A Standardized Extract of the Fruit of Ziziphus jujuba (Jujube) Induces


Feb 12, 2014 - The fruit of Ziziphus jujuba Mill., known as Chinese date or jujube, is consumed as a health supplement worldwide. To study the role of...
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A Standardized Extract of the Fruit of Ziziphus jujuba (Jujube) Induces Neuronal Differentiation of Cultured PC12 Cells: A Signaling Mediated by Protein Kinase A Jianping Chen, Maitinuer Maiwulanjiang, Kelly Y. C. Lam, Wendy L. Zhang, Janis Y. X. Zhan, Candy T. W. Lam, Sherry L. Xu, Kevin Y. Zhu, Ping Yao, David T. W. Lau, Tina T. X. Dong, and Karl W. K. Tsim* Division of Life Science and Center for Chinese Medicine, The Hong Kong University of Science and Technology, Hong Kong, China S Supporting Information *

ABSTRACT: The fruit of Ziziphus jujuba Mill., known as Chinese date or jujube, is consumed as a health supplement worldwide. To study the role of jujube in brain benefits, its effects on neuronal differentiation of PC12 cells were studied. Application of jujube water extract induced neurite outgrowth of PC12 cells, >25% of which were differentiated; this effect was similar to that of nerve growth factor. In parallel, the expressions of neurofilaments (NFs) in jujube-treated cultures showed a dose-dependent increase, with the highest inductions by ∼150% for NF68 and NF160 and by ∼100% for NF200. Application of H89, a protein kinase A inhibitor, attenuated jujube-induced neurite outgrowth of the cultures. Besides, using jujube extract induced the phosphorylation of cAMP responsive element binding protein on PC12 cells, which was blocked by H89. These results support the use of jujube as a food supplement for the prevention of neurodegenerative diseases in which neurotrophin deficiency is involved. KEYWORDS: jujube, Ziziphus jujuba Mill., neuronal differentiation, neurite outgrowth, neurofilament expression, PKA signaling



INTRODUCTION In the pathophysiology of neurodegenerative disorders, the levels of neurotrophic factors are altered. Their decreased levels result in malfunction of development, survival, and functional maintenance of neuronal cells.1 Currently, there are no effective medicines in controlling neurodegenerative diseases, and using neurotrophic factor is thought to be one of the potential therapeutic approaches.2 However, it is difficult to deliver neurotrophins, that is, nerve grow factor (NGF) into the brain, as the large proteins hardly pass the blood−brain barrier.3 Therefore, a search for neurotrophic factor-stimulating agents with small molecules is needed. Jujube is the fruit of Ziziphus jujuba Mill., commonly known as Chinese date or red date. In the ancient Chinese book on herbal medicine Huangdi Neijing (475−221 BC), the fruit of Z. jujuba was considered as one of the five most valuable fruits in China. In addition, jujube was identified as one of the superior herbal medicines in Shennong Bencao Jing (300 BC−200 AD). Traditionally, one of the main benefits of jujube was considered to benefit our brain by calming the mind and improving the quality of sleep, as such prolonging one’s life-span. Current nutrition research has indicated that jujube contains a variety of nutrients, including carbohydrates, minerals, vitamins, and amino acids.4,5 Furthermore, pharmacological results have revealed that the fruit possesses antioxidant activities,6,7 immunological activities,8−12 and potential sedative effects.13,14 Here, we speculated that jujube might possess multitarget small molecules that could pass the blood−brain barrier in achieving its traditional brain benefits. © 2014 American Chemical Society

In a previous study, we have chemically standardized the jujube extract and identified its protective effect on PC12 cells against oxidative insult.15 Here, we extend our effort in evaluating the brain benefits of jujube. The neurotrophic properties of jujube in inducing neurite outgrowth and neurofilament expression were determined. In addition, the possible role of the protein kinase A (PKA)−cAMP responsive element binding protein (CREB) signaling pathway in the induction of neuronal differentiation was also revealed.



MATERIALS AND METHODS

Preparation of Standardized Jujube Extract. The fruits of Z. jujuba from Hebei, China, were collected in November 2012. The plant materials were authenticated by Dr. Tina Dong on the basis of their morphological characteristics. A voucher specimen (12-11-A) is kept at the Center for Chinese Medicine, The Hong Kong University of Science and Technology. After the kernel had been removed and the fruit had been chopped into slices, the fruits were dried at 40 °C for 1 week. The water content of dried fruits was 90%, the culture medium was changed to DMEM without serum over 5 h. The cells were treated with jujube extract or forskolin at different time points in the absence or presence of H89 (0, 5, 10, and 20 min). Then, the cells were harvested, digested with 200 μL of 2× direct lysis buffer, and boiled for 10 min before an 8% gel electrophrosesis was performed. Western Blot Analysis. Following the electrophoresis, proteins that were separated on an 8% SDS-PAGE were transferred to the nitrocellulose membrane, using a Mini Trans-Blot cell at 40 V and 0.1 A for 16 h in 1× transfer buffer containing 24 mM Tris, 192 mM glycine, 15% ethanol, and 0.1% SDS. Transfer and equal loading of the samples were confirmed by staining with Ponceau-S. The nitrocellulose was blocked with 5% fat-free milk in Tris-buffer saline/0.1% Tween 20 (TBS-T) and then incubated in the primary antibody diluted in 2.5% fat-free milk in TBS-T overnight in a cold room (4 °C). The primary antibodies used were antineurofilament 200 (NF200; Sigma, St. Louis, MO, USA), anti-NF160 (Sigma), antiNF68 (Sigma), antiphospho-CREB (Cell Signaling Technology, Beverly, MA, USA), antitotal CREB (Cell Signaling Technology), and antiglyceraldehyde 3-phosphate dehydrogenase (GAPDH; Abcam Ltd., Cambridge, UK). Following that, the nitrocellulose was rinsed with TBS-T and incubated for 2 h at room temperature in horseradish peroxidase (HRP)-conjugated anti-mouse secondary antibody (Invitrogen) diluted in the 2.5% fat-free milk in TBS-T. After intensive

washing with TBS-T, the immune complexes were visualized using the enhanced chemiluminescence (ECL) method (GE Healthcare, Piscateway, NJ, USA). The intensities of the bands in the control and different samples running on the same gel and under strictly standardized ECL conditions were compared on an image analyzer, using a calibration plot constructed from a parallel gel with serial dilutions of one of the samples. Statistical Analysis. Individual data were expressed as the mean ± standard error of the mean (SEM). Statistical tests were performed with t test (version 13.0, SPSS, IBM Corp., Armonk, NY, USA). Statistically significant changes were classified as significant (∗) when p < 0.05, more significant (∗∗) when p < 0.01, and highly significant (∗∗∗) when p < 0.001.



RESULTS Jujube Induces the Neurite Outgrowth of PC12 Cells. Before the application of jujube extract onto cell cultures, the water extract was chemically standardized.15 The amounts of nucleobases, including uracil, xanthine, hypoxanthine, guanine, and/or adenine in water extract, should be no less than 80 μg/g of dried extract. The amounts of nucleosides, including cytidine, uridine, and/or guanosine in jujube extract should be no less than 150 μg/g. The amounts of cyclic nucleotides, including cGMP and cAMP, in the extract should be no less than 100 μg/g. The amounts of flavonoids, including quercetin 3-O-rutinoside, quercetin 3-O-galactoside, quercetin 3-O-β-Dglucoside, and kaempferol 3-O-rutinoside, in jujube extract should be no less than 35 μg/g. In addition, the extraction efficiency of the extract was 77.3 ± 0.1% (w/w, mean ± SEM, n = 4). PC12 cells are a widely employed model for the study of neuronal differentiation.18 To investigate the efficacies of jujube on PC12 differentiation, water extract of jujube was applied onto cultured PC12 cells for 72 h before examination under microscope. As shown in Figure 1A, the water extract of jujube induced neurite outgrowth of PC12 cells. To quantify the change of morphology, 10 areas in the cultures were randomly selected, and then the number of differentiated cells and neurite length were calculated. The results indicated that after the treatment with water extract on PC12 cells for 3 days, the differentiated cells were counted to about 25% of the total cells in cultures (Figure 1B, upper panel). The length of neurite was also measured. The number of cells possessing neurite length at 15−30 μm was more significantly increased in water extracttreated cells (Figure 1B, lower panel). In addition to the morphological exploration, the differentiation could also be evaluated by a biochemical analysis in determining the expression of neurofilament that is the neuronal cell-specific cytoskeleton proteins, including NF68 (at ∼68 kDa), NF160 (at ∼160 kDa), and NF200 (at ∼200 kDa). NGF served as positive control, which could induce the expression of neurofilament in a dose-dependent manner (Supporting Information Figure S1). As for jujube water extract, the treatment of 72 h at various concentrations (0.75, 1.5, and 3.0 mg/mL) induced the expressions of NF68, NF160, and NF200, with the highest induction by ∼150, 150, and 100%, respectively (Figure 2). PKA Signaling Mediates Jujube-Induced Neuronal Differentiation on PC12 Cells. An activation of the cAMP pathway is essential for neuronal differentiation on PC12 cells.19 Thus, the potency of jujube extract in stimulating neurite outgrowth and neurofilament expression via the cAMP pathway was investigated. Forskolin, a CREB phosphorylation inducer, was reported to induce neurite outgrowth and protein 1891

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Figure 2. Jujube induces the expression of neurofilament on cultured PC12 cells. (A) Cultured PC12 cells were treated with jujube water extracts (0.75−3.0 mg/mL) for 72 h. The cell lysates were collected to determine the expressions of NF68, NF160, and NF200. NGF (50 ng/ mL) served as the positive control. GAPDH served as loading control. (B) Quantification plot is shown in histograms. Values are expressed as the percentage of increase to basal reading (untreated culture), mean ± SEM, n = 5.

Figure 1. Jujube induces neurite outgrowth in cultured PC12 cells. (A) Jujube water extract (2.0 mg/mL) was applied into cultured PC12 cells for 72 h, with fresh medium or jujube extract every 24 h. NGF (50 ng/ mL) served as the positive control. Cells were fixed with ice-cold 4% paraformaldehyde, and then the neurite outgrowth was examined under microscope. Representative images are shown, n = 5. Bar = 20 μm. Arrowheads indicate cell neurite. (B) To quantify the differentiation effect, the percentage of differentiated cell numbers (upper panel) and length of neurite (lower panel) were counted as described under Materials and Methods. Data are expressed as the percentage of cells in 100 counted cells, mean ± SEM, n = 5. Statistical comparison was made with the control; (∗) p < 0.05; (∗∗) p < 0.01.

were also attenuated by H89 (Figure 4). The blockage did not apply to the induction by NGF. Besides, the application of H89 did not fully abolish the jujube effect in both cases of neurite outgrowth and neurofilament expression. The nuclear transcription factor CREB has been welldocumented to play a crucial role in neuronal differentiation on PC12 cells.21 The phosphorylated CREB recruiting the coactivator CREB-binding protein allows its binding at the promoter and further stimulates target gene transcription that leads to neuronal differentiation. Therefore, the role of jujube in inducing the phosphorylation of CREB was investigated. As indicated in Figure 5A, the jujube extract could induce the phosphorylation of CREB at ∼43 kDa, and its inductive role could be fully abolished by H89 (Figure 5B). On the basis of the aforementioned studies, the jujube extract has been demonstrated to induce neuronal differentiation in terms of neurite outgrowth and neurofilament expression. Still it is less known which active component accounts for the neuronal differentiation. On the one hand, cAMP was found in jujube.22 Interestingly, its content was determined to be much higher in comparison with all other horticultural fruits, that is,

phosphorylation on PC12 cells. The effect of forskolin could be inhibited by H89, a cyclic AMP-dependent20 and a marketed selective PKA inhibitor. To explore the possible mechanism of jujube-induced neuronal differentiation, PC12 cells were pretreated with H89 for 3 h. Then, the culture was treated with jujube extract for 72 h. Their effects on neurite outgrowth and neurofilament expression were evaluated. The forskolininduced neurite outgrowth and neurofilament expression were inhibited by H89 (Figures 3 and 4). Similarly, the jujubeinduced neurite outgrowth could be blocked by H89 (Figure 3). In addition, neurofilament expressions, induced by jujube, 1892

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Figure 3. Inhibition of PKA signaling attenuates jujube-induced neurite outgrowth on cultured PC12 cells. Cultured PC12 cells were pretreated with or without H89 (5 μM; a PKA inhibitor) for 3 h and then treated with forskolin (10 μM), NGF (50 ng/mL), and jujube extract (2.0 mg/mL) for 72 h, with fresh medium or extract every 24 h. Cells were fixed with ice-cold 4% paraformaldehyde, and then the neurite outgrowth was examined under a microscope. To quantify the differentiation effect, the percentage of differentiated cell numbers (A) and length of neurite (B) were counted as described in Figure 1. Data are expressed as the percentage of cells in 100 counted cells, mean ± SEM, n = 5. Statistical comparison was made with the H89-treated group; (∗) p < 0.05; (∗∗) p < 0.01.

apple and pear.15,23,24 On the other hand, it is well-known that the increase in cAMP level could activate PKA and subsequently phosphorylate CREB. Thus, we speculated that the cAMP in the jujube extract might contribute to its effect on neuronal differentiation. Here, cAMP at 2 μM (approximately the amount in 2 mg/mL extract) was applied to PC12 cells. As shown in Figures 6 and 7, it could also induce both neurite outgrowth and neurofilament expression on cultured PC12 cells. In comparison with cAMP, the effect of jujube extract was better than that of cAMP in inducing neurite outgrowth, especially in neurite length between 15 and 30 μm (Figure 6). In addition, other possible ingredients including cGMP and a flavonoid mixture containing (−)-catechin, procyanidin B2, (−)-epicatechin, quercetin 3-O-galactoside, quercetin 3-Orutinoside, quercetin 3-O-β-D-glucoside, and kaempferol 3-Orutinoside (approximately the amount in 2 mg/mL extract)

Figure 4. Inhibition of PKA signaling suppresses jujube-induced neurofilament expression on cultured PC12 cells. (A) Cultured PC12 cells were pretreated with or without H89 (5 μM; a PKA inhibitor) for 3 h and then treated with forskolin (10 μM), NGF (50 ng/mL), and jujube extract (2.0 mg/mL) for 72 h. The cell lysates were collected to determine the expressions of NF68, NF160, and NF200. GAPDH served as loading control. (B) Quantification plot (NF68, NF160, and NF200) is shown in histograms. Data are expressed as the fold of change (× Basal) against the control (no treatment; set as 1), mean ± SEM, n = 5. Statistical comparison was made with the H89-treated group; (∗) p < 0.05; (∗∗) p < 0.01.

were also investigated, yet no significant effect was observed (Figure 6). The jujube extract performed better effect on the expression of NF200 than that of cAMP, whereas cAMP possessed better activity in the expression of NF160 than jujube 1893

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Figure 6. cAMP found in jujube induces neurite outgrowth in cultured PC12 cells. Jujube extract (2 mg/mL), cAMP at 2 μM, cGMP at 1.2 μM, or flavonoid mixture including (−)-catechin (52 nM), procyanidin B2 (49 nM), (−)-epicatechin (40 nM), quercetin 3-Ogalactoside (2 nM), quercetin 3-O-rutinoside (157 nM), quercetin 3O-β-D-glucoside (6 nM) and kaempferol 3-O-rutinoside (4 nM) was applied into cultured PC12 cells for 72 h, with fresh medium or drugs every 24 h. The concentrations of cAMP, cGMP, and flavonoids were the same as in 2 mg/mL of jujube extract. NGF (50 ng/mL) served as the positive control. Cells were fixed with ice-cold 4% paraformaldehyde, and then the neurite outgrowth was examined under a microscope. To quantify the differentiation effect, the percentage of differentiated cell numbers (A) and length of neurite (B) were counted as described under Materials and Methods. Data are expressed as the percentage of cells in 100 counted cells, mean ± SEM, n = 5. Statistical comparison was made between extract and cAMP; (∗) p < 0.05.

Figure 5. Jujube extract promotes CREB phosphorylation on PC12 cells. (A) Cultured PC12 cells were serum starved over 5 h before treatment with jujube (2 mg/mL) or forskolin (10 μM) for different times. Total CREB and phosphorylated CREB (both at ∼43 kDa) were revealed by using specific antibodies. (B) Cultured PC12 cells, serum starved for over 5 h, were pretreated with or without H89 (5 μM; a PKA inhibitor) for 3 h prior to treatment with forskolin (10 μM) and jujube (2.0 mg/mL) for 10 min. Total CREB and phosphorylated CREB were revealed by using specific antibodies (upper panel). Quantification plot is shown in histograms (lower panel). Data are expressed as the fold of change (× Basal) against the control (no treatment; set as 1), mean ± SEM, n = 5. Statistical comparison was made with the H89-treated group; (∗) p < 0.05; (∗∗) p < 0.01.



DISCUSSION In the developing neuron, neuronal differentiation is a vital and first stage of development, in which the axon and neurite are promoted to prepare for synapse formation. Recently, morphological observation and biochemical detection were considered as the main approaches to evaluate neuronal differentiation. Here, we found that treatment with jujube extract in cultured PC12 cells could robustly stimulate the expression levels of NF68 and NF160. Both NF68 and NF160 are protein markers for the early stage of the differentiation. In

extract. No significant difference was found on NF68 between the extract and cAMP (Figure 7). 1894

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ment of PKA−CREB signaling in jujube-induced neuronal differentiation on PC12 cells. As reported previously, the water extract contained a higher amount of cyclic nucleotides, as well as their derivatives,15 which could be one of the crucial ingredients in contributing to the jujube-induced beneficial effect. In addition, cyclic nucleotides and their derivatives also have been reported to participate in the regulation and modulation of numerous physiological processes in the human body.28−32 In parallel, 20 of these chemicals have been identified from jujube.33 Among many of these chemicals, cAMP showed a relatively high amount in jujube, and interestingly its content was much higher than in other horticultural fruits, that is, apple and pear.15,22−24 In line with this notion, the amount of cAMP at approximately the amount contained in 2 mg/mL of jujube extract possessed neuronal differentiation, that is, neurite outgrowth and neurofilament expression. This strongly suggested that jujube cAMP played a role in neuronal differentiation, at least in cultured PC12 cells. In support of this hypothesis, a study revealed that the jujube extract could increase the level of cAMP in the plasma and hippocampus of jujube-treated animals.34 However, the amount of cAMP within jujube did not fully account for its functions, as the jujube extract showed better neuronal differentiation function when compared to the same amount of cAMP. Thus, other active ingredients of jujube should not be eliminated. Flavonoids have been reported to possess biological activities in promoting neuronal differentiation.17 The flavonoids within jujube, including (−)-catechin, (−)-epicatechin, kaempferol 3O-rutinoside, and quercetin 3-O-rutinoside, were also included in this screening study, yet no effect was found among them, which suggested that flavonoids were not the active ingredients for jujube-induced neuronal differentiation. Furthermore, the water extract of jujube was found to contain a higher amount of cGMP; however, it did not induce differentiation. Thus, the effect of other possible ingredients in jujube should not be eliminated. We speculated that other chemicals such as saponins might also contribute to the effect of jujube, which will be investigated in the future.

Figure 7. cAMP found in jujube induces the expression of neurofilament on cultured PC12 cells. (A) Cultured PC12 cells were treated with jujube extract (2 mg/mL) and cAMP (2 μM) for 72 h. The cell lysates were collected to determine the expressions of NF68, NF160, and NF200. NGF (50 ng/mL) served as the positive control. GAPDH served as loading control. (B) Quantification plot is shown in histograms. Data are expressed as the fold of change (× Basal) against the control (no treatment; set as 1), mean ± SEM, n = 5. Statistical comparison was made between extract and cAMP; (∗) p < 0.05.

contrast, the expression of NF200, a marker for the late stage of neuron differentiation,24 was also altered but to a lesser extent. Thus, the involvement of jujube in neuronal differentiation could be mainly at the early stage, but might not fully support the entire differentiation process at late stage.17 In addition to neurofilament expression, the jujube-induced neurite outgrowth on PC12 cells was also observed morphologically, which was in line with the role of neurofilaments being the structural components of differentiated neurons.25 In the cases of other neurite-inducing agents, however, the robust inductive effect on neurofilament expression was not consistent with neurite outgrowth, suggesting that the neurofilament expression and the neurite outgrowth could be two independent events.17 An increase of cAMP level activates PKA, followed by subsequent CREB phosphorylation, and the transcription factor binding on the promoter finally triggers target gene expression and leads to neurite outgrowth.26 Here, we found that the inhibition of PKA signaling by H89 suppressed the jujubeinduced neuronal differentiation, but not that of NGF-treated cultures. Our study was in good agreement with a previous report that NGF-induced neurite outgrowth could not be blocked by H89.20 This implied that the differentiation mechanisms could be different between the inductions triggered by jujube and NGF.27 Further study also revealed that jujube could also promote the phosphorylation of CREB. The aforementioned studies therefore elucidated the involve-



ASSOCIATED CONTENT

S Supporting Information *

Dose-dependent effect of NGF in neuronal differentiation on cultured PC12 cells. (A) Cultured PC12 cells were treated with NGF (0.5−50 ng/mL) for 72 h. The cell lysates were collected to determine the expressions of NF68, NF160, and NF200. GAPDH served as loading control. (B) Quantification plot (NF68, NF160 and NF200) is shown in histograms. Data are expressed as the fold of change (× Basal) against the control (no treatment; set as 1), mean ± SEM, n = 4 (Figure S1). This material is available free of charge via the Internet at http:// pubs.acs.org.



AUTHOR INFORMATION

Corresponding Author

*(K.W.K.T.) Mail: Division of Life Science and Center for Chinese Medicine, The Hong Kong University of Science and Technology, Clear Water Bay Road, Hong Kong, China. Phone: +852-2358 7332. Fax: +852-2358 1559. E-mail: [email protected] 1895

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Funding

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This research was supported by the Hong Kong Research Grants Council Theme-based Research Scheme (T13-607/ 12R), GRF (661110, 662911, 663012, 662713), and the Foundation of the Awareness of Nature (TAON12SC01) to K.W.K.T. J.C. received a scholarship from the Hong Kong Chiu Chow Chamber of Commerce. Notes

The authors declare no competing financial interest.



ABBREVIATIONS USED



REFERENCES

PKA, protein kinase A; H89, N-[2-(p-bromocinnamylaminl)ethyl]-5-isoquinolinesulfonamide; cAMP, adenosine 3′,5′-cyclic monophosphate; cGMP, guanosine 3′,5′-cyclic monophosphate; CREB, cAMP responsive element binding protein; SDS-PAGE, sodium dodecyl sulfate−polyacrylamide gel electrophrosesis; NFs, neurofilaments; NGF, nerve growth factor; GAPDH, glyceraldehyde 3-phosphate dehydrogenase

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