A method for extracting kaempferol from camellia oil byproduct oil cake

Abstract

The invention discloses a method for extracting high-purity kaempferol from camellia oil byproduct oil cake, including: camellia oil cake is first degreased by n-hexane, then extracted with methanol as extraction reagent, filtered to remove residue, obtain extract, extract is decompressed and distilled to remove solvent, and crude extract is obtained; crude extract is subjected to forward silica gel column chromatography, and petroleum ether: acetone = 20: 1, 1: 1V / V are used successively as eluents for elution, petroleum ether: acetone = 1: 1V / V fractions are merged, decompressed and distilled to remove solvent, and crude fractions are obtained; reverse silica gel is used as filler, reversed-phase silica gel column chromatography is carried out on crude fractions, gradient elution is carried out with methanol-water as eluent, fractions are collected according to elution gradient, 20% methanol-water, 40% methanol-water, 60% methanol-water elution fractions are merged, decompressed and distilled to remove solvent, and kaempferol is obtained. The inventive method can obtain high-purity kaempferol, and product purity can reach 97.4%.

Description

Technical Field

[0001] The invention belongs to the field of extraction and separation, and specifically relates to a method for extracting high-purity kaempferol from oil cake, a byproduct of camellia oil Background Art

[0002] Kaempferol is an organic compound belonging to the flavonoids class of compounds with a chemical formula of C 15 H 10 O6, the pure monomer is a yellow crystalline powder, slightly soluble in water, soluble in hot ethanol, ether, DMSO and other organic solvents, alias: kaempferol-3, kaempferol flavonol. Kaempferol is also a flavonoid active substance, and its structure is similar to kaempferol, but the two cannot be classified as the same substance.

[0003] Kaempferol has excellent biological activity:

[0004] Cancer prevention and treatment: Long-term diet can reduce the risk of cancer. High intake of kaempferol can reduce the recurrence of advanced colorectal adenoma and significantly reduce the risk of pancreatic cancer.

[0005] Kaempferol has good anti-infection and anti-inflammatory activity, and is also effective against methicillin-resistant Staphylococcus aureus (MRSA), Vibrio cholerae and Enterococcus faecalis. In terms of anti-inflammatory, kaempferol mainly achieves its anti-inflammatory effect by inhibiting the expression of inflammatory factors.

[0006] Diabetes prevention and treatment: Diabetes is an endocrine metabolic disease that seriously threatens human health, and its incidence is increasing at an unprecedented rate. Studies have shown that kaempferol has the same efficacy as the hypoglycemic drug rosiglitazone as a PPAR agonist, but its adverse reactions are far weaker than rosiglitazone, and it is expected to become a new generation of insulin sensitizer.

[0007] Kaempferol is mainly derived from the rhizome of Kaempferol galanga L., a plant of the ginger family. After enrichment, a mixed solvent of methanol and acetonitrile is generally used as an eluent for full gradient elution separation, but this method is too complicated and cumbersome and cannot be effectively separated and purified. Kaempferol is also widely present in various fruits, vegetables and beverages. People have extracted its pure products from tea, cabbage, witch hazelnut, propolis, grapefruit and other green plants. In industry, the dried mature fruits of the leguminous plant Sophora japonica are usually used as raw materials, and the kaempferol product is obtained by steam distillation and recrystallization after concentration, enrichment, separation and purification. In the laboratory, high performance liquid chromatography is usually used directly for separation and purification. These operations are complicated, time-consuming, and the raw material and production line costs are also high.

[0008] Camellia oil is extracted from the seeds of the Camellia oleifera Abel. of the Theaceae family. The by-product of camellia oil production, camellia oil cake, is mainly sold on the market as a raw material for detergents or shampoos, and its economic value is also very low.

[0009] At present, there is no relevant research on isolating pure kaempferol from camellia oil cake. Summary of the invention

[0010] The purpose of the present invention is to effectively utilize agricultural wastes, and extract high-purity natural active substance kaempferol from camellia seed oil cake produced after oil is squeezed from oil tea tree seeds as raw materials. The lipids remaining in the camellia seed oil cake after oil is squeezed will seriously interfere with the separation effect of other active substances, and there are also a large number of various flavonoid glycosides, polyphenols, and various polar saponins and glycosyl substances similar to kaempferol in the oil cake, especially the oil cake contains kaempferol, which is close to kaempferol in structure, and the above chemical components will greatly affect the separation and purification of kaempferol.

[0011] The objective of the present invention is achieved through the following technical solutions:

[0012] A method for extracting high-purity kaempferol from camellia oil byproduct oil cake, comprising:

[0013] Step (1), the camellia oil cake is first degreased with n-hexane, then extracted with methanol as an extraction reagent, the residue is filtered to remove the extract, and the extract is distilled under reduced pressure to remove the solvent to obtain a crude extract, which is a liquid extract;

[0014] Step (2), the crude extract is subjected to normal silica gel column chromatography, using petroleum ether:acetone = 20:1 and 1:1 V / V as eluents for elution, combining the fractions of petroleum ether:acetone = 1:1 V / V, and removing the solvent by reduced pressure distillation to obtain a crude fraction;

[0015] Step (3), using reverse silica gel as filler, performing reverse phase silica gel column chromatography on the crude fraction, using methanol-water as eluent for gradient elution, collecting fractions according to the elution gradient, combining 20% ​​methanol-water, 40% methanol-water, and 60% methanol-water elution fractions, and removing the solvent by reduced pressure distillation to obtain kaempferol.

[0016] In step (1), preferably, the camellia oil cake is soaked in n-hexane for 24 hours, and the usage ratio of camellia oil cake to n-hexane is 1:2 kg / L.

[0017] Preferably, methanol is used as the extraction reagent, and the extraction is repeated 2 to 3 times. During each extraction, the usage ratio of camellia oil cake to methanol is 1:2 kg / L, and the extraction time is 12 hours.

[0018] In step (2), the crude extract is loaded by a wet method, specifically: normal phase silica gel is mixed with the crude extract until no obvious liquid extract is visible, and the mixture is completely dried to obtain loading silica gel; blank silica gel is taken according to a thickness ratio of the sample layer to the blank silica gel layer of 1:2, the blank silica gel is shaken with methanol, and then poured into a column chromatography column, and then the loading silica gel is added.

[0019] The volume ratio of eluent to column volume for each elution gradient was 5:1.

[0020] The mesh number of the normal phase silica gel is 80 meshes.

[0021] In step (3), the crude fraction is loaded by dry method, specifically: the crude fraction is dissolved in methanol and mixed with reversed-phase silica gel to obtain loading silica gel, and blank reversed-phase silica gel is added to the chromatographic column according to the thickness ratio of the sample layer to the blank reversed-phase silica gel layer of 1:4, and the loading silica gel is added after ensuring that the upper surface of the blank reversed-phase silica gel layer is flat.

[0022] The reverse silica gel is C18 bonded silica gel.

[0023] The gradient elution is: pure water, 20% methanol-water, 40% methanol-water, 60% methanol-water, 80% methanol-water, and pure methanol are used for gradient elution in sequence. Only the 20% methanol-water, 40% methanol-water, and 60% methanol-water elution fractions contain high-purity kaempferol, and kaempferol is present in large quantities in the 80% methanol-water elution fraction.

[0024] Considering that high-purity kaempferol is mainly in the 40% methanol-water elution fraction, as a further preferred technical solution of the present invention, the 40% methanol-water elution fraction is collected, and the solvent is removed by reduced pressure distillation to obtain kaempferol.

[0025] Beneficial effects of the present invention:

[0026] The method uses n-hexane to remove residual oil components, uses normal phase silica gel column chromatography and uses petroleum ether acetone methanol solvents with different ratios to preliminarily enrich kaempferol, and then uses reverse phase silica gel column chromatography to remove impurities through pure water elution, thereby obtaining high-purity kaempferol under 20% methanol-water, 40% methanol-water and 60% methanol-water elution conditions, the product purity can reach 97.4%, and the yield is about 0.422‰, indicating that the method is suitable for extracting kaempferol from camellia seed oil cake. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is a separation flow chart of the present invention.

[0028] Figure 2 It is the high performance liquid chromatogram of the 20% methanol-water elution fraction;

[0029] Figure 3 It is the high performance liquid chromatogram of the 40% methanol-water elution fraction;

[0030] Figure 4 It is the high performance liquid chromatogram of the 60% methanol-water elution fraction;

[0031] Figure 5 It is the high performance liquid chromatogram of the 80% methanol-water elution fraction;

[0032] Figure 6 The figure is the HPLC chromatogram of the purified kaempferol. DETAILED DESCRIPTION

[0033] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples.

[0034] Example 1

[0035] like Figure 1 As shown, the camellia oil cake is used as a raw material, and high-purity kaempferol is extracted and separated from the raw material, and the steps are as follows:

[0036] Step (1), extraction

[0037] Take 2.5 kg of camellia oil cake, pour it into a conical flask, add 5 L of n-hexane and soak for 24 hours to remove residual lipid components, pour out the n-hexane; then add 5 L of analytical grade methanol, soak overnight, filter, repeat extraction with methanol twice, combine the methanol extracts, remove the solvent by distillation to obtain 104 g of crude extract.

[0038] Step (2), separation and purification

[0039] The crude extract was wet-loaded and subjected to normal phase silica gel column chromatography, specifically: an appropriate amount of normal phase silica gel was mixed with the crude extract until no obvious liquid extract was visible, and the mixture was completely dried to obtain the loading silica gel; blank silica gel (silica gel mesh number was 80 mesh) was taken according to the thickness ratio of the sample layer to the blank silica gel layer of 1:2, the blank silica gel and excess methanol (analytical grade) were shaken and poured into a column chromatography column, and then the loading silica gel was added; a mixed solvent of petroleum ether: acetone = 20: 1 V / V, a mixed solvent of petroleum ether: acetone = 1: 1 V / V, and pure methanol were used as eluents for elution, and thin layer chromatography was used for detection (the elution of the next elution gradient can be started only when no obvious points appear on the thin layer chromatography plate), the amount of each eluent was 5 L, and the loading column volume was 1 L;

[0040] When thin layer chromatography was used for detection, a silica gel plate (specification: 50 mm×100 mm, thickness: 0.2-0.25 mm) was used, and the elution fraction of petroleum ether:acetone=20:1 V / V used petroleum ether:acetone=5:1 V / V as a developing solvent, and the elution fraction of petroleum ether:acetone=1:1 V / V used petroleum ether:acetone=3:2 V / V as a developing solvent;

[0041] The fraction obtained by elution with petroleum ether:acetone = 1:1 V / V was evaporated under reduced pressure to obtain about 8 g of solid, which was the crude fraction; the fraction eluted with pure methanol contained sugars and a large amount of flavonoid glycosides.

[0042] The crude fraction was dry-loaded and subjected to reverse-phase silica gel column chromatography: the crude fraction was dissolved in methanol and then mixed with C18 bonded silica gel (reverse-phase silica gel) to obtain the loading silica gel; dry loading was used, blank reverse-phase silica gel was taken according to the thickness ratio of the sample layer and the blank reverse silica gel layer of 1:4, and added to the chromatographic column, the column wall was knocked until its surface was flat, and then the loading silica gel was added, and clean absorbent cotton was inserted to prevent the silica gel from being washed up, and the gradient elution was carried out with pure water, 20% methanol-water, 40% methanol-water, 60% methanol-water, 80% methanol-water, and 100% methanol in turn, and thin layer chromatography was used (silica gel plate specification 50mm×100mm, thickness 0.2-0.25mm ) was used for detection until no obvious spots appeared on the thin layer chromatography plate before starting the next elution gradient elution. Fractions were collected according to the elution gradient, namely: water elution fraction, 20% methanol-water elution fraction, 40% methanol-water elution fraction, 60% methanol-water elution fraction, 80% methanol-water elution fraction, 100% methanol elution fraction. The 20% methanol-water elution fraction, 40% methanol-water elution fraction, 60% methanol-water elution fraction, and 80% methanol-water elution fraction were analyzed by high performance liquid chromatography. It was found that although the polarity of 60% methanol-water and 80% methanol-water was close, the 60% methanol-water elution fraction ( Figure 4 ) contained high-purity kaempferol (0.226 g, purity 93.4%), while the 80% methanol-water elution fraction ( Figure 5 ) contains flavonoid glycosides and kaempferol, but almost no kaempferol; using 20% ​​methanol-water ( Figure 2 ) elution can obtain high-purity kaempferol (0.245g, purity 97.6%), but the content is less than 60% methanol-water elution fraction, and high-purity kaempferol (0.615g, purity 98.7%) is mainly in the 40% methanol-water elution fraction; and usually, high performance liquid chromatography is required to achieve the technical effect of separating kaempferol and kaempferol. The 20% methanol-water elution fraction, the 40% methanol-water elution fraction, and the 60% methanol-water elution fraction are combined and distilled under reduced pressure to obtain a yellow powder, and the yield of kaempferol is about 0.422‰.

[0043] Step (3), purity test and structure identification

[0044] The yellow powder was dissolved in chromatographic methanol and transferred into an EP tube, filtered through an organic filter membrane and injected into a 1.5 ml liquid phase bottle. The purity of the separated product was tested by high performance liquid chromatography.

[0045] In step (2), an Agilent 1290 Infinity II was used, and in step (3), a Merrill Instruments LC-100 liquid chromatograph was used; in step (2) and step (3), the conditions of high performance liquid chromatography were the same, as follows:

[0046] C8 chromatographic column (Agilent, Eclipse Plus C8, 5μm, 4.6×250mm), mobile phase was water and acetonitrile, flow rate was 4mL / min, column temperature was room temperature, injection volume was 150μL, elution gradient was as shown in Table 1, HPLC chromatogram is shown in Figure 6 , the product purity is 97.4%.

[0047] Table 1. Elution gradient

[0048]

[0049] The yellow powder was dissolved in deuterated methanol and its hydrogen spectrum and carbon spectrum were detected by nuclear magnetic resonance (the carbon spectrum attribution is shown in Table 2). The yellow powder was determined to be kaempferol.

[0050]

[0051] Table 2. Carbon spectrum of kaempferol

[0052] serial number Measured data <![CDATA[Micro-spectrum standard data 13 C-NMR]]> C-2 147.55 146.7 C-3 136.71 135.8 C-4 176.93 175.9 C-5 157.76 156.5 C-6 98.75 98.5 C-7 165.1 163.9 C-8 93.94 93.5 C-9 162.04 161.1 C-10 104.05 103.5 C-1’ 123.25 121.8 C-2’6’ 130.18 129.6 C-3’5’ 115.5 115.5 C-4’ 160.06 159.6

Claims

1. A method for extracting high-purity kaempferol from camellia oil byproduct oil cake, characterized in that include: Step (1), firstly degreasing camellia seed oil cake with n-hexane, then extracting with methanol as an extraction agent, filtering to remove residues to obtain an extract, and distilling the extract under reduced pressure to remove the solvent to obtain a crude extract; Step (2), the crude extract is subjected to normal silica gel column chromatography, using petroleum ether:acetone = 20:1 and 1:1 V / V as eluents for elution, combining the fractions of petroleum ether:acetone = 1:1 V / V, and removing the solvent by reduced pressure distillation to obtain a crude fraction; Step (3), using C18 bonded silica gel as filler, performing reverse phase silica gel column chromatography on the crude fraction, and using methanol-water as eluent for gradient elution, wherein the gradient elution is: using pure water, 20% methanol-water, 40% methanol-water, 60% methanol-water, 80% methanol-water, and pure methanol for gradient elution in sequence, collecting the 40% methanol-water elution fraction, and removing the solvent by reduced pressure distillation to obtain kaempferol.

2. The method for extracting high-purity kaempferol from camellia oil byproduct oil cake according to claim 1, characterized in that In step (1), methanol is used as an extraction reagent and the extraction is repeated 2 to 3 times. During each extraction, the usage ratio of camellia oil cake to methanol is 1:2 kg / L, and the extraction time is 12 hours.

3. The method for extracting high-purity kaempferol from camellia oil byproduct oil cake according to claim 1, characterized in that In step (2), the crude extract is loaded by wet method, and the thickness ratio of the sample layer to the blank silica gel layer is 1:2; the mesh size of the normal phase silica gel is 80 mesh.

4. The method for extracting high-purity kaempferol from camellia oil byproduct oil cake according to claim 1, characterized in that In step (2), the volume ratio of the eluent to the loading column volume in each elution gradient is 5:

1.

5. The method for extracting high-purity kaempferol from camellia oil byproduct oil cake according to claim 1, characterized in that In step (3), the crude fraction is loaded by dry method, and the thickness ratio of the sample layer to the blank reverse silica gel layer is 1:4.

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