Tea saponin E3, its isolation and purification methods, and its application as a polyp killer in moon jellyfish.

Abstract

This invention discloses tea saponin E3, its isolation and purification method, and its application as a killer of moon jellyfish polyps, belonging to the field of natural product chemistry. The structure of tea saponin E3 is shown below. Derived from plants, it is a natural triterpenoid saponin compound. Compared with tea saponin, it exhibits stronger killing activity against moon jellyfish polyps. Furthermore, its single component and well-defined structure facilitate standardized application and evaluation of potential ecological risks in the biocontrol of moon jellyfish polyps.

Description

Technical Field

[0001] This invention relates to a saponin compound, its separation and purification method, and its application, specifically to tea saponin E3, its separation and purification method, and its application as a polyp killer of moon jellyfish, belonging to the field of natural medicinal chemistry technology. Background Technology

[0002] Moon jellyfish outbreaks have become a significant ecological disaster threatening my country's nearshore marine ecosystems, easily triggering serious socio-economic problems. Moon jellyfish are dioecious and have an extremely complex life cycle; the mortality rate and reproductive rate during the polyp stage are key factors determining the adult population size. Therefore, effectively controlling the population size and reproductive capacity of moon jellyfish polyps is crucial for preventing jellyfish outbreaks.

[0003] Plant-derived pesticides are a class of biological pesticides developed based on natural plant metabolites. Due to their eco-friendliness and specificity, they have been widely used for pest control. Studies have found that tea saponins extracted from camellia seed meal can effectively inhibit the growth and development of the polyps of *Meretrix meretrix*, showing great promise in the development of biological pesticides targeting this organism. Tea saponins are natural nonionic surfactants with excellent emulsifying, foaming, and dispersing properties. Their main components are oleanane-type pentacyclic triterpenoid saponins, composed of hydrophobic aglycones, hydrophilic sugars, and organic acids. In addition, tea saponins typically contain other components such as tea seed polysaccharides and flavonoids. However, the main monomer structure and mechanism of action that have a killing effect on *Meretrix meretrix* remain unclear. Meanwhile, due to factors such as the source of raw materials, pretreatment methods, and extraction methods, the proportions of various components in tea saponins vary greatly between different batches. This problem greatly limits the standardized application of tea saponin-related products in the field of biological control of moon jellyfish polyps and the assessment of their potential ecological risks. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide a tea saponin monomer compound with a single component, a well-defined structure, and a killing effect on the polyps of the moon jellyfish, as well as a method for separating and purifying the tea saponin monomer compound and its applications.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] Tea saponin E3 has the following structure:

[0007] .

[0008] The aforementioned method for separating and purifying tea saponin E3 includes the following steps:

[0009] (1) Crush the camellia seed meal, extract it with an ethanol solution with a volume concentration of 80%, concentrate the extract under reduced pressure to obtain crude extract paste;

[0010] (2) The crude extract was fractionally extracted using petroleum ether, ethyl acetate and n-butanol. The extract was concentrated under reduced pressure to obtain petroleum ether fraction, ethyl acetate fraction and n-butanol fraction, respectively.

[0011] (3) Take n-butanol extract and dissolve it in methanol. Pack the column dry and load the sample dry. Use dichloromethane and methanol as eluents for gradient elution. The volume ratios of dichloromethane and methanol are 20:1, 15:1 and 10:1, respectively. Elute with each eluent gradient for 3 column volumes to obtain eluent A, eluent B and eluent C, respectively.

[0012] (4) Eluent C was concentrated under reduced pressure and then separated and purified by an ODS column. The sample was loaded by wet method and eluted with methanol and water as eluents in a gradient. The volume ratios of methanol and water were 1:1, 2:1, 3:1 and 4:1, respectively. Each eluent was used to elute 3 times the column volume to obtain eluent I, eluent II, eluent III and eluent IV.

[0013] (5) Concentrate the eluent IV under reduced pressure, then separate and purify it using a gel chromatography column, load the sample by wet method, elute with 3 column volumes of methanol, collect one tube of eluent every 5 min, determine the components of the eluent by thin-layer chromatography, combine the eluents with the same components, allow the solvent to evaporate naturally, and obtain tea saponin E3.

[0014] Preferably, in step (1), the extraction is carried out at room temperature for 48 hours.

[0015] Preferably, in step (3), 200-mesh silica gel is used for dry packing.

[0016] Preferably, in step (4), the ODS column is filled with 50 μm reversed-phase silica gel filler.

[0017] Preferably, in step (5), the gel chromatography column is filled with Sephadex LH-20 gel.

[0018] The aforementioned application of tea saponin E3 in the preparation of a polyp killer for moon jellyfish.

[0019] The advantages of this invention are:

[0020] (1) The tea saponin E3 provided by the present invention is derived from plants and is a natural triterpenoid saponin compound. Compared with tea saponin, it has stronger killing activity against the polyps of the moon jellyfish. Its mechanism of action is clear, and its components are simple and its structure is clear, which is beneficial for its standardized application and evaluation of potential ecological risks in the field of biological control of the polyps of the moon jellyfish.

[0021] (2) The method for separating and purifying tea saponin E3 provided by the present invention is simple to operate, low in cost, and has good separation and purification effect, making it suitable for large-scale batch production. Attached Figure Description

[0022] Figure 1 This is a single-crystal ellipsoid diagram of tea saponin E3, in which the white ellipsoid represents hydrogen atoms, the black ellipsoid represents carbon atoms, and the red ellipsoid represents oxygen atoms;

[0023] Figure 2 These are the HPLC-MS spectra of tea saponin E3, where A is the ultra-high performance liquid chromatogram of tea saponin E3 and B is the mass spectrum of tea saponin E3.

[0024] Figure 3 This is a statistical chart showing the mortality rate of moon jellyfish polyps after treatment with different concentrations of tea saponin E3;

[0025] Figure 4 This is a regression curve of toxicity of moon jellyfish polyps after treatment with tea saponin E3 for 48 h and 96 h.

[0026] Figure 5 These are the staining results of tissue sections of the polyps of the control group and the 3μM tea saponin E3 treatment group. Among them, A and C are the staining results of tissue sections of the polyps of the control group, and B and D are the staining results of tissue sections of the polyps of the 3μM tea saponin E3 treatment group.

[0027] Figure 6 This is a graph showing the transcriptomic analysis results of the polyps of the moon jellyfish in the control group and the 3μM tea saponin E3 treatment group;

[0028] Figure 7 This is a graph showing the results of KEGG pathway enrichment analysis of genes expressing significant differences. Detailed Implementation

[0029] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0030] I. Structure of tea saponin monomer compounds

[0031] The structure of the tea saponin monomer compound (tea saponin E3) provided by this invention is shown below:

[0032] .

[0033] II. Isolation and purification of tea saponin E3

[0034] The tea saponin E3 provided by this invention is isolated from camellia seed meal, and the specific isolation and purification method is as follows:

[0035] (1) The camellia seed meal was crushed and extracted with an 80% ethanol solution at room temperature for 48 hours. The extract was concentrated under reduced pressure to obtain crude extract.

[0036] (2) The crude extract was fractionally extracted using petroleum ether, ethyl acetate and n-butanol. The extract was concentrated under reduced pressure to obtain petroleum ether fraction, ethyl acetate fraction and n-butanol fraction, respectively.

[0037] (3) Take 200g of n-butanol extract and dissolve it in methanol. Mix it with an equal mass of 200-mesh silica gel. Pack the same 200-mesh silica gel into a column by dry loading. Use dichloromethane and methanol as eluents for gradient elution. The volume ratios of dichloromethane and methanol are 20:1, 15:1 and 10:1, respectively. Elute with each eluent gradient for 3 times the column volume to obtain eluent A, eluent B and eluent C, respectively.

[0038] (4) Eluent C (obtained in an elution system with a volume ratio of dichloromethane to methanol of 10:1) was concentrated under reduced pressure and then separated and purified using an ODS column (filled with 50 μm reversed-phase silica gel). The sample was loaded by wet method and eluted with methanol and water as eluents in gradients. The volume ratios of methanol and water were 1:1, 2:1, 3:1 and 4:1, respectively. Each eluent gradient was used to elute 3 times the column volume to obtain eluent I, eluent II, eluent III and eluent IV, respectively.

[0039] (5) Eluent IV (obtained in an elution system with a methanol and water volume ratio of 4:1) was concentrated under reduced pressure and then separated and purified by a gel chromatography column (filled with Sephadex LH-20 gel). The sample was loaded by wet method and eluted with methanol (3 column volumes). One tube of eluent was collected every 5 min. The composition of the eluent was determined by thin-layer chromatography. Eluents with the same composition were combined and the solvent was allowed to evaporate naturally at 25°C to obtain colorless crystals.

[0040] The above-mentioned colorless crystals with intact morphology were selected and subjected to single-crystal X-ray diffraction (SC-XRD) to determine the structure of the crystal. The resulting single-crystal ellipsoid diagram is shown in [Figure number missing]. Figure 1 .

[0041] Additionally, approximately 100 μg of the above-mentioned colorless crystals with intact morphology were dissolved in 1 mL of methanol-water solution (v:v = 1:9), and the molecular weight of the crystals was analyzed using ultra-high performance liquid chromatography-mass spectrometry (HPLC-MS / MS). The HPLC-MS spectrum of the crystals is shown below. Figure 2 .

[0042] Depend on Figure 1 and Figure 2 It can be seen that the colorless crystals prepared above, in HPLC-MS negative ion mode (ESI), -The molecular weight is 1188, and based on database information, its molecular formula is predicted to be C. 57 H 88 O 26 Its structure contains a pentacyclic triterpenoid aglycone and a sugar chain composed of glucuronic acid, arabinose, xylose and galactose. Based on the above, the crystal is identified as tea saponin E3.

[0043] III. Cultivation of Moon Jellyfish Polyps

[0044] The moon jellyfish polyps used in the experiment were cultured in the laboratory of the Muping Integrated Environmental Experiment Station, Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences. The polyps were attached to 5mL PET bottles, which were then placed in an incubator at a temperature of 18.5±0.3℃ with a light:dark ratio of 12h:12h. The seawater used was natural seawater that had undergone sand filtration and UV treatment, with a pH of 8.12±0.03 and a salinity of 31.4±0.4. During the laboratory culture period, the nauplii of Artemia were fed every two days, and the culture seawater was replaced.

[0045] IV. Experiment on the killing of moon jellyfish polyps by tea saponin E3

[0046] The experiment consisted of 6 treatment groups, including 5 tea saponin E3 concentration levels (1μM, 2μM, 3μM, 4μM, 5μM) and a control group, with 3 replicates for each treatment group.

[0047] 252 moon jellyfish polyps with the same developmental stage were randomly divided into groups, with 14 moon jellyfish polyps cultured in each parallel.

[0048] A 1 mM tea saponin E3 stock solution was prepared, and the corresponding dose of tea saponin E3 stock solution was added to each PET bottle with attached polyps to achieve the preset concentration of tea saponin E3 in each treatment group. After 48 h and 96 h of treatment, the number of dead polyps was observed and recorded. The absence of response to acupuncture was used as the criterion for determining death. The mortality rate was statistically analyzed, and the toxicity regression curve was plotted and the median lethal concentration (LC50) was calculated using probability analysis.

[0049] The mortality rates of moon jellyfish polyps treated with different concentrations of tea saponin E3 for 48 h and 96 h are shown in the table below. Figure 3 .

[0050] Depend on Figure 3 It can be seen that after treatment with tea saponin E3 for 48 hours, 4 μM tea saponin E3 can kill 71.4% of the polyps of the moon jellyfish, and 5 μM tea saponin E3 can kill all the polyps of the moon jellyfish; after treatment with tea saponin E3 for 96 hours, 4 μM tea saponin E3 can kill 88.1% of the polyps of the moon jellyfish.

[0051] The toxicity regression curves of moon jellyfish polyps treated with tea saponin E3 for 48 h and 96 h are shown in the figure. Figure 4 .

[0052] Depend on Figure 4 Calculations show that: when tea saponin E3 was treated with moon jellyfish polyps for 48 h, the median lethal concentration (LC50) was 2.47 μM, with a 95% confidence interval of [1.97, 3.02]; when tea saponin E3 was treated with moon jellyfish polyps for 96 h, the median lethal concentration was 1.98 μM, with a 95% confidence interval of [1.69, 2.26].

[0053] The above results show that tea saponin E3 has strong killing activity against moon jellyfish polyps, and it can be identified as the main component of tea saponin with killing activity against moon jellyfish polyps. It can be applied to the standardized application of tea saponin-related products in the field of biological control of moon jellyfish polyps and the study of the killing mechanism.

[0054] V. Observation of tissue sections of the polyps of *Lycoris radiata* after treatment with tea saponin E3

[0055] After 48 hours of treatment with tea saponin E3, three polyps of *Meretrix meretrix* were collected from both the control group and the 3 μM tea saponin E3 treatment group. The polyps were fixed in Bonn's solution for 12 hours. After fixation, the polyps were transferred to a 70% ethanol solution. A semi-automatic benchtop tissue dehydrator was used for dehydration, clearing, and paraffin embedding. The samples were then embedded using a heated paraffin embedding system and sectioned using a fully automated tissue sectioner. After hematoxylin-eosin staining, the tissue sections were examined under a microscope (magnification 400×).

[0056] The staining results of tissue sections of the polyps of the moon jellyfish in the control group and the 3μM tea saponin E3 treatment group are shown in the figure. Figure 5 .

[0057] Depend on Figure 5 It can be seen that: the control group of moon jellyfish polyps consists of two parts, ectoderm and endoderm, which are separated by mesoglea. The cells are arranged in a tight and orderly manner, and the cell nuclei are clear. Compared with the control group, the endoderm of moon jellyfish polyps treated with 3μM tea saponin E3 showed edema, vacuoles of different sizes appeared in the endoderm cells, the cells were arranged in a disordered manner, and the tissue structure was severely damaged.

[0058] The above results indicate that exposure to 3 μM tea saponin E3 can cause severe physiological and morphological damage to the polyp epithelial cells of the moon jellyfish.

[0059] VI. Transcriptomic analysis of the polyps of *Lycoris radiata* treated with tea saponin E3.

[0060] After 48 hours of treatment with tea saponin E3, total RNA was extracted from the polyps of *Jellyfish* in both the control and 3 μM tea saponin E3-treated groups using the Trizol method. After confirming the quality of the total RNA, cDNA libraries were constructed, and high-throughput transcriptome sequencing was performed on the Illumina platform. Sequences were assembled and compared with the *Jellyfish* reference genome to obtain transcriptome annotation information and expression matrices. Based on the significance and fold change of gene expression levels, differentially expressed genes between the 3 μM tea saponin E3-treated and control groups were screened, and KEGG pathway enrichment analysis was performed on genes with significant differences in expression.

[0061] Transcriptomic analysis results of the control group and the 3μM tea saponin E3 treatment group of moon jellyfish polyps are shown in the figure. Figure 6 .

[0062] Depend on Figure 6 It was found that, compared with the control group, the expression levels of 270 genes and 230 genes in the polyp of the moon jellyfish were significantly upregulated in the 3μM tea saponin E3 treatment group.

[0063] The results of the KEGG pathway enrichment analysis of genes with significant differences in expression are shown below. Figure 7 .

[0064] Depend on Figure 7 It can be seen that exposure to tea saponin E3 significantly affects multiple programmed cell death-related signaling pathways, such as the p53 signaling pathway, which is involved in cell necrosis and apoptosis in the polyps of the moon jellyfish.

[0065] The above results indicate that tea saponin E3 can act as a cellular stressor, disrupting the cellular homeostasis of the moon jellyfish polyp, thereby leading to reduced polyp vitality, inhibited growth, and even death.

[0066] In summary, tea saponin E3 can disrupt the tissue structure of moon jellyfish polyps, interfere with key programmed cell death signaling pathways, trigger cellular stress responses, and lead to abnormal physiological functions or even death. Therefore, tea saponin E3 has a strong killing effect on moon jellyfish polyps and can effectively control their population size. As a natural plant-derived insecticidal compound, tea saponin E3 can effectively prevent moon jellyfish outbreaks and has great potential as a green pesticide.

[0067] It should be noted that the above embodiments are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art can make other variations or modifications based on the above description. It is impossible to exhaustively list all possible implementations here. All obvious variations or modifications derived from the technical solutions of this invention are still within the scope of protection of this invention.

Claims

1. Application of tea saponin E3 in the preparation of a polyp killer for moon jellyfish, wherein the structure of tea saponin E3 is shown below: 。

Images