Notes
Black pepper (Piper nigrum) oleoresin has a neuroprotective effect on apoptosis induced by activity deprivation
2023 年 29 巻 6 号 p. 567-573
詳細
2023 年 29 巻 6 号 p. 567-573
Neuronal activity plays a key role in the development and maintenance of the central nervous system. In cultured cerebellar granule neurons (CGNs), activity deprivation by treatment with a low concentration of KCl induces neuronal apoptosis. We found that black pepper (Piper nigrum) oleoresin significantly increased the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide signal on activity-deprived CGNs, whereas piperine, a major compound in black pepper extract, did not. Consistently, induction of cleaved caspase-3, a hallmark of apoptosis was inhibited by black pepper oleoresin upon neuronal activity deprivation. These results show that black pepper oleoresin protects CGNs from apoptosis induced by activity deprivation, and suggest that components other than piperine might contribute to this effect.
Piper nigrum, known as black pepper, is one of the major spice plants in the world and is used for a variety of medicinal applications (Ahmad et al., 2012; Butt et al., 2013). Piperine is a major alkaloid found in black pepper, which exhibits pharmacological properties, including antioxidant, anti-tumor, and anti-inflammatory effects (Tiwari et al., 2020; Haq et al., 2021). Piperine inhibits glutamate-induced apoptosis in cultured hippocampal neurons (Fu et al., 2010) as well as in cerebral ischemic injury (Hua et al., 2019). In animal models of Alzheimer's disease, piperine (Chonpathompikunlert et al., 2010) and black pepper extracts (Rajashri et al., 2020; Mostafa et al., 2021) inhibited neurodegeneration and improved memory impairment. In addition to piperine, compounds such as phenolic compounds, alkaloids, flavonoids, carotenoids, and terpenoids are found in black pepper extracts (Parmar et al., 1997; Kapoor et al., 2009; Lee et al., 2020). The neuroprotective function of these compounds other than piperine remains to be clarified.
Neuronal activity plays pivotal roles in neuronal survival and development. Cerebellar granule neurons (CGNs) in culture require membrane depolarization for their survival, and activity deprivation caused by lowering KCl concentration induces remarkable neuronal cell death. Upon activity deprivation, cell cycle factors are reactivated in postmitotic CGNs that activate proapoptotic BCL-2-like proteins (Padmanabhan et al., 1999; Konishi et al., 2002). Reactivation of cell-cycle factors has also been reported in neurodegenerative disorders such as Alzheimer's disease and Parkinson's disease (Frade and Ovejero-Benito 2015). Thus, the system of activity deprivation on CGNs could be a model not only for neuronal death during development, but also for neurodegenerative diseases. In this study, we carried out screening of natural products, and found that black pepper oleoresin, and not piperine alone, can protect CGNs from apoptosis induced by activity deprivation.
Extracts and compounds Black pepper oleoresin (4010001646) and chili oleoresin (4010000036) were purchased from Synthite Industries Private Ltd. (Kerala, India). Piperine (PL0187006) was purchased from Plant Lipids Private Limited (Kerala, India). Capsaicin and dihydrocapsaicin were purified by preparative highperformance liquid chromatography (LC-8A system, Shimadzu Co., Ltd., Kyoto, Japan) of chili oleoresin (CAP1187, Synthite Industries Private Ltd.).
Preparation of neuronal cell cultures Animals were treated according to the institutional ethics guidelines, and experiments were approved by the animal ethics committees of the University of Fukui (Approval No.: R04083). Primary cultures of CGNs were prepared from Jcl:ICR mice (CLEA Japan, Tokyo, Japan) as described previously (Matsumoto et al., 2022). Cerebella collected from a litter of mice at postnatal days 4 to 6 after euthanizing by decapitation were digested with trypsin (TRL, Worthington, Lakewood, NJ). Neurons were plated on cover glass that had been coated with poly-L-ornithine (P2533, Sigma Aldrich, St. Louis, MO), and maintained in Minimal Essential Medium (11090-081, Thermo Fisher Scientific, Waltham, MA) supplemented with 10% calf serum (SH30072.03, Thermo Fisher Scientific), penicillin (100 units/ml, P7794, Sigma-Aldrich), streptomycin (0.1 mg/ml, P9137, Sigma-Aldrich), and glutamine (2 mM, G8540, Sigma-Aldrich). The concentration of KCl in the media was adjusted to 30 mM or 5 mM. To prevent the proliferation of non-neuronal cells, cytosine β-D-arabinofuranoside (10 µM, 034–11,954, Sigma-Aldrich) was added to the culture at 1 day in vitro (DIV). For the experiments shown in Fig. 1, 1 × 105 cells were placed in each well of a 96-well plate. For the experiments shown in Figs. 2 and 3, 5 × 105 cells were spread in each well of a 24-well plate containing cover glasses. For the experiment shown in Fig. 4A, 2 × 104 cells were spread in each well of a flexiPERM micro insert (94.6011.436, Sarstedt, Nümbrecht, Germany) attached to a cover glass. For the experiment shown in Fig. 4C, 4 × 104 cells were maintained in each well of a 24-well plate by the low-density culture method as described previously (Kubota et al., 2013).
Oleoresin black pepper increased the MTT signal on activity-deprived CGNs. (A) CGNs at 5 DIV that were maintained in media containing 5 mM KCl and extracts/oils from plants were subjected to MTT assay. The following concentrations were used: oleoresin chili pepper: 0.005 %, oleoresin black pepper: 0.001 %, holy basil extract: 0.0002 %, elemi oil: 0.0002 %, citrus unshiu oil: 0.001 %, java galangal oil: 0.001 %, calamus oil: 0.0002 %, clove leaf oil: 0.0002 %, and juniper berry oil: 0.001 % (n = 9 culture wells). The signal of 30 mM KCl is used as reference and values were shown as relative to the average at 5 mM KCl. Dashed line corresponds to signal value at 30 mM. Oleoresin black pepper significantly increased the MTT signal (Dunnett's test). (B–D) CGNs at 5 DIV that were treated with different concentrations of piperine (B), capsaicin (C), or dihydrocapsaicin (D) were subjected to MTT assay as in (A) (n = 7 culture wells for piperine, n = 8 culture wells for capsaicin and dihydrocapsaicin). No significant increase in MTT signals was observed upon treatment with these compounds. **p < 0.01, ***p < 0.001.
Oleoresin black pepper prevented neuronal loss and activation of caspase-3 of CGNs induced by activity deprivation. (A) CGNs were maintained in media containing 30 mM or 5 mM KCl with or without 0.001 % oleoresin black pepper. Neurons were fixed at 5 DIV and subjected to immunocytochemistry using antibodies against α-tubulin and cleaved caspase-3. Nuclear staining with Hoechst dye is also shown. Arrowheads indicate representative apoptotic cells with pyknotic nuclei and signals for cleaved caspase-3. (B) The relative density of surviving neurons was significantly reduced by KCl deprivation (5 mM), which was partially rescued with oleoresin black pepper (n = 5 experiments except for 30 mM KCl with oleoresin black pepper (n = 4), Turkey's test). (C) Oleoresin black pepper significantly reduced the number of cleaved caspase-3-positive cells induced by KCl deprivation (n = 5 experiments except for 30 mM KCl with oleoresin black pepper (n = 4), Turkey's test). Scale bar = 50 μm. **p < 0.01, ***p < 0.001.
Oleoresin chili prevented neuronal loss of CGNs induced by activity deprivation. (A) CGNs were maintained in media containing 30 mM or 5 mM KCl with or without 0.005 % oleoresin chili. Neurons were fixed at 5 DIV and subjected to immunocytochemistry as in Fig. 2. Arrowheads indicate representative apoptotic cells with pyknotic nuclei and signals for cleaved caspase-3. (B) The relative density of surviving neurons was significantly reduced by KCl deprivation (5 mM), which was partially rescued with oleoresin chili (n = 4 experiments, Turkey's test). (C) No significant effect of oleoresin chili was observed on the number of cleaved caspase-3-positive cells (n = 4 experiments, Turkey's test). Scale bar = 50 μm. *p < 0.05, ***p < 0.001.
Oleoresin black pepper had no significant effect on the neurite length of CGNs. (A) CGNs at 4 DIV that were maintained in the presence or absence of 0.001 % oleoresin black pepper were subjected to immunocytochemistry with anti-α-tubulin antibody. Neurons were also treated with BIO, a glycogen synthase kinase 3 inhibitor. Scale bar = 50 μm. (B) No significant effect of oleoresin black pepper on the neurite length was observed, whereas BIO significantly reduced the neurite length (n = 12, 10, 6, and 6 culture wells, for DMSO, 1 µM BIO, 10 µM BIO, and oleoresin black pepper, respectively, Dunnett's test). (C) Low-density cultures of CGNs at 7 DIV that were maintained in the presence or absence of cultured 0.001% oleoresin black pepper were subjected to immunocytochemistry with anti-α-MAP2 antibody. (D) Lengths of dendrites were quantified based on MAP2 staining. No significant effect of oleoresin black pepper on the dendritic length was observed (n = 4 culture wells, Welch's test). Scale bar = 50 μm. ***p < 0.001.
Analysis of neuroprotective functions Surviving neurons were detected by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Upon culture preparation, natural products diluted in dimethyl sulfoxide (DMSO) (0.5 µl in 100 µl of media for each well of a 96 wellplate) were added to wells containing CGNs. To reduce the pH change during treatment, the culture medium was supplemented with 20 mM Hepes-KOH pH 7.2. At 5 DIV, fresh culture medium was added to the neurons along with 10 μl of 5 mg/ml MTT (M009, Dojindo, Kumamoto, Japan) in PBS. After standing for 2 h in a CO2 incubator, precipitates were suspended with 40 mM HCl/isopropyl alcohol. MTT signals were measured by a plate reader (MTP-450Lab, Corona Electric, Hitachinaka, Japan) with a test wavelength of 570 nm and a reference wavelength of 650 nm.
Immunocytochemistry Immunocytochemistry of cultured CGNs was performed as described previously (Saga et al., 2020). A mouse monoclonal antibody against α-tubulin (12G10 at 1:1000; Developmental Studies Hybridoma Bank of University of Iowa) and rabbit polyclonal antibodies against cleaved caspase-3 (9661 at 1:400, Cell Signaling Technology, Danvers, Massachusetts, USA), and MAP2 (AB5622 at 1:1000, Merk KGaA, Darmstadt, Germany) were used. For secondary antibodies, goat anti-mouse-IgG conjugated to Alexa Fluor 488/568 (1:1000; ab150113/ab175473, Abcam, Cambridge, UK) and goat anti-rabbit-IgG conjugated to Alexa Fluor 488/568 (1:1000; A11008/A11011, Thermo Fisher Scientific) were used. Nuclei were stained with Hoechst 33258 (861405, Merk KGaA). Images were acquired using an Axiovert 200M fluorescence microscope equipped with an MRm monochromatic digital camera (Carl Zeiss, Oberkochen, Germany).
Data analysis and statistics Image data were processed using Fiji software (Schindelin et al., 2012). No calculation for sample size predetermination was performed. In the neuronal survival analysis, all data from an experiment were excluded when inhibition of depolarization did not induce neuronal cell death. Neurite length was measured automatically using the neurite outgrowth module in MetaMorph software (Molecular Devices, San Jose, California, USA). Statistical analysis was performed using the statistical software R version 4.2.2i). BoxplotRii) was used to generate boxplots. The box represents the 25–75th percentiles, and the median is indicated. The whiskers show the smallest and largest values within a distance of 1.5 times the interquartile range above and below the limits of the box. Data points are represented by dots. Outliers were excluded according to values 1.5 times outside of the interquartile range. The rest of the data met the assumption of normality according to the Shapiro–Wilk test. In this study, we used Dunnett's test, Tukey's test, or Welch's test to assess the significance of differences between groups. The levels of significance are denoted as follows: *p < 0.05, ** p < 0.01, *** p < 0.001. The values are presented as the mean ± SEM.
To identify neuroprotective functions of aromatic plants, activity-deprived CGNs produced by lowering KCl to 5 mM were treated with 50 kinds of essential oils or plant extracts at different concentrations (i.e., 0.0002, 0.001, 0.005, and 0.025 % (v/v)). At 5 DIV, culture plates were subjected to an MTT assay to assess cell viability by measuring cellular metabolic activity. From the first screening (n = 4 experiments), we selected 9 extracts that revealed a maximum of more than 1.5-fold signals in the MTT assay (Supplemental Materials, Table S1) and subjected them to the same assay at the same concentration. The whole screening process was conducted in a blinded manner. In the second screening, black pepper oleoresin (0.001 %), but no other essential oils/extracts, showed a significant increase in MTT signals in activity-deprived CGNs (p < 0.001, Dunnett's test, n = 9 experiments) (Fig. 1A). Although not significant, chili pepper oleoresin (Capsicum annuum) tended to increase the MTT signals at 0.005 % (p = 0.16). No effect was observed in holy basil oil, elemi oil, citrus unshiu oil, java galangal oil, calamus oil, clove leaf oil, and juniper berry oil (Fig. 1A).
Because piperine is the major bioactive component of black pepper oleoresin (Fig. S1), we treated CGNs with different concentrations (0.16 to 100 ng/ml) of piperine and subjected them to the MTT assay (Fig. 1B). We estimated that approximately 3 ng/ml of piperine is contained in 0.001 % of black pepper oleoresin. Since it has been reported that piperine activates the capsaicin receptor, the TRPV1 ion channel (Okumura et al., 2010), and chili pepper oleoresin tended to increase the MTT signals, we also treated neurons with different concentrations (0.48 to 300 ng/ml) of capsaicin and dihydrocapsaicin, which are major bioactive components of chili pepper (Butnariu et al., 2012; Sora et al., 2015) (Fig. 1C, D). No significant increase in MTT signals in activity-deprived CGNs was detected by treating with these compounds, suggesting that the inhibition of apoptosis by black pepper oleoresin is not likely related to TRPV1 receptor. These results suggest that components in black pepper oleoresin other than piperine contribute to the increase of MTT signals in activity-deprived CGNs.
To investigate whether black pepper oleoresin protects neurons from apoptosis, we stained neurons with a specific antibody for a cleaved (active) form of caspase-3. Neurons were simultaneously stained with antibody against tubulin, and nuclei were visualized with Hoechst 33258 (Fig. 2A). Consistent with a previous study (Saga et al., 2020), the density of neurites stained positive for tubulin tended to be low in the culture medium containing 5 mM KCl compared with that in the culture medium containing 30 mM KCl. The proportion of cells with pyknotic nuclei and signals for the cleaved caspase-3, both of which are hallmarks of apoptotic cells, tended to be high in the culture medium containing 5 mM KCl and were often overlapped (Fig. 2A). When we counted the number of surviving neurons by nuclear staining, we found a reduction in the density of neurons caused by KCl deprivation, which was significantly increased by black pepper oleoresin (52 % ± 4 % in KCl withdrawal vs. 75 % ± 4 % in KCl withdrawal with black pepper oleoresin, p < 0.01, Tukey's test) (Figs. 1A, 2B), which is consistent with the results of the MTT assay. Likewise, activity-deprivation increased the number of cleaved caspase-3-positive neurons (2.0 % ± 0.3 % in control vs. 6.1 % ± 0.8 % in KCl withdrawal, p < 0.01, Tukey's test), and black pepper oleoresin significantly reduced the number of cleaved caspase-3-positive neurons induced by activity withdrawal (3.7 % ± 0.6 %, p < 0.05, Tukey's test) (Fig. 2C). These results showed that black pepper oleoresin protected neurons from apoptosis induced by activity deprivation, possibly by acting upstream of caspase-3.
Chili pepper oleoresin significantly increased the density of neurons in the 5 mM KCl medium assay (59 % ± 3 % in KCl withdrawal vs. 72 % ± 4 % in KCl withdrawal with chili pepper oleoresin, p < 0.05, Tukey's test) (Fig. 3A, B). Thus, it is likely that the tendency of an increase in MTT signals (Fig. 1A) reflects the increased survival of CGNs by chili pepper oleoresin compared to black pepper oleoresin. However, no significant reduction in cleaved caspase-3 signals was detected by chili pepper oleoresin. Although we could not conclude whether chili pepper oleoresin acted on the caspase-3 independent pathway, or whether the neuroprotective effect was too small to detect caspase-3 activation, chili pepper oleoresin may also have a neuroprotective function.
Finally, we investigated whether black pepper oleoresin affected the morphogenesis of CGNs. To analyze neritic length, CGNs were cultured at lower density (Fig. 4A). Black pepper oleoresin had no significant effect on neurite length (212 ± 9 |im in control vs. 209 ± 7 µm in black pepper oleoresin, p = 0.99, Dunnett's test), whereas treatment with 6-bromoindirubin-3′-oxime (BIO), a glycogen synthase kinase 3 inhibitor, significantly reduced axonal lengths, as reported previously (Kubota et al., 2013) (Fig. 4A, B). Because CGN neurites stained with tubulin mostly represent axonal length under this culture condition, we selectively stained dendrites using an antibody for MAP2, a marker for dendrites (Fig. 4C). Compared with control neurons, no significant change was observed in the length of neurites or dendrites per neuron (21.6 ± 1.5 µm in control vs. 19.1 ± 0.9 µm in black pepper oleoresin, p = 0.17, Welch's test) (Fig. 4D). Thus, at this concentration, black pepper oleoresin did not affect the axonal and dendritic structures in the culture and prevent apoptosis.
In the current study on the MTT assay and immunocytochemistry, we report the neuroprotective function of black pepper oleoresin on caspase-3 mediated apoptosis of CGNs upon activity deprivation in vitro. Recent studies have described the anticancer effects of a piperine-free P. nigrum extract that contains a number of alkaloids and terpenes, including caryophyllene (Tedasen et al., 2020). As caryophyllene is also contained in chili pepper (Lei et al., 2021), it may be useful to test whether it has a neuroprotective function. Although caryophyllene is found in other oils, it is possible that the toxicity of other compounds masked the neuroprotective function in our screening experiments. Bioavailability of functional components should be considered as next steps. We previously detected several bioactive food factors in the brain of mice (Takemoto et al., 2021a, 2021b, 2022b). In addition, combination with functional components and essential oils can enhance incorporation into the brain (Iwamoto et al., 2019). Further studies are needed to investigate bioavailability of functional components.
Acknowledgements The authors thank Dr. Shinichi Matsumura (Inabata Koryo, Co., Ltd.) for helpful discussions. Y.K. received research grants from JSPS KAKENHI (20K06889) related, in part, to this study.
Conflict of interest This study was supported in part by a research grant from Inabata Koryo, Co., Ltd.
cerebellar granule neuron
MTT3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide
DIVday in vitro
DMSOdimethyl sulfoxide
BIO6-bromoindirubin-3’-oxime
| Natural products | Concentration (%) | MTT signal | Natural products | Concentration (%) | MTT signal | ||
|---|---|---|---|---|---|---|---|
| 1 | Cinnamon leaf oil | 0.001 | 1.3 ± 0.2 | 26 | Chili oleoresin | 0.005 | 2.0 ± 0.2 |
| 2 | Peppermint oil | 0.0002 | 1.1 ± 0.3 | 27 | Black pepper oleoresin | 0.001 | 2.0 ± 0.6 |
| 3 | Cumin oil | 0.005 | 1.0 ± 0.2 | 28 | Dementholised corn mint oil | 0.0002 | 1.8 ± 0.3 |
| 4 | Orange oil | 0.0002 | 1.4 ± 0.3 | 29 | Rosemary oil | 0.0002 | 1.8 ± 0.2 |
| 5 | Ginger oil DIST | 0.001 | 1.3 ± 0.2 | 30 | Holy basil oil | 0.0002 | 1.9 ± 0.8 |
| 6 | Turmeric oil | 0.0002 | 1.1 ± 0.2 | 31 | El emi o i l | 0.0002 | 1.9 ± 0.3 |
| 7 | Triglyceride | 0.001 | 1.1 ± 0.2 | 32 | Myrrh oil | 0.0002 | 1.9 ± 0.6 |
| 8 | Galangal oil | 0.001 | 1.1 ± 0.2 | 33 | Olibanum oil | 0.001 | 1.7 ± 0.3 |
| 9 | Sesame oil | 0.0002 | 1.1 ± 0.2 | 34 | Lime oil CP | 0.001 | 1.7 ± 0.4 |
| 10 | Parsley seed oil | 0.001 | 1.0 ± 0.1 | 35 | Lime oil DIST | 0.0002 | 1.2 ± 0.2 |
| 11 | Wild turmeric oil | 0.0002 | 0.8 ± 0.1 | 36 | Hassaku orange oil | 0.001 | 1.6 ± 0.3 |
| 12 | Fennel oil | 0.0002 | 1.0 ± 0.1 | 37 | Citrus unshiu oil | 0.001 | 1.8 ± 0.2 |
| 13 | Vetiver oil | 0.001 | 0.3 ± 0.1 | 38 | Yuzu oil | 0.001 | 1.2 ± 0.1 |
| 14 | Eucalyptus oil | 0.001 | 1.0 ± 0.1 | 39 | Leech lime oil | 0.0002 | 1.4 ± 0.2 |
| 15 | Cassia oil | 0.0002 | 0.5 ± 0.2 | 40 | Clove bud oil | 0.0002 | 1.5 ± 0.3 |
| 16 | Lemon oil | 0.005 | 0.7 ± 0.3 | 41 | Java galangal oil | 0.001 | 1.6 ± 0.3 |
| 17 | Nutmeg oil | 0.0002 | 1.1 ± 0.1 | 42 | Mace oil | 0.0002 | 1.3 ± 0.2 |
| 18 | Black pepper oil | 0.0002 | 1.1 ± 0.4 | 43 | Calamus oil | 0.0002 | 1.6 ± 0.1 |
| 19 | Lavender oil | 0.001 | 0.4 ± 0.1 | 44 | Celery seed oil | 0.001 | 1.3 ± 0.1 |
| 20 | Coriander oil | 0.001 | 1.4 ± 0.4 | 45 | Clove leaf oil | 0.0002 | 1.8 ± 0.5 |
| 21 | Sesamin | 0.025 | 1.2 ± 0.2 | 46 | Lemon glass oil | 0.0002 | 1.4 ± 0.3 |
| 22 | Ginger oil | 0.0002 | 1.4 ± 0.2 | 47 | Juniper berry oil | 0.001 | 2.4 ± 0.3 |
| 23 | Curry leaf oleoresin | 0.001 | 1.4 ± 0.4 | 48 | Ajowan oil | 0.001 | 1.1 ± 0.3 |
| 24 | Mustard (Brassica sp.) oil | 0.0002 | 0.2 ± 0.1 | 49 | Palmarosa oil | 0.001 | 1.0 ± 0.2 |
| 25 | Mustard (Brassica juncea) oil | 0.001 | 0.6 ± 0.5 | 50 | Dill oil | 0.001 | 1.1 ± 0.3 |
*MTT signal of each product represents the value relative to the signal with 5 mM KCl, and shows the maximam among the mean values (n = 4) at concentrations of 0.0002, 0.001, 0.005 and 0.025% (v/v).
HPLC chromatograms of oleoresin black pepper. Piperine was detected at 3.859 minutes. The analysis was performed on a Shimadzu UHPLC system (Shimadzu, Kyoto, Japan) that included two LC-30AD infusion pumps, a CTO-20AC column oven, a SIL-30AC automatic sampling device, a DGU-20A5R Solvent Degasser, CBM-20A system controller. Chromatographic conditions: column, L-Column 2ODS (Chemicals Evaluation and research Institute, Japan, Tokyo, Japan); mobile phase, acetonitrile/water/tetrahydrofuran = 45:55:7(v/v); injection volume; 2 µL; flow rate, 0.3 mL/min; temperature, 40°C; detection, UV at 343 nm with a PDA detector (SPD-M20A).
