Bioprospecting the Metabolome of Plant Urtica dioica L.: A Fast Dereplication and Annotation Workflow in Plant Metabolomics.

Plants have a pivotal role in ethnopharmacology, and their preparations are in use globally. However, getting down to the structure requires an effective workflow and mostly requires a time-consuming isolation process. Although bioassay-guided approaches are widely popular, they face a massive problem of rediscovery in recent times, especially in plant metabolomics. Mass spectrometry (MS)-based approach incorporated molecular networking via Global Natural Product Social Molecular Networking (GNPS) is considered here for the benefit of the fast screening of secondary metabolites. This study uses direct crude extracts obtained from various parts of the Urtica dioica plant for the characterization of secondary metabolites. The crude extract of the plant initially displayed promising antioxidant and anti-diabetic activities. Then, we employed mass spectrometry-based dereplication to identify the phytochemical components in the extracts. This led to the discovery of 7 unknown and 17 known secondary metabolites, which were further verified with the SIRIUS 4 platform, a computational tool for the annotation of compounds using tandem MS data. On the other hand, chasing the antioxidant activity of methanolic extract of U. dioica leaves, we employed a bioassay-guided isolation approach. With this method, we isolated and characterized compound 13, a known molecule, which possessed strong antioxidant activity without showing much toxicity in the brine shrimp lethality test at the test concentration of 1 mg/mL. With our results, we advocate the MS-based approach as a good starting point for the dereplication of compounds from the complex crude extracts of plants.

Investigation of antioxidant, antimicrobial and toxicity activities of lichens from high altitude regions of Nepal.

BACKGROUND: Several lichen species are reported to be used tradiationally in many theraupatic practices. Many lichen species are reported as sources of several bioactive natural compounds. Several lichen species of Nepal are so far chemically unexplored. METHODS: The morphological, anatomical and phytochemical characteristics of lichens were compared for the taxonomic identification of the species. Methanol- water extract of lichens were sub fractionated into hexane, dichloromethane and methanol fractions for bioactivity assays. Antimicrobial activities of extracts were evaluated agaisnt pathogenic bacteria and fungal species. DPPH test was used for antioxidant potential evaluation. Brineshrimp test was perfermed to evaluate toxicity of the extracts. RESULTS: A total of 84 lichen specimens were collected and identified from Annapurna Conservation Area (ACA) Nepal. The specimens were identified as belonging to 19 genera and 47 species. Methanol fractions of 16 specimens and dichloromethane (DCM) fractions of 21 lichens specimens showed antioxidant activities comparable with commercial standards (BHA, Butylated hydroxyanisole, IC50=4.9±0.9 µg/mL) even at crude extract level. Similarly, the DCM fraction of 17 lichens showed potential antimicrobial activity against a Gram-positive bacterium (Staphylococcus aureus KCTC3881) and DCM fractions of 45 lichens showed antimicrobial activity against a Gram-negative bacterium (Klebsiella pneumoniae KCTC2242). DCM fractions of three lichens showed antifungal activity against the yeast, Candida albicans KCTC 7965. Likewise, methanol fractions of 39 lichens and DCM fractions of 74 lichens showed strong toxicity against brine shrimp nauplii with more than 80% mortality. CONCLUSION: Such biological activity-rich lichen specimens warrant further research on exploration of natural products with antioxidant, antimicrobial and anti cancer (toxic) potential.

Estimation of antioxidant, antimicrobial activity and brine shrimp toxicity of plants collected from Oymyakon region of the Republic of Sakha (Yakutia), Russia.

BACKGROUND: Several plants are reported to be produced various biological active compounds. Lichens from the extreme environments such as high altitude, high UV, drought and cold are believed to be synthesized unique types of secondary metabolites than the other one. Several human pathogenic bacteria and fungi have been muted into drug resistant strains. Various synthetic antioxidant compounds have posed carcinogenic effects. This phenomenon needs further research for new effective drugs of natural origin. This manuscript aimed to screen new source of biological active compounds from plants of subarctic origin. RESULTS: A total of 114 plant species, including 80 species of higher plants, 19 species of lichens and 15 species of mosses, were collected from Oymyakon region of the Republic of Sakha (Yakutia), Russia (63˚20'N, 141˚42'E-63˚15'N, 142˚27'E). Antimicrobial, DPPH free radical scavenging and brine shrimp (Artemia salina) toxicity of all crude extract were evaluated. The obtained result was analyzed and compared with commercial standards. A total of 28 species of higher plants showed very strong antioxidant activity (DPPH IC50, 0.45-5.0 µg/mL), 13 species showed strong activity (DPPH IC50, 5-10 µg/mL), 22 species showed moderate antioxidant activity (DPPH IC50,10-20 µg/mL) and 17 species showed weak antioxidant activity (DPPH IC50 more than 20 µg/mL). Similarly, 3 species of lichen showed strong antioxidant activity, one species showed moderate and 15 species showed weak DPPH reducing activity. In addition, 4 species of mosses showed moderate antioxidant activity and 11 species showed weak antioxidant activity. Similarly, extracts of 51 species of higher plants showed antimicrobial (AM) activity against Staphylococcus aureus and 2 species showed AM activity against Candida albicans. Similarly, 11 species of lichen showed AM activity against S. aureus and 3 species showed AM activity against Escherichia coli. One species of moss showed AM activity against S. aureus. And finally, one species of higher plant Rheum compactum and one species of lichen Flavocetraria cucullata showed the toxicity against Brine shrimp larvae in 100 µg/mL of concentration. CONCLUSION: The experimental results showed that subarctic plant species could be potential sources of various biologically active natural compounds.

Estimation of antioxidant, antimicrobial activity and brine shrimp toxicity of plants collected from Oymyakon region of the Republic of Sakha (Yakutia), Russia

BACKGROUND: Several plants are reported to be produced various biological active compounds. Lichens from the extreme environments such as high altitude, high UV, drought and cold are believed to be synthesized unique types of secondary metabolites than the other one. Several human pathogenic bacteria and fungi have been muted into drug resistant strains. Various synthetic antioxidant compounds have posed carcinogenic effects. This phenomenon needs further research for new effective drugs of natural origin. This manuscript aimed to screen new source of biological active compounds from plants of subarctic origin. RESULTS: A total of 114 plant species, including 80 species of higher plants, 19 species of lichens and 15 species of mosses, were collected from Oymyakon region of the Republic of Sakha (Yakutia), Russia (63˚20′N, 141˚42′E - 63˚15′N, 142˚27′E). Antimicrobial, DPPH free radical scavenging and brine shrimp (Artemia salina) toxicity of all crude extract were evaluated. The obtained result was analyzed and compared with commercial standards. A total of 28 species of higher plants showed very strong antioxidant activity (DPPH IC50, 0.45-5.0 µg/mL), 13 species showed strong activity (DPPH IC50, 5-10 µg/mL), 22 species showed moderate antioxidant activity (DPPH IC50,10-20 µg/mL) and 17 species showed weak antioxidant activity (DPPH IC50 more than 20 µg/mL). Similarly, 3 species of lichen showed strong antioxidant activity, one species showed moderate and 15 species showed weak DPPH reducing activity. In addition, 4 species of mosses showed moderate antioxidant activity and 11 species showed weak antioxidant activity. Similarly, extracts of 51 species of higher plants showed antimicrobial (AM) activity against Staphylococcus aureus and 2 species showed AM activity against Candida albicans. Similarly, 11 species of lichen showed AM activity against S. aureus and 3 species showed AM activity against Escherichia coli. One species of moss showed AM activity against S. aureus. And finally, one species of higher plant Rheum compactum and one species of lichen Flavocetraria cucullata showed the toxicity against Brine shrimp larvae in 100 µg/mL of concentration. CONCLUSION: The experimental results showed that subarctic plant species could be potential sources of various biologically active natural compounds.

Ramalin, a novel nontoxic antioxidant compound from the Antarctic lichen Ramalina terebrata.

Ramalin (γ-glutamyl-N'-(2-hydroxyphenyl)hydrazide), a novel compound, was isolated from the methanol-water extract of the Antarctic lichen Ramalina terebrata by several chromatographic methods. The molecular structure of ramalin was determined by spectroscopic analysis. The experimental data showed that ramalin was five times more potent than commercial butylated hydroxyanisole (BHA) in scavenging 1-diphenyl-2-picryl-hydazil (DPPH) free radicals, 27 times more potent in scavenging 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid free radicals (ABTS(+)) than the vitamin E analogue, trolox, and 2.5 times more potent than BHT in reducing Fe(3+) to Fe(2+) ions. Similarly, ramalin was 1.2 times more potent than ascorbic acid in scavenging superoxide radicals and 1.25 times more potent than commercial kojic acid in inhibiting tyrosinase enzyme activity, which ultimately leads to whitening of skin cells. Ramalin showed no or very little cytotoxicity in human keratinocyte and fibroblast cells at its antioxidant concentration. Furthermore, ramalin was assessed to determine its antioxidant activity in vivo. One microgram per milliliter ramalin significantly reduced the released nitric oxide (NO) and 0.125 µg/ml ramalin reduced the produced hydrogen peroxide (H(2)O(2)) in LPS (lipopolysaccharide)-stimulated murine macrophage Raw264.7 cells. Considering all the data together, ramalin can be a strong therapeutic candidate for controlling oxidative stress in cells.

Antioxidant activity of Sanionia uncinata, a polar moss species from King George Island, Antarctica.

Antioxidant agents counter reactive oxygen species (ROS) and can be used in cosmetic and medicinal applications. The goal of this study was to evaluate the antioxidant activity of an Antarctic moss species from King George Island (Antarctica), tentatively designated as KSJ-M5. On the basis of morphological characteristics, KSJ-M5 was identified as Sanionia uncinata (Hedw.) Loeske (Amblystegiaceae). The identification was confirmed by comparing the partial sequence of the ITS (internal transcribed spacer) region with that in GenBank. The antioxidant activity of an ethanol extract of KSJ-M5 was evaluated by analyzing its reducing power, superoxide scavenging activity, ABTS [2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)] cation scavenging activity, and DPPH (1,1-diphenyl-2-picrylhydrazyl) free-radical scavenging activity. The reducing power of 1 mg of KSJ-M5 extract was equivalent to 31.9 +/- 0.9 microg (Mean +/- SD, n = 3) of the commercial standard, BHT (butylated hydroxytoluene). IC(50) values of the KSJ-M5 extract for DPPH free-radical scavenging activity, superoxide scavenging activity, and ABTS cation scavenging activity were found as 356 +/- 26.8 microg/mL, 466.2 +/- 43.4 microg/mL, and 181.3 +/- 12.2 microg/mL, respectively. The total phenolic content in 1 mg of KSJM5 extract was equivalent to 12.7 +/- 2.7 microg of pyrocatechol. These results clearly showed that KSJ-M5 could be an important source of natural antioxidant agents for improved medicinal and cosmetic applications.

Antibacterial potential of Antarctic lichens against human pathogenic Gram-positive bacteria.

Extracts from five Antarctic lichens (L3, Stereocaulon alpinum; L5, Ramalina terebrata; L6, Caloplaca sp.; L8, Lecanora sp.; and L17, Caloplaca regalis) were tested for antimicrobial activities against several clinically important microbes by disk diffusion. The minimum inhibitory concentration (MIC) of each extract was determined by a broth dilution method. Extracts from L3, L5, L6 and L8 were active against two Gram(+) strains. B. subtilis was more sensitive to lichen extracts (except L5) than S. aureus. The MIC of lichen extracts against B. subtilis and S. aureus was observed from 36.7 +/- 0.3 to 953.8 +/- 85.8 microg/mL and 68.5 +/- 0.6 to >1000 microg/mL, respectively. Comparisons of MIC values of Antarctic lichen crude extracts to previously published MIC values of some reported lichen metabolites against Gram(+) bacteria indicated that Antarctic lichens might be an enriched source of effective antibacterial agents against clinically relevant Gram(+) species.

Thin layer chromatography analysis of antioxidant constituents of lichens from Antarctica.

Antioxidant agents against reactive oxygen species can be used for several cosmetic and medicinal applications. Methanol-water (90:10 v/v) extracts of five polar lichen species--namely Stereocaulon alpinum Laurer (Stereocaulaceae); Ramalina terebrata Hook and Taylor (Ramalinaceae); Caloplaca sp. (Teloschistaceae); Lecanora sp. (Lecanoraceae); and Caloplaca regalis (Vain.)Zahlbr (Teloschistaceae) from King George Island (Antarctica)--were analyzed using thin layer chromatography (TLC) followed by a DPPH (2,2-diphenyl-1-picrylhydrazyl) spray technique. The experimental data showed that 33-50% of the major constituents of the test extracts were active antioxidants. Stereocaulon alpinum and R. terebrata showed a higher number (50%) of antioxidant constituents, although their activities were comparatively weak. The strength of antioxidant activity in terms of discoloration of DPPH was shown to be stronger by the constituents of S. alpinum, C. regalis and C. sp. In addition, phenolic content in these Antarctic lichen extracts was in the range of 17-47 mg/g, supporting the antioxidant data of TLC analysis. Thus, these results suggest that Antarctic lichen contains a variety of strong antioxidant constituents. Therefore, further study of the laboratory culture of lichen is warranted to investigate possible commercial production, followed by isolation and characterization of the active antioxidant agents, which can be used against various oxidative stress-related diseases.