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Aim To explore the diuretic effect, diuretic mechanism and pharmacokinetics of Phytolacca acinosa Roxb., and clarify its "quantity-time-effect" relationship.Methods Firstly, qualified rats were modeled by water load model, given different doses of Phytolacca acinosa Roxb.aqueous extract, then the diuretic effect was investigated.Secondly, Western blot was used to detect the protein expression of aquaporins AQP2, AQP4 and the angiotensin II receptors ATGR1, ATGR2, and renin in the RAAS system in kidney tissues.Thirdly, the established LC-MS/MS biological analysis method was used to detect the esculentoside A(EsA)content in the plasma, calculate the pharmacokinetic parameters and analyze the correlation between the blood concentration and the drug effect.Results The water load model was successfully established.Compared with the model group, hydrochlorothiazide had a significant diuretic effect(P<0.01).Low, medium and high dose groups of Phytolacca acinosa Roxb.all had obvious diuretic effects(P<0.01), EsA also had a significant diuretic effect(P<0.05).Phytolacca acinosa Roxb.aqueous extract and EsA significantly down-regulated the expression of AQP2, AQP4, ATGR1 and renin protein.The pharmacokinetic results showed that the Cmax and AUC0-t of EsA in the plasma of rats in the low, medium, and high dose groups of aqueous extract increased with the increase of the dose.Conclusions Phytolacca acinosa Roxb.had a diuretic effect, which is related to inhibiting the expression of aquaporins AQP2 and AQP4 and inhibiting the expression of angiotensin II type 1 receptor and renin, thereby inhibiting the reabsorption of renal tubules and collecting ducts.
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This paper summarizes the herbal textual research, authenticity identification, processing, composition and pharmacology, clinical studies, industry and agriculture resources development of Phytolaccae Radix based on the ancient herbology works and references. The herbal textual research showed that Phytolaccae Radix had different names like Shanglu or Danglu or Zhanglu. The effects are external application of carbuncle, swelling and sore toxin, internal administration of diuretic and hydroncus. The main producing area is transferred from northwest to southeast. The root has the abnormal structure of concentric ring, which is different from the eight kinds of common adulterants. It is mainly processed with vinegar to reduce toxicity and increase efficacy. Triterpenoid saponins, polysaccharides and pokeweed antiviral proteins (PAPs) are the main effective components of Phytolaccae Radix, which have the functions of diuresis and diarrhea, improving inflammation of kidney, liver, respiratory tract and neuroinflammatory diseases, as well as antibacterial, antiviral, antitumor and other pharmacological activities. It is mainly used for the treatment of cirrhosis ascites, nephrotic syndrome and external application for the treatment of constipation clinically. Phytolaccae Radix has important application value in plant disease resistance, insect resistance and environmental restoration. The clinical efficacy of Phytolaccae Radix is clear and it has made important progress in the research of components, pharmacology, industrial and agricultural resources development, which provides support for the rational development and comprehensive utilization of it.
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Objective:To explore the effect and mechanism of esculentoside on lipopolysaccharide-induced mastitis in mice. Method:Female BALB/c mice were randomly divided into control group, model group, dexamethasone group (DEX, 5.0 mg·kg-1) and esculentoside group (50, 25, 12.5 mg·kg-1). The mastitis model of postpartum female mice (BALB/c) induced by lipopolysaccharide (LPS) was used to analyze the pathological conditions of breast tissue and the activity of recombinant myeloperoxidase（MPO）, factor content and oxidative stress level. Western blot was used to evaluate the effect of esculentoside on Toll-like receptor4 (TLR4)/nuclear transcription factor (NF-κB) signaling pathway proteins. Result:Compared with the normal group, the breast tissue of the model group had typical mastitis changes, such as hyperemia and congestion, the level of MPO increased, and the expression levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) increased significantly (P<0.01). Compared with LPS model group, esculentoside groups could significantly improve the inflammatory damage of mammary gland tissue, reduce the secretion of neutrophils and the activity of MPO, the expression levels of pro-inflammatory factors TNF-α, IL-1β and IL-6 were also significantly down-regulated, the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were increased, while the level of malondialdehyde (MDA) was decreased, and the activation of TLR4/NF-κB signaling pathway was inhibited by esculentoside（P<0.05，P<0.01）. Conclusion:Esculentoside have a protective effect on lipopolysaccharide-induced mastitis in mice, which may be related to the inhibition of TLR4/NF-κB signaling pathway protein expression.
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Objective: To compare the protective effect of Phytolaccae Radix and its processed products on nephropathy induced by doxorubicin (DOX) in rats, and explore its mechanism. Method: A rat model of nephropathy was established by a single tail intravenous injection of DOX hydrochloride. Content of esculentoside A (EsA) in Phytolaccae Radix and its processed products was determined by HPLC-ELSD. Contents of serum total protein (TP), albumin (Alb), urea nitrogen (BUN), serum creatinine (SCr), total cholesterol (TC) and urine protein (UP) were determined by enzyme-linked immunosorbent assay (ELISA). The mRNA and protein expression of transforming growth factor-β (TGF-β) in renal tissue of rats was examined by real-time quantitative polymerase chain reaction (Real-time PCR) and immunohistochemistry. Result: A single intravenous injection of DOX could induce a severe nephrotic syndrome associated with decreased serum TP, Alb and elevated serum BUN, SCr, TC, and a high urinary excretion of protein (Pβ in renal tissue of model group rats was significantly higher than that of blank group (PPPConclusion: Phytolaccae Radix and its processed products can improve the symptoms of DOX nephropathy model rats in different degrees, among which the vinegar prepared products have the strongest effect, and this effect may be related to the reduction of TGF-β expression in renal tissue.
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A new triterpenoid saponin named esculentoside U(1), along with the five known compounds, was isolated and characterized from the roots of Phytolacca acinosa, a commonly used traditional Chinese medicine with anti-inflammatory and anti-rheumatoid activities. The structure of the new saponin was elucidated as 3-O-[β-D-glucopyranosyl-(1→4)]-β-D-xylopyranosyl]-2, 23-dihydroxyolean-11, 13(18)-diene-28, 29-dioic acid 29-methyl ester(1). The assignment of all NMR signals of 1 was performed by means of 2D-NMR experiments.
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In order to study the biosynthesis pathway of esculentoside A, the Illumina HiSeq 4000 highthroughput sequencing method was used to analyze the transcriptome of Phytolacca americana seedlings. The 9.60 Gb clean data were obtained after the transcriptome of P. americana assembled by Trinity software. The total 63 957 unigenes were obtained after assembly and the average length was 988.82 bp, among them 24 517 unigenes (38.33%) were annotated in the public databases Nr, Swiss-Prot, COG, KOG, Pfam, GO and KEGG. According to the assignment of KEGG pathway, 53 unigenes were involved in terpenoid backbone biosynthesis and 8 unigenes involved in triterpenoid biosynthesis. Additionally, there were 417 unigenes assigned to other secondary metabolic pathways in P. americana. The post-modification enzyme genes involved in the esculentoside A biosynthesis were also analyzed in the transcriptome of P. americana. The results indicated that 130 unigenes may have the function of CYP450 which was involved in oxidation/hydroxylation modification of P. americana secondary metabolites. Furthermore, 46 unigenes had the function of glycosyltransferase UGT. The transcriptome data of P. americana laid a foundation for studying the biosynthesis pathway of esculentoside A and other secondary metabolites, and also provided theoretical basis for formation of medicinal materials quality.
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Objective To observe the influence of serum containing esculentoside A(EsA) on the proliferation of glomerular mesangial cell (GMC) and the activation of ERK1/2-AP-1 pathway of glomerular mesangial cell induced by IL-1β.Methods SD rats were gavaged by different doses of EsA(5,10,20,40 mg/kg) for getting medicated sera.The control group was set (gavage by 0.5sodium carboxymethylcellulose);the EsA medicated serum was used to treat rGMC.The control serum group was set.The influence of EsA medicated serum in each group on rGMC proliferation was detected by MTT;the rGMC was divided into the blank control group,IL-1β single action group,IL-1β+ EsA double action group,IL-1β+ U016 double action group and IL-1β+ U0126 + EsA combined action group,which were synchronized and then cultured for 48 h.Western blot was used to detectthe expression of p-ERK1/2 and AP-1 an the imaging analysis was performed.Results The EsA medicated serum(5-10 mg/kg gavage) inhibited the cellular proliferation(P＜0.05 or P＜0.01);the IL-1β group promoted the expression of p-ERK1/2 and AP-1 in rGMC(P＜ 0.05),after acting on rGMC in the IL-1β+EsA double action group,IL-1β+U0126 double action group and IL-1β+U0126+EsA combined action group,the expression of p-ERK1/2 and AP-1 was decreased(P＜0.05).Conclusion Serum containing EsA (5～10 mg/kg gavage) significantly inhibits the rGMC proliferation;EsA inhibits IL-1β induced rGMC proliferation,its action pathway on ERK1/2-AP-1 is one of mechanisms for inhibiting rGMC proliferation.
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Objective To understand the intensity and characteristics of acute toxicity of esculentoside A on mice and measure relevant parameters and observe its diuresis effect on rat. Methods After intraperitoneal injection of different concentrations of esculentoside A to mice, toxic reactions were observed. Rats with water load were intraperitoneally injected with different doses of esculentoside A. Total urine volume in six consecutive hours after the injection was determined. Results The LD50 of esculentoside A calculated by Bliss method was 26. 19 mg · kg-1 , and the 95% confidence interval was 23. 11-29. 85 mg·kg-1 . The mortality and acute toxicity of esculentoside A appeared to be dose-dependent while the blank control group had no abnormal reaction. The urine volume was significantly different between high dose group and the negative control group. No significant difference in urine volume was found between middle and the negative control group, and between low dose group and the negative control group. Conclusion Esculentoside A is poisonous to mice when single dose was intraperitoneally injected, and high dose of esculentoside A has diuresis effect on rat.
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Objective To observe the effect of esculemoside A (EsA)on the proliferation and expression of CDK2 and P27 of rats glomerular mesangial cell .Methods rGMC was cultured in vitro ,The cell growth and cell toxicity were detected by MTT assay ;rGMC proliferation was observed by Rnase/PI-Flous ;The expression of CDK2 and P27 were measured by Western blotting .Results EsA at observed dose has not apparent cytotoxicity effect on rGMC .EsA(2 .5-5 .0 mg/L) inhibited the proliferation of rGMC after 48h .EsA increased the number of the G1 phase and reduced the number of the S phase of IL-1βinduced rGMC .At the same time ,EsA inhibited the expression of CDK2 and promoted the expression of P27 of IL-1βinduced rGMC .Conclusion The GMC is one of the mainly target cell which EsA bing therapeu tical action .EsA inhibited proliferation of IL-1βinduced the GMC ,inhibited the expression of CDK2 and activated the expression of P27 may be its mechanism .
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Objective: To optimize the best processing technique of Phytolaccae Radix stir-baked with vinegar. Methods L9(34) orthogonal test was used with three factors: baked temperature, time, and vinegar amount. The content of esculentoside A was determined by RP-HPLC, and the mucosa irritation test of mice was detected. The processing technology was optimized by comprehensive evaluation. Results: The optimized processing technology was satisfied with some conditions as the following: stir-baked with 30% vinegar for 30 min at the temperature of 120 °C. Phytolaccae Radix was moistened with vinegar to be exhausted. Conclusion: The optimized processing technology of Phytolaccae Radix is stable and feasible with reliable repetition.
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Objective:To investigate the effect of esculentoside (EsA) on apoptosis of murine thymocyte. Methods:Using electronic microscope, DNA agarose electrophoresis and flow cytometry analysis,the effect of EsA on apoptosis of murine thymocyte was examined. Results:The result showed that apoptosis of activated thymocyte by ConA was markedly promoted by 2.5, 5, 10 ?g/ml EsA in murine thymocyte culture for 3 h, but the spontaneous apoptosis was not affected by EsA. Conclusion:The results suggest that EsA has the positive effect on apoptosis of activated murine thymocyte.
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Interferon-gamma(IFN-?)was induced by PWM or esculentoside in human splenic lymphocytes. PWM-induced IFN-r was partially purified to a spe. act. of 106 RU/mg protein by batch adsorption on controlled-pore glass (CPG) beads and desorption by ethylene glycol in PBS.Use of native-made micro-pore glass(MPG)instead of CPG to purify IFN-r showed similar purification efficacy.Both CPG and MPG recovered could be regenerated for repeated use.The yield from glass beads was further purified to a spe.act.of 107 RU/mg protein, with about 78%recovery, by the anti-new-born calf serum antibodies-Sepharose 4B(ACS)column chromatography.Almost all the antiviral activity induced by PWM or esculentoside was destroyed by pH 2 or heat at 56℃ and neutralized by monoclonal antibody against HuIFN-r, thereby identified as HuIFN-r. Bio-Gel P-200 chromatography resulted in two peaks of activity for PWM-induced IFN, a major one with molecular weight of 40,000-45,700 and a minor one of 25,700 dalton, but only one peak with molecular weight of 45,700-49,000 for esculentoside-induced IFN.
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The effects of pokeweed mitogen (PWM) and esculentoside (Es) in inducing immune interferon (IFN-?) from normal traumatic spleen and patient's spleen cells were studied.The interferon induced was proved to be human immune interferon (HUIFN-?) by determination of its sensitivity to pH 2 and unstabitity at 56℃ as well as neutraliztion by monoclonal antibody to HUIFN-?.The average titres of IFN-?,induced with PWM or Es,from the sphen cells of 10 traumatic pesons were 3.40?0.59 and 2.92?0.42 Log10 CPI50 U/ml respectively,and those from the spleen cells of 8 patients were 3.69?0.35 and 3.01?0.35 LogloCPI50 U/ml respectively.The effec of Es in inducing IFN-Y from either traumatic or patient's spleen cells was lower than that of PWM (P