Physalactone and 4Î²-Hydroxywithanolide E Isolated from Physalis

Article Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX

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Physalactone and 4β-Hydroxywithanolide E Isolated from Physalis peruviana Inhibit LPS-Induced Expression of COX‑2 and iNOS Accompanied by Abatement of Akt and STAT1 Eun-Jung Park,†,‡ Mayuramas Sang-Ngern,‡,§ Leng Chee Chang,‡ and John M. Pezzuto*,†,‡ †

J. Nat. Prod. Downloaded from pubs.acs.org by TULANE UNIV on 01/16/19. For personal use only.

Arnold and Marie Schwartz College of Pharmacy and Health Sciences, Long Island University, Brooklyn, New York 11201, United States ‡ The Daniel K. Inouye College of Pharmacy, University of Hawaìi at Hilo, Hilo, Hawaii 96720, United States § School of Cosmetic Science, Mae Fah Luang University, Tasud, Muang, Chiang Rai, Thailand ABSTRACT: In previous studies, withanolides isolated from Physalis peruviana were found to exhibit anti-inflammatory potential by suppressing nitrite production induced by lipopolysaccharide (LPS) treatment. Currently, we selected two of the most potent compounds, 4βhydroxywithanolide E (1) and physalactone (2), to examine the underlying mechanism of action. With LPS-stimulated RAW 264.7 cells in culture, the compounds inhibited the mRNA and protein expression of iNOS and COX-2. To determine which upstream signaling proteins were involved in these effects, phosphorylation levels of three mitogen-activated protein kinases (MAPKs) including ERK1/2, JNK1/2, and p38, were examined, but found unaffected. Similarly, the degradation of IκBα was not attenuated by the compounds. However, phosphorylation of Akt at the Ser-473 residue was inhibited, as was the phosphorylation of STAT1. Interestingly, the compounds also reduced the protein level of total STAT1, possibly by ubiquitin-dependent protein degradation. In sum, these results indicate the potential of 1 and 2 to mediate anti-inflammatory effects through the unexpected mechanism of inhibiting the transcription of iNOS and COX-2 via Akt- and STAT1-related signaling pathways.

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was shown to induce the production of nitrite/nitrate, thereby suggesting the use of an in vitro cell line based model to measure the endogenous nitrite/nitrate synthesis in nitrosamine-induced carcinogenesis.6 Of further importance, nitric oxide (NO) was established as an unstable intermediate in the conversion of L-arginine to nitrite and nitrate,7 and therefore this procedure is known interchangeably as a NO or nitrite assay, even though a nitrite/nitrate assay involves one additional step in which nitrate is converted to nitrite catalyzed by nitrate reductase. Over the years, the assay has been frequently used to evaluate the anti-inflammatory activity of samples since macrophages (RAW 264.7) represent the first line of defense against bacterial infection (LPS from Gram-negative bacteria) by inducing an acute “inflammatory” immune response. In fact, based on a search of PubMed (“RAW 264.7, LPS, and nitric oxide”), 2352 articles have been published using this methodology (Figure 1). Since 2008, more than 20 articles have appeared each year in two natural-product-related

nflammation is an essential defense mechanism to protect the host from various exogenous and endogenous stimuli. However, if the inflammatory response is not curtailed in a timely manner, sustained inflammatory signals can damage adjacent tissues (e.g., causing tissue fibrosis) or serve as an etiological factor in the genesis of conditions such as asthma, atherosclerosis, cancer, cardiovascular diseases, inflammatory bowel diseases, mood disorders, neurological disorders, and periodontal disease.1,2 Accordingly, it is meaningful to explore novel anti-inflammatory compounds, including those associated with edible sources, which may be of relevance for preventing or reducing the risk of the aforementioned diseases. One method for establishing the potential of antiinflammatory activity involves a nitrite colorimetric assay. This assay allows for the indirect estimation of nitrite concentration utilizing the Griess reaction, first described in 1879,3 in which nitrite forms a purple azo chromophore with an aryl amine. In 1982, employing the Griess reaction, Green et al. established an automated system to measure nitrate and nitrite in biological fluids, including gastric juice, milk, plasma, and saliva.4 Meanwhile, it was reported that the RAW 264.7 cell line (Abelson murine leukemia virus-transformed macrophage cells derived from male BALB/c mice) showed sensitivity to an endotoxin from Gram-negative bacteria termed lipopolysaccharide (LPS), along with cell growth inhibition.5 Subsequently, treatment of RAW 264.7 with LPS © XXXX American Chemical Society and American Society of Pharmacognosy

Special Issue: Special Issue in Honor of Drs. Rachel Mata and Barbara Timmermann Received: October 15, 2018

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DOI: 10.1021/acs.jnatprod.8b00861 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Figure 1. Chronological landmarks of scientific publications related to the nitrite assay using LPS-stimulated RAW 264.7 cells. The bibliographic search was conducted using PubMed with three keywords: RAW 264.7, LPS, and nitric oxide (open bars). In addition, using the same keywords, the number of publications appearing in J. Nat. Prod. and J. Agric. Food Chem. is shown (solid bars) (access date: 09-27-2018).

Figure 2. Chemical structures of 4β-hydroxywithanolide E (1) and physalactone (2).

Figure 3. Effect of 1 and 2 on iNOS and COX-2 protein expression (A) and nitrite production (B) in LPS-stimulated RAW 264.7 cells. RAW 264.7 cells were pretreated with 1 (50 μM) or 2 (50 μM) for 30 min and stimulated with 1 μg/mL LPS for an additional 16 h. Protein expression levels of iNOS and COX-2 were examined by Western blot analyses (A), and the nitrite levels (μM) in the culture media were estimated using Griess reagent (B) (n = 3).

retic, and sedative effects.8 Using the nitrite assay, we demonstrated the anti-inflammatory potential of a series of withanolides obtained from the aerial parts of this plant,9,10 the most potent of which were 4β-hydroxywithanolide E (1; CAS registry number: 54334-04-2) and physalactone (2) (Figure 2). Compound 1 is known to be present in P. peruviana,11 P. viscosa,12 P. cinerascens,13 P. angulata,14 and P. pruinosa,15 and 2

journals including Journal of Natural Products and Journal of Agricultural Food and Chemistry. We have been interested in exploring the constituents and potential medicinal value of Physalis peruviana Linnaeus (Solanaceae). The medicinal properties of P. peruviana L. have been reported to include analgesic, antiamoebic, antiparasitic, antiseptic, antispasmodic, cataract-cleaning, diuB



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Figure 4. Concentration-dependent effect of 1 and 2 on nitrite production (A) and iNOS and COX-2 protein expression (B) in LPS-stimulated RAW 264.7 cells. RAW 264.7 cells were pretreated with three different concentrations of 1 (0.8, 2, and 5 μM) or 2 (6.4, 16, and 40 μM) for 30 min and stimulated with 1 μg/mL LPS for an additional 18 h. Protein expression levels of iNOS and COX-2 were examined by Western blot analyses (A), and the nitrite levels in the culture media were estimated using Griess reagent (B) (n = 3). An asterisk (*) indicates a significant difference between LPS-treated control and 1- or 2-pretreated/LPS-treated control with p values less than 0.05.

Figure 5. Effect of 1 and 2 on the relative mRNA expression levels of iNOS and COX-2 in LPS-activated RAW 264.7 cells. RAW 264.7 cells were pretreated with the indicated concentrations of 1 or 2 for 30 min, followed by LPS treatment for 3, 6, 9, and 12 h. RNA was extracted and used in the qRT-PCR reaction. After normalization to the internal standard, β-actin, fold-changes relative to LPS-nontreated control are presented (n = 3). An asterisk (*) indicates a significant difference between LPS-treated control and 1- or 2-pretreated/LPS-treated control in each time point with p values less than 0.05.

was isolated from P. alkekengi L.,16 P. cinerascens,13 and P. peruviana.9 Compound 1 demonstrated anti-inflammatory activity in diabetic mouse adipose tissue15 and antiproliferative

or apoptotic activities toward various cancer cell lines including breast,17 colon,18 pancreas,19 gingiva,20 liver,21 and lung.22 C



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Figure 6. Overview of the LPS signaling pathways.

Next, we examined the concentration dependence of the inhibitory effects of 1 and 2. Based on previously reported IC50 values,9,10 pretreatment concentrations in the range of 0.128− 5.0 and 1.0−40 μM were selected for 1 and 2, respectively. As shown in Figure 4A, nitrite levels were reduced with IC50 values of approximately 1.8 and 13.8 μM with 1 and 2, respectively. These values differ from previous results,9,10 most likely due to the different assay conditions [e.g., different assay plates (24-well vs 96-well) and incubation times (18 h vs 20 h)]; nonetheless, the relative potency remains the same. Further, as shown in Figure 4B, both 1 and 2 suppressed LPS upregulated expression of iNOS and COX-2 in a concentration-dependent manner. The iNOS protein bands were nearly undetectable with cells pretreated with higher concentrations of 1 (2 and 5 μM) and 2 (16 and 40 μM). The effect on COX-2 protein expression was less pronounced but concentration-dependent. Next, the mRNA levels of iNOS (gene symbol: Nos2) and COX-2 (gene symbol: Ptgs2) were examined using quantitative polymerase chain reaction (qPCR). As shown in Figure 5, the mRNA expression of iNOS was significantly upregulated after 3, 6, 9, and 12 h of incubation with LPS, by 294-, 1068-, 1434-, and 3821-fold, respectively, relative to that of unstimulated cells. Likewise, the mRNA expression of COX-2 was increased in response to LPS in a time-dependent manner, but to a lower extent than iNOS. Pretreatment with 1 and 2 at concentrations of 5 and 40 μM, respectively, attenuated iNOS and COX-2 mRNA expression at each indicated time. Notably, although both compounds suppressed LPS-induced mRNA expression of iNOS and COX-2, greater inhibitory effects were observed with iNOS. It may be of relevance that superoxide dismutase differentially regulates iNOS and COX-2 in LPS-stimulated RAW 264.7 cells,24 so in future studies it could be of value to investigate the potential of 1 and 2 to protect against oxidative stress induced by LPS.

However, the underlying mechanism by which 1 and 2 inhibit the nitrite-inflammatory actions in LPS-stimulated RAW 264.7 cells has not been reported. Therefore, following our previous reports demonstrating the fundamental activity of 1 and 2,9,10 we now further unravel underlying mechanisms of inhibitory effects on the production of nitrite.

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RESULTS AND DISCUSSION In previous work conducted with RAW 264.7 cells, of the withanolides tested, the greatest inhibition of NO production was mediated by 1,10 followed by 2.9 In addition, however, at the highest concentration tested (50 μM), treatment with 1 led to a cytotoxic response, whereas 2 was not active in this regard. With unprimed RAW 264.7 cells, NO found in the culture media is less than 1 μM. Following challenge with LPS, this can surge up to around 40 μM, due to the induction of inducible nitric oxide synthase (iNOS). As such, the LPSinduced response is suppressed by S-methyl isothiourea, a selective inhibitor of iNOS, or direct knockdown of iNOS.23 Therefore, as a first attempt to understand the inhibitory mechanism of 1 and 2, we examined the effect of treatment on iNOS protein expression. As shown in Figure 3A, protein levels of iNOS in RAW 264.7 cells incubated with LPS for 16 h increased in comparison to quiescent cells, but this was significantly attenuated by treatment with 1 or 2 (50 μM) 30 min prior to LPS exposure. Consistent with this response, nitrite levels in resting cells (0.9 ± 0.2 μM) were greatly elevated by LPS treatment (32.4 ± 0.9 μM). As anticipated, pretreatment with 1 or 2 significantly reduced nitrite in culture media to 0.9 ± 0.1 μM and 1.1 ± 0.2 μM, respectively (Figure 3B). In addition, treatment with 1 or 2 inhibited LPS-induced protein expression of cyclooxygenase-2 (COX-2), a key inflammatory mediator (Figure 3A). D



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Given these results, to elucidate the inhibitory mechanism by 1 and 2 on LPS-stimulated iNOS and COX-2 gene expression, cell signaling pathways related to transcription were investigated. An overview of the LPS signaling pathway is presented in Figure 6. The signal is initiated as LPS binds to Toll-like receptor 4 (TLR4)/myeloid differentiation factor 2 (MD-2) complex in the plasma membrane, followed by the activation of intracellular signals through two adaptors, i.e., myeloid differentiation marker 88 (MyD88) and Tollinterleukin-1 (IL-1) receptor domain-containing adaptorinducing interferon-β (TRIF), and two sorting adaptors, i.e., Toll/IL-1 receptor domain-containing adaptor protein (TIRAP) and TRIF-related adaptor molecule (TRAM).25 There are two distinct downstream pathways: MyD88/TIRAPdependent and TRAM/TRIF-dependent signaling pathways. In the MyD88/TIRAP pathway, MAPK and nuclear factorkappa B (NF-κB) signaling pathways activate transcription factors, which subsequently promote the expression of proinflammatory genes including Nos2 and Ptgs2. In the TRAM/ TRIF-dependent pathway, phosphorylated Akt can induce the phosphorylation of interferon regulatory factor 3 (IRF-3),26 which is a transcription factor of type I interferons (IFNs) such as IFNβ. Then, secreted IFNβ transfers a secondary signal via binding to type I interferon receptor (IFNAR) complex, which initiates a signaling cascade through Janus kinase 1 (JAK1) and tyrosine kinase 2 (TYK2) to activate signal transducer and activator of transcription 1 (STAT1) and STAT2 and subsequent transcription of Nos2. Taking this into account, we first examined the phosphorylation levels of MAPKs in the MyD88/TIRAP-dependent pathway. The activation of MAPKs has been implicated in LPS-stimulated iNOS expression and NO production; blockade of MAPKs by treatment with inhibitors (e.g., MAPK p38 inhibitor SB203580,27 ERK inhibitor PD98059,27 and JNK inhibitor SP60012528), or knockdown of p38 and JNK by siRNA methods,29 reduced LPS-induced expression of iNOS, or production of NO in murine macrophage cell lines.27,28 The phosphorylation of MAPKs at amino acid residues of Thr180 and Tyr182 for p38 MAPK,30 Thr183 and Tyr185 for JNK,31 and Thr202 and Tyr204 for ERK32 leads to the functional activation of MAPKs. Therefore, the phosphorylation level of each MAPK was examined to evaluate the effects of 1 and 2 on activation. As illustrated in Figures 7 and 8, the phosphorylated active forms of MAPKs in quiescent cells were essentially undetectable. In comparison, the phosphorylation levels of p38, ERK, and JNK peaked within 10 to 30 min after LPS treatment, whereas total levels of MAPKs were unchanged. However, the increased phosphorylation of MAPKs was unaffected by the treatment with 1 (5 μM) or 2 (40 μM) prior to LPS exposure. Alternatively, with LPS-stimulated RAW 264.7 cells, NF-κB can function as a transcription factor for the expression of iNOS. As such, expression is diminished by treatment with known inhibitors of NF-κB, such as pyrrolidine dithiocarbamate.33 There are two distinct NF-κB signaling pathways: the canonical and noncanonical pathways. In general, the canonical pathway is associated with innate immunity, whereas the noncanonical/alternative pathway is related to adaptive immunity.34,35 Typically, NF-κB exists in an inactivated state due to binding with the inhibitor of κB (IκB).36 Of the three classical IκBs (IκBα, IκBβ, and IκBε), IκBα is known to play a pivotal role in sequestering the major NF-κB heterodimer, p65/p50, in the inactive state.37,38 Activation of IκB kinase

Figure 7. Effect of pretreatment of 1 on LPS-induced phosphorylation MAPKs, Akt, and STAT1 and degradation of IκBα. RAW 264.7 cells were treated with 5 μM 1 for 30 min prior to LPS (1 μg/mL) treatment for the indicated times. Protein expression levels were determined by Western blot analysis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is used as an internal standard.

Figure 8. Effect of pretreatment of 2 on LPS-induced phosphorylation MAPKs, Akt, and STAT1 and degradation of IκBα. RAW 264.7 cells were treated with 5 μM 1 for 30 min prior to LPS (1 μg/mL) treatment for the indicated times. Protein expression levels were determined by Western blot analysis.

(IKK) initiates ubiquitin-dependent degradation of IκB. Upon the degradation of IκBα, NF-κB dimers (e.g., p65/p50) can bind to the cis-regulatory element in the promoter region for the subsequent transcription.39,40 Thus, we examined total protein levels of IκBα at various times after LPS treatment, based on previous observations regarding the complete degradation and recovery of IκBα.41 E



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suggesting these components play a critical role in Akt activation,48 and Akt phosphorylation was markedly reduced after depletion of Rab8a.49 Therefore, it would be of interest to study the potential of 1 and 2 to modulate these components. Another potential target is nuclear factor of activated T-cells 5 (NFAT5), which is required for the transcription of Nos2 and subsequent NO production in LPS-activated RAW 264.7.50 In sum, it is worth emphasizing the vast potential value of controlling inflammation51 and the distinctive approach of targeting iNOS. In regard to the latter, aberrant levels of NO can damage neurons and oligodendrocytes,52 and targeted inhibition of STATs and IRFs has been described as a treatment strategy for cardiovascular disease.53 Also, decreased iNOS expression mediated by distamycin A improved survival from endotoxemia.54 It remains to be seen if 1 and 2, or derivatives thereof, will have any practical utility for inflammatory conditions. It is apparent that the overall potency of 1 is greater than 2, almost certainly due to the α,β-unsaturated ketone moiety associated with 1. Nonetheless, it is notable that the activity of 2 closely parallels that of 1, suggesting the α,β-unsaturated ketone is not essential for mediating the response and may in fact lead to less specificity or greater toxicity. It would be of interest to explore these thoughts in future studies.

As illustrated in Figures 7 and 8, complete IκBα degradation was observed around 10 min after LPS treatment, followed by recovery. However, pretreatment with 1 (5 μM) (Figure 7) or 2 (40 μM) (Figure 8) did not alter this pattern of degradation or recovery, suggesting that targeting of the NF-κB pathway is not responsible for the iNOS inhibitory activity mediated by these compounds. It may be noted, however, that 1 has been reported to decrease inflammatory responses by inhibiting NFκB signaling in diabetic mouse adipose tissue,15 so further investigation may be necessary. The next possibility examined was whether 1 and 2 inhibited Nos2 expression via the TRAM/TRIF-dependent pathway. TRIF activates TRIF/TANK binding kinase 1 (TBK1) and, subsequently, activated TBK1 enhances the phosphorylation of Akt, which can ultimately enhance iNOS. Knockdown of TBK1 in RAW 264.7 cells mitigated LPS-stimulated phosphorylation levels of Akt at Ser473.42 In addition, Akt itself is involved in iNOS and COX-2 expression: Blockage of Akt causes reduced expression of COX-2 and iNOS,29 and dominant-negative-Akt expression in cells attenuates iNOS expression.43 Therefore, we can assume that Akt plays a crucial role in iNOS and COX-2 expression, and the potential of 1 and 2 to alter the status of Akt was examined. As shown in Figures 7 and 8, the phosphorylation levels of Akt began to increase from 10 min after LPS challenge and surged at 2 and 4 h as judged by strong Akt bands phosphorylated at the Ser473 residue. Both 1 (5 μM) and 2 (40 μM) attenuated LPS-induced phosphorylated activation of Akt without attenuating total Akt levels. In the context of iNOS expression, Akt induces IRF-3 phosphorylation in RAW264.7 cells,26 and TBK1 and Akt work in concert to induce the maximal activation of IRF-3 and subsequent expression of IFN-β.42 IFN-β, in turn, initiates the JAK/STAT signaling pathway. Activated JAK1 and TYK2 phosphorylate STAT1 at the Tyr701 residue,44 leading to Nos2 expression. In the current study, following treatment with LPS, STAT1 levels remained relatively constant, whereas phosphorylation of STAT1 at Tyr701 was greatly enhanced after 2 and 4 h of incubation. Treatment with 1 (5 μM) (Figure 7) or 2 (40 μM) (Figure 8) strongly attenuated the phosphorylation levels of STAT1 while producing only a slight decrease in total STAT1. Similarly, it has been reported that hydroquinone suppresses IFN-β expression by targeting the Akt/IRF-3 pathway.26 Taken together, these data indicate that 1 and 2 inhibit iNOS and COX-2 by an unexpected mechanism; that is, rather than affecting the MAPK and NF-κB pathways, the compounds work through modulation of the Akt-STAT1 axis. This seems feasible since previous work has shown a major contribution of MyD88-independent pathways on the expression of iNOS in LPS-activated macrophages.45 Also, a recent study demonstrated a fundamental contribution of the JAK/STAT1 signaling pathway on iNOS expression based on using STAT1 (fludarabine) and JAK (AG-490) inhibitors.46 In addition, Nos2 expression is drastically reduced in macrophages from type I IFN and STAT1 knockout mice.47 Therefore, we currently demonstrate that 1 and 2 regulate LPS-induced iNOS expression by inactivation of the TRIFAkt-IRF3-IFNβ-JAK-STAT1 axis, but further work is yet required to reveal the complete story. In particular, additional components along the networks shown in Figure 6 are worth examination. For example, reduced phosphorylation of Akt was observed in macrophages lacking TRIF and MyD88,

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EXPERIMENTAL SECTION

General Experimental Procedures. Compounds 1 and 2 were isolated from the methanol extract of the aerial parts of Physalis peruviana L. as previously described.9,10 The RAW 264.7 murine macrophage-like cell line was purchased from American Type Culture Collection (Lot# 62094971; Manassas, VA, USA). Dulbecco’s modified Eagle’s medium (DMEM) with 4.5 g/L glucose without Lglutamine and phenol red was purchased from Lonza Group Ltd. (Basel, Switzerland). Penicillin−streptomycin solution (50×) was purchased from Mediatech, Inc. (Manassas, VA, USA). LPS from Escherichia coli O111:B4 was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Primary and secondary antibodies and cell lysis buffer (10×) for Western blot analysis were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Luminata Forte Western HRP substrate was purchased from EMD Millipore (Billerica, MA, USA). Bradford reagent and iScript Reverse Transcription Supermix for RT-qPCR were purchased from BioRad Laboratories, Inc. (Hercules, CA, USA). Oligonucleotide PCR primers and Trizol reagent were purchased from Invitrogen (Carlsbad, CA, USA). PerfeCTa SyBR Green FastMix, ROX was purchased from Quanta Biosciences (Gaithersburg, MD, USA). Cell Culture. The RAW 264.7 murine macrophage-like cell line was cultured in DMEM containing 10% (v/v) heat-inactivated fetal bovine serum (FBS), penicillin G potassium salt (100 IU/mL), and streptomycin sulfate (100 μg/mL) in a 37 °C humidified incubator with 5% CO2 in air. Measurement of Nitrite Production. The concentration of nitrite in the cultured media was used as a measure of NO production. The assay was performed as previously described.55 RAW 264.7 cells were plated at a density of 5 × 105 per well in a 24-well culture plate and incubated in a 37 °C humidified incubator with 5% CO2 in air for 24 h. The incubated cells were pretreated with phenol-red-free medium containing various concentrations of 1 or 2 for 30 min, followed by 1 μg/mL of LPS treatment for an additional 16 or 18 h. Aliquots of supernatant from each well (100 μL) were transferred to a 96-well plate, and nitrite concentration was measured using Griess reagent. Western Blot Analysis. The total cell lysates were prepared according to the manufacturer’s instructions as described previously56 and stored at −80 °C until use. After determining the protein concentration of the lysate using the Bradford protein assay, an equal amount of total protein in each cell lysate was separated by sodium F



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dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (8− 12%) and electrotransferred to PVDF membranes. After being blocked by Tris-buffered saline with 0.1% Tween 20 (TBST) containing 5% (w/v) skimmed milk for an hour at room temperature, membranes were washed three times with TBST and further incubated with corresponding primary antibodies in TBS with 3% skimmed milk overnight at 4 °C. After the wash step with TBST to remove the unbound antibodies, membranes were incubated with secondary antibodies conjugated with horseradish peroxidase (HRP) for 2 h at room temperature. After TBST wash, HRP substrate was applied to the membrane and chemiluminescence emanating from each immunoblot was detected using a charge-coupled device-based digital image analysis system (Geliance 1000 imager, PerkinElmer, Inc., Waltham, MA, USA). Quantitative Polymerase Chain Reaction. Total RNA extracted from cells using Trizol reagent57 was dissolved in RNasefree water. After the measurement of concentration and purity of isolated RNA using a BioSpec-nano spectrophotometer (Shimadzu Scientific Instruments, Inc., Columbia, MD, USA), 1 μg of total RNA was reverse transcribed using iScript Reverse Transcription Supermix for RT-qPCR on an ABI 7300 thermocycler (Applied Biosystems Inc., Foster City, CA, USA). The cDNA amplification of iNOS, COX-2, and β-actin was performed with the SyBR green method as the fluorescence signal as previously described.56 Expression, calculated using the 2−ΔΔCT method, was performed to analyze the results.58 Statistical Analysis. Data are presented as means ± standard deviation for the indicated number of independent experiments. Statistical analysis was performed using GraphPad Prism version 7.03 (GraphPad Software, Inc., San Diego, CA, USA). p values less than 0.05 (p < 0.05) were considered statistically significant.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +1-808-933-2960. Fax: +1-808-933-2974. E-mail: [email protected]. Tel: +1-718-488-1004. Fax: +1-718488-1213. E-mail: [email protected]. ORCID

Eun-Jung Park: 0000-0001-9486-8692 Notes

The authors declare the following competing financial interest(s): The authors have submitted a provisional patent application.

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ACKNOWLEDGMENTS This research project was supported by grants 2G12RR003061-26 from the National Center for Research Resources, 8G12MD7601-27 from the National Institute on Minority Health and Health Disparities, and Medical Research Program of the Hawaii Community Foundation (HCF), 15ADVC-74402, and DKICP start-up funds (LCC). The authors would like to thank Mr. S. E. Lorch (Lani ko Honua Berry Farm, Pepeekeo) for providing poha plant berries for this study, and DKICP for research facilities (NMR and LC-MS).

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DEDICATION Dedicated to Dr. Rachel Mata, National Autonomous University of Mexico, Mexico City, Mexico, and Dr. Barbara N. Timmermann, University of Kansas, for their pioneering work on bioactive natural products.

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REFERENCES

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