Preparative HPLC Research Articles

N-acetyldopamine oligomers from Periplaneta americana with anti-inflammatory and vasorelaxant effects and their spatial distribution visualized by mass spectrometry imaging

Ethnopharmacological relevancePeriplaneta americana is a medicinal insect that has been applied to promote blood circulation and remove blood stasis based on traditional Chinese medicine (TCM) for a long time. Its modern preparation, Xinmailong injection, was adopted for the treatment of congestive heart failure (CHF). The bioactive constituents of P. americana and their correlation with its traditional uses need further investigation. Aim of the studyThis study aimed to elucidate the N-acetyldopamine (NADA) oligomers from P. american, determine their spatial distribution, and investigate their anti-inflammatory and vasorelaxant effects to provide scientific evidence supporting the clinical use of this medicinal insect. Material and methodsNADA oligomers were isolated from the CH2Cl2: CH3OH (2:1) extract of P. americana, through sequential chromatographic methods including silica gel, Sephadex LH-20, preparative HPLC, and chiral-phase separation. Their structures were determined by HRESIMS, 1D and 2D NMR spectroscopic analysis, chiral resolution, and calculated electronic circular dichroism analysis. With the aid of atmospheric pressure scanning matrix-assisted laser desorption/ionization mass spectrometry imaging (AP-SMALDI MSI), the isolated compounds in a spatial profile within P. americana were identified. NO production was measured to assess anti-inflammatory activity. Vasorelaxant activity assessments were performed on the norepinephrine-precontracted 3rd-order mesenteric arteries. ResultsSeven new NADA trimers, peridopamines A−G (1−7), two new NADA dimers, peridopamines H and I (8 and 9), and six known NADA derivatives (10−15), were obtained from P. americana. The trimers and dimers were detected and showed similar pattern of distribution with accumulation in peripheral and rigid parts of P. americana, based on quasimolecular ion AP-SMALDI MS images of sections from the whole body and dissected areas of the insect. Furthermore, the anti-inflammatory and vasorelaxant effects of isolated compounds were investigated. Compounds 8 and 9 presented significant and moderate anti-inflammatory potentials, respectively. Compounds 8, 10, 12 and 15 possess significant vasorelaxant potentials at concentrations correlated with EC50 values of 6.7–23.7 μM. ConclusionFifteen NADA oligomers were isolated from P. americana. The distribution of these compounds was visualized by AP-SMALDI imaging experiments and NADA oligomers were mainly observed in peripheral parts. Bioassays showed that the tested compounds had anti-inflammatory and vasorelaxant activities, which indicated that NADA oligomers are active ingredients of this insect-based TCM and have potential for the treatment of cardiovascular disease.

CH3 CN@open-C60 : An Effective Inner-Space Modification and Isotope Effect Inside a Nano-Sized Flask.

Despite several small molecules being encapsulated inside cage-opened fullerene derivatives, such species have not considerably affected the structures and properties of the outer carbon cages. Herein, we achieved an effective inner-space modification for an open-cage C60 derivative by insertion of a neutral CH3 CN molecule into the cavity. The CH3 CN@open-C60 thus obtained showed an enhanced polarity, thus affording an easy separation from a mixture containing the empty cage by column chromatography on silica gel, without the preparative HPLC that was needed for previous cases. The less negative reduction potentials with respect to those of empty cage reflect the decreased energy level of the LUMO, which is supported by the DFT calculations. NMR spectroscopy, single-crystal X-ray analysis, and theoretical calculations revealed that both the presence of the encapsulated CH3 CN and cage deformation caused by the CH3 CN play an essential role in the change of the electronic properties. Furthermore, the favored binding affinity of deuterated acetonitrile CD3 CN with internal C60 surface is discussed.

Integrated strategy for the screening of cyclooxygenase-2 inhibitors from triterpenoid saponins in Clematis tangutica.

Screening of novel cyclooxygenase-2 (COX-2) inhibitors from complex natural products is not an easy task. To establish an efficient and feasible strategy for screening COX-2 inhibitors from triterpenoid saponins (TPSs) in Clematis tangutica. Taking TPSs in C.tangutica as example, an optimized macroporous resin (MR) method was established for the enrichment of TPSs. High-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (HPLC-QTOFMS) was performed to establish the phytochemical profiling of TPSs. Molecular docking was performed to predict the ligand-target interactions and discover the active substances. Chemometric techniques were performed to visualize the structure-effect relationships. High-speed countercurrent chromatography and preparative HPLC were performed to prepare the targets. In vitro activity experiment of COX-2 was performed to verify the virtual screening results. TPSs in C.tangutica were well enriched with the recovery rate of (80.22 ± 2.37)%. Thirty-four kinds of TPSs of oleanane type were deduced by HPLC-QTOFMS. Five TPSs of clematangoside C, clematangoside D, clematangoticoside J, hederoside H1 , and hederasaponin B showed stronger binding abilities with COX-2. The structure with more sugar groups at C-28 may be more conducive to the combination with COX-2. Targets were prepared with purities all above 98%. The IC50 values of target TPSs were 6.03 ± 0.24, 12.44 ± 0.15, 9.36 ± 0.19, 4.78 ± 0.13, and 2.59 ± 0.11 μmol/L, respectively. The integrated strategy using MR, HPLC-QTOFMS, molecular docking, chemometrics, target preparation, and in vitro verification was feasible for rapidly screening COX-2 inhibitors from TPSs in C.tangutica.

Capsaicin derived from endemic chili landraces combats Shigella pathogen: Insights on intracellular inhibition mechanism

Ethnic tribals in northeast India have been growing and maintaining local chili landraces for ages. These chilies are known for their characteristic pungency and immense therapeutic properties. Capsaicin, a significant chili metabolite, is recognized as a natural drug for pain relief, diabetic neuropathy, psoriasis, arthritis, etc. In this study, we tried to observe the influence of locality factors on the pungency and bioactive features of Capsicum annuum L. landraces. We also checked the gastro-protective ability of these chilies, especially in the cure of shigellosis. Phytometabolite characterization and estimation were done through spectrophotometric methods. Preparative and analytical HPLC techniques were employed for extracting and purifying capsaicin-enriched fractions. Shigella flexneri growth retardation was determined through the broth dilution method. Gentamicin protection assay and ELISA were done to assess the intracellular invasion and IL-1β inflammasome production by S.flexneri. The correlation analyses postulated that phenols, flavonoids, chlorophylls, β-carotene, and capsaicin synthase upregulation strongly influenced capsaicin biosynthesis in chili cultivars. Correspondingly, the inhibitory efficacy of the HPLC-purified Balijuri-derived capsaicin was more effective than the Raja-derived capsaicin in inhibiting intracellular Shigella growth. Reduced levels of pro-inflammatory cytokine (IL1β) in capsaicin-treated Shigella-infected cells probably reduced inflammation-mediated intestinal damage, limiting bacterial spread. This investigation advocates the unique potential of local chilies in curing deadly ‘shigellosis’ with mechanistic evidence. Our observation justifies the traditional healing practices of the ethnic people of NE India.

Antifungal Activity of Hop Leaf Extracts and Xanthohumol on Two Strains of Venturia inaequalis with Different Sensitivities to Triazoles.

Hop cones are well-known for their antimicrobial properties, attributed to their specialized metabolites. Thus, this study aimed to determine the in vitro antifungal activity of different hop parts, including by-products such as leaves and stems, and some metabolites against Venturia inaequalis, the causal agent of apple scab. For each plant part, two types of extracts, a crude hydro-ethanolic extract and a dichloromethane sub-extract, were tested on spore germination of two strains with different sensitivities to triazole fungicides. Both extracts of cones, leaves and stems were able to inhibit the two strains, whereas rhizomes did not show activity. The apolar sub-extract of leaves appeared as the most active modality tested with half maximal inhibitory concentrations (IC50) of 5 and 10.5 mg·L-1 on the sensitive strain and the strain with reduced sensitivity, respectively. Differences in activity level between strains were noticed for all active modalities tested. Sub-extracts of leaves were then separated into seven fractions by preparative HPLC and tested on V. inaequalis. One fraction, containing xanthohumol, was especially active on both strains. This prenylated chalcone was then purified by preparative HPLC and showed significant activity against both strains, with IC50 of 1.6 and 5.1 mg·L-1. Therefore, xanthohumol seems to be a promising compound to control V. inaequalis.

Chemical composition and cardiotropic activity of Ziziphora clinopodioides subsp. bungeana (Juz.) Rech.f.

Ethnopharmacological relevanceZiziphora clinopodioides subsp. bungeana (Juz.) Rech.f. is a subshrub that is widely distributed in China, Kazakhstan, Kyrgyzstan, Mongolia, Russia, Tajikistan, Turkmenistan, and Uzbekistan. The species is used in traditional medicine for the relief of symptoms connected to cardiovascular diseases like coronary heart disease or hypertension. Aim of the studywas to validate traditional use of Z. clinopodioides subsp. bungeana for the treatment of coronary hearth diseases using in vivo models and to find active compounds responsible for the activity. Materials and methodsMultiple extracts were obtained from the aerial parts of Z. clinopodioides subsp. bungeana using maceration, liquid-liquid extraction, CO2 extraction and ultrasound-assisted extraction. Preliminary screening studies for the evaluation of the efficacy of Z. clinopodioides subsp. bungeana extracts on the model of hemic hypoxia were performed. The most effective samples were selected and included in the main study. Stage 2 of the study evaluated the cardiotropic activity of the selected extracts on a model of chronic heart failure. Preparations were administered to animals intragastrically once a day for 28 days. For the isolation of individual compounds plant material was extracted with 96% ethanol. The obtained crude extract was sequentially extracted with n-hexane and dichloromethane and separated by chromatography on a Diaion HP-20 column. The obtained fractions were further subjected to Sephadex LH-20 column chromatography and eluted isocratically with 96% ethanol (EtOH) to yield subfractions, which were further separated by preparative HPLC to obtain 13 individual compounds. ResultsExtracts obtained from Ziziphora clinopodioides subsp. bungeana (Juz.) Rech.f. herb were subjected to pharmacological screening for the evaluation of their efficacy on hemic hypoxia. Based on the obtained results, out of the sixteen tested extracts two (AR and US 60%) were selected for further evaluation of their cardiotropic activity. Modeling of chronic heart failure was carried out in accordance with the following stages: 1) anesthesia with chloral hydrate at a dose of 450 mg/kg, intraperitoneally, 2) artificial ventilation of the lungs, 3) thoracotomy, 4) modeling of permanent ischemic or ischemic-reperfusion damage. Both extracts effected the indicators of contraction and output, comparable to the reference drug - Monopril. Based on the extraction methods used to obtain RAF and US60 and data from the literature, it can be assumed that they contain compounds with medium polarity, including polyphenols and terpenoids. At the next stage three previously undescribed monoterpenoid derivatives – Ziziphoric acid (1), Ziziphoroside D (2) and 6′-malonylziziphoroside A (3), along with two previously described megastigmane glucosides – blumenol C glucoside (4), blumenol C 9-O-(6′-O-malonyl-beta-D-glucopyranoside (5) and two previously described monoterpenoids 7a-hydroxymintlactone (6), 7-hydroxypiperitone (7) together with six polyphenols – pinocembrine-7-O-rutinoside (8), chrysine-7-O-rutinoside (9), acacetin-7-O-rutinoside (10), luteolin-7-O-rutinoside (11), rutin (12) and rosmarinic acid (13) were isolated from Z. clinopodioides subsp. bungeana extracts. ConclusionOur results support the traditional use of Z. clinopodioides subsp. bungeana for the treatment of coronary diseases. As a result of Z. clinopodioides subsp. bungeana extracts screening in vivo, two extracts were selected as potential cardiotropic agents. Phytochemical analysis of the plant material led to the isolation of five terpenoid derivatives, two megastigmane glycosides, five flavonoids and one cinnamic acid derivative, which could be responsible for the reported biological activity. Future experiments are required to understand the mechanisms of action for the isolated compounds.

Isolation of Alpha-linolenic acid (ALA) from tasar, Antherea mylitta pupal oil and its anticancer activity

Previously, we reported that tasar silkworm pupal oil (TPO) is a rich source of alpha-linolenic acid (ALA) (37%). In this study, we have used three chromatographic techniques, namely preparative HPLC, gas chromatography-mass spectroscopy (GC-MS), and gas chromatography flame ionization detection (GC-FID), to purify, determine, and quantify alpha-linolenic acid (ALA), respectively, from TPO. Additionally, to determine the potential of the purified ALA as an anticancer drug, the sulforhodamine B (SRB) assay was utilised for in vitro investigation. Results revealed that ALA was successfully purified in a fraction of TPO by preparatory HPLC. It was discovered that only ALA was present in the purified fraction of ALA when it underwent GC-MS analysis, further confirming its purity. In addition, the quantification analysis using GC-FID showed that the concentration of ALA was 11760 ppm. Furthermore, anticancer analysis revealed purified ALA significantly inhibited the growth of a colon cancer cell line, (COLO-205) with a GI 50 ≤20. This is the initial investigation into isolating and purifying ALA from Tasar pupal oil.
 Keywords: Alpha-linolenic acid; GC-FID; HPLC; Omega-3Preparative, Tasar pupal oil.

Inverse Polyamidoamine (i‐PAMAM) Dendrimer Antimicrobials

AbstractHere we redesigned the branches of polyamidoamine (PAMAM) dendrimers by moving the amide carbonyl group on the other side of the amide nitrogen atom, transforming the β‐alaninyl‐amidoethylamine branch, which easily undergoes retro‐Michael reactions and renders PAMAMs intrinsically unstable, into a more stable glycyl‐amidopropylamine branch. The resulting inverse PAMAM (i‐PAMAM) dendrimers have the same carbon framework as PAMAMs and only differ by the position of the carbonyl group. In contrast to PAMAMs which are prepared in solution and are difficult to purify, we synthesize i‐PAMAMs using high‐temperature solid‐phase peptide synthesis by iterative coupling and deprotection of the commercially available N,N‐bis(N′‐Fmoc‐3‐aminopropyl)glycine and purify them preparative reverse phase HPLC. Our i‐PAMAM dendrimers show no detectable degradation over time. We demonstrate this new class of dendrimers with the synthesis of antimicrobial dendrimers with potent yet non‐membrane disruptive activities against both Gram‐negative and Gram‐positive bacteria.

Metabolite Chemical Composition of the Bletilla striata (Thunb.) Reichb. f. Endophyte Penicillium oxalicum

Penicillium oxalicum strain can be isolated from the Bletilla striata (Thunb.) Reichb. f. tubers. Its solid-state fermentation products are concentrated by percolation extraction. Separation and purification have been conducted to the ethyl acetate extracts by preparative HPLC. Based on the use of spectrometry, we have determined 17 known compounds, 12,13-dihydroxy-fumitremorgin C (1), pseurotin A (2), tyrosol (3), cyclo-(L-Pro-L-Val) (4), cis-4-hydroxy-8-O-methylmellein (5), uracil (6), cyclo-(L-Pro-L-Ala) (7), 1,2,3,4-tetrahydro-4-hydroxy-4-quinolin carboxylic acid (8), cyclo-(Gly-L-Pro) (9), 2'-deoxyuridine (10), 1-(β-D-ribofuranosyl)thymine (11), cyclo-(L-Val-Gly) (12), 2'-deoxythymidine (13), cyclo-(Gly-D-Phe) (14), cyclo-L-(4-hydroxyprolinyl)-D-leucine (15), cyclo-(L)-4-hydroxy-Pro-(L)-Phe (16), uridine (17). Here, we report compounds 1–3, 5, 7–8, 11–12, 14–17 are first found and isolated from this endophyte.

Identification of Potential Insulinotropic Cytotoxins from Indian Cobra Snake Venom Using High-Resolution Mass Spectrometry and Analyzing Their Possible Interactions with Potassium Channel Receptors by In Silico Studies.

Snake venoms are a potential source of bioactive peptides, which have multiple therapeutic properties in treating diseases such as diabetes, cancer, and neurological disorders. Among bioactive peptides, cytotoxins (CTXs) and neurotoxins are low molecular weight proteins belonging to the three-finger-fold toxins (3FTxs) family composed of two β sheets that are stabilized by four to five conserved disulfide bonds containing 58-72 amino acid residues. These are highly abundant in snake venom and are predicted to have insulinotropic activities. In this study, the CTXs were purified from Indian cobra snake venom using preparative HPLC and characterized using high-resolution mass spectrometry (HRMS) TOF-MS/MS. Further SDS-PAGE analysis confirmed the presence of low molecular weight cytotoxic proteins. The CTXs in fractions A and B exhibited dose-dependent insulinotropic activity from 0.001 to 10µM using rat pancreatic beta-cell lines (RIN-5F) in the ELISA. Nateglinide and repaglinide are synthetic small-molecule drugs that control sugar levels in the blood in type 2 diabetes, which were used as a positive control in ELISA. Concluded that purified CTXs have insulinotropic activity, and there is a scope to use these proteins as small molecules to stimulate insulinotropic activities. At this stage, the focus is on the efficiency of the cytotoxins to induce insulin. Additional work is ongoing on animal models to see the extent of the beneficial effects and efficiency to cure diabetes using streptozotocin-induced models.

A new dihydrochalcone from Humulus scandens

To study the chemical constituents from the stems and leaves of Humulus scandens, this study isolated thirteen compounds by different chromatographic methods including silica gel column, ODS, Sephadex LH-20 and preparative HPLC. Based on comprehensive analysis, the chemical structures were elucidated and identified as citrunohin A(1), chrysosplenetin(2), casticin(3), neoechinulin A(4), ethyl 1H-indole-3-carboxylate(5), 3-hydroxyacetyl-indole(6),(1H-indol-3-yl) oxoacetamide(7), inonotusic acid(8), arteannuin B(9), xanthotoxol(10), α-tocopherol quinone(11), eicosanyl-trans-p-coumarate(12), and 9-oxo-(10E,12E)-octadecadienoic acid(13). Among them, compound 1 was a new dihydrochalcone, and the other compounds were obtained from H. scandens for the first time.

Inhibition of NO Production in LPS-Stimulated Primary Rat Glial Cells by Gnidilatimonoein and Extract of Daphne mucronata.

In the CNS, glial cells are involved in neuroinflammation and neuropathic pain. The glial cells are activated by a variety of pathological conditions and release pro-inflammatory mediators, including nitric oxide (NO). Overexpression of iNOS (inducible nitric oxide synthase) and extra NO is detrimental to neurophysiology and neuronal viability. This study aimed to examine the effect of Gnidilatimonein isolated from D. mucronata and its leaves extract (as natural phytochemicals) on NO production in the LPS-induced primary glial cells. A preparative HPLC method was used to isolate gnidilatimonoein from leaves ethanolic extract. Various doses of Gnidilatimonoein, the ethanolic extract were applied to primary glial cells inflamed by lipopolysaccharide. A Colorimetric test, an MTT assay, and a RT-PCR analysis were then performed to analyze and compare NO production, cell viability, and iNOS expression. Gnidilatimonoein treatment of pretreated primary glial cells significantly inhibited iNOS expression and decreased NO synthesis. Plant extracts also reduced NO production in inflamed microglial and glial at 0.1-3 mg.mL-1. At these concentrations, none of these compounds exerted a cytotoxic effect, suggesting that their anti-inflammatory effects were not due to the death of cells. This study indicates that D. mucronata and its active compound, Gnidilatimonoein, could have restrained effects on the expression of iNOS on the induced glial cells; however, further investigation is warranted.

RP-HPLC hyphenated with ESI -Q-TOF –MS for the detection of potential degradants in dolutegravir

Dolutegravir is an antiviral agent, a second-generation HIV integrase strand transfer inhibitor (INSTI). A precise liquid chromatography-tandem mass spectrometry method has been developed and validated for the detection of potential degradants in Dolutegravir. The goal of this study was to detect major and minor potential degradants that can develop during the various stages of the drug molecule, start from acquiring of raw material for the synthesis, manufacturing, formulation and storage of the final product. With this objective in mind preparative scale HPLC was used to detect possible oxidative degradants in Dolutegravir. The oxidative degradation sample was isolated using Prominence preparative high performance liquid chromatograph equipped with a reverse phase C18 Inertsil ODS (300mm×19mm ×7µ) column. Mobile phase was 0.1% trifluoro acetic acid in water (A) and acetonitrile (B) (50:50). Ethyl acetate was used for the extraction of aqueous layer and the same was distilled and the collected fractions were evaporated to dryness for LCMS studies. To detect the degradation products mass spectra of the drug was established by direct infusion of degradant into mass spectrometry systems. The LC-MS spectrum of Dolutegravir was recorded by Shimadzu LCMS spectrometer in the electron spray ionization mode. The outcome was intense protonated molecular ion peak at m/z 304.27 and 249.06 in the positive ionization mode, scan range 120–560 amu and the other possible degradant structures with m/z 320.32, m/z 207.18, m/z 185.17, m/z 385.41, m/z 155.14. The information obtained from degradation behaviour of dolutegravir by stress degradation studies, under peroxide oxidation and the degradation products formed were identified by HPLC, LCMS analytical techniques are useful information regarding manufacturing, formulation and storage of the drug.

Phytoconstituents from Piliostigma foveolatum (Dalzell) Thoth. leaves induce antiproliferative effect, apoptosis, and cell cycle arrest in Hop-62 cells

The study evaluated the therapeutic potential of ethanolic leaf extract of Piliostigma foveolatum (Dalzell) Thoth. (EEBF), its toluene, ethylacetate, methanol soluble fractions (viz. TFBF, EFBF, MFBF), and isolated phytoconstituents against lung cancer. Four compounds were isolated from MFBF by column chromatography and preparative HPLC. Structures were elucidated by IR, 13C-NMR, 1H-NMR, mass spectroscopy and identified as Quercetin, Kaempferol, Isorhamnetin, and ß-glucogallin. EEBF and its biofractions exhibited remarkable antiproliferative activity with GI50<85µg/mL, while isolated Quercetin, Kaempferol, Isorhamnetin, and ß-Glucogallin displayed GI50 values of 56.15 ± 1.16 μ M, 68.41 ± 3.98 μ M, 55.08 ± 0.57 μ M and 58.99 ± 12.39 μ M respectively. MFBF demonstrated significant apoptotic activity with 42.24 ± 0.57% cells in early and 4.61 ± 0.88% cells in late apoptosis comparable to standard Doxorubicin. Kaempferol exhibited 23.03 ± 0.37% cells in early and 2.11 ± 0.55% cells in late apoptosis, arresting Hop-62 cells in S-phase. In silico molecular docking, revealed that isolated constituents effectively bound to the same binding site of caspase-3 as Doxorubicin, highlighting their apoptotic mode of action.

Investigation on phytochemical constituents of Achillea aucheri Boiss. Endemic to Iranian flora accompanied by antioxidant and antimicrobial evaluations

In traditional medicine, the genus Achillea is utilized for its anti-inflammatory, antispasmodic, and diuretic properties. Achillea aucheri Boiss., species native to Iranian flora was studied due to the Achillea genus's rich supply of phytochemical substances and the absence of essential phytochemical investigations.The methanolic extract (ME) from aerial parts of A. aucheri was fractionated using a C-18 pre-packed cartridge and vacuum liquid chromatography, followed by chromatographic separation using a reversed-phase preparative HPLC. The isolated components were structurally elucidated using 1D and 2D-NMR spectroscopy. Furthermore, essential oil from A. aucheri was isolated through hydrodistillation. Analysis of the essential oil was done through gas chromatography–mass spectrometry (GC-MS) and GC-FID. Free-radical scavenging activity, total flavonoid and phenolic content of the ME were determined using the DPPH assay, AlCl3, and Folin-Ciocalteu reagents, respectively. Anti-microbial activity of the A. aucheri essential oil was investigated against different bacteria and fungus via disk diffusion and micro-dilution methods.Structure elucidation of the purified compounds revealed presence of one major flavonoid (1) and a sesquiterpene (2) in ME. From the A. aucheri essential oil, α-thujone (45.6%), artemisia alcohol (26.5%), and yomogi alcohol (8.8%) were the major constituents. The total flavonoid, phenolic content of the ME were 39.70 ± 0.05 mgQE/gE and 66.70 ± 0.08 mgGAE/gE, respectively, while IC50 value in DPPH assay was 86.28 ± 0.01 μg/mL. Concerning the achieved antimicrobial activity, maximum diameter of inhibition zone of the essential oil was obtained against S. aureus (31.5 ± 0.5 mm) followed by S. epidermidis (13.5 ± 0.7 mm) and M. luteus (11.5 ± 0.5 mm). Additionally, the minimum inhibitory concentration (MIC) values for S. aureus, S. epidermidis, and M. luteus were recorded as 2.5, 2.5 and 1.25% (v/v), respectively. Exhibited activity could be explained to the presence of α-thujone, artemisia alcohol and yomogi alcohol, the most abundant phytochemicals of A. aucheri essential oil.

An efficient isolation and purification of broad partition coefficient range ginsenosides from roots of Panax quinquefolium L. by linear gradient counter-current chromatography coupled with preparative high-performance liquid chromatography.

As a famous health food, roots of Panax quinquefolium L. possessed immune regulation and enhancement of the central nervous system, in which ginsenosides are the main active component with different numbers and positions of sugars, causing different chemical polarities with a challenge for the separation and isolation. In this study, a fast and effective bilinear gradient counter-current chromatography was proposed for preparative isolation ginsenosides with a broad partition coefficient range from roots of Panax quinquefolium L. In terms of the established method, the mobile phases comprising n-butanol and ethyl acetate were achieved by adjusting the proportion. Coupled with the preparative HPLC, eleven main ginsenosides were successfully separated, including ginsenoside Rg1 (1), Re (2), acetyl ginsenoside Rg1 (3), Rb1 (4), Rc (5), Rg2 (6), Rb3 (7), quinquefolium R1 (8), Rd (9), gypenoside X VII (10) and notoginsenoside Fd (11), with purities exceeding 95% according to the HPLC results. Tandem mass spectrometry and electrospray ionization mass spectrometry were adopted for recognizing the isolated compound architectures. Our study suggests that linear gradient counter-current chromatography effectively separates the broad partition coefficient range of ginsenosides compounds from the roots of Panax quinquefolium L. In addition, it can apply to active compound isolation from other complicated natural products.

Cytotoxic Activity of Alkaloids Isolated from Stephania glandulifera Miers

From the caudex of Stephania glandulifera Miers, five alkaloids were isolated using a cold percolation method with 90% methanol. The methanol extract was fractionated into three different fractions from where the alkaloids were purified using preparative HPLC. Structure elucidations were performed by modern spectral analysis including Mass and 1D and 2D-NMR spectroscopy. The purified compounds were tested against the LN-229 human brain glioblastoma cell line by performing a Sulforhodamine B assay (SRB). The isolated compounds were confirmed to be tetrahydropalmatine (1), palmatine (2), stepharanine (3), stepholidine (4), and stepharine (5). Compound 5 showed anticancer activity in the LN-229 human brain glioblastoma cell line with an IC50 value of 8.1 μg/mL. In this study, five alkaloids belonging to three classes namely proaporphine, protoberberine, and tetrahydroprotoberberine were isolated for the first time from Stephania glandulifera Miers. Among them, stepharine showed anticancer activity in the LN-229 human brain glioblastoma cell line which can be further developed as a potential anticancer agent.

Molecular Assessment of Metabolome Alterations in Lotus japonicus Roots Induced by Arbuscular Mycorrhiza

AbstractAn arbuscular mycorrhiza symbiosis, which is established by 80% of all vascular plants and Glomeromycota fungi, can boost the growth, stress tolerance, and yield of agricultural crop plants by increasing the mineral supply of the host plant [1]. Mineral nutrients absorbed by the fungus are exchanged for photoassimilates from the plant via highly branched fungal structures called arbuscules which are formed within root cortex cells [2]. The process of arbuscule formation and degradation is highly dynamic and accompanied by considerable cell reprogramming [3], but there is only limited knowledge on the influence of arbuscular mycorrhiza on the host metabolome.We investigated alterations of the host plant's root metabolome induced by arbuscular mycorrhizal symbiosis using the model legume Lotus japonicus. Methanol extracts of mycorrhizal (myc) and non‐mycorrhizal roots (mock) were analyzed employing ultra‐highperformance liquid chromatography–electrospray ionization–time‐of‐flight–ion mobility–mass spectrometry (UPLC–ESI–ToF–IM–MS) in an untargeted metabolomics approach. Up‐regulated marker metabolites were identified via principal components analysis (PCA) and orthogonal partial least squares–discriminant analysis (OPLS–DA) depicted as S‐plots, and their structures elucidated by co‐chromatography with authentic standards or by isolation from L. japonicus roots using preparative HPLC and one‐ and two‐dimensional nuclear magnetic resonance spectroscopy (NMR) experiments. We isolated three previously unknown mycorrhizal marker polyphenols from L. japonicus roots, one being a coumaronochromone, one pterocarp‐6a‐ene, and one aryl benzofuran carboxaldehyde. Additionally, we identified three more coumaronochromones, three flavonoids, three isoflavonoids, and one coumestan in mycorrhized L. japonicus.

Manipulation in Culture Conditions of Nanofrustulum shiloi for Enhanced Fucoxanthin Production and Isolation by Preparative Chromatography.

Microalgae produce a variety of high-value chemicals including carotenoids. Fucoxanthin is also a carotenoid that has many physiological functions and biological properties. For this reason, the cost-effective production of fucoxanthin at an industrial scale has gained significant attention. In the proposed study, fucoxanthin production was aimed to be increased by altering the culture conditions of N. shiloi. The effect of light intensity aeration rate, different nitrogen sources, and oxidative stress on the biomass and fucoxanthin productivity have been discussed. Based on these results, the fucoxanthin increased to 97.45 ± 2.64 mg/g by adjusting the light intensity to 50 µmol/m2s, and aeration rate at 5 L/min using oxidative stress through the addition of 0.1 mM H2O2 and 0.1 mM NaOCl to the culture medium. Fucoxanthin was then purified with preparative HPLC using C30 carotenoid column (10 mm × 250 mm, 5 μm). After the purification procedure, Liquid chromatography tandem mass spectrometry (LC-MS/MS) and UV-vis spectroscopy were employed for the confirmation of fucoxanthin. This study presented a protocol for obtaining and purifying considerable amounts of biomass and fucoxanthin from diatom by manipulating culture conditions. With the developed methodology, N. shiloi could be evaluated as a promising source of fucoxanthin at the industrial scale for food, feed, cosmetic, and pharmaceutical industries.

Identification and Characterization of the Isomeric Impurity of the Fungicide "Cyazofamid".

Identification and characterization of biproducts/ impurities present in agrochemicals are critical in view of their efficacy and safety towards public health. We herein present our study on identification and characterization of an impurity, 5-chloro-2-cyano-N,N-dimethyl-4-p-tolylimidazole-1-sulfonamide (2) present in the fungicide, "cyazofamid". Intermittent HPLC analysis of the reaction of substituted imidazole (1) with N,N-dimethylsulfamoyl chloride suggested that 2 is formed during the reaction. Isolation by preparative HPLC and characterization by NMR, LC/HRMS, MS/MS and single crystal XRD analysis confirmed 2 as an isomer of cyazofamid, wherein the N,N-dimethyl sulfonamide group was positioned on the other nitrogen of imidazole in close proximity to chloride group. Computational studies further supported the formation of 2 and ruled out the other possible isomeric structures.