Rapid preparation and proton NMR fingerprinting of polysaccharides from Radix Astragali.

The purity, content, and structure of the polysaccharides prepared from a specific medicinal plant are the fundamental basis to interpret the observed biological activities. An ultrafiltration-based method has been developed for rapid preparation of total and fractional polysaccharides from Radix Astragali in high yield and purity. This method involves extraction of plant material by hot water, treatment with Sevag reagent, and ultrafiltration using molecular weight cutoff concentrators. The prepared polysaccharides were assessed by 1H NMR spectroscopy, providing general purity, fingerprinting, and structural information. This method may be used to efficiently screen polysaccharides in plants.

Phytochemical and Micro-Morphological Characterization of Atraphaxis pyrifolia Bunge Growing in the Republic of Kazakhstan.

Atraphaxis pyrifolia is a native species of Central Asia, known for curing several disorders. The species has little knowledges about its chemical composition and any information about its morphological characteristics despite its importance in traditional Asian medicine. This is one of the first approaches to the phytochemical and morphological characterization of this species. Micro-morphology was performed on the stem, and leaf parts of this plant to profile the morpho-anatomical characters using brightfield, fluorescence, polarized and scanning electron microscopy. Leaves were extracted with hexane and methanol. The hexane extract was analyzed using GC-MS analysis revealing the major presence of γ-sitosterol and nonacosane. The methanolic extract was submitted to Vacuum Liquid Chromatography and Sephadex LH-20. HPTLC, HR-ESI-MS and NMR techniques were used to identify the main compounds. Four glycosylated flavonoids were isolated: 8-O-acetyl-7-O-methyl-3-O-α-l-rhamnopyranosylgossypetin (Compound 1), and 7-O-methyl-3-O-α-l-rhamnopyranosylgossypetin (Compound 3), and two other compounds reported for the first time in the literature (Compounds 2 and 4). The findings presented herein furnish pertinent information essential for the identification and authentication of this medicinal plant. Such insights are invaluable for facilitating robust quality control measures and serve as a foundational framework for subsequent endeavours in metabolic, pharmacological, and taxonomical analyses.

Flavones isolated from Pseudognaphalium liebmannii with tracheal smooth muscle relaxant properties.

The inflorescences of Pseudognaphalium liebmannii are used as folk medicine to treat various respiratory diseases. In this work, we report the isolation of seven known flavones: 5-hydroxy-3,7-dimethoxyflavone 1, 5,8-dihydroxy-3,7-dimethoxyflavone 2, 5,7-dihydroxy-3,8-dimethoxyflavone 3 (gnaphaliin A), 3,5-dihydroxy-7,8-dimethoxyflavone 4 (gnaphaliin B), 3,5-dihydroxy-6,7,8-trimethoxyflavone 5, 3,5,7-trimethoxyflavone 6 and 3-O-methylquercetin 7. All these flavones except 1 and 6 showed a relaxant effect on guinea pig tracheal preparation with EC50 between 69.91 ± 15.32 and 118.72 ± 7.06 µM. Aminophylline (EC50 = 122.03 ± 7.05 µM) was used as a relaxant reference drug. The active flavones shifted the concentration-response curves of forskolin and nitroprusside leftward, and significantly reduced the EC50 values of these drugs. Furthermore, these flavones dose-dependently inhibited phosphodiesterase (PDE) in an in vitro assay. This reveals that the inflorescences of P. liebmannii contain several flavones with relaxant effect on airway smooth muscle and with PDEs inhibition that contribute to supporting the anti-asthmatic traditional use.

Identification and Functional Characterization of Oxidosqualene Cyclases from Medicinal Plant Hoodia gordonii.

Oxidosqualene cyclases (OSCs) are the key enzymes accountable for the cyclization of 2,3-oxidosqualene to varied triterpenoids and phytosterols. Hoodia gordonii (from the family Apocynaceae), a native of the Kalahari deserts of South Africa, Namibia, and Botswana, is being sold as a prevalent herbal supplement for weight loss. The appetite suppressant properties are attributed to P57AS3, an oxypregnane steroidal glycoside. At the molecular level, the enzymes involved in the biosynthesis of triterpenes and phytosterols from H. gordonii have not been previously reported. In the current study, predicted transcripts potentially encoding oxidosqualene cyclases were recognized first by searching publicly available H. gordonii RNA-seq datasets. Two OSC-like sequences were selected for functional analysis. A monofunctional OSC, designated HgOSC1 which encodes lupeol synthase, and HgOSC2, a multifunctional cycloartenol synthase forming cycloartenol and other products, were observed through recombinant enzyme studies. These studies revealed that distinct OSCs exist for triterpene formation in H. gordonii and provided opportunities for the metabolic engineering of specific precursors in producing phytosterols in this plant species.

Identification of Botanicals that Unmask ß-Glucan from the Cell Surface of an Opportunistic Fungal Pathogen.

Dectin-1 expressed on host immune cells recognizes ß-glucans within the cell walls of fungal pathogens and plays an important role in the clearance of fungal infections. However, because ß-glucan is masked by an outer layer of mannoproteins, fungal pathogens can evade detection by host immune cells. In this study, a microplate-based screen was developed to identify ß-glucan unmasking activity exhibited by botanicals. This screen measures the activity of a reporter gene in response to the transcriptional activation of NF-κB due to the interaction between ß-glucan on the fungal cell surface and Dectin-1 present on host immune cells. In this proof-of-concept study, we screened a collection of botanicals (10 plants and some of their reported pure compound actives) used in traditional medicine for their antifungal properties. Several hits were identified in samples that unmasked ß-glucan at sub-inhibitory concentrations. The hit samples were confirmed by fluorescent staining with a ß-glucan antibody, verifying that the samples identified in the screen did indeed unmask ß-glucan. These results indicate that the purported antifungal activities attributed to some botanicals may be due, at least in part, to the presence of compounds that exhibit ß-glucan unmasking activity. Enhanced exposure of cell wall ß-glucans would allow the host to build resilience against fungal infections by helping the immune system to detect the pathogen and mount a more effective clearance mechanism. This screen, together with direct killing/growth inhibition assays, may therefore serve as a valuable tool for substantiating the use of botanicals in preventing and/or treating fungal infections.

Phytochemical study of Seriphidium khorassanicum (syn. Artemisia khorassanica) aerial parts: sesquiterpene lactones with anti-protozoal activity.

Two new eudesmane-type sesquiterpene lactones, 1ß,3α,8α-trihydroxy-11ß,13-dihydroeudesma-4(15)-en-12,6α-olide (1) and 1ß,4α,8α-trihydroxy-11ß,13-dihydroeudesma-12,6α-olide (2), and an unprecedented elemane-type sesquiterpene lactone, 1ß,2ß,8α-trihydroxy-11ß,13-dihydroelema-12,6α-olide (3) along with a known eudesmanolide artapshin (4) were isolated from Seriphidium khorassanicum. Structures were elucidated by NMR, HR-ESI-MS, and ECD spectral data analysis. The anti-protozoal activity was evaluated against Leishmania major promastigotes and amastigote-infected macrophages. They showed dose- and time-dependent activity against L. major amastigotes with IC50 values in the range of 4.9 to 25.3 µM being favourably far below their toxicity against normal murine macrophages with CC50 values ranging from 432.5 to 620.7 µM after 48 h of treatment. Compound 3 exhibited the strongest activity and the highest selectivity index (SI) with IC50 of 4.9 ± 0.6 µM and SI of 88.2 comparable with the standard drug, meglumine antimoniate (Glucantime), with IC50 and SI values of 15.5 ± 2.1 µM and 40.0, respectively.

The Cardiorenal Effects of Piper amalago Are Mediated by the Nitric Oxide/Cyclic Guanosine Monophosphate Pathway and the Voltage-Dependent Potassium Channels.

Piper amalago L. is used in Brazilian traditional medicine to treat inflammation, chest pain, and anxiety. This study aimed to investigate the safety and the renal and cardiovascular effects of the volatile oil (VO) and the aqueous (AE) and hydroalcoholic (HE) extracts from P. amalago. The gas chromatography-mass spectrometry analyses identified 47 compounds in the VO, with ß-cyclogermacrene, spathulenol, ß-phellandrene, and α-pinene standing out. Among the 47 compounds also found in AE and HE by liquid chromatography-mass spectrometry, glycosylated flavones, organic acids, amino acids, and amides were highlighted. Some examples of these compounds are methoxy-methylenedioxy cis-cinnamoyl pyrrolidine, methoxy-methylenedioxy trans-cinnamoyl pyrrolidine, and cyclobutene-2,4-bis-(1,3-benzodioxol-5-methoxy-6-yl)-1,3-dicarboxapyrrolidide. The acute toxicity experiments were conducted on female rats (n = 5). The cardiorenal assays (n = 8) and evaluations of vasodilatory effects on the mesenteric vascular bed (n = 5) were conducted on male rats. In either extract or VO, there were no mortality or changes in relative weights or histopathological analysis of the organs. Urinary volume and renal electrolyte excretion were elevated significantly during repeated dose 7-day treatment with different preparations from P. amalago. None of the preparations induced hypotension or changes in cardiac electrical activity. Only HE promoted significant vasodilatory effects in rats' isolated mesenteric vascular beds. These effects were completely abolished in the presence of L-NAME plus 4-aminopyridine. Therefore, P. amalago leaves are safe and present diuretic activity after acute and repeated dose administration over 7 days. Moreover, the HE induced significant vasodilator response in rats' mesenteric vascular beds by NO/cGMP pathway and voltage-dependent K+ channels activation.

Fatimanols Y and Z: two neo-clerodane diterpenoids from Teucrium yemense.

Teucrium yemense (Defl.), a medicinal plant, grows in Yemen and Saudi Arabia and is also referred to as Reehal Fatima. The plant has a long history of use in these regions for the treatment of diabetes, rheumatism, and renal conditions. Phytochemical investigation of the aerial parts of T. yemense yielded two previously undescribed neo-clerodane diterpenoids, namely fatimanols Y and Z (1 and 2) along with the known teulepicephin (3), 8-acetylharpagide (4) and teucardosid (5). Structure elucidation was accomplished from their 1D and 2D NMR, ECD, and MS characteristics as well as by comparing them to related reported compounds. The new molecules expand understanding of secondary metabolites of this genus. Compounds 1-5 did not show antimicrobial activity against various bacterial and fungal strains.

Development of a Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) Method for Characterizing Linalool Oral Pharmacokinetics in Humans.

Lavender (Lavandula angustifolia Miller or Lavandula officinalis Chaix) is an ethnopharmacological plant commonly known as English lavender. Linalool and linalyl acetate are putative phytoactives in lavender essential oil (LEO) derived from the flower heads. LEO has been used in aroma or massage therapy to reduce sleep disturbance and to mitigate anxiety. Recently, an oral LEO formulation was administered in human clinical trials designed to ascertain its anxiolytic effect. However, human pharmacokinetics and an LC-MS/MS method for the measurement of linalool are lacking. To address this deficiency, a rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the analysis of linalool in human serum. Prior to the analysis, a simple sample preparation protocol including protein precipitation and liquid-liquid extraction of serum samples was created. The prepared samples were analyzed using a C18 reversed-phase column and gradient elution (acetonitrile and water, both containing 0.1% formic acid). A Waters Xevo TQ-S tandem mass spectrometer (positive mode) was used to quantitatively determine linalool and IS according to transitions of m/z 137.1→95.1 (tR 0.79 min) and 205.2→149.1 (tR 1.56 min), respectively. The method was validated for precision, accuracy, selectivity, linearity, sensitivity, matrix effects, and stability, and it was successfully applied to characterize the oral pharmacokinetics of linalool in humans. The newly developed LC-MS/MS-based method and its application in clinical trial serum samples are essential for the characterization of potential pharmacokinetic and pharmacodynamic interactions.

Authentication and Quality Control of the Brazilian Traditional Herb "Espinheira-Santa" (Monteverdia ilicifolia) by Morpho-Anatomy and Microscopy.

The leaves of Monteverdia ilicifolia (syn. Maytenus ilicifolia), commonly called espinheira-santa, are widely used in South American traditional medicines to treat gastritis and ulcers. Several products labeled as espinheira-santa are sold as dietary supplements in retail stores and via e-commerce. Many different species with similar leaf morphology are often mistaken for Monteverdia ilicifolia and used as espinheira-santa, including Monteverdia aquifolia (Celastraceae), Citronella gongonha (Cardiopteridaceae), Jodina rhombifolia (Santalaceae), Sorocea bonplandii (Moraceae), and Zollernia ilicifolia (Fabaceae). This study aimed to characterize M. ilicifolia and distinguish it from adulterants using morphological and microscopic techniques. In addition, foreign matter and powder characteristics of botanical materials sold as "espinheira-santa" were analyzed. The morphoanatomical studies of the leaves and stems of M. ilicifolia and its five adulterant species have revealed noteworthy features that can help species identification and quality control of commercial espinheira-santa. This study showed that many commercial espinheira-santa materials were adulterated and of inferior quality.

Immulina as an Immunostimulatory Supplement: Formulation and Pharmacological Studies.

Immulina is a commercially available extract of Arthrospira platensis enriched with bacterial lipoproteins that acts as a potent Toll-like receptor 2 agonist. However, the immunostimulatory effect of Immulina is not well understood in vivo. Here, to devise an Immulina formulation suitable for in vivo oral gavage dosing, Immulina nanosuspension was prepared and freeze-dried to yield lyophilized nano-Immulina, which had an average particle size of around 300 nm and fully retained the bioactivity as a Toll-like receptor 2 agonist. Compared to the regular Immulina powder, lyophilized nano-Immulina notably accelerated the dissolution in aqueous media. Immulina nanosuspension was found to stimulate the production of proinflammatory cytokines in murine bone marrow-derived dendritic cells and macrophages. The immune response to Immulina was investigated in healthy mice by longitudinally monitoring the phagocytic activity of circulating neutrophils as a surrogate marker. Following daily oral ingestion of Immulina nanosuspension (10 mg/mouse/day), the phagocytic activity of circulating neutrophils was significantly elevated, suggesting an important mechanism for Immulina to enhance innate immunity.

Chemical Profiles and Cytotoxic Activities of Essential Oils from Six Species of Baccharis Subgenus Coridifoliae (Asteraceae).

Several Baccharis species are popularly known in traditional medicine as "carquejas", "vassouras", "ervas-santas" and "mio-mios", and are used as anti-inflammatories, digestives, and diuretics. This study aimed to investigate the chemical compositions and cytotoxic activities of essential oils (EOs) of six Baccharis species belonging to subgenus Coridifoliae, namely B. albilanosa, B. coridifolia, B. erigeroides, B. napaea, B. ochracea, and B. pluricapitulata. GC/MS analyses of the EOs showed that the oxygenated sesquiterpenes spathulenol (7.32-38.22 %) and caryophyllene oxide (10.83-16.75 %) were the major components for all the species. The EOs of almost all species were cytotoxic against cancer (BT-549, KB, SK-MEL and SK-OV-3) and normal kidney (VERO and LLC-PK1) cell lines, whereas B. erigeroides EO showed cytotoxicity only against LLC-PK1. This article augments the current knowledge about the chemical-biological properties of Baccharis subgenus Coridifoliae and discusses the therapeutic potentials of these economically unexploited plants.

Presence and Quantity of Botanical Ingredients With Purported Performance-Enhancing Properties in Sports Supplements.

This case series study examines the accuracy of labels of dietary sports supplements containing botanical ingredients.

Deoxyribonucleic Acid Barcoding for the Identification of Botanicals.

The Natural Herbal Products industry uses botanicals or herbs as raw materials for production of herbal products or dietary supplements. Recently, the demand for natural herbal products has increased tremendously and this has led to adulteration and to counterfeit herbal products. The present chapter deals with currently used molecular methods from "simple" single genomic regions to high-throughput whole genome or transcriptome sequencing methods used in the identification of botanicals.

Quality Consistency of Herbal Products: Chemical Evaluation.

The widespread utility of herbal products has been rising considerably worldwide, including both developed and developing countries, leading to the rapid growth of their availability in the United States and globally. This substantial increase in consumption of herbal products has witnessed the emergence of adverse effects upon oral administration of certain of these products, and thus has raised safety concerns. The adverse effects caused by the consumption of certain botanical medicines occur primarily as a result of the poor quality of plant raw materials or the finished products, which inherently may affect safety and/or efficacy. The poor quality of some herbal products can be attributed to a lack of proper quality assurance and quality control. A high demand for herbal products that surpasses production, combined with a desire for maximizing profits, along with a lack of rigorous quality control within some manufacturing facilities have led to the emergence of quality inconsistencies. The underlying causes for this involve the misidentification of plant species, or their substitution, adulteration, or contamination with harmful ingredients. Analytical assessments have revealed there to be frequent and significant compositional variations between marketed herbal products. The inconsistency of the quality of herbal products can be ascribed essentially to the inconsistency of the botanical raw material quality used to manufacture the products. Thus, the quality assurance and the quality control of the botanical raw materials is may contribute significantly to improving the quality and consistency of the quality of the end products. The current chapter focuses on the chemical evaluation of quality and consistency of herbal products, including botanical dietary supplements. Different techniques, instruments, applications, and methods used in identifying, quantifying, and generating chemical fingerprints and chemical profiles of the ingredients of the herbal products will be described. The strengths and weaknesses of the various techniques available will be addressed. Limitations of the other approaches including morphological or microscopic analysis and DNA-based analysis will be presented.

6-Oxofurostane and (iso)Spirostane Types of Saponins in Smilax sieboldii: UHPLC-QToF-MS/MS and GNPS-Molecular Networking Approach for the Rapid Dereplication and Biodistribution of Specialized Metabolites.

Identifying novel phytochemical secondary metabolites following classical pharmacognostic investigations is tedious and often involves repetitive chromatographic efforts. During the past decade, Ultra-High Performance Liquid Chromatography-Quadrupole Time of Flight-Tandem Mass Spectrometry (UHPLC-QToF-MS/MS), in combination with molecular networking, has been successfully demonstrated for the rapid dereplication of novel natural products in complex mixtures. As a logical application of such innovative tools in botanical research, more than 40 unique 3-oxy-, 3, 6-dioxy-, and 3, 6, 27-trioxy-steroidal saponins were identified in aerial parts and rhizomes of botanically verified Smilax sieboldii. Tandem mass diagnostic fragmentation patterns of aglycones, diosgenin, sarsasapogenin/tigogenin, or laxogenin were critical to establishing the unique nodes belonging to six groups of nineteen unknown steroidal saponins identified in S. sieboldii. Mass fragmentation analysis resulted in the identification of 6-hydroxy sapogenins, believed to be key precursors in the biogenesis of characteristic smilaxins and sieboldins, along with other saponins identified within S. sieboldii. These analytes' relative biodistribution and characteristic molecular networking profiles were established by analyzing the leaf, stem, and root/rhizome of S. sieboldii. Deducing such profiles is anticipated to aid the overall product integrity of botanical dietary supplements while avoiding tedious pharmacognostic investigations and helping identify exogenous components within the finished products.

Ultrastructural, Energy-Dispersive X-ray Spectroscopy, Chemical Study and LC-DAD-QToF Chemical Characterization of Cetraria islandica (L.) Ach.

The lichen Cetraria islandica (L.) Ach. has been used in traditional and modern medicines for its many biological properties such as immunological, immunomodulating, antioxidant, antimicrobial, and anti-inflammatory activities. This species is gaining popularity in the market, with interest from many industries for selling as medicines, dietary supplements, and daily herbal drinks. This study profiled the morpho-anatomical features by light, fluorescence, and scanning electron microscopy; conducted an elemental analysis using energy-dispersive X-ray spectroscopy; and phytochemical analysis was performed using high-resolution mass spectrometry combined with a liquid chromatography system (LC-DAD-QToF) of C. islandica. In total, 37 compounds were identified and characterized based on comparisons with the literature data, retention times, and their mass fragmentation mechanism/s. The identified compounds were classified under five different classes, i.e., depsidones, depsides, dibenzofurans, aliphatic acids, and others that contain simple organic acids in majority. Two major compounds (fumaroprotocetraric acid and cetraric acid) were identified in the aqueous ethanolic and ethanolic extracts of C. islandica lichen. This detailed morpho-anatomical, EDS spectroscopy, and the developed LC-DAD-QToF approach for C. islandica will be important for correct species identification and can serve as a useful tool for taxonomical validation and chemical characterization. Additionally, chemical study of the extract of C. islandica led to isolation and structural elucidation of nine compounds, namely cetraric acid (1), 9'-(O-methyl)protocetraric acid (2), usnic acid (3), ergosterol peroxide (4), oleic acid (5), palmitic acid (6), stearic acid (7), sucrose (8), and arabinitol (9).

Evaluation of the Herb-Drug Interaction (HDI) Potential of Zingiber officinale and Its Major Phytoconstituents.

Ginger is currently one of the most popular herbs commonly added to diverse foods, beverages, and dietary supplements. We evaluated the ability of a well-characterized ginger extract, and several of its phytoconstituents, to activate select nuclear receptors as well as modulate the activity of various cytochrome P450s and ATP-binding cassette (ABC) transporters because phytochemical-mediated modulation of these proteins underlies many clinically relevant herb-drug interactions (HDI). Our results revealed ginger extract activated the aryl hydrocarbon receptor (AhR) in AhR-reporter cells and pregnane X receptor (PXR) in intestinal and hepatic cells. Among the phytochemicals investigated, (S)-6-gingerol, dehydro-6-gingerdione, and (6S,8S)-6-gingerdiol activated AhR, while 6-shogaol, 6-paradol, and dehydro-6-gingerdione activated PXR. Enzyme assays showed that ginger extract and its phytochemicals dramatically inhibited the catalytic activity of CYP3A4, 2C9, 1A2, and 2B6, and efflux transport capabilities of P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP). Dissolution studies with ginger extract conducted in biorelevant simulated intestinal fluid yielded (S)-6-gingerol and 6-shogaol concentrations that could conceivably exceed cytochrome P450 (CYP) IC50 values when consumed in recommended doses. In summary, overconsumption of ginger may disturb the normal homeostasis of CYPs and ABC transporters, which in turn, may elevate the risk for HDIs when consumed concomitantly with conventional medications.

Is Baikiain in Tara Flour a Causative Agent for the Adverse Events Associated with the Recalled Frozen French Lentil & Leek Crumbles Food Product? - A Working Hypothesis.

The French Lentil & Leek Crumbles frozen food product was recently recalled due to reports of gastrointestinal issues. So far, 393 adverse illness complaints and 133 hospitalizations have been reported from consumption of this food, and the tara (Tara spinosa) protein flour ingredient is hypothesized to be responsible. A multipronged approach resulted in identification of (S)-(-)-baikiain in tara as a compound of interest due to its abundance, possible metabolic fate, and close resemblance to irreversible inhibitors of L-pipecolate oxidase. Oral administration of baikiain in ND4 mice showed a statistically significant increase in blood ALT levels and a reduction in liver GSH.