阅读说明：本技术 络石藤中具抗炎活性的甘油酯类化合物及其制备方法 (Glyceride compounds with anti-inflammatory activity in trachelospermi caulis and preparation method thereof ) 是由 谭钦刚 宋鸿志 赖春华 于 2020-07-13 设计创作，主要内容包括：本发明公开了络石藤中具有抗炎活性的甘油酯类化合物及其制备方法,所述的甘油酯类化合物有两个,分别为1-hexadecanoyl propan-2,3-diol化合物1和1-heptadecanoyl propan-2,3-diol化合物2。其中化合物2为新化合物,并通过Griess法、ELISA等实验进一步研究化合物1的抗炎效果,结果显示化合物1对NO抑制作用的IC<Sub>50</Sub>值为15.76μM,对炎症因子TNF-<I>α</I>和IL-6也均具有一定的抑制作用,其中对IL-6的抑制作用强于TNF-<I>α</I>。实验还通过Western Blot考察了化合物1对RAW 264.7细胞中诱导型iNOS的蛋白表达,结果显示化合物1能够明显抑制RAW 264.7细胞中诱导型iNOS的蛋白表达,表明化合物1通过抑制炎症相关的iNOS的表达,从而抑制炎症的发生。本发明从络石藤中发现结构新颖的、具有抗炎活性的甘油酯类化合物,为制备抗炎新药奠定基础。(The invention discloses a glyceride compound with anti-inflammatory activity in trachelospermi caulis and a preparation method thereof, wherein the glyceride compound comprises 1-hexadecanoyl propan-2,3-diol compound 1 and 1-heptadecenoyl propan-2,3-diol compound 2. Wherein the compound 2 is a new compound, the anti-inflammatory effect of the compound 1 is further researched by experiments such as Griess method, ELISA and the like, and the result shows that the IC of the compound 1 on the NO inhibition effect 50 The value is 15.76 mu M, and the anti-inflammatory factor TNF- α And IL-6 also have certain inhibitory effects, wherein the inhibitory effect on IL-6 is stronger than that of TNF- α . Experiment also examined the induction of compound 1 in RAW264.7 cells by Western BlotThe results show that the compound 1 can obviously inhibit the protein expression of inducible iNOS in RAW264.7 cells, and show that the compound 1 can inhibit the generation of inflammation by inhibiting the expression of iNOS related to the inflammation. The invention discovers the glyceride compound with novel structure and anti-inflammatory activity from the Chinese starjasmine stem, and lays a foundation for preparing new anti-inflammatory drugs.)

1. The glyceride compound with anti-inflammatory activity in the trachelospermi caulis is characterized in that: two glyceride compounds are 1-hexadecanoyl propan-2,3-diol compound 1 and 1-heptadecanoyl propan-2,3-diol compound 2, and have the structures shown in the following formulas:

1-hexadecanoyl propan-2,3-diol

1-heptadecanoyl propan-2,3-diol 。

2. the method for extracting and separating glycerides compounds having antiinflammatory activity from trachelospermi caulis according to claim 1, comprising the steps of:

(1) drying and crushing Chinese starjasmine stem, adding a 95% ethanol water solution into the crushed Chinese starjasmine stem to immerse the surface of the Chinese starjasmine stem for 7-10 days, concentrating under reduced pressure to recover ethanol to obtain a total extract, suspending the total extract in water, placing the suspension in a separating funnel, adding ethyl acetate for extraction, and recovering a solvent under reduced pressure to obtain an ethyl acetate part;

(2) passing the ethyl acetate part through a normal phase silica gel column, setting a concentration gradient for elution, and sequentially obtaining elution fractions I-XIII;

(3) performing MCI chromatographic column chromatography on the fraction VI, performing methanol-water gradient elution to obtain nine fractions VI-1 to VI-9, performing Sephadex LH-20 gel column chromatography on VI-3, performing methanol elution, and recrystallizing to obtain a 1-heptadecenyl propan-2,3-diol compound 2;

VI-9 passing through Sephadex LH-20 gel column, eluting with 50% methanol water, and passing through ODS C₁₈The reverse phase column yielded 1-hexadecanoyl propan-2,3-diol compound 1.

3. The method for extracting and separating glycerides compounds having antiinflammatory activity from trachelospermi caulis according to claim 2, wherein: and (3) carrying out gradient elution on the normal-phase silica gel column in the step (2) by using an eluent with the volume ratio of petroleum ether to acetone =10:1 to chloroform to methanol =1: 1.

4. The method for extracting and separating glycerides compounds having antiinflammatory activity from trachelospermi caulis according to claim 2, wherein: and (3) performing gradient elution on the methanol water, wherein the concentration of the methanol water is subjected to gradient elution according to 30%, 50%, 70%, 90% and 100%.

5. The use of glycerides compounds with anti-inflammatory activity in trachelospermi caulis as claimed in claim 1, characterized in that: can be used for preparing antiinflammatory medicine.

Technical Field

The invention relates to glyceride compounds, in particular to glyceride compounds with anti-inflammatory activity, which are extracted and separated from Chinese starjasmine stem.

Background

Trachelospermi caulis is derived from Trachelospermi caulis of Apocynaceae (Trachelospermi caulis)Trachelospermum jasminoides(Lindl) Lem) dried leafy rattan. In ancient times, Trachelospermi caulis was used for treating rheumatism, arthritis of heat type, spasm of tendons and vessels, soreness of waist and knees, sore throat, pharyngitis, sore, swelling, traumatic injury, external hemorrhage, etc.; at present, the Chinese starjasmine stem is mainly used for treating rheumatic arthritis and myalgia, particularly for patients with limb contracture and difficulty in flexion and extension, and has obvious curative effect.

According to the literature report, the anti-inflammatory component of the trachelospermi caulis is mainly a lignan component, especially the dibenzyl butyrolactone lignan, and the reports of glyceride compounds in the trachelospermi caulis are very few, but the glyceride compounds with anti-inflammatory activity are not reported.

In order to further discover a new anti-inflammatory active ingredient from trachelospermi caulis and lay a foundation for the development of a new drug, the inventors conducted intensive studies on chemical ingredients of trachelospermi caulis. Combining with the screening of anti-inflammatory pharmacodynamic activity, separating and extracting the trachelospermi caulis and identifying the glyceride compounds with anti-inflammatory activity.

Disclosure of Invention

The invention discloses two glyceride compounds extracted and separated from Chinese starjasmine stem, an extraction and separation method, structure identification and an experiment on the anti-inflammatory activity of one compound.

The technical scheme for realizing the purpose of the invention is as follows:

glyceride compounds with anti-inflammatory activity in trachelospermi caulis, the glyceride compounds are 1-hexadecanoyl propan-2,3-diol compound 1 and 1-heptadecanoyl propan-2,3-diol compound 2, and have the following structures:

。

the method for extracting and separating the glyceride compounds of the trachelospermi caulis comprises the following steps:

(1) drying and crushing Chinese starjasmine stem, taking 20kg of crushed Chinese starjasmine stem, adding a 95% ethanol water solution to immerse the surface of the Chinese starjasmine stem for 7-10 days, concentrating under reduced pressure to recover ethanol to obtain a total extract, suspending the total extract in water, placing the suspension in a separating funnel, adding ethyl acetate for extraction, and recovering a solvent under reduced pressure to obtain 479.8 g of an ethyl acetate part;

(2) passing the ethyl acetate part through a normal phase silica gel column, setting a concentration gradient for elution, and sequentially obtaining elution fractions I-XIII;

(3) subjecting the fraction VI to MCI chromatographic column chromatography, gradient eluting with methanol water to obtain nine fractions VI-1 to VI-9, subjecting VI-3 to Sephadex LH-20 gel column chromatography, eluting with methanol, and recrystallizing to obtain 1-heptadecanoyl propan-2,3-diol compound 2 (20.0 mg);

VI-9 passing through Sephadex LH-20 gel column, eluting with 50% methanol water, and passing through ODS C₁₈The reverse phase column gave 1-hexadecanoyl propan-2,3-diol compound 1 (1.0 mg).

And (3) carrying out gradient elution on the normal-phase silica gel column in the step (2) by using an eluent with the volume ratio of petroleum ether to acetone =10:1 to chloroform to methanol =1: 1.

And (3) performing gradient elution on the methanol water, wherein the concentration of the methanol water is subjected to gradient elution according to 30%, 50%, 70%, 90% and 100%.

Compound 1 structural identification by mass spectrometry (ESI-MS M/z 353 [ M + Na ]]⁺) The molecular formula is presumed to be C₁₉H₃₈O₄；

¹HNMR (400 MHz, CD₃OD) _H : 0.87 (3H, t,J= 6.0 Hz, H-16'), 1.26 (24H,sbr, H-4' to H-15'), 1.62 (2H, t,J= 6.5Hz, H-3'), 2.33 (2H, t,J= 6.5 HzH-2'), 4.16 (1H, dd,J= 6.0,10.0Hz H-l), 3.93 (1H, p,J= 5.5 Hz, H-2), 3.63(dd,J= 6.0, 10.0 Hz, H-3);¹³CNMR (100 MHz, CD₃OD) _C : 14.45 (C-16'), 23.75(C-15'), 33.09 (C-14'), 30.22, 30.43, 30.49, 30.61, 30.73, 30.79, 30.81 (C-4'to C-13'), 26.00 (C-3'), 34.94 (C-2'), 175.49 (C-l') 66.47 (C-l), 71.15 (C-2), 64.06 (C-3); the above data and literature (Misra L, Siddiqi SA. n-Alkanes andα-PalmitinfromPaspalum scrobiculatumthe report by Seeds, Zeitschrift fur Naturforschung C, 2000, 55(C): 500-502.) agreed, and was identified as 1-hexanecanoyl propan-2, 3-diol.

Identifying the structure of compound 2 by high resolution mass spectrometry (HR-ESI-MS)m/z: 389 [M + COOH]^-) The molecular formula is presumed to be C₂₀H₄₀O₄There are 2 unsaturations;

¹HNMR (400 MHz, CD₃OD) _H : 0.87 (3H, t,J= 6.0 Hz, H-17'), 1.25 (26H,sbr, H-4' to H-16'), 1.62 (2H, t,J= 6.5Hz, H-3'), 2.34 (2H, t,J= 6.5 HzH-2'), 4.16 (2H, dd,J= 6.0, 10.0 Hz H-l), 3.92 (1H, p,J= 5.5Hz,H-2), 3.62(2H, dd,J= 6.0,10.0Hz, H-3);¹³CNMR (100 MHz, CD₃OD) _C : 14.44 (C-17'),23.75 (C-16'), 33.09 (C-15'), 30.22, 30.42, 30.48, 30.61, 30.72, 30.76,30.79, 30.80 (C-4' to C-14'), 26.00 (C-3'), 34.95 (C-2'), 175.51 (C-l'),66.47 (C-l), 71.15 (C-2), 64.07 (C-3)。

compound 2 in comparison with Compound 1, of Compound 2¹H NMR and¹³c NMR data was similar to compound 1 except there was a strong peak of 26H (CH) in the H spectrum at =1.62₂) Since 2 more H than compound 1 and 14 more molecular weight than compound 1 in compound 2 by high-resolution mass spectrometry, it was found that 1 more methylene was present in the long chain of compound 2 than compound 1, compound 2 was named 1-heptadecanoyl propan-2,3-diol, and this compound was a novel compound.

The invention discovers the glyceride compounds of the Chinese starjasmine stem with novel structure and anti-inflammatory activity from the Chinese starjasmine stem, and lays a foundation for preparing new anti-inflammatory drugs.

Drawings

FIG. 1 shows the inhibitory effect of compound 1 (abbreviated as HPD) on the production of NO by RAW264.7 cells induced by LPS, note: in comparison to the set of models,^**P<0.01；

FIG. 2 is a graph showing the effect of HPD on the viability of RAW264.7 cells after 24 h;

FIG. 3 shows TNF-once production by LPS-induced RAW264.7 cells 24 h after HPD actionαInhibition, note: in comparison with the normal group,^##P<0.01; in comparison to the set of models,^*P<0.05，^**P<0.01；

FIG. 4 shows the inhibition of LPS-induced IL-6 production by RAW264.7 cells after 24 h of HPD, in which: in comparison with the normal group,^##P<0.01; in comparison to the set of models,^*P<0.05，^**P<0.01；

FIG. 5 shows the effect of HPD on the expression level of iNOS in RAW264.7 cells induced by LPS 24 h, in which: in comparison with the normal group,^##P<0.01; in comparison to the set of models,^**P<0.01。

Detailed Description

The anti-inflammatory effect of the glycerol ester compound 1 of trachelospermi caulis is further illustrated by the following experiments.

The anti-inflammatory activity of Compound 1 in Trachelospermi caulis was measured by Griess method, and 6 concentration gradients of 200, 100, 50, 25, 12.5, and 6.25 μ M were set, respectively, and the NO inhibition rate is shown in FIG. 1, which is compared with Indomethacin (IC) as a positive control₅₀26.36 μ M), compound 1 has obvious inhibition effect on LPS-induced NO production by RAW264.7 cells, IC₅₀The value was 15.76. mu.M, and thus HPD was further investigated.

The influence of compound HPD on the cell survival rate is detected by adopting an MTT colorimetric method, as shown in figure 2, 200, 100, 50, 25, 12.5 and 6.25 mu M concentration gradients are respectively set, and the incubation is carried out for 24 hours, so that the cells are non-toxic when the drug concentration of each dose group of the compound is detected, and the cell survival rate is more than 90%.

TNF-activated compound HPD in RAW264.7 cellsαAnd IL-6 production inhibition results, as can be seen from Table 1, FIG. 3 and FIG. 4, the normal group TNF-αAnd IL-6 expression levels were 285.00 and 41.72 Pg/mL, respectively, while the model group TNF-αAnd the expression level of IL-6 reaches 4414.46 and 2130.86 Pg/mL, which indicates that the model of inflammation model is successfully modeled; second, TNF-once with increasing sample concentration, compared to the normal and model groupsαAnd IL-6 expression levelShows that the HPD induces TNF-once on RAW264.7 cells induced by LPSαHas certain inhibitory effect on IL-6, but compared with the IL-6, the inhibitory effect of HPD on the inflammatory factor is more obvious than that of TNF-α；

Table 1: 24 h after HPD action, LPS induces RAW264.7 cells to generate TNF-αAnd IL-6 inhibition

Note: in comparison with the normal group,^##P<0.01; comparing with model group<0.01

As a result of the expression of iNOS protein in RAW264.7 cells by the compound HPD, as shown in FIG. 5, the iNOS protein expression level in the model group RAW264.7 cells was significantly increased (P < 0.01) compared with that in the normal group; compared with the model group, the relative protein expression amount of iNOS in RAW264.7 cells is remarkably reduced by HPD along with the increase of the concentration (P < 0.01).

Nitric Oxide (NO) is a biologically active gas molecule, an important regulator of intercellular information transfer, and functions to mediate cellular immunity and inflammatory toxicity. Excessive production of NO is closely related to inflammation, and at acute inflammation sites, inflammatory substances and inflammatory mediators can induce or increase the synthesis and release of NO, and NO itself has cytotoxicity and can react with free radicals to produce, for example, ONOO^-Plasma, leading to increased toxicity, promoting edema and pustules at the site of inflammation. In the experiment, the trachelospermi caulis compound 1 is screened through NO inhibition activity, and compared with a positive control drug indometacin, HPD can obviously inhibit the generation of NO, which shows that HPD has good anti-inflammatory effect. Inflammatory factor TNF-through ELISA assayαAnd IL-6 expression level are measured, and the result shows that HPD induces TNF-once by RAW264.7 cells induced by LPSαAnd IL-6, wherein the inhibition effect of HPD on the inflammatory factor IL-6 is better than that of TNF-α。

Nitric Oxide Synthase (NOS) is a key enzyme catalyzing NO synthesis, and mainly includes neural (nNOS), endothelial (eNOS) and Inducible (iNOS), which are collectively called constructsShaped NOS (cNOS), which produces very low normal concentrations; inducible (iNOS) is hardly expressed under normal physiological conditions and is induced by Lipopolysaccharide (LPS) and interferon-αInterleukin-1, tumor necrosis factor-βAnd the like, and can be induced and activated, so that L-arginine can be used as a substrate, and is converted into L-citrulline under the action of iNOS, and a large amount of NO is released at the same time, thereby generating inflammatory reaction. In the experiment, protein expression of inducible iNOS in RAW264.7 cells by HPD is investigated by Western Blot, and the result shows that the HPD can obviously inhibit the protein expression of inducible iNOS in RAW264.7 cells, which indicates that the HPD can inhibit the activity of iNOS related to inflammation, thereby inhibiting the generation of inflammation.