A method for increasing the content of terpinolene in oenanthe javanica

Abstract

The application provides a method for increasing the content of terpinolene in water celery, which comprises: spraying methyl jasmonate during the growth of water celery. Through the spraying treatment of methyl jasmonate used in the application, the content of terpenes in water celery is sharply increased, and the content of terpinolene is extremely outstandingly increased, which is a plant cultivation method for comprehensively increasing the content of terpenes in water celery and greatly increasing terpinolene.

Description

Technical Field

[0001] This invention belongs to the field of agricultural planting technology, specifically relating to a method for increasing the content of volatile terpenoid oils in water celery. Background Technology

[0002] Water celery (Oenanthejavanica (Blume) DC) is a perennial aquatic herb (Yoshida et al., 2013). It contains abundant nutrients, minerals, polyphenols, carotenoids, and a large amount of lutein (Kongkachuichai, Charoensiri, Yakoh, Kringkasemsee, & Insung, 2015), possessing rich potential value in pharmacology, nutrition, and phytochemistry (Lu & Li, 2019). Water celery and its extracts are effective in treating jaundice, hypertension, and hepatitis (Han, Huang, Yang, Liu, & Wu, 2008; Wang, Yang, Liu, Huang, & Wu, 2005). Water celery also has a unique flavor, with volatile compounds mainly consisting of terpenes (Feng et al., 2023; Seo & Baek, 2005).

[0003] Terpenes are natural compounds whose basic structural unit is isoprene and its derivatives (Bouvier, Rahier, & Camara, 2005). Terpenes are formed by the sequential condensation of the five-carbon monomer isopentyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP) (Kuzuyama, 2017). Essential oils made from terpenes can be used in aromatherapy for anxiety (Agatonovic-Kustrin, Kustrin, Gegechkori, & Morton, 2020), and due to their antioxidant activity, they can also be used as food preservatives (Kobenan et al., 2022; Mahato et al., 2019) and have antibacterial activity (Mustafa, 2015). Terpenoids also possess antitumor properties (El-Baba et al., 2021), anti-inflammatory activity (Ge et al., 2022), neuroprotective activity (Zhang, Zhou, Song, Yao, & Song, 2020), and anticancer potential (Jain, Dhingra, Narsinghani, & Sharma, 2016). Terpinene is one such monoterpene compound with important industrial and pharmaceutical uses, and its aroma also makes it widely used as a flavoring agent.

[0004] Jasmonic acid (JA) and its derivative methyl jasmonic acid (MeJA) are ubiquitous endogenous plant hormones in the plant kingdom (Wasternack & Hause, 2002). MeJA is a plant growth regulator that can induce the JA signaling pathway to regulate plant secondary metabolites, further regulating terpene biosynthesis (Li et al., 2020; Paponov, Antonyan, Slimestad, & Paponov, 2021). Exogenous MeJA treatment promotes the biosynthesis of volatile terpenoids in tea, lavender, and grapes (D'Onofrio, Matarese, & Cuzzola, 2018; Dong et al., 2022; Shi et al., 2021). The methods for enhancing the flavor of watercress are currently unclear, but spraying with MeJA may improve its flavor. Summary of the Invention

[0005] The purpose of this section is to outline some aspects of embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

[0006] In view of the problems existing in the above and / or prior art, the present invention is proposed.

[0007] Therefore, the purpose of this invention is to overcome the shortcomings of the prior art and provide a method for increasing the content of terpene oil in watercress.

[0008] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a method for increasing the content of terpene oil in water celery, comprising: spraying methyl jasmonate during the growth of water celery.

[0009] As a preferred embodiment of the method for increasing the terpene oil content of water celery according to the present invention, the growth process of water celery includes the following steps:

[0010] Planting preparation: Deeply till the soil and apply fertilizer, keeping the soil moist;

[0011] Seedling selection and planting: Prepare water celery seedlings, keeping them intact when digging them up, and after dividing them, select and plant them in the pond;

[0012] Water celery growth management: During the growth of water celery, maintain a shallow water layer, remove weeds, and ensure sufficient fertilizer during the growth process;

[0013] Methyl jasmonic acid treatment: Spray exogenous methyl jasmonic acid on water celery that has grown for a certain number of days;

[0014] As a preferred embodiment of the method for increasing the content of terpene oil in water celery according to the present invention, the methyl jasmonate is sprayed at a time when the weather is sunny and cool, from 5:00 to 6:00 pm.

[0015] As a preferred embodiment of the method for increasing the terpene oil content of water celery according to the present invention, in the methyl jasmonate treatment, the water celery that has grown for a certain number of days is 35 days after transplanting.

[0016] As a preferred embodiment of the method for increasing the content of terpene oil in water celery according to the present invention, the amount of methyl jasmonate sprayed is 50 mL per water celery plant.

[0017] As a preferred embodiment of the method for increasing the content of terpene oil in water celery according to the present invention, wherein the concentration of methyl jasmonate is 0-3 mmol / L.

[0018] As a preferred embodiment of the method for increasing the content of terpene oil in water celery according to the present invention, the method further includes harvesting water celery, wherein the harvesting time is 24 hours or 48 hours after treatment with methyl jasmonate.

[0019] As a preferred embodiment of the method for increasing the content of terpene oil in water celery according to the present invention, wherein: when the harvest time is 24h, the concentration of methyl jasmonate is 0.5 mmol / L.

[0020] As a preferred embodiment of the method for increasing the content of terpene oil in water celery according to the present invention, wherein: when the harvest time is 48h, the concentration of methyl jasmonate is 0.1mm / L.

[0021] As a preferred embodiment of the method for increasing the content of terpene oils in water celery according to the present invention, the content of terpenoids in water celery is increased after treatment with methyl jasmonate.

[0022] Beneficial effects of this invention:

[0023] The methyl jasmonate spraying method used in this invention results in a dramatic increase in the terpene content of water celery, with a particularly prominent increase in the content of terpinene. This is a comprehensive plant cultivation method that can significantly increase the terpene content in water celery and substantially enhance terpinene content. Attached Figure Description

[0024] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Wherein:

[0025] Figure 1 GC-MS analysis of volatile substances in water celery in this embodiment of the invention;

[0026] Figure 2 The values ​​of MeJA and JA in watercress after spraying with 0.1 mmol / L methyl jasmonate in this embodiment of the invention are shown. Detailed Implementation

[0027] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0028] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0029] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0030] The water celery variety used in this embodiment of the invention is "Fuqin No. 1", and all plant materials are preserved at the Yangzhou University Aquatic Vegetable Experiment Base.

[0031] Example 1

[0032] Planting preparation: First, deeply plow the land, then apply 20 catties of organic fertilizer per mu, and keep the soil moist.

[0033] Seedling selection: In April, when the water celery seedlings in the resource container have grown to 10 cm in height and have 7 true leaves, dig them out with soil attached, ensuring the roots are intact. Gently divide the seedlings and select those with intact leaves, free from pests and diseases, and growing vigorously.

[0034] Planting: Plant individual plants into the prepared pond at a depth of 3.5-4.5cm, with a plant spacing of 6cm and a row spacing of 3cm.

[0035] Water celery growth management: After transplanting, monitor the water level daily to maintain a shallow water layer of 2cm and remove weeds. 20 days after transplanting, apply 20 catties of urea per mu. 30 days after transplanting, apply 20 catties of other nitrogen fertilizer per mu as top dressing and remove duckweed from the water.

[0036] The specific settings for different concentrations of exogenous methyl jasmonate are as follows: CK: 0 mmol / L; T1: 0.1 mmol / L; T2: 0.5 mmol / L; T3: 1 mmol / L.

[0037] Methyl jasmonic acid treatment: Spray the entire water celery plant that has grown for 35 days with exogenous methyl jasmonic acid, 50 ml per plant, once every other day, for a total of three treatments.

[0038] Treatment time: Spray the entire water celery plant with a spray bottle on a sunny and cool afternoon between 5:00 and 6:00.

[0039] The volatile substances of water celery were determined at 24 h and 48 h after the last treatment.

[0040] The flavor determination of watercress was performed using GC-MS: A total of 5 g of watercress sample was placed in the appropriate extraction flask, and 3-heptanol (Sigma-Aldrich) was added. Volatile compounds were extracted and collected by placing an SPME fiber (65 μm PDMS / DVB) in a headspace at 50 °C for 30 min. The collected volatile compounds were then injected into a GC-MS (DSQ-II Thermo) equipped with a DB-5 mass spectrometry column (30 m × 0.25 mm × 0.25 μm) for further analysis. Helium was used as the carrier gas at a constant flow rate of 0.8 mL / min. The oven temperature was set at 50 °C for 2 min, increased to 75 °C at 5 °C / min and held for 3 min, then increased to 155 °C at 10 °C / min and held for 4 min, and finally increased to 210 °C at 5 °C / min and held for 2 min. Mass spectrometry analysis was performed with an ionization energy of 70 eV and a scan range of 33–350 amu. The volatile compounds in watercress were identified by mass spectrometry comparison with the NIST 05 library. 3-Heptanol was used as an internal standard.

[0041] Example 2

[0042] In Example 1, 19 terpenoid compounds and their contents in watercress were identified using gas chromatography-mass spectrometry with different concentrations of methyl jasmonate. The volatile components and their contents after different treatments are shown in Table 1.

[0043] Table 1. Volatile components and their contents obtained with or without spraying and with different concentrations of methyl jasmonate.

[0044]

[0045] In Table 1, treatments T1, T2, and T3 all increased the content of terpenoids in water celery, with treatment T1 showing the best effect (Table 1). The most abundant flavor compound in water celery is terpinene (…). Figure 1 The content of all terpenoid compounds increased, with terpinene having the highest content. After 24 hours of treatment, the T1 content was 8.44 μg / g, which was 4 times that of the control. After 48 hours of treatment, its content was 42.43 μg / g, which was 20 times that of the control.

[0046] The results of hormone analysis in water celery showed that, compared with the control group (CK), exogenous MeJA treatment significantly increased the contents of MeJA and JA in water celery. Figure 1 Before treatment, the JA content was 11.53 ng / g. After 24 hours of T1 treatment, the JA content was 136.09 ng / g, a 12-fold increase compared to the control (CK); the MeJA content was 29.18 ng / g, a 13-fold increase compared to the CK. After 48 hours of T1 treatment, the JA content was 109.62 ng / g, a 9-fold increase compared to the CK, but the decrease was not significant compared to 24 hours. The MeJA content was 28.61 ng / g, a 13-fold increase.

[0047] As shown in Table 1, spraying methyl jasmonate can increase the content of all types of terpenoids, with a particularly significant increase in the content of terpinene. The total terpenoid content increased to 460.53% after 24 hours of treatment with T2 and to 1975.98% after 48 hours of treatment with T1. The content of terpinene increased to 472.68% and 2187.11% respectively. When the treatment time was 24 hours, the T2 concentration treatment had a significant advantage, while when the treatment temperature was 48 hours, the T1 concentration had a significant advantage.

[0048] If harvesting time is within acceptable limits, treat with T1 concentration for 48 hours. If harvesting time is too tight, the concentration should be slightly increased compared to T1 concentration.

[0049] After 48 hours of treatment, a significant increase in terpinene content was observed in the T1 concentration treatment compared to the T2 and T3 concentration treatments. It is speculated that at other concentrations, methyl jasmonate regulates protease activity, while at the T1 concentration after 48 hours, it regulates at the gene level, promoting gene expression and thus achieving higher terpinene expression levels.

[0050] The above experimental method was used to detect some terpenoid compounds at 10 days and 20 days, and the data obtained are shown in Tables 2 and 3.

[0051] Table 2. Contents of some volatile components obtained after spraying with different concentrations of methyl jasmonate 10 days later.

[0052]

[0053] Table 3. Terpenoid content obtained after spraying with different concentrations of methyl jasmonate 20 days later.

[0054]

[0055] As shown in Tables 2 and 3, our invention, when sprayed with methyl jasmonate, can still achieve a higher content of terpinene compared to other terpenoids at 10 days, which is a relatively long time, and can still maintain a certain content of terpinene at 20 days, which is an even longer time. The solution proposed in this invention has a very significant effect on increasing the content of terpinene.

[0056] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for increasing the content of terpenoid oil in watercress, characterized in that: Methyl jasmonate was sprayed during the growth of water celery. The growth process of the water celery includes the following steps: Planting preparation: Deeply till the soil and apply fertilizer, keeping the soil moist; Seedling selection and planting: Prepare water celery seedlings, keeping them intact when digging them up, and after dividing them, select and plant them in the pond; Water celery growth management: During the growth of water celery, maintain a shallow water layer, remove weeds, and ensure sufficient fertilizer during the growth process; Methyl jasmonic acid treatment: Spray exogenous methyl jasmonic acid on water celery that has grown for a certain number of days; The methyl jasmonate was to be sprayed during a sunny and cool afternoon, between 5:00 and 6:

00. In the methyl jasmonate treatment, the water celery that has grown for a certain number of days is 35 days after transplanting. The methyl jasmonate was sprayed at a rate of 50 mL per water celery plant. The concentration of methyl jasmonate in the methyl jasmonate is 0~3 mmol / L; It also includes the harvesting of water celery, wherein the harvesting time is 24 hours or 48 hours after treatment with methyl jasmonate; When the harvest time is 24 hours, the concentration of methyl jasmonate is 0.5 mmol / L. When the harvest time was 48 hours, the concentration of methyl jasmonate was 0.1 mmol / L.

2. The method for increasing the content of terpene oil in watercress according to claim 1, characterized in that: The methyl jasmonate treatment increased the terpene content in water celery.

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