Phenolic Composition of the Leaves of Pyrola rotundifolia L. and Their Antioxidant and Cytotoxic Activity.

The leaves of Pyrola rotundifolia L. were extracted in the mixed solvent of methanol/acetone/water (2:2:1, v/v/v) and investigated for their phytochemical analysis and biological activity. Total phenolic and flavonoid contents were determined spectrophotometrically. A high content of phenols (208.35 mg GAE/g of dry extract), flavonoids (38.90 mg QE/g of dry extract) and gallotannins (722.91 GAE/g of dry extract) was obtained. Ultra-high performance liquid chromatography diode array detector tandem mass spectrometry (UHPLC-DAD-MS) allowed for the detection of 23 major peaks at 254 nm. The extract was analyzed for its antioxidant capacity using 2,2-diphenyl-1-picryl-hydrazyl (DPPH•) and 2,2'-azinobis[3-ethylbenzthiazoline]-6-sulfonic acid (ABTS•+) radical scavenging, metal chelating power and ß-carotene-linoleic acid bleaching assays. The examined extract showed moderate radical scavenging and chelating activity, and good inhibiting ability of linoleic acid oxidation (EC50 = 0.05 mg/mL) in comparison to standards. The cytotoxic effect in increasing concentration on five types of leukemic cell lines was also investigated using trypan blue vital staining. It was found that the analyzed extract induced the apoptosis of all the tested cell lines. Our findings suggest that the leaves of P. rotundifolia are a source of valuable compounds providing protection against oxidative damage, hence their use in traditional medicine is justified.

A Comprehensive Review of the Genus Pyrola Herbs in Traditional Uses, Phytochemistry and Pharmacological Activities.

Pyrola (Pyrolaceae), also known as Luxiancao/in China, was recorded in Sheng Nong's Herbal Classic listed in top grade. Pyrola herbs were used as medicinal plants for a long history with wide-ranging activities such as nourishing kidney-yang, strengthening muscles and bones, activating blood, stopping bleeding, dispelling rheumatism, and eliminating dampness. Currently, the research on Pyrola plants is increasing year by year but there is no comprehensive and detailed review concerning genus Pyrola. This review aims to sum up the updated and comprehensive information about botany and traditional use, phytochemistry, pharmacological activities and safety by analyzing the information available on Pyrola plants via internationally accepted scientific databases. Collectively, more than 100 compounds have been isolated from the Pyrola plants. Furthermore, a total of 33 prescriptions containing Pyrola plants are compiled in this review. Pyrola plants are used as indispensable agents in traditional Chinese medicine due to its activities of antimicrobial, anti-inflammatory, antioxidant, lipidlowering, cardiovascular and cerebrovascular protection, proliferation of osteoblasts promoting, antineoplastic and etc. Further work should be developed on the elucidation of structure-function relationship, understanding of multi-target pharmacological effects, as well as developing its application both in clinical usage and functional food for research and development of Pyrola plants.

Pyrola incarnata demonstrates neuroprotective effects against ß-amyloid-induced memory impairment in mice.

This study aims to investigate the neuroprotective effects of Pyrola incarnata against ß-amyloid-induced memory impairment in mice. Ethanol extract of Pyrola incarnata (EPI) was obtained and led to eleven phytochemicals successfully by isolation and purification, which were elucidated by spectroscopic analysis (1H NMR, 13C NMR and HR-ESI-MS). Thereinto, ursolic acid was gained as most abundant monomer. C57BL/6 mice were intracerebroventricular injected with aggregated Aß25-35. Open-field test, Barnes maze test and Morris water maze were conducted for evaluating cognition processes of EPI and ursolic acid. EPI significantly improved learning and memory deficits, attenuated the Aß25-35 level of deposition immunohistochemically. Further studies revealed that ursolic acid as bioactive phytochemical of P. incarnata improved spatial memory performance and ameliorated Aß25-35 accumulation by activating microglia cells and up-regulating Iba1 level in the hippocampus. These findings suggest P. incarnata could improve the cognition of mice and be a promising natural source for the treatment of neurodegenerative disease.

Antioxidant and Cytoprotective effects of Pyrola decorata H. Andres and its five phenolic components.

BACKGROUND: Pyrola decorata H. Andres, is exclusively distributed in China and a source of traditional Chinese herbal medicine Luxiancao for more than 2000 years. Here, we evaluated the antioxidant and cytoprotective effects of P. decorata and its five phenolic components (protocatechuic acid, gallic acid, hyperoside, 2''-O-galloylhyperin, and quercetin), and discussed their antioxidant chemistry. METHODS: A lyophilized aqueous extract of P. decorata (LAEP) was prepared and analyzed with high-performance liquid chromatography (HPLC). LAEP and its five phenolic components were comparatively investigated using five antioxidant assays, including ferric-reducing antioxidant power, cupric ion-reducing antioxidant capacity, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide radical (PTIO•)-scavenging, 1,1-diphenyl-2-picryl-hydrazl radical (DPPH•)-scavenging, and 2,2'-azino-bis(3-ethylbenzo-thiazoline-6-sulfonic acid) radical (ABTS+•)-scavenging activities. The reaction products of the five phenolic components with 4-methoxy-2,2,6,6-tetramethylpiperidine-1-oxyl radical (4-methoxy-TEMPO•) were determined with ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UPLC-ESI-Q-TOF-MS/MS) analysis. LAEP and its five phenolic components were incubated with bone marrow-derived mesenchymal stem cells (bmMSCs) subjected to oxidative stress to demonstrate their cytoprotective effects with a flow cytometry assay. RESULTS: In the five antioxidant assays, LAEP and its five phenolic components dose-dependently increased the radical-scavenging (or reducing power) activities. However, the IC50 values of hyperoside were consistently higher than those of 2''-O-galloylhyperin. UPLC-ESI-Q-TOF-MS/MS analysis results indicated that the five phenolics could yield dimer products in the presence of 4-methoxy-TEMPO• via the radical adduct formation (RAF) pathway. Flow cytometry assay results confirmed the cytoprotective activity of LAEP and its five phenolic components toward stressed bmMSCs. In particular, 2''-O-galloylhyperin could more effectively reduce the percentage of damaged bmMSCs than hyperoside. CONCLUSION: LAEP and its five phenolic components may undergo redox-based pathways (such as electron transfer and H+ transfer) and covalent-based pathway (i.e., RAF) to exhibit antioxidant activity. One consequence of RAF is the generation of phenolic-phenolic dimer. In both organic and aqueous media, 2''-O-galloylhyperin exhibited higher redox-based antioxidant levels (or cytoprotective levels) than those with hyperoside. The differences could be attributed to 2''-O-galloylation reaction.

[Chemical constituents, biological activities and quality control of plants from genus Pyrola].

Plants from the genus Pyrola are widely distributed in North Temperate zone. The quinones, phenol glycosides, terpenoids, flavonoids and volatile oil compounds have been identified from these plants. The in vivo and in vitro studies have shown that the genus Pyrola plants exhibit a wide range of pharmacological properties, including antioxidant, antitumor, antibacterial, anti-ischemia and anti-inflammatory activities. Based on analysis of the literature of the genus Pyrola plant, this review summarized the research on chemical constituents, pharmacology and quality control in recent years which can provide evidences for further investigation on the genus Pyrola plants.

Antioxidant activity against H2O2-induced cytotoxicity of the ethanol extract and compounds from Pyrola decorate leaves.

CONTEXT: The leaves of Pyrola decorate H. Andr (Pyrolaceae), known as Luxiancao, have long been used for treating kidney deficiency, gastric haemorrhage and rheumatic arthritic diseases in traditional Chinese medicine. OBJECTIVE: The phytochemicals and antioxidant capacities in vitro of P. decorate leaves were investigated. MATERIALS AND METHODS: Ethanol, petroleum ether, acetidin, n-butyl alcohol and aqueous extracts of Pyrola decorate leaves were prepared by solvent sequential process, and then isolated and purified to obtain phytochemicals. Cell viability was measured by MTT assay. PC12 cells were pretreated for 24 h with different extractions of P. decorate leaves at concentrations of 0.1, 0.5, 1, 5 and 10 mg/mL, then H2O2 of 0.4 mM was added in all samples for an additional 2 h. The antioxidant capacities of betulin, ursolic acid and monotropein were determined in PC12 cells against H2O2 induced cytotoxicity in vitro as well. RESULTS: Nine compounds (1-9) were isolated and structurally determined by spectroscopic methods, especially 2D NMR analyses. Ethanol extract treated groups showed inhibitory activity with IC50 value of 10.83 mg/mL. Betulin, ursolic acid and monotropein were isolated from P. decorate, and demonstrated with IC50 values of 6.88, 6.15 and 6.13 µg/mL, respectively. DISCUSSION AND CONCLUSIONS: In conclusion, Pyrola decorate is a potential antioxidative natural plant and worth testing for further pharmacological investigation in the treatment of oxidative stress related neurological disease.

Chemical Composition of the Essential Oil of the Boreal Relict of Pyrola rotundifolia L. from Northern Kazakhstan.

In Kazakhstan Pyrola rotundifolia L. is the plant-relict in the flora of insular pine forests of the region of low hillocks and declivities in Kazakhstan - a group of insular pine forests of Kokshetau, Bayanaul and Karkaralinsk. In this study, the essential oils from dried aerial parts of P. rotundifolia, collected in natural habitats of the State National Natural Park "Burabay" (Akmola oblast, Northern Kazakhstan), were extracted by hydrodistillation and analyzed by gas chromatography - mass spectrometry. The yield of the essential oil amounted to 0.057 % in relation to the mass of the air-dry raw material. The major components in dried plant oil were 2,6-dimethyl-1,4-naphthoquinone (12.99-93.49%) and dibutyl phthalate (4.42-40.48%), depending on the growth conditions.

An effective homogenate-assisted negative pressure cavitation extraction for the determination of phenolic compounds in pyrola by LC-MS/MS and the evaluation of its antioxidant activity.

A novel extraction method, homogenate-assisted negative pressure cavitation extraction (HNPCE), was designed for the extraction and determination of the main phenolic compounds of Pyrola incarnata Fisch. by LC-MS/MS. The particle sizes and extraction yields in the process of homogenization were compared with conventional pulverization. The results showed that homogenization for less than 120 s could produce more suitable particle size powders for analyte extraction. The following NPCE parameters were optimized by a BBD test and under the optimal conditions, the maximum extraction yields of arbutin, epicatechin, hyperin, 2'-O-galloylhyperin and chimaphilin increased by 68.7%, 72.0%, 43.3%, 62.5% and 34.5% with respect to normal NPCE. The LC-MS/MS method was successfully applied for the quantification of five target compounds in pyrola, and the results of the precision test indicated a high accuracy of the present method for the quantification of the target compounds in pyrola. Furthermore, the antioxidant activities of the pyrola extracts were also determined. The results showed that pyrola had good antioxidant activities and it was a valuable antioxidant natural source.

Negative pressure cavitation-microwave assisted preparation of extract of Pyrola incarnata Fisch. rich in hyperin, 2'-O-galloylhyperin and chimaphilin and evaluation of its antioxidant activity.

A novel and effective extraction method, namely negative pressure cavitation-microwave assisted extraction technique (NMAE), was developed for the preparation of extracts of Pyrola incarnata Fisch., which are rich in the main constituents hyperin, 2'-O-galloylhyperin and chimaphilin. Single factor experiments and Box-Behnken design (BBD) were combined with a response surface methodology to examine factors affecting extraction. Maximum extraction yields of hyperin, 2'-O-galloylhyperin and chimaphilin (1.339±0.029, 4.831±0.117 and 0.329±0.011mg/g, respectively) were achieved under the following optimised conditions: 700W microwave power, 50°C extraction temperature, 30:1mL/g liquid-solid ratio, -0.05MPa negative pressure, 55% ethanol concentration and 12min extraction time. First-order kinetics equation demonstrated that NMAE offered significant savings in extraction time, and enhancing extraction efficiency. Furthermore, NMAE extracts yielded excellent antioxidant activity (IC50 0.121mg/mL for DPPH 2.896mmol FeSO4/g DW FRAP).

Phytochemical composition, antioxidant activity and HPLC fingerprinting profiles of three Pyrola species from different regions.

The present study was performed to investigate the variation of phytochemical composition, antioxidant activity and High Performance Liquid Chromatography (HPLC) fingerprinting profiles of three Pyrola species. Thirteen samples (eight P. decorata, three P. calliantha and two P. renifolia) were collected from different regions in China. The tannin, hyperoside and quercetin contents of all samples were determined by reverse-phase HPLC and varied within the range 9.77-34.75, 0.34-2.16 and 0.062-0.147 mg/g dry weigh, respectively. Total flavonoid content was evaluated and varied within the range 16.22-37.82 mg/g dry weight. Antioxidant activity was determined by DPPH assay, with IC50 ranging from 7.96 to 50.33 µg/ml, ABTS•+ and FRAP assay, within the range 612.66-1021.05 and 219.64-398.12 µmol equiv. Trolox/g, respectively. These results revealed that there were significant variations in phytochemical profiles and antioxidant activity among all samples. Due to the higher phytochemical content and significant antioxidant activity, P. calliantha was selected as the most valuable species, and the P. calliantha sample from Left banner of Alxa even possessed the strongest antioxidant activity among all the thirteen samples. Futhermore, Emei Mountain was proved to be the most suitable region for producing P. decorata. Moreover, in order to further evaluate the diversities and quality of Pyrola, HPLC fingerprint analysis coupled with hierarchical cluster and discrimination analyses were introduced to establish a simple, rapid and effective method for accurate identification, classification and quality assessment of Pyrola. Thirteen samples were divided into three groups consistent with their morphological classification. Two types of discriminant functions were generated and the ratio of discrimination was 100%. This method can identify different species of Pyrola and the same species from different regions of origin. Also, it can be used to compare and control the quality of Pyrola and other natural products prepared from them.

An effective negative pressure cavitation-microwave assisted extraction for determination of phenolic compounds in P. calliantha H. Andr.

A novel negative pressure and microwave assisted extraction technique (NMAE) was first proposed and applied for extraction of phenolic compounds from pyrola. [C4MIM]BF4 aqueous solution was selected as extraction solvent. Optimal extraction conditions were microwave power 700 W, negative pressure -0.07 MPa, temperature 40 °C, liquid-solid ratio 20 : 1, ionic liquid (IL) concentration 0.5 M, extraction time 15 min. The predominance of NMAE was investigated by comparing with microwave-assisted extraction (MAE) and negative pressure cavitation extraction (NPCE) using a first-order kinetics equation. The C∞ values of the target compounds by NMAE were from 0.406 to 5.977 mg g⁻¹ higher than these by MAE and NPCE, which indicated that NMAE had higher extraction yields. The K values of NMAE were also the highest; it was testified that the target compounds could be transferred from matrix into solvent much more effectively by NMAE than by MAE and NPCE. In addition, the NMAE method was validated in terms of repeatability and reproducibility, the relative standard deviation for relative recovery was lower than 5.43 and 8.78%, respectively. Therefore, NMAE was a developed extraction technique for analytical sample preparation. The RP-HPLC-UV method was also successfully applied for the quantification of six target compounds in pyrola.

Chemical constituents of volatile oil from Pyrolae herba and antiproliferative activity against SW1353 human chondrosarcoma cells.

The objective of the present study was to identify chemical constituents of volatile oil from Pyrolae herba (PHVO) and evaluate the antiproliferative activity of PHVO against SW1353 human chondrosarcoma cells. The volatile oil from Pyrolae herba was prepared by hydrodistillation and characterized by gas chromatography-mass spectroscopy (GC-MS). A total of 12 components in PHVO were identified representing 81.62% of the total integrated chromatographic peaks. The major compounds were found to be n-hexadecanoic acid (29.29%), cedrol (17.08%), 6,10,14-trimethyl-2-pentadecanone (9.59%) and cis-9-octadecadienoic acid (8.23%). The antiproliferative activity of PHVO against SW1353 cells was investigated using MTT assay, flow cytometry and western blot analysis. Our results demonstrated that PHVO inhibited SW1353 cell viability in a dose- and time-dependent manner. Furthermore, PHVO treatment decreased the number of cells entering the S phase and caused a reduction in the expression of cyclin D1, cyclin-dependent kinase (CDK)4 and CDK6, whereas it caused an increase in the expression of p21. PHVO demonstrated potent antitumor activity against SW1353 cells, suggesting its potential use as a therapeutic agent in the treatment of chondrosarcoma.

Simultaneous determination of seven major triterpenoids in Pyrola decorata H. Andres by LC-MS method.

A liquid chromatography-mass spectrometry method was developed and validated for the quantitative determination of seven triterpenoids, 3-beta-O-alpha-L-arabinopyranosylsiaresinolic acid-28-O-beta-D-glucopyranosyl ester, ziyuglycoside I, pomolic acid, maslinic acid, colosic acid, oleanolic acid and ursolic acid in Pyrola decorata H. Andres. Chromatographic separation was achieved on a Hypersil C18 column using isocratic elution followed by a linear gradient elution of methanol and water as mobile phase. The analytes were ionized by atmospheric pressure chemical ionization source and determined on selected ion monitoring mode. All analytes showed good linearity (r2 > or = 0.9984) within the test ranges and the recovery rates were 94.5% - 103.3%. Satisfactory precision and reproducibility were obtained with relative standard deviation less than 5%. The method was simple, accurate and performed well in application to the determination of twenty commercial samples of P. decorata collected from different regions of China. It could be used for the quality control of both plant materials and preparations of P. decorata.

The 1,4-naphthoquinone derivative from Pyrola rotundifolia activates AMPK phosphorylation in C2C12 myotubes.

An aqueous ethanol extract of Pyrola rotundifolia L. induced AMP-activated protein kinase (AMPK) phosphorylation in C2C12 myotubes. The bioassay-guided fractionation of the extract led to the isolation a 2-methyl-7-hydroxymethyl-1,4-naphthoquinone, or a 7'-hydroxy-chimaphilin, which showed concentration-dependent AMPK phosphorylation activity at 2.5-20 µg/ml. At a concentration of 10 µg/ml (50 µM), an approximately four-fold increase in the AMPKα(Thr¹7²) phosphorylation level was observed. The stimulatory effect of naphthoquinone on AMPK activity was comparable to that of known compounds found in natural sources that activate the AMPK signaling pathway.

[Studies on separation, purification and structure characteristics of a polysaccharide LTC-II from Pyrola corbieri].

OBJECTIVE: To characterize the structure of polysaccharide LTC-II obtained from Pyrola corbieri. METHOD: The polysaccharide was extracted from P. corbieri by hot water and ethanol precipitation. Crude polysaccharide was purified by DEAE-Cellulose chromatography and Sephacryl S-300 HR column chromatography. The purity and molecular weight of polysaccharide was determined by gel permeation chromatography. UV, IR, optical rotation, complete acid hydrolysis, periodate oxydation, Smith degradation, partial acid hydrolysis and methylation analysis were applied to determine the structural features. RESULT: A homogeneous fraction LTC-II was obtained and its relative molecular mass was 22 000 Da. It consisted of arabinose, mannose, glucose, galactose in the molar ratio of 35. 2: 1.0: 13. 4: 4. 2. LTC-II had a backbone consisting glucose, mannose, galactose and mainly contained (1 --> 6)-linkaged glucose. The side chain possessed arabinose, glucose, galactose and mainly contained (1 --> 5)-linkaged arabinose. The terminal sugar were mainly glucose and galactose. CONCLUSION: Studies on the preliminary characterization of polysaccharide LTC-II from P. corbieri for the first time.

[Protective effect of total flavonoid of Herba Pyrolae on acute myocardial ischemic injury induced by isoproterenol in rats].

OBJECTIVE: To investigate the protective effect of total flavonoid of Herba Pyrolae (TFHP) on acute myocardial ischemic injury induced by isoproterenol in rats and the primary mechanisms. METHODS: Acute myocardial ischemic models were established by i. s. of isoproterenol. ECG of the rats were recorded, activities of creatine phosphokinase (CK), lactate dehydrogenase (LDH) and the superoxide dismutase (SOD), and the levels of malondialdehyde (MDA), NO and FFA in rat serum were determined, heart index were measured and histopathological changes of myocardium were observed. RESULTS: In comparison with model group, TFHP raised the height of T wave, reduced CK, LDH activities and FFA levels in serum, decreased heart index and relieved myocardial ischemic injury. The primary study of its mechanism showed that TFHP decreased MDA level, increased SOD activity and NO levels in the rat serum. CONCLUSION: TFHP has protective effect on acute myocardial ischemic injury possibly via antioxidation and increasing the release and production of NO.

[A new naphthaquinone derivative from Pyrola calliantha H. Andres].

To investigate the chemical constituents of hemostatic extract of Pyrola calliantha H. Andres, the extract was subjected to chromatographic separation and purification. Along with some known compounds, a new naphthaquinone derivative was isolated and identified as 2-(1, 4-dihydro-2, 6-dimethyl-1, 4-dioxo-3-naphthalenyl)-3, 4, 5-trihydroxylbenzoic acid by physicochemical and spectroscopic analysis.

Two new isomeric alpha-tetralones from Pyrola calliantha.

Callianthones A ( 1) and B ( 2), a pair of new isomeric alpha-tetralones, together with a known alpha-tetralone ( 3) and four known flavonoids ( 4 - 7) were isolated from the 50 % EtOH extract of Pyrola calliantha. The structures and absolute configurations of the two new isomers were established to be (2 S,4 R) - and (2 S,4 S)-2,4-dihydroxy-2,7-dimethyl-3,4-dihydronaphthalen-1(2 H)-one ( 1 and 2, respectively) on the basis of spectral analysis, including 2 D NMR, model studies, and CD spectra.

[Studies on chemical constituents of in herb Pyrola calliatha].

OBJECTIVE: To investigate the chemical constituents of Pyrola calliatha. METHOD: The chemical constituents were isolated by various column chromatographic methods. The structures were identified by spectral data. RESULT: Ten compounds were isolated and identified as chimaphilin (1), uvaol(2), ursolic acid (3), 2beta,3beta,23-trihydroxy-12-ene-28-ursolic acid (4), daucosterol (5), 2alpha,3beta,23,24-tetrahydroxy-12-ene-28-ursolic acid (6), emodin (7), gallic acid (8), monotropein (9), adenosine (10). CONCLUSION: Compounds 2,4,6,7,10 were obtained from this genus for the first time, compounds 5, 9 were obtained from this species for the first time. Antifungal activity of compounds 1-4, 6-9 were evaluated. Compound 1 showed the strong activity.

[Protective effect of total flavonoid of Herba Pyrolae on acute myocardial ischemic injury in rats].

OBJECTIVE: To investigate the protective effect of Total Flavonoid of Herba Pyrolae(TFHP) on acute myocardial ischemic injury in rats. METHODS: Acute myocardial ischemic models were established by i. v. injection of pituitrin and the ligation of left descending coronary artery in rats. ECG of the rat was recorded, myocardial ischemic size was evaluated, and activites of creatine phosphokinase( CK), lactate dehydrogenase (LDH) and superoxide dismutase (SOD), level of malondialdehyde (MDA) in the rat serum were determined. RESULTS: In comparison with the model control, TFHP significantly reduced the incidence of ischemic arrhythmia induced by pituitrin, size of myocardial infarction, release of myocardial CK and LDH caused by the ligation of left descending coronary artery in rats. The primary study of action mechanism showed that TFHP decreased MDA level and increased SOD activity in the rat serum. CONCLUSION: TFHP provide a protective effect on acute myocardial ischemic injury via antioxidation.