Inhibition Of Nitric Oxide Production Research Articles

Inhibition of nitric oxide production enhances the activity of facial nerve tubulin polymerization and the ability of tau to promote microtubule assembly after neurorrhaphy

We previously reported that inhibition of nitric oxide (NO) production promotes rat reconnected facial nerve regeneration. However, the underlying mechanism is obscure. Microtubule assembly is known to be essential to axon regeneration; nevertheless, tubulins and microtubule-associated proteins (MAPs) have been demonstrated as targets for NO and peroxynitrite. Thus, we hypothesized that NO and/or peroxynitrite may affect facial nerve regeneration via influencing on microtubule assembly. First, tubulins and tau (a MAP) were extracted from facial nerves of normal rats, treated with NO donor or peroxynitrite, and processed for microtubule assembly assay. We found that peroxynitrite, DEA NONOate, and Angeli's salt reduced the tubulin polymerization activity to a greater extent than GSNO, SIN-1, and SNAP. Additionally, SIN-1, peroxynitrite, and Angeli's salt impaired the ability of tau to promote microtubule assembly. Next, nitrosative stress biomarkers 3-nitrotyrosine (3-NT) and S-nitrosylated cysteine (SNO-Cys) were immunolabeled in facial nerves. Both biomarkers were highly upregulated in proximal and distal stumps of reconnected facial nerves at 3 days and 1 week after neurorrhaphy. Notably, the expression of 3-NT was greatly reduced at 2 weeks, whereas that of SNO-Cys was maintained. Conversely, inhibition of NO production with L-NAME prevented the upregulation of SNO-Cys. Further, we used tubulins and tau extracted from facial nerves of sham-operated, nerve suture + vehicle treatment, and nerve suture + L-NAME treatment rats to perform microtubule assembly assay. We found that L-NAME treatment enhanced polymerization activity of tubulins and ability of tau to promote microtubule assembly. It is noteworthy that α-tubulin plays a more important role than β-tubulin since the activity of microtubule assembly using α-tubulin extracted from L-NAME-treated rats was greatly elevated, whereas that using β-tubulin extracted from L-NAME-treated rats was not. Overall, our findings support that inhibition of NO production reduces nitrosative stress, and may thus facilitate microtubule assembly and facial nerve regeneration.

Trimeric chalchonoids from the total phenolic extract of Chinese dragon's blood (the red resin of Dracaena cochinchinensis)

Four new chalchonoid trimers, named cochinchinenins N-Q (1-4), along with a pair of known enantiomers (5-6), were isolated from the total phenolic extract of Chinese dragon's blood (the red resin of Dracaena cochinchinensis). The planar structures of 1-4 were elucidated by extensive spectroscopic analysis including HRESIMS and 1D/2D NMR. The absolute configurations of new compounds were established by ECD data. Compound 1 exhibited significant inhibition of nitric oxide production in lipopolysaccharide-stimulated BV-2 microglial cells with IC50 value of 11.5±1.7μM.

Oxidative stress in the skin: Impact and related protection.

Skin, our first interface to the external environment, is subjected to oxidative stress caused by a variety of factors such as solar ultraviolet, infrared and visible light, environmental pollution, including ozone and particulate matters, and psychological stress. Excessive reactive species, including reactive oxygen species and reactive nitrogen species, exacerbate skin pigmentation and aging, which further lead to skin tone unevenness, pigmentary disorder, skin roughness and wrinkles. Besides these, skin microbiota are also a very important factor ensuring the proper functions of skin. While environmental factors such as UV and pollutants impact skin microbiota compositions, skin dysbiosis results in various skin conditions. In this review, we summarize the generation of oxidative stress from exogenous and endogenous sources. We further introduce current knowledge on the possible roles of oxidative stress in skin pigmentation and aging, specifically with emphasis on oxidative stress and skin pigmentation. Meanwhile, we summarize the science and rationale of using three well-known antioxidants, namely vitamin C, resveratrol and ferulic acid, in the treatment of hyperpigmentation. Finally, we discuss the strategy for preventing oxidative stress-induced skin pigmentation and aging.

In vitro Cytotoxicity and Antioxidant Study of Rhodomyrtus tomentosa (Aiton) Hassk. Ethanolic Leaf Extract on LPS-induced RAW 264.7 Macrophage Cells

Aims: Ethanol extract of Rhodomyrtus tomentosa leaves found specifically on Malaysian soil was used to further investigate the antioxidant properties and cytotoxicity against RAW 264.7 macrophage cells in the search for a safer and effective natural antioxidant agent.
 Study Design: Antioxidant potential of R. tomentosa were analyzed through series of spectrometric assays and cell-based bioassays model.
 Place and Duration of Study: This study was carried out at Faculty of Pharmacy, Universiti Teknologi MARA (UiTM), Puncak Alam Campus, 43200, Selangor, Malaysia from the year of 2019 to 2021.
 Methodology: R. tomentosa leaves were subjected to extraction with 95% ethanol. The extracts were then denoted as ethanolic leaves extract of R. tomentosa or EtRT extract. EtRT extract were then screen for its antioxidant activity (AOA) and total antioxidant capacity (TAC) through DPPH radical scavenging assay and ferric reducing antioxidant power (FRAP) assay. After that, EtRT extract were brought to observe its toxicity against RAW 264.7 macrophage cells in MTT assay. Once their toxicity was obtained, EtRT extracts were finally tested for their ability to inhibit intracellular reactive oxygen species (ROS) and nitric oxide (NO) inhibition in RAW 264.7 macrophage cells to further analyze their antioxidant properties.
 Results: In this study, EtRT extracts dose dependently showed the ability to scavenge DPPH radicals and reduce ferric ions during DPPH radicals scavenging activity assay and ferric reducing antioxidant power assay (FRAP), respectively. In DPPH radical scavenging activity assay, EtRT extracts showed EC50 value at 12.37 ± 1.73 µg/mL with ARP value of 0.08 almost as near as ascorbic acid’s ARP value which is 0.09. Further into the study, EtRT extract were not cytotoxic to RAW 264.7 macrophage cells at concentrations 3.91 µg/mL and lower which showed more than 86.4% cell viability with IC50 value at 204.70 ± 5.30 µg/mL. EtRT extract possessed the ability to inhibit ROS production on LPS-induced RAW 264.7 macrophage cells at 7.813 µg/mL and lower, with the highest concentration can reduce up to 30.20% ± 1.01 out of the total ROS produced by the induced cells. Furthermore, EtRT extract also have evidenced that it is able to significantly inhibit NO production by the LPS-induced RAW 264.7 macrophage cells at 7 µg/mL and lower being the highest at 56.73% ± 0.11 inhibition of the highest concentration tested.
 Conclusions: This study suggests that EtRT extracts have the potential to reduce LPS-induced oxidative stress due to the antioxidant activities of phenolic compounds in the extracts, and that at low doses, EtRT extracts had low to no cytotoxicity on RAW 264.7 macrophage cells. As a result, EtRT extract could be a promising natural medicinal agent for the treatment of oxidative stress.

Anti-Inflammatory Activity of 4-((1R,2R)-3-Hydroxy-1-(4-hydroxyphenyl)-1-methoxypropan-2-yl)-2-methoxyphenol Isolated from Juglans mandshurica Maxim. in LPS-Stimulated RAW 264.7 Macrophages and Zebrafish Larvae Model.

Juglans mandshurica Maxim., a traditional folk medicinal plant, is widely distributed in Korea and China. In our previous study, we isolated a new phenylpropanoid compound, 4-((1R,2R)-3-hydroxy-1-(4-hydroxyphenyl)-1-methoxypropan-2-yl)-2-methoxyphenol (HHMP), from J. mandshurica. In the present study, we evaluated the anti-inflammatory activity of HHMP on lipopolysaccharide (LPS)-stimulated RAW 264.7 cells and zebrafish larvae. HHMP significantly inhibited LPS-induced nitric oxide (NO) and prostaglandin E2 production in a dose-dependent manner. Moreover, HHMP treatment considerably suppressed LPS-induced expression of inducible nitric oxide synthase and cyclooxygenase-2. We also demonstrated the mechanisms of HHMP inhibition of inflammatory responses in LPS-stimulated RAW 264.7 cells via Western blot analysis and immunofluorescence staining. Furthermore, HHMP significantly inhibited NO production in LPS-stimulated zebrafish larvae. Consequently, we established that HHMP significantly inhibited the LPS-induced activation of NF-κB and MAPK and the nuclear translocation of p65 in RAW 264.7 cells. Taken together, our findings demonstrate the effect of HHMP on LPS-induced inflammatory responses in vitro and in vivo, suggesting its potential to be used as a natural anti-inflammatory agent.

Anti-Inflammatory Effects of Morinda citrifolia Extract against Lipopolysaccharide-Induced Inflammation in RAW264 Cells.

Leaves of Morinda citrifolia (noni) have been used in Polynesian folk medicine for the treatment of pain and inflammation, and their juice is very popular worldwide as a functional food supplement. This study aimed to demonstrate that M. citrifolia seed extract exerts anti-inflammatory effects on RAW264 cells stimulated by lipopolysaccharide. To confirm the inhibitory effect of M. citrifolia seed extract, we assessed the production of nitric oxide (NO) and inflammatory cytokines. The M. citrifolia seed extract showed a significant inhibition of NO production, with no effect on cell viability, and was more active than M. citrifolia seed oil, leaf extract, and fruit extract. The M. citrifolia seed extract was found to reduce the expression of inducible NO synthase and tumor necrosis factor-alpha of pro-inflammatory cytokines. These results suggest that the anti-inflammatory effect of M. citrifolia seed extract is related to a reduction in the expression of inflammatory mediators and support its potential therapeutic use.

Nitric oxide is induced and required for efficient Kaposi's sarcoma-associated herpesvirus lytic replication.

Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic virus associated with several human malignancies. KSHV lytic replication promotes the spread of infection and progression of KSHV-associated malignancies; however, the mechanism regulating KSHV lytic replication remains unclear. In this study, we investigated the role of nitric oxide (NO) in KSHV lytic replication. In the TREx BCBL1-RTA KSHV lytic replication cell system, induction of KSHV lytic replication increased intracellular and extracellular NO. Chemical inhibition of NO production resulted in a lower level of KSHV lytic replication as shown by a reduced level of infectious virions, and decreased levels of viral lytic transcripts and proteins. In a second KSHV lytic replication system of iSLK-RGB-BAC16 cells, we confirmed that KSHV lytic replication increased NO production. Chemical inhibition of NO production resulted in reduced numbers of cells expressing enhanced green fluorescent protein and blue fluorescent protein, two reporters that closely track the expression of KSHV early and late genes, respectively. Consistent with these results, inhibition of NO production resulted in reduced levels of infectious virions, and viral lytic transcripts and proteins. Importantly, exogenous addition of a NO donor was sufficient to enhance the full KSHV lytic replication program. These results demonstrate that NO is required for efficient KSHV lytic replication, and NO playsa crucial role in the KSHV life cycle and KSHV-induced malignancies.

Effect of Anti-vascular Endothelial Growth Factor on the Matrix Metalloproteinase Activity in Trabecular Meshwork Cells

Purpose: To investigate the effects of anti-vascular endothelial growth factor (anti-VEGF) on the activity of matrix metalloproteinase (MMP) in human trabecular meshwork cells (HTMC). Methods: Primary HTMC cultures were exposed to 0, 0.25, and 0.50 mg/mL anti-VEGF bevacizumab (BV) for 24 hours. The permeability through the trabecular meshwork cell monolayer was assessed using carboxyfluorescein and trans-epithelial endothelial electrical resistance (TEER). The levels of MMP-1/-2 were measured by Western blotting and the production of nitric oxide (NO) was assessed with the Griess assay. Results: Bevacizumab at 0.50 mg/mL decreased the permeability of carboxyfluorescein significantly (p = 0.017) and did not affect TEER (p = 0.308). Administration of 0.50 mg/mL BV decreased MMP-1 and MMP-2 activities (p = 0.014, p = 0.016, respectively) and inhibited NO production significantly (p = 0.023). Conclusions: Anti-VEGF decreased the permeability through the HTMC monolayer, which was accompanied by decreased MMP-1/-2 activity and limited NO production.

The neurotrophic and antineuroinflammatory effects of phenylpropanoids from Zanthoxylum nitidum var. tomentosum (Rutaceae)

Three novel lignans (1, 5 and 6) and two novel quinic acids (16 and 17) along with 15 known phenylpropanoids were obtained from the ethanol extract of Zanthoxylum nitidum var. tomentosum (Rutaceae). Their structures were confirmed by comprehensive spectroscopic data (NMR and HRESIMS), and the absolute configurations of all novel compounds were elucidated based on electronic circular dichroism (ECD) spectroscopic data. The production of nitric oxide (NO) in BV-2 microglial cells induced through lipopolysaccharide (LPS) was used to evaluate in vitro anti-neuroinflammatory activity of compounds 1–20. Compound 2, 3, 7 and 16 showed excellent inhibition of LPS-induced NO production. The structure-activity relationships of the isolates were investigated. In addition, the mechanism of action of 2 was elucidated by RT-PCR and Western blotting analysis, which indicated that it reduced neuroinflammatory mainly through NLRP3/caspase1 signaling pathways in LPS-induced BV2 microglial cells.

Hispidin inhibits LPS-induced nitric oxide production in BV-2 microglial cells via ROS-dependent MAPK signaling.

Neuroinflammation is associated with many neurodegenerative diseases. Abnormal activation of microglial cells in the central nervous system (CNS) is a major characteristic of neuroinflammation. Nitric oxide (NO) free radicals are produced by activated microglia and prolonged presence of large quantities of NO in the CNS can lead to neuroinflammation and disease. Hispidin is a polyphenol derived from Phellinus linteus (a valuable medicinal mushroom) with strong antioxidant, anticancer and antidiabetic properties. A previous study demonstrated that hispidin significantly inhibited NO production via lipopolysaccharide (LPS)-induced RAW264.7 macrophages. Therefore, the present study used MTT assay was used to detect the effect of hispdin on cell viability. Griess reagent analysis was used to measure NO production. Reverse transcription-semi quantitative PCR and western blotting were used to evaluate the effects of hispdin on iNOS mRNA and MAPK/ERK/JNK protein levels. Fluorescence microscopy and flow cytometry were used to detect the effects of hispdin on the production of ROS and phagocytosis of cells. The present results indicated that hispidin could significantly inhibit the increase of NO production and iNOS expression in BV-2 microglial cells stimulated by LPS. The inhibitory effect of hispidin on NO production was similar to that of S-methylisothiourea sulfate, an iNOS inhibitor. Signaling studies demonstrated that hispidin markedly suppresses LPS-induced mitogen activated protein kinases and JAK1/STAT3 activation, although not the NF-κB signaling pathway. The present observations in LPS-stimulated BV-2 microglial cells indicated that hispidin might serve as a therapeutic candidate for the treatment of NO-induced neuroinflammation and, potentially, as a novel iNOS inhibitor.

In vitro evaluation of selligueain A effects on the proinflammatory mediators production in RAW264.7 murine macrophages

Introduction: Selligueain A derived from the roots of Polypodium feei was shown to have anti-inflammatory activity, which was tested in vivo on the rats’ paw edema induced by carrageenan. The aim of this study was to evaluate the anti-inflammatory mechanism of selligueain A in vitro against the production of pro-inflammatory mediators. Methods: In this study, RAW264.7 cells were used as an inflammatory cell model, and observations were made on the inflammatory mediators nitric oxide (NO), inducible nitric oxide synthase (iNOS), and tumour necrosis factor-α (TNF-α). The NO concentration was measured by the Griess reaction, and the iNOS enzyme and the TNF-α concentrations were determined by the ELISA method. Cell viability was assessed by the [3-(4,5-dimethylthiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] (MTS) test. Results: Selligueain A at concentrations of 100 and 150 µM suppressed the production of NO, iNOS, and TNF-α in RAW264.7 cells stimulated by lipopolysaccharide (LPS). The concentration of 150 µM showed the highest inhibition of NO, iNOS, and TNF-α mediators with the percentage inhibition of 64.85, 55.01, and 48.54%, respectively. Conclusion: This study shows that selligueain A has anti-inflammatory activity through inhibition of NO, iNOS, and TNF-α production in RAW264.7 macrophage cells.

Eupomatenes A – E: Neolignans isolated from the leaves of Australian rainforest plant Eupomatia laurina

A detailed phytochemical investigation of the leaves of the Australian rainforest tree Eupomatia laurina, led to the discovery of five new neolignans, eupomatenes A – E and eight known compounds, eupomatenoid-2, trans-(2′S)-2-[1′-(4-methoxyphenyl)prop-2′-yl]anethol, chlorogenic acid, chlorogenic acid-methyl ester, tyrosol-1-O-β-xylopyranosyl-1(1 → 6)-O-β-glucopyranoside, leucoside, kaempferol-3-O-neohesperidoside, and pachypodol. The structures of all the compounds were determined by detailed spectroscopic analysis. All compounds were also evaluated for their anti-inflammatory properties by assessing their inhibitory effects on nitric oxide (NO) production and TNF- α release in RAW 264.7 macrophages. Whilst slight anti-inflammatory activity (in terms of inhibition of NO production) was observed with eupomatenes A – E, this was also associated with high levels of cell growth inhibition.

Panabipinoside A and panabipinoside B, two new oleanane triterpenoid saponins from the roots of Panax bipinnatifidus with nitric oxide inhibitory activity

Panax bipinnatifidus belongs to the ginseng genus and it is used in traditional Vietnamese and Chinese medicine. Phytochemical studies of the roots of this plant led to the isolation of two new oleanane triterpenoid saponins, panabipinoside A and panabipinoside B, and three known compounds, ginsennoside Ro, 3- O- β-D-glucopyranosyl-(1→3)- β-glucuronopyranosyl oleanolic acid, and spinasaponin A 28- O-glucoside. Their structures are established by extensive spectroscopic analysis (IR, high-resolution electrospray ionization mass spectrometry, and nuclear magnetic resonance) and by comparison of the spectral data with those reported in the literature. The anti-inflammatory activity of the isolated compounds is evaluated by their inhibition of nitric oxide production in lipopolysaccharide stimulated RAW 264.7 cells. Compounds 2–5 showed inhibitory effects on nitric oxide production with IC50 values of 0.62 ± 0.09, 0.21 ± 0.04, 0.30 ± 0.03, and 0.45 ± 0.05 µg/mL, respectively, compared to value of 8.08 ± 0.09 µg/mL for the positive control compound, NG-monomethyl-L-arginine.

Ginkgolide B‑mediated therapeutic effects on perioperative neurocognitive dysfunction are associated with the inhibition of iNOS‑mediated production of NO.

Perioperative neurocognitive dysfunction (PND) is a prevalent neurological complication after anesthesia and surgery. Ginkgolide B (GB) has been suggested to improve lipopolysaccharide-induced learning and memory impairment. The present study aimed to investigate whether GB serves a protective role against PND by inhibiting inducible nitric oxide synthase (iNOS) and nitric oxide (NO). Abdominal surgery was performed on 10- to 12-week-old male C57BL/6 mice under isoflurane anesthesia. Prior to surgery, 1400W (a specific iNOS inhibitor) and GB were administered via intraperitoneal injection. Open field and fear conditioning tests were conducted to assess cognitive function on postoperative days 1 and 3. Biochemical assays were performed to evaluate alterations in NO, malondialdehyde (MDA) and superoxide dismutase (SOD) levels. Western blotting was performed to measure iNOS expression in the hippocampus on postoperative day 1. In addition, hematoxylin and eosin staining was performed to detect the neuronal morphology in the hippocampus. Following treatment with 1400W or GB, surgery-induced cognitive dysfunction was improved. Compared with the control group, the surgery group exhibited significant overproduction of iNOS and MDA in the hippocampus on postoperative day 1. Higher levels of NO were also detected in the hippocampus and prefrontal cortex of the surgery group on postoperative day 1. Furthermore, pretreatment with 1400W or GB significantly inhibited the surgery-induced elevation of NO and MDA in brain tissues. Moreover, GB pretreatment significantly inhibited surgery-induced downregulation of SOD and upregulation of iNOS. Surgery-induced increases in neuronal loss and the Bax/Bcl-2 ratio in the hippocampus were significantly inhibited by pretreatment with GB. Collectively, the results of the present study demonstrated that the therapeutic effects of GB on PND were associated with inhibition of iNOS-induced NO production, increased SOD, and the alleviation of neuronal loss and apoptosis.

Aspilia africana C. D. Adams inhibits the production of lipopolysaccharide-induced inflammatory mediators in murine macrophage RAW264.7 cells by suppressing the NF-κB and PI3K/Akt pathways

Aspilia africana C. D. Adams (A. africana) is used in African folk medicine as a wound healing remedy. In this study, the potential anti-inflammatory effects of A. africana were investigated in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. The cytotoxicity of A. africana was assessed in RAW264.7, human embryonic kidney 293, and HaCaT cells using the WST-1 assay. The results indicated that concentrations up to 400 μg/mL were safe for all cell lines studied. Further analyses, such as the nitric oxide (NO) assay, quantitative reverse transcription polymerase chain reaction (qRT-PCR), and western blotting, were performed to evaluate the anti-inflammatory potential of A. africana. The results of NO analysis showed that A. africana inhibited NO production, while the results of western blot analysis showed that the expression levels of important inflammatory enzymes, such as inducible nitric oxide synthase and cyclooxygenase-2, and pro-inflammatory cytokines (tumor necrosis factor-α, interleukin [IL]-6, and IL-1β) were significantly suppressed by A. africana. The results also demonstrated that A. africana prevents the activation of the nuclear factor κB (NF-κB) and phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) pathways by inhibiting phosphorylated (p)-p65 NF-κB, p-Akt, p-IκBα, and p-PI3K. qRT-PCR analysis revealed that A. africana affects the expression of pro-inflammatory cytokines at the gene level. The results of this study showed that A. africana prevents the production of inflammatory mediators in RAW264.7 cells stimulated with LPS by regulating the NF-κB and PI3K/Akt signaling pathways.

In Silico Analysis of Laccifer Lacca as a Potential Therapeutic Agent for Cervical, Breast and Lung Cancers

Laccifer lacca has generally been used as pigmenting, coloring agent and dying in chemical industry. Although, it has wide range of industrial applications, but inappropriately, due to lesser availability of data, it has been ignored. Keeping in mind, the wide application of Laccifer lacca, we tried to report the in-silico anti-cancer effects. The experimental techniques used to determine the structure was X-RAY diffraction. The reported resolution of this entry is 2.80 Å. Percentile scores (ranging between 0-100) for global authentication metrics of the record. In silico have a good pool to explore various parameters in molecular docking. We have performed in silico analysis of the active components of Laccifer lacca against the cervical, breast and lung cancer proteins and also found that lac extract enhances the production of anti-inflammatory markers and the increase is significant when compared to the standard vinblastine. It has been demonstrated by Lala and colleagues that a short lived molecule nitric oxide can result in the progression of human tumours. Therefore, the prominent antioxidant activity of phytochemical that can act as inhibitors of nitric oxide production can act as anticancer therapeutics. Both methanolic and aqueous extract shows significant anticancer effect on the hela, MCF-7 & A549 cells suggesting them as potential anticancer therapeutics for future.
 Keywords: Laccifer lacca, In-vitro & In-silico analysis, Carcinogenesis, Anti-inflammatory, Molecular Docking.

Discovery of a novel inhibitor of nitric oxide production with potential therapeutic effect on acute inflammation

Inflammation as a host’s excessive immune response to stimulation, is involved in the development of numerous diseases. To discover novel anti-inflammatory agents and based on our previous synthetic work on marine natural product Chrysamide B, it and a series of derivatives were synthesized and evaluated for their anti-inflammatory activity on inhibition of LPS-induced NO production. Then the preliminary structure–activity relationships were conducted. Among them, Chrysamide B is the most potent anti-inflammatory agent with low cytotoxicity and strong inhibition on the production of NO (IC50 = 0.010 μM) and the activity of iNOS (IC50 = 0.082 μM) in LPS-stimulated RAW 264.7 cells. Primary studies suggested that the mechanism of action may be that it interfered the formation of active dimeric iNOS but not affected transcription and translation. Furthermore, its good performance of anti-inflammatory effect on LPS-induced multiple inflammatory cytokines production, carrageenan-induced paw edema, and endotoxin-induced septic mice, was observed. We believe that these findings would provide an idea for the further modification and research of these analogs in the future.

Validation of an HPLC method for quantification of anti-inflammatory markers in an ethanolic extract of Sahastara and its anti-inflammatory activity in vitro.

Background and purpose:Sahastara (SHT) is a traditional Thai medicine for the treatment of musculoskeletal and joint pain. It consists of 21 plant components. A previous study demonstrated the anti-inflammatory activity of SHT on inhibition of nitric oxide production and prostaglandin E2 (PGE2) production, however, inhibitory effects on tumor necrosis factor-alpha (TNF-α) has not been reported. In this study, we evaluated the anti-inflammatory activity of SHT on inhibitory effects on TNF-α and PGE2 production and presented an analytical method for validation of SHT.Experimental approach:Anti-inflammatory activity was evaluated by inhibitory activity on TNF-α and PGE2 production in RAW264.7 cells. The validated procedure was conducted according to ICH guidelines. The validated parameters were specificity/selectivity, linearity, range, the limit of detection (LOD), and limit of quantitation (LOQ).Findings/Results:Ethanolic extract of SHT exerted inhibitory activity on PGE2 production in RAW264.7 cells with IC50 16.97 ± 1.16 μg/mL. Myristica frangrans seed extract showed the highest inhibitory activity on PGE2 production. Piper retrofractum extract showed the highest inhibitory activity on TNF-α production. For the HPLC method, all validated parameters complied with standard requirements. Each analyzed peak showed good selectivity with a baseline resolution greater than 1.51. The linearity of all compounds was > 0.999. The % recovery of all compounds was within 98.0-102.0%. The precision of all compounds was less than 2.0% CV.Conclusion and implications:Ethanolic extracts of SHT possess anti-inflammatory activity by inhibition of TNF-α and PGE2 production in vitro. This study provides support for the traditional use of SHT. The validated results showed good specificity/selectivity, linearity, precision, and accuracy with appropriate LOD and LOQ. This study is the first report on the validation of the HPLC method of SHT for use as quality control of the SHT extract.

Ethnopharmacology, biological activities and chemical compounds of Canarium strictum: An important resin-yielding medicinal tree in India

The resin of Canarium strictum Roxb. is used for rheumatism and asthma; the bark is used as a mosquito repellent. The major compounds in the resin are triterpenoids, but as no studies have been performed on the bark, this study investigated this economically important resource. Ten folk healers were interviewed about their medicinal uses of C. strictum. Resin and bark were extracted with dichloromethane followed by methanol using accelerated solvent extraction. The extracts were fractionated using different chromatographic methods, and isolated compounds were identified by NMR spectroscopy and GC–MS. Resin and bark extracts were investigated for DPPH radical scavenging, 15-lipoxygenase inhibition, effects on nitric oxide (NO) production in LPS-activated dendritic D2SC/I cells and toxicity against Artemia salina nauplii. Traditional healers used resin to treat colds, airway afflictions and rheumatoid arthritis. α-Amyrin and β-amyrin were identified as the major constituents in the dichloromethane resin extract. From the stem bark, procyanidins, gallic acid, methyl gallate, scopoletin, 3,3′-di-O-methylellagic acid 4-O-α-arabinofuranoside and elephantorrhizol (3,3′,4′,5,6,7,8-heptahydroxyflavan) were isolated and identified. By GC–MS, α-amyrin and β-amyrin and their acetates, lupeol, and taraxasterol were identified. Radical scavenging, 15-lipoxygenase inhibitory activity and inhibition of NO production was observed from resin and bark extracts, and no toxicity towards Artemia salina nauplii was found. Triterpenoids and procyanidins are the major compounds in C. strictum resin and stem bark, respectively. The high content of triterpenoids might contribute to anti-inflammatory effects and give a rationale for the widespread usage of the resin in India.

Adipose Stromal Vascular Fraction Restores Coronary Microvascular Flow‐Mediated Dilation in Aging Female Rats via Enhanced Peroxynitrite Signaling

Introduction Coronary Microvascular Disease (CMD) presents in a majority of aging post-menopausal women and is defined by hyperconstricted tone. CMD can lead to chronic ischemia exacerbated by reduced perfusion with aging. Flow-mediated dilation (FMD) primarily regulates vascular patency via endothelial shear stress, but the signaling mechanism of FMD alters throughout the lifespan from vasoprotective nitric oxide (NO) in youth to the less efficacious hydrogen peroxide (H2O2) in advancing age. Reactive Oxygen Species (ROS), including H2O2, suppress NO, cause cellular damage and are pro-atherogenic. Adipose Stromal Vascular Fraction (SVF) is a heterogenous cell population that has antioxidant and regenerative properties. We hypothesize that coronary microvascular FMD is reduced with age, but aged rats treated with SVF (OSVF) will exhibit restored FMD via increased NO and decreased ROS at baseline and with flow. Methods Isolated coronary microvessels from young (4 months), old (24 months), or OSVF (tail vein injection 4 weeks prior to sacrifice at 24 months) from female Fischer 344 rats were assessed via pressure myography. FMD was assessed at flow rates of 5, 10, 15, 20, and 25 μL/min at physiologic pressure (45 mmHg) and temperature (37°C). Mean Fluorescence Intensity (MFI) of NO, H2O2, superoxide (O2-), peroxynitrite (ONOO-) and antioxidant glutathione (GSH) were assessed via intraluminally-infused fluorescence dyes (2,3-diaminonapthalene, mitopy1, mitosox, biotracker 515, and SEMKUR-IM, respectively) at baseline and during flow at 10 and 25 μL/min. Results Percent relaxation to intraluminal flow (FMD) is reduced with aging and restored to youthful levels by SVF (Figure 1a). At baseline, H2O2 and ONOO- MFI are significantly increased with aging compared to young and OSVF (p = < .001, .01 vs. young, < .001, .024 vs. OSVF). O2- MFI is reduced at baseline in OSVF compared to aging alone (p = .004). NO and GSH MFI are significantly reduced at baseline with aging (p = .01, < .001) and increased slightly (non-significant) with OSVF. Intraluminal flow preferentially increases MFI of NO in youth, H2O2 in aging, and ONOO- in OSVF. These data correspond functionally as FMD was significantly attenuated in youth via L-NAME inhibition of NO production, inhibited in OSVF via uric acid scavenging of ONOO-, but was unaffected by reducing H2O2 signaling via catalase in aging controls (Figure 1b-d). Conclusions FMD is abrogated with aging, contributing to CMD hyperconstriction associated with increased ROS and loss of NO and GSH. FMD can be restored with therapeutic SVF associated with increased ONOO- signaling in response to intraluminal flow. Although ONOO- is considered a ROS, it could be considered a NO reservoir inducing FMD as a NO donor since O2- and NO combine to form ONOO-. Alternatively, ONOO- can elicit FMD as an Endothelium-Derived Hyperpolarizing Factor. Future directions will differentiate between these alternative mechanisms. SVF represents a plausible future clinical direction to alleviate vascular oxidative stress and restore microvascular patency and perfusion.