WO2009105912A1 - 一种灵芝孢子油脂肪乳剂及其质量控制方法与应用 - Google Patents
一种灵芝孢子油脂肪乳剂及其质量控制方法与应用 Download PDFInfo
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- WO2009105912A1 WO2009105912A1 PCT/CN2008/000396 CN2008000396W WO2009105912A1 WO 2009105912 A1 WO2009105912 A1 WO 2009105912A1 CN 2008000396 W CN2008000396 W CN 2008000396W WO 2009105912 A1 WO2009105912 A1 WO 2009105912A1
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- ganoderma lucidum
- spore oil
- lucidum spore
- fat emulsion
- peak
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
-
- 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
- A61K36/06—Fungi, e.g. yeasts
- A61K36/07—Basidiomycota, e.g. Cryptococcus
- A61K36/074—Ganoderma
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
Definitions
- the invention relates to the technical field of ganoderma spore oil, in particular to a ganoderma spore oil fat emulsion, a quality control technique thereof and a medicament use.
- Ganoderma lucidum is a general term for the Polyporaceae Ganoderma fungus Glucidum karst and Gjaponicum Lloyd. It has the functions of strengthening the body and fixing it, and is called the top grade by the book.
- Ganoderma lucidium spore is an extremely fine spore that ejects from the cap of the ganoderma lucidum during the growth and mature period.
- Ganoderma lucidum spores are complex in chemical composition, such as fatty acids, alcohols, triterpenoids, alkaloids, lactones, proteins and amino acids, glycopeptides, vitamins, carotene, and inorganic ions. Modern pharmacological studies have shown that Ganoderma lucidum spore oil is the main anti-tumor effect of Ganoderma lucidum spores.
- Ganoderma lucidum spore oil The research group of Ganoderma lucidum spore oil research and test analysis showed that the main components of Ganoderma lucidum spore oil are composed of various components such as Ganoderma lucidum spore oil ester, fatty acid, Ganoderma lucidum and ergosterol.
- Ganoderma lucidum spore oil soft capsule containing Ganoderma lucidum spore oil as a main component has been put on the market, which is suitable for tumor suppression and prevention, and post-surgical recovery and immunity enhancement.
- it is an oral agent, which acts through the gastrointestinal tract after administration and cannot directly enter the blood of the human body.
- Ganoderma lucidum spore oil is water-insoluble, and it is quite difficult to prepare a common injection. This problem can be solved by preparing a ganoderma lucidum spore oil fat emulsion.
- the name previously filed by the applicant is "Ganoderma lucidum spore oil fat emulsion", and the Chinese invention patent No. ZL200410051661.3 solves the problem.
- the patent application number is 200510068335.8, and the Chinese invention patent entitled "Ganoderma lucidum spore oil intravenous emulsion injection and its preparation method" also discloses a ganoderma lucidum spore oil intravenous emulsion injection.
- the Ganoderma lucidum spore oil fat emulsions disclosed in these two patents include fatty oils or injectable oils, and the process is complicated, the active ingredients are unclear, and there is no refining process for the ganoderma spore oil for injection and the chemical composition of the fat emulsion. And the quality control method to further elaborate and other shortcomings.
- Ganoderma lucidum spores such as Ganoderma lucidum spore oil, Ganoderma lucidum spores and their preparations, including Ganoderma lucidum spore oil soft capsule, Ganoderma lucidum spore oil fat emulsion injection, Ganoderma lucidum spore oil fat emulsion oral preparation, Ganoderma lucidum spore capsule, Ganoderma lucidum spore Tablets, Ganoderma lucidum spore powder, etc., but true and false are difficult to distinguish, quality is difficult to distinguish.
- the object of the present invention is to overcome the deficiencies of the existing Ganoderma lucidum spore oil fat emulsion, and to provide a Ganoderma lucidum spore oil fat emulsion having clear active ingredients and strong physiological activity.
- Another object of the present invention is to provide a quality control method for the above-mentioned ganoderma lucidum spore oil fat emulsion.
- a further object of the present invention is to provide the use of the above-mentioned ganoderma lucidum spore oil fat emulsion for the preparation of a medicament for treating a tumor, improving an immune effect, and attenuating a chemotherapeutic drug.
- a ganoderma spore oil fat emulsion consisting of the following components and weight percentages:
- Emulsifier 0.5 ⁇ 10%
- the balance is water; the final P H of the fat emulsion is adjusted to 6 to 9.
- the ganoderma lucidum spore oil is preferably refined ganoderma lucidum spore oil, and the purification method is as follows: centrifuging the ganoderma spore oil, removing water, adding 0.5% ⁇ 10% of the weight of the ganoderma lucidum spore oil Tanning agent, stirring evenly, heating to 40 ⁇ 70 ° C, keeping warm for 20 ⁇ 40 minutes, centrifuging, fine filtration, obtaining refined ganoderma spore oil; the adsorbent is activated carbon, silica gel, neutral alumina, diatomaceous earth, One or several mixtures in white soil.
- the emulsifier is preferably a mixture of one or more of soybean lecithin, egg yolk lecithin, pulverolone, polyglyceryl palmitate diol or alginate. The best is egg yolk lecithin or soy lecithin.
- the isotonic agent is preferably a mixture of one or more of glycerin, glucose, xylitol, maltose or sorbitol, most preferably glycerin.
- the function of the isotonic agent is to bring the osmotic pressure of the preparation close to the physiological osmotic pressure of the human body.
- the first quality control method of the above-mentioned ganoderma lucidum spore oil fat emulsion is determined by high performance liquid chromatography to determine the content of 1,2-oleic acid-3-palmitic acid triglyceride and/or glycerol trioleate in the product.
- the amount of 1,2-oleic acid-3-palmitic acid triglyceride per lg of Ganoderma spore oil fat emulsion is 2 mg ⁇ 62.5 mg and/or the amount of glycerol trioleate is 1.6 mg ⁇ 50.0 mg;
- the high performance liquid chromatography method is according to the following chromatographic conditions: using octadecyl-bonded silica gel as a filler; using acetonitrile, isopropanol or dichloromethane as a binary or ternary in any ratio
- the composition of the mixture is a mobile phase, the flow rate of the mobile phase is 0.5 ⁇ 2.0ml / min ; detected by evaporative light scattering detector or differential refractive index detector; the number of theoretical plates is 1,2-oleic acid-3-palmitic acid
- the peak of triglyceride or triolein should be no less than 2000; the column temperature is 10 ⁇ 50 °C.
- the second quality control method of the above-mentioned ganoderma lucidum spore oil fat emulsion is determined by high performance liquid chromatography for the content of ergosterol in the product, and the amount of ergosterol per lg of the ganoderma lucidum spore oil fat emulsion is 0.04 mg ⁇ 7.5 mg ; Ergosterol is a unique component in fungi.
- Ganoderma lucidum spore oil is the only fungus vegetable oil known at present. Therefore, ergosterol can be distinguished from other vegetable oils as a component of ganoderma spore oil.
- the chromatographic conditions are: using octadecyl bonded silica as a filler; using methanol, ethanol, acetonitrile, aqueous methanol, aqueous ethanol or acetonitrile as mobile phase, or ternary or methanol, ethanol, acetonitrile and water
- the quaternary composition mixture is a mobile phase, or a mixture of tetrahydrofuran and water is used as a mobile phase.
- the volume ratio of tetrahydrofuran to water is 75:25; the detection wavelength is 280 ⁇ 2mn; the number of theoretical plates is calculated based on the peak of ergosterol. Less than 2000.
- the chromatographic conditions of the high-performance liquid phase are preferably: the column is made of octadecyl silane-bonded silica gel as a filler; the mobile phase is acetonitrile-isopropanol, and the volume ratio is 53:47; evaporative light scattering detector; reference substance : Take glycerol trioleate reference substance;
- the fingerprint of the ganoderma lucidum spore oil fat emulsion there are 15 common peaks, wherein there are 4 fingerprint peaks exceeding 5% of the total peak area, and the other retention peaks are calculated by the relative retention time of the chromatographic peak of the triolein.
- the relative retention time and the relative peak area are calculated.
- the above four fingerprint peaks are the peak average relative retention time RT of 0.778, the relative peak area is 9.54 ⁇ 15.36%, and the average peak of the 10th peak is 0.832, the relative peak area.
- the range is 5.76 ⁇ 9.43%; the peak of glycerol trioleate of peak 11 is 1.000, the relative peak area is 22.29 ⁇ 27.80; the average RT of peak 12 is 1.075, and the relative peak area is 26.82 ⁇ 37.76%.
- the pH of the ganoderma lucidum spore oil of the present invention can be adjusted during the preparation using a sodium hydroxide or phosphate buffer solution.
- the final pH is between 6 and 9.
- ganoderma lucidum spore oil emulsion can also be determined by methods known in the art, wherein: the particle size of the milk particles is evenly distributed between 100 and 500 nm; the number of milk particles larger than 1 mm must not exceed 1%. Do not detect milk particles larger than 5mm.
- Bacterial endotoxin should contain less than 0.5 EU of endotoxin per 1 ml of fat emulsion.
- the ganoderma lucidum spore oil fat emulsion of the invention has good medicinal activity and can be mixed with other drugs alone or in combination to prepare a medicament for treating tumors, improving immunity and attenuating chemotherapeutic drugs.
- the ganoderma spore pool f fat emulsion of the invention is particularly suitable for intravenous and intravenous injection, and the ganoderma spore oil directly enters human blood.
- the preferred dosage form is an injectable emulsion.
- the present invention has the following beneficial effects:
- the ganoderma lucidum spore oil fat emulsion of the invention has clear active ingredients and has good biological activity, and can be used alone or in combination with other anti-tumor drugs for treating tumors, improving immunity, and performing tumors for radiotherapy and chemotherapy. Patients can improve their quality of life and reduce the side effects of drugs after treatment.
- the ganoderma lucidum spore oil fat emulsion of the invention is particularly suitable for intravenous and intravenous injection, and the ganoderma spore oil directly enters the human blood, has quick effect and complete absorption.
- the ganoderma lucidum spore oil injection fat emulsion of the invention is a product with high safety, reliable quality and low toxicity.
- the preparation of the ganoderma lucidum spore oil fat emulsion of the invention is easy to obtain, the process is simple, and can be prepared by conventional production equipment and processes, and is suitable for large-scale production into mass production by a pharmaceutical factory producing injections with G M P.
- the quality control method of the present invention is an easy method for determining the content of 1,2-oleic acid-3-palmitic acid triglyceride and glycerol trioleate in the preparation as a method and basis for quality control of the product. Strong maneuverability and high reproducibility.
- the quality control method of the present invention can accurately determine the content of ergosterol in the preparation as a method and basis for quality control of the product, and has high operability, high reproducibility and specificity.
- the quality control method of the invention utilizes a fingerprint, the method is simple, stable, high precision, reproducible, easy to grasp, and can grasp the variety and quality of the ganoderma spore oil fat emulsion from the overall feature of the chromatogram.
- the quality control and authenticity identification of Ganoderma lucidum spore oil fat emulsion provides a new method.
- Figure 1 is a high-performance liquid chromatogram of the triglyceride reference substance for 0 to 60 minutes;
- Figure 2 is a standard fingerprint of 0 to 60 minutes of Ganoderma lucidum spore oil fat emulsion
- Figure 3 shows the fingerprint of 10 batches of Ganoderma lucidum spore oil fat emulsion for 0 ⁇ 60 minutes.
- Ganoderma lucidum spore oil refining Centrifuge ganoderma lucidum spore oil, remove water, add 5% activated carbon of Ganoderma lucidum spore oil, stir it, heat it to 50 ⁇ , keep for 30 minutes, centrifuge, finely filter, and obtain refined Ganoderma lucidum spore oil .
- Preparation method Under the condition of nitrogen gas, the emulsifier is added to the refined ganoderma lucidum spore oil, and the emulsifier is completely dissolved at high speed; the oil phase is slowed down under the condition of constant temperature water bath 50 ° C and high speed dispersion emulsification 12000 rpm.
- Example 7 Determination of 1,2-linoleic acid-3-palmitic acid triglyceride and glycerol trioleate in ganoderma lucidum spore oil fat emulsion
- octadecyl-bonded silica gel was used as a filler; column: Kromasil ClSASmn ⁇ SOmn um column; column temperature: 30 ° C; mobile phase: acetonitrile: isopropanol volume ratio 40:60; evaporative light scattering Detector detection; Flow rate: 0.5 ml/min.
- the number of theoretical plates is calculated as 1,2-oleic acid-3-palmitic acid triglyceride or glycerol trioleate peak, which are all greater than 2000;
- Preparation of reference solution Take 1,2-oleic acid-3-palmitic acid triglyceride, glycerol trioleate reference substance 2.98mg, 2.67mg, placed in a 25ml volumetric flask, dissolved in methanol and diluted to the mark. Prepare a solution containing 0.119 mg and 0.107 mg per lml, respectively, to obtain a reference solution;
- test solution Precision weighed ganoderma lucidum spore oil fat emulsion (Example 1) 1.025g, added 0.2g anhydrous sodium sulfate, heated on a water bath to rupture the emulsion, transferred to a separatory funnel, extracted with ether 3 Second, each time 2.0ml, combined with ether solution, evaporated to dryness, add mobile phase: acetonitrile: isopropanol (40:60) dissolved, transferred to a 100ml volumetric flask, and dilute to the mark, that is, the test solution;
- the amount of 1,2-oleic acid-3-palmitic acid triglyceride per lg of ganoderma spore oil fat emulsion is 2 mg ⁇ 62.5 mg and I or glycerol trioleate is 1.6 mg ⁇ 50.0 mg;
- Example 8 Determination of 1,2-oleic acid-3-palmitic acid triglyceride and glycerol trioleate in Ganoderma lucidum spores
- Preparation of reference solution Take 1,2-oleic acid-3-palmitic acid triglyceride, glycerol trioleate reference substance 2.98mg, 2.67mg, placed in a 25ml volumetric flask, dissolved in methanol and diluted to the mark. Prepare a solution containing 0.119 mg and 0.107 mg per lml, respectively, to obtain a reference solution;
- test solution Precision weighed Ganoderma lucidum spore oil fat emulsion (Example 2) 302.45mg, in a Soxhlet extractor, add 30ml of ether, cold soak overnight, then add 50ml of ether, heat extraction in water bath for 8 hours, The extract is recovered to the end of the ether, and the residue is dissolved in the mobile phase: acetonitrile: dichloromethane (59:41), transferred to a 50 ml volumetric flask, and dissolved to the mark to obtain a test solution;
- Measurement results The reference solution and the test solution were respectively accurately extracted by 10 ⁇ l, injected into a high performance liquid chromatograph, and determined by logarithm.
- Each lg Ganoderma lucidum spore contains 1,2-oleic acid-3-palmitic acid triglyceride, glycerol triglyceride The acid ester was 59.28 mg and 46.52 mg.
- the amount of 1,2-oleic acid-3-palmitic acid triglyceride per lg of Ganoderma lucidum spore oil fat emulsion is 2 mg ⁇ 62.5 mg and I or glycerol trioleate is 1.6 mg ⁇ 50:0 mg ;
- Example 9 Determination of ergosterol in Ganoderma lucidum spore oil emulsion
- octadecyl bonded silica was used as a filler; methanol was used as the mobile phase; the detection wavelength was 280 nm.
- the number of theoretical plates shall be calculated based on the peak of ergosterol and shall not be less than 2000;
- Preparation of reference solution take the appropriate amount of ergosterol reference substance, add methanol to make 0.08mg solution per lml, that is, the reference solution;
- Measurement results 10 ⁇ 1 of each of the reference solution and the test solution are accurately extracted and injected into a high performance liquid chromatograph, and measured and calculated. Ergosterol 0.6 mg per lg of emulsion.
- the amount of ergosterol in the ganoderma lucidum spore oil fat emulsion per lg is 0.004 mg to 7.5 mg, which indicates that the quality of the fat emulsion of the embodiment 1 meets the requirements.
- Example 10 Ganoderma lucidum spore oil fat emulsion HPLC standard fingerprint
- Preparation of reference solution using glycerol trioleate as reference material, taking appropriate amount of glycerol trioleate reference substance, and diluting with mobile phase to prepare a solution containing 0.15 mg of triolein per 1 ml as a reference substance. Solution.
- test solution Precision weighed Ganoderma lucidum spore oil fat emulsion (Example 4) 0.533g, added 0.2g anhydrous sodium sulfate, heated on a water bath to rupture the emulsion, transferred to a separatory funnel, extracted with ether 3 times , each time 2.0ml, combined with ether solution, evaporated to dryness, add mobile phase: acetonitrile: isopropanol (40:60) dissolved, transferred to a 100ml volumetric flask, and dilute to the mark, that is, the test solution;
- the reference solution and the ganoderma lucidum spore oil emulsion were respectively injected into the sample solution, and determined by high performance liquid chromatography, and the chromatogram of 60 minutes was recorded, as shown in Figs. 1, 2 and 3.
- the relative retention time and relative peak area of the other peaks were calculated as the relative retention time and relative peak area of the chromatographic peak (S peak) of glycerol trioleate.
- the common peak is determined:
- the fingerprints of the obtained Ganoderma lucidum spore oil emulsion can be compared by HPLC chromatogram to determine the common characteristic peaks, and the HPLC standard of Ganoderma lucidum spore oil composed of the common characteristic peaks is obtained.
- the standard fingerprint has 15 characteristic peaks, and the relative retention time of each peak The standard deviation RSD is less than 2%.
- the average RT of the peak No. 1 is 0.133, the RSD is 0.31%, and the relative peak area is 0.10 ⁇ 2.40%; the average RT of the peak No.
- the average peak of the 12th peak is 1.075, and the RSD is 0.10%.
- the area ranged from 26.82 to 37.76%; the average T of the 13th peak was 1.158, the RSD was 0.21%, and the relative peak area ranged from 1.31 to 2.03%; the average RT of the 14th peak was 1.370, the RSD was 0.13%, and the relative peak area range It is 1.22 ⁇ 1.72%; the average R of the 15th peak is 1.479, the RSD is 0.15%, and the relative peak area ranges from 0.54 to 1.03%.
- Example 11 Ganoderma lucidum spore oil emulsion stability test:
- Example 4 The sample of Example 4 was subjected to an accelerated test for 6 months, and the conditions were as follows: a standing temperature of 37 Torr and a relative humidity of 90%.
- test results show that the test sample of the present invention is substantially stable in quality during the accelerated inspection period.
- Example 12 Ganoderma lucidum spore oil emulsion safety test
- Guinea pig systemic active allergy test 24 Hartley guinea pigs were randomly divided into 4 groups according to gender and body weight, 3 males and 3 females in each group. A negative control group, a positive control group, and a low- and high-dose group of test subjects were set. The drug was administered intraperitoneally, and the administration volume was 0.5 mLA once every other day for five consecutive times. Animals in each group were challenged on the 12th day after the last intraperitoneal injection, and the volume of the drug was 2.0 mIJ, and the reaction of the animals was observed. According to the incidence and degree of allergic reactions, the systemic active allergies are judged.
- ASA Guinea pig systemic active allergy test
- Rat passive skin sensitization test 24 SPF grade SD rats were randomly divided into 4 groups according to gender and body weight, and 3 males and 3s in each group.
- the negative control group, the positive control group, the low-dose group and the high-dose group (125 and 250 mg /kg*bw, respectively, were equivalent to 0.14 and 0.28 times of the clinically-prepared dose of 10 g, respectively).
- the drug was administered intraperitoneally, once every other day for five consecutive times, and blood was collected on the 10th day after the last sensitization to prepare an antiserum.
- Another 24 SPF-class SD rats were selected. The grouping and grouping were the same as those of the above-mentioned antibody preparation.
- Each group of animals was intradermally injected with 0.1 ml of antiserum of each corresponding group, and challenged 48 hours after passive sensitization.
- the negative control group and the low-dose and high-dose groups no blue spots were found in the inner layer of the back skin, and the positive control group showed obvious blue spots. It is suggested that under the conditions of this experiment, there is no obvious allergic reaction to the passive skin allergy test of SD rats by Ganoderma lucidum spore oil injection.
- Rabbit vascular irritation test 4 healthy New Zealand rabbits were used in the experiment, and the left and right ears were used to compare themselves. The left ear was injected with the test drug (100 mg/mL, which is equivalent to the clinical intravenous infusion). At the concentration, the right ear was given an equal volume of 0.9% sodium chloride injection as a control, once a day for 7 consecutive days. At the end : 2 animals were dissected 48 hours after the second administration, and the remaining 2 animals were subjected to necropsy 14 days after the last administration. Visual and microscopic examination results: No pathological changes were observed in the venous vessels and surrounding tissues of the rabbit ears and rabbits on the donor side and the control side. It is suggested that under the conditions of this experiment, Ganoderma lucidum spore oil injection has no obvious irritative reaction on rabbit ear vein and surrounding tissues.
- Rabbit Muscle Stimulation Test Four healthy New Zealand rabbits were used for the experiment. The four-head muscles of the right and left muscles were compared with each other. The test substance was administered to the left side (100 mg/mL, which is equivalent to the clinical intravenous infusion). Concentration), an equal volume of 0.9% sodium chloride injection was administered to the right as a control, l.OmlJ side, single administration. Two animals were dissected 48 hours after administration, and the remaining two animals were subjected to necropsy 14 days after administration. No obvious abnormalities were observed in the muscles of the injection sites on each side of the four rabbits.
- miceroscopic examination results One animal at 48 hours after administration and two animals at 14 days of necropsy were arranged neatly at the left and right side of the injection site, and no abnormal changes were observed.
- the local muscle tissue in the right side showed mild degeneration of muscle fibers, and a large number of inflammatory cells infiltrated in the muscle fiber interstitial of the lesion.
- the left part of the muscle tissue showed flaky degeneration, necrosis and even disappearance.
- the negative control side on the right side mild mechanical stimulation also occurred.
- the stimulation of the local muscle tissue on the left side may be mechanical stimulation. It is suggested that under the conditions of this experiment, Ganoderma lucidum spore oil injection has no obvious irritating reaction on the quadriceps of rabbits.
- the test consisted of 4 doses of 4, 6, 8, 12, 16 g/kg.bw (24, 36, 48, 72, 96 times of the clinically-used doses, respectively), one dose for each dose, one single vein for Beagle dogs. Dosing. The results showed that: Beagle dogs had no obvious abnormalities in electrocardiogram, hematology and urine examination after a single administration, and some changes occurred in blood biochemistry. The longer the dose, the longer the duration; the dose was 12, 16g kg. At .bw, the animals developed symptoms such as quietness, decreased activity, and decreased appetite. The single-injection of Ganoderma lucidum spore oil was a non-toxic dose of 4g/kg.bw to Beagle dogs, and the tolerance was greater than 16 g/kg.bw.
- Example 14 Experimental therapeutic effect of ganoderma lucidum spore oil injection iv administration on mouse H 22 liver cancer
- the well-grown 7-22 day H 22 tumors were inoculated subcutaneously into the right temporal part of the mice, about 4.5 ⁇ 5 ⁇ 10 6 cells/cell, and were randomly divided into cages 24 hours after inoculation.
- the body weight was measured every day during the administration, and the animals were sacrificed on the 8th day, and the tumor weight was weighed.
- the average tumor weight of each group was calculated, and the tumor inhibition rate was determined and subjected to t-test.
- Blank milk 10 10 19.9 ⁇ 1.0 26.5 ⁇ 3.2 1.66 ⁇ 0.33
- CTX 30 10 10 18.9 ⁇ 1.2 24.5 ⁇ 3.1 0.69 ⁇ 0.21** 58.4 Ganoderma lucidum spore oil
- Example 15 Experimental therapeutic effect of Ganoderma lucidum spore oil injection milk iv on human gastric adenocarcinoma SGC-7901 nude mice xenografts The growth of human gastric adenocarcinoma SGC-7901 nude mice xenograft tumor tissue was cut into 1.5 mm 3 Under sterile conditions, inoculated subcutaneously in the right axilla of nude mice.
- the nude mice xenografts were measured with a vernier caliper to measure the diameter of the transplanted tumors, and the animals were randomly divided into groups after the tumors were grown to 100-300 mm 3 .
- the method of measuring the tumor diameter is used to dynamically observe the antitumor effect of the test substance.
- the number of tumor diameters measured is 3 times a week, and each measurement is also required to weigh the mouse.
- the experimental group was administered intravenously once every other day; the positive control drug was Taxol (TAX) lOmg kg, once every other day; negative control group To give an equal volume of blank milk.
- the growth inhibition effect of the drug on the tumor was evaluated by the relative tumor inhibition rate T/C (%).
- the test results are as follows: Table 4 Ganoderma lucidum spore oil injection milk iv administration experimental treatment of human gastric adenocarcinoma SGC-7901 nude mice xenografts
- mice intravenous control group (20 ml/kg, blank group), Ganoderma lucidum spore oil injection emulsion high, medium and low dose groups (1 , 0.5, 0.25 g kg) iv administration, Maipuxin injection group (0.42 mg / kg) Sc administration; except for Maipuxin once every other day, the other groups were administered once a day, a total of 7
- the hair was removed from the abdomen of the mouse in the range of 3 ⁇ 3 cm 2 ; on the second day of administration, 1% DNFB was evenly applied to the abdomen of the mouse (DNFB is a hapten, which binds to the skin protein to form a whole antigen.
- DNFB is a hapten, which binds to the skin protein to form a whole antigen.
- Ganoderma lucidum spore oil injection high and medium dose group can significantly increase the swelling degree and swelling rate of mice (P ⁇ 0.05). That is, Ganoderma lucidum spore oil injection can enhance the specificity of H 22 tumor-bearing mice. Sexual cellular immune function.
- Example 17 Ganoderma lucidum spore oil injection iv administration on the phagocytic function of the reticuloendothelial system (RES) of H 22 tumor-bearing mice
- mice 50 ICR mice were taken and inoculated with H 22 solid tumors. The next day, the body weight was randomly divided into 5 groups, 10 in each group, male and female, and the experiment was set up: intravenous control group (20ml/kg, blank milk) ), Ganoderma lucidum spore oil injection high, medium and low dose groups (1, 0.5, 0.25g kg) iv administration, positive control drug Maipuxin injection group (0.42mg kg) Sc administration. Except for Maipuxin, which was administered once every other day, the other groups were administered once a day for a total of 11 doses.
- Example 18 Effect of Ganoderma lucidum spore oil injection iv administration on cyclophosphamide (Cy) chemotherapy in H22 tumor-bearing mice - Experimental method: 60 ICR mice of the above specifications were inoculated with H 22 solid tumor, after inoculation The rats were weighed in 24 hours, and were randomly divided into 6 groups, 10 in each group, half male and half female.
- the experiment was set up: blank control group, intravenous control group (20mVkg, blank milk), ganoderma spore oil injection milk high, medium,
- the low dose group (1, 0.5, 0.25 g kg) was administered iv, and the positive control drug, Maipuxin injection group (0.42 mg/kg), was administered. Except that Maipuxin was administered once every other day, the other groups were administered once a day for 7 times. On the 4th and 5th day after the administration, except for the blank control group, the other groups started ip Cy (100 mg/kg).
- the animals were sacrificed 24 hours after stopping the drug, weighed before sacrifice, and blood was taken from the orbital venous vein. The total number of peripheral blood leukocytes was microscopically examined.
- the Ganoderma lucidum spore oil injection high-dose group and the Maipuxin injection group can significantly counteract the peripheral blood leukocytes, bone marrow nucleated cells, thymus coefficient and the H 22 tumor-bearing mice caused by Cy.
- the spleen coefficient decreased (PO.05, P ⁇ 0.01).
- mice of the above specifications were randomly divided into 6 groups according to body weight, 10 rats in each group, half male and half female.
- Experimental settings blank control group, intravenous control group (20ml/kg, blank milk), Ganoderma lucidum
- the spores were administered in the high, medium and low doses of the injected milk (1, 0.5, 0.25 g/kg), and the positive control group, Maipuxin injection group (0.42 mg kg), was administered. Except for Maipuxin, which was administered once every other day, the other groups were administered once a day for a total of 7 doses. On the 4th and 5th day after administration, except for the blank control group, the other groups started ip Cy (100mg kg) for 2 consecutive days.
- the animals were sacrificed 24 hours after stopping the drug, weighed before sacrifice, and blood was taken from the orbital venous vein. The total number of white blood cells in the peripheral blood was simultaneously taken from one side of the complete femur. The number of nucleated cells in the bone marrow was measured, and the thymus and spleen were weighed. The thymus and spleen coefficients were calculated and statistically processed (t-test). Experimental results: The results showed that compared with the blank control group, the number of peripheral blood leukocytes, bone marrow nucleated cells, thymus and spleen were significantly decreased in the blank milk + Cy model (100 mg/kg, ip) group (P ⁇ 0.01).
- the Ganoderma lucidum spore oil injection high-dose group and the Maipuxin injection group significantly inhibited the decrease of peripheral blood leukocytes, bone marrow nucleated cells and spleen coefficient of normal mice caused by Cy ( P ⁇ 0.05)
- the Ganoderma lucidum spore oil injection emulsion group significantly inhibited the decrease of the number of nucleated cells and spleen coefficient of normal mouse bone marrow caused by Cy (PO.05).
- the results are shown in Table 8.
- mice of the above specifications were inoculated, and H 22 solid tumors were inoculated according to the transplanted tumor research method. 24 hours after inoculation, except for 10 animals in the blank control group, the other 50 mice were irradiated with 6Q Co. 500 rad; ⁇ Co irradiated animals were randomly divided into 5 groups according to body weight, 10 rats in each group, male and female, experimental settings: blank control group, intravenous control group (20ml kg, blank milk), ganoderma spore oil injection milk high In the middle, low-dose group (1, 0.5, 0.25 g/kg), iv was administered, and the positive control drug, Maipuxin injection group (0.42 mg k g ), was administered.
- mice of each group were administered 24 hours after inoculation. Except that Maipuxin was administered once every other day, the other groups were administered once a day for 7 times. The animals were sacrificed 24 hours after stopping the drug, and weighed before sacrifice. And taking blood from the orbital vein, microscopic examination of the total number of peripheral white blood cells, while picking the spleen Dirty, thymus and one side of the complete femur, the number of bone marrow nucleated cells were measured, the spleen and thymus were weighed, the spleen index and thymus index were calculated, and statistical processing (t test) was performed.
- Ganoderma lucidum spore oil injection iv high dose group can significantly reduce the number of peripheral blood leukocytes, bone marrow nucleated cells and spleen coefficient of H 22 tumor-bearing mice caused by ⁇ Co (P ⁇ 0.05), Ganoderma lucidum spores
- the medium dose group of oil-injected milk iv was significantly resistant to the decrease in the number of peripheral blood leukocytes in H 22 tumor-bearing mice caused by 6Q Co (P ⁇ 0.05).
- Table 9 Ganoderma lucidum spore oil injection milk iv administration attenuation effect on H 22 tumor-bearing mice 60 Co radiotherapy (_?
- mice of the above specifications were taken, except for 10 animals in the blank control group, the other 50 mice were irradiated with 6Q Co at a dose of 500 rad .
- 6Q C 0 irradiation the animals were randomly divided into 5 groups according to their body weight.
- Group 10 female Male and female, experimental settings: blank control group, intravenous control group (20ml/kg, blank milk), ganoderma lucidum spore oil injection high, medium and low dose group (1, 0.5, 0.25g kg) iv administration, positive
- the control drug Maipuxin injection group (0.42 mg kg, ) Sc was administered. Animals of each group were administered 24 hours after inoculation.
- results The results showed that, compared with the control group, 60 blank milk ten. .
- the peripheral blood leukocytes, bone marrow nucleated cells, thymus and spleen coefficient of the model (500 rad) group were significantly decreased (P ⁇ 0.01).
- the Maipuxin injection group significantly inhibited the decrease of peripheral blood leukocytes, bone marrow nucleated cells, spleen and thymus coefficient in normal mice caused by 6Q Co (P ⁇ 0.05).
- the H-spore-injected milk high-dose group significantly inhibited the decrease of peripheral blood leukocyte count, bone marrow nucleated cell count d and spleen coefficient caused by ⁇ C 0 (P ⁇ 0.05).
- Table 10 The results are shown in Table 10.
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Description
种灵芝孢子油脂肪乳剂及其质量控制方法与应用 技术领域
本发明涉及灵芝孢子油技术领域，具体的说，涉及一种灵芝孢子油脂肪乳剂及其质 量控制技术和药物用途。
背景技术
从八十年代开始， 日本己开始对灵芝进行抗肿瘤方面的基础研究； 到九十年代， 美 国、 日本等国将灵芝应用至临床， 用于癌症及 AIDS (艾滋病）治疗方面， 得到了国际 研究学者的广泛注意。 灵芝是担子菌纲多孔菌科 (Polyporaceae)灵芝属 (Ganoderma)真菌 赤芝 Glucidum.karst和紫芝 GjaponicumLloyd的总称，具有扶正固本等功效,被《本经》 称为上品。 灵芝孢子 (Ganoderma lucidiumspore)是灵芝生长成熟期从菌盖弹射出来极其 细小的孢子,为灵芝的生殖细胞， 具有灵芝的全部遗传活性物质，其药用价值也正日益受 到重视。 灵芝孢子的化学成分复杂， 有以下几类：脂肪酸类、 醇类、 三萜类、 生物碱 类、 内酯、 蛋白质和氨基酸类、糖肽类、 维生素类、 胡萝卜素、和无机离子类等。 现代 药理研究表明灵芝孢子油是灵芝孢子抗肿瘤的的主力。本研究组通过对灵芝孢子油研究 和测试分析表明，灵芝孢子油主要成分为灵芝孢子油酯类、脂肪酸类、灵芝酸类和麦角 甾醇类等多种成分组成。
现已有以灵芝孢子油为主成分的灵芝孢子油软胶囊上市， 适用于肿瘤的抑制与预 防， 肿瘤术后恢复增强免疫力等。但它是口服剂，服后通过肠胃道吸收起作用，不能直 接进入人体的血液。灵芝孢子油是非水溶性的，制备普通的注射剂是有相当难度的。通 过制备灵芝孢子油脂肪乳剂可解决该问题，本申请人之前申报的名称为 "灵芝孢子 油脂肪乳剂"，专利号为 ZL200410051661.3的中国发明专利正好解决了该问题。专利申 请号为 200510068335.8，名称为"灵芝孢子油静脉乳注射液及其制备方法"的中国发明专 利也公开了一种灵芝孢子油静脉乳注射液。但是这两个专利所公幵的灵芝孢子油脂肪乳 剂均包括了脂肪油或注射用油，存在工艺复杂，活性成分不清楚，没有对注射用的灵芝 孢子油的精制工艺和脂肪乳的化学成分以及质量控制方法做进一步的阐述等缺点。
关于灵芝孢子的产品越来越多，如灵芝孢子油、灵芝孢子及其它们的制剂有灵芝孢 子油软胶囊、灵芝孢子油脂肪乳注射剂、灵芝孢子油脂肪乳口服剂、灵芝孢子胶囊、灵 芝孢子片、灵芝孢子散剂等，但真假难辩，质量优劣难分。关于灵芝孢子油的控制质量 方法已经有不少文献报道，本申请人也申请了几项关于灵芝孢子油质量控制方法的发明 专利， 但是关于灵芝孢子油脂肪乳剂的质量控制方法却未见相关报道。
发明内容
本发明的目的是克服现有灵芝孢子油脂肪乳剂存在的不足， 提供一种活性成 分清楚、 生理活性强的灵芝孢子油脂肪乳剂。
本发明的另一个目的是提供上述灵芝孢子油脂肪乳剂的质量控制方法。
本发明的进一步目的是提供上述灵芝孢子油脂肪乳剂在制备用于治疗肿瘤、提高免 疫作用、 对放化疗药物减毒作用的药物中的应用。
为了实现上述目的， 本发明采用如下技术方案：
一种灵芝孢子油脂肪乳剂， 由如下组分和重量百分数组成：
灵芝孢子油 2〜25%；
乳化剂 0.5〜10%；
等渗剂 0.2〜5¾>；
余量为水; 调节脂肪乳剂的最终 PH为 6〜9。
在上述灵芝孢子油脂肪乳剂中，所述灵芝孢子油优选经过精制的灵芝孢子油，其精 制方法为： 将灵芝孢子油离心， 脱去水分， 加入占灵芝孢子油重量 0.5%〜10%的吸咐 剂， 搅拌均匀， 加热到 40〜70°C， 保温 20〜40分钟， 离心， 精过滤， 得到精制灵芝孢 子油；所述吸附剂为活性碳、硅胶、中性氧化铝、硅藻土、白土中的一种或几种混合物。
在上述灵芝抱子油脂肪乳剂中， 所述乳化剂优选为大豆卵磷脂、 蛋黄卵磷脂、 普 流罗尼、聚甘油棕榈酸二醇或海藻酸盐中的一种或几种的混合物。最佳为蛋 黄卵磷脂或大豆卵磷脂。
在上述灵芝孢子油脂肪乳剂中，所述等渗剂优选为甘油、葡萄糖、木糖醇、麦芽糖 或山梨醇中的一种或几种的混合物， 最佳是甘油。 等渗剂的作用是使制剂的渗 透压接近于人体的生理渗透压。
上述灵芝孢子油脂肪乳剂的第一种质量控制方法，采用高效液相色谱法测定其产品 中含 1,2-油酸 -3-棕榈酸甘油三酯和 /或甘油三油酸酯的含量， 每 lg灵芝孢子油脂肪乳 剂中含 1,2-油酸 -3-棕榈酸甘油三酯的量为 2mg〜62.5mg和 /或含甘油三油酸酯的量为 1.6mg〜50.0mg;
所述的高效液相色谱法是按下述色谱条件： 用十八垸基键合硅胶为填充剂； 以乙 腈、异丙醇、二氯甲垸三种溶剂以任意比例的二元或三元组成的混合液为流动相，流动 相的流速为 0.5〜2.0ml/min; 用蒸发光散射检测器或差示折光率检测器检测； 理论板数 按 1，2-油酸 -3-棕榈酸甘油三酯或甘油三油酸酯峰计算， 均应不低于 2000; 色谱柱温为 10〜50°C。
上述灵芝孢子油脂肪乳剂的第二种质量控制方法，采用高效液相色谱法测定其产品 中麦角甾醇的含量， 每 lg的灵芝孢子油脂肪乳剂中含麦角甾醇的量为 0.04mg〜7.5mg; 麦角甾醇类是菌类植物中特有成分， 灵芝孢子油是目前所了解的唯一的菌类植物油， 因 此麦角留醇可以作为灵芝孢子油区别于其它植物油的特征成分。
所述色谱条件为： 用十八烷基键合硅胶为填充剂； 以甲醇、 乙醇、 乙腈、 甲醇水溶 液、 乙醇水溶液或乙腈水溶液为流动相， 或以甲醇、 乙醇、 乙腈与水的三元或四元组成 混合液为流动相， 或以四氢呋喃和水的混合液为流动相， 四氢呋喃和水的体积比为 75: 25； 检测波长为 280±2mn; 理论板数按麦角甾醇峰计算， 应不低于 2000。
上述灵芝孢子油脂肪乳剂的第三种质量控制方法，釆用高效液相的方法，通过比较
若干批灵芝孢子油脂肪乳剂色谱图，由其共有特征峰构成的灵芝孢子油脂肪乳剂标准指 纹图谱。 所述高效液相的色谱条件优选为:色谱柱采用十八垸基硅烷键合硅胶为填料； 流动相为乙腈-异丙醇， 体积配比为 53: 47； 蒸发光散射检测器； 参照物： 取甘油三油 酸酯对照品；
所述灵芝孢子油脂肪乳剂指纹图谱中， 共有峰有 15个， 其中超过总峰面积 5 %的 指纹峰有 4个，以甘油三油酸酯的色谱峰的相对保留时间为 1计算其它色谱峰的相对保 留时间并计算相对峰面积，上述 4个指纹峰分别是 9号峰平均相对保留时间 RT为 0.778， 相对峰面积范围为 9.54〜15.36%； 10号峰的平均 ΚΓ为 0.832， 相对峰面积范围 5.76〜 9.43 %； 11号峰甘油三油酸酯即参照峰的 ΚΓ为 1.000，相对峰面积范围为 22.29〜27.80 ； 12号峰的平均 RT为 1.075， 相对峰面积范围为 26.82〜37.76%。
本发明所述灵芝孢子油乳的 pH值可以在制备过程中使用氢氧化钠或磷酸盐缓冲溶 液来调节。 最终 pH值在 6〜9之间。
所述灵芝孢子油乳的其它常规质量指标还可以通过本领域已知的方法进行测定，其 中:乳粒的粒径平均分布在 100〜500nm之间;大于 1mm的乳粒数不得过 1%， 并不得检 出大于 5mm的乳粒。
过氧化值:低于 2mmol/kg。
细菌内毒素每 1ml脂肪乳中含内毒素的量应小于 0.5EU。
本发明的灵芝孢子油脂肪乳剂具有很好的药物活性，能单独或与其他药物混合，制' 备用于治疗肿瘤、提高免疫作用、对放化疗药物减毒作用的药物。本发明的灵芝孢子池 f 脂肪乳剂特别适用于动、 静脉注射， 灵芝孢子油直接进入人体血液。 最佳剂型 为注射用乳剂。
与现有技术相比， 本发明具有如下有益效果：
1.本发明的灵芝孢子油脂肪乳剂活性成分清楚，具有很好的生物活性，能单独使用 或与其他抗肿瘤药物联合应用，用于治疗肿瘤、提高机体免疫能力，对于进行放疗和化 疗的肿瘤患者， 可提高其生存质量并减轻治疗后的药物毒副反应。
2.本发明的灵芝孢子油脂肪乳剂特别适用于动、 静脉注射， 灵芝孢子油直 接进入人体血液， 起效快， 吸收较完全。
3.本发明的灵芝孢子油注射脂肪乳是安全性高、 质量可靠， 毒副反应低的产品。
4.制备本发明的灵芝孢子油脂肪乳剂的原料易得， 工艺简单， 可用常规的生 产设备和工艺制备， 适合于具 G M P生产注射剂的药厂大规模地投入批量 生产。
5.本发明的质量控制方法通过准确测定制剂中 1,2-油酸 -3-棕榈酸甘油三酯和甘油 三油酸酯的含量， 作为该产品质量控制的方法和依据， 方法简便， 可操作性强， 重现性 高。
6.本发明的质量控制方法通过准确测定制剂中的麦角甾醇的含量，作为该产品质量 控制的 法和依据， 可操作性强， 重现性高， 特异强。
7.本发明的质量控制方法利用指纹图谱， 方法简便、 稳定、 精密度高、 重现性好、 易于掌握，能从色谱的整体特征面貌上把握灵芝孢子油脂肪乳剂的品种和质量情况，为 灵芝孢子油脂肪乳剂的质量控制和真伪鉴别提供了一种全新的方法。
附图说明
图 1为甘油三酸脂对照品 0~60分钟的高效液相图；
图 2为灵芝孢子油脂肪乳的 0~60分钟的标准指紋图谱；
图 3为 10批灵芝孢子油脂肪乳 0~60分钟的指纹图谱。
具体实施方式
实施例 1〜6
灵芝孢子油的精制： 将灵芝孢子油离心， 脱去水分， 加入占灵芝孢子油重量 5%的 活性碳， 搅拌均勾， 加热到 50Ό，保温 30分钟， 离心， 精过滤， 得到精制灵芝孢子油。
. 制备方法： 在通氮气的条件下， 将乳化剂加入到精制灵芝孢子油中， 高速捣碎至乳化剂完全溶解； 在恒温水浴 50 °C和高速分散乳化 12000rpm 的条件下， 将油相缓缓加入等渗剂水溶液中， 至混合均匀； 用氢氧化钠调 节 pH6〜9 ; 立即将制好的初乳投入勾质机中， 调节均质压力 60Mpa、 均质 次数 8次、 均质温度 60 °C， 均质后用微孔滤膜滤过， 灌装于输液瓶中， 通 入氮气， 加丁基胶塞， 铝盖密封； 灭菌， 即得。 制得产品符合上述各项质 检规定， 并符合药典相关制剂标准。
参照上述制备方法， 实施例 1〜6的配方如表 1所示， 表 1 中所述重量 百分数为重量百分数。 实施例 1〜6的配方
用高效液相色谱法测定：
色谱条件 用十八垸基键合硅胶为填充剂 ； 色谱柱： Kromasil ClSASmn ^SOmn um柱； 柱温： 30°C ; 流动相： 乙腈:异丙醇的体积比为 40:60; 蒸 发光散射检测器检测； 流速： 0.5ml/min。 理论板数按 1,2-油酸 -3-棕榈酸甘油三酯或甘 油三油酸酯峰计算， 均大于 2000;
对照品溶液的制备： 取 1,2-油酸 -3-棕榈酸甘油三酯、甘油三油酸酯对照品 2.98mg、 2.67mg, 置 25ml的量瓶中， 加甲醇溶解并稀释至刻度， 制成每 lml分别含 0.119mg、 0.107 mg的溶液， 即得对照品溶液；
供试品溶液的制备： 精密称取灵芝孢子油脂肪乳剂 （实施例 1 ) 1.025g， 加入 0.2g 无水硫酸钠，于水浴上加热使乳剂破裂，转移至分液漏斗中，以乙醚萃取 3次，每次 2.0ml， 合并乙醚液， 蒸干， 加流动相： 乙腈:异丙醇 （40:60) 溶解， 转移至 100ml量瓶中， 并 定容至刻度， 即得供试品溶液；
测定结果： 分别精密吸取对照品溶液与供试品溶液各 10μ1， 注入高效液相色谱仪， 测定， 取对数计算， 即得。 每 lg乳剂中含 1,2-油酸 -3-棕榈酸甘油三酯、 甘油三油酸酯 分别为 4.21mg、 3.43mg。
符合每 lg 灵芝孢子油脂肪乳剂中含 1,2-油酸 -3-棕榈酸甘油三酯的量为 2mg〜 62.5mg和 I或含甘油三油酸酯的量为 1.6mg〜50.0mg;
这说明实施例 1的脂肪乳剂质量符合要求。 实施例 8 灵芝孢子中 1,2-油酸 -3-棕榈酸甘油三酯、 甘油三油酸酯的含量测定
用高效液相色谱法测定：
色谱条件 用十八垸基键合硅胶为填充剂 ； 色谱柱 ： Kromasil ClSAGmmxSSOmm^um柱； 柱温： 室温； 流动相： 乙腈:二氯甲垸 (59:41), 蒸发光散射 检测器检测； 流速： 1.0ml/min。 理论板数按 1,2-油酸 -3-棕榈酸甘油三酯或甘油三油酸 酯峰计算， 均大于 2000;
对照品溶液的制备： 取 1,2-油酸 -3-棕榈酸甘油三酯、甘油三油酸酯对照品 2.98mg、 2.67mg， 置 25ml的量瓶中， 加甲醇溶解并稀释至刻度， 制成每 lml分别含 0.119mg、 0.107 mg的溶液， 即得对照品溶液；
供试品溶液的制备： 精密称取灵芝孢子油脂肪乳剂（实施例 2 ) 302.45mg, 置索氏 提取器中， 加入乙醚 30ml， 冷浸过夜， 再加入乙醚 50ml， 于水浴加热提取 8小时， 提 取液回收乙醚至尽， 残渣以流动相： 乙腈:二氯甲焼 (59:41 ) 溶解， 转移至 50ml量瓶中， 并定溶至刻度， 即得供试品溶液；
测定结果： 分别精密吸取对照品溶液与供试品溶液各 10μ1， 注入高效液相色谱仪， 测定， 取对数计算， 即得。 每 lg灵芝孢子中含 1,2-油酸 -3-棕榈酸甘油三酯、 甘油三油
酸酯为 59.28mg、 46.52mg。
符合每 lg灵芝孢子油脂肪乳剂中含 1,2-油酸 -3-棕榈酸甘油三酯的量为 2mg〜 62.5mg和 I或含甘油三油酸酯的量为 1.6mg〜50:0mg;
这说明实施例 2的脂肪乳剂质量符合要求。 实施例 9 灵芝孢子油乳剂中麦角甾醇的含量测定
用高效液相色谱法测定：
色谱条件与波长选择用十八烷基键合硅胶为填充剂; 甲醇为流动相； 检测波长为 280nm。 理论板数按麦角甾醇峰计算， 应不低于 2000;
对照品溶液的制备：取麦角甾醇对照品适量，加甲醇制成每 lml含 0.08mg的溶液， 即得对照品溶液；
供试品溶液的制备： 精密称取含灵芝孢子油脂肪乳剂（实施例 1 ) 10g，.加入 2g无 水硫酸钠，于水浴上加热使乳剂破裂，转移至分液漏斗中，以乙醚萃取 3次，每次 20ml， 合乙醚液， 蒸干， 以石油醚 10ml溶解， 加于已处理好的硅胶柱（100〜200目， 10g， 内径 15mm)上， 用石油醚： 乙酸乙酯 （90: 10) 120ml洗脱， 弃去洗脱液， 再用石油 醚： 乙酸乙酯 （80: 20) 120ml洗脱， 收集洗脱液， 蒸干， 残渣用甲醇溶解并转移至 10ml量瓶中， 加甲醇至刻度， .摇匀， 即得供试品溶液。
测定结果： 分别精密吸取对照品溶液与供试品溶液各 10μ1， 注入高效液相色谱仪， 测定， 计算， 即得。 每 lg乳剂中含麦角甾醇 0.6mg。
符合每 lg的灵芝孢子油脂肪乳剂中含麦角甾醇的量为 0.004mg〜7.5mg，这说明实 施例 1的脂肪乳剂质量符合要求。 实施例 10灵芝孢子油脂肪乳剂 HPLC标准指纹图谱
参照物溶液的制备：选用甘油三油酸酯为参照物，取甘油三油酸酯对照品适量，用 流动相稀释， 制成每 lml中含甘油三油酸酯 0.15mg的溶液， 作为参照物溶液。
供试品溶液制备： 精密称取灵芝孢子油脂肪乳剂 （实施例 4) 0.533g， 加入 0.2g无 水硫酸钠，于水浴上加热使乳剂破裂，转移至分液漏斗中，以乙醚萃取 3次，每次 2.0ml， 合并乙醚液， 蒸干， 加流动相： 乙腈:异丙醇 (40:60)溶解， 转移至 100ml量瓶中， 并 定容至刻度， 即得供试品溶液；
分别取参照物溶液及灵芝孢子油乳供试品溶液各 ΙΟμΙ进样， 照高效液相色谱法测 定， 记录 60分钟的色谱图， 见图 1、 2和图 3。 以甘油三油酸酯的色谱峰 (S峰)的相对 保留时间和相对峰面积为 1计算其它色谱峰的相对保留时间和相对峰面积。
共有峰确定：
通过 10批灵芝孢子乳指纹图谱测定， 所得到的灵芝孢子油乳指纹图谱， 可通过 HPLC色谱图进行比较，确定其共有特征峰，得到了由其共有特征峰构成的灵芝孢子油 乳的 HPLC标准指纹图谱。 该标准指纹图谱有 15个特征峰， 各峰的相对保留时间的相
对标准偏差 RSD均小于 2%。 其中 1号峰的平均 RT为 0.133， RSD为 0.31 %, 相对峰 面积范围为 0.10〜2.40%； 2号峰的平均 RT为 0.152， RSD为 0.32%,相对峰面积范围 为 0.31〜17.51 %； 3号峰的平均 RT为 0.239， RSD为 1.18%，相对峰面积范围为 0.14〜 1.20%； 4号峰的平均 ΚΓ为 0.285， RSD为 0.11 %，相对峰面积范围为 0.10〜2.16%； 5 号峰的平均 R 为 0.296， RSD为 0.71 %，相对峰面积范围为 0.19〜2.05%； 6号峰的平 均 T为 0.479， RSD为 0.12%，相对峰面积范围为 0.47〜3.56%； 7号峰的平均 RT为 0.608， RSD为 0.11 %,相对峰面积范围为 1.70〜4.06%； 8号峰的平均 ΚΓ为 0.648, RSD为 0.11 %,相对峰面积范围为 0.34〜1.80%； 9号峰的平均 RT为 0.778， RSD为 0.12%,相对峰面积范围为 9.54〜15.36%； 10号峰的平均 RT为 0.832, RSD为 0.10%， 相对峰面积范围为 5.76〜9.43%； 11号峰甘油三油酸酯即参照峰的 ΚΓ为 1.000，相对峰 面积范围为 22.29〜27.80%； 12号峰的平均 RT为 1.075， RSD为 0.10%，相对峰面积 范围为 26.82〜37.76%； 13号峰的平均 T为 1.158， RSD为 0.21 %，相对峰面积范围 为 1.31〜2.03 %； 14号峰的平均 RT为 1.370， RSD为 0.13%,相对峰面积范围为 1.22〜 1.72%； 15号峰的平均 R 为 1.479， RSD为 0.15%，相对峰面积范围为 0.54〜1.03%。 实施例 11.灵芝孢子油乳稳定性试验：
对实施例 4样品进行 6个月的加速试验，考察条件：放置温度 37Ό ,相对湿度 90%。
(内毒素检查和无菌检查方法按中国药典 2005年版有关规定进行）试验结果见表 2:
表 2 6个月加速试验考察结果
试验结果表明， 本发明的试验样品在加速考察期内质量基本稳定。 实施例 12. 灵芝孢子油乳安全性试验
1.豚鼠全身主动过敏试验（ASA): 试验用 Hartley豚鼠 24只， 按性别和体 重随机分为 4组， 每组雌雄各 3只。 设阴性对照组、 阳性对照组、 受试物低、 高 剂量组。 腹腔注射给药， 给药体积为 0.5mLA ， 隔日一次， 连续五次。 各组动 物分别于末次腹腔注射后第 12天激发，给药体积 2.0mIJ只，并观察动物的反应，
根据过敏反应发生率和发生程度判断其全身主动过敏性。 结果： 阴性对照组 1 例豚鼠出现排尿现象 （判断为生理性的排尿）， 余下 5例豚鼠均未见异常， 所以 阴性对照组过敏反应发生率为 0; 阳性对照组啄鼠全部死亡， 过敏反应发生率为 100%； 受试物低、 高剂量组各有 1例豚鼠出现排尿或排粪现象 （判断为生理性 的排尿或排粪）， 余下豚鼠均未见异常， 所以受试物低、 高剂量组的过敏反应发 生率均为 0; 提示在本试验条件下， 灵芝孢子油注射乳低、 高剂量组 （62.5、 125.0mg/kg.bw ) 对 Hartley豚鼠进行的全身主动过敏性试验未见明显过敏反应。
2.大鼠被动皮肤过敏试验 （PCA): 试验用 SPF级 SD大鼠 24只， 按性别和体重 随机分为 4组， 每组雌雄各 3只。 设阴性对照组、 阳性对照组、 受试物组低、 高剂量 组 (分别为 125和 250mg/kg*bw，按等效剂量折算约分别相当于临床拟用剂量 10g 的 0.14和 0.28倍）。 腹腔注射给药， 隔日一次， 连续五次， 末次致敏后第 10天 采血， 制备抗血清。 另取 SPF级 SD大鼠 24只， 分组和组别设置与上述制备抗体的 方法相同， 各组动物分别皮内注射各对应组的抗血清 O.lmL, 被动致敏 48小时后 进行激发， 结果阴性对照组和受试物低、高剂量组大鼠背部皮肤内层均未见蓝斑， 阳性对照组可见明显蓝斑。 提示在本试验条件下， 灵芝孢子油注射乳对 SD大鼠进 行被动皮肤过敏试验未见明显过敏反应。
3.兔血管刺激性试验： 试验用 4只健康新西兰兔， 采用同体左右侧耳朵自身 · 对比法， 左侧耳缘静咏注射受试药物 （100mg/mL， 约相当于临床静咏滴注拟用 浓度）， 右耳给予等体积的 0.9 %氯化钠注射液作对照， 每天一次， 连续 7天。 末 : 次给药后 48小时剖检 2只动物， 余下 2只动物在末次给药后 14天进行剖检。 肉 眼和镜检结果： 给药侧与对照侧兔耳兔耳缘静脉血管及周围组织均未见病理改 变。提示在本试验条件下， 灵芝孢子油注射乳对兔耳缘静脉及周围组织未见明显 刺激性反应。
4.兔肌肉刺激性试验： 试验用 4只健康新西兰兔， 采用同体左右侧肌肉股四 头肌自身对比法， 左侧给予受试物 （100mg/mL， 约相当于临床静咏滴注拟用浓 度）， 右侧给予等体积 0.9 %氯化钠注射液作对照， l.OmlJ侧， 单次给药。 给药后 48小时剖检 2只动物， 余下 2只动物在给药后 14天进行剖检。 肉眼观察 4只兔 的各侧注射部位肌肉均未见明显异常。 镜检结果： 给药后 48小时剖检的 1只动 物和 14天进行剖检的 2只动物左右侧注射部位肌肉肌纤维排列整齐， 均未见异常变 化。余下一只兔在末次给药后 48小时，右侧局部肌肉组织出现肌纤维小灶性轻度变性， 病灶肌纤维间质可见大量炎细胞浸润，左侧局部肌肉组织出现肌纤维片状变性、坏死甚 至消失，病灶及肌纤维间有大量炎细胞浸润， 局部可见少量红细胞。根据右侧为阴性对 照侧， 亦出现轻度机械性刺激， 结合肉眼观察结果， 左侧局部肌肉组织出现的刺激可 能是机械性刺激。 提示在本试验条件下， 灵芝孢子油注射乳对兔的股四头肌未见 明显刺激性反应。
5.体外溶血试验： 向盛有 2%红血球混悬液的各支药液管中分别加入不等量
的灵芝孢子油注射乳 (lOOmg/mL, 约相当于临床静咏滴注拟用浓度)，灵芝孢子油 注射乳的各支药液管在 3 小时内均未见溶血或红细胞凝集现象。 提示在本试验条件 下， 三个批号的灵芝孢子油注射乳体外溶血性试验为阴性。 实施例 13 灵芝孢子油 Beagle犬急性毒性试验
试验设 4、 6、 8、 12、 16g/kg.bw (分别相当人临床拟用量的 24、 36、 48、 72、 96 倍） 5个剂量， 每个剂量用 Beagle犬一只， 单次静脉给药。 试验结果表明： Beagle犬 单次给药后， 其心电图、 血液学及尿液检查未见明显异常， 血液生化学发生一些改变， 剂量越大持续时间越长； 在给药剂量达到 12、 16g kg.bw时， 动物出现安静、活动减少、 食欲下降等症状。灵芝孢子油乳单次静脉滴注对 Beagle犬的无毒反应剂量为 4g/kg.bw， 耐受量大于 16 g/kg.bw。
实施例 14 灵芝孢子油注射乳 iv给药对小鼠 H22肝癌的实验治疗作用
选用生长良好的 7〜11天的 H22瘤种，接种于小鼠右侧腋部皮下，约 4.5〜5xl06 细 胞 /只， 接种 24小时后随机分笼， 静脉给药。 给药期间每天测定体重， 第 8天处死动 物， 称瘤重， 计算各组平均瘤重， 求出肿瘤抑制率并进行 t检验。
表 3.灵芝孢子油注射乳 iv给药对小鼠 H22肝癌的肿瘤生长抑制作用 土
组别 动物数 体重 （
剂量 mg kg- g) ，田看里 抑制率 （％)'
dl d8 dl d8 (g)
空白乳 10 10 19.9±1.0 26.5±3.2 1.66±0.33
CTX 30 10 10 18.9±1.2 24.5±3.1 0.69±0.21** 58.4 灵芝孢子油
1.0g/kg 10 10 19.0±0.7 26.4±3.3 0.92±0.33** 44.6 , 注射乳
灵芝孢子油
0.5g/kg 10 10 19.6±0.9 27.8±2.8 1.05±0.29** 36.7 注射乳
灵芝孢子油
025g kg 10 10 19.7土 1.2 29.1±2.8 1.31±0.52 21.1 注射乳
dl、 d8= 接种后第 1、 8天；
**尸<0.05 ， 与空白乳组比较。
由表 3的数据表明，灵芝孢子油注射乳能抑制小鼠移植瘤 H22 (肝癌）的肿瘤生长。 实施例 15灵芝孢子油注射乳 iv对人胃腺癌 SGC-7901裸小鼠移植瘤的实验治疗作用 取生长旺盛期的人胃腺癌 SGC-7901裸小鼠移植瘤瘤组织剪切成 1.5 mm3左右， 在 无菌条件下， 接种于裸小鼠右侧腋窝皮下。 裸小鼠移植瘤用游标卡尺测量移植瘤直径， 待肿瘤生长至 100— 300mm3后将动物随机分组。 使用测量瘤径的方法， 动态观察被试 物抗肿瘤的效应。肿瘤直径的测量次数为每周 3次， 每次测量同时还需称鼠重。实验组 静脉给药， 隔天 1次； 阳性对照药为泰素 （TAX) lOmg kg, 隔天 1次； 阴性对照组
为给等体积的空白乳。以相对肿瘤抑制率 T/C (%)来评价药物对肿瘤的生长抑制作用。 试验结果为： 表 4 灵芝孢子油注射乳 iv 给药对人胃腺癌 SGC-7901 裸小鼠移植瘤的实验治疗作
组别 动物数 体重 （g) TV(x±SD) RTV T/C P值 剂量
mg kg
开始 开始 最后 d12 d33 (%)
空白乳 12 12 18.4±1.3 21.8±1.0 157±131 3055±2804 19.01±5.95
TAX 10 6 6 18.0±1.9 19.1±0.8 144+103 1021±537 8.71±3.55 45.8 <0.05 灵芝孢
子油注 lg kg 6 6 19.0±1.0 24.0±1.6 143±56 1508±415 11.02±2.37 58.0 <0.05 射乳
灵芝孢
子油注 0.5g/kg 6 6 18.6±1·1 22.9±1.6 156192 2013±863 14.04±3.24 73.9 射乳
灵芝孢
0.25g k
子油注 6 6 18.2±1.4 23.2±1.2 142 26 2420±630 16.98±2.43 89.3
g
射乳
分笼给药时间
由表 4的数据表明，灵芝孢子油注射乳对人胃腺癌 SGC-7901裸小鼠移植瘤具有抑制作 用。 实施例 16灵芝孢子油注射乳 iv对 H22荷瘤小鼠特异性细胞免疫功能的影响
取 ICR小鼠 50只， 按移植性肿瘤研究法接种 H22实体型瘤 (在无菌操作下取瘤块， 称重， 用玻璃组织匀浆器研磨， 磨匀后放人无菌容器内， 加生理盐水稀释成 1:3的细胞 悬液， 每只小鼠右前肢腋窝皮下接种 0.2 ml)。 次日按体重随机分为 5组， 每组 10只， 雌雄各半， 实验设： 静注对照组 (20 ml/kg， 空白组)， 灵芝孢子油注射乳剂高、 中、 低 剂量组 (1、 0.5、 0.25 g kg) iv给药， 迈普新注射液组 (0.42 mg/kg)Sc给药； 除迈普新为隔 日给药一次外， 其余各组每天给药一次， 共给药 7次， 给药第 1天每鼠腹部去毛,范围 为 3X3 cm2; 给药第 2天,将 1% DNFB均匀涂抹于小鼠腹部 (DNFB为一种半抗原，与皮 肤蛋白结合成全抗原后可刺激 T淋巴细胞增殖成致敏淋巴细胞)。 末次给药后半小时， 将 1% DNFB均匀涂抹于小鼠右耳进行攻击， 使局部产生迟发型变态反应 (水肿)， 攻击 后 24 h， 脱颈椎处死小鼠， 剪下左右耳壳， 用打孔器取下直径 8 mm 的耳片， 称重， 以左右耳片重量之差作为肿胀度， 并计算肿胀抑制率， 比较各组的差别。
灵芝孢子油注射乳剂 iv对 H22荷瘤小鼠延迟性超敏反应的影响 ( X ± SD , η=ιο) 剂量
组别 给药前体重 给药后体重 肿胀度 (mg) 肿胀率 (<¾)
(mg kg) 空白乳对照 19.50±1.35 25.60±2.12 9.16±3.68 62.06±24.11 灵芝孢子油
1000 19.40±1.35 25.50±2.12 12.73±3.70* 88.52±28.94* 注射乳剂
500 19.80±1.69 25.30±2.11 12.84±3.79* 76.53±15.19
250 19.90±1.60 25.10±2.13 11.87±2.30 72.74±11.33 迈普新 0.42 19.30±1.34 25.10±2.18 13.50±3.44* 89.12±19.76*
*P<0.05， **P<0.01 与 H22荷瘤空白乳对照组比较
由表 5的数据表明，灵芝孢子油注射乳高、中剂量组可显著提高小鼠的肿胀度和肿胀率 (P<0.05)o 即灵芝孢子油注射乳可增强 H22荷瘤小鼠的特异性细胞免疫功能。 实施例 17灵芝孢子油注射乳 iv给药对 H22荷瘤小鼠网状内皮系统 (RES)吞噬功能的影） 响
实验方法： 取 ICR小鼠 50只， 接种 H22实体型瘤， 次日按体重随机分为 5组， 每 组 10只， 雌雄各半， 实验设： 静注对照组 (20ml/kg， 空白乳)， 灵芝孢子油注射乳高、 中、 低剂量组 (1、 0.5、 0.25g kg) iv给药， 阳性对照药迈普新注射液组 (0.42mg kg)Sc给 药。 除迈普新为隔日给药一次外， 其余各组每天给药一次， 共给药 11次。 于末次 iv、 ig给药后 24h，尾静脉注射印度墨水 (1 :3稀释) 0.1ml 1 10g，于注入墨水后 1分钟及 5分 钟时用定量采血管从小鼠眼眶后静脉丛取血 20μ1， 立刻吹入 0.1 % Na2C03液 2ml中， 于 680nm处比色， 于 5min采血后立即处死小鼠， 并取肝、 脾、 胸腺称重， 按下式计 算廓清指数 K及吞噬系数 α值； 肝脏系数和脾脏系数。 所得数据进行统计学处理 (t检 验)。
表 6.灵芝孢子油注射乳 iv给药对 H22荷瘤小鼠网状内皮系统 (RES)吞噬功能的影响
( ±5»， n=10) 剂量 肝脏系数 脾脏系数 胸腺系数 组别 廓清指数 k 吞噬系数 α
(mg/kg) (g 100g) (g 100g) (g 100g) 空白乳对照 0.05±0.02 4.98±0.76 6.52 0.98 0.85±0.14 0.24±0.04 灵芝孢子油
注射乳 1000 0.10±0.03** 5.91±1.16* 7.03±1.40 1.00±1.32 0.33±0.12*
500 0.11 ±0.02** 5.38±0.71 7.76±1.56* 1.18±0.46* 0.32±0.10*
250 0.10±0.02** 5.51±1.13 7.03士 1.52 1.55±0.58** 0.27±0.08 阳性对照：
迈普新 0.42 0.09±0.02** 5.71±0.76* 6.91±1.08 1.16±0.44* 0.33±0.11*
*P<0.05, **P<0.01 与 H22荷瘤空白乳对照组比较
由表 6的数据表明， 灵芝孢子油注射乳可显著提高 H22荷瘤小鼠的廓清指数 K 、 吞噬 · 系数 α和脾脏系数， 可增强 Η22荷瘤小鼠网状内皮系统 (RES)吞噬功能。 ' 实施例 18 灵芝孢子油注射乳 iv给药对 H22荷瘤小鼠环磷酰胺 (Cy)化疗的减毒作用 - 实验方法： 取上述规格 ICR小鼠 60只， 接种 H22实体瘤， 接种后 24小时称鼠重， - 并随机分为 6组， 每组 10只， 雌雄各半， 实验设： 空白对照组， 静注对照组 (20mVkg， 空白乳)， 灵芝孢子油注射乳高、 中、 低剂量组 (1、 0.5、 0.25g kg) iv给药， 阳性对照药 迈普新注射液组 (0.42mg/kg)Sc给药。 除迈普新为隔日给药一次外， 其余各组每天给药 一次，共给药 7次，于给药第 4、 5天除空白对照组外，其余各组开始 ip Cy(100mg/kg)， 连续 2d， 于停药后 24h处死动物， 处死前称重， 并由眼眶静脉取血， 镜检外周血白细 胞总数， 同时摘取一侧完整股骨， 分别测定骨髓有核细胞数， 并取胸腺、脾称重， 计算 胸腺、 脾脏器系数， 并进行统计学处理 (t检验)。
实验结果： 结果表明，与 H22荷瘤空白对照组相比， 空白乳 +Cy模型 (100mg kg， ip) 组的外周血白细胞、 骨髓有核细胞数； 胸腺、 脾系数均明显下降 (P<0.01)。 与环磷酰胺
(Cy)模型组相比， 灵芝孢子油注射乳高剂量组及迈普新注射液组皆可显著对抗由 Cy造 成的 H22荷瘤小鼠外周血白细胞、骨髓有核细胞数、胸腺系数和脾脏系数的下降 (PO.05 , P<0.01)。 结果见表 7。
表 7. 灵芝孢子油注射乳 iv给药对 H22荷瘤小鼠化疗的减毒作用 ( 土 ,n=10) 绚别 剂 量 小鼠体重 (g) 脾脏指数 胸腺指数 WBC 骨髓有核细胞
(mg /kg)给药前给药后 (g 100g) (g/100g) (109/L) 数 (105 /根）
19.80士 24.00± 1.29土 0.20± 5.61士 191.03± 空白对照
1.75 2.11 0.47** 0.06** 1.14** 30.65** 空白乳 +Cy 100 19.90士 22.70± 0.48土 0.08土 1.63土 60.23±
1.60 2.16 0.13 0.04 0.40 11.18 灵芝孢子油 1000+100 19.60± 22.30士 0.60± 0.12± 2.02土 73.15土 注射乳 + Cy 1.43 2.11 0.12* 0.04* 0.43*
5C H100 20.00士 23.00± 0.57土 0.10± 1.73士 68.79士
1.83 2.16 0.21 0.03 0.61 12.51
250+100 19.70± 23.00± 0.58土 0.11土 1.91土 61.48土
1.57 2.26 0.16 0.04 0.65 12.50 迈普新 + Cy 0.42+100 19.70± 23.60± 0.64土 0.12土 2.03土 73.31土 - n i
1.57 2.27 0.21* 0.04* 0.44*
*P< 0.05， **P< 0.01与空白乳 +Cy组比较
由表 7的数据表明， 灵芝孢子油注射乳对 H22荷瘤小鼠环磷酰胺 (Cy)化疗具有减毒作 用。 实施例 19灵芝孢子油注射乳 给药对正常小鼠化疗 (Cy)的减毒作用
实验方法： 取上述规格 ICR小鼠 60只， 按体重随机分为 6组， 每组 10只， 雌雄 各半， 实验设： 空白对照组， 静注对照组 (20ml/kg， 空白乳)， 灵芝孢子有注射乳高、 中、 低剂量组 (1、 0.5、 0.25g/kg) iv给药， 阳性对照药迈普新注射液组 (0.42mg kg)Sc给药。 除迈普新为隔日给药一次外， 其余各组每天给药一次， 共给药 7次。 于给药第 4、 5天 除空白对照组外， 其余各组开始 ip Cy(100mg kg)， 连续 2d， 于停药后 24h处死动物， 处死前称重， 并由眼眶静脉取血， 镜检外周血白细胞总数， 同时摘取一侧完整股骨， 分 别测定骨髓有核细胞数， 并取胸腺、 脾称重， 计算胸腺、 脾脏器系数， 并进行统计学处 理 (t检验)。
实验结果： 结果表明， 与空白对照组相比， 空白乳 +Cy模型 (100mg/kg， ip)组的外 周血白细胞、 骨髓有核细胞数， 胸腺、 脾系数均明显下降 (P<0.01)。 与空白乳 +Cy组相 比，灵芝孢子油注射乳高剂量组及迈普新注射液组皆可显著对抗由 Cy造成的正常小鼠 外周血白细胞、 骨髓有核细胞数和脾脏系数的下降 (P<0.05)， 灵芝孢子油注射乳中剂量 组可显著对抗由 Cy造成的正常小鼠骨髓有核细胞数和脾脏系数的下降 (PO.05), 结果 见表 8。
表 8. 灵芝孢子油注射乳 iv给药对正常小鼠化疗的减毒作用 ±S )， n=10) 组别 剂 量 小鼠体重 (g) 脾脏指数胸腺指数 WBC 骨髓有核细胞数
(g 100g) (g 100g) (109 L) (105 /根）
(mg kg) 给贿 给药后
0.55土 0.32土 10.67士 200.18士 宇白对昭
19.40±1.0725.20±2.15 3.03** 21.17** p p
0.28 *土 * 0.12土 62· 14土 空白乳 +Cy *
y 100 19.8G±1.3223.20±2.20 0.06 0.04 14.57
p
灵芝孢子油 0.34土 0.12土 * 1.98土 77.29土 注射乳 + Cy 1000+10019.1Ό±1.2023.00±2.11 0.06* 0.06 0.26*
p
0.35土 0.13士 1.75士 * 74.89士
500+100 19.90±1.3723.30±2.11 0.08* 0.07 0.55 11.44*
0.35土 0.13士 1.61土 65.寸73士
25O -10O 19.30±1.1623.10±2.13 0.08* 0.05 0.46 19.41
0.36士 0.18土 2.04土 75.43土 迈普新 + Cy 0.42+10019.60±1.2622.90±2.18 0.03** 13.32*
*P< 0.05， **P< 0.01与空白乳 +Cy组比较
由表 8的数据表明， 灵芝孢子油注射乳对正常小鼠环磷酰胺 (Cy)化疗具有减毒作用。 实施例 20.灵芝孢子油注射乳 iv给药对 H22荷瘤小鼠 6QCo放疗的减毒作用
实验方法:取上述规格 ICR小鼠 60只， 按移植性肿瘤研究法接种 H22实体瘤， 于接 种后 24^， 除空白对照组 10只动物外， 其余 50只小鼠进行 6QCo照射， 剂量为 500rad; ^Co照射后动物按体重随机分为 5组， 每组 10只， 雌雄各半， 实验设： 空白对照组， 静注对照组 (20ml kg， 空白乳)， 灵芝孢子油注射乳高、 中、低剂量组 (1、 0.5、 0.25g/kg) iv给药， 阳性对照药迈普新注射液组 (0.42mg kg)Sc给药。各组动物于接种后 24h给药， 除迈普新为隔日给药一次外， 其余各组均为每天给药一次， 共给药 7次， 并于停药后 24h处死动物， 处死前称重， 并由眼眶静脉取血， 镜检外周血白细胞总数， 同时摘取脾
脏、 胸腺和一侧完整股骨， 分别测定骨髓有核细胞数， 称脾脏、 胸腺重， 计算脾脏指数 和胸腺指数， 并进行统计学处理 (t检验)。
实验结果:结果表明， 与 H22荷瘤空白对照组相比， 空白乳十60。模型 (500rad)组的 外周血白细胞、 骨髓有核细胞数、 胸腺、 脾系数均明显下降 (P<0.01)。 与60 Co模型组相 比， 迈普新注射液组可显著地对抗由 6GC0造成的 H22荷瘤小鼠外周血白细胞、 骨髓有 核细胞数、脾脏和胸腺系数的下降 (P<0.05)， 灵芝孢子油注射乳 iv给药高剂量组可显著 对抗由 ^Co造成的 H22荷瘤小鼠外周血白细胞数、 骨髓有核细胞数和脾脏系数的下降 (P<0.05)，灵芝孢子油注射乳 iv给药中剂量组可显著对抗由 6QCo造成的 H22荷瘤小鼠外 周血白细胞数的下降 (P<0.05)。 结果见表 9。 表 9灵芝孢子油注射乳 iv 给药对 H22荷瘤小鼠60 Co放疗的减毒作用(_? ±^， n=10) 剂 量 小鼠体重 (g)脾脏指数胸腺指数 WBC 骨髓有核细胞数 (mg /kg) ^贿 ^ (g/100g) (g/100g) (109 /L) (105 /根）
19.40± 24.50± 0.62土 P o 239.48士 空白对照
1.26 2.37 0.20** 53.92** 空白乳 60。。 100 19.90± 24.10± 0.25士 0.12土 1.63士 68.31±
1.45 2.13 0.08 0.04 0.40 16.68
^Co 灵芝孢
1000+10020.00± 24.60± 0.33士 0.14土 2.02士 85.60± 子油注射乳
1.63 2.41 0.08* 0.05 0.41* 18.60*
500+100 19.70± 24.10± 0.29士 0.14土 2.03士 79.05士
1.57 2.13 0.03 0.03 0.42* 16.51
250+100 19.60± 24.30± 0.29土 0.12± 1.94± 79.93土
1.07 2.21 0.05 0.03 0.54 17.37
^Co +迈普新 0.42+100 19.60± 24.20士 0.33土 0.17土 1.98士 82.71土
1.51 2.25 0.04* 0.07* 0.31*
*Ρ< 0.05， **Ρ< 0.01与空白乳 +6()Co组比较 由表 9的数据表明，灵芝孢子油注射乳 iv给药对 H22荷瘤小鼠 ""Co放疗具有减毒作用。
实施例 21灵芝孢子油注射乳 iv给药对正常小鼠 δ()α>放疗的减毒作用
实验方法:取上述规格 ICR小鼠 60只， 除空白对照组 10只动物外，其余 50只小鼠 进行 6QCo照射， 剂量为 500rad; 6QC0照射后动物按体重随机分为 5组， 每组 10只， 雌
雄各半， 实验设： 空白对照组， 静注对照组 (20ml/kg， 空白乳)， 灵芝孢子油注射乳高、 中、 低剂量组 (1、 0.5、 0.25g kg) iv给药， 阳性对照药迈普新注射液组 (0.42mg kg， )Sc 给药。各组动物于接种后 24h给药，除迈普新为隔日给药一次外，其余各组均为每天给 药一次， 共给药 7次， 并于停药后 24h处死动物， 处死前称重， 并由眼眶静脉取血， 镜 检外周血白细胞总数，同时摘取脾脏、胸腺和一侧完整股骨，分别测定骨髓有核细胞数， 称脾脏、 胸腺重， 计算脾脏指数和胸腺指数， 并进行统计学处理 (t检验)。
实验结果： 结果表明， 与空白对照组相比， 空白乳十60。。模型 (500rad)组的外周血 白细胞、骨髓有核细胞数、 胸腺、 脾系数均明显下降 (P<0.01)。 与 ^Co模型组相比， 迈 普新注射液组可显著地对抗由 6QCo造成的正常小鼠外周血白细胞、 骨髓有核细胞数和 脾脏、胸腺系数的下降 (P<0.05)，灵芝 * H孢- 子油注射乳高剂量组可显著地对抗由∞C0造成 的正常小鼠外周血白细胞数、骨髓有核细胞数 d和脾脏系数的下降 (P<0.05)，结果见表 10。
p o
表 10.灵芝孢子油注射乳 iv给药对正常小鼠 * *
H *- 60 Co放疗的减毒作用 ( n=10) 组别 剂 量 ： 小鼠体重 (g) 脾脏指数胸腺指数 WBC 骨髓有核细
(mg/kg) 给药前 给药后 (g 100g) (g 100g) (109 /L) 胞数 (105 /根）
19.60± 0.50± 0.30± 4.27士 221.90士 空白对照
1.35 0.16** 0.13** 1.22** 63.09**
100 19.40± 22.60± 0.18士 0.09士 0.87士 54.95士 '
1.43 2.12 0.05 0.04 0.16 14.22
60Co +灵
芝孢子油 1000+100 19.70土 23.40± 0.26士 0.13士 1.09± 66.99士 注射乳 1.25 2.12 0.09* 0.06 0.29* 11.21*
50(H100 19.70±1. 22.60± 0.28士 0.09士 0.96± 63.04土
42 2.27 0.04 0.35 13.17
250+100 19.50± 22.80± 0.28土 0.10± 0.98± 55.89士
1.65 2.25 0.04 0.15 13.43
60Co +迈
0.42+100 19.90士 23.30士 0.14士 1.21士 67.83土 普新
1.66 2.11 0.05* 0.38* 13.09* 叩< 0.05， **P< 0.01与 ^Co +空白乳组比较
由表 10的数据表明， 灵芝孢子油注射乳 iv给药对正常小鼠 6QCo放疗具有减毒作用。
Claims
1.一种灵芝孢子油脂肪乳剂， 其特征在于由如下组分和重量百分数组成：
精制灵芝孢子油 2〜25%;
乳化剂 0.5〜10%；
等渗剂 0.2〜5%；
余量为水；
其中所述脂肪乳剂的最终 pH为 6〜9。
2.根据权利要求 1所述的灵芝孢子油脂肪乳剂，其特征在于所述精制灵芝孢子油是 指将提取后的灵芝孢子油进行精制， 其精制方法为：
将灵芝孢子油离心， 脱去水分， 加入占灵芝孢子油重量 0.5%〜10%的吸咐剂， 搅 拌均匀， 加热到 40~70Ό， 保温 20〜40分钟， 离心， 精过滤， 得到精制灵芝孢子油； 所述吸附剂为活性碳、 硅胶、 中性氧化铝、 硅藻土、 白土中的一种或几种混合物。
3.根据权利要求 1所述的灵芝孢子油脂肪乳剂，其特征在于所述乳化剂为大豆卵磷 脂、 蛋黄卵磷脂、 普流罗尼、 聚甘油棕榈酸二醇或海藻酸盐中的一种或几种 的混合物。
4.根据权利要求 1所述的灵芝孢子油脂肪乳剂， 其特征在于所述等渗剂为甘油、葡 萄糖、 甘露醇、 麦芽糖或山梨醇中的一种或几种的混合物。
5.—种权利要求 1所述灵芝孢子油脂肪乳剂的质量控制方法， 其特征在于， 采用高 效液相色谱法测定其产品中含 1,2-油酸 -3-棕榈酸甘油三酯和 I或甘油三油酸酯的含量， 每 lg灵芝孢子油脂肪乳剂中含 1, 2-油酸- 3-棕榈酸甘油三酯的量分别为 2mg〜62. 5mg和' I或含甘油三油酸酯的量分别为 1. 6mg〜50. Omg;
所述的高效液相色谱法是按下述色谱条件： 用十八垸基键合硅胶为填充剂； 以乙 腈、异丙醇、二氯甲烷三种溶剂以任意比例的二元或三元组成的混合液为流动相，流动 相的流速为 0.5〜2.0πι1/πιΐη; 用蒸发光散射检测器或差示折光率检测器检测； 理论板数 按 1,2—油酸一 3—棕榈酸甘油三酯或甘油三油酸酯峰计算， 均应不低于 2000; 色谱柱 温为 10〜50°C。
6.—种权利要求 1所述灵芝孢子油脂肪乳剂的质量控制方法，其特征在于，采用高 效液相色谱法测定其产品中麦角甾醇的含量，每 lg的灵芝孢子油脂肪乳剂中含麦角甾醇 的量为 0. 04mg~7. 5mg;
所述色谱条件为： 用十八烷基键合硅胶为填充剂； 以甲醇、 乙醇、 乙腈、 甲醇水溶 液、 乙醇水溶液或乙腈水溶液为流动相， 或以甲醇、 乙醇、 乙腈与水的三元或四元组成 混合液为流动相， 或以四氢呋喃和水的混合液为流动相， 四氢呋喃和水的体积比为 75: 25； 检测波长为 280±2nm; 理论板数按麦角 醇峰计算， 应不低于 2000。
7.—种权利要求 1所述灵芝孢子油脂肪乳的质量控制方法， 其特征在于， 采用高效 液相的方法，通过比较若干批灵芝孢子油脂肪乳剂色谱图， 由其共有特征峰构成的灵芝 孢子油脂肪乳剂标准指纹图谱。
8.根据权利要求 7所述的标准指紋图谱，其特征在于，所述高效液相的色谱条件为: 色谱柱采用十八綜基硅烷键合硅胶为填料；流动相为乙腈-异丙释， #积配¾¾5 ： 47 ; 蒸发光散射检测器； 参照物： 取甘油三油酸酯对照品； ' .
所述灵芝孢子油脂肪乳剂指纹图谱中， 共有峰有 15个， 其中超过总峰面积 5 %的 指纹峰有 4个，以甘油三油酸酯的色谱峰的相对保留时间为 1计算其它色谱峰的相对保 留时间并计算相对峰面积，上述 4个指纹峰分别是 9号峰平均相对保留时间 ΚΓ为 0.778， 相对峰面积范围为 9.54-15.36% ; 10号峰的平均 ΚΓ 为 0.832， 相对峰面积范围为 5.76-9.43 %； 11 号峰甘油三油酸酯即参照峰的 RT 为 1.000， 相对峰面积范围为 22.29-27.80%； 12号峰的平均 R 为 1.075， 相对峰面积范围为 26.82~37.76%。
9.权利要求 1所述灵芝孢子油脂肪乳剂在制备用于治疗肿瘤、提高免疫作用、对放 化疗药物减毒作用的药物中的应用。
10.根据权利要求 9所述的应用， 其特征在于， 所述药物的剂型为注射用乳剂。
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| JP2010547022A JP2011513208A (ja) | 2008-02-26 | 2008-02-26 | レイシ胞子油脂肪乳剤、その品質管理方法および薬物調整への応用方法 |
| PCT/CN2008/000396 WO2009105912A1 (zh) | 2008-02-26 | 2008-02-26 | 一种灵芝孢子油脂肪乳剂及其质量控制方法与应用 |
| US12/918,949 US20110059124A1 (en) | 2008-02-26 | 2008-02-26 | The quality control method and application of a kind of ganoderma lucidum spore oil fat emulsion |
| KR1020107020997A KR20100129303A (ko) | 2008-02-26 | 2008-02-26 | 영지 포자유 지방 유제류의 품질 관리 방법 및 용도 |
| DE112008003744T DE112008003744T5 (de) | 2008-02-26 | 2008-02-26 | Fettemulsion, umfassend Ganoderma-Sporenöl, Verfahren zur Qualitätskontrolle und Verwendung davon |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102967682A (zh) * | 2012-12-14 | 2013-03-13 | 广州白云山汉方现代药业有限公司 | 一种灵芝孢子油或灵芝孢子粉或其制剂的质量检测方法 |
| CN109212085A (zh) * | 2018-10-22 | 2019-01-15 | 广州白云山汉方现代药业有限公司 | 一种灵芝孢子油的指纹图谱及其构建方法和应用 |
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| CN106501439B (zh) * | 2016-09-13 | 2019-03-12 | 广州神农生物技术有限公司 | 一种赤灵芝提取物对照品及其制备方法和应用 |
| CN109932441B (zh) * | 2019-03-01 | 2022-08-19 | 贵州瑞和制药有限公司 | 一种舒肝宁注射液hplc指纹图谱的建立方法 |
| CN110261506A (zh) * | 2019-06-26 | 2019-09-20 | 广东省微生物研究所（广东省微生物分析检测中心） | 灵芝挥发性成分的hs-spme/gc-ms特征图谱及其构建方法和应用 |
| CN111579662A (zh) * | 2020-05-11 | 2020-08-25 | 上海市农业科学院 | 一种灵芝孢子粉及其产品中甾醇和脂肪酸类成分的hplc测定方法 |
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| WO2024192708A1 (zh) * | 2023-03-22 | 2024-09-26 | 北京同仁堂国药有限公司 | 一种灵芝活性成分在制备治疗皮肤氧化损伤的药物中的应用 |
| CN117030871B (zh) * | 2023-06-30 | 2024-02-27 | 济南辰欣医药科技有限公司 | 一种检测棕榈酸帕利哌酮中棕榈酸的方法 |
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| CN109212085A (zh) * | 2018-10-22 | 2019-01-15 | 广州白云山汉方现代药业有限公司 | 一种灵芝孢子油的指纹图谱及其构建方法和应用 |
| CN109212085B (zh) * | 2018-10-22 | 2021-05-25 | 广州白云山汉方现代药业有限公司 | 一种灵芝孢子油的指纹图谱及其构建方法和应用 |
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| Publication number | Publication date |
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| US20110059124A1 (en) | 2011-03-10 |
| JP2011513208A (ja) | 2011-04-28 |
| KR20100129303A (ko) | 2010-12-08 |
| DE112008003744T5 (de) | 2011-04-14 |
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