A method for simultaneously separating and purifying Tellimagrandin I and II from the shell of Camellia oleifera fruits
Through solvent extraction and multi-step extraction technology, Tellimlegrandin I and II were isolated and purified from the oleifera fruit shell, solving the problem of unutilized oleifera fruit shell resources and achieving efficient and environmentally friendly separation effect.
Patent Information
- Application Number
- CN202211562149.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-12-07
- Publication Date
- 2025-06-17
- Estimated Expiration
- 2042-12-07
AI Technical Summary
The prior art fails to effectively utilize the hydrolyzable tannins in the oleifera fruit shell, resulting in the underdevelopment and utilization of its resources, and the discarding or combustion of the oleifera fruit shell causes pollution to the environment.
Tellimgrandin I and II were simultaneously isolated and purified from the oleifera fruit shell by solvent extraction, alkalization, acidification, extraction agent extraction and activated carbon decolorization to achieve high purity and high yield separation.
It has achieved efficient and simple separation and purification of Tellimgrandin I and II from the oil tea fruit shell, with a purity of more than 98%, and a yield of more than 0.5%. It is suitable for large-scale production and reduces environmental pollution.
Smart Images
Figure CN115850352B_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of extraction of effective active ingredients from plants, and particularly relates to a method for simultaneously separating and purifying Tellimagrandin I and II from camellia oleifera fruit shells. Background Art
[0002] Camellia oleifera Abel Camellia oleifera Abel. is a unique woody oil-bearing tree species in China and is also one of the world's four major woody oil sources, along with oil palm, olive, and coconut. Camellia oleifera has a long cultivation history in China. The fruit of Camellia oleifera is called tea fruit, which consists of three parts from outside to inside: camellia oleifera fruit shell, camellia oleifera seed shell, and camellia oleifera seed. It is mainly concentrated in southern regions of China such as Guangxi, Hunan, and Jiangxi provinces (autonomous regions). Previous bioactivity studies have revealed that the whole plant of Camellia oleifera has significant neuroprotective, anti-inflammatory, antibacterial, anthelmintic, antitussive, antioxidant, and anticancer activities. The camellia oleifera fruit shell is a residue produced during the processing of Camellia oleifera, but it has not been fully developed and utilized at present. However, most of the camellia oleifera fruit shells are discarded or burned, which will pollute the air, water bodies, and the ecosystem, and the saponin in them even causes water bodies to foam and produce toxicity. Some studies have shown that the camellia oleifera fruit shell contains a certain amount of natural antioxidants, with a content of 1.21%. Thin layer chromatography and HPLC-Uv analysis show that its components are natural gallic acid, catechin substances, tea saponin, and flavonoid compounds, and the total flavonoid content in the camellia oleifera fruit shell is between 4.95% and 6.84%.
[0003] Tannic acid, also known as tannin, tannin, and tannic acid, is a plant secondary metabolite and is widely present in plants such as Chinese gall, chestnut wood, tara, sorghum, and coffee. Since tannic acid can combine with proteins, polysaccharides, metal ions, etc. to form precipitates, reducing the digestion and absorption of feed nutrients by animal bodies, it was once considered an antinutritional factor. However, more and more studies have found that tannic acid has good functions such as anti-diarrhea, antibacterial, and antioxidant, and has received extensive attention in animal nutrition. Although the chemical composition of the camellia oleifera fruit shell has been studied in depth at present, there is no report on the extraction and purification of hydrolysable tannins in the camellia oleifera fruit shell. Summary of the Invention
[0004] Aiming at the raw material of agricultural and forestry waste, camellia oleifera fruit shell, the present invention provides a method for simultaneously separating and purifying Tellimagrandin I and II. This method has convenient process operation, high product content, high recovery rate, and the solvents used have little harm, and is suitable for large-scale production operation.
[0005] To achieve the above object, the technical solution adopted by the present invention is as follows: A method for simultaneously separating and purifying Tellimagrandin I and II from Camellia oleifera fruit shells. The Camellia oleifera fruit shell raw material is crushed and extracted with an organic solvent. After the first extract is filtered and concentrated, the concentrated solution is alkalized, heated, allowed to stand, and then filtered to obtain the second extract. After the second extract is acidified, it is extracted with an extractant. After the extract is concentrated, a mixture containing low contents of Tellimagrandin I and II is obtained. The mixture containing low contents of Tellimagrandin I and II is decolorized and eluted with solvents in different ratios to obtain compounds with high contents of Tellimagrandin I and II. The obtained Tellimagrandin I and II have a purity greater than 98% and a yield greater than 0.5%.
[0006] The structural formulas of the obtained Tellimagrandin I and II are as follows:
[0007]
[0008] Further preferably, the organic solvent is an ethanol aqueous solution with a volume percentage concentration of 30% - 90%. The extraction process is as follows: The material-liquid ratio is 1:10 - 30 g / mL, the extraction temperature is 60 - 80 °C, and extraction is carried out with heat reflux and stirring, and then filtered to obtain the first extract.
[0009] Further preferably, the first extract is concentrated until there is no alcohol smell, and a sodium hydroxide solution with a mass fraction of 5% is added to the concentrated solution for alkalization, adjusting the pH value of the concentrated solution to 8 - 10, and heating to a temperature of 78 °C to obtain the second extract.
[0010] Further preferably, a hydrochloric acid solution with a mass fraction of 5% is added to the extract for acidification, adjusting the pH value of the second extract to 3 - 6.
[0011] Further preferably, extraction is carried out using an extractant, and the number of extraction times is 3 - 5 times, and it is concentrated under reduced pressure to obtain an extract after extraction.
[0012] Further preferably, the organic solvent used as the extractant is: saturated n-butanol, ethyl acetate, dichloromethane, petroleum ether, n-hexane or n-propanol. After the extract is concentrated, a mixture containing low contents of Tellimagrandin I and II is obtained.
[0013] Further preferably, the mixture containing low contents of Tellimagrandin I and II is redissolved in hot water, activated carbon is added, and after heating and stirring, it is first eluted with 0 - 50% ethanol to obtain Tellimagrandin I, and eluted with 50 - 100% ethanol to obtain Tellimagrandin II.
[0014] Due to the above-described technical solution, the present invention has the following advantages:
[0015] By using methods such as solvent extraction, alkali dissolution and acid precipitation of the extract, solvent extraction of the acid solution, and stepwise elution of activated carbon decolorization, Tellimagrandin I and II are rapidly separated and purified from camellia oleifera fruit shells simultaneously. On the premise of ensuring purity and yield, this process is simple, efficient, requires less investment, and is suitable for large-scale production. Description of the Drawings
[0016] Figure 1 It is the mass spectrum of Tellimagrandin I obtained in Example 1.
[0017] Figure 2 It is the mass spectrum of Tellimagrandin II obtained in Example 1.
[0018] Figure 3 It is the hydrogen spectrum of Tellimagrandin I obtained in Example 1.
[0019] Figure 4 It is the hydrogen spectrum of Tellimagrandin II obtained in Example 1.
[0020] Figure 5 It is the carbon spectrum of Tellimagrandin I obtained in Example 1.
[0021] Figure 6 It is the carbon spectrum of Tellimagrandin II obtained in Example 1. Detailed Description of the Invention
[0022] The present invention can be further described in detail with reference to the following examples; however, the following examples are merely illustrative, and the present invention is not limited to these examples.
[0023] A method for simultaneously separating and purifying Tellimagrandin I and II from camellia oleifera fruit shells is as follows:
[0024] (1) The camellia oleifera fruit shell raw material is dried and pulverized, and an ethanol aqueous solution with a volume percentage concentration of 30%-90% is added according to a solid-liquid ratio of 1:10-30 g / mL, and heat reflux stirring extraction is carried out at an extraction temperature of 60-80 °C for 60 minutes each time, and then filtered to obtain the first extract.
[0025] (2) The first extract is concentrated until there is no alcohol smell, and a sodium hydroxide solution with a mass fraction of 5% is added to the concentrated solution for alkalization, and the pH value of the concentrated solution is adjusted to 8-10, and then heated to a temperature of 78 °C to obtain the second extract.
[0026] (3) Acidify the second extract by adding a hydrochloric acid solution with a mass fraction of 5%, and adjust the pH value of the second extract to 3 - 6.
[0027] (4) Extract using an organic solvent for 3 - 5 times, and concentrate the extract under reduced pressure to obtain an extractive paste after extraction. The organic solvent used as the extractant is: saturated n-butanol, ethyl acetate, dichloromethane, petroleum ether, n-hexane or n-propanol. After concentration of the extract, a mixture containing low contents of Tellimagrandin I and II is obtained.
[0028] (5) The mixture containing low contents of Tellimagrandin I and II is redissolved in hot water, activated carbon is added, and after heating and stirring, it is cooled. First, elute with 0 - 50% ethanol to obtain Tellimagrandin I, and elute with 50 - 100% ethanol to obtain Tellimagrandin II.
[0029] Example 1
[0030] Take 500 g of crushed camellia oleifera fruit shells, add an ethanol aqueous solution with a volume percentage concentration of 30% according to the ratio of solid to liquid of 1:10 (g / mL), and perform hot reflux stirring extraction at a temperature of 60 °C for 60 minutes each time. Filter to obtain the first extract.
[0031] Concentrate the first extract until it has no alcohol smell. Alkalize the concentrated solution by adding a sodium hydroxide solution with a mass fraction of 5%, adjust the pH value of the concentrated solution to 8, and heat to a temperature of 78 °C to obtain the second extract.
[0032] Acidify the extract by adding a hydrochloric acid solution with a mass fraction of 5% and adjust the pH value to 3.
[0033] Extract using saturated n-butanol for 3 times, and after concentration of the extract, a mixture containing low contents of Tellimagrandin I and II is obtained.
[0034] The mixture containing low contents of Tellimagrandin I and II is redissolved in hot water, decolorized by adding 10% activated carbon by mass, first eluted with a 10% ethanol aqueous solution, and the eluate is filtered and dried after standing cold to obtain 2.62 g of Tellimagrandin I (yield 0.524%, purity 98.12%). Then elute with a 60% ethanol aqueous solution, and the eluate is filtered and dried after standing cold to obtain 2.31 g of Tellimagrandin II (yield 0.462%, purity 98.05%).
[0035] The structural formula of the obtained light yellow powdery substance is as follows:
[0036]
[0037] Tellimagrandin I has the molecular formula C 34 H 26 O 22 , is a light yellow powder, and its mass spectrum is as shown in Figure 1 , ESI-MS m / z [M-H] - 785. The spectral data of compound Tellimagrandin I are as shown in Figure 2 and Figure 3 , 1 H NMR 3.82 (1H, dd, J = 1.8, 13.2 Hz, glu H-6β), 3.91 (1H, dd, J = 1.8, 13.2 Hz, glu H-6α), 4.20 (1H, dd, J = 6.6, 9.6 Hz, gluH-4α), 4.63 (1H, dd, J = 6.6, 9.6 Hz, glu H-4β), 4.96 (1H, d, J = 7.7 Hz, glu H-1β), 5.08 (1H, m, gluH-5α), 5.11 (1H, m, glu H-5β), 5.19 (1H, d, J = 9.8 Hz, glu H-2α), 5.20 (1H, m, glu H-2β), 5.30 (1H, t, J = 6.8 Hz, glu H-6β), 5.34 (1H, t, J = 6.7 Hz, glu H-6α), 5.48 (1H, t, J = 9.6 Hz, glu H-1α), 5.58 (1H, t, J = 9.1 Hz, glu H-3β), 5.81 (1H, t, J = 9.8 Hz, glu H-3α), 6.46 (1H, s, HHDP D, H-6β), 6.50 (1H, s, HHDP C, H-6β), 6.61 (1H, s, HHDP D, H-6α), 6.61 (1H, s, HHDP C, H-6α), 6.89 (2H, s, galloyl B, H-2, 6β), 6.93 (2H, s, galloyl A, H-2, 6β), 6.99 (2H, s, galloyl B, H-2, 6α), 7.02 (2H, s, galloyl A, H-2, 6α); 1313C NMR 64.34 (glu C-6α), 64.41 (glu C-6β), 67.74 (glu C-4β), 71.82 (glu C-5β), 72.11 (glu C-3α), 72.13 (glu C-2α), 72.90 (glu C-4α), 73.74 (glu C-5α), 74.46 (glu C-2β), 74.94 (glu C-3β), 91.93 (glu C-1α), 97.26 (glu C-1β), 108.35 (HHDP C, C-6β), 108.38 (HHDP D, C-6β), 108.73 (HHDP C, C-6α), 108.77 (HHDP D, C-6α), 110.48 (2C, galloyl B, C-2, 6β), 110.53 (2C, galloyl A, C-2, 6β), 110.58 (2C, galloyl B, C-2, 6α), 110.60 (2C, galloyl A, C-2, 6α), 116.53 (HHDP C, C-2β), 116.55 (HHDP D, C-2β), 116.57 (HHDP C, C-2α), 116.59 (HHDP D, C-2α), 120.71 (galloyl A, C-1β), 120.73 (galloyl B, C-1β), 120.91 (galloyl A, C-1α), 121.06 (galloyl B, C-1α), 125.94 (HHDP C, C-1β), 126.02 (HHDP D, C-1β), 126.08 (HHDP C, C-1α), 126.37 (HHDP D, C-1α), 137.76 (HHDP C, C-4β), 140.07 (HHDP D, C-4β), 140.09 (HHDP C, C-4α), 140.15 (HHDP D, C-4α), 144.94 (HHDP C, C-3α,β), 145.96 (HHDP D, C-3α,β), 146.01 (HHDP C, C-5α,β), 146.04 (HHDP D, C-5α,β), 146.06 (galloyl A, C-4β), 146.07 (galloyl B, C-4β), 146.33 (galloyl A, C-4α), 146.36 (galloyl A, C-4α), 146.52 (2C, galloyl A, C-3, 5αβ), 146.55 (2C, galloyl B, C-3, 5αβ), 167.24 (galloyl A, C-7α), 167.62 (galloyl A, C-7β), 167.85 (galloyl B, C-7β), 168.08 (galloyl B, C-7α), 169.38 (HHDP C, C-7β), 169.46 (HHDP C, C-7α), 169.81 (HHDP D, C-7β), HHDP D, C-7α).
[0038] The molecular formula of Tellimagrandin II is C 41 H 30 O 26 , a light yellow powder, and the mass spectrum is as shown in Figure 1 . ESI-MS m / z [M+H] + 939. The spectral data of compound Tellimagrandin I are as shown in Figure 2 and Figure 3 . 1 1H NMR 3.92 (1H, d, J = 12.6 Hz, glu H-6), 4.44 (1H, m, glu H-5), 5.21 (1H, t, J = 9.8 Hz, glu H-4), 5.37 (1H, dd, J = 6.4, 13.2 Hz, glu H-6), 5.75 (1H, t, J = 9.8, 18.9 Hz, glu H-3), 5.53 (1H, dd, J = 8.4, 9.2 Hz, glu H-2), 6.11 (1H, d, J = 8.4 Hz, glu H-1), 6.49 (1H, s, HHDP D, H-6’), 6.62 (1H, s, HHDP E, H-6”), 6.92 (2H, s, galloyl C, H-2, 6), 6.95 (s, 2H, galloyl B, H-2, 6), 7.05 (s, 2H, galloyl A, H-2, 6); 1313C NMR 63.76 (glu C-6), 71.31 (glu C-4), 72.46 (glu C-2), 73.64 (glu C-5), 74.06 (glu C-3), 94.18 (glu C-1), 108.28 (HHDP D, C-3), 108.65 (HHDP E, C-3), 110.43 (2C, galloyl C, C-2,6), 110.55 (2C, galloyl B, C-2,6), 110.62 (2C, galloyl A, C-2,6), 116.54 (HHDP D, C-1), 116.70 (HHDP E, C-1), 119.75 (galloyl A, C-1), 120.32 (galloyl B, C-1), 120.45 (galloyl C, C-1), 125.76 (HHDP D, C-2), 126.27 (HHDP E, C-2), 137.71 (HHDP D, C-5), 137.73 (HHDP E, C-5), 140.12 (galloyl, C-4), 140.33 (galloyl B, C-4), 140.81 (galloyl A, C-4), 144.90 (2C, HHDP D, E, C-6), 145.95 (HHDP D, C-4), 145.98 (HHDP E, C-4), 146.28 (2C, galloyl C, C-3,5), 146.43 (2C, galloyl B, C-3,5), 146.62 (2C, galloyl A, C-3,5), 166.21 (galloyl A, C-7), 166.94 (galloyl B, C-7), 167.59 (galloyl C, C-7), 169.22 (HHDP D, C-7), 169.58 (HHDP E, C-7).
[0039] Example 2
[0040] Take 500 g of crushed camellia oleifera fruit shells, add an aqueous ethanol solution with a volume percentage concentration of 40% according to the solid-liquid ratio of 1:15 (g / mL), carry out hot reflux stirring extraction, the extraction temperature is 60 °C, 60 minutes each time, filter to obtain the first extract.
[0041] The first extract is concentrated until there is no alcohol smell, add a sodium hydroxide solution with a mass fraction of 5% to the concentrated solution for alkalization, adjust the pH value of the concentrated solution to 8.5, and heat to 78 °C to obtain the second extract.
[0042] Add a hydrochloric acid solution with a mass fraction of 5% to the second extract for acidification, and adjust the pH value of the second extract to 3.5.
[0043] Extract with ethyl acetate three times. After concentrating the extract, a mixture containing low contents of Tellimagrandin I and II is obtained.
[0044] The mixture containing low contents of Tellimagrandin I and II is redissolved in hot water, decolorized with 10% activated carbon by mass, first eluted with 20% aqueous ethanol solution, and the eluate is filtered and dried after standing cold to obtain 2.75 g of Tellimagrandin I (yield 0.55%, purity 98.04%). Then it is eluted with 60% aqueous ethanol solution, and the eluate is filtered and dried after standing cold to obtain 2.82 g of Tellimagrandin II (yield 0.564%, purity 98.36%).
[0045] Example 3
[0046] Take 500 g of crushed camellia oleifera fruit shells, add 50% aqueous ethanol solution with a volume percentage concentration according to the solid-liquid ratio of 1:20 (g / mL), extract by hot reflux stirring at an extraction temperature of 70 °C for 60 minutes each time, and filter to obtain the first extract.
[0047] The first extract is concentrated until there is no alcohol smell, 5% sodium hydroxide solution by mass is added to the concentrated solution for alkalization, the pH value of the concentrated solution is adjusted to 9.0, and it is heated to 78 °C to obtain the second extract.
[0048] Add 5% hydrochloric acid solution by mass to the second extract for acidification, and adjust the pH value of the second extract to 4.0.
[0049] Extract with dichloromethane four times. After concentrating the extract, a component containing low contents of Tellimagrandin I and II is obtained.
[0050] The component containing low contents of Tellimagrandin I and II is redissolved in hot water, decolorized with 10% activated carbon by mass, first eluted with 30% aqueous ethanol solution, and the eluate is filtered and dried after standing cold to obtain 2.56 g of Tellimagrandin I (yield 0.512%, purity 98.31), then eluted with 70% aqueous ethanol solution, and the eluate is filtered and dried after standing cold to obtain 2.67 g of Tellimagrandin II (yield 0.534%, purity 98.08%).
[0051] Example 4
[0052] Take 500 g of crushed camellia oleifera fruit shells, add an ethanol aqueous solution with a volume percentage concentration of 60% according to the solid-liquid ratio of 1:25 (g / mL), carry out hot reflux stirring extraction, the extraction temperature is 80 °C, 60 minutes each time, filter to obtain the first extract.
[0053] The first extract is concentrated until there is no alcohol smell, add a sodium hydroxide solution with a mass fraction of 5% to the concentrated solution for alkalization, adjust the pH value of the concentrated solution to 9.5, and the heating temperature of the alkalized solution is 78 °C to obtain the second extract.
[0054] Add a hydrochloric acid solution with a mass fraction of 5% to the second extract for acidification, and adjust the pH value of the second extract to 4.5.
[0055] Use petroleum ether for extraction, extract 5 times, and after concentrating the extract, obtain a component containing low contents of Tellimagrandin I and II.
[0056] The component containing low contents of Tellimagrandin I and II is redissolved with hot water, add activated carbon with a mass of 10% for decolorization, first elute with an ethanol aqueous solution with a concentration of 40%, and after the eluate is left to stand, filtered and dried to obtain 2.73 g of Tellimagrandin I (yield 0.546%, purity 98.24%), then elute with an ethanol aqueous solution with a concentration of 80%, and after the eluate is left to stand, filtered and dried to obtain 2.98 g of Tellimagrandin II (yield 0.596%, purity 98.57%).
[0057] Example 5
[0058] Take 500 g of crushed camellia oleifera fruit shells, add an ethanol aqueous solution with a volume percentage concentration of 90% according to the solid-liquid ratio of 1:30 (g / mL), carry out hot reflux stirring extraction, the extraction temperature is 60 °C, 60 minutes each time, filter to obtain the first extract.
[0059] The first extract is concentrated until there is no alcohol smell, add a sodium hydroxide solution with a mass fraction of 5% to the concentrated solution for alkalization, adjust the pH value of the concentrated solution to 10, and heat to 78 °C to obtain the second extract.
[0060] Add a hydrochloric acid solution with a mass fraction of 5% to the second extract for acidification, and adjust the pH value of the second extract to 6.
[0061] Use n-propanol for extraction, extract 3 times, and after concentrating the extract, obtain a component containing low contents of Tellimagrandin I and II.
[0062] The components containing low contents of Tellimagrandin I and II were redissolved with hot water, 10% by mass of activated carbon was added for decolorization, elution was first carried out with 50% aqueous ethanol solution, and the eluate was filtered by cold setting and dried to obtain 2.71 g of Tellimagrandin I (yield 0.542%, purity 98.33%). Then, elution was carried out with 90% aqueous ethanol solution, and the eluate was filtered by cold setting and dried to obtain 2.96 g of Tellimagrandin II (yield 0.592%, purity 98.54%).
[0063] Finally, it should be noted that the above are only preferred examples of the present invention and are not used to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or perform equivalent replacements for some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.
Claims
1. A method for simultaneously separating and purifying Tellimagrandin I and II from the shell of Camellia oleifera fruit, characterized in that, Crush the raw material of camellia oleifera fruit shell and extract it with an organic solvent. The organic solvent is an ethanol aqueous solution with a volume percentage concentration of 30%-90%. The extraction process is as follows: the material-liquid ratio is 1:10-30 g / mL, the extraction temperature is 60-80°C, and extract with heat reflux and stirring to obtain the first extract; filter the first extract, concentrate it until there is no alcohol smell, add a sodium hydroxide solution with a mass fraction of 5% for alkalization, adjust the pH value of the concentrated solution to 8-10, heat the alkalized solution, the heating temperature of the alkalized solution is 78°C, let it stand, filter to obtain the second extract, add a hydrochloric acid solution with a mass fraction of 5% to the second extract for acidification, adjust the pH value of the second extract to 3-6, and perform extraction with an extractant after acidifying the second extract. The extractant is: saturated n-butanol, ethyl acetate, dichloromethane, petroleum ether, n-hexane or n-propanol; after concentrating the extract, a mixture containing Tellimagrandin I and II is obtained. After the mixture containing Tellimagrandin I and II is redissolved in hot water, activated carbon is added, and after heating and stirring, it is first eluted with 0-50% ethanol to obtain Tellimagrandin I, and eluted with 50-100% ethanol to obtain Tellimagrandin II; The structural formula of the obtained Tellimagrandin I is as follows: ； The structural formula of the obtained Tellimagrandin II is as follows: 。 2. The method for simultaneously separating and purifying Tellimagrandin I and II from the shell of Camellia oleifera fruit according to claim 1, characterized in that, Use the extractant for extraction, the extraction times are 3-5 times, and concentrate under reduced pressure to form an extract after extraction.
3. The method for simultaneously separating and purifying Tellimagrandin I and II from the shell of Camellia oleifera fruit according to claim 1, characterized in that, The dosage of the activated carbon is 10% of the mass of the mixture containing Tellimagrandin I and II.