Growth environment and organ specific variation in in-vitro cytoprotective activities of Picea mariana in PC12 cells exposed to glucose toxicity: a plant used for treatment of diabetes symptoms by the Cree of Eeyou Istchee (Quebec, Canada)
verfasst von:
Ashleigh D. Downing, Hoda M. Eid, Andrew Tang, Fida Ahmed, Cory S. Harris, Pierre S. Haddad, Timothy Johns, John T. Arnason, Steffany A. L. Bennett, Alain Cuerrier
The Cree of Eeyou Istchee (James Bay area of northern Quebec) suffer from a high rate of diabetes and its complications partly due to the introduction of the western lifestyle within their culture.
As part of a search for alternative medicine based on traditional practice, this project evaluates the biological activity of Picea mariana (Mill.) Britton, Sterns & Poggenb. needle, bark, and cone, in preventing glucose toxicity to PC12-AC cells in vitro (a diabetic neurophathy model) and whether habitat and growth environment influence this activity.
Methods
Three different organs (needle, bark, and cone) of P. mariana were collected at different geographical locations and ecological conditions and their 80% ethanolic extracts were prepared. Extracts were then tested for their ability to protect PC12-AC cells from hyperglycaemic challenge at physiologically relevant concentrations of 0.25, 0.5, 1.0 and 2.0 μg/mL. Folin-Ciocalteu method was used to determine the total phenolic content of P. mariana extracts.
Results
All extracts were well-tolerated in vitro exhibiting LD50 of 25 μg/mL or higher. Extracts from all tested organs showed a cytoprotective concentration-dependent response. Furthermore, the cytoprotective activity was habitat- and growth environment-dependent with plants grown in bog or forest habitats in coastal or inland environments exhibiting different cytoprotective efficacies. These differences in activity correlated with total phenolic content but not with antioxidant activity. In addition, this paper provides the first complete Ultra-Performance Liquid Chromatography-quadrupole time-of-flight (UPLC-QTOF) mass spectrometry analysis of Picea mariana’s bark, needles and cones.
Conclusions
Together, these results provide further understanding of the cytoprotective activity of Canadian boreal forest plants identified by the Cree healers of Eeyou Istchee in a cell model of diabetic neuropathy. Their activity is relevant to diabetic peripheral neuropathic complications and shows that their properties can be optimized by harvesting in optimal growth environments.
Begleitmaterial
Additional file 1:Figure S1. (A-T). Comparison of cytoprotective, mitogenic and cytotoxic activities of P. mariana pooled needle extracts from forest and bog populations in a PC12-AC model of high glucose stress. Populations are listed in ascending order from coastal west [1] to inland east [21] with forest (F) and bog (B) populations next to one another for comparison. Organ type is specified as the last number in the code; needle (N). Therefore, each graph has a code identifying population number, habitat type and organ type above it in that order. Bioactivity was assessed using the formazan dye WST which measures mitochondrial dehydrogenase activity. Treatment wells containing extract were standardized to the normoglucose control (C) and compared to this and the high glucose control (HG) for the determination of protective, mitogenic or toxic effects. A students t-test was used in order to determine the significant difference between the normoglucose control (100%) and the high glucose control (56.799%, n = 54 wells/condition, bar graph, p ≤ 0.05). Anova analysis was employed in order to compare the % viability for each concentration 0.25, 0.50, 1.00 and 2.00 μg/mL to the normoglucose and high glucose controls. Differences were deemed significant (*cytoprotection, #mitogenic) when p ≤ 0.05 (n = 3 treatment wells/concentration). (PDF 973 kb)
Additional file 2:Figure S2. (A-I). Comparison of cytoprotective, mitogenic and cytotoxic activities of P. mariana pooled bark extracts from forest and bog populations in a PC12-AC model of high glucose stress. Populations are listed in ascending order from coastal west [1] to inland east [14] with forest (F) and bog (B) populations next to one another for comparison. Organ type is specified as the last number in the code; bark (B). Therefore each graph has a code identifying population number, habitat type and organ type above it in that order. Bioactivity was assessed using the formazan dye WST which measures mitochondrial dehydrogenase activity. Treatment wells containing extract were standardized to the normoglucose control (C) and compared to this and the high glucose control (HG) for the determination of protective, mitogenic or toxic effects. A students t-test was used in order to determine the significant difference between the normoglucose control (100%) and the high glucose control (56.799%, n = 54 wells/condition, bar graph, p ≤ 0.05). Anova analysis was employed in order to compare the % viability for each concentration 0.25, 0.50, 1.00 and 2.00 μg/mL to the normoglucose and high glucose controls. Differences were deemed significant (*cytoprotection, #mitogenic) when p ≤ 0.05 (n = 3 treatment wells/concentration). (PDF 476 kb)
Additional file 3:Figure S3. (A-F). Comparison of cytoprotective, mitogenic and cytotoxic activities of P. mariana pooled cone extracts from bog populations in a PC12-AC model of high glucose stress. Populations are listed in ascending order from coastal west [3] to inland east [15] Habitat is the second letter in the code (bog = B) while Organ type is specified as the last number in the code (cone = C). Therefore each graph has a code identifying population number, habitat type and organ type above it in that order. Bioactivity was assessed using the formazan dye WST which measures mitochondrial dehydrogenase activity. Treatment wells containing extract were standardized to the normoglucose control (C) and compared to this and the high glucose control (HG) for the determination of protective, mitogenic or toxic effects. A students t-test was used in order to determine the significant difference between the normoglucose control (100%) and the high glucose control (56.799%, n = 54 wells/condition, bar graph, p ≤ 0.05). Anova analysis was employed in order to compare the % viability for each concentration 0.25, 0.50, 1.00 and 2.00 μg/mL to the normoglucose and high glucose controls. Differences were deemed significant (*cytoprotection, #mitogenic) when p ≤ 0.05 (n = 3 treatment wells/concentration). (PDF 171 kb)
Ashleigh D. Downing and Hoda M. Eid contributed equally to this work.
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Abkürzungen
ATCC
American Type Culture Collection
DPPH
1,1-diphenyl-2-picryl-hydrazyle radical
SIV
Syndromic Importance Value
UPLC-QTOF MS
Ultra-Performance Liquid Chromatography-quadrupole time-of-flight mass spectrometry
Background
In recent years, there has been increasing recognition of the deteriorating health status of Canadian First Nation peoples with respect to chronic diseases [1, 2]. Among the chronic diseases afflicting these populations, diabetes has stood out as a literal epidemic [3‐5]. Low levels of compliance due to treatment incompatibility have led to high levels of severe complications in these populations [3, 6‐8].
Picea mariana (Mill.) Britton, Sterns & Poggenb., commonly known as black spruce or Inaatuk in Cree has been used as a folk remedy for skin and soft tissues conditions and as analgesic [9‐11]. Poultice of inner bark has been applied for infected inflammations [10]. Infusion of bark has been used for pain relief [9] and the gum soaked in hot water has been used in burn dressing or smeared on painful areas of the body [11]. Decoction of needles has been applied to cure skin sores and to speed wound healing [12].
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The proposed solution to the rejection of western medication, as part of the CIHR Team in Aboriginal Antidiabetic Medicines (TAAM), is to make traditional treatment options readily available alongside conventional treatment [7, 13]. To accomplish this goal, semi-structured ethnobotanical surveys have been conducted in Misstisini, Whapmagoostui [14, 15], Waskaganish and Nemaska [16]. Using the Syndromic Importance Value (SIV) calculation, P mariana emerged in ethnobotanical surveys as a top ranking plant [15]. Specifically, it is mentioned as a possible treatment for symptoms related to diabetic neuropathy (pain, loss of sensation which increases risk of wounds to the extremities, slow-healing wounds and greater risk of wound infections due to weakened immune response) [14, 15].
To date, only antidiabetic activities of a cone extract have been investigated. The extract showed high antioxidant activity, insulin sensitizing and glitazone-like effects. It also conferred protection to PC12 cells, a model of peripheral neuronal precursors from high glucose insult [14, 17, 18]. Given these data and that extracts from needles of Picea glauca (Moench) Voss (a closely related species) also protect PC12 cells from hyperglycemic challenge [19], we compared the cytoprotective efficacy of extracts prepared from three different organs (needle, bark and cone) of P. mariana collected at different geographical locations and ecological conditions. Our primary goal was to establish whether cytoprotective activity was dependent upon habitat and growth environment and to elucidate what the ideal collection conditions might be for this plant. Here we describe how cytoprotective and mitogenic activities differ between extracts sampled from coastal and inland populations (Waskaganish to Mistissini) in areas where bog (low land) meets forest (high land). Six of nine Eeyou Istchee communities across a varied northern Quebec landscape have already participated in ethnobotanical studies that may lead to the incorporation of traditional pharmacopeia into clinics. Communities are situated inland (Mistissini, Nemaska, Oujé-Bougoumou) or on the coast of James or Hudson Bay (Waskaganish, Wemindji, and Whapmagoostui). The evaluation of local harvesting is essential to determine whether plant organs will yield similar benefits under different growing conditions, knowing especially that this region is vast. The development of this research question has come directly from the elders of Eeyou Istchee who emphasized the importance of plant selection and regional variation.
Methods
Plant material
P. mariana organs (needle, bark and cone) were harvested in proximity to the Cree communities of Waskaganish, Nemaska and Mistissini located within the 50° and 51° N latitudes (Quebec, Canada). Twenty-one populations were selected (Fig. 1). Plants were harvested at the boundaries of high land (forest) and low land (bog) to enable collection from two distinct habitats (bog or forest) within the same geographical growth environment (measured as proximity to the James Bay coast). Growth environment was considered coastal if it was < 50 km from the coast and inland if further. To ensure that habitats were distinct, bog stands were defined as trees growing in > 30 cm sphagnum moss. Forests were defined as having trees growing in organic soil with < 20 cm sphagnum moss ground cover. Separate populations had to be at least 5 km apart and in most cases were much further in order to sample across all three communities. Needles and bark were harvested from 10 trees within a 50 m radius for each habitat. Cones were collected from the same trees when available however, they were often unreachable or not present. All bulk materials were air dried upon collection and dried to completion in a plant drier at 40 °C prior to shipment. All samples were collected and dried within a few weeks. The dried samples were extracted at the University of Ottawa. Voucher specimens were deposited at the Marie-Victorin Herbarium (MT) located at the Jardin botanique de Montréal (PM001 to PM040).
Fig. 1
Map representing plant sampling locations (white number in black circle). The markers represent both the high and low land locations since they were next to each other. For example the circle labelled “3” represents bog and forest populations. At this location materials for B3N, B3B, B3C, F3N, F3B and F3C were collected. Cree communities are represented by a ▲ symbol. This map was simplified using photoshop, from “The Far North Nunavik and James Bay” tourist map which was sourced from the Base de données géographiques et administratives du Québec (BDGA)
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Extract preparation
Twenty populations of needles were extracted. Pooled samples were made by weighing out 2 g of material from each tree (10x) resulting in a total of 2 g per location. The 2 g sample was placed in a magic bullet blender (Homeland Housewares) along with 20 mL of 80% ethanol (10 mL/g material) and collected in 50 mL Fisher falcon tubes. After blending, the sample was sonicated for 20 min and the first extraction mixture was stored at 4 °C for 1 week. The ethanol extract was centrifuged for 15 min at 1811 g and the liquid supernatant was removed by pipette and stored at 4 °C. Ten mL of fresh 80% ethanol was added to the pellet, sonicated and stored at 4 °C. The second supernatant was then removed and added in the same fashion as the first. Alcohol was then removed from the pooled supernatant using a Labconco Centrivap (Fisher Scientific, Ottawa, Canada) at 40 °C for 72 h. The alcohol-free extract was lyophilized using a freeze dryer (Edwards Pirani 501, Fisher Scientific, Ottawa, Canada) for 72 h. The result was a dry stable extract that was homogenized and stored in a freezer at − 20 °C. Nine of the 20 populations were included for the analysis of bark extracts. It was necessary in the case of bark and cone to grind samples in a Wiley mill (2 mm mesh) tree by tree before evenly pooling the populations. As before 2 g of the pooled sample was processed using the same protocol as for the needles. In the case of cones, however, only five extracts were prepared by pooling 4 trees per population due to a lack of material. A total of 64 different extracts were prepared (20 needle bog, 20 needle forest, 9 bark bog, 9 bark forest and 6 cone bog = 64).
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Cell culture and bioactivity assays
PC12-AC cells of the clonal derivate of the rat pheochromocytoma cell line (American Type Culture Collection (ATCC), Manassas, VA, USA) [20] were maintained in 10 cm dishes with 10 mL complete media (RPMI, 10% horse serum, 5% new born calf serum) at 37 °C with 5% atmospheric CO2. Complete media provided a normoglucose environment (11 mM glucose). Bioactivity was assessed as previously described [19, 21]. To elicit glucose toxicity (hyperglycemia), complete medium was replaced with this same serum-free media supplemented with 150 mM glucose and 0.025% Bovine Serum Albumin (BSA). Following 96 h (hyperglycemia assays) of treatment, the cell proliferation reagent WST-1 (Roche Diagnostics, Laval, Quebec, Canada) was added to each well and incubated for 60 min before spectrophotometric analysis at 420 nm (formazan) and 620 nm [22]. Cultures were compared to cell-free treatment media incubated for the same period. Cell number per well was calculated from standard curves derived from wells containing known cell densities. Standardization allowed data to be compared across replicates. For LD50 assessments, each extract was tested at eight different concentrations in quintuplicate over two separate experiments (n = 15 per concentration). For hyperglycemia assays, maximal test concentrations were at least one log concentration below their LD50 in normoglucose media. Each extract was tested at four different concentrations in a minimum of triplicate measures (n = 3–6). Data from control cultures were combined across plates (n = 54). Percent viability was calculated as follows: % viability = cell number (treatment well) / mean cell number(control). Assigned bioactivities (cytotoxic, cytoprotective, or mitogenic) based on this screen were validated by direct assessment of viable cell number using Trypan Blue hemocytometer counts.
DPPH antioxidant assay
Extracts were tested for their radical scavenging activity using the 1,1-diphenyl-2-picryl-hydrazyle radical (DPPH) test as in [23]. Ascorbic acid (AA) was used as a control to generate a standard curve for the calculation of its IC50: the concentration where 50% of the DPPH radical is quenched by AA. The following Least squares regression equation was used [\( {\hat{y}}_i-{y}_0=\frac{\sum \limits_i\left\{\left({x}_i-{x}_0\right)\left({y}_i-{y}_0\right)\right\}}{\sum \limits_i{\left({x}_i-{x}_0\right)}^2}\left({x}_i-{x}_0\right) \)] in order to determine the line of best fit for the IC50 calculation where y = absorbance at 517 nm and x = concentration in ppm. This method is employed over the traditional method of discerning the linear portion of the line visually and is a more accurate method of extrapolation. Pure catechin and epicatechin compound was used as positive controls for comparison to crude plant extract samples. Extracts identified as having bioactivity in both screening paradigms and following direct validation were tested to determine whether antioxidant activity correlated with cytoprotection or mitogenicity in hyperglycemic media. Three independent assays were run with samples tested in duplicate.
Total phenolics test
The Folin-Ciocalteu method was used as previously described [24], and later modified by Harris et al. (2008). A standard curve was constructed using dilutions of quercetin (Sigma Aldrich Co) at concentrations of 1.0, 0.75, 0.5, 0.25, 0.10, and 0.05 mg/mL. Eight hundred micro liters of Folin reagent was added to 160 μL of sample with 540 μL 7.5% NaHCO3 and stored in the dark for 2 h. Absorbance was recorded at 725 nm using a spectrophotometer (Molecular Devices SpectraMax 5, Molecular Devices, Sunnyvale, CA, USA). Fifteen milligrams of crude extract was re-solubilized in 80% ethanol at 10 mg/mL inside 1.5 mL centrifuge tubes. The samples were then vortexed until totally dissolved and sonicated for 15 min. These extracts were dyed using the same procedure and the concentration determined using the standard curve.
Ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF)
Samples were prepared by dissolving 40 mg of crude extract in 1 mL of methanol (Sigma Aldrich, St Louis, MO, USA). A standard of 600 μL of resveratrol (1 mg/mL) (Sigma) was added to 500 μL of extract to make a total volume of 1100 μL. The mixture was filtered through a 0.2 μm PTFE nylon membrane filter (Chromatographic Specialties Inc., Brockville, ON, Canada) and sonicated for 5 min before injection.
Analyses were undertaken on an Acquity UPLC coupled with XevoG2 QTOF system (Waters Inc., Milford, MT, USA). UPLC analyses were performed on a Waters Acquity system. Separations were performed on a BEH C18 1.7 μm, 2.1 × 100 mm column (part #186002352; serial #02113226415705, LANSET# General Purpose 2.1 × 100 BEH) connected with a VanGuard pre-column 2.1 × 5 mm. Mobile phases: A, water+ 0.1% formic acid, and B-acetonitrile + 0.1% formic acid (Fisher Optima LC-MS). Flow rate 0.8 ml/min (back pressure at starting conditions = 10,000 PSI). Column temperature, 65 °C, sample temperature 4 °C. Mobile phase composition of B, 0–2 min 2% B isocratic, 2–5 min linear gradient 2–100% B, 5–6 min 100% B isocratic, 6–8 min return to initial mobile phase composition of 2% B isocratic. A 5 μL injection was performed through a 10 μL loop followed by strong wash 200 μL (50% acetonitrile+ 50% water) and weak wash 600 μL (10% acetonitrile + 90% water). Q-TOF was operated in positive and negative electrospray ionization modes. MassLynx software (Version 4.1) was used to acquire high and low energy spectra in MSe ESI+ and MSe ESI- modes within the mass range of 100–1500 Da. Lock mass was set with Leucine Enkephalin C12 at 556.2615 Da [M + H]+ 1 and 554.261 Da [M-H]− 1, source and desolvation temperatures were 150 °C and 500 °C respectively. Cone gas and desolvation gas (nitrogen) were set at 50 and 1200 L/hr). The identification of the compounds is based on matching the molecular ion acquired in low fragmentation setting and the major fragments acquired in high fragmentation setting within the mass accuracy of 5 PPM.
Results
Percentage yield
Upon extraction, 18, 25 and 30% of dried needles, bark and cone were sequestered.
Bioactivity
LD50 values for all extracts were 25 μg/mL or higher. Consequently, each extract was screened at 0.25, 0.5, 1 and 2 μg/mL for capacity to alter glucotoxicity in vitro (Tables 1, 2, 3, Figs. 2, 3, Additional files 1-3: Figures S1-S3). Maximal concentrations of extracts were at least one log concentration below their LD50 in normoglucose media. Glucotoxicity (high glucose, HG) reduced the number of viable cells to 56.8% that of normoglucose controls (C) (Figs. 2 and 3). Samples were stated as biologically active if at least two or more concentrations elicited a statistically significant change in viable cell number. Compounds that increased viable cell number to values significantly higher than the normoglucose controls (> 100%) were considered mitogenic. Compounds that significantly protected cells from high glucose toxicity (56.8%) without exceeding normoglucose viability values were considered cytoprotective; compounds that enhanced glucotoxicity at any concentration were considered cytotoxic (Tables 1, 2, 3).
Table 1
Bioactivity of Picea mariana needle extracts in PC12-AC grown in hyperglycemic media
Extract IDa
Bioactivity in hyperglycemic mediab
1FN
Cytoprotective
1BN
None
2FN
None
2BN
None
3FN
None
3BN
None
4FN
None
4BN
None
5FN
None
5BN
None
6FN
Cytoprotective
6BN
Cytoprotective
7FN
None
7BN
Cytoprotective
8FN
Mitogenic
8BN
Mitogenic
9FN
None
9BN
Cytoprotective
10FN
Cytoprotective
10BN
None
11FN
None
11BN
None
13FN
None
13BN
None
14FN
None
14BN
None
15FN
None
15BN
None
16FN
Cytoprotective
16BN
None
17FN
Cytoprotective
17BN
Cytoprotective
18FN
Cytoprotective
18BN
Cytoprotective
19FN
None
19BN
Cytoprotective
20FN
None
20BN
None
21FN
Cytoprotective
21BN
None
aThe extract ID code defines the growth environment (Area#1–21, Fig. 1), habitat (B Bog or F forest) and organ (N needle). For example, 1FN is an extract prepared from needles collected in a forest habitat at location #1. Populations are listed in ascending order from coastal west (1) to inland east (21) with forest (F) and bog (B) populations next to one another for comparison
bBioactivity was classified comparing a 96 h treatment in hyperglycemic (150 mM) serum-free media. Viable cell number following extract treatment was established using the WST-1 assay compared to standard curves of known cell number. Vehicle control was 0.1% DMSO. Extracts that at two or more concentrations increased viable cell number to values significantly higher than the normoglucose controls (> 100%) were classified as mitogenic, those which significantly protected cells from high glucose toxicity without apparent mitogenic activity were classified as cytoprotective, and those which enhanced glucotoxicity were classified as cytotoxic. Statistics were ANOVA, post-hoc Tukey tested vs. vehicle-treated cultures in normo- or high glucose media. All concentration-response data and statistical analyses are presented in Additional file 1: Figure S1
Table 2
Bioactivity of Picea mariana bark extracts in PC12-AC grown in hyperglycemic media
Extract IDa
Bioactivity in hyperglycemic mediab
1FB
Mitogenic
1BB
Mitogenic
2FB
Cytoprotective
2BB
Cytoprotective
3FB
Cytoprotective
3BB
Cytoprotective
4FB
Cytoprotective
4BB
Cytoprotective
5FB
None
5BB
None
6FB
Cytoprotective/Mitogenic
6BB
Cytoprotective/Mitogenic
7FB
Cytoprotective
7BB
Cytoprotective
11FB
None
11BB
Cytoprotective
14FB
None
14BB
None
aThe extract ID code defines the growth environment (Area#1–21, Fig. 1), habitat (B Bog or F forest) and organ (B bark). For example, 1FB is an extract prepared from bark collected in a forest habitat at location #1
bBioactivity was classified using a 96 h treatment in hyperglycemic (150 mM) serum-free media. Viable PC12-AC cell number following extract treatment was established using the WST-1 assay compared to standard curves of known cell number. Vehicle control was 0.1% DMSO. Extracts that at two or more concentrations increased viable cell number to values significantly higher than the normoglucose controls (> 100%) were classified as mitogenic, those which significantly protected cells from high glucose toxicity without apparent mitogenic activity were classified as cytoprotective, and those which enhanced glucotoxicity were classified as cytotoxic. Samples classified with dual bioactivities (ie. Cytoprotective/Mitogenic) are possible when two concentrations satisfy our definition of one bioactivity (ie. Cytoprotective) and two other concentrations from the same sample are in another category (ie. Mitogenic). Statistics were ANOVA, post-hoc Tukey tested vs. vehicle-treated cultures in normo- or high glucose media. All concentration-response data and statistical analyses are presented in Additional file 2: Figure S2
Table 3
Bioactivity of Picea mariana cone extracts in PC12-AC grown in hyperglycemic media
Extract IDa
Bioactivity in hyperglycemic mediab
3BC
Cytoprotective
7BC
Cytoprotective
11BC
Mitogenic
12BC
Mitogenic
14BC
None
15BC
None
aThe extract ID code defines the growth environment (Area#1–21, Fig. 1), habitat (B Bog or F forest) and organ (C cone). For example, 1BC is an extract prepared from cones collected in a bog habitat at location #1
bBioactivity was classified comparing a 96 h treatment in hyperglycemic (150 mM) serum-free media. Viable cell number following extract treatment was established using the WST-1 assay compared to standard curves of known cell number. Vehicle control was 0.1% DMSO. Extracts that at two or more concentrations increased viable cell number to values significantly higher than the normoglucose controls (> 100%) were classified as mitogenic, those which significantly protected cells from high glucose toxicity without apparent mitogenic activity were classified as cytoprotective, and those which enhanced glucotoxicity were classified as cytotoxic. Statistics were ANOVA, post-hoc Tukey tested vs. vehicle-treated cultures in normo- or high glucose media. All concentration-response data and statistical analyses are presented in Additional file 3: Figure S3
Fig. 2
Comparison of cytoprotective and mitogenic activities of P. mariana needle (●), bark (■) and cone (▲) extracts in PC12-AC grown in hyperglycemic media. MANOVA analysis was used to average all extracts based on their organ type and concentration. Significant cytoprotection (*) or mitogenic activity (#) was afforded when Dunnett post hoc tests were p ≤ 0.05 as compared to the normoglucose (C) and high glucose (HG) controls. All organs showed significant protection at all concentrations except cone at 2 μg/mL (needle n = 120, bark n = 60, cone n = 18)
Fig. 3
Comparison of cytoprotective and mitogenic activities of P. mariana extracts on PC12-AC by growth environment. MANOVA analysis was used to determine the mean viability with respect to habitat (forest/ bog), growth environment (coastal/inland), organ type (Needle = ●, Bark = ■) and concentration (0.25, 0.50, 1.00, 2.00 μg/mL) interactions. Significant protection (*) or mitogenic activity (#) was identified by Dunnett post hoc tests (p ≤ 0.05) by comparison to the (C) normoglucose or (HG) high glucose controls respectively. The same p value was used to denote a significant difference between needle and bark (+) at specified concentrations. a Comparison of bark and needle extract protection in coastal forest environments (bark n = 12, needle n = 12). b Comparison of bark and needle extract protection in coastal bog environments (bark n = 12, needle n = 12). c Comparison of bark and needle extract protection in inland forest environments (bark n = 18, needle n = 51). d Comparison of bark and needle extract protection in inland bog environments (bark n = 18, needle n = 45)
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×
We found that 50% of all organ extracts improved viability of PC12 cultures under hyperglycemic conditions (Tables 1, 2, 3, Additional files 1-3: Figures S1-S3). Moreover, all extracts were well-tolerated in that none of them enhanced glucose cytotoxicity. We identified 24 cytoprotective (38% of total) and seven mitogenic (9% of total) extracts. Two extracts (3% of total) exhibited both concentration-dependent cytoprotective and mitogenic activities. When comparing the overall bioactivity of extracts prepared from needle, bark and cone collected in all regions and habitats, we found that protective effects were maximal at the lowest tested concentrations in all cases and were not organ-specific with the exception of bark and cone which were more effective mitogens in high glucose media than needle extracts (Tables 1, 2, 3, Additional files 1-3: Figures S1-S3, Fig. 2).
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Regional variation
A primary concern of the TAAM team is to provide laboratory data assessing the importance of plant selection and regional variation emphasized by the elders and healer of the Eeyou Istchee in their traditional preparations. Their insights are crucial to ensuring that plant usage will yield similar benefits in all communities. We found that 50 % of P. mariana needle, bark, and cone extracts (32 extracts) did not exhibit significant biological activity in our screening paradigm. To test whether extract efficacy depended upon the growth habitat where the plant had been harvested, we compared efficacy of needle and bark extracts collected in bog and forest habitats. Cone extracts were not included in this analysis as we were only able to collect cones from bog sites.
We did not detect any statistically significant differences between the bioactivities of needle and bark extracts grown in different habitats (data not shown). To test whether growth environment and habitat interacted to produce organs with different bioactivities, samples collected in different growth habitats were grouped into coastal or inland environments (Fig. 1). This analysis revealed a statistically significant interaction among growth environment, habitat, and organ bioactivity (Fig. 3). Extracts prepared from bark collected in coastal environments showed cytoprotection where needles extracts did not (Fig. 3a and b). Moreover, in coastal-derived extracts, activity was maximal (and concentration-dependent) at low concentrations in extracts prepared from bark collected in a forest habitat (Fig. 3a) and at high concentrations in extracts prepared from bark collected in a bog habitat (Fig. 3b). At their highest percent of cell viability, extracts were even mitogenic. This interaction was organ-specific. Neither growth environment nor habitat altered the cytoprotective efficacy of needle extracts (Fig. 3a-d).
Total phenolics and antioxidant activity
These data suggest that the amounts of biologically active compounds are responsible for organ-specific, habitat-dependent cytoprotective bioactivities of the extracts. It has previously been suggested [17] that antioxidant activity is responsible for antidiabetic activities in P. mariana cone extracts. To further explore this hypothesis, we measured total phenolic content in the needle, bark and cone extracts (Fig. 4) and correlated this content with the antioxidant activity of selected extracts using the Folin-Ciocalteu total phenolics method and DPPH antioxidant tests employed in previous TAAM studies [17].
Fig. 4
Total phenolic content in terms of quercetin equivalents, of P. mariana needle, bark and cone extracts. Needle, bark and cone phenolic content was found to be significantly different from each other at p ≤ 0.05 ((*) = significantly different from needle; (#) = significantly different from bark; (%) = significantly different from cone). Cone had the highest phenolic content with 398.68 (SE = 48.77, n = 5), followed by bark at 244.08 (SE = 14.51, n = 18) and needle with 146.34 μg/mg extract (SE = 3.78, n = 40)
×
We found that the combined phenolic content was highest in cone extracts and lowest in needle. For each milligram of extract, 146.5 ± 4.2 μg (needles), 270.3 ± 13.9 μg (bark) and 398.7 ± 48.8 l μg (cone) were phenolic compounds (Fig. 4).
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PH antioxidant assays were completed on 14 selected extracts and plotted with respect to phenolic content (Fig. 5). A significant correlation between antioxidant activity and total phenolic content was detected (r2 = 0.63, p < 0.05, Fig. 5).
Fig. 5
Regression analysis of total phenolic content of P. mariana extracts with respect to antioxidant activity. Total phenolic content is measured in terms of quercetin equivalents and antioxidant activity is calculated with respect to the ascorbic acid standard curve. The trend shows that as phenolic content increases the amount of plant extract (EC50 in ppm) necessary to reach the IC50 of ascorbic acid decreases and therefore, antioxidant activity increases with increasing phenolic content (r2 = 0.63, p ≤ 0.05, n = 14). The data passed a normality test in order to ensure that it follows a normal distribution
×
Total phenolic content positively correlated with bioactivity (r2 = 0.30, p = 0.05, Fig. 6, Tables 1, 2, 3). Antioxidant activity, however, was not correlated with bioactivity (r2 = 0.046, p = 0.460, Fig. 7). Taken together, these results suggest that, while the phenolic content of organ extracts prepared from plants harvested in different habitats and growth environments likely underlies bioactivity, the oxyradical scavenging capacity of these phenolics is not the primary mechanisms of action.
Fig. 6
Regression analysis of total phenolic content of of P. mariana extracts with respect to bioactivity in PC12-AC in high glucose media. The trend shows that as phenolic content increases the percent viability increases (p ≤ 0.05, n = 13)
Fig. 7
Regression analysis of antioxidant activity of P. mariana extracts with respect to PC12-AC bioactivity in high glucose media. Antioxidant activity is measured with respect to ascorbic acid and bioactivity is represented as percent viability with respect to normoglucose control. We do not see any trend; therefore, the antioxidant activity is not likely the main mechanism of protection (p = 0.89, n = 13)
×
×
UPLC-QTOF analyses
Analyses of the methanol extracts of needle, bark and cone extracts were completed by Dr. Ammar Saleem at the LANSET core facility, University of Ottawa and yielded the identification of two classes of compounds – phenolics (Additional file 4: Figure S4-a) and terpenes (Additional file 4: Figure S4-b). Using the natural products data base that we have created, a total of 32 phenolic and terpene compounds were detected in P mariana needle, bark and cone extracts (Table 4, Additional file 5: Figure S5a-c). All plant parts contained proanthocyanidins b6 and b3, as well as abietic acid, dehyroabietic acid and oxodehydroabietic acid. Eleven compounds were confirmed as present in needles, with an additional 10 tentatively compounds and four in trace quantities. Kaemferol-7-O- glucoside was unique to the needles. The bark contained 8 confirmed compounds and 6 tentatively present and 2 in trace quantities with picealactone b as a unique component. The cones have not been previously analysed and revealed 10 confirmed compounds, 7 tentatively present and 5 trace compounds. Two significant compounds here are resveratrol, with antioxidant, antidiabetic and other biological activities [25], as well as taxifolin, also an antioxidant but structurally related to quercetin, which has previously been reported to protect against oxidative stress induced apoptosis [26]. Additional information on the new UNIFI based identification system used here and based on diagnostic low and high energy spectra will be published elsewhere.
Table 4
List of compounds in P. mariana needle, bark and cone extracts identified by ESI-QTOF positive electrospray ionization. C (Retention time match ±0.02 min, low and high energy spectra match within ±5 ppm mass accuracy); P (low and high energy spectra match within 5 ppm mass accuracy); T (signal intensity of quasi molecular ion below 1500 counts); A (absent)
#
Compound
Molecular formula
Accurate mass
[M + H]+ 1
Rt. (min)
Needle
Bark
Cone
Phenolics
1
3′,4′-Dihydroxyacetophenone
C8H8O3
152.0473
153.0553
2.71
C
C
A
2
4-Hydroxybenzoic acid
C7H6O3
138.0317
139.0396
2.66
C
T
T
3
4-Methylcatechol
C7H8O2
124.0524
125.0604
2.98
T
T
T
4
Coumarin
C9H6O2
146.0368
147.0447
2.95
C
C
A
5
Dihydroquercetin
C15H12O7
304.0583
305.0662
3.00
A
T
C
6
Isorhapontigenin
C15H14O4
258.0892
259.0971
3.11
T
A
C
7
Isorhapontigenin 3-O-beta-D-glucopyranoside
C21H24O9
420.1420
421.1500
3.12
A
A
P
8
Kaempferol
C15H10O6
286.0477
287.0557
3.08
A
A
C
9
Kaempferol 3-(3″-p-coumaryl-6″-ferulylglucoside)
C40H34O16
770.1847
771.1926
3.74
P
A
A
10
Kaempferol-7-O-glucoside
C21H20O11
448.1006
449.1085
2.94
C
A
A
11
Piceatannol
C14H12O4
244.0736
245.0815
2.91
C
A
C
12
Procyanidin B3
C30H26O12
578.1424
579.1504
3.92
C
C
C
13
Procyanidin B6
C30H26O12
578.1424
579.1504
2.65
C
C
C
14
Resveratrol
C14H12O3
228.0786
229.0866
3.04
T
A
C
Terpenes
15
Picealactone C
C20H22O4
326.1518
327.1597
3.58
A
P
A
16
Isolariciresinol
C20H24O6
360.1573
361.1652
3.70
P
A
A
17
Picealactone A
C20H22O3
310.1569
311.1648
4.48
A
P
T
18
Dehydroabietic acid
C20H28O2
300.2089
301.2169
3.91
C
C
C
19
12-Hydroxydehydroabietic acid
C20H28O3
316.2038
317.2118
4.37
P
P
P
20
Abieta-8,11,13-trien-7-one
C20H28O
284.2140
285.2219
4.19
A
P
P
21
7-Oxodehydroabietic acid
C20H26O3
314.1882
315.1961
4.42
C
C
C
22
Abietic acid
C20H30O2
302.2246
303.2325
4.97
C
C
C
23
Sandaracopimaric acid
C20H30O2
302.2246
303.2325
4.52
C
C
A
24
Typhasterol
C28H48O4
448.3553
449.3632
5.20
P
A
A
25
(+)-Picealactone B
C20H22O4
326.1518
327.1597
3.15
P
A
A
26
(+)-Lariciresinol
C20H24O6
360.1573
361.1652
3.73
P
A
A
27
Gibberellin A1
C19H24O6
348.1573
349.1652
4.63
P
A
T
28
Gibberellin A15
C20H26O4
330.1831
331.1910
3.83
P
P
P
29
Gibberellin A51
C19H24O5
332.1624
333.1703
4.01
P
P
P
30
Gibberellin A29
C19H24O6
348.1573
349.1652
4.66
P
A
T
31
Gibberellin A7
C19H22O5
330.1467
331.1546
3.10
T
A
P
32
Neocembrene
C20H32
272.2504
273.2583
5.12
A
A
P
Bioactivity of taxifolin
To investigate whether taxifolin elicits cytoprotective effects and may contribute to the observed activity of cone extracts, a pure taxifolin standard (Sigma Aldrich, Oakville, ON) was administered at increasing non-toxic concentrations (0.8, 1.6 and 3.2 μM) to PC12 cells exposed to glucotoxicity (Additional file 6: Figure S6). Cytoprotection was significant at 1.6 μM with nearly identical concentration-dependent response as seen for quercetin [22].
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Discussion
There is a paucity of strategies effective in managing chronic pain, infection, paralysis, and loss of sensation in association with peripheral diabetic neuropathy [8, 27]. Based on our ethnobotanical studies, we have observed that preparations from various organs of P. mariana are used to treat slow healing infections, sores and numbness (particularly of the extremities), all symptoms related to diabetic neuropathy. We demonstrate for the first time that P. mariana needles and bark are effective protectors of peripheral neuronal precursor survival when exposed to hyperglycemic conditions. We confirm the protection of cones, which was first demonstrated by our team [17]. We have also shown that harvest location can impact the cytoprotective activities of P. mariana, particularly for bark extracts, which tended to be more effective when obtained from inland forest populations than from coastal and inland bog populations. In a similar fashion, [28, 29] showed a latitudinal trend in key phenolic concentration but failed to find a correlation with bioactivity. In general, the strength of the observed activity of needle, bark and cone is correlated with the total phenolic concentration of the extracts. The one notable exception was a cone extract that was suspected to have caused oxidation, a negative response, in PC12-AC cells. The presence of too many antioxidants can actually be harmful, resulting in oxidation and therefore a decrease in cell survival. This situation was observed in preliminary DPPH tests at higher extract concentrations [30].
These in vitro findings validate the importance of P. mariana’s role as part of the Cree pharmacopoeia and underline the importance of plant selection and extract strength. In a review of Canadian aboriginal plant use, [31], it has been reported that this plant is used by the Cree to treat many ailments including heart problems, high blood pressure as well as many infectious and inflammatory conditions. To TAAM’s ethnobotanical and in vitro findings [14, 15, 17, 32], we have contributed additional positive evidence that boreal plants from the Cree pharmacopoeia are capable of treating diabetes neuropathy symptoms. Specifically, we have shown at the cellular level, that this traditional medicine may be effective in the treatment of this “new” disease afflicting First Nation peoples, namely the potential to manage diabetic peripheral symptoms by protecting peripheral neurons and their precursors from hyperglycemic insult.
These results are similar to those found in Harris et al. (2008) for P. glauca, except that all P. mariana organs showed protection. In contrast to Harris et al. (2008) findings, P. mariana needles were generally the lowest protector against diabetic insult. One factor that should be re-examined in the case of P. glauca is concentration range. It may be necessary to test bark and cone at much lower concentrations than 10 μg/mL. This is recommended because our graphs often showed a decreasing dose response from the lowest concentration to the highest (Fig. 3). Conversely, given strong correlations in both studies with total phenolic content, these data could also suggest species differences in the phenolic constituents of the different organs that underlie the cytoprotective and mitogenic bioactivities. This is a good argument for the comprehensive fractionation-based assays of organs collected at different locations and under different growth habitats in order to identify active components.
We further show that extract efficacy depends upon careful selection of plant organs depending upon growth environment and habitat as indicated by the Cree elders and healers of Eeyou Istchee. This is an important finding as it supports the efforts of traditional practitioners in ensuring equivalent benefits across communities. Variation in activity by growth environment was observable mainly for bark. In this case, we recommend inland populations or, if harvesting in proximity to the coast, to choose forest sites instead of bog sites. Conveniently, this is easier ground to navigate and a less fragile habitat. However, if a bog is the only site available for harvesting, it may be possible to achieve the same level of protection in coastal sites, for example, by simply using more plant material per treatment preparation. In the case of needles, neither growth environment nor habitat altered the cytoprotective efficacy of extracts, so no special recommendations are warranted. A lack of specificity should allow collectors to avoid overharvesting in one area and, therefore, decreases the possibility of harmful environmental impacts.
Using UPLC-QTOF method, a total of 14 phenolics and 18 terpenes were detected or tentatively identified in needle, bark and cones extracts. Among these phenolics, piceatannol was detected in cone and needle extracts, yet absent from bark extract. On the other hand, the identities of resveratrol and isorhapontigenin were only confirmed in the cone extract. These three compounds belong to a group of compounds called stilbenes and have shown a wide range of biological activities such as antidiabetic, antioxidant, anti-aging, anti-cancer and anti-inflammatory activities [33‐36]. Of note, resveratrol, is the most studied stilbene and is a known cytoprotector found in red wine [37] while isorhapontigenin is a novel stilbene that was recently identified in red wine. Stilbenes were previously detected in a number of unrelated gymnosperm and angiosperm species such as Abies, Picea, Pinus, Juniperus, Rheum and Morus species [38]. Besides the properties or activities listed above, stilbenes have been shown to improve blood circulation and have antimicrobial activities [38, 39]. Another interesting phenolic group is the proanthocyanidins; they constitute a subclass of flavonoids that has attracted growing attention due to their potential health promoting effects [40]. Procyanidin B3 and Procyanidin B6 are detected in needle, bark and needle extracts. On the other hand, the flavanonols dihydroquercetin or taxifolin was only confirmed in the cone extract but may nonetheless contribute to cytoprotective effects elicited by these extracts (Additional file 5: Figure S5). Concerning terpenes, the conifer biomarkers abietic and dehydroabietic acids as well as 7-Oxodehydroabietic acid were detected in the extracts of the three plant parts while sandaracopimaric acid was only identified in the needle extract. Importantly, abietic acid and dehydroabietic acid were reported to exhibit a potent inflammatory activity and have antioxidant as well as antiobesity and regulation of glucose metabolism [41]. While still preliminary, this approach can identify the bioactive compounds that underlie species differences as well as reflect the observed growth environment and habitat impact upon extract efficiency.
Conclusion
P. mariana is an effective protector of peripheral neuronal precursor survival when cells are subjected to high glucose conditions. All organs tested in this diabetic neuropathy model (needles, bark and cone) proved to be efficacious. As all organ parts are protective and the boreal forest of Canada is highly populated by this tree species, we believe P. mariana is an excellent candidate for a renewable source of medicine. Further in vitro investigations are necessary to elucidate the underlying mechanisms of action and phenolic variation. In vivo tests using diabetes specific animal models are also needed to confirm that bioactivity remains upon ingestion. Of course, the successful application of this study’s findings will also depend on the continuation of an already interdependent relationship developed by the TAAM team, health care professionals and the Eeyou Istchee communities. As the bonds we have built are strong, we are confident that not only P. mariana but many of the plants from the Cree pharmacopeia will become part of a new holistic form of diabetes treatment, culturally acceptable to the Cree of Eeyou Istchee and supported by traditional practice and evidence-based inquiry.
Acknowledgements
We thank Research Associate Ammar Saleem, Lanset Facility University of Ottawa for the identification of compounds using the new UNIFI phytochemical database. Very special thanks are due to Josephine Diamond, Alice Jacob and Jimmy Trapper from Waskaganish First Nation, Isabel Wapachee, Louise Jolly Wapachee, Bessie Blackned, Annie Jolly, Charlie Jolly, Abel Jolly, Alice Ottereyes Jolly, Helen Jolly Metaweskum, Mary Jolly, Helen Jolly Wapachee and Edith Jolly Matoush from Nemaska First Nation, Smally Petawabano, Laurie Petawabano, Charlie Coon, Sophie Coon, Mabel Gunner, Abel Mark, Kathleen Mark, Elizabeth Coon Come, Herriett Matoush, Sandy Matoush, Rene Coon and Emma Coon Come from the Cree Nation of Mistissini and Abraham Mamianskum, Agnes Kawapit, Andrew Natachequan, Anne Sandy, Eliza George Mamianskum, Eliza Kawapit, James Kawapit, Jeanny Masty, John Petagumskum Sr., Lucy Rupert, Maggie Natachequan and Mathew Natachequan from Whapmagoostui First Nation as well as to 66 other Cree Elders from all four communities who kindly agreed to be interviewed but chose not to be specifically mentioned. They made this research possible by allowing us to use their knowledge relating to medicinal plants, transmitted to them by their elders. Their trust has also enabled a useful exchange between indigenous knowledge and western science. We thank Mr. Steven Rennick for his technical assistance.
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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Additional file 1:Figure S1. (A-T). Comparison of cytoprotective, mitogenic and cytotoxic activities of P. mariana pooled needle extracts from forest and bog populations in a PC12-AC model of high glucose stress. Populations are listed in ascending order from coastal west [1] to inland east [21] with forest (F) and bog (B) populations next to one another for comparison. Organ type is specified as the last number in the code; needle (N). Therefore, each graph has a code identifying population number, habitat type and organ type above it in that order. Bioactivity was assessed using the formazan dye WST which measures mitochondrial dehydrogenase activity. Treatment wells containing extract were standardized to the normoglucose control (C) and compared to this and the high glucose control (HG) for the determination of protective, mitogenic or toxic effects. A students t-test was used in order to determine the significant difference between the normoglucose control (100%) and the high glucose control (56.799%, n = 54 wells/condition, bar graph, p ≤ 0.05). Anova analysis was employed in order to compare the % viability for each concentration 0.25, 0.50, 1.00 and 2.00 μg/mL to the normoglucose and high glucose controls. Differences were deemed significant (*cytoprotection, #mitogenic) when p ≤ 0.05 (n = 3 treatment wells/concentration). (PDF 973 kb)
Additional file 2:Figure S2. (A-I). Comparison of cytoprotective, mitogenic and cytotoxic activities of P. mariana pooled bark extracts from forest and bog populations in a PC12-AC model of high glucose stress. Populations are listed in ascending order from coastal west [1] to inland east [14] with forest (F) and bog (B) populations next to one another for comparison. Organ type is specified as the last number in the code; bark (B). Therefore each graph has a code identifying population number, habitat type and organ type above it in that order. Bioactivity was assessed using the formazan dye WST which measures mitochondrial dehydrogenase activity. Treatment wells containing extract were standardized to the normoglucose control (C) and compared to this and the high glucose control (HG) for the determination of protective, mitogenic or toxic effects. A students t-test was used in order to determine the significant difference between the normoglucose control (100%) and the high glucose control (56.799%, n = 54 wells/condition, bar graph, p ≤ 0.05). Anova analysis was employed in order to compare the % viability for each concentration 0.25, 0.50, 1.00 and 2.00 μg/mL to the normoglucose and high glucose controls. Differences were deemed significant (*cytoprotection, #mitogenic) when p ≤ 0.05 (n = 3 treatment wells/concentration). (PDF 476 kb)
Additional file 3:Figure S3. (A-F). Comparison of cytoprotective, mitogenic and cytotoxic activities of P. mariana pooled cone extracts from bog populations in a PC12-AC model of high glucose stress. Populations are listed in ascending order from coastal west [3] to inland east [15] Habitat is the second letter in the code (bog = B) while Organ type is specified as the last number in the code (cone = C). Therefore each graph has a code identifying population number, habitat type and organ type above it in that order. Bioactivity was assessed using the formazan dye WST which measures mitochondrial dehydrogenase activity. Treatment wells containing extract were standardized to the normoglucose control (C) and compared to this and the high glucose control (HG) for the determination of protective, mitogenic or toxic effects. A students t-test was used in order to determine the significant difference between the normoglucose control (100%) and the high glucose control (56.799%, n = 54 wells/condition, bar graph, p ≤ 0.05). Anova analysis was employed in order to compare the % viability for each concentration 0.25, 0.50, 1.00 and 2.00 μg/mL to the normoglucose and high glucose controls. Differences were deemed significant (*cytoprotection, #mitogenic) when p ≤ 0.05 (n = 3 treatment wells/concentration). (PDF 171 kb)
Growth environment and organ specific variation in in-vitro cytoprotective activities of Picea mariana in PC12 cells exposed to glucose toxicity: a plant used for treatment of diabetes symptoms by the Cree of Eeyou Istchee (Quebec, Canada)
verfasst von
Ashleigh D. Downing Hoda M. Eid Andrew Tang Fida Ahmed Cory S. Harris Pierre S. Haddad Timothy Johns John T. Arnason Steffany A. L. Bennett Alain Cuerrier
