Separation, extraction and identification of natural small molecule compound hevo from carob pod and its application

Abstract

The application obtains a new natural small molecule compound from the fruit pod of Caesalpinia eriostachys by solvent extraction, various column separation and purification. 1 H-NMR, 13 C-NMR nuclear magnetic data and molecular weight determination, combined with literature data, identify the structure of the compound, and clarify its chemical structure. Through activity experiments, it is found that the natural small molecule compound HEVO of Caesalpinia eriostachys pod helps to repair the damaged hair caused by chemical factors or external factors.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of daily chemicals, in particular to a kind of new carob pod natural small molecule compound HEVO extraction method and application. BACKGROUND

[0002] Hair is mainly composed of medulla, cortex and cuticle, wherein cuticle is the main protective effect, which gives hair luster and elasticity. Cuticle can be damaged due to daily life combing, perming, hair dyeing, shampooing, and ultraviolet irradiation, etc. Chemical perming and dyeing is almost the most direct cause of hair damage. With the development of society, the number of people perming and dyeing hair is increasing, and it is of great significance to develop products with nourishing and care functions for hair damage. SUMMARY

[0003] The present application aims to provide a preparation method of carob pod natural small molecule compound, and another object of the present application is to provide an application of carob pod natural small molecule compound.

[0004] Specifically, the present application provides a carob pod natural small molecule compound HEVO having the effect of repairing hair damage.

[0005] The chemical formula of the carob pod natural small molecule compound HEVO is C 18 H 20 O2N6

[0006] The structure of the carob pod natural small molecule compound HEVO is as follows:

[0007]

[0008] The present application separates, analyzes the spectrum and identifies the structure of the natural small molecule components in the carob pod of Ceratonia siliqua L. guided by LC-MS / MS.

[0009] The present application separates and extracts the carob pod of Ceratonia siliqua L. as raw material; the specific steps are as follows:

[0010] The present application adopts alcohol extraction method, concentrates the extract to obtain extract; disperses the extract with water, performs resin adsorption and elution; sequentially uses petroleum ether and ethyl acetate for extraction.

[0011] The natural small molecule HEVO is separated and purified from the carob pod of Ceratonia siliqua L.

[0012] The present application uses silica gel column chromatography, ODS column chromatography, Sephadex LH-20 and semi-preparative liquid chromatography to separate and purify the carob pod of Ceratonia siliqua L. step by step.

[0013] In the above-mentioned method, the silica gel column chromatography preferably uses a 200-300 silica gel column; and the elution procedure preferably uses petroleum ether-ethyl acetate (1:1→0:1) gradient elution, followed by dichloromethane-methanol (7:1→0:1) gradient elution.

[0014] In the above-mentioned method, the ODS column preferably uses ODS (40-63 μm); and the mobile phase preferably uses methanol-water solution.

[0015] In the above-mentioned method, the Sephadex LH-20 column preferably uses dichloromethane-methanol elution at a volume ratio of 1:1.

[0016] In the above-mentioned method, the semi-preparative liquid phase separation first uses a 250×20 mm YMC-Pack ODS-A column for separation, and then uses a 250×10 mm YMC-Pack ODS-A column for separation.

[0017] The obtained monomer is subjected to nuclear magnetic data (HNMR, CNMR) and molecular weight determination. 1 HNMR, 13 C NMR) and molecular weight determination.

[0018] In some embodiments of the present application, the carob pod is the dried fruit pod of the legume carob tree (Latin name: Ceratonia siliqua Linn.).

[0019] The present application proves by experiments that the carob pod natural small molecule HEVO provided in the present application has the effect of repairing damaged hair. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 High-resolution mass spectrum of the carob pod natural small molecule compound HEVO.

[0021] Figure 2 HNMR of the carob pod natural small molecule compound HEVO 1 HNMR.

[0022] Figure 3 CNMR of the carob pod natural small molecule compound HEVO 13 CNMR.

[0023] Figure 4 HMBC spectrum of the carob pod natural small molecule compound HEVO.

[0024] Figure 5 Chemical structure of the carob pod natural small molecule compound HEVO. DETAILED DESCRIPTION

[0025] The following examples are intended to illustrate the present application but not to limit the scope of the present application.

[0026] The following carob pods are the dried fruit pods of the plant Ceratonia siliqua Linn. (Leguminosae) and were provided by Shenzhen Aimin Pharmaceutical Technology Co., Ltd.

[0027] Example 1 Preparation of natural small molecule compounds from carob pods

[0028] (1) Extraction of natural small molecule compound HEVO from carob pods

[0029] The seed-free carob pod powder 20.0 kg was refluxed twice (1.5 h each time) with 8 times the weight of 95% ethanol solution and then refluxed for 1.5 h with 7 times the weight of 70% ethanol solution. The combined extract was concentrated under reduced pressure to recover the solvent to obtain the total extract. The extract was dispersed in 12 times the weight of water and then adsorbed on a macroporous adsorption resin D101. The sugar and other impurities were removed by eluting with 3 column volumes of distilled water, and then the non-sugar small molecule fraction was obtained by eluting with 3 column volumes of 95% ethanol and recovering the solvent.

[0030] The obtained non-sugar small molecule fraction was dispersed in 50% methanol solution and successively extracted with petroleum ether and ethyl acetate for 5 times, respectively, to obtain the petroleum ether fraction, the ethyl acetate fraction and the water fraction, respectively. HPLC analysis (chromatographic column: ACE 5C18 column (4.6 x 250 mm, 5 μm); mobile phase system: water (A) - methanol (B); flow rate: 1.0 mL / min; column temperature: 30 °C; injection volume: 10 μL; detection wavelength: 254 nm; gradient elution) showed that the non-sugar small molecule compounds were mainly concentrated in the ethyl acetate fraction.

[0031] The dried ethyl acetate fraction extract was successively subjected to gradient elution on a 300 mesh silica gel column with elution systems of petroleum ether-ethyl acetate and dichloromethane-methanol in the order of petroleum ether-ethyl acetate (volume ratio 50:50, 40:60, 25:75, 5:95, 0:100) and dichloromethane-methanol (volume ratio 88:12, 75:25, 70:30, 60:40, 0:100), and an equal portion of sample was injected. The eluate was detected by TLC (GF 254 silica gel plate) and similar fractions were combined to produce 10 components (A to J in order of decreasing polarity). Component C was the fraction eluted with petroleum ether-ethyl acetate 25:75-0:100.

[0032] The fraction C obtained by silica gel column chromatography was separated by ODS column with gradient elution of 15% to 100% methanol solution as mobile phase to obtain four sub-fractions (C1-C4), C4 (75-92%) was separated by Sephadex LH-20 column with 1:1 dichloromethane-methanol as mobile phase, and then purified by semi-preparative liquid chromatography (column I: YMC-Pack ODS-A, 250 x 20 mm, 5 μm) with 39% methanol-water solution as mobile phase to obtain white powder compound, which is natural small molecule compound HEVO from Caesalpinia sepiaria.

[0033] (2) Structure identification of natural small molecule compound from Caesalpinia sepiaria

[0034] The natural small molecule compound HEVO was subjected to nuclear magnetic data (H NMR, CNMR) and HR-ESI-MS (high resolution mass spectrometry) molecular weight determination. 1 H NMR, 13 CNMR) and HR-ESI-MS (high resolution mass spectrometry) molecular weight determination. 1 H NMR was performed using CD3OD, 600 MHz; 13 C NMR was performed using (CD3OD, 150 MHz);

[0035] Detection results of small molecule compound HEVO:

[0036] The small molecule compound HEVO is white powder, and the HR-ESI-MS result is shown in Figure 1 , the positive ion mode HR-ESI-MS gives the quasi-molecular ion peak m / z 353 [M+H] + , deduces the molecular formula of the compound C 18 H 20 O2N6, and the unsaturation degree is 12. As shown in Figure 2 , a group of olefinic proton signals [δH 8.06 (1H, d, J = 9.6 Hz, H-4), 6.03 (1H, d, J = 9.6 Hz, H-3)] are observed in 1 H NMR, three singlet aromatic proton signals [δH 7.05 (2H, s, H-12, 16), 6.22 (1H, s, H-6), 6.21 (1H, s, H-8)], and a group of ethyl signals [δH 4.27 (2H, q, J = 7.2 Hz, H2-18), 1.35 (3H, t, J = 7.2 Hz, H3-19)]. As shown in Figure 3 , 13The 12C NMR spectrum showed that compound A contained 18 carbon signals, including 14 aromatic or olefinic carbon signals (δC 163.9, 158.0, 157.6, 146.4, 146.4, 141.6, 139.7, 121.7, 110.0, 110.0, 109.3, 103.7, 99.4, 95.3), 2 carbonyl carbon signals (δC 168.5, 164.4), 1 methylene carbon signal (δC 61.7), and 1 methyl carbon signal (δC 14.6). Comparison of these NMR data with those of ethyl gallate and 5,7-dihydroxycoumarin suggests that compound HEVO is a polymer of two similar fragments: ethyl gallate and 5,7-dihydroxycoumarin.

[0037] like Figure 4 As shown, in the HMBC spectrum, hydrogen-carbon long-range correlation signals were observed at H2-18 and C-17, indicating that the ethyl group is connected to C-17 via N; hydrogen-carbon long-range correlation signals were observed at H-16 and C-15 / C-17, and at H-12 and C-11 / C-13 / 14 / 16, verifying that compound HEVO has a structure similar to that of ethyl gallate skeleton. Furthermore, hydrogen-carbon long-range correlation signals were observed at H-3 and C-2 / C10; H-4 and C-2 / C-5 / C-9; H-6 and C-5 / C-10; and H-8 and C-6 / C-7 / C9 / 10, verifying that compound HEVO has a structure similar to that of coumarin. Based on the above analysis and combined with the molecular formula of this compound, C... 18 H 20 O2N6, structural identification is as follows Figure 5 As shown.

[0038] A literature review revealed no compounds with the same structure; therefore, the natural small molecule compound HEVO is a novel compound.

[0039] Example 2: Shampoo containing HEVO, a natural small molecule compound from carob pods

[0040] This embodiment provides a shampoo with the following composition (by weight): 2% HEVO, a natural small molecule compound from carob pods in Example 1, and 98% shampoo base.

[0041] The shampoo base is composed of the following (by weight percentage): 15% ammonium lauryl ether sulfate, 3% sodium methyl myristoyl taurate, 7% lauramidopropyl betaine, 0.5% cocamide MEA, 2% polyquaternium-47, 0.1% cocoyl hydrolyzed soy protein, 0.2% dilinoleamide propyl PG-dimethylammonium chloride phosphate, 0.2% guar hydroxypropyltrimethylammonium chloride, 0.1% fragrance, 0.1% iodopropynyl butylcarbamate, and 71.8% deionized water.

[0042] Example 3

[0043] This comparative example provides a shampoo with the following composition (mass percentage): natural small molecule compound HEVO of Caesalpinia sappan pod of Example 1 1%, shampoo base 99%. The composition of the shampoo base is the same as that of Example 1.

[0044] Example 4

[0045] This comparative example provides a shampoo with the following composition (mass percentage): natural small molecule compound A of Caesalpinia sappan pod of Example 1 0.5%, shampoo base 99.5%. The composition of the shampoo base is the same as that of Example 1.

[0046] Experiment Example 1: Repairing effect of shampoo containing natural small molecule compound HEVO of Caesalpinia sappan pod on hair damaged by permanent dyeing

[0047] In order to verify the repairing effect of the shampoo of the examples on hair damaged by permanent dyeing, 100 volunteers aged 25-50 years old who had dyed their hair in the past year and whose hair was damaged during the dyeing process, causing problems such as brittle, dry and easy to break hair, were selected and divided into 10 groups, with 30 people in each group. The shampoo of Example 2, 3 and 4 was provided respectively, and the use time was 3 months and the use frequency was once every 2 days. The degree of hair breakage of the volunteers before and after 3 months of use was observed and analyzed to test the repairing effect of the shampoo on hair damaged by permanent dyeing. The hair samples to be tested were washed with distilled water and dried under the same conditions before being tested, and were balanced for 24 h under the conditions of 20°C and 45% relative humidity. A blank control group of 10 people was set up, and the same shampoo containing 100% shampoo base was used under the same conditions. The determination method was as follows: under the conditions of 20°C and 45% relative humidity, an INSTRON 4466 strength testing machine was used to test the breaking strength of the hair samples in dry state, with a stretching speed of 30 mm / min and a force resolution of 0.01 N. In order to ensure the accuracy of the stretching test results, hair samples with a length of more than 10 cm were selected. The samples were accurately weighed (accurate to 0.0001 g) and the length (mm) of the samples was measured with a steel ruler to measure the linear density (p x ). The samples were clamped in the upper and lower clamps of the strength machine.

[0048] The samples were stretched at a constant speed until they broke, and the breaking strength (F) and breaking elongation (e L ) were recorded, and the breaking strength (Fr) was calculated.

[0049] The linear density calculation formula is: p x = 10 6 ×M / L (where M is the weight and L is the length)

[0050] The breaking strength calculation formula is: Fr = F x 100 / px x 10 (where Fr is the sample breaking strength in units of CN / dtex, F is the sample breaking force in units of N, p is the sample linear density in units of tex) x x 10 (where Fr is the sample breaking strength in units of CN / dtex, F is the sample breaking force in units of N, p is the sample linear density in units of tex)

[0051] Table 1 Hair breaking strength

[0052]

[0053] The results show that the shampoos in Examples 2, 3 and 4 can all improve the breaking strength of permed and dyed damaged hair. The repairing effect of permed and dyed damaged hair of Example 2 is remarkable.

[0054] In summary, the natural small molecule HEVO of Albizia Lebbeck pod provided by the present application has the efficacy of repairing permed and dyed damaged hair, and can be used for the development of cosmetics.

[0055] Although the present application has been described in detail with general description and specific embodiments above, some modifications or improvements can be made on the basis of the present application, which is obvious to those skilled in the art. Therefore, these modifications or improvements made on the basis of not deviating from the spirit of the present application, all belong to the scope of the present application claimed.

Claims

1.A natural small molecule compound HEVO of Albizia Lebbeck represented by the following formula (I), 2. Process for the preparation of a compound according to claim 1, characterized in that, The method comprises the following steps: Firstly, 95% ethanol is used for extraction twice, and 70% ethanol is used for extraction once, and the extract is concentrated to obtain an extract; the extract is dispersed with water, and subjected to macroporous adsorption resin D101 adsorption, water elution, 95% ethanol elution, and recovery of non-sugar small molecule parts; petroleum ether and ethyl acetate are used for extraction for 5 times respectively, and the ethyl acetate part is collected; The ethyl acetate part is subjected to silica gel column chromatography separation, and elution systems are petroleum ether-ethyl acetate and dichloromethane-methanol in sequence; then, ODS column chromatography separation is performed, and a mobile phase is 15%-100% methanol solution; finally, Sephadex LH-20 separation and semi-preparative HPLC separation and purification are performed. 3.The method of claim 2, characterized in that: When petroleum ether and ethyl acetate are used for extraction, 50% methanol is used for dispersion; When silica gel column chromatography separation is performed, a silica gel column is 200-300 mesh, and eluent petroleum ether-ethyl acetate is in a volume ratio of 1:1→0:1, and eluent dichloromethane-methanol is in a volume ratio of 7:1→0:1; When ODS column chromatography is used, ODS filler is 40-63 μm, and a mobile phase is 15%-100% methanol solution; When Sephadex LH-20 separation is performed, dichloromethane-methanol is used for elution in a volume ratio of 1:1; When semi-preparative HPLC separation and purification are performed, a chromatographic column is YMC-Pack ODS-A (250×20 mm, 5 μm), and 39% methanol aqueous solution is used for elution. 4.Use of the compound of claim 1 in the field of hair cosmetics for repairing hair damaged by hair dyeing.

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