Method for simultaneously enriching and preparing ultrahigh-purity caffeic acid and narcissoside from ginkgo leaves

Abstract

The invention discloses a method for simultaneously enriching and preparing ultra-high-purity caffeic acid and narcissoside from ginkgo leaves, which comprises the following steps: quickly extracting crushed ginkgo leaves by using a pure methanol solvent in combination with an ultrasonic wall breaking manner at room temperature. Carrying out vacuum filtration on the extracting solution, carrying out rotary evaporation and concentration until no alcohol smell exists, and treating and removing impurities by using an ultrafiltration membrane to obtain a clear solution; and carrying out vacuum drying on the solution to obtain the solid ginkgo leaf extract. The narcissoside and the caffeic acid are prepared from the extract redissolved by methanol through high performance liquid chromatography column fixed peak collection, and the HPLC (high performance liquid chromatography) purities of the narcissoside and the caffeic acid can reach 30-50%. After the first liquid chromatography conditions are adjusted, the solution rich in narcissoside and caffeic acid is subjected to second high performance liquid chromatography accurate peak determination collection, and the HPLC purity of the obtained narcissoside and caffeic acid can reach 99.50% or above. The method is simple and convenient to operate, short in extraction and enrichment time and high in selectivity and reproducibility, and the prepared product is ultrahigh in purity.

Description

Technical Field

[0001] This invention relates to the field of extraction and purification technology of chemical active ingredients from natural products, and specifically to a method for preparing enriched narcissin and caffeic acid. Background Technology

[0002] Ginkgo, belonging to the Ginkgoaceae family and the Ginkgo genus, is also known as maidenhair tree, duck's foot tree, or grandfather-grandson tree. It is one of the world's most precious tree species, hailed as a "living fossil" of the plant kingdom, and is considered one of the world's four major garden trees, along with cedar, Norfolk Island pine, and golden larch. In China, ginkgo is often compared to peony and orchid by horticulturists, hailed as one of the "Three Treasures of the Garden," and revered as the national tree. Globally, ginkgo trees are mainly distributed in China, Japan, North Korea, South Korea, Canada, New Zealand, Australia, and the United States. China has the richest ginkgo resources and holds an important position in the global distribution of ginkgo trees. Ginkgo trees are highly adaptable to climate and soil, allowing them to grow and reproduce in diverse geographical environments.

[0003] Ginkgo biloba extract possesses numerous advantages, including abundant active ingredients, broad pharmacological effects, high safety, ease of extraction and purification, and wide-ranging applications. These advantages make ginkgo biloba extract exhibit enormous application potential and market prospects in the pharmaceutical, health food, and cosmetic fields. Ginkgo biloba extract is remarkably effective in treating coronary heart disease, angina pectoris, and hyperlipidemia, improving related symptoms, optimizing heart health and blood lipid levels, and preventing cardiovascular and cerebrovascular diseases. It also possesses antibacterial, anti-adhesion, and anticancer potential in the oral cavity.

[0004] Caffeic acid is an organic compound belonging to the hydroxycinnamic acid family, possessing both phenolic hydroxyl and acrylic acid functional groups. It is widely found in the plant kingdom, including fruits, vegetables, grains, herbs, and spices; it is also a component of the organic acids in ginkgo leaves. Narcissin, also known as isorhamnetin-3-O-rutin, is a flavonoid compound. While commonly found in plants like daffodils, it is not limited to daffodil sources. It is primarily extracted from the whole herb of *Hedyotis chrysotricha*, a plant in the Rubiaceae family, and studies have also revealed its successful isolation from *Morinda citrifolia* (noni fruit).

[0005] Caffeic acid possesses a variety of effects, including hemostasis, coagulation, anti-inflammation, antibacterial properties, immunomodulation, antitumor activity, cardiovascular protection, and the ability to increase white blood cell and platelet counts. Narcissin (especially colchicine) is a compound with diverse biological activities, exhibiting significant antioxidant, anti-inflammatory, and neuroprotective capabilities. It helps prevent disease, maintain health, and may play a positive role in anti-cancer and cardiovascular protection.

[0006] The molecular weight of narcisin is 624.56, CAS: 604-80-8, and its structural formula is as follows:

[0007] Caffeic acid has a molecular weight of 180.16, CAS number 331-39-5, and its structural formula is as follows:

[0008] Currently, with the development of science and technology, extraction and purification techniques are constantly improving. Modern extraction techniques such as ultrasound-assisted extraction and microwave-assisted extraction have been applied to improve the extraction rate and purity of narcissin from ginkgo leaves. These techniques can more effectively separate narcissin from ginkgo leaves while reducing damage to heat-sensitive components. In terms of purification, advanced techniques such as high-performance liquid chromatography (HPLC) are widely used in the purification process of ginkgo leaf extracts to ensure the acquisition of high-purity narcissin. Caffeic acid is not one of the main components in ginkgo leaf extracts. Although caffeic acid is not a core or in-depth research subject in ginkgo leaf polyphenol research, its low content makes the extraction process complex and challenging, requiring the use of advanced extraction techniques and precise purification processes to optimize extraction efficiency.

[0009] Existing patents and literature primarily report methods for identifying narcissin and caffeic acid in Ginkgo biloba extract, or studies and applications of their activity, with relatively few studies on enrichment and purification. Chinese patents 202110540716.0 and 202011417214.0 both disclose methods for identifying components in Ginkgo biloba extract, proving the presence of narcissin, but without separation and purification, resulting in relatively complex substances. Chinese patent 201611217062.3 discloses a method for simultaneously determining the content of multiple components in Ginkgo biloba extract and its preparations. However, this method does not involve freeze-drying or ultrafiltration of the Ginkgo biloba extract, resulting in numerous impurity peaks and difficulty in long-term preservation. While it can detect the content of narcissin, it cannot obtain high-purity narcissin through a single liquid chromatography analysis. Chinese Patent 201710887699.1 discloses a method for determining the content and uses of flavonoids in Ginkgo biloba leaves and their preparations. This method uses a water bath reflux extraction, which has a long extraction time. Liquid chromatography analysis confirmed that the total flavonoid content was 5.047 mg / g, with a percentage content between 0.21% and 0.72%, making it difficult to obtain high-purity naringin. All the above methods use a single liquid chromatography detection to prove the presence of naringin in Ginkgo biloba leaf extract, but cannot obtain a high-purity product. Chinese Patent 202210953454.5 discloses an integrated extraction, enrichment, separation, and purification method for the entire industrial chain of effective components from Ginkgo biloba leaves. It first separates water-soluble and lipid-soluble components. Based on the different physicochemical properties, the complex separation and purification process of Ginkgo biloba leaf extract can obtain more abundant active substances (such as ginkgolides) with a purity of over 90%, but it is difficult to separate caffeic acid, which has a low content, into a single component.

[0010] Researchers have used different macroporous adsorption resins to enrich narcisin in Sophora japonica buds and determined its content to be between 4% and 8% using high-performance liquid chromatography (HPLC). Other researchers extracted Sophora japonica buds with ethanol three times after pulverizing them, and then extracted the combined extracts with petroleum ether, chloroform, ethyl acetate, and n-butanol, followed by evaporation to dryness. The ethyl acetate and chloroform fractions were separated by silica gel column chromatography, then eluted with chloroform and methanol, combined, and eluted with methanol again by silica gel column chromatography to obtain a yellow powder. This method is complex, time-consuming, and inefficient. Researchers have prepared Sophora japonica flower extract using water extraction and determined the narcisin content to be between 3% and 6% using HPLC. Numerous studies have been conducted on extracting caffeic acid from other raw materials. Researchers have used common extraction methods such as boiling, ultrasonic extraction, alcohol extraction, and Soxhlet extraction to extract caffeic acid from samples including Cynanchum paniculatum, Lysimachia christinae, Malva verticillata, and Duchesnea indica. HPLC analysis showed that the purity of these extracts was below 50%. However, research on extracting caffeic acid from Ginkgo biloba leaves is relatively limited.

[0011] Based on the different polarities of narcisin and caffeic acid, this invention proposes a method using high-performance liquid chromatography (HPLC) to simultaneously enrich and purify narcisin and caffeic acid from Ginkgo biloba extract. This method is simple to operate, and the final HPLC purity of narcisin and caffeic acid both reach over 99.50%. Summary of the Invention

[0012] This invention provides a method for enriching and purifying narcissin and caffeic acid from Ginkgo biloba extract, which solves the problems of complex extraction and separation methods, low efficiency, and long time in the production process of narcissin and caffeic acid with a certain purity. It is a new production process.

[0013] The technical solution adopted in this invention is as follows: A method for simultaneously enriching and preparing ultra-high purity caffeic acid and narcisin from ginkgo leaves, comprising the following steps: Step 1, extraction: Weigh the crushed ginkgo leaves and extract them using an ultrasonic method at a certain material-to-liquid ratio. The ultrasonic power is 100~300W, the extraction solvent is pure methanol, the extraction temperature is room temperature, and the extraction time is 5~30min. The second step is filtration: the extract obtained in the first step is vacuum filtered, and the clear liquid is collected. The third step is concentration and ultrafiltration: the extract obtained in the second step is concentrated by rotary evaporation, the concentrate is added with water and then evaporated by rotary evaporation, the concentrate is diluted with water again and then ultrafiltered using an ultrafiltration membrane, and the solution obtained by ultrafiltration is collected. Step 4, drying: The extract obtained in step 3 is dried under vacuum to obtain solid ginkgo leaf extract; Step 5, HPLC column separation: The Ginkgo biloba extract obtained in step 4 was completely dissolved in methanol. HPLC analysis was performed, and under specific wavelength, temperature, flow rate, time, and injection volume, gradient elution was used to collect the peaks of narcisin and caffeic acid in a specific mobile phase. HPLC detection showed that the HPLC purity of narcisin and caffeic acid reached 30%–70%. The first collection was re-collected and analyzed, and the HPLC purity reached 99.50%.

[0014] The material-to-liquid ratio extracted in the first step is 1:10 or 1:30.

[0015] The temperature of rotary evaporation in the second step is 50~60℃.

[0016] The ultrafiltration membrane used in the third step is an Ultracel 1kDa ultrafiltration membrane with a pore diameter of 44.5mm and made of regenerated cellulose.

[0017] The vacuum freeze drying in the fourth step is set at a temperature of -5°C, a pressure of 1000 mtorr, and a drying time of 48 h.

[0018] In the fifth step, the liquid phase conditions for the first liquid phase collection are as follows: gradient elution 0-6 min: 90-70% B, 6-13 min: 70-60% B, 13-17 min: 60-55% B, 17-19 min: 55-51.7% B, 19-27 min: 51.7-48.5% B, 27-28 min: 48.5-45% B, 28-35 min: 45-23% B, 35-40 min... The elution parameters were: 23-95% B, 40-45 min: 95-90% B, 45-60 min: 90-90% B, wavelength 280 nm, injection volume 20 μL, elution time 60.01 min, flow rate 0.8 mL / min, and an Agilent TC-C18 column. For liquid chromatography detection and the second liquid chromatography, isocratic elution was used, with phase A being pure methanol and phase B being 0.2% acetic acid in water, with phase A comprising 25% and phase B comprising 75%. The flow rate was 0.8 mL / min, wavelength 280 nm, injection volume 20 μL, time 30 min, and an Agilent TC-C18 column was used. High-quality silica gel was used as the main porous packing material, with an inner diameter of 4.6 mm and a particle size of 5 μm. In the fifth step, the first peak determination collection took place at times of 17.51–18.10 min and 23.46–23.95 min, with collection heights of 400,000 μV and 350,000 μV, respectively. In the second peak determination collection, naringin was collected at times of 9.73–10.60 min with a collection height of 25,000 μV, and caffeic acid was collected at times of 20.01–21.50 min with a collection height of 3,000 μV.

[0019] Beneficial effects: 1. This invention utilizes ultrasonic rapid cell disruption to improve the release and extraction efficiency of narcissin and caffeic acid, significantly shortening the extraction time (3-5 min) and the production cycle; 2. In this invention, ultrafiltration is used before freeze-drying to remove macromolecular impurities such as polysaccharides and proteins, which reduces impurity peaks in liquid phase separation and is beneficial for separating and purifying the target compound; 3. The production process of this invention is simple, and extraction and separation are carried out at a lower temperature, which effectively protects the active substances from thermal shrinkage, decomposition, oxidation or polymerization during the extraction process, and the obtained product is easy to store for a long time. 4. This invention is the first to simultaneously enrich and prepare ultra-high purity narcisin and caffeic acid from Ginkgo biloba leaves. The process employs two high-performance liquid chromatography (HPLC) separations, resulting in a stable, selective, accurate, and reproducible preparation method. HPLC analysis shows that the enriched narcisin and caffeic acid both reached 99.50%. 5. This invention uses widely available and inexpensive raw materials, and prepares narcissin and caffeic acid more economically than other methods. Attached Figure Description

[0020] Figure 1 A flowchart provided for this invention; Figure 2 The liquid chromatogram of enriched narcisin; Figure 3 The liquid chromatogram of enriched caffeic acid; Figure 4 The mass spectrum (negative charge mode) of the enriched narcisin. Figure 5 This is the mass spectrum (positive charge mode) of the enriched caffeic acid. Detailed Implementation

[0021] A method for simultaneously enriching caffeic acid and narcisin from ginkgo leaves (1) Extraction: Accurately weigh a certain amount of crushed ginkgo leaves as the starting material, and use organic solvent to perform ultrasonic extraction under the conditions that the ratio of ginkgo leaves to solvent is 1:10~1:30, the ultrasonic power range is 100-300W, the extraction environment temperature is room temperature, and the extraction time is 5-30min.

[0022] (2) Vacuum filtration: The extract obtained in step (1) is vacuum filtered to remove solid particles and impurities, and the filtrate is retained to obtain the initial extract.

[0023] (3) Concentration and ultrafiltration: The extract from step (2) is rotary evaporated at 50-60°C to obtain a concentrate. The concentrate is subjected to a second rotary evaporation and diluted with pure water. The diluted concentrate is ultrafiltered using a 1 kDa ultrafiltration membrane, and the extract that passes through the ultrafiltration membrane is collected.

[0024] (4) Drying: Place the ultrafiltration solution obtained in step (3) in a freeze dryer and vacuum dry it to a solid at -5℃ to 5℃ to obtain Ginkgo biloba extract.

[0025] (5) Liquid chromatography column separation: The Ginkgo biloba extract obtained in step four was completely dissolved in methanol. Liquid chromatography analysis was performed at a wavelength of 280 nm, a column temperature of 30 °C, a flow rate of 0.8 mL / min, and an injection volume of 40 μL. A gradient elution method was used to collect the peaks of narcissin and caffeic acid in a specific mobile phase. Liquid chromatography detection showed that the HPLC purity of narcissin and caffeic acid was 30%–70%. A second peak collection and detection was performed, and the HPLC purity reached 99.50%. The collected samples were verified by liquid chromatography-mass spectrometry.

[0026] The solvent used in step (1) is pure methanol solvent with a purity of 99%.

[0027] In step (3), during rotary evaporation, the amount of water added is 1 / 5 of the original solution volume.

[0028] The vacuum drying time in step (4) is 48 hours.

[0029] The target solution collected in step (5) should be stored in an environment of 4-8℃ in a timely manner to facilitate the second liquid phase separation.

[0030] In step (5), reversed-phase high-performance liquid chromatography (RP-HPLC) was used for detection and collection. The conditions for the first collection were gradient elution: 0-6 min: 90-70% B, 6-13 min: 70-60% B, 13-17 min: 60-55% B, 17-19 min: 55-51.7% B, 19-27 min: 51.7-48.5% B, 27-28 min: 48.5-45% B, 28-35 min: 45-23% B. %B, 35-40min: 23-95%B, 40-45min: 95-90%B, 45-60min: 90-90%B, wavelength: 280nm, injection volume: 20µL, elution time: 60.01min, flow rate: 0.8mL / min, column used: Agilent TC-C18 column; liquid chromatography detection and second liquid chromatography collection conditions: isocratic elution, phase A: pure methanol, phase B: 0.2% acetic acid in water, phase A: 25%, phase B: 75%. Flow rate: 0.8mL / min, wavelength: 280nm, injection volume: 20µL, time: 30min, column used: Agilent TC-C18 column; The liquid chromatography-mass spectrometry conditions used in step (5) were as follows: phase A was pure methanol, phase B was 0.2% acetic acid water, phase A accounted for 25%, and phase B accounted for 75%. The flow rate was 0.3 mL / min, the wavelength was 280 nm, the injection volume was 10 μL, the column temperature was 30 °C, the scanning mode was positive ion mode and negative ion mode, the scanning range was 100~1000 m / z, and the scanning voltage was +4500 V or -4500 V.

[0031] Example 1 (1) Extraction: Accurately weigh 16.600g of crushed ginkgo leaves as the starting material. Add 500mL of methanol as the extraction solvent, i.e., the material-to-liquid ratio is 1:30, and ultrasonic extraction is used. Set the ultrasonic power to 300W, control the extraction temperature to room temperature, and the extraction time to 10 min; (2) Vacuum filtration: The extract obtained in step (1) is vacuum filtered to remove solid particles and impurities, and the filtrate is retained to obtain the initial extract; (3) Concentration and ultrafiltration: The extract from step (2) is rotary evaporated at 50°C to obtain a concentrate. The concentrate is subjected to a second rotary evaporation and diluted with pure water. The diluted concentrate is ultrafiltered using a 1 kDa ultrafiltration membrane, and the extract that passes through the ultrafiltration membrane is collected; (4) Drying: The ultrafiltration solution obtained in step (3) is placed in a freeze dryer and vacuum dried to a solid at -5°C to obtain Ginkgo biloba extract; (5) Liquid chromatography column separation: The Ginkgo biloba extract obtained in step 4 was completely dissolved in methanol. After liquid chromatography analysis, under the conditions of wavelength of 280 nm, column temperature of 30 °C, flow rate of 0.8 mL / min and injection volume of 40 μL, the peaks of narcissin and caffeic acid were collected in a specific mobile phase by gradient elution. The HPLC purity of narcissin and caffeic acid was 50.8% and 41.3%, respectively. After a second peak collection and detection, the HPLC purity was 99.50% and 99.52%, respectively.

[0032] Example 2 (1) Extraction: Accurately weigh 3.000g of crushed ginkgo leaves as the starting material. Add 30mL of methanol as the extraction solvent, with a material-to-liquid ratio of 1:10, and use ultrasonic extraction. Set the ultrasonic power to 150W, the extraction temperature to room temperature, and the extraction time to 5min; (2) Vacuum filtration: The extract obtained in step (1) is vacuum filtered to remove solid particles and impurities, and the filtrate is retained to obtain the initial extract; (3) Concentration and ultrafiltration: The extract from step (2) is rotary evaporated at 50°C to obtain a concentrate. The concentrate is subjected to a second rotary evaporation and diluted with pure water. The diluted concentrate is ultrafiltered using a 1 kDa ultrafiltration membrane, and the extract that passes through the ultrafiltration membrane is collected; (4) Drying: The ultrafiltration solution obtained in step (3) is placed in a freeze dryer and vacuum dried to a solid at -5°C to obtain Ginkgo biloba extract; (5) Liquid chromatography column separation: The Ginkgo biloba extract obtained in step 4 was completely dissolved in methanol. After liquid chromatography analysis, under the conditions of wavelength of 280 nm, column temperature of 30 °C, flow rate of 0.8 mL / min and injection volume of 40 μL, the peaks of narcisin and caffeic acid were collected in a specific mobile phase by gradient elution. The HPLC purity of narcisin and caffeic acid was 53.2% and 32.1%, respectively. After a second peak collection and detection, the HPLC purity was 99.51% and 99.53%, respectively.

[0033] Example 3 (1) Extraction: Accurately weigh 50.000g of crushed ginkgo leaves as the starting material. Add 0.5L of methanol as the extraction solvent, i.e., the material-to-liquid ratio is 1:10, and ultrasonically extract for 30min. The ultrasonic power is 1200W, and the extraction temperature is 40℃; (2) Vacuum filtration: The extract obtained in step (1) is vacuum filtered to remove solid particles and impurities, and the filtrate is retained to obtain the initial extract; (3) Concentration and ultrafiltration: The extract from step (2) is rotary evaporated at 60°C to obtain a concentrate. The concentrate is subjected to a second rotary evaporation and diluted with pure water. The diluted concentrate is ultrafiltered using a 1 kDa ultrafiltration membrane, and the extract that passes through the ultrafiltration membrane is collected; (4) Drying: Place the ultrafiltration solution obtained in step (3) in a freeze dryer and vacuum dry it to a solid at 5°C to obtain Ginkgo biloba extract; (5) Liquid chromatography column separation: The Ginkgo biloba extract obtained in step 4 was completely dissolved in methanol. After liquid chromatography analysis, under the conditions of wavelength of 280 nm, column temperature of 30 °C, flow rate of 0.8 mL / min and injection volume of 40 uL, the peaks of narcissin and caffeic acid were collected in a specific mobile phase by gradient elution. The HPLC purity of narcissin and caffeic acid was 73.6% and 71.8%, respectively. After a second peak collection and detection, the HPLC purity was 99.51% and 99.50%, respectively.

[0034] In the above three implementation cases, reversed-phase high-performance liquid chromatography (RP-HPLC) was used for detection and collection. The conditions for the first liquid phase collection were gradient elution: 0-6 min: 90-70% B, 6-13 min: 70-60% B, 13-17 min: 60-55% B, 17-19 min: 55-51.7% B, 19-27 min: 51.7-48.5% B, 27-28 min: 48.5-45% B, 28-35 min: 45- 23% B, 35-40 min: 23-95% B, 40-45 min: 95-90% B, 45-60 min: 90-90% B, wavelength 280 nm, injection volume 20 μL, elution time 60.01 min, flow rate 0.8 mL / min, using an Agilent TC-C18 column; liquid chromatography detection and second liquid chromatography collection conditions: isocratic elution, phase A: pure methanol, phase B: 0.2% acetic acid in water, phase A: 25%, phase B: 75%. Flow rate 0.8 mL / min, wavelength 280 nm, injection volume 20 μL, time 30 min, using an Agilent TC-C18 column.

Claims

1. A method for simultaneously enriching and preparing ultra-high purity caffeic acid and narcisin from Ginkgo biloba leaves, characterized in that, The steps are as follows: Step 1, extraction: Weigh the crushed ginkgo leaves and extract them using an ultrasonic method at a certain material-to-liquid ratio. The ultrasonic power is 100~300W, the extraction solvent is pure methanol, the extraction temperature is room temperature, and the extraction time is 5~30min. The second step is filtration: the extract obtained in the first step is vacuum filtered, and the clear liquid is collected. The third step is concentration and ultrafiltration: the extract obtained in the second step is concentrated by rotary evaporation, the concentrate is added with water and then evaporated by rotary evaporation, the concentrate is diluted with water again and then ultrafiltered using an ultrafiltration membrane, and the solution obtained by ultrafiltration is collected. Step 4, drying: The extract obtained in step 3 is dried under vacuum to obtain solid ginkgo leaf extract; Step 5, liquid chromatography column separation: The ginkgo leaf extract obtained in step 4 was completely dissolved in methanol. After liquid chromatography analysis, narcissin and caffeic acid were collected in a specific mobile phase by gradient elution under certain wavelength, temperature, flow rate, time and injection volume. The purity of narcissin and caffeic acid was found to be 30%~70% by liquid chromatography detection. The first collection solution was collected and detected again for a second time. The HPLC purity of both reached more than 99.50%.

2. The method for preparing Ginkgo biloba extract as described in claim 1, characterized in that, The material-to-liquid ratio extracted in the first step is 1:10 or 1:

30.

3. The temperature of rotary evaporation in the second step as described in claim 1 is 50~60℃.

4. The ultrafiltration membrane in the third step as described in claim 1 is an Ultracel ultrafiltration membrane with a pore diameter of 44.5 mm, made of regenerated cellulose, and of one type among 1 kDa, 3 kDa, 5 kDa, and 30 kDa.

5. The vacuum freeze drying in the fourth step as described in claim 1, wherein the temperature is set to -5°C, the pressure is 1000 mtorr, and the drying time is 48 h.

6. The liquid phase conditions in the fifth step as described in claim 1, wherein the conditions for the first liquid phase collection are gradient elution: 0-6 min: 90-70% B, 6-13 min: 70-60% B, 13-17 min: 60-55% B, 17-19 min: 55-51.7% B, 19-27 min: 51.7-48.5% B, 27-28 min: 48.5-45% B, 28-35 min: 45% B. -23% B, 35-40 min: 23-95% B, 40-45 min: 95-90% B, 45-60 min: 90-90% B, wavelength 280 nm, injection volume 20 μL, elution time 60.01 min, flow rate 0.8 mL / min; liquid chromatography detection and second liquid chromatography collection conditions are isocratic elution, phase A is pure methanol, phase B is 0.2% acetic acid water, phase A accounts for 25%, phase B accounts for 75%. Flow rate 0.8 mL / min, wavelength 280 nm, injection volume 20 μL, time 30 min. The columns used include, but are not limited to, one of the following columns: Agilent TC-C18 liquid chromatography column, Agilent ZORBAXSB-Aq liquid chromatography column, and XBridge BEH C18 column.

7. In the fifth step as described in claim 1, the first peak determination collection takes place at times of 17.51–18.10 min and 23.46–23.95 min, with collection heights of 400,000 μV and 350,000 μV, respectively. In the second peak determination collection, the collection time for naringin is 9.73–10.60 min, with a collection height of 25,000 μV, and the collection time for caffeic acid is 20.01–21.50 min, with a collection height of 3,000 μV.

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