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Findings

In fifty patients with endometriosis and fifty age-matched controls confirmed by laparoscopy or laparotomy, whole blood samples were collected and digested using supra pure 65% HNO3. Whole blood levels of nickel and lead were measured using Total Reflection X-ray Fluorescence (TXRF) while cadmium levels were evaluated using graphite furnace atomic absorption spectroscopy (GFASS). Women with endometriosis had significantly higher (P=0.016) geometric mean (95% CI) whole blood nickel levels [2.6(1.9-3.3) μg/L] as compared to women without endometriosis [0.8 (0.7-0.9) μg/L]. Whole blood levels of cadmium and lead were similar between the two groups.

Methods

This case control study was conducted among women of the reproductive age group, at the Professorial Gynecology Unit of the National Hospital, Colombo, Sri Lanka. Patients awaiting elective laparotomy or laparoscopy for diagnostic and/or treatment purposes were included. Those who were diagnosed visually as having endometriosis subsequent to laparotomy or laparoscopy were selected as cases (n=50). Apart from the ectopic endometrial tissue sample that was collected as described in the previous study [], simultaneous venous blood sample was collected from each case. Women matched for age in whom endometriosis had been excluded by laparoscopy or laparotomy were recruited as controls (n=50). Indications for laparoscopy or laparotomy in controls were subfertility, dysmenorrhea, chronic pelvic pain or detection of an ovarian mass in ultrasound scan.

TNF-α, IL-6, and IL-8/CXCL1 cytokine levels

BEAS-2B cells were pretreated for 1 hour with 0–200 nM 7(S)-MaR1 or 7(R)-MaR1, followed by 5% HDE for 24 hours. Cell supernatants were collected and IL-6 and IL-8 enzyme-linked immunosorbent assays (ELISAs) were performed as previously described []. Alternatively, mouse lung slices were pre-treated for 1 hour with 0–200 nM MaR1, followed by 5% HDE treatment, or given 5% HDE treatment followed by 0–200 nM 7(S)-MaR1 1 hr after the HDE treatment was given. At 24 hours following HDE treatment, lung slice supernatants were collected and assayed for murine TNF-α, IL-6, and the murine IL-8 cognate CXCL1 using ELISAs.

MaR1 reduces the release of HDE-induced pro-inflammatory cytokines (IL-6 and IL-8) by airway epithelial cells (BEAS-2B cell line)

Previous studies have shown treatment of BECs with 5% HDE for 24 hours leads to significant increases in IL-6 and IL-8 release [,]. To test the effects of MaR1 pretreatment on cytokine release from BECs in response to HDE, BEAS-2B cells were pretreated with 0, 1, 10, 100, or 200 nM MaR1 (in all studies, the 7[S] form of MaR1 was used, unless otherwise stated). After 1 hr, 5% HDE was added to the cultures and allowed to incubate for 24 hours. In cells receiving MaR1 pretreatment, IL-6 and IL-8 cytokine levels were dose-dependently inhibited compared to cells receiving HDE alone, as measured at 24 hours post-HDE exposure (Figure ). In the absence of HDE, MaR1 alone had no effect on IL-6 or IL-8 levels at any concentration tested. These studies were repeated using the 7(R) isoform of MaR1, achieving similar results (data not shown).

Abstract

The present study deals with the preparation of polyurethane (PU) films impregnated with a nonsteroidal anti-inflammatory drug, tolfenamic acid (TA). Solvent evaporation technique has been employed for the preparation of TA-PU films in two different ratios of 1 : 2 and 1 : 5 in Tetrahydrofuran (THF) or THF-ethanol mixtures. The prepared films were characterized using X-Ray Diffraction (XRD), Differential Scanning Calorimetry (DSC), Fourier Transform Infrared Spectroscopy (FTIR), Scanning Electron Microscopy (SEM), and release studies. The results indicate transformation of crystalline TA to its amorphous form. The degree of crystallinity changes both by increasing the polymer concentration and solvent used for the film preparations. The release profiles of TA were also found to be affected, showing a decrease from approximately 50% to 25% from 1 : 2 to 1 : 5 ratios, respectively.

3.1. Appearance of TA-PU Films

Equal volumes of the solutions were used in the preparation of all films. However, TA-PU films prepared by two different methods showed some differences in their appearance. The films prepared by method I (both drug and polymer in THF) were found to be more transparent with smooth surface whereas the films prepared by method II (drug in ethanol and polymer in THF) appeared opaque with slightly rough surface. No signs of recrystallization or changes in appearance took place in all the films during storage for up to 3 months in a desiccator.

3.2.1. XRD Studies

The pure crystalline TA shows characteristic diffraction peaks at 2θ values of 5.21°, 11.58°, 15.72°, 18.70°, 19.77°, 24.90°, 25.32°, 25.90°, and 26.81°, respectively (). In case of PU, a hallow diffraction pattern with no sharp peaks has been observed in the pure as well as in the control films thus indicating its amorphous nature. A typical diffraction pattern has been presented in . The diffraction patterns of the 1 : 2 TA-PU films prepared by method I indicated a marked decrease in the peaks intensity of the crystalline TA and show very small peaks at around 15.72–18.70° and 24.90–26.81° whereas the remaining peaks of the drug have not been detected (). The diffraction pattern of 1 : 5 TA-PU films also shows similar type of result with comparatively less intense peaks in the same region (). This decrease in peak intensity clearly indicates that majority of drug has been transformed to the amorphous form.

3.2.1. XRD Studies

The films prepared using ethanol for drug solubilisation (method II) showed similar type of diffraction patterns for 1 : 2 and 1 : 5 ratios, respectively, but the intensity of the peaks is comparatively higher to the films prepared in THF only (method I) (Figures and ). Therefore, method I could be considered as more effective comparatively in converting crystalline TA to its amorphous form. Similarly, the difference in the diffraction pattern of the films indicates that the solvent also plays an important role in modifying the solid state properties of the drug in addition to the technique used and the nature of the polymer. THF evaporates more rapidly as compared to ethanol-THF mixture [] and therefore the use of THF alone has resulted in greater amorphicity of the drug. The samples were further subjected to analysis by DSC, FTIR, and SEM techniques in order to confirm the results obtained by XRD.

Abstract

Snakebites are a major Collective Health problem worldwide. In Brazil, Bothrops jararaca snake venom (BjV) evokes hemostatic disturbances, bleeding manifestations, and redox status imbalance. Specific antivenom therapy, although efficacious to revert most snakebite-induced manifestations, is incapable of treating secondary manifestations, such as oxidative/nitrosative stress. Searching for new complementary therapies that could attenuate physiological derangements triggered by envenomation, we elected to test quercetin-3-rutinoside (rutin) by its potential as both a potent antioxidant and a hemostasis modulatory compound. The activity of rutin was evaluated both on the biological activities of crude BjV in vitro, and in vivo by the ability of rutin (14.4 mg/kg b.w.) to modulate hematological, hemostatic and redox status markers altered by BjV injection (1.6 mg/kg b.w., s.c.) in mice. In vitro, rutin failed to inhibit BjV-induced platelet aggregation and biological activities of major BjV enzymes (metalloproteinases, phospholipases A2, serine proteases, and L-amino acid oxidases). On the other hand, rutin attenuated local hemorrhage, and the increase in reactive species, prevented the fall in RBC counts and fibrinogen levels, diminished tail bleeding and shortened prothrombin time (PT) evoked by envenomation. Furthermore, rutin reduced tissue factor (TF) activity and altered the protein expression of TF in liver, lungs, heart and skin. In conclusion, the disturbances in redox status and hemostatic system induced by B. jararaca envenomation were modulated by rutin, suggesting it has a great potential to be used as an ancillary therapeutic agent for snakebites.

Ethics statement

Male Swiss mice, weighing 30–35 g, were obtained from the Animal Facility of Instituto Butantan, and were maintained with free access to food and water. The experimental procedures involving human donors, rats, and mice were in accordance with National Guidelines, and were approved, respectively, by the National Human Research Ethics Committee (Plataforma Brasil, CAAE: 51368615.5.0000.0065), and the Institutional Animal Care and Use Committee from Instituto Butantan (CEUAIB 4388061115) and the Faculdade de Medicina, Universidade de São Paulo (protocol 188/15). All procedures involving animals were in accordance with the National Guidelines of Conselho Nacional de Controle de Experimentação Animal (CONCEA) [].

Venom and rutin solutions

BjV was dissolved in sterile saline (0.8 mg/mL); rutin (7.2 mg/mL) was dissolved in a solution containing 1 vol. of propylene glycol and 1 vol. of saline. The solutions were prepared immediately before use. The dose of BjV (1.6 mg/kg b.w., s.c. route) was selected based on previous tests to determine a dose that evoked hemostatic disturbances in mice similar to those observed in rats [, ] and humans []. The dose of rutin (14.4 mg/kg b.w, s.c. route) was established based on previous experiments, showing that this dose was effective to modulate redox status and hemostatic parameters.

Experimental groups and procedures

Animals were randomly allocated in four experimental groups (n = 6/group/time interval) () and received (4.0 mL/kg b.w., s.c.): saline (saline control, i.e., the negative control); rutin (rutin control, 14.4 mg/kg b.w. of rutin); BjV+saline (positive control, 1.6 mg/kg b.w. of BjV); and BjV+rutin (rutin treatment, 14.4 mg/kg b.w. of rutin and 1.6 mg/kg b.w. of BjV). The solutions were incubated at 37°C for 30 min before administration.

Experimental groups and procedures

At 3, 6 and 24 h after injection, animals (5–6 mice/group) were anesthetized with isoflurane (induction and maintenance at 2.5%), and blood and tissues were collected (). Blood was collected from the caudal vena cava into plastic syringes to obtain complete blood cell counts (CBC), blood smears and plasma. Blood was added to flasks containing the following anticoagulants: CTAD (75 mM trisodium citrate, 42 mM citric acid, 139 mM dextrose, 15 mM theophylline, 3.7 mM adenosine, 0.2 mM dipyridamole, and 2 μM imipramine), 3.2% trisodium citrate, or 269 mM Na2EDTA. Bothrops antivenom (1 vol. to 100 vol. of blood) was added to all flasks to avoid in vitro clotting of the blood induced by BjV. Fragments of liver, lungs, heart (50–100 mg) and skin (0.5–0.75 cm2 around the site of s.c. injection) were collected in flasks containing RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 2 mM Na2-EDTA, and a protease inhibitor cocktail, pH 7.5) or HBSA buffer (20 mM HEPES-NaOH buffer, pH 7.5, containing 100 mM NaCl, 0.02% NaN3 and 1 mg/mL bovine albumin), both containing Bothrops antivenom, and frozen at -80°C. Tissues were disrupted using FastPrep-24 (#6004–500, MP Biomedicals) for 60 s at 6.5 m/s, followed by 3 cycles of freezing in dry ice and thawing in a water bath at 37°C. Finally, samples were centrifuged at 13000 g for 10 min at 4°C, and supernatants were collected and frozen at -80°C. Protein concentration in the supernatants was determined by bicinchoninic acid assay []. As reference samples, pools of normal plasma and each organ (liver, lungs, heart and skin) were obtained and assayed on each determination.

Abstract

Conclusion: Incorporation of lysine is supported by the comparatively high concentration of cytoplasmic lysine, not enzyme specificity.

Abstract

Formation of the peptidoglycan stem pentapeptide requires the insertion of both l and d amino acids by the ATP-dependent ligase enzymes MurC, -D, -E, and -F. The stereochemical control of the third position amino acid in the pentapeptide is crucial to maintain the fidelity of later biosynthetic steps contributing to cell morphology, antibiotic resistance, and pathogenesis. Here we determined the x-ray crystal structure of Staphylococcus aureus MurE UDP-N-acetylmuramoyl-l-alanyl-d-glutamate:meso-2,6-diaminopimelate ligase (MurE) (E.C. 6.3.2.7) at 1.8 Å resolution in the presence of ADP and the reaction product, UDP-MurNAc-l-Ala-γ-d-Glu-l-Lys. This structure provides for the first time a molecular understanding of how this Gram-positive enzyme discriminates between l-lysine and d,l-diaminopimelic acid, the predominant amino acid that replaces l-lysine in Gram-negative peptidoglycan. Despite the presence of a consensus sequence previously implicated in the selection of the third position residue in the stem pentapeptide in S. aureus MurE, the structure shows that only part of this sequence is involved in the selection of l-lysine. Instead, other parts of the protein contribute substrate-selecting residues, resulting in a lysine-binding pocket based on charge characteristics. Despite the absolute specificity for l-lysine, S. aureus MurE binds this substrate relatively poorly. In vivo analysis and metabolomic data reveal that this is compensated for by high cytoplasmic l-lysine concentrations. Therefore, both metabolic and structural constraints maintain the structural integrity of the staphylococcal peptidoglycan. This study provides a novel focus for S. aureus-directed antimicrobials based on dual targeting of essential amino acid biogenesis and its linkage to cell wall assembly.

2.1. Q3R Protects Cultured Cells from CT

We hypothesized Q3R would block the cytopathic activity of CT through its inhibitory action on PDI. To test this prediction, cAMP levels were quantified from CHO cells incubated with CT for 2 h in the absence or presence of Q3R. As shown in A, Q3R-treated cells were protected from the cytopathic activity of CT. In contrast, Q3R did not inhibit the cAMP response generated from cells expressing an ER-localized CTA1 construct (B). Transfection with the pCDNA3.1/ssCTA1 plasmid results in co-translational insertion of CTA1 into the ER lumen, proteolytic cleavage of its N-terminal ER-targeting signal sequence (ss), and dislocation of CTA1 back into the cytosol where it elicits a cAMP response beginning 2 h post-transfection []. We found the cAMP response from Q3R-treated cells was 11% greater than the response from untreated cells that were also expressing CTA1. The basis for this mild sensitization remains to be determined, but the result clearly shows that Q3R does not block (i) the ER-to-cytosol export of free CTA1 or (ii) the enzymatic activity of cytosolic CTA1. The inhibitory effect of Q3R on CT intoxication thus occurs at a step preceding translocation of free CTA1 to the cytosol.

2.2. Q3R Does Not Prevent PDI from Binding or Reducing CTA1

Surface plasmon resonance (SPR) was used to examine the impact of Q3R on PDI binding to CTA1 (A). For this experiment, CTA1 was coupled to a SPR sensor. A baseline measurement corresponding to the mass of sensor-bound CTA1 was set at 0 refractive index units (RIU), and Q3R-treated PDI was then added to the perfusion buffer. The elevated signal following this injection indicated Q3R-treated PDI had bound to CTA1, thereby adding mass to the sensor surface and altering the RIU. The pattern of binding was similar to what was previously reported for untreated PDI []. Thus, Q3R did not disrupt the physical association between PDI and CTA1.

2.3. Q3R Disrupts PDI-Driven Disassembly of the CT Holotoxin

Our collective data indicated Q3R does not block PDI binding to CTA1, the PDI-induced shift in CTA1 protease sensitivity, or reduction of the CTA1/CTA2 disulfide bond by PDI. We next examined the effect of Q3R on disassembly of the CT holotoxin by PDI (). For this experiment, CT was appended to a GM1-coated SPR sensor slide. A baseline measurement corresponding to the mass of the captured holotoxin was then set at 0 RIU. Subsequent addition of PDI to the perfusion buffer resulted in an elevated RIU signal indicative of PDI binding to CT. In the absence of Q3R (A), the rise in RIU was followed by a rapid drop in signal to a point below the baseline value corresponding to the mass of the CT holotoxin. This drop occurred despite the continued presence of PDI in the perfusion buffer and was attributed to the PDI-mediated release of CTA1 from the sensor-bound CTA2/CTB5 complex. Both PDI and its displaced CTA1 binding partner would be removed from the sensor by the flow of the perfusion buffer, thus lowering the RIU below its initial baseline value. After removing PDI from the perfusion buffer, sequential antibody injections confirmed our interpretation: no signal was obtained from the PDI and CTA1 antibodies, whereas the CTB antibody produced a positive result. This result was consistent with previous reports that used SPR to monitor the PDI-driven disassembly of CT [,].

2.3. Q3R Disrupts PDI-Driven Disassembly of the CT Holotoxin

As with untreated PDI, injection of Q3R-treated PDI into the perfusion buffer produced an elevated RIU (B). However, no loss of signal occurred while Q3R-treated PDI was in the perfusion buffer. The RIU only dropped when Q3R-treated PDI was removed from the perfusion buffer, which indicated PDI had been released from the intact CT holotoxin. Yet the signal remained above the initial baseline measurement and never dropped below 0 RIU. It thus appeared that some PDI remained associated with the sensor-bound holotoxin for an extended time. This interpretation was confirmed by the positive signals generated from each sequential perfusion of anti-PDI, anti-CTA1, and anti-CTB antibodies over the sensor. Q3R-treated PDI could thus bind to the CT holotoxin but could not efficiently separate CTA1 from CTA2/CTB5.

1. Introduction

Buckwheat is a pseudocereal with high nutritional value and beneficial health properties widely described in the recent years [,,]. Rutin (Ru, quercetin-3-rutinoside) is the main buckwheat flavonoid antioxidant present in seeds, groats, hull, flours, processed buckwheat products, and sprouts whereas quercetin (Q) is present in significantly lower concentrations []. The beneficial health effects of buckwheat bioactive compounds as well as Ru and Q is dependent on their absorption in the gut and catabolism by the gut microbiota [,]. Ru and Q have antioxidant activity and inhibits low-density lipoprotein peroxidation and has anti-inflammatory and vasoactive properties as well []. The bioaccessibility, usually evaluated by in vitro digestion procedures, is the quantity or fraction released from the food matrix in the gastrointestinal lumen and available for intestinal absorption [,]. In food science in vitro digestion models let us predict a compound’s bioaccessibility with several advantages such as relative inexpensiveness, simplicity, controlled conditions and reproducible results [,]. However, significant amounts of bioactive compounds may be found in the insoluble fraction left after digestion in vitro which is in vivo moved to the colon, fermented by colon microbiota and then generate derived phenolic metabolites []. Therefore, for the first time, we expanded the definition of bioaccessibility when its evaluation is based on the content of bioactive compounds in the soluble and insoluble fraction left after digestion in vitro. The bioaccessibility of Ru and Q depends on the material, processing, and the food matrix []. There is no available information on potentially affecting factors of the defined in this work expanded bioaccessibility of Ru and Q such as fermentation of buckwheat flours by lactic acid bacteria (LAB), biscuits baked and controlled digestion in vitro may change the functional properties of flours and the derived bakery products [,]. For example, the anti-nutritional factors are degraded after fermentation of buckwheat flours and their nutritional value is increased []. The aim of this study was to find out: (1) the effect of fermentation of raw and roasted common buckwheat flours (F. esculentum Moench) by select LAB on the Ru and Q contents; (2) the effect of BBs baking on the Ru and Q contents; and (3) the expanded bioaccessibility of Ru and Q from BBs determined after digestion in vitro.

2.2. Fermentation of Buckwheat Flours by LAB

Raw buckwheat flour and roasted buckwheat groats produced from Polish commercial common buckwheat (Fagopyrum esculentum Moench var. Kora) were purchased from a local industry plant (Melvit S.A., Kruki, Poland). Raw flour was produced from the milling of purified buckwheat whereas roasted buckwheat groats was obtained after roasting the whole raw seeds whilst simultaneously steaming (overheat water vapor at 588 kPa) and heating them at 160 °C for 30 min. The roasted groats were ground in a laboratory mill and sifted through a sieve with a diameter of 450 μm, which gave the roasted flour.

2.2. Fermentation of Buckwheat Flours by LAB

Two types of pre-treated buckwheat flour and 14 strains of LAB (L. acidophilus (145, La5, V), L. casei (LcY, 2K), L. delbruecki subsp. bulgaricus (151, K), L. plantarum (W42, IB), L. rhamnosus (GG, 8/4, K), L. salivarius AWH, and Streptococcus thermophilus Mk-10) were subjected to a liquid-state fermentation (LSF). The origin of the LAB was recently described by Wronkowska et al. []. The 5% suspensions of buckwheat flour in distilled water were inoculated with 8.00 log CFU mL−1 of selected LAB and fermented at 37 °C for 24 h. After fermentation, flours were freeze-dried (Christ-Epsilon 2-6D LSC plus, Osterode am Harz, Germany) and retrieved for BBs preparation. The buckwheat flours not subjected to the fermentation process was used as a control flours and after baking as control BBs.

3.1. The Effect of Fermentation on the Ru and Q Content of Raw and Roasted Buckwheat Flour

A LAB-dependent variation in the Ru and Q content of fermented buckwheat flours was noted. The average Ru content in fermented raw buckwheat flours was 355.7 μg g−1 d.m. compared to 376 μg g−1 d.m. for control non-fermented flour. A slightly increased Ru concentration was noted in raw buckwheat flour fermented by L. plantarum W42 (by 26%), L. casei LcY (by 7.6%), L. acidophilus La5 (by 6.3%), and L. salivarius AWH (by 5.5%), whereas the lowest was observed in flour fermented by L. acidophilus V (decrease by 34.8%) compared to non-fermented raw flour (). The average Ru content in fermented roasted buckwheat flour was 139.9 μg g−1 d.m. compared to 220 μg g−1 d.m. for control unfermented roasted flour (). In contrast to raw buckwheat flour, the fermentation of roasted buckwheat flour caused a significant decrease in Ru concentration of up to 53% compared to non-fermented roasted flour (). Our results are in accordance to those reported recently that also solid-state fermentation (SSF) with Rhizopus oligosporus caused a decrease in Ru content compared to non-fermented buckwheat groats []. Starzyńska-Janiszewska et al. [] found significantly increased levels of Ru in a quinoa product obtained after 40 h of SSF with R. oligosporus compared to cooked quinoa seeds.

Abstract

Dietary intake of linoleic acid (LNA, 18:2n-6) has increased dramatically during the 20th century and is associated with greater prevalence of obesity. The endocannabinoid system is involved in regulation of energy balance and a sustained hyperactivity of the endocannabinoid system may contribute to obesity. Arachidonic acid (ARA, 20:4n-6) is the precursor for 2-AG and anandamide (AEA), and we sought to determine if low fat diets (LFD) could be made obesogenic by increasing the endocannabinoid precursor pool of ARA, causing excessive endocannabinoid signaling leading to weight gain and a metabolic profile associated with obesity. Mice (C57BL/6j, 6 weeks of age) were fed 1 en% LNA and 8 en% LNA in low fat (12.5 en%) and medium fat diets (MFD, 35 en%) for 16 weeks. We found that increasing dietary LNA from 1 to 8 en% in LFD and MFD significantly increased ARA in phospholipids (ARA–PL), elevated 2-AG and AEA in liver, elevated plasma leptin, and resulted in larger adipocytes and more macrophage infiltration in adipose tissue. In LFD, dietary LNA of 8 en% increased feed efficiency and caused greater weight gain than in an isocaloric reduction to 1 en% LNA. Increasing dietary LNA from 1 to 8 en% elevates liver endocannabinoid levels and increases the risk of developing obesity. Thus a high dietary content of LNA (8 en%) increases the adipogenic properties of a low fat diet.

Animals

Male mice (n = 36), 6 weeks of age (C57BL/6j, Taconic, Denmark) were randomly assigned to four experimental diets (n = 9) (Table ) and housed individually. The animals were maintained on a 12:12 h light–dark cycle at 29 ± 1 °C. All animals were sacrificed at 22 weeks of age. Table 1Diet composition and fatty acid profile of dietsLow fat diets (12.5 en%)Medium fat diets (35 en%)1 en% LNA8 en% LNA1 en% LNA8 en% LNAg/kg Cocosa (coconut oil)3–12985 Safflower oil144–42 Olive oil44–3331 Flaxseed oil2637 Total oil added (g/kg)5050165165% energy derived from Fat11113234 Carbohydrate71714544 Protein18182322(% of energy) Sum SFA2.51.52517 Sum MUFA9278 18:2n-61818 20:4n-6–––– Sum n-61818 18:3n-30.30.90.41 20:5n-3–––– 22:6n-3–––– Sum n-30.30.90.31 Total n-6/n-3 ratio3938 Estimated n-6 HUFA1 (%)56725372 Estimated Omega-3 Index2 11511.55.5All diets were supplemented with 60 mg/k ethoxyquin, 2.5 g/kg choline bitartrate, 3 g/kg l-cystein, 13 g/kg vitamin mix, 47 g/kg mineral mix, 50 g/kg sucrose, 50 g/kg cellulose, 100 g/kg corn starch. Energy (kcal) of diets; LF diets; 3,900, MF diets; 4,500 LNA linoleic acid, SFA saturated fatty acids, MUFA monounsaturated fatty acids, HUFA highly unsaturated fatty acids (>20 carbon) 1 Calculated from the Lands equation [], n-3 HUFA = 100 − n-6 HUFA 2 Omega-3 index: (omega-3 HUFA × 0.32) − 3.5 [, ]

Abstract

Non-transformed wild type (NTWT) and hairy root cultures of Rhodiola kirilowii were grown in medium supplemented with 2.5 mM cinnamyl alcohol as a precursor and/or sucrose (1 %) on the day of inoculation or on the 14th day of culture. Rosarin, rosavin, and rosin were produced by NTWT root culture but only rosarin and rosavin by hairy roots. Approximately 80 and 95 % of the glycosides were released into the medium for NTWT and hairy root cultures, respectively. The highest rosavin yield, 505 ± 106 mg/l, was in hairy root culture when cinnamyl alcohol was applied on the day of inoculation with the addition of sucrose on the 14th day of culture. For rosin production, supplementation with cinnamyl alcohol alone on day 14 was more favourable with the highest amount 74 ± 10 mg/l in NTWT root culture. Only traces of rosarin were detected.

Enhancement of rosavin production

To enhance rosavin production, a cinnamyl alcohol (CA) and/or sucrose feeding strategy was applied. Four types of experiments were performed: (1) CA was added to the medium on the day of inoculation to give 2.5 mM; (2) CA (2.5 mM) was added to the medium on the day of inoculation and the medium was further supplemented with 1 %, (w/v) sucrose on the 14th day of culture; (3) CA (2.5 mM) was added to the medium on the 14th day of culture; (4) sucrose (1 %) and CA (2.5 mM) were both added on the 14th day of culture.

Enhancement of rosavin production

NTWT and hairy root cultures growing in unsupplemented medium were used as control cultures. Each experiment was performed in triplicate. Ten flasks were taken for each type of culture at the end of the 28 day growth cycle.