METHODOLOGY

4. MATERIAL AND METHOD

4.1 Collection of the Cucumis sativus leaves.

The leaves of Cucumis sativus (cucumber leaves) were collected in and around

Hoysalakatte village, Chikkanayakanahally taluk, Tumkur, Karnataka in July 2024

Fresh leaves were collected and dried under shade for 7-8days. They were powdered

Using a mechanical grinder and were further used for other processes.

4.2 Organoleptic Evaluation.

Organoleptic evaluation refers to evaluation of Cucumis sativus leaves by colour,

odour, taste, texture etc. The organoleptic characters of Cucumis sativus leaves were

carried out based on official method16.

4.2.1 Macroscopic analysis of Leaves:

Macroscopic analysis of the Cucumis sativus leaves was done. The shape,

size, surface characters of the plants were noted.

4.3 Microscopical Studies.

4.3.1 Leaf Microscopy:

The outer epidermal membrane layer was cleared in KOH, mounted with glycerin and

observed under microscope. The presence or absence of the following was observed:

parasitic type of stomata on abaxial (dorsal surface), secretory cells among ground

tissue of midrib portion, xylem, phloem, fibers (stained with phloroglucinol + HCL,

iodine and safranin) and epidermal hairs (type of trichome and distribution). The

transverse sections of the fresh leaves through the lamina and midrib were observed

after clearing and staining, respectively17.

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4.3.2 Powder Microscopy:

Coarse powder of the leaves was used to study the microscopical characters of the

leaf powder. Powder is treated with potassium hydroxide, Hydrochloric acid and

Phloroglucinol for staining and was observed under the microscope18.

4.4 Extraction of Cucumis sativus Leaves.

Soxhlet extraction is the continuous process of extraction with hot organic solvent

(Ethanolic). The collected leaves of Cucumis sativus leaves are grind into fine powder

then the 25gms of powdered plant material is taken in a thimble which is placed in the

Soxhlet extractor. The extractor which has a siphoning system is fitted on top of a

round bottom flask. A condenser is fitted at the top of the extractor. Enough quantity

of the extracting solvent is poured in to the flask placed on the heating mantle. On

heating the solvent evaporates, rises to the condenser, where it condenses and drained

back to the extractor holding the thimble with the plant material. When the extractor

becomes full with the hot solvent the solvent siphons down to the flask along with the

extracted constituents. The recycling of the evaporated solvent allowed to continue

until the extraction is completed (i.e.; Till all the constituents from the siphons tube

were removed and the thimble becomes decolored)19.

4.5 Phytochemical Investigation.

Following chemical tests were carried out for extract of Cucumis sativus

Leaves to identify the presence of various phytochemical constituent. Following

chemical tests were carried out for extract of Cucumis sativus leaves to identify the

presence of various phytochemical constituent20.

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4.5.1 Test for Alkaloids:

Mayer's Test: Few ml of extract was treated with Mayer's reagent. Formation of

cream or pale-yellow precipitate indicates the presence of alkaloids.

Wagner's Test: Few ml of extract was treated with Wagner's test. Formation of

reddish brown precipitate indicates the presence of alkaloids.

Dragendorff's Test: Few ml of extract was treated with dragendorff's reagent.

Formation of reddish brown precipitate indicates the presence of alkaloids.

Hager's Test: Few ml of extract was treated with Hager's test reagent. Formation of

yellow precipitate indicates the presence of alkaloids.

4.5.2 Test for Carbohydrates:

Molisch's Test: Filtrate was treated with 2 drops of alcoholic α-naphthol solution in a

test tube and 2 ml of conc. Sulphuric acid was added carefully along the sides of the

test tube. Formation of violet ring at the junction indicates the presence of

Carbohydrates.

Fehling's Test: Filtrate was hydrolyzed with dilute hydrochloric acid, neutralized

with alkali and heated with Fehlings- A and Fehlings- B solutions. Formation of red

precipitate indicates the presence of reducing sugars.

Benedict's Test: Filtrate was treated with Benedict's reagent and heated on waterbath.

Formation of orange red precipitate indicates the presence of reducing sugars.

4.5.3 Test for Glycosides:

Keller-Killani's Test: Extract the powder with water glacial acetic acid add ferric

chloride and add conc. Sulphuric acid through the side of the test tube. Brown ring at

the junction of liquids indicates the presence of glycosides.

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4.5.4 Test for Flavonoids:

Shinoda Test: Extract was treated with the mixture containing piece of magnesium

ribbon and HCL Formation of red color indicates presence of flavonoids.

Lead Acetate Test: To the extract add 10% Lead acetate solution was added.

Formation of yellow colour ppt. indicates presence of flavonoids.

4.5.5 Test for tannins:

Test with Ferric Chloride: To the solution of sample added ferric chloride solution.

A blue, black, violet or green precipitate or colour confirms the presence of tannins.

4.5.6 Test for Steroids:

Libermann Burchard test: To 2mg of dried extract was dissolved in acetic

anhydride, heated to boiling, cooled and then 1ml of concentrated sulphuric acid was

added along the sides of the test tube, Formation of green colour indicated the

presence of steroids.

4.5.7 Test for Saponins:

Foam test: 1ml solution of extract was diluted with distill water to 20ml and shaken

in a graduated cylinder for 15 min. development of stable foam suggests the presence

of saponins.

Lead acetate test: 1 ml extract was treated with 1% lead acetate solution. Formation

of white ppt indicated the presence of saponins.

4.5.8 Test for proteins:

Biuret's test: To 1ml of hot extract, 5-8 drops of 10% w/v sodium hydroxide solution,

followed by dl or 2 drops 3% w/v copper sulphate solution were added. Formation of a

violet red colour indicated the presence of proteins.

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Millon's test: To 1ml of extract dissolved in 1ml of distilled water and 5-6 drops of

Millon's reagent were added. Formation of white ppt, which turns red on heating,

indicated the presence of proteins.

4.6 Physio-chemical Evaluation of the plants.

4.6.1 Loss on Drying (LOD):

About 2-5 g of the prepared air dried Cucumis sativus leaves powder were accurately

weighed in a previously dried and tared flat weighing bottles. The samples were

placed in the drying chamber (Oven). Drying was carried out by heating to 100-105°C

for 15 minutes. Then bottles were removed from the oven and then weighed. The

experiment was repeated till two consecutive weighing did not differ by more than

5µg, unless otherwise stated in the test procedure. The loss on drying was studied21.

4.6.2 Ash Value:

Ash content of the crude drug is generally taken to be the residue remaining after

incineration. It represents the inorganic salts naturally occurring in the drug and

adhering to it, but may also include inorganic matter added for the purpose of

adulteration. Total ash is the remaining after incineration. Total Acid insoluble ash is

the part of the total ash which is insoluble in dilute HCL. Water-soluble is the part of

total ash which is soluble in hot water22.

4.6.3 Total Ash Value:

About 2g of the powdered ingredient of Cucumis sativus leaves powder were

accurately weighed in a tarred silica crucible. The powdered drug was spread as a fine

layer at the bottom of the crucible. The crucible was incinerated at a temperature not

exceeding 450°C until free from carbon. The crucible was cooled and weighed. The

procedure was repeated till a constant weight was observed. The percentage of the

total ash was calculated in triplicate with reference to the air-dried drug22.

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4.6.4 Extractive Value:

Extractive value is a measure of the content of the drug extracted by solvents

Extractive value can be water soluble, ethanol soluble, methanol soluble and

chloroform soluble extractives. The Extractive value unless and otherwise prescribed,

is carried out by maceration.

Approximately 4g of previously weighed air-dried Cucumis sativus leaves powdered

were taken in glass stopper flask and macerated with 100 ml of chloroform: ethanol

(75:25). It was shaken frequently and then allowed to stand for 24h It was filtered

rapidly taking precautions against loss of the solvent.25ml of filtrate was evaporated

to dryness in a tarred flat-bottomed petri dish, dried at 105°C, cooled in a desiccator

and weighed. The percentage of water- soluble extractive was calculated with

reference to air-dried drug23.

Note – Chloroform and alcohol soluble extractive, follow same procedure of water

soluble extractive excepting the solvent (water) which is replaced by chloroform and

alcohol.

4.7 Standardization by Analytical Techniques.

Thin Layer Chromatography:

Thin layer chromatography is mainly used for qualitative screening of plant extract

which serve as a very important tool in the overall phytochemical research studies.

Ethanol extract was standardized by using thin layer co- chromatography technique.

Procedure:

Slurry of silica gel G was prepared in distilled water and poured over a glass plate to

form a thin film. The prepared plates were allowed for setting (air-drying). After

setting, the plates were kept in an oven at100 to 120° C (30 min) for activation. The

test extract was applied to the activate plates (1cm above from the bottom). It was then

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kept in previously saturated developing chamber containing mobile phase, and

allowed to run 3/4th of the height of the plate. The developed plate was removed, air

dried and observed under ultraviolet light and fluorescent compound identified and by

keeping it in an Iodine saturated chamber. Then compared with standard drug spot and

calculate the Value using following formula5.

Distance travelled by solute

Rf = Distance travelled by solvent

Mobile phase for ethanol extract of Cucumis sativus – Chloroform.

4.8 Performing nucleation assay

First of all, in-vitro growth of stone nucleus in the absence of any inhibitor was done.

For this, a volume of 1.0 ml of 0.025M CaCl2 and 2ml of Tris-buffer (pH 7.4) was

added to a test tube. Formation of the turbidity results immediately after mixing of the

above chemicals (up to 10 min) and then the measurement of turbidity formed (in

terms of absorption at 620 nm in Shimadzu UV 17000 UV/Visible spectrophotometer

of model). Absorptions were noted down and obtained data were used as the

uncontrolled growth of the stone nucleus for the comparison of growth in the presence

of the standard drugs and plant extracts. For the study of the effect of plants extracts

against stone nucleus formation, four sets of test tubes with 1ml of 0.025M Calcium

Chloride, 2ml Tris-buffer and 1ml (50 mg/ml solution) of three plant extracts were

taken. Two more sets of test tubes were prepared same as the above in which synthetic

drug of the polyherbal formulation, Cystone was administered. Then 1 ml volume of

0.025M sodium oxalate was added to each test tube.

Immediately after the mixing of sodium oxalate, measurement of change in turbidity

of the solution was done up to the period of 10 min post mixing. Inhibition in stone

nucleus formation was calculated by the graphical method using the following

mathematical formula: s𝐼𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 % = 1 – 𝑆𝑖/ 𝑆𝑐 𝑥100 10.

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