Haplotype-resolved chromosome-level genome assembly of Ehretia macrophylla.

Ehretia macrophylla Wall, known as wild loquat, is an ecologically, economically, and medicinally significant tree species widely grown in China, Japan, Vietnam, and Nepal. In this study, we have successfully generated a haplotype-resolved chromosome-scale genome assembly of E. macrophylla by integrating PacBio HiFi long-reads, Illumina short-reads, and Hi-C data. The genome assembly consists of two haplotypes, with sizes of 1.82 Gb and 1.58 Gb respectively, and contig N50 lengths of 28.11 Mb and 21.57 Mb correspondingly. Additionally, 99.41% of the assembly was successfully anchored into 40 pseudo-chromosomes. We predicted 58,886 protein-coding genes, of which 99.60% were functionally annotated from databases. We furthermore detected 2.65 Gb repeat sequences, 659,290 rRNAs, 4,931 tRNAs and 4,688 other ncRNAs. The high-quality assembly of the genome offers a solid basis for furthering the fields of molecular breeding and functional genomics of E. macrophylla.

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).

Exploring the Biological and Phytochemical Potential of Jordan's Flora: A Review and Update of Eight Selected Genera from Mediterranean Region.

Jordan's flora is known for its rich diversity, with a grand sum of 2978 plant species that span 142 families and 868 genera across four different zones. Eight genera belonging to four different plant families have been recognized for their potential natural medicinal properties within the Mediterranean region. These genera include Chrysanthemum L., Onopordum Vaill. Ex. L., Phagnalon Cass., and Senecio L. from the Asteraceae family, in addition to Clematis L. and Ranunculus L. from the Ranunculaceae family, Anchusa L. from the Boraginaceae family, and Eryngium L. from the Apiaceae family. The selected genera show a wide variety of secondary metabolites with encouraging pharmacological characteristics including antioxidant, antibacterial, cytotoxic, anti-inflammatory, antidiabetic, anti-ulcer, and neuroprotective actions. Further research on these genera and their extracts will potentially result in the formulation of novel and potent natural pharmaceuticals. Overall, Jordan's rich flora provides a valuable resource for exploring and discovering new plant-based medicines.

Healing of Skin Wounds in Rats Using Creams Based on Symphytum Officinale Extract.

Rosmarinic acid is a well-known natural antioxidant and anti-inflammatory compound, and it is one of the polyphenolic compounds found in comfrey plants. Comfrey root also contains allantoin, which helps with new skin regeneration. This study aimed to investigate the healing and skin regeneration process of skin wounds in Wistar rats using creams based on comfrey extract and to correlate the results with active compounds in the extract. The obtained results showed that comfrey root is rich in bioactive compounds, including allantoin, salvianolic acid, and rosmarinic acid, which are known for their great free radical scavenging activity, and the high antioxidant activity of the extract may be mainly due to these compounds. The obtained extract has an antimicrobial effect on Staphylococcus aureus (1530.76/382.69), Escherichia coli (6123.01/6123.01), and Pseudomonas aeruginosa (6123.01/6123.01). The macroscopic evaluation and the histological analysis of the skin defects 14 days after the intervention showed faster healing and complete healing in the skin excisions treated with oil-in-water cream with 20% extract of comfrey as the active ingredient.

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.

Effect of robbing intensity on reproductive success of Symphytum officinale (Boraginaceae).

The intervention of nectar robbers in plant pollination systems will cause some pollinators to modify their foraging behavior to act as secondary robbers, consequently adopting a mixed foraging strategy. The influence of nectar robbing on pollinator behavior may be affected by spatio-temporal difference of robbing intensity, and consequently, may have different effects on the pollination of host plants. However, whether and how the nectar robbing might influence pollinators under different robbing intensity still needs further investigation. In this study, Symphytum officinale was used to detect the effect of nectar robbers on pollinators under different robbing intensity as well as their effects on plant reproductive success. Six robbing levels and three bumblebees with mixed foraging behaviors were used to evaluate the effect of different robbing intensity on pollinator behavior, visitation rate, flower longevity and pollen deposition. Our results indicated that the robbing rate increased gradually with the proportion of robbed flowers, but which did not affect the frequency of legitimate visits. The increase of robbing rate promoted the corolla abscission, and then enhanced the self-pollen deposition, but which had no significant effect on cross-pollen deposition. These results indicate that the overall fitness of S. officinale was improved by combined self and cross-pollination modes when visited by both pollinators and nectar robbers simultaneously. Although nectar robbing is not uncommon, its consequences for pollination in the interaction web have not been well studied. Our results emphasize the significance of indirect impacts in mediating the adaptive outcomes of species interactions.

Non-destructive environmental safety assessment of threatened and endangered plants in weed biological control.

Assessing the risk of nontarget attack (NTA) for federally listed threatened and endangered (T&E) plant species confamilial to invasive plants targeted for classical biological control, is one of the most important objectives of pre-release environmental safety assessments in the United States. However, evaluating potential NTA on T&E species is often complicated by restrictive agency requirements for obtaining propagules, or the ability to propagate plants and rear agents to the appropriate phenostages synchronously for testing, or both. Here, we assessed whether plant cues associated with a host recognition can be used for testing the attractiveness of four T&E and one rare single population plant species non-destructively for a candidate biocontrol agent. We used the seed-feeding weevil, Mogulones borraginis, a candidate for the biological control of the invasive plant, Cynoglossum officinale (Boraginaceae) as the study system. We collected olfactory and visual cues in the form of flowering sprigs from T&E plant species confamilial to the invasive plant in a non-destructive manner and used them to measure behavioral responses and searching time of weevils. Female weevils preferred C. officinale to all tested plant species in dual-choice bioassays using either olfactory or visual cues in a modified y-tube device. Furthermore, female weevils were repelled by the combined olfactory and visual cues from all tested T&E plant species in a dual-choice test against controls (e.g., purified air in an empty arm), indicating that it would be extremely unlikely for the weevil to attack any of these species upon release in the United States. Principal component analysis based on 61 volatile organic compounds effectively separated the five confamilial plant species and C. officinale, corroborating the results of behavioral bioassays. We conclude that studies on pre-alighting host selection behavior and the underlying physiological mechanisms of how organisms select host plants they exploit can aid in environmental safety testing of weed biological control agents.

The healing effect of a mixture of Arnebia euchroma and animal fat on burn wounds in rats in comparison with sulfadiazine.

OBJECTIVE: Thermal burn is a serious cause of morbidity and mortality that affects millions of people worldwide. The aim of this experimental study was to investigate the efficacy of Arnebia euchroma (AE) to treat burn wounds in a rat model. METHOD: A total of 80 male rats (200-250g) were shaved over the back of the neck (2×3cm2) and a second-degree burn wound was induced at this site under general anaesthesia. The rats were then randomly assigned to one of four groups (each n=20) and the burns were treated daily for 14 days as follows: (1) dressed with animal fat; (2) dressed with sulfadiazine; (3) dressed with a mixture of AE and animal fat; (4) no treatment (control). Five rats from each group were sacrificed on days 3, 5, 9 and 14 post-burn and the wounds were evaluated histologically and immunohistochemically for the expression of interleukin (IL)-1 and IL-6. RESULTS: There was a significant increase at day 3 and decrease on day 5 samples for the expression of IL-1 in the AE plus fat group and IL-6 in the AE plus fat and sulfadiazine groups, compared to the control and fat treatment groups, respectively. Both AE plus fat and sulfadiazine treatments reduced inflammation and granulation tissue formation by day 5 post-burn, while re-epithelialisation commenced by day 9 post-burn. In addition, burns treated with AE plus fat exhibited keratinised epidermis, associated with regular collagen fibres, compared to moderately dense collagen fibres without vascularisation in the sulfadiazine group. CONCLUSION: These findings suggested that AE plus fat was superior to sulfadiazine in enhancing burn wound healing in rats.

Comparative Analyses of Complete Chloroplast Genomes of Microula sikkimensis and Related Species of Boraginaceae.

The present study provides a detailed analysis of the chloroplast genome of Microula sikkimensis. The genome consisted of a total of 149,428 bp and four distinct regions, including a large single-copy region (81,329 bp), a small single-copy region (17,261 bp), and an inverted repeat region (25,419 bp). The genome contained 112 genes, including 78 protein-coding genes, 30 tRNA genes, and 4 rRNA genes, and some exhibited duplication in the inverted repeat region. The chloroplast genome displayed different GC content across regions, with the inverted repeat region exhibiting the highest. Codon usage analysis and the identification of simple sequence repeats (SSRs) offer valuable genetic markers. Comparative analysis with other Boraginaceae species highlighted conservation and diversity in coding and noncoding regions. Phylogenetic analysis placed M. sikkimensis within the Boraginaceae family, revealing its distinct relationship with specific species.

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.

Anoikis and cancer cell differentiation: novel modes of shikonin derivatives anticancer action in vitro.

BACKGROUND: Shikonin is a naturally occurring naphthoquinone found in the roots of several genera of the Boraginaceae family, widely known for its numerous biological activities, such as antiinflammatory, antioxidant, antimicrobial and anticancer. In this study, the antitumor effect of six naphthoquinones isolated from the roots of Onosma visianii was evaluated using two cell lines, mouse melanoma B16 and highly aggressive rat glioma cell line C6. METHODS AND RESULTS: All examined shikonins dose-dependently decreased the viability of tested cells, with compounds 5 and 6 being the most potent ones and hence subjected to further analysis. The diminished viability of B16 melanoma cells was in correlation with detected caspase-mediated apoptosis. Importantly, observed altered cell morphology along with the loss of dividing potential upon exposure to both shikonins implied reprogram of B16 cell phenotype. Elevated expression of myelin basic protein indicated the acquirement of Schwann-like cell phenotype, while detected autophagy might be connected to this phenomenon. On the contrary, upon exposure to both agents, C6 cells underwent specific cell death-anoikis, provoked by detachment from the extracellular matrix and compromised integrin signaling. Oppositely to compound 5, compound 6 realized anoikis in a caspase-independent manner and under sustained ERK1/2 activation, indicating the deviation from standard proanoikis signaling. CONCLUSIONS: Herein, we have pointed out the diversity and novelty in the mode of action of shikonin derivatives depending on the tumor cell features, which represents a good platform for new investigations of these promising natural compounds.

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.

The extraction and anti-inflammatory screening of Onosma glomeratum Y. L. Liu.

"Zicao" has a long medicinal history and has a variety of pharmacological activities. As the main resource of "zicao" in Tibet, Onosma glomeratum Y. L. Liu (tuan hua dian zi cao), usually used for treating pneumonia in Tibet, has not been reported deeply. In order to determine the main anti-inflammatory active ingredients of Onosma glomeratum Y. L. Liu, in this study, the extracts enriched in naphthoquinones and polysaccharides were optimized prepared form Onosma glomeratum Y. L. Liu by ultrasonic extraction, and reflux extraction, respectively, with Box-Behnken design effect surface method. And their anti-inflammatory abilities were screened on LPS induced A549 cells model, for figuring out the anti-inflammatory active ingredients from Onosma glomeratum Y. L. Liu.The extract enriched naphthoquinone was obtained under following condition: extract with 85% ethanol in a liquid to material ratio of 1:40 g/mL at 30 °C for 30 minutes using ultrasound, leading to the extraction rate of total naphthoquinone as 0.98 ± 0.017%; the extract enriched polysaccharides was prepared as follows: extract 82 minutes at 100 °C with distilled water in a liquid to material ratio of 1:50 g/mL, with extraction rate of polysaccharide as 7.07 ± 0.02%.On the LPS-induced A549 cell model, the polysaccharide extract from Onosma glomeratum Y. L. Liu showed better anti-inflammatory effects than the naphthoquinone extract, indicating the extract enriched in polysaccharides is the anti-inflammatory extract of Onosma glomeratum Y. L. Liu, which could serve as a potential anti-inflammatory extract in medical and food industries in the future.

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.

Development and Validation of a Method to Quantify Flavonoids in Leaves of Lithospermum officinale (Boraginaceae).

The common gromwell Lithospermum officinale L. is a valuable medicinal plant that has been used in traditional medicine since ancient times. A method to quantify flavonoids in L. officinale leaves by differential spectrophotometry was developed taking advantage of the flavonoid reaction with aluminum chloride. The optimum duration of the reaction was determined, as well as the optimum volume-to-volume ratio between an aqueous ethanolic extract of L. officinale leaves and 2% aluminum chloride (aqueous ethanolic solution). Rutin was used as a standard. The method was validated in terms of specificity, linearity, precision, and accuracy and proved suitable for analytical purposes. The flavonoid content expressed in terms of rutin was found to exceed 2% of the absolutely dry weight in L. officinale leaves over different years of cultivation.

[Correlation between rhizosphere environment and content of medicinal components of Arnebia euchroma].

This study aimed to explore the correlation between rhizosphere soil microorganisms of wild Arnebia euchroma and the content of medicinal components to provide guidance for the selection of the ecological planting base. The total DNA of rhizosphere soil microorganisms of wild A. euchroma was extracted, and the microbial community structure of rhizosphere soil microorganisms was analyzed by IlluminaMiseq high-throughput sequencing technology. The content of total hydroxynaphthoquinone pigment and ß,ß'-dimethylacrylalkannin in medicinal materials was determined by high-performance liquid chromatography(HPLC). The physicochemical pro-perties of rhizosphere soil of wild A. euchroma in main producing areas were determined, and the correlation of soil microbial abundance with index component content and soil physicochemical properties was analyzed by SPSS software. The results showed that the species composition of rhizosphere fungi and bacteria in A. euchroma from different habitats was similar at the phylum and genus levels, but their relative abundance, richness index(Chao1), and community diversity(Simpson) index were different. Correlation analysis showed that the content of available phosphorus in soil was positively correlated with the content of total hydroxynaphthoquinone pigment and ß,ß'-dimethylacrylalkannin, and the abundance of five fungal genera such as Solicoccozyma and six bacterial genera such as Pseudo-nocardia and Bradyrhizobium was positively correlated with the content of medicinal components in medicinal materials. The abundance of Bradyrhizobium was significantly positively correlated with the content of ß,ß'-dimethylacrylalkanin. The abundance of fungi such as Archaeorhizomyces was significantly positively correlated with the content of available phosphorus in rhizosphere soil, and Bradyrhizobium was significantly negatively correlated with soil pH. Therefore, the abundance of fungi and bacteria in the rhizosphere of A. euchroma has a certain correlation with the medicinal components and the physicochemical properties of the rhizosphere soil, which can provide a scientific basis for the selection of ecological planting bases in the later stage.

Using extract from alkanet (Alkanna tinctoria) as a source of both a red lipid stain and a blue counterstain for histology.

Alkanet (Alkanna tinctoria) is a plant native to and cultivated in parts of Europe, Asia and the Middle East. It has been used for thousands of years as a medicinal agent and as a colorant for textiles, food and cosmetics. An extract from the root of this plant has been used with a mordant to stain nuclei. We describe here the versatility of different extracts from this plant to stain lipids red and to counterstain certain other tissue elements blue. The color variation and selective differential staining is due to solvent polarity and pH. Extracts contain numerous chemical species, all of which are derivatives of the indicator dye, naphthazurin. Our red extract is nonionic below pH 7 and partitions from its somewhat polar solvent of 100% isopropanol to nonpolar lipids. The blue extract is dianionic at high pH in 70% isopropanol and binds ionically to cationic tissue structures such as collagen, muscle and cytoplasm of other cells.

[Effect of acetylalkannin from Arnebia euchroma on proliferation, migration, and invasion of human melanoma A375 cells].

This study aimed to explore the effect and mechanism of acetylalkannin from Arnebia euchroma on the proliferation, migration, and invasion of human melanoma A375 cells. A375 cells were divided into a blank group, and low-, medium-, and high-dose acetylalkannin groups(0.5, 1.0, and 2.0 µmol·L~(-1)). The MTT assay was used to detect cell proliferation. Cell scratch and transwell migration assays were used to detect cell migration ability, and the transwell invasion assay was used to detect cell invasion ability. Western blot was used to detect the protein expression of migration and invasion-related N-cadherin, vimentin, matrix metalloproteina-se-9(MMP-9), and Wnt/ß-catenin pathway-related Wnt1, Axin2, glycogen synthase kinase-3ß(GSK-3ß), phosphorylated GSK-3ß(p-GSK-3ß), ß-catenin, cell cycle protein D_1(cyclin D_1), and p21. Real-time fluorescence-based quantitative polymerase chain reaction(real-time PCR) was used to detect the mRNA expression of E-cadherin, matrix metalloproteinase-2(MMP-2), N-cadherin, vimentin, ß-catenin, snail-1, and CD44. MTT results showed that the cell inhibition rates in the acetylalkannin groups significantly increased as compared with that in the blank group(P<0.01). The results of cell scratch and transwell assays showed that compared with the blank group, the acetylalkannin groups showed reduced cell migration and invasion, and migration and invasion rates(P<0.05, P<0.01) and weakened horizontal and vertical migration and invasion abilities. Western blot results showed that compared with the blank group, the high-dose acetylalkannin group showed increased expression of Axin2 protein(P<0.05), and decreased expression of N-cadherin, vimentin, MMP-9, Wnt1, p-GSK-3ß, ß-catenin, cyclin D_1, and p21 proteins(P<0.05, P<0.01). The expression of GSK-3ß protein did not change significantly. PCR results showed that the overall trend of MMP-2, N-cadherin, vimentin, ß-catenin, snail-1, and CD44 mRNA expression was down-regulated(P<0.01), and the expression of E-cadherin mRNA increased(P<0.01). Acetylalkannin can inhibit the proliferation, migration, and invasion of human melanoma A375 cells, and its mechanism of action may be related to the regulation of Wnt/ß-catenin signaling pathway.

LC-MS/MS Evaluation of Pyrrolizidine Alkaloids Profile in Relation to Safety of Comfrey Roots and Leaves from Polish Sources.

Comfrey (Symphytum officinale L.) has a long tradition of use in the treatment of musculoskeletal disorders. However, due to hepatotoxic pyrrolizidine alkaloids (PAs), the EMA restricts the use of comfrey root (CR) to external use only and for short periods of time. Recent studies indicate a low permeability of PAs across the skin, calling into question the safety of topical application of products containing comfrey preparations. The aim of our work was to develop and validate an HPLC method enabling the separation of isomeric PAs from comfrey and, on this basis, to assess the potential toxicity of CR and comfrey leaf (CL) obtained from various Polish sources. The qualitative and quantitative analysis of PAs via HPLC-MS/MS was performed in MRM mode. The results obtained confirmed a lower content of PAs in CL than in CR and showed a wide variation in the composition of PAs in CR, with a much more stable profile of PAs in CL. Factor analysis confirmed that CRs and CLs differ in PA content, which is influenced by the growth conditions and geographical origin. The determined concentrations of PAs prove that in some CRs available on the Polish herbal market, the content of PAs may exceed the daily dose considered safe.