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.

Phytochemical profiling and bioactivity assessment of underutilized Symphytum species in comparison with Symphytum officinale.

BACKGROUND: Symphytum (comfrey) genus, particularly Symphytum officinale, has been empirically used in folk medicine mainly for its potent anti-inflammatory properties. In an attempt to shed light on the valorization of less known taxa, the current study evaluated the metabolite profile and antioxidant and enzyme inhibitory effects of nine Symphytum species. RESULTS: Phenolic acids, flavonoids and pyrrolizidine alkaloids were the most representative compounds in all comfrey samples. Hierarchical cluster analysis revealed that, within the roots, S. grandiflorum was slightly different from S. ibericum, S. caucasicum and the remaining species. Within the aerial parts, S. caucasicum and S. asperum differed from the other samples. All Symphytum species showed good antioxidant and enzyme inhibitory activities, as evaluated in DPPH (up to 50.17 mg Trolox equivalents (TE) g-1), ABTS (up to 49.92 mg TE g-1), cupric reducing antioxidant capacity (CUPRAC, up to 92.93 mg TE g-1), ferric reducing antioxidant power (FRAP, up to 53.63 mg TE g-1), acetylcholinesterase (AChE, up to 0.52 mg galanthamine equivalents (GALAE) g-1), butyrylcholinesterase (BChE, up to 0.96 mg GALAE g-1), tyrosinase (up to 13.58 mg kojic acid equivalents g-1) and glucosidase (up to 0.28 mmol acarbose equivalents g-1) tests. Pearson correlation analysis revealed potential links between danshensu and ABTS/FRAP/CUPRAC, quercetin-O-hexoside and DPPH/CUPRAC, or rabdosiin and anti-BChE activity. CONCLUSIONS: By assessing for the first time in a comparative manner the phytochemical-biological profile of a considerably high number of Symphytum samples, this study unveils the potential use of less common comfrey species as novel phytopharmaceutical or agricultural raw materials. © 2024 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

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.

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.

Comfrey polysaccharides modulate the gut microbiota and its metabolites SCFAs and affect the production performance of laying hens.

Effects of dietary supplementation of comfrey polysaccharides (CPs) on production performance, egg quality, and microbial composition of cecum in laying hens were evaluated. A total of 240 laying hens were allocated into 4 groups with 6 replicates per group. The laying hens were fed diets containing CPs at levels of 0, 0.5, 1.0, and 1.5 %, respectively. The results showed that the egg production rate increased by 5.97 %, the egg mass improved by 6.71 %, and the feed conversion rate reduced by 5.43 % in the 1.0 % supplementation group of CPs compared with those in the control group. The digestibility of ash, crude fat, and phosphorus was notably improved by the addition of CPs at 1.0 % (P < 0.05). The relative abundances of Bacteroidetes at the phylum level, Bacteroidaceae, Rikenellaceae, and Prevotellaceae at the family level were increased by CPs (P < 0.05). The relative abundances of Bacteroides, Megamonas, Rikenellaceae_RC9_gut_group, [Ruminococcus]_torques_group, Methanobrevibacter, Desulfovibrio, Romboutsia, Alistipes, and Intestinimonas at the genus level were increased by CPs (P < 0.05). Dietary supplementation of CPs could enhance the production performance of laying hens, which might be related to the improvement of nutrient digestibility and microbial community modulations in the cecum. Therefore, CPs have potential application value as prebiotics in laying hens.

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.

Streptomyces pactum and Bacillus consortium influenced the bioavailability of toxic metals, soil health, and growth attributes of Symphytum officinale in smelter/mining polluted soil.

Soil microbes influence the uptake of toxic metals (TMs) by changing soil characteristics, bioavailability and translocation of TMs, and soil health indicators in polluted environment. The potential effect of Streptomyces pactum (Act12) and Bacillus consortium (B. subtilis and B. licheniformis; 1:1) on soil enzymes and bacterial abundance, bioavailability and translocation of Zn and Cd by Symphytum officinale, and physiological indicators in soil acquired from Fengxian (FX) mining site. Act12 and Bacillus consortium were applied at 0 (CK), 0.50 (T1), 1.50 (T2), and 2.50 (T3) g kg-1 in a split plot design and three times harvested (H). Results showed that soil pH significantly dropped, whereas, electrical conductivity increased at higher Act12 and Bacillus doses. The extractable Zn lowered and Cd increased at each harvest compared to their controls. Soil ß-glucosidase, alkaline phosphatase, urease and sucrase improved, whereas, dehydrogenase reduced in harvest 2 and 3 (H2 and H3) as compared to harvest 1 (H1) after Act12 and Bacillus treatments. The main soil phyla individually contributed ∼5-55.6%. Soil bacterial communities' distribution was also altered by Act12 and Bacillus amendments. Proteobacteria, Acidobacteria, and Bacteroidetes increased, whereas, the Actinobacteria, Chloroflexi, and Gemmatimonadetes decreased during the one-year trial. The Zn and Cd concentration significantly decreased in shoots at each harvest, whereas, the roots concentration was far higher than the shoots, implicating the rhizoremediation by S. officinale. Accumulation factor (AF) and bioconcentration ratio (BCR) of Zn and Cd in shoots were lower and remained higher in case of roots than the standard level (≥1). BCR values of roots indicated that S. officinale can be used for rhizoremediation of TMs in smelter/mines-polluted soils. Thus, field trials in smelter/mines contaminated soils and the potential role of saponin and tannin exudation in metal translocation by plant will broaden our understanding about the mechanism of rhizoremediation of TMs by S. officinale.

Bacillus subtilis and saponin shifted the availability of heavy metals, health indicators of smelter contaminated soil, and the physiological indicators of Symphytum officinale.

Bacillus subtilis and saponin were tested for the uptake of heavy metals (HMs) by Symphytum officinale grown in a smelter-contaminated soil in completely randomized design. Soil pH and electrical conductivity increased by 0.11 unit (T3) and 754 mS cm-1 (T2), respectively. The bioavailable Zn decreased by 5.80% (T2); Cd and Pb increased by 6.21% (T2) and 13.46% (T3), respectively. Soil urease increased by 24% (T3) and alkaline phosphatase, ß-glucosidase, and dehydrogenase decreased by 20% (T2), 27.70% (T2), and 21% (T1), respectively. Soil amendments altered the microbial diversity. Fourier-transform infrared spectroscopy and X-ray diffraction reported no obvious changes, except saponin application, which led to possible release of HMs in soil. The fresh weight of Symphytum officinale increased by 21.3 and 5.50% in T2 and T3, respectively. Chlorophyll (a) and carotenoid decreased by the sole application of B. subtilis and saponin and vice-versa for chlorophyll (b). Mono-application of B. subtilis efficiently increased the peroxidase (POD: 27%) and polyphenol oxidase (PPO: 13.56%), whereas, co-application enhanced the phenylalanine ammonia-lyase (PAL: 6.50%) level in shoots. Zn concentration in the shoots and roots declined by 12.75 and 27.32% in T1, respectively. Cd increased (3.92%, T3) in shoots and decreased (39.25%, T1) in roots; Pb concentration remained below detection in shoots and increased by 40% (T3) in roots due to accumulation in dead cells and cell vacuoles. Overall, B. subtilis and saponin influenced the bioavailability of HMs, enzymatic activities, and bacterial abundance in the soil; plant growth indicators, antioxidants activities, and metal uptake in shoots and roots.

Comfrey (Symphytum spp.) as a feed supplement in pig nutrition contributes to regional resource cycles.

In smallholder agriculture, the fast-growing and perennial accumulator plant comfrey (Symphytum spp.) was used to supply pigs with protein and minerals. Comfrey leaves show similar values in dry matter as soybean or blue lupine in crude protein content, but much higher levels of calcium and phosphorus. However, in terms of increased efficiency in animal husbandry, comfrey has been displaced by mainly soybean and cereals. Due to its profile of macro- and micronutrients the use of comfrey could have the potential to re-establish local resource cycles and help remediate over-fertilized soils. The aim of the study was to evaluate whether a modern pig breed accepts a continuous feed supplement of dried comfrey leaves. After an initial adaptation period post-weaning, German Landrace piglets were subjected to either a standard control diet or a diet supplemented with 15% dried comfrey leaves for 4 weeks. Body weight was reduced in comfrey-supplemented piglets compared to controls, which might be attributed to reduced palatability in the experimental setting. Nevertheless, comfrey-supplemented piglets exhibited adequate bone mineralization and intestinal integrity. The microbiome profile in feces and digesta revealed higher diversity in comfrey-supplemented piglets compared to controls, with pronounced effects on the abundances of Treponema and Prevotella. This may be due to described bio-positive components of the comfrey plant, as data suggest that the use of comfrey leaves may promote intestinal health. Digestive tract phosphorus levels were reduced in piglets receiving comfrey supplementation, which may ultimately affect phosphorus levels in manure. Results indicate that comfrey leaves could serve as a feed component in integrated agricultural systems to establish regional nutrient cycles. The trial provides a basis for further work on comfrey as a regionally grown protein source and effective replacement for rock mineral supplements.

LC-HRMS/MS phytochemical profiling of Symphytum officinale L. and Anchusa ochroleuca M. Bieb. (Boraginaceae): Unveiling their multi-biological potential via an integrated approach.

This study was aimed at providing a comprehensive phytochemical characterization and multi-biological assessment of Symphytum officinale L., a medicinal plant with a noteworthy traditional use, and Anchusa ochroleuca M. Bieb., a Boraginaceae species from the Romanian flora. The dichloromethane, methanol and 65 % ethanol extracts obtained from the roots and aerial parts of both plants revealed the presence of numerous phenolic acids, oxygenated fatty acids, pyrrolizidine alkaloids (PAs) and flavonoids, as assessed by LC-HRMS/MS analysis. Consistent with their higher total phenolic content, the polar aerial part extracts of S. officinale and root extracts of A. ochroleuca showed the most significant antioxidant activities, as evaluated by DPPH (173.22-216.98 mg TE/g) and ABTS (219.41-311.97 mg TE/g) radical scavenging, CUPRAC (387.18-626.40 mg TE/g), FRAP (199.36-299.86 mg TE/g) and total antioxidant capacity (2.28-2.68 mmol TE/g). Furthermore, both plants exhibited good tyrosinase (19.11-43.89 mg KAE/g) and α-glucosidase (2.45-12.54 mmol ACAE/g) inhibitory effects. The orthogonal projections to latent structures discriminant analysis (OPLS-DA) allowed the objective differentiation between the roots and aerial parts of the two investigated species based on their phytochemical and biological profiles. The partial least square (PLS) analysis showed that several individual phenolic acids, such as danshensu, rabdosiin and rosmarinic acid, significantly contributed to the antioxidant potential of both Boraginaceae species, whilst the relative levels of sucrose were positively correlated with the anti-enzymatic properties. Overall, S. officinale and A. ochroleuca could be regarded as rich sources of bioactive phytochemicals that could further lead to developing novel phyto-pharmaceutical commodities.

CRISPR/Cas9-Mediated Genome Editing in Comfrey (Symphytum officinale) Hairy Roots Results in the Complete Eradication of Pyrrolizidine Alkaloids.

Comfrey (Symphytum officinale) is a medicinal plant with anti-inflammatory, analgesic, and proliferative properties. However, its pharmaceutical application is hampered by the co-occurrence of toxic pyrrolizidine alkaloids (PAs) in its tissues. Using a CRISPR/Cas9-based approach, we introduced detrimental mutations into the hss gene encoding homospermidine synthase (HSS), the first pathway-specific enzyme of PA biosynthesis. The resulting hairy root (HR) lines were analyzed for the type of gene-editing effect that they exhibited and for their homospermidine and PA content. Inactivation of only one of the two hss alleles resulted in HRs with significantly reduced levels of homospermidine and PAs, whereas no alkaloids were detectable in HRs with two inactivated hss alleles. PAs were detectable once again after the HSS-deficient HRs were fed homospermidine confirming that the inability of these roots to produce PAs was only attributable to the inactivated HSS and not to any unidentified off-target effect of the CRISPR/Cas9 approach. Further analyses showed that PA-free HRs possessed, at least in traces, detectable amounts of homospermidine, and that the PA patterns of manipulated HRs were different from those of control lines. These observations are discussed with regard to the potential use of such a CRISPR/Cas9-mediated approach for the economical exploitation of in vitro systems in a medicinal plant and for further studies of PA biosynthesis in non-model plants.

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

INTRODUCTION: 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. OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Safety of medicinal comfrey cream preparations (Symphytum officinale s.l.): The pyrrolizidine alkaloid lycopsamine is poorly absorbed through human skin.

European Union guidelines indiscriminately discuss a permitted daily exposure (PDE) for pyrrolizidine alkaloids (PA) of up to 0.007 µg/kg body weight for oral and for topical exposure to herbal medicinal products. In this study, lycopsamine served as a model substance for measuring the extent of skin permeation of PAs following the application of a spiked comfrey cream (Symphytum officinale s.l.) to abdominal skin from human donors in Franz diffusion cells. PAs could be excluded in the non-spiked cream with a limit of detection of 8 µg/kg. Only small amounts of the applied quantity of lycopsamine had migrated through the skin sample into the receptor cell side of the diffusion cell after 24 h. In five of six diffusion cells, there was no detectable lycopsamine within the skin and only 0.6 ± 0.4% of the applied dose in the receptor fluid. The theoretical skin penetration of 4.9% of the applied quantity of lycopsamine largely resulted from the worst case approach of assuming the presence of at least a quantity corresponding to the limit of detection - the true penetration is probably considerably lower. Even with the worst-case calculation, the currently discussed guidelines on PA overestimate the risk related to topical 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 RELEVANCE: Symphytum 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 STUDY: The 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 METHODS: Four 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)-1ß, IL-8 and tumor necrosis factor alpha (TNF-α), in lipopolysaccharide (LPS)-stimulated neutrophils was determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: The 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-1ß 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%). CONCLUSIONS: The 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-1ß stimulated human umbilical vein endothelial cells (HUVEC), with EC values of 64 and 50 µM, respectively.

Comfrey (Symphytum spp.) as an alternative field crop contributing to closed agricultural cycles in chicken feeding.

Local cultivars of comfrey (Symphytum spp.) have been used to cover protein and mineral requirements of farm animals in low-input systems. Due to its known health-promoting (e.g. allantoin), but also anti-nutritive ingredients (e.g. pyrrolizidine alkaloids), multidisciplinary approaches are essential in order to quantify the nutritional value and the potential of its use in poultry and farm animals in terms of meeting animal needs, using local resources as well as remediating over-fertilized soils. Focusing on animal effects, here one-day old sexed Cobb500 broiler chickens were subjected to either a standard control diet or a standard diet supplemented with 4% dried comfrey leaves for 32 days. Performance traits indicate good acceptance of supplementation with comfrey leaves. Parameters for liver function, mineral homeostasis, bone mineral density as well as intestinal microanatomy revealed no signs of impairment. Quantified pyrrolizidine alkaloids were below the detection limit in liver and breast muscle (<5 µg/kg tissue). Comfrey supplemented male broiler chickens showed higher ash content in breast muscle and revealed altered gene expression profiles for metabolic pathways in blood cells. In healthy broiler chickens, the transcriptome analyses revealed no aberrations in the immune-related pathways due to comfrey supplementation. The results imply that the use of comfrey leaves in a high-performance broiler line seems feasible and offers the potential for closed nutrient cycles in site-adapted local agricultural systems. Further analyses need to focus on possible growth-promoting and health-improving components of comfrey and the safe use of chicken products for human consumption.

Epidermal Regeneration Induced by Comfrey Extract: A Study by Light and Electron Microscopy.

INTRODUCTION: An accelerated healing of superficial wounds was demonstrated in clinical trials with a topical comfrey preparation (Symphytum × uplandicum Nyman). The effect has previously not been examined in skin models. METHODS: An established in vitro model of epidermal cells with the typical strata was used for the observation of effects of applied substances on skin regeneration. Damage corresponding to a typical abrasion was created on day 1 by punching an opening into the epidermal fine structure down to the stratum basale. Samples were either untreated (controls) or exposed to comfrey cream on days 2, 3, 5, and 6. Tissue samples were taken for light and electron microscopy on days 1, 4, and 7. RESULTS AND CONCLUSIONS: Application of comfrey cream led to a quicker regeneration of skin cells and to an earlier differentiation of the cells towards a normal fine structure with a visible distinction of epidermal strata, keratin, and corneocyte formation within 4-7 days. The study covered the early days of skin regeneration and confirms the benefits observed in published clinical trials and non-interventional studies in patients with abrasions.

A systematic review and quality assessment of case reports of adverse events for borage (Borago officinalis), coltsfoot (Tussilago farfara) and comfrey (Symphytum officinale).

Symphytum officinale (comfrey), Tussilago farfara (coltsfoot) and Borago officinalis (borage) have long histories of therapeutic use, but their safety has been questioned due to the presence of unsaturated pyrrolizidine alkaloids (PAs). The evidence base underlying these concerns relies in part on case reports. This systematic review assesses these case reports for their reliability to inform this debate. METHOD: Study selection was restricted to case reports describing possible pyrrolizidine alkaloid related harm and ingestion of comfrey, coltsfoot or borage. An extensive search of academic databases was conducted. Papers meeting the criteria were critically appraised. RESULTS: The search resulted in 11 appropriate case reports, none of which involved borage. Nine reports were assessed for causality and indicated some degree of association between the material ingested and the adverse event. Lack of unequivocal identification of the species ingested compromised attribution and was a significant source of uncertainty. Three levels of identity confusions were found; misidentification or substitution at the level of the whole herb; omission of appropriate botanical identification and attribution of a specific PA to either comfrey or coltsfoot when it is a constituent found in other plants of established toxicity. CONCLUSION: These cases are an unreliable body of evidence on which to draw conclusions about the safety of the oral consumption of Symphytum officinale and Tussilago farfara. Toxicological studies based on oral ingestion of phytochemically-complex preparations of these herbs may be the most accurate methodology for assessing clinical risk.

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.

Symphytum officinale augments osteogenesis in human bone marrow-derived mesenchymal stem cells in vitro as they differentiate into osteoblasts.

ETHNOPHARMACOLOGICAL RELEVANCE: Mesenchymal stem cells (MSCs) are multipotent stem cells possessing regenerative potential. Symphytum officinale (SO) is a medicinal plant and in homoeopathic literature, believed to accelerate bone healing. AIM OF THE STUDY: This study aimed to determine if homoeopathic doses of SO could augment osteogenesis in MSCs as they differentiate into osteoblasts in vitro. MATERIALS AND METHODS: Bone marrow samples were obtained from patients who underwent bone grafting procedures (n = 15). MSCs were isolated, expanded and characterized by flow cytometry (CD90, CD105). Cytotoxicity of SO was evaluated by MTT assay. Osteogenic differentiation was induced in MSCs with ß-glycerophosphate, ascorbic acid and dexamethasone over 2 weeks. Different homoeopathic doses of SO (MT, 3C, 6C, 12C and 30C) were added to the basic differentiation medium (BDM) and efficiency of MSCs differentiating into osteoblasts were measured by evaluating expression of Osteocalcin using flow cytometry, and alkaline phosphatase activity using ELISA. Gene expression analyses for osteoblast markers (Runx-2, Osteopontin and Osteocalcin) were evaluated in differentiated osteoblasts using qPCR. RESULTS: Flow cytometry (CD90, CD105) detected MSCs isolated from bone marrow (93-98%). MTT assay showed that the selected doses of SO did not induce any cytotoxicity in MSCs (24 hours). The efficiency of osteogenic differentiation (2 weeks) for different doses of Symphytum officinale was determined by flow cytometry (n = 10) for osteoblast marker, Osteocalcin, and most doses of Symphytum officinale enhanced osteogenesis. Interestingly, gene expression analysis for Runx-2 (n = 10), Osteopontin (n = 10), Osteocalcin (n = 10) and alkaline phosphatase activity (n = 8) also showed increased osteogenesis with the addition of Symphytum officinale to BDM, specially mother tincture. CONCLUSIONS: Our findings suggest that homoeopathic dose (specially mother tincture) of Symphytum officinale has the potential to enhance osteogenesis.