells and maintain liver function through reducing in-flammatory mediator levels and improving microcirculation. Salvia is also able to significantly im-prove the liver function at the early postoperative stage. Salvia is able to reduce endotoxemia, regulate the production and secretion of vasoactive substances, and im-prove renal blood perfusion, thereby exerting protective effects against damage to kidney function (Ye et al., 2010).

Conversely, the lower doses of the S. Leriifolia extract (50 mg/kg and 100 mg/kg) did not reduce the elevated activities of ALT and AST significantly when administrated 1hr after established hepatoxins, carbon, tetrachloride and acetamino-phen. In addition, treatment of mice with a single oral dose (2g/kg) of alcoholic seed extract of S. leriifolia caused a significant reduction in blood glucose level (Hosseinzadeh et al., 1998). Moreover the higher content of antioxidant substances in sage and barley led to improvement the serum clinical chemistry in rats treated with acrylamide (Abd El-Aziem, et al., 2004). Also, several


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reports indicated that blood choles-terol and lipoprotein concentration can be reduced in human (Braaten et al., 1994) and animal (Kalra and Jood 2001) by aglucan from barley. Moreover, the change of plasma tes-tosterone levels in male mice treated with CP may result from a local in-hibition of the fetal testicular func-tion. Elangovan, et al., (2006), also reported that CP treatment causes impairment of sperm and its fertiliz-ing ability in mice.

I n conclusion, the present study revealed that CP induced cyto-

genic, biochemical and histopa-thological alterations typically to those reported in the literature. Both two concentrations of sage have a protective role against these deleteri-ous effects possibly due to their con-tents of antioxidant substances.

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issn 1110-7537 protective effect of sage ( salvia officinalis l.) on...

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