| Aim:Abnormal Savda Munziq (ASMq) is a herbal formula composed of ten medicinal herbs, namely Cordia dichotoma, Ziziphus jujuba, Glycyrrhiza uralensis, Anchusa italica, Foeniculum vulgare, Euphorbia humifusa, Lavandula angustifolia, Melissa officinalis and Alhagi pseudoalhagi. It is a patent medicine (Patent No.: C082130082.8) and used in Traditional Uighur Medicine in Xinjiang region of China for the treatment and prevention of cancer, diabetes and cardiovascular diseases. Recent studies have shown that ASMq can scavenge free radicals and protect mitochondria and DNA from oxidative damage, and significantly inhibit the growth and viability of human hepatoma cell line, HepG2, by increasing cytoplasmic leakage and inhibiting protein, DNA and RNA synthesis. This thesis aims to: (a) study the chemical composition of ASMq and quantify its main components; (b) find the in-vitro anticancer active components of ASMq; (c) find preparation methods which suitable for manufacturing of different extracts from ASMq; (d) establish a HPLC method for analyzing ASMq and its enriched active components; (e) evaluate the in-vitro anticancer activity of single herbs of ASMq.Methods:1.A preliminary phytochemical screening test has been done to confirm the presence or absence of some chemical components in ASMq; The content of total flavonoids, expressed as rutin equivalents, was measured by using the NaNO2-Al(NO3)3-NaOH colorimetric assay; The content of total phenolics, expressed as gallic acid equivalents, was measured by using the Folin-Ciocalteu assay; The content of total saccharides, expressed as glucose equivalents, was measured by the phenol-sulfuric acid method; The content of total saponins, expressed as ammonium glycyrrhizinate equivalents, was measured by the vanillin–perchloric acid method; The content of total alkaloids was measured by gravimetry.2.Four extracts, namely Extr-1, Extr-2, Extr-3 and Extr-4, were prepared from ASMq. Among these extracts, Extr-1?Extr-2 and Extr-3 were obtained by macroporous resin chromatography as well as Extr-4 by polyamide chromatography; Thin-layer chromatography (TLC) analysis and preliminary chemical tests were performed on each extract of ASMq and the content of main components of each extract was determined; the in-vitro antiproliferative activity of these extracts on HL-60 cells were compared; the IC50 value was determined for Extr-4; Thin-layer chromatography (TLC) analyses of ASMq extracts were performed by using fast blue salt B reagent (FBS) and anisaldehyde/ sulphuric acid reagent (AS) as spray reagents, respectively. Silicagel 60 F254 plates were used as stationary phase.3.Fractionation of Extr-4 was performed using solid phase extraction (SPE). Extr-4 was dissolved in 40% MeOH and applied onto a SPE column, which had been preconditioned with MeOH and H2O. The unretained components were collected to yield the first subfraction (SPE-p). The compounds retained on the sorbent beds were eluted sequentially with H2O and 20%, 40%, 60% and 80% MeOH. The resulting eluates were also collected separately and evaporated under reduced pressure to dryness to yield SPE-0, SPE-20, SPE-40, SPE-60 and SPE-80.HL-60 cells were collected and plated in wells at a density of 1.0×106 cells per ml and and allowed to attach for 24 hours. The wells were divided into 7 groups. Cells in the first group were treated with blank solvent, while cells in the other six groups were treated with SPE-p, SPE-0, SPE-20, SPE-40, SPE-60 and SPE-80, respectively. The final concentration of each SPE fraction in HL-60 cell medium was 50μg/ml. The cell viability was determined by trypan blue exclusion method after 24h, 48h and 72h of incubation (37°C, 5% CO2). Assay was repeated in two succesive trials, each of which was performed in triplicate.4.Accelarated solvent extraction (ASE) was applied to the extraction of single herbs in the formula of ASMq. The extraction of each herb was performed as follow: First, a filter was placed at the bottom of a cell, then powder of each herb was placed inside the cell and weighed. With the top cell cap secured, the cell was placed on the ASE autosampler carousel. The extraction of each herb was performed using CH2Cl2 followed by MeOH as extraction solvents under the ASE conditions. After the extraction process is completed, the instrument will automatically shut down. The resulting extracts were separately transferred into 50 ml round bottom flasks, evaporated at 40℃under reduced pressure and dried under a stream of air to obtain CH2Cl2 and MeOH extract of each herb.HL-60 cells were collected during the logarithmic growth phase, plated in wells at a density of 1.0×106 cells per ml and and allowed to attach for 24 hours. The wells were divided into 19 groups. Cells in the first group were treated with blank solvent, while cells in the other 18 groups were treated with CH2Cl2 extract and MeOH extract of each herb, respectively. The final concentration of an extract of each herb in HL-60 cell medium was 100μg/ml. The cell viability was determined by trypan blue exclusion method after 24h, 48h and 72h of incubation (37°C, 5% CO2). Assay was performed in triplicate.TLC analyses of herbs and ASMq extracts were performed by using fast blue salt B reagent (FBS) and natural products/ polyethylene glycol reagent (NP/PEG) as spray reagents, respectively. Silicagel 60 F254 plates were used as stationary phase.5.The phenolic compounds exist in ASMq were analyzed by means of HPLC-DAD. A Hypersil BDS-C18 (4.0 mm×250 mm, 5μm; Agilent, USA) was used throughout this study. The guard column was LiChospher? 100 RP-18 (4.0 mm×4.0 mm, 5μm; Hewlett Packard, Germany). Detection was performed at 254 nm and the absorption spectra of compounds were recorded between 190 and 450 nm. The elution solvents used were A (H2O-HAc, pH 2.8) and B (MeCN with an aliquot of HAc). The solvent gradient elution program was as follows: a linear increase from 15% to 50% B (0 to 60 min), increase up to 99% B (60 to 70 min), isocratic elution until 75 min, a linear decrease back to 15% B from 75 to 76 min. The flow-rate was 1.2 ml/min. The column was operated at room temperature. The sample injection volume was 10μl.Identification of compounds was achieved by comparing retention time values and UV spectra as well as spiking samples with reference compounds.Results:1.The phytochemical screening of ASMq gave positive tests for saccharides, saponins, phenolics, flavonoids, alkaloids, coumarins and/or lactones, organic acids, amin- oacids and negative tests for tannins, anthocyanins, anthraquinones, cardiac glycosides.The content of total flavonoids, total phenolics, total saccharides and total alkaloids were measured. As a result, a good linearity between concentrations and absorbances of rutin was found in the range of 0.011～0.130 mg/ml, LOD and LOQ were estimated as 0.06 and 0.18μg/ml respectively, recovery ranged from 95.1% to 97.5%, and precision was evaluated as 1.34%, the content of total flavonoids in the water extract and ethanol extract of ASMq were quantified as 16.2mg/g and 19.6mg/g, respectively; A good linearity between concentrations and absorbances of gallic acid was found in the range of 2.2～7.7μg /ml, LOD and LOQ were estimated as 0.01 and 0.03μg/ml respectively, recovery was ranged from 99.5% to 104.8%, and precision was evaluated as 3.39%. The content of total phenolics in the water extract and ethanol extract of ASMq were quantified as 38.28mg/g and 21.54mg/g respectively; A good linearity between concentrations and absorbances of glucose was found in the range of 0.0023-0.0146 mg/ml, the recovery was determined to be 97.5% (n=9), and precision was evaluated as 1.34% (n=5), and the content of total saccharides in Abnormal Savda Munziq were quantified as 57.99±1.36% (n=3) ; A good linearity between concentrations and absorbances of ammonium glycyrrhizinate was found in the range of 0.0115～0.0577mg/ml, the recovery was 90.4% (n=5), and the precision was evaluated as 1.14% (n=5). The content of total saponins in Abnormal Savda Munziq were determined to be 4.69±0.08% (n=3) ; The content of total alkaloids in ASMq should be 0.44% (0.21%+0.23%) at the most.2.The result of TLC analysis and preliminary chemical assay on ASMq extracts have indicated that Extr-1, Extr-3 and Extr-4 contain saccharides, saponins and phenolics, respectively. Extr-2 was also proved to contain phenolics and saponins, but their respective spots are not so distinct, comparing to those observed in Extr-3 and Extr-4. The quantification tests have shown that the content of flavonoids and phenolics in Extr-4 were 33.3%±0.7% and 13.9%±0.7%, respectively, whereas the content of saccharides in Extr-1 and the content of saponins in Extr-3 were 51.4%±0.9% and 60.7%±0.1%, respectively. The total amount of main components of Extr-2 has remained undetermined due to their uncertainty.Extr-4 reduced the cell viability of HL-60 cells remarkably compared with other extracts. Its IC50 value after 24 h, 48 h and 72 h cell exposure were determined to be 105.7μg/ml, 95.1μg/ml and 72.3μg/ml, respectively.3.The result of in-vitro anticancer test has revealed that all SPE fractions showed various degrees of antiproliferative activity on HL-60 cells. Among them, SPE-40 was found to exhibit the strongest inhibitory effect on the proliferation of HL-60 cells by inducing a time-dependent decline in cell viability.After spraying the TLC plates with NP/PEG reagent, all SPE fractions except SPE-80 showed yellow spots. More yellow spots were detected in SPE-40 than in other fractions. On the TLC plate of Extr-4, SPE-40 and rutin, a common spot with same color and same Rf value was detected.4. The extraction of single herbs of ASMq was performed using ASE. Data generated from cell test clearly showed that CH2Cl2 extracts of eight herbs, namely Cordia dichotoma, Euphorbia humifusa, Adiantum capillus-veneris, Glycyrrhiza uralensis, Ziziphus jujuba, Lavandula angustifolia, Foeniculum vulgare, Anchusa italica, Melissa officinalis and MeOH extracts of two herbs, namely Glycyrrhiza uralensis and Euphorbia humifusa, significantly reduced the viability of HL-60 cells with a time-dependent response relationship, respectively. The antiproliferative activity of CH2Cl2 extract of each herb was shown to be stronger than that of its MeOH extract.Spots belong to phenolics were detected in Anchusa italica, Adiantum capillus- veneris, Euphorbia humifusa and Glycyrrhiza uralensis. CH2Cl2 extract of Glycyrrhiza uralensis showed stronger spots compared with other extracts. Besides, Extr-4 and Glycyrrhiza uralensis have shown similar phenolic spots with same color at same position. In addition, SPE-40 and five herbs, namely Euphorbia humifusa, Adiantum capillus-veneris, Anchusa italica, Foeniculum vulgare, Melissa officinalis, have same flavonoid spots with same color (yellow) at same position.5.Using HPLC-DAD, caffeic acid (1), rutin (2), isoquercitrin (3), isorhamnetin 3-O-rutinoside (4), apigenin 7-O-glucoside (5), rosmarinic acid (6), luteolin (7) and formononetin (8), were found in Extr-4. Among these phenolic compounds, 3 and 6 were identified in SPE-p; 1, 3, 5 and 6 were present both in SPE-0 and SPE-20, 2 - 6, were identified in SPE-40, whereas 4, 7 and 8, were identified only in SPE-60.Conclusion:1.The main components of ASMq and their contents were systematically analyzed; methods of determining total phenolics, total flavonoids, total sugars and total saccharides in ASMq were established to evaluate its main composition. The result will provide a scientific basis for the fractionation and quality control of ASMq. As a result, ASMq was found to contain mainly sugars, saponins, phenolics (including flavonoids) and alkaloids. In the order of decreasing content, they can be arranged as sugars, saponins, phenolics and alkaloids. The sum of whole amounts of total saccharides, total saponins and total phenolics account for two thirds of whole weight of ASMq.2.Four extracts, namely Extr-1, Extr-2, Extr-3 and Extr-4, were prepared from ASMq. The qualification and quantification assays have revealed that the main components of Extr-1, Extr-3 and Extr-4 were polysaccharides, saponins and phenolics, respectively. The preparation method of each extract was found to be simple and suitable for industrialization. In-vitro anticancer test and TLC analysis have implied a possible correlation between phenolic compounds and their antiproliferative activity on HL-60 cells. Among the four extracts, Extr-4 was shown to possess the strongest antiprolifertive activity on HL-60 cells.3.Extr-4 was further fractionated into six subfractions by using solid phase extraction. Among these fractions, SPE-40 has shown the strongest inhibitory effect on HL-60 cells. The result of TLC analysis revealed the presence of more flavonoids in this fraction than in any other fractions, indicating that flavonoid could be an active component or one of the active components in ASMq.4. The cell test clearly showed that CH2Cl2 extracts of eight herbs, namely Cordia dichotoma, Euphorbia humifusa, Adiantum capillus-veneris, Glycyrrhiza uralensis, Ziziphus jujuba, Lavandula angustifolia, Foeniculum vulgare, Anchusa italica, Melissa officinalis and MeOH extracts of two herbs, namely Glycyrrhiza uralensis and Euphorbia humifusa, have antiproliferative activity against HL-60 cells. The antiproliferative activity of CH2Cl2 extract of each herb was shown to be stronger than that of its MeOH extract, indicating that the in-vitro anticancer effects of organic compounds with low polarity may be stronger than those of organic compounds with high polarity.5.HPLC-DAD analysis has revealed the presence of caffeic acid, rutin, isoquercitrin, isorhamnetin 3-O-rutinoside, apigenin 7-O-glucoside, rosmarinic acid, luteolin and formononetin in Extr-4. Among these phenolic compounds, rutin, isoquercitrin, isorhamnetin 3-O-rutinoside, apigenin 7-O-glucoside and rosmarinic acid were also exist in SPE-40 (the most active SPE fraction).In conclusion, the bioassay guided fractionation of ASMq led to the isolation of the most active fraction rich in phenolics. |
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