Disc-diffusion method

The disc diffusion susceptibility method is simple and practical and has been well-standardized. The test is performed by applying bacterial to the surface media of Mueller-Hinton agar (MHA) ^[61]. For the antimicrobial test using this method first the media and Petri dishes incubated with steam Autoclave at 121^oC and 1.5 pressures for 15-20 minute. The microorganisms using sterile cotton swab to media. Then filter paper discs (about 6mm in diameter), containing the test compounds of fixed concentration (50 ppm, 100 ppm, 200 ppm, 400 ppm of EOs) are placed on the agar surface using sterile pair of forceps. The Petri dishes are incubated under suitable conditions 37^oC for 24 Hours. Generally the antimicrobial agent (test) diffuses into the agar and inhibits growth of the test microorganism and then the diameters of minimum inhibition zones (MIZ)are measured using ruler ^[62,63] .

16

CHAPTER3: MATERIALS AND METHODS 3.1. Materials

3.1.1. Raw materials

The fresh leaves of Cymbopogon citratus and Thymus schimperi were collected in April, 2011EC from Fagita district, Amhara National Regional State, North West of Ethiopia.

The plants were identified and authenticated by Dr. Ali Seidu Botanist of Bahir Dar University in Biology department. The plant leaves were cleaned and air (shadow) dried until constant weight was achieved. The air dried leaves of Cymbopogon citratus and Thymus schimperi were ground by coffee grinder and packed in polyethylene bags to avoid oxidation. Then, it was made ready to essential oil extraction

3.1.2. Chemicals and Reagents

The chemicals and reagents used for this study were anhydrous Sodium sulphate (Na2SO4), Dichloromethane (CH2Cl2), Dimethyl sulfoxide (DMSO) [(CH3)2SO], Methanol (97.5%, Finechem ,Ethiopia), Ethanol, Ferric chloride (FeCl3), (Blulux, India),), hydrochloric acid (HCl) (LOBAChemie), Sodium hydroxide (NaOH), 1%sodium phosphate buffer (0.2M of PH 6.6) , Trichloroacetic acid (CCl3COOH) (Blulux, India), potassium hexacyanoferrate (II) (K3[Fe(CN)6], Muller Hinton agar (Himedia, India), Ascorbic acid (Blulux,India), Gallic acid, DPPH, Sodium carbonate (Na2CO3) and Ferric chloride (FeCl3.6H2O)^[64].

3.1.3 Instruments and apparatuses

The apparatuses and instruments used in this study include Whatman No 1 filter paper, Electronic digital beam balance, Measuring cylinder, Pestle and mortar Coffee grinder, Funnel, Tong, Water bath, reparatory funnel (pyrex, England), condenser, Round bottom flask, Erlenmeyer flask, Volumetric flask Aluminum foil,Centrifuge, UV-vis spectrometer , Autoclave, electrical incubator plate and beaker.

17 3.2. Experimental Methods

3.2.1. Extraction of essential oil

Fresh leaves of C. citratus and T.schimperi were harvested, air-dried under the shade and stored at room temperature in darkness until hydro distillation.

Figure 8: Graphical representation of essential oil preparation from C: Citratus and T:schimperi leaves

Extraction of essential oil from subjected plant was carried out using Clevenger apparatus with the following conditions: 50-100 g of air-dried plant material, 500 ml distilled water and 2-3 hours hydro distillation. The essential oil was taken from the upper layer by using separator funnel and essential oil was dried over anhydrous sodium sulfate to

18

remove water after extraction. The resulting oil placed into sealed vials and was stored in refrigerator at 4 ^o C ^[4,50].

3.3. Qualitative phytochemical analysis

Essential oil extracts from the hydro distillation of C: citratus and T:schimperi essential oil were used for qualitative analysis of different phytochemicals based on standard procedures described on different literatures[65,66,67,68]

.

3.3.1. Test for terpenoids (Salkowskis Test)

2 mL of plant extracts in a test tube then concentrated sulfuric acid was added to this mixture that was resulted in reddish brown interface confirming the presence of terpenoids.

3.3.2. Test for steroids (Salkowski test)

2 mL of extract with 1 mL of concentrated H2SO4 acid were added carefully along the sides of the test tubes. A red color was produced confirms the presence of steroids.

3.3.3. Test for flavonoids

2 mL of the extract was treated with 2 mL of dilute NH3 solution and a few drops of concentrated H2S04 acid. Formations of yellow color indicated the presence of flavonoid.

3.3.4. Test for saponins

To a little amount of each of the sample in a test tube, 2 mL of distilled water was added and Vigorously shaken for 15 minutes. Formation of 1 cm foam confirms a positive result.

3.3.5. Test for phenols (ferric chloride test)

19

1 mL solution of the extract with 2 mL of distilled water followed by drops of 10% aqueous FeCl3 solution were added. Formation of dark green or blue green indicated the presence of phenols.

3.4. Procedure for Total Phenolic Content Determination

The total phenolic content was determined by Folin-Ciocalteu method as described by different concentration of the standard Gallic acid (20 ppm, 40 ppm, 60 ppm 80 ppm and 100 ppm) to construct the calibration curve was prepared in similar manner.

Measurements of the blue color formed were carried out after 1 h at 765 nm on a UV-visible spectrometer against the reagent blank(pure solvent).

3.5. Standard Solution, essential oil and reagent preparation

Standard Solutions ,essential oil and different reagents used in this study for antioxidant activity ,phenolic content and antimicrobial activity determination were prepared using in various literatures as follows.

3.5.1. Solution Preparation for Antioxidant Assay 3.5.1.1. DPPH Radical Scavenging Assay

The antioxidant activity of the extracts essential oil determined with scavenging activity of the stable 2, 2-diphenyl-1-Picrylhydrazyl (DPPH) free radical assay. Stock solution of ascorbic acid was prepared by dissolving 0.02 g (20 mg) of ascorbic acid in 100 ml of volumetric flask and water was added up to the mark to prepare a stock solution. From this stock solution of ascorbic acid 25 ppm, 50 ppm, 75 ppm, 100 ppm and 125 ppm concentrations were prepared to construct the calibration curve. Similarly 0.002 g (2 mg) of C: Citratus and T:schimperi essential oils were dissolved in 10 mL of

20

volumetric flask individually and 1:1ratio of methanol distilled H2O was added up to the mark to prepare 200 stock solutions. From these stock solutions 25 ppm, 50 ppm, 75 ppm, 100 ppm and 125 ppm concentrations were prepared to study the DPPH scavenging activity of both essential oil . 0.004 g of DPPH powder was taken into 100 mL of volumetric flask and methanol was added up to the mark in the flask to prepare 0.004% DPPH solution and 1 ml of DPPH solution was added to 3 ml of various concentrations of extracts and ascorbic acid to be tested. After 30 min, absorbance was measure at 517 nm. Ascorbic acid with a series of concentration used as a reference material. The percent inhibition (% I) of DPPH radical scavenging activity calculated using the following formula ^{[13, 52]}.

Percent inhibition (% I) =

Where Ac is the absorbance of the control (methanol + DPPH) and As is the absorbance of the sample

3.5.1.2 Ferric reducing antioxidant power (FRAP) assay

Reducing power was determined by the method prescribed. 2.5 ml of different concentrations of essential oil and ascorbic acid (50 ppm,100 ppm, 150 ppm, 200 ppm) was mixed with 2.5ml of phosphate buffer (0.2 M, p^H =6.6) and potassium ferricyanide (1%) ([K3 Fe (CN)6]). The mixture was incubated at 50°C for 20 min. After the incubation, 2.5ml of 10% trichloroacetic acid added to the reaction mixture. The upper layer of 4ml solution mixed with 4ml of distilled water and added 0.5 ml of ferric chloride (0.1%) solution . The absorbance was measured at 700 nm ^[51]. The result was expressed as:

(%) inhibition=

,

where As and Ac are the absorbance value of the test sample and control respectively.

21

3.6. Procedure for Antioxidant Capacity Determination

3.6.1. Scavenging activity against 2, 2-diphenyl-1-pecrylhydrazyl (DPPH)

The quantitative measurement of free radical scavenging activities of C. citrates and T.

Schimperi essential oils was done in terms of hydrogen donating or radical scavenging ability of the stable DPPH (2, 2-diphenyl-1-picryl-hydrazyl) with slight modification^[56]. 1 mL of DPPH solution was added to 3 mL of essential oils of C.

citrates and T. Schimperi leaves of the test sample at different concentrations (25 ppm, 50 ppm, 75 ppm, 100 ppm and 125 ppm) and after 30 min of incubation and Absorbance was measured at 517 nm using UV spectroscopy (Lambda 35, Perkin Elmer)

3.6.2. Ferric Reducing Antioxidant Power (FRAP) Assay

The reducing power of essential oils of C. citrates and T. Schimperi leaves was determined according to the method described ^[69]. 2.5 mL of different concentrations of essential oils of C. citrates and T. Schimperi leaves (50 ppm, 100 ppm, 150 ppm and 200 ppm) individually was mixed with 2.5 mL of phosphate buffer solution ( PH=6.6, 0.2 M) and 2.5 mL of potassium hexacyanoferrate (1%) (K3 [Fe (CN)6]) . The mixture was incubated at 50^oC for 20 min in water bath. Then 2.5 mL of (10%) Trichloroacetic acid was added to the mixture to terminate the reaction. After incubation 4 mL of the upper layer of the solution was mixed with 4 mL of distilled water and 0.5 mL of (0.1%) FeCl3 solution and the absorbance of the blue color developed was measured at 700 nm using spectrophotometer (Lambda 35, Perkin Elmer) against a blank solution.

3.7. Procedures for Antimicrobial Susceptibility Testing 3.7.1. Source of Microorganism

Muller Hinton Agar media used for antibacterial test was prepared by dissolving 3.8 g of Muller Hinton agar in 100 mL of volumetric flask and distilled water was added up to the mark. Essential oils of C. citrates and T. Schimperi leaves were evaluated in vitro for antimicrobial assay by using the disc diffusion method.

22

The antimicrobial activities of all samples were tested against two gram positive bacterial species, Staphylococcus aureus (S.aureus) and Streptococcus pyogenes (S.Pyogenes), two gram negative bacterial species, Escherichia coli (E.coli) and Klebsiella pneumoniae (K.pneumoniae) using Muller Hinton agar (MHA) medium. All the microbial species were obtained from microbiology laboratory of Biology Department in Bahir Dar University and Gentamycin was used as standard drug against bacteria. The old culture strain was refreshed and used for the antimicrobial test.

3.7.2. Incubation

The prepared culture medium, specifically the Muller Hinton agar and petri dishes were incubated with steam autoclave at 121^oC and 1.5 pressure for 15-20 minute and the refreshed test organisms using sterile swab moistened with the bacterial suspensions. What man No1 filter paper was used to prepare 6 mm disc with punching machine. Then disc papers was sterilized in autoclave (oven dry) and loaded with suitable concentration of test essential oils was placed in bacterial culture nutrient agar plates. Similarly standard antibiotic discs, Gentamycin was placed on the surface of the medium in the plates as positive control and incubated at 37 ⁰C for 24 hours and at room temperature[4, 51, 71]

.

After 24 hours incubation, if the organism is susceptible to the test solution of essential oil, the growth of bacteria in the media is inhibited and clear zone (zone of inhibition) was observed. Then the clear zone was measured in millimeter and taken as inhibition zone. Similarly the inhibition zone of the standard antibiotics was measured and compared with plant essential oil ^[71].

3.7.3. Preparation of test solution

The crude essential oils 2 mg of C. citrates and T. Schimperi leaves were dissolved with 5 ml DMSO. A series of essential oil (50 ppm, 100 ppm, 200 ppm, and 400 ppm) concentrations were prepared for antimicrobial activity test. The experiment was

23

Where Ac is the absorbance of the control and As is the absorbance of the sample

………..2

Where Asample and Acontrol are the absorbance value of the test sample and blank respectively.

A calibration curve of standards was constructed to determine total phenolic content.

Linear equations was also derived from calibration curves in the form of Y= ax +c……….3

Where, a = slope c = y-intercept

The total phenolic content of the extracts EOs were expressed in terms of Gallic acid equivalence per gram of dry sample.

………..………..4

X -is Activity= in the equation of the standard Y= is the absorbance of the sample

c= is the y intercept of the standard graph

24 3.9. Over all methods of the experiment

Figure 9: Over all methods of the experiment

25 schimperi leaves yield are 0,58% w/w and 0.47 % w/w, respectively as shown in (Table 1). The relative yield (% w/w) of essential oils of Cymbopogon citratus is higher (0.58%) that of Thymus schimperi ( 0.47%). This study results showed that hydro distillation of C.citratus EOs extraction gave higher yields than T. schemperi EOs extract.

Table 1: Percentage yields of extracts.

26

Figure 10: Percentage yield of Cymbopogon citratus and Thymus schimperi essential oil extract

4.2. Qualitative phytochemical analysis

The qualitative analysis of bioactive compounds present in hydro distillation extracts essential oil had been analyzed as follows. The present study showed that the presence of different bioactive compounds in Cymbopogon citratus and Thymus schimperi essential oil by using color change as a confirmatory test. From this result Cymbopogon citratus EOs extract was found to have a wide range of bioactive compounds like flavonoids, phenols and terpinoids .Thymus schimperi EO extract was also positive for flavonoids, Steroids, phenols and terpinoids. Saponin was however abscent in both extracts of EOs.

Table 2: Qualitative analysis of phytochemicals analysis in C. citratus and T. schimperi essential oil extracts

Phytochemicals Types of test or Reagent Cymbopogon citratus

Thymus schimperi

Phenols Ferric chloride test ++ ₊

Flavonoids Lead acetate test ++ +

Steroids Salkowski test + +

Terpinoids Lieberman-Burchard test ++ +

Saponins Foam test - -

Key:- (++) = highly present, (+) = present, (-) = not present

27

Saponin Phenol Flavonoids Steroids Terpinoids

Figure 11: Color observed in some phytochemical test

4.3. Quantitative phytochemical analysis 4.3.1. Determination of total phenolic content 4.3.1.1. Calibration Curve of Gallic Acid

The Folin- Ciocalteu reagent (FCR) or Folin phenol reagent or folin-Denis reagent, also called the Gallic acid equivalent method (GAE) is a mixture of phosphomolybdate and phosphotungstate used for the colorimetric in vitro assay of phenolic and polyphenolic antioxidants ^[62]. The assay is simple and relies on the transfer of electron in alkaline medium from phenolic /antioxidant/ to phosphomolybdic and phosphotungstic acid complexes, manifested in the formation of blue color complexes [possibly (PMoW₁₁O₄₀)⁴⁻] which can be determined by measuring the absorbance at 765 nm using UV-Vis spectrophotometer ^[72].

Mo(VI) (yellow) + e^- (from anti-oxidant) Mo(V)(blue)

Scheme 7: Chemistry of oxidation of phenolic compounds by molybdotungstate reagent As followed from (Table 3) shown below, for phenolic content determination in the essential oils Cymbopogon citratus and Thymus schimperi, Gallic acid as a standard solution ranging from 20 to 100 ppm were used to construct a calibration curve for the determination of the total phenolic content of each sample. The equation of the graph

28

obtained from the calibration curve of Gallic acid was Y=0.0172x-0.2495 with linear regression coefficient (R) and R=0.9906 (Figure 12).

Table 3: Absorbance of the standard Gallic acid (at 765 nm)

Conc. of Gallic acid in ppm Absorbance at 765 nm

20 0.1540

40 0.3995

60 0.7129

80 1.1265

100 1.5059

Figure 12: Calibration curve of Gallic acid for TPC

The total phenolic content of the leaf extracts was reported in terms of milligram Gallic acid equivalent (mgGAE). As shown in (Table 4) the absorbance of each extracted EOs sample found in the range of Gallic acid standard solution. Quantitatively the total phenolic content of each plant extract in (mgGAE) calculated using equation (4).

As observed in (Table 6). the highest concentration of total phenolic content of leaf extract Cymbopogon citratus and Thymus schimperi essential oils were observed (40.6075 and 27.811) mgGAE/g EOs extract respectively. Therefore, this result show that the EOs Of Cymbopogon citratus had higher total phenolic content than Thymus schimperi EOs.

29

Table 4: Total phenolic content of leaf extract of C. citratus and T. schimperi essential oil in mgGAE/ g sample

Plant extract Absorbance at 765 nm mgGAE/g essential oil

C. Citratus essential oil 1.1474 40.6075

T. Schemperi essential oil 0.7072 27.811

Figure 13: Total phenolic content of leaf extract C. citratus and T. schimperi essential oil in mgGAE/g extract

4.4. Antioxidant assay

4.4. 1. DPPH Scavenging Assay

The antioxidant scavenging capacity of the plant extract can be measured by DPPH radical scavenging assay. The working principle of DPPH is based on the reduction of DPPH in alcoholic solution in the presence of a hydrogen-donating antioxidant due to the formation of the non-radical form DPPH-H in the reaction. Freshly prepared DPPH solution exhibits a deep purple color with an absorption maximum at 517 nm using methanol solvent. This purple color generally fades when antioxidant molecules quench DPPH free radicals (i.e. by providing hydrogen atoms or by electron donation, via a free-radical attack on the DPPH molecule) and convert them into a colorless /yellow product (Scheme 8) resulting in a decrease in absorbance at 517 nm band ^{[73, 74]}.

30

Free radical form (purple color) non-radical form (pale yellow) Scheme 8: Reaction of DPPH with an antioxidant

4.4.1.1. Calibration Curve for Ascorbic Acid

For the DPPH radical scavenging assay different concentrations of Ascorbic acid (25 ppm, 50 ppm, 75 ppm, 100 ppm and 125 ppm) were used to construct the calibration curve. The Calibration curve was constructed as a function of Absorbance versus concentration of Ascorbic acid. The absorbance at 517 nm and the percentage inhibition of Ascorbic acid was given below in (table 5).

Table 5: Absorbance and % inhibition value of Ascorbic acid (Absorbance at 517 nm) Concentration (ppm) Absorbance of AA at 517 nm % Inhibition of AA

25 0.17365 61.41968

50 0.13569 69.85337

75 0.10167 77.41169

100 0.06983 84.48567

125 0.03507 92.2084

The equation obtained from the calibration curve of ascorbic acid (Figure 14) for DPPH radical scavenging assay was y = -0.0014x + 0.2061 with linear regression coefficient (R²) 0.999

31

Figure 14: Calibration curve of Ascorbic acid for DPPH scavenging assay at 517 nm For the antioxidant activity study, different concentrations of Cymbopogon citratus and Thymus schimperi essential oils (25 ppm, 50 ppm, 75 ppm, 100 ppm and 125 ppm) were screened with DPPH, a stable free radical, and shows strong radical scavenging activities.

The activities of the test sample in DPPH scavenging assay can be expressed as a decrease in absorbance . (Table 6 and. Figure 15) showed that decreased in absorbance of the extract sample as the concentration of the each extract increased from 25 ppm to125 ppm respectively.

The change in absorbance of DPPH radicals caused by antioxidants is due to the reaction between the antioxidant molecules of the EOs of plant extract and the free radicals. As the absorbance decreases, the free radical scavenging activity becomes high. The scavenging activities of the EOs of Cymbopogon citratus and Thymus schimperi percentage inhibition increases with increasing EOs of plant concentration.

The result (Table 6), showed that the percentage inhibition of DPPH free radicals of Cymbopogon citratus and Thymus schimperi have the value ranges from (28.23%-42.9%

and 26.32%-38.86%) respectively when the concentrations of plant extract increases from 25 ppm to 125 ppm.

The previously published data reported as radical scavenging activity of 36.71% and 64.29 % for T.schemperi vulgaris EO at concentrations of 5 and 10 mg/ml, respectively

[75]

. The previously reported as radical scavenging activity of 55.5% for Cymbopogon y = -0.0014x + 0.2061

32

citratus EO at concentrations of 0.44 mg/ml ^[76].The antioxidant and antibacterial activity of EOs are associated with their phenolics content ^[77] .

The result obtained in the present study showed that Cymbopogon citratus extract have higher free radical scavenging activity than Thymus schimperi EOs extract. This antioxidant activity was associated with TPC. Lower absorption of the reaction mixture indicated higher free radical scavenging activity. The differences scavenging activity of previous report and this study may be factors such as geographic location, environmental and climate conditions, season, soil type, and the method of drying and extraction of the oil. Percentage (%) of inhibition of Cymbopogon citratus and Thymus schimperi essential oils and ascorbic acid at each concentration can be calculated by using the equation (1).

Table 6: Absorbance of DPPH scavenging activities of essential oils of C. citratus and T.schimperi at 517 nm

Concentration of essential oil In ppm

Absorbance at 517 nm % inhibition of essential oi

C. citratus T. schimperi C. citratus T.schimperi

25 0.562 0.582 28.23 26.32

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Figure 15: Graph of DPPH scavenging activity plants extract EOs and standards

4.4.2 Reducing power assay (FRAP assay)

In this study the antioxidant activity of Cymbopogon citratus and Thymus schimperi was determined using reducing power assay. The assay works based on the reduction of the yellow color of ferric complex (Fe(CN^-)6)^-3 to a Prussian blue ferrous complex (Fe (CN -)6)^-4 by the action of electron donating antioxidants. The Ferric reducing antioxidant method works based on the reduction of Fe³⁺ to Fe²⁺ due to the action of antioxidants present (Scheme 9) ^{[68, 78]}.

Scheme 9: Reduction of Iron (III) to Iron (II) using extracts

For this study C. citratus and T. schimperi EOs extracts and potassium hexacyanoferrate (K3[Fe(CN)6]) in the presence of phosphate buffer solution forms light yellow color after incubation for 20 minutes at 50^oC. This light yellow color turns to Prussian blue color after addition of 0.5 mL of 0.1% Ferric chloride (FeCl3) solution, which is an indication for the reduction of ferric complex to ferrous form. This may probably due to the presence of antioxidant compound in the extract and confirmed by measuring the absorbance of the Prussian blue color formed at 700 nm using UV-Vis spectrophotometer

0

34

[78]. The intensity of Prussian blue color formed for different concentration of EOs leaf extracts of C. citratus and T. schimperi were different. Color intensity depends on the type of plant and concentration of the plant used. For the different intensity of color, different absorbance was recorded and leads to different reducing power C. citratus EOs has strong reducing power than T. schimperi EOs extracts.

For quantification of reducing power of Cymbopogon citratus and Thymus schimperi essential oils, Ascorbic acid as a standard in different concentrations (50 ppm, 100 ppm, 150 ppm and 200 ppm) were used to construct a calibration curve. The calibration curve was plotted as absorbance (UV responses) at 700 nm versus the

For quantification of reducing power of Cymbopogon citratus and Thymus schimperi essential oils, Ascorbic acid as a standard in different concentrations (50 ppm, 100 ppm, 150 ppm and 200 ppm) were used to construct a calibration curve. The calibration curve was plotted as absorbance (UV responses) at 700 nm versus the

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