HPLC同时测定不同产地茄根中2种苯丙酰胺类成分的含量

建立同时测定茄根中2种苯丙酰胺类成分N-反式-对香豆酰基去甲辛弗林和N-反式-对香豆酰基酪胺含量的高效液相色谱方法。采用Agilent Eclipse XDB C₁₈色谱柱（4.6 mm × 250 mm,5 μm）;以甲醇-0.1%甲酸水溶液为流动相梯度洗脱,流速1.0 mL·min^-1;柱温30 ℃;检测波长300 nm。结果表明,N-反式-对香豆酰基去甲辛弗林和N-反式-对香豆酰基酪胺色谱峰分离良好,分别在2.84~68.16,3.10~74.40 mg·L^-1呈良好的线性关系,平均加样回收率（n=9）分别为99.30%,102.8%。该方法快速、准确、重复性好,可用于茄根药材的质量控制。     

阿萨伊油中β-谷甾醇及总甾醇含量测定和抗氧化活性研究

建立阿萨伊油中甾醇类成分的含量测定方法,并对阿萨伊油的抗氧化活性进行评价。采用高效液相色谱法建立了阿萨伊油中β-谷甾醇、总甾醇(β-谷甾醇、豆甾醇之和,以β-谷甾醇计)的含量测定方法。采用Purosper STAR LP C₁₈(4.6 mm×250 mm,5 μm)色谱柱,以乙腈-异丙醇(30:70)为流动相,流速1 mL·min^-1,柱温为室温,蒸发光散射检测器漂移管温度70 ℃,雾化器流速2.0 L·min^-1。结果表明β-谷甾醇在0.766~6.38 μg线性关系良好,回归方程为Y=1.639X+0.702 7(r=0.999 2),平均加样回收率为95.7%(RSD 2.8%)。阿萨伊油中β-谷甾醇的质量分数为1.85 mg·g^-1,总甾醇的质量分数为1.93 mg·g^-1。同时采用总氧自由基清除能力(TOSC)法对阿萨伊油的抗氧化活性进行评价,结果表明样品具有显著的抗氧化能力,样品浓度和抗氧化能力具有较好的剂量-效应关系。     

高效液相色谱法测定三七地上部分γ-氨基丁酸的含量

采用高效液相色谱法测定不同产地的三七地上部分中γ-氨基丁酸的含量。色谱条件采用C₁₈柱色谱柱（4.6 mm×250 mm,5 μm）,以乙腈-4.1 g·L^-1醋酸钠缓冲液为流动相,检测波长为254 nm。γ-氨基丁酸在0.01~1.03 g·L^-1线性范围内线性关系良好;三七茎叶和花中γ-氨基丁酸的平均加样回收率分别为101.7%（RSD 1.1%,n=3）和97.3%（RSD=0.38% ,n=3）。结果表明,不同产地的三七茎叶和花样品GABA含量差异较小,但三七花中GABA平均含量高于茎叶,分别为0.49%,0.53%。该方法操作简便、结果稳定,适合于三七地上部分中γ-氨基丁酸含量测定,为三七地上部分资源综合开发提供依据。     

HPLC同时测定山腊梅清感茶中4种黄酮苷的含量

目的: 建立高效液相色谱方法同时测定山腊梅清感茶中4种黄酮苷含量的方法. 方法: 采用依利特hypersil ODS2色谱柱(4.6 mm× 250 mm,5 μm),以乙腈-0.1%冰醋酸为流动相进行梯度洗脱,流速1.0 mL·min^-1,检测波长344 nm,柱温35 ℃. 结果: 芦丁在7.012~70.12 mg·L^-1线性关系良好(r=0.999 6),平均回收率为100.68%,RSD 3.3%,山柰酚-3-O-β-D-半乳糖-(6-1)-α-L-鼠李糖苷在5.126~51.26 mg·L^-1线性关系良好(r=0.999 9),平均回收率为98.48%,RSD 1.7%,山柰酚-3-O-芸香糖苷在2.631~26.31 mg·L^-1线性关系良好(r=0.999 8),平均回收率为101.82%,RSD 1.9%,紫云英苷在2.634~26.34 mg·L^-1线性关系良好(r=0.999 9),平均回收率为97.02%,RSD 1.7%. 结论: 该方法操作简便,准确,重复性好,可用于山腊梅清感茶的质量控制.     

HPLC同时测定正天丸中芍药苷、阿魏酸、升麻素苷、5-O-甲基维斯阿米醇苷的含量

该研究以高效液相色谱同时测定正天丸(钩藤、白芍、川芎、当归、防风等)中芍药苷、阿魏酸、升麻素苷、5-O-甲基维斯阿米醇苷的含量。试验中采用Insertil ODS-C₁₈色谱柱(4.6 mm×250 mm,5 μm),以乙腈-0.05%磷酸溶液为流动相,梯度洗脱,流速1.0 mL·min^-1,柱温30℃,检测波长230 nm。由试验结果可知,芍药苷、阿魏酸、升麻素苷、5-O-甲基维斯阿米醇苷在11.37~170.5,2.188~32.82,2.896~43.44,3.000~45.00 mg·L^-1峰面积与进样量线性关系良好,r分别为 0.999 9,0.999 9,1.000 0,1.000 0;回收率(n = 6)分别为102.3%(RSD 1.2%),99.71%(RSD 1.9%),101.2%(RSD 1.2%),99.40%(RSD 2.4%)。按所建方法对5批样品中上述4种成分的含量进行测定,结果均令人满意。该方法简便、准确,重复性好,可用于正天丸中芍药苷、阿魏酸、升麻素苷、5-O-甲基维斯阿米醇苷的质量控制。     

UPLC-PDA-ESI-MS分析杜仲中化学成分

目的： 建立杜仲药材中化学成分的超高效液相色谱-电喷雾质谱（UPLC-PDA-ESI-MS）分析方法。 方法： 采用ACQUITY UPLC系统,BEH C₁₈色谱柱（2.1 mm×100 mm,1.7 μm）,0.1%甲酸乙腈-0.1%甲酸水溶液为流动相,流速0.3 mL·min^-1,梯度洗脱,检测波长190~400 nm,柱温45 ℃。Waters电喷雾三重四级杆质谱仪（TQD）,负离子模式检测,ESI喷雾电压3 kV,去溶剂气温度350 ℃,去溶剂气（N₂）流量650 L·h^-1,锥孔气（N₂）流量50 L·h^-1,扫描范围m/z 100~1 000。 结果： 杜仲中化学成分获得了较好的分离和检测,共鉴定出3个木脂素类、4个环烯醚萜类和4个苯丙素类成分。 结论： 所建方法灵敏度高、分离度好,适用于杜仲药材中化学成分的快速定性鉴定。     

HPLC测定紫草提取物中β,β'-二甲基丙烯酰阿卡宁的含量

目的: 建立HPLC测定紫草提取物中β,β'-二甲基丙烯酰阿卡宁的含量。 方法: 采用HPLC,Agilent HC-C₁₈色谱柱 (4.6 mm ×250 mm,5 μm);流动相乙腈-水-甲酸(70:30:0.05),检测波长275 nm,柱温40 ℃,流速1 mL·min^-1。 结果: β,β'-二甲基丙烯酰阿卡宁在50~200 μg具有良好的线性关系(r=0.999 8 ),平均加样回收率103.31%,RSD 1.44%(n=5)。 结论: 方法准确,重复性好,可作为紫草提取物的含量控制。     

UPLC-Q-TOF-MS法快速分析石楠叶中化学成分

目的 采用超高效液相色谱串联飞行时间质谱（UPLC-Q-TOF-MS）法快速分析石楠Photinia serrulata叶化学成分。方法 色谱柱为Waters BEH C₁₈色谱柱（100 mm×2.1 mm,1.7 μm）,流动相为乙腈（A）-0.1%甲酸水溶液（B）,梯度洗脱,体积流量0.3 mL/min。采用peakview 1.2软件解析样品和混合对照品质谱图,得到化合物的精确相对分子质量、多级质谱数据、质谱碎片离子信息等,结合文献报道进行成分鉴定。结果 共鉴定了98个化学成分,主要包括30个黄酮类成分,24个有机酸类成分,15个苯丙素类成分,14个萜类成分和15个其他类成分,其中95个化合物是首次从该植物中发现的化学成分。结论 建立的UPLC-Q-TOF-MS法能快速、准确、较全面地鉴定石楠叶药材化学成分,为石楠叶药效物质基础研究提供依据。     

扶正解毒颗粒对染镍大鼠肾组织核因子-κB活化的影响

目的: 研究中药扶正解毒颗粒(FJG)对硫酸镍(NiSO₄)染毒大鼠肾组织核因子-κB (NF-κB)活化的影响。方法: 50只Wistar大鼠采用NiSO₄ 2.5 mg ·kg^-1 ip(连续7 d,此后间日1次)制备肾损伤模型,随机分为NiSO₄组(模型组)、 FJG高、中、低剂量组(分别为20,10,5 g ·kg^-1 ·d^-1),二硫基丁二酸(DMSA),50 mg ·kg^-1 ·d^-1)组。另设NS组(空白对照)及FJG对照组(FJG 10 g ·kg^-1 ·d^-1)。各组大鼠ig 1次/d,共4周。测定各组大鼠血尿素氮(BUN)、肌酐(SCr)及24 h尿蛋白定量(24 h-UP),免疫组化SP法观察肾组织NF-κB活化。结果: NiSO₄组大鼠出现肾功能损伤,NF-κB活化:肾小球(34.62±9.11)%,肾小管(62.45±14.78)%; FJG大剂量组NF-κB活化:肾小球(2.08±0.64)%,肾小管(11.48±3.39)%,明显低于NiSO₄组(P<0. 01),肾功能损伤亦明显减轻(P<0.05,P<0. 01)。结论: FJG可能通过抑制肾组织NF-κB活性的异常升高而发挥拮抗镍性肾损伤的作用。     

花旗松素对β-淀粉样肽损伤神经元的保护作用及机制

目的: 研究花旗松素对 β-淀粉样肽(β-amyloid peptide,Aβ)损伤神经元的保护作用及其机制. 方法: 从新生乳鼠大脑皮层中分离纯化神经元,与Aβ(5 μmol·L^-1)、不同浓度花旗松素(20~100 μmol·L^-1)共同孵育24 h,CCK8法检测细胞存活率,Hoechst 33258染色和Annexin V-FITC/PI检测细胞凋亡,分光光度法检测半胱天冬酶-3(caspase-3)活性的变化. 结果: 在细胞活力实验中,与正常组相比,模型组细胞活力显著下降(49.2±1.3) %,不同浓度的花旗松素可以提高神经元活力,其中80 μmol·L^-1花旗松素给药组细胞活力增至(68.7±3.2) %,与模型组相比有极显著差异;细胞凋亡结果显示,与正常组相比较,模型组细胞凋亡率为(50.8±1.5) %(P<0.01),不同浓度的花旗松素给药组可以降低细胞凋亡率,其中40 μmol·L^-1 花旗松素凋亡率为(41.5±2.7) %,与模型组比较呈显著性差异(P<0.05);凋亡酶caspase-3的活性与正常组(0.12±0.02) U·μg^-1比较,模型组的caspase-3的活性升高(2.37±0.16) U·μg^-1,P<0.01),与模型组相比,40 μmol·L^-1花旗松素组caspase-3显著降低(1.77±0.07) U·μg^-1, P<0.01).花旗松素能明显提高Aβ损伤神经元的细胞活力,抑制神经元的凋亡,降低凋亡酶caspase-3的活性. 结论: 花旗松素对Aβ损伤神经元具有保护作用,其机制可能与抑制caspase-3活性从而实现抗凋亡作用有关.     

The anti-ulcer activities of bisabolangelone from Angelica polymorpha

Aim of the study

Evaluate the anti-ulcer effects of bisabolangelone from Angelica polymorpha Maxim and provide the basic data to further study for the Angelica polymorpha and bisabolangelone.

Materials and methods

Bisabolangelone was isolated from Angelica polymorpha Maxim collected from Shennongjia Forest District of China. The structure of bisabolangelone was elucidated by NMR and MS spectrums. The anti-ulcer effects were evaluated with length of lesion (mm) and activity of H⁺/K⁺-ATPase in two models induced by ethanol and Pylorus ligation. Experimental groups were administered with different doses of bisabolangelone (3.8, 7.6 and 15.3 mg/kg). The positive control group was administered omeprazole with a dose of 3.3 mg/kg.

Results

Bisabolangelone significantly reduced the length of lesion (3.8, 7.6 and 15.3 mg/kg, P < 0.01), inhibited the activity of H⁺/K⁺-ATPase (3.8, 7.6 and 15.3 mg/kg, P < 0.01), decreased the volume of gastric juice (7.6 and 15.3 mg/kg, P < 0.05), and increased the pH value of gastric juice (7.6 and 15.3 mg/kg, P < 0.01, 3.8 mg/kg, P < 0.05).

Conclusions

Bisabolangelone is the main anti-ulcer active compound of Angelica polymorpha, and remarkably preventive and therapeutic action on gastric ulcer. It is possible that bisabolangelone inhibited the activity of the H⁺/K⁺-ATPase, then reducing the secretion of H⁺, and the anti-ulcer mechanism of bisabolangelone was deserved to be further studied.     

Mechanisms underlying the diuretic effects of Tropaeolum majus L. extracts and its main component isoquercitrin

Ethnopharmacological relevance

Previous studies have shown that the extracts obtained from Tropaeolum majus L., and its main compound isoquercitrin (ISQ), exhibit pronounced diuretic effects, supporting the ethnopharmacological use of this plant. The aim of this study was to evaluate the efficacy and mechanisms underlying the diuretic action of an ethanolic extract of Tropaeolum majus (HETM), its purified fraction (TMLR), and its main compound ISQ, in spontaneously hypertensive rats (SHR).

Materials and methods

The diuretic effects of HETM (300 mg/kg; p.o.), TMLR (100 mg/kg; p.o.), and ISQ (10 mg/kg; p.o.), were compared with classical diuretics in 7 days repeated-dose treatment. The urinary volume, sodium, potassium, chloride, bicarbonate, conductivity, pH and density were estimated in the sample collected for 15 h. The plasmatic concentration of sodium, potassium, urea, creatinine, aldosterone, vasopressin, nitrite and angiotensin converting enzyme (ACE) activity were measured in samples collected at the end of the experiment (seventh day). Using pharmacological antagonists or inhibitors, we determine the involvement of bradykinin, prostaglandin and nitric oxide (NO) in ISQ-induced diuresis. In addition, reactive oxygen species (ROS) and the activity of erythrocytary carbonic anhydrase and renal Na⁺/K⁺/ATPase were evaluated in vitro.

Results

HETM, TMLR and ISQ increased diuresis similarly to spironolactone and also presented K⁺-sparing effects. All groups presented both plasmatic aldosterone levels and ACE activity reduced. Previous treatment with HOE-140 (a B2-bradykinin receptor antagonist), or indomethacin (a cyclooxygenase inhibitor), or L-NAME (a NO synthase inhibitor), fully avoided the diuretic effect of ISQ. In addition, the 7 days treatment with ISQ resulted in increased plasmatic levels of nitrite and reducing ROS production. Moreover, the renal Na⁺/K⁺/ATPase activity was significantly decreased by ISQ.

Conclusion

Our results suggest that the mechanisms through ISQ and extracts of Tropaeolum majus increase diuresis in SHR rats are mainly related to ACE inhibition, increased bioavailability of bradykinin, PGI2, and nitric oxide, besides an inhibitory effect on Na⁺/K⁺-ATPase.     

基于1H-NMR的代谢组学方法对醋制甘遂解毒作用的研究

甘遂为有毒中药,临床上常经醋制后入药以降低其毒性。该文采用代谢组学方法比较生甘遂和醋甘遂对正常大鼠的损伤情况,研究醋制对甘遂毒性的缓解作用。连续7 d以生甘遂（EK）、醋甘遂（VEK）的水提取物对大鼠灌胃给药,药量均为9 g·kg^-1·d^-1（按生药量计）,对照组给予生理盐水。停止灌胃后继续观察7 d,收集14 d白天尿液,用于¹H-NMR检测;于第8天将每组大鼠处死一半,第15天处死另一半,采集肝脏,一部分用于¹H-NMR检测,另一部分用于病理学切片检测。病理学切片检测结果显示在本实验剂量下甘遂及醋制甘遂对大鼠肝脏没有造成损伤,但代谢组学方法分析发现在实验的第2周甘遂造成大鼠肝、肾及消化系统内部内源性代谢产物谱的紊乱;同时发现醋甘遂的毒性要比生甘遂低得多,传统醋制可以降低甘遂的毒性。该研究显示代谢组学方法有助于评价甘遂的毒性及醋制对甘遂的解毒作用。     

Effects of strictosamide on mouse brain and kidney Na+, K+-ATPase and Mg2+-ATPase activities

Present study reports on the general bioactivity of strictosamide and on its effects on Na(+),K(+)-ATPase and Mg(2+)-ATPase activities of Charles River male mouse. Strictosamide is the main glycoalkaloid of Sarcocephalus latifolius (Rubiaceae) leaves and roots, used as medicinal plant in folk medicine. In this work, we studied the in vitro effects of various concentrations of strictosamide (0.25, 0.5, 1 or 2 mg/mL) and the in vivo effects of single doses (50, 100 or 200 mg/kg, i.p.) of this compound on kidney and brain Na(+),K(+)-ATPase and Mg(2+)-ATPase activities. Results of general study showed that strictosamide is slightly toxic to Charles River mouse (LD(50)=723.17 mg/kg), producing CNS depression and kidney toxicity, but the exact mechanism of these effects could not be defined. Strictosamide inhibited the in vitro and in vivo Mg(2+)-ATPase activity on kidney but had nonsignificant effect on brain. Furthermore, strictosamide had nonsignificant in vitro and in vivo effect on kidney Na(+),K(+)-ATPase activity but produced an in vivo increase of Na(+),K(+)-ATPase activity of brain, these findings suggesting that strictosamine may be related to the induction of alpha(2) isoform of Na(+),K(+)-ATPase and may account for the folk use of Sarcocephalus latifolius root infusion on hypertension.     

Antiulcer and gastric antisecretory effects of dichloromethane fraction and piplartine obtained from fruits of Piper tuberculatum Jacq. in rats

Ethnopharmacological relevance

Piper tuberculatum Jacq. (Piperaceae) is medicinally used as an analgesic and as a treatment for gastric complaints. Thus, the current study aimed to investigate the gastroprotective and antisecretory properties of the dichloromethane fraction of the fruit of Piper tuberculatum (DFPT) and piplartine, a compound isolated from the DFPT, in rats.

Materials and methods

Gastric ulcers were induced in fasted rats by oral administration of absolute ethanol and then mucus content and glutathione (GSH) levels were measured. Mechanisms underlying the antisecretory action were studied through gastric H⁺,K⁺-ATPase activity of highly purified rabbit gastric microsomes and pylorus ligature method in rats.

Results

In the acute toxicity test the values of estimated LD₅₀ for oral and intraperitoneal administration of DFPT were 1.6266 and 0.2684 g/kg, respectively. The DFPT (ED₅₀=29 mg/kg, p.o.) and piplartine (4.5 mg/kg, p.o.) promoted gastroprotection against acute lesions induced by ethanol, effect that could be related with the maintenance of GSH levels in the gastric mucosa. However, only DFPT stimulated gastric mucus secretion. In vitro, the DFPT and piplartine inhibited the H⁺,K⁺-ATPase activity and, in vivo DFPT and piplartine also reduced basal gastric acid secretion, as well as that stimulated by pentagastrin.

Conclusions

These results demonstrate that DFPT and piplatine cause marked gastroprotective effects accompanied by the increase and maintenance of gastric mucus and GSH levels, as well as a reduction in gastric acid secretion through the gastrinergic pathway.     

In vivo inhibition of gastric acid secretion by the aqueous extract of Scoparia dulcis L. in rodents

The freeze-dried aqueous extract (AE) from the aerial parts of Scoparia dulcis was tested for its effects on experimental gastric hypersecretion and ulcer in rodents. Administration of AE to animals with 4h pylorus ligature potently reduced the gastric secretion with ED(50)s of 195 mg/kg (rats) and 306 mg/kg (mice). The AE also inhibited the histamine- or bethanechol-stimulated gastric secretion in pylorus-ligated mice with similar potency suggesting inhibition of the proton pump. Bio-guided purification of the AE yielded a flavonoid-rich fraction (BuF), with a specific activity 4-8 times higher than the AE in the pylorus ligature model. BuF also inhibited the hydrolysis of ATP by H(+),K(+)-ATPase with an IC(50) of 500 microg/ml, indicating that the inhibition of gastric acid secretion of Scoparia dulcis is related to the inhibition of the proton pump. Furthermore, the AE inhibited the establishment of acute gastric lesions induced in rats by indomethacin (ED(50)=313 mg/kg, p.o.) and ethanol (ED(50)=490 mg/kg, p.o.). No influence of the AE on gastrointestinal transit allowed discarding a possible CNS or a cholinergic interaction in the inhibition of gastric secretion by the AE. Collectively, the present data pharmacologically validates the popular use of Scoparia dulcis in gastric disturbances.     

泽泻麸制前后健脾作用研究

目的:对比泽泻炮制前后对大鼠胃泌素含量以及十二指肠Na+ -K+ -ATP酶活性的影响,探讨泽泻炮制前后健脾作用的变化.方法:取50只雄性SD大鼠随机分为空白组、泽泻生品低剂量组、泽泻生品高剂量组、泽泻麸制品低剂量组、泽泻麸制品高剂量组,空白组ig给予生理盐水,各低、高剂量组每次ig给予对应药物2.0,6.0 g·kg-1.每天ig给药2次,7d后测定大鼠血清胃泌素水平及十二指肠中Na+ -K+ -ATP酶活性.采用离体器官实验法,以大鼠离体十二指肠平滑肌的收缩幅度、相对张力及收缩频率为指标,观察在克氏液中泽泻炮制前后提取液对大鼠离体十二指肠平滑肌的影响.结果:泽泻麸制品高剂量组血清胃泌素含量为(3.77±0.49) ng·L-1,明显高于相同剂量生品组(3.24±0.13)ng·L-1(P＜0.05);麸制品高剂量组Na+ -K+-ATP酶含量为(13.31±1.99)pmol·g-1,明显高于相同剂量生品组(11.77±1.37) pmol· g-1(P＜0.05);生品和麸制品在高浓度时,对大鼠离体十二指肠收缩振幅及相对张力都有增强(P＜0.05).结论:泽泻能增加大鼠血清胃泌素含量、提高十二指肠Na+ -K+ -ATP酶活性,以及大鼠离体十二指肠肠管的运动功能,且呈剂量依赖关系,炮制后作用增强.     

Flavonoid-rich fraction of Maytenus ilicifolia Mart. ex. Reiss protects the gastric mucosa of rodents through inhibition of both H+,K+ -ATPase activity and formation of nitric oxide

Maytenus ilicifolia Mart. ex. Reissek (Celastraceae), a medicinal plant known in Brazil as "espinheira-santa" is commonly used to treat gastric disorders. The effect of the flavonoid-rich fraction separated from the leaves was evaluated for its gastroprotective properties and the mechanism(s) involved in this activity. Intraperitoneal administration of the flavonoid-rich fraction potently protected rats from experimentally induced chronic (ED(50)=79 mg/kg) and acute gastric lesions by ethanol (ED(50)=25mg/kg) and indomethacin (ED(50)=4 mg/kg) without altering the decreased amount of cytoprotective glutathione and mucus amount in the injured gastric mucosa. A potent reduction of gastric acid hypersecretion (ED(50)=7 mg/kg, i.p.) was accompanied by a reduction of nitric oxide release (ED(50)=1.6 mg/kg, i.p.) in the gastric secretion of 2h pylorus ligated rats which suggests an important role for nitric oxide-dependent mechanisms. Inhibition of gastric acid secretion in vivo was correlated with the in vitro inhibition of rabbit gastric H(+),K(+)-ATPase activity (IC(50)=41 microg/mL). Chemical investigation of the fraction showed galactitol (25%), epicatechin (3.1%) and catechin (2%) as the majoritary components. Collectively, the results show that the flavonoid-rich fraction of Maytenus ilicifolia potently protects animals from gastric lesions with high potency through inhibition of gastric acid secretion.     

Anti-secretory,anti-inflammatory and anti-Helicobacter pylori activities of several fractions isolated from Piper carpunya Ruiz & Pav.

Ethnopharmacological relevance

The leaves of Piper carpunya Ruiz & Pav. (syn Piper lenticellosum C.D.C.) (Piperaceae), are widely used in folk medicine in tropical and subtropical countries of South America as an anti-inflammatory, anti-ulcer, anti-diarrheal and anti-parasitical remedy as well as an ailment for skin irritations.

Aims of the study

To study the anti-inflammatory, anti-secretory and anti-Helicobacter pylori activities of different fractions isolated from an ethanolic extract of the leaves of Piper carpunya, in order to provide evidence for the use of this plant as an anti-ulcer remedy. Moreover, to isolate the main compounds of the extract and relate their biological activity to the experimental results obtained with the fractions.

Materials and methods

Sixteen fractions were obtained from the ethanolic extract (F I–XVI) and 16 pure compounds were isolated and identified from these fractions. We studied the effects of the fractions (0.1–400 μg/mL) on the release of myeloperoxidase (MPO) enzyme from rat peritoneal leukocytes, on rabbit gastric microsomal H⁺, K⁺-ATPase activity and anti-Helicobacter pylori anti-microbial activity using the microdilution method (MM). The main compounds contained in the fractions were isolated and identified by ¹H- and ¹³C NMR spectra analysis and comparison with the literature data.

Results

Eight fractions showed inhibition of MPO enzyme (F I–IV, X, XII, XIV and XV). The highest inhibition was observed with F XIV (50 μg/mL, 60.9%, p < 0.001). F X and XII were the most active ones, inhibiting the gastric H⁺, K⁺-ATPase activity with IC₅₀ values equal to 22.3 μg/mL and 28.1 μg/mL, respectively. All fractions, except F XV, presented detectable anti-Helicobacter pylori activity, with a diameter of inhibition zones ranging from 11 mm up to 50 mm. The best anti-Helicobacter pylori activity was obtained with F III and V. Both fractions killed Helicobacter pylori with lowest concentration values, about 6.25 μg/mL. Sixteen pure compounds were isolated, five of them are flavonoids that possess strong anti-oxidant and free radical scavenging activity, e.g. vitexin, isovitexin, and rhamnopyranosylvitexin. Terpenoids like sitosterol, stigmasterol and phytol, which have shown gastroprotective activity, and dihydrochalcones, like asebogenin, with anti-bacterial activity, were also isolated. Furthermore, the rare neolignan 1, that is a DNA polymerase β lyase inhibitor, and (6S, 9S)-roseoside, that shows strong anti-bacterial activity, were isolated, for the first time, from the genus Piper.

Conclusions

We suggest that the flavonoids isolated from F I and II (vitexin, isovitexin, rhamnopyranosylvitexin and isoembigenin) contribute to the anti-MPO activity, as well as to their anti-Helicobacter pylori activity. These flavonoids may also be responsible for the important inhibition of H⁺, K⁺-ATPase activity. Also the phytosterols and phytol obtained from F XIV and XV could be involved in these gastroprotective activities. These results encourage us to continue phytochemical studies on these fractions in order to obtain full scientific validation for this species.     

Hepatotoxic constituents and toxicological mechanism of Xanthium strumarium L. fruits

Ethnopharmacological relevance

In the recent years, the international community has attached increasing importance to possible toxicity associated with Traditional Chinese Medicine (TCM). And hepatotoxicity is one of the major concerns, a fundamental pathological process induced by toxicant. This paper is in an attempt to identify the hepatotoxic components in Xanthium strumarium L. fruits (XSF) and interpret the toxicological mechanism induced by XSF.

Materials and methods

XSF extract was prepared and seven characteristic components were isolated and identi?ed in XSF water extracts. We evaluated their hepatotoxicity effect on cell proliferation and lactate dehydrogenase (LDH) activity in L-02 and BRL liver cell line. An integrated metabonomics study using high-resolution ¹H nuclear magnetic resonance (¹H NMR) spectroscopy combined with multivariate statistical analysis was undertake to elucidate the hepatotoxicity mechanism induced in rats by XSF. The urine and serum metabolites were measured after treatment of rats with XSF (7.5, 15.0 and 30.0 g/kg/day) for 5 days.

Results

The results showed that atractyloside, carboxyatractyloside, 4'-desulphate-atractyloside and XSF induced significant cytotoxic effects in both L-02 and BRL liver cell lines, indicating that atractyloside, carboxyatractyloside, and 4'-desulphate-atractyloside were the toxic components of XSF. When rats were treated with XSF at 30.0 g/kg the hepatotoxicity was reflected in the changes observed in serum biochemical profiles and by the histopathological examination of the liver. The levels of VLDL/LDL, 3-HB, lactate, acetate, acetone and glutamate in serum were increased in this group, while d-glucose, choline and valine were decreased. The elevation in the levels of succinate, citrate, 2-oxo-glutamate, glycine, 3-HB, acetate, lactate, hippurate, dimethylglycine, methylamine, dimethylamine, phenylalanine and tryptophan was observed in urine, in contrast a reduction in the intensities of taurine, d-glucose, N-acetyl-glucoprotein and trimethylamine-N-oxide (TMAO) was observed.

Conclusions

The results demonstrate that the major hepatotoxicity constituents are atractyloside, carboxyatractyloside and 4'-desulphate-atractyloside, and the hepatotoxicity of XSF involves mitochondrial inability, fatty acid metabolism, and some amino acids metabolism. This integrated ¹H NMR -based metabolic profiling approach has been able to capture and probe the metabolic alterations associated with the onset and progression of hepatotoxicity induced by XSF, and permits a comprehensive understanding of systemic toxicity for phytochemicals and other types of xenobiotic agents.