Anti At Hero Sclerotic

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VSP 2001/04/20 Prn:26/04/2004; 14:00 {RA} F:iph2138.tex; VTeX/NJ p. 1 (48-129)

Inammopharmacology , Vol. 00, No. 0, pp. 122 (2004)

VSP 2004.Also available online - www.vsppub.com

Anti-inammatory properies of BHUx, a polyherbalformulation to prevent atherosclerosis

YAMINI B. TRIPATHI 1,, M. MALLIKARJUNA REDDY 2 , R. S. PANDEY 1 ,J. SUBHASHINI 2, O. P. TIWARI 1 , B. K. SINGH 1 and P. REDDANNA 2

1 Department of Medicinal Chemistry, Institute of Medical Sciences, Banaras Hindu University,Varanasi-221005, India

2 Department of Animal Sciences, University of Hyderabad, Hyderabad-500 046, India

Received 8 January 2004; revised 28 March 2004; accepted 29 March 2004

Abstract BHUx is a polyherbal formulation consisting of water-soluble fractions of ve medicinalplants ( Commiphoran mukul, Terminalia arjuna, Bosvelia serata, Semicarpus anacardium andStrychnos nuxvomica ). The present study was undertaken to evaluate its antioxidant and anti-inammatory effects. BHUx, standardized by HPLC ngerprinting and ltered through 0.2 mlter paper, was employed for different studies under in vivo and in vitro conditions. Under in vivoconditions, BHUx signicantly reduced inammation in the carrageenan-induced rat paw oedemamodel of inammation, suggesting its anti-inammatory properties. In order to test the mechanismof action of BHUx, further in vitro studies were undertaken on cumene-hydroperoxide-induced lipidperoxidation in liver homogenate, LPS-induced NO production in peritoneal macrophages and onkey enzymes of arachidonic acid cascade, involved in the mediation of inammation. Under theconditions, BHUx showed concentration-dependent inhibition of CHP-induced lipid peroxidation inliver homogenate, suggesting its antioxidant properties. Similarly the potent anti-inammatory effectsof BHUx are evident by (a) preferential inhibition of COX-2 (IC 50 for COX-2 = 80 g/ml and IC 50for COX-1 = 169 g/ml), (b) low ratios in the IC 50 values of COX-2/COX-1 (0.47), (c) decreasedproduction of NO in LPS-induced peritoneal macrophages and (d) inhibition of 5-LOX (IC 50 = 795 g/ml). BHUx also showed a preference for 15-lipoxygenase (IC 50 = 44 g/ml), a key enzyme

implicated in LDL oxidation. These studies suggest that BHUx is acting mainly at three levels, i.e., asa potent natural antioxidant, by reduction of key inammatory mediators of arachidonic acid cascadeand by preventing 15-LOX-mediated LDL oxidations, to prevent atherosclerosis.

Key words : Atherosclerosis; inammation; cyclooxygenase; lipoxygenase; nitric oxide; antioxidant;herbal; Ayurveda.

To whom correspondence should be addressed. Tel.: (91-542) 236-6577 or 236-9659; Fax: (91-542) 236-6566; e-mail: [email protected]


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2 Y. B. Tripathi et al.

1. INTRODUCTION

Atherosclerosis is a progressive inammatory disease characterized by lipid inltra-tion in the wall of large arteries (atherosclerotic plaques) (Bailey and Butler, 1973;Rauch et al ., 2001). Platelet and leukocyte recruitment on endothelial cells con-stitutes an early mechanism of vascular inammatory damage and consequent ves-sel occlusion (Ross, 1999). Recently, it was shown that enzymes that metabolizearachidonic acid (lipoxygenases and cyclooxygenases) play a major role in the ini-tiation and promotion of atherosclerosis (Bhagat et al ., 1997; Vallance et al ., 1997;Biasucci et al ., 1999).

Cyclooxygenase (COX) converts arachidonic acid to prostaglandin H 2 (PGH 2)

which, in turn, is transformed, tissue specically, into a series of nal active prod-ucts like prostaglandins (E 2 , D2 , F2 ), thromboxanes (TXA 2 & B 2) and prostacy-clin (PGI 2). Two COX isoforms, cyclooxygenase-1 (COX-1) and cyclooxygenase-2(COX-2), have been identied. While COX-1 is constitutively expressed and main-tains homeostatic processes, COX-2 is the inducible isoform of cyclooxygenase,which plays a major role in the inammatory and other pathological conditions(Smith et al ., 1996).

Platelet aggregation is known to play a crucial role in thrombosis. The COX-1enzyme in platelets, is responsible for the formation of thromboxane A 2 (TxA 2),which initiates platelet aggregation. Because, inhibition of COX-1 in the gastricmucosa also prevent the formation of cytoprotective prostaglandins; therefore,the benecial anti-platelet effects of COX-1 inhibitors appear to be inseparablefrom its gastric side effects. COX-2 is expressed largely in circulating bloodleukocytes, vascular cells and macrophages that inltrate atherosclerotic plaques,which contribute directly to vascular disease and thrombus formation (Burleigh et al ., 2002).

Lipoxygenases (LOXs) constitute a heterogeneous family of lipid peroxidizingenzymes, capable of oxygenating polyunsaturated fatty acids to their correspondinghydroperoxy derivatives. In mammals, LOXs are classied with respect to theirpositional specicity of arachidonic acid oxygenation into 5-, 8-, 12- and 15-LOXs.Arachidonate 15-LOXs may be sub-classied into a reticulocyte-type (type-1) andan epidermis-type (type-2) enzyme.

Arachidonic acid metabolites of 5-LOX pathway are leukotrienes (LTs). As

they are expressed in diseased arteries, the roles of LTs in atherogenesis meritconsideration (Spanbroek and Habenicht, 2003; Spanbroek et al., 2003). 5-LOXcontributes importantly to the atherogenic process and reduced 5-LOX expressionis partly responsible for the resistance to atherosclerosis in mice (Meharabian et al. , 2002). Recently, 5-LOX gene was identied as an important contributor of atherosclerosis in mice and humans at different levels, such as lesion initiation,growth and cellular proliferation within the lesion, and/or destabilization of plaquesthat can lead to their rupture (Mehrabina et al. , 2002; Mehrabian and Allayee, 2003).The hydroperoxide product of 15-LOX, 15-HPETE, acts as an activator of the freeradical mediated non-enzymatic lipid peroxidation of LDL (Yamamoto, 1991).


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Anti-inammatory properies of BHUx 3

Thus, it can be suggested that a promising pharmacological approach to reducecardiovascular events associated with atherosclerosis as effectively as possibleshould include: (a) inhibition of COX-1 to prevent platelet TXA 2 formation;(b) inhibition of COX-2 to down regulate leukocyte activation and wide-spreadvascular inammation; (c) inhibition of 5-LOX to further and specically reduceleukocyte inammatory and thrombogenic potential; (d) inhibition of 15-LOX toprevent the lipid peroxidation of LDL; (e) inhibition of lipid peroxidation by theuse of antioxidants; (f) to raise the serum HDL levels; (g) to stabilize the existingplaque by inhibiting the factors responsible for its bursting.

The aim of this study was to investigate the effects of BHUx on rat paw model

of inammation, in vitro effects on lipid peroxidation, Degree of LPS induced NOproduction by the activated macrophages and on the enzymes of COX-1, COX-2,5-LOX and 15-LOX. BHUx was also compared with the commonly used COX andLOX inhibitors.

Thus, bearing in mind the multi-etiological factors for atherosclerosis, a combina-tion drug was formulated and named BHUx. It is a patented polyherbal formulation,consisting of the specic water-soluble fraction of ve medicinal plants (Tripathi et al ., 2002). These plants are Commiphora mukul (Tripathi et al ., 1988a,b), Ter-menalia arjuna (Tripathi et al ., 1989), Bosvelia serata (Kimmatkar et al ., 2003),Semicarpus anacardium (Tripathi and Singh, 2001) and Strychnos nuxvomica (Tri-pathi and Chaurasia, 1996) in a particular ratio. CaCO 3 (Shankha Bhasma) has beenadded to the nished product to reduce the gastric irritation, if any. These plants aretime tested and in clinical use in the Ayurvedic system of medicine for centuries(Pandey et al ., 1967). Several phytochemicals have been isolated form these plnats,from time to time and their pharmacological properties on different experimentalmodels have also been reported (Table 1). Thus, based on the basic information andlong clinical use for various claims, these plant extracts were combined in a specicratio and tried for the prevention of diet-induced atherosclerosis in rabbits. Usingthe techniques of HPLC and TLC nger printing the nished product has been stan-dardized to avoid batch-to-batch variation. These plants with key phytochemicalsmay target several signaling pathways and may bring benecial effects through asynergistic or additive approach. Recently, Moore and co-workers have shown thatZ-guggulsterone of Commiphora mukul acts through the FXR (Farnesoid X Recep-tors) in the liver to lower the raised cholesterol level in the blood (Urizar et al .,2002).

We have found that in diet-induced atherosclerosis model of rabbits BHUxreduces plaque formation, signicantly along with elevation in serum HDL levels,but without effects on other lipids in the blood (Tripathi et al ., 2002). However,the mechanism of action of BHUx is not clearly dened. Thus, the present work has been undertaken to evaluate the mechanism of action of BHUx and providescientic evidence for its anti-atherogenic effects.


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Table 1.List of phytochemicals and their pharmacological claims, isolated from different ingredients of BHUx

Component Plant Principal components Pharmacological effect

A Commiphoramukul

Oleoresin, diterpene hydro-carbon, diterpene alcohol[A1], Z-guggulsterone,E-guggulsterone [A2], gug-gulsterone VI, guggulsterol-I,guggulsterol-II and guggul-sterol-III, guggulsterol-

IVl, sesamin, camphorene,quercetin, quercetin-3- O-a -L-arabinoside, quercetin-3- O-b-D-galactoside, quercetin-3-O-a-L-rhamnoside, quercetin-3-O-b-D-glucuronide [A3],ellagic acid and pelargonidin-3,5-di- O-glucoside, sitosteroland stigmasterol, with 20 a -hydroxy-4-pregnen-3-one, 20 b-hydroxy-4-pregnen-3-one, 16 b-hydroxy-4,17-(20 z)-pregnadien-3-one and16a -hydroxy-4-pregnen-3-one

Anti-inammatory [A4], hy-polipidemic, thyroid stimulant[A5] demulcent, immune sys-tem stimulant, diuretic, carni-native, antispasmodic, emme-nagogue, astingent and antisep-tic

B Terminaliaarjuna

Triterpnoid saponins, anjanticacid, arjunone, arjunolone, leu-teolin [B1], gallic acid al-legic acid, oligomeric proan-thocyanidins (OPCs); phytos-terols, calcium, magnesium,zinc and copper [B2]

Wound healing [B3], ischemicheart disease [B4], cardiovas-cular disease [B5], myocar-dial neurosis [B6], angina,hypolipidemic, antimutagenic[B7], anti-oxidant [B8]

C Semecarpusanacardium

Anacardic acid [C1], avones(jeediavanone [C2], carpu-avanone [C3], semecarpua-vanone, gallaavanone [C4])

bhilawanol diene, phenolic glu-coside and anacardocides [C5],semecarpetine

Wound healing, hypolipidemic[C6], anti-inammatory [C7],anti-oxidant, anti-oedema,hemicarnia, sprain, anticancer

[C8], anti-tumour [C9],rheumatoid arthritis

D Boswelliaserata

Triterpene [D1] (acetyl-11keto-beta-boswellic acid(AKBA) [D2], keto-beta-boswellic acid (KBA) [D3])

Anti-inammatory [D4],analgestic [D5], antiarthritic,antiproliferative, chronic colitis[D6], ulcerative colitis [D7],Crohns disease [D8], bronchialasthma [D9], brain edemas


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Table 2.(Continued)

Component Plant Principal components Pharmacological effect

E Strychnosnux vomica

Brucine (C 23 H26 O4N2) [E1],strychnine (C 21 H22 O2N2)[E2], pseudobrucine (3-hydr-oxybrucine), pseudostrychnine(3-hydroxystrychnine), 4-hydr-oxy-3-methoxystrychnine,4-hydroxystrychnine, nor-macusine, O -methylmacusine,

-colubrine [E3], -colubrine3-hydroxy -colubrine,isostrychnine (novocine)mavacurine, alpha-colubene,vomicine, icajine [E4]

Gastric problem, antiviral[E5], anti-ulcer [E6], anaemia,asthma, bronchitis, consti-pation, diabetes, insomnia,cadiopolmus, nervous disorder,eczema [E7], rheumatism [E8]

References:[A1] Rucker (1972). [C8] Gothoskar and Ramadive (1971).[A2] Urizar et al . (2002). [C9] Chitnis et al . (1980).[A3] Dekebo A et al . (2002). [D1] Culioli et al . (2003).[A4] Arora et al. (1971). [D2] Park et al . (2002).[A5] Tripathi et al. (1984). [D3] Altmann et al . (2002).[B1] Pettit et al. (1996). [D4] Krohn et al . (2001).[B2] Shaila et al . (1998). [D5] Menon et al . (1971).

[B3] Mukherjee et al. (2003). [D6] Gupta et al . (2001).[B4] Khan et al. (2002). [D7] Gupta et al . (1997).[B5] Miller (1998). [D8] Ammon (2002).[B6] Sumitra et al . (2001). [D9] Gupta et al . (1998).[B7] Kaur et al . (2002). [E1] Malone (1992).[B8] Gupta et al . (2001). [E2] Baser et al . (1979).[C1] Paramashivappa et al . (2002). [E3] Bratati and Dutta (1988).[C2] Horowitz and Jurd (1961). [E4] Bratati and Dutta (1991).[C3] Murthy (1988). [E5] Singh and Gupta (1991).[C4] Rao et al . (1973). [E6] Panda and Panda (1993).[C5] Gil et al . (1995). [E7] Masilamani et al . (1981).[C6] Sharma et al . (1995). [E8] Choudhuri (1977).[C7] Satyavati et al . (1969).

2. MATERIALS AND METHODS

2.1. Materials

Arachidonic acid, N , N , N , N -tetramethyl- p-phenylenediamine (TMPD), lipopoly-saccharide and carrageenan were purchased from Sigma (St. Louis, MO, USA).Nordihydroguaretic acid (NDGA) and indomethacin were purchased from Cayman(Ann Arbor, MI, USA). Celecoxib was a generous gift from Unichem Laboratories,Mumbai, India. Phosphotungstic acid, thiobarbituric acid, trichloroacetic acid,acetic acid, sodium salicylate and EDTA were purchased from Central Drug House


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(India). Fetal bovine serum and RPMI 1640 antibiotics were purchased from HiMedia (Mumbai, India). Ascorbate, FeCl 3 , sodium tungstate, sodium nitrite andother reagents were of analytical grade. CF albino rats (body weight 125150 g)were purchased from the Central Animal Facility of Institute of Medical Sciences,Banarus Hindu University. They were maintained with rat pellets (Hindustan Lever,Bombay, India) and given tap water ad libitum . The protocol was approved by theInstitutional Animal Ethics Committee.

2.2. Preparation of BHUx

100 mg of BHUx was extracted into 10 ml of boiling water and centrifuged at10 000 rpm for 5 min. The supernatant was collected, ltered through 0.2 m ltersand used to study the effect on the activities of LOXs and cyclooxygenases.

2.3. Effects of BHUx: in vivo studies

2.3.1. Effect on carrageenan-induced paw oedema. Drug was given orally asper protocol described in Table 2. A single injection of 0.1 ml of 1% carrageenansolution (a polygalactose sulphate, extracted from fresh moss, which produceslocalized acute inammation) was injected locally in the hind paw of the rat underthe plantar aponeurosis. It produced acute inammatory oedema leading to markedincrease in volume of the limb. Control animals received the drug vehicle (10%

Tween-20 in water) and experimental animals received BHUx suspended in 10%Tween-20 in water at the dose of 400 mg/kg body weight up to 6 days. On day 7,the anti-inammatory response was monitored in terms of mercury displacement onhourly interval up to 4 h after the carrageenan injection. Percentage inhibition wascalculated as per the method described by Winter et al . (1962).

% Inhibition = (V c V t) 100 /V c ,

where V c and V t were average oedema volume of control and treated grouprespectively.

Table 2.Effect of BHUx on carrageenan-induced rat paw oedema model of inammation

S.N. Group Change in paw % Inhibition Weight of % Inhibitionoedema after 4 h cotton pellet(mmHg, n = 6) (mg, n = 6)

1 Sham control 1 .85 0.14 28 .4 1.82 BHUx (400 mg/kg 0 .7 0.12 62 19 .6 1.47 31

body weight, 6 days)

Anti-inammatory effects of BHUx were measured in terms of paw oedema volume and weightof cotton pellet, as described in the methodology. A single injection of 0.1 ml of 1% carrageenansolution was injected locally and the effect of BHUx was checked.


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2.3.2. Cotton pellet granuloma. For this experiment, dry sterilized cotton pellets(10 0.5 mg) were implanted subcutaneous in rats (125150 g body weight) anaes-thetized by intraperitoneal injection of sodium pentobarbitone (30 mg/kg body wt).A small incision was made in the midline of the dorsal surface and a pocket was cre-ated by inserting a blunt-ended pair of scissors into the incision, taking care that nobleeding occurred. Four cotton pellets (pre-weighed) were implanted (two on eachside of the midline incision) into each animal, and then the cut skin was stitchedunder antibiotics (Bailey, 1988). BHUx was orally given in the dose described inTable 2, daily for 7 days. The pellets were taken out on day 8, washed and driedat 60 C for 24 h. The granuloma weight obtained from control (where only drugvehicle was given for 7 days) and BHUx-treated animals were used to calculate per-centage inhibition in the increase of weight as described earlier (Chaurasia et al .,1995).

2.4. Effects of BHUx: in vitro studies

2.4.1. Antioxidant properties. Antioxidant properties of BHUx were evalu-ated by cumene hydroperoxide (CHP)-induced lipid peroxidation in rat liver ho-mogenates.

2.4.1.1. Preparation of rat liver homogenate. Liver from a healthy rat un-der diethyl-ether anaesthesia was perfused with phosphate-buffered saline (PBS),

through hepatic portal vein and then isolated. Its lobes were dried between blottingpapers (to remove excess of blood) and were cut into small pieces with a heavy-duty blade. They were then homogenized in glass-Teon homogenizing tube inphosphate buffer saline (pH 7.4) in cold condition. It was centrifuged at 2000 rpmfor 10 min and supernatant was diluted with PBS up to a nal concentration of protein of 0.81.5 mg/0.1 ml. Protein concentration was measured by using theFolin-phenol method (Lowry et al ., 1951).

2.4.1.2. Assay of lipid peroxidation as thiobarbituric acid reactive substances(TBARS). An aliquot of 3 ml liver homogenate (5%) was taken to each 35-mmglass Petri dishes. In the control plates, different volumes of vehicle were added andin experimental plates suspension of BHUx was added in different concentrations

(Tripathi and Chaurasia, 1996). The plates were mixed gently and pre-incubated for20 min at 37 C. Lipid peroxidation was induced by adding 1.5 mM CHP to eachplate and incubated for another 20 min and then 0.1 ml incubation mixture wastransferred to a tube containing 1.5 ml of 10% trichloroacetic acid (TCA). After10 min, tubes were centrifuged and the TCA-soluble fraction was kept safely todevelop the colour reaction. Absorbance was monitored at 535 nm as describedearlier (Okhawa et al ., 1979) with slight modication (Tripathi et al ., 1995).The values were calculated on comparison with the standard curve prepared byusing 1,1,3,3,-tetra-ethoxy-propane (TEP) and expressed as nmol malondialdehyde(MDA)/100 mg protein.


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2.4.2. Effect on NO production. In another set of experiments, the effect of BHUx on activated rat peritoneal macrophages, isolated from the normal healthyrats, was studied. To get the activated macrophages, 1 ml thioglycolate (4%)was injected intraperitoneally into rats and after 4 days macrophages were isolatedfrom the peritoneal uid, washed two times and cultured in 50-mm glass plates, asdescribed below. The plates were randomly divided in three different groups. GroupA was kept as normal, Group B was treated with 25 ng/ml LPS and Group C wasfurther divided into 5 sub-groups and treated with different concentrations of BHUxextract along with 25 ng/ml LPS. After 24 h of incubation, the culture mediumwas isolated to determine the NO level by Griess reagent (Ding et al ., 1998).In brief, 100- l aliquots were removed from conditioned medium and incubatedwith an equal volume of Griess reagent (1:2 of 1% sulphaphanilamide in 2.5%H3PO 4 and 0.1% naphthylethylene-diamine dihydrochloride) at room temperaturefor 10 min. The absorbance at 540 nm was taken to calculate the concentration of nitrite. NaNO 2 was used as the internal standard. The attached cells were carefullysubjected to the methylene viability test (Tripathi and Pandey, 2003).

2.4.3. Macrophage culture. An equal number of cells isolated from the peri-toneal uid were plated in 50-mm glass plates and kept for 2 h in a humidiedincubator maintained with 5% CO 2 at 37

C to attach the cells. Attached cells werenally washed three times with normal saline, and then cultured in RPMI-1640medium, supplemented with of 2.0 g/l NaHCO 3 , 100 IU/ml penicillin, 100 ug/ml

streptomycin, 20 g/ml gentamycin and 10% foetal calf serum (FCS) (Jessup et al.,1992).

2.4.4. Effects of BHUx on cyclooxygenases and lipoxygenases. Partially-puriedfractions of 5-LOX (Reddanna et al ., 1990), 15-LOX (Zschocke and Van Staned,2000), COX-1 and COX-2 (Reddy et al ., 2000) were employed for testing the invitro effects of BHUx.

2.4.4.1. Assay for cyclooxygenases. Enzymatic activity of COX-1 and COX-2was measured as described earlier (Solomon et al ., 2003) with slight modicationsusing a chromagenic assay based on the oxidation of N , N , N , N -tetramethyl- p-phenylenediamine (TMPD) during the reduction of PGG 2 to PGH 2. The assay

mixture in a nal volume of 1 ml contained Tris-HCl buffer (pH 8.0, 100 mM),hematin (15 M), EDTA (3 M), enzyme (COX-1 or COX-2, 100 g) and testcompound (BHUx/celecoxib/indomethacin at different concentrations in 12 l of buffer). The mixture was pre-incubated at 25 C for 15 min and then the reaction wasinitiated by the addition of arachidonic acid (100 M) and TMPD (120 M). Theenzyme activity was measured by estimating the initial velocity of TMPD oxidationfor the rst 25 s of the reaction, following the increase in absorbance at 603 nm. Alow rate of non-enzymatic oxidation observed in the absence of COX-1 and COX-2was subtracted from the experimental value while calculating the percent inhibition.The IC 50 values for these compounds were calculated.


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2.4.4.2. Lipoxygenase assay. A polarographic method was used to measure theenzyme activities with a Clarks oxygen electrode on Gilson model 5/6 oxygraphas per the method described earlier (Grossman et al ., 1968). A typical reactionmixture contained 1.6 ml of assay buffer (potassium phosphate buffer, pH 6.3 for5-LOX and pH 7.4 for 15-LOX) and 100 l of enzyme. The reaction was initiatedby addition of 10 l of arachidonic acid with 133 M nal concentration. Thereaction was allowed to proceed at 25 C and the rate of decrease in oxygen wastaken as a measure of enzyme activity. Enzyme activity is expressed as moloxygen consumed/min per mg protein. Assays were performed with addition of different concentrations of BHUx or LOX inhibitor (NDGA) to the reaction mixtureand IC 50 values were calculated (Tripathi et al ., 1995).

2.5. HPLC ngerprinting of BHUx

HPLC ngerprinting of BHUx was done as per the method described earlier(Tripathi et al ., 1989). BHUx was dissolved in HPLC grade water in a boiling waterbath. Then it was cooled and centrifuged at 12 000 g for 20 min. The supernatantwas saved and ltered through 0.2- m lter paper. 100 l of the above ltratewas injected into a RP-18 HPLC column and eluted isocratically by employingwater/acetonitrile (70:30, v/v) for 20 min. The eluate was monitored at a wavelengthof 254 nm.

3. RESULTS

3.1. Antioxidant properties of BHUx on cumene hydroperoxide (CHP) induced lipid peroxidation in rat liver homogenate

The results show concentration-dependent inhibition in the CHP-induced lipidperoxidation in the liver homogenate. The IC 50 for BHUx was calculated to be102 g/ml of liver homogenate (Table 3).

3.2. Anti-inammatory effects of BHUx on carrageenan-induced rat paw oedemaand granuloma pouch model

BHUx at a concentration of 400 mg/kg body weight showed inhibition in theoedema (62%), induction in the rat paw oedema model, and in the enhancementof the weight of cotton pellet (31%) in the granuloma pouch model (Table 2). Theresponse was statistically signicant.

3.3. Effect of BHUx on cyclooxygenase and lipoxygenase activity

BHUx showed dose-dependent inhibition of COX-1 in vitro as measured by TMPDassay and data were compared with indomethacin and celecoxib (Fig. 1). The IC 50values were calculated for the above compounds and data presented in Table 4. As


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Table 3.Antioxidant properties of BHUx on cumene hydroperoxide (CHP)-induced lipid peroxidation in ratliver homogenates

S.N. Group Lipid peroxidation * % Inhibition(nmol/100 mg protein)

1 Sham control 118 4.141a Sham control with CHP (1.5 mM) 576 6.42 CHP + BHUx ( g/ml of homogenate)2a 50 436 .7 7.2 242b 100 341 8.4 402c 150 228 7.8 60

2d 200 195 5.9 66* Lipid peroxidation was measured in terms of TBARS.

Figure 1. The inhibitory effect of BHUx (25200 g), indomethacin (110 g) and celecoxib (1

50 g) on COX-1 activity. The values expressed as % inhibition of COX-1 activity are mean SD of three independent observations.

shown in Table 4, the IC 50 value for BHUx was 169 g/ml, compared to 3.8 g/mlof indomethacin, a non-specic inhibitor, and 20.8 g/ml for celecoxib, a selectiveCOX-2 inhibitor. BHUx inhibited COX-2 with an IC 50 value of 80 g/ml, whereasindomethacin inhibited COX-2 at 23.15 g/ml and celecoxib at 3.75 g/ml (Fig. 2,Table 4). The COX-2/COX-1 ratio for BHUx is 0.47 and is comparable to 0.18 of celecoxib, a COX-2-specic inhibitor. With indomethacin, a non-specic inhibitorof cyclooxygenases, the COX-2/COX-1 ratio was 6.18. The effect of BHUx on


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Table 4.Comparative IC 50 values of BHUx and various standard inhibitors for cyclooxygenases and lipoxy-genases ( in vitro assay)

S.N. Enzyme BHUx NDGA Indomethacin Celecoxib( g/ml) ( g/ml) ( g/ml) ( g/ml)

1 COX-1 169 3.85 20.22 COX-2 80 24.4 3.753 COX-2/COX-1 0.47 6.18 0.184 5-LOX 795 7.5 5 15-LOX 44 23.5

In vitro effects of BHUx and other standard inhibitors were measured on cyclooxygenases andlipoxygenases and the IC 50 values determined.

Figure 2. The inhibitory effect of BHUx (25150 g), indomethacin (550 g) and celecoxib (110 g) on COX-2 activity. The values expressed as % inhibition of COX-2 activity are mean SD of three independent observations.

5-LOX and 15-LOX, in comparison with NDGA, a known inhibitor of LOXs, ispresented in Fig. 3 and Fig. 4, respectively. The IC 50 values were calculated andpresented in Table 4. As shown in Table 4, BHUx inhibited both 5- and 15-LOX,but with higher specicity towards 15-LOX. The IC 50 for 5-LOX is 795 g/ml andthat of 15-LOX is 44 g/ml.


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Figure 3. The inhibitory effect of BHUx (101000 g) and NDGA (2.525 g) on 5-LOX activity.The values expressed as % inhibition of 5-LOX activity are mean SD of three independentobservations.

Figure 4. The inhibitory effect of BHUx (10100 g) and NDGA (10100 g) on 15-LOX activity.The values expressed as % inhibition of 15-LOX activity are mean SD of three independentobservations.


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Table 5.Effect of BHUx extract on LPS-induced NO production by activated peritoneal macrophage cells

Parameter Group LPS (25 ng/ml) + BHUx (ng/ml)

Normal LPS 5 50 250 500 5000(25 ng/ml)

NO 10 .24 35.94 33.43 30.50 25.67 20.16 17.35 2.31 3.24a 2.513 2.512 2.236 1.596 3.044

MB 0.693 0.774 0.745 0.730 0.727 0.0.713 0.689 0.02 0.11 c 0.01a 0.01a 0.009 a 0.005 b 0.090

NO, nitric oxide production in terms of mol NO 2 / 3 106 macrophage cells. MB, Methylene

Blue uptake in terms of absorbance at 660 nm. Values are mean SD of eight different experiments.Statistical comparison was made with normal. P value: a P < 0.001, b P < 0.01, cP < 0.05.

Figure 5. HPLC ngerprint of BHUx water extract. The HPLC ngerprint shows 19 peaks ondifferent retention times.

3.4. Effect of BHUx on NO production

In vitro results indicate that the thioglycolate activated macrophages are hyper-sensitive to LPS and producing NO in the range of 3336 mol/3 106 cells,whereas macrophages isolated from normal animals produce NO in the range of 911 mol/3 106 cells under similar conditions. However, this NO production wassignicantly inhibited by the simultaneous and pre-incubation with BHUx extractin a concentration-dependent manner. This indicates the strong anti-inammatoryproperty of BHUx with an IC 50 value at 50 ng/ml (Table 5).


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4. DISCUSSION

Epidemiological and experimental studies have suggested an association betweenacute and chronic-inammation and risk of numerous pathological disorders, in-cluding cardiovascular disease (Vallance et al ., 1997). The changes in endothe-lial function may underlie this association. Mild systemic inammation impairsendothelium-dependent dilation in humans. Certain pro-inammatory cytokines(TNF- and Il-1 ) induce endothelial dysfunction in humans (Bhagat et al ., 1997).It is also evident that unstable angina is associated with inammation, which mightprecede the onset of the syndrome (Biasucci et al ., 1999).

Inammatory cells produce a highly complicated mixture of growth and differen-

tiation factors as well as biologically active arachidonic acid metabolites, includinglipid hydroperoxides, leukotrienes and prostanoids, produced via the lipoxygne-nase and cyclooxygenase pathways, respectively. Some of these arachidonic acidmetabolites, in particular leukotriene B 4 (LTB 4) and prostaglandin E 2 (PGE 2), areimportant inammatory mediators. Inhibition of biosynthesis of inammatory me-diators by blocking the activities of those enzymes would be an important treatmentof many inammatory disease states (Zshocke et al ., 2000).

Natural compounds, obtained from medicinal plants, have been used as traditionalremedies for hundreds of years (Pandey et al ., 1967). Many medicinal herbs arewidely used for treatment of various inammatory diseases. Recently, we haveshown that the anti-inammatory property of C-phycocyanin, a biliprotein fromSpirulina platensis , is due to selective inhibition of COX-2 (Reddy et al ., 2000).It was also shown to induce apoptosis in a mouse macrophage cell line (Bobbiliet al ., 2003) and chronic myeloid leukemia cell line (K562) (Subhashini et al .,2004). In the present study BHUx, which is a mixture of ve medicinally importantplant extracts (these individual plants have been in clinical use for centuries in theAyurvedic system of medicine) in a particular ratio, has shown a potent inhibitoryeffect against enzymes of arachidonic acid metabolism, along with antioxidantproperty that play major role in inammation.

BHUx has also shown signicant reduction in the aortic lesions in the atherogenic-diet-fed rabbits. The raised serum HDL and comparatively less response to the low-ering in triglyceride and cholesterol accompanied this reduction. Specic stainingof the histological section of aorta and coronary artery has shown the intactness of

the collagen cap on the plaque surface (Mehrabian et al ., 2002; Mehrabian and Al-layee, 2003). Inammation is known to induce endothelial dysfunction in humans,involving IL-1, and aspirin can prevent this effect (Kharbanda et al ., 2002). Thepreferential inhibition of COX-2 by BHUx, observed in the present study, could beresponsible for its anti-inammatory properties. The mean lesion area in the prox-imal aorta was shown to be decreased by 25% ( P = 0.02) and 37% ( P = 0.003)in mice receiving rofecoxib and indomethacin, respectively (Burleigh et al ., 2002).However, there was no signicant difference in serum cholesterol and triglyceridelevels, but small amount of collagen was present in the lesions. These data indicatethat inhibition of prostaglandin synthesis with a selective COX-2 inhibitor delays


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the progression of atherogenesis during fatty streak lesion. The results describedherewith BHUx, show the inhibition of inammation induced by carrageenan andalso in granuloma formation in rats, which indicates its net anti-inammatory prop-erty. The mechanism of its action could also be through its antioxidant propertybecause it inhibits the CHP-induced production of lipid peroxides.

The selective inhibition of COX-2 by rofecoxib or suppression of the geneencoding COX-2 resulted in the prevention of atherosclerotic lesion formationwithout any modication of serum lipids in LDL receptor decient mice, whichare fed on a lipid-enriched athrosclerotic diet (Pitt et al ., 2002). Celecoxib,another COX-2 inhibitor, was shown to improve endothelial function in patientswith coronary artery disease (Chenvard et al ., 2003). Inhibition of COX-2 wasshown to be particularly benecial in those patients with arthritis or other chronicinammatory diseases who have additional cardiovascular risk (Solomon et al .,2003). Furthermore, an intact platelet function in the presence of COX-2 inhibitorsmight reduce bleeding complications, which are associated with non-specic COXinhibitor treatment.

Another important cascade of COX-2 production is the activation of macrophagesby free radicals and oxidized LDL. This COX-2 not only causes inammation butalso induces the expression of matrix metalloproteins (MMPs), which destabilizethe atherosclerotic plaque. Therefore, COX-2 inhibitors in the physiological rangemay interfere with macrophage migration by reducing release and activation of MMPs, thereby stabilizing the plaques and avoid bursting (Wesley et al ., 1998).Together these data suggest that COX-2 inhibitors might reduce the inammatorycontribution to vascular damage and atherothrombosis and have the potentialadvantage over non-specic COX inhibitors with gastric side effects.

The IC 50 ratios of COX-2/CXO-1 provide a useful comparison of relative valuesfor a series of NSAIDs tested in the same system. However, this ratio for aparticular NSAID will vary according to whether it is measured using intact cells,cell homogenates, puried enzymes, or recombinant proteins expressed in bacterial,insect, or animal cells. Studies indicate that a high degree of in vitro biochemicalselectivity for COX-2 will be required in order to achieve effective functionalselectivity in vivo. The ratio demonstrates the relative selectivity of NSAIDstowards the two COX isoforms and low ratios indicate a preferential inhibition of

COX-2. In the present study the COX-2/COX-1 ratio of the IC 50 values calculatedfor BHUx in vitro with the partially-puried enzymes is 0.47, which is comparableto the COX-2-specic inhibitor celecoxib, with 0.18 as against 6.18 recorded forindomethacin, a non-specic COX inhibitor. Figure 1, shows the effect of celecoxibon COX-1 to be more potent than that of COX-2, but it is already reported thatthis agent is a known COX-2-selective inhibitor. Here celecoxib, which is aselective COX-2 inhibitor, has inhibited COX-1 with an IC 50 of 20.2 g/l, whereasindomethacin, which is a preferential COX-1 inhibitor, inhibited COX-1 with anIC 50 of 3.85 g/ml. However, the ratio of IC 50 of COX-2/COX-1 for indomethacinis 6.18, whereas that for celecoxib is 0.18, as shown in Table 4. This shows that


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the celecoxib is a selective COX-2 inhibitor. Since the inhibitory concentrationsof COX inhibitors vary from assay system to assay system and from laboratoryto laboratory, the IC 50 ratios of compounds are compared for studying the selectiveinhibitory properties of the compounds. Based on this logic, the selectivity of BHUxfor COX-2 has been proposed here.

The leukotrienes (LTs) formed by 5-LOX, which is expressed in leukocytesmainly, possess potent pro-inammatory activities and, thus, might be involvedin cardiovascular disease. The unstable LTA 4 generated in neutrophils by theactivity of 5-LOX is converted to LTB 4, a compound with potent chemo-attractantand pro-inammatory properties. The unstable LTA 4 is also transferred fromneutrophils to platelets and endothelial cells, which possess LTC

4synthase activity.

The formation of cysteinyl leukotrienes by cellcell interaction would then causecoronary contraction. Thus, inhibition of neutrophil function by inhibiting 5-LOXcould not only suppress the direct contribution of these cells to inammation, butalso downregulate the contribution of platelets and other interacting cells. Duringinammatory disease the arachidonic acid metabolism represents an importantaspect of platelet/polymorphonuclear leukocyte (PMNL) cross talk, relevant in thepathogenesis (Cerletti et al ., 1999). In vitro activated platelets signicantly increasePMNL leukotriene biosynthesis, and PMNLs increase platelet TxB 2 synthesis byproviding eachother with free arachidonic acid (Marcus et al ., 1982). Interestingly,PMNLs synthesize various mediators, which cause cellular injury by initiating lipidperoxidation, altering vascular permeability and activating vascular and circulatingcells. The 5-LOX pathway is abundantly expressed in arterial walls of patientsaficted with various lesion stages of atherosclerosis of the aorta and of coronaryand carotid arteries. 5-LOX is localized to macrophages, dendritic cells, foam cells,mast cells and neutrophilic granulocytes, and the number of 5-LOX expressing cellsmarkedly increased in advanced lesions. 5-LOX cascade-dependent inammatorycircuits, consisting of several leukocyte lineages and arterial wall cells, evolvewithin the blood vessel wall during critical stages of lesion development. They raisethe possibility that anti-leukotriene drugs may be an effective treatment regimen inlate-stage disease process (Spanbroek and Habenicht, 2003; Spanbroek et al ., 2003).Even though the IC 50 of BHUx towards 5-LOX is very high, regular usage of thismixture during therapy could help to maintain the therapeutic dose and inhibit the

enzyme.Apart from inhibition of 5-LOX and cyclooxygenase-2, BHUx inhibited 15-LOXwith relatively higher specicity. 15-LOX is thought to play the key step in theoxidation of phospholipid moiety of the LDL and inhibition of 15-LOX could bethe novel therapeutic approach for the management of atherosclerosis. The 12/15-LOX expressed in macrophages is capable of oxygenating linoleic acid, esteriedto cholesterol in the LDL particle, and thus this enzyme is presumed to initiate LDLoxidation (Zhu et al ., 2003). 12/15-LOX-gene disruption attenuates atherogenesisin LDL receptor-decient mice (George et al ., 2001). In the present study theinhibition of 15-LOX is comparable with that of the unspecic LOX inhibitor


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NDGA, and this inhibition will help to control the oxidation of phospholipid moietyof LDL, which undergoes oxidation under the effect of free radical attack mediatedby 15- and 5-LOX.

Despite signicant protection afforded by some non-steroidal anti-inammatorydrugs (NSAIDs) like aspirin in groups of patients with thrombotic cardiovasculardisease, many patients do not derive any clinical benet and might even experienceside effects (De Gaetano, 2001). The limited protection afforded by these drugsis explained by genetic variability in response to drug, differing inuences of concomitant vascular risk factors and their severity, such as hypertension, thepossibility that TxA 2-mediated platelet activation is crucially involved in a limited,but still dened, set of thrombotic events (De Gaetano, 2001; De Gaetano et al .,2002). In any case, the new anti-thrombotic approaches should not only reducethe risk of adverse reactions but also successfully treat patients who are resistant tothese drugs. Thus, BHUx, because of its multi-targeted action and being a naturalextract, could be a suitable candidate that could reduce the toxicities associated withcurrently available NSAIDs. Inhibition of COX-1, COX-2, 5-LOX and 15-LOX byBHUx could inhibit platelet TXA 2 formation, down regulate leukocyte activationand wide spread vascular inammation and reduce leukocyte inammatory andthrombogenic potential. Thus, BHUx is acting on mainly at two levels, one directlyas free radical scavenger and other at the inammatory mediators level to preventatherosclerosis.

HPLC ngerprint was consistent and this was used to avoid the batch-to-batch

variation during the experiment. The peaks show that BHUx has different com-pounds, which might be having different biological responses. This gives a leadfor the development of specic compounds for specic actions. For clinical useof BHUx as herbal medicine, BHUx, however, would be preferable because of itsholistic approach in action. It is true to especially for those diseases, which havemulti etiological factors, like atherosclerosis.

5. CONCLUSIONS

This study suggests that BHUx, a polyherbal formulation, possesses potent anti-inammatory and antioxidant activity. BHUx being a natural source, withoutany side effects, can be used to control atherosclerosis. Thus, the present studyprovides a mechanism and scientic evidence for the therapeutic potential of BHUx.Further studies, however, should be taken up to isolate and characterize the activecompounds of this mixture.

Acknowledgements

This work was supported by grants from the Department of Biotechnology, Govern-ment of India through a project at BHU, Varanasi, India. The authors are thankfulto Surya Pharmaceuticals, Varanasi, for preparing BHUx as per our specication.


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We are thankful to the administrative staff at the Department of Medicinal Chem-istry who allowed us to carry out the experiment. The CSIR fellowship granted toM. Mallikarjuna Reddy is gratefully acknowledged.

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