A method for rapid resin formation in Dalbergia odorifera from Guangdong and Guangxi

Abstract

This invention provides a rapid resin-forming method for Dalbergia odorifera (Guangdong and Guangxi), comprising the following steps: selecting Dalbergia odorifera plants and drilling holes horizontally or downwards along the main trunk; after drilling, injecting a resin-forming liquid, fermented twice, into each hole. The raw materials for the resin-forming liquid include rice, Eclipta prostrata, Polygonum hydropiper, and Pandanus leaves. The raw materials for the resin-forming liquid are mixed, crushed, and fermented with water to obtain a preliminary fermentation mixture; the preliminary fermentation mixture is filtered, and after filtration, it undergoes fungal fermentation again, followed by filtration again to obtain the resin-forming liquid. This rapid resin-forming method for Dalbergia odorifera allows for re-formation of resin 1-1.2 years after initial resin formation, increases the volatile oil components and content of the extract, and exhibits good antibacterial and anti-inflammatory effects.

Description

Technical Field

[0001] This invention relates to the field of resin formation technology, and more particularly to a method for rapid resin formation in Dalbergia odorifera from Guangdong and Guangxi. Background Technology

[0002] Dalbergia benthamii Prain, a vine belonging to the genus Dalbergia in the legume family (Fabaceae), is a perennial woody vine. The name "Dalbergia benthamii" first appeared in the book *Illustrated Flora of Major Chinese Plants - Legumes*. It is mainly distributed in Guangdong, Hainan, and Guangxi provinces of China. Dalbergia benthamii (large-leaved variety) is a perennial woody vine. When injured, it secretes resin to repair wounds, producing a fragrant substance called *jiangzhenxiang*, also known as *zitengxiang* or *jiguxiang*. *Jiangzhenxiang* from Hainan typically takes over fifty years to produce this fragrance. The resin in *jiangzhenxiang* is mainly concentrated in injured areas, such as the forks of the vine, infected parts, and wounds. It is rich in resin and has a strong, clear aroma. *Jiangzhenxiang* contains abundant flavonoids, possessing various biological activities, including analgesia, hemostasis, antibacterial, and anti-inflammatory properties. Its unique fragrance and medicinal value are highly valued by consumers. Currently, the main methods for rapid resin formation in Dalbergia odorifera include: bacterial infusion, cutting, drilling, and burning. However, these methods have the following problems: First, the resin formation cycle required by natural and artificial methods is very long, generally more than fifty years for natural resin formation; second, the quality of agarwood obtained by traditional methods is inconsistent, making it difficult to meet the market demand for high-quality Dalbergia odorifera; third, improper artificial methods can cause irreversible damage to the Dalbergia odorifera plants. Therefore, this invention provides a method for rapid resin formation in Dalbergia odorifera. Summary of the Invention

[0003] In view of this, the present invention develops a method for rapid resin formation of Dalbergia odorifera in Guangdong and Guangxi.

[0004] The technical solution of this invention is implemented as follows:

[0005] A method for rapid resin formation in Dalbergia odorifera from Guangdong and Guangxi provinces includes the following steps:

[0006] S1. Select Dalbergia odorifera plants from Guangdong and Guangxi, and drill holes on the main trunk in a horizontal or downward direction.

[0007] S2. After drilling, inject the fermented resin into each hole. The resin ingredients include: 2-3 parts rice, 1-2 parts Eclipta prostrata, 1-1.5 parts Polygonum hydropiper, and 0.8-1.2 parts Pandanus tinctoria leaves.

[0008] The method for preparing the resinous liquid includes: mixing the resinous liquid raw materials, crushing them, adding water for the first fermentation to obtain a fermentation initial mixture; filtering the fermentation initial mixture, performing a second fungal fermentation after filtration, filtering again to obtain the resinous liquid.

[0009] Preferably, the water-added fermentation is anaerobic fermentation, with a fermentation time of 38-42 days and a fermentation temperature of 28-32℃.

[0010] Preferably, the initial fermentation mixture obtained by the first addition of water is composed of rice, Eclipta prostrata, Polygonum hydropiper, Panax notoginseng leaves and water in a mass-to-volume ratio of (2-3)g:(1-2)g:(1-1.5)g:(0.8-1.2)g:(25-35)mL.

[0011] Preferably, the fungus is Trichoderma atroviride and Trichoderma harziauum, and the mass ratio of the initial fermentation mixture, Trichoderma atroviride and Trichoderma harziauum is (100-90):(3-6):(2-5).

[0012] Preferably, the fungal fermentation is carried out at a temperature of 24-26℃, for a fermentation time of 6-7 days, with a pH value of 5-5.2, and under sealed anaerobic fermentation conditions.

[0013] Preferably, in S1, the drilling is performed on two Dalbergia odorifera plants with a height of 18-22cm and a trunk diameter of >5cm. At a height of 20-30cm from the ground, a 1.7-2.0mm diameter drill bit is used to drill holes horizontally or downwards at a 3-6° angle along the trunk, penetrating the trunk. The horizontal distance between adjacent drill holes is 1.7-2.0cm, and the distance between adjacent vertical rows is 15-20cm.

[0014] Preferably, in step S2, the incense liquid is injected into each hole in one go within 25-30 minutes after drilling, with a dose of 0.2-0.25g.

[0015] Compared with the prior art, the beneficial effects of the present invention are:

[0016] This invention selects Dalbergia odorifera plants from Guangdong and Guangxi, drills holes in the main trunk, and injects twice-fermented resin into each hole. The resin raw materials are a compound of rice, Eclipta prostrata, Polygonum hydropiper, and Panax notoginseng leaves. This compound resin raw material improves the resin production efficiency and extract content. The resin is then subjected to a two-stage anaerobic fermentation process. The raw materials are mixed, crushed, and water is added for fermentation to obtain a preliminary fermentation mixture. After filtration, it undergoes a second fungal fermentation, and the filtered final product is the resin finished product. The rapid resin formation method for Dalbergia odorifera using the method of this invention allows for re-scent formation 1-1.2 years after initial resin formation, increasing the variety and content of volatiles in the extract. The volatile oil includes elemol, methyl eugenol, whiskey lactone, isoelemene, linalool, and cis-cobaene. Whiskey lactone, linalool, and cis-B-cobaene were detected, with elemol accounting for 73%-89% and methyl eugenol accounting for 7.6%-20.0%. It also includes whiskey lactone, isoelemene, linalool, and cis-B-cobaene. In addition, a total of 8 fragrance components were detected, all of which contain methyl eugenol and isoelemene. The resin products of the rapid resin formation method for Dalbergia odorifera according to Examples 1-3 of this invention have a rich variety of volatile oils and an increased content. Attached Figure Description

[0017] Figure 1 After resin formation treatment, significant oil accumulation appears in the xylem of the tree trunk. With increasing age, the black resin covers the entire trunk. This black resin is the fragrance of Dalbergia odorifera, also known as sandalwood, and can be used in medicine, to extract essential oils, or to make aromatic products. The intermittent distribution of the black resin in the image is because it first forms at the resin-forming pores and gradually extends along the trunk, becoming more extensive over time until the entire trunk is almost entirely covered in resin.

[0018] Figure 2 The trunks of plants that have not undergone resin formation treatment are light-colored and have no oil accumulation. Detailed Implementation

[0019] To better understand the technical content of this invention, specific embodiments are provided below to further illustrate the invention. Unless otherwise specified, the experimental methods used in the embodiments of this invention are conventional methods.

[0020] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available.

[0021] Trichoderma atroviride: strain sourced from the Institute of Microbiology, Chinese Academy of Sciences, original number E115, effective viable count ≥12 billion / gram; Trichoderma harziauum: strain sourced from the China Microbial Culture Collection Center, effective viable count ≥2.5 billion / gram.

[0022] Preparation Example 1

[0023] The preparation method of the incense-forming liquid includes: mixing 2 parts rice, 1 part Eclipta prostrata, 1 part Polygonum hydropiper, and 1 part Pandan leaves, pulverizing them, adding water, and anaerobic fermenting for 38 days at a temperature of 28℃ to obtain the initial fermentation mixture. The mass-to-volume ratio of rice, Eclipta prostrata, Polygonum hydropiper, Pandan leaves, and water in the initial fermentation mixture is 2g:1g:1g:1g:25mL. The initial fermentation mixture is filtered, and then subjected to fungal fermentation again. The fungi are Trichoderma atroviride and Trichoderma harziauum, with a mass ratio of the initial fermentation mixture, Trichoderma atroviride, and Trichoderma harziauum of 100:3:2. The fermentation temperature is 24℃, the fermentation time is 6 days, the pH value is 5, and the fermentation conditions are sealed anaerobic fermentation. The mixture is then filtered again to obtain the incense-forming liquid.

[0024] Preparation Example 2

[0025] The preparation method of the incense-forming liquid includes: mixing 3 parts rice, 1.2 parts Eclipta prostrata, 1 part Polygonum hydropiper, and 1.3 parts pandanus leaves, pulverizing them, adding water, and anaerobic fermenting for 42 days at 32℃ to obtain the initial fermentation mixture. The mass-to-volume ratio of rice, Eclipta prostrata, Polygonum hydropiper, pandanus leaves, and water in the initial fermentation mixture is 3g:1.2g:1.2g:1.2g:35mL. The initial fermentation mixture is filtered, and then subjected to fungal fermentation again. The fungi used are Trichoderma atroviride and Trichoderma harziauum, with a mass ratio of the initial fermentation mixture, Trichoderma atroviride, and Trichoderma harziauum of 90:6:5. The fermentation temperature is 26℃, the fermentation time is 7 days, the pH value is 5.2, and the fermentation conditions are sealed anaerobic fermentation. The mixture is filtered again to obtain the incense-forming liquid.

[0026] Preparation Example 3

[0027] The preparation method of the incense-forming liquid includes: mixing 2.5 parts of rice, 1.1 parts of Eclipta prostrata, 1.1 parts of Polygonum hydropiper, and 1 part of Pandanus tinctoria leaves, pulverizing them, adding water, and anaerobic fermenting for 40 days at a temperature of 30℃ to obtain the initial fermentation mixture. The mass-to-volume ratio of rice, Eclipta prostrata, Polygonum hydropiper, Pandanus tinctoria leaves, and water in the initial fermentation mixture is 2.5g:1.1g:1.1g:1g:28mL. The initial fermentation mixture is filtered, and then fermented again with fungi, namely Trichoderma atroviride and Trichoderma harziauum. The mass ratio of the initial fermentation mixture, Trichoderma atroviride, and Trichoderma harziauum is (100-90):(3-6):(2-5). The fermentation temperature is 25℃, the fermentation time is 6.5 days, the pH value is 5.1, and the fermentation conditions are sealed anaerobic fermentation. The mixture is filtered again to obtain the incense-forming liquid.

[0028] Example 1

[0029] A method for rapid resin formation in Dalbergia odorifera from Guangdong and Guangxi provinces includes the following steps:

[0030] Step 1: Select two Dalbergia odorifera plants with a height of 18cm and a trunk diameter of 5.5cm. At a height of 22cm from the ground, use a 1.8mm diameter drill bit to drill holes horizontally or horizontally downward at a 3° angle along the trunk, penetrating the trunk. The horizontal distance between adjacent drill holes should be 1.7cm, and the distance between adjacent vertical rows should be 15cm.

[0031] Step 2: Within 25 minutes after drilling, inject 0.2g of resin-forming liquid into each hole at once. The resin-forming liquid is prepared according to the method in Preparation Example 1.

[0032] Example 2

[0033] Step 1: Select two Dalbergia odorifera plants with a height of 22cm and a trunk diameter of 6cm. At a height of 30cm from the ground, use a 2.0mm diameter drill bit to drill holes horizontally or horizontally downward at a 6° angle along the trunk, penetrating the trunk. The horizontal distance between adjacent drill holes should be 2.0cm, and the distance between adjacent vertical rows should be 20cm.

[0034] Step 2: Within 30 minutes after drilling, inject 0.25g of resin-forming liquid into each hole at once. The resin-forming liquid is prepared according to the method in Preparation Example 2.

[0035] Example 3

[0036] Step 1: Select two Dalbergia odorifera plants with a height of 20cm and a trunk diameter of 5.5cm. At a height of 25cm from the ground, use a 1.8mm diameter drill bit to drill holes horizontally or horizontally downward at a 4° angle along the trunk, penetrating the trunk. The horizontal distance between adjacent drill holes should be 1.9cm, and the distance between adjacent vertical rows should be 18cm.

[0037] Step 2: Within 28 minutes after drilling, inject 0.23g of resin-forming liquid into each hole at once. The resin-forming liquid is prepared according to the method in Preparation Example 3.

[0038] Comparative Example 1

[0039] The main difference between Comparative Example 1 and Example 1 is that the rice and Eclipta prostrata, the raw materials for preparing the resinous liquid in Example 1, are removed. The other methods and steps are the same as in Example 1.

[0040] Comparative Example 2

[0041] The main difference between Comparative Example 2 and Example 1 is that Polygonum hydropiper and Pandanus leaves were removed from the preparation method of the agarwood liquid in Example 1, while the other methods and steps were the same as in Example 1.

[0042] Comparative Example 3

[0043] The main difference between Comparative Example 3 and Example 1 is that in the preparation method of the incense liquid in Example 1, both anaerobic fermentations were changed to aerobic fermentation, while the other methods and steps were the same as in Example 1.

[0044] Comparative Example 4

[0045] The main difference between Comparative Example 4 and Example 1 is that the fungal fermentation step was removed from the preparation method of the incense liquid in Example 1, while the other methods and steps are the same as in Example 1.

[0046] Experiment 1

[0047] Each example was divided into three groups: Example 1 (Examples 1-1, 1-2, 1-3), Example 2 (Examples 2-1, 2-2, 2-3), and Example 3 (Examples 3-1, 3-2, 3-3). The comparative examples were grouped in the same way. The volatile oils from the 13-month extracts obtained in Examples 1-3 and Comparative Examples 1-4 were extracted using steam distillation. The mass-to-volume ratio of extract to water was 1 g: 5-8 mL, the extraction time was 30-50 min, and the temperature was 20-30℃. The distilled volatile oils were dried with anhydrous sodium sulfate and weighed. GC-MS analysis and qualitative analysis of chemical components were performed on the volatile oils. The experimental results showed that the average volatile oil content in Examples 1-3 ranged from 2.48% to 2.56%, while that in Comparative Examples 1-4 ranged from 0.96% to 1.10%. The volatile oil content in Examples 1-3 was significantly higher than that in the comparative examples (see Table 1).

[0048] Table 1

[0049] Group Sample type Average volatile oil content (%) Example 1 raw materials 2.56a Example 2 raw materials 2.48a Example 3 raw materials 2.53a Comparative Example 1 raw materials 1.02b Comparative Example 2 raw materials 0.98b Comparative Example 3 raw materials 1.10b Comparative Example 4 raw materials 0.96b

[0050] The extracted volatile oil was analyzed by GC-MS. The results showed that a total of 81 components were detected, including 24 sesquiterpenoids. Table 2 lists the data of the volatile oil components with higher content. The main components of the volatile oil were elemol, methyl eugenol, whiskey lactone, isoelemene, linalool, and cis-cobaene. Whiskey lactone, linalool, and cis-β-cobaene were detected, with relative percentages ranging from 73%-89% (elemol), 7.6%-20.0% (methyl eugenol), 0.03%-2.51% (whiskey lactone), 0.15-0.79% (isoelemene), 0.01%-0.55% (linalool), and 0.01%-0.66% (cis-β-cobaene), respectively. In addition, eight fragrance components were detected, all of which contained methyl eugenol and isoelemene, as detailed in Table 2. This indicates that the resin products produced by the rapid resin formation method of Dalbergia odorifera in Guangdong and Guangxi according to Examples 1-3 are rich in volatile oils and have increased content.

[0051] Table 2

[0052]

[0053]

[0054] Experiment 2:

[0055] Xylene-induced ear swelling experiment in mice. Forty-five mice were randomly divided into a blank control group, a dexamethasone positive control group, extracts of Dalbergia odorifera from Examples 1-3, and extracts of Dalbergia odorifera from Comparative Examples 1-4, with five equilibrium tests in each group. Except for the blank control group, which was administered distilled water by gavage, the other groups were given the corresponding dose of the drug once a day at a dose of 15 mL / kg for 7 consecutive days. The mice were fasted for 12 hours before the last administration but allowed free access to water. Sixty minutes after administration, 0.02 mL of xylene was dripped into the right ear of each mouse in each group to induce inflammation. The mice were euthanized 15 minutes later. Ear flaps of the inflamed mice were punched out using a 5 mm diameter punch, weighed, and the degree of swelling and the rate of swelling inhibition were calculated.

[0056] Ear swelling (mg) = Weight of right earpiece - Weight of left earpiece

[0057] Examples 1-3 showed that the extracts from *Dalbergia odorifera* effectively inhibited ear swelling in mice, with better inhibitory effects than the comparative and control groups. This indicates that the *Dalbergia odorifera* extract obtained by the raw materials and methods of this invention has good anti-inflammatory activity and can reduce ear inflammation.

[0058] Table 3

[0059]

[0060]

[0061] Experimental Results: The raw materials for the resin-forming liquid of this invention are a compound of rice, Eclipta prostrata, Polygonum hydropiper, and Pandanus leaves. A two-stage fermentation process is used for anaerobic fermentation of the resin-forming liquid. The raw materials are mixed, crushed, and water is added for fermentation to obtain a preliminary fermentation mixture. After filtration, it undergoes another fungal fermentation, and the filtered final resin-forming liquid is obtained. Dalbergia odorifera plants are selected, and holes are drilled in the main trunk. The resin-forming liquid after two fermentations is injected into each hole to induce resin formation. Using the rapid resin-forming method of Dalbergia odorifera of this invention, resin formation can occur again 1-1.2 years after the initial formation, improving the resin production efficiency and extract content of Dalbergia odorifera. The compound of rice, Eclipta prostrata, Polygonum hydropiper, and Pandanus leaves in the resin-forming liquid increases the types and content of volatiles in the extract. The combined use of the two-stage fermentation process improves the antibacterial and anti-inflammatory activity of the extract. When the raw materials of the resin-forming liquid are changed in Comparative Examples 1-2, and the fermentation process is changed in Comparative Examples 3-4, the antibacterial effect in the extract is significantly reduced. The raw materials, proportions, and methods of the resin-forming liquid of this invention enable rapid resin formation in Dalbergia odorifera (Guangdong and Guangxi), resulting in volatile oils containing fragrance components such as elemol, methyl eugenol, and whiskey lactone. The resin-forming products of Dalbergia odorifera (Guangdong and Guangxi) produced by the rapid resin formation method of this invention according to Examples 1-3 have increased the variety and content of volatile oils.

[0062] 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 principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for fast knotting of Dalbergia hupeana, characterized in that, The method comprises the following steps: S1, selecting two Dalbergia hupeana plants, drilling holes in the main stem in a horizontal direction or downwardly inclined direction; S2, after drilling the holes, injecting fermented incense-forming liquid into each hole; The incense-forming liquid raw material comprises 2-3 parts of rice, 1-2 parts of Eclipta prostrata, 1-1.5 parts of Polygonum hydropiper, and 0.8-1.2 parts of Pothos leaves; The preparation method of the incense-forming liquid comprises the following steps: mixing the incense-forming liquid raw material, crushing, and first-time water fermentation to obtain a fermented preliminary mixed liquid; filtering the fermented preliminary mixed liquid, second-time fungal fermentation after filtering, and third-time filtering to obtain the incense-forming liquid; The first-time water fermentation is anaerobic fermentation, the fermentation time is 38-42 days, and the fermentation temperature is 28-32 DEG C. The fermented preliminary mixed liquid obtained by the first-time water fermentation is composed of rice, Eclipta prostrata, Polygonum hydropiper, Pothos leaves and water in a mass / volume ratio of (2-3) g:(1-2) g:(1-1.5) g:(0.8-1.2) g:(25-35) mL. The fungi are Trichoderma atroviride (ATCC 20495) Trichoderma atroviride and Trichoderma harzianum (ATCC 20847) Trichoderma harziauum The mass ratio of the initial fermentation mixture, Trichoderma atroviride and Trichoderma harzianum is (100-90):(3-6):(2-5). The fungal fermentation is carried out under the following conditions: the fermentation temperature is 24-26 DEG C, the fermentation time is 6-7 days, the pH value is 5-5.2, and the fermentation condition is sealed anaerobic fermentation.

2. The method for rapid resin formation of Dalbergia odorifera in Guangdong and Guangxi according to claim 1, characterized in that, S1, the drilling, selecting two Dalbergia hupeana plants, drilling holes in the main stem in a horizontal direction or horizontal downwardly inclined direction at a distance of 20-30 cm from the ground level by using a drill bit with a diameter of 1.7-2.0 mm, penetrating the trunk, the horizontal distance between adjacent holes is 1.7-2.0 cm, and the distance between adjacent upper and lower rows is 15-20 cm.

3. The method for rapid resin formation of Dalbergia odorifera in Guangdong and Guangxi according to claim 1, characterized in that, S1, the two Dalbergia hupeana plants, the plant height is 18-22 cm, and the main stem diameter is greater than 5 cm.

4. The method for rapid resin formation of Dalbergia odorifera in Guangdong and Guangxi according to claim 1, characterized in that, S2, the incense-forming liquid is injected into each hole at one time within 25-30 min after drilling the holes.

5. The method for rapid resin formation of Dalbergia odorifera in Guangdong and Guangxi according to claim 1, characterized in that, The plants can form incense again after 1-1.2 years after the incense formation in the same year.

Images