Component separation method of xanthoceras sorbifolia bunge tea leaf dichloromethane extract

Abstract

The invention provides a component separation method of a xanthoceras sorbifolia bunge tea leaf dichloromethane extract. Comprising the following steps: step 1, preparing materials and equipment; step 2, extracting and separating: weighing shiny-leaved yellowhorn tea leaf powder, heating, refluxing and extracting by using an ethanol solution, combining extracting solutions, and concentrating under reduced pressure to obtain a total extract; extracting with dichloromethane, concentrating under reduced pressure to obtain 26.1 g of dichloromethane, dissolving the residual methanol extract with 70% methanol water, filtering, performing preparative liquid chromatography, and eluting; 3, through separation and analysis, the four compounds are determined to be quercitrin, quercetin, myricetin and rutin respectively. According to separation results, the xanthoceras sorbifolia bunge tea contains more flavonoid components, and the flavonoid components have the effects of resisting inflammation, resisting oxidation, reducing blood fat and the like and are important components for playing pharmacological effects of the xanthoceras sorbifolia bunge tea.

Description

Technical Field

[0001] The invention relates to the field of preparation of dichloromethane extracts of Xanthoceras sorbifolia tea leaves, and in particular to a component separation method of dichloromethane extracts of Xanthoceras sorbifolia tea leaves. Background Art

[0002] Xanthoceras sorbifolia is a plant with both medicinal and edible properties. Domestic and foreign researchers have studied the chemical components of various parts of Xanthoceras sorbifolia. The main phytochemical components of different parts of Xanthoceras sorbifolia include triterpenes, flavonoids, phenolic acids, coumarins, lignans, hemiterpenes, monoterpenes, alkaloids and sterols. Among them, triterpenes, flavonoids and phenolic acids are the main compounds.

[0003] In an in vitro study, Wang et al. used ultrasound-assisted extraction of flavonoids from Xanthoceras sorbifolia leaves to study their antibacterial activity against Escherichia coli, Staphylococcus aureus, and Bacillus subtilis. Moreover, the inhibitory effects of total flavonoids and tea polyphenols on the three bacteria increased with their concentrations (2.14, 4.28, and 8.56 mg / mL). At a concentration of 8.56 mg / mL, total flavonoids, tea polyphenols, and 95% ethanol had significant effects on Escherichia coli (inhibition zones of 7.72 ± 0.43 mm, 6.60 ± 0.21 mm, and 7.30 ± 0.35 mm, respectively), Staphylococcus aureus (inhibition zones of 6.70 ± 0.24 mm, 6.40 ± 0.31 mm, and 6.62 ± 0.23 mm, respectively), and Bacillus subtilis (inhibition zones of 6.60 ± 0.39 mm, 6.22 ± 0.28 mm, and 6.10 ± 0.33 mm, respectively). The results showed that flavonoids had inhibitory effects on all three bacteria.

[0004] Many flavonoids have been isolated and identified from Xanthoceras sorbifolia. So far, statistics show that the main types are brass, flavanone, dihydroflavonoid, biflavonoid and other compounds. It can be seen that the preparation of dichloromethane extract from Xanthoceras sorbifolia tea leaves is a crucial step in the above applications. Summary of the invention

[0005] The invention aims to provide a method for separating components of a dichloromethane extract of Xanthoceras sorbifolia tea leaves.

[0006] The present invention is achieved through the following technical solutions:

[0007] The present invention relates to a method for separating components of a dichloromethane extract of Xanthoceras sorbifolia tea leaves, which specifically comprises the following steps:

[0008] 1. Materials and Methods

[0009] 1.1 Instruments and materials Bruker AVAIVCEIII-500 NMR spectrometer (Bruker, Germany); LC-20AT preparative liquid chromatograph C18 (21.2 mm × 250 mm, 10 μm) (Shimadzu, Japan); column chromatography silica gel (200-300 mesh, Qingdao Hailang Silica Gel Desiccant Co., Ltd.); dextran gel (Sephadex LH-20, Pharmacia, Sweden); silica gel prefabricated plate (GF254, 100 mm × 200 mm, Qingdao Yonghai Silica Gel Co., Ltd.); chromatographic methanol and other reagents are all analytical grade (Tianjin Damao Chemical Reagent Factory). Xanthoceras sorbifolia leaves were collected in Tongliao City, Inner Mongolia Autonomous Region in July 2023 and identified as Xanthoceras sorbifolia leaves by Professor Laxinamujila of Inner Mongolia University for Nationalities. The specimens were stored in Laboratory 306, College of Mongolian Medicine, Inner Mongolia University for Nationalities.

[0010] 1.2 Extraction and separation

[0011] Weigh 150g of Xanthoceras sorbifolia tea powder, extract it with 75% ethanol solution by heating under reflux for 3 times, each time for 3h, combine the extracts and concentrate under reduced pressure to obtain the total extract as ethanol extract (62.75g), add a certain amount of water to a part of it to make a suspension, extract it with dichloromethane, and concentrate it under reduced pressure to obtain a dichloromethane extract (26.1g), and dissolve the other part of the remaining extract with 70% methanol water, filter it and conduct preparative liquid chromatography, use water (A)-75% formic acid (B pump), the flow rate is 5ml / min, and isocratic elution is performed at 274nm.

[0012] 1.3 Analysis

[0013] Four compounds were isolated from the dichloromethane extract of Xanthoceras sorbifolia tea leaves. 1 H-NMR, 13 C-NMR) data analysis and literature data comparison, the chemical structural formulas of four compounds were determined. All compounds were isolated from Xanthoceras sorbifolia tea leaves for the first time. The structural formulas of the isolated compounds are shown in Table 1.

[0014] Table 1

[0015]

[0016]

[0017] The present invention has the following advantages:

[0018] The method of the present invention is to prepare the dichloromethane extract of Xanthoceras sorbifolia tea leaves. From the types of compounds obtained by extraction and separation, it can be concluded that Xanthoceras sorbifolia tea leaves contain more flavonoid components, and 4 compounds are determined to be quercetin, quercetin, myricetin, and rutin; and flavonoid compounds, as products of secondary metabolism in plants, have anti-inflammatory, anti-oxidative, hypolipidemic, and hypolipidemic effects, and are important components for the pharmacological effects of Xanthoceras sorbifolia tea leaves. The test results of the present invention lay a foundation for the research on the quality standard of Xanthoceras sorbifolia tea leaves and the research on the basic material of pharmacological effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a process diagram for the preparation of the dichloromethane extract of Xanthoceras sorbifolia tea leaves and the method for separating its components. DETAILED DESCRIPTION

[0020] The present invention is described in detail below in conjunction with specific embodiments. It should be noted that the following embodiments are only further descriptions of the present invention, but the protection scope of the present invention is not limited to the following embodiments.

[0021] Example

[0022] This embodiment relates to a method for separating the components of a dichloromethane extract of Xanthoceras sorbifolia tea leaves. Figure 1 As shown, the specific steps are:

[0023] 1. Materials and Methods

[0024] 1.1 Instruments and materials Bruker AVAIVCEIII-500 NMR spectrometer (Bruker, Germany); LC-20AT preparative liquid chromatograph C18 (21.2 mm × 250 mm, 10 μm) (Shimadzu, Japan); column chromatography silica gel (200-300 mesh, Qingdao Hailang Silica Gel Desiccant Co., Ltd.); dextran gel (Sephadex LH-20, Pharmacia, Sweden); silica gel prefabricated plate (GF254, 100 mm × 200 mm, Qingdao Yonghai Silica Gel Co., Ltd.); chromatographic methanol and other reagents are all analytical grade (Tianjin Damao Chemical Reagent Factory). Xanthoceras sorbifolia leaves were collected in Tongliao City, Inner Mongolia Autonomous Region in July 2023 and identified as Xanthoceras sorbifolia leaves by Professor Laxinamujila of Inner Mongolia University for Nationalities. The specimens were stored in Laboratory 306, College of Mongolian Medicine, Inner Mongolia University for Nationalities.

[0025] 1.2 Extraction and separation

[0026] Weigh 150g of Xanthoceras sorbifolia tea powder, extract it with 75% ethanol solution by volume for 3 times under heating and reflux, each time for 3h, combine the extracts and concentrate under reduced pressure to obtain the total extract as ethanol extract (62.75g), add a certain amount of water to a part of it to make a suspension, extract it with dichloromethane, and concentrate it under reduced pressure to obtain a dichloromethane extract (26.1g), and dissolve the remaining extract in 70% methanol water, filter it, and conduct preparative liquid chromatography, use water (A)-75% formic acid (B pump), the flow rate is 5ml / min, and the elution isocratic at 274nm;

[0027] 1.3 Analysis

[0028] Four compounds were isolated from the dichloromethane extract of Xanthoceras sorbifolia tea leaves. 1 H-NMR, 13 C-NMR) data analysis and literature data comparison, identified four compounds are: quercetin, quercetin, myricetin, rutin; all compounds are isolated from Xanthoceras sorbifolia tea leaves for the first time.

[0029] The structural formula (I) of the quercetin is:

[0030]

[0031] The structural formula of the quercetin is (II):

[0032]

[0033] The structural formula (III) of the myricitrin is:

[0034]

[0035] The structural formula (IV) of the rutin is:

[0036]

[0037] 2. Results of the above NMR data analysis:

[0038] Compound 1: yellow powder.

[0039] 1 H-NMR (500MHz, Methanol-d4) δ: 7.31 (d, J = 11.6 Hz, 4H), 6.89 (s, 2H), 6.36 (s, 2H), 6.19 (s, 2H), 5.34 (s, 2H), 0.92 (s, 7H). 13C-NMR (126MHz, MeOD) δ: 179.79, 166.25, 163.36, 159.44, 158.70, 146.58, 136.38, 123.15, 123.01, 117.11, 116.54, 106.00, 103.70, 100.05, 94.92, 74.01, 73.44, 72.30, 64.59, 17.80. The above data are basically consistent with the literature, so compound 1 is identified as quercitrin.

[0040] Compound 2: 1 H-NMR (500MHz, Methanol-d4): 7.25 (d, J = 16.3 Hz, 4H), 6.32 (s, 2H), 6.13 (s, 2H), 5.01 (d, J = 7.8 Hz, 3H), 4.46 (s, 2H). 13 C-NMR (126 MHz, MeOD) δ:

[0041] 179.50,166.33,159.49,158.60,146.55,146.46,135.89,110.51,104.99,102.56,100.13,95.01,78.34,77.35,75.81,74.09,73.99,72.32,72.25,71.48,69.84,68.71,64.55,17.99. The above data are basically consistent with the literature, so compound 2 is identified as quercetin.

[0042] Compound 3: Yellow granular crystals.

[0043] 1 H-NMR(500MHz,Methanol-d4)δ: 6.77(s,9H),6.22(s,5H),6.02(s,4H),5.14(s,4H),4.05(s,4H),3.7 3–3.64(m,4H),3.63(s,4H),3.51–3.45(m,3H),3.41(dd,J=11.4,4.7Hz,5H),0.78(d,J=17.8Hz,17H). 13C-NMR (126MHz, MeOD) δ: 179.79, 166.18, 163.34, 159.56, 158.65, 146.99, 138.05, 136.43, 122.04, 109.70, 105.96, 103.75, 99.99, 94.86, 74.00, 73.49, 72.25, 72.18, 72.02, 64.56, 17.81. The above data are basically consistent with the literature, so compound 3 is identified as myricitrin.

[0044] Compound 4: 1 H-NMR (500MHz, Methanol-d4) δ: 7.70 (s, 1H), 7.62 (d, J = 8.4Hz, 3H), 7.58 (d, J = 8.2Hz, 1H), 6.86 (d, J=8.5Hz,2H),6.38(s,2H),6.20(s,2H),5.09(d,J=7.6Hz,2H),4.51(s,2H),1.11(d,J=6.1Hz,5H). 13 C-NMR (126MHz, MeOD) δ: 179.55, 166.44, 163.14, 159.48, 158.70, 149.99, 146.01, 135.78, 123.72, 117.84, 116.22, 105.72, 104.91, 102.59, 100.19, 95.08, 78.35, 77.38, 75.89, 74.10, 74.03, 72.40, 71.56, 69.87, 68.72, 64.58, 18.05. The above data are basically consistent with the literature, so compound 4 is identified as rutin.

[0045] From the types of compounds obtained by extraction and separation, it can be seen that Xanthoceras sorbifolia tea contains more flavonoids, and flavonoids, as products of secondary metabolism in plants, have anti-inflammatory, antioxidant, hypolipidemic, and hypolipidemic effects, and are important components of Xanthoceras sorbifolia tea to exert pharmacological effects. The experimental results of the present invention lay the foundation for the research on the quality standard of Xanthoceras sorbifolia tea and the research on the basic material of pharmacological effects.

[0046] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various modifications or variations within the scope of the claims, which will not affect the essence of the present invention.

Claims

1. A method for separating components of a dichloromethane extract of Xanthoceras sorbifolia tea leaves, characterized in that: The following steps are involved: Step 1, preparation of materials and equipment; Step 2, extraction and separation Weigh 150 g of Xanthoceras sorbifolia tea powder, extract it with 75% ethanol solution by heating and refluxing for 3 times, each time for 3 h, combine the extracts and concentrate under reduced pressure to obtain 62.75 g of total extract; 62.75g of the extract was extracted with dichloromethane, and after reduced pressure concentration, 26.1g of dichloromethane was obtained. The remaining extract was dissolved in 70% methanol water, filtered and subjected to preparative liquid chromatography, using water-75% formic acid, a flow rate of 5ml / min, and isocratic elution at a wavelength of 274nm; Step 3, Analysis A total of four compounds were isolated from the dichloromethane extract of Xanthoceras sorbifolia tea leaves. Through the analysis of nuclear magnetic resonance data and comparison with literature data, the four compounds were determined to be quercetin, quercetin, myricetin and rutin.

2. The method for separating components of the dichloromethane extract of Xanthoceras sorbifolia tea leaves according to claim 1, characterized in that: The structural formula (I) of the quercetin is:

3. The method for separating components of the dichloromethane extract of Xanthoceras sorbifolia tea leaves according to claim 1, characterized in that: The structural formula (II) of the quercetin is:

4. The method for separating components of the dichloromethane extract of Xanthoceras sorbifolia tea leaves according to claim 1, characterized in that: The structural formula (III) of the myricitrin is:

5. The method for separating components of the dichloromethane extract of Xanthoceras sorbifolia tea leaves according to claim 1, characterized in that: The structural formula (IV) of the rutin is:

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