發(fā)布時間：2018-05-12 00:38

  本文選題：蘋果花 + 槲皮素��； 參考：《河北醫(yī)科大學(xué)》2017年碩士論文

【摘要】：蘋果花來源于薔薇科植物蘋果Malus pumila Mill的干燥花。4-5月間采摘,陰干,放置干燥處貯存。蘋果花性平,歸脾、胃、腎經(jīng)。蘋果花茶有治療神經(jīng)痛,解毒,補血,明目,祛痘,美白的作用。經(jīng)查閱,未找到有關(guān)蘋果花的文獻。目前市場上蘋果花以花茶的形式銷售,但未收載于任何質(zhì)量標準中,其質(zhì)量難以有效控制。因此,本試驗對蘋果花性狀、顯微鑒別、薄層鑒別、雜質(zhì)、水分、總灰分、酸不溶性灰分、醇溶性浸出物(熱浸法)等項目進行研究,并進行了有效成分根皮素的含量測定方法研究。在此基礎(chǔ)上對8個省份收集到的12批蘋果花進行了HPLC指紋圖譜研究分析,可為蘋果花的品種鑒別及質(zhì)量標準制定提供一定的依據(jù)。目的:根據(jù)質(zhì)量標準的制定方法要求,對各個項目進行研究,為蘋果花的質(zhì)量標準制定奠定基礎(chǔ)。方法:1對8個省份收集到的12批蘋果花樣品進行性狀鑒別、顯微鑒別研究,測定12批樣品中雜質(zhì)、水分、總灰分、酸不溶性灰分、浸出物的含量。2薄層色譜方法的建立(1)提取條件:實驗過程中摸索了不同的提取溶劑及提取方法,篩選最佳的提取溶劑和提取方法。(2)展開條件:根據(jù)蘋果花成分的特點,確定合適的展開劑、固定相和顯色劑等。3含量測定方法的建立(1)提取條件:考察不同提取方法、不同提取溶劑及其濃度、不同提取時間、不同濃度鹽酸水解液、不同粉末粒度對提取蘋果花中專屬性有效成分根皮素的影響,選擇根皮素提取率最高的提取條件。(2)色譜條件:篩選最佳檢測波長,選擇合適的固定相,調(diào)整流動相的組成、比例,選擇槲皮素峰、根皮素峰、山柰素峰與相鄰雜質(zhì)峰分離度較好的色譜條件。(3)系統(tǒng)適用性試驗:在上述色譜條件下,考察根皮素色譜峰的理論板數(shù)和分離度。(4)標準曲線:配制系列濃度的根皮素對照品溶液,分別進樣,記錄根皮素的峰面積,以根皮素濃度為橫坐標,相應(yīng)峰面積為縱坐標,繪制標準曲線。(5)精密度試驗:分別取同一份對照品溶液與同一份供試品溶液,連續(xù)進樣6次,測定根皮素的峰面積,計算RSD值。(6)重復(fù)性試驗:精密稱取同一批蘋果花樣品6份,配制供試品溶液,分別進樣,測定根皮素的峰面積,計算RSD值。(7)穩(wěn)定性試驗:分別取同一份對照品溶液與同一份供試品溶液,分別于0、2、4、6、12、24、48h進樣分析,測定根皮素峰面積,計算RSD值。(8)回收率試驗:精密稱取已知根皮素含量的蘋果花適量,分別加入相當于樣品中所含根皮素含量的80%、100%、120%的根皮素對照品溶液,配制供試品溶液,分別進樣,測定根皮素的含量,計算其回收率和RSD值。(9)檢測限與定量限的測定:將根皮素對照品溶液逐級稀釋,當信噪比大于等于3時,為檢測限;當信噪比大于等于10時,為定量限。(10)耐用性試驗:采用三根不同品牌的色譜柱、兩臺不同品牌的高效液相色譜儀分別測定1批樣品的含量,計算RSD值及RD值。(11)樣品測定:在上述色譜條件下,測定12批蘋果花中根皮素的含量。4指紋圖譜方法的建立(1)提取條件和色譜條件的選擇除檢測波長為260nm外,其他條件同含量測定項下。(2)精密度試驗:取同一份供試品溶液連續(xù)進樣6次,記錄保留時間和峰面積。(3)重復(fù)性試驗:精密稱取同一批蘋果花6份,配制供試品溶液,分別進樣,記錄保留時間和峰面積。(4)穩(wěn)定性試驗:取同一份供試品溶液,分別在0、2、4、6、12、24、48h進樣分析,記錄保留時間和峰面積。(5)指紋圖譜的建立:取不同省份來源的蘋果花12個批次,配制供試品溶液,進行指紋圖譜分析及相似度評價。結(jié)果:1確定了蘋果花性狀和粉末顯微鑒別特征;雜質(zhì)測定結(jié)果為1%~3%;水分測定結(jié)果為5.9%~7.9%;總灰分測定結(jié)果為6.2%~8.5%;酸不溶性灰分測定結(jié)果為0.6%~2.0%;醇溶性浸出物(熱浸法)測定結(jié)果為34.9%~41.7%。2薄層色譜法(1)提取方法:取本品粉末0.5g,置具塞三角燒瓶中,加乙醇40m L,密塞,超聲處理20分鐘,放冷,搖勻,濾過。取續(xù)濾液20 m L,置250 m L三角燒瓶中,加乙醇20 m L,25%鹽酸溶液10 m L,搖勻,置85℃水浴中加熱回流30分鐘,蒸干。殘渣加乙醇4m L使溶解,作為供試品溶液。(2)展開條件:薄層板為硅膠G板,以甲苯-乙酸乙酯-甲酸(7∶2∶1)的上層溶液為展開劑,展開,取出,晾干,噴以3%三氯化鋁乙醇溶液,在105℃加熱約10分鐘,置紫外光燈(365nm)下檢視,供試品色譜中,在與對照樣品色譜和對照品色譜相應(yīng)的位置上,顯相同顏色的熒光斑點。3含量測定方法(1)提取條件:取本品粉末(過四號篩)約0.2g,精密稱定,置具塞三角燒瓶中,精密加入乙醇20m L,密塞,稱定重量,超聲處理20分鐘,放冷,再稱定重量,用乙醇補足減失的重量,搖勻,濾過,精密量取續(xù)濾液10m L,置100m L三角燒瓶中,加乙醇10m L,25%鹽酸溶液5m L,搖勻,置85℃水浴中加熱回流1小時,冷卻至室溫,轉(zhuǎn)移至50m L量瓶中,用乙醇稀釋至刻度,搖勻,濾過,即得。(2)色譜條件:采用YMC ODS-A C18(250 mm*4.6 mm,5μm)色譜柱;流動相:甲醇-0.4%磷酸溶液(50:50);檢測波長為286nm;流速1.0m L·min-1;柱溫為30℃,進樣量為10μL。(3)系統(tǒng)適用性試驗:在此色譜條件下,理論板數(shù)按根皮素峰計算應(yīng)不低于5000,分離度大于1.5。(4)標準曲線的繪制:根皮素在1.23137~246.274μg/m L范圍內(nèi),線性關(guān)系良好,回歸方程為y=40466x-6026,r=1.0000(n=6)。(5)精密度試驗:儀器和方法精密度良好,RSD均為0.1%。(6)重復(fù)性試驗:樣品重復(fù)性良好,RSD為0.2%。(7)穩(wěn)定性試驗:對照品溶液和供試品溶液在48h內(nèi)穩(wěn)定性良好,RSD值分別為0.2%和0.1%。(8)回收率試驗:根皮素的平均回收率為104%,RSD為1.7%。(9)根皮素的檢測限為0.4ng,定量限為1.2ng。(10)耐用性試驗:色譜柱和儀器耐用性良好。(11)蘋果花中根皮素的含量結(jié)果為2.3%~4.2%。4指紋圖譜方法(1)提取條件和色譜條件的選擇除檢測波長為260nm外,其他條件同含量測定項下。(2)精密度試驗:根皮素峰為參照峰,計算各共有峰相對保留時間和占總峰面積2%以上共有峰的相對峰面積,其RSD值分別為0.03%~0.11%和0.25%~0.98%,精密度良好。(3)重復(fù)性試驗:12個主要共有峰相對保留時間和占總峰面積2%以上色譜峰的相對峰面積均無明顯變化,其RSD值分別為0.02%~0.10%和0.19%~1.36%,重復(fù)性良好。(4)穩(wěn)定性試驗:樣品在48h內(nèi),12個共有峰相對保留時間和占總峰面積2%以上色譜峰的相對峰面積均無明顯變化,其RSD值分別為0.04%~0.19%和0.10%~1.04%,穩(wěn)定性良好。(5)指紋圖譜的建立:得到不同產(chǎn)地蘋果花的指紋圖譜及12個共有峰。(6)數(shù)據(jù)分析:運用“中藥色譜指紋圖譜相似度評價系統(tǒng)軟件”2004年A版(國家藥典委員會開發(fā))”對所得數(shù)據(jù)進行分析,所得結(jié)果能為蘋果花的品種鑒別及質(zhì)量標準的制定提供依據(jù)。結(jié)論:1性狀鑒別、顯微鑒別、檢查項、醇溶性浸出物方法穩(wěn)定。2薄層色譜該方法斑點清晰可見,分離度、重復(fù)性、穩(wěn)定性良好,能夠很好的作為蘋果花的定性鑒別手段。3含量測定以專屬性有效成分根皮素的含量為主要指標,建立了蘋果花的定量分析方法,該方法精密度、重復(fù)性、穩(wěn)定性良好,可用于評價蘋果花質(zhì)量,為蘋果花的質(zhì)量標準的制定奠定基礎(chǔ)。4指紋圖譜通過建立蘋果花的高效液相色譜指紋圖譜,生成對照圖譜,計算相似度,該方法精密度和重復(fù)性良好,能夠控制蘋果花的質(zhì)量,同時為蘋果花的真?zhèn)舞b別提供了數(shù)據(jù)支持。
[Abstract]:Apple flowers are derived from the dried flowers of the Rosaceae plant apple Malus pumila Mill for.4-5 months, dry and dry, stored in the dry place. Apple flower is flat, spleen, stomach, kidney meridian. Apple flower tea has the effect of treating nerve pain, detoxification, blood supplement, eyesight, acne and whitening. No literature on apple flower is found. Apple flowers are in the market at present. Tea is sold in the form of sale, but it is not taken in any quality standard, its quality is difficult to control effectively. Therefore, this experiment has studied the characteristics of apple flower, microscopic identification, TLC identification, impurities, moisture, total ash, acid insoluble ash, alcohol soluble extract (hot leaching) and so on, and studied the method for the determination of the content of the effective component of the root bark. On this basis, the HPLC fingerprint of 12 batches of apple flowers collected in 8 provinces was studied and analyzed, which could provide a certain basis for the identification and quality standard of apple flower. Objective: To study the various items according to the requirements of quality standard formulation and establish the foundation for the quality standard of apple flower. Method: 1 to 8. The 12 batch of apple samples collected in the province were identified, microscopic identification, determination of impurities, moisture, total ash, acid insoluble ash, and the content of the extract content by.2 thin layer chromatography (1) extraction conditions in 12 batches of apple (1) extraction conditions: different extraction solvents and extraction methods were explored during the experiment, and the best extraction solvent and extraction were selected. Methods: (2) expansion conditions: according to the characteristics of the apple flower composition, determine the suitable expansion agent, fixed phase and color reagent and other.3 content determination methods (1) extraction conditions: different extraction methods, different extraction solvents and their concentration, different extraction time, different concentration of hydrochloric acid hydrolysate, different powder granularity to the extraction of apple flower exclusive. The extraction conditions of the highest extraction rate of rhizotin were selected. (2) chromatographic conditions: selecting the best detection wavelength, selecting the appropriate stationary phase, adjusting the composition of the mobile phase, the ratio of the quercetin peak, the rhizin peak, the kaempferol peak and the adjacent complex peaks. (3) the system applicability test: Under the above chromatographic conditions, the theoretical plate number and the separation degree of the chromatographic peak of the root bark were investigated. (4) the standard curve: a series of concentration of the root skin hormone control solution was prepared, and the peak area of the root skin element was recorded respectively. The root bark concentration was taken as the horizontal coordinate, the corresponding peak area was the vertical coordinate, and the standard curve was drawn. (5) the precision test: the same control respectively. Product solution and the same sample solution, continuously sample 6 times, determine the peak area of the root of the root, calculate the RSD value. (6) repeatability test: precisely called the same batch of apple samples 6, preparation of the sample solution, sample respectively, determine the peak area of the root bark, calculate the RSD value. (7) stability test: the same control solution and the same supply, respectively. Test product solution, 0,2,4,6,12,24,48h analysis, determine the area of the peak of root bark and calculate the RSD value. (8) recovery rate test: a proper amount of the apple flower, known as the known root bark content, is added to the solution of 80%, 100%, 120% of the root skin element in the sample, respectively. The content of the root skin element, calculate its recovery and RSD value. (9) determination of detection limit and quantitative limit: dilute the root bark control solution step by step, when the signal to noise ratio is greater than 3, the detection limit; when the signal to noise ratio is greater than 10, the quantitative limit. (10) durability test: using three different brands of chromatographic column, two different brands of high performance liquid color color The content of 1 batches of samples was measured by the spectrometer, and the value of RSD and RD were calculated. (11) the determination of the content of.4 fingerprints in the 12 batches of apple flowers under the above chromatographic conditions (1) the selection of the extraction conditions and chromatographic conditions except the detection wavelength was 260nm, the other conditions and the content determination. (2) precision test: the same one 6 times for the sample solution, record retention time and peak area. (3) repeatability test: 6 copies of the same batch of apple flowers were accurately weighed, and the sample solution was prepared. The retention time and peak area were recorded respectively. (4) the stability test: the same sample solution was taken in the 0,2,4,6,12,24,48h sample analysis to record the retention time and peak surface. (5) (5) establishment of fingerprints: 12 batches of apple flowers from different provinces, preparation of sample solution, fingerprint analysis and similarity evaluation. Results: 1 determine apple flower character and powder microscopic identification; impurity determination result is 1%~3%; water determination result is 5.9%~7.9%; total ash determination result is 6.2%~8.5%; acid not The result of soluble ash determination is 0.6%~2.0%, and the result of alcohol soluble extract (hot leaching) is 34.9%~41.7%.2 thin layer chromatography (1) extraction method: take the powder 0.5g, insert the plug triangle flask, add ethanol 40m L, the dense plug, the ultrasonic treatment for 20 minutes, put cold, shake well, filter through 20 m L, set up 250 m L triangle flask, and add ethanol 20 m L, 25% Hydrochloric acid solution 10 m L, shake well, heat reflux for 30 minutes in 85 centigrade water bath, evaporate dry. Residue plus ethanol 4m L to dissolve. (2) expansion condition: thin plate is silica gel G plate, toluene ethyl acetate formic acid (7: 2: 1) supersolution is expanded, spread, remove, dry, and spray with 3% aluminum chloride ethanol solution at 105 C About 10 minutes, under the ultraviolet light (365nm) inspection, in the sample chromatography, in the corresponding position of the control sample chromatography and the control product chromatography, the same color fluorescence spot.3 content determination method (1) extraction conditions: the powder (over four sieves) is about 0.2g, the fine density is called, the plug triangle flask, precision adding ethanol 20m L, dense plug, called Set weight, ultrasonic treatment for 20 minutes, put cold, then weigh the weight, use ethanol to reduce the weight of lost, shake well, filter, and take the continuous filtrate 10m L, set 100m L triangle flask, add ethanol 10m L, 25% hydrochloric acid 5m L, shake well, heat back for 1 hours at 85 C water bath, cool to room temperature, transfer to 50m L measuring bottle, and shake to scale, shake with ethanol to scale, shake to scale, shake with ethanol to scale, shake with ethanol to scale, shake to scale, shake with ethanol to scale, shake to scale, shake with ethanol to scale, shake to scale, shake with ethanol to scale, shake to scale, shake with ethanol to scale, shake to scale, shake with ethanol to scale, shake and shake to scale, shake with ethanol to scale, shake to scale, shake with ethanol to scale, shake to scale, shake with ethanol to scale, shake to scale, shake with ethanol to scale, shake to scale, shake with ethanol to scale, shake to scale, shake with ethanol to scale, shake to scale, shake with ethanol to scale, shake scale, shake with ethanol to scale, shake to scale, shake with ethanol to scale, shake to scale, shake with ethanol to scale, shake to scale, shake with ethanol to scale, shake to scale, shake with ethanol to scale, shake Uniform, filtration, that is, (2) chromatographic conditions: using YMC ODS-A C18 (250 mm*4.6 mm, 5 m) column; mobile phase: methanol -0.4% phosphoric acid solution (50:50); detection wavelength 286nm; flow rate 1.0m L min-1; column temperature 30 C, sample quantity is 10 micron (3) system applicability test: under this chromatographic condition, the theoretical plate number should not be less than 5000 according to the root pepein peak calculation. Draw the standard curve more than 1.5. (4) standard curve: the linear relationship is good in the range of 1.23137~246.274 g/m L, the regression equation is y=40466x-6026, r=1.0000 (n=6). (5) precision test: the precision of the instrument and method is good, RSD is 0.1%. (6) repeatability test: the sample reproducibility is good, RSD is 0.2%. (7) stability test: the control solution solution The stability of the test solution in 48h was good, the RSD value was 0.2% and 0.1%. (8) recovery test respectively: the average recovery rate of the root skin pigment was 104%, the RSD was 1.7%. (9), the limit of 1.7%. (9) was 1.2ng. (10) durability test: the chromatographic column and the instrument had good durability. (11) the result of the content of the root bark in the apple flower was 2.3%~4.2%.4 finger. The pattern method (1) the selection of the extraction conditions and the chromatographic conditions except the detection wavelength is 260nm, the other conditions and the content determination items. (2) the precision test: the root bark peak is the reference peak, the relative peak area of the total peak relative retention time and the total peak area over 2% of the total peak is calculated, and the RSD value is 0.03%~0.11% and 0.25%~0.98%, respectively. (3) repeatability test: the relative peak area of the 12 main peak relative retention time and the total peak area above 2% was no obvious change, and the RSD value was 0.02%~0.10% and 0.19%~1.36%, and the repeatability was good. (4) the stability test: the sample was in the 48h, the relative retention time of the 12 common peaks and the total peak area above 2% chromatography The relative peak area of the peak was no obvious change, and its RSD value was 0.04%~0.19% and 0.10%~1.04%, and the stability was good. (5) fingerprint establishment: fingerprint and 12 common peaks of apple flower from different habitats. (6) data analysis: application of "chromatographic fingerprint similarity evaluation system software of traditional Chinese Medicine" (National Pharmacopoeia Committee) 2004 (National Pharmacopoeia Committee) Analysis of the obtained data, the results can provide the basis for the identification and quality standard of apple flower. Conclusion: 1 character identification, microscopic identification, inspection item, alcohol soluble extract method stable.2 thin layer chromatography, the spot is clearly visible, the degree of separation, reproducibility, stability are good, and it can be used as apple flower well. The quantitative analysis method of.3 was established by qualitative identification method. The quantitative analysis method of apple flower was established. This method is precise, repeatable and stable. It can be used to evaluate the quality of apple flower, and lay the foundation of.4 fingerprint for the quality standard of apple flower to establish the height of apple flower. The HPLC fingerprint was used to generate the control map and calculate the similarity. The precision and repeatability of the method were good. It could control the quality of apple flower and provide data support for the authenticity identification of apple flower.

【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R284.1

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10 方玲;陳方亮;余翠琴;金朱明;黃瑞平;;不同產(chǎn)地烏藥的HPLC指紋圖譜研究[J];中草藥;2013年02期

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1 龐偉;蘋果多酚的分離純化及抗氧化性研究[D];西北大學(xué);2007年

2 楊建榮;蘋果多酚的高效液相色譜檢測方法的建立[D];山東大學(xué);2006年

3 任靜;蘋果多酚類物質(zhì)的提取分離及活性研究[D];西北大學(xué);2005年

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