A kind of schizonepeta bitter compound and preparation method thereof

Abstract

The present invention provides a schizophyllum bitter compound and a preparation method thereof. The structure of the schizophyllum bitter compound of the present invention is: The separation and preparation method of the compound of the present invention is to segment the ethyl acetate extract of the calyx of Lantern Festival with a normal phase silica gel column, elute with chloroform: methanol (90: 1-0: 1), and then perform normal phase column chromatography with chloroform: methanol (1: 0-20: 1) to elute the obtained 90: 1 fraction, and then perform gel column chromatography, ODS column chromatography, and semi-preparative HPLC to separate and purify. The present invention provides a theoretical basis for further enriching the research on the components of the calyx of Lantern Festival, and the preparation method provided is simple in steps and has strong operability.

Description

Technical Field

[0001] The invention belongs to the research field of natural drug chemistry, and specifically relates to a schizophrenia bitter compound and a preparation method thereof. Background Art

[0002] Physalis alkekengi L.var.franchetii (Mast.)Makino is a perennial herb of the genus Physalis in the family Solanaceae. It is also known as the Red Girl and the Hanging Golden Lantern. It is widely distributed in East Asia. In my country, it is distributed in all provinces except Tibet. It mostly grows in woodlands, fields, and streams and ditches. At the same time, some provinces and cities also cultivate it as an economic crop. In the 2020 edition of the "Pharmacopoeia of the People's Republic of China", the dried calyx or the calyx with fruit is used as the medicinal part, which has the effects of clearing away heat and detoxifying, relieving sore throat and reducing phlegm, and promoting diuresis and relieving stranguria. It is used to treat sore throat and hoarseness, cough with phlegm and heat, difficulty urinating, and painful stranguria due to heat; it can also be used externally to treat pemphigus and eczema.

[0003] The compounds in the Chinese lantern are mainly steroidal components and flavonoid components. As the characteristic steroidal active ingredient in the Chinese lantern, physalis bitters have a special 13,14-cleaved-16,24-ring ergostane-type skeleton structure and account for a large proportion of the isolated steroidal compounds. In further pharmacological studies, it was found that the physalis bitters components all showed good anti-inflammatory, antibacterial, anti-tumor and other pharmacological activities. Summary of the invention

[0004] In the first aspect, the present invention provides a schizophrenia bitter compound and a preparation method thereof. The present invention separates a schizophrenia bitter compound from the calyx of Lantern Festival, providing a theoretical basis for further clarifying the research and development and utilization of the active ingredients of Lantern Festival.

[0005] In the second aspect, the structure of the schizonepeta bitter compound of the present invention is:

[0006]

[0007] The compound name is: physalinol B

[0008] (1) In a third aspect, the preparation method of the bitter compounds of the schizonepeta tenuifolia provided by the present invention is as follows: 4.5 kg of dried calyx of Lantern Festival is cut into 2 cm, 95% and 75% (volume fraction) ethanol of 10 times the weight of the medicinal material is added, and refluxed and extracted three times, each extraction time is 2 hours, the solvent is recovered under reduced pressure, and the 6 concentrates are combined to obtain a total extract (1.45 kg);

[0009] (2) adding distilled water to the total extract to form a suspension, sequentially performing polar gradient extraction with petroleum ether, ethyl acetate, and water-saturated n-butanol, and recovering the solvent;

[0010] (3) The ethyl acetate extract was separated by silica gel column chromatography and gradient eluted with a chloroform-methanol system, with the volume ratio of the elution solvents being 90:1, 80:1, 60:1, 50:1, 30:1, 20:1, 10:1, 5:1, 1:1, and 0:1;

[0011] (4) the 90:1 elution portion in step (3) was further separated by silica gel column chromatography, and gradient elution was performed using a chloroform-methanol system, with the elution solvent volume ratio being 1:0, 80:1, 50:1, and 20:1;

[0012] (5) combining the 1:0 and 80:1 eluates in step (4), separating the mother liquor after filtering out the crystalline solid through a sephadex LH-20 gel column, and combining the four components under TLC monitoring, separating the third component through ODS column chromatography, and using a methanol-water system for gradient elution, with a volume ratio of 30:70, 40:60, 50:50, 60:40, 70:30, and 80:20;

[0013] (6) The 40%-70% methanol-water elution fractions in step (5) are combined and concentrated, and separated and purified by semi-preparative HPLC to obtain the schizonepeta tenuifolia bitter compound.

[0014] It is further defined that the conditions of semi-preparative HPLC in step (6) are: using methanol-water with a volume ratio of 60:40 as the mobile phase, a flow rate of 4 ml / min, a detection wavelength of 254 nm, 0.8 ml of sample per injection, and a retention time of 46 min.

[0015] The invention separates a novel schizophytin compound from the persistent calyx of Physalis alkekengi L var.franchetii (Mast.) Makino, and provides a preparation method and a structure identification method of the compound.

[0016] In order to further understand the features and technical contents of the present invention, please refer to the following detailed description and drawings of the present invention. However, the attached drawings are only provided for reference and illustration and are not intended to limit the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The present invention provides a schizophyllum bitter 1 H- 1 HCOSY and key HMBC-related signals;

[0018] Figure 2 The present invention provides a schizophyllum bitter 1 H-NMR spectrum;

[0019] Figure 3 The present invention provides a schizophyllum bitter 13 C-NMR spectrum;

[0020] Figure 4 It is the HMQC spectrum of schizonepeta tenuifolia provided by the present invention;

[0021] Figure 5 It is the HMBC spectrum of schizonepeta tenuifolia provided by the present invention;

[0022] Figure 6 It is the HRESIMS spectrum of the schizonepeta tenuifolia provided by the present invention. DETAILED DESCRIPTION

[0023] The above contents are further supplemented by specific embodiments below, but it should not be understood that the scope of the present invention is limited to the following examples. Any changes or improvements based on the above ideas of the present invention and according to common technical knowledge and conventional operating means in the field belong to the scope of the present invention.

[0024] Example 1 This example provides a schizone bitter compound and a preparation method thereof. The schizone bitter compound is isolated from the extract of the calyx of Lantern Festival, and the structural formula is

[0025]

[0026] The preparation method is as follows:

[0027] (1) Cut the dried Lantern Festival calyx (4.5 kg) into 2 cm, add 95% and 75% (volume fraction) ethanol 10 times the weight of the medicinal material, and reflux extract 3 times, each extraction time is 2 hours, soak overnight after reflux extraction, recover the solvent under reduced pressure, and combine the 6 concentrates to obtain Lantern Festival calyx total extract (1.45 kg);

[0028] (2) adding 1.5 times the volume of distilled water to the total extract of Lantern Festival calyx to form a 2L suspension, and sequentially using 2L of petroleum ether, ethyl acetate, and water-saturated n-butanol for polarity gradient extraction until no obvious extract is left after the extraction solvent is recovered, thereby obtaining a petroleum ether fraction, an ethyl acetate fraction, and a water-saturated n-butanol fraction;

[0029] (3) The ethyl acetate fraction (130 g) was mixed with an equal weight of 100-200 mesh silica gel and subjected to silica gel column chromatography. A chloroform-methanol system was used for gradient elution. The volume ratios of the elution solvents were 90:1, 80:1, 60:1, 50:1, 30:1, 20:1, 10:1, 5:1, 1:1, and 0:1. Eleven components were obtained by TLC monitoring and merging.

[0030] (4) The elution fraction with a volume ratio of 90:1 in step (3) was separated by silica gel column chromatography, and gradient elution was performed using a chloroform-methanol system with a volume ratio of 1:0, 80:1, 50:1, and 20:1, and five components were obtained by combining the components after TLC monitoring;

[0031] (5) The 1:0 and 80:1 eluates in step (4) were combined, and the mother liquor D3 (560 mg) after filtering out the crystalline solid was separated by sephadex LH-20 gel column, using chloroform:methanol=1:1 as the elution solvent, and the same components were combined to obtain four components under TLC monitoring. The third component D3C (320 mg) was separated by ODS column chromatography, using a methanol-water system for gradient elution, and the elution solvent volume ratio was 30:70, 40:60, 50:50, 60:40, 70:30, 80:20; the 40%-70% methanol-water elution fractions were combined and concentrated (78 mg), and separated and purified by semi-preparative HPLC, using methanol-water with a volume ratio of 60:40 as the mobile phase, a flow rate of 4 ml / min, a detection wavelength of 254 nm, 0.8 ml of each injection, and a retention time of 46 min. The fractions containing the target compound were collected and the solvent was recovered under reduced pressure to obtain the target compound (23 mg).

[0032] The schizophylline in this example is a colorless needle-shaped crystal, soluble in dichloromethane; TLC thin layer was developed with dichloromethane: methanol = 50: 1, R f =0.31, dark spots were observed under ultraviolet light at 254nm, and purple spots appeared after heating after color development with concentrated sulfuric acid-vanillin reagent. High-resolution mass spectrometry (HRESIMS) showed that the quasi-molecular ion peak of the compound was m / z 563.1894 [M+Na] + , its molecular formula is estimated to be C 29 H 32 O 10 , its unsaturation is calculated to be 14. 1 H-NMR spectrum ( Figure 2 ) can be found in three characteristic angular methyl signals of bitter components of Physalis H :1.35(3H, s, H-21), 1.54(3H, s., H-19), 1.71(3H, s, H-28); 1 oxygen-linked methyl signal δ H:3.59 (3H, s, H-29); 1 hydroxyl hydrogen signal δ H :6.89(1H,s,OH-13); 4 olefinic hydrogen signals δ H : 5.61 (1H, dt, J=7.3, 11.3Hz, H-3), 5.59 (1H, dd, J=2.8, 7.1Hz; H-4), 6.06 (1H, d, J=7.25, 11.3Hz, H-6), 5.73 (1H, dt, J=3.7, 10.1Hz, H-7).

[0033] exist 13 C-NMR( Figure 3 ) were observed, and combined with HMQC and HMBC signals, they can be attributed to 3 methyl signals (δ C 18.34, C-19; 21.25, C-21; 29.01, C-28); 1 methyl signal connected to the oxygen of the ester group (δ C 51.51, C-29); 5 methylene signals including (δ C 26.67, C-11; 26.87, C-12; 29.31, C-23), 1 methylene signal connected to the carbonyl group in the ester group (δ C 33.73, C-2) and a methylene signal connected to the oxygen of the ester group (δ C 60.98, C-27); 5 methine signals including (δ C 46.81, C-8; 40.10, C-9; 47.53, C-16), one methine signal connected to the carbonyl group in the ester group (δ C 76.43, C-22) and a methine signal connected to the oxygen of the ester group (δ C 47.53, C-25); 5 quaternary carbon signals including (δ C 28.37, C-24), 4 quaternary carbon signals connected to oxygen (δ C 78.93, C-13; 82.59, C-14; 82.78, C-17; 81.30, C-20); 6 olefinic carbon signals (δ C 123.76, C-3; 123.52, C-4; 122.9, C-5; 130.42, C-6; 119.9, C-7; 130.77, C-10); 4 ester carbon signals (δ C 171.32, C-1; 171.75, C-15; 172.94, C-18; 170.25, C-26). Compound 1 H-NMR, 13 C-NMR, HMBC, 1The H-1H COSY and NOESY data and signal assignments are shown in Table 1.

[0034] Table 1 1 H-NMR 300MHz, 13 C-NMR 125MHz, HMBC 500MHz, 1 H- 1 H COSY 500MHz, NOESY500MHz, in DMSO-d 6 )

[0035]

[0036] By comprehensively utilizing modern spectroscopy methods and combining Table 1 with relevant data from the literature, it was determined that its structure was a new compound that had not been reported in the literature. The specific structure of schizonepeta tenuifolia is shown below:

[0037]

Claims

1. A schizonepeta bitter compound, characterized in that: The structural formula of the cleavage physalis bitter compound is 2. The method for preparing the bitter compounds of schizonepeta tenuifolia according to claim 1, characterized in that: The preparation method comprises the following steps: cutting the dried Lantern Festival calyx to 2 cm, extracting with 95% and 75% (volume fraction) ethanol by refluxing respectively, recovering the solvent under reduced pressure to obtain a total extract, adding distilled water to the total extract to form a suspension, sequentially using petroleum ether, ethyl acetate and water-saturated n-butanol for polar gradient extraction, recovering the solvent to obtain extracts of various parts, subjecting the ethyl acetate extract to two consecutive silica gel column chromatography separations, using a chloroform-methanol system for gradient elution, combining the 1:0 and 80:1 eluates of the second silica gel column chromatography, filtering out the crystalline solid, and separating and purifying the mother liquor by gel column chromatography, ODS column chromatography and semi-preparative HPLC to obtain the schizophyllum bitter compounds.

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