A natural polyphenol composition of yellow skin fruit peel and a preparation method thereof

Abstract

The application provides a natural polyphenol composition of phellodendri cortex and a preparation method thereof, relates to the technical field of biological medicines, and comprises the following steps: constructing a standard curve of target polyphenol compounds by using high performance liquid chromatography, analyzing and detecting the content of the target polyphenol compounds in phellodendri cortex extract, and determining a collection time period; using preparative liquid chromatography to elute and separate the natural polyphenol composition in the phellodendri cortex extract, and collecting elution components in the collection time period to obtain the natural polyphenol composition of phellodendri cortex. The prepared natural polyphenol composition has a purity of 75% or more; and contains target polyphenol compound components and polyphenol compound components with unknown structures that are not compared with standard products; compared with the phellodendri cortex extract before purification, part of polyphenol compound monomers and polyphenol monomer composition, the natural polyphenol composition has better anti-inflammatory activity. The technical problem that there is no effective purification and preparation method for the natural polyphenol composition in phellodendri cortex in the prior art is solved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to a Clausena lansium peel natural polyphenol composition and a preparation method thereof. BACKGROUND

[0002] Clausena lansium (Lour.) Skeels is a tree of Rutaceae Clausena, which is a characteristic crop in Lingnan region such as Guangdong, Guangxi, Hainan, Yunnan and Fujian. The fruit of C. lansium is often used for fresh food or processed into juice, jam and other products due to its unique flavor and rich nutritional active ingredients. However, the peel of C. lansium is less directly eaten and used for processing food due to its hard taste and slight bitterness, and is mostly discarded. C. lansium was first recorded in Compendium of Materia Medica, and its roots, leaves, fruits and seeds can be used as medicine. The peel has high medicinal value, has the effects of diuresis, swelling reduction and qi movement and pain relief, and is used for treating symptoms related to inflammation such as abdominal pain, phlegm and cough.

[0003] Polyphenols are the most widely distributed components in medicinal plant secondary metabolites, which affect color, flavor and nutritional efficacy. Studies have shown that polyphenolic compounds have anti-inflammatory effects, and the peel of C. lansium contains more polyphenolic compounds than the pulp and the fruit core.

[0004] At present, the fruit peel of Phellodendri amurense Rupprecht has been studied to obtain the extract, and a variety of polyphenolic compounds such as rutin, quercitrin and isoquercitrin have been identified from the extract, and the extract also contains polyphenolic compounds with unknown structures. The anti-inflammatory activity of the fruit peel extract of Phellodendri amurense Rupprecht and some polyphenolic compound monomers has been verified by experiments. The natural polyphenolic composition prepared from the fruit peel of Phellodendri amurense Rupprecht may exert a specific degree of anti-inflammatory effect through synergistic or antagonistic effects between different types and different contents of polyphenolic compounds. Compared with the crude extract of the fruit peel of Phellodendri amurense Rupprecht, the natural polyphenolic composition after purification has a higher concentration of effective polyphenolic compounds, and the interference of impurities on the activity is excluded. Compared with polyphenolic compound monomers, the purification of polyphenolic composition can effectively save the purification time and economic cost, and can cover some polyphenolic compounds with unknown structures but significant effects, which may make the product have better anti-inflammatory effect through synergistic or antagonistic effects. However, there is currently a lack of effective purification and preparation method for the natural polyphenolic composition in the fruit peel of Phellodendri amurense Rupprecht. High performance liquid chromatography (HPLC) is the most common method for separating and quantifying polyphenolic compounds in samples, and preparative high performance liquid chromatography (prep-HPLC) is mainly used for the separation and recovery of target monomer compounds. However, for complex extracts, the separation and detection efficiency is low due to the interference of a large amount of impurities and the limitation of instrument detection limit and sensitivity. Therefore, sample pretreatment is usually required before entering the liquid chromatography separation to improve the purity of the target components, including liquid liquid extraction (LLE), solid phase extraction (SPE), column chromatography (CC) and the like. The separation process is complicated, time-consuming and has low product yield.

[0005] In view of this, the present application is proposed. SUMMARY

[0006] One of the purposes of the present application is to provide a preparation method of the natural polyphenolic composition of the fruit peel of Phellodendri amurense Rupprecht, so as to at least solve the technical problem that there is a lack of effective purification and preparation method for the natural polyphenolic composition in the fruit peel of Phellodendri amurense Rupprecht in the prior art.

[0007] The second purpose of the present application is to provide a natural polyphenolic composition of the fruit peel of Phellodendri amurense Rupprecht.

[0008] In order to achieve the above purposes of the present application, the following technical solutions are adopted:

[0009] In a first aspect, the present application provides a preparation method of natural polyphenol composition of Chinese wax tree bark, comprising the following steps:

[0010] A, using high performance liquid chromatography to construct standard curve of target polyphenols, analyzing and detecting the content of target polyphenols in Chinese wax tree bark extract, and determining the collection time period;

[0011] B, using preparative liquid chromatography to elute and separate natural polyphenol composition in Chinese wax tree bark extract, and collecting elution components in the collection time period to obtain natural polyphenol composition of Chinese wax tree bark;

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

[0013] The elution and separation includes using water and methanol as mobile phase, water as A and methanol as B; 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.

[0014] Further, the elution and separation further includes detection wavelength of 200-400nm;

[0015] Preferably, the elution and separation further includes injection amount of 50-70μL;

[0016] Preferably, the elution and separation further includes flow rate of 1.0-2.0mL / min;

[0017] Preferably, the elution and separation further includes chromatographic column of C18 column;

[0018] Preferably, the elution and separation further includes column temperature of 35-40℃.

[0019] Further, the analysis and detection further includes detection wavelength of 200-400nm;

[0020] Preferably, the analysis and detection further includes injection amount of 35-45μL;

[0021] Preferably, the analysis and detection further includes flow rate of 0.5-1.0mL / min;

[0022] Preferably, the analysis and detection further includes chromatographic column of C18 column;

[0023] Preferably, the analysis detection further comprises a column temperature of 35-40℃.

[0024] Further, the polyphenol compounds at least comprise myricetin-3-O-galactoside, flosin, rutin, isorhamnetin-3-O-neohesperidoside, isoquercitrin, quercitrin and 8-hydroxy psoralen.

[0025] The collection time period in the elution separation is 10-20 min.

[0026] Further, the preparation method of the Clausena lansium peel extract is ethanol extraction.

[0027] Further, the ethanol extraction method comprises adding Clausena lansium peel sample powder into an ethanol solution for extraction, and obtaining the Clausena lansium peel extract after concentration and drying.

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

[0029] Preferably, the volume concentration of the ethanol solution is 65%-75%.

[0030] Preferably, the extraction temperature is 60-70℃.

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

[0032] Preferably, the extraction times is 3.

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

[0034] Further, the processing method of the Clausena lansium peel extract for the elution separation comprises using 80%-90% methanol solution (V / V) to prepare a Clausena lansium peel extract solution of 10-30 mg / ml, and then filtering through a microporous filter membrane.

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

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

[0037] In the second aspect, the application provides a Clausena lansium peel natural polyphenol composition prepared by the above preparation method.

[0038] The application provides a preparation method of a natural polyphenol composition of phellodendri cortex, compared with a traditional macroporous resin chromatography and other polyphenol purification methods, 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 and the content ratio of the target polyphenol compounds; the separation condition of the preparative liquid chromatography separation natural polyphenol composition is not required to be too high, and the cost of separation and preparation is effectively saved; the prepared natural polyphenol composition has a purity of 75% or above, while the purity of the phellodendri cortex extract before purification is only about 37% to 45%; and the natural polyphenol composition contains target polyphenol compound components and polyphenol compound components with unknown structures which are not compared with standard products, compared with the phellodendri cortex extract before purification, part of polyphenol compound monomers and polyphenol monomer composition, the natural polyphenol composition has better anti-inflammatory activity. The technical problem that there is no effective purification and preparation method for the natural polyphenol composition in the phellodendri cortex in the prior art is solved. BRIEF DESCRIPTION OF DRAWINGS

[0039] In order to more clearly illustrate the specific embodiments of the application or the technical solutions in the prior art, the drawings needed in the specific embodiments or the prior art description will be briefly introduced below. Obviously, the drawings in the following description are some embodiments of the application, and other drawings can also be obtained by those skilled in the art without creative labor.

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

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

[0042] Figure 3 The HPLC ultraviolet absorption spectrum of different phellodendri cortex extracts provided by the embodiment of the application at 254nm;

[0043] Figure 4 The HPLC ultraviolet absorption spectrum of different phellodendri cortex extracts provided by the embodiment of the application at 320nm;

[0044] Figure 5 The prep-HPLC ultraviolet absorption spectrum of the phellodendri cortex extract provided by the embodiment 2 of the application at 254nm;

[0045] Figure 6 The HPLC ultraviolet absorption spectrum of the natural polyphenol composition of phellodendri cortex provided by the embodiment 2 of the application at 320nm;

[0046] Figure 7The HPLC ultraviolet absorption spectrum of the natural polyphenol composition of the phaeodendron amapaense peel provided for the present application at 280 nm. DETAILED DESCRIPTION

[0047] Unless otherwise defined, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear; however, in the event of any latent ambiguity, definitions provided herein take precedent. In this application, the use of "or" means "and / or" unless specifically stated otherwise, e.g., "comprises at least one of A and B" means that the composition can comprise A, or B, or both A and B. Moreover, the use of "including" and other forms of the word "comprise" is intended to be non-limiting.

[0048] Unless otherwise indicated, the methods and techniques of the present application 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 throughout the present specification.

[0049] One aspect of the present application provides a method for preparing a natural polyphenol composition of phaeodendron amapaense peel, comprising the following steps:

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

[0051] B. Using preparative liquid chromatography to elute and separate the natural polyphenol composition in the phaeodendron amapaense peel extract, and collect the elution components within the collection time period to obtain the natural polyphenol composition of phaeodendron amapaense peel;

[0052] The analysis and detection includes using 0.05% formic acid water and acetonitrile as the mobile phase, 0.05% formic acid water as A and acetonitrile as B; the elution program 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 and separation includes using water and methanol as the mobile phase, water as A and methanol as B; the elution program 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.

[0054] Compared with traditional macroporous resin chromatography and other polyphenol purification methods, 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, and the content ratio of target polyphenol compounds; the separation conditions of the natural polyphenol composition as the target do not need to be too high, which effectively saves the cost of separation and preparation; the natural polyphenol composition prepared has a purity of 75% or more, while the purity of the Huangpi fruit peel extract before purification is only about 37% to 45%; and it ensures that the target polyphenol compound components and the polyphenol compound components with unknown structures not compared with the standard are contained; compared with the Huangpi fruit peel extract before purification, part of the polyphenol compound monomers and the polyphenol monomer composition, it has better anti-inflammatory activity. The technical problem of lacking an effective purification and preparation method for the natural polyphenol composition in the Huangpi fruit peel in the prior art is solved.

[0055] In some specific embodiments, the collection time can be determined according to the peak time of the target polyphenol compound and the separation time distance of the impurity peak region.

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

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

[0058] In order to ensure the normal separation capacity of the chromatographic column and the collection efficiency during the elution and separation of the natural polyphenol composition of Huangpi fruit peel, in some specific embodiments, the elution separation further includes an injection amount of 50-70 μL.

[0059] Specifically, the injection amount 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, or can be any value between 50 μL and 70 μL.

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

[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, or any value between 1.0 and 2.0 mL / min.

[0062] In some specific embodiments, the elution separation further comprises that the column is a C18 column.

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

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

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

[0066] In the analysis detection, in order to achieve accurate quantitative detection of natural polyphenolic compounds in the fruit peel of Xanthoceras sorbifolia Bunge, in some specific embodiments, the analysis detection further comprises that the injection volume is 35-45 μL.

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

[0068] In some specific embodiments, the analysis detection further comprises that the flow rate is 0.5-1.0 mL / min.

[0069] Specifically, the flow rate in the analysis 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, or any value between 0.5 and 1.0 mL / min.

[0070] In some specific embodiments, the analysis detection further comprises that the column is a C18 column.

[0071] In some embodiments, the analysis further comprises a column temperature of 35-40 °C.

[0072] In some embodiments, the target polyphenols include myricetin-3-O-galactoside, rhodionoside, rutin, isorhamnetin-3-O-neohesperidoside, isoquercitrin, quercitrin and 8-hydroxy-pulchinenoside. By selecting polyphenols as standard, a plurality of polyphenols in the fruit peel of Phellodendron amurense Rupr. can be detected at one time. Separation and comparison of a plurality of polyphenols in the fruit peel of Phellodendron amurense Rupr. can ensure the accuracy of the collection period, so as to improve the collection efficiency.

[0073] According to the types of polyphenols that may be contained in the fruit peel extract of Phellodendron amurense Rupr. determined by experiments, polyphenol standard solutions of various types were prepared and injected into a high performance liquid chromatograph, respectively. It was found that the retention times of polyphenols of various types were close and concentrated in the interval of 7-15 min. As can be seen from the high performance liquid chromatogram of the fruit peel extract of Phellodendron amurense Rupr. Figure 3 ), there are continuous multiple peaks in the interval of 7-15 min, and 7 peaks correspond to the retention times of myricetin-3-O-galactoside, rhodionoside, rutin, isorhamnetin-3-O-neohesperidoside, isoquercitrin, quercitrin and 8-hydroxy-pulchinenoside, respectively. Therefore, it can be basically determined that the interval of 7-15 min in the high performance liquid chromatograph is the characteristic fingerprint region of the polyphenols in the fruit peel of Phellodendron amurense Rupr. The interval is far apart from the impurity peak region (2-4 min, 20-25 min) by more than 3 min, and the polyphenols and impurities can be well separated under this method. In some embodiments, the collection period in the elution and separation is 10-20 min. On the basis of the elution program of the high performance liquid chromatograph, the elution method of the preparative liquid chromatograph is set. The elution programs of the two are basically the same, and the elution time of the polyphenol corresponding elution gradient (i.e. about 60% B) is appropriately extended in the preparative liquid chromatograph. As can be seen from the elution diagram of the preparative liquid chromatograph, Figure 5 ), the distribution of the polyphenol characteristic fingerprint region and the impurity peak region is basically consistent with that in the high performance liquid chromatograph. The polyphenol characteristic fingerprint region and the impurity region are well separated, and therefore it is determined that the collection period is the time period corresponding to the polyphenol characteristic fingerprint region, i.e. 10-20 min. After the collected natural polyphenol composition is prepared into a solution, it is injected into a high performance liquid chromatograph for analysis. As can be seen from the high performance liquid chromatogram, Figure 6 ), the polyphenol composition is mainly distributed in the interval of 7-15 min, and the purity is obviously improved, which further proves that the collection period of 10-20 min determined in the preparative liquid chromatograph is correct.

[0074] The polyphenols are easily soluble in ethanol. In some embodiments, the preparation method of the fruit peel extract of Phellodendron amurense Rupr. is ethanol extraction.

[0075] In some embodiments, the ethanol extraction method comprises adding ethanol solution to the sample powder of the fruit peel of Clausena lansium to extract, and then drying the concentrated extract to obtain the fruit peel extract of Clausena lansium.

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

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

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

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

[0080] In some embodiments, the extraction is performed for 3 times.

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

[0082] The fruit peel extract solution of Clausena lansium prepared by using the methanol solution can effectively remove the sugar impurities after the microfiltration treatment, thereby improving the efficiency of elution, separation, and analysis and detection. In some embodiments, the treatment method of the fruit peel extract of Clausena lansium for elution and separation comprises using 80%-90% methanol solution (V / V) to prepare a fruit peel extract solution of Clausena lansium at a concentration of 10-30 mg / ml, and then performing microfiltration. In some embodiments, the treatment method of the fruit peel extract of Clausena lansium for analysis and detection comprises using 80%-90% methanol solution (V / V) to prepare a fruit peel extract solution of Clausena lansium at a concentration of 8-12 mg / ml, and then performing microfiltration.

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

[0084] Specifically, the pore size of the microfiltration membrane can 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 within the range of 0.20-0.50 μm, and is preferably 0.22 μm or 0.45 μm.

[0085] In some embodiments, the preparation method of the fruit peel of Clausena lansium comprises taking fresh fruit peel of Clausena lansium and drying and crushing the same.

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

[0087] According to another aspect of the present application, a natural polyphenol composition of yellow skin fruit peel is also provided, which is prepared by the above preparation method.

[0088] The technical solutions of the present application will be described clearly and completely in combination with the embodiments. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0089] Myricetin-3-O-galactoside, quercitrin, 8-hydroxy-podophyllotoxin were purchased from MedChemExpress, batch numbers were RM2096, RM0926, RM1147 respectively; Isorhamnetin-3-O-neohesperidoside, Flos Lonicerae was purchased from ChemFaces, batch numbers were CFN99744, CFN99830 respectively; Isoquercitrin was purchased from Macklin, batch number was Q817141; Rutin was purchased from Source Leaf, batch number was B20771.

[0090] A standard curve was established: myricetin-3-O-galactoside, flos lonicerae, rutin, isorhamnetin-3-O-neohesperidoside, isoquercitrin, quercitrin and 8-hydroxy-podophyllotoxin were used as standard samples to prepare a mixed standard sample solution. The concentration of isorhamnetin-3-O-neohesperidoside, quercitrin and 8-hydroxy-podophyllotoxin was 50 μg / ml, the concentration of myricetin-3-O-galactoside, flos lonicerae and isoquercitrin was 100 μg / ml, and the concentration of rutin was 200 μg / ml. The elution was performed in a high-performance liquid chromatograph. A C18 column was used. The maximum absorption wavelength for rutin and quercitrin detection was 254 nm, and the maximum absorption wavelength for myricetin-3-O-galactoside, flos lonicerae, isorhamnetin-3-O-galactoside, isoquercitrin and 8-hydroxy-podophyllotoxin detection was 320 nm. The injection amount was 1 μL, 2 μL, 5 μL, 10 μL and 20 μL. The flow rate was 1.0 mL / min. The column temperature was 40°C. 0.05% formic acid water (A) and acetonitrile (B) were used as the mobile phase. The elution program was as follows: 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 results are shown in Table 1, where the peak numbers correspond to Table 1. The standard curve fitting is shown in Table 1. Figures 1-2 Table 1: Standard curve fitting

[0091] Table 1 Linear standard curve of polyphenol content

[0092]

[0093] Example 1

[0094] Preparation of natural polyphenol composition in the peel of No. I variety of Xingpi: fresh Xingpi was washed and peeled. The peel of Xingpi was dried in a heat pump drying machine at 40 for 48 h, and the moisture content was 4.52%. The dried peel was then ground into powder.

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

[0096] Separation and analysis of polyphenols in Xingpi peel extract: 80% methanol solution (V / V) was used to prepare a 10 mg / ml Xingpi peel extract solution, which was filtered through a 0.22 μm microporous filter. In high performance liquid chromatography (HPLC), a C18 column (Zorbax SB-C18, 4.6 mm x 250 mm 5-Micro, Agilent) was used, the detection wavelength was 254 / 320 nm, the injection volume was 40 μL, the flow rate was 1.0 mL / min, the column temperature was 40, and 0.05% formic acid water (A) and acetonitrile (B) were used as the mobile phase, with the following elution program: 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 results are shown in Table 1 and Figure 1, wherein the peak numbers correspond to Table 1. The results showed that the contents of myricetin-3-O-galactoside, rhodomyrtol, rutin, isorhamnetin-3-O-neohesperidoside, isoquercitrin, quercitrin, and 8-hydroxy psoralen 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. Figure 3 and Figure 4

[0097] ​Preparation of natural polyphenol composition from the fruit peel of Clausena lansium: 80% methanol solution (V / V) was used to prepare 10 mg / ml solution of the fruit peel extract of Clausena lansium, which was filtered through a 0.22 μm microfiltration membrane. In the preparation liquid chromatography (prep-HPLC), a C18 column (Zorbax Eclipse XDB-C18, 4.6 mm x 250 mm 5-Micro, Agilent) was used; the detection wavelength was 254 nm; the injection volume was 50 μL; the flow rate was 1.0 mL / min; the column temperature was 40; water (A) and methanol (B) were used as the mobile phase, and the elution program was as follows: 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; the elution components were collected from 10-20 min, and then concentrated and freeze-dried to obtain the natural polyphenol composition from the fruit peel of Clausena lansium, with a purity of 75.47%.

[0098] Example 2

[0099] Preparation of natural polyphenol composition from the fruit peel of Clausena lansium (II): Fresh Clausena lansium was washed and peeled. The fruit peel was dried in a heat pump drying machine at 50 for 36 h, and the moisture content was 4.38%. The dried fruit peel was crushed.

[0100] Extraction of the fruit peel of Clausena lansium: 15 g of the fruit peel powder was added to 20 times the volume of 75% ethanol solution (V / V), and extracted for 3 times at 60 for 1 h each time. The extract was filtered, concentrated, and freeze-dried to obtain the fruit peel extract of Clausena lansium, with a purity of 40.87% of polyphenols.

[0101] Separation and analysis of polyphenols in the fruit peel extract of Clausena lansium: 85% methanol solution (V / V) was used to prepare 8 mg / ml solution of the fruit peel extract of Clausena lansium, which was filtered through a 0.22 μm microfiltration membrane. In the high performance liquid chromatography, a C18 column (Zorbax SB-C18, 4.6 mm x 250 mm 5-Micro, Agilent) was used; the detection wavelength was 254 / 320 nm; the injection volume was 45 μL; the flow rate was 1.0 mL / min; the column temperature was 40; 0.05% formic acid water (A) and acetonitrile (B) were used as the mobile phase, and the elution program was as follows: 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 results are shown in Table 1. Figure 3 and Figure 4The peak numbers correspond to Table 1. The results show that the contents of myricetin-3-O-galactoside, rhodaxanthoside, rutin, isorhamnetin-3-O-neohesperidoside, isoquercitrin, quercitrin and 8-hydroxy-pulchinenoside in the extract are 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 Clausena lansium peel: 30 mg / ml Clausena lansium peel extract solution was prepared using 85% methanol solution (V / V) and filtered through a 0.22 μm microporous membrane. In the preparation of liquid chromatography, a C18 column (Zorbax Eclipse XDB-C18, 4.6 mm x 250 mm 5-Micro, Agilent) was used; the detection wavelength was 254 nm; the injection volume was 70 μL; the flow rate was 1.5 mL / min; the column temperature was 40; and water (A) and methanol (B) were used as the mobile phase, and the elution program was as follows: 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. The elution components in 10-20 min were collected, concentrated and freeze-dried to obtain the natural polyphenol composition from Clausena lansium peel, with a purity of 78.65%. The chromatogram is shown in Figure 2. Figure 5 and Figure 6 As shown, four sample bottles (sample bottle 9 to sample bottle 12) were used, and each sample bottle collected eluent for 2.5 min, and the elution components in 10-20 min were obtained after being combined.

[0103] Example 3

[0104] Preparation of natural polyphenol composition from Clausena lansium peel of variety III: Fresh Clausena lansium fruits were washed and peeled. The Clausena lansium peel was dried in a heat pump drying machine at 45 for 40 h, and the water content was 2.98%. The dried peel was crushed.

[0105] Extraction of Clausena lansium peel: 20 g of Clausena lansium peel powder was added to 25 times the volume of 65% ethanol solution (V / V), and extracted for 3 times, 1 h each time. The extract was filtered, concentrated and freeze-dried to obtain Clausena lansium peel extract, with a polyphenol purity of 45.06%.

[0106] Isolation and analysis of polyphenols in the extract of the fruit peel of Phyllanthus emblica: 12 mg / ml of the extract of the fruit peel of Phyllanthus emblica was prepared using 90% methanol solution (V / V) and filtered through a 0.22 μm microfiltration membrane. In high performance liquid chromatography, a C18 column (Zorbax SB-C18, 4.6 mm x 250 mm 5-Micro, Agilent) was used; the detection wavelength was 280 / 330 nm; the injection volume was 35 μL; the flow rate was 0.8 mL / min; the column temperature was 35; and 0.05% formic acid water (A) and acetonitrile (B) were used as the mobile phase, and the elution program was as follows: 0-3 min, 10%-20% B; 3-15 min, 20%-60% B; 15-18 min, 60%-90% B; 18-22 min, 90%-10% B; and 22-25 min, 10% B. The results are shown in Table 1 and FIG. 1, in which the peak numbers correspond to Table 1. The results of the determination showed that the contents of myricetin-3-O-galactoside, rhodaxanthoside, rutin, isorhamnetin-3-O-neohesperidoside, isoquercitrin, quercitrin, and 8-hydroxy-pulchinenoside 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. Figure 3 and Figure 4 The results of the determination showed that the contents of myricetin-3-O-galactoside, rhodaxanthoside, rutin, isorhamnetin-3-O-neohesperidoside, isoquercitrin, quercitrin, and 8-hydroxy-pulchinenoside 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 the natural polyphenol composition of the fruit peel of Phyllanthus emblica: 20 mg / ml of the extract of the fruit peel of Phyllanthus emblica was prepared using 90% methanol solution (V / V) and filtered through a 0.22 μm microfiltration membrane. In preparative liquid chromatography, a C18 column (Zorbax Eclipse XDB-C18, 4.6 mm x 250 mm 5-Micro, Agilent) was used; the detection wavelength was 280 nm; the injection volume was 60 μL; the flow rate was 2.0 mL / min; the column temperature was 35; and water (A) and methanol (B) were used as the mobile phase, and the elution program was as follows: 0-5 min, 10%-20% B; 5-15 min, 20%-60% B; 15-20 min, 60% B; 20-25 min, 60%-90% B; and 25-30 min, 90%-10% B. The elution components collected from 10 to 20 min were concentrated and freeze-dried to obtain the natural polyphenol composition of the fruit peel of Phyllanthus emblica, which had a purity of 81.90%.

[0108] Comparative Example 1

[0109] Different from example 1, if the separation and analysis of polyphenols in the high performance liquid chromatography is not carried out at all, the extract of the fruit peel of Phellodendron amurense Rupr. is directly separated in the preparative liquid chromatography. This needs to explore the separation conditions of polyphenol components and impurity components in the preparative liquid chromatography from zero, and the retention time interval of polyphenol compounds cannot be quickly known, so that the suitable elution and collection time cannot be accurately selected to prepare the natural polyphenol composition. For different samples of the fruit peel of Phellodendron amurense Rupr., although the collection time of the preparation of the natural polyphenol composition is fixed at 10-20 min, the content and proportion of each target polyphenol compound in the composition need to be known at the same time, so the analysis and detection procedure of the high performance liquid chromatography is essential before each preparation.

[0110] Comparative example 2

[0111] Different from example 1, the separation and analysis conditions of the high performance liquid chromatography are as follows:

[0112] The conditions of the analytical high performance liquid chromatography are as follows: Agilent XDB C18 column: 18 mm x 250 mm, 5 μm; mobile phase A is acetonitrile + 0.01% formic acid, and mobile phase B is ultrapure water + 0.01% formic acid; the gradient elution procedure is as follows: 0-30 min, the volume ratio of A phase and B phase is 5:95-25:75; 30 min-60 min, the volume ratio of A phase and B phase is 25:75-45:55; the flow rate is 1.0 mL / min, and the ultraviolet absorbance is detected at 280 nm. The results are shown in Table 1. Figure 7 Under the separation conditions of the analytical high performance liquid chromatography, the polyphenols in the fruit peel of Phellodendron amurense Rupr. cannot be eluted, and the effective separation and content determination of different types of polyphenols cannot be achieved. Therefore, for the fruit peel extract of Phellodendron amurense Rupr., it is expected to use the analytical high performance liquid chromatography to view the peak occurrence and trend of polyphenols, and then use the preparative high performance liquid chromatography to separate the main polyphenol chromatographic peak, and then obtain the purified polyphenols, which cannot be achieved under the separation conditions of the analytical high performance liquid chromatography.

[0113] Experimental example 1: NO content determination

[0114] In this experiment, the fruit peel extract of Phellodendron amurense Rupr. and the natural polyphenol composition prepared in example 2 are selected, and the following operations are carried out.

[0115] After the RAW264.7 cells are cultured, 10 6 / mL cell suspension, 100 μL per well was added to a 96-well plate. After 24 h of incubation at 37, 5% CO2incubator, the culture medium was discarded. Different concentrations of the extract of the fruit peel of Clausena lansium or the natural polyphenol composition of the fruit peel of Clausena lansium were added. After 3 h of drug intervention, 1 μg / mL lipopolysaccharide (LPS) was added to induce inflammation of the cells. After 16 h, the cell culture supernatant was collected, and the content of NO therein was determined according to the Griess kit instructions. The method for using the Griess kit was as follows: 50 μL of the cell culture supernatant, 50 μL of Griess reagent A and 50 μL of Griess reagent B were sequentially added to a 96-well plate, and after being thoroughly mixed, the absorbance value was determined at 540 nm, and the concentration of NO was calculated according to the standard curve of the standard sample 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 extract of the fruit peel of Clausena lansium group: 25 μg / mL extract of the fruit peel of Clausena lansium intervention + LPS induction; ④ high-concentration extract of the fruit peel of Clausena lansium group: 50 μg / mL extract of the fruit peel of Clausena lansium intervention + LPS induction; ⑤ low-concentration natural polyphenol composition of the fruit peel of Clausena lansium group: 25 μg / mL extract of the fruit peel of Clausena lansium intervention + LPS induction; ⑥ high-concentration natural polyphenol composition of the fruit peel of Clausena lansium group: 50 μg / mL extract of the fruit peel of Clausena lansium intervention + LPS induction; and ⑦ positive control group: 10 μg / mL dexamethasone + LPS induction.

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

[0118] Group NO content (μM) Control group 2.47±0.30a Model group 47.22±0.75g 25 μg / mL of Clausena lansium peel extract group 41.61±0.97f 50 μg / mL of Clausena lansium peel extract group 38.58±0.46e 25 μg / mL of natural polyphenol composition of Clausena lansium peel group 36.31±1.10d 50 μg / mL of natural polyphenol composition of Clausena lansium peel group 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 can be seen from Table 2, compared with the extract of the fruit peel of Clausena lansium at the same concentration, the natural polyphenol composition of the fruit peel of Clausena lansium has a significantly stronger ability to inhibit NO secretion of LPS-induced RAW 264.7 cells and has better anti-inflammatory activity. It can be known that the elution and separation process can effectively remove impurity components interfering with the activity in the extract, and the natural polyphenol composition can more efficiently exert the anti-inflammatory effect.

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

[0122] In this experiment, the natural polyphenol composition of the fruit peel of Clausena lansium prepared in Example 2 was used, and the following operations were performed:

[0123] After the RAW 264.7 cells were cultured, 10 6The cells were seeded in 60 mm diameter culture dishes at a density of 1 x 106 / mL of cell suspension. After 24 h incubation at 37 °C in a 5% CO2incubator, the culture medium was discarded. Different concentrations of the natural polyphenol composition of the skin of P. glabra, polyphenol monomers or polyphenol monomer composition were added. After 3 h of drug intervention, 1 μg / mL LPS was added to induce inflammation in the cells. After 16 h, the culture medium was discarded, the cells were washed with phosphate buffered saline (PBS), and then RIPA cell lysis solution was added. After centrifugation, the supernatant was taken, and the total protein content was determined using a BCA kit. After denaturation, the protein samples were separated in a sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF membrane in a transmembrane instrument. The PVDF membrane was blocked in 5% skim milk for 2 h, and then, according to the antibody instructions, the primary antibody was added, and the 4 shaker was incubated overnight. After washing with Tris buffered saline with Tween 20 (TBST), the secondary antibody was added, and the shaker was incubated at room temperature for 1 h. After TBST washing, the enhanced chemiluminescence (ECL) developing agent was prepared, evenly dropped on the membrane to cover the whole membrane, and placed in the automatic chemiluminescence image analysis system for imaging and photographing. The gray value software Gelpro32 was used to analyze the gray value of the bands, and the relative expression amount of the target protein was calculated according to 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 natural polyphenol composition of the skin of P. glabra group: 50 μg / mL natural polyphenol composition of the skin of P. glabra intervention + LPS induction; ④ rutin group: 50 μg / mL rutin intervention + LPS induction; ⑤ quercitrin group: 50 μg / mL quercitrin 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, smyrilagin, rutin, isorhamnetin-3-O-neohesperidoside, isoquercitrin, quercitrin and 8-hydroxy psoralen, with the same ratio as the natural polyphenol composition of the skin of P. glabra prepared in Example 2) intervention + LPS induction. The proportion of the expression amount of the phosphorylated protein to the expression amount of the total protein (p-IκBα / IκBα, p-ERK / ERK) in the model group was 100%.

[0125] Table 3: Effect of each group on the inhibition of LPS-induced phosphorylation of inflammatory pathway proteins in RAW 264.7 cells

[0126] Group p-IκBα / IκBα (%) p-ERK / ERK (%) Control group 4.83±0.08a 14.08±2.20a Model group 100.00±6.70c 100.00 ± 9.76de Natural polyphenol composition of Clausena lansium peel group 55.77±4.80b 57.66±12.61b Rutin group 431.54±26.64d 87.10 ± 4.27cd Quercitrin 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] Wherein, different letters between groups represent significant differences (p <0.05).

[0128] As shown in Table 3, the ability of the natural polyphenol composition of the fruit peel of Clausena lansium to inhibit the phosphorylation of IκBα and ERK proteins is equivalent to that of quercitrin, but significantly stronger than that of rutin, isorhamnetin-3-O-neohesperidoside and the polyphenol monomer composition, and it can more effectively inhibit the activation of the inflammatory signaling pathway in RAW 264.7 cells by LPS, and has good anti-inflammatory efficacy. Therefore, it can be known that the known and unknown polyphenol compounds contained in the natural polyphenol composition of the fruit peel of Clausena lansium may exert better anti-inflammatory activity through specific interactions. Compared with the polyphenol monomer, the natural polyphenol composition has lower preparation cost and wider application prospect.

[0129] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present application, but not to limit them; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing embodiments, or make equivalent replacement for part or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A method of preparing a natural polyphenol composition of yellow skin fruit, characterized by, The method comprises the following steps: A. Constructing a standard curve of target polyphenols by using high performance liquid chromatography, analyzing and detecting the content of target polyphenols in the extract of Clausena lansium peel, and determining the collection time period; B. Eluting and separating the natural polyphenol composition in the extract of Clausena lansium peel by using preparative liquid chromatography, and collecting the elution components in the collection time period to obtain the natural polyphenol composition of Clausena lansium peel; The analysis and detection comprises using 0.05% formic acid water and acetonitrile as the mobile phase, 0.05% formic acid water as A and acetonitrile as B; and the elution program is 10%-20% B for 0-3 min, 20%-60% B for 3-15 min, 60%-90% B for 15-18 min, 90%-10% B for 18-22 min, and 10% B for 22-25 min; The elution and separation comprises using water and methanol as the mobile phase, water as A and methanol as B; and the elution program is 10%-20% B for 0-5 min, 20%-60% B for 5-15 min, 60% B for 15-20 min, 60%-90% B for 20-25 min, and 90%-10% B for 25-30 min; The chromatographic column for the elution and separation is a C18 column; The preparation method of the extract of Clausena lansium peel is an ethanol extraction method, which comprises adding an ethanol solution to Clausena lansium peel sample powder for extraction, and obtaining the extract of Clausena lansium peel after concentration and drying; the volume concentration of the ethanol solution is 65%-75%; the extraction temperature is 60-70 DEG C; The said natural pomace of yellow skin includes at least myricetin-3-O- O - galactoside, nobiletin, rutin, isorhamnetin-3-O- O - neohesperidin, isoquercitrin, quercitrin and 8-hydroxy- psoralen; The collection time period in the elution and separation is 10-20 min.

2. The production method according to claim 1, characterized by, The elution and separation further comprises a detection wavelength of 200-400 nm.

3. The production method according to claim 2, characterized by, The elution and separation further comprises an injection amount of 50-70 mu L.

4. The production method according to claim 2, characterized by, The elution and separation further comprises a flow rate of 1.0-2.0 mL / min.

5. The preparation method according to claim 2, characterized in that, The elution and separation further comprises a column temperature of 35-40 DEG C.

6. The production method according to claim 1, characterized by, The analysis and detection further comprises a detection wavelength of 200-400 nm.

7. The production method according to claim 6, characterized by The analysis and detection further comprises an injection amount of 35-45 mu L.

8. The production method according to claim 6, characterized by, The analysis and detection further comprises a flow rate of 0.5-1.0 mL / min.

9. The preparation method according to claim 6, characterized in that, The analysis and detection further comprises a chromatographic column of C18.

10. The preparation method according to claim 6, characterized in that, The analysis and detection further comprises a column temperature of 35-40 DEG C.

11. The method of claim 1, wherein, The volume ratio of the Clausena lansium peel sample powder to the ethanol solution is 1:20-30.

12. The method of claim 1, wherein, The extraction time is 1 h.

13. The method of claim 1, wherein, The extraction is performed for 3 times.

14. The method of claim 1, wherein, The drying mode is freeze drying.

15. The method of claim 1, wherein The treatment method of the extract of Clausena lansium peel for the elution and separation comprises filtering a solution of 10-30 mg / ml of the extract of Clausena lansium peel prepared by using 80%-90% v / v methanol solution through a microporous filter membrane.

16. The method of claim 1, wherein The treatment method of the extract of Clausena lansium peel for the analysis and detection comprises filtering a solution of 8-12 mg / ml of the extract of Clausena lansium peel prepared by using 80%-90% v / v methanol solution through a microporous filter membrane.

17. The method of manufacturing according to claim 15 or 16, wherein, The microporous filter membrane has a pore size of 0.20-0.50 mu m.

18. The method of claim 17, wherein, The microporous filter membrane has a pore size of 0.22 mu m or 0.45 mu m.

19. A natural polyphenol composition of yellow skin fruit, characterized by, Prepared by the preparation method in any one of claims 1-18.

Images