Two Ecdysteroids Isolated from Micropropagated Lychnis flos-cuculi and the Biological Activity of Plant Material.

Genetically uniform plant material, derived from Lychnis flos-cuculi propagated in vitro, was used for the isolation of 20-hydroxyecdysone and polypodine B and subjected to an evaluation of the antifungal and antiamoebic activity. The activity of 80% aqueous methanolic extracts, their fractions, and isolated ecdysteroids were studied against pathogenic Acanthamoeba castellani. Additionally, a Microtox® acute toxicity assay was performed. It was found that an 80% methanolic fraction of root extract exerts the most potent amoebicidal activity at IC50 of 0.06 mg/mL at the 3rd day of treatment. Both ecdysteroids show comparable activity at IC50 of 0.07 mg/mL. The acute toxicity of 80% fractions at similar concentrations is significantly higher than that of 40% fractions. Crude extracts exhibited moderate antifungal activity, with a minimum inhibitory concentration (MIC) within the range of 1.25-2.5 mg/mL. To the best of our knowledge, the present report is the first to show the biological activity of L. flos-cuculi in terms of the antifungal and antiamoebic activities and acute toxicity. It is also the first isolation of the main ecdysteroids from L. flos-cuculi micropropagated, ecdysteroid-rich plant material.

Micropropagation and Production of Somatic Seeds for Short-Term Storage of the Endangered Species Eryngium alpinum L.

Eryngium alpinum L. is a high-value herb and a source of important compounds that include phenolics, triterpenoid saponins, and essential oils. The present report indicates successful micropropagation of this species. In our study, medium supplemented with BAP 2.0 mg/L, IAA 1.0 mg/L, and GA3 1.0 mg/L was found to be the most suitable for long-term culture and for effective proliferation, irrespective of the passage number. Roots induction, without basal callus formation, was observed when individual microshoots were placed on Murashige & Skoog medium augmented with auxin, and formation was the most advantageous in the presence of NAA alone or when combined with IAA or IBA. The encapsulated propagules were tested for their capability to endure different storage periods under low temperature. Therefore, we developed an efficient method for synseeds production by encapsulation of axillary buds in the sodium alginate matrix, storage for 2, 4, and 6 months, as well as the regeneration process. The maximum regeneration rate of 74% ± 2.72% was observed for axillary buds encapsulated in 4% sodium-alginate complexed with 300 mM calcium chloride after 2 months of storage at low temperature. This is the first report on E. alpinum micropropagation and somatic seeds production.

Phytochemical Screening and Acanthamoebic Activity of Shoots from in Vitro Cultures and in Vivo Plants of Eryngium alpinum L.-The Endangered and Protected Species.

Genetically uniform shoots of Eryngium alpinum L. cultured in vitro were subjected to the qualitative analysis applying the UPLC-HESI-HRMS technique. In vitro cultures give the opportunity to perform the phytochemical studies on the protected species without harvesting the plant material from the natural environment. The phytochemical screening of the crude methanolic extracts of shoots, both from in vitro cultures and in vivo plants, revealed the presence of phenolic acids, coumarins, flavonoids, triterpenoid saponins, amino acids, or dipeptides. Active compounds detected are known to have medicinal importance, and for this reason, the present study represents a preliminary investigation of the extracts against pathogenic and opportunistic amoeba. Among the extracts tested, the extract of shoots from in vitro cultures exhibited remarkable amoebicidal action against trophozoites. On the second day of treatment, the extract at the concentrations of 5 mg/mL, 2.5 mg/mL, and 0.5 mg/mL showed the highest antiamoebicidal effect: the inhibition of trophozoites reached 81.14%, 66.38%, and 54.99%, respectively. To our best knowledge, the present report is the first to show the phytochemical screening and to discuss the antiamoebic activity of Eryngium alpinum L. shoots, both from in vitro cultures and in vivo plants.

Micropropagation of Chaenomeles japonica: A Step towards Production of Polyphenol-rich Extracts Showing Antioxidant and Antimicrobial Activities.

A protocol for C. japonica micropropagation with a confirmation of genome size stability of the in vitro-propagated plantlets was developed. The highest number of shoots multiplied in vitro was obtained on Murashige & Skoog medium (MS) with 1.0 mg L-1 N6-benzyladenine plus 1.0 mg L-1 indole-3-acetic acid. The highest number of roots was observed for the shoots on MS with 15 g L-1 sucrose plus 1.0 mg L-1 indole-3-acetic acid. The acclimatization rate was significantly high. The qualitative HPLC analyses confirmed the presence of phenolic acids and flavonoids in the extracts. The extracts from both shoot cultures and the leaves from field-grown plants revealed antioxidant activity and they exhibited moderate antimicrobial activity. The conducted research confirmed the regeneration potential of genetically-stable plants of C. japonica under in vitro conditions, the ability of the plantlets to produce polyphenols as those present in field-grown plants, as well as their antioxidant potential.

Effect of Pentacyclic Triterpenoids-Rich Callus Extract of Chaenomeles japonica (Thunb.) Lindl. ex Spach on Viability, Morphology, and Proliferation of Normal Human Skin Fibroblasts.

The effect of the well-characterized callus extract of Chaenomeles japonica on viability, morphology, and proliferation of normal human skin fibroblasts was investigated. The phytochemical analysis was performed using the ultra-high performance liquid chromatography method. The total phenolic, phenolic acid, and flavonoid contents were determined spectrophotometrically. The antioxidant activity was investigated using the DPPH (1,1-Diphenyl-1-picrylhydrazyl Radical Scavenging), FRAP (Ferric Reducing Antioxidant Power), and CUPRAC (CUPric Reducing Antioxidant Capacity) assays. The callus growth index during passages was high as well as the content of pentacyclic triterpenoids. The microscopic observations of the fibroblast viability, morphology and the evaluation of the proliferation ratio (xCELLigence system) proved that the influence of callus extract on the fibroblasts was dose-dependent. The evaluated level of fibroblasts proliferation rate after 72 h of incubation with callus extract at concentration 12.5 µg L-1 was the highest compared to all the analyzed ligands. Moreover, callus extract administrated for 72 h caused a significant increase in the proliferation rate in comparison with the control group (5.7 ± 0.1 vs. 4.4 ± 0.9; p < 0.01). The preliminary studies carried out may suggest that the callus extract rich in triterpenoids may be a potential source of cosmetic ingredients with a beneficial effect on human skin.

Comparison of bioactive compounds content in leaf extracts of Passiflora incarnata, P. caerulea and P. alata and in vitro cytotoxic potential on leukemia cell lines

ABSTRACT Passiflora caerulea L., P. alata Curtis and P. incarnata L. (synonym for P. edulis Sims), are the most popular representatives of the Passiflora genus in South America. In recent years, a growing attention is paid to the biological activity and phytochemical profiles of crude extracts from various species of Passiflora in worldwide. The aim of this study was to evaluate and to compare of anti-leukemic activity of the dry crude extracts from leaves of three Passiflora species from greenhouse of Poland in two human acute lymphoblastic leukemia cell lines: CCRF-CEM and its multidrug resistant variant. Two systems of liquid chromatography in order to assessment of phytochemical composition of extracts were applied. Extracts of P. alata and P. incarnata showed the potent inhibitory activity against human acute lymphoblastic leukemia CCRF-CEM, while P. caerulea not showed activity (or activity was poor). Despite similarities in quality phytochemical profile of extracts from P. caerulea and P. incarnata, differences in quantity of chemical compounds may determine their various pharmacological potency. For the activity of P. alata extract the highest content of terpenoids and a lack of flavones C-glycosides are believed to be crucial. Summarizing, the crude extract from P. alata leaves may be considered as a substance for complementary therapy for cancer patients.

THE USE OF EXTRACTS FROM PASSIFLORA SPP. IN HELPING THE TREATMENT OF ACANTHAMOEBIASIS.

Chronic progressive diseases of the central nervous system such as granulomatous amoebic encephalitis, amoebic keratitis, amoebic pneumonitis and also skin infections caused by free-living amoebae (Acanhamoeba spp.) are a significant challenge for pharmacotherapy. This is due to the lack of effective treatment because of encystation, which makes the amoebae highly resistant to anti-amoebic drugs. A very inter- esting and promising source of future drugs in this area are plant materials obtained not only from the habitat but also from plant in vitro culture as an alternative source of biomaterials. Alcoholic extracts from leaves of Passiflora incarnata, P. caerulea, P. alata (Passifloraceae) and from callus cultures were evaluated in vito for amoebicidal activity. Phytochemical analysis showed that all extracts contained phenolic compounds including flavonoids? Biological study revealed that all extracts showed amoebostatic and amoebicidal properties in concentrations from 4 to 12 mg/mL. Extracts of P. alata leaf and callus showed the most effective activities (IC5, 4.01 mg/mL, IC,5 7.29 mg/mL, respectively) after 48 h of exposure, which was correlated with the highest concentration of total phenolics and flavonoids in comparison with other extracts.

Eryngium creticum – ethnopharmacology, phytochemistry and pharmacological activity. A review

Abstract Eryngium creticum Lam. (E. cyaneum Sibth. & Sm., E. syriacum Lam.), Saniculoideae, Apiaceae is of great importance in the traditional Greco – Arab medicine. This study was carried out in order to contribute to the ethnopharmacological knowledge of this medicinal species. This review describes the botanical characterization and distribution, as well as critically assesses the phytochemical properties and biological activities of E. creticum, a species that has been used in traditional medicine for many decades. Possible trends and perspectives for future research of this plant are discussed, as well. E. creticum has been found to contain several chemical constituents, mostly sesquiterpenes, monoterpenes, aldehydes, coumarins, sitosterols and sugars. Eryngo with its bioactive compounds possesses a wild range of biological activities. It was reported that in traditional medicine E. creticum was applied mainly as the remedy for snake and scorpion bites. Some published studies have shown a broad spectrum of biological and pharmacological activities, including anti-snake and anti-scorpion venom, as well as antibacterial, antifungal and antileishmanial effects. Other have indicated antihyperglycemic, hypoglycemic and antioxidant activities of this species. The in vitro studies and in vivo models have provided a simple bioscientific explanation for its various ethnopharmacological uses.

Improvement in Long-Term Memory following Chronic Administration of Eryngium planum Root Extract in Scopolamine Model: Behavioral and Molecular Study.

Eryngium planum L. (EP) is as a rare medicinal plant with a lot of potentials as pharmaceutical crops. The aim of our study was to assess the effect of subchronic (28-fold) administration of a 70% ethanol extract of EP roots (200 mg/kg, p.o.) on behavioral and cognitive responses in Wistar rats linked with acetylcholinesterase (AChE), butyrylcholinesterase (BuChE), and beta-secretase (BACE-1) mRNA levels and AChE and BuChE activities in the hippocampus and frontal cortex. On the last day of experiment, 30 min after the last dose of EP or Huperzine A (HU), scopolamine (SC) was given at a dose of 0.5 mg/kg b.w. intraperitoneally. The results of a passive avoidance test showed an improvement in long-term memory produced by the EP extract in both scopolamine-induced rats and control group. EP caused an insignificant inhibition of AChE and BuChE activities in the frontal cortex and the hippocampus. EP decreased mRNA AChE, BuChE, and BACE-1 levels, especially in the cortex. Our results suggest that the EP extract led to the improvement of the long-term memory in rats coupled with total saponin content. The mechanism of EP action is probably complicated, since HPLC-MS analysis showed 64 chemical compounds (phenolics, saponins) in the extract of EP roots.

Methyl jasmonate, yeast extract and sucrose stimulate phenolic acids accumulation in Eryngium planum L. shoot cultures.

Eryngium planum L. has been reported as a medicinal plant used in traditional medicine in Europe. The tissue cultures may be an alternative source of the biomass rich in desired bioactive compounds. The purpose of this study was to investigate the influence of the biotechnological techniques on the selected phenolic acids accumulation in the agitated shoot cultures of E. planum. Qualitative and quantitative analyses of those compounds in 50% aqueous - methanolic extracts from the biomass were conducted by applying the HPLC method. Methyl jasmonate (MeJA), yeast extract (YE) and sucrose (Suc) stimulated accumulation of the phenolic acids: rosmarinic (RA), chlorogenic (CGA) and caffeic (CA) in in vitro shoot cultures. Cultivation of shoots in liquid MS media supplemented with 1.0 mg L(-1) 6-benzyladenine and 0.1 mg L(-1) indole-3-acetic acid in the presence of 100 µM MeJA for 48h was an optimum condition of elicitation and resulted in approximately 4.5-fold increased content of RA + CGA + CA in plant material compared to the control (19.795 mg g(-1) DW, 4.36 mg g(-1) DW, respectively). The results provide the first evidence that the selected phenolic acids can be synthesized in elicited shoot cultures of flat sea holly in higher amount than in untreated shoots.

Ragged Robin (Lychnis flos-cuculi) - a plant with potential medicinal value

Lychnis flos-cuculi L., Caryophyllaceae, contains a number of active compounds belonging to several chemical groups. Previous studies have led to the identification of phytoecdysteroids, triterpenoids saponins, volatile compounds, fatty acid derivatives, phenolic acids and flavonoids. Research on pharmacological activity showed that plant extracts inhibited the growth of bacteria and fungi. The antimitotic properties of preparations from the herb L. flos-cuculi were also reported. The phytochemical analyses demonstrated that this taxon contains pharmaceutically promising compounds, but more phytochemical and pharmacological studies of L. flos-cuculi are needed for further information regarding this plant. This review summarizes reports regarding chemical composition and biological activity of L. flos-cuculi as well as several cognate species, which pose opportunities related to in vitro propagation and cell and tissue cultures. In vitro-regenerated plantlets could be a good source of genetically uniform plant material for future research.

Progress in micropropagation of Passiflora spp. to produce medicinal plants: a mini-review

Micropropagation of Passiflora species and its hybrids may play an important role in the production of healthy and disease-free plants which can be a source of medicinal herbal products, nutritional fruits and ornamental flowers. The rapid multiplication of elite plants to obtain pharmacognostic material, containing valuable flavonoid C-glycosides, is possible by usingcontrolled in vitro conditions, constituents of the medium and the interactions of plant growth regulators (1-naphtaleneacetic acid, benzyladenine, gibberellin GA3,kinetin, indole-3-acetyl-L-aspartic acid, indole-3-butyric acid, thidiazuron) and influencing various chemical additives (silver nitrate, coconut water, activated charcoal). Investigations of specific requirements during stages of micropropagation, such as the establishment of primary cultures (including type of explants, age of donor plant), shoot multiplication (by direct and indirect organogenesis and embryogenesis), rooting and acclimatization of regenerated plants are summarized in this review. The following species were recently studied for micropropagation: P. alata, P. caerulea, P. cincinnata, P. edulis, P. foetida, P. setacea, P. suberosa. It seems that for awide range of applications of in vitro clones of Passiflora, interdisciplinary studies including genetic and phytochemical aspects are needed.

The evaluation of the amebicidal activity of Eryngium planum extracts.

Selected fractions of ethanolic extracts obtained from leaves and roots of Eryngium planum (Apiaceae) were evaluated in vitro for amebicidal activity against Acanthamoeba castellanii. This free-living ameba is the cause of Acanthamoeba keratitis, which is a painful, vision-impairing disease of the eyes, and chronic granulomatous amebic encephalitis. Treatment is very difficult and not always effective because of encystation, which makes the amebae highly resistant to anti-amebic drugs. The search for novel natural amebicidal agents is still of current interest. Fractions of E. planum ethanolic extract from basal leaves: flavonoid fraction (Lf), flavonoid-saponin fraction (Lf-s), saponin fraction (Ls) and phenolic acids fraction (La) and from roots: saponin fraction (Rs) and phenolic acids fraction (Ra) were assayed for antiamebic activity. In the presence of the saponin fractions and phenolic acid fractions (ranging from 1-5 mg/mL), the number of the trofozoites of Acanthamoeba castellanii viable strain 309 decreased during the experimental period (0-72 h). On the other hand, the flavonoid fraction from leaves showed a stimulating activity on the amebae. Almost all fractions (except the flavonoid fraction) showed a time- and dose-dependent amebistatic activity on the trophozoites. Of the fractions tested, the phenolic acid fraction from roots at the concentration of 5 mg/L showed the amebicidal activity on the trophozoites.

[Medicinal plants in the phytotherapy of alcohol or nicotine addiction. Implication for plants in vitro cultures]. / Rosliny lecznicze stosowane w fitoterapii uzaleinien od nikotyny lub alkoholu--implikacje dla zastosowania roslinnych kultur in vitro.

The increasing problem of nicotine and alcohol addiction, and small availability of drugs in the pharmacologic treatment causes that there are still looking for new drugs that could be used in addiction prevention and relief of withdrawal symptoms. Currently, attention has focused on a number of species possessed above mentioned pharmacological profile that do not occur naturally in moderate climate in Poland, including Passiflora incarnata, Pueraria lobata, Salvia miltiorrhiza, Salvia przewalskii. A rich source of biologically active compounds showing their possible benefit against addiction are plant derived both from its natural state as well as by biotechnological methods. Studies using in vitro plant cultures allow receiving material containing interesting secondary metabolites (active compounds) in the of shoots, root, callus and suspension cultures. Overview of pharmacological studies showed that several experiments carried out in animal models of alcoholism, and only few studies have been done on nicotine addiction using herbs. It has been shown that an extract of the herb Passiflora incarnata (and its benzoflavone derivative-BZF) can be an interesting plant material that could reduce the intensity of nicotine or alcohol withdrawal symptoms, however, only few studies have been published in this area. A larger amount of evidence has been provided to the anti-alcohol effect of the extract from the root of Pueraria lobata (kudzu). It is known that kudzu root extract is effective at reducing alcohol intake in animals and in humans. The three major isoflavones present in kudzu extracts, daidzin, daidzein and puerarin are responsible for the beneficial effects in reduction of alcohol consumption, although the exact mechanism by which kudzu suppresses ethanol intake remains to be clarified. It has been proven that daidzin in vitro is a strong, selective and reversible inhibitor of aldehyde dehydrogenase. Moreover, studies on the CNS receptor gene expression showed that the extract of kudzu possibly acts through opioid system and exhibits antagonist activity by influencing the opioid receptors mi, delta and the expression of endogenous opioid precursors (proopiomelanocortin) similarly as naltrexone. Besides kudzu, also pre-clinical data suggest that extracts from Salvia miltiorrhiza and Salvia przewalskii are effective in reducing voluntary alcohol intake in animal models of excessive alcohol drinking and their main active compounds - tanshinones and miltiron are responsible for this effect. In summary, there is a need for further studies on the mechanisms of plant extracts and their active compounds action that are valuable alternative way for the prevention and treatment of various drug dependences.

Plant extracts as natural amoebicidal agents.

Strains of Acanthamoeba sp. constitute a factor contributing to the occurrence of chronic granulomatous amoebic encephalitis, keratitis, pneumonia, as well as inflammations of other organs. Treatment of these diseases is very difficult and not always effective. A majority of these infections have been fatal. The aim of our study was to examine the amoebicidal or amoebistatic activity of plant extracts from Rubus chamaemorus, Pueraria lobata, Solidago virgaurea and Solidago graminifolia. For the purpose of isolation of pharmacologically active substances, we used the aboveground parts of plants, together with flowers, roots and leaves. It was established that extracts from S. virgauera, P. lobata and R. chamaemorus displayed chemotherapeutic properties in vitro in concentrations of approximately 0.01-0.05 mg extract/mL, i.e., in concentrations of 0.350 microg/mL expressed in ellagic acid for R. chamaemorus and 0.053 microg/mL expressed in puerarin for P. lobata. Therapeutic index values is 3.5-20. As a result of in vivo experiments, it was found out that, following therapy using the extracts, animals infected with Acanthamoeba sp. survived for an extended period (2.5-3 times longer). It was determined that plant extracts may be used both externally and internally in the case of a combined therapy for acanthamoebiasis. The tested extracts are not toxic for animals.

[Amebicidal plants extracts]. / Pelzakobójcze ekstrakty roslinne.

The free-living amoebae from genus Acanthamoeba are the causative agents of granulomatous amebic encephalitis (GAE), a chronic progressive disease of the central nervous system; amebic keratitis (AK), a chronic eye infection; amebic pneumitis (AP), a chronic lung infection, and skin infection. Chemotherapy of Acanthamoeba infection is problematic. The majority of infections have been fatal. Only a few cases are reported to have been treated successfully with very highly toxic drugs. The therapy might be succeed, if the diagnosis and therapy is made at very early stage of infection. In our experiments we used the following plant extracts: Solidago virgaurea, Solidago graminifolia, Rubus chamaemorus, Pueraria lobata, and natural plants products as ellagic acid and puerarin. Those therapeutic agents and plants extracts have been tested in vitro for amebicidal or amebostatic activity against pathogenic Acanthamoeba spp. Our results showed that methanol extracts obtained from plants are active against axenic pathogenic Acanthamoeba sp. trophozoites in vitro at concentration below 0.1 mg/ml. Further studies are needed to investigate whether these extracts are also effective in vivo in animal model of infection with Acanthamoeba sp.

Antimicrobial activity of Solidago virgaurea L. from in vitro cultures.

The antimicrobial activity of ethanolic and methanolic extracts of Solidago virgaurea herb from micropropagated plants and callus tissue was evaluated.