Direct analysis in real time mass spectrometry (DART-MS) for rapid screening of Carpaine in Carica papaya leaf products.

Carpaine, a major alkaloid present in Carica papaya leaves, has been shown to increase platelet counts in patients suffering from thrombocytopenia. Numerous commercial papaya leaf products are available, but few provide proper bioactive ingredient information. We present herein a technique for rapid screening of carpaine in these products using DART-MS. The results indicate that carpaine was detected in various forms (powder, solution) of papaya leaves. Its presence was confirmed by examining the mass pattern when conducted on a standard solution at both low and high voltages (+10 V and +90 V), using MS1 and MS2 data obtained from LC-QTOF-MS/MS. The protonated molecule was identified at m/z = 479.38, with a fragment ion at m/z = 240.20. LOD for identifying carpaine in powder and solution matrices were 5.0 × 10-5 %w/w and 0.05 µg/mL, respectively. The proposed method has been successfully validated with the AOAC International standards and can be used to identify carpaine in papaya leaf products.

Identification of pancreatin inhibitors from Thai medicinal Piper plants for antidiabetic and anti-obesity activities using high-performance thin-layer chromatography-bioautographic assay.

Exploring the potential of natural products against diabetes and obesity is in demand nowadays. Pancreatic α-amylase and pancreatic lipase are the drug targets to minimize the absorption of glucose from starch and fatty acids from lipids, respectively. In this study, five Piper species, namely P. sarmentosum (Ps), P. wallichii (Pw), P. retrofractum (Pr), P. nigrum (Pn), and P. betle (Pb), which are commonly used as food ingredients and traditional medicines, were evaluated for their inhibitory activities against pancreatin using the microtiter plate method. Additionally, pancreatin inhibitors were identified through a cost-effective high-performance thin-layer chromatography (HPTLC)-bioautography developed using red starch and p-nitrophenyl palmitate, corresponding to anti-amylase and -lipase activities, respectively. Of the 15 samples tested, leaf samples from Pb, which had the highest total phenolic and total flavonoid contents, exhibited remarkable inhibitory activity against pancreatin, with a relative amylase inhibitory capacity (RAIC) ranging between 4.260 × 10-5 and 4.861 × 10-5 and a reciprocal half-maximal inhibitory concentration (1/IC50, PTL) of 0.390-0.510 (mg/mL)-1. Additionally, Ps samples demonstrated the second-ranked anti-pancreatin activity. Principal component analysis indicated that total phenolic content contributed to the anti-pancreatin activities of Pb samples. The anti-pancreatin bands were isolated and identified as caffeic acid, myricetin, genistein, piperine, and eugenol. Myricetin, in the roots of Ps samples, showed notable anti-pancreatin activity, which was consistent with results from the in silico prediction toward pancreatic α-amylase and pancreatic lipase. Caffeic acid and eugenol were present in Pb samples. In conclusion, the developed cost-effective pancreatin HPTLC-bioautography efficiently identified amylase and lipase inhibitors from Piper herbs, which supported the use of these plants for antidiabetes and anti-obesity.

Quantifying fecal and plasma short-chain fatty acids in healthy Thai individuals.

Short-chain fatty acids (SCFAs) are involved in important physiological processes such as gut health and immune response, and changes in SCFA levels can be indicative of disease. Despite the importance of SCFAs in human health and disease, reference values for fecal and plasma SCFA concentrations in healthy individuals are scarce. To address this gap in current knowledge, we developed a simple and reliable derivatization-free GC-TOFMS method for quantifying fecal and plasma SCFAs in healthy individuals. We targeted six linear- and seven branched-SCFAs, obtaining method recoveries of 73-88% and 83-134% in fecal and plasma matrices, respectively. The developed methods are simpler, faster, and more sensitive than previously published methods and are well suited for large-scale studies. Analysis of samples from 157 medically confirmed healthy individuals showed that the total SCFAs in the feces and plasma were 34.1 ± 15.3 µmol/g and 60.0 ± 45.9 µM, respectively. In fecal samples, acetic acid (Ace), propionic acid (Pro), and butanoic acid (But) were all significant, collectively accounting for 89% of the total SCFAs, whereas the only major SCFA in plasma samples was Ace, constituting of 93% of the total plasma SCFAs. There were no statistically significant differences in the total fecal and plasma SCFA concentrations between sexes or among age groups. The data revealed, however, a positive correlation for several nutrients, such as carbohydrate, fat, iron from vegetables, and water, to most of the targeted SCFAs. This is the first large-scale study to report SCFA reference intervals in the plasma and feces of healthy individuals, and thereby delivers valuable data for microbiome, metabolomics, and biomarker research.

Volatile organic compounds for diagnosis of early hepatocellular carcinoma in at-risk patients.

BACKGROUND: Volatile organic compounds (VOCs) have been shown as promising biomarkers for hepatocellular carcinoma (HCC) diagnosis. We aimed to investigate the performance of VOCs for diagnosing early-stage HCC in patients at-risk for HCC. METHODS: VOCs were identified in exhaled breath samples collected from 87 early-stage HCC patients, 90 cirrhotic patients, and 72 HBV-infected patients using thermal desorption-gas chromatography/field-asymmetric ion mobility spectrometry. The VOC levels were compared between the three groups. An association between VOCs and HCC was determined using logistic regression analysis. Diagnostic performance of VOCs was estimated using the AUROC and compared to serum alpha-fetoprotein (AFP). RESULTS: The levels of acetone monomer, dimethyl sulfide, 1,4-pentadiene, isopropyl alcohol, and acetone dimer were significantly different between the three groups. After adjusting for liver function test and AFP, acetone dimer was significantly associated with HCC. Acetone dimer significantly outperformed AFP with 86.2 % vs. 61.2 % sensitivity, 87.6 % vs. 66.2 % specificity, 86.9 % vs. 63.5 % for accuracy, and AUROC of 0.908 vs. 0.665, p = 0.007, <0.001, <0.001, and 0.001, respectively, for differentiating between HCC and cirrhosis. CONCLUSION: Acetone showed a better performance than AFP for diagnosing early HCC in at-risk patients. Further studies to validate the utility of VOCs as an HCC surveillance tool are needed.

Untargeted metabolomics reveal pathways associated with neuroprotective effect of oxyresveratrol in SH-SY5Y cells.

Oxyresveratrol has been documented benefits for neurodegenerative disease. However, the specific molecular mechanisms and pathways involved is currently limited. This study aimed to investigate the potential neuroprotective mechanisms of oxyresveratrol using rotenone-induced human neuroblastoma SH-SY5Y cytotoxicity. Cells were divided into the following groups: control, rotenone, and oxyresveratrol pre-treated before being exposed to rotenone. Cellular assays were performed to investigate neuroprotective effects of oxyresveratrol. The results showed that 20 µM oxyresveratrol was effective in preventing rotenone-induced cell death and decreasing ROS levels in the cells. The alteration of metabolites and pathways involved in the neuroprotective activities of oxyresveratrol were further investigated using LC-QTOF-MS/MS untargeted metabolomics approach. We hypothesized that oxyresveratrol's neuroprotective effects would be associated with neurodegenerative pathways. A total of 294 metabolites were identified. 7,8-dihydrobiopterin exhibited the highest VIP scores (VIP > 3.0; p < 0.05), thus considered a biomarker in this study. Our results demonstrated that pretreatment with oxyresveratrol upregulated the level of 7,8-dihydrobiopterin compared to the positive control. Pathway analysis verified that 7,8-dihydrobiopterin was primarily associated with phenylalanine, tyrosine, and tryptophan metabolism (impact = 1, p < 0.001), serving as essential cofactors for enzymatic function in the dopamine biosynthesis pathway. In conclusion, oxyresveratrol may be benefit for the prevention of neurodegenerative diseases by increasing 7,8-dihydrobiopterin concentration.

How do prolonged anchorage-free lifetimes strengthen non-small-cell lung cancer cells to evade anoikis? - A link with altered cellular metabolomics.

BACKGROUND: Malignant cells adopt anoikis resistance to survive anchorage-free stresses and initiate cancer metastasis. It is still unknown how varying periods of anchorage loss contribute to anoikis resistance, cell migration, and metabolic reprogramming of cancerous cells. RESULTS: Our study demonstrated that prolonging the anchorage-free lifetime of non-small-cell lung cancer NCI-H460 cells for 7 days strengthened anoikis resistance, as shown by higher half-life and capability to survive and grow without anchorage, compared to wild-type cells or those losing anchorage for 3 days. While the prolonged anchorage-free lifetime was responsible for the increased aggressive feature of survival cells to perform rapid 3-dimensional migration during the first 3 h of a transwell assay, no significant influence was observed with 2-dimensional surface migration detected at 12 and 24 h by a wound-healing method. Metabolomics analysis revealed significant alteration in the intracellular levels of six (oxalic acid, cholesterol, 1-ethylpyrrolidine, 1-(3-methylbutyl)-2,3,4,6-tetramethylbenzene, ß-alanine, and putrescine) among all 37 identified metabolites during 7 days without anchorage. Based on significance values, enrichment ratios, and impact scores of all metabolites and their associated pathways, three principal metabolic activities (non-standard amino acid metabolism, cell membrane biosynthesis, and oxidative stress response) offered potential links with anoikis resistance. CONCLUSIONS: These findings further our insights into the evolution of anoikis resistance in lung cancer cells and identify promising biomarkers for early lung cancer diagnosis.

Metabolomics approach to identify key volatile aromas in Thai colored rice cultivars.

In addition to white jasmine rice, Thailand has many native-colored rice varieties with numerous health benefits and the potential to become a global economic crop. However, the chemical characteristics of aromatic substances in native-colored rice are still mostly unknown. This study aimed to identify the key volatile aroma compounds and the biosynthetic pathways possibly involved in their formation in Thai native-colored rice varieties, and thus leading to the search for potential genetic markers for breeding colored rice with better aromatic properties. Twenty-three rice varieties in four categories: aromatic white, aromatic black, non-aromatic black, and non-aromatic red, were investigated (n=10 per variety). Seed husks were removed before the analysis of rice volatile aromas by static headspace gas chromatography-mass spectrometry. Untargeted metabolomics approach was used to discover the key volatile compounds in colored rice. Forty-eight compounds were detected. Thirty-eight of the 48 compounds significantly differed among groups at p<0.05, 28 of which at p<0.0001, with the non-aromatic black and red rice containing much lower content of most volatile constituents than the aromatic black and white rice. Focusing on the aromatic black rice, the samples appeared to contain high level of both compound groups of aldehydes (3-methylbutanal, 2-methylbutanal, 2-methylpropanal, pentanal, hexanal) and alcohols (butane-2,3-diol, pentan-1-ol, hexan-1-ol). Biosynthetically, these distinctive black-rice volatile compounds were proposed to be formed from the metabolic degradation of branched-chain amino acids (L-leucine, L-isoleucine and L-valine) and polyunsaturated fatty acids (linoleic acid and α-linolenic acid), involving the branched-chain aminotransferases and keto-acid decarboxylases and the 9-lipoxygonases and 13-lipoxygeases, respectively. The proposed degradative pathways of amino acids and fatty acids were well agreed with the profiles key volatile compounds detected in the Thai native-colored rice varieties.

Exhaled volatile organic compounds for diagnosis of hepatocellular carcinoma.

Volatile organic compounds (VOCs) profile for diagnosis and monitoring therapeutic response of hepatocellular carcinoma (HCC) has not been well studied. We determined VOCs profile in exhaled breath of 97 HCC patients and 111 controls using gas chromatography-mass spectrometry and Support Vector Machine algorithm. The combination of acetone, 1,4-pentadiene, methylene chloride, benzene, phenol and allyl methyl sulfide provided the highest accuracy of 79.6%, with 76.5% sensitivity and 82.7% specificity in the training set; and 55.4% accuracy, 44.0% sensitivity, and 75.0% specificity in the test set. This combination was correlated with the HCC stages demonstrating by the increased distance from the classification boundary when the stage advanced. For early HCC detection, d-limonene provided a 62.8% sensitivity, 51.8% specificity and 54.9% accuracy. The levels of acetone, butane and dimethyl sulfide were significantly altered after treatment. Patients with complete response had a greater decreased acetone level than those with remaining tumor post-treatment (73.38 ± 56.76 vs. 17.11 ± 58.86 (× 106 AU, p = 0.006). Using a cutoff of 35.9 × 106 AU, the reduction in acetone level predicted treatment response with 77.3% sensitivity, 83.3% specificity, 79.4%, accuracy, and AUC of 0.784. This study demonstrates the feasibility of exhaled VOCs as a non-invasive tool for diagnosis, monitoring of HCC progression and treatment response.

Pharmacokinetics and metabolomics investigation of an orally modified formula of standardized Centella asiatica extract in healthy volunteers.

The formula of a standardized extract of Centella asiatica (ECa 233) was modified to improve its dissolution, with implications for pharmacokinetics and metabolomic profile. This study aimed to understand the resultant changes in disposition kinetics of ECa 233 and alterations to human metabolome after oral administration. This study was a two-sequence of dosages (250 and 500 mg), with an open-label phase I clinical trial. The modified formula was administered in single and multiple doses to twelve healthy Thai volunteers. The major parent compounds, madecassoside and asiaticoside, were rarely absorbed, instead undergoing biotransformation into active metabolites, madecassic acid and asiatic acid with possibility to be eliminated via fecal route. Increasing the dose of ECa 233 resulted in significantly greater plasma levels of those active metabolites, with accumulation of asiatic acid after multiple oral administration for seven days. Examining the impacts of accumulation behavior on metabolomics, the study traced changes in levels pre- and post-dose of five relevant human metabolites. Administration of ECa 233 was found to be significantly associated with an increase of choline, an endogenous metabolite with documented benefits for learning and memory. Therefore, ECa 233 may be useful in mitigating cognitive impairment, through its role in modulating human metabolites.

Effects of Centella asiatica extract on antioxidant status and liver metabolome of rotenone-treated rats using GC-MS.

Centella asiatica has been used as a culinary vegetable or medicinal herb. In this study, the hepatoprotective effect of the standardized extract of C. asiatica (ECa233) in rotenone-treated rats was examined using a GC-MS-based metabolomic approach. ECa233 contains >80% triterpenoids with a ratio of madecassoside to asiaticoside of 1.5(±0.5):1. Rats were randomly divided into three groups (with six rats/group): sham negative control, rotenone positive control and the ECa233 test group. Rats in the ECa233 group received 10 mg/kg ECa233 orally for 20 days, followed by 2.5 mg/kg intraperitoneal rotenone injection to induce toxicity before being sacrificed. Metabolomic analysis showed that supplementation of ECa233 protected rat liver against rotenone toxicity. Pipecolinic acid was one of the most important metabolites; its level was decreased in the rotenone group as compared with the control. Supplementation with ECa233 before administration of rotenone raised pipecolinic acid to levels intermediate between controls and rotenone alone. The metabolomics approach also helped discover a possible new genuine epimetabolite in the present work. Antioxidant tests revealed that ECa233 inhibited lipid peroxidation and increased catalase activities in liver tissue.

Neuroprotective effect of a standardized extract of Centella asiatica ECa233 in rotenone-induced parkinsonism rats.

BACKGROUND: Mitochondrial dysfunction and reactive oxygen species (ROS) generation cause dopaminergic neurodegeneration in Parkinson's disease. The neuroprotective approach is a promising strategy to slow disease progression in Parkinson's disease. A standardized extract of Centella asiatica ECa233 has been previously reported to have pharmacological effects in the central nervous system. PURPOSE: This study aimed to determine the neuroprotective effect and mechanisms of ECa233 in rotenone-induced parkinsonism rats. METHODS: Rats were orally given either vehicle or ECa233 (10, 30 and 100 mg/kg) for 20 consecutive days. Rotenone (2.5 mg/kg i.p.) was given to parkinsonism (PD) and ECa-treated rats from day 15 to 20. Locomotor activity was recorded on day 1, 14, 17 and 20. Tyrosine-hydroxylase (TH) immunohistological staining was used to determine dopaminergic neurons in the substantia nigra and striatum. Furthermore, mitochondrial complex I activity, malondialdehyde (MDA) levels, superoxide dismutase (SOD) and catalase protein expression were measured in brain tissue. RESULTS: Rats receiving ECa233 30 mg/kg showed a significant increase in distances (p < 0.01) together with a higher number and intensity of dopaminergic neurons in the substantia nigra and striatum (p < 0.001) compared to PD rats. ECa233 (30 mg/kg) protected against mitochondrial complex I inhibition, decreased MDA levels (p < 0.05) and increased SOD (p < 0.01) and catalase (p < 0.05) expression. CONCLUSION: ECa233 can protect against rotenone-induced motor deficits and dopaminergic neuronal death. These effects are mediated through the protection of mitochondrial complex I activity, the effects of antioxidants and the enhancement of antioxidant enzyme expression.

Development of a beef flavor lexicon and its application to compare the flavor profile and consumer acceptance of rib steaks from grass- or grain-fed cattle.

Ten panelists were selected from the local community to develop a meat lexicon composed of 18 terms that describe flavor attributes found in red meats. This flavor lexicon was used to compare the flavor profile of meat from beef cattle finished on grass or grain. Steaks from grass-fed animals were significantly (P<0.05) higher in barny, bitter, gamey, and grassy flavor, and lower in juicy and umami notes. Gamey, barny, bitter and grassy were some of the attributes inversely correlated to the degree of liking of the meat and therefore can be classified as "negative" attributes. Brothy, umami, roast beef, juicy, browned, fatty and salty are some of the attributes positively correlated to the degree of liking of beef and therefore can be identified as attributes that drive consumers' acceptance. Steaks from grass-fed cattle were rated by consumers as slightly liked (6.08 on a 9-point scale), while steaks from grain-fed animals were rated as moderately liked (7.05 on a 9-point scale).