ABSTRACT
ObjectiveTo establish the identification method of Dalbergiae Odoriferae Lignum（DOL） and its counterfeits by nuclear magnetic resonance hydrogen spectrum（1H-NMR） combined with multivariate statistical analysis. Method1H-NMR spectra of DOL and its counterfeits were obtained by NMR， and the full composition information was established and transformed into a data matrix， and the detection conditions were as follows：taking dimethyl sulfoxide-d6（DMSO-d6） containing 0.03% tetramethylsilane（TMS） as the solvent， the constant temperature at 298 K（1 K=-272.15 ℃）， pulse interval of 1.00 s， spectrum width of 12 019.23 Hz， the scanning number of 16 times， and the sampling time of 1.08 s. Similarity examination and hierarchical cluster analysis（HCA） were performed on the data matrix of DOL and its counterfeits， and orthogonal partial least squares-discriminant analysis（OPLS-DA） was used to analyze the data matrix and identify the differential components between them. In the established OPLS-DA category variable value model， the category variable value of DOL was set as 1， and the category variable value of the counterfeits was set as 0， and the threshold was set as ±0.3， in order to identify the commercially available DOL. The OPLS-DA score plot was used to determine the types of counterfeits in commercially available DOL， and it was verified by thin layer chromatography（TLC）. ResultThe results of similarity analysis and HCA showed that there was a significant difference between DOL and its counterfeits. OPLS-DA found that the differential component between DOL and its counterfeits was trans-nerolidol. The established category variable value model could successfully identify the authenticity of the commercially available DOL. The results of the OPLS-DA score plot showed that there were heartwood of Dalbergia pinnata and D. cochinchinensis in the commercially available DOL， and were consistent with the TLC verification results. ConclusionThere is a phenomenon that heartwood of D. pinnata and D. cochinchinensis are sold as DOL in the market. 1H-NMR combined with multivariate statistical analysis can effectively distinguish DOL and its counterfeits， which can provide a reference for the identification of them.
ABSTRACT
The present study detected the component content in Dalbergiae Odoriferae Lignum by HPLC fingerprint and the multi-component determination method. HPLC analysis was performed on the Agilent ZORBAX SB-C_(18) column(4.6 mm×250 mm, 5 μm). Acetonitrile-0.5% phosphoric acid aqueous solution with gradient elution was employed as the mobile phase. The flow rate was 1.0 mL·min~(-1) and the column temperature was maintained at 30 ℃. The detection wavelength was 210 nm and the sample volume was 10 μL. The similarity of 18 batches of Dalbergiae Odoriferae Lignum was 0.343-0.779, indicating that there were great differences between different batches of Dalbergiae Odoriferae Lignum. Eighteen common peaks were identified, including eight flavonoids such as liquiritigenin and latifolin. The mass fractions of liquiritigenin, luteolin, naringenin, isoliquiritigenin, formononetin, dalbergin, latifolin, and pinocembrin were in the ranges of 0.134 1%-0.495 2%, 0.028 2%-0.167 0%, 0.016 3%-0.591 3%, 0.053 5%-0.188 0%, 0.142 4%-0.640 1%, 0.068 0%-0.590 7%, 0.003 2%-1.980 7%, and 0.009 6%-0.740 2%, respectively. Eighteen batches of Dalbergiae Odoriferae Lignum were divided into three categories by cluster analysis and eight differential components in Dalbergiae Odoriferae Lignum were marked by partial least-squares discriminant analysis(PLS-DA). The cumulative variance contribution rate was 90.5%. The HPLC fingerprint combined with the multi-component determination method for Dalbergiae Odoriferae Lignum is easy in operation and accurate in results, with good repeatability and reliability. The quality of Dalbergiae Odoriferae Lignum can be evaluated and analyzed by the PLS-DA model. This study is expected to provide a reference for the quality control and clinical application of Dalbergiae Odoriferae Lignum.
Subject(s)
Chromatography, High Pressure Liquid/methods , Drugs, Chinese Herbal/analysis , Flavonoids/analysis , Quality Control , Reproducibility of ResultsABSTRACT
Fourteen compounds were isolated from the ethanol extract of Dalbergiae Odoriferae Lignum by various chromatographic techniques, including column chromatographies on silica gel, Sephadex LH-20 and semi-preparative HPLC. Their structures were determined by spectroscopic techniques as S-3'-hydroxy-7,2',4'-trimethoxyisoxane(1), 2-(2',4'-dimethoxyphenyl)-6-hydroxybenzofuran(2), 2-(2'-hydroxy-4'-methoxyphenyl)-6-methoxybenzofuran(3), 7,2',4'-trimethoxydihydroisoflavone(4), sativanone(5), 3,9-dimethoxy-6H-benzofuro[3,2-c]chromen-6-one(6),(6 aS,11 aS)-homopterocarpin(7),(6 aS,11 aS)-8-hydroxy-3,9-dimethoxypterocarpan(8),(6 aS,11 aS)-3,8,9-trimethoxypterocarpan(9), isodalbergin(10), isoliquiritigenin(11), butein(12), butin(13) and 3,7,4'-trihydroxyflavone(14). Among them, compound 1 was a new compound, while 2 and 3 were new natural products, 6, 8, 9 and 14 were isolated for the first time from Dalbergiae Odoriferae Lignum. Compounds 1-14 were tested for their cytotoxic activity against human hepatoma cell line BEL-7402, human gastric cancer cell line SCG-7901, human lung cancer cell line A549, human chronic myeloid leukemia cell line K562 and HeLa human cervical cancer cellline by MTT method. Compound 1 exhibited significant cytotoxicity with IC_(50) values ranging from 2.85 to 11.62 μg·mL~(-1). In addition, 2, 11 and 12 showed weak cytotoxic activities.
Subject(s)
Humans , Antineoplastic Agents , Chromatography, High Pressure Liquid , HeLa CellsABSTRACT
Objective::Dalbergiae Odoriferae Lignum is a rare traditional Chinese medicine material in China. However, there are many varieties of various sources and different qualities in the market at present. In order to further define the pharmacodynamic substance basis, electrospray time-of-flight tandem mass spectrometry (UPLC-Q-TOF-MS/MS) was used to rapidly analyze chemical constituents of methanol extract of Dalbergiae Odoriferae Lignum. Method::Chromatographic separation was performed on an UPLC RRHD SB-C18(3.0 mm×100 mm, 1.8 μm)for gradient elution, with mixtures of acetonitrile and 0.1%formic acid-water as mobile phases at a flow rate of 0.3 mL·min-1. The column temperature was maintained at 40 ℃. The data was collected in a negative ion mode with electro-spray ionization source(ESI). Result::According to molecular ion peaks and MS2 mass spectrometry characteristic fragment ions, Mass Bank databases, as well as the mass spectrometry information of reference substances and relevant literatures, a total of 83 constituents were identified, including 18 flavones, 31 isoflavones, 10 neoflavonoids, 9 isoflavanones, 7 other flavonoids and 8 other components. Conclusion::UPLC-Q-TOF-MS/MS can quickly, accurately and comprehensively identify chemical constituents in methanol extract of Dalbergiae Odoriferae Lignum, and isoflavones, flavones, neoflavonoids and isoflavanones are the main chemical constituents, which laid a foundation for the basic research of medicinal substances of Dalbergiae Odoriferae Lignum, and provided theoretical basis and technical support for the improvement of quality standards of Dalbergiae Odoriferae Lignum.
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Objective: To reveal the extraction, distribution, and influencing factors of volatile components in the extraction process of Dalbergiae Odoriferae Lignum (DOL). Methods: Volatile oil was extracted by steam distillation, and the extract was collected every 30 min to separate the aromatic water part and the volatile oil part, of which chemical composition was analyzed by GC-MS. Results: A total of 136 volatile constituents were obtained, of which trans-nerolidol, (E)-β-famesene, and α-farnesene were the main characteristic constituents. A total of 22 kinds of components distributed only in water, 13 kinds only in oil and 101 kinds in both oil and water. The specific components in water were positively correlated with the dissolution/diffusion of the main characteristic components in aromatic water, while negatively correlated with the main components in volatile oil. The water solubility of the unique components in water is the highest. The results of principal component analysis showed that PC1 (melting point, surface tension, polar surface, negative correlation of refractive index) and PC2 (polarizability, molecular weight, boiling point positive correlation, negative correlation of water solubility) were the principal components that lead to the difference of component distribution. Conclusion: Affected by the physical and chemical properties of volatile component itself, some components specifically distributed in water which increased the content of main components in the aromatic water, resulting in volatile oil extraction process easy to “emulsification”, in turn, leading to an important reason for the declining quality of volatile oil.
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Dalbergiae Odoriferae Lignum is derived from heartwood of Dalbergia odorifera,which is national Ⅱ level of rare and endangered protective plants in China. Its resources are scarce and its price is high. In order to find substitutes of D. odorifera,the chemical constituents of 70% ethanol extract of heartwood of D. catifolia were systematically studied by using silica gel,Sephadex LH-20 column chromatography,and semi-preparative HPLC. Sixteen neoflavanoids were isolated and identified as eight dalbergiphenols( 1-8),three dalbergiones( 9-11),two dalbergins( 12,13),two benzophenones( 14,15) and one other type neoflavanoids( 16) based on spectroscopic data analyses and/or comparing the spectroscopic data with those in literature. Among them,compounds 3,7 and 11 were isolated from the genus Dalbergia for the first time,and compounds 2,4-6,8,14 and 15 were isolated from the D. latifolia for the first time. Ten neoflavonoids were both discovered from D. latifolia and D. odorifera.
Subject(s)
Benzophenones , China , Chromatography, High Pressure Liquid , Dalbergia , Plant ExtractsABSTRACT
Objective To analyze similarities and differences of volatile components of Dalbergiae Odoriferae Lignum (DOL) and its adulterants, so as to provide a scientific basis for the identification of the authenticity of the pros and cons of DOL, and offer the reference for die further study of DOL. Methods With headspace solid phase microextraetion (HS-SPME) combined with gas chromatography-mass spectrometry (GC-MS), volatile components of DOL and its adulterants were analyzed and identified, and the relative content of each component was calculated by area normalization method. Results There were 70 compounds detected, in which 38 compounds were identified. They were mainly alkenes, alcohols, aldehydes and ketones. A total of 42 compounds were detected from DOL volatile components, and 25 components were identified, accounting for 96.54% of the volatile components; 28 peaks were detected from its adulterants, and 19 compounds were identified, accounting for 80.7% of the volatile components. Conclusion There were huge differences in volatile components between DOL and its adulterants. Only 6 common components were defected both in the DOL and it’s adulterants, with their quite different contents. The main component of DOL was nerolidol, accounting for 61.47% of volatile components; the main component of its adulterants was beta selinene, accounting for 59.04% of total volatile components. The present result provides a scientific guidance for the identification of true or false DOL and also a reference for the further development of Dalbergia wood.
ABSTRACT
Objective To analyze similarities and differences of volatile components of Dalbergiae Odoriferae Lignum(DOL) and its adulterants,so as to provide a scientific basis for the identification of the authenticity of the pros and cons of DOL,and offer the reference for the further study of DOL. Methods With headspace solid phase microextraction(HS-SPME)combined with gas chromatography-mass spectrometry(GC-MS),volatile components of DOL and its adulterants were analyzed and identified,and the relative content of each component was calculated by area normalization method. Results There were 70 compounds detected,in which 38 compounds were identified. They were mainly alkenes,alcohols,aldehydes and ketones. A total of 42 compounds were de?tected from DOL volatile components,and 25 components were identified,accounting for 96.54% of the volatile components;28 peaks were detected from its adulterants,and 19 compounds were identified,accounting for 80.7%of the volatile components. Con?clusion There were huge differences in volatile components between DOL and its adulterants. Only 6 common components were de?fected both in the DOL and it's adulterants,with their quite different contents. The main component of DOL was nerolidol,accounting for 61.47%of volatile components;the main component of its adulterants was beta selinene,accounting for 59.04%of total volatile components. The present result provides a scientific guidance for the identification of true or false DOL and also a reference for the fur?ther development of Dalbergia wood.
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OBJECTIVE: To identify Dalbergiae Odoriferae Lignum and its adulterants using DNA barcoding. METHODS: ITS2 is one of the popular DNA barcoding in the identification of traditional Chinese medicine. In this paper, the ITS2 regions of Dalbergiae Odoriferae Lignum and its adulterants were amplified and sequenced bi-directional. The length and GC content of ITS2 sequence were analyzed through MEGA5.0 software. The genetic distances were computed by kimura 2-parameter (K2P) model. Dalbergiae Odoriferae Lignum and its adulterants have been identified through the species identification system for traditional Chinese medicine and neighbor-joining (NJ) phylogenetic tree. RESULTS: The sequence lengths of ITS2 of Dalbergiae Odoriferae Lignum were 216 bp, and the GC content was 68.5%. The minimum K2P interspecific genetic distances of Dalbergiae Odoriferae Lignum and its adulterants were 0.009, which was larger than that of the intraspecific genetic distances of Dalbergiae Odoriferae Lignum. The Dalbergiae Odoriferae Lignum and its adulterants can be obviously identified using the Species identification System and NJ phylogenetic trees. CONCLUSION: ITS2 Regions as DNA barcode can identify Dalbergiae Odoriferae Lignum and its adulterants accurately.