The Application of 13C NMR and Untargeted Multivariate Analysis for Classifying Virgin Coconut Oil

Virgin coconut oil (VCO) is produced from fresh mature coconut meat without the use of chemicals or high heat. VCO can be made using three processes: fermentation, centrifuge, and expeller. To determine quality, it is important to be able to differentiate control VCO (fresh) from old VCO, refined bleached and deodorized coconut oil (RBDCO), and VCO which has been adulterated with RBDCO. Differentiating these types of samples has remained a challenge because of their chemical similarity. This study investigated the ability of 13C NMR and multivariate analysis to differentiate these different coconut oil samples. The methodology used the standard 13C NMR pulse sequence with broadband 1H decoupling with dioxane as the internal standard (IS). After pre-processing of the spectra (alignment, bucketing/binning, normalization with respect to dioxane IS peak), untargeted multivariate analyses, both unsupervised and supervised, were done on the bins of the 13C peaks. Principal components analysis (PCA), a linear unsupervised method, was able to differentiate control VCO (n = 57) from RBDCO (n = 21), adulterated VCO (n = 9), and old VCO (n = 11). Partial least squares–discriminant analysis (PLS–DA) was used as the supervised linear binary classifier. Using overall accuracy and AUC-ROC curves (by 100 cross validation and single validation using manual holdout), the supervised dataset with an optimized model gave performances that were 99%, 95%, and 80% improved in differentiating control VCO vs. RBDCO, old VCO, and adulterated VCO (one vs. one), respectively. Predictive ability (Q2 \u3c 0.20) and overall accuracy (\u3c0.80) were poor compared to the previous models for binary classifier models (one vs. rest) to differentiate among the three VCO processes. This may be due to the variations in production conditions and methods that different VCO producers use. We conclude that 13C NMR combined with linear techniques can be used to accurately differentiate fresh VCO from RBDCO, old VCO, and adulterated VCO

Untargeted Bioassay Strategy for Medicinal Plants: In Vitro Antidiabetic Activity and 13C NMR Profiling of Extracts from Vitex negundo L

Bioassay-guided fractionation is the principal method for the identification of active constituents in medicinal plants. By design, this method aims to identify the most active compound in a complex mixture with the objective of discovering novel drug candidates. Described here is a complementary method for the identification of known bioactive compounds in medicinal plants which is untargeted and which takes advantage of the large NMR database of known natural products and availability of statistical software. This untargeted bioassay strategy is demonstrated as a proof of principle in the determination of the antidiabetic compounds in Vitex negundo L. Crude methanol and ethanol extracts, and chloroform, ethyl acetate and aqueous fractions of V. negundo L. were prepared and tested for their in vitro antidiabetic potential using the glucose diffusion retardation assay and the in vitro starch-amylase inhibition assay. The same crude extracts and fractions were profiled using 13C nuclear magnetic resonance (NMR) spectroscopy. The 13C NMR spectra of twelve known compounds from the semi-polar fraction of V. negundo – two iridoids, seven iridoid glucosides, two flavonoids and one flavonoid C-glucoside – were matched from the 13C NMR spectra of the extracts and fractions. The 13C NMR match factor values of the twelve compounds were used in the multivariate correlation analysis with antidiabetic activity using the glucose diffusion retardation activity and the starch-amylase inhibition assay. This method was able to correlate the seven iridoid glucosides with the antidiabetic activity, a result that would have been difficult to obtain using bioassay-guided fractionation

Chemical profiling and chemical standardization of Vitex negundo using 13C NMR

Chemical profiling and standardization of the defatted methanol extract of the leaves of Vitex negundo L. were carried out using 13C nuclear magnetic resonance (NMR) analysis followed by chemometric analysis of the chemical shift data. Chemical profile was obtained using a k-means cluster profile and chemical standardization which was achieved using a multivariate control chart. The V. negundo samples were made up of four groups: the training set, submitted samples from production farms, commercial samples, such as tablets, capsules and teas, and experimental samples (samples which were allowed to degrade). Four groups were generated in k-means cluster, which generally corresponded to the four types of samples. The multivariate control chart identified samples whose quality exceeded the upper control limit, all of which were commercial samples and experimental samples. The samples were also analyzed by quantitative thin layer chromatography (qTLC) using agnuside as marker compound. Comparison of the qTLC results with the k-means cluster and the multivariate control chart showed poor correspondence. This means that a univariate analysis of a plant sample using a marker compound is useful only for quantification of the target compound. On the other hand, chemical profiling and standardization of medicinal plants should use a multivariate method

13C Nuclear Magnetic Resonance Phytochemical Profiling of the In Vitro Antidiabetic Potential of Vitex negundo L.

Vitex negundo has been known since ancient times as a medicinal plant. The objective of this study is to investigate the effect of methanol and ethanol extracts, and ethyl acetate, chloroform and aqueous fractions of Vitex negundo using an in vitro model to test glucose diffusion and to determine the phytochemical profile of the extracts and fractions using 13C nuclear magnetic resonance (NMR) spectroscopy. The chloroform fractions, ethyl acetate-EtOH and ethyl acetate-MeOH gave the highest inhibitory effect on both the diffusion activities in vitro. Retardation of glucose diffusion suggests that negundo has the potential to lower postprandial glucose. Correlation analysis of the 13C NMR profile with retardation activity suggests that compounds containing glycosidic residues may be responsible for the glucose retardation activity. This is the first example where activity has been correlated with specific structural features of compounds from a crude extract using 13C NMR chemical shifts to assist in the identification of active compounds

Identification of four iridoids in the pharmacologically-active fraction of Vitex negundo, L.

Four iridoids--2\u27-p-hydroxybenzoyl mussaenosidic acid, 6\u27-p-hydroxybenzoyl mussaenosidic acid, agnuside and Lagundinin--were identified in the pharmacologically-active fraction of the leaves of Vitex negundo L. The data obtained from 2\u27-p-hydroxybenzoyl mussaenosidic acid modified a previous assignment while lagundinin is a newly identified iridoid

13C Nuclear Magnetic Resonance Phytochemical Profiling of the In Vitro Antidiabetic Potential of Vitex negundo L.

Vitex negundo has been known since ancient times as a medicinal plant. The objective of this study is to investigate the effect of methanol and ethanol extracts, and ethyl acetate, chloroform and aqueous fractions of Vitex negundo using an in vitro model to test glucose diffusion and to determine the phytochemical profile of the extracts and fractions using 13C nuclear magnetic resonance (NMR) spectroscopy. The chloroform fractions, ethyl acetate-EtOH and ethyl acetate-MeOH gave the highest inhibitory effect on both the diffusion activities in vitro. Retardation of glucose diffusion suggests that negundo has the potential to lower postprandial glucose. Correlation analysis of the 13C NMR profile with retardation activity suggests that compounds containing glycosidic residues may be responsible for the glucose retardation activity. This is the first example where activity has been correlated with specific structural features of compounds from a crude extract using 13C NMR chemical shifts to assist in the identification of active compounds

Chemical profiling of Philippine Moringa oleifera leaves

Chemical profiling can be applied to medicinal plant raw materials and commercial products to verify identity and to monitor quality. In this paper, we describe the chemical profiles of the volatile oil and methanol extract of Moringa oleifera plant samples collected from various regions of the Philippines. The volatile oil was collected by hydro-distillation and analysed using gas chromatography-mass spectrometry (GCMS). A total of 71 compounds were identified from all of the samples; however, the profile of compounds was very variable. Leaves and stalks produced different volatile profiles: benzyl aldehyde was present in all leaves, but was not detected in any of the stalks. The volatile oil from the stalks did not contain any terpenes. The methanol extract was defatted using hexane and analysed by thin layer chromatography (TLC) and 13C nuclear magnetic resonance (13C NMR) spectroscopy. Quantitative TLC analysis was performed using afzelin and kaempferol as marker compounds. Afzelin was measured in the range 0.02-0.66%, but kaempferol was not detected in any of the samples. No trend was found for afzelin concentration. PCA analysis of the 13C NMR chemical profile indicates that Philippine moringa is highly variable