Materials Methods
Collections:
Bryophyllum pinnatum plant leaves were collected from BVVS Ayurvedic Medical
College and Hospital Bagalkot.
Methods:
The collected leaves were washed with running tap water and wiped by a muslin cloth.
Cleaned leaves were divided into four parts of each 600g for various drying methods.
Drying methods:
For the removal of water content we have followed different drying methods like oven
drying, sun drying, shade drying and blanched shade drying.
Hot air oven drying method - For this drying method 600g weighed leaves were placed
evenly on oven tray and placed in oven at the temperature 50ºC.
Sun drying method - In this drying 600g leaves were kept in a tray and placed outside to
expose sun light.
Shade drying method - The weighed leaves were placed on a blotting paper and kept in
a room temperature where light is absent.
Blanched and shade drying method – In this method the leaves are added to a large pot
containing Luke warm water for 4 minutes and then removed and allowed to cool. Then
kept for shade drying.
EXTRACTION METHODS (Prashant Tiwari et al 2011):
The extraction method involved in the separation of medicinally active portions of plant
tissues by different solvents. For this extraction method Methanol, Chloroform and water
is used as solvents.
Method 1:
Maceration: For this extraction method 4g powder from each drying method is macerated
with 95% of 200ml methanol and 99% of 200ml chloroform and then filtered. The extract
used for further qualitative tests.
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Method 2:
Magnetic stirring: for magnetic stirring method 4g dried leaf powder is decocted with
250 ml of water at 35ºC at 120 rpm for 5 hours and then filtered.
QUALITATIVE SCREENING OF PHYTOCHEMICALS(Prashant
Tiwari et al 2011):
Dried, finely powdered leaf was extracted successively with water, methanol and
chloroform using maceration and Magnetic stirring method. All the extracts were further
screened for phytochemical tests ()
Test for alkaloid:
Wagner’s test: For this test 3ml extract is taken in a clean test tube and added with 1ml
of Wagner’s reagent. This result in the formation of reddish brown precipitate indicates
the presence of alkaloids.
Test for saponin:
Froth test: 1ml o f solvent extract is added with 2ml of water and shaken frequently for
few minutes. As a result the formation of 1cm layer of foam gives indication of presence
of saponins.
Test for tannin:
For this test 3 ml of extract is added with 1 ml of FeCl 3. As a result bluish black color is
appeared indicates the presence of tannin.
Test for terpenoid:
Salkwoski test: To the 1ml of solvent extract 2ml of chloroform and 2ml of H 2SO4 is
added which results in the formation of golden yellow color indicates the presence of
terpenoids.
Test for protein:
Xanthoprotein test: 2ml of extract were treated with few drops of Conc. HNO 3 the
formation of yellow color indicates the presence of protein.
Test for flavanoid: To the test tube 2ml of extract is taken to this few drops of NaoH is
added. Formation of yellow color which becomes colorless on addition of [Link]
indicates the presence of flavanoids.
Test for Phenols:
Ferric –chloride test: 3ml of extract is taken in test tube and added by few drops of
FeCl3. The appearance of bluish black color indicates the presence of phenols.
Test for amino acid:
Nin-hydrin test: to the 2ml of extract few drops of nin-hydrin reagent is added and
boiled for few minutes. Appearance of blue color indicates the presence of amino acids.
Quantitative tests for phenols(S Sadashivam et al Biochemical methods
page no 203-204):
For the quantitative test the phenols react with phosphomolybdic acid in Folin-ciocaltaeau
reagent in alkaline medium and produce blue color complex. In a clean test tube with
extracts of concentration 0.1 and 0.2 and make up the volume to 1ml by adding distilled
water and then add o.5 ml folin-ciocalteau reagent shake it well to homogenize after this
add 2ml of NA2Co3 and then these are incubated in dark for 30 minutes after incubation
the absorbance was read at the wavelength of 650nm. For standard the galic acid solution
was prepared by mixing 100mg (0.01g) with 100ml distilled water in volumetric flask as
a stock solution for the preparation of working solution 10ml of stock solution and make
up the volume to 100ml. as similar for sample tests the galic acid is taken in
concentration of 0.2-1.0 and remaining FC reagent and Na 2Co3 and incubated in dark for
30 minutes and absorbance is readed at [Link] determination of total phenolic
content is in the sample from the standard curve by taking Gallic acid as standard.
α-Amylase inhibition screening assay(Ishnav K B 2018 ref 13)
In this assay, 160μl of α-Amylase enzyme and 120μl of plant extract were mixed
and incubated at 37ºC for 45minutes. After, incubation the mixture was powdered into
well made petriplate containing 3% agar and 1.2% starch. And the plates could stand for
3 days at 25ºC in incubator and then flooded with iodine solution and allowed to stand for
15mins. The diameter of zone of starch hydrolysis was measured. As a control, the
enzyme was added into the well of the plate without plant extract.
Diameter of Control – Diameter of test
% 0 α- Amylase inhibition = ------------------------------------------------------- x 100
Diameter of Control
Quantitative test for α- Amylase inhibition by DNSA method (Mohshina F
P et al 2022):
The effect of α-Amylase on the rate of reaction as well as the inhibitory activity of
different solvent extracts by in- vitro testing was carried out spectophotometrically by
amylase inhibitory method to identify α- Amylase enzyme which helps in digestion and
metabolism of carbohydrates which are majourly polysaccharides.
1ml of α- Amylase was mixed with 1ml of extract in a test tube and kept at a temperature
of 37º C for 10 minutes. After prior incubation, 1ml of 1% solution of starch was added to all
the test tubes and incubated at 37ºC temperature for 15 minutes. The reaction was completed
with 2ml of DNSA reagent, which was kept in boiling water bath for 5 minutes the brought
back to room temperature and diluted with 3ml of water, the absorbance was checked at
546nm. The control is kept without plant extract.
% Inhibition = Enzyme activity of control- Enzyme activity of extract × 100
Enzyme activity of control
Antioxidant Assay by DPPH method (Chang et al 2001):
The radical scavenging activity of different extracts was determined by using DPPH assay
according to chang et al,. (2001). The decrease in the absorption of the DPPH solution after
the addition of an antioxidant was measured at 517nm. Ascorbic acid (10mg\ml DMSO) was
used as reference.
Different volumes (2-20μl) of plant extracts were made up to 40μl with DMSO and 2.96ml
DPPH (0.1mm) solution was added. The reaction mixture was incubated in dark condition at
room temperature for 20 min, the absorbance of the mixture was read at 517nm. 3ml of
DPPH was taken as control. The % radical scavenging activity of the plant extracts was
calculated using the following formula,
% RSA = Abs control – Abs sample × 100
Abs control
Where, RSA is the Radical Scavenging Activity; Abs control is the absorbance of DPPH
radical + ethanol]; Abs sample is the absorbance of DPPH radical + plant extract.