3.1. Collection of plant materials
Plant parts of Madhuca longifolia were collected from 25-30 year old trees, from a temple-owned grove, in a village, Rajendrum Arcot of Thanjavur district, Tamilnadu. Basella alba and Cissus quadrangularis are collected from the plants maintained at
Collected plant specimens were identified with the use of local Floras viz. Gamble (1967) and Matthew (1983). Identity of plant specimens was confirmed in consultation with Botanical Survey of India (BSI)
3.2. Anatomical methods
Collected plant parts were fixed in FAA (95% Ethyl alcohol 50 parts + Glacial acetic acid 5 parts + Formalian 10 parts + Distilled water 35 parts) in the field itself. Free-hand sections paraffin embedded sections and macerations were employed wherever necessary. Drawings were made with the use of prism type camera Lucida and microphotographs were taken using Leitz-Orthoplan microscope. Calcium oxalates were photographed with polarized light in the same microscope. Micro-measurements were made by calibrating a stage micrometer with ocular micrometer.
Fixed plant parts were dehydrated in N-butyl alcohol (NBA) or tertiary butyl alcohol (TBA) series.
NBA dehydration series was prepared as follows:
| Sl. No. | Ethyl Alcohol ml | NBA ml | Distilled water ml | Time in hours |
| 1 | 20 | 10 | 70 | 1 |
| 2 | 25 | 15 | 60 | 1 |
| 3 | 30 | 25 | 45 | 1 |
| 4 | 30 | 40 | 30 | 1 |
| 5 | 25 | 55 | 20 | 2 |
| 6 | 20 | 70 | 10 | 2 |
| 7 | 15 | 85 | -- | 2 |
| 8 | -- | 100 | -- | 2 |
TBA dehydration series was prepared as follows:
S1. No | Distilled Water ml | TBA ml | Ethyl Alcohol 95% Ml | Ethyl Alcohol 100% ml | Times in hours |
| 1 | 50 | 10 | 40 | -- | 2 |
| 2 | 30 | 20 | 50 | -- | 12 |
| 3 | 15 | 35 | 50 | -- | 1 |
| 4 | -- | 55 | 45 | -- | 1 |
| 5 | -- | 75 | -- | 25 | 1 |
| 6 | -- | 100 | -- | -- | 1 |
| 7 | -- | 100 | -- | -- | 12 |
| 8 | -- | 100 | -- | -- | 1 |
Dehydrated materials were infiltrated at 55-60° C and embedded in paraffin wax of melting point 56-58° C. Transverse and longitudinal sections were cut at 5-15 1.1 thickness using a rotary microtome. Haupt's adhesive with formalin (4%) was used for affixing the paraffin sections on the slides.
Staining:
The staining schedules followed were,
Toluidine Blue '0'
Safranin 'a' and Fast Green 'FCF'
Toluidine Blue '0':
Differential staining of sections was achieved without removing the paraffin by using 0.05% toluidine blue in water. After staining and washing in distilled water, the slides were dried, de-waxed in xylene and mounted in DPX.
Safranin '0' and Fast Green 'FCF'
2.25 gm of safranin was dissolved in 225 ml of 95% alcohol. One gm of fast green was dissolved in a mixture of clove oil and absolute alcohol (in the ratio of 75:25). The de-waxed slides were stained in safranin for sufficient time and washed in alcohol until the excess stain was removed. Then the slides were stained in fast green and differentiated in clove oil and alcohol. The slides were brought down to xylene and mounted in DPX.
Histochemical stains
Phloroglucinol
This stain was used to localize lignified cells. Sectioned plant parts were flooded in 1 % solution of phloroglucinol in ethanol for 1-2 minutes. Excess stain was drained off. To this few drops of HCI was added. It was added and excess HCI was drained off. It was mounted in week glycerin and observed.
Ferric chloride
Localization of tannin was achieved by use of ferric chloride. Sectioned materials were kept in 10% formalin containing 2% ferric chloride. Stained sections were washed and mounted in water.
Iodine - Potassium Iodide
It was used to stain starch grains in the cells. 2 gm of potassium iodide and 0.2 gm of iodine was dissolved in 100 ml of water and used as reagent. Sections of plant materials were mounted in the above reagent and observed.
Maceration Technique
Jeffrey's maceration method was adopted. Small pieces of specimens not more than one nun in thickness were kept in a solution of equal parts of 10% chromic acid and 10% nitric acid for overnight or until the materials became' mushy' in texture. After maceration, the tissues were washed in water and stained with safranin. The macerated and stained tissues were spread on slides and mounted in glycerin for observation.
3.3. Quantitative microscopy
Peelings of upper and lower leaf epidermal layers were used for calculating the stomatal index. Leaf segments of 2 X 10 mm in size were soaked in sodium hypochlorite solution, until they got bleached. These bleached materials were used for calculating the palisade ratio, vein islet number and vein termination number. Calcium oxalate crystals were also observed in these materials and measured.
3.4. Preparation of powder
The necessary plant parts were collected and dried under shade. These dried materials were mechanically powdered after keeping them in an oven at 35°C for 24 hours. These powdered materials were used for further physicochemical, phytochemical and fluorescent analysis.
3.5. Analytical Methods
The procedures recommended in Indian Pharmacopoeia (1966) were followed for calculating total ash, acid-insoluble ash, loss on drying at 110°C and alcohol and water-soluble extractive percentages. The percentage of extractive value in different solvents was calculated by successive extraction of the sample in a soxhlet extractor with petroleum ether (60-80°C), benzene, chloroform, alcohol and water.
3.5.1. Total Ash
5 gm of plant powder was ignited in an electric furnace at 600°C in silica crucible until the sample reach a constant weight.
3.5.2. Acid - Insoluble Ash Value
Total ash obtained was heated with addition of 25 ml of dilute HCl for 10 minutes. It was filtered in an ash-less filter paper (Whatman No. 41) and the residue was ignited in the furnace to get a constant weight.
3.5.3. Loss on drying
Freshly collected and pre-weighed samples were dried in an electric oven at 110°C until reaching a constant weight.
3.5.4. Solubility percentage
Alcohol
5 gm of powdered material along with 100 ml of alcohol are shaken well occasionally for the first 6 hours and kept undisturbed for 18 hours. The liquefied extract thus obtained was concentrated in an vacuum oven and the percentage was calculated with the weight of the drug powder taken.
Water
The procedure adopted for solubility percentage of alcohol is used with chlorofonn water instead of alcohol to get the water solubility percentage.
3.6. Powder Analysis
Fluorescent analysis was carried out by using the method of Chase and Pratt, 1949. Behavior of powdered plant materials with different chemical reagents was carried out as mentioned by Kay (1938) and Johansen (1940).
3.7. Qualitative phytochemical studies
Qualitative phytochemical analyses were done using the procedures of Kokate (1994). Alkaloids, carbohydrates, tannins and phenols, fixed oils and fats, saponins and gums and mucilage’s were qualitatively analyzed. Shinoda's test was followed to analyze flavonoids.
3.7.1. Alkaloids
The extracts were dissolved in diluted sulphuric acid and filtered. The filtrate was treated with Mayer's, Dragendorfrs, Hager's and Wagner's reagents separately. Appearance of cream, orange brown, yellow and reddish brown precipitates in response to the above reagents respectively indicate the presence of alkaloids.
3.7.2. Carbohydrates
300 gm of aqueous and alcoholic extracts were dissolved in water and filtered. The filtrate was treated with concentrated sulphuric acid and then with Molisch's reagent. Appearance of pink to violet color indicates the presence of carbohydrates.
The filtrate was boiled with Fehling's and with Benedict's solutions. Fonnation of brick red precipitate in Fehling's and Benedict's solutions is the positive result for reducing sugars and non-reducing sugars respectively.
3.7. 3. Tannins and phenols
Small quantity of alcoholic and aqueous extracts were dissolved in water and to that ferric chloride solution (5%) or gelatin solution (1%) or lead acetate solution (10%) was added. Appearance of blue color with ferric chloride or precipitation with other reagents indicates the presence of tannins and phenols.
3.7.4. Flavonoids
The extract with few ml of alcohol was heated with magnesium and then concentrated hydrochloric acid was added under cooling. Appearance of pink color indicates the presence of flavonoids.
3.7.5. Gums and Mucilage’s
About 10 ml of extract was slowly added to 25 ml of absolute alcohol with constant stirring. Precipitation indicates the presence of gums and mucilage’s.
3.7.6. Fixed oils and fats
A drop of concentrated extract was pressed in-between two filter papers and kept undisturbed. Oil stains on the paper indicate the presence of oils and fats.
3.7.7. Saponins
About one ml of the alcoholic and aqueous extracts were dissolved separately in 20 ml of water and shacked in a graduated cylinder for 15 minutes. Formation of one cm layer offoam indicates the presence of saponins.
3.8. Quantitative phytochemical tests
3.8.1. Estimation of Ascorbic acid or Vitamin 'C'
Ascorbic acid or vitamin 'C' is estimated following the procedure in AOAC (Anonymous, 1980).
Reagents
Oxalic acid: 4 % concentration in water.
Sulphuric acid: 0.5 N solutions.
Thio urea: 10% concentration in water.
DNPH: 2 % concentration was prepared by dissolving 2 gm of dinitrophenyl hydroxine in 100 ml of 0.5 N sulphuric acid and filtered.
Sulphuric acid: 80 % concentration in water.
Bromine water: Few drops of liquid bromine was dissolved in water under cooling.
Standard solution: 100 mg of ascorbic acid was dissolved in 100 ml of 4% oxalic acid in a standard flask. Working standard was prepared by dissolving 10 ml of standard solution with 90 ml of 4% oxalic acid. The concentration was 100 mg/ml. It was converted to dehydro- form by adding bromine water. When it turns orange in color, it was blown with air and the excess of bromine was removed.
Standard Curve
Different aliquots (0.2 to 2 ml) of dehydro- form of working standard was taken in test tubes and their volume was made to 3 ml with water. To each tube was added 1 ml of DNPH and 1 or 2 drops of thio urea. The tubes were incubated at 37°C for 3 hours. After incubation the orange-red oxazone crystals formed was dissolved by adding 7 ml of 80% sulphuric acid. The absorbance was measured at 540 urn and a standard grape was plotted.
Sample preparation
0.5 gm of sample was ground well in a pestle and mortar with oxalic acid and filtered. The filtrate was made-up to known volume with oxalic acid. Known volume of (10 ml) the above was changed to dehydro form using the procedure adopted for working standard.
Estimation in sample
Dehydro-form of sample was taken in aliquots and preceded as for plotting the standard curve. The absorbance was compared with the standard grape and the percentage of ascorbic acid was calculated.
3.8.2. Estimation of tannins
Estimation of tannins was carried out by using Folin-Denis reagent (Anonymous,
1980).
Reagents
Folin-Denis reagent: To 750 ml of water 100 gm of sodium tungstate, 20 gm of phosphomolybdic acid and 50 ml of 85% phosphoric acid were added. The whole mixture was refluxed for 2 hours. It was cooled and diluted to 1000 mI.
Saturated sodium carbonate solution: 35 gm of anhydrous sodium carbonate was dissolved in 100 ml of water at 70-80°C and cooled for overnight. Clear liquid was decanted and used.
Standard solution: 100 mg of tannic acid was dissolved in 1000 ml of water. Fresh solutions were prepared for each test.
Preparation of sample
5 gm of sample was boiled with 400 ml of water for 30 minutes. The extract was cooled and transferred to 500 ml flask and made-up the volume.
Preparation of standard curve
10 ml of standard solution was made up to 100 ml with distilled water. 1-10 ml aliquots were taken in clear test tubes. 0.5 ml of Folin-Denis reagent and one ml of sodium carbonate solution were added to each tube. Each tube was leveled to 10 ml with distilled water. All the reagents in each tube were mixed well, kept undisturbed for about 30 minutes and read at 760 nm against reagent blank.
Estimation in sample
An aliquot of the sample extract containing not more than 0.1 mg of tannic acid was used and the percentage of tannin was determined.
Calculation
| Tannins as tannic acid % | = | mg of tannic acid X dilution X 100 ____________________________________________________ | ||
| ml of sample taken for color development | X Weight of sample taken | X 1000 | ||
3.8.3. Estimation of phenol
Phenol was estimated using the method of Bray and Thorpe, 1954.
Reagents
Folin-Ciocalteau reagent: Standard Qualigens reagent was bought from scientific supplier.
Sodium carbonate solution: 20% solution in water.
Standard solution: 100 mg of catechol in 100 mI of water. It was diluted to 10 times for working standard.
Preparation of standard curve
Different aliquots of working standard were taken in test tubes and their volume was made up to 3 mI with water. 0.5 mI of Folin-Ciocalteau reagent was added to each tube and kept for 3 minutes. To each tube, 2 ml of 20% sodium carbonate solution was added and mixed thoroughly. The test tubes were kept in boiling water exactly for one minute and cooled down. The absorbance was measured at 650 nm against blank and a standard grape was plotted.
Preparation of sample
One gm of sample was ground in a pestle and mortar with 80% alcohol. Alcoholic extract was centrifuged at 10000 RPM for 20 minutes. The supernatant was saved separately and the residue was re-extracted with 80% alcohol and centrifuged for more than five times. The supernatants collected was evaporated to dryness and dissolved in known volume of water.
Estimation in sample
Aliquots of sample were taken in test tubes diluted with water and absorbance read as above and using the standard grape concentration of phenol (as mg/100 gm sample) was calculated by using the standard graph.
3.8.4. Estimation of Sucrose As Invert Sugar
Estimation of sucrose as invert sugar was carried out by using modified Fehling's solution (Soxhlet) (Anonymous, 1980).
Reagents
1) Soxhlet modification of Fehling's solution was prepared freshly by mixing equal quantity of copper sulphate solution and alkaline tartrate solution (soxhlet reagent).
Copper sulphate solution: 34.639 gm of copper sulphate was dissolved in water and diluted to 500 ml.
Alkaline tartrate solution: 17 3 gm of potassium sodium tartrate and 50 gm of sodium hydroxide were dissolved in water and diluted to 500 mi. Let it stand for two days and the clear solution was decanted and used.
2) Standard solution: 9.5 gm of pure sucrose with 5 mI of HCI was dissolved in water and made up to 100 mi. It was stored for three days at 20-25OC and then diluted to 1000 ml. Necessary quantity can be neutralised with I
Standardization of standard solution
10 or 25 ml of mixed Soxhlet solution was pipette out in a conical flask. To this was added neutralized and diluted standard solution as to reduce the copper content of reagent. It was boiled on a wire-gauge at moderate boiling for 2 minutes. One ml of (0.02%) methylene blue was added and ended the titration by adding few drops of sugar solution to get faintest blue precipitate.
Preparation of sample and estimation
The powder was boiled in alcohol, filtered and made up to 100 ml. The volume (ml) of Soxhlet reagent required to reduce all the copper content as per the procedure for standardization was calculated.
Calculations
| mg of sugar in 100 ml of sample | = | Total reducing sugar required X 100 _______________________________ |
| Titer value |
3.8.5. Estimation of Total Saponins
Saponins were quantitatively estimated by the procedure followed by Bamuji et al.
(1985) with some modifications.
Estimation
Known quantity of the sample powder was extracted with alcohol in a Soxhlet extractor. The extract was concentrated in vacuum and re-extracted with petroleum ether, ether and ethyl acetate sequentially. The defatted residue was dissolved in least amount of ethanol and poured in drops in excess acetone with constant stirring. The precipitate was filtered and re-extracted for many times to get the purified saponins in acetone. The final precipitate was filtered in a pre-weighed filter paper and the percentage was calculated with the weight of the sample taken.
3.9 TLC studies
Alcoholic extracts were spotted on Silica Gel' G' coated plates. N-butanol, glacial acetic acid and water in the ratio of 4: 1:2 are used as mobile phase for identification of anthocyanins (Wagner et al., 1984).
3.10. Standardization of oil from seed kernels
Best solvent for extracting more percentage of oil from kernels was determined, by extracting the coarsely powdered seed kernels in a Soxhlet extractor with different solvents like hexane, petroleum ether, and chloroform.
Refractive index was found out with an Abbe's type refractometer and specific gravity was calculated with a standard pycnometer. Saponificaion value and iodine value are calculated by the standard procedures (Anonymous, 1980).
3.10.1. Saponificaion value
One gm of oil was dissolved in 10 ml of ether/ethanol (2: 1 v/v) mixture. To this, 25 ml of 0.5 N alcoholic potassium iodide was added and boiled in water bath for 30 minutes, with an air condenser, which was used to avoid loss by evaporation. After boiling, it was cooled and titrated against 0.5 N HCl, using phenolphthalein indicator. Simultaneously another flask was processed with out oil, as blank.
| Saponificaion number | = | 28.05 (B-S) _______________ g |
Where,
B= Blank titer value; S= Sample titer value; g= weight of sample in gram;
3.10.2. Iodine value
Hanus iodine solution: 13.2 gm of pure iodine was dissolved in 1000 ml of glacial acetic acid and to that 3 ml of bromine water was added.
Determination
One gm of oil was dissolved in .10 ml of chloroform. To that, 25 ml of Hanus iodine solution was added and kept in dark for 30 minutes with occasional shaking. After 30 minutes, 10 ml of 15% potassium iodide and 100 ml of freshly boiled and cooled water was added and mixed well. It was titrated against 0.1 N sodium thio sulphate, until the solution turns colorless. A drop of starch solution was added and titrated again, until it turns colorless. Simultaneously, an empty flask without oil is also processed, as blank.
| Iodine value | = | (B-S) X N X 12.69 ________________ g |
Where,
B = Blank titer value;
S = Sample titer value;
N = Normality of Na2S203;
g = Weight of the sample in gram
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