TWI558697B - Preparation of monomeric form of polymethoxyflavone from citrus peel - Google Patents

Description

Method for preparing polymethoxylated flavonoids from citrus peel to monomethyl form

The invention relates to a method for preparing a polymethoxylated flavonoid to a monomethyl form in a citrus peel, in particular to a method for preparing a polymethoxylated flavonoid to a monomethyl form by using a microwave heating method or a high temperature and high pressure heating method for promoting citrus peel .

Citrus is an evergreen fruit tree of the Rutaceae family, native to the tropical and subtropical regions of Southeast Asia. The main economic production areas are distributed between 40 degrees south and north latitude. This range is called the citrus production belt. It has a wide variety, including: broad-skinned mandarin orange, hybrid mandarin, sweet orange, pomelo, grapefruit, lemon, lime and other miscellaneous. Citrus...etc.

The components of the citrus peel include essential oils, pectin, Phenolic acid, Flavonoid, and the like. It has been confirmed in the literature that citrus peel has antibacterial, anti-oxidant, anti-inflammatory, blood lipid-regulating and melanin-inhibiting effects. Flavonoids in citrus peels can be roughly classified into Flavanone glycosides and Polymethoxyflavones (PMFs) depending on their structure. The flavanone glycoside is most representative and widely studied by Hesperidin, and previous studies have been considered as the main source of anti-inflammatory components in orange peel, but In recent years, many studies have pointed out that the source of active ingredients in orange peel is PMFs, rather than the commonly known flavanone glycoside. PMFs, ie, flavonoid structures, contain multiple methoxy groups (-OMe), mostly present in the citrus fruit peel, and the main structure is Benzo-γ-pyrone (15-carbon, C6-C3-C6) (Li et al., 2009). ). More than 20 PMFs have been extracted and identified from orange peel, and the content and type of PMFs vary depending on the citrus fruit (Jayaprakasha et al., 2000). The 5-OH-PMFs are one methyl group (Me) reduced by the methoxy group at the carbon number 5 position in the above PMFs structure to form 5-OH-PMFs in the form of a monomethyl group. The structures of the six major PMFs and 5-OH-PMFs are shown in Figures 1 and 2, wherein the six PMFs contain 4 to 7 methoxy groups on the carbon skeleton.

The physiological activities of PMFs include anti-inflammatory, anti-tumor, prevention of arterial atherosclerosis, regulation of blood sugar, protection of joints and neuroprotection. Many literatures indicate that 5-OH-PMFs in the monomethyl form have better biological activity than PMFs, and have been confirmed to include anti-cancer, anti-inflammatory, etc. (Li et al., 2007c; Lai et al., 2007; Pan et al., 2007; Xiao et al., 2009; Qiu et al., 2010). However, the content of 5-OH-PMFs in most citrus peels is very small. If the content of 5-OH-PMFs is increased, the physiological activity of citrus peel can be enhanced.

The synthesis of 5-OH-PMFs generally requires a multi-step synthesis reaction, but the acid treatment can be used to demethylate the methoxy group at the carbon number 5 position in the Nobiletin structure to form 5-Demethylnobiletin, which is a one-step synthesis. Reaction, and has high yield characteristics (Li et al., 2009); or use dimethyl ketone (Dimethyldioxirane) and acetone solution at -20 ° C to form 3-Demethylnobiletin (Li et al., 2007); or use of Aspergillus niger to produce 4 ' -Demethylnobiletin (Okuno and Miyazawa, 2004); also literature indicates that Nobiletin is biotransformed in rats to produce 3 ' - demethylnobiletin, 4 '-Demethylnobiletin and 3', 4 '-Didemethylnobiletin other metabolites (Murakami et al, 2002;. Yasuda et al, 2003;. Li et al, 2006.).

However, the above methods for synthesizing 5-OH-PMFs still have much room for improvement in rate and productivity. Therefore, in this study, microwave treatment and high temperature and high pressure system were used to acidify PMFs, compared with the prior art oil bath heating. The yield and production rate of PMFs converted to 5-OH-PMFs can be increased, and the biological activity and utilization of citrus peel can be increased.

In the prior art, the PMFs in the citrus peel are demethylated by the acid treatment of the methoxy group on the carbon No. 5 in the PMFs structure to form 5-OH-PMFs with better biological activity and anti-cancer and anti-inflammatory effects. However, the acid treatment process needs to be heated to promote the reaction. However, the prior art heating method mainly utilizes the oil bath heating method, and the heat energy is transferred from the container to the reaction solution by contact conduction, and the heat energy is dispersed in the reaction solution by convection, so that the temperature of the reaction solution rises. However, the heating method of the oil bath is slow in heating speed and uneven in temperature distribution, so that the yield of PMFs converted to 5-OH-PMFs is low. The invention adopts the microwave heating method for the first time, and the radiant heating causes the reaction solution to directly absorb the microwave energy and rapidly heats up. Compared with the conventional heating method, the microwave heating has the advantages of fast, uniform and high efficiency. In addition, the invention also uses a high temperature and high pressure method to heat the reaction solution to form saturated steam, so that the temperature in the reactor reaches 121 ° C and the pressure reaches 1.5 kg / m ² , so that the probability of intermolecular collision increases under high temperature and high pressure, and the reaction is more It is easy to carry out, and effectively increases the reaction rate and yield of PMFs converted to 5-OH-PMFs.

In order to achieve the above needs and purposes, the technical means adopted by the present invention is to provide a method for preparing PMFs to remove monomethyl form of 5-OH-PMFs from citrus peel, which comprises the following steps: (a) acid treatment, and PMF setting In the container, hydrochloric acid (Hydrochloric acid, HCl) and ethanol (Ethanol, EtOH) are mixed into the container to form a reaction solution; (b) heat treatment, the reaction solution is microwave-heated by a microwave instrument to promote the reaction solution. The methoxy group at the carbon position of the PMF structure is subjected to a demethylation reaction; (c) cooling treatment, the reaction solution is allowed to stand and allowed to cool to room temperature; (d) acid-base neutralization, using sodium hydroxide (NaOH) as Neutralizing the reaction solvent with acid and alkali, adding NaOH to the cooled reaction solution for acid-base neutralization reaction, and then removing the reaction solvent by acid-base neutralization under reduced pressure concentration, and then adding distilled water to the reaction solution; and (e The extraction step is carried out by using ethyl acetate (Ethyl acetate, EtOAc) as an extract, and the reaction solution obtained by the neutralization reaction of the acid-base is subjected to layered extraction. After the extraction is completed, the extract is removed by a vacuum concentration method; The foregoing extraction steps were carried out twice to obtain PMF and 5-OH-PMF dried extract powder.

The invention has the advantages of using a microwave instrument to react the solution Microwave heating is performed to directly absorb the microwave energy by the reaction solution to rapidly increase the temperature. Compared with the conventional oil bath heating mode, the microwave heating has the advantages of rapid temperature rise, uniform heat, high efficiency, etc., and the reaction solution can be made. The chemical molecules in the molecule perform the demethylation reaction more efficiently, achieving the effect of rapidly preparing 5-OH-PMFs.

Preferably, the microwave instrument is an open system of a focusing microwave synthesizer equipped with a condenser tube, wherein the focused microwave synthesizer is equipped with a condensing tube and the open system has a set power of 150 W, a temperature of 105 ° C, and a heating time. It is 0.5 to 2 hours. This technical means only uses multiple experimental parameter adjustments to find the best reaction for power and temperature, and then adjusts the heating time to find the optimal conditions for the production of 5-OH-PMFs.

Preferably, the (b) heat treatment step may be replaced by a high temperature and high pressure heating method, wherein the high temperature and high pressure heating method has a set temperature of 121 ° C, a pressure of 1.5 kg/m ² , and a heating time of 15-30 minutes. This technical means uses a high-temperature and high-pressure heating method to increase the probability of collision of chemical molecules in the reaction solution, thereby making the reaction easier. This high temperature and high pressure heating method uses the adjustment of the heating time to find out the optimum conditions for producing 5-OH-PMFs.

More preferably, wherein the PMF is taken in an amount of 50 mg (mg); the HCl concentration is 6 N (equivalent concentration), the EtOH concentration is 100%, and the two are mixed in a volume ratio of 1:1 and added to the container; The concentration was 6 N; the volume of distilled water was 20 mL; the volume of EtOAc used in each extraction step was 20 mL. The selection of each of the above solvents (solutions) was the best reaction conditions found after several experimental tests.

Another citrus peel of the present invention prepares PMFs to monomethyl form The method of 5-OH-PMFs, wherein the step further comprises: (f) quantitative analysis, dissolving the PMF and 5-OH-PMF dry extract powder with EtOH, configuring to form a solution to be tested, and then waiting The solution was centrifuged, and then quantitative analysis was performed by high performance liquid chromatography (HPLC) with ultraviolet-visible (UV-Vis) spectrometer to calculate the respective contents of PMF and 5-OH-PMF in the extract.

In addition to the use of microwave heating and high temperature and high pressure heating to promote the PMF demethylation reaction, the present invention also uses HPLC/UV-Vis to quantitatively analyze the produced extract, and will be produced by microwave heating and high temperature and high pressure heating. The 5-OH-PMFs yield and rate are compared with the prior art oil bath heating mode, so that the efficacy of the 5-OH-PMFs of the present invention can be more clearly manifested.

Preferably, the PMF and the 5-OH-PMF dry extract powder are taken as 5 mg, and the concentration of 100% EtOH is used to form 25 mL of the solution to be tested; and the gravity acceleration ratio of the centrifuge is 6800 g, the time of centrifugation. It is 5 minutes. The selection of each of the above solvents (solutions) and the speed and time of centrifugation, but the optimal reaction conditions found after several experimental tests.

Figure 1 shows the chemical structure of six major PMFs and their molecular weights.

Figure 2 shows the chemical structure of six major 5-OH-PMFs and their molecular weights.

The following together with the drawings and the preferred embodiment of the present invention, further The technical means by which the present invention is achieved for the purpose of achieving a predetermined creation are set forth.

Example 1: Microwave Heating Method - Open Condensing System

Take 50mg Nobiletin or Tangeretin in a round bottom flask, add 20mL of 6N HCl and 100% EtOH mixed solution (1:1, v/v) for acid treatment, and use a focused microwave synthesizer with condensation The reaction was carried out in an open system for demethylation. The set power was 150 W, the temperature was 105 ° C, and the microwave reaction time was 0.5, 1, 1.5 and 2 hours, respectively. After the reaction was completed, the reaction solution was allowed to cool to room temperature. The mixture was neutralized with acid and alkali with an equivalent concentration of 6N NaOH, and concentrated under reduced pressure to remove the solvent. Then, 20 mL of distilled water was added, and then the reaction solution obtained by neutralizing the acid-base neutralization reaction was separated by 20 mL of EtOAc. After the extraction was completed, the extraction was completed. The EtOAc extract was concentrated under reduced pressure to remove the extract, and the mixture was evaporated.

Example 2: High temperature and high pressure heating method

50 mg of Nobiletin or Tangeretin was placed in a serum bottle, and 20 mL of a 6 N HCl solution and a 100% EtOH mixed solution (1:1, v/v) were added for acid treatment at a temperature of 121 ° C and a pressure of 1.5 kg / m ^{2 is} subjected to high temperature and high pressure heat treatment for 15 and 30 minutes to carry out a demethylation reaction. After the reaction is completed, the reaction solution is allowed to cool to room temperature, and then neutralized with an alkalinity of 6 N NaOH, and concentrated by decompression under reduced pressure. After the solvent, 20 mL of distilled water was added, and the reaction solution of the acid-base neutralization reaction was separated and extracted with 20 mL of EtOAc. After the extraction was completed, the EtOAc extract was concentrated under reduced pressure to remove the extract, and the above extraction step was repeated twice, EtOAc. The extract was concentrated under reduced pressure to give a dried extract powder.

Example 3: Quantification of acid treated samples

5 mg of the above acid-treated EtOAc extract powder was dissolved in 100% EtOH and quantified to 25 mL. The appropriate amount of the solution was centrifuged at a gravity acceleration rate of 6800 g for 5 minutes, and then quantitatively analyzed by HPLC/UV-Vis to integrate the area. The results were substituted into the calibration curve to calculate the respective contents of PMF and 5-OH-PMF in the acid-treated extract.

Comparative example: oil bath heating method

50 mg of Nobiletin or Tangeretin was placed in a round bottom flask, and 20 mL of a 6 N HCl solution and a 100% EtOH mixed solution (1:1, v/v) were added for acid treatment, and refluxed in an oil bath for 15 hours. Demethylation reaction, the reaction solution was allowed to cool to room temperature, then added with an equivalent concentration of 6N NaOH for acid-base neutralization, and concentrated under reduced pressure to remove the solvent, then added 20 mL of distilled water, and then 20 mL of EtOAc in acid and alkali The reaction solution after completion of the reaction was subjected to a layered extraction. After the extraction was completed, the EtOAc extract was concentrated under reduced pressure to remove the extract, and the above-mentioned extraction step was repeated twice, and the EtOAc extract was concentrated under reduced pressure to give a dried extract powder.

The experimental results of the above examples and comparative examples are shown in Tables 1 and 2. "O 15hr" indicates that the oil bath of the comparative example was heated for 15 hours; "M 0.5hr" indicates that the microwave of Example 1 was heated for 0.5 hours. Such a push; "H 0.25 hr" means high temperature and high pressure heating of Example 2 for 0.25 hours, and so on. Table 1 shows the contents of Nobiletin, 5-OH nobiletin, Tangeretin, and 5-OH tangeretin in each dry extract powder. Table 2 further analyzes the quantitative results, and calculates the yield of 5-OH tangeretin and 5-OH nobiletin. Rate (%) [yield = mg of 5-OH tangeretin (or 5-OH nobiletin) / mg of EtOAc extracts × 100%]. As can be seen from the analysis, the yield of 5-OH tangeretin formed by heating in an oil bath for 15 hours was 61.14±0.28%, and the yield of 5-OH nobiletin was 50.23±0.65%. The yields of 5-OH tangeretin at microwave heating for 0.5, 1, 1.5 and 2 hours were 21.19±0.16, 26.39±0.17, 33.58±0.31 and 40.79±0.77%, respectively, and the yields of 5-OH nobiletin were 20.51±0.25, 28.43, respectively. ±0.46, 37.97±0.10 and 44.77±0.7%. The yields of 5-OH tangeretin at high temperature and high pressure for 0.25 and 0.5 hours were 62.7 ± 1.53 and 83.87 ± 0.71%, respectively, and the yields of 5-OH nobiletin were 49.87 ± 0.13 and 61.56 ± 1.9%, respectively.

Then, the above analytical quantitative results were further converted into an hourly yield [hourly yield = yield / heating time (hour)], and the results are shown in Table 3, 5-OH produced per hour by heating in an oil bath. The yield of tangeretin was 4.08 ± 0.02%, and the hourly yield of 5-OH nobiletin was 3.35 ± 0.04%. The yields of 5-OH tangeretin generated by microwave heating were 42.39±0.32, 26.39±0.17, 22.39±0.21 and 20.39±0.39%, respectively, and the hourly yields of 5-OH nobiletin were 41.02±0.5 and 28.43±, respectively. 0.46, 25.31 ± 0.07 and 22.38 ± 0.35%. The yield of 5-OH tangeretin generated by high temperature and high pressure heating was 250.8±6.12 and 167.74±1.42%, respectively, and the hourly yield of 5-OH nobiletin was 199.48±0.52 and 123.13±3.8%, respectively.

After finishing comparison, it can be found that the 5-OH tangeretin converted by microwave heating treatment is 5.0-10.4 times of oil bath heat treatment [microwave treatment yield / heating time (hour) / oil bath treatment yield / heating time (hour) )], 5-OH nobiletin is 6.7-12.2 times that of oil bath heat treatment. 5-OH tangeretin converted to high temperature and high pressure per hour for oil bath treatment 41.1-61.5 倍 [high temperature and high pressure treatment yield / reaction time (hour) / oil bath treatment yield / reaction time (hour)], 5-OH nobiletin is 36.8-59.6 times the oil bath treatment. The statistical results show that the acid treatment of the high temperature and high pressure heating acid treatment and the microwave heating has better yield than the prior art acid treatment with oil bath heating, so as to rapidly carry out the organic synthesis test of 5-OH-PMFs. Especially, the acid treatment with high temperature and high pressure heating has the best effect.

In summary, the present invention utilizes the microwave heating method to convert the PMFs into 5-OH-PMFs. The chemical molecules can rapidly absorb the microwave energy by radiant heating, and at the same time have the advantages of uniform heating and high efficiency. In addition, the present invention also utilizes a high-temperature and high-pressure heating method to convert the PMFs into 5-OH-PMFs, and the probability of collision between chemical molecules is greatly increased, the reaction is more easily performed, and the reaction rate of converting PMFs into 5-OH-PMFs is effectively improved. As well as the yield, the biological activity, anticancer and anti-inflammatory effects of 5-OH-PMFs are further utilized, and the utilization of citrus peel is increased.

The above is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention. Although the present invention has been disclosed in the above preferred embodiments, it is not intended to limit the invention, and A person skilled in the art can make some modifications or modifications to equivalent embodiments by using the above-disclosed technical contents without departing from the technical scope of the present invention. The present invention is not limited to any simple modifications, equivalent changes and modifications of the above embodiments.

Claims (4)

A method for preparing polymethoxyflavones (PMFs) to monomethyl form (5-OH-PMFs) by citrus peel, comprising the following steps: (a) acid treatment, placing PMF in a container, and in a container Hydrochloric acid (HCl) and ethanol (Ethanol, EtOH) are added to form a reaction solution; (b) heat treatment, microwave heating of the reaction solution to promote the No. 5 carbon position of the PMF structure in the reaction solution The methoxy group is subjected to a demethylation reaction; (c) cooling treatment, the reaction solution is allowed to stand and allowed to cool to room temperature; (d) acid and alkali are neutralized, and sodium hydroxide (NaOH) is used as an acid base to neutralize the reaction solvent. Adding NaOH to the cooled reaction solution for acid-base neutralization reaction, after completion of the reaction, removing the acid-base neutralization reaction solvent by using a reduced pressure concentration method, and then adding distilled water to the reaction solution; and (e) extracting step to acetic acid B The ester (Ethyl acetate, EtOAc) is used as an extract, and the reaction solution completed by the acid-base neutralization reaction is subjected to layered extraction. After the extraction is completed, the extract is removed by a vacuum concentration method; and the above extraction step is repeated at least twice to obtain P The extract powder was dried with MF and 5-OH-PMF. A method for preparing a polymethoxyflavonol to a monomethyl form according to the citrus peel of claim 1, wherein the microwave instrument is an open system of a focusing microwave synthesizer coupled with a condenser. A method for preparing a polymethoxyflavonol to a monomethyl form according to the citrus peel of claim 2, wherein the focused microwave synthesizer is equipped with a condenser tube The open system has a set power of 150 W, a temperature of 105 ° C, and a heating time of 0.5 to 2 hours. A method of preparing a polymethoxyflavonate to a monomethyl form according to the citrus peel of claim 3, wherein the heating time is 0.5 hours.