Genetic diversity and main functional composition of Lingzhi strains from main producing areas in China.

In this study, we used genotyping by sequencing (GBS) to examine the genetic diversity of 22 strains of Lingzhi and the quality differences in 15 fruit bodies of Lingzhi from different Chinese regions. The phylogenetic trees of 22 strains were constructed based on ITS (Internal transcribed spacer) and SNP (single nucleotide polymorphism). Moisture, ash, water-soluble extracts, alcohol-soluble extracts, polysaccharides, and triterpenoids from 15 fruit bodies of Lingzhi were detected and analyzed based on Chinese Pharmacopoeia and the US Pharmacopoeia references. Moreover, the monosaccharide composition of polysaccharides was studied using PMP-HPLC, and the effect of polysaccharides on the proliferation rate of splenocytes was investigated in vitro. The identification results of these strains by the phylogenetic trees which were constructed based on ITS sequences and SNPs showed that most of the strains applied in the main producing areas of Lingzhi in China were accurate except for a few inaccurate strains. The moisture, ash, water and alcohol soluble extractive, polysaccharide and triterpenoid content of all samples were meet the requirements of the Chinese Pharmacopoeia, while the polysaccharide and triterpenoid content of less than half of the samples meet the requirements of the U.S. Pharmacopoeia. The polysaccharide extracted from these samples have different effects on the proliferation rate of spleen cells. To sum up, this is the first study that reported on the differences in Lingzhi strains from the main producing areas in China. The quality of some fruit bodies did not meet the pharmacopeia requirements, and wrong strains were used in some production areas; thus, strains should be given special attention before legal processing.

Inhibitory effect of triterpenoids from the mushroom Inonotus obliquus against α-glucosidase and their interaction: Inhibition kinetics and molecular stimulations.

Bioassay-guided fractionation led to the isolation of a series of triterpenoids (1-46) including 12 new ones (1-12) from the mushroom Inonotus obliquus. The structures of all the compounds were elucidated by spectroscopic analysis as well as by comparison with literature data. Triterpenoids 1-3, 6, 7, 16, 24, 25, 27, 38, 43, 44 and 46 showed strong α-glucosidase inhibition, with IC50 values from 11.5 to 81.8 µM. Their structure-activity relationships were discussed. Inonotusol F (24) showed the strongest inhibitory activity and it presented noncompetitive inhibition against α-glucosidase. Molecular docking and molecular dynamics stimulation further demonstrated that GLU302 and PHE298 were key amino acids for the inhibition of inonotusol F (24) towards α-glucosidase. This study indicates the vital role of triterpenoids in explaining hypoglycemic effect of Inonotus obliquus and provides important evidence for further development and utilization of this mushroom.

4-Methoxydalbergione is a potent inhibitor of human astroglioma U87 cells in vitro and in vivo.

Astroglioma is the most common primary tumor in the central nervous system without effective treatment strategies. Temozolomide (TMZ) is a chemotherapeutic drug to treat astroglioma but exhibits low potency and has side effects. Therefore, there is an urgent need to develop new compounds to treat astroglioma. Dalbergia sissoo Roxb was the source of Dalbergia odorifera in traditional Chinese medicine (TCM) and has been clinically used as an anti-tumor medicine. 4-Methoxydalbergione (4MOD) is purified from Dalbergia sissoo Roxb., and shows an inhibitory effect on osteosarcoma, but its effects on astroglioma have not been reported. Here, we evaluate its anti-astroglioma effects on both in vitro and in vivo models. In cultured astroglioma U87 cells, 4MOD inhibited cell proliferation and induced cell apoptosis in a time- and concentration-dependent manner. Compared with TMZ, 4MOD exhibited a tenfold greater potency of anti-astroglioma effects. 4MOD effectively stalled the cell cycle in G2 phase. Transcriptome sequencing (RNA-seq) showed that 4MOD upregulated 158 genes and downregulated 204 genes that are mainly enriched in cell membrane, cell division, cell cycle, p53, TNF, and MAPK signaling pathways, which may underlie its anti-tumor mechanisms. In a nude mouse xenograft model transplanted with U87 cells, 10 mg/kg 4MOD slowed down tumor growth rate, while at 30 mg/kg dose, it reduced tumor size. Collectively, this study demonstrates that 4MOD is a potent native compound that remarkably inhibits U87 astroglioma growth in both in vitro and in vivo models.

Relationship between gut microbiota and type 2 diabetic erectile dysfunction in Sprague-Dawley rats.

In order to investigate the relationship between gut microbiota and type 2 diabetic erectile dysfunction (T2DED), we analyzed the characteristics of gut microbiota in the Sprague-Dawley (SD) rats with T2DED. Thirty-five SD rats were randomly divided into two groups: control group (n=15) with normal diet, and experimental group (n=20) with construction of T2D model. Faecal and serum samples were collected at 2nd and 8th week after establishment of T2D model, respectively. Faecal samples were used for analysis of gut microbiota, and serum samples for detection of trimethylamine N-oxide (TMAO), lipopolysaccharide (LPS), and inflammatory factors like interleukin-1 (IL-1), IL-2, IL-10, and monocyte chemoattractantprotein-1 (MCP-1). The main compositions of gut microbiota were Bacteroidetes, Proteobacteria and Firmicutes at the phylum level, and Oscillospira, Allobaculum, Bacteroides, Ruminococcus, SMB53, Prevotella, Coprococcus, Sutterella and Blautia at the genus level with relatively higher abundance in all SD rats. The relative abundance of Enterococcus, Corynebacterium, Aerococcus, Facklamia (opportunistic pathogens in most case) increased, and that of Allobaculum, Bifidobacterium, Eubacterium, Anaerotruncus (beneficial bacteria) decreased in T2DED group as compared with that at 2nd week after establishment of T2D model (T2D2 group). The serum contents of TMAO, LPS, IL-1, IL-2, IL-10 and MCP-1 in T2DED group were significantly higher than those in control group. The gut microbiota of T2DED rats was inhibited. The gut microbiota of T2DED rats had changed, as the relative abundance of beneficial bacterium was decreased while that of opportunistic pathogens was increased. The variations of gut microbiota might lead to inflammation and prompt the emergence of erectile dysfunction in the rats with T2D. TMAO might play an important role in the formation of T2DED.

Relationship between Gut Microbiota and Type 2 Diabetic Erectile Dysfunction in Sprague-Dawley Rats / 华中科技大学学报（医学）（英德文版）

In order to investigate the relationship between gut microbiota and type 2 diabetic erectile dysfunction (T2DED),we analyzed the characteristics of gut microbiota in the Sprague-Dawley (SD) rats with T2DED.Thirty-five SD rats were randomly divided into two groups:control group (n=15)with normal diet,and experimental group (n=20) with construction of T2D model.Faecal and serum samples were collected at 2nd and 8th week after establishment of T2D model,respectively.Faecal samples were used for analysis of gut microbiota,and serum samples for detection of trimethylamine N-oxide (TMAO),lipopolysaccharide (LPS),and inflammatory factors like interleukin-1 (IL-1),IL-2,IL-10,and monocyte chemoattractantprotein-1 (MCP-1).The main compositions of gut microbiota were Bacteroidetes,Proteobacteria and Firmicutes at the phylum level,and Oscillospira,Allobaculum,Bacteroides,Ruminococcus,SMB53,Prevotella,Coprococcus,Sutterella and Blautia at the genus level with relatively higher abundance in all SD rats.The relative abundance of Enterococcus,Corynebacterium,Aerococcus,Facklamia (opportunistic pathogens in most case) increased,and that ofAllobaculum,Bifidobacterium,Eubacterium,Anaerotruncus (beneficial bacteria) decreased in T2DED group as compared with that at 2nd week after establishment of T2D model (T2D2 group).The serum contents of TMAO,LPS,IL-1,IL-2,IL-10 and MCP-1 in T2DED group were significantly higher than those in control group.The gut microbiota of T2DED rats was inhibited.The gut microbiota of T2DED rats had changed,as the relative abundance of beneficial bacterium was decreased while that of opportunistic pathogens was increased.The variations of gut microbiota might lead to inflammation and prompt the emergence of erectile dysfunction in the rats with T2D.TMAO might play an important role in the formation of T2DED.

Total triterpenoids from Ganoderma Lucidum suppresses prostate cancer cell growth by inducing growth arrest and apoptosis.

In this study, one immortalized human normal prostatic epithelial cell line (BPH) and four human prostate cancer cell lines (LNCaP, 22Rv1, PC-3, and DU-145) were treated with Ganoderma Lucidum triterpenoids (GLT) at different doses and for different time periods. Cell viability, apoptosis, and cell cycle were analyzed using flow cytometry and chemical assays. Gene expression and binding to DNA were assessed using real-time PCR and Western blotting. It was found that GLT dose-dependently inhibited prostate cancer cell growth through induction of apoptosis and cell cycle arrest at G1 phase. GLT-induced apoptosis was due to activation of Caspases-9 and -3 and turning on the downstream apoptotic events. GLT-induced cell cycle arrest (mainly G1 arrest) was due to up-regulation of p21 expression at the early time and down-regulation of cyclin-dependent kinase 4 (CDK4) and E2F1 expression at the late time. These findings demonstrate that GLT suppresses prostate cancer cell growth by inducing growth arrest and apoptosis, which might suggest that GLT or Ganoderma Lucidum could be used as a potential therapeutic drug for prostate cancer.

Effect of bajijiasu isolated from Morinda officinalis F. C. how on sexual function in male mice and its antioxidant protection of human sperm.

ETHNOPHARMACOLOGICAL RELEVANCE: In recent years, the physiological aspects of human fertility have been seriously influenced by the interactions of genetic and environmental factors. Almost one in 20 males has been affected by male infertility, providing a great challenge and an opportunity to use natural compounds as alternatives to chemical drugs with comprehensive adverse effects. However, ample evidences are scanty to support the physiological mechanisms of natural compounds used to treat male infertility. In traditional Chinese medicine, Morinda officinalis F. C. How is widely used as a herb that invigorates the kidneys and supports yang, the original energy in the human body, to resist diseases and in treating male infertility. In this study, we evaluated whether bajijiasu isolated from the roots of M. officinalis F.C. How is a potential agent for the treatment of male infertility. MATERIALS AND METHOD: In this study, both normal and kidney-yang-deficient mice were administered bajijiasu orally at different concentrations. To determine the pharmacological mechanism of bajijiasu, we observed the sexual behavior and genital organ coefficients, determined their serum hormone levels, analyzed their sperm quality parameters, and examined histopathological sections from them. We also used enzymatic assays to determine the effects of bajijiasu on superoxide dismutase, glutathione peroxidase, and malondialdehyde. Confocal micro-Raman spectroscopy was used to investigate the changes in the DNA of H2O2-damaged human sperm after treatment with bajijiasu in vitro. RESULTS: Our results showed that bajijiasu enhanced the sexual behavior of both normal and kidney-yang-deficient mice. It also markedly increased the testosterone concentrations, reduced the levels of cortisol, improved the quality of the sperm, and counteracted the histopathological impairment induced by hydroxyurea in the kidney-yang-deficient mice. The enzymatic assay and Raman spectra showed that bajijiasu protects the DNA of sperm from damage by H2O2. CONCLUSION: Bajijiasu is a potential androgen-like drug that modulates hormone levels to some extent without producing reproductive-organ lesions, enhances the sexual function of male mice, and protects the DNA of human sperm from H2O2 damage. Thus, bajijiasu is an active ingredient of M. officinalis F.C. How that improves the human reproductive capacity.

Rare earths exposure and male infertility: the injury mechanism study of rare earths on male mice and human sperm.

The weight; testis/body coefficient; levels of LDH, SDH, SODH, G-6PD, and testosterone; cell cycle; and cell apoptosis of the male mice were influenced after being treated with 200 mg/[kg/day] of rare earths suspension for 3 weeks. The "Raman fingerprints" of the human sperm DNA exposed to 0.040 mg/ml CeCl3 were very different from those of the untreated; the Raman bands at 789 cm(-1) (backbone phosphodiester), PO4 backbone at 1,094 cm(-1), methylene deformation mode at 1,221 cm(-1), methylene deformation mode at 1,485 cm(-1), and amide II at 1,612 cm(-1), of which intensities and shifts were changed, might be the diagnostic biomarkers or potential therapeutic targets. The injury mechanism might be that the rare earths influence the oxidative stress and blood testosterone barrier, tangle the big biomolecule concurrently, which might cause the testicular cells and vascular system disorder and/or dysfunction, and at the same time change the physical and chemical properties of the sperm directly.

Antioxidative protective effect of icariin on the FeSO4/H 2O 2-damaged human sperm based on confocal raman micro-spectroscopy.

Oxidative stress is implicated in male infertility and significantly higher reactive oxygen species are detected in 25% of infertile males. Although different agents of various alternative medicines, including traditional Chinese medicine, have been tried with varying success, evidence remains limited on whether and how much herbs or supplements might help increase the anti-oxidant ability of the sperm. This study examined the anti-oxidative effects of icariin, a flavonoid isolated from Herba Epimedii, on the human sperm. We prepared the FeSO4/H2O2-damaged human sperms, which were co-cultured with icariin in vitro, and then observed the changes of the sperm by employing Raman micro-spectroscopy. The results showed that Raman mapping with a 514 nm excitation laser allowed clear differentiation of the nucleus, neck, and, in particular, the mitochondria-rich middle piece of a human sperm cell. The effect of icariin on different organelles of the sperm was quantified by localized spectral Raman signatures obtained within milli-seconds, and icariin could keep the "Raman fingerprint" of the human sperm the same as the control groups, suggesting that icariin could protect the human sperm from being damaged by FeSO4/H2O2. Icariin may serve as a tonifying and replenishing agent of herbal origin for enhancing reproductive functions.

Protective effects of bajijiasu in a rat model of Aß25₋35-induced neurotoxicity.

ETHNOPHARMACOLOGICAL RELEVENCE: Neurodegenerative diseases (NDs) caused by neurons and/or myelin loss lead to devastating effects on patients׳ lives. Although the causes of such complex diseases have not yet been fully elucidated, oxidative stress, mitochondrial and energy metabolism dysfunction, excitotoxicity, inflammation, and apoptosis have been recognized as influential factors. Current therapies that were designed to address only a single target are unable to mitigate or prevent disease progression, and disease-modifying drugs are desperately needed, and Chinese herbs will be a good choice for screening the potential drugs. Previous studies have shown that bajijiasu, a dimeric fructose isolated from Morinda officinalis radix which was used frequently as a tonifying and replenishing natural herb medicine in traditional Chinese medicine clinic practice, can prevent ischemia-induced neuronal damage or death. MATERIALS AND METHODS: In order to investigate whether bajijiasu protects against beta-amyloid (Aß25₋35)-induced neurotoxicity in rats and explore the underlying mechanisms of bajijiasu in vivo, we prepared an Alzheimer׳s disease (AD) model by injecting Aß25-35 into the bilateral CA1 region of rat hippocampus and treated a subset with oral bajijiasu. We observed the effects on learning and memory, antioxidant levels, energy metabolism, neurotransmitter levels, and neuronal apoptosis. RESULTS: Bajijiasu ameliorated Aß-induced learning and memory dysfunction, enhanced antioxidative activity and energy metabolism, and attenuated cholinergic system damage. Our findings suggest that bajijiasu can enhance antioxidant capacity and prevent free radical damage. It can also enhance energy metabolism and monoamine neurotransmitter levels and inhibit neuronal apoptosis. CONCLUSION: The results provide a scientific foundation for the use of Morinda officinalis and its constituents in the treatment of various AD. Future studies will assess the multi-target activity of the drug for the treatment of AD.

Confocal mirco-Raman spectroscopic analysis of the antioxidant protection mechanism of the oligosaccharides extracted from Morinda officinalis on human sperm DNA.

ETHNOPHARMACOLOGICAL RELEVENCE: Male infertility is a stressful and frustrating problem for the society, but a number of male infertility treatments are available as traditional Chinese medicine strategies which have been tried with variable success, while evidence is still limited on whether-or how much-herbs or supplements might help increase fertility, so the aim of this study was to investigate if the oligosaccharides extracted from Morinda officialis, a Chinese herb, is the active constituents to the fertility. MATERIALS AND METHODS: In this study, we prepared the H2O2-demaged human sperm, cocultured with the oligosaccharides in vitro, then observed the changes of the DNA using confocal micro-Raman spectroscopy, and comparative analysis the differences of the spectra of different treated groups. RESULTS: The results showed that the oligosaccharides extracted from Morinda officialis can keep the "Raman fingerprints" of the human sperm DNA almost the same as those of the control groups, but very different from the H2O2-induced groups, especially the intensity of bands at 787, 993, 1094, 1254, 1340, 1376, 1421, 1443, 1487, 1577 and 1662cm(-1) which could be as potential targets for the drugs finding, and further principal component analysis was successfully used to classify the Raman spectra of normal control and model groups. CONCLUSION: This results suggested that the oligosaccharides can protect the DNA of human sperm from being damaged by H2O2, and which was one of the active constituents of Morinda officialis on treating infertility. It was also demonstrated that Morinda officialis as a tonifying and replenishing natural herb medicine can be used to enhance reproductive functions, and the Raman spectroscopy could be an applicable technology for screening active components in vitro from herbs.

Method to detect the variants of the erythrocyte in a rat model of Aß25-35-induced neurotoxicity based on micro-Raman spectroscopy.

Alzheimer's disease irreversibly and progressively damages the brain, but the treatments in clinical trials are too slow. So, we hypothesized that the presence of erythrocyte variants with AD could be used as a noninvasive means to predict or trigger for administration of the preventive therapeutics, and the aim of this study is to develop a method using Raman spectroscopy in a rat model of Aß25-35-induced neurotoxicity, and then evaluate the protective effect of bajijiasu by this method. Results showed that the Raman spectra fingerprints of the erythrocyte of model group were obvious different from those of the normal control, as peaks around the region 650 cm(-1) belonged to the s-s makers, 1605 cm(-1) corresponded to the high spin (deoxygenated-Hb) marker, 1374 cm(-1) arises from ν4 as a sign of concentration of O2, and 1123 and 1033 cm(-1) are associated with the trans stretching vibrations of CAC skeleton. Results also showed that bajijiasu can make these changes recover. Our study also suggested that erythrocyte variants detected using Raman spectroscopy should be tested in a specific longitudinal study for the association with AD diagnosis, and if positive, can be used as a prognostic marker.

[The analysis and identification of fritillaria cirrhosa by Raman spectra].

Laser Raman spectrum technology was used to distinguish fritillaria cirrhosa from its adulterants rapidly and accurately. The study is based on that different traditional Chinese medicine contains different chemical compositions, and the differences could be displayed in Raman spectra. The Raman spectra of fritillaria cirrhosas shows that several characteristic strong peaks could be found at 442, 480, 863, 941, 1 083, 1 129, 1 342, 1 463 and 2 910 cm(-1), and a few obvious peaks appear at 111, 302, 360, 409, 527, 579, 618, 718, 767, 1 052, 1 083, 1 207 and 1 261 cm(-1). According to the Raman spectra, a Raman fingerprint of fritillaria cirrhosa was set up, which could be used to distinguish its adulterants. This analysis could be used in identifying fritillaria cirrhosa rapidly, accurately and nondestructively.

[Protective effect of oligosaccharides from Morinda officinalis on beta-amyloid-induced dementia rats].

OBJECTIVE: To observe the effect of oligosaccharides of Morinda officinalis (OMO) on beta-amyloid-induced dementia rats, and study its pharmacological mechanism in treatment of dementia. METHOD: The dementia model rats were established by injecting Abeta25-35 10 microLg into bilateral hippocampus. OMO high-dose (60 mg . kg-1 . d-1) group, OMO low-dose (20 mg . kg-1 . d-1 ) groups, the blank group, the sham operation group and the positive donepezil HC1 group (0. 125 mg kg-1 . d-1) were designed for the experiment. They were continuously administered with drugs at the 15th day after operation for 25 days. Kit microplate method was used to detect the contents of super oxide dismutase (SOD), malondialdehyde (MDA), catalase (CAT), glutathione reductase (GSH-Px), acetylcholine (ACh) , acetylcholinesterase (AChE) and Na+ /K+ -ATPase. RESULT: Compared with the model group, all of administration groups showed higher SOD, CAT and GSH-Px levels, and lower MDA in the brain tissues. Besides, they also showed rise in the activities of ACh and Na+ /K+ -ATPase. CONCLUSION: OMO can ameliorate on beta-amyloid-induced dementia rats by enhancing oxidation resistance, activating brain energy metabolism and improving the injury of cholinergic system.

Protective effect of Bajijiasu against ß-amyloid-induced neurotoxicity in PC12 cells.

Beta-amyloid peptide (Aß), a major protein component of senile plaques associated with Alzheimer's disease (AD), is also directly neurotoxic. Mitigation of Aß-induced neurotoxicity is thus a possible therapeutic approach to delay or prevent onset and progression of AD. This study evaluated the protective effect of Bajijiasu (ß- D-fructofuranosyl (2-2) ß- D-fructofuranosyl), a dimeric fructose isolated from the Chinese herb Radix Morinda officinalis, on Aß-induced neurotoxicity in pheochromocytoma (PC12) cells. Bajijiasu alone had no endogenous neurotoxicity up to 200 µM. Brief pretreatment with 10-40 µM Bajijiasu (2 h) significantly reversed the reduction in cell viability induced by subsequent 24 h exposure to Aß25-35 (21 µM) as measured by MTT and LDH assays, and reduced Aß25-35-induced apoptosis as indicated by reduced annexin V-EGFP staining. Bajijiasu also decreased the accumulation of intracellular reactive oxygen species and the lipid peroxidation product malondialdehyde in PC12 cells, upregulated expression of glutathione reductase and superoxide dismutase, prevented depolarization of the mitochondrial membrane potential (Ψm), and blocked Aß25-35-induced increases in [Ca(2+)] i . Furthermore, Bajijiasu reversed Aß25-35-induced changes in the expression levels of p21, CDK4, E2F1, Bax, NF-κB p65, and caspase-3. Bajijiasu is neuroprotective against Aß25-35-induced neurotoxicity in PC12 cells, likely by protecting against oxidative stress and ensuing apoptosis.