NMR fingerprinting of depolymerised durian rind pectin and its antibacterial activity.

This study investigated the effect of commercial sterilisation treatment (121 °C for 15 min) on durian rind pectin. The change of structure was observed by NMR (nuclear magnetic resonance) and the antibacterial activity was assessed by microdilution method to obtain the minimum inhibitory concentration (MIC) value. NMR spectra revealed the rhamnogalacturonan-I and homogalacturonan structure, with lower methyl-ester in sterilised pectin. Native pectin was unable to inhibit Escherichia coli and Staphylococcus aureus at the highest tested concentration (MIC > 25 mg/mL), but sterilised pectin showed inhibitory effect against E.coli (MIC 12.5 mg/mL) and S. aureus (MIC 6.25 mg/mL). Membrane filtration to obtain fraction < 20 kDa enhanced the inhibition against S. aureus further, but not for E.coli. The antibacterial effect was possibly correlated to the decrease of molecular weight (MW) and degree of esterification (DE) of durian rind pectin. E. coli was more resistant to pectin than S. aureus.

Untargeted metabolomics using liquid chromatography-high resolution mass spectrometry and chemometrics for analysis of non-halal meats adulteration in beef meat.

OBJECTIVE: The adulteration of raw beef (BMr) with dog meat (DMr) and pork (PMr) becomes a serious problem because it is associated with halal status, quality, and safety of meats. This research aimed to develop an effective authentication method to detect non-halal meats (dog meat and pork) in beef using metabolomics approach. METHODS: Liquid chromatography-high resolution mass spectrometry (LC-HRMS) using untargeted approach combined with chemometrics was applied for analysis non-halal meats in BMr. RESULTS: The untargeted metabolomics approach successfully identified various metabolites in BMr DMr, PMr, and their mixtures. The discrimination and classification between authentic BMr and those adulterated with DMr and PMr were successfully determined using partial least square-discriminant analysis (PLS-DA) with high accuracy. All BMr samples containing non-halal meats could be differentiated from authentic BMr. A number of discriminating metabolites with potential as biomarkers to discriminate BMr in the mixtures with DMr and PMr could be identified from the analysis of variable importance for projection value. Partial least square (PLS) and orthogonal PLS (OPLS) regression using discriminating metabolites showed high accuracy (R2>0.990) and high precision (both RMSEC and RMSEE <5%) in predicting the concentration of DMr and PMr present in beef indicating that the discriminating metabolites were good predictors. The developed untargeted LC-HRMS metabolomics and chemometrics successfully identified non-halal meats adulteration (DMr and PMr) in beef with high sensitivity up to 0.1% (w/w). CONCLUSION: A combination of LC-HRMS untargeted metabolomic and chemometrics promises to be an effective analytical technique for halal authenticity testing of meats. This method could be further standardized and proposed as a method for halal authentication of meats.

Exploration of the main active metabolites from Tinospora crispa (L.) Hook. f. & Thomson stem as insulin sensitizer in L6.C11 skeletal muscle cell by integrating in vitro, metabolomics, and molecular docking.

ETHNOPHARMACOLOGICAL RELEVANCE: Tinospora crispa (L.) Hook. f. & Thomson stem (TCS) has long been used as folk medicine for the treatment of diabetes mellitus. Previous study revealed that TCS possesses multi-ingredients and multi-targets characteristic potential as insulin sensitizer activity. However, its mechanisms of action and molecular targets are still obscure. AIM OF THE STUDY: In the present study, we investigated the effects of TCS against insulin resistance in muscle cells through integrating in vitro experiment and identifying its active biomarker using metabolomics and in molecular docking validation. MATERIALS AND METHODS: We used centrifugal partition chromatography (CPC) to isolate 33 fractions from methanolic extract of TCS, and then used UHPLC-Orbitrap-HRMS to identify the detectable metabolites in each fraction. We assessed the insulin sensitization activity of each fraction using enzyme-linked immunosorbent assay (ELISA), and then used confocal immunocytochemistry microscopy to measure the translocation of glucose transporter 4 (GLUT4) to the cell membrane. The identified active metabolites were further simulated for its molecular docking interaction using Autodock Tools. RESULTS: The polar fractions of TCS significantly increased insulin sensitivity, as measured by the inhibition of phosphorylated insulin receptor substrate-1 (pIRS1) at serine-312 residue (ser312) also the increasing number of translocated GLUT4 and glycogen content. We identified 58 metabolites of TCS, including glycosides, flavonoids, alkaloids, coumarins, and nucleotides groups. The metabolomics and molecular docking simulations showed the presence of minor metabolites consisting of tinoscorside D, higenamine, and tinoscorside A as the active compounds. CONCLUSIONS: Our findings suggest that TCS is a promising new treatment for insulin resistance and the identification of the active metabolites in TCS could lead to the development of new drugs therapies for diabetes that target these pathways.

Analysis of Pork in Beef Sausages Using LC-Orbitrap HRMS Untargeted Metabolomics Combined with Chemometrics for Halal Authentication Study.

Beef sausage (BS) is one of the most favored meat products due to its nutrition and good taste. However, for economic purposes, BS is often adulterated with pork by unethical players. Pork consumption is strictly prohibited for religions including Islam and Judaism. Therefore, advanced detection methods are highly required to warrant the halal authenticity of BS. This research aimed to develop a liquid chromatography-high-resolution mass spectrometry (LC-HRMS) method to determine the halal authenticity of BS using an untargeted metabolomics approach. LC-HRMS was capable of detecting various metabolites in BS and BS containing pork. The presence of pork in BS could be differentiated using principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA) with high accuracy. PLS-DA perfectly classified authentic BS and BS containing pork in all concentration levels of pork with R2X = (0.821), R2Y(= 0.984), and Q2 = (0.795). The level of pork in BS was successfully predicted through partial least squares (PLS) and orthogonal PLS (OPLS) chemometrics. Both models gave high R2 (>0.99) actual and predicted values as well as few errors, indicating good accuracy and precision. Identification of discriminating metabolites' potential as biomarker candidates through variable importance for projections (VIP) value revealed metabolites of 2-arachidonyl-sn-glycero-3-phosphoethanolamine, 3-hydroxyoctanoylcarnitine, 8Z,11Z,14Z-eicosatrienoic acid, D-(+)-galactose, oleamide, 3-hydroxyhexadecanoylcarnitine, arachidonic acid, and α-eleostearic acid as good indicators to detect pork. It can be concluded that LC-HRMS metabolomics combined with PCA, PLS-DA, PLS, and OPLS was successfully used to detect pork adulteration in beef sausages. The results imply that LC-HRMS untargeted metabolomics in combination with chemometrics is a promising alternative as an analytical technique to detect pork in sausage products. Further analysis of larger samples is required to warrant the reproducibility.

Dietary supplementation of black rice bran to colon carcinogen-induced mice: Examining the development of colorectal cancer by improving environmental colon conditions.

This research aims to identify the effects of the administration of a black rice bran diet on colorectal cancer in dextran sodium sulfate and azoxymethane-induced BALB/c mice. The research was conducted on three groups consisting of eight Balb/c mice: two groups were fed with carcinogens, and the third group, referred to as the normal group, was supplied with Isotonic NaCl 0.9% intraperitoneally. One group fed with carcinogens was supplied a standard AIN 1993 M diet modified with black rice bran as a substitute of fibre source, while the other two mice groups were fed the standard diet (AIN-93M) containing cellulose fibre. At the 17th week, all mice were euthanized; their colonic sections were taken for histopathological evaluation, and cecum for short-chain fatty acids concentration, total lactic acid bacteria, pH and ß-glucuronidase activity evaluations. The results show an increase in the total lactic acid bacteria and short-chain fatty acids in the mice group fed with rice bran. Consequently, pH value and ß-glucuronidase activity had decreased. Histopathological evaluation of mucosal tissue exhibited inhibition of the tumor growth rate in the mice groups fed rice bran compared to the group supplied with the standard diet. Furthermore, the proliferating cell nuclear antigen expression had decreased significantly, while expression of caspase-8 and caspase-3 had increased notably, in the group fed with a rice bran diet. These results suggest that black rice bran can effectively inhibit colon carcinogenesis. The potential of black rice bran as a source of fibre has not been studied in detail regarding the inhibition mechanism of colorectal cancer cells; further investigation in this field could provide valuable information about new strategies to prevent colorectal cancer. This strand of research is very important to developing preventive methods against cancer and promoting the concept of healthy products, including functional foods.

Metabolomics Study Reveals Biomarker L-Proline as Potential Stress-Protectant Compound for High-Temperature Bioethanol Fermentation by Yeast Pichia kudriavzevii 1P4.

High-temperature ethanol fermentation (> 40 °C) can be applied as effective bioprocess technology to increase ethanol production. Thermotolerant yeast Pichia kudriavzevii 1P4 showed the ability to produce ethanol at optimum 37 °C. Thus, this study evaluated the ethanol productivity of isolate 1P4 at high-temperature ethanol fermentation (42 and 45 °C) and the identification of metabolite biomarkers using untargeted metabolomics with liquid chromatography-tandem mass spectrometry (LC-MS/MS). 1P4 showed tolerance to temperature stress up to 45 °C and thus relevant for high-temperature fermentation. As measured by gas chromatography (GC), bioethanol production of 1P4 at 30, 37, 42, and 45 °C was 5.8 g/l, 7.1 g/l, 5.1 g/l, and 2.8 g/l, respectively. The classification of biomarker compounds was based on orthogonal projection analysis to latent structure discriminant analysis (OPLS-DA), resulting in L-proline being a suspected biomarker compound for isolate 1P4 tolerance against high-temperature stress. Indeed, supplementation of L-proline on fermentation medium supported the growth of 1P4 at high temperatures (> 40 °C) than without L-proline. The bioethanol production with the addition of the L-proline resulted in the highest ethanol concentration (7.15 g/l) at 42 °C. Supplementation of L-proline as a stress-protective compound increased ethanol productivity at high-temperature fermentation of 42 and 45 °C by 36.35% and 83.33%, respectively, compared without the addition of L-proline. Preliminary interpretation of these results indicates that bioprocess engineering through supplementation of stress-protective compounds L-proline increases the fermentation efficiency of isolate 1P4 at higher temperatures (42 °C and 45 °C).

Detection of Pork in Beef Meatballs Using LC-HRMS Based Untargeted Metabolomics and Chemometrics for Halal Authentication.

Adulteration of high-quality meat products using lower-priced meats, such as pork, is a crucial issue that could harm consumers. The consumption of pork is strictly forbidden in certain religions, such as Islam and Judaism. Therefore, the objective of this research was to develop untargeted metabolomics using liquid chromatography-high resolution mass spectrometry (LC-HRMS) combined with chemometrics for analysis of pork in beef meatballs for halal authentication. We investigated the use of non-targeted LC-HRMS as a method to detect such food adulteration. As a proof of concept using six technical replicates of pooled samples from beef and pork meat, we could show that metabolomics using LC-HRMS could be used for high-throughput screening of metabolites in meatballs made from beef and pork. Chemometrics of principal component analysis (PCA) was successfully used to differentiate beef meatballs and pork meatball samples. Partial least square-discriminant analysis (PLS-DA) clearly discriminated between halal and non-halal beef meatball samples with 100% accuracy. Orthogonal projection to latent structures-discriminant analysis (OPLS-DA) perfectly discriminated and classified meatballs made from beef, pork, and a mixture of beef-pork with a good level of fitness (R2X = 0.88, R2Y = 0.71) and good predictivity (Q2 = 0.55). Partial least square (PLS) and orthogonal PLS (OPLS) were successfully applied to predict the concentration of pork present in beef meatballs with high accuracy (R2 = 0.99) and high precision. Thirty-five potential metabolite markers were identified through VIP (variable important for projections) analysis. Metabolites of 1-(1Z-hexadecenyl)-sn-glycero-3-phosphocholine, acetyl-l-carnitine, dl-carnitine, anserine, hypoxanthine, linoleic acid, and prolylleucine had important roles for predicting pork in beef meatballs through S-line plot analysis. It can be concluded that a combination of untargeted metabolomics using LC-HRMS and chemometrics is promising to be developed as a standard analytical method for halal authentication of highly processed meat products.

Volatile compounds, texture, and color characterization of meatballs made from beef, rat, wild boar, and their mixtures.

The purpose of this research was to characterize the volatile compounds, texture, and color profile of meatballs made from beef, rat, wild boar, and their combinations. Volatile compounds were analyzed using SPME/GC-MS and multivariate data analysis (PCA, PLS-DA). Additionally, several textural features such as hardness, gumminess, chewiness, cohesiveness, and colour (L, a∗, b∗, C, and h) were also analyzed. The findings revealed that texture and color characteristics can only be used to differentiate meatballs based on their raw meat materials when meat adulterants are used in high concentrations (≥50%). PLS-DA analysis of volatile data revealed distinct groupings among various types of meatballs, including meatballs adulterated with rat or wild boar meat at the lowest percentage used in this study (20%). By using VIP and correlation coefficient, the strongest markers in beef, rat, and wild boar meatballs were identified as (Z)-2-amino-5-methyl-benzoic acid, 2-heptenal, and cyclobutanol, respectively. Nonanal was consistently found as a significant marker in the meatballs made from a mixture of beef-rat and beef-wild boar at different ratios. This study demonstrated that the volatile profile of meat is more reliable than physicochemical profiles for developing an analytical tool for quickly identifying undesired meat in meat-derived products.

Using metabolomics to discover the immunomodulator activity of food plants.

Many edible plants exhibit immunomodulator activities that have beneficial effects on human health. These activities include the ability to activate, multiply, or suppress elements of the immune response. Some of these plants promote health by strengthening host defences against different diseases. In this article, we provide a comprehensive review of the constituents of several edible plants, their immunomodulatory activity, and mechanism of actions for Carica papaya, Coffea sp, Asparagus cochinchinensis, Dioscorea alata, beans, mushrooms, herbs, spices, and several vegetables. The studies reported here are pre-clinical (in vitro and in vivo) and clinical studies (limited in number). The bioactive compounds responsible for the immunomodulator activity of these plants were yet to be identified. This is because the plant is naturally a complex mixture, whilst the immune system is also an intricate system involving many cells and cytokines/chemokines. Metabolomics is a key tool for conducting global profiling of metabolites in a complex system. Therefore, it offers the ability to identify the presence of compounds in plant extracts associated with their immunomodulation effects. Likewise, metabolomics can also be used to detect any changes to metabolites in the cell as a response to treatment. Therefore, affected metabolic pathways that lead to the activation of certain immune responses can be determined from one single experiment. However, we found in this review that the use of a metabolomics approach is not yet fully developed for an immunomodulator study of food plants. This is important for the direction of future research in this field because unlike medicinal plants, food plants are consumed on a regular basis in small amounts with more obvious effects on the immune system. Information about possible bioactive compounds, their interactions (synergism, antagonism), and how the human body responds to them should be studied in a more holistic way.

Network pharmacology integrated molecular dynamics reveals the bioactive compounds and potential targets of Tinospora crispa Linn. as insulin sensitizer.

Insulin resistance is a metabolic disorder characterized by the decreased response to insulin in muscle, liver, and adipose cells. This condition remains a complex phenomenon that involves several genetic defects and environmental stresses. In the present study, we investigated the mechanism of known phytochemical constituents of Tinospora crispa and its interaction with insulin-resistant target proteins by using network pharmacology, molecular docking, and molecular dynamics (MD) simulation. Tinoscorside A, Makisterone C, Borapetoside A and B, and ß sitosterol consider the main phytoconstituents of Tinospora crispa by its binding with active sites of main protein targets of insulin resistance potential therapy. Moreover, Tinoscorside A was revealed from the docking analysis as the ligand that binds most strongly to the target protein, PI3K. This finding was strengthened by the results of MD simulation, which stated that the conformational stability of the ligand-protein complex was achieved at 15 ns and the formation of hydrogen bonds at the active site. In conclusion, Tinospora crispa is one of the promising therapeutic agent in type 2 diabetes mellitus management. Regulation in glucose homeostasis, adipolysis, cell proliferation, and antiapoptosis are predicted to be the critical mechanism of Tinospora crispa as an insulin sensitizer.

Measuring the health effects of food by metabolomics.

Metabolomics of human biological fluids or tissues is used to discover markers for diseases by comparing the metabolome of the patients against healthy individuals. Ultimately, these markers can be used in drug discovery to determine how medications normalize (at least in part) the human metabolome at specific disease stages to homeostatic. Likewise, the health effects of food can be studied. Even metabolomics of the food can be combined with metabolomics of the treated patients to correlate compounds from food with measurable health effects from clinical studies. Various chemometric analyses of these metabolomics data are used to identify markers for diseases and to obtain evidence for health effects. This review discusses recent researches (published from 2013 to 2021) on whether specific dietary intervention to humans suffering from metabolic disorders may improve their pathological status. The scope is limited to those associated with major lifestyle diseases such as diabetes, obesity, and cardiovascular diseases, for which food is thought may have detrimental as well as beneficial effects on human health. It includes metabolites characterization of different biological samples such as the human serum/plasma, urine, saliva, feces, or ileal fluid. Whether the study results supported the claimed health benefits and whether the research was conducted with appropriate study design, was criticized.

α-glucosidase inhibitors from Syzygium polyanthum (Wight) Walp leaves as revealed by metabolomics and in silico approaches.

ETHNOPHARMACOLOGICAL RELEVANCE: Syzygium polyanthum (Wight) Walp leaves are traditionally used to cure diabetes in many regions of Indonesia. Traditional use involves boiling the leaves until the water is reduced to half volume, and then the decoction is taken 1-2 times daily. Despite several studies reporting the antidiabetic activity of this plant, bioactive compounds have not been well identified. AIM OF THE STUDY: Indonesia is one of the countries with the highest diabetes cases, particularly type 2 diabetes mellitus (T2DM). Few people have access to modern medicinal treatment; thus, the role of antidiabetic traditional medicine has become increasingly important. This research aimed to identify α-glucosidase inhibitors from S. polyathum leaves using a metabolomics approach. When the active compounds of S. polyathum are properly identified, the quality of the herb can be more easily controlled. MATERIALS AND METHODS: The dried leaves of S. polyanthum were extracted by a comprehensive extraction method using a solvent combination of n-hexane, acetone, and water in a gradient, resulting in a total of 42 fractions. All fractions were subjected to an in vitro α-glucosidase inhibition test and chemical profile analysis using Nuclear Magnetic Resonance (NMR) and high performance liquid chromatography (HPLC). Orthogonal projection least square (OPLS) analysis was used to correlate the two data to identify NMR signals, and HPLC chromatogram peaks correlated to the activity. 2D NMR and ultra-high-performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS) analyses were also used to give more precise compound identification. The activity of the identified active compounds was confirmed by an in silico technique. RESULTS AND DISCUSSION: The results of the α-glucosidase activity test showed that the most active fractions were obtained from solvents with medium polarity: Fractions 9 and 10 (F9 and F10), obtained from gradient acetone-water 4:1 and 3:2, respectively. The IC50 values of F9 and F10 were 24.8 and 31.8 µg/mL, respectively. NMR data showed that F9 had more intense and diverse signals in the aromatic region than F10. OPLS analysis results showed that some typical flavonoid signals abundant in F9 positively correlated with α-glucosidase activity. 2D NMR and UHPLC-HRMS analysis of F9 led to the conclusion that these signals could be attributed to myricetin-3-O-rhamnoside (myricitrin) and epigallocatechin-3-gallate (EGCG). In silico analysis confirmed these results, as myricitrin and EGCG had binding energies resembling acarbose as a positive control (-8.47, -8.19, and -10.13, respectively). CONCLUSIONS: NMR and HPLC-metabolomics successfully identified myricitrin and EGCG as α-glucosidase inhibitors from S. polyanthum leaves, and docking analysis validated their inhibitory activity. The results of this study justified the traditional use of S. polyanthum as an antidiabetes herbal.

Investigation of Yacon Leaves (Smallanthus sonchifolius) for α-Glucosidase Inhibitors Using Metabolomics and In Silico Approach.

Yacon (Smallanthus sonchifolius (Poepp.) H. Robinson) leaves is traditionally consumed as herbal tea in many countries including Indonesia. This plant's antidiabetic properties have been extensively researched, but studies on the responsible active compound identification are scarce. Information on the active compounds is critical for the consistency of Yacon herbal tea quality. The aim of this study was to identify α-glucosidase inhibitors in Indonesian Yacon leaves grown in two different locations using FTIR- and LC-MS/MS-based metabolomics in combination with in silico technique. Yacon leaves ethanol (50 and 95%) and water extracts were tested for α-glucosidase inhibitory activity, with the 95% ethanol extract being the most active. Geographical origins were found to have no major impact on the activity. In parallel, chemical profile of Yacon leaves extract was determined using FTIR and LC-MS/MS. Orthogonal Projection to Latent Structure (OPLS) was used to analyze both sets of data. OPLS analysis of FTIR data showed that compounds associated to α-glucosidase inhibitor activity included those with functional groups -OH, stretched CH, carbonyl, and alkene. It was consistent with the result of OPLS analysis of LC-MS/MS data, which revealed that based on their VIP and Y-related coefficient value, nystose, 1-kestose, luteolin-3'-7-di-O-glucoside, and 1,3-O-dicaffeoilquinic acid isomers, strongly linked to Yacon's α-glucosidase inhibitor activity. In silico study supported these findings, revealing that the four compounds were potent α-glucosidase inhibitors with docking score in the range of - 100.216 to - 115.657 kcal/mol, which are similar to acarbose (- 115.774 kcal/mol) as a reference drug.

Immunomodulatory and Antioxidant Activities of Select Indonesian Vegetables, Herbs, and Spices on Human Lymphocytes.

Edible plants have attracted increasing attention as functional foods as they are rich in bioactive compounds with health benefits, including antioxidant and immunomodulatory activities. However, scientific evidence of these health effects is limited. This study is aimed at determining antioxidant and immunomodulatory activities of 25 select vegetables, herbs, and spices commonly consumed in Indonesia. Phytochemical profiles were determined by measuring total flavonoid content and 1H-NMR. Human blood lymphocyte cells were used to probe the immunomodulatory potency and treated with the methanol extract of these vegetables, herbs, and spices. The results showed the enhanced propensity for all tested plant extracts to stimulate lymphocyte proliferation, except Pandanus amaryllifolius. Etlingera elatior, Ocimum xcitriodorum, Kaempferia galanga, and Apium graveolens had the highest lymphocyte cell proliferation stimulation index (SI) at concentrations of 41.67, 16.67, 4.17, and 2.5 mg/mL culture, respectively (SI 2.21 ± 0.05, 2.62 ± 0.12, 3 ± 0.05, and 2.64 ± 0.07, respectively). The NMR spectra of these four most potent plants showed low peaks in the aromatic/phenolic area and several other peaks indicating the presence of terpenoid, steroid, amino acid, and sugar compounds. The results demonstrate the immunomodulatory potential of all vegetables, herbs, and spices, except P. amaryllifolius, although this potential did not necessarily correlate with flavonoid content and antioxidant activity. Nevertheless, this research showed promising health effect, particularly immunomodulation, of the various local plants. Further elaboration on the specific immunomodulatory activity will be interesting.

Emulsifying properties and antioxidant activity of soy protein isolate conjugated with tea polyphenol extracts.

Functional properties of proteins, such as emulsification, foam formation and antioxidant activity, can be improved by conjugating the proteins with phenolic substances. We reported here changes in the structural, physicochemical and functional properties of Soy Protein Isolate (SPI) after conjugation with phenolic substances in the green tea and black tea extracts. Conjugation of SPI with tea extracts were conducted using alkaline treatment (pH 9.0) followed by air exposure. The results showed that conjugation of SPI increased the protein molecular size and decreased the protein hydrophobicity. The hydrophobic decreasing effect by the treatment was larger with the black tea extract than by green tea extract. SPI-tea polyphenol conjugates significantly (p < 0.05) increased the emulsifying ability of SPI up to 43% and the emulsifying stability of SPI up to 59%. SPI-tea polyphenol conjugates which was prepared using 0.75% (w/w SPI) green tea polyphenol extract showed the best emulsifying properties with strong repulsion forces between the droplets, smaller emulsion droplet size and lower polydispersity index of droplets size distribution. Although the conjugation product is still inferior to egg lecithin in emulsion stability, antioxidant activity of SPI was significantly (p < 0.05) improved in a concentration dependant manner. SPI-black tea polyphenol conjugates showed greater antioxidant activity than SPI-green tea polyphenol conjugate. The present study shows the feasibility and benefits of the use of SPI-tea polyphenol conjugates as a food emulsifier.

GC-MS metabolomics revealed protocatechuic acid as a cytotoxic and apoptosis-inducing compound from black rice brans.

GC-MS metabolomics was used to discriminate the phytochemicals profile of Indonesian white, red, and black rice brans, and Japanese white rice brans. This technique was used for the first time to identify compounds in rice brans having cytotoxic activity against WiDr colon cancer cells. Orthogonal Projection to the Latent Structure (OPLS) analysis showed that protocatechuic acid (PA) was a discriminating factor found in black rice brans which strongly correlated with its cytotoxicity (IC50 8.53 ± 0.26 µM). Real time-PCR data demonstrated that PA cytotoxicity at different concentrations (1, 5, 10, 25 and 50 µg/mL) was mediated through different pathways. Bcl-2 expression was downregulated at all tested concentrations indicating apoptosis stimulation. At 1-10 ppm concentration, PA activated both intrinsic and extrinsic apoptosis pathways since the expression of p53, Bax, caspase-8, and caspase-9 were upregulated. At a higher dose (25 and 50 µg/mL), PA possibly involved in pyroptosis-mediated pro-inflammatory cell death by upregulating the expression of caspase-1 and caspase-7.

Methanol Extract of Coleus amboinicus (Lour) Exhibited Antiproliferative Activity and Induced Programmed Cell Death in Colon Cancer Cell WiDr.

Coleus amboinicus(Lour) (CA) has been reported to possess many pharmacological activities. In this study, evaluation of cytotoxicity using brine shrimp lethality bioassay and MTT assay using WiDr cell lines was carried out. The expression of several genes responsible for programmed cell death of the methanol extract of CA was also investigated. The morphology of the cells undergoing apoptosis was detected using Hoechst staining assay. The gene expression of BAX, BCL2, P53, Caspase 1, 7, 8, and 9 of treated samples with different concentrations (10, 15, 25 & 50 µg/ml) were measured with RT PCR. The phytochemical profiles were investigated using LC MS. The results showed that the lethality concentration (LC50) of methanol extract using brine shrimp was 34.545 µg/ml and the extract exhibited good antiproliferative activity against cancer cells WiDr with IC50 value (8.598 ± 2.68 µg/ml) as compared to standard drug 5-fluorouracil (IC50 value 1.839 ± 0.03 µg/ml). There was apoptotic evidences from the morphology of treated cells. The expressions of BAX,P53, and Caspase 9 were upregulated in lower concentration of the extract (10 and 15 µg/ml) but downregulated in higher concentration (25 and 50 µg/ml). BCL2 as anti-apoptotic gene was downregulated in all concentrations. Caspase 1 and Caspase 7 were upregulated in high concentration (25 and 50 µg/ml), but downregulated in lower concentrations. These data provide a mode of cell death for the methanol extract of CA in low concentrations corresponding to apoptosis with intrinsic pathway. Many valuable compounds identified including caffeic acid, rosmarinic acid, malic acid, eicosapentanoic acid, benserazide, alpha-linolenic acid, betaine, Salvanolic B, 4-hydroxibenzoic acid and firulic acid have been previously reported as being active agents against many cancer cells. This study suggested that CA might become an effective ingredient for health-beneficial foods to prevent colon cancer.

Metabolomics approach for determining potential metabolites correlated with sensory attributes of Melaleuca cajuputi essential oil, a promising flavor ingredient.

Melaleuca cajuputi subsp. cajuputi is one of the Australian Melaleuca species commonly found in Pulau Buru (Maluku, Indonesia). Its oil, the M. cajuputi essential oil (MCEO), has been utilized as the main flavor of the Indonesian functional food, Cajuputs Candy. However, the availability of MCEO is becoming limited. On the other hand, Indonesia has many other potential MCEO sources which can be developed as flavor ingredient. Thus, it is noteworthy to explore these new MCEO sources by studying their sensory characteristics and metabolite profiles. This study was conducted to identify potential metabolites that are correlated to sensory attributes of MCEO by using the metabolomics approach. The metabolite profiles of thirteen MCEOs from different origins were analyzed by gas chromatography-mass spectrometry while sensory analyses on Cajuputs Candy were conducted by difference-from-control and rate-all-that-apply tests. Sixty metabolites from the MCEO were annotated that includes 1,8-cineole, α-terpineol, caryophyllene, α-pinene, and γ-terpinene. Sensory analysis revealed cooling aftertaste and sweet taste as favorable attributes. Further analysis using Orthogonal Partial Least Square indicated that 1,8-cineole and γ-terpinene were correlated with cooling aftertaste, while 1,8-cineole and caryophyllene were also correlated with sweet taste. In contrast, linalool and nerolidol were associated with the feature of the most characteristic manufacturer's products which have unfavorable attributes such as floral, iodophor-like, metallic, and soapy attributes. The identification of these metabolites will be useful for the selection of MCEOs that can potentially be used as flavor.

Cajuputs candy impairs Candida albicans and Streptococcus mutans mixed biofilm formation in vitro.

Background:Cajuputs candy (CC), an Indonesian functional food, utilizes the bioactivity of Melaleuca cajuputi essential oil (MCEO) to maintain oral cavity health. Synergistic interaction between Candida albicans and Streptococcus mutans is a crucial step in the pathogenesis of early childhood caries. Our recent study revealed several alternative MCEOs as the main flavors in CC. The capacity of CC to interfere with the fungus-bacterium relationship remains unknown. This study aimed to evaluate CC efficacy to impair biofilm formation by these dual cariogenic microbes. Methods: The inhibition capacity of CC against mixed-biofilm comprising C. albicans and S. mutans was assessed by quantitative (crystal violet assay, tetrazolium salt [MTT] assay, colony forming unit/mL counting, biofilm-related gene expression) and qualitative analysis (light microscopy and scanning electron microscopy). Result: Both biofilm-biomass and viable cells were significantly reduced in the presence of CC. Scanning electron microscopy imaging confirmed this inhibition capacity, demonstrating morphology alteration of C. albicans, along with reduced microcolonies of S. mutans in the biofilm mass. This finding was related to the transcription level of selected biofilm-associated genes, expressed either by C. albicans or S. mutans. Based on qPCR results, CC could interfere with the transition of C. albicans yeast form to the hyphal form, while it suppressed insoluble glucan production by S. mutans. G2 derived from Mojokerto MCEO showed the greatest inhibition activity on the relationship between these cross-kingdom oral microorganisms (p < 0.05). Conclusion: In general, all CC formulas showed biofilm inhibition capacity. Candy derived from Mojokerto MCEO showed the greatest capacity to maintain the yeast form of C. albicans and to inhibit extracellular polysaccharide production by S. mutans. Therefore, the development of dual-species biofilms can be impaired effectively by the CC tested.

Plant Sterol Esters in Extruded Food Model Inhibits Colon Carcinogenesis by Suppressing Inflammation and Stimulating Apoptosis.

Plant sterols in their free forms are known to inhibit colon cancer. Whether these activities persist when compounds are incorporated into processed food is not reported yet. This study aimed to test the ability of plant sterol esters (PSE) incorporated into a nonpuffed extruded food (NPE) model to inhibit colon carcinogenesis. PSE was added into NPE at four concentrations (0.0%, 0.7%, 1.4%, and 2.1%). PSE-NPE activity was tested in azoxymethane/dextran sodium sulfate-induced Balb/c mice. The groups given PSE-NPE did not show any colon tumor formation. Immunohistochemistry results revealed that the group fed with 1.4% PSE had the lowest histoscore for cyclooxygenase-2 expression and the highest histoscore for cleaved caspase-3, cleaved caspase-8, and cleaved caspase-9expressions. The results of this study indicated that even after incorporation into a food system, which is processed using high pressure and temperature, PSE retained its chemopreventive activity. The proposed mechanisms are by suppressing inflammation and inducing apoptosis.