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CHAPTER 4
INHIBITION OF COLLAGENASE BY WITHANIA
SOMNIFERA AND CARDIOSPERMUM HALICACABUM
EXTRACT AGAINST COLLAGENOLYTIC
DEGRADATION OF COLLAGEN
4.1 INTRODUCTION
Tissue engineering and regenerative medicine is a rapidly evolving
interdisciplinary field that aims to regenerate new tissue to replace damaged
tissues or organs. The extracellular matrix (ECM) is a complex mixture of
macromolecules that play an essential instructional role in the development of
tissues and organs. Therefore, tissue engineering approaches rely on the need
to present the correct cues to cells, to guide them to maintain tissue-specific
functions. The matrix metalloproteinases (MMPs) are a large family of
calcium-dependent zinc-containing endopeptidases, which are responsible for
remodeling and degradation of the ECM. Over expression of MMPs-1, 8, 13,
18 (collagenase I, II, III and IV) are capable of degrading native interstitial
collagens I, II and III and at the cellular surface producing changes in cellular
behavior and subsequent pathological responses. To devise strategies for
using collagen in the development of advanced biomaterials for biomedical
engineering, it is necessary to confer resistance to proteolytic (MMPs-
collagenase) degradation (Anderson et al 2008).
Controlled synthesis and degradation of ECM of fibrous protein of
collagen is an important feature in a variety of physiological conditions such
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as embryonic development, blastocyst implantation, nerve growth, tissue
remodeling and tissue repair. In pathological conditions such as uncontrolled
collagen breakdown and synthesis an essential part of arthritis and fibrosis,
invasion and metastasis of malignant tumors, apoptosis, atherosclerosis,
aneurysm, periodontal diseases, skin, corneal and gastric ulceration, liver
fibrosis and among others, generally in processes including ECM degradation
(Cawton et al 1996; Sternlicht et al 2001). The MMPs are a large family of
calcium-dependent zinc-containing endopeptidases (also called matrixins),
which are responsible for the tissue remodeling and degradation of the ECM
components, including collagens, elastins, gelatin, matrix glycoproteins and
proteoglycan (Kleiner et al 1993) are shown in the Table 4.1. Most of the
normal and abnormal cells including fibroblasts, macrophages, neutrophils,
osteoclasts, etc. can synthesize and release MMPs (Rao et al 1999).
Over expression of MMPs also play a role in pathological
conditions involving untimely and accelerated turnover of ECM, including
RA and OA, invasion and metastasis of malignant tumors. Recent
observations suggest that MMPs also play a role in cancer cell survival
(Hanemaaijer et al 1997; Konttinen et al 1998; Stahle-Backdahl etal 1997;
Reboul et al 1996; Wallach et al 2005; Chang et al 2001; Egeblad et al 2002).
The rheumatoid tissue produces the excess amount of collagenases destroying
the articular cartilage and invading the autologous joint (Hanemaaijer et al
1997). Possible involvement of chondrocytes in destruction of cartilage in OA
has been suggested by the finding of collagenase of OA cartilage (Ehrlich et
al 1977). Wounding of skin was also shown to induce collagenase formation
by both epithelial and mesenchymal cells at the wound margin (Wu et al
2007). Collagenases I, II, III and IV (MMP-1, 8, 13 and 18) and MMP-18 are
capable of degrading native interstitial collagens I, II, III, IV and X at a
specific site between Gly775–Ile776 of the 1 chains and Gly775-Leu776
residues of 2 chains at the ¾ length from the N-terminus of the molecule
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(Simionescu et al 1996; Fernandez et al 2006; Visse et al 2003). MMP-1 is
expressed by various normal cells, for example, fibroblast, keratinocytes,
endothelial cells, macrophages, chondrocytes and osteoblasts, as well as by
many different types of tumor cells (Brinckerhoff et al 2005).
Table 4.1 List of matrix metalloproteinases
Gene Name Location
MMP1 Interstitial collagenase secreted
MMP2Gelatinase-A, 72kDa
gelatinasesecreted
MMP3 Stromelysin 1 secreted
MMP7 Matrilysin, PUMP 1 secreted
MMP8 Neutrophil collagenase secreted
MMP9Gelatinase-B, 92 kDa
gelatinasesecreted
MMP10 Stromelysin 2 secreted
MMP11 Stromelysin 3 secreted
MMP12Macrophage
metalloelastasesecreted
MMP13 Collagenase 3 secreted
MMP14 MT1-MMP membrane-associated
MMP15 MT2-MMP membrane-associated
MMP16 MT3-MMP membrane-associated
MMP17 MT4-MMP membrane-associated
MMP18Collagenase 4, xenopus
collagenase-
MMP19RASI-1, occasionally
referred to as stromelysin-4-
MMP20 Enamelysin secreted
MMP21 X-MMP secreted
MMP23A CA-MMP membrane-associated
MMP23B - membrane-associated
MMP24 MT5-MMP membrane-associated
MMP25 MT6-MMP membrane-associated
MMP26 Matrilysin-2, endometase -
MMP27 MMP-22, C-MMP -
MMP28 Epilysin secreted
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Human MMP-8 is expressed by various types of cells, including
chondrocytes, rheumatoid synovial fibroblasts, gingival fibroblasts, and oral
mucosa keratinocytes and melanoma cells (Hanemaaijer et al 1997; Palosaari
et al 2003). MMP-13 expressions are also detected in pathological conditions
that are characterized by the destruction of normal collagenous tissue, e.g. in
OA cartilage, rheumatoid synovium, chronic cutaneous ulcers, intestinal
ulcerations, chronic periodontitis, atherosclerosis and aortic aneurysms (Lindy
et al 1997; Golub et al 1997). MMPs have recently become interesting
targets for drug design in the search for novel types of anti-cancer, anti-
arthritis, other pharmacological agents useful in the management of
osteoporosis, aortic aneurysm, glomerulonephritis, and multiple sclerosis
among others. Several synthetic MMP inhibitors have been tested in clinical
trials evaluating their ability to control the arthritis and growth and invasion
of malignant tumors in vivo (Baker et al 2002). However, limited
performance of MMP inhibitors in phase III clinical trials has been
disappointing. Therefore, using the broad spectrum of MMP inhibitors
selectively targeting certain MMPs may be preferable. Novel inhibition
strategies for MMPs may serve as an important approach for targeted therapy
for pathologies and also stabilization of collagen based matrix tissue
engineering.
Many pharmacological studies have been carried out to describe
multiple biological properties of Withania somnifera root, leaves and fruit
extract (Mishra et al 2000; Scartezzini et al 2000). W. somnifera is one of the
most widely used herbs in Ayurvedic medicine named Ashwaghanda and
contain several alkaloids, withanolides, a few flavanoids and reducing sugars,
a rich source of bioactive compounds (Kandil et al 1994; Umadevi et al
1996). The various alkaloids present include withanine, somniferine, somnine,
withananine, psuedo-withanine, tropine, psuedo-tropine, 3- -gloyloxytropane,
choline, cuscohygrine, isopelletierine, anaferine and anahydrine (Dhar et al
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2006). Similar to many other plant extracts, these extracts possessed anti-
oxidant, anti-microbial, anti-enzymatic, effective reduction of protein
antigenicity, anti-mutagenic and anticarcinogenic properties (Russo et al
2001; Jayaprakasam et al 2003; Girish et al 2006; Bhattacharya et al 2001;
Visavadiya et al 2007; Rasool et al 2007). It has been recently reported to be
the inhibitor of angiogenesis and thus protective in certain types of cancers
(Mohan et al 20040. It has been linked with inhibitory and preventive effects
in various human cancers and cardiovascular diseases (Christina et al 2004;
Mohanty et al 2004). Chondroprotective potential of root extracts of W.
somnifera in OA had been studied (Sumantran et al 2007). W. somnifera
extract (WSE) was reported to be the most effective chemo-preventive agents
against the initiation, promotion and progression of multistage carcinogenesis
(Leyon et al 2004; Davis et al 2001). Cancer-preventing activity has been
demonstrated for this extract and their constituents in many animal models of
tumorgenesis (Padmavathi et al 2005). In vitro, this extract inhibited the
proliferation of various cancer cell lines and induced cancer cell apoptosis
(Widodo et al 2007). The diverse active constituents present in different parts
of the plant are believed to be responsible for the multiple medicinal
properties of W. somnifera (Scartezzini et al 2000). It could have the
therapeutic role in the prevention of glycation-induced pathogenesis in
diabetes mellitus and aging (Babu et al 2007).
Cardiospermum halicacabum (Linn), family Sapindaceae, is a
deciduous, branching, herbaceous climber, which is distributed throughout the
plains of India and used in the treatment of arthritis, chronic rheumatism,
stiffness of limbs, snake bite; its roots for nervous diseases, as a diaphoretic,
diuretic, laxative, refrigerant, etc. Phytochemical constituents of C.
halicacabum such as flavones, aglycones, triterpenoids, glycosides and a
range of fatty acids and volatile ester have been reported from the various
parts of extracts from this plant (Ahmed et al 1993; Srinivas et al 1998).
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Although scientific evidence is mounting to support the claims that C.
halicacabum may be a remedy for RA, its use is still limited to traditional
rural cultures.
The inhibition of MMP-1, MMP-8 and MMP-13 caused by plant
secondary metabolites such as flavonoids and polyphenol was confirmed by
gelatin zymography, and was observed for MMPs associated various
connective tissues (Folgueras et al 2004; Yang et al 2008). Plant flavonoids
were tested for their ability to inhibit the prokaryotic and eukaryotic cell
derived collagenase activities. Preincubation of bacterial collagenases with
flavonoid and polyphenol reduced the collagenase activity as well (Makimura
et al 1993).
Figure 4.1 This figure is focused on the catalytic site of ChC
In addition to mammalian MMPs, there are also some other
enzymes such as bacterial collagenases, which can degrade ECM. One such
bacterial collagenase known as Clostridium histolyticum collagenase (ChC)
(Figure 4.1), which belongs to the family of M-31 metalloproteinase family, is
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capable of hydrolyzing triple helical region of collagen under physiological
conditions as well as many synthetic peptides (Miller et al 1976).
In fact, the crude homogenate of ChC, which contains several
distinct collagenase isozymes, is the most efficient system known for the
degradation of ECM. MMPs-Collagenases and ChC, though relatively
different, are considered to act through the same mechanism of action in the
hydrolysis of proteins and collagen like synthetic peptides (Miller et al 1976;
Mandl et al 1958; Ryan et al 1971). Similar compositions of AAs are present
in the active site of mammalian and bacterial collagenases. Moreover,
commercially available bacterial collagenases represent a mixture of various
iso-enzymes with different activities.
4.1.1 Scope of the Work
In the present study, the effects of W. somnifera extract (WSE) and
C. halicacabum extract (CHE) on ChC activity using the native enzyme
associated with bovine achilles tendon (BAT) collagen were evaluated. An
attempt to understand the role of both these plant extracts in the mode and
degree of inhibition of ChC has now been made.
4.2 MATERIALS AND METHODS
4.2.1 Materials
All reagents and chemicals used were of analytical grade.
Collagenase (Type IA) and N-[3-(2-furyl) acryloyl]-Leu-Gly-Pro-Ala
(FALGPA) were sourced from Sigma chemicals Co., USA. All other reagents
and chemicals used for the study were sourced from SRL Ltd., India.
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4.2.2 Preparation and Purification of WSE and CHE
Commercial available Ayurvedic powders of W. somnifera and C.
halicacabum were percolated four times with ethanol: water (1:3) at room
temperature (Dhar et al 2006). The combined extracts were filtered,
centrifuged and concentrated to 1/6th of the original volume under reduced
pressure at a temperature of 50 ± 5 °C. Finally, the extract was completely
dried under vacuum in desiccator. Total yield of the WSE and CHE are 15
and 17% respectively. For the isolation of flavonoids, steroidal glycosides and
withanolides/glucowithanolides, the aqueous ethanolic extract (1:3) was
dissolved in water and the solution was successively extracted with
chloroform in a separating funnel. Both chloroform fractions were separately
concentrated under reduced pressure to yield the residues containing
flavonoids, steroidal glycosides and withanolides/glycowithanolides.
4.2.3 Preparation of BAT
Tendons from bovine Achilles legs were collected and washed with
0.9% NaCl at 4 °C to remove the adhering muscles and other soluble proteins.
The BAT was subsequently washed extensively in double distilled water and
treated with 0, 5, 10, 20, 40 and 80 g WSE and CHE for 24 hrs at room
temperature (27 °C) without any agitation. After 24 hrs both the extract
treated BAT were stored in water at 4 °C before testing for resistance to ChC.
4.2.3.1 ChC hydrolysis of plant extracts treated BAT
% collagen degradation was calculated by the following method.
The native, WSE and CHE treated BAT were further treated with ChC. ChC
treatment was carried out in 0.04 M CaCl2 solution buffered at pH 7.2 with
0.05 M tris HCl. The collagen: ChC ratio (25 mg BAT: 0.5 mg ChC) was
maintained at 50:1. The samples were incubated at a temperature of 37°C.
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Samples were collected at various time intervals ranging from 6 to 96 hrs and
stored in freezer. The cleavages of native and treated BAT were monitored by
the release of soluble form of Hyp from insoluble collagen (Woessner et al
1961). Aliquots of 750 l of supernatant were withdrawn after centrifuging at
10,000 rpm for 10 min. The ChC hydrolysates were hydrolyzed 6 N HCl in
sealed hydrolysis tubes for 16 h. The hydrolysates were evaporated to dryness
in a porcelain dish over a water bath to remove excess acid. The residues of
free acid were made up to a known volume and the % Hyp was determined
using the method (Ryan et al 1971). Hyp is an unique AA for collagen and it
offers itself as a useful marker for identifying collagen in the presence of non-
collagenous proteins. The method of determining hyp involves the oxidation
of Hyp to pyrrole-2-carboxylic acid, which complexes with p-
dimethylaminobenzaldehyde exhibiting maximum absorbance at 557 nm.
% Soluble collagen = % Hyp 7.4
Based on the soluble (solubilized due to enzymatic hydrolysis)
collagen content in the supernatant solution of the ChC treated BAT the %
degradation of collagen for native and both the extract treated tendons are
calculated as
100xcollagenInitial
collagenlelubSocollagenInitial100redationdegcollagen%
4.2.3.2 Monitoring of BAT Degradation by Inhibition of ChC Activity
% of inhibition of ChC by WSE and CHE were calculated by the
following method. A known amount of ChC in 1 ml of 0.1 M Tris-HCl (pH
7.4) containing 0.05 M CaCl2 at 25 °C was incubated with 0, 5, 10, 20, 40
and 80 g of both the extracts for 18 h. Subsequently the achilles tendons
were treated with the incubated samples of native and both the extract treated
133
100deg%
deg%deg%%
extract
ChCnative
plantChCnative
radationcollagen
radationcollagenradationcollagenInhibition
ChC. The ratio of collagen: ChC was maintained at 50: 1 and the reaction
buffered at pH 7.4 using 0.1 M Tris-HCl and 0.05 M CaCl2. The treated
samples were incubated at 37°C and after 72 h the reaction was stopped and
the mixture was centrifuged for 15 min at 10000 rpm. The supernatant was
analyzed for Hyp (Ryan et al 1971) and % collagen degradation was
determined. % inhibition by both this plant extract was calculated as
differences in the % degradation of BAT collagen treated by native and both
the plant extracts treated ChC
4.2.4 Extraction of BAT Collagen
The BAT, collected fresh from local slaughter house, were
manually dissected out from surrounding fascia, followed by washing in
distilled water. They were cut into small bits of 3-4 mm each with a sharp
knife and were solubilized by a patented procedure developed at CLRI
(Sehgal et al 2001).
4.2.4.1 Inhibition of ChC activity by zymography analysis
The ChC is treated with varying concentrations viz., 0, 5, 10, 20, 40
and 80 g of aqueous solution of WSE and CHE for 24 h at 25°C. Both the
extracts treated ChC were directly applied to a 7.5% SDS-PAGE that
contained 0.1% collagen. After electrophoresis, the gel was washed twice
with 2.5% Triton X-100 for 30 min, three times with water for 10 min, and
incubated in a solution of 50 mmol/l Tris-HCl, 5 mmol/l CaCl, 1 mol/l
ZnSO4 (pH 8.0) for 5 days at 37°C. The gel was stained with Coomassie blue.
134
4.2.5 Kinetic Investigations on the Assay of Native ChC
ChC assay using FALGPA as substrate was performed according to
the method reported earlier (Van Wart et al 1981). Assays were carried out
spectrophotometrically by continuously monitoring the decrease in
absorbance of FALGPA after the addition of ChC. The FALGPA
(at concentrations of 0.1–1.6 mM) was taken in appropriate amount of Tricine
buffer (0.05 M Tricine, 0.4 M NaCl and 10 mM CaCl2, pH 7.5) and ChC (100
l of 0.4 mg/ml) was added and the final volume was adjusted to 1 mL. The
course of hydrolysis of FALGPA was monitored using Varian Cary 100 UV–
visible spectrophotometer by following the decrease in absorbance at 324 nm
when [FALGPA] = 0.1 mM. At higher concentrations of FALGPA viz. (0.2
mM) and (0.4–1.6 mM), the decrease in absorbance were measured at 338
and 345 nm, respectively. An initial rate treatment was adopted by treating the
first 10% of hydrolysis according to standard methods (Espenson et al 1981).
4.2.5.1 Kinetic Investigations on Inhibition of ChC
The reaction of WSE and CHE treated ChC with FALGPA were
performed under the same conditions mentioned as employed for the assay of
native ChC in this study. The ChC is treated with varying concentrations viz.,
0, 10, 20, 40, 80 and 160 g of aqueous solution of both the extracts for 18 h
at 25 °C. The final concentrations of the ChC in all the treatments are
maintained constant (0.4 mg/mL). The FALGPA (at concentrations of 0.1–1.6
mM) was taken in appropriate amount of Tricine buffer (0.05 M Tricine, 0.4
M NaCl and 10 mM CaCl2, pH 7.5) and WSE incubated ChC (100 l) was
added and the final volume is adjusted to 1mL. The hydrolysis of substrate
was monitored at the corresponding wavelengths (immediately after the
addition of both of the extract incubated ChC) as done in the case of native
ChC. The concentrations of substrate (FALGPA) used were in the range of
0.1–1.6 mM. Rates of hydrolysis were calculated employing initial rate
135
methods. The rate data were analyzed in terms of Michaelis–Menten
treatment. From the Linewaver-burk plots of v-1
vs [S] -1 the kinetic
parameters such as Vmax, maximum velocity and Km, the Michaelis-Menten
constant of the enzyme were calculated. Initial velocities were calculated from
the slope of the absorbance changes during the first 10% of hydrolysis and
converted into units of microkatals ( mol/s) by dividing to full hydrolysis and
multiplying by the substrate concentration.
4.2.6 Circular Dichroism Spectral Analysis of Plant Extracts Treated
ChC
Circular dichroism spectrum of Type IA ChC of concentration
0.18 mg/ml in 1 mM acetate buffer (pH 4.0) was acquired at 25 °C using
Jasco-J715 spectropolarimeter. The conformational changes in ChC on
interaction with WSE and CHE were investigated after incubating the enzyme
with varying concentrations (10–80 g) of both these extracts.
4.2.7 Statistics
Results were expressed as mean values and standard deviations (±SD).
4.2 RESULTS
Even though several MMPs are involved in ECM degradation in
both RA and OA, the two collagenases, MMP-1 and MMP-13 and also some
other bacterial collagenases are probably the major mediators of collagen
degradation and the irreversible joint destruction. The antiproliferative,
proapoptotic, anti-invasive, antiosteoclastogenic, antiangiogenic,
antimetastatic, radiosensitizing, antiarthritic, and cardioprotective effects
assigned to sterols and withanolides may be mediated in part through the
suppression of NF- B and NF- B-regulated gene products (Ichikawa et al
2006). A study was carried out to determine the inhibition of ChC activity for
binding of WSE and CHE to the active site of ChC. It is of profound interest
136
to establish how the plant extracts exhibit dose dependent inhibition to the
ChC activity against collagen breakdown.
4.3.1 Stability of WSE and CHE Treated BAT Against ChC
The % of collagen degradation (based on Hyp released) for the
native, WSE and CHE treated BAT by ChC at various time periods has been
determined (Figure 4.2(a) and (b)). Significant reduction in the degradation of
collagen is observed for the fibres treated with the plant extract viz., both the
extracts compared to native BAT collagen. WSE treated BAT collagen
exhibited 37.64 % (@ 80) degradation of collagen at 72 h period of
incubation, whereas CHE treated BAT collagen exhibited only 33.35 %
collagen degradation for the same time period of incubation. Both these
extracts can interact with collagen through H-bonding and hydrophobic
interactions. The hydrophobic core of the steroidal lactone molecule
incorporates itself likely into hydrophobic areas while hydroxyl moieties of
steroids may establish multiple H-bonds with neighboring collagen
molecules, resulting in improved stabilization of collagen and prevent the free
access of ChC to active sites on the collagen chains. The stability of plant
extracts such as flavonoids, alkaloids and withanolides treated collagen fibres
against ChC would have been brought about by protecting the active sites in
collagen recognized by ChC. The significant differences in the enzymatic
stability offered by both of the plant extracts such as flavonoids, alkaloids and
withanolides could be due to the effectiveness of the latter in exhibiting better
interaction with collagen through multiple H-bonded crosslinks. It is
important to probe further the ability of these plant extract flavonoids,
alkaloids and withanolides extract in the direct inhibition of ChC.
137
Figure 4.2 The % of degradation of (a) WSE and (b) CHE treated BAT
collagen by ChC
138
4.3.2 Inhibition of ChC by Plant Extracts Against BAT Collagen
Inhibition of ChC activity was tested using BAT collagen as a substrate.
WSE and CHE treatment to ChC exhibited dose dependent inhibition on the
collagenolytic activity against BAT collagen degradation (Figure 4.3a and b). The
inhibition increased with increase in concentration of both the plant extracts at 80
g concentration 76.66 and 86.66% inhibition has been observed against ChC
hydrolysis (@ 72 h respectively). Withanolides/ Glycowithanolides having unit of
Steroidal lactone, glycoside and hydroxyl groups can exhibit better H- bonding
and hydrophobic interactions with ChC and hence can influence significant
changes in the conformation of ChC resulting in high inhibition in the activity of
ChC against ECM degradation. The difference in the inhibition of ChC was
statistically significant (Student's t test, p < 0.0005). Because steroidal compounds
demonstrated higher inhibitory properties against ChC, this enzyme was selected
to test the inhibitory activity of flavonoids, alkaloids and withanolides.
4.3.3 Effect of WSE and CHE on ChC by Collagen Zymography
These study conducted by isolated BAT collagen which was
confirmed by SDS-PAGE analysis (Figure 4.4a). In the ChC inhibition assay
of native, WSE and CHE treated ChC determined by collagen zymography,
collagen degradation by native ChC was shown as clear area, and the
intensities of the area were quantified by densitometric scanning and
expressed in relative intensity to collagen as a substrate. Regarding clear band
intensity of degraded collagen by native ChC as 100 after incubation for 24 h,
both the extracts treated ChC had much fewer clear bands, whose relative
clear band intensity was 11.2±2.7.
139
Figure 4.3 The % of inhibition of ChC by (a) WSE and (b) CHE at
various concentrations
140
Figure 4.4 SDS-PAGE of (a) BAT collagen, and gelatin zymography of
(b) WSE and (c) CHE treated ChC
141
From the ChC inhibition assay, it was known that the extracts
treated with ChC decrease the zone intensity on collagen zymography against
collagen degradation by ChC up to about 80%. Both extracts exhibited a
dose-dependent inhibitory effect on ChC activity (20% at 10 g/ml, 60% at
40 g/ml and 75% at 80 g/ml), and complete inhibition was observed at C.
halicacabum concentrations above 80 g/ml (Figure 4.3b and c).
4.3.4 Kinetic Analysis of the Inhibition of ChC
In order to establish the mechanism of inhibition of ChC activity by
plant extracts ChC treated with varying concentrations of WSE and CHE has
been studied for their ability to hydrolyze FALGPA. To analyze the inhibition
of ChC by WSE and CHE, Michaelis–Menten plot, Lineweaver–Burk plot i.e.
double-reciprocal plot and Dixon plot for the hydrolysis of FALGPA by both
plant extracts treated ChC have been determined (Figure 4.5a and b and 4.6 a
and b). The kinetic parameters like Km, Vmax and Ki are calculated and
tabulated (Table 4.2).
4.3.5 Inhibition Kinetics of ChC
To determine the nature of the inhibition of ChC by WSE and
CHE, experiments were carried out using known amounts of ChC in the
presence or absence of various concentrations of both plant extracts
respectively. The velocity (v) of the reaction was calculated for each substrate
concentration (S), and the data were presented in a Michaelis menton plot (v
versus S) and Lineweaver-Burk plot (1/v versus 1/S) to determine the kinetic
parameters. The Michaelis–Menten plot obtained for FALGPA hydrolysis by
ChC incubated with different concentrations of both the plant extracts
revealed (Figure 4.5 (a) and 4.6(a)) competitive and mixed type inhibition.
142
Figure 4.5 Lineweaver–Burk of FALGPA hydrolysis by ChC in the
presence of a) WSE and b) CHE. Assays were performed in
Tricine buffer pH 7.5, with varying concentrations of
FALGPA (0.1–1.6 mM)
143
Figure 4.6 Dixon plots of FALGPA hydrolysis by ChC in the presence
of a) WSE and b) CHE respectively. Assays were performed
in Tricine buffer pH 7.5, with varying concentrations of
FALGPA (0.1–1.6 mM)
144
The Lineweaver–Burk plots i.e. double-reciprocal plots obtained
for FALGPA hydrolysis by ChC incubated in the presence of different
concentrations of WSE revealed that there was no change in the 1/Vmax
compared to control, it is known that Vmax is unchanged by the presence of a
competitive inhibitor and CHE revealed that there was change in the 1/Vmax
by the presence of a mixed type of inhibitor. Increasing concentration of
inhibitor [I] results in a family of lines with different slopes because the
intercept on the 1/V axis is equal to 1/Vmax. That is, regardless of the
concentration of a competitive inhibitor, a sufficiently high substrate
concentration will always displace the inhibitor from the enzymes active site.
Both the extracts exhibited dose-dependent inhibition on CHC activity, and
Lineweaver–Burk plots clearly showed a competitive and mixed type of
inhibition respectively (Figure 4.5 (a) and 4.6 (b)).
4.3.6 Determination of Michaelis–Menten Constant
The Michaelis–Menten constant (Km) obtained from Lineweaver–
Burk plots for native ChC was Km=0.55 mM for the substrate FALGPA at 25
°C pH=7. This Km value of the FALGPA hydrolysis by ChC is found to be
similar to that obtained earlier (Ryan et al 1971). The Vmax was found to be
0.2439±0.05 m mol s 1. From the Michaelis–Menten constants (Table 4.2),
the addition of 160 g of both these extracts to ChC resulted in Km values of
3.33 0.007 and 1.612 0.002 mM respectively. And then six lines obtained
with enzyme only and five concentration of WSE intercept at the same
position on the y-axis, indicating that Vmax is the same in all cases (Table
4.2). Furthermore, the intercept of the lines on the x-axis demonstrates that
the Km value increases when the WSE concentration increases. This is
characteristic of a competitive mode of inhibition. While Vmax and Kcat values
obtained for ChC were unchanged in the presence of W. somnifera , clear
changes were observed in the Km and kcat/Km values. These results further
verify that the inhibition of ChC by W. somnifera is competitive mode,
145
indicating that in the presence of withanolides, ChC is less effective and
therefore has a lower specific activity and C. halicacabum has a mixed type of
inhibition.
Table 4.2 Michaelis-Menten parameters for ChC hydrolysis of
FALGPA at 25 °C, pH 7.5 in the presence of varying
concentrations of WSE and CHE
Nature of
Treatment
Plant
extract
[µg]
Km
(mM)
Vmax
(mmol s-1
)
Type of
inhibition
Ki
(µg)
Control - 0.555 0.2439
WSE
10
20
40
80
160
0.625
0.714
0.833
1.666
3.333
0.2439Competitive
type20
CHE
5
10
20
40
80
0.349
0.709
0.893
1.351
1.612
0.1551
0.2059
0.1902
0.2418
0.1598
Mixed type
There are 3 main types of inhibition (competitive, noncompetitive,
and uncompetitive) that are most commonly used to describe the binding of
an inhibitor to ChC. A schematic representation of these types of inhibition is
provided.
Figure 4.7(a) Competitive inhibition where substrate (S) and inhibitor
(I) binding events are mutually exclusive
146
Figure 4.7 (b) Examples of noncompetitive inhibition where Inhibitor (I)
binding occurs at a site distinct from the substrate (S)
binding site and the catalytic center (c) of the active site
Figure 4.7(c) Uncompetitive inhibition where inhibitor (I) only binds in
the presence of substrate (S)
4.3.7 Determination of the Inhibition Constant
The inhibition constant Ki is obtained from a Dixon plot of V-1
vs.
various concentrations of W somnifera and substrate (Figure 4.6a). For the
determination of the Ki of W somnifera against ChC, the velocity of cleavage
of FALGPA was measured at WSE concentrations ranging from 10 to 160 µg.
In this plot, the reciprocals of velocity were plotted against inhibitor
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concentration at different concentrations of substrate. A series of straight lines
was obtained, converging to the same point, and that value represents -Ki. It
was found that the Ki for W somnifera is 20 µg. The competitive mode of
inhibition exhibited by W. somnifera may work through direct competition
with the substrate by binding to the active site, or binding to a remote site and
causing a conformational change in the ChC. Both the mechanisms give
identical positive results. It is important to study if there are any changes in
the conformation of ChC on the binding of the WSE and CHE due to
competitive and mixed type of inhibitions.
4.3.8 Conformational Changes of WSE and CHE Treated ChC
The effect of the both the extracts on the conformation of ChC has
been investigated by monitoring CD spectral changes (Figure 4.8a and b). In
the far UV region, ChC exhibits double minima at about 210 and 220 nm and
a maximum at 195 nm with a crossover point at about 200 nm. ChC has
helix, sheet, turn and random coil structure. CD spectrum of the native
ChC exhibits double negative bands at 208 and 222 nm and a positive band
around 195 nm. These results are characteristics of helix, whereas a
peptide/protein having sheet conformation alone will exhibit a single
negative band between 215 and 225 nm.
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Figure 4.8 CD spectral analysis of ChC treated with different
concentration of (a) WSE and (b) CHE
ChC has both helix and sheet conformation almost equally
distributed as its secondary structure, which may not be clearly
distinguishable through the CD spectrum. The structure of ChC changes
significantly to sheet after interaction with both the plant extracts (80 g),
which is observed in the spectra where a negative band at 208 nm shifts to
215 nm, which is characteristic of sheet (Figure 4.8 (a)). Treatment of low
concentration (40 g) of C. halicacabum changes the structure of ChC to
sheet and with further increase in concentration of C. halicacabum, the
structure is shifted more towards random coil conformation (Figure 4.8 (b)).
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4.4 DISCUSSION
MMP inhibitors were investigated recently in several animal
models of human diseases, mainly cancer and arthritis, and promising
pharmacological effects have been observed in many cases. Thus, as the
controlled degradation of ECM is crucial for growth, invasive capacity
metastasis, and angiogenesis in human tumors inhibition of some of the
enzymes involved, such as the MMPs, can lead to the interrelation of novel
anti-cancer therapies based on inhibitors of the proteases. The controlled
degradation of collagen in both healthy and diseased conditions has received
much attention in recent years. MMP-1, 8, 13, 18 and MMP-14 collagenases
(MT1-MMP) are known to target Type I collagen degradation (Salminen et al
2002; Giambernardi et al 2001). The collagenolytic activity of collagenase on
collagen fibrils and reflect a depolymerization on the ECM, and than
inhibition of collagenase I and III activity is very important for the controlled
breakdown of ECM of collagen in RA and OA and multi-stage carcinogenesis
condition (Wallach et al 2005; Chang et al 2001; Foda et al 2001; Rasool et al
2007). In both the diseases, inflammatory cytokines such as interleukin-1 beta
(IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) stimulate production
of MMPs. Expression of other MMPs such as MMP-2, MMP-3 and MMP-9,
is also elevated in arthritis and these enzymes degrade non-collagen matrix
components of the joints tissue. Many researches have demonstrated that
collagen stability on ECM and inhibition of MMPs activity could be enhanced
through various plant secondary metabolites such as plant flavonoids,
alkaloids, phenolics and polyphenolics (Lim et al 2007). Collagen breakdown
in the uteri of rats was blocked by oestradiol (Woessner et al 1961; Woessner
et al 1973). The anti-arthritic effect of green tea ployphenol has also been
studied (Singh et al 2010; Haqqi et al 1999). Various studies indicate that, W.
somnifera and C. halicacabum possess antioxidant, anti-microbial, anti-
mutagenic and anti-carcinogeneic, anti-stress effect, immunomodulatory,
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hemopoetic, rejuvenating activity, as well as exerting an influence on the
endocrine, nervous and cardiopulmonary systems has also been already
reviewed (Mishra et al 2000; Gopalakrishnan et al 1976).
The possible use of inhibitors for collagenase in degenerative
diseases such as RA and OA is also being investigated. There are lot of
hydroxyl group present in plant flavonoids that inhibit the collagenase activity
and interact with collagen backbone (Cervantes-Laurean et al 2006; Ronziere
et al 1981). The nature of interaction of the flavonoids, alkaloids and
vegetable tannin with collagen and collagenases are currently under
investigation. While most of the current interest in the WSE and CHE relates
to its antioxidant and anti-carcinogenic potential (Davis et al 2000; Sheeba et
al 2009), the focus of the present work is on the potential for both the extracts
to control the ChC activity against ECM degradation. The loss of ECM
elements, including Type I, II and III collagen, reflects MMP mediated
degradation of collagen and control of growth factor activation and in
downstream inhibition of gene expression such as matrixins are reviewed
(Newby et al 2000).
This investigation was undertaken to evaluate the inhibitory effect
of WSE and CHE on the ChC activity in a dose dependent manner and
associated with ECM of BAT collagen degradation. Both the extracts
exhibited strong inhibitory effect against ChC activity. Inhibitory effect of
flavonoids on the host enzymes MMP-2 and MMP-9 have been already
studied (Demeule et al 2000; Hazgui et al 2008; Kim et al 2008). W.
somnifera roots powders detected mainly its anti-inflammatory activity in
animal models of joint diseases has been studied (Mishra et al 2000). It has
also been shown effective in the treatment of experimentally-induced gouty
arthritis in rats (Rasool et al 2007) and the chondroprotective effect potential
in OA have been studied (Sumantran et al 2007). A case study of C.
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halicacabum as a traditional herb for treating RA has been studied
(Ragupathy et al 2007). That inhibition increased with increasing
concentration of both the extracts has been observed against ChC hydrolysis
of ECM of Achilles tendon collagen, and which is indicative effectiveness of
the flavonoids, alkaloids and withanolides in direct inhibition of ChC.
Secondly, these ChC inhibition kinetic parameters characterize
different aspects of WSE and CHE treated ChC interaction with FALGPA.
The inhibition of ChC occurs through binding of both the extracts such as
flavonoids, alkaloids and withanolides/ Glycowithanolides, saponin and
tannins to the active part of the enzyme or inhibiting the activation by binding
to the ChC. This kinetic study indicates the inhibition of ChC activity and
provides an insight into possible roles of the extracts in the stabilization of
collagen against ChC promoted hydrolysis of FALGPA. The ability of
different concentrations of these extracts to function as inhibitors is deduced
from the values of Vmax, Km and Ki obtained for the hydrolysis of FALGPA
by ChC. Both the extracts behaves as a competitive and mixed type of
inhibitor respectively against ChC activity and such behavior was also elicited
by flavonoids, withanolides/ glycowithanolides, glycosides, tannins and
polyphenols. These MMPs cleave the three chains of native triple-helical
Type I, II and III collagens after Gly in a particular sequence (Gln/Leu)–
Gly#(Ile/Leu)–(Ala/Leu) (# indicates the bond cleaved) located approximately
three quarters away from the N-terminus of the collagen molecule. The action
of these enzymes is critical for the initiation of collagen breakdown, as once
collagens are cleaved into 3/4 and 1/4 fragments, they denature at body
temperature and are degraded by gelatinases and other nonspecific tissue
proteinases (Figure 4.9 (a)). The primary structures of the collagenases show
that the proteins are similar and modular, each possessing a signal peptide of
20 residues, a propeptide domain (80 AAs) conferring latency, a zinc-
containing catalytic domain of approximately 180 residues, and a substrate
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specificity domain (210 AAs). The three-dimensional scaffold of the catalytic
domain is composed of a five-stranded -sheet and three -helices (Figure 4.9
(b)), similar to the -sandwich architecture observed in zinc-dependent
endopeptidases of this superfamily (Borkakoti 2004).
Figure 4.9 Cleavage sites in Collagen I by (a) MMP-1 or ChC and (b)
Catalytic domain of ligand-free fibroblast collagenase
(MMP-1) shown in a simplified form
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A deep cleft around the catalytic zinc located towards the
C-terminus of the domain delineates the active site and substrate-binding
region of the protease. Three histidine residues of the protein and atoms of the
bound inhibitor chelate the catalytic zinc. Additionally, there is second,
structural zinc and several calcium atoms that stabilize the structure.
Withanolides and flavonoids were found to be the preferred chelator for the
preparation of collagenase inhibitors. The observations of the present study on
the behavior of these extracts may be interpreted as follows. It has been
shown experimentally that the side chain carboxyl groups of Aspartic (Asp)
and glutamic acids (Glu) participate in conjunction with tyrosine (Tyr)
residues in the stabilization of the active conformation of the native ChC. The
implication of lysine (Lys) residues in the active site of ChC has been shown
earlier using experiments with specific reagents. The competitive and the
mixed type of ChC inhibition elicited by these two plant extracts may arise
from the binding of withanolides, alkaloids and tannins to the side chain
carboxyl residues of aspartic (Asp) or glutamic acids (Glu), with the net result
of change in the conformation of ChC. It was found that these extracts are a
competitive and mixed type of inhibitor of the ChC activity against FALGPA.
The inhibition of collagenase I by green tea polyphenol and then anticancer
effects of plant flavonoids from green tea have been already reported (Kim et
al 2008). The structure of ChC changes significantly the conformation after
interaction with withanolides and tannins etc., These results suggest that the
steric structure of flavonoids, withanolides and tannins containing steroidal
lactone unit is important for the inhibition of ChC activity.
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4.5 CONCLUSION
This study has provided convincing evidence that WSE and CHE
facilitated against collagen degradation through collagenase inhibition. The
exact type of mode of inhibition of ChC seems to be influenced strongly by
the nature and structure of both the extracts. From these results it is clearly
indicated that the change in the conformation of ChC by these extracts is a
major factor in the inhibition of collagenolytic activity. The hydroxyl groups
of withanolides and alkaloids can act as H-bond acceptor/donors with the
backbone amide and other side chain functional groups viz., hydroxyl, amino
and carboxyl groups of ChC. Therefore these extracts provide selective
inhibition of ChC and could constitute an important aid in the prevention of
collagen scaffold degradation.
