Phytoplankton community and HAB species in the South China Sea detected by morphological and metabarcoding approaches.

The southern Chinese coast is one of the most developed regions in China and is an area where harmful algal blooms (HABs) have occurred frequently. In this study, differences in the phytoplankton community between microscopic observations and 18S rDNA metabarcoding were compared in 89 surface water samples collected from the southern Chinese coast and the western South China Sea (SCS). This is the first report investigating the phytoplankton community and HAB species using a combination of morphological and metabarcoding approaches in this sea area. There were substantial differences in phytoplankton community structure detected by the two methods. Microscopic observation revealed diatom predominance in the phytoplankton community, while metabarcoding indicated dinoflagellate dominance. The phytoplankton community structure obtained by microscopic observation better reflects the real situation in the water column. Metabarcoding annotated more species than morphospecies observed by microscopy. Haptophyta and Cryptophyta were the specific phyla detected in metabarcoding but were missed in microscopy due to their small size. Conversely, some taxa were found in microscopic analysis alone, such as species in Dinophysis, Prorocentrum, and Scrippsiella, suggesting some biases during metabarcoding and gaps in sequence databases. Metabarcoding is superior for detecting morphologically cryptic, small-sized and HAB taxa, such as unarmored dinoflagellates, nanosized hatophytes and chlorophytes, as well as multiple species in Alexandrium, Pseudonitzschia, and Chaetoceros in our study. A total of 62 HAB taxa were identified in this study, including blooming and potentially toxic species. Diatom abundances generally decreased southward, while those of dinoflagellates and haptophytes showed the opposite trend. Chlorophytes were mainly distributed in coastal waters, especially in the Pearl River Estuary. Phytoplankton community structures were shaped by nutrients and salinity, and phosphorus was the most limiting factor for phytoplankton growth. The phytoplankton community in the western SCS showed unique characteristics away from those in the coastal sea areas. The results suggest that the combination of morphological and metabarcoding approaches comprehensively reveals the phytoplankton community structure and diversity of HAB species.

Effects of Jian Pi Qu Shi Formula on intestinal bacterial flora in patients with idiopathic membranous nephropathy: A prospective randomized controlled trial.

BACKGROUND: The incidence of idiopathic membranous nephropathy (IMN) has recently increased remarkably. Immune dysfunction caused by disordered intestinal flora might be an important factor affecting IMN. The Jian Pi Qu Shi Formula (JPQSF) shows promise in treating IMN. Here, we sequenced 16S rRNA genes to compare intestinal flora between patients with IMN and healthy persons. We also conducted a randomized controlled clinical trial to further compare the intestinal flora of patients with IMN treated with traditional Chinese medicine (TCM) and western medicine (WM). METHODS: Among 40 patients with IMN treated at Department of Nephrology in Xiyuan Hospital, Chinese Academy of Traditional Chinese Medicine between July 2016 and December 2018, we compared 30 of them with 10 healthy persons (controls). The IMN group was randomly assigned to receive JPQSF (TCM) or immunosuppressant WM therapy in (n = 15 per group) for 6 months. Intestinal microbiota diversity was analyzed using alpha diversity and beta diversity. Intestinal flora that significantly differed between the groups was analyzed using MetaStat. The effects and safety of the therapies were determined based on the values for plasma albumin, 24-h urine protein excretion, serum creatinine, urea nitrogen, estimate glomerular filtration rate (eGFR), complete blood count, and liver enzymes. All data were statistically analyzed using Statistical Package for the Social Sciences (SPSS) 20.0 statistical software. RESULTS: Baseline characteristics did not significantly differ between the IMN and healthy groups, or the TCM and WM groups. After six months of treatment, 24-h urinary protein significantly declined in the TCM and WM groups (before and after treatment: 3.24 ± 1.74 vs. 1.73 ± 1.85 g, P < 0.05 and 3.94 ± 1.05 vs. 1.91 ± 1.18 g, P < 0.05, respectively). Plasma albumin was significantly increased in the TCM group (before vs. after treatment: 32.44 ± 9.04 vs. 39.99 ± 7.03 g/L, P < 0.05), but did not significantly change in the WM group (31.55 ± 4.23 vs. 34.83 ± 9.14 g/L, P > 0.05). Values for urea nitrogen, serum creatinine, and eGFR did not significantly change in either group. The alpha diversity index for intestinal flora differed between the IMN and healthy groups, and the TCM and WM groups. Comparisons of multiple samples (beta diversity) revealed differences in intestinal flora between the IMN and healthy groups, and the TCM and WM groups. The Metastat analysis findings showed that the main genera that differed between the IMN group before treatment and the healthy group were Christensenellaceae_R-7_group, Bifidobacterium (77), Dorea, Escherichia-Shigella, Parabacteroides, Bifidobacterium, and Coprococcus_3. After TCM therapy, the main differential genera were Butyricimonas, Bacteroides, Alistipes, and Lachnospira, and after WM therapy, these were Ruminococcus_2, Lachnospiraceae_ND3007_group, Lachnospira, Bifidobacterium, Alistipes, and [Eubacterium]_ventriosum_group. CONCLUSION: Patients with IMN might have disordered intestinal flora, and JPQSF can regulate intestinal flora in patients with IMN.

Bioactive Components of Chinese Propolis Water Extract on Antitumor Activity and Quality Control.

To understand the material basis of antitumor activity of Chinese propolis water extract (CPWE), we developed a simple and efficient method using macroporous absorptive resin coupled with preparative high performance liquid chromatography and separated and purified eleven chemical components (caffeic acid, ferulic acid, isoferulic acid, 3,4-dimethoxycinnamic acid, pinobanksin, caffeic acid benzyl ester, caffeic acid phenethyl ester, apigenin, pinocembrin, chrysin, and galangin) from CPWE; then we tested the antitumor activities of these eleven components using different human tumor cell lines (MCF-7, MDA-MB-231, HeLa, and A549). Furthermore, cell migration, procaspase 3 level, and reactive oxygen species (ROS) of effective components from CPWE were investigated. Our data showed that antitumor activities of the eleven components from CPWE were different from each other. CPWE and its effective components induced apoptosis by inhibiting tumor cell migration, activating caspase 3, and promoting ROS production. It can be deduced that the antitumor effects of propolis did not depend on a single component, and there must exist "bioactive components," which also provides a new idea for Chinese propolis quality control.

[Chemical constituents of Trichosanthes kirilowii peels].

OBJECTIVE: To study the chemical constituents of the peels of Trichosanthes kirilowii. METHODS: Many chromatographic techniques were used including repeated silica column chromatography, polyamide resin and semi-preparative high performance liquid chromatography. According to the physical and chemical properties and spectral analysis, the chemical structures of the compounds were determined. RESULTS: Thirteen compounds were isolated and identified as quercetin-3-O-[alpha-L-rhamnose(1 --> 2)-beta-D-glucopyranosyl]-5-O-beta-D-glucopyranoside (1), quercetin-3-O-rutinoside (2), apigenin-7-O-beta-D-glucopyranoside (3), diosmetin-7-O-beta-D-glucopyranoside (4), luteolin (5), apigenin (6), diosmetin (7), methyl palmitate (8), methyl stearate (9), palmitic acid (10), beta-sitosterol (11), alpha-spin-asterol (12) and stigmasterol (13). CONCLUSION: Compounds 1 - 3, 5 - 7 are isolated from this plant for the first time.

An efficient preparative procedure for main flavonoids from the peel of Trichosanthes kirilowii Maxim. using polyamide resin followed by semi-preparative high performance liquid chromatography.

In this study, a simple and efficient preparative procedure was developed for preparation of seven flavonoids from the peel of Trichosanthes kirilowii Maxim. using polyamide resin followed by semi-preparative high performance liquid chromatography (SPHPLC). First, the ethyl acetate fraction from the peel of T. kirilowii Maxim. obtained "prefractionation" using polyamide resin, which yielded two subfractions. And then the two subfractions were isolated by SPHPLC with an isocratic elution of methanol-water. Finally, seven known flavonoids were purified from 35 g of ethyl acetate extract including quercetin-3-O-[α-l-rhamnose (1→2)-ß-d-glucopyranosyl]-5-O-ß-d-glucopyranoside (19 mg), quercetin-3-O-rutinoside (24 mg), apigenin-7-O-ß-d-glucopyranoside (10mg), diosmetin-7-O-ß-d-glucopyranoside (45 mg), luteolin (21 mg), apigenin (15 mg), and diosmetin (56 mg). The purities of the compounds were determined by HPLC and the chemical structures were confirmed by UV and NMR analysis. In the present study, a simple, effective, and rapid procedure was established for preparative separation of multiple components from the peel of T. kirilowii Maxim. Furthermore, it was scalable and economical, so it was a promising basis for large-scale preparation of flavonoids from other plant extracts.

Extraction and preparative purification of tanshinones from Salvia miltiorrhiza Bunge by high-speed counter-current chromatography.

A method for extraction and preparative separation of tanshinones from Salvia miltiorrhiza Bunge was successfully established in this paper. Tanshinones from Salvia miltiorrhiza Bunge were extracted using ethyl acetate as the extractant under reflux. The extracts were then purified by high speed counter-current chromatography (HSCCC) with light petroleum-ethyl acetate-methanol-water (6:4:6.5:3.5, v/v) as the two phase solvent system. The upper phase was used as the stationary phase and the lower phase as the mobile phase. 8.2mg of dihydrotanshinone I, 5.8 mg of 1,2,15,16-tetrahydrotanshiquinone, 26.3mg of cryptotanshinone, 16.2mg of tanshinone I, 25.6 mg of neo-przewaquinone A, 68.8 mg of tanshinone IIA and 9.3mg of miltirone were obtained from 400mg of extracts from Salvia miltiorrhiza Bunge in one-step HSCCC separation, with the purity of 97. 6%, 95.1%, 99.0%, 99.1%, 93.2%, 99.3% and 98.7%, respectively, as determined by HPLC area normalization method. Their chemical structures were identified by ¹H NMR.

Preparative isolation of flavonoid compounds from Oroxylum indicum by high-speed counter-current chromatography by using ionic liquids as the modifier of two-phase solvent system.

A preparative high-speed counter-current chromatography method for isolation and purification of flavonoid compounds from Oroxylum indicum was successfully established by using ionic liquids as the modifier of the two-phase solvent system. Two flavonoid compounds including baicalein-7-O-diglucoside and baicalein-7-O-glucoside were purified from the crude extract of O. indicum by using ethyl acetate-water-[C(4)mim][PF(6)] (5:5:0.2, v/v) as two-phase solvent system. 36.4 mg of baicalein-7-O-diglucoside and 60.5 mg of baicalein-7-O-glucoside were obtained from 120 mg of the crude extract. Their purities were 98.7 and 99.1%, respectively, as determined by HPLC area normalization method. The chemical structures of the isolated compounds were identified by (1)H-NMR and (13)C-NMR.

Preparative isolation of neomangiferin and mangiferin from Rhizoma anemarrhenae by high-speed countercurrent chromatography using ionic liquids as a two-phase solvent system modifier.

A preparative high-speed countercurrent chromatography method for isolation and purification of neomangiferin and mangiferin from Rhizoma anemarrhenae was successfully established by using ionic liquids as the modifier of the two-phase solvent system. Neomangiferin and mangiferin were purified from the crude extract of R. anemarrhenae by using ethyl acetate-water-[C(4)mim][PF(6)] (5:5:0.2 v/v) as two-phase solvent system. In total, 22.5 mg of neomangiferin and 70.6 mg of mangiferin were obtained from 150 mg of the crude extract. The purities of neomangiferin and mangiferin were 97.2 and 98.1%, respectively, as determined by HPLC. The chemical structures of the isolated compounds were identified by (1)H-NMR and (13)C-NMR.

[In situ dynamics of phosphorus in the rhizosphere solution and organic acids exudation of two aquatic plants].

A mini-rhizotron experiment with Alternanthera philoxeroides and Typha latifolia was conducted to measure the spatial and temporal dynamics of phosphorus in the rhizosphere solution. The organic acids in the in situ rhizosphere soil solution were analyzed. A decreasing phosphorus concentration gradient in soil solution toward the root was observed for both A. philoxeroides and T. latifolia. The phosphorus concentration in the rhizosphere soil solution of A. philoxeroides (2.53 mg x L(-1)) was lower than that of T. latifolia (5.43 mg x L(-1)) in the forth sampling day. Compared to T. latifolia, A. philoxeroides released more malic acid (27.33 umol x L(-1)) which was more efficient in phosphorus mobilization. A. philoxeroides was more effective in phosphorus uptake in the rhizosphere than T. latifolia.

Preparative isolation and purification of costunolide and dehydrocostuslactone from Aucklandia lappa Decne by high-speed counter-current chromatography.

A preparative high-speed counter-current chromatography (HSCCC) method for isolation and purification of costunolide and dehydrocostuslactone from the Chinese medicinal plant Aucklandia lappa Decne (Muxiang in Chinese) was successfully established by using light petroleum-methanol-water (5:6.5:3.5, v/v/v) as the two-phase solvent system. The upper phase of light petroleum-methanol-water (5:6.5:3.5, v/v/v) was used as the stationary phase of HSCCC. 35.7 mg of costunolide and 43.6 mg of dehydrocostuslactone with the purity of 100% and 99.6%, respectively, were separated successfully in one-step separation from 110 mg of crude sample from Aucklandia lappa Decne. The structures of costunolide and dehydrocostuslactone were identified by 1H NMR and 13C NMR.

Preparative isolation and purification of three flavonoids from the Chinese medicinal plant Epimedium koreamum Nakai by high-speed counter-current chromatography.

A preparative high-speed counter-current chromatography (HSCCC) method for isolation and purification of flavonoids from the Chinese medicinal plant Epimedium koreamum Nakai was successfully established by using chloroform-methanol-water (4:3.5:2, v/v) as the two-phase solvent system. The method yielded 11.4 mg of epimedokoreanoside I, 46.5 mg of icariin and 17.7 mg of icariside II from 200 mg of the crude sample in one-step separation with the purity of 98.2%, 99.7% and 98.5%, respectively, as determined by high-performance liquid chromatography (HPLC). The structures of the flavonoids were identified by 1H NMR and 13C NMR.

Preparative isolation and purification of hydroxyanthraquinones and cinnamic acid from the chinese medicinal herb Rheum officinale Baill. by high-speed counter-current chromatography.

A high-speed counter-current chromatography (HSCCC) method for preparative separation and purification of five hydroxyanthraquinones and cinnamic acid from the Chinese medicinal herb Rheum officinale Baill. was developed by using pH-gradient elution. The purities of rhein, emodin, aloe-emodin, chrysophanol, physcion and cinnamic acid were all over 98%, as determined by high performance liquid chromatography (HPLC). The structures of them were identified by 1H NMR.

Preparative isolation and purification of coumarins from Angelica dahurica (Fisch. ex Hoffn) Benth, et Hook. f (Chinese traditional medicinal herb) by high-speed counter-current chromatography.

A preparative high-speed counter-current chromatography (HSCCC) method for isolation and purification of coumarins from Angelica dahurica (Fisch. ex Hoffin) Benth, et Hook. f(Baizhi in Chinese) was successfully established by using n-hexane-methanol-water as the two-phase solvent system in gradient elution mode. The upper phase of n-hexane-methanol-water (5:5:5, v/v) was used as the stationary phase of HSCCC. The mobile phase used in HSCCC was the lower phase of n-hexane-methanol-water (5:5:5, v/v) and n-hexane-methanol-water (5:7:3, v/v) that was changed in gradient. Three major components including imperatorin, isoimperatorin and oxypeucedanine were isolated, each at over 98% purity as determined by high-performance liquid chromatography (HPLC). The peak fractions of HSCCC were identified by 1H-NMR and 13C-NMR.