The present study aimed to explore the chemical constituents from the stems and leaves of Cephalotaxus fortunei. Seven lignans were isolated from the 75% ethanol extract of C. fortunei by various chromatographic methods, including silica gel, ODS column chromatography, and HPLC. The structures of the isolated compounds were elucidated according to physicochemical properties and spectral data. Compound 1 is a new lignan named cephalignan A. The known compounds were identified as 8-hydroxy-conidendrine(2), isolariciresinol(3), leptolepisol D(4), diarctigenin(5), dihydrodehydrodiconiferyl alcohol 9'-O-β-D-glucopyranoside(6), and dihydrodehydrodiconiferyl alcohol 4-O-β-D-glucopyranoside(7). Compounds 2 and 5 were isolated from the Cephalotaxus plant for the first time.
Cephalotaxus ; Lignans/analysis* ; Plant Leaves/chemistry* ; Ethanol ; Chromatography, High Pressure Liquid

The present study optimized the ethanol extraction process of Ziziphi Spinosae Semen-Schisandrae Sphenantherae Fructus drug pair by network pharmacology and Box-Behnken method. Network pharmacology and molecular docking were used to screen out and verify the potential active components of Ziziphi Spinosae Semen-Schisandrae Sphenantherae Fructus, and the process evaluation indexes were determined in light of the components of the content determination under Ziziphi Spinosae Semen and Schisandrae Sphenantherae Fructus in the Chinese Pharmacopoeia(2020 edition). The analytic hierarchy process(AHP) was used to determine the weight coefficient of each component, and the comprehensive score was calculated as the process evaluation index. The ethanol extraction process of Ziziphi Spinosae Semen-Schisandrae Sphenantherae Fructus was optimized by the Box-Behnken method. The core components of the Ziziphi Spinosae Semen-Schisandrae Sphenantherae Fructus drug pair were screened out as spinosin, jujuboside A, jujuboside B, schisandrin, schisandrol, schisandrin A, and schisandrin B. The optimal extraction conditions obtained by using the Box-Behnken method were listed below: extraction time of 90 min, ethanol volume fraction of 85%, and two times of extraction. Through network pharmacology and molecular docking, the process evaluation indexes were determined, and the optimized process was stable, which could provide an experimental basis for the production of preparations containing Ziziphi Spinosae Semen-Schisandrae Sphenantherae Fructus.
Ethanol ; Molecular Docking Simulation ; Network Pharmacology ; Seeds/chemistry* ; Ziziphus/chemistry* ; Plant Extracts/chemistry* ; Schisandra/chemistry* ; Fruit/chemistry* ; Technology, Pharmaceutical

β-glucosidase has important applications in food, pharmaceutics, biomass conversion and other fields, exploring β-glucosidase with strong adaptability and excellent properties thus has received extensive interest. In this study, a novel glucosidase from the GH1 family derived from Cuniculiplasma divulgatum was cloned, expressed, and characterized, aiming to find a better β-glucosidase. The amino acid sequences of GH1 family glucosidase derived from C. divulgatum were obtained from the NCBI database, and a recombinant plasmid pET-30a(+)-CdBglA was constructed. The recombinant protein was induced to express in Escherichia coli BL21(DE3). The enzymatic properties of the purified CdBglA were studied. The molecular weight of the recombinant CdBglA was 56.0 kDa. The optimum pH and temperature were 5.5 and 55 ℃, respectively. The enzyme showed good pH stability, 92.33% of the initial activity could be retained when treated under pH 5.5-11.0 for 1 h. When pNPG was used as a substrate, the kinetic parameters K_m, V_max and K_cat/K_m were 0.81 mmol, 291.99 μmol/(mg·min), and 387.50 s^-1 mmol^-1, respectively. 90.33% of the initial enzyme activity could be retained when CdBglA was placed with various heavy metal ions at a final concentration of 5 mmol/L. The enzyme activity was increased by 28.67% under 15% ethanol solution, remained unchanged under 20% ethanol, and 43.68% of the enzyme activity could still be retained under 30% ethanol. The enzyme has an obvious activation effect at 0-1.5 mol/L NaCl and can tolerate 0.8 mol/L glucose. In conclusion, CdBglA is an acidic and mesophilic enzyme with broad pH stability and strong tolerance to most metal ions, organic solvents, NaCl and glucose. These characteristics may facilitate future theoretical research and industrial production.
beta-Glucosidase ; Sodium Chloride ; Temperature ; Glucose ; Ethanol/chemistry* ; Ions ; Hydrogen-Ion Concentration ; Enzyme Stability ; Substrate Specificity

The effective components of flavonoids in the "Pueraria lobata-Hovenia dulcis" drug pair have low bioavailability in vivo due to their unstable characteristics. This study used microemulsions with amphoteric carrier properties to solve this problem. The study drew pseudo-ternary phase diagrams through titration compatibility experiments of the oil phase with emulsifiers and co-emulsifiers and screened the prescription composition of blank microemulsions. The study used average particle size and PDI as evaluation indicators, and the central composite design-response surface method(CCD-RSM) was used to optimize the prescription; high-dosage drug-loaded microemulsions were obtained, and their physicochemical properties, appearance, and stability were evaluated. The results showed that when ethyl butyrate was used as the oil phase, polysorbate 80(tween 80) as the surfactant, and anhydrous ethanol as the cosurfactant, the maximum microemulsion area was obtained. When the difference in results was small, K_(m )of 1∶4 was chosen to ensure the safety of the prescription. The prescription composition optimized by the CCD-RSM was ethyl butyrate(16.28%), tween 80(9.59%), and anhydrous ethanol(38.34%). When the dosage reached 3% of the system mass, the total flavonoid microemulsion prepared had a clear and transparent appearance, with average particle size, PDI, and potential of(74.25±1.58)nm, 0.277±0.043, and(-0.08±0.07) mV, respectively. The microemulsion was spherical and evenly distributed under transmission electron microscopy. The centrifugal stability and temperature stability were good, and there was no layering or demulsification phenomenon, which significantly improved the in vitro dissolution of total flavonoids.
Polysorbates/chemistry* ; Flavonoids ; Pueraria ; Surface-Active Agents/chemistry* ; Ethanol ; Emulsions ; Particle Size ; Solubility

Nine compounds were isolated from the 90% ethanol extract of Salacia polysperma by silica gel, Sephadex LH-20 column chromatography, together with preparative HPLC methods. Based on HR-ESI-MS, MS, 1D and 2D NMR spectral analyses, the structures of the nine compounds were identified as 28-hydroxy wilforlide B(1), wilforlide A(2), 1β,3β-dihydroxyurs-9(11),12-diene(3),(-)-epicatechin(4),(+)-catechin(5),(-)-4'-O-methyl-ent-galloepicatechin(6), 3-hydroxy-1-(4-hydroxy-3-methoxy-phenyl)propan-1-one(7),(-)-(7S,8R)-4-hydroxy-3,3',5'-trimethoxy-8',9'-dinor-8,4'-oxyneoligna-7,9-diol-7'-aldehyde(8), and vanillic acid(9). Compound 1 is a new oleanane-type triterpene lactone. Compounds 1, 3, 4, 7-9 were isolated from the Salacia genus for the first time. All compounds were assayed for their α-glucosidase inhibitory activity. The results suggested that compound 8 exhibited moderate α-glucosidase inhibitory activity, with an IC_(50) value of 37.2 μmol·L~(-1), and the other compounds showed no α-glucosidase inhibitory activity.
Salacia/chemistry* ; alpha-Glucosidases ; Triterpenes/pharmacology* ; Magnetic Resonance Spectroscopy ; Ethanol ; Molecular Structure

OBJECTIVE: To investigate the anti-oxidant and anti-inflammatory effects of ethanol extract of Polygala sibirica L. var megalopha Fr. (EEP) on RAW264.7 mouse macrophages. METHODS: RAW264.7 cells were pretreated with 0-200 µg/mL EEP or vehicle for 2 h prior to exposure to 1 µg/mL lipopolysaccharide (LPS) for 24 h. Nitric oxide (NO) and prostaglandin (PGE₂) production were determined by Griess reagent and enzyme-linked immunosorbent assay (ELISA), respectively. The mRNA levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor α (TNF-α), interleukin-1beta (IL-1β), and IL-6 were determined using reverse transcription polymerase chain reaction (RT-PCR). Western blot assay was used to determine the protein expressions of iNOS, COX-2, phosphorylation of extracellular regulated protein kinases (ERK1/2), c-Jun N-terminal kinase (JNK), inhibitory subunit of nuclear factor Kappa B alpha (Iκ B-α) and p38. Immunofluorescence was used to observe the nuclear expression of nuclear factor-κ B p65 (NF-κ B p65). Additionally, the anti-oxidant potential of EEP was evaluated by reactive oxygen species (ROS) production and the activities of catalase (CAT) and superoxide dismutase (SOD). The 2,2-diphenyl-1-picrylhydrazyl (DPPH), hydroxyl (OH), superoxide anion (O₂^-) radical and nitrite scavenging activity were also measured. RESULTS: The total polyphenol and flavonoid contents of EEP were 23.50±2.16 mg gallic acid equivalent/100 g and 43.78±3.81 mg rutin equivalent/100 g. With EEP treatment (100 and 150 µg/mL), there was a notable decrease in NO and PGE₂ production induced by LPS in RAW264.7 cells by downregulation of iNOS and COX-2 mRNA and protein expressions (P<0.01 or P<0.05). Furthermore, with EEP treatment (150 µg/mL), there was a decrease in the mRNA expression levels of TNF-α, IL-1β and IL-6, as well as in the phosphorylation of ERK, JNK and p38 mitogen-activated protein kinase (MAPK, P<0.01 or P<0.05), by blocking the nuclear translocation of NF-κ B p65 in LPS-stimulated cells. In addition, EEP (100 and 150 µg/mL) led to an increase in the anti-oxidant enzymes activity of SOD and CAT, with a concomitant decrease in ROS production (P<0.01 or P<0.05). EEP also indicated the DPPH, OH, O₂^- radical and nitrite scavenging activity. CONCLUSION EEP inhibited inflammatory responses in activated macrophages through blocking MAPK/NF-κ B pathway and protected against oxidative stress.
Animals ; Mice ; Antioxidants/pharmacology* ; Lipopolysaccharides/pharmacology* ; Polygala ; Transcription Factor RelA/metabolism* ; Tumor Necrosis Factor-alpha/metabolism* ; Ethanol/chemistry* ; Interleukin-6/metabolism* ; Anti-Inflammatory Agents/chemistry* ; Reactive Oxygen Species/metabolism* ; Cyclooxygenase 2/metabolism* ; Nitrites/metabolism* ; NF-kappa B/metabolism* ; Nitric Oxide/metabolism* ; Superoxide Dismutase/metabolism* ; RNA, Messenger ; Nitric Oxide Synthase Type II/metabolism*

This study employed Box-Behnken design combined with flux attenuation to explore the nanofiltration conditions for separation of alcohol precipitation liquid during the preparation of Reduning Injection and discussed the applicability of nanofiltration in the separation of the liquid with high-concentration ethanol. The effects of nanofiltration molecular weight cut-off(MWCO) and pH on the rejection of chlorogenic acid, 3,5-dicaffeoylquinic acid, and 4,5-dicaffeoylquinic acid were consistent with the principles of pore size sieving and charge effect, respectively. The rejection of the three phenolic acids was reduced by concentration polarization effect caused by trans-membrane pressure(TMP). The swelling of membrane surface decreased the pore size and membrane flux for effective separation. Chlorogenic acid and 4,5-dicaffeoylquinic acid were more sensitive to pH and ethanol concentration than 3,5-dicaffeoylquinic acid. A certain correlation existed between the compound structure and the separation factors of nanofiltration, and the separation rules were associated with the comprehensive effect of charge effect, pore size sieving, concentration polarization, steric hindrance and so on.
Chlorogenic Acid ; Drugs, Chinese Herbal/chemistry* ; Ethanol ; Injections

The present study investigated the chemical constituents from Uncaria sessilifructus and their neuroprotective activities. The compounds were separated and purified from the 90% ethanol extract of U. sessilifructus by various chromatographic methods, including silica gel, Sephadex LH-20, and semi-preparative HPLC. Seven compounds were obtained, and their structures were identified as uncanidine J(1), uncanidine K(2), 17-O-ethylhirsutine(3), tetrahydroalstonine(4), akuammigine(5), hirsutine(6), and hirsuteine(7) by physicochemical properties and various spectral techniques, including UV, IR, MS, and NMR. Compounds 1 and 2 are two new compounds. Compound 3 is a new natural product, and compound 4 was isolated from U. sessilifructus for the first time. In addition, the isolated compounds were evaluated for their neuroprotective effects on oxygen and glucose deprivation/reoxygenation(OGD/R) injury in primary cortical neurons in rats. The results showed that compounds 1-7 had different degrees of protective effects on OGD/R injury. The EC_(50) values of compounds 2-4 were(0.17±0.03),(1.70±0.38), and(1.79±0.23) μmol·L~(-1), respectively.
Animals ; Biological Products ; Drugs, Chinese Herbal/pharmacology* ; Ethanol ; Glucose ; Indole Alkaloids ; Neuroprotective Agents/pharmacology* ; Oxygen ; Rats ; Secologanin Tryptamine Alkaloids ; Silica Gel ; Uncaria/chemistry*

Twelve flavonoids were isolated and purified from the ethyl acetate fraction of 95% ethanol extract of Dalbergia odorifera by heat reflux extraction, solvent extraction, recrystallization, normal phase silica gel, Sephadex LH-20, MCI gel and HPLC methods. The structures were identified with multiple spectroscopic methods, including 1 D-NMR, 2 D-NMR and MS. The compounds were identified as 6,7,8-trimethoxy-5,4'-dihydroxy isoflavone(1), medicarpin(2), 7,2'-dihydroxy-4'-methoxy-isoflavanol(3), biochanin A(4), prunetin(5), genistein(6), pratensein(7), 3-(4-hydroxyphenyl)-6-isopentenyl-7-methoxy-4H-chromen-4-one(8), tectorigenin(9), irisolidone(10), vestitol(11), and formononetin(12). Compound 1 was a new isoflavone, and compound 8 was isolated from D. odorifera for the first time. The results showed that compounds 1-3 had inhibitory effects on tyrosinase, with inhibition rates of 35.58%, 38.63% and 51.34% at the concentration of 1.0 mmol·L~(-1), respectively.
Dalbergia/chemistry* ; Ethanol ; Flavonoids/chemistry* ; Genistein ; Isoflavones/pharmacology* ; Monophenol Monooxygenase ; Plant Extracts/pharmacology* ; Silica Gel ; Solvents

Two previously undescribed steroidal alkaloids, compounds 1-2, along with two known ones(3-4), were isolated from the 80% ethanol extract of ripe berries of Solanum nigrum by chromatographic methods, including silica gel, ODS, and HPLC. Based on spectroscopic and chemical evidence, including IR, NMR, and HR-ESI-MS data, the structures of the isolated compounds were identified as 12β,27-dihydroxy solasodine-3-O-β-D-glucopyranoside(1), 27-hydroxy solasodine-3-O-β-D-glucopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-[α-L-rhamnopyranosyl-(1→4)]-β-D-glucopyranoside(2), solalyraine A(3), and 12β,27-dihydroxy solasodine(4). Compounds 1-2 were tested for their potential effects against the proliferation of A549 cells, which revealed that compounds 1-2 had weak cytotoxic activity.
Alkaloids/analysis* ; Ethanol ; Fruit/chemistry* ; Molecular Structure ; Plant Extracts/chemistry* ; Saponins/analysis* ; Silica Gel/analysis* ; Solanum/chemistry* ; Solanum nigrum/chemistry* ; Steroids/pharmacology*