TH26723Y - Methods for preparing extracts containing diterpenoid compounds with anti-inflammatory activity from the leaves, rhizomes, roots, or mixtures of these of Kaempferia koratensis Picheans., Zingiberaceae. - Google Patents

Abstract

This invention relates to a method for preparing extracts containing anti-inflammatory diterpenoid compounds from the leaves, rhizomes and roots, or mixtures of these of *Kaempferia galanga*. This involves raw material preparation, extraction using fermentation or reflux extraction under the invented conditions, evaporation, and separation, including the use of High performance liquid chromatography (HPLC) to analyze the fingerprints and chemical composition for use in controlling the quality of herbal raw materials, enabling accurate harvesting and purchasing of herbs, as well as the separation of pure compounds and analysis of the pure diterpenoid compound structures using both one- and two-dimensional nuclear magnetic resonance spectroscopy and high-resolution mass spectrometry (HRMS). The extracts obtained from this method contain the anti-inflammatory active compounds roscorane B, 1alpha,11alpha-dihydroxyhydropimara-8(14),15-diin. (1alpha,11alpha-dihydroxypimara-a-8(14),15-diene), kaempulchraol I, and geranylgeraniol derivative are the main components and compounds 1-9 are components.

Claims (10)

1. The preparation method for extracts containing diterpenoid compounds with anti-inflammatory activity from the leaves, rhizomes, roots, or mixtures of Kaempferia koratensis involves the following steps: a. Selecting mature Kaempferia koratensis plants, cleaning them, separating the leaves, rhizomes, roots, or mixtures, and drying them. Then, the remaining leaves, rhizomes, roots, or mixtures of Kaempferia koratensis that are not completely dried are further processed to obtain finely minced pieces. b. Maceration of the finely minced leaves, rhizomes, roots, or mixtures of Kaempferia koratensis from step a in a 50% to 95% by weight ethanol solution. The resulting solution is then filtered to remove the solids, and the solvent is evaporated using an evaporator. This step is repeated at least twice. To obtain substances from the leaves, rhizomes, and roots, or mixtures thereof, of *Kaempferia galanga*, significantly free of ethanol as a solvent: C. Add a mixture of chloromethane and methanol (1:1) to the substances from the leaves, rhizomes, and roots, or mixtures thereof, of *Kaempferia galanga*, significantly free of ethanol as a solvent, from step B. Then, ferment and evaporate the mixture of chloromethane and methanol (1:1) using the conditions specified in step B. to obtain crude extracts from the leaves, rhizomes, and roots, or mixtures thereof, of *Kaempferia galanga*. 2. The process under Patent 1, whereby the age of the galangal used in the preparation of the extract is 8-10 months from the time of planting until the date of harvest; cleaning of the galangal is done using water; and grinding of the leaves, rhizomes and roots, or a mixture of those of the still-dry galangal to a small size. 3. The process under claim 1 or 2 in which the leaves, rhizomes and roots, or mixtures of those small pieces of Curcuma longa from claim a. are fermented in an ethanol solution for at least 48 hours. 4. Any of the procedures under claims 1 through 3 in which step b involves evaporation of the solvent using an evaporator at a temperature of 40 to 50 degrees Celsius. 5. Any one of the three methods 1 through 4 in which the ratio of leaves, rhizomes and roots, or a mixture of those of *Pterocarpus indicus* to ethanol solvent or a mixture of dichloromethane and methanol (1:1) is 1:2-5 by weight to volume. 6. Any one of the processes under claims 1 through 5 may be selected to dry the crude extract using a vacuum press for 8 to 16 hours. 7. Any one of the six claims under the following process: In step B, the fermentation of small-sized leaves, rhizomes, and roots, or mixtures thereof of Curcuma longa from claim A, may be selected to be extracted by retlux extraction at a temperature in the range of 40 to 80 degrees Celsius for a continuous retlux period of at least 8 to 12 hours. 8. Any of the procedures under claims 1 through 7, which include the following steps: i. Separation of active compounds from the leaves, rhizomes, and roots, or mixtures of these of *Kaempferia galanga*, using chromatography techniques, both normal phase and reverse phase, and elution by molecular filtration through a Scphadex LH-20 column, gel, and high-performance liquid chromatography (HPLC). The mobile phase uses a solvent mixture for elution through the column. ii. Collection of fractions by identifying the presence of diterpenoid compounds in each fraction using thin-layer chromatography (TLC) and staining with Godin reagent stain, where the substance exhibits the characteristic shading properties. The dyes and UV absorption at 254 nm and 366 nm are the same, allowing for the formation of a single fraction to obtain diterpenoid compounds. iii. Chemical structure determination using spectroscopic techniques including nuclear magnetic resonance spectroscopy (VMR) and high-precision mass spectrometry (HRMS) in the active fraction from the leaves, rhizomes and roots, or mixtures of these of *Phra Khorat*, which are diterpenoid compounds. 9. The procedure under claim 7 where the mixture of solvents of the mobile phase for column elution shall be hexane with dichloromethane, hexane with ethyl acetate, dichloromethane with methanol, ethyl acetate with methanol, methanol with water, or acetonitrile with water in proportions between 0 and 100% by volume. 10. A compound containing diterpenoid compounds from the leaves, rhizomes, and roots, or a mixture of those compounds of Curcuma longa obtained by any of the methods specified in the prior claims, and pharmaceutically acceptable additives.