A method for efficiently separating high-purity conjugated linoleic acid from tetrastigma hemsleyanum

Abstract

The application discloses a method for efficiently separating high-purity conjugated linoleic acid from Tetrastigma hemsleyanum, which takes stems and leaves of Tetrastigma hemsleyanum as raw materials, and sequentially carries out ethanol extraction, ethyl acetate extraction, normal-phase silica gel column chromatography, gel column chromatography and semi-preparative reverse-phase liquid chromatography coupling purification process, so that 10E, 12Z-CLA monomers with chemical purity of greater than or equal to 99% are efficiently prepared. Experiments prove that the compound can significantly inhibit the inflammatory reaction of macrophages induced by lipopolysaccharide, and the mechanism is that the compound down-regulates TLR4 protein expression and inhibits NF-kappa B p65 phosphorylation, so as to block the activation of the TLR4 / NF-kappa B signal pathway. The application realizes high-value utilization of the abandoned resources of Tetrastigma hemsleyanum, and the obtained product can be used as a definite active ingredient for developing anti-inflammatory drugs, functional foods or cosmetics.

Description

Technical Field

[0001] This invention relates to the fields of natural product chemistry and pharmaceutical technology, and in particular to a method for efficiently separating high-purity conjugated linoleic acid from *Trifolium repens*, while also providing corresponding conjugated linoleic acid and its anti-inflammatory applications. Background Technology

[0002] Natural products are a vital resource for the development of innovative drugs and functional ingredients. *Tetrastigma hemsleyanum* Diels et Gilg, a perennial herbaceous vine belonging to the genus *Tetrastigma* in the family Vitaceae, has seen its potential value recognized as a traditional Chinese medicine for clearing heat and detoxifying. However, in the process of modern research and industrial transformation, *Tetrastigma hemsleyanum* still faces a series of intertwined scientific bottlenecks and technological challenges, mainly in the following three aspects: First, there are significant shortcomings in resource utilization, hindering the sustainable development of the industry. Currently, the utilization of *Tripterygium wilfordii* relies almost entirely on its underground tubers, which have a long harvesting cycle and cause severe damage to wild resources. Meanwhile, the vast quantities of above-ground parts (stems and leaves) are discarded as agricultural waste after harvesting, resulting in resource waste and environmental burden. Existing technologies have not yet found a high-value utilization pathway for these "waste parts," which limits the sustainable supply of raw materials and leads to high product costs, becoming a key constraint on the large-scale development of the industry.

[0003] Secondly, research on the material basis and mechanism of action remains in a "black box" state, hindering product quality control and upgrading. Although existing studies have shown that crude extracts of *Tripterygium wilfordii* possess anti-inflammatory activities, current analyses mostly focus on activity evaluation at the level of mixtures such as total flavonoids and total extracts. This research model fails to answer core questions such as "which specific components are at work," "what are their precise chemical structures," and "through which molecular pathways do they exert their effects." Due to the unclear material basis, product quality standards are difficult to establish based on clearly defined active ingredients; the unclear mechanism of action also means that product efficacy lacks modern scientific evidence, thus severely limiting its transformation from traditional medicinal materials to modern standardized and precise products (such as herbal medicines or functional food ingredients with clearly defined chemical compositions).

[0004] Finally, the known sources and preparation methods for active ingredients are limited, lacking innovation and intellectual property advantages. Conjugated linoleic acid (CLA), as a fatty acid known to have anti-inflammatory and other health benefits, currently relies mainly on chemical synthesis or extraction from ruminant animal products and a few oilseed plants. These sources typically yield mixtures of multiple isomers, and isolating and purifying them to obtain specific configurations is costly. More importantly, to date, no research has systematically discovered and isolated high-purity 10E,12Z-CLA monomers from *Trifolium repens*—especially its discarded aerial parts. Developing efficient, renewable processes for preparing high-value compounds with specific configurations from this novel plant source remains a gap in research.

[0005] In summary, the lack of systematic development technology for non-traditional medicinal parts of *Tripterygium wilfordii* limits the discovery channels for high-value active ingredients. Furthermore, the inability to obtain high-purity, clearly defined monomeric compounds hinders in-depth pharmacological mechanism research, which in turn restricts the development of high-end products based on well-defined components and the establishment of intellectual property rights. Therefore, breaking this closed loop, innovatively discovering new sources from waste resources, establishing a technical system for the precise preparation of specific active monomers, and elucidating their mechanisms of action have become urgent tasks for promoting technological breakthroughs and industrial upgrading in this field. Summary of the Invention

[0006] The primary objective of this invention is to solve the following problems existing in the prior art: The above-ground resources of *Trifolium repens* have not been fully utilized. There is a lack of methods for systematically isolating high-purity, specific anti-inflammatory monomeric compounds from *Tripterygium wilfordii*. The precise molecular targets and signaling pathways underlying the anti-inflammatory effects of Tripterygium wilfordii are unclear.

[0007] To this end, this invention provides a targeted extraction and enrichment method for converting above-ground stem and leaf waste of *Trifolium repens* into high-value raw materials, achieving resource utilization. Specifically, it provides a dedicated purification process coupled with multi-mode chromatography, capable of efficiently and reproducibly preparing 10E,12Z-CLA monomers with a chemical purity ≥99% from complex plant matrices, solving the problems of difficult separation and low purity from this new source. The core molecular mechanism of action of 10E,12Z-CLA from this specific source is clarified, providing irrefutable experimental evidence for its precise application as a TLR4 / NF-κB pathway inhibitor, thereby supporting its in-depth development in fields such as new drugs and functional foods.

[0008] Specifically, the present invention adopts the following technical solution: A method for efficiently separating high-purity conjugated linoleic acid from *Trifolium repens* includes the following steps: (1) After drying and pulverizing the aerial parts of Trifolium repens, use an ethanol aqueous solution with a volume fraction of 65-75% at a material-liquid ratio of 1:8-1:12 (g / mL) and stir at 50-60℃ for 2-4 times, each time for 2-4 hours. Combine the extracts and concentrate under reduced pressure until there is no alcohol taste to obtain a total extract rich in polar active ingredients. (2) The total extract was dispersed in water and extracted in a stepwise manner with petroleum ether, ethyl acetate and n-butanol. The ethyl acetate extract was collected, concentrated and dried to obtain the ethyl acetate fraction extract which showed the strongest anti-inflammatory activity in the initial screening. (3) Multimode coupled column chromatography purification: a. Preliminary separation using normal-phase silica gel column chromatography: The ethyl acetate extract was dry-loaded onto 200-300 mesh silica gel as the stationary phase, and eluted using a gradient of petroleum ether-ethyl acetate system. The elution gradient was gradually changed from 20:1 (v / v) to 1:1. The fractions with Rf values ​​between 0.3 and 0.5 (petroleum ether:ethyl acetate = 5:1 as the developing solvent) were monitored by thin-layer chromatography (TLC) and the major fractions were combined to obtain the core anti-inflammatory active component Fr.5. b. Dual purification by gel column molecular sieve and adsorption: The core component Fr.5 was dissolved in methanol and subjected to Sephadex LH-20 gel column chromatography with methanol as the mobile phase for isocratic elution. Fractions with strong UV absorption at 210 nm and showing as a single spot on TLC were collected and combined into the highly active subcomponent Fr.5.1. c. Reversed-phase preparative liquid chromatography purification and target compound capture: The subfraction Fr.5.1 was finally purified by semi-preparative high-performance liquid chromatography. The chromatographic conditions were: C18 reversed-phase column, mobile phase of acetonitrile-water system, isocratic or micro-gradient elution at a volume ratio of 75:25 to 85:15, flow rate of 8-15 mL / min, detection wavelength of 210 nm, and specific collection of chromatographic peaks with retention times in the range of 20.5-21.5 min. After concentration, a colorless oily crude product was obtained. (4) Recycling and Finishing: Dissolve the crude product in methanol again and repeat the reverse-phase preparation liquid phase purification process of step (3)c 1-2 times until the main peak area ratio is ≥99% and there is no interference from other impurity peaks when detected by analytical HPLC (C18 column, acetonitrile:water = 80:20, detection wavelength 210 nm), thus obtaining high-purity 10E,12Z-conjugated linoleic acid.

[0009] Furthermore, in step (1), the volume fraction of the ethanol aqueous solution is preferably 70%, and the extraction is performed 3 times, each time for 3 hours.

[0010] Furthermore, in step (3)a, the specific procedure for gradient elution is as follows: elution is performed in stages using solvent ratios of petroleum ether:ethyl acetate = 20:1, 10:1, 5:1, 2:1, 1:1 (v / v), with each ratio having an elution volume of 3-5 column volumes.

[0011] Furthermore, in step (3)c, the mobile phase is preferably acetonitrile:water = 80:20 (v / v), and isocratic elution is performed, with the retention time of the target compound 10E,12Z-CLA being 20.8-21.2 minutes.

[0012] Furthermore, in step (4), the purity criteria for the analytical HPLC are: at a detection wavelength of 210 nm, the normalized area percentage of the target chromatographic peak is ≥99.0%, and there are no co-eluting impurity absorption peaks in the three-dimensional scan of the full wavelength of 190-400 nm.

[0013] A 10E,12Z-conjugated linoleic acid, prepared by the above method, has a chemical purity ≥99%, and its 1H-NMR spectrum shows characteristic peak intensities at chemical shifts δ5.60 ppm (dd, J=15.4, 7.1 Hz, H-10) and δ5.96 ppm (d, J=11.6 Hz, H-12) conforming to the 10E,12Z configuration.

[0014] A pharmaceutical composition or health food for the prevention or relief of inflammation, comprising an effective dose of 10E,12Z-conjugated linoleic acid as described in claim 6, and pharmaceutically or food-grade excipients.

[0015] Application of 10E,12Z-conjugated linoleic acid in the preparation of formulations for inhibiting the overactivation of the TLR4 / NF-κB signaling pathway in macrophages.

[0016] A method for screening and enriching specific configuration conjugated linoleic acid in plants, characterized in that: using non-traditional medicinal parts of plants as raw materials, after sequentially performing directional extraction in step (1) of claim 1 and enrichment of the ethyl acetate fraction in step (2), a three-step column chromatography coupling technique including normal phase silica gel column (petroleum ether-ethyl acetate gradient), Sephadex LH-20 gel column (methanol) and semi-preparative C18 reversed phase chromatography (acetonitrile-water system) is used to finally collect the target component at a specific retention time window.

[0017] Compared with the prior art, the beneficial effects of the present invention are: (1) For the first time, the above-ground parts (stems and leaves) of *Tripterygium wilfordii* were systematically developed as raw materials for extracting 10E,12Z-CLA, which improved the comprehensive utilization rate of resources and reduced the cost of raw materials; (2) A systematic extraction, extraction and multi-stage column chromatography purification process was established, which can stably obtain 10E,12Z-CLA monomer compounds with a purity of not less than 99%, laying the foundation for quality control; (3) The 10E,12Z-CLA obtained in this invention was confirmed through an in vitro cell model (LPS-induced RAW264.7 macrophages) to have significant anti-inflammatory activity. It can dose-dependently inhibit the secretion of pro-inflammatory factors such as NO, TNF-α, IL-1β, and IL-6, and promote the release of IL-10. Its mechanism of action is to inhibit the expression of TLR4 protein and the phosphorylation of the NF-κB p65 subunit, thereby downregulating the expression of key inflammatory mediators such as iNOS and COX-2. (4) This compound can be used as a clearly defined active ingredient to develop novel natural anti-inflammatory drugs, functional foods that help regulate immunity, or soothing and repairing cosmetics, and has good market potential and development value. Attached Figure Description

[0018] Figure 1 shows the 13C-NMR (A), 1H-NMR (B), HSQC (C), HMBC (D), and COSY (E) spectra of the product of Example 1 of the present invention, the 13C-NMR attribution diagram (F), and the correlation analysis diagram of HMBC and COSY. Detailed Implementation

[0019] The representative embodiments shown in the accompanying drawings will now be further refined. It should be understood that the following description is not intended to limit the embodiments to a single preferred embodiment. Rather, it is intended to cover alternatives, modifications, and equivalents that may be included within the substance and scope of the embodiments defined by the appended claims.

[0020] Example 1: Preparation and structural identification of 10E,12Z-CLA Raw material processing and extraction: Take 5.0 kg of dried above-ground stems and leaves of *Trifolium repens*, pulverize and pass through a 40-mesh sieve. Use 70% ethanol solution at a material-to-liquid ratio of 1:10, and extract three times at 55℃ with stirring, each time for 3 hours. Combine the extracts, recover the ethanol under reduced pressure at 50℃, and concentrate to obtain the total extract.

[0021] Solvent extraction enrichment: The total extract was dispersed in 2L of water and extracted three times each with equal volumes of petroleum ether, ethyl acetate, and n-butanol. The ethyl acetate phase was collected, concentrated under reduced pressure, and freeze-dried to obtain 85g of the yellow powdery ethyl acetate fraction.

[0022] Silica gel column chromatography (preliminary separation): 60g of the ethyl acetate fraction was mixed with 120g of crude silica gel (100-200 mesh) and dry-loaded onto a glass column (Φ6 cm × L 60 cm) packed with 1.2kg of 200-300 mesh silica gel. Elution was performed using a gradient of petroleum ether-ethyl acetate mixed solvent (20:1→10:1→5:1→2:1→1:1, v / v), with approximately 3 column volumes eluted per gradient. Thin-layer chromatography (TLC) was used for monitoring. Fractions with Rf≈0.4 (petroleum ether:ethyl acetate = 5:1 development) were combined to obtain fraction Fr.5 (2.6g).

[0023] Gel column chromatography (purification and concentration): Dissolve 800 mg Fr.5 in a small amount of methanol and load the solution onto a methanol-equilibrated Sephadex LH-20 column (Φ3.2 cm × L 45 cm). Use methanol as the mobile phase and elute isocratically. Collect fractions that show strong absorption at 210 nm and a single main spot on TLC, and combine them to obtain Fr.5.1 (485 mg).

[0024] Semi-preparative HPLC purification (target capture): 45 mg of Fr. 5.1 was dissolved in methanol and filtered through a 0.22 μm filter membrane. A semi-preparative HPLC system (C18 column, 5 μm, 10 × 250 mm) was used with acetonitrile-water (80:20, v / v) as the mobile phase, isocratic elution, a flow rate of 12 mL / min, and a detection wavelength of 210 nm. Chromatographic peaks with retention times in the range of 20.8–21.2 min were accurately collected, injected repeatedly, and combined. Acetonitrile was removed by rotary evaporation under reduced pressure, followed by lyophilization to obtain the crude product.

[0025] Cyclic purification and purity determination: The crude product was purified again under the conditions described in step 5. The final product was analyzed by analytical HPLC (C18 column, acetonitrile-water = 80:20, 1.0 mL / min, 210 nm), showing a single chromatographic peak with a normalized area percentage of 99.3%. The product was a colorless oily substance, weighing 6.5 mg.

[0026] The following is the specific structural identification: Nuclear magnetic resonance (NMR) analysis was performed on the product. Compound 1 is a colorless oil, as can be seen from its 1D NMR and 2D NMR spectra (…). Figure 1 The molecular formula of the product is C 18 H 32O3. 13C-NMR (150 MHz, CD3OD) chemical shifts (δ): 178.5 (C-1), 35.6 (C-2), 26.3 (C-3), 30.7 (C-4), 30.6 (C-5), 30.5 (C-6), 26.5 (C-7), 38.4 (C-8), 73.4 (C-9), 137.4 (C-10), 126.5 (C-11), 129.3 (C-12), 132.9 (C-13), 28.6 (C-14), 30.3 (C15), 32.6 (C-16), 23.6 (C-17), 14.4 (C-18). 1H-NMR (600 MHz, CD3OD) data are shown in Table 2-3. The above data is basically consistent with the published research results, so the product was identified as 10E,12Z-linoleic acid (10E,12Z-CLA).

[0027] Specifically, the 1H-NMR (600 MHz, CD3OD) spectrum showed key characteristic peaks: δ 5.60 (dd, J = 15.4, 7.1 Hz, 1H, H-10) and δ 5.96 (d, J = 11.6 Hz, 1H, H-12). Combined with 13C-NMR and two-dimensional spectral data, the compound was confirmed to be 10E,12Z-conjugated linoleic acid.

[0028] Table of 1H NMR spectrum analysis of the product Anti-inflammatory activity and mechanism of 10E,12Z-CLA (in vitro cell experiments) Cell viability assay: The effect of 10E,12Z-CLA (0-25 μg / mL) on the viability of mouse macrophages RAW264.7 was detected by CCK-8 assay. The results showed that the cell viability was 102.3%, 96.8%, and 85.8% at concentrations of 5, 10, and 20 μg / mL, respectively, indicating no significant toxicity at concentrations ≤20 μg / mL.

[0029] The cytotoxicity of 10E,12Z-CLA (0-25 μg / mL) on RAW264.7 cells was evaluated using the CCK-8 assay. Solvent-treated cells served as a control (set at 100% viability). Results showed that the metabolic activity of cells treated with 5 μg / mL and 10 μg / mL was comparable to the control group (P>0.05), while the cell viability of the 20 μg / mL treatment group remained above 85%. This indicates that 10E,12Z-CLA has no significant inhibitory effect on cell growth at concentrations ≤20 μg / mL, i.e., no significant cytotoxicity. This safe concentration range will be used in subsequent anti-inflammatory experiments.

[0030] Anti-inflammatory phenotype validation: NO inhibition: An LPS (1 μg / mL) inflammation model was established. Compared with the LPS group, pretreatment with 10E,12Z-CLA (5, 10, 20 μg / mL) dose-dependently inhibited NO secretion, with inhibition rates of 35.2%, 68.7%, and 92.9%, respectively (P<0.01 for the 20 μg / mL group).

[0031] Cytokine regulation: ELISA showed that 10E,12Z-CLA (10 μg / mL) significantly reduced the levels of TNF-α, IL-1β, and IL-6 in the supernatant (by about 40-60%), while increasing the secretion of the anti-inflammatory factor IL-10 by about 1.8 times.

[0032] Gene expression: RT-qPCR showed that 10E,12Z-CLA can downregulate the mRNA expression of pro-inflammatory genes such as iNOS, COX-2, and IL-6 in a dose-dependent manner.

[0033] Molecular mechanisms elucidated: Western blot analysis showed that LPS stimulation significantly upregulated TLR4 protein expression and induced p65 phosphorylation in RAW264.7 cells. Intervention with 10E,12Z-CLA (10, 20 μg / mL) significantly reversed this effect, dose-dependently reducing TLR4 protein levels and significantly inhibiting p65 phosphorylation. Simultaneously, the protein expression of its downstream effector molecules iNOS and COX-2 also decreased synchronously. These results directly demonstrate that 10E,12Z-CLA exerts its anti-inflammatory effect by inhibiting the TLR4 / NF-κB signaling pathway.

[0034] Preparation of 10E,12Z-CLA tablets: Take 10g of 10E,12Z-CLA prepared in Example 1, add 150g of microcrystalline cellulose, 80g of corn starch, 20g of crospovidone, and 3g of magnesium stearate, and mix well. Perform wet granulation, dry, and then compress into tablets to produce 1000 oral tablets, each containing 50mg of 10E,12Z-CLA. This tablet can be used as an adjunct to improve chronic inflammatory conditions.

[0035] Comparative Example 1: Comparison of conventional plant oil extraction methods: The total oil was obtained by directly extracting the aerial parts of *Trifolium repens* powder with n-hexane using Soxhlet extraction. After methylation, GC-MS analysis was performed. The results showed that the total oil contained low levels of CLA compounds (<0.5%), and consisted of a mixture of multiple isomers such as 9c,11t-CLA, 10t,12c-CLA, and 10t,12t-CLA, making it impossible to obtain a single 10E,12Z-CLA monomer. This comparison highlights the necessity and inventive advantage of the multi-step coupled chromatographic process of this invention in obtaining high-purity monomers with specific configurations.

[0036] It will be apparent to those skilled in the art that modifications, combinations, and variations can be made to the teachings described above.

Claims

1. A method for efficiently separating high-purity conjugated linoleic acid from Tetrastigma hemsleyanum Diels et Lu, characterized in that: Includes the following steps: ​ (1) After drying and pulverizing the aerial parts of Trifolium repens, use an ethanol aqueous solution with a volume fraction of 65-75% at a material-liquid ratio of 1:8-1:12 (g / mL) and stir at 50-60℃ for 2-4 times, each time for 2-4 hours. Combine the extracts and concentrate under reduced pressure until there is no alcohol taste to obtain a total extract rich in polar active ingredients. (2) The total extract was dispersed in water and extracted in a stepwise manner with petroleum ether, ethyl acetate and n-butanol. The ethyl acetate extract was collected, concentrated and dried to obtain the ethyl acetate fraction extract which showed the strongest anti-inflammatory activity in the initial screening. (3) Multimode coupled column chromatography purification: a. Preliminary separation using normal-phase silica gel column chromatography: The ethyl acetate extract was loaded onto a dry plate using 200-300 mesh silica gel as the stationary phase. Gradient elution was performed using a petroleum ether-ethyl acetate system, with the elution gradient gradually changing from 20:1 (v / v) to 1:

1. Thin-layer chromatography monitoring was used to combine the major fractions with Rf values ​​between 0.3 and 0.5 to obtain the core anti-inflammatory active component Fr.

5. b. Dual purification by gel column molecular sieve and adsorption: The core component Fr.5 was dissolved in methanol and subjected to Sephadex LH-20 gel column chromatography with methanol as the mobile phase for isocratic elution. Fractions with strong UV absorption at 210 nm and showing as a single spot on TLC were collected and combined into the highly active subcomponent Fr.5.

1. c. Reversed-phase preparative liquid chromatography purification and target compound capture: The subfraction Fr.5.1 was finally purified by semi-preparative high-performance liquid chromatography. The chromatographic conditions were: C18 reversed-phase column, mobile phase of acetonitrile-water system, isocratic or micro-gradient elution at a volume ratio of 75:25 to 85:15, flow rate of 8-15 mL / min, detection wavelength of 210 nm, and specific collection of chromatographic peaks with retention times in the range of 20.5-21.5 min. After concentration, a colorless oily crude product was obtained. (4) Recycling and finishing: Dissolve the crude product in methanol again and repeat the reverse-phase preparation liquid phase purification process of step (3)c 1-2 times until the main peak area ratio is ≥99% and there are no other impurity peaks to interfere with it, thus obtaining high-purity 10E,12Z-conjugated linoleic acid.

2. The method of claim 1, wherein: In step (1), the volume fraction of the ethanol aqueous solution is preferably 70%, and the extraction is performed 3 times, each time for 3 hours.

3. The method of claim 1, wherein: In step (3)a, the specific procedure for gradient elution is as follows: elution is carried out in stages using solvent ratios of petroleum ether:ethyl acetate = 20:1, 10:1, 5:1, 2:1, 1:1 (v / v), with each ratio having an elution volume of 3-5 column volumes.

4. The method of claim 1, wherein: In step (3)c, the mobile phase is preferably acetonitrile:water = 80:20 (v / v), and isocratic elution is performed. The retention time of the target compound 10E,12Z-CLA is 20.8-21.2 minutes.

5. The method of claim 1, wherein: In step (4), the purity criteria for the analytical HPLC are: at a detection wavelength of 210 nm, the normalized area percentage of the target chromatographic peak is ≥99.0%, and there are no co-eluting impurity absorption peaks in the three-dimensional scan of the full wavelength of 190-400 nm.

6. A conjugated linoleic acid characterized by: Prepared by the method described in any one of claims 1-5, the chemical purity is ≥99%, and the characteristic peak intensity ratio at chemical shifts δ5.60 ppm (dd, J=15.4, 7.1 Hz, H-10) and δ5.96 ppm (d, J=11.6 Hz, H-12) in its 1H-NMR spectrum conforms to the 10E,12Z configuration.

7. A pharmaceutical composition or health food for preventing or relieving inflammation, characterized by: It includes an effective dose of the 10E,12Z-conjugated linoleic acid of claim 6, and pharmaceutically or food-grade excipients.

8. The use of the 10E,12Z-conjugated linoleic acid of claim 6 in the preparation of a formulation for inhibiting the overactivation of the TLR4 / NF-κB signaling pathway in macrophages.

9. A method for screening and enriching specific configurational conjugated linoleic acid in plants, characterized in that: Using non-traditional medicinal parts of plants as raw materials, the target components are collected by sequentially performing directional extraction in step (1) and enrichment of the ethyl acetate fraction in step (2) as described in claim 1, and then using a three-step column chromatography coupling technique including normal-phase silica gel column (petroleum ether-ethyl acetate gradient), Sephadex LH-20 gel column (methanol) and semi-preparative C18 reversed-phase chromatography (acetonitrile-water system) to collect the target components within a specific retention time window.

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