A Descriptive Chemical Composition of Concentrated Bud Macerates through an Optimized SPE-HPLC-UV-MS2 Method-Application to Alnus glutinosa, Ribes nigrum, Rosa canina, Rosmarinus officinalis and Tilia tomentosa.

Concentrated bud macerates (CBMs) are obtained from meristematic tissues such as buds and young shoots by maceration in a solvent composed of glycerin, water and ethanol (1/1/1/, v/v). Their traditional utilization in gemmotherapy has gained interest in the past years, and the knowledge of their chemical characterization can provide commercial arguments, particularly to secure their quality control. Therefore, an optimized method for phytochemical analysis including glycerol removal by a preliminary solid phase extraction (SPE) followed by compound identification using high performance liquid chromatography coupled with ultra-violet and tandem mass detectors (HPLC-UV-MS2) was developed. This method was applied on 5 CBMs obtained from Alnus glutinosa, Ribesnigrum, Rosmarinus officinalis, Rosa canina and Tilia tomentosa in order to determinate their chemical composition. Their antioxidant effects were also investigated by radical scavenging activity assays (DPPH and ORAC). Glycerol removal improved the resolution of HPLC chemical profiles and allowed us to perform TLC antioxidant screening. Our approach permitted the identification of 57 compounds distributed in eight major classes, three of them being common to all macerates including nucleosides, phenolic acids and glycosylated flavonoids. Quantification of the later class as a rutin equivalent (RE) showed a great disparity between Rosa canina macerate (809 mg RE/L), and the other ones (from 175 to 470 mg RE/L). DPPH and ORAC assays confirmed the great activity of Rosa canina (4857 and 6479 µmol TE/g of dry matter, respectively). Finally, phytochemical and antioxidant analysis of CBMs strengthened their phytomedicinal interest in the gemmotherapy field.

Does larch arabinogalactan enhance immune function? A review of mechanistic and clinical trials.

The common cold is a viral infection with important economic burdens in Western countries. The research and development of nutritional solutions to reduce the incidence and severity of colds today is a major focus of interest, and larch arabinogalactan seems to be a promising supportive agent. Arabinogalactan has been consumed by humans for thousands of years and is found in a variety of common vegetables as well as in medicinal herbs. The major commercial sources of this long, densely branched, high-molecular-weight polysaccharide are North American larch trees. The aim of this article is to review the immunomodulatory effects of larch arabinogalactan derived from Larix laricina and Larix occidentalis (North American Larix species) and more specifically its role in the resistance to common cold infections. In cell and animal models, larch arabinogalactan is capable of enhancing natural killer cells and macrophages as well as the secretion of pro-inflammatory cytokines. In humans a clinical study demonstrated that larch arabinogalactan increased the body's potential to defend against common cold infection. Larch arabinogalactan decreased the incidence of cold episodes by 23 %. Improvements of serum antigen-specific IgG and IgE response to Streptococcus pneumoniae and tetanus vaccination suggesting a B cell dependent mechanism have been reported in vaccination studies with larch arabinogalactan, while the absence of response following influenza vaccination suggests the involvement of a T cell dependent mechanism. These observations suggest a role for larch arabinogalactan in the improvement of cold infections, although the mode of action remains to be further explored. Different hypotheses can be envisaged as larch arabinogalactan can possibly act indirectly through microbiota-dependent mechanisms and/or have a direct effect on the immune system via the gut-associated lymphoid tissue (GALT).

Evaluation of the anti-inflammatory and antioxidative potential of four fern species from China intended for use as food supplements.

Inflammation plays a major role in many diseases, for instance in arteriosclerosis, rheumatoid arthritis, autoimmune disorders and cancer. Since many plants contain compounds with anti-inflammatory activity, their consumption may be able to prevent the development of inflammatory-based diseases. Edible ferns are some of the most important wild vegetables in China and have traditionally been used both for dietary and therapeutic purposes. In this study we investigated the anti-inflammatory and antioxidant potential of fern extracts from Matteuccia struthiopteris, Osmundajaponica, Matteuccia orientalis and Pteridium aquilinum intended for use as nutraceuticals. Two modes of action were investigated: the inhibition of the pro-inflammatory gene expression of interleukin-1 beta (IL1-ß) and interleukin-6 (IL6), and the gene expression of iNOS by LPS-elicited macrophages. The results showed a decrease of IL1-ß gene expression for the five fern extracts. This effect was more pronounced for the extracts prepared from the roots of O. japonica (IC50 of 17.8 µg/mL) and the young fronds of M orientalis (50.0 µg/mL). Regarding the indirect measurement of NO, via iNOS gene expression, an interesting decrease of 50% was obtained with the extract of M. orientalis fronds at a low concentration (20 µg/mL) compared with P. aquilinum fronds (160 µg/mL) and leaves of O. japonica. The latter showed a higher decrease but at a high concentration of extract (160 µg/mL). The five fern extracts were also evaluated for their ability to scavenge 2,2-diphenyl-l-picrylhydrazyl (DPPH) radicals and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). All fern extracts exhibited antioxidant effects but the roots of O. japonica and the fronds of M orientalis were most efficient. The HPLC-MS analysis of the constituents of the fern extracts confirmed the presence of chlorogenic acid, caffeic acid, p-coumaric acid, ferulic acid, kaempferol and apigenin, molecules known to exhibit antiinflammatory and/or antioxidant properties.

Acute effect of Ceylon cinnamon extract on postprandial glycemia: alpha-amylase inhibition, starch tolerance test in rats, and randomized crossover clinical trial in healthy volunteers.

BACKGROUND: Postprandial hyperglycemia is a known risk factor for the development of several health disorders including type 2 diabetes, obesity, oxidative stress, and cardiovascular diseases. One encouraging approach for a better control of postprandial glycemia is to reduce carbohydrate digestion. Cinnamon extracts have been known for managing blood glucose. However, their effects on inhibiting digestion of carbohydrate have been poorly analyzed to date. The aim of this study was to investigate the acute effect of a specific Ceylon cinnamon hydro-alcoholic extract (CCE) on carbohydrate digestion and post-meal blood glucose reduction. METHODS: In vitro enzymatic assays and in vivo starch tolerance tests in rats were designed as preclinical assays. Then, a randomized, double-blind, placebo-controlled, cross-over clinical trial was conducted in 18 healthy female and male volunteers. Following the intake of 1 g of CCE, the subjects ate a standardized meal. Blood samples were collected during the 2 hours following the meal to measure glucose and insulin concentrations. Areas under the curves were calculated and statistical differences between the CCE and placebo groups were analyzed using the Mann Whitney-Wilcoxon test. RESULTS: CCE has demonstrated in the in vitro study that it inhibited pancreatic alpha-amylase activity with an IC50 of 25 µg/mL. In the in vivo study, CCE was shown to acutely reduce the glycemic response to starch in a dose-dependent manner in rats. This effect was significant from the dose of 12.5 mg/kg of body weight. In both, the in vitro and in vivo studies, the hydro-alcoholic extract has shown to be more efficacious than the aqueous extract. In the human clinical trial, 1 g of CCE lowered the area under the curve of glycemia between 0 and 120 min by 14.8% (P = 0.15) and between 0 and 60 min by 21.2% (P < 0.05) compared to the placebo. This effect occurred without stimulating insulin secretion. No adverse effects were reported. CONCLUSION: These results suggest that Ceylon cinnamon hydro-alcoholic extract (CCE) may provide a natural and safe solution for the reduction of postprandial hyperglycemia and therefore help to reduce the risks of developing metabolic disorders. TRIAL REGISTRATION: ClinicalTrials.gov NCT02074423 (26/02/2014).

Carnosic acid-rich rosemary (Rosmarinus officinalis L.) leaf extract limits weight gain and improves cholesterol levels and glycaemia in mice on a high-fat diet.

Rosemary (Rosmarinus officinalis L.) extracts (RE) are natural antioxidants that are used in food, food supplements and cosmetic applications; exert anti-inflammatory and anti-hyperglycaemic effects; and promote weight loss, which can be exploited to develop new preventive strategies against metabolic disorders. Therefore, the aim of the present study was to evaluate the preventive effects of rosemary leaf extract that was standardised to 20 % carnosic acid (RE) on weight gain, glucose levels and lipid homeostasis in mice that had begun a high-fat diet (HFD) as juveniles. The animals were given a low-fat diet, a HFD or a HFD that was supplemented with 500 mg RE/kg body weight per d (mpk). Physiological and biochemical parameters were monitored for 16 weeks. Body and epididymal fat weight in animals on the HFD that was supplemented with RE increased 69 and 79 % less than those in the HFD group. Treatment with RE was associated with increased faecal fat excretion but not with decreased food intake. The extract also reduced fasting glycaemia and plasma cholesterol levels. In addition, we evaluated the inhibitory effects of RE in vitro on pancreatic lipase and PPAR-γ agonist activity; the in vitro findings correlated with our observations in the animal experiments. Thus, the present results suggest that RE that is rich in carnosic acid can be used as a preventive treatment against metabolic disorders, which merits further examination at physiological doses in randomised controlled trials.

Importance of extract standardization and in vitro/ex vivo assay selection for the evaluation of antioxidant activity of botanicals: a case study on three Rosmarinus officinalis L. extracts.

The overproduction of free radicals and oxygen reactive species is suspected to be implicated in a wide range of metabolic reactions that can have pernicious consequences in the development of a variety of human diseases. Botanical extracts are sources of antioxidants that counteract both free radicals and oxygen reactive species. The processing conditions used in the botanical extraction may influence the antioxidant composition; therefore, different extracts from the same plant may have different antioxidant properties. To illustrate this fact, we conducted a study using three commercial rosemary (Rosmarinus officinalis L.) leaf extracts. The three extracts were standardized to contain, respectively, 20% carnosic acid, 40% ursolic acid, or 20% rosmarinic acid. They were evaluated for their total (hydrophilic + lipophilic) antioxidant effects on oxygen radical absorbance capacity (ORAC), their ferric reducing/antioxidant power (FRAP), and their capacity to inhibit Cu(2+)-induced low-density lipoprotein (LDL) oxidation ex vivo. The ursolic acid extract showed the lowest antioxidant capacity on all models. The rosmarinic acid extract had an antioxidant capacity 1.5 times higher on ORAC and four times higher on FRAP than the carnosic acid extract. However, the carnosic acid extract was better than the rosmarinic acid extract in inhibiting the oxidation of LDL ex vivo. These results encourage conducting further studies to evaluate the carnosic acid and rosmarinic acid extracts in vivo. Our study offers an example of the importance of the extraction procedures, on which depends the nature of the antioxidant composition, and highlights interest to proceed with in vitro/ex vivo assay selection for the evaluation of the antioxidant properties of botanical extracts.

Gastrointestinal tolerance to an inulin-rich soluble roasted chicory extract after consumption in healthy subjects.

OBJECTIVES: We aimed to assess in healthy subjects 1) the effect of two doses of a new naturally inulin-rich soluble chicory extract (IRSCE) on overall gastrointestinal discomfort after short-term ingestion and 2) the effect on gastrointestinal symptoms of long-term consumption of IRSCE administered at a dose compatible with its future commercial use. METHODS: First, the effect of IRSCE was assessed on overall gastrointestinal discomfort in a double-blind, crossover study where 18 subjects received in a randomized order a morning coffee drink including 10 g of sucrose alone (control period) or with IRSCE at two doses (8.9 and 14.0 g containing 5.0 and 7.8 g of inulin, respectively) during three consecutive 6-d periods. Second, 35 subjects were followed during a randomized, double-blind protocol where they were asked to take twice a day an instant coffee drink containing IRSCE (8.1 g/d containing inulin 5.0 g/d) or sucrose 8.1 g/d during 4 wk. The effects of the treatment on flatulence, bloating, abdominal pain, stool consistency, and number were recorded. RESULTS: In the first study a significant slight increase (P = 0.05) in overall abdominal discomfort was observed with the morning coffee drink containing 7.8 g of inulin after 1 wk of consumption. In the second study, no significant differences between the IRSCE and placebo groups were evidenced with respect to gastrointestinal symptoms during the consumption period. CONCLUSION: Short- and long-term consumptions of IRSCE, given at a daily dose containing 5 g of inulin, are well tolerated by healthy subjects.

Plant extracts from central Asia showing antiinflammatory activities in gene expression assays.

Plant natural products remain a good resource for the discovery of novel pharmaceuticals. A mouse macrophage-based quantitative, reverse transcription polymerase chain reaction (qRT-PCR) system was optimized to screen plant extracts for antiinflammatory activities using three well known genetic markers of inflammation. Plants used for extraction were taxonomically identified and vouchered species from two Central Asian countries, Uzbekistan and Kyrgyzstan, collected through the International Cooperative Biodiversity Groups (ICBG) program. The mRNA expression of cyclooxygenase-2, interleukin 1beta and inducible nitric oxide synthase genes in RAW macrophages was determined quantitatively in response to treatment with plant extracts applied at 100 microg/mL. The screening of 1000 extracts from 449 plant species belonging to 68 plant families resulted in 75 extracts (7.5%) showing strong (75% or higher inhibition) activity against at least one target gene. Many extracts showed qualitative and quantitative differences in the levels of activities against each target gene. Extracts identified from this screen were able to reduce inflammatory symptoms in vivo, thereby validating the screening approach.

Can an apple a day keep the doctor away?

The modern pharmaceutical industry based on synthetic chemistry severed the historical connection between plants, food and medicines. The growing costs of discovering new chemical entity-based drugs through high throughput screening methods may yet again reconnect plants and human health at a new level of technological sophistication. Multi-component botanical therapeutics that comprise functional foods, dietary supplements and botanical drugs hold several advantages over conventional drugs that may earn them a more prominent place in the medicine of the future. They can deliver mixtures of multi-functional molecules with potentiating and synergistic effects and pleiotropic targeting at a reasonable cost and with fewer regulatory constraints. They are well suited for long-term disease prevention in an era of genetic testing and increased life expectancy. They also provide additional vehicles for delivering health and wellness. Technologies that address the needs of discovery, development and manufacturing of multi-component botanical therapeutics are emerging. They include computational and bioinformatics approaches, cell based gene expression and high-content screening systems, and phytochemical elicitation and unique plant cultivation / extraction methods designed to optimize the production of bioactives, standardize overall extract composition and assure batch-to-batch product consistency. Nevertheless, multi-component botanical therapeutics carry risks associated with potential interactions with conventional drugs and adverse reactions, which are difficult to detect and diagnose. They face problems of acceptance by the medical community and pharmaceutical industry, safety and efficacy validation, poor standardization and quality control, and difficulties in identifying active ingredients and determining their complex mode(s) of action. Solving these problems will accelerate the merger of grocery stores with pharmacies and agriculture with chemical manufacturing and provide physicians and patients with broader and more individualized choices for disease prevention and treatment.

Phloem specific expression driven by wheat dwarf geminivirus V-sense promoter in transgenic dicotyledonous species.

Wheat dwarf geminivirus (WDV) is a single-stranded DNA Mastrevirus. The large intergenic region (LIR) of WDV contains cis-acting elements essential for the replication of the genome as well as for the bidirectional transcription of virus genes. The LIR was fused to the GUS (uidA) reporter gene and the WDV viral sense (V-sense) promoter activity derived from the stable integration of that promoter was analysed in transgenic dicot plants. Various dicot species were tested, including Nicotiana tabacum, Nicotiana benthamiana, Arabidopsis thaliana and Cucumis melo. The GUS activity driven by the WDV promoter was also compared to that obtained in plants transformed with the GUS gene controlled by the CaMV 35S promoter as well as two phloem-specific promoters derived from the Arabidopsis thaliana AtSUC2 and AtAHA3 genes. Histochemistry showed that the WDV V-sense promoter consistently induced an expression pattern restricted to the vascular tissues, predominantly in the phloem of all organs. This promoter exhibited levels of GUS activity comparable to that driven by AtSUC2 and AtAHA3 promoters. A vascular expression pattern was observed in the four dicots tested. This was stable during plant development and was not altered following viral infection by an unrelated geminivirus. The uses of such a promoter are discussed regarding the targeting to the phloem of molecules active against vascular pests or pathogens.

Plants and human health in the twenty-first century.

The concept of growing crops for health rather than for food or fiber is slowly changing plant biotechnology and medicine. Rediscovery of the connection between plants and health is responsible for launching a new generation of botanical therapeutics that include plant-derived pharmaceuticals, multicomponent botanical drugs, dietary supplements, functional foods and plant-produced recombinant proteins. Many of these products will soon complement conventional pharmaceuticals in the treatment, prevention and diagnosis of diseases, while at the same time adding value to agriculture. Such complementation can be accelerated by developing better tools for the efficient exploration of diverse and mutually interacting arrays of phytochemicals and for the manipulation of the plant's ability to synthesize natural products and complex proteins. This review discusses the history, future, scientific background and regulatory issues related to botanical therapeutics.