沉香、檀香评价体系的优化及对沉香结香方法的评价
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摘要
正品沉香药材分为国产沉香和进口沉香,是中国、日本、印度及其他东南亚国家的传统名贵药材和天然香料。沉香具有行气止痛、温中止呕、纳气平喘的功效。白木香是国产沉香的唯一植物资源,现为国家二级保护植物。白木香树因受到虫蚁、雷劈、伤病等损害会分泌树脂并浸润沉积于木质部中,经多年结聚而形成沉香,获得高品质的沉香一般需要30年以上。商业上的巨大利润和非法贸易正导致世界各地的沉香资源越来越少,沉香属大部分植物被列入世界自然保护联盟(IUCN,2002)濒危植物红皮书中的“受威胁(threatened)”等级,属于世界限制贸易的野生濒危树种。因此加快沉香生产速度、提高沉香品质成为目前急需解决的问题。但迄今为止,沉香的生物合成途径和形成机理尚未阐明,人工造香技术生产的沉香与天然沉香的品质差异有待探讨,目前市售沉香混伪、制伪情况严重。
檀香为檀香科檀香属的树种,是名贵香料、药材和高级工艺雕刻品原料,其药用部分是具有芳香性的心材和从心材中提取的檀香油。在檀香自然状态下生长十分缓慢,8年左右才开始形成心材,一般需20～25年左右才能完全形成高等级的檀香心材。檀香属下的多个品种都被称为檀香,包括印度檀香、大果澳洲檀香、大花澳洲檀香、滨海夏威夷檀香和斐济檀香等,但品质最佳的是印度檀香,《中国药典》2010年版(一部)也仅收载印度檀香一个品种。种源差异、成材时间过长等因素使得檀香药材品质差异极大,市场价值差异极大,混伪情况严重。
目前沉香、檀香质量评价方法均缺乏有效、专属的评价指标。鉴于此,本文利用TLC、GC-MS、DNA分子测序等技术优化了沉香和檀香质量评价体系,应用数学处理模型和数理统计方法对沉香GC-MS数据进行挖掘,找出与沉香品质相关的特征性成分,并以优化后的沉香评价体系对不同人工造香技术的结香效果进行比较研究。对引种檀香的rDNAITS序列进行分析。旨在为识别伪制沉香和檀香、完善沉香和檀香的质量标准提供依据,并指导人工造香技术快速生产沉香药材。
本文采用《中国药典》2010年版(一部)方法对收集的34个沉香样品进行检验,检验结果结合药材的外观和来源,可将34个沉香样品分为三类：天然沉香、人工沉香和伪品。天然沉香按照商品沉香的分类标准分为三个等级：两个一等品(油格占80%),三个二等品(油格占60%)和三个三等品(油格占40%以下)。人工沉香根据来源分为三个物理刺激样品、两个化学刺激样品和三个化学与生物联合刺激所得的样品。按照中国药典检验的结果表明只有质量较优的天然沉香完全符合规定,而人工沉香的颜醇浸出物微量升华试验与中国药典的规定有差异。
取8个天然沉香和8个人工沉香样品,在《中国药典》2010年版(一部)TLC鉴别方法的基础上进行优化,分别考察了不同展开溶剂系统、不同检视方法,构建了沉香的TLC指纹图谱,经方法学考察该指纹图谱具有一定的重复性、稳定性,符合指纹图谱研究的相应要求。TLC指纹图谱可以直观、快速地区分天然沉香和人工沉香,但无法区别不同等级天然沉香和不同结香方法生产的人工沉香。
为解决上述问题,本文构建了沉香的GC-MS特征图谱,并从天然沉香品质较优、人工沉香品质较差的差异入手,以GC-MS数据为基础进行由浅入深的数据分析。
具体分析过程如下：
1.取16个天然与人工沉香样品的GC-MS指纹图谱中的431个色谱峰数据进行聚类分析,发现该指纹图谱可以有效地区分天然沉香和人工沉香。
2.检索结果表明沉香中主要存在5大类化合物：2-(2-苯乙基)色酮类(后面简称色酮类)、倍半萜类、脂肪族、芳香族和甾体类化合物。取色酮类、倍半萜类、脂肪族、芳香族和甾体类化合物的总相对百分含量加上醇溶性浸出物含量6组数据进行聚类分析,聚类结果显示采用分类数据可以将所有16个样品分成2个大类,第一类包含所有8个天然沉香以及30号人工沉香,且品质较好的天然一等品和二等品都先各自聚为一支再与其他天然沉香聚成一支；30号虽然为刀砍物理刺激所得样品,但因其结香时间较长,其品质接近天然沉香,与天然沉香聚为一支；第二类包含7个人工沉香,且按照不同结香方法进行聚类。说明不同类别化合物及其含量能更好地体现了不同品质沉香与不同结香方法所得沉香之间的差异。
取天然沉香和人工沉香的不同类别化合物总含量的平均值进行两独立样品t检验,了解到色酮类化合物和倍半萜类化合物的含量在天然沉香与人工沉香中具有显著性差异；然后将色酮类化合物、倍半萜类化合物的含量和醇浸出物含量进行双变量相关分析,发现色酮类化合物含量、倍半萜化合物含量与醇浸出物含量分别呈负相关和正相关,且两者之间具有较强的负线性相关。由于倍半萜类化合物为沉香的主要活性成分,说明醇溶性浸出物含量与沉香的品质存在一定的相关性。色酮类化合物与倍半萜类化合物的含量比值在天然沉香中比较稳定,可以明显区分人工沉香与天然沉香,且可以有效区别不同结香方法所得的样品。因此,该比值可以作为沉香品质评价的一个主要指标。
3.采用遗传算法-支持向量机联用(GA-SVM)数学模式优选影响天然沉香与人工沉香分类的主要成分。本文将天然沉香和人工沉香的GC-MS结果(各成分的峰面积)作为支持向量机的输入特征参数,采用遗传算法与支持向量机耦合,对特征参数进行优选,得到49个对天然沉香和人工沉香分类最具影响力的成分。对这49个成分进行独立样本双侧t检验,取具有统计学差异的22个化合物的百分含量与醇溶性浸出物含量进行相关分析。发现其中有13个化合物的含量与醇溶性浸出物含量之间呈现中等强度以上的相关性,且具有统计学意义。其中含量最高的成分是白木香醛和斯巴醇。取16个样品的白木香醛、斯巴醇含量和醇溶性浸出物含量进行聚类分析,结果以上述三个指标进行聚类可以将沉香一等品和二等品聚为一类；其他人工沉香以及一些品质较差的天然沉香聚为一类。结果表明,白木香醛与斯巴醇的含量确实能够有效地区分不同品质的沉香。
结合上述实验和数据分析结果,确定优化后的沉香评价体系包括外观性状、醇溶性浸出物的微量升华试验、醇溶性浸出物含量、优化后的TLC指纹图谱和GC-MS指纹图谱,并且可以用醇溶性浸出物含量、色酮类与倍半萜类化合物的含量比值以及白木香醛和斯巴醇的含量等数据对沉香品质进行评价。
取优化后的评价体系对不同结香技术所得的动态样品进行分析,并以醇溶性浸出物含量、色酮类与倍半萜类化合物的含量比值、白木香醛和斯巴醇相对含量为指标,对结香效果进行评价。发现甲酸与拟盘多毛孢属内生真菌(Pestalotiopsis sp.)混合诱导的白木香结香效果最好,而甲酸与多节孢内生真菌(Nodulisporium sp.)混合诱导的白木香结香效果最差。
本文构建了檀香的TLC、GC-MS指纹图谱。取檀香样品的GC-MS指纹图谱数据,结合文献数据进行统计分析,发现总檀香醇%及α-/β-檀香醇%比值在不同檀香品种之间具有一定的变化规律,与品质具有一定相关性,可与挥发油含量一起用于檀香药材的质量评价。优化后的檀香评价体系包括外观性状、挥发油含量测定、优化后的TLC指纹图谱和GC-MS指纹图谱,并且可以用挥发油含量、总檀香醇含量和α-檀香醇和β-檀香醇的含量比值为评价指标对檀香的品质进行评价。
采集不同培育代数、不同来源的印度、印尼檀香,采用PCR扩增-测序技术对其rDNA ITS序列进行测定,了解了我国广东地区所引种檀香的基因变异情况,并为后续的药材分子鉴定提供基础。DNA测序结果表明,檀香的rDNA ITS序列在种内稳定,不受引种代数、寄主等因素的影响。通过GenBank数据库中最近源序列相似率分析,构建系统发育树,结果表明檀香属植物种间存在较大差异,可应用DNA分子测序技术建立檀香分子鉴定方法,对市售檀香品种正本清源。
本研究通过沉香和檀香TL、GC-MS指纹图谱、rDNA ITS序列系统发育树的构建和分析,综合运用药物分析学、化学计量学和分子系统学的方法,探讨了与沉香和檀香品质相关的特征成分和评价指标,优化了沉香和檀香的质量评价体系。并深入分析天然沉香和人工沉香品种差异,揭示了不同人工造香技术的优劣。为加快沉香生产速度、提高沉香和檀香的品质,开发和利用人工种植沉香和檀香资源奠定基础。
Agarwood. the resinous portions of Aquilaria plants, have been used as medicines and incenses in Asia. Aquilaria sinensis (Lour) Gilg.(Thymeleaceae), distributed in Fujian, Guangdong, Guangxi and Hainan provinces of China, is the major producer of agarwood in China, which had been recommended to be the grade Ⅱ protected plant in China (1999). In nature, agarwood was produced only when the plant was wounded or attacked by pathogens or insects. Black and fragrant resin produced under occasional stimulation is deposited in the stem or main branches of the trees. It is generally considered that the more the resin, the better the quality of agarwood. With increased consumption of agarwood in recent years, over-exploitation of agarwood in forest caused depletion of the natural resources. The International Union for the Conservation of Nature (IUCN) in2002classified all species of Aquilaria including A. sinensis as "vulnerable"(IUCN2002). So, improving the yield and quality of agarwood is very important now. However, the formation of agarwood has long been mysterious and associated with arcane forces, and the difference in quality between natural agarwood and artificial agarwood is not clear. There is a serious adulterants of agarwood in market.
Sandalwood belongs to the Santalaceae family, highly valued for its fragrant heartwood, which contains sandal oil that is used in perfumes, medicine. In natural condition, Sandal grows very slowly; about8years begin to form heartwood, which normally takes20to25years to fully form high grade sandalwood. The species in Santalaceae family such as S. album, S. yasi, S. spicatum, S. paniculatum, S. austrocaledonicum etc can form fragrant heartwood. But, there are great quality difference among the species. This is true for S.album L. from India, Indonesia and East Timor which is the bench mark of commercial sandalwood species, owing to its high quality. S.album L is the only specie in the Pharmacopoeia of the People's Republic of China (Chinese Pharmacopoeia,2010). The variation of quality among species and the long time to form fragrant heartwood caused the quality of sandalwood vary greatly; market value difference is great, and leading to a severe mixed false situation.
In order to meet the increasing demand, Aquilaria sinensis trees and sandalwood trees were widely cultivated in the south of china. However, the lack of reasonable and feasible evaluation system of quality hinders the development and utilization of agarwood and sandalwood.
So, in this study, we intended to find out the quality-related evaluation index of agarwood and sandalwood by using the statistical models, and to optimize the evaluation system of agarwood and sandalwood by using thin layer chromatography (TLC) and gas chromatography-mass spectrometry (GC-MS) techniques. And the rDNA ITS sequences of S. album L. introduced from India and Indonesia were amplified by PCR technology and were sequenced. The study can distinguish the fake from commercial agarwood and sandalwood, and can improve the standard of quality control. It is useful in rapid production of agarwood by artificial technology.
34Samples collected from market were detected by the methods in Chinese Pharmacopoeia. Combined with the appearance and sources, the34samples can be divided into three groups by the test results:natural agarwood, artificial agarwood and fake agarwood. Follow the commercial classification criteria, the natural agarwoods were categorized into three grades. The1st grade has the best quality, the2nd grade has medium quality and the3rd grade has the poorest quality. The artificial agarwoods were categorized into three groups by the different artificial inducing technology. Three samples were artificial induced by physical methods, two samples were artificial induced by chemical methods, and three samples were artificial induced by chemical and biological joint stimulation. All the experimental results of the natural agarwoods met Chinese pharmacopoeia (edition2010) regulations, but the color reaction results of the artificial agarwoods were different from the regulations of Chinese pharmacopoeia (edition2010).
An optimized TLC chromatographic fingerprint was established on the basis of TLC identification method of Chinese Pharmacopoeia.8Natural agarwoods and8artificial agarwoods were performed, and the results indicated that the TLC chromatographic fingerprint could distinguish the natural agarwoods and artificial agarwoods easily, but could not distinguish different grade in natural agarwoods and the artificial agarwoods from different artificial inducing technology.
In order to distinguish the agarwoods with different quality and from different artificial inducing technology. A GC-MS chromatographic fingerprint was established. The data of8natural agarwood samples and8artificial agarwood samples in GC-MS chromatograph were studied by Genetic algorithm-Support vector machine (GA-SVM)model, two independent samples t test, correlation analysis and hierarchical clustering analysis. Specific analysis of the process are as follows:
1. The relative contents of431peaks were cluster by hierarchical clustering analysis method. The clustering results indicated that the GC-MS chromatographic fingerprint could distinguish the natural agarwoods and artificial agarwoods.
2. The components identified in the GC-MS chromatographic fingerprint were divided into five classes:2-(2-phenylethyl) chromanone derivatives, sesquiterpenes, aromatic compounds,aliphatic compounds and steroids. The major constituents were2-(2-phenylethyl) chromanone derivatives in all samples. The total relative contents of five compounds combined with the content of ethanol extraction were cluster by hierarchical clustering analysis method. The clustering results indicated that the different grade in natural agarwoods and the artificial agarwoods from different artificial inducing technology could be distinguished.
The average total relative contents of five compounds were analyzed using two independent samples t test. It was found that the total relative contents of2-(2-phenylethyl) chromanone derivatives and sesquiterpenes had significant difference between natural agarwoods and artificial agarwoods. The correlation analysis among the total relative contents of2-(2-phenylethyl) chromanone derivatives and sesquiterpenes and the content of ethanol extraction revealed that there was a positive correlation between the total relative contents of sesquiterpenes and the content of ethanol extraction and a negative correlation between the total relative contents of2-(2-phenylethyl) chromanone derivatives and the content of ethanol extraction. There was a negative linear correlation between the total relative contents of2-(2-phenylethyl) chromanone derivatives and sesquiterpenes. Owing to sesquiterpenes was the active components of agarwood, the quality of agarwood should be related with the content of ethanol extraction and the relative content of2-(2-phenylethyl) chromone derivatives and sesquiterpenes.
3. The Genetic algorithm-Support vector machine (GA-SVM)model was used to mine the data of8natural agarwood samples and8artificial agarwood samples in order to extract the the most influential ingredient which can evaluate the quality of agarwood.49ingredients extracted were analyzed using two independent samples t test. Only22ingredients had significant difference between natural agarwoods and artificial agarwoods. After correlation analysis among the relative contents of22ingredients and the content of ethanol extraction, it was found only13ingredients had significantly correlation with the content of ethanol extraction. Among the13ingredients, the relative contents of baimuxinal and spathulenol were higher than the others. The finding supposed that the relative contents of baimuxinal and spathulenol should be closely related with the quality of agarwood. In order to verify the suppose, the relative contents of baimuxinal and spathulenol combined with the content of ethanol extraction were cluster by hierarchical clustering analysis method. The clustering results indicated that the different grade in natural agarwoods and the artificial agarwoods from different artificial inducing technology could be distinguished almost.
So, the optimized evaluation system of agarwood includes description, color reaction, ethanol extraction content detection and the TLC, GC-MS analysis. Four quality-related evaluation index, including the content of ethanol extraction, the relative content of2-(2-phenylethyl) chromone derivatives and sesquiterpenes, the contents of baimuxinal and spathulenol, can be used in the quality evaluation of agarwood.
Then the evaluation system of agarwood with the four evaluation indexes was used in evaluating the quality of agarwood from different artificial inducing technology. A pinholes-infusion method to induce the generation of agarwood by chemically stimulated and/or inoculate combined method was established.1-2years after the artificial inoculation, resinous wood were collected and the inoculating effects were detected by the four evaluation indexes mentioned above. The results revealed that the artificial agarwood produced by chemically stimulated with formic acid combined with infected by Pestalotiopsis sp. method had higher quality than other resinous wood produced by other methods. In contrast, the artificial agarwood produced by chemically stimulated with formic acid combined with infected by Nodulisporium sp.method had poorest quality in all resinous wood.
The TLC and GC-MS chromatographic fingerprint were also developed for evaluating and controlling the quality of sandalwood. Combined with literature data, some regulation was found by statistical analysis, such as there was some regularly variation of total content of santalol and α-/β-santalol percent ratio among different santalum species. So the total content of santalol and α-/β-santalol percent ratio combined with the heartwood oil content can be used to evaluate the quality of sandalwood.
The optimized evaluation system of sandalwood includes description, heartwood oil content detection and the TLC, GC-MS analysis. Three quality-related evaluation index, including the content of heartwood oil, the relative content of2-(2-phenylethyl) chromone derivatives and sesquiterpenes, the total content of santalol and α-/β-santalol percent ratio can be used in the quality evaluation of sandalwood.
The optimized evaluation system of sandalwood is not good in distinguishing the different species in Santalaceae family. So to develop a molecular identification of Santalum may be useful.
Different fresh leaves samples of Santalum album L. introduced from India and Indonesia were collected and their rDNA ITS sequences were amplified by PCR technology and were sequenced by the Beijing Genomics institute. All Santalum reference sequences in GenBank were collected for further phylogenetic analysis. It was foud that the rDNA ITS sequence data of Santalum is stable in same species, but exist difference inter-species. The result should provide the basis for molecular identification of Santalum.
The optimized evaluation system of agarwood and sandalwood, combined with evaluation indexes which were found by data mining technology are reasonable and feasible in the quality control of agarwood and sandalwood. And the evaluation systems can promote the efficient artificial techniques to increase the production and improve the quality of agarwood and sandalwood.
檀香为檀香科檀香属的树种,是名贵香料、药材和高级工艺雕刻品原料,其药用部分是具有芳香性的心材和从心材中提取的檀香油。在檀香自然状态下生长十分缓慢,8年左右才开始形成心材,一般需20～25年左右才能完全形成高等级的檀香心材。檀香属下的多个品种都被称为檀香,包括印度檀香、大果澳洲檀香、大花澳洲檀香、滨海夏威夷檀香和斐济檀香等,但品质最佳的是印度檀香,《中国药典》2010年版(一部)也仅收载印度檀香一个品种。种源差异、成材时间过长等因素使得檀香药材品质差异极大,市场价值差异极大,混伪情况严重。
目前沉香、檀香质量评价方法均缺乏有效、专属的评价指标。鉴于此,本文利用TLC、GC-MS、DNA分子测序等技术优化了沉香和檀香质量评价体系,应用数学处理模型和数理统计方法对沉香GC-MS数据进行挖掘,找出与沉香品质相关的特征性成分,并以优化后的沉香评价体系对不同人工造香技术的结香效果进行比较研究。对引种檀香的rDNAITS序列进行分析。旨在为识别伪制沉香和檀香、完善沉香和檀香的质量标准提供依据,并指导人工造香技术快速生产沉香药材。
本文采用《中国药典》2010年版(一部)方法对收集的34个沉香样品进行检验,检验结果结合药材的外观和来源,可将34个沉香样品分为三类：天然沉香、人工沉香和伪品。天然沉香按照商品沉香的分类标准分为三个等级：两个一等品(油格占80%),三个二等品(油格占60%)和三个三等品(油格占40%以下)。人工沉香根据来源分为三个物理刺激样品、两个化学刺激样品和三个化学与生物联合刺激所得的样品。按照中国药典检验的结果表明只有质量较优的天然沉香完全符合规定,而人工沉香的颜醇浸出物微量升华试验与中国药典的规定有差异。
取8个天然沉香和8个人工沉香样品,在《中国药典》2010年版(一部)TLC鉴别方法的基础上进行优化,分别考察了不同展开溶剂系统、不同检视方法,构建了沉香的TLC指纹图谱,经方法学考察该指纹图谱具有一定的重复性、稳定性,符合指纹图谱研究的相应要求。TLC指纹图谱可以直观、快速地区分天然沉香和人工沉香,但无法区别不同等级天然沉香和不同结香方法生产的人工沉香。
为解决上述问题,本文构建了沉香的GC-MS特征图谱,并从天然沉香品质较优、人工沉香品质较差的差异入手,以GC-MS数据为基础进行由浅入深的数据分析。
具体分析过程如下：
1.取16个天然与人工沉香样品的GC-MS指纹图谱中的431个色谱峰数据进行聚类分析,发现该指纹图谱可以有效地区分天然沉香和人工沉香。
2.检索结果表明沉香中主要存在5大类化合物：2-(2-苯乙基)色酮类(后面简称色酮类)、倍半萜类、脂肪族、芳香族和甾体类化合物。取色酮类、倍半萜类、脂肪族、芳香族和甾体类化合物的总相对百分含量加上醇溶性浸出物含量6组数据进行聚类分析,聚类结果显示采用分类数据可以将所有16个样品分成2个大类,第一类包含所有8个天然沉香以及30号人工沉香,且品质较好的天然一等品和二等品都先各自聚为一支再与其他天然沉香聚成一支；30号虽然为刀砍物理刺激所得样品,但因其结香时间较长,其品质接近天然沉香,与天然沉香聚为一支；第二类包含7个人工沉香,且按照不同结香方法进行聚类。说明不同类别化合物及其含量能更好地体现了不同品质沉香与不同结香方法所得沉香之间的差异。
取天然沉香和人工沉香的不同类别化合物总含量的平均值进行两独立样品t检验,了解到色酮类化合物和倍半萜类化合物的含量在天然沉香与人工沉香中具有显著性差异；然后将色酮类化合物、倍半萜类化合物的含量和醇浸出物含量进行双变量相关分析,发现色酮类化合物含量、倍半萜化合物含量与醇浸出物含量分别呈负相关和正相关,且两者之间具有较强的负线性相关。由于倍半萜类化合物为沉香的主要活性成分,说明醇溶性浸出物含量与沉香的品质存在一定的相关性。色酮类化合物与倍半萜类化合物的含量比值在天然沉香中比较稳定,可以明显区分人工沉香与天然沉香,且可以有效区别不同结香方法所得的样品。因此,该比值可以作为沉香品质评价的一个主要指标。
3.采用遗传算法-支持向量机联用(GA-SVM)数学模式优选影响天然沉香与人工沉香分类的主要成分。本文将天然沉香和人工沉香的GC-MS结果(各成分的峰面积)作为支持向量机的输入特征参数,采用遗传算法与支持向量机耦合,对特征参数进行优选,得到49个对天然沉香和人工沉香分类最具影响力的成分。对这49个成分进行独立样本双侧t检验,取具有统计学差异的22个化合物的百分含量与醇溶性浸出物含量进行相关分析。发现其中有13个化合物的含量与醇溶性浸出物含量之间呈现中等强度以上的相关性,且具有统计学意义。其中含量最高的成分是白木香醛和斯巴醇。取16个样品的白木香醛、斯巴醇含量和醇溶性浸出物含量进行聚类分析,结果以上述三个指标进行聚类可以将沉香一等品和二等品聚为一类；其他人工沉香以及一些品质较差的天然沉香聚为一类。结果表明,白木香醛与斯巴醇的含量确实能够有效地区分不同品质的沉香。
结合上述实验和数据分析结果,确定优化后的沉香评价体系包括外观性状、醇溶性浸出物的微量升华试验、醇溶性浸出物含量、优化后的TLC指纹图谱和GC-MS指纹图谱,并且可以用醇溶性浸出物含量、色酮类与倍半萜类化合物的含量比值以及白木香醛和斯巴醇的含量等数据对沉香品质进行评价。
取优化后的评价体系对不同结香技术所得的动态样品进行分析,并以醇溶性浸出物含量、色酮类与倍半萜类化合物的含量比值、白木香醛和斯巴醇相对含量为指标,对结香效果进行评价。发现甲酸与拟盘多毛孢属内生真菌(Pestalotiopsis sp.)混合诱导的白木香结香效果最好,而甲酸与多节孢内生真菌(Nodulisporium sp.)混合诱导的白木香结香效果最差。
本文构建了檀香的TLC、GC-MS指纹图谱。取檀香样品的GC-MS指纹图谱数据,结合文献数据进行统计分析,发现总檀香醇%及α-/β-檀香醇%比值在不同檀香品种之间具有一定的变化规律,与品质具有一定相关性,可与挥发油含量一起用于檀香药材的质量评价。优化后的檀香评价体系包括外观性状、挥发油含量测定、优化后的TLC指纹图谱和GC-MS指纹图谱,并且可以用挥发油含量、总檀香醇含量和α-檀香醇和β-檀香醇的含量比值为评价指标对檀香的品质进行评价。
采集不同培育代数、不同来源的印度、印尼檀香,采用PCR扩增-测序技术对其rDNA ITS序列进行测定,了解了我国广东地区所引种檀香的基因变异情况,并为后续的药材分子鉴定提供基础。DNA测序结果表明,檀香的rDNA ITS序列在种内稳定,不受引种代数、寄主等因素的影响。通过GenBank数据库中最近源序列相似率分析,构建系统发育树,结果表明檀香属植物种间存在较大差异,可应用DNA分子测序技术建立檀香分子鉴定方法,对市售檀香品种正本清源。
本研究通过沉香和檀香TL、GC-MS指纹图谱、rDNA ITS序列系统发育树的构建和分析,综合运用药物分析学、化学计量学和分子系统学的方法,探讨了与沉香和檀香品质相关的特征成分和评价指标,优化了沉香和檀香的质量评价体系。并深入分析天然沉香和人工沉香品种差异,揭示了不同人工造香技术的优劣。为加快沉香生产速度、提高沉香和檀香的品质,开发和利用人工种植沉香和檀香资源奠定基础。
Agarwood. the resinous portions of Aquilaria plants, have been used as medicines and incenses in Asia. Aquilaria sinensis (Lour) Gilg.(Thymeleaceae), distributed in Fujian, Guangdong, Guangxi and Hainan provinces of China, is the major producer of agarwood in China, which had been recommended to be the grade Ⅱ protected plant in China (1999). In nature, agarwood was produced only when the plant was wounded or attacked by pathogens or insects. Black and fragrant resin produced under occasional stimulation is deposited in the stem or main branches of the trees. It is generally considered that the more the resin, the better the quality of agarwood. With increased consumption of agarwood in recent years, over-exploitation of agarwood in forest caused depletion of the natural resources. The International Union for the Conservation of Nature (IUCN) in2002classified all species of Aquilaria including A. sinensis as "vulnerable"(IUCN2002). So, improving the yield and quality of agarwood is very important now. However, the formation of agarwood has long been mysterious and associated with arcane forces, and the difference in quality between natural agarwood and artificial agarwood is not clear. There is a serious adulterants of agarwood in market.
Sandalwood belongs to the Santalaceae family, highly valued for its fragrant heartwood, which contains sandal oil that is used in perfumes, medicine. In natural condition, Sandal grows very slowly; about8years begin to form heartwood, which normally takes20to25years to fully form high grade sandalwood. The species in Santalaceae family such as S. album, S. yasi, S. spicatum, S. paniculatum, S. austrocaledonicum etc can form fragrant heartwood. But, there are great quality difference among the species. This is true for S.album L. from India, Indonesia and East Timor which is the bench mark of commercial sandalwood species, owing to its high quality. S.album L is the only specie in the Pharmacopoeia of the People's Republic of China (Chinese Pharmacopoeia,2010). The variation of quality among species and the long time to form fragrant heartwood caused the quality of sandalwood vary greatly; market value difference is great, and leading to a severe mixed false situation.
In order to meet the increasing demand, Aquilaria sinensis trees and sandalwood trees were widely cultivated in the south of china. However, the lack of reasonable and feasible evaluation system of quality hinders the development and utilization of agarwood and sandalwood.
So, in this study, we intended to find out the quality-related evaluation index of agarwood and sandalwood by using the statistical models, and to optimize the evaluation system of agarwood and sandalwood by using thin layer chromatography (TLC) and gas chromatography-mass spectrometry (GC-MS) techniques. And the rDNA ITS sequences of S. album L. introduced from India and Indonesia were amplified by PCR technology and were sequenced. The study can distinguish the fake from commercial agarwood and sandalwood, and can improve the standard of quality control. It is useful in rapid production of agarwood by artificial technology.
34Samples collected from market were detected by the methods in Chinese Pharmacopoeia. Combined with the appearance and sources, the34samples can be divided into three groups by the test results:natural agarwood, artificial agarwood and fake agarwood. Follow the commercial classification criteria, the natural agarwoods were categorized into three grades. The1st grade has the best quality, the2nd grade has medium quality and the3rd grade has the poorest quality. The artificial agarwoods were categorized into three groups by the different artificial inducing technology. Three samples were artificial induced by physical methods, two samples were artificial induced by chemical methods, and three samples were artificial induced by chemical and biological joint stimulation. All the experimental results of the natural agarwoods met Chinese pharmacopoeia (edition2010) regulations, but the color reaction results of the artificial agarwoods were different from the regulations of Chinese pharmacopoeia (edition2010).
An optimized TLC chromatographic fingerprint was established on the basis of TLC identification method of Chinese Pharmacopoeia.8Natural agarwoods and8artificial agarwoods were performed, and the results indicated that the TLC chromatographic fingerprint could distinguish the natural agarwoods and artificial agarwoods easily, but could not distinguish different grade in natural agarwoods and the artificial agarwoods from different artificial inducing technology.
In order to distinguish the agarwoods with different quality and from different artificial inducing technology. A GC-MS chromatographic fingerprint was established. The data of8natural agarwood samples and8artificial agarwood samples in GC-MS chromatograph were studied by Genetic algorithm-Support vector machine (GA-SVM)model, two independent samples t test, correlation analysis and hierarchical clustering analysis. Specific analysis of the process are as follows:
1. The relative contents of431peaks were cluster by hierarchical clustering analysis method. The clustering results indicated that the GC-MS chromatographic fingerprint could distinguish the natural agarwoods and artificial agarwoods.
2. The components identified in the GC-MS chromatographic fingerprint were divided into five classes:2-(2-phenylethyl) chromanone derivatives, sesquiterpenes, aromatic compounds,aliphatic compounds and steroids. The major constituents were2-(2-phenylethyl) chromanone derivatives in all samples. The total relative contents of five compounds combined with the content of ethanol extraction were cluster by hierarchical clustering analysis method. The clustering results indicated that the different grade in natural agarwoods and the artificial agarwoods from different artificial inducing technology could be distinguished.
The average total relative contents of five compounds were analyzed using two independent samples t test. It was found that the total relative contents of2-(2-phenylethyl) chromanone derivatives and sesquiterpenes had significant difference between natural agarwoods and artificial agarwoods. The correlation analysis among the total relative contents of2-(2-phenylethyl) chromanone derivatives and sesquiterpenes and the content of ethanol extraction revealed that there was a positive correlation between the total relative contents of sesquiterpenes and the content of ethanol extraction and a negative correlation between the total relative contents of2-(2-phenylethyl) chromanone derivatives and the content of ethanol extraction. There was a negative linear correlation between the total relative contents of2-(2-phenylethyl) chromanone derivatives and sesquiterpenes. Owing to sesquiterpenes was the active components of agarwood, the quality of agarwood should be related with the content of ethanol extraction and the relative content of2-(2-phenylethyl) chromone derivatives and sesquiterpenes.
3. The Genetic algorithm-Support vector machine (GA-SVM)model was used to mine the data of8natural agarwood samples and8artificial agarwood samples in order to extract the the most influential ingredient which can evaluate the quality of agarwood.49ingredients extracted were analyzed using two independent samples t test. Only22ingredients had significant difference between natural agarwoods and artificial agarwoods. After correlation analysis among the relative contents of22ingredients and the content of ethanol extraction, it was found only13ingredients had significantly correlation with the content of ethanol extraction. Among the13ingredients, the relative contents of baimuxinal and spathulenol were higher than the others. The finding supposed that the relative contents of baimuxinal and spathulenol should be closely related with the quality of agarwood. In order to verify the suppose, the relative contents of baimuxinal and spathulenol combined with the content of ethanol extraction were cluster by hierarchical clustering analysis method. The clustering results indicated that the different grade in natural agarwoods and the artificial agarwoods from different artificial inducing technology could be distinguished almost.
So, the optimized evaluation system of agarwood includes description, color reaction, ethanol extraction content detection and the TLC, GC-MS analysis. Four quality-related evaluation index, including the content of ethanol extraction, the relative content of2-(2-phenylethyl) chromone derivatives and sesquiterpenes, the contents of baimuxinal and spathulenol, can be used in the quality evaluation of agarwood.
Then the evaluation system of agarwood with the four evaluation indexes was used in evaluating the quality of agarwood from different artificial inducing technology. A pinholes-infusion method to induce the generation of agarwood by chemically stimulated and/or inoculate combined method was established.1-2years after the artificial inoculation, resinous wood were collected and the inoculating effects were detected by the four evaluation indexes mentioned above. The results revealed that the artificial agarwood produced by chemically stimulated with formic acid combined with infected by Pestalotiopsis sp. method had higher quality than other resinous wood produced by other methods. In contrast, the artificial agarwood produced by chemically stimulated with formic acid combined with infected by Nodulisporium sp.method had poorest quality in all resinous wood.
The TLC and GC-MS chromatographic fingerprint were also developed for evaluating and controlling the quality of sandalwood. Combined with literature data, some regulation was found by statistical analysis, such as there was some regularly variation of total content of santalol and α-/β-santalol percent ratio among different santalum species. So the total content of santalol and α-/β-santalol percent ratio combined with the heartwood oil content can be used to evaluate the quality of sandalwood.
The optimized evaluation system of sandalwood includes description, heartwood oil content detection and the TLC, GC-MS analysis. Three quality-related evaluation index, including the content of heartwood oil, the relative content of2-(2-phenylethyl) chromone derivatives and sesquiterpenes, the total content of santalol and α-/β-santalol percent ratio can be used in the quality evaluation of sandalwood.
The optimized evaluation system of sandalwood is not good in distinguishing the different species in Santalaceae family. So to develop a molecular identification of Santalum may be useful.
Different fresh leaves samples of Santalum album L. introduced from India and Indonesia were collected and their rDNA ITS sequences were amplified by PCR technology and were sequenced by the Beijing Genomics institute. All Santalum reference sequences in GenBank were collected for further phylogenetic analysis. It was foud that the rDNA ITS sequence data of Santalum is stable in same species, but exist difference inter-species. The result should provide the basis for molecular identification of Santalum.
The optimized evaluation system of agarwood and sandalwood, combined with evaluation indexes which were found by data mining technology are reasonable and feasible in the quality control of agarwood and sandalwood. And the evaluation systems can promote the efficient artificial techniques to increase the production and improve the quality of agarwood and sandalwood.
引文
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