Discovery of a new polymorph of clotrimazole through melt crystallization: Understanding nucleation and growth kinetics.

Clotrimazole (CMZ) is a classical antifungal drug for studying crystallization. In this study, a new CMZ polymorph (Form 2) was discovered during the process of nucleation and growth rate determination in the melt. High-quality single crystals were grown from melt microdroplets to determine the crystal structure by x-ray diffraction. Form 2 is metastable and exhibits a disordered structure. The crystal nucleation and growth kinetics of the two CMZ polymorphs were systematically measured. Form 2 nucleates and grows faster than the existing form (Form 1). The maximum nucleation rate of Forms 1 and 2 was observed at 50 °C (1.07 Tg). The summary of the maximum nucleation rate temperature of CMZ and the other six organic compounds indicates that nucleation near Tg in the supercooled liquid is a useful approach to discovering new polymorphs. This study is relevant for the discovering new drug polymorphs through an understanding of nucleation and growth kinetics during melt crystallization.

Bio-nanocomplexes with autonomous O2 generation efficiently inhibit triple negative breast cancer through enhanced chemo-PDT.

As one kind of aggressive cancer, triple-negative breast cancer (TNBC) has become one of the major causes of women mortality worldwide. Recently, combinational chemo-PDT therapy based on nanomaterials has been adopted for the treatment of malignant tumor. However, the efficacy of PDT was partly compromised under tumor hypoxia environment due to the lack of sustainable O2 supply. In this study, CeO2-loaded nanoparticles (CeNPs) with peroxidase activity were synthesized to autonomously generate O2 by decomposing H2O2 within tumor region and reprogramming the hypoxia microenvironment as well. Meanwhile, the compound cinobufagin (CS-1) was loaded for inhibiting TNBC growth and metastasis. Moreover, the hybrid membrane camouflage was adopted to improve the biocompatibility and targeting ability of nanocomplexes. In vitro assay demonstrated that decomposition of H2O2 by CeO2 achieved sustainable O2 supply, which accordingly improved the efficacy of PDT. In turn, the generated O2 improved the cytotoxicity and anti-tumor migration effect of CS-1 by downregulating HIF-1α and MMP-9 levels. In vivo assay demonstrated that the combination of CS-1 and PDT significantly inhibited the growth and distance metastasis of tumor in MDA-MB-231 bearing mice. Thus, this chemo-PDT strategy achieved satisfactory therapeutic effects by smartly utilizing the enzyme activity of nanodrugs and special micro-environment of tumor.

Preparative isolation and purification of 12 main antioxidants from the roots of Polygonum multiflorum Thunb. using high-speed countercurrent chromatography and preparative HPLC guided by 1,1'-diphenyl-2-picrylhydrazyl-HPLC.

A hyphenated strategy by off-line coupling of 1,1'-diphenyl-2-picrylhydrazyl-high-performance liquid chromatography, high-speed countercurrent chromatography, and preparative high-performance liquid chromatography was established to screen and separate antioxidants from ethyl acetate fraction of the roots of Polygonum multiflorum. Under the targeted guidance of 1,1'-diphenyl-2-picrylhydrazyl-high-performance liquid chromatography experiment, 12 compounds were identified as potential antioxidants and readily isolated by high-speed counter-current chromatography and preparative high-performance liquid chromatography. Ultraviolet spectroscopy, mass spectrometry, and 1 H NMR spectroscopy were employed to identify their structures, which were assigned as gallic acid (1, 6.2 mg, 98.28%), catechin (2, 8.8 mg, 90.69%), epicatechin (3, 4.1 mg, 96.71%), polydatin (4, 5.3 mg, 94.91%), 2,3,5,4'-tetrahydroxy stilbene-2-Ο-ß-D-glucoside (5, 20.2 mg, 95.23%), piceatannol (6, 5.3 mg, 96.85%), rutin (7, 5.4 mg, 97.92%), resveratrol (8, 5.2 mg, 96.94%), isorhapontigenin (9, 11.4 mg, 94.81%), hyperoside (10, 9.7 mg, 98.52%), rhein (11, 4.9 mg, 97.46%), and emodin (12, 8.2 mg, 95.74%). Notably, compounds 6 and 9 were isolated from Polygonum multiflorum for the first time. In addition, antioxidant activity of compounds 1-12 were evaluated, and compounds 1-8 and 10 exhibited stronger antioxidant activity than ascorbic acid (positive control). These results indicated that the proposed method is a highly efficient strategy to screen and isolate antioxidants from complex natural products.

Large-scale separation of acetylcholinesterase inhibitors from Zanthoxylum nitidum by pH-zone-refining counter-current chromatography target-guided by ultrafiltration high-performance liquid chromatography with ultraviolet and mass spectrometry screening.

A new strategy by converging ultrafiltration high-performance liquid chromatography with ultraviolet and mass spectrometry and pH-zone-refining counter-current chromatography was developed for the rapid screening and separation of potential acetylcholinesterase inhibitors from the crude alkaloidals extract of Zanthoxylum nitidum. An optimized two-phase solvent system composed of chloroform/methanol/water (4:3:3, v/v) was used in this study. And, in the optimal solvent system, 45 mM hydrochloric acid was added to the aqueous stationary phase as the retainer, while 5 mM triethylamine was added to the organic mobile phase as the eluter. As a result, with the purity of over 95%, five alkaloids including jatrorrhizine (1, 340 mg), columbamine (2, 112 mg), skimmianine (3, 154 mg), palmatine (4, 226 mg), and epiberberine (5, 132 mg) were successfully purified in one step from 3.0 g crude alkaloidals extract. And their structures were identified by ultraviolet, mass spectrometry, 1 H and 13 C NMR spectroscopy. Notably, compounds 2, 4 and 5 were firstly reported in Z. nitidum. In addition, acetylcholinesterase inhibitory activities of compounds 1-5 were evaluated, and compounds 3, 4 and 5 exhibited stronger acetylcholinesterase inhibitory activity (IC50 values at 8.52 ± 0.64, 14.82 ± 1.21 and 3.12 ± 0.32 µg/mL, respectively) than berberine (IC50 value at 32.86 ± 2.14 µg/mL, positive control). The results indicated that the proposed method is an efficient technique to rapidly screen acetylcholinesterase inhibitors from complex samples, and could be served as a large-scale preparative technique for separating ionizable active compounds.

Separation of α-glucosidase inhibitors from Potentilla kleiniana Wight et Arn using solvent and flow-rate gradient high-speed counter-current chromatography target-guided by ultrafiltration HPLC-MS screening.

INTRODUCTION: Potentilla kleiniana Wight et Arn is widely used as a herbal medicine to treat type 2 diabetes. However, detailed information about its active compounds is lacking. OBJECTIVE: To develop an efficient method for the rapid screening and separation of α-glucosidase inhibitors from Potentilla kleiniana Wight et Arn. METHODOLOGY: Potential α-glucosidase inhibitors from Potentilla kleiniana Wight et Arn were rapidly screened out through ultrafiltration high-performance liquid chromatography mass spectrometry (HPLC-MS), and then followed by a target-guided high-speed counter-current chromatography (HSCCC) separation using two-phase solvent systems composed of n-hexane/ethyl acetate/methanol/water (1:10:1:10, v/v/v/v and 1:10:5:6, v/v/v/v), and adopting increasing flow-rate from 1.5 to 3.0 mL/min after 200 min. Their structures were identified by ultraviolet (UV), MS, proton nuclear magnetic resonance (1 H-NMR) and carbon-13 (13 C)-NMR, and their α-glucosidase inhibitory activities were assessed by in vitro assay. RESULTS: Five α-glucosidase inhibitors including gallic acid (25.7 mg, 98.2%, 1), brevifolincarboxylic acid (9.86 mg, 95.3%, 2), ethyl evifolincarboxylate (13.26 mg, 97.6%, 3), 3,3'-di-O-methylellagic acid-4'-O-ß-d-glucopyranoside (16.26 mg, 95.1%, 4), and 3,3'-di-O-methylellagic acid (10.54 mg, 96.8%, 5) were successfully purified from 250 mg n-butanol extract in a single run. Compounds 1, 2, 4 and 5 exhibited stronger α-glucosidase inhibitory activities[half maximal inhibition concentration (IC50 ) values at 173.41 ± 6.35, 323.46 ± 8.08, 44.63 ± 2.50, and 20.73 ± 2.56 µM, respectively] than acarbose (IC50 value at 332.12 ± 5.52 µM, reference compound). CONCLUSIONS: Notably, compounds 2-5 were reported in the Potentilla kleiniana Wight et Arn for the first time. The results indicated that the proposed method could be applied for the rapid screening and preparative separation of α-glucosidase inhibitors from a complex matrix.

Simultaneous In Situ Extraction and Fabrication of Surface-Enhanced Raman Scattering Substrate for Reliable Detection of Thiram Residue.

We report a novel strategy of simultaneous in situ extraction and fabrication of surface-enhanced Raman scattering substrate (IE-SERS) to perform selective and reliable on-site determination of thiram residue in soil, fruits, and vegetables. In this protocol, the thiram residue on complex surfaces can facilely diffuse into the solvent (dichloromethane (DCM)) and specifically bind to gold nanoparticles (AuNPs), affording the SERS substrate through the embedding of the thiram-trapped AuNPs into the cellulose p-toluenesulfonates (CTSAs) film through the evaporation of DCM. SERS signals of the specifically prepared CTSAs could be used as an internal standard to calibrate the absolute signal of thiram, which can avoid the fluctuation of SERS intensities caused by uneven and irregular morphology of SERS substrate. Thus, reliable quantitation of thiram through SERS detection and superior reproducibility in the SERS measurement (RSD = 4.21%) were achieved. As for directly sensing the thiram residue in soil, the established method shows strong anti-interference ability and a good linear response from 0.1 to 12 µg/g with a low limit of detection (LOD) of 50 ng/g, which is lower than that of all the previously reported methods. The recoveries range from 91.76 to 112.3% for thiram in paddy soils, indicating that the established IE-SERS method is reliable and applicable to the detection of thiram residue in real soil samples. In addition, the measurement of the residual thiram on strawberry and cucumber surface was also successfully accomplished by this strategy, indicating that the established method also has great potential in the in situ ultrasensitive detection of thiram on irregular fruits and vegetables.

Rapid screening and identification of antioxidants in the leaves of Malus hupehensis using off-line two-dimensional HPLC-UV-MS/MS coupled with a 1,1'-diphenyl-2-picrylhydrazyl assay.

The leaves of Malus hupehensis have a strong antioxidant activity and are commonly consumed as a healthy tea. However, detailed information about its antioxidants is incomplete. Herein, we developed an effective strategy based on combining off-line two-dimensional high-performance liquid chromatography with ultraviolet and tandem mass spectrometry detection with a 1,1'-diphenyl-2-picrylhydrazyl assay to rapidly screen and identify the antioxidants from the leaves of M. hupehensis. In the orthogonal two-dimensional liquid chromatography system, a Venusil HILIC column was used for the first dimension, while a Universil XB-C18 column was installed in the second dimension. As a result, 32 antioxidants, including ten dihydrochalcones, two flavanones, nine flavonols, four flavones, and seven phenolic acids were tentatively identified, out of which 23 compounds, as far as we know, were isolated and characterized from the leaves of M. hupehensis for the first time. To the best of our knowledge, this is the first systematic investigation of the antioxidants from the leaves of M. hupehensis. The results indicated that the proposed method is an efficient technique to rapidly investigate antioxidants, especially for coeluted and minor compounds in a complex system.

Study on the material basis and immunological enhancement activity of dangdi oral liquid.

Studies have shown that a lot of traditional Chinese medicines could improve the immunity of the body. Dangdi oral liquid (DDO) was mainly composed of Angelica sinensis (Oliv.) Diels (Danggui), Rehmannia glutinosa Libosch. (Dihuang), Achyranthes bidentata Bl. (Niuxi), Glycyrrhiza uralensis Fisch. (Gancao). In this study, the rapid ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) method was used to identify the potentially effective compounds of DDO. Then the immune activity of DDO was measured by lymphocyte proliferation, macrophage phagocytic function, NK cell activity, delayed type hypersensitivity reaction, hemolytic plaque number, sIgA content and immune organ index. The results showed that a total of 51 compounds were identified. In addition, DDO could significantly promote the lymphocyte proliferation, improve macrophage phagocytic ability, NK cell activity, hemolytic plaque number, sIgA content and immune organ index compared with control group, and the medium dose possessed the best efficacy (P＜0.05). These results indicated that DDO could enhance the immunity of mice.

Impact of Poloxamer on Crystal Nucleation and Growth of Amorphous Clotrimazole.

Surfactants have been widely used as effective additives to increase the solubility and dissolution rates of amorphous solid dispersions (ASDs). However, they may also generate adverse effects on the physical stability of ASDs. In this study, we systematically investigated the impacts of poloxamer, a frequently used surfactant, on the crystallization of amorphous clotrimazole (CMZ). The added poloxamer significantly decreased the glass transition temperature (Tg) of CMZ and accelerated the growth of Form 1 and Form 2 crystals. It was found that the poloxamer had an accelerating effect on Form 1 and Form 2 but showed a larger accelerating effect on Form 1, which resulted from a combined effect of increased mobility and local phase separation at the crystal-liquid interface. Additionally, the added poloxamer exhibited different effects on nucleation of the CMZ polymorphs, which was more complicated than crystal growth. The nucleation rate of Form 1 was significantly increased by the added poloxamer, and the effect increased with increasing P407 content. However, for Form 2, nucleation was slightly decreased or unchanged. The nucleation of Form 2 may have been influenced by the Form 1 crystallization, and Form 2 converted to Form 1 during nucleation. This study increases our understanding of poloxamer and its impacts on the melt crystallization of drugs.

Rubus suavissimus S. Lee Extract Alleviates Oxidative Stress and Inflammation in H2O2-Treated Retinal Pigment Epithelial Cells and in High-Fat Diet-Fed Mouse Retinas.

BACKGROUND: Age-related macular degeneration (AMD) is the most common cause of visual disorders in the aged population and is characterized by the formation of retinal pigment epithelium (RPE) deposits and dysfunction/death of the RPE and photoreceptors. It is supposed that both oxidative stress and inflammation play a critical role in the pathogenesis of AMD. The development of therapeutic strategies against oxidative stress and inflammation in AMD is urgently needed. Rubus suavissimus S. Lee (RS), a medicinal plant growing in the southwest region of China, has been used as an herbal tea and medicine for various diseases. METHODS: In this project, we evaluate the therapeutic potential of RS extract for AMD. We prepared RS extracts from dried leaves, which contained the main functional compounds. RESULTS: RS extract significantly increased cell viability, upregulated the expression of antioxidant genes, lowered the generation of malondialdehyde and reactive oxygen species, and suppressed inflammation in H2O2-treated human RPE cells. In the in vivo study, treatment with RS extract attenuated body weight gain, lowered cholesterol and triglyceride levels in the liver and serum, increased antioxidant capacity, and alleviated inflammation in the retina and RPE/choroid of mice fed a high-fat diet. CONCLUSIONS: Our findings suggest that RS extract offers therapeutic potential for treating AMD patients.

The antisolvent coprecipitation method for enhanced bioavailability of poorly water-soluble drugs.

In recent years, poorly water-soluble drug candidates in the drug development pipeline have been a challenging issue for the pharmaceutical industry. Many delivery systems, such as nanocrystals, cocrystals, nanoparticles, and amorphous solid dispersions (ASDs) have been developed to overcome these problems. A large number of methods are utilized to realize the above delivery systems. Among all the preparation methods, the antisolvent coprecipitation method is a relatively simple, cost-effective method, offering many advantages over conventional methods. An overview of recent developments for each solubility enhancement approach using the antisolvent coprecipitation method is presented. This current review details a comprehensive overview of the antisolvent coprecipitation process and its properties, as well as the fundamentals for enhancing the solubility and bioavailability of poorly water-soluble drugs by nanotization, polymorph control with polymers and/or surfactants. Furthermore, this review also presents insights into the factors affecting the antisolvent coprecipitation process.

Anti-inflammatory activities of Gardenia jasminoides extracts in retinal pigment epithelial cells and zebrafish embryos.

Age-related macular degeneration (AMD) is the most common cause of visual impairment in developed countries. Inflammation serves a critical role in the pathogenesis of AMD. Gardenia jasminoides is found in several regions of China and is traditionally used as an organic yellow dye but has also been widely used as a therapeutic agent in numerous diseases, including inflammation, depression, hepatic and vascular disorders, which may reflect the variability of functional compounds that are present in Gardenia jasminoides extracts (GJE). To investigate the therapeutic potential of GJE for AMD, ARPE-19 cells were treated with lipopolysaccharide (LPS) or LPS plus GJE. GJE significantly decreased LPS-induced expression of proinflammatory cytokines, including IL-1ß, IL-6 and TNF-α. In the in vivo study, GJE inhibited CuSO4-induced migration of primitive macrophages to the lateral line in zebrafish embryos. GJE also attenuated expression of cytokines (IL-1ß, IL-6 and TNF-α), NFKB activating protein (nkap) and TLR4 in ARPE-19 cells. The results of the present study demonstrated the anti-inflammatory potential of GJE in vitro and in vivo, and suggested GJE as a therapeutic candidate for AMD.

Inhibition of resveratrol glucosides (REs) on advanced glycation endproducts (AGEs) formation: inhibitory mechanism and structure-activity relationship.

The study on inhibitory effects of resveratrol glucosides (REs) on advanced glycation endproducts (AGEs) formation is still unmet. Herein, for the first time, the antiglycation activities of five REs in the fetal bovine serum proteins (FBS)/fructose system were evaluated, and its structure-activity relationship and antiglycation mechanism were further explored. These REs showed remarkable inhibition toward AGEs formation. Among them, Piceatannol-3'-O-glucoside (PG) exhibited highest antiglycation activity as reflected in approximately 80% inhibition of fluorescent AGEs at the concentration of 1.0 mM. The structure-activity relationship analysis indicated that glucoside attached to the B ring of resveratrol displays a superior antiglycation activity. Moreover, the results of antiglycation mechanism showed that the antiglycation activity of REs was proportional to their antioxidant capacity and methylglyoxal (MGO) trapping capacity. Therefore, the REs are promising candidates worthy of further exploration for preventing AGEs accumulation in vivo, thereby treating AGEs-associated diseases.

4-Mercaptophenylboronic acid-modified spirally-curved mesoporous silica nanofibers coupled with ultra performance liquid chromatography-mass spectrometry for determination of brassinosteroids in plants.

Herein, for the first time, thiol-functionalized mesoporous silica (mSiO2-SH) nanofibers with a spirally-curved twisted hexagonal morphology were synthesized via a simple one-pot protocol. 4-Mercaptophenylboronic acids (4-MPBA) were attached onto the mSiO2-SH nanofibers via disulfide bond, serving as boronate affinity sorbent to selectively capture brassinosteroids (BRs) from plant extract. The resulting BRs-MPBA derivatives were easily eluted from the sorbent by cleaving the disulfide bond, which was subsequently subjected to ultra performance liquid chromatography-mass spectrometry (UPLC-MS) analysis. Thus, the in situ extraction/derivatization/desorption method coupled with UPLC-MS was established for the fast, sensitive and selective detection of BRs in plant tissues. Finally, based on the developed method, endogenous BRs were successfully detected in leaf of H. lupulus L., silique of A. thaliana, and panicle of O. sativa L.

In situ fabrication of label-free optical sensing paper strips for the rapid surface-enhanced Raman scattering (SERS) detection of brassinosteroids in plant tissues.

A surface-enhanced Raman scattering (SERS)-functionalized, gold nanoparticle (AuNPs)-immobilized paper strip was developed to realize label-free rapid detection of brassinosteroids (BRs) in plant tissues. The AuNPs paper strip was fabricated through utilizing poly(γ-glutamic acid) (γ-PGA) as the linker and concentrator for the directly immobilized AuNPs on paper, which can circumvent many drawbacks of the existing SERS substrates. An enhancement factor of 1.13×108, high reproducibility (relative standard deviation of 4.78%), highly sensitive detection limit with a correlation coefficient of 0.99 (1×10-11M 4-mercaptophenylboronic acid), and long-term stability for the responses of the SERS-functionalized AuNPs paper strip to Raman intensities were achieved by optimizing reaction conditions including the concentrations of γ-PGA and HAuCl4/NaBH4. Ultimately, this developed paper strip was applied to the determination of BRs in plant tissues, obtaining precisions with RSD values in a range of 3.0-13.8%. The SERS-functionalized AuNPs paper strip featuring label-free rapid analysis, and high sensitivity could serve as an excellent alternative for on-site rapid detections.

A new ellagic acid derivative from Polygonum runcinatum.

A new ellagic acid derivative, 3,3'-dimethylellagic acid-4'-O-(6″-galloyl)-ß-D-glucoside, named runcinatside (5), together with four known compounds 3,3'-dimethylellagic acid (1), 3,3',4'-trimethylellagic acid (2), 3,3'-dimethylellagic acid-4'-O-ß-D-glucoside (3) and 3-methylellagic acid-4'-O-α-L-rhamno-pyranoside (4), was isolated from the roots of Polygonum runcinatum Buch.-Ham. ex D.Don Var. sinense Hemsl and the structures of these compounds were established by spectroscopic methods and comparison with previously reported data. All compounds showed antioxidant activities in vitro and compound 5 possessed the highest activity.

A new taxane diterpenoid from Taxus chinensis var. mairei.

Taxadiene (3), a new taxane diterpenoid with an unusual hydroxy substituting at C-17, and six known compounds including two taxane diterpenoids (1 and 2) and four flavonoids (4-7) were isolated from the whole seedling of the Taxus chinensis var. mairei. Among them, compound 7 was isolated from T. chinensis var. mairei for the first time. Structures of these compounds were elucidated on the basis of spectroscopic data and by comparison with reported literature data.

[Isolation and structure identification of C19 diterpenoid alkaloids from Aconitum carmichaeli by high-speed counter-current chromatography].

Three C19 diterpenoid alkaloids were isolated and purified from the lateral roots of Aconitum carmichaeli Debx. (Fuzi in Chinese) by high-speed counter-current chromatography (HSCCC). A mixture of n-hexane-ethyl acetate-methanol-water (3:5:4:5, v/v/v/v) was used as the two phase solvent system. The lower phase was used as the mobile phase and was operated at a flow rate of 2.0 mL/min, while the apparatus was rotated at 850 r/min, and the detection wavelength was at 235 nm. Under these conditions, 15.3 mg of beiwutine, 35.1 mg of mesaconitine and 22.7 mg of hypaconitine were obtained from 90 mg of crude extract in one-step separation with the purities of 97.9%, 96.2% and 99.2%, respectively, determined by high performance liquid chromatography. The structures of these three compounds were identified by electrospray ionization mass spectrometry (ESI-MS), 1H-nuclear magnetic resonance (1H-NMR) and 13C-NMR. The results indicate that HSCCC is a powerful technique for the purification of diterpenoid alkaloids from the lateral roots of Aconitum carmichaeli Debx.