IN VITRO ANTI-INFLAMMATORY AND

ANTIMICROBIAL ACTIVITIES OF
PHENYLPROPANOIDS FROM
Piper belle L. (PIPERACEAE)

L. A. D. WILLIAMS,aE. A. VASQUEZ, a,b p . P. MILAN,b

C. ZEBITZ AND W. KRAUS a

alnstitut fUr Chemie, Universitat Hohenheim, Garbenstr 30, 70593 Stuttgart,

Germany
bPhilippine Root Crop Research, Visayas State Colleage ofAgriculture, 6521-A,
Baybay, Leyte, Philippine
Clnstitut fur Phytomedizin Universitat Hohenheim, 70593 Stuttgart, Germany

Abstract. The present study evaluates the in vitro anti-microbial and anti-inflammatory activities of some
phenylpropanoids isolated from Piper betle L. The study revealed that both the antimicrobial and anti-
inflammatory activities depend on the position of the hydroxyl functionality associated with the aromatic
moiety. In order to validate the usefulness of the Bovine Serum Albumin (BSA) assay as a tool for the
screening of non-steroidal anti-inflammatory compounds, which is the main focus of the present
investigation, 10 NMR interactions were conducted on the BSA-phenylpropanoid mixtures. The NMR
data obtained revealed that the interactions were localized at two regions: (1) at 3.0 - 3.8 ppm, which can
be assigned to lysine, an amino acid residue capable of inducing reactive oxygen species (Sadler &
Tucker, 1992; Kawabata & Packer, 1994), and (2) at 6.3 - 7.8 ppm, a region associated with tyrosine
amino acid residues, which are aimportant components (binding sites) in the receptor motifs regulating
various signal transduction cascades involved in the release of important anti-inflammatory cytokines.
Using these two important biochemical findings we hereby proposed a hypothesis why BSA could be a
useful in vitro test system for finding natural products with anti-inflammatory properties.

l. INTRODUCTION

The secondary metabolites biosynthesized by tropical and sub-tropical plants have

provided interesting prototype molecules which have served for the development of
important medicinal and agrochemical agents (McChesnay & Clark, 1992). Piper
betle L. is a tropical medicinal plant widely grown in Southeast Asia, the Pacific
islands and the Caribbean basin. Various therapeutic uses have been reported for
P. betle, which include: antibacterial, anti-inflammatory and anti-hypertension
(Saaed et al., 1993; Nguyen & Doan, 1990).
In the present article we hereby reports on the bioassay-guided isolation of the
active anti-inflammatory and antimicrobial compounds from the leaves of P. betle
using in vitro methods.

221
A.P. Rauter et al. (eds.),
Natural Products in the New Millennium: Prospects and Industrial Application, 221-227.
© 2002 Kluwer Academic Publishers.
222 L. A. D. WILLIAMS et al.

2. MATERIALS AND METHODS

2.1.1solalion of Compounds

The dried leaves of P. belle were crushed and serially extracted with petrol ether
(30-50 0c), ethyl acetate and methanol. The petrol ether extract was found to be
positive for both antimicrobial and antidenaturation activity of heat treated bovine
serum albumin.
The petrolether extract was further worked up with repeated column
chromatography, while purification was done using HPLC-Diol with 1-3% ethanol
in petrol ether. The active compounds were identified using NMR and MS analyses
in conjunction with reported data.
These compounds were found to be the following six known phenylpropanoids:
chavicol (1), chavibetol (2), chavibetol acetate (3), eugenol (4) iso-eugenol (5) and
methyl eugenol (6) (Figure 1).

HO ~
~I

(1)
MeO
~~I
OH
(2)

MeO
~ OAc
HO
~~I
OMe
(3) (4)

M~
HO ~
(5)
I
MeO
~~I
OMe
(6)

Figure 1. Compounds found in Piper betle.

 IN VITRO ANTI-INFLAMMATORY AND ... 223

2.2. Biological Testing

2.2.1. In vitro Anti-injlammatory Assay

Currently, the methods used for evaluating anti-inflammatory properties of
compounds and extracts are all strongly based on the utilization of animal models
e.g. the rat paw and ear oedema assays. These animal based assays are strongly
criticized by various animal right groups from around the world. Thus, we hereby
proposed the stabilization of heat-treated bovine serum albumin by non-steroidal
anti-inflammatory compounds, which was first reported by Grant et al. (1970) as a
screening method for finding these important medicinal agents (the non-steriodal
anti-inflammatory compounds). The present proposal is based on the findings
obtained in this study for some known phenylpropanoids with established anti-free
radical and anti-inflammatory properties.
Stock solution of 5.0% (w/v) bovine serum albumin (BSA), fraction V of 96%
purity (Sigma Chemical Co.), without further purification was prepared in a mixture
of 0.05 M Tris-phosphate buffer saline which was adjusted to pH 6.5 with glacial
acetic acid. Triton X-I00; 0.01% (w/v, in distilled water) was added to the Tris-
acetate buffer in a ratio of 5:95 (V/V).i
The following stock solutions 50.0 ppm, 5.0 ppm, 0.5 ppm and 0.05 ppm of the
phenylpropanoids 1 - 6 were prepared in methanol. Methanol was selected for
dissolving the test compounds based on the fact that it will not affect the gross
molecular configuration of albumin up to a concentration of 30% (wlv) (Zakrzewski
& Goch, 1968). From each of the above mentioned stock solutions 500 !JL was
added to 5.0 mL of the stock BSA solution to produce concentrations ranging from
10 - 0.1 IlglmL of each test compound. The control consisted of 500 !JL of methanol
in 5.0 mL of the 0.5% (w/v) stock BSA in Tris acetate bufferTriton-X 100 mixture.
Each sample was heated for 5.0 minutes at 70°C, cooled and its turbidity measured
at 660 nm on a Beckman Spectrophotometer. The degree of precipitation (inhibition
or promotion) of the BSA from the solution by each compound was calculated on a
percentage basis, relative to the control.

2.2.2. 1D Proton NMR Interaction between Denaturated BSA and Phenyl-

propanoids
After spectrophotometric analyses of the various phenylpropanoids-BSA mixtures,
they were diluted by a factor of 1: 10 (v/v) with a solution of Tris-phosphate buffer
saline (PH 6.5)-deuterium oxide (020) (9:1; v/v) and analyzed using a VARIAN
UNITY INOVA 300 MHz instrument for new interaction 1D proton signals.

2.2.3. Biological Testing (ILe Bioautography Assay)

2.2. 3.1. Antibacterial Testing

Bacillus subtilis (Gram positive) and Pseudomonas fluorescens (Gram negative)
bacteria were used as the test microbes. From stock solutions of each compounds
10 J.1L were applied onto TLC plates (silica gel 60 F24i AI sheet, Merck) to give
concentrations of 200 - 0.5 Ilglspot.
The treated TLC plates were then sprayed with suspensions of B. subtilis and
P. jluorescens in nutrient broth and incubated at 37°C for 24 h. Compounds having
224 L. A. D. WILLIAMS et al.

strong inhibitory action on the growth of the bacteria appeared as clear zones against
a pinkish/purplish background after spraying with iodotetrazolium salt solution
(Homan & Fusch, 1970). Ampicillin and Isoniazid were used as the positive
controls.

2.2.3.2. Antifungal Testing

TLC plates of similar concentrations as mentioned above in the antibacterial assay
were prepared and then sprayed with spore suspension of Cladosporium
cucumerinum in nutrient medium. The treated plates were then incubated in a
humidifier in the dark for 48 h under laboratory conditions. Active compounds
appeared as clear zones against a dark background on the TLC plates (Hamburger &
Cordell, 1987). Ketoconazole was used as the positive control.

3. RESULTS AND DISCUSSION

All the phenylpropanoids tested were capable of protecting the BSA from heat
induced denaturation according to the data presented in Figure 2. However, the
effects seem to depend both on the structures and concentration of the compounds.
Grants et al. (1970) have reported similar phenomena with BSA for the following
non-steroidal anti-inflammatory compounds; indomethacin, ibufenac, flufenamic
acid and salicylic acid in vitro.
Further structure activity analyses of the data presented in Figure 2 revealed that
replacement of hydroxyl groups by methoxy or acetate e.g. methyl eugenol and
chavibetol acetate, reduced the anti-denaturation potential of the phenylpropanoids
on the BSA. The data also revealed that the following compounds chavicol,
chavibetol, eugenol and iso-eugenol were equally effective in protecting the
denaturation of the BSA by 80 - 95% (Figure 2). Williams and colleagues (2001,
unpublished results) have used the present BSA methods to identify various crude
extracts and compounds from plants of the Papaveraceae and Labiatae families,
which also show potent anti-inflammatory activity in the rat-paw oedema assay. It is
of interest to note that eugenol is now formulated with voltaren to enhance its anti-
inflammatory potentials.
The ID proton NMR interaction data presented in Table 1, revealed that two
interaction regions were observed between these phenylpropanoids and the BSA.
These signals were observed at 3.2 - 3.8 ppm and 6.8 - 7.0 ppm in the aliphatic and
aromatic regions of the lD NMR spectra, respectively. It is reported that heat
induced denaturation of BSA produced distinct immunological aggregates, which
are different from the untreated (parent) protein. These heat induced aggregates have
demonstrated properties which are closely related to some immunomodulatory
diseases e.g. serum sickness nephritis (Maurer, 1959; Lireman et aI., 1967). The
receptor motifs e.g. the Fc-epsilon-RI which regulates the signal transduction
cascade of several immunomodulatory and anti-inflammatory cytokines are
composed of tyrosine amino acid residues (Turner & Kinet, 1999). The tyrosine
amino acid residue complex of BSA resonates at about 6.3 - 7.8 ppm at pH 6.5
(Sadler & Tucker, 1992). Recently, we have used this lD proton NMR
BSA-compound interaction analyses to successfully predict and determined the
signal transduction pathway for some biologically active molecules (Rosner et al.,
2001).
 Effects of phenylpropanoids on the denaturation
of bovine serum albumin
120
~
100 ~ ::s
--- ;;5
~- . ;'1'; <:)
80 '"
c -+- Chavicol
0 60 §
:.;::; ---Cahvibetol
:0 40
.c. ---Chavibetol acetate
c / --- """*- Eugenol
20 ---------.
<J.) -.
0) // """"*""" Iso-eugenol
m 0
-+- Methyl eugenol
0~ -20 //
i
~
-40 Y
-
-60 -

0.1 1.0 10.0

concentration (ppm)
N
N
VI
Figure 2.
226 L. A. D. WILLIAMS et al.

Table 1. Co"elation oJphenylpropanoids-denaturated BSA interaction from lD proton NMR.

1. No significant interaction between methly eugenol and chavibetol acetate

withBSA.
2. Eugenol and iso-eugenol produced new signals at 3.2.- 3.8 ppm with the
BSA.
3. Strong suppression signals for eugenol at 3.2 ppm.
4. Eugenol and chavibetol gave two sharp new signals at 6.8 and 7.0 ppm,
while isoeugenol and chavicol gave one broad signal with the BSA.

Similarly, BSA contains amino acid residues e.g. lysine (epsilon) which
resonates at 3.0-3.8 ppm and are capable of binding with anti-inflammatory active
compounds having redox properties e.g. diclofenac (voltaren), Kawabata & Packer
(1994). Molecules such as dic10fenac have the ability of preventing free radical
induced damaged in BSA from processes such as glycation (Glycation =
non-enzymatic binding of glucose to proteins e.g. BSA in vitro, on these lysine
amino acid residues). Glycation is one of the fundamental pathological processes
associated with the on set of many forms of degenerative diseases (Kawabata &
Packer, 1994). The phenylpropanoids also show very strong interaction signals with
BSA in regions of the ID proton NMR spectra that are assigned to these active free
radical amino acid clusters. Thus, BSA in its in vitro state does possess
immunomodulatory and redox biological properties which are fundamental in the
inflammatory processes and could be the reason for its useful application for the
screening of anti-inflammatory non-steroidal molecules.
The antimicrobial activities of the phenylpropanoids were influenced by the
types of functional groups and their positions. Thus, the overall antimicrobial
activity of phenylpropanoids with a propenyl side chain e.g. 1, 2 and 4 , were less
active than those with an isopropenyl function as in s. The position of the hydroxyl
functionality was also crucial to the activity observed. Thus, when the hydroxyl
function was para to the propenyl group e.g. 1, the activity was less than when it is
in position meta. The antimicrobial activity of all the phenylpropanoids was smaller
than that of the commercial antibiotics tested (Table 2).

Table 2. Antimicrobial activities oJphenylpropanoids.

Compounds Microorganisms and MIC values (J.1g/spot)

B. subtilis P. fluorescens C. cucumerinum
Chavicol > 50.0 45.0 50.0
Chavibetol 50.0 > 50.0 25.0
Chavibetol acetate N/A > 100.0 N/A
Eugenol 50.0 40.0 10.0
Isoeugenol 50.0 25.0 5.0
Methyl eugenol > 100.0 > 100.0 > 100.0
Positive controls
Ketoconazole 1.5
Ampicillin 2.5 5.0
Isoniazid 2.5 0.5
TLC autobiographic assay.
 IN VITRO ANTI-INFLAMMATORY AND ... 227

Bever-Oliver (1986) has suggested that the effective in vivo antimicrobial

activity associated with various oleoresins containing phenylpropanoids cannot be
justified based on the minute quantity that is used in correcting the infectious state
that is associated with these pathogens, and may be attributed to an indirect
activation of the immune system. The strong interactions signals observed in the
aromatic region in the 10 proton NMR spectra for the BSAI phenylpropanoids
mixture suggest that these phenolics may up-regulate the immune system by an
interaction with tyrosine based receptor motifs for some important
immunomodulatory receptors, e.g. the Fc-epsilon-Rl receptor complex, which are
important in cytokine release.

Acknowledgments

The authors thank Ms. Sonja Ringer, Inorganic Chemistry Universitlit Hohenheim,
for conducting the BSAIphenylpropanoids spectrophotometric analyses, Ms. Sabine
Reeb and Iris Klaiber, Organic Chemistry for NMR and MS measurements,
Universitlit Hohenheim and fmancial support from the Humboldt Foundation (Dr.
L.A.D. Williams) and the European Union (Brussels, Belgium) (Dr. Erlinda
Vasquez).

4. NOTES
I. Triton X-I 00 (0.01% w/v) can be omitted from the carrier solution. If this is done, then a stock solution
of 0.2% (w/v) BSA in Tris-acetate phosphate buffer pH 6.5 is ideal for evaluating the compounds. The
test compounds should then be applied to 5.0 mL BSA in 50 j.lL of methanol, and the mixture heated for
3.0 min at 70 -73 ·C prior to spectrophotometric analyses.

5. REFERENCES
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