CN107746397B - Compound Oleracone C and its extraction separation method in purslane - Google Patents

Abstract

The invention relates to the field of extraction and separation of traditional Chinese medicines, in particular to a new compound extracted, separated and identified from purslane medicinal material and an extraction and separation method thereof, specifically a compound Oleracone C in purslane and an extraction and separation method thereof. The molecular formula of the new compound provided by the present invention is C ₁₇ H ₁₆ O ₅ , and it is named oleracone C; it is found through research that the new compound of the present invention has anti-inflammatory and anti-oxidative effects, and at the same time provides a simple, A fast, environmentally friendly, high-purity extraction and separation method.

Description

Compound oleracone C in purslane and its extraction and separation method

technical field

The invention relates to the field of extraction and separation of traditional Chinese medicines, in particular to a new compound extracted, separated and identified from purslane medicinal material and an extraction and separation method thereof, specifically a compound Oleracone C in purslane and an extraction and separation method thereof.

Background technique

Purslane (Portulaca oleracea L.), also known as longevity dish, horse amaranth, is a plant of the family Amaranthaceae. Purslane is drought and waterlogging resistant, light and shade resistant, widely distributed, and rich in resources. It has attracted much attention as a wild plant for both medicine and food. The 2015 edition of "Pharmacopia of the People's Republic of China" recorded the dry aerial parts of purslane as medicine. , has the functions of clearing away heat and detoxification, cooling blood to stop bleeding, and stopping dysentery.

Modern pharmacological studies of purslane have shown that it has the functions of anti-inflammatory and analgesic, antibacterial and antiviral, lowering blood pressure, lowering blood fat, anti-oxidation, anti-cancer, relaxing skeletal muscle and smooth muscle, and regulating immune function. Studies have shown that many chemical components of purslane provide a material basis for its diverse pharmacological effects. The main chemical components of purslane include flavonoids, coumarins, terpenoids, steroids, alkaloids, amino acids, various pigments and minerals. Substance etc. Among them, alkaloids are a main class of chemical components in purslane. The alkaloid components that have been reported so far include norepinephrine, dopamine, a small amount of dopa, adenosine, uracil, adenine, N, N-bicyclic Hexylurea, allantoin, N-trans-feruloyltyramide; also cyclic dipeptide alkaloids and amide alkaloids: purslane amides A-I, K, L, N-S.

At present, most of the chemical components isolated from purslane are known, and their structural novelty is low. Therefore, the development and separation of new compounds in purslane is urgently needed.

Contents of the invention

In view of the above problems, the present invention provides a compound Oleracone C in purslane and its extraction and separation method, specifically a new compound extracted from purslane. It is found through research that the new compound of the present invention has anti-inflammatory and antioxidant effects and provide a simple, rapid, environmentally friendly and high-purity extraction and separation method for the new compound of the present invention.

In order to achieve the above-mentioned purpose of the present invention, the molecular formula of the new compound provided by the present invention is C ₁₇ H ₁₆ O ₅ , named oleracone C, and the chemical structural formula is:

In order to achieve the above object of the present invention, the present invention also provides a method for extracting and separating compound oleracone C in purslane, the specific steps are as follows.

Step 1. Take the dried medicinal material of purslane, extract it with alcohol (the amount of ethanol is 8 to 16 times that of the medicinal material), filter the alcohol extract, and combine the filtrate to directly heat and concentrate, let it cool to room temperature, and obtain the medicinal solution for later use;

Step 2. Evaporate the traditional Chinese medicine liquid in step 1, put it on a silica gel column, elute with ethyl acetate, recover ethyl acetate under reduced pressure to extract, and obtain ethyl acetate extract;

Step 3. Separate the ethyl acetate extract in step 2 through a polyamide column, use ethanol-water gradient elution, evaporate 50% ethanol to dryness, put it on a silica gel column, and sequentially use ethyl acetate-methanol gradient elution to obtain several elutions The part is detected by thin-layer chromatography, the color is developed, and the color-developed elution parts are combined, and the combined elution parts are concentrated to dryness under reduced pressure, and set aside;

Step 4. Chromatographically separate the product obtained in step 3 on a pretreated ODS column (Octadecylsilyl, octadecylsilane-bonded silica gel packing), and elute with methanol-water gradient to obtain several elution sites, which are separated by thin layer Chromatography is used for detection and color development, and the elution parts of each color development are respectively concentrated to dryness under reduced pressure to obtain concentrates for subsequent use;

Step 5. The new compound obtained in step 4 is separated and prepared by HPLC (high performance liquid phase), and methanol-0.1% formic acid is used as the mobile phase for isocratic elution, and finally the new compound of the present invention is obtained.

The pretreatment process of the ODS is to soak in methanol for 24 hours, put it on the column, wash with methanol until it drops into water without turbidity, and then equilibrate with the initial mobile phase.

Compared with the prior art, the present invention has beneficial effects.

The separation and pharmacological activity research of purslane novel compound described in the present invention are not reported by existing paper periodicals; Alcohol extraction, polyamide column, silica gel column chromatography, ODS medium pressure column and HPLC were used for separation, purification and preparation, and new compounds were successfully extracted and separated. The method has only five steps, and the operation method is simple and fast. The extraction and separation process Alcohol extraction and ethyl acetate elution are mainly used, and the process method is environmentally friendly, and the purity of the compounds separated by this method is higher than 90%. In addition, studies have shown that the above compounds have anti-inflammatory and anti-oxidative effects. Therefore, the new compound of the present invention Its salts and derivatives can be used as synthetic leads of other compounds, raw materials for new drug development and pharmacological activity research, and can also be used to prepare anti-inflammatory and anti-oxidative drugs.

Description of drawings

Fig. 1 is the ultraviolet spectrogram of the new compound oleracone C of the present invention.

Fig. 2 is the high-resolution mass spectrum of the new compound oleracone C of the present invention.

Fig. 3 is a C ¹ H-NMR spectrogram of the new compound oleracone of the present invention.

Fig. 4 is a spectrum chart of the new compound oleracone C ¹³ C-NMR of the present invention.

Fig. 5 is the carbon nuclear magnetic resonance spectrum (DEPT) spectrogram of the new compound oleracone C of the present invention.

Fig. 6 is the NMR ¹ H- ¹ HCOSY spectrum of the new compound oleracone C of the present invention.

Fig. 7 is the nuclear magnetic resonance HMBC spectrogram of the new compound oleracone C of the present invention.

Fig. 8 is the nuclear magnetic resonance HSQC spectrogram of the new compound oleracone C of the present invention.

Fig. 9 is the NMR NOESY spectrum of the new compound oleracone C of the present invention.

Detailed ways

The present invention will be further described below in conjunction with specific embodiments.

Example 1.

The present invention provides a new compound, the molecular formula is C ₁₇ H ₁₆ O ₅ , named oleracone C and the chemical structural formula is:

The new compound is named oleracone C according to the structure, and Table 1 shows the NMR data of the new compound: ¹ H-NMR and ¹³ C-NMR in DMSO.

Table 1: NMR data of the new compound oleracone C of the present invention.

For structural identification, see Figures 1-9.

Oleracone C: Brown yellow powder, easily soluble in methanol, slightly soluble in chloroform. After spotting the sample on the silica gel thin-layer plate, spray the ferric chloride test solution and the spot turns blue. UV(MeOH)λ _max : 214,286nm. HRESI(+)TOFMS gave a quasi-molecular ion peak of m/z: 301.1039[M+H] ⁺ , with a molecular weight of 300.0998. Combining ¹ H-NMR, ¹³ C-NMR and DEPT data, it is speculated that the possible molecular formula of the compound is C ₁₇ H ₁₆ O ₅ , and the degree of unsaturation is 10. ¹³ C-NMR spectrum and DEPT spectrum showed 17 carbon signals, respectively 1 CH ₃ (δ: 55.98), 2 CH ₂ (δ: 27.20; 69.41), 7 CH (δ: 44.19; 93.64; 94.81; 115.17; 119.01; 127.81; 130.90), seven quaternary carbons (one carbonyl carbon, δ: 198.66; four double-bonded carbons with O, δ: 167.38; 163.63; 162.85; 155.59; two double-bonded carbons, δ: 124.24 ; 102.18).

The ¹ H-NMR spectrum showed two active H signals δ9.59(1H, bs) and δ12.12(1H, bs), indicating the possible presence of two hydroxyl groups. 1 methyl signal, δ3.79(3H, s); 2 methylene signals, δ2.60(1H, m), δ3.13(1H, m); δ4.16(1H, m ), δ4.27(1H, m); 7 methine signals are δ3.07(1H, m), δ6.06(1H, d, J=2.3), δ6.07(1H, d, J =2.3), δ6.73 (1H, m), δ6.82 (1H, m), δ7.05 (1H, m), δ7.06 (1H, m). According to the H-HCOSY spectrum, it can be known that Hδ4.16, δ4.27, δ2.60, δ3.13 in methylene are coupled with methine δ3.09 respectively; two methines δ6.07 and δ6.08 phase coupling; methine δ6.82, δ6.73, δ7.05, δ7.06 are coupled with each other, indicating the existence of benzene ring. According to the correlation peaks of the HMBC spectrum, H-6 and H-8 are respectively coupled with C-7 and C-10, and H-6 and H-8 are coupled with each other, indicating that they are associated with C-7 and C-10; methoxy Hδ3.79 in the group is coupled to C-7, and C-7 (δ167.38) is located in the low field region, suggesting that it is connected to O, indicating that the methoxy group is connected to C-7 on the benzene ring; according to the NOE spectrum , H in the methoxy group is coupled with H-6, H-8, indicating that it is associated with C-6, C-9; H-8 is coupled with C-9, H-6 is coupled with C-5, And C-5 (δ163.63), C-9 (δ162.85) is located in the low field area, suggesting that it is connected to O, and there is an OH connected to C-5 on the benzene ring; H-2, H-3, H -11 is coupled, and C-2 (δ69.41) is located in the low field region, suggesting that it is connected to O. At the same time, H-2 is coupled to C-9, indicating that C-2 and C-9 are connected to O; H -2, H-3, H-11 are coupled to the carbonyl C of C-4, indicating association with C-4. H-3', H-4', H-5' and H-6' are coupled with each other, where H-3', H-5', H-6' are coupled with C-1', H-3', H-4', H-5', H-6' are coupled with C-2', suggesting the presence of a benzene ring and C-1' and C-2' being ortho-substituted; where C-2' (δ155. 59) It is in the low field region, suggesting that it is connected to O, indicating that C-2' is connected with a hydroxyl group; H-2, H-3, H-11 are coupled with C-1', indicating that it is related to C-1' , H-11 is coupled with C-2', C-6', indicating that it is related to C-2', C-6', H-6' is coupled with C-1', indicating that it is related to C-1', In summary, C-11 is connected to C-1'. According to the above information, it can be determined that this new compound has the above structure.

The present invention also provides an extraction and separation method for the above-mentioned new compound, the specific steps are:

Step 1: Weigh 80 kg of dried purslane medicinal material, extract with 50% ethanol under reflux, the amount of 50% ethanol (v/v) is 10 times that of the medicinal material, reflux extract twice, each time for 2 hours, combine the filtrates, heat and reduce pressure to recover ethanol , let cool to room temperature, and obtain the medicinal solution for later use.

Step 2: Evaporate the liquid medicine obtained in step 1 to dryness, then separate it by silica gel column chromatography, and elute isocratically with ethyl acetate (120L), wherein the silica gel is 100-200 mesh, and recover ethyl acetate under reduced pressure below 40°C to Extraction to obtain ethyl acetate extract.

Step 3: Separate the ethyl acetate extract in step 2 through a polyamide column, and use ethanol-water (0/100, 30/70, 50/50, 70/30, 100/0, v/v) gradient elution , evaporated to dryness with 50% ethanol, and separated by silica gel column chromatography, wherein the silica gel was 200-300 mesh, followed by gradient elution with ethyl acetate-methanol (5:1, 2:1, 1:2, v:v), A total of 15 parts were obtained (that is, a total of 15 bottles were obtained, each bottle was 400 mL), detected by thin-layer chromatography, and the color was developed, and the 1-3 elution parts of the color were combined, and the combined 1-3 parts were heated at 40 ° C. Concentrate to dryness under reduced pressure and set aside.

Step 4: Separate the product obtained in step 3 by pretreated ODS medium-pressure column chromatography (Octadecylsilyl, octadecylsilane bonded silica gel filler), wherein the particle size of the filler is 20-40 μm, and use methanol-water (84/ 16, 93/7, 97/3, 100/0, v/v) gradient elution (pressurization, so that the flow rate is 1mL/min, the temperature is room temperature), to obtain 10 sites (that is, gradient elution to obtain 10 bottles , 200 mL per bottle), detected by thin-layer chromatography, color developed, and the 1-5 parts of the color were retained, concentrated to dryness under reduced pressure below 50 ° C, and set aside to obtain a new compound. The pretreatment process of the ODS is soaked in methanol for 24 hours, put on the column, washed with methanol until dripped into water without turbidity, and then equilibrated with the initial mobile phase.

Step 5: The new compound obtained in step 4 was separated and prepared by HPLC, using methanol: 0.1% formic acid (61:39, v/v) as the mobile phase, and the detection wavelength was 210, 280nm, and the new compound of the present invention was obtained by separation and preparation. The purity measured by one method is 90-99%.

Anti-inflammatory effects of the novel compounds of the invention.

1. Main materials.

1.1. Drugs and reagents: The new compounds used in the experiment were prepared by the above-mentioned method, with a purity of 90-99%, weighed accurately, and diluted with DMSO to the required solutions for the following dosage groups. DMEM high-glucose medium, fetal bovine serum (U.S. Hyclone Company); penicillin and streptomycin (Hangzhou Sijiqing Company); LPS (U.S. Sigma Company); IL-6, TNF-α, PGE ₂ ELISA kits (U.S. Cayman Company); cell lysate, Griess reagent (Beiyuntian Biotechnology Co., Ltd.).

1.2 Cell line: RAW264.7 macrophages (US ATCC cell bank).

1.3 Grouping: divided into control group, LPS group and experimental group, each group.

2 Experimental methods.

2.1 Cell culture, DMEM high-glucose medium, add 10% fetal bovine serum, 1% antibiotics (100U/mL penicillin and 100 μg/mL streptomycin), place 37.5%, CO ₂ incubator culture.

2.2 MTT colorimetric method was used to measure cell viability. RAW264.7 macrophages in the logarithmic growth phase were inoculated in 96-well culture plates in the above three groups, the cell density was ¹ ×104/mL, 100 μL per well, and the temperature was 37°C. After cultivating overnight under 5% _CO2 conditions, the experimental group added different concentrations of the new compound Oleracone C (1-50 μM) of the present invention, and after incubation for 1 h, added LPS with a final concentration of 1 μg/mL to the LPS group and the experimental group respectively. A zero-adjustment group (culture solution containing DMSO solvent) was set up, and three replicate wells were set up in each group to investigate the effect of adding drugs on the cells. After the above-mentioned groups of cells were cultured for 24 hours, 20 μL of 5 mg/mL MTT was added to the cells in each well, and the temperature was 37 ° C, and the incubation was continued for 4 hours under the condition of 5% CO ₂ . Sulfone (DMSO) was shaken for 10 minutes to fully dissolve the intracellular crystals, and the absorbance value of each well was measured at a wavelength of 570 nm with a microplate reader.

2.3 The content of NO was measured by Griess method, and the inhibitory effect of the new compound of the present invention on the NO production of mouse macrophage RAW264.7 induced by LPS was investigated. Mouse macrophage RAW264.7 was cultured in the high-glucose cell culture medium DMEM containing 10% fetal bovine serum after passage, and the new compound oleracone C (1-20 μ M) of the present invention was added in different concentrations in the experimental group, at 37 ° C, 5 After incubation for 1 h under the condition of %CO ₂ , the inflammatory response was induced with LPS (final concentration: 1 μg/mL). After 24 h, the supernatant was collected, and each treatment group was repeated for 3 wells. The NO content in the cell supernatant was measured by the Griess method, and the effect of the novel compound of the present invention on LPS-induced NO release from RAW264.7 cells at different concentrations was used to reflect the NO level.

2.4 Determination of inflammatory factors IL-6, TNF-α and inflammatory mediator PGE ₂ by ELISA: RAW264.7 macrophages in the logarithmic growth phase were seeded in 24-well culture plates at a cell density of 1×10 ⁵ cells/mL, and each Well 1mL, temperature 37 ℃, 5% _CO Under the condition of cultivating overnight, the experimental group was added the new compound oleracone C (1-20μM) of the present invention, and after incubation for 1h, LPS (final concentration was 1μg/mL) was added to each well. Incubate for 24 hours, and repeat 3 wells for each group of treatments. The contents of IL-6, TNF-α and PGE ₂ secreted by RAW264.7 macrophages treated with new compounds derived from purslane were determined by ELISA.

3 Experimental results.

The experimental results show that the special compound of the present invention has no effect on the proliferation of LPS-induced macrophage RAW264.7, is safe and non-toxic; and can effectively inhibit the excessive inflammatory cytokines IL-6 and TNF produced by LPS-induced macrophage RAW264.7 -α and inflammatory mediators NO and PGE ₂ in a concentration-dependent manner.

The results of the relative cell viability experiment are shown in Table 2.

Table 2: The effect of the present invention on the relative survival rate of RAW264.7 macrophages.

Note: ^* P<0.05 Compared with the control group (the high concentration group has a significant difference),

Table 3 shows the experimental results of NO content determination by Griess method.

Table 3: Effect of the present invention on LPS-induced NO release from RAW264.7 cells (mean ± standard deviation, n=3)

Note: ^* P<0.05 compared with the control group, ^# P<0.05 compared with the LPS group.

Table 4 shows the results of the determination of inflammatory factors IL-6, TNF-α and inflammatory mediator PGE ₂ by ELISA.

Table 4: Effects of the present invention on the contents of IL-6, TNF-α and PGE ₂ secreted by LPS-induced RAW264.7 cells (mean ± standard deviation, n=3).

Note: ^* P<0.05 compared with the control group, ^# P<0.05 compared with the LPS group.

Antioxidative effects of the novel compounds of the present invention.

1 main material.

1.1 Drugs and reagents: The new compounds used in the experiment were prepared by the above-mentioned method, with a purity of 90-99%, weighed accurately, and diluted with methanol to the required solutions for the following dosage groups. DPPH (1,1-diphenyl-2-picrylhydrazine radical) (Sigma-Fluka); BHA (tert-butyl hydroxyanisole) (Shanghai Xiangrui Technology Co., Ltd.); methanol, chromatographically pure (Changtai Industrial Co., Ltd. company).

1.2 Grouping: control group, experimental group, blank group.

2. Experimental method.

Colorimetric method was used to determine the ability to eliminate DPPH free radicals. For the sample group, 1 mL of DPPH solution (126.80 μM) was added to a 4 mL cuvette, and then 1 mL of sample solutions of different concentrations (8.32, 16.61, 33.31, 50.02, 66.61 μM) were added; For the control group, 1 mL of methanol solution was added to a 4 mL cuvette, and then 1 mL of sample solutions of different concentrations were added; for the blank group, 1 mL of DPPH solution was added to a 4 mL cuvette, and then 1 mL of methanol solution was added. The three groups were fully mixed, and kept at room temperature in the dark for 10 minutes, and the absorbance value was measured at 517 nm. After standing for 30 minutes, the operation was performed in the same way. Each sample was measured three times to get the average value, and the positive control was BHA solution with different concentrations. Calculate the scavenging rate of the sample for DPPH radicals according to the following formula, and further calculate the _IC50 value of the free radical scavenging rate.

DPPH clearance rate (%) = 1 - (A ₁ - A ₂ )/A ₀ × 100%, where A ₀ is the absorbance value of the blank group; A ₁ is the absorbance value of the sample group; A ₂ is the absorbance value of the control group value.

3. Experimental results.

Experimental results show that the new compound of the present invention has a scavenging effect on DPPH free radicals, and the scavenging rate is also significantly increased with the increase of drug concentration. See Table 5 for the IC ₅₀ values of the new compounds of the present invention on DPPH free radicals.

Table 5 The scavenging effect of the new compounds of the present invention on DPPH free radicals.

group IC₅₀(μM) BHA 59.14 Oleracone C 23.27

In summary, the present invention provides a special compound and its extraction and separation method, sequentially adopts 50% ethanol reflux extraction, polyamide column chromatography, silica gel column chromatography, ODS medium pressure column separation and purification, and successfully separates and obtains a new compound , the method is simple, fast, and environmentally friendly, and the compound obtained through separation by this method has a high purity. Due to the unique chemical structure of the obtained compound, it is extracted from the commonly used traditional Chinese medicine purslane, which has anti-inflammatory and anti-oxidative effects. Therefore, the present invention Special compounds and their salts and derivatives can be used as natural products to develop new traditional Chinese medicines, which have broad prospects.

Claims (5)

1. compound Oleracone C in purslane, it is characterized in that, the molecular formula of described compound is C ₁₇ H ₁₆ O ₅ , and chemical structural formula is: . 2. the extraction separation method of compound Oleracone C in purslane as claimed in claim 1, is characterized in that, concrete steps are: Step 1. Take the dried medicinal material of purslane, extract it with alcohol, filter the alcohol extract, heat and concentrate the combined filtrate directly, let it cool to room temperature, and obtain the medicinal solution for later use; Step 2. Evaporate the Chinese medicinal liquid in step 1 to dryness, put it on a silica gel column, elute with ethyl acetate, recover the ethyl acetate under reduced pressure to the extract, and obtain the ethyl acetate eluate; Step 3, the ethyl acetate eluate in step 2 is separated through a polyamide column, and ethanol-water gradient elution is adopted, and the volume ratio of ethanol-water is 0/100, 30/70, 50/50, 70/30 and 100/0, 50% ethanol was evaporated to dryness, then applied to a silica gel column, sequentially eluted with ethyl acetate-methanol gradient to obtain several elution sites, detected by thin-layer chromatography, color development, combined color elution sites, combined The last eluted part was concentrated to dryness under reduced pressure and set aside; wherein, the volume ratio of ethyl acetate-methanol was 5:1, 2:1 and 1:2; Step 4, separate the product obtained in step 3 by pretreated ODS column chromatography, and elute with methanol-water gradient to obtain several eluted parts, which are detected by thin-layer chromatography, and the color is developed, and each color-developed wash is The decompressed parts are respectively concentrated to dryness under reduced pressure to obtain a concentrate for use; wherein, the volume ratio of methanol to water is 84/16, 93/7, 97/3 or 100/0; Step 5, the concentrate obtained in step 4 is separated and prepared by HPLC, and methanol-0.1% formic acid is used as the mobile phase for isocratic elution, and finally the compound Oleracone C in purslane is obtained; wherein, the volume ratio of methanol:0.1% formic acid is 61:39. 3. the extraction separation method of compound Oleracone C in purslane as claimed in claim 2, it is characterized in that, the pretreatment process of described ODS is that methanol soaks 24 hours, puts on the column, washes to drop into water without Turbid, then equilibrate with the initial mobile phase. 4. The method for extracting and separating the compound Oleracone C in purslane as claimed in claim 2, wherein the alcohol extraction in step 1 is ethanol extraction, and the amount of ethanol is 8 to 16 times that of medicinal materials. 5. The compound Oleracone C as claimed in claim 1 is used for the preparation of anti-inflammatory or anti-oxidative medicine.