Agnieszka Bazylko

Determination of Macrocyclic Ellagitannin Oenothein B in Plant Materials by HPLC‐DAD‐MS: Method Development and Validation

INTRODUCTION Oenothein B (OeB) is a dimeric macrocyclic ellagitannin occurring mainly in the genus Oenothera. In the literature no validated HPLC method for quantification of this compound has been reported. OBJECTIVE To develop and validate an efficient HPLC method for quantification of OeB in plant materials. METHODOLOGY Extraction conditions of OeB from plant material were optimised. Then the first validated HPLC method for quantification was developed. RESULTS We have shown that ultrasonic-assisted extraction at 40 °C in 15 min using water are the most optimal conditions for OeB extraction. We have quantified OeB in several plant materials belonging to Oenotheraceae family. The lowest amount of OeB was present in Circaea lutetiana herb (27.64 ± 0.26 mg/g) and the highest was quantified in Epilobium hirsutum aerial parts (72.91 ± 1.38 mg/g). CONCLUSION The first HPLC method for quantification of OeB in plant materials was developed and validated. We also for the first time optimised the extraction conditions for OeB.

In vitro antioxidant and anti-inflammatory activities of extracts from Potentilla recta and its main ellagitannin, agrimoniin.

ETHNOPHARMACOLOGICAL RELEVANCE Potentilla recta is one of the numerous cinquefoil species growing in Poland. It is used in traditional medicine e.g. in the treatment of skin inflammation. AIM OF THE STUDY The purpose of the present study is to evaluate antioxidant and anti-inflammatory activities of extracts and subfractions of the P. recta herb (obtained by using solvents of different polarity) in in vitro systems as well as to examine their chemical composition. MATERIALS AND METHODS Antioxidant activities of the extracts, subfractions and agrimoniin were evaluated using DPPH and three other radicals (O2(-), H2O2, and HClO) generated in cell-free systems. Anti-hyaluronidase activity was measured by using the turbidimetric method. Inhibition of lipoxidase activity was measured spectrophotometrically, using linoleic acid as a substrate. The composition of the most active subfraction was determined using the HPLC-DAD-MS(n) method. RESULTS All tested samples showed scavenging activity against all the examined reactive species in a concentration-dependent manner. The highest scavenging activity against DPPH, H2O2 and HClO was observed in the ethyl acetate subfraction (PRE3) (SC50 ± SEM [μg/mL]: 25.39 ± 2.49, 1.79 ± 0.25 and 8.52 ± 1.16 respectively). It was only in the xanthine/xanthine oxidase system that the antioxidation potential of the diethyl ether subfraction (PRE2) (SC50 ± SEM [μg/mL]: 6.59 ± 1.33) was higher than that of the subfraction PRE3 (SC50 ± SEM [μg/mL]: 8.57 ± 1.37). Also, in the studies of lipoxidase and hyaluronidase inhibition activity the strongest effect was observed for PRE3, with IC50 [μg/mL] = 86.31 ± 5.46, and 12.99 ± 1.31, respectively. The chromatographic method (HPTLC-DPPH) revealed that the principal substance responsible for the activity, is a tannin like compound. Isolated agrimoniin showed significant reactive oxygen species scavenging activity and significant enzyme inhibition activity (including xanthine oxidase inhibition activity). Agrimoniin exerted the strongest scavenging activity against H2O2 (SC50 ± SEM [μM]: 0.20 ± 0.01). This compound also significantly inhibited the enzymatic activity of lipoxidase (IC50 [μM] = 36.47 ± 1.29), and, particularly, of hyaluronidase (IC50 [μM] = 2.65 ± 0.40). CONCLUSIONS The strong scavenging activity against H2O2, and the inhibition of the enzymatic activity of lipoxidase, and particularly, hyaluronidase observed for the tested subfractions and agrimoniin, partly explain the beneficial effects of P. recta in treatment of skin inflammation.

Inhibition of NF-κB-Dependent Cytokine and Inducible Nitric Oxide Synthesis by the Macrocyclic Ellagitannin Oenothein B in TLR-Stimulated RAW 264.7 Macrophages

Immunomodulatory effects of oenothein B (1), a macrocyclic ellagitannin from various Onagraceae species, have been described previously. However, the mechanisms underlying the anti-inflammatory activity of 1 have not been fully clarified. The effects of 1 were investigated on inducible nitric oxide synthase, TLR-dependent and TLR-independent signal transduction cascades, and cytokine expression using murine macrophages (RAW 264.7). Compound 1 (10-60 μg/mL) reduced NO production, iNOS mRNA, and iNOS protein levels in a dose-dependent manner, without inhibition of iNOS enzymatic activity. It reduced the binding of the NF-κB p50 subunit to the biotinylated-consensus sequence and decreased nuclear p65 translocation. Gallic acid as a subunit of the macrocyclic ellagitannin 1 showed a far lower inhibitory activity. Nitric oxide production was reduced by 1 after stimulation using TLR2 (Pam2CSK4) and TLR4 (Kdo2) agonists, but this compound did not inhibit inducible nitric oxide synthesis after stimulation using interferon-gamma. IL-1beta, IL-6, and TNF-alpha mRNA synthesis was clearly reduced by the addition of 1. Oenothein B (1) inhibits iNOS after stimulation with LPS, TLR2, and TLR4 agonists via inhibition of TLR/NF-κB-dependent inducible nitric oxide and cytokine synthesis independent from IFN-gamma/JAK/STAT pathways. The full molecular structure of this macrocyclic ellagitannin seems to be required for its immunomodulatory actions.

Polyphenolic Profile, Antioxidant and Anti-Inflammatory Activity of Eastern Teaberry (Gaultheria procumbens L.) Leaf Extracts.

Dry leaf extracts of eastern teaberry (Gaultheria procumbens L.) were evaluated as a source of bioactive phytocompounds through systematic activity testing and phytochemical profiling. The antioxidant efficiency was tested using five complementary in vitro models (DPPH; FRAP; linoleic acid (LA) peroxidation assay; O2•− and H2O2 scavenging tests) in parallel with standard antioxidants. The 75% methanol extract and its diethyl ether, ethyl acetate (EAF), n-butanol and water fractions exhibited the dose-dependent responses in all assays, with the highest capacities found for EAF (DPPH EC50 = 2.9 μg/mL; FRAP = 12.8 mmol Fe2+/g; IC50 for LA-peroxidation = 123.9 μg/mL; O2•− SC50 = 3.9 μg/mL; H2O2 SC50 = 7.2 μg/mL). The EAF had also the highest anti-inflammatory activity in the inhibition tests of lipoxygenase and hyaluronidase (60.14% and 21.83% effects, respectively, at the concentration of 100 μg/mL). Activity parameters of the extracts correlated strongly with the levels of total phenolics (72.4–270.7 mg GAE/g), procyanidins, and phenolic acids, whereas for flavonoids only moderate effects were observed. Comprehensive UHPLC-PDA-ESI-MS3 and HPLC-PDA studies led to the identification of 35 polyphenols with a procyanidin A-type trimer, quercetin 3-O-glucuronide, isomers of caffeoylquinic acids, and (‒)-epicatechin being the dominant components. Significant activity levels, high phenolic contents and high extraction yields (39.4%–42.5% DW for defatted and crude methanol extracts, respectively) indicate the value of eastern teaberry leaves as bioactive products.

Determination of antioxidant activity of extracts and fractions obtained from Galinsoga parviflora and Galinsoga quadriradiata, and a qualitative study of the most active fractions using TLC and HPLC methods.

Taking into account the role of reactive oxygen species in the development of inflammation, and the application of the plants of genus Galinsoga Ruiz & Pav. in folk medicines for inflammatory states, we investigated and compared the antioxidant activities of particular Galinsoga extracts and fractions. The compositions of the most active fractions were studied using thin layer chromatography (TLC) and high-performance liquid chromatography (HPLC) methods. The extracts and fractions from Galinsoga parviflora Cav. and Galinsoga quadriradiata Ruiz et Pav. possess dose-dependent free radical-scavenging ability against DPPH• and superoxide radicals, as well as inhibitory effects on linoleic acid peroxidation in a manner comparable to gallic acid. In the most active fractions, flavonoids, patulitrin, quercimeritrin, quercitagetrin and caffeoyl derivatives were detected. Our research demonstrates that the investigated herbs are an interesting source of preparations with significant antioxidant effects. Our results justify the use of both raw materials in inflammatory diseases, among others, due to their ability to prevent free radical-induced deleterious effects.

Determination of in vitro antioxidant and UV-protecting activity of aqueous and ethanolic extracts from Galinsoga parviflora and Galinsoga quadriradiata herb

Galinsoga species are used in folk medicine as anti-inflammatory agents and accelerators for wound healing. They also have reported antioxidant activity. We examined aqueous and ethanolic extracts derived from the Galinsoga herb as potential photoprotectors, as the role of reactive oxygen species (ROS) has implicated in skin damage. The extracts used in the study were standardized by determining the sum of flavonoids, and the amount of caffeic acid and its derivatives. The antioxidant activity of the extracts was evaluated by examining the scavenging of two radicals (O2(-) and H2O2) generated in cell-free systems. We also examined the effect on ROS generation by human skin fibroblasts after UV irradiation. In addition we determined the cytotoxicity of the extracts and their protective effect against damage caused by UV irradiation (MTT test, LDH release test and staining with annexine V-FITC/PI). Our findings show that the ethanolic extracts from the herb have cytotoxic effects, while the aqueous extracts from Galinsoga herb have protective activity, in part due to their ability to inhibit ROS generation. In the conclusion the aqueous extracts from the both tested species may be effective as photoprotectors.

Chemical fingerprint of Potentilla species by using HPTLC method

Chromatographic fingerprints are often used to establish the optimal conditions of extraction, standardize extracts and detect changes or degradation of the plant material during the extract preparation. This method can also be optimized to identify and quantify specific markers (chemotaxonomy) [1–3]. Several chromatographical methods including thin-layer chromatography (TLC) have been developed to analyze polyphenols, for example, tannins and flavonoids in various matrices [4–8]. Recently, ultrathin layer chromatography (UTLC), a new, miniaturized form of TLC, has also become one of the most useful approaches towards quality control of polyphenolics in plant material [9].

Densitometric determination of flavonoids in methanolic and aqueous extracts of Epilobii angustifolii herba by use of HPTLC

Separation and quantitative analysis of quercetin glycosides in methanolic and aqueous extracts of Epilobii angustifolii herba have been achieved by HPTLC with densitometric detection. The compounds were separated on silica gel 60F254 HPTLC plates with ethyl acetate—formic acid—water, 68 + 2.5 + 3 (v/v), as mobile phase; densitometric detection of the flavonoids was performed at λ = 350 nm. The amounts of the compounds were calculated by use of regression equations obtained from calibration plots. The flavonoids were more abundant in the aqueous extract than in the methanolic extract. Quercetin glucuronide was the most abundant compound in both extracts (21.22 mg g−1 and 17.85 mg g−1, respectively). The method is rapid, easy, and selective, particularly for quercetin glucuronide analysis.

Quantitative Determination of Secoiridoids and Phenylpropanoids in Different Extracts of Ligustrum Vulgare L. Leaves by a Validated HPTLC–Photodensitometry Method

INTRODUCTION The genus Ligustrum (Oleaceae) is distributed in Europe and Asia (south China and Korea), where it is used to prevent hypertension, sore throats, inflammation and diabetes. The main groups of compounds in extracts of Ligustrum vulgare are biologically active secoiridoids and phenylpropanoids. OBJECTIVES The aim of the study was primarily the development and validation of a HPTLC-photodensitometry method for separation and determination of secoiridoids (oleacein, oleuropein) and phenylpropanoids (echinacoside) in different extracts prepared from leaves of L. vulgare. A secondary issue was the quantitative screening of oleacein, oleuropein and echinacoside in extracts from leaves collected at different stages of plant growth (from May to September). METHODS A HPTLC-photodensitometry method was developed and validated for quantification of oleuropein, oleacein and echinacoside in plant extracts (aqueous and ethanolic extract, decoction, infusion). Silica gel was used as the stationary phase and dichloromethane:methanol:formic acid:water (80:25:1.5:4, v/v/v/v) as the mobile phase. RESULTS The HPTLC-photodensitometry method developed for quantification of oleacein, oleuropein and echinacoside was specific, accurate and precise. The presence of oleacein was detected in aqueous extracts, whereas oleuropein was present, in particular, in ethanolic extracts, decoctions and infusions. Echinacoside was detected in all the extracts prepared. The content of secoiridoids was variable from May to September, whereas the amount of echinacoside increased in this term. CONCLUSION The developed and validated HPTLC-photodensitometry method allowed performing fast screening of quantitative profiles of oleacein, oleuropein and echinacoside in preparations of privet leaves.

Anthocyans-rich Aronia melanocarpa extract possesses ability to protect endothelial progenitor cells against angiotensin II induced dysfunction.

BACKGROUND Endothelial progenitor cells (EPC) may provide protection against atherosclerosis and plaque rupture by their innate ability to replace dysfunctional or damaged endothelial cells in plaque microvessels. There is evidence that angiotensin II may impair the angiogenic functions of EPCs in the atherosclerotic plaque by accelerating senescence and inhibiting their proliferation through oxidative stress induction. PURPOSE In this study, we examined whether chokeberry (Aronia melanocarpa) fruit extract, containing mainly anthocyanins with potent antioxidative properties, could protect EPCs against angiotensin-induced oxidative stress. METHODS EPCs were isolated from peripheral blood of young healthy volunteers and cultivated on fibronectin-coated plates in the presence or absence of angiotensin II (1 µM) and chokeberry extract (1-25 µg/ml). RESULTS EPCs exposed to chokeberry extract prior to angiotensin II showed a significant increase of proliferation and telomerase activity, and a decrease in the percentage of senescent cells and intracellular ROS formation in comparison to angiotensin II treated cells. Furthermore, extract increased migration ability, adhesion to fibronectin and the angiogenic potential of EPC in vitro diminished by angiotensin II in a concentration-dependent manner. That effect was related to the activation of the Nrf2 transcription factor and the increase of HO-1 expression. CONCLUSIONS Our results suggested that chokeberry extract may protect EPCs against angiotensin II-induced dysfunction and could play a potential role in the prevention of coronary artery disease.