Agilawood inducer, preparation method thereof and agilawood formation method

Abstract

The invention discloses an agilawood inducer, a preparation method of the agilawood inducer and an agilawood forming method, and relates to the technical field of medicinal plant cultivation. Active components in the agilawood inducer comprise a stimulant and an agilawood producing agent; the stimulant is prepared from 14-hydroxyl brassinosterol and dihydromyricetin; the edgeworthia chrysantha agent comprises ampelopsis grossedentata, berba aristolochiae mollissimae, kalimeris indica, caltrop, corydalis impatiens, sarcopyramis nepalensis, saxifraga stolonifera and piper The stimulants are all natural plant components, the agilawood forming agents are all selected traditional Chinese medicine components, and the agilawood inducer is not added with any artificially synthesized chemical substances, so that the agilawood inducer is safe to use; meanwhile, the agilawood inducer can be used for producing agilawood meeting the national pharmacopoeia 2020 edition standard, the quality is high, the agilawood producing speed is high, and the agilawood can be formed within only 5 months.

Description

Technical Field

[0001] The invention relates to the technical field of medicinal plant cultivation, and in particular to an agarwood inducer and a preparation method thereof and an agarwood formation method. Background Art

[0002] Agarwood is a resinous wood of the plant Aquilaria Lam. of the Thymelaeaceae family, which is widely used in the fields of spices, medicine, and cultural relics collection. Agarwood has the effects of promoting qi and relieving pain, warming the middle and stopping vomiting, and relieving asthma. Modern pharmacological studies have shown that agarwood has a wide range of pharmacological effects such as analgesia, sedation, antibacterial, anti-inflammatory, improvement of myocardial ischemia, and anti-tumor. The existing 21 species of Aquilaria are mainly distributed in Southeast Asia, among which Aquilaria sinensis (Lour.) Gilg. and Aquilaria yunnanensis S.C. Huang are native to China.

[0003] Healthy Aquilaria plants do not produce agarwood. They only secrete secondary metabolites when they are damaged by lightning strikes, broken branches, etc., and agarwood is formed after years of accumulation. Because the resources of Aquilaria plants are endangered and the formation of agarwood takes a long time, the yield of agarwood is low, which seriously fails to meet market demand. Commonly used artificial agarwood formation methods mainly include physical damage methods such as cutting, drilling holes, and nailing, chemical induction methods that inject agarwood inducers such as plant hormones and salts, and artificial inoculation methods. The physical damage method takes a long time to form agarwood and has low yields. The chemical induction method forms agarwood quickly, but there are problems with chemical reagent pollution and toxicity. The artificial inoculation method has high requirements for the operating environment and process management. Therefore, there is an urgent need for a safe, efficient, and convenient agarwood inducer to promote the formation of agarwood in order to meet market demand. Summary of the invention

[0004] In view of the above-mentioned defects of the prior art, the present invention proposes an agarwood inducer and a preparation method thereof and an agarwood formation method to solve the problems raised by the above-mentioned background technology.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] A kind of agarwood inducer, comprising a stimulant and a fragrance-binding agent; the stimulant comprises 20-50 parts of 14-hydroxybrassinosteroid and 10-25 parts of dihydromyricetin by weight; the fragrance-binding agent comprises 30-50 parts of rattan tea, 25-40 parts of rhizoma atractylodis macrophylla, 25-35 parts of amaranth, 20-28 parts of tribulus terrestris, 12-25 parts of corydalis saibeiensis, 10-16 parts of mulberry head red, 7-17 parts of saxifrage and 5-15 parts of konjac by weight.

[0007] Preferably, the rattan tea is the stems and leaves of Ampelopsis grossedentata (Hand.-Mazz) WT Wang of the Vitaceae family, the whole plant contains chemical components such as flavonoids, phenols, steroids, terpenes, polysaccharides, volatile oils, among which the content of flavonoids is as high as 45%; the bone-seeding wind is the whole herb of Aristolochia mollissima Hance of the Aristolochiaceae family, containing alkaloids, aristolochic acid, volatile oils, lactones and sugars, etc.; the amaranth is the amaranth of the Asteraceae family

[0008] The whole herb of Kalimeris indica (Linn.) Sch.-Bip. [Astertndicus L.]; Tribulus terrestris is the dried mature fruit of Tribulus terrestris L., a plant of the Tribulaceae family; Corydalis impatiens is the whole herb of Corydalis impatiens (Pall.) Fisch., a plant of the Poppy family; Sarcopyramis nepalensis Wall. is the whole herb of Sarcopyramis nepalensis Wall., a plant of the Melastomataceae family; Saxifragaceae is Saxifragas tolonifera Curt, a plant of the genus Saxifraga of the family Saxifragaceae; Piperhancei Maxim. is the stems, leaves or roots of Piperhancei Maxim., a plant of the family Piperhanceaceae.

[0009] Preferably, the stimulant comprises, by weight, 30 parts of 14-hydroxybrassinosteroid and 20 parts of dihydromyricetin; the aroma-binding agent comprises, by weight, 40 parts of rattan tea, 30 parts of rhizoma amarantii, 30 parts of amaranth, 25 parts of tribulus terrestris, 20 parts of corydalis saibei, 15 parts of mulberry head red, 12 parts of saxifrage and 8 parts of konjac.

[0010] Preferably, the preparation method of the stimulant comprises the following steps: dihydromyricetin and water are mixed and dissolved at a solid-liquid ratio of 1:(5-10) g / mL, cooled to 25-30°C indoors, 14-hydroxybrassinosteroid and ferulic acid with a mass concentration of 5-12 mg / mL are added, and the mixture is rotated at 2500-4000 r / min for 20-30 min to obtain the stimulant.

[0011] Preferably, in the preparation method of the stimulant, the temperature of the water is 85-100° C., and the solid-liquid ratio of 14-hydroxybrassinosteroid and ferulic acid is 1:(2-7) g / mL.

[0012] Preferably, the method for preparing the flavoring agent comprises the following steps:

[0013] S1. Weigh the raw materials by weight, wash and crush the rattan tea, rhizoma spatholobi, rhizoma amarantii, rhizoma terrestris, rhizoma corydalis, rhizoma chudalis, rhizoma saxifrage and rhizoma konjac to obtain a mixture;

[0014] S2, adding 2 to 4 times the weight of deionized water to the mixture obtained in step S1 and grinding it into a slurry to obtain a first slurry for later use;

[0015] S3, subjecting the first slurry to ultrasonic vibration treatment and then centrifuging to obtain a first supernatant and a first precipitate; adding 3 to 5 times the weight of water to the first precipitate, subjecting the first precipitate to ultrasonic vibration treatment and then centrifuging to obtain a second supernatant and a second precipitate;

[0016] S4, inoculating the second precipitate with 1-6% of Bifidobacterium longum bacterial solution according to the mass ratio, adjusting the pH to 6.5-7.0, and fermenting at a temperature of 30-35° C. for 5-10 days to obtain a fermentation product;

[0017] S5. Combine the first supernatant, the second supernatant and the fermented product, heat to 90-100°C, boil for 15-25 minutes, and then filter to obtain the aroma agent.

[0018] Preferably, the ultrasonic frequency of the ultrasonic oscillation treatment in step S3 is 30-45 kHz, and the ultrasonic time is 30-40 min.

[0019] Preferably, the concentration of the Bifidobacterium longum bacterial solution in step S4 is 1.0×10 6 ~2.5×10 8 CFU / mL, Bifidobacterium longum was purchased from Xi'an Precision Bioengineering Co., Ltd., model PRS0311.

[0020] Preferably, a method for making agarwood comprises the following steps:

[0021] (1) Drilling treatment: Select Aquilaria plants that are 3 to 5 years old and have a breast diameter of 7 to 12 cm. Remove excess branches, branches and tops, retain the main trunk, and drill holes 35 to 45 cm from the ground. Drill 20 to 25 holes horizontally around the trunk with an equal interval of 1 to 3 cm. Then, perform the same treatment every 15 to 20 cm along the trunk vertically upward. Select the horizontal and vertical center lines of the top wound surface and drill holes with an equal interval of 1 to 3 cm.

[0022] (2) Irritant injection: inject the irritant into the slot, and seal the slot with a plastic film to perform the first sealing process;

[0023] (3) Injection of aroma agent: After 7 to 10 days of sealing treatment in step (2), unwrap the plastic film, inject the aroma agent, seal the slot with a sealing agent, and perform a second sealing treatment;

[0024] (4) Extracting the agarwood: 5 to 8 months after the second sealing treatment, remove the brown oily substance in the trunk and the oily trunk, which is the agarwood.

[0025] Preferably, the Aquilaria plant is selected from Aquilaria sinensis.

[0026] Preferably, in step (1), the slot hole has a depth of 3 to 5 cm and a width of 1 to 2 cm.

[0027] Preferably, the injection amount of the stimulant in each slot of step (2) is 2 to 5 mL, and the injection amount of the aroma agent is 4 to 8 mL. Preferably, the sealing agent is a plant healing agent sealing glue purchased from Shandong Jinshitai Agricultural Technology Co., Ltd.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] (1) The agarwood inducer of the present invention comprises a stimulant and an aroma-forming agent, wherein the stimulant comprises 14-hydroxybrassinosteroid and dihydromyricetin; the aroma-forming agent comprises rattan tea, safflower, amaranth, thornleaf terrestris, Corydalis saibeiensis, red mulberry, saxifrage and konjac; the agarwood inducer is a natural plant component without adding any chemical substances, and is safe and convenient to use. At the same time, when the agarwood is physically damaged, the agarwood inducer provides the agarwood plant with sufficient plant hormones and growth nutrients required for growth and healing, as well as vitamins, polysaccharides, polypeptides, microorganisms, etc. that can effectively stimulate the tree to form aroma, thereby promoting the secretion of resin on the wound surface. After the resin is secreted, it is precipitated in the xylem and continuously diffuses along the wood vessels, finally forming agarwood with high quality and fast aroma-forming speed.

[0030] (2) The arrangement of the drill holes of the present invention allows the tree to be in full contact with the agarwood inducer in both the longitudinal and transverse directions, so that the agarwood of the tree is produced evenly and the contact area between the wound surface and the agarwood inducer is increased.

[0031] (3) The agarwood resin formation method of the present invention is to first inject a stimulant and then inject the resin-forming agent. By setting the order of the stimulant and the resin-forming agent, the stimulant can produce molecular oxygen, causing strong stimulation to the tree, thereby promoting the secretion of resin; the resin-forming agent can provide the necessary nutrients for the formation of resin, accelerate the stress response inside and outside the tree, and promote the production of more resin.

[0032] (4) The content of alcohol-soluble extract ranges from 26.0 to 26.5%, and the content of agarwood tetraol ranges from 0.32 to 0.37%. After 5 months of agarwood formation, the standard of the 2020 edition of the Chinese Pharmacopoeia can be met. The content of alcohol-soluble extract is not less than 10.0%, and the content of agarwood tetraol is not less than 0.10%. After the agarwood inducer is used on the white agarwood tree, the growth rates of the content of alcohol-soluble extract, agarwood tetraol, volatile oil and sesquiterpenoid content in the volatile oil are 419.61%, 270%, 377.78% and 407.59%, respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Drill hole diagram for the top of the tree. DETAILED DESCRIPTION

[0034] The technical solution of the present invention is further described in detail below in conjunction with specific implementation methods.

[0035] Example 1

[0036] A kind of agarwood inducer, comprising a stimulant and a fragrance-binding agent; the stimulant comprises 20 parts of 14-hydroxybrassinosteroid and 10 parts of dihydromyricetin by weight; the fragrance-binding agent comprises 30 parts of rattan tea, 25 parts of rhizoma atractylodis macrophylla, 25 parts of amaranth, 20 parts of tribulus terrestris, 12 parts of corydalis saibeiensis, 10 parts of mulberry head red, 7 parts of saxifrage and 5 parts of konjac by weight.

[0037] The preparation method of the stimulant comprises the following steps: dihydromyricetin and water at a temperature of 85°C are mixed and dissolved at a solid-liquid ratio of 1:5 g / mL, and after being placed in a room and cooled to 25°C, 14-hydroxybrassinosteroid and ferulic acid with a mass concentration of 5 mg / mL are added, and the mixture is rotated at 2500 r / min for 20 minutes to obtain the stimulant; wherein the solid-liquid ratio of 14-hydroxybrassinosteroid and ferulic acid is 1:2 g / mL.

[0038] The preparation method of the flavoring agent comprises the following steps:

[0039] S1. Weigh the raw materials by weight, wash and crush the rattan tea, rhizoma spatholobi, rhizoma amarantii, rhizoma terrestris, rhizoma corydalis, rhizoma chudalis, rhizoma saxifrage and rhizoma konjac to obtain a mixture;

[0040] S2, adding 2 times the weight of deionized water to the mixture obtained in step S1 and grinding it into slurry to obtain a first slurry for later use;

[0041] S3, subjecting the first slurry to ultrasonic oscillation treatment and then centrifuging to obtain a first supernatant and a first precipitate; adding 3 times the weight of water to the first precipitate, subjecting the first precipitate to ultrasonic oscillation treatment and then centrifuging to obtain a second supernatant and a second precipitate; wherein the ultrasonic frequency is 30KHz and the ultrasonic time is 30min;

[0042] S4, inoculating 1% of Bifidobacterium longum bacterial solution into the second precipitate according to the mass ratio, adjusting the pH to 6.5, and fermenting at 30°C for 5 days to obtain a fermentation product; the concentration of the Bifidobacterium longum bacterial solution is 1.0×10 6 CFU / mL;

[0043] S5. Combine the first supernatant, the second supernatant and the fermented product, heat to 90°C, cook for 15 minutes, and then filter to obtain the flavoring agent.

[0044] A method for forming agarwood, comprising the following steps:

[0045] (1) Drilling treatment: A 3-year-old Aquilaria sinensis tree with a breast diameter of 7 cm was selected. Excess branches, twigs and tops were removed, and the main trunk was retained. A hole was drilled at 35 cm from the ground in the main trunk. Twenty slots were drilled horizontally around the trunk with an equal spacing of 1 cm. Then, the same treatment was performed every 15 cm along the trunk. The depth of the slots was 3 cm and the width was 1 cm. The horizontal and vertical center lines of the top wound surface were selected, and slots were drilled at an equal spacing of 1 cm.

[0046] (2) Stimulant injection: 2 mL of stimulant was injected into each slot, and the slot was sealed with a plastic film for the first sealing process;

[0047] (3) Injection of aroma agent: After 7 days of sealing treatment in step (2), unwrap the plastic film, inject 4 mL of aroma agent into each slot, seal the slot with sealing agent, and perform the second sealing treatment;

[0048] (4) Extracting the agarwood: 5 months after the second sealing treatment, cut off the brown oily substance in the trunk and the oily trunk, which is the agarwood.

[0049] Example 2

[0050] A kind of agarwood inducer, comprising a stimulant and a fragrance-binding agent; the stimulant comprises 50 parts of 14-hydroxybrassinosteroid and 25 parts of dihydromyricetin by weight; the fragrance-binding agent comprises 50 parts of rattan tea, 40 parts of rhizoma atractylodis macrophylla, 35 parts of amaranth, 28 parts of tribulus terrestris, 25 parts of corydalis saibei, 16 parts of mulberry head red, 17 parts of saxifrage and 15 parts of konjac by weight.

[0051] The preparation method of the stimulant comprises the following steps: dihydromyricetin and water at a temperature of 100°C are mixed and dissolved at a solid-liquid ratio of 1:10 g / mL, and after being placed in a room and cooled to 30°C, 14-hydroxybrassinosteroid and ferulic acid with a mass concentration of 12 mg / mL are added, and the mixture is rotated at 4000 r / min for 30 minutes to obtain the stimulant; the solid-liquid ratio of 14-hydroxybrassinosteroid and ferulic acid is 1:7 g / mL.

[0052] The preparation method of the flavoring agent comprises the following steps:

[0053] S1. Weigh the raw materials by weight, wash and crush the safflower, safflower, amaranth, tribulus, corydalis saibei, red mulberry, saxifrage and konjac to obtain a mixture;

[0054] S2, adding 4 times the weight of deionized water to the mixture obtained in step S1 and grinding it into slurry to obtain a first slurry for later use;

[0055] S3, subjecting the first slurry to ultrasonic oscillation treatment and then centrifuging to obtain a first supernatant and a first precipitate; adding 5 times the weight of water to the first precipitate, subjecting the first precipitate to ultrasonic oscillation treatment and then centrifuging to obtain a second supernatant and a second precipitate; the ultrasonic frequency is 45KHz, and the ultrasonic time is 40min;

[0056] S4, inoculating 6% of Bifidobacterium longum bacterial solution into the second precipitate according to the mass ratio, adjusting the pH to 7.0, and fermenting at 35°C for 10 days to obtain a fermentation product; the concentration of the Bifidobacterium longum bacterial solution is 2.5×10 8 CFU / mL;

[0057] S5. Combine the first supernatant, the second supernatant and the fermented product, heat to 100°C, cook for 25 minutes, and then filter to obtain the aroma agent.

[0058] A method for forming agarwood, comprising the following steps:

[0059] (1) Drilling treatment: A 5-year-old Aquilaria sinensis tree with a breast diameter of 12 cm was selected. Excess branches, twigs, and tops were removed, and the main trunk was retained. A hole was drilled 45 cm from the ground at the main trunk. 25 slots were drilled horizontally around the trunk at an equal interval of 3 cm. The slot depth was 5 cm and the width was 2 cm. The same treatment was then performed every 20 cm along the vertical direction of the trunk. The horizontal and vertical center lines of the top wound surface were selected, and slots were drilled at an equal interval of 3 cm.

[0060] (2) Stimulant injection: 5 mL of stimulant was injected into each slot, and the slot was sealed with a plastic film for the first sealing process;

[0061] (3) Injection of aroma agent: After 10 days of sealing treatment in step (2), unwrap the plastic film, inject 8 mL of aroma agent into each slot, seal the slot with sealing agent, and perform the second sealing treatment;

[0062] (4) Extracting the agarwood: 8 months after the second sealing treatment, cut off the brown oily substance in the trunk and the oily trunk, which is the agarwood.

[0063] Example 3

[0064] An agarwood inducer, wherein the stimulant comprises, by weight, 30 parts of 14-hydroxybrassinosteroid and 20 parts of dihydromyricetin; the aroma-binding agent comprises, by weight, 40 parts of rattan tea, 30 parts of rhizoma amarantii, 30 parts of amaranth, 25 parts of tribulus terrestris, 20 parts of corydalis saibeiensis, 15 parts of mulberry head red, 12 parts of saxifrage and 8 parts of konjac.

[0065] The preparation method of the stimulant comprises the following steps: dihydromyricetin and water at a temperature of 95°C are mixed and dissolved at a solid-liquid ratio of 1:8 g / mL, and after being placed in a room and cooled to 26°C, 14-hydroxybrassinosteroid and ferulic acid with a mass concentration of 10 mg / mL are added, and the mixture is rotated at 3000 r / min for 25 minutes to obtain the stimulant; the solid-liquid ratio of 14-hydroxybrassinosteroid and ferulic acid is 1:3 g / mL.

[0066] The preparation method of the flavoring agent comprises the following steps:

[0067] S1. Weigh the raw materials by weight, wash and crush the safflower, safflower, amaranth, tribulus, corydalis saibei, red mulberry, saxifrage and konjac to obtain a mixture;

[0068] S2, adding 3 times the weight of deionized water to the mixture obtained in step S1 and grinding it into slurry to obtain a first slurry for later use;

[0069] S3, subjecting the first slurry to ultrasonic oscillation treatment and then centrifuging to obtain a first supernatant and a first precipitate; adding 4 times the weight of water to the first precipitate, subjecting the first precipitate to ultrasonic oscillation treatment and then centrifuging to obtain a second supernatant and a second precipitate; the ultrasonic frequency is 40KHz, and the ultrasonic time is 35min;

[0070] S4, inoculating 4% of Bifidobacterium longum bacterial solution into the second precipitate according to the mass ratio, adjusting the pH to 6.8, and fermenting at 32°C for 6 days to obtain a fermentation product; the concentration of the Bifidobacterium longum bacterial solution is 2.0×10 7 CFU / mL.

[0071] S5. Combine the first supernatant, the second supernatant and the fermented product, heat to 95°C, boil for 20 minutes, and then filter to obtain the flavoring agent.

[0072] A method for forming agarwood, comprising the following steps:

[0073] (1) Drilling treatment: A four-year-old Aquilaria sinensis tree with a breast diameter of 10 cm was selected. Excess branches, twigs and tops were removed, and the main trunk was retained. A hole was drilled at 40 cm from the ground. 22 slots were drilled horizontally around the trunk at an equal interval of 2 cm. The slot depth was 4 cm and the width was 1.5 cm. The same treatment was then performed vertically upward at intervals of 18 cm. The horizontal and vertical center lines of the top wound surface were selected, and slots were drilled at an equal interval of 2 cm.

[0074] (2) Stimulant injection: 3 mL of stimulant was injected into each slot, and the slot was sealed with a plastic film for the first sealing process;

[0075] (3) Injection of aroma agent: After 8 days of sealing treatment in step (2), unwrap the plastic film, inject 5 mL of aroma agent into each slot, seal the slot with sealing agent, and perform the second sealing treatment;

[0076] (4) Extracting the agarwood: 6 months after the second sealing treatment, cut off the brown oily substance in the trunk and the oily trunk, which is the agarwood.

[0077] Comparative Example 1

[0078] The difference between this comparative example and Example 3 is that no agarwood inducer is injected after drilling, and the other components and steps remain unchanged.

[0079] A method for forming agarwood, comprising the following steps:

[0080] (1) Drilling treatment: A four-year-old Aquilaria sinensis tree with a breast diameter of 10 cm was selected. Excess branches, twigs and tops were removed, and the main trunk was retained. A hole was drilled at 40 cm from the ground. 22 slots were drilled horizontally around the trunk at an equal interval of 2 cm. The slot depth was 4 cm and the width was 1.5 cm. The same treatment was then performed every 18 cm along the trunk. The horizontal and vertical center lines of the top wound surface were selected and slots were drilled at an equal interval of 2 cm. The slots were sealed with a sealing agent.

[0081] (2) Extracting the agarwood: After the sealing treatment for 6 months, the brown oily substance and the oily trunk are cut off, which is the agarwood.

[0082] Comparative Example 2

[0083] The difference between this comparative example and Example 3 is that no irritant is added after drilling, and the other components and steps remain unchanged.

[0084] The invention discloses an agarwood inducing agent, i.e. an agarwood-forming agent, which comprises, by weight, 40 parts of rattan tea, 30 parts of rhizoma amarantii, 30 parts of amaranth, 25 parts of tribulus terrestris, 20 parts of corydalis saibeiensis, 15 parts of mulberry leaf, 12 parts of saxifrage and 8 parts of konjac.

[0085] The preparation method of the flavoring agent comprises the following steps:

[0086] S1. Weigh the raw materials by weight, wash and crush the rattan tea, rhizoma spatholobi, rhizoma amarantii, rhizoma terrestris, rhizoma corydalis, rhizoma chudalis, rhizoma saxifrage and rhizoma konjac to obtain a mixture;

[0087] S2, adding 3 times the weight of deionized water to the mixture obtained in step S1 and grinding it into slurry to obtain a first slurry for later use;

[0088] S3, subjecting the first slurry to ultrasonic oscillation treatment and then centrifuging to obtain a first supernatant and a first precipitate; adding 4 times the weight of water to the first precipitate, subjecting the first precipitate to ultrasonic oscillation treatment and then centrifuging to obtain a second supernatant and a second precipitate; the ultrasonic frequency is 40KHz, and the ultrasonic time is 35min;

[0089] S4, inoculating 4% of Bifidobacterium longum bacterial solution into the second precipitate according to the mass ratio, adjusting the pH to 6.8, and fermenting at 32°C for 6 days to obtain a fermentation product; the concentration of the Bifidobacterium longum bacterial solution is 2.0×10 7 CFU / mL.

[0090] S5. Combine the first supernatant, the second supernatant and the fermented product, heat to 95°C, boil for 20 minutes, and then filter to obtain the flavoring agent.

[0091] A method for forming agarwood, comprising the following steps:

[0092] (1) Drilling treatment: A four-year-old Aquilaria sinensis tree with a breast diameter of 10 cm was selected. Excess branches, twigs and tops were removed, and the main trunk was retained. A hole was drilled at 40 cm from the ground. 22 slots were drilled horizontally around the trunk at an equal interval of 2 cm. The slot depth was 4 cm and the width was 1.5 cm. The same treatment was then performed vertically upward at intervals of 18 cm. The horizontal and vertical center lines of the top wound surface were selected, and slots were drilled at an equal interval of 2 cm.

[0093] (2) Injection of aroma agent: 5 mL of aroma agent is injected into each slot, and the slot is sealed with a sealing agent to perform a sealing process;

[0094] (3) Extracting the agarwood: After the sealing treatment for 6 months, cut off the brown oily substance in the trunk and the oily trunk, which is the agarwood.

[0095] Comparative Example 3

[0096] The difference between this comparative example and Example 3 is that no aroma agent is added after drilling, and the other components and steps remain unchanged.

[0097] An agarwood inducer, i.e. a stimulant, comprises 30 parts of 14-hydroxybrassinosteroid and 20 parts of dihydromyricetin by weight.

[0098] The preparation method of the stimulant comprises the following steps: dihydromyricetin and water at a temperature of 95°C are mixed and dissolved at a solid-liquid ratio of 1:8 g / mL, and after being placed in a room and cooled to 26°C, 14-hydroxybrassinosteroid and ferulic acid with a mass concentration of 10 mg / mL are added, and the mixture is rotated at 3000 r / min for 25 minutes to obtain the stimulant; the solid-liquid ratio of 14-hydroxybrassinosteroid and ferulic acid is 1:3 g / mL.

[0099] A method for forming agarwood, comprising the following steps:

[0100] (1) Drilling treatment: A four-year-old Aquilaria sinensis tree with a breast diameter of 10 cm was selected. Excess branches, twigs and tops were removed, and the main trunk was retained. A hole was drilled at 40 cm from the ground. 22 slots were drilled horizontally around the trunk at an equal interval of 2 cm. The slot depth was 4 cm and the width was 1.5 cm. The same treatment was then performed vertically upward at intervals of 18 cm. The horizontal and vertical center lines of the top wound surface were selected, and slots were drilled at an equal interval of 2 cm.

[0101] (2) Stimulant injection: 3 mL of stimulant was injected into each slot, and the slot was sealed with a plastic film;

[0102] (3) Extracting the agarwood: After the sealing treatment for 6 months, cut off the brown oily substance in the trunk and the oily trunk, which is the agarwood.

[0103] Comparative Example 4

[0104] The difference between this comparative example and Example 3 is that: using the existing technology, the agarwood inducer is converted into a chemical component, and the other ingredients and steps remain unchanged.

[0105] With reference to patent application number CN202210727631.8 "A method for producing agarwood by sawing and clamping a tree trunk", the agarwood-forming conditions and methods of Example 4 were adopted. A white agarwood tree with an age of 8 years and a breast diameter greater than 10 cm was selected. At 20 cm from the ground, a clamp with a depth of 4 cm was cut with an electric saw. A clamp was sawed every 60 cm, and branches without clamps were cut off. Prepare 1L of agarwood inducer containing 0.0% phenylalanine and 0.05% p-hydroxyphenylpropionic acid, and spray it evenly in the clamp with a spray bottle until the surface of the clamp is moistened. After an interval of 1 month, re-prepare the above-mentioned agarwood inducer and spray it evenly into all the clamps. After 6 years, cut down the whole tree, cut the discolored woody part ranging from yellowish brown to brownish black, and obtain agarwood after drying.

[0106] Comparative Example 5

[0107] The difference between this comparative example and Example 3 is that: using the existing technology, the agarwood inducer is changed to inoculum, and the other components and steps remain unchanged.

[0108] Referring to the patent application number CN201510170409.2 "A method for producing agarwood", the agarwood formation conditions and methods of Example 5 were adopted. A 7-year-old agarwood tree was selected, and 6 holes with a depth of 5 cm were drilled using an ordinary electric drill with a drill bit diameter of 0.6 cm at a distance of 10 cm from the top of the tree, and each hole was filled with 7 g of microcapsules (the Rhizoctonia solani was used to produce agarwood).

[0109] Rhizoctonia sp. (CPCC480164), Fusarium oxysporum (CPCC810100), Acremonium strictum (ACCC30554), Acremonium endophytium (CCTCCM206118), and Penicillium sp. (CPCC441453) were inoculated into potato liquid culture medium, and cultured in shake flasks at 25°C until the bacteria grew vigorously to obtain culture fluid. Acremonium furcatum (CGMCC3.5370) was inoculated into potato liquid culture medium, and cultured in shake flasks at 27°C until the bacteria grew vigorously to obtain culture fluid. The culture solution of the above strains was mixed with 3% sodium alginate (mass fraction) solution at a volume ratio of 1:30, and then an equal volume of 4.5% calcium chloride solution was slowly dripped, slowly stirred, and vacuum dried to obtain microcapsules of each strain, and the microcapsules of each strain were mixed in equal volumes and loaded into capsules to obtain microcapsules encapsulating mixed strains. ), and then the holes were sealed with rubber plugs. After 6 months, the brown oily substance formed around the wound and the yellow-brown discolored wood were cut off and dried to obtain agarwood.

[0110] Experimental example

[0111] Refer to the test method of the efficacy of agarwood in the 2020 edition of the Chinese Pharmacopoeia, and test the hot soaking method under the determination of alcohol-soluble extracts (General Rule 2201) and agarwood tetraol (C 17 H 18 O6);

[0112] Agarwood oil content detection: The agarwood sample was crushed and passed through a 26-mesh sieve. 100 g was accurately weighed and placed in a 5000 ml round-bottom flask. 2500 ml of distilled water was added and soaked overnight. A steam distillation device Clevenger was installed and the extraction was performed at a low boiling temperature for 12 hours. The volatile oil was collected, and an appropriate amount of anhydrous sodium sulfate was added. The volatile oil was obtained after using a 0.45 μm organic phase microporous membrane filter.

[0113] Detection of sesquiterpenoid content in agarwood oil: The volatile oil of the sample was subjected to GC-MS analysis. The instrument was Varian 450GC / 300MS coupling instrument from Varian, USA. The chromatographic column was a quartz capillary column VF-5MS, 30m×0.25mm, with a film thickness of 0.25μm. Temperature program: from 50℃ to 155℃ at 10℃ / min, retained for 15min; then raised to 280℃ at 8℃ / min, retained for 10min, carrier gas was He, column flow rate was 1ml / min, and injection volume was 1μl. The injection port temperature was 250℃, without splitting. Mass spectrometry conditions: EI source; ionization voltage: 70eV; ion source temperature: 250℃; full scan range: 50~500amu. 1μl of C8-C40 normal alkane mixed standard was detected in the same way. The relative percentage of each peak compound was calculated by peak area normalization method, and the RI index corresponding to each peak was directly calculated according to AMDIS software. The mass spectra obtained from each peak were retrieved by searching the NIST08 standard mass spectrum library and reference reports. The RI values ​​of compounds with high similarity were then compared on NIST Chemistry WebBook (http: / / webbook.nist.gov / chemistry / ), sesquiterpenoid compounds were identified, and the total sesquiterpenoid percentage content was calculated.

[0114] The content of chromone compounds can be tested by referring to the method given by Wu Zeqing et al., Determination of chromone content of agarwood medicinal materials formed by three different methods (2015). Visible spectrophotometry (VS) was used with molybdenum phosphotungstate-lithium sulfate as the color developer and Aquilarone E chromone as the reference solution to determine the content of total chromones in agarwood medicinal material test solution at a wavelength of 760nm. The HPLC method was used to determine the content of 5 chromones in agarwood, using a Waters Symmetry Shield C18 column (4.6mm×250mm, 5μm), a mobile phase of acetonitrile (A)-0.1% phosphoric acid water (B), a flow rate of 1mL / min, a detection wavelength of 252nm, and a column temperature of 30℃.

[0115] The test results are shown in Table 1:

[0116] Table 1 Agarwood quality test

[0117]

[0118]

[0119] It can be seen from Table 1 that the agarwood quality obtained by injecting the agarwood inducer of Example 3 into the white agarwood tree is the best. The growth rates of the alcohol-soluble extract content, agarwood tetraol content, volatile oil content and sesquiterpene content in volatile oil in Example 3 are 419.61%, 270%, 377.78% and 407.59% respectively compared with Comparative Example 1. It can be seen that the injection of the agarwood inducer of the present invention into the white agarwood tree has a significant improvement in the quality of the obtained agarwood; compared with Comparative Examples 2 and 3, it can be seen that the stimulant and the fragrance agent in the agarwood inducer complement each other and jointly promote the fragrance of the white agarwood tree, and the agarwood obtained in 5 months meets the standards of the 2020 edition of the Chinese Pharmacopoeia, the content of alcohol-soluble extract is not less than 10.0%, and the content of agarwood tetraol is not less than 10.0%. Not less than 0.10%; By comparing Example 3 with Comparative Examples 4 and 5, it can be seen that the volatile components of agarwood mainly contain two components: 2-(2-phenylethyl) chromones and sesquiterpenes, both of which are related to the formation of the special fragrance and efficacy of agarwood. The sesquiterpenes content and 2-(2-phenylethyl) chromones content account for 80.6% in Example 3, 64.2% in Comparative Example 4, and 55.6% in Comparative Example 5. It can be seen that the medicinal value and economic value of the agarwood obtained in Example 3 are higher, and the ingredients used in the agarwood inducer of Implementation 3 are all natural plant ingredients, which have the lowest degree of damage to the effective ingredients of the white wood tree itself, which is conducive to the large-scale use of this method to promote the formation of agarwood to meet market demand.

[0120] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. An agarwood inducing agent, characterized in that: The agarwood inducer includes a stimulant and a fragrance-binding agent; the stimulant includes 20-50 parts of 14-hydroxybrassinosteroid and 10-25 parts of dihydromyricetin by weight; the fragrance-binding agent includes 30-50 parts of rattan tea, 25-40 parts of rhizoma atractylodis macrophylla, 25-35 parts of amaranth, 20-28 parts of tribulus terrestris, 12-25 parts of corydalis saibei, 10-16 parts of mulberry head red, 7-17 parts of saxifrage and 5-15 parts of konjac by weight.

2. The agarwood inducing agent according to claim 1, characterized in that: The stimulant includes 30 parts of 14-hydroxybrassinosteroid and 20 parts of dihydromyricetin by weight; the aroma-binding agent includes 40 parts of rattan tea, 30 parts of rhizoma amarantii, 30 parts of amaranth, 25 parts of tribulus terrestris, 20 parts of corydalis saibei, 15 parts of mulberry head red, 12 parts of saxifrage and 8 parts of konjac by weight.

3. An agarwood inducer according to claim 1, characterized in that: The preparation method of the stimulant comprises the following steps: dihydromyricetin and water are mixed and dissolved at a solid-liquid ratio of 1:(5-10) g / mL, cooled to 25-30°C indoors, 14-hydroxybrassinosteroid and ferulic acid with a mass concentration of 5-12 mg / mL are added, and the mixture is rotated at 2500-4000 r / min for 20-30 minutes to obtain the stimulant.

4. An agarwood inducer according to claim 3, characterized in that: The temperature of the water is 85-100° C., and the solid-liquid ratio of 14-hydroxybrassinosteroid to ferulic acid is 1:(2-7) g / mL.

5. An agarwood inducer according to claim 1, characterized in that: The preparation method of the flavoring agent comprises the following steps: S1. Weigh the raw materials by weight, wash and crush the safflower, safflower, amaranth, tribulus, corydalis saibei, red mulberry, saxifrage and konjac to obtain a mixture; S2, adding 2 to 4 times the weight of deionized water to the mixture obtained in step S1 and grinding it into a slurry to obtain a first slurry for later use; S3, subjecting the first slurry to ultrasonic vibration treatment and then centrifuging to obtain a first supernatant and a first precipitate; adding 3 to 5 times the weight of water to the first precipitate, subjecting the first precipitate to ultrasonic vibration treatment and then centrifuging to obtain a second supernatant and a second precipitate; S4, inoculating 1-6% of Bifidobacterium longum bacterial solution into the second precipitate according to the mass ratio, adjusting the pH to 6.5-7.0, and fermenting at a temperature of 30-35° C. for 5-10 days to obtain a fermentation product; S5. Combine the first supernatant, the second supernatant and the fermented product, heat to 90-100°C, boil for 15-25 minutes, and then filter to obtain the aroma agent.

6. An agarwood inducer according to claim 5, characterized in that: The ultrasonic frequency of the ultrasonic oscillation treatment in step S3 is 30-45 KHz, and the ultrasonic time is 30-40 min.

7. An agarwood inducer according to claim 5, characterized in that: The concentration of the Bifidobacterium longum bacterial solution in step S4 is 1.0×10 6 ~2.5×10 8 CFU / mL.

8. A method for producing agarwood, characterized in that: The steps include: (1) Drilling treatment: Select Aquilaria plants with an age of 3 to 5 years and a diameter at breast height of 7 to 12 cm, remove unnecessary branches, branches and tops, retain the main trunk, drill slots at an equal interval of 1 to 3 cm in the cross section of the top of the main trunk, and drill holes at a distance of 35 to 45 cm from the ground in the main trunk. Drill 20 to 25 slots at an equal interval of 1 to 3 cm around the trunk horizontally, and then perform the same treatment every 15 to 20 cm along the trunk upward; (2) Stimulant injection: inject the stimulant described in claim 1 into the slot, and seal the slot with a plastic film to perform the first sealing process; (3) Injection of aroma-binding agent: After 7 to 10 days of sealing treatment in step (2), the plastic film is unwrapped, and the aroma-binding agent according to claim 1 is injected, and the slot holes are sealed with a sealing agent, and a second sealing treatment is performed; (4) Extracting the agarwood: 5 to 8 months after the second sealing treatment, remove the brown oily substance in the trunk and the oily trunk, which is the agarwood.

9. The method for making agarwood according to claim 8, characterized in that: In step (1), the slot hole has a depth of 3 to 5 cm and a width of 1 to 2 cm.

10. The method for forming agarwood according to claim 8, characterized in that: In step (2), the injection amount of the stimulant into each slot is 2 to 5 mL, and the injection amount of the aroma agent is 4 to 8 mL.

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