PHA 010 Pharmacognosy & Plant Chemistry SAS 4 PDF

Course Code: PHA 010 (Pharmacognosy and Plant Chemistry) Student Activity Sheet SAS # 4 Name: _____________________...

Course Code: PHA 010 (Pharmacognosy and Plant Chemistry) Student Activity Sheet SAS # 4 Name: ___________________________________________________________ Class number: _______ Section: ____________ Schedule: ____________________________________ Date: ____________ Lesson title: Tannins Materials: 1. Student activity sheet Lesson Objectives: 2. Pen At the end of this module, you are expected to; 1. Identify the Pharmaceutical and Pharmacological uses of Tannins References: related Crude drugs -Shah, biren. (latest ed.). Textbook of 2. Identify the sources of the Tannins related Crude drugs pharmacognosy and phytochemistry. 1. India : elsevier. -Tyler, varro. Pharmacognosy, , phila. : lea & febiger, latest ed. Productivity Tip: Success isn’t always about greatness, Its About Consistency. Consistent Hard work Leads to success, Greatness will then follow. A. LESSON PREVIEW/REVIEW Introduction (5mins) The name ‘tannin’ is derived from the French ‘tanin’ (tanning substance) and is used for a range of natural polyphenols. Tannins are complex organic, non-nitrogenous plant products, which generally have astringent properties. These compounds comprise a large group of compounds that are widely distributed in the plant kingdom. The term ‘tannin’ was first used by Seguin in 1796 to denote substances which have the ability to combine with animal hides to convert them into leather which is known as tanning of the hide. According to this, tannins are substances which are detected by a tanning test due to its absorption on standard hide powder. The test is known as Goldbeater’s skin test. Figure 1. Examples of different Polyphenols Activity 1: What I Know Chart, part 1 (5 mins) What I Know Questions: What I Learned (Activity 4) 1. What is a Polyphenol? 2. What commercial products contain Polyphenols as their Component... This document is the property of PHINMA EDUCATION  Course Code: PHA 010 (Pharmacognosy and Plant Chemistry) Student Activity Sheet SAS # 4 B.MAIN LESSON Activity 2: Content Notes (90mins) The tannin compounds can be divided into two major groups on the basis of Goldbeater’s skin test. A group of tannins showing the positive tanning test may be regarded as true tannins, whereas those, which are partly retained by the hide powder and fail to give the test, are called as pseudotannins. Most of the true tannins are high molecular weight compounds. These compounds are complex polyphenolics, which are produced by polymerization of simple polyphenols. They may form complex glycosides or remains as such which may be observed by their typical hydrolytic reaction with the mineral acids and enzymes. Two major chemical classes of tannins are usually recognized based on this hydrolytic reaction and nature of phenolic nuclei involved in the tannins structure. The first class is referred to as hydrolysable tannins, whereas the other class is termed as condensed tannins. Hydrolysable Tannins As the name implies, these tannins are hydrolysable by mineral acids or enzymes such as tannase. Their structures involve several molecules of polyphenolic acids such as gallic, hexahydrodiphenic, or ellagic acids, bounded through ester linkages to a central glucose molecule. On the basis of the phenolic acids produced after the hydrolysis, they are further categorized under gallotannins composed of gallic acid or ellagitannins which contains hexahydrodiphenic acid which after intraesterification produces ellagic acid. Hydrolysable tannins are sometimes referred to as pyrogallol tannins as the components of phenolic acids on dry distillation are converted to pyrogallol derivatives. The hydrolysable tannins are soluble in water, and their solution produces blue colour with ferric chloride. Condensed tannins, unlike the previously explained group are not readily hydrolysable to simpler molecules with mineral acids and enzymes, thus they are also referred to as nonhydrolysable tannins. The term proanthocyanidins is sometimes alternatively used for these tannins. The compounds containing condensed tannins contain only phenolic nuclei which are biosynthetically related to flavonoids. Catechin which is found in tannins is flavan-3-o1, whereas leucoanthocyanidins are flavan-3,4-diol structures. These phenolics are frequently linked to carbohydrates or protein molecules to produce more complex tannin compounds. When treated with acids or enzymes, they tend to polymerize yielding insoluble red coloured products known as phlobaphens. The phlobaphens give characteristic red colour to many drugs such as cinchona and wild cherry bark. On dry distillation, they yield catechol derivatives. Condensed tannins are also soluble in water and produces green colour with ferric chloride. The families of the plants rich in both of the above groups of tannins include Rosaceae, Geraniaceae, Leguminosae, Combretaceae, Rubiaceae, Polygonaceae, Theaceae, etc. The members of families Cruciferae and Papaveraceae on the other hand are totally devoid of tannins. In the plants in which tannins are present, they exert an inhibitory effect on many enzymes due to their nature of protein precipitation and therefore contribute a protective function in barks and heartwood. Catechin Leucoanthocyanidin Figure 2. Example of condensed tannins Pseudotannins, are simple phenolic compounds of lower molecular weight. They do not respond to the tanning reaction of Goldbeater’s skin test. Gallic acid, Chlorogenic acid, or the simple phenolics such as catechin are pseudoannins which are abundantly found in plants, especially in dead tissues and dying cells. CHARACTERISTICS OF TANNINS 1. Tannins are colloidal solutions with water. 2. Non-crystalline substance. 3. Soluble in water (exception of some high molecular weight structures), alcohol, dilute alkali, and glycerin. 4. Sparingly soluble in ethyl acetate. 5. Insoluble in organic solvents, except acetone. This document is the property of PHINMA EDUCATION  Course Code: PHA 010 (Pharmacognosy and Plant Chemistry) Student Activity Sheet SAS # 4 6. Molecular weight ranging from 500 to >20,000. 7. Oligomeric compounds with multiple structure units with free phenolic groups. 8. Can bind with proteins and form insoluble or soluble tannin—protein complexes. Tannins belong to the phenolics class of secondary metabolites. All phenolic compounds; either primary or secondary are in one way or another formed through the shikimic acid pathway (phenylpropanoid pathway). Other phenolics such as isoflavones, coumarins, lignins, and aromatic amino acids (tryptophan, phenylalanine, and tyrosine) are also formed by the same pathway. Hydrolyzable tannins (Hts) and condensed tannins (proanthocyanidins) are the two main categories of tannins that impact animal nutrition. Common tannins are formed as follows: - Gallic acid is derived from quinic acid. - Ellagotannins are formed from hexahydroxydiphenic acid esters by the oxidative coupling of neighbouring gallic acid units attached to a D-glucose core. - Further oxidative coupling forms the hydrolysable tannin polymers. - Proanthocyanidin (PA) biosynthetic precursors are the leucocyanidins (flavan-3,4-diol and flavan-4-ol) which on autoxidation, in the absence of heat, form antho- cyanidin and 3-deoxyanthocianidin, which, in turn, polymerize to form PAs. Both hydrolysable and condensed tannins are highly soluble in water and alcohol but insoluble in organic solvents such as solvent ether, chloroform, and benzene. Tannin compounds can be easily extracted by water or alcohol. The general method for the extraction of tannic acid from various galls is either with water-saturated ether, or with mixture of water, alcohol, and ether. In such cases, free acids such as Gallic and ellagic acid go along with ether, whereas true tannin gets extracted in water. If the drug consists of chlorophyll or pigment, it may be removed by ether. After extraction, the aqueous and ethereal layers are separately concentrated, dried, and subjected to further isolation and purification using various separation techniques of chromatography. Tannins can be isolated from various plant sources, however chemical tests are needed to be conducted to further determine its presence in the crude extract. Such as; 1. Goldbeater’s skin test: Goldbeater’s skin is a membrane produced from the intestine of Ox. It behaves just like untanned animal hide. A piece of goldbeaters skin previously soaked in 2% hydrochloric acid and washed with distilled water is placed in a solution of tannin for 5 minutes. It is then washed with distilled water and transferred to 1% ferrous sulphate solution. A change of the colour of the goldbeater’s skin to brown or black indicates the presence of tannin. Hydrolysable and condensed tannins both give the positive goldbeater’s test, whereas pseudotannins show very little colour or negative test. 2. Phenazone Test: To 5 ml of aqueous solution of tannin containing drug, add 0.5 g of sodium acid phosphate. Warm the solution, cool, and filter. Add 2% phenazone solution to the filtrate. All tannins are precipitated as bulky, coloured precipitate. 3. Gelatin Test: To a 1% gelatine solution, add little 10% sodium chloride. If a 1% solution of tannin is added to the gelatine solution, tannins cause precipitation of gelatine from solution. 4. Test for Catechin (Matchstick Test): Catechin test is the modification of the well-known phloroglucinol test for lignin. Matchstick contains lignin. Dip a matchstick in the dilute extract of the drug, dry, moisten it with concentrated hydrochloric acid, and warm it near a flame. Catechin in the presence of acid produces phloroglucinol which stains the lignified wood pink or red. 5. Test for chlorogenic acid: A dilute solution of chlorogenic acid-containing extract, if treated with aqueous ammonia and exposed to air, slowly turns green indicating the presence of chlorogenic acid. 6. Vanillin-hydrochloric acid test: The drug shows pink or red color with a mixture of vanillin: alcohol: dilute HCl in the ratio 1:10:10. The reaction produces phloroglucinol which along with vanillin gives pink or red Tannins occur in crude drugs either as major active constituents as in oak bark, hamamelis leaves, and bearberry leaves, etc. or as subsidiary components as in clove, cinnamon, peppermint, or garden sage. In many cases, they synergistically increase the effectiveness of active principles. Tannins are medicinally significant due to their astringent properties. They promote rapid healing and the formation of new tissues on wounds and inflamed mucosa. Tannins are used in the treatment of varicose ulcers, hemorrhoids, minor burns, frostbite, as well as inflammation of the gums. This document is the property of PHINMA EDUCATION  Course Code: PHA 010 (Pharmacognosy and Plant Chemistry) Student Activity Sheet SAS # 4 Internally tannins are administered in cases of diarrhea, intestinal catarrh, and in cases of heavy metal poisoning as an antidote. In recent years, these compounds have demonstrated their antiviral activities for the treatment of viral diseases including AIDS. Tannins are used as mordant in dyeing, manufacture of ink, sizing paper and silk, and for printing fabrics. It is used along with gelatine and albumin for the manufacture of imitation horns and tortoise shells. They are widely used in the leather industry for the conversion of hiding into leather, the process is known as tanning. Tannins are also used for clarifying beer or wine, in photography, or as a coagulant in rubber manufacture. Tannins are used for the manufacture of gallic acid and pyrogallol, and sometimes as a reagent in analytical chemistry. CRUDE DRUGS Nutgalls (Nutgalls, blue galls, Turkish galls. ) Nutgall consists of the pathological outgrowth obtained from the young twigs of the dyers oak, Quercus infectoria Olivier, belonging to family Fagaceae. Outgrowth is caused by the puncture of ovums of insect Cynips tinctoria or Adleria gallaetinctoriae Olivier Family Cynipidae. Oak galls are obtained principally from Asiatic Turkey. Dyers oak is found in Turkey, Syria, Iran, Cyprus, and Greece. Larvae of the insect C. tinctoria after emerging from the eggs, pierces the delicate epidermis near the growing point of the twigs where the eggs are deposited by the insect. The gall begins to enlarge, when the chrysalis stage is reached, starch disappears from the neighbourhood of insect and is replaced by gallic acid, whereas central cells consist of tannic acid. The insect passes through the larval and pupal stages. If the galls are not collected and dried at this stage the mature insect comes out of the gall and escapes, and during this stage galls changes the colour from a bluish grey, through olive-green to almost white. After the escape of the insect, a central cavity is formed, and the tannic acid is oxidized in the presence of moisture and air. The more porous gall is the white gall of commerce. In Asiatic Turkey, galls are collected before the escape of the insect in the months of August and September. After drying, they are sorted out according to colour into three grades, that is, blue, green, and white and exported. Fig. 3 Twigs of Quercus infectoria Nutgalls contains about 50–70% tannin mainly gallotannic acid which is official tannic acid. It also consists of 2– 4% gallic acid, ellagic acid, sitosterol, methyl belulate and methyl oleanolate which are methyl esters of betulic and oleanolic acid. Recently few more compounds such as Nyctanthic acid, roburic acid, and syringic acids have been reported from galls. It contains abundant starch. Tannic acid of commerce is a hydrolysable tannin which yields gallic acid and glucose. The molecule of tannic acid may contain the gallic acid up to pentagalloyl glucose. It is isolated by fermentation and subsequent extraction of galls with water-saturated ether. Nutgall is the major source of tannic acid, which is largely used in tanning and dyeing industry and for the manufacture of ink. It is used medicinally as a local astringent in ointments and suppositories. Various types of galls are produced on plants by insects of the genera Cynips and Aphis. Chinese and Japanese galls are of commercial interest. These galls are formed on Rhuschinensis Mill, family Anacardiaceae by an aphis, Schlectendaliachinensis. These galls are knoty, grey, irregular and breaks easily to show irregular cavities. They contain 57–77% of tannins. These drugs have been used in China and Japan since time immemorial as astringent and styptic. Pale Catechu (Gambier, pale catechu, catechu.) Gambier or pale catechu is a dried aqueous extract produced from the leaves and young twigs of Uncaria gambier Roxburgh., belonging to family Rubiaceae.U. gambier is a native of erstwhile Malaya. It is cultivated in Indonesia, Malaysia, Sumatra, Bornea, and Singapore at elevation up to 150m. The plant is used mostly for the production of the drug, which is marketed through Singapore. Propagation of U. gambier is This document is the property of PHINMA EDUCATION  Course Code: PHA 010 (Pharmacognosy and Plant Chemistry) Student Activity Sheet SAS # 4 done by seeds. Seeds are sown in the nursery to raise the seedlings, which after about 9 months are planted out in the clearing about 3 meters apart. Leaves and young shoots are collected as the first crop during the second year’s growth. Later the crop is taken every year. The plant continues to give sufficient leaves and twigs for up to 20 years, but the maximum yield is obtained during the eighth year of growth. The collected leaves and twigs are transported to the factory as a loose material. The material is put into large drums with about three-quarters of boiling water. It is boiled for about three hours with intermittent stirring. The marc is subsequently removed by large wooden forks and lodged on the surface to drain the liquor back into the vessels. It is pressed and washed. The washing is added to the extract. The combined total aqueous extract is then concentrated for one and half-hour till it becomes a thick, yellowish-green paste. It is transferred from the vessels to wooden tubs, stirred while it is hot, and cooled in a stream of water to crystallize tannins. Semi-crystallized paste is again transferred to wooden trays in which it sets. They are cut into cubes by a wooden knife and dried in sum. The drug is also made into large blocks in kerosene tins. Figure 4. Image of U. gambier Figure 5. Structures of constituents of U. gambier Pale catechu contain from about 7 to 30% of pseudotannin catechin and 22 to 55% of a phlobatannin catechutannic acid. Both of the about component constitute over 60% of the drug. It also contains catechu red, gambier fluorescin and quercetin. It contains indole alkaloid up to 0.05%, which includes gambirtannin and its derivatives.Gambirtannin gives a strong fluorescence under UV light. Catechin forms white, needle-like crystals, which dissolves in alcohol and hot water. Catechutannic acid gives green color with ferric chloride. Pale catechu is medicinally used as local astringent. In diarrhea, it is used as a general astringent. It is largely used in various countries of east for chewing with betel leaf. A large proportion of gambier is used in the dyeing and tanning industries. It is used for tanning of animal hides to convert it to leather. Black Catechu (Cutch, black catechu, kattha).Black catechu is the dried aqueous extract prepared from the heartwood of Acacia catechu Willdenow, belonging to family, Leguminosae. Catechu is common throughout the tract from Punjab to Assam ascending to an altitude of 300 m. It is also quite common in drier regions of peninsula such as Madhya Pradesh, Maharashtra, Gujarat, Rajasthan, Bihar, and Tamil Nadu. Possibly, the use of black catechu could be traced back in history from the time of chewing betel leaf, in which it has been used as adjuvant. In old days, it was used by women as a colouring agent for the feet. Since 15th century, this natural material has been exported to Europe. The old information about catechu is by a Portuguese writer Garcia de Orta in 1574. Dr. Wrath first used the scientific process to extract catechu, and showed that catechu consists of two parts, such as, kattha and cutch. This document is the property of PHINMA EDUCATION  Course Code: PHA 010 (Pharmacognosy and Plant Chemistry) Student Activity Sheet SAS # 4 Cutch or black catechu resembles pale catechu or gambier in its composition. It contains about 2–12% of catechin and about 25 to 33% of phlobatannin catechutannic acid. The principle fraction of cutch has been identified as a mixture of catechin isomers which includes (-) epicatechin, acatechin, DL-acacatechin, L-acacatechin, and D-isoacacatechin. It also contains 20–30% gummy matter, catechin red, quercetin, and querecitin. It yields 2–3% of ash. Cutch is used in medicine as an astringent. It cures troubles of the mouth, diseases of the throat, and diarrhea. It also increases appetite. In India and eastern countries, it is used in betel leaves for chewing. In dyeing industries, cutch is used for dyeing fabrics brown or black. It is also used in calico printing. Figure 6. Image of Catechu Activity 3: Skill-building Activities (30 mins) Look for a pharmaceutical products that may contain tannins as its main ingredient. You may go to the nearest drugstore from you or search via the internet. In the first column, place the picture and name of the pharmaceutical product, for the second one is the crude drug(plant) that is present in the pharmaceutical product, please note that the crude drug should be the active ingredient of the product. The third one is the secondary metabolite present in the crude drug, and the last column will be the intended use of the pharmaceutical product. COMPLETE THE CONTENTS OF THE TABLE Secondary metabolite present in the Pharmaceutical product Crude drug present Use crude drug This document is the property of PHINMA EDUCATION  Course Code: PHA 010 (Pharmacognosy and Plant Chemistry) Student Activity Sheet SAS # 4 Activity 4: What I Know Chart, part 2 (5 mins) Instruction: Please go back to the What I Know table which is placed in Activity 1. Please do read again the question and answer the question allotted for activity 4 “What I learned” Activity 5: Check for Understanding (30mins) Instruction. Answer the following questions, this is based on the modules that you have just covered. Please take note that the intention of this is to measure your knowledge and learning on the covered module. So please do well. 1. Tannins are a plant secondary metabolite that is vastly known to contain which structure? a. Anthracene c. Polyphenols b. Aldehyde d. Cyclohexanes 2. Tannins are vastly known to contain what pharmacological activity? a. Astringent c. Diuretic b. Carminative d. Antiseptic 3. The following are tests that are done to detect the presence of tannins ECEPT: a. Gold Beater’s Test c. Lead Acetate Test b. Ferric Chloride Test d. Ninhydrine Test 4. What enzyme can hydrolyze tannins? a. Emulsin c. Myrosin b. Tannase d. Tannic Acid 5. Hydrolysable tannins are also known as: a. Condensed Tannins c. Non-Hydrolysable tannins b. Pyrogallols d. Phlobatannins 6. Condensed Tannins is also known as the following EXCEPT: a. Non-Hydrolysable tannins c. Phlobatannins b. Catechol d. Pyrogallols 7. A Flavan-3-ol condensed tannin is also known as: a. Catechin c. Leucoanthocyanidin b. Gallic Acid d. Ellagic Acid 8. The following are characteristics of tannins EXCEPT: a. Forms solution with Water b. Non crystalline substance c. binds to protein d. Soluble with Acetone 9. The pathway wherein tannins are synthesized. a. Acetate Pathway c. Mevalonate Pathway b. Light Dependent Pathwat d. Shikimic acid Pathway This document is the property of PHINMA EDUCATION  Course Code: PHA 010 (Pharmacognosy and Plant Chemistry) Student Activity Sheet SAS # 4 10. Guava is leaf extract was undergone with phytochemical screening for the contents, the conducted test was a Gelatin test, what is the Expected Positive result for this? a. presence of Precipitate c. Change of color b. Evolution of Gas d. Inconclusive B. LESSON WRAP-UP Activity 6: Thinking about Learning (5 mins) A. Work Tracker You are done with this session! Let’s track your progress. Shade the session number you just completed. PHARMACOGNOSY AND PLANT CHEMISTRY P1 P2 P3 1 2 3 4 5 6 7 8 9 10 B. Think about your Learning. Today was a good start, it was quite hard but the important thing is that you learned new things. Please do tell me what is the highlight of the session for you and what are the thingsyou encountered in this session that inspired you to become The Best Pharmacist. _____________________________________________________________________ _____________________________________________________________________ _____________________________________________________________________ _____________________________________________________________________ _____________________________________________________________________ ________________. This document is the property of PHINMA EDUCATION