Materials And Method For Extracting LC And BC Biology Essay

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Solvents like ethanol, petroleum ether, ethyl acetate, butanol, methanol, toluene, formic acid, glacial acetic acid, acetonitrile, dimethyl sulphoxide and Folin- Ciocalteu's were purchased from SD fine chem. Ltd, Mumbai. Quercetin, Gallic acid, Rutin, 1,1-Diphenyl-2-picrylhydrazyl (DPPH) and ascorbic acid (vitamin C) Dulbecco's modified eagle's medium (DMEM), Eagles minimum essential medium (EMEM), Trypsin, EDTA, Sulphorhodamine blue, Trichloro acetic acid, Fetal calf serum and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma Aldrich, St Louis, USA. Human cervical cancer cell lines (HeLa), Human skin cancer cell lines (A431) and Human laryngeal epithelial carcinoma cell lines (HEp2) were obtained from NCCS, Pune. Precoated TLC aluminum sheets silica gel 60F254 (10 x10 cm, 0.2 mm thick) were obtained from E. Merck Ltd, Mumbai. All other chemicals and solvents used in this study were of the analytical grade.

6.1.1. Collection and authentication of plant materials

Fresh leaves and Bark of Albizia lebbeck L. were collected locally from Tamilnadu, India and authenticated by Botanical survey of India (BSI), Coimbatore, TamilNadu. Leaves and Bark were separated from adulterants, shade dried and powdered coarsely.

6.1.2. Extraction procedure

The air dried, powered leaves (100g) and bark (200 g) were taken in 1000 ml soxhlet apparatus and extracted with absolute alcohol until the colour disappeared (7 days). The extract was then concentrated by rotary evaporator under reduced pressure and stored in vacuum desiccators to remove the residual solvent. Finally a deep green colored sticky mass of leaf crude extract (LC) and dark brown colored free flow coarse powder of bark crude extract (BC) was obtained and stored in desiccators until use. Further the extract was weighed and the % yield was determined.

Fig 8: Extraction of Albizia lebbeck L. bark in soxhlet apparatus

6.1.3. Fractionation procedure

The ethanolic crude extract of both LC and BC (10g) was dissolved or dispersed in water (100 ml) and extracted with petroleum ether (100 ml) using a separating funnel for 30 min. The ether layer was then removed and fresh petroleum ether (100 ml) was added to the flask and continued the extraction. This was repeated until to get the colourless fraction of petroleum ether and all the ether fractions were pooled and concentrated under vacuum to yield the petroleum ether fraction of leaf (LPE) and bark (BPE) respectively. Similarly the subsequent fractionation in the order of ethyl acetate and then with n-butanol was carried out. The resulted ethyl acetate fractions (LEA &BEA), n-butanol fractions (LBT &BBT) and the remaining aqueous fractions (LAQ &BAQ) were concentrated and dried under vacuum desiccators. The % yield of each fraction were determined and reported.

Fig 9: Fractionation of extracts by using separating funnel

6.2. Phytochemical analysis of extracts

6.2.1. Preliminary phytochemical screening

A) Tests for carbohydrates

A small quantity of extract was dissolved separately in 5ml of distilled water and filtered. The filtrate was tested to detect the presence of carbohydrates.

Molish's test:

A small amount of filtrate was treated with few drops of alcoholic α-naphthol solution and then 2ml of concentrated sulphuric acid was added along the sides of the tes tube. Appearance of purple colour or reddish violet colour ring at the junction of two liquids infers the presence of carbohydrates.

Fehling's test:

Equal volume of Fehling's A [copper sulphate in distilled water] and Fehling's B [potassium tartarate and sodium hydroxide in distilled water] reagents were mixed few drops of filtrate and heated gently. Formation of brick red precipitate indicates the presence of reducing sugars.

Benedict's test:

The filtrate was heated with few drops of Benedict's reagent (alkaline solution containing cupric citrate complex). Formation of reddish brown precipitate infers the presence of reducing sugars.

Barfoed's test:

To few ml of the filtrate, few drops of Barfoed's reagent was added and boiled. Formation of red precipitate of copper oxide indicates the presence of monosaccharide.

B) Test for alkaloids

A small portion of the solvent free extract was stirred separately with a few drops of dilute hydrochloric acid and filtered. The filtrate was used for the following tests.

Mayer's test:

To the filtrate, add few drops of Mayer's reagent [potassium mercuric iodide solution]. Cream (dull white) precipitate was obtained.

Dragendroff's test:

To few ml of the filtrate, 1 ml of Dragendroff's reagent [potassium bismuth iodide solution] was added. Formation of orange red precipitate indicates the presence of alkaloids.

Wagner's test:

A small amount of filtrate was treated with Wagner's reagent [solution of iodine in potassium iodide]. Reddish brown precipitate was obtained.

Hager's test:

To the filtrate, few drops of Hager's reagent were added. Yellow precipitate was obtained.

C) Tests for glycosides

A small amount portion of the extract was hydrolysed with dilute hydrochloric acid for few hours on a water bath and the hydrolysate was tested to detect the presence of glycosides.

Legal's test:

To the hydrolysate, 1 ml of sodium nitroprusside solution was added and it was made alkaline with sodium hydroxide solution. Appearance of pink to red colour infers the presence of glycosides.

Borntrager's test:

Hydrolysate was treated with an equal volume of organic solvents [ether or chloroform]. The organic layer was separated, to this equal quantity of dilute ammonia was added. Ammoniacal layer acquires rose pink colour, infers the presence of glycosides.

Killer-Killani test:

Hydrolysate was dissolved in acetic acid containing trace amount of ferric chloride and transferred to the surface of concentrated sulphuric acid. A reddish brown colour was produced at the junction of two liquids gradually became blue, indicates the presence of glycosides.

D) Tests for proteins and amino acids

Ninhydrin test:

A small quantity of extract solution was boiled with 0.2% solution of Ninhydrin. Blue colour indicates the presence of free amino acids.

Million's test:

To the extract solution, 2ml Million's reagent [mercuric nitrate in nitric acid containing traces of nitrous acid] was added. Appearance of red colour indicates the presence of proteins and free amino acids.

Biuret test:

The extract was treated with equal volume of 40% sodium hydroxide and 2 drops of 1% copper sulphate solution. Pink or purple colour indicates the presence of proteins.

E) Tests for phytosterols and triterpenoids

Liebermann - Buchard test:

A small quantity of extract was treated with few drops of acetic anhydride, followed by a few drops of concentrated sulphuric acid. A brown ring was formed at the junction of two layers and the upper layer turns green colour, infers the presence of phytosterols and formation of deep red colour indicates the presence of triterpenoids.

Salkowski test:

A small quantity of the extract was treated with chloroform and few drops of concentrated sulphuric acid and allowed to stand for few minutes. Red colour appears at the lower layer indicates the presence of phytosterols and if it is yellow in colour at the lower layer indicates the presence of triterpenoids.

Zak's test:

To 1 ml of the chloroform solution of the extract, mixture of glacial acetic acid, ferric chloride and concentrated sulphuric acid was added. Formation of purple colour indicates the presence of phytosterols.

F) Tests for flavonoids

Zinc hydrochloride reduction test:

The extract was treated with mixture of zinc dust and concentrated hydrochloric acid. Formation of red colour indicates the presence of flavanoids.

Shinoda test: [Magnesium hydrochloride reduction test]

The extracts were dissolved in alcohol and a piece of magnesium (magnesium turnings) was added followed by a drop of concentrated hydrochloric acid and heated. Appearance of crimson red or occasionally green to blue colour infers the presence of flavanoids.

Alkaline reagent test:

To the extract, a few drops of sodium hydroxide solution were added. Formation of an intense yellow colour, which turns to colourless on addition of few drops of dilute hydrochloric acid, indicates the presence of flavanoids.

G) Test for saponins

Foam test:

To the extract, 20ml of distilled water was added and agitated on a graduated cylinder for 15 min. The formation of about 1cm layer of foam indicates the presence of saponins.

H) Tests for steroids and sterols

Liebermann - Buchard test:

The test sample was dissolved in 2ml of chloroform in a dry test tube. Few drops of acetic anhydride were added followed by 2 drops of concentrated sulphuric acid. The solution became red, then blue and finally bluish green in colour.

Salkowski test:

Extract was dissolved in chloroform and concentrated sulphuric acid was added. Bluish red, cherry red and purple colour was noted in chloroform layer whereas acid assumed marked green fluorescence.

I) Tests for phenols

Ferric chloride test:

To 1 ml of the alcoholic solution of the extract, 2 ml of distilled water followed by few drops of 10% ferric chloride was added. Formation of blue or green colour indicates the presence of phenols.

Lead acetate test:

1 ml of alcoholic solution of extract was diluted with 5 ml of distilled water and to this few drops of 1% aqueous solution of lead acetate was added. A yellow colour precipitate was formed which indicates the presence of phenols.

J) Tests for tannins

Lead acetate test:

In a test tube containing about 5 ml of an aqueous extract, few drops of lead acetate was added. A yellow or red colour precipitate was formed indicates the presence of tannins.

Gelatine test:

The extracts were treated with 1% gelatine solution containing 10% sodium chloride. Formation of white precipitate indicates the presence of tannins (Khandelwal, 2004).

6.2.2. Estimation of total phenolic content

Reagents:

Folin-Ciocalteu

20% Sodium Carbonate

Procedure:

The total polyphenols of the extract were determined by the Folin-Ciocalteu assay method (Singleton et al., 1999). An aliquot (0.25 ml) of extract or standard solution of gallic acid (0.5, 1, 2, 4 and 8 μg/ml) was mixed with 0.25 ml of Folin-Ciocalteu reagent. After 5 min, 0.25 ml of distilled water and 1.25 ml of 20% aqueous sodium carbonate solution (Na2CO3) were added to the above mixture. Distilled water was used as reagent blank. After incubation at room temperature for 120 min, the absorbance of the mixture was measured against prepared reagent blank at 765 nm. The sample was tested in triplicate and a calibration curve with six data points for gallic acid was obtained. The results were compared to a gallic acid calibration curve and the total phenolic content was expressed as mg of gallic acid equivalents (GAE) per gram of extract.

6.2.3. Identification and quantification of phenolic compounds

6.2.3.1 High performance liquid chromatography (HPLC)

A) Optimization of separation conditions

Mobile phase

The mobile phase of Acetonitrile: Methanol in various ratios 50:55, 55:45, 60:40, 70:30 were tried and the chromatograms were recorded at 365 nm with a flow rate of 1ml/min. At the ratio of 60:40 of Acetonitrile and Methanol was selected as the ideal ratio for the estimation of rutin.

Effect of flow Rate:

Keeping the mobile phase ratio 60:40 Acetonitrile: Methanol, the chromatograms were recorded at various flow rate 1 - 1.5 ml/ min but at 1 ml/ min the peaks were sharp with good resolution.

B) Optimization of chromatographic condition for the estimation of rutin

Preparation of standard

10 mg of the rutin was accurately weighed and transferred into 10 ml standard flask, and then the solution made up to 10 ml with the mobile phase Acetonitrile: Methanol (30:70). From this 10 µg/ ml solution was prepared and injected into the HPLC column without any further treatment and chromatogram was recorded.

Preparation of sample solution

10mg of the respective sample extract was accurately weighed and transferred into 10ml standard flask, and then the solution made up to 10ml with the mobile phase (1mg/ ml). From this 200µg/ ml solution was prepared and injected into the HPLC column without any further treatment and chromatogram was recorded.

Selection of wavelength:

An UV spectrum of 10 µg/ ml rutin in water was recorded by scanning in the range of 200 nm to 400 nm. The rutin was shown max at 365 nm.

Selection of chromatographic method:

Proper selection of the method depends upon the nature of the sample (ionic/ion sable/neutral molecule), its molecular weight and solubility. The drug selected in the present study was in polar nature and hence reverse phase or ion-pair or ion-exchange chromatography method may be used. The reverse phase HPLC was selected for the initial separations because of its simplicity and suitability.

Fixed chromatographic conditions:

Based on the above studies, the following chromatographic conditions were finally optimized for the simultaneous estimation of rutin in Albizia lebbeck L. Benth extract.

Stationary phase : Phenomenex C18 Column

Mobile phase : Solvent A: HPLC water

Solvent B: Acetonitrile and Methanol (30: 70)

Solvent ratio : 60: 40

Detection : 365 nm

Flow rate : 1.0 ml/ min

Sample size : 20 μl

Needle wash : HPLC water

Column temperature : Room temperature (20°C)

6.2.3.2 High performance thin layer chromatography (HPTLC)

High performance thin layer chromatography (HPTLC) is a suitable quality assessment tool for the evaluation of herbal medicines and natural drug. Additionally, numerous samples can be run in a single analysis thereby it will reduce the analytical time. With HPTLC, the same analysis can be viewed single and different wavelengths of light thereby providing a more complete profile of the plant then it is typically observed with more specific types of analysis. Quantitative analysis can be determined by using the formula:

Sample Peak Area

Amount to be present = X Concentration of standard

(Sample) Standard Peak Area

Preparation of standard quercetin solution

Quercetin (10 mg) was accurately weighed and dissolved in methanol in 10 ml volumetric flask, and then the solution was made up to 10 ml.

Preparation of extracts solution

Both the extracts and fractions (100 mg) were accurately weighed into 10ml volumetric flask, and dissolved in methanol and the volume was made up to 10ml.

A) Analytical specification

Selection of wavelength:

The sensitivity of HPTLC method depends upon the proper selection of wavelength by UV detector. An ideal wavelength is the one that gives maximum absorbance and good response for the drug detected at lower concentration. The drug was scanned under UV and this displayed the max at 254 nm. Hence the wavelength 254 nm was selected for quercetin.

Selection of Mobile Phase:

The following mobile phase systems (Table 2) were tried for selecting the suitable chromatogram for quercetin.

Table 2: Mobile phase for quercetin

Mobile Phase

Rf Value

Observation

Toluene: Ethyl Acetate: Glacial Acetic acid (6:3:1)

0.30

Broad Peak

Ethyl Acetate: Formic acid: Toluene: Methanol (4:2:3:1)

0.59

Spot Migrated with the solvent front

Toluene: Ethyl Acetate: Formic acid: Glacial Acetic acid: Methanol (3:4:1:1:1)

0.25

Symmetrical Peak

Toluene: Ethyl Acetate: Formic acid: Glacial Acetic acid (2.5:5:0.5:2)

0.63

Asymmetrical Peak with tailing

B) Optimization of the proposed method:

Information:

Application Port : Linomat V

Detector : CAMAG TLC scanner

Experimental Parameters:

Stationary phase : Precoated silica gel 60F254 aluminium sheets

TLC Plate 10x10 cm x0.2mm thickness

Mobile phase for Quercetin : Toluene: Ethyl acetate: Formic acid: Glacial

Acetic acid: Methanol (3:4:1:1:1 V/V)

Chamber saturation : 15 min

Band length : 8 mm

Application position : 10 mm

Solvent front position : 80.0 mm

Instrumental Parameters:

Number of track : 8

Position of first track X : 12 mm

Distance between tracks : 14.5 mm

Scan start position Y : 5.0 mm

Scan end position : 85.0 mm

Slit dimensions : 6 x 0.3 mm, micro

Optimize optical system : light

Scanning speed : 20 mm/S

Data resolution : 100 µm/steps

Measurement table:

Wavelength : 254 nm for quercetin

Lamp : D2

Measurement type : Remission

Measurement mode : Absorption

Optical filter : Second order

Detector mode : Automatic

6.3. Pharmacological studies

6.3.1 In vitro antioxidant activity

DPPH radical scavenging assay

Reagents

Diphenyl-2-picryl-hydrazyl (DPPHË™)

Methanol

Procedure:

The DPPH radical scavenging assay was performed according to the method reported by Brand-Williams et al. (1995) with some modification. Briefly, 1ml of sample or MeOH (control) was added to the same volume of methanolic solution of a 0.3 mM DPPHË™. Mixtures were shaken vigorously and left to stand in the dark at room temperature for 30min then absorbance was read at 515 nm.

Antiradical activity was expressed as inhibition percentage (I%) and calculated using the following equation :

Abscontrol - Abssample

Inhibition percentage (I%) = X 100

Abscontrol

Extracts having colour in the case UV absorptions that overlap DPPHË™ at 515 nm, blank solution (without DPPHË™) was prepared to correct any influence due to colour. So, in the previous equation the term Abssample was substituted with (Abssample - Absblank). Different sample concentrations were used in order to obtain calibration curves and to calculate the IC 50 values (IC 50: inhibition concentration required to obtain a 50% radical scavenging activity) (Monica et al., 2009).

6.3.2 In vitro anti cancer activity

The extracts and fractions that showed high polyphenol content and flavonoids were studied for its cytotoxic effects on three human cancer cell lines namely human cervical cancer cell line (HeLa), human skin cancer cell line (A431) and human laryngeal epithelial carcinoma cell line (Hep2). All the above cells were purchased from National Centre for Cell Sciences (NCCS), Pune.

The cell lines HeLa and Hep2 were grown in Eagles Minimum Essential Medium containing 10% fetal bovine serum (FBS) where as A431 was grown in Dulbeccos modified eagles medium (DMEM) containing 10% FBS. All cells were maintained at 370C, 5% CO2, 95% air and 100% relative humidity. Maintenance cultures were passaged weekly, and the culture medium was changed twice a week.

Cell treatment procedure

Cells were detached with trypsin-ethylenediaminetetraacetic acid (EDTA) to make single cell suspensions and viable cells were counted using a hemocytometer and diluted with medium with 5% FBS to give final density of 1x105 cells/ml. one hundred microlitres per well of cell suspension were seeded into 96-well plates at plating density of 10,000 cells/well and incubated to allow for cell attachment at 370C, 5% CO2, 95% air and 100% relative humidity. After 24 h the cells were treated with serial concentrations of the extracts and fractions. They were initially dissolved in neat dimethylsulfoxide (DMSO) and further diluted in serum free medium to produce five concentrations. One hundred microlitres per well of each concentration was added to plates to obtain final concentrations of 100, 50, 25, 12.5 and 6.25 µg/ml. The final volume in each well was 200 µl and the plates were incubated at 370C, 5% CO2, 95% air and 100% relative humidity for 48h. A standard flavonoids quercetin was used as standard and the medium containing without samples were served as control. Triplicate was maintained for all concentrations.

MTT assay

MTT is a yellow water soluble tetrazolium salt. A mitochondrial enzyme in living cells, succinate-dehydrogenase, cleaves the tetrazolium ring, converting the MTT to an insoluble purple formazan. Therefore, the amount of formazan produced is directly proportional to the number of viable cells.

After 48h of incubation, 15µl of MTT (5mg/ml) in phosphate buffered saline (PBS) was added to each well and incubated at 370C for 4h. The medium with MTT was then flicked off and the formed formazan crystals were solubilized in 100µl of DMSO and then measured the absorbance at 570 nm using micro plate reader. The % cell inhibition was determined using the following formula.

% cell Inhibition = 100- Abs (sample)/Abs (control) x100.

Nonlinear regression graph was plotted between % Cell inhibition and Log10 concentration and IC50 was determined using GraphPad Prism software.

SRB assay

After 48h of incubation, the assay was terminated by the addition of cold trichloroacetic acid (TCA). Cells were fixed in situ by the gentle addition of 50 µl of cold 50% (w/v) TCA (final concentration, 10% TCA) and incubated for 60 min at 40C. The supernatant was discarded, and the plates were washed five times with tap water and air dried. Sulphorhodamine B (SRB) solution (100 µl) at 0.4 % (w/v) in 1% acetic acid was added to each well and plates were incubated for 10 min at room temperatire. After staining, unbound dye was removed by washing five times with 1% acetic acid and the plates were air dried. Bound stain was subsequently solubilized with 10mM trizma base and the absorbance was read on microplate reader at 515 nm. The % cell inhibition was determined using the following formula.

% cell Inhibition = 100- Abs (sample)/Abs (control) x100.

Nonlinear regression graph was plotted between % Cell inhibition and Log10 concentration and IC50 was determined using GraphPad Prism software.

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