Kinetic optimisation of the reversed phase liquid chromatographic separation of rooibos tea (Aspalathus linearis) phenolics on conventional high performance liquid chromatographic instrumentation

Rooibos tea, produced from the endemic South African shrub Aspalathus linearis, has various health-promoting benefits which are attributed to its phenolic composition. Generating reliable, quantitative data on these phenolic constituents is the first step towards documenting the protective effects associated with rooibos tea consumption. Reversed phase liquid chromatographic (RP-LC) methods currently employed in the quantitative analysis of rooibos are, however, hampered by limited resolution and/or excessive analysis times. In order to overcome these limitations, a systematic approach towards optimising the RP-LC separation of the 15 principal rooibos tea phenolics on a 1.8 μm phase using conventional HPLC instrumentation was adopted. Kinetic plots were used to obtain the optimal configuration for the separation of the target analytes within reasonable analysis times. Simultaneous optimisation of temperature and gradient conditions provided complete separation of these rooibos phenolics on a 1.8 μm C18 phase within 37 min. The optimised HPLC–DAD method was validated and successfully applied in the quantitative analysis of aqueous infusions of unfermented and fermented rooibos. Major phenolic constituents of fermented rooibos were found to be a phenylpropanoid phenylpyruvic acid glucoside (PPAG), the dihydrochalcone C-glycoside aspalathin, the flavones isoorientin and orientin, and a flavonol O-diglycoside tentatively identified as quercetin-3-O-robinobioside. Content values for PPAG, ferulic acid and quercetin-3-O-robinobioside in rooibos are reported here for the first time. Mass spectrometric (MS) and tandem MS detection were used to tentatively identify 13 additional phenolic compounds in rooibos infusions, including a new luteolin-6-C-pentoside-8-C-hexoside and a novel C-8-hexosyl derivative of aspalathin reported here for the first time.     

Fast method development of rooibos tea phenolics using a variable column length strategy

The development of a method for the separation of standard compounds of the 15 main phenolics found in rooibos tea is presented. The separation of these compounds in a single HPLC analysis is particularly challenging due to the similarity of rooibos phenolics. As a result, multiple methods are often required to analyze all major phenolics in rooibos tea samples. The method development process is significantly enhanced in this study by using the recently introduced automated column coupler in combination with the variable column length strategy. This strategy consists of performing the initial scouting runs, wherein the best separation conditions are determined, on a short column and subsequently fine-tuning the separation on longer columns to benefit from their higher separation performance. It is demonstrated that the method development process can further be expedited by operating each column length at the maximum pressure, in this case 1000 bar. Although this holds in general, it is even more the case for the presently considered sample, since the selectivity of the sample is more pressure- than temperature-dependent. Applying the optimized method to unfermented and fermented aqueous rooibos tea extracts in combination with Q-TOF mass spectrometry, some 30 phenolic compounds are tentatively identified.     

Antiliperoxidant activity of pomegranate peel extracts on lard

Phenolics were extracted from powdered pomegranate peel using water, methanol, acetone and ethyl acetate (EtOAc), respectively. The antiliperoxidant activity of the extracts on lard was studied by peroxide value method. All the extracts showed enhanced inhibitory effect on lard peroxidation with the increase of phenolic concentrations. Acetone extract exhibited the highest antiliperoxidant activity followed by water, methanol and EtOAc extracts. Acetone extract at 0.1% (w/w) and water extract at 0.2% (w/w) demonstrated an antiliperoxidant effect close to that of tea polyphenols (0.02%, w/w) and higher than that of BHT (0.02%, w/w). At 0.2% (w/w), acetone extract showed a higher inhibitory activity on lard oxidation than that of tea polyphenols and BHT. Owing to the high antiliperoxidant property, acetone extract may have possible application in the food industry.     

Antioxidant, diuretic activities and polyphenol content of Stereospermum kunthianum Cham. (Bignoniaceae)

Stereospermum kunthianum was used for biological and phytochemical investigations. In biological studies, antioxidant activities were investigated with water, methanol and aqueous acetone extracts. Furthermore, the xanthine oxidase inhibitory activity and the diuretic activity of an aqueous acetone extract were evaluated. In the phytochemical investigations, the flavonoids and polyphenols were quantified spectrophotometrically in all extracts followed by high-performance liquid chromatography-mass spectrometry (HPLC-MS) analysis of an aqueous acetone extract. The 2,2-diphenyl-1-picrylhydrazyl (DPPH), ferric reducing/antioxidant power (FRAP) and 2,2'-azinobis (3-ethylbenzoline-6-sulphonate (ABTS) methods have shown that the aqueous acetone extract presents the best antioxidant activities. This aqueous acetone extract was further proven to have interesting xanthine oxidase inhibitory activity, but only a weak diuretic activity. This aqueous acetone extract also possessed the highest phenolic and flavonoid contents. HPLC-MS analysis allowed identifying and quantifying, rutin, isoquercitrin, quercetin, hyperoside, quercitrin and luteolin and the glycosides of ferulic, sinapic p-coumaric acids and kaempferol, apigenin in aqueous-acetone extract.     

Analysis of heartsease (Viola tricolor L.) flavonoid glycosides by micro-liquid chromatography coupled to multistage mass spectrometry

Micro-liquid chromatography (microLC) in conjunction with multistage mass spectrometry (MSn) was introduced to study several major heartsease flavonoid glycosides. High-resolution microLC separation was achieved by using a monolithic poly(p-methylstyrene-co-1,2-bis(p-vinylphenyl)ethane) column under reversed-phase conditions. The MS/MS and MS3 analysis of the flavonoid components of interest provided data about their glycosylation type and position, nature of their aglycones, and the structure/linkage information of their glycan moieties. With our microLC-MSn approach, four flavonol O-glycosides, nine flavone-C-glycosides, and three flavone C,O-glycosides were characterized in heartsease methanol extract. All of these glycoconjugates were found to be the derivatives of six aglycones: apigenin, chrysoeriol, isorhamnetin, kaempferol, luteolin, and quercetin.     

Chemical constituents of the aerial parts of Scutellaria lateriflora and their alpha-glucosidase inhibitory activities

MeOH extracts of 37 herbs were tested in screening experiments for rat intestinal alpha-glucosidase. The MeOH extract of the aerial parts of Scutellaria lateriflora L. (Lamiaceae) significantly inhibited sucrase and maltase activities, using sucrose and maltase as the substrates. Enzyme inhibition guided-fractionation of the MeOH extract of S. lateriflora resulted in the isolation of a new diterpene glucoside, deacetylajugarin-IV 18-O-beta-D-glucopyranoside (1), along with 20 known phenolics (2-21). The structures of 1-21 were elucidated on the basis of MS and NMR data analyses. Baicalein (4) and baicalin (10), a glycoside of 4, showed moderate sucrase inhibitory activities at IC50 values of 14.9 and 36.3 microM, respectively, whereas luteolin (3), acteoside (16), leucosceptoside A (18), and isoacteoside (20) showed weak inhibitory activities at IC50 values of 811, 522, 727, and 443 microM, respectively. This is the first report on mammalian alpha-glucosidase inhibitory activities of S. lateriflora extract and identification of the constituents responsible for the activities. Apigenin (2), luteolin (3), 6-methoxyluteolin 4'-methyl ether (6), isoscutellarin 8-O-beta-D-glucuronide (7), luteolin 7-O-beta-D-glucuronide (9), wogonin 7-O-beta-D-glucuronide methyl ester (12), eriodictyol (13), naringenin (14), naringenin 7-O-beta-D-glucuronide (15), jionoside D (17), leucosceptoside A (18), and (+)-syringaresinol 4'-O-beta-D-glucopyranoside (21) were isolated from this plant for the first time. The inhibitory properties of S. lateriflora extract against alpha-glucosidase provide a prospect for its antidiabetic usage.     

Comprehensive two-dimensional liquid chromatographic analysis of rooibos (Aspalathus linearis) phenolics

Rooibos tea is an unique beverage prepared from unfermented and fermented plant material of the endemic Cape fynbos plant, Aspalathus linearis. The well-known health-promoting benefits of rooibos are partly attributed to its phenolic composition. Detailed investigation of the minor phenolic constituents of rooibos is, however, hampered by the limitations associated with conventional HPLC methods used for its analysis. In this study, the applicability of comprehensive two-dimensional liquid chromatographic methods for the in-depth analysis of rooibos phenolics was investigated. Phenolic compounds were separated according to polarity by hydrophilic interaction chromatography (HILIC) in the first dimension, whilst reversed-phase liquid chromatography (RP-LC) provided separation according to hydrophobicity in the second dimension. Ultraviolet photodiode array and electrospray ionisation mass spectrometry were used to identify phenolic compounds. Comprehensive HILIC × RP-LC demonstrated its applicability for the analysis of a diverse range of phenolic compounds in unfermented and fermented rooibos samples, in which large qualitative differences in the phenolic composition were established. The combination of these orthogonal separations provided a significant improvement in resolution, as exemplified by practical peak capacities in excess of 2000 and 500 for off-line and on-line methods, respectively.     

Identification and mass spectrometric characterization of glycosylated flavonoids in Triticum durum plants by high-performance liquid chromatography with tandem mass spectrometry

A mass spectrometric method for extensive detection and semi-quantitative determination of flavonoid glycosides in stem and leaves of young Triticum durum plants is presented. About 100 g of sample were lyophilized and ground, and the compounds of interest were then extracted, cleaned-up, and fractionated using high-performance liquid chromatography (HPLC). Tandem mass spectrometry analyses were performed using a quadrupole-linear ion trap instrument with an information-dependent data acquisition (IDA) protocol that looped two experiments, enhanced MS scan and enhanced product ion scan. Various glycoconjugates, which are all derivatives of only four flavones, apigenin, luteolin, chrysoeriol and tricin, were identified and belong to the following categories: 7 monoglycosides, 31 diglycosides, 15 triglycosides and 1 tetraglycoside. Among these some acylated glycosides were found. Tricin derivatives are present exclusively as O-glycosides, while apigenin and luteolin are present always as C-glycosides. Semi-quantitative estimation was performed by using the monoglycoside and diglycoside of quercetin as internal standards.     

A new flavone C-glycoside from the style of Zea mays L. with glycation inhibitory activity

A new flavone C-glycoside, chrysoeriol 6-C-beta-fucopyranoside (1), and a known flavone C-glycoside (2) were isolated from the style of Zea mays L. These structures were elucidated on the basis of spectroscopic analysis. Compound 1 exhibited glycation inhibitory activity.     

Phytochemical Fingerprinting and In Vitro Antimicrobial and Antioxidant Activity of the Aerial Parts of Thymus marschallianus Willd. and Thymus seravschanicus Klokov Growing Widely in Southern Kazakhstan

The chemical composition of the hydroethanolic extracts (60% v/v) from the aerial parts of Thymus marschallianus Willd (TM) and Thymus seravschanicus Klokov (TS) from Southern Kazakhstan flora was analyzed together with their hexane fractions. Determination of antibacterial, antifungal and antioxidant activities of both extracts was also performed. RP-HPLC/PDA and HPLC/ESI-QTOF-MS showed that there were some differences between the composition of both extracts. The most characteristic components of TM were rosmarinic acid, protocatechuic acid, luteolin 7-O-glucoside, and apigenin 7-O-glucuronide, while protocatechuic acid, luteolin 7-O-glucoside, luteolin 7-O-glucuronide, and eriodictyol predominated in TS. The content of polyplenols was higher in TS than in TM. The GC-MS analysis of the volatile fraction of both examined extracts revealed the presence of thymol and carvacrol. Additionally, sesquiterpenoids, fatty acids, and their ethyl esters were found in TM, and fatty acid methyl esters in TS. The antioxidant activity of both extracts was similar. The antibacterial activity of TS extract was somewhat higher than TM, while antifungal activity was the same. TS extract was the most active against Helicobacter pylori ATCC 43504 with MIC (minimal inhibitory concentration) = 0.625 mg/mL, exerting a bactericidal effect. The obtained data provide novel information about the phytochemistry of both thyme species and suggest new potential application of TS as a source of bioactive compounds, especially with anti-H. pylori activity.     

In Vitro Alpha-Glucosidase Inhibitory Activity and the Isolation of Luteolin from the Flower of Gymnanthemum amygdalinum (Delile) Sch. Bip ex Walp.

Diabetes mellitus is a major health issue that has posed a significant challenge over the years. Gymnanthemum amygdalinum is a well-known plant that can be potentially used to treat this disease. Therefore, this study aimed to evaluate the inhibitory effect of its root, stem bark, leaves, and flower extracts on alpha-glucosidase using an in vitro inhibition assay to isolate the bioactive compounds and determine their levels in the samples. The air-dried plant parts were extracted by maceration using methanol. The results showed that the flower extract had the greatest inhibitory effect (IC50 47.29 ± 1.12 µg/mL), followed by the leaves, roots, and stem bark. The methanolic flower extract was further fractionated with different solvents, and the ethyl acetate fraction showed the strongest activity (IC50 19.24 ± 0.12 µg/mL). Meanwhile, acarbose was used as a positive control (IC50 73.36 ± 3.05 µg/mL). Characterization based on UV, 1H-, and 13C-NMR established that the ethyl acetate fraction yielded two flavonoid compounds, namely, luteolin and 2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-3-methoxy-4H-chromen-4-on, which had IC50 values of 6.53 ± 0.16 µg/mL and 39.95 ± 1.59 µg/mL, respectively. The luteolin levels in the crude drug, methanolic extract, and ethyl acetate fraction were 3.4 ± 0.2 mg (0.3%), 32.4 ± 0.8 mg (3.2%), and 68.9 ± 3.4 mg (6.9%) per 1 g samples, respectively. These results indicated that the G. amygdalinum flower extract exerted potent inhibitory alpha-glucosidase activity.     

茶皂素―金属复合抗菌剂的制备及抗菌性能研究

通过抑菌环法探讨了茶皂素、金属离子、茶皂素―金属复合抗菌剂对大肠杆菌和金黄色葡萄球菌的抗菌活性,考察了茶皂素纯度、浓度、金属离子种类等因素对抗菌剂抗菌活性的影响。结果表明,茶皂素的抗菌活性与单一的金属离子的抗菌活性相当,茶皂素对大肠杆菌的最低抑制浓度为5 mg/mL,对金黄色葡萄球菌的最低抑制浓度是10 mg/mL;茶皂素与金属离子复配抗菌活性具有协同效应,尤其茶皂素―锌复合抗菌剂对大肠杆菌的抑制效果大大加强。     

Shelf-Life Stability of Ready-to-Use Green Rooibos Iced Tea Powder—Assessment of Physical,Chemical, and Sensory Properties

Green rooibos extract (GRE), shown to improve hyperglycemia and HDL/LDL blood cholesterol, has potential as a nutraceutical beverage ingredient. The main bioactive compound of the extract is aspalathin, a C-glucosyl dihydrochalcone. The study aimed to determine the effect of common iced tea ingredients (citric acid, ascorbic acid, and xylitol) on the stability of GRE, microencapsulated with inulin for production of a powdered beverage. The stability of the powder mixtures stored in semi-permeable (5 months) and impermeable (12 months) single-serve packaging at 30 °C and 40 °C/65% relative humidity was assessed. More pronounced clumping and darkening of the powders, in combination with higher first order reaction rate constants for dihydrochalcone degradation, indicated the negative effect of higher storage temperature and an increase in moisture content when stored in the semi-permeable packaging. These changes were further increased by the addition of crystalline ingredients, especially citric acid monohydrate. The sensory profile of the powders (reconstituted to beverage strength iced tea solutions) changed with storage from a predominant green-vegetal aroma to a fruity-sweet aroma, especially when stored at 40 °C/65% RH in the semi-permeable packaging. The change in the sensory profile of the powder mixtures could be attributed to a decrease in volatile compounds such as 2-hexenal, (Z)-2-heptenal, (E)-2-octenal, (E)-2-nonenal, (E,Z)-2,6-nonadienal and (E)-2-decenal associated with “green-like” aromas, rather than an increase in fruity and sweet aroma-impact compounds. Green rooibos extract powders would require storage at temperatures ≤ 30 °C and protection against moisture uptake to be chemically and physically shelf-stable and maintain their sensory profiles.     

Synthesis and a-glucosidase inhibitory activity of chrysin,diosmetin,apigenin,and luteolin derivatives

Several derivatives have been synthesized from chrysin, diosmetin, apigenin, and luteolin, which were isolated from diverse natural plants. The a-glucosidase inhibitory activity of these compounds was evaluated. The glucosidase inhibitory activity of all derivatives(IC50 24.396 mmol/L) was higher compared with that of the reference drug, acarbose(IC50= 563.601 40.492 mmol/L), and 1-deoxynojirimycin(IC50= 226.912 12.573 mmol/L). O30,O7-Hexyl diosmetin(IC50= 2.406 0.101 mmol/L)was the most potent inhibitor identified. These compounds showed a higher inhibitory ability compared with their precursors except the luteolin derivatives. In general, the inhibitory activity of the synthetic derivatives was enhanced with long alkyl chains at positions 30, 40 and 7 of the flavonoid.     

Evaluation inhibitory activity of catechins on trypsin by capillary electrophoresis–based immobilized enzyme microreactor with chromogenic substrate

In this study, a capillary electrophoresis‐based online immobilized enzyme microreactor was developed for evaluating the inhibitory activity of green tea catechins and tea polyphenol extracts on trypsin. The immobilized trypsin activity and other kinetic parameters were evaluated by measuring the peak area of the hydrolyzate of chromogenic substrate S‐2765. The results indicated that the activity of the immobilized trypsin remained approximately 90.0% of the initial immobilized enzyme activity after 30 runs. The value of Michaelis–Menten constant (K_m) was (0.47 ± 0.08) mM, and the half‐maximal inhibitory concentration (IC₅₀) and inhibition constant (K_i) of benzamidine were measured as 3.34 and 3.00 mM, respectively. Then, the inhibitory activity of four main catechins (epicatechin, epigallocatechin, epicatechin gallate, and epigallocatechin gallate) and three tea polyphenol extracts (green tea, white tea, and black tea) on trypsin were investigated. The results showed that four catechins and three tea polyphenol extracts had potential trypsin inhibitory activity. In addition, molecular docking results illustrated that epigallocatechin gallate, epicatechin gallate, epicatechin, and epigallocatechin were all located not only in the catalytic cavity, but also in the substrate‐binding pocket of trypsin. These results indicated that the developed method is an effective tool for evaluating inhibitory activity of catechins on trypsin.     

Bioactive constituents from Chinese natural medicines. XX. Inhibitors of antigen-induced degranulation in RBL-2H3 cells from the seeds of Psoralea corylifolia

The ethyl acetate-soluble fraction of the methanolic extract from the seeds of Psoralea corylifolia was found to inhibit the release of beta-hexosaminidase, as a marker of antigen-IgE-induced degranulation in RBL-2H3 cells. Sixteen constituents were isolated from the ethyl acetate-soluble fraction and several constituents, Delta(1),3-hydroxybakuchiol (IC(50) = 49 microM), Delta(3),2-hydroxybakuchiol (69 microM, bavachin (58 microM), and psoralidin (ca. 100 microM), showed inhibitory activities against the antigen-induced degranulation.     

Pancreatic lipase inhibition by C-glycosidic flavones Isolated from Eremochloa ophiuroides

Activity-guided fractionation of a methanolic extract of the leaves of Eremochloa ophiuroides (centipede grass) using a pancreatic lipase inhibitory assay led to the isolation and identification of a new C-glycosidic flavone, luteolin 6-C-?-D-boivinopyranoside (1), as well as eight known compounds. The structures of these compounds were established on the basis of interpretation of their spectroscopic data. Among these isolates, the C-glycosidic flavones 1-5 showed potent inhibitory effects on pancreatic lipase, with IC?? values ranging from 18.5 ± 2.6 to 50.5 ± 3.9 μM.     

Chemical constituents and allelopathic activity of Machaerium eriocarpum Benth.

Abstract

The analysis by HPLC-PDA of the hydroalcoholic extract of the leaves of M. eriocarpum together with the injection of the fractions containing the already identified metabolites allowed the detection of at least 5 flavonoids, of which two are derived from apigenin and three from luteolin. After isolating larger amounts of isovitexin (I), assays were performed to evaluate the allelopathic activity together with the crude extract. The results show that the initial inhibition indexes were very similar to those observed in the treatments with F17 (Fraction enriched in isovitexin) and F18 (isovitexin), mainly in the concentrations of 500 and 1000?mg L^?1. The index of the number of lateral roots, an increase of the inhibitory effect is observed with the increase of the concentration of M. eriocarpum extract.     

Polyphenolic characterisation and bioactivity of an Oxalis pes-caprae L. leaf extract

The present work is focused on the characterisation of the polyphenolic content of an Oxalis pes-caprae L. leaf extract and on the evaluation of its bioactivity with particular interest on its vascular activity and antioxidant potential. The polyphenolic content was characterised by HPLC-DAD and LC-MS/MS. The vascular activity was evaluated according to the influence on the serotonergic and adrenergic systems of the human internal mammary artery (HIMA). Antioxidant and neuroprotective studies were also conducted. Several luteolin and apigenin derivatives were identified as main constituents of the extract, which did not present any contractile effect nor had any effect on the serotonergic system of HIMA. However, it showed antagonistic effect on the adrenergic system, inhibiting the contraction to noradrenaline (reduction of 58.44% of maximum contraction). The extract showed antioxidant activity and standardised luteolin and apigenin derivatives showed neuroprotective potential, particularly homoorientin.     

Isolation and characterization of phenolic compounds from the leaves of Salix matsudana

A bioassay-guided in vitro screen has revealed that a 70% methanol extract of the leaves of Salix matsudana shows considerable inhibitory activity against cyclooxygenases (COX-1 and COX-2). A subsequent phytochemical study led to the isolation of a new flavonoid, matsudone A (1), together with five known flavonoids--luteolin (2), isoquercitrin (3), 7-methoxyflavone (4), luteolin 7-O-glucoside (5), 4',7-dihydroxyflavone (6)--and two phenolic glycosides, leonuriside A (7) and piceoside (8). Their structures were elucidated on the basis of extensive 1D- and 2D-NMR studies, high resolution ESI mass spectroscopic analyses and comparisons with literature data. The isolated compounds 1-8 were tested for their inhibitory activities against COX-1 and COX-2. Compounds 1, 5 and 6 were found to have potent inhibitory effect on COX-2 and compounds 3-5 exhibited moderate inhibition against COX-1.