Association analysis and molecular tagging of phytochemicals in the endangered medicinal plant licorice (Glycyrrhiza glabra L.).

Licorice (Glycyrrhiza glabra L.) is a medicinal plant species valued in many countries in Asia and Europe for its phytochemical characteristics. Licorice biodiversity is becoming threatened nowadays in Iran due to increasing demand and a drastic decline of its natural habitats. Therefore, licorice domestication would be necessary in the near future, and molecular breeding would help to introduce genotypes suitable for cultivation. The present study was carried out with 170 individual licorice plants sampled in the wild in 59 localizations in 21 provinces of Iran. The association of 436 polymorphic AFLP markers, produced by 15 primer combinations (EcoRI/MseI), with six phenotypic phytochemical traits was studied. The AMOVA analysis show gene diversity among and within localizations. The population structure analysis identified two main sub-populations with significant genetic variation. Significant associations were identified between three markers (E3/M40-4, E34/M4-12 and E12/M31-15) and glycyrrhizin concentration, and between four markers (E11/M34-12, E11/M34-15, E9/M7-29, and E9/M7-30) and phenolic compounds contents. Markers detected can be useful in the domestication of licorice as well as in breeding programs. Licorice sampled in four localizations (KBA1, KBA2, SKh2 and Fa1) were found to be superior in terms of glycyrrhizin and antioxidants content, and therefore they can be considered as elite genotypes which could be included in the domestication process.

Effect of different quality of light on growth and production of secondary metabolites in adventitious root cultivation of Hypericum perforatum.

Naphthodianthrone derivatives that produced in Hypericum perforatum (St. John's Wort) are valuable secondary metabolites for depression treatment and photodynamic therapy. However, the traditional cultivation of this plant does not meet both quantitatively and qualitatively the high demand of the pharmaceutical industry. So, the adventitious root culture along with elicitation has been introduced as an alternative for production of such valuable bioactive compounds. The aim of this study was to evaluate the effect of darkness and red, blue and fluorescent light on growth and production of secondary metabolites in the adventitious root cultivation of H. perforatum. Our results showed that biomass production was significantly higher in the cultures grown under dark and red light, but in terms of hypericins production, red light was the best. Despite the inhibitory effect of five weeks blue light treatment on both biomass and secondary metabolite production of adventitious roots, one-week blue light treatment of four-weeks grown roots is an effective stimulator for increasing total phenolic compounds and hypericins. Interestingly, the roots were regenerated under red light and stems and leaves were formed.

Ultrasound-assisted packed-bed extraction of hypericin from Hypericum perforatum L. and optimization by response surface methodology.

This paper presents the successful application of ultrasound-assisted packed-bed (UAE-PB) method for the extraction of hypericin from the Hypericum perfuratum L. The Soxhlet system was utilized for the determination of suitable solvent from ethanol, methanol or from the mixture of different proportions of ethanol-methanol. The mixture of 50:50 v/v ethanol-methanol was obtained to be the most suitable solvent since it led to the highest extraction amount of hypericin. The extraction amount of hypericin increased by 13.6% and 21.4% when the solvent changed from pure methanol to the mixture of 50:50 v/v ethanol-methanol for the extraction time of 3 and 8 h, respectively. Subsequently, the extraction was conducted through the UAE-PB, and the effects of temperature, time, and the ratio of solvent to the dried plant were studied. The response surface method (RSM) was used to investigate the effect of parameters on the extraction in the UAE-PB system. At the temperature of 60 °C, extraction time of 105 min, and the solvent to plant ratio of 15.3, the maximum extraction yield of hypericin was achieved. In the optimal conditions, the amount of extraction was 0.112 mg hypericin/g dried plant, which was in accordance with the optimized predicted value (0.111 mg hypericin/g dried plant) from Design-Expert software.

Effect of drought stress on growth parameters, osmolyte contents, antioxidant enzymes and glycyrrhizin synthesis in licorice (Glycyrrhiza glabra L.) grown in the field.

Glycyrrhiza glabra L. (licorice) is a medicinal species rich in the specialised plant metabolite glycyrrhizin. It has been previously proposed that drought, which is increasing in importance due to the climatic change and scarcity of water resources, can promote the synthesis of glycyrrhizin. The effects of slight, moderate and intense drought (70, 35 and 23% of the regular irrigation, respectively) on growth parameters, osmolyte content, oxidative stress markers, antioxidant enzymes, glycyrrhizin biosynthesis genes and root glycyrrhizin concentration and contents, have been assessed in five Iranian licorice genotypes grown in the field. Drought decreased progressively biomass and leaf relative water contents, and increased progressively osmolyte (proline, glycine-betaine and soluble sugars) concentrations in leaves and roots. Drought caused oxidative stress in leaves, as indicated by lipid peroxidation and hydrogen peroxide concentrations, and increased the activities of antioxidant enzymes in leaf extracts (catalase, peroxidase, superoxide dismutase and pholyphenoloxidase). Drought promoted the synthesis of glycyrrhizin, as indicated by the increases in the expression of the glycyrrhizin biosynthesis pathway genes SQS1, SQS2, bAS, CYP88D6, CYP72A154 and UGT73, and increased the root concentrations of glycyrrhizin with drought in some genotypes. However, the large decreases in root biomass caused by drought led to general decreases in the amount of glycyrrhizin per plant with moderate and intense drought, whereas the slight drought treatment led to significant decreases in glycyrrhizin content in only one genotype. Under intense drought two of the genotypes were still capable to maintain half of the control glycyrrhizin yield, whereas in the other three genotypes glycyrrhizin yield was 22-33% of the control values. Results indicate that under intense drought, with only 23% of the normal water dose being applied, an appropriate choice of genotype can still lead to acceptable glycyrrhizin yields.

Reverse micellar extraction of fungal glucoamylase produced in solid-state fermentation culture.

Partial purification of glucoamylase from solid-state fermentation culture was, firstly, investigated by reverse micellar extraction (RME). To avoid back extraction problems, the glucoamylase was kept in the original aqueous phase, while the other undesired proteins/ enzymes were moved to the reverse micellar organic phase. The individual and interaction effects of main factors (i.e., pH and NaCl concentration in the aqueous phase, and concentration of sodium bis-2-ethyl-hexyl-sulfosuccinate (AOT) in the organic phase) were studied using response surface methodology. The optimum conditions for the maximum recovery of the enzyme were pH 2.75, 100 mM NaCl, and 200 mM AOT. Furthermore, the optimum organic to aqueous volume ratio (Vorg/Vaq) and appropriate number of sequential extraction stages were 2 and 3, respectively. Finally, 60% of the undesired enzymes including proteases and xylanases were removed from the aqueous phase, while 140% of glucoamylase activity was recovered in the aqueous phase and the purification factor of glucoamylase was found to be 3.0-fold.