Phenolic Composition and Antioxidant Activity of Prunes and

Prune J uice (Prunus domesti ca)

Jenni fer L. Donovan,

Anne S. Meyer,

and Andrew L. Waterhouse*

,
Department of Vi ti cul ture and Enol ogy, Uni versi ty of Cal i forni a at Davi s, Davi s, Cal i forni a 95616, and
Department of Bi otechnol ogy, Techni cal Uni versi ty of Denmark, Lyngby, Denmark
Phenol i c compounds i n foods have been associ ated wi th reduced i nci dences of heart di sease by acti ng
as anti oxi dants for l ow-densi ty l i poprotei n (LDL). Commerci al prune and prune jui ce extracts
(Prunus domestica cv. French) were anal yzed for phenol i cs by reversed phase HPLC wi th di ode
array detecti on and tested for the abi l i ty to i nhi bi t the Cu
2+
-catal yzed oxi dati on of human LDL.
The mean concentrati ons of phenol i cs were 1840 mg/kg, 1397 mg/kg, and 441 mg/L i n pi tted prunes,
extra l arge prunes wi th pi ts, and prune jui ce, respecti vel y. Hydroxyci nnamates, especi al l y
neochl orogeni c aci d, and chl orogeni c aci d predomi nated, and these compounds, as wel l as the prune
and prune jui ce extracts, i nhi bi ted the oxi dati on of LDL. The pi tted prune extract i nhi bi ted LDL
oxi dati on by 24, 82, and 98% at 5, 10, and 20 M gal l i c aci d equi val ents (GAE). The prune jui ce
extract i nhi bi ted LDL oxi dati on by 3, 62, and 97% at 5, 10, and 20 M GAE. These data i ndi cate
that prunes and prune jui ce may provi de a source of di etary anti oxi dants.
Keywords: Low-density lipoprotein (LDL); antioxidants; phenolics; neochlorogenic acid; prune;
plum; Prunus domestica
I NTRODUCTI ON
Phenol i c compounds are i mportant components of
many frui ts, vegetabl es, and beverages, i n whi ch they
contri bute to col or and sensory properti es such as
bi tterness and astri ngency (Machei x et al ., 1990).
Epi demi ol ogi cal studi es have shown that consumpti on
of foods and beverages ri ch i n phenol i c content i s
correl ated wi th reduced i nci dences of heart di sease
(Hertog et al ., 1993, 1995; Cri qui and Ri ngel , 1994;
Renaud and deLorgeri l , 1992). One possi bl e expl ana-
ti on i s that phenol i c compounds sl ow the progressi on
of atheroscl erosi s by acti ng as anti oxi dants toward l ow-
densi ty l i poprotei ns (LDL) (Frankel et al ., 1993; Ki nsel l a
et al ., 1993). The oxi dati ve modi fi cati on of LDL i s a
cri ti cal step i n the devel opment of atheroscl erosi s, and
preventi on of thi s step i s thought to sl ow the progressi on
of the di sease (Stei nberg, 1993; Esterbauer et al ., 1992).
Studi es have shown that phenol i c compounds contai ned
i n frui ts are potent i nhi bi tors of the i n vi tro oxi dati on
of LDL (Meyer et al ., 1997; Tei ssedre et al ., 1996; Ri ce-
Evans et al ., 1996; Frankel et al ., 1995; Vi nson et al .,
1995; deWhal l ey et al ., 1990). There are al so studi es
suggesti ng that phenol i c compounds have anti oxi dant
acti vi ty i n vi vo (Fuhrman et al ., 1995; Whi tehead et al .,
1995; Zl och, 1969; Rusznyak and Sszent-Gyorgi , 1936).
Cal i forni an prunes are produced from a cul ti var of
Prunus domestica pl ums referred to by several names
i ncl udi ng French, Peti te, dAgen, and dEnte (Chaney,
1981). Cal i forni a produces 67% of the worl ds prune
suppl y (Cal i forni a Prune Board, 1997). The pri mary
objecti ve of thi s i nvesti gati on was to determi ne the l evel
of total phenol i cs, the concentrati on of each phenol i c
compound, and the vari abi l i ty of phenol i c compounds
i n Cal i forni an prunes and prune jui ce. To i denti fy
changes that resul t from processi ng, freshl y harvested
unprocessed prune-maki ng pl ums were al so anal yzed
for phenol i cs. The second mai n objecti ve of thi s i nves-
ti gati on was to determi ne i f prune and prune jui ce
extracts had anti oxi dant acti vi ty toward human LDL.
MATERI ALS AND METHODS
Phenolic Standards. (+)-Catechi n, (-)-epi catechi n, and
p-coumari c aci d were obtai ned from Al dri ch Chemi cal Co.
(Mi l waukee, WI ). Ruti n, (hydroxymethyl )furfural (HMF), and
caffei c aci d were purchased from Si gma Chemi cal Co. (St.
Loui s, MO). Chl orogeni c aci d (5-caffeoyl qui ni c aci d) was
purchased from Fl uka Chemi cal Corp. (Buchs, Swi tzerl and).
Gal l i c aci d was obtai ned from MCB Manufacturi ng Chemi sts
I nc. (Ci nci nnati , OH), sorbi c aci d was obtai ned from Eastman
Kodak Co. (Rochester, NY), and mal vi n was purchased from
Pfal tz & Bauer (Waterbury, CT). Neochl orogeni c aci d (3-
caffeoyl qui ni c aci d) was ki ndl y provi ded by Dr. Murray I sman
(Department of Pl ant Sci ences, Uni versi ty of Bri ti sh Col umbi a,
Vancouver).
PrunesandPruneJ uiceSamples. Twenty-one sampl es
of pi tted prunes, 12 sampl es of extra l arge unpi tted prunes,
and 9 sampl es of prune jui ce from di fferent orchards and
processi ng faci l i ti es throughout Cal i forni a were obtai ned from
the Cal i forni a Prune Board (Pl easonton, CA) over a 6 month
peri od duri ng 1995-1996. These sampl es were commerci al l y
packaged and sel ected from l ots of prunes and prune jui ce
desti ned for the market. Four sampl es of freshl y harvested
unprocessed prune-maki ng pl ums were al so obtai ned from the
Cal i forni a Prune Board.
Extraction of Phenolic Compounds. Ten prunes were
randoml y sel ected from one freshl y opened package, pi tted by
hand, and cut i nto smal l pi eces; a 10.0 g porti on was homog-
eni zed i n 75 mL of methanol (HPLC grade, Fi sher Sci enti fi c,
Spri ngfi el d, NJ) contai ni ng 50 mg/L sodi um metabi sul fi te
(Mal l i nckrodt I nc., Chesterfi el d, MO). After a 30 mi n extrac-
* Author to whom correspondence shoul d be addressed
[tel ephone (916) 752-4777; fax (916) 752-0381; e-mai l
al waterhouse@ucdavi s.edu].

Uni versi ty of Cal i forni a.

Techni cal Uni versi ty of Denmark.

1247 J. Agric. Food Chem. 1998, 46, 12471252
S0021-8561(97)00831-5 CCC: $15.00 1998 American Chemical Society
Published on Web 03/13/1998
ti on peri od (whi l e agi tati ng), the sampl e was centri fuged
(2000g, 5 mi n), the supernatant was removed, and the
preci pi tate was re-extracted a second ti me wi th 75 mL of
methanol for 30 mi n and then a thi rd ti me wi th 75 mL of 80%
aqueous methanol for 30 mi n. The supernatants were com-
bi ned, rotary evaporated at 35 C to 50 mL, and then di l uted
to 100 mL wi th water. Phenol i cs were extracted from prune
jui ce by mi xi ng 25 mL of prune jui ce wi th 75 mL of 80%
aqueous methanol saturated wi th sodi um chl ori de (Fi sher
Sci enti fi c). After a 30 mi n extracti on peri od (whi l e agi tati ng),
the sol uti on was centri fuged to obtai n two separate l i qui d
phases. The methanol fracti on was removed, and the remai n-
i ng l i qui d was extracted two addi ti onal ti mes wi th 75 mL of
80% aqueous methanol for 30 mi n each. The methanol -
contai ni ng fracti ons were combi ned, rotary evaporated, and
then di l uted to 100 mL wi th water.
Analysisof Phenolic Compounds. Pri or to anal ysi s, the
extracts were fi l tered through 0.45 m pol y(tetrafl uoroethyl -
ene) (PTFE) syri nge ti p fi l ters (Gel man Sci ences, Ann Arbor,
MI ) i nto fl i nt gl ass HPLC vi al s equi pped wi th PTFE-l i ned
cri mp caps. The phenol i c composi ti on of the prunes and
prunes jui ces was anal yzed by hi gh-performance l i qui d chro-
matography (HPLC) as descri bed previ ousl y (Lamuel a-Raven-
tos and Waterhouse 1994), wi th sl i ght changes i n the mul ti -
l i near mobi l e phase gradi ent (Tabl e 1). Four wavel engths
were moni tored for quanti tati on: 280 nm for catechi ns and
benzoi c aci ds, 316 nm for hydroxyci nnamates, 365 nm for
fl avonol s, and 520 nm for anthocyani ns.
Compounds were quanti fi ed by cal i brati on wi th the de-
scri bed standards. Compounds that were not avai l abl e were
quanti fi ed usi ng the peak areas of standards wi th si mi l ar
spectral characteri sti cs and quanti ti es were reported as equi va-
l ent amounts of that commerci al standard.
Statistical Analysis. A t test was to used to obtai n a
confi dence i nterval for the mean l evel of total phenol s i n the
popul ati on of Cal i forni an prunes and prune jui ce. The cal cu-
l ati on of the confi dence i nterval was descri bed by Moses
(1986): ) x ( (ts)/ n, where was the popul ati on mean
(unknown), xwas the sampl e mean, swas the sampl e standard
devi ati on, n was the number of sampl es, and t was the t val ue
at n - 1 degrees of freedom.
Preparation of LDL. Pl asma was prepared from bl ood
col l ected by veni puncture from fi ve heal thy vol unteers i n 10
mL Vacutai ner tubes (Becton Di cki nson I nc., Frankl i n Lakes,
NJ) contai ni ng ethyl enedi ami netetraaceti c aci d (K
3EDTA).
LDL was prepared by sequenti al densi ty preparati ve ul tra-
centri fugati on (Orr et al ., 1991). Pri or to oxi dati on, EDTA was
removed by di al ysi s usi ng Spectra/Por membrane tubi ng
(MW cutoff of 12-14 kDa; Spectrum Medi cal I ndustri es, I nc.,
Los Angel es, CA) i nto pH 7.4 deoxygenated phosphate buffered
sal i ne at 4 C. LDL protei n concentrati on was determi ned
wi th a Lowry protei n anal ysi s ki t (Si gma) and was di l uted to
1000 g/mL protei n pri or to oxi dati on.
Antioxidant Activity for LDL. To assess the anti oxi dant
acti vi ty of the prunes and prune jui ce, the extracts were
prepared wi thout the addi ti on of sodi um metabi sul fi te. After
the methanol was compl etel y removed by rotary evaporati on,
total phenol s were measured usi ng the Fol i n-Ci ocal teu (FC)
method (Si ngl eton and Rossi , 1965). The extracts were stored
at 4 C and used before any measurabl e degradati on of
phenol i cs had occurred (12-24 h).
I nhi bi ti on of LDL oxi dati on was determi ned by moni tori ng
hexanal producti on by stati c headspace gas chromatography
from copper-catal yzed LDL oxi dati on (2 h at 37 C, 80 M
CuSO4) as previ ousl y descri bed (Frankel et al ., 1992). Resul ts
were cal cul ated after repl i cate anal yses and expressed as
percent i nhi bi ti on of the control LDL: [(C -S)/C] 100, where
C was hexanal formed from the LDL wi th no extract added
and S was hexanal formed from the LDL wi th the prune or
prune jui ce extract added.
RESULTS AND DI SCUSSI ON
Extraction and Analysis Procedure. The extrac-
ti on procedures were suffi ci ent to extract phenol i c
compounds from prunes and prune jui ce. The extent
of extracti on was i nvesti gated by performi ng two ex-
tracti ons wi th 100% methanol i n addi ti on to the de-
scri bed procedure. For both prunes and prune jui ce, l ess
than an addi ti onal 4%of chl orogeni c and neochl orogeni c
aci d (the two predomi nant phenol i cs i n prunes) was
obtai ned i n these extracts. The HPLC method descri bed
here provi ded separati on of the major peaks i n these
extracts. The peaks that were speci fi cal l y i denti fi ed
general l y made up 90%of the total area at 280 nm. The
peaks that were tentati vel y i denti fi ed by cl ass (that i s,
ci nnamates and fl avonol s) made up onl y 1-2% of the
total amount of phenol s i denti fi ed i n prunes. The peaks
that were not i denti fi ed general l y made up <5% of the
area at 280 nm. A chromatogram of a pi tted prune
extract i s shown i n Fi gure 1. The extracti on and HPLC
anal ysi s were al so very reproduci bl e. Reproduci bi l i ty
was assessed by separate extracti ons and HPLC anal y-
ses of si x homogeneous sampl es of prunes and prune
jui ce. The coeffi ci ents of vari ati on were <3%for neochl o-
rogeni c and chl orogeni c aci d i n both prunes and prune
jui ce.
Phenolic Composition of Prunes. The phenol i c
composi ti on and the amount of total phenol s as deter-
mi ned by HPLC were si mi l ar i n the pi tted prunes and
the extra l arge prunes wi th pi ts (Tabl e 2). The confi -
Table 1. Conditions Used for HPLC Analysis of Phenolic
Compounds in Prunes and Prune J uice
i nstrument Hewl ett-Packard 1090 l i qui d chromatograph
col umn Li Chrosphere C18, 4 250 mm, 5 m
parti cl e si ze
i njecti on vol ume 25 L
fl ow rate 0.5 mL/mi n
detecti on photodi ode array (190-600 nm)
mobi l e phase A ) 50 mM NaH4H2PO4 at pH 2.60
B ) 80% acetoni tri l e, 20% A
C ) 200 mM o-phosphori c aci d at pH 1.50
gradi ent
5.0 mi n %A, 100.0; %B, 0.0; %C, 0.0
8.0 mi n %A, 92.0; %B, 8.0; %C, 0.0
20.0 mi n %A, 0.0; %B, 14.0; %C, 86.0
25.0 mi n %A, 0.0; %B, 16.5; %C, 82.0
35.0 mi n %A, 0.0; %B, 21.5; %C, 78.5
70.0 mi n %A, 0.0; %B, 50.0; %C, 50.0
75.0 mi n %A, 100.0; %B, 0.0; %C, 0.0
80.0 mi n %A, 100.0; %B, 0.0; %C, 0.0
Figure1. HPLC chromatogram of pi tted prune extract: (A)
HMF; (B) neochl orogeni c aci d; (C) ci nnamate; (D) 3-p-couma-
royl qui ni c aci d (tentati ve); (E) chl orogeni c aci d; (F) caffei c aci d;
(G) ci nnamate; (H) coumari c aci d; (I ) fl avonol ; (J) ruti n; (K)
sorbi c aci d. The si gnal at 520 nm was al so moni tored; however,
i t i s not shown here because no si gnal s were obtai ned at that
wavel ength.
1248 J. Agric. Food Chem., Vol. 46, No. 4, 1998 Donovan et al.
dence i nterval s for the l evel of total phenol i cs were 1840
( 390 mg/kg i n the popul ati on of Cal i forni an pi tted
prunes and 1397 ( 190 mg/kg i n the popul ati on of
Cal i forni an extra l arge prunes wi th pi ts (t test, p <
0.05, mass does not i ncl ude the pi t).
Hydroxyci nnamates made up 98% of the phenol i c
materi al , and neochl orogeni c aci d general l y accounted
for >65% of the total phenol i cs. Fl avonol s accounted
for 2% of total phenol i cs and were the onl y fl avonoi ds
i denti fi ed i n the prune sampl es.
Fl avan-3-ol s and anthocyani ns were absent i n the
sampl es (al though there was 54 mg/kg catechi n i n the
fresh prune-maki ng pl ums, as wi l l be di scussed bel ow).
The l ack of fl avan-3-ol s i s i n contrast to a previ ousl y
publ i shed report that water extracts of Cal i forni an
prunes, normal i zed to 18.5%sol i ds, contai ned 126-179
mg/L catechi n (van Gorsel et al ., 1992). I f catechi n was
present at these l evel s, i t woul d have been detected i n
thi s study, so the reason for thi s i nconsi stency i s not
apparent. However, other researchers have reported
that fresh prune-maki ng pl ums (that i s, not dehydrated)
contai n l ow amounts of catechi n, that i t predomi nates
i n the ski n of the pl um, and that l evel s si gni fi cantl y
decrease duri ng the dehydrati on process (Raynal et al .,
1989). The present resul ts agree wi th previ ous reports
that neochl orogeni c aci d i s the pri nci pal phenol i c com-
pound i n prunes, fol l owed by i ts i somer chl orogeni c aci d.
Ruti n has al so been previ ousl y i denti fi ed as a predomi -
nant fl avonol , and prunes have been characteri zed by
the absence of anthocyani ns (Raynal and Moutounet,
1989; van Gorsel et al ., 1992).
Level s of phenol i cs i n prunes are di ffi cul t to compare
wi th other commerci al frui ts because most previ ous
studi es di d not anal yze l arge, representati ve sampl es
of commerci al products. Many factors can affect phe-
nol i c l evel s (Machi ex et al ., 1990) so the amount found
i n one sampl e may not refl ect the average l evel on the
market. However, the l evel reported here for prunes
surpasses the l evel s reported for many other popul ar
frui ts. Red Fl ame seedl ess tabl e grapes were recentl y
reported to contai n <250 mg of phenol i cs/kg of grape
and whi te tabl e vari eti es <50 mg/kg of grape (Meyer et
al ., 1997), al though a mi l d extracti on procedure was
used duri ng thi s study. Appl es were reported to contai n
1200 mg/kg (Kuhnau, 1976; Herrmann, 1989), oranges
830 mg/kg (Kuhnau, 1976; Bi l yk, 1986), pears 265 mg/
kg (Kuhnau, 1976; Herrmann, 1989), and cherri es 850
mg/kg of frui t (Kuhnau, 1976; Herrmann, 1989), whi l e
bl ueberri es were excepti onal l y hi gh i n phenol i cs and
surpassed the amount found i n prunes, contai ni ng 4500
mg of phenol i cs/kg of frui t (Kuhnau, 1976; Pel eg, 1991;
Bi l yk, 1986).
Phenolic Compositionof PruneJ uice. The mean
concentrati on of total phenol i c compounds, i denti fi ed by
HPLC, i n prune jui ce was 441 mg/L. The confi dence
i nterval for the average concentrati on of total phenol i cs
i n the popul ati on of Cal i forni an prune jui ce was 441 (
48 mg/L (t test, p < 0.05). The speci fi c phenol i c
compounds i n the prune jui ce were the same as the
phenol i cs i n the prunes (Tabl e 2). Hydroxyci nnamates
made up al most 99% of the phenol i cs i n prune jui ce.
Neochl orogeni c aci d accounted for 51%of the phenol i cs,
and chl orogeni c aci d accounted for 44%of the phenol i cs.
The rati o of these two compounds i s i n contrast to the
prune sampl es, i n whi ch chl orogeni c aci d was found at
l ess than hal f of the l evel of neochl orogeni c aci d. The
present i nvesti gati on does not, however, permi t us to
determi ne the reason for thi s di fference. Fl avonol s were
found at 6 mg/L and accounted for 1%of phenol i cs. Li ke
the prunes, fl avan-3-ol s and anthocyani ns were absent
i n al l of the sampl es tested.
The presence of l arge quanti ti es of neochl orogeni c aci d
i n prune jui ce sets i t apart from other popul ar jui ces
such as appl e, orange, pear, and grape (Machei x et al .,
1990). Fl avonoi ds and phl ori dzi n i n appl e jui ce (Spanos
et al ., 1990), tartari c aci d esters of hydroxyci nnamates
i n grape jui ce, anthocyani ns i n red grape jui ce (Spanos
and Wrol stad, 1990a; Mazza and Mi ni ati , 1993; Fernan-
dez de Si mon et al ., 1992), and catechi ns i n pear jui ce
(Spanos and Wrol stad, 1990b) are components that
make these jui ces di fferent from prune jui ce. Al so,
prune jui ce contai ned hi gher l evel s of phenol i c com-
pounds than the l evel s reported for many other com-
merci al jui ces. Whi te grape jui ces were reported to
contai n <100 mg/L (Fernandez de Si mon et al ., 1992;
Spanos and Wrol stad, 1990a), but a comprehensi ve
anal ysi s of phenol i cs i n commerci al red grape jui ce has
not been reported. Whi te wi nes usual l y contai n <200
mg/L phenol i c compounds, whereas red wi nes typi cal l y
contai n between 400 and 700 mg/L monomeri c phenol i c
compounds i n addi ti on to 1000 mg/L pol ymeri c pol yphe-
nol s (Si ngl eton, 1980). Pear jui ce has been reported to
Table 2. Phenolic Composition of Pitted Prunes, Extra Large Prunes with Pits, Prune J uice, and Fresh Unprocessed
Prunes
a
compound
pi tted prunes
(mg/kg) (n ) 21)
extra l arge
prunes wi th pi ts
(mg/kg) (n ) 12)
prune jui ce
(mg/L) (n ) 9)
fresh
prune-maki ng
pl ums (n ) 4)
neochl orogeni c aci d 1306 ( 629 928 ( 219 225 ( 34 807 ( 103
3-coumaroyl qui ni c aci d
b
15 ( 13 10 ( 4 4 ( 1 10 ( 1
catechi n nd
c
nd nd 54 ( 14
chl orogeni c aci d 436 ( 201 411 ( 126 193 ( 26 144 ( 23
caffei c aci d 9 ( 8 10 ( 5 3 ( 1 nd
coumari c aci d 10 ( 5 10 ( 5 4 ( 1 nd
other ci nnamates
b
24 ( 54 4 ( 6 7 ( 2 nd
ruti n 33 ( 25 14 ( 6 4 ( 1 25 ( 5
other fl avonol s
b
9 ( 13 14 ( 3 2 ( 1 2 ( 2
anthocyani ns
b
nd nd nd 76 ( 14
HMF
d
220 ( 189 291 ( 205 528 ( 91 nd
sorbi c aci d
d
818 ( 310 425 ( 177 nd nd
total phenol i cs 1840 ( 855 1397 ( 299 441 ( 59 1107 ( 114
a
Val ues are expressed as mean concentrati ons ( the standard devi ati on.
b
Other ci nnamates are reported i n caffei c aci d equi val ents,
other fl avonol s i n ruti n equi val ents, and anthocyani ns i n mal vi n equi vel ents; the i denti fi cati on of 3- coumaroyl qui ni c aci d i s tentati ve
and reported i n coumari c aci d equi val ents.
c
nd, not detected (<3 mg/kg of prune).
d
HMF and sorbi c aci d are not phenol i c compounds.
Phenolic Antioxidants in Prunes J. Agric. Food Chem., Vol. 46, No. 4, 1998 1249
contai n up to 300 mg/L and appl e jui ce up to 330 mg/L,
but some processi ng procedures reduced the l evel s i n
both these jui ces to <1 mg/L (Spanos and Wrol stad,
1990b, Spanos et al ., 1990).
Phenolic Composition of Prune-makingPlums.
The amount of total phenol i cs, i denti fi ed by HPLC, i n
prune-maki ng pl ums ranged from 948 to 1219 mg/kg
wi th an average of 1107 mg/kg (Tabl e 2). Hydroxyci n-
namates were found onl y i n thei r esteri fi ed form and
accounted for 84-90% of total phenol s, wi th neochl o-
rogeni c aci d predomi nati ng. The free ci nnamates found
i n the prunes and prune jui ce were probabl y formed
duri ng processi ng by ester hydrol ysi s, whi ch may have
been aci d-catal yzed or perhaps due to an esterase
natural l y present i n the pl ums. Fl avonol s accounted
for 2-3% of phenol i cs. The fl avan-3-ol , catechi n, ac-
counted for 4-8% of total phenol i cs; however, epi cat-
echi n was not detected i n any of the sampl es, and thi s
resul t agrees wi th previ ous reports that thi s cul ti var of
pl ums does not contai n epi catechi n (Raynal et al ., 1989;
van Gorsel et al ., 1992). Anthocyani ns were al so found
i n the fresh pl ums and accounted for 4-9%of phenol i cs.
The edi bl e porti on of fresh prune-maki ng pl ums
contai ns 24% sol ubl e sol i ds, whereas commerci al
prunes contai n 62% sol ubl e sol i ds. Prune jui ce i s an
aqueous extract of dehydrated prunes normal i zed to
18.5% sol i ds (Mi l l er, 1981; Cal i forni a Prune Board,
1990). The water content and mass of each product
change duri ng processi ng. Thi s must be consi dered
when the phenol i c l evel s of these products are compared
because l evel s are reported on the basi s of fi nal mass
or vol ume. Duri ng processi ng, the total amount of
sol ubl e sol i ds remai ns constant, and when phenol i c
l evel s are expressed on the basi s of sol ubl e sol i ds, i t i s
cl ear that the processi ng of prunes from fresh pl ums
degrades approxi matel y hal f of the phenol i c compounds.
Because prunes and prune jui ce had si mi l ar l evel s of
phenol i cs on the basi s of sol ubl e sol i ds, the processi ng
of prune jui ce from prunes does not appear to si gni fi -
cantl y degrade phenol i c compounds. Phenol i c l evel s i n
each product are expressed on the basi s of mass or
vol ume and sol ubl e sol i ds content i n Tabl e 3.
HMF andSorbic AcidLevelsinPruneProducts.
HMF i s not a phenol i c compound; however, because i t
was extracted and separated usi ng the descri bed ana-
l yti cal procedure for phenol i cs, i t i s reported i n thi s
study. HMF forms from the dehydrati on of sugars i n
the presence of aci d and heat and al so duri ng the
Mai l l ard browni ng reacti on. Thi s compound i s found
i n many common foods and other thermal l y processed
frui t jui ces (Lang, 1970). As expected, al l of the prune
sampl es contai ned HMF (Tabl e 2). However, on aver-
age, the amount of HMF i n the prune sampl es was
<10% of the amount of total phenol i cs. Prune jui ce, on
the other hand, contai ned approxi matel y twi ce as much
HMF as the prune sampl es, and the amount surpassed
the l evel of phenol i cs i n the jui ce. The i ncreased l evel
of HMF i n the prune jui ce shoul d be expected because
of the addi ti onal exposure to heat duri ng the extracti on
procedure. HMF was not detected i n any sampl es of
fresh unprocessed prunes. Al though l i ttl e i nformati on
i s avai l abl e on the nutri ti onal i mportance of thi s
compound, no adverse effects were observed when rats
were fed l evel s of 450 mg/kg of di et (Lang, 1970).
Sorbi c aci d i s a preservati ve, used to prevent un-
wanted mi crobi al growth, whi ch i s routi nel y used i n
prune processi ng (Cal i forni a Prune Board, 1990). The
average concentrati ons of sorbi c aci d were 818 mg/kg
i n pi tted prunes and 425 mg/kg i n the extra l arge prunes
wi th pi ts (Tabl e 2). As expected, sorbi c aci d was not
detected i n any of the sampl es of fresh unprocessed
prunes or prune jui ce. Because prune jui ce i s heat
treated pri or to bottl i ng (Jackson, 1981), there i s no need
to add thi s preservati ve.
Inhibition of LDL Oxidation in Vitro. The prune
and the prune jui ce extracts si gni fi cantl y i nhi bi ted the
oxi dati on of l i pi ds i n i sol ated human LDL when tested
at mi cromol ar phenol concentrati ons (Tabl e 4). At
equi val ent total phenol l evel s accordi ng to the FC assay,
the prune extract was a more powerful i nhi bi tor of LDL
oxi dati on than the prune jui ce extract. Total phenol s
by the FC assay i n the prune jui ce extract were 4 ti mes
the amount i denti fi ed by HPLC [1640 mg/L gal l i c aci d
equi val ents (GAE)]. Conversel y, total phenol s by the
FC assay i n the prune extract were onl y 1.3 ti mes the
amount i denti fi ed by HPLC (2370 mg/kg GAE). The
i ncreased FC response of the prune jui ce extract may
be due to i nterferences whi ch i ncreased the apparent
phenol concentrati on and thus the di l uti on factor of the
jui ce extract for the anti oxi dant test. I f so, thi s may
expl ai n the decreased anti oxi dant acti vi ty of the jui ce
extract compared wi th the prune extract. Reductones
formed from Mai l l ard browni ng reacti ons may have
been i nterferences i n these assays. HMF, however, i s
not thought to be an i nterference because i t di d not have
a response i n the FC assay or appreci abl e anti oxi dant
acti vi ty toward LDL.
The anti oxi dant acti vi ty of the prune extract i s si mi l ar
to that of other foods that contai n phenol i c phytochemi -
cal s such as grapes, wi ne, and chocol ate. These foods
were shown to si gni fi cantl y i nhi bi t LDL oxi dati on at
5-10 M (Frankel et al ., 1993, 1995; Tei ssedre et al .,
1996; Waterhouse et al ., 1996; Meyer et al ., 1997).
The predomi nant phenol i c compounds i n prunes al so
i nhi bi ted LDL oxi dati on (Tabl e 4). The anti oxi dant
Table 3. Total Amount of Phenolic Compounds
Identified by HPLC in Prune Products Expressed on the
Basis of Total Mass and Total Soluble Solids
sampl e
sol ubl e
sol i ds
b
(%)
total
phenol s
(mg/kg)
total phenol s
(mg/kg of
sol ubl e sol i ds)
pi tted prunes 62 1840 2698
extra l arge prunes wi th pi ts
a
62 1397 2253
prune jui ce
a
18.5 441 2384
fresh unprocessed pl ums 24 1107 4613
a
Val ues do not i ncl ude the mass of the pi t; phenol i cs i n prune
jui ce are expressed as mg/L.
b
Sol ubl e sol i ds contents are based
on previ ousl y reported l evel s (Mi l l er, 1981; Cal i forni a Prune
Advi sory Board, 1990).
Table 4. Inhibition of Cu
2+
-Catalyzed LDL Oxidation in
Vitro by Prune and Prune J uice Extracts and Selected
Compounds Found in These Products
% i nhi bi ti on at
sampl e 5 M
a
10 M
a
20 M
a
prune extract 24 ( 3 82 ( 6 98 ( 1
prune jui ce extract 3 ( 3 62 ( 1 97 ( 1
neochl orogeni c aci d 87 ( 3 99 ( 1 99 ( 1
chl orogeni c aci d 91 ( 6 99 ( 1 99 ( 1
HMF 3 ( 4 5 ( 2 0 ( 6
sorbi c aci d nd
b
nd 15 ( 7
a
I nhi bi ti on data are gi ven as mean val ues ( the standard
devi ati on of repl i cate resul ts; the prune and prune jui ce extracts
were tested at l evel s reported i n gal l i c aci d equi val ents (GAE).
b
nd, not determi ned.
1250 J. Agric. Food Chem., Vol. 46, No. 4, 1998 Donovan et al.
acti vi ty of neochl orogeni c aci d toward LDL has not been
previ ousl y reported; however, the strong anti oxi dant
acti vi ty of chl orogeni c aci d toward LDL i s si mi l ar to
previ ousl y descri bed reports (Ri ce-Evans et al ., 1996;
Nardi ni et al ., 1995). Other phenol i c components i n
prunes such as ruti n and caffei c aci d have al so been
reported to be acti ve i nhi bi tors of human LDL oxi dati on
(Tei ssedre et al ., 1996; Ri ce-Evans et al ., 1996; Nardi ni
et al ., 1995).
Rel ati vel y l i ttl e i s known about the absorpti on and
metabol i sm of hydroxyci nnami c aci ds, and no i nforma-
ti on i s avai l abl e on the exi stence of these compounds
i n human bl ood or ti ssues. However, both caffei c and
ferul i c aci d are thought to be absorbed, at l east i n part,
by humans because thei r metabol i tes have been de-
tected i n human uri ne (Jacobson et al ., 1983).
CONCLUSI ONS
Prunes and prune jui ce were characteri zed by hi gh
concentrati ons of hydroxyci nnami c aci ds, especi al l y
neochl orogeni c aci d. The processi ng of prunes from
fresh pl ums degraded phenol i c compounds. Approxi -
matel y hal f of the fl avonol s and hal f of the hydroxyci n-
namates were degraded after commerci al processi ng.
Furthermore, anthocyani ns and fl avan-3-ol s had been
compl etel y degraded after processi ng. Conversel y, the
processi ng of prune jui ce from prunes di d not appear to
si gni fi cantl y degrade phenol i c compounds.
The amount of phenol i cs i n one servi ng of prune jui ce
(240 mL) i s 106 mg. Thi s val ue i s hi gher than the
amount i n one servi ng of prunes (42 g), whi ch i s 73 mg.
Prunes and prune jui ce contai n hi gher l evel s of phenol i c
compounds than many other frui ts, and commerci al
jui ces and dai l y consumpti on of ei ther of these products
woul d i ncrease the di etary i ntake of hydroxyci nnamates.
The prune extracts, as wel l as neochl orogeni c aci d and
chl orogeni c aci d, the two predomi nant phenol i c com-
pounds contai ned i n prunes, were anti oxi dants toward
i sol ated human LDL. I f these compounds have si mi l ar
acti vi ti es i n vi vo, consumpti on of prunes woul d be a good
source of di etary anti oxi dants. Hydroxyci nnamates are
abundant i n many foods, and the rol e of these com-
pounds i n human nutri ti on requi res further research.
ACKNOWLEDGMENT
We are grateful to Murray I sman (Department of
Pl ant Sci ences, Uni versi ty of Bri ti sh Col umbi a, Van-
couver) for the donati on of an authenti c sampl e of
neochl orogeni c aci d. We thank Edwi n Frankel , Debra
Pearson, and Rosemary Wal zem for hel p wi th the LDL
experi ments.
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Recei ved for revi ew September 26, 1997. Revi sed manuscri pt
recei ved January 26, 1998. Accepted January 27, 1998. We
thank the Prune Advi sory Board for fi nanci al support and the
suppl y of prunes and prune jui ce throughout thi s project.
JF970831X
1252 J. Agric. Food Chem., Vol. 46, No. 4, 1998 Donovan et al.