2.1 CLASSIFICATION & NOMENCLATURE OF PLANT 2.1.1 Scientific Classification of Tecomella undulata13 Kingdom Plantae (Plants) Subkingdom Tracheobionta (Vascularplants) Super division Spermatophyta (Seed plants) Division Magnoliophyta (Flowering plants) 2.1.2 Class Magnoliopsida (Dicotyledons) Subclass Asteridae Order Scrophulariales Family Bignoniaceae Genus Tecomella Nomenclatures13 Botanical name: Tecomella undulata Common name: English: Roheda, Honey Tree, Desert Teak, Marwar Teak Hindi: Roheda, Rohida Sanskrit: Chalachhada, Dadimacchada, Dadimapuspaka 34
2.2 STANDARDIZATION OF PLANT 2.2.1 Plant Collection and Identification The bark of Tecomella undulata was collected from the fields of Nohar, Hanumangarh (Rajasthan), in the month of November 2009 at morning time. The bark was identified by Dr. HB Singh (Scientist Incharge), NISCAIR (National Institute of Science Communication and Information Resources), New Delhi. (Ref. No. NISCAIR/RHMD/ Consult/2009-10/1326/128) 2.2.2 2.2.2.1 Material Solvents: Acetic acid, Acetone, Benzene, n-butanol, Chloroform, Dichloromethane, Ethanol, Ethyl acetate, n-hexane, Methanol, Pyridine, Petroleum ether, Tween 80, Toluene, Xylene are used. All solvents were purchased from Sd fine chemicals Pvt. Ltd. 2.2.2.2 Chemicals: Ammonia solution, Bismuth carbonate, Calcium chloride, Copper sulphate, Ferric chloride, Formic acid, Glacial acetic acid, Hydrochloric acid, Iodine, Lead acetate, Magnesium chloride, Mercuric chloride, Ninhydrin, Niric acid, Phloroglucinol, Potassium iodide, Potassium dichromate, Potassium sodium tartarate, Safranine, Sodium acetate, Sodium iodode, Sodium hydroxide, Sodium nitroprusside, Suden red III, Sulphuric acid, Tannic acid, vanillin. All the chemicals were purchased from Sd fine chemicals Pvt Ltd., Mimbai. Tecomin was purchased from Clearsynth Labs (P) Ltd. mumbai 2.3 PHARMACOGNOSTIC STUDY128-133 2.3.1 Morphological studies Macroscopic evaluation: Medicinal plant materials are categorized according to sensory, microscopic characteristics. Taking into consideration the variations in sources of crude drug and their chemical nature, they are standardized by using different techniques including the methods of estimation of chief active constituents. 35
Organoleptic evaluation of drugs refers to the evaluation of drugs by colour, odour, size, shape, taste and special features including touch and texture etc. They are of primary importance before any further testing can be carried out. Organoleptic evaluations can be done means of organs of sense which includes the above parameters and thereby define some specific characteristics of material which can be considered as a first step towards establishment of identity and degree of purity. The following Organoleptic investigations were done. Colour: The untreated sample was properly examined under diffused sunlight or artificial light source with wavelengths similar to that of daylight. Shape and Size: The length, breadth and thickness of the drug are of great importance while evaluating a crude drug. A graduating ruler in millimeter is adequate for the measurement. Small piece of bark were measured by aligning ten of them on a sheet of a calibrated paper 1 mm apart between the line and the result was divided 10. Average length, breadth and thickness were determined. Surface characteristics, texture and fracture: The study of morphology of bark was done by taking ten samples and observed for various qualitative and quantitative macroscopic characters viz. internal and external texture, fracture, thickness of bark and length width of the same. The texture was best examined by taking a small quantity of material and rubbing it between the thumb and forefingers, it was usually described as smooth, rough and gritty. The touch of the material determined the softness or hardness. 2.3.2 Microscopic studies The bark of Tecomella undulata was boiled with water until soft. Free hand sections of bark parts was cut, transferred on slides, cleared by warming with chloral hydrate and mounted in glycerine. 2.3.2.1 Powder analysis Bark were washed, cut into small pieces and were allowed to dry in the shade. Dried materials were ground in a mixer to a coarse powder and used for following investigation. 36
Organoleptic properties of Powder: Powder of bark of Tecomella undulata was examined for colour, odour and taste. Microscopy of powder: Powdered (# 60) bark of Tecomella undulata was cleared with chloral hydrate, mounted in glycerin and observed under the compound microscope. 2.3.3 Physiochemical Studies Physiochemical investigations were done to identify the amount of inorganic and moisture content and estimate dry weight of drug. 2.3.3.1 Determination of ash values a) Total ash value Method: Weighed accurately 2 to 3 gm of air-dried bark, in a tarred platinum or silica dish and incinerated at a temperature not exceeding 4500 C until free from carbon, cooled and weighted. If a carbon free residue could not be obtained then exhausted the charred mass with hot water, collected the residue on an ash less filter paper incinerated the residue and filter paper until the ash was white or nearly so, added the filtrate, evaporated to dryness and ignited at a temperature not exceeding 4500 C. Percentage of ash with reference to the air dried drug was calculated. b) Determination of acid insoluble ash Method: Boiled the ash with 25 ml of 2M Hydrochloric acid for 5 min, collected the insoluble matter in a Gooch crucible or on an ash less filter paper, washed with hot water, ignited, cooled in desiccator and weighed. Percentage of acid insoluble ash with reference to air-dried drug was calculated. c) Determination of water soluble ash Method: Boiled the ash for 5min. with 25 ml of water, collected the insoluble in a Gooch crucible or on an ash less filter paper, washed with hot water, and ignited for 15min. at a temperature not exceeding 4500 C. Then Subtracted the weight of insoluble matter from the weight of the ash, the difference in weight represents the water soluble ash and percentage of water soluble ash with reference to the air dried drug was calculated. 37
2.3.4 Loss on drying Accurately weighted quantity of sample was taken in a tarred L.O.D. The sample was heated at 1050 C in an oven and weighed. This procedure was repeated until a constant weight was obtained. The moisture content of sample was calculated with reference air dried drug. 2.3.5 Treatment of powder drug with different chemical reagents Powdered drug treated with various chemical reagent of different strength and then observed for change in colour. 2.3.6 Fluorescence analysis of powder drug The powdered drug was treated with different reagents and was examined under UV light in UV chamber and observed for fluorescence 2.3.7 ph determination ph 1% Solution: 1 gm (1%) and 10 gm (10%) of the accurately weighed powder of Tecomella undulata bark was dissolved in 100 ml water and filtered. ph of filtrate was determined by using ph meter. 2.3.8 Determination of extractive values The water soluble and alcohol soluble extractive values of air dried sample were evaluated using the procedure described in IP 1996. Ø Water soluble extractives Ø Alcohol soluble extractives Ø Water soluble extractives 5 gm of air dried plant material was added to 50 ml of boiled water at 80 C in a stoppered flask. It was shaken well and allowed to stand for 10 minutes so as to cool it and then it was filtered. 5 ml of filtrate was transferred to a tarred evaporating dish, which is 7.5 cm in diameter, the solvent was evaporated on water bath, allowed to dry for 30 minutes, 38
finally dried in an oven for 2 hours at 100 C and residue was weighed. Percentage of water soluble extractives was calculated with reference to the air dried drug. Ø Alcohol soluble extractives 5 grams of air dried plant material was macerated with 100 ml of methanol in a closed flask, shaking frequently during the first 6 hours and allowed to stand for 18 hours. Thereafter it was filtered rapidly taking precaution against loss of methanol. Evaporate 25 ml of filtrate to dryness in a tarred flat bottom shallow dish dried at 105 C and weighed. Percentage methanol soluble extractive was calculated with reference to the air-dried plant material. 2.3.9 Phytochemical Investigation 2.3.9.1 Preparation of extracts and Determination % yield Preparation of different extracts of Tecomella undulata powdered bark had been done successively in a continuous soxhlet extractor with Hexane, Petroleum ether, Chloroform, Ethyl acetate and Methanol. Aqueous extract was prepared by decoction method. A definite weight of crude drug powder was taken in a soxhlet apparatus thimble after making moderately coarse followed by continuous hot soxhlet extraction was done by various solvent in a sequence of increasing polarity as follows: 1. Hexane 2. Petroleum ether 3. Chloroform 4. Ethyl acetate 5. Methanol Concentrate the prepared liquid mass in Rota Evaporator and the percentage yield were calculated on the basis of dry weight. For preparation of aqueous extract the powder drug was heated with sufficient distilled water for 1 hr, solvent was evaporated in water bath and % yield was calculated on the basis of dry weight. 39
2.3.9.2 Preliminary Phytochemical Screening Chemical tests: Presence of type of constitutes in Petroleum ether, Ethyl acetate, Methanol and Water extract were determined by using various chemical diagnostic agents as follows: 2.3.9.2.1 a. Test for alkaloids Mayer s test: Extract/fraction on treatment with Mayer s reagent [Potassium mercuric iodide solution] should give cream coloured precipitate if alkaloids are present. b. Dragendorffs test: Extract/fraction on treatment with Dragondroff s reagent [Potassium bismuth iodide solution] should give reddish brown precipitate if alkaloids are present. c. Wagner s test: Extract or fraction on treatment with Wagner s reagent [solution of iodine in potassium iodide] should give reddish brown precipitate if alkaloids present. d. Hager s test: Extract or fraction on treatment with Hager s reagent [saturated solution of Picric acid] should give yellow precipitate if alkaloids are present. 2.3.9.2.2 Tannic acid test Extract or fraction on treatment with 10% Tannic acid solution should give buff coloured precipitate if alkaloids are present. 2.3.9.2.3 Test for Glycosides I. General test Part A: Extracted 200 mg of the drug by warming in test tube with 5ml of dilute (10%) sulfuric acid on a water bath at 1000 C for 2 min, centrifuged or filtered, pipette off supernatant or filtrate. Neutralized the acid extract with 5% solution of sodium hydroxide (noted the volume of NaOH added). Added 0.1 ml of fehling s solution A and B until alkaline (test with ph paper) and heated on water bath for 2 min. noted the quantity of red precipitate formed and compared with that formed in Part-B. Part B: Extracted 200mg of the drug using 5 ml of water instead of sulfuric acid. After boiling added volume of water equal to the volume of NaOH used in the above test. Added.01 ml of Fehling s solution A and B until alkaline (test with ph paper) and heated on water bath for 2 min. noted the quantity of red precipitate formed. 40
The quantity of precipitate formed in Part-B with that of formed in Part-A was compared, if the precipitate in Part-A greater than in Part-B then Glycoside may be present. Since Part-B represents the amount of free reducing sugar already present in the crude drug. Whereas Part-A represents free reducing sugar plus those related on acid hydrolysis of any sides in the crude drug. II. Chemical test for specific glycosides A. Test for saponin glycosides a) Froth Test: Placed solution of drug in water in a semi micro tube shaken well and noted the stable froth. b) Haemolysis test: Added 0.2 ml solution of saponin (prepared in 1% normal saline) to 0.2 ml of v/v blood in normal saline and mixed well, centrifuged and noted the red supernatant; compared with control tube containing 0.2 ml of 10% blood in normal saline diluted with 0.2 ml of normal saline. B. Test for anthraquinone glycosides a) Borntrager s test: Boiled extract or fraction with 1 ml of dil. Sulphuric acid in test tube for 5 min (anthracene glycosides are hydrolyzed to aglycone and sugars by boiling with acids) centrifuged or filtered while hot (if centrifuged hot, the plant material can be removed while anthracene aglycones are still sufficientlysoluble in hot water, they are however insoluble in cold water), pipette out the supernatant or filtrate, cooled and shaken with an equal volume of dichloromethane (the aglycones will dissolve referably in dichloromethane) separated the lower dichloromethane layer and shaken with half its volume with dilute ammonia. A rose pink to red color is produced the ammonical layer (aglycones based on anthroquinones give red color in the presence of alkali) if anthraquinone is present. b) Modified Borntrager s test: boiled 200 mg of the extract with 2ml of dilute sulphuric acid, 2ml of 5% aqueous ferric chloride solution for 5min and continued the test as above. As some plant contains anthracene aglycone in reduced form, if ferric chloride is used during the extraction, oxidation to anthraquinones takes place, which shows response to the Borntrager s test if anthraquinone is present. 41
C. Test for cardiac glycosides a) Kedde s test: Extracted the drug with chloroform, evaporated to dryness and added one drop of 90% alcohol and 2 drops of 2% 3,5-dinitro benzoic acid (3, 5-dinitro benzene carboxylic acid-kedde s reagent) in 90% alcohol. Made alkaline with 20% sodium hydroxide solution. If purple color is produced it shows the color reaction with 3,5-dinitrobenzoic acids depends upon the presence of an α,β-unsaturated-o lactones in glycone. b) Keller-Killiani test [test for deoxy sugars]: Extracted the drug with chloroform and evaporated it to dryness. Added 0.4ml of glacial acetic acid containing a trace amount of ferric chloride. Transferred to a small test tube and added carefully 0.5 ml of concentrated sulphuric acid by the side of the test tube, blue color appears in the acetic acetic acid layer if cardiac glycosides are present. D. Test for cyanogenetic glycosides Placed 200 mg of drug in a conical flask and moistened with few drops of water, (there should be no free liquid at the bottom of the flask the test will not work if there is any liquid in flask as the hydrogen cyanide produced will dissolve in the water rather than, come off as a gas to react with the paper). Moistened a piece of picric acid paper with sodium carbonate solution (5% aqueous) then suspended by means of cork in the neck of the flask, warmed gently at about 370 C and observed the change in color. Hydrogen cyanide is liberated from cyanogenetic glycoside by the enzyme activity and reacts with sodium picrate to from the reddish purple sodium isopicrate if cyanogenetic glycoside is present. 2.3.9.2.4 a) Test for tannins & phenolic compounds Gelatin test: Extract or fraction with 1% gelatin solution containing 10% sodium chloride gives white precipitate if tannins are present. b) Ferric chloride test: Extract or fraction gives blue green color with ferric chloride if phenols are present. c) Vanillin Hydrochloride test: Extract or fraction when treated with few drops of vanillin hydrochloride reagent gives purplish red color if the test is positive. 42
d) Alkaline reagent test: Extract or fraction with sodium hydroxide solution gives yellow to red precipitate within short time if tannins are present. e) Mitchell s test: with iron and ammonium citrate or iron and sodium tartarate extract or fraction gives a water-soluble iron-tannin complex, which is insoluble in soluble of ammonium acetate if tannins are present. f) Extract or fraction when treated with heavy metals; precipitates tannins if present. g) Extract or fraction yield bulky precipitate with phenazone especially in the presence of sodium and phosphate provided tannins are present. 2.3.9.2.5 a) Test for flavonoids Shinoda test (Magnesium hydrochloride reduction test): To the extract or fraction, added few fragments of magnesium ribbon and added cone. Hydrochloric acid drop wise, pink scarlet, crimson red or occasionally green to blue color appears few minutes if flavonoids are present. b) Zinc Hydrochloride reduction test: To the extract or fraction, added a mixture of zinc dust and conc. Hydrochloric acid acid. It gives red color after few minutes if flavonoids are present. c) Alkaline reagent test: To the extract or fraction, added few drops of sodium hydroxide solution; formation of an intense yellow color, which turns to colorless on addition of few drops of dilute acid, indicates presence of flavonoids. 2.3.9.2.6 Test for proteins & amino acids a) Millons test: Extract or fraction when treated with 2 ml of millons reagent (Mercuric nitrate in nitric acid containing traces of nitrous acid), gives a white precipitate which turns red upon gentle heating indicates presence of proteins and amino acids. b) Ninhydrin test: Extract or fraction when boiled with 0.2% solution of ninhydrin (Indane 1,2,3 trione hydrate), gives violet color if proteins and amino acids are present. 2.3.9.2.7 Test for sterols & triterpenoids a) Liebermann Burchard test: Extract or fraction when treated with few drops of acetic anhydride and then boiled and cooled, Conc. Sulfuric acid was then added from the sides of the test tube, shows brown ring at the junction of two layers and the upper layer 43
turns green which shows the presence of steroids and formation of deep red color indicates the presence of triterpenoids. b) Salkowski test: Extract or fraction when treated with chloroform and few drops of con. Sulfuric acid, shaken well and allowed to stand for some time, red color appears at lower layer indicates the presence of steroids and formation of yellow colored lower layer indicates the presence of triterpenoids. 2.3.9.2.8 Test for carbohydrates a) Molisch s test: Treated the extract or fraction with few drops of alcoholic alpha naphthol and added 0.2 ml of conc. Sulfuric acid slowly through the sides of the test tube when a purple to violet color ring at the junction indicates presence of carbohydrates. b) Benedict s test: Treated the extract or fraction with few drops of Benedict s reagent (alkaline solution containing cupric citrate complex) and upon boiling on water bath, reddish brown precipitate forms if reducing sugars are present. c) Barfoed s test: It is a general test for monosaccharide. Heat the test tube containing 1 ml of reagent and 1 ml of extract fraction in a beaker of boiling water. If red Cuprous oxide is formed within 2 min, monosaccharide present. Disaccharide on prolonged heating (about 10 min) may also cause reduction, owing to partial hydrolysis to monosaccharide. d) Camnelisation: Extract or fraction when treated with strong sulfuric acid, undergoes charring with the dehydration along with burning sugar smell indicating presence of carbohydrates. e) Selwinoff s test: Hydrochloric acid reacts with ketose sugar to form derivatives of furfuraldehyde, which gives red colored compound when linked with resorcinol. Added extract or fraction to about 5 ml of reagent and boiled. Fructose gives red color within half minute. The test is sensitive to 5.5 mmol/ ltr if glucose is absent, but if glucose is presents it is less sensitive and in addition of large amount of glucose can give similar color. f) Tollen s test: To 100 ml of extract or fraction, added 2 ml of Tollen s reagent and heated gently, a silver mirror is obtained onside the wall of the test tube, indicates the presence of aldose sugar. 44
g) Fehling s test: Equal volume of Fehling s A (Copper sulfate in distilled water) and Fehling s B (Potassium tartarate and sodium hydroxide in distilled water) reagents are mixed and few drops of extract or fraction was added and boiled, brick red precipitate of cuprous oxide forms, if reducing sugars are present. 2.3.9.2.9 Test for free sugars: After complete removal of free sugars, the extract was hydrolyzed with mineral acid and then tested for the glycone and aglycones moieties. a) Raymond s test: Extract fraction when treated with dinitro benzene in hot methanolic alkaline, should give violet color. b) Legal s test: Extract fraction when treated with pyridine and added alkaline sodium nitroprusside solution, blood red color should appear. c) Bromine water test: Extract fraction when treated with bromine water should give precipitate. 2.3.9.2.10 a) Test for fats & fixed oils Stain test: Pressed the small quantity of extract between two filter papers, the stain on first filter paper indicates the presence of fixed oils. b) Saponification test: Added a few drops of 0.5 N of alcoholic potassium hydroxide to small quantities of various extracts along with a drop of phenolphthalein separately and heated in water bath for 1-2 hours. The formation of soap or partial neutralization of alkali indicates the presence of fixed oils and fats. 2.3.10 HPTLC Study for Tecomella undulata134-135 The HPTLC was carried out using a Hamilton 100 µl HPTLC syringe, Camag LinomatIV semiautomatic sample aplicator, Camag twin trough chamber (20x20 cm), Camag TLC Scanner-3, Camag CATS IV integration software, Silica gel- G60F254, 20 X 20cm TLC plate of 0.2 mm thickness was used as stationary Phase. Development of plates, chromatograms and calibration curve was produced for both plant extracts and formulation. 45
2.4 RESULTS 2.4.1 Morphological studies Results obtained from macroscopic examination were tabulated in table 2.1. Figure 2.1 showed the macroscopic structure of Tecomella undulata stem bark. Table 2.1: Macroscopical examination of bark of Tecomella undulata S.N. Parmrter Bark 1 Shape & Structure Curved 2 ColourOuter Surface Dull Brown in colour Inner Surface Dark Brownish in colour 3 Taste Slightly bitter 4 Odour Odourless 5 Fracture Short 6 Thickness 5-8 mm 7 Surface texture- 2.4.2 Outer surface Rough with transverse irregular cracks Inner surface Smooth with transverse concentric striations Microscopic studies of Tecomella undulata stem bark The bark of Tecomella undulata was boiled with water until soft. Free hand sections of bark parts was cut, transferred on slides, cleared by warming with chloral hydrate and mounted in glycerine. Results are shown in Figure 2.2 Qualitative and quantitative observations were made using X10 eye piece and X10 objectives. Photomicrographs were taken using Binocular photo- microscopic apparatus (LEICA, Italy) attached with Nikon camera. 46
(A) Outer bark (B) Inner bark Figure 2.1: Macroscopic structure of bark of Tecomella undulata Figure 2.2: Photomicrographs of transverse sections of stem bark of T. undulata 47
2.4.3 2.4.3.1 Powder Analysis Organoleptic properties Colour: Dark brown Odour: Odourless Taste: 2.4.4 Tasteless Powder characteristics of bark of Tecomella undulata Powdered (# 60) bark of Tecomella undulata was cleared with chloral hydrate, mounted in glycerin and observed under the compound microscope (Figure 2.3). (A) (B) (C) (D) Figure 2.3 Photomicrographs shows powder characteristics of bark of T.undulata. (A) Phloem fibre (B) Calcium oxalate crystals & Tanninferous cells (C) Medullary rays (D) Cork cells 48
2.4.5 Physical evaluation of powder drug Powder of bark was treated with standard procedures and results obtained were tabulated in table 2.2 Table 2.2: Physical evaluation of powdered drug of bark of Tecomella undulata S.N. 1 Parameter Loss on Drying 2 9.5 Extractive Values Ethanol soluble extractive 32 Water soluble extractive 7.2 3 2.4.6 Values (%)w/w Ash Values Total ash 13.5 Water soluble ash 5.0 Acid insoluble ash 7.5 Sulphated ash 3.0 Treatment of powder drug with different chemical reagents Powder material was treated with different chemicals and observations were tabulated in table 2.3 Table 2.3: Powdered drug analysis of bark of Tecomella undulata with different chemical reagents S.N. Reagents Colour observed 1 Powder + Conc. HCl Brownish 2 Powder + Conc. HNO3 Reddish Brownish 3 Powder + Conc. H2 SO4 Blackish 4 Powder + Glacial acetic acid Brownish 5 Powder + 5% NaOH Dark Brownish 6 Powder + 5% KOH Dark Brownish 49
7 Powder + 5% Ferric chloride Dark Greenish 8 Powder + Picric acid (saturated aq. sol.n ) Brownish 9 Powder + Ammonia solution Blackish 2.4.7 Fluorescence analysis of powder drug The fluorescence analysis of the bark Tecomella undulata powder was examined under UV light in UV chamber and observed for fluorescence were tabulated in table 2.4 Table 2.4: Fluorescence analysis of powdered bark of T. undulata S.N. Treatment Observation Observation (under long (under short wavelength) wavelength) 1 Powder as such 2 Powder + 1 N NaOH in methanol Light Greenish 3 Powder + 1 N NaOH in water Light Greenish 4 Powder + 50% HCl Light Greenish 5 Powder + 50% HNO3 Light Greenish 6 Powder + 50% H2SO4 Light Greenish 7 Powder + Petroleum ether 8 Powder +Chloroform Light Greenish 9 Powder + Picric Acid Greenish 10 Powder + 5% Ferric chloride sol.n Light Greenish 11 Powder + 5% iodine solution Light Greenish 12 Powder + Methanol Light Greenish 13 Powder + HNO3 + NH3 Greenish 2.4.8 ph determination ph of aqueous extract of Tecomella undulata was checked with ph meter and results were tabulated in table 2.5 50
Table 2.5: ph determination of powdered bark of Tecomella undulata ph 2.4.9 2.4.9.1 1% SOLUTION 10% SOLUTION 6.7 6.1 Phytochemical Investigation Preparation of extracts 100g of the air dried powdered bark was extracted successively with hexane, petroleum ether (40-60 C), ethyl acetate, chloroform and methanol in a Soxhlet extractor until siften tube (side tube) was completely colourless. Each time before extracting with the next solvent, the powdered material was dried in the tray drier below 50 C. The extract was concentrated by distilling off the solvent using rota-evaporator. For preparation of aqueous extract powder of bark was decocted with distilled water for 1 hour and Extract was then filtered using Whatman filter paper (size no.1). Solvent was evaporated in water bath. Percentage yield values were calculated on the basis of dry weight tabulated in table 2.6 Table 2.6: Percentage yield of extracts of bark of Tecomella undulata S.N. Extracts % Yield w/w 1 Hexane Extract 0.16 2 Petroleum ether Extract 10.03 3 Chloroform Extract 1.80 4 Ethyl Acetate Extract 16.91 5 Methanolic Extract 18.57 6 Aqueous extract 8.85 2.4.10 Qualitative Phytochemical Test The results of various quantitative phytochemical tests were tabulated in table 2.7 51
Table 2.7: Qualitative phytochemical tests of extracts Tecomella undulata bark S. Test N. Powder Pet. Ethyl Metha- drug Ether acetate nolic Water 1 Carbohydrates 2 Protiens and amino acids 3 Alkaloids 4 Flavanoids 5 Tanins and phenolic cpds. 6 Fixed oil and Fats 7 Phytosterols 8 Glycosides 9 Gum and Mucilage 10 Saponins 11 Volatile oil 2.4.11 HPTLC Study of Tecomella undulata a) Preparation of Sample solution Accurately weighted 10 mg of aqueous extract of Tecomella undulata was dissolved in 10 ml distilled water (1000 µg/ml) and passed through 0.45 Millipore filters. b) Preparation of Tecomin solution The standard solution was prepared by dissolving 10 mg tecomin in 10 ml purified water (1000µg/ml). The working standard of 200 µg/ml was prepared from standard solution by 52
diluting with purified water. Different concentrations of 10, 20, 30, 40, 50 µg/ml were prepared from standard solutions. c) Chromatographic conditions Analysis was performed on 20 cm 20 cm HPTLC silica gel-g60f254 plates. The plate cleaned by predevelopment to the top with methanol, and dried in an oven 1050 C for 5 min. Sample and standard zones were applied to the layer as bands by means of a CAMAG. Linomat-4 semiautomatic sample applicator equipped with a 100 µl syringe and operated with the settings band length 6 mm, application rate 150 µl/sec, distance between bands 8 mm, distance from the plate side edge 6.5 mm, and distance from the bottom of the plate 2 cm. d) Calibration curve of tecomin 10, 20, 30, 40 and 50 µl standard solution of tecomin applied onto TLC plate to generate Calibration curve. The plate was developed in the mobile phase Toluene: Acetone: Formic acid (2.5: 0.5:0.2 v/v/v) and dried in an oven 1050 C for 5 min. The standard zones were quantified by linear scanning at 251 nm by use of a TLC Scanner III CAMAG. Data of peak height and peak area of each spot was recorded. The calibration curve was prepared by plotting concentration (mg/spot) versus peak area (Figure 2.6). e) Recovery Studies Recovery Study was performed by spiking 10, 20, 30 and 40 µg/spot of standard drug externally to the pre-spotted (10 µg/spot) samples. The experiment was conducted in triplicate and applied onto the plate in duplicate. 53
Figure 2.4: HPTLC chromatogra m of Tecomella undulata e xtract Figure 2.5: HPTLC chromatogra m of Tecomin 54
Table 2.8: Comparative data of Standard and T. undulata bark extract Track Conc. of Tecomin ((µg/spot) Rf Mean Peak 1 10 0.65 5656.6±90.93 2 20 0.65 6625.8±150.83 3 30 0.65 7428.0±178.35 4 40 0.65 8225.5±174.92 5 50 0.65 9162.3±165.27 6 Extract of T. undulata 0.65 5801.1±118.35 Table 2.9: Method validation parameters for calibration curve S.N. Parame te rs Tecomin 1 Correlation- coe ffic ie nt (r) 0.9993 2 Repeatability (% CV) 0.6352 3 Range (µg/spot) 10-50 4 Slope 86.111 Table 2.10: Re cove ry s tudy of marke r compound by propos e d HPTLC me thod Marker Conc. Conc. Peak Area Amount Taken Added Mean±SD (n=3) found (µg/spot) (µg/spot) Recovery Avg. Recovery Mean±SD (n=3) 10 0 5654.6±110.15 9.99±0.43 99.90 10 10 6612.5±180.25 19.79±0.36 98.95 Tecomin 10 20 7421.2±225.10 29.52±0.49 98.40 10 30 8245.4±155.85 39.66±1.10 99.15 10 40 9159.3±189.88 49.57±1.15 99.14 99.10 55
Figure 2.6: Calibration curve of Tecomin Table 2.11: S.N. 1 Content of Tecomin present in Extract Plant extract Tecomin content (µg/ml) Aqueous extract Tecomella undulata 1.10 56
2.5 DISCUSSION Tecomella undulata is belongs from family Bignoniaceae. In pharmacognostical study the microscopical evaluation, macroscopical evaluation, p hysicochemical studies, p hytochemica l screening and chromatograp hic studies were performed for characterization and standardization of bark. It was found that bark of plant brown in colour, curved and 5-8 mm in diameter. Stone cells and oxalate crystals, tanninferous clls were viewed in microscopical examination. In physiochemical studies, Tecomella undulata was evaluated for total Ash value (13.5% w/w), acid insoluble ash (7.5% w/w), water soluble ash (5.0% w/w), sulphated ash (3.0% w/w), loss on drying (9.5% w/w), Ethanol soluble extractive (32% w/w) and water soluble extractive (7.2% w/w) values. In phytochemical screening studies, various extract of Tecomella undulata was studied for carbohydrates, proteins & amino acids, flavonoids, tannin & phenolic compounds, alkaloids, glycoside and sterols etc. It was found that stem bark of Tecomella undulata was contains glycosides, tannins and phenolic compound, saponins and phytosterols. In HPTLC analysis, different concentrations of standard solution of tecomin were applied in triplicate on HPTLC p lates and aqueous extract of Tecomella undulata applied o n HPTLC plate for estimation of tecomin. 57