Pharmaceutical Biology

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Antioxidant, anti-collagenase and anti-elastase

activities of Phyllanthus emblica, Manilkara zapota
and silymarin: an in vitro comparative study for
anti-aging applications

Sirinya Pientaweeratch, Vipaporn Panapisal & Anyarporn Tansirikongkol

To cite this article: Sirinya Pientaweeratch, Vipaporn Panapisal & Anyarporn Tansirikongkol
(2016) Antioxidant, anti-collagenase and anti-elastase activities of Phyllanthus�emblica, Manilkara
zapota and silymarin: an in�vitro comparative study for anti-aging applications, Pharmaceutical
Biology, 54:9, 1865-1872, DOI: 10.3109/13880209.2015.1133658

To link to this article: https://doi.org/10.3109/13880209.2015.1133658

Published online: 24 Feb 2016.

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PHARMACEUTICAL BIOLOGY, 2016
VOL. 54, NO. 9, 1865–1872
http://dx.doi.org/10.3109/13880209.2015.1133658

RESEARCH ARTICLE

Antioxidant, anti-collagenase and anti-elastase activities of Phyllanthus

emblica, Manilkara zapota and silymarin: an in vitro comparative study
for anti-aging applications
Sirinya Pientaweeratch, Vipaporn Panapisal and Anyarporn Tansirikongkol
Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand

ABSTRACT ARTICLE HISTORY

Context Phyllanthus emblica L. (Euphorbiaceae) (amla), Manilkara zapota L.P. Royen (Sapotaceae) Received 3 April 2015
(sapota) and silymarin are reported to contain antioxidant effects. However, information on other Accepted 14 December 2015
biological activities relating to the anti-aging properties is limited. Revised 6 October 2015
Objective To compare in vitro antioxidants, anti-collagenase (MMP-1 and MMP-2) and anti-elastase Published online
properties as well as the phenolic and flavonoid contents of amla, sapota and silymarin as potential 12 February 2016
anti-aging ingredients. KEYWORDS
Materials and methods The ethanol amla and sapota fruit extracts were prepared by three cycles Amla; MMPs total flavonoid
of maceration with 24 h duration each. The total phenolic (TPC) and flavonoid (TFC) contents were content; sapota; total
determined. The antioxidant capacity was evaluated by DPPH and ABTS assays. The effects of phenolic content
MMP-1, MMP-2 and elastase inhibitions were determined by using the EnzChekÕ assay kits
(Molecular-Probes, Eugene, OR).
Results Amla exhibited the highest in TPC (362.43 ± 11.2 mg GAE/g) while silymarin showed the
highest in TFC (21.04 ± 0.67 mg QE/g). Results of antioxidant activity by DPPH and ABTS methods
showed that amla possessed the most potent capacity with IC50 values of 1.70 ± 0.07 and
4.45 ± 0.10 mg/mL, respectively. Highest inhibitions against MMP-1, MMP-2 and elastase were
detected for sapota with IC50 values of 89.61 ± 0.96, 86.47 ± 3.04 and 35.73 ± 0.61 mg/mL,
respectively.
Discussion and conclusion Test extracts offered anti-aging properties in different mechanisms.
Amla showed the highest phenolic content and antioxidant property with moderate anti-
collagenase. Silymarin exhibited measurable flavonoid content with anti-elastase effect. Sapota
showed the highest collagenase and elastase inhibitions with moderate antioxidant effect. Thus,
extracts might be added as a mixture to gain the overall anti-aging effects.

Introduction proteinases categorized into five sub-groups based on

Aging of skin is a complex biological phenomenon due
to intrinsic and extrinsic factors. Intrinsic factors are
caused by time passage whereas extrinsic factors are
mainly from sun exposure (Assaf et al. 2010). UV
exposure causes physical changes of the skin through
complex pathways and finally generates reactive oxygen
species or ROS, matrix metalloproteinases (MMPs) and
elastase secretion (Demeule et al. 2000). ROS directly
causes skin aging by involving the oxidative damage of
lipids, proteins, and DNA of the skin. ROS can also
indirectly induce the production of MMPs via MAP-
kinase pathway (Fisher et al. 2002; Sim et al. 2007).
MMPs are a group of zinc-dependent extracellular
their substrate: collagenase, gelatinase, stromelysins,
membrane-type MMPs (MT-MMPs) and others.
Collagenase is responsible for extracellular matrix
(ECM) remodelling including collagen breakdown.
Elastase, a serine proteinase, is primarily responsible
for the breakdown of elastin in ECM. Since collagen and
elastin mainly maintain skin structural integrity and
elasticity, the depletions of collagen and elastin contri-
bute to undesired wrinkles and aging skin.
Plant extracts, such as white tea, have been widely
investigated and found to possess free radical scavenging,
anti-collagenase and anti-elastase activities (Thring et al.
2009). The tea contains various chemical constituents

CONTACT Anyarporn Tansirikongkol anyarporn.t@chula.ac.th Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmaceutical
Sciences, Chulalongkorn University, Phayathai Road, Bangkok 10330, Thailand
ß 2016 Informa UK Limited, trading as Taylor & Francis Group
1866 S. PIENTAWEERATCH ET AL.
such as epigallocatechin-3-gallate (EGCG), quercetin,
kaempferol and gallic acid, which are classified in the
groups of tea catechin, flavonoids and phenolic acid.
Plant extracts, containing these structural-related com-
ponents, therefore, might show anti-aging benefits.
Phyllanthus emblica (L.) (Euphorbiaceae), commonly
known as amla, is a medicinal plant that has been
reported to be a good source of antioxidants due to being
rich in ascorbic acid, polyphenols and phenolic acids
such as gallic and ellagic acids (Kim et al. 2005;
Yokozawa et al. 2007). Moreover, recent studies revealed
its cosmetic benefits including anti-tyrosinase, anti-
wrinkle, antibacterial and anti-inflammatory properties
(Joseph et al. 2013). Amla extract contains a large
amount of functional tannins such as emblicanin,
pedunclagin and puniglucoin and, therefore, it may
exert its activities through a mechanism similar to that
of EGCG (Fujii et al. 2008).
Manilkara zapota (L.) P.Royen (Sapotaceae), or
sapota, has been used as herbal Indian medicine for a
decade. The sapota or sapodilla fruit is known to be a
good source of antioxidants. Ethanol pulp extract
contains alkaloids, saponins, terpenoids, flavonoids,
tannins, leucoanthocyanidins, anthroquinones, gluco-
sides and catechol (Gomathy et al. 2013). Ma et al.
(2003) found bioactive flavonoids from methanol extract
of sapota fruit such as ()-epicatechin, (+)-gallocatechin,
gallic acid, quercetin, myricitrin and (+)-catechin, of
which the latter three pure compounds were reported to
inhibit enzyme collagenase and elastase activities (Sim
et al. 2007; Thring et al. 2009).
Silymarin is a standardized flavonoid extract from the
milk thistle seeds, Silybum marianum (L.) Gaertner. The
main active constituent in silymarin is silybin. Silymarin
demonstrates antioxidant and anti-inflammatory proper-
ties in mouse skin which prevents skin disorders such as
photoaging or UV-related skin cancer (Katiyar et al.
2008).
The present study determines and compares total
phenolic and flavonoid contents together with the
investigation of in vitro antioxidants, anti-collagenase
and anti-elastase activities of the extracts. Ethanol amla
(P. emblica) extract, ethanol sapota (M. zapota) extract
and silymarin were selected on the basis of their different
phytochemical compositions. These plant extracts might
be a candidate for novel natural anti-aging ingredients.
Materials and methods
Materials
Dried amla fruits were supplied by Herbal pharmacy,
Chao Phraya Abhaibhubejhr Hospital (Thailand) during
May 2013. Sapota fruits were purchased in November
2011 from Thai vegetable and fruit market in Pathum
Thani (Thailand). The yellowish standardized extract of
silymarin was gifted from Berlin Pharmaceutical
Industry (Thailand), which were purchased from IVAX
Pharmaceutical s.r.o. (Czech Republic). The voucher
specimens were deposited in the Department of
Pharmacognosy and Pharmaceutical Botany,
Chulalongkorn University, Thailand, where identifica-
tion of plant specimens was confirmed by Associate
Professor Boonchoo Sritularak. Folin-Ciocalteau reagent,
standardized gallic acid, quercetin, DPPH (2,2-diphenyl-
2-picrylhydrazyl hydrate), EGCG, ABTS (2,20 -azino-bis
(3-ethylbenzthiazoline-6-sulphonic acid)) and potassium
persulphate were purchased from Sigma Aldrich (St.
Louis, MO). AR sodium carbonate was obtained from
Ajax Finechem Pty Ltd, Taren Point, Australia.
Aluminium chloride solution and potassium acetate
were purchased from Merck (Darmstadt, Germany) and
May & Baker Ltd. (Wandsworth, UK), respectively.
Ascorbic acid was obtained from Carlo Erba (Rodano,
Italy). EnzChekÕ collagenase/gelatinase assay kit
(E-12055) and EnzChekÕ elastase assay kit (E-12056)
were purchased from Molecular-Probes (Eugene, OR).
Methods
Plant extracts and stock solution preparations
The dried amla fruits were ground and soaked in 95%
ethanol (1:4) for 24 h at room temperature. The solvent
was collected and the process was repeated over three
cycles. All collected solvents were combined and evapo-
rated to dryness under reduced pressure at 40  C. The
crude ethanol amla extract was further purified by
dissolving the extract in 95% ethanol. The mixture was
concentrated under reduced pressure giving the yield of
17.59%.
Sapota fruits were peeled and the seeds were discarded.
The fruit pulp was cut into small slices and soaked in
absolute ethanol for 4 d at room temperature. The process
was repeated over four cycles. Then, all collected solvents
were combined and evaporated to dryness under reduced
pressure giving the yield of 11.09%.
Both extracts were kept in tight and light-resistant
containers and stored in a refrigerator at 4  C for further
study. Silymarin powder was stored in a desiccator at
room temperature.
Total phenolic content
Total phenolic content of all samples was assessed by
using a modified Folin–Ciocalteau method (Miliauskas
et al. 2004). Folin–Ciocalteau (FC) reagent is mixture of
phosphomolydate and phosphotungstate, which oxidizes

phenolates and reduces the heteropoly acids to a blue
complex. Diluted sample (20 mL) in 95% ethanol, 10%
FC reagent (100 mL) and 75 g/L of sodium carbonate
(80 mL) were mixed in a 96-well plate. Deionized water
was used as a blank. After a 60 min incubation period at
room temperature and light protected, the absorbance
of the reaction mixture was measured at 765 nm with
a microplate spectrophotometer (Spectramax M5,
Molecular Devices, Sunnyvale, CA). The standard
curve for quantifying phenolic contents was prepared
by using gallic acid as a reference. All samples were
performed in triplicate. The results were expressed as
milligrams of gallic acid equivalent (GAE) per gram of
the extract.
Total flavonoid content
Total flavonoid content was measured by the aluminium
chloride colorimetric assay with slight modification
(Chang et al. 2002). Diluted sample in 95% ethanol
(80 mL) were mixed with 10% aluminium chloride
solution (4 mL), 1 M potassium acetate (4 mL) and
deionized water (112 mL) in a 96-well plate. 10%
Aluminium chloride was substituted by distilled water
as a blank. After 30 min of incubation, the absorbance of
the reaction mixture was measured at 415 nm. The
standard curve for quantifying flavonoid contents was
prepared by using quercetin as a reference. All samples
were performed in triplicate. The results were expressed
as milligrams of quercetin equivalent (QE) per gram of
the extract.
Free radical scavenging activity
DPPH and ABTS free radical scavenging activities were
determined to assess antioxidant activities for all test
samples. DPPH free radical scavenging activities of
tested extracts were determined based on a protocol
modified from Marinova and Batchvarov (2011).
A volume of 0.06 mM DPPH solution in absolute
ethanol was prepared. Samples with different concentra-
tions in absolute ethanol were equally mixed with
ethanol DPPH solution to obtain a total of 200 mL.
After a 30 min incubation period in the dark, the
absorbance was measured at 517 nm by using the
microplate spectrophotometer. Each measurement was
corrected with its background, which was the sample
without DPPH solution. L-(+)-ascorbic acid was
employed as a standard. All samples were performed in
triplicate. The ability to scavenge DPPH radical was
calculated as the following:
DPPH inhibition ð%Þ ¼ ½ðAcontrol  Atest Þ=Acontrol   100:
PHARMACEUTICAL BIOLOGY 1867
The antioxidant activities of all extracts were finally
expressed as an IC50 value that is defined as the
concentration (mg/mL) of extract showing 50%
inhibition.
The ABTS radical method (Lee et al. 2014) was
slightly modified in order to evaluate the antioxidant
effect of the samples. The ABTS reagent was prepared by
adding 140 mM potassium persulphate (88 mL) in 5 mL
of 7 mM ABTS solution. The mixture was light-protected
and incubated at room temperature for 12–16 h to allow
free radicals to be fully generated, after which, it was
diluted with distilled water (1:49 v/v). To determine
antioxidant activity, diluted samples (20 mL) and ABTS
solution (180 mL) were mixed in 96-well microplate and
kept in the dark for 6 min. The absorbance was then
measured at 734 nm. Distilled water and L-(+)-ascorbic
acid were used as negative and positive controls,
respectively. Each absorbance was corrected with its
background, which was the sample without ABTS
solution. The ability to scavenge ABTS radical was
calculated using the same equation as DPPH.
MMP-1 and MMP-2 inhibitions
The in vitro collagenase inhibitions focusing on MMP-1
and MMP-2 were determined using EnzChekÕ collage-
nase/gelatinase kits (Molecular Probes, Eugene, OR). The
substrates for MMP-1 and MMP-2 inhibition assays
were DQTM collagen 1 and DQTM gelatin, respectively.
Collagenase (ChC) from Clostridium histolyticum, DQTM
substrate and various concentrations of the sample
extract were separately dissolved in pH 7.4 Tris-HCL
buffer. Diluted extract (80 mL), DQTM gelatin (20 mL) and
ChC (100 mL) were mixed in a 96-well plate. The final
concentrations of ChC and DQTM substrate were 0.2
units/mL and 12.5 mg/mL, respectively. After 90 min of
incubation, light protected at room temperature, the
fluorescence intensity was measured with the excitation
and the emission wavelength at 485 nm and 538 nm,
respectively, using a fluorescent microplate reader
(Spectramax M5, Molecular Devices, Sunnyvale, CA).
1,10-Phenanthroline and EGCG were used as a positive
inhibitor and a standard, respectively. The ability to
inhibit against gelatinase/collagenase was calculated as
following:
Collagenase inhibition (%) ¼ {[(AB)–(CD)]/
(AB)}  100, where A is the fluorescent intensity
without the test sample (control), B is the fluorescent
intensity without the test sample and enzyme (blank of
A), C is the fluorescent intensity with the test sample,
and D is the fluorescent intensity with the test sample
without enzyme. The anti-collagenase activities of all
samples were finally expressed as an IC50 value.
1868 S. PIENTAWEERATCH ET AL.

Inhibition of elastase inhibited DPPH and ABTS free radicals in a dose-

The assay was followed EnzChekÕ elastase assay kit
(Molecular Probes, Eugene, OR). Briefly, porcine pan-
creatic elastase (PE), DQTM elastin substrate and various
concentrations of the sample extract were separately
dissolved in pH 8 Tris-HCL buffer. Diluted extract
(50 mL) was preincubated with 0.4 U/mL PE (100 mL) for
15 min. 0.1 mg/mL DQTM elastin (50 mL) was then added
into the mixture. After 30 min incubation with light
protection, the fluorescence intensity was measured with
the excitation and the emission wavelength at 485 nm
and 538 nm, respectively. EGCG and N-methoxysucci-
nyl-Ala-Ala-Pro-Val-chloromethyl ketone were used as
a standard and a positive inhibitor, respectively. The
negative control was Tris-HCl buffer containing the
substrate. The ability to inhibit against elastase was
calculated using the equation mentioned in the section of
MMP-1 and MMP-2 inhibitions. The anti-elastase
activities of all samples were finally expressed as an
IC50 value.
Results
Total phenolic and flavonoid content
The results of total phenolic content (TPC) and total
flavonoid content (TFC) among three extracts; ethanol
P. emblica (amla) extract, ethanol M. zapota (sapota)
extract and silymarin are summarized in Table 1.
Amla extract showed the highest total phenolic
content followed by silymarin and sapota extract.
Silymarin exhibited the highest total flavonoid content,
whereas amla extract contained a lower amount of
flavonoid. Total flavonoid content was, however,
undetectable in the sapota extract.
DPPH and ABTS free radical scavenging activities
The antioxidant capacities of the test extracts were
determined by DPPH and ABTS based methods which
are widely used in plant and food research for screening
antioxidant activity (Karadag et al. 2009; Floegel et al.
2011; Jain et al. 2011). The study revealed that all extracts
dependent manner with similar order. Amla showed the
most potent ability to scavenge DPPH and ABTS free
radicals, which was comparable with ascorbic acid. The
inhibitory activities on both free radicals of silymarin
and sapota were much lower than that of amla. All 50%
inhibition concentrations (IC50) against ABTS were
higher than that of DPPH, except for silymarin (Table 2).
Inhibition of collagenase and elastase
MMP-1 and MMP-2 collagenase and elastase inhibitory
effects were performed using the test kits, and the results
are shown in Table 3. The test extracts inhibited both
collagenase and elastase in a dose-dependent manner
(Figure 1). Among the test extracts, amla and sapota
showed the most effect in collagenase inhibition at
almost one-fold higher than silymarin. However, their
effects were 10 times lower than a standard catechin,
EGCG. Regarding anti-elastase activity, sapota and
silymarin possessed a comparable effect with an IC50
value of 35.73 ± 0.61 mg/ml and 38.57 ± 0.04 mg/ml,
respectively. Interestingly, their inhibition activities were
almost three times higher than EGCG. Amla showed the
poorest anti-elastase activity among the extracts.
Discussion
Test extracts used in the study were selected based on
the group of chemical constituents. Major chemical
components of amla have been reported as ascorbic acid,
Table 2. Antioxidative capacity of the extracts measured by
DPPH- and ABTS-based methods (mean ± SD).
IC50 (mg/mL)
Plant/extract DPPH ABTS
P. emblicaa (Amla) 1.70 ± 0.07 4.45 ± 0.10
M. zapotaa (Sapota) 37.63 ± 1.18 73.14 ± 2.84
Silymarin 27.85 ± 0.98 12.34 ± 0.21
Ascorbic acid 1.38 ± 0.05 3.45 ± 0.06
a
The ethanolic extracts.

Table 3. Biological activities of the extracts on MMP-1, MMP-2

Table 1. The total phenolic and flavonoid contents of the test
and elastase inhibitions, data expressed as IC50 (mean ± SD).
extracts.
IC50 (mg/mL)
Total phenolic Total flavonoid
content content MMP-1 MMP-2 Elastase
Plant/extract (mg GAE/g) (mg QE/g) Plant/extract inhibition inhibition inhibition
P. emblicaa (Amla) 362.43 ± 11.22 6.40 ± 0.88 P. emblicaa (Amla) 95.97 ± 3.28 89.32 ± 0.88 387.85 ± 8.78
M. zapotaa (Sapota) 38.56 ± 1.98 UD M. zapotaa (Sapota) 89.61 ± 0.96 86.47 ± 3.04 35.73 ± 0.61
Silymarin 233.37 ± 7.53 21.04 ± 0.67 Silymarin N/A 133.69 ± 1.15 38.57 ± 0.04
EGCG 9.73 ± 0.18 8.19 ± 0.40 93.99 ± 3.44
UD: undetectable; GAE: gallic acid equivalent; QE: quercetin equivalent. a
a
The ethanolic extracts. The ethanolic extracts.
 PHARMACEUTICAL BIOLOGY 1869

% Collagenase (MMP-2) Inhibition

90 80
80 70

% Elastase Inhibition
70 60
60
50
50
40
40
30 30

20 20
10 10
0 0
0 100 200 300 400 0 200 400 600
Amla concentration (µg/ml) Amla concentration (µg/ml)
% Collagenase (MMP-2) Inhibition

90 80
80 70

% Elastase Inhibition
70 60
60
50
50
40
40
30 30
20 20
10 10
0 0
0 100 200 300 400 500 0 10 20 30 40 50
Sapota concentration (µg/ml) Sapota concentration (µg/ml)
% Collagenase (MMP-2) Inhibition

90 80
80 70
% Elastase Inhibition

70 60
60
50
50
40
40
30 30
20 20
10 10
0 0
0 50 100 150 200 250 300 0 10 20 30 40 50 60
Silymarin concentration (µg/ml) Silymarin concentration (µg/ml)

Figure 1. Effects of ethanolic amla extract, ethanolic sapota extract and silymarin on collagenase and elastase inhibitions. The percent
inhibitions expressed as mean ± SD.

and phenolic compounds with some flavonoid content 4.45 mg/mL, respectively. Its effect was comparable with
(Majeed et al. 2009). Silymarin contained high flavonoid ascorbic acid which is a well-known antioxidant widely
content (Cai et al. 2009). Sapota was selected due to the used in cosmetic products. The present study revealed a
presence of some phenolic compounds, flavonoid and new alternative potent antioxidant for anti-aging appli-
tannin (Gomathy et al. 2013). cation. This finding is in accordance with the findings of
Phenolic and flavonoid compounds have been Spiridon et al. (2011) and Floegel et al. (2011) presenting
reported to present significant antioxidant properties a linear correlation between phenolic content and DPPH
(Miliauskas et al. 2004). In the present study, amla scavenging activity. Samples with high content of
extract exhibited the highest total phenolic content. This phenolic compound tend to express high DPPH
result might be due to the fact that major components of scavenging activity. The factors behind the correlation
phenolic compounds in amla are gallic and ellagic acids between TPC and DPPH free radical activity might be
which are in the group of phenolic compounds due to the same principle that utilizes an electron-
(Yokozawa et al. 2007; Majeed et al. 2009; Amir et al. transfer mechanism (Karadag et al. 2009). Regarding the
2011). Amla extract also showed the most potent DPPH ABTS-based test system, the results show the same rank
and ABTS free radical scavenging activity among order of antioxidant property compared with data
the test extracts with an IC50 value of 1.70 and obtained from the DPPH method due to similarity of
1870 S. PIENTAWEERATCH ET AL.
mechanisms. However, silymarin showed moderately
higher antioxidant capacity as measured by the ABTS
assay relative to the DPPH assay.
With respect to TFC, silymarin unsurprisingly
showed the highest total flavonoid content since it is
clearly known as a flavonolignan, one of the flavonoid
compounds. Such flavonoid compound also showed a
DPPH scavenging ability due to its reducing ability and
metal-chelating property (Malesev & Kuntic 2007;
Pallab et al. 2013). Total phenolic and total flavonoid
content might be screening parameters of the antioxi-
dant activity of test substance. However, the degree of
antioxidant property depends not only on the concen-
tration of phenolic or flavonoid content but also on the
type of chemical entity as well as its presenting
structure in the substance. Therefore, a compound
with low total phenolic and flavonoid content might
also possess an antioxidant effect due to the presence of
other bioactive compounds. Substances with such
properties might be included to prevent skin aging
associated with oxidative damage.
MMPs are a group of zinc-containing proteinases.
MMP-1 or interstitial collagenase initiates the break-
down mostly of type I, II and III collagens which are the
most abundant interstitial collagens in dermis while
MMP-2 is responsible for breakdown of type I–III, IV
and VII collagens in which the latter two are most
abundant in the dermal–epidermal junction. In addition
to MMPs, elastase is an enzyme that digests another
interstitial fibre in the skin, called elastin. Depletion of
both structural fibres in skin results in the lack of skin
integrity and elasticity contributing to wrinkle formation
and aging skin. Amla extract was previously reported
to inhibit collagenase activity, determined by using
the EnzChekÕ gelatinase/collagenase assay kit
(Chanvorachote et al. 2009) and which conformed to
the results of this experiment. It significantly inhibited
MMP-1 and MMP-2 with activity comparable with that
of sapota. The inhibition effect of amla extract might
involve several mechanisms. Hydroxyl groups of poly-
phenol could interact with the backbone or other
functional group side chain of collagenase. In addition,
hydrophobic interaction between the benzene ring of
polyphenol and collagenase could also result in the
conformational changes leading to unfunctioned enzyme
(Madhan et al. 2007). Another mechanism involves the
Zn ion active site on collagenase. Collagenase contains a
structural Zn ion at its active site which plays a major
role in facilitating interaction with an inhibitor (Bigg
et al. 1994). Phyllanthus emblica fruit contains polyphe-
nol compounds including gallic acid and hydrolyzable
tannin which are known to be metal chelators and, thus,
may bind to a Zn ion active site and prevent the
substrate from enzyme digestion (McDonald et al. 1996).
In addition to the polyphenol, flavonoid also chelated Zn
metal by its 3-hydroxyflavon structure (Malesev &
Kuntic 2007). An ability of silymarin for collagenase
inhibition might also be caused by binding to a Zn active
site. Significant inhibition on MMP-1 and MMP-2 of
amla and sapota suggested their ability to delay break-
down of collagen fibre, hence, maintaining integrity of
skin layer.
Sapota and silymarin exhibited significant inhibition
in elastase activity. They showed a potent anti-elastase
property that was nearly three-fold superior to EGCG,
while amla extract exhibited weak elastase inhibition.
The data are consistent with previous reports from
several groups where certain phenolic compounds and
flavonoid possess anti-elastase activity in dose depend-
ency. Phenols, such as epicatechin, catechin, resveratrol
and procyanidin B2 (Hrenn et al. 2006; Wittenauer et al.
2015), and flavonoid such as kaempferol, quercetin and
myricetin (Kanashiro et al. 2007) significantly inhibited
elastase activity. In agreement with Wittenauer et al.
(2015), amla extract containing gallic acid as major
component possessed poor inhibitory property toward
elastase enzyme. Surprisingly, sapota showed significant
collagenase inhibition comparable with amla, and was
significantly higher in elastase inhibition although it
contains lower total phenolic and undetectable flavonoid
content. Sapota, thus, might contain a different type of
phenolic compound or other bioactive components
which perform other mechanism of inhibition.
Compounds with anti-proteinase activity could, there-
fore, be included in anti-aging formulations in order to
delay the breakdown of skin fibres.
Skin aging involves many complex pathways. The
current study suggests that TPC and/or TFC might refer
to antioxidant properties to some extent but are not well
correlated with anti-proteinase activity. The single test
parameter of TPC, TFC, DPPH or ABTS free radical
scavenging assay frequently used to screen antioxidant
properties might not provide enough information to
determine the potential anti-aging agent. Amla
exhibited the potent antioxidant effect, but it was
not a good source of elastase inhibitor. Although
sapota had undetected flavonoid content and possessed
the moderate antioxidant activity, it showed excellent
collagenase and elastase inhibition among test extracts.
Often, extracts might be ignored when the results
indicate poor antioxidant activity. However, those
extracts might present other benefits as seen with
sapota. Thus, collagenase and elastase inhibitions
should be additionally investigated for screening of
anti-aging agents.

Conclusions
Among the test samples, ethanol amla extract contained
high phenolic content and showed the most potent
antioxidant with moderate collagenase and poor elastase
inhibition. Sapota showed the highest collagenase and
elastase inhibition with a slightly antioxidant effect.
Silymarin showed high flavonoid content and inhibited
the elastase comparable with sapota but was poor in anti-
collagenase activity compared with others. All test
extracts presented anti-aging property with different
mechanisms. There is hardly an ‘‘all in one’’ component
that exhibits total anti-aging effects on each of the skin-
aging pathways. Thus, extracts might be added as a
mixture to gain the overall effect in cosmetic products.
Purification of each extract and investigation into the
effect of different combinations shall be further studied.
Disclosure statement
The authors report that they have no conflicts of interest.
Funding information
This research was financially supported by a research grant
from the Faculty of Pharmaceutical Sciences, Chulalongkorn
University, Thailand.
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