A method of minimally invasive knot tying

Abstract

The present application provides a kind of minimally invasive ligation method, utilize 1-3mm drill bit in white lignum tree transverse and longitudinal drilling, do not drill through the bark, the horizontal distance between adjacent holes is 2-4cm, vertical distance is 5-8cm, then sequentially inject ligation liquid in each hole, after 3 months, agarwood can be condensed, with the passage of time, agarwood oil and fat accumulate in large quantities.The present application adopts minimally invasive ligation technology, reduces the harm to tree body, maintains the overall growth trend of tree body, does not cause large area damage to tree, ensures its normal growth level, effectively absorbs ligation agent, conforms to the natural growth law of agarwood, and promotes the formation of agarwood.

Description

Technical Field

[0001] This invention relates to the field of agricultural biotechnology, and more specifically to a minimally invasive method for resin formation. Background Technology

[0002] Agarwood refers to the resinous wood of two species, *Aquilaria agallocha* Roxb. and *Aquilaria sinensis* (Lour.) Gilg, belonging to the genus *Aquilaria* of the family Thymelaeaceae. *Aquilaria sinensis* is the main species of agarwood produced in China. Normally growing *Aquilaria sinensis* does not produce resin. Only when affected by natural factors (windfall, fire, insect infestation, microbial invasion, or lightning strike) or human factors (cutting, drilling, nailing, inoculation, etc.), and accumulated over a long period, can high-quality agarwood form. Due to the extremely low probability of natural agarwood formation and the long formation period, wild agarwood resources are becoming increasingly scarce.

[0003] In recent years, large-scale planting of Aquilaria sinensis has been carried out in Guangdong, Hainan, Guangxi, and other regions, utilizing modern resin-inducing techniques to promote resin production. Traditional resin-inducing techniques involve injuring the Aquilaria sinensis tree through methods such as cutting, burning, nailing, drilling, or artificially inoculating the wounds with microorganisms using modern biotechnology to stimulate the tree's defense mechanisms and promote resin production. However, these techniques are considered to have low yields and inconsistent quality, are cumbersome to operate, and negatively impact the tree's healthy growth. A new technique developed in recent years, called whole-body resin-inducing, involves repeatedly injecting resin-inducing agents into the trunk to induce a defense response, causing resin production throughout the entire plant. This technique is currently being widely adopted and promoted. It is claimed that resin production can occur within six months after treatment, with yields dozens of times higher than traditional methods, and meeting national pharmacopoeia standards. While this method can produce resin relatively quickly, it requires drilling large and deep holes with close spacing, still causing significant damage to the entire plant and hindering the long-term healthy growth of the Aquilaria sinensis tree. Currently, there are few technical solutions for obtaining high-quality agarwood through minimally invasive techniques to introduce resin-forming agents. Building upon minimally invasive techniques, selecting a suitable resin-forming agent is crucial for rapidly inducing the formation of high-quality agarwood. Research on resin-forming agents used in minimally invasive techniques is also very limited. Summary of the Invention

[0004] In view of the shortcomings of the existing technology, the present invention provides a method for inducing resin formation in agarwood trees using minimally invasive techniques.

[0005] The technical solution of this invention is as follows:

[0006] A minimally invasive method for resin formation includes the following steps: using a 1-3 mm drill bit to make horizontal and vertical holes in aquilaria trees (preferably over 3 years old), without drilling through the bark, with a horizontal distance of 2-4 cm and a vertical distance of 5-8 cm between adjacent holes, and injecting 5-8 mL of resin-forming agent into each hole.

[0007] The resin-forming agent consists of: methyl jasmonic acid, salicylic acid, sodium chloride, formic acid, a culture of *Boswellia sulphurea*, and sodium sulfite.

[0008] Preferably, the amount of *Boswellia sulphurea* culture used in the fragrance-forming agent is 100 ml / L-300 ml / L.

[0009] Preferably, the method for preparing the sulfur-oxidizing botulism culture is as follows: take a single colony of activated sulfur-oxidizing botulism, transfer it to a high-magnesium medium containing sodium thiosulfate, and culture it in a shaker for at least 72 hours.

[0010] Preferably, the high-magnesium culture medium containing sodium thiosulfate contains the following components: 10-15 g / L peptone, 3-5 g / L beef extract, 5-8 g / L sodium chloride, 1-2 g / L sodium thiosulfate, and 5-7 g / L magnesium chloride, and is cultured at 30±2℃ and 200±50 rpm for 72-96 h.

[0011] Preferably, the fragrance-forming agent comprises: methyl jasmonate 10-16 mg / L, salicylic acid 25-40 mg / L, sodium chloride 0.4-0.8 mg / L, formic acid 6-9 mg / L, 100-300 ml / L of *Boswellia sulphurea* culture, and sodium sulfite 7-10 mg / L.

[0012] Preferably, the fragrance-forming agent comprises: methyl jasmonate 12 mg / L, salicylic acid 40 mg / L, sodium chloride 0.5 mg / L, formic acid 8 mg / L, 100 ml / L of *Boswellia sulphurea* culture, and sodium sulfite 8 mg / L.

[0013] Preferably, the drilling location is a part of the tree that is 50cm or taller and has a trunk diameter of 8cm or more.

[0014] Preferably, the resin formation treatment is carried out under sunny outdoor temperatures of 20±2℃ to 30±2℃. Specifically, when the resin formation treatment is carried out at around 20℃, the resulting agarwood exhibits a better inhibitory effect on Shigella flexneri.

[0015] Preferably, the amount of the fragrance-forming agent used is at least 5 mL. More preferably, the amount of the fragrance-forming agent used is 5-8 mL.

[0016] On the other hand, the fragrance-forming agent described in this invention plays an important auxiliary role in the implementation of the minimally invasive technique of this invention. The fragrance-forming agent comprises: methyl jasmonate 10-16 mg / L, salicylic acid 25-40 mg / L, sodium chloride 0.4-0.8 mg / L, formic acid 6-9 mg / L, *Boswellia sulphureus* culture 100-300 ml / L, and sodium sulfite 7-10 mg / L.

[0017] Compared with the prior art, the beneficial results of the present invention are as follows:

[0018] The minimally invasive agarwood formation method of this invention involves small drill holes and a relatively small number of holes, resulting in less damage to the tree. It also promotes rapid agarwood formation and produces high-quality agarwood, yielding premium agarwood material in approximately three months. As time progresses, the agarwood accumulates and its quality improves. The resulting agarwood possesses excellent pharmaceutical properties and exhibits a significant inhibitory effect on pathogenic microorganisms.

[0019] The amount of fragrance-forming agent used in this invention is small, requiring only 5-8 mL to be injected into each well. Detailed Implementation

[0020] The *Boswellia sulfadiazine* strain described in this invention is not particularly limited; similar strains belonging to the same class of *Boswellia sulfadiazine* achieve similar effects. These similar strains include *Boswellia sulfadiazine* strains with accession numbers 1.9081, 1.5383, and 1.5360 from the China General Microbiological Culture Collection Center (CGMCC), strains isolated using conventional methods, or strains obtained from other commercial sources. This invention has verified that *Boswellia sulfadiazine* strains from various sources can achieve similar effects. The following examples provide the results of experiments using a commercial strain (brand YLKBIO, catalog number YLK-JZ1071). These examples are merely partial examples of this invention and are not intended to limit the scope of the invention.

[0021] Example 1 - A minimally invasive method for resin formation

[0022] Using minimally invasive techniques, drill horizontally and vertically into Aquilaria sinensis trees (over 3 years old) at a height of 50cm and a trunk diameter of 8cm or more, using a 1-3mm drill bit. Do not drill through the bark. The horizontal distance between adjacent holes should be 2-4cm, and the vertical distance should be 5-8cm. Inject 5-8mL of resin-forming agent into each hole.

[0023] The resin-forming agent consists of: methyl jasmonate 12 mg / L, salicylic acid 40 mg / L, sodium chloride 0.5 mg / L, formic acid 8 mg / L, 100 ml / L of *Boswellia sulphurea* culture, and sodium sulfite 8 mg / L.

[0024] Boswellia sulfoxylate culture: Take a single colony of activated Boswellia sulfoxylate and transfer it to a high magnesium medium containing sodium thiosulfate (10 g / L peptone, 3 g / L beef extract, 5 g / L sodium chloride, 1 g / L sodium thiosulfate, 5 g / L magnesium chloride, with the remainder being distilled water, pH 7.4). Incubate at 30±2℃ and 200±50 rpm for 96 h (at least 72 h, preferably 96 h in this example).

[0025] In another embodiment of the present invention, the high-magnesium culture medium containing sodium thiosulfate is: peptone 10-15 g / L, beef extract 3-5 g / L, sodium chloride 5-8 g / L, sodium thiosulfate 1-2 g / L, and magnesium chloride 5-7 g / L, and cultured at 30±2℃ and 200±50 rpm for 72-96 h. That is, the aroma-forming effect achieved within this range is similar, with no significant difference.

[0026] In another embodiment of the present invention, the fragrance-forming agent comprises: methyl jasmonate 10-16 mg / L, salicylic acid 25-40 mg / L, sodium chloride 0.4-0.8 mg / L, formic acid 6-9 mg / L, *Boswellia sulphureus* culture 100 ml / L, and sodium sulfite 7-10 mg / L. That is, the fragrance-forming effect achieved within this range is similar, with no significant difference.

[0027] Example 2 - A minimally invasive method for resin formation

[0028] Using minimally invasive techniques, drill horizontally and vertically into Aquilaria sinensis trees (over 3 years old) at a height of 50cm and a trunk diameter of 8cm or more, using a 1-3mm drill bit. Do not drill through the bark. The horizontal distance between adjacent holes should be 2-4cm, and the vertical distance should be 5-8cm. Inject 5-8mL of resin-forming agent into each hole.

[0029] The resin-forming agent consists of: methyl jasmonate 12 mg / L, salicylic acid 40 mg / L, sodium chloride 0.5 mg / L, formic acid 8 mg / L, 200 ml / L of *Boswellia sulphurea* culture, and sodium sulfite 8 mg / L.

[0030] Boswellia sulfoxylate culture: Take a single colony of activated Boswellia sulfoxylate and transfer it to a high magnesium medium containing sodium thiosulfate (10 g / L peptone, 3 g / L beef extract, 5 g / L sodium chloride, 1 g / L sodium thiosulfate, 5 g / L magnesium chloride, with the remainder being distilled water, pH 7.4-7.6). Incubate at 30±2℃ and 200±50 rpm for 96 h (at least 72 h, preferably 96 h in this example).

[0031] Example 3 - A minimally invasive method for resin formation

[0032] Using minimally invasive techniques, drill horizontally and vertically into Aquilaria sinensis trees (over 3 years old) at a height of 50cm and a trunk diameter of 8cm or more, using a 1-3mm drill bit. Do not drill through the bark. The horizontal distance between adjacent holes should be 2-4cm, and the vertical distance should be 5-8cm. Inject 5-8mL of resin-forming agent into each hole.

[0033] The resin-forming agent consists of: methyl jasmonate 12 mg / L, salicylic acid 40 mg / L, sodium chloride 0.5 mg / L, formic acid 8 mg / L, 300 ml / L of *Boswellia sulphurea* culture, and sodium sulfite 8 mg / L.

[0034] Boswellia sulfoxylate culture: Take a single colony of activated Boswellia sulfoxylate and transfer it to a high magnesium medium containing sodium thiosulfate (10 g / L peptone, 3 g / L beef extract, 5 g / L sodium chloride, 1 g / L sodium thiosulfate, 5 g / L magnesium chloride, with the remainder being distilled water, pH 7.4-7.6). Incubate at 30±2℃ and 200±50 rpm for 96 h (at least 72 h, preferably 96 h in this example).

[0035] Example 4 - A minimally invasive method for resin formation

[0036] Using minimally invasive techniques, drill horizontally and vertically into Aquilaria sinensis trees (over 3 years old) at a height of 50cm and a trunk diameter of 8cm or more, using a 1-3mm drill bit. Do not drill through the bark. The horizontal distance between adjacent holes should be 2-4cm, and the vertical distance should be 5-8cm. Inject 5-8mL of resin-forming agent into each hole.

[0037] The resin-forming agent consists of: methyl jasmonate 12 mg / L, salicylic acid 40 mg / L, sodium chloride 0.5 mg / L, formic acid 8 mg / L, 200 ml / L of *Boswellia sulphurea* culture, and sodium sulfite 8 mg / L.

[0038] Bosporum sulfoxylate culture: Single colonies of activated Bosporum sulfoxylate were transferred to a solution containing sodium thiosulfate. Low magnesium The medium (10 g / L peptone, 3 g / L beef extract, 5 g / L sodium chloride, 1 g / L sodium thiosulfate, 1 g / L magnesium chloride, balance distilled water, pH 7.4-7.6) was incubated at 30±2℃ and 200±50 rpm for 96 h (at least 72 h, preferably 96 h in this example).

[0039] Example 5 - A minimally invasive method for resin formation

[0040] Using minimally invasive techniques, drill horizontally and vertically into Aquilaria sinensis trees (over 3 years old) at a height of 50cm and a trunk diameter of 8cm or more, using a 1-3mm drill bit. Do not drill through the bark. The horizontal distance between adjacent holes should be 2-4cm, and the vertical distance should be 5-8cm. Inject 5-8mL of resin-forming agent into each hole.

[0041] The resin-forming agent consists of: methyl jasmonate 12 mg / L, salicylic acid 40 mg / L, sodium chloride 0.5 mg / L, formic acid 8 mg / L, and sodium sulfite 8 mg / L.

[0042] All examples 1-5 were carried out under sunny outdoor temperature conditions of 30±2℃.

[0043] Example 6 - A minimally invasive method for resin formation

[0044] The operation method in this embodiment is the same as that in embodiment 2, except that the resin formation treatment is carried out under the condition of an outdoor temperature of 20±2℃ on a sunny day.

[0045] In the above embodiments, agarwood material was collected from the resin-forming area three months after the resin-forming agent was injected, and then tested. The testing was conducted in accordance with the requirements for agarwood in the 2020 edition of the Chinese Pharmacopoeia.

[0046] The results showed that the agarwood obtained in Examples 1-4 and Example 6 exhibited mottled patterns of alternating dark brown resin and yellowish-white wood, while the resin in Example 5 was lighter in color. The main component detection results showed that the high-performance liquid chromatograms of the samples in Examples 1-6 all showed six characteristic peaks, corresponding to the six characteristic peaks in the chromatogram of the control material (wild agarwood). The content of alcohol-soluble extractives in all agarwood samples was greater than 10%, with the content of alcohol-soluble extractives in Examples 1-4 and Example 6 being significantly higher than that in Example 5, increasing by at least 50%; calculated on a dried basis, the content of agaritol in each example was greater than 0.10%.

[0047] Antibacterial test:

[0048] The test bacteria were Shigella flexneri, Escherichia coli, and Staphylococcus aureus. Single colonies of the test bacteria were inoculated into LB medium, cultured, and diluted to a concentration of 1×10⁻⁶. 6 Bacterial suspension at CFU / mL.

[0049] According to the method under the "Determination of Extractives" section 2201 of the Chinese Pharmacopoeia 2020, the alcohol-soluble extractives of the agarwood samples in each example were obtained by extraction with 60% ethanol as solvent.

[0050] The alcohol-soluble extract was diluted to different concentrations, and 200 μL of each solution was added to melted culture medium (3 g beef extract, 10 g peptone, 5 g NaCl, 15 g agar, diluted with water to 1000 mL, pH 7.4-7.6) and mixed well, so that the concentrations of the alcohol-soluble extract in the culture medium were 500, 250, 125, 62.5, and 31.25 mg / mL, respectively. 200 μL of 60% ethanol was added as a blank control. 200 μL of bacterial suspension was inoculated into culture plates with different drug concentrations, one bacterial strain per plate, and incubated at 37 ℃ for 24 h. Colony growth was observed, and the lowest concentration at which no colonies grew was the minimum inhibitory concentration (MIC). The experiment was repeated 5 times, and the results are shown in Table 1.

[0051] Table 1

[0052]

[0053] Examples 1-3 employed the minimally invasive resin-forming method of this invention, combined with a matching resin-forming agent. The *Boswellia sulphureus* culture in Example 4 was obtained using a low-magnesium culture medium. The resin-forming agent in Example 5 contained no *Boswellia sulphureus* culture. Example 6 involved resin-forming treatment at approximately 20°C. The results showed that Examples 1-3 and Example 6 exhibited the best antibacterial effects. Although the resin-forming speed in Example 6 was slower than in Examples 1-4 at 20±2°C, the antibacterial experiment showed a better inhibitory effect against *Shigella flexneri*. This may be because the slower transmission rate of the resin-forming agent at 20°C facilitates repeated stimulation of the tree, leading to an increase in the content of active ingredients in the secreted resin.

Claims

1. A minimally invasive method for resin formation, characterized in that, Includes the following steps: Use a 1-3mm drill bit to drill horizontally and vertically into the Aquilaria sinensis tree, without drilling through the bark. The horizontal distance between adjacent holes should be 2-4cm, and the vertical distance should be 5-8cm. Inject a resin-forming agent into each hole. The resin-forming agent comprises: methyl jasmonate 10-16 mg / L, salicylic acid 25-40 mg / L, sodium chloride 0.4-0.8 mg / L, formic acid 6-9 mg / L, 100-300 ml / L of *Boswellia sulphurea* culture, and sodium sulfite 7-10 mg / L. The method for preparing the sulfur-oxidizing botulinum culture is as follows: take a single colony of activated sulfur-oxidizing botulinum, transfer it to a high magnesium medium containing sodium thiosulfate, and culture it in a shaker for at least 72 hours. The high-magnesium culture medium contains the following components: peptone 10-15 g / L, beef extract 3-5 g / L, sodium chloride 5-8 g / L, sodium thiosulfate 1-2 g / L, and magnesium chloride 5-7 g / L.

2. The method for minimally invasive resin formation according to claim 1, characterized in that, In the resin-forming agent, the amount of *Boswellia sulphurea* culture used is 100 ml / L-300 ml / L.

3. The method for minimally invasive resin formation according to claim 1, characterized in that, The resin-forming agent comprises: methyl jasmonate 12 mg / L, salicylic acid 40 mg / L, sodium chloride 0.5 mg / L, formic acid 8 mg / L, 300 ml / L of *Boswellia sulphurea* culture, and sodium sulfite 8 mg / L.

4. The method for minimally invasive resin formation according to claim 1, characterized in that, The drilling site is a part of the tree that is 50cm or taller and has a trunk diameter of 8cm or more.

5. The method for minimally invasive resin formation according to claim 1, characterized in that, Agarwood formation treatment was carried out under sunny outdoor temperatures of 20±2℃~30±2℃.

6. A resin-forming agent suitable for minimally invasive resin formation, characterized in that, The resin-forming agent comprises: methyl jasmonate 10-16 mg / L, salicylic acid 25-40 mg / L, sodium chloride 0.4-0.8 mg / L, formic acid 6-9 mg / L, 100-300 ml / L of *Boswellia sulphurea* culture, and sodium sulfite 7-10 mg / L. The method for preparing the sulfur-oxidizing botulinum culture is as follows: take a single colony of activated sulfur-oxidizing botulinum, transfer it to a high magnesium medium containing sodium thiosulfate, and culture it in a shaker for at least 72 hours. The high-magnesium culture medium contains the following components: peptone 10-15 g / L, beef extract 3-5 g / L, sodium chloride 5-8 g / L, sodium thiosulfate 1-2 g / L, and magnesium chloride 5-7 g / L.

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