Exploring the wound healing potential of Lobostemon fruticosus using in vitro and in vivo bioassays.

ETHNOPHARMACOLOGICAL RELEVANCE: Lobostemon fruticosus (L.) H.Buek is a perennial and woody shrub of the Boraginaceae family, found in the Cape region of South Africa. The leaves and twigs are used to treat dermatological conditions such as wounds, burns, ringworm, erysipelas and eczema. Anti-inflammatory, antibacterial, antiviral and anti-proliferative activities of L. fruticosus have been reported. However, there is a void in research which reports on the wound healing properties of this plant. AIM OF THE STUDY: Aligned with the traditional use of L. fruticosus, our study aimed to use in vitro and in vivo bioassays to confirm the wound healing potential of the plant. MATERIALS AND METHODS: An aqueous methanol extract (80% v/v) of L. fruticosus was prepared using a sample collected from the Western Cape Province of South Africa and chromatographically profiled by ultra-performance liquid chromatography coupled to mass spectrometry (UPLC-MS). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assay was performed to determine the non-toxic concentrations of the extract for subsequent use in the in vitro scratch assay. Both the human keratinocyte (HaCaT) and fibroblast (BJ-5ta) cell lines were employed in the in vitro scratch assay. The in vivo caudal fin amputation assay was used to assess the wound healing potential of L. fruticosus, by monitoring fin regeneration in zebrafish larvae treated with the plant extract at various concentrations. RESULTS: Six major compounds were tentatively identified in the L. fruticosus extract namely; globoidnan A, globoidnan B, rutin, rabdosiin, sagerinic acid and rosmarinic acid. The potentially toxic pyrrolizidine alkaloids were also identified and quantitatively confirmed to be present at a low concentration of 119.58 ppm (m/m). Treatment of HaCaT and BJ-5ta cells with the plant extract in the scratch assay resulted in an increase in cell migration, which translates to accelerated wound closure. After 24 hr treatment with 100 µg/mL of extract, wound closure was recorded to be 91.1 ± 5.7% and 94.1 ± 1.3% for the HaCaT and BJ-5ta cells, respectively, while the untreated (medium) controls showed 72.3 ± 3.3% and 73.0 ± 4.3% for the two cell lines, respectively. Complete wound closure was observed between 24 and 36 hr, while the untreated control group did not achieve 100% wound closure by the end of the observation period (48 hr). In vivo, the crude extract at 100 µg/mL accelerated zebrafish caudal fin regeneration achieving 100.5 ± 3.8% regeneration compared to 68.3 ± 6.6% in the untreated control at two days post amputation. CONCLUSIONS: The study affirms the wound healing properties, as well as low toxicity of L. fruticosus using both in vitro and in vivo assays, which supports the traditional medicinal use. Other in vitro assays that target different mechanisms involved in wound healing should be investigated to support the current findings.

Phytochemical Characteristics and Antimicrobial Effects of Nonea rossica (Boraginaceae) Extracts.

Phytochemical characteristics and antimicrobial properties of extracts were studied in Nonea rossica Steven (Boraginaceae), which is widespread in Russia. The aerial part (herb) of N. rossica was harvested from a steppe meadow in the Novosibirsk region during flowering. The qualitative composition of biologically active compounds (BACs) was determined by thin-layer chromatography. Quantitative assays were carried out by spectrophotometry; flavonoids, hydroxycinnamic acids, and coumarin-like compounds were measured with reference to rutin, caffeic acid, and coumarin, respectively. Antimicrobial activity was determined by the serial dilution method. Gram-positive bacterial (Staphylococcus aureus ATCC 6538 FDA 209P and Bacillus cereus ATCC 10702) and fungal (Candida albicans NCTC 885-653) strains were used as test cultures. Phenolic BACs (hydroxycinnamic acids, flavonoids, and coumarins) were detected, and their quantitative contents determined. The highest yield of phenolic BACs was achieved using 40-70% ethanol as an extractant. Antimicrobial activity against S. aureus and B. cereus and antifungal activity against C. albicans were detected in N. rossica herb extracts prepared using 40-70% ethanol. The extracts were tested for the contents of caffeic acid and coumarin. Synergistic interactions of these compounds determined the bactericidal and fungistatic properties of the extracts.

A class of geranylquinol-derived polycyclic meroterpenoids from Arnebia euchroma against heart failure by reducing excessive autophagy and apoptosis in cardiomyocytes.

Ten new B-ring aromatized 6/6/6-tricyclic dearomatized benzocogeijerene-based meroterpenoids with unusual methyl 1,2-shift or demethylation (2-9b), and two new geranylquinol derivatives (1 and 10), together with two known compounds (11 and 12), were isolated from the roots of Arnebia euchroma. Their structures were elucidated by extensive spectroscopic methods, X-ray diffraction crystallography, and ECD calculations. The plausible biosynthetic pathways including the unusual methyl 1,2-shfit and demethylation for B-ring aromatized 6/6/6-tricyclic meroterpenoids were discussed. Compounds 1, 2, 5, 6, 11, and 12 showed significant cardioprotective activities comparable to diltiazem against isoprenaline (ISO)-induced H9C2 cell damage in vitro. Compound 11 probably exerted heart-protective effect on ISO-induced H9C2 cells by modulating the PI3K-AKT-mTOR pathway, reducing excessive autophagy, and decreasing myocardial apoptosis.

Identification, quantification and nutraceutical evaluation of the extracts from Arnebia benthamii roots of Himalayan regions of J&K, India.

Arnebia benthamii is one of the important sources of biologically active naphthoquinone pigments. The present study aimed at extraction of shikonin from Arnebia benthamii roots and its characterization. In order to identify and quantify shikonin, the extracts were evaluated using HPLC, LCMS, GCMS, NP-HPTLC and FTIR. Furthermore, nutraceutical evaluation was also done. It was found that the amount of shikonin was very low in the extracts obtained by using aqueous ethanol as it was not detected through chromatographic techniques. However, when hexane was used for extraction, a significant amount of shikonin (4.55 mg/g) was detected. The shikonin showed a linear range from 2-55 µg/mL with LOD and LOQ of 2.65 and 8.02 respectively, with a retention time of 3.64 min. The results of FTIR revealed that hexane extract had the intensity of functional groups similar to that of the standard. The values of DPPH radical inhibition were observed as 82.98 ± 0.01, 65.09 ± 0.23 %, 62.28 ± 0.86 % and 54.09 ± 0.23 % for Std, Ehex, Eus and Evs, respectively. The hexane extract showed the highest antioxidant activity as compared to other samples. Moreover, the hexane extracted shikonin displayed significantly (p > 0.05) high α-amylase and pancreatic lipase inhibition, indicating its high anti-diabetic and anti-lipidemic potential. It can be concluded that hexane is the best solvent for the extraction of shikonin and has better nutraceutical potential compared to ethanolic extracts.

Rapid identification and determination of adulteration in medicinal Arnebiae Radix by combining near infrared spectroscopy with chemometrics.

The medicinal Arnebia Radix (AR) is one of widely-used Chinese herbal medicines (CHMs), usually adulterated with non-medicinal species that seriously compromise the quality of AR and affect patients' health. Detection of these adulterants is usually performed by using expensive and time-consuming analytical instruments. In this study, a rapid, non-destructive, and effective method was proposed to identify and determine the adulteration in the medicinal AR by near-infrared (NIR) spectroscopy coupled with chemometrics. 37 batches of medicinal AR samples originated from Arnebia euchroma (Royle) Johnst., 11 batches of non-medicinal AR samples including Onosma paniculatum Bur. et Franch and Arnebia benthamii (Wall. ex G. Don) Johnston, and 72 batches of adulterated AR samples were characterized by NIR spectroscopy. The data driven-soft independent modeling by class analogy (DD-SIMCA) and partial least squares-discriminant analysis (PLS-DA) were separately used to differentiate the authentic from adulterated AR samples. Then the PLS and support vector machine (SVM) were applied to predict the concentration of the adulteration in the adulterated AR samples, respectively. As a result, the classification accuracies of DD-SIMCA and PLS-DA models were 100% for the calibration set, and 96.7% vs. 100% for the prediction set. Moreover, the relative prediction deviation (RPD) values of PLS models reached 11.38 and 7.75 for quantifying two adulterants species, which were obviously superior to the SVM models. It can be concluded that the NIR spectroscopy coupled with chemometrics is feasible to identify the authentic from adulterated AR samples and quantify the adulteration in adulterated AR samples.

Pleonotoquinones, Cytotoxic Oxepinenaphthoquinones from Pleonotoma jasminifolia.

Two unusual naphthoquinones, named here as pleonotoquinones A (1) and B (2), were isolated along with two known anthraquinones (3 and 4) via chromatographic separations of an ethyl acetate extract of the roots of Pleonotoma jasminifolia. Compounds 1 and 2 are the first examples of quinones bearing a 2-methyloxepine moiety. The compounds were isolated with the aid of mass spectrometry and molecular networking, and their structures were resolved using 1D and 2D NMR and HRESIMS data. The isolated compounds were evaluated for their antiproliferative activity against human cancer cell lines, and compounds 1 and 2 displayed cytotoxicity against human colon cancer HCT116 cells (IC50 = 2.6 µM for compound 1 and IC50 = 4.3 µM for compound 2) and human liver cancer HepG2 cells (IC50 = 1.9 µM for compound 1 and IC50 = 6.4 µM for compound 2).

A natural approach to breast cancer treatment: investigation of chemical features of aerial parts of endemic Onosma sintenisii Hausskn. ex Bornm and its antioxidant properties, in vitro cytotoxic and apoptosis induction on MCF-7 cells.

Onosma sintenisii Hausskn. ex Bornm. (O. sintenisii) belongs to the Boraginaceae family and it is an endemic species from Irano-turanian phytogeographical region (central and eastern Anatolia) that distributes in steppe areas. This study aimed to investigate the chemical composition, antioxidant, in vitro cytotoxic and apoptosis induction of methanol extract of aerial parts of O. sintenisii. As a result of GC/MS analysis, 14 components were identified, and the major compounds of the extracts are retronecine (13.94%), α.-D-Glucopyranosiduronic acid (10.86%), melaniline (7.5%) and 1,2-Butanediol (4.02%), respectively. Antioxidant properties of O. sintenisii were determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, ferric reducing antioxidant power (FRAP) and superoxide radical scavenging activity methods. While the DPPH free radical scavenging activity results of O. sintenisii extract varied between 62.49% and 32.27%, reducing power activity and superoxide radical scavenging activity were found to be low. The result of the MTT assay revealed strong anticancer activity of O. sintenisii extract. The most significant cytotoxic effect was noted at a concentration of 1000 µg/mL after 48 hours. These findings together with flow cytometry analysis suggest that apoptosis can be the main mechanism underlying cell death after O. sintenisii extract treatment.

Effects of Naphthoquinones from the Roots of Onosma Armeniacum Klokov on Wound Healing.

In Turkish folk medicine, the roots of Onosma armeniacum Klokov are used to heal wounds, burns, hemorrhoids, hoarseness, dyspnea, stomach ulcers, and abdominal aches. The objective was to evaluate the plant's ethnopharmacological applications using in vivo pharmacological experimental models. In vivo linear incision and circular excision the wound models were used to assess the wound healing activity along with histopathological investigation. The active component(s) were isolated and identified after being exposed to several chromatographic separation procedures on the primary extract. The n-hexane-dichloromethane mixture extract was subjected to chromatographic separation after the wound-healing activity was confirmed. Deoxyshikonin (1), ß,ß-dimethylacrylshikonin (2), α-methyl-n-butylshikonin (3), isovalerylshikonin (4), acetylshikonin (5), ß-hydroxyisovalerylshikonin (6), and 5,8-O-dimethylacetylshikonin (7) were identified as the structures of the isolated compounds. The efficacy of O. armeniacum to heal wounds was investigated in this study. Shikonin derivatives were identified as the primary active components of the roots by bioassay-guided fractionation and isolation procedures.

Pyrrolizidine alkaloids are synthesized and accumulated in flower of Myosotis scorpioides.

Pyrrolizidine alkaloids (PAs) are specialized metabolites that are produced by various plant families that act as defense compounds against herbivores. On the other hand, certain lepidopteran insects uptake and utilize these PAs as defense compounds against their predators and as precursors of their sex pheromones. Adult males of Parantica sita, a danaine butterfly, convert PAs into their sex pheromones. In early summer, P. sita swarms over the flowers of Myosotis scorpioides, which belongs to the family Boraginaceae. M. scorpioides produces PAs, but the organs in which PAs are produced and whether P. sita utilizes PAs in M. scorpioides are largely unknown. In the present study, we clarified that M. scorpioides accumulates retronecine-core PAs in N-oxide form in all organs, including flowers. We also identified two M. scorpioides genes encoding homospermidine synthase (HSS), a key enzyme in the PA biosynthetic pathway, and clarified that these genes are expressed in all organs where PAs accumulate. Phylogenetic analysis suggested that these two HSS genes were originated from gene duplication of deoxyhypusine synthase gene like other HSS genes in PA-producing plants. These results suggest that PAs are synthesized and accumulated in the flower of M. scorpioides and provide a possibility for a PA-mediated interaction between P. sita and M. scorpioides.

Comparative Analysis of Shikonin and Alkannin Acyltransferases Reveals Their Functional Conservation in Boraginaceae.

Shikonin and its enantiomer, alkannin, are bioactive naphthoquinones produced in several plants of the family Boraginaceae. The structures of these acylated derivatives, which have various short-chain acyl moieties, differ among plant species. The acylation of shikonin and alkannin in Lithospermum erythrorhizon was previously reported to be catalyzed by two enantioselective BAHD acyltransferases, shikonin O-acyltransferase (LeSAT1) and alkannin O-acyltransferase (LeAAT1). However, the mechanisms by which various shikonin and alkannin derivatives are produced in Boraginaceae plants remain to be determined. In the present study, evaluation of six Boraginaceae plants identified 23 homologs of LeSAT1 and LeAAT1, with 15 of these enzymes found to catalyze the acylation of shikonin or alkannin, utilizing acetyl-CoA, isobutyryl-CoA or isovaleryl-CoA as an acyl donor. Analyses of substrate specificities of these enzymes for both acyl donors and acyl acceptors and determination of their subcellular localization using Nicotiana benthamiana revealed a distinct functional differentiation of BAHD acyltransferases in Boraginaceae plants. Gene expression of these acyltransferases correlated with the enantiomeric ratio of produced shikonin/alkannin derivatives in L. erythrorhizon and Echium plantagineum. These enzymes showed conserved substrate specificities for acyl donors among plant species, indicating that the diversity in acyl moieties of shikonin/alkannin derivatives involved factors other than the differentiation of acyltransferases. These findings provide insight into the chemical diversification and evolutionary processes of shikonin/alkannin derivatives.

Euchronin A-F isolated from the Arnebia euchroma (Royle) Johnst. and their anti-proliferative activities in vitro.

Six new naphthoquinones, euchronin A-F (1-6) and nine known naphthoquinones (7-15), were isolated from the roots of Arnebia euchroma (Royle) Johnst. The structures of the new compounds were confirmed by extensive spectroscopic analyses, including UV, IR, HR-ESI-MS, 1D and 2D NMR. In the present study, we estimated the anti-proliferative activities of these compounds with HaCaT cells. The results indicated that compounds 2 and 4 showed strong anti-proliferative activities at 25 µM, with relative viability at 38.83% and 68.44%, respectively.

Extraction, Distribution and Diversity of Phenolic Compounds in Most Widespread Boraginaceae Species from Macedonia.

A systematic study of extraction efficiency of polyphenolic compounds from the most widespread Boraginaceae species was carried out. The optimal extraction was achieved with 50 % (V/V) methanol for phenolic acids and flavonoids, 0.2 % (V/V) HCl in 50 % (V/V) methanol for anthocyanins and pure water for flavan-3-ols. The distribution and diversity of polyphenolic compounds in plant material obtained from wild-growing Anchusa officinalis, Cynoglossum creticum Mill., Echium vulgare, Echium italicum, and Onosma heterophylla Griseb. species from Macedonia was also assessed. These widespread Boraginaceae species contain phenolic acid derivatives, flavonoids, flavan-3-ols and anthocyanins and in total 31 of them were identified, from which 22 were first identified in the representative species, and 6,8-di-C-glucosides of apigenin and luteolin were identified for the first time in Boraginaceae. The profiles of polyphenolic compounds for each sample were obtained and their phytochemical profile established. The potential for further bioactivity studies of Anchusa officinalis and Cynoglossum creticum containing up to 24577.05 µg/g and 14304.15 µg/g of total polyphenols were assumed to be highest, followed by Echium vulgare (from 6382.61 to 14114.33 µg/g), Onosma heterophylla (9463.97 µg/g) and Echium (4108.14 µg/g).

Influence of pyrrolizidine alkaloids depletion upon the biological activity of Symphytum officinale L. extracts.

ETHNOPHARMACOLOGICAL RELEVANCE: Comfrey (Symphytum officinale L., Boraginaceae) root preparations are used as both traditional remedies and therapeutic agents in treating pain and inflammation associated with joint, bone, and muscle ailments. Even though numerous phytochemicals contribute to the beneficial effects of comfrey, the presence of toxic pyrrolizidine alkaloids (PAs) overshadows its uses. AIM OF THE STUDY: In this work, different PA-/mucilage-depleted/undepleted comfrey root extracts were subjected to detailed phytochemical characterization and biological evaluation. MATERIALS AND METHODS: The phytochemical profiling was performed by LC-HRMS/MS. The quantification of PAs and major phenolic compounds was carried out by LC-MS/MS and LC-DAD. Antioxidant and enzyme inhibitory activity was determined by in vitro free radical scavenging, ion reducing, metal chelating, cholinesterase, tyrosinase, amylase, and glucosidase assays. Using an ex vivo model of LPS-stimulated neutrophils, their viability (as measured by flow cytometry) and the release of IL-1ß, IL-8, and TNF-α were determined (ELISA assay). RESULTS: 12 phenolic acids, six PAs, three organic acids, two fatty acids, and two sugars were identified in the obtained comfrey extracts. The PA-depleted materials contained PAs levels below 2 ppm, whereas the removal of mucilage increased the content of rosmarinic acid, globoidnan A, globoidnan B, and rabdosiin. PA-depletion did not significantly affect the antioxidant potential. However, the radical scavenging and metal reducing properties were higher in the mucilage-depleted extracts. Neither PA-depletion nor mucilage-depletion had considerable effects on the in vitro inhibitory activity of cholinesterases, tyrosinase, amylase, and glucosidase or release of ex vivo pro-inflammatory cytokines (e.g., IL-1ß, IL-8, and TNF-α) in LPS-stimulated neutrophils. CONCLUSIONS: In light of their superior safety profiles, PA-depleted comfrey extracts can be utilized further in cosmetic and pharmaceutical products.

Naphthoquinones and triterpenoids from Arnebia euchroma (Royle) Johnst and their hypoglycemic and lipid-lowering effects.

A new pentacyclic triterpenoid, 2-hydroxy-1-ene-hydroxyhopanone (19), and a new benzoxepin-5-one, 3-(4-methyl-3-penten-1-yl)-6-hydroxy-9-methoxy-2H-1-benzoxepin-5-one (25), along with 26 known compounds (1-18, 20-24, 26-28), were isolated from the roots of Arnebia euchroma (Royle) Johnst. The structures of the new compounds were elucidated by extensive spectroscopic analyses. The absolute configurations of shikonofurans 9-13 were determined by quantum chemical ECD calculations and CD spectra comparison for the first time. Pharmacological study revealed that naphthoquinones 1-5, 7, and 8 had obvious cytotoxicity toward human lung adenocarcinoma A549 cell line. Meanwhile, the hypoglycemic and lipid-lowering effects of isolated compounds were assessed by checking their inhibitory effects on key enzymes regulating glucose and lipid metabolism. Results showed that compounds 1, 3, 5, 6, 8, 18, and 19 could inhibit the activity of ATP-citrate lyase (ACL); compound 7 could inhibit the activity of acetyl-CoA carboxylase (ACC1); while compounds 8 and 19 showed inhibitory effects on protein tyrosine phosphatase 1B (PTP1B). Among them, the naphthoquinone 6, steroid 18, and triterpenoid 19 showed moderate inhibitory effects on ACL and PTP1B, but didn't exhibit obvious cytotoxicity. This study demonstrated that compounds 6, 18, and 19 show great promising for the development of new agents for the treatment of metabolic diseases.

Eight new arnebinol B-based meroterpenoids with planar chirality in the constrained 6/10/5 tricyclic skeleton from Arnebia euchroma and their cytotoxicities.

Eight new arnebinol B-based meroterpenoids ((-)-1, 2, 3, (-)-5, and 7-10) with a constrained 6/10/5 tricyclic backbone were isolated from the roots of Arnebia euchroma. The planar and steric structures of these new compounds were unambiguously elucidated by extensive spectroscopic analyses, X-ray diffraction crystallography, and ECD calculations. The predominant relative orientation between H-7 and the Z double bond with a methyl substituent in the rigid 10-membered carbocycle, along with the planar chirality of the Z double bond was analyzed and discussed for the first time. The illustration of the planar chirality derived from the Z double bond should be paid great importance during the structure elucidation on these homologous meroterpenoids. All the isolated meroterpenoids were screened for their cytotoxicities against the HCT-8, PANC-1, HGC-27, HepG2, and PC9 cell lines, and compounds (+)-5 and (-)-5 exhibited the most potent cytotoxicity.

Relayed chromatography - Countercurrent chromatography in series with liquid chromatography for the separation of natural products.

Chromatography is an essential method for separating natural products. In this study, we proposed the concept of 'relayed chromatography', based on the strategy of combining different chromatography with relayed resolution by in-situ concentration technique. The following chromatographic methods were used: high-speed countercurrent chromatography (HSCCC), silica gel liquid chromatography (silica gel LC), and reverse phase liquid chromatography (reverse phase LC). The proposed strategy was effectively applied to the preparative separation of naturally existing naphthaquinones. After the first separation stage (silica gel LC), acetylalkannin (1) was directly collected, while fractions 1, 4 and 5 were collected and respectively subjected to recycling CCC separation after concentration. Thus, deoxyshikonin (2), 8-O-methyl-11-O-acetylshikonin (6), ß-acetoxyisovalerylalkannin (7) and alkannin (8) were collected. Fraction 2 was concentrated and injected in reverse phase LC separation. After collection of isobutyrylalkannin (3), the remaining effluent from reverse phase LC retained the peak resolution (R4,5=0.45) and was injected into a recycling CCC elution. Finally, ß, ß-dimethylacrylalkannin (4), and isovalerylalkannin (5) were collected with sufficient resolution (R4,5=1.25). Eight naturally occurring naphthaquinones were thus isolated from Arnebia euchroma. The purities of all the compounds were determined by HPLC to be > 90%, and the chemical structures were determined by spectral method. Among the aforementioned compounds, 8-O-methyl-11-O-acetylshikonin (6) was separated as a new compound from A. euchroma. In conclusion, the relayed strategy that retains the resolution of the previous chromatographic stage can improve CCC separation efficiency, which may expand the range of application of CCC combined with different chromatography to the separation of natural products.

Buglossoides spp. seeds, a land source of health-promoting n-3 PUFA and phenolic compounds.

Ahiflower oil© is extracted from the seeds of Buglossoides arvensis, which contains high amounts of stearidonic acid (SDA, 18:4n-3), while its phenolic composition still is unreported. Moreover, several Buglossoides taxa remain unstudied and could become natural sources of SDA. In this work, seeds of several Buglossoides taxa and Ahiflower oil© were screened for fatty acids, phenolic compounds, and in vitro antiproliferative activities against colorectal cancer cells. Four flavonoids and 16 phenolic acids were identified and quantified. Among Buglossoides taxa, the highest amounts of phenolic compounds were found in samples collected in Spain, under a warm Mediterranean climate. Rosmarinic and lithospermic acids were the main phenols found in Buglossoides seeds. The MTT assay showed dose- and time-dependent inhibitory effects of B. arvensis extracts on HT-29 cancer cells, with a GI50value of ∼280 µg/mL after 72 h of cell exposure to seed extracts. The latter showed lower antiproliferative activity than that of pure phenolics due to the simultaneous presence of other compounds in the extracts, as evidenced by 1H NMR. This work constitutes the first approach to evaluate the seeds of several Buglossoides taxa as functional oils-providers to use them as functional foods.

Systematic Screening of Chemical Constituents in the Traditional Chinese Medicine Arnebiae Radix by UHPLC-Q-Exactive Orbitrap Mass Spectrometry.

Arnebiae Radix (dried root of Arnebia euchroma (Royle) Johnst.) has been used in traditional Chinese medicine (TCM) to treat macular eruptions, measles, sore throat, carbuncles, burns, skin ulcers, and inflammation. Previous studies have shown that shikonins and shikonofurans are two of their main bioactive ingredients. However, systematic investigations of their constituents have rarely been conducted. It is necessary to establish a rapid and effective method to identify the chemical constituents of Arnebiae Radix. This will help to further improve the effective resource utilization rate of this plant. In this study, a rapid and effective UHPLC-Q-Exactive Orbitrap mass spectrometry method was established to simultaneously analyze chemical ingredients in Arnebiae Radix within a short period of time. Based on the results of a full scan MS, the MS2 database (mzVault and mzCloud), the diagnostic fragment ions, the retention time, and the bibliography, a total of 188 compounds were identified, with 114 of those being reported from Arnebiae Radix for the first time. The results of this study lay the foundation for obtaining a thorough understanding of the active ingredients in Arnebiae Radix and its quality control. This method may be widely used for the chemical characterization of different samples.

Hieracium waldsteinii (Asteraceae) and Onosma stellulata (Boraginaceae) as a Source of Antioxidant and Antimicrobial Agents.

The current study aimed to phytochemically characterize (including a detailed phenolic profile) two endemic Balkan's species (Hieracium waldsteinii and Onosma stellulata) and determine their possible application as a source of natural antioxidant and antimicrobial agents. The main phenolic compound in both species (in all examined parts) was chlorogenic acid. Eriodictyol, genistein and naringenin were quantified only in H. waldsteinii while isorhamnetin-3-O-rutinoside and sinapic acid were characteristic for O. stellulata. The highest antioxidant activity (98 mg AAE/g dry weight for TAC assay) was ascribed to the flower extract of H. waldsteinii while the lowest results (∼4.3 mg AAE/g dry weight for FRP assay) were exhibited by the extracts obtained from the plant's stem. Antimicrobial assays showed moderate antibacterial, i. e., moderate/strong activity against several tested fungi (in particular Trichoderma viride). Correlation analysis revealed strong positive connection between phenolic compounds and reducing power of extracts as well as between total phenolic and flavonoid content and the obtained minimal inhibitory concentration recorded in antibacterial assays.

Antimicrobial and Antioxidant Properties of Total Polyphenols of Anchusa italica Retz.

Anchusa italica Retz has been used for a long time in phytotherapy. The aim of the present study was to determine the antioxidant and antibacterial activities of extracts from the leaves and roots of Anchusa italica Retz. We first determined the content of phenolic compounds and flavonoids using Folin-Ciocalteu reagents and aluminum chloride (AlCl3). The antioxidant activity was determined using three methods: reducing power (FRAP), 2.2-diphenyl-1-picrylhydrazyl (DPPH), total antioxidant capacity (TAC). The antimicrobial activity was investigated against four strains of Escherichia coli, two strains of Klebsiella pneumoniae and coagulase-negative Staphylococcus, and one fungal strain of Candida albicans. The results showed that the root extract was rich in polyphenols (43.29 mg GAE/g extract), while the leave extract was rich in flavonoids (28.88 mg QE/g extract). The FRAP assay showed a strong iron reduction capacity for the root extract (IC50 of 0.11 µg/mL) in comparison to ascorbic acid (IC50 of 0.121 µg/mL). The DPPH test determined an IC50 of 0.11 µg/mL for the root extract and an IC50 of 0.14 µg/mL for the leaf extract. These values are low compared to those for ascorbic acid (IC50 of 0.16 µg/mL) and BHT (IC50 0.20 µg/mL). The TAC values of the leaf and root extracts were 0.51 and 0.98 mg AAE/g extract, respectively. In vitro, the extract showed inhibitory activity against all strains studied, with diameters of zones of inhibition in the range of 11.00-16.00 mm for the root extract and 11.67-14.33 mm for the leaf extract. The minimum inhibitory concentration was recorded for the leaf extract against E. coli (ATB:57), corresponding to 5 mg/mL. Overall, this research indicates that the extracts of Anchusa italica Retz roots and leaves exert significant antioxidant and antibacterial activities, probably because of the high content of flavonoids and polyphenols.