Quality control of Aloe vera (Aloe barbadensis) and Aloe ferox using band-selective quantitative heteronuclear single quantum correlation spectroscopy (bs-qHSQC)

In the present study, band-selective quantitative heteronuclear single quantum correlation spectroscopy (bs-qHSQC) was applied for the quality control of the two Aloe species present in the European Pharmacopeia. After development and validation of a complete spectral range (csr-) qHSQC assay, a specific pulse program with selective excitation was applied and the measuring time was reduced from 135 to 32 min, while maintaining the same resolution. This bs-qHSQC method (method I) showed slightly higher RSD values compared to the csr-qHSQC method (maximum RSD of 2.80%), but better recovery rates in comparison to the assay of the Pharmacopeia (97.3% for Aloe vera and 96.6% for Aloe ferox). Apart from quantifying the total anthranoid content, the method moreover allows the quantitation of aloin among other aloin derivatives, such as 7-hydroxyaloin, as well as the differentiation of the two investigated species. Additionally, a second bs-qHSQC method (method II) for the fast (4 min) determination of the aloin A/B ratio was developed and compared to 13C qNMR measurements. Showing the same results in much less analysis time, the latter approach contributes to a general problem in natural product chemistry, the co-occurrence of structurally similar compounds and their analysis in complex matrices, e.g. plant extracts

Two-dimensional qNMR of anthraquinones in Frangula alnus (Rhamnus frangula) using surrogate standards and delay time adaption

In the absence of adequate reference material, a problem often encountered in natural product chemistry, we investigated the use of surrogate standards in two-dimensional qNMR for the quantification of anthraquinones in the bark of alder buckthorn (Frangula alnus). Using the integrals of cross signals in the HSQC spectrum obtained from commercial standards rutin and duroquinone and adapting the delays for the 1JCH coupling, we quantified the total amount of anthraquinones and anthraquinone glucosides, as well as the content of the value-determining glucofrangulins and frangulins. Thereby, duroquinone was used as an external standard to establish the calibration curve for the methylated anthraquinone scaffold, whereas calibration curves for the glycosides were obtained using the anomeric proton signals of the rutinose disaccharide. The method was validated for accuracy, precision, specificity, linearity and limit of quantitation and shows clear advantages over the method of the European Pharmacopeia, especially in terms of specificity and meaningfulness of the results. Apart from being a useful alternative in the quality control of alder buckthorn, the presented approach demonstrates, moreover, the versatility of sophisticated 2D measurements in quantitative NMR

Prediction of the sweetening effect of Siraitia grosvenorii (luo han guo) fruits by two-dimensional quantitative NMR

During the last decade, dried fruits of Siraitia grosvenorii (luo han guo), also known as monk fruit, have become popular food ingredients. Luo han guo extracts, which are promoted as non-caloric natural sweeteners, are now incorporated into dietary supplements, soft drinks, and energy shakes. The compounds responsible for the sweetening effect are glycosylated cucurbitane-type triterpenoids, the so-called mogrosides. However, of the more than 40 known mogroside compounds, only 11-α-hydroxy-mogrosides exhibit a sweetening effect, whereas the other triterpenoids are non- or bitter-tasting. We have used two-dimensional quantitative NMR to determine selectively the content of 11-α-hydroxy-mogrosides in these dried fruits and thus to predict their sweetening potential. Homonuclear (H,H COSY) and heteronuclear (HSQC) methods were developed, validated, and compared. Both techniques were found suitable for the quality control of luo han guo fruits and extracts, the COSY experiment being advantageous with regard to accuracy, precision, and limit of quantification

Quantification of diterpene acids in Copaiba oleoresin by UHPLC-ELSD and heteronuclear two-dimensional qNMR

In this study, we present the quantitation of eight diterpene acids in the oleoresin of Copaifera reticulata Ducke by UHPLC-ELSD and quantitative HSQC (heteronuclear single quantum correlation spectroscopy). UHPLC was performed using reversed phase material and external calibration and showed RSD values of ≤ 3% (repeatability) and ≤ 4% (precision), and mean recovery rates of 91.2 to 104.8%. LOQs were determined with 10 and 20 µg/mL, and LODs with 4 and 8 µg/mL, respectively. For the qHSQC method, calibration curves of eight different NMR cross-peaks (furylic, endo- and exocyclic methine signals, exocyclic methylene and methyl signals) were established and normalized with dimethyl terephthalate, which served as internal standard. This approach allowed the direct quantification of four major and one minor diterpene, whereas simple calculation procedures led to the contents of the remaining minor compounds. Comparison with the results of the UHPLC assay showed good agreement for seven of the eight diterpene acids. In terms of precision, the qHSQC method was advantageous for the quantification of the three main compounds, whereas UHPLC-ELSD was superior in the determination of the minor components. In contrast to previous reports, kolavenic acid was identified as a major diterpene acid in the oleoresin of Copaifera reticulata, with amounts of 4.0 ± 0.3%

Determination of Total Sennosides and Sennosides A, B, and A1 in Senna Leaflets, Pods, and Tablets by Two-Dimensional qNMR

In the present work, a two-dimensional qNMR method for the determination of sennosides was established. Using band-selective HSQC and the cross correlations of the characteristic 10-10' bonds, we quantified the total amount of the value-determining dianthranoids in five minutes, thus, rendering the method not only fast, but also specific and stability indicating. The validation of the method revealed excellent accuracy (recovery rates of 98.5 to 103%), precision (RSD values of 3.1%), and repeatability (2.2%) and demonstrated the potential of 2D qNMR in the quality control of medicinal plants. In a second method, the use of 2D qNMR for the single analysis of sennosides A, B, and A1 was evaluated with acceptable measurement times (31 min), accuracy (93.8%), and repeatability (5.4% and 5.6%) for the two major purgatives sennoside A and B. However, the precision for sennoside B and A1 was not satisfactory, mainly due to the low resolution of the HSQC signals of the two compounds

Biological Activities of Two Major Copaiba Diterpenoids and Their Semi-synthetic Derivatives

The oleoresin of Copaifera reticulata Ducke, Fabaceae, is a traditional Brazilian remedy used for a wide range of applications. Commonly named copaiba, the oleoresin has been found to exhibit strong antimicrobial effects in our previous study, which could be attributed to some of its diterpenoid constituents. In order to find new biological activities and to eventually enhance the before observed effects, (−)-polyalthic acid (1) and kaurenoic acid (2), together with eight prepared semi-synthetic derivatives (1a–1c and 2a–2e) were evaluated for their cytotoxic, antibacterial and antifungal properties. Regarding the gram-positive bacteria Enterococcus faecium and methicillin-resistant Staphylococcus aureus, we found that both the exocylic methylene group and the carboxyl group were crucial for the activity against these two clinically relevant bacterial strains. Investigation of the antifungal activity, in contrast, showed that the carboxyl group is unnecessary for the effect against the dermatophytes Trichophyton rubrum and Cryptococcus neoformans, indicated by low micromolar IC50 values for both (−)-polyalthic acid diethylamide (1a) as well as (−)-polyalthic acid methyl ester (1b). Apart from studying the biological activity, the structure of one semi-synthetic derivative, compound 1c, is being reported for the first time. During the course of the structure elucidation of the new compound, we discovered inconsistencies regarding the stereochemistry of polyalthic acid and its stereoisomers, which we clarified in the present work. [Figure not available: see fulltext.]PeerReviewe

Quantification of the total amount of black cohosh cycloartanoids by integration of one specific 1H NMR signal

Quantitative analysis is an important field in the quality control of medicinal plants, aiming to determine the amount of pharmacologically active constituents in complex matrices. Often biological effects of herbal drugs are not restricted to single compounds, but are rather caused by a number of often biogenetically related plant metabolites. Depending on the complexity of the analyzed plant extract, conflicts between accuracy, such as total content assays using photometric or colorimetric methods, and comprehensiveness, e.g. quantification of one or a few lead compounds can occur. In this study, we present a qHNMR approach determining the total amount of cycloartanoids in black cohosh (Actaea racemosa) rhizomes. Perdeuterated methanol containing 1,2,4,5-tetrachloro-3-nitrobenzene as an internal standard was used for extraction. Amounts of cycloartanoids were then measured by integrating 1H NMR signals of all cycloartenoids' H-19 exo protons. Due to their unusually low chemical shifts, these signals are well separated from all remaining signals in crude extracts. Thus, accurate (recovery rates of 99.5-102.5%) and precise (relative standard deviations below 2.5%) quantification of cycloartanoids was accomplished. To the best of our knowledge, this is the first example of a quantification of the total amount of a pharmacologically relevant compound class by integration of one 1H NMR signal characteristic for all members of this particular compound class. Additionally, we propose a new term and unit for the evaluation of medicinal plants and herbal medicinal products: the "specific partial amount of substance" of pharmacologically active constituents, indicated in mmol/g

METABOLISM OF N-HYDROXYGUANIDINES (N-HYDROXYDEBRISOQUINE) IN HUMAN AND PORCINE HEPATOCYTES: REDUCTION AND FORMATION OF GLUCURONIDES

ABSTRACT: The biotransformation of N-hydroxydebrisoquine, a model substrate for N-hydroxyguanidines, was studied in vitro with cultured and characterized porcine and human hepatocytes. The objective of the present work was to compare the N-oxidative and N-reductive metabolism of this compound using a monolayer culture system with previously described microsomal studies and to investigate the phase 2 metabolism, in particular, the glucuronidation of this class of compounds. At the same time, the suitability of pig hepatocytes as a model system for the human metabolism could be investigated. Two glucuronides of the parent compound Nhydroxydebrisoquine were analyzed. For the first time, one of these phase 2 metabolites could be identified as an O-glucuronide of an N-hydroxyguanidine by comparing it to a synthesized authentic compound. The involvement of certain human UDP-glucuronosyltransferases (UGTs) was evaluated by incubating the substrate with eight human hepatic recombinant UGT enzymes. Metabolites were determined by a newly developed LC-MS (liquid chromatography/mass spectrometry) analysis using electrospray ionization (ESI). The known microsomal reduction of the N-hydroxylated compound was also demonstrated with hepatocytes. The N-hydroxylation of the corresponding reduced compound (debrisoquine), which was previously described with microsomes, could not be detected in hepatocytes. There was no qualitative difference in the formation of the described derivatives by human and porcine hepatocytes. All phase 2 metabolites identified in hepatocyte culture were also formed by glucuronosyltransferases. In culture, the N-reduction of the N-hydroxylated substrate is the dominating reaction, indicating a predominance of N-reduction in vivo

Development of an HPLC-MS/MS Method for Chiral Separation and Quantitation of (R)- and (S)-Salbutamol and Their Sulfoconjugated Metabolites in Urine to Investigate Stereoselective Sulfonation

The aim of this study was to develop and optimize a chiral HPLC-MS/MS method for quantitative analysis of (R)-/(S)-salbutamol and (R)-/(S)-salbutamol-4′-O-sulfate in human urine to allow for bioanalytical quantitation of the targeted analytes and investigations of stereoselectivity in the sulfonation pathway of human phase Ⅱ metabolism. For analytical method development, a systematic screening of columns and mobile phases to develop a separation via enantiomerically selective high performance liquid chromatography was performed. Electrospray ionization settings were optimized via multiple-step screening and a full factorial design-of-experiment. Both approaches were performed matrix-assisted and the predicted values were compared. The full factorial design was superior in terms of prediction power and knowledge generation. Performing a longitudinal excretion study in one healthy volunteer allowed for the calculation of excretion rates for all four targeted analytes. For this proof-of-concept, either racemic salbutamol or enantiopure levosalbutamol was administered perorally or via inhalation, respectively. A strong preference for sulfonation of (R)-salbutamol for inhalation and peroral application was found in in vivo experiments. In previous studies phenol sulfotransferase 1A3 was described to be mainly responsible for salbutamol sulfonation in humans. Thus, in vitro and in silico investigations of the stereoselectivity of sulfotransferase 1A3 complemented the study and confirmed these findings

Elucidation of the function of two glycosyltransferase genes (lanGT1 and lanGT4) involved in landomycin biosynthesis and generation of new oligosaccharide antibiotics

AbstractBackground: The genetic engineering of antibiotic-producing Streptomyces strains is an approach that became a successful methodology in developing new natural polyketide derivatives. Glycosyltransferases are important biosynthetic enzymes that link sugar moieties to aglycones, which often derive from polyketides. Biological activity is frequently generated along with this process. Here we report the use of glycosyltransferase genes isolated from the landomycin biosynthetic gene cluster to create hybrid landomycin/urdamycin oligosaccharide antibiotics.Results: Production of several novel urdamycin derivatives by a mutant of Streptomyces fradiae Tü2717 has been achieved in a combinatorial biosynthetic approach using glycosyltransferase genes from the landomycin producer Streptomyces cyanogenus S136. For the generation of gene cassettes useful for combinatorial biosynthesis experiments new vectors named pMUNI, pMUNII and pMUNIII were constructed. These vectors facilitate the construction of gene combinations taking advantage of the compatible MunI and EcoRI restriction sites.Conclusions: The high-yielding production of novel oligosaccharide antibiotics using glycosyltransferase gene cassettes generated in a very convenient way proves that glycosyltransferases can be flexible towards the alcohol substrate. In addition, our results indicate that LanGT1 from S. cyanogenus S136 is a D-olivosyltransferase, whereas LanGT4 is a L-rhodinosyltransferase