摘要
沉香[Lignum Aquilariae Resinatum]为瑞香科(Thymelaeceae)沉香属(Aquilaria)或拟沉香属(Gyrinops)植物含树脂的木材,是树体受伤后分泌出来的油脂成分和木质成分的固态凝聚物。白木香(Aquilaria sinensis)为中国特有的珍贵药用植物,是我国生产名贵芳香类药材沉香的主要资源植物,也是中药沉香的正品来源。白木香所形成的沉香为中国药典(2010版)所收载,具有行气止痛、温中止呕、纳气平喘等功效。本文以白木香为研究对象,对其木质部结构及诱导形成沉香的结构防御、化学防御、不同来源的沉香的质量评价、信号分子及与防御反应相关的酶类进行了系统的研究,主要结果如下：
1.白木香的木质部结构特征
健康的3年生白木香的木质部由五部分组成：髓心、放射状木射线、岛型内涵韧皮部、纤维管胞、导管。髓心内分布着大量的淀粉粒,且易塌陷；木射线和内涵韧皮部的薄壁细胞内含有大量淀粉粒、还原糖和蛋白质等储藏物质；导管内未见任何形态的内含物。纤维管胞内存在串联十数个细胞的棒状径列条。各种细胞内壁上具有不同类型的纹孔：导管内壁上有附物纹孔,互列纹孔式,与相邻的射线薄壁细胞、导管分别形成半具缘纹孔对和具缘纹孔对。纤维管胞内壁上有单斜附物纹孔。导管和纤维管胞内存在栓塞。
2.沉香的形成是对外界伤害的物理防御
通过对半断干法诱导白木香形成的沉香进行解剖结构研究,发现在沉香形成过程中,木质部结构发生了明显的变化：导管内出现2种内含物：侵填体和树胶,堵塞了导管；在伤口下16-20mm侵填体和树胶所占百分比最大,树胶数量为侵填体的8倍,白木香是可被诱导产生侵填体与树胶而树胶占优势的树种；出现了明显的分层特征：从上至下依次为腐烂层、沉香层、过渡层、白木层；腐烂层的各种组织内都含有大量的菌丝,内涵韧皮部和木射线薄壁细胞内的淀粉粒消失,内涵韧皮部细胞破裂形成孔洞,导管内壁出现裂隙状纹孔口,可见导管内含物；沉香层内涵韧皮部和木射线薄壁细胞内的淀粉粒消失,大量油脂状物质出现,并充满内涵韧皮部的薄壁细胞,且有较多的草酸钙柱晶产生,导管内发现侵填体与树胶,此层内只在靠上的位置发现有少量菌丝。过渡层内木射线和内涵韧皮部的淀粉粒减少,夹杂有油脂状物质,内涵韧皮部内偶见结晶,未见菌丝；导管内常见内含物。白木层与健康的白木香结构特征一致。菌丝最密集的位置并不是导管内含物数量最多的地方。
3.沉香的形成是对外界伤害的化学防御
通过对不同处理时间的不同层的沉香样品进行GC/MS分析,共鉴定出65种化合物。化合物的相对含量和种类在每层中是不同的。在对照样品中仅有11种烷烃类成分,而伤害处理的样品中大部分化合物是倍半萜与芳香族成分；随着处理时间的延长,烷烃类物质的相对含量急剧减少,而倍半萜和芳香族成分的种类和相对含量迅速增加。在处理15d、30d和60d的样品中分别有20种(11种烷烃类和9种倍半萜和芳香族类物质)、28种化合物(14种烷烃类物质和14种倍半萜和芳香族化合物)和53种(17种烷烃类和36种倍半萜和芳香族类物质)化合物被鉴定出来。已知这些倍半萜和芳香族类化合物对真菌具有抑菌、抗菌活性,形成对外界伤害的化学防御。
4.沉香的形成是代谢途径的改变
通过对伤害处理不同时间的白木香的淀粉、糖和挥发性成分分析,结果发现随处理时间的增加,淀粉含量逐渐减少,而糖含量呈现先增加后减少的趋势,在对照白木中有12种烷烃类成分,伤害处理的样品中增加了8种倍半萜和芳香族化合物。随着处理时间的延长,烷烃类物质的相对含量急剧减少,而倍半萜和芳香族成分的相对含量迅速增加,反映了代谢方向发生了改变。
5.沉香形成过程中信号分子的变化
通过酶联免疫分析技术和生理生化分析测定等方法,检测伤害不同时间的白木香中信号分子(过氧化氢、水杨酸、茉莉酸、乙烯、一氧化氮)的浓度,揭示这些信号分子的变化规律,结果可知：伤害后信号分子的浓度短时间内发生了较大变化,达到峰值的时间依次为一氧化氮,乙烯,水杨酸,过氧化氢,茉莉酸。
6.与防御反应有关的酶的变化
通过试剂盒法和常规生理生化分析测定方法,检测处理不同时间的白木香样品中与防御反应有关的酶及脂质过氧化物的浓度变化,由结果可知：SOD,CAT,PAL,和NOs各酶的活力在极短的时间内达到峰值,酶活性到达峰值的时间顺序依次为：H202酶、NO合成酶、PAL、SOD:同时MDA浓度也发生极大的变化,并一直高于对照。
7.通体结香法沉香的显微特征与质量评价
对通体结香法、野生、半断干和接菌法产生的沉香进行感官评价和显微结构、醇提物、TLC、GC/MS分析,发现通体结香法处理白木香20个月所得沉香达到了中国药典(2010版)规定要求,且与野生沉香在化学成分种类、醇溶性浸出物以及挥发油得率方面均极为接近,优于半断干和接菌法产生的沉香。除此之外,通体结香法沉香的显微结构具有明显的区窒化特征,在沉香层的外圈分裂产生了一层细胞,阻断了沉香层与外面木质部的物质交流,限制了沉香层的扩大,影响了沉香的产量。
Lignum Aquilariae Resinatum (agarwood) is a solid-state condensate with mixed with resin and lignin that forms in Aquilaria and Gyrinops trees of the family Thymelaeaceae. Aquilaria sinensis is the most important plant resource which produces agarwood and peculiar precious medicinal plant in China. According to Chinese Pharmacopoeias (2010edition), agarwood have many therapeutic properties, such as anti-emetic, carminative, sedative and so on. In this article, we systematically investigated the change of microstructure, quality evaluation, and chemical compositions of the agarwood with increasing treatment time, Main findings are as follows:
1Structure characteristic of xylem in A. sinensis
The stem of A. sinensis was composed of five parts:piths, included phloems, xylem rays, vessels, and fibre tracheids; The included phloem, which was foraminate type, evenly distributed in the xylem on the transverse section; Xylem rays belonged to heterogeneous Ⅲ; Alignment of vessels pore were generally in radial pore cluster(two to four), pore multiple, pore chain (five to ten), and pore solitary; Vestured pits were alternately arranged in the vessels'lateral wall; Simple acclivitous vestured pits were present in the lateral wall of fibre tracheid; Trabeculas crossed through ten fibre tracheids neighboring pith. Embolisms were observed in the vessels and fibre tracheid. Trabeculas and embolisms were firstly observed in A. sinensis. The contents and storage tissues of the starch grains, reducing sugar, and volatile constituents existed significant difference in different aged stem.
2Agarwood formation is physical blocks to damages
In the process of agarwood formation from A. sinensis with partial-stem-breaking method, gels and tyloses in vessels both originated from xylem ray parenchyma cells and neighboring paratracheal parenchyma cells. Tyloses are outgrowths of parenchyma cells into the neighboring vessels, while gels are amorphous substances into the vessel from the parenchyma cells. Obvious four distinct layers including decomposed layer, agarwood layer, transition layer, and white-timber layer were observed below the wound surface days after wounded. The typical agarwood chemical components were detected on days after wounded. Among chemicals assayed, previous notable wound inducible constituents in other plant species were detected in different zones at different time points after wounded. Every layer has unique structural features and layer depth depended on the different treatment time. The stratified structure characteristics from wound surface to agarwood layer and gradual transition to normal layer showed formation of agarwood layer was to defense against expansion of microorganisms and external damage. Distribution of each region in the vertical axis was not horizontal, presented mutual interleaving phenomenon.
3Agarwood formation is chemical defenses to damage
Chemical composition of A. sinensis being injured compared with that of healthy A. sinensis, the type and relative content of alkaness harply decline, while the type and relative content of aromatics and sesquiterpene compounds rapidly increased with the increasing wounding time, especially in the agarwood layer, and these substances generally have antibacterial and antimicrobial effect, agarwood layer prevented from the expansion of damage from the other side. A total of sixty-five compounds were identified from the three wood samples with different wounding time. Types of constituents were different in each layer of sample, the major constituents being alkanes, scsquiterpenes and aromatics in wounded sample, while all eleven components being alkanes in control samples. This suggested agarwood formation was chemical defenses to damage.
4Chang of metabolic pathways in in the process of agarwood formation
Content of starch, sugar content, and volatile component were examined through anthrone-sulfuric acid methods, I2-KI colorimetry and GC-MS analysis, respectively. The starch content decreased, while the sugar content increased gradually with the different treatment time. Twelve kinds alkane component were identified in the control and eight kinds sesquiterpene and aromatic compounds were identified in the treated sample. The relative content of alkanes was drastically reduced and the relative content of sesquitcrpene and aromatic component.was rapidly increased. This suggested metabolic pathway has took changes
5Content change of signal molecules in the process of agarwood formation
Methods such as antibody enzyme-linked immunosorbent assay (ELISA), physiological and biochemical analysis were used to examine the content of signal molecules such as H2O2, SA, JA, NO, etliylene, variations in physiological indexes, and interaction between signal molecules in the samples of A. sinensis with different treatment time. Response time of NO, ethylene, SA, H2O2, NO, and JA increased in turns.
6Changes of protective Enzymes activity in the process of agarwood formation
Methods such as assay kit, conventional methods of physiological and biochemical analysis were used to examine the protective Enzymes activity, such as SOD, CAT, PAL, and NOs in the samples of A. sinensis with different treatment time. The main results were shown as follows:Enzymes activity of CAT, NOs, PAL, and SOD reached peak value in a short period in turns, and activated defence response. While content of MDA changed greatly in a short period, keep high level all the while.
7Value investigation of agarwood with different induction methods
Agarwood characteristics through Agar-wit were similar to those of high-grade wild agarwood in terms of texture, chemical constituents, essential oil and ethanol-soluble extract content, and superior to that of agarwood with partly-breaking stem method and inoculated-fungus method, with the lattermost quality far surpassing the requirement of traditional Chinese medicine agarwood, as indicated in Chinese Pharmacopoeia2010. The obvious boundary divided stem into some different compartments at the transverse section. Boundary layer and the area on both side of boundary have significant different characteristics. The area inside boundary characterized by decay of tissues where hyphae were observed in the vessels and parenchyma cell of included phloem, while the area outside boundary characterized by living tissues where a large number of starch grains were observed in the parenchyma cell of included phloem and xylem ray and occlusions was not in the vessels. Boundary layer, which formed an obvious barrier and separated the decay area and white wood, was filled with oily substance in the vessels, fiber tracheids, and parenchyma cell of included phloem and xylem ray at the cross and longitudinal section. At the mean time, boundary layer limited the development of agarwood layer and influenced the output of agarwood.
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