Yuzhi Du

Anti-fatigue activity of polysaccharides from the fruits of four Tibetan plateau indigenous medicinal plants

ETHNOPHARMACOLOGICAL RELEVANCE The fruits of Hippophae rhamnoides L., Lycium barbarum L., Lycium ruthenicum Murr. and Nitraria tangutorum Bobr. are traditional medicinal food of Tibetans and used to alleviate fatigue caused by oxygen deficiency for thousands of years. The present study focused on exploiting natural polysaccharides with remarkable anti-fatigue activity from the four Qinghai-Tibet plateau characteristic berries. MATERIALS AND METHODS The fruits of Hippophae rhamnoides, Lycium barbarum, Lycium ruthenicum and Nitraria tangutorum were collected from Haixi national municipality of Mongol and Tibetan (N 36.32°, E98.11°; altitude: 3100 m), Qinghai, China. Their polysaccharides (HRWP, LBWP, LRWP and NTWP) were isolated by hot-water extraction, and purified by DEAE-Cellulose ion-exchange chromatography. The total carbohydrate, uronic acid, protein and starch contents of polysaccharides were determined by a spectrophotometric method. The molecular weight distributions of polysaccharides were determined by gel filtration chromatography. Their monosaccharide composition analysis was performed by the method of 1-phenyl-3-methyl-5-pyrazolone (PMP) pre-column derivatization and RP-HPLC analysis. HRWP, LBWP, LRWP and NTWP (50, 100 and 200 mg/kg) were orally administrated to mice once daily for 15 days, respectively. Anti-fatigue activity was assessed using the forced swim test (FST), and serum biochemical parameters were determined by an autoanalyzer and commercially available kits; the body and organs were also weighted. RESULT LBWP, LRWP and NTWP were mainly composed of glucans and some RG-I pectins, and HRWP was mainly composed of HG-type pectin and some glucans. All the four polysaccharides decreased immobility in the FST, and the effects of LBWP and NTWP were demonstrated in lower doses compared with HRWP and LRWP. There was no significant difference in liver and heart indices between non-treated and polysaccharide-treated mice, but the spleen indices were increased in LBWP and NTWP (200mg/kg) group. Moreover, the FST-induced reduction in glucose (Glc), superoxide dismutase (SOD) and glutathione peroxidase (GPx) and increase in creatine phosphokinase (CK), lactic dehydrogenase (LDH), blood urea nitrogen (BUN), triglyceride (TG) and malondialdehyde (MDA) levels, all indicators of fatigue, were inhibited by HRWP, LBWP, LRWP and NTWP to a certain extent while the effects of LBWP and NTWP were much better than that of HRWP and LRWP at the same dosage. CONCLUSION Water-soluble polysaccharides HRWP, LBWP, LRWP and NTWP, from the fruits of four Tibetan plateau indigenous berry plants, significantly exhibited anti-fatigue activities for the first time, through triglyceride (TG) (or fat) mobilization during exercise and protecting corpuscular membrane by prevention of lipid oxidation via modifying several enzyme activities. Moreover, it is demonstrated that LBWP and NTWP are more potent than HRWP and LRWP, which were proposed to be applied in functional foods for anti-fatigue and antioxidant potential.

Anticancer and immunostimulating activities of a novel homogalacturonan from Hippophae rhamnoides L. berry

Our previous study isolated an anti-fatigue polysaccharide (HRWP) from the Hippophae rhamnoides berry. In this study, using ion-exchange chromatography and gel filtration chromatography in turn, a water-soluble homogenous polysaccharide HRWP-A was isolated from HRWP. Structural analysis determined that HRWP-A was a polysaccharide with repeating units of (1→4)-β-d-galactopyranosyluronic residues, of which 85.16% were esterified with methyl groups. An antitumor activity assay showed that HRWP-A could significantly inhibit the Lewis lung carcinoma (LLC) growth in tumor-bearing mice. Further experiments suggested that the antitumor effect of HRWP-A might be mediated through immunostimulating activity, as it enhances the lymphocyte proliferation, augments the macrophage activities, as well as promoting NK cell activity and CTL cytotoxicity in tumor-bearing mice. To our knowledge, this is the first report on a natural antitumor high-methoxyl homogalacturonan pectin from the H. rhamnoides berry-a compound that acts as a potential immunostimulant and anticancer adjuvant.

Mercury sulfides are much less nephrotoxic than mercury chloride and methylmercury in mice

Mercury sulfides (α-HgS, β-HgS) are frequently included in traditional medicines. Mercury is known for nephrotoxicity, their safety is of concern. To address this question, mice were orally administrated with Zuotai (54% β-HgS, 30mg/kg), α-HgS (HgS, 30mg/kg), HgCl2 (33.6mg/kg), or MeHgCl (3.1mg/kg) for 7days, and nephrotoxicity was examined. Animal body weights were decreased by HgCl2 and to a lesser extent by MeHg, but unaltered after Zuotai and HgS. HgCl2 and MeHg produced renal tubular vacuolation, interstitial inflammation and cell degeneration with protein cysts in the tubular lumen, while these pathological lesions were mild in Zuotai and HgS-treated mice. Electron microscopy showed that HgCl2 and MeHg produced spotted swelling endothelium reticulum, while these lesions were mild or absent in Zuotai and HgS-treated mice. Renal Hg contents reached 250-300ng/mg kidney in HgCl2 and MeHg groups as compared to 2-3ng/mg in Zuotai and HgS groups. The expression of kidney injury biomarkers, kidney injury molecule-1 (Kim-1) and neutrophil gelatinase-associated lipocalin (Ngal), were increased after HgCl2 and MeHg, but unaltered after Zuotai and HgS. The expression of renal influx transporters Oat3 and Oatp4c1 was decreased, while the expression of renal efflux transporter such as Mrp2, Mrp4, and Mate2 was increased following HgCl2 and MeHg. These gene expressions were unchanged after Zuotai and HgS. In summary, both α-HgS and β-HgS are less nephrotoxic than HgCl2 and MeHg, indicating that chemical forms of mercury are a major determinant of mercury disposition and toxicity.

Physicochemical and functional properties of gelatin extracted from Yak skin

Different molecular weight distribution (MWD) gelatin was extracted from Yak skin after enzymatic pretreatments and their physicochemical and functional properties (SDS-PAGE, UV-vis absorption spectra, DSC, FT-IR, Amino acid analysis, AFM, emulsibility and foamability) were analyzed. The gelatin was extracted by pepsin and got different MWD of Yak skin gelatin by controlling the enzymolysis time. The SDS-PAGE showed the MWD of the Yak skin gelatin. The UV-vis absorption turned out that the broad MWD of Yak skin gelatin had a higher maximum absorption peaks. The FT-IR and AFM indicated that the gelatin structures and microstructures changed with the change of the MWD. The broad MWD of the Yak skin gelatin had a higher denaturation temperature (TD), and it was higher than most of the other mammals and marine biological gelatin. The broad MWD gelatin also had higher imino acids (proline and hydroxyproline) contents and lower foamability and emulsibility compared to the narrow MWD gelatin. These findings, obtained for the first time for Yak skin gelatin, showed that it has great potential for application as an alternative to commercial gelatin due to its good thermotolerance, particularly in the applications of the biological materials, stabilizer of thermo-tolerant and so on.

Chemical Species, Micromorphology, and XRD Fingerprint Analysis of Tibetan Medicine Zuotai Containing Mercury

Zuotai (gTso thal) is one of the famous drugs containing mercury in Tibetan medicine. However, little is known about the chemical substance basis of its pharmacodynamics and the intrinsic link of different samples sources so far. Given this, energy dispersive spectrometry of X-ray (EDX), scanning electron microscopy (SEM), atomic force microscopy (AFM), and powder X-ray diffraction (XRD) were used to assay the elements, micromorphology, and phase composition of nine Zuotai samples from different regions, respectively; the XRD fingerprint features of Zuotai were analyzed by multivariate statistical analysis. EDX result shows that Zuotai contains Hg, S, O, Fe, Al, Cu, and other elements. SEM and AFM observations suggest that Zuotai is a kind of ancient nanodrug. Its particles are mainly in the range of 100–800 nm, which commonly further aggregate into 1–30 μm loosely amorphous particles. XRD test shows that β-HgS, S8, and α-HgS are its main phase compositions. XRD fingerprint analysis indicates that the similarity degrees of nine samples are very high, and the results of multivariate statistical analysis are broadly consistent with sample sources. The present research has revealed the physicochemical characteristics of Zuotai, and it would play a positive role in interpreting this mysterious Tibetan drug.

Hormesis of methylmercury-human serum albumin conjugate on N9 microglia via ERK/MAPKs and STAT3 signaling pathways

ABSTRACT Methylmercury (MeHg+) is an extremely toxic organomercury cation that can induce severe neurological damage. Once it enters the body, methylmercury binds to amino acids or proteins containing free sulfhydryl groups. In particular, methylmercury is known to bind with human serum albumin (HSA) in human plasma; however, the effects of methylmercury‐HSA conjugate (MeHg‐HSA) on the central nervous system (CNS) are not fully understood. In the present study, we used the microglial cell line N9 as the target cells to evaluate the effect of MeHg‐HSA on physiological function of the CNS preliminarily. The various factors in the cell culture were monitored by MTT assay, total lactate dehydrogenase assay, ELISA, qPCR, Western blot and flow cytometry techniques. The results showed that low‐dose treatment with MeHg‐HSA activated N9 cells, promoting cell proliferation and total cell number, enhancing NO and intracellular Ca2+ levels, and suppressing the release of TNF&agr; and IL1&bgr; without cytotoxic effects; while high‐dose MeHg‐HSA exhibited cytotoxic effects on N9 cells, including promoting cell death and increasing the secretion of TNF&agr; and IL1&bgr;. These results indicate that MeHg‐HSA causes hormesis in microglia N9 cells. Furthermore, ERK/MAPKs and STAT3 signaling pathways related to the hormesis of MeHg‐HSA on N9 cells. In addition, low dose of MeHg‐HSA might be viewed as something very close to a lowest observed adverse effect level (LOAEL) for N9 cells. These findings will be useful for investigating the hormesis mechanism of MeHg+ and exploring the specific functions of MeHg‐sulfhydryl conjugates on the central nervous system. HIGHLIGHTSMethylmercury‐human serum albumin adduct causes hormesis in N9 microglia cells.ERK/MAPKs and STAT3 signaling pathways might be related to the hormesis of MeHg‐HSA on N9 cells.2ng/mL of MeHg‐HSA was close to a NOAEL for N9 cells.

Hormesis of mercuric chloride-human serum albumin adduct on N9 microglial cells via the ERK/MAPKs and JAK/STAT3 signaling pathways

Mercury chloride (HgCl2), a neurotoxicant that cannot penetrate the blood-brain barrier (BBB). Although when the BBB are got damaged by neurodegenerative disorders, the absorbed HgCl2, mainly in form of Hg (II)-serum albumin adduct (Hg-HSA) in human plasma, can penetrate BBB and affect central nervous system (CNS) cells. Current study planned to evaluate the effect of Hg-HSA on the physiological function of N9 microglial cells. At low dosage (15 ng/mL) of Hg-HAS, the observed outcomes was: promoted cell propagation, Nitric Oxide (NO) and intracellular Ca2+ levels enhancement, suppressed the release of TNF-α and IL-1β and inhibited cell proliferation. At high dosage (15 μg/mL) we observed decline in NO and intracellular Ca2+ levels, and increment in the release of TNF-α and IL-1β. These biphasic effects are similar to hormesis, and the hormesis, in this case, was executed through ERK/MAPKs and JAK/STAT3 signaling pathways. Study of quantum chemistry revealed that Hg2+ could form stable coordination structures in both Asp249 and Cys34 sites of HSA. Although five-coordination structure in Asp249 site is more stable than four-coordination structure in Cys34 site but four-coordination structure is formed easily in-vivo in consideration of binding-site position in spatial structure of HSA.

The depressive-like behaviors of chronic unpredictable mild stress-treated mice ameliorated by Tibetan medicine Zuotai: involvement in the hypothalamic–pituitary–adrenal (HPA) axis pathway

Background Zuotai, a famous Tibetan medicinal mixture containing metacinnabar, is traditionally used for the purpose of tranquilizing minds and soothing nerves. However, it still lacks substantial experimental data for it to be approved for use. Aim This study was designed to assess the effects of Zuotai on depressive-like symptoms in a chronic unpredictable mild stress (CUMS) mouse model, and to explore its potential mechanism, particularly the hypothalamic–pituitary–adrenal (HPA) axis pathway. Materials and methods First, Kunming mice were exposed to the CUMS procedure and simultaneously administered Zuotai or imipramine (positive control) by gavage continuously for 6 weeks. Then, depressive-like behaviors of mice in each group were tested with the sucrose preference test, forced swimming test, tail suspension test, and open field test. Meanwhile, the three key neuroendocrine hormones (corticotropin releasing hormone, adrenocorticotropic hormone and corticosterone) in HPA axis pathway, and the level of the emotion-related monoamine neurotransmitters (5-hydroxytryptamine and norepinephrine) were measured using enzyme-linked immunosorbent assay. Furthermore, total mercury in the hypothalamus and hippocampus were determined using an automatic, direct mercury analyzer. Results Zuotai or imipramine significantly increased the body weight and the sucrose preference ratio in sucrose preference test, and dramatically improved motor activity in forced swimming test, tail suspension test, and open field test in CUMS mice. Zuotai or imipramine remarkably decreased levels of corticotropin-releasing hormone, adrenocorticotropic hormone, and corticosterone in the HPA axis, and increased levels of 5-hydroxytryptamine and norepinephrine in the serum in CUMS mice. However, a small amount of mercury was deposited in the hypothalamus and hippocampus in Zuotai-treated mice, which may pose a potential risk to the central nervous system. Conclusion Zuotai has a strong ability to ameliorate depressive-like behaviors in CUMS-treated mice through inhibition of the HPA axis and upregulation of monoamine neurotransmitters. These findings provide new insight into the pharmacological effect of Zuotai on depression.

An antioxidant α-glucan from Cladina rangiferina (L.) Nyl. and its protective effect on alveolar epithelial cells from Pb 2+ -induced oxidative damage

Air pollution is a serious global health problem nowadays. So, it is an emergency to pay sufficient attention to treat and prevent the diseases caused by air pollution, especially respiratory disease and lung damage. Cladina rangiferina (L.) Nyl. is an edible lichen that has been used in medicinal diets to treat respiratory and other diseases for over 500 years. In this study, a water-soluble polysaccharide, CRWP-P, was obtained from C. rangiferina by hot-water extraction, freeze-thawing separation, and Fehling reagent purification. Structural analysis showed that CRWP-P is a linear α-(1 → 3),(1 → 4)-d-glucan without branches. Its Mw was determined to be 1.05 × 105 Da. Its (1,3)-α-d-glucopyranosyl: (1,4)-α-d-glucopyranosyl ratio is approximately 1:2. Antioxidant activity assay showed that C. rangiferina polysaccharides, especially CRWP-P, had appreciable DPPH radical-scavenging activity and reducing power. Notably, they could effectively decrease cell breakdown and ROS generation, inhibit lipid peroxidation, increase key antioxidase activity, and promote glutathione redox cycling in Pb2+-oxidative injured A549 alveolar epithelium cells. Overall, the results of this study indicated that C. rangiferina polysaccharides, especially CRWP-P, have the potential to be natural antioxidants for the treatment of lung oxidative damage induced by lead of air pollutants.

Molecular structural properties of extracted gelatin from Yak skin as analysed based on molecular weight

ABSTRACT This paper dealt with the physicochemical, functional and antioxidative properties of Yak skin gelatins with various molecular weights. The gelatin was extracted by alkaline process, and different molecular weight gelatin was achieved by ultrafiltration with different molecular weight cut membrane. The SDS-PAGE showed the molecular weight of the yak skin gelatin. The UV-Vis absorption turned out that the high molecular weight (HMW) of yak skin gelatin had a higher maximum absorption peaks. The FT-IR indicated the change of gelatin structures with the change of the molecular weight. No matter the HMW or the low molecular weight (LMW), the Yak skin gelatin had a high denaturation temperature (Td), and it was higher than most of the other mammals and marine biological gelatin. The HMW gelatin had higher imino acids contents and lower oxidation resistance, foamability and emulsibility compared to the LMW gelatin. These findings showed that a certain molecular weight range of Yak skin gelatin has great potential for application as an alternative to commercial gelatin due to its good antioxidative, foamability emulsibility and thermotolerance.