Rats exposed to Solanum lycocarpum fruit in utero and during lactation: neurochemical, behavioral and histopathological effects

Solanum lycocarpum St. Hil (Solanaceae) is a native shrub very common in the Brazilian savannah. This plant contains steroidal glycoalkaloids that can be transformed into an intermediate for steroidal drug production. In this way, it is very possible that these glycoalkaloids and its aglycone, once in the body by ingestion of S. lycocarpum fruits, may act by disrupting the endocrine system. Because its fruits may be consumed by pregnant animals in the fields, the present study determined the possible toxic effects of exposure to S. lycocarpum fruit (10% added in the diet) from gestation day (GD) 6 to postnatal day (PND) 07 in rat dams. The unripe fruits contained 0.6% of solamargine and 0.9% of solasonine. S. lycocarpum, 10% in the diet, during gestation and the beginning of lactation reduced intrauterine growth. In addition, 20% of the treated dams showed some dead pups at birth. Reduced body weight was observed from birth through adulthood in male and female offspring exposed to 10% S. lycocarpum unripe fruits. During adulthood, female offspring showed impaired sexual behavior and male offspring showed prominent degeneration of testis germinative cells, characterized by a reduced number of germ cells and vacuolation. Also, the exposed offspring showed reduced hypothalamic norepinephrine (NOR), vanillylmandelic acid (VMA), 3-methoxy-4-hydrophenylglycol (MHPG) and homovanillic acid (HVA) levels, and reduced striatum NOR, HVA, VMA, MHPG, dopamine (DA), dihydroxyphenylacetic acid (DOPAC) and 5-hydroxyindolacetic acid (5-HIAA) levels. These results suggest that the fruit may act as an estrogen, with a long-term effect, impairing the receptive lordosis behavior of female offspring and promoting testis abnormalities in male offspring at adulthood. Finally, it appears to disrupt brain organization since important central monoamine level alterations were also observed.     

Simultaneous determination of three steroidal glycoalkaloids in Solanum xanthocarpum by high performance thin layer chromatography

A new high-performance thin-layer chromatographic (HPTLC) method has been developed for the simultaneous quantitation of three bioactive steroidal glycoalkaloid (SGA) markers, solasonine (SN), solamargine (SM) and khasianine (KN) in the plant Solanum xanthocarpum. Extraction efficiency of targeted SGAs from plant matrix using methanol and acidified methanol were studied using percolation, ultrasonication and microwave techniques. The separation was achieved on silica gel 60F(254) TLC plates using chloroform-methanol-water as mobile phase. The quantitation of SGAs was carried out using the densitometric reflection/absorption mode at 520 nm after post chromatographic derivatization using Dragendorff's reagent. The method was validated for peak purity, precision, accuracy, robustness, limit of detection (LOD) and quantitation (LOQ). Method specificity was confirmed using retention factor (R(f)), Vis spectral correlation and electrospray ionization mass spectra (ESI-MS) of marker compounds in the sample track.     

澳洲茄边碱对前列腺癌细胞增殖的抑制作用及机制研究

目的：研究澳洲茄边碱(solamargine,SM)对人前列腺癌激素非依赖细胞增殖的影响及可能的作用机制.方法用不同浓度 SM(0、1、2、4、6、8、10μmol/L)处理 DU145和 PC3细胞, MTT法检测 SM 对细胞生长的影响,Western blot 检测 SM 对相关信号通路蛋白 p38 MAPK、ERK1/2 MAPK、MUC1表达的影响.结果6μmol/L SM作用24 h后DU145和PC3细胞活力分别为(52.53±9.05)％、(56.28±2.36)％,并具有时间和剂量依赖;10μmol/L SM作用24 h后细胞活力分别为(27.36±2.72)％、(32.07±2.53)％.流式细胞术分析显示不同浓度 SM(0、4、6、8μmol/L)处理 PC3细胞能引起 PC3细胞阻滞在 G1期,G1期细胞比例分别为(52.61±0.50)％、(52.96±1.49)％、(66.16±2.84)％和(69.03±2.38)％.且能激活 MAPK信号通路减少下游蛋白 MUC1的表达.结论 SM能明显抑制DU145和PC3细胞生长,该作用可能与 MAPK信号通路的激活以及下游 MUC1蛋白表达下调有关.     

Sensitive and robust UPLC-MS/MS method to determine the gender-dependent pharmacokinetics in rats of emodin and its glucuronide

A sensitive and simple liquid chromatography-mass spectrometry (LC-MS) method has been developed and validated for the quantification of solamargine, a steroidal glycoalkaloid, in rat plasma. Vincristine was selected as the internal standard. Sample preparation involved simple liquid-liquid extraction by ethyl acetate with high efficiency. The chromatographical separation was performed on a Shimadzu C(18) column (150mm×2.0mm, 5μm) with a gradient elution of acetonitrile and 0.02% (v/v) formic acid. The elutes were detected under positive electrospray ionization (ESI) and the target analytes quantified by selected ion monitoring (SIM) mode. The method was sensitive with the lowest limit of quantitation (LLOQ) at 0.5ng/mL in 50μL of rat plasma. Good linearity (r(2)=0.9996) was obtained covering the concentration of 0.5-2000.0ng/mL. The intra- and inter-day assay precision ranged from 2.87 to 3.60% and 0.52 to 6.81%, respectively. In addition, the stability, extraction recovery and matrix effect involved in the method were also validated. The practical utility of the aforementioned method was successfully confirmed in the pharmacokinetic evaluation of solamargine in Sprague-Dawley rats after intravenous administration.     

Toxicological effects of alpha-solamargine in experimental animals

Alpha-solamargine isolated from the fresh fruits of Solanum americanum Miller was studied for its toxicity. Lethality studies in rats showed a dose-mortality relationship with a LD(50) of 42 mg/kg body weight intraperitoneally. The chronic and subchronic toxicity investigations indicated that the size of the glycoalkaloid dose was more important than the total glycoalkaloid intake. No appreciable toxic effects were observed at doses below 35 mg/kg body weight as indicated by blood parameters, enzyme levels and histological sections of kidney, liver and cardiac muscle. Alpha-solamargine did not affect the weight of the testes and epididymis or the number of spermatozoa but produced a slight irritation and congestion in the epididymis and testis at doses up to 50 mg/kg body weight.     

Solanaceous steroidal glycoalkaloids and poisoning by Solanum torvum, the normally edible susumber berry

Ingestion of immature, environmentally stressed, or cultivar-specific Solanum species (particularly the potato) has been previously associated with gastrointestinal and neurological symptoms caused by solanaceous steroidal glycoalkaloids (SGAs). We report on two geographically, temporally disparate outbreaks of poisoning by susumber berries (Solanum torvum- Solanaceae) and on detection of alkaloids not present in non-toxic berries. Five family members in New York City participated in a traditional evening meal containing Jamaican susumber berries. All those consuming berries were symptomatic the following morning with varying degrees of gastrointestinal distress, dizziness, slurred speech, cranial nerve deficits, and ataxia. The most seriously afflicted patient developed hypertension, confusion, proximal upper extremity weakness, and hypercapnic respiratory failure requiring prolonged mechanical ventilation. A separate cohort of six patients in Toronto ate unripe Jamaican susumber berries. They presented 14 h post-ingestion with varying degrees of diarrhea, weakness, facial paralysis, slurred speech, ataxia, early hypertension, and proximal weakness. Two patients had ventilatory decompensation; one required intubation. Poisonous berries appeared indistinguishable from non-toxic varieties. We isolated solasonine, larger amounts of solamargine, and other steroidal glycoalkaloids in the toxic berry strains. S. torvum poisoning can produce significant neurological and gastrointestinal effects which appear to be mediated by SGAs present in the berries.     

高效液相色谱法测定龙葵中澳洲茄碱与澳洲茄边碱含量的研究

目的利用高效液相色谱法以澳洲茄碱与澳洲茄边碱为指标对中药龙葵进行定量分析,建立龙葵的含量测定方法。方法采取反相高效液相色谱法,利用C18色谱柱,以乙腈-0.5%磷酸溶液为流动相梯度洗脱,在203 nm波长下测定龙葵中澳洲茄碱与澳洲茄边碱的含量。结果在上述测定条件下,澳洲茄碱在1.002～20.04μg范围内线性关系良好,澳洲茄边碱在1.023～20.46μg范围内线性关系良好。测定方法重复性良好,龙葵中澳洲茄碱回收率为99.29%,RSD为1.13%;澳洲茄边碱回收率为98.94%,RSD为1.28%。结论该方法准确性、灵敏度、重复性高,简便,准确,易操作,可作为龙葵的质量控制方法。     

澳洲茄边碱对肝癌 HepG2细胞增殖及凋亡的影响

目的：探讨澳洲茄边碱（SM ）对 HepG2细胞增殖、凋亡的影响及其可能作用机制。方法用不同浓度SM 5、10、15和20μg／ml分别处理 HepG2细胞3、6、12和24 h ,并设不加药的对照组。采用MTT法检测 HepG2细胞增殖,DAPI染色法观察细胞核形态的变化,流式细胞术检测细胞凋亡和周期,Western blot法检测B细胞淋巴瘤‐白血病2（Bcl‐2）、Bcl相关X蛋白（Bax）、半胱天冬氨酸蛋白酶3（Caspase‐3）及Ki67的蛋白表达。结果与对照组相比,SM 呈剂量依赖性地抑制HepG2细胞增殖,促进HepG2细胞凋亡,将细胞周期阻滞于G2／M期,并且上调Bax和Caspase‐3蛋白表达,下调Bcl‐2和Ki67蛋白表达（P＜0．05或P＜0．01）。结论 SM 能有效抑制 HepG2细胞的增殖,促进细胞凋亡的发生;SM上调Bax和Caspase‐3表达,下调Bcl‐2和Ki67表达,细胞周期阻滞于G2／M期可能是其发挥上述作用的机制。     

pH梯度法制备澳洲茄边碱脂质体

目的:研究制备澳洲茄边碱脂质体的最佳工艺,并建立起包封率的测定方法。方法:采用pH梯度法制备澳洲茄边碱脂质体,以包封率为指标进行工艺优化,通过SephadexG-50葡聚糖凝胶柱分离脂质体,高效液相色谱法测定澳洲茄边碱的包封率。结果:制备澳洲茄边碱脂质体的最佳条件,药物与卵磷脂的质量比为1∶30,胆固醇与卵磷脂质量比为1∶4,脂质体外水相pH值最终调至6.5,孵育温度为55℃,此时的包封率可以达到70%以上。结论:用pH梯度法能制备包封率较高的澳洲茄边碱脂质体。     

Toxicological evaluation of acute and sub-chronic ingestion of hydroalcoholic extract of Solanum cernuum Vell. in mice

Ethnopharmacological relevance

Solanum cernuum Vellozo is a Brazilian shrub or small tree, restricted to Southwest states of the country. It has been widely used for the treatment of many ailments. The pharmacological activity of the extract on gastric ulcer has been the major therapeutic target proposed by the population investigated.

Materials and methods

In the acute toxicity test was used increasing doses of the extract (2, 4, 8, 12, 16, 20 and 25 g of extract per kilogram of body weight). The animal behavior was observed from 5 h after a single administration of the extract and subsequently monitored daily until the fourteenth day, beyond the calculation of the estimated LD50 of the extract. In the test sub-chronic toxicity was used two doses of the extract (0.1 and 1.4 g/kg) and the parameters analyzed over 31 days were: body weight, food intake, behavior, respiratory rate, movement and mortality of animals. After anesthesia, blood samples were collected for hematological and biochemical analysis. The animals were euthanized followed by macroscopic analysis of the stomach and intestine. Liver, lungs and kidneys were removed, weighed and analyzed histopathologically.

Results

In the acute toxicity test was observed a dose-dependent mortality and the value of estimated LD50 was 14.50 g/kg. In the hematological and biochemical analyses there were significant increase in the activities of AST and ALT indicating liver toxicity, but the extract was not able to alter food intake, body weight and organ weights after 31 days of treatment and it did not produce significant histopathological changes.

Conclusion

Therefore we can consider the hydroalcoholic extract of Solanum cernuum Vell as practically non-toxic in acute administration and safe in the sub-chronic administration, as hepatotoxicity was observed only with the highest dose used, not with the dose routinely used by the native population.