An anti-fatigue osmanthus standard active substance pheg50 and a preparation method and application thereof

Abstract

The application discloses an anti-fatigue osmanthus standard active substance PHEG50 and a preparation method and application thereof, and particularly relates to the field of plant extracts. The preparation method of the osmanthus standard active substance PHEG50 comprises the following steps: mixing osmanthus dried flowers with water, adding acid to adjust the pH value, and extracting under certain temperature conditions to obtain osmanthus crude extract; filtering the osmanthus crude extract to obtain a filtrate, and treating the filtrate through a ceramic membrane, an ultrafiltration membrane and a nanofiltration membrane, and then concentrating the filtrate through an RO membrane and drying to obtain the osmanthus standard active substance PHEG50. The scheme disclosed by the application effectively reduces the degradation and isomerization of verbascoside into isoverbascoside, and efficiently prepares the osmanthus standard active substance with high total phenylethanol glycoside content. The osmanthus standard active substance PHEG50 provided by the application has the effect of obviously relieving physical fatigue.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of plant extracts, and particularly relates to an anti-fatigue osmanthus fragrans standard active substance PHEG50 and a preparation method and application thereof. BACKGROUND

[0002] Osmanthus fragrans (Thunb.) Lour. is a plant of Oleaceae and Osmanthus, which grows between rock ridges and is also known as shanguili or yanguili. There are 31 species of Osmanthus plants in the world, 26 of which are endemic to China, and 21 of which are endemic to China. China is the distribution center of the genus and the country that first cultivated and used osmanthus flowers in the world. Osmanthus fragrans is one of the top ten traditional flowers in China and has been cultivated for more than 2,500 years in China. There are many varieties of osmanthus flowers, with about 154 varieties in China, which are divided into four variety groups, namely, four seasons, silver osmanthus, gold osmanthus, and red osmanthus.

[0003] According to Chinese Herbal Medicine, osmanthus flower is also known as Osmanthus fragrans, which is the flower of Osmanthus fragrans of Oleaceae. It is harvested during flowering in September-October, impurities are removed, and it is dried in the shade and stored in airtight containers. Osmanthus flower has a bitter taste and a warm nature. It belongs to the lung, spleen, and kidney meridians. It has the effects of warming the lungs, resolving dampness, dispelling cold, and relieving pain. It is used to treat cold pain in the abdomen, cold abdominal pain, and painful menstruation.

[0004] Invention patent CN116159089A, a phenylethanoid glycoside extract, its preparation method and application in anti-glycation, discloses a preparation method for preparing phenylethanoid glycosides (PHEG) 23-49% from osmanthus; the osmanthus extract prepared by the technical solution contains less than 50% phenylethanoid glycosides, and the contents of verbascoside and rhodioside are low, which cannot meet the standard of phenylethanoid glycoside content of osmanthus standard active substance.

[0005] Ding Lixin et al. used HPLC to detect the contents of rhodioside and verbascoside in osmanthus from different production areas (Journal of Pharmaceutical Analysis, 2013, 33(5), 894-897), which showed that osmanthus contains a high content of rhodioside and verbascoside. Wu Gaili et al. studied the degradation and isomerization of verbascoside into isoverbascoside under the conditions of pH 5-7, boiling water heating for 0-300 min (Chinese Herbal Drugs, 2022, 53(11), 3295-3305), which suggested that verbascoside may not be stable under certain pH and temperature conditions.

[0006] Pharmacological literature shows that although verbascoside and isoverbascoside have similar antioxidant and fatigue relief effects, there are obvious differences in their activities such as reducing uric acid. Therefore, it is necessary to optimize the process conditions and inhibit the isomerization of verbascoside into isoverbascoside. SUMMARY

[0007] In view of the deficiencies of the prior art, the present application aims to use Osmanthus fragrans as raw material, control reasonable parameter conditions, avoid the degradation and isomerization of verbascoside into isoverbascoside, and use a pure physical processing technology to efficiently prepare Osmanthus fragrans standard active substance PHEG50 with a total phenylethanol glycoside content of greater than or equal to 50%.

[0008] The first aspect of the present application provides a preparation method of Osmanthus fragrans standard active substance PHEG50, comprising the following steps:

[0009] (1) mixing Osmanthus fragrans dried flowers with water at a liquid material ratio of greater than or equal to 22 mL:1 g, adjusting the pH value to 3-4 by adding an acid, and extracting at 55-80 DEG C for 1-4 h to obtain an Osmanthus fragrans crude extract;

[0010] (2) filtering the Osmanthus fragrans crude extract to obtain a filtrate, and concentrating and drying the filtrate to obtain the Osmanthus fragrans standard active substance PHEG50.

[0011] Preferably, the Osmanthus fragrans dried flowers are Dan Gui dried flowers, Jin Gui dried flowers and / or Yin Gui dried flowers.

[0012] Preferably, the Osmanthus fragrans dried flowers in step (1) are crushed and then sieved through a sieve with a mesh size of greater than or equal to 10, and then mixed with water.

[0013] Preferably, the acid used to adjust the pH value to 3-4 in step (1) is at least one of citric acid, malic acid, phosphoric acid, hydrochloric acid and acetic acid.

[0014] Preferably, the pH value is 3-4, the temperature is 60-80 DEG C, and the extraction time is 1-2 h in step (1).

[0015] Preferably, the Osmanthus fragrans crude extract in step (2) is filtered through a ceramic membrane while hot to obtain a ceramic membrane filtrate.

[0016] The ceramic membrane filtrate is filtered through an ultrafiltration membrane with a molecular weight cut-off of 5000-10000 daltons to remove macromolecules such as proteins and polysaccharides, and obtain an ultrafiltration membrane permeate.

[0017] The ultrafiltration membrane permeate is filtered through a nanofiltration membrane with a molecular weight cut-off of 200-250 daltons to remove monosaccharides, free amino acids and other small molecule components, and obtain a nanofiltration membrane retentate.

[0018] The nanofiltration membrane retentate is concentrated through an RO membrane and dried to obtain the Osmanthus fragrans standard active substance PHEG50.

[0019] The second aspect of the present application provides an anti-fatigue Osmanthus fragrans standard active substance PHEG50, which is prepared by the above preparation method.

[0020] Preferably, the anti-fatigue Osmanthus fragrans standard active substance PHEG50 contains greater than or equal to 50% total phenylethanol glycosides by mass percentage.

[0021] Preferably, the anti-fatigue osmanthus standard active PHEG50 contains verbascoside ≥40%, salidroside ≥8%, and isoverbascoside ≤4% by mass percentage.

[0022] The third aspect of the present application provides the use of the aforementioned anti-fatigue osmanthus standard active PHEG50 in the preparation of anti-fatigue medicines, foods and / or health products.

[0023] The fourth aspect of the present application provides the use of the aforementioned anti-fatigue osmanthus standard active PHEG50 in the preparation of detection standards.

[0024] Advantages of the present application:

[0025] 1. The technical solution provided by the present application effectively reduces the degradation and isomerization of verbascoside into isoverbascoside, and efficiently prepares the osmanthus standard active PHEG50 with total phenylethanol glycosides ≥50%, and the verbascoside in the total phenylethanol glycosides is ≥40%, the salidroside is ≥8%, and the isoverbascoside is less than 4%.

[0026] 2. The osmanthus standard active PHEG50 provided by the present application has obvious effect of relieving physical fatigue. DETAILED DESCRIPTION

[0027] The technical solutions in the present application will be described clearly and completely in combination with the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0028] The technical solutions of the present application will be further described in combination with specific embodiments. It should be understood that the following embodiments are only used to explain the present application, and are not used to limit the present application.

[0029] Preparation of test examples and detection of extraction rate

[0030] Osmanthus fragrans L. dry flower powder was crushed and passed through a 20-mesh sieve. Several portions of 10.0 g were weighed and placed in 250-ml conical flasks. 220 ml of pure water was added to each flask, and 165 mg of citric acid was added to adjust the pH to 3, or 27.5 mg of citric acid was added to adjust the pH to 4. No citric acid was added to the Osmanthus fragrans L. water extract, and the pH was 6. The water bath was heated at different temperatures, and the mixture was stirred every 10 min for 1 min. The extraction was performed for 4 h, and samples were taken every hour. The samples were filtered through a 0.45-μm membrane, and HPLC detection was performed. The extraction yields of verbascoside and isoverbascoside were calculated. Verbascoside and isoverbascoside are isomers. After the Osmanthus fragrans L. dry flower was crushed, 1.0 g was weighed, and 50 ml of 70% ethanol was added. The mixture was extracted at room temperature for 45 min by ultrasonic extraction. The sample was detected, and the extract was used as a reference. The detection results are shown in Tables 2-4.

[0031] Table 1 Extraction yields at different temperatures (55, 60, 70, 80, and 90°C) at pH 3

[0032]

[0033]

[0034] Table 2 Extraction yields at different temperatures (55, 60, 70, 80, and 90°C) at pH 4

[0035]

[0036] Table 3 Extraction yields at different temperatures (55, 60, 70, 80, and 90°C) at pH 6

[0037]

[0038]

[0039] HPLC detection method

[0040] A Waters e2695 HPLC was used, and the detector was a 2998 PDA Detector. The chromatographic column was an Atlantis TM 3.5 μm, 4.6×250 mm; the detection wavelengths were 327 nm, 280 nm, 254 nm, and 230 nm, the flow rate was 1 ml / min; the column temperature was 30±2°C; and the mobile phase gradient is shown in Table 4.

[0041] Table 4 Mobile phase gradient table

[0042] Time / min 0.1% formic acid in acetonitrile / % 0.1% formic acid in water / % 0 20 80 10 35 65 12 20 80 15 20 80

[0043] Test example result analysis:

[0044] Table 2 shows that at pH = 3, the temperature below 60℃, the verbascoside is stable, even a small amount of isoverbascoside is converted to verbascoside, but the temperature is too low, the extraction time is long, and the extraction efficiency is low. At 70-80℃, the extraction time is 1 hour, the verbascoside is stable and is not isomerized to isoverbascoside. The extraction time is more than 2 hours, and a small amount of verbascoside is isomerized to isoverbascoside. When the extraction temperature exceeds 90℃, the verbascoside is isomerized to isoverbascoside obviously.

[0045] Table 3 shows that at pH = 4, the temperature below 60℃, the verbascoside is stable and is not isomerized to isoverbascoside. The extraction time is 3 hours below 55℃, and the extraction yield of verbascoside is less than 80%. When the temperature exceeds 70℃, the verbascoside is isomerized to isoverbascoside obviously.

[0046] Table 4 shows that without adjusting the acid, at pH = 6, the temperature above 55℃, the verbascoside is partially isomerized to isoverbascoside. The isomerization ratio increases obviously above 70℃.

[0047] Preparation of examples and comparative examples

[0048] Preparation of Example 1

[0049] Step (1), 3000g of dried Dangui flower was crushed through a 10-mesh sieve to obtain dried Dangui flower powder for use;

[0050] Step (2), 45L of pure water was added to a 100L extraction barrel, and citric acid was added to adjust the pH to 3. After heating to 80℃, the dried Dangui flower powder was added, and intermittent stirring extraction was performed for 60min. Filtration was performed through a 100-mesh stainless steel sieve, and the obtained filter residue was continuously added into 30L of water solution with pH = 3, and intermittent stirring extraction was performed at 80℃ for 30min. Filtration was performed through a 100-mesh sieve.

[0051] Step (3), the filtrates of the two extractions were combined, and ceramic membrane filtration was performed while hot. The ceramic membrane filtrate was filtered through a 10000-dalton ultrafiltration membrane, and the ultrafiltration membrane permeate was further filtered through a 200-dalton nanofiltration membrane. The nanofiltration membrane retentate was collected. The 200-dalton nanofiltration membrane retentate was further concentrated through an RO membrane to a solid content of 25%, and vacuum concentration and drying were performed under reduced pressure to obtain the sample of Example 1.

[0052] Preparation of Example 2

[0053] Step (1), 3000g of dried Dangui flower was crushed through a 10-mesh sieve to obtain dried Dangui flower powder for use;

[0054] Step (2), 100L extraction barrel is added into pure water 45L, and ascorbic acid (VC) is added, pH is adjusted to 4, and after heating to 60℃, dani guihua dry flower powder is added, and intermittent stirring extraction is carried out for 60 min; 100 mesh stainless steel screen is filtered, and the obtained filter residue is continuously put into 30L of water solution with pH = 4, 60℃, intermittent stirring extraction is carried out for 30 min, and 100 mesh screen is filtered.

[0055] Step (3), the filtrates of the two times of extraction are combined, ceramic membrane is filtered while hot, ceramic membrane filtrate is filtered through 10000 dalton ultrafiltration membrane, the ultrafiltration membrane permeate is further filtered through 200 dalton nanofiltration membrane, and the nanofiltration membrane retentate is collected; the 200 dalton nanofiltration membrane retentate is further concentrated through RO membrane to 25% of solid content, and vacuum concentration is carried out under reduced pressure to obtain the sample of example 2.

[0056] Preparation of example 3

[0057] Step (1), 3000g of dani guihua dry flower is crushed through 10 mesh screen to obtain dani guihua dry flower powder for standby use;

[0058] Step (2), 100L extraction barrel is added into pure water 45L, and ascorbic acid (VC) is added, pH is adjusted to 4, and after heating to 60℃, dani guihua dry flower powder is added, and intermittent stirring extraction is carried out for 60 min; 100 mesh stainless steel screen is filtered, and the obtained filter residue is continuously put into 30L of water solution with pH = 4, 60℃, intermittent stirring extraction is carried out for 30 min, and 100 mesh screen is filtered.

[0059] Step (3), the filtrates of the two times of extraction are combined, ceramic membrane is filtered while hot, ceramic membrane filtrate is filtered through 10000 dalton ultrafiltration membrane, the ultrafiltration membrane permeate is further filtered through 200 dalton nanofiltration membrane, and the nanofiltration membrane retentate is collected; the 200 dalton nanofiltration membrane retentate is further concentrated through RO membrane to 25% of solid content, and vacuum concentration is carried out under reduced pressure to obtain the sample of example 2.

[0060] Preparation of example 4

[0061] Step (1), 3000g of dani guihua dry flower is crushed through 10 mesh screen to obtain dani guihua dry flower powder for standby use;

[0062] Step (2), 100L extraction barrel is added into pure water 45L, and ascorbic acid (VC) is added, pH is adjusted to 4, and after heating to 60℃, dani guihua dry flower powder is added, and intermittent stirring extraction is carried out for 60 min; 100 mesh stainless steel screen is filtered, and the obtained filter residue is continuously put into 30L of water solution with pH = 4, 60℃, intermittent stirring extraction is carried out for 30 min, and 100 mesh screen is filtered.

[0063] Step (3), combine the two times of extraction filtrate, ceramic membrane hot filtration, ceramic membrane filtrate, 10000 Dalton ultrafiltration membrane filtration, ultrafiltration membrane permeate, through the 300 Dalton nanofiltration membrane, collect nanofiltration membrane retentate; 300 Dalton nanofiltration membrane retentate, through the RO membrane to concentrate to solid content 25%, freeze-drying, get the sample of Comparative Example 1.

[0064] Preparation of Comparative Example 1

[0065] Step (1), 3000g of dried Dangui flower was crushed through a 10-mesh sieve to obtain dried Dangui flower powder for use.

[0066] Step (2), 45L of pure water was added to a 100L extraction barrel, heated to 80℃, and then the dried Dangui flower powder was added, and intermittent stirring extraction was performed for 60min; 100-mesh stainless steel sieve filtration was performed, and the obtained filter residue was continuously added to 30L of pure water, 80℃, intermittent stirring extraction was performed for 30min, and 100-mesh sieve filtration was performed.

[0067] Step (3), combine the two times of extraction filtrate, ceramic membrane hot filtration, ceramic membrane filtrate, 10000 Dalton ultrafiltration membrane filtration, ultrafiltration membrane permeate, through the 300 Dalton nanofiltration membrane, collect nanofiltration membrane retentate; 300 Dalton nanofiltration membrane retentate, through the RO membrane to concentrate to solid content 25%, freeze-drying, get the sample of Comparative Example 1.

[0068] Preparation of Comparative Example 2

[0069] Step (1), 3000g of dried Dangui flower was crushed through a 10-mesh sieve to obtain dried Dangui flower powder for use.

[0070] Step (2), 45L of pure water was added to a 100L extraction barrel, heated to 80℃, and then the dried Dangui flower powder was added, and intermittent stirring extraction was performed for 60min; 100-mesh stainless steel sieve filtration was performed, and the obtained filter residue was continuously added to 30L of pure water, 80℃, intermittent stirring extraction was performed for 30min, and 100-mesh sieve filtration was performed.

[0071] Step (3), combine the two times of extraction filtrate, ceramic membrane hot filtration, ceramic membrane filtrate, 10000 Dalton ultrafiltration membrane filtration, ultrafiltration membrane permeate, through the 300 Dalton nanofiltration membrane, collect nanofiltration membrane retentate; 300 Dalton nanofiltration membrane retentate, through the RO membrane to concentrate to solid content 25%, freeze-drying, get the sample of Comparative Example 1.

[0072] Preparation of Comparative Example 3

[0073] Step (1), 3000g of dried Dangui flower was crushed through a 10-mesh sieve to obtain dried Dangui flower powder for use.

[0074] Step (2), 100L extraction barrel is added with 45L pure water, and citric acid is added to adjust pH to 3. After heating to 80℃, dan gui flower dry pollen is added, and intermittent stirring extraction is carried out for 60min. 100 mesh stainless steel screen is used for filtration, and the obtained filter residue is continuously put into 30L water solution with pH of 3, and intermittent stirring extraction is carried out for 30min at 80℃. 100 mesh screen is used for filtration.

[0075] Step (3), the two times of extraction filtrates are combined, and ceramic membrane is used for hot filtration. The ceramic membrane filtrate is concentrated to solid content of 25% by RO membrane, and spray drying is carried out to obtain the sample of the comparative example 3.

[0076] The yield of the sample of the example and the comparative example and the content analysis of the main components in the sample are shown in table 5:

[0077] Table 5 HPLC detection results

[0078]

[0079] Anti-fatigue mouse experiment

[0080] Test sample: PHEG50 prepared by example 1, physiological saline;

[0081] 40 healthy male SPF level mice of Kunming species with 5 weeks of age and weighing 29.00±1.65g are randomly divided into exercise group, PHEG50 low dose (15mg / kg, test sample mass to mouse weight ratio) + exercise group, medium dose (30mg / kg) + exercise group, high dose (60mg / kg) + exercise group after adaptive feeding of experimental animals for 2 days. There are 10 mice in each group, and the mice are free to eat at room temperature of 18-24℃. PHEG50 is dissolved with physiological saline, and the mice are gavaged once a day. The normal control group and the exercise group are gavaged with physiological saline with the same volume. The experimental time is 4 weeks. The general conditions of the mice are observed and recorded every day, and the mice are weighed once a week.

[0082] Except for the normal control group, the mice in each group are subjected to non-weighted swimming at 3h after gavage. The water temperature is 25℃, and the water depth is 40cm. The mice are subjected to 30min every day in the first week, 60min every day in the second week, 90min every day in the third week, and 120min every day in the fourth week. The mice are subjected to the swimming for 6 days every week, and the swimming is carried out for 4 weeks. On the 29th day, the mice in each group except for the normal control group are subjected to one-time exhaustive swimming, and the exhaustive time is recorded.

[0083] Table 6 body weight and exhaustive swimming time of mice in each group

[0084] Group Body weight before experiment / g Body weight at 4th week / g Swimming time to exhaustion / min Exercise group 29.36±1.28 31.19±2.36# 78.60±26.38* Low dose group 29.47±1.34 32.37±2.19# 118.32±29.27* Medium dose group 29.24±1.27 32.45±2.28# 145.11±30.25* High dose group 29.35±1.36 32.68±2.44# 157.24±35.48*

[0085] Note: "#" represents P<0.01; "*" represents 0.01

[0086] Result analysis:

[0087] The exhaustive swimming experiment of mice shows that compared with the exercise group, the exhaustive swimming time of the low, medium and high dose PHEG50+exercise groups is significantly prolonged, and the exhaustive swimming time is obviously prolonged with the increase of the dose, indicating that the osmanthus standard active substance PHEG50 provided by the application has obvious effect of relieving physical fatigue.

[0088] Finally, it should be noted that: the above only for the preferred embodiments of the present application, and not for limiting the present application, although the foregoing embodiments of the present application are described in detail, for those skilled in the art, it still can be modified, or equivalent replacement of part of the technical features recorded in the foregoing embodiments. Any modification, equivalent replacement, improvement, etc. within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A process for the preparation of Osmanthus fragrans standard active PHEG 50, characterized by, It comprises the following steps: (1) mixing Osmanthus fragrans Lour. dried flowers with water, with a liquid-material ratio of 15 mL:1 g, adding citric acid to adjust the pH value to 3, and then extracting at 80℃ for 1 h to obtain a crude extract of Osmanthus fragrans Lour.; (2) filtering the crude extract of Osmanthus fragrans Lour. to obtain a filtrate, and then concentrating and drying the filtrate to obtain a standard active substance PHEG50 of Osmanthus fragrans Lour.; The crude extract of Osmanthus fragrans Lour. is filtered with a ceramic membrane while hot to obtain a ceramic membrane filtrate; the ceramic membrane filtrate is filtered with a 5000-10000 dalton ultrafiltration membrane to obtain an ultrafiltration membrane permeate; the ultrafiltration membrane permeate is filtered with a 200-250 dalton nanofiltration membrane to obtain a nanofiltration membrane retentate; and the nanofiltration membrane retentate is concentrated with an RO membrane and dried to obtain the standard active substance PHEG50 of Osmanthus fragrans Lour. The standard active substance PHEG50 of Osmanthus fragrans Lour. contains 58.87% total phenylethanol glycosides by mass percentage, and contains 45.36% verbascoside, 11.32% salidroside, and 2.19% isoverbascoide by mass percentage.

2. The process for the preparation of Osmanthus fragrans standard active PHEG 50 as claimed in claim 1, wherein, The dried flowers of Osmanthus fragrans Lour. in step (1) are crushed to pass through a sieve with a mesh size of ≥10.

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