Indian Jou rn al of Experimental Biology Vol. 41. ovember 2003, pp. 1294- 1299

Effect of a polyherbal formulation , Ambrex , on butylated hydroxy toluene (BHT) induced toxicity in rats

* R S Dev i. Shoba araya n. K Vija i Mohan . K E Sabi tha & C S ShYLlma la Dev i

Department or BiocheJ1l istry and Molecular Biology. Un i ve rsit y or Madras. Gu indy Carnpu s. Chennai 600 025. India

Neccil 'l'ti 24 Febru({rv 2003: r(, I'iser/ 21 April 2003

Erf'cet of po lyherhal rormulation Arnbrex was eva luated in buty lated hydroxy lOluene (BIIT ) inducedlOxici ty or lungs and li ver in rats. Tox icity was produced hy adm inister ing BHT (500 mg/kg/day ) ror :l days. Lung damage was ev idcnced by elevated leve ls of broneho al veolar lavage fluid (BAL) parameters such as protein. lac tate. lactate dehydrogena, e (LDII ). alka line phosphatase (ALP). acid pho,phatase (ACP) and glucose-6-phosphate dehydrogenase (G6PD H). Liver damagc was proved hy eleva ted levels or serum protein and markers such as LDI-1. ALP. aspartate amino transrerase (A T ). alanine iI/nino Iransf'crase (A L T), decreased leve l or l ipid peroxidcs (LpO) in se rum and gluta lhione (GS H) in li ver. Administration of aqueous ."uspcnsion or Arnbrex (SO rng/kg ora ll y) retained these ekvatcd level, of BAL-protein. lactate, LDH. ALP. ACp. (i6PDH and sc rulll -pmlein. LD H. ALP. AST and ALT at ncar normal va lues . Decreased level of Ii er GSII was rClained alnea r normalcy ill Ambrc x pretreated I3 HT -admini stercd an imals. T here was no change in li ver LPO in ali lhe rou r groups.

Key words : Alllbrex. BAL (Bronci1oa lveolar lavage). BHT (Buty lated hydroxytolucne). Hepatotox icity. Lung tox icity. Serum marker,

8HT, a syntheti c phenoli c an ti ox idant. w iddy used as a food add iti ve, confers substanti al bencf'its to man by preserving and improv ing th e palatab ilit y of rood l

.

Preserva ti ves may inhi bit growth and act ivit y of microorgan isms by interfering w ith their cel l mcmbranes. enzyme act i vi ty or genet ic mechan isms. Other preserva ti ves may be used as an ti ox idants to hinder the ox idat ion of unsaturated fats, as neutral izers or acid i ty, stabi I izers to prevent phys ica l changes, firm ing agents, and as coat ing or wrappers to keep out microorganisms. prevent loss or water, or hinder undes irab le microbial , enzYlllatic and chemi c;J1 reactions2

. 8 11T compou nd has been the subjec t of ex lensi ve tox ico logica l in vesti gati on in vari ous animals. Though generall y considered to be safe at the concentrations present in foods (the acceptab le dail y intake of 8HT for man O.Smg/kg).1. high doses of 81 1T cause haemorrhag ic death accompani ed by inhibiti on of hepati c prothrombin synthes is in rats'\ pulmonary injury in mice). 8HT has been shown to induce reversible mi xed fu nct ion ox idases and li ver enlargement in rats6 and pelios is, hepatoce llul ar vacuo lat ion , degeneration and necrosis in the li ver of mice7 and found to increase the mitotic ac ti vity of

Corrc'TXlIIdent author: E- Illall : c"clevi @yahoo.colll

Phone: 044-244 12575.

li ver ce ll s in ratsx. In some in vest igat ions on the mechani slll of 8HT-induced hepatotoxic ity. plasma transaminase leve ls were used to indi cate tox icit /. 8HT, i f gi ven at high doses to mice causes lung (i<lInage IO

T rad iti onal drugs have been the starling point ror the discovery of many important drugs. Thi s rac t has led to chemical and pharmacologica l investigat ions of plants and the undenaking of general biologica l screen ing programmes of plants not onl y in India. but all over the world 'l . Alnbrex (trade nanle) IS a polyherbaJ formulat ion (a coarse powder partly soluble in water) consisting of Aswagandha ( Wifhania sOJl/nifcra ).

A mber, Shalaill ishi ri (Orchis 1I1C1sellla), Roomi mastagi (S/lOreli robusfa) and M adanakamappu (Cyeas

eireinalis ). Shorea robu.\'(a IS glv n In ulcers, haemorrhoids, bleeding, anaemia and weak d igesti on l

". Amber is an ; nti septi c, ant ispasillod ic and also gi ven in delirium and infec ti ous diseases l 3 In olden days, Amber-based med icine was used ror prevenling premature ag ing and for increasing longev ity . The medicine is used by the sick to recover hea l th and increase resistance to c1i . e::lses. stamina, endurance of athelets ancl for tremendous boost to

their performance by increasing the energy in the human body. Thi s is due to the pres~nce of succ inic ac id in Amber l

.l. Amber brace let e::lses rheumatic pain

DEV I I'{ 01. : EFFECT OF AMB REX 0 BHT - I DUCED TOX IC ITY 1295

and amber coral beads supposedly help in cases of thyroid illncsses l5 Cycas c ircilla /is is good for hyperdipsia, burning sensa ti on, sores and swellings l6. With ania sOi/lnijera has anti ox idant effect and reponed to have anti stress, immunomodulatory, antiinflammatory ef fec ts by free radical scavenging ac ti vit/ 7 O rchis lIIascu/a has antidi arrhoeal and antidyscntery etrectsl8. Hence, an attempt has been made to eva luate the potency of A mbrex In modulating BHT-induced tox icit y in rats.

Materials and Methods M ale albino rats of Wi star strain ( I OO- 150g) were

used as experimental animal model in the present study. Rats were fed with commercial pellet diet and water ad libitlllll. BHT was purchased from Sisco Research Laboratory , Chennai and A mbrex was girted by Care and Cure Herbs Pvt. Ltd. , Chennai. A nimals were di vided into four groups o \" six animals cac ho A ll animal experiments were carri ed out as per the guidelines prov ided by Instituti onal A nimals Ethi cs Committee (I AEC) .

Group I was gi ven sunflower o il intraperitonea ll y for 3 days (contro l), group II animals were injected (ip) with BHT (500 mg/kg/day in sunflower oil ) for 3 days I'!, group III animals were pretreated with aqueous suspension of Ambrex (50 mg/kg/day, po) fo r 18 days and the animal s were treated with BHT for 3 days pri or to sacri f ice and Group I V - drug control rats were admini stered aqueous suspension of A mbrex al one (50 mg/kg/day, orall y) for 18 days and sunflower oi l was administered ( ip) for 3 days pri or to sacrifi ce ( the total experimental peri od was 18 days) .

The ex perimental animal s were sacrifi ced after overni ght fasting. Blood was collected by culting the jugular vein and serum was separated by centri fugation. Li ver was removed, homogeni sed with Tri s-HCI buffer and the supernatant was used for the estimati on

'0 ' I of LPO- and GSH- . The lungs were removed and washed with ice-cold saline to remove blood.

Iso/a tio /l (~(lmill ch (}a" 'eo/({ r/a\lage (BA L ).flll id The lungs were lavaged two times with 5 ml of

lavage fluid (0. 15M NaCI- 0.0 I M Tri s HC!. I'H 7.4). The lungs were connec ted to a sy ringe via a trachea l tubing and allowed the lavage fluid to pass till al l the alveoli adjacent to the visceral pleura were fill ed. T he lavage i"luid was withdrawn and again infused. The recovered fluid was stored in icc. The combined lavages were centrifuged at 1000 g for 10 min and the supernatant flui d was used for es timati on of vari ous

" marker enzy mes--. E · . f' . ' 3 " LDI-I ' 4 d A LP») - slJm<1 tlon o · proteJll -' , assay 0 - an -.

in both serum and BAL were performed. Estimat ion o f iactate26

, ACP25 and G6PDH 27 were assayed in BA L fluid and AST2R, A LT2x and lipid perox ides21l

were es timated in serum . Intergroup differences were compared using Student 's t-tes t.

Results Table I shows the levels of protein , lactate and

ac ti viti es of LDH, A LP, ACP and G6PDH in BAL fluid. A signifi cant increase (P <O.OO I ) ill the levels of protein , lactate and the acti viti es o f LDH , A LP, ACP and G6PDH was observed in the BA L flu id of" BHT-treated group II rats when compared to group I

Tahle I - Levels of prote in. lactatc and act ivities nf LDH. A L P. G6PI)H and ACP in bronchoalvcolar lavage Iluid of experi mcntal anilllais

I Valucs are mcan ± SD for six animals in each group l

Parameters Group I Group II Group III Protein 35-U \6 ± no I 469.75 ± :16.66" .. •• h358.03 ± 35.39d • •••

( l11g/ml ) Laclalc 1.701 ± O. IS 3. 19 ± D.22" .... h 1.73 ± 0.08d ....

(11 III 0 1/111 I ) LDH 5.32 ± 0.5S 11 .6 ± U.8" .. •• h5.87 ± 0.46d

••••

(I U/dl ) ALP 13.9 1 ± 1.27 2 1.37 ± 1.65" .... hI 4.29± 1.29d

.. • •

(~lm()lc of phcnol l iberateel/min/e1 I) G6PDH 1.33 ± 0.44 2.28 ± 0.12" .. •• h 1.45 ± 0.09d ....

(IU/d l) ACP 3.22 ± 0.28 7.47 ± 0.37" .. •• h3.54 ± 0.32d

.. • •

(fll11ole of phenol li berated/ l11in/e1I )

"Grou p I Vs group II ; hGroup I Vs group III: cGroup I Vs group IV: dGroup II Vs group III. **** P < 0.00 I .

Group IV 365.98 ± 36.54"

1.9 1 ±O. 14'·

5.92 ± 0.4S'

12.85 ± 0.84<

1.2 1±0 11 <

3.3 1 ± 0.39'

1296 I IDI A J EX P 131 0 L. OVEMI3ER 20m

control rats whereas the leve ls of protein and lactate and the acti viti es of marker enzymes were retained al near normal status in A mbrex pretreated SilT administered group III an imals when compared to group I on tra l animals and decreased significantly (P< O.OO I ) when compared to group II BHT treated anima ls. 0

significant changes in these parameters were observed in the Ambrex alone trea ted group IV animal s.

Table 2 shows the leve l of protein and ac ti viti es of LDH. AST. ALT . ALP and LPO in serum. Level of serum protein, acti viti es o f LDH, AST, ALT and ALP increased significan tl y (P <O.OO I ) in SHT trea ted group I I when compared to group I control rat s. These parameters were reta ined al near norIl1alcy in Ambrex pretrea ted SI-IT admini stered group III wh en compared to :; roup I control rats and sign ifi cantl y decreased (P < 0.00 I ) when compared to group II SHT treated rat s. No signifi cant changes were observed in Ambrex alone trea ted group IV animal s. Lipid perox ide levels in SHT trca ted group II ,"ere signifi cantl y (IJ < O.OO I ) decreased when compared to contro l group I, whereas Ambrex pretreated SilT admin istered group III anima ls showed signifi ca nt decrease (P < 0.00 I ) in LPO leve ls when compared to group I control a nim ~t1 s. In AIl1brex pretreated SHT admin istered grou p III animal s. LPO level was not

changed signifi cantl y when compared 10 group II SHT Ireated rats. In A mbrex alone group IV animals. the LPO level showed no signifi can t change whcn compared to cont ro l group I an imals.

T ab le 3 shows the leve ls of LPO and GS H in li ver. The level of hepatic LPO was not changed signifi cantl y in all the four groups. whereas the leve l o f hepatic GSH was decreased signifi cantl y (P <O. f)OI ) in SHT trea ted group II rats when compared to contro l group I rats. In Ambrex p retre~lted SHT adm inistered group III rats , the GSH leve l was increased sign i fi cCl ntl y (P < 0.00 I ) whcn compared to SHT trea ted group II animal s and decreased significantl y (P <O.OOI) when compared to control group I animal s. Ambrex alone admini stcred grLlUp IV animals showed a increase (1) < 0.00 1) in GS H content when compared to cont ro l group I rats.

Discussion S HT is biotransformed into a reacti ve. electroph i I ic

metabolit e SI-IT-quinone ll1ethide by cy tochrome P.jSII mediated metabo li : 1l1 and causes tox icil/'! which can covalen tl y bind to va ri ous ce llular Ilucleophil es, espec iall y those containing su lnl yd ry l groups such as g lu ta thi one and cys teine' O SHT quinone methide has been detec ted ill I' i l'o in li ver and lung. 31.

Table 2 - ACli vitics or seruill l1larkers or normal and expcri lllcntal groups

I Valucs arc Illca n ± SD 1'01' ~ i x an imals in cach group l

Paralllctcrs

Protein (g/dl ) LD II ( l Ull .) AST ( l UlL) ALT ( I ILl ALP ( l Ul L) LPO

Croup I

5.n±0.6 1

7948 ± 7.4 1

:1:1.28 ± 3.58

17.07± 1.25

3.57 ± OA8

1.86 ± 0. 1-1

Group II Group III

9.15 ± 0.804" .. •• h5 .72 ± 0.44" .. ••

11 -1. 2 ± 12.08" .... 1'98.06 ± 9.39" ....

60.77 ± -1 .DS" .... h49.67 ± 3.0<)" ....

25 .67 ± 1.7"·· .. h2145 ± 2.25" ....

5.73 ± n. I7" .... h3.7 ± 0. 16" ....

1. 18±0. 1-1" .... h"" I .03 ± O. 15" (p lIl<l/c, or MDA/hr/ l1lg prote in )

"Croup I V, group II : hGroup I Vs group III: ' Group I Vs group IV: "Group II V;-. group III. **** P < O.OO I.

Paraillcter, LI'O (nl1lolc,/g Wet tissuc) CiS H (plllok;-./g wct ti ssuc)

Tahlc 3 - Lcvel, of LPO and GSH in livcr or rour dirfercnt groups

I Values are Illea n ± SD for 6 ani llla ls in cac h groupl

Group I Group II Group III 342.50 ± 7.85 355.50 ± 6.55" h339.66 ± 3.04<1

5.33 ± 0.38 1.79 ± 0.26" .... 11 .... 3.35 ± 0.32<1····

"C,I\Jllp I Vs group II : hCroup I Vs group III : ' C roup I Vs group IV: "Group II Vs group III. **** IJ < n.no I.

Group IV

5.g8 ± 028'

76 .. B ± J .D9'

3 1.73±3.14'·

18.67 ± 1.25"

3.86 ± D. 17'

1.62 ± 0.36'

Group IV 348.66 ± :'>.65'

8.08 ± 0.5<)'· ....

DI:::Y I (,ll1l . : EFFECT OF AMBREX ON BHT-INDUCED TOX ICIT Y 1297

An increase in BAL protein and lactate contents in BHT-induced rats may indicate an increase in al veo lar capillary permeability and metaboli sm respecti ve ly'9. BHT has been reported to produce acute lung injury'2. Lavage LDH is a marker for ce ll lys is ' 9 and a stati sti ca lly signi f icant increase in BAL-LDH indicates lung cell damage. In the present study, oral pretreatment with Ambrex decreased the BHT­induced alterations.

G6PDH is a cy toplasmic enzyme whi ch could occur ex trace llularl y only in the presence of increased ce ll membrane permeability". G6PDH has been reported to occur in type I pneumocy tes whi ch are most vulnerable to injury. It is also a marker enzyme for hexose monophosphate shunt path way of glucose metabol ism which increases in ac ti vated macroph ages and in ti ssues where sy ntheti c process or repair is needed13 G6PDH has been increased signifi cantl y in the present study which denotes the increased cell membrane permeabilit y due to BHT-induced toxicity.

Ac id phosphatase is a lysosomal enzyme released by polymorphonuclear leucocytes during phagocytos is

" and to some e-x tent by macrophages·· . Damage or death of these ce ll s would release thi s enzyme into the lavage flui d. A lkaline phosphatase is associated with pl asma membrane of mammalian cell s. In lungs, type II ce ll s contain alkaline phosphatase but not type I or macrophages. A n increase in lavage fluid A LP is a use ful index for detec ting type I pneumocyte hypertrophy'" . T ype II ce ll s are involved in the storage and synthes is of pulmonary surfactant and injury to these ce ll s may result in loss of proper oxygen exchange function of the lungJ

) . Increase in ACP, ALP and G6PDH is indicati ve of an inflammatory response which is due to BHT-induced tox icit y. Acti vities of ACP, A LP and G6PDH were retained by Ambrex pretreatment. The observed results may be due to the prote-cti ve effi cacy of ingredients present in A mbrex.

Alterati ons in serum constitu ent. deri ved from the li ver may occur as a result of anyone o f the several morpholog ica l changes. The appearance o f ce llular constituents in the blood strea m is probabl y the most accurate refl ecti on of ti ssue damage.l6. Determination of the acti vity of hepati c enzy mes released into the blood by the damaged liver is one- of the most use ful too ls in the study of hepatotox icity'7

Enzymes that are most commonly employed as indicators o f li ver damage are AST and AL T. Both enzymes are present in hi gh concentrations in the li ver' . Aminotransferases are released from hepati c

cell s as a consequence of increased membrane permeability and hepatocellular reactions to tox ic agents. The increase in aminotransferase is generall y more striking witti chemical s producing necrosis, for example carbon tetrachl oride36

. A n elevated acti vity of ALT in BHT-injected rats were reported earlier3

7..l'J

and the above data coincide with the results of the present study. A LP is mostl y used as an index of li ver functi on and increased levels were found in serum of most patients with li ver damage4o

. Many chemica l carcinogens have been reported to increase the acti vity of serum A Lp4oA' . BHT produces puzzling data. BHT, if gi ven (in diet or by injec ti on) before a chemical carcinogen would af fo rd pro tection aga inst chemica l carcinogenes is. On the other hand, exposure to BHT after exposure to a carcinogen would sometimes enhance tumour deve lopment. Thc capability of BHT to produce ce ll hyperpl as ia in the target organ was thought to be instrumental in the tumour-enhancing or ' promoting' activ ity of BHT"2 . Present study showed an elevated serum A LP acti vit y, an index of li ver damage and it may be due to the possible carcinogenic effec t o f BHT. Pretrea tment with Ambrex modul ates the changes produced by BHT tox icity.

The lipid peroxidation of ce llul ar membrane has been proposed as one of the mechani sms by which a number of foreign compounds produce structu ra l ti ssue injury"3, However, for certain chemica ls such as acetami nophen and bromobenzene, the importance o f perox idati ve damage is a matter of continued discu ss ion4

". Lipid perox idati on which occurred during chemica ll y induced hepatoce l lular nec ros is was due to the depleti on o f GSH"5. The level of lipid perox ides in serum decreased after BHT induct ion"!>. Similar observa ti ons have been recorded in the present in vesti gations. The hepati c clamage produced by BHT was associated with a consequence of GSH depletion rather than with li pid peroxidation"7 The results of the present stucly also impl y the same. In Group (/I A mbrex pretrea ted BHT' administered rats, the leve l of serum LPO decreased when compared to BHT trea ted Group IJ , thi s may be due to BHT itself being an antiox idant and hepati c damage prod uced by BHT was assoc iated with GSH depleti on rather than with LP047. The decrease in serum LPO in group III when compared to group I might be due to the antioxidant property of A mbrex. There was an increase in li ver GSH in Group III Ambrex pretrea ted BHT administered rats when compared to BHT treated group II. There wa. no change in li ver LPO in

1298 I DIAN J EXP BIOL, OVEMBER 2(0)

all the four groups. Thi s may be due to the antioxidant property of the whole formulation, particularl y it may be due to Wilhallia sOl/lIl/fera l 7

, ShorNI rol)//Sla~ x and also be due to the synergist ic act ivity of the components present in A mbrex. The decrease in li ver GSH in group III Ambrex pretrea ted BHT­administered animals when compared to contro l group I might he due to the utili za ti on or GSH by damaged ce ll s. GS H plays an important ro le in the detox ificati on of acti va ted metaho lites of BHT~7 . When the hepati c GSH level is low for a long time or when excessi ve quantiti es of the activated metabolit es arc produced, the metabo lites bind to essential ce llular macromolecules causi ng hepati c dal11age~7 . Therefore, the alteration s by BHT is prevented by A illbrex whi ch i~ ev ident from the resul ts.

The augmented acti v iti es or some or the d iagnostic marker enzy mes in serum, such as AST, A L T, ALP and LDH may be related to the hepatotox icity induced by BHT andlor its metabo lites . Thererore, the result suggests that the hepatic damage produced by BHT was decreased by Ambrex. One of the constituents or Ambrex , Wilhallia sOll/lllfem has free rad ica l scavenging acti vity' 7 and contains a bitter alkaloid ' Somni ferin '. It also contains reducing sugar, phy tosterol, ipuranol and mixture of saturated and unsaturated ac ids. The vari ous acti ve principl es in Wilhallio so/Illl(fera show, neuroprotect i ve, anx ioly ti c antidepressant (putati ve an tis tress) and adaptogenic activ it/ ~ . The other constituent , SIlO rea robllsta

contains fl avonoids4l). Flavonoids inhibit cy tochrome

P450"o and are potent an ti ox idants and rree radi ca l scavengers51 . It can be concluded that, the ingredients present in the Ambrex might be responsible 1'01' the observed protec ti ve efrec t against BHT-induced tox icit y. However, fUrlher study is needed in some more models of experimental hepati c and lung damage to elucidate the exact molecular and biochemica l mechanisms in vo lved to es tab li sh its therapeutic role as a hepatoprotec ti ve and pulmonary preventive agent.

Acknowledgement One of the authors (KES) acknowledges the

financial ass istance rendered by CSIR, New Delhi .

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DEVI ef al.: EFFECT OF A MBR EX 0 BHT-INDUCED TOXICITY 1299

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