A method for obtaining sesquiterpenes containing beta-cedrene and cedrol from the aerial roots of Dalbergia odorifera

Abstract

This invention discloses a method for obtaining sesquiterpenoids containing β-juniperene and juniperol from the aerial roots of *Dalbergia odorifera*, comprising the following steps: (S1) establishing a *Dalbergia odorifera* aerosol culture system; (S2) inducing the production of sesquiterpenoids from the aerial roots of *Dalbergia odorifera* under stress; (S3) extracting the sesquiterpenoids from the aerial roots of *Dalbergia odorifera* using ethyl acetate and identifying them using GC-MS. This invention also discloses the application of β-juniperene or juniperol in the preparation of reagents that promote the growth of *Arabidopsis thaliana* and in the preparation of drugs that inhibit lung cancer cells.

Description

Technical Field

[0001] This invention belongs to the field of sandalwood technology, specifically relating to a method for obtaining sesquiterpenoids containing β-juniperene and juniperol from the aerial roots of sandalwood. Background Technology

[0002] Dalbergia odorifera T.chen, also known as Hainan rosewood, is a rare and valuable hardwood with both medicinal and economic value. Currently, wild resources of Dalbergia odorifera are scarce. Its slow growth rate means that the flourishing of seedling cultivation can only initially address the problem of resource depletion and is insufficient to drive the development of downstream industries such as pharmaceuticals, spices, and antiques. Therefore, shortening the cultivation cycle and exploring new medicinal parts and components are crucial for the in-depth development of Dalbergia odorifera resources.

[0003] Introducing modern agricultural and biotechnological technologies into the cultivation of traditional Chinese medicinal herbs is an effective way to protect traditional resources and develop new ones, and it can also provide a good material system for subsequent basic research. Aeroponics is a modern agricultural cultivation technique with advantages such as promoting root organ growth and development and a short cultivation cycle. Currently, my country's aeroponics industry is mostly concentrated on vegetable cultivation, with few reports on the cultivation of woody medicinal plants. Therefore, applying aeroponics to the cultivation of *Dalbergia odorifera* roots is a beneficial attempt to develop new resources of *Dalbergia odorifera*. Furthermore, secondary metabolites in plant roots possess various biological activities, representing a major avenue for the development of new traditional Chinese medicines. As the source plant of *Dalbergia odorifera*, the composition, activity, and synthesis mechanism of secondary metabolites in the roots of *Dalbergia odorifera* seedlings deserve our close attention. Discussions on these issues will provide a certain reference for understanding the active components of *Dalbergia odorifera* and expanding the medicinal parts.

[0004] Current research on the chemical composition of Dalbergia odorifera mainly focuses on its heartwood. Related studies indicate that the heartwood is rich in volatile oils and flavonoids. The main component of the volatile oil is trans-nerolidol, and it also contains compounds such as (E)-β-farnesene, (3Z,6E)-α-farnesene, bisabolene, safflowerin, bisabolene, α-farnesene, dihydro-3-(2-methyl-2-propenyl)-2,5-furandione, 7-(2,6-dimethyl-hepta-1,5-dienyl)-3,8,8-trimethyl-bicyclo[4.2.0]oct-2-en luteolin, 2,4-dimethyl-2,6-heptadienal, and (±)α-bisabolol. It is noteworthy that current research on the chemical composition of the roots of Dalbergia odorifera seedlings is limited and requires further investigation.

[0005] Therefore, this invention aims to explore the application prospects of aeroponic cultivation in the development of Dalbergia odorifera resources, using the aerial roots of Dalbergia odorifera seedlings as the object, and at the same time provide reference information for understanding the medicinal components of Dalbergia odorifera. Summary of the Invention

[0006] The purpose of this invention is to provide a method for obtaining sesquiterpenoids containing β-juniperene and juniperol from the aerial roots of Dalbergia odorifera.

[0007] The present invention also aims to provide the use of β-juniperene or juniperol in the preparation of reagents that promote the growth of Arabidopsis thaliana, and the use of β-juniperene or juniperol in the preparation of drugs that inhibit lung cancer cells.

[0008] The first objective of this invention can be achieved by the following technical solution: a method for obtaining sesquiterpenoids containing β-juniperene and juniperol from the aerial roots of *Dalbergia odorifera*, comprising the following steps:

[0009] (S1) Select one-year-old sandalwood seedlings grown in soil as transplanting seedlings, cut off all lateral roots and part of the main root, disinfect and wash them, dip the roots with IBA+NAA, and transfer them into the aerosol culture system for culture. In the early stage, pure aquatic root induction culture is used, and in the later stage, nutrient solution is used for nutrient culture.

[0010] (S2) Add ethephon solution to the aeroponic nutrient solution to stress the aeroponic seedlings after nutrient culture.

[0011] (S3) Select the lateral roots of sandalwood aerosol seedlings after stress treatment, freeze-dry them, grind them into powder, add ethyl acetate for vortex extraction multiple times, centrifuge to collect the supernatant, concentrate and obtain the sample to be tested.

[0012] (S4) The sample to be tested was detected by GC-MS to identify volatile substances containing β-cedrene and cedrene alcohol.

[0013] In the above method for obtaining sesquiterpenoids containing β-juniperene and juniperol from the aerial roots of Dalbergia odorifera:

[0014] Optionally, in step (S1), disinfection is performed using a KMnO4 solution with a mass percentage of 0.08–0.12% for 55–65 seconds.

[0015] More preferably, or optionally, in step (S1), disinfection is performed using a KMnO4 solution with a mass percentage of 0.10% for 60 seconds.

[0016] Optionally, in step (S1), the roots are dipped in IBA+NAA at a concentration of 0.8-1.2 g / L for 8-12 seconds, wherein the mass ratio of IBA to NAA is 1:1.

[0017] More preferably, in step (S1), the roots are dipped in IBA+NAA at a concentration of 1.0 g / L for 10 seconds, wherein the mass ratio of IBA to NAA is 1:1.

[0018] Optionally, in step (S1), the culture is transferred to an aerosol culture system for 28 to 32 days, with pure aquatic rooting induction culture used for the first 6 to 8 days and nutrient solution used for the remaining days for nutrient culture.

[0019] More preferably, in step (S1), the culture is transferred to an aerosol culture system for 30 days, with pure aquatic rooting induction culture for the first 7 days and nutrient solution for nutrient culture for the last 23 days.

[0020] Optionally, the nutrient solution in step (S1) is Flower More No. 15 nutrient solution with a concentration of 0.4 to 0.6 g / L.

[0021] More preferably, the nutrient solution in step (S1) is Flower More No. 15 nutrient solution with a concentration of 0.5g / L.

[0022] Optionally, in step (S2), the final concentration of the ethephon solution added to the aeroponic nutrient solution is 4-6 wt%, and the stress treatment time for the aeroponic seedlings after nutrient culture is 13-16 days.

[0023] More preferably, in step (S2), the final concentration of the ethephon solution added to the aeroponic nutrient solution is 5 wt%, and the stress treatment time for the aeroponic seedlings after nutrient culture is 13 to 16 days.

[0024] The experimental results of this invention show that 5 wt% ethephon can induce the accumulation of volatile components in the aerial roots of sandalwood.

[0025] Optionally, in step (S2), the ratio of the amount of lateral roots of sandalwood aerosol seedlings to ethyl acetate is: 1g: 4-6mL, the extraction temperature is 3-5℃, the extraction time is 22-26h each time, and the number of extractions is 2-4 times.

[0026] More preferably, in step (S2), the ratio of the amount of lateral roots of sandalwood aerosol seedlings to ethyl acetate is 1g:5mL, the extraction temperature is 4℃, the extraction time is 24h each time, and the number of extractions is 3.

[0027] Optionally, the GC-MS detection conditions in step (S3) are as follows: column HP-5ms, 30m×0.25mm×0.25μm; initial temperature 35℃, held for 5min, then increased to 300℃ at 8℃ / min, held for 5min; injection volume 1μL; injection port temperature 250℃; quadrupole temperature 150℃; ion source temperature 230℃; ionization mode EI; electron energy 70eV; carrier gas He, purity 99.99%, flow rate 1mL / min; mass scan range 35~450AMU; solvent delay 3min. The obtained chromatograms and mass spectrometry data are compared using the Willy and NIST standard mass spectrometry libraries to identify each component, and the relative content of each component is calculated using the area normalization method.

[0028] In a preferred embodiment of the present invention, a complete aerosol culture system for Dalbergia odorifera was established through screening of culture conditions: One-year-old soil-grown Dalbergia odorifera seedlings were selected as transplanting seedlings, all lateral roots and part of the taproot were removed, the roots were disinfected with 0.1% KMnO4 solution for 1 minute, rinsed thoroughly with pure water, and then dipped in 1.0 g / L IBA + NAA (equal mass ratio) for 10 seconds before being transferred to the aerosol culture system for cultivation. The cultivation period was 30 days, with the first 7 days being pure water rooting induction culture, and the following 23 days using 0.5 g / L Flower More No. 15 nutrient solution for nutrient cultivation. This culture system can harvest a sufficient quantity of high-quality Dalbergia odorifera aerosol roots within a relatively short cultivation period, making it a relatively ideal culture system.

[0029] The aerosol culture system can be a conventional aerosol culture system disclosed in the prior art, preferably an aerosol culture system invented by the inventor of this application, application number CN202120640461.0, etc.

[0030] The second objective of the present invention can be achieved by the following technical solution: the application of β-cucurbitene or cucurbitol in the preparation of a reagent that promotes the growth of Arabidopsis thaliana.

[0031] Optionally, the concentrations of β-cedrene and cedrene are less than 5 μmol / L, more preferably 5 μmol / L.

[0032] This invention verified the growth-promoting activities of β-cucurbitene and cucurbitol through experiments. The results showed that 5 μmol / L of β-cucurbitene and cucurbitol could promote the growth of Arabidopsis thaliana, specifically by increasing the total biomass and the number of lateral roots. Moreover, high concentrations of β-cucurbitene did not inhibit the growth of Arabidopsis thaliana, but instead promoted it, and can be used as a growth regulator.

[0033] The present invention further provides the use of β-juniperene or juniperol in the preparation of drugs that have an inhibitory effect on lung cancer cells.

[0034] Optionally, the concentration of β-cedrene and cedrene is greater than 200 μmol / L, more preferably 400–600 μmol / L.

[0035] In terms of anti-tumor effects, the present invention selected lung cancer cell lines H1299 and A549 for investigation. The experimental results showed that high concentrations (greater than 200 μmol / L, preferably 400-600 μmol / L) of β-cedrol and cedrol had significant inhibitory effects on lung cancer cells, and the inhibitory effect increased with the increase of administration time.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] (1) This invention establishes an aerosol culture system for sandalwood by screening hormone induction conditions and nutrient solution culture conditions;

[0038] (2) This invention also explores the feasibility of stress-induced production of volatile components in the aerial roots of Dalbergia odorifera. The results show that 5 wt% ethephon can significantly promote the accumulation of sesquiterpenes in the aerial roots of Dalbergia odorifera.

[0039] (3) The present invention further verifies the bioactivity of β-juniperene and juniperol. The experimental results show that low concentrations of β-juniperene and juniperol promote the growth of Arabidopsis thaliana, and high doses can inhibit the proliferation of lung cancer cells.

[0040] (4) Therefore, this invention establishes a sandalwood aerosol culture system to obtain a large number of high-quality aerial roots in a short culture period, stresses sandalwood aerial roots, preliminarily explores the effect of ethephon on the volatile components of aerial roots, and verifies the bioactivity of the main volatile components, sesquiterpenoids β-juniperene and juniperol. Attached Figure Description

[0041] The invention will be further described below with reference to the accompanying drawings.

[0042] Figure 1 This refers to the pretreatment before aeroponic transplanting of sandalwood in Example 1;

[0043] Figure 2 This shows the rooting of Dalbergia odorifera under different hormone induction conditions in Example 1;

[0044] Figure 3 This shows the rooting of *Dalbergia odorifera* under different types of nutrient solutions in Example 1.

[0045] Figure 4 This shows the rooting of *Dalbergia odorifera* under different concentrations of nutrient solution in Example 1;

[0046] Figure 5The rooting status of lateral roots of *Dalbergia odorifera* seedlings under 5wt% ethephon stress on days 10, 13, 16, 19, and 25 in Example 1.

[0047] Figure 6 This is a superimposed image of the total ion concentrations of each sample in Example 1;

[0048] Figure 7 The total sesquiterpenoid content in the aerial roots of Dalbergia odorifera in Example 1 changes over time;

[0049] Figure 8 The effect of β-cucurbitene on Arabidopsis thaliana growth in Example 2 is shown in Figure (A), which is a diagram showing the root-promoting effect of β-cucurbitene; Figure (B) is a statistical diagram of the main root length of Arabidopsis thaliana; Figure (C) is a statistical diagram of the number of lateral roots of Arabidopsis thaliana; and Figure (D) is a statistical diagram of the total biomass of Arabidopsis thaliana.

[0050] Figure 9 The effect of β-juniperol on Arabidopsis thaliana growth in Example 2 is shown in Figure (A), which is a diagram showing the root-promoting effect of β-juniperol; Figure (B) is a statistical diagram of the main root length of Arabidopsis thaliana; Figure (C) is a statistical diagram of the number of lateral roots of Arabidopsis thaliana; and Figure (D) is a statistical diagram of the total biomass of Arabidopsis thaliana.

[0051] Figure 10 The images show the anti-lung cancer activity of β-cedrene and cedrene in Example 2. Figure (A) shows the cell viability of H1299 cells in the β-cedrene treatment group, Figure (B) shows the cell viability of A549 cells in the β-cedrene treatment group, Figure (C) shows the cell viability of H1299 cells in the cedrene treatment group, and Figure (D) shows the cell viability of A549 cells in the cedrene treatment group. Detailed Implementation

[0052] Unless otherwise specified, all raw materials or reagents used below are commercially available products or obtained through conventional methods.

[0053] Example 1

[0054] The following raw materials and equipment are preferred, but not limited to:

[0055] The one-year-old seedlings of Dalbergia odorifera were purchased from Guangzhou Da Senlin Nursery.

[0056] For the water mist cultivation machine, please refer to application number: CN202120640461.0.

[0057] Potassium permanganate, indolebutyric acid (IBA, Sigma), naphthaleneacetic acid (NAA, Sigma), Flower More 1 (Flower More Yinyu Brand Store), Flower More 10 (Flower More Yinyu Brand Store), Flower More 15 (Yongchun Horticulture).

[0058] 1. Pre-transplanting treatment of aerosol boxes and seedlings

[0059] The planting cups and planting cotton were disinfected by soaking in chlorine dioxide disinfectant, and the aerosol chamber and its circulation system were also disinfected by spraying. Afterwards, they were rinsed with pure water to ensure the aerosol chamber was as sterile as possible. Healthy *Dalbergia odorifera* seedlings were selected, and their leaves were pruned to adjust their height. The terrestrial roots of the seedlings were soaked and cleaned with water, and excess lateral roots and part of the taproot were trimmed. The treated roots were then disinfected with 0.1% KMnO4 solution for 1 minute for subsequent experimental treatment.

[0060] Pre-transplanting treatment of sandalwood aeroponics, such as Figure 1 As shown.

[0061] 2. Induction of aerial roots in sandalwood

[0062] The experiment designed a total of 9 induction treatment groups with 3 types of hormones (IBA, NAA, and IBA+NAA in equal mass ratios) and 3 concentration gradients (0.5 g / L, 1 g / L, 1.5 g / L), and 1 blank control group as the induction scheme for aerial roots.

[0063] The specific procedure was as follows: the disinfected sandalwood seedlings were treated with hormone-dipped roots according to the experimental groups for 10 seconds. Then the sandalwood seedlings were transferred to the aerosol culture system for rooting culture. After 20 days, the rooting rate ( / %) and the number of roots ( / strips) were counted.

[0064] Rooting of Dalbergia odorifera under different hormone induction conditions, as follows Figure 2 As shown in Table 1, among the hormone treatment groups, the IBA+NAA compound group in equal proportions had the best induction effect, followed by the IBA group, and the NAA group was slightly worse. The optimal induction scheme was 1.0 g / L IBA+NAA, and the number of roots per aerosol seedling was 87.5 (75.3-96.3), as shown in Table 1.

[0065] Table 1. Statistical table of hormone-induced rooting (M(P25~P75))

[0066]

[0067] 3. Screening of nutrient solution conditions for aerial roots of Dalbergia odorifera

[0068] The selection of nutrient solution conditions mainly involves considerations of nutrient element ratio and nutrient solution concentration. Firstly, to investigate the effect of nutrient element ratio on the growth of aerial roots of *Dalbergia odorifera*, the experiment selected *Huaduoduo* general-purpose fertilizer No. 1, *Huaduoduo* high-nitrogen fertilizer No. 10, and *Huaduoduo* high-phosphorus fertilizer No. 15 as research subjects. *Dalbergia odorifera* aeroponic seedlings were cultured for 30 days, with the first 7 days in pure water and the following 23 days in different nutrient solutions at 0.5 g / L. The rooting status of *Dalbergia odorifera* under different nutrient solutions is shown below. Figure 3 As shown.

[0069] The rooting of sandalwood under different concentrations of nutrient solution is as follows: Figure 4 As shown, the evaluation indicators included the underground parts, including total root length and fresh root weight. The experiment screened for the optimal nutrient solution concentration for promoting growth, designing three concentrations: 0.5 g / L, 1.0 g / L, and 1.5 g / L. The culture period was extended to 40 days, with the first 20 days using a low concentration (0.1 g / L) of nutrient solution and the subsequent 20 days using the designed concentration. The evaluation indicators included total root length ( / m) and fresh weight ( / g).

[0070] For the statistics of the number of roots, total root length and fresh weight, the experiment selected a biological statistical method with single-plant replication.

[0071] The results showed that the culture effect of Huaduoduo No. 15 group was significantly better than that of Huaduoduo No. 10 and Huaduoduo No. 1 group. The fresh weight of the aerial roots of a single plant was 0.88 (0.71-1.10) g, and the total root length was 3.08 (2.27-5.67) m (Table 2).

[0072] Table 2 Effects of nutrient solution type on aerial root growth (M(P25~P75))

[0073] Number of examples Fresh weight / g Total root length / m Huaduoduo 15 10 0.88(0.71～1.10) 3.08(2.27～5.67) Huaduoduo 10 10 0.09(0.03～0.25) 0.35(0.22～0.78) Flower More 1 10 0.19(0.03～0.44) 0.69(0.20～1.09) <![CDATA[x 2 ]]> <![CDATA[18.880 a ]]> <![CDATA[18.614 a ]]> P 0.000 0.000

[0074] Nutrient solution concentration is crucial for the growth of aeroponic seedlings. For example, excessively high phosphorus concentrations can be toxic to plants and inhibit growth. To achieve better cultivation results, the concentration of Flower More No. 15 was screened, with the M (P25–P75) values ​​of fresh weight and total root length as the evaluation indicators. The results showed that under a 0.5 g / L culture condition, the fresh weight of the aerial roots of *Dalbergia odorifera* was 1.84 (1.36–2.56) g, and the total root length was 6.41 (5.60–8.58) m, significantly higher than other culture groups, indicating a more suitable culture concentration (Table 3).

[0075] Table 3 Effects of nutrient solution concentration on aerial root growth (M(P25~P75))

[0076] Number of examples Fresh weight / g Total root length / m 0.5g / L 10 1.84(1.36～2.56) 6.41(5.60～8.58) 1.0g / L 10 1.17(0.77～1.73) 4.07(3.38～4.95) 1.5g / L 10 0.31(0.08～1.61) 1.399(0.70～7.09) <![CDATA[x 2 ]]> 8.841a 7.946a P 0.012 0.019

[0077] Aeroponics is a novel soilless cultivation technology, with core components including seedling selection, rooting induction, and nutrient culture. In this invention, the aeroponic cultivation of Dalbergia odorifera used one-year-old seedlings as transplanting material, retaining some of the taproot to shorten the time for aerial root development.

[0078] Regarding rooting induction, the experiment mainly evaluated the induction effects of IBA, NAA, and IBA+NAA in equal proportions. The optimal induction scheme was 1.0 g / L of IBA+NAA.

[0079] It is noteworthy that among the three induction schemes mentioned above, the induction effect of the NAA treatment group was weaker. NAA is an important hormone for inducing adventitious roots from callus tissue. Observation of the formation process of aerial roots of Dalbergia odorifera revealed that aerial roots of Dalbergia odorifera originate from the stele of the main root and break through the cortex to grow into the aerosol environment. Callus tissue is not involved in this process. Therefore, it is speculated that the formation mode of aerial roots of Dalbergia odorifera is the main reason for the weakening effect of NAA induction. When selecting induction hormones, the rooting mode of cuttings should be fully considered. If roots are used as cuttings for propagation, NAA should be avoided alone or the amount of NAA in the compound hormone should be appropriately reduced.

[0080] In addition, during the nutrient culture stage, the effects of different element ratios and nutrient solution concentrations on the growth of aerial roots of *Dalbergia odorifera* were evaluated. The results showed that 0.5 g / L of Flower More No. 15 (high phosphorus) water-soluble fertilizer had a better culture effect. Phosphorus is an important element involved in plant energy metabolism and nucleic acid metabolism, and can promote early plant development. Since the plant's phosphorus requirement changes with its growth state, dynamically adjusting the nutrient solution concentration will be a direction for optimizing the subsequent culture conditions of *Dalbergia odorifera*. It is noteworthy that during the long-term (40-day) culture process of *Dalbergia odorifera* aerial roots, some aerial roots showed varying degrees of fungal contamination and aging as the culture time increased, which was very detrimental to the subsequent experiments. Therefore, the final culture period was shortened to 30 days.

[0081] In summary, a complete aerosol culture system for *Dalbergia odorifera* was established through screening of culture conditions. Specifically, one-year-old soil-grown *Dalbergia odorifera* seedlings were selected as transplanting seedlings. All lateral roots and part of the taproot were removed. The seedlings were disinfected with 0.1% KMnO4 solution for 1 minute, rinsed thoroughly with pure water, and then dipped in 1.0 g / L IBA+NAA solution for 10 seconds before being transferred to the aerosol culture system for cultivation. The cultivation period was 30 days, with the first 7 days used for pure water root induction culture, and the following 23 days using 0.5 g / L Flower More No. 15 nutrient solution for nutrient cultivation. This culture system can harvest a sufficient quantity of high-quality *Dalbergia odorifera* aerosol roots within a relatively short cultivation period, making it a relatively ideal culture system.

[0082] 4. Stress treatment of aerial roots of sandalwood

[0083] A 40wt% ethephon solution was added to the aeroponic nutrient solution, adjusting the concentration to 5wt%. This nutrient solution was then applied to aeroponic seedlings that had grown for 30 days to induce stress. To maintain the ethephon concentration, the nutrient solution was changed every 3 days. Samples were taken and chemically analyzed on days 10, 13, 16, 19, and 25 of the stress period. Each stress sample consisted of a mixture of lateral roots from three seedlings. Rooting status was as follows... Figure 5 As shown.

[0084] 5. Sample preparation

[0085] Sampling: Select 3 sandalwood aerosol seedlings after stress treatment, wash the lateral roots, absorb the moisture with filter paper, quickly cut them and place them in cryovials, flash freeze them with liquid nitrogen for 10 minutes and place them in a -80 degree freezer for subsequent testing.

[0086] Sample preparation: The cryopreserved sample was ground into powder using liquid nitrogen. 1.00 g of powder was weighed into a 10 mL centrifuge tube, 5 mL of ethyl acetate was added, and the mixture was extracted at 4 °C for 24 h. To ensure extraction efficiency, the mixture was vortexed 3 times during the extraction. After extraction, the sample was centrifuged and the supernatant was collected and concentrated to 1 mL using a nitrogen blower to obtain the sample to be tested. The experiment was repeated three times.

[0087] 6. GC-MS detection conditions

[0088] The chromatographic column was HP-5ms (30m × 0.25mm × 0.25μm); the initial temperature was 35℃, held for 5 min, then increased to 300℃ at 8℃ / min and held for 5 min; the injection volume was 1μL; the injection port temperature was 250℃; the quadrupole temperature was 150℃; the ion source temperature was 230℃; the ionization mode was EI; the electron energy was 70eV; the carrier gas He (99.99%) flow rate was 1mL / min; the mass scan range was 35–450 AMU; and the solvent delay was 3 min. The obtained chromatograms and mass spectrometry data were compared using the Willy and NIST standard mass spectrometry libraries to qualitatively identify each component, and the relative content of each component was calculated using the area normalization method.

[0089] GC-MS results showed that ethephon induced the accumulation of sesquiterpenes in the aerial roots of *Dalbergia odorifera*, and the total sesquiterpenes content in the aerial roots of *Dalbergia odorifera* showed a trend of first increasing and then decreasing with the increase of stress time. Figure 6-7 The structural formulas of (+)-β-cucurbitene and cucurbitol are as follows:

[0090]

[0091] Comparison of sesquiterpene content under different stress durations: The sesquiterpene components with a relative content greater than 0.5% in each group of samples were summarized and analyzed. The experiment screened out 17 sesquiterpene substances, including β-juniperene and juniperol. Overall, the 16-day stress treatment group induced the most diverse range of sesquiterpene species and the highest content, followed by the 13-day stress group.

[0092] Table 4 Comparison of sesquiterpenoid components in the aerial roots of Dalbergia odorifera

[0093]

[0094] This invention investigated the stress treatment and volatile component analysis of aerial roots of *Dalbergia odorifera* using 5 wt% ethephon, aiming to provide a reference for the further development of *Dalbergia odorifera* aerial root resources. Experimental results showed that ethephon stress promoted the senescence of *Dalbergia odorifera* aerial seedlings, specifically manifested as leaf drop and root yellowing. Furthermore, with increasing stress duration, the total sesquiterpene content in *Dalbergia odorifera* aerial roots exhibited a trend of first increasing and then decreasing.

[0095] Example 2

[0096] Of the 17 sesquiterpenoids obtained in Example 1, two components with promising application prospects, β-juniperene and juniperol, were screened for efficacy experiments. β-juniperene and juniperol were obtained by column chromatography of the supernatant from Example 1, followed by elution with an eluent, and then column chromatography of the fractions for chromatographic identification. Since the amount of β-juniperene and juniperol obtained in Example 1 was very small, β-juniperene and juniperol standards were used instead of those obtained in Example 1 for the following experiments:

[0097] Among them, the seeds of wild-type Arabidopsis thaliana (L.) Heynh were donated to Professor He Rui's research group at Guangzhou University of Chinese Medicine; the H1299 and A549 cell lines were donated to Professor Liu Yongqiang's research group at Guangzhou University of Chinese Medicine; and β-cucurbitene standard (Pusi Technology) and cucurbitol standard (Maclean).

[0098] Data Statistics and Analysis: Experimental data were initially processed using Excel 2019 and subjected to the Shapiro-Wilk normality test using SPSS 26.0 software. Subsequently, one-way ANOVA was performed on data that met the requirements of normality and homogeneity of variance. Multiple comparisons were performed using the LSD test. All results are expressed as mean ± standard deviation. The Kruskal-Wallis H test was performed on comparison groups that did not conform to normality, and the results are expressed as M (P25–P75). Furthermore, statistical graphs were created using GraphPad Prism 8.

[0099] 2.1 Drug co-culture in Arabidopsis thaliana

[0100] The effects of β-cucurbitene and cucurbitol on the growth of Arabidopsis thaliana were evaluated using a co-culture method. First, β-cucurbitene was added to sterilized 1 / 2 MS medium to prepare inoculation plates with final concentrations of 0 μmol / L, 5 μmol / L, 10 μmol / L, 15 μmol / L, 20 μmol / L, 25 μmol / L, 100 μmol / L, and 300 μmol / L. Then, Arabidopsis thaliana seeds were disinfected by soaking in 2% NaClO for 15 min, followed by washing six times with sterile water to ensure no NaClO residue. Finally, the treated seeds were inoculated onto the inoculation plates and vernalized at 4°C for two days, followed by 15 days of cultivation in a light incubator. After cultivation, the taproot length ( / cm), lateral root number ( / root), and total biomass ( / mg) of Arabidopsis thaliana seedlings in each treatment group were recorded. A single-plant replicate biostatistical method was used, selecting the 10 most representative samples from each treatment group for statistical analysis. The operation of cypressin is the same as that of β-cypressene.

[0101] 2.2 Effects of β-Cupressene and Cupressol on Arabidopsis thaliana Growth

[0102] In the β-cucurbitene treatment group, drug concentrations of 5 μmol / L, 10 μmol / L, 15 μmol / L, 100 μmol / L, and 300 μmol / L significantly promoted the development of lateral roots in Arabidopsis thaliana and increased the total biomass of the plants. The optimal concentration was 5 μmol / L, with the number of lateral roots reaching 39.50 (27.50–51) and the total biomass reaching 14.90 (12.05–24.63) mg (mg / L). Figure 8 ).

[0103] In the cypressin treatment group, only a concentration of 5 μmol / L promoted the growth of Arabidopsis thaliana, but the effect was not significant. At this concentration, the number of lateral roots in Arabidopsis thaliana was 19.5 (8.75–27.25), and the total biomass was 11.5 (6.70–18.33) mg. Furthermore, when the concentration of cypressin was greater than 15 μmol / L, the growth of the taproot of Arabidopsis thaliana was significantly inhibited; when it was greater than 50 μmol / L, the occurrence of lateral roots and the total biomass of the plant were significantly inhibited. Figure 9 ).

[0104] 2.3 Evaluation of anti-lung cancer activity using the MTT assay

[0105] The effects of β-juniperene and juniperol on the proliferation of cell lines H1299 and A549 were evaluated using the MTT assay to explore their anti-lung cancer activity. The experiment was divided into a blank group, a control group, and a drug-treated group, with three replicates for each group. The specific experimental procedures are as follows:

[0106] Two cells in the logarithmic growth phase were digested and resuspended, and the suspension concentration was adjusted to 8 × 10⁻⁶ cells by cell counting. 4 Cells were cultured at a concentration of 100 μL / mL, and then the resulting cell suspension was seeded into 96-well plates at a rate of 100 μL / well. After 24 h of culture, the original culture medium was replaced with drug-containing medium at concentrations of 600 μmol / L, 400 μmol / L, 200 μmol / L, 100 μmol / L, 50 μmol / L, 25 μmol / L, 12.5 μmol / L, 6.25 μmol / L, and 0 μmol / L for continued co-culture for 24 h and 48 h. After the culture period, 10 μL of MTT solution (5 mg / mL) was added to each well and the plates were incubated for 4 h. After the reaction, the culture medium was discarded, and 100 μL of DMSO was added to each well. The mixture was shaken to dissolve the cells, and the absorbance at 490 nm was measured. Cell viability was then calculated as follows: Cell viability = (drug-treated group - blank group) / (control group - blank group) × 100%. The half-maximal inhibitory concentration (IC50) of each drug-treated group after 48 h of drug administration was calculated using SPSS. 50 ).

[0107] 2,4-β-Cupressene and Cupressol's anti-lung cancer activity

[0108] MTT assay results showed that β-cedrene and cedrene at concentrations greater than 200 μmol / L, especially 400–600 μmol / L, significantly inhibited the proliferation of lung cancer cells H1299 and A549, and prolonged administration time enhanced the inhibitory effect. Figure 10 The calculated half-maximal inhibitory concentrations (IC50) showed that β-cucurbitene had IC50 values ​​of 1043.644 μmol / L and 461.749 μmol / L against H1299 and A549, respectively; while cucurbitol had IC50 values ​​of 147.463 μmol / L and 202.516 μmol / L against H1299 and A549, respectively. In comparison, cucurbitol showed better anti-lung cancer activity.

[0109] Therefore, this embodiment investigated the bioactivity of β-junctene, a key secondary metabolite in the roots of *Dalbergia odorifera* seedlings, and related compound junctene alcohol, mainly including the evaluation of growth regulation and antitumor activity. Firstly, regarding growth regulation, the growth-promoting activities of β-junctene and junctene alcohol were verified. The results showed that β-junctene and junctene alcohol at concentrations below 5 μmol / L could promote the growth of *Arabidopsis thaliana*, specifically manifested as an increase in total biomass and the number of lateral roots. High concentrations of β-junctene did not inhibit the growth of *Arabidopsis thaliana*, but rather promoted it. Secondly, regarding antitumor activity, lung cancer lines H1299 and A549 were selected for investigation. The experimental results showed that high concentrations of β-junctene and junctene alcohol had a significant inhibitory effect on lung cancer cells, and the inhibitory effect increased with increasing administration time.

[0110] The above embodiments are merely preferred embodiments of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-substantial changes and substitutions made by those skilled in the art based on the present invention shall fall within the scope of protection claimed by the present invention.

Claims

1. A method for obtaining sesquiterpenoids containing β-juniperene and juniperol from the aerial roots of *Dalbergia odorifera*, characterized in that... Includes the following steps: (S1) Select one-year-old sandalwood seedlings grown in soil as transplanting seedlings, cut off all lateral roots and part of the main root, disinfect and wash them, dip the roots with IBA+NAA, and transfer them into the aerosol culture system for culture. In the early stage, pure water root induction culture is used, and in the later stage, nutrient solution is used for nutrient culture. (S2) Add ethephon solution to the aeroponic nutrient solution to stress the aeroponic seedlings after nutrient culture; (S3) Select the lateral roots of sandalwood aerosol seedlings after stress treatment, freeze-dry them, grind them into powder, add ethyl acetate for vortex extraction multiple times, centrifuge to collect the supernatant, concentrate and obtain the sample to be tested. (S4) The sample to be tested was detected by GC-MS to identify volatile substances containing β-juniperene and juniperol; In step (S1), the roots are dipped in IBA+NAA at a concentration of 0.8~1.2g / L for 8~12s, wherein the mass ratio of IBA to NAA is 1:

1. In step (S1), the culture is transferred to an aerosol culture system for 28-32 days. The first 6-8 days are induced by pure aquatic rooting, and the remaining days are nutrient cultured in nutrient solution. In step (S1), the nutrient solution is Flower More No. 15 nutrient solution with a concentration of 0.4~0.6g / L; In step (S2), the final concentration of ethephon solution added to the aeroponic nutrient solution is 4-6 wt%, and the stress treatment time for the aeroponic seedlings after nutrient culture is 13-16 days.

2. The method for obtaining sesquiterpenoids containing β-juniperene and juniperol from the aerial roots of *Dalbergia odorifera* according to claim 1, characterized in that: In step (S1), disinfection is performed using a KMnO4 solution with a mass percentage of 0.08-0.12% for 55-65 seconds.

3. The method for obtaining sesquiterpenoids containing β-juniperene and juniperol from the aerial roots of *Dalbergia odorifera* according to claim 1, characterized in that: The ratio of the amount of lateral roots of sandalwood aerosol seedlings to ethyl acetate in step (S2) is as follows: 1g: 4~6mL, the extraction temperature is 3~5℃, the extraction time is 22~26h each time, and the number of extractions is 2~4 times.

4. The method for obtaining sesquiterpenoids containing β-juniperene and juniperol from the aerial roots of *Dalbergia odorifera* according to claim 1, characterized in that: In step (S3), the GC-MS detection conditions were as follows: column HP-5ms, 30 m × 0.25 mm × 0.25 µm; initial temperature 35℃, held for 5 min, then increased to 300℃ at 8℃ / min, held for 5 min; injection volume 1 µL; injection port temperature 250℃; quadrupole temperature 150℃; ion source temperature 230℃; ionization mode EI; electron energy 70 eV; carrier gas He, purity 99.99%, flow rate 1 mL / min; mass scan range 35–450 AMU; solvent delay 3 min. The obtained chromatograms and mass spectrometry data were compared using the Willy and NIST standard mass spectrometry libraries to identify each component, and the relative content of each component was calculated using the area normalization method.

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