Comparative analysis of chloroplast genomes of Pulsatilla species reveals evolutionary and taxonomic status of newly discovered endangered species Pulsatilla saxatilis.

BACKGROUND: Pulsatilla saxatilis, a new species of the genus Pulsatilla has been discovered. The morphological information of this species has been well described, but its chloroplast genome characteristics and comparison with species of the same genus remain to be reported. RESULTS: Our results showed that the total length of chloroplast (cp.) genome of P. saxatilis is 162,659 bp, with a GC content of 37.5%. The cp. genome contains 134 genes, including 90 known protein-coding genes, 36 tRNA genes, and 8 rRNA genes. P. saxatilis demonstrated similar characteristics to other species of genus Pulsatilla. Herein, we compared cp. genomes of 10 species, including P. saxatilis, and found that the cp. genomes of the genus Pulsatilla are extremely similar, with a length of 162,322-163,851 bp. Furthermore, The SSRs of Pulsatilla ranged from 10 to 22 bp in length. Among the four structural regions of the cp. genome, most long repeats and SSRs were detected in the LSC region, followed by that in the SSC region, and least in IRA/ IRB regions. The most common types of long repeats were forward and palindromic repeats, followed by reverse repeats, and only a few complementary repeats were found in 10 cp. genomes. We also analyzed nucleotide diversity and identified ccsA_ndhD, rps16_trnK-UUU, ccsA, and rbcL, which could be used as potential molecular markers for identification of Pulsatilla species. The results of the phylogenetic tree constructed by connecting the sequences of high variation regions were consistent with those of the cp. gene phylogenetic tree, and the species more closely related to P. saxatilis was identified as the P. campanella. CONCLUSION: It was determined that the closest species to P. saxatilis is P. campanella, which is the same as the conclusion based on pollen grain characteristics, but different from the P. chinensis determined based on morphological characteristics. By revealing information on the chloroplast characteristics, development, and evolution of the cp. genome and the potential molecular markers, this study provides effective molecular data regarding the evolution, genetic diversity, and species identification of the genus Pulsatilla.

Pharmacokinetic studies of soyalkaloid A from Portulaca oleracea L. using ultra high-performance liquid chromatography electrospray ionization quadrupole-time of flight mass spectrometry and its antioxidant activity.

Soyalkaloid A was isolated from Portulaca oleracea L. for the first time in our laboratory and then a rapid and sensitive ultra-high-performance liquid chromatography electrospray ionization quadrupole-time of flight mass spectrometry (UHPLC-ESI-Q-TOF/MS) method with hesperidin as internal standard (IS) was developed and validated to investigate the pharmacokinetics of soyalkaloid A in rats after oral and intravenous administrations. The analysis was achieved on an Agilent Zorbax Eclipse Plus C18 Column (2.1 × 50 mm, 1.8 µm) by elution with acetonitrile and water (containing 0.1% formic acid), at a flow rate of 0.3 mL/min. The MS analysis was performed in the positive ion mode with monitored ion m/z 227.0814 [M + H]+ and 611.1971 [M + H]+ for soyalkaloid A and IS, respectively. The linear range was established over the concentration range 7.5-6000 ng/mL (r = 0.9951). The intra- and inter-assay accuracy and precision were between -4.86-4.49 and 1.93-9.66, respectively. The lower limits of detection and quantitation observed were 2.1 and 7.4 ng/mL, respectively. The rapid, sensitive and specific UHPLC-ESI-Q-TOF/MS method was successfully applied to a pharmacokinetic study of soyalkaloid A. Moreover, its antioxidant was studied via a 1,1-diphenyl-2-picryl-hydrazyl radical scavenging assay, the IC50 value being 20.73 ± 0.51 µM.

Investigating the bioavailabilities of olerciamide A via the rat's hepatic, gastric and intestinal first-pass effect models.

Olerciamide A (OA) is a new alkaloid isolated from Portulaca oleracea L. that has been proved to possess a low bioavailability (F) after oral administration in rats in our previous study. Hence, to clarify the reasons for its low bioavailability, hepatic, gastric and intestinal first-pass effect models were established, and a rapid, sensitive UHPLC method was validated and applied for the determination after dosing via the femoral, portal, gastric and intestinal routes. As inhibitors of CYP3A and P-gp, verapamil, midazolam and borneol in low and high dose groups were selected to improve the low bioavailability of olerciamide A. Moreover, a rectal administration method was also carried out to improve the bioavailability of olerciamide A. The results showed that the bioavailability of olerciamide A using hepatic, gastric and intestinal routes were 92.16%, 84.88% and 5.76%, respectively. The areas under the plasma concentration-time curve from zero to infinity (AUC0 → ∞ ) were increased a little after being dosed with 10 and 30 mg/kg verapamil (p > 0.05), but markedly increased after being dosed with 0.4 and 1.2 mg/kg midazolam as well as 8 and 24 mg/kg borneol (p < 0.05). Besides, the AUC0 â†’ ∞ values after the lower and upper rectal administrations were separately similar to the intravenous and intraportal administrations. Our study showed that the intestinal first-pass effect mainly contributed to the low bioavailability of olerciamide A in rats due to it being a substrate of CYP3A and P-gp as well as to its poor intestinal absorption.

Pharmacokinetics and metabolism of olerciamide A from Portulaca oleracea L. in rats by UHPLC-UV and UHPLC-ESI-Q-TOF/MS.

The aim of this study was to elucidate the pharmacokinetics of olerciamide A in rats after oral and intravenous administration of Portulaca oleracea L. extract by a simple and rapid ultra high-performance liquid chromatography method with bergapten as internal standard. The pharmacokinetic results indicated that olerciamide A was rapidly distributed with a time to peak concentration of 30 min after oral administration and presented a low oral absolute bioavailability of 4.57%. The metabolism of olerciamide A in rats was also investigated using ultra-high-performance liquid chromatography electrospray coupled with quadrupole-time of flight mass spectrometry to elucidate the reason for the low absolute bioavailability of olerciamide A and seven metabolites of oleraciamide A were found in rat plasma and urine.

Hepatic, gastric and intestinal first-pass effects of vitexin-2''-O-rhamnoside in rats by ultra-high-performance liquid chromatography.

Previous research in our laboratory found that the absolute bioavailability of vitexin-2''-O-rhamnoside (VR) was quite low at 4.89%. A rapid and sensitive UHPLC method using hesperidin as an internal standard was therefore developed and validated to investigate the reasons for this by determining VR in rat plasma after administering intravenously, intraportally (5 mg/kg), intraduodenally and intragastrically (40 mg/kg) to the rat model of the hepatic, gastric and intestinal first-pass effects. As only a high intestinal first-pass effect of VR was found, that is, there existed a low bioavailability of VR (2.40%), inhibitors of P-glycoprotein (P-gp) and cytochrome P450 3A (CYP3A), including verapamil, cyclosporin A and midazolam, and absorption enhancers, including bile salts and borneol, combined with VR, were instilled into duodenum to evaluate the effects on bioavailability of VR. The results demonstrated that area under the concentration-time curve (AUC) values of VR slightly increased after administration of verapamil, cyclosporin A and midazolam, indicating that CYP3A and P-gp do not play an important role in the first-pass effect in the intestine. AUC values of VR significantly increased after administering bile salts or borneol, indicating that the low bioavailability of VR was mainly related to its poor absorption in the intestine.

Tissue distribution comparison between healthy and fatty liver rats after oral administration of hawthorn leaf extract.

Hawthorn leaves, a well-known traditional Chinese medicine, have been widely used for treating cardiovascular and fatty liver diseases. The present study aimed to investigate the therapeutic basis treating fatty liver disease by comparing the tissue distribution of six compounds of hawthorn leaf extract (HLE) in fatty liver rats and healthy rats after oral administration at first day, half month and one month, separately. Therefore, a sensitive and specific HPLC method with internal standard was developed and validated to determine chlorogenic acid, vitexin-4''-O-glucoside, vitexin-2''-O-rhamnoside, vitexin, rutin and hyperoside in the tissues including heart, liver, spleen, kidney, stomach and intestine. The results indicated that the six compounds in HLE presented some bioactivity in treating rat fatty liver as the concentrations of the six compounds varied significantly in inter- and intragroup comparisons (healthy and/or fatty liver group).

Hepatic, gastric, and intestinal first-pass effects of vitexin in rats.

CONTEXT: Recent research has demonstrated that vitexin exhibits a prominent first-pass effect. In this light, it is necessary to investigate the causes of this distinct first-pass effect. OBJECTIVE: The aim of this study was to evaluate hepatic, gastric, and intestinal first-pass effects of vitexin in rats and, furthermore, to investigate the role of P-glycoprotein (P-gp) and cytochrome P450 3A (CYP3A) in the absorption and secretion of vitexin in the duodenum. MATERIALS AND METHODS: Vitexin was infused into rats intravenously, intraportally, intraduodenally, and intragastrically (30 mg/kg). In addition, verapamil (50 mg/kg), a common substrate/inhibitor of P-gp and CYP3A, was also instilled with vitexin into the duodenum to investigate the regulatory action of P-gp and CYP3A. The plasma concentrations of vitexin were measured by the HPLC method using hesperidin as an internal standard. RESULTS: The hepatic, gastric, and intestinal first-pass effects of vitexin in rats were 5.2%, 31.3%, and 94.1%, respectively. In addition, the total area under the plasma concentration-time curve from zero to infinity (AUC) of the vitexin plus verapamil group and of the normal saline group was 44.9 and 39.8 µgc min/mL, respectively. DISCUSSION AND CONCLUSION: The intestinal first-pass effect of vitexin was considerable, and gastric and hepatic first-pass effects also contribute to the low absolute oral bioavailability of vitexin. The AUC of the vitexin plus verapamil group was slightly higher than that of the vitexin plus normal saline group (by approximately 1.13-fold), suggesting that verapamil does not play an important role in the absorption and secretion of vitexin.

Four alkaloids from Portulaca oleracea L. and their anti-inflammatory.

Four alkaloids were isolated from Portulaca oleracea L., including two new compounds, 2-(4-amino-6-hydroxy-1,6-dihydropyrimidin-5-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol, named Olerapyrimidine (1) and (2 R,3S,4S,5R,6S)-2-(hydroxymethyl)-6-((6-hydroxypyridin-3-yl)oxy)tetrahydro-2H-pyran-3,4,5-triol, named Olerapyridine (2), and two known compounds including 1H-imidazole (3) and (5S, 6 R, 7S, 8 R)-5-amino-(2Z,4Z)-1,2,3-trihydroxybuta-2,4-dienyloxy-pentane-6,7,8,9-tetraol (4) from P. oleracea for the first time. Their structures were determined by spectroscopic methods, including UHPLC-ESI-Q-TOF/MS, 1D and 2D NMR spectra. Both Olerapyrimidine and Olerapyridine at 20 µM could inhibit the inflammatory factors, IL-1ß and TNF-α in the RAW 264.7 cells induced by LPS.

Two novel alkaloids from Portulaca oleracea L. and their anti-inflammatory bioactivities.

Two novel alkaloids were identified as (E)-N-(4-3,4-dihydroxy-6-(hydroxymethyl)-5-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran-2-yl)oxy)-2,5-dihydroxyphenyl)-3-(4-hydroxyphenyl)acrylamide (1), named Oleracrylimide D, (E)-N-(4-3,4-dihydroxy-6-(hydroxymethyl)-5-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran-2-yl)oxy)-2,5-dihydroxyphenyl)-3-(4-hydroxy-3-methoxyphenyl)acrylamide (2), named Oleracrylimide E, isolated from Portulaca oleracea L. The structures were identified by spectroscopic methods, including 1D NMR, 2D NMR, and UHPLC-ESI-QTOF/MS methods, also, the anti-inflammatory bioactivities of the compounds were studied by ELISA method.

Two novel ketone alkaloids from Portulaca oleraceaL. and their anti-inflammatory activities.

Eleven compounds were obtained from Portulaca oleracea L., including two novel ketone alkaloids, (1, 2), 4-hydroxy-3-methoxybenzamide (3) (isolated for the first time), ß-adenosine (4), oleracrylimide A and B (5, 6), oleracein H, C, D, Q and A (7-11). The two novel ketone alkaloids were identified as 5-acetyl-5-methylcyclopent-2-ene-1-carboxamide (1), named oleraciamide H, and (2 R,3S,4R,5R)-5-((R)-1,2-dihydroxyethyl)-3,4-dihydroxytetrahydrofuran-2-yl glycinate (2), named oleracone Q by spectroscopic methods, including 1D, 2D NMR and compound fingerprints. Additionally, their anti-inflammatory activities were tested via RAW 264.7 cells induced by LPS and found that they could significantly inhibit the release of IL-1ß and TNF-α.

A new alkaloid and two organic acids from Portulaca oleracea L. and their bioactivities.

A new alkaloid, identified as 1-benzyl-2-nitroso-1,2,3,4-tetrahydroisoquinoline-6,7-diol, named oleraisoquinoline (1), and five organic acids and two esters, identified as 5-(hydroxymethyl)furan-2-carboxylic acid (2), 1H-pyrrole-2,5-dicarboxylic acid (3), (7E,10E)-octadeca-7,10-dienoic acid (4), (10E,13E)-octadeca-10,13-dienoic acid (5), (7E,10E)-hexadeca-7,10-dienoic acid (6), methyl tridecanoate (7) and methyl (9E,12E)-octadeca-9,12-dienoate (8), were isolated from Portulaca oleracea L., among which compounds 2 and 4‒7 were isolated for the first time. Moreover, the anti-inflammatory activities of compounds 1‒3 were studied, especially, compound 1 presented good inhibitory effects on the production of inflammatory factors IL-1ß and TNF-α.

Two novel carbon skeleton compounds from Portulaca oleracea L. and their effects on nitric oxide.

Two new carbon skeleton compounds, identified as 2-(1-(3-hydroxy-2,2,6-trimethyl-6-vinyltetrahydro-2H-pyran-3-yl)-2-methylpropoxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol named Olerapyran A (1), and (E)-3-(6-acetyl-2-methyl-5-((3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)cyclooctylidene)butan-2-one named Oleraoctyl (2), were first isolated from Portulaca oleracea L., then their structures were determined using spectroscopic methods, including UHPLC-ESI-QTOF/MS, 1D and 2D NMR. In addition, the activities of Olerapyran A and Oleraoctyl inhibiting nitric oxide (NO) were studied.

Two new alkaloids from Portulaca oleracea L. and their anti-inflammatory and anticholinesterase activities.

Two new alkaloids identified as 2-(((S,Z)-1-(1H-azirin-1-yl)-5-methylhex-1-en-3-yl)oxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol and (S,Z)-1-(1H-azirin-1-yl)-5-methylhex-1-en-3-ol, named olerazirin A (1), olerazirin B (2), together with five known alkaloids, identified as cyclo (L-Val-L-Ala) (3), cyclo-(glycyl-L-leucine) (4), cyclo-(Gly-Phe) (5), cyclo (Ser-Phe) (6), (3S,6S)-3-[(1R)-1-hydroxyethyl]-6-(phenyl-methyl)-2,5-piperazinedione (7) were obtained from Portulaca oleracea L. using a range of chromatographic techniques, 1D and 2D NMR, and high-resolution electrospray ionisation time-of-flight mass spectroscopic methods, in which the compounds 3-7 were isolated from P. oleracea for the first time. In addition, the results showed that the compounds 1 and 2 have anti-inflammatory activities and compounds 1-3 and 5-7 exhibit the anticholinesterase activities.

Comparative study on the excretion of vitexin-4''-O-glucoside in mice after oral and intravenous administration by using HPLC.

The aim of the present study was to characterize the excretion of pure vitexin-4"-O-glucoside (VOG) in mice following oral and intravenous administration at a dose of 30 mg/kg. A sensitive and specific HPLC method with hespridin as internal standard, a Diamonsil C18 column protected with a KR C18 guard column and a mixture consisting of methanol-acetonitrile-tetrahydrofuran-0.1% glacial acetic acid (6:2:18:74, v/v/v/v) as mobile phase was developed and validated for quantitative analysis in biological samples. VOG could be excreted as prototype in excreta including urine and feces after both routes of administration, and the cumulative excretion of VOG was 24.31 ± 11.10% (17.97 ± 5.59% in urinary excretion; 6.34 ± 5.51% in fecal excretion) following oral dosing and 5.66 ± 3.94% (4.78 ± 3.13% in urinary excretion; 0.88 ± 0.81% in fecal excretion) following intravenous dosing. The results showed that the elimination of VOG after the two routes was fairly low, which meant that VOG was metabolized as other forms and the elimination after oral dosing was almost 4.3-fold that after intravenous dosing. For both routes of administration, VOG excreted as prototype in urine was much more than that in feces, nearly 2.83-fold for oral administration and 5.43-fold for intravenous administration, which should be attributed to enterohepatic circulation. Taken together, renal excretion was the dominant path of elimination of VOG for oral and intravenous administration in mice and biliary excretion contributed less.

Two new ester alkaloids from Portulaca oleracea L. and their bioactivities.

Two new ester alkaloids were isolated from Portulaca oleracea L., identified as (5-aminofuran-2-yl) methyl acetate (1) named oleracone N and 4(S)-ethyl 3-acetamido-3-(dihydroxyamino) propanoate (2) named oleracone O. The structures were elucidated via spectroscopic methods, including 1 D and 2 D NMR, UHPLC-ESI-QTOF/MS and CD spectrometry technique. It was suggested that both oleracone N and oleracone O could significantly inhibit inflammatory factors IL-1ß and TNF-α in RAW 264.7 cells induced by LPS.

Two new natural products from Portulaca oleracea L. and their bioactivities.

Two new natural products, belonging to alkaloids, identified as ((2R,3S,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl acetate (1) and (5-hydroxypyridin-2-yl)methyl acetate (2), were isolated from Portulaca oleracea L. The structures were identified by spectroscopic methods, including 1D, 2D NMR, and UHPLC-ESI-QTOF/MS methods. Meanwhile, the anti-inflammatory and anticholinesterase bioactivities were found in these two compounds.

A novel skeleton alkaloid from Portulaca oleracea L. and its bioactivities.

A novel skeleton alkaloid was obtained from Portulaca oleracea L., which was identified as 10,11-dihydroxybenzo[5',6'] pentaleno[1',2':3,4]pyrrolo[2,1-b]oxazol-7(11bH)-one, named oleracone M, and its structure was determined using UHPLC-ESI-QTOF/MS, 1D NMR and 2D NMR spectroscopy, and circular dichroism. Then the bioactivities of the compound were investigated including the anti-inflammatory, anti-acetylcholinesterase and antioxidant activities. The results showed that the novel skeleton alkaloid exhibited the potent effect on inhibiting the secretion of IL-1ß at 10 µM, anticholinesterase activity with IC50 value of 49.58 µM, and antioxidant activity with IC50 value of 66.43 µM.

A new alkaloid from Portulaca oleracea L. with its anti-inflammatory activity.

A new isoindole alkaloid, 6-hydroxy-2-(4'''-hydroxy-3'''-methoxyphenethyl)-4-(4'-hydroxy-3'-methoxyphenyl)-7-methoxy-1H-benzo[f]isoindole-1,3(2H)-dione, named oleraisoindole B was isolated from Portulaca oleracea L., its structure was elucidated using NMR and UHPLC-ESI-Q-TOF/MS spectroscopic methods, and presented anti-inflammatory activity at 5 µM.

Three novel alkaloids from Portulaca oleracea L. and their anti-inflammatory bioactivities.

Three novel alkaloids, identified as (E)-N-((2R)-3-(2,5-dihydroxy-4-((3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)phenyl)-2-hydroxypropanoyl)-3-(4-hydroxyphenyl)acrylamide (1), named oleracrylimide A, (E)-N-((2R)-3-(2,5-dihydroxy-4-((3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)phenyl)-2-hydroxypropanoyl)-3-(4-hydroxy-3-methoxyphenyl)acrylamide (2), named oleracrylimide B, and (E)-N-((2R)-3-(2,5-dihydroxy-4-((3,4,5-trihydroxy-6-(((3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)phenyl)-2-hydroxypropanoyl)-3-(4-hydroxy-3-methoxyphenyl)acrylamide (3), named oleracrylimide C were isolated from Portulaca oleracea L. and the structures of the three novel compounds were determined by 1D and 2D NMR, circular dichroism, and UHPLC-ESI-QTOF/MS spectroscopic methods. Moreover, the bioactivities of anti-inflammation of the three compounds were investigated via testing RAW 264.7 macrophage cell stimulated by Lipopolysaccharide.