Porphyrin compound in Camellia chrysantha and preparation method thereof

Abstract

The present invention belongs to the technical field of natural drug chemistry, and specifically relates to a porphyrin compound in Camellia chrysantha and a preparation method thereof. In the present invention, Camellia chrysantha leaves are used as raw materials, and four porphyrin compounds are obtained from Camellia chrysantha leaves for the first time through the steps of extraction, extraction, separation and purification, which is beneficial to the development and quality control of Camellia chrysantha medicinal materials and preparations thereof, and provides a chemical basis and material reference for the comprehensive development of Camellia chrysantha medicinal materials and the research on the pharmacological activity of natural plants.

Description

Technical Field

[0001] The invention belongs to the technical field of natural drug chemistry, and specifically relates to a porphyrin compound in Camellia chrysantha and a preparation method thereof. Background Art

[0002] Camellia chrysantha has rich medicinal value. Studies have shown that its flowers are rich in vitamins and minerals that can lower blood sugar, blood lipids, and blood pressure, and regulate the body's immunity. Its leaves have anti-inflammatory, liver-protecting, choleretic, and anti-tumor effects. In-depth research on Camellia chrysantha has broad market prospects and development value.

[0003] Camellia chrysantha has obvious pharmacological effects, but its composition is relatively complex. Currently, terpenes, flavonoids, alkaloids, anthraquinones and other compounds have been reported. Chlorophyll compounds responsible for photosynthesis in green leaf plants have high research value. However, there is currently a gap in the research on porphyrins (chlorophylls) in Camellia chrysantha. In order to further understand the chlorophyll components in Camellia chrysantha, it is particularly necessary to enrich, separate and purify the new chlorophyll (chlorophyllide) components. Summary of the invention

[0004] The present invention aims to solve the above technical problems and provides a porphyrin compound in Camellia chrysantha and a preparation method thereof.

[0005] The technical solution of the present invention is:

[0006] A porphyrin compound of Camellia chrysantha, the structure of which is shown in Formula I, Formula II, Formula III and / or Formula IV:

[0007]

[0008]

[0009] They are named respectively: Camellia Chlorophyllide Ⅰ (Formula I), Camellia Chlorophyllide Ⅱ (Formula II), Camellia Chlorophyllide Ⅲ (Formula III), and Camellia Chlorophyllide Ⅳ (Formula IV).

[0010] Further, the following steps are included:

[0011] 1) extracting golden camellia leaves with 95-100% ethanol by volume to obtain an extract, and concentrating the extract under reduced pressure to remove ethanol to obtain golden camellia extract;

[0012] 2) adding water to the camellia chrysantha extract prepared in step 1) and stirring until a suspension is formed;

[0013] 3) extracting the suspension in step 2) with petroleum ether, petroleum ether-chloroform, chloroform, and ethyl acetate in sequence, and concentrating the ethyl acetate portion under reduced pressure to obtain an ethyl acetate extract;

[0014] 4) subjecting the ethyl acetate extract of step 3) to silica gel column chromatography, using petroleum ether-chloroform-ethyl acetate as an eluent for gradient elution to obtain a fraction containing a porphyrin compound;

[0015] 5) subjecting the fraction containing the porphyrin compound obtained in step 4) to an alumina chromatography column, and performing gradient elution using petroleum ether-ethyl acetate as an eluent to obtain subdivided fractions containing the porphyrin compound;

[0016] 6) subjecting the subdivided fraction containing the porphyrin compound obtained in step 5) to Sephadex LH-20 column chromatography and isocratically eluting with chloroform-methanol to obtain subdivided subfractions containing the porphyrin compound;

[0017] 7) Purifying the sub-fraction containing porphyrin in step 6) by semi-preparative high performance liquid chromatography to obtain the porphyrin compound.

[0018] Furthermore, in step 4), in the gradient elution, the volume ratios of petroleum ether, chloroform and ethyl acetate are 1:0:0, 20:1:0, 10:1:0, 5:1:0, 1:1:0, 0:1:0, 0:20:1, 0:10:1, 0:8:1, 0:5:1 and 0:0:1 respectively.

[0019] Furthermore, in step 5), in the gradient elution, the volume ratio of petroleum ether to ethyl acetate is 100:1 to 1:1.

[0020] Preferably, in the gradient elution, the volume ratio of petroleum ether to ethyl acetate is 100:1, 50:1, 20:1, 10:1, 8:1, 5:1, 1:1.

[0021] Furthermore, in step 6), in the isocratic elution, the volume ratio of chloroform to methanol is 3:1 to 1:1.

[0022] Furthermore, in step 7), the semi-preparative HPLC includes normal phase semi-preparative HPLC or reverse phase semi-preparative HPLC.

[0023] Furthermore, the conditions of the normal phase semi-preparative high performance liquid chromatography are: the chromatographic column is selected as an Agilent ZORBAXRX-SIL semi-preparative column, and the mobile phase is selected as n-hexane-isopropanol with a volume ratio of 98:2.

[0024] Furthermore, the reverse phase semi-preparative HPLC conditions are as follows: a Welch UltimateXB-C18 semi-preparative column is selected as the chromatographic column, and acetonitrile-methanol-water with a volume ratio of 90:9:1 or acetonitrile-methanol with a volume ratio of 95:5 is selected as the mobile phase.

[0025] The beneficial effects of the present invention are:

[0026] 1. The present invention uses Camellia chrysantha leaves as raw materials, and obtains 4 new porphyrin compounds from Camellia chrysantha leaves for the first time through the steps of extraction, extraction, separation and purification, which is beneficial to the development and quality control of Camellia chrysantha medicinal materials and their preparations, and provides a chemical basis and material reference for the comprehensive development of Camellia chrysantha medicinal materials and the research on the pharmacological activity of natural plants.

[0027] 2. In the present invention, four new porphyrin compounds were obtained from Camellia chrysantha leaves. They were difficult to separate due to their small polarity differences. The present invention explored more practical separation conditions with simple operation and low cost, and was suitable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The novel compound of the present invention is of formula I 1 H-NMR spectrum;

[0029] Figure 2 The novel compound of the present invention is of formula I 13 C-NMR spectrum;

[0030] Figure 3 DEPT 135 spectrum of the novel compound of formula I of the present invention;

[0031] Figure 4 The novel compound of the present invention is of formula I 1 H- 1 H COSY spectrum;

[0032] Figure 5 is the HSQC spectrum of the novel compound of formula I of the present invention;

[0033] Figure 6 is the HMBC spectrum of the novel compound of formula I of the present invention;

[0034] Figure 7 The novel compound of the present invention is of formula II 1 H-NMR spectrum;

[0035] Figure 8 The novel compound of the present invention is of formula II 13 C-NMR spectrum;

[0036] Fig. 9DEPT 135 spectrum of the novel compound of formula II of the present invention;

[0037] Fig.10 The novel compound of formula II of the present invention is 1 H- 1 H COSY spectrum;

[0038] Fig.11 HSQC spectrum of the novel compound of formula II of the present invention;

[0039] Fig.12 It is the HMBC spectrum of the novel compound of formula II of the present invention;

[0040] Fig.13 The novel compound of the present invention is of formula III 1 H-NMR spectrum;

[0041] Fig.14 The novel compound of the present invention is of formula III 13 C-NMR spectrum;

[0042] Fig.15 DEPT 135 spectrum of the novel compound of formula III of the present invention;

[0043] Fig.16 The novel compound of the present invention is of formula III 1 H- 1 H COSY spectrum;

[0044] Fig.17 HSQC spectrum of the novel compound of formula III of the present invention;

[0045] Fig.18 It is the HMBC spectrum of the novel compound of formula III of the present invention;

[0046] Fig.19 The novel compound of the present invention is of formula IV 1 H-NMR spectrum;

[0047] Fig. 20 The novel compound of the present invention is of formula IV 13 C-NMR spectrum;

[0048] Fig.21 DEPT 135 spectrum of the novel compound of formula IV of the present invention;

[0049] Fig. 22 The novel compound of the present invention is of formula IV 1 H- 1 H COSY spectrum;

[0050] Fig.23HSQC spectrum of the novel compound of formula IV of the present invention;

[0051] Fig.24 is the HMBC spectrum of the novel compound of formula IV of the present invention;

[0052] Fig.25 The semi-preparative liquid chromatograms of the novel compounds of formula I and formula II of the present invention;

[0053] Fig.26 This is a semi-preparative liquid chromatogram of the new compounds of formula III and formula IV of the present invention. DETAILED DESCRIPTION

[0054] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0055] Example 1

[0056] The method for preparing porphyrin compounds in Camellia chrysantha comprises the following steps:

[0057] 1) Take 5 kg of dried golden camellia tea leaves, cut them into pieces of about 1×1 cm, add 95% ethanol by volume, extract them under 65°C reflux for 3 times, each time for 3 hours, and concentrate under reduced pressure at 40°C to remove ethanol, to obtain 892 g of golden camellia extract;

[0058] 2) The camellia chrysantha extract obtained in step 1) was fully stirred with 40° C. hot water and ultrasonicated for 30 minutes to obtain a suspension;

[0059] 3) extracting the suspension of step 2) with petroleum ether, petroleum ether-chloroform with a volume ratio of 5:1, petroleum ether-chloroform with a volume ratio of 1:1, chloroform, and ethyl acetate three times respectively, and concentrating the ethyl acetate portion under reduced pressure at 50° C. to obtain an ethyl acetate extract;

[0060] 4) loading the ethyl acetate extract in step 3) into a silica gel chromatography column, eluting with a gradient of petroleum ether-chloroform-ethyl acetate in a volume ratio of 1:0:0, 20:1:0, 10:1:0, 5:1:0, 1:1:0, 0:1:0, 0:20:1, 0:10:1, 0:8:1, 0:5:1, 0:0:1, combining the same fractions with the TLC detection method, and separating 15 fractions Fr.1 to Fr.15 according to the TLC detection results, wherein five fractions Fr.1, Fr.2, Fr5, Fr9, and Fr.12 were found to contain porphyrin compounds;

[0061] 5) The five fractions Fr.1, Fr.2, Fr5, Fr9 and Fr.12 in step 4) were combined and loaded into an alumina chromatography column, and gradient eluted with petroleum ether-ethyl acetate at a volume ratio of 100:1, 50:1, 20:1, 10:1, 8:1, 5:1 and 1:1, combined with the TLC detection method, the same fractions were combined, and four fractions Fr.1.1 to Fr.1.4 were separated according to the TLC detection results, of which Fr.1.2 and Fr.1.4 were found to contain porphyrin compounds after detection;

[0062] 6) The Fr.1.2 fraction in step 5) is loaded into a Sephadex LH-20 column, and isocratically eluted with chloroform-methanol in a volume ratio of 2:1, and the gray band under visible light in the column is observed and collected to obtain the Fr.1.2.1 fraction;

[0063] 7) The Fr.1.4 fraction in step 5) was loaded into a Sephadex LH-20 column, and eluted isocratically with chloroform-methanol in a volume ratio of 2:1, and the gray band under visible light in the column was observed and collected to obtain the Fr.1.4.1 fraction;

[0064] 8) The Fr.1.2.1 fraction in step 6) was purified by normal phase semi-preparative HPLC. The conditions of the normal phase semi-preparative HPLC were as follows: the chromatographic column was Agilent ZORBAX RX-SIL semi-preparative column, the mobile phase was n-hexane-isopropanol with a volume ratio of 98:2, the detection wavelength was 418 nm, and two chromatographic peaks at 23.1 to 23.9 minutes and 25.8 to 26.5 minutes were collected respectively (e.g. Fig.25 As shown), two new compounds of formula I and formula II can be obtained;

[0065] 9) The Fr.1.4.1 fraction of step 7) was purified by reverse phase semi-preparative HPLC. The reverse phase semi-preparative HPLC conditions were as follows: Welch Ultimate XB-C18 semi-preparative column was selected as the chromatographic column, acetonitrile-methanol-water with a volume ratio of 90:9:1 was selected as the mobile phase, and the detection wavelength was 418 nm; two chromatographic peaks at 9.5 to 9.9 minutes and 10.1 to 10.5 minutes were collected respectively (e.g. Fig.26 As shown), two new compounds of formula III and formula IV can be obtained.

[0066] The TLC detection conditions of the present invention are as follows: the developing solvent is a petroleum ether-chloroform-toluene system, the liquid in the colorless transparent glass bottle containing the fraction should be green to dark green; the TLC thin layer silica gel plate shows dark green to gray spots or color bands under visible light; the fluorescence is observed under UV 365nm to show red to purple spots or color bands; the detection under visible light and ultraviolet must meet the above three conditions at the same time, and the presence of porphyrin substances in the fraction can be identified.

[0067] It should be noted that: the extraction in the above step 1) uses 95% to 100% ethanol or 95% to 100% methanol by volume, and refluxes for 2 or 4 times; the extraction in step 3) is performed 2 or 4 times; in step 6), chloroform-methanol with a volume ratio of 3:1 or chloroform-methanol with a volume ratio of 1:1 is used to replace chloroform-methanol with a volume ratio of 2:1; in step 8), the detection wavelength is selected at 210nm; in step 9), the detection wavelength is selected at 210nm, and the mobile phase is selected at a volume ratio of 95:5 of acetonitrile-methanol. The other steps are the same as in Example 1, and the beneficial effects described in the present invention can also be achieved.

[0068] Example 2 Structural Identification

[0069] Using one-dimensional nuclear magnetic resonance ( 1 H-NMR, 13 C-NMR, DEPT 135) and two-dimensional techniques ( 1 H- 1 The structures of the compounds were identified by H COSY, HSQC, and HMBC.

[0070] The results are as follows:

[0071] The compound of formula I is a dark green powdery solid, which is purple-red under UV 365nm. The structure of the compound is determined by nuclear magnetic resonance technology, and its molecular formula is C 37 H 40 N 4 O 6 , NMR data are shown in Table 1, and the spectrum is shown in Figure 1-6 shown.

[0072] Table 1 NMR data of the compound of formula I (600 / 151 MHz, CDCl 3 ):

[0073]

[0074]

[0075] Active protons: NH: -1.73 (1H, br s); 0.42 (1H, br s); OH: 5.35 (1H, br s).

[0076] The compound of formula II is a dark green powdery solid, which is red under UV 365nm. The structure of the compound is determined by nuclear magnetic resonance technology, and its molecular formula is C 38 H 42 N 4 O 5 , NMR data are shown in Table 2, and the spectrum is shown in Figure 7-12 shown.

[0077] Table 2 NMR data of compound of formula II (600 / 151MHz, CDCl 3 ):

[0078]

[0079]

[0080] Active protons: NH: -1.84 (1H, br s), 0.26 (1H, br s); OH: 5.51 (1H, br s).

[0081] The compound of formula III is a dark green powdery solid, which is red under UV 365nm. The structure of the compound is determined by nuclear magnetic resonance technology, and its molecular formula is C 37 H 38 N 4 O 8 , NMR data are shown in Table 3, and the spectrum is shown in Figure 13-18 shown.

[0082] Table 3 NMR data of compound III (600 / 151 MHz, CDCl 3 ):

[0083]

[0084] Active protons: NH: -1.17 (1H, br s); -0.71 (1H, br s); OH: 6.34 (1H, br s).

[0085] The compound of formula IV is a dark green powdery solid, which is red under UV 365nm. The structure of the compound is determined by nuclear magnetic resonance technology, and its molecular formula is C 37 H 38 N 4 O 7 , NMR data are shown in Table 4, and the spectrum is shown in Figure 19-24 shown.

[0086] Table 4 NMR data of compound IV (600 / 151 MHz, CDCl 3 ):

[0087]

[0088] Active protons: NH: -1.62 (1H, br s); 0.44 (1H, br s); OH: 5.36 (1H, br s).

[0089] The above description is a detailed description of the preferred feasible embodiments of the invention, but the embodiments are not intended to limit the scope of the patent application of the present invention. All equivalent changes or modified changes completed under the technical spirit suggested by the present invention should fall within the patent scope covered by the present invention.

Claims

1. A method for preparing porphyrin compounds in Camellia chrysantha. It is characterized in that The structure of the porphyrin compound is shown in Formula I, Formula II, Formula III and / or Formula IV: The preparation method comprises the following steps: 1) extracting golden camellia leaves with 95-100% ethanol by volume to obtain an extract, and concentrating the extract under reduced pressure to remove ethanol to obtain golden camellia extract; 2) adding water to the camellia chrysantha extract prepared in step 1) and stirring until a suspension is formed; 3) extracting the suspension in step 2) with petroleum ether, petroleum ether-chloroform, chloroform, and ethyl acetate in sequence, and concentrating the ethyl acetate portion under reduced pressure to obtain an ethyl acetate extract; 4) subjecting the ethyl acetate extract of step 3) to silica gel column chromatography, using petroleum ether-chloroform-ethyl acetate as an eluent for gradient elution to obtain a fraction containing a porphyrin compound; 5) subjecting the fraction containing the porphyrin compound obtained in step 4) to an alumina chromatography column, and performing gradient elution using petroleum ether-ethyl acetate as an eluent to obtain subdivided fractions containing the porphyrin compound; 6) subjecting the subdivided fraction containing the porphyrin compound obtained in step 5) to Sephadex LH-20 column chromatography and isocratically eluting with chloroform-methanol to obtain subdivided subfractions containing the porphyrin compound; 7) Purifying the sub-fraction containing porphyrin in step 6) by semi-preparative high performance liquid chromatography to obtain the porphyrin compound.

2. The method for preparing porphyrin compounds in Camellia chrysantha according to claim 1, It is characterized in that In step 4), in the gradient elution, the volume ratios of petroleum ether, chloroform and ethyl acetate are 1:0:0, 20:1:0, 10:1:0, 5:1:0, 1:1:0, 0: 1：0、0： 20：1、0： 10：1、0： 8：1、0： 5：1、0： 0：1。 3. The method for preparing porphyrin compounds in Camellia chrysantha according to claim 1, It is characterized in that In step 5), in the gradient elution, the volume ratio of petroleum ether to ethyl acetate is 100:1 to 1:

1.

4. The method for preparing porphyrin compounds in Camellia chrysantha according to claim 3, It is characterized in that In the gradient elution, the volume ratio of petroleum ether to ethyl acetate is 100:1, 50:1, 20:1, 10:1, 8:1, 5:1, and 1:

1.

5. The method for preparing porphyrin compounds in Camellia chrysantha according to claim 1, It is characterized in that In step 6), in the isocratic elution, the volume ratio of chloroform to methanol is 3:1 to 1:

1.

6. The method for preparing porphyrin compounds in Camellia chrysantha according to claim 1, It is characterized in that In step 7), the semi-preparative HPLC includes normal phase semi-preparative HPLC or reverse phase semi-preparative HPLC.

7. The method for preparing porphyrin compounds in Camellia chrysantha according to claim 6, It is characterized in that The conditions of the normal phase semi-preparative high performance liquid chromatography are as follows: the chromatographic column is selected as an Agilent ZORBAX RX-SIL semi-preparative column, and the mobile phase is selected as n-hexane-isopropanol with a volume ratio of 98:

2.

8. The method for preparing porphyrin compounds in Camellia chrysantha according to claim 6, It is characterized in that The reverse phase semi-preparative high performance liquid chromatography conditions are as follows: a Welch Ultimate XB-C18 semi-preparative column is selected as the chromatographic column, and acetonitrile-methanol-water with a volume ratio of 90:9:1 or acetonitrile-methanol with a volume ratio of 95:5 is selected as the mobile phase.

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