| Lymphoma is one of the top ten common malignancies in China. According to statistics, about 8.4 million people would be found suffering from lymphoma per year, between them more than 4.7 million dead, and the number is rising at a rate of 5% per year. About 90% of the patients suffering from non-Hodgkin’s lymphoma, this type of lymphoma have a higher degree of malignancy and a worse prognosis. The treatment effect of the commonly used chemotherapy drugs is obvious, but often accompanied by severe adverse reactions.Compared with general anti-tumor chemotherapy drugs, traditional Chinese medicine was considered to have multiple components and multiple targets, can act on tumor’s different segments, and some other characters such as low toxicity, antagonizing drug-resistance, enhancing the body immunity and higher life quality of patients. In recent years, Traditional Chinese medicine anti-tumor research has become a hotpot in anti-tumor drug research area.At present, the research on anti-tumor effect of Houttuyniae Herba mostly focused on extractions rather than the volatile oil, such as leukemia, lung cancer, colon cancer, Ehrlich ascites carcinoma, ovarian cancer, and so on. While there was no report about the anti-NHL effect of Houttuyniae Herba.Our previous studies showed that volatile oil from Houttuyniae Herba has good activity against NHL, and there was no relevant report at home and abroad. Therefore, to control the increasing prevalence of NHL, we used lymphoma Raji cells and studied the following aspects:Optimization of the extraction process of volatile oil from Houttuyniae Herba; Spectrum-effect relationship of GC-MS spectrum of volatile oil from Houttuynia cordata Thunb with anti lymphoma effect; Effect of volatile oil from houttuyniae herba on apoptosis of Raji cells; Preparation and evaluation of volatile oil from Houttuyniae Herba nanostructured lipid carriers delivery systems. These studies will provide a reliable reference for the future development and clinical application of volatile oil from Houttuyniae Herba as a kind of anti-tumor medicine.1. Optimization of the extraction process of volatile oil from Houttuyniae HerbaWith extraction rate of volatile oil, factors such as grinding granularity, soaking time, solid-liquid ratio and refluxing time were optimized by orthogonal test. The optimized extraction conditions were:raw herbs crushed into crude powder, adding 10-fold distilled water, soaking for 10 hours and refluxing for 10 hours. The result of verification experiment shows that the extraction technology is reasonable and feasible, could be used for the extraction of volatile oil from Houttuyniae Herba.2. Study on Spectrum-effect Relationship of GC-MS Spectrum of Volatile Oil from Houttuynia cordata Thunb with Anti lymphoma EffectThe inherent connections were studied between GC-MS Chromatogram and Inhibition rate, with Partial Least Squares Regression Analysis (PLS). There were 7 peaks X6、X14、X15、X22、X25、X26、X27 of the GC-MS Chromatogram play the most important role in the anti-tumor effect, with relatively high positive correlation coefficient:0.192、0.099、0.201、0.281、0.393、0.137、0.101,accounting for 89.88% of the total positive correlation coefficient, determined as effective compounds. The anti-tumor effect of volatile oil from Houttuynia cordata Thunb is the result of combined effect of several component such as (Benzene, 1-methyl-4-(1-methylethyl)-), ((R)-3-Cyclohexen-1-ol,4-methyl-1-(1-methylethyl)-), (Benzenemethanol,.alpha.,.alpha.,4-trimethyl-), (2-Undecanone), Peak cluster ((1-Undecanol), (n-Decanoic acid) and (2,6-Octadien-l-ol,3,7-dimethyl-, acetate, (E)-)), (2-Dodecanone), (Caryophyllene), these components constitute the effective component group.3. Effect of volatile oil from houttuyniae herba on apoptosis of Raji cellsWe observed the inhibition rate induced by volatile oil from Houttuyniae Herba on Raji cell proliferation by MTT, the apoptosis rate by Annexin V-FITC/PI double staining method, and the cell cycle by PI staining method. By those experiments, we studied the inducing Raji cells apoptosis effect and the relevant mechanisms.The MTT results showed that volatile oil from Houttuyniae Herba has a significant growth inhibitory effect on Raji cells, the 50% inhibitory concentration (IC50) of 24h,48h and 72h were 0.1258μL/mL,0.0996μL/mL and 0.1052μL/mL, the inhibition showed significant dose-response relationship in a certain concentration range, the difference between groups was statistically significant (P<0.05). The apoptosis rate test results showed that volatile oil from Houttuyniae Herba can induce Raji cells apoptosis in a dose-dependent manner, the Raji cells apoptosis rate was (3.27±0.74)%, (13.10±0.82)%, (33.33±3.50)%, (55.53±0.90)%, (75.23± 0.87)% after been treated by different concentrations as 0.00μL/mL,0.05μL/mL, 0.1μL/mL,0.15μL/mL and 0.2μL/mL of volatile oil, the difference between groups was statistically significant (F=878.629,P<0.05).The cell cycle analysis showed that the percentage of cells in G0/G1 phase was gradually increased with the increase of volatile oil’s concentration (F=645.930, P<0.05), this pointed that volatile oil from Houttuyniae Herba was able to arrest Raji cells in G0/G1 phase. The results showed that volatile oil from Houttuyniae Herba can induce Raji cells apoptosis, and the mechanism may be related to the block of synthesis of DNA and protein in cells.4. Preparation and Evaluation of nanostructured lipid carrier loaded with volatile oil from Houttuyniae HerbaWe prepared nanostructured lipid carrier loaded with volatile oil from Houttuyniae Herba by melt emulsification-ultrasonic dispersion method. With particle size and Zeta potential as the indicators, the formula and technology were optimized by the single factor exploration and orthogonal design. The method for determination of component content was established by gas chromatographic (GC). The Pharmacy nature of the formulation was evaluated by detected its appearance, shape, size distribution, encapsulation efficiency, drug loading and stability and so on.The optimal formula:monostearin 0.12g, caprylic capric triglyceride 0.08g, poloxamer 188 0.533g, egg yolk lecithin 0.267g, volatile oil from Houttuyniae Herba 0.1g, double distilled water was added to 20mL; Preparation:the colostrum was stirred for 20min, and then ultrasonic dispersing for lOmin, with 100% amplitude.The nanostructured lipid carrier particles were spherical, the particle size was (70.76±1.74)nm, zeta potential was (-25.40±1.08) mv, encapsulation efficiency was 90.33% and drug loading 5.76%. Stability test results showed that high temperature environments would change the color of volatile oil from Houttuyniae Herba nanostructured lipid carrier water dispersion system, while the nanostructured lipid carrier system would become unstable in low temperature environment, thus, nanostructured lipid carrier loaded with volatile oil from Houttuyniae Herba should be stored at room temperatures. |
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