CN106946975B - Triptolide derivative and preparation method and preparation thereof - Google Patents

Abstract

The invention discloses a triptolide derivative, a preparation method and a preparation thereof, wherein the chemical structure of the triptolide derivative is shown as the formula (I):

Description

Triptolide derivative and preparation method and preparation thereof

Technical Field

The invention relates to the technical field of medicines, in particular to a triptolide derivative and a preparation method and a preparation thereof.

Background

Triptolide (TP), also known as Triptolide and Triptolide, is an epoxy diterpenoid lactone compound isolated from Chinese medicinal Tripterygium wilfordii Hook F plants, and has strong biological activities including anti-inflammation, immunosuppression, anti-tumor, antifertility and the like (Liu Q. Triptolide and its expanded multiple pharmaceutical functions [ J ]. International immunological pharmacology,2011,11(3):377 383). The antitumor value of tripterygium wilfordii as a main active component is widely researched, and at present, at least 60 tumor cell lines can be inhibited by TP (luoyongwei, shichang, Liaoming yang. triptolide antitumor action mechanism research progress [ J ] Chinese traditional medicine journal, 2009,34(16):2024-2026) through research. TP has the following four characteristics as an anti-tumor medicament: the compound has broad-spectrum antitumor activity in a mouse transplantation tumor animal model, has the inhibition rate of 50-90 percent on primary tumors of breast cancer, bladder cancer, gastric cancer and melanoma, and is effective on the primary tumors and secondary tumors; secondly, the growth and the metastasis of various solid tumors can be inhibited, and the growth and the metastasis of various solid tumors are not related to the state of a tumor inhibition gene (P53), so that the tumor inhibition gene can still play a role in P53-deficient tumor cells; ③ the tumor cells with high expression of multidrug resistance protein (MDR1), and the cells are resistant to common chemotherapy drugs such as paclitaxel, therefore, TP can be used with chemotherapy drugs to increase the curative effect; has stronger antitumor activity than that of the traditional antitumor drugs, and generates very strong antitumor activity at low concentration (Yang S, Chen J, Guo Z, et al. triptolide inhibitors the Growth and Metastasis of Solid Tumors [ J ]. Molecular cancer therapeutics,2003,2(1): 65-72). The specific mechanism of the TP antitumor effect is unknown, and induction of tumor cell apoptosis through various signaling pathways is probably the most important mechanism of its antitumor effect. TP can also inhibit tumor growth and metastasis by inhibiting angiogenesis, blocking cell cycle progression, inhibiting extracellular matrix degradation, etc. (Meng C, Zhu H, Song H, et al. targets and molecular mechanisms of triptolide in Cancer therapy [ J ]. Chinese Journal of Cancer Research,2014,26(5): 622-626). The advantages of broad spectrum, high efficiency and the like of the TP in the aspect of tumor resistance enable the TP to have wide clinical application prospect.

Although TP has been shown to be a therapeutic effect in leukemia as early as 1972 (Kupchan S M, Coort W A, Dailey R G, et al. Tumor inhibitors. LXXIV. triptolide and tripdiolide, novelatileukemic diagnostic peptides from Tripterygium wilfordii [ J ]. journal of the American Chemical Society,1972,94(20):7194 with 7195), and in more than ten years a number of in vitro and in vivo studies have shown well that TP has an inhibitory effect on the growth of a variety of solid tumor cells and solid tumors. However, a highly effective drug for TP application in antitumor therapy has not yet been developed for marketing. The difficulties are mainly reflected in the following aspects:

TP has high toxicity: the drug effect is exerted, meanwhile, the drug effect is accompanied by serious toxic and side effects, and the narrow treatment window is shown, so that the clinical application of the drug is greatly limited. Its toxic action is mainly reflected in several aspects of general toxicity, target organ toxicity, genetic toxicity, reproductive system toxicity, local irritation toxicity, etc. Whereas target organ toxicity relates to almost all organs with physiological functions, even therapeutic doses of TP show suppression of immune function, and overdose and poisoning cause atrophy of lymphoid organs and necrosis of lymphocytes (Li X, Jiang Z, Zhang L. triptolide: progress on research in pharmacology and pathology [ J ]. Journal of Ethnopharmacology,2014,155(1): 67-79).

Short half-life of TP: the in vivo elimination rate is high, the half-life period is short, the due anti-tumor effect of the in vivo elimination agent is weakened to a great extent, the distribution of the in-blood volume is wide, the targeting property is poor, and the adverse reaction is aggravated. Shao et al have studied the pharmacokinetic characteristic of TP in rat in vivo with the liquid chromatography/mass spectrometry method (LC/MS), the result shows that SD rat single oral administration TP dosage is 0.6, 1.2 and 2.4mg/kg respectively, the peak time of blood concentration is about 10min, the elimination half-life is 16.81-21.70 min, the pharmacokinetic parameter of comparing the three shows that AUC and Cmax value can not be improved with the increase of dosage, suggest that the clinical application may present nonlinear kinetic characteristic when high dosage; SD rats have a half-life of about 15min at a single intravenous TP dose of 0.6 mg/kg. TP can be rapidly distributed to the tissues being examined (plasma, heart, liver, spleen, lung, kidney) and the changes in tissue paralleled the changes in plasma concentration (Shao F, Wang G, Xie H, et al Pharmaceutical study, clinical of immunological therapy and clinical medicine, in rates [ J ]. Biological & Pharmaceutical Bulletin,2007,30(4): 702-707).

The TP preparation has poor drug property: TP solubility was less than 0.3mg/mL in either distilled water, 0.1mol/L HCl or PBS pH6.8, as specified by solubility terminology described in the Chinese pharmacopoeia 2015 edition, and belongs to a very slightly water soluble substance (Zhang Smart triptolide lipid nanoparticles research [ D ]. university of science and technology, 2014). In addition, the solubility of TP in some other pharmaceutically acceptable solvents and oils is also not more than 0.5% (w/w), which makes it difficult to prepare conventional solutions for intravenous injection. Even if the liposome or nanoparticle preparation is developed into a plurality of novel liposome or nanoparticle preparations, the defects of low drug loading, poor encapsulation rate, unstable preparation and the like still exist. The forest seiil et al try to prepare TP nano liposome, the maximum drug-loading rate still only reaches 0.016% (Chinese patent CN 105816428A); zhang smart preparation of TP lipid nanoparticles, lipid/drug 50: 1-100: under the condition of 1, the encapsulation rate still does not exceed 60 percent and is far lower than the specified requirements of Chinese pharmacopoeia (Zhang Smart, triptolide lipid nanoparticle research [ D ] Huazhong university of science and technology, 2014); stability examination of the prepared liposome by the Wang Shujuan shows that the newly prepared TP liposome aggregates after being placed at room temperature for 10 days, basically precipitates after one month, and even if the freeze-drying treatment is carried out, the entrapment rate of the liposome reconstructed by rehydration after freeze-drying is still reduced to different degrees compared with that before freeze-drying, and is less than 40% (Wang Shujuan, preliminary study on the preparation and quality of triptolide liposome [ D ]. Yangzhou university, 2010). The properties make it difficult to develop TP into a highly effective in vivo injection preparation beneficial to tumor treatment.

How to prolong the action time in vivo and reduce the toxic and side effects on the premise of keeping the bioactivity of TP is a key problem which needs to be solved urgently and effectively in clinic, and TP can be safely and effectively applied. Prodrugs (prodrugs) are biologically inactive or poorly active per se and are metabolized in vivo to become active, a process which alters the pharmacokinetic properties of the parent drug in vivo. Thus, in the search for TPs, many researchers have been tempted to structurally modify TPs to obtain TP prodrugs with good pharmacokinetic properties. Due to their poor water solubility, most researchers tend to design and synthesize novel water-soluble TP derivatives with good pharmacokinetic properties in order to reduce the toxic side effects of the drugs. Such as: PG490-88Na, WilGraf, Minnelide, etc., are water-soluble prodrugs of TP (Fidler J M, Li K, Chung C, et al PG490-88, a derivative of triptolide, consumers tune Molecular regression and transmitters to chemotherapy [ J ]. Molecular Cancer Therapeutics,2003,2(9): 855: 862; Zhou Z, YangY, Ding J, et al triptolide: structural modifications, structural-activity modifications, biological activities, clinical details and mechanisms [ J ]. Natural products, 2012,29(4):457,475). Although the pro-drug salifying TP well improves the preparation property, the aim of reducing toxicity and improving the pharmacokinetic property is not expected to be achieved, and the possible defects of too fast degradation release or incomplete conversion and the like still exist. Even more so, in a dose escalation study in a phase I clinical trial of PG490-88Na, subjects were patients with advanced solid tumors administered by intravenous injection once a week with a two week rest week. Adverse reactions frequently occurring in the test include anemia, hypodynamia, nausea, vomiting, diarrhea, constipation and the like, and the grades are all 1-2. However, pharmacokinetic studies show that there is a high inter-individual difference (2-3 times) in the pharmacokinetic properties of PG490-88Na, the degree of conversion to TP is difficult to predict, and the conversion process is slow and incomplete. Thus, PG490-88Na is not an ideal TP derivative, and the clinical trial has been forced to pause (Kitzen J, de Joge M J, lasers C H, et al. phase I dose-interpretation of F60008, a novel apoptosis indicator, in Patients with advanced soluble tissues [ J ]. European Journal of Cancer,2009,45(10): 1764-1772). Thereafter, there was no renewal of the report associated with the PG490-88Na clinical trial. It is clear that the improvement of water solubility of TP salts is an ideal modification to meet the clinical requirements.

On the other hand, as a hot spot of current research and development, the nano preparation has shown wide application prospect, and has the advantages that: the nano-drug can reduce the toxicity through the targeting effect, and further improve the curative effect on the basis of ensuring the effectiveness; the medicament is wrapped in the nanoparticles, particularly the long-circulating nanoparticles have obvious sustained and controlled release effects, so that the elimination rate of the medicament in vivo is greatly slowed down, the half-life period is prolonged, the medicament has enough time to target the medicament at a tumor part, the distribution in normal tissues is reduced, and the safety of clinical medication is improved. The above characteristics are ideal effects that TP is urgently needed to achieve. And with the development of preparation technology, a plurality of nano preparations are sequentially applied to clinic at present, such as adriamycin liposome

Paclitaxel for injection (albumin-bound type), trade name

Paclitaxel polymer micelle

Etc. (Barenholz Y.

—The first FDA-approved nano-drug:Lessons learned[J]Journal of Controlled Release,2012,160(160): 117-; gongyuan, Luming, Lijie, etc. paclitaxel (albumin bound) for injection to treat advanced gastric cancer [ J]144-148 of the journal of Beijing university, 2014,46 (1); lee K S, Chung H C, Im S A, et al, Multi center phase II tertiary of Genex-PM, a Cremophor-free, polymeric micro-particle formation of paclitaxel, in-tissues with a statistical break cancer [ J ]]Breast cancer research and Treatment,2008,108(2): 241-. The preparation fully illustrates the feasibility of the nano preparation for marketing antitumor drugs, can provide important reference for the development of TP, or the development of the TP into the nano preparation is a more correct research direction.

However, as determined by the defect of drug property of the TP preparation, the direct preparation of TP into liposome, micelle, nanoparticle and other encapsulated nano-preparations has poor feasibility, and the main reason is that the lipid solubility of TP is too low and the compatibility with the encapsulated material is not matched. How to improve the lipid solubility of TP will be the focus of the nano-formulation development. Since the chemical properties of the parent drug can be significantly improved by the prodrug, it is important to determine whether the prodrug can satisfy the pharmaceutical requirements of the preparation. Chinese patent CN105263475A discloses a TP prodrug "MRx 102", but MRx102 has no or poor druggability, and is mainly reflected in: in the embodiment, the amount of the oil phase used in the MRx102 emulsion formulation is large, and the majority of the oil phase is 40%, as described in the patent, "the classical emulsion formulation contains 10-30% triglyceride", and 40% oil phase directly results in the formulated emulsion being thick cream (filler J M, An J, Carter B Z, et al. clinical anti-inflammatory activity, toxicology, toxi-kinetics and formation level of ternary derivative MRx102[ J ]. Cancer chemo and Pharmacology,2014,73(5): 961-; ② the solubility in oil is too poor, for the moment, regardless of the safety of tricaprylin, even if 29.4% tricaprylin is used as the oil phase to prepare the emulsion in the patent table 4, the solubility of MRx102 is 0.681 mug/mL, which directly results in too low drug loading; ③ the stability is too poor, in the patent table 5, even if 40% tricaprylin is used as the emulsion prepared from the oil phase (taking E-3 as an example), the drug-loading amount of the emulsion with 2mg/mL is prepared, the drug content (solubility) is 1529, 1514, 1353 and 1176 μ g/mL respectively from 0 hour, 1 hour, 24 hours and 8 days, namely the drug-loading amount of the prepared emulsion is reduced by 41.2% after the prepared emulsion is placed for 8 days, and the prepared emulsion does not have drug-forming property; MRx102 emulsion formula contains high content of tricaprylin, and the patent shows that partial or complete replacement of tricaprylin by soybean oil reduces the solubility of MRx102 in the emulsion, which indicates that tricaprylin cannot be lacked, and the component is mainly used in skin care products and cosmetics at present and has potential safety hazard in intravenous administration preparations; in order to increase the dissolution of MRx102 in the emulsion, ionic surfactant sodium cholate is added in the formula for solubilization, which also shows that the prodrug does not have the characteristic of obviously improving lipid solubility. The ionic surfactant has high toxicity and strong hemolytic effect (treford. pharmacy [ M ]. Beijing: people health Press 2011: 42). In addition, the patent does not relate to the preparation of other nano preparations such as liposome, micelle and the like, or the development of fat emulsion.

In summary, despite the research on triptolide, the problem of drug potency of triptolide preparations still stands out, and further research and development are urgently needed to improve the problem and develop efficient drugs for TP application in antitumor therapy.

Disclosure of Invention

In order to overcome the defect of TP drug property, the invention develops a large amount of structural modification work aiming at TP, starts with a fat-soluble prodrug in a targeted manner, finds that the fat solubility is obviously improved after TP is bonded with saturated fatty acid, synthesizes triptolide fatty acid ester modified by saturated fatty acid with different chain lengths within the range of 2n carbon atoms (n is 2-9) through further screening, and shows that the drug property of the triptolide fatty acid ester preparation is superior to that of TP. The liposome, the micelle, the nanoparticle and the fat emulsion which are tried to be prepared have good entrapment effect. Further in vivo and in vitro studies show that the prepared nano preparation has the advantages of remarkably reducing toxicity, prolonging half-life period and the like.

The invention aims to provide a triptolide derivative, in particular to triptolide fatty acid ester, which has a chemical structure shown in a formula (I):

in the formula (I), R represents a C2 n linear alkyl acyl group, and n is 2-9, preferably 4-9, and more preferably 7-9.

The second objective of the invention is to provide a preparation method of the triptolide fatty acid ester, wherein the triptolide fatty acid ester can be obtained by esterification reaction of triptolide and saturated fatty acid, and the synthetic route is as follows:

wherein ROH is a saturated fatty acid, and R is as defined in any one of claims 1 to 3;

the method comprises the following specific steps:

(a) dissolving saturated fatty acid, a condensing agent and a catalyst in an organic solvent, and cooling to 0-10 ℃ under stirring to obtain a mixed solution;

(b) dissolving triptolide in an organic solvent, dripping the triptolide into the mixed solution, reacting for 15-45 minutes at 0-10 ℃, continuing to react at room temperature, and separating to obtain triptolide fatty acid ester after the esterification reaction is finished.

In the preparation method of the triptolide fatty acid ester,

the condensing agent is p-nitrobenzoyl chloride, N, N '-Dicyclohexylcarbodiimide (DCC), N, N' -Diisopropylcarbodiimide (DIC), 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC. HCl), 2-chloro-1-methylpyridinium iodide (CMPI), benzotriazole-1-yl-oxy-tripyrrolidinyl phosphate (PyBOP), 2- (7-azobenzotriazol) -N, N, N ', N' -tetramethylurea Hexafluorophosphate (HATU), O- (5-chlorobenzotriazol-1-yl) -di (dimethylamino) carbonium Hexafluorophosphate (HCTU), O- (benzotriazole-1-yloxy) -dipiperidine carbonium hexafluorophosphate (HBPipU), One or more of N-hydroxysuccinimide (NHS) and N-hydroxythiosuccinimide (sulfo-NHS), preferably DCC or EDC & HCl;

the catalyst is one or more than two of 1-Hydroxybenzotriazole (HOBT), 4-Dimethylaminopyridine (DMAP), triethylamine and N, N-Diisopropylethylamine (DIPEA), and preferably DMAP or DIPEA;

the organic solvent is one or more than two of dichloromethane, trichloromethane, N-Dimethylformamide (DMF) and N, N-Dimethylacetamide (DMA), and the organic solvent is preferably anhydrous.

The molar ratio of the saturated fatty acid to the triptolide is 1.2-4: 1, preferably 2-3: 1; the molar ratio of the condensing agent to the triptolide is 1.2-4: 1, preferably 2-3: 1.

The triptolide fatty acid ester can be used for preparing anti-tumor or anti-inflammatory drugs.

The third purpose of the invention is to provide the nanometer preparation of triptolide fatty acid ester, wherein the nanometer preparation is liposome, polymer micelle, albumin nanoparticle or fat emulsion, and liposome is preferable.

In some preferred embodiments of the present invention, the liposome is prepared from the following components in parts by weight and volume:

the above formula is preferably:

wherein the content of the first and second substances,

the phospholipid is one or more of egg yolk phospholipid, soybean phospholipid, phospholipids from various animal sources, hydrogenated egg yolk phospholipid, hydrogenated soybean phospholipid, dipalmitoyl phosphatidylcholine, dimyristoyl phosphatidylcholine, distearoyl phosphatidylcholine, phosphatidylethanolamine, distearoyl phosphatidylglycerol, dipalmitoyl phosphatidylglycerol, dioleoyl phosphatidylcholine, cardiolipin and sphingomyelin; egg yolk phospholipids or soybean phospholipids are preferred.

The PEGylated phospholipid is one or more than two of PEGylated distearoyl phosphatidyl ethanolamine, PEGylated dipalmitoyl phosphatidyl ethanolamine and PEGylated dimyristoyl phosphatidyl ethanolamine, preferably PEGylated distearoyl phosphatidyl ethanolamine; the PEG average molecular weight of the PEG phospholipid is 1000-8000, preferably 1500-3500, and more preferably 2000.

The freeze-drying protective agent is one or more than two of trehalose, sucrose, maltose, lactose, mannitol and glucose; trehalose or sucrose is preferred.

The pH regulator is one or more than two of sodium hydroxide, sodium acetate, acetic acid, phosphate, carbonate, hydrochloric acid, citric acid and the like; sodium hydroxide, hydrochloric acid or citric acid is preferred.

The liposome can be prepared by the following steps:

(A) preparing a liposome crude product, namely weighing triptolide fatty acid ester, phospholipid, PEGylated phospholipid and cholesterol according to a formula, adding a proper amount of organic solvent I, heating and dissolving at 25-75 ℃ to obtain an organic phase, slowly injecting the organic phase into a proper amount of water for injection at 25-75 ℃, and uniformly stirring while injecting to obtain the liposome crude product;

(B) preparing a liposome solution, namely placing the crude liposome in a high-pressure homogenizer for homogenizing and emulsifying, or placing the crude liposome in an extruder for extruding sequentially through extrusion films with different apertures, or placing the crude liposome in the high-pressure homogenizer for homogenizing and then extruding to obtain the liposome solution;

(C) weighing a freeze-drying protective agent with a formula amount, and dissolving the freeze-drying protective agent in the liposome solution; adding water for injection to a constant volume, and adjusting the pH value to a specified value; filtering with 0.22 μm filter membrane, packaging, freeze drying, sealing, and capping to obtain the final product;

alternatively, the first and second electrodes may be,

replacing the step (A) with: (A') weighing triptolide fatty acid ester, phospholipid, PEG phospholipid and cholesterol according to the formula, dissolving in an organic solvent II, performing vacuum rotary evaporation at 40-60 ℃ to remove the solvent to obtain a lipid film, and hydrating with a proper amount of water for injection to obtain a crude liposome.

The organic solvent I is selected from one or more than two of absolute ethyl alcohol, propylene glycol and tertiary butanol; the dosage is 1-10% g/ml, the organic solvent I is retained in the liposome, or the crude liposome product is emulsified and then removed by an ultrafiltration or scraper film evaporator.

The organic solvent II is one or more than two of dichloromethane, trichloromethane and absolute ethyl alcohol, and the dosage of the organic solvent II is 1-10% g/ml.

The lyoprotectant can be directly added into the liposome solution prepared in the step (B), or can be dissolved in the water for injection and then mixed with the liposome solution (i.e. dissolved in the water phase for adding).

The pore diameter of the extrusion film in the step (B) is selected from one or more than two of 2.0 μm, 1.0 μm, 0.8 μm, 0.6 μm, 0.4 μm, 0.2 μm, 0.1 μm and 0.05 μm, and the pore diameter is changed from large to small in sequence during extrusion.

The invention has the beneficial effects that:

the invention develops a large amount of structural modification work aiming at TP, starts from the perspective of improving the lipid solubility, bonds saturated fatty acid at C14 OH of TP, reduces the polarity of hydroxyl into ester, introduces fatty acid at the same time, can effectively enhance the lipid solubility, has stable property and difficult oxidation, and can obviously improve the drug forming property of TP nano-preparation. The triptolide fatty acid ester preparations modified by saturated fatty acids with different chain lengths within the range of 2n carbon atoms (n is 2-9) are further screened and synthesized to have better drug properties than TP, the nano preparations such as liposome, micelle, nanoparticle and fat emulsion prepared from the triptolide fatty acid ester have good entrapment effects, and the prepared nano preparations have the characteristics of large drug loading rate, high entrapment rate, stable properties and the like. Further in vivo and in vitro studies show that the prepared nano preparation has the advantages of remarkably reducing toxicity, prolonging half-life period and the like. The invention provides possibility for successfully developing triptolide nano-preparations and can lay a solid foundation for the research and application of triptolide.

Detailed Description

The present invention will be further described with reference to the following examples. It should be understood that the following examples are illustrative only and are not intended to limit the scope of the present invention.

The TP of the invention is purchased from Jiangsu Beida pharmaceutical science and technology Limited; saturated fatty acids were purchased from the national pharmaceutical group chemical agents limited.

The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers.

Example 1 preparation of different triptolide fatty acid esters

1.1.1 preparation of Tripterygium wilfordii ester of stearic acid (TP-SA)

Adding 3mmol of stearic acid, 3mmol of DCC and 3mmol of DMAP into a reaction vessel, adding 20mL of anhydrous dichloromethane for dissolving, and stirring for 30 minutes under the ice bath condition; dissolving 1mmol of TP in a proper amount of anhydrous dichloromethane, slowly dropwise adding into a reaction system, reacting for 30 minutes under an ice bath condition, continuously reacting overnight at room temperature, and separating and purifying the reactant by a silica gel column to obtain 532.2mg of triptolide stearate. The yield was 84.9%.

¹H NMR(DMSO-_d6,600MHz)4.98(1H,s,14-CH),4.86(1H,d,J＝18.88Hz,19-CH),4.77(1H,d,J＝18.88Hz,19-CH),3.95(1H,d,11-CH),3.69(1H,d,12-CH),3.56(1H,d,7-CH),2.28-2.39(2H,m,2ˊ-CH₂),1.73-2.00(1H,m,15-CH),1.80-1.85(2H,m,3ˊ-CH₂),1.57-1.59(2H,m,2-CH₂),1.24-1.34(28H,m,14×CH₂),0.92(3H,s,20-CH₂),0.85-0.87(6H,m,16、17-CH₂),0.76(3H,t,18ˊ-CH₂).

¹³C NMR(DMSO,150MHz)175.52,170.59,71.08,70.66,63.74,63.08,61.30,59.84,55.29,54.93,35.51,34.19,31.78,29.57,29.51,29.37,29.23,29.19,28.71,28.11,25.06,22.85,22.57,17.90,17.05,17.00,14.39,14.13.

ESI-MS(m/z)：627.8[M+H]⁺.

The chemical structural formula is as follows:

1.1.2 preparation of Tripterygium wilfordii ester of stearic acid (TP-SA)

Adding 2.5mmol of stearic acid, 2.5mmol of EDC & HCl and 2.5mmol of DIPEA into a reaction vessel, adding 20mL of anhydrous dichloromethane for dissolving, and stirring for 30 minutes under the ice bath condition; dissolving 1mmol of TP in a proper amount of anhydrous dichloromethane, slowly dropwise adding into the reaction system, reacting for 30 minutes under an ice bath condition, continuously reacting overnight at room temperature, and separating and purifying the reactant by a silica gel column to obtain 524.0mg of triptolide stearate. The yield was 83.6%.

1.2.1 preparation of triptolide palmitate (TP-PA)

Adding 3mmol of palmitic acid, 3mmol of DCC and 3mmol of DMAP into a reaction vessel, adding 20mL of anhydrous dichloromethane for dissolving, and stirring for 30 minutes under the ice bath condition; dissolving 1mmol of TP in a proper amount of anhydrous dichloromethane, slowly dropwise adding into the reaction system, reacting for 45 minutes under an ice bath condition, continuously reacting overnight at room temperature, and separating and purifying the reactant by a silica gel column to obtain 504.7mg of purified triptolide palmitate. The yield was 84.3%.

¹H NMR(DMSO-_d6,600MHz)4.96(1H,s,14-CH),4.86(1H,d,J＝18.88Hz,19-CH),4.73(1H,d,J＝18.86Hz,19-CH),3.98(1H,d,11-CH),3.69(1H,d,12-CH),3.57(1H,d,7-CH),2.28-2.42(2H,m,2ˊ-CH₂),1.73-2.00(1H,m,15-CH),1.80-1.85(2H,m,3ˊ-CH₂),1.57-1.61(2H,m,2-CH₂),1.24-1.35(24H,m,12×CH₂),0.93(3H,s,20-CH₂),0.85-0.88(6H,m,16、17-CH₂),0.78(3H,t,18ˊ-CH₂).

¹³C NMR(DMSO,150MHz)178.52,173.59,70.88,70.26,63.64,63.18,61.30,59.34,55.29,55.03,35.91,34.21,31.78,29.77,29.23,29.17,28.71,28.05,25.13,22.80,22.57,17.90,17.35,17.09,14.42,14.11.

ESI-MS(m/z)：599.8[M+H]⁺.

The chemical structural formula is as follows:

1.2.2 preparation of triptolide palmitate (TP-PA)

Adding 2mmol of palmitic acid, 2mmol of EDC & HCl and 2mmol of DIPEA into a reaction vessel, adding 20mL of anhydrous dichloromethane for dissolving, and stirring for 30 minutes under the ice bath condition; dissolving 1mmol TP in a proper amount of anhydrous dichloromethane, slowly dropwise adding into the reaction system, reacting for 30 minutes under an ice bath condition, continuously reacting overnight at room temperature, and separating and purifying the reactant by a silica gel column to obtain 495.2mg of purified triptolide palmitate. The yield was 82.7%.

1.3.1 preparation of Tripterygium Wilfordii myristate (TP-MA)

Adding 3mmol of myristic acid, 3mmol of DCC and 3mmol of DMAP into a reaction container, adding 20mL of anhydrous trichloromethane for dissolving, and stirring for 15 minutes under the ice bath condition; dissolving 1mmol TP in an appropriate amount of anhydrous chloroform, slowly adding dropwise into the reaction system, reacting for 30 minutes under ice bath condition, continuing to react overnight at room temperature, and separating and purifying the reactant by a silica gel column to obtain 464.5mg of triptolide myristate. The yield was 81.4%.

¹H NMR(DMSO-_d6,600MHz)4.97(1H,s,14-CH),4.84(1H,d,J＝18.88Hz,19-CH),4.73(1H,d,J＝18.83Hz,19-CH),3.97(1H,d,11-CH),3.66(1H,d,12-CH),3.61(1H,d,7-CH),2.27-2.41(2H,m,2ˊ-CH₂),1.73-2.00(1H,m,15-CH),1.80-1.85(2H,m,3ˊ-CH₂),1.57-1.61(2H,m,2-CH₂),1.22-1.35(20H,m,10×CH₂),0.90(3H,s,20-CH₂),0.83-0.87(6H,m,16、17-CH₂),0.74(3H,t,18ˊ-CH₂).

¹³C NMR(DMSO,150MHz)178.92,173.10,70.90,62.73,61.64,59.34,58.43,55.29,55.03,49.97,46.13,46.05,35.91,34.21,34.19,32.22,31.88,31.52,29.57,29.23,29.17,28.71,28.05,25.13,22.80,22.57,17.90,17.35,17.09,14.42,14.08.

ESI-MS(m/z)：571.7[M+H]⁺.

The chemical structural formula is as follows:

1.3.2 preparation of Tripterygium Wilfordii myristate (TP-MA)

2mmol of myristic acid, 2mmol of EDC & HCl and 2mmol of DIPEA are added into a reaction vessel, 20mL of anhydrous dichloromethane is added for dissolution, and stirring is carried out for 15 minutes under the ice bath condition; dissolving 1mmol of TP in a proper amount of anhydrous chloroform, slowly adding dropwise into the reaction system, reacting for 30 minutes under an ice bath condition, continuously reacting overnight at room temperature, and separating and purifying the reactant by a silica gel column to obtain 476.5mg of triptolide myristate. The yield was 83.5%.

1.4.1 preparation of triptolide laurate (TP-LA)

Adding 1.2mmol of lauric acid, 1.2mmol of DIC and 1.2mmol of HOBT into a reaction vessel, adding 20mL of anhydrous DMF for dissolving, and stirring for 20 minutes under the ice bath condition; dissolving 1mmol of TP in a proper amount of anhydrous dichloromethane, slowly dropwise adding into the reaction system, reacting for 30 minutes under an ice bath condition, continuously reacting overnight at room temperature, and separating and purifying the reactant by a silica gel column to obtain 397.3mg of triptolide laurate. The yield was 73.2%.

¹H NMR(DMSO-_d6,600MHz):4.78(1H,s,14-CH),4.50(1H,d,J＝18.77Hz,19-CH),4.43(1H,d,J＝18.77Hz,19-CH),3.35(2H,s,11、12-CH),3.21(1H,t,7-CH),2.56-2.60(1H,m,4-CH),2.32-2.37(2H,m,2ˊ-CH₂),2.16-2.21(1H,m,3-CH),1.80-1.85(2H,m,3ˊ-CH₂),1.73-1.77(1H,m,15-CH),1.57-1.61(4H,m,1、2-CH₂),1.44-1.50(2H,m,6-CH₂),1.25-1.30(16H,m,8×CH₂),1.04-1.10(1H,m,5-CH),0.93(3H,s,20-CH₂),0.85-0.88(6H,m,16、17-CH₂),0.78(3H,t,12ˊ-CH₂).

¹³C NMR(150MHz,DMSO)：178.9,173.3,71.0,62.6,61.5,59.3,58.7,52.4,49.7,46.0,34.9,34.2,31.6,29.6,29.3,22.7,21.6,19.3,18.5,14.3.

ESI-MS(m/z)：543.7[M+H]⁺.

The chemical structural formula is as follows:

1.5.1 preparation of triptolide caprate (TP-DA)

1.5mmol of capric acid, 1.5mmol of paranitrobenzoyl chloride and 1.5mmol of triethylamine are added into a reaction vessel, 20mL of anhydrous dichloromethane is added for dissolution, and the mixture is stirred for 5 minutes under the ice bath condition; dissolving 1mmol of TP in a proper amount of anhydrous dichloromethane, slowly dropwise adding into a reaction system, reacting for 30 minutes under an ice bath condition, continuously reacting overnight at room temperature, and separating and purifying the reactant by a silica gel column to obtain 390.6mg of triptolide decanoate. The yield was 75.9%.

¹H NMR(DMSO-_d6,600MHz):4.88(1H,s,14-CH),4.54(1H,d,J＝18.71Hz,19-CH),4.33(1H,d,J＝18.71Hz,19-CH),3.35(2H,s,11、12-CH),3.21(1H,t,7-CH),2.56-2.60(1H,m,4-CH),2.32-2.37(2H,m,2ˊ-CH₂),2.16-2.25(1H,m,3-CH),1.82-1.85(2H,m,3ˊ-CH₂),1.73-1.77(1H,m,15-CH),1.57-1.61(4H,m,1、2-CH₂),1.44-1.50(2H,m,6-CH₂),1.25-1.30(12H,m,6×CH₂),1.04-1.10(1H,m,5-CH),0.93(3H,s,20-CH₂),0.85-0.88(6H,m,16、17-CH₂),0.78(3H,t,10ˊ-CH₂).

¹³C NMR(150MHz,DMSO)：178.9,173.5,71.7,62.6,61.6,59.3,58.4,52.3,49.9,46.0,34.8,34.2,31.5,29.5,26.3,25.0,22.7,21.6,19.1,14.0.

ESI-MS(m/z)：515.6[M+H]⁺.

The chemical structural formula is as follows:

1.6.1 preparation of Tripterygium wilfordii A caprylate (TP-CA)

Adding 4mmol of octanoic acid, 4mmol of DCC and 4mmol of DMAP into a reaction vessel, adding 20mL of anhydrous DMA for dissolving, and stirring for 30 minutes under the ice bath condition; dissolving 1mmol of TP in a proper amount of anhydrous dichloromethane, slowly dropwise adding into a reaction system, reacting for 45 minutes under an ice bath condition, continuously reacting overnight at room temperature, and separating and purifying the reactant by a silica gel column to obtain 411.2mg of triptolide caprylate. The yield was 84.5%.

¹H NMR(DMSO-_d6,600MHz):4.81(1H,s,14-CH),4.47(1H,d,J＝18.68Hz,19-CH),4.22(1H,d,J＝18.68Hz,19-CH),3.37(2H,s,11、12-CH),3.17(1H,t,7-CH),2.56-2.60(1H,m,4-CH),2.33-2.37(2H,m,2ˊ-CH₂),2.16-2.21(1H,m,3-CH),1.80-1.86(2H,m,3ˊ-CH₂),1.73-1.78(1H,m,15-CH),1.57-1.61(4H,m,1、2-CH₂),1.39-1.50(2H,m,6-CH₂),1.25-1.30(8H,m,4ˊ、5ˊ、6ˊ、7ˊ-CH₂),1.04-1.10(1H,m,5-CH),0.93(3H,s,20-CH₂),0.85-0.88(6H,m,16、17-CH₂),0.78(3H,t,18ˊ-CH₂).

¹³C NMR(150MHz,DMSO)：178.8,173.2,71.1,62.6,61.3,59.6,58.2,52.3,49.9,46.0,34.9,34.2,31.5,29.0,26.3,25.0,22.7,21.6,19.0,13.9.

ESI-MS(m/z)：487.6[M+H]⁺.

The chemical structural formula is as follows:

1.7.1 preparation of triptolide caproate (TP-HA)

2.5mmol of caproic acid, 2.5mmol of HATU and 2.5mmol of DMAP are added into a reaction vessel, 20mL of anhydrous DMF is added for dissolution, and the mixture is stirred for 15 minutes under the ice bath condition; dissolving 1mmol TP in a proper amount of anhydrous chloroform, slowly adding dropwise into the reaction system, reacting for 30 minutes under an ice bath condition, continuously reacting overnight at room temperature, and separating and purifying the reactant by a silica gel column to obtain 352.6mg triptolide caproate. The yield was 76.9%.

¹H NMR(DMSO-_d6,600MHz):4.88(1H,s,14-CH),4.45(1H,d,J＝18.78Hz,19-CH),4.15(1H,d,J＝18.78Hz,19-CH),3.43(2H,s,11、12-CH),3.20(1H,t,7-CH),2.66-2.76(1H,m,4-CH),2.28-2.35(2H,m,2ˊ-CH₂),2.17-2.20(1H,m,3-CH),1.80-1.85(2H,m,3ˊ-CH₂),1.73-1.79(1H,m,15-CH),1.57-1.61(4H,m,1、2-CH₂),1.39-1.50(2H,m,6-CH₂),1.25-1.30(4H,m,4ˊ、5ˊ-CH₂),1.04-1.10(1H,m,5-CH),0.93(3H,s,20-CH₂),0.85-0.88(9H,m,16、17、6ˊ-CH₂).

¹³C NMR(150MHz,DMSO)：178.5,172.0,71.3,63.1,61.8,59.8,58.6,53.0,49.6,46.3,34.9,34.2,32.2,31.8,26.5,22.4,21.7,19.3,14.1.

ESI-MS(m/z)：459.5[M+H]⁺.

The chemical structural formula is as follows:

1.8.1 preparation of triptolide butyrate (TP-BA)

Adding 3mmol of butyric acid, 3mmol of EDC & HCl and 3mmol of DIPEA into a reaction container, adding 20mL of anhydrous trichloromethane for dissolving, and stirring for 30 minutes under the ice bath condition; dissolving 1mmol of TP in a proper amount of anhydrous trichloromethane, slowly dripping into a reaction system, reacting for 45 minutes under an ice bath condition, continuously reacting overnight at room temperature, and separating and purifying the reactant by a silica gel column to obtain 341.8mg of triptolide butyrate. The yield was 79.4%.

¹H NMR(DMSO-_d6,600MHz):4.96(1H,s,14-CH),4.86(1H,d,J＝18.88Hz,19-CH),4.73(1H,d,J＝18.86Hz,19-CH),3.98(1H,d,11-CH),3.93(1H,d,12-CH),3.70(1H,d,7-CH),2.66-2.76(1H,m,4-CH),2.28-2.42(2H,m,2ˊ-CH₂),2.17-2.20(1H,m,3-CH),1.80-1.85(2H,m,3ˊ-CH₂),1.73-1.78(1H,m,15-CH),1.57-1.61(4H,m,1、2-CH₂),1.39-1.50(2H,m,6-CH₂),1.04-1.10(1H,m,5-CH),0.99(3H,t,4ˊ-CH₃),0.93(3H,s,20-CH₂),0.85-0.88(6H,m,16、17-CH₂).

¹³C NMR(150MHz,DMSO)：178.9,173.1,70.9,62.7,61.7,59.3,58.4,52.4,49.9,46.1,36.4,34.1,32.2,31.9,26.3,21.6,19.2,18.9,13.5.

ESI-MS(m/z)：431.5[M+H]⁺.

The chemical structural formula is as follows:

example 2: preparation of different nanometer preparations of triptolide fatty acid ester

The different triptolide fatty acid esters prepared in example 1 are used for pharmaceutical investigation, and the preparations are examined to include liposome, polymer micelle, albumin nanoparticle and fat emulsion.

2.1 preparation of different triptolide fatty acid ester liposomes

2.1.1a preparation of Tripterygium wilfordii ester stearate (TP-SA) liposome

Weighing 0.2g of triptolide stearate, 2g of yolk phospholipid (PC-98T) and 0.2g of cholesterol, adding into 5g of absolute ethyl alcohol, and heating and dissolving at 60 ℃ to obtain an organic phase; slowly injecting the organic phase into 80mL of 60 ℃ water for injection, and uniformly stirring while injecting to obtain a liposome crude product; putting the liposome crude product into an extruder, and sequentially extruding through extrusion films with the aperture of 0.2 mu m, 0.1 mu m and 0.05 mu m to obtain a liposome solution; removing ethanol in the liposome by ultrafiltration; weighing 25g of sucrose, and dissolving in the liposome solution; adding water for injection to a constant volume of 100mL, and adjusting the pH value to 4.0 by using sodium hydroxide and hydrochloric acid; filtering with 0.22 μm filter membrane, packaging, freeze drying, sealing, and capping.

2.1.1b preparation of Tripterygium wilfordii ester stearate (TP-SA) liposomes

Weighing 0.2g of triptolide stearate, 2g of yolk phospholipid (EPCS), 0.2g of PEGylated distearoyl phosphatidyl ethanolamine (DSPE-PEG2000) and 0.2g of cholesterol, dissolving in a proper amount of dichloromethane, performing vacuum rotary evaporation at 45 ℃ to remove the solvent to obtain a lipid film, and hydrating with 80mL of water for injection to obtain a liposome crude product; homogenizing and emulsifying the liposome crude product in a high-pressure homogenizer to obtain a liposome solution; weighing 25g of trehalose, and dissolving in the liposome solution; adding water for injection to a constant volume of 100mL, and adjusting the pH value to 6.0 by using sodium hydroxide and hydrochloric acid; filtering with 0.22 μm filter membrane, packaging, freeze drying, sealing, and capping.

2.1.1c preparation of Tripterygium wilfordii stearate (TP-SA) liposomes

Weighing 0.3g of triptolide stearate, 3g of hydrogenated phospholipid (HSPC), 0.3g of PEG distearoyl phosphatidyl ethanolamine (DSPE-PEG2000) and 0.3g of cholesterol, dissolving in a proper amount of chloroform, performing vacuum rotary evaporation at 45 ℃ to remove the solvent to obtain a lipid film, and hydrating with 75mL of injection water to obtain a liposome crude product; homogenizing and emulsifying the liposome crude product in a high-pressure homogenizer to obtain a liposome solution; weighing 30g of sucrose, and dissolving in the liposome solution; adding water for injection to a constant volume of 100mL, and adjusting the pH value to 8.0 by using sodium hydroxide and citric acid; filtering with 0.22 μm filter membrane, packaging, freeze drying, sealing, and capping.

2.1.1d preparation of Tripterygium wilfordii ester stearate (TP-SA) liposome

Weighing 0.2g of triptolide stearate, 2g of yolk phospholipid (PC-98T), 0.2g of PEGylated distearylethanolamine (DSPE-PEG2000) and 0.2g of cholesterol, dissolving in a proper amount of dichloromethane, performing vacuum rotary evaporation at 45 ℃ to remove the solvent to obtain a lipid film, weighing 25g of sucrose, and dissolving in 80mL of water for injection to hydrate the lipid film to obtain a crude liposome; homogenizing and emulsifying the liposome crude product in a high-pressure homogenizer to obtain a liposome solution; weighing 25g of sucrose, and dissolving in the liposome solution; adding water for injection to a constant volume of 100mL, and adjusting the pH value to 6.5 by using sodium hydroxide and hydrochloric acid; filtering with 0.22 μm filter membrane, packaging, freeze drying, sealing, and capping.

2.1.2a preparation of triptolide palmitate (TP-PA) liposome

Weighing 0.2g of triptolide palmitate, 2g of yolk phospholipid (PC-98T) and 0.2g of cholesterol, adding 5g of absolute ethyl alcohol, and heating and dissolving at 60 ℃ to obtain an organic phase; slowly injecting the organic phase into 80mL of 60 ℃ water for injection, and uniformly stirring while injecting to obtain a liposome crude product; putting the liposome crude product into an extruder, and sequentially extruding through extrusion films with the aperture of 0.2 mu m, 0.1 mu m and 0.05 mu m to obtain a liposome solution; removing ethanol in the liposome by ultrafiltration; weighing 25g of sucrose, and dissolving in the liposome solution; adding water for injection to a constant volume of 100mL, and adjusting the pH value to 4.0 by using sodium hydroxide and hydrochloric acid; filtering with 0.22 μm filter membrane, packaging, freeze drying, sealing, and capping.

2.1.2b preparation of triptolide palmitate (TP-PA) liposome

Weighing 0.2g of triptolide palmitate, 2g of yolk phospholipid (EPCS), 0.2g of PEGylated distearoyl phosphatidyl ethanolamine (DSPE-PEG2000) and 0.2g of cholesterol, dissolving in a proper amount of dichloromethane, performing vacuum rotary evaporation at 45 ℃ to remove the solvent to obtain a lipid film, and hydrating with 80mL of water for injection to obtain a liposome crude product; homogenizing and emulsifying the liposome crude product in a high-pressure homogenizer to obtain a liposome solution; weighing 25g of trehalose, and dissolving in the liposome solution; adding water for injection to a constant volume of 100mL, and adjusting the pH value to 6.0 by using sodium hydroxide and hydrochloric acid; filtering with 0.22 μm filter membrane, packaging, freeze drying, sealing, and capping.

2.1.2c preparation of triptolide palmitate (TP-PA) liposome

Weighing 0.3g of triptolide palmitate, 3g of hydrogenated phospholipid (HSPC), 0.3g of PEG distearoyl phosphatidyl ethanolamine (DSPE-PEG2000) and 0.3g of cholesterol, dissolving in a proper amount of chloroform, performing vacuum rotary evaporation at 45 ℃ to remove the solvent to obtain a lipid film, and hydrating with 75mL of injection water to obtain a liposome crude product; homogenizing and emulsifying the liposome crude product in a high-pressure homogenizer to obtain a liposome solution; weighing 30g of sucrose, and dissolving in the liposome solution; adding water for injection to a constant volume of 100mL, and adjusting the pH value to 8.0 by using sodium hydroxide and citric acid; filtering with 0.22 μm filter membrane, packaging, freeze drying, sealing, and capping.

2.1.2d preparation of triptolide palmitate (TP-PA) liposome

Weighing 0.2g of triptolide palmitate, 2g of yolk phospholipid (PC-98T), 0.2g of PEGylated distearoylethanolamine (DSPE-PEG2000) and 0.2g of cholesterol, dissolving in a proper amount of dichloromethane, performing vacuum rotary evaporation at 45 ℃ to remove the solvent to obtain a lipid film, weighing 25g of sucrose, and dissolving in 80mL of water for injection to obtain a liposome crude product; homogenizing and emulsifying the liposome crude product in a high-pressure homogenizer to obtain a liposome solution; weighing 25g of sucrose, and dissolving in the liposome solution; adding water for injection to a constant volume of 100mL, and adjusting the pH value to 6.5 by using sodium hydroxide and hydrochloric acid; filtering with 0.22 μm filter membrane, packaging, freeze drying, sealing, and capping.

2.1.3a preparation of Tripterygium Wilfordii myristate (TP-MA) liposome

Weighing 0.2g of triptolide myristate, 2g of egg yolk phospholipid (EPCS), 0.2g of PEGylated distearoyl phosphatidyl ethanolamine (DSPE-PEG2000) and 0.2g of cholesterol, dissolving in a proper amount of dichloromethane, performing vacuum rotary evaporation at 45 ℃ to remove the solvent to obtain a lipid film, and hydrating with 80mL of water for injection to obtain a liposome crude product; homogenizing and emulsifying the liposome crude product in a high-pressure homogenizer to obtain a liposome solution; weighing 25g of trehalose, and dissolving in the liposome solution; adding water for injection to a constant volume of 100mL, and adjusting the pH value to 6.0 by using sodium hydroxide and hydrochloric acid; filtering with 0.22 μm filter membrane, packaging, freeze drying, sealing, and capping.

2.1.3b preparation of Tripterygium Wilfordii myristate (TP-MA) liposomes

Weighing 0.3g of triptolide myristate, 3g of hydrogenated phospholipid (HSPC), 0.3g of PEG distearoyl phosphatidyl ethanolamine (DSPE-PEG2000) and 0.3g of cholesterol, dissolving in a proper amount of chloroform, performing vacuum rotary evaporation at 45 ℃ to remove the solvent to obtain a lipid film, and hydrating with 75mL of injection water to obtain a liposome crude product; homogenizing and emulsifying the liposome crude product in a high-pressure homogenizer to obtain a liposome solution; weighing 30g of sucrose, and dissolving in the liposome solution; adding water for injection to a constant volume of 100mL, and adjusting the pH value to 8.0 by using sodium hydroxide and citric acid; filtering with 0.22 μm filter membrane, packaging, freeze drying, sealing, and capping.

2.1.4a preparation of Tripterygium wilfordii laurate (TP-LA) liposome

Weighing 0.5g of triptolide laurate, 5g of yolk phospholipid (E80), 0.7g of PEG distearoyl phosphatidyl ethanolamine (DSPE-PEG5000) and 0.5g of cholesterol, adding 4g of propylene glycol, and heating and dissolving at 75 deg.C to obtain an organic phase; slowly injecting the organic phase into 80mL of 75 ℃ water for injection, and uniformly stirring while injecting to obtain a liposome crude product; putting the liposome crude product into an extruder, and sequentially extruding through extrusion films with the aperture of 1.0 mu m, 0.6 mu m, 0.4 mu m, 0.2 mu m, 0.1 mu m and 0.05 mu m to obtain a liposome solution; removing propylene glycol from the liposome by ultrafiltration; weighing 25g of lactose, and dissolving in the liposome solution; adding water for injection to a constant volume of 100mL, and adjusting the pH to 7.0 by using hydrochloric acid and phosphate; filtering with 0.22 μm filter membrane, packaging, freeze drying, sealing, and capping.

2.1.4b preparation of Tripterygium wilfordii laurate (TP-LA) liposome

Weighing 0.2g of triptolide laurate, 4g of soybean phospholipid (PL-100M), 0.4g of PEG dimyristoyl phosphatidylethanolamine (DMPE-PEG3500) and 0.4g of cholesterol, dissolving in chloroform, performing vacuum rotary evaporation at 45 ℃ to remove the solvent to obtain a lipid film, and hydrating with 80mL of water for injection to obtain a liposome crude product; putting the liposome crude product into an extruder, and sequentially extruding through extrusion films with the aperture of 0.2 mu m, 0.1 mu m and 0.05 mu m to obtain a liposome solution; weighing 25g of sucrose, and dissolving in the liposome solution; adding water for injection to a constant volume of 100mL, and adjusting the pH value to 7.0 by using phosphate; filtering with 0.22 μm filter membrane, packaging, freeze drying, sealing, and capping.

2.1.5a preparation of Tripterygium wilfordii Decoate (TP-DA) liposomes

Weighing 0.7g of triptolide caprate, 7g of soybean phospholipid (S100), 1g of PEGylated distearoyl phosphatidyl ethanolamine (DSPE-PEG2000) and 1g of cholesterol, adding 10g of absolute ethyl alcohol, and heating and dissolving at 30 ℃ to obtain an organic phase; slowly injecting the organic phase into 75mL of 30 ℃ water for injection, and uniformly stirring while injecting to obtain a liposome crude product; putting the liposome crude product into an extruder, and sequentially extruding through extrusion films with the aperture of 0.2 mu m, 0.1 mu m and 0.05 mu m to obtain a liposome solution; evaporating to remove ethanol from liposome with scraper type film; weighing 30g of mannitol, and dissolving in the liposome solution; adding water for injection to a constant volume of 100mL, and adjusting the pH to 6.0 by using acetic acid and phosphate; filtering with 0.22 μm filter membrane, packaging, freeze drying, sealing, and capping.

2.1.5b preparation of Liposome of triptolide caprate (TP-DA)

Weighing 0.2g triptolide caprate, 2g yolk phospholipid (EPCS), 0.2g PEG distearoyl phosphatidyl ethanolamine (DSPE-PEG8000) and 0.2g cholesterol, adding 8g tert-butyl alcohol, and heating and dissolving at 50 deg.C to obtain organic phase; slowly injecting the organic phase into 80mL of 50 ℃ water for injection, and uniformly stirring while injecting to obtain a liposome crude product; homogenizing and emulsifying the liposome crude product in a high-pressure homogenizer to obtain a liposome solution; evaporating to remove tert-butyl alcohol from liposome by scraper film; weighing 20g of trehalose, and dissolving in the liposome solution; adding water for injection to a constant volume of 100mL, and adjusting the pH value to 6.0 by using sodium hydroxide and hydrochloric acid; filtering with 0.22 μm filter membrane, packaging, freeze drying, sealing, and capping.

2.1.6a preparation of Tripterygium wilfordii A caprylate (TP-CA) liposome

Weighing 0.05g of triptolide caprylate, 0.5g of yolk phospholipid (EPCS) and 0.4g of PEG dipalmitoyl phosphatidylethanolamine (DPPE-PEG2000), dissolving in a proper amount of dichloromethane, performing vacuum rotary evaporation at 50 ℃ to remove a solvent to obtain a lipid film, and hydrating with 90mL of water for injection to obtain a liposome crude product; putting the liposome crude product into an extruder, and sequentially extruding through extrusion films with the aperture of 0.4 mu m, 0.2 mu m, 0.1 mu m and 0.05 mu m to obtain a liposome solution; weighing 4g of maltose, and dissolving in the liposome solution; adding water for injection to a constant volume of 100mL, and adjusting the pH value to 6.0 by using sodium hydroxide and hydrochloric acid; filtering with 0.22 μm filter membrane, packaging, freeze drying, sealing, and capping.

2.1.6b preparation of Tripterygium wilfordii A caprylate (TP-CA) liposome

Weighing 0.1g of triptolide caprylate, 1g of soybean phospholipid (DOPC), 1g of PEGylated distearoyl phosphatidyl ethanolamine (DSPE-PEG1500) and 0.1g of cholesterol, adding 3g of tert-butyl alcohol, and heating to dissolve at 25 deg.C to obtain an organic phase; slowly injecting the organic phase into 95mL of 25 ℃ water for injection, and uniformly stirring while injecting to obtain a liposome crude product; putting the liposome crude product into an extruder, and sequentially extruding through extrusion films with the aperture of 0.2 mu m, 0.1 mu m and 0.05 mu m to obtain a liposome solution; removing tert-butyl alcohol in the liposome by ultrafiltration; weighing 10g of lactose, and dissolving in the liposome solution; adding water for injection to a constant volume of 100mL, and adjusting the pH value to 4.0 by using sodium hydroxide and hydrochloric acid; filtering with 0.22 μm filter membrane, packaging, freeze drying, sealing, and capping.

2.1.6c preparation of Tripterygium wilfordii A caprylate (TP-CA) liposome

Weighing 0.4g of triptolide caprylate, 4g of yolk phospholipid (EPCS), 0.4g of PEG distearoyl phosphatidyl ethanolamine (DSPE-PEG2000) and 0.4g of cholesterol, dissolving in a proper amount of absolute ethyl alcohol, performing vacuum rotary evaporation at 45 ℃ to remove the solvent to obtain a lipid film, and hydrating with 85mL of water for injection to obtain a liposome crude product; homogenizing and emulsifying the liposome crude product in a high-pressure homogenizer to obtain a liposome solution; weighing 15g of trehalose, and dissolving in the liposome solution; adding water for injection to a constant volume of 100mL, and adjusting the pH value to 7.0 by using sodium hydroxide and hydrochloric acid; filtering with 0.22 μm filter membrane, packaging, freeze drying, sealing, and capping.

2.1.7a preparation of Tripterygium wilfordii Hexate (TP-HA) liposome

Weighing 1g of triptolide caproate, 10g of yolk phospholipid (PC-98T), 0.5g of PEGylated distearoyl phosphatidyl ethanolamine (DSPE-PEG2000) and 0.5g of cholesterol, dissolving in a proper amount of dichloromethane, performing vacuum rotary evaporation at 45 ℃ to remove the solvent to obtain a lipid film, weighing 30g of sucrose, and dissolving in 80mL of water for injection to obtain a liposome crude product; homogenizing and emulsifying the liposome crude product in a high-pressure homogenizer to obtain a liposome solution; weighing 30g of trehalose, and dissolving in the liposome solution; adding water for injection to a constant volume of 100mL, and adjusting the pH value to 8.0 by using sodium hydroxide and hydrochloric acid; filtering with 0.22 μm filter membrane, packaging, freeze drying, sealing, and capping.

2.1.7b preparation of Tripterygium Wilfordii Hexate (TP-HA) liposomes

Weighing 0.2g of triptolide caproate, 2g of soybean phospholipid (SPC-3) and 0.2g of PEGylated distearoyl phosphatidyl ethanolamine (DPPE-PEG2000), dissolving in a proper amount of absolute ethanol, performing vacuum rotary evaporation at 50 ℃ to remove a solvent to obtain a lipid film, and hydrating with 75mL of water for injection to obtain a liposome crude product; putting the liposome crude product into an extruder, and sequentially extruding through extrusion films with the aperture of 2.0 mu m, 1.0 mu m, 0.4 mu m, 0.1 mu m and 0.05 mu m to obtain a liposome solution; weighing 40g of sucrose, and dissolving in the liposome solution; adding water for injection to a constant volume of 100mL, and adjusting the pH to 5.0 by using acetic acid and phosphate; filtering with 0.22 μm filter membrane, packaging, freeze drying, sealing, and capping.

2.1.8A preparation method of triptolide butyrate (TP-BA) liposome

Weighing 0.2g of triptolide butyrate, 3g of hydrogenated phospholipid (HSPC), 0.3g of PEG distearoyl phosphatidyl ethanolamine (DSPE-PEG6000) and 0.3g of cholesterol, adding 6g of propylene glycol, and heating and dissolving at 55 ℃ to obtain an organic phase; slowly injecting the organic phase into 85mL of 55 ℃ water for injection, and uniformly stirring while injecting to obtain a liposome crude product; homogenizing and emulsifying the liposome crude product in a high-pressure homogenizer, then placing in an extruder, and extruding through an extrusion film with the aperture of 0.05 μm to obtain a liposome solution; removing propylene glycol from the liposome by ultrafiltration; weighing 9g of glucose and 6g of mannitol, and dissolving in the liposome solution; adding water for injection to a constant volume of 100mL, and adjusting the pH value to 10.0 by using sodium hydroxide; filtering with 0.22 μm filter membrane, packaging, freeze drying, sealing, and capping.

2.1.8b preparation of triptolide butyrate (TP-BA) liposome

Weighing 0.2g of triptolide butyrate, 2g of phospholipid (DSPC), 0.2g of PEG distearoyl phosphatidyl ethanolamine (DSPE-PEG2000) and 0.8g of cholesterol, adding 1g of absolute ethyl alcohol, and heating and dissolving at 50 ℃ to obtain an organic phase; slowly injecting the organic phase into 80mL of 50 ℃ water for injection, and uniformly stirring while injecting to obtain a liposome crude product; putting the liposome crude product into an extruder, and sequentially extruding through extrusion films with the aperture of 2.0 mu m, 1.0 mu m, 0.4 mu m and 0.05 mu m to obtain a liposome solution; removing ethanol in the liposome by ultrafiltration; weighing 25g of trehalose, and dissolving in the liposome solution; adding water for injection to a constant volume of 100mL, and adjusting the pH value to 6.0 by using sodium hydroxide and hydrochloric acid; filtering with 0.22 μm filter membrane, packaging, freeze drying, sealing, and capping.

2.2 preparation of different triptolide fatty acid ester polymer micelles

2.2.1a preparation of Tripterygium wilfordii ester stearate (TP-SA) Polymer micelle

Weighing 0.2g of triptolide stearate and 1.2g of polyethylene glycol monomethyl ether-polylactic acid (mPEG-PDLLA), and dissolving in an appropriate amount of acetonitrile; removing the solvent by vacuum rotary evaporation at the temperature of 60 ℃ to form a film; pre-heating to 60 ℃ with 90mL of water for injection, shaking and hydrating; adding 20g trehalose, dissolving, diluting to 100mL volume, filtering with 0.22 μm filter membrane, packaging, freeze drying, sealing, and capping.

2.2.1b preparation of triptolide palmitate (TP-PA) Polymer micelle

Weighing 0.3g of triptolide palmitate and 1.5g of polyethylene glycol monomethyl ether-polylactic acid (mPEG-PDLLA) and dissolving in a proper amount of acetonitrile; removing the solvent by vacuum rotary evaporation at the temperature of 60 ℃ to form a film; pre-heating to 60 ℃ with 90mL of water for injection, shaking and hydrating; adding 20g sucrose, dissolving, diluting to 100mL volume, filtering with 0.22 μm filter membrane, packaging, freeze drying, sealing, and capping.

2.2.2 preparation of Tripterygium Wilfordii myristate (TP-MA) Polymer micelle

Weighing 0.2g of triptolide myristate and 1.4g of polyethylene glycol monomethyl ether-polylactic acid (mPEG-PDLLA), and dissolving in an appropriate amount of acetonitrile; removing the solvent by vacuum rotary evaporation at the temperature of 60 ℃ to form a film; 85mL of water for injection is used for shaking and hydrating after being preheated to 60 ℃; adding 12g glucose and 8g mannitol, dissolving, diluting to 100mL volume, filtering with 0.22 μm filter membrane, packaging, freeze drying, sealing, and capping.

2.2.3 preparation of Tripterygium wilfordii A caprylate (TP-CA) Polymer micelle

Weighing 0.4g of triptolide caprylate and 5g of polyethylene glycol monomethyl ether-polylactic acid (mPEG-PDLLA) and dissolving in a proper amount of acetonitrile; removing the solvent by vacuum rotary evaporation at 55 ℃ to form a film; 85mL of water for injection is used for shaking and hydrating after being preheated to 55 ℃; diluting to 100mL, filtering with 0.22 μm filter membrane, packaging, freeze drying, sealing, and capping.

2.2.4 preparation of Tripterygium wilfordii butyrate ester (TP-BA) polymer micelle

Weighing 0.5g of triptolide butyrate and 2.5g of polyethylene glycol monomethyl ether-polylactic acid (mPEG-PDLLA), and dissolving in a proper amount of acetonitrile; removing the solvent by vacuum rotary evaporation at 50 ℃ to form a film; 85mL of water for injection is used for shaking and hydrating after being preheated to 60 ℃; adding 20g maltose to dissolve, diluting to 100mL, filtering with 0.22 μm filter membrane, packaging, freeze drying, sealing, and capping.

2.3 preparation of triptolide fatty acid ester albumin nanoparticles

2.3.1 preparation of Tripterygium wilfordii methylstearate (TP-SA) Albumin nanoparticles

Weighing 4.5g of human serum albumin, adding 100mL of water for injection, stirring at low temperature to mix, dropping 1mL of chloroform when the temperature is reduced to 2 ℃, and shearing at 0 ℃ at 10000rpm for 5min to obtain a water phase; weighing 0.5g of triptolide stearate, dissolving in 3mL (1: 2) of an absolute ethyl alcohol-chloroform mixed solvent to obtain an organic phase; slowly dripping the organic phase under the condition of high-speed shearing and dispersing the water phase, and shearing at 10000rpm for 5min to obtain primary emulsion; transferring the primary emulsion to a high-pressure homogenizer, and respectively circulating for 2 times at homogenizing pressure of 5000psi, 10000psi, 15000psi and 20000 psi; diluting with 5 times of 2 deg.C water for injection after homogenizing, removing solvent by scraper film evaporation, concentrating to 100 mL; filtering with 0.22 μm filter membrane for sterilization, packaging, freeze drying, sealing, and capping.

2.3.2 preparation of Tripterygium wilfordii palmitate (TP-PA) Albumin nanoparticles

Weighing 4.5g of human serum albumin, adding 100mL of water for injection, stirring at low temperature to mix, dropping 1mL of chloroform when the temperature is reduced to 2 ℃, and shearing at 0 ℃ at 10000rpm for 3min to obtain a water phase; weighing 0.4g of triptolide palmitate, dissolving in 3mL (1: 4) of an absolute ethyl alcohol-chloroform mixed solvent to obtain an organic phase; slowly dripping the organic phase under the condition of high-speed shearing and dispersing the water phase, and shearing at 10000rpm for 5min to obtain primary emulsion; transferring the primary emulsion to a high-pressure homogenizer, and respectively circulating for 2 times at homogenizing pressure of 5000psi, 10000psi, 15000psi and 20000 psi; diluting with 5 times of 2 deg.C water for injection after homogenizing, removing solvent by scraper film evaporation, concentrating to 100 mL; filtering with 0.22 μm filter membrane for sterilization, packaging, freeze drying, sealing, and capping.

2.3.3 preparation of Tripterygium Wilfordii myristate (TP-MA) Albumin nanoparticles

Weighing 4.5g of human serum albumin, adding 100mL of water for injection, stirring at low temperature to mix, dropping 1mL of chloroform when the temperature is reduced to 2 ℃, and shearing at 0 ℃ at 10000rpm for 3min to obtain a water phase; weighing 0.45g of triptolide myristate, dissolving in 2.5mL (1: 4) of absolute ethanol-chloroform mixed solvent to obtain an organic phase; slowly dripping the organic phase under the condition of high-speed shearing and dispersing the water phase, and shearing at 12000rpm for 8min to obtain primary emulsion; transferring the primary emulsion to a high-pressure homogenizer, and respectively circulating for 2 times at homogenizing pressures of 10000psi, 15000psi and 20000 psi; diluting with 5 times of 2 deg.C water for injection after homogenizing, removing solvent by scraper film evaporation, concentrating to 100 mL; filtering with 0.22 μm filter membrane for sterilization, packaging, freeze drying, sealing, and capping.

2.3.4 preparation of Tripterygium wilfordii A caprylate (TP-CA) albumin nanoparticles

Weighing 4.5g of human serum albumin, adding 100mL of water for injection, stirring at low temperature to mix, dropping 1mL of chloroform when the temperature is reduced to 2 ℃, and shearing at 0 ℃ at 10000rpm for 5min to obtain a water phase; weighing 0.5g of triptolide caprylate, dissolving in 4mL (1: 3) of an absolute ethyl alcohol-chloroform mixed solvent to obtain an organic phase; slowly dripping the organic phase under the condition of high-speed shearing and dispersing the water phase, and shearing at 10000rpm for 5min to obtain primary emulsion; transferring the primary emulsion to a high pressure homogenizer, and circulating for 2 times at a homogenizing pressure of 15000psi and 4 times at 20000 psi; diluting with 5 times of 2 deg.C water for injection after homogenizing, removing solvent by scraper film evaporation, concentrating to 100 mL; filtering with 0.22 μm filter membrane for sterilization, packaging, freeze drying, sealing, and capping.

2.3.5 preparation of Tripterygium wilfordii butyrate (TP-BA) albumin nanoparticles

Weighing 4.5g of human serum albumin, adding 100mL of water for injection, stirring at low temperature to mix, dropping 1mL of chloroform when the temperature is reduced to 2 ℃, and shearing at 0 ℃ at 10000rpm for 3min to obtain a water phase; weighing 0.4g of triptolide butyrate, dissolving in 2mL (1: 5) of an absolute ethyl alcohol-chloroform mixed solvent to obtain an organic phase; slowly dripping the organic phase under the condition of high-speed shearing and dispersing the water phase, and shearing at 10000rpm for 3min to obtain primary emulsion; transferring the primary emulsion to a high-pressure homogenizer, and respectively circulating for 2 times at homogenizing pressure of 5000psi, 10000psi, 15000psi and 20000 psi; diluting with 5 times of 2 deg.C water for injection after homogenizing, removing solvent by scraper film evaporation, concentrating to 100 mL; filtering with 0.22 μm filter membrane for sterilization, packaging, freeze drying, sealing, and capping.

2.4 preparation of fatty acid triptolide ester emulsion

2.4.1 preparation of triptolide stearate (TP-SA) fat emulsion

Weighing 5g of medium chain triglyceride for injection and 0.2g of triptolide stearate, heating in water bath to 70 ℃, stirring and dissolving to obtain an oil phase; weighing 3g of phospholipid (PL-100M) and adding into 90mL of water for injection, and shearing to disperse to obtain a water phase; mixing the oil phase and the water phase at 70 deg.C, emulsifying for 5min with a shear emulsifying machine to obtain primary emulsion, and diluting to 100mL with injectable water; homogenizing and emulsifying the primary emulsion in a high pressure homogenizer, adjusting pH to 5.0 with hydrochloric acid, filtering with 0.22 μm filter membrane, packaging, sealing, and sterilizing at 121 deg.C for 15 min.

2.4.2 preparation of triptolide palmitate (TP-PA) fat emulsion

Weighing 2.5g of medium chain triglyceride for injection, 2.5g of soybean oil and 0.2g of triptolide palmitate, heating in water bath to 60 deg.C, stirring for dissolving to obtain oil phase; weighing 3g of phospholipid (PL-100M) and adding into 90mL of water for injection, and shearing to disperse to obtain a water phase; mixing the oil phase and the water phase at 60 deg.C, emulsifying for 5min with a shear emulsifying machine to obtain primary emulsion, and diluting to 100mL with injectable water; homogenizing and emulsifying the primary emulsion in a high pressure homogenizer, adjusting pH to 7.0 with phosphate, filtering with 0.22 μm filter membrane, packaging, sealing, and sterilizing at 100 deg.C for 30 min.

2.4.3 preparation of triptolide myristate (TP-MA) fat emulsion

Weighing 6g of soybean oil for injection and 0.3g of triptolide myristate, heating in water bath to 70 ℃, stirring and dissolving to obtain an oil phase; weighing 3g of phospholipid (PL-100M) and adding into 90mL of water for injection, and shearing to disperse to obtain a water phase; mixing the oil phase and the water phase at 70 ℃, emulsifying for 8min by using a shearing emulsifying machine to obtain primary emulsion, and metering the volume to 100mL by using water for injection; homogenizing and emulsifying the primary emulsion in a high pressure homogenizer, adjusting pH to 6.5 with phosphate, filtering with 0.45 μm and 0.22 μm filter membrane respectively for sterilization, packaging, and sealing.

2.4.4 preparation of triptolide caprylate (TP-CA) fat emulsion

Weighing 4g of medium chain triglyceride for injection, 4g of soybean oil and 0.4g of triptolide caprylate, heating in water bath to 80 ℃, stirring and dissolving to obtain an oil phase; weighing 4g of phospholipid (PC-98T) and 1g of glycerol, adding into 85mL of water for injection, and shearing to disperse to obtain a water phase; mixing the oil phase and the water phase at 80 deg.C, emulsifying for 5min with a shear emulsifying machine to obtain primary emulsion, and diluting to 100mL with injectable water; homogenizing and emulsifying the primary emulsion in a high pressure homogenizer, adjusting pH to 6.0 with hydrochloric acid, filtering with 0.45 μm filter membrane, packaging, sealing, and sterilizing at 121 deg.C for 15 min.

2.4.5 preparation of triptolide butyrate (TP-BA) fat emulsion

Weighing 4g of medium chain triglyceride for injection, 0.01g of oleic acid and 0.2g of triptolide butyrate, heating in water bath to 80 ℃, stirring for dissolving to obtain an oil phase; weighing 3g of phospholipid (SPC-3) and adding into 90mL of water for injection, and shearing to disperse to obtain a water phase; mixing the oil phase and the water phase at 65 deg.C, emulsifying for 6min with a shear emulsifying machine to obtain primary emulsion, and diluting to 100mL with injectable water; homogenizing and emulsifying the primary emulsion in a high pressure homogenizer, adjusting pH to 5.0 with lactic acid, filtering with 0.22 μm filter membrane, packaging, sealing, and sterilizing at 121 deg.C for 15 min.

The results show that: the triptolide fatty acid ester can be effectively prepared into liposome, polymer micelle, albumin nanoparticle, fat emulsion and other nanometer preparations, and has good pharmacy.

Example 3: measurement of liposome particle size and encapsulation efficiency

Using the lipid ratio of the drugs of example 2.1.1b as the unified prescription to prepare different triptolide fatty acid esters and triptolide liposomes, and measuring the particle size and encapsulation efficiency of the liposomes. The results are shown in Table 1 below,

TABLE 1 measurement results of liposome particle size and encapsulation efficiency

Experimental results show that the triptolide fatty acid ester can be prepared into liposome under the same prescription conditions, the particle size distribution is 100-130 nm, and the entrapment rate is more than 90%; in addition, due to drug precipitation in the triptolide preparation process, liposome cannot be effectively prepared, and the entrapment rate is less than 50%. Meanwhile, comprehensive comparison shows that the triptolide fatty acid ester obtained by modifying the saturated fatty acid with the carbon number more than or equal to 8 has smaller particle size and more uniform distribution, so that the saturated fatty acid with the carbon number of 2n (n is 4-9) is preferred.

Example 4: cytotoxicity test

Based on the optimization of the embodiment 3, the lipid ratio of the medicine given in the embodiment 2.1.1b is used as a unified prescription to prepare triptolide fatty acid ester liposomes modified by saturated fatty acids with different chain lengths and 2n carbon atoms (n is 4-9), and the TP (DMSO) solution is used as a control to perform the anti-tumor toxicity research of MCF-7 and A549 cells, and the specific operation steps are as follows:

1) cells in good growth state were diluted to an appropriate concentration with a certain medium, and then seeded into 96-well culture plates at a density of 5000 cells per well, 100. mu.L per well. The plates were pre-incubated in an incubator for 24h (37 ℃, 5% CO)₂)。

2) To the plates were added 10. mu.L each of different concentrations of drug (equimolar dosing, 3 duplicate wells per concentration). The plates were incubated in an incubator for 48 h.

3) To each well was added 10. mu.L of CCK-8 medium. The plates were incubated in an incubator for 4 h.

4) Absorbance at 450nm was measured with a microplate reader. And (4) taking an average value and calculating the inhibition rate.

Calculating the formula: the inhibition rate is [ (Ac-As)/(Ac-Ab) ]. times.100%

As: experimental well (cell-containing medium, CCK-8, drug)

Ac: control wells (Medium with cells and CCK-8, no drug)

Ab: blank well (Medium without cells and drugs, CCK-8)

As a result: the cytostatic IC was calculated separately for each drug concentration₅₀The values are shown in Table 2.

TABLE 2 cytotoxic Effect IC₅₀Value result

The experimental result shows that compared with TP, the liposome prepared from triptolide fatty acid ester has lower cytotoxicity. Embodied in the aspect of inhibiting cell growth, the drug concentration IC required by the triptolide fatty acid ester liposome for reaching half inhibition₅₀The triptolide fatty acid lipid liposome modified by saturated fatty acid with carbon number more than or equal to 14 has higher IC than other triptolide fatty acid lipid liposomes₅₀In view of the concentration, a saturated fatty acid having 2n carbon atoms (n is 7 to 9) is more preferable.

Example 5: pharmacokinetics study in rats

Based on the optimization of the embodiment 4, C is prepared by taking the lipid ratio of the medicines given in the embodiment 2.1.1b as a unified prescription_2n(n is 7-9) saturated fatty acid modified triptolide fatty acid ester (TP-MA, TP-PA and TP-SA) liposome with different chain lengths in the range, and performing pharmacokinetic study on rats in vivo by taking a TP (DMSO) solution as a reference. The rat is administrated intravenously at equivalent TP3mg/kg, the blood sampling time points are set to be 2, 5, 10, 15, 30, 45, 60, 90, 120 and 180min, and each group of the drug comprises 5 SD rats (plus).

Sample processing and analysis methods: adding 4 times of methanol into plasma to precipitate protein, centrifuging at 12000r/min for 10min, collecting supernatant, removing methanol, extracting the residual liquid with 400 μ L methyl tert-butyl ether twice, and centrifuging at 12000r/min for 10 min. Organic extraction phases are collected, concentrated and volatilized to be dry, 100 mu L of methanol is added for redissolution, supernatant liquid is centrifugally taken, the liquid phase content is measured, the blood concentration is calculated, pharmacokinetic fitting is carried out by adopting DAS2.0 software, and the half-life period result is shown in table 3.

TABLE 3 drug half-life in vivo results

The experimental result shows that compared with TP, the lipidosome prepared by triptolide fatty acid ester can effectively prolong the half-life period of the drug action and obviously improve the pharmacokinetic property.

While the preferred embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that the invention is not limited thereto, and that various changes and modifications may be made without departing from the spirit of the invention, and the scope of the appended claims is to be accorded the full scope of the invention.

Claims (15)

1. A triptolide fatty acid ester has a chemical structure shown in formula (I):

in the formula (I), R represents C2 n straight chain alkyl acyl, and n is 7-9.

2. The method for preparing the triptolide fatty acid ester of claim 1, wherein the triptolide fatty acid ester is obtained by esterification of triptolide and saturated fatty acid, and the synthetic route is as follows:

wherein ROH is a saturated fatty acid, R is as defined in claim 1;

the method comprises the following specific steps:

(a) dissolving saturated fatty acid, a condensing agent and a catalyst in an organic solvent, and cooling to 0-10 ℃ under stirring to obtain a mixed solution;

(b) dissolving triptolide in an organic solvent, dripping the triptolide into the mixed solution, reacting for 15-45 minutes at 0-10 ℃, continuing to react at room temperature, and separating to obtain triptolide fatty acid ester after the esterification reaction is finished.

3. The method of claim 2,

the condensing agent is paranitrobenzoyl chloride, N, N ' -dicyclohexylcarbodiimide, N, N ' -diisopropylcarbodiimide, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, 2-chloro-1-methylpyridinium iodide, benzotriazol-1-yl-oxy-tripyrrolidinyl phosphorus hexafluorophosphate, 2- (7-azo-benzotriazol) -N, N, N ', one or more than two of N' -tetramethylurea hexafluorophosphate, O- (5-chlorobenzotriazol-1-yl) -di (dimethylamino) carbenium hexafluorophosphate, O- (benzotriazol-1-yloxy) -dipiperidino carbenium hexafluorophosphate, N-hydroxysuccinimide and N-hydroxythiosuccinimide;

the catalyst is one or more than two of 1-hydroxybenzotriazole, 4-dimethylaminopyridine, triethylamine and N, N-diisopropylethylamine;

the organic solvent is one or more than two of dichloromethane, trichloromethane, N-dimethylformamide and N, N-dimethylacetamide.

4. The method of claim 2, wherein the molar ratio of saturated fatty acids to triptolide is 1.2-4: 1, and the molar ratio of condensing agent to triptolide is 1.2-4: 1.

5. The use of the triptolide fatty acid ester of claim 1 in the preparation of an anti-tumor or anti-inflammatory drug.

6. A nano-preparation of triptolide fatty acid ester according to claim 1, which is liposome, polymer micelle, albumin nanoparticle or fat emulsion.

7. The nano-preparation according to claim 6, wherein the liposome is prepared from the following components in percentage by weight and volume:

8. the nano-formulation according to claim 7, wherein the liposome is prepared from the following components in weight-to-volume ratio:

9. the NanoPrepration according to claim 7 or 8,

the phospholipid is one or more of yolk phospholipid, soybean phospholipid, hydrogenated yolk phospholipid, hydrogenated soybean phospholipid, dipalmitoyl phosphatidylcholine, dimyristoyl phosphatidylcholine, distearoyl phosphatidylcholine, phosphatidylethanolamine, distearoyl phosphatidylglycerol, dipalmitoyl phosphatidylglycerol, dioleoyl phosphatidylcholine, cardiolipin and sphingomyelin;

the PEG phospholipid is one or more than two of PEG distearoyl phosphatidyl ethanolamine, PEG dipalmitoyl phosphatidyl ethanolamine and PEG dimyristoyl phosphatidyl ethanolamine, and the average PEG molecular weight of the PEG phospholipid is 1000-8000;

the freeze-drying protective agent is one or more than two of trehalose, sucrose, maltose, lactose, mannitol and glucose;

the pH regulator is one or more of sodium hydroxide, sodium acetate, acetic acid, phosphate, carbonate, hydrochloric acid, citric acid, etc.

10. The NanoPrepration according to claim 7 or 8,

the phospholipid is egg yolk phospholipid or soybean phospholipid;

the PEGylated phospholipid is PEGylated distearoyl phosphatidyl ethanolamine with the average molecular weight of 2000;

the freeze-drying protective agent is trehalose or sucrose;

the pH regulator is sodium hydroxide, hydrochloric acid or citric acid.

11. A process for the preparation of liposomes according to claim 7 or 8, said process comprising the steps of:

(A) preparing a liposome crude product, namely weighing triptolide fatty acid ester, phospholipid, PEGylated phospholipid and cholesterol according to a formula, adding a proper amount of organic solvent I, heating and dissolving at 25-75 ℃ to obtain an organic phase, slowly injecting the organic phase into a proper amount of water for injection at 25-75 ℃, and uniformly stirring while injecting to obtain the liposome crude product;

(B) preparing a liposome solution, namely placing the crude liposome in a high-pressure homogenizer for homogenizing and emulsifying, or placing the crude liposome in an extruder for extruding sequentially through extrusion films with different apertures, or placing the crude liposome in the high-pressure homogenizer for homogenizing and then extruding to obtain the liposome solution;

(C) weighing a freeze-drying protective agent with a formula amount, and dissolving the freeze-drying protective agent in the liposome solution; adding water for injection to a constant volume, and adjusting the pH value to a specified value; filtering with 0.22 μm filter membrane, packaging, freeze drying, sealing, and capping to obtain the final product;

alternatively, the first and second electrodes may be,

replacing the step (A) with: (A') weighing triptolide fatty acid ester, phospholipid, PEG phospholipid and cholesterol according to the formula, dissolving in an organic solvent II, performing vacuum rotary evaporation at 40-60 ℃ to remove the solvent to obtain a lipid film, and hydrating with a proper amount of water for injection to obtain a crude liposome.

12. The method for preparing liposomes according to claim 11, wherein the organic solvent I is one or more selected from the group consisting of absolute ethanol, propylene glycol and t-butanol; the organic solvent I is retained in the liposome, or is removed by ultrafiltration or a scraper film evaporator after the crude liposome is emulsified.

13. The method for preparing the liposome according to claim 11, wherein the organic solvent II is one or more of methylene chloride, chloroform and absolute ethyl alcohol.

14. The method of claim 11, wherein the lyoprotectant is added externally to the liposome solution or internally to the aqueous phase.

15. The method for preparing liposomes according to claim 11, wherein the pore size of the extruded membrane in the step (B) is one or more selected from the group consisting of 2.0 μm, 1.0 μm, 0.8 μm, 0.6 μm, 0.4 μm, 0.2 μm, 0.1 μm and 0.05 μm, and the pore size is sequentially changed from large to small during extrusion.