WO2018161507A1 - 低毒雷公藤新多苷、其制备方法及其应用 - Google Patents

低毒雷公藤新多苷、其制备方法及其应用 Download PDF

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WO2018161507A1
WO2018161507A1 PCT/CN2017/099589 CN2017099589W WO2018161507A1 WO 2018161507 A1 WO2018161507 A1 WO 2018161507A1 CN 2017099589 W CN2017099589 W CN 2017099589W WO 2018161507 A1 WO2018161507 A1 WO 2018161507A1
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triptolide
tripterygium
tripterygium wilfordii
ethanol
glycosides
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PCT/CN2017/099589
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English (en)
French (fr)
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刘博�
毛炜
刘旭生
徐鹏
韩晓东
周文
徐方方
刘敬功
李援朝
杨祎琦
邓金宝
吴利兰
吴云山
陈伟英
田瑞敏
陆金健
张玉琴
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广东省中医院
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Priority to US16/492,725 priority Critical patent/US10925913B2/en
Priority to JP2019528662A priority patent/JP6872614B2/ja
Publication of WO2018161507A1 publication Critical patent/WO2018161507A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/37—Celastraceae (Staff-tree or Bittersweet family), e.g. tripterygium or spindletree
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
    • A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/50—Methods involving additional extraction steps
    • A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying

Definitions

  • the invention relates to the field of medicine, in particular to a low-toxic tripterygium wilfordii glycosides, to a preparation method of the tripterygium wilfordii glycosides, and a medical use thereof.
  • Syndrome purpura and lupus nephritis, lupus erythematosus, subacute and chronic severe hepatitis, chronic active hepatitis; can also be used for allergic cutaneous vasculitis, dermatitis and eczema, as well as psoriatic arthritis, leprosy reaction , thiophene disease, recurrent aphthous ulcer, ankylosing spondylitis, etc.
  • Tripterygium glycosides Since the introduction of tripterygium glycosides into clinical use, related adverse reactions have occurred from time to time, involving the blood system, reproductive system, urinary system and digestive system. The reason is that the main active ingredients in Tripterygium wilfordii, such as triptolide, triptolide and other diterpenoids, tripterygium and triptolide, tripterygium Alkali, triptolide and other alkaloids have greater toxicity; on the other hand, the standard extract or ratio extract of Tripterygium wilfordii on the market often only uses triptolide as a single evaluation index, resulting in A species of Tripterygium wilfordii glycosides is mixed with fish.
  • triptolide triol As early as 1991, Ma Pengcheng and others first isolated and identified the structure of triptolide triol. However, the content of triptolide triol in tripterygium was extremely low, and only 720 mg of triptolide was obtained in 20 kg of tripterygium. In the same year, Zheng Jiarun et al. also studied the alleviation experiment and serum hemolytic hormone assay of triptolide triol on mouse ear swelling experimental model, and the results showed that triptolide triol has only superficial anti-inflammatory activity, and There is no immunosuppressive activity. Therefore, triptolide triol has not been popularized in the medical field.
  • Another object of the present invention is to provide a process for the preparation of the above-mentioned Tripterygium wilfordii.
  • Another object of the present invention is to provide a pharmaceutical use of the above-mentioned Tripterygium wilfordii.
  • the present invention provides a low-toxic Tripterygium wilfordii polyglycoside prepared by the following method:
  • tripterygium glycosides (retaining samples for detecting the content of triptolide) are placed in an oil bath at 90 to 120 ° C.
  • the hydrolysis reaction is carried out by heating under reflux for 18 to 96 hours (preferably 30 hours).
  • the solvent is removed under reduced pressure (the solvent can also be removed by extraction), reconstituted with absolute ethanol, centrifuged, and the insoluble matter is removed, and the solution is further added with absolute ethanol to determine the content of triptolide. The amount, and the reduction in triptolide.
  • step (2) adding triptolide-triethanol-anhydrous ethanol solution to the reaction product of step (1), mixing and then drying the solvent to obtain a low-toxic tripterygium wilfordii; the addition amount of triptolide is tripterygium
  • the amount of alpha reduction is 0 to 20 times the number of moles (mol).
  • tripterygium glycosides are prepared by the following method:
  • tripterygium glycosides retaining sample for detecting the content of triptolide
  • a tripterygium glycosides retaining sample for detecting the content of triptolide
  • the hydrolysis reaction is carried out by heating under reflux for 18 to 96 hours (preferably 30 hours).
  • the solvent is removed under reduced pressure (the solvent can also be removed by extraction), reconstituted with absolute ethanol, centrifuged, and the insoluble matter is removed, and the solution is further added with anhydrous ethanol to determine the content of triptolide. The amount, and the reduction in triptolide.
  • step b) adding triptolide-triethanol-anhydrous ethanol solution to the reaction product of step b), mixing and then drying the solvent to obtain a low-toxic tripterygium wilfordii; triptolide triol is added to triptolide The number of moles (mol) is reduced by 0 to 20 times.
  • the present invention provides the above-mentioned low-toxic Tripterygium wilfordii glycosides in the preparation of a treatment for nephrotic syndrome, primary glomerular nephropathy, purpuric and lupus nephritis, rheumatoid Inflammation, lupus erythematosus, subacute and chronic severe hepatitis, chronic active hepatitis; application in allergic cutaneous vasculitis, dermatitis, eczema, psoriatic arthritis, ankylosing spondylitis.
  • triptolide and lactone are the diterpenoids of Tripterygium wilfordii.
  • the chemical structures are similar and can be transformed into each other under certain conditions.
  • the triptolide by converting the triptolide into a triptolide triol at a pH of 3.0 to 5.0 by a refluxing reaction between sodium dihydrogen phosphate and phosphoric acid, the tripterygium glycosides are treated as above.
  • the chemical processing of the conditions reduces the content of triptolide in the polyglycoside, thereby reducing the toxicity of the preparation.
  • the efficacy of the formulation is correspondingly reduced.
  • Tripterygium wilfordii polyglycoside can effectively alleviate renal pathological damage and proteinuria in nephrotic syndrome, improve the body's inflammation level, have obvious therapeutic effect on nephrotic syndrome, and have sustained curative effect.
  • the Tripterygium wilfordii glycosides provided by the present invention can also have therapeutic effects on other immune-related diseases, especially inflammation-mediated immune-related diseases, and have attenuating effects.
  • Figure 1 is a chromatogram of tripterygium glycosides and tripterygium wilfordii I, II, III.
  • Figure 3 is a graph showing the toxicity results of Tripterygium wilfordii in each cell line.
  • Figure 4 is a graph showing the results of inhibition of normal T/B lymphocyte proliferation by triptolide monomer.
  • Figure 5 is a graph showing the results of inhibition of T/B lymphocyte proliferation induced by triptolide triol monomer.
  • Figure 6 is a graph showing the results of inhibition of inflammatory factors in Raw264.7 cells by triptolide triol monomer.
  • Figure 9 is a graph showing the results of inhibition of podocyte apoptosis by triptolide triol monomer.
  • Figure 11 is a graph showing the results of laser confocal microscopy of podocyte marker protein expression.
  • Figure 12 is a graph showing the effect of each experimental group on the urine protein of NS rats.
  • Figure 13 is a graph showing the effect of each experimental group on albumin of NS rats.
  • Figure 14 is a graph showing the effect of each experimental group on total cholesterol and triglyceride in NS rats.
  • Figure 15 shows the results of pathological light microscopy.
  • Ultra Performance Liquid Chromatograph Waters Acquity, Waters, USA), one ten thousandth balance (AL104-IC, METTLER TOLEDO, Switzerland), one hundred thousandth of a balance (AB135-S, METTLER TOLEDO, Switzerland), acidity meter (PHS-3C, Shanghai Jingke Instrument Co., Ltd.), magnetic stirrer (HS7, IKA, Germany), rotary film evaporator (RV 10 basic V, IKA, Germany), desktop microcentrifuge (Microfuge16, BACKMAN, USA) , full-wavelength microplate reader (1510, Thermo Fisher Scientific (China) Co., Ltd.), Thermo Scientific pipetting gun, 1-10 ⁇ L, 10-100 ⁇ L, 100-1000 ⁇ L (Thermo Fisher Scientfic.inc), vertical protein electrophoresis Transfer membrane system (Bio-Rad), automatic immunoassay analyzer (Abbott), Lycra slicer (Leica).
  • the prepared tripterygium glycosides were sampled and tested for the content of triptolide, and compared with the commercially available tripterygium glycosides by their fingerprints and characteristic components. After comparison, the obtained tripterygium glycosides and mainstream products on the market, Shanghai Fudan Fuhua Pharmaceutical Co., Ltd., Hunan Qianjin Xieli Pharmaceutical Co., Ltd., China Resources Sanjiu Pharmaceutical Co., Ltd., Tripterygium wilfordii, Hunan Synergy Pharmaceutical Co., Ltd. The fingerprints of the produced tripterygium glycosides are quite similar.
  • HPLC detection conditions column: BEH Shield RP18 (2.1 ⁇ 100 mm, 1.7 ⁇ m); mobile phase: acetonitrile-water system; gradient elution, elution procedure see Table 1; flow rate: 0.3 mL / min; detection wavelength: 220 nm; Column temperature: 35 ° C; injection volume: 3 ⁇ L; results are shown in Figure 1, Figure 2.
  • the control product is a mixture of triptolide and triptolide triol. It can be seen from Fig. 1 and Fig. 2 that the triptolide in Tripterygium wilfordii I, II and III is less than that in Tripterygium wilfordii; Tripterygium wilfordii I and Tripterygium wilfordii in Tripterygium wilfordii The content of ester triol is extremely small, and the content of triptolide triol in Tripterygium wilfordii II and III is relatively high.
  • triptolide and triptolide triol as standard, quantitative analysis by HPLC can obtain triptolide and triptolide Content, as shown in Table 2
  • HK-2, HL-7702, GC1, GC2, TM4 are all purchased from ATCC; HK-2: 37°C high glucose DMEM+10% FBS+1% double antibody; HL-7702, GC1, GC2, TM4 are both 37°C DMEM+10%FBS+1% double antibody; finally cultured in an incubator containing 5% CO 2 , cultured cells to 80% adherent growth, cells in logarithmic growth phase, after 0.25% trypsin digestion Adjust the appropriate cell density to a 96-well plate. After the cell monolayer is attached, switch to a synchronized medium and synchronize for 12 hours.
  • the left side is an enlarged view of the IC 50 value in the 0-200 segment
  • the right side is an enlarged view of the IC 50 value in the 0-1 segment (the vertical axis is truncated by two magnifications due to the large difference in activity).
  • the toxicity of the Tripterygium wilfordii polyglycoside group in each cell line is significantly reduced, and the ratio of each compound is about 1000 times lower than that of A.
  • triptolide triol: triptolide is 10:1 (close to the ratio of tripterygium wilfordii II), the toxicity of the mixed monomer is reduced by nearly 100 times, and the toxicity is significantly reduced.
  • mice were sacrificed by cervical dislocation, soaked in 75% ethanol, and the spleen of the mice was taken out in a clean bench.
  • the spleen was washed 3 times in DPBS, fully moistened, and 200 gauze was placed on a 35 mm dish, and the piston of a 10 mL syringe was placed.
  • Uniformly grind and grind in one direction wet with DPBS while grinding, centrifuge the collected lymphocyte suspension at 1500 rpm for 3 min, add 7 mL of red blood cell lysate to each spleen, centrifuge at room temperature for 30 min, centrifuge at 1500 rpm for 3 min, discard the supernatant, and then use DPBS.
  • T lymphocyte specific value-inducing inducer Con A model group (5ug/mL, corresponding model group) and B lymphocyte specific value-inducing inducer LPS model group (5ug/mL, corresponding model group) were added 200 ⁇ L;
  • Con A administration group was first added T lymphocyte specific value-inducing inducer Con A (10ug/mL, corresponding model group) 100 ⁇ L, and then add different concentrations of triptolide triol (100, 200, 300, 400ug / mL) 100 ⁇ L;
  • LPS administration group first added B lymphocyte specific 100 ⁇ L of LPS (10ug/mL, corresponding model group), and add 100 ⁇ L of different concentrations of triptolide (100, 200, 300, 400ug/mL); each group at 37 ° C, 5%
  • the cells were cultured in a CO 2 incubator, and the cell
  • triptolide triol has the effect of inhibiting the proliferation of normal T and B lymphocytes in vitro, and its inhibitory effect is dose-dependent, and the cell activity of each administration group is significantly lower than that of the control group and the model group, p ⁇ 0.05; 50ug/mL triptolide triol and 200ug/mL triptolide triol had significant difference in cell activity inhibition p ⁇ 0.05.
  • Fig. 5 It can be seen from Fig. 5 that after adding the T cell-specific inducer ConA and the B cell-specific inducer Lps, respectively, the treatment is carried out with different concentrations of triptolide triol, and the results are shown in Fig. 5, and the cell activity of the administration group is shown. They were all higher than the blank control group, but they were lower than the model group. Therefore, it was found that triptolide did have a low immunosuppressive effect, and the inhibition of T lymphocytes was significantly higher than that of B cells. 0.05. Cellular inhibition alone was not dose dependent.
  • the other groups used LPS (1 ug/mL) 1 mL as a modeling agent to induce the cell inflammatory model, and the drug treatment was carried out: the model group plus 1 mL of the medium and the drug-administered group (Legong)
  • the vine lactone triol 50, 100, 150, 200 ⁇ g / mL dose group) were added 1 mL of the corresponding concentration of the drug solution, while the blank control group was directly added 2 mL of the medium; Western blot, qPCR, ELISA, etc. were used to detect the inflammation of different treatment groups.
  • TNF- ⁇ is mainly secreted by mesangial cells, epithelial cells, or mononuclear macrophages on inflamed glomeruli, which not only stimulates mesangial cell division and proliferation, but also stimulates the system.
  • Membrane cells secrete a variety of inflammatory cytokines, aggravate the proliferation of mesangial cells, and the cytokine TNF- ⁇ has an important proinflammatory effect.
  • triptolide has improved cytokine secretion in a mouse model of macrophage inflammatory injury induced by LPS.
  • NF- ⁇ B P65 regulates the expression of inflammatory cytokine genes such as TNF- ⁇ , IL-6, and IL-10, thereby affecting the body's inflammatory response and regulating the proliferation and growth of T and B cells.
  • Differentiation plays an important role in humoral and cellular immunity. It can be seen from the figure that triptolide triol can effectively reduce the effects of the inflammatory factors TNF- ⁇ and IL-10. At high concentrations, the expression of the anti-inflammatory factor IL-10 is increased. This suggests that the drug can effectively reduce the transcription of inflammatory factors and has some immunomodulatory activity.
  • the mouse immortal podocyte line MPC5 was donated by the Department of Nephrology, Guangdong Institution of Traditional Chinese Medicine.
  • the treatment of MCP5 was as follows: Western blot, IFC, qPCR, ELISA, etc. were used to detect the expression levels of inflammation-related proteins, genes and cytokines in different treatment groups. It is a temperature sensitive cell, which is increased under the condition of 33°C RPMI-1640+10%FBS+1% double antibody+10U/mL ⁇ -IFN, and 37°C RPMI-1640+10%FBS+1% double-antibody culture differentiation 10 - 14 days spare.
  • the treatment of MCP5 was as follows: except for the blank control group, the other groups were treated with ADM (0.8 ⁇ g/mL) 1 mL as a modeling agent to induce apoptosis model, and the drug treatment was carried out: the model group was added with 1 mL of medium and the drug-administered group ( Triptolide triol 50, 100, 150, 200 ⁇ g / mL dose group) respectively added 1mL of the corresponding concentration of the drug solution, while the blank control group directly added 2mL of the medium; Western blot, IFC, qPCR, ELISA and other methods to detect different treatments The expression levels of inflammation-related proteins, genes, and cytokines.
  • ADM 0.8 ⁇ g/mL
  • the drug treatment was carried out: the model group was added with 1 mL of medium and the drug-administered group ( Triptolide triol 50, 100, 150, 200 ⁇ g / mL dose group) respectively added 1mL of the corresponding concentration
  • podocyte membrane protein Podocin and apoptosis-related gene Bax was detected by flow cytometry using Annexin V and Propidium iodide (PI) labeled with FITC, and the cells of early apoptosis were The late cells and dead cells are distinguished, and the results are shown in Fig. 9, Fig. 10, and Fig. 11.
  • mice 100 male SD rats were healthy. After 7 days of feeding, 8 rats were randomly selected as blank control group. The remaining rats were injected with doxorubicin in 5.0 mg/kg rats under non-anesthetic conditions to induce adriamycin nephropathy. Animal model of the syndrome.
  • Rattan polyglycoside III group After 3 weeks of doxorubicin injection, 100 rats were subjected to 24-hour urine protein excretion measurement. Successfully modeled rats were determined by comparison with a blank group (injection of the same amount of normal saline). Rats with 24-hour urinary protein excretion between 70-180 mg were randomly divided into model group, tripterygium glycosides group, tripterygium wilfordii glycosides group I, tripterygium wilfordii II group and Leigong. Rattan polyglycoside III group. The content ratio results are the same as in Table 2.
  • Preparation of the drug Take 0.4g of tripterygium glycosides, first add 5mL of absolute ethanol to dissolve the drug, then slowly add the absolute ethanol solution to the sodium carboxymethyl cellulose solution and dilute to 423mL, while adding Shake well in the hour hand direction and mix by ultrasound.
  • the drug preparation method of Tripterygium wilfordii I, II and III was the same as that of Tripterygium wilfordii, and the concentration was 0.95g/L.
  • tripterygium glycosides group was given tripterygium glycosides (0.95g/L); Tripterygium wilfordii I, II, II, In the group III (0.95 g/L), tripterygium wilfordii I, II and III were administered separately for 8 weeks.
  • the perfused dose of each rat was 1 mL / (100 g rat)
  • Animal condition Each group of rats was weighed twice a week, and the mental state, body hair, edema, and activity of the rats were observed.
  • rat serum was taken by abdominal aorta blood sampling, and serum total protein, albumin, urea nitrogen, creatinine, total cholesterol, and serum were determined by automatic biochemical analyzer.
  • Blood biochemical indicators such as triglycerides, the results are shown in Table 5, Figure 13, and Figure 14.
  • Triglyceride levels Compared with the blank group, the triglyceride level in the model group was significantly increased (P ⁇ 0.01); compared with the model group, the polyglycoside group, the new polyglycoside II, III group Total cholesterol levels were significantly lower (P ⁇ 0.05 or P ⁇ 0.01).
  • NS rats are impaired by the barrier function of the glomeruli, causing a large amount of albumin to leak into the urine, forming a large amount of proteinuria. Therefore, an important indicator for the treatment of NS disease It is to reduce the amount of urinary protein excretion in NS rats. When glomerular sclerosis is severely present, urinary protein excretion may also be significantly reduced, resulting in false positive results. Therefore, in order to judge the effect of drugs on NS diseases, in addition to observing the excretion of urinary protein, it should be combined with the results of serum creatinine, urea nitrogen levels and renal pathological examination results.
  • Tripterygium wilfordii and Tripterygium wilfordii have significant therapeutic effects on NS rats, and the two drugs have similar effects, but Tripterygium wilfordii II acts faster.
  • the new glucosides of Tripterygium wilfordii obtained in this experiment have achieved the goal of maintaining efficacy in the new process of attenuating and maintaining efficacy.
  • the novel polyglycosides of Tripterygium wilfordii Hook the novel polyglycosides of Tripterygium wilfordii Hook.
  • nephrotic syndrome primary glomerular nephropathy, purpuric and lupus nephritis, rheumatoid arthritis, lupus erythematosus, subacute and chronic severe Hepatitis, chronic active hepatitis; the application of allergic cutaneous vasculitis, dermatitis, eczema, psoriatic arthritis, ankylosing spondylitis provides the basis and confirms its feasibility.

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Abstract

一种低毒的雷公藤新多苷，由雷公藤多苷经化学炮制并添加雷公藤内酯三醇得到。该低毒雷公藤新多苷能有效缓解肾病综合征的肾脏病理损伤及蛋白尿产生，改善机体炎症水平，对肾病综合征具有明显疗效。

Description

低毒雷公藤新多苷、其制备方法及其应用 技术领域
本发明涉及医药领域，具体来说，是涉及一种低毒的雷公藤新多苷，还涉及该雷公藤新多苷的制备方法，以及其医药用途。
背景技术
雷公藤多苷(Tripterygium wilfordii Hook.f.)是从卫矛科植物雷公藤的根、皮、叶中提取的一组混合苷，其主要化学成分包括二萜内酯、生物碱、三萜等。药理研究证明，雷公藤具有抗炎、免疫抑制或免疫调节、抗肿瘤等作用，是目前国内外热点研究的免疫调节药，可用于治疗类风湿性关节炎、原发性肾小球肾病、肾病综合征、紫瘢性及狼疮性肾炎、红斑狼疮、亚急性及慢性重症肝炎、慢性活动性肝炎；亦可用于过敏性皮肤脉管炎、皮炎和湿疹，以及银屑病性关节炎、麻风反应、白噻氏病、复发性口疮、强直性脊柱炎等。
雷公藤多苷投入临床使用以来，相关的不良反应还是时有发生，累及系统涉及血液系统、生殖系统、泌尿系统和消化系统等。究其原因，一方面是雷公藤多苷中的主要有效成分如雷公藤甲素、雷公藤内酯酮等二萜类成分，雷公藤红素、雷公藤内酯醇等三萜类成分，雷公藤晋碱、雷公藤次碱等生物碱类成分，均具有较大的毒性；另一方面，市场上雷公藤多苷标准提取物或比例提取物往往仅以雷公藤甲素为单一评价指标，导致各种雷公藤多苷提取物鱼目混杂。
早在1991年马鹏程等人首次分离和鉴定了雷公藤内酯三醇结构，但是，雷公藤内酯三醇在雷公藤属植物中的含量极低，20kg雷公藤仅可以获得720mg雷公藤内酯三醇。同年，郑家润等人同时研究了雷公藤内酯三醇对小鼠耳肿胀实验模型的缓解实验和血清血容素测定实验，其结果表明，雷公藤内酯三醇仅具浅表的抗炎活性，而并无免疫抑制活性。因此，雷公藤内酯三醇一直未能在医药领域得到推广应用。
发明内容
本发明的目的是提供一种全新的、低毒雷公藤多苷。
本发明的另外一个目的是提供上述雷公藤新多苷的制备方法。
本发明的另外一个目的是提供上述雷公藤新多苷在制药上的应用。
根据本发明的一个方面，本发明提供了一种低毒雷公藤新多苷，由下述方法制备得到：
(1)在pH3.0～5.0的由磷酸二氢钠与磷酸配置的缓冲溶液中，将雷公藤多苷(留样，用于检测雷公藤甲素的含量)置于90～120℃油浴中加热回流进行水解反应18～96h(优选30h)。
反应完成后，减压移除溶剂(也可以通过萃取的方式移除溶剂)，加无水乙醇复溶，离心，去掉不溶物，溶液再加无水乙醇定容，取样检测雷公藤甲素的量，并得出雷公藤甲素的减少量。
(2)往步骤(1)的反应产物中添加雷公藤内酯三醇-无水乙醇溶液，混合后挥干溶剂，得到低毒雷公藤新多苷；雷公藤内酯三醇的添加量为雷公藤甲素减少量摩尔(mol)数的0～20倍。
步骤(1)和(2)中的无水乙醇，也可以选用其他无毒的有机溶剂来代替，如：异丙醇。
上述的雷公藤多苷通过下述方法制备得到：
以雷公藤的干燥根茎为原料，以质量分数为95％的乙醇(乙醇与雷公藤干燥根茎的体积质量比为4～16：1)回流提取3～12h，提取液减压浓缩得粗浸膏，粗浸膏用中性氧化铝吸附，挥干乙醇，再用三氯甲烷提取，减压浓缩得到雷公藤多苷。
据本发明的另一个方面，本发明提供了上述低毒雷公藤新多苷的制备方法，其特征在于，包括以下步骤：
a)以雷公藤的干燥根茎为原料，以质量分数为95％的乙醇(乙醇与雷公藤干燥根茎的体积质量比4～16倍)回流提取3～12h，提取液减压浓缩得粗浸膏，粗浸膏用中性氧化铝吸附，挥干乙醇，再用三氯甲烷提取，减压浓缩得到雷公藤多苷；
b)在pH3.0～5.0的由磷酸二氢钠与磷酸配置的缓冲溶液中，将雷公藤多苷(留样，用于检测雷公藤甲素的含量)置于90～120℃油浴中加热回流进行水解反应18～96h(优选30h)。
反应完成后，减压移除溶剂(也可以通过萃取的方式移除溶剂)，加无水乙醇复溶，离心，去掉不溶物，溶液再加无水乙醇定容，取样计算雷公藤甲素的量，并得出雷公藤甲素的减少量。
c)往步骤b)的反应产物中添加雷公藤内酯三醇-无水乙醇溶液，混合后挥干溶剂，得到低毒雷公藤新多苷；雷公藤内酯三醇的添加量为雷公藤甲素减少量摩尔(mol)数的0～20倍。
步骤(1)和(2)中的无水乙醇，也可以选用其他无毒的有机溶剂来代替，如：异丙醇。
据本发明的另一个方面，本发明提供了上述低毒雷公藤新多苷在制备治疗肾病综合征、原发性肾小球肾病、紫瘢性及狼疮性肾炎、类风湿性关 节炎、红斑狼疮、亚急性及慢性重症肝炎、慢性活动性肝炎；过敏性皮肤脉管炎、皮炎、湿疹、银屑病性关节炎、强直性脊柱炎药物中的应用。
雷公藤甲素与内酯三醇都是雷公藤的二萜类成分，二者化学结构相似，在一定条件可相互转化。本发明中，通过在pH3.0～5.0由磷酸二氢钠与磷酸配置的缓冲溶液中回流反应，将雷公藤甲素转化为雷公藤内酯三醇的基础上，将雷公藤多苷整体进行同上条件的化学炮制，使多苷中的雷公藤甲素含量降低，从而降低制剂的毒性。但在降低毒性的同时，制剂的药效也相应减弱。为此，本发明补加一定量低毒有效(治疗窗大)的雷公藤内酯三醇，以增加制剂的药效。本发明首次通过减少雷公藤甲素含量，并相应增加雷公藤内酯三醇含量保证了相当的抗炎活性，同时减少了因免疫抑制带来的毒性，为其在其它药物制剂中的应用及在临床疾病的治疗中提供物质基础。
本发明的低毒雷公藤新多苷，具有如下优点：
1、雷公藤新多苷相比市售雷公藤制剂来说毒性大大降低(p<0.05)；
2、雷公藤新多苷能有效缓解肾病综合征的肾脏病理损伤及蛋白尿的产生，改善机体炎症水平，对肾病综合征具有明显的治疗效果，具有持续的疗效。
3、由于免疫相关疾病具有机理的类似性，本发明提供的雷公藤新多苷也可以对其它免疫相关疾病尤其是炎症介导型的免疫相关疾病起到治疗作用，并有减毒效果。
附图说明
图1为雷公藤多苷及雷公藤新多苷Ⅰ、Ⅱ、Ⅲ的色谱图。
图2为图1增加对照品后的色谱图。
图3为雷公藤新多苷在各细胞系中的毒性结果图。
图4为雷公藤内酯三醇单体抑制正常T/B淋巴细胞增值结果图。
图5为雷公藤内酯三醇单体抑制诱导的T/B淋巴细胞增值结果图。
图6为雷公藤内酯三醇单体抑制Raw264.7细胞炎症因子产生结果图。
图7为雷公藤内酯三醇单体抑制Raw264.7细胞炎症基因产生结果图。
图8为雷公藤内酯三醇单体抑制Raw264.7细胞NF-κB蛋白表达结果图。
图9为雷公藤内酯三醇单体抑制足细胞凋亡结果图。
图10为足细胞标志蛋白podocin及凋亡蛋白Bax蛋白表达结果图。
图11为足细胞标志蛋白表达激光共聚焦结果图。
图12为各实验组对NS大鼠尿蛋白的影响趋势图。
图13为各实验组对NS大鼠白蛋白的影响示意图。
图14为各实验组对NS大鼠总胆固醇、甘油三酯的影响示意图。
图15为病理学光镜检查结果。
具体实施方式
下面通过实施例对本发明作进一步详细的说明。
下述制备例中所用常用试剂，均为市售试剂，所使用的仪器为：
超高效液相色谱仪(Waters Acquity，美国Waters公司)，万分之一天平(AL104-IC，瑞士METTLER TOLEDO公司)，十万分之一天平(AB135-S，瑞士METTLER TOLEDO公司)，酸度计(PHS-3C，上海精科仪器有限公司)，磁力搅拌器(HS7，德国IKA公司)，旋转薄膜蒸发仪(RV 10 basic V，德国IKA公司)，台式微量离心机(Microfuge16，美国BACKMAN公司)，全波长酶标仪(1510，赛默飞世尔科技(中国)有限公司)，Thermo Scientific移液枪，1-10μL、10-100μL、100-1000μL(Thermo Fisher Scientfic.inc)，蛋白垂直电泳转膜系统(Bio-Rad)，全自动免疫分析仪(Abbott)，莱卡切片机(Leica)。
1、雷公藤新多苷的制备
以雷公藤的干燥根茎1500g为原料，以12L质量分数为95％的乙醇回流提取10h，提取液减压浓缩得粗浸膏，粗浸膏用中性氧化铝吸附，挥干乙醇，再用三氯甲烷5L提取，减压浓缩得到雷公藤多苷。
制得的雷公藤多苷，取样检测雷公藤甲素的含量，并同通过其指纹图谱和特征性成分与市售的雷公藤多苷进行比较。经比较，所得的雷公藤多苷和市场上的主流产品上海复旦复华药业有限公司、湖南千金协力药业有限公司、华润三九药业有限公司的雷公藤制剂、湖南协力药业有限公司生产的雷公藤多苷片指纹图谱相当。
取磷酸二氢钠，加水溶解，在室温下，加体积分数为1％的磷酸溶液调pH为4.0，即为pH4.0的磷酸二氢钠/磷酸缓冲溶液。
在pH4.0的磷酸二氢钠/磷酸缓冲溶液中，取部分制得的雷公藤多苷置于90～120℃油浴中加热回流进行水解反应30h，减压移除溶剂后，加无水乙醇复溶，离心，去掉不溶物，加无水乙醇定容至1L。取样10mL，计算雷公藤甲素的减少量。再取300mL直接挥干溶剂得到雷公藤新多苷Ⅰ；另取300mL，添加10倍(mol倍数)雷公藤甲素减少量的雷公藤内酯三醇-乙醇溶液，挥干溶剂后得到雷公藤新多苷Ⅱ；另取300mL，添加20倍(mol倍数)雷公藤甲素减少量的雷公藤内酯三醇-乙醇溶液，挥干溶剂后得到雷公藤新多苷Ⅲ，于-20℃冰箱保存。通过以上方式获得了雷公藤新多苷Ⅰ、Ⅱ、Ⅲ。
具体操作流程如下图：
Figure PCTCN2017099589-appb-000001
2、雷公藤新多苷Ⅰ、Ⅱ、Ⅲ中雷公藤甲素与内酯三醇的含量测定
HPLC检测条件：色谱柱：BEH Shield RP18(2.1×100mm，1.7μm)；流动相：乙腈-水体系；梯度洗脱，洗脱程序见表1；流速：0.3mL/min；检测波长：220nm；柱温：35℃；进样体积：3μL；结果见图1、图2。
表1
Figure PCTCN2017099589-appb-000002
图2中，对照品为雷公藤甲素和雷公藤内酯三醇的混合品。由图1和图2可见，雷公藤新多苷Ⅰ、Ⅱ、Ⅲ中的雷公藤甲素都比雷公藤多苷中的要少；雷公藤新多苷Ⅰ、雷公藤多苷中的雷公藤内酯三醇，含量极其微小，而雷公藤新多苷Ⅱ、Ⅲ中的雷公藤内酯三醇含量较多。
以雷公藤甲素和雷公藤内酯三醇单体为标准品，用HPLC定量分析可得雷公藤多苷与雷公藤新多苷Ⅰ、Ⅱ、Ⅲ中雷公藤甲素和雷公藤内酯三醇的含量，具体如表2
表2 雷公藤多苷与雷公藤新多苷中标志性成分的含量(n＝3)
Figure PCTCN2017099589-appb-000003
3、细胞实验
3.1HK-2、HL-7702、GC1、GC2、TM4细胞培养及毒性检测
HK-2、HL-7702、GC1、GC2、TM4均购自ATCC；HK-2：37℃高糖DMEM+10％FBS+1％双抗；HL-7702、GC1、GC2、TM4均为37℃DMEM+10％FBS+1％双抗；最终均置于含5％CO2的温箱中培养，培养细胞至贴壁生长80％，取处于对数生长期的细胞，0.25％胰蛋白酶消化后，调整合适的细胞密度至96孔板中，待细胞单层贴壁后，换为同步化培养基培养，同步化12h。细胞进入静止期后加药处理，分别为:雷公藤多苷(LGT 0、1、5、10、20、30、50、100μg/mL)、雷公藤新多苷Ⅱ(LGT-2，0、1、5、10、20、30、50、100μg/mL)、雷公藤新多苷Ⅲ(LGT-4，0、5、10、20、30、50、75、100μg/mL)、LGT-5(0、0.1、2.5、5、10、15、25、50μg/mL)、雷公藤甲素(0、1、5、15、25、40、80、150ng/mL)、雷公藤内酯三醇(0、50、100、200、250、300、400、500μg/mL)，mix(甲素：三醇＝1：10，0、22、88、110、330、660、990、2200ng/mL)，每次给药200μL，给药后继续培养24h，进行MTT检测细胞活性并计算给药24h后的IC50值，其结果见图3。
图3中，左边为IC50值在0-200段的放大图，右边为IC50值在0-1段的放大图(由于活性差别大，纵轴采用两种放大倍数截断)。由图3可知，雷公藤新多苷组在各个细胞系毒性显著降低，各配比平均低于甲素毒性的近1000倍左右。其中雷公藤内酯三醇：雷公藤甲素为10：1(接近雷公藤新多苷Ⅱ的比例)时，混合单体毒性降低近100倍左右，毒性显著降低。
3.2 免疫抑制活性检测：
断颈处死小鼠，浸泡于75％的乙醇中，在超净台中取出小鼠脾脏，将脾脏在DPBS中洗3遍，充分湿润，将200目得纱布至于35mm的皿上，10mL注射器的活塞按一个方向均匀研磨研磨，边研磨边用DPBS湿润，将收集到的淋巴细胞悬液1500rpm离心3min，每个脾脏加入7mL红细胞裂解液，室温作用30min，1500rpm离心3min弃去上清，再用DPBS洗涤3遍，除去多余血清，取4mL的70％Percoll加在15mL离心管的底层，4mL 40％Percoll加在15mL离心管的上层，在水平离心机上800g室温离心20min，吸取40％percoll和70％percoll之间的细胞，用4mL DPBS洗涤3遍；接种至96孔培养板中，加药处理，分别为：空白对照组(只加培养基)、不同浓度的雷公藤内酯三醇(50、100、150、200ug/mL)、T淋巴细胞特异性增值诱导剂Con A(10ug/mL，对应正常抑制组)和B淋巴细胞特异性增值诱导剂LPS(10ug/mL，对应正常抑制组)，各加入200μL；于37℃、5％CO2培养箱培养，24h后MTT方法检测细胞活性和抑制率，结果如图4所示。
特异性抑制实验淋巴细胞前处理操作同上，在淋巴细胞接种至96孔培养板后，进行加药处理，其中空白对照组(只加培养基)、T淋巴细胞特异性增值诱导剂Con A模型组(5ug/mL，对应模型组)和B淋巴细胞特异性增值诱导剂LPS模型组(5ug/mL，对应模型组)各加入200μL；Con A给药组先分别加入T淋巴细胞特异性增值诱导剂Con A(10ug/mL，对应模型组)100μL多份，再分别加入不同浓度的雷公藤内酯三醇(100、200、300、400ug/mL)100μL；LPS给药组先分别加入B淋巴细胞特异性增值诱导剂LPS(10ug/mL，对应模型组)100μL多份，再分别加入不同浓度的雷公藤内酯三醇(100、200、300、400ug/mL)100μL；各组于37℃、5％CO2培养箱培养，24h后MTT方法检测细胞活性和抑制率，结果如图4和图5所示。
由图4可知：雷公藤内酯三醇具有抑制体外正常T、B淋巴细胞增值的作用，并且其抑制作用呈剂量依赖性，各给药组细胞活性显著低于对照组和模型组p<0.05；50ug/mL雷公藤内酯三醇与200ug/mL雷公藤内酯三醇对细胞活性抑制程度具有显著差异p<0.05。
由图5可知：在分别加入T细胞特异性诱导剂ConA和B细胞特异性诱导剂Lps之后，再用不同浓度雷公藤内酯三醇进行处理，所得结果如图5所示，给药组细胞活性均高于空白对照组，但均低于模型组，由此可知雷公藤内酯三醇确实存在较低的免疫抑制作用，其中对T淋巴细胞的抑制作用显著高于对B细胞的抑制作用p<0.05。对单独的细胞抑制并未呈现剂量依赖性。
3.3 抗炎活性检测结果
Raw264.7 37℃、5％CO2RPMI-1640+10％FBS+1％双抗。最终置于培养箱中培养。培养细胞至贴壁生长至80％，取处于对数生长期的细胞，0.25％ 胰蛋白酶消化后，调整合适的细胞密度至6孔板中。待细胞单层贴壁后，换为同步化培养基培养，同步化12h。细胞进入静止期后,除空白对照组外，其它各组以LPS(1ug/mL)1mL为造模剂，诱导细胞炎症模型，同时加药处理：模型组加1mL培养基、给药组(雷公藤内酯三醇50、100、150、200μg/mL剂量组)分别加入1mL相应浓度药液，而空白对照组直接加2mL培养基；分别用Western blot、qPCR、ELISA等方法检测不同处理组的炎症相关蛋白、基因、细胞因子的表达水平、NF-κB转录相关因子的表达及IL-6、IL-10、TNF-α等炎症细胞因子的表达。
如图6所示，体内外研究表明，TNF-ɑ主要是由发炎肾小球上的系膜细胞、上皮细胞，或者单核巨噬细胞分泌，不仅刺激系膜细胞分裂增殖，同时可刺激系膜细胞分泌多种炎性细胞因子，加重系膜细胞的增殖，细胞因子TNF-ɑ具有重要致炎作用。本课题研究结果可知：雷公藤内酯三醇具有改善由LPS诱导的小鼠巨噬细胞炎症损伤模型中细胞因子的分泌。
如图7、8所示，NF-κB P65通过调控TNF-ɑ、IL-6、IL-10等炎症细胞因子基因的表达，进而影响机体的炎症反应，调控T、B细胞的增殖、生长和分化，在体液和细胞免疫中发挥重要的作用。从图中可知：雷公藤内酯三醇可有效降低炎症因子TNF-ɑ和IL-10的作用。在高浓度时可升高抑炎因子IL-10的表达。这提示：药物可有效降低炎性因子的转录，并有一定的免疫调节活性。
4 雷公藤内酯三醇保护足细胞凋亡实验
小鼠永生性足细胞系MPC5由广东省中医院肾病科馈赠。MCP5处理方式如下：分别用Western blot、IFC、qPCR、ELISA等方法检测不同处理组的炎症相关蛋白、基因、细胞因子的表达水平。其为温度敏感性细胞，在33℃RPMI-1640+10％FBS+1％双抗+10U/mLγ-IFN条件下增值，37℃RPMI-1640+10％FBS+1％双抗培养基分化10-14天备用。MCP5处理方式如下：除空白对照组外，其它各组以ADM(0.8μg/mL)1mL为造模剂，诱导细胞凋亡模型，同时加药处理：模型组加1mL培养基、给药组(雷公藤内酯三醇50、100、150、200μg/mL剂量组)分别加入1mL相应浓度药液，而空白对照组直接加2mL培养基；分别用Western blot、IFC、qPCR、ELISA等方法检测不同处理组炎症相关蛋白、基因、细胞因子的表达水平。足细胞裂孔膜蛋白Podocin和凋亡相关基因Bax的表达，利用标记了FITC的Annexin Ⅴ和Propidium iodide(PI)对细胞进行双染并进行流式细胞仪检测，以此将凋亡早期的细胞和晚期的细胞以及死细胞区分开来，结果见图9、图10、图11。
如图9-11所示，足细胞受到损伤后，发生凋亡、自噬、坏死，导致其数目减少，最终产生蛋白尿，影响肾功能。由结果可知全分化的足细胞利用0.8μg/mL的ADM造成凋亡细胞模型，再给予雷公藤内酯三醇处理后，细胞的凋亡情况有所改善，这提示，雷公藤内酯三醇具有保护足细胞损伤的作用，可用于临床足细胞病的治疗。由实验结果可知：雷公藤内酯三醇具有保护足细胞凋亡的作用，但在高浓度时有上升趋势。
5 动物实验
5.1 采用阿霉素(ADR)诱导大鼠肾病综合症疾病动物模型的构建
雄性健康的SD大鼠100只，饲养7天后，随机抽取8只作为空白对照组，余下大鼠在非麻醉条件下一次性尾静脉按5.0mg/kg大鼠注射阿霉素诱导阿霉素肾病综合征动物模型。
分组：在注射阿霉素3周后，对100只大鼠进行24小时尿蛋白排泄量的测定。通过与空白组(注射等量生理盐水)比较，确定建模成功的大鼠。选择24小时尿蛋白排泄量在70-180mg之间的大鼠按尿蛋白排泄量随机平均分成模型组、雷公藤多苷组、雷公藤新多苷Ⅰ组、雷公藤新多苷Ⅱ组及雷公藤新多苷Ⅲ组。含量配比结果同表2
表2雷公藤多苷与雷公藤新多苷中二者的含量(n＝3)
Figure PCTCN2017099589-appb-000004
5.2 给药剂量的选取
药物的制备:取雷公藤多苷0.4g，先加5mL无水乙醇溶解药物，再将无水乙醇溶液缓慢滴加到羧甲基纤维素钠溶液中并定容至423mL，边加边按顺时针方向摇匀，再超声混匀。雷公藤新多苷Ⅰ、Ⅱ、Ⅲ的药物配制方法同雷公藤多苷,浓度均为0.95g/L。
各组于造模成功次日起，每日灌胃，空白组与模型组给予生理盐水；雷公藤多苷组给予雷公藤多苷(0.95g/L)；雷公藤新多苷Ⅰ、Ⅱ、Ⅲ(0.95g/L)组分别给予雷公藤新多苷Ⅰ、Ⅱ、Ⅲ，连续给药8周。每只大鼠的灌胃剂量均为1mL/(100g大鼠)
5.3观察指标的检测
动物情况：各组大鼠每周称重2次，并观察大鼠的精神状态、体毛、浮肿、活动情况等。
尿蛋白含量的测定：各组大鼠于造模前，造模第3周及给药后第2、4、6、8周单独置于代谢笼中，收集24小时尿液，记录尿量，并采用考马斯亮蓝法测定尿蛋白浓度，计算24小时尿蛋白排泄量，结果见表3、表4、图12。
表3给药前各组大鼠24小时尿蛋白的排泄量(mg，n＝8，
Figure PCTCN2017099589-appb-000005
)
Figure PCTCN2017099589-appb-000006
与空白组比较，*P<0.05，**P<0.01
表4 给药后各组大鼠24小时尿蛋白的排泄量(mg，n＝8，
Figure PCTCN2017099589-appb-000007
)
Figure PCTCN2017099589-appb-000008
与空白组比较，＊P<0.05，＊ ＊P<0.01；与模型组比较，#P<0.05，# #P<0.01
由实验结果(表3、表4、图12)可知：在整个试验过程中，空白组大鼠精神状态良好，体毛有光泽，活动、饮食正常，尿量正常。大鼠造模2周后，造模大鼠均出现饮水减少，尿量减少，部分大鼠还有烂尾现象。给药后，模型组大鼠始终躁动不安，体毛凌乱，尿量减少，消瘦。与模型组相比，给药各组上述表现有所改善。
由实验结果可知：经SPSS 20.0统计软件分析，各时间点的数据都符合正态分布。造模前，各组大鼠尿蛋白排泄量无明显差别。造模第3周，各造模组大鼠尿蛋白排泄量与空白组相比，都具有显著性的差异(P<0.01)，提示造模成功。在治疗过程中，各治疗组大鼠尿蛋白排泄量均有不同程度的 减少。给药结束后，与模型组相比，雷公藤多苷组与新多苷Ⅱ组尿蛋白排泄量有显著性减少(P<0.05)。而雷公藤多苷组与新多苷Ⅱ组之间尿蛋白排泄量无显著性差别。
血液生化指标的检测：在实验第8周结束时，采用腹主动脉取血的方式取大鼠血清，用全自动生化分析仪测定血清中总蛋白、白蛋白、尿素氮、肌酐、总胆固醇、甘油三酯等血液生化指标，结果见表5、图13、图14。
表5 各组大鼠血液生化指标的测定结果(n＝8，
Figure PCTCN2017099589-appb-000009
)
Figure PCTCN2017099589-appb-000010
与空白组比较，*P<0.05，**P<0.01；与模型组比较，#P<0.05，# #P<0.01
由实验结果可知：经SPSS 20.0统计软件分析，各组数据均符合正态分布。实验结果显示：1)各组大鼠总蛋白水平无显著性差异。模型组大鼠白蛋白水平显著降低，与空白组相比，具有显著性差异(P<0.01)；与模型组相比，新多苷Ⅱ组白蛋白含量显著提高，具有显著性差异(P<0.01)。2)各组大鼠血清尿素氮、肌酐水平均无显著性差异。3)总胆固醇水平：模型组大鼠总胆固醇水平显著升高，与空白组相比，具有显著性差异(P<0.01)；与模型组相比，新多苷Ⅱ组总胆固醇水平显著降低，具有显著性差异(P<0.05)。4)甘油三酯水平：与空白组相比，模型组大鼠甘油三酯水平有显著性的升高(P<0.01)；与模型组相比，多苷组、新多苷Ⅱ、Ⅲ组总胆固醇水平均有显著性降低(P<0.05或P<0.01)。
组织病理学检查：腹主动脉取血后，摘取大鼠的肾脏，用10％的福尔马林溶液固定，石蜡包埋并切片，HE染色，进行病理学检查。结果见图15。由结果可知：光镜下显示：空白组肾小球、肾小管及肾间质未见明显异常。模型组可见系膜细胞和基质增生，肾间质纤维化，部分肾小管上皮细胞可见颗粒变性，有的肾小球出现萎缩。各治疗组的肾脏病理情况较模型组有所改善。
综上所述：NS大鼠由于肾小球的屏障功能受损，致使大量白蛋白渗漏到尿液中，形成大量蛋白尿。因此，对于NS疾病的治疗，一个重要的指标 就是降低NS大鼠的尿蛋白排泄量。而当肾小球损伤严重出现肾小球硬化的时候，尿蛋白的排泄量也可能显著降低，从而出现假阳性的结果。因此，判断药物对NS疾病的疗效，除了观察尿蛋白的排泄量之外，还应结合血肌酐、尿素氮的水平以及肾脏的病理学检查结果进行综合评价。本实验结果表明：雷公藤新多苷Ⅱ能显著降低NS大鼠24h尿蛋白的排泄量，且肌酐、尿素氮水平并无异常，新多苷Ⅱ组大鼠肾脏病理切片中也未见明显的肾小球硬化等病理变化。由此可知，雷公藤新多苷Ⅱ对NS大鼠具有显著地治疗作用。药效学结果表明：给大鼠一次性尾静脉注射5mg/kg的阿霉素可成功的建立NS动物模型。从药物的干预情况可以看出，雷公藤多苷与雷公藤新多苷Ⅱ对NS大鼠具有显著地治疗作用，二者药效相似，但雷公藤新多苷Ⅱ起效更快。本实验中获得的雷公藤新多苷Ⅱ达到了减毒持效新工艺研究中持效的目的。由此，为本发明的雷公藤的新多苷在制备治疗肾病综合征、原发性肾小球肾病、紫瘢性及狼疮性肾炎、类风湿性关节炎、红斑狼疮、亚急性及慢性重症肝炎、慢性活动性肝炎；过敏性皮肤脉管炎、皮炎、湿疹、银屑病性关节炎、强直性脊柱炎药物中的应用提供了基础，并证实了其可行性。
以上所述的仅是本发明的一些实施方式。对于本领域的普通技术人员来说，在不脱离本发明创造构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。

Claims (3)

  1. 低毒雷公藤新多苷，其特征在于，由下述方法制备得到：
    (1)在pH3.0～5.0的由磷酸二氢钠与磷酸配置的缓冲溶液中，将雷公藤多苷置于90～120℃油浴中加热回流进行水解反应18～96h；
    反应前后，均取样检测雷公藤甲素的量，并得出雷公藤甲素的减少量；
    (2)往步骤(1)的反应产物中添加雷公藤内酯三醇-无水乙醇溶液，混合后挥干溶剂，得到低毒雷公藤新多苷；雷公藤内酯三醇的添加量为雷公藤甲素减少量摩尔数的0～20倍；
    上述的雷公藤多苷通过下述方法制备得到：
    以雷公藤的干燥根茎为原料，以质量分数为95％的乙醇回流提取3～12h，提取液减压浓缩得粗浸膏，粗浸膏用中性氧化铝吸附，挥干乙醇，再用三氯甲烷提取，减压浓缩得到雷公藤多苷；乙醇与雷公藤干燥根茎的体积质量比为4～16：1。
  2. 根据权利要求1所述的低毒雷公藤新多苷的制备方法，其特征在于，包括以下步骤：
    a)以雷公藤的干燥根茎为原料，以质量分数为95％的乙醇回流提取3～12h，提取液减压浓缩得粗浸膏，粗浸膏用中性氧化铝吸附，挥干乙醇，再用三氯甲烷提取，减压浓缩得到雷公藤多苷；乙醇与雷公藤干燥根茎的体积质量比为4～16：1；
    b)在pH3.0～5.0的由磷酸二氢钠与磷酸配置的缓冲溶液中，将雷公藤多苷置于90～120℃油浴中加热回流进行水解反应18～96h；
    反应前后，均取样检测雷公藤甲素的量，并得出雷公藤甲素的减少量；
    c)往步骤b)的反应产物中添加雷公藤内酯三醇-无水乙醇溶液，混合后挥干溶剂，得到低毒雷公藤新多苷；雷公藤内酯三醇的添加量为雷公藤甲素减少量摩尔数的0～20倍。
  3. 低毒雷公藤新多苷在制备治疗肾病综合征、原发性肾小球肾病、紫瘢性及狼疮性肾炎、类风湿性关节炎、红斑狼疮、亚急性及慢性重症肝炎、慢性活动性肝炎；过敏性皮肤脉管炎、皮炎、湿疹、银屑病性关节炎、强直性脊柱炎药物中的应用。
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