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To cite this article: Z. Mohd Zin , A. Abdul Hamid , A. Osman , N. Saari & A. Misran (2007)
Isolation and Identification of Antioxidative Compound from Fruit of Mengkudu (Morinda citrifolia L.),
International Journal of Food Properties, 10:2, 363-373, DOI: 10.1080/10942910601052723
Z. Mohd Zin
Department of Food Science, Faculty of Agrotechnology and Food Science, Kolej
Universiti Sains dan Teknologi Malaysia (KUSTEM), Terengganu, Malaysia
This study was conducted to isolate and identify the antioxidative compound from fruit
extracts of Mengkudu (Morinda citrifolia L.). The extract was fractionated on a Sephadex
LH-20 column chromatography using ethanol as eluate. Six major fractions were isolated
according to UV absorption intensity of phenolic contents at 725 nm. Antioxidative activity
of these fractions was evaluated in a ferric thiocyanate method (FTC) and thiobarbituric
acid test (TBA). The antioxidative activities were then compared to that of a -tocopherol
(natural antioxidant) and butylated hydroxytoulene or BHT (synthetic antioxidant). Results
showed that all isolated fractions demonstrated high antioxidative activity compared to
either BHT or a -tocopherol. Further separation of these fractions on a high-performance
liquid chromatography (HPLC) procedure based on water-methanol gradient with trifluo-
reacetic acid (TFA) for simultaneous analysis of flavonoids, indicated that they contain
several active compounds. The major flavonoids that have been detected in M. citrifolia are
catechin and epicatechin.
INTRODUCTION
Traditional cultures have been using the fruit, bark, leaves and roots of noni
(Morinda citrifolia L.) for a very long time. It is said that Polynesian Islanders first culti-
vated and domesticated the noni tree over 2000 years ago for food and medical purposes.
The plant has been reported to have broad effects including antimicrobial,[1] anti-cancer
activity,[2] antioxidant properties,[3] anti-inflammatory activity,[4] analgesic activity,[5] and
cardiovascular activity.[6]
About 160 phytochemical compounds have been identified in the noni plant, and the
major micronutrients are phenolic compounds, organic acids, and alkaloids.[7] The phe-
363
364 ZIN ET AL.
nolic compounds were reported to be the major groups of functional micronutrient in noni.
They include damnacanthal, scopoletin, morindone, alizarin, aucubin, nordamnacanthal,
rubiadin, rubiadin-1-methyl ether, and other anthraquinone glycosides.[8–10]
The presence of phenolic compounds in noni plant may have significant effect on
their total antioxidant activity. Recent report by Su et al.[10] revealed that neolignan and
americanin A[3] from n-BuOH-soluble partition part of MeOH extract of noni fruits was
found to have significant antioxidant activity in two antioxidant bioassays. Similarly,
Mohd et al.,[3] reported that antioxidant properties of noni fruit extract exhibited strong
inhibition of lipid oxidation. In other report by Wang and Su,[7] found that the superoxide
anion radicals (SAR) scavenging activity of noni juice was shown to be 2.8 times higher
than that of vitamin C, 1.4 times of that pycnogenol (PYC), and almost of the same order
as that of grape seed powder.
The Morinda citrifolia plant especially its fruit has been the object of many claims
concerning its nutritional and functional properties. These beneficial effects may result
from some compounds, in particular, scopoletin, nitric oxide, alkaloids, flavonoids, and
sterols that attributed to the antioxidant potential of noni.[11] As a result, consumption of
this fruit is currently high. In Malaysia, the commercial interest in noni’s fruit is tremen-
dous and some local companies has started commercializing it as healthy juice as well as
dietary supplements.
In this article, we describe the isolation and identification of antioxidative com-
pounds from fruit of Mengkudu (Morinda citrifolia L.), as these may provide considerable
contribution to ascertain the individual potency of the compounds, which have the poten-
tial of being incorporated into foods as functional ingredients.
Milford, MA, USA). Pure flavonoids standards (flavanols i.e., (+)-catechin, (−)- epicat-
echin; flavonols i.e., quercetin, myricetin, kaempferol and rutin; flavones i.e., apigenin
and luteolin; flavanones i.e., naringin), Na DEDTC and trifluoroacetic acids were
obtained from Sigma Chemical Co. (St. Louis, MO, USA), methanol (HPLC grade) were
purchased from Merck (Germany), hydrochloric acid was purchased from Malinckrodt
Baker, Inc. (Kentucky, USA). Determination of antioxidative activity of the fractions was
determined using FTC method adapted from Osawa and Namiki.[13] The Thiobarbituric
acid test (TBA) was conducted according to the method of Ottolenghi[15] and Kikuzaki
and Nakatani.[16]
Statistical Analysis
All experiments were conducted in triplicates and statistical analysis was done
according to the SAS[17] User’s Guide. Analysis of Variance was performed by ANOVA
procedure. Duncan’s multiple range tests were used to determine significant differences
between the means.
0.25
0.2 III
Absorbance of TPC at 725 nm
0.15
II
0.1
I
IV
0.05
V VI
0
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49
Fraction Number (5 ml/tube)
Figure 1 Eluates following Sephadex LH-20 column chromatography of fruit fraction of M. citrifolia.
Figures 2 and 3 show the antioxidative activities of the six isolated fractions from
fruit extract of M. citrifolia as measured by FTC and TBA method, respectively. The
activity in decreasing order was BHT, I, α-tocopherol, V, IV, VI, III, and II. Fraction I dis-
played the strongest antioxidative activity, with activity that was not significantly (p <
0.05) different to that of BHT or α-tocopherol. This was true for both assay tested. Wang
et al.,[24] and Hertog et al.,[25] reported that the antioxidant property of some vegetables
ANTIOXIDANT COMPOUND FROM MENGKUDU 367
2.5
I
II
2 III
a
IV
V
ab
VI
BHT bc
Tocopherol
Absorbance at 500 nm
1.5
Control c
d
1
0.5
f
fg
0
0 1 2 3 4 5 6 7
Incubation time (day)
Figure 2 Antioxidative activity of Sephadex LH 20 column chromatographic fractions obtained from fruit
extracts of M. citrifolia as measured by FTC method. Absorbance values represent triplicates of different sam-
ples analysed. Values with same letter (abc) are not significantly different (p < 0.05), between samples.
and fruits were partly due to low molecular weight phenolic compounds, particularly the
flavonoids, which were known to be potent antioxidant. Two known glycosides (rutin and
asperulosidic acid) and a novel trisaccharide fatty acid ester [2,6-di-O-(β-D-glucopyrano-
syl)-1-O-octanoyl-β-D-glucopyranose] were isolated from the n-butanol-soluble fraction
obtained from the ethanol extracts of the fruit of M. citrifolia.[24]
Interestingly, Fraction I, exhibited the highest antioxidative activity, although it con-
tained only 2.21 mg/100 g of phenolic compounds (Table 1) which was comparatively lower
than that found in either fraction V or VI. This is probably due to the more potent antioxidant
present in fraction I compared to that of fraction V or VI. The result also showed a lack of
correlation between antioxidative activity and their content of phenolic compounds. Accord-
ing to Shahidi and Naczk,[14] Folin-Ciocalteu method measured other constituents than phe-
nolic, and its specificity is poor. The Folin-Ciocalteu reagent detected all phenolic groups
found in the extract, including those found in the extractable proteins. These results suggested
that factors other than total phenolic compounds may be playing a role in the antioxidant
368 ZIN ET AL.
Control a
a-Tocopherol cde
BHT e
I de
II ab
III ab
IV bcd
V abcd
VI abc
Figure 3 Antioxidative activity of Sephadex LH 20 column chromatographic fractions obtained from fruit
extracts of M. citrifolia as measured by TBA method. Absorbance values represent triplicates of different sam-
ples analysed. Values with same letter (abc) are not significantly different (p < 0.05), between samples.
activity in the fruit fraction of M. citrifolia, and that their molecular structures play an impor-
tant role in their antioxidant activity by developing antagonistic or synergistic effects in
combination with themselves or with other constituents of the extracts.[26–29] M. citrifolia
fruits were also a good source of potassium and vitamin C. The high levels of potassium
indicated the usefulness of the fruit as a survival food.[26–29]
The presence of terpenoids in the fruits has also been reported suggesting that it may
account for the biological activity of the fruit.[26–29] The chemical composition of the fruit
varied depending upon its stage of development. The green fruits contained high amounts
of the medium sized fatty acids, palmitic and octadecanoic acids, while the ripe ones con-
tained larger quantities of the short chain fatty acids, octanoic and hexanoic acids, and the
rotten fruits contained greater levels of esterified octanoic and hexanoic acids, possibly
from ethanolic fermentation.[30–31]
ANTIOXIDANT COMPOUND FROM MENGKUDU 369
Gardner et al.,[32] suggested that phenolic compounds are the major contributor to
the antioxidative activity of apple, pineapple, and vegetable juice. According to Rice-
Evans et al.,[33] phenolic compounds are found to be an effective hydrogen donor, which
makes them good antioxidants. Even though the phenolic compound was reported to be
highly responsible for the antioxidative activity in herbs, possible synergism of phenolic
compounds with one another or with other components present in the extract may be
responsible for observation in this study. On the basis of these results, all fractions were
used for the identification of the antioxidants compounds.
The chromatograms of six fractions of fruit of M. citrifolia measured at 370 nm are
given in Fig. 4. The flavonoids content of fraction I, II, III, IV, V, and VI of fruit of M. cit-
rifolia is shown in Fig. 5. It is interesting to note that total catechin was the major fla-
vonoid found in all the fractions collected. According to Balentine et al.,[34] catechins were
structurally, primarily, flavonols, and form 20–30% of dry weight of green tea. Major cat-
echins found in fresh tea and green tea are (−) gallate (EGCG), (−) epigallocatechin
(EGC), (−) epicatechin gallate (ECG), and (−) epicatechin (EC). In this study, results show
that Fraction VI was found to contain the highest amount of total catechin (1111.2 mg/100
g), which was significantly (p < 0.05) higher than any other fractions. This result corre-
lated well with the highest content of total phenolic found in Fraction VI of fruit extract of
M. citrifolia (Table 1).
It is intriguing that Fraction VI exhibited lower antioxidative activities compared to
other fractions tested, even though it contained the highest amount of catechin. Interest-
ingly, Fraction I, exhibited the highest antioxidative activity, although it contained lower
amount of catechin, as compared to that found in fraction VI. This is probably due to the
more potent antioxidant present in fraction I compared to that of fraction VI. On the other
hand, other flavonoids (naringin, rutin, and quercetin) are found only in trace amounts.
Balentine et al.[34] also reported that all green tea catechin were not equally active. Galloyl
esters of catechins were more active than non-galloylated catechins because they had
lower redox potentials. Some catechins, for example epicatechin, were found to be less
effective against particular cancer cell lines, although they did show synergistic effects
with other catechins and caffeine.[35]
In vitro and in vivo experiments have shown that catechins were potentially benefi-
cial to human health: they were potent antioxidants, anticarcinogens, antiinflammatory
agents, and inhibitors of platelet aggregation.[35] Scott et al.,[36] stated that (+)-catechin of
green tea leaves contributed to its antioxidative activity. According to Meyer et al.,[37] cat-
echins showed high activity in inhibiting copper-catalyzed oxidation of human LDL
in vitro. Thus, catechins may be responsible for the therapeutic activities, such as antibac-
terial, anti-aging, antiarthritic, antituberculotic and antitumor in M. citrifolia.
CONCLUSIONS
It is encouraging to see that all the fractions studied demonstrated high antioxidative
activity compared to either BHT or α-tocopherol. The potency of some of these com-
pounds could provide scientific basis for the health benefits claims regarding M. citrifolia
fruits in folk medicine and warrant further studies to assess their potential as effective nat-
ural remedies. The major flavonoids that have been detected in M. citrifolia fruits were
catechin and epicatechin and were expressed as total catechin. Total catechins occurred at
different concentrations in different fractions of the plant’s fruit. Thus, fruit of M. citrifo-
lia has great potential as an excellent source of catechin and should be studied more
370 ZIN ET AL.
Fraction I
Fraction II
Fraction III
Fraction IV
Fraction V
Fraction VI
Figure 4 Chromatogram of fraction I, II, III, IV, V, VI obtained from fruit extracts of M. citrifola monitored at
370 nm. The chromatographic conditions are described in text.
thoroughly to distinguish the exact catechins that were present. The information can be
used as a guide to formulate a product from these species and also to serve as a reference
point for future research. The present study also suggested that consumption of fruit of M.
citrifolia might have potential health effects. In conclusion, these results further support
the use of this fruit by the indigenous population as a traditional medicine. However, fur-
ther investigation on toxicity needs to be carried out in future before it can be recom-
mended as a natural source of antioxidants.
ANTIOXIDANT COMPOUND FROM MENGKUDU 371
1200 A
Total Catechin
Naringin
1000
Rutin
Quercetin
800
Flavanoid Content (mg/100g)
600
B
C
400
E D
F
200
0
Frac. I Frac. II Frac. III Frac. IV Frac. V Frac. VI
Figure 5 Flavonoid content of Sephadex LH 20 column chromatographic fractions obtained from fruit extracts
of M. citrifolia. Values with same letter (ABC) are not significantly different at (p < 0.05).
ACKNOWLEDGMENT
The authors would like to thank The Ministry of Science, Technology and Innovation of Malaysia
for financing the project and the laboratory facilities for Universiti Putra Malaysia.
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