Anti-inflammatory properties of BHUx, a polyherbal formulation to prevent atherosclerosis

Inflammopharmacology Vol 12 No 2 pp 131ndash152 (2004) VSP 2004Also available online - wwwvsppubcom

Anti-inflammatory properties of BHUx a polyherbalformulation to prevent atherosclerosis

YAMINI B TRIPATHI 1lowast M MALLIKARJUNA REDDY 2 R S PANDEY 1J 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 UniversityVaranasi-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

AbstractmdashBHUx is a polyherbal formulation consisting of water-soluble fractions of five medicinalplants (Commiphora mukul Terminalia arjuna Boswellia serrata Semecarpus anacardium andStrychnos nux vomica) The present study was undertaken to evaluate its antioxidant and anti-inflammatory effects BHUx standardized by HPLC fingerprinting and filtered through 02 micromfilter paper was employed for different studies under in vivo and in vitro conditions Under in vivoconditions BHUx significantly reduced inflammation in the carrageenan-induced rat paw oedemamodel of inflammation suggesting its anti-inflammatory properties In order to test the mechanismof action of BHUx further in vitro studies were undertaken on cumene-hydroperoxide-induced lipidperoxidation (CHP) in liver homogenate LPS-induced NO production in peritoneal macrophages andon key enzymes of arachidonic acid cascade involved in the mediation of inflammation Under theconditions BHUx showed concentration-dependent inhibition of CHP-induced lipid peroxidation inliver homogenate suggesting its antioxidant properties Similarly the potent anti-inflammatory effectsof BHUx are evident by (a) preferential inhibition of COX-2 (IC50 for COX-2 = 80 microgml and IC50for COX-1 = 169 microgml) (b) low ratios in the IC50 values of COX-2COX-1 (047) (c) decreasedproduction of NO in LPS-induced peritoneal macrophages and (d) inhibition of 5-LOX (IC50 = 795microgml) BHUx also showed a preference for inhibiting 15-lipoxygenase (IC50 = 44 microgml) a keyenzyme implicated in LDL oxidation These studies suggest that BHUx is acting mainly at threelevels ie as a potent natural antioxidant by reduction of key inflammatory mediators of arachidonicacid cascade and by preventing 15-LOX-mediated LDL oxidations to prevent atherosclerosis

Key words Atherosclerosis inflammation cyclooxygenase lipoxygenase nitric oxide antioxidantherbal Ayurveda

lowastTo whom correspondence should be addressed Tel (91-542) 236-6577 Fax (91-542) 236-6566e-mail Yamini30sifycom

132 Y B Tripathi et al

1 INTRODUCTION

Atherosclerosis is a progressive inflammatory disease characterized by lipid infiltra-tion in the wall of large arteries (atherosclerotic plaques) (Bailey and Butler 1973Rauch et al 2001) Platelet and leukocyte recruitment on endothelial cells con-stitutes an early mechanism of vascular inflammatory 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 1997Biasucci et al 1999)

Cyclooxygenase (COX) converts arachidonic acid to prostaglandin H2 (PGH2)which in turn is transformed tissue specifically into a series of final active prod-ucts like prostaglandins (E2 D2 F2α) thromboxanes (TXA2 and B2) and prostacy-clin (PGI2) Two COX isoforms cyclooxygenase-1 (COX-1) and cyclooxygenase-2(COX-2) have been identified While COX-1 is constitutively expressed and main-tains homeostatic processes COX-2 is the inducible isoform of cyclooxygenasewhich plays a major role in the inflammatory 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 A2 (TxA2)which initiates platelet aggregation Because inhibition of COX-1 in the gastricmucosa also prevents the formation of cytoprotective prostaglandins thereforethe beneficial 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 infiltrate atherosclerotic plaqueswhich contribute directly to vascular disease and thrombus formation (Burleighet 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 classified with respect to theirpositional specificity of arachidonic acid oxygenation into 5- 8- 12- and 15-LOXsArachidonate 15-LOXs may be sub-classified into a reticulocyte-type (type-1) andan epidermis-type (type-2) enzyme

Arachidonic acid metabolites of 5-LOX pathway are leukotrienes (LTs) Asthey 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 al2002) Recently the 5-LOX gene was identified as an important contributor ofatherosclerosis in mice and humans at different levels such as lesion initiationgrowth and cellular proliferation within the lesion andor destabilization of plaquesthat can lead to their rupture (Mehrabian 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)

Anti-inflammatory properties of BHUx 133

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 TXA2 formation(b) inhibition of COX-2 to down regulate leukocyte activation and wide-spreadvascular inflammation (c) inhibition of 5-LOX to further and specifically reduceleukocyte inflammatory and thrombogenic potential (d) inhibition of 15-LOX toprevent the lipid peroxidation of LDL (e) inhibition of lipid peroxidation by the useof antioxidants (f) to raise the serum HDL levels and (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 modelof inflammation in vitro effects on lipid peroxidation degree of LPS-induced NOproduction by the activated macrophages and on the enzymes of COX-1 COX-25-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 formulationconsisting of the specific water-soluble fraction of five medicinal plants (Tripathiet al 2002) These plants are Commiphora mukul (Tripathi et al 1988ab) Termi-nalia arjuna (Tripathi et al 1989) Boswellia serrata (Kimmatkar et al 2003)Semecarpus anacardium (Tripathi and Singh 2001) and Strychnos nux vomica(Tripathi and Chaurasia 1996) in a particular ratio CaCO3 (Shankha Bhasma)has been added to the finished product to reduce the gastric irritation if any Theseplants are time tested and in clinical use in the Ayurvedic system of medicine forcenturies (Pandey et al 1967) Several phytochemicals have been isolated fromthese plants from time to time and their pharmacological properties on differentexperimental models have also been reported (Table 1) Thus based on the basicinformation and long clinical use for various claims these plant extracts were com-bined in a specific ratio and tried for the prevention of diet-induced atherosclerosisin rabbits Using the techniques of HPLC and TLC finger printing the finished prod-uct has been standardized to avoid batch-to-batch variation These plants with keyphytochemicals may target several signaling pathways and may bring beneficial ef-fects through a synergistic or additive approach Recently Moore and co-workershave shown that Z-guggulsterone of Commiphora mukul acts through the FXR(Farnesoid X Receptors) in the liver to lower the raised cholesterol level in the blood(Urizar et al 2002)

We have found that in the diet-induced atherosclerosis model of rabbits BHUxreduces plaque formation significantly along with elevation in serum HDL levelsbut without effects on other lipids in the blood (Tripathi et al 2002) Howeverthe mechanism of action of BHUx is not clearly defined Thus the present workhas been undertaken to evaluate the mechanism of action of BHUx and providescientific evidence for its anti-atherogenic effects


Table 1List 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-guggulsteroneE-guggulsterone [A2] gug-gulsterone VI guggulsterol-Iguggulsterol-II and guggul-sterol-III guggulsterol-IVl sesamin camphorenequercetin 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-35-di-O-glucoside sitosteroland stigmasterol with 20a-hydroxy-4-pregnen-3-one 20b-hydroxy-4-pregnen-3-one 16b-hydroxy-417-(20z)-pregnadien-3-one and16a-hydroxy-4-pregnen-3-one

Anti-inflammatory [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 magnesiumzinc and copper [B2]

Wound healing [B3] ischaemicheart disease [B4] cardiovas-cular disease [B5] myocar-dial neurosis [B6] anginahypolipidemic antimutagenic[B7] anti-oxidant [B8]

C Semecarpusanacardium

Anacardic acid [C1] flavones(jeediflavanone [C2] carpu-flavanone [C3] semecarpufla-vanone gallaflavanone [C4])bhilawanol diene phenolic glu-coside and anacardocides [C5]semecarpetine

Wound healing hypolipidemic[C6] anti-inflammatory [C7]anti-oxidant anti-oedemahemicarnia sprain anticancer[C8] anti-tumour [C9]rheumatoid arthritis

D Boswelliaserrata

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

Anti-inflammatory [D4]analgestic [D5] antiarthriticantiproliferative chronic colitis[D6] ulcerative colitis [D7]Crohnrsquos disease [D8] bronchialasthma [D9] brain edemas


Table 1(Continued)


E Strychnosnux vomica

Brucine (C23H26O4N2) [E1]strychnine (C21H22O2N2)[E2] pseudobrucine (3-hydr-oxybrucine) pseudostrychnine(3-hydroxystrychnine) 4-hydr-oxy-3-methoxystrychnine4-hydroxystrychnine nor-macusine Oprime-methylmacusineβ-colubrine [E3] α-colubrine3-hydroxy β-colubrineisostrychnine (novocine)mavacurine alpha-colubenevomicine icajine [E4]

Gastric problems antiviral[E5] anti-ulcer [E6] anaemiaasthma bronchitis consti-pation diabetes insomniacadiopolmus nervous disordereczema [E7] rheumatism [E8]

References[A1] Rucker (1972) [C8] Gothoskar and Ramadive (1971)[A2] Urizar et al (2002) [C9] Chitnis et al (1980)[A3] Dekebo 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

21 Materials

Arachidonic acid NNN primeN prime-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 LaboratoriesMumbai India Phosphotungstic acid thiobarbituric acid trichloroacetic acidacetic acid sodium salicylate and EDTA were purchased from Central Drug House


(India) Fetal bovine serum RPMI 1640 and antibiotics were purchased from HiMedia (Mumbai India) Ascorbate FeCl3 sodium tungstate sodium nitrite andother reagents were of analytical grade CF albino rats (body weight 125ndash150 g)were purchased from the Central Animal Facility of Institute of Medical SciencesBanarus Hindu University They were maintained with rat pellets (Hindustan LeverBombay India) and given tap water ad libitum The protocol was approved by theInstitutional Animal Ethics Committee

22 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 filtered through 02 microm filtersand used to study the effect on the activities of LOXs and cyclooxygenases

23 Effects of BHUx in vivo studies

231 Effect on carrageenan-induced paw oedema Drug was given orally asper protocol described in Table 2 A single injection of 01 ml of 1 carrageenansolution (a polygalactose sulphate extracted from fresh moss which produceslocalized acute inflammation) was injected locally in the hind paw of the rat underthe plantar aponeurosis It produced acute inflammatory oedema leading to markedincrease in volume of the limb Control animals received the drug vehicle (10Tween-20 in water) and experimental animals received BHUx suspended in 10Tween-20 in water at the dose of 400 mgkg body weight up to 6 days On day 7the anti-inflammatory 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 = (Vc minus Vt) times 100Vc

where Vc and Vt were average oedema volume of control and treated grouprespectively

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

SN Group Change in paw Inhibition Weight of Inhibitionoedema after 4 h cotton pellet(mmHg n = 6) (mg n = 6)

1 Sham control 185 plusmn 014 284 plusmn 182 BHUx (400 mgkg 07 plusmn 012 62 196 plusmn 147 31

body weight 6 days)

Anti-inflammatory effects of BHUx were measured in terms of paw oedema volume and weightof cotton pellet as described in the methodology A single injection of 01 ml of 1 carrageenansolution was injected locally and the effect of BHUx was checked


232 Cotton pellet granuloma For this experiment dry sterilized cotton pel-lets (10 plusmn 05 mg) were implanted subcutaneously in rats (125ndash150 g body weight)anaesthetized by intraperitoneal injection of sodium pentobarbitone(30 mgkg body wt) A small incision was made in the midline of the dorsal surfaceand a pocket was created by inserting a blunt-ended pair of scissors into the inci-sion taking care that no bleeding occurred Four cotton pellets (pre-weighed) wereimplanted (two on each side of the midline incision) into each animal and then thecut skin was stitched under antibiotics (Bailey 1988) BHUx was orally given inthe dose described in Table 2 daily for 6 days The pellets were taken out on day 7washed and dried at 60C for 24 h The granuloma weight obtained from control(where only drug vehicle was given for 6 days) and BHUx-treated animals wereused to calculate percentage inhibition in the increase of weight as described earlier(Chaurasia et al 1995)

24 Effects of BHUx in vitro studies

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

2411 Preparation of rat liver homogenate Liver from a healthy rat un-der diethyl-ether anaesthesia was perfused with phosphate-buffered saline (PBS)through the hepatic portal vein and then isolated Its lobes were dried betweenblotting papers (to remove excess of blood) and were cut into small pieces witha heavy-duty blade They were then homogenized in glass-Teflon homogenizingtube in phosphate buffer saline (pH 74) in cold condition It was centrifuged at2000 rpm for 10 min and supernatant was diluted with PBS up to a final concentra-tion of protein of 08ndash15 mg01 ml Protein concentration was measured by usingthe Folin-phenol method (Lowry et al 1951)

2412 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 37C Lipid peroxidation was induced by adding 15 mM CHP to eachplate and incubated for another 20 min and then 01 ml incubation mixture wastransferred to a tube containing 15 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 modification (Tripathi et al 1995)The values were calculated on comparison with the standard curve prepared byusing 1133-tetra-ethoxy-propane (TEP) and expressed as nmol malondialdehyde(MDA)100 mg protein


242 Effect on NO production In another set of experiments the effect ofBHUx 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 fluid 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 ngml LPS and Group C wasfurther divided into 5 sub-groups and treated with different concentrations of BHUxextract along with 25 ngml 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-microl aliquots were removed from conditioned medium and incubatedwith an equal volume of Griess reagent (12 of 1 sulphaphanilamide in 25H3PO4 and 01 naphthylethylene-diamine dihydrochloride) at room temperaturefor 10 min The absorbance at 540 nm was taken to calculate the concentration ofnitrite NaNO2 was used as the internal standard The attached cells were carefullysubjected to the methylene viability test (Tripathi and Pandey 2003)

243 Macrophage culture An equal number of cells isolated from the peri-toneal fluid were plated in 50-mm glass plates and kept for 2 h in a humidifiedincubator maintained with 5 CO2 at 37C to attach the cells Attached cellswere finally washed three times with normal saline and then cultured in RPMI-1640 medium supplemented with 20 gl NaHCO3 100 IUml penicillin 100 ugmlstreptomycin 20 microgml gentamycin and 10 foetal calf serum (FCS) (Jessup et al1992)

244 Effects of BHUx on cyclooxygenases and lipoxygenases Partially-purifiedfractions of 5-LOX (Reddanna et al 1990) 15-LOX (Zschocke and Van Staned2000) COX-1 and COX-2 (Reddy et al 2000) were employed for testing the invitro effects of BHUx

2441 Assay for cyclooxygenases Enzymatic activity of COX-1 and COX-2was measured as described earlier (Solomon et al 2003) with slight modificationsusing a chromogenic assay based on the oxidation of NNN primeN prime-tetramethyl-p-phenylenediamine (TMPD) during the reduction of PGG2 to PGH2 The assaymixture in a final volume of 1 ml contained Tris-HCl buffer (pH 80 100 mM)hematin (15 microM) EDTA (3 microM) enzyme (COX-1 or COX-2 100 microg) and testcompound (BHUxcelecoxibindomethacin at different concentrations in 12 microl ofbuffer) The mixture was pre-incubated at 25C for 15 min and then the reaction wasinitiated by the addition of arachidonic acid (100 microM) and TMPD (120 microM) Theenzyme activity was measured by estimating the initial velocity of TMPD oxidationfor the first 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 inhibitionThe IC50 values for these compounds were calculated


2442 Lipoxygenase assay A polarographic method was used to measure theenzyme activities with a Clarkrsquos oxygen electrode on Gilson model 56 oxygraphas per the method described earlier (Grossman et al 1968) A typical reactionmixture contained 16 ml of assay buffer (potassium phosphate buffer pH 63 for5-LOX and pH 74 for 15-LOX) and 100 microl of enzyme The reaction was initiatedby addition of 10 microl of arachidonic acid with 133 microM final concentration Thereaction was allowed to proceed at 25C and the rate of decrease in oxygen wastaken as a measure of enzyme activity Enzyme activity is expressed as micromoloxygen consumedmin per mg protein Assays were performed with addition ofdifferent concentrations of BHUx or LOX inhibitor (NDGA) to the reaction mixtureand IC50 values were calculated (Tripathi et al 1995)

25 HPLC fingerprinting of BHUx

HPLC fingerprinting 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 times g for 20 min The supernatantwas saved and filtered through 02-microm filter paper 100 microl of the above filtratewas injected into a RP-18 HPLC column and eluted isocratically by employingwateracetonitrile (7030 vv) for 20 min The eluate was monitored at a wavelengthof 254 nm

3 RESULTS

31 Anti-inflammatory effects of BHUx on carrageenan-induced rat paw oedemaand granuloma pouch model

BHUx at a concentration of 400 mgkg 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 significant

32 Antioxidant properties of BHUx on cumene hydroperoxide (CHP) inducedlipid peroxidation in rat liver homogenate

The results show concentration-dependent inhibition in the CHP-induced lipidperoxidation in the liver homogenate The IC50 for BHUx was calculated to be102 microgml of liver homogenate (Table 3)

33 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 IC50

values were calculated for the above compounds and data are presented in Table 4


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

SN Group Lipid peroxidation Inhibition(nmol100 mg protein)

1 Sham control 118 plusmn 4141a Sham control with CHP (15 mM) 576 plusmn 642 CHP + BHUx (microgml of homogenate)2a 50 4367 plusmn 72 242b 100 341 plusmn 84 402c 150 228 plusmn 78 602d 200 195 plusmn 59 66

Lipid peroxidation was measured in terms of TBARS

Figure 1 The inhibitory effect of BHUx (25ndash200 microg) indomethacin (1ndash10 microg) and celecoxib (1ndash50 microg) on COX-1 activity The values expressed as inhibition of COX-1 activity are mean plusmn SD ofthree independent observations

As shown in Table 4 the IC50 value for BHUx was 169 microgml compared to 38microgml of indomethacin a non-specific inhibitor and 208 microgml for celecoxib aselective COX-2 inhibitor BHUx inhibited COX-2 with an IC50 value of 80 microgmlwhereas indomethacin inhibited COX-2 at 2315 microgml and celecoxib at 375 microgml(Fig 2 Table 4) The COX-2COX-1 ratio for BHUx is 047 and is comparable to018 of celecoxib a COX-2-specific inhibitor With indomethacin a non-specificinhibitor of cyclooxygenases the COX-2COX-1 ratio was 618 The effect of


Table 4Comparative IC50 values of BHUx and various standard inhibitors for cyclooxygenases and lipoxy-genases (in vitro assay)

SN Enzyme BHUx NDGA Indomethacin Celecoxib(microgml) (microgml) (microgml) (microgml)

1 COX-1 169 mdash 385 2022 COX-2 80 mdash 244 3753 COX-2COX-1 047 mdash 618 0184 5-LOX 795 75 mdash mdash5 15-LOX 44 235 mdash mdash

In vitro effects of BHUx and other standard inhibitors were measured on cyclooxygenases andlipoxygenases and the IC50 values determined


BHUx on 5-LOX and 15-LOX in comparison with NDGA a known inhibitor ofLOXs is presented in Fig 3 and Fig 4 respectively The IC50 values werecalculated and presented in Table 4 As shown in Table 4 BHUx inhibited both5- and 15-LOX but with higher specificity towards 15-LOX The IC50 for 5-LOXis 795 microgml and that of 15-LOX is 44 microgml


Figure 3 The inhibitory effect of BHUx (10ndash1000 microg) and NDGA (25ndash25 microg) on 5-LOX activityThe values expressed as inhibition of 5-LOX activity are mean plusmn SD of three independentobservations



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

Parameter Group LPS (25 ngml) + BHUx (ngml)

Normal LPS 5 50 250 500 5000(25 ngml)

NO 1024 3594 3343 3050 2567 2016 1735plusmn 231 plusmn 324a plusmn 2513 plusmn 2512 plusmn 2236 plusmn 1596 plusmn 3044

MB 0693 0774 0745 0730 0727 0713 0689plusmn 002 plusmn 011c plusmn 001a plusmn 001a plusmn 0009a plusmn 0005b plusmn 0090

NO nitric oxide production in terms of micromol NOminus2 3 times 106 macrophage cells MB Methylene

Blue uptake in terms of absorbance at 660 nm Values are mean plusmn SD of eight different experimentsStatistical comparison of the LPS group was made with normal and of the BHUx experimental groupwith the LPS group P value aP lt 0001 bP lt 001 cP lt 005

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

34 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 33ndash36 micromol3 times 106 cellswhereas macrophages isolated from normal animals produce NO in the range of 9ndash11 micromol3 times 106 cells under similar conditions However this NO production wassignificantly inhibited by the simultaneous and pre-incubation with BHUx extractin a concentration-dependent manner This indicates the strong anti-inflammatoryproperty of BHUx with an IC50 value at 50 ngml (Table 5)


4 DISCUSSION

Epidemiological and experimental studies have suggested an association betweenacute and chronic-inflammation 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 inflammation impairsendothelium-dependent dilation in humans Certain pro-inflammatory cytokines(TNF-α and IL-1β) induce endothelial dysfunction in humans (Bhagat et al 1997)It is also evident that unstable angina is associated with inflammation which mightprecede the onset of the syndrome (Biasucci et al 1999)

Inflammatory cells produce a highly complicated mixture of growth and differ-entiation factors as well as biologically active arachidonic acid metabolites includ-ing lipid hydroperoxides leukotrienes and prostanoids produced via the lipoxyge-nase and cyclooxygenase pathways respectively Some of these arachidonic acidmetabolites in particular leukotriene B4 (LTB4) and prostaglandin E2 (PGE2) areimportant inflammatory mediators Inhibition of biosynthesis of inflammatory me-diators by blocking the activities of those enzymes would be an important treatmentof many inflammatory 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 inflammatory diseases Recently we haveshown that the anti-inflammatory 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 al2004) In the present study BHUx which is a mixture of five 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 inflammation

BHUx has also shown significant 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 Specific stainingof the histological section of aorta and coronary artery has shown the intactness ofthe collagen cap on the plaque surface (Mehrabian et al 2002 Mehrabian and Al-layee 2003) Inflammation is known to induce endothelial dysfunction in humansinvolving 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-inflammatory properties The mean lesion area in the prox-imal aorta was shown to be decreased by 25 (P = 002) and 37 (P = 0003)in mice receiving rofecoxib and indomethacin respectively (Burleigh et al 2002)However there was no significant 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


the progression of atherogenesis during fatty streak lesion The results describedherewith BHUx show the inhibition of inflammation induced by carrageenan andalso in granuloma formation in rats which indicates its net anti-inflammatory 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 modification of serum lipids in LDL receptor deficient mice whichare fed on a lipid-enriched athrosclerotic diet (Pitt et al 2002) Celecoxibanother 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 beneficial in those patients with arthritis or other chronicinflammatory diseases who have additional cardiovascular risk (Solomon et al2003) Furthermore an intact platelet function in the presence of COX-2 inhibitorsmight reduce bleeding complications which are associated with non-specific 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 inflammation 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 ofMMPs thereby stabilizing the plaques and avoid bursting (Wesley et al 1998)Together these data suggest that COX-2 inhibitors might reduce the inflammatorycontribution to vascular damage and atherothrombosis and have the potentialadvantage over non-specific COX inhibitors with gastric side effects

The IC50 ratios of COX-2COX-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 cellscell homogenates purified enzymes or recombinant proteins expressed in bacterialinsect 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 ofCOX-2 In the present study the COX-2COX-1 ratio of the IC50 values calculatedfor BHUx in vitro with the partially-purified enzymes is 047 which is comparableto the COX-2-specific inhibitor celecoxib with 018 as against 618 recorded forindomethacin a non-specific 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 IC50 of 202 microgl whereasindomethacin which is a preferential COX-1 inhibitor inhibited COX-1 with anIC50 of 385 microgml However the ratio of IC50 of COX-2COX-1 for indomethacinis 618 whereas that for celecoxib is 018 as shown in Table 4 This shows that


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 IC50 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-inflammatory activities and thus might be involvedin cardiovascular disease The unstable LTA4 generated in neutrophils by theactivity of 5-LOX is converted to LTB4 a compound with potent chemo-attractantand pro-inflammatory properties The unstable LTA4 is also transferred fromneutrophils to platelets and endothelial cells which possess LTC4 synthase activityThe formation of cysteinyl leukotrienes by cellndashcell interaction would then causecoronary contraction Thus inhibition of neutrophil function by inhibiting 5-LOXcould not only suppress the direct contribution of these cells to inflammation butalso downregulate the contribution of platelets and other interacting cells Duringinflammatory disease the arachidonic acid metabolism represents an importantaspect of plateletpolymorphonuclear leukocyte (PMNL) cross talk relevant in thepathogenesis (Cerletti et al 1999) In vitro activated platelets significantly increasePMNL leukotriene biosynthesis and PMNLs increase platelet TxB2 synthesis byproviding eachother with free arachidonic acid (Marcus et al 1982) InterestinglyPMNLs 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 patientsafflicted with various lesion stages of atherosclerosis of the aorta and of coronaryand carotid arteries 5-LOX is localized to macrophages dendritic cells foam cellsmast cells and neutrophilic granulocytes and the number of 5-LOX expressing cellsmarkedly increased in advanced lesions 5-LOX cascade-dependent inflammatorycircuits 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 IC50 of BHUx towards 5-LOX is very high regular usage of thismixture during therapy could help to maintain the therapeutic dose and inhibit theenzyme

Apart from inhibition of 5-LOX and cyclooxygenase-2 BHUx inhibited 15-LOXwith relatively higher concentration for inhibiting effect 15-LOX is thought to playthe key step in the oxidation of phospholipid moiety of the LDL and inhibition of15-LOX could be the novel therapeutic approach for the management of atheroscle-rosis The 1215-LOX expressed in macrophages is capable of oxygenating linoleicacid esterified to cholesterol in the LDL particle and thus this enzyme is presumedto initiate LDL oxidation (Zhu et al 2003) 1215-LOX-gene disruption attenuatesatherogenesis in LDL receptor-deficient mice (George et al 2001) In the presentstudy the inhibition of 15-LOX is comparable with that of the unspecific LOX in-


hibitor NDGA and this inhibition will help to control the oxidation of phospholipidmoiety of LDL which undergoes oxidation under the effect of free radical attackmediated by 15- and 5-LOX

Despite significant protection afforded by some non-steroidal anti-inflammatorydrugs (NSAIDs) like aspirin in groups of patients with thrombotic cardiovasculardisease many patients do not derive any clinical benefit and might even experienceside effects (De Gaetano 2001) The limited protection afforded by these drugsis explained by genetic variability in response to drug differing influences ofconcomitant vascular risk factors and their severity such as hypertension thepossibility that TxA2-mediated platelet activation is crucially involved in a limitedbut still defined set of thrombotic events (De Gaetano 2001 De Gaetano et al2002) 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 TXA2 formation down regulate leukocyte activationand wide spread vascular inflammation and reduce leukocyte inflammatory andthrombogenic potential Thus BHUx is acting on mainly at two levels one directlyas free radical scavenger and other at the inflammatory mediators level to preventatherosclerosis

HPLC fingerprint (Fig 5) was consistent and this was used to avoid the batch-to-batch variation during the experiment The peaks show that BHUx has differentcompounds which might be having different biological responses This gives alead for the development of specific compounds for specific actions For clinicaluse of BHUx as herbal medicine BHUx however would be preferable because ofits holistic 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-inflammatory and antioxidant activity BHUx being a natural source withoutany side effects can be used to control atherosclerosis Thus the present studyprovides a mechanism and scientific evidence for the therapeutic potential of BHUxFurther 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 specification


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

REFERENCES

Altmann A Fischer F Schubert-Zsilavecz M et al (2002) Boswellic acids activate p42MAPK andp38 MAPK and stimulate Ca2+ mobilization Biochem Biophys Res Commun 290 185ndash190

Ammon H P (2002) Boswellic acids (components of frankincense) as the active principle intreatment of chronic inflammatory diseases Wien Med Wochenschr 152 373ndash378

Arora R B Kapoor V Gupta S K et al (1971) Isolation of a crystalline steroidal compoundfrom Commiphora mukul and its anti-inflammatory activity Ind J Exp Biol 9 403ndash404

Bailey J M and Butler J (1973) Anti-inflammatory drugs in experimental atherosclerosis I relativepotencies for inhibiting plaque formation Atherosclerosis 17 517ndash522

Bailey P J (1988) Sponge implants as models Methods Enzymol 162 327ndash334Baser K H C Bisset N G and Hylands P J (1979) Protostrychnine a new alkaloid from

Strychnos nux-vomica Phytochemistry 18 512ndash514Bhagat K and Vallance P (1997) Inflammatory cytokines impair endothelium dependent dilation in

human veins in vivo Circulation 96 3042ndash3047Biasucci L M Liuzzo G and Fantuzzi G (1999) Increasing levels of interleukin (IL)-I Ra and

IL-6 during the first 2 days of hospitalization in unstable angina are associated with increased riskof in-hospital coronary events Circulation 99 2079ndash2084

Bobbili V V Parthasarathi A Mubarak Ali A et al (2003) Phycocyanin mediated apoptosis inAK-5 tumor cells involves down-regulation of Bcl-2 and generation of ROS Mol Cancer Ther 21165ndash1170

Bratati D and Dutta P C (1988) Alkaloid of Strychnos mix-vomica flower Planta Med 54 363Bratati D and Dutta P C (1991) Alkaloid in floral parts of Strychnos mix-vomica Planta Med 57

19ndash20Burleigh M E Babaev V R Oates J A et al (2002) Cyclooxygenase-2 promotes early

atherosclerotic lesion formation in LDL receptor-deficient mice Circulation 105 1816ndash1823Cerletti C Evangelista V and de Gaetano G (1999) P-selectin-beta 2-integrin cross talk a

molecular mechanism for polymorphonumclear leukocyte recruitment at the site of vasculardamage Thromb Haemost 82 787ndash793

Chaurasia S Tripathi P and Tripathi Y B (1995) Antioxidant and anti-inflammatory property ofSandhika A compound herbal drug Ind J Exp Biol 33 428ndash432

Chenevard R Huumlrlimann D Beacutechir M et al (2003) Selective COX-2 inhibition improvesendothelial function in coronary artery disease Circulation 107 405ndash409

Chitnis M P Bhatia K G Pathak M K et al (1980) Anti-tumour activity of the extract ofSemecarpus anacardium L nuts in experimental tumour models Ind J Exp Biol 18 6ndash8

Choudhuri R C (1977) Role of some indigenous drugs in sandhigata vata Rheumatism 13 10ndash15Cornicelli J A and Trivedi B K (1999) 15-Lipoxygenase and its inhibition A novel therapeutic

target for vascular diseases Curr Pharm Design 5 11ndash20Culioli G Mathe C Archier P et al (2003) A lupane triterpene from frankincense (Boswellia sp

Burseraceae) Phytochemistry 62 537ndash541De Gaetano G (2001) Aspirin and the prevention of ischemic heart disease A Socratic dialogue

between a cardiologist a clinical pharmacologist and an expert of blood platelets Ital Heart J 2582ndash588


De Gaetano G et al (2002) Pharmacogenetics as a new antiplatelet strategy in Platelets inThrombotic and Non Thrombotic Disorders Gresele P Page C P Fuster V and VermylenJ (Eds) pp 964ndash977 Cambridge University Press Cambridge

Dekebo A Dagne E and Sterner O (2002) Furanosesquiterpenes from Commiphora sphaerocarpaand related adulterants of true myrrh Fitoterapia 73 48ndash55

Ding A H Nathan C F and Stuehr D J (1998) Release of reactive nitrogen intermediatesand reactive oxygen intermediates from mouse peritoneal macrophages comparison of activatingcytokines and evidence for independent production J Immunol 141 2407ndash2412

George J Afek A Shaish A et al (2001) 1215-Lipoxygenase gene disruption attenuatesatherogenesis in LDL receptor-deficient mice Circulation 104 1646ndash1650

Gil R R Lin L Cordell G A et al (1995) Anacardoside from the seeds of Semecarpusanacardium Phytochemistry 39 405ndash407

Gothoskar S V and Ranadive K J (1971) Anticancer screening of SAN-AB An extract of markingnut Semicarpus anacardium Ind J Exp Biol 9 372ndash375

Grossman S Ben Aziz A Budowski P et al (1968) Enzymic oxidation of carotene and linoleateby alfalfa Extraction and separation of active reactions Phytochemistry 8 2287ndash2293

Gupta I Parihar A Malhotra P et al (1997) Effects of Boswellia serrata gum resin in patientswith ulcerative colitis Eur J Med Res 2 37ndash43

Gupta I Gupta V Parihar A et al (1998) Effects of Boswellia serrata gum resin in patients withbronchial asthma results of a double-blind placebo-controlled 6-week clinical study Eur J MedRes 3 511ndash514

Gupta I Parihar A Malhotra P et al (2001) Effects of gum resin of Boswellia serrata in patientswith chronic colitis Planta Med 67 391ndash395

Gupta R Singhal S et al (2001) Antioxidant and hypocholesterolaemic effects of Terminaliaarjuna tree-bark powder a randomised placebo-controlled trials J Ass Phys Ind 49 231ndash235

Horowitz R M and Jurd L (1961) Spectral studies on flavonoid compounds II Isoflavones andflavanones J Org Chem 26 2446ndash2449

Jessup W Mander E L and Dean R T (1992) The intercellular storage and turnover ofapolipoprotein B of oxidized LDL in macrophages Biochim Biophys Acta 1126 167ndash177

Kaur K Arora S Kumar S et al (2002) Modulatory effect of phenolic fractions of Terminaliaarjuna on the mutagenicity in Ames assay J Environ Pathol Toxicol Oncol 21 45ndash56

Khan M T Lampronti I Martello D et al (2002) Identification of pyrogallol as an antiprolifera-tive compound present in extracts from the medicinal plant Emblica officinalis effects on in vitrocell growth of human tumor cell lines Int J Oncol 21 187ndash192

Kharbanda R K Walton B Allen M et al (2002) Prevention of inflammation-inducedendothelial dysfunction a novel vasculo-protective action of aspirin Circulation 105 2600ndash2604

Kimmatkar N Thawani V Hingorani L et al (2003) Efficasy and tolerability of Boswellia serrataextract in treatment of osteoarthitis of knee mdash a randomized double blind placebo controlled trialPhytomedicine 10 3ndash7

Krohn K Rao M S Raman N V et al (2001) High-performance thin layer chromatographicanalysis of anti-inflammatory triterpenoids from Boswellia serrata Roxb Phytochem Anal 12374ndash376

Lowry O H Rosebrough N J Farr A L et al (1951) Protein determination using folin-ciocalteaureagent J Biol Chem 193 438ndash448

Malone M H (1992) Brucine lethality in mice J Ethnopharmacol 35 295ndash297Marcus A J Broekman M J Safier L B et al (1982) Formation of leukotrienes and other

hydroxy acids during platelet neutrophil interactions in vitro Biochem Biophys Res Commun109 130ndash137

Masilamani G Ali S and Subbulakshmi V (1981) Study of karappan (eczema) J Res AyurvedaSiddha 2 109ndash121


Mehrabian M and Allayee H (2003) 5-lipoxygenase and atherosclerosis Curr Opin Lipidol 14447ndash457

Mehrabian M Allayee H Wong J et al (2002) Identification of 5-lipoxygenase as a major genecontributing to atherosclerosis susceptibility in mice Circ Res 91 120ndash126

Menon M K and Kar A (1971) Analgesic and psychopharmacological effects of the gum resin ofBoswellia serrata Planta Med 19 333ndash341

Miller A L (1998) Botanical influences on cardiovascular disease Altern Med Rev 3 422ndash431Mukherjee P K Mukherjee K Rajesh Kumar M et al (2003) Evaluation of wound healing

activity of some herbal formulations Phytother Res 17 265ndash268Murthy S S N (1988) Semecarpetin a biflavanone from Semecarpus anacardium Phytochemistry

27 3020ndash3022Okhawa H Ohishi N and Yagi K (1979) Assay for lipid peroxides in animal tissues by

thiobarbituric reaction Anal Biochem 95 351ndash358Qiao J-H Tripathi J Mishra N K et al (1997) Role of macrophage colony-stimulating factor in

atherosclerosis mdash studies of osteopetrotic mice Am J Pathol 150 1678ndash1699Panda P K and Panda D P (1993) Antiulcer activity of nux vomica and its comparison with

cimetidine in shay rat Ind Drugs 30 53ndash56Pandey G S and Chunekar K C (1967) In Bhavaprakash Bhava prakash nighantu pp 139ndash141

Chaukhambha Vidya Bhawan VaranasiPandey S Sharma M Chaturvedi P et al (1994) Protective effect of Rubia Cardifolia on lipid

peroxide formation in isolated rat liver homogenate Ind J Exp Biol 32 180ndash183Paramashivappa R Phani Kumar P Subba Rao P V et al (2002) Synthesis of sildenafil analogues

from anacardic acid and their phosphodiesterase-5 inhibition J Agric Food Chem 18 7709ndash7713

Park Y S Lee J H Harwalkar J A et al (2002) Acetyl-11-keto-beta-boswellic acid (AKBA) iscytotoxic for meningioma cells and inhibits phosphorylation of the extracellular-signal regulatedkinase 1 and 2 Adv Exp Med Biol 507 387ndash393

Pettit G R Hoard M S Doubek D L et al (1996) Antineoplastic agents 338 The cancer cellgrowth inhibitory Constituents of Terminalia arjuna (Combretaceae) J Ethnopharmacol 53 57ndash63

Pitt B Pepine C and Willerson J T (2002) Cyclooxygenase-2 inhibition and cardiovascularevents Circulation 106 167ndash169

Pratico D Tillmann C Zhang Z B et al (2001) Acceleration of atherogenesis by COX-1dependent prostanoid formation in low-density lipoprotein receptor knockout mice Proc NatlAcad Sci USA 98 3358ndash3363

Rao N S P Row L R and Brown R T (1973) Phenolic constituents of Semecarpus anacardiumPhytochemistry 12 671ndash675

Rauch U Osende J I Fuster V et al (2001) Thrombus formation on atherosclerotic plaquespathogenesis and clinical consequences Ann Intern Med 134 224ndash238

Reddanna P Whelan J Maddipati K R et al (1990) Purification of arachidonate 5-lipoxygenasefrom potato tubers Methods Enzymol 187 268ndash277

Reddy C M Bhat V B Kiranmai G et al (2000) Selective inhibition of cyclooxygenase-2by C-phycocyanin a biliprotein from Spirulina platensis Biochem Biophys Res Commun 277599ndash603

Ross R (1999) Atherosclerosis an inflammatory disease New Engl J Med 340 115ndash126Rucker G (1972) Monocyclic diterpenes from Indian gugul resin (Commiphora mukul) Arch

Pharm 305 486ndash493Satyavati G V Prasad G V Das P K et al (1969) Anti-inflammatory activity of Semecarpus

anacardium Linn mdash A preliminary study Ind J Physiol Pharmacol 13 37ndash45


Sendobry S M Cornicelli J A Welch K et al (1997) Attenuation of diet-induced atheroscle-rosis in rabbits with a highly selective 15-lipoxygenase inhibitor lacking significant antioxidantproperties Br J Pharmacol 120 1199ndash1206

Shaila H P Udupa S L Udupa A L et al (1998) Hypolipidemic activity of three indigenousdrugs in experimentally induced atherosclerosis Int J Cardiol 67 119ndash124

Sharma A Mathur R and Dixit V P (1995) Hypocholesterolemic activity of nutshell extract ofSemecarpus anacardium (Bhilawa) in cholesterol fed rabbits Ind J Exp Biol 33 444ndash448

Singh L M and Gupta G (1991) Research on antiviral efficacy on homeopathic drugs againstanimal viruses Homeopathic Heritage 16 113ndash117

Smith W L Garavito R M and Dewitt D L (1996) Prostaglandin endoperoxide H synthases(cyclooxygenase-)-1 and -2 J Biol Chem 271 33157ndash33160

Solomon D H Karlson E W Rimm E B et al (2003) Cardiovascular morbidity and mortalityin women diagnosed with rheumatoid arthritis Circulation 107 1303ndash1307

Spanbroek R and Habenicht A J (2003) The potential role of antileukotriene drugs in atheroscle-rosis Drug News Perspect 16 485ndash489

Spanbroek R Grabner R Lotzer K et al (2003) Expanding expression of the 5-lipoxygenasepathway within the arterial wall during human atherogenesis Proc Natl Acad Sci USA 1001238ndash1243

Subhashini J Mahipal S V K Reddy M C et al (2004) Molecular mechanisms involved inC-phycocyanin induced apoptosis in human chronic myeloid leukemia cell line K562 BiochemPharmacol in press

Sumitra M Manikandan P Kumar D A et al (2001) Experimental myocardial necrosis inrats role of arjunolic acid on platelet aggregation coagulation and antioxidant status Mol CellBiochem 224 135ndash142

Tripathi Y B and Chaurasia S (1996) Effect of S nuxvomica alcoholic extract on lipid peroxidationin rat liver Int J Pharmacol 34 295ndash299

Tripathi Y B and Pandey R S (2003) Semecarpus anacardium L nuts inhibit lipopolysaccharideinduced NO production in rat macrophages along with its hypolipidemic property Ind J ExpBiol in press

Tripathi Y B and Singh A V (2001) Effect of Semecarpus anacardium nuts on lipid peroxidationInd J Exp Biol 39 798ndash801

Tripathi Y B Malhotra O P and Tripathi S N (1984) Thyroid stimulating action of Z-guggulsterone obtained from Commiphora mukul Planta Med 1 78ndash80

Tripathi Y B Tripathi V P and Tripathi P (1989) Effect of T arjuna-extract on KCl-inducedcontraction on rat vas deferens Phytother Res 13 162ndash164

Tripathi Y B Tripathi P Reddy M V R et al (1988a) Effect of Semicarpus anacardium on cellcycle of DU-145 cells Phytomedicine 5 383ndash388

Tripathi Y B Tripathi P Malhotra O P et al (1988b) Thyroid stimulatory action of gugguls-terone mechanism of action Planta Med 4 271ndash276

Tripathi Y B Sharma M Shukla S et al (1995) Rubia cordifolia inhibits potato-lipoxygenasesInd J Exp Biol 33 109ndash112

Tripathi Y B Singh B K Pandey R S et al (2002) Anti atherogenic role of BHUx a patentpolyherbal formulation 15th Annual conference of Indian Society for Atherosclerosis ResearchTirupati 2002 (abstract)

Urizar N L Liverman A B Dodds D T et al (2002) A natural product that lowers cholesterolas an antagonist ligand for FXR Science 296 1703ndash1706

Vallance P Collier J and Bhagat K (1997) Infection inflammation and infarction does acuteendothelial dysfunction provide a link Lancet 349 1391ndash1392

Wesley R B Meng X Godin D et al (1998) Extracellular matrix modulates macrophagefunctions characteristic to atheroma collagen type I enhances acquisition of resident macrophagetraits by human peripheral blood monocytes in vitro Arterioscler Thromb Vasc Biol 18 432ndash440


Winter C A Risley E A and Nuss G W (1962) Carragenan induced oedema in hind paw of therat as an assay for anti-inflammatory drugs Proc Soc Exp Biol Med 111 544ndash547

Yamamoto S (1991) ldquoEnzymaticrdquo lipid peroxidation Reactions of mammalian lipoxygenases FreeRadic Biol Med 10 149ndash159

Zhu H Takahashi Y Xu W et al (2003) Low density lipoprotein receptor-related protein-mediated membrane translocation of 1215-lipoxygenase is required for oxidation of low densitylipoprotein by macrophages J Biol Chem 278 13350ndash13355

Zschocke S and Van Staned J (2000) Cryptocarya species substitute plants for Ocotea bullataA pharmacological investigation in terms of cyclooxygenase-1 and -2 inhibition J Ethnopharma-col 71 473ndash478


1 INTRODUCTION

Atherosclerosis is a progressive inflammatory disease characterized by lipid infiltra-tion in the wall of large arteries (atherosclerotic plaques) (Bailey and Butler 1973Rauch et al 2001) Platelet and leukocyte recruitment on endothelial cells con-stitutes an early mechanism of vascular inflammatory 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 1997Biasucci et al 1999)

Cyclooxygenase (COX) converts arachidonic acid to prostaglandin H2 (PGH2)which in turn is transformed tissue specifically into a series of final active prod-ucts like prostaglandins (E2 D2 F2α) thromboxanes (TXA2 and B2) and prostacy-clin (PGI2) Two COX isoforms cyclooxygenase-1 (COX-1) and cyclooxygenase-2(COX-2) have been identified While COX-1 is constitutively expressed and main-tains homeostatic processes COX-2 is the inducible isoform of cyclooxygenasewhich plays a major role in the inflammatory 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 A2 (TxA2)which initiates platelet aggregation Because inhibition of COX-1 in the gastricmucosa also prevents the formation of cytoprotective prostaglandins thereforethe beneficial 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 infiltrate atherosclerotic plaqueswhich contribute directly to vascular disease and thrombus formation (Burleighet 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 classified with respect to theirpositional specificity of arachidonic acid oxygenation into 5- 8- 12- and 15-LOXsArachidonate 15-LOXs may be sub-classified into a reticulocyte-type (type-1) andan epidermis-type (type-2) enzyme

Arachidonic acid metabolites of 5-LOX pathway are leukotrienes (LTs) Asthey 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 al2002) Recently the 5-LOX gene was identified as an important contributor ofatherosclerosis in mice and humans at different levels such as lesion initiationgrowth and cellular proliferation within the lesion andor destabilization of plaquesthat can lead to their rupture (Mehrabian 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)









A Commiphoramukul



B Terminaliaarjuna






D Boswelliaserrata




Table 1(Continued)







21 Materials













body weight 6 days)

















3 RESULTS




























Normal LPS 5 50 250 500 5000(25 ngml)









4 DISCUSSION


















5 CONCLUSIONS


Acknowledgements




REFERENCES








































































































A Commiphoramukul



B Terminaliaarjuna






D Boswelliaserrata




Table 1(Continued)







21 Materials













body weight 6 days)

















3 RESULTS




























Normal LPS 5 50 250 500 5000(25 ngml)









4 DISCUSSION


















5 CONCLUSIONS


Acknowledgements




REFERENCES

































































































Table 1(Continued)







21 Materials













body weight 6 days)

















3 RESULTS




























Normal LPS 5 50 250 500 5000(25 ngml)









4 DISCUSSION


















5 CONCLUSIONS


Acknowledgements




REFERENCES











































































































body weight 6 days)

















3 RESULTS




























Normal LPS 5 50 250 500 5000(25 ngml)









4 DISCUSSION


















5 CONCLUSIONS


Acknowledgements




REFERENCES















































































































3 RESULTS




























Normal LPS 5 50 250 500 5000(25 ngml)









4 DISCUSSION


















5 CONCLUSIONS


Acknowledgements




REFERENCES




















































































































Normal LPS 5 50 250 500 5000(25 ngml)









4 DISCUSSION


















5 CONCLUSIONS


Acknowledgements




REFERENCES

































































































4 DISCUSSION


















5 CONCLUSIONS


Acknowledgements




REFERENCES













































































































5 CONCLUSIONS


Acknowledgements




REFERENCES


































































































REFERENCES
































































































Anti-inflammatory properties of BHUx, a polyherbal formulation to prevent atherosclerosis

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Transcript of Anti-inflammatory properties of BHUx, a polyherbal formulation to prevent atherosclerosis