The secret of the white wood incense incense technique is revealed, find out!
Zhang Libao et al
National Natural Science Foundation of China (32060357).
Aquilaria sinensis belongs to the genus Agarwood of the Ruixiang family, because of its rich balsam, it is named after sinking into the water, also known as soil agarwood, tooth incense tree, daughter incense, cliff incense, green cinnamon incense, guan incense, rue incense, when the trunk of the plant is affected by external adverse factors, the pathological tissue that remains is agarwood, if the balsam is less, can not be immersed in water, it is called yellow ripe incense, stack incense; It is distributed in the evergreen broad-leaved mixed forests of low-altitude mountains and hills in Guangdong, Hainan, Guangxi, Fujian, Yunnan, Taiwan and other low-altitude mountains and hills in mainland China, and is the only plant source of domestic agarwood by the Pharmacopoeia of the People's Republic of China: 2020 Edition, Part I, and is a unique and precious medicinal source plant in mainland China. Agarwood made of white wood incense has the activities of regulating the central nervous system, anti-inflammatory, antibacterial, anti-tumor, anti-depressant, antioxidant, hypoglycemic, regulating blood lipids, antipyretic and analgesic, etc., and can be used to treat symptoms such as chest and abdominal distension and pain, stomach cold, vomiting, hiccups, cough, rheumatism, high fever, kidney deficiency, qi reversal, and shortness of breath, and also has a potential therapeutic effect on diabetes, Alzheimer's disease and other diseases, so it has been sought after as a top-grade medicinal material by famous doctors throughout the ages. In addition, agarwood is mostly used to make incense or high-end cosmetics or handicrafts with collectible value, etc., and the leaves can be made into tea brewing and drinking.
With the predatory felling of wild agarwood resources and the destruction of habitats, wild agarwood species are endangered and extinct, and it is urgent to improve the efficiency and quality of incense through artificial incense technology. The existing research results show that artificial incense technology is mainly divided into physical trauma method (hole digging method, cutting method, fire nailing method, burning tree trunk method, cutting off the stem or breaking branch method), chemical induction (ethephon, methyl jasmonate, salicylic acid, sodium chloride, formic acid, ferrous chloride, ferric chloride and sodium sulfite), artificial inoculation induction (Melanotus flavolives, Fusarium laseritum, and Dispora cocoa-hair). Lasiodiplodia theobromae, Penicilluma meleagrinum, Trichoderma atroviride, Trichoderma koningiopsis, Penicilliumitalicum, Fusarium solani, and Citrus grapes Botryosphaeria rhodina, Fomitopsissp., Botryos rhodina) and other methods induce incense. However, the existing incense formation methods have shortcomings, and the physical trauma method mainly uses different ways to carry out artificial physical damage to the body of the white wood incense tree, forcing the white wood incense tree body to produce a plant defense response, and the brown to dark black pathological tissue - agarwood produced under the action of secondary metabolism. Different physical treatment methods have their own advantages, the advantages are simple operation, low cost, the disadvantage is that the physical method of incense formation speed is slow, the incense collection time is long, the incense only gathers near the wound and the yield is limited, and even will cause the death of the tree. The chemical method of inducing incense formation greatly improves the speed of incense formation and is simple to operate, which mainly uses chemical reagents or their combination forms to induce or force white wood incense to produce stress reaction and then produce agarwood substances. The disadvantage is that the agarwood produced is more irritating, the price is low, and it is not yet recognized by the market, and whether the chemical agents used will cause pollution to the environment, whether the agarwood produced can meet the requirements of the national pharmacopoeia, and whether it is toxic to the human body after use and other factors are still unclear. Artificial inoculation induces incense formation is the use of endophytic fungi or exogenous fungi that can induce white wood incense to produce agarwood substances, this method further improves the efficiency of incense formation compared with the two, ensures the quality of incense, and does not have any chemical additives will not cause pollution to the tree body and the environment, which is currently widely used in artificial incense formation.
The whole body incense technology is the world's leading incense technology developed on the basis of the hypothesis of white wood incense defense-induced reaction incense formation first proposed by researcher Wei Jianhe, which provides great theoretical and practical value for the incense formation mechanism of white wood incense, provides strong core technical support for the artificial incense technology of white wood incense, and takes an important step to solve the bottleneck of agarwood artificial incense technology. In this paper, according to the mechanism of defense induced incense formation of white wood incense, and drawing on the principle of whole-body incense infusion method, the fragrance liquid was filled into a pointed mouth bottle in advance, inserted into the hole made in advance, and the plant's own metabolism was used to transport the fragrance solution to all parts of the tree through the xylem, and then the whole tree could induce incense formation. At the same time, the insect resident feeding effect can be used to carry the fragrance liquid, which can be infected in the form of insect carriers, and compared with the two incense forming methods of insect moth and mechanical perforation, the infection effect can be verified.
1 Materials and methods
1.1 Ingredients
1.1.1 Overview of Baimuxiang Base Baimuxiang Base is located in the Nabanhe Nature Reserve of Xishuangbanna Dai Autonomous Prefecture, Yunnan Province, and selects Baimuxiang trees with an average stem diameter of more than 20 cm, an age of 15 years, and good tree condition.
1.1.2 Literature visual analysis data The literature data comes from the CNKI (http://www.cnki.net) database, and as of February 16, 2023, a total of 49 documents have been retrieved with the theme of "whole body incense", and all search results are exported to Refworks format for later use.
1.1.3 Fungus and borer insects were all from the Institute of Forest Protection, Yunnan Academy of Forestry and Grassland Sciences, with internal numbers YB-1, YB3-1, HLH, YML, and are now preserved by the Institute of Forest Protection, Yunnan Academy of Forestry and Grassland Sciences.
1.1.4 Preparation of white wood incense liquid White wood incense liquid is a fermentation broth independently developed by our research group according to the principle of infusion method of incense formation, which can significantly induce the fungus of white wood incense to inoculate in the culture broth. The mycelium in the fragrance solution was cut by a shearing machine at a speed of 15 000r/min for 2 min, and was divided into a special sharp bottle with a capacity of 15 mL/bottle for later use.
1.2 Test Methods
1.2.1 Visual analysis of literature After using CiteSpace 6.1R6 [34] to convert the search results, according to the literature search results, the analysis time is set from December 2010 to January 2023, with keywords as the analysis target, and the network graph cropping (Pathfinder) and pruning slice d networks algorithms are used, and the rest are default.
1.2.2 Artificial incense treatment of white wood incense Mechanical perforation treatment is used as the CK control of this test, and at the same time it is the premise of the other three treatments, mainly using a hand-held lithium drill 50 cm from the ground, from bottom to top, with a spacing of 10~15 cm, a row spacing of 20 cm, a drilling diameter of 10 mm, and an inclination angle of 45 ° to punch holes, and the hole depth is dry diameter 1/3;The treatment of the incense liquid is to directly insert the pointed bottle filled with the fragrance liquid into the hole made by the electric drill; the insect moth treatment is to use tweezers to put the larvae of the longhorn beetle directly into the hole, and wrap the hole in the trunk of the tree with plastic wrap from bottom to top to prevent it from escaping and causing biological disasters, and at the same time use the insect needle to pierce the hole to ensure ventilation and avoid its suffocation and death; the difference between the insect carrier treatment and the insect moth treatment is that the treatment needs to use tweezers to clamp the larvae of the orange longhorn longhorn in a beaker containing the fragrance liquid and dip it evenly in 1~2 s, and then quickly put it into the well, and the rest is the same as the moth-eaten treatment. Each tree had one treatment and three duplicates, uniformly numbered and tagged on the tree (Table 1).
1.2.3 Sample sampling After 2 months of artificial incense treatment, use a hole punch to sample the incense part of the tree, dry it and set it aside.
1.2.4 Alcohol extract extraction Each sample weighed 2 g and added it to an Erlenmeyer flask, added 50 mL of absolute ethanol and let it stand for 1 h, then placed in a water bath at 80 °C for 1 h, dried the evaporation dish 3 h in advance and weighed the mass of the empty dish, immediately added 25mL of the filtered extract, steamed in a water bath for 1 h, measured the quality of the evaporation dish, and then calculated the content of the alcohol extract.
1.2.5 Data analysis and cartography Two months after the end of the experiment, the infection effect of all treatments was measured with a ruler and the infected area was measured (Table 2). Based on the experimental data, Graphpad prism 8.3.0 was used to plot the infection area of different treatments (Fig. 2).
Table 1 Numbers of different treatments of white wood incense
Table 2 The infection area of different treatments of white wood
2 Results and Analysis
2.1 Literature analysis of the whole body incense
According to the results of CiteSpace analysis (Fig. 1), there were 32, 21, and 7 articles containing keywords such as agarwood, white wood, and all-body incense, respectively. The results showed that the whole body incense technology was mainly applied to white wood incense, and the incense method, anti-inflammatory activity, chemical composition, quality, pharmacological effect, acute toxicity, differential composition, quality evaluation, In recent years, molecular omics and other technical means have accelerated the research on the mechanism of incense, but the efficiency of the existing artificial incense formation still needs to be improved, the quality of incense formation still needs to be improved, and whether the content of the alcohol extract of agarwood meets the requirements of the national "Pharmacopoeia" and the mechanism of incense formation is particularly important.
Figure 1 Cluster analysis of keywords in the whole body
2.2 Analysis of infection effect
According to the statistics of Graphpad prism 8.3.0, the infection area from large to small was as follows: Zomycetes, insects, fragrance liquid, and CK control. The results showed that the CK control had the smallest infection area and the infection effect of all treatments was higher than that of the CK control, which had the effect of accelerating fragrance formation, the overall infection area of the insect-coyod treatment was the largest, indicating that the insect-inocchi treatment was the best combination of all treatments in this experiment, and the insect-eaten treatment was higher than the perfume solution treatment, indicating that the key factor in accelerating the tree aroma formation was insects (Fig. 2).
Fig. 2 Infection area of different treatments of white wood
2.3 Analysis of alcohol extract content
According to the measured content of agarwood formed by various artificial incense forming techniques, it can be seen that the tetraol content of agarwood in all treatments was higher than that of the mechanical control method, indicating that the three artificial incense forming methods could promote the incense formation of the white wood incense tree, and compared with the traditional incense forming methods, the alcohol extract content obtained by YB-1 was significantly stronger than that of all other incense forming methods, indicating that YB-1 It was the dominant strain that induced the formation of incense in the white wood, and the insect-bearing method was the best combination, while there was no significant difference between the other strains and different treatments.
Figure 3 Agarwood tetraglycol content
3 Discussion
The annual number of published papers is an important indicator to measure the research progress of a certain academic field [35], and the latest research results show that the keywords "agarwood", "white wood" and "whole body incense" have the highest frequency, and the literature related to the quality, pharmacodynamic effect, and technology and mechanism of incense is a relatively large proportion of the current hot research contents.
The content of alcohol extract contained in agarwood formed by whole-body incense technology is one of the key factors affecting the quality of incense, and agarwood tetraol is one of the main components of alcohol extract, which is the key index for identifying authenticity and fake, and the research on the quality evaluation of agarwood mainly focuses on trait description, microscopic identification, thin layer identification, physicochemical identification, extract content, volatile oil, etc. [36-39], so improving the content of alcohol extract of agarwood is one of the important contents of the current research.
The research on the efficacy of the whole body incense mainly focuses on regulating gastrointestinal motility, protecting the liver and heart, sedation, anti-inflammatory and other research. However, at present, the mechanism of incense formation is not clear, and the current main trend is to the hypothesis that the defense response induces incense formation in Agarwood alba hypothesis, which is believed to be the motivation for the initiator to induce the defense response of A. alba as an ecitrator, which in turn forces the tree to produce defense substances with antibacterial activity, that is, the main chemical components of the medicinal herb Agarwood, which are compounded with other cellular components to form an invading body that blocks the ducts and vascular bundles of the secondary xylem to resist further damage to Agarwood by external physical and chemical damage or fungal infection [40]. ] 。 However, this study has certain limitations: compared with other databases, the CNKI database has fewer literatures on the theme of "whole body incense", and secondly, due to the short time, the content of the alcohol extract of agarwood is small, which does not meet the requirements of the Pharmacopoeia.
Through this study, it was found that the incense liquid, the insect carrier, and the insect 3 Compared with the general physical incense technology, the treatment method has a promoting effect, and the combined form of insecticide has the best infection effect, which is stronger than the incense liquid and insect moth treatment, indicating that the fungus induces incense itself to have the advantages of fast incense formation, high incense quality, and the natural agarwood produced by the agarwood incense, coupled with the strong independent diffusion ability of the borer insects, which is convenient for custody (artificial breeding or assisted migration and breeding), and is convenient for the release and artificial control of the forest, and finally achieve the effect of all-over fragrance. If we can find out the process and mechanism of insect decay and fungal infection to induce the incense formation of white wood incense, and then propose the corresponding "insect fungus" incense forming technology or method, it will be a major innovation in incense forming technology.
Although the existing incense method accelerates the efficiency and quality of incense to a certain extent, but the overall output is still low, the cycle is long, the quality is uneven, etc., the best effect of white wood incense is the whole body incense effect, the industry generally believes that worm leakage agarwood is the best quality natural agarwood, but also the best way to achieve the whole body incense. Therefore, in order to know the main insects and their associated fungi that cause "insect leakage", this research group uses insects, fungi or their associated bacteria, and borrowing insects to carry incense-forming fungi (e.g., Fusarium sp., Penicillium sp., Trichoderma sp., Murphyllus aureus, and Dipspora coccus). Botryodiplodia sp., et al.) synergistically induced the formation of white wood incense, that is, through human intervention, with the help of "insect + fungus" combined infection to induce the formation of white wood incense. In this paper, the research status of the whole body fragrance formation technology was analyzed, and the effect of the whole body fragrance formation was verified by the insect carrier fungus induced fragrance formation test, in order to finally achieve the effect of the whole body fragrance formation.