Claims for Patent: 9,849,118

Summary for Patent: 9,849,118

Title:	Protein tyrosine phosphatase inhibitor, preparation method and uses thereof
Abstract:	Disclosed in the present invention is a protein tyrosine phosphatase inhibitor. The preparation method therefor is: extracting the crude product from the Isaria Fumosorosea Wize solid or liquid fermentation broth using ethyl acetate, ethanol, methanol, or a mixed solvent of chloroform and methanol; separating the obtained extract using column chromatography on silica gel; and obtaining the target product. The inhibitor can be used to prepare pharmaceutical compositions for treating and preventing diabetes, obesity and cancers.
Inventor(s):	Luo; Duqiang (Baoding, CN), Zhang; Jun (Baoding, CN), Liu; Zhiqin (Baoding, CN)
Assignee:	Heibei University (CN)
Application Number:	14/403,113
Patent Claims:	1. A method for preparing a substantially isolated protein tyrosine phosphatase inhibitor having the following structural formula: ##STR00003## the method comprising: extracting a solid or liquid fermentation broth of entomogenous fungus Isaria Fumosorosea Wize with ethyl acetate, ethanol, methanol, or a mixed solvent of chloroform and methanol; separating the resulting extract by column chromatography on silica gel; and obtaining the substantially isolated protein tyrosine phosphatase inhibitor, Fumosorinone. 2. The method for preparing the protein tyrosine phosphatase inhibitor according to claim 1, further comprising: A. cultivating and preparing a fermentation broth by: inoculating the Isaria Fumosorosea Wize strain on a modified potato dextrose culture medium with an inoculation needle; culturing the mixture in a shaker at 150 rpm and at 26.degree. C. for 7 days to obtain a seed broth; further inoculating the seed broth on a rice culture medium and culturing under light at 26.degree. C. for 30 days to obtain a fermented product; wherein, the modified potato dextrose culture medium is formulated by the following steps: dissolving 200 parts by weight of peeled potato, 20 parts by weight of dextrose, 3 parts by weight of KH2PO.sub.4, 1.5 parts by weight of MgSO.sub.4, 0.1 parts by weight of citric acid, and 0.01 parts by weight of vitamin Bi in 1000 parts by weight of sterile water, and adjusting pH to 6.5 and sterilizing at 121.degree. C. for 20 minutes; and the rice culture medium is formulated by the following steps: dissolving 80 parts by weight of rice in 100 parts by weight of sterile water, soaking for 12 hours, and then sterilizing at 121.degree. C. for 30 minutes; B. extracting and separating the fermented product by: extracting the fermented product by soaking it in an equal volume of ethyl acetate at room temperature for 48 hours; evaporating to dryness by vacuum distillation with a rotatory evaporator at 45.degree. C. to obtain an extractum sample; mixing said extractum sample with 1.2 times by weight of silica gel having a mesh size of 100 to 200 under stirring; drying and grinding uniformly to obtain a powder, thereby absorbing said extractum sample onto particles of the silica gel; then performing atmospheric pressure column chromatography on silica gel having a mesh size ranging from 300 to 400, and eluting gradiently with petroleum ether/ethyl acetate system in ratios of 100:0, 95:5, 90:10, 80:20, 60:40 and 50:50 (v/v); collecting the eluents, evaporating by vacuum distillation at 45.degree. C. concentrating and washing with an organic solvent; further subjecting the eluents with 80:20 of petroleum ether/ethyl acetate to the atmospheric pressure column chromatography on silica gel, and eluting gradiently with petroleum ether/ethyl acetate system in ratios of 100:0, 50:1, 20:1, 15:1, 10:1 and 5:1 (v/v) again; collecting the eluents, evaporating by vacuum distillation at 45.degree. C. to obtain the protein tyrosine phosphatase inhibitor, Fumosorinone, at the 5:1 elution section. 3. The method for preparing the protein tyrosine phosphatase inhibitor according to claim 1, further comprising: A. cultivating and preparing a fermentation broth of Isaria Fumosorosea Wize by: inoculating the Isaria Fumosorosea Wize strain on a modified potato dextrose culture medium with an inoculation needle; culturing the mixture in a shaker at 150 rpm and at 26.degree. C. for 7 days to obtain a seed broth; further inoculating the seed broth on a rice culture medium and culturing under light at 26.degree. C. for 30 days to obtain a fermented product; wherein, the modified potato dextrose culture medium is formulated by the following steps: dissolving 200 parts by weight of peeled potato, 20 parts by weight of dextrose, 3 parts by weight of KFI2PO.sub.4, 1.5 parts by weight of MgSO.sub.4, 0.1 parts by weight of citric acid, and 0.01 parts by weight of vitamin B-1 in 1000 parts by weight of sterile water, and adjusting pH to 6.5 and sterilizing at 121.degree. C. for 20 minutes; and the rice culture medium is formulated by the following steps: dissolving 80 parts by weight of rice in 100 parts by weight of sterile water, soaking for 12 hours, and then sterilizing at 121.degree. C. for 30 minutes; B. extracting and separating the fermented product by: extracting the fermented product by soaking it in equal volume of ethyl acetate at room temperature for 48 hours; evaporating to dryness by vacuum distillation with a rotatory evaporator at 45.degree. C. to obtain an extractum sample; mixing said extractum sample with 1.2 times by weight of silica gel having a mesh size of 100 to 200 under stirring; drying and grinding uniformly to obtain a powder, thereby absorbing said extractum sample onto particles of the silica gel particles; then performing atmospheric pressure column chromatography on silica gel having a mesh size ranging from 300 to 400, and eluting gradiently with petroleum ether/ethyl acetate system in ratios of 100:0, 95:5, 90:10, 80:20, 60:40 and 50:50 (v/v); collecting the eluents, evaporating by vacuum distillation at 45.degree. C., concentrating and washing with an organic solvent; combining the eluents from each elution section, and performing the atmospheric pressure column chromatography on silica gel again, and eluting gradiently with petroleum ether/ethyl acetate system in ratios of 100:0, 50:1, 20:1, 15:1, 10:1 and 5:1 (v/v) again; collecting the eluents, evaporating by vacuum distillation at 45.degree. C.; combining the eluents from each elution section again, and repeating the gradient elution with petroleum ether/ethyl acetate in ratios of 100:0, 50:1, 20:1, 15:1, 10:1 and 5:1 (v/v) to obtain the protein tyrosine phosphatase inhibitor, Fumosorinone, at the 5:1 elution section. 4. The method for preparing the protein tyrosine phosphatase inhibitor according to claim 1, further comprising: A. cultivating and preparing a fermentation broth of Isaria Fumosorosea Wize by: inoculating the Isaria Fumosorosea Wize strain on a modified potato dextrose culture medium with an inoculation needle; culturing the mixture in a shaker at 150 rpm and at 26.degree. C. for 7 days to obtain a seed broth; further inoculating the seed broth on a rice culture medium and culturing under light at 26.degree. C. for 30 days to obtain a fermented product; wherein, the modified potato dextrose culture medium is formulated by the following steps: dissolving 200 parts by weight of peeled potato, 20 parts by weight of dextrose, 3 parts by weight of KH.sub.2PO.sub.4, 1.5 parts by weight of MgSO.sub.4, 0.1 parts by weight of citric acid, and 0.01 parts by weight of vitamin Bi in 1000 parts by weight of sterile water, and adjusting pH to 6.5 and sterilizing at 121.degree. C. for 20 minutes; and the rice culture medium is formulated by the following steps: dissolving 80 parts by weight of rice in 100 parts by weight of sterile water, soaking for 12 hours, and then sterilizing at 121.degree. C. for 30 minutes; B. extracting and separating the fermented product by: extracting the fermented product by soaking it in equal volume of ethyl acetate at room temperature for 48 hours; evaporating to dryness by vacuum distillation with a rotatory evaporator at 45.degree. C. to obtain an extractum sample; mixing said extractum sample with 1.2 times by weight of silica gel having a mesh size of 100 to 200 under stirring; drying and grinding uniformly to obtain a powder, thereby absorbing said extractum sample onto particles of the silica gel particles; then performing atmospheric pressure column chromatography on silica gel having a mesh size ranging from 300 to 400, eluting gradiently with petroleum ether/ethyl acetate system in ratios of 100:0, 95:5, 90:10, 80:20, 60:40 and 50:50 (v/v); collecting the eluents, evaporating by vacuum distillation at 45.degree. C., concentrating and washing with an organic solvent; further subjecting the eluents obtained from the above elution section with 80:20 of petroleum ether/ethyl acetate to the atmospheric pressure column chromatography on silica gel, and eluting gradiently with chloroform/methanol system in ratios of 100:0, 95:5 and 90:10 (v/v) to obtain the protein tyrosine phosphatase inhibitor, Fumosorinone, at the 90:10 elution section.

Details for Patent 9,849,118

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Merck Sharp & Dohme Corp.	ZOSTAVAX	zoster vaccine live	For Injection	125123	05/25/2006		2032-05-22