Macathioureas A-D, four new thiourea analogues from the roots of Lepidium meyenii.

Macathioureas A-D (1-4), four new thiourea derivatives with a carbamothioylpyrrolidine-2-carboxamide framework, were isolated from the roots of Lepidium meyenii (Maca) collected from Qujing, Yunnan Province of China. Their structures were identified based on extensive spectroscopic data, including 1D NMR, 2D NMR, and HRESIMS techniques. Their absolute configurations were assigned as 7S by the comparison of the experimental and predicted electronic circular dichroism (ECD) spectra. All the thiourea analogues were tested for their cytotoxicities against five human cancer cell lines. However, no significant activities were detected at concentrations up to 40 µM.

Isolation, structure elucidation, and high-performance liquid chromatography quantification of photolytic degradation impurities in acrivastine.

Acrivastine is a second-generation H1-receptor antagonist, and its structure is sensitive to ultraviolet. Four unknown and one reported degradation products can be detected in the ultraviolet radiation solutions of acrivastine. To improve the quality control of acrivastine, the photodegradation impurities were isolated and structurally elucidated. There are four new impurities (1-3 and 5), and one reported compound (4). The isolation strategy was designed as preparative high-performance liquid chromatography using a reversed phase column with volatile acid addition in the mobile phase, combined with preparative thin-layer chromatography using silica gel with alkaline addition in the mobile phase. Using the developed methods, five impurities (1-5) were efficiently purified after two or three chromatography runs with purities > 95%. The structures of compounds 1-5 are elucidated based on spectroscopy analysis of MS, and nuclear magnetic resonance spectroscopy. Using the impurity standard, the high-performance liquid chromatography method was developed and validated. The method was proved to be sensitive, accurate (Recovery% 96.1-107.7%), linear (0.15-0.75 µg/mL, R2 > 0.996), robust, and specific, and it was successfully used to determine the degradation impurities of acrivastine and its formulation.

New ent-Kaurane and cleistanthane diterpenoids with potential cytotoxicity from Phyllanthus acidus (L.) Skeels.

Six diterpenoids including three ent-kauranes (1-2, 4) and three cleistanthanes (3, 5-6) were isolated from the roots and stems of Phyllanthus acidus (L.) Skeels. Of them, (16S)-ent-16,17,18-tri-hydroxy-19-nor-kaur-4-en-3-one (1), phyllanthone A (2), and 6-hydroxycleistanthol (3) are new compounds, while the ent-kaurane diterpenoids were reported from the titled plant for the first time. Their structures were elucidated on the basis of the extensive spectroscopic analyses. Compounds 2 and 4-6 displayed cytotoxic potential with IC50 values ranging from 1.96 to 29.15 µM. They also showed moderate anti-inflammatory activities (IC50 = 6.30-12.05 µM). Particularly, the new ent-kaurane 2 displayed cytotoxic potential against HL-60 (IC50 = 2.00 µM) and MCF-7 (IC50 = 3.55 µM) cells, and anti-inflammatory activity (IC50 = 6.47 µM).

Anti-Fatigue Peptides from the Enzymatic Hydrolysates of Cervus elaphus Blood.

Red deer (Cervus elaphus) blood is widely used as a health product. Mixed culture fermentation improves the flavor and bioavailability of deer blood (DB), and both DB and its enzymatic hydrolysates exhibit anti-fatigue activities in vivo. To elucidate the bioactive ingredients, enzymatic hydrolysates were fractioned into different peptide groups using reversed phase resin chromatography, and then evaluated using an exhaustive swimming mice model to assess swimming time and biochemical parameters. The structures of the bioactive peptides were elucidated by high performance liquid chromatography with tandem mass detection. Thirty-one compounds were identified as glutamine or branched-chain amino acids containing short peptides, of which Val-Ala-Asn, Val-Val-Ser-Ala, Leu(Ile)-Leu(Ile)-Val-Thr, Pro-His-Pro-Thr-Thr, Glu-Val-Ala-Phe and Val-Leu(Ile)-Asp-Ala-Phe are new peptides. The fractions containing glutamine or valine short peptides, Ala-Gln, Val-Gln, Val-Val-Ser-Ala, Val-Leu(Ile)-Ser improved exercise endurance by increasing hepatic glycogen (HG) storage. The peptides group containing Leu(Ile)-Leu(Ile), Asp-Gln, Phe- Leu(Ile), Val-Val-Tyr-Pro contributed to decreased muscle lactic acid (MLA)accumulation and to an increase in HG. The anti-fatigue activities of DB hydrolysates were attributed to the synergistic effects of different types of peptides.

Axial Chiral Binaphthoquinone and Perylenequinones from the Stromata of Hypocrella bambusae Are SARS-CoV-2 Entry Inhibitors.

A new axial chiral binaphtoquinone, hypocrellone (1), and a new perylenequinone, hypomycin F (2), were isolated from the stromata of Hypocrella bambusae, together with five known compounds, 3-7. The structures of 1 and 2 were assigned by spectroscopic and HRESIMS data analyses. The axial chirality of 1 was determined by electronic circular dichroism data analysis, and the absolute configurations of 2 and 3 were determined by X-ray crystallography. The axial chirality of 7 was determined by UV-induced photooxidation from 4. Compounds 1, 4, and 5 showed inhibitory activity against pseudotyped SARS-CoV-2 infection in 293T-ACE2 cells with IC50 values of 0.17, 0.038, and 0.12 µM. Compounds 4 and 5 were also active against live SARS-CoV-2 infection with EC50 values of 0.22 and 0.21 µM, respectively. Further cell-cell fusion assays, surface plasmon resonance assays, and molecular docking studies revealed that 4 and 5 could bind with the receptor-binding domain of SARS-CoV-2 S protein to prevent its interaction with human angiotensin-converting enzyme II receptor. Our results revealed that 4 and 5 are potential SARS-CoV-2 entry inhibitors.

The identification and isolation of anti-inflammatory ingredients of ethno medicine Breynia fruticosa.

ETHNOPHARMACOLOGICAL RELEVANCE: Breynia fruticosa is a folk medicine in China, traditionally used to treat gastroenteritis, sore throat, eczema and arthritis. However, the bioactive ingredients are unknown. AIM OF THE STUDY: To identify and isolate the anti-inflammatory ingredients of B. fruticosa. MATERIALS AND METHODS: B. fruticosa extracts were fractioned by Amberchrom CG161M and Toyopearl HW40C resins. Acetic acid-induced capillary permeability mice model was used to evaluate the anti-inflammation activities of fractions. The anti-inflammatory ingredients were identified by high performance liquid chromatography/mass spectroscopy (HPLC-MS). On-line two dimensional liquid chromatography system was constructed to remove the tannins and enrich the breynins. The breynins were purified by preparative HPLC and evaluated for their anti-arthritis activities using complete Freund's adjuvant (CFA) induced arthritis rats model. RESULTS: The anti-inflammatory ingredients of B. fruticosa are sulfur containing sesquiterpenoids (breynins). The on-line two dimensional preparative liquid chromatography system can effectively remove the tannins and enrich the bioactive ingredients in large scale within 1 h. Four major breynins were purified, and their structures were elucidated by analysis of MS and NMR data. Breynins can significantly prevent the rats' arthritis deterioration, with inhibition ratio 50% at dose 0.2 mg kg-1, comparable with that of indomethacin at dose 2 mg kg-1. CONCLUSION: The breynins have strong anti-arthritis activities, which is responsible to the anti-inflammatory effects of B. fruticosa. However, breynins are also toxic components of B. fruticosa.

A fluorescent probe composed of quantum dot labeled aptamer and graphene oxide for the determination of the lipopolysaccharide endotoxin.

Endotoxins are complex lipopolysaccharides (LPS) and key components of the outer cell membrane of Gram-negative bacteria. The authors report on a fluorescent aptamer-based probe for the determination of LPS of Gram-negative bacteria. An aptamer against LPS was fluorescently labeled with CdSe/ZnS quantum dots. Its emission is quenched on addition of graphene oxide (GO). On addition of LPS, the aptamer binds LPS and GO is released. This results in the recovery of fluorescence, typically measured at excitation/emission wavelengths of 495/543 nm. The probe responds to LPS in the 10-500 ng·mL-1 concentration range, and the detection limit is 8.7 ng·mL-1. It can be used for selective detection of LPS from different Gram-negative bacteria, in the presence of biological interferents. Graphical abstract Schematic presentation of a green fluorescent probe comprised of an aptamer labelled with CdSe/ZnS quantum dots and of graphene oxide. Lipopolysaccharides bind to the aptamer and release graphene oxide to result in fluorescence recovery, which is measured at an emission wavelength 543 nm.

Monitoring of bacteria biofilms forming process by in-situ impedimetric biosensor chip.

A biosensor chip integrated interdigital microelectrodes was proposed and applied to monitor the formation process of Salmonella and E. coli biofilms in this paper. The biosensor chip was composed of a glass substrate with interdigital microelectrodes and PDMS layer with micro cavities. The electrochemical impedance spectroscopy (EIS) of Salmonella and E. coli biofilms was measured by the biosensor chip using alternating voltage of 100 mv in the frequency range from 1 Hz to 100 kHz for 48 h. It was illustrated that the changes of impedance spectroscopy of biofilms occurred with culture time. Furthermore, impedance spectroscopy of biofilms was fitted by an equivalent circuit model including the biofilms capacitance (Cb) and the biofilms resistance (Rb) parameters. The results indicated that the Cb presented a tendency to decrease first and then rise with culture time, while the Rb was in the opposite direction. These changing trends were consistent with the formation process of biofilms that bacteria adhered to electrodes surface, and then formed mature biofilms, finally escaped from biofilms. In addition, it was also demonstrated that the changing trends of Cb and Rb with culture time were quite different between Salmonella and E. coli. The results obtained by impedance detection were in accordance with the results of using crystal violet staining to analyze biofilms formation process, under the same conditions for bacterial culture. The biosensor chip provided a promising platform for further study of biofilms owing to its unique advantages of real time, continuity, and non-invasion for bacteria biofilms detection and in-situ monitoring.

New utilization of Polygonum multiflorum polysaccharide as macromolecular carrier of 5-fluorouracil for controlled release and immunoprotection.

WPMP-2 is an acid polysaccharide isolated from Polygonum multiflorum, which demonstrated excellent immunomodulatory activity. In order to reduce immunosuppression of 5-fluorouracil (5-Fu), WPMP-2 was utilized as a macromolecular carrier to conjugate the 5-Fu derivatives 5-fluorouracil-1-acetic acid (5-FUAC) through ester bond. The conjugate showed controlled drug release behaviour in vitro at 37°C in phosphate buffer (pH7.4), and only 5-FUAC was detected in the media. The cytotoxicity test in vitro showed that the conjugate exhibited different cytotoxicity to HepG-2 and HT-29 cells. In addition, immunization study in vivo illustrated that the conjugate displayed immunoprotective effect by mitigating inhibition and damage effects of 5-Fu on secretion of cytokines, proliferation of splenocytes, and phagocytosis of peritoneal macrophages. It was indicated that the conjugation of 5-Fu and WPMP-2 could be a potential double effective drug delivery system.

Structure characterization of two functional polysaccharides from Polygonum multiflorum and its immunomodulatory.

Two purified polysaccharides named WPMP-1 and WPMP-2 were obtained from Polygonum multiflorum with an average molecular weight of 2.04kDa and 92.13kDa, respectively. Structural characterization revealed that WPMP-1was supposed to be a glucan composed of 1,4-linked α-d-Glcp, 1,4,6-linked α-d-Glcp, and terminal α-d-Glcp. WPMP-2 contained a complex branched structre formed by 1,2-linked α-d-Rhap, 1,2,4-linked α-d-Rhap, 1,3,5-linked α-l-Ara, 1,5- linked α-l-Araf, terminal α-l-Araf, 1,4-linked ß-d-Galp, 1,4-linked α-d-GalpA, and 4-linked α-d-GalpA. In vitro, WPMPs were of activation effect on splenocyte and macrophages, and could also protect immunocyte against 5-Fu induced immunosupression by restoring the proliferation rate, phagocytic index and cytokines secretion level. Moreover, the acid polysaccharide WPMP-2 exhibited better immunomodulatory activity than neutral polysaccharide WPMP-1, which indicated that the immunomodulatory activity of WPMPs were positively associated with higher content of uronic acid, percentage of rhamnose, arabinose and galactose, and branching degree. These findings could promote the polysaccharides from Polygonum multiflorum to be attractive functional food supplement and immunomodulatory adjuvant.

Phyllaciduloids A-D: Four new cleistanthane diterpenoids from Phyllanthus acidus (L.) Skeels.

Four new cleistanthane diterpenoids, phyllaciduloids A-D (1-4), were isolated from the roots and stems of Phyllanthus acidus (L.) Skeels (Phyllanthaceae). Their structures were elucidated on the basis of extensive spectroscopic analysis. Phyllaciduloids B-D (2-4) present in their structures with ether bond between C-7 and C-16, which were rarely reported. All the isolates were evaluated for their cytotoxic activities against five human cancer cell lines.

A new dimeric stilbene from the lianas of Gnetum parvifolium.

A new dimeric stilbene, gnetifolin P (1), was isolated from the lianas of Gnetum parvifolium, together with seven known compounds 2-8. The structure of compound 1 was determined by extensive NMR and HRESIMS data analyses, and quantum chemical calculations. All the compounds were evaluated for their anti-inflammatory activity. Compounds 1 and 6 inhibited the expression of IL-1ß on LPS induced THP-1 cells with the inhibition rate of 35.78 and 64.67%, respectively, at concentration of 25 µg/mL.

Sensitive determination of pyrrolizidine alkaloids in Tussilago farfara L. by field-amplified, sample-stacking, sweeping micellar electrokinetic chromatography.

Pyrrolizidine alkaloids are the toxic components in Tussilago farfara L. Due to the lack of standard substances for quantitative analysis and traces of pyrrolizidine alkaloids in total alkaloids, the full quality control of Tussilago farfara L has been limited. In this study, we aimed to solve the difficulty of determination of pyrrolizidine alkaloids and identify more components in the total alkaloids. An on-line preconcentration method has been applied to improve determining sensitivity of pyrrolizidine alkaloids in Tussilago farfara L. in which included field-amplified sample stacking and sweeping in micellar electrokinetic capillary chromatography. The main parameters that affected separation and stacking efficiency were investigated in details. Under the optimal conditions, the sensitivity enhancement factors obtained by the developed method for the analytes were from 15- to 12-fold, the limits of detection of senkirkine and senecionine were 2∼5 µg/L. Senkirkine and senecionine have been detected in alkaloids (c) of Tussilago farfara L, along ferulic acid methyl ester and methyl caffeate. The developed method was also applied to the analysis of acid extraction (a) of Tussilago farfara L, and senkirkine could be detected directly. The results indicated that the developed method is feasible for the analysis of pyrrolizidine alkaloids in Tussilago farfara L with good recoveries.

New cytotoxic lignan glycosides from Phyllanthus glaucus.

During the process of exploring bioactive lead compounds from Phyllanthus species, two new glycosides including an arylnaphthalene lignan, diphyllin 4-O-α-L-arabinopyranosyl-(1 → 3)-α-L-arabinopyranoside (1), and a phenolic compound, 3,4,5-trimethoxybenzyl alcohol 7-O-α-L-arabinofuranosyl-(1 → 6)-ß-D-glucopyranoside (2), were isolated from the methanol extract of the whole plants of Phyllanthus glaucus Wall. ex Müll. Arg. In addition, 31 known compounds, including 19 lignan derivatives (3-21), four phenylpropanoids (22-25), seven simple phenolics (26-32) and one monoterpenoid (33) were obtained. Their structures were determined on the basis of the HR-ESI-MS, 1D and 2D NMR spectroscopic analysis, and pre-column derivative/chiral HPLC analysis in case of 1 for the absolute configurations. All these compounds were obtained from P. glaucus for the first time. Moreover, the known lignan glycoside, phyllanthusmin C (5) showed in vitro cytotoxicities against HL-60, MCF-7 and SW480 cells with IC50 values of 9.2 ± 0.2, 19.2 ± 1.7 and 20.5 ± 0.9, respectively.

Triterpenoids with Promoting Effects on the Differentiation of PC12 Cells from the Steamed Roots of Panax notoginseng.

The roots of Panax notoginseng, an important Chinese medicinal plant, have been used traditionally in both the raw and processed forms, due to the different chemical constituents and bioactivities found. Thirty-eight dammarane-type triterpenoid saponins were isolated from the steam-processed roots of P. notoginseng, including 18 new substances, namely, notoginsenosides SP1-SP18 (1-18). The structures of 1-18 were determined on the basis of spectroscopic analysis and acidic hydrolysis. The absolute configuration of the hydroxy group at C-24 in 1-4, 19, and 20 was determined in each case by Mo2(AcO)4-induced circular dichroism. The new compounds were found to feature a diversity of highly oxygenated side chains, formed by hydrolysis of the C-20 sugar moiety followed by dehydration, dehydrogenation, epoxidation, hydroxylation, or methoxylation of the main saponins in the raw roots. The new saponins 1, 2, 6-8, 14, and 17 and the known compounds 20-27 showed promoting effects on the differentiation of PC12 cells, at a concentration of 10 µM.

Anti-viral and cytotoxic norbisabolane sesquiterpenoid glycosides from Phyllanthus emblica and their absolute configurations.

In an effort to identify anti-viral and cytotoxic compounds from Phyllanthus spp., 14 highly oxygenated norbisabolane sesquiterpenoids, phyllaemblicins H1-H14, were isolated from the roots of Phyllanthus emblica Linn, along with phyllaemblicins B and C and glochicoccinoside D. Their structures were determined on the basis of detailed spectroscopic analysis and chemical methods. Determination of absolute configurations of these compounds was facilitated by theoretical calculations of electronic circular dichroism (ECD) spectra using time-dependent density functional theory (TDDFT) for the aglycone components, and pre-column derivative/chiral HPLC analysis for the monosaccharides. The known glochicoccinoside D displayed potent activity against influenza A virus strain H3N2 and hand, foot and mouth virus EV71, with IC50 values of 4.5±0.6 and 2.6±0.7 µg/ml, respectively. Phyllaemblicin H1 showed moderate cytotoxicity against human cancer cell lines A-549 and SMMC-7721, with IC50 values of 4.7±0.7 and 9.9±1.3 µM, respectively.

Fistulains A and B, New Bischromones from the Bark of Cassia fistula, and Their Activities.

Fistulains A and B (1 and 2), two novel bischromones with unique coupling patterns, alone with their biosynthetic related compound 3, were isolated from the bark of Cassia fistula. Fistulain A represents a new type of dimeric chromone alkaloid biogenetically derived from a chromone and a tricyclic alkaloid through an unusual C-14-N linkage. Fistulain B has a new carbon skeleton with a C-14-C-5' linkage formed between two different chromone units. Fistulain A displayed anti-TMV activity, and both 1 and 2 showed weak cytotoxicities.

Minor dehydrogenated and cleavaged dammarane-type saponins from the steamed roots of Panax notoginseng.

Nine new minor dehydrogenated and cleavaged dammarane-type triterpenoid saponins, namely notoginsenosides ST6-ST14 (1-9) were isolated from the steamed roots of Panax notoginseng, together with 14 known ones. Among them, 5-7 and 21-22 were protopanaxadiol type and the left 18 compounds, including 1-4, 8-20, and 23 were protopanaxatriol type saponins. Their structures were identified by extensive analysis of MS, 1D and 2D NMR spectra, and acidic hydrolysis. Resulted from the side chain cleavage, the new saponins 1 and 2 featured in a ketone group at C-25, and 3-5 had an aldehyde unit at C-23. The known saponins 12, 16 and 18 displayed the enhancing potential of neurite outgrowth of NGF-mediated PC12 cells at a concentration of 10 µM, while 20 exhibited acetyl cholinesterase inhibitory activity, with IC50 value of 13.97 µM.

Anti-hepatitis B virus activities and absolute configurations of sesquiterpenoid glycosides from Phyllanthus emblica.

During the process exploring anti-viral compounds from Phyllanthus species, eight new highly oxygenated bisabolane sesquiterpenoid glycoside phyllaemblicins G1­G8 (1­8) were isolated from Phyllanthus emblica, along with three known compounds, phyllaemblicin F (9), phyllaemblic acid (10) and glochicoccin D (11). Phyllaemblicin G2 (2), bearing a tricyclo [3.1.1.1] oxygen bridge ring system, is an unusual sesquiterpenoid glycoside, while phyllaemblicins G6­G8 (6­8) are dimeric sesquiterpenoid glycosides with two norbisabolane units connecting through a disaccharide. All the structures were elucidated by the extensive analysis of HRMS and NMR data. The relative configuration of phyllaemblicin G2 was constructed based on heteronuclear coupling constants measurement, and the absolute configurations for all new compounds were established by calculated electronic circular dichroism (ECD) using time dependent density functional theory. The sesquiterpenoid glycoside dimers 6­9 displayed potential anti-hepatitis B virus (HBV) activities, especially for the new compound 6 with IC50 of 8.53 ± 0.97 and 5.68 ± 1.75 µM towards the HBV surface antigen (HBsAg) and HBV excreted antigen (HBeAg) secretion, respectively.

Anti-hepatitis B virus norbisabolane sesquiterpenoids from Phyllanthus acidus and the establishment of their absolute configurations using theoretical calculations.

Nineteen new highly oxygenated norbisabolane sesquiterpenoids, phyllanthacidoid acid methyl ester (1), and C-T (4-21), were isolated from Phyllanthus acidus Skeels, together with two known ones, phyllanthusols A (2) and B (3), whose sugar moiety was revised as glucosamine-N-acetate, rather than the previously assigned mannosamine-N-acetate. Compounds 2 and 3 were renamed respectively as phyllanthacidoids A (2) and B (3) to avoid confusion. All of the isolates except for 1 are glycosides, whose saccharide moieties possess a pentaoxy cyclohexane (scyllo quercitol) connecting with glucosamine-N-acetate or glucosyl moieties, which are first examples in natural products. Phyllanthacidoids N-R (15-19) with 8R configurations and/or 5,8-diketal skeleton, are unprecedented structures among norbisabolane sesquiterpenoids. Phyllanthacidoids S (20) and T (21) have the unusual tricyclo [3.1.1.1] oxygen bridge skeleton formed by a diketal system, of which the relative configurations of the aliphatic chain were assigned on the basis of heteronuclear coupling constants. The absolute configurations of compounds (1-21) were established by means of calculated electronic circular dichroism (ECD) and coupling constants. Compounds 1-5, 7-9, 10, and 14 displayed potential anti-hepatitis B virus (HBV) activities, with IC50 values of 0.8-36 µM against HBV surface antigen (HBsAg) and HBV excreted antigen (HBeAg), and the results indicated that the 5-ketal group and sugar moieties had contributions to the selectivity of HBsAg and HBeAg.