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ABSTRACT
Fifteen medicinal plant extracts were investigated for: total phenolic content and free radical scavenging effect
by DPPH and ABTS assays; anti-lipid peroxidation activity by TBARS; and for antiglycation activity. The
results revealed that the total phenolic content showed good correlation with free radical scavenging by ABTS
(r = 0.721) and anti-lipid peroxidation by TBARS (r = -0.659), but showed no correlation with antiglycation.
Three extracts from Tamarindus indica, Camellia sinensis and Artocarpus lakoocha demonstrated a significant
antioxidant effect, and also showed a promising antiglycation effect. The IC50 (mg/ml) were 0.9-0.16 for the
DPPH method; TEAC values (mg Trolox/mg sample) of 1.722.83 for the ABTS method; IC50 (mg/ml) of 0.64-
1.22 for the TBARS method; and IC50 ranging from 0.01 to 3.20 for the antiglycation method. These three herbs
were found to possess effective antioxidant and antiglycation activities, and could be further developed for use
in anti-aging cosmetics.
D-ribose; thiobarbituric acid (TBA); butylated concentrations within the range of 1090% reduction in
hydroxytoluene (BHT); quercetin; kaempferol; Trolox; absorbance, as calculated in Eq. (1):
gallic acid; D-glucose; and sodium azide were obtained
from Sigma Chemical, USA. Other chemicals used in the DPPH radical scavenging activity (%)
study were 2,2'-azobis (2-amidinopropane) hydrochloride = [Ao - (A1 As)]/Ao x 100 (1)
(AAPH) (Wako Pure Chemical Industries, Japan); D- where Ao is the absorbance of the solution, A1 is the
fructose (Merck, Germany); Triton X-100 absorbance in the presence of the plant extract in DPPH
(C14H22O(C2H4O)n) (Dow Chemical, USA); soybean solution, and As (which is used for error correction arising
phosphatidylcholine (Degussa, Germany); and cholesterol from unequal color of the sample solutions) is the
(lanolin, C27H46O; Fluka Chemie, Switzerland) absorbance of the sample extract solution without DPPH.
The IC50 value (mg/ml) of each extract was estimated by
Preparation of plant extracts sigmoid non-linear regression using SigmaPlot 2000
All plants were dried at 50C and then ground into Demo.
powder. A powder sample of each plant (100 g) was
mixed with 95% ethanol (2,000 ml) in the dark at room Evaluation of free radical scavenging activity using
temperature and shaken during the 4.5 h extraction time to ABTS assay
ensure complete extraction. The extracts were filtered ABTS radical cat ions (ABTS+) were produced by
through Whatman No.4 paper and then centrifuged reacting ABTS stock solution with 2.45 mM potassium
(1500x g for 15 min). Ethanol was removed from the persulfate and allowing the mixture to stand in the dark at
supernatants on a rotary evaporator at 50 mm Hg, 50oC, to room temperature for 1216 h before use. After the
yield thick and viscous residues as ethanolic crude addition of 1.0 ml of diluted ABTS+ solution to 10 ml of
extracts. antioxidant compounds or Trolox standards (final
concentration 0-15 mM) in ethanol, absorbance readings
were taken at 30C exactly 30 min later. Determinations
Determination of total phenolic content
of percentage inhibition by spectrophotometry at 734 nm
The total phenolic content of each ethanolic extract was
were carried out in triplicate.
determined using Folin-Ciocalteu reagent (Khknen et
al., 1999). Each sample of extract (0.8 to 0.9 g 0.01 Lipid peroxidation assay
mg) was diluted with 5 ml methanol; then the sample This assay was modified from the method of Prabhakar
solution (200 l) was transferred into a test tube and (2007). A solution of soybean phosphatidylcholine
mixed thoroughly with 1 ml of Folin-Ciocalteu reagent. (247.36 mg) and cholesterol (30.92 mg) in chloroform (20
After mixing for 3 min, 0.8 ml of 7.5% (w/v) sodium ml) was dried under vacuum in a rotary evaporator
carbonate was added. Each sample mixture was agitated (<50C) to yield a thin, homogenous film, which was
with a vortex mixer, allowed to stand for a further 30 min placed in a desiccator for 24 h. The film was then
in the dark, and centrifuged at 3300 g for 5 min. The dispersed in phosphate buffered saline (PBS) solution (pH
absorbance of each plant extract and a prepared blank 7.2, 20 ml) in a water bath (50C). The mixture was
were measured at 765 nm using a Genesys 20 sonicated to obtain a homogeneous suspension of
spectrophotometer (Thermo Fisher Scientific, USA). liposome. Lipid peroxidation was initiated by adding 60
Total phenolic content was expressed as mg/g gallic acid l of AAPH to the mixture containing liposome (600 l).
equivalent using the equation obtained from the The reaction mixture was incubated at 50C for 24 h.
calibration curve: y = 4.3308x + 0.0.0336, R2 = 0.9994 as After incubation, 250 l of thiobarbituric acid (0.6% w/v),
shown in fig.1. Data are reported as mean standard 100 l of Triton X-100 (3% v/v) and 500 l of BHT (20%
deviation of three replications. v/v) were added to terminate the reaction. The samples
were heated at 90C for 30 min, and then allowed to cool.
Evaluation of free radical scavenging activity using The absorbance of the upper organic layer was measured
DPPH assay by a multimode detector at 540 nm.
The experiment was carried out according to the method
of Blois (1958) with a slight modification. Briefly, 20 l Protein glycation assay
of each sample solution dissolved in 95% ethanol was Protein glycation was assayed following Kim and Kim
mixed with 180 l of 167 M DPPH in methanol. The (2003) as modified from McPherson. Bovine serum
mixtures were incubated at 37C for 30 min, and then the albumin (20 mg/ml) was incubated with D-fructose (235
absorbance was measured at 540 nm using a DTX 880 mM) and D-glucose (235 mM) in potassium phosphate
multimode detector (Beckman Coulter, USA). A control buffer (200 mM; pH 7.4). Ethanol (98%) was used to
solution (containing only DPPH) underwent the same dissolve the sample extracts. All of the reagents and
process. All measurements were performed in triplicate. extracts were filtered through No. 4 filter paper, and each
The percentage of DPPH radical scavenging activity of of the mixtures was incubated at 60C for 6 d.
each plant extract was determined at five sample Fluorescence intensity was measured by a multimode
404 Pak. J. Pharm. Sci., Vol.23, No.4, October 2010, pp.403-408
Nasapon Povichit et al.
detector at an excitation of 370 nm and an emission of Free radical scavenging activity using DPPH assay
440 nm. Ethanolic solution of aminoguanidine was used The results showed that P. emblica extract exhibited the
as a known inhibitor (Gillery, 2006). highest activity in scavenging DPPH radicals, followed by
T. indica, C. sinensis, T. bellerica and A. lakoocha (fig.
All results are presented as mean standard deviation 3). These results were comparable to kaempferol; the best
(SD). Data correlations were obtained by Pearson standard for inhibiting this reaction was quercetin,
correlation, using the SPSS program version 12.0. A followed by Trolox (table 1). No significant correlation
value of P < 0.01 was considered statistically significant. was found between phenolic content and DPPH at P <
0.01 (data not shown).
RESULTS AND DISCUSSION
16
The results of testing the total phenolic content, free
14
radical scavenging, anti-lipid peroxidation, and
antiglycation activities of 15 medicinal plants, as well as 12
(m g /m l)
standard substances, are presented in table 1. 10
1.6
50
6
IC
1.4 y = 4.3308x + 0.0336 4
R 2 = 0.9994
1.2
absorbance(nm)
2
1 0
.
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0
0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 Fig. 3: Anti-free radical activity (IC50) of ethanol extracts
concentration of gallic acid(mg/ml) from medicinal plants determined by DPPH assay
Fig. 1: Linear correlation of absorbance (nm) versus total Free radical scavenging activity using ABTS assay
phenolic content. Results were expressed as Trolox equivalent antioxidant
capacity (TEAC). P. emblica demonstrated the highest
Total phenolic content free radical scavenging activity, followed by C. sinensis
The results revealed that C. sinensis contained the highest and T. indica, all of which were higher than gallic acid
phenolic content, followed by A. lakoocha and T. indica. (table 1 and fig. 4). They were also superior to
Their GAE values were 0.560.02, 0.480.01, and chlorogenic acid and quercetin, whereas the extracts from
0.440.04, respectively. The extract of G. atroviridis A. lakoocha and T. bellerica were comparable to Trolox.
presented the lowest phenolic content (0.040.03); A. Additionally, their total phenolic contents were
ascalonicum (0.050.01) and C. indica (0.070.01) also significantly correlated with antioxidant activity: r =
showed low phenolic content, as shown in table 1 and fig. 0.721 (P < 0.01), as shown in fig. 5.
2. Phenolic content might be considered to correlate with
their activities.
3.5
Total phenolic content as GAE
3
0.6
2.5
0.5
TE A C value
0.4 2
0.3 1.5
0.2 1
0.1 0.5
0 0
in s i a
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ia
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ar
hr
Fig. 2: Total phenolic contents of 15 medicinal plant Fig. 4: Anti-free radical activity (ABTS) of ethanol
extracts, determined using Folin-Ciocalteu reagent extracts from various plant parts of herbs.
1.50
1.00
phenolic content and glycation tests (data not shown).
0.50
0.00
Phenolic
Fig. 5: Correlation between total phenolic content and
anti-oxidant activity (ABTS) of 15 Medicinal plants.
14
12
10
possesses antioxidant activity. The constituents of C.
8 sinensis include large amounts of (-)-epigallocatechin, (-)-
6
4 epicatechin, (+)-gallocatechin, and (+)-catechin and their
2
0
derivatives, which have been shown to have positive
effects on human health (Flutter and Gao, 2004). Our
in s c a
T a
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ol
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nt rus ia
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ss n
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hr
Table 1: Total phenolic content and antioxidant, anti-lipid peroxidation, and antiglycation activities of 15 medicinal plants and 6
standard substances.
Total phenolic content
Calculated GAE DPPH TBARS Glycation
Medicinal plants Part of ABTS
(0.05 mg/ml) based on IC 50 IC 50 (mg/ml) a IC 50 (mg/ml) a
sample TEAC a
sample weight (mg/ml) a
(0.1 mg/ml)a
Allium ascalonicum Bulb 0.050.01 1.330.03 0.010.04 13.740.01 0.900.08
Allium sativum Bulb 0.160.03 5.300.04 0.060.03 9.750.04 43.920.06
Angiopteris evecta Rhizome 0.100.03 0.420.01 0.360.04 7.300.04 0.280.07
Artocarpus lakoocha Heartwood 0.480.01 0.160.01 1.720.04 1.220.02 3.200.06
Camellia sinensis Leaf 0.560.02 0.100.02 2.830.05 1.110.01 0.040.05
Chromolaena odorata Leaf 0.090.02 1.330.03 0.290.03 10.610.03 0.600.08
Citrus indica Fruit 0.070.01 5.200.06 0.130.02 7.600.02 17.640.10
Curcuma comosa Rhizome 0.150.02 0.500.03 0.090.02 2.020.03 12.300.08
Garcinia atroviridis Fruit 0.040.03 15.200.02 0.010.03 18.120.03 0.320.07
Garcinia mangostana Fruit Peel 0.310.02 0.380.01 0.520.05 4.300.04 31.330.07
Jatropha gossypifolia Rhizome 0.300.01 1.450.04 0.420.03 0.420.03 8.400.06
Morus alba Leaf 0.110.03 0.850.03 0.100.03 2.600.02 0.620.07
Phyllanthus emblica Fruit 0.290.05 0.060.02 2.940.03 1.370.03 0.020.05
Tamarindus indica Seed Coat 0.440.04 0.090.02 2.790.06 0.640.04 0.010.06
Terminalia bellerica Fruit 0.170.02 0.120.06 1.200.04 0.870.03 0.020.06
Gallic acid - - - 2.180.02 - -
Chlorogenic acid - - - 1.050.03 - -
Trolox - - 0.090.01 1.000.00 4.010.02 -
Quercetin - - 0.060.02 0.280.04 0.380.03 -
Kaempherol - - 0.130.02 - 1.210.02 -
Aminoguanidine - - - - - 0.050.02
a
MeanSD
were determined to have low phenolic content. Their products. However, this in vitro evaluation should be
antiglycation effect may be due to other constituents corroborated with an in vivo study. Since the active
contained in the extracts; this requires further compounds are phenolic with low molecular weight and
investigation. are lipophilic, they are expected to exhibit good
penetration of the stratum corneum barrier of the skin. An
All of the results indicate that the extracts from C. in vivo activity test could confirm skin penetration. The
sinensis, P. emblica, T. indica, A. lakoocha and T. findings of this study warrant further development of anti-
bellerica may prove to be a promising source of anti- aging cosmetic products based on some of these plant
aging ingredients for skin-care products. extracts.
CONCLUSION ACKNOWLEDGEMENTS
The results from DPPH, ABTS and TBARS assays of The authors thank the Department of Pharmaceutical
some plant extracts indicated high antioxidative activities, Sciences, Faculty of Pharmacy, Chiang Mai University,
especially the ethanolic extracts of T. indica, T. bellerica, Thailand, for financial support.
P. emblica, A. lakoocha and C. sinensis. Total phenolic
contents presented as GAE were also the highest among REFERENCES
these plant extracts, which confirmed that phenolic
compounds play an important role in exhibiting Blois MS (1958). Antioxidant determinations by the use
antioxidative activity. In addition to their high of a stable free radical. Nature, 181: 1198-1200.
antioxidative activities, which were comparable to those Dyer DG, Dunn JA, Thorpe SR, Bailie KE, Lyons TJ,
of quercetin and kaempferol and greater than that of McCance DR and Baynes JW (1993). Accumulation of
Trolox, these plant extracts also exhibited protein Maillard reaction products in skin collagen in diabetes
glycation inhibitory activity comparable to that of and aging. J. Clin. Invest., 91: 2463-2469.
aminoguanidine. The results also indicated that extracts Flutter B and Gao B (2004). MHC class I antigen
from C. sinensis, T. indica and A. lakoocha have the presentation recently trimmed and well presented.
potential for providing anti-aging properties in skin care Cell Mol. Immunol., 1(1): 22-30.
Gillery P (2006). Oxidative stress and protein glycation in and Unnikrishnan MK (2007). Antioxidant and
diabetes mellitus. Ann. Biol. Clin. (Paris), 64(4): 309- radioprotective effect of the active fraction of Pilea
314. microphylla (L.) ethanolic extract. Chem. Biol.
Khknen MP, Hopia AI, Vuorela HJ, Rauha J-P, Pihlaja Interact., 165(1): 22-32.
K, Kujala TS and Heinonen M (1999). Antioxidant Rabe JH, Mamelak AJ, McElgunn PJ, Morison WL and
activity of plant extracts containing phenolic Sauder DN (2006). Photoaging: mechanisms and
compounds. J. Agric. Food Chem., 47(10): 3954-3962. repair. J. Am. Acad. Dermatol., 55(1): 1-19.
Kim HY and Kim K (2003). Protein glycation inhibitory Rahbar S and Figarola JL (2002). Inhibitors and breakers
and antioxidative activities of some plant extracts in of advanced glycation endproducts (AGEs): A review.
vitro. J. Agric. Food Chem., 51(6): 1586-1591. Curr. Med. Chem. Immunol., Endocr. Metab. Agents,
Komutarin T, Azadi S, Butterworth L, Keil D, 2: 135-161.
Chitsomboon B, Suttajit M and Meade BJ (2004). Rusak G, Krajai M and Plee N (1997). Inhibition of
Extract of the seed coat of Tamarindus indica inhibits tomato bushy stunt virus infection using a
nitric oxide production by murine macrophages in vitro quercetagetin flavonoid isolated from Centaurea
and in vivo. Food Chem. Toxicol., 42(4): 649-658. rupestris L. Antiviral Res., 36: 125-129.
Maisuthisakul P, Suttajit M and Pongsawatmanit R Vioux-Chagnoleau C, Lejeune F, Sok J, Pierrard C,
(2007). Assessment of phenolic content and free Marionnet C and Bernerd F (2006). Reconstructed
radical-scavenging capacity of some Thai indigenous human skin: from photodamage to sunscreen
plants. Food Chem., 100: 1409-1418. photoprotection and anti-aging molecules. J. Dermatol.
Prabhakar KR, Veerapur VP, Bansal P, Parihar VK, Sci. (Supplement), 2(1): S1-S12.
Reddy Kandadi M, Bhagath Kumar P, Priyadarsini KI