Extraction Optimization and Qualitative/Quantitative Determination of Bioactive Abietane-Type Diterpenes from Three Salvia Species (Common Sage, Greek Sage and Rosemary) by 1H-qNMR.

The objective of this study was the optimization of the extraction process and the qualitative and quantitative determination of the bioactive metabolites: 12-O-methylcarnosic acid (12MCA), carnosic acid (CA), carnosol (CS), 7-O-methyl-epi-rosmanol (7MER) and rosmanol (RO) in infusions, decoctions, turbulent flow extracts, tinctures and oleolites from three Salvia species: Salvia officinalis L. (common sage, SO), Salvia fruticosa Mill. (Greek sage, SF) and Salvia rosmarinus Spenn (syn Rosmarinus officinalis L.) (rosemary, SR), using Quantitative Proton Nuclear Magnetic Resonance Spectroscopy (1H-qNMR). Regarding the aqueous extracts, decoctions appeared to be richer sources of the studied metabolites than infusions among the three plants. For SR, the turbulent flow extraction under heating was the most efficient one. The optimum time for the preparation of decoctions was found to be 5 min for SF and SO and 15 min for SR. It is noteworthy that SR tinctures were not stable in time due to decomposition of the abietane-type diterpenes CA and CS because of the polar solvent used for their preparation. Contrary to this finding, the oleolites of SR appeared to be very stable. Olive oil as a solvent for extraction was very protective for the contained abietane-type diterpenes. A preliminary stability study on the effect of the storage time of the SF on the abietane-type diterpenes content showed that the total quantity of abietanes decreased by 16.51% and 40.79% after 12 and 36 months, respectively. The results of this investigation also demonstrated that 1H-qNMR is very useful for the analysis of sensitive metabolites, like abietane-type diterpenes, that can be influenced by solvents used in chromatographic analysis.

Genetic Evaluation of In Vitro Micropropagated and Regenerated Plants of Cannabis sativa L. Using SSR Molecular Markers.

Simple sequence repeat (SSR) markers were used to evaluate the genetic stability of the acclimatized micropropagated and regenerated plants of a high cannabidiol (H-CBD) and a high cannabigerol (H-CBG) variety of Cannabis sativa L. Shoot regeneration and proliferation were achieved by culturing calli in Murashige and Skoog basal medium (MS) supplemented with several concentrations of 6-benzyladenine (BA) or thidiazuron (TDZ). Calli derived mostly from stem explants, rather than leaves, cultured on MS supplemented with 2,4-Dichlorophenoxyacetic acid (2,4-D) or combination of kinetin (KIN) with 1-Naphthaleneacetic acid (NAA) or 2,4-D. Rooting of the regenerated plantlets accomplished on half-strength MS medium supplemented with indole-3-butyric acid (IBA). Previous studies performed have developed an efficient in vitro micropropagation protocol for mass production. Both in vitro methodologies can be employed in genetic breeding via molecular techniques. The genetic stability of micropropagated and regenerated plants was accomplished using twelve SSR primer pairs that produced reproducible and clear bands, ranging from 90 to 330 bp in size, and resulted in amplification of one or two alleles, corresponding to homozygous or heterozygous individuals. The SSR amplification products were monomorphic across all the micropropagated and regenerated plants and comparable to mother plants. The monomorphic banding pattern confirmed the genetic homogeneity of the in vitro cultured acclimatized and mother plants as no somaclonal variation was detected in clones for these specific SSRs. Our results evidently suggest that the developed culture protocols for in vitro multiplication is appropriate and applicable for clonal mass propagation of the C. sativa varieties and demonstrate the reliability of this in vitro propagation system.

An Alternative In Vitro Propagation Protocol of Cannabis sativa L. (Cannabaceae) Presenting Efficient Rooting, for Commercial Production.

An alternative in vitro propagation protocol for medical Cannabis sativa L. cultivars for pharmaceutical industrial use was established. The aim of the protocol was to reduce the culture time, offering healthy and aseptic propagating material, while making the whole process more economic for industrial use. The propagation procedure was performed using plastic autoclavable vented and non-vented vessels, containing porous rooting fine-milled sphagnum peat moss-based sponges, impregnated in ½ Murashige and Skoog liquid growth medium, supplemented with indole-3-butyric acid (IBA) at various concentrations (0, 2.46, 4.92, and 9.84 µM) or by dipping nodal cuttings into 15 mM IBA aqueous solution. The highest average root numbers per cutting, 9.47 and 7.79 for high cannabidiol (H_CBD) and high cannabigerol (H_CBG) varieties, respectively, were achieved by dipping the cuttings into IBA aqueous solution for 4 min and then placing them in non-vented vessels. The maximum average root length in H_CBD (1.54 cm) and H_CBG (0.88 cm) was ascertained using 2.46 µM filter sterilized IBA in non-vented vessels. Filter-sterilized IBA at concentrations of 2.46 µM in vented and 4.92 µM in non-vented vessels displayed the maximum average rooting percentages in H_CBD (100%) and H_CBG (95.83%), respectively. In both varieties, maximum growth was obtained in non-vented vessels, when the medium was supplemented with 4.92 µM filter-sterilized IBA. Significant interactions between variety and vessel type and variety and IBA treatments were observed in relation to rooting traits. Approximately 95% of plantlets were successfully established and acclimatized in field. This culture system can be used not only for propagating plant material at an industrial scale but also to enhance the preservation and conservation of Cannabis genetic material.

A New Definition of the Term "High-Phenolic Olive Oil" Based on Large Scale Statistical Data of Greek Olive Oils Analyzed by qNMR.

In the last few years, a new term, "High-phenolic olive oil", has appeared in scientific literature and in the market. However, there is no available definition of that term regarding the concentration limits of the phenolic ingredients of olive oil. For this purpose, we performed a large-scale screening and statistical evaluation of 5764 olive oil samples from Greece coming from >30 varieties for an eleven-year period with precisely measured phenolic content by qNMR. Although there is a large variation among the different cultivars, the mean concentration of total phenolic content was 483 mg/kg. The maximum concentration recorded in Greece reached 4003 mg/kg. We also observed a statistically significant correlation of the phenolic content with the harvest period and we also identified varieties affording olive oils with higher phenolic content. In addition, we performed a study of phenolic content loss during usual storage and we found an average loss of 46% in 12 months. We propose that the term high-phenolic should be used for olive oils with phenolic content > 500 mg/kg that will be able to retain the health claim limit (250 mg/kg) for at least 12 months after bottling. The term exceptionally high phenolic olive oil should be used for olive oil with phenolic content > 1200 mg/kg (top 5%).

Biotechnological Approaches on Two High CBD and CBG Cannabis sativa L. (Cannabaceae) Varieties: In Vitro Regeneration and Phytochemical Consistency Evaluation of Micropropagated Plants Using Quantitative 1H-NMR.

High cannabidiol (CBD) and cannabigerol (CBG) varieties of Cannabis sativa L., a species with medicinal properties, were regenerated in vitro. Explants of nodal segments including healthy axillary bud, after sterilization, were placed in Murashige-Skoog (MS) culture medium. The shoots formed after 30 days were subcultured in full- or half-strength MS medium supplemented with several concentrations of 6-benzyl-amino-purine (BA) or thidiazuron (TDZ). The highest average number and length of shoots was achieved when both full and half-strength MS media were supplemented with 4.0 µM BA. The presence of 4.0 µM TDZ showed also comparable results. BA and TDZ at concentrations of 4.0, 8.0 µM and 2.0, 4.0 µM respectively, displayed the maximum shooting frequency. The new shoots were transferred on the same media and were either self-rooted or after being enhanced with different concentrations of indole-3-butyric acid (IBA) or α-naphthalene acetic acid (NAA). Presence of 2.0 or 4.0 µM IBA or 4.0 µM NAA resulted to the optimum rooting rates. The maximum average number and length of roots per shoot was observed when the culture media was supplemented with 4.0 µM IBA or NAA. Approximately 92% of the plantlets were successfully established and acclimatized in field. The consistency of the chemical profile of the acclimatized in vitro propagated clones was assessed using quantitative 1H-NMR high throughput screening. In each variety, analysis of the micropropagated plant in comparison with the mother plant showed no statistically significant differences (p ≤ 0.05) in CBD+ cannabidiolic acid (CBDA) and CBG+ cannabigerolic acid (CBGA) content respectively, thus indicating stability of their chemical profile.

High-Throughput 1H-Nuclear Magnetic Resonance-Based Screening for the Identification and Quantification of Heartwood Diterpenic Acids in Four Black Pine (Pinus nigra Arn.) Marginal Provenances in Greece.

A high-throughput quantitative Nuclear Magnetic Resonance 1H-NMR method was developed and applied to screen the quantity of the diterpenic resin acids in the heartwood of black pine, due to the renewed scientific interest in their medicinal properties and use in various diseases treatment. The 260 samples were taken from Pinus nigra clones, selected from four provenances of the Peloponnese (Greece), participating in a 35-year-old clonal seed orchard. Total resin acids per dry heartwood weight (dhw) varied greatly, ranging from 30.05 to 424.70 mg/gdhw (average 219.98 mg/gdhw). Abietic was the predominant acid (76.77 mg/gdhw), followed by palustric acid (47.94 mg/gdhw), neoabietic acid (39.34 mg/gdhw), and pimaric acid (22.54 mg/gdhw). Dehydroabietic acid was at moderate levels (11.69 mg/gdhw), while levopimaric, isopimaric, and sandaracopimaric acids were in lower concentrations. The resin acid fraction accounted for 72.33% of the total acetone extractives. Stilbenes were presented in significant quantities (19.70%). The resin acid content was composed mainly of the abietane type resin acids (83.56%). Peloponnesian Pinus nigra heartwood was found to be the richest source of resin acids identified to date and is considered the best natural source for the production of such bioactive extracts. The results indicate a high potential for effective selection and advanced breeding of pharmaceutical and high economic value bioactive substances from Pinus nigra clones.

Identification of black pine (Pinus nigra Arn.) heartwood as a rich source of bioactive stilbenes by qNMR.

BACKGROUND: Recently published studies have demonstrated the strong anti-inflammatory properties of Scots pine (Pinus sylvestris L.) heartwood extracts, related to its stilbene content. In order to find alternative sources of Pinus heartwood extracts rich in stilbenes, a large number of samples were investigated, using a new developed high-throughput screening method based on quantitative nuclear magnetic resonance. RESULTS: The new method enabled us to measure the levels of pinosylvin, pinosylvin monomethyl ether and pinosylvin dimethyl ether in heartwood extracts in only 45 s per sample. The method was applied to 260 Pinus nigra trees originating from Peloponnese (southern Greece) from four different natural populations of the species. The results obtained showed that the total stilbenoids per dry heartwood weight varied greatly, ranging from 10.9 to 128.2 mg g-1drywood (average 59.92 ± 21.79 mg g-1drywood ). The major stilbene in all cases was pinosylvin monomethyl ether (40.32 ± 15.55 mg g-1drywood ), followed by pinosylvin (17.07±6.76 mg g-1drywood ) and pinosylvin dimethyl ether (2.54 ± 1.22 mg g-1drywood ). The highest stilbene content of P. nigra samples was found to be 6.3 times higher than the highest reported figure for P. sylvestris L. CONCLUSION: Pinus nigra heartwood is the richest source of pinosylvin and pinosylvin monomethyl ether identified to date and can be considered the best natural resource for production of bioactive extracts. © 2016 Society of Chemical Industry.

The effect of chronically increased intra-abdominal pressure on rectus abdominis muscle histology an experimental study on rabbits.

BACKGROUND: The aim of this study was to specify the histologic response of the rectus abdominis muscle of the rabbit, to the chronically increased intra-abdominal pressure. MATERIALS AND METHODS: Forty-five New Zealand white rabbits were divided into three groups. In all groups, a rubber bag was implanted into the peritoneal cavity. In group A (n=15) the bags were kept empty. In group B (n=15) the bags were filled with normal saline in order to achieve an intra-abdominal pressure of over 12 mmHg. This pressure was kept at this level for 8 wk. In group C (n=15) the intra-abdominal rubber bags were filled with lead covered by silicone, equiponderant to the mean weight of the normal saline insufflated in group B. After 8 wk we took biopsies of the rectus abdominis muscle and counted the proportion of the different types of muscular fibers (type I, IIA, and IIB/X). RESULTS: Significant difference was found in the proportion of the three types of muscle fibers. Intra-abdominal hypertension led to an increase in type I fibers (P=0.008). No difference was noticed between groups A and C. CONCLUSIONS: The histologic response to the increased intra-abdominal pressure was an increase in type I muscle fibers. Charging with lead did not cause any significant change in the proportion of muscular fibers.