TW202000192A - Method for preparing anti-oxidative ingredient of kaempferol from Taiwan indigenous cinnamon with hydrolysis technology capable of avoiding use of highly volatile organic solvents that are harmful to human body - Google Patents

Abstract

The present invention provides a method for preparing an anti-oxidative ingredient of kaempferol from Taiwan indigenous cinnamon by hydrolysis technology, wherein an extraction step is divided into six steps. The process includes extracting a first solvent of the Taiwan indigenous cinnamon pieces at high temperature; performing filtering to obtain an extract; concentrating the extract in vacuum to obtain a concentrated extract; adding the concentrated extract to an acidic substance; performing hydrolysis at 40 DEG C to 60 DEG C for 6 to 10 hours; after standing for 15.5 to 16.5 hours, performing neutralization by adding an alkaline substance to obtain a hydrolysate; filtering the hydrolysate and washing the filter residues with a second solvent; and concentrating the hydrolysate in vacuum into a paste so as to obtain a hydrolysate containing kaempferol.

Description

Method for preparing kaempferol, an antioxidant component, from Taiwan native soil cinnamon by hydrolysis technology

The invention relates to the field of plant extraction, in particular to a method for extracting kaempferol from Taiwan native seed cinnamon.

Kaempferol (Kaempferol) is a natural flavonoid compound found in plants, such as tea, broccoli, delphinium, witch hazel, grapefruit, Brussels sprouts, apples and other plants. Pathological studies have found that there is a positive correlation between the intake of kaempferol and the reduction of the incidence of many diseases, such as cancer and cardiovascular diseases. A large number of preclinical studies have shown that kaempferol and some kaempferol glycosides have a wide range of pharmacological activities, including antioxidant, anti-inflammatory, antibacterial, anti-cancer, heart protection, nerve protection, anti-diabetes, anti-osteoporosis, anti-estrogen, anti-anxiety , Analgesic and anti-allergic effects ( JMCalderon-Montano, E. Burgos-Moron, C. Perez-Guerrero, M. Lopez-Lazaro , 2011). More studies have found that kaempferol, quercetin and myricetin can reduce the risk of smokers suffering from pancreatic cancer and reduce the incidence of lung cancer.

However, the extraction technology of kaempferol still has much to be improved. Chinese patent CN101845467A discloses a method for extracting kaempferol, which uses enzymes to convert the ingredients in the raw materials into kaempferol, and then extracts using highly volatile organic solvents (ethyl acetate). However, because organic solvents are harmful to humans, In the extraction process, it may cause long-term harm to the extractor's body; and Taiwan Patent No. TWI352611 discloses an extraction method of the flavonoid glycoside component of earth cinnamon leaf, which uses supercritical carbon dioxide fluid extraction, brine extraction, and salt removal, although the flavonoids The glycoside component contains kaempferol, and the previous case did not use ethyl acetate extraction, but it is not known whether the extract component does contain kaempferol, and the extraction process is complicated, which may take a lot of time and money.

Therefore, it is a problem to be solved in the art to provide a simple and safe method for extracting kaempferol.

In view of this, the purpose of the present invention is to provide a simple and safe extraction method by extracting cinnamon from Taiwan native soil to convert kaempferol to kaempferol by hydrolysis.

In order to achieve the above-mentioned object of the invention, the present invention provides a method for extracting kaempferol from Taiwan native soil cinnamon. The method includes the following steps. First, add the native Taiwan soil cinnamon pieces to the first solvent for high temperature extraction, filter and concentrate in vacuo to obtain a concentrated extract, add the concentrated extract to an acidic substance, hydrolyze at 40 ℃ ~ 60 ℃ for 6 to 10 hours, let stand After 14 to 18 hours, add an alkaline substance to neutralize to obtain a hydrolysate, filter the hydrolysate and wash the filter residue with a second solvent, and concentrate in vacuo to extract to prepare the hydrolysate containing kaempferol.

The first solvent is ethanol with a volume concentration of 40% to 60%; the second solvent is ethanol with a volume concentration of 92 to 98%. In addition, the acidic substance is selected from the group consisting of hydrochloric acid, acetic acid and sulfuric acid; the alkaline substance is selected from the group consisting of sodium bicarbonate, sodium percarbonate and sodium carbonate.

In order to achieve the above object of the invention, the present invention provides a Taiwan native seed soil cinnamon extract, wherein the Taiwan native seed soil cinnamon extract contains kaempferol 5-10 mg/g.

S1~S6‧‧‧Step flow

Figure 1 is a flow chart of the method for extracting kaempferol from Taiwan native seed cinnamon; Figure 2 is an HPLC chromatogram of standard kaempferol and kaempferol; Figure 3 is an HPLC chromatogram of Taiwan native seed cinnamon cinnamon extract before hydrolysis Figure 4 is the HPLC chromatogram of Taiwan native soil cinnamon extract after hydrolysis.

Unless otherwise defined, all technical and scientific terms mentioned in this manual are the meanings that can be commonly understood by the field of the professional to which they belong.

As used herein, the term "extract" refers to a product prepared by extraction. The extract can be presented as a solution dissolved in a solvent, or the extract can be presented as a concentrate or essence that contains no or substantially no solvent. As described further below, the extract can also be formulated in pharmaceutical compositions or foods. The term extract may be a single extract obtained from a specific extraction step or a series of extraction steps, or the extract may also be a combination of extracts obtained from independent extraction steps. Therefore, these combined extracts are also covered by the term "extract."

As used herein, "raw materials" generally refer to plant raw materials, including individual whole plants or a combination of one or more component parts of plants, including leaves, roots (including but not limited to main roots, tail roots, and fiber roots), stems, bark , Berries, seeds, and flowers, where the plant or component may include raw, dried, cooked, heated, or otherwise physically processed materials to facilitate processing, which may further include whole, minced, and diced , Milled, ground or otherwise processed to affect the size and physical integrity of the plant material. The term "raw material" can be used to denote the extraction product of the material source used in the additional extraction process.

The present invention is exemplified and illustrated by the following examples, but the present invention is not limited by the following examples. The materials used in the present invention are all commercially available, and the following are only examples of pipes that can be obtained.

Embodiment 1. Method for hydrolyzing process of cinnamon from Taiwan native soil

To extract kaempferol, the extraction step is shown in Figure 1. Step S1 is to prepare the native Taiwan soil cinnamon; in step S2, the first solvent is added for high temperature extraction to obtain an extract; then in step S3, vacuum concentration is performed to obtain a concentrated extract; step S4 is to add an acidic substance to hydrolyze, and then add an alkaline substance to obtain hydrolysis S5 step is to filter the hydrolysate and wash the filter residue with a second solvent; then in step S6 step vacuum concentration to extract, to prepare the hydrolysate containing kaempferol.

In a preferred embodiment, a Taiwan native seed soil cinnamon is prepared, and the Taiwan native seed soil cinnamon pieces are added to ethanol with a volume concentration of 40%~60% for high temperature extraction, and the selected high temperature temperature is 60°C~90 After filtration, an extract is obtained, and the extract is concentrated in vacuo to obtain a concentrated extract. The concentrated extract is added with an acidic substance and hydrolyzed at a temperature of 40°C to 60°C for 6 to 10 hours. After standing for a period of time , Add an alkaline substance to neutralize to obtain a hydrolysate, filter the hydrolysate to wash the filter residue with ethanol with a volume concentration of 70% to 100%, and then concentrate in vacuo to extract to prepare the hydrolysis of kaempferol. Thing.

In a preferred embodiment, Taiwan native soil cinnamon branches and leaves are prepared, weighed 1 kg, ethanol is added, 10 L of 50% ethanol (EtOH) is taken, and extracted at 70°C for 4 hours to obtain For the extract, the extract is filtered to remove the filter residue, and the filtered filtrate is concentrated in vacuo to 1 L to obtain a concentrated extract.

Take the aforementioned 1L concentrated extract, the acidic substance added is hydrochloric acid, the 12N hydrochloric acid (HCl) 43.75mL, hydrolyzed at 50 ℃ for 8 hours, stand at room temperature for 16 hours, and add an alkaline substance for neutralization The alkaline substance is sodium bicarbonate, and hydrochloric acid (HCl) is neutralized with 44.1 g of sodium bicarbonate to obtain a hydrolysate. The hydrolysate is filtered, and the filter residue is washed with 95% ethanol by volume, and then filtered The hydrolysate afterwards is concentrated in vacuo to a extract, and the product obtained after freeze-drying the extract is the hydrolysate containing kaempferol.

Example 2. Analysis of kaempferol content in cinnamon hydrolysate of Taiwan native soil

To understand whether the cinnamon hydrolysate of Taiwan native seed soil extracted by the Taiwan native seed cinnamon extraction method in this case contained kaempferol, Taiwan native seed soil was analyzed by high-performance liquid chromatograph (HPLC) The kaempferol content of cinnamon hydrolysate.

Before performing HPLC, the sample must be processed first. 102.4 mg of the native Taiwan soil cinnamon hydrolysate was dissolved in anhydrous methanol, quantified to 100 mL, and filtered with a 0.45 μm filter membrane.

In HPLC analysis, the column is inertsil (250×4mm, 5μm), the column temperature is 35 ℃; and the mobile phase system is gradient elution with water (0.1% TFA), acetonitrile (AeCN) and methanol (MeOH) (its movement The phase gradient table is shown in Table 1); flow rate 0.5mL/min; wavelength 342nm; injection volume 10μL.

Figure 2 shows the HPLC chromatograms of kaempferitrin and kaempferol, wherein the retention time (RT) of kaempferitrin is 42.095 minutes; the kaempferit residence time is 61.952 minutes. Figure 3 shows that the retention time of kaempferol in the native Taiwan soil cinnamon extract before the hydrolysis was 41.777 minutes, and kaempferol was not detected; however, after the native Taiwan soil cinnamon extract was hydrolyzed, the kaempferol reaction occurred and its kaempferol The retention time of glycoside is 41.917 minutes, while the retention time of kaempferol is 61.940 minutes (as shown in Figure 4). According to the results shown in Figures 3 and 4, kaempferol was not originally present in the natural ingredients of the native Taiwan soil cinnamon , Through the Taiwan native soil cinnamon extraction method in this case, hydrolyzed kaempferol into kaempferol. The hydrolysis rate and conversion rate are calculated by the following formula:

Where W ₁ is the content of kaempferol after hydrolysis (mg/g); W ₀ is the content of kaempferol before hydrolysis (mg/g);

Wherein W ₀ is the content of kaempferol before hydrolysis (mg/g); W ₂ is the content of kaempferol after hydrolysis (mg/g); 286.23 is the molecular weight of kaempferol; 578.52 is the molecular weight of kaempferol.

It can be seen from the experimental results that 1 kg of Taiwan native soil cinnamon (branch, leaf) is taken, and the dry weight of the product is 55.03 g after extraction, and the dry weight of the product after hydrolysis is 57.48 g. It can be seen from Table 2 that the content of kaempferol in Taiwan native soil cinnamon before hydrolysis was 26.1 mg/g, and the content after hydrolysis was 5.9 mg/g. After hydrolysis, the content of kaempferol was 6.52 mg/g. According to the above calculation, after the native Taiwan soil cinnamon extract is hydrolyzed, the hydrolysis rate of kaempferol is 77.3%; the conversion rate of kaempferol is 50.4%.

To sum up, the extraction method in this case converts the kaempferol that is not originally present in Taiwan native soil, through the hydrolysis step, the kaempferol in the Taiwan native soil cinnamon extract is converted into kaempferol, and no further enzyme conversion is needed in the process. No need to use organic solvent ethyl acetate extraction, the extraction step is safer and simpler than the current kaempferol extraction method.

The above-mentioned embodiments are only to illustrate the technical ideas and features of the present invention, and its purpose is to enable those skilled in the art to understand the content of the present invention and implement it accordingly, when the patent scope of the present invention cannot be limited, that is Equal changes or modifications made in accordance with the spirit disclosed by the present invention should still be covered by the patent scope of the present invention.

S1~S6‧‧‧Step flow

Claims (17)

A method for extracting kaempferol from Taiwan native seed cinnamon, which is prepared by the following steps: Step 1: Prepare a Taiwan native seed cinnamon; Step 2: Add a first solvent to perform high temperature extraction at 60℃~90℃ After filtration, an extract is obtained; Step 3: The extract is concentrated in vacuo to obtain a concentrated extract; Step 4: The concentrated extract is added with an acidic substance and hydrolyzed at a temperature of 40°C to 60°C for 6 to 10 hours After a period of time, add an alkaline substance to neutralize to obtain a hydrolysate; Step 5: Filter the hydrolysate and wash the filter residue with a second solvent; Step 6: Concentrate the hydrolysate in vacuo to extract Hydrolysate containing kaempferol. The method as described in item 1 of the patent application scope, wherein the first solvent is ethanol with a volume concentration of 40% to 60%. The method as described in item 2 of the patent application, wherein the first solvent is preferably ethanol with a volume concentration of 48-52%. The method as described in item 1 of the patent application scope, wherein the ratio of the native Taiwanese cinnamon cinnamon to the weight volume of the first solvent is 1:5~1:20. The method as described in item 1 of the patent application, wherein the preferred high-temperature extraction temperature in step 1 is 70°C. The method as described in item 1 of the patent application scope, wherein the high temperature extraction time of step 1 is 2 to 6 hours. The method as described in item 1 of the patent application scope, wherein the preferred high-temperature extraction time in step 1 is 3.5 to 4.5 hours. The method according to item 1 of the patent application scope, wherein the acidic substance is selected from the group consisting of hydrochloric acid, acetic acid, and sulfuric acid. The method according to item 1 of the patent application scope, wherein the alkaline substance is selected from the group consisting of sodium bicarbonate, sodium percarbonate, and sodium carbonate. The method as described in item 1 of the patent application scope, wherein the preferred hydrolysis time in step 2 is 7.5 to 8.5 hours. The method as described in item 1 of the patent application scope, wherein the standing time of step 2 is 14 to 18 hours. The method as described in item 11 of the patent application scope, wherein the preferred standing time in step 2 is 15.5~16.5 hours. The method as described in item 1 of the patent application scope, wherein the second solvent is ethanol with a volume concentration of 70% to 100%. The method as described in item 13 of the patent application scope, wherein the second solvent is preferably ethanol with a volume concentration of 92-98%. The method as described in item 1 of the patent application scope, wherein the ratio of the hydrolysate to the second solvent is 1:5~1:20. The method as described in item 1 of the patent application scope, wherein the extract can be further freeze-dried in step three. A native Taiwan soil cinnamon extract is obtained by the method of any one of patent applications 1 to 16, wherein the Taiwan native soil cinnamon extract contains kaempferol 5-10mg/g.

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