Clausena lansium peel natural polyphenol composition and preparation method thereof

Abstract

The invention provides a clausena lansium peel natural polyphenol composition and a preparation method thereof, and relates to the technical field of biological medicines.The preparation method comprises the steps that a target polyphenol compound standard curve is constructed through high performance liquid chromatography, the content of target polyphenol compounds in clausena lansium peel extract is analyzed and detected, and a collection time period is determined; and eluting and separating the natural polyphenol composition in the clausena lansium peel extract by using preparative liquid chromatography, and collecting eluted components within a collection time period to obtain the clausena lansium peel natural polyphenol composition. The purity of the prepared natural polyphenol composition reaches 75% or above; the target polyphenol compound components and the polyphenol compound components with unknown structures, which are not compared by using the standard substance, are ensured to be contained; compared with a clausena lansium peel extract, a part of polyphenol compound monomer and a polyphenol monomer composition before purification, the clausena lansium peel extract has better anti-inflammatory activity. The technical problem that an effective purification preparation method for the natural polyphenol composition in the clausena lansium peel is lacked in the prior art is solved.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to a natural polyphenol composition of yellow peel and a preparation method thereof. Background Art

[0002] Clausena l'Aglaonema (Lour.) Skeels is a tree of the genus Clausena l'Aglaonema in the Rutaceae family. It is a specialty crop in the Lingnan region, including Guangdong, Guangxi, Hainan, Yunnan and Fujian. Clausena l'Aglaonema fruit is often eaten fresh or processed into juice, jam and other products due to its unique flavor and rich nutritional active ingredients. However, the peel of Clausena l'Aglaonema fruit has a hard texture and a slightly bitter taste, so it is rarely eaten directly and is rarely used to be processed into food, and is mostly discarded. Clausena l'Aglaonema fruit was first recorded in the Compendium of Materia Medica. Its roots, leaves, fruits and seeds can all be used as medicine. Among them, the peel has a higher medicinal value, has the effects of diuresis and swelling, promoting qi and relieving pain, and is mainly used to treat symptoms related to inflammation, such as abdominal pain, phlegm and cough.

[0003] Polyphenols are the most widely distributed components of secondary metabolites of medicinal plants, which affect color, flavor and nutritional effects. Studies have shown that polyphenols have anti-inflammatory effects, and kumquat peel contains more polyphenols than the pulp and core.

[0004] At present, studies have been conducted to obtain extracts from the peel of Phellodendron chinense, and various polyphenolic compounds such as rutin, quercetin and isoquercetin have been identified from them, and polyphenolic compounds with unknown structures are also contained. The anti-inflammatory activity of the extract of Phellodendron chinense and some of the phenolic compound monomers therein have been experimentally confirmed. A natural polyphenol composition is isolated and prepared from the peel of Phellodendron chinense, in which polyphenolic compounds of different types and contents may exert a certain degree of anti-inflammatory effect through synergistic or antagonistic effects. Compared with the crude extract of Phellodendron chinense peel, the concentration of effective phenolic compounds in the purified natural polyphenol composition is higher, and the interference of impurity components on the activity is eliminated. Compared with the phenolic compound monomers, the purified polyphenol composition can effectively save purification time and economic costs, and can also cover some phenolic compounds with unknown structures but significant effects, and may make the product have better anti-inflammatory effects through synergistic or antagonistic effects. However, there is currently a lack of effective purification and preparation methods for natural polyphenol compositions in Phellodendron chinense peel. High performance liquid chromatography (HPLC) is the most common method for separating and quantifying polyphenolic compounds in samples, while preparative high performance liquid chromatography (prep-HPLC) is mostly used for the separation and recovery of target monomer compounds. However, for extracts with complex components, they are subject to interference from a large number of impurities and limitations of instrument detection limits and sensitivity, resulting in low separation and detection efficiency. Therefore, sample pretreatment is usually required before entering liquid chromatography separation to improve the purity of the target component, including solvent extraction (Liquid liquid extraction, LLE), solid phase extraction (SPE), macroporous resin adsorption chromatography (Column chromatography, CC), etc. The separation process is cumbersome, time-consuming, and the product yield is low.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] One of the purposes of the present invention is to provide a method for preparing a natural polyphenol composition in the peel of Wampee, so as to at least solve the technical problem in the prior art of lacking an effective purification preparation method for the natural polyphenol composition in the peel of Wampee.

[0007] The second object of the present invention is to provide a natural polyphenol composition of wampee peel.

[0008] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0009] In a first aspect, the present invention provides a method for preparing a natural polyphenol composition of wampee peel, comprising the following steps:

[0010] A. Use high performance liquid chromatography to construct a standard curve of target polyphenolic compounds, analyze and detect the content of target polyphenolic compounds in the yellow peel peel extract, and determine the collection time period;

[0011] B. using preparative liquid chromatography to elute and separate the natural polyphenol composition in the wampee peel extract, collecting eluted components within a collection time period to obtain the wampee peel natural polyphenol composition;

[0012] The analysis and detection comprises using 0.05% formic acid water and acetonitrile as mobile phases, 0.05% formic acid water as A, acetonitrile as B; the elution procedure is 0-3 min, 10%-20% B; 3-15 min, 20%-60% B; 15-18 min, 60%-90% B; 18-22 min, 90%-10% B; 22-25 min, 10% B;

[0013] The elution separation includes water and methanol as mobile phases, water as A, methanol as B; the elution procedure is: 0-5min, 10%-20% B; 5-15min, 20%-60% B; 15-20min, 60% B; 20-25min, 60%-90% B; 25-30min, 90%-10% B.

[0014] Further, the elution separation also includes a detection wavelength of 200 to 400 nm;

[0015] Preferably, the elution separation further comprises an injection volume of 50 to 70 μL;

[0016] Preferably, the elution separation further comprises a flow rate of 1.0 to 2.0 mL / min;

[0017] Preferably, the elution separation further comprises that the chromatographic column is a C18 column;

[0018] Preferably, the elution separation further comprises a column temperature of 35-40°C.

[0019] Further, the analysis and detection also includes a detection wavelength of 200 to 400 nm;

[0020] Preferably, the analytical detection also includes an injection volume of 35 to 45 μL;

[0021] Preferably, the analytical detection also includes a flow rate of 0.5 to 1.0 mL / min;

[0022] Preferably, the analysis and detection further comprises that the chromatographic column is a C18 column;

[0023] Preferably, the analytical detection also includes a column temperature of 35-40°C.

[0024] Further, the polyphenolic compounds include at least myricetin-3-O-galactoside, nicotin, rutin, isorhamnetin-3-O-neohesperidoside, isoquercitrin, quercitrin and 8-hydroxypsoralen;

[0025] The collection time period in the elution separation is 10 to 20 minutes.

[0026] Furthermore, the preparation method of the Wampee peel extract is an ethanol extraction method.

[0027] Furthermore, the ethanol extraction method comprises taking a powder of a Wampee peel sample and adding an ethanol solution to extract the Wampee peel extract after concentration and drying;

[0028] Preferably, the volume ratio of the kumquat peel powder to the ethanol solution is 1:20-30;

[0029] Preferably, the volume concentration of the ethanol solution is 65% to 75%;

[0030] Preferably, the extraction temperature is 60-70°C;

[0031] Preferably, the extraction time is 1 h;

[0032] Preferably, the extraction is performed 3 times;

[0033] Preferably, the drying method is freeze drying.

[0034] Furthermore, the treatment method for the eluted and separated Wampee peel extract comprises preparing a 10-30 mg / ml Wampee peel extract solution using 80%-90% methanol solution (V / V), and then filtering the solution through a microporous filter membrane.

[0035] Furthermore, the processing method of the Wampee peel extract for the analysis and detection comprises using 80% to 90% methanol solution (V / V) to prepare 8 to 12 mg / ml Wampee peel extract solution, and then filtering it through a microporous filter membrane.

[0036] Furthermore, the pore size of the microporous filter membrane is 0.20-0.50 μm, preferably 0.22 μm or 0.45 μm.

[0037] In a second aspect, the present invention provides a natural polyphenol composition of wampee peel, which is prepared by the above-mentioned preparation method.

[0038] The invention provides a method for preparing a natural polyphenol composition of kumquat peel. Compared with traditional polyphenol purification methods such as macroporous resin chromatography, the combination of high performance liquid chromatography and preparative liquid chromatography separation technology can directly obtain the separation of target polyphenol compounds and impurity components, as well as the content ratio of target polyphenol compounds; the separation of natural polyphenol composition by preparative liquid chromatography is the target, and the requirements for separation conditions do not need to be too high, which effectively saves the cost of separation and preparation; the purity of the prepared natural polyphenol composition reaches 75% or more, while the purity of the kumquat peel extract before purification is only about 37% to 45%; and it is ensured that the target polyphenol compound components and the polyphenol compound components with unknown structures that are not compared with the kumquat peel extract before purification, part of the polyphenol compound monomers and the polyphenol monomer composition have better anti-inflammatory activity. The invention solves the technical problem that there is a lack of effective purification and preparation methods for natural polyphenol compositions in kumquat peel in the prior art. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0040] Figure 1 The HPLC ultraviolet absorption spectrum of the mixed standard provided by the present invention at 254nm;

[0041] Figure 2 The HPLC ultraviolet absorption spectrum of the mixed standard provided by the present invention at 320nm;

[0042] Figure 3 HPLC ultraviolet absorption spectra at 254 nm of different wampee peel extracts provided in the embodiments of the present invention;

[0043] Figure 4 HPLC ultraviolet absorption spectra at 320 nm of different wampee peel extracts provided in the embodiments of the present invention;

[0044] Figure 5 The prep-HPLC ultraviolet absorption spectrum of the wampee peel extract provided in Example 2 of the present invention at 254 nm;

[0045] Figure 6 The HPLC ultraviolet absorption spectrum of the natural polyphenol composition of the yellow peel provided in Example 2 of the present invention at 320 nm;

[0046] Figure 7The HPLC ultraviolet absorption spectrum of the natural polyphenol composition of wampee peel provided in Comparative Example 2 of the present invention at 280 nm. DETAILED DESCRIPTION

[0047] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, however, in the case of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. In this application, unless otherwise stated, the use of "or" means "and / or". In addition, the use of the term "including" and other forms is non-limiting.

[0048] The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated.

[0049] In one aspect, the present invention provides a method for preparing a natural polyphenol composition of wampee peel, comprising the following steps:

[0050] A. Use high performance liquid chromatography to construct a standard curve of target polyphenolic compounds, analyze and detect the content of target polyphenolic compounds in the yellow peel peel extract, and determine the collection time period;

[0051] B. using preparative liquid chromatography to elute and separate the natural polyphenol composition in the wampee peel extract, collecting eluted components within a collection time period to obtain the wampee peel natural polyphenol composition;

[0052] The analysis and detection comprises using 0.05% formic acid water and acetonitrile as mobile phases, 0.05% formic acid water as A, acetonitrile as B; the elution procedure is 0-3 min, 10%-20% B; 3-15 min, 20%-60% B; 15-18 min, 60%-90% B; 18-22 min, 90%-10% B; 22-25 min, 10% B;

[0053] The elution separation includes water and methanol as mobile phases, water as A, methanol as B; the elution procedure is: 0-5min, 10%-20% B; 5-15min, 20%-60% B; 15-20min, 60% B; 20-25min, 60%-90% B; 25-30min, 90%-10% B.

[0054] Compared with traditional polyphenol purification methods such as macroporous resin chromatography, the combination of high performance liquid chromatography and preparative liquid chromatography separation technology can directly obtain the separation of target polyphenol compounds and impurity components, as well as the content ratio of target polyphenol compounds; the separation of natural polyphenol compositions by preparative liquid chromatography is targeted, and the requirements for separation conditions do not need to be too high, effectively saving the cost of separation and preparation; the purity of the prepared natural polyphenol composition is 75% or more, while the purity of the yellow peel peel extract before purification is only about 37% to 45%; and it is ensured that the target polyphenol compound components and the unknown polyphenol compound components that have not been compared with the standard are contained; compared with the yellow peel peel extract before purification, some polyphenol compound monomers and polyphenol monomer compositions, it has better anti-inflammatory activity. The technical problem of the lack of an effective purification and preparation method for the natural polyphenol composition in the yellow peel peel in the prior art is solved.

[0055] The collection time can be determined based on the peak time of the target polyphenol compound and the time distance separating it from the impurity peak area.

[0056] In some specific embodiments, the elution separation further comprises a detection wavelength of 200-400 nm. Within this wavelength range, polyphenol compounds can be effectively identified, the retention time distribution of polyphenol compounds can be determined, and then the collection time period can be determined.

[0057] Specifically, the detection wavelength in the elution separation may be, but is not limited to, 200 nm, 240 nm, 280 nm, 320 nm, 360 nm or 400 nm, and may also be any value between 200 and 400 nm.

[0058] In order to ensure the normal separation ability and collection efficiency of the chromatographic column during the elution and separation of the natural polyphenol composition of the wampee peel, in some specific embodiments, the elution and separation also includes an injection volume of 50 to 70 μL.

[0059] Specifically, the injection volume in the elution separation can be but is not limited to 50 μL, 52 μL, 54 μL, 56 μL, 58 μL, 60 μL, 62 μL, 64 μL, 66 μL, 68 μL or 70 μL, and can also be any value between 50 and 70 μL.

[0060] In some specific embodiments, the elution separation further comprises a flow rate of 1.0 to 2.0 mL / min to ensure the separation effect of the polyphenol component and the impurity component.

[0061] Specifically, the flow rate in the elution separation can be but is not limited to 1.0 mL / min, 1.1 mL / min, 1.2 mL / min, 1.3 mL / min, 1.4 mL / min, 1.5 mL / min, 1.6 mL / min, 1.7 mL / min, 1.8 mL / min, 1.9 mL / min or 2.0 mL / min, and can also be any value between 1.0 and 2.0 mL / min.

[0062] In some specific embodiments, the elution separation further comprises a chromatographic column being a C18 column;

[0063] In some specific embodiments, the elution separation further comprises a column temperature of 35-40°C.

[0064] In some specific embodiments, the analysis and detection further comprises a detection wavelength of 200-400 nm. Within this wavelength range, polyphenol compounds can be effectively identified, the retention time distribution of polyphenol compounds can be determined, and then the collection time period can be determined.

[0065] Specifically, the detection wavelength in the analysis and detection may be, but is not limited to, 200 nm, 240 nm, 280 nm, 320 nm, 360 nm or 400 nm, and may also be any value between 200 and 400 nm.

[0066] In the analysis and detection, in order to achieve accurate quantitative detection of natural polyphenol compounds in the kumquat peel, in some specific embodiments, the analysis and detection also includes an injection volume of 35 to 45 μL.

[0067] Specifically, the injection volume in the analysis and detection can be but is not limited to 35μL, 36μL, 37μL, 38μL, 39μL, 40μL, 41μL, 42μL, 43μL, 44μL, 45μL, 46μL, 47μL, 48μL, 49μL or 70μL, and can also be any value between 35 and 45μL.

[0068] In some specific embodiments, the analytical detection also includes a flow rate of 0.5 to 1.0 mL / min.

[0069] Specifically, the flow rate in the analytical detection can be but is not limited to 0.5 mL / min, 0.6 mL / min, 0.7 mL / min, 0.8 mL / min, 0.9 mL / min or 1.0 mL / min, and can also be any value between 0.5 and 1.0 mL / min.

[0070] In some specific embodiments, the analysis and detection further comprises a chromatographic column being a C18 column;

[0071] In some specific embodiments, the analytical detection also includes a column temperature of 35-40°C.

[0072] In some specific embodiments, the target polyphenolic compounds include at least myricetin-3-O-galactoside, nicotin, rutin, isorhamnetin-3-O-neohesperidoside, isoquercitrin, quercitrin and 8-hydroxypsoralen. By selecting polyphenolic compounds as standard substances, multiple polyphenolic compounds of Phellodendron chinense peel can be detected at one time. Separating and comparing multiple polyphenolic compounds of Phellodendron chinense peel can ensure the accuracy of the collection time period to improve the collection efficiency.

[0073] According to the types of polyphenolic compounds that may be contained in the extract of the yellow peel, various types of polyphenolic compound standards were prepared into solutions and injected into high performance liquid chromatography. The results showed that the retention time of various types of polyphenolic compounds was close, and the peaks were concentrated in the range of 7 to 15 minutes. The high performance liquid chromatogram of the yellow peel extract ( Figure 3 ) It can be seen that there are continuous multiple peaks in the interval of 7 to 15 minutes, and 7 peaks correspond to the retention times of 7 polyphenolic compounds, namely, myricetin-3-O-galactoside, nicotin, rutin, isorhamnetin-3-O-neohesperidin, isoquercitrin, quercetin and 8-hydroxypsoralen. Therefore, it can be basically concluded that the interval of 7 to 15 minutes in the high performance liquid chromatography is the characteristic fingerprint area of ​​yellow peel peel polyphenols, and the separation time from the impurity peak area (2 to 4 minutes, 20 to 25 minutes) is far, reaching more than 3 minutes. Under this method, polyphenols and impurities can be well separated. In some specific embodiments, the collection time period in the elution separation is 10 to 20 minutes. On the basis of the elution procedure of high performance liquid chromatography, the elution method of preparative liquid chromatography is set. The elution procedures of the two are basically the same. The elution time of the corresponding elution gradient (i.e., about 60% B) of the polyphenolic compounds is appropriately extended in the preparative liquid chromatography. It can be seen that the elution diagram of the preparative liquid chromatography ( Figure 5 ) is basically consistent with the distribution of the polyphenol characteristic fingerprint region and the impurity peak region in the high performance liquid chromatography, and the polyphenol characteristic fingerprint region and the impurity region are well separated. Therefore, the collection time period is determined to be the time period corresponding to the polyphenol characteristic fingerprint region, that is, 10-20 minutes. After the collected natural polyphenol composition is prepared into a solution, it is injected into the high performance liquid chromatography again for analysis. From the high performance liquid chromatography ( Figure 6 ) It can be seen that the polyphenol composition is still mainly distributed in 7-15min, and the purity is significantly improved, which further confirms that the collection time period of 10-20min determined in the preparative liquid chromatography is correct.

[0074] Polyphenol compounds are easily soluble in ethanol. In some specific embodiments, the preparation method of the Phellodendron chinense peel extract is an ethanol extraction method.

[0075] In some specific embodiments, the ethanol extraction method comprises taking a powder of a Wampee peel sample and adding it to an ethanol solution for extraction, and then concentrating and drying to obtain a Wampee peel extract.

[0076] Appropriate ethanol content can improve the extraction efficiency of the wampee peel extract. In some specific embodiments, the volume ratio of the wampee peel powder to the ethanol solution is 1:20-30.

[0077] In some specific embodiments, the volume concentration of the ethanol solution is 65% to 75%.

[0078] In some specific embodiments, the extraction temperature is 60-70°C.

[0079] In some specific embodiments, the extraction time is 1 hour.

[0080] In some specific embodiments, the extraction is performed three times.

[0081] In some specific embodiments, the drying method is freeze-drying.

[0082] The methanol solution is used to prepare the Wampee peel extract solution, which can effectively remove sugar impurities after being filtered through a microporous filter membrane, thereby improving the efficiency of elution separation and analytical detection. In some specific embodiments, the method for treating the Wampee peel extract for elution separation comprises using 80% to 90% methanol solution (V / V) to prepare a 10 to 30 mg / ml Wampee peel extract solution, and then filtering it through a microporous filter membrane. In some specific embodiments, the method for treating the Wampee peel extract for analytical detection comprises using 80% to 90% methanol solution (V / V) to prepare an 8 to 12 mg / ml Wampee peel extract solution, and then filtering it through a microporous filter membrane.

[0083] In some specific embodiments, the pore size of the microporous filter membrane is 0.20-0.50 μm.

[0084] Specifically, the pore size of the microporous filter membrane may be, but is not limited to, 0.20 μm, 0.22 μm, 0.30 μm, 0.40 μm, 0.45 μm, 0.50 μm, or any value between 0.20 and 0.50 μm, preferably 0.22 μm or 0.45 μm.

[0085] In some specific embodiments, the method for preparing the kumquat peel raw material includes taking fresh kumquat peel, drying it and then crushing it.

[0086] The moisture content of the dried kumquat peel powder is controlled at 2.5% to 5.0%. The drying conditions include a temperature of 40 to 50° C. and drying for 36 to 48 hours. Specifically, a heat pump dryer can be used for drying. The effect of the processing process on the natural polyphenol composition in the kumquat peel raw material can be reduced, and the extraction efficiency can be improved.

[0087] According to another aspect of the present invention, a natural polyphenol composition of wampee peel is provided, which is prepared by the above-mentioned preparation method.

[0088] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0089] Myricetin-3-O-galactoside, quercetin, and 8-hydroxypsoralen were purchased from Medson with batch numbers RM2096, RM0926, and RM1147, respectively; isorhamnetin-3-O-neohesperidoside and nicotinoids were purchased from ChemFaces with batch numbers CFN99744 and CFN99830, respectively; isoquercetin was purchased from Maclean with batch number Q817141; and rutin was purchased from Yuanye with batch number B20771.

[0090] Establish a standard curve: use myricetin-3-O-galactoside, nicotin, rutin, isorhamnetin-3-O-neohesperidoside, isoquercetin, quercetin and 8-hydroxypsoralen as standard substances to prepare a mixed standard solution, wherein the concentrations of isorhamnetin-3-O-neohesperidoside, quercetin and 8-hydroxypsoralen are 50 μg / ml, the concentrations of myricetin-3-O-galactoside, nicotin, isoquercetin are 100 μg / ml, and the concentration of rutin is 200 μg / ml. Elution was performed in high performance liquid chromatography. The spectral column used was a C18 column; the maximum absorption wavelength for the detection of rutin and quercetin was 254nm, and the maximum absorption wavelength for the detection of myricetin-3-O-galactoside, nicotin, isorhamnetin-3-O-galactoside, isoquercetin, and 8-hydroxypsoralen was 320nm; the injection volume was 1μL, 2μL, 5μL, 10μL, and 20μL; the flow rate was 1.0mL / min; the column temperature was 40℃; 0.05% formic acid water (A) and acetonitrile (B) were used as the mobile phases, and the elution program was 0-3min, 10%-20% B; 3-15min, 20%-60% B; 15-18min, 60%-90% B; 18-22min, 90%-10% B; 22-25min, 10% B. The results are as follows Figure 1-2 As shown, the peak numbers correspond to those in Table 1. The standard curve fitting is shown in Table 1.

[0091] Table 1 Linear standard curve of polyphenol content

[0092]

[0093] Example 1

[0094] Preparation of natural polyphenol composition in the peel of kumquat I: Raw materials preparation: Wash and peel fresh kumquats. Dry the kumquat peels in a heat pump dryer at 40°C for 48 hours until the moisture content is 4.52%, and then crush.

[0095] Preparation of the extract of the yellow peel: 10 g of the yellow peel powder was added with 30 times the volume of 70% ethanol solution (V / V), and extracted 3 times at 70%, each time for 1 hour. The extract was filtered, concentrated, and freeze-dried to obtain the yellow peel extract, in which the polyphenol purity was 37.13%.

[0096] Separation and analysis of polyphenols in the extract of Phellodendron chinense peel: Use 80% methanol solution (V / V) to prepare a 10mg / ml solution of Phellodendron chinense peel extract, and pass it through a 0.22μm microporous filter membrane. In high performance liquid chromatography (HPLC), the chromatographic column used was a C18 column (ZorbaxSB-C18, 4.6mm×250mm 5-Micro, Agilent); the detection wavelength was 254 / 320nm; the injection volume was 40μL; the flow rate was 1.0mL / min; the column temperature was 40; 0.05% formic acid water (A) and acetonitrile (B) were used as the mobile phases, and the elution program was: 0-3min, 10%-20% B; 3-15min, 20%-60% B; 15-18min, 60%-90% B; 18-22min, 90%-10% B; 22-25min, 10% B. The results are as follows: Figure 3 and Figure 4 As shown, the peak numbers correspond to Table 1. The measurement results showed that the contents of myricetin-3-O-galactoside, nicotin, rutin, isorhamnetin-3-O-neohesperidoside, isoquercitrin, quercitrin and 8-hydroxypsoralen in the extract were 66.51 mg / 100 g, 57.37 mg / 100 g, 440.03 mg / 100 g, 57.05 mg / 100 g, 45.24 mg / 100 g, 124.47 mg / 100 g and 16.70 mg / 100 g, respectively.

[0097] Preparation of natural polyphenol composition from Wampee peel: 80% methanol solution (V / V) was used to prepare a 10 mg / ml Wampee peel extract solution, which was filtered through a 0.22 μm microporous filter membrane. In preparative liquid chromatography (prep-HPLC), a C18 column (Zorbax Eclipse XDB-C18, 4.6 mm × 250 mm 5-Micro, Agilent) is used as a chromatographic column; the detection wavelength is 254 nm; the injection volume is 50 μL; the flow rate is 1.0 mL / min; the column temperature is 40°C; water (A) and methanol (B) are used as mobile phases, and the elution procedure is: 0-5 min, 10%-20% B; 5-15 min, 20%-60% B; 15-20 min, 60% B; 20-25 min, 60%-90% B; 25-30 min, 90%-10% B, and the elution components of 10-20 min are collected, concentrated, and freeze-dried to obtain a natural polyphenol composition of kumquat peel with a purity of 75.47%.

[0098] Example 2

[0099] Preparation of natural polyphenol composition in the peel of No. II variety kumquats Raw materials preparation: fresh kumquats were washed and peeled. The kumquat peels were dried in a heat pump dryer at 50°C for 36 hours until the moisture content was 4.38%, and then crushed.

[0100] Extraction of Wampee peel: Take 15g of Wampee peel powder, add 20 times volume of 75% ethanol solution (V / V), extract 3 times at 60°C, each time for 1 hour. The extract is filtered, concentrated, and freeze-dried to obtain Wampee peel extract, in which the purity of polyphenols is 40.87%.

[0101] Separation and analysis of polyphenols in the extract of Phellodendron chinense peel: Use 85% methanol solution (V / V) to prepare 8mg / ml Phellodendron chinense peel extract solution, and pass it through a 0.22μm microporous filter membrane. In the high performance liquid chromatography, the chromatographic column used was a C18 column (Zorbax SB-C18, 4.6mm×250mm 5-Micro, Agilent); the detection wavelength was 254 / 320nm; the injection volume was 45μL; the flow rate was 1.0mL / min; the column temperature was 40; 0.05% formic acid water (A) and acetonitrile (B) were used as the mobile phases, and the elution program was: 0-3min, 10%-20% B; 3-15min, 20%-60% B; 15-18min, 60%-90% B; 18-22min, 90%-10% B; 22-25min, 10% B. The results are as follows Figure 3 and Figure 4As shown, the peak numbers correspond to Table 1. The measurement results showed that the contents of myricetin-3-O-galactoside, nicotin, rutin, isorhamnetin-3-O-neohesperidoside, isoquercitrin, quercitrin and 8-hydroxypsoralen in the extract were 89.90 mg / 100 g, 107.71 mg / 100 g, 735.74 mg / 100 g, 93.59 mg / 100 g, 77.23 mg / 100 g, 111.32 mg / 100 g and 22.66 mg / 100 g, respectively.

[0102] Preparation of natural polyphenol composition from Wampee peel: 85% methanol solution (V / V) was used to prepare a 30 mg / ml Wampee peel extract solution, which was filtered through a 0.22 μm microporous filter membrane. In the preparative liquid chromatography, a C18 column (Zorbax EclipseXDB-C18, 4.6mm×250mm 5-Micro, Agilent) was used as the chromatographic column; the detection wavelength was 254nm; the injection volume was 70μL; the flow rate was 1.5mL / min; the column temperature was 40; water (A) and methanol (B) were used as the mobile phases, and the elution procedure was: 0-5min, 10%-20% B; 5-15min, 20%-60% B; 15-20min, 60% B; 20-25min, 60%-90% B; 25-30min, 90%-10% B, and the elution fractions of 10-20min were collected, concentrated, and freeze-dried to obtain a natural polyphenol composition of the yellow peel with a purity of 78.65%. The chromatogram is shown in FIG. Figure 5 and Figure 6 As shown, 4 sample bottles (sample bottle 9 to sample bottle 12) are used, and each sample bottle continuously collects eluate for 2.5 minutes, and after merging, elution components of 10-20 minutes are obtained.

[0103] Example 3

[0104] Preparation of natural polyphenol composition in the peel of kumquat III: Raw materials preparation: Wash and peel fresh kumquats. Dry the kumquat peels in a heat pump dryer at 45°C for 40 hours until the moisture content is 2.98%, and then crush.

[0105] Extraction of Wampee peel: Take 20g of Wampee peel powder, add 25 times the volume of 65% ethanol solution (V / V), and extract 3 times at 65, each time for 1 hour. The extract is filtered, concentrated, and freeze-dried to obtain Wampee peel extract, in which the purity of polyphenols is 45.06%.

[0106] Separation and analysis of polyphenols in the extract of kumquat peel: Use 90% methanol solution (V / V) to prepare a 12mg / ml kumquat peel extract solution, and pass it through a 0.22μm microporous filter membrane. In the high performance liquid chromatography, the chromatographic column used was a C18 column (Zorbax SB-C18, 4.6mm×250mm 5-Micro, Agilent); the detection wavelength was 280 / 330nm; the injection volume was 35μL; the flow rate was 0.8mL / min; the column temperature was 35; 0.05% formic acid water (A) and acetonitrile (B) were used as the mobile phases, and the elution program was: 0-3min, 10%-20% B; 3-15min, 20%-60% B; 15-18min, 60%-90% B; 18-22min, 90%-10% B; 22-25min, 10% B. The results are as follows Figure 3 and Figure 4 As shown, the peak numbers correspond to Table 1. The measurement results showed that the contents of myricetin-3-O-galactoside, nicotin, rutin, isorhamnetin-3-O-neohesperidoside, isoquercitrin, quercitrin and 8-hydroxypsoralen in the extract were 138.93 mg / 100 g, 98.90 mg / 100 g, 761.62 mg / 100 g, 99.56 mg / 100 g, 75.29 mg / 100 g, 172.20 mg / 100 g and 52.29 mg / 100 g, respectively.

[0107] Preparation of natural polyphenol composition from Wampee peel: 90% methanol solution (V / V) was used to prepare a 20 mg / ml Wampee peel extract solution, which was filtered through a 0.22 μm microporous filter membrane. In the preparative liquid chromatography, a C18 column (Zorbax EclipseXDB-C18, 4.6mm×250mm 5-Micro, Agilent) is used as a chromatographic column; the detection wavelength is 280nm; the injection volume is 60μL; the flow rate is 2.0mL / min; the column temperature is 35; water (A) and methanol (B) are used as mobile phases, and the elution program is: 0-5min, 10%-20% B; 5-15min, 20%-60% B; 15-20min, 60% B; 20-25min, 60%-90% B; 25-30min, 90%-10% B, and the elution components of 10-20min are collected, concentrated, and freeze-dried to obtain a natural polyphenol composition of kumquat peel with a purity of 81.90%.

[0108] Comparative Example 1

[0109] Different from Example 1, if the polyphenols of the Wampee peel extract are not separated and analyzed in high performance liquid chromatography, and the Wampee peel extract is directly separated in preparative liquid chromatography, it is necessary to explore the separation conditions of the polyphenol components and the impurity components in the preparative liquid chromatography from scratch, and it is impossible to quickly know the retention time interval of the polyphenol compounds, so it is impossible to accurately select the appropriate elution collection time to prepare the natural polyphenol composition. For different Wampee peel extract samples, although the collection time for the subsequent preparation of the natural polyphenol composition is fixed at 10-20min, it is necessary to know the content and ratio of each target polyphenol compound in the composition at the same time, so before each preparation, the high performance liquid chromatography analysis and detection procedure is indispensable.

[0110] Comparative Example 2

[0111] The HPLC separation and analysis conditions different from those in Example 1 were used, and the specific conditions were as follows:

[0112] The conditions of analytical HPLC were Agilent XDB C18 column: 18 mm × 250 mm, 5 μm; mobile phase A was acetonitrile + 0.01% formic acid, and phase B was ultrapure water + 0.01% formic acid; the gradient elution program was 0 to 30 min, the volume ratio of phase A to phase B was 5:95 to 25:75; 30 min to 60 min, the volume ratio of phase A to phase B was 25:75 to 45:55; the flow rate was 1.0 mL / min, and the ultraviolet absorbance was detected at 280 nm. The results are as follows Figure 7 As shown, this separation method cannot elute the polyphenols of the yellow peel, and cannot achieve effective separation and content determination of different types of polyphenols. Therefore, for the yellow peel extract, it is expected to use analytical HPLC to check the peak conditions and trends of polyphenols, and then use preparative HPLC to separate the main polyphenol chromatographic peaks, and then obtain purified polyphenols, which cannot be achieved under the separation conditions of this analytical HPLC.

[0113] Experimental Example 1 Determination of NO Content

[0114] This experiment selected the kumquat peel extract and the kumquat peel natural polyphenol composition prepared in Example 2, and was specifically conducted according to the following operations.

[0115] After culturing RAW264.7 cells, 10 6 / mL cell suspension, 100 μL per well was added to a 96-well plate. Incubate in an incubator at 37 with 5% CO2 for 24 hours, then discard the culture medium. Add different concentrations of Phellodendron chinense peel extract or Phellodendron chinense peel natural polyphenol composition. After 3 hours of drug intervention, add 1 μg / mL lipopolysaccharide (LPS) to induce inflammatory response in the cells. After 16 hours, collect the cell culture supernatant, and determine the NO content according to the instructions of the Greiss kit. The method of using the Griess kit is as follows: add 50 μL of cell culture supernatant, 50 μL of Griess reagent A and 50 μL of Griess reagent B to a 96-well plate, mix thoroughly, measure the absorbance at a wavelength of 540 nm, and calculate the NO concentration based on the standard curve of the standard Nitrites.

[0116] The experimental groups were as follows: ① blank control group: culture medium incubation; ② model group: culture medium incubation + LPS induction; ③ low concentration of Phellodendron chinense peel extract group: 25 μg / mL Phellodendron chinense peel extract intervention + LPS induction; ④ high concentration of Phellodendron chinense peel extract group: 50 μg / mL Phellodendron chinense peel extract intervention + LPS induction; ⑤ low concentration of Phellodendron chinense peel natural polyphenol composition group: 25 μg / mL Phellodendron chinense peel extract intervention + LPS induction; ⑥ high concentration of Phellodendron chinense peel natural polyphenol composition group: 50 μg / mL Phellodendron chinense peel extract intervention + LPS induction; ⑦ positive control group: 10 μg / mL dexamethasone + LPS induction.

[0117] Table 2 Effects of each group of samples on inhibiting LPS-induced NO secretion in RAW 264.7 cells

[0118] Grouping NO content (μM) Blank control group 2.47±0.30a Model Group 47.22±0.75g 25 μg / mL Phellodendron chinense peel extract group 41.61±0.97f 50 μg / mL Phellodendron chinense peel extract group 38.58±0.46e 25 μg / mL natural polyphenol composition group of yellow peel 36.31±1.10d 50μg / mL natural polyphenol composition group of yellow peel 32.07±0.32c Positive control group 28.03±0.17b

[0119] Among them, different letters between groups represent significant differences (p<0.05).

[0120] As shown in Table 2, compared with the same concentration of the Wampee peel extract and the Wampee peel natural polyphenol composition, the Wampee peel natural polyphenol composition has a significantly stronger ability to inhibit LPS-induced RAW264.7 cells from secreting the inflammatory mediator NO, and has better anti-inflammatory activity. It can be seen that the elution separation process can effectively remove the impurities that interfere with the activity in the extract, and the natural polyphenol composition can more efficiently exert its anti-inflammatory effect.

[0121] Experimental Example 2 Western blot determination of inflammatory pathway proteins

[0122] This experiment uses the natural polyphenol composition of yellow peel prepared in Example 2 for the experiment, and specifically follows the following operations:

[0123] After culturing RAW 264.7 cells, 10 6 / mL cell suspension was inoculated in a 60mm diameter culture dish. After culturing for 24 hours in an incubator at 37 with 5% CO2, the culture medium was discarded. Different concentrations of natural polyphenol composition, polyphenol compound monomer or polyphenol monomer composition of yellow peel were added. After 3 hours of drug intervention, 1μg / mL LPS was added to induce inflammatory response in cells. After 16 hours, the culture medium was discarded, and phosphate buffered saline (PBS) was added to wash the cells, and then RIPA cell lysis buffer was added. After centrifugation of each group, the supernatant was taken and the total protein content was determined by BCA kit. After the protein sample was denatured, it was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membrane in a membrane transfer instrument. The PVDF membrane was blocked in 5% skim milk for 2 hours, and then the primary antibody was added according to the antibody instructions, and incubated overnight at 4 shaking. After washing with Tris buffered saline and detergent Tween 20 (TBST), add secondary antibody and continue incubation on a shaker at room temperature for 1 hour. After washing with TBST, prepare enhanced chemiluminescence (ECL) colorimetric reagent, evenly drop it on the membrane to cover the entire membrane, place it in the fully automatic chemiluminescence image analysis system for imaging, and take pictures. Use the gray value software Gelpro32 to analyze the gray value of the bands, and calculate the relative expression of the target protein based on the internal standard protein.

[0124] The experimental groups were as follows: ① blank control group: culture medium incubation; ② model group: culture medium incubation + LPS induction; ③ wampee peel natural polyphenol composition group: 50 μg / mL wampee peel natural polyphenol composition intervention + LPS induction; ④ rutin group: 50 μg / mL rutin intervention + LPS induction; ⑤ quercetin group: 50 μg / mL quercetin intervention + LPS induction; ⑥ isorhamnetin-3-O-neohesperidoside group: 50 μg / mL isorhamnetin-3-O-neohesperidoside intervention + LPS induction; ⑦ polyphenol monomer composition group: 50 μg / mL polyphenol monomer composition (including myricetin-3-O-galactoside, nicotinidine, rutin, isorhamnetin-3-O-neohesperidoside, isoquercetin, quercetin and 8-hydroxypsoralen, the proportion of which is consistent with that in the wampee peel natural polyphenol composition prepared in Example 2) intervention + LPS induction. The ratio of phosphorylated protein expression to total protein expression in the model group (p-IκBα / IκBα, p-ERK / ERK) was 100%.

[0125] Table 3 Effects of each group on inhibiting phosphorylation of inflammatory pathway proteins induced by LPS in RAW 264.7 cells

[0126] Grouping p-IκBα / IκBα(%) p-ERK / ERK(%) Blank control group 4.83±0.08a 14.08±2.20a Model Group 100.00±6.70c 100.00±9.76de Natural polyphenol composition of yellow peel 55.77±4.80b 57.66±12.61b Rutin Group 431.54±26.64d 87.10±4.27cd Quercetin group 51.38±3.16b 49.74±3.94b Isorhamnetin-3-O-neohesperidoside group 100.82±13.59c 106.95±12.00e Polyphenol monomer composition group 85.96±4.41c 76.87±2.31c

[0127] Among them, different letters between groups represent significant differences (p<0.05).

[0128] As shown in Table 3, compared with polyphenol monomers (rutin, quercetin and isorhamnetin-3-O-neohesperidoside) and polyphenol monomer compositions, the ability of the natural polyphenol composition of the yellow peel peel to inhibit the phosphorylation of IκBα and ERK proteins is comparable to quercetin, but significantly stronger than rutin, isorhamnetin-3-O-neohesperidoside and polyphenol monomer compositions, and can more effectively inhibit the activation of LPS on the inflammatory signaling pathway in RAW 264.7 cells, with good anti-inflammatory efficacy. It can be seen that the polyphenol compounds with known and unknown structures contained in the natural polyphenol composition of the yellow peel peel may exert better anti-inflammatory activity through specific interactions. Compared with phenolic compound monomers, the natural polyphenol composition has a lower preparation cost and a broader application prospect.

[0129] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a natural polyphenol composition from Wampee peel, characterized in that: The following steps are involved: A. Use high performance liquid chromatography to construct a standard curve of target polyphenolic compounds, analyze and detect the content of target polyphenolic compounds in the yellow peel peel extract, and determine the collection time period; B. using preparative liquid chromatography to elute and separate the natural polyphenol composition in the wampee peel extract, collecting eluted components within a collection time period to obtain the wampee peel natural polyphenol composition; The analysis and detection comprises using 0.05% formic acid water and acetonitrile as mobile phases, 0.05% formic acid water as A, acetonitrile as B; the elution procedure is 0-3 min, 10%-20% B; 3-15 min, 20%-60% B; 15-18 min, 60%-90% B; 18-22 min, 90%-10% B; 22-25 min, 10% B; The elution separation comprises water and methanol as mobile phases, water as A, methanol as B; the elution procedure is: 0-5min, 10%-20% B; 5-15min, 20%-60% B; 15-20min, 60% B; 20-25min, 60%-90% B; 25-30min, 90%-10% B.

2. The preparation method according to claim 1, characterized in that: The elution separation also includes a detection wavelength of 200 to 400 nm; Preferably, the elution separation further comprises an injection volume of 50 to 70 μL; Preferably, the elution separation further comprises a flow rate of 1.0 to 2.0 mL / min; Preferably, the elution separation further comprises that the chromatographic column is a C18 column; Preferably, the elution separation further comprises a column temperature of 35-40°C.

3. The preparation method according to claim 1, characterized in that: The analytical detection also includes a detection wavelength of 200 to 400 nm; Preferably, the analytical detection also includes an injection volume of 35 to 45 μL; Preferably, the analytical detection also includes a flow rate of 0.5 to 1.0 mL / min; Preferably, the analysis and detection further comprises that the chromatographic column is a C18 column; Preferably, the analytical detection also includes a column temperature of 35-40°C.

4. The preparation method according to claim 1, characterized in that: The target polyphenolic compounds include at least myricetin-3-O-galactoside, nicotin, rutin, isorhamnetin-3-O-neohesperidoside, isoquercitrin, quercitrin and 8-hydroxypsoralen; The collection time period in the elution separation is 10 to 20 minutes.

5. The preparation method according to any one of claims 1 to 4, characterized in that: The preparation method of the wampee peel extract is an ethanol extraction method.

6. The preparation method according to claim 5, characterized in that: The ethanol extraction method comprises taking a powder of a yellow peel peel sample, adding an ethanol solution to extract, and concentrating and drying to obtain a yellow peel peel extract; Preferably, the volume ratio of the kumquat peel powder to the ethanol solution is 1:20-30; Preferably, the volume concentration of the ethanol solution is 65% to 75%; Preferably, the extraction temperature is 60-70°C; Preferably, the extraction time is 1 h; Preferably, the extraction is performed 3 times; Preferably, the drying method is freeze drying.

7. The preparation method according to claim 6, characterized in that: The treatment method for the eluted and separated Wampee peel extract comprises preparing a 10-30 mg / ml Wampee peel extract solution with 80%-90% methanol solution (V / V), and then filtering the solution through a microporous filter membrane.

8. The preparation method according to claim 6, characterized in that: The processing method of the Wampee peel extract for the analytical detection comprises preparing 8-12 mg / ml Wampee peel extract solution with 80%-90% methanol solution (V / V), and filtering the solution through a microporous filter membrane.

9. The preparation method according to claim 7 or 8, characterized in that: The pore size of the microporous filter membrane is 0.20-0.50 μm, preferably 0.22 μm or 0.45 μm.

10. A natural polyphenol composition of wampee peel, characterized in that: The compound is prepared by the preparation method according to any one of claims 1 to 9.

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