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Abstract

Results: Polysulfide-bridged allergen dimers exhibit different allergenic properties compared with the monomer.

Abstract

Many allergens share several biophysical characteristics, including the capability to undergo oligomerization. The dimerization mechanism in Bet v 1 and its allergenic properties are so far poorly understood. Here, we report crystal structures of dimeric Bet v 1, revealing a noncanonical incorporation of cysteine at position 5 instead of genetically encoded tyrosine. Cysteine polysulfide bridging stabilized different dimeric assemblies, depending on the polysulfide linker length. These dimers represent quaternary arrangements that are frequently observed in related proteins, reflecting their prevalence in unmodified Bet v 1. These conclusions were corroborated by characteristic immunologic properties of monomeric and dimeric allergen variants. Hereby, residue 5 could be identified as an allergenic hot spot in Bet v 1. The presented results refine fundamental principles in protein chemistry and emphasize the importance of protein modifications in understanding the molecular basis of allergenicity.

Abstract

Many allergens share several biophysical characteristics, including the capability to undergo oligomerization. The dimerization mechanism in Bet v 1 and its allergenic properties are so far poorly understood. Here, we report crystal structures of dimeric Bet v 1, revealing a noncanonical incorporation of cysteine at position 5 instead of genetically encoded tyrosine. Cysteine polysulfide bridging stabilized different dimeric assemblies, depending on the polysulfide linker length. These dimers represent quaternary arrangements that are frequently observed in related proteins, reflecting their prevalence in unmodified Bet v 1. These conclusions were corroborated by characteristic immunologic properties of monomeric and dimeric allergen variants. Hereby, residue 5 could be identified as an allergenic hot spot in Bet v 1. The presented results refine fundamental principles in protein chemistry and emphasize the importance of protein modifications in understanding the molecular basis of allergenicity.

Identification of the Recombinant Bet v 1 Dimer

During purification, we observed WT Bet v 1a to elute as a double peak in ion exchange chromatography. Although the dominant peak (∼95% in all analyzed batches) was monomeric wild type Bet v 1a (as evidenced by MS analysis), the high salt fraction contained both monomeric and dimeric Bet v 1a, as judged by size exclusion chromatography (a). This interpretation was confirmed by clear native PAGE and SDS-PAGE (b). The SDS-PAGE analysis also revealed the Bet v 1a dimer to be reduction-sensitive. This observation was surprising, given the lack of cysteine residues in Bet v 1a. More intriguingly, the redox sensitivity of the Bet v 1a dimer can be further distinguished: one dimer species is completely redox stable under native PAGE conditions, whereas a second dimer species dissociates under reducing native PAGE conditions (c). We conclude that the Bet v 1a bridging is inherently redox-sensitive; depending on the quaternary structure, the bridging can be protected from reducing agents by the native dimer structure.

Abstract

5-Hydroxy-l-tryptophan (5-HTP) is a naturally occurring aromatic amino acid present in the seeds of the African plant Griffonia simplicifolia. Although 5-HTP has therapeutic effects in various symptoms, efficient method of producing 5-HTP has not been established. In this study, we developed a novel cofactor regeneration process to achieve enhanced synthesis of 5-HTP by using modified l-phenylalanine 4-hydroxylase of Chromobacterium violaceum. For the synthesis of 5-HTP using Escherichia coli whole cell bioconversion, l-tryptophan and 5-HTP degradation by E. coli endogenous catabolic enzymes should be considered. The tryptophanase gene was disrupted using the λ red recombination system, since tryptophanase is postulated as an initial enzyme for the degradation of l-tryptophan and 5-HTP in E. coli. For regeneration of the cofactor pterin, we screened and investigated several key enzymes, including dihydropteridine reductase from E. coli, glucose dehydrogenase from Bacillus subtilis, and pterin-4α-carbinolamine dehydratase from Pseudomonas syringae. Genes encoding these three enzymes were overexpressed in an E. coli tryptophanase-deficient host, resulting in the synthesis of 0.74 mM 5-HTP in the presence of 0.1 mM pterin and the synthesis of 0.07 mM 5-HTP in the absence of regeneration of pterin. These results clearly indicated the successful regeneration of pterin. Following optimization of the reaction conditions, 2.5 mM 5-HTP was synthesized with cofactor regeneration, while 0.8 mM 5-HTP was recovered without cofactor regeneration under the same reaction conditions, suggesting that the principle described here provides a new method for cofactor regeneration.

Experimental elucidation of the function of Rv0132c

We have tested whether Rv0132c represents an F420-dependent hydroxymycolic acids dehydrogenase (fHMAD) by introducing this gene and hma into M. smegmatis. As expected, the plasmid pEP-hma, which was constructed based on the E. coli-mycobacterium shuttle vector pSMT3 and carried Mtb hma gene (rv0642c) under the control of its native promoter element, produced H-MAs in M. smegmatis mc2155 or wild-type (wt) strain (). The expression of both hma and rv0132c genes from pEP- rv0132c/hma led to the production of K-MAs (); rv0132c alone (pEP- rv0132c) did not provide either H-MAs or K-MAs (). M. smegmatis, as such (host control; ) or while carrying pSMT3 (vector control; ), also did not produce either H-MAs or K-MAs; they contained α-, α′-, and E-MAs.

Abstract

This study determined if 6-weeks vitamin D2 supplementation (vitD2, 3800 IU/day) had an influence on muscle function, eccentric exercise-induced muscle damage (EIMD), and delayed onset of muscle soreness (DOMS) in National Association for Stock Car Auto Racing (NASCAR) NASCAR pit crew athletes. Subjects were randomized to vitD2 (n = 13) and placebo (n = 15), and ingested supplements (double-blind) for six weeks. Blood samples were collected and muscle function tests conducted pre- and post-study (leg-back and hand grip dynamometer strength tests, body weight bench press to exhaustion, vertical jump, 30-s Wingate test). Post-study, subjects engaged in 90 min eccentric-based exercise, with blood samples and DOMS ratings obtained immediately after and 1- and 2-days post-exercise. Six weeks vitD2 increased serum 25(OH)D2 456% and decreased 25(OH)D3 21% versus placebo (p < 0.001, p = 0.036, respectively), with no influence on muscle function test scores. The post-study eccentric exercise bout induced EIMD and DOMS, with higher muscle damage biomarkers measured in vitD2 compared to placebo (myoglobin 252%, 122% increase, respectively, p = 0.001; creatine phosphokinase 24 h post-exercise, 169%, 32%, p < 0.001), with no differences for DOMS. In summary, 6-weeks vitD2 (3800 IU/day) significantly increased 25(OH)D2 and decreased 25(OH)D3, had no effect on muscle function tests, and amplified muscle damage markers in NASCAR pit crew athletes following eccentric exercise.

1. Introduction

Limited evidence suggests that treatment of vitamin D insufficient athletes may improve performance [,]. In the UK, 5000 IU/day vitamin D3 supplementation for eight weeks improved 10 m sprint times and vertical jump performance in athletes who started the study with a mean serum 25(OH)D level of 12 ng/mL []. Maintaining adequate vitamin D status may also reduce inflammation and aid in recovery from injury or intensive workouts, but data in humans are inconsistent [,]. One study showed that vitamin D3-treated rats experienced attenuation in plasma creatine kinase (CK) and inflammation biomarkers following high-intensity exercise, with an increase in muscle VDR protein expression []. No previous human study has been published regarding the effect of vitamin D supplementation in countering eccentric exercise-induced muscle damage (EIMD) and delayed onset of muscle soreness (DOMS). We hypothesized that 6-weeks supplementation with vitamin D (3800 IU/day) using vitamin D2 Portobello mushroom powder would improve muscle function and strength, and attenuate EIMD and DOMS in NASCAR pit crew athletes during their off-season in December and January.

2.4. Supplement

Fresh Portobello mushrooms (Agaricus bisporus) were air dried at 71–76 °C for 48–72 h in a convection dryer []. Once dried, the mushrooms pieces were milled to approximately 35 mesh, or 500 µm. The powder was then treated on a vibrating conveyor with pulsed light (UVB) from a Xenon broad spectrum lamp (100–800 nm) operating at 3 pulses per second for a total of thirty 2 ms pulses which converted ergosterol to ergocalciferol (vitamin D2). Subjects were given Portobello mushroom powder with or without vitamin D2 mixed in soymilk powder (non-vitamin D fortified) in six plastic containers (one for each week of the study). Subjects ingested one level teaspoon of the product each day (with or without 3800 IU vitamin D2) during breakfast.

3. Results

Total serum 25(OH)D was 43.7 ± 2.7 and 39.6 ± 1.6 ng/mL in the placebo and vitamin D groups, respectively, during October, 40.7 ± 2.1 and 36.6 ± 1.7 ng/mL in December (pre-supplementation), and 38.6 ± 1.8 and 37.4 ± 1.9 ng/mL in January (post-supplementation) (time effect p = 0.001, interaction effect p = 0.238). Supplementation with mushroom vitamin D2 powder for 6 weeks caused no significant change in 25(OH)D (p = 0.127), a significant increase in serum 25(OH)D2 (8.14 ± 1.96 ng/mL, p < 0.001) and a significant decrease in serum 25(OH)D3 (−7.48 ± 2.28 ng/mL, p = 0.036) compared to placebo (0.076 ± 1.19 ng/mL and −2.11 ± 1.09 ng/mL, respectively) (A,B). Serum 25(OH)D3 was highest in October and then decreased to levels measured in December and January in the placebo group (within group contrasts, p < 0.01).

3. Results

The post-study eccentric training bout caused significant increases in serum myoglobin (), LDH (), CK (), and DOMS () (all time effects, p < 0.001). Significantly higher post-exercise serum levels for myoglobin and CK were measured in the vitamin D2 compared to placebo group (both interaction effects, p < 0.001), with a trend for higher serum LDH levels (interaction effect, p = 0.065). The pattern of change in DOMS did not differ between groups (interaction effect, p = 0.490). For the whole group, change in serum 25(OH)D2 correlated significantly with change in post-exercise serum myoglobin (r = 0.57, p = 0.001).

4. Discussion

Contrary to our hypothesis, high-dose vitamin D2 supplementation in NASCAR pit-crew athletes during a 6-week period in December and January amplified EIMD and had no effect on muscle function. Vitamin D2 supplementation increased serum 25(OH)D2 ~8 ng/mL but decreased serum 25(OH)D3 ~7.5 ng/mL, with no significant change in total 25(OH)D.

Animals

Male Sprague–Dawley rats (250–300 g) were purchased from Sankyo Labo Service Co. Inc. (Tokyo, Japan). Three or four animals were housed per cage in a temperature-controlled environment (23 ± 0.5°C) with a 12-h light–dark cycle (light on at 7 am) with food and water ad libitum. One hundred and thirty-three rats (forced swimming test, 25; open-field test, 21; elevated plus-maze test, 28; contextual fear-conditioning test, 23; 5-HTP-induced head-twitches, 18; DOI-induced head-twitches, 18) were used in the present study.

Head-twitch test

The head-twitch test was carried out according a method described by Rojas-Corrales et al. [] with minor modifications. Rats were orally treated with MAK (1 g/kg) or fluvoxamine (10 mg/kg) 60 min before intraperitoneal injection of 5-HTP (200 mg/kg). Immediately after 5-HTP administration, the rat was placed into an observation cage (30 cm × 25 cm × 18 cm) and the cumulative number of head twitches (i.e., rapid movements of the head with little or no involvement of the trunk) counted by an observer blinded to the study protocol for 30 min. Similarly, the effect of MAK on DOI (3 mg/kg)-induced head-twitch responses was measured.

5-HTP- or DOI-induced head twitches

Figure shows the effect of MAK (1 g/kg) on 5-HTP-induced head twitches. One-way ANOVA revealed significant differences in 5-HTP-induced head twitches ([F(2,15) = 33.4, P < 0.01]). Post hoc analyses indicated significant differences between the control group and fluvoxamine-treated group (P < 0.01), but the difference between the control group and MAK-treated group was not significant (P = 0.33). Figure demonstrates the effect of MAK on DOI-induced head twitches. One-way ANOVA revealed significant differences in DOI-induced head twitches ([F(2,15) = 25.2, P < 0.01]). Post hoc analyses indicated significant differences between the control group and MAK-treated group (P < 0.01).

Objective(s):

We performed a phase IIa clinical trial of trans-1-amino-3-18F-fluoro-cyclobutane carboxylic acid (anti-18F-FACBC), a synthetic amino acid analog for PET, in patients with metastatic prostate cancer.

Subjects

Ten male patients with prostate cancer aged 69.9 ± 7.9 years (mean ± SD) were enrolled in this study between November 2010 and March 2011 (). The inclusion criteria were: a) histological positivity for prostate cancer on two or more cores obtained by needle biopsy of the prostate; and b) presence of lymph node metastasis and/or bone metastasis demonstrated on conventional imaging such as computed tomography (CT), magnetic resonance (MR) imaging, and bone scintigraphy. Patients, who had undergone therapy for prostate cancer, including surgical resection, radiation therapy, hormonal therapy, and chemotherapy, were excluded. All patients underwent anti-18F-FACBC PET after an interval of more than 3 weeks after biopsy to avoid possible effects of biopsy on PET images. The study was performed after approval by the institutional review boards of the host institution and according to Good Clinical Practice (GCP Ordinance No. 28, 27, March 1997). Written informed consent was obtained from all patients before participation. This clinical trial was registered as JapicCTI-101326.

PET/CT Procedures

Anti-18F-FACBC was injected after fasting for 4 h or longer, followed by PET/CT imaging using a TruePoint Biograph 6 PET/CT scanner (Siemens Medical Solutions, Hoffman Estates, IL). For CT, 5 mm thick contiguous slices were acquired without the administration of contrast medium. The initial five patients underwent whole-body PET imaging (whole-body group), and the injected dose ranged from 93.8 to 287.8 MBq. Whole-body PET imaging was performed 5 min (early) and 30 min (late) after injection. Data were acquired from the mid-thigh level to the top of the head for 2.5 min per bed position. The remaining five subjects underwent 60 min dynamic PET imaging of the pelvic region immediately after injection (dynamic group), and the injected dose was fixed at approximately 200 MBq (194.3–202.4 MBq). Images for visual evaluation were reconstructed using data of 2.5 min duration and centered at 5, 15, 30, and 58 min after injection. Additionally, images were reconstructed at 15 s/frame × 4, 30 s/frame × 4, 2 min/frame × 6, 3 min/frame × 5, and 5 min/frame × 6 to assess the time course quantitatively.

Safety Assessment

The following assessments were performed before and 24 h after injection to determine the safety of a single injection of anti-18F-FACBC: interview of subjective symptoms, physical examinations, 12 lead electrocardiography, measurements of blood pressure and pulse rate, hematology, blood chemistry, and urinalysis. An interview of subjective symptoms and physical examinations were also performed at the completion of PET/CT imaging.

Discussion

The phase I clinical trial of anti-18F-FACBC in Japan indicated its safety in six young adults (). In the present phase IIa clinical trial, safety was tested in 10 patients with metastatic prostate cancer. Mild adverse events were noted in two patients, however, they resolved spontaneously without treatment, suggesting the safety of a single intravenous injection of anti-18F-FACBC in patients with prostate cancer. PET images of diagnostic quality were acquired after injection of anti-18F-FACBC ranging from 93.8 to 287.8 MBq in dose. Avidity of anti-18F-FACBC to primary prostate lesions, metastatic lymph nodes, and metastatic bone lesions was indicated. The study population was small, predominantly included patients with high Gleason scores, and did not include control subjects. Despite these limitations, the results of the present study, in combination with those of previous studies (-), warrant further investigation of diagnostic performance of anti-18F-FACBC PET in comparison with conventional imaging.

Abstract

CO2 permeability of plasma membrane intrinsic protein 2 (PIP2) aquaporins of Hordeum vulgare L. was investigated. Five PIP2 members were heterologously expressed in Xenopus laevis oocytes. CO2 permeability was determined by decrease of cytosolic pH in CO2-enriched buffer using a hydrogen ion-selective microelectrode. HvPIP2;1, HvPIP2;2, HvPIP2;3 and HvPIP2;5 facilitated CO2 transport across the oocyte cell membrane. However, HvPIP2;4 that is highly homologous to HvPIP2;3 did not. The isoleucine residue at position 254 of HvPIP2;3 was conserved in PIP2 aquaporins of barley, except HvPIP2;4, which possesses methionine instead. CO2 permeability was lost by the substitution of the Ile254 of HvPIP2;3 by methionine, while water permeability was not affected. These results suggest that PIP2 aquaporins are permeable to CO2. and the conserved isoleucine at the end of the E-loop is crucial for CO2 selectivity.

Isoleucine 254 (I-254) is an important factor for CO2 permeability of HvPIP2;3

Simultaneous substitution of four amino acids of HvPIP2;3, A-268, L-281, R-289 and S-290, to L-268, F-281, S-289 and R-290 [HvPIP2;3(LFSR)] did not affect the acidification of the cytosol of HvPIP2;3(LFSR) cRNA-injected oocytes compared with HvPIP2;3-injected oocytes (). The osmotic water permeability (Pf) of HvPIP2;3 and HvPIP2;3(LFSR) was not significantly different (P > 0.05, ). This indicates that the four amino acids in the C-terminal stretch are not involved in the CO2 permeability of HvPIP2;3. Substitution of I-254 of HvPIP2;3, which is localized at the edge of the E-loop, by methionine (C) resulted in substantial repression of the PCO2 (D). Substitution of methionine 254 (M-254) of HvPIP2;4 by isoleucine, HvPIP2;4(M254I) (B) caused activation of PCO2 (D). The Pf of HvPIP2;3(I254M) and HvPIP2;4(M254I) was examined to test whether the mutated aquaporins were functional (E). The substitution of I-254 of HvPIP2;3 and M-254 of HvPIP2;4 did not show any apparent effect on Pf. This indicates that I-254 of HvPIP2;3 is crucial to the CO2 permeability.

Methods

Young, mature and old differentiated 3T3-L1 adipocytes were cultured for 48 h in the presence of 100 μM EPA, or 50 μM DHA complexed to albumin, whereas in control conditions only albumin was added to the medium. The Oil Red O staining was used to confirm adipocytes differentiation, and measure triglycerides content in cells. The concentration of adipokines (interleukin 6, adiponectin and leptin) in conditioned media was measured using mouse-specific ELISA kits.

Results

The fat accumulation in 3T3-L1 adipocytes was positively correlated with their age; however, EPA and DHA did not affect lipid accumulation on any stage of maturation. EPA and DHA increased the concentration of secreted adiponectin when compared with control, but only in the case of young adipocytes (58% and 35%, respectively). Moreover, EPA supplementation increased interleukin 6 concentration in conditioned medium, while DHA exerted an opposite effect on all stages of cellular maturation. Furthermore, EPA treatment increased leptin release from young cells, while DHA did not affect the secretion of this adipokine. In mature 3T3-L1 adipocytes both experimental factors decreased synthesis of leptin; however, in old cells no impact of these PUFA was noted.

Methods

Young, mature and old differentiated 3T3-L1 adipocytes were cultured for 48 h in the presence of 100 μM EPA, or 50 μM DHA complexed to albumin, whereas in control conditions only albumin was added to the medium. The Oil Red O staining was used to confirm adipocytes differentiation, and measure triglycerides content in cells. The concentration of adipokines (interleukin 6, adiponectin and leptin) in conditioned media was measured using mouse-specific ELISA kits.

Results

The fat accumulation in 3T3-L1 adipocytes was positively correlated with their age; however, EPA and DHA did not affect lipid accumulation on any stage of maturation. EPA and DHA increased the concentration of secreted adiponectin when compared with control, but only in the case of young adipocytes (58% and 35%, respectively). Moreover, EPA supplementation increased interleukin 6 concentration in conditioned medium, while DHA exerted an opposite effect on all stages of cellular maturation. Furthermore, EPA treatment increased leptin release from young cells, while DHA did not affect the secretion of this adipokine. In mature 3T3-L1 adipocytes both experimental factors decreased synthesis of leptin; however, in old cells no impact of these PUFA was noted.

Methods

Young, mature and old differentiated 3T3-L1 adipocytes were cultured for 48 h in the presence of 100 μM EPA, or 50 μM DHA complexed to albumin, whereas in control conditions only albumin was added to the medium. The Oil Red O staining was used to confirm adipocytes differentiation, and measure triglycerides content in cells. The concentration of adipokines (interleukin 6, adiponectin and leptin) in conditioned media was measured using mouse-specific ELISA kits.

Results

The fat accumulation in 3T3-L1 adipocytes was positively correlated with their age; however, EPA and DHA did not affect lipid accumulation on any stage of maturation. EPA and DHA increased the concentration of secreted adiponectin when compared with control, but only in the case of young adipocytes (58% and 35%, respectively). Moreover, EPA supplementation increased interleukin 6 concentration in conditioned medium, while DHA exerted an opposite effect on all stages of cellular maturation. Furthermore, EPA treatment increased leptin release from young cells, while DHA did not affect the secretion of this adipokine. In mature 3T3-L1 adipocytes both experimental factors decreased synthesis of leptin; however, in old cells no impact of these PUFA was noted.

Methods

Young, mature and old differentiated 3T3-L1 adipocytes were cultured for 48 h in the presence of 100 μM EPA, or 50 μM DHA complexed to albumin, whereas in control conditions only albumin was added to the medium. The Oil Red O staining was used to confirm adipocytes differentiation, and measure triglycerides content in cells. The concentration of adipokines (interleukin 6, adiponectin and leptin) in conditioned media was measured using mouse-specific ELISA kits.

Results

The fat accumulation in 3T3-L1 adipocytes was positively correlated with their age; however, EPA and DHA did not affect lipid accumulation on any stage of maturation. EPA and DHA increased the concentration of secreted adiponectin when compared with control, but only in the case of young adipocytes (58% and 35%, respectively). Moreover, EPA supplementation increased interleukin 6 concentration in conditioned medium, while DHA exerted an opposite effect on all stages of cellular maturation. Furthermore, EPA treatment increased leptin release from young cells, while DHA did not affect the secretion of this adipokine. In mature 3T3-L1 adipocytes both experimental factors decreased synthesis of leptin; however, in old cells no impact of these PUFA was noted.

Methods

Young, mature and old differentiated 3T3-L1 adipocytes were cultured for 48 h in the presence of 100 μM EPA, or 50 μM DHA complexed to albumin, whereas in control conditions only albumin was added to the medium. The Oil Red O staining was used to confirm adipocytes differentiation, and measure triglycerides content in cells. The concentration of adipokines (interleukin 6, adiponectin and leptin) in conditioned media was measured using mouse-specific ELISA kits.

Effects of EPA and DHA on lipid accumulation and lipolysis

Oil Red O was extracted from adipocytes and absorbance was measured to evaluate triglyceride contents in cells. The accumulation of lipids in differentiated 3T3-L1 cells was positively correlated with the day of culture (Figure ). However, examined polyunsaturated fatty acids EPA and DHA did not affect lipid accumulation on any stage of maturation. Glycerol release was measured to determine the influence of EPA and DHA on the intensity of lipolysis in cultured adipocytes. Experimental factors had no impact on lipolysis in used concentrations (that constituted the highest concentrations showing no toxic effect in the cells, as evaluated in a preliminary study – data not shown).

Abstract

Jieduquyuziyin prescription (JP), a traditional Chinese medicine (TCM) prescription, has been widely used for the clinical treatment of systemic lupus erythematosus (SLE). However, the complex chemical constituents of JP and the multifactorial pathogenesis of SLE make research on the therapeutic mechanism of JP in SLE challenging. In this paper, a serum metabolomics approach based on rapid resolution liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (RRLC-Q-TOF/MS) was employed to acquire the metabolic characteristics of serum samples obtained from mice in the SLE model group, JP-treated group, prednisone acetate (PA)-treated group and control group. The orthogonal partial least squares (OPLS) was applied to recognize metabolic patterns, and an obvious separation of groups was obtained. Thirteen metabolites, namely, phosphatidylethanolamine (PE 20:3), hepoxilin B3, lyso- phosphatidylethanolamine (lyso-PE 22:6), 12S-hydroxypentaenoic acid (12S-HEPE), traumatic acid, serotonin, platelet-activating factor (PAF), phosphatidylcholine (PC 20:5),eicosapentaenoic acid (EPA), 12(S)-hydroxyei- cosatetraenoic acid (12S-HETE), 14-hydroxy docosahexaenoic acid (14-HDOHE), lyso-phosphatidylcholine (lyso-PC 20:4), and indole acetaldehyde, were identified and characterized as differential metabolites involved in the pathogenesis of SLE. After treatment with JP, the relative content of 12(S)-HETE, PAF, 12(S)-HEPE, EPA, PE (20:3), Lyso-PE(22:6), and 14-HDOHE were effectively regulated, which suggested that the therapeutic effects of JP on SLE may involve regulating disturbances to the metabolism of unsaturated fatty acid, tryptophan and phospholipid. This research also demonstrated that metabolomics is a powerful tool for researching complex disease mechanisms and evaluating the mechanism of action of TCM.

Raw herbal medicines and JP extract

The following raw herbal medicines were purchased from the Chinese Herbal Medicine Co. Ltd. of Zhejiang Chinese Medical University (Hangzhou, China). JP was a combination of dried root of Radix Rehmanniae, Carapax Trionycis, Herba Artemisiae Annuae, Rhizoma Cimicifugae foetidae, Herba Hedyotidis, Radix Paeoniae Rubra, Herba Centellae Asiaticae, Semen Coicis, Fructus Citri Sarcodactylis and Radix Glycyrrhizae (at ratios of 5∶4∶4∶9∶5∶4∶5∶5∶3∶2). Next, 120 g JP was cut into small pieces and soaked in water (1/10 w/v) for 1 h, then boiled for 2 h. The filtrate was collected and the residue was extracted again with the same volume of water for another 2 h. The filtrates were combined and concentrated under vacuum to make a 1200 mL volume of JP.

Animals and treatments

40 female C57BL/6J mice, 8 weeks old and weighing 22 to 26 g, were purchased from the Experimental Animal Center of Zhejiang Chinese Medical University and were housed in four cages. The mice were housed in a specific-pathogen-free (SPF) environment and had free access to a standard diet and tap water. All animals were kept in an animal room with suitable conditions: temperature at 20 ± 2°C, relative humidity at 55% ± 10%, and 12 h dark-to-light cycle. All animals were allowed to acclimate for 1 week before the experiment. The mice were randomly divided into four groups (n = 10): JP-treated group, PA-treated group, SLE model group, and control group; and JP-treated group, PA-treated group, SLE model group were given a single 0.5 mL intraperitoneal injection of pristine (Sigma-Aldrich, Louis, MO, USA). JP and PA were given orally (daily) to JP-treated group and PA-treated group, respectively, from 1 month after pristane injection and continued till 5 months . PA was dissolved in physiological saline as a concentration of 0.69 mg mL−1, and the dosage was 18 mL kg−1 per day. The dosage of JP was also 18 mL kg−1 per day, and the SLE model group and control group were administered orally with an equivalent volume of physiological saline.

Abstract

Purslane (Portulaca oleracea L.) is an important plant naturally found as a weed in field crops and lawns. Purslane is widely distributed around the globe and is popular as a potherb in many areas of Europe, Asia, and the Mediterranean region. This plant possesses mucilaginous substances which are of medicinal importance. It is a rich source of potassium (494 mg/100 g) followed by magnesium (68 mg/100 g) and calcium (65 mg/100 g) and possesses the potential to be used as vegetable source of omega-3 fatty acid. It is very good source of alpha-linolenic acid (ALA) and gamma-linolenic acid (LNA, 18 : 3 w3) (4 mg/g fresh weight) of any green leafy vegetable. It contained the highest amount (22.2 mg and 130 mg per 100 g of fresh and dry weight, resp.) of alpha-tocopherol and ascorbic acid (26.6 mg and 506 mg per 100 g of fresh and dry weight, resp.). The oxalate content of purslane leaves was reported as 671–869 mg/100 g fresh weight. The antioxidant content and nutritional value of purslane are important for human consumption. It revealed tremendous nutritional potential and has indicated the potential use of this herb for the future.

2.2. Omega-3 Fatty Acid

Purslane leaves contain higher contents of alpha-linolenic acid (18 : 3 w3), alpha-tocopherol, ascorbic acid and glutathione than the leaves of spinach. It grows in growth chambers containing seven times higher contents of alpha-tocopherol than that found in spinach. One hundred grams of fresh purslane leaves (one serving) contains about 300–400 mg of 18 : 3 w3; 12.2 mg of alpha-tocopherol, 26.6 mg of ascorbic acid, 1.9 mg of beta-carotene, and 14.8 mg of glutathione [].