Effect of madecassoside on hippocampal NR2B expression and cognitive function in rat traumatic brain injury model / 国际外科学杂志

Objective:To investigate the Effect of madecassoside (MC) on hippocampal NR2B expression and cognitive function following traumatic brain injury (TBI).Methods:One hundred and sixteen SD rats were provided by the experimental animal center of Medical College in Xi′an Jiao Tong University. Rats were randomly divided into four groups ( n=29) as following: Control group (Con group), TBI group, MC treated TBI rats group (TBI+ MC group) and MC treated control rats group (Con+ MC group). NR2B protein levels in hippocampus were detected by Western-blot at 12 h, 1 d, 3 d, 7 d, 14 d and 21 d post trauma. Hippocampal NR2B positive cells were studied by immunohistochemical (IHC) staining at 21 d post trauma. Cognitive functions of rats were evaluated by Morris water maze (MWM) test from 21 d to 25 d post trauma. Results:Expression of hippocampal NR2B in TBI group at 12 h, 1 d and 3 d post injury were 3.31±0.28, 2.17±0.31 and 1.96±0.31, which presented statistical difference among the three time-points ( P<0.05) and were significantly increased compared to Con group ( P<0.05). However, there was no difference of NR2B level between TBI group (0.93±0.13) and Con group ( P>0.05) at 7 d post injury. In addition, NR2B expression in TBI group at 14 d and 21 d post injury were 0.45±0.04 and 0.21±0.04, which were much lower than that in Con group. IHC staining revealed that the number of hippocampal NR2B positive cells in TBI group (33.26±9.71) were lesser than that of Con group (86.62±17.05). Increased Escape latency, decreased platform crossing times and target quadrant duration were found in TBI group compared with Con group ( P<0.05). Hippocampal NR2B expression in TBI+ MC group at 12 h, 1 d and 3 d post injury were 2.37±0.34, 1.38±0.22 and 1.14±0.16. Difference among the three time-points exhibited statistical significance ( P<0.05). In addition, they were much lower than that in TBI group at the same time-points ( P<0.05), but no difference was found between TBI+ MC group (0.97±0.06) and TBI group at 7 d post injury ( P>0.05). Moreover, NR2B expression in TBI+ MC group at 14 d and 21 d post injury were 0.86±0.08 and 0.52±0.06, which were much higher than that in TBI group ( P<0.05). IHC staining showed the number of hippocampal NR2B positive cells in TBI+ MC group (69.08±12.24) were much more than that of TBI group (33.26±9.71). A decrease of escape latency, an increase of platform crossing times and target quadrant duration were found in TBI+ MC group compared to TBI group ( P<0.05). Conclusion:MC treatment could inhibit the up-regulation of NR2B in hippocampus at early period of TBI and prevent the down-regulation of NR2B at advanced stage of TBI, which led to a remarkable improvement for the cognitive dysfunction caused by TBI.

Effect of different compatibility ratios of Centella asiatica-Rhubarb on the dissolution rate of effective components of Centella asiatica / 国际中医中药杂志

Objective@#To study the effects of different compatibility ratios of Centella asiatica- Rhubarb on the dissolution rates of madecassoside and asiaticoside.@*Methods@#By using the HPLC method to analyze the content of madecassoside and asiaticoside in the Centella asiatica extract solution as well as Centella asiatica - Rhubarb extract solution with 6 different kinds of compatibility ratios. The tests were carried out by Thermo C18 (250 mm×4.6 mm, 5 μm) by isocratic elution with acetonitrile and 2 mmol/L of beta-cyclodextrin solution as mobile phase at a flow rate of 1 ml/min, the column temperature was 30 ℃ and the detection wavelength was 205 nm.@*Results@#Compared with the Centella asiatica extract solution, the dissolution rate of madecassoside decreased significantly with the increase of the proportion of rhubarb. The combination of Centella asiatica and Rhubarb had little effect on the content of asiaticoside.@*Conclusions@#Considering the clinical application and efficacy of Centella asiatica, the optimal ratio of Centella asiatica-Rhubarb is 10:1 and 15:1.

Validated high performance liquid chromatography for simultaneous determination of stability of madecassoside and asiaticoside in film forming polymeric dispersions

ABSTRACT The objective of the work was to validate the high performance liquid chromatography for simultaneous determination of stability of madecassoside and asiaticoside in Centella asiatica (L.) Urb., Apiaceae, extract-loaded film forming polymeric dispersions. High performance liquid chromatography method was validated in five topics: linearity and range, limit of detection and limit of quantitation, specificity, precision, and accuracy. Results showed the method had a good linearity (R2 > 0.9990) in the range of 5-150 µg/ml and specific. The limit of detection and limit of quantitation of madecassoside were 81 and 245 ng/ml and asiaticoside were 21 and 64 ng/ml, respectively. The percent relative standard deviation of intraday and interday precision were less than 1 and 3%, respectively. The accuracy presented as percent recovery was 101.54-103.29% for madecassoside and 100.39-102.58% for asiaticoside. This validated high performance liquid chromatography method was used to determine the stability of the formulation containing Centella asiatica extract. Centella asiatica extract-loaded film forming polymeric dispersions used Eudragit® RS 30D and Eudragit® RL 30D as film former, glycerin as plasticizer, and absolute ethanol as solvent and penetration enhancer. Three formulations with different ratio of Eudragit® RS 30D and Eudragit® RL 30D were prepared and stored for 90 days at 4 ºC, 25 ºC, and 40 ºC. Stability results showed that almost all of the formulations were unstable at 25 ºC and 40 ºC. Except, two of three formulations were stable at 4 ºC. However, the formulation was further developed to improve the stability of madecassoside and asiaticoside in the formulation.

Madecassoside impedes invasion of rheumatoid fibroblast-like synoviocyte from adjuvant arthritis rats via inhibition of NF-κB-mediated matrix metalloproteinase-13 expression / 中国天然药物

Fibroblast-like synoviocytes (FLS) play a pivotal role in Rheumatoid arthritis (RA) pathogenesis through aggressive migration and invasion. Madecassoside (Madec), a triterpenoid saponin present in Centella asiatica herbs, has a potent anti-inflammatory effect. In the present study, Madec exerted an obvious therapeutic effect in reversing the histological lesions in adjuvant-induced arthritis (AIA) rats. To recognize the anti-rheumatoid potentials of Madec, we further investigated whether Madec interfered with FLS invasion and metalloproteinase (MMP) expression. In cultures of primary FLS isolated from the AIA rats, Madec (10 and 30 μmol·L) was proven to considerably inhibit migration and invasion of FLS induced by interleukin 1β (IL-1β), but exhibiting no obvious effect on cell proliferation. Madec repressed IL-1β-triggered FLS invasion by prohibiting the expression of MMP-13. Additionally, Madec suppressed MMP-13 transcription via inhibiting the MMP-13 promoter-binding activity of NF-κB. Our results further showed that Madec down-regulated the translocation and phosphorylation of NF-κB as demonstrated by Western blotting and immunofluorescence assays. In conclusion, our results suggest that Madec exerts anti-RA activity via inhibiting the NF-κB/MMP-13 pathway.

Madecassoside impedes invasion of rheumatoid fibroblast-like synoviocyte from adjuvant arthritis rats via inhibition of NF-κB-mediated matrix metalloproteinase-13 expression / 中国天然药物

Fibroblast-like synoviocytes (FLS) play a pivotal role in Rheumatoid arthritis (RA) pathogenesis through aggressive migration and invasion. Madecassoside (Madec), a triterpenoid saponin present in Centella asiatica herbs, has a potent anti-inflammatory effect. In the present study, Madec exerted an obvious therapeutic effect in reversing the histological lesions in adjuvant-induced arthritis (AIA) rats. To recognize the anti-rheumatoid potentials of Madec, we further investigated whether Madec interfered with FLS invasion and metalloproteinase (MMP) expression. In cultures of primary FLS isolated from the AIA rats, Madec (10 and 30 μmol·L) was proven to considerably inhibit migration and invasion of FLS induced by interleukin 1β (IL-1β), but exhibiting no obvious effect on cell proliferation. Madec repressed IL-1β-triggered FLS invasion by prohibiting the expression of MMP-13. Additionally, Madec suppressed MMP-13 transcription via inhibiting the MMP-13 promoter-binding activity of NF-κB. Our results further showed that Madec down-regulated the translocation and phosphorylation of NF-κB as demonstrated by Western blotting and immunofluorescence assays. In conclusion, our results suggest that Madec exerts anti-RA activity via inhibiting the NF-κB/MMP-13 pathway.

Preliminary study on artificial planting technique of Centella asiatica / 中草药

Objective: To study the artificial planting technique of Centella asiatica. Methods: The yield and the total content of asiaticoside and madecassoside in C.asiatica were used as evaluating indicators. And the influences of different factors such as soil type, planting density, base fertilizer, fertilization, and shading degree on planting effect were investigated. Results: C.asiatica was suitable to be planted in peat soil/perlite (1∶1), the planting density was 20 cm × 20 cm, the base fertilizer was organic fertilizer/ compound fertilizer (1∶1) and its consumption was 7.5 g/m2, urea (18 g/m2) was applied after 4 months and compound fertilizer (45 g/m2) was applied after 5 months, the appropriate shading degree was 75%. Conclusion: The research results could be used to establish the GAP planting base of C.asiatica.

Determination of madecassoside and asiaticoside in Centella asiatica formula granules by HPLC method / 药学实践杂志

Objective To establish a HPLC method for determination of madecassoside and asiaticoside in Centella asiatica formula granules.Methods Chromatographic separation was performed on Ultimate AQ-C18 column(4.6 mm×250 mm,5 μm) with mobile phase of acetonitrile(A)-2 mmol/L β-cyclodextrin(0~30 min:21% A→23% A;30~60 min:23% A→25% A).The flow rate was set at 1.0 ml/min, the column temperature at 30 ℃ and detection wavelength at 205 nm.Results Madecassoside and asiaticoside showed good linearity (r>0.9995) in the ranges of 0.187 7~3.754 μg and 0.184 3~3.686 μg respectively.The specificity, repeatability, precision,recovery and stability were satisfied to the method validation requirements of China Pharmacopoeia.Conclusion The method can determine madecassoside and asiaticoside in Centella asiatica formula granules.

Effect of madacassoside on D-galactose-induced learning and memory impairment in mice / 中国药理学通报

Aim To investigate the protective effect and mechanism of madecassoside from hydrocotyle sibthorpioides (MHS)on learning and memory impair-ment induced by D-galactose (D-gal)in mice.Meth-ods Totally 75 SPF Kunming male mice were divided into normal control group,model group,low-,middle-and high-doses of MHS treated groups.The dementia models were induced with D-gal.The learning and memory functions were tested by Morris water maze, the level of Aβ1 -42 and its related proteins in the hip-pocampus was determined by Western blot,and the ex-pression of Aβ-related genes were determined by RT-PCR.Results Compared with model group,MHS markedly decreased the content of Aβ1 -42 ,inhibited the expression of APP,BACE1 and CatB,but promo-ted the expression of NEP and IDE.In addition,AHS significantly increased the expression of plasticity-relat-ed proteins including PSD-95,p-NMDAR1,p-CaMKII, p-PKACβ,PKCγ,p-CREB and BDNF.Conclusions MHS could remarkably ameliorate the learning and memory impairment induced by D-gal in mice,which may be due to its ability to inhibit the Aβgeneration and deposition and promote synaptic plasticity related protein expression.

Inhibitory effect of madecassoside on LPS-stimulated microglia / 中国病理生理杂志

[ ABSTRACT] AIM:To observe the inhibitory effect of madecassoside on the LPS-stimulated microglia and to inves-tigate its possible mechanism.METHODS:Microglia cells of neonatal Sprague-Dawley ( SD) rats were cultured, isolated and purified.Microglia cells were activated with lipopolysaccharide ( LPS) .The inhibitory effect of madecassoside on micro-glia was measured by MTT assay.Tumor necrosis factor alpha (TNF-α), interleukin 1β(IL-1β) were detected by ELISA. Cell cycle and apoptotic rate were evaluated by flow cytometry.The expression of TLR4 was detected by Western blotting. The expression of NF-κB was detected by RT-PCR.RESULTS: LPS induced the proliferation of microglia and release in-flammatory cytokines significantly.Compared with LPS group, madecassoside inhibited the proliferation of microglia induced by LPS in a dose dependent manner.The IC50 value of madecassoside was 10.97 nmol/L to microglia after incubation for 48 h.Madecassoside also decreased the levels of TNF-αand IL-6, increased the ratios of microglia at the G2 phase and the ap-optotic rate, decreased the expression of TLR4 and NF-κB significantly (P<0.05).CONCLUSION:Madecassoside has in-hibitory effects on the proliferation of LPS-stimulated microglia, by which the mechanism may be related to inhibition of the expression of TLR4 and NF-κB, change of cell cycle distribution and induction of microglia apoptosis.

Preparing technology of asiaticosides with high-purity / 中草药

Objective: To study the preparing technology of asiaticosides with high purity. Methods: Macroreticular resin column chromatography and recrystallization were used to optimize the preparing technology of asiaticosides. HPLC method was established for the contcent determination of asiaticosides. Results: Centella asiatica was subjected to 75% ethanol distilment, water and acid precipitation, HPD100 macroreticular resin separation, and recrystallization in acetic ester. Then asiaticosides with high purity over 90% were obtained. Conclusion: The process is stable and feasible, and it could be used for the preparation of high-purity asiaticosides.

A LC-ESI-MS method for the simultaneous determination of madecassoside and its metabolite madecassic acid in rat plasma: comparison pharmacokinetics in normal and collagen-induced arthritic rats / 中国天然药物

To develop a simple and highly sensitive high performance liquid chromatography with electrospray ionization mass spectrometric (LC-ESI-MS) method for the simultaneous determination of madecassoside and its major metabolite madecassic acid in rat plasma, and compare the pharmacokinetics of the two compounds in normal and collagen-induced arthritis (CIA) rats. Glycyrrhetinic acid was used as the internal standard (IS). Chromatographic separation was accomplished on an Inertsil ODS-3 column, using a gradient elution with the mobile phase composed of acetonitrile and water acidified with 0.1% (V/V) formic acid. Detection was achieved by ESI-MS under the negative selected ion monitoring (SIM) mode. In normal and CIA rats, madecassoside (30 mg·kg(-1)) was orally administered for 21 consecutive days from the day of arthritis onset. For madecassoside, the linear range was 10-1 000 ng·mL(-1) with the square regression coefficient (r) of 0.998 9, while for madecassic acid, the linear range was 10-500 ng·mL(-1) with the square regression coefficient (r) of 0.996 1. The lower limit of quantification was 10 ng·mL(-1) for both analytes. The intra- and inter-day precision ranged from 1.78% to 13.42% for madecassoside and 2.30% to 14.90% for madecassic acid, and the accuracy was between -0.95% and 6.30% for madecassoside and between -1.48% and 5.34% for madecassic acid. The average recoveries of madecassoside, madecassic acid and IS from spiked plasma samples were > 81%. The developed method was successfully applied to the pharmacokinetic study of madecassoside and madecassic acid in rats after an oral administration of madecassoside. During initial 7 days of dosing, the cmax and AUC of madecassoside were greatly decreased and Vd/F was markedly increased in CIA rats, and no significant difference was observed on the first day of dosing. In contrast, the T1/2, cmax and AUC of madecassic acid were significantly increased, and Ke of madecassic acid was greatly decreased in CIA rats compared with normal rats. Along with repeated administration of madecassoside, the differences of pharmacokinetic parameters of both madecassoside and madecassic acid between CIA and normal rats gradually subsided. The pharmacokinetic characteristics of both madecassoside and madecassic acid in rats were significantly altered by arthritis status, and the differences of pharmacokinetic parameters between arthritis and normal rats coincide with the severity of arthritis.

Quantitative analysis and HPLC specific chromatogram of Centellae Herba and its total glucosides by chiral mobile phase additives / 中草药

Objective: To separate the isomers of madecassoside in Centellae Herba and its total glucosides, determine madecassoside and asiaticoside in them, and establish an HPLC specific chromatogram for Centellae Herba total glucosides by using β-cyclodextrin as mobile phase additives. Methods: The Dikma Diamonsil™ C18 column (250 mm x 4. 6 mm, 5 μm) was served as analytical column and acetonitrile-2 mmol/L β-cyclodextrin (24 : 76) as mobile phase at a flow rate of 1. 0 mL/min, the detection wavelength was 205 nm and column temperature was 30°C. Theoretical plate number of asiaticoside was over 4 000. Results: The isomers in Centellae Herba and its total glucosides were separated with the additives of β-cyclodextrin, meeting the requirements of quantitative analysis. In which, madecassoside and asiaticoside showed a good linearity in the range of 0.181-18. 1 μg (r=0. 999 98) and 0.195-19. 5 μg (r=0. 999 98) each. The mean recovery of madecassoside and asiaticoside in Centellae Herba was 101. 2% and 101. 6% (n=9), and that of its 11 batches of total glucosides was 100. 3% and 99. 3%, respectively. Under the qualitative conditions, the six common peaks in HPLC specific chromatogram of its 11 batches of total glucosides could be used as marker peaks for qualitative identification. Conclusion: The analytical method is proved to be convenient and accurate, which can be used for the quality control of Centellae Herba and its total glucosides.

Determination of madecassoside in SD rats plasma by solid-phase extraction RP-HPLC / 重庆医科大学学报

Objective:To establish a method for madecassoside determination by solid-phase extraction RP-HPLC in SD rats plasma. Methods:The sample was cleaned with an Strata-X solid-phase extraction . HPLC conditions were as follows:Welchrom-C18 column (250 mm?4.6 mm,5 ?m),water-acetonitrile(24∶76)as mobile phase(flowrate:1.0 ml/min),cucurbitacin B as internal standard, detection wavelength of 225 nm,and column temperature of 30℃. Results:In this situation,the remaining times of the madecassoside and cucurbitacin B were 5.11 min and 8.14 min,respectively. The linear range of the method was 5 ?g/ml～1.5 mg/ml (r=0.999 8).The low limit of detection was 1 ?g/ml. The method recoveries and the extraction recoveries were 90.21%～95.65% and 80.65%～87.04%,respectively. The within-day RSD and between-day RSD were between 1.31%～3.07%(n=5). Conclusion:Themethod is simple,convenient,accurate, reproducible,and suitable for studying the pharmacokinetics of madecassoside in SD rats .

Therapeutic potential of madecassoside in transgenic mice of amyotrophic lateral sclerosis / 中草药

Objective To investigate the therapeutic potential of madecassoside in mice expressing a mutant human Cu,Zn superoxide dismutase(SOD1)-G93A linked to human amyotrophic lateral sclerosis(ALS).Methods Effects of madecassoside on onset of clinical disease,survival of mice,decline of motor function,and motor neuron(MN) degeneration were observed by behavioral analysis and histological eva-(luation.) Results Madecassoside(61.1?11.0) and(185.6?18.7) mg/(kg?d) ig administration,beginning at 70 d of age until death,prolonged survival of SOD1-G93A ALS mice by 11.4 and 9.4 d,respectively(P