Determination of Stomatal Index

The Plant material of Viscum capitellatum Smith. parasitism on Dendrophthoe falcata which is itself parasitic on M. indica was collected from Amba Ghat, Kolhapur, Western Ghat region of Maharashtra from India in November 2009. The collection are lies [Latitude 16o 58′ 0.59″N and Longitude 73° 48′ 36.61″E at altitude 1100m]. The plant specimen (Voucher no. 550) was authenticated by Dr. Vinay Raole, Reader, Department of Botany, M.S. University, Baroda, India.
Pharmacognostical Study
Macroscopical Study[68]
It includes the shape, size, colour, texture, surface and odour of the drug in crude or powered form and often sufficient to enable to identify the whole drugs.
Microscopical Study
Histochemistry
It gives the idea about the colour reaction of specific chemical reagent towards plant tissues [68]. Microscopical images are given in Figure no. 2.
Quantitative Microscopy [66-69]
Transverse sections of scale and stems were obtained by means of a microtome and stained with different staining reagents as per standard procedures [66, 70-71]. All observations were performed using Motic Digital Photomicroscope.
Histological study of leaves and stem were performed by reported method [69]. Leaves were boiled in a 5% aqueous solution of NaOH for 5 min while stems were boiled with 10% aqueous solution of NaOH for 10 min. After cooling and washing with water, pieces were treated with a 25% aqueous solution of chromic acid for 30 min at room temperature. Washed pieces of both leaf and stem were pressed in between two slides and slides coves.
Determination of Stomatal Number
The average number of stomata per square millimeter of epidermis is termed the stomatal number.
Determination of Stomatal Index
The percentage proportional to the ultimate divisions of the epidermis of a leaf, which has been converted into stomata, is termed the stomatal index.
SI = S Ã- 100
E + S
Where SI = Stomatal index, S = number of stomata per unit area and E = number of ordinary epidermal cells in the same unit area.
Procedure [68]
Pieces of leaf between margin or midrib was cleared and mounted, and the lower surface examined by means of a microscope with a 4mm objective and an eyepiece containing a 5mm square micrometer disc. Counts were made of the numbers of the epidermal cells and of stomata within a square grid, a cell being counted if at least half of its area lies within the grid. The stomata index was determined for both leaf surfaces. Results pertaining to quantitative microscopical study are given in table no. 8.

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Analytical Study
Ash Value
1.1 Total ash
Total ash gives the idea about the residue obtained after ignition. It consist of physiological ash obtain by ignition of plant tissues and non physiological ash obtain by ignition of extraneous matter adhering to the surface of Plant. 2 gm of accurately weighed air dried powdered drug was taken in silica crucible. This silica crucible with drug material was kept in muffle furnace and ignited at temperature 4500C. The material was heated till the white coloured ash and constant weight is obtained. The procedure was performed in triplicate. Result is given in table No. 9.
The total ash was calculated by subtracting the weight of crucible with ash of drug after ignition from weight of crucible with drug powder before ignition. Percentage of total ash was calculated with reference to air-dried drug.
Acid insoluble ash
Acid insoluble ash gives the idea about the presence of inorganic material such as calcium oxalate present in plant material.
The ash obtained in the total ash method was boiled with 25 ml of 2N hydrochloric acid for 5 min. Insoluble matter was collected on ash less filter paper (Whatman paper) and washed with hot water. The material retained on filter paper and along with filter paper, was further ignited and weighed. Percentage of acid insoluble ash was calculated with reference to air dried material. Result is given in table No. 9.
Water soluble ash
The ash obtained from total ash was boiled with 25 ml water for 5 min. All insoluble matter was collected on ash less filter paper, washed with hot water and ignited for 15 min at the temperature not exceeding 4500C.
The percentage of water soluble ash was calculated by subtracting weight of insoluble matter from weight of total ash. The difference between weights represents water soluble ash. Percentage of water soluble ash was calculated with reference to air dried drug. Result is given in table No. 9.
Extractive Value
Extraction by cold maceration
It is the process of extraction of crude drugs with solvents with several daily shakings or stirring at room temperature.1 kg of powdered plant was extracted with 5 lit of methanol by cold maceration method. The extract was concentrated on rotary vacuum evaporator (Roteva Equitron, Mumbai) and further dried in vacuum dryer [73].
Successive extraction by using Soxhlet apparatus
Weighed accurately 200gm of dried, powered crude drug and kept in a filter paper cover which was already placed in thimble. Then the solvent was slowly poured onto it. The solvent from thimble goes to lower round bottom flask via siphon tube due to the siphoning or syphon cycle. Such 2-3 cycles of solvent were performed and then drug powder was kept for 12 hours with solvent for imbibitions. After 12 hours imbibitions, solvent from flask heated to form vapors. Due to heat the solvent from RBF gets converted into its vapors, and then these vapors pass via side tube into the condenser where it gets condensed. This solvent dripped again on to drug material, which was placed in thimble. This process was continued till thimble gets filled with solvent and when level of solvent reaches to syphon tube, pulling of whole solvent into the flask is taken place. All this events repeated several times and drug material gets extracted continuously with fresh solvent. This process was performed for 3 days and when syphon solution showed negative test for phytoconstituents, extraction was completed. Then the heating was stopped and the mixture was collected and cooled. Then this mixture was filtered and concentrated by using rotary flash vacuum evaporator. The extract was dried in vacuum dryer and was stored in freeze. Then this marc obtained after pet ether extraction and subjected again to extraction by following solvents (Table 10) [73].
Moisture content by Loss on Drying
2 g of air powdered drug was placed in a silica crucible. Before that, crucible was cleaned and dried and weight of empty crucible was taken. The powder was spread in a thin uniform layer. The crucible was then placed in the oven at 1050C. The powder was dried for 4 h and cooled in a desiccator to room temperature and weight of the cooled crucible plus powder was noted. Result is given in table no. 9.
Analysis of inorganic constituents (Elemental analysis)
Ash of drug material was prepared and adds 50% v/v HCl or 50% v/v HNO3 to ash. Keep it for 1 hour. Filtered and with the filtrate performed the test as per method reported [74]. The results of analysis of inorganic constituents are given in (Table 11).
 
Test for calcium
a) Add dil. NH4OH and saturated ammonium oxalate solution to filtrate.
White ppt of calcium oxalate forms which is soluble in HCl.
Calcium present.
b) Add ammonium carbonate to filtrate.
White ppt which is insoluble in NH4Cl.
Calcium present.
Tests for iron
a) Add 2% potassium ferricyanide to filtrate.
Dark blue coloration.
Iron present.
b) To filtrate, add 5% ammonium thiocyanate.
Blood red color.
Iron present.
c) To filtrate, add dil. HCl and sol. of KMnO4.
Pink color.
Iron present.
Tests for magnesium
a) To filtrate add NaOH.
White ppt.
Magnesium present.
b) To filtrate add (NH4)2CO3.
White ppt, redissolve in NH4Cl.
Magnesium present.
Tests for potassium
a) Add sodium cobalt nitrite to filtrate.
Yellow ppt.
Potassium present.
b) Flame test.
Violet color to flame.
Potassium present.
Tests for sodium
a) Add uranyl zinc acetate to filtrate, shake well.
Yellow crystalline ppt.
Sodium present.
Tests for carbonate
a) Add HgCl2 to filtrate.
Brownish red ppt.
Carbonate present.
b) Add dil. Acid to the filtrate.
Effervescence of CO2
Carbonate present.
c) Add MgSO4 to filtrate.
White ppt.
Carbonate present.
Tests for Sulphate
a) Add BaCl2 to filtrate.
White crystalline ppt
Sulphate present.
b) Add filtrate to lead acetate sol.
White ppt.
Sulphate present.
Tests for phosphate
a) Add HNO3 and ammonium molybdate to filtrate, heat 10 min. cool.
b) Add silver ammonium- nitrate to filtrate
Yellow crystalline ppt.
Light yellow ppt
Phosphate present.
Phosphate present.
Tests for chloride
a) Add AgNO3 to filtrate.
b) To filtrate, add manganese dioxide and H2SO4
White curd ppt, soluble in dil. NH3.
Odour of chlorine
Chloride present.
Chloride present.
Tests for nitrate
a) Add water to filtrate, add H2SO4 from side of test tube.
b) Add H2SO4 and copper to filtrate, warm
Brown color at junction of two liquid
Liberation of red fumes
Nitrate present.
Nitrate present.
Determination of Type of Starch Grains
The shape of starch grains present was determined according to the reported method [68]. Size of starch grains were measured with the help of calibrated Photomicroscope using Motic software. Starch grains were identified by staining with Iodine solution. The Motic digital Photomicroscope was calibrated with images obtained with various magnifications (10x, 40x and 100x) by using standard slide in 1.3 software. The images obtained in triplicate and average figures calculated from 20 readings in each parameter (Table no. 12).
Crude Fiber Content
Pre-weighed dried powder material was extracted with Petroleum ether (b.p. 40- 600C) using soxhlet apparatus for 8 h. The marc obtained after extraction was utilized for determination of Crude Fiber Content.
Crude fiber was investigated by acid-base digestion with H2SO4 (1.25%) and of NaOH (1.25%) solution. The marc after extraction was taken into a 500ml beaker and 200ml of boiling H2SO4 added. The content was boiled for 30 minutes, cooled, filtered and the residue washed three times with 50ml of boiling water. The washed residue was further boiled in 200ml of NaOH for 30 minutes. The digest was filtered to obtain residue. This was washed three times with 50ml of boiling water and lastly with 25ml of ethanol.
The washed residue was dried in an oven at 1250C to constant weight and cooled in dessicator. The residue was scraped into a pre-weighed porcelain crucible, weighed, ashed at 5500C for 2 hours, cooled in a dessicator and weighed. Crude fiber content was expressed as percentage loss in weight on ignition. Result is given in table No. 13.
Phyto-chemical Analysis
Extracts
Petroleum ether, benzene, chloroform, acetone and methanol extract obtained by successive extraction method and aqueous extract by maceration method [68, 95].
Qualitative analysis
All the extracts were subjected to proximate chemical analysis and its result is given in table no. 14.
 
Tests for Acidic compounds:
a) To the test solution add sodium bi-carbonate
b) Test solution treated with warm water and filter. Test the filtrate with litmus paper.
Tests for Alkaloids:
a) Dragendorff’s Test: Test solution treated with Dragendorff’s reagent (potassium bismuth iodide)
b) Mayer’s Test: Test solution treated with Mayer’s reagent (Potassium mercuric iodide).
c) Wagner’s Test: Test solution treated with Wagner’s reagent (Iodine in potassium iodide).
d) Hager’s Test: To the test solution add gives with Hager’s reagent (Saturated picric acid solution).
e) Tannic acid test: Test solution treated with Tannic acid solution.
f) Picrolonic acid test: Test solution treated with Picrolonic acid.
Test for amino acids:
a) Million’s Test: Test solution treated with Million’s reagent and heated on a water bath.
b) Ninhydrin Test: Test solution boiled with Ninhydrin reagent.
Test for Carbohydrates:
a) Molisch’s Test: To the test solution add with few drops of Molisch’s reagent (Alcoholic-naphthol) and 2ml of conc. sulphuric acid is added slowly from the sides of the test tube.
b) Barford’s Test: Test solution heated with Barford’s reagent on water bath.
c) Selivanoffs test (Test for Ketones): To the test solution add crystals of resorcinol and equal volumes of concentrated hydrochloric acid and heat on a water bath.
d) Test for pentose: To the test solution add equal volumes of hydrochloric acid containing small amount of Phloroglucinol and heat.
e) Osazone formation test: Heat the test solution with the solution of phenyl hydrazine hydrochloride, sodium acetate, and acetic acid.
Test for Flavonoids:
a) Shinoda Test: Test solution treated with fragments of magnesium ribbon and conc. Hydrochloric acid.
b) Alkaline Reagent Test: Test solution treated with sodium hydroxide solution
c) Zinc-Hydrochloride test: Treat test solution with zinc dust and few drops of HCL
Test for glycosides:
General test: Extract 200 mg of drug with 5 ml of dilute sulphuric acid by warming on a water bath, filter it, and neutralize the acid extract with 5 % solution of sodium hydroxide. Add 0.1 ml of Fehling’s solution A and B until it becomes alkaline (test with pH paper) and heat on water bath for 2 minutes.
Test B: Repeat Test A procedure by using 5 ml of water instead of dilute sulphuric acid. Note the quantity of red precipitate formed.
Chemical tests for specific glycosides:
Tests for Anthraquinone glycosides:
a) Borntrager’s test: Boil the test material with 1ml of sulphuric acid for 5minutes. Filter while hot. Cool the filtrate; shake with equal volume of dichloromethane or chloroform. Separate the lower layer of dichloromethane or chloroform; shake it with half of its volume of dilute ammonia.
b) Modified Borntrager’s test: Boil 200 mg of test material with 2ml of sulphuric acid. Treat with 2 ml of 5 % aqueous ferric chloride solution (freshly prepared) for 5 minutes, shake it with equal volume of chloroform and continue the test as above.
c) Test for hydroxy anthraquinones: treat the sample with potassium hydroxide solution.
Tests for cardiac glycosides:
a) Kedde’s test: Extract the drug with chloroform, evaporate to dryness. Add one drop of 90 % alcohol and 2 drops of 2 % sodium hydroxide solution.
b) Keller-Killiani Test: (Test for deoxy sugars) Extract the drug with chloroform and evaporate it to dryness. Add 0.4 ml of glacial acetic acid containing ferric chloride, add carefully 0.5 ml of conc. sulphuric acid by the side of test tube.
c) Raymond’s number: treat the test solution with hot methanolic alkali.
d) Baljet’s Test: The test solution treated with sodium picrate or picric acid.
e) Legal’s Test: Test solution treated with pyridine [made alkaline by adding sodium nitroprusside solution].
f) Tests for coumarins glycosides: Place small amount of sample in test tube and covered it with a filter paper, moistened with dilute sodium hydroxide solution. Placed the covered test tube on water bath for several minutes. Remove the paper and expose it to ultraviolet (UV) light.
Cynogentic glycosides:
Place 200 mg of drug in conical flask and moisten with few drops of water.( Flask should be completely dry because hydrogen cyanide produced will dissolve in the water rather than come off as gas to react with paper) moisten a piece of picric acid paper with 5% aqueous sodium carbonate solution and suspended in neck of flask. Warm gently at about 37oC. Observe the change in color.
Saponin glycosides:
Froth test: Place 2 ml solution of drug in water in a test tube, shake well.
Tests for steroids and triterpenoids:
a) Liebermann Burchard Test: Treat the extract with few drops of acetic anhydride, boil and cool, add conc. sulphuric acid from the sides of test tube.
b) Salkowski test: Treat the extract with few drops of conc. sulphuric acid.
c) Sulfur powder test: Add small amount of sulfur powder to the test solution.
d) Tests for inulin: To the test solution add the solution of -naphthol and sulphuric acid.
e) Tests for Lignin: Treat the sample with hydrochloric acid and Phloroglucinol.
Tests for Mucilage:
Treat the sample with thionine solution. After 15 min wash with alcohol
Tests for tannins:
a) Ferric-Chloride Test: Treat test solution with few drops of ferric chloride solution.
b) Gelatin test: To the test solution add 1 % gelatin solution containing 10 % sodium chloride.
Tests for proteins:
a) Heat test: Heat the test solution in boiling water bath.
b) Biuret Test: Test solution treated with Biuret reagent (40% sodium hydroxide and dilute copper sulfate solution).
c) Xanthoproteic test: To the test solution, add 1 ml of conc. nitric acid and boil yellow precipitate is formed. After cooling it, add 40 % sodium hydroxide solution.
d) Test for starch: To the test solution, add weak aqueous iodine solution. Blue color indicates presence of starch, which disappears on heating and reappears on cooling.
Effervescence produces
Litmus paper turns blue
Gives reddish brown colored precipitate
Gives cream colored precipitate
Gives reddish brown colored precipitate
Gives yellow colored precipitate
Gives buff colored precipitate
Gives yellow colored precipitate
White colored precipitate
Gives violet color
Purple to violet ring appears at the junction of two liquids
If red cupric oxide is formed
Rose color is produced
Red color produced.
Yellow crystals formed.
Observe under microscope.
Shows pink scarlet, crimson red or occasionally green to blue color after few minutes.
Shows increase in the intensity of yellow color on addition of few drops of dilute acid.
Shows red color after few minutes.
Red Precipitate formed
compared with precipitate of test A
A rose pink to red color is produced in ammonical layer.
A rose pink to red color is produced in ammonical layer.
Red color produced
Purple color is produced.
Acetic acid layer shows blue colour.
Violet colour produced
Gives yellow to orange color
Gives blood red color
Paper shows green fluorescence.
Reddish purple color
Stable froth (foam) formed
Brown ring is formed at the junction of two layers,
If upper layer turns green
If upper layer turns deep red
Red color at lower layer
Yellow color at lower layer
It sinks at the bottom
Brownish red color formed
Pink color formed
Mucilage turns violet red.
Gives dark blue color
Green color appears
Precipitate formed
Proteins gets coagulated
Gives violet color
Orange color formed
Blue color, which disappears on heating and reappears on cooling
Acidic compounds present
Acidic compounds present
Alkaloids present
Alkaloids present
Alkaloids present
Alkaloids present
Alkaloids present
Alkaloids present
Amino acids present
Amino acids present
Carbohydrates present
Monosaccharides are present.
Carbohydrates present
Carbohydrates present
Carbohydrates present
Flavonoids present
Flavonoids present
Flavonoids present
If the precipitate in Test A is greater than in Test B then glycoside may be present.
Anthraquinone glycosides present
Anthraquinone glycosides present
Hydroxy anthraquinones present
Cardiac glycosides present
Cardiac glycosides present
Cardiac glycosides present
Cardiac glycosides present
Cardiac glycosides present
Coumarins glycosides present
Cynogentic glycosides present
Saponin glycosides
Present
Steroids present
Triterpenoids present
Steroids present
Triterpenoids present
Steroids present
Inulin Present
Lignin Present
Mucilage present
Hydrolysable tannins
Condensed tannins
Tannins present
Proteins present
Proteins present
Proteins present
Starch present
Floroscence Analysis of various extracts
Petroleum ether, Benzene, Chloroform, Acetone, Methanol and Aqueous extracts were screened for fluorescence characteristic. The observation pertaining to their colour in day light and under ultra-violet light were noticed and represented in table. Many substances for example quinine in solution in dilute sulphuric acid when suitably illuminated emit light of a different wavelength or colour from that which falls on them. This emitted light (fluorescence) ceases when the exciting light is removed [68].Results given in Table No. 15.
HPLC Analysis of sample drug
The chromatographic pattern of plant was obtained as per report with some modifications for which the HPLC conditions are as follows.
Extract: The methanol extract diluted with HPLC grade methanol and filtered through whatman filter paper and used for analysis
Instrument: Shimadzu LC-20AT with UV/visible detector
Stationary Phase: Bonda- pack C-18 column with 250Ã-4mm
Mobile Phase: Methanol (80): Water (20)
Detection wave length: 350 nm
Flow Rate: 2 ml/min.
HPLC Chromatogram is given in Fig. 3 and its retention time is given in Table no. 16
HPTLC Analysis of sample drug
The chromatographic pattern of plant was obtained as per report with some modifications for which the HPTLC conditions are as follows.
Extract: Methanolic Extract
Instrument: HPTLC (Camag, Switzerland)
Stationary Phase: pre-coated silica gel plates
Mobile Phase: Ethyl acetate: Formic acid: Glacial acetic acid: water (100:05:10:20)
Spraying Reagent: Natural Product Reagent (NP reagent)
Detection: 365 nm.
HPTLC Chromatogram is given in Fig. 4 and its retention time is given in Table no. 17.
Isolation and characterization of chemical principle
Compound I
The methanol extract was dissolved in water and partitioned with ethyl acetate and n- butanol. The ethyl acetate fraction was subjected to column chromatography for isolation of compounds.
Column chromatography: The separation of extract constituents was done by column chromatography. The clean and dried glass column was used. The silica gel for column chromatography (#60-120) was activated at 1100c.The column was filled with silica gel and mobile phase without formation of any air bubbles. The silica gel was then allowed to stabilize in the column. Mixture of two or three compounds was isolated from the ethyl acetate fraction of methanol extract of the plant with following experimental conditions [73].
Height of column: 20 cm
Diameter of column: 3.5 cm.
Stationary phase: Silica gel (#60-120).
Mobile phase: Benzene†’ Chloroform †’ Ethyl acetate†’ Methanol with variant Proportions
Elution: Gradient elution.
Fraction quantity: 25 ml
Preparative TLC:
20 X 20 glass plates were coated with the thick layer of silica gel or any other adsorbent material. The plates were then activated at 1100c.The sample-containing mixture of two or more compounds were applied in the form of thin band on the plate. The plate was then developed. The different bands separated on the plate were scratched and recovered with methanol. Purity of dried sample was checked by TLC method. One single compound was isolated with the help of preparative chromatography from fractions 54- 58. The compound is given for spectral analysis. FTIR spectra, Mass spectra and 1HNMR are given in fig. no. 5, 6 and 7 respectively. The spectral data of FTIR and 1HNMR are given in Table no. 18 and 19 respectively. The assumed structure of the compound (Quercetin) is given in Fig. No. 8.
Compound II
Petroleum ether extract obtained is processed for separation of the unsaponifiable and saponifiable matter. Extract is allowed to saponify using alcoholic KOH with reflux and then it is extracted with solvent ether for separation of unsaponifiable matter. The aqueous phase is acidified with concentrated H2SO4 and then again extracted with the solvent ether for separation of the saponifiable matter [73].
Fractionation of unsaponifiable matter
Experimental:
Height of column: 25 cm
Diameter of column: 3.5 cm.
Stationary phase: Silica gel for column chromatography (#60-120).
Mobile phase: Benzene†’ Ethyl acetate
Elution: Gradient elution.
Fraction quantity: 30 ml
Fractions No. 24-27 were subjected for thin layer chromatography with following experimental conditions.
Stationary phase: Silica gel H
Mobile phase: Ethyl acetate: Benzene (1: 9)
Detection: Vanilin-sulphuric acid reagent
Identification: Whitish Purple colour
Fraction was concentrated and single band was applied. After plate development; developed band was scraped (Rf. 0.62). After separation of single compound from the silica, it is dried. This sample was further given for spectroscopic analysis. FTIR spectra, Mass spectra and 1HNMR are given in fig. no. 9, 10 and 11 respectively. The spectral data of FTIR and 1HNMR are given in Table no. 20 and 21 respectively. The assumed structure of the compound (Quercetin) is given in Fig. No. 12.
Biochemical Estimations
a) Estimation of Total carbohydrate content
The estimation of carbohydrate was done using the method acid base digestion.
Principle:
In hot acidic media glucose is converted to hydroxy methyl furfural by dehydration. This forms a green colour product with phenol.
Procedure:
100mg of the aqueous extract was taken and it was hydrolyzed by keeping it on water bath for 3 hours with 5 ml of HCl (2.5N) and cooled at room temperature. Neutralized it with sodium carbonate and volume was made up to 100 ml and from this centrifuge 10 ml of the solution. Then 0.2, 0.4, 0.6, 0.8 and 1ml of working standard was pipetted out into a series of test tube and in separate test tubes 0.1 and 0.2 ml of sample solution was pipetted out and the volume was make up to 1ml with water. The blank was prepared with 1 ml distilled water. Then 1ml phenol solution and 5ml of sulphuric acid (96%) was added to each test tube and shaken well. After 10 min the test tube was placed in water bath at 25-30ï‚°C for 20 min. The absorbance was read at 490 nm. And the amount of total carbohydrate present was calculated in the sample using standard graph. Result pertaining to Total carbohydrate content is given in Table no. 22 and Calibration curve of standard glucose dilutions are given in Fig. No. 13.
Estimation of Bitterness value
The bitterness value of plant material was compared with diluted solution of Quinine hydrochloride.
Preparation of Solutions
Preparation of Quinine hydrochloride solution
The stock solution of 100µg/ml was prepared from which a series of dilutions 42, 44, 46, 48, 50, 52, 54, 56 and 58 µg/ml were prepared.
Preparation of Sample Preparation
Form the stack solution of 1000 µg/ml, 100, 200, 300 and 400µg/ml dilutions were prepared.
Method
Tasted all the dilutions of sample and Quinine sulphate by taking the solution in mouth and swirled it for 30 secs in mouth mainly near to the tongue. After tasting each dilution the mouth wash rinsed thoroughly with drinking water and taken the interval of 10 mins. Until the bitter sensation of previous dilution was no more remain. Then compared the dilution of sample which produced the same bitterness equivalent to the dilution of Quinine sulphate. Then bitterness value was calculated according to following formula.
Bitterness value in units per gram = 2000 Ã- A
B Ã- C
Where A= quantity of Quinine sulphate (mg) having higher bitterness
B= the concentration of stock solution (mg/ml)
C= Volume of sample in ml having higher bitterness
Result pertaining to estimation of bitterness value is given in Table no. 22
Total Phenolic content
The total phenolic content of methanol extract of V. capitellatum Smith. (VCM) was estimated using Folin-Ciocalteu reagent. In this method, the blue colour formed due to the polyphenol was measured at 760 nm using UV spectrophotometer.
Chemicals
Folin- Ciocalteu reagent (Merck Co.)
Gallic acid (Sigma Ltd., USA)
Sodium carbonate (SISCO Research Laboratory Pvt. Ltd., Mumbai, India)
Reagent preparation
Folin-Ciocalteu (phenol) reagent
The reagent was prepared by diluting 1ml with 5ml of distilled water.
Sodium carbonate
15% solution was prepared in distilled water.
Gallic acid solution
The stock solution was prepared by dissolving 1mg gallic acid in 10ml of water from which different concentrations (20-100µg/ml) were prepared.
Sample preparation
Sample solution was prepared by dissolving 10 mg of the extract in 100 ml of methanol to give (100 µg/ml) solution.
Procedure
0.1ml of extract was mixed with the 0.2ml of Folin-Ciocalteu reagent, 2 ml water and 1 ml of sodium carbonate solution, and absorbance was measured at 760 nm after 10 min incubation at 50 0C. The total phenolic was expressed as µg gallic acid equivalent. Result pertaining to Total phenolic content is given in Table no. 22 and Calibration curve of standard gallic acid dilutions are given in Fig. No. 14.
Total Flavonoid Content
Total flavonoid content of VCM was determined using method reported [79].
 

Investigation of the Effects of Conditions on the Size and density of Stomatal Pores

The aim of this report is to investigate the effect of conditions: temperature, light, moisture, sugar and there effect on the size and density of stomatal pores. The experimental hypothesis is that the greater the light intensity the bigger size and lower density of stomatal pores and the more water there is the smaller the pores and the higher number of stomatal pores. Samples from two plants were experimented on, the Geranium plant and the Kalanchoe daigremontiana plant. From both plants 1cm by 1cm samples of the leaves were taken to measure the stomata sizes and to count the number of stomatal pores. The results showed that the condition with the greatest effect is the light conditions which showed greater stomatal size and density.
Introduction
Although sotmatal opening may seem like … it could contribute in climate change, as we know climate change is due to the rapid increase of green house gasses and in particular carbon dioxide (CO2) in our atmosphere and thus this is a serious current global concern.
Stomata
Stomata are tiny structures found on the epidermis of a plant. The tiny pore called the stoma is surrounded by guard cells which are specialised cells. The main function of the stomata is to allow gases such as carbon dioxide, water vapour and oxygen to move rapidly into and out of the leaf. Since most of the water (90%) is lost through stomata, plants regulate the degree of stomatal opening to reduce the water loss. The guard cells have unevenly thickened walls. The cell wall around stoma is tough and flexible and the one away from stoma is thinner.
Stomata are openings generally present on the lower surface of the leaves through which the gases and water vapour diffuse in and out easily. The oxygen diffuses in through the stomata and then enters the leaf cells. Similarly, the carbon dioxide produced by the leaf cells diffuses out through the stomata.
Stomata are generally open during the day so that COâ‚‚ can enter he plant for photosynthesis (which needs light). They are usually closed at night to save water, because COâ‚‚ is no longer needed (photosynthesis can’t happen in the dark).
What is photosynthesis?
Photosynthesis is the process by which plants, some bacteria, and some protistans use the energy from sunlight to produce sugar, which cellular respiration converts into ATP, the “fuel” used by all living things. The conversion of unusable sunlight energy into usable chemical energy, is associated with the actions of the green pigment chlorophyll. Most of the time, the photosynthetic process uses water and releases the oxygen.
Plants are the only photosynthetic organisms to have leaves (and not all plants have leaves). A leaf may be viewed as a solar collector crammed full of photosynthetic cells.
The raw materials of photosynthesis, water and carbon dioxide, enter the cells of the leaf, and the products of photosynthesis, sugar and oxygen, leave the leaf.
Cross section of a leaf, showing the anatomical features important to the study of photosynthesis: stoma, guard cell, mesophyll cells, and vein (Farabee, 2007)Water enters the root and is transported up to the leaves through specialized plant cells known as xylem (pronounces zigh-lem). Land plants must guard against drying out (desiccation) and so have evolved specialized structures known as stomata to allow gas to enter and leave the leaf. Carbon dioxide cannot pass through the protective waxy layer covering the leaf (cuticle), but it can enter the leaf through an opening (the stoma; plural = stomata; Greek for hole) flanked by two guard cells. Likewise, oxygen produced during photosynthesis can only pass out of the leaf through the opened stomata. Unfortunately for the plant, while these gases are moving between the inside and outside of the leaf, a great deal water is also lost. Cottonwood trees, for example, will lose 100 gallons of water per hour during hot desert days. Carbon dioxide enters single-celled and aquatic autotrophs through no specialized structures.
Stomata opening and closing
most plants do not have the aforementioned facility and must therefore open and close their stomata during the daytime in response to changing conditions, such as light intensity, humidity, and carbon dioxide concentration. It is not entirely certain how these responses work. However, the basic mechanism involves regulation of osmotic pressure.
When conditions are conducive to stomatal opening (e.g., high light intensity and high humidity), a proton pump drives protons (H+) from the guard cells. This means that the cells’ electrical potential becomes increasingly negative. The negative potential opens potassium voltage – gated channels and so an uptake of potassium ions (K+) occurs. To maintain this internal negative voltage so that entry of potassium ions does not stop, negative ions balance the influx of potassium. In some cases chloride ions enter, while in other plants the organic ion malate is produced in guard cells. This in turn increases the osmotic pressure inside the cell, drawing in water through osmosis. This increases the cell’s volume and turgor pressure. Then, because of rings of cellulose microfibrils that prevent the width of the guard cells from swelling, and thus only allow the extra turgor pressure to elongate the guard cells, whose ends are held firmly in place by surrounding epidermal cells, the two guard cells lengthen by bowing apart from one another, creating an open pore through which gas can move.

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When the roots begin to sense a water shortage in the soil, abscisic acid (ABA) is released. ABA binds to receptor proteins in the guard cells’ plasma membrane and cytosol, which first raises the pH of the cytosol of the cells and cause the concentration of free Ca2+ to increase in the cytosol due to influx from outside the cell and release of Ca2+ from internal stores such as the endoplasmic reticulum and vacuoles. This causes the chloride (Cl-) and inorganic ions to exit the cells. Secondly, this stops the uptake of any further K+ into the cells and subsequentally the loss of K+. The loss of these solutes causes a reduction in osmotic pressure, thus making the cell flaccid and so closing the stomatal pores.
Interestingly, guard cells have more chloroplasts than the other epidermal cells from which guard cells are derived. Their function is controversial. (stomata opening, 2008)
This diagram shows normal responses of stomata to light, CO2, pH, K+ ion and Water Deficiency
Transpiration
Transpiration is the term used to describe the transport of water through an actual, vegetated plant into the atmosphere. Transpiration is an important part of the evapotranspiration process, and a major mechanism of the water cycle in the atmosphere. Transpiration may also refer to the rate of the water vapor transport through the whole vegetative canopy (that is, through the group of plants).
Just as you release water vapor when you breathe, plants do, too-although the term “transpire” is more appropriate than “breath.” During this process individual water molecules are released from the surface of the plant body through tiny structures called stomata. There are many more individual water vapor molecules inside the air spaces between the tissues of a plant than in the air surrounding the plant body. Consequently water vapor will always exit the plant along a concentration gradient. As more water vapor molecules exit the plant, the remaining water molecules tug on each other and will pull an entire column of water throughout the plant body through special tissues called xylem during the process of transpiration. One way to visualize transpiration is to put a plastic bag around some plant leaves. As Figure 1 shows, transpired water will condense on the inside of the bag. If the bag had been wrapped around the soil below it, too, then even more water vapor would have been released, as water also evaporates from the soil. During a growing season, a leaf will transpire many times more water than its own weight. An acre of corn gives off about 3,000-4,000 gallons (11,400-15,100 liters) of water each day, and a large oak tree can transpire 40,000 gallons (151,000 liters) per year.
Factors affecting transpiration
The amount of water that plants transpire varies greatly geographically and over time. There are a number of factors that determine transpiration rates:
Temperature: Transpiration rates go up as the temperature goes up, especially during the growing season, when the air is warmer due to stronger sunlight and warmer air masses. Higher temperatures cause the plant cells which control the openings (stoma), where water is released to the atmosphere, to open, whereas colder temperatures cause the openings to close.
Relative humidity: As the relative humidity of the air surrounding the plant rises the transpiration rate falls. It is easier for water to evaporate into dryer air than into more saturated air.
Wind and air movement: Increased movement of the air around a plant will result in a higher transpiration rate. This is somewhat related to the relative humidity of the air, in that as water transpires from a leaf, the water saturates the air surrounding the leaf. If there is no wind, the air around the leaf may not move very much, raising the humidity of the air around the leaf. Wind will move the air around, with the result that the more saturated air close to the leaf is replaced by drier air.
Soil-moisture availability: When soil moisture is lacking, plants can begin to senesce (premature ageing, which can result in leaf loss) and transpire less water.
Type of plant: Plants transpire water at different rates. Some plants which grow in arid regions-for example, cacti and succulents-conserve precious water by transpiring less water than other plants.
(Burba & Pidwirny, 2007)
Kalanchoe Daigremontiana
Kalanchoe daigremontiana and also sometimes called Mother of Thousands, is a plant from southwest Madagascar. The plant generally would reach up to 3 feet (1m) tall and consist of leaves that reach 6-8 inches long and about 1.25 inches wide. These are medium green above and blotched with purple underneath. The most interesting feature of this plant is the fact that it has spoon-shaped bulbiliferous spurs that bear young plants on its margins. This plant is distinguished by its ability to propagate via vegetative propagation. All parts of the plant are poisonous, which can even be fatal if ingested by infants or small pets.

Blooming: In the greenhouse, the plants bloom sporadically in late winter. The compound cymes have 1 inch (2.5 cm) long purplish flowers.
Culture: Kalanchoe daigremontiana needs full sun to partial shade with a well-drained soil mix. In the greenhouse, soil is mixed consisting of 1 part peat moss to 2 parts loam and sand. The plants are watered and allowed to dry slightly before watering again. They are generally fertilised only once during the season with a balanced fertiliser. During the winter months, water is somewhat restricted, but the plants are not allowed to dry out completely. The plants can become very weedy, so these plants should not be used around other plants. Plantlets are drought resistant, root readily, and if allowed to establish, can easily create a plant epidemic wherever the plantlets land (hence their common name).
Propagation: Kalanchoe daigremontiana is easily propagated from plantlets formed on the edges of leaves or from cuttings. Cuttings must be kept very dry to root.

(Kalanchoe daigremontiana – Mother of Thousands, 2006)
Geranium
Geraniums are just one of the members of the family Geraniaceae. Pelargonium are also a member of the same family so that confusion has arisen by both of them being referred to as Geraniums
The Geranium as it is commonly known, is actually of the genus Pelargonium. Geranium is the correct botanical name of the separate genus that contains the related Cranesbills. Both genera are in the Family Geraniaceae. Which originally included all the species in one genus, Geranium, but they were later separated into two genera by Charles L’Héritier in 1789. The first species of Pelargonium known to be cultivated was Pelargonium triste, a native of South Africa. The cranesbills make up the genus Geranium of 422 species of annual, biennial, and perennial plants found throughout the temperate regions of the world and the mountains of the tropics, but mostly in the eastern part of the Mediterranean. One can make the distinction between the two by looking at the flowers : Geranium has symmetrical flowers, while Pelargonium has irregular or maculate petals. The name “cranesbill” derives from the appearance of the seed-heads, which have the same shape as the bill of a Crane. The genus name is derived from the Greek word geranos, meaning ‘crane’.
All geranium species are perennials and generally winter hardy plants, and are generaly grown for their attractive flowers. They generally have a long lifespan and most have a mounding habit, with palmately lobed foliage. Some species have spreading rhizomes. They are normally grown in part shade to full sun, in well draining but moisture retentive soils, that are rich in humus. They are generally found in Mediterranean areas. (The Origin of the Geranium, 2006)
Propagation: is by semi-ripe cuttings in summer, by seed, or by division in autumn or spring. (Geranium, 2009)
Experiment
Aim
To investigate the effect of conditions: temperature, light, moisture, sugar, can effect on the size and density of stomatal pores.
Hypothesis
The greater the light intensity the bigger size and lower density of stomatal pores; the more water there is the smaller the pores and the higher number of stomatal pores.
Apparatus

Microscope
Slides
stage micrometer
Leaves
Sugar solution
Iodine (optional)
Tweezers
Petri dish
Clear nail vanish
Beakers

Method

Select the leaves which will be used
Put them in the different conditions over night
Next day, polish all the leaves with a rarther thick layer of clear nail vanish
Allow to dry for about 2 hours
When completely dry, gently peal of the nail polish
Put it on the slide and observe with the microscope, also insert the eye piece graticule to measure the size of the stomata
(optional) if not very visible, apply iodine on the slide put the piece of nail polish on top and press down with tissue to clear of excess solution
Count the number of stomata visible and measure the size

Results
We see here that in the conditions with light and sugar the stomata appears to have the greatest size whereas in water conditions and dark condition stomatal size appear to be very small.
Here we see that stomatal size appear to be generally larger with the lowest size to be 0.02 mm which is for the condition with sugar placed in the dark. The condition with the highest stomatal size is the condition with water placed in the light with 0.08mm in size, followed by the condition with sugar placed in light with a stomatal size of 0.07.
The graph shows that the condition with sugar placed in the dark had the lowest stomatal density followed by the dark condition with 55 stomatal pores in the 1 cm by 1 cm sample. The conditions with light seem to have the highest number of stomatal pores with the highest condition, water placed in light having 120 stomatal pores followed by the condition in purely light having 98 stomatal pores. On the other hand the condition with sugar placed in light has the lowest conditions with 30 pores per 1cm square.
Generally the number of stomatal pores in the 1 cm by 1 cm sample are fairly quite low with the highest number of pores present in the condition presented with light with a number of 99 pores. The condition with the lowest number of pores was the condition in water placed in the dark with 25 followed by the condition in the water placed in the light with followed by the condition in the water placed in the light with 28 pores.
Discussion
From the results it is clear that the stomatal size is greatly dependent on the conditions in which it is located. For the geranium we can see that the stomata has the greatest size of 0.1 mm (see appendix 1) in the condition with light, here it is possible to accept the hypothesis, the fact that it has a greater stomatal density in conditions with the most light. On the other hand the Mexican hat showed lower numbers such as 0.04 mm. This may be because of the origin of this sort of plant and the fact that they are generally found in hot countries where there is generally more light could have ment that these sort of plants undergo various adaptation and therefore have adapted to have smaller stomatal sizes even thought there is a lot of light present.
We also notice that the geranium has the most stomatal numbers (120 stomatal pores in a 1cm by 1cm sample of leaf) in the water condition; this shows that stomatal numbers increase in the presence of more water. Here also the Mexican hat leaf shows the opposite, here it has a very low stomatal number, this may be an anomaly or may be purely due to the fact that the plant is found in areas with water shortages and therefore here again it may have adapted to maintain low numbers of stomata pores in water.
In the conditions with sugar we see that sugar doesn’t have much effect on the Mexican Hat since the results for the conditions with sugar placed in a area with light had results very close to the results for the conditions in water placed in light conditions. Bearing in mind the fact that the sugar solution was made up of water mixed with sugar, for the results to be similar to the results with water only we can assume that what is causing the effect is in fact the water and not so much of the sugar. On the other hand from the graphs we see that it may have some effect on the Geranium plant since the results alter for conditions in only water than the results for conditions in sugar.
The conditions in the fridge showed quite average to low results, this is because in the fridge it had lower temperatures and therefore colder temperatures cause the openings to close.
During the investigation it was almost impossible to find the actual stomatal pore size between the guard cells and you rarely get the chance to measure the diameter of the hole therefore vague assumptions had to be drawn by just having to measure the length of the closed hole and make some assumptions from there.
Another way to find out whether stomata are open or closed, or more accurately, how open they are, is by measuring leaf gas exchange. A leaf is enclosed in a sealed chamber and air is driven through the chamber. By measuring the concentrations of carbon dioxide and water vapor in the air before and after it flows through the chamber, one can calculate the rate of carbon gain (photosynthesis) and water loss (transpiration) by the leaf.
However, because water loss occurs by diffusion, the transpiration rate depends on two things: the gradient in humidity from the leaf’s internal air spaces to the outside air, and the diffusion resistance provided by the stomatal pores. Stomatal resistance (or its inverse, stomatal conductance) can therefore be calculated from the transpiration rate and humidity gradient. (The humidity gradient is the humidity inside the leaf, determined from leaf temperature based on the assumption that the leaf’s air spaces are saturated with vapor, minus the humidity of the ambient air, which is measured directly.) This allows scientists to learn how stomata respond to changes in environmental conditions, such as light intensity and concentrations of gases such as water vapor, carbon dioxide, and ozone.
Reference
Image 1: http://www.tutorvista.com/search/stomata-opening
Image 2: http://www.tutorvista.com/search/stomata-opening
Image 3: http://www.tutorvista.com/search/stomata-opening
Bibliography
Burba, G., & Pidwirny, M. (2007, April 1). Transpiration. Retrieved December 15, 2009, from Encyclopedia of Earth : http://www.eoearth.org/article/Transpiration
Farabee, M. (2007, June 6). PHOTOSYNTHESIS. Retrieved December 15, 2009, from http://www.emc.maricopa.edu/faculty/farabee/BIOBK/BioBookPS.html
Geranium. (2009, November 10). Retrieved December 15, 2009, from Wikipedia, the free encyclopedia: http://en.wikipedia.org/wiki/Geranium
Kalanchoe daigremontiana – Mother of Thousands. (2006, September 21). Retrieved December 15, 2009, from Plant of the week: http://www.plantoftheweek.org/week375.shtml
stomata opening. (2008, – -). Retrieved December 15, 2009, from tutorvista.com: http://www.tutorvista.com/search/stomata-opening
The Origin of the Geranium. (2006, October 8). Retrieved December 15, 2009, from A SouthWest Oasis: http://aswoasis.wordpress.com/2006/10/08/the-origin-of-the-geranium/