A Test For Carbohydrates Biology

Standardization of herbal drugs is complex process since biotype; ecotype and genotypic factors are known to affect the level of secondary metabolites of plants.However information on external morphology, anatomy and micrometric data of organized plant material can be suitably used in identification of plant material. In addition, certain physico-chemical parameters such as extractive value, ash values etc can provide rough idea about quality and purity of crude drugs.

5.1.1 Collection and Authentication of plant material

The plant material was collected from the local area of Adgaone of Dist. Nasik, Maharashtra, in the month of September- 2009 at the time of collection, plant including flowers and roots were collected. The plant materialwasobtained from Nasik district (M.S.) and authenticated by Dr. D. A. Patil, reader and the authorized plant identifier of Department of Botany, SSVPS College, North Maharashtra University, Dhule (M.S) India; a specimen is preserved in the college herbarium (KBHSS/PCG/2009/12). During plant material collection, infected parts of plants were carefully separated. The plant material was thoroughly washed with water to remove adhered particles and debris and dried in shade.

5.1.2 Preparation and Storage

The plant parts were placed in open place for 3-4 daysfor drying. Dried material were homogenized and passed through sieve no 40. Powdered drug was stored in airtight container and protected from light for further use.

5.1.3 Morphological study

The external morphology was studied according to reported methods.96 Khandelwal Macroscopic study was performed by using simple microscope. The colour, odour, taste, size and shape of flowers and roots were determined.

5.1.4 Microscopical analysis

The free-hand section of scale of flowers and root were prepared from fresh plant material and finally stained with various staining reagents as per standard procedures. The disaggregation of plant material was performed by reported method. In brief, scales were disaggregated by means of boiling in an aqueous solution of NaOH (5 % w/v) for 5 min. After cooling and washing with water, pieces were treated with an aqueous solution of chromic acid (25 % v/v) for 30 min at room temperature. The section where cleared with chloral hydrates solution, stained with phloroglucinol-hydrochloric acid (1:1) and toludine blue. 97,98 (O'BrieTP,)Kokate C. K., 2008.Powdered drug were used for the observation of power microscopical character. The powdered drug were separately treated with phloroglucinol-hydrochloric acid(1:1)solution, acetic acid and iodine solution to determine the presence of the lignified fibers, Calcium oxalate crystals and starch grains respectively. A series of digital images captured using a Motic Digital microscope fitted with 1/3'' CCD camera imaging accessory and using Motic Images 2000 (1.3 version) analysis software. The micrometric data were generated from average of 30 measurements for each sample and expressed as lower limit mean+SD.99,100(Brain KR, Kokate C.K. 2010..)

5.1.5 Fluorescence analysis: 101, 102(Gokhale S.B.,) Pratt

The fluroscence analysis is carried out on the powdered sample. Dry powdered sample was placed on a slide, treated with several drops of specified reagents and observed within minute under UV lamp.

5.1.6 Behaviour of powder towards specific reagents:103, 104(Sing VK, 2002,Kalaskar)

Behaviour of flower and root powders of T. populanea with different chemical reagents were performed to detect occurrence of phytoconstituents along with colour changes under ordinary daylight by standard method.

5.1.7. Physiochemical constants 105-107 (Khandelwal KR. 2005, Indian Pharmacopoeia. 1996, Mukherjee PK. 2002.)

5.1.7.1. Ash Value

Ash values are indicative to some extent of care taken in collection and preparation of drug for market and of foreign matter content of natural drug. The object of determining ash value of vegetable drugs is to remove all traces of organic matter which may otherwise interfere in analytical determination. On incineration, crude drugs normally leave an ash usually consisting of carbonates, phosphates and silicates of sodium, potassium, calcium and magnesium.

The total, acid-insoluble, water soluble and sulphated ash values were determined according to Indian Pharmacopoeial methods.

5.1.7.1.1. Elemental analysis of Ash 104.

The powdered drug was incinerated in muffle furnance to obtain ash. The ash was treated with 50% hydrochloric acid for 30 minutes and filtered. The filtrate was used for the detection of elements by specific test.

5.1.7.1.2. Test for aluminium: Test solution was treated with dilute ammonia solution observed gelatinous precipitation, soluble in hydrochloric acid, acetic acid and sodium hydroxide solution.

5.1.7.1.3. Test for Copper

a. Test solution treated with hydrogen sulphide produced brownish -black precipitate.

b. Test solution was treated with sodium hydroxide produced light blue precipitation.

5.1.7.1.4. Test for Calcium

a. Test solution treated with ammonium carbonate solution shows white precipitate which after boiling and cooling is insoluble in solution of ammonium sulphide.

b. Test solution treated with potassium chromate observed yellow crystalline precipitate.

5.1.7.1.5. Test for Iron

a. Test solution mixed with dilute hydrochloric acid and potassium permanganate produced faint pink coloration.

b. Test solution mixed with dilute hydrochloric acid and solution of ammonium thiocynate produced blood red coloration.

5.1.7.1.6. Test for Magnesium

a. Test solution shown white precipitation with after boiling with ammonium carbonate.

b. test solution, dilute ammonia and sodium phosphate solution produced white crystalline precipitate.

5.1.7.1.7. Test for Potassium: Test solution and perchloric acid observed white precipitation

5.1.7.1.8. Test for Sodium: Sample moistens with HCl and introduced on platinum wire into the flame of bursen burner shows yellow colour.

5.1.7.2. Moisture Content

Loss on drying is the loss in weight in percent w/w resulting from loss of water and volatile matter of any kind that can be driven off under specific conditions.

5.1.7.3. Extractive value

The alcohol and water soluble extractive values were obtained according to Indian Pharmacopoeial methods. In brief, 5 gm of coarsely powdered and air dried powder was macerated in 100 ml of ethanol (90 % v/v) in a closed container for 24 h. After 24 h, content was filtered rapidly to minimize alcohol loss. Accurately 25 ml of filtrate was evaporated to dryness in a tarred flat bottomed shallow evaporating dish and dried at 1050C and weighed.

Similarly water soluble extractive was determined. In brief, 5 gm of coarsely powdered and air dried sample was macerated in 100 ml of water in a closed container for 24 h, with frequently shaking during the first 6 h and allowed to stand for 18 h. After 24 h, content was filtered rapidly to minimize water loss. Accurately 25 ml of filtrate was evaporated to dryness in a tarred evaporating dish and dried at 1050C and weighed.

5.1.8. Phytochemical Evaluation108 (khadbadi) 98,109 (Kokate, Trease)

50 g of powdered plantmaterialwas packed in white cotton bags and were then extracted successively using different solvents in a Soxhlet apparatus. The solvents used for extraction were petroleum ether (60-80 0C), benzene, chloroform, ethyl acetate, methanol and water.Each time before extracting with the next solvent, the powdered material was dried in an air oven below 500C. The extractive values and preliminary phytochemical studies were carried out on these successive extracts.

In the present study, the qualitative chemical tests were performed on obtained successive extractsto ensure the presence ofphytoconstituents such as alkaloids, glycosides, tannins, flavonoids, terpenoids, steroids, carbohydrates, etc.

5.1.8.1. Test for Alkaloids:

Dragendorff's test: To the extract add Dragendorff's reagent, orange brown precipitate indicates the presence of alkaloids.

Mayer's test: To the extract add Mayer's reagent, cream colored precipitate indicates the presence of alkaloids.

Wagner's test: To the extract add Wagner's reagent, reddish brown precipitate indicates the presence of alkaloids.

Hager's test: To the extract add Hager's reagent, yellow precipitate indicates the presence of alkaloids.

5.1.8.2. Test for Carbohydrates

Molish's test: To the extract add few drops of alcoholic a-naphthol, then add few drops of concentrated sulphuric acid through sides of test tube; purple to violet color ring appeared at the junction of two liquids.

Barfoed's test: 1ml of extract is heated with 1ml of Barfoed's reagent, if red cupric oxide is formed, indicates the presence of monosaccharide. Disaccharides on prolong heating (about 10 min.) may also cause reduction, owing to partial hydrolysis to monosaccharide.

5.1.8.3. Test for Flavonoids

Shinoda test: To the extract add few magnesium turnings and concentrated hydrochloric acid drop wise, pink scarlet, crimson red or occasionally green to blue color appears after few minutes.

Lead acetate test: To small quantity of residue add lead acetate solution yellow colour precipitate is formed indicates the presence of flavonoids.

5.1.8.4. Test for GlycosidesGeneral test:

Test A: Extract 200 mg of drug with 5ml of dilute sulphuric acid by warming on a water bath and filtered. Then neutralize the acid extract with 5% solution of sodium hydroxide. Add 0.1ml of Fehling's solution A and B and heat on a water bath for 5 minutes. First yellow then brick red precipitate is formed indicating the presence of glycosides.

Test B: Extract 200 mg of the drug using 5 ml of water instead of sulphuric acid. After boiling add equal amount of water as used for sodium hydroxide in the above test. Add 0.1 ml Fehling's A and B until alkaline (test with pH paper) and heat on water bath for 2 min. Note the quantity of red precipitate formed.

Compare the quantity of precipitate formed in Test B with that of formed in test A. If the precipitate in Test A is greater than in Test B then glycoside may be present. Since Test B represents the amount of free reducing sugar already present in the crude drug, whereas Test A represents free reducing sugar and those getting after acid hydrolysis of any glycoside in the crude drug.

Legal's test: aqueous or alcoholic extract, add 1 ml pyridine and 1 ml of sodium nitroprusside, pink colour appears indicates the presence of glycosides

Baljet's test: A thick section shows yellow to orange colour with sodium picrate indicates the presence of glycosides.

5.1.8.5. Test for Phenolic compounds (Tannins)

Ferric chloride test: Treat the extract with ferric chloride solution, blue color appears if hydrolysable tannins present and green color appears if condensed tannins present.

Lead acetate test: To small quantity of aqueous extract add lead acetate solution. White colour precipitate is formed indicates the presence of tannins.

5.1.8.6. Test for Proteins:

Biuret test: To the 2 ml extract 2 ml add 4 % NaOH and few drops of 1 % CuSO4 solution Biuret reagent was added, violet color indicates the presence of proteins.

Xanthoproetic test: To the (5 ml) extract, add 1 ml of concentrated H2SO4 and boil, yellow precipitate is formed. After cooling it, add 40% NH4OH solution, orange color is formed.

5.1.8.7. Test for Steroids and Triterpenoids:

Libermann-Burchard test: - Mix the extract with chloroform, add few drops of acetic anhydride boiled and cooled. Then add concentrated sulphuric acid from the side of the test tube, brown ring is formed at the junction of two layers and upper layer turns green which shows presence of steroids and formation of deep red color indicates presence of triterpenoids.

Salkowski test: Treat the extract with few drops of concentrated sulphuric acid red colour at chloroform layer and acid layer shows greenish yellow fluorescence indicates the presence of steroids and formation of yellow coloured lower layer indicates presence of triterpenoids.

5.1.9. Extraction Methodology5.1.9.1. Extraction of flowers powder

Dried powder of flower was extracted with methanol (4.5 L) for 6 h at room temperature. The methanolic extract of flower (TPF) was combined and evaporated under reduced pressure at 400C to about 50mL. The TPF was partitioned three times with n-butanol saturated with H2O (50 mL each time). Further, n-butanol fraction was partitioned two times with diluted ammonia solution (50 mL each time). Then the ammonia layer was concentrated under reduced pressure at 40°C. The extracts were freezed-dry to yield about 210g.While, the aqueous fraction was treated with chloroform (3 times, 50 mL) to obtain the chloroform fraction (165 g) 110 (He-Long Bai, 2010)

Fig 5.1: Extraction scheme for Thespesia populnea flower powder

5.1.9.2. Extraction of root powder

The dried roots powder (1.5kg) was extracted with methanol (4 L) by using soxhlet method for 7 days at 45°C.Theethanolic extract of root (TPR) was filtered and concentrated. Further, TPR was dissolved in water and extracted with n-hexane for 3hr to obtained n-hexane fraction (240 g).Theaqueous layer treated with carbon tetrachloride (3 times, 100 mL) lower layer of carbone tetrachloride was collected and concentrated (35 g); while, the separated aqueous layer was concentrated using evaporator (110 g).111 (Md.AbdulMuhit, 2010.)

Fig. 5.2: Extraction scheme of root powered of T. populnea Linn.

5.1.10. Chromatographic study5.1.10.1. Thin layer chromatography (TLC)

TLC study was done with TPF and TPR to find out the probable active compounds present in them. On the precoated silica gel TLC plate, test samples (dissolved in respective solvents) were applied. Then the plate was properly marked on the top for their identification. To avoid insufficient chamber saturation and undesirable edge effect, a smooth sheet of filter paper was placed (in U shape) in rectangular TLC chamber and was allowed to be in the developing solvent. Having been moistened, the paper was then pressed against the walls of the chamber, so that it adhered to the walls. Several solvent systems were tried during the study. The plate was sprayed withanisaldehydesulphuric acid (AS) reagent, Vanillin sulphuric acid (VS) reagent or Libermann- Burched (LB)reagent and heated at 1000C for 5 minutes. The most informative and satisfactory resolution was taken as final solvent system.

5.1.10.1.1 Thin layer chromatography of TPF.112(Cetkovic,).

TLC profile

Stationary phase

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