Simultaneous quantitative analysis of 11 flavonoid derivatives with a single marker in persimmon leaf extraction and evaluation of their myocardium protection activity

An improved, reliable and comprehensive method for assessing the quality of the ethyl acetate extract from persimmon leaves (EAPL) and its commercial preparation, Naoxinqing (Brain and Heart Clear capsules), has been developed and validated. Based on HPLC–DAD-ESI-Q-TOF–MS analysis, myricetin-3-O-β-d-galactoside (1), myricetin-3-O-glucoside (2), quercetin-3-O-β-d-galactoside (3), quercetin-3-O-β-d-glucoside (4), quercetin-3-O-(2″-O-galloyl-β-d-galactoside) (5), quercetin-3-O-(2″-O-galloyl-β-d-glucoside) (6), kaempferol-3-O-β-d-galactoside (7), kaempferol-3-O-β-d-glucoside (8), kaempferol-3-O-(2″-O-galloyl-β-d-galactoside) (9), kaempferol-3-O-(2″-O-galloyl-β-d-glucoside) (10), quercetin (11) and kaempferol (12) were identified from 15 batch samples. A HPLC fingerprint analytical method was established. All compounds, with the exception of compound 2, were simultaneously quantified by the single standard to determine multi-components (SSDMC) method, using kaempferol-3-O-β-d-glucoside as the internal standard. The rate of analysis was found to be faster with the SSDMC method than with current acid hydrolysis method (Pharmacopoeia of the People's Republic of China 2015 edition) and the results were more intuitive and reliable. Three-dimensional principal component analysis revealed that there were similar characteristics in persimmon leaf from same district. Analysis of the myocardial cell protection activity of 11 monomeric compounds showed that compounds 12, 11 and 10 were the main active ingredients that produce pharmacologic functions in EAPL. Among these compounds, the bioactive constituent of myricetin-3-O-β-d-galactoside was determined for the first time in Diospyros khaki. Thus, we have established an effective assessment method that can be applied to the comprehensive quality evaluation of EAPL extract and Naoxinqing capsule.

     

New acylated flavonoid glycosides from flowers of Aerva javanica

Chromatographic purification of ethyl acetate soluble fraction of the methanolic extract of the flowers of Aerva javanica yielded three new acylated flavone glycosides: kaempferol-3-O-β-d-[4?-E-p-coumaroyl-α-l-rhamnosyl(1 → 6)]-galactoside (1), kaempferol-3-O-β-d-[4?-E-p-coumaroyl-α-l-rhamnosyl(1 → 6)]-(3″-E-p-coumaroyl)galactoside (2), and kaempferol-3-O-β-d-[4?-E-p-coumaroyl-α-l-rhamnosyl(1 → 6)]-(4″-E-p-coumaroyl)galactoside (3), along with p-coumaric acid (4), caffeic acid (5), gallic acid (6), eicosanyl-trans-p-coumarate (7), hexadecyl ferulate (8), and hexacosyl ferulate (9). The compounds 1–9 were characterized using 1D (¹H, ¹³C) and 2D NMR (HMQC, HMBC, and COSY) spectroscopy and mass spectrometry (EI-MS, HR-EI-MS, FAB-MS, and HR-FAB-MS) and in comparison with the reported data in the literature. Compound 1 showed weak inhibitory activity against enzymes, such as acetylcholinesterase, butyrylcholinesterase, and lipoxygenase with IC₅₀ values 205.1, 304.1, and 212.3 μM, respectively, whereas compounds 2 and 3 were only weakly active against the enzyme acetylcholinesterase.     

Polyphenolic compounds from the leaves of Koelreuteria paniculata Laxm

From the fresh leaves of Koelreuteria paniculata Laxm (Sapindaceae), four new compounds, named ethyl p-trigallate (1), 3"-O-galloyl-4'-O-galloyl-4-O-galloyl-gallic acid (2), ethyl p-heptagallate (3) and 3"-galloylquercitrin (4), together with 12 known compounds namely catechin (5), galloylepicatechin (6), isorhamnetin (7), kaempferol-3-O-arabinopyranoside (8), quercetin-3'-O- g -D-arabinopyranoside (9), quercitrin (10), methyl p-digallate (11), methyl m-digallate (12), p-digalloyl acid (13), m-digalloyl acid (14), hyperin (15) and kaempferol-3-O- f -L-rhamnoside (16) were isolated by extensive column chromatographic separation. Their structures were elucidated on the basis of chemical and spectroscopic methods. Compound 9 was not reported previously with pyranoside of arabinose at C-3'. Compounds 4 and 9 possessed the activity for PTK inhibition.     

Flavonoids of Calligonum polygonoides and their cytotoxicity

Context Calligonum polygonoides L. subsp. comosum L’ Hér. (Polygonaceae), locally known as “arta”, is a slow-growing small leafless desert shrub.

Objective Isolation, structure elucidation and evaluation of cytotoxic activity of flavonoids from C. polygonoides aerial parts.

Materials and methods Flavonoids in the hydroalcoholic extract of the of C. polygonoides were isolated and purified using column chromatography and preparative HPLC. The structures of the isolated flavonoids were elucidated on the basis of spectroscopic data including 2D NMR techniques. The cytotoxic activity of the isolated flavonoids (6.25, 25, 50 and 100?μg/mL) was evaluated against liver HepG2 and breast MCF-7 cancer cell lines using sulphorhodamine-B assay.

Results A new flavonoid, kaempferol-3-O-β-D-(6″-n-butyl glucuronide) (1), and 13 known flavonoids, quercetin 3-O-β-D-(6″-n-butyl glucuronide) (2), kaempferol-3-O-β-D-(6″-methyl glucuronide) (3), quercetin-3-O-β-D-(6″-methyl glucuronide) (4), quercetin-3-O-glucuronide (5), kaempferol-3-O-glucuronide (6), quercetin-3-O-α-rhamnopyranoside (7), astragalin (8), quercetin-3-O-glucopyranoside (9), taxifolin (10), (+)-catechin (11), dehydrodicatechin A (12), quercetin (13), and kaempferol (14), were isolated from the aerial parts of C. polygonoides. Quercetin showed significant cytotoxic activity against HepG2 and MCF-7 cell lines with IC₅₀ values of 4.88 and 0.87?μg/mL, respectively. Structure–activity relationships were analyzed by comparing IC₅₀ values of several pairs of flavonoids differing in one structural element.

Discussion and conclusion The activity against breast cancer cell lines decreased by glycosylation at C-3. The presence of 2,3-double bond in ring C, carbonyl group at C-4 and 3’,4’-dihydroxy substituents in ring B are essential structural requirements for the cytotoxic activity against breast cancer cells.     

Flavonoid constituents of Dobera glabra leaves: amelioration impact against CCl4-induced changes in the genetic materials in male rats

Context: Dobera glabra (Forssk.) Poir (Salvadoraceae) is a highly valued tree with diverse importance as special mineral sourced feed and a folkloric tool for forecasting droughts. However, there are no reports on its phytochemical and biological investigations.

Objective: Phytochemical investigation of D. glabra leaves and its protective potential against CCl₄ inducing changes in the genetic materials.

Materials and methods: D. glabra extract, DGE (70% MeOH/H₂O), was applied to polyamide column chromatography, eluting with MeOH/H₂O of decreasing polarities, followed by preparative chromatographic tools, yielded seven compounds. Three DGE doses (50, 100 and 200?mg/kg bw/d) were administrated for 8 weeks intragastrically to male albino rats prior treated with CCl₄ (0.5?mL/kg/bw). The reactive oxygen species (ROS) levels, expression changes of glutamate transporters (GLAST, GLT-1 and SNAT3) mRNA, DNA fragmentation and glutathione peroxidase (GPx) activity were investigated in the liver tissues of these rats.

Results: Isorhamnetin-3-O-β-glucopyranoside-7-O-α-rhamnopyranoside, isorhamnetin-3-O-α-rhamnopyranoside-7-O-β-glucopyranoside, kaempferol-3,7-di-O-α-rhamnopyranoside, isorhamnetin-3-O-β-glucopyranoside, kaempferol-3-O-β-glucopyranoside, isorhamnetin and kaempferol were identified. DGE (200?mg/kg bw)?+?CCl₄ exhibited the most significant reduction in ROS levels and DNA fragmentation with 251.3% and141% compared to 523.1% and 273.2% for CCl₄, respectively. Additionally, it increased significantly the mRNA expression of GLAST, GLT-1 and SNAT3 to 2.16-, 1.72- and 2.09-fold, respectively. Also, GPx activity was increased to 4.8?U/mg protein/min compared to CCl₄ (1.8?U/mg protein/min).

Discussion and conclusion: Flavonoid constituents, antioxidant effect and genotoxic protection activity of D. glabra were first reported. DGE may be valuable in the treatment and hindrance of hepatic oxidative stress and genotoxicity.     

Schistosomicidal evaluation of flavonoids from two species of Styrax against Schistosoma mansoni adult worms

Context: Schistosomiasis is a major health problem worldwide. Thus, the search for new schistosomicidal agents from natural sources can provide prototypes for drug discovery.

Objective: The present study investigated the chemical composition of the EtOAc fractions of Styrax pohlii Pohl (Styracaceae) (EF-SP) aerial parts and S. camporum A. DC. leaves (EF-SC), as well as schistosomicidal activities against Schistosoma mansoni adult worms, which have not yet been studied.

Materials and methods: The crude ethanol extracts of S. camporum leaves and S. pohlii aerial parts (EE-SC and EE-SP) were partitioned with n-hexane, EtOAc, and n-BuOH. The EtOAc fractions were purified by preparative HPLC. The crude extracts, EtOAc fractions and pure compounds were tested against S. mansoni adult worms in vitro.

Results: The purification procedure resulted in the isolation of kaempferol-3-O-(2′′,4′′-di-O-(E)-p-coumaroyl)-β-d-glucopyranoside (1), kaempferol-3-O-(2′′,6′′-di-O-(E)-p-coumaroyl)-β-d-glucopyranoside (2), quercetin (3), and kaempferol (4). The bioassay results indicated that EE-SC, EF-SC, EF-SP, and compounds 2 and 4 are able to separate coupled S. mansoni adult worms. Additionally, EE-SC, EF-SP, and compound 4 killed the adult schistosomes in vitro at 100 µg/mL and 100 µM.

Discussion and conclusion: This is the first time that the presence of compounds 1–2 in S. pohlii and 3–4 in S. camporum has been reported. Additionally, biological results indicated that S. pohlii and S. camporum have great potential as a source of active compounds.     

Antiulcerogenic Activity of Alhagi maurorum.

Abstract

Six main flavonoid glycosides were isolated, for the first time, from the ethanol extract of Alhagi maurorum Boiss (Leguminosae).. They were identified as kaempferol, chrysoeriol, isorhamnetin, chrysoeriol-7-O.-xylosoid, kaempferol-3-galactorhamnoside, and isorhamnetin 3-O.-β-D-apio-furanosyl (1-2) β-D-galactopyranoside. Their identities were established by m.p., UV, EI-mass, Fab-mass, 600 MHz ¹H and ¹³C NMR. The total extract (300 and 400 mg/kg) and two of the isolated compounds (chrysoeriol 7-O.-xylosoid and kaempferol-3-galactorhamnoside, 100 mg/kg each) showed a very promising antiulcerogenic activity with curative ratios 66.31%, 69.57%, 75.49%, and 77.93%, respectively.     

Chemical constituents from Mahkota dewa

A new phenolic glycoside (1), mahkoside A, together with six known compounds including mangiferin (2), kaempferol-3-O-β-d-glucoside (3), dodecanoic acid (4), palmitic acid (5) ethyl stearate (6) and sucrose (7), were isolated from the pit of Mahkota dewa. Their structures were identified on the basis of spectroscopic analysis. All the compounds were isolated from the title plant for the first time.     

Polyphenolic compounds isolated from the leaves of Myrtus communis

Four hydrolyzable tannins [oenothein B (1), eugeniflorin D₂ (2), and tellimagrandins I (3) and II (4)], two related polyphenolic compounds [gallic acid (5) and quinic acid 3,5-di-O-gallate (6)], and four myricetin glycosides [myricetins 3-O-β-d-xyloside (7), 3-O-β-d-galactoside (8), 3-O-β-d-galactoside 6″-O-gallate (9), and 3-O-α-l-rhamnoside (10)] were isolated from the leaves of Myrtus communis. Antioxidant activities of the isolated compounds were evaluated by 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay.     

Flavonoids from Camptosorus sibiricus Rupr.

Three new flavonol glycosides, kaempferol-3-O-(6-trans-caffeoyl)-β-d-glucopyranosyl-(1 → 2)-β-d-glucopyranoside (1), kaempferol-3-O-(6-trans-caffeoyl)-β-d-glucopyranosyl-(1 → 2)-β-d-glucopyranoside-7-O-β-d-glucopyranoside (2), and kaempferol-3-O-(6-trans-p-coumaroyl)-β-d-glucopyranosyl-(1 → 2)-β-d-glucopyranoside-7-O-β-d-glucopyranoside (3), were isolated from the aerial part of Camptosorus sibiricus. Their structures were elucidated by spectroscopic methods, including 2D NMR spectral techniques.     

Kaempferol-3-O-rutinoside from Afgekia mahidoliae promotes keratinocyte migration through FAK and Rac1 activation

The restoration of the epidermal epithelium through re-epithelialization is a critical process in wound healing. Directed keratinocyte migration to the wound is required, and the retardation of this process may result in a chronic, non-healing wound. The present study contributes to research aiming to identify promising compounds that promote wound healing using a human keratinocyte model. The effects of three kaempferol glycosides from an Afgekia mahidoliae leaf extract, kaempferol-3-O-arabinoside, kaempferol-3-O-glucoside, and kaempferol-3-O-rutinoside, on keratinocyte migration were determined. Interestingly, kaempferol-3-O-rutinoside exhibited a pronounced effect on wound closure in comparison to the parental kaempferol and other glycosides. The mechanism by which kaempferol-3-O-rutinoside enhances cell migration involves the induction of filopodia and lamellipodia formation, increased cellular levels of phosphorylated FAK (Tyr 397) and phosphorylated Akt (Ser 473), and up-regulation of active Rac1-GTP. The data obtained in this study may support the development of this compound for use in wound healing therapies.

     

鼠掌老鹳草抗菌活性成分的研究

从鼠掌老鹳草中分得一个具有抗菌活性的新化合物—短叶苏木酚酸乙酯(7),通过光谱分析测定了结构。另外还分得10个已知成分。     

Compounds from the pods of Astragalus armatus with antioxidant,anticholinesterase, antibacterial and phagocytic activities

Context: The phytochemical study and biological activities of Astragalus armatus Willd. subsp. numidicus (Fabaceae) pods, an endemic shrub of Maghreb, are reported.

Objective: This study isolates the secondary metabolites and determines the bioactivities of Astragalus armatus pods.

Materials and methods: The chloroform, ethyl acetate and n-butanol extracts of hydro-ethanolic extracts were studied. Antioxidant activity was investigated using DPPH and ABTS radical scavenging, CUPRAC and ferrous chelating assays at concentrations ranging from 3 to 200?μg/mL. Anticholinesterase activity was determined against acetylcholinesterase and butyrylcholinesterase enzymes at 50, 100 and 200?μg/mL. Antibacterial activity was performed according to minimum inhibitory concentration (MIC) method. Carbon clearance method in albino mice was used for the phagocytic activity at concentrations 50, 70 and 100?mg/kg body weight. Spectroscopic techniques were used to elucidate the compounds.

Results: Ethyl acetate extract afforded a flavonoid (1) while the n-butanol extract gave four flavonoids (2–5), a cyclitol (6) and a cycloartane-type saponin (7). The ethyl acetate extract exhibited highest antioxidant activity in DPPH (IC₅₀: 67.90?±?0.57?μg/mL), ABTS (IC₅₀: 11.30?±?0.09?μg/mL) and CUPRAC (A_0.50: 50.60?±?0.9?μg/mL) assays. The chloroform extract exhibited the best antibacterial activity against Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa, each with 80?μg/mL MIC values. The n-butanol extract enhanced phagocytic activity.

Discussion and conclusion: Isorhamnetin (1), isorhamnetin-3-O-α-l-rhamnopyranosyl-(1 → 6)-β-d-galactopyranoside (2), isorhamnetin-3-O-β-d-apiofuranosyl-(1 → 2)-[α-l-rhamnopyranosyl-(1 → 6)]-β-d-galactopyranoside (3), kaempferol-3-O-(2,6-di-O-α-l-rhamnopyranosyl)-β-d-galactopyranoside (4), kaempferol-3-O-(2,6-di-O-α-l-rhamnopyranosyl)-β-d-glucopyranoside (5), pinitol (6) and cyclomacroside D (7) were isolated whereas 1, 2, 6 and 7 are reported for the first time from A. armatus.     

Two new glycosides from Carduus acanthoides

Two new glycosides, syringic acid-4-O-β-l-arabinopyranoside (1) and kaempferol-3-O-α-l-rhamnopyranosyl-7-O-β-d-glucuronopyranoside (2), were isolated from whole plants of Carduus acanthoides (Asteraceae), and their structures were elucidated on the basis of spectroscopic analysis.     

Metabolism of 6-Chloro-5-cyclohexylindane-1-carboxylic Acid (TAI-284), a New Non-steroidal Anti-inflammatory Agent. II. Isolation and Identification of Metabolites in the Perfusate of the Isolated Liver Perfusion in Rats

Abstract

1. Biotransformation of 6-chloro-5-cyclohexylindane-1-carboxylic acid labelled with tritium and deuterium was studied in isolated perfused rat liver. Five metabolites were isolated by column and thin-layer chromatography.

2. The metabolites and their methyl esters were characterized by mass and proton magnetic resonance spectrometry with aid of a shift reagent, tris(dipivalomethanato)europium. The following metabolites were identified : 6-chloro-5-(4′-oxocyclohexyl)indane-1-carboxylic acid(metabolite I),6-chloro-5-(cis-4′-hydroxycyclohexyl)indane-1-carboxylic acid (metabolite IIa), 6-chloro-5-(trans-4′-hydroxycyclohexyl)indane-1-carboxylic acid (metabolite III), 6-chloro-5-(cis-3′-hydroxycyclohexyl)indane-1-carboxylic acid (metabolite IV or IIb). Metabolites IV and IIb are diastereoisomers.

3. The compound was metabolized primarily by hydroxylation producing metabolites IIa (cis-4′-ol), IIb (cis-3′-ol), III (trans-4′-ol) and IV (cis-3′-ol). Metabolites IIa and III seemed to be further metabolized to I (4′-oxo).     

Three new flavonoid glycosides from Urena lobata

Three new flavonoid glycosides, kaempferol-3-O-β-d-apiofuranosyl(1 → 2)-β-d-glucopyranosyl-7-O-α-l-rhamnopyranoside (1), kaempferol-4′-O-β-d-apiofuranosyl-3-O-β-d-glucopyranosyl-7-O-α-l-rhamnopyranoside (2), and 5,6,7,4′-tetrahydroxy-flavone-6-O-β-d-arabinopyranosyl-7-O-α-l-rhamnopyranoside (3), were isolated from the aerial parts of Urena lobata L., along with 10 known compounds (4–13). Their structures were determined based on spectroscopic methods including 1D and 2D NMR spectroscopy as well as HR-ESI-MS.     

Protein tyrosine phosphatase 1B (PTP1B) inhibitory activity and glucosidase inhibitory activity of compounds isolated from Agrimonia pilosa

Context: Despite phytochemical studies of Agrimonia pilosa Ledeb. (Rosaceae), the antidiabetic effects of this plant are unknown.

Objective: This study characterizes the isolated compounds from the aerial parts of A. pilosa and evaluates their PTP1B and α-glucosidase inhibitory properties.

Materials and methods: Ethanol extract of A. pilosa was found to inhibit 64% PTP1B activity at 30?μg/mL. The ethanol extract was partitioned with methylene chloride, ethyl acetate, n-butanol, and water fractions. Among these, the ethyl acetate fraction displayed the most potent PTP1B activity. The ethyl acetate extract was separated by chromatographic methods to obtain flavonoids and triterpenoids (1–11); which were evaluated for their inhibitory effects on PTP1B activity with p-nitrophenyl phosphate (p-NPP) as a substrate, and also α-glucosidase enzyme.

Results: Compounds 1–11 were identified as apigenin-7-O-β-d-glucuronide-6″-methyl ester, triliroside, quercetin-7-O-β-d-glycoside, quercetin-3-O-β-d-glycoside, kaempferol, kaempferol-3-O-α-l-rhamnoside, β-sitosterol, ursolic acid, tormentic acid, methyl 2-hydroxyl tricosanoate, and palmitic acid. Compounds 8, 9, and 11 displayed inhibitory effects on PTP1B activity with IC₅₀ values of 3.47?±?0.02, 0.50?±?0.06, and 0.10?±?0.03?μM, respectively. Compounds 3, 4, 6, and 9 exhibited inhibition of the α-glucosidase activity with IC₅₀ values of 11.2?±?0.2, 29.6?±?0.9, 28.5?±?0.1, and 23.8?±?0.4?μM, respectively.

Discussion and conclusion: As major ingredients of A. pilosa, compounds 1, 6, 8, and 9 showed the greatest inhibitory potency on PTP1B activity. Compounds 3, 6, 8, and 9 also showed potent inhibitory effects on α-glucosidase enzyme. This result suggested the potential of these compounds for developing antidiabetic agents.     

Ellagic acid glycosides with hepatoprotective activity from traditional Tibetan medicine Potentilla anserina

Two new gallic acid glycosides, potentillanosides G (1) and H (2), were newly isolated from the methanol extract of the tuberous roots of Potentilla anserina (Rosaceae), together with a known compound, ellagic acid 3-O-α-l-rhamnopyranoside (3). Their structures were elucidated on the basis of chemical and physicochemical evidence. Among the constituents, potentillanoside H (2, IC₅₀ = 99.5 μM) was found to show hepatoprotective activity.

     

A new ferulic acid ester and other constituents from Dracocephalum peregrinum

A new ferulic acid ester, 1′-methyl-2′-hydroxyethyl ferulate (1), together with methylcaffeate (2), 4-hydroxy cinnamic acid (3), ferulic acid (4), caffeic acid (5), diosmetin (6), luteolin (7), 5,3′,4′-trihydroxy-3,7-dimethoxyflavone (8), eriodictyol (9), kaempferol (10), quercetin (11), acacetin-7-O-glcopyranoside (12), 4-(β-glucopyranosyloxy) benzoic acid (13), luteolin-7-O-(6″-feruloyl) glucopyranoside (14), luteolin-7-O-glucopyranoside (15), kaempferide-3-O-rhamnopyranoside (16), quercitrin (17), kaempferol-3-O-glucopyranoside (18), prunasin (19), quercetin-7-O-glucopyranoside (20), quercetin-3-O-glucopyranoside (21), plantaginin (22), linarin (23), luteolin-7-O-rutinoside (24), and chlorogenic acid (25) were isolated from the aerial parts of Dacocephalum peregrinum. The structure of 1 was elucidated on the basis of spectroscopic and HR-ESI-MS analyses. In addition, compound 1 exhibited mild inhibitory effect on NO production in LPS-stimulated RAW264.7 cells.     

Biotransformation of diethenylbenzenes. III: Identification of metabolites of 1,3-diethenylbenzene in rat

1. Biotransformation of 1,3-diethenylbenzene (1) in rat gave four major metabolites, namely, 3-ethenylphenylglyoxylic acid (2), 3-ethenylmandelic acid (3), N-acetyl-S-[2-(3-ethenylphenyl)-2-hydroxyethyl]-L-cysteine (4) and N-acetyl-S-[1-(3-ethenylphenyl)-2-hydroxyethyl]-L-cysteine (5) were isolated from urine and identified by n.m.r. and mass spectrometry.

2. Four minor metabolites, 3-ethenylbenzoic acid (6), 3-ethenylphenylacetic acid (7), 3-ethenylbenzoylglycine (8) and 2-(3-ethenylphenyl)ethanol (9) were identified by g.l.c.-mass spectrometric analysis of urine extract derivatized in two different ways.

3. All identified metabolites are derived from 3-ethenylphenyloxirane (10), a reactive metabolic intermediate. No product of any metabolic transformation of second ethenyl group has been identified. However, several minor unidentified metabolites were detected by g.l.c.-mass spectrometry.

4. Total thioether excretion in 24h urine after a single i.p. dose of 1 amounted to 28·3±3·5 dose (mean±SD). No significant differences in the thioether fraction were observed in the dose range 100-300mg/kg.

5. Thioether metabolites consisted mainly of mercapturic acids 4 and 5. The ratio of metabolites 5 to 4 was 62:38. Each mercapturic acid consisted of two diastereomers. Their ratio, as determined by quantitative ¹³C-n.m.r. measurement was 95:5 and 79:21 for mercapturic acids 4 and 5, respectively.