| I The innovation research of sulfur fumigated Angelicae Dahuricae Radix (Baizhi) based on some analytical methodsAngelicae dahuricae radix (Baizhi) is the dried roots of Angelica dahurica (Fisch. ex Hoffm.) Benth. et Hook. f. or A dahurica (Fisch. ex Hoffm.) Benth. et Hook. f. var. formosana (Boiss.) Shan et Yuan (family Umbelliferae). It is reported that Baizhi can be used for common cold, headache, supra-orbital bone pain, nasal congestion, nasal discharge, allergic rhinitis, swelling and pain, and so on.Baizhi is processed by post-harvest drying process before market circulation. The traditional method of drying process is sulfur fumigation. Though sulfur fumigation has the effects of rapid drying and insect prevenion, this method can cause chemical transformation of original bioactive components, efficiency reduction. The quality and drug safety of Baizhi were impacted by sulfur fumigation. In this condition, it is important to determinate sulfur fumigated Baizhi, evaluate the quality of Baizhi, and improve its quality control. In this paper, the rapid separation of chemical constituents from Baizhi was conducted by high-speed counter-current chromatography (HSCCC). The obtained chemical constituents can be used as reference substance for qualitative and quantitative analysis in this study. The rapid identification of sulfur fumigated Baizhi and the quantified model of imperatorin were developed by near-infrared spectrum (NIR). The effect of sulfur fumigation on the chemical consitituents of Baizhi was evaluated by nuclear magnetic resonance (NMR) and high performance liquid chromatography (HPLC). The quality evaluation of Baizhi commercial slices was conducted by HPLC. In addition, two alternative methods to sulfur fumigation were developed in order to avoid the harmful effect and ensure the quality of Baizhi during post-harvest drying process.A novel three-phase solvent system of n-hexane, methyl acetate, acetonitrile, and water at a volume ratio of 4:3:4:4 was prepared for the first time. Baizhi petroleum ether extract (Baizhi:3.4 kg, petroleum ether extrac:18.7 g [yield:0.55%]) was separated with upper phase (UP)/lower phase (LP) of this three-phase solvent system by HSCCC for the first time. Eight coumarins named oxypeucedanin hydrate (B1, 18.9 mg, purity>98.0%), byakangelicin (B2,8.4 mg, purity>98.0%), byakangelicol (B3,33.5 mg, purity 75.6%), bergapten (B4,10.3 mg, purity> 98.0%), oxypeucedanin (B5,29.7 mg, purity>98.0%), imperatorin (B6,78.6 mg, purity> 98.0%), phellopterin (B7,40.4 mg, purity>98.0%), and isoimperatorin (B8,30.6 mg, purity 93.3%) were obtained all at once. Byakangelicol (B3) was isolated by HSCCC for the first time. Comparing the HPLC chromatogram with HSCCC chromatogram of Baizhi petroleum ether extract, we found the peak order of compound B3 was located after that of compound B4 in HPLC chromatogram, but the peak order of compound B3 was located prior to that of compound B4 in HSCCC chromatogram. The irreversible adsorption might take place between compound B3 and the solid support in HPLC column. In addition, the retention times of compounds B3 (43.1 min) and B5 at (43.6 min) were very close in HPLC chromatogram, but compounds B3 and B5 were successfully separated with HSCCC operation because of the perfect partition coefficients of B3 (KD=0.48) and B5 (KD=0.94) in this three-phase solvent system.The rapid identification method of sulfur fumigated Baizhi and the quantitative model of imperatorin from Baizhi were established by NIR for the first time. The samples are 29 batches of non-sulfur fumigated Baizhi and 29 batches of sulfur fumigated Baizhi. The process of rapid identification of sulfur fumigated Baizhi was described as follows:all NIR spectrograms were pretreated by the method of first derivative derivation and vector normalization in the range of 3811.0-8806.0 cm-1. The Ward’s Algorithm method was used for clustering analysis of non-sulfur fumigated and sulfur fumigated Baizhi. Non-sulfur fumigated and sulfur fumigated Baizhi can be successfully classified by using this rapid and simple method. The development of quantitative model of imperatorin was described as follows:the randomly selected 30 batches of Baizhi were divided into calibration set (n=22) and validation set (n=8) by cross-validation method. The NIR spectrograms of calibration set were pretreated by first derivative derivation and a straight line subtraction in the range of 6102.2-5446.3cm-1. The first 10 principal components were chosen. The quantitative model of imperatorin was established by partial least squares regression analysis. According to the quantitative model, the average recovery rate of validation set is 102.5%. The results indicate that the quantitative model of imperatorin can be applied to determine the content of imperatorin in Baizhi accurately.The proton nuclear magnetic resonance (1H-NMR) fingerprints of petroleum ether extract of non-sulfur fumigated Baizhi (15 batches) and that of sulfur fumigated Baizhi (15 batches) were developed by NMR for the first time, respectively. The results indicate that 1H-NMR fingerprints of non-sulfur fumigated Baizhi and that of sulfur fumigated Baizhi were significantly different. There are ten characteristic peaks which are significantly different, i.e.δ3.20-3.25 ppm,δ3.60-3.70 ppm, δ3.90-3.95 ppm,δ4.10-4.25 ppm,δ4.40-4.66 ppm,δ6.24-6.42 ppm, δ7.00-7.10 ppm,δ7.10-7.25 ppm,δ7.60-7.70 ppm, and δ8.10-8.28 ppm. By comparing with’H-NMR of imperatorin, isoimperatorin, osthole, oxypeucedanin, bergapten, and byakangelicol, it indicates that the relatively integral areas of these reference substances were lower in sulfur fumigated Baizhi. We could speculate that sulfur fumigation may affect the contents of thses reference substances. In addition, we successfully distinguished non-sulfur fumigated Baizhi with sulfur fumigated Baizhi by principal component analysis (PCA). The results of 1H-NMR-PCA and NIR clustering analysis are consistent. It indicats that both methods can be used for determinon of sulfur fumigated Baizhi.The quality consisitence of Baizhi commercial slices were evaluated by HPLC for the first time. The samples are 18 batches of non-sulfur fumigated Baizhi,18 batches of sulfur fumigated Baizhi and 31 batches of Baizhi commercial slices. HPLC analysis were carried on Diamonsil(R)-C18(4.6 mm×250 mm,5 μm), maintaining at 35℃. The mobile phase consisted of a combination of A (acetonitrile) and B (water) with a liner gradient. The flow rate was 1.0 mL/min. The detector wavelength was set at 210-800 nm (Max Plot). The fingerprints of non-sulfur fumigated Baizhi, sulfur fumigated Baizhi, and Baizhi commercial slices were developed, respectively. The similarity values were calculated by Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine (2004A). The HPLC fingerprints of non-sulfur fumigated Baizhi were similar, and their similarity values were 0.92-0.98. The HPLC fingerprints of sulfur fumigated Baizhi were similar, and their similarity values were 0.88-0.96. The results indicate that non-sulfur fumigated Baizhi and sulfur fumigated Baizhi are quality consistence, respectively. However, the HPLC fingerprints of non-sulfur fumigated Baizhi and sulfur fumigated Baizhi were significantly different. The result of HPLC fingerprints is consistent with the result of 1H-NMR fingerprints, which indicats that the chemical consitutents of Baizhi is significantly different before and after sulfur fumigation. The HPLC fingerprints of 31 baatches of Baizhi commercial slices were different. The HPLC fingerprints of No.2 (Hubei) and No.15 (Ningxia) were similar with that of non-sulfur fumigated Baizhi. The HPLC fingerprints of other 29 batches of commercial slices were similar with that of sulfur fumigated Baizhi. In addition, the ratio of similarity value b/similarity value c(similarity value b:the corrections between HPLC fingerprints of commercial slices and reference fingerprints of non-sulfur fumigated Baizhi; similarity value c:the corrections between HPLC fingerprints of commercial slices and reference fingerprints of sulfur fumigated Baizhi) was calculated to deduce whether the commercial slices were processed by sulfur fumigation. For one batch of commercial slices, it was more similar to non-sulfur Baizhi when the ratio is bigger than 1. The ratios of No.2 (1.08) and No.15 (1.24) are bigger than 1. The result of similarity analysis is consistent with HPLC fingerprints. In the end, two batches of commercial slices (No.2 and No.15) were determined as non-sulfur fumigated commercial slices by using the NIR clustering analysis which was developed in this paper. The result verified the results of similarity analysis and HPLC fingerprints.The contents of 9 coumarins in Baizhi commercial slices were simultaneously measured by area normalization method for the first time. The effect of sulfur fumigation on the contents of imperatorin, isoimperatorin and osthole were simultaneously investigated for the first time. It indicates that byakangelicin, imperatorin, isoimperatorin and osthole were detected in all commercial slices; pimpinellin was only detected in non-sulfur fumigated commercial slices (No.2 and No.15); xanthotoxin, byakangelicol, bergapten, and oxypeucedanin were detected in non-sulfur fumigated commercial slices and some sulfur fumigated commercial slices. The contents of byakangelicin, imperatorin, isoimperatorin and osthole were analysed by statistics analysis. The contents of imperatorin, isoimperatorin and osthole were significantly lower in sulfur fumigated commercial slices than that in non-sulfur fumigated commercial slices (p<0.05), but the contents of byakangelicin were not (p>0.05). The effect of sulfur fumigation on the contents of imperatorin, isoimperatorin and osthole were further evaluated. The results indicate the sulfur fumigation can affect the contents of imperatorin, isoimperatorin and osthole. The contents of imperatorin, isoimperatorin and osthole were reduced by 39.20%,34.91% and 35.47%, respectively. The results of quantitative analysis and 1H-NMR fingerprints analysis were consistent.The effect of surfur fumigation on Baizhi was comprehensively studied by some analysis method. The results of 1H-NMR-PCA and NIR clustering analysis are consistent, which indicates the chemical constituents of Baizhi were singnificantly different before and after surfur fumigation. The results of 1H-NMR and HPLC analysis are consistent, which indicates the contents of imperatorin, isoimperatorin and osthole were reduced after sulfur fumigation. The analysis method of NIR was simple, rapid, and environmental. The analysis method of NMR has advantages of rapid analysis and small sample amount. The analysis method of HPLC was simple and correct in quantitative analysis.To avoid the harmful effect of sulfur fumigation, we developed two alternative methods to sulfur fumigation, and evaluated the alternative methods by the appearance identification, the rate of production, and the contents of alcohol extract and imperatorin (at 0 month and 12 month). The average rates of production are 24.79% and 25.17%. The average contents of alcohol extract are 19.89% and 20.43%. The average contents of imperatorin are 0.18% and 0.15% at 0 month, and 0.17% and 0.15% at 12 month. All productions processed by alternative methods meet the standards of the pharmacopoeia of People’s Republic of China of 2010 edition, and all of them are similar with the productions which processed by natural drying method (the average rate of production:24.26%, the average content of alcohol extract: 18.97%, the average contents of imperatorin:at 0 month,0.15%; at 12 month,0.14%). These alternative methods take the place of sulfur fumigation, and have a wide range of practical values.Ⅱ The certified reference material development of β-sitosterolβ-Sitosterol, one of the most abundant and wide physterols in plants and plant products, exists in many edible plants, such as, fruit of Cucurbita moschata, seed of Avena sativa, leaf of Spinacia oleracea. It also exists in many traditional Chinese medicines, such as fruit of Panax ginseng, and seeds of Linum usitatissimum, Perilla frutescens, and Platycladus orientalis.β-Sitosterol can reduce the LDL-C level in hypercholesterolemic children and adult, has exerts antiproliferative effect and so on. In a word,β-sitosterol is considered as a main functional compound in medicinal and edible plants. In the foundation of "Demstration project and technical standardization of reference materials from natural products" (No.201210209), we obtained a batch of β-sitosterol crystals as a certified reference material that used for quality evaluation index of some traditional Chinese medicine and agricultural products based on ISO Guide 35:2006/GB/T 15000-2008 Reference materiels work guide (3).The crude β-sitosterol was from the seeds of Sinapis alba L. (Cruciferae) S. and recrystallized in methanol. The obtained β-sitosterol is colorless and soft columnar crystal The elemental analysis, high resolution mass spectrometry, nuclear magnetic resonance (NMR) spectroscopy, infrared spectrum, and X-ray crystal diffraction are used to identify the constructure. Based on the data, there are two molecular C29H50O and one molecular H2O in a symmetric unit. The HPLC/ELSD was uesd to detect the purity. The analysis is performed using a HPLC system with Kromasil 100-5C18 (4.6 × 250 mm,5 μm). The mobile phase is methanol-water (99:1, v/v) with a flow rate of 1.0 mL/min. The column temperature is controlled at 30℃. The flow tube temperature is 80℃, and the flow rate is 1.6 mL/min. Sample preparation was as follows:the sample (1 mg) is dissolved by methanol (5 mL), and filered through a 0.45 μm millipore filter; the injection volume is 10 μL; the purity is calculated by area normalization method. The purity of β-sitosterol is ≥95%. After structure determination and purity testing, the pure samples were subpackaged into brown bottle (each bottle 10 mg,300 bottles, maintained at 0-8℃).According to ISO Guide 35:2006/GB/T 15000-2008 Reference materiels work guide (3) "the general principles and statistical methods of reference materiels characterization", the uniformity examination of β-sitosterol is performed according to the requirements in chapture 7. The F test is used to determine whether the uniformity data conforms to the normal distribution. The F value of β-sitosterol is 1.94. The value is less than critical value (2.22) which indicates the compound is homogeneous.The stabilities of β-sitosterol are investigated by stability test and accelerated stability test. In the stability test, the compunds are packaged in simulated market sales, and store in the condition of 0-8℃ (temperature) for 2 years. At 0,1,2,3,6,9, 12,18, and 24 month, take three samples and measure the purity, respectively. In the end, using t test and F test to analysis the data of long term stability test. The absolute value of slope (0.0014%) is less than 0.010%(the multiplication of t factor (2.37, v=8, P=0.95) and the uncertainty related to the slope. The result of t test has shown that the slope is not significant, and there is not observed instability. The F value of β-sitosterol is 0.12. It is lower than the critical value (5.59). In that case,β-sitosterol is stable for 24 months. In the accelerated test, the compunds are packaged in simulated market sales, and store in the condition of 40℃±2℃ (temperature) and 75%±5% (relative humidity) for 6 months. At 0,1,2,3 and 6 month, we take three samples and measure the purity, respectively. In the end, using t test and F test to analysis the data of accelerated stability test. The absolute value of slope (0.0077%) is less than 0.067%(the multiplication of t factor (3.18, v=3, P=0.95) and the uncertainty related to the slope). The results of t test have shown that the slope is not significant, and there is not observed instability. The F value of β-sitosterol is 0.1330. It is lower than the critical value (7.71). In that case,β-sitosterol is stable for at least 6 months in the accelerated condition. So the validity of β-sitosterol is 2 years, and the validity of β-sitosterol is 6 months in hot and humid condition.According to ISO Guide 35:2006/GB/T 15000-2008, we select 8 laboratories to fix values. The samples are randomly assigned to each laboratory, and are detected by HPLC-ELSD. The abnormal date of each laboratory is tested by Grubb’s test. The Grubb’s statistics G of β-sitosterol in 8 laboratories (1.073,1.231,1.278,1.730,1.354, 1.780,1.497,1.648) are all lower than G0.99 (6) (1.944) and G0.95 (6) (1.832). The result shows that the data are all effective and there is not abnormal date. The normalities of data are test by kurtosis. The results show that the bk values of β-sitosterol from eight laboratories data (2.66,1.49,1.28,2.62,1.77,2.77,1.81,2.47) are all smaller than the critical value (3.70), so there isn’t abnormal value. According to GB/T 15000.3-2008, the results of characterization are composed by standard values and uncertainty. For reference materials, the measured uncertainty of special standard values (UCRM,0.28%) is composed by the uncertainty of standard values (u(X)) (0.01%), homogeneity test (ubb) (0.05%) and stability test(ults) (0.13%). According to all measured results, the special standard value and expanded uncertainty of β-sitosterol is 95.41% and 0.28. |
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