OBJECTIVE: To establish the method of quality control for traditional Tibetan Medicine Zsuotai.METHODS: Collecting the samples of Tsuotai from Qinghai, Tibet, Sichuan, and Gansu province, to detect Hg2+ by Zsuotai reacted with HCl-HNO3 (3:1), and to determine the quantity of HgS in Zsuotai by sulfocyanate volumetric method.
RESULTS: The method for the determination of HgS in Zsuotai was in good reproducibility (RSD = 0.68%). The calibration curve was linear (r = 0.9999) within -0.0002 - 0.2123 g of mercuric sulfide. The recovery was 100.94% (RSD = 0.66%).
CONCLUSIONS: This method is convenient and accurate, so it can be used to establish quality control of the medicinal material.
In this work, a hyphenated technique of dual ultrasound-assisted dispersive liquid-liquid microextraction combined with microwave-assisted derivatization followed by ultra high performance liquid chromatography tandem mass spectrometry has been developed for the determination of phytosterols in functional foods and medicinal herbs. Multiple reaction monitoring mode was used for the tandem mass spectrometry detection. A mass spectrometry sensitive reagent, 4'-carboxy-substituted rosamine, has been used as the derivatization reagent for five phytosterols, and internal standard diosgenin was used for the first time. Parameters for the dual microextraction, microwave-assisted derivatization, and ultra high performance liquid chromatography tandem mass spectrometry were all optimized in detail. Satisfactory linearity, recovery, repeatability, accuracy and precision, absence of matrix effect, extremely low limits of detection (0.005-0.015 ng/mL) and limits of quantification (0.030-0.10 ng/mL) were achieved. The proposed method was compared with previously reported methods. It showed better sensitivity, selectivity, and accuracy. The matrix effect was also significantly reduced. The proposed method was successfully applied to the determination of five phytosterols in vegetable oil (sunflower oil, olive oil, corn oil, peanut oil), milk and orange juice (soymilk, peanut milk, orange juice), and medicinal herbs (Ginseng, Ganoderma lucidum, Cordyceps, Polygonum multiflorum) for the quality control of functional foods and medicinal herbs.
ETHNOPHARMACOLOGICAL RELEVANCE: Rhodiola crenulata, a traditional Tibetan medicine, has shown promise in the treatment of hypobaric hypoxia (HH)-induced brain injury. However, the underlying mechanisms remain unclear. This study investigated the protective effects of R. crenulata aqueous extract (RCAE) on HH-induced brain injury in rats.MATERIALS AND METHODS: An animal model of high-altitude hypoxic brain injury was established in SD rats using an animal decompression chamber for 24 h. Serum and hippocampus levels of superoxide dismutase (SOD), malondialdehyde (MDA), glutathione (GSH), oxidized glutathione (GSSG), and lactate dehydrogenase (LDH) were then determined using commercial biochemical kits. Neuron morphology and vitality were also evaluated using H&E and Nissl staining, and TUNEL staining was used to examine apoptosis. Gene and protein expression of HIF-1α, microRNA 210, ISCU1/2, COX10, Apaf-1, cleaved Caspase-3, Caspase-3, Bax, Bcl-2, and Cyto-c were determined by western blot, immunohistochemical and qRT-PCR analysis.
RESULTS: RCAE administration attenuated HH-induced brain injury as evidenced by decreased levels of MDA, LDH, and GSSG, increased GSH and SOD, improvements in hippocampus histopathological changes, increased cell vitality and ATP level, and reduced apoptotic cell numbers. RCAE treatment also enhanced HIF-1α, ISCU1/2, COX10, and Bcl-2 protein expression, while dramatically inhibiting expression of Apaf-1, Bax, Cyto-c, and cleaved Caspase-3. Treatment also increased gene levels of HIF-1α, microRNA 210, ISCU1/2, and COX10, and decreased Caspase-3 gene production.
CONCLUSIONS: RCAE attenuated HH-induced brain injury by regulating apoptosis and mitochondrial energy metabolism via the HIF-1α/microRNA 210/ISCU1/2 (COX10) signaling pathway.
Identifying and protecting “keystone structures” is essential to maintain biodiversity in an increasingly human-dominated world. Sacred forests, i.e. natural areas protected by local people for cultural or religious regions, may be keystone structures for forest birds in the Greater Himalayas, but there is limited understanding of their use by bird communities. We surveyed birds and their habitat in and adjacent to six Tibetan sacred forests in northwest Yunnan China, a biodiversity hotspot. Our goal was to understand the ecological and conservation role of these remnant forest patches for forest birds. We found that sacred forests supported a different bird community than the surrounding matrix, and had higher bird species richness at plot, patch, and landscape scales. While we encountered a homogeneous matrix bird community outside the scared forests, the sacred forests themselves exhibited high heterogeneity, and supported at least two distinct bird communities. While bird community composition was primarily driven by the vegetation vertical structure, plots with the largest-diameter trees and native bamboo groves had the highest bird diversity, indicating that protecting forest ecosystems with old-growth characteristics is important for Himalayan forest birds. Finally, we found an increased bird use of the sacred forests and their edges during 2010, a severe drought year in Yunnan, indicating that sacred forests may serve as refuges during extreme weather years. Our results strongly indicate that sacred forests represent an important opportunity for Himalayan bird conservation because they protect a variety of habitat niches and increase bird diversity at multiple spatial scales.
Sub-acute and chronic toxic effects of total steroidal saponins (TSSN) extracts from Dioscorea zingiberensis C.H. Wright on various internal organs and biochemical indicators have never been studied before and this study is the first of its kind to demonstrate sub-acute and chronic toxicities of TSSN on dogs. Administration of TSSN extracts at doses up to 3000 mg/Kg daily for 14 days, no biochemical and organ changes were observed on the experimental groups of dogs. Further, chronic toxicity study through oral administration of TSSN extracts at the gradual doses of 50, 250 and 500 mg/Kg for 90 days followed by a 2-week recovery assay revealed absence of significant architectural and morphological changes in internal organs which were confirmed through histopathological examination and merely no significant alteration in the biochemical indicators including hematologic and urine analysis and electrocardiogram compared to the control dogs. This toxicological evaluation came across with the finding that the herbal preparation can be considered as nontoxic and animals could tolerate the extracts at doses up to 500 mg/Kg with LD50 greater than 3000 mg/Kg. It may serve as a preliminary scientific evidence for further therapeutic investigations.
Sub-acute and chronic toxic effects of total steroidal saponins (TSSN) extracts from Dioscorea zingiberensis C.H. Wright on various internal organs and biochemical indicators have never been studied before and this study is the first of its kind to demonstrate sub-acute and chronic toxicities of TSSN on dogs. Administration of TSSN extracts at doses up to 3000 mg/Kg daily for 14 days, no biochemical and organ changes were observed on the experimental groups of dogs. Further, chronic toxicity study through oral administration of TSSN extracts at the gradual doses of 50, 250 and 500 mg/Kg for 90 days followed by a 2-week recovery assay revealed absence of significant architectural and morphological changes in internal organs which were confirmed through histopathological examination and merely no significant alteration in the biochemical indicators including hematologic and urine analysis and electrocardiogram compared to the control dogs. This toxicological evaluation came across with the finding that the herbal preparation can be considered as nontoxic and animals could tolerate the extracts at doses up to 500 mg/Kg with LD50 greater than 3000 mg/Kg. It may serve as a preliminary scientific evidence for further therapeutic investigations.
Erigeron multiradiatus (Lindl.) Benth is a traditional Tibetan medicine herb long used to treat various diseases related to inflammation. Our previous phytochemical studies on E. multiradiatus resulted in the isolation of scutellarin, which is a known flavone glucuronide with comprehensive pharmacological actions. In present study, we investigated the inhibition action of scutellarin on high glucose-induced vascular inflammation in human endothelial cells (ECV304 cells). Consistent with previous reports, exposure of ECV304 cells to high glucose for 24 h caused an increase of intercellular adhesion molecule-1 (ICAM-1) and monocyte chemoattractant protein 1 (MCP-1), and promoted cell adhesion between monocyte and ECV304 cells. However, pretreatment with scutellarin (0.1 and 1 microM) reversed these effects in a concentration-dependent manner. Scutellarin was able to inhibit the activation of NF-kappaB induced by high glucose in ECV304 cells. Furthermore, although oral administration of scutellarin (10 and 50 mg/kg) did not produce significant antihyperglycemic action, it lowered the serum MCP-1 levels significantly in alloxan-induced diabetic mice. Therefore, our results suggest that scutellarin has anti-inflammation effect that may afford some protection against hyperglycemia-induced vascular inflammatory both in vitro and in vivo.
A reversed-phase high-performance liquid chromatographic method with diode array detection was established to simultaneously determine the seven bioactive lignans in <i>Herpetospermum caudigerum</i>, namely ent-isolariciresinol (<b>1</b>), dehydrodiconiferyl alcohol (<b>2</b>), herpetrione (<b>3</b>), herpetin (<b>4</b>), herpetetrone (<b>5</b>), herpetotriol (<b>6</b>) amd herpetal (<b>7</b>). The HPLC assay was performed on a Restek Pinnacle DB C<sub>18</sub> column (250 × 4.6 mm, 5 µm) with gradient elution of acetonitrile and 0.1% phosphoric acid within 65 min. The detection wavelength was 240 nm. The flow-rate was 1.0 mL/min. All calibration curves showed good linearity (<i>r</i>² > 0.9998) within test ranges. The method was reproducible with intra- and inter-day variation of less than 1.98%. The method provided good accuracy with recoveries in the range 95.19-102.64% with RSDs less than 1.52%. The method was successfully applied to the quantification of seven constituents in 15 <i>H. caudigerum</i> samples collected from different cities. The results indicated that the developed assay could be considered as a suitable quality control method for <i>H. caudigerum</i>. Copyright © 2008 John Wiley & Sons, Ltd.
Purpose: To develop an ultra-high performance liquid chromatography (UPLC) - photodiode array (PDA) method to compare the chemical composition of two different medicinal components of Pterocephalus hookeri. Methods: Samples were chromatographically separated in succession using Waters Acquity UPLCR BEH C18 column (2.1 × 100 mm, 1.7 µm) and gradient elution (0.2% phosphoric acid aqueous - acetonitrile). Using partial least squares discriminant analysis and one-way analysis of variance, attempts were made to distinguish different medicinal parts of P. hookeri. Results: Regression equation for 10 compounds showed good linear regression (R² > 0.9994). The relative standard deviations of precision, stability, repeatability and recovery were under 5%. Compared with the aerial plant part, the root had significantly higher levels of sylvestroside I (p < 0.01), cantleyoside (p < 0.001), dipsanosides B (p < 0.01) and dipsanosides A (p < 0.01), but significantly lower levels of loganic acid (p < 0.001), chlorogenic acid (p < 0.01), and isochlorogenic acid (p < 0.01). There were no significant differences between loganin, sweroside and isochlorogenic acid C. Conclusion: The described method is simple, accurate and reproducible, and can be used for the simultaneous determination of 10 major compounds of P. hookeri. The results demonstrate that there is variation in the chemical composition of the aerialpart and root of P. hookeri and that loganic acid and cantleyoside are the primary chemical biomarkers.
This study is to develop an UPLC-PDA method for determination of 10 major components in Pterocephalus. The UPLC-PDA assay was performed on a Waters Acquity UPLCR BEH C₁₈（2.1 mm ×100 mm,1.7 μm）, and the column temperature was at 30 ℃. The mobile phase consists of water containing 0.2% phosphoric acid (A) and acetonitrile (B) in gradient elution at a flow rate of 0.4 mL•min⁻¹. The detection wave length was set at 237 and 325 nm, and the injection volume was 1 μL in the UPLC system. The linear range of 10 detected compounds were good (r≥0.999 7), and the overall recoveries ranged from 96.30% to 103.0%, with the RSD ranging from 0.72% to 2.9%. The method was simple, accurate and reproducible, which can be used for the simultaneous determination of the content of ten major components in P. hookeri.
• In situ derivatization-UADLLME was firstly reported for NTs in rat brain microdialysates. • Lissamine rhodamine B sulfonyl chloride was firstly used as derivatization reagent. • The method was simple, rapid, green, efficient, sensitive and low matrix effect. • This method was successfully applied for Parkinson’s rat brain microdialysates.<br>Simultaneous monitoring of several neurotransmitters (NTs) linked to Parkinson’s disease (PD) has important scientific significance for PD related pathology, pharmacology and drug screening. A new simple, fast and sensitive analytical method, based on in situ derivatization-ultrasound-assisted dispersive liquid-liquid microextraction (in situ DUADLLME) in a single step, has been proposed for the quantitative determination of catecholamines and their biosynthesis precursors and metabolites in rat brain microdialysates. The method involved the rapid injection of the mixture of low toxic bromobenzene (extractant) and acetonitrile (dispersant), which containing commercial Lissamine rhodamine B sulfonyl chloride (LRSC) as derivatization reagent, into the aqueous phase of sample and buffer, and the following in situ DUADLLME procedure. After centrifugation, 50 μL of the sedimented phase (bromobenzene) was directly injected for ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) detection in multiple reaction monitoring (MRM) mode. This interesting combination brought the advantages of speediness, simpleness, low matrix effects and high sensitivity in an effective way. Parameters of in situ DUADLLME and UHPLC-MS/MS conditions were all optimized in detail. The optimum conditions of in situ DUADLLME were found to be 30 μL of microdialysates, 150 μL of acetonitrile containing LRSC, 50 μL of bromobenzene and 800 μL of NaHCO3-Na2CO3 buffer (pH 10.5) for 3.0 min at 37 °C. Under the optimized conditions, good linearity was observed with LODs (S/N > 3) and LOQs (S/N > 10) of LRSC derivatized-NTs in the range of 0.002-0.004 and 0.007-0.015 nmol/L, respectively. It also brought good precision (3.2-12.8%, peak area CVs%), accuracy (94.2-108.6%), recovery (94.5-105.5%) and stability (3.8-8.1%, peak area CVs%) results. Moreover, LRSC derivatization significantly improved chromatographic resolution and MS detection sensitivity of NTs when compared with the reported studies through the introduction of a permanent charged moiety from LRSC into NTs. Taken together, this in situ DUADLLME method was successfully applied for the simultaneous determination of six NTs in biological samples.
To evaluate the efficacy and safety associated with anti-hypoxia effect and establish the quality standard for Brassicea Radix extract, the investigations of acute toxicity and subacute toxicity were carried out to preliminarily appraise the toxicity, and the models of normal pressure hypoxia, acute cerebral ischemia and sodium nitrite poisoning in mice were used to evaluate the effect of enhancing anoxia endurance. Then according to the methods described in the Appendix of Chinese Pharmacopoeia (2010 edition), the sulfuric acid-phenol method was applied to determine the content of polysaccharide, and the water, ash and insoluble matter in water inspections were carried out and the control medicinal herb was identified with the samples by qualitative TLC. The results indicated that ① the toxic effects (LD₅₀) of mice was 56.73 g•kg⁻¹ by oral administration of Brassicea Radix extract, while Dm and Dn were respective 86.80 g•kg•d⁻¹ and 35.55 g•kg•d⁻¹;②the determined effective dosage of Brassicea Radix extract which could enhance anoxia endurance was 0.388 g•kg⁻¹•d⁻¹; ③ the methods of TLC and the content of polysaccharide were established. The method of quality control has been recorded in Sichuan Province Standard for Tibetan Medicine, which is reliable, accurate and simple, with good reproducibility. Meanwhile, given the prominent effect on anti-hypoxia and good safety, it provided important basis for clinic safe and effective usage and the development of health products.
Objective: To study the chemical constituents of the volatile oils from the flowers of Rhododendron anthopogon D. Don.; Methods: The volatile oils from the flowers of Rhododendron anthopogon D. Don were extracted by water steam distillation and its chemical constituents were separated and identified by GC-MS. The content of each constituent was determined by area normalizetion method.; Results: Fifty peaks were separated and forty seven components were identified, which constituted about 97.44% of the total essential oils.; Conclusions: The main compounds are N-acetyl-1, 2, 3, 4-tetrahydro-isoquinoline (29.23%), 2-Ethoxypropane (12.47%), 3-Methyl-6-tert-butylphenol (10.83%), 3-Methyl-5-phenyl-isothiazole (6.38%), Diphenylamine (4.20%), N-ethyl-1, 2,3,4-tetrahydro-naphthalenamine (3.62%), Pentacosane (3.12%) and Tricosane (3.06%).;
Zuotai (gTso thal) is a typical representative of Tibetan medicines containing heavy metals, but there is still lack of modem safety evaluation data so far. In this study, acute toxicity test, sub-acute toxicity test, one-time administration mercury distribution experiment, long-term mercury accumulative toxicity experiment and preliminary study on clinical safety of Compound Dangzuo were conducted in the hope of obtain the medicinal safety data of Zuotai. In the acute toxicity test, half of KM mice given the lethal dose of Zuotai were not died or poisoned, and LD50 was not found. The maximum tolerated dose of Zuotai was 80 g x kg(-1). In the subacute toxicity test, Zuotai could reduce ALT, AST, Crea levels in serums under low dose (13.34 mg x kg(-1) x d(-1)) and medium dose (53.36 mg x kg(-1) x d(-1)), with significant difference under low dose, and increase the levels of ALT, AST, MDA, Crea in serums under high dose (2 000 mg x kg(-1) x d(-1)); besides, the levels of BUN and GSH in serums reduced with the increase in dose of Zuotai, indicating a significant dose-effect relationship. In the one-time administration distribution experiment, the content of mercury in rat kidney, liver and lung increased after the one-time administration with Zuotai, with a significant dose-dependent relationship in kidney. In the long-term mercury accumulative toxicity experiment, KM mice were administered with equivalent doses of Zuotai for 4.5 months and then stopped drug administration for 1.5 months. Since the 2.5th month, they showed significant mercury accumulation in kidney, which gradually reduced after drug withdrawal, without significant change in mercury content in liver, spleen and brain and ALT, AST, TBIL, BUN and Crea in serum. At the 4.5th month after drug administration, KM mice showed slight structural changes in kidney, liver and spleen tissues, and gradually recovered to normal after drug withdrawal. Besides, no significant difference in weight gain was found between the Zuotai group and the control group. According to the findings of the clinical safety study of Dangzuo, after subjects administered Dangzuo under clinical dose for one month, their serum biochemical indicators, blood routine indicators and urine routine indicators showed no significant adverse change. This study proved that traditional Tibetan medicine Zuotai was slightly toxic, with a better safety in clinical combined administration and no adverse effects on bodies under the clinical dose and clinical medication cycle. However, long-term high-dose administration of Zuotai may have a certain effect on kidney.;
Two new prenylated indole diterpenoids, tolypocladins K and L (1 and 2), together with a known analog terpendole L (3), were isolated from the solid fermentation culture of a mine soil-derived fungus Tolypocladium sp. XL115. Their structures and relative configurations were determined by comprehensive spectroscopic data analysis, as well as by comparison of their NMR data with those related known compounds. Compound 3 exhibited remarkable antibacterial activity against Micrococcus luteus with an MIC value of 6.25 μg/mL, and compounds 1 and 3 displayed moderate antifungal activity selectively against tested strains with MIC values of 25-50 μg/mL.
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