Development of Antioxidant Film Based on Blends of

Stenochlaena palustris Flour and Poly(Vinyl Alcohol)

Nazira Mahmud, Khairul Farihan Kasim, Akmal Hadi Ma’ Radzi

School of Bioprocess Engineering, Universiti Malaysia Perlis, Perlis, Malaysia.

Abstract - The aim of this work was to develop antioxidant films based on blends of Stenochlaena
palustris flour and poly(vinyl alcohol) (PVA). Four different PVA types were tested. All films
produced were analyzed for their antioxidant activity by diphenyl-picrylhydrazyl radical
scavenging (DPPH) method. S. palustris flour – PVA 88% blends film contains the highest
antioxidant activity. The kinetic study of the selected film shows the EC 50, TEC50 and Antioxidant
Efficiency value of 20 mg/ml, 55 min and 0.91×10 -3 respectively. The use of low concentration PVA
and addition of glycerol made the film more flexible with greater elongation at break but low tensile
strength. The thermal analysis shows the thermal degradation of the film produced.

I. INTRODUCTION

In recent years, antioxidants have gained a lot of importance because of their potential as
prophylactic and therapeutic agents in many diseases. Antioxidants are substances which can greatly
reduce the adverse damage due to oxidants by crumbling them before they react with biologic targets,
preventing chain reactions or preventing the activation of oxygen to highly reactive products [1]. Plant
sourced food antioxidants like vitamin C, vitamin E, carotenes, phenolic acids, phytate and phytoestrogens
have been recognized as having the potential to reduce disease risk [2]. The use of natural blends of
protein, polysaccharides and lipids directly obtained from agricultural sources, takes advantage of each
component in the original system and appears to be a new opportunity for materials in the area of
biodegradable films [3]. Recently, many attentions had been paid on preparing the totally biodegradable
blends with the natural polymers and their derivatives, such as gelatin/poly(vinyl alcohol) [4], starch/
poly(vinyl alcohol) [5], plant-derived natural biodegradable material/ poly(vinyl alcohol) [6],etc. These
blends present good compatibility and retain biodegradability. However, there is no further research
conducted on determining the antioxidant content of film produced from the blends of polymer and natural
material.

Stenochlaena palustris (Burm) Bedd. a scrambling fern, is distributed in a large part of the tropical
areas from southern and northern India through Malaysia to Polynesia and Australia [7]. The red young
fronds are used as spinach and are said to be particularly beneficial when eaten at breakfast-time during
the fasting month. The juice is used to cure fevers, the long rhizomes to bind baskets and fish traps, and a
very durable rope can be made from the stems [8].

Poly(vinyl alcohol) (PVA) is a semi-crystalline, highly biocompatible, non-toxic hydrophilic

polymer with good chemical and thermal stability. PVA is recognized as a biodegradable polymer [9]. PVA
has the capability of H-bonding with its solvents and decreases their surface tension as other surface active
agents [10].
 Antioxidants which have been used in diet or as formulated agents have some common problems in
their efficacy as therapeutic agents which are related to their physicochemical and biopharma-ceutical
properties [11]. Many of the antioxidants have poor oral bioavailability, due to low solubility,
permeability, stability and/or drug biotransformation before they reach systemic circulation [12].
Development of antioxidant films based on blends of S. palustris flour and poly(vinyl alcohol) (PVA) will
eliminate the utilization of antioxidant formulation through oral route. This antioxidant films can be used
mainly in cosmetic applications and a potential material in production of biodegradable packaging or
plastic.

II. MATERIALS

A. Chemicals
Poly(vinyl alcohol)(99-100%, 95%, 88%, & 75%) were purchased from Acros Organics. 2,2-
diphenyl-1-picrylhydrazyl free radical (DPPH) was purchased from Merck. Kaempferol, myricetin,
quercetin dihydrate, and rutin hydrate were purchased from Sigma-aldrich Co. Glycerol and methanol
were purchased from HmBG.

Characteristics of the PVA used were tabulated in Table 1.

Table 1: Characteristics of poly(vinyl alcohol) used.

PVA types Molecular weight range
99-100% 86000
95% 95000
88% 88000
75% 2000
*PVA types indicate the percentage hydrolysis degree of each PVA.

B. Plant materials
S. palustris were collected from open area at Wang Tepus, Jitra, Kedah. Fresh S. palustris leaves were
dried in the oven at 60˚C for 24 hrs. The dried samples then ground and the resulting flour were sieved at
45 µm to produce fine flour.

III. PROCEDURES

A. Film preparation
S. palustris flour/PVA blended films were made by the casting technique. The two solutions were first
prepared separately.

The S. palustris flour solution was prepared with 4 g of S. palustris flour/100 g solution. The
S. palustris flour and distilled water were mixed for 2 hrs with a magnetic stirrer. The S. palustris flour
dispersion was then heated to 75˚C and glycerol (20 g glycerol/100 g polymers) was added. The
temperature was increased at 85˚C for 60 min with constant magnetic stirring [9].
 The PVA solution was prepared with PVA (4 g/100 g solution) and distilled water. The PVA was
stirred for at least 4 hrs at room temperature, and then heated at 85 to 95˚C depending of the PVA type,
until the PVA was completely dissolved [9]. Films were produced by varying the PVA content from 10%
to 100% (w/w) of the dry matter.

Blends of S. palustris flour and PVA solutions were prepared by mixing these two solutions. The
blends were heated at 85˚C for 1 hr with constant stirring but avoid frothing to increase the interaction
between the components [9]. Water was added to maintain the volume. The film forming solutions were
cast onto a glass plate with thickness of about 0.5 - 1 mm, after which air bubbles were removed by
flaming and each solution was dried for 24 hrs at 55 ˚C to form the desired films. Complete drying was
avoided as some moisture was required for films to remain flexible and not to crack. The films were
finally removed (by peeling) from the glass [5].

B. Antioxidant assay
The antioxidant properties of the films were carried out by using DPPH method as described in [13]
with slight modification. An aliquot of 22 µL of the sample was added to 200 µL of DPPH (60 µM) in a
96-well flat-bottom microplate. The absorbance was recorded in a VersaMax microplate reader at 517 nm
after 30 min incubation time at 37˚C [14]. The radical scavenging activity was calculated by the
following equation:

DPPH activity ( % ) = 1 -
[ Absorbance of sample at 517nm
Absorbance of DPPH at 517nm
×100
]
C. Efficiency analysis
Efficiency analysis involves antioxidant determination of serial film concentration. An aliquot of
22 µL of the sample was added to 200 µL of DPPH (60 µM) in a 96-well flat-bottom microplate. The
absorbance was recorded in a VersaMax microplate reader at 517 nm after 30 min incubation time until
plateau at 37˚C [14]. The remaining level of DPPH (% DPPH REM) in the reaction medium was calculated
using the following relation [15]:

% of remaining DPP H . =100 . ( A s 517 nm ( t=30 ) / A c 517 nm ¿

Where As 517 nm is the absorbance of the sample measured at t = 30 min and A c 517 nm is the absorbance of the
control. The percentage of remaining DPPH against the standard concentration was then plotted to obtain
the amount of antioxidant necessary to decrease the initial DPPH concentration by 50 %. The time
needed to reach the steady state to EC 50 concentration (TEC50) was calculated graphically. Taking into
account that both, EC50 and TEC50, affect the antiradical capacity, a new parameter: antiradical efficiency
(AE), which combines these two factors, was defined [16]:

AE = 1/ EC50 T EC 50

D. Film characterization
i. Thermal analysis
The thermal analysis methods used were the thermogravimetric analysis (TGA) and differential
scanning calorimetry (DSC).

TGA analysis was carried our using a Perkin Elmer (TGA 7) under nitrogen atmosphere. Samples
(about 3 mg) were heated from room temperature to 900 ˚C at a heating rate of 10 ˚C/min.

Thermal analysis instruments DSC model Q100 was used for measurement of glass transition
temperatures. 3-4 mg of the sample was crimped in aluminum pan and loaded along with reference pan
without sample. The heating rate was 10 ˚C/min, and heating was carried out up to 900˚C.

ii. Mechanical properties measurement

The mechanical properties were determined using tensile and elongation at break test. The tensile
strength (TS) and elongation at break (EB) of the films in both dry and wet states were measured on a
universal testing machine Hounsfield (H10KS), at a tensile rate of 10 mm/min. The wet films were
measured immediately after immersed in water for 30 min. Three dumbbell shaped specimens (ASTM
D882) were cut from film. Specimens had a width of 3.73 mm. The specimen’s average thickness was
about 2.15 mm. The TS and EB values are the averages of three measurements.

IV. RESULTS AND DISCUSSION

A. PVA selection
Four different types of PVA were tested in the formulation of blended films with equal proportions of
S. palustris flour – PVA. All films were homogeneous and easily peel off from the support except for
PVA 75%. The characteristics of the film produced were simplified in Table 2. Even though it had been
dry for 72 hrs, the S. palustris flour – PVA 75% film was unable to peel smoothly. This may due to the
low compatibility between this partially hydrolyzed and low molecular weight PVA and the S. palustris
flour [9].

Table 2: Characteristics of film produced

PVA Types Film produced
99-100% Easily peeled at high conc. and peelable at low conc.
95% Easily peeled at high conc. and peelable at low conc.
88% Easily peeled at high conc. and peelable at low conc.
75% Not peeled at high and low conc., tend to rupture when
separate from support
*PVA types indicate the percentage hydrolysis degree of each PVA.

There were inter and intra chain bonding between the polar hydroxyl groups in the PVA molecules
which will be disrupted during dissolution process but may reform during cooling or solvent evaporation.
At high molecular weight PVA, the solution viscosity increases significantly and hence reduced rate of
solvent evaporation [17]. When using low molecular weight PVA, the viscosity of solution was much
lower and so increased the rate of solvent evaporation. This conditions cause the inter and intra chains
bonding of hydroxyl group in PVA reform and consequently, less formation of hydrogen bond between
the PVA chains and water molecules. Low molecular weight PVA comparatively has low ability of film
producing [17].

B. Antioxidant activity
The antioxidant activity of the S. palustris flour – PVA films produced were carried out after 1 hr
incubation time at 37 ˚C at five different PVA concentration (10, 32.5, 55, 77.5 & 100%) with three
different film concentration (25, 50 & 100 mg/ml) for each type of PVA. Figure 1, 2 & 3, shows the
antioxidant activity of S. palustris flour – PVA 99-100% blends film, S. palustris flour – PVA 95% blends
film & S. palustris flour – PVA 88% blends film respectively. The antioxidant analysis conducted on PVA
88%, PVA 95% and PVA 99-100% films without S. palustris flour (control film) gave the result of 0%
antioxidant activity. It is proven that S. palustris species contain natural antioxidant and the antioxidant
source of S. palustris flour – PVA blend film was from S. palustris flour itself.

Antioxidant activities were lowest at 100% PVA concentration for all type of PVA. Comparing all
three types of PVA, PVA 88% shows highest antioxidant content for all concentration used. While PVA
95% shows lowest antioxidant content compare to the other two types of PVA, might be due to the higher
molecular weight of PVA 95%.

It is well known that in general plastic industry, oxidative stabilizers were added in order to improve
the properties of plastic produced. The reason for low antioxidant content of S. palustris flour – PVA 95%
film could be at high concentration of PVA, films act as oxygen carrying agent [18]. Thus, S.
palustris flour stabilizes PVA structure acting as radical scavengers (stabilization effect). Thus, S.
palustris flour stabilizes PVA structure acting as radical scavengers (stabilization effect). Substitution of
H+ by S. palustris flour leads to the increasing electron donating effect of the hydroxyl group, which
induce the decreasing antioxidant activity of S. palustris [19].

The highest antioxidant activities were recorded from film developed from PVA 88% at 55% PVA
concentration.

100 25 mg/ml dilution rate

50 mg/ml dilution rate
100 mg/ml dilution rate

80
Antioxidant activity (%)

60

40

20

PVA conc. (%)

 Figure 1: Antioxidant activity of S. palustris flour – PVA 99-100% blends film at different concentrations of the
PVA and different dilution rate of the film. Values are the means ± SEM (n = 4).

100
25 mg/ml dilution rate
50 mg/ml dilution rate
100 mg/ml dilution rate

80

Antioxid ant activity (%)

60

40

20

PVA conc. (%)

Figure 2: Antioxidant activity of S. palustris flour – PVA 95% blends film at different concentrations of the PVA
and different dilution rate of the film. Values are the means ± SEM (n = 4).
100
25 mg/ml dilution rate
50 mg/ml dilution rate
100 mg/ml dilution rate

80
Antioxidant activity (%)

60

40

20

PVA conc. (%)

Figure 3: Antioxidant activity of S. palustris flour – PVA 88% blends film at different concentrations of the PVA
and different dilution rate of the film. Values are the means ± SEM (n = 4).

C. Efficiency analysis
Efficiency analyses were conducted on S. palustris – PVA 88% (55% PVA concentration) blend film
at varies film concentration (Figure 4). Reacting DPPH solution with each dilution rate of sample films
for every 30 min incubation time until plateau at 37 ˚C facilitates the extraction of antioxidant
compounds from the sample thereby increasing the measured antioxidant activity of the sample. The
results showed that the absorbance decreased as the radical was scavenged by antiradicals, through
donation of hydrogen, to give the reduced form DPPH-H.

Basically, the antioxidant activities of the blends films were increased with concentration up to
20 mg/ml and future increased up to 100 mg/ml resulted in significant decrease of antioxidant activity.
This might be explained by the fact that at higher concentrations, films act as oxygen carrying agent and
serves as pro-oxidant in the oxidation of lipid. Chlorophylls are known to be well known
photosensitisers, capable of converting triplet oxygen to singlet oxygen, and might also have acted as
prooxidants and ultimately would have reduced the antioxidant capacity of the film [18].

Antioxidant activities for 20 mg/ml film concentration were highest at all incubation time show that
it is the most suitable film concentration to be used in this antioxidant assay.

120

110 30 min
60 min
100 90 min
120 min
90 150 min
180 min
80
Antioxidant activity (%)

70

60

50

40

30

20

10

0
Kaempferol Myricetin Quercetin Rutin 10 15 20 25 40 50 75 100

Dilution rate (mg/ml)

Figure 4: Effect of dilution rate (mg/ml) on antioxidant activity of S. palustris – PVA 88% blends films. Values
show means ± SEM (n = 4).

i. Antioxidant behavior
Figure 5 illustrates kinetic behavior of S. palustris – PVA blend film, the values of % DPPH
remaining as a function of time are presented at a concentration of sample in the reaction mixture varies
from 10 mg/ml to 100 mg/ml. In the presence of film solutions a rapid initial decrease of DPPH content
was followed by slow subsequent disappearance of DPPH. There were significant differences between
the slopes after the end of the initial fast step. These differences were related to the role of secondary
slow reactions (dimerization or disproportionation) of the radicals formed [15].
 110 10 mg/ml
100 15 mg/ml
90 20 mg/ml

% DPPH remaining
80
25 mg/ml
70
60 40 mg/ml
50 50 mg/ml
40 75 mg/ml
30 100
20 mg/ml
10
0
0 20 40 60 80 100 120 140 160 180 200
time (min)

Figure 5: Antioxidant behavior of different film concentration.

ii. EC50
Antioxidants in blends film may not necessarily freely available to react with DPPH, hence they react
at different rates and the reaction will often not go to completion in a reasonable assay time. Therefore,
the sample size that can lower the initial absorbance of DPPH solution by 50% has been chosen as the
endpoint for measuring the antioxidant activity [2]. This sample size was called EC 50. The lower EC50, the
higher the antioxidant activity of a compound is [20]. Figure 6 shows the EC50 value was 20 mg/ml.

250

200
% DPPH remaining

f(x) = 1.79x + 13.24

150 R² = 0.94

100

50

0
0 20 40 60 80 100 120
sample conc (mg/ml)

Figure 6: Determination of EC50

iii. TEC50
TEC50 was the time needed to reach a steady state at the concentration corresponding to EC 50. This
parameter was obtained by plotting the times at steady state against the concentration for each antioxidant
compound, as illustrated in Figure 7. Based on TEC50 values determined, the kinetic behavior of the
antioxidant compound can be classified as follows: < 5 min (intermediate); 5 – 100 min (rapid) and > 100
min (slow). The TEC50 value of S. palustris – PVA blend film was about 55 minute.
 20

Time at steady state (min)

0
0 f(x) = 20 40 60 80 100 120
R² = 0
Film conc. (mg/ml)

Figure 7: Determination of the time needed to reach the steady state to EC 50 concentration.

iv. Antioxidant efficiency

DPPH method permits to evaluate not only the electron or hydrogen atom donating properties of
antioxidants, but also the rate of their reaction towards the free radicals. Antioxidant Efficiency (AE) was
defined, which comprises these two aspects in order to characterize easily the behavior of a substance as
antioxidant [16]. The lower the EC50, the lower the T EC and the higher the AE. From calculation, the
50

AE of S. palustris/PVA blend film was 0.91 × 10-3. According to the following classification (AE
-3 -3 -3 -3 -3 -3
≤ 1 × 10 low; 1 × 10 < AE ≤ 5 × 10 medium; 5 × 10 < AE ≤ 10 × 10 high and AE > 10 × 10 very
high) [15], the AE of S. palustris/PVA blend film was suggest the slow rate of reaction.

D. Film characterization
i. Thermal analysis

Thermogravimetric analysis

Figure 8 & 9 shows thermogravimetric curve obtained for PVA 88% film and S. palustris – PVA
88% blend film. TGA curves for S. palustris – PVA 88% blend film show a weight loss in 4 stages. The
first stage ranges between 30 – 105 ˚C and shows about 7.2% weight loss. The second stage of weight
loss starts at 105 ˚C and continues up to 305 ˚C during which there was 81% weight loss due to
degradation of starch [21]. The third and fourth stage which begin at 305 ˚C and 375 ˚C are the small
stage degradation at which the weight loss were 3.6% and 3.4% corresponding to the ending of
degradation process. Decomposition of PVA proceeds in three stages. The first weight loss at
50 – 200 ˚C is due to the moisture vaporization. The second stage, which begins at 250 ˚C, mainly
involves dehydration accompanied by the formation of some volatile products. In the third stage, the
polyene residues are further degraded at 450 ˚C to yield carbon and hydrocarbons [21].
 Figure 8: TGA thermogram of PVA 88% (55 % PVA conc.) film.

On the basis of analysis of the thermogravimetric curves some characteristic parameters of blend
thermodegradation were determined as: the temperature of initial decomposition (Tdi) and the temperature
at maximum decomposition rate (Tmax) [21].

Figure 9: TGA & DSC thermogram of the S. palustris – PVA 88% (55 % PVA conc.) blends film: ( ) TGA; (---)
DSC.

Differential scanning calorimetry (DSC) analysis

The DSC curves obtained for PVA 88% film and S. palustris – PVA 88% blend film are shown in
Figure 9 & 10. The PVA 88% film and S. palustris – PVA 88% blend film showed Tg at 168 ˚C and 138
˚C respectively. An endothermic peak, due to the melting (Tm) of the crystalline phase present in PVA
88% film and S. palustris – PVA 88% blend film can be observed at 197 ˚C and 243 ˚C respectively. The
differences in the observed Tm values were due to the addition of glycerol to the S. palustris – PVA
88% blend film. The addition of S. palustris flour reduces the PVA crystallinity and as a consequence the
decreases of PVA melting point were observed on S. palustris – PVA 88% blend film. The values of
temperature of initial decomposition (Tdi), temperature at maximum decomposition rate (T max), glass
transition temperature (Tg), and melting temperature (Tm) were simplified in Table 3.

Figure 10: DSC thermogram of PVA 88% (55 % PVA conc.) film.
Table 3: The values of temperature of initial decomposition (T di), temperature at maximum decomposition rate
(Tmax), glass transition temperature (Tg), and melting temperature (Tm).
Films Tmax Tg Tm (˚C)
Tdi (˚C) (˚C)
(˚C)

PVA 88% 50 250 – 450 168 170

S. palustris – 30 105 – 305 138 243
PVA 88%

ii. Mechanical properties

Figure 11 shows the variations of tensile strength and elongation at break of the film. The values of
tensile strength and elongation at break after performed the experiment at triplet were
0.0977 ± 0.0144 MPa and 266 ± 19.95% using 0.5500 ± 0.3606 N load at break. In [22], the tensile
strength and elongation at break for 88% PVA film were 3 Mpa and 155 % respectively.

Comparing these values, it can be seen that the tensile strength of 88% PVA was higher than S.
palustris flour – 88% PVA while the elongation at break value of 88% PVA was lower than S.
palustris flour – 88% PVA. It shows that film produced were actually low in elasticity due to effect of
low concentration of PVA and plasticization. The use of glycerol as plasticizer caused a reduction in the
resistance (decrease in TS) and an increase in the flexibility (increase in EB) of the films [23]. This
behavior, typical of the plasticization phenomenon, is a consequence of the increase in molecular
mobility with the addition of plasticizer.
 0.12 Tensile strength (MPa) 290
Elongation at break (%)
0.1 280

Elongation at break (%)

Tensile Strength (MPa)
270
0.08
260
0.06
250
0.04
240

0.02 230

0 220
0.65 0.85 0.15
Load at break (N)

Figure 11: The tensile strength and elongation at break of S. palustris – PVA 88% (55 % PVA conc.) blend film
for different load at break.

V. CONCLUSION
S. palustris flour-PVA blend film had been successfully developed by using three types of PVA;
88%, 95%, and 99-100%, however unsuccessfully by using PVA 75%. The highest antioxidant activity
was show by the film formulated with PVA 88% at 55% PVA concentration. The efficiency analyses
conducted shows that 20 mg/ml of the film concentration gives the highest antioxidant activity. The EC 50
value also shows the concentration of 20 mg/ml was needed to reduce radical concentration by 50%.
Antioxidant efficiency (AE) of the film, gives the value of 0.91 × 10 -3 indicate the slow rate of reaction as
antioxidant. The use of glycerol plasticizer gives high elongation at break but low tensile strength of
films produced. The TGA and DSC analysis shows the thermal degradation of polymer. The degradation
of PVA 88% film and S. palustris – PVA 88% blend film were highest at the temperature range of
250 – 450 ˚C and 105 – 305 ˚C respectively.

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