Icariside compound, preparation method thereof, and application thereof

Abstract

An icariside compound as shown in Formula I wherein the compound is a natural chemical component in the traditional Chinese herbal epimedium or a chemically modified or a totally synthetic product based on the natural component. The compound can be used for preparing pharmaceuticals, health care products, cosmetic and skin care products and the like for improvement of immunity in a human body.

Claims

1. A method for treating ascites cancer or tuberculosis comprising administering to a subject in need thereof a therapeutically effective amount of icariside compounds having the following formula: ##STR00002## wherein: R.sub.1 is OH, R.sub.2 is OH, and R.sub.3 is OCH3: wherein the icariside compounds improve subject immunity by promoting subject cells to produce γ-interference.

2. A method for treating ascites cancer or tuberculosis comprising administering to a subject in need thereof a therapeutically effective amount of icariside compounds having the following formula: ##STR00003## wherein: R.sub.1 is OH, R.sub.2 is OH, R.sub.3 is OH; wherein the icariside compounds improve subject immunity by promoting subject cells to produce γ-interference.

3. A method for treating ascites cancer or tuberculosis comprising administering to a subject in need thereof a therapeutically effective amount of icariside compounds having the following formula: ##STR00004## wherein: R.sub.1 is OCH.sub.3, R.sub.2 is OH, R.sub.3 is OCH.sub.3; or; R.sub.1 is OH, R.sub.2 is OCH.sub.3, R.sub.3 is OCH.sub.3; wherein the icariside compounds improve subject immunity by promoting subject cells to produce γ-interference.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows the reaction of enzymatic conversion of icariin to icariside I in Example 1.

(2) FIG. 2 shows the reaction of enzymatic conversion of epimedoside A to icariside C in Example 2.

(3) FIG. 3 shows chemical and enzymatic conversion of icariin to icariside compound (Y-5XS-1) in Example 3.

(4) FIG. 4 shows chemical conversion of icariside I to another icariside compound (Y-3XS-1) in Example 4.

(5) FIG. 5 shows the pharmaceutical icariside I (PBMCs subjected to anti-CD3 treatment) stimulating and promoting the generation of IFN-γ with promotion percentage in Example 6.

(6) FIG. 6 shows the pharmaceutical icariside I (subjected to the combined treatment of anti-CD3 and anti-CD28) stimulating and promoting the generation of IFN-γ with promotion percentage in Example 6.

(7) FIG. 7 shows the pharmaceutical icariside I promoting IFN-γ production in a mixed lymphocytes culture in Example 7.

(8) FIG. 8 shows the effect of different icariside I compounds on levels of IFN-γ produced by mixed lymphocytes [icariside I compounds: (1) icariin; (2) icariside I; (3) icariside C; (4) Y-3XS-1; (5) Y-5XS-1; (6) Y-4′ XS-1] in Example 8.

(9) FIG. 9 shows the effect of the pharmaceutical icariside I on level of IFN-γ produced by PBMCs and PFMCs in Example 9.

(10) FIG. 10 shows the effect of icariside I on xenograft tumor in nude mice (growth curve) in Example 11.

(11) FIG. 11 shows the effect of icariside I on xenograft tumor in nude mice (final change of tumor weight) in Example 11

DETAILED DESCRIPTION

(12) The following examples further illustrate the embodiments of the present invention.

Example 1

(13) Preparation of Icariside I

(14) (1) Preparation of Icariside I by Separation from Epimedium.

(15) According to the method reported by Li Wenkui, et al. (Herbal, 1995, 26 (9), 453-455), icariside I was separated and purified with a content of more than 95% from the whole plant of epimedium. The chemical structure of the product was characterized by 1H NMR, Mass spectroscopy, and other instrumentations.

(16) (2) Preparation of Icariside I by Enzymatic Conversion of Icariin as Raw Material.

(17) A. Preparation of icariin: According to the method reported by Li Wenkui, et al. (Herbal, 1995, 26 (9), 453-455), icariin was separated and purified with a content of more than 95% from the whole plant of epimedium. The chemical structure of the product was characterized by .sup.1H NMR, Mass spectroscopy, and other instrumentations.

(18) B. Preparation of icariside I: 2 g of icariin (purity 98%) was dissolved in a pH6.8 phosphate buffer, and then 1 g of immobilized rhamnosidase was added. The mixture was stirred at 60° C. for 24 hours for reaction. The mixture was filtered to collect solids after crystallization. Then the solid were recrystallized with methanol to obtain 1.3 g icariside I (purity 95%). The chemical structure of the product was characterized by .sup.1H NMR, Mass spectroscopy, and other instrumentations. The specific reaction formula was shown in FIG. 1.

Example 2

(19) Preparation of Icariside C

(20) (1) Preparation of Icariside C by Separation from Epimedium.

(21) According to the method reported by Li Wenkui, et al. (Herbal, 1995, 26 (9), 453-455), icariside C with a content of more than 95% was obtained after separation and purification from the whole plant of epimedium. The chemical structure of the product was characterized by .sup.1H NMR, Mass spectroscopy, and other instrumentations.

(22) (2) Preparation of Icariside I from Epimedoside a as Raw Material by Enzymatic Conversion.

(23) A. Preparation of epimedoside A: epimedoside A is one of the components with a higher content in epimedium. According to the method reported by Xu Sui Xu et al. (Herbs, 1981, 14, 24-26), epimedoside A with a content of more than 95% was obtained by separation and purification from the whole plant of epimedium. The chemical structure of the product was characterized by .sup.1H NMR, Mass spectroscopy, and other instrumentations.

(24) B. Preparation of icariside C: Icariside C was obtained by removal of 3-O-rhamnosyl group from epimedoside A with rhamnosidase by the method in Examples 1 (2) using epimedoside A as raw material. The chemical structure of the product was characterized by .sup.1H NMR, Mass spectroscopy, and other instrumentations. The specific reaction formula was shown in FIG. 2.

Example 3

(25) Preparation of Icariside Compounds Based on Modification of the R.sub.1 Group

(26) Using icariin as raw material, the OH group of R.sub.1 was selectively transformed into another group by a chemical method (the process does not affect glycosyl groups present in the molecule). Then, by applying the method in Example 1 (2), the R.sub.2 Rha group of the modified icariin was removed with an enzymatic method to obtain the desired icariside compounds.

(27) Preparation Example: 2 ml chloroform was added to 50 mg icariin followed by addition of 10 mg iodomethane (CH.sub.3I) and addition of 10 mg silver oxide (Ag.sub.2O). Then, the solution was stirred at room temperature for 24 hours. After reaction, chloroform was removed by rotary evaporation. The residue was re-dissolved with 95% ethanol and was purified by column chromatography (silica gel column) to obtain the product with R.sub.1 being a methoxy group. Finally, by applying the method in Example 1 (2), the R.sub.2 Rha group in the modified icariin was removed with an enzymatic method. The desired icariside compound (Y-5XS-1) with R.sub.1 being a methoxy group was obtained and the chemical structure of the product was characterized by .sup.1H NMR, Mass spectroscopy, and other instrumentations. The specific reaction formula was shown in FIG. 3.

Example 4

(28) Preparation of Icariside Compounds Based on Modification of the R.sub.2 Group

(29) Using icariside I as raw material, the OH group at position 3 of icariside I (i.e. R.sub.2=0H) was selectively modified to obtain the desired icariside compound.

(30) Preparation Example: 5 ml of ethanol (95%), and 30 mg dimethyl sulfate were added to 100 mg of icariside I, and then 15 mg sodium hydroxide was added. The mixture was stirred at room temperature for 5 hours. After completion of the reaction, 20 ml of water was added to the mixture and then extraction with ether was performed. The separated ether layer was dried with anhydrous sodium sulfate. After removal of ether, the crude product was dissolved in 95% ethanol and then purified by column chromatography (silica gel column) to obtain the desired icariside compound (Y-3XS-1) with R.sub.2 being a methoxy group. The chemical structure of the product was characterized by .sup.1H NMR, Mass spectroscopy, and other instrumentations. A small amount of icariside compound (Y-3XS-1) with R.sub.1 being a methoxy group can also be obtained by this method. The specific reaction formula is shown in FIG. 4.

Example 5

(31) Preparation of Icariside Compounds Based on Modification of the R.sub.3 Group

(32) Using epimedoside A as starting material, the OH group of R.sub.3 was selectively transformed into another group by a chemical method (the process does not affect glycosyl groups present in the molecule). Finally, by applying the method in Example 1 (2), the R.sub.2 Rha group in the modified icariin product was removed with an enzymatic method. The desired icariside compounds were obtained.

(33) Preparation Example: 5 ml of isopropanol containing 5% water and 30 mg diisopropyl sulfate were added to 100 mg epimedoside A followed by addition of 15 mg sodium hydroxide. The mixture was stirred at room temperature for 10 hours. After completion of the reaction, 20 ml of water was added and then extraction with ether was performed. The ether layer was dried with anhydrous sodium sulfate. After removal of ether, the crude product was dissolved in ethanol and then purified by column chromatography (silica gel column) to obtain the modified product with R.sub.3 being an isopropoxy group. Finally, by applying the method in Example 1 (2), the R.sub.2 Rha group in the modified icariin product was removed with an enzymatic method. The desired icariside compound (Y-4′ XS-1) with R.sub.3 being an isopropoxy group was obtained and the chemical structure of the product was characterized by .sup.1H NMR, Mass spectroscopy, and other instrumentations.

Example 6

(34) Test of Using Icariside I to Promote Human γ-Interferon Production and to Improve Human Immunity (I)

(35) Effect of icariside I on promoting normal human peripheral blood mononuclear cells (PBMCs subjected to mAb treatment) to produce γ-interferon (interferon-gamma, IFN-γ) (an experimental model mimicking enhancing immunity in human body).

(36) (1) Preparation of icariside I solution for the test. 10 mg icariside I was dissolved in 1 ml DMSO (with initial concentration of 10 mg/ml). The solution was then transferred into small vials and stored in a freezer at −80° C. for future use.

(37) (2) Preparation of normal human peripheral blood mononuclear cells (PBMCs).

(38) The whole blood samples were drawn from peripheral vein of healthy volunteers. Heparin (blood:heparin=10 ml: 80 μl) was used for anticoagulation. Then, it is diluted with equal volume of Hank's solution. The diluted blood samples were slowly laid on the Ficoll separation solution (Ficoll to diluted blood ratio is 3:4) followed by Ficoll density gradient centrifugation (22° C. 2200 r/min, 20 min). After centrifugation, samples were divided into three layers in the tube. At the interface between the top and middle layers, there was a narrow band which is a mononuclear cell-based yellow-white cloud layer. The peripheral blood mononuclear cells (PBMCs) were drawn and transferred into another centrifuge tube with a pipette. The obtained PBMCs were washed with Hank's solution twice (1800 r/min, 8 min). The supernatant was removed after the last centrifugation and the residue was added with RPMI 1640 complete medium to re-suspend the cells followed by mixing. The mononuclear cells were counted with trypan blue solution. Desired concentration was obtained by adjusting cell number by adding RPMI 1640 complete medium.

(39) (3) in vitro culture of lymphocytes with stimulation by a pharmaceutical

(40) The cell concentrations of freshly isolated peripheral blood mononuclear cells (PBMCs) were adjusted to 2×10.sup.6/ml with RPMI 1640 complete medium. Then a stimulatory molecule monoclonal Anti-CD3 (1 μg/ml) or Anti-CD3 (1 μg/ml)/Anti-CD28 (1 μg/ml) was added or not added, and three different concentrations (at a gradient) of the pharmaceutical icariside I (at a final concentration of 1 ng/ml, 10 ng/ml, 100 ng/ml) were added and then well-mixed. The cells after stimulation with the pharmaceutical were seeded and cultured in 96-well round-bottom plates. The cell concentration per well was 4×10.sup.5/ml (200 μl/well) with each of the stimulation conditions repeated for three wells. The cells were incubated at 37° C. with 5% CO.sub.2 in an incubator for four different time periods: 12 h, 24 h, 36 h and 48 h. Finally, the supernatant of each well with different culture time was taken respectively for test and measurement.

(41) (4) ELISA Analysis of Production of Cytokine IFN-γ

(42) The collected cell culture supernatants with different culture time were detected with ELISA to measure the amount of cytokine IFN-γ produced. The reagents, antibodies, and testing methods used for ELISA were based on the operational instructions for use provided by the manufacturer. All data analysis and graph were made using the software GraphPad Prism 5 version. The results were shown in FIGS. 5 and 6. FIGS. 5 and 6 showed that icariside I significantly enhanced the IFN-γ level produced by PBMCs induced by a monoclonal stimulatory molecule and the enhancement was dose-dependent with icariside I.

(43) Different concentrations (ng/ml) of the pharmaceutical icariside I were added to PBMCs treated with anti-CD3. FIG. 5A: icariside I significantly increased the production of the cytokine IFN-γ in a time and dose dependent way. FIG. 5B: percentage of IFN-γ production promoted by icariside I at different concentrations and at different time points.

(44) Different concentrations (ng/ml) of the pharmaceutical icariside I were added to PBMCs treated with anti-CD3 and anti-CD28. FIG. 6A: curve of change of the amount of produced cytokine IFN-γ with the pharmaceutical icariside I at different concentrations and at different time points. FIG. 6B: promotion rate by the pharmaceutical icariside I at different concentrations and at different time points. *P<0.05; **-***P<0.01.

Example 7

(45) Test of Using Icariside I to Promote Human γ-Interferon Production and to Improve Human Immunity (II)

(46) Effect of icariside I on promotion of γ-interferon (interferon-gamma, IFN-γ) production by human peripheral blood mixed lymphocytes during culture (an experimental model mimicking enhancing immunity in human body).

(47) (1) Preparation of human peripheral blood mononuclear cells (PBMCs): following the method in Example 5 (2), peripheral blood PBMCs from two normal human were prepared.

(48) (2) Mixed lymphocyte culture (MLC) reaction with stimulation of pharmaceuticals:

(49) Cell concentrations of freshly isolated peripheral blood mononuclear cells (PBMCs) were adjusted to 5×10.sup.5/ml with RPMI1640 complete medium. Then, lymphocytes from two healthy human with equal volumes were mixed to maintain a total cell concentration of 1×10.sup.6/ml. Different concentrations (in a gradient) of or no icariside I were added thereto (with final concentration of 100 ng/ml, 1000 ng/ml) followed by fully mixing. Then the cells after stimulation were seeded and cultured in a 96-well round-bottom culture plate. The cell concentration per well was maintained at 2×10.sup.5/ml (200 μl/well) and was incubated at 37° C. with 5% CO.sub.2 for 120 h in an incubator. The levels of cytokine IFN-γ were detected.

(50) (3) ELISA Detection for the Levels of Cytokine IFN-γ Production

(51) According to the method stated in Example 5 (4), the γ-interferon generated from human peripheral blood mixed lymphocyte culture (MLC) was detected.

(52) The results were shown in FIG. 7. FIG. 7 showed that after mixed culture of the lymphocytes from two normal human, IFN-γ level was increased. After addition of icariside I, generation of IFN-γ in the mixed lymphocyte culture was significantly promoted in a dose dependent way (up to 300%).

(53) Equal cell concentrations of lymphocytes from healthy human were mixed cultured for five days (120 h). The pharmaceutical icariside I was added (in a gradient of different pharmaceutical concentrations) or not added. Experimental results showed that the pharmaceutical icariside I can significantly promote the production of cytokine IFN-γ. Among them, the human lymphocyte A control is a group of samples containing only one kind of human lymphocyte, while human lymphocyte B control is a group of samples containing two kinds of human lymphocytes, that is, a control group in which two kinds of human lymphocytes were mixed for culture without adding icariside I. The concentrations of icariside I added were 0.1 and 1 μg/ml respectively. *P<0.05; **P<0.01.

Example 8

(54) Screening Test of the Ability of Icariside Compounds in Promoting Human γ-Interferon Production

(55) According to the test stated in Example 7, i.e. using mixed lymphocyte culture (MLC) reaction with stimulation of pharmaceuticals as a screening model, different icariside compounds were subjected to screening to evaluate the ability thereof in promoting human γ-interferon production.

(56) Cell concentrations of freshly isolated peripheral blood mononuclear cells (PBMCs) were adjusted to 5×10.sup.5/ml with RPMI1640 complete medium. Then, equal volumes of lymphocytes from two healthy people were mixed for culture and the total cell concentration was maintained at 1×10.sup.6/ml. Different icariside I compounds (final concentration at 1000 ng/ml) were added and well-mixed. After stimulation with the pharmaceuticals, the cells were seeded and cultured in a 96-well round-bottom culture plate. The cell concentration per well was maintained at 2×10.sup.5/ml (200 μl/well) and the cells were cultured at 37° C. with 5% CO.sub.2 for 120 h in an incubator. The level of cytokine IFN-γ produced was then detected.

(57) The results were shown in FIG. 8. FIG. 8 showed that IFN-γ was increased after culture of mixed lymphocytes from two normal people. After the addition of different icariside I compounds (with final concentration 1000 ng/ml), the IFN-γ production levels in mixed lymphocyte culture were found to have a relatively large difference. This illustrated that different compounds showed different effect on cell immunity.

(58) These test results showed that icariside I had the best ability to promote human γ-interferon production, while icariin showed almost no effect and other analogs showed different degrees of promotion.

Example 9

(59) Test of Icariside I in Promoting Pleural Effusion Cells of Patients Suffering from Tuberculosis to Produce Human γ-Interferon and Thus Enhancing Human Ability to Resist TB Infection.

(60) (1) Isolation of human peripheral blood mononuclear cells (PBMCs): according to the method stated in Example 5 (2), normal human peripheral blood PBMCs were prepared.

(61) (2) Isolation of Pleural fluid cells (PFMCs, namely cells stimulated by tuberculosis bacteria infection) from patients suffering from tuberculosis: blood samples were taken from volunteers (vein blood of TB patients) and then anticoagulated with heparin and diluted with an equal volume of Hank's solution. The solution was centrifuged once (1800 rpm, 22° C. 8 minutes). After removal of the supernatant, an appropriate amount of ELS (erythrocyte lysate) was added for re-suspension. After 3-5 minutes, an appropriate amount of Hank's solution was added for stop and then the mixture was centrifuged twice (1800 rpm, 22° C. 8 minutes), followed by addition of RPMI1640 complete medium to adjust the PBMC cell concentration to 2×10.sup.9 cells/L.

(62) (3) PBMCs and PFMCs cell culture (MLC) with stimulation by a pharmaceutical: Icariside I was used at concentrations of 0.1 μg/ml, 0.5 μg/ml, and 1 μg/ml respectively to stimulate PBMCs and PFMCs under the incubation condition at 37° C. with 5% CO.sub.2 for 48 hours.

(63) (4) ELISA assay for analysis of IFN-γ: according to the method stated in Example 5 (4), the levels of γ-interferon produced by PBMCs and PFMCs after culture with pharmaceutical stimulations were measured.

(64) The results were shown in FIG. 9. FIG. 9 showed that after using icariside I for stimulation in culture, no significant promoting effect was shown for PBMCs in producing IFN-γ, while there was a significant and dose dependent effect for PFMCs in producing IFN-γ. It indicated that icariside I can significantly promote the production of IFN-γ by TB bacteria infected PFMCs (pleural effusion cells of patients suffering from tuberculosis). Results of this test suggest very clearly that, icariside I can improve the body's ability to fight against TB infection and thus it has important promising applications in immunotherapy of tuberculosis.

(65) PBMCs from healthy human and PFMCs from TB infected patients were respectively cultured for 2 days (48 h), then added or not added with the pharmaceutical icariside I (at a gradient of different concentrations). Experimental results showed that icariside I can significantly promote the production level of cytokine IFN-γ in PFMCs of TB infected patients. Among the tests, the concentrations of icariside I added were 0.1, 0.5 and 1 μg/ml. *P<0.05; **-***P<0.01.

Example 10

(66) Test of Icariside I in Inhibiting Tumorigenesis and Tumor Progression in Mice

(67) NIH mice used for the tests were about 18-22 grams and divided into 10 mice per group. The liquid (0.2 ml) of S180 ascites tumor of mice was inoculated subcutaneously into the armpit region of mice. The next day after the inoculation, each group of mice were injected intraperitoneally: (1) physiological saline group; (2) solvent control group (DMSO); (3) positive control group [cyclophosphamide 60 mg/(kg.Math.d)]; (4) low-dose group 1: icariside I 2 mg/(kg.Math.d); (5) low-dose group 2: icariside I 5 mg/(kg.Math.d); (6) middle-dose group: icariside I 10 mg/(kg.Math.d); (7) high-dose group: icariside I 20 mg/(kg.Math.d). After administration for 10 continuous days, the administration was stopped and on the next day, the mice were weighed and sacrificed with tumor mass stripped and weighed. The physiological saline group and solvent group were used as a control for calculation of tumor inhibition rate. The results are shown in Table 1.

(68) TABLE-US-00001 TABLE 1 Experimental results of inhibition of tumor growth with icariside I in mice tumor inhibition tumor rate (%) inhibition with rate (%) weight of change of physiological with solvent number final tumor body saline group as group as Group for start number (x ± SD, g) weight (g) control control physiological saline group 10 10 1.04 ± 0.54 +16.1 — — solvent group (25% DMSO) 10 10 1.34 ± 0.60 +12 — — positive control 10 10  0.6 ± 0.06 +4.5 42.3 55.2 cyclophosphamide group (60 mg/Kg) icariside I low dose group 10 10 0.98 ± 0.47 +11.6 5.8 26.9 (2 mg/Kg) icariside I low dose group 11 11 0.77 ± 0.47 +12.5 26.0 42.5 (5 mg/Kg) icariside I middle dose group 10 10 0.64 ± 0.25 +10.6 38.5 52.2 (10 mg/Kg) icariside I high dose group 10 10 0.34 ± 0.12 +9.0 67.3 74.6 (20 mg/Kg)

(69) The results in Table 1 show that icariside I can significantly inhibit tumorigenesis and tumor progression in mice with an effect better than that of the positive control pharmaceutical cyclophosphamide.

Example 11

(70) Test of Effect of Icariside I on Xenograft Tumor Growth in Nude Mice

(71) The test used 4-6 weeks old BALB/c (nu/nu) female nude mice weighed 18-22 g fed in a SPF clean room with free access to food and water. Models were established with 40 nude mice by transplanting tumor mass. When the tumor grew to the initial volume for administration with a pharmaceutical (100 mm.sup.3), these mice were divided into groups with 10 mice for each group. For each group, nude mice were injected intraperitoneally: (1) the blank control group was injected with 0.1 ml/10 g solvent (5% DMSO+95% physiological saline); (2) the positive pharmaceutical injection group [cyclophosphamide (0.6 mg/10 g)]; (3) administration group (icariside I): high concentration group injected with: 0.1 mg/10 g; low concentration group injected with: 0.02 mg/10 g. The administration was conducted every 2 days in a 30-day period. Vernier caliper was used to measure the largest longitudinal diameter (L) and transverse diameter (S) of tumor every two days. The approximate volume of the tumor was calculated with the equation of V (cm.sup.3)=0.5×L× S.sup.2 to plot a tumor growth curve. After the pharmaceutical treatment, the tumor was stripped and weighed for record. The results were shown in FIGS. 10 and 11. It is shown that icariside I can significantly inhibit growth and development of the transplanted tumor mass in nude mice with an effect better than that of the positive control pharmaceutical cyclophosphamide.

Example 12

(72) Acute Toxicity Test for Icariside I

(73) Mice weighed 18-22 grams were randomly divided into six groups with 10 mice each group. Each individual group of mice were treated (orally) with: physiological saline, DMSO 2.5 ml/kg, and icariside I: 2000 mg/kg, 1000 mg/kg, 500 mg/kg, 100 mg/kg respectively. After observation for 14 days, none of the test mice died. The results show that the acute toxicity LD50 value of icariside I in mice is greater than 2000 mg/kg. Therefore, the acute toxicity of icariside I is very low which is safe for use in preparation of pharmaceuticals, health products, food, and cosmetic and skin care products for prevention and treatment of immunity-related diseases.