Eleutherococcus divaricatus Fruits Decrease Hyaluronidase Activity in Blood Serum and Protect from Antioxidative Damages in In Vitro Model.

Fruits are very important dietary components and a source of biologically active compounds used in nutritional pharmacology. Particularly due to the presence of polyphenolic compounds, fruits play an important role in the prevention of diseases of civilization. Therefore, it is important to study the phytochemicals and biological activity of fruits, especially those with a long-standing use in ethnomedicine. In this study, we determined the chemical profile and biological activity of a methanolic extract of the Eleutherococcus divaricatus fruits. Amongst nine polyphenols studied, only chlorogenic acid, protocatechuic acid, and eleutheroside E have been detected. The extract showed a weak anti-hyaluronidase activity from bovine testicular in a range of 9.06-37.70% and quite high for human serum hyaluronidase from children diagnosed with acute leukemia in a range of 76-86%. A weak anti-tyrosinase activity was obtained in a range of 2.94-12.46%. Moreover, the extract showed antioxidant properties against DPPH radical, ABTS radical, and O2•-. In addition, the antioxidant activity of the extract was evaluated by FRAP assay and Fe2+ ion chelation assay. These preliminary studies partially justify the traditional use of the plant in inflammatory- and immune-related diseases, in which hyaluronidase and free radicals can participate. A difference in human serum hyaluronidase inhibition may result from the inter-patient variability. Regardless of that, the results mean that polyphenolic compounds may stimulate activity of hyaluronidase, as well as to protect cells from the oxidative damages. However, further studies in ex vivo and in vivo models are needed, including blood isolated from a larger number of patients.

Phenolic Compounds and Antioxidant and Anti-Enzymatic Activities of Selected Adaptogenic Plants from South America, Asia, and Africa.

Despite the fact that there are many studies related to the adaptogenic and pro-healthy activities of plant-based compounds, there are some adaptogenic plants whose activities are not fully known, especially those coming from the wild regions of Asia, Africa, and South America. The aim of these studies was to examine the contents of non-nutritional compounds, such as polyphenols, flavonoids, and phenolic acids in ten adaptogenic species (Astragalus membranaceus (AM), Uncaria rhynchophylla (UR), Polygonum multiflorum (PM), Angelica sinensis (AS), Andrographis paniculatea (AP), Tinospora cordifolia (TC), Uncaria tomentosa (UT), Pfaffia paniculate (PP), Sutherlandia frutescens (SF), and Rhaponticum carthamoides (RC)). Considering biological activity, their antioxidant (DPPH, ABTS, FRAP, and ferrous-ion-chelating ability assays), anti-acetylcholinesterase, anti-hyaluronidase, and anti-tyrosinase activities were evaluated. The richest in polyphenols, flavonoids, and phenolic acids was UR (327.78 mg GAE/g, 230.13 mg QE/g, and 81.03 mg CA/g, respectively). The highest inhibitions of acetylcholinesterase, hyaluronidase, and tyrosinase were observed for TC, UR, and PM, respectively. In the case of antioxidant properties, extract from PM appeared to most strongly reduce DPPH, extract from UR inhibited ABTS, and extract from SF showed the best chelating properties. It should be noted that a particularly interesting plant was Ulcaria rhynchophylla. The results mean that there were compounds in UR with broad biological activities, and this species should be explored in more detail. Additionally, our results justify the traditional use of these species in the nutripharmacological or ethnopharmacological care systems of different regions.

Paving the way towards effective plant-based inhibitors of hyaluronidase and tyrosinase: a critical review on a structure-activity relationship.

Human has used plants to treat many civilisation diseases for thousands of years. Examples include reserpine (hypertension therapy), digoxin (myocardial diseases), vinblastine and vincristine (cancers), and opioids (palliative treatment). Plants are a rich source of natural metabolites with multiple biological activities, and the use of modern approaches and tools allowed finally for more effective bioprospecting. The new phytochemicals are hyaluronidase (Hyal) inhibitors, which could serve as anti-cancer drugs, male contraceptives, and an antidote against venoms. In turn, tyrosinase inhibitors can be used in cosmetics/pharmaceuticals as whitening agents and to treat skin pigmentation disorders. However, the activity of these inhibitors is stricte dependent on their structure and the presence of the chemical groups, e.g. carbonyl or hydroxyl. This review aims to provide comprehensive and in-depth evidence related to the anti-tyrosinase and anti-Hyal activity of phytochemicals as well as confirming their efficiency and future perspectives.

Effectiveness of Volatile Natural Deep Eutectic Solvents (VNADESs) for the Green Extraction of Chelidonium majus Isoquinoline Alkaloids.

The Chelidonium majus plant is rich in biologically active isoquinoline alkaloids. These alkaline polar compounds are isolated from raw materials with the use of acidified water or methanol; next, after alkalisation of the extract, they are extracted using chloroform or dichloromethane. This procedure requires the use of toxic solvents. The present study assessed the possibility of using volatile natural deep eutectic solvents (VNADESs) for the efficient and environmentally friendly extraction of Chelidonium alkaloids. The roots and herb of the plant were subjected three times to extraction with various menthol, thymol, and camphor mixtures and with water and methanol (acidified and nonacidified). It has been shown that alkaloids can be efficiently isolated using menthol-camphor and menthol-thymol mixtures. In comparison with the extraction with acidified methanol, the use of appropriate VNADESs formulations yielded higher amounts of protopine (by 16%), chelidonine (35%), berberine (76%), chelerythrine (12%), and coptisine (180%). Sanguinarine extraction efficiency was at the same level. Additionally, the values of the contact angles of the raw materials treated with the tested solvents were assessed, and higher wetting dynamics were observed in the case of VNADESs when compared with water. These results suggest that VNADESs can be used for the efficient and environmentally friendly extraction of Chelidonium alkaloids.

Phenolic Profile, Antioxidant, Anti-Enzymatic and Cytotoxic Activity of the Fruits and Roots of Eleutherococcus senticosus (Rupr. et Maxim.) Maxim.

Eleutherococcus senticosus (Rupr. et Maxim.) Maxim. is well-known for its adaptogenic properties in traditional Eastern medicine. It has been categorized as an endangered species due to the over-exploitation of the roots. As a result, alternatives must be found, including the usage of renewable aerial parts such as fruits. The goal of this research was to determine the phenolic compounds and the enzymatic, antioxidant, and cytotoxic activities of the intractum gained from the E. senticosus fruits and the mixture of chloroform-methanol roots extract with naringenin (3:7:5). The obtained results showed, that the intractum contained 1.02 mg/g ext. of polyphenols, 0.30 mg/g ext. of flavonoids, and 0.19 mg/g ext. of phenolic acids. In turn, the mixture of chloroform-methanol roots extract with naringenin (3:7:5) contained 159.27 mg/g ext. of polyphenols, 137.47 mg/g ext. of flavonoids, and 79.99 mg/g ext. of phenolic acids. Regarding the anti-enzymatic assay, the IC50 values for tyrosinase and hyaluronidase were equal to 586.83 and 217.44 [µg/mL] for the intractum, and 162.56 and 44.80 [µg/mL] for the mixture, respectively. Both preparations have possessed significant antioxidant activity in the ABTS, DPPH, and ferrozine tests. No cytotoxic effect on the FaDu and HEP G2 cancer cell lines was observed. Our findings support the traditional use of fruits and roots. Moreover, the results indicate also that adaptogens are rather nontoxic for normal and cancer cells, which corresponds with some hypotheses on adaptogens activity.

Pharmacognostic Evaluation and HPLC-PDA and HS-SPME/GC-MS Metabolomic Profiling of Eleutherococcus senticosus Fruits.

Eleutherococcus senticosus (Rupr. et Maxim.) Maxim. is a medicinal plant used in Traditional Chinese Medicine (TCM) for thousands of years. However, due to the overexploitation, this species is considered to be endangered and is included in the Red List, e.g., in the Republic of Korea. Therefore, a new source of this important plant in Europe is needed. The aim of this study was to develop pharmacognostic and phytochemical parameters of the fruits. The content of polyphenols (eleutherosides B, E, E1) and phenolic acids in the different parts of the fruits, as well as tocopherols, fatty acids in the oil, and volatile constituents were studied by the means of chromatographic techniques [HPLC with Photodiode-Array Detection (PDA), headspace solid-phase microextraction coupled to gas chromatography-mass spectrometry (HS-SPME/GC-MS)]. To the best of our knowledge, no information is available on the content of eleutherosides and phenolic acids in the pericarp and seeds. The highest sum of eleutheroside B and E was detected in the whole fruits (1.4 mg/g), next in the pericarp (1.23 mg/g) and the seeds (0.85 mg/g). Amongst chlorogenic acid derivatives (3-CQA, 4-CQA, 5-CQA), 3-CQA was predominant in the whole fruits (1.08 mg/g), next in the pericarp (0.66 mg/g), and the seeds (0.076 mg/g). The oil was rich in linoleic acid (C18:3 (n-3), 18.24%), ursolic acid (35.72 mg/g), and α-tocopherol (8.36 mg/g). The presence of druses and yellow oil droplets in the inner zone of the mesocarp and chromoplasts in the outer zone can be used as anatomical markers. These studies provide a phytochemical proof for accumulation of polyphenols mainly in the pericarp, and these structures may be taken into consideration as their source subjected to extraction to obtain polyphenol-rich extracts.

Morphological, Anatomical, and Phytochemical Studies of Carlina acaulis L. Cypsela.

Carlina acaulis L. has a long tradition of use in folk medicine. The chemical composition of the roots and green parts of the plant is quite well known. There is the lowest amount of data on the cypsela (fruit) of this plant. In this study, the microscopic structures and the chemical composition of the cypsela were investigated. Preliminary cytochemical studies of the structure of the Carlina acaulis L. cypsela showed the presence of substantial amounts of protein and lipophilic substances. The chemical composition of the cypsela was investigated using spectrophotometry, gas chromatography with mass spectrometry, and high-performance liquid chromatography with spectrophotometric and fluorescence detection. The cypsela has been shown to be a rich source of macro- and microelements, vegetable oil (25%), α-tocopherol (approx. 2 g/kg of oil), protein (approx. 36% seed weight), and chlorogenic acids (approx. 22 g/kg seed weight). It also contains a complex set of volatile compounds. The C. acaulis cypsela is, therefore, a valuable source of nutrients and bioactive substances.

Extracts from Eleutherococcus senticosus (Rupr. et Maxim.) Maxim. Roots: A New Hope Against Honeybee Death Caused by Nosemosis.

Pollinators, the cornerstones of our terrestrial ecosystem, have been at the very core of our anxiety. This is because we can nowadays observe a dangerous decline in the number of insects. With the numbers of pollinators dramatically declining worldwide, the scientific community has been growing more and more concerned about the future of insects as fundamental elements of most terrestrial ecosystems. Trying to address this issue, we looked for substances that might increase bee resistance. To this end, we checked the effects of plant-based adaptogens on honeybees in laboratory tests and during field studies on 30 honeybee colonies during two seasons. In this study, we have tested extracts obtained from: Eleutherococcus senticosus, Garcinia cambogia, Panax ginseng, Ginkgo biloba, Schisandra chinensis, and Camellia sinensis. The 75% ethanol E. senticosus root extract proved to be the most effective, both as a cure and in the prophylaxis of nosemosis. Therefore, Eleutherococcus senticosus, and its active compounds, eleutherosides, are considered the most powerful adaptogens, in the pool of all extracts that were selected for screening, for supporting immunity and improving resistance of honeybees. The optimum effective concentration of 0.4 mg/mL E. senticosus extract responded to c.a. 5.76, 2.56 and 0.07 µg/mL of eleutheroside B, eleutheroside E and naringenin, respectively. The effect of E. senticosus extracts on honeybees involved a similar adaptogenic response as on other animals, including humans. In this research, we show for the first time such an adaptogenic impact on invertebrates, i.e., the effect on honeybees stressed by nosemosis. We additionally hypothesised that these adaptogenic properties were connected with eleutherosides-secondary metabolites found exclusively in the Eleutherococcus genus and undetected in other studied extracts. As was indicated in this study, eleutherosides are very stable chemically and can be found in extracts in similar amounts even after two years from extraction. Considering the role bees play in nature, we may conclude that demonstrating the adaptogenic properties which plant extracts have in insects is the most significant finding resulting from this research. This knowledge might bring to fruition numerous economic and ecological benefits.

Polysaccharide-Rich Fractions from Rosa rugosa Thunb.-Composition and Chemopreventive Potential.

The huge health-beneficial potential of polysaccharides encourages the search for novel sources and applications of these compounds. One poorly explored source of polysaccharides is the rose. The content and biological activity of polysaccharides in rose organs is an almost completely unaddressed topic, therefore, polysaccharide-rich extracts (crude polysaccharides, CPLs) from petals, leaves, hips, and achenes of Rosa rugosa Thunb. were studied for their composition and the influence on various cellular processes involved in the development of cancer and other civilization diseases. The study revealed the presence of water-soluble and -insoluble polysaccharides (including ß-glucans) and protein-polysaccharide conjugates in rose organs. Rose hips were found to be the most abundant source of polysaccharides. Different polysaccharide-rich extracts showed the ability to inhibit pro-inflammatory enzymes (COX-1, COX-2, hyaluronidase), a radical scavenging effect (against DPPH• and ABTS•+), and antiproliferative activity (in the A549 lung and SW480 colon cancer cell lines) in in vitro assays. Therefore, rose crude polysaccharides are very promising and can potentially be used as natural chemopreventive agents.

Effects of Glutathione on Hydrolytic Enzyme Activity in the Mouse Hepatocytes.

In this study, the effect of glutathione (GSH) on the activity of hydrolytic enzymes of lysosomal, microsomal, and cytosolic fractions was studied in the mouse hepatocytes. The experiments involved 30 Swiss male mice, divided into the experimental and control groups of 15 mice each. The former group received injections of 12 µL/g of GSH solution at a dose of 100 µg/g body weight, whereas the latter received 12 µL/g of physiological saline, all given intraperitoneally daily for 7 days. Then, fragments of liver tissue were collected from the euthanized animals and processed to obtain lysosomal, microsomal, and cytosolic fractions of hepatocytes. The activity of the following enzymes was investigated in vitro: ß-glucuronidase, ß-galactosidase, ß-glucosidase, N-acetyl-hexosaminidase, lysosomal esterase and lipase, acid phosphatase, cathepsin D and L, leucine aminopeptidase, and alanine aminopeptidase. We found that GSH, administered in vivo in the mouse, in the main (73% cases), increased the in vitro activity of the majority enzymes abovementioned, although the effect was somehow variable, depending on the fraction of hepatocytes and the type of enzyme. The findings imply that GSH supplementation may intensify the rate of cellular hydrolytic degradation, i.e., the rate of disposal by the cell of unwanted materials.

LC-ESI-MS/MS profiling of phenolics from Eleutherococcus spp. inflorescences, structure-activity relationship as antioxidants, inhibitors of hyaluronidase and acetylcholinesterase.

Nature is a source of many plant-based molecules used as pro- or drugs. Eleutherococcus species are native to Asia and the North Russia, and are traditionally used to treat various diseases. In turn, neither secondary metabolites of the species cultivated in the West Europe nor the bioactivity is known. No differences in the phenols and flavonoids content in the inflorescences were found. The richest in polyphenols was E. giraldii (5.18 mg/g), while in flavonoids it was E. gracilistylus (1.80 mg/g). Using LC-ESI-MS/MS, protocatechuic and trans-caffeic acids have been identified as the most abundant compounds in E. gracilistylus, E. giraldii, E. senticosus (833.4; 855.6; 614.7 and 280.8; 156.0; 167.6 µg/g DE). It was observed that all species were able to chelate Fe2+ with the EC50 value of 0.2, 0.6, 0.3 mg/mL for E. gracilistylus, E. giraldii, E. senticosus, respectively. E. gracilistylus exhibited the strongest antiperoxidation and anti-DPPH∗ activity (EC50 3.2 and 0.48 mg/mL). The weak inhibitory potential has been observed in case of AChE inhibition at the level of 16.17 and 12.2% for E. gracilistylus, E. giraldii. We report for the first time that the extracts inhibited Hyal activity in the range from 16.4 to 60.7%. To our best knowledge, no information was available on this activity of the inflorescences and this provides a background to study inflorescences in more detail. Considering the SAR, an antioxidant activity may be correlated with a high amount of protocatechuic and trans-caffeic acids and their chemical structure.

TLC profiling, nutritional and pharmacological properties of Siberian ginseng (Eleutherococcus senticosus) cultivated in Poland.

The chemical composition and pharmacological activity of E. senticosus cultivated in Poland were investigated. Studies included the assay of TPC and TFC, 2D-TLC identification of phenolic acids, HPTLC-detection of antioxidants, and antioxidative, antileukemic, anti-MMPs properties of E. senticosus. The ethanolic extracts from the roots, spring leaves, fruits, and the chloroform extract from the roots were tested. The richest in polyphenols are the fresh fruits (57.5 mg/g), while in flavonoids the spring leaves (27.4 mg/g). The antioxidant ability both in extracts and single phenolic constituents were checked out by the measurement of the DPPH radical scavenging activity, iron (II) chelating and lipid peroxidation inhibitory activity. Using HPTLC-DB test eleutherosides B and E1 have been found as the phenolic antioxidants. Thirty six percent of apoptotic cells have been observed in Jurkatt 45 line by the treatment with the chloroform extract from the roots. Only the chloroform extract from the roots and the ethanolic one from the dried fruits have shown the inhibitory activities against MMPs. It is noteworthy, that our studies have been done for the first time, and the plant material has come from another geographical zone (Poland) than native (Asia).

The Eleutherococcus senticosus fruits' intractum affects changes in the transepithelial electric potential in the distal section of the rabbit's large intestine and inhibits hyaluronidase.

ETHNOPHARMACOLOGICAL RELEVANCE: Eleutherococcus senticosus (Rupr. et Maxim.) Maxim. has been used in traditional Russian medicine due to its recognized immunostimulant and anti-inflammatory activities. Compounds present in the fruits have demonstrated the capability to modulate the activity of enzymes such as hyaluronidase, suggesting their potential value in the development of effective therapies for various conditions where anti-inflammatory properties are beneficial, such as gastrointestinal diseases and tumor growth. AIM OF THE STUDY: In order to support the use of the fruits in folk medicine, this study is aimed to evaluate, post-mortem, the impact of E. senticosus fruits intractum (40 % extract made from fresh fruits) on the transepithelial electrogenic transport of sodium ions in the colon. The objective of this study was also to examine the impact of the intractum on proinflammatory serum hyaluronidase in children diagnosed with acute leukemia. METHODS: The study employed the Ussing technique to examine electrophysiological characteristics of isolated epithelial tissue, using the distal colon wall isolated from 10 New Zealand white male rabbits. The effect of the intractum on the inhibition of human serum hyaluronidase was examined with turbidimetric screening methods, using the blood samples collected from patients diagnosed with acute leukemia. RESULTS: For the first time, we discovered that the intractum used in the stimulation fluid, caused hyperpolarization reactions in colon tissue. Statistical analysis showed that these reactions were significantly different in relation to the control. The intractum significantly inhibited hyaluronidase activity with the mean value by group of 60 %, and 40 % for aescin used as a control. CONCLUSION: The results support the traditional use of the fruits in inflammatory-related diseases. The use of intractum of E. senticosus on the distal colon wall demonstrates its protective effect on the wall integrity and in a relation to hyaluronidase inhibition may additionally indicate its anti-inflammatory property. Thus, the results mean that the intractum may be used in colon-related diseases.

Evaluation of the yield, chemical composition and biological properties of essential oil from bioreactor-grown cultures of Salvia apiana microshoots.

Microshoot cultures of the North American endemic Salvia apiana were established for the first time and evaluated for essential oil production. Stationary cultures, grown on Schenk-Hildebrandt (SH) medium, supplemented with 0.22 mg/L thidiazuron (TDZ), 2.0 mg/L 6-benzylaminopurine and 3.0% (w/v) sucrose, accumulated 1.27% (v/m dry weight) essential oil, consisting mostly of 1,8-cineole, ß-pinene, α-pinene, ß-myrcene and camphor. The microshoots were adapted to agitated culture, showing biomass yields up to ca. 19 g/L. Scale-up studies demonstrated that S. spiana microshoots grow well in temporary immersion systems (TIS). In the RITA bioreactor, up to 19.27 g/L dry biomass was obtained, containing 1.1% oil with up to ca. 42% cineole content. The other systems employed, i.e. Plantform (TIS) and a custom made spray bioreactor (SGB), yielded ca. 18 and 19 g/L dry weight, respectively. The essential oil content of Plantform and SGB-grown microshoots was comparable to RITA bioreactor, however, the content of cineole was substantially higher (ca. 55%). Oil samples isolated from in vitro material proved to be active in acetylcholinesterase (up to 60.0% inhibition recorded for Plantform-grown microshoots), as well as hyaluronidase and tyrosinase-inhibitory assays (up to 45.8 and 64.5% inhibition observed in the case of the SGB culture).

Effectiveness and patient satisfaction of dental emergencies in Pitié Salpêtrière Hospital, Paris, during the COVID 19 pandemic.

Background: A previous study reported the effectiveness and patient satisfaction in the dental emergency unit (DEU) of the Pitie Salpetrière Hospital in Paris before coronavirus disease 2019 (COVID-19). The same methodology was used during the COVID-19 pandemic to compare pain, anxiety, and patient satisfaction during the two periods. Methods: This prospective study was conducted in 2020 (NCT04354272) on adult patients. Data were collected on day zero (D0) on site and then by phone during the daytime on day one (D1), day three (D3), and day seven (D7). The primary objective was to assess the pain intensity at D1. Secondary objectives were to assess pain intensity at D3 and D7, anxiety intensity at D1, D3, and D7, and patient satisfaction. Patients were evaluated on a 0-10 numeric scale on D1, D3, and D7; mean scores were compared with non-parametric statistics (ANOVA, Dunn's). Results: A total of 445 patients were given the opportunity to participate in the study, and 370 patients consented. Seventy-one were lost during follow-up. Ultimately, 299 patients completed all the questionnaires and were included in the analysis. In the final sample (60% men, 40% women, aged 39 ± 14 years), 94% had health insurance. The mean pain scores were: D0, 6.1 ± 0.14; D1, 3.29 ± 0.16; D3, 2.08 ± 0.16; and D7, 1.07 ± 0.35. This indicates a significant decrease of 46%, 67%, and 82% at D1, D3, and D7, respectively, when compared to D0 (P < 0.0001). The mean anxiety scores were D0, 4.7 ± 0.19; D1, 2.6 ± 0.16; D3, 1.9 ± 0.61; and D7, 1.4 ± 0.15. This decrease was significant between D0 and D7 (ANOVA, P < 0.001). Perception of general health improved between D1 and D7. The overall satisfaction was 9.3 ± 0.06. Conclusion: DEU enabled a significant reduction in pain and anxiety with high overall satisfaction during COVID-19, which was very similar to levels observed pre-COVID-19 pandemic.

Phenolic acids as chemotaxonomic markers able to differentiate the Euphrasia species.

The objective of this study was to get the answer on the question, which phenolic compounds may serve as chemical markers in taxonomy of Euphrasia spp. (E. nemorosa, E. rostkoviana, E. stricta) collected from the wild. Moreover, it is still unknown how and which environmental parameters can impact on the quantity of polyphenols, flavonoids and phenolic acids. To the authors' knowledge, this is the first study that analyses such a broad spectrum of phenolic compounds in the genus Euphrasia and takes into account the habitat conditions of their synthesis. The species of Euphrasia differ significantly in the content of phenolic compounds. Euphrasia rostkoviana has the highest total content of polyphenols and flavonoids. Euphrasia stricta contains the largest amounts of phenolic acids, such as 5-O-caffeoylquinic, p-coumaric, protocatechuic and salicylic acid. It has been evidenced that the content of metabolites in the Euphrasia herb depends on a number of habitat parameters. The increased content of phenolic acids in E. stricta can be attributed to the fact that this species occupies drier habitats, with lower content of nitrogen and organic carbon compared to E. nemorosa and E. rostkoviana. The compounds that can be considered as chemotaxonomic markers are salicylic and protocatechuic acid for E. stricta, 5-O-caffeoylquinic acid for E. rostkoviana and ferulic acid for E. nemorosa. These findings provide theoretical and empirical basis for a chemotaxonomic classification of those species, which taken together with morphological characteristics should prevent misidentification.

Eleutherosides in aerial parts of Eleutherococcus species cultivated in Poland.

Many Eleutherococcus species grow in Siberia, China, Korea, Japan, and the Philippines. The most well known is Eleutherococcus senticosus, which contains pharmacologically active compounds, such as eleutherosides, flavonoids, vitamins, and complex polysaccharides. E. senticosus owes its medicinal properties mainly to eleutherosides. The objective of this study was to determine eleutherosides B, E, and E1 in the aerial parts of different Eleutherococcus species. The eleutherosides were extracted with ethanol, and the extracts were cleaned by SPE on C18 columns, and then analyzed by HPTLC. Silica gel plates with fluorescence indicator, designated F254, were used with chloroform-methanol-water (70 + 30 + 4, v/v/v) and chloroform-methanol-toluene-ammonium hydroxide (9 + 6 + 3 + 2, v/vv/v) mobile phases. Two-step elution with these mobile phases was used for the development of chromatograms. Eleutherosides were visualized by derivatization with Liebermann-Burchard reagent. This reagent was used for the first time to detect eleutherosides. Eleutherosides B, E, and E1 were detected in the fruits of the investigated species. E. senticosus contained three of the investigated compounds, and E. sessiliflorus, E. gracilistylus, and E. divaricatus two compounds each.

The intractum from the Eleutherococcus senticosus fruits affects the innate immunity in human leukocytes: From the ethnomedicinal use to contemporary evidence-based research.

ETHNOPHARMACOLOGICAL RELEVANCE: In the ethnomedicine of Russia, the Eleutherococcus senticosus (Rupr. et Maxim.) Maxim. fruits and roots are used to treat immune-related diseases. Because of the overexploitation of the roots, the species is considered to be endangered and is put on the Red List in some countries (e.g. the Republic of Korea). Therefore, the aerial parts of E. senticosus might be explored as a new sustainable source of compounds with an adaptogenic activity. AIM OF THE STUDY: This study is aimed to evaluate the adaptogenic activity of the Eleutherococcus senticosus fruits intractum to support the use of the fruits in folk medicine of Russia. MATERIALS AND METHODS: The effect on IL-2 and IL-10 release by peripheral blood leukocytes (PBLs) was measured by the ELISA, the CPE on the A549 and PBLs were determined with trypan blue and the MTT. The innate immunity assay was done in the VSV-PBLs model. Metabolic profiling was done using HPLC-DAD and HPLC-RID. RESULTS: We report for the first time that the intractum (300 µg/mL) and eleutheroside E (100 µg/mL) and B (100 µg/mL) do not act as a virucidal agent (VSV). The intractum and eleutherosides E and B caused the increase of the PBLs proliferation up to 24.61 and 100%, resp. The decreased viral replication in the VSV-PBLs-Int model might be associated with an increased secretion of IL-10 (328 pg/mL). Eleutheroside E and B did not affect the innate immunity. No eleutherosides were determined in the intractum, the ethyl acetate layer contained caffeic and protocatechuic acids. A large amount of myo-inositol and D-mannitol was found (267.5 and 492.5 mg/g DE). CONCLUSIONS: Our observations justify the traditional use of the fruits in Russia in immune-related diseases. The results mean that there are other compounds than eleutherosides responsible for the adaptogenic effect, probably myo-inositol and caffeic acid, for which an immunostimulatory activity has already been confirmed.

Multidirectional anti-melanoma effect of galactolipids (MGDG-1 and DGDG-1) from Impatiens parviflora DC. and their synergy with doxorubicin.

The anti-melanoma potential of galactolipids: MGDG-1 and DGDG-1, isolated from Impatiens parviflora, and their synergistic effect with anticancer drug - doxorubicin (DOX) was investigated. Both compounds demonstrated time- and dose-dependent cytotoxicity against human melanoma cells of different metastatic potential. MGDG-1 was more effective than DGDG-1, with the highest activity against A375 cell line (IC50 = 15.14 µg/mL). Both compounds acted selectively, were devoid of hepatotoxicity or mutagenicity. Additionally, MGDG-1 proved to be a tyrosinase inhibitor. Co-administration of MGDG-1 and DGDG-1 with DOX revealed a synergistic cytotoxic effect on melanoma cells. The cytotoxicity of all tested MGDG-1/DOX and DGDG-1/DOX cocktails was considerably higher than that of each agent administered alone. MGDG-1/DOX (Mix3) reduced the viability of A375 melanoma cells almost totally and this effect was 2-fold more potent as compared to DOX alone. Our study indicates that the overall effect is enhanced with the increasing concentration of MGDG-1 in the cocktail. These results open up a possibility for lowering therapeutic doses of chemotherapeutics such as doxorubicin when co-administrated with galactolipids. Thus, MGDG-1 can be prospectively considered as multidirectional anti-melanoma agent and can be recommended for further in vitro and in vivo studies, especially in search for effective combined therapy.

Amplicon Sequencing of Variable 16S rRNA from Bacteria and ITS2 Regions from Fungi and Plants, Reveals Honeybee Susceptibility to Diseases Results from Their Forage Availability under Anthropogenic Landscapes.

European Apis mellifera and Asian Apis cerana honeybees are essential crop pollinators. Microbiome studies can provide complex information on health and fitness of these insects in relation to environmental changes, and plant availability. Amplicon sequencing of variable regions of the 16S rRNA from bacteria and the internally transcribed spacer (ITS) regions from fungi and plants allow identification of the metabiome. These methods provide a tool for monitoring otherwise uncultured microbes isolated from the gut of the honeybees. They also help monitor the composition of the gut fungi and, intriguingly, pollen collected by the insect. Here, we present data from amplicon sequencing of the 16S rRNA from bacteria and ITS2 regions from fungi and plants derived from honeybees collected at various time points from anthropogenic landscapes such as urban areas in Poland, UK, Spain, Greece, and Thailand. We have analysed microbial content of honeybee intestine as well as fungi and pollens. Furthermore, isolated DNA was used as the template for screening pathogens: Nosema apis, N. ceranae, N. bombi, tracheal mite (Acarapis woodi), any organism in the parasitic order Trypanosomatida, including Crithidia spp. (i.e., Crithidia mellificae), neogregarines including Mattesia and Apicystis spp. (i.e., Apicistis bombi). We conclude that differences between samples were mainly influenced by the bacteria, plant pollen and fungi, respectively. Moreover, honeybees feeding on a sugar based diet were more prone to fungal pathogens (Nosema ceranae) and neogregarines. In most samples Nosema sp. and neogregarines parasitized the host bee at the same time. A higher load of fungi, and bacteria groups such as Firmicutes (Lactobacillus); γ-proteobacteria, Neisseriaceae, and other unidentified bacteria was observed for Nosema ceranae and neogregarine infected honeybees. Healthy honeybees had a higher load of plant pollen, and bacteria groups such as: Orbales, Gilliamella, Snodgrassella, and Enterobacteriaceae. Finally, the period when honeybees switch to the winter generation (longer-lived forager honeybees) is the most sensitive to diet perturbations, and hence pathogen attack, for the whole beekeeping season. It is possible that evolutionary adaptation of bees fails to benefit them in the modern anthropomorphised environment.