Add 1 mL 1M sutfiric * Pipette off the clear 0! ‘id (H,SO,) and shake well. Let stand; Test one portion lear upper layer into two sm Positive results for alkaloids With Dragendorff’s reagent, a Positive test is indicated by an orange ; itive t gi © test is indicated by an orang: ‘With Mayer's reagent, a positive testi indicated by a white precipitate, 2.1.2 Laboratory test tube method for alkaloid analysis @: .2.1 Preliminary assay Hydrochloric acid, HCl, 2M Add 17 mL of cone. HCI (12M) to enough distilled water to make 100 mL solution. Take an equivalent of 20 g plant material from the stock plant extract prepared in Section 1.1 and place in an evaporating dish; Evaporate to a syrupy consistency over a steam bath; Add 5 mL of 2M hydrochloric acid (HCI); heat with stirring for about 5 min. and cool; + Add about 0.5 g sodium chloride; stir and filter; + Wash the residue with enough 2M HCI to bring the filtrate to a volume of about 5 mL; Take 1 mL of the filtrate and test with 2 to 3 drops of Dragendorft’s reagent; ‘Take another 1 mL. of the filtrate and test with 2 to 3 drops of Mayer's reagent; Record the relative amount of precipitation observed as follows: (+) slight turbidity (++) definite turbidity (+++) heavy precipitationPia Confirm test results by the following confirmatory test. 2.2 Cpnfirmatory test * To the remaining 3 mL, of the filtrate in Section 2.1.2.1, enough 28% ammonia (CAUTION: causes burns, v. irvitating!) until the solution is alkaline to litmus; + Extract the alkaline solution three times with small portions of less than 10 mL. of chloroform (CAUTION: carcinogenic!). Combine the lower chloroform extracts and reserve the upper aqui 21.23; + Evaporate the chloroform extracts to dryness under the hood steam bath; + Take up the residue with § mL of 2M HCI, and stir over a steam bath for about 2 min., cook; + Filter and divide filtrate into two portions; + Test one portion with Mayers reagent and the other with Dragendorff’s reagents *+ Record the results observed as (+), (++) or (e+), add dropwise apors extremely ieous layer for Section and over @ Positive results indicate the presence of primary, secondary or tertiary alkaloids .3 Test for quaternary bases and/or amine oxide + Acidify the alkaline aqueous layer obtained in Section 2.1.2.2 above with 2M HCI, + Filter and divide the filtrate into two portions; + To one portion add Mayer's reagent and to the other, Dragendor#’s reagent; + Observe the results and record as above. A score of (++) or (+++) in both Mayer’s and Dragendorf’s tests may be taken as an indication of the presence of quaternary and/or amine oxide bases; a (+) score ‘ay be the result of incomplete chloroform extraction, thus should be considered negative for quaternary bases. 2.1.3 Thin-layer chromatographic analysis for alkaloids: The Farnsworth- Euler method ‘Thin-layer chromatography is a powerful tool for separating and detecting ‘small quantities of the different constituents in a mixture, This method is capable of detecting secondary, tertiary alkaloids and water-soluble bases as well as eliminating interfering materials that give false positive results, Based on the number and the color intensity of the spots, this method can indicate the number of possible alkaloids present in the plant extract and the relative amount of these alkaloids. Thus, this method of analysis can be used to confirm the 2932 Reminder before proceeding to Section 22, logbook entry shou! suchas Important details, material and other botanical details: + Name of plan Concentration of plant extrac as grams dried sample/ml. extra + Results observed in the different tests; + Documentation of the chromatogram, specifying: = advorbent used, = developing solvent used, = visualizing agent used, = thesample origin, solvent front ‘and respective bR,, obtained in the bracing and rns observed with visualizing ems ofthe chromatogra 2.2 Steroids: Cardenolides and Bufadienolides jdal glycosides and are referred to as i the heart muscle. These cardiac '5-membered lactone Both types have an Cardenolides and bufadienolides are ste “cardiac glycosides” since they have a dramatic effect on the lyosides differ onthe steroidal skeleton, the cardenoides have 2 fing while the bufadienolides have a 6-numbered lactone #n8- unsaturated lactone ring and one or more 2-deoxysugars. “The presence of eardiae glycosides may be detected by in-vitro or in-vivo assays. For phytochemical screening ofthe plant extracts, the test maf be done on the crude leokolfc extract or the partially purified extract. The chemical test should detect the presence ofthe deoxy-sugas the steroid nucleus and the ‘unsaturated lactones. ‘An initial positive result with the Keller-Killiani test indicates only the presence of deoxysugars. The extract should also be tested with the Liebermann Burchard test to er the presence ofthe aglycone steroidal group; andthe Kedde test, for the presence ce rtd lactones, Positive esults with all the three test reagents indicate the presence of cardiac glycosides. ‘A confirmatory in-vivo assay results for cardiac glycosides + Take an equivalent of 10 g plant material from the pl: previously prepared. Refer to Seetion 1-1; + Evaporate this to incipient dryness over temperature; + Defat by taking up the residue with 6 mL of hexane and water, 2:1 v/s = Partition by gently shaking the mixture in a test tube; - Pipette out the upper hexane layer; ~ Repeat the treatment with hexane until most of the colored pigments have been removed. Discard all the hexane extracts properly; of the plant extract will eliminate the false-positive lant extract stock solution a water bath, Cool to room+ Heat the defated aqueous layer over a water bath to remove the residual hexane Cool to room temperature; sent + Divide to 3 portions for Sections 2.2.1.1, 2.2.1.2 and 2.2.1.3, Gautthe Keller-Kiliani test: for 2-deoxysugars Jron({I) chloride, FeCl, reagent Dissolve 3 mL of 19 FeCl, in 50 mL glacial acetic acid Iron(III) chloride solution, 19 Dissolve 1.0 g FeCl, in 100 mL distilled water, Positive result: A reddish-brown color, which may turn blue or purple, indicates the presence of 2-deoxysugars. PHYTOCHEMISTRY + Toone portion of the defatted aqueous layer, free from hexane, add 3 mL, of ferric chloride, and stir, + With the test tube in an inclined position, add cautiously from a pipette, 1 mL of cone. sulfuric acid (CAUTION: very corrosive!) letting the acid tickle down along the insides of the test tube; + Allow the mixture to stand upright and observe for any coloration at the interface of the acid and the aqueous layers. (Garting Liebermann-Burchard test: for unsaturated steroids Lieberman-Burchard test for unsaturated steroids and triterpenes ‘The positive results give colors ranging from blue to green, red, pink, purple or violet, because of the steroid/triterpenoid skeleton. + To another portion of the defitted aqueous layer free from hexane (refer to Seetion 2.2.1) add 10 mL. dichloromethane and stir the mixture with a glass rod for a few minutes allow to stand; + Pipette off the lower dichloromethane extract; + Dry the dichloromethane extract by passing this extract through about 100 mg anhydrous sodium sulfate (Na,SO,) placed over dry filter paper in a funnel, Refer to Figure P2; + Divide the filtrate into two portions; use one for the control; + Treat the other portion with 3 drops of acetic anhydride (CAUTION: corrosive!) then one drop of conc. sulfuric acid (CAUTION: highly corrosive!); Figure P2. Set-up for filtration through anhydrous sodium sulfate 33imnmediate color changes foray and observe for further color changes, Cony, ey, i + Observe inhourand Let stand for at the control. (ove Keade test: for unsaturated lactones apace! gof 3Sdintrobenzoie acid in a mixture of 50 mL methyl alcoho) (CH,OH) and 30 mL. 2M potassium hydroxide, jum hydroxide, KOH, 2M Poca yn OT in enoagh ile water © make 100 mL, Positive standard for ‘unsaturated lactone i ‘Use 2 mL of 0.025% digitoxin dissolved in methanol. [Abive- violet color indicates presence of unsaturated lactone, + ‘Tothe third portion of the defatted aqueous layer free from hexane (refe, to Section 2.2.1) add 2 mL. dichloromethane (CH,Cl,) and mix well; “Take the lower dichloromethane layer and teat this with 4 drops of Kee reagent, Compare the color with the positive standard treated as in the above, 2.2.2 Thin-layer chromatographic method for unsaturated Inctones For chromatographic techniques, refer tothe Appendix, Phytochemistry Section for the following: Preparation of thin-layer plates, Preparation of the developing chamber; Sporting (applying sample) on plates; Developing the plate; ‘sualizing the chromatogram; Documenting the chromatogram. : Dil i of ck lt xr vith 04 CHLCL: CHO (1) and 1 Sexton 2213 abr) tbh nde pg ene ene ee + Deco th pte wth CH,CI:CHLOH Ga), + Dry the chromatogram and spray with ; Pui ram and spray with Kedde reagent: Refer to Section 2.2.1.3 + Observe the color of the spots and or Dene ett pt andcompc ih ato te oie sndww “Ss sedatives aren orange-red compound tha ae sll in ho Anite TL They may be detected by the Borntrigr’ test however, the water and in le for very sabe anhraquinone gleosies or reduced derivatives of the antheanol types. Hence, the test must be ‘modified to first hydrolyze and oxidize such compounds. ‘Anthraquinones give a characteristic red, viol ‘Through spectral methods, they may be distingu! since they give four or five absorption bands in the ultraviol [Atleast three ofthese bands lie between 215 and 300 nm, an Jet, green or purple color with a base ished from other classes of quinones let and the visible regions. dd another above 430 nm, 2.4.1 Test tube screening methods f"Tentoies C2411 The Borneriger’s Ammonia solution Dilute 35.7 mL. of 28% ammonia with enough distilled water to make a total volume of 100 mL. ‘Take an equivalent of 1 g plant material from the stock plant extract prepared in Section 1.1 and evaporate to incipient dryness over a steam bath; Take up the residue with 10 mL. distilled water and filter discarding the residue; Extract the aqueous filtrate twice with 5 mL portions of benzene (CAUTION: carcinogenic!), combining the benzene extracts; Divide the combined benzene extracts into two portions, Reserve one portion as the control; ‘Treat the other portion with § mL ammonia solution and shake; ‘Compare with the control tube. Positive result: A red coloration in the lower ammoniacal layer indicates the presence of anthraquinones. th coloration occurs, proceed to Section 2. 2.4.1.2 The modified Borntriiger’s test Te, Take an equivalent of 1 g plant material from the stock plant extract prepared in Section 1.1 and evaporate to incipient dryness over a steam bath; ‘Add 10 mL 0.5 M potassium hydroxide and 1 mL of 5% hydrogen peroxide (H,O,) and stirsAlternative spray reagent 1 4o make 100 ml, ole gh met Magnesium acetate, 0.5% methanolle enous esi acetal ive result. Refer Dissolve 0.5 g magne sr indicates pomr solution eine 00 dar nce stan Positive reslt‘The formation of to the color obtained with the rfé 5 olution of magnesi 10.536 methanolic mn wit Jried chromatogra" : hot pl Be orc over a acetate; pout 1 ; Heat de sprayed plate for mis 270% oference standard ompare + Observe color produced and comP: late; 2.5 Flavonoids Recent reports of the antiviral offlavonoide have generated interes are phenolic plant pigments generally ¢ ‘Fionoid include the anthoyains 204 the es. : are the cathechins, aurones and ch: - and wien group of coving mate ‘Anthocyanins make up the mos hydrolysis yield sugars and colored : a alycosides and on hydrolysis yield in plans The anthocyanin Sins. Extracting the plant material with 1% hydrochloric a a ae tan identify anthocyanins. An orange-red to blue-red color acid, then boiling the extract,can i : Sponbotingindees th pene aabocainss | a Ieacoanthoeyanins have as ther aglycone the leucoanthocyanidins. These are Lo aaa ni gre clor when bolled with an acid. Leucoanthocyaing fivean intence red or volt color after boiling the acid-treated extract fora longer time Chalcones and auroncs ive an immediate red color when the defatted ethanolic extay is treated with just the acd. -anflammatory and cytotoxic activities anf contiing plants. Flavonoids ns te been yone nes ly Jeucoanthocyanins. Other flavonoids One of the most useful tests for flavonoids is the Wilstatter cyanidin reaction which detects compounds having the ¥-benzopyrone nucleus, Treatment of the defatted alcoholic plant extract with concentrated hydrochloric acid and subsequent reduction with magnesium metal give colors ranging from orange to red to crimson and magenta, and occasionally to green orblue. Favonoids may be separated and identified by mean of chromatography combined with color reactions and the use of ultraviolet light. 1 Test tube screening methods ‘Take an equivalent of 10g plant material from the stock plant extract prepared in Section 1.1 and evaporate to incipient dryness over a stearn bath, Cool to room temperature defatby taking up the esidue with 9 mL hexaneand water 2:1) or with petrole es um ether (CAUTION: solvents are inflammable). 'scard the hexane or petroleum ether extrac;+ Dilute the defatted aqueous layer wit F wus layer with 10 mL of 80% ethyl al + Filter and divide the filtrate into four test tubes aa + Take one portion as control. Ce: sate for leucoanthocyanins: Bate-Smith and Metcalf method aed one portion of the above alcohol filtrate with 0.5 mL. hydrochloric acid (12M); . + Observe for any color change; ‘Warm for 15 min. ina water bath. Observe for f a serve for further color change within an hourand compare withthe cont nn uerstanes with + Record your results Positive result: A strong red or violet color indicates the Jeucoanthocyanins. icates the presence of Gass for y-benzopyrone nucleus: Wilstatter “cyanidin" test > Take another portion of the alcohol filtrate above, refer to Section 2.5.1 and treat with 0.5 mL conc. hydrochloric acid (12M); r + Add 3-4 pieces of magnesium turnings; + Observe any color change within 10 min, and compare withthe contro tube; + If definite coloration occurs, dilute with an equal volume of warerand add 1 mL octyl alcohol; + Shake well and allow to stand; + Note the color in each layer. Record your results Positive result: Colors ranging from orange to red, to crimson and magenta, and occasionally to green or blue may be observed. 25.2 Two-dimensional thin-layer chromatographic (TLC) sereening method For chromatographic techniques, refer to the Appendix. Two dimensional chromatography |= Use glass plates 12 x 12 cm. coated with silica gel G; 3-4 cm from the left corner of the silica coated plate; + Spot the extract about oe the solvent front; + Develop in equilibrated chamber using the frst solvent up to + Airdry; tum the plate 90° counterclockwise and develop in the secon! solent allowing the solvent co travel in a perpendicular direction as that of dhe Fis solvent; + Airedry and spray. 39‘The steroidal saponins are of great importance because oftheir rela compounds a8 sx hormones, cortisone, Vitamin D and the wane Because of these strctures, saponins produce characteristic ec nn the Liebermann-Burchard test for unsaturated sterols and triterpenes Then from blue to green, red, pink, purple or violet. ppenes, The colors range ‘Asimple test forthe saponins, however i the froth upon shaking an aqueous alcoholic solution of be detected by their ability to lower the surface te ionship to such formation of persistent honeycomb nt honeycom the plant extract. Saponins can also pee sion of water as wel as their ability Confirmatory tests can be conducted by thin-layer chrom: measurements. ry thin-layer chromatography and by spectral 2.6.1 The screening methods for saponins 26.1.1 The froth test —Saponins in plant materials can cause a persistent foam when the aqueous solution is agitated. " ee Vark Preparation of gogo’ extract - “Take about 1 g of the bark of Entada phaseoloides (L.) Mere, locally known as ‘gogo’. Cut in small strips and soak irr 10 mL of 80% ethyl alcohol. Allow to stand for 30 minutes. ‘Take a volume of the plant extract prepared in Section 1.1 equivalent to 2g plant material and transfer this to a test tube; Ina separate test tube have 1 mL of ‘gogo! extract to stand as the standards ‘Add 10 mL of distilled water to each of the test tubes, stopper and shake both test tubes vigorously for 30 seconds, + Allow to stand for 10 min. and observe for a honeycomb froth; + Compare the result of the plant extract with that of the standard, t: Ifthe ‘honeycomb’ froth greater than 2 cm height from the su persists after 10 min., the sample is considered positive for saponins. ts with poor frothing effects . '5% sodium carbonate solution to basfy the extract. The formation dense froth indicates the presence of free fatty acids 2.6.1.2 The capillary tube test Saponins tend to lower the surface tension in water. 41 > i = wn 3 oo oa) 3) 2) SI fe 0+ Allow the covered plats to stand undisturbe + Observe for any clear zones of hemolysis» + Record the diameter of the halo in mm, cups for one hour “hemolytical halo", 4 observed around all he agar 246.14 Determination of saponins in the presence ofan ining Tannins and certain plant acids have hemolytic test for the detection of sapon present in a plant extrac, no hemolyis test results. IFtannins and othe plant polyphenolic compound jnthe plant extnct then these ae enone bye ec aa cent magnum Ree Secton2a Tika et to be effective in removing interfering tannins and other compounds. other plant polyphenolic been shown to interfere with the ins. If saponins and tannins were both would occur. false-negative saponin : i) E =I : IS 5 Sodium chloride, 109% Dissolve 10 g NaClin enough distilled water to make 100 ml. solution, + Take volume ofthe plant extract prepared in Section 1.1 equivalent to 10 g plant material and evaporate this to incipient dryness ler gcc bath; ‘ + Extract the residue with 20 mL of hot distilled water. Add 5 drops of 10% sodium chloride solution and filter; + Treat the saline filtrate with 2 g of magnesium oxide to form a sur and heat over a steam bath for about 10 min; + Bxerat the slurry with hor 80% ethanol, then iter; + Subject the detannated aqueous-ethanol filtrate to the hemolytic tes, given in Section 2.6.1.3. 2.6.2 Semi-quantitative assays for saponins (WHO, 1998) ‘The hemolytic activity of plant materials is determined by comparison with a reference material, Saponin R, a commercially available reference material Saponin R has a hemolytic activity of 1000 units per gram. ‘A suspension of erythrocytes is mixed with equal volumes ofa serial dilution of the extract of the plant material. The lowest concentration to effect complete hemolysis is determined after allowing the mixtures to stand for a given period of time.7 Calculate the hemolytic activity of the plant material using the fay, Win formula; 1000 a/b Where: 1000 = the defined hemolytic activity of saponin R in relay to cow's blood ion ity of saponin R that produces total hemolysis (g qua quantity of plant mate hat produces total hemos Ss /- 1623'Thel icbermann-Burchard test for unsaturated sterols and tri terpeng, he Liebermann-Burchard test for unsaturated sterols and triterpene, range of colors from blue to green, red, pink, purple or violet. give Take the equivalent of 10 g plant material from the stock plant extract prepared in Section 1.1 and evaporate to dryness over a steam bath. Cool to room. temperature; + Defat the material as in Section 2.2.1; + Treat the aqueous layer with 10 mL chloroform (CAUTION: carcinogenie!), and gently shake the mixture; + Allow to stand and pipette off the chloroform extract; + Dry the chloroform extract by filtering the mixture through about 100 mg anhydrous sodium sulfate held over dry filter paper. Refer to Figure P3; + Divide the filtrate into two portions. Use one portion for control; + Treat the other portion with 3 drops of aceticanhydride (CAUTION: highly corrosive!) then one drop of conc. sulfuric acid (CAUTION: highly corrosive!); + Observe for any immediate color change. Let stand for an hour; + Observe for further color changes; Figure P3. Setupfor + Compare with the control and record the results. ‘faa 2.6.4 Thin-layer chromatographic (TLC) test for sapogenins Sapogenins are the aglycones, oF the non-sugar groups of the saponins. The aglycone part ofthe saponin molecule is not considered to be hemolytic: If the plant extract gives a positive est for saponins, the saponins in the wk mire are hyde with te a. The resulting sapogenin a detected ymain groups of tannins, the hydrolysable tannin, eal are heterogeneous ae containing phengh the condensed ta Tinkages. ‘The las condensed ang a compounds united Jsrelated to flavonoid pigments frequent consinn ‘ers of phenolic compound nsed tannins are decoy ‘ney are pole of reatmient with acid, the condensed ins are decomposed in eh wesc Be hlohaphene or tannin reds. to ‘reall compounds known sp Geabay nins pea fan) implant erat y the gelatin rs. In SUCH, the ag peste lain protein The eacton made sensitive By the addin of rae ace the calting-out of the protein-tannin complex. Positive ex peer ee eee ride west ydolyzable tannins give blue Orble con while condensed tannins give a brownish-green color to oe : slyphenols,on the other handalso give characteristic color reactions with the, aoa Polyphenols include a wide range of plant substances, which Posten common, an aromatic ring bearing two or more hydroxyl groups. These substance. a to be water-soluble since they most frequently occur combined with sugar gleoxg Polyphenols, however, give no precipitate with the gelatin test. Polyphenols, because oftheir similarity to tannins in terms of structure, are ye additives in tanning agents in the leather industry. Chemically, there are 690 cosed tannins. The fOr cose units by ester oa 4 B Q Pe a fl 2) le) & a i 2.7.1 Screening methods for tannins \2.7-1.Lthe test tube method Sodium chloride, NaCl, 10% Dissolve 10 g NaCl in enough distilled water to make 100 mL solution, + Takean sulentof 10g plant materi fom the stock planestnet prepa in Section 1.1 and evaporate ths to incipient dryness over a stam be + Extract the residue with 20 mL of hot distilled water. Add 5 drops sf sodium chloride solution; =“ + Fer and divide the filtrate into three test tubes. Take one portion sth control; 3 + Take an aqueous solution of tannic acid as reference standard, Gelatin test Preparation of the tes solutions Gelatin-salt reagent pene an equal amount of 1% gelatin solution with 10% sodium chloride Positive result for tannins ‘The formation of ajelly-precipitate indicate the presence of tannins.+ Treatone portion ofthe filtrate of the plant ext saereagent. Do likewise othe tannic acd solution fn latin ‘olution, the reference standard + Compare with the control and the references ference standard ron (II) chloride, FeCl, reagent Dissolve 3 mL of 1% FeCl, solution in 100. ml. distilled water. Iron (III) chloride solution, 1% Dissolve 1.0 g FeCl, in 100 mL distilled water, Poser jue-black color indicates the peng eo ate in Note: Polyphenols also give colored products with the ferric chloride test eazent. loride test reagent, + Treat another portion of the plant filtrate with with three drops of ferric chlor reagent.Do likewise othe tannic acid sahion Moe + Compare with the control and the reference standard. 2.7.1.2 Spot test for tannins Preparation of the para-nitrosodimethylaniline yellow reagent paper “Take a strip of filter paper, "x 1"in size. Completely wet the paper ina solution 0f0.2gofp-nitrosodimethylaniline and 1 g p-dimethylamino-benzaldehyde dissolved in 100 mL ethanol. Dry the paper in ar to leave a yellow strip of reagent papes. Positive result for tannins ‘The formation of a red or red-brown stain on the yellow reagent paper is indicative of a positive response. Confirmatory result ‘A more intense red stain on the white background shows the presence of + Take 10 goffinely cut fresh leaf sample and macerate with 10 mL. of 0.1M hydrochloric acid, filter off the residue; + Evaporate the filtrate to 1 mL volume; + Place a drop of the prepared acid plant filtrate on the yellow reagent papers + Heat the spotted paper on top of a hot plate, reaction and dry the paper in about 3 minutes; + Observe the color of the spot; hot enough to hasten the 49‘Toinsure that hydrolysis takes place, scamistue ofemulsin and flax Tatts the or etimesecommended. The miatureof stdeshaving romaticand aliphatic tna SPHIN from any one of « numberof tones and ‘cyanogens that Gas iqnarte Preparation of the picrate paper Sodium picrate solution Dissolve 5 g sodium carbonate and 0.5 g picrc acid 100 mL solution. acid in enough water to make Picrate paper Dip strips of filter paper (1m x5 em) ito a freshly prepared solution of sc lution of sodium jcrate. Drip dry and then finally dry between sheets of newspr eS eas veen sheets of newsprints. Prepare picrate addition o ‘ast . ofalond erin mes with a buffer, is imarase enzymes will hy \erefore, insure ena ‘might be present = + Place 2 to 5 g of the crushed plant material in a test tube; «+ Moisten with enough water, then add a few drops of chloro i toenhance enzyme activity. One mL. orienta sleion Oe) ny also be added to insure hydrolysis ofthe glycoside, cere + Stopperthe tube with acork from which is suspended a strip of yellow picrate paper, taking care that the paper strip does not touch the inner side of the test tube Refer to Figure P5; ‘© Warm the tube at 35-40°C in a water bath or keep at room temperature for 2103 hours; + Observe for any color change inthe picrate papers + If no change in color is observed after 3 hours, the test is considered negative. 1e Guignard Test Figure PS. Test tube set-up for the st Pl i = a 3 = a [o>] ie) o) (= > e &