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1.  Distinguishing Vaccinium Species by Chemical Fingerprinting Based on NMR Spectra, Validated with Spectra Collected in Different Laboratories 
Planta medica  2014;80(0):732-739.
A method was developed to distinguish Vaccinium species based on leaf extracts using nuclear magnetic resonance spectroscopy. Reference spectra were measured on leaf extracts from several species, including lowbush blueberry (Vaccinium angustifolium), oval leaf huckleberry (Vaccinium ovalifolium), and cranberry (Vaccinium macrocarpon). Using principal component analysis, these leaf extracts were resolved in the scores plot. Analysis of variance statistical tests demonstrated that the three groups differ significantly on PC2, establishing that the three species can be distinguished by nuclear magnetic resonance. Soft independent modeling of class analogies models for each species also showed discrimination between species. To demonstrate the robustness of nuclear magnetic resonance spectroscopy for botanical identification, spectra of a sample of lowbush blueberry leaf extract were measured at five different sites, with different field strengths (600 versus 700 MHz), different probe types (cryogenic versus room temperature probes), different sample diameters (1.7 mm versus 5 mm), and different consoles (Avance I versus Avance III). Each laboratory independently demonstrated the linearity of their NMR measurements by acquiring a standard curve for chlorogenic acid (R2 = 0.9782 to 0.9998). Spectra acquired on different spectrometers at different sites classifed into the expected group for the Vaccinium spp., confirming the utility of the method to distinguish Vaccinium species and demonstrating nuclear magnetic resonance fingerprinting for material validation of a natural health product.
PMCID: PMC4242896  PMID: 24963620
NMR fingerprinting; Vaccinium spp; Ericaceae; nontargeted screening; multilab study; metabolomics
2.  Advances in the Research and Development of Natural Health Products as Main Stream Cancer Therapeutics 
Natural health products (NHPs) are defined as natural extracts containing polychemical mixtures; they play a leading role in the discovery and development of drugs, for disease treatment. More than 50% of current cancer therapeutics are derived from natural sources. However, the efficacy of natural extracts in treating cancer has not been explored extensively. Scientific research into the validity and mechanism of action of these products is needed to develop NHPs as main stream cancer therapy. The preclinical and clinical validation of NHPs would be essential for this development. This review summarizes some of the recent advancements in the area of NHPs with anticancer effects. This review also focuses on various NHPs that have been studied to scientifically validate their claims as anticancer agents. Furthermore, this review emphasizes the efficacy of these NHPs in targeting the multiple vulnerabilities of cancer cells for a more selective efficacious treatment. The studies reviewed here have paved the way for the introduction of more NHPs from traditional medicine to the forefront of modern medicine, in order to provide alternative, safer, and cheaper complementary treatments for cancer therapy and possibly improve the quality of life of cancer patients.
PMCID: PMC4391654  PMID: 25883673
3.  Evaluation of the Efficacy & Biochemical Mechanism of Cell Death Induction by Piper longum Extract Selectively in In-Vitro and In-Vivo Models of Human Cancer Cells 
PLoS ONE  2014;9(11):e113250.
Currently chemotherapy is limited mostly to genotoxic drugs that are associated with severe side effects due to non-selective targeting of normal tissue. Natural products play a significant role in the development of most chemotherapeutic agents, with 74.8% of all available chemotherapy being derived from natural products.
To scientifically assess and validate the anticancer potential of an ethanolic extract of the fruit of the Long pepper (PLX), a plant of the piperaceae family that has been used in traditional medicine, especially Ayurveda and investigate the anticancer mechanism of action of PLX against cancer cells.
Materials & Methods
Following treatment with ethanolic long pepper extract, cell viability was assessed using a water-soluble tetrazolium salt; apoptosis induction was observed following nuclear staining by Hoechst, binding of annexin V to the externalized phosphatidyl serine and phase contrast microscopy. Image-based cytometry was used to detect the effect of long pepper extract on the production of reactive oxygen species and the dissipation of the mitochondrial membrane potential following Tetramethylrhodamine or 5,5,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine chloride staining (JC-1). Assessment of PLX in-vivo was carried out using Balb/C mice (toxicity) and CD-1 nu/nu immunocompromised mice (efficacy). HPLC analysis enabled detection of some primary compounds present within our long pepper extract.
Our results indicated that an ethanolic long pepper extract selectively induces caspase-independent apoptosis in cancer cells, without affecting non-cancerous cells, by targeting the mitochondria, leading to dissipation of the mitochondrial membrane potential and increase in ROS production. Release of the AIF and endonuclease G from isolated mitochondria confirms the mitochondria as a potential target of long pepper. The efficacy of PLX in in-vivo studies indicates that oral administration is able to halt the growth of colon cancer tumors in immunocompromised mice, with no associated toxicity. These results demonstrate the potentially safe and non-toxic alternative that is long pepper extract for cancer therapy.
PMCID: PMC4234676  PMID: 25401766
4.  Lingonberry (Vaccinium vitis-idaea L.) Exhibits Antidiabetic Activities in a Mouse Model of Diet-Induced Obesity 
Vaccinium vitis-idaea, commonly known as lingonberry, has been identified among species used by the Cree of Eeyou Istchee of northern Quebec to treat symptoms of diabetes. In a previous study, the ethanol extract of berries of V. vitis-idaea enhanced glucose uptake in C2C12 muscle cells via stimulation of AMP-activated protein kinase (AMPK) pathway. The purpose of this study was to examine the effect of plant extract in a dietary mouse model of mild type 2 diabetes. C57BL/6 mice fed a high-fat diet (HFD, ∼35% lipids) for 8 weeks that become obese and insulin-resistant (diet-induced obesity, DIO) were used. Treatment began by adding V. vitis-idaea extract to HFD at 3 different concentrations (125, 250, and 500 mg/Kg) for a subsequent period of 8 weeks (total HFD, 16 weeks). The plant extract significantly decreased glycemia and strongly tended to decrease insulin levels in this model. This was correlated with a significant increase in GLUT4 content and activation of the AMPK and Akt pathways in skeletal muscle. V. vitis-idaea treatment also improved hepatic steatosis by decreasing hepatic triglyceride levels and significantly activated liver AMPK and Akt pathways. The results of the present study confirm that V. vitis-idaea represents a culturally relevant treatment option for Cree diabetics and pave the way to clinical studies.
PMCID: PMC4072050  PMID: 25013446
5.  Ovulation but not milt production is inhibited in fathead minnows (Pimephales promelas) exposed to a reproductively inhibitory pulp mill effluent 
A 5-day fathead minnow (FHM) spawning assay is used by industry to monitor pulp mill effluent quality, with some mill effluents capable of completely inhibiting spawning. The purpose of this report is to characterize the effect of an inhibitory effluent on egg and milt production in FHM.
Eight tanks were treated with an inhibitory effluent while eight were kept with clean water. Each tank contained two males and four females as per the 5-day FHM spawning assay used by industry. Females were stripped of ovulated eggs and males of milt in four effluent-exposed and four control tanks. Eggs oviposited in every tank were also counted and checked for fertilization and data analyzed with 2-way ANOVA.
We show that female, but not male, fathead minnow reproductive function is impaired in the 5-day fathead minnow spawning assay used by industry to evaluate pulp mill effluent quality in Canada. Milt production was not changed in the control or exposed males mid-way and at the end of the five day exposure (p > 0.05; n = 8). Total egg production (stripped + oviposited) was impaired (p < 0.05) in fathead minnows exposed to effluent (288 eggs/tank, n = 4 tanks) compared to those in control tanks (753 eggs/tank, n = 4 tanks).
Our results indicate that males are able to detect female signals and prepare appropriately for spawning while in females inhibition of ovulation is occurring somewhere along the hypothalamus-pituitary-gonad reproductive axis. These results suggest female-specific neuroendocrine disruption and provide mechanistic insight into an assay used by industry to assess pulp mill effluent quality.
PMCID: PMC4035717  PMID: 24884628
Fathead minnow; Pulp mill effluent; Reproductive inhibition; 5-day fathead minnow spawning assay; Ovulation; Milt
6.  Pharmacy study of natural health product adverse reactions (SONAR): a cross-sectional study using active surveillance in community pharmacies to detect adverse events associated with natural health products and assess causality 
BMJ Open  2014;4(3):e003431.
To investigate the rates and causality of adverse event(s) (AE) associated with natural health product (NHP) use, prescription drug use and concurrent NHP-drug use through active surveillance in community pharmacies.
Cross-sectional study of screened patients.
10 community pharmacies across Alberta and British Columbia, Canada from 14 January to 30 July 2011.
The participating pharmacy staff screened consecutive patients, or agents of patients, who were dropping or picking up prescription medications.
Primary outcome measures
Patients were screened to determine the proportions of them using prescription drugs and/or NHPs, as well as their respective AE rates. All AEs reported by the screened patients who took a NHP, consented to, and were available for, a detailed telephone interview (14%) were adjudicated fully to assess for causality.
Over a total of 105 pharmacy weeks and 1118 patients screened, 410 patients reported taking prescription drugs only (36.7%; 95% CI 33.9% to 39.5%), 37 reported taking NHPs only (3.3%; 95% CI 2.4% to 4.5%) and 657 reported taking prescription drugs and NHPs concurrently (58.8%; 95% CI 55.9% to 61.6%). In total, 54 patients reported an AE, representing 1.2% (95% CI 0.51% to 2.9%), 2.7% (95% CI 0.4% to 16.9%) and 7.3% (95% CI 5.6% to 9.6%) of each population, respectively. Compared with patients who reported using prescription drugs, the patients who reported using prescription drugs and NHPs concurrently were 6.4 times more likely to experience an AE (OR; 95% CI 2.52 to 16.17; p<0.001). Combined with data from Ontario, Canada, a national proportion was calculated, which found that 45.4% (95% CI 43.8% to 47.0%) of Canadians who visit community pharmacies take NHPs and prescription drugs concurrently, and of those, 7.4% (95% CI 6.3% to 8.8%) report an AE.
A substantial proportion of community pharmacy patients use prescription drugs and NHPs concurrently; these patients are at a greater risk of experiencing an AE. Active surveillance provides a means of detecting such AEs and collecting high-quality data on which causality assessment can be based.
PMCID: PMC3975764  PMID: 24682573
Complementary Medicine; Toxicology
7.  Investigating Wild Berries as a Dietary Approach to Reducing the Formation of Advanced Glycation Endproducts: Chemical Correlates of In Vitro Antiglycation Activity 
Evidence supports the health promoting benefits of berries, particularly with regard to the prevention and management of chronic diseases such cardio- and cerebrovascular disease, diabetes and Alzheimer’s disease. Two related pathophysiological features common to many of these conditions are oxidative stress and the accumulation of advanced glycation endproducts (AGEs). Whereas antioxidant properties are well-established in several species of berries and are believed central to their protective mechanisms, few studies have investigated the effects of berries on AGE formation. Here, employing a series of complementary in vitro assays, we evaluated a collection of wild berry extracts for 1) inhibitory effects on fluorescent-AGE and Nε- (carboxymethyl)lysine-albumin adduct formation, 2) radical scavenging activity and 3) total phenolic and anthocyanin content. All samples reduced AGE formation in a concentration-dependent manner that correlated positively with each extract’s total phenolic content and, to a lesser degree, total anthocyanin content. Inhibition of AGE formation was similarly related to radical scavenging activities. Adding antiglycation activity to the list of established functional properties ascribed to berries and their phenolic metabolites, our data provide further insight into the active compounds and protective mechanisms through which berry consumption may aid in the prevention and treatment of chronic diseases associated with AGE accumulation and toxicity. As widely available, safe and nutritious foods, berries represent a promising dietary intervention worthy of further investigation.
PMCID: PMC3930840  PMID: 24448675
Fruits; Aging; Chronic disease; Oxidative stress; Radical scavenging; Polyphenolics; Anthocyanins
8.  Associations between toxic metals in follicular fluid and in vitro fertilization (IVF) outcomes 
We previously reported associations between trace concentrations of Hg, Cd and Pb in blood and urine and reproductive outcomes for women undergoing in-vitro fertilization (IVF). Here we assess measurements in single follicular fluid (FF) specimens from 46 women as a presumably more relevant marker of dose for reproductive toxicity.
FF specimens were analyzed for Hg, Cd and Pb using sector field-inductively coupled plasma-mass spectrometry (SF-ICP-MS). Variability sources were assessed by nested ANOVA. Multivariable regression was used to evaluate associations for square root transformed metals with IVF outcomes, adjusting for confounders.
An inverse association is detected for FF Pb and fertilization (relative risk (RR) = 0.68, P = 0.026), although positive for Cd (RR = 9.05, P = 0.025). While no other statistically significant associations are detected, odds ratios (OR) are increased for embryo cleavage with Hg (OR = 3.83, P = 0.264) and Cd (OR = 3.18, P = 0.644), and for embryo fragmentation with Cd (OR = 4.08, P = 0.586) and Pb (OR = 2.22, P = 0.220). Positive estimates are observed for Cd with biochemical (RR = 19.02, P = 0.286) and clinical pregnancies (RR = 38.80, P = 0.212), yet with very low precision.
We have identified associations between trace amounts of Pb and Cd in FF from a single follicle, and oocyte fertilization. Yet, the likelihood of biological variation in trace element concentrations within and between follicles, coupled with levels that are near the limits of detection suggest that future work should examine multiple follicles using a ‘one follicle-one oocyte/embryo’ approach. A larger study is merited to assess more definitively the role that these environmental factors could play with respect to egg quality in IVF programs.
PMCID: PMC3528869  PMID: 23229520
Cadmium (Cd); Follicular fluid (FF); in vitro fertilization (IVF); Lead (Pb); Mercury (Hg); Reproductive outcomes
9.  The Action of Antidiabetic Plants of the Canadian James Bay Cree Traditional Pharmacopeia on Key Enzymes of Hepatic Glucose Homeostasis 
We determined the capacity of putative antidiabetic plants used by the Eastern James Bay Cree (Canada) to modulate key enzymes of gluconeogenesis and glycogen synthesis and key regulating kinases. Glucose-6-phosphatase (G6Pase) and glycogen synthase (GS) activities were assessed in cultured hepatocytes treated with crude extracts of seventeen plant species. Phosphorylation of AMP-dependent protein kinase (AMPK), Akt, and Glycogen synthase kinase-3 (GSK-3) were probed by Western blot. Seven of the seventeen plant extracts significantly decreased G6Pase activity, Abies balsamea and Picea glauca, exerting an effect similar to insulin. This action involved both Akt and AMPK phosphorylation. On the other hand, several plant extracts activated GS, Larix laricina and A. balsamea, far exceeding the action of insulin. We also found a significant correlation between GS stimulation and GSK-3 phosphorylation induced by plant extract treatments. In summary, three Cree plants stand out for marked effects on hepatic glucose homeostasis. P. glauca affects glucose production whereas L. laricina rather acts on glucose storage. However, A. balsamea has the most promising profile, simultaneously and powerfully reducing G6Pase and stimulating GS. Our studies thus confirm that the reduction of hepatic glucose production likely contributes to the therapeutic potential of several antidiabetic Cree traditional medicines.
PMCID: PMC3707264  PMID: 23864882
10.  Populus balsamifera Extract and Its Active Component Salicortin Reduce Obesity and Attenuate Insulin Resistance in a Diet-Induced Obese Mouse Model 
Populus balsamifera L. (BP) is a medicinal plant stemming from the traditional pharmacopoeia of the Cree of Eeyou Istchee (CEI—Northern Quebec). In vitro screening studies revealed that it strongly inhibited adipogenesis in 3T3-L1 adipocytes, suggesting potential antiobesity activity. Salicortin was identified, through bioassay-guided fractionation, as the active component responsible for BP's activity. The present study aimed to assess the potential of BP and salicortin at reducing obesity and features of the metabolic syndrome, in diet-induced obese C57Bl/6 mice. Mice were subjected to high fat diet (HFD) for sixteen weeks, with BP (125 or 250 mg/kg) or salicortin (12.5 mg/kg) introduced in the HFD for the last eight of the sixteen weeks. BP and salicortin effectively reduced whole body and retroperitoneal fat pad weights, as well as hepatic triglyceride accumulation. Glycemia, insulinemia, leptin, and adiponectin levels were also improved. This was accompanied by a small yet significant reduction in food intake in animals treated with BP. BP and salicortin (slightly) also modulated key components in signaling pathways involved with glucose regulation and lipid oxidation in the liver, muscle, and adipose tissue. These results confirm the validity of the CEI pharmacopoeia as alternative and complementary antiobesity and antidiabetic therapies.
PMCID: PMC3678421  PMID: 23781256
12.  Characterizing the cytoprotective activity of Sarracenia purpurea L., a medicinal plant that inhibits glucotoxicity in PC12 cells 
The purple pitcher plant, Sarracenia purpurea L., is a widely distributed species in North America with a history of use as both a marketed pain therapy and a traditional medicine in many aboriginal communities. Among the Cree of Eeyou Istchee in northern Québec, the plant is employed to treat symptoms of diabetes and the leaf extract demonstrates multiple anti-diabetic activities including cytoprotection in an in vitro model of diabetic neuropathy. The current study aimed to further investigate this activity by identifying the plant parts and secondary metabolites that contribute to these cytoprotective effects.
Ethanolic extracts of S. purpurea leaves and roots were separately administered to PC12 cells exposed to glucose toxicity with subsequent assessment by two cell viability assays. Assay-guided fractionation of the active extract and fractions was then conducted to identify active principles. Using high pressure liquid chromatography together with mass spectrometry, the presence of identified actives in both leaf and root extracts were determined.
The leaf extract, but not that of the root, prevented glucose-mediated cell loss in a concentration-dependent manner. Several fractions elicited protective effects, indicative of multiple active metabolites, and, following subfractionation of the polar fraction, hyperoside (quercetin-3-O-galactoside) and morroniside were isolated as active constituents. Phytochemical analysis confirmed the presence of hyperoside in the leaf but not root extract and, although morroniside was detected in both organs, its concentration was seven times higher in the leaf.
Our results not only support further study into the therapeutic potential and safety of S. purpurea as an alternative and complementary treatment for diabetic complications associated with glucose toxicity but also identify active principles that can be used for purposes of standardization and quality control.
PMCID: PMC3556504  PMID: 23216659
Diabetic neuropathy; Glucose toxicity; Traditional medicine; Quercetin-3-O-galactoside; Morroniside
13.  Study of Natural Health Product Adverse Reactions (SONAR): Active Surveillance of Adverse Events Following Concurrent Natural Health Product and Prescription Drug Use in Community Pharmacies 
PLoS ONE  2012;7(9):e45196.
Many consumers use natural health products (NHPs) concurrently with prescription medications. As NHP-related harms are under-reported through passive surveillance, the safety of concurrent NHP-drug use remains unknown. To conduct active surveillance in participating community pharmacies to identify adverse events related to concurrent NHP-prescription drug use.
Methodology/Principal Findings
Participating pharmacists asked individuals collecting prescription medications about (i) concurrent NHP/drug use in the previous three months and (ii) experiences of adverse events. If an adverse event was identified and if the patient provided written consent, a research pharmacist conducted a guided telephone interview to gather additional information after obtaining additional verbal consent and documenting so within the interview form. Over a total of 112 pharmacy weeks, 2615 patients were screened, of which 1037 (39.7%; 95% CI: 37.8% to 41.5%) reported concurrent NHP and prescription medication use. A total of 77 patients reported a possible AE (2.94%; 95% CI: 2.4% to 3.7%), which represents 7.4% of those using NHPs and prescription medications concurrently (95%CI: 6.0% to 9.2%). Of 15 patients available for an interview, 4 (26.7%: 95% CI: 4.3% to 49.0%) reported an AE that was determined to be “probably” due to NHP use.
Active surveillance markedly improves identification and reporting of adverse events associated with concurrent NHP-drug use. Although not without challenges, active surveillance is feasible and can generate adverse event data of sufficient quality to allow for meaningful adjudication to assess potential harms.
PMCID: PMC3461007  PMID: 23028841
14.  Larix laricina, an Antidiabetic Alternative Treatment from the Cree of Northern Quebec Pharmacopoeia, Decreases Glycemia and Improves Insulin Sensitivity In Vivo 
Larix laricina K. Koch is a medicinal plant belonging to traditional pharmacopoeia of the Cree of Eeyou Istchee (Eastern James Bay area of Canada). In vitro screening studies revealed that, like metformin and rosiglitazone, it increases glucose uptake and adipogenesis, activates AMPK, and uncouples mitochondrial function. The objective of this study was to evaluate the antidiabetic and antiobesity potential of L. laricina in diet-induced obese (DIO) C57BL/6 mice. Mice were subjected for eight or sixteen weeks to a high fat diet (HFD) or HFD to which L. laricina was incorporated at 125 and 250 mg/kg either at onset (prevention study) or in the last 8 of the 16 weeks of administration of the HFD (treatment study). L. laricina effectively decreased glycemia levels, improved insulin resistance, and slightly decreased abdominal fat pad and body weights. This occurred in conjunction with increased energy expenditure as demonstrated by elevated skin temperature in the prevention study and improved mitochondrial function and ATP synthesis in the treatment protocol. L. laricina is thus a promising alternative and complementary therapeutic approach for the treatment and care of obesity and diabetes among the Cree.
PMCID: PMC3408811  PMID: 22888363
15.  Comprehensive Evidence-Based Assessment and Prioritization of Potential Antidiabetic Medicinal Plants: A Case Study from Canadian Eastern James Bay Cree Traditional Medicine 
Canadian Aboriginals, like others globally, suffer from disproportionately high rates of diabetes. A comprehensive evidence-based approach was therefore developed to study potential antidiabetic medicinal plants stemming from Canadian Aboriginal Traditional Medicine to provide culturally adapted complementary and alternative treatment options. Key elements of pathophysiology of diabetes and of related contemporary drug therapy are presented to highlight relevant cellular and molecular targets for medicinal plants. Potential antidiabetic plants were identified using a novel ethnobotanical method based on a set of diabetes symptoms. The most promising species were screened for primary (glucose-lowering) and secondary (toxicity, drug interactions, complications) antidiabetic activity by using a comprehensive platform of in vitro cell-based and cell-free bioassays. The most active species were studied further for their mechanism of action and their active principles identified though bioassay-guided fractionation. Biological activity of key species was confirmed in animal models of diabetes. These in vitro and in vivo findings are the basis for evidence-based prioritization of antidiabetic plants. In parallel, plants were also prioritized by Cree Elders and healers according to their Traditional Medicine paradigm. This case study highlights the convergence of modern science and Traditional Medicine while providing a model that can be adapted to other Aboriginal realities worldwide.
PMCID: PMC3247006  PMID: 22235232
16.  In Vivo Anti-Diabetic Activity of the Ethanolic Crude Extract of Sorbus decora C.K.Schneid. (Rosacea): A Medicinal Plant Used by Canadian James Bay Cree Nations to Treat Symptoms Related to Diabetes 
A number of potential anti-diabetic plants were identified through an ethnobotanical survey of the traditional pharmacopeia of the Cree of Eeyou Istchee (CEI—Northeastern Canada) used against symptoms of diabetes and their biological activity assessed by in vitro bioassays. Among these, Sorbus decora C.K.Schneid. (Rosacea) ranked highly and increased the transport of glucose in skeletal muscle cells in culture. The present study thus aimed at confirming the antidiabetic potential of S. decora in in vivo models of insulin resistance and diabetes, notably the streptozotocin Type 1 diabetic rat (STZ), the genetic KK-Ay Type 2 diabetic mouse and the rat rendered insulin resistant with 10% glucose water consumption for 6 weeks. Sorbus decora ethanolic crude extract (SDEE) was administered orally (200 mg kg−1) and compared to metformin (150 or 500 mg kg−1). The intragastric (i.g.) gavage of SDEE transiently decreased glycemia in STZ rats in a bi-phasic manner but the effect was cumulative over several days. In KK-Ay mice, SDEE incorporated in food (0.12%) decreased glycemia by 15% within 1 week as compared to vehicle controls. In pre-diabetic insulin-resistant rats, SDEE fed daily by i.g. gavage for 2 weeks significantly decreased the slight hyperglycemia and hyperinsulinemia, without affecting sugar water intake. Using the HOMA insulin resistance parameter, the effect of SDEE was equivalent to that of metformin. In conclusion, the ethanolic crude extract of S. decora demonstrates both anti-hyperglycemic and insulin-sensitizing activity in vivo, thereby confirming anti-diabetic potential and validating CEI traditional medicine.
PMCID: PMC3163474  PMID: 19887507
17.  Combined analysis of transcriptome and metabolite data reveals extensive differences between black and brown nearly-isogenic soybean (Glycine max) seed coats enabling the identification of pigment isogenes 
BMC Genomics  2011;12:381.
The R locus controls the color of pigmented soybean (Glycine max) seeds. However information about its control over seed coat biochemistry and gene expressions remains limited. The seed coats of nearly-isogenic black (iRT) and brown (irT) soybean (Glycine max) were known to differ by the presence or absence of anthocyanins, respectively, with genes for only a single enzyme (anthocyanidin synthase) found to be differentially expressed between isolines. We recently identified and characterized a UDP-glycose:flavonoid-3-O-glycosyltransferase (UGT78K1) from the seed coat of black (iRT) soybean with the aim to engineer seed coat color by suppression of an anthocyanin-specific gene. However, it remained to be investigated whether UGT78K1 was overexpressed with anthocyanin biosynthesis in the black (iRT) seed coat compared to the nearly-isogenic brown (irT) tissue.
In this study, we performed a combined analysis of transcriptome and metabolite data to elucidate the control of the R locus over seed coat biochemistry and to identify pigment biosynthesis genes. Two differentially expressed late-stage anthocyanin biosynthesis isogenes were further characterized, as they may serve as useful targets for the manipulation of soybean grain color while minimizing the potential for unintended effects on the plant system.
Metabolite composition differences were found to not be limited to anthocyanins, with specific proanthocyanidins, isoflavones, and phenylpropanoids present exclusively in the black (iRT) or the brown (irT) seed coat. A global analysis of gene expressions identified UGT78K1 and 19 other anthocyanin, (iso)flavonoid, and phenylpropanoid isogenes to be differentially expressed between isolines. A combined analysis of metabolite and gene expression data enabled the assignment of putative functions to biosynthesis and transport isogenes. The recombinant enzymes of two genes were validated to catalyze late-stage steps in anthocyanin biosynthesis in vitro and expression profiles of the corresponding genes were shown to parallel anthocyanin biosynthesis during black (iRT) seed coat development.
Metabolite composition and gene expression differences between black (iRT) and brown (irT) seed coats are far more extensive than previously thought. Putative anthocyanin, proanthocyanidin, (iso)flavonoid, and phenylpropanoid isogenes were differentially-expressed between black (iRT) and brown (irT) seed coats, and UGT78K2 and OMT5 were validated to code UDP-glycose:flavonoid-3-O-glycosyltransferase and anthocyanin 3'-O-methyltransferase proteins in vitro, respectively. Duplicate gene copies for several enzymes were overexpressed in the black (iRT) seed coat suggesting more than one isogene may have to be silenced to engineer seed coat color using RNA interference.
PMCID: PMC3163566  PMID: 21801362
18.  Fataluku medicinal ethnobotany and the East Timorese military resistance 
An ethnobotanical study of medicinal and poisonous plants used by the East Timor resistance was undertaken in the Lautem District of East Timor to study medicinal plant use in the region. Interviews were conducted with a single key consultant from the resistance army who belonged to the Fataluku culture. This study is of importance as a historical document and because no previous medicinal ethnobotanical studies on this region exist.
A rapid ethnobotanical survey of medicinal and poisonous plants was conducted through the proposed Conis Santana National Park in the Lautem district of East Timor. Medicinal and poisonous plants were identified by a Consultant and data was collected by the authors using classical descriptive ethnobotanical techniques (i.e. no quantitative measures) through an unstructured open ended interview.
During the survey 40 medicinal and poisonous plants were identified by the Consultant and collected by the authors. Defining characteristics of the Consultant's knowledge include a high frequency use of trees, heavily forested habitats, leaves, decoctions and drinks for a range of conditions relevant to a resistance army.
Despite limitations of the study, important contributions of this study include preservation of a part of the cultural history of the resistance movement and traditional botanical knowledge of the Fataluku. Furthermore, initial findings may indicate that traditional botanical knowledge is unique amongst different East Timorese cultures in terms of plant selection.
PMCID: PMC1796860  PMID: 17241473

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