PROTECTIVE EFFECTS OF PHENOLICS FROM JUJUBE (ZIZIPHUS JUJUBA) LEAF AGAINST H2O2-INDUCED OXIDATIVE STRESS IN NEURONAL PC-12 CELLS - New Century ...

 
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CURRENT TOPICS IN NUTRACEUTICAL RESEARCH Vol. 16, No.2, pp. 129-138, 2018
                                              ISSN 1540-7535 print, Copyright © 2018 by New Century Health Publishers, LLC
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Research Article

             PROTECTIVE EFFECTS OF PHENOLICS FROM JUJUBE (ZIZIPHUS JUJUBA)
           LEAF AGAINST H2O2-INDUCED OXIDATIVE STRESS IN NEURONAL PC-12 CELLS

       1
        Jun Ho Jung, 1Young Min Chi, 2Chang-Ho Jeong, 2Jong-Kwang Lee,3Young-Il Bae and 4Sun Jin Hur

  Division of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul 02841, Korea; 2JK
   1

 Nutra Co. Ltd., #101-601, Hanwha Ggumegreen bldg, 97 Hyowon-ro, Suwon-si, 442-835, Korea; 3Jinju Bioindustry
     Foundation, 001 Wolasanro, Munsaneup, Jinju-si, 660-844, Korea; and 4Department of Animal Science and
 Technology, Chung-Ang University, 4726 Seodong-daero, Daedeok-myeon, Anseong-si, Gyeonggi-do 17546, Korea

                                  [Received October 29, 2017; Accepted January 3, 2018]

                                             [Communicated by Prof. Zhiling Yu]

ABSTRACT: We aimed to determine the antioxidant and                INTRODUCTION
neuronal cell protective effects of jujube (Ziziphus jujuba)          Patients with Alzheimer’s disease (AD) have a high sensitivity to
leaf solvent fractions. The butanol fraction from jujube           (reactive oxygen species) ROS. Accumulated intracellular H2O2,
showed potent antioxidant activity in each assay, and its          which is an ROS, causes lipid peroxidation of cell membranes
ABTS radical scavenging effect, FRAP, and MDA inhibition           and triggers apoptotic cell death via the activation of caspases
increased in a dose-dependent manner. Following H2O2               (Chun et al., 2005; Chang et al., 2008). AD is one of the most
treatment, intracellular ROS accumulation significantly            serious threats to human health in aging societies of developed
reduced when butanol fraction from jujube leaf was present         countries. In particular, AD is a major neurodegenerative disease
in the PC12 cell media. In the MTT cell viability assay, the       and is characterized by loss of memory and cognition. Around
butanol fraction showed a protective effect against H2O2-          18 million people in the world suffer from AD, and this number
induced neurotoxicity and inhibited LDH release into the           is expected to rise to 34 million by 2025 (Hong et al., 2007).
medium (7.13-43.89%). Total phenols of the three solvent           Many studies have demonstrated that the brains of patients with
fractions were 72.50 (chloroform), 297.18 (butanol),               AD are subjected to increased oxidative stress due to free radical
and 17.93 (water) mg/g GAE. The predominant phenolic               damage (Murakami et al., 2000).
compounds in jujube leaves are rutin (57.07 mg/100 g) and             Many phenolics protect neuronal cells from oxidative stress
quercetin (9.27 mg/100 g). These data suggest that butanol         induced by ROS or amyloid-β (Aβ) protein, and this may be
fraction from jujube leaf including phenolics may be useful        related to the pathogenesis of AD (Om et al., 2008; Park et
natural antioxidants to reduce the risk of neurodegenerative       al., 2001). Some phytochemicals from natural plant sources,
diseases including Alzheimer’s disease.                            including fruits and vegetables, may reduce the risk of AD because
                                                                   of their antioxidant properties that reduce oxidative insults
KEY WORDS: Antioxidants, Cell viability, Jujube leaf,              (Pinzo´n-Arango et al., 2009). Epidemiological observation has
Neuronal protective effect                                         revealed that increased antioxidant uptake is inversely related to
                                                                   the risk of incidence of AD (Psotová et al., 2003). Antioxidants
Corresponding Author: Sun Jin Hur, Department of Animal            are vital substances that have the ability to protect the body
Science and Technology, Chung-Ang University, 4726                 from damage caused by free radical-induced oxidative stress
Seodong-daero, Daedeok-myeon, Anseong-si, Gyeonggi                 (Ozsoy et al., 2008). There is an increasing interest in natural
17456, Republic of Korea. Tel.: + 82 31 670 4673; Fax: + 82        antioxidants (e.g., polyphenols) present in medicinal and dietary
31 670 3108. E-mail address: hursj@cau.ac.kr                       plants, which might help prevent oxidative damage (Silva et al.,
130 Neuronal cell protective effect of jujube leaf

2005). Phenolics have the ideal structural chemistry for free          diacetate     (DCF-DA),       3-[4,5-dimethythiazol-2-yl]-2,5-
radical-scavenging activity and have been shown to be more             diphenyl tetrazolium bromide (MTT) assay kit, and the lactate
effective antioxidants than tocopherols and ascorbate in in vitro      dehydrogenase (LDH) assay kit were purchased from Sigma
studies. A few phenolics have also been reported to prevent a          Chemical Co. (St. Louis, MO, USA). All solvents used were of
decrease in the activities of the antioxidant enzymes superoxide       analytical grade and purchased from Sigma Chemical Co. (St.
dismutase (SOD) and glutathione peroxidase (GSH-Px), and               Louis, MO, USA). RPMI 1640 medium and fetal bovine serum
to prevent a significant depletion of glutathione (GSH) (Yu et         were obtained from Gibco BRL (Grand Island, NY, USA).
al., 2009). Therefore, recognition of the importance and role
of non-nutrient compounds, particularly phenolics as natural           Plant material
antioxidants, has greatly increased (Hagerman et al., 1998). To          Jujube leaves were collected from Jinju in Korea in September
find new natural sources of physiologically active compounds,          2015, and were authenticated by Institute of Agriculture and
we studied the antioxidant and protective effects of jujube leaves     Life Sciences, Gyeongsang National University where voucher
on neuronal cells. Jujube (Ziziphus jujuba) is a tree belonging        specimens were maintatined. These samples were stored at -20
to the Rhamnaceae family. It has been domesticated worldwide           ℃ until use.
and is found primarily in the subtropical regions of Asia and
America (Mahajan et al., 2009). Jujube is consumed in both             Jujube leaf extracts
its fresh and dried forms and is used in a variety of products            Each solvent fraction of freeze-dried jujube leaf was obtained
including breads, cakes, candies, and compotes. In Korea, jujube       in the following manner. Powdered jujube leaves (500 g)
is used as an ingredient in porridge, rice cakes, and Yakbap (a        were put in suspension and extracted using 500 ml of 80%
sweet Korean dish whose name literally means medicinal rice).          ethanol at 80 ℃ for 3 h. The extracts were filtered through
In Korea, jujube has recently come into prominent use as an            Whatman No. 2 filter paper (Whatman International Limited,
ingredient in processed foods such as drinks and tea granules          Kent, England) and evaporated to dryness. The dried material
(Choi et al., 2015). Furthermore, jujube is also used as traditional   was dissolved in 200 mL of distilled water. The solution
medicine in Korea (Kwon et al.,1993) and is reported to possess        was consecutively portioned in a separation funnel with the
numerous health-promoting benefits such as antioxidant activity        equivalent amount of chloroform, butanol, and water. Each
(Kim et al., 2005), anticancer effects (Vahedi et al., 2008),          fraction was concentrated in a vacuum evaporator (Eyela
and hepatoprotective effects (Lee et al., 1995). Although it           NE, Tokyo Rikakikai Co., Ltd., Tokyo, Japan) at 40 ℃. The
has already been demonstrated that the jujube fruit and seed           water filtrate was frozen and lyophilized. The fractions were
contain phenolic compounds, little is known about the effect           placed in a glass bottle and stored at -20 ℃ until use. The
of jujube leaves on antioxidant activities and oxidative stress-       lyophilized fractions were dissolved in 10% DMSO to obtain
induced neurotoxicity. Because the antioxidant activities and          a concentration of 1000 μg/mL.
protective effects of jujube leaves on neuronal cells have not been
previously reported, the objectives of this study were to assess       ABTS radical scavenging activity
the various antioxidant activities and protective effects of jujube       To test the free radical scavenging ability of the three jujube
leaf extract on neuronal cells and identify the active compounds       leaf extracts, a 2,2’-azino-bis(3-ethylbenzthiazoline-6-sulfonic
contained in jujube leaves that are usually discarded during           acid) assay was used. ABTS was dissolved in water to obtain
frequent pruning to obtain information that might enable their         a stock concentration of 7 mM. ABTS working solution was
utilization in functional foods. This is in keeping with efforts to    prepared by allowing the ABTS stock solution to react with 2.45
maintain a balance between the conservation of biodiversity and        mM potassium persulfate (final concentration) and further
encouragement of controlled exploitation of plant resources for        allowing the mixture to stand in the dark at room temperature
economic gains, specifically in the field of biopharming, in which     for 12-16 h before use. For the study of leaf extract sample,
the waste of valuable natural resources should be minimized.           the ABTS stock solution was diluted with 5 mM phosphate-
                                                                       buffered saline (pH 7.4) to obtain an absorbance of 0.70 at
MATERIAL AND METHODS                                                   734 nm. After the addition of 980 μL of diluted ABTS to 20
Chemicals                                                              μL of the sample, the absorbance reading was taken 5 min after
                                                                       the initial mixing (Jeong et al., 2010). This antioxidant activity
   Folin-Ciocalteu’s    phenol       reagent, 2,2’-azino-bis(3-        is stated as percent ABTS scavenging that is calculated as:
ethylbenzthiazoline-6-sulfonic acid (ABTS), potassium                  % ABTS scavenging activity = [(control absorbance – sample
persulfate, 2,4,6-tripyridyl-S-triazine (TPTZ), trichloroacetic        absorbance) / (control absorbance)] × 100
acid (TCA), thiobarbituric acid (TBA), vitamin C,
α-tocopherol, catechin, 2-[4-(2-hydroxyethyl)piperazin-1-yl]           Ferric reducing antioxidant power (FRAP) of jujube leaf
ethanesulfonic acid (HEPES), sodium bicarbonate, penicillin,           extracts
streptomycin, myricetin, quercetin, kaempferol, ferrous sulfate          The FRAP assay used in this study is described in Jeong et al.
(FeSO4), hydrogen peroxide (H2O2), dimethyl sulfoxide                  (2010). Briefly, 1.5 mL of working, prewarmed FRAP reagent
(DMSO), penicillin, streptomycin, 2’,7’-dichlorofluorescein            (10 volumes 300 mM acetate buffer, pH 3.6 + 1 vol of 10 mM
Neuronal cell protective effect of jujube leaf 131

2,4,6-tripyridyl-S-triazine in 40 mM HCl + 1 vol of 20 mM           and if the presence of the jujube leaf butanol fraction had any
FeCl3) at 37 ℃ was mixed with 50 μL of the leaf extract fractions   effect on it. Neuronal PC12 cells were plated at a density of
and standards. This was vortex mixed and the absorbance read        106 cells/well on 96-well plates in 100 μL of RPMI. The cells
at 593 nm against a reagent blank at a predetermined time after     were pre-incubated with the butanol fraction for 48 h before
sample-reagent mixing. The test was performed at 37 ℃ and the       200 μM of H2O2 were added. The cells were treated with or
0-4 minutes reaction time window was used.                          without H2O2 for 2 h. The amount of MTT formazan product
                                                                    was determined by measuring absorbance using a microplate
Malondialdehyde (MDA) assay using mouse brain                       reader (680, Bio-Rad, Tokyo, Japan) at a test wavelength of 570
homogenates                                                         nm and a reference wavelength of 690 nm.
   This assay was carried out using the method described by
Chang et al. (2001). The brains of young adult male Balb/c          Lactate dehydrogenase assay
mice were dissected and homogenized in ice-cold Tris-HCl               Neuronal PC12 cells, treated as described in the above
buffer (20 mM, pH 7.4) to produce a 1/10 homogenate. The            paragraph, were precipitated by centrifugation at 250 × g for
homogenate was centrifuged at 12,000 × g for 15 minutes at          4 min at room temperature, 100 μL of the supernatant were
4 ℃. A 1 mL aliquot of the supernatant was incubated with           transferred into new wells, and lactate dehydrogenase (LDH)
the test leaf extract samples in the presence of 10 μM FeSO4        concentration was measured using the in vitro toxicology assay
and 0.1 mM vitamin C at 37 ℃ for 1 h. The reaction was              kit (TOX-7, Sigma Co., St. Louis, MO, USA). Damage to the
terminated by the addition of 1 mL TCA (28%, w/v) and               plasma membrane was evaluated by measuring the amount of
1.5 mL TBA (1%, w/v) in succession, and the solution was            the intracellular enzyme LDH released into the medium.
then heated at 100 ℃. After 15 min, the absorbance of the
MDA-TBA complex was measured at 532 nm. (+)-Catechin,               Determination of total phenolics
a well-known antioxidant, was used as a positive control. The         Total phenolic content was determined using
inhibition ratio (%) was calculated as follows:                     spectrophotometric analysis (Jeong et al., 2010). Briefly, a 1
                                                                    mL portion of the appropriately diluted extract was added to a
% inhibition = [(control absorbance – sample absorbance) /          25 mL volumetric flask containing 9 mL of deionized distilled
control absorbance] × 100                                           water (ddH2O). A reagent blank using ddH2O was prepared.
                                                                    One mL of Folin-Ciocalteu’s phenol reagent was added to the
Neuronal cell culture                                               mixture and shaken. After 5 min, 10 mL of a 7% Na2CO3
  The PC12 cell line was derived from a transplantable rat          solution was added and the solution was mixed. The mixed
pheochromocytoma. The cells respond reversibly to nerve             solution was then immediately diluted to a volume of 25 mL
growth factor (NGF) by induction of the neuronal phenotype.         with ddH2O and mixed thoroughly. After 90 min at 23 ℃, the
PC12 cells (KCLB 21721, Korea Cell Line Bank, Seoul, Korea)         absorbance was read at 750 nm. The standard curve for total
were propagated in RPMI 1640 medium containing 10% fetal            phenolics was made using gallic acid standard solution (0-100
bovine serum, 25 mM HEPES, 25 mM sodium bicarbonate,                mg/L) following the same procedure as above. Total phenolics in
50 units/mL penicillin, and 100 μg/mL streptomycin.                 each solvent fraction were expressed as milligrams of gallic acid
                                                                    equivalents (mg GAE/g) of the sample.
Measurement of intracellular oxidative stress
   Levels of intracellular reactive oxygen species (ROS) were       Determination of phenolics
measured using the 2’,7’-dichlorofluorescein diacetate (DCF-           Phenolic compounds in the butanol fraction were measured at
DA; fluorescent probe) assay (Heo et al., 2001). Briefly, cells     280 nm using 11 phenolics standard solution using a diode array
(104 cells/well on 96-well) were treated for 10 min with the        UV-visible detector (Agilent 1100 series, Agilent Co., Santa Clara,
indicated concentrations of the butanol fraction (jujube leaf       CA, USA). Separation was achieved with a LiChrospher 100 RP-
extract) or vitamin C. The cells were then treated with or          18 column (250 mm × 4.6 mm id, 5 μm, Merck Co., Darmstadt,
without 200 μM hydrogen peroxide for 2 h. At the end of the         Germany). The elution solvents were (A) 0.01 M-potassium
treatment, cells were incubated in the presence of 50 μM DCF-       phosphate buffer adjusted to pH 3.0 by phosphoric acid, and,
DA in phosphate buffered saline (PBS). Fluorescence was then        (B) methanol. The solvent gradient elution program used was as
quantified using the Tecan SER-NR 94572 fluorometer (Tecan          follows: initial 90% (A), hold for 9.5 min; linear gradient to 68%
Systems Inc., San Jose, CA, USA) using 485 nm excitation and        (A) in 3.5 min; linear gradient to 67% (A) 17 min; linear gradient
530 nm emission filters.                                            to 20% (A) 1 min; linear gradient to 90% (A) 1 min, and hold for
                                                                    10 min. The flow rate was 1.5 ml/min. Phenolics were identified
Cell viability assay                                                by comparison of their retention time (RT) values and UV spectra
   The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-              with those of known standards and quantified by peak areas from
tetrazoliumbromide]-based TOX1 in vitro toxicology assay kit        the chromatograms. All analyses were run in triplicate and mean
(Sigma Co., St. Louis, MO, USA) was used to determine cell          values were calculated. The content of phenolic compounds was
viability in PC12 cells under H2O2-induced oxidative stress         expressed in mg/100 g extract.
132 Neuronal cell protective effect of jujube leaf

Statistical analysis                                                                                                                             the chloroform and water fractions showed low radical-
   All data are expressed as mean ± SD (n = 3). Data from                                                                                        scavenging activity; these were approximately 3-4 times lower
each experimental were analyzed using a one-way analysis of                                                                                      than the activity of the butanol fraction. Fig. 1A shows a steady
variance (ANOVA) and Duncan’s multiple-range test (p
Neuronal cell protective effect of jujube leaf 133

in jujube leaf were the most likely candidate compounds            FIGURE 3. Effect of the butanol fraction of jujube leaves
capable of reducing ferric ions. According to recent reports,      on ROS production in the presence and absence of H2O2
there was a strong correlation between total phenolics and         in PC12 cells. Data are presented as the mean ± SD of 3
antioxidant activity in many plant species (Dasgupta et al.,       independent experiments in triplicate; different letters represent
2004; Dorman et al., 2004). In addition, many phenolics            significant differences between means. *p < 0.05
have shown high levels of antioxidant activity.                                         160
                                                                                                                          a
                                                                                                          a
Inhibitory effect of jujube leaf extract on lipid peroxidation                          140                                     b
    There has been increasing interest in lipid peroxidation                            120                                             c
                                                                                                 d

                                                                 Oxidative stress (%)
because the formation of cytotoxic products such as MDA
                                                                                                                   e                           d
and 4-hydroxynonenal can influence cellular apoptosis and                               100

several human diseases (Sevanian et al., 2000). Therefore,                                                                                             f
                                                                                         80
in this assay, antioxidant activities of three fractions from
jujube leaf on both ferric ion- and vitamin C-induced                                    60
lipid peroxidation on mouse brain homogenates were also
                                                                                         40
confirmed. The results, shown in Fig. 2, reveal that the
three fractions of jujube leaf had inhibitory effects against                            20
lipid peroxidation of the mouse brain homogenates. The
                                                                                          0
inhibitory effects of all fractions against lipid peroxidation                                Control   200 µM   200 µM   12    25     50     100     200
decreased in the following order: butanol fraction>water                                                 H2O2     Vit.C
                                                                                                                               Concenration (µg/mL)
fraction>chloroform fraction. The butanol fraction showed
                                                                 production of amyloid β protein (Aβ). Aβ levels can trigger
excellent suppression activity against lipid peroxidation in
                                                                 cell death through a mechanism involving hydrogen peroxide
mouse brain homogenates. The butanol fraction had a stronger
                                                                 (Behl et al., 1994).
inhibitory effect than (+)-catechin at all concentrations
                                                                     These results suggest that the butanol fraction of jujube
tested and more than 50% of inhibitory activity against lipid
                                                                 leaf has antioxidant activity and may be able to play an
peroxidation was observed at the 25 μg/mL concentration. It
is also noteworthy that (+)-catechin, which has an EC50 value    important role in reducing oxidative stress, an important risk
of 24.08 μg/mL, showed less inhibitory activity than the         factor for neurodegenerative diseases such as AD.
butanol fraction (EC50 value of 19.94 μg /mL). Therefore, the
active compounds in the butanol fraction might be potential      Influence of the jujube leaf butanol fraction on the viability
natural antioxidant supplements for food and pharmaceutical      of neuronal cells treated with H2O2
products. They might also be used to stabilize foods against         Alteration in the mitochondrial permeability transition
oxidative deterioration.                                         pore protein occurs in cells undergoing apoptosis (Salet et
                                                                 al., 1997) and this is related to the release of cytochrome c
Measurement of intracellular oxidative stress                    (Ott et al., 2001). Mitochondria might be one of the main
    To examine the intracellular accumulation of ROS in PC12     targets of oxidative stress causing neuronal cell death. Fig. 4
cells, used as neuronal cell models, 2’,7’-dichlorofluorescein   shows the improved cell viability of PC12 cells under oxidative
diacetate (DCF-DA) was used. The DCF-DA probe, which             stress, measured using an MTT assay, being mainly attributed
is freely permeable across cell membranes, is hydrolyzed by      to bioactive phenolics derived from the jujube leaf butanol
cytosolic esterases to non-fluorescent dichlorofluorescein       fraction. MTT is converted to purple formazan by living
(DCFH). Then, DCFH interacting with ROS is oxidized              cells, in part, because of mitochondrial processes. A significant
to a highly fluorescent substance, 2’,7’-dichlorofluorescein     color difference was observed between control neuronal cells
(DCF). Exposure of PC12 cells to H2O2 for 2 h resulted           with no H2O2 treatment and groups treated with the butanol
in a 139.21% increase of ROS levels compared to controls         fraction at 100-200 μg/mL followed by H2O2 exposure (Fig.
(Fig. 3). Pretreatment of PC12 cells with the butanol            4). The treatment with H2O2 for 2 h decreased the viability of
fraction significantly prevented them from intracellular ROS     PC12 cells up to 65.16% compared to the control (100%).
accumulation in comparison to control PC12 cells that were       At a concentration of 100-200 μg/mL, the butanol fraction
treated with only H2O2.                                          provided effective protection that resulted in an acceptable
    Vitamin C is one of the naturally occurring major            PC12 cell viability against oxidative stress. At 200 μg/mL
nutrients having antioxidant activity (Kim et al., 2002).        concentration of the jujube leaf butanol fraction, the viability
Vitamin C was used as a positive control. Pretreatment with      of PC12 cells significantly increased up to 99.00% that of
200 μM vitamin C of PC12 cells resulted in significantly         the control cells. Therefore, these results also suggest that
lower oxidative stress compared to PC12 cells with H2O2          neuronal cell protection conferred by the butanol fraction of
treatment alone (Fig. 3). Oxidative stress in AD may result      jujube leaf extract is partially due to mitochondrial protective
from aging, energy deficiency, inflammation or excessive         mechanisms.
134 Neuronal cell protective effect of jujube leaf

  FIGURE 4. Protective effect of the butanol fraction of jujube                            FIGURE 5. Inhibitory effect of butanol fraction on LDH
  leaves against H2O2-induced cell death in PC12 cells. PC12                               release in H2O2-treated PC12 cells. Cells, first treated with
  cells were pretreated for 48 h with various concentrations of                            the butanol fraction, were then treated with 200 μM H2O2 for
  the butanol fraction. The cells were then treated with 200 μM                            2 h. The LDH activity in culture supernatants was measured
  H2O2 for 2 h. Cell viability was measured using the MTT assay                            with a colorimetric LDH assay kit. Vitamin C (200 μM) was
  kit. Vitamin C (200 μM) was used as a positive control. Data                             used as a positive control. Data are presented as the mean ±
  are presented as the mean ± SD of 3 independent experiments                              SD of 3 independent experiments in triplicate; different letters
  in triplicate; different letters represent significant differences                       represent significant differences between means. *p < 0.05
  between means. *p < 0.05                                                                                              100
                     120                                                                                                                  a

                                                                                          LDH release into medium (%)
                              a                                                     a
                                                                                                                         80                               b
                     100
                                                                               b
                                                c                       c
                                                                d                                                                                                  c
                      80                                                                                                                                                   d
Cell viability (%)

                                                       e                                                                 60
                                       f                                                                                                                                          e
                      60                                                                                                                                                               f
                                                                                                                         40
                                                                                                                                                   g
                      40
                                                                                                                                 h
                                                                                                                         20
                      20

                       0                                                                                                  0
                           Control   200 µM   200 µM   12       25     50     100   200                                       Control   200 µM   200 µM   12       25     50     100   200
                                      H2O2     Vit.C                                                                                     H2O2
                                                            Concentration (µg/mL)                                                                 Vit.C
                                                                                                                                                               Concentration (µg/mL)

Protective effect of the butanol fraction of jujube leaves                                  the cellular membrane at all the concentrations tested (Fig. 5).
against H2O2-induced membrane damage                                                        After oxidative treatment with H2O2 for 2 h, the amount of
    The neuronal membrane, containing polyunsaturated                                       LDH release from PC12 cells increased to 63.88% compared
fatty acids, is vulnerable to oxidative stress induced by ROS                               to that from the untreated controls. However, the various
such as H2O2. Lipid peroxidation can alter the fluidity of the                              levels of butanol fraction treatment showed protective effects
plasma membrane. The LDH assay provided an estimate of                                      against oxidative stress in neuronal cells and followed a dose-
the percentage of surviving PC12 cells whose cell membranes                                 dependent pattern (Fig. 5). Treatment of PC12 cells with 100-
were intact. The butanol fraction protected the integrity of                                200 μg/mL concentration of the butanol fraction resulted in
FIGURE 6. HPLC chromatogram of standards (A) and butanol fraction of jujube leaves (B). Retention time 19.916 min: rutin,
20.578 min: quercetin.
                                  (A) Standard
Neuronal cell protective effect of jujube leaf 135

                (B) Butanol fraction

TABLE 1. Total phenolics of three fractions and phenolics content of butanol fraction from jujube leaf. The data are
presented as mean ± SD (n=3).
                                                                                                       Content
                                                                             Fractions
                                                                                                     (mg/g GAE)
                                                                           Chloroform                 72.50±0.37
                                Total phenolics                            Butanol                   297.18±1.06
                                                                              Water                   17.93±0.16
                                                                            Retention                   Content
                                                  Compounds
                                                                            time (min)                  (mg/g)
                                           Procatechuic acid                   5.512                        -
                                           Catechin                            7.796                        -
                                           Syringic acid                       9.863                        -
                                           Caffeic acid                       10.021                        -
                                           Vanillic acid                      11.467                        -
                    Phenolics              Epicatechin                        11.853                        -
                                           p-Coumaric acid                    14.032                        -
                                           Ferulic acid                       15.039                        -
                                           Rutin                              18.916                     57.07
                                           Quercitrin                         20.578                      9.27
                                           Quercetin                          22.933                        -
significant protection compared to the group treated with H2O2      fraction may provide an added health benefit by reducing the
only (Fig. 5). This finding suggests that the butanol fraction of   risk of neurodegenerative diseases such as AD.
jujube leaf might attenuate the extent of oxidative stress damage
from H2O2 insult in neuronal PC12 cells and also protect the        Total phenolics of three fractions and individual phenolic
integrity of the cell’s biological membrane. Our results suggest    composition of butanol fraction from jujube leaf
that the phenolics of the butanol fraction might be inhibiting         Phenolic compounds, such as flavonoids, phenolic
neuronal apoptosis, which is the ultimate consequence of all        acid, and tannins are considered major contributors to the
these cellular dysfunctions. Therefore, phenolics of the butanol    antioxidant activity of natural plants. These antioxidants also
136 Neuronal cell protective effect of jujube leaf

possess diverse biological activities, such as anti-inflammatory,      (1994). Hydrogen peroxide mediates amyloid β protein
anti-carcinogenic, and anti-atherosclerotic activities that may        toxicity. Cell, 77: 817-827.
be associated with their antioxidant activity (Chung et al.,
1998). The total phenolics of three fractions from the 80%             Chang, H. J., Choi, E. H. and Chun, H. S. (2008).
ethanol extract of jujube leaf are presented in Table 1. The           Quantitative structure-activity relationship (QSAR) of
butanol fraction had the highest phenolic content (297.18 mg           antioxidative anthocyanidins and their glycosides. Food Science
GAE/g). It is approximately 4-fold more than the phenolic              and Biotechnology, 17: 501-507.
content of the chloroform fraction (72.50 mg GAE/g) and
17-fold more than the phenolic content of the water fraction           Chang, S. T., Wu, J. H., Wang, S. Y., Kang, P. L., Yang, N. S.
(17.93 mg GAE/g).                                                      and Shyur, L. F. (2001). Antioxidant activity of extracts from
    Jujube leaf extract fractions were subjected to further analysis   Acacia confusa bark and heartwood. Journal of Agricultural and
by HPLC. The butanol fraction from jujube leaf contained               Food Chemistry, 49: 3420-3424.
two phenolic compounds. By comparing the retention time
and UV spectra of these compounds to those of standards,               Choi, J., An, X., Lee, B. H., Lee, J. S., Heo, H. J., Kim, T. and
rutin was identified as the main phenolic compound (Fig. 6).           Kim, D. O. (2015). Protective effects of bioactive phenolics
Furthermore, the HPLC results indicated that rutin (57.07              from jujube (Ziziphus jujuba) seeds against H2O2–induced
mg/g) was the predominant phenolic compound in the butanol             oxidative stress in neuronal PC-12 cells. Food Science and
fraction of jujube leaf extract (Fig. 6 and Table 1). Based on         Biotechnology, 24: 2219-2227.
the results of the phenolic composition of the butanol fraction,
we can conclude that these compounds (particularly rutin and           Chun, O. K., Kim, D. O., Smith, N., Schroeder, D., Han,
quercitrin) contribute to the antioxidant and cell protective          J. T. and Lee, C. Y. (2005). Daily consumption of phenolics
effects of jujube leaf in neuronal cells. The results obtained         and total antioxidant capacity from fruit and vegetables in the
from this study are noteworthy, not only with respect to the           American diet. Journal of the Science of Food and Agriculture, 85:
antioxidant and neuronal cell protective effects of jujube leaves      1715-1724.
but also with respect to its rutin content. The activity of jujube
is attributed to these phenolic compounds and specifically rutin.      Chung, K. T., Wong, T. Y., Wei, C. I., Huang, Y. W. and
The main phenolic compound found in onion and buckwheat                Lin, Y. (1998). Tannins and human health: a review. Critical
is rutin. Rutin is a compound of interest because it has it            Reviews in Food Science and Nutrition, 38: 421-464.
has a wide range of biological effects that include including
antioxidant, anticarcinogenic, antimicrobial activities, and anti-     Dasgupta, N. and De, B. (2004). Antioxidant activity of Piper
neurodegenerative effects (Dreosti et al., 1997; Jankun et al.,        betle L. leaf extract in vitro. Food chemistry, 88: 219-224.
1997; Almajano et al., 2008; Heo et al., 2004).
    In this context, the increased cell viability associated with      De-Hong, Y. U., Yong-Ming, B. A. O., Li-Jia, A. N. and
jujube leaf extract may be mainly attributed to rutin, and to          Ming, Y. A. N. G. (2009). Protection of PC12 cells against
a lesser extent, to the other antioxidant phenolics. Finally, our      superoxide-induced damage by isoflavonoids from Astragalus
results verified that compounds in the jujube leaf have very           mongholicus. Biomedical and Environmental Sciences, 22: 50-
strong antioxidant activities, and suggest that the jujube leaf        54.
can be utilized as an effective natural antioxidant source and
chemopreventive agent against neurodegenerative disease                Dorman, H. J. D. and Hiltunen, R. (2004). Fe (III)
such as Alzheimer’s disease. Further studies are needed to             reductive and free radical-scavenging properties of summer
determine the relationship between the specific antioxidant            savory (Satureja hortensis L.) extract and subfractions. Food
and neuroprotection using in vivo tests.                               Chemistry, 88: 193-199.

CONFLICT OF INTEREST DISCLOSURE                                        Dreosti, I. E., Wargovich, M. J. and Yang, C. S. (1997).
   We confirm that there are no known conflicts of interest            Inhibition of carcinogenesis by tea: the evidence from
associated with this publication and there has been no                 experimental studies. Critical Reviews in Food Science &
significant financial support for this work that could have            Nutrition, 37: 761-770.
influenced its outcome.
                                                                       Hagerman, A. E., Riedl, K. M., Jones, G. A., Sovik, K. N.,
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138 Neuronal cell protective effect of jujube leaf
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