Anti at Hero Sclerotic

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Inflammopharmacology, Vol. 00, No. 0, pp. 1 – 22 (2004)

 VSP 2004.
Also available online - www.vsppub.com
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Anti-inflammatory properies of BHUx, a polyherbal
formulation to prevent atherosclerosis
YAMINI B. TRIPATHI 1,∗ , M. MALLIKARJUNA REDDY 2 , R. S. PANDEY 1 ,
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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
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Received 8 January 2004; revised 28 March 2004; accepted 29 March 2004
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Abstract—BHUx is a polyherbal formulation consisting of water-soluble fractions of five medicinal

plants (Commiphoran mukul, Terminalia arjuna, Bosvelia serata, Semicarpus anacardium and
Strychnos nuxvomica). The present study was undertaken to evaluate its antioxidant and anti-
inflammatory effects. BHUx, standardized by HPLC fingerprinting and filtered through 0.2 µm
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filter paper, was employed for different studies under in vivo and in vitro conditions. Under in vivo
conditions, BHUx significantly reduced inflammation in the carrageenan-induced rat paw oedema
model of inflammation, suggesting its anti-inflammatory properties. In order to test the mechanism
of action of BHUx, further in vitro studies were undertaken on cumene-hydroperoxide-induced lipid
peroxidation in liver homogenate, LPS-induced NO production in peritoneal macrophages and on
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key enzymes of arachidonic acid cascade, involved in the mediation of inflammation. Under the
conditions, BHUx showed concentration-dependent inhibition of CHP-induced lipid peroxidation in
liver homogenate, suggesting its antioxidant properties. Similarly the potent anti-inflammatory effects
of BHUx are evident by (a) preferential inhibition of COX-2 (IC50 for COX-2 = 80 µg/ml and IC50
for COX-1 = 169 µg/ml), (b) low ratios in the IC50 values of COX-2/COX-1 (0.47), (c) decreased
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production of NO in LPS-induced peritoneal macrophages and (d) inhibition of 5-LOX (IC50 = 795
µg/ml). BHUx also showed a preference for 15-lipoxygenase (IC50 = 44 µg/ml), a key enzyme
implicated in LDL oxidation. These studies suggest that BHUx is acting mainly at three levels, i.e., as
a potent natural antioxidant, by reduction of key inflammatory mediators of arachidonic acid cascade
and by preventing 15-LOX-mediated LDL oxidations, to prevent atherosclerosis.
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Key words: Atherosclerosis; inflammation; 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: Yamini30@sify.com

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2 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, 1973;
Rauch et al., 2001). Platelet and leukocyte recruitment on endothelial cells con-
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stitutes an early mechanism of vascular inflammatory damage and consequent ves-
sel occlusion (Ross, 1999). Recently, it was shown that enzymes that metabolize
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arachidonic 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 H2 (PGH2 )
which, in turn, is transformed, tissue specifically, into a series of final active prod-
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ucts like prostaglandins (E2 , D2 , F2α ), thromboxanes (TXA2 & 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 cyclooxygenase,
which plays a major role in the inflammatory and other pathological conditions
(Smith et al., 1996).
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Platelet aggregation is known to play a crucial role in thrombosis. The COX-1
enzyme in platelets, is responsible for the formation of thromboxane A2 (TxA2 ),
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which initiates platelet aggregation. Because, inhibition of COX-1 in the gastric

mucosa also prevent the formation of cytoprotective prostaglandins; therefore,
the beneficial anti-platelet effects of COX-1 inhibitors appear to be inseparable
from its gastric side effects. COX-2 is expressed largely in circulating blood
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leukocytes, vascular cells and macrophages that infiltrate atherosclerotic plaques,

which contribute directly to vascular disease and thrombus formation (Burleigh et
al., 2002).
Lipoxygenases (LOXs) constitute a heterogeneous family of lipid peroxidizing
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enzymes, capable of oxygenating polyunsaturated fatty acids to their corresponding

hydroperoxy derivatives. In mammals, LOXs are classified with respect to their
positional specificity of arachidonic acid oxygenation into 5-, 8-, 12- and 15-LOXs.
Arachidonate 15-LOXs may be sub-classified into a reticulocyte-type (type-1) and
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an 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 merit
consideration (Spanbroek and Habenicht, 2003; Spanbroek et al., 2003). 5-LOX
contributes importantly to the atherogenic process and reduced 5-LOX expression
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is partly responsible for the resistance to atherosclerosis in mice (Meharabian et

al., 2002). Recently, 5-LOX gene was identified 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 plaques
that 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 free
radical mediated non-enzymatic lipid peroxidation of LDL (Yamamoto, 1991).
Anti-inflammatory properies of BHUx 3
Thus, it can be suggested that a promising pharmacological approach to reduce

cardiovascular events associated with atherosclerosis as effectively as possible
should include: (a) inhibition of COX-1 to prevent platelet TXA2 formation;
(b) inhibition of COX-2 to down regulate leukocyte activation and wide-spread
vascular inflammation; (c) inhibition of 5-LOX to further and specifically reduce
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leukocyte inflammatory and thrombogenic potential; (d) inhibition of 15-LOX to
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prevent the lipid peroxidation of LDL; (e) inhibition of lipid peroxidation by the
use of antioxidants; (f) to raise the serum HDL levels; (g) to stabilize the existing
plaque 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 inflammation, in vitro effects on lipid peroxidation, Degree of LPS induced NO
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production 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 and
LOX 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,
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consisting of the specific water-soluble fraction of five medicinal plants (Tripathi et
al., 2002). These plants are Commiphora mukul (Tripathi et al., 1988a,b), Ter-
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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. CaCO3 (Shankha Bhasma) has been
added to the finished product to reduce the gastric irritation, if any. These plants are
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time 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 experimental
models have also been reported (Table 1). Thus, based on the basic information and
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long clinical use for various claims, these plant extracts were combined in a specific
ratio and tried for the prevention of diet-induced atherosclerosis in rabbits. Using
the techniques of HPLC and TLC finger printing the finished product has been stan-
dardized to avoid batch-to-batch variation. These plants with key phytochemicals
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may target several signaling pathways and may bring beneficial effects through a
synergistic or additive approach. Recently, Moore and co-workers have shown that
Z-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.,
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2002).
We have found that in diet-induced atherosclerosis model of rabbits BHUx
reduces plaque formation, significantly 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 defined. Thus, the present work
has been undertaken to evaluate the mechanism of action of BHUx and provide
scientific evidence for its anti-atherogenic effects.
Table 1.
List of phytochemicals and their pharmacological claims, isolated from different ingredients of BHUx
Component Plant Principal components Pharmacological effect
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A Commiphora Oleoresin, diterpene hydro- Anti-inflammatory [A4], hy-
mukul carbon, diterpene alcohol polipidemic, thyroid stimulant
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[A1], Z-guggulsterone, [A5] demulcent, immune sys-
E-guggulsterone [A2], gug- tem stimulant, diuretic, carni-
gulsterone VI, guggulsterol-I, native, antispasmodic, emme-
guggulsterol-II and guggul- nagogue, astingent and antisep-
sterol-III, guggulsterol- tic
IVl, sesamin, camphorene,
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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, sitosterol
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and stigmasterol, with 20a-
hydroxy-4-pregnen-3-one, 20b-
hydroxy-4-pregnen-
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3-one, 16b-hydroxy-4,17-
(20z)-pregnadien-3-one and
16a-hydroxy-4-pregnen-3-one
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B Terminalia Triterpnoid saponins, anjantic Wound healing [B3], ischemic

arjuna acid, arjunone, arjunolone, leu- heart disease [B4], cardiovas-
teolin [B1], gallic acid al- cular disease [B5], myocar-
legic acid, oligomeric proan- dial neurosis [B6], angina,
thocyanidins (OPCs); phytos- hypolipidemic, antimutagenic
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terols, calcium, magnesium, [B7], anti-oxidant [B8]

zinc and copper [B2]
C Semecarpus Anacardic acid [C1], flavones Wound healing, hypolipidemic

anacardium (jeediflavanone [C2], carpu- [C6], anti-inflammatory [C7],
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flavanone [C3], semecarpufla- anti-oxidant, anti-oedema,

vanone, gallaflavanone [C4]) hemicarnia, sprain, anticancer
bhilawanol diene, phenolic glu- [C8], anti-tumour [C9],
coside and anacardocides [C5], rheumatoid arthritis
semecarpetine
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D Boswellia Triterpene [D1] (acetyl-11 Anti-inflammatory [D4],

serata keto-beta-boswellic acid analgestic [D5], antiarthritic,
(AKBA) [D2], keto-beta- antiproliferative, chronic colitis
boswellic acid (KBA) [D3]) [D6], ulcerative colitis [D7],
Crohn’s disease [D8], bronchial
asthma [D9], brain edemas
Table 2.
(Continued)
Component Plant Principal components Pharmacological effect

E Strychnos Brucine (C23 H26 O4 N2 ) [E1], Gastric problem, antiviral
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nux vomica strychnine (C21 H22 O2 N2 ) [E5], anti-ulcer [E6], anaemia,
[E2], pseudobrucine (3-hydr- asthma, bronchitis, consti-
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oxybrucine), pseudostrychnine pation, diabetes, insomnia,
(3-hydroxystrychnine), 4-hydr- cadiopolmus, nervous disorder,
oxy-3-methoxystrychnine, eczema [E7], rheumatism [E8]
4-hydroxystrychnine, nor-
macusine, O -methylmacusine,
β-colubrine [E3], α-colubrine
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3-hydroxy β-colubrine,
isostrychnine (novocine)
mavacurine, alpha-colubene,
vomicine, icajine [E4]
References:
[A1] Rucker (1972). [C8] Gothoskar and Ramadive (1971).
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[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).
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[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).
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[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).
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[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).
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[C7] Satyavati et al. (1969).
2. MATERIALS AND METHODS
2.1. Materials
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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
(India). Fetal bovine serum and RPMI 1640 antibiotics were purchased from Hi
Media (Mumbai, India). Ascorbate, FeCl3 , sodium tungstate, sodium nitrite and
other reagents were of analytical grade. CF albino rats (body weight 125–150 g)
were purchased from the Central Animal Facility of Institute of Medical Sciences,
Banarus Hindu University. They were maintained with rat pellets (Hindustan Lever,
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Bombay, India) and given tap water ad libitum. The protocol was approved by the
Institutional Animal Ethics Committee.
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2.2. Preparation of BHUx
100 mg of BHUx was extracted into 10 ml of boiling water and centrifuged at
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10 000 rpm for 5 min. The supernatant was collected, filtered through 0.2 µm filters
and 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 as
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per protocol described in Table 2. A single injection of 0.1 ml of 1% carrageenan
solution (a polygalactose sulphate, extracted from fresh moss, which produces
localized acute inflammation) was injected locally in the hind paw of the rat under
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the plantar aponeurosis. It produced acute inflammatory oedema leading to marked

increase 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,
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the anti-inflammatory response was monitored in terms of mercury displacement on

hourly interval up to 4 h after the carrageenan injection. Percentage inhibition was
calculated as per the method described by Winter et al. (1962).
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% Inhibition = (Vc − Vt ) × 100/Vc ,

where Vc and Vt were average oedema volume of control and treated group
respectively.
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Table 2.
Effect of BHUx on carrageenan-induced rat paw oedema model of inflammation
S.N. Group Change in paw % Inhibition Weight of % Inhibition

oedema after 4 h cotton pellet
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(mmHg, n = 6) (mg, n = 6)
1 Sham control 1.85 ± 0.14 28.4 ± 1.8
2 BHUx (400 mg/kg 0.7 ± 0.12 62 19.6 ± 1.47 31
body weight, 6 days)
Anti-inflammatory effects of BHUx were measured in terms of paw oedema volume and weight
of cotton pellet, as described in the methodology. A single injection of 0.1 ml of 1% carrageenan
solution was injected locally and the effect of BHUx was checked.
2.3.2. Cotton pellet granuloma. For this experiment, dry sterilized cotton pellets
(10 ± 0.5 mg) were implanted subcutaneous in rats (125–150 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 no
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bleeding occurred. Four cotton pellets (pre-weighed) were implanted (two on each
side of the midline incision) into each animal, and then the cut skin was stitched
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under antibiotics (Bailey, 1988). BHUx was orally given in the dose described in
Table 2, daily for 7 days. The pellets were taken out on day 8, washed and dried
at 60◦ C for 24 h. The granuloma weight obtained from control (where only drug
vehicle was given for 7 days) and BHUx-treated animals were used to calculate per-
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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-
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ated by cumene hydroperoxide (CHP)-induced lipid peroxidation in rat liver ho-
mogenates.
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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 blotting
papers (to remove excess of blood) and were cut into small pieces with a heavy-
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duty blade. They were then homogenized in glass-Teflon homogenizing tube in

phosphate buffer saline (pH 7.4) in cold condition. It was centrifuged at 2000 rpm
for 10 min and supernatant was diluted with PBS up to a final concentration of
protein of 0.8–1.5 mg/0.1 ml. Protein concentration was measured by using the
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Folin-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-mm
glass Petri dishes. In the control plates, different volumes of vehicle were added and
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in experimental plates suspension of BHUx was added in different concentrations

(Tripathi and Chaurasia, 1996). The plates were mixed gently and pre-incubated for
20 min at 37◦ C. Lipid peroxidation was induced by adding 1.5 mM CHP to each
plate and incubated for another 20 min and then 0.1 ml incubation mixture was
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transferred to a tube containing 1.5 ml of 10% trichloroacetic acid (TCA). After

10 min, tubes were centrifuged and the TCA-soluble fraction was kept safely to
develop the colour reaction. Absorbance was monitored at 535 nm as described
earlier (Okhawa et al., 1979) with slight modification (Tripathi et al., 1995).
The values were calculated on comparison with the standard curve prepared by
using 1,1,3,3,-tetra-ethoxy-propane (TEP) and expressed as nmol malondialdehyde
(MDA)/100 mg protein.
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 healthy
rats, was studied. To get the activated macrophages, 1 ml thioglycolate (4%)
was injected intraperitoneally into rats and after 4 days macrophages were isolated
from the peritoneal fluid, washed two times and cultured in 50-mm glass plates, as
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described below. The plates were randomly divided in three different groups. Group
A was kept as normal, Group B was treated with 25 ng/ml LPS and Group C was
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further divided into 5 sub-groups and treated with different concentrations of BHUx
extract along with 25 ng/ml LPS. After 24 h of incubation, the culture medium
was isolated to determine the NO level by Griess reagent (Ding et al., 1998).
In brief, 100-µl aliquots were removed from conditioned medium and incubated
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with an equal volume of Griess reagent (1:2 of 1% sulphaphanilamide in 2.5%
H3 PO4 and 0.1% naphthylethylene-diamine dihydrochloride) at room temperature
for 10 min. The absorbance at 540 nm was taken to calculate the concentration of
nitrite. NaNO2 was used as the internal standard. The attached cells were carefully
subjected to the methylene viability test (Tripathi and Pandey, 2003).
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2.4.3. 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 humidified
incubator maintained with 5% CO2 at 37◦ C to attach the cells. Attached cells were
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finally washed three times with normal saline, and then cultured in RPMI-1640
medium, supplemented with of 2.0 g/l NaHCO3 , 100 IU/ml penicillin, 100 ug/ml
streptomycin, 20 µg/ml gentamycin and 10% foetal calf serum (FCS) (Jessup et al.,
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1992).
2.4.4. Effects of BHUx on cyclooxygenases and lipoxygenases. Partially-purified

fractions 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 in
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vitro effects of BHUx.

2.4.4.1. Assay for cyclooxygenases. Enzymatic activity of COX-1 and COX-2
was measured as described earlier (Solomon et al., 2003) with slight modifications
using a chromagenic assay based on the oxidation of N,N,N ,N -tetramethyl-p-
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phenylenediamine (TMPD) during the reduction of PGG2 to PGH2. The assay

mixture in a final volume of 1 ml contained Tris-HCl buffer (pH 8.0, 100 mM),
hematin (15 µM), EDTA (3 µM), enzyme (COX-1 or COX-2, 100 µg) and test
compound (BHUx/celecoxib/indomethacin at different concentrations in 12 µl of
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buffer). The mixture was pre-incubated at 25◦ C for 15 min and then the reaction was
initiated by the addition of arachidonic acid (100 µM) and TMPD (120 µM). The
enzyme activity was measured by estimating the initial velocity of TMPD oxidation
for the first 25 s of the reaction, following the increase in absorbance at 603 nm. A
low rate of non-enzymatic oxidation observed in the absence of COX-1 and COX-2
was subtracted from the experimental value while calculating the percent inhibition.
The IC50 values for these compounds were calculated.
2.4.4.2. Lipoxygenase assay. A polarographic method was used to measure the

enzyme activities with a Clark’s oxygen electrode on Gilson model 5/6 oxygraph
as per the method described earlier (Grossman et al., 1968). A typical reaction
mixture contained 1.6 ml of assay buffer (potassium phosphate buffer, pH 6.3 for
5-LOX and pH 7.4 for 15-LOX) and 100 µl of enzyme. The reaction was initiated
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by addition of 10 µl of arachidonic acid with 133 µM final concentration. The
reaction was allowed to proceed at 25◦ C and the rate of decrease in oxygen was
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taken as a measure of enzyme activity. Enzyme activity is expressed as µmol
oxygen consumed/min per mg protein. Assays were performed with addition of
different concentrations of BHUx or LOX inhibitor (NDGA) to the reaction mixture
and IC50 values were calculated (Tripathi et al., 1995).
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2.5. 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 water
bath. Then it was cooled and centrifuged at 12 000 × g for 20 min. The supernatant
was saved and filtered through 0.2-µm filter paper. 100 µl of the above filtrate
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was injected into a RP-18 HPLC column and eluted isocratically by employing
water/acetonitrile (70:30, v/v) for 20 min. The eluate was monitored at a wavelength
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of 254 nm.
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3. RESULTS
3.1. Antioxidant properties of BHUx on cumene hydroperoxide (CHP) induced

lipid peroxidation in rat liver homogenate
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The results show concentration-dependent inhibition in the CHP-induced lipid

peroxidation in the liver homogenate. The IC50 for BHUx was calculated to be
102 µg/ml of liver homogenate (Table 3).
3.2. Anti-inflammatory effects of BHUx on carrageenan-induced rat paw oedema

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and granuloma pouch model

BHUx at a concentration of 400 mg/kg body weight showed inhibition in the
oedema (62%), induction in the rat paw oedema model, and in the enhancement
of the weight of cotton pellet (31%) in the granuloma pouch model (Table 2). The
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response was statistically significant.
3.3. Effect of BHUx on cyclooxygenase and lipoxygenase activity

BHUx showed dose-dependent inhibition of COX-1 in vitro as measured by TMPD
assay and data were compared with indomethacin and celecoxib (Fig. 1). The IC50
values were calculated for the above compounds and data presented in Table 4. As
Table 3.
Antioxidant properties of BHUx on cumene hydroperoxide (CHP)-induced lipid peroxidation in rat
liver homogenates
S.N. Group Lipid peroxidation* % Inhibition

(nmol/100 mg protein)
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1 Sham control 118 ± 4.14
576 ± 6.4
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1a Sham control with CHP (1.5 mM)
2 CHP + BHUx (µg/ml of homogenate)
2a 50 436.7 ± 7.2 24
2b 100 341 ± 8.4 40
2c 150 228 ± 7.8 60
2d 200 195 ± 5.9 66
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* Lipid peroxidation was measured in terms of TBARS.
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Figure 1. The inhibitory effect of BHUx (25–200 µg), indomethacin (1–10 µg) and celecoxib (1–
50 µg) on COX-1 activity. The values expressed as % inhibition of COX-1 activity are mean ± SD of
three independent observations.
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shown in Table 4, the IC50 value for BHUx was 169 µg/ml, compared to 3.8 µg/ml
of indomethacin, a non-specific inhibitor, and 20.8 µg/ml for celecoxib, a selective
COX-2 inhibitor. BHUx inhibited COX-2 with an IC50 value of 80 µg/ml, whereas
indomethacin inhibited COX-2 at 23.15 µg/ml and celecoxib at 3.75 µg/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-specific inhibitor. With indomethacin, a non-specific inhibitor
of cyclooxygenases, the COX-2/COX-1 ratio was 6.18. The effect of BHUx on
Table 4.
Comparative IC50 values of BHUx and various standard inhibitors for cyclooxygenases and lipoxy-
genases (in vitro assay)
S.N. Enzyme BHUx NDGA Indomethacin Celecoxib

(µg/ml) (µg/ml) (µg/ml) (µg/ml)
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1 COX-1 169 — 3.85 20.2
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2 COX-2 80 — 24.4 3.75
3 COX-2/COX-1 0.47 — 6.18 0.18
4 5-LOX 795 7.5 — —
5 15-LOX 44 23.5 — —
In vitro effects of BHUx and other standard inhibitors were measured on cyclooxygenases and
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lipoxygenases and the IC50 values determined.
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Figure 2. The inhibitory effect of BHUx (25–150 µg), indomethacin (5–50 µg) and celecoxib (1–
10 µg) on COX-2 activity. The values expressed as % inhibition of COX-2 activity are mean ± SD of
three independent observations.
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5-LOX and 15-LOX, in comparison with NDGA, a known inhibitor of LOXs, is

presented in Fig. 3 and Fig. 4, respectively. The IC50 values were calculated and
presented in Table 4. As shown in Table 4, BHUx inhibited both 5- and 15-LOX,
but with higher specificity towards 15-LOX. The IC50 for 5-LOX is 795 µg/ml and
that of 15-LOX is 44 µg/ml.
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Figure 3. The inhibitory effect of BHUx (10–1000 µg) and NDGA (2.5–25 µg) on 5-LOX activity.
The values expressed as % inhibition of 5-LOX activity are mean ± SD of three independent
observations.
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Figure 4. The inhibitory effect of BHUx (10–100 µg) and NDGA (10–100 µg) on 15-LOX activity.
The values expressed as % inhibition of 15-LOX activity are mean ± SD of three independent
observations.
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
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(25 ng/ml)
NO 10.24 35.94 33.43 30.50 25.67 20.16 17.35
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± 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 ± 0.11c ± 0.01a ± 0.01a ± 0.009a ± 0.005b ± 0.090
NO, nitric oxide production in terms of µmol NO− 2 /3 × 10 macrophage cells. MB, Methylene
6
Blue uptake in terms of absorbance at 660 nm. Values are mean ± SD of eight different experiments.
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Statistical comparison was made with normal. P value: a P < 0.001, b P < 0.01, c P < 0.05.
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Figure 5. HPLC fingerprint of BHUx water extract. The HPLC fingerprint shows 19 peaks on
different retention times.
3.4. Effect of BHUx on NO production

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In vitro results indicate that the thioglycolate activated macrophages are hyper-
sensitive to LPS and producing NO in the range of 33–36 µmol/3 × 106 cells,
whereas macrophages isolated from normal animals produce NO in the range of 9–
11 µmol/3 × 106 cells under similar conditions. However, this NO production was
significantly inhibited by the simultaneous and pre-incubation with BHUx extract
in a concentration-dependent manner. This indicates the strong anti-inflammatory
property of BHUx with an IC50 value at 50 ng/ml (Table 5).
4. DISCUSSION
Epidemiological and experimental studies have suggested an association between
acute and chronic-inflammation and risk of numerous pathological disorders, in-
cluding cardiovascular disease (Vallance et al., 1997). The changes in endothe-
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lial function may underlie this association. Mild systemic inflammation impairs
endothelium-dependent dilation in humans. Certain pro-inflammatory cytokines
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(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 might
precede the onset of the syndrome (Biasucci et al., 1999).
Inflammatory cells produce a highly complicated mixture of growth and differen-
tiation factors as well as biologically active arachidonic acid metabolites, including
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lipid hydroperoxides, leukotrienes and prostanoids, produced via the lipoxygne-
nase and cyclooxygenase pathways, respectively. Some of these arachidonic acid
metabolites, in particular leukotriene B4 (LTB4) and prostaglandin E2 (PGE2 ), are
important inflammatory mediators. Inhibition of biosynthesis of inflammatory me-
diators by blocking the activities of those enzymes would be an important treatment
of many inflammatory disease states (Zshocke et al., 2000).
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Natural compounds, obtained from medicinal plants, have been used as traditional
remedies for hundreds of years (Pandey et al., 1967). Many medicinal herbs are
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widely used for treatment of various inflammatory diseases. Recently, we have

shown that the anti-inflammatory property of C-phycocyanin, a biliprotein from
Spirulina 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 (Bobbili
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et al., 2003) and chronic myeloid leukemia cell line (K562) (Subhashini et al.,
2004). In the present study BHUx, which is a mixture of five medicinally important
plant extracts (these individual plants have been in clinical use for centuries in the
Ayurvedic system of medicine) in a particular ratio, has shown a potent inhibitory
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effect against enzymes of arachidonic acid metabolism, along with antioxidant

property 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-
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ering in triglyceride and cholesterol accompanied this reduction. Specific staining

of 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). Inflammation is known to induce endothelial dysfunction in humans,
involving IL-1, and aspirin can prevent this effect (Kharbanda et al., 2002). The
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preferential inhibition of COX-2 by BHUx, observed in the present study, could be

responsible for its anti-inflammatory 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 significant difference in serum cholesterol and triglyceride
levels, but small amount of collagen was present in the lesions. These data indicate
that inhibition of prostaglandin synthesis with a selective COX-2 inhibitor delays
the progression of atherogenesis during fatty streak lesion. The results described
herewith BHUx, show the inhibition of inflammation induced by carrageenan and
also in granuloma formation in rats, which indicates its net anti-inflammatory prop-
erty. The mechanism of its action could also be through its antioxidant property
because it inhibits the CHP-induced production of lipid peroxides.
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The selective inhibition of COX-2 by rofecoxib or suppression of the gene
encoding COX-2 resulted in the prevention of atherosclerotic lesion formation
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without any modification of serum lipids in LDL receptor deficient mice, which
are fed on a lipid-enriched athrosclerotic diet (Pitt et al., 2002). Celecoxib,
another COX-2 inhibitor, was shown to improve endothelial function in patients
with coronary artery disease (Chenvard et al., 2003). Inhibition of COX-2 was
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shown to be particularly beneficial in those patients with arthritis or other chronic
inflammatory diseases who have additional cardiovascular risk (Solomon et al.,
2003). Furthermore, an intact platelet function in the presence of COX-2 inhibitors
might reduce bleeding complications, which are associated with non-specific COX
inhibitor treatment.
Another important cascade of COX-2 production is the activation of macrophages
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by free radicals and oxidized LDL. This COX-2 not only causes inflammation but
also induces the expression of matrix metalloproteins (MMPs), which destabilize
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the atherosclerotic plaque. Therefore, COX-2 inhibitors in the physiological range

may 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 inflammatory
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contribution to vascular damage and atherothrombosis and have the potential

advantage over non-specific COX inhibitors with gastric side effects.
The IC50 ratios of COX-2/CXO-1 provide a useful comparison of relative values
for a series of NSAIDs tested in the same system. However, this ratio for a
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particular NSAID will vary according to whether it is measured using intact cells,
cell homogenates, purified enzymes, or recombinant proteins expressed in bacterial,
insect, or animal cells. Studies indicate that a high degree of in vitro biochemical
selectivity for COX-2 will be required in order to achieve effective functional
selectivity in vivo. The ratio demonstrates the relative selectivity of NSAIDs
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towards 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 IC50 values calculated
for BHUx in vitro with the partially-purified enzymes is 0.47, which is comparable
to the COX-2-specific inhibitor celecoxib, with 0.18 as against 6.18 recorded for
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indomethacin, a non-specific COX inhibitor. Figure 1, shows the effect of celecoxib

on COX-1 to be more potent than that of COX-2, but it is already reported that
this agent is a known COX-2-selective inhibitor. Here celecoxib, which is a
selective COX-2 inhibitor, has inhibited COX-1 with an IC50 of 20.2 µg/l, whereas
indomethacin, which is a preferential COX-1 inhibitor, inhibited COX-1 with an
IC50 of 3.85 µg/ml. However, the ratio of IC50 of COX-2/COX-1 for indomethacin
is 6.18, whereas that for celecoxib is 0.18, as shown in Table 4. This shows that
the celecoxib is a selective COX-2 inhibitor. Since the inhibitory concentrations

of COX inhibitors vary from assay system to assay system and from laboratory
to laboratory, the IC50 ratios of compounds are compared for studying the selective
inhibitory properties of the compounds. Based on this logic, the selectivity of BHUx
for COX-2 has been proposed here.
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The leukotrienes (LTs) formed by 5-LOX, which is expressed in leukocytes
mainly, possess potent pro-inflammatory activities and, thus, might be involved
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in cardiovascular disease. The unstable LTA4 generated in neutrophils by the
activity of 5-LOX is converted to LTB4, a compound with potent chemo-attractant
and pro-inflammatory properties. The unstable LTA4 is also transferred from
neutrophils to platelets and endothelial cells, which possess LTC4 synthase activity.
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The formation of cysteinyl leukotrienes by cell–cell interaction would then cause
coronary contraction. Thus, inhibition of neutrophil function by inhibiting 5-LOX
could not only suppress the direct contribution of these cells to inflammation, but
also downregulate the contribution of platelets and other interacting cells. During
inflammatory disease the arachidonic acid metabolism represents an important
aspect of platelet/polymorphonuclear leukocyte (PMNL) cross talk, relevant in the
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pathogenesis (Cerletti et al., 1999). In vitro activated platelets significantly increase
PMNL leukotriene biosynthesis, and PMNLs increase platelet TxB2 synthesis by
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providing eachother with free arachidonic acid (Marcus et al., 1982). Interestingly,
PMNLs synthesize various mediators, which cause cellular injury by initiating lipid
peroxidation, altering vascular permeability and activating vascular and circulating
cells. The 5-LOX pathway is abundantly expressed in arterial walls of patients
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afflicted with various lesion stages of atherosclerosis of the aorta and of coronary
and carotid arteries. 5-LOX is localized to macrophages, dendritic cells, foam cells,
mast cells and neutrophilic granulocytes, and the number of 5-LOX expressing cells
markedly increased in advanced lesions. 5-LOX cascade-dependent inflammatory
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circuits, consisting of several leukocyte lineages and arterial wall cells, evolve
within the blood vessel wall during critical stages of lesion development. They raise
the possibility that anti-leukotriene drugs may be an effective treatment regimen in
late-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 this
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mixture 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-LOX
with relatively higher specificity. 15-LOX is thought to play the key step in the
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oxidation of phospholipid moiety of the LDL and inhibition of 15-LOX could be

the novel therapeutic approach for the management of atherosclerosis. The 12/15-
LOX expressed in macrophages is capable of oxygenating linoleic acid, esterified
to cholesterol in the LDL particle, and thus this enzyme is presumed to initiate LDL
oxidation (Zhu et al., 2003). 12/15-LOX-gene disruption attenuates atherogenesis
in LDL receptor-deficient mice (George et al., 2001). In the present study the
inhibition of 15-LOX is comparable with that of the unspecific LOX inhibitor
NDGA, and this inhibition will help to control the oxidation of phospholipid moiety
of LDL, which undergoes oxidation under the effect of free radical attack mediated
by 15- and 5-LOX.
Despite significant protection afforded by some non-steroidal anti-inflammatory
drugs (NSAIDs) like aspirin in groups of patients with thrombotic cardiovascular
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disease, many patients do not derive any clinical benefit and might even experience
side effects (De Gaetano, 2001). The limited protection afforded by these drugs
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is explained by genetic variability in response to drug, differing influences of
concomitant vascular risk factors and their severity, such as hypertension, the
possibility that TxA2 -mediated platelet activation is crucially involved in a limited,
but still defined, set of thrombotic events (De Gaetano, 2001; De Gaetano et al.,
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2002). In any case, the new anti-thrombotic approaches should not only reduce
the risk of adverse reactions but also successfully treat patients who are resistant to
these drugs. Thus, BHUx, because of its multi-targeted action and being a natural
extract, could be a suitable candidate that could reduce the toxicities associated with
currently available NSAIDs. Inhibition of COX-1, COX-2, 5-LOX and 15-LOX by
BHUx could inhibit platelet TXA2 formation, down regulate leukocyte activation
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and wide spread vascular inflammation and reduce leukocyte inflammatory and
thrombogenic potential. Thus, BHUx is acting on mainly at two levels, one directly
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as free radical scavenger and other at the inflammatory mediators level to prevent
atherosclerosis.
HPLC fingerprint was consistent and this was used to avoid the batch-to-batch
variation during the experiment. The peaks show that BHUx has different com-
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pounds, which might be having different biological responses. This gives a lead
for the development of specific compounds for specific actions. For clinical use
of BHUx as herbal medicine, BHUx, however, would be preferable because of its
holistic approach in action. It is true to especially for those diseases, which have
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multi etiological factors, like atherosclerosis.
5. CONCLUSIONS
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This study suggests that BHUx, a polyherbal formulation, possesses potent anti-
inflammatory and antioxidant activity. BHUx being a natural source, without
any side effects, can be used to control atherosclerosis. Thus, the present study
provides a mechanism and scientific evidence for the therapeutic potential of BHUx.
Further studies, however, should be taken up to isolate and characterize the active
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compounds 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 thankful
to 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 to
M. Mallikarjuna Reddy is gratefully acknowledged.
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VSP 2001/04/20 Prn:26/04/2004; 14:00 {RA} F:iph2138.tex; VTeX/NJ p.

Inflammopharmacology, Vol. 00, No. 0, pp. 1 – 22 (2004)

YAMINI B. TRIPATHI 1,∗ , M. MALLIKARJUNA REDDY 2 , R. S. PANDEY 1 ,

1 Department of Medicinal Chemistry, Institute of Medical Sciences, Banaras Hindu University,

Abstract—BHUx is a polyherbal formulation consisting of water-soluble fractions of five medicinal

Key words: Atherosclerosis; inflammation; cyclooxygenase; lipoxygenase; nitric oxide; antioxidant;

542) 236-6566; e-mail: Yamini30@sify.com

which initiates platelet aggregation. Because, inhibition of COX-1 in the gastric

leukocytes, vascular cells and macrophages that infiltrate atherosclerotic plaques,

enzymes, capable of oxygenating polyunsaturated fatty acids to their corresponding

an epidermis-type (type-2) enzyme.

is partly responsible for the resistance to atherosclerosis in mice (Meharabian et

Anti-inflammatory properies of BHUx 3

Thus, it can be suggested that a promising pharmacological approach to reduce

Component Plant Principal components Pharmacological effect

B Terminalia Triterpnoid saponins, anjantic Wound healing [B3], ischemic

terols, calcium, magnesium, [B7], anti-oxidant [B8]

C Semecarpus Anacardic acid [C1], flavones Wound healing, hypolipidemic

flavanone [C3], semecarpufla- anti-oxidant, anti-oedema,

D Boswellia Triterpene [D1] (acetyl-11 Anti-inflammatory [D4],

Anti-inflammatory properies of BHUx 5

Component Plant Principal components Pharmacological effect

[A5] Tripathi et al. (1984). [D3] Altmann et al. (2002).

[B5] Miller (1998). [D8] Ammon (2002).

[C7] Satyavati et al. (1969).

2. MATERIALS AND METHODS

Arachidonic acid, N,N,N ,N -tetramethyl-p-phenylenediamine (TMPD), lipopoly-

2.3. Effects of BHUx: in vivo studies

the plantar aponeurosis. It produced acute inflammatory oedema leading to marked

the anti-inflammatory response was monitored in terms of mercury displacement on

% Inhibition = (Vc − Vt ) × 100/Vc ,

S.N. Group Change in paw % Inhibition Weight of % Inhibition

Anti-inflammatory properies of BHUx 7

2.4. Effects of BHUx: in vitro studies

duty blade. They were then homogenized in glass-Teflon homogenizing tube in

Folin-phenol method (Lowry et al., 1951).

in experimental plates suspension of BHUx was added in different concentrations

transferred to a tube containing 1.5 ml of 10% trichloroacetic acid (TCA). After

2.4.2. Effect on NO production. In another set of experiments, the effect of

2.4.4. Effects of BHUx on cyclooxygenases and lipoxygenases. Partially-purified

vitro effects of BHUx.

phenylenediamine (TMPD) during the reduction of PGG2 to PGH2. The assay

Anti-inflammatory properies of BHUx 9

2.4.4.2. Lipoxygenase assay. A polarographic method was used to measure the

3.1. Antioxidant properties of BHUx on cumene hydroperoxide (CHP) induced

The results show concentration-dependent inhibition in the CHP-induced lipid

3.2. Anti-inflammatory effects of BHUx on carrageenan-induced rat paw oedema

and granuloma pouch model

response was statistically significant.

3.3. Effect of BHUx on cyclooxygenase and lipoxygenase activity

S.N. Group Lipid peroxidation* % Inhibition

Anti-inflammatory properies of BHUx 11

S.N. Enzyme BHUx NDGA Indomethacin Celecoxib

5-LOX and 15-LOX, in comparison with NDGA, a known inhibitor of LOXs, is

Anti-inflammatory properies of BHUx 13

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

3.4. Effect of BHUx on NO production

widely used for treatment of various inflammatory diseases. Recently, we have

effect against enzymes of arachidonic acid metabolism, along with antioxidant

ering in triglyceride and cholesterol accompanied this reduction. Specific staining

preferential inhibition of COX-2 by BHUx, observed in the present study, could be

Anti-inflammatory properies of BHUx 15