Method of preparing a therapeutic composition using a preparation from epidermal gel secretions of catfish

Abstract

A method of preparing a therapeutic composition from the epidermal gelatinous secretion of catfish includes mixing catfish epidermal gel secretions (EGS) with phosphate buffered saline (pH 7.5) to provide an extract, homogenizing the extract to provide a homogenized extract, and centrifuging the homogenized extract to provide a soluble protein fraction and an insoluble protein fraction. The therapeutic composition can include about 87% soluble proteins and about 13% lipids.

Claims

1. A method for preparing a therapeutic composition adapted for administration to a patient in need thereof by intraperitoneal (IP) or sub-cutaneous (SC) injection, the method consisting of: collecting epidermal gel secretions of an Arabian Gulf catfish; fractionating the epidermal gel secretions to provide a soluble protein fraction and an insoluble protein fraction, the fractionation comprising: mixing the epidermal gel secretions with an extraction buffer to provide an extract, wherein the extraction buffer includes 0.05 M phosphate buffered saline, pH 7.5 containing 0.14 M NaCl; homogenizing the extract with a homogenizer to provide a homogenate; and centrifuging the homogenate to provide the soluble fraction and the insoluble fraction, wherein the centrifugation was at 15,000 rpm for 15 minutes, the therapeutic composition comprising the soluble fraction without any insoluble filamentous proteins, cellular debris and microorganisms; freeze-drying the soluble fraction; determining whether the freeze-dried soluble fraction includes 87% soluble proteins and 13% lipids; and if the freeze-dried soluble fraction includes less than 13% lipids, supplementing the freeze-dried soluble fraction with a lipid fraction extracted from the epidermal gel secretions of the catfish to obtain a therapeutic composition containing 87% soluble proteins and 13% lipids; freeze-drying the therapeutic composition containing 87% soluble proteins and 13% lipids; and dissolving the therapeutic composition containing 87% soluble proteins and 13% lipids in saline for administration by intraperitoneal or subcutaneous injection.

Description

EXAMPLE 1

Preparation of SPF and Calculation of Soluble Protein in Solution

(1) EGS was collected from the catfish skin and kept at 80 C. until use. Frozen EGS was thawed to 4 C., mixed with an equal volume of extraction buffer [phosphate buffered saline (PBS), 0.05 M containing (NaH.sub.2PO.sub.4/Na.sub.2HPO.sub.4) and 0.14 M NaCl, pH 7.5], and homogenized with an Ultra Truex (IKA) homogenizer. This step and all subsequent purification procedures were carried out at 4C unless otherwise indicated. The homogenate was centrifuged at 15,000 rpm for 15 min. The supernatant was collected, and the pellet (insoluble protein fraction) was extracted with extraction buffer (2-4 times for any undissolved soluble compositions). Each time, the soluble fraction was separated from the insoluble fraction by centrifugation as described above, the extracted soluble fractions were pooled. The combined extracted soluble fractions provided the soluble protein fraction (SPF) (Fraction B).

(2) To find the concentration of catfish soluble proteins in the SPF (Fraction B), the SPF was diluted with PBS (1:50). 0.1 ml of the diluted sample was mixed well with 5 ml of Coomassie Brilliant Blue solution and kept in tubes at room temperature for about 10 minutes. Absorbance was read at 595 nm for the sample, and its protein concentration was determined by comparing its absorbance against absorbance for a standard curve for different bovine serum albumin concentrations. Fraction B was found to include about 87% soluble proteins and about 13% lipids.

(3) The SPF (Fraction B) was then dissolved in the extraction buffer for injection.

(4) It is to be understood that the method for preparing a therapeutic composition using epidermal gel secretion of catfish is not limited to the specific embodiments described above, but encompasses any and all embodiments within the scope of the generic language of the following claims enabled by the embodiments described herein, or otherwise shown in the drawings or described above in terms sufficient to enable one of ordinary skill in the art to make and use the claimed subject matter.