Enhancement of Macarpine Production in Eschscholzia Californica Suspension Cultures under Salicylic Acid Elicitation and Precursor Supplementation.

Macarpine is a minor benzophenanthridine alkaloid with interesting biological activities, which is produced in only a few species of the Papaveraceae family, including Eschscholzia californica. Our present study was focused on the enhancement of macarpine production in E. californica suspension cultures using three elicitation models: salicylic acid (SA) (4; 6; 8 mg/L) elicitation, and simultaneous or sequential combinations of SA and L-tyrosine (1 mmol/L). Sanguinarine production was assessed along with macarpine formation in elicited suspension cultures. Alkaloid production was evaluated after 24, 48 and 72 h of elicitation. Among the tested elicitation models, the SA (4 mg/L), supported by L-tyrosine, stimulated sanguinarine and macarpine production the most efficiently. While sequential treatment led to a peak accumulation of sanguinarine at 24 h and macarpine at 48 h, simultaneous treatment resulted in maximum sanguinarine accumulation at 48 h and macarpine at 72 h. The effect of SA elicitation and precursor supplementation was evaluated also based on the gene expression of 4'-OMT, CYP719A2, and CYP719A3. The gene expression of investigated enzymes was increased at all used elicitation models and their changes correlated with sanguinarine but not macarpine accumulation.

Evaluation of Manganese Chloride's Effect on Biosynthetic Properties of In Vitro Cultures of Eschscholzia californica Cham.

The basal production of secondary metabolites in medicinal plants is limited. One of the effective approaches that encourages plants to produce a remarkable amount of precious compounds is an application of elicitors. Our work was focused on the elicitation of Eschscholzia californica Cham. suspension cultures using various concentrations of MnCl2 (5; 10; 15 mg/L) with the aim of evaluating its effect on sanguinarine, chelerythrine, and macarpine production and gene expression of enzymes involved in the biosynthesis of mentioned secondary metabolites (BBE, 4'-OMT, CYP80B1) or in defense processes (LOX). Suspension cultures were exposed to elicitor for 24, 48, and 72 h. The content of alkaloids in phytomass was determined on the basis of their fluorescence properties. The relative mRNA expression of selected genes was analyzed using the ΔΔCt value method. PCR products were evaluated by melting curve analysis to confirm the specific amplification. Our results demonstrated that Eschscholzia californica Cham. cell suspension cultures evince sensitivity to the presence of MnCl2 in growth media resulting in the increased production of benzophenanthridine alkaloids and gene expression of selected enzymes. Manganese chloride seems to be a potential elicitor supporting natural biosynthetic properties in plant cell cultures and can be applied for the sustained production of valuable secondary metabolites.

Agrimonia eupatoria L. and Cynara cardunculus L. Water Infusions: Comparison of Anti-Diabetic Activities.

Diabetes mellitus (DM) is frequently diagnosed at a time when patients already suffer from several cardiovascular complications. Our previously published data (Molecules 201520 (11): 20538-50) on the anti-oxidative properties of Agrimonia eupatoria L. (AE) and Cynara cardunculus L. (CC) prompted us to extend the available evidence on their possible protective activities on selected DM-related parameters in a streptozotocin-induced DM rat model and in a series of in vitro experiments. Male rats were divided into four groups: control group, untreated diabetic group, AE and CC treated diabetic groups. During a five-week period, changes in blood glucose and body weight were monitored. Then, rats were sacrificed and subjected to the assessment of changes in the reactivity of aortas and measurement of butyrylcholinesterase activity. To complete the panel of experiments, α-glucosidase activity was assessed in vitro. Our results demonstrate that both tested extracts exert similar anti-diabetic activities. However, better anti-oxidant activity of the A. eupatoria extract indicates its higher clinical potential in the prevention and/or adjuvant therapy of developing cardiovascular complications related to DM and diseases associated with oxidative stress.

[Effect of abiotic elicitation on the sanguinarine production and polyphenol oxidase activity in the suspension culture of Eschscholtzia californica CHAM]. / Vplyv abiotickej elicitácie na produkciu sanguinarínu a aktivitu polyfenoloxidázy v suspenznej kultúre Eschscholtzia californica CHAM.

Elicitation of plant in vitro cultures represents a biotechnological tool to improve the production of secondary metabolites. In this study, the effect of AgNO3 and CdCl2 on the sanguinarine production by the suspension culture of Eschscholtzia californica CHAM. was investigated. Elicitors were added to the cultures at the 14th day of subcultivation and their effect on the sanguinarine production was evaluated after a 48 h exposure. AgNO3 at the concentration of 0.075 mmol.l-1 and CdCl2 at the concentration of 4 mmol.l-1 induced a ca. 5.2- and 5.6-multiple increase in sanguinarine synthesis, respectively. This amount represents probably the maximal production, because a further increase in the elicitors concentrations did not increase sanguinarine production. Both abiotic elicitors induced a polyphenol oxidase specific activity increase. Polyphenol oxidase is probably involved in the biosynthesis of sanguinarine at the level of dopamine formation. Dopamine is a precursor of (S)-norcoclaurine, the first intermediate with the benzylisoquinoline structure.

[Testing of the potentially probiotic lactobacilli for use in food supplements]. / Testovanie potenciálne probiotických laktobacilov pre pouzitie vo výzivových doplnkoch.

UNLABELLED: Some lactobacilli strains are used as probiotics in food industry and in dietary supplements. Eight lactobacilli strains, originated from the stomach of the lamb and goatling, are tested for their potential use in human and/or veterinary medicine in our department. Sanguinarine is a major alkaloid of Papaveraceae suspension cultures. For its antimicrobial, antifungal, anti-plaque and anti-inflammatory properties sanguinarine found application in dental preparations. A mixture of sanguinarine with other alkaloids is used for appetite stimulation of livestock. This paper is focused on testing of selected lactobacilli sensitivity to sanguinarine, isolated from the opium poppy suspension cultures. L. reuteri KO5 demonstrated the lowest sensitivity (MIC 0.6 mg.l-1). Other less sensitive strains were L. murinus C, L. mucosae D and L. plantarum KG4 (MIC 0.3 mg.l-1). For a combination of more strains of microorganisms in one preparation it is necessary to know their ability to influence their growth and survival. To test the mutual influence of lactobacilli, the streak line method on agar plates was used. The results show that the strains L. reuteri E, L. plantarum KG1 a L. reuteri KO4m are the most suitable ones for the use in combinations. KEYWORDS: Lactobacillus spp. probiotics food supplements sanguinarine.

[Comparison of sanguinarine production in suspension cultures of the Papaveraceae plants]. / Porovnanie produkcie sanguinarínu suspenznými kultúrami rastlín celade Papaveraceae.

Intact plants of the Papaveraceae family are producers of a whole range of benzylisoquinoline alkaloids, which are used in pharmaceutical industry. In vitro cultures derived from plants of the Papaveraceae do not have the ability to produce such a broad spectrum of alkaloids, only the biosynthetic pathway leading to sanguinarine is active. This study deals with the preparation of in vitro cultures of Papaver somniferum, Eschscholtzia californica, Chelidonium majus and Macleaya cordata. Their sanguinarine production abilities were tested and compared. The lowest amounts of sanguinarine from all cultures tested were accumulated in suspension cultures of the opium poppy (0.45-0.55 µg in 1 g of fresh weight). Eschscholtzia californica, Chelidonium majus and Macleaya cordata cultures produced similar amounts of sanguinarine (18.0-22.7 µg; 20.5-26.3 µg; 15.4-20.3 µg in 1 g of fresh weight, resp.). The elicitation study used a biotic stressor, Botrytis cinerea hydrolysate. In all cultures treated, an increase in sanguinarine accumulation was observed. Of all cultures tested, the most intensive response was observed in the opium poppy cultures, although the amount of sanguinarine in the elicited poppy cultures was lower than in the non-elicited samples of the other cultures.

[Bacteriocins produced by lactic acid bacteria]. / Bakteriocíny produkované baktériami mliecneho kvasenia.

Lactic acid bacteria comprise several genera of gram-positive bacteria that are known for the production of structurally different antimicrobial substances. Among them, bacteriocins are nowadays in the centre of scientific interest. Bacteriocins, proteinaceous antimicrobial substances, are produced ribosomally and have usually a narrow spectrum of bacterial growth inhibition. According to their structure and the target of their activity, they are divided into four classes, although there are some suggestions for a renewed classification. The most interesting and usable class are lantibiotics. They comprise the most widely commercially used and well examined bacteriocin, nisin. The non-pathogenic character of lactic acid bacteria is advantageous for using their bacteriocins in food preservation as well as in feed supplements or in veterinary medicine.

Involvement of lipoxygenase in elicitor-stimulated sanguinarine accumulation in Papaver somniferum suspension cultures.

The involvement of lipoxygenase (LOX, EC 1.13.11.12) in elicitor-induced opium poppy defense response was investigated. Papaver somniferum L. suspension cultures were treated with abiotic elicitor methyl jasmonate (MJ), fungal elicitor (Botrytis cinerea homogenate) and phenidone (specific inhibitor of LOX) to determine the involvement of this enzyme in production of sanguinarine, the major secondary metabolite of opium poppy cultures. P. somniferum suspension cultures responded to elicitor treatment with strong and transient increase of LOX activity followed by sanguinarine accumulation. LOX activity increased in elicited cultures, reaching 9.8 times of the initial value at 10 h after MJ application and 2.9 times after B. cinerea application. Sanguinarine accumulated to maximal levels of 169.5 ± 12.5 µg g⁻¹ dry cell weight in MJ-elicited cultures and 288.0 ± 10.0 µg g⁻¹ dry cell weight in B. cinerea-elicited cultures. The treatment of cells with phenidone before elicitor addition, significantly reduced sanguinarine production. The relative molecular weight of P. somniferum LOX (83 kDa) was estimated by using immunobloting and its pH optimum was shown to be pH 6.5.