Pharmaceutical Biology, 2010; 48(4): 469–481

RESEARCH ARTICLE

In vitro anti-aging activities of Terminalia chebula gall

extract
Aranya Manosroi1,2, Pensak Jantrawut1, Toshihiro Akihisa3, Worapaka Manosroi4, and
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Jiradej Manosroi1,2
1
Faculty of Pharmacy, Chiang Mai University, Chiang Mai, Thailand, 2Natural Products Research and Development
Center (NPRDC), Science and Technology Research Institute (STRI), Faculty of Pharmacy, Chiang Mai University,
Chiang Mai, Thailand, 3College of Science and Technology, Nihon University, 1-8 Kanda Surugadai, Chiyoda-ku,
Tokyo, Japan, and 4Faculty of Medicines, Chiang Mai University, Chiang Mai, Thailand

Abstract
Context: The Thai Lanna region has its own folklores and wisdoms in various fields such as traditional medi-
cines. The galls of Terminalia chebula Retz. (Combretaceae) frequently appear in many Thai Lanna medicinal
plant recipes for promoting longevity.
Objectives: To investigate the in vitro anti-aging activities of the extracts from 15 plants including T. chebula
For personal use only.

gall selected from the Thai medicinal plant recipes that have been traditionally used for longevity.
Materials and methods: The plant extracts were prepared by four extraction methods including hot (HW) and
cold (CW) aqueous processes and hot (HM) and cold (CM) methanol processes. These extracts were tested
for antioxidative and tyrosinase inhibition activity as well as the proliferative and MMP-2 inhibition activity
on early aging human skin fibroblasts in order to evaluate their in vitro anti-aging activity.
Results: At 0.1 mg/mL, the CW extract of T. chebula gall exhibited the highest DPPH radical scavenging
activity with scavenging of 84.64% ± 2.22%, whereas ascorbic acid, α-tocopherol and butylated hydroxyl
toluene gave 96.50% ± 0.1%, 35.74% ± 0.2% and 27.43% ± 0.1%, respectively. The CW extract of T. chebula
gall indicated the highest stimulation index (SI) on normal human fibroblast proliferation of 1.441 which was
more active than ascorbic acid (SI 1.21). This extract has also demonstrated MMP-2 inhibition on fibroblasts
determined by zymography 1.37 times more potent than ascorbic acid.
Discussion and conclusion: This study has confirmed the traditional use of T. chebula gall in many Thai medici-
nal plant recipes for longevity which will be beneficial for further development of anti-aging products.
Keywords:  Antioxidative activity; in vitro anti-aging assays; human skin fibroblasts; Terminalia chebula gall

Introduction reactive molecules with unpaired electrons which can

The global market values of anti-aging products which
help the body fight off the damage caused by aging are
increasing continuously. The largest group of com-
pounds in anti-aging products is antioxidants. Aging is
a very complex biological process including the dam-
age from free radicals and the dark spots of the skin
from melanin overproduction. Free radicals are highly
cause damage to cell membranes, lipids, proteins, and
DNA. Damage to DNA eventually results in collagen
breakdown. Dark spots of the aged skin are from mela-
nin overproduction which may be caused by chronic
sun exposure, melasma, or other hyperpigmentation
diseases (Briganti et  al., 2003). Tyrosinase, a copper-
containing monooxygenase, is a key enzyme that cata-
lyzes melanin synthesis in melanocytes (Sturm et  al.,

Address for Correspondence: Professor Aranya Manosroi, Natural Products Research and Development Center (NPRDC), Science and Technology Research
Institute (STRI), Chiang Mai University, Chiang Mai 50200, Thailand. Tel.: +66 53 894806; Fax: +66 53 894169. E-mail: pmpti005@chiangmai.ac.th

(Received 15 October 2009; revised 23 December 2009; accepted 29 December 2009)

ISSN 1388-0209 print/ISSN 1744-5116 online © 2010 Informa UK Ltd

DOI: 10.3109/13880200903586286 http://www.informahealthcare.com/phb
 470   Aranya Manosroi et al.
2001). Applications of tyrosinase inhibitors may be the
least invasive procedure for maintaining skin white-
ness (Kadekaro et al., 2003). Fibroblasts which produce
collagens, glycosaminoglycans, reticular and elastic
fibers, and glycoproteins are found in the extracellular
matrix. Collagen, the major structural component of the
skin in the dermis has been suggested to be the cause
of the clinical changes observed in naturally aged and
photo-aged skin (Millis et al., 1989; Chung et al., 2001).
Collagen synthesis has been studied by serial cultures
of human dermal fibroblasts (Millis et  al., 1989). The
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effects on skin collagen synthesis for anti-aging evalu-
ation can be determined from the proliferation activity
of the human fibroblasts by the sulforhodamine B (SRB)
assay. Gelatinase A (MMP-2) digests native collagen
types I, II and III in a similar manner to the collagenases
(Aimes & Quigley, 1995; Patterson et al., 2001). MMP-2
induction was mediated by phenomena accelerated in
aged human skin. Increased expression of MMP-2 is
involved with collagen degradation in aged human skin
(Steinbrenner et al., 2003), leading to wrinkle formation
and aged appearance. Thus, MMP-2 inhibitors which
delay collagen degradation can be used to evaluate anti-
aging activity.
For personal use only.
Traditional herbs provide interesting and largely
unexplored sources for the development of potential
new cosmetic and pharmaceutical products. The Lanna
region covered many provinces and cities in China,
Myanmar and Thailand. It was an independent country
about 700 year ago. In Thailand, this area called “Thai
Lanna” which included seven provinces of Chiang Mai,
Chiang Rai, Lamphun, Lampang, Phayao, Phrae and
Nan. The Thai Lanna region has its own folklore and
wisdoms in various fields such as politics, agriculture
and traditional medicines. The Thai Lanna medicinal
plant recipes are still currently used by the people in
the northern part of Thailand. The Thai Lanna medici-
nal plant recipes were recorded as Lanna scripts in
palm leaves, mulberry paper or Streblus asper Lour.
(Moraceae) paper. The Natural Products Research and
Development Center (NPRDC) at Chiang Mai University
in Thailand has developed a database containing the
Thai Lanna medicinal plant recipes collected from many
institutes, temples and folklore doctors (Manosroi et al.,
2006). Twenty-one Thai Lanna medicinal textbooks with
over 19,161 recipes, 2,556 diseases, and 4,672 medicinal
plants were collected. Many Thai Lanna medicinal plant
recipes have implied the anti-aging application of being
slow down the aging process and promoting vitality and
well being. Terminalia chebula Retz. (Combretaceae) is
one of the Thai Lanna medicinal plants which appear
in many of these recipes. It is widely grown in tropical
regions. The dried galls of T. chebula are frequently sold
at fresh markets in Southeast Asia. Its medical applica-
tions include astringent, purgative, supplements for
anti-aging and imparting of longevity as well as boosting
of the immune system (Pharmacopoeia Commission of
PRC, 1997; Zhu, 1998; Lemmens & Bunyapraphatsara,
2003). Moreover, T. chebula fruits have been shown
to have antioxidant (Chen et  al., 2003), antimicrobial
(Burapadaja & Bunchoo, 1995), and anticancer activities
(Lee et al., 1995; Saleem et al., 2002).
In this present study, 15 plants including T. chebula
gall were selected from the Thai Lanna medicinal plant
recipes database and their extracts were prepared by the
aqueous and methanol extraction using hot and cold
processes. These extracts were tested for antioxidative
and tyrosinase inhibition activity as well as the prolifera-
tive and MMP-2 inhibition activity on early aging human
skin fibroblasts in order to evaluate their in vitro anti-
aging activity.
Materials and methods
Materials
L-(+)-Ascorbic acid (vitamin C), α-tocopherol, butylated
hydroxytoluene (BHT), 2,2-diphenyl-1-picryhydrazyl
radical (DPPH), EDTA, sulforodamine B, dimethyl sul-
foxide (DMSO), kojic acid, ferrozine and ferric chloride
(FeCl2) were purchased from Sigma (St. Louis, MO).
Tyrosinase from mushroom (4187 U/mg) and L-tyrosine
were purchased from Fluka (Buchs, Switzerland). Alpha-
modified Eagle’s culture medium, antibiotics penicillin
and streptomycin, fetal bovine serum and tripsin were
purchased from Hyclone (Logan, Utah). All other chemi-
cals and reagents were analytical grade.
Plant selection
Fifteen medicinal plants including T. chebula gall were
selected from the Thai Lanna Medicinal Plant Textbook
Database version “Manosroi 2” developed by NPRDC,
Science and Technology Research Institute (STRI),
Chiang Mai University in Thailand. The frequent tra-
ditional use and scientific evidence for anti-aging and
longevity indicated in the recipes were used to select the
plants. The selected plants were collected from Chiang
Mai Province in Thailand during January to February in
2008 (Table 1). The specimen was authenticated by Suda
Saowakhon, a botanist at Faculty of Pharmacy, Chiang
Mai University, Thailand and deposited at NPRDC, STRI;
Chiang Mai University in Thailand.
Preparation of the extracts
The plants were washed, cut into pieces, dried at 40° ± 2°C
in a hot air oven, ground to powder and kept in an airtight
plastic bag at 4° ± 2°C until use. For the extraction process,
 Anti-aging of  Terminalia chebula   471

Table 1.  Comparison of percentage yields of the 60 extracts from the 15 selected Thai Lanna plants including T. chebula gall prepared by aqueous
and methanol cold and hot processes.
% yield
Scientific name (family) Thai Lanna name Part used CM HM CW HW
Acorus gramineus L. (Araceae) Wan Nam Whole plant 7.1 7.05 10.45 14.85
Cassia fistula L. (Leguminosae) Ratcha Phruek Fruit 15.45 15.1 25.39 27.54
Cyperus rotundus L. (Cyperaceae) Ya Haeo Mu Root 5.2 6.0 13.03 21.71
Dregea volubilis (L.f.) Benth. Ex Hook.f. (Asclepiadaceae) Kra Thung Ma Ba Leaf 11.3 6.16 15.0 25.26
Eclipta prostrata L. (Asteraceae) Ka Meng Whole plant 6.95 2.64 8.4 17.7
Myristica fragrans Houtt. (Myristicaceae) Chan Thet Stem 4.87 3.94 1.2 5.07
Nigella sativa L. (Ranunculaceae) Thian Daeng Seed 12.1 12.61 1.2 12.86
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Plumbago indica L. (Plumbaginaceae) Chetta Mun Phloeng Daeng Whole plant 17.38 16.13 14.02 29.3
Piper nigrum L.(Piperaceae) Phrik Thai Fruit 4.46 3.08 7.54 9.12
Pellacalyx parkinsonii Fisch. ST (Rhizophoraceae) Kan Phlu Flower 31.41 29.25 22.7 36.7
Piper sarmentosum Roxb. (Piperaceae) Cha Phlu Leaf 7.81 4.1 22.7 25.03
Plumbago zeylanica L. (Plumbaginaceae) Chetta Mun Phloeng Khao Whole plant 5.58 2.13 8.3 11.95
Terminalia chebula Retz. (Combretaceae) Kot Phung Pla Gall 59.02 23.28 49.85 60.0
Tinospora crispa L. (Menispermaceae) Boraphet Stem 8.95 6.8 15.87 18.76
Zingiber officinale Roscoe (Zingiberaceae) Khing Rhizome 12.3 12.6 15.74 22.98
Note: CM, cold methanol process; HM, hot methanol process; CW, cold aqueous process; HW, hot aqueous process

the dried plant powder (100 g) was extracted using four The filtrate was acidified with dilute HCl, filtered, cooled
different conditions. For hot methanol (HM), the powder and shaken with benzene. The benzene layer was sepa-
was extracted by continuous Soxhlet extraction for 1 h rated and put into a clean test tube and shaken with 2 mL
For personal use only.

in 400 mL methanol until exhausted (65° ± 2°C). For hot
water (HW), the powder was heated for 1 h with 400 mL
distilled water at 100° ± 2°C and then cooled to room
temperature (27° ± 2°C). For cold methanol (CM) and
cold water (CW), 100 g of the powder was macerated in
400 mL of methanol or distilled water and sonicated in
a bath sonicator for 1 h at room temperature (27° ± 2°C).
The mixtures were filtered through Whatman No. 1 fil-
ter paper and the plant residues were re-extracted twice
under the same conditions. The filtrates were pooled
and concentrated under vacuum by a rotary evaporator
(R-124 Buchi, Flawil, Switzerland), and lyophilized. The
dried extracts were stored at 4° ± 2°C prior to use. Sixty
extracts were obtained and the percentage yields were
calculated on a dry weight basis.
Phytochemical test of the extracts
The extract (20 mg), dissolved in 20 mL of 80% metha-
nol, was used for detecting the presence of alkaloids,
flavonoids, glycosides, saponins, tannins and xantho-
nes according to the methods previously described. For
alkaloid, 2 mL of the extract solution mixed with 1 mL
of 1% HCl was boiled over a water bath and 6 drops
of Dragendorff’s reagent were added. Cream-ish or
­brownish-red or orange precipitate indicated the pres-
ence of alkaloids (Brimer et al., 1989). Quinine sulfate
(Sigma) was used as a positive control. For anthraqui-
none (Borntrager’s test) determination, 0.1 g of the
­powder extract was boiled with 4 mL of alcoholic KOH for
2–3 minutes and diluted with 4 mL of water and filtered.
of the dilute ammonia solution. Extracts consisting of
anthraquinones gave an orange-red to deep orange-red
color in the aqueous layer (Allen, 1974; Harbone, 1976).
Anthraquinone from Fluka was used as a positive con-
trol. For the presence of flavonoids (Shinoda test), 2 mL
of the extract solution mixed with 1 mL of concentrated
HCl and magnesium ribbon gave the pink tomato-red
color (Allen, 1974). Luteolin from Sigma was used as a
positive control. For the qualitative assay of glycoside
(Fehling’s test for reducing sugars), 2 mL of the extract
solution mixed with 1 mL of Fehling’s solution was
heated in a water bath for 10 min. The brick-red precipi-
tate indicated the presence of reducing sugar contained
in glycosides (Harbone, 1976; Onwukaeme et al., 2007).
Glucose, fructose and galactose from Sigma were used
as positive controls. For saponin (Frothing test), 0.5 mL
of the extract solution was mixed with 5 mL of distilled
water. The frothing persistence indicated the presence of
saponins (Allen, 1974; Harbone, 1976). Saponaria offici-
nalis extract from NPRDC, STRI, Chiang Mai University
was used as a positive control. For tannins, 2 mL of the
extract solution were mixed with 2 mL of 15% FeCl3 solu-
tion. The blue-black precipitate indicated the presence of
tannins (VanMiddlesworth & Cannell, 1998; Onwukaeme
et al., 2007). Tannic acid from Fluka was used as a posi-
tive control. For xanthones, 2 mL of the extract solution
was mixed with 1 mL 5% KOH reagent. The formation
of yellow precipitate indicated the presence of xantho-
nes (Allen, 1974; Harbone, 1976). Garcinia mangostana
Linn. extract from NPRDC, STRI, Chiang Mai University
was used as a positive control.
 472   Aranya Manosroi et al.
Antioxidative assays
DPPH radical scavenging activity
The DPPH radical scavenging activity of all extracts
was determined by a modified method previously
described (Tachibana et  al., 2001). Briefly, 50 µL of
the five serial concentration extracts, 0.001-10 mg/mL
dissolved in methanol and 20%v/v DMSO (1:1), and
50 µL of ethanol solution of DPPH were put into each
well of a 96-well microplate (Nalge Nunc International,
NY). The reaction mixture was allowed to stand for
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30 min at 27° ± 2°C, and the absorbance was meas-
ured at 515 nm by a well reader (Bio-Rad, model 680
microplate reader, Philadelphia, PA 19102-1737, USA)
against a blank (methanol mixed with 20% v/v DMSO,
1:1). Ascorbic acid, BHT and α-tocopherol (0.001-
10 mg/mL) were used as positive controls. The experi-
ments were done in triplicate. The IC50 value which was
the concentration of the sample that scavenged 50% of
the DPPH radical was determined. The percentages of
DPPH radical scavenging activity were calculated:
Scavenging (%) = (Abscontrol − Abssample )/Abscontrol × 100%
For personal use only.
The histogram of the percentages of DPPH radical
scavenging activity of the extract at 0.1 mg/mL was
presented.
Chelating assay
The Fe2+ chelating ability of the extract was measured
by the ferrous iron-ferrozine complex method (Decker
& Welch, 1990). Briefly, the reaction mixture contain-
ing 2 mM FeCl2 (10 µL) and 5 mM ferrozine (10 µL) and
100 µL of the five serial concentration extracts (0.001-
10 mg/mL dissolved in methanol and 20% v/v DMSO 1:1
solution) were mixed in a 96-well plate and incubated
for 10 min at 27° ± 2°C. The absorbance was recorded by
a well reader at 570 nm. The absorbance of the control
was determined by replacing the extract with methanol.
EDTA (0.001-10 mg/mL) was used as a positive control.
The experiments were done in triplicate. The IC50 value
which was the concentration of the sample that chelated
50% of the ferrous iron was determined. The ability of the
sample to chelate ferrous ion was calculated:
Chelating effect (%) = [1 − (Abssample /Abscontrol )]× 100%
The histogram of the percentages of chelating effect of the
extract at 0.1 mg/mL was presented.
Tyrosinase inhibition assay
The extracts were assayed by the modified tyrosinase
inhibition method previously described (Shimizu et al.,
1998). Briefly, 120 µL of 1.66 mM of tyrosine solution
in 0.1 M phosphate buffer (pH 6.8), 60 µL of five serial
concentrations of the extracts (0.001-10 mg/mL dis-
solved in methanol and 20% v/v DMSO 1:1) and 60 µL
phosphate buffer were mixed in a 96-well plate and incu-
bated at 37° ± 2°C for 60 min. Then, 60 µL of tyrosinase
enzyme solution (0.6 mg/mL) in phosphate buffer were
added. The enzyme activity at 37° ± 2°C was measured
by a well reader at 450 nm. Ascorbic acid and kojic acid
(0.001–10 mg/mL) were used as positive controls. The
experiments were done in triplicate. The IC50 value which
was the concentration of the sample that inhibited 50%
of the enzyme activity was determined. The inhibition
percentage of tyrosinase was calculated:
Tyrosinase inhibition (%) = [(Abscontrol − Abssample )
/Abscontroll ]× 100%
The histogram of the percentages of the inhibition effect
of the extract at 0.1 mg/mL was presented.
Cell culture
The normal human skin fibroblasts were provided by
Natthanej Luplertlop at the Department of Tropical
Hygiene, Faculty of Tropical Medicine, Mahidol
University, Bangkok, Thailand. Cells were cultured
under standard conditions in the complete culture
medium containing α-modified Eagle’s culture medium
(MEM-Alpha) supplemented with 10% (v/v) fetal bovine
serum (FBS), penicillin (100 U/ml) and streptomycin
(100 mg/ mL). Cells were incubated in a temperature-
­controlled, humidified incubator (Shel Lab, model
2123TC, Cornelius, OR 97113, USA) with 5% CO2 at 37°C.
Cells were used at the 15th passage.
Cell proliferation activity by the SRB assay
The extracts from the three plants showing the highest
activity of DPPH scavenging, chelating and tyrosinase
inhibition activities were selected to test for cell prolif-
eration activity of the 15th passage normal human skin
fibroblasts by SRB assay according to the method of
Papazisis et al. (1997). Ascorbic acid (0.001–10 mg/mL)
was used as a positive control. The cells were plated at a
density of 1 × 105 cells/well in 96-well plates and left for
cell attachment on the plate overnight in 5% CO2 at 37°C.
Cells were then exposed to five serial concentrations of
the extracts (0.001–10 mg/mL) for 24 h. After incuba-
tion, the adherent cells were fixed in situ, washed and
dyed with SRB. The bound dye was solubilized and the
absorbance was measured at 540 nm by a well reader.
The assays were done in three independent and separate
 Anti-aging of  Terminalia chebula   473

experiments. The percentage of cell growth was calcu- Statistical analysis

lated using the follow equation:
%G = ( Asample − Ablank /Acontrol − Ablank ) ×100
Stimulation index (SI) which was the ratio between the
percentages of %G treated with the extracts at 0.1 mg/mL
and the control (no treatment) was presented.
All assays were performed in triplicate in three inde-
pendent and separate experiments. The data were pre-
sented as means ± standard deviations (SD) from three
independent analyses, separate experiments.
Results and discussion

Percentage yields of the plant extracts prepared by

Gelatinolytic activity on MMP-2 inhibition of the plant
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different processes
extracts (Zymography)
The percentage yields of the 60 extracts prepared by four
The CM, CW, HM and HW extract of the three plants
different extraction processes (CM, HM, CW and HW)
which showed the highest activity of human fibroblast
of the 15 selected Thai Lanna medicinal plants were
proliferation activity were selected to assay for gelati-
presented in Table 1. Aqueous extracts of most plants
nolytic activity of MMP-2 inhibition in comparing with
gave higher percentage yield than the methanol extracts.
ascorbic acid. A monolayer of 5 × 105 cells at the 15th pas-
The plants may contain more water-soluble than water-
sage normal human skin fibroblasts was maintained in
insoluble constituents. The highest percentage yields
the culture medium without FBS for 24 h, treated with the
were from T. chebula gall by CM, CW and HW at 59.02%,
extracts and ascorbic acid at concentration 0.1 mg/mL
49.85% and 60% respectively. For HM, P. parkinsonii
and incubated for 48 h. The culture supernatants were
flower gave the highest percentage yield at 29.25% while
collected. To assess the gelatinolytic activities of MMP-2
T. chebula gall gave 23.28%. HW of all plants gave higher
in culture media, SDS-PAGE zymography using gelatin as
yields than CW, while CM showed higher yields than
For personal use only.

a substrate was performed. Briefly, 20 µL of cell culture

HM. Most plants may contain heat labile water-insoluble
supernatant were suspended in loading buffer,0.125M
components. Thus, the hot process appeared to be the
Tris (pH 6.8), 4% SDS and 0.04% bromophenol blue,
superior process for water-soluble components, whereas
and, without prior denaturation, run on 10% SDS-
the cold process was advantageous for water-insoluble
polyacrylamide gel containing 1 mg/mL gelatin. After
substances.
electrophoresis, gels were washed to remove SDS and
incubated for 20 min in renaturing buffer (50 mM Tris,
5 mM CaCl2, 0.02% NaN3, 2.5% Triton X-100). The gels Phytochemical tests of the extracts
were then incubated for 24 h at 37°C in developing buffer
Table 2 shows the phytochemical constituents of the
50 mM Tris (pH 7.5), 5 mM CaCl2, 0.02% NaN3 and 1%
60 extracts. Alkaloid was a basic secondary metabo-
Triton X-100. Gels were subsequently stained with 0.5%
lite in all extracts, while glycosides and tannin were in
Coomassie brilliant blue G-250 and de-stained in 30%
most extracts. Few extracts contained anthraquinones.
methanol and 10% acetic acid (v/v) to detect gelatino-
Interestingly, the extracts of C. fistula fruit prepared by
lytic activity (Kim et al., 2007). The gel was documented
all processes (CM, HM, CW and HW) contained all phy-
by a gel documentation system (Bio-Rad Laboratories,
tochemical compounds including alkaloids, anthraqui-
UK) and analyzed by Quantity 1-D analysis software.
nones, flavonoids, glycosides, saponins, tannins and
The area multiplied by intensity (mm2) of the bands on
xanthones. All extracts by all extraction processes con-
the gel was determined as the relative MMP-2 content
tained thesame phytochemicals, except for anthraqui-
(Carmeliet et  al., 1997; Arican & Ceylan, 1999). The
nones in M. fragrans, P. indica and P. zeylanica which
percentages of MMP-2 inhibition in comparing to the
were found in HM and CM, but not found in HW and
control (the untreated systems) were calculated by using
CW. Saponins in P. nigrum and tannins in A. gramineus
the following equation:
were found in CW and HW; CM and HM, but not found in
CM and HM; CW and HW, respectively. For all T. chebula
MMP - 2 inhibition (%) = 100 − [( MMP − 2 content of sample
gall extracts, they contained alkaloids, flavonoids, sapon-
/MMP − 2 content of control) × 100] ins, tannins and xanthones but no anthraquinones and
glycosides. Phytochemicals in the extracts appeared
The assays were done in three independent separate to depend on types of plants more than the extraction
experiments. The potency of MMP-2 inhibition of the conditions. Generally, solvents and temperatures are
samples was compared with the positive control (ascor- important parameters to obtain high yields. Temperature
bic acid). has a positive effect on the extraction yields and rates by
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For personal use only.

474   Aranya Manosroi et al.

Table 2.  Qualitative determination of constituents by phytochemical tests in 60 extracts from the 15 selected Thai Lanna plants including T. chebula gall prepared by various extraction
processes.
Alkaloids Anthraquinones Flavonoids Glycosides Saponins Tannins Xanthones
Extracts of plants CM HM CW HW CM HM CW HW CM HM CW HW CM HM CW HW CM HM CW HW CM HM CW HW CM HM CW HW
A. gramineus + + + + − − − − − − − − + + + + − − − − + + − − − − − −
C. fistula + + + + + + + + + + + + + + + + + + + + + + + + + + + +
C. rotundus + + + + − − − − − − − − + + + + − − − − + + + + − − − −
D. volubilis + + + + − − − − − − − − + + + + + + + + − − − − − − − −
E. prostrata + + + + − − − − − − − − + + + + + + + + + + + + + + + +
M. fragrans + + + + + + − − − − − − + + + + − − − − + + + + − − − −
N. sativa + + + + − − − − + + + + + + + + − − − − + + + + − − − −
P. indica + + + + + + − − − − − − + + + + − − − − + + + +
− − − −
P. nigrum + + + + − − − − − − − − − − − − − − + + + + + + − − − −
P. parkinsonii + + + + − − − − + + + + + + + + − − − − + + + + + + + +
P. sarmentosum + + + + − − − − − − − − − − − − − − − − − − − − − − − −
P. zeylanica + + + + + + − − − − − − + + + + − − − − + + + + − − − −
T. chebula + + + + − − − − + + + + − − − − + + + + + + + + + + + +
T. crispa + + + + − − − − − − − − + + + + − − − − + + + + − − − −
Z. officinale + + + + − − − − − − − − + + + + − − − − + + + + − − − −
Note: CM, cold methanol process; HM, hot methanol process; CW, cold aqueous process; HW, hot aqueous process; + presence; − absence.
 Anti-aging of  Terminalia chebula   475

enhancing the solubility of the compounds. However, were 2.37 and 3.09 times more potent than α-tocopherol
some heat labile phytochemicals may be degraded by (IC50 value of 0.038 ± 0.002 mg/mL) and BHT (IC50 value
high temperature as CW gave higher yield than HW of of 0.049 ± 0.002 mg/mL), respectively. The phytochemi-
most plants. cals found in this extract including alkaloids, flavonoids,
saponins, tannins and xanthones (Table 2) may be syn-
ergistic and responsible for this activity. T. chebula and
DPPH radical scavenging activity
its galls have been used traditionally in combination
Table 3 shows the IC50 values of the DPPH radical scav- with other Thai Lanna medicinal plants in many recipes
enging assay of the 60 extracts. Figure 1 demonstrated for promoting longevity. Thus, this result has supported
the percentages of DPPH radical scavenging activity of the folklore wisdom application of T. chebula gall in Thai
the 60 extracts at 0.1 mg/mL. The standard antioxidants Lanna medicines.
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(ascorbic acid, α-tocopherol and BHT at 0.1 mg/ml) gave

the scavenging activity of 96.5% ± 0.1%, 35.74% ± 0.2%
Chelating activity
and 27.43% ± 0.1% with IC50 values of 0.014 ± 0.001,
0.038 ± 0.002 and 0.049 ± 0.002 mg/mL, respectively. Table 3 shows the IC50 values of chelating activity assay
The methanol extract of most plants exhibited higher of the 60 extracts. Figure 2 presents the percentages of
activity than the aqueous extracts. Alcoholic solvents the chelating effect of 60 extracts at 0.1 mg/mL. None
have been commonly employed to extract phenolic of the extracts indicated better chelating activity than
compounds from plants, because of obtaining high EDTA at 0.1 mg/mL which gave 87.74% ± 0.1% and the
yield although they were not highly selective for phenols IC50 value of 0.079 ± 0.001 mg/mL. The CM, HM, CW
(Spigno & De Faveri, 2007). The phenolic compounds and HW extracts of T. chebula gall gave the IC50 val-
which have been reported to scavenge DPPH include ues of 0.282 ± 0.002, 0.217 ± 0.002, 2.287 ± 0.275 and
flavonoids, anthraquinones, anthocyanidins, xantho- 1.151 ± 0.006, respectively. The highest chelating activity
nes, and tannins. They also scavenged superoxide and of 79.49% ± 1.1% was found in HM of C. rotundus root
For personal use only.

hydroxyl radical by single electron transfer (Ho et  al., with the IC50 value of 0.094 ± 0.001 mg/mL. In fact, C.
1999; Choi et  al., 2002). Extracts from T. chebula gall rotundus has been reported to contain many phenolic
prepared by all processes exhibited higher DPPH radi- compounds, such as gallic acid, p-coumaric acid and
cal scavenging activity than those of other plants. The epicatechin (Proestos et al., 2005) which are potent anti-
highest scavenging activity of T.chebula gall extract at oxidants by ferric reducing antioxidant power and Trolox
84.64% ± 2.22% was found (at 0.1 mg/mL) by CW proc- equivalent antioxidant capacity assays along with metal
ess with the IC50 value of 0.016 ± 0.001 mg/mL, which chelating properties (Amin & Razieh, 2007). From our

100.00
90.00
DPPH radical scavenging activity (%)

CM
80.00 HM
70.00 CW
60.00 HW

50.00
40.00

30.00
20.00

10.00
0.00
A. gramineus

C. fistula

C. rotundus

E. prostrata

P. indica

P. nigrum

P. sarmentosum

P. zeylanica

T. chebula

T. crispa

Z. officinale
D. volubilis

M. fragrans

N. sativa

Ascorbic acid

alpha-tocopherol
P. parkinsonii

BHT

Figure 1.  Comparison of the percentages of DPPH radical scavenging activity of the 60 extracts at 0.1 mg/mL from the 15 selected Thai Lanna
plants including T. chebula gall and the standard antioxidants (ascorbic acid, α-tocopherol and BHT at 0.1 mg/mL). CM, cold methanol process;
HM, hot methanol process; CW, cold aqueous process; HW, hot aqueous process.
 Pharmaceutical Biology Downloaded from informahealthcare.com by Karolinska Institutet University Library on 06/08/14
For personal use only.

Table 3.  IC50 values of the 60 extracts from the 15 selected Thai Lanna plants including T. chebula gall determined by the DPPH radical scavenging, chelating and tyrosinase inhibition assays.
Extracts of IC50 of DPPH radical scavenging assay (mg/mL) IC50 of chelating assay (mg/mL) IC50 of tyrosinase inhibition assay (mg/mL)
476   Aranya Manosroi et al.

plants CM HM CW HW CM HM CW HW CM HM CW HW
A. gramineus 0.065 ± 0.008 0.065 ± 0.006 0.159 ± 0.016 0.076 ± 0.009 0.152 ± 0.002 0.138 ± 0.006 4.148 ± 0.149 0.598 ± 0.002 0.132 ± 0.002 0.184 ± 0.002 1.251 ± 0.003 0.598 ± 0.002
C. fistula 0.044 ± 0.001 0.045 ± 0.006 0.057 ± 0.003 0.054 ± 0.003 0.476 ± 0.001 0.375 ± 0.002 1.571 ± 0.088 0.15 ± 0.008 0.076 ± 0.001 0.084 ± 0.002 2.507 ± 0.019 0.15 ± 0.008
C. rotundus 0.035 ± 0.002 0.028 ± 0.001 0.066 ± 0.005 0.033 ± 0.001 0.104 ± 0.001 0.094 ± 0.001 2.125 ± 0.006 1.117 ± 0.001 0.104 ± 0.001 0.129 ± 0.005 3.192 ± 0.007 1.117 ± 0.001
D. volubilis 0.085 ± 0.021 0.102 ± 0.011 0.052 ± 0.002 0.127 ± 0.002 0.487 ± 0.008 0.355 ± 0.001 1.05 ± 0.029 0.436 ± 0.001 0.087 ± 0.008 0.156 ± 0.004 0.696 ± 0.001 0.436 ± 0.001
E. prostrate 0.04 ± 0.011 0.060 ± 0.002 0.057 ± 0.019 0.066 ± 0.001 0.642 ± 0.036 0.548 ± 0.030 0.727 ± 0.01 0.634 ± 0.048 0.142 ± 0.036 0.174 ± 0.007 0.385 ± 0.017 0.334 ± 0.048
M. fragrans 0.042 ± 0.001 0.042 ± 0.001 0.064 ± 0.003 0.041 ± 0.009 0.233 ± 0.002 0.121 ± 0.024 0.669 ± 0.001 1.573 ± 0.132 0.093 ± 0.002 0.106 ± 0.006 2.166 ± 0.068 1.573 ± 0.132
N. sativa 0.085 ± 0.003 0.053 ± 0.003 0.062 ± 0.008 0.074 ± 0.006 0.161 ± 0.010 0.15 ± 0.023 1.18 ± 0.155 3.04 ± 0.039 0.161 ± 0.01 0.189 ± 0.004 3.353 ± 0.018 3.04 ± 0.039
P. indica 0.036 ± 0.002 0.033 ± 0.003 0.06 ± 0.01 0.047 ± 0.007 0.175 ± 0.004 0.165 ± 0.034 2.578 ± 0.105 0.179 ± 0.021 0.145 ± 0.004 0.175 ± 0.002 0.524 ± 0.027 0.179 ± 0.021
P. nigrum 0.058 ± 0.029 0.079 ± 0.003 0.034 ± 0.004 0.054 ± 0.004 6.376 ± 0.031 6.36 ± 0.255 6.742 ± 0.027 1.485 ± 0.064 1.076 ± 0.031 1.137 ± 0.015 1.64 ± 0.004 1.485 ± 0.064
P. parkinsonii 0.026 ± 0.003 0.027 ± 0.003 0.027 ± 0.009 0.036 ± 0.003 0.306 ± 0.008 0.163 ± 0.009 0.886 ± 0.015 0.443 ± 0.012 0.106 ± 0.008 0.129 ± 0.002 0.541 ± 0.03 0.443 ± 0.012
P. sarmentosum 0.028 ± 0.003 0.032 ± 0.001 0.118 ± 0.005 0.07 ± 0.001 0.324 ± 0.003 0.266 ± 0.019 1.743 ± 0.033 1.06 ± 0.005 0.124 ± 0.003 0.14 ± 0.005 2.26 ± 0.198 1.060 ± 0.005
P. zeylanica 0.045 ± 0.002 0.049 ± 0.004 0.063 ± 0.003 0.04 ± 0.005 0.436 ± 0.001 0.125 ± 0.003 2.335 ± 0.449 0.795 ± 0.063 0.096 ± 0.001 0.129 ± 0.003 1.473 ± 0.161 0.795 ± 0.063
T. chebula 0.021 ± 0.004 0.017 ± 0.001 0.016 ± 0.001 0.018 ± 0.007 0.282 ± 0.002 0.217 ± 0.002 2.287 ± 0.275 1.151 ± 0.006 0.082 ± 0.002 0.088 ± 0.001 0.393 ± 0.008 0.151 ± 0.006
T. crispa 0.142 ± 0.016 0.09 ± 0.015 0.171 ± 0.018 0.15 ± 0.01 0.332 ± 0.022 0.124 ± 0.005 0.775 ± 0.005 1.645 ± 0.134 0.152 ± 0.022 0.23 ± 0.016 1.484 ± 0.187 1.645 ± 0.134
Z. officinale 0.035 ± 0.004 0.036 ± 0.002 0.044 ± 0.001 0.087 ± 0.003 0.52 ± 0.001 0.352 ± 0.004 0.865 ± 0.004 1.664 ± 0.013 0.12 ± 0.001 0.174 ± 0.006 2.969 ± 0.022 1.664 ± 0.013
Standards IC 50 of DPPH radical scavenging assay (mg/mL) IC50 of chelating assay (mg/mL) IC50 of tyrosinase inhibition assay (mg/mL)
Ascorbic acid 0.014 ± 0.001 − 0.046 ± 0.001
α-Tocopherol 0.038 ± 0.002 − −
BHT 0.049 ± 0.002 − −
EDTA − 0.079 ± 0.001 −
Kojic acid − − 0.03 ± 0.001
Note: CM, cold methanol process; HM, hot methanol process; CW, cold aqueous process; HW, hot aqueous process.
 Anti-aging of  Terminalia chebula   477

phytochemical test, C. rotundus extract by HM contained 2S-configuration together with the common flavan-3-
alkaloids, glycosides and tannins (Table 2). ols and proanthocyanidins like catechin, epicatechin,
procyanidin B-2 and epiafzelechin which are strong
tyrosinase inhibitors have also been found in the pods
Tyrosinase inhibition activity
of C. fistula (Kashiwada et al., 1990).
Table 3 shows the IC50 values of the tyrosinase inhibition
activity of 60 extracts. Figure 3 demonstrates the percent-
Proliferation of normal human skin fibroblasts by the
ages of tyrosinase inhibition of 60 extracts at 0.1 mg/ mL.
SRB assay
The CM, HM, CW and HW extracts of T. chebula gall
gave the IC50 values of 0.082 ± 0.002, 0.088 ± 0.001, From the DPPH radical scavenging, chelating and
0.393 ± 0.008 and 0.151 ± 0.006 respectively. The CM tyrosinase inhibition assays, three plants including T.
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extract of C. fistula fruit gave the highest tyrosinase chebula gall, C. rotundus root and C. fistula fruit were
inhibition activity of 63.55% ± 0.16% with the IC50 value selected to investigate for human skin fibroblasts prolif-
of 0.076 ± 0.001 mg/mL, but lower than ascorbic acid and erative assay. The stimulation index (SI) of the extracts
kojic acid (at 0.1 mg/mL) which gave 71.53% ± 0.2% and at 0.1 mg/mL on normal human skin fibroblasts (15th
88.63% ± 0.1% with the IC50 values of 0.046 ± 0.001 and passage) is shown in Table 4. Extracts of all three plants
0.03 ± 0.001 mg/mL, respectively. From Table 2, the CM by HW and CW showed higher SI than by HM and CM.
extract of C. fistula contained anthraquinones, flavo- The methanol extracts appeared to be more toxic to
noids, glycosides, saponins and tannins. The methanol the cells than the aqueous extracts. The CW extract of
extract of C. fistula has been evaluated in in vitro models T. chebula gall gave the highest cell stimulative effect
in protecting free radical-induced lipid peroxidation in with the SI value of 1.441 ± 0.084 which was more potent
model membranes (Sunil & Muller, 1998). Flavonoids in than ascorbic acid (at 0.1 mg/mL) that gave the SI value
this plant have also been reported to inhibit tyrosinase of 1.21 ± 0.033. The higher cell stimulative effect on
due to their ability to chelate copper in the active site fibroblasts of the aqueous extracts in comparing to the
For personal use only.

(Maity et al., 1998; Cuellar et al., 2001). The fruit tissue methanol extracts might be due to the presence of more
of this plant was found to be a rich source of potassium, polar compounds in the aqueous extracts (CW and
calcium, iron and manganese (Barthakur et  al., 1995) HW) which are usually more bioactive and less toxic to
which may be competitive inhibitors of the tyrosinase cells. This result can anticipate the effect of the extract
enzyme. Proanthocyanidins including flavan-3-ol (epi- on collagen synthesis from the stimulation of human
afzelechin and epicatechin) units with an abnormal fibroblast proliferation.

100

90
CM
80
HM
70 CW
Chelating effect (%)

60 HW

50
40

30

20
10

0
EDTA
C. fistula

E. prostrata

M. fragrans

N. sativa

P. indica

P. nigrum

P. sarmentosum

P. zeylanica

T. chebula

T. crispa

Z. officinale
A. gramineus

C. rotundus

D. volubilis

P. parkinsonii

Figure 2.  Comparison of the percentages of the chelating effect (%) by the ferrous iron-ferrozine complex method of the 60 extracts at
0.1 mg/mL from the 15 selected Thai Lanna plants including T. chebula gall and the standard chelating agent (EDTA at 0.1 mg/mL). CM, cold
methanol process; HM, hot methanol process; CW, cold aqueous process; HW, hot aqueous process.
 478   Aranya Manosroi et al.

100
90
CM
80
Tyrosinase inhibition (%)

HM
70 CW
60 HW
50
40
30
20
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10
0
C. fistula

E. prostrata
A. gramineus

C. rotundus

D. volubilis

M. fragrans

N. sativa

P. indica

P. nigrum

P. sarmentosum

P. zeylanica

T. chebula

T. crispa

Z. officinale

Ascorbic acid

Kojic acid
P. parkinsonii
Figure 3.  Comparison of the percentages of tyrosinase inhibition of the 60 extracts at 0.1 mg/mL from the 15 selected Thai Lanna plants including
T. chebula gall and the standard whitening agents (ascorbic acid and kojic acid at 0.1 mg/mL). CM, cold methanol process; HM, hot methanol
process; CW, cold aqueous process; HW, hot aqueous process.

Table 4.  Comparison of the stimulation index (SI) of the 12 extracts at be not only from the cell proliferation stimulation, but
0.1 mg/mL of the three selected Thai Lanna plants including T. chebula also from the phytochemicals existing in the CW extract
For personal use only.

gall on normal human skin fibroblasts (15th passage). of this plant including alkaloids, flavonoids, tannins and
SI at 0.1 mg/mL xanthones. This result has also supported the traditional
Extracts CM HM CW HW use for longevity of T. chebula gall since the indication of
C. fistula 1.289 ± 0.181 1.292 ± 0.026 1.366 ± 0.11 1.34 ± 0.123 this plant in many Thai Lanna recipes was by cold aque-
C. rotundus 1.158 ± 0.07 1.172 ± 0.15 1.25 ± 0.199 1.233 ± 0.167 ous extraction (maceration).
T. chebula 1.364 ± 0.057 1.411 ± 0.188 1.441 ± 0.084 1.393 ± 0.104
Ascorbic acid 1.21 ± 0.033
Note: CM, cold methanol process; HM, hot methanol process; CW,
cold aqueous process; HW, hot aqueous process. SI is the ratio cal-
Conclusion
culated between the percentages of cell growth of the systems treated
and not treated (control) with the extract. This present study has demonstrated the in vitro anti-
aging activities of T. chebula gall in comparison to 14
Thai Lanna medicinal plants with the indication for
Gelatinolytic activity on MMP-2 inhibition of the plant
longevity selected from the database of Thai Lanna
extracts (Zymography)
medicinal plant recipes. The biological activities which
Although many studies have reported on the biological are related to anti-aging including antioxidative, tyrosi-
activities of T.chebula (Lee et  al., 1995; Burapadaja & nase inhibition and proliferative stimulation as well as
Bunchoo, 1995; Saleem et al., 2002; Chen et al., 2003), the MMP-2 expression inhibition on human skin fibrob-
there was no report on the effects of the extract of this lasts were used to evaluate the 60 extracts prepared by
plant on MMP-2 expression in human dermal fibrob- aqueous and methanol hot and cold processes. For
lasts. Figure 4 shows the comparison of the inhibition of all 15 plants including T. chebula gall, an aqueous hot
MMP-2 between T. chebula gall extracts (CW, CM, HW extraction process (HW) gave higher yields than the
and HM) and ascorbic acid by zymography. All extracts aqueous cold process (CW), whereas the methanol cold
of T. chebula gall as well as ascorbic acid inhibited the process (CM) gave better yield than the methanol hot
MMP-2 expression. The CW extract of T. chebula gall at process (HM). The methanol extract either by the hot
0.1 mg/mL showed the inhibition of MMP-2 at 89.94% or cold process of most plants exhibited higher DPPH
of the control which was about 1.37 times more potent radical scavenging, chelating and tyrosinase inhibi-
than ascorbic acid that gave 65.79% of the control. This tion activities than the aqueous extracts. The three Thai
has suggested that the CW extract of T. chebula gall was Lanna medicinal plants, including T. chebula gall, C.
a potent inhibitor of MMP-2 expression on early aging rotundus root and C. fistula fruit which gave the high-
human skin fibroblasts (15th passage). This effect may est DPPH radical scavenging, chelating and tyrosinase
 Anti-aging of  Terminalia chebula   479

A (0.1 mg/ml) CM CW HM HW Ascorbic Control

1st run MMP-2

2nd run

3rd run
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B 1000000
797800.89
900000

800000
MMP-2 contents [area x intensity (mm2)]

700000
567304.36
600000

500000

400000 356743.80 344809.75

300000 270929.91
For personal use only.

200000
79963.40
100000

0
CM CW HM HW Ascorbic Control
acid
120.0

89.94
100.0

80.0
65.79
% MMP-2 inhibition

54.92 56.44
60.0

40.0 28.32

20.0

0.0
CM CW HM HW Ascorbic
acid

Figure 4.  Comparison of the gelatinolytic activity on MMP-2 inhibition between T. chebula gall extracts (CW, CM, HW and HM) at 0.1 mg/mL
and ascorbic acid at 0.1 mg/mL. (A) zymograms of three independent separate experiments, (B) MMP-2 contents [area × intensity; (mm2)] (left)
and the percentages of MMP-2 inhibition (right). CM, cold methanol process; HM, hot methanol process; CW, cold aqueous process; HW, hot
aqueous process).
 480   Aranya Manosroi et al.
inhibition activity respectively, were selected to test for
proliferation stimulation and MMP-2 expression inhibi-
tion on normal human skin fibroblasts (15th passage).
The aqueous extracts exhibited stronger proliferation
stimulation activity on normal human fibroblasts than
the methanol extracts. For the CW extracts, T. chebula
gall showed more stimulative effect than ascorbic acid,
while C. fistula fruit and C. rotundus root gave almost
the same effect as ascorbic acid. Moreover, the inhibi-
tion of MMP-2 expression determined by zymography
of T.chebula gall CW extract was 1.37 times more potent
Pharmaceutical Biology Downloaded from informahealthcare.com by Karolinska Institutet University Library on 06/08/14
than ascorbic acid. This present study has not only dem-
onstrated the potent in vitro anti-aging effects of the cold
aqueous extract of T.chebula gall, but also supported the
traditional indication for longevity of this plant in the
Thai Lanna medicinal plant recipes as well.
Acknowledgements
The authors would like to thank Dr. Natthanej Luplertlop
at Department of Tropical Hygiene, Faculty of Tropical
Medicine, Mahidol University in Thailand for providing
the human skin fibroblasts.
For personal use only.
Declaration of interest
This work was supported by the Thailand Research Fund
(TRF) under the RGJ-PhD program and Natural Products
Research and Development Center (NPRDC), Science
and Technology Research Institute (STRI), Faculty of
Pharmacy, Chiang Mai University, Chiang Mai 50200,
Thailand. The authors report no conflicts of interest. The
authors alone are responsible for the content and writing
of the paper.
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