Evaluation of estrogenic activity of Trifolium pratense L. and Cimicifuga racemosa L. plant extracts and formulations using the planar-YES assay

Supplementation with natural estrogens, known as phytoestrogens are desirable for the treatment of women suffering from climacteric complaints after menopause. However, in patients with hormone-dependent cancers, such as specific types of breast cancer, the intake of phytoestrogens may be contraindicated, since some compounds are claimed to trigger the formation of metastasis-initiating cancer cells [1]. HPTLC is an analytical tool in quality control of medicinal plants extracts. Separated compounds from multicompound extracts are fixed on the solid silica phase like a compound library. By direct coupling of the Yeast Estrogen Screen (YES) on the HPTLC plate, this compound library can also be used for screening of estrogenic activity. The reporter gene YES assay encoding the human estrogen receptor alpha (ERα) was adapted for the screening of Trifolium pratensis L. and Cimcifuga racemosa L. extracts and formulations [2,3]. The method is rapid and simple with no need for special sample preparation. Excipients such as microcrystalline cellulose, carboxymethylcellulose or polyvinyl pyrrolidone did not interfere with the method. In samples of T. pratensis, pronounced fluorescent zones of Genistein and Biochanin A could be identified, indicating estrogen receptor affinity. The half maximal effective concentration (EC50) of Genistein and Biochanin A was 2.13 and 3.34 ng, respectively. Cimicifugin showed weak estrogenic activity visible as fluorescent zones at applied masses of 4 µg. However, no ERα affinity was detected in C. racemosa samples. Formononetin interacted with ERα but was not detected in C. racemosa samples or commercial products. [1] Virk-Baker MK, Nagy TR, Barnes S. Planta medica 2010; 76: 1132 – 1142. [2] McDonnell DP, Nawaz Z, O'Malley BW. Mol Cell Biol 1991; 11: 4350 – 4355. [3] McDonnell DP, Nawaz Z, Densmore C, Weigel NL, Pham TA, Clark JH, O'Malley BW. J Steroid Biochem Mol Biol 1991; 39: 291 – 297

Recent advances in effect-directed enzyme assays based on thin-layer chromatography

Thin-layer chromatography (TLC) together with its more modern form HPTLC is a rapid and cost effective analytical tool with a long tradition in quality control of medicinal plants, extracts and natural products. Separated compounds are fixed on the solid silica phase to form a compound library. Through direct coupling of visualizable enzyme reactions on the TLC plate, this compound library can also be used for activity screening. Such TLC-based bioautographic enzyme and enzyme inhibition assays complement first stage development activity screening assays. They provide not only phytochemical results by chromatographic separation, but also additional information about the activity of constituents or fractions in multi-compound mixtures, and thus can reveal and distinguish artefacts generated by certain compound classes. This review summarizes recently introduced TLC bioautographic enzyme assays as well as advances in already existing procedures. Bioautographic enzyme and enzyme inhibitory assays offer a rapid, high-throughput method for screening of secondary metabolite profiles for potential enzyme and enzyme inhibitory activities

Unprecedented sensitivity of the planar yeast estrogen screen by using a spray-on technology

The planar yeast estrogen screen (p-YES) can serve as a highly valuable and sensitive screening tool for the detection of estrogenic compounds in various sample matrices such as water and wastewater, personal care products and foodstuff. The method combines the separation of sample constituents by thin layer chromatography with the direct detection of estrogenic compounds on the surface of the HPTLC-plate. The previous protocol using the immersion of a normal phase silica HPTLC-plate in a cell suspension for bio-autography resulted in blurred signals due to the accelerated diffusion of compounds on the wet surface of the HPTLC-plate. Here, the application of the yeast cells by spraying on the surface of the HPTLC-plate is described as an alternative approach. The presented method for the hyphenation of normal phase thin layer chromatography with a yeast estrogen screen results in much sharper signals compared to reports in previous publications. Satisfying results were achieved using cultures with cell densities of 1000 FAU. Due to the reduced signal broadening, lower limits of quantification for estrogenic compounds were achieved (Estrone (E1) = 2 pg/zone, 17β-estradiol (E2) = 0.5 pg/zone, 17α-ethinylestradiol (EE2) = 0.5 pg/zone and Estriol (E3) = 20 pg/zone). As demonstrated, it is possible to characterize profiles of estrogenic activity of wastewater samples with high quality and reproducibility. The improved sensitivity opens the stage for applications using native samples from waste- or even surface water directly applied on HPTLC-plates without the need for prior sample treatment by e.g. solid phase extraction

Is comfrey root more than toxic pyrrolizidine alkaloids? Salvianolic acids among antioxidant polyphenols in comfrey (Symphytum officinale L.) roots

Comfrey root preparations are used for the external treatment of joint distortions and myalgia, due to its analgesic and anti-inflammatory properties. Up to date, key activity-determining constituents of comfrey root extracts have not been completely elucidated. Therefore, we applied different approaches to further characterize a comfrey root extract (65% ethanol). The phenolic profile of comfrey root sample was characterized by HPLC-DAD-QTOF-MS/MS. Rosmarinic acid was identified as main phenolic constituent (7.55 mg/g extract). Moreover, trimers and tetramers of caffeic acid (isomers of salvianolic acid A, B and C) were identified and quantified for the first time in comfrey root. In addition, pyrrolizidine alkaloids were evaluated by HPLC-QQQ-MS/MS and acetylintermedine, acetyllycopsamine and their N-oxides were determined as major pyrrolizidine alkaloids in the comfrey root sample. Lastly, the antioxidant activity was determined using four assays: DPPH and ABTS radicals scavenging assays, reducing power assay and 15-lipoxygenase inhibition assay. Comfrey root extract exhibited significant antioxidant activities when compared to known antioxidants. Thus, comfrey root is an important source of phenolic compounds endowed with antioxidant activity which may contribute to the overall bioactivity of Symphytum preparations

Functional and Radiologic Outcomes of Degenerative Versus Traumatic Full-Thickness Rotator Cuff Tears Involving the Supraspinatus Tendon.

BACKGROUND Arthroscopic rotator cuff repair (ARCR) is among the most commonly performed orthopaedic procedures. Several factors-including age, sex, and tear severity-have been identified as predictors for outcome after repair. The influence of the tear etiology on functional and structural outcome remains controversial. PURPOSE To investigate the influence of tear etiology (degenerative vs traumatic) on functional and structural outcomes in patients with supraspinatus tendon tears. STUDY DESIGN Cohort study; Level of evidence, 2. METHODS Patients undergoing ARCR from 19 centers were prospectively enrolled between June 2020 and November 2021. Full-thickness, nonmassive tears involving the supraspinatus tendon were included. Tears were classified as degenerative (chronic shoulder pain, no history of trauma) or traumatic (acute, traumatic onset, no previous shoulder pain). Range of motion, strength, the Subjective Shoulder Value, the Oxford Shoulder Score (OSS), and the Constant-Murley Score (CMS) were assessed before (baseline) and 6 and 12 months after ARCR. The Subjective Shoulder Value and the OSS were also determined at the 24-month follow-up. Repair integrity after 12 months was documented, as well as additional surgeries up to the 24-month follow-up. Tear groups were compared using mixed models adjusted for potential confounding effects. RESULTS From a cohort of 973 consecutive patients, 421 patients (degenerative tear, n = 230; traumatic tear, n = 191) met the inclusion criteria. The traumatic tear group had lower mean baseline OSS and CMS scores but significantly greater score changes 12 months after ARCR (OSS, 18 [SD, 8]; CMS, 34 [SD,18] vs degenerative: OSS, 15 [SD, 8]; CMS, 22 [SD, 15]) (P < .001) and significantly higher 12-month overall scores (OSS, 44 [SD, 5]; CMS, 79 [SD, 9] vs degenerative: OSS, 42 [SD, 7]; CMS, 76 [SD, 12]) (P≤ .006). At the 24-month follow-up, neither the OSS (degenerative, 44 [SD, 6]; traumatic, 45 [SD, 6]; P = .346) nor the rates of repair failure (degenerative, 14 [6.1%]; traumatic 12 [6.3%]; P = .934) and additional surgeries (7 [3%]; 7 [3.7%]; P = .723) differed between groups. CONCLUSION Patients with degenerative and traumatic full-thickness supraspinatus tendon tears who had ARCR show satisfactory short-term functional results. Although patients with traumatic tears have lower baseline functional scores, they rehabilitate over time and show comparable clinical results 1 year after ARCR. Similarly, degenerative and traumatic rotator cuff tears show comparable structural outcomes, which suggests that degenerated tendons retain healing potential