13 Protein GELATIN Synonyms : Gelatinum. Biological souree : Gelatin is a product obtained by the partial hydrolysis of collagen, derived from the skin, white connective tissue, tendons, ligament and bones of ox (Bostourus Linn.), sheep (Ovisaries Linn), ete. Family : Bovidae. Preparation of gelatin : The insoluble collagens are converted into soluble gelatin which is then purified and concentrated to a solid form. Commercially, gelatin is obtained from by-product of slaughtered cattle, sheep and hogs. The starting materials, e.g. bones, are defatted with an organic solvent. Sometimes these are decalcified with hydrochloric acid. ‘The material is then heated with water at 85°C to convert collagens to gelatin. The solution is decolorized, filtered by electro-osmosis, concentrated under reduced pressure, allowed to set into gel in shallow trays and dried rapidly in drying-room at 30, 40, 50 and 60° for some weeks. Characters : Gelatin is occurs in thin sheets, strips or as granular powder. High grade gelatin light yellow, semi crystalline substance. It is Odourless and tasteless. In cold water it swells up and slowly dissolves on warming to form viscous solution. . Chemical constituents : Gelatin mainly consists of protein glutin, which on hydrolysis gives a mixture of amino acids like glycine, alanine, leucine, aspartic acid, argenine, lysine, isoleucine, valine, tyrosine, cystine, cysteine, glutamic acid, argenine, etc. Adhesive property of gelatin is due to the presence of glutin. CH;-COOH (CH3-CH-COOR Ceiee NH NH2 NH Glycine Alanine "valine192 Textbook of Pharmacognosy and Phytochemistry-1 ce cu,cHicooH A i Sy NH ] SU] an Histidine HOOC-CH;CH-COOH be woe : NH Tyrosine Uses: SVP PPP a Chemical Tests : Ie 5. 6. In capsule preparation. ‘As culture medium in Bacteriology. In making suppositories. _ Source of protein in nutritional experiments. As a substitute for blood plasma. Zinc oxide is added to form zinc gelatin which is used in topical protectant. |. Itis used as suspending agent and binding agent. |. Gelatin is also widely used in food products and photographic emulsions. . Gelatin is also used for the microencapsulation of drugs, where the active drug is sealed inside a microsized capsule or beadlet, which may then be handled as a powder. When gelatin is heated with soda lime in dry test tube, ammonia is evolved due to the presence of nitrogenous compound in gelatin. Gelatin solution is added Million’s reagent to give a white ppt, which turns red on heating. Gelatin gives buff white ppt with tannic acid solution. |. Biuret test: to 3 ml of test solution of gelatin. NaOH (1 ml of 5%) is added whereby white to whitish buff coloured ppt is formed which does not dissolve on heating. Yellow ppt is formed on adding picrie acid to solution of gelatin. It gives yellow ppt with trinitrophenol in aqueous solution. CASEIN Biological source: Casein is a principal phosphor protein in milk and constitutes 3% milk. It is comprise about 80% total protein of milk.Protein 193 Description: 1. It is white, slightly yellow, tasteless, odourless, amorphous solid, hygroscopic, stable when dry but deteriorates rapidly when damp. 2. It is insoluble in water, soluble in dilute alkali, concentrated acid, but precipitates from dilute acid solution. 3. It does not coagulate on heating. Chemical constituents: 1. It contains 80% proteins. 2. The main casein contents are Si and S: caseins. B-casein and k- casein. All these caseins have less solubility at pH 4.6. 3. Caseins are conjugated protein with phosphate groups which are responsible for binding of calcium, Preparation of caseii 1. Place 20 ml (20 g) of milk into a 125 ml flask and heat at 40°C in a water bath, 2. Add 5 drops of glacial acetic acid and stir for about 1 min. 3. Filter the resulting mixture through filter paper held in a funnel and gently squeeze out most of liquid. 4. Remove the solid (casein and fat) from the cheesecloth, place it into a 100 ml beaker and add 10 ml of 95% ethanol. 5. Stir well to break up the product. Pour off the liquid and add 10 ml of 1:1 ether-ethanol mixture to the solid. 6. Stir well and filter through filer paper. 7. Let the solid drain well, then scrape it into a weighed filter paper and let it dry in the air. Uses: 1. It is use as adhesive, fabrics, food additives and binder. 2. Casein used by body builder for practices because if its slow digestion source of amino acid nature. 3. It is uses as dietary stipplement source of protein in pre and post- operative care. 4, It is uses in sizing of textile and paper industry. As an adhesive, in preparation of casein plastic and casein pains. gaa14 Enzymes Enzymes are organic catalyst, proteinous in nature, non living (life less), high molecular weight substances produced by living organism. It serves as catalyst in many complex chemicals reactions that make up life processes. The enzymes contain several common properties. (a) Enzymes are colloidal in nature, soluble in water and dilute alcohol but are precipitated by concentrated alcohol. (b) These are most active in temperature between 35°C to 40°C. (c) These are destroyed at temperature between above 65°C especially in the presence of moisture and it becomes inactive at orc. (d) They are sensitive to pH and presence of other substances in medium. The enzymes are classified into following categories : (1) Hydrolase’s : For catalysis of hydrolytic reactions. (2) Transferases : For the transfer of chemical group from one molecule to another. (3) Oxido-redductases : catalyze the oxidation-reduction reactions. (4) Lyses : Catalyze the addition of groups to double bonds or vice versa. (5) Isomerases they are responsible for intermolecular rearrangements. (6) Synthetases : Catalyze the condensation of two molecules coupled with the cleavage pyrophosphate bond of ATP or similar triphosphate. ENZYMES S. | Name of | Part ofthe | Biological | Chemical Uses N. | the Drug plant source constituents 1 | Papain [Latex of unripe| Cariya papaya | Papain, clarification of fruit chrymopapain | beverages and as a meat tenderizer; cheese manufacture as a substitute of rennin 2 |Bromelain |” mixture of | Ananascomosus.| peptidase, | treatment of soft proteolytic Family: | cosmosein and tissue enzymes from | Bromeliaceae anain inflammation and stem and ripen oedema due to fruits of surgery and injury, pineapple plantEnzymes 195 3 | Serratiope |“ proteolytic _ | Serratiamarcoac treatment of Ptidase enzyme ens rheumatoid isolated from arthritis and the non osteoarthritis pathogenicente robacteria 4 | Urokinase |" protease | protease enzyme ‘treatment of enzyme isolated from pulmonary isolated from | human urine embolism, human urine [and from human thrombosis, andfrom | kidney cells by coronary artery human kidney | tissue culture or cells by tissue | by recombinant culture or by |DNA technology. recombinant DNA technology. 5 | Streptokin culture of the |” purified | the meatment of ase Group CB strain} bacterial pulmonary of Streptococcus | protein with | embolism, deep haemolyticus | about 484 | vein thrombosis, that is capable | amino acid arterial of solely residue | thrombosis and ‘converting ‘embolism, plasminogen to arteriovenous plasmin cannula occlusion, and coronary artery thrombosis, 6 | Pepsin Stomach | ox (Bostaurus), artificial agent to the sheep assist digestion (Ovisaries), or the hog (Susscrofa) PAPAIN Biological Source : It is a mixture of proteolytic enzymes devised from the latex of unripe fruit of tropical melon tree, Carica papaya, Family : Caricaceae, Method of preparation For processing of papain, the latex of these fruits is collected in aluminium trays, to the collected latex; potassium metabisulphite (5g/hkg of latex) is added. The extraneous matter is cleared out by passing through sieves and latex is dried in vacuum shelf dryer at 55-60°C, It is also processed by spray drying methods. his dried latex is called papain, Description Papain is available as light brown or white colour amorphous powder with typical odour and taste. It shows maximum proteolytic activity between pH 5 to 6. It is soluble in water and glycerin,196 . Textoook of Pharmacognosy and Phytochemistry1 Chemical nature: The different proteolytic enzymes present in papain are the mixture of papain and chrymopapain, proteolytic enzymes act on polypeptides and amides. Uses: 1. Itis used in clarification of beverages and as a meat tenderizer. 2. It is employed in cheese manufacture as a substitute of rennin. 8. It is also used for degumming of silk fabrics in textile industry and in leather industry for dehairing of skins and hides. 4, Medicinally, it is used as an anti-inflammatory agent. 5. It is employed to relieve the symptoms of episiotomy (Surgical incision of the vulva for obstetric purpose). BROMELAIN Biological source : It is a protein digesting and milk clotting enzyme. Bromelain is a mixture of proteolytic enzymes from stem and ripen fruits of pineapple plant Ananascomosus. Family : Bromeliaceae Geographical source: pineapple is found in India, Taiwan, Brazil, China, Thialand and USA, ete. Chemical constituents : 1. It contains peptidase, cosmosein and anain. 2. Fruits are rich in soluble mono and disaccharides, vitamins and inorganic acids. Preparation of Bromelain from fruits : 1. Fruits were cut into small piece, weighed, macerated and juice was obtained. 2, Juice wad pressed and filtered through cheese cloth. 3. pH of the juice was adjusted to 6. 4, Ammonium sulphate was added until saturated to precipitated the enzyme. 5, Partial purification was done by redissolving crude enzyme in NaCN and repeatedly precipitating it, firstly with 0.6 ammonium sulphate and then with acetone. 6. The precipitate is thoroughly washed with acetone and ether and dried in vacuum oven at low temperature. Description : 1, It is brown coloured powder. 2, It is optimum pH 5-8. 3. It has slightly soluble in water.Enzymes 197 4. It is insoluble in organic solvents like ether, chloroform and alcohol. Uses: 1, Itis used in the treatment of soft tissue inflammation and oedema due to surgery and injury. 2. It is used certain’ cosmetics such facial cleansers and bath preparations. 3. Itis used as digestive enzyme after pancreatectomy. 4, It is also prevents and minimizes the severity of angina pectoris and ischemic attacks. SERRATIOPEPTIDASE Serratiopeptidase, also known as Serrapeptase or Serratiapeptidase, is a proteolytic enzyme isolated from the non pathogenicenterobacteria Serratiamarcescens. This enzyme is found naturally in the intestine of the silkworm, which is used by the silkworm to dissolve the cocoon and emerge as a moth. Serratiopeptidase is a proteolytic enzyme isolated from the micro-organism Serratiamarcescens. Serratiamarcescensis a gram negative, bacillus shaped bacteria that belongs to the family Enterobacteriaceae. Uses: 1. The use of enzyme with fibrinolytic, proteolytic and anti-endemic activities has gained increasing support in recent years for the treatment of inflammatory ear, nose and throat (ENT) conditions. 2. It is also used-in the treatment of rheumatoid arthritis and osteoarthrit : 3. Serratiopeptidase dissolves blood clots. It is used to treat cardiovascular diseases, Arthritis, Eye problems, etc. 4. Serratiopeptidase reduces internal tissue edema and inflammation caused at post-operative handling. Reduction in edema reduces chances of rupture at tissue as well as risk in case of plastic surgery and graft rejection, 5. Serratiopeptidase has mucolytic activity in sinuses, ear cavities and anti-inflammatory activity in upper respiratory tract organs and helps in faster resolution, better antibiotic bioavailability and faster cure rates, 6. An anti-inflammatory agent (particularly for post traumatic swelling). 7. For Bronchitis (Serratiopeptidase loosens and expels mucous) 8, Serratiopeptidase digests dead tissue, blood clots, cysts, and arterial plaque.198 ‘Textbook of Pharmacognosy and Phytochemistry-1 9. Serratiopeptidase reduces inflammation and helps in faster healing and repair. 10. Serratiopeptidase is used in acute painful inflamed dermatitis. URIKINASE Synonyms : urokinase-type plasminogen activator (uPA) Biological source : It is protease enzyme isolated from human urine and from human kidney cells by tissue culture or by recombinant DNA technology. Preparation of urikinase: 1. It isa fibrilytic enzyme produced by recombinant technology using genetically modified E. coli cells. 2. It is firstly produced by prourikinase which is converted into active form by plamin and kallikarien, 3. Itcan also be purified directly from human urine. 4, It bind with absorbent like silica gel or kaolin for the purification of product. 5. It is further purified with sodium chloride or ethanol or by technique like chromatography. 6, Human urinakinase needs sterile filtration, aseptic filling and freeze drying. Description: 1. It is white, lyophilized powder 2. It is soluble in water. 3. It is an activator of endogeneousfibrinolytic system, which converts plasminogen to plasmin and degrades fibrinogen, fibrin clots and other plasma proteins. Uses: It is used in the treatment of pulmonary embolism, thrombosis, coronary artery and in restoring the potency of intravenous catheters. STREPTOKINASE Streptokinase is a proteolytic enzyme derived from the culture of hemolytic Streptococci. The protein molecule contains no cystine and has ‘an isoelectric point of pH-4.7. The protein molecule possesses thrombolytic capability, catalyzing the transformation of human plasminogen to plasmin by hydrolytic cleavage of the argval bond. Its enzymatic action based upon 1:1 non-covalent complex with the zymogen from which active plasmin is released. This complex has been dissociated into two components, one with plasmin activity along with other having plasminogen activity, in which complexation first takes place betweenEnzymes 199 streptokinase and the plasmin contaminant of plasminogen. This complex, which is then free to transform a second plasminogen molecule. Streptokinase is a single chain coenzyme obtained from culture of the Group CB strain of Streptococcus haemolyticus that is capable of solely converting plasminogen to plasmin. It is used extensively as a predominant fibinolytic agent to help in a big way for the specific removal of fibrin thrombi from arteries. A mixture of streptokinase and streptodornase, as produced by a hemolytic streptococcus grown in a specific environment of: aerated- submerged culture, is used meticulously to clean up the debris from wounds and burns effectively. Properties: It is a hygroscopic, white powder of friable solid. It is freely soluble in an aqueous medium; and quite unstable in concentrations of less than 10,0001U/ml. Applications of streptokinase: 1. Use of streptokinase is indicated in the treatment of pulmonary embolism, deep vein thrombosis, arterial thrombosis and embolism, arteriovenous cannula occlusion, and coronary artery thrombosis. 2. It is recommended for the management and control of myocardial infarction (AMI) in adults to bring about various therapeutic benefits, such as : * Specific lysis of intracoronary thrombi. ¢ Reduction of infect size and congestive heart failure associated with AMI, when administered by the IV route. * Improvement in ventricular function. : + Remarkable reduction of mortality associated with AMI. 3. It is also indicated for the adequate lysis of objectively diagnosed pulmonary emboli, involving obstruction of blood flow to a lobe or multiple segments, with or without unstable haemodynamics. PEPSIN A mixture containing a proteolytic ferment or enzyme obtained from the glandular layer of fresh stomachs of healthy pigs, and capable of digesting not less than 3000 times its own weight of freshly coagulated and disintegrated egg albumen. Source: Pepsin is prepared from the stomach of the ox (Bostaurus), the sheep (Ovisaries), or the hog (Susscrofa), the mucous membrane being the part used. Methods of preparation: following methods are: 1. Pepsin is prepared by digesting the minced stomach linings with hydrochloric acid. This solution is clarified, partially evaporated, dialyzed, concentrated and either poured on glass plates to dry,200 Textbook of Pharmacognosy and Phytochemistry-1 thus forming scale pepsin or carefully evaporated in vacuum, forming spongy pepsin. 2. The extraneous matter is first removed from the inner surface of the stomach by washing, and the mucous membrane scraped off with a blunt instrument; the pulp thus obtained is placed on glass or porcelain and dried and finally reduced to a powder. This forms a rather poor quality, owing to the presence of mucus and inert matter. 3,-'The finely chopped mucous coat is macerated in dilute hydrochloric acid (about 2 per cent.) and to the filtered solution, common salt is added; the floating precipitate which results is carefully washed, then dried, and the dried residue mixed with sugar of milk until the strength of the article is such that 1 grain will dissolve 3000 grains of coagulated albumen, the strength directed by the United States Pharmacopoeia. 4. A scale pepsin is made by digesting the mucous lining at the temperature of about 100°C with about 0.2 per cent of HCl (or water acidulated with other acids to the same degree of acidity) until the membrane is completely or nearly all dissolved. The solution is neutralized by a suitable alkali and the filtered product, after reduction by evaporation at a low temperature (sometimes in vacuo) to a syrupy consistence, is spread on plates of glass and dried in a current of warm air, care being taken not to allow the temperature to exceed 40°C (104°F). The dried, transparent film is then scraped from the plates and broken into more or less fine lamella. Description : A yellowish-white amorphous powder or thin, pale yellowish, somewhat transparent scales, with faint odor and slight saline ‘or acidulous taste, but no indication of decomposition; should not be hygroscopic. It invariably contains some rennin; its solutions, therefore, will coagulate milk. Incompatible with alkalies, alcohol, and heat renders it inert. Action and use : Pepsin has a digestive action upon the food taken. jnto the stomach, and is employed as an artificial agent to assist digestion when there is functional derangement of the stomach. gaa