Ghulam Mustafa Kamal

Compositional differences among Chinese soy sauce types studied by 13C NMR spectroscopy coupled with multivariate statistical analysis

Soy sauce a well known seasoning all over the world, especially in Asia, is available in global market in a wide range of types based on its purpose and the processing methods. Its composition varies with respect to the fermentation processes and addition of additives, preservatives and flavor enhancers. A comprehensive (1)H NMR based study regarding the metabonomic variations of soy sauce to differentiate among different types of soy sauce available on the global market has been limited due to the complexity of the mixture. In present study, (13)C NMR spectroscopy coupled with multivariate statistical data analysis like principle component analysis (PCA), and orthogonal partial least square-discriminant analysis (OPLS-DA) was applied to investigate metabonomic variations among different types of soy sauce, namely super light, super dark, red cooking and mushroom soy sauce. The main additives in soy sauce like glutamate, sucrose and glucose were easily distinguished and quantified using (13)C NMR spectroscopy which were otherwise difficult to be assigned and quantified due to serious signal overlaps in (1)H NMR spectra. The significantly higher concentration of sucrose in dark, red cooking and mushroom flavored soy sauce can directly be linked to the addition of caramel in soy sauce. Similarly, significantly higher level of glutamate in super light as compared to super dark and mushroom flavored soy sauce may come from the addition of monosodium glutamate. The study highlights the potentiality of (13)C NMR based metabonomics coupled with multivariate statistical data analysis in differentiating between the types of soy sauce on the basis of level of additives, raw materials and fermentation procedures.

Optimization protocol for the extraction of antioxidant components from Origanum vulgare leaves using response surface methodology.

In the present work, the response surface methodology (RSM) based on a central composite rotatable design (CCRD), was used to determine optimum conditions for the extraction of antioxidant compounds from Origanum vulgare leaves. Four process variables were evaluated at three levels (31 experimental designs): methanol (70%, 80%, and 90%), the solute:solvent ratio (1:5, 1:12.5, 1:20), the extraction time (4, 10, 16 h), and the solute particle size (20, 65, 110 micron). Using RSM, a quadratic polynomial equation was obtained by multiple regression analysis for predicting optimization of the extraction protocol. Analysis of variance (ANOVA) was applied and the significant effect of the factors and their interactions were tested at 95% confidence interval. The antioxidant extract (AE) yield was significantly influenced by solvent composition, solute to solvent ratio, and time. The maximum AE was obtained at methanol (70%), liquid solid ratio (20), time (16 h), and particle size (20 micron). Predicted values thus obtained were closer to the experimental value indicating suitability of the model. Run 25 (methanol:water 70:30; solute:solvent 1:20; extraction time 16 h and solute particle size 20) showed highest TP contents (18.75 mg/g of dry material, measured as gallic acid equivalents) and DPPH radical scavenging activity (IC50 5.04 μg/mL). Results of the present study indicated good correlation between TP contents and DPPH radical scavenging activity. Results of the study indicated that phenolic compounds are powerful scavengers of free radical as demonstrated by a good correlation between TP contents and DPPH radical scavenging activity.

Variation in Phenolic Profile, β-Carotene and Flavonoid Contents, Biological Activities of Two Tagetes Species from Pakistani Flora

The objective of present study was to evaluate the variation in phenolic profile, β‐carotene, flavonoid contents, antioxidant and antimicrobial properties of Tagetes erecta and Tagetes patula (T. erecta and T. patula) through different in vitro assays. Antioxidant activity was determined through 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) free radical scavenging and inhibition of linoleic acid peroxidation assays and antibacterial and antifungal activities studied using the disc diffusion and resazurin microtiter‐plate assays against bacterial and fungal strains. Moreover, total phenolics (TP), total carotenoids (TC) and total flavonoids (TF) were also determined. Highest (TP 35.8 mg GAE/g) and TF (16.9 mg CE/g) contents were found in MeOH extract of T. patula. T. erecta extract showed higher TC contents (6.45 mg/g) than T. patula extract (6.32 mg/g). T. erecta exhibited the highest DPPH radical‐scavenging activity (IC50) (5.73 μg/mL) and inhibition of linoleic acid peroxidation (80.1%). RP‐HPLC revealed the presence of caffeic acid, sinapic acid and ferulic acid in Tagetes extracts, m‐coumaric acid in T. erecta whereas chlorogenic acid in T. patula extract only. Both extracts possessed promising antimicrobial activity compared to the ciprofloxacin and flumequine (+ve controls) against Bacillus subtilis and Alternaria alternate. Both extract were rich source of polyphenols exhibiting excellent biological activities.

Chemical Composition and Biological Activities of Essential Oil and Extracts from Ocimum sanctum

ABSTRACT The objective of the present research work was to evaluate the antioxidant, antimicrobial and antimalarial activities of essential oil and various extracts from O. sanctum. Gas chromatography-mass spectrometry analysis identified the following major compounds with their quantification as: eugenol (22.0%), β-elemene (19.2%), β-caryophyllene (19.1%), and Germacrene D (5.03%). HPLC analysis of O. sanctum extracts revealed that gallic acid, chlorogenic acid, p-hydroxy benzoic acid, caffeic acid, vanillic acid, p-coumeric acid, sinapic acid, and ferulic acid were the important phenolic acids. The methanol extract exhibited highest level of total phenolic (1.36 g/100 g dry plant material) and total flavonoid (0.67 g/100 g dry plant material) followed by ethanol and n-hexane extracts. The oil and extracts exhibited excellent free radical scavenging potential as measured by 2,2-diphenyl-1-picrylhydrazyl radical free radical-scavenging ability, and antioxidant activity as measured by inhibition of linoleic acid oxidation. Essential oil, n-hexane, methanol, and ethanol extracts exhibited moderate antimalarial potential in term of anti-haem biocrystallization activity. In the resazurin microtitre plate and disc diffusion assays, the essential oil of O. sanctum showed better antibacterial activity than various extracts. The results of the present investigation demonstrated significant (p < 0.05) variations in the antioxidant, antimicrobial, and antimalarial activities of essential oil and extracts from O. sanctum.

Specific patterns of spinal metabolites underlying α-Me-5-HT-evoked pruritus compared with histamine and capsaicin assessed by proton nuclear magnetic resonance spectroscopy

The mechanism behind itching is not well understood. Proton nuclear magnetic resonance (1H-NMR) spectroscopic analysis of spinal cord extracts provides a quick modality for evaluating the specific metabolic activity of α-Me-5-HT-evoked pruritus mice. In the current study, four groups of young adult male C57Bl/6 mice were investigated; one group treated with saline, while the other groups intradermally injected with α-Me-5-HT (histamine independent pruritogen), histamine (histamine dependent pruritogen) and capsaicin (algogenic substance), respectively. The intradermal microinjection of α-Me-5-HT and histamine resulted in a dramatic increase in the itch behavior. Furthermore, the results of NMR studies of the spinal cord extracts revealed that the metabolites show very different patterns for these different drugs, especially when comparing α-Me-5-HT and capsaicin. All the animals in the groups of α-Me-5-HT and capsaicin were completely separated using the metabolite parameters and principal component analysis. For α-Me-5-HT, the concentrations of glutamate, GABA, glycine and aspartate increased significantly, especially for GABA (increased 17.2%, p=0.008). Furthermore, the concentration of NAA increased, but there was no significant difference (increased 11.3%, p=0.191) compared to capsaicin (decreased 29.1%, p=0.002). Thus the application of magnetic resonance spectroscopy technique, coupled with statistical analysis, could further explain the mechanism behind itching evoked by α-Me-5-HT or other drugs. It can thus improve our understanding of itch pathophysiology and pharmacological therapies which may contribute to itch relief.

Assessment of Amino Acid Neurotransmitters in Rat Brain Microdialysis Samples by High-Performance Liquid Chromatography with Coulometric Detection

A simple method was developed for simultaneous determination of five amino acid neurotransmitters (taurine, glutamate, aspartate, glycine, and γ-aminobutyric acid) in brain microdialysates by high-performance liquid chromatography with coulometric detection. The microdialysates were derivatized with o-phthalaldehyde in 5 mM sodium sulfite and separated within 17 min using isocratic elution. Detection parameters for these transmitters were linearity (r2) > 0.999 over the concentration range from 0.1 to 15.00 µM (0.01–1.00 µM for GABA); reproducibility 2.88%–11.67% (relative standard deviation); the limits of detection 4.4–17.2 fmol; and recovery 86.66%–103.42%. This method was successfully applied to analyze these neurotransmitters in the brain. The changes of neurotransmitters were also investigated via local administration of potassium chloride and bicuculline, which was a γ-aminobutyric acid receptor antagonist. It was found that the developed method is more efficient as compared to previously reported methods in simultaneously determining five major neurotransmitters because it shows quick separation and sensitive detection, and is easy to operate. This method can be a practical choice for continuous monitoring of the amino acid neurotransmittersin vivo.

Evaluation of metabolites extraction strategies for identifying different brain regions and their relationship with alcohol preference and gender difference using NMR metabolomics

Metabolomics generate a profile of small molecules from cellular/tissue metabolism, which could directly reflect the mechanisms of complex networks of biochemical reactions. Traditional metabolomics methods, such as OPLS-DA, PLS-DA are mainly used for binary class discrimination. Multiple groups are always involved in the biological system, especially for brain research. Multiple brain regions are involved in the neuronal study of brain metabolic dysfunctions such as alcoholism, Alzheimers disease, etc. In the current study, 10 different brain regions were utilized for comparative studies between alcohol preferring and non-preferring rats, male and female rats respectively. As many classes are involved (ten different regions and four types of animals), traditional metabolomics methods are no longer efficient for showing differentiation. Here, a novel strategy based on the decision tree algorithm was employed for successfully constructing different classification models to screen out the major characteristics of ten brain regions at the same time. Subsequently, this method was also utilized to select the major effective brain regions related to alcohol preference and gender difference. Compared with the traditional multivariate statistical methods, the decision tree could construct acceptable and understandable classification models for multi-class data analysis. Therefore, the current technology could also be applied to other general metabolomics studies involving multi class data.

Proteomics and metabolomics analysis of hepatic mitochondrial metabolism in alcohol-preferring and non-preferring rats

Alcohol preference induced tolerance in humans and animals when their bodily functions adapt to compensate for the disruption caused by alcohol consumption. This was thought to be an important component of the genetic predisposition to alcoholism. To investigate the underlying mechanisms of hepatic metabolic tolerance during alcohol preference, the alcohol preferring and alcohol non-preferring rats were used in this study. The liver mitochondria were purified for comparative quantitative proteomics analysis, and the liver metabolite extracts were collected for metabolomics analysis. Our study identified 96 differentially expressed hepatic mitochondrial proteins that associated with alcohol preference, the further gene ontology and protein interaction network analysis suggest a down-regulation of amino acid metabolism and up-regulation of lipid metabolism. We found alcohol preference induced a series of enzymes decreased (e.g. SSADH and GABA-T) and several amino acids increased (e.g. glutamate and aspartate) in rat liver, indicating down-regulations of glutamate degradation occurred during alcohol preference. Most of these changes were due to the genetic differences between alcohol preferring and non-preferring animals. Furthermore, this study would provided new insights to further clarify the mechanisms of hepatic metabolic tolerance during alcohol preference.

Reconstructing diffusion ordered NMR spectroscopy by simultaneous inversion of Laplace transform

2D diffusion-ordered NMR spectroscopy (DOSY) has been widely recognized as a powerful tool for analyzing mixtures and probing inter-molecular interactions in situ. But it is difficult to differentiate molecules with similar diffusion coefficients in presence of overlapped spectra. Its performance is susceptible to the number of chemical components, and usually gets worse when the number of components increases. Here, to alleviate the problem, numerical simultaneous inversion of Laplace transform (SILT) of many related variables is proposed for reconstructing DOSY spectrum (SILT-DOSY). The advantage of the proposed method in comparison to other methods is that it is capable of estimating the number of analytes more accurately and deriving corresponding component spectra, which in turn leads to the more reliable identification of the components.

13C-NMR-Based Metabolomic Profiling of Typical Asian Soy Sauces

It has been a strong consumer interest to choose high quality food products with clear information about their origin and composition. In the present study, a total of 22 Asian soy sauce samples have been analyzed in terms of 13C-NMR spectroscopy. Spectral data were analyzed by multivariate statistical methods in order to find out the important metabolites causing the discrimination among typical soy sauces from different Asian regions. It was found that significantly higher concentrations of glutamate in Chinese red cooking (CR) soy sauce may be the result of the manual addition of monosodium glutamate (MSG) in the final soy sauce product. Whereas lower concentrations of amino acids, like leucine, isoleucine and valine, observed in CR indicate the different fermentation period used in production of CR soy sauce, on the other hand, the concentration of some fermentation cycle metabolites, such as acetate and sucrose, can be divided into two groups. The concentrations of these fermentation cycle metabolites were lower in CR and Singapore Kikkoman (SK), whereas much higher in Japanese shoyu (JS) and Taiwan (China) light (TL), which depict the influence of climatic conditions. Therefore, the results of our study directly indicate the influences of traditional ways of fermentation, climatic conditions and the selection of raw materials and can be helpful for consumers to choose their desired soy sauce products, as well as for researchers in further authentication studies about soy sauce.