Natural products isolation studies of the paleoendemic plant species Nothofagus gunnii and Nothofagus cunninghamii.

The first natural product isolation studies of Nothofagus gunnii (Hook.f.) Oerst and Nothofagus cunninghamii (Hook.f.) Oerst have been undertaken. A previously unreported stilbene derivative, pinosylvin monoacetate, was isolated from the leaves of N. gunnii, in addition to 14 known compounds; including the flavonoids galangin, pinobanksin, catechin and quercetin; sesquiterpenoids such as, ilicol and (+)-ß-costol acetate; 2,4-dihydroxy-6-methoxychalcone and pinosylvin. Four known flavonoid natural products, catechin, quercetin, ayanin, and avicularin were isolated from the leaves of N. cunninghamii. This study reveals that N. gunnii is a rich source of flavonoid, chalcone and stilbene compounds, while primarily hydroxyflavonoid compounds are found in N. cunninghamii. The isolated phytochemicals are consistent with the evolutionary relationships suggested to exist among Nothofagus species.

Radical scavenging activity and metabolomic profiling study of ylang-ylang essential oils based on high-performance thin-layer chromatography and multivariate statistical analysis.

Ylang-ylang (YY) essential oil (EO) is distilled from the fresh-mature flowers of the Annonaceae family tropical tree Cananga odorata [Lam.] Hook. f. & Thomson, and is widely used in perfume and cosmetic industries for its fragrant character. Herein, two different metabolomic profiles obtained using high-performance thin-layer chromatography (HPTLC), applying different stains, namely 2,2-diphenyl-1-picrylhydrazyl (DPPH·) and p-anisaldehyde, were used for discrimination of 52 YY samples across geographical origins and distillation grades. The first profile is developed using the DPPH· stain based on the radical scavenging activity (RSA) of YY EOs. Results of the HPTLC-DPPH· assay confirmed that RSA of YY EOs is in proportion to the length of distillation times. Major components contributing to the RSA of YY EOs were tentatively identified as germacrene D and α-farnesene, eugenol and linalool, by gas chromatography-mass spectrometry (GC-MS) and GC-flame ionisation detector (GC-FID). The second profile was developed using the general-purpose p-anisaldehyde stain based on the general chemical composition of YY EOs. Untargeted metabolomic discrimination of YY EOs from different geographical origins was performed based on the HPTLC-p-anisaldehyde profiles, followed by principal component analysis (PCA). A discrimination and prediction model for identification of YY distillation grade was developed using PCA and partial least squares regression (PLS) based on binned HPTLC-ultraviolet (254 nm) profiles, which was successfully applied to distillation grade determination of blended YY Complete EOs.

Asperuloside Enhances Taste Perception and Prevents Weight Gain in High-Fat Fed Mice.

Asperuloside is an iridoid glycoside found in many medicinal plants that has produced promising anti-obesity results in animal models. In previous studies, three months of asperuloside administration reduced food intake, body weight, and adipose masses in rats consuming a high fat diet (HFD). However, the mechanisms by which asperuloside exerts its anti-obesity properties were not clarified. Here, we investigated homeostatic and nutrient-sensing mechanisms regulating food intake in mice consuming HFD. We confirmed the anti-obesity properties of asperuloside and, importantly, we identified some mechanisms that could be responsible for its therapeutic effect. Asperuloside reduced body weight and food intake in mice consuming HFD by 10.5 and 12.8% respectively, with no effect on mice eating a standard chow diet. Fasting glucose and plasma insulin were also significantly reduced. Mechanistically, asperuloside significantly reduced hypothalamic mRNA ghrelin, leptin, and pro-opiomelanocortin in mice consuming HFD. The expression of fat lingual receptors (CD36, FFAR1-4), CB1R and sweet lingual receptors (TAS1R2-3) was increased almost 2-fold by the administration of asperuloside. Our findings suggest that asperuloside might exert its therapeutic effects by altering nutrient-sensing receptors in the oral cavity as well as hypothalamic receptors involved in food intake when mice are exposed to obesogenic diets. This signaling pathway is known to influence the subtle hypothalamic equilibrium between energy homeostasis and reward-induced overeating responses. The present pre-clinical study demonstrated that targeting the gustatory system through asperuloside administration could represent a promising and effective new anti-obesity strategy.

Distinct Drimane Chemotypes in Tasmanian Mountain Pepper (Tasmannia lanceolata): Differences in the Profiles of Pungent Leaf Phytochemicals Associated with Altitudinal Cline.

This study assesses whether the distinct altitudinal cline in leaf morphology (decreased leaf width and length with increased altitude) in Tasmanian mountain pepper (Tasmannia lanceolata) is associated with changes in the leaf chemistry of wild populations from different ecological landscapes and altitudes. The presence of distinct pungent drimane sesquiterpenoid chemotypes was identified: subalpine woodland and wet sclerophyll forest chemotypes. Isolation studies and analysis of extracts revealed that wet sclerophyll forest T. lanceolata populations featured polygodial as the principal terpenoid, with profiles similar to the commercial cultivars sampled. In contrast, the subalpine woodland populations contained the drimane sesquiterpenoids 1ß-acetoxy-9-deoxyisomuzigadial and 3ß-acetoxydrimenin and the conspicuous absence of the pungent principle polygodial.

Pressurized Hot Water Extraction and Capillary Electrophoresis for Green and Fast Analysis of Useful Metabolites in Plants.

The search for useful compounds from plants is an important research area. Traditional screening that involves isolation and identification/quantitation is tedious, time consuming, and generates a significant amount of chemical waste. Here, we present a simple, fast, and green strategy to assess ≥0.1% wt/wt quantities of useful compounds in plants/spices using pressurized hot water extraction using a household espresso machine followed by chemical analysis using capillary electrophoresis. Three demonstrations with polygodial, cinnamaldehyde, coumarin, and shikimic acid as target metabolites are shown. Direct analysis of extracts was by the developed micellar electrokinetic chromatography and capillary zone electrophoresis methods. The approach, which can be implemented in less developed countries, can process many samples within a day, much faster than traditional techniques that would normally take at least a day. Finally, 0.8-1.1% wt/wt levels of shikimic acid were found in Tasmanian-pepperberry and Tasmanian-fuschia leaves via the approach.

Employing Pressurized Hot Water Extraction (PHWE) to Explore Natural Products Chemistry in the Undergraduate Laboratory.

A recently developed pressurized hot water extraction (PHWE) method which utilizes an unmodified household espresso machine to facilitate natural products research has also found applications as an effective teaching tool. Specifically, this technique has been used to introduce second- and third-year undergraduates to aspects of natural products chemistry in the laboratory. In this report, two experiments are presented: the PHWE of eugenol and acetyleugenol from cloves and the PHWE of seselin and (+)-epoxysuberosin from the endemic Australian plant species Correa reflexa. By employing PHWE in these experiments, the crude clove extract, enriched in eugenol and acetyleugenol, was obtained in 4-9% w/w from cloves by second-year undergraduates and seselin and (+)-epoxysuberosin were isolated in yields of up to 1.1% w/w and 0.9% w/w from C. reflexa by third-year students. The former exercise was developed as a replacement for the traditional steam distillation experiment providing an introduction to extraction and separation techniques, while the latter activity featured guided-inquiry teaching methods in an effort to simulate natural products bioprospecting. This primarily derives from the rapid nature of this PHWE technique relative to traditional extraction methods that are often incompatible with the time constraints associated with undergraduate laboratory experiments. This rapid and practical PHWE method can be used to efficiently isolate various classes of organic molecules from a range of plant species. The complementary nature of this technique relative to more traditional methods has also been demonstrated previously.

Novel polygodial analogs P3 and P27: Efficacious therapeutic agents disrupting mitochondrial function in oral squamous cell carcinoma.

Polygodial, a drimane sesquiterpenoid dialdehyde isolated as a pungent component of the water pepper Persicaria hydropiper, exhibits antifeedant, antimicrobial, anti-inflammatory and anticancer effects. Polygodial also activates transient receptor potential vanilloid subtype 1 (TRPV1) channels. Previously, we described the synthesis of a C12-Wittig derivative of polygodial, termed P3, with significant antiproliferative effects against multiple cancer types including oral squamous cell carcinoma (OSCC). In the present study, a more potent derivative, P27, with superior anti-proliferative effects in vitro and antitumor effects in Cal-27 derived xenografts is described. Polygodial, P3, and P27 all significantly decreased OSCC tumor growth, with P27 being equipotent with polygodial and P3 being the least efficacious. However, neither analog elicited the adverse effect observed with polygodial: Profound transient inflammation. Although P3 and P27 pharmacophores are based on polygodial, novel effects on OSCC cell cycle distribution were identified and shared anticancer effects that are independent of TRPV1 activity were observed. Polygodial elicits an S-phase block, whereas P3 and P27 lead to G2/M phase arrest. Pretreatment of OSCC cells with the TRPV1 antagonist capsazepine does not affect the antiproliferative activity of P3 or P27, indicating that TRPV1 interactions do not regulate OSCC cell proliferation. Indeed, calcium imaging studies identified that the analogs neither activate nor antagonize TRPV1. Behavioral studies using a rat model for orofacial pain confirmed that these analogs fail to induce nocifensive responses, indicating that they are non-noxious in vivo. All compounds induced a significant concentration-dependent decrease in the mitochondrial transmembrane potential and corresponding apoptosis. Considering that P27 is equipotent to polygodial with no TRPV1-associated adverse effects, P27 may serve as an efficacious novel therapy for OSCC.

Identifying the "Mushroom of Immortality": Assessing the Ganoderma Species Composition in Commercial Reishi Products.

Species of Ganoderma, commonly called reishi (in Japan) or lingzhi (in China), have been used in traditional medicine for thousands of years, and their use has gained interest from pharmaceutical industries in recent years. Globally, the taxonomy of Ganoderma species is chaotic, and the taxon name Ganoderma lucidum has been used for most laccate (shiny) Ganoderma species. However, it is now known that G. lucidum sensu stricto has a limited native distribution in Europe and some parts of China. It is likely that differences in the quality and quantity of medicinally relevant chemicals occur among Ganoderma species. To determine what species are being sold in commercially available products, twenty manufactured products (e.g., pills, tablets, teas, etc.) and seventeen grow your own (GYO) kits labeled as containing G. lucidum were analyzed. DNA was extracted, and the internal transcribed spacer (ITS) region and translation elongation factor 1-alpha (tef1α) were sequenced with specific fungal primers. The majority (93%) of the manufactured reishi products and almost half of the GYO kits were identified as Ganoderma lingzhi. G. lingzhi is native to Asia and is the most widely cultivated and studied taxon for medicinal use. Illumina MiSeq sequencing of the ITS1 region was performed to determine if multiple Ganoderma species were present. None of the manufactured products tested contained G. lucidum sensu stricto, and it was detected in only one GYO kit. G. lingzhi was detected in most products, but other Ganoderma species were also present, including G. applanatum, G. australe, G. gibbosum, G. sessile, and G. sinense. Our results indicate that the content of these products vary and that better labeling is needed to inform consumers before these products are ingested or marketed as medicine. Of the 17 GYO kits tested, 11 kits contained Ganoderma taxa that are not native to the United States. If fruiting bodies of exotic Ganoderma taxa are cultivated, these GYO kits will likely end up in the environment. The effects of these exotic species to natural ecosystems needs investigation.

Extraction of carboxylic acid-containing diterpenoids from Dodonaea viscosa via pressurised hot water extraction.

Pressurised hot water extraction (PHWE) was employed to effect the extraction of two carboxylic acid-containing ent-labdane diterpenoids from Dodonaea viscosa. The different extraction profile provided by PHWE in this case suggests that this recently developed method also has applications as a complementary tool for natural products extraction.

Bitter melon protects against ER stress in LS174T colonic epithelial cells.

BACKGROUND: Bitter Melon (BM) has been used as a functional food in traditional Chinese and Indian medicine for many generations and has gained a great deal of attention due to its apparent benefits in moderating some of the pathogenic processes in a variety of inflammatory conditions. BM extract (BME) has been shown to possess strong anti-oxidant properties. In addition, it can ameliorate oxidative stress and potentially ER stress. There is increasing evidence that oxidative and ER stress are major contributors for intestinal secretory cell dysfunction which leads to local inflammation and disease pathogenesis that are hallmarks of inflammatory bowel diseases (IBD). Hence, the search for potential therapeutics against ER stress and oxidative stress in intestinal epithelial secretory cells may provide valuable resources for the management of IBD. The aim of the present study was to investigate the effects of BME in ameliorating ER stress in colonic epithelial cells. METHODS: Human colonic adenocarcinoma LS174T cells were used for the assessment of BME effects on colonic epithelial cells in vitro. Cell viability was assessed using trypan blue exclusion and the effect of BME in ameliorating tunicamycin (TM)-induced ER stress was determined by analysing the mRNA expression of the common ER stress markers; ATF6, XBP1, GRP78, CHOP and PERK by quantitative RT-PCR and GRP78 and CHOP by western blot. RESULTS: In the absence of ER stress, BME exhibited no cell toxicity up to 2.0% w/v and no significant effect on the basal mRNA expression of ER stress markers in LS174T cells. In contrast, pre-treatment of LS174T cells with BME followed by induction of ER stress resulted in a significant decrease in mRNA expression of ATF6, XBP1, GRP78, CHOP and PERK and protein expression of GRP78 and CHOP. Co-treatment during induction of ER stress and post- treatment following induction of ER Stress in LS174T cells resulted in a lower but still significant reduction in mRNA expression levels of most ER stress markers. CONCLUSIONS: This is one of the first studies demonstrating the efficacy of BME in reducing expression of ER stress markers in colonic epithelial cells suggesting the potential of BME as a dietary intervention in ameliorating ER stress and oxidation in IBD. Interestingly, while the most significant effect was seen with pre-treatment of cells with BME there was a reduced but still significant effect when co-treated or even post-treated. This suggests that BME may even be effective in modulating ER stress in the face of an existing cell stress environment.

Auxin biosynthesis in pea: characterization of the tryptamine pathway.

One pathway leading to the bioactive auxin, indole-3-acetic acid (IAA), is known as the tryptamine pathway, which is suggested to proceed in the sequence: tryptophan (Trp), tryptamine, N-hydroxytryptamine, indole-3-acetaldoxime, indole-3-acetaldehyde (IAAld), IAA. Recently, this pathway has been characterized by the YUCCA genes in Arabidopsis (Arabidopsis thaliana) and their homologs in other species. YUCCA is thought to be responsible for the conversion of tryptamine to N-hydroxytryptamine. Here we complement the genetic findings with a compound-based approach in pea (Pisum sativum), detecting potential precursors by gas chromatography/tandem-mass spectrometry. In addition, we have synthesized deuterated forms of many of the intermediates involved, and have used them to quantify the endogenous compounds, and to investigate their metabolic fates. Trp, tryptamine, IAAld, indole-3-ethanol, and IAA were detected as endogenous constituents, whereas indole-3-acetaldoxime and one of its products, indole-3-acetonitrile, were not detected. Metabolism experiments indicated that the tryptamine pathway to IAA in pea roots proceeds in the sequence: Trp, tryptamine, IAAld, IAA, with indole-3-ethanol as a side-branch product of IAAld. N-hydroxytryptamine was not detected, but we cannot exclude that it is an intermediate between tryptamine and IAAld, nor can we rule out the possibility of a Trp-independent pathway operating in pea roots.

Total synthesis of (+/-)-rhazinal, an alkaloidal spindle toxin from Kopsia teoi.

The title alkaloid 1 and its B-nor-congener 3, both of which display potent and unusual anti-mitotic properties, have been synthesized in racemic form and characterised by single-crystal X-ray analysis.