Comparative pharmacokinetics of six major compounds in normal and insomnia rats after oral administration of Ziziphi Spinosae Semen aqueous extract

Ziziphi Spinosae Semen(ZSS),a traditional Chinese medicine,is used in clinics for the treatment of insomnia in China and other Asian countries.Herein,we described for the first time a comparative pharmacokinetics study of the six major compounds of ZSS in normal control(NC)and para-chlorophenylalanine(PCPA)-induced insomnia model(IM)rats that were orally administered the aqueous extract of ZSS.An ultra-high-performance liquid chromatography coupled with quadrupole orbitrap mass(UHPLC-Q-Orbitrap-MS)method was developed and validated for the simultaneous determination of coclaurine,magnoflorine,spinosin,6000-feruloylspinosin,jujuboside A(JuA),and jujuboside B(JuB)in ZSS in rat plasma.The established approach was successfully applied to a comparative pharmacokinetic study.The systemic exposures of spinosin and 6000-feruloylspinosin were decreased in the IM group compared to the NC group,while plasma clearance(CL)was significantly increased.The Tmax values of JuA and JuB in IM rats were significantly lower than those in NC rats.The T1/2 of JuA in the IM group was significantly accelerated.The pharmacokinetic parameters of coclaurine and magnoflorine were not evidently affected between the two groups.These results indicate that the pathological state of insomnia altered the plasma pharmacokinetics of spinosin,6000-feruloylspinosin,JuA,and JuB in the ZSS aqueous extract,providing an experimental basis for the role of ZSS in insomnia treatment.The comparative pharmacokinetics-based UHPLC-Q-Orbitrap-MS using full-scan mode can therefore provide a reliable and suitable means for the screening of potentially effective substances applied as quality markers of ZSS.

Quality analysis of commercial samples of Ziziphi spinosae semen(suanzaoren) by means of chromatographic fingerprinting assisted by principal component analysis

Due to the scarcity of resources of Ziziphi spinosae semen （ZSS）, many inferior goods and even adulterants are generally found in medicine markets. To strengthen the quality control, HPLC fingerprint common pattern established in this paper showed three main bioactive compounds in one chromatogram simultaneously. Principal component analysis based on DAD signals could discriminate adulterants and inferiorities. Principal component analysis indicated that all samples could be mainly regrouped into two main clusters according to the first principal component （PC1, redefined as Vicenin II） and the second principal component （PC2, redefined as zizyphusine）. PC1 and PC2 could explain 91.42%of the variance. Content of zizyphusine fluctuated more greatly than that of spinosin, and this result was also confirmed by the HPTLC result. Samples with low content of jujubosides and two common adulterants could not be used equivalently with authenticated ones in clinic, while one reference standard extract could substitute the crude drug in pharmaceutical production. Giving special consideration to the well-known bioactive saponins but with low response by end absorption, a fast and cheap HPTLC method for quality control of ZSS was developed and the result obtained was commensurate well with that of HPLC analysis. Samples having similar fingerprints to HPTLC common pattern targeting at saponins could be regarded as authenticated ones. This work provided a faster and cheaper way for quality control of ZSS and laid foundation for establishing a more effective quality control method for ZSS.

Quality control of Semen Ziziphi Spinosae standard decoction based on determination of multi-components using TOF-MS/MS and UPLC-PDA technology

A sensitive,fast and comprehensive method for the quality assessment of Semen Ziziphi Spinosae(SZS)standard decoction with characterization of its chemical components was developed and validated.UPLC-Q/TOF-MS/MS system was used to identify thirty-six chemical components of SZS standard decoction which included nucleosides,phenolic acids,alkaloids,and flavonoids.Furthermore,a UPLC-PDA method was validated to simultaneously determine adenosine,protocatechuic acid,magnoflorine,catechin,protocatechin,vicenin II,spinosin,kaempferol-3-rutinoside,and 6'''-feruloylspinosin which represent four species of characteristic compounds.The qualitative method had been validated according to Chinese Pharmacopoeia(2015 edition)in terms of lineary,repeatability,recovery and stability for all analytes,with the results showing good precision,accuracy and stability.In conclusion,the method using UPLC combined with MS and PDA provided a novel way for the standardization and identification of SZS standard decoction,and also offered a basis for qualitative analysis and quality assessment of the preparations for SZS standard decoction.

Primary Study of Novel Method for Determination of Saponin in Ziziphi Spinosae Semen (<i>Suan-Zao-Ren</i>) by Digitally Scanning the Photo of the HPTLC Image without Configuration of TLC Scanner Facility

Conventional quantity determination of Chinese herbal medicines by High-Performance Thin-Layer Chromatography (HPTLC) must be carried out on a TLC scanner for measuring the marker component on the HPTLC plate. Generally the HPTLC image of the herbal drug always takes the photograph as the permanent visual document. Considering the photo of the HPTLC image preserving all the visual quality-related parameters, there might be an alternative cost/effective approach worth studying. It would be presumably applicable to use the exclusively developed software as a virtual scanner instead of the TLC Scanner instrument to do digital scanning, integrating and calculating target components in the photo of the HPTLC image. It means digitally scanning the photo of the HPTLC image solely with software does the same thing as the instrumental scanner does. Based on this protocol, we primarily conducted the assay of the contents of the major saponins, jujuboside a and jujuboside b, in Ziziphi Spinosae Semen (Suan-Zao-Ren). Using home-developed software “digiscan” for acquiring the raw data and integrating the peaks area of the target components with “Origin Pro”, the contents of jujuboside a and jujuboside b can then be calculated respectively. The primary investigated experiment results were well compatible with official HPLC-ELSD method in Chinese Pharmacopoeia. The methodology validation and comparative analysis with HPLC confirmed that the unprecedented method is acceptable. The advantage of the recommended approach is far more simple, flexible and cost/effective than the conventional method, and it can be done at anytime and anywhere once only if the high quality HPTLC image photo is available. There would be a bright perspective for application to other herbal medicines. And the one-stop software for merging digital scanning, integration and calculation together is in progress.

A study of storage conditions for pre-mashed fried Semen Zizyphi Spinosae using a multi-factor orthogonal design

Background:To facilitate the preparation of traditional Chinese medicines they are pre-mashed,i.e.,mashed in advance.However,storage conditions for pre-mashed traditional Chinese medicines are based on subjective judgments of pharmacists,and the best storage conditions have not generally been determined.Semen Zizyphi Spinosae is a commonly used traditional Chinese medicine,and it is usually used after it is fried.The medicine needs to be mashed in a timely manner to ensure its effectiveness.The Chinese Pharmacopoeia provides a limit for its aflatoxin content.Methods:The orthogonal experimental design method optimizes the best plan for pre-mashed fried Semen Zizyphi Spinosae.Experimental conditions were defined using the L^(9)(3^(4))orthogonal design table.Four factors and three levels were used to study storage conditions.The four factors and three levels are as follows:storage temperature(10°C,20°C,and 30°C),storage humidity(45%,60%,and 75%),storage times(10,20 and 30 days),and particle sizes for the powder(coarsest,coarse and medium powders).The contents of jujuboside A,spinosin,aflatoxin B1,total aflatoxins(aflatoxins B1,B2,G1,and G2),moisture,total ash,acid value,and saponification values were measured.Results:The results demonstrated that the highest jujuboside A and spinosin contents were obtained using a storage temperature of 20°C,a storage humidity of 75%,a storage time of 10 days,and with a coarse powder particle size.Aflatoxin B1 and total aflatoxins(aflatoxins B1,B2,G1 and G2)were not detected under these conditions.Conclusion:There is no requirement for traditional Chinese medicines to be pre-mashed.This study evaluated various storage conditions for pre-mashed fried Semen Zizyphi Spinosae,and considered the influence of four factors on the contents of jujuboside A,spinolin and aflatoxin for quality control to provide a reference for other pre-mashed traditional Chinese medicines.

Determination of five neurotransmitters in the rat brain for the study of the hypnotic effects of Ziziphi Spinosae Semen aqueous extract on insomnia rat model by UPLC-MS/MS

Ziziphi Spinosae Semen(ZSS) has been used for treatment of insomnia in China for centuries. To reveal the influence of insomnia on the levels of the neurotransmitters including serotonin(5-HT), glutamic acid(Glu),γ-aminobutyric acid(GABA),noradrenaline(NE) and dopamine(DA), and to study the role of ZSS aqueous extract in the treatment of insomnia, an UPLC-ESIMS/MS method was developed and validated for simultaneous determination of five neurotransmitters in the rat brain. The brain samples were pretreated by one-step direct protein precipitation with acetonitrile. The analytes were detected in positive mode with multiple reaction monitoring(MRM) and the procedure was completed in less than 10 min. The method showed a good linearity(R^2 >0.9967) with the other validation parameters were within acceptance range. The results indicated that the concentration of 5-HT, GABA and DA is significantly lower(P < 0.01) in para-chlorophenylalanine(PCPA)-induced insomnia rat model group, while Glu and NE significantly higher than those in control group(P < 0.01). Treatment with ZSS aqueous extract(4 or 8 g·kg^1·d^-1 for seven days) could ameliorate the symptoms of insomnia by significantly changing the levels of the neurotransmitter parameters mentioned above. The data obtained in this study demonstrate that ZSS aqueous extract could ameliorate the symptoms of insomnia by modulating the levels of monoamines and amino acid neurotransmitters in the brain.

Identification and discrimination of Ziziphi Spinosae Semen and Ziziphi Mauritianae Semen based on HPLC fingerprint analysis

A simple, reproducible and reliable HPLC fingerprint analysis method of Ziziphi Spinosae Semen(ZSS) and Ziziphi Mauritianae Semen(ZMS) was established, and then it was applied to analyze samples collected from different locations. A total of 12 common fingerprint peaks were designated in ZSS and ZMS, respectively, and five of which were definitely identified. Of these common peaks, 10 peaks were presented in both ZSS and ZMS, whereas two were characteristic for ZSS and ZMS, respectively. Furthermore, the two characteristic peaks of ZMS were definitely assigned as 6′′′-sinapoylspinosin and frangufoline by HPLC guided purification and structural elucidation. The HPLC fingerprint analysis was proved to be useful in identifying and discriminating ZSS and ZMS, which was beneficial for quality control of ZSS and its products.

Capparis spinosa L waste activated carbon as an efficient adsorbent for crystal violet toxic dye removal: Modeling, optimization by experimental design, and ecological analysis

Textile dyes are dramatic sources of pollution and non-aesthetic disturbance of aquatic life and therefore represent a potential risk of bioaccumulation that can affect living species.It is imperative to reduce or eliminate these dyes from liquid effluents with innovative biomaterials and methods.Therefore,this research aims to highlight the performance of Capparis spinosa L waste-activated carbon(CSLW-AC)adsorbent to remove crystal violet(CV)from an aqueous solution.The mechanism of CV adsorption on CSLW-AC was evaluated based on the coupling of experimental data and different characterization techniques.The efficiency of the CSLW-AC material reflected by the equilibrium adsorption capacity of CV could reach more than 195.671 mg·g^(–1) when 0.5 g·L^(–1) of CSLW-AC(Particle size≤250μm)is introduced into the CV of initial concentration of 100 mg·L^(–1) at pH 6 and temperature 65℃ and in the presence of potassium ions after 60 min of contact time according to the one parameter at a time studies.The adsorption behavior of CV on CSLW-AC was found to be consistent with the pseudo-second-order kinetic model and Frumkin's linear isothermal model.The thermodynamic aspects indicate that the process is physical,spontaneous,and endothermic.The optimization of the process by the Box Behnken design of experiments resulted in an adsorption capacity approaching 183.544 mg·g^(–1)([CV]=100 mg·L^(–1) and[CSLW-AC]=0.5 g·L^(–1) at 35 min).The results of the Lactuca sativa seeds germination in treated CV(70%),adsorbent solvent and thermal regeneration(more than 5 cycles),and process cost analysis(1.0484 USD·L^(–1))tests are encouraging and promising for future exploitations of the CSLW-AC material in different industrial fields.

Aszonapyrone A Isolated from Neosartorya spinosa IFM 47025 Inhibits the NF-κB Signaling Pathway Activated by Expression of the Ependymoma-Causing Fusion Protein ZFTA-RELA

Ependymoma is a rare and chemotherapy-resistant brain tumor, which has resulted in a delay in the development of drugs to treat it. A subclass of supratentorial ependymomas (ST-EPN), designated ST-EPN-zinc finger-translocation-associated (ZFTA, ST-EPN-ZFTA), exhibits the expression of a fusion protein comprising ZFTA and v-rel reticuloendotheliosis viral oncogene homolog A (RELA), an effector transcription factor of the nuclear factor-kappa B (NF-κB) pathway (ZFTA-RELA). The expression of ZFTA-RELA results in the hyperactivation of the oncogenic NF-κB signaling pathway, which ultimately leads to the development of ST-EPN-ZFTA. To identify inhibitors of the NF-κB signaling pathway activated by the expression of ZFTA-RELA, we used a doxycycline-inducible ZFTA-RELA-expressing NF-κB reporter cell line and found that extracts of the fungus Neosartorya spinosa IFM 47025 exhibited NF-κB inhibitory activity. We identified eight compounds [aszonapyrone A (2), sartorypyrone A (3), epiheveadride (4), acetylaszonalenin (5), (R)-benzodiazepinedione (6), aszonalenin (7), sartorypyrone E (8) and (Z, Z)-N,N’-(1,2-bis[(4-methoxyphenyl)methylene]-1,2-ethanediyl)bis-formamide (9)] from N. spinosa IFM 47025 culture extract using a variety of chromatographic techniques. The structures of these compounds were identified through the analysis of various instrumental data (1D, 2D-NMR, MS, and optical rotation). The NF-κB responsive reporter assay indicated that compounds 2, 3, 5, 7, and 9 exhibited inhibitory activity. We further evaluated the inhibitory activity of these compounds against the expression of endogenous NF-κB responsive genes (CCND1, L1CAM, ICAM1, and TNF) and found that compound 2 showed significant inhibitory activity. Further studies are required to elucidate the mechanism of action of compound 2, which may serve as a lead compound for the development of a novel therapy for ST-EPN-ZFTA.

ICP-AES法测定陕北地区酸枣仁中微量元素的含量

[目的]建立测定酸枣仁中Mn、Fe、Cu、Zn、Ca、Mg、Sr含量的方法。[方法]以陕北酸枣仁为试材,用HNO3+HClO4混合酸作为消解剂消化样品,利用电感耦合等离子发射光谱法进行测定样品中微量元素的含量。ICP-AES的工作条件:功率为1.2 kW;冷却气(He)流量为14 L/min;载气流量为0.7 L/min;辅助气流量为1.2 L/min;光谱温度为38℃;线性电压为104%;检测高度较低。各元素分析线波长分别为257.610(Mn)、259.940(Fe)、327.396(Cu)、213.856(Zn)、393.366(Ca)、279.553(Mg)和407.771(Sr)nm。[结果]在此消解条下件,测得酸枣仁中Mn、Fe、Cu、Zn、Ca、Mg、Sr的含量分别为31.70、59.86、8.14、53.38、1 358.45、2 624.80和13.62μg/g;与其他地区相比陕北酸枣仁微量元素含量适中。[结论]该方法准确可靠,可用于酸枣仁中Mn、Fe、Cu、Zn、Ca、Mg、Sr的含量测定。

酸枣仁的等级划分及制作方法研究

对酸枣仁的质量指标及等级划分、制作方法、检验方法和包装运输贮存等内容进行了研究,为酸枣仁的进一步开发利用提供依据。

棘胸蛙烂皮病奇异变形杆菌的分离、鉴定及对药物敏感性研究

从患烂皮病的棘胸蛙(Rana spinosa)中分离到一株致病菌,通过形态学、生理生化试验等方法鉴定该致病菌株为奇异变形杆菌(Proteus mirabilis)。同时,通过抑菌圈法研究了该菌株对11种抗生素、11种中草药、4种消毒剂的敏感性。结果显示:该菌株对头孢曲松、阿莫西林、链霉素、诺氟沙星、庆大霉素、卡那霉素、氯霉素等抗生素敏感,对四环素、红霉素不敏感;未观察到黄芪、茵陈对该菌的抑制作用,忍冬藤抑菌效果最好,其次依次为紫花地丁、金银花与甘草、鱼腥草、大青叶与柴胡、陈皮、黄连;致病菌株对消毒剂的敏感性依次为:高锰酸钾﹥三氯异氰脲酸﹥硫酸铜﹥氧化钙。

酸枣活性成分分析及药理作用研究进展

对酸枣中的主要活性成分黄酮类、皂苷类、生物碱类物质、脂肪酸、氨基酸及矿物质等进行分析,并对其主要药理作用的研究进展进行综述,为酸枣仁的进一步开发利用提供依据。

HPLC法测定维药刺山柑种子中没食子酸和芦丁的含量

目的：建立刺山柑种子中没食子酸和芦丁的含量测定方法。方法采用Phnomenex色谱柱（250 mm ＆#215;4．6 mm ，5μm），以乙腈-2 mL · L -1磷酸溶液为流动相，流速为1．0 mL · min-1，检测波长为254 nm ，梯度洗脱。结果没食子酸在1.4～14．0μg、芦丁在1.4～28．0μg范围内呈良好的线性关系，没食子酸平均回收率为99．9％，RSD为2．0％；芦丁平均回收率为100．6％， RSD为1．3％。结论 HPLC法测定没食子酸和芦丁的含量，方法简便可行，重复性和分离效果好，可为刺山柑的进一步开发利用及质量控制提供依据。

金银花叶提取物对棘胸蛙烂皮病治疗效果的探讨

研究了金银花(Lonicera japonica)叶不同溶剂提取物对棘胸蛙(Rana spinosa)烂皮病治疗效果的影响。首先制备金银花叶的水提物与醇提物,分别用终浓度为16 mg/m L的水提物和醇提物浸浴患病蝌蚪,比较蝌蚪的存活量和平均体重。结果表明,金银花叶醇提物治疗组的蝌蚪存活数量远高于金银花叶水提物治疗组。说明金银花叶的醇提物对患病蝌蚪有较好的治疗效果。

酸枣仁、覆盆子对酒精戒断焦虑征的治疗作用

研究酸枣仁、覆盆子对酒精戒断焦虑症的治疗作用,并探讨其作用机制。研究方法选取健康SD大鼠随机分组,腹腔注射酒精28天,给药治疗3天,最后一次给药1h后,高架十字迷宫测其焦虑行为,HPLC测海马中去甲肾上腺素的含量。结果与模型组相比,大剂量覆盆子提取物、大剂量炒制酸枣仁醇提物在高架十字迷宫测试中能显著增加其次数、时间(P<0.05),在HPLC检测中可降低海马中去甲肾上腺素含量(P<0.05),且酸枣仁作用更为显著。由此得出结论,覆盆子提取物、炒制酸枣仁醇提物能减轻酒精戒断时大鼠所表现出的焦虑样行为,其抗焦虑症的作用机制可能与降低大鼠海马组织中去甲肾上腺素含量有关。

美国西部不同地区Grayia spinosa核DNA ITS序列分析

藜科植物Grayia spinosa是美国西部地区的特有种,多生长在干旱盐碱地,具有重要的生态价值。该研究测定了采自美国西部犹他州G.spinosa的nrDNA ITS序列,与Gen Bank中已提交的G.spinosa的所有ITS序列以及G.spinosa的四个近缘种作为外类群进行比较,分析了美国西部不同地区G.spinosa ITS序列的一级结构与其RNA二级结构的变异规律。结果表明:所有G.spinosa样品的nrDNA ITS序列长度在611~623 bp之间,GC含量在60.35%~61.0%之间,序列间共存在22个变异位点,5个为简约信息位点。各样品间的遗传距离在0.001 8~0.008 9之间,不同样品间的遗传距离与地理距离的相关性不显著。邻接法构建的系统发育树显示所有G.spinosa聚为一大支,与外类群形成明显分支。此外,利用RNAfold在线软件预测了G.spinosa ITS序列的RNA二级结构,将8个G.spinosa样品的RNA二级结构根据构型差异大体上分为四类,分别记为type A,B,C和D四类,主要变异出现在ITS1和ITS2区。所不同的是在G.spinosa ITS的一级结构分析中GSNE1与GSWA8体现出更近的亲缘关系,但二者的RNA二级结构差异明显,同时GSNE2、GSUT3、GSUT4、GSCA5、GSCA6、GSCO7在ITS序列一级结构分析中也体现出较近的亲缘关系,但是他们的RNA二级结构差异明显。这可能与ITS序列的RNA二级结构在进化中体现出更大的保守性有关。

The advertisement call of the giant spiny frog Paa spinosa

We analyzed the advertisement call of Paa spinosa at Yuliang Mountain,Lanxi,Zhejiang Province,in eastern China.Temporal and spectral call parameters were analyzed,along with call intensity.Calls comprised of three to seven notes,the last of which had the longest duration.Three formants(harmonics)were clearly distinguishable from the audio spectrogram.The dominant frequency ranged from 411-1534 Hz,and was either the first or the second formant.The number of notes within a call was positively correlated to air temperature,so that calls contained more notes during the day.Also,the dominant frequency appeared to be lower at the highest temperature.

Isolation and identification of bioactive antibacterial components in leaf extracts of Vangueria spinosa(Rubiaceae)

Objective:The column chromatographic fraction of ethyl acetate(EA1,EA2,EA3,EA4 and EA5) leaf extracts of Vangueria spinosa(V.spinosa) were screened for antibacterial activity and phytochemical analysis.Methods:EA3 fraction was isolated and identified by column chromatography,thin layer chromatography,spectral data analysis and phytochemical screening were used for analysis.Results:EA3 fraction was significantly active at 4 to 64 mg/L against Staphylococcus aureus,Escherichia coli,Klebsiella pneumoniae and Pseudomonas aeruginosa with minimum inhibitory concentration of 1.5625 to 3.1250 mg/mL.The active fraction(EA3) revealed the presence of flavonoid with retention factor value(R_f) of 0.39.The active antibacterial agent in the most potent fraction(EA3) was isolated and identified as flavonoid(-)-epicatechin-3-O-β-glucopyranoside by thin layer chromatography(TLC) and phytochemical screening.EA1 and EA2 show inhibitory activity at 4 to 64 mg/L against Staphylococcus aureus only where as fraction EA4 and EA5 do not shows any inhibitory activity within that range of concentration against any bacteria.Conclusions:The results support the ethnomedicinal use of leaf of V.spinosa for the treatment of bacterial diseases.

Genetic Diversity and Population Structure for the Conservation of Giant Spiny Frog(Quasipaa spinosa)Using Microsatellite Loci and Mitochondrial DNA

The giant spiny frog （Quasipaa spinosa） is an endangered species with a relatively small distribution limited to southern China and Northern Vietnam. This species is becoming increasingly threatened because of over-exploitation and habitat degradation. This study provides data on the genetic diversity and population genetic structure of the giant spiny frog to facilitate the further development of effective conservation recommendations for this economically important but threatened species. We examined 10 species-specific microsatellite loci and Cyt b genes （562 bp） collected from 13 wild populations across the entire range of this species. Results of 10 microsatellite loci analysis showed a generally high level of genetic diversity. Moreover, the genetic differentiation among all 12 populations was moderate to large （overall Fs7= 0.1057）. A total of 51 haplotypes were identified for Cyt b, which suggests high haplotype nucleotide diversities. Phylogeographic and population structure analyses using both DNA markers suggested that the wild giant spiny frog can be divided into four distinct major clades, i.e., Northern Vietnam, Western China, Central China, and Eastern China. The clades with significant genetic divergence are reproductively isolated, as evidenced by a high number of private alleles and strong incidence of failed amplification in microsatellite loci. Our research, coupled with other studies, suggests that Q. spinosa might be a species complex within which no detectable morphological variation has been revealed. The four phylogenetic clades and some subclades with distinct geographical distribution should be regarded as independent management units for conservation purposes.