Peter Mouatt

Adulteration of Ginkgo biloba products and a simple method to improve its detection

Extracts of ginkgo (Ginkgo biloba) leaf are widely available worldwide in herbal medicinal products, dietary supplements, botanicals and complementary medicines, and several pharmacopoeias contain monographs for ginkgo leaf, leaf extract and finished products. Being a high-value botanical commodity, ginkgo extracts may be the subject of economically motivated adulteration. We analysed eight ginkgo leaf retail products purchased in Australia and Denmark and found compelling evidence of adulteration with flavonol aglycones in three of these. The same three products also contained genistein, an isoflavone that does not occur in ginkgo leaf. Although the United States Pharmacopeia - National Formulary (USP-NF) and the British and European Pharmacopoeias stipulate a required range for flavonol glycosides in ginkgo extract, the prescribed assays quantify flavonol aglycones. This means that these pharmacopoeial methods are not capable of detecting adulteration of ginkgo extract with free flavonol aglycones. We propose a simple modification of the USP-NF method that addresses this problem: by assaying for flavonol aglycones pre and post hydrolysis the content of flavonol glycosides can be accurately estimated via a simple calculation. We also recommend a maximum limit be set for free flavonol aglycones in ginkgo extract.

Kava for the Treatment of Generalized Anxiety Disorder RCT: Analysis of Adverse Reactions, Liver Function, Addiction, and Sexual Effects

Presently, little is known about a number issues concerning kava (Piper methysticum), including (i) whether kava has any withdrawal or addictive effects; (ii) if genetic polymorphisms of the cytochrome (CYP) P450 2D6 liver enzyme moderates any potential adverse effects; and (iii) if medicinal application of kava has any negative or beneficial effect on sexual function and experience. The study design was a 6‐week, double‐blind, randomized controlled trial (n = 75) involving chronic administration of kava (one tablet of kava twice per day; 120 mg of kavalactones per day, titrated in non‐response to two tablets of kava twice per day; 240 mg of kavalactones) or placebo for participants with generalized anxiety disorder. Results showed no significant differences across groups for liver function tests, nor were there any significant adverse reactions that could be attributed to kava. No differences in withdrawal or addiction were found between groups. Interesting, kava significantly increased females sexual drive compared to placebo (p = 0.040) on a sub‐domain of the Arizona Sexual Experience Scale (ASEX), with no negative effects seen in males. Further, it was found that there was a highly significant correlation between ASEX reduction (improved sexual function and performance) and anxiety reduction in the whole sample. Copyright

Green and Black Cardamom in a Diet-Induced Rat Model of Metabolic Syndrome

Both black (B) and green (G) cardamom are used as flavours during food preparation. This study investigated the responses to B and G in a diet-induced rat model of human metabolic syndrome. Male Wistar rats were fed either a corn starch-rich diet (C) or a high-carbohydrate, high-fat diet with increased simple sugars along with saturated and trans fats (H) for 16 weeks. H rats showed signs of metabolic syndrome leading to visceral obesity with hypertension, glucose intolerance, cardiovascular remodelling and nonalcoholic fatty liver disease. Food was supplemented with 3% dried B or G for the final eight weeks only. The major volatile components were the closely related terpenes, 1,8-cineole in B and α-terpinyl acetate in G. HB (high-carbohydrate, high-fat + black cardamom) rats showed marked reversal of diet-induced changes, with decreased visceral adiposity, total body fat mass, systolic blood pressure and plasma triglycerides, and structure and function of the heart and liver. In contrast, HG (high-carbohydrate, high-fat + green cardamom) rats increased visceral adiposity and total body fat mass, and increased heart and liver damage, without consistent improvement in the signs of metabolic syndrome. These results suggest that black cardamom is more effective in reversing the signs of metabolic syndrome than green cardamom.

Anti-Inflammatory Activity and Structure-Activity Relationships of Brominated Indoles from a Marine Mollusc

Marine molluscs are rich in biologically active natural products that provide new potential sources of anti-inflammatory agents. Here we used bioassay guided fractionation of extracts from the muricid Dicathais orbita to identify brominated indoles with anti-inflammatory activity, based on the inhibition of nitric oxide (NO) and tumour necrosis factor α (TNFα) in lipopolysaccharide (LPS) stimulated RAW264.7 macrophages and prostaglandin E2 (PGE2) in calcium ionophore-stimulated 3T3 ccl-92 fibroblasts. Muricid brominated indoles were then compared to a range of synthetic indoles to determine structure-activity relationships. Both hypobranchial gland and egg extracts inhibited the production of NO significantly with IC50 of 30.8 and 40 μg/mL, respectively. The hypobranchial gland extract also inhibited the production of TNFα and PGE2 with IC50 of 43.03 µg/mL and 34.24 µg/mL, respectively. The purified mono-brominated indole and isatin compounds showed significant inhibitory activity against NO, TNFα, and PGE2, and were more active than dimer indoles and non-brominated isatin. The position of the bromine atom on the isatin benzene ring significantly affected the activity, with 5Br > 6Br > 7Br. The mode of action for the active hypobranchial gland extract, 6-bromoindole, and 6-bromoisatin was further tested by the assessment of the translocation of nuclear factor kappa B (NFκB) in LPS-stimulated RAW264.7 mouse macrophage. The extract (40 µg/mL) significantly inhibited the translocation of NFκB in the LPS-stimulated RAW264.7 macrophages by 48.2%, whereas 40 µg/mL of 6-bromoindole and 6-bromoistain caused a 60.7% and 63.7% reduction in NFκB, respectively. These results identify simple brominated indoles as useful anti-inflammatory drug leads and support the development of extracts from the Australian muricid D. orbita, as a new potential natural remedy for the treatment of inflammation.

Extraction and Quantification of Bioactive Tyrian Purple Precursors: A Comparative and Validation Study from the Hypobranchial Gland of a Muricid Dicathais orbita.

Muricidae are marine molluscs known for the production of Tyrian purple and bioactive precursor compounds. A validation study for the extraction and analysis of secondary metabolites found in the hypobranchial gland of the muricid Dicathais orbita is reported, using high performance liquid chromatography–mass spectrometry (HPLC-MS) with diode array detector (DAD). Quantification of the dominant secondary metabolites from D. orbita is described, followed by a comparison of solvent extraction procedures and stability studies. The intra- and inter-day relative standard deviation (RSD) for tyrindoxyl sulphate was 0.46% and 0.17%, respectively. The quantification was linear for standards murexine, 6-bromoisatin, and tyrindoxyl sulphate. The limits of detection were 0.03, 0.004, and 0.07 mg/mL, respectively, and the limits of quantification were 0.09, 0.01, and 0.22 mg/mL, respectively. The results showed that alcoholic solvents were better for extracting choline ester and indoxyl sulphate ultimate precursors, while chloroform was more suitable for the extraction of the intermediate precursors. Multivariate analysis revealed significant differences in extract composition according to the solvent used. Stability testing showed an increase of the oxidative compounds 6-bromoisatin and putative tyrindoxyl S-oxide sulphate in the ethanol extracts while more degradation products were seen in the chloroform extracts after months of cold storage. The validated method was found to be simple, reproducible, precise, and suitable for quantification of the secondary metabolites of muricid molluscs for dye precursor and nutraceutical quality control, as well as applications in marine chemical ecology.

Achacha (Garcinia humilis) Rind Improves Cardiovascular Function in Rats with Diet-Induced Metabolic Syndrome

Garcinia humilis is a fruit known as achachairú. It is native to South American countries such as Bolivia, Peru, and Brazil, but it is also cultivated as achacha in northern Australia. The aim of this study was to determine the phytochemicals in achacha rind and pulp and to investigate these components as potential treatments for the symptoms of metabolic syndrome. Both rind and pulp contain procyanidins and citric acid rather than hydroxycitric acid. Male Wistar rats (8–9 weeks old) were fed with either high-carbohydrate, high-fat, or corn starch diets for 16 weeks. Intervention groups were fed with either diet supplemented with 1.5% G. humilis rind powder or 2.0% G. humilis pulp for the last 8 weeks of the protocol. Rats fed a high-carbohydrate, high-fat diet exhibited hypertension, dyslipidemia, central obesity, impaired glucose tolerance, and non-alcoholic fatty liver disease. G. humilis rind decreased systolic blood pressure, diastolic stiffness, left ventricular inflammatory cell infiltration, and collagen deposition in high-carbohydrate, high-fat diet-fed rats. However, there was no change in glucose tolerance, body weight, or body composition. Therefore, G. humilis rind, usually a food by-product, but not the edible pulp, showed potential cardioprotection with minimal metabolic changes in a rat model of diet-induced metabolic syndrome.

Effect of cooking on nutrient composition and anticancer indoles of the marine whelk Dicathais orbita – Can it be another high-value seafood product?

The Australian marine mollusc Dicathais orbita has been identified as a functional food with potential for use in the prevention of colon cancer. This study investigated the effect of cooking on the biochemical profile of the edible flesh and extracts containing anticancer brominated indoles. The whelk flesh was high in protein (>75 mg/100 mg dry weight) and there was a significant increase in protein and amino acids after cooking, associated with a loss of moisture and lipids. The flesh also has a high proportion of polyunsaturated fatty acids (>49%), with omega-3:omega-6 around 1. The flesh contains unusually high levels of Docosapentaenoic acid and over 1000 mg/100 g serve of Eicosapentaenoic acid and Docosahexaenoic acid. Cooking resulted in a quantitative decrease in the bioactive compounds, however the main anticancer compound 6-bromoisatin can be retained after boiling. This provides evidence that cooking does not negatively impact the functional food properties of these muricids whelks.

Volatile and bioactive compounds in opercula from Muricidae molluscs supports their use in ceremonial incense and traditional medicines

Muricidae molluscs are the source of a valuable purple dye that was traded as a luxury item in the Mediterranean region and by the late Byzantine was reserved for royalty and priests. Less well known is the use of muricid opercula in sacred incense and traditional medicines, although they are still used as rare ingredients today. This study provides the first chemical assessment of opercula from Muricidae, based on several traditional preparation procedures. Chemical analysis of opercula smoke revealed aromatic phenols, which act as fragrance stabilisers and produce a “medicinal” odour. Analysis of lipid extracts revealed pharmaceutically active compounds, including brominated indoles, choline esters and adenosine, consistent with their traditional medical applications. Depending on the preparation procedures, toxic pyridine was also detected. ICP-MS analysis of muricid opercula shows the presence of essential macro and microelements, as well as metals, some of which exceed the recommended safe levels for human use. Nevertheless, these findings support the Muricidae as an historically important marine resource, providing Biblical dyes, medicines and perfume. The opercula contains biologically active compounds and produces smoke containing volatile scent compounds, consistent with their identification as the most likely source of onycha, a controversial ingredient in sacred incense.

A simple HPLC method for detecting adulteration of ginkgo extracts with flavonol glycosides

We have shown that current USP and EP/BP methods for quantifying ginkgo flavonoids cannot detect adulteration with free flavonol aglycones. We propose a simple modification of these methods, based on the measurement of aglycones preand post-hydrolysis, to allow for the accurate estimation of the amounts of both glycosides and free aglycones in ginkgo leaf extracts. The presence of genistein in products with high levels of free aglycones is consistent with adulteration with Sophora japonica L., which contains genistein, quercetin and kaempferol glycosides [5], and has previously been reported as a suspected adulterant of ginkgo extracts [4]. Pharmacopoeial tests for ginkgo extracts should be improved in order that they might effectively ensure the authenticity, efficacy and safety of this important phytomedicine.