Zizyphi Spinosi Semen Research Articles

Comparative Study of the Bioactive Constituents of Raw and Ripe Zizyphi Spinosi Semen

High-performance liquid chromatographic-tandem mass spectrometric (HPLC-MS-MS) methods were developed for the analysis of 13 bioactive constituents of Zizyphi Spinosae Semen ( ZSS), and for samples of ZSS fried at different temperatures and from different production areas. The results showed that the content of bioactive constituents increased with the increase in frying time from 65 to 80 min at 150 °C; among the 14 samples of ZSS from different production areas, those from Shanxi Changzhi, Shandong Jining, and Hebei Xingtai had a higher content of bioactive constituents. This analytical method can accurately and quickly analyze the bioactive constituents of ZSS and provide a scientific basis for its quality control.

Chromatographic Determination of the Absolute Configuration in Sanjoinine A That Increases Nitric Oxide Production.

A chiral derivatization strategy with phenylglycine methyl ester (PGME) was employed to develop a straightforward method to determine the absolute configurations of N,N-dimethyl amino acids. The PGME derivatives were analyzed using liquid chromatography-mass spectrometry to identify the absolute configurations of various N,N-dimethyl amino acids based on their elution time and order. The established method was applied to assign the absolute configuration of the N,N-dimethyl phenylalanine in sanjoinine A (4), a cyclopeptide alkaloid isolated from Zizyphi Spinosi Semen widely used as herbal medicine for insomnia. Sanjoinine A displayed production of nitric oxide (NO) in LPS-activated RAW 264.7 cells.

Anxiolytic-like effect of Suanzaoren–Wuweizi herb-pair and evidence for the involvement of the monoaminergic system in mice based on network pharmacology

BackgroundSuanzaoren-Wuweizi herb-pair (SWHP), composed of Zizyphi Spinosi Semen (Suanzaoren in Chinese) and Schisandrae Chinensis Fructus (Wuweizi in Chinese), is a traditional herbal formula that has been extensively used for the treatment of insomnia. The study aimed to explore the targets and signal pathways of Suanzaoren-Wuweizi (S-W) in the treatment of anxiety by network pharmacology, and to verify the pharmacodynamics and key targets of SWHP in mice.MethodsThe Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) as well as literature mining were used to obtain the main chemical ingredients of Suanzaoren and Wuweizi. The SwissTargetPrediction platform was used to predict drug-related targets. The GeneCards, TTD, DisGeNET and OMIM databases were used to obtain potential targets for the treatment of anxiety with the chemical components of S-W. Drug-disease intersection genes were selected, and a protein-protein interaction (PPI) network was constructed using STRING. The core targets of S-W in the treatment of anxiety were selected according to the topological parameters, and GO functional enrichment as well as KEGG pathways enrichment analyses were performed for potential targets. The relationship network of the “drug-active ingredient-disease-target-pathway” was constructed through Cytoscape 3.8.0. The pharmacodynamics of SWHP in the treatment of anxiety was evaluated by the elevated plus maze (EPM), the light/dark box test (LDB) and the open field test (OFT). The mechanisms were examined by measuring monoamine neurotransmitters in brain of mice.ResultsThe results showed that there were 13 active ingredients for the treatment of anxiety in the network. This includes sanjoinenine, swertisin, daucosterol, schizandrer B, wuweizisu C and gomisin-A. Additionally, there were 148 targets, such as AKT1, TNF, SLC6A4, SLC6A3, EGFR, ESR1, HSP90AA1, CCND1, and DRD2, mainly involved in neuroactive ligand-receptor interactions, the Serotonergic synapse pathway and the cAMP signaling pathway. After 1 week of treatment, SWHP (2 and 3 g/kg) induced a significant increase on the percentage of entries into and time spent on the open arms of the EPM. In the LDB test, SWHP exerted anxiolytic-like effect at 2 g/kg. In the open-field test, SWHP (2 g/kg) increased the number of central entries and time spent in central areas. The levels of brain monoamines (5-HT and DA) and their metabolites (5-HIAA, DOPAC) were decreased after SWHP treatment.ConclusionsThe anti-anxiety effect of SWHP may be mediated by regulating 5-HT, DA and other signaling pathways. These findings demonstrated that SWHP produced an anxiolytic-like effect and the mechanism of action involves the serotonergic and dopaminergic systems, although underlying mechanism remains to be further elucidated.

Jujuboside B Inhibited High Mobility Group Box Protein 1-Mediated Severe Inflammatory Responses in Human Endothelial Cells and Mice.

High mobility group box protein 1 (HMGB1) is a biomolecule that acts as an alerting signal of late sepsis by accelerating the production of proinflammatory cytokines, and eventually leads to various inflammation-related symptoms. When released into plasma at high concentration, it disrupts precise diagnosis and prognosis and worsens the survival of patients with systemic inflammatory conditions. Jujuboside B (JB) is a natural compound pressed from the seed of Zizyphi Spinosi Semen, which is known for its medical efficacies in treating various conditions such as hyperlipidemia, hypoxia, and platelet aggregation. Nevertheless, the medicinal activity of JB on HMGB1-involved inflammatory response in vascular cells in the human body is still ambiguous. Therefore, we hypothesized that JB could regulate the lipopolysaccharide (LPS)-induced dynamics of HMGB1 and its mediated cascade in inflammatory responses in human umbilical vein endothelial cells (HUVECs). In this experiment, JB and HMGB1 were administered in that order. In vitro and in vivo permeability, and cell viability, adhesion, and excavation of leukocytes, development of cell adhesion molecules, and lastly production of proinflammatory substances were investigated on human endothelial cells and mouse disease models to investigate the efficacy of JB in inflammatory condition. JB substantially blocked the translocation of HMGB1 from HUVECs and controlled HMGB1-induced adhesion and extravasation of the neutrophils through LPS-treated HUVECs. Moreover, JB decreased the formation of HMGB1 receptors and continually prevented HMGB1-induced proinflammatory mechanisms by blocking transcription of nuclear factor-κB and synthesis of tumor necrosis factor-α. In conclusion, JB demonstrated preventive effects against inflammatory pathologies and showed the potential to be a candidate substance for various inflammatory diseases by regulating HMGB1-mediated cellular signaling.

Hepatoprotective functions of jujuboside B.

Jujuboside B (JB) found in the seeds of Zizyphi Spinosi Semen possesses pharmacological functions, such as anti-inflammatory, antiplatelet aggregation, and antianxiety potentials. This study evaluated the effect of JB on liver failure in cecal ligation and puncture (CLP)-induced sepsis. First, we observed histopathological changes in the liver by optical microscopy and the activity of enzymes in serum such as alanine aminotransferase (ALT) and aspartate aminotransferase (AST). We further measured the levels of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, nitric oxide (NO), and antioxidative parameters in liver homogenate. The expression of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2), and glucocorticoid receptor (GR) in the liver was observed by Western blotting. CLP enhanced the migration of inflammatory cells, ALT and AST concentrations, and necrosis, which were reduced by JB. In addition, JB reduced 11β-HSD2 expression and levels of inflammatory mediators (TNF-α, IL-1β, and NO) in the liver, increased GR expression, enhanced endogenous antioxidative capacity. These results further suggest that JB may protect the liver against CLP-induced damage by regulating anti-inflammatory responses, downregulating 11β-HSD2 expression and antioxidation, and up-regulating GR expression.

Jujuboside B post-treatment attenuates PM2.5-induced lung injury in mice

ABSTRACT Fine particulate matter (PM2.5) is an air pollutant that causes severe lung injury. We investigated the effects of Jujuboside B (JB), a component of Zizyphi Spinosi Semen, on lung toxicity caused by PM2.5, and we identified the mechanism of its protective effect. Lung injury in an animal model was induced by intratracheal administration of a PM2.5 suspension. After 2 days of PM2.5 pretreatment, mice were administered JB via the tail vein three times over a 2-day period. JB significantly reduced the histological lung damage as well as the lung wet/dry weight ratio. JB also considerably reduced PM2.5-induced autophagy dysfunction, apoptosis, inflammatory cytokine levels, and the number of PM2.5-induced lymphocytes in the bronchial alveolar fluid. We conclude that by regulating TLR2, 4-MyD88, and mTOR-autophagy pathways, JB exerts a protective effect on lung injury. Thus, JB can be used as a potential therapeutic agent for PM2.5-induced lung damage.

Jujuboside B Inhibits the Proliferation of Breast Cancer Cell Lines by Inducing Apoptosis and Autophagy.

Jujuboside B (JB) is one of the main biologically active ingredients extracted from Zizyphi Spinosi Semen (ZSS), a widely used traditional Chinese medicine for treating insomnia and anxiety. Breast cancer is the most common cancer and the second leading cause of cancer-related death in women worldwide. The purpose of this study was to examine whether JB could prevent breast cancer and its underlying mechanism. First, we reported that JB induced apoptosis and autophagy in MDA-MB-231 and MCF-7 human breast cancer cell lines. Further mechanistic studies have revealed that JB-induced apoptosis was mediated by NOXA in both two cell lines. Moreover, the AMPK signaling pathway plays an important role in JB-induced autophagy in MCF-7. To confirm the anti-breast cancer effect of JB, the interaction of JB-induced apoptosis and autophagy was investigated by both pharmacological and genetic approaches. Results indicated that autophagy played a pro-survival role in attenuating apoptosis. Further in vivo study showed that JB significantly suppressed the growth of MDA-MB-231 and MCF-7 xenografts. In conclusion, our findings indicate that JB exerts its anti-breast cancer effect in association with the induction of apoptosis and autophagy.

Mechanism of the Intestinal Absorption of Six Flavonoids from Zizyphi Spinosi Semen Across Caco-2 Cell Monolayer Model.

Flavonoid compounds are one kind of active ingredients isolated from a traditional Chinese herb Zizyphi spinosae semen (ZSS). Studies have shown that ZSS flavonoids have significant antioxidant effects. In this study, the Caco-2 cell monolayer model was constructed to investigate the intestinal absorption characteristics and mechanism of Isovitexin (IV), Swertisin (ST), Isovitexin-2''-O-β-D-glucopyranoside (IVG), Spinosin (S), 6'''-p-coumaroylspinosin (6-CS) and 6'''-feruloylspinosin (6-FS). The results of the bidirectional transport assay showed that the six flavonoids have good intestinal absorption in a near-neutral and 37°C environment, and the absorbability in descending order was 6-FS>6- CS>IVG>S>IV>ST. The results of carrier inhibition experiments and transport kinetics indicated that the absorption mechanism of six flavonoids was energy-dependent monocarboxylate transporter (MCT)-mediated active transport. In particular, the para-cellular pathway also participated in the transport of IV, ST, IVG and S. Furthermore, the efflux process of six flavonoids was mediated by P-glycoprotein (P-gp) and multidrug resistance protein (MRP), which may result in a decrease of bioavailability. Our findings provide significant information for revealing the relationship between the intestinal absorption mechanism of flavonoids and its structure as well as laying a basis for the research of flavonoid preparations.

Jujuboside B promotes the death of acute leukemia cell in a RIPK1/RIPK3/MLKL pathway-dependent manner

Initiation of necroptosis has been considered as a promising strategy for anticancer therapies, especially for eradicating apoptosis-resistant malignant cells. Jujubisode B is a natural saponins extracted from the seeds of Zizyphi Spinosi Semen, and possesses multiple pharmacological activities, including antianxiety, anti-inflammation, antiplatelet aggregation and induction of apoptosis. This study aims to explore the effect of jujuboside B on acute leukemic cells and the underlying mechanisms. Our results showed that jujuboside B inhibited leukemia cell growth in a dose-dependent manner and attenuated the clonogenic ability of U937 cells, concomitant with activation of RIPK1/RIPK3/MLKL pathway; these phenomena were evidently blocked by necroptosis inhibitor (Nec-1). With the help of Molecular Operating Environment (MOE) program, we identified that RIPK1, RIPK3 and MLKL are potential targets of jujuboside B. To the best of our knowledge, this is the first study to provide evidence that jujuboside B possesses antileukemic activity via a mechanism involving activation of RIPK1/RIPK3/MLKL pathway.

Separation and Purification of Zizyphusine, Spinosin, and 6′′′-Feruloylspinosin From Zizyphi Spinosi Semen

The methods of extraction, separation, and purification of zizyphusine, spinosin, and 6′′′-feruloylspinosin from Zizyphi Spinosi Semen ( ZSS) were investigated. From 31.2 g defatted ZSS, 47.7 mg zizyphusine, 57.8 mg spinosin, and 80.5 mg 6′′′-feruloylspinosin were obtained after ultrasonic extraction, purification with macroporous resin, separation by flash chromatography, and purification by high-pressure preparative chromatography. The purities of zizyphusine, spinosin, and 6′′′-feruloylspinosin were 98.6%, 98.2%, and 99.0%, respectively, and their yields 85.0%, 82.8%, and 83.0%. The methods provide stable samples of these compounds for further study of their physiological function and application.

The pharmacokinetics and tissue distribution of coumaroylspinosin in rat: A novel flavone C-glycoside derived from Zizyphi Spinosi Semen.

Zizyphi Spinosi Semen (ZSS) has a long history of sedative-hypnotic use in China. As a novel flavone C-glycoside, coumaroylspinosin is a main flavonoid only found in ZSS. Up to now, its pharmacokinetic information and tissue distribution in vivo are not available yet. With a simple, rapid and sensitive HPLC-MS/MS method, the pharmacokinetics and tissue distribution of coumaroylspinosin were investigated in Sprague-Dawley (SD) rats after its intravenous administration. Puerarin was used as the internal standard (IS). The samples were extracted by a simple protein precipitation method with methanol. The MS analysis was performed with multiple reaction monitoring (MRM), and the transitions were set at m/z 753.3→427.0 for coumaroylspinosin and m/z 415.3→295.3 for IS, respectively. The method was successfully applied for investigating the pharmacokinetics and tissue distribution of coumaroylspinosin in Sprague Dawley (SD) rats after tail vein injection with 4.0mg/kg of the flavonoid. The calibration curves covered over the range of 0.02-10μg/mL in plasma and various tissues samples with good linearity(r≥0.9956). The lower limit of quantification (LLOQ) in all samples was less than 20ng/mL. The intra- and inter-day precisions were below 15% and accuracy was from -3.78% to 4.68%. No significant matrix effect was observed, and the average extraction recovery was acceptable. Coumaroylspinosin could be cleared quickly from the rat plasma with the half-life (t1/2) of 1.86±0.15h. It was distribute widely in vivo, and the main tissue depots of coumaroylspinosin in rats were found to be intestine, muscle and lung. With the method, the pharmacokinetic parameters and tissue distribution of coumaroylspinosin in SD rats were investigated for the first time. The results demonstrated that coumaroylspinosin was distributed widely and rapidly in various rat tissues after intravenous administration.

Tissue Distribution of Jujuboside A in Sprague-Dawley Rats Determined by an Efficient HPLC–ESI-MS/MS Method

Jujuboside A (JuA), a dammarane-type triterpene glycoside, is a main bioactive saponin in Zizyphi Spinosi Semen (a traditional Chinese herbal food). In this research, the distribution of JuA in Sprague-Dawley rats was investigated with a new and efficient HPLC–ESI-MS/MS method. The results showed that JuA distributed rapidly and widely in various rat tissues (including heart, liver, spleen, kidney, and lung) after intravenous administration. In addition, it was initially found that JuA could pass through the blood-brain barrier and reach various areas of the brain quickly. At 0.25 hr, the concentration of JuA in the hippocampus (204.10 ng · g−1) was significantly higher than in the cerebrum (144.27 ng · g−1), olfactory bulb (98.42 ng · g−1), and corpus striatum (76.04 ng · g−1) (p < 0.05), indicating that the hippocampus was the main accumulation area of JuA in the brain.

불면증에서 산조인의 유무에 따른 약물배오의 경향성 고찰

Objectives : Zizyphi spinosi Semen is widely used for treating insomnia in korea and other oriental country. This study is analyzing combination tendency of Zizyphi spinosi Semen in herbal formula for treating insomnia. Methods : Herbal formulas for treating insomia were searched in "The Prescription Dictionary of Traditional Chinese Medicine", and checked in each literary source. Then, the herbal formula was divided in two groups : FCZ(The formula group containing Zizyphi spinosi Semen) and FWZ(The formula group without Zizyphi spinosi Semen). FCZ and FWZ were compared by following criteria; i) Prevalence of herbal formula by period ii) Prevalence of medicinal material's effects iii) Prevalence of medicinal material iv) Prevalence of the nature of midicinals v) Prevalence of flavor of medicinals vi) Prevalence of meridian entry. Results : 116 Herbal formula met our criteria, and herbal formulas were divided in two groups : FCZ(n=59), FWZ(n=57). The result of prevalence in FCZ is following; Herbal formula prevalence increased as time passed to the Qing(淸) dynasty. Tonifying and replenishing medicinal(補益藥), tranquillizing medicinal (安神藥), and inducing diuresis to drain dampness medicinal(利水滲濕藥) are the three most prevalent types of materia medica. In specifically, Panax ginseng radix(人蔘), Liriopis Tuber(麥門冬), Angelicae Gigantis Radix(當歸), Poria Cocos(茯神), Glycyrrhizae Radix(甘草), Poria cocos(茯神), Polygalae Radix(遠志), Thuja Orientalis L.(柏子仁), Paeonia Japonica(白芍藥), and HgS(硃砂) are combined frequently. The most prevalent nature of medicinals were 'warm microwarm(溫 微溫)' and flavor of medicinals was normal(平). Meridian entrys that frequently combined were heart(心), spleen(脾), and liver(肝). Conclusion : We found combination tendency of Zizyphi Semen in herbal formula for treating insomnia.

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY COUPLED WITH TANDEM MASS SPECTROMETRY APPLIED FOR METABOLIC STUDY OF SPINOSIN BY RAT INTESTINAL FLORA

A rapid, sensitive, and selective liquid chromatography–tandem mass spectrometric (HPLC-MS/MS) method was developed and validated for the determination of spinosin, a pharmacologically active constituent isolated from Zizyphi Spinosi Semen (ZSS). This method was used to examine the degradation kinetics of spinosin by rat intestinal flora and to elucidate the structure of its metabolite. The analysis was performed using an aqueous mobile phase (containing 0.1% formic acid) modified by acetonitrile at a flow rate of 0.30 mL/min. Triple quadrupole mass spectrometric detection with an electrospray ionization (ESI) interface was conducted via multiple reaction monitoring (MRM) and single ion monitor (SIM) under negative ionization mode. The result of degradation experiment showed that spinosin can be metabolized in vitro by rat intestinal bacteria and its metabolite was swertisin. This may affect bioavailability of spinosin.

A New Saponin from the Seeds of Zizyphus jujuba var. spinosa

Sour jujube (Zizyphus jujuba Miller var. spinosa Hu ex H. F. Chou) is a thorny, rhamnaceous deciduous plant distributed widely throughout tropical and subtropical regions. The dried seeds are named sanjoin (Zizyphi Spinosi Semen) in Korean and have a thousand-year history of use in traditional medicine as a sedative for treating mild anxiety, nervousness and sleep-related problems. Previous phytochemical investigations on the seeds of this plant have resulted in the isolation of saponins, flavone C-glycosides, cyclopeptide alkaloids, triterpenoids, and others. These compounds exhibit multiple activities, such as hypnotic, histamine-release inhibitory, anti-inflammatory and sedative, inhibition of foam cell formation, adjuvant, and cardiotonic effects. Our previous studies focused on isolating the flavonoid-C-glycosides and the sedative activities of these flavonoids. In the continuing search for new chemicals and biomarkers from Z. jujuba var. spinosa for quality control studies of related herbal medicines, 8 flavonoids, along with 3 other known components, were isolated. In the present investigation, we report the isolation and structural elucidation of a new saponin, named jujuboside A2 (1), together with three known saponins, zizyphus saponin III (2), jujuboside B (3), jujuboside A (4), and a known sesquiterpene glucoside, dihydrophaseic acid 3-O-β-D-glucopyranoside (5), from the seeds of Z. jujuba var. spinosa. Compound 1 was obtained as a colorless amorphous powder. Its molecular formula was established as C58H94O26 from the [M−H] peak at m/z 1205.5961 (Calcd for C58H93O26: 1205.5955) in the HR-FAB-MS. The IR absorption bands at 3414, 1647, and 1043 cm implied the presence of hydroxyl, double bond, and glycosidic C-O functionalities. Acid hydrolysis of compound 1 with 5% HCl afforded a mixture of ebelin lactone and 17(Z)-ebelin lactone as the aglycon and D-glucose, 6-deoxy-L-talose, D-xylose, and L-arabinose as the sugar components identified on GLC analysis of the thiazolidine derivatives. The Hand C-NMR spectra of 1 suggested the presence of a jujubogenin moiety [δH 0.72, 0.97, 1.09, 1.10, 1.38, 1.67, and 1.70 (all s, CH3-19, CH3-29, CH3-18, CH3-28, CH3-21, CH3-26, and CH3-27), 3.18 (dd, J = 4.0, 11.7 Hz, H-3), 5.20 (br t, J = 9.5 Hz, H-23), and 5.53 (br d, J = 7.9 Hz, H-24)] and five monosaccharide units through easily identifiable signals for anomeric protons and carbons [δH 4.86 (d, J = 5.2 Hz), 4.90 (d, J = 7.7 Hz), 5.00 (d, J = 7.6 Hz), 5.43 (d, J = 7.3 Hz), and 6.11 (br s); δC 104.4, 105.2, 103.6, 106.0, and 101.7)]. The (−)-FAB-MS showed an [M−H] ion at m/z 1205, which was consistent with a pentasaccharide glycoside carrying two glucose, one 6-deoxyL-talose, two pentose (xylose and arabinose) units, and an aglycon with a molecular mass of 472. The presence of other

Genotoxicity of Zizyphi Spinosi Semen in Bacterial Reverse Mutation (Ames) Test, Chromosomal Aberration and Micronucleus Test in Mice

Zizyphi spinosi semen (Z. spinosi) has been used in traditional Chinese medicine for the treatment of rheumatoid arthritis and wounds. However, toxicity in high doses was often observed due to the presence of alka- loids. This study was conducted to investigate the potential genotoxicity of Z. spinosi in vitro and in vivo. This was examined by the Bacterial reverse mutation (Ames) test using Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coli WP2uvrA, Chromosomal aberration was investigated using Chinese hamster lung cells and the micronucleus test using mice. Z. Spinosi did not induce mutagenicity in the Ames test, and it did not produce chromosomal aberration in Chinese hamster lung cells with and without metabolic activation, nor in the micronucle- ated polychromatic erythrocytes in the bone marrow cells in mice. Based on these results, it is concluded that Z. spi- nosi does not have mutagenic potential under the conditions examined in each study.

Preparative Chromatographic Purification and Separation of Individual Jujubasaponins from Zizyphi Spinosi Semen

Zizyphi Spinosi Semen is well known as a kind of herb for the treatment of insomnia and anxiety. However, due to the lack of purified compounds for functional tests, limited data proves pharmacological activities of each jujubasaponin monomer. In this study, we developed a novel method for the preparative separation and purification of jujuboside A, A1, B, and B1, simultaneously, from Zizyphi Spinosi Semen. Macroporous resin and reversed-phase flash chromatography was successfully used and gave a good resolution of each jujubasaponins monomer. Jujuboside A, A1, B, and B1 with a purity of over 98% were yielded.

Betulinic acid ameliorates endothelium-dependent relaxation in L-NAME-induced hypertensive rats by reducing oxidative stress

ObjectiveModern pharmacological studies reveal that betulinic acid (BA), the key active constituent of traditional Chinese cardiovascular herbs Zizyphi Spinosi semen, up-regulated endothelial nitric oxide synthase (NOS) and down-regulated NADPH oxidase,...

Betulinic acid ameliorates endothelium-dependent relaxation in l-NAME-induced hypertensive rats by reducing oxidative stress

Zizyphi Spinosi semen (ZSS) is one of the most widely used traditional Chinese herbs with protective effects on the cardiovascular system. It is not clear whether betulinic acid (BA), the key active constituent of ZSS, has beneficial cardiovascular effects on N ω-nitro- l-arginine methyl ester hydrochloride ( l-NAME)-induced hypertensive rats. The objective of this study was to investigate the effect of BA on endothelium-dependent vasorelaxation in isolated aortic rings from l-NAME-induced hypertensive rats and its underlying mechanisms. Male Sprague–Dawley rats were injected with l-NAME (15 mg/kg/d, i.p.) for 4 weeks to induce hypertension. After treatment with l-NAME for 2 weeks, rats with mean blood pressure >120 mm Hg measured by tail-cuff method were considered hypertensive and then injected with BA (0.8, 4, 20 mg/kg/d, i.p.) for the last 2 weeks. The effect of BA on the tension of rat thoracic aortic rings was measured in an organ bath system. The levels of nitric oxide (NO), reactive oxygen species (ROS), and the activity of superoxide dismutase (SOD) and nitric oxide synthase (NOS) in aortas were assayed. We found that BA (0.1–100 μM) evoked a concentration-dependent vasorelaxation in endothelium-intact normal rat aortic rings, which was significantly attenuated by pretreatment with l-NAME (100 μM) or methylene blue (MB, 10 μM), but not by indomethacin (10 μM). Pretreatment with EC 50 (1.67 μM) concentration of BA enhanced the acetylcholine (ACh)-induced vasorelaxation, which was also markedly reversed by both l-NAME and MB. The blood pressure in hypertensive rats increased to 135.22 ± 5.38 mm Hg ( P < 0.01 vs. control group), which was markedly attenuated by high dose of BA. The ACh-induced vasorelaxation in hypertensive rat aortic rings was impaired, which was markedly improved by chronic treatment with BA (20 mg/kg/d) for 2 weeks. The increase of ROS level and the decrease of NO level, SOD and eNOS activities in hypertensive rat aortas were all markedly inhibited by BA. These results indicate that BA decreased blood pressure and improved ACh-induced endothelium-dependent vasorelaxation in l-NAME-induced hypertension rats, which may be mediated by reducing oxidative stress and retaining the bioavailability of NO in the cardiovascular system.

RAPID LARGE‐SCALE PURIFICATION OF BETULINIC ACID

ABSTRACTA rapid and efficient method for large‐scale purification of natural betulinic acid from Zizyphi Spinosi Semen was described. The procedure involved reflux extraction, preparative separation using low‐pressure reverse‐phase liquid chromatography and purification by crystallization. High purity (over 90%) and recovery of betulinic acid were obtained by preparative low‐pressure reverse‐phase liquid chromatography. Betulinic acid with the purity of over 99% was produced by crystallization. The method described was a useful tool in the preparative‐scale isolation of betulinic acid with low cost.PRACTICAL APPLICATIONSThe production of betulinic acid is easier and the yield and purity of betulinic acid produced this way are higher than before. This method can be used not only for experiment in the laboratory but also for industrial applications. The material – Zizyphi Spinosi Semen is a by‐product of wild jujube juice industry, so it is easy to get. High‐purity betulinic acid produced this way was sold to many companies at home and abroad. It is used as food additive and cosmetic material because of its insomnia and anxiety function. Reagents are common and inexpensive, some of them are reusable.