J Herbmed Pharmacol. 2018; 7(2): 88-93.

http://www.herbmedpharmacol.com doi: 10.15171/jhp.2018.15

Journal of Herbmed Pharmacology

Antioxidants, anticollagenase and antielastase potentials of

ethanolic extract of ripe sesoot (Garcinia picrorrhiza Miq.)
fruit as antiaging
Sri Utami1*, Qomariyah Romadhiyani Sachrowardi1, Ndaru Andri Damayanti1, Arroyan Wardhana1, Irfan Syarif1,
Said Nafik2, Betharie Cendera Arrahmani3, Hanna Sari Widya Kusuma4, Wahyu Widowati5
1
Faculty of Medicine, YARSI University, Central Jakarta, Indonesia
2
Directorate General of Intellectual Property, Ministry of Law and Human Rights, Republic of Indonesia, South Jakarta, Indonesia
3
Faculty of Business and Engineering, University of Applied Sciences Würzburg-Schweinfurt, Schweinfurt, Germany
4
Aretha Medika Utama, Biomolecular and Biomedical Research Center, Bandung, Indonesia
5
Faculty of Medicine, Maranatha Christian University, Bandung, Indonesia

ARTICLE INFO ABSTRACT

Article Type: Introduction: Nitric oxide (NO) is a free radical that belongs to reactive nitrogen species
Original Article (RNS). The excess amount of NO in body generates physical changing on skin as a
consequence of alteration in connective tissue through formation of lipid peroxides, cell
Article History: content, and enzymes. These free radicals induce damage to extracellular matrix (ECM) and
Received: 26 September 2017 are responsible in reducing skin elasticity. Antioxidants possess significant role in delaying
Accepted: 10 January 2018 aging process by scavenging free radicals and preventing collagenase and elastase enzymes
activities. This study aimed to evaluate
antioxidants, anticollagenase and antielastase potentials of ethanolic extract of ripe sesoot
Keywords: (Garcinia picrorrhiza Miq.) fruit (GpKar) as antiaging remedy.
GpKar Methods: Antioxidant activity was performed by NO scavenging activity assay, while anti-aging
Antioxidant activity was performed through inhibitory effects of collagenase and elastase activities.
Anticollagenase Results: In antioxidant activity, GpKar had lower NO scavenging activity (IC50=1530.34 µg/
Antielastase mL) compared to xanthone (IC50=85.40 µg/mL). In collagenase inhibitory activity, GpKar also
Antiaging had lower inhibition collagenase activity (IC50= 1169.31 µg/mL) compared to xanthone (IC50=
286.32 µg/mL). In elastase inhibitory activity, GpKar had lower inhibition elastase activity
(IC50= 152.93 µg/mL) compared to xanthone (IC50= 21.26 µg/mL).
Conclusion: In summary, GpKar and its compounds possess antioxidant, anticollagenase, and
antielastase activities for antiaging, and might be beneficial in these subjects.

Implication for health policy/practice/research/medical education:

Extract of G. picrorrhiza has potential to increase antioxidant, anticollagenase, and antielastase activities. Therefore, more
investigations need for identification of the active compounds in G. picrorrhiza extract involved in antioxidant and antiaging
activities.
Please cite this paper as: Utami S, Sachrowardi QR, Damayanti NA, Wardhana A, Syarif I, Nafik S, et al. Antioxidants,
anticollagenase and antielastase potentials of ethanolic extract of ripe sesoot (Garcinia picrorrhiza Miq.) fruit as antiaging. J
Herbmed Pharmacol. 2018;7(2):88-93. doi: 10.15171/jhp.2018.15

Introduction (RNS). NO is produced essentially as bio-regulatory

Skin aging process is divided into two categories i.e. molecule in several physiological processes such as neural
intrinsic aging and extrinsic aging (1,2). Intrinsic aging signal transmission, immune response, vasodilatation,
is naturally caused by the alteration of skin elasticity as and blood pressure regulation. On certain conditions, the
time goes by, whereas extrinsic aging generally is caused concentration of NO can be increased as a result of photo
by accumulation of free radicals (1,3). Nitric oxide (NO) radiation or chemical exposures (4,5).
is a free radical belonging to reactive nitrogen species In addition, the excessive NO in body may result in premature

*Corresponding author: Dr. Sri Utami, S.Si., M.Si., S.H.;

Faculty of Medicine, YARSI University, Jl. Letjend Suprapto, Cempaka
Putih, Jakarta Pusat, 10510, Indonesia. Email: uutsuyono@yahoo.com
 Antioxidants, anticollagenase and antielastase potentials of sesoot

aging. The excessive NO leads to structural changes of Griess reagent was used in NO assay (13).
cells and stimulates matrix metalloproteinases (MMPs) In this study the NO scavenging activity was estimated
enzymes such as collagenase and elastase that may induce using Griess Illosvoy reaction based on the method that
collagen and elastin loss (6-8). Collagenase is an enzyme was performed by Parul et al with minor modifications
that plays an important role in degradation of collagen. (14). Sodium nitroprusside (10 mM) (Merck 106541,
Collagen is the main component with percentage of 70%- Germany) in phosphate buffered saline (PBS) (Gibco
80% of the total skin weight. The increasing degradation 1740576, Canada) was mixed with different concentrations
of collagen is significant in aging (9). of GpKar and xanthone (20.83–666.67 µg/mL; µM). The
Aging can be prevented by scavenging free radicals. The mixtures were incubated for 2 hours in 25˚C. After the
excessive free radicals like NO can be reduced by escalating incubation period, Griess reagent (1% sulfanilamide
antioxidant intake through food or supplements. [Merck 111799, Germany], 2% H3PO4 [Merck 100573,
Furthermore, the other way to retard aging is inhibiting Germany] and 0.1% N-(1-napththyl) ethylenediamine
collagenase and elastase activities. As collagenase and dihydrochloride) (Sigma 222488, USA) were added.
elastase increase significantly with age, inhibiting their The absorbance of the chromophore that was formed
activities may retard skin aging without interfering their during diazotization of the nitrite with sulfanilamide
abilities to breakdown damaged skin components. In and subsequent coupling with Naphthylethylenediamine
other words, the use of inhibiting agents helps restore the dihydrochloride was read at 546 nm with microplate
balance that the skin possessed when it was younger. reader (Thermo Scientific Multiscan GO). The same
Nowadays, the use of natural substances to prevent reaction mixture without the extract yet equivalent in
premature aging is more preferable in food, cosmetic, amount of ethanol served as the control.
and therapeutic industry. These would be promising
alternatives for synthetic antioxidants in respect of low Collagenase inhibitory activity assay as antiaging potential
cost, high compatibility with dietary intake and low Inhibition of collagenase activity was measured based
harmful effects inside the human body. Many compounds on the method that was elaborated by Sigma Aldrich
in plants have been identified as free radical or active and Thring et al with some modifications. The assay
oxygen scavengers (10). It has been investigated that was performed by dissolving 10 µL collagenase from
Garcinia picrorrhiza fruit contains secondary metabolites Clostridium histolyticum (Sigma C8051, USA) (0.01 U/
that act as antioxidant such as xanthone, biflavonoidsa, mL in cold distilled water), 60 µL buffer Tricine (50 mM,
and benzophenons and their derivatives. Based on a pH 7.5, contains 10 mM CaCl2 and 400 mM NaCl), 30
primary research, fruit extract of G. picrorrhiza contains µL sample (0-250 µg/mL in DMSO). The mixtures were
camboginol or garcinol, a derivative of benzophenones. incubated for 20 minutes at 37˚C. After incubation time,
In the present study, NO scavenging activities of GpKar 20 µL substrate N-[3-(2-Furyl)acryloyl]-leu-gly-Pro-Ala
and its compounds as well as their inhibitory activities on (Sigma F5135, USA) (1 mM in buffer Tricine) was added.
collagenase and elastase were evaluated. Absorbance at 335 nm was measured immediately after
adding the substrate (11,15,16).
Materials and Methods % Collagenase inhibition = (1-B/A) x 100%
Preparation of GpKar A = Sample absorbance
Sesoot fruits were collected from Bogor Botanical B = Control absorbance
Garden, Bogor, West Java. The plant was authenticated by
herbarium staff of Bogor Botanical Garden. Sesoot fruits Elastase inhibitory activity assay
were mashed (500 g) and extracted using ethanol 70% Elastase inhibitory activity was measured by modified
by maceration method. Every 24 hours the ethanol was method of Sigma Aldrich and Thring et al with some
filtered and the wastes were re-macerated in triplicate. The modifications by Widowati et al (11,15). A mixture of 10
ethanol filtrate was collected and condensed in 50˚C using µL of various levels of samples (4.17–133.33 µg/mL), 5 µL
rotary evaporator (Hitachi) to obtain GpKar. The extract elastase from porcine pancreas (Sigma 45124, USA) (0.5
in paste form was stored at -20˚C and used for the assay. mU/mL in the cold distilled water) and 125 µL Tris buffer
Standard compound used in this study was xanthone was pre-incubated for 15 minutes at 25°C. Mix solution
(Sigma X0626, USA) (11,12). was added N-Sucanyl-Ala-Ala-Ala-p-Nitroanilide
substrate [Sigma 54760, USA] and then incubated for 15
Nitric oxygen scavenging activity assay minutes at 25˚C. Absorbance was measured immediately
Sodium nitroprusside is soluble in aqueous solution at after incubation time with 410 nm wavelength.
physiological pH of 7.2 producing NO. NO reacts with % Elastase inhibition = (1-B/A) x 100%
oxygen to produce stable products (nitrate and nitrite) A = Sample absorbance
under aerobic conditions. Scavengers of NO compete with B = Control absorbance
oxygen leading to reduced production on nitrite ions.

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 Utami et al

Results that GpKar has lower collagenase inhibitory activity

The effect of GpKar on nitric oxide scavenging activity compared to xanthone.
Inhibition potentials of nitrite formation by GpKar and
the standard antioxidant xanthone were calculated relative The effect of GpKar on elastase inhibitory activity
to the control. Inhibition data (percentage inhibition) The percentage of elastase inhibitory activities of GpKar
were linearized against the concentration of each extract and xanthone can be seen in Figure 3. Figure 3 shows that
and standard antioxidant. IC50 which is an inhibitory the elastase inhibitory activities of GpKar and xanthone are
concentration of each extract required to reduce 50% of concentration-dependent. At the highest concentration
the NO formation was determined (14). (133.33 µg/mL), the elastase inhibitory activity of GpKar
Figure 1 shows that the NO scavenging activity was (40.37 ± 3.11%) was lower than xanthone (59.55 ± 0.48%).
concentration dependent. At the highest concentration Furthermore, IC50 value of GpKar (152.93 µg/mL) was
(666.67 µg/mL) NO scavenging activity of GpKar (20.26 higher than xanthone (21.26 µg/mL) (Table 3). These
± 0.47%) was higher than xanthone (63.38 ± 0.12%). results indicate that GpKar has lower elastase inhibitory
Furthermore, IC50 value of GpKar (1530.34 µg/mL) was activity compared to xanthone.
higher than xanthone (85.40 µg/mL) (Table 1). These
results indicate that GpKar has lower NO scavenging Discussion
activity compared to xanthone. The natural substances from plants have been widely
used for treating aging. It has been investigated that
The effect of GpKar on collagenase inhibitory activity the investigated plant contains active antioxidants that
A spectrophotometric method was performed to find out are related to its phytochemical compounds (17,18). In
collagenase inhibitory activity. The collagenase inhibitory several studies, some plants have shown the ability to
activity of GpKar and xanthone can be seen in Figure 2 reduce free radicals having anti-aging potential through
and Table 2. Figure 2 shows that collagenase inhibitory their components such as polyphenols from blackberry,
activities of GpKar and xanthone are concentration- curcumin from C. longa (19), procyanidins from V.
dependent. At the highest concentration (2500 µg/mL) vinifera (20) and garcinol from G. indica (21).
collagenase inhibition activity of GpKar (68.08 ± 4.18%) Sesoot belongs to genus of Garcinia. Recent studies have
was lower than xanthone (68.91 ± 1.72%). Furthermore, shown that sesoot contains high levels of xanthone,
IC50 value of GpKar (1169.31 µg/mL) was higher than bioflavonoids, and prenylated benzophenones. Previous
xanthone (286.32 µg/mL) (Table 2). These results indicate studies have shown that the extract of sesoot fruit

Concentration (µg/mL; µM)

Concentration (µg/mL; µM)

Figure 1. Nitric Oxide (NO) scavenging activity of GpKar and

xanthone. Figure 2. Collagenase inhibitory activity of GpKar and Xanthone.
*GpKar and xanthone were diluted using DMSO to reach the final *GpKar and Xanthone were diluted using DMSO to reach the final
concentration of 20.83; 41.67; 83.33; 166.67; 333.33; 666.67 (µg/ concentration of 78.125; 156.25; 312.5; 625.00; 1250.00; 2500.00
mL; µM). (µg/mL; µM).

Table 1. IC50 values of nitric oxide scavenging activities by GpKar Table 2. IC50 values of of collagenase inhibitory activities by
and xanthone GpKar and xanthone

Samples Equation r2 IC50 (µM) IC50 (µg/mL) Samples Equation r2 IC50 (µM) IC50 (µg/mL)
GpKar y = 0.03x – 0.51 0.97 - 1530.34 GpKar y = 0.01x + 34.28 0.92 - 1169.31
Xanthone y = 0.08x + 14.48 0.90 435.28 85.40 Xanthone y = 0.02x + 24.69 0.96 1459.35 286.32
*GpKar= G. picrorrhiza extracts, IC50= The half maximal inhibitory *GpKar= G. picrorrhiza extracts, IC50= The half maximal inhibitory
concentration. Linear equations, coefficient of regression (r2) and IC50 concentration. Linear equations, coefficient of regression (r2) and IC50
of the samples were calculated. IC50 of GpKar in µg/mL xanthone was of the samples were calculated. IC50 of GpKar in µg/mL xanthone was
presented in µM and µg/mL. presented in µM and µg/mL.

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 Antioxidants, anticollagenase and antielastase potentials of sesoot

aerobic conditions, NO is very unstable and reacts with

oxygen to produce NO2, N2O4, N3O4, the stable products
of nitrate and nitrite and peroxynitrite when reacted
with superoxide. These progenitors are highly genotoxic.
Besides, NO may affect enzymatic activities that lead
to mutagenesis (26). NO is responsible for altering the
structural and functional behavior of many cellular
components. Antioxidants have the ability to reduce
Concentration (µg/mL; µM) NO by donating their electron. This may be due to the
antioxidant principles in the extract, which compete
with oxygen to react with NO, thereby inhibiting the
production of nitrite (14).
Figure 3. Elastase inhibitory activity of GpKar and Xanthone.
*GpKar and xanthone were diluted using DMSO to reach the final Skin aging is a natural process that occurs in organisms
concentration of 4.17; 8.33; 16.67; 33.33; 66.67; 133.33 (µg/mL; µM). with characteristic of losing skin elasticity implicated
in formation of wrinkle, uneven pigmentation, brown
Table 3. IC50 values of of elastase inhibitory activities by GpKar spots, laxity and leathery appearance (11). Though aging
and xanthone will occur naturally but at some conditions it can be
accelerated by some factors like free radicals. Aging can be
Samples Equation r2 IC50 (µM) IC50 (µg/mL)
characterized by its physical and psychosocial changing.
GpKar y = 0.33x – 0.25 0.94 - 152.93
Physical changing in visual can lessen skin-elasticity,
Xanthone y = 0.36x + 11.28 0.99 108.38 21.26
causing wrinkle, uneven pigmentation, brown spots, laxity
*GpKar= G. picrorrhiza extracts, IC50= The half maximal inhibitory
concentration. Linear equations, coefficient of regression (r2) and IC50 and leathery appearance (16). The excess of free radicals
of the samples were calculated. IC50 of GpKar in µg/mL xanthone was in body may overturn natural cellular antioxidant defense
presented in µM and µg/mL. and lead to oxidation and further contributing to cellular
functional impairment (27).
has camboginol or garcinol compound, a derivate of During aging process, there is also imbalance between
benzophenones that has various therapeutic activities. collagen production and degradation. Collagen production
Xanthone compounds in sesoot are oxygenated xanthones decreases whereas level of collagenase increases. Exact
and prenylated ones. Due to the xanthone itself that mechanism of wrinkles formation is still unknown but it
has phenolic functional group on linear tri-cyclic ring, has been observed that free radicals cause enhancement in
xanthone has a wide range of biological activities such as the expression of MMPs which is collagenase and elastase,
antioxidant property (22). which in turn causes repetitive breakdown of collagen
In this study, GpKar was tested to find out its potential and elastine (28). However, undesirable aging can also
in NO scavenging potential, as anticollagenase and be retarded by scavenging free radical with antioxidant
antielastase agent. In NO scavenging assay, GpKar has and inhibiting collagenase and elastase activitie. Other
higher IC50 value compared to xanthone. It means that than that, in collagenase and elastase inhibition activities,
GpKar has lower NO scavenging activity than xanthone GpKar has lower inhibitory activity compared with
does. Even though GpKar has lower IC50 than xanthone xanthone. In collagenase inhibitory activity assay, the
but GpKar still indicates antioxidant potential. The IC50 value of collagenase inhibitory activity of GpKar was
Garcinia contains camboginol that structurally is similar higher than xanthone (Table 2) and in elastase inhibitory
to a well-known antioxidant, curcumin, which contains activity IC50 value of GpKar was higher than Xanthone
both phenolic hydroxyl groups and β-diketone moiety. (Table 3). These results indicate that GpKar has lower
Several studies have demonstrated that garcinol exhibits collagenase and elastase inhibitory activity compared to
significantly the antioxidative property and possesses xanthone. Collagen and elastin are the main components
inhibitory activity on lipid peroxidation (23,24). of skin that possess important role in maintaining skin
NO is an important chemical mediator generated by structure. Collagenase and elastase enzymes cause
endothelial cells, macrophages and neurons which is repetitive collagen breakdown and are responsible for
involved in regulation of physiological processes (14). structural defect in dermis and wrinkle development (29).
NO is generated in biological tissues by a specific NO Collagenase and elastase contribute to production and
synthase, which metabolizes arginine to citraline with the degradation of collagen and elastin (30).
formation of NO via five electrons of oxidative reaction
(25). Nevertheless, the excess amount of NO in body may Conclusion
result in aging acceleration or even degenerative chronic In summary, GpKar and its compounds possess
ailments (6). In aging-acceleration process, NO does not antioxidant and antiaging activities and might be used to
interact directly with DNA or proteins. However, under prevent aging. However, xanthone has higher antioxidant

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 Utami et al

power through NO scavenging activity and has higher phosphorus and potassium deficiency. Plant Cell Physiol.
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Research Center, Aretha Medika Utama, Bandung,
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