Symphytum Research Articles

Pectin from comfrey roots alleviate DSS-induced ulcerative colitis in mice through modulating the intestinal barrier.

The objective of this study was to investigate whether pectin extracted from comfrey roots (CRPs) could alleviate ulcerative colitis (UC) induced by sodium dextran sulfate (DSS) in mice by improving the intestinal barrier. CRP was able to relieve symptoms associated with weight loss, diarrhea, and colon length in UC mice. CRP inhibited the excessive secretion of TNF-α and IL-6 and increased the level of IL-10 in the serum. After CRP treatment, the mRNA expression levels of ZO-1 and Muc2 increased. The colonic epithelial cells recovered well, and the mucous layer was relatively intact in the CRP group. CRP and 5-aminosalicylic acid (ASA) alleviated UC symptoms in mice by reducing the abundance of Oscillibacter, Alistipes, and Anaeroplasma and increasing the abundance of Lachnospiraceae_UCG-001 to regulate the intestinal microflora. The abundance of Rikenellaceae was positively correlated with the mRNA expression level of ZO-1, and the abundance of Monoglobus was positively correlated with the mRNA expression level of Muc2. These results suggested that CRP could repair the intestinal barrier and mitigate DSS-induced colon damage in mice, indicating that CRP may be a potential functional component in combating UC.

The Profile and Content of Polyphenolic Compounds and Antioxidant and Anti-Glycation Properties of Root Extracts of Selected Medicinal Herbs

The objective of our study was to analyse the extracts from six medicinal herb roots (marshmallow, dandelion, liquorice, angelica, burdock, and comfrey) in terms of antioxidant capacity (ABTS, DPPH) and inhibition of advanced glycation end product (AGEs) formation. The quantification of phenolic acids and flavonoids was analysed using the UHPLC-DAD-MS method. Fifteen polyphenolic compounds were detected in the studied herbs. The higher number of polyphenols were found in marshmallows (ten polyphenols), while the lowest was in comfrey (five compounds). Liquorice root revealed the highest individual phenolic concentration (382 µg/g dm) with the higher contribution of kaempferol-3-O-rutinoside. Comfrey root extract was characterised by the most abundant TPC (Total Phenolic Content) value (29.79 mg GAE/ g dm). Burdock and comfrey showed the strongest anti-AGE activity studies with the BDA-GLU model. Burdock root was also characterised by the highest anti-AGE activity in the BSA-MGO model. The highest antioxidant capacity was determined by ABTS (72.12 µmol TE/g dw) and DPPH (143.01 µmol TE/g dw) assays for comfrey extract. The p-coumaric acid content was significantly correlated with anti-AGE activity determined by the BSA-MGO model. This research sheds new light on the bioactivity of root herbs, explaining the role of p-coumaric acid in preventing diabetes.

Enrichment of rosmarinic acid from comfrey (Symphytum officinale L.) root extract by macroporous adsorption resins and molecular docking studies

The rosmarinic acid (RA) is known as the major phenolic compound in comfrey roots possessing notable biological properties. This study aimed to enhance the polyphenol content of the comfrey root extract through the use of six different types of macroporous resins chromatography including DM-130, LXA-88, LSD-001, LXA-10, HP-20, and LXA-17. The LSD-001 demonstrated the highest separation efficiency, resulting in a 7-fold increase of RA content from 11.44 mg.g−1 in the extract to 75.79 mg.g−1 for the desorbed extract, as confirmed by HPLC analysis. In optimization of the static adsorption, 40 min contanct time, 11.8 g resin dosage, and 10000 µg mL−1 extract concentration were attained for maximum amount of adsorption capacity and ratio. Also in desorption process the minmum time for maximum recovery was 7 min with 60 mL eluent volume. The isotherm and kinetic studies of RA adsorption on LSD-001 resin showed reasonable agreement with Langmuir suggesting monolayer adsorption machenism, and the pseudo-second-order models (R2=0.98). Morover, the enriched extract containg the highest amount of RA showed an antioxidant activity of almost 3.5 times more than the comfrey root extract. Molecular modeling revealed stacking interactions and hydrogen bonding between the polar sites of RA and the benzene ring of LSD-001 resin. Furthermore, molecular docking of the ligand with the polymer yielded a docking score of −5.75 kcal.mol−1 revealed the incorporation of RA and polymeric structure of LSD-001 ion exchange resin.

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.

Assessment of allantoin concentrations in comfrey root available on the Polish market

Comfrey (<i>Symphytum officinale</i> L.) root is a commonly known rich source of allantoin. This compound is widely used in pharmacy and cosmetology, but mainly in a synthetic form. However, the properties of natural allantoin in products of biological origin may be different from those of synthetic allantoin, especially due to the presence of other bioactive substances. Moreover, even in the same raw material, the level of this compound may vary depending on the plant age and environmental conditions. Therefore, we compared the content of allantoin in selected <i>Symphyti Radix</i> raw materials available on the Polish market. The allantoin concentrations determined using the HPLC method were found to be in the range of 15.14-36.46 mg/g DW. Although comfrey root can be an important source of natural allantoin in dermocosmetics and dermatological formulations, fluctuations in the level of this compound depending on the origin of the raw material should be taken into consideration.

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.

Skin microbiota metabolism of natural products from comfrey root (Symphytum officinale L.)

Ethnopharmacological relevanceComfrey root (Symphytum officinale L., Boraginaceae) has been used in folk medicine for a long time to treat different diseases. It is recommended for swellings, phlebitis, contusions, gastro-duodenal ulcers, respiratory diseases, and metrorrhagia. Currently, preparations from S. officinale are only topically used due to its wound-healing effects, and for reducing inflammation and the treatment of broken bones, tendon damage, painful joints and muscles. Although it is a widespread plant material, little is known about the interaction of externally applied preparations of comfrey with the human skin microbiome. Aim of the studyThe study aims to determine the interaction between human skin microbiota and the comfrey root extracts, by monitoring the biotransformation of the constituents present in the extract and evaluating changes in the population of the skin microbiota in an ex vivo setting. Material and methodsThe comfrey root extract was incubated with the human skin microbiota from ten healthy donors. The UHPLC-DAD-MSn analysis determined the composition of the raw extract and the microbial metabolites. Bacterial genomic DNA was extracted and examined by amplification sequencing of the 16S rDNA to determine changes in the bacterial composition. ResultsThe hydroethanolic extract of comfrey root primarily consists of phenolic acids, pyrrolizidine alkaloids, and their derivatives, and lignans. The natural products present in the extract underwent biodegradation by the skin microbiota, leading to the formation of smaller molecules. It was observed that the skin microbial metabolism primarily focused on modifying the derivatives of pyrrolizidine alkaloids. It resulted in the production of deacetylated and deesterificated compounds. However, it did not lead to the conversion of these compounds into free alkaloids. ConclusionsThe microbiota-triggered biotransformation of the comfrey root extract was observed. A few N-oxides were metabolized to deacetylated and deesterificated forms in ex vivo conditions. It suggests that the intermittent external applications of comfrey preparations perchance are unlikely to pose a substantial risk. While it even may serve as a potential factor influencing the extract activity in treating skin diseases.

β, β-Dimethylacrylshikonin potentiates paclitaxel activity, suppresses immune evasion and triple negative breast cancer progression via STAT3Y705 phosphorylation inhibition based on network pharmacology and transcriptomics analysis

BackgoundTriple negative breast cancer (TNBC) is an extremely aggressive and rapidly progressing cancer, wherein existing therapies provide little benefit to patients. β, β-Dimethylacrylshikonin (DMAS), an active naphthoquinone derived from comfrey root, has potent anticancer activity. However, the antitumor function of DMAS against TNBC remains to be proved. PurposeExplore effects of DMAS on TNBC and clarify the mechanism. Study designNetwork pharmacology, transcriptomics and various cell functional experiments were applied to TNBC cells to explore the effects of DMAS on TNBC. The conclusions were further validated in xenograft animal models. MethodsMTT, EdU, transwell, scratch tests, flow cytometry, immunofluorescence, and immunoblot were utilized to assess the activity of DMAS on three TNBC cell lines. The anti-TNBC mechanism of DMAS was clarified by overexpression and knockdown of STAT3 in BT-549 cells. In vivo efficacy of DMAS was analysed using a xenograft mouse model. ResultsIn vitro analysis revealed that DMAS inhibited the G2/M phase transition and suppressed TNBC proliferation. Additionally, DMAS triggered mitochondrial-dependent apoptosis and reduced cell migration by antagonizing epithelial-mesenchymal transition. Mechanistically, DMAS exerted its antitumour effects by inhibiting STAT3Y705 phosphorylation. STAT3 overexpression abolished the inhibitory effect of DMAS. Further studies showed that treatment with DMAS inhibited TNBC growth in a xenograft model. Notably, DMAS potentiated the sensitivity of TNBC to paclitaxel and inhibited immune evasion by downregulating the immune checkpoint PD-L1. ConclusionsFor the first time, our study revealed that DMAS potentiates paclitaxel activity, suppresses immune evasion and TNBC progression by inhibiting STAT3 pathway. It has the potential as a promising agent for TNBC.

The use of babosa (Aloe vera) in treating burns: a literature review.

Alo vera is a centenary remedy use for minor wounds and burns, but its mechanism of wound healing has not been know since. This article will evaluate and gather evidence of the effectiveness and safety of the use of aloe vera in the treatment of burns. A systematic review was carried out on the databases: MEDLINE, LILACS, DECS, SCIELO, in the last 7 years, with the descriptors: "Aloe", "Burns" and "treatment". 16 articles were found. After using the exclusion criteria; research in non-humans and literature review; 5 articles were selected. The article Teplick et al. (2018) performed an in vitro clinical experiment in A. Vera solution, and demonstrated that there was proliferation and cell migration of human skin fibroblasts and keratinocytes, in addition to being protective in the death of keratonocytes. That is, it accelerates the healing of wounds. Muangman et al. (2016), evaluated 50 patients with 20% of the total body surface area burned with second-degree burns, between 18-60 years old, with half of the group receiving gauze dressings with soft paraffin containing 0.5% chlorhexidine acetate and the other half receiving polyester dressings containing extracts of medicinal plants mainly Aloe Vera. It had positive results, a higher healing speed and shorter hospital stay compared to the control group. Hwang et al. (2015) investigated the antioxidant effects of different extracts from 2,4,6,8,12 months of Aloe Vera. And the 6-month concentrated extract of 0.25 mg / mL had a higher content of flavonoids (9.750 mg catechin equivalent / g extract) and polyphenols (23.375 mg gallic acid equivalent / g extract) and the greater ferric reducing antioxidant power (0.047 mM equivalent ferrous sulfate / mg extract), that is, greater potential for free radical scavenging and also a protective effect against oxidative stress induced by tert-butyl hydroperoxide (t-BHP), suggesting evidence of a bioactive potential of A. vera . However, in the article Kolacz et al. (2014) suggested as an alternative treatment the use of Aloe Vera dressing in combination with honey, lanolin, olive oil, wheat germ oil, marshmallow root, wormwood, comfrey root, white oak bark, lobelia inflata, glycerin vegetable oil, beeswax and myrrh, without obtaining significant and conclusive results that would allow the conventional treatment of burns to be subsidized. Finally, in the article by Zurita and Gallegos (2017), it carried out a descriptive cross-sectional study with 321 people, both sexes between 17-76 years of age, of an inductive nature, exploring the experience of this population and their behavioral attitudes regarding the treatment of dermatoses. Aloe vera had 13.8% cited by individuals in the treatment of acne and 33.6% in the treatment of burns. Even with evidence that suggests the efficacy in the treatment of burns with the use of Aloe Vera extract, further clinical trials with larger sample space on the use of Aloe vera dressings in medium burns are suggested for further conclusions.

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

Ethnopharmacological relevanceComfrey (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 studyIn this work, different PA-/mucilage-depleted/undepleted comfrey root extracts were subjected to detailed phytochemical characterization and biological evaluation. Materials and methodsThe 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-1β, IL-8, and TNF-α were determined (ELISA assay). Results12 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-1β, IL-8, and TNF-α) in LPS-stimulated neutrophils. ConclusionsIn light of their superior safety profiles, PA-depleted comfrey extracts can be utilized further in cosmetic and pharmaceutical products.

Shikonin Attenuates Cochlear Spiral Ganglion Neuron Degeneration by Activating Nrf2-ARE Signaling Pathway.

The molecular mechanisms that regulate the proliferation and differentiation of inner ear spiral ganglion cells (SGCs) remain largely unknown. Shikonin (a naphthoquinone pigment isolated from the traditional Chinese herbal medicine comfrey root) has anti-oxidation, anti-apoptosis and promoting proliferation and differentiation effects on neural progenitor cells. To study the protective effect of shikonin on auditory nerve damage, we isolated spiral ganglion neuron cells (SGNs) and spiral ganglion Schwann cells (SGSs) that provide nutrients in vitro and pretreated them with shikonin. We found that shikonin can reduce ouabain, a drug that can selectively destroy SGNs and induce auditory nerve damage, caused SGNs proliferation decreased, neurite outgrowth inhibition, cells apoptosis and mitochondrial depolarization. In addition, we found that shikonin can increase the expression of Nrf2 and its downstream molecules HO-1 and NQO1, thereby enhancing the antioxidant capacity of SGNs and SGSs, promoting cells proliferation, and inhibiting cells apoptosis by activating the Nrf2/antioxidant response elements (ARE) signal pathway. However, knockdown of Nrf2 rescued the protective effect of shikonin on SGNs and SGSs damage. In addition, we injected shikonin pretreatment into mouse that ouabain-induced hearing loss and found that shikonin pretreatment has a defensive effect on auditory nerve damage. In summary, the results of this study indicate that shikonin could attenuate the level of oxidative stress in SGNs and SGSs through the Nrf2-ARE signaling pathway activated, induce the proliferation and differentiation of SGNs, and thereby improve the neurological hearing damage in mice. Therefore, shikonin may be a candidate therapeutic drug for endogenous antioxidants that can be used to treat neurological deafness.

Allantoin from Valuable Romanian Animal and Plant Sources with Promising Anti-Inflammatory Activity as a Nutricosmetic Ingredient

Helix aspersa (HA), Helix pomatia (HP) and Symphytum officinale are common organisms in Romania’s biosphere, widely known for their allantoin content and their therapeutic properties. Herein, the allantoin was separated and quantified from the aqueous extracts of Romanian comfrey root and the secretions of HA and HP snails. This study also focused on determining the antioxidant and anti-inflammatory activities of these Romanian allantoin-rich samples. The plant extracts were obtained through two methods: ultrasonic extraction and enzymatic ultrasonic extraction. A microplate method was used for the quantitative determination of allantoin content. The antioxidant activity was measured by using the DPPH radical scavenging method. The antioxidant capacity of the samples was studied in order to observe the type of interactions generated by the chemical complex present in their composition. High concentrations of allantoin were obtained by enzymatic ultrasonic extraction method (EUE—102 ± 0.74 μg/mL), and also in the water-soluble fraction of the snail secretion (FS1—22.051 μg/mL). The antioxidant screening suggests that Symphytum officinale and snail mucus extracts could be used as promising natural substitutes for synthetic antioxidants in products used for therapeutic purposes. The evaluation of anti-inflammatory activity was also investigated, allantoin-rich samples showing a promising action (FS1—81.87 ± 2.34%). In future, the inclusion of allantoin-rich extracts in various novel pharmaceutical forms for new therapeutic applications could be achieved. The study will continue with the formulation of a nutricosmetic product with snail mucus and Symphytum officinale extract as principal bioactive ingredients.

Influence of the Post-Harvest Storage Time on the Multi-Biological Potential, Phenolic and Pyrrolizidine Alkaloid Content of Comfrey (Symphytum officinale L.) Roots Collected from Different European Regions.

Comfrey (Symphytum officinale L.) roots are well-known bioactive ingredients included in various cosmeceutical and pharmaceutical preparations. In this study, the influence of the post-harvest storage on the chemico-biological potential of roots collected from different European regions and stored for up to six months was investigated. Total phenolic content (TPC) and total phenolic acid content (TPAC) were spectrophotometrically estimated, whereas the levels of individual phenolic and pyrrolizidine alkaloidal markers were determined by HPLC-DAD and HPLC-MS/MS, respectively. The changes in the biological potential was tracked via antioxidant (DPPH, ABTS, CUPRAC, and FRAP) and anti-enzymatic (cholinesterase, tyrosinase, glucosidase, and amylase) assays. TPC and TPAC varied from 6.48–16.57 mg GAE/g d.w. root and from 2.67–9.03 mg CAE/g, respectively. The concentration of the four phenolics (rosmarinic acid, globoidnan A, globoidnan B, rabdosiin) and six pyrrolizidine alkaloids generally showed maximum values at 1–3 months, after which their levels significantly decreased. With respect to the bioassays, the samples showed a wide range of antioxidant and anti-enzymatic effects; however, a direct storage time–bioactivity relationship was not observed. Similar conclusions were also revealed by the multivariate and correlation analyses. Our study could improve the current knowledge of the shelf-life properties of comfrey-based products and enhance their industrial exploitation.

Support for Regulatory Assessment of Percutaneous Absorption of Retronecine-type Pyrrolizidine Alkaloids through Human Skin.

1,2-unsaturated pyrrolizidine alkaloids are found naturally in Symphytum officinale, well known as comfrey, which has a longstanding use for the topical treatment of painful muscle and joint complaints. Pyrrolizidine alkaloids (PA) are a relevant concern for the safety assessment due to their liver genotoxicity profile, and close attention is paid during manufacturing to minimizing their levels. Current regulatory risk assessment approaches include setting limits that derive from toxicity data coming from the oral route of exposure. This study investigated to what extent pyrrolizidine alkaloids are bioavailable following topical exposure, assessing penetration of retronecine-type PAs in an in vitro human skin model. A single comfrey root formulation was spiked with 3 different congeners (a 7R-monoester, an open-chained 7R-diester, and a cyclic diester) and percutaneous absorption measured per OECD guidelines and good laboratory practices. The measured penetration for all 3 PAs was low and compared favourably with existing in vitro data. Although consideration of different regulatory guidance influences the determination of dermally absorbed dose, these data facilitate the understanding of absorption differences following topical exposure, which in turn can be taken into account in the risk assessment.

Phytochemical Analysis of Symphytum officinale Root Culture Extract

Comfrey (Symphytum officinale) root preparations are known for their analgesic and anti-inflammatory properties. In this study, the phenolic profile of extract samples of Symphytum officinale callus root cultures (up to 70% of ethanol) was determined using various approaches. For the first time, a unique polymer of two types was isolated from samples of comfrey root cultures (the caffeic acid derivative is a new class of natural polyether with 3-(3,4-dihydroxyphenyl) glyceric acid as a repeating unit). M-methoxybenzoic acid (30.05 μg/mL extract) or rosmarinic acid (45.70 μL/mL extract) were identified as the main phenolic component. Rosmarinic, chlorogenic, and caffeic acids have anti-inflammatory, anti-apoptotic, antitumor, neuroprotective, antioxidant, and other properties, which also determine the characteristics of comfrey and preparations made from it. The studied phenolic profile and the analysis of published data showed that the extract samples of comfrey callus cultures had similar characteristics, which makes them an important source of phenolic compounds with pronounced antioxidant activity.

Skuteczność i bezpieczeństwo stosowania korzenia Symphytum officinale L. – przegląd danych literaturowych

Comfrey root – Symphyti radix has been present in medicine since ancient times. Nowadays, European Medicine Agency (EMA) restricts its only for external use, in short therapies not exceeding 10 days. The last decade has been characterized by the progress in research on the chemical composition and metabolism of biologically active compounds present in the root and leaves. The result is the presence of herbal medicinal products containing comfrey root extracts with reduced content or free of toxic pyrrolizidine alkaloids on the European market of herbal products. Moreover, the medicinal properties of leaves from another Symphytum species, namely Russian comfrey, are also in the use. The results of clinical trials indicate the beneficial effects of plant raw materials obtained from comfreys in musculoskeletal system disorders – comfrey root and skin inflammations – leaves of Russian comfrey. The paper presents the history of use, chemical composition, therapeutic effect and toxicity of comfrey, as well as characterizes clinical trials on products containing comfrey extracts from root and leaves.

Globoidnan A, rabdosiin and globoidnan B as new phenolic markers in European-sourced comfrey (Symphytum officinale L.) root samples.

Symphytum officinale L. (comfrey, Boraginaceae) is a cultivated or spontaneously growing medicinal plant that is traditionally used for the treatment of bone fractures, hematomas, muscle pains and joint pains. A wide range of topical preparations and dried roots for ex tempore applications are marketed in European drug stores or pharmacies. The aim of this study was to perform the qualitative and quantitative analysis of pyrrolizidine alkaloids (PAs) and phenolic compounds in the hydroethanolic extracts of 16 commercial comfrey root batches purchased from 12 different European countries. Liquid chromatography hyphenated with high-resolution tandem mass spectrometry (LC-HRMS/MS) was used for the profiling of PAs and phenolic compounds, whereas LC-MS/MS and liquid chromatography with diode array detection (LC-DAD) were used for their quantification. 20 PAs (i.e. intermedine, lycopsamine, acetylintermedine, acetyllycopsamine, symphytine, symphytine-N-oxide), 17 phenolic compounds (i.e. caffeic and rosmarinic acids, rabdosiin, globoidnan A, globoidnan B) and 9 nonphenolic compounds (sugars, organic and fatty acids) were fully or partly annotated in the analysed samples. In addition, the quantitative analyses revealed that globoidnan B, rabdosiin and globoidnan A are new phenolic markers that can be used together with rosmarinic acid and PAs for the quality control of commercial comfrey root batches. This study brings new insights into the phytochemical complexity of S. officinale, revealing not only numerous toxic PAs, but also a significant number of valuable phenolic compounds that could contribute to the bioactivities of comfrey-based preparations.

Symphytum officinale L.: Liquid-liquid chromatography isolation of caffeic acid oligomers and evaluation of their influence on pro-inflammatory cytokine release in LPS-stimulated neutrophils

Ethnopharmacological relevanceSymphytum officinale L. (comfrey, Boraginaceae) has been traditionally used for millennia in joint distortions, myalgia, bone fractures and hematomas. However, key activity-determining constituents and molecular mechanisms underlying its use have not been completely elucidated. Aim of the studyThe objective of this study was to isolate and identify the major compounds from a hydroethanolic root extract of S. officinale and evaluate their antioxidant potential, alongside their effect on the cytokine production of ex vivo stimulated neutrophils, thus providing scientific support for the traditional use of comfrey root. Material and methodsFour caffeic acid oligomers were isolated from comfrey roots by liquid-liquid chromatography, their structures being established by MS and NMR analyses. In vitro antioxidant evaluation was performed by DPPH and ABTS assays. The cytotoxicity of isolated compounds was established by flow cytometry. The effect on cytokine release, such as interleukin (IL)-1β, IL-8 and tumor necrosis factor alpha (TNF-α), in lipopolysaccharide (LPS)-stimulated neutrophils was determined by enzyme-linked immunosorbent assay (ELISA). ResultsThe main constituents found in comfrey root were represented by four caffeic acid oligomers, namely globoidnan B (1), rabdosiin (2), rosmarinic acid (3) and globoidnan A (4). Rabdosiin, globoidnans A and B were isolated for the first time from S. officinale. In the in vitro antioxidant tests, compound 2 was the most active, with EC50 values in DPPH and ABTS assays of 29.14 ± 0.43 and 11.13 ± 0.39, respectively. Neutrophils’ viability over the tested concentration domain of 12.5–50 μM was not altered. At 50 μM, all compounds significantly inhibited IL-1β release, with compound 3 (45.60% release vs. LPS stimulated neutrophils) being the most active, followed by compounds 1 (53.85%), 2 (69.89%) and 4 (60.68%). ConclusionsThe four caffeic acid oligomers reported in S. officinale root may contribute to the overall anti-inflammatory activity for which comfrey preparations are used in traditional medicine.

LC-ESI-FT-MSn Metabolite Profiling of Symphytum officinale L. Roots Leads to Isolation of Comfreyn A, an Unusual Arylnaphthalene Lignan.

Preparations of comfrey (Symphytum officinale L.) roots are used topically to reduce inflammation. Comfrey anti-inflammatory and analgesic properties have been proven in clinical studies. However, the bioactive compounds associated with these therapeutic activities are yet to be identified. An LC–ESI–Orbitrap–MSn metabolite profile of a hydroalcoholic extract of comfrey root guided the identification of 20 compounds, including a new arylnaphthalene lignan bearing a rare δ-lactone ring, named comfreyn A. Its structure was determined using extensive 2D NMR and ESI–MS experiments. Additionally, the occurrence of malaxinic acid, caffeic acid ethyl ester, along with the lignans ternifoliuslignan D, 3-carboxy-6,7-dihydroxy-1-(3′,4′-dihydroxyphenyl) -naphthalene, globoidnan A and B, and rabdosiin was reported in S. officinale for the first time. These results helped to redefine the metabolite profile of this medicinal plant. Finally, caffeic acid ethyl ester and comfreyn A were found to significantly inhibit E-selectin expression in IL-1β stimulated human umbilical vein endothelial cells (HUVEC), with EC values of 64 and 50 µM, respectively.

Comparative Assessment of Phytochemical Profiles of Comfrey (Symphytum officinale L.) Root Extracts Obtained by Different Extraction Techniques.

In this work a comparative study on phytochemical profiles of comfrey root extracts obtained by different extraction approaches has been carried out. Chemical profiles of extracts obtained by supercritical fluid (SFE), pressurized liquid (PLE), and conventional solid/liquid extraction were compared and discussed. Phytochemical composition was assessed by high-performance liquid chromatography coupled with electrospray time-of-flight mass spectrometry (HPLC-ESI-QTOF-MS/MS) identifying 39 compounds reported for the first time in comfrey root, mainly phenolic acids and fatty acids. The influence of different extraction parameters on phytochemical profiles of S. officinale root was investigated for all applied techniques. PLE and maceration, using alcohol-based solvents (aqueous methanol or ethanol), were shown to be more efficient in the recovery of more polar compounds. Greater numbers of phenolics were best extracted by PLE using 85% EtOH at 63 °C. The use of SFE and 100% acetone for 30 min enabled good recoveries of nonpolar compounds. SFE using 15% EtOH as a cosolvent at 150 bar produced the best recoveries of a significant number of fatty acids. The main compositional differences between extracts obtained by different extraction techniques were assigned to the solvent type. Hence, these results provided comprehensive approaches for treating comfrey root enriched in different phytochemicals, thereby enhancing its bioaccessibility.