R. Suri, A. Crozier, H. Zaharinah and A.R. ZuraidaJ. Trop. Agric. and Fd. Sc. 44(1)(2016): 81 – 94
On-line high-performance liquid chromatography analysis of the antioxidant activity of phenolic compounds in selected tropical citrus
R. Suri1, A. Crozier2, H. Zaharinah1 and A.R. Zuraida1
1Food Science Technology Research Centre, MARDI Headquarters, Persiaran MARDI-UPM,43400 Serdang, Selangor, Malaysia2Plant Products and Human Nutrition Group, Division of Environmental and Evolutionary Biology, Graham Kerr Building, Institute of Biomedical and Life Science, University of Glasgow, G12 8QQ, Scotland, UK
AbstractCitrus hystrix, Citrus microcarpa and Citrus suhuiensis fruits were analysed by high-performance liquid chromatography (HPLC) with photo-diode array and MS2 detector (HPLC-PDA-MS2) to identify flavone, flavanone and dihydrochalcone. The antioxidant potential on each tropical citrus flavonoid was performed using HPLC with post-column on-line antioxidant detection based on 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical scavenging assay. Phloretin-3′,5′-di-C-glucopyranoside present in C. microcarpa fruit peel and flesh possess high Trolox Equivalent Antioxidant Ratio (TEAR) values by 3.4 and 3.1 respectively. Quercetin-3-O-rutinoside, hesperetin-7-O-neohesperidoside and hesperetin-7-O-rutinoside also showed high TEAR values in C. hystrix and C. suhuiensis fruit, but have lower than phloretin-3′,5′-di-C-glucopyranoside. Levels of TEAR values of flavonoids in C. microcarpa, C. suhuiensis and C. hystrix fruits were as follows; phloretin-3′,5′-di-C-glucopyranoside > quercetin-3-O-rutinoside > hesperetin-7-O-neohesperidoside > hesperetin-7-O-rutinoside = luteolin-7-O-rutinoside = naringenin-7-O-rutinoside. Citrus microcarpa has been widely used in Asian food and beverages or as a preserved snack, is consider to be a good source of phloretin-33′,5′-di-C-glucopyranoside. This compound is structurally almost similar to aspalathin and nothofagin, the potent antioxidant compounds in rooibos tea.
Keywords: C. microcarpa, C. suhuiensis, C. hystrix, HPLC with on-line antioxidant assay, phloretin- 3′,5′-di-C-glucopyranoside
'
IntroductionThe antioxidant activity of citrus flavonoids depends upon their chemical structures, which involve the presence and configuration of hydroxyl groups, a 2 – 3 double bond and a 4-oxo function and the degree of polymerisation. For example, the antioxidant activity of flavones and
flavanones increases according to the total number of hydroxyl groups and the 3′4′-catechol structure (Heim et al. 2002). Methoxylation, glycosylation including types of carbohydrate moieties could also affect the antioxidant activity of citrus flavanones and flavones (Kim and Lee 2004). Generally flavonoid aglycone has more potent
antioxidant than flavonoid with glycoside moiety (Heim et al. 2002). Citrus microcarpa or musk lime is a small “orange” type fruit with a loose skin and has a sweet musky smell. Unlike other tropical citrus, C. microcarpa has no thorns. The fruit is 2 – 4 cm in diameter and yellow when ripe. The pulp is juicy, sour and has 6 – 8 segments. On average, the seed content is 3 – 4% per fruit weight and the juice recovery is between 35 – 38% per fruit weight (Burkill 1966). Studies showed 6,7-Dimethoxycoumarin (Tatum and Berry 1977), lutein, cryptoxanthin and b-carotene (Tee and Lim 1991) were identified in C. microcarpa fruit. Various flavonoids such as isosakuranetin-7-O-rutinoside, eriodictyol-7-O-rutinoside, hesperetin-7-O-rutinoside, naringenin-7-O-rutinoside, eriodictyol-7-O-neohesperidoside, naringenin-7-O-neohesperidoside, hesperetin-7-O-neohesperidoside and naringenin-7-O-neohesperidoside-6’’-malonate were also reported in C. microcarpa fruit (Kanes et al. 1993). Citrus suhuiensis (limau langkat or madu) is grown mainly in Terengganu, Malaysia. The fruit, one of the finest mandarin oranges in the country, is oblate, globose and smooth and produces juice with a weak aroma. The fruit typically contains 12 – 15 seeds/fruit. The Cleopatra root stock is used and more than 2000 hectares are planted with C. suhuiensis in Malaysia. Citrus hystrix fruit and leaf have been widely used as food flavouring or as a drink. According to Murakami et al. (1999), bergamottin, oxypeucedanin and 5-(6’,7’-dihydroxy-3’,7’-dimethyl-2-octenyl)oxypsolaren were among the coumarins present in the fruit. Bergamottin may inhibit interferon-g-(IFN-g-) induced nitric oxide generation in murine macrophage cell line (RAW 264.7). The antioxidant activity of individual polyphenolic compounds can be accessed using HPLC with an on-line antioxidant detection system. This technique requires ABTS radical with radical scavenging
compound accessed by comparing to the synthetic vitamin E derivative (6-hydroxy-2,5,7,8-tetramethylchloroman-2-carboxyl acid) known as Trolox (Stewart et al. 2005). ABTS•- is generated by persulfate oxidation of ABTS2-. In this study, TEAR, which is based on decolouration of radical cation ABTS•- and forms ABTS2-, was used. TEAR is defined as ratio concentration (mM) of Trolox in equivalent to a 1 mm solution of the compound under investigation and it reflects the ability of the hydrogen-donating antioxidant compounds to scavenge the ABTS•- radical. At present, there are no available data on the antioxidant capacity of single phenolic compounds in tropical citrus species such as ‘limau purut’(C. hystrix), ‘limau kasturi, (C. microcarpa) and ‘limau madu’ (C. suhuiensis). Therefore, the present study was to identify and quantify flavonoids in tropical citrus that contributes to the total antioxidant activity.
Materials and methodsThe three species of tropical citrus fruits (C. hystrix, C. microcarpa and C. suhuiensis) were bought from Kampung Baru Market, Kuala Lumpur, Malaysia. Whenever possible, citrus from the same farmer were chosen. Leaf, peel and flesh were dried using an oven at 40 °C and stored at –20 °C prior to analysis. Diosmetin, apigenin, phloretin, hesperetin-7-O-rutinoside, naringenin-7-O-rutinoside, diosmetin-7-O-rutinoside and quercetin-3-O-rutinoside were obtained from AASC Ltd. (Southampton, U.K.). Apigenin-8-C-glucoside-2’-rhamnoside (vitexin-2″-O-rhamnoside), quercetin and diosmetin-7-O-neohesperidoside were purchased from Extrasynthase (Genay, France). Luteolin was obtained from Apin Chemical Ltd. (Abingdon, Oxon, U.K.). Hesperetin-7-O-neohesperidoside, isosakuranetin-7-O-rutinoside, eriodictyol-7-O-rutinoside, eriodictyol-7-O-neohesperidoside, formic acid, Trolox and ABTS were supplied from
83
R. Suri, A. Crozier, H. Zaharinah and A.R. Zuraida
Sigma-Aldrich (Poole, Dorset, U.K.). HPLC solvents were obtained from Rathburn Chemicals (Walkerburn, Scotland, U.K.). Methanol was supplied from Rathburn Chemicals (Walkerburn, Scotland,U.K.). All other chemicals and reagents were obtained from Sigma-Aldrich (Poole, Dorset, U.K.) unless otherwise stated. For the extraction of phenolics in citrus, 5 g of three replicates of dried citrus were extracted according to the method developed by Roowi and Crozier (2011) by using 10 ml acidified methanol (0.1% HCL) and centrifuged at 4,000 g for 20 min at 4 °C. The pellet was extracted twice more with the same solvent and the combined methanolic extract reduced to dryness in vacuo using a rotary evaporator and redissolved in 10 ml of acidified methanol. All samples were subdivided into 2 ml aliquots and stored at –20 °C before analysis.
HPLC-diode array and MS-MS analysisMethanolic extracts of citrus were analysed in triplicate on a Surveyor HPLC comprising HPLC pump, an AS 3000 auto sampler (cooled at 6 °C) and a UV6000 photo diode array detector (PDA) scanning from 250 – 700 nm (Thermo Finnigan, San Jose, USA). Separation of flavonoids was carried out using a Synergi RP-Max (Torrance, CA, USA) 4 μm, 250 x 4.6 mm i.d. C12 reverse-phase column at 40 °C, eluted at 1 ml/min with a gradient of acetonitrile in water containing 0.1% formic acid. Flavanones were detected at 280 and 365 nm, while dihydrochalcone was monitored at 280 nm. After the extract passed through the flow cell of the PDA monitor, column eluate was split and 20% was directed to a Finnigan LCQ Decca mass spectrometer with an electrospray interface (ESI), operating in full scan data dependent MS mode from 100 – 2000 amu. Samples were analysed using the negative ion mode. ESI-MS parameters were as follows: potential of ESI source, 4 kV;
capillary temperature 400 °C. Eriodictyol-7-O-rutinoside, naringenin-7-O-rutinoside, isosakuranetin-7-O-rutinoside, diosmetin-7-O-rutinoside, hesperetin-7-O-rutinoside and hesperetin-7-O-neohesperidoside were quantified by reference to standard calibration curves obtained with the PDA at lmax values. Quantifications of phloretin-3′,5′-di-C-glucopyranoside, apigenin-6,8-di-C-glucopyranoside, diosmetin-6,8-di-C-glucopyranoside, quercetin-3-O-rutinoside-7-O-glucoside, luteolin-7-O-rutinoside and diosmetin-6-C-glucoside were based on their aglycones.
Determination of antioxidant activity using the ABTS•- decolouration assayThe antioxidant activity of selected tropical citrus was determined using the ABTS•- assay based on the methods developed by Dapkevicius et al. (2001), Koleva et al. (2001) and Stewart et al. (2005). A 2 mM ABTS•- stock solution containing 3.5 mm potassium persulphate was prepared and incubated in darkness at room temperature. This allows for stabilisation of the radical. ABTS reagent was prepared by diluting the stock 8-fold in 0.1 M potassium phosphate buffer at pH 8.0. Aliquots (5 μl) of citrus extracts were injected into a Surveyor HPLC System (Thermofinigan). HPLC eluate from the PDA was mixed with the ABTS reagent at a “T” piece at a flow rate of 0.5 ml/min delivered by a Shimadzu LC-10 AD VP Liquid Chromatography pump. A Shimadzu GT-154 Vacuum degasser was used to remove any oxygen in the reagent. After mixing by passing through a 1.5 M x 0.4 mm loop maintained at 40 °C, the absorbance was measured at 720 nm (Nemphlar Bioscience, Lanark, UK). Data were analysed using Thermo Finnigan ChromquestTM chromatography software Version 4.0. Antioxidant potential was quantified by reference to a Trolox standard calibration curve. Results were expressed as TEAR values. All samples were analysed in triplicate.
84
Antioxidant activities of tropical citrus
Results and discussionThe present study focuses on the antioxidant potential of tropical citrus phenolics and further discusses the involvement of the number and position of hydroxyl groups, O-glycosylation and O-methylation, the carbonyl group and double bond carbons. Tropical citrus were analysed using HPLC with PDA and MS2 detection allowing the identification of flavanones, flavanol and dihydrochalcone (Tables 1 – 2 and Figure 1 – 4). Chalcones are the biochemical precursor of all flavonoids produced from 4-coumaroyl CoA and three molecules of malonyl CoA by chalcone synthase activity (Dixon and Steele 1999). Phloretin-3′,5′-di-C-glucopyranoside, which is a dihydroxychalcone, was the most potent antioxidant compound in C. microcarpa peel and flesh by 3.4 and 3.1 respectively. This hydroxychalcone was responsible for 76% and 81% respectively to the total antioxidant activity of the extract in C. microcarpa peel and flesh (Table 3 and Figure 2). High TEAR value (3.1) was also found for C. hystrix flesh (Table 4 and Figure 3). High antioxidant activity of phloretin has been previously reported (Rezk et al. 2002; Nakamura et al. 2003; Kim and Lee 2004). Phloretin had a potent antioxidant activity to scavenge the peroxynitrile radical, hydroxyl radical and 1,1-diphenyl-2-picrylhydrazyl radical and was also able to scavenge lipid peroxidation (Rezk et al. 2002). The TEAR value of phloretin-3′,5′-di-C-glucopyranoside is higher than that of flavanones is most probably due to the hydroxyl group on the B ring that makes the 2,6-dihydroxyacetophenone or 2,4,6-trihydroxyacetophenone structure (Figure 4) comparable to phloroglucinol (Rezk et al. 2002; Kim and Lee 2004). The antioxidant activity of 2,4,6-trihydroxyacetophenone not only depends exclusively on the three hydroxyl groups of ring A, but on the keto-enol transformation of the carbonyl groups that stabilises the radical after hydrogen
abstraction. Among the three B ring hydroxyl groups, the 2′ position is essential for radical scavenging activity (Rezk et al. 2002; Nakamura et al. 2003). As there is also no sugar moiety substitute ion at any of the hydroxyl groups adjacent to the carbonyl group, as in phloretin-2-b-D-glucoside (phloridzin), this may well explain the high TEAR value of phloretin-3′,5′-di-C-glucopyranoside. For example, occupation of two-hydroxyl by glucose decreased the antioxidant activity of phloridzin in comparison to phloretin (Rezk et al. 2002; Hijova 2006). Phloretin-3′,5′-di-C-glucopyranoside is structurally almost similar to dihydrochalcone-2′,2,3,4′,6′-pentahydroxy-3-C-glucopyranoside (aspalathin) and dihydrochalcone-2′,2,4′,6′-tetrahydroxy-3-C-glucopyranoside (nothofagin), potent antioxidant compounds in unfermented Rooibos tea Aspalathus linearis (Figure 4) (Von Gadow et al. 1997; Joubert et al. 2004; Joubert et al. 2005). Nothofagin and aspalathin have one sugar unit attached at C3′ of the B ring, whereas phloretin-3′,5′-di-C-glucopyranoside has 2 sugar units attached at the C3′ and C5′. Quercetin-3-O-rutinoside was the only flavonol found in tropical citrus occurring in C. hystrix leaf. This compound showed a higher TEAR value than most flavanones in tropical citrus, but lower than that of phloretin-3′,5′-di-C-glucopyranoside. Kim and Lee (2004) also reported that quercetin-3-O-rutinoside has a lower antioxidant activity than phloretin. The antioxidant activity of quercetin-3-O-rutinoside is most probably due to the presence of a C4 hydroxyl group and a n2,3 double bond on the C ring (Amic et al. 2003). However, attachment of rutinose at the 3-hydroxyl adjacent to the 4-carbonyl group of quercetin leads to co-planarity loss of the B-ring that decreases the delocalisation potential. This will reduce the antioxidant activity of quercetin-3-O-rutinoside compared to quercetin (van Acker et al. 1996).
85
R. Suri, A. Crozier, H. Zaharinah and A.R. Zuraida
Tabl
e 1.
Sum
mar
y of
phe
nolic
com
poun
ds in
Citr
us s
uhui
ensi
s an
d C
itrus
mic
roca
rpa
frui
t with
HPL
C w
ith d
iode
arr
ay d
etec
tion
and
MS2
dete
ctio
n
Peak
t R (m
in)
(pee
l)at R
(min
)(fl
esh)
bC
ompo
unds
l max
(nm
)[M
-H]-
(m/z
)M
S2 fr
agm
ents
ions
(m/z
)
C. s
uhui
ensis
117
.417
.4A
pige
nin-
6,8-
di-C
-gl
ucop
yran
osid
e33
059
347
3 ([
0,2 X
1]- ;
[M-H
]- -120
), 50
3 ([
0,3 X
1]- ;
[M-H
]- -90)
, 533
([1,
3 X1]
- ; [M
-H]- -
60) a
nd 5
75
([M
-H]- -
H2O
)2
18.4
n.d.
Dio
smet
in-6
,8-d
i-C-
gluc
opyr
anos
ide
335
623
503
([0,
2 X1]
)- ; [M
-H]- -
120)
, 383
([0,
2 X0
0,2 X
1]- [
M-H
]- -12
0-12
0), 5
33 ([
0,3 X
1]-
[M-H
]- -90
), 60
5 ([
M-H
]- -H
2O).
319
.0n.
d.D
iosm
etin
-6,8
-di-C
-gl
ucop
yran
osid
e (is
omer
)33
062
350
3 ([
0,2 X
1])- ;
[M-H
]- -12
0), 3
83 ([
0,2 X
0 0,
2 X1]
- [M
-H]- -
120-
120)
, 533
([0,
3 X1]
-
[M-H
]- -90
), 60
5 ([
M-H
]- -H
2O).
4n.
d.26
.1N
arin
geni
n-7-
O-r
utin
osid
e28
057
927
1[N
arin
] ([M
-H]- -
Rut
)5
28.2
28.3
Hes
pere
tin-7
-O-r
utin
osid
e28
560
930
1 [H
esp]
([M
-H]- -
Rut
)6
35.5
35.5
Isos
akur
anet
in-7
-O-r
utin
osid
e28
059
328
5 ([
M-H
]- - R
ut)
C. m
icro
carp
a
1n.
d25
.4A
pige
nin-
6,8-
di-C
-gl
ucop
yran
osid
e33
559
347
3([0,
2 X0]
- ; [M
-H]- -
120
), 50
3 ([
0,3 X
1]- ;
[M-H
]- - 9
0)53
3 ([
1,3 X
1]- ;
[M-H
]- - 6
0) a
nd 5
75 ([
M-H
]- -H
2O)
230
.544
.5A
pige
nin-
8-C
-glu
cosi
de-2
’-rh
amno
side
340
577
457
[0,2 X
1]- ,
413
[Z1]
-
332
.545
.4U
nkno
wn
apig
enin
con
juga
te34
057
745
7 [0,
2 X1]
- , 41
3 [Z
1]-
435
.351
.2Ph
lore
tin-3′,5′-d
i-C-
gluc
opyr
anos
ide
285
597
477
([0,
2 X1]
- ; [M
-H]- -
120
), 38
7 ([
0,2 X
1 0,
3 X0]
- ; [M
-H]- -
120
; [M
-H]- -
120
), 35
7 ([
0,2 X
1 0,
2 X0]
- ; [M
-H]- -
120
; [M
-H]- -
120)
417
([0,
3 X1
0,3 X
0]- ;
[M-H
]- -90
; [M
-H]- -
90)
538
.556
.0D
iosm
etin
-7-O
-neo
hesp
erid
osid
e34
560
744
3 [0,
2 X1]
- , 32
3 [Y
0]-
648
.8n.
d.H
espe
retin
-7-O
-rut
inos
ide
285
609
301
[Hes
p] ([
M-H
]- -R
ut),
343,
325
753
.169
.2H
espe
retin
-7-O
-ne
ohes
perid
osid
e28
560
930
1 [H
esp]
([M
-H]- -
Neo
hesp
)
*Pea
k nu
mbe
rs a
nd H
PLC
rete
ntio
n tim
es re
fer t
o H
PLC
trac
e in
Fig
ure
1 an
d 2.
[M-H
]- = n
egat
ivel
y ch
arge
d m
olec
ular
ion;
Rut
= ru
tinos
ide;
Hes
p =
hesp
eret
in;
t R =
rete
ntio
n tim
eab
Sam
ples
wer
e an
alys
ed u
sing
5 –
30%
ace
toni
trile
in 0
.1%
form
ic a
cid
for 7
0 m
in; c S
ampl
e w
ere
anal
ysed
usi
ng 1
0 –
30%
ace
toni
trile
in 0
.1%
form
ic a
cid
for 7
0 m
in;
d Sam
ple
wer
e an
alys
ed u
sing
5 –
20%
ace
toni
trile
in 0
.1%
form
ic a
cid
for 1
10 m
inn.
d. =
not
det
ecte
d
86
Antioxidant activities of tropical citrusTa
ble
2. S
umm
ary
of p
heno
lic c
ompo
unds
in C
itrus
hys
trix
with
HPL
C w
ith d
iode
arr
ay d
etec
tion
and
MS2
dete
ctio
n
Peak
t R (m
in)-
leaf
at R
(min
)-pe
elb
t R (m
in)-
flesh
cC
ompo
unds
λ max
(nm
)[M
-H]- (
m/z
)M
S2 fr
agm
ents
ions
(m/z
)
112
.9n.
d.n.
d.A
pige
nin-
6,8-
di-C
-gl
ucop
yran
osid
e33
059
347
3 ([
0,2 X
1]- ;
[M-H
]- -120
), 50
3 ([
0,3 X
1]- ;
[M
-H]- -
90),
533
([1,
3 X1]
- ; [M
-H]- -
60)
and
575
([M
-H]- -
H2O
) 2
n.d.
n.d.
32.7
Erio
dict
yol-7
-O-r
utin
osid
e28
559
528
7 [E
rid] (
[M-H
]- - R
ut)
3n.
d.n.
d.35
.4Er
iodi
ctyo
l-7-O
-ne
ohes
perid
osid
e28
559
545
9 ([
1,3 A
]- ; [M
-H]- -
136
), 23
5 ([
Y0
0,3 X
0 +
H]- ),
286
([Y
0]- ;
[M-H
]- - 3
08)
4n.
d.n.
d.35
.5Ph
lore
tin-3′,5′-d
i-C-
gluc
opyr
anos
ide
285
597
477
([0,
2 X1]
- ; [M
-H]- -
120
), 38
7 ([
0,2 X
1 0,
3 X0]
- ; [M
-H]- -
120
; [M
-H]- -
120
), 35
7 ([
0,2 X
1 0,
2 X0]
- ; [M
-H]- -
120
; [M
-H]-
-120
) 417
([0,
3 X1
0,3 X
0]- ;
[M-H
]- -90
; [M
-H]- -
90)
519
.0n.
d.n.
d.Q
uerc
etin
-3-O
-rut
inos
ide-
7-O
-glu
cosi
de33
077
160
9 ([
M-H
]- -G
lu),
463
([M
-H]- -
Rut
), 30
1 6
22.6
n.d.
n.d.
Que
rcet
in-3
-O-r
utin
osid
e35
060
930
1 [H
esp]
([M
-H]- -
Rut
), 34
3, 3
25 7
25.9
n.d.
n.d.
Lute
olin
-7-O
-rut
inos
ide
340
593
285
([M
-H]- -
Rut
) 8
32.9
28.1
47.6
Hes
pere
tin-7
-O-r
utin
osid
e28
060
930
1 [H
esp]
([M
-H]- -
Rut
), 34
3, 3
25 9
34.7
28.7
n.d.
Dio
smet
in-7
-O-r
utin
osid
e34
560
729
9 ([
M-H
]- - R
ut)
1035
.729
.349
.8H
espe
retin
-7-O
-ne
ohes
perid
osid
e28
060
930
1[H
esp]
([M
-H]- -
Rut
)
1136
.8n.
d.n.
d.U
nkno
wn
dios
met
in
conj
ugat
e34
560
729
9 ([
M-H
]- - R
ut)
*Pea
k nu
mbe
rs a
nd H
PLC
rete
ntio
n tim
es re
fer t
o H
PLC
trac
e in
Fig
ure
3. [M
-H]- =
neg
ativ
ely
char
ged
mol
ecul
ar io
n; G
lu =
glu
cosi
de; R
ut =
rutin
osid
e;
Hes
p =
hesp
eret
in; t
R =
rete
ntio
n tim
ea S
ampl
es w
ere
anal
ysed
usi
ng 1
0 –
65%
ace
toni
trile
in 0
.1%
form
ic a
cid
for 6
5 m
in; b S
ampl
es w
ere
anal
ysed
usi
ng 5
= 3
0% a
ceto
nitri
le in
0.1
% fo
rmic
aci
d fo
r 70
min
; a Sam
ples
wer
e an
alys
ed u
sing
10
– 60
% a
ceto
nitri
le in
0.1
% fo
rmic
aci
d fo
r 50
min
n.d.
= no
t det
ecte
d
87
R. Suri, A. Crozier, H. Zaharinah and A.R. Zuraida
Tabl
e 3.
Ant
ioxi
dant
pot
entia
l of i
ndiv
idua
l phe
nolic
com
poun
ds in
C. m
icro
carp
a an
d C
. suh
uien
sis
frui
ts
Peak
Com
poun
dsPe
elFl
esh
Con
c.(μ
m)
Trol
ox q
ui.(μ
m)
TEA
RC
onc.
(μm
)Tr
olox
equ
i.(μ
m)
TEA
R
C. m
icro
carp
aFl
avon
e1
Api
geni
n-6,
8-di
-C-g
luco
pyra
nosi
de
n.d.
n.
d.n.
d.
37 ±
6
9 ±
10.
22
Api
geni
n-8-
C-g
luco
side
-2’-
rham
nosi
de 3
6 ±
4
n.d.
n.d.
94
± 6
n.
d.n.
d.3
Unk
own
apig
enin
con
juga
te 4
7 ±
7
n.d.
n.d.
112
± 8
n.
d.n.
d.5
Dio
smet
in -7
-O-n
eohe
sper
idos
ide
45
± 10
8
± 0
0.2
42
± 1
0 1
1 ±
10.
3To
tal
129
8 (2
.4%
) 3
2219
(4.4
%)
Cha
lcon
e4
Phlo
retin
-3′,5′-d
i-C-g
luco
pyra
nosi
de 7
4 ±
1025
1 ±
103.
4 1
13 ±
6 35
4 ±
133.
1To
tal
74
251
(75.
6%)
113
354
(81.
4%)
Flav
anon
e6
Hes
pere
tin-7
-O-r
utin
osid
e 5
2 ±
9 2
7 ±
10.
5
n.d.
n.
d.n.
d.7
Hes
pere
tin-7
-O-n
eohe
sper
idos
ide
52
± 10
46
± 2
0.9
49
± 6
62
± 1
1.3
Tota
l 10
473
(22%
)
4962
(14.
2%)
C. s
uhui
ensis
Flav
one
1A
pige
nin-
6,8-
di-C
-glu
copy
rano
side
38
± 0
n.
d.n.
d.
35 ±
1 1
0 ±
00.
32
Dio
smet
in-6
,8-d
i-C-g
luco
pyra
nosi
de 15
7 ±
0
7 ±
20.
0
n.d.
n.
d.n.
d.3
Dio
smet
in-6
,8-d
i-C-g
luco
pyra
nosi
de (i
som
er)
84
± 2
n.
d.n.
d.
n.d.
n.
d.n.
d.To
tal
279
7 (3
.2%
)10
(3.4
%)
Flav
anon
e4
Nar
inge
nin-
7-O
-rut
inos
ide
n.
d.
n.d.
n.d.
2261
± 4
6 2
0 ±
10.
05
Hes
pere
tin-7
-O-r
utin
osid
e 45
4 ±
3 22
0 ±
70.
5 4
49 ±
8 26
0 ±
30.
66
Isos
akur
anet
in-7
-O-r
utin
osid
e 1
8 ±
0
n.d.
n.d.
7
± 0
n.
d.n.
d.To
tal
472
220
(96.
9%)
2717
280
(96.
6%)
TEA
R =
Tro
lox
Equi
vale
nt A
ntio
xida
nt R
atio
; n.d
.= n
ot d
etec
ted.
For
pea
k nu
mbe
rs s
ee F
igur
es 1
and
2C
once
ntra
tions
and
Tro
lox
equi
lava
nts
expr
esse
d as
mea
n va
lues
± S
D (n
= 3
)
88
Antioxidant activities of tropical citrus
Among flavanones, hesperetin-7-O-neohesperidoside and hesperetin-7-O-rutinoside exhibited a TEAR value lower than that of phloretin-3′,5′-di-C-glucopyranoside and quercetin-3-O-rutinoside in C. microcarpa. The major contributor to the antioxidant activity of C. suhuiensis peel and flesh was from hesperetin-7-O-rutinoside, which accounted for 97% and 90% of the total antioxidant activity respectively (Table 3 and Figure 1). This compound had a TEAR value of 0.5 for peel and 0.6 for flesh. Hesperetin-7-O-neohesperidoside and hesperetin-7-O-rutinoside had an antioxidant ratio substantially higher than
0 10 20 30 40 50 60 70
0 10 20 30 40 50 60 70
720 nm
280 nm
1
45
6
D
A
1 2 3
5
6
720 nm
280 nm
Abs
orba
nce
Retention time (min)
Figure 1. Online HPLC-ABTS assay of Citrus suhuiensis fruit peel (A) and fruit flesh (B). Peak 1 = apigenin-6-8-di-C-glucopyranoside; peak 2 = diosmetin-6-8-di-C-glucopyranoside; peak 3 = diosmetin-6-8-di-C-glucopyranoside (isomer); peak 4 = naringenin-7-O-rutinoside; peak 5 = hesperetin-7-O-rutinoside; peak 6 = isosakuranetin-7-O-rutinoside
other compounds in C. hystrix fruit and leaf (Table 4 and Figure 3). The antioxidant activities of hesperetin-7-O-rutinoside and hesperetin-7-O-neohesperidoside may be due to the presence of 3′-hydroxyl and 4′-methoxy groups on the B ring. Although hesperetin-7-O-rutinoside and hesperetin-7-O-neohesperidoside have no B ring catechol structure that is able to scavenge free radicals, the methyl substitution will activate the C-3′ or C-4′ hydroxyl and result in enhanced antioxidant activity (van Acker et al. 1996). Hesperetin-7-O-neohesperidoside had a slightly higher TEAR value than hesperetin-7-O-rutinoside in C. hystrix leaf and C. microcarpa peel,
89
R. Suri, A. Crozier, H. Zaharinah and A.R. Zuraida
Tabl
e 4.
Ant
ioxi
dant
pot
entia
l of i
ndiv
idua
l phe
nolic
com
poun
ds in
Citr
us h
ystr
ix fr
uit a
nd le
af
Peak
Com
poun
dsLe
afPe
elFl
esh
Con
c.(μ
m)
Trol
ox e
qui.
(μm
)TE
AR
Con
c.(μ
m)
Trol
ox e
qui.
(μm
)TE
AR
aC
onc.
(μm
)Tr
olox
equ
i.(μ
m)
TEA
R
Flav
one
1A
pige
nin-
6,8-
di-C
-gl
ucop
yran
osid
e 5
3 ±
8
n.d.
n.d.
n.
d.
n.d.
n.d.
n.
d.
n.d.
n.d.
5Q
uerc
etin
-3-O
-rut
inos
ide-
7-O
-gl
ucos
ide
48
± 8
n.
d.n.
d.
n.d.
n.
d.n.
d.
n.d.
n.
d.n.
d.
6Q
uerc
etin
-3-O
-rut
inos
ide
93
± 18
60
± 0
0.6
n.
d.
n.d.
n.d.
n.
d.
n.d.
n.d.
7Lu
teol
in-7
-O-r
utin
osid
e 4
0 ±
6 2
0 ±
10.
5
n.d.
n.
d.n.
d.
n.d.
n.
d.n.
d. 9
Dio
smet
in-7
-O-r
utin
osid
e 11
5 ±
21 1
1 ±
20.
1 2
0 ±
1
3 ±
00.
2
n.d.
n.
d.n.
d.11
Unk
now
n di
osm
etin
con
juga
te 17
4 ±
30 1
9 ±
30.
1
n.d.
n.
d.n.
d.
n.d.
n.
d.n.
d.
Tot
al52
211
0 (2
8.6%
) 2
03
(1.2
%)
00
Cha
lcon
e 4
Phlo
retin
-3′,5′-d
i-C-
gluc
opyr
anos
ide
n.
d.
n.d.
n.d.
n.
d.
n.d.
n.d.
111
± 4
345
± 31
3.1
Tota
l 1
1134
5 (5
7.7%
)
Flav
anon
e 2
Erio
dict
yol-7
-O-r
utin
osid
e
n.d.
n.
d.n.
d.
n.d.
n.
d.n.
d. 6
48 ±
26
250
± 1
0.4
3Er
iodi
ctyo
l-7-O
-neo
hesp
erid
osid
e
n.d.
n.
d.n.
d.
n.d.
n.
d.n.
d.
24 ±
1
3 ±
10.
1 8
Hes
pere
tin-7
-O-r
utin
osid
e 13
8 ±
26 4
9 ±
10.
4 24
1 ±
1 11
5 ±
50.
5 2
70 ±
21
n.
d.n.
d.10
Hes
pere
tin-7
-O-n
eohe
sper
idos
ide
464
± 77
225
± 31
0.5
251
± 3
124
± 8
0.5
211
± 1
9
n.d.
n.d.
Tota
l60
227
4 (7
1.4%
)49
223
9 (9
8.8%
) 11
5325
3 (4
2.3%
)
TEA
R =
Tro
lox
Equi
vale
nt A
ntio
xida
nt R
atio
; n.d
. = n
ot d
etec
ted.
For
pea
k nu
mbe
rs s
ee F
igur
e 3
Con
cent
ratio
ns a
nd T
rolo
x eq
uiva
lent
s ex
pres
sed
as m
ean
valu
es ±
SD
(n =
3)
90
Antioxidant activities of tropical citrus
thus is in keeping with the report by Kim and Lee (2004). This may be a reflection of the different sugar moieties at the C7 position. For instance, the combination of a neohesperidose and a methoxy group of the A and C-ring noticeably increased the antioxidant power. It could be hypothesised that attachment of neohesperidoside at the 7 position of the A-ring and a methoxyl group at the 3′ or 4′ position of the C-ring influenced the ability of hesperetin-7-O-neohesperidoside to delocalise electrons and enhanced the antioxidant potential (Di Majo et al. 2005). On the other hand, hydroxyl groups at the 5- and 7-position of the A-ring of flavanone might not take part in scavenging radicals, and thus conjugation of a sugar at this position
may not affect the antioxidant activity of the flavanone (Nakamura et al. 2003). TEAR values for hesperetin-7-O-rutinoside and hesperetin-7-O-neohesperidoside in C. hystrix fruit flesh cannot be calculated because these compounds were not separated when analysed using HPLC-online antioxidant assay. Naringenin-7-O-rutinoside was the major flavonoid in C. suhuiensis fruit flesh, but made a lower contribution to the antioxidant activity and had a lower TEAR value than hesperetin-7-O-neohesperidoside and hesperetin-7-O-rutinoside (Table 3). This is in keeping with the observations by Miller and Rice-Evans (1997). The low antioxidant activity of naringenin-7-O-rutinoside is most probably due to glycosylation at the 7-hydroxyl group in the A ring in conjunction with the saturated heterocyclic C ring. The presence of only one B-ring hydroxyl, at the 4′ position, results in a marked lowering of the antioxidant potential of naringenin-7-O-rutinoside (Rice-Evans et al. 1996). Apigenin-6,8-di-C-glucopyranoside also contributed to the antioxidant activity in C. microcarpa flesh, but had a lower TEAR value than phloretin-3′,5′-di-C-glucopyranoside (Table 3). The C. suhuiensis flesh contained high amounts of naringenin-7-O-rutinoside and diosmetin-6,8-di-C-glucopyranoside, but these compounds exhibited low TEAR values of 0.01. A trace amount of isosakuranetin-7-O-rutinoside was also detected in C. suhuiensis, but did not show any antioxidant activity. Eriodictyol-7-O-rutinoside, which has a catechol moiety and a n2,3 double bond in the B ring, showed higher antioxidant potential than apigenin-8-C-glucoside-2’-rhamnoside in C. hystrix fruit flesh (Table 3 and Table 4), but had almost similar TEAR value to luteolin-7-O-rutinoside and hesperetin-7-O-rutinoside (Table 4). The apigenin-6,8-di-C-glucopyranoside, diosmetin-6,8-di-C-glucopyranoside and diosmetin-6-C-glucoside had low TEAR
91
R. Suri, A. Crozier, H. Zaharinah and A.R. Zuraida
values ranging form 0.1 – 0.3, demonstrating that C-glycosylation at the A-ring decreases antioxidant activity. In general, levels of TEAR for tropical citrus phenolics were as follows; phloretin-3′,5′-di-C-glucopyranoside > quercetin-3- O-rutinoside > hesperetin-
7-O-neohesperidoside > hesperetin-7-O-rutinoside = luteolin-7-O-rutinoside = naringenin-7-O-rutinoside. In this study, the individual antioxidant capacities of each flavonoid were added together to give a total HPLC-derived antioxidant capacity (Table 5). The total
Table 5. Total antioxidant activity of tropical citrus
Total antioxidant capacitya HPLC antioxidant capacityb
a = Trolox equivalent concentration of tropical citrus; b = Addition of the Trolox equivalent concentrations calculated for each citrus peak separated by HPLC; P = peel; L = leaf and F = fleshValues in parentheses represent data expressed as a percentage of the value for total antioxidant capacity
O O
OO
OO
O O
OO
OO
O
H
O
O
O
O
OO
OO
A
HOR
O
B A
6
1
23451'
2'3'4'
5'6'
O OO
O
O O
A C
B
OO
OO
OH
R = OH - Dihydrochalcone-2', 2,3,4',6'- pentahydroxy-3-C-glucopyranoside (Aspalathin)R = H - Dihydrochalcone-2',2,4',6'-tetrahydroxy-3-C-glucopyranoside (Nothofagin)
Figure 4. Major antioxidant flavonoids in tropical citrus and comparison of phloretin-3′,5′-di-C-glucopyranoside to aspalathin and nothofagin (A), potent antioxidant compounds in rooibos tea
92
Antioxidant activities of tropical citrus
antioxidant activity was determined on-line after removing the HPLC column. This enabled the antioxidant activity of compounds that are retained and do not elute from the reverse phase HPLC column to be included in the estimate of total antioxidant activity. Addition of the antioxidant potential of all individual components varied from 14% – 52% (Table 5). The big differences between HPLC-derived and total antioxidant activity of tropical citrus extract probably represents the antioxidant activity of unknown or other antioxidant compounds such as vitamin C, E, essential oil and carotenoids, which appear not to elute from the HPLC column. Green tea phenolics such as (–)-epigallocatechin gallate, (–)-epicatechin gallate and gallic acid are known for their potent antioxidant properties (Stewart et al. 2005). However, studies by Yen et al. (2002) and Azam et al. (2004) found that while green tea catechins are able to reduce Fe3+ to Fe2+ they also exihibit strong pro-oxidant activity in a deoxyribose degradation assay. Other study also reported that compounds with multiple hydroxyl groups such as (–)-epigallocatechin gallate and gallic acid increased the H2O2 radicals in a Fenton system (Hanaski et al. 1994). Thus, C. microcarpa fruit, which is high in phloretin-3′,5′-di-C-glucopyranoside, may be a potentially rich source of dietary antioxidants. The question of whether phloretin-3′,5′-di-C-glucopyranoside is also a pro-oxidant has yet to be answered. Joubert et al. (2005) reported aspalathin, which is structurally very similar to phloretin-3′,5′-di-C-glucopyranoside, also showed pro-oxidant activity, but in the presence of vitamin C it was a potent antioxidant. This study provided useful information on antioxidant activities of tropical citrus, which due to their molecular structure and number of hydroxyl groups. The identified compounds may be responsible for at least some of the health benefits due to tropical citrus intake.
AcknowledgementMalaysian Agricultural Research and Development Institute is acknowledged for funding this project.
ReferencesAmic, D., Davidovic-A., Reslo, D. and Trinajstic,
N. (2003). Structure-radical scavenging activity, relationship of flavonoids. Crotia Chemica Acta 76(1): 55 – 61
Burkill, I.H. (1966). A Dictionary of The Economic Product of The Malay Peninsular, Vol. 1 Kuala Lumpur: Ministry of Agriculture and Cooperative
Azam, S., Hadi, N., Khan, N.U. and Hadi, S.M. (2004). Pro-oxidant property of green tea polyphenols epicatechin and epigallocatechin-3-gallate: implications for anticancer properties. Toxicol. In Vitro 18: 556 – 561
Dapkevicius, A., van Beek, T.A. and Niederlander, H.A.G. (2001). Evaluation and comparison of two improved techniques for the on-line detection of antioxidants in liquid chromatography elutes. J. Chromatography 912: 73 – 82
Di Majo, D., Giammanco, M., Guardia, M.L., Tripoli, E., Giammanco, S. and Finotti, E. (2005). Flavanones in Citrus fruit: structure-antioxidant activity relationships. Food Res. Int. 38: 1161 – 1166
Dixon, R.A. and Steele, C.L. (1999). Flavonoids and isoflavonoids – a gold mine formetabolic engineering. Trends Plant Sci. 4: 394 – 400
Hanaski, Y., Ogawa, S. and Fukui, S. (1994). The correlation between active oxygenscavenging of oxidative effects of flavonoids. Free Radic Biol. Med. 16: 845 – 850
Heim, K., Tagliaferro, A.R. and Bobilya, D.J. (2002). Flavonoid antioxidants: chemistry,metabolism and structure-activity relationships. J. Nutr. Biochem. 13: 572 – 584
Hijova, E., (2006). Bioavalability of chalcones. Bratisl. Lek. Listy 107(3): 80 – 84
Joubert, E., Winterton, P., Britz, T.J. and Ferreira, D. (2004). Superoxide anion radical and a,a-diphenyl-b-picrylhydrazyl radical scavenging capacity of rooibos (Aspalathus linearis) aqueous extracts, crude phenolic fractions, tannin and flavonoids. Food Res. Int. 37: 133 – 138
Joubert, E., Winterton, P., Britz, T.J. and Gelderblom, W.C.A. (2005). Antioxidant and pro-oxidant activities of aqueous extract and crude polyphenolic fractions of Rooibos (Aspalathus linearis). J. Agric. Food Chem. 53: 10260 – 10267
93
R. Suri, A. Crozier, H. Zaharinah and A.R. Zuraida
Kanes, K., Tisserat, B., Berhow, M. and Vandercook, C. (1993). Phenolic compositions of various tissues of rutaceae species. Phytochemistry 4: 967 – 974
Kim, D-O. and Lee, C.Y. (2004). Comprehensive study on the vitamin C equivalent antioxidant capacity (VCEAC) of various polyphenolic in scavenging a free radical and its structural relationship. Crit. Rev. in Food Sci. Nutr. 44: 253 – 273
Koleva, I.I., Niederlander, H.A.G. and van Beek, T.A. (2001). Application of ABTS radical cation for selective on-line detection of radical scavengers in HPLC eluates. Anal. Chem. 73: 3373 – 3381
Miller, N.J. and Rice-Evans, C.A. (1997). The relative contributions of ascorbic acid andphenolic antioxidants to the total antioxidant activity of orange and apple fruit juice and blackcurrant drink. Food Chem. 60(3): 331 – 337
Murakami, A., Gao, G., Kim, O.K., Omura, M., Yano, M., Ito, C., Furukawa, H., Jiwajinda, S., Koshimizu, K. and Ohigashi, H. (1999). Identification of coumarins from the fruit of Citrus hystrix DC as inhibitors of nitric oxide generation in mouse macrophage RAW 264.7 cells. J. Agric. Food Chem. 47(1): 333 – 339
Nakamura, Y., Watanabe, S., Miyake, N., Kohno, H. and Osawa, T., (2003). Dihydrochalcones: Evaluation as novel radical scavenging antioxidants. J. Agric. Food Chem. 51: 3309 – 3312
Rezk, B.M., Haenen, G.R.M.M., Van der Vijgh, W. and Bast, A., (2002). The antioxidant activity of phleoretin: the disclosure of new antioxidant pharmacophore in flavonoids. Biochem. and Biophysical Res. Comm. 295: 9 – 13
Rice-Evans, C., Miller, N. and Paganga, G., (1996). Structure-antioxidant activity relationships of flavonoids and phenolic acids. Free Radic. Biol. Med. 20: 933 – 956
Roowi, S. and Crozier, A. (2011). Flavonoids in tropical citrus species. Journal of Agricultural and Food Chemistry 59(22): 12217 – 12225
Stewart, A.J., Mullen, W. and Crozier, A. (2005). On-line high-performance liquid chromatography analysis of the antioxidant activity of phenolic compounds of green tea and black tea. Mol. Nutr. Food Res. 49: 52 – 60
Tatum, H. and Berry, R. (1977). 6,7-Dimethoxycoumarin in the peels of Citrus. Phytochemistry 16(7): 1091 – 092
Tee, E.S. and Lim, C.L. (1991). Carotenoid compositions content of Malaysian vegetables and fruits by the AOAC and HPLC methods. Food Chem. 41: 309 – 339
van Acker, S.A.B.E., De Groot, M.J., Van den Berg, D-J., Tromp, M.N.J.L., Den Kelder, G.D-O., Van der Vijgh, W.J.F. and Bast, A. (1996). Quantum chemical explanation of the antioxidant activity of flavonoids. Chem. Res. Toxicol. 9(8): 1305 – 1312
Von Gadow, A., Joubert, E. and Hansmann, C.F. (1997). Comparison of the antioxidant activity of aspalathin with that of other plant phenols of rooibos tea (Aspalathuslinearis), a tocopherol, BHT and BHA. J. Agric. Food Chem. 45: 632 – 638
Yen, G-C., Duh, P-D. and Tsai, H-L. (2002). Antioxidant and pro-oxidant properties of ascorbic acid and gallic acid. Food Chem. 79: 307 – 313
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Antioxidant activities of tropical citrus
AbstrakBuah limau purut (Citrus hystrix), limau kasturi (Citrus microcarpa) dan limau madu (Citrus suhuiensis) telah dianalisis kandungan bahan fenolik menggunakan alat kromatografi turus berprestasi tinggi dengan pengesan ‘diode array’ dan spektrum jisim MS2 (HPLC-PDA-MS2). Potensi aktiviti antioksida pada tiap-tiap bahan flavonoid telah ditentukan menggunakan kromatografi turus berprestasi tinggi secara dalam talian dengan cara mengadakan tindak balas dengan bahan reagen asid 2,2¢-azinobis-(3-etilbenzotiazolina-6-sulfonik (ABTS). Phloretin-3′,5′-di-C-glucopiranosida hadir dalam kulit buah dan isi limau kasturi (C. microcarpa) dan menunjukkan nilai aktiviti nisbah antioksidan berasaskan Trolox atau ‘Trolox Equivalent Antioxidant Ratio’ (TEAR) yang tinggi iaitu 3.4 dan 3.1. Quercetin-3-O-rutinosida, hesperetin-7-O-neohesperidosida and hesperetin-7-O-rutinosida yang hadir dalam buah C. hystrix dan C. suhuiensis mempunyai nilai TEAR yang lebih rendah berbanding phloretin-3′,5′-di-C-glucopiranosida. Tahap nilai TEAR bahan flavonoid dalam buah C. microcarpa, C. suhuiensis dan C. hystrix adalah seperti berikut; phloretin-3′,5′-di-C-glucopyranosida > quercetin-3-O-rutinosida > hesperetin-7-O-neohesperidosida > hesperetin-7-O-rutinosida = luteolin-7-O-rutinosida = naringenin-7-O-rutinosida. Citrus microcarpa biasanya digunakan secara meluas dalam makanan, minuman dan jeruk di kalangan masyarakat Asia dan mempunyai kandungan bahan phloretin-3′,5′-di-C-glucopyranosida yang tinggi. Bahan ini mempunyai struktur yang hampir sama dengan aspalatin dan notofagin, iaitu sejenis bahan antioksidan yang kuat dalam teh rooibos.
