SUPPLEMENTARY MATERIAL

Phytochemical analysis, Antimicrobial, Antioxidant and Urease inhibitory Potential of Cyphostemma digitatum Lam.

Rasool Khan*, Abdullah Qasem Saif, Mohammad Mansour Quradha, Jawad Ali and Abdur Rauf

Institute of Chemical Sciences, University of Peshawar, Peshawar-25120, KPK, Pakistan

In this paper we report the antimicrobial, antiradical and urease inhibitory potential along with photochemical investigation of the crude extracts of Cyphoctemma digitatum Lam. Phytochemical screening ofboth the crude (Hot/ Cold) alcoholic and aqueous extracts of C. digitatum showed the presence of alkaloids, flavonoids, saponins, coumarins , steroids, terpenoids and tannins. The crude methanolic extract (Hot/Cold) exhibited good antioxidant activity, while the aqueous extract was a weak antioxidant. The crude methanolic extract was found more active against Bacillus subtilis,while both the extracts showed moderate antifungal potential, the methanolic crude extract showed good activity urease inhibitory than aqueous crude extract.

Key WordsCyphostemmadigitatum . Phytochemical screening . Antimicrobial . Antioxidant activity . Urease inhibition.

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*Corresponding author. Email:

3. Experimental

3.1. Material and Methods

Plant Material

Cyphostemma digitatum were collected from Sharab Al Salam, Taiz city (Yemen) in the month of July 2013. The plant was identified by Prof. Dr Abdur Rashid,Department of Botany University of Peshawar, Peshawar, Pakistan. A voucher specimen No 20053(pup) was deposited in the herbarium of the same department.

3.2. Extraction

Finely grinded leaves (4 kg) of C. digitatum were extracted with hot methanol using Soxhlet extractor. After complete extraction, excess of methanol was evaporated under reduced pressure in rotary evaporator at500Cand 600g extract was obtained. The shade dried and grinded leaves of C. digitatum (2 kg) were also subjected to cold extraction with methanol for six days (X3). The solvent extract was concentrated under reduced pressure in rotary evaporator at45 0C and 266 g dark extract was obtained.The crushed plant material (2 kg) was also assessed to distal water to obtained aqueous crude extract a dark syrup 244g according to reported methods (Chatterjee et al. 2004). All extracts were separately analyzed for their phytochemical constituents and biological potential.

3.3. Chemicals and reagents used

Dragendroff’s reagent, Fehling solution A and B, acetic anhydride, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical, FeCl3.

4. Pharmacological tests

4.4.1 Phytochemical screening

The methanolic and aqueous crudeextracts was evaluated for phytochemicalscreening to identify the secondary metabolitesby using standard qualitative methods (Rashid et al.2013; Yadav et al.2011; Uddin& Rauf2012a)

4.4.2Antibacterial activity

Antibacterial potential was evaluated by agar well diffusion method using Muller-Hinton agar (MHA) as medium according to standard methods (Uddin & Rauf2012b; Jigna et al. 2007). In typical experimental procedure, the cultures were equipped in triplicates at 37°C for 24 to 72 hours. 0.6 ml of the broth culture of the tested organism was put in a sterile Petri-dish and added 20 ml of the sterile molten MHA. Wells were bored into the medium using 0.2 ml of each extract while Streptomycin (2 mg/ml) was used as a standard drug. Inoculation was done for an hour to ensure the diffusion of the antimicrobial agent into the medium. The inoculation plates were incubated for 24 hour at 37°C and the diameters of the zone of inhibition of microbial growth were measured in millimeters. (Mahesh et al. 2008). The bacterial strains which are used are Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Klebsiella pneumonia.

4.4.3. Antifungal assays

Antifungal influence of crude methanolic and aqueous extracts of leaves of C. digitatum against two fungal strains bloodline were appointed by the tube dilution procedure according to reported method (Rauf et al. 2012). 22mg of each methanolic and aqueous crudes extracts were dissolved in 1ml sterile DMSO which serve as a stock solution. Sterile Sabouraud’s dextrose agar medium (5ml) was placed in a test tube and inoculated with the samplesolution (400 µg /ml) kept in slanting position at room temperature overnight. The fungal culture was then inoculated on the slant. The samples were incubated for 7 days at 29 °C and growth inhibition was observed(Duraipandiyan et al. 2011).

4.5. Antioxidant activity

Antioxidant activity of crud extracts of (methanol and aqueous) leaves of C. digitatum were performed according to standard protocol (Brand et al. 1995)using 2,2-Diphenyl-1-picrylhydrazyl (DPPH) was determined by UV spectrophotometry at 517 nm .Briefly, 25 mg of each crudes extracts were dissolved in 50 ml analytical grade methanol to prepared stock solution. Different concentrations i.e. 10 µg/ml, 20 µg/ml, 40 µg/ml, 60 µg/ml, 80 µg/ml, 100 µg/ml are prepared by dilution formula. 9.5 gm of DPPH was dissolved in 25ml of methanol to prepare 1 mM solution of DPPH. 1ml of this solution was mixed with 4ml of sample solutions in methanol (containing 10 -100 µg/ml ) and control (without sample). Kept each sample in dark for 30 min; the absorbance was measured at 517nm. Decreasing of the DPPH solution absorbance refers an increase of the DPPH radical-scavenging activity. Scavenging of free radicals by DPPH as percent radical scavenging activities (%RSA) was calculated as follows:

DPPH = (A0)Control abs - ( A1) Extract abs *100

Control

Where, A0 control is the absorbance of the blank sample and A1 sample is the absorption of sample standard sample. (Waliullah at al.2012). Data were presented as mean and standard error of means. The statistical analysis was performed using Prism Graphed.

4.6. Urease inhibition assay

Reaction mixtures containing 25 μl of enzyme (jack bean urease) solution and 55 μl of buffers containing 100 mM urea were incubated with 5 μl of test extract (0.5 mM concentration) at 30 0C for 15 min in 96-well plates. Urease activity was determined by measuring ammonia production using the indophenol method as described by weather burn. Briefly, 45 μl each phenol reagent (1% 2036 J. Med. Plants Res. w/v phenol and 0.005% w/v sodium nitroprussside) and 70 μl of alkali reagent (0.5% w/v NaOH and 0.1% active chloride NaOCl) were added to each well. The increasing absorbance at 630 nm was measured after 50 min, using a microplate reader (Molecular Device, USA). All reactions were performed in triplicate in a final volume of 200 μl. The results (change in absorbance per min) were processed by using softMax Pro software (molecular Device, USA). The whole tests were performed at pH 6.8. Percentage inhibitions were calculated from the following formula

% inhibition = 100-(ODtestwell/ODcontrol) ×100.

Thiourea was used as the standard inhibitor of urease (Khan et al. 2013; Arfan et al.2010).

Table S1. Phytochemical analysis of crude extract and aqueous extract of Cyphostemma digitatum

Chemical consitutants

/

Methanol (Cold)

/

Methanol (Hot)

/

Aqueous

Alkaloids / + / + / +
Tannins / + / + / -
Anthraquinones / - / - / -
Glycosides / - / - / -
Reducing Sugars / - / - / -
Saponins / + / + / +
Flavonoids / + / + / +
Phlobatanins / - / - / -
Steroids / + / + / +
Terpenoids / + / + / +
Cardiac Glycosids / - / - / -
Coumarins / + / + / +
Emodins / - / - / -
Anthocyanins / - / - / -
Betacyanins / + / + / +
Carbohydrates / - / - / -
Monosaccharide / - / - / -
Free Reducing sugars / - / - / -
Combined reducing sugar / - / - / -
Shinoda’s test for flavonoids / + / + / +
Lead ethanoate test for flavonoids / + / + / +
Soluble starch / - / - / -
Phenolic compounds / + / + / +

Table S-2. Antioxidant activity of crude extract and aqueous extract of Cyphostemma digitatum

Concentration / % DPPH MeOH(Hot) / % DPPH MeOH
( Cold) / % DPPH of Aqueous / Ascorbic acid
10 µg/ml / 20.53±1.12 / 19.3±1.01 / 18.62±1.21 / 91.34±1.21
20 µg/ml / 24.87±1.23 / 21.97±1.87 / 19.47±1.53 / 91.39±1.28
40 µg/ml / 37.19±1.95 / 27.49±1.00 / 20.00±1.94 / 92.59±1.90
60 µg/ml / 41.062±1.64 / 29.04±1.22 / 21.24±1.84 / 94.47±1.92
80 µg/ml / 46.85±1.22 / 32.85±1.52 / 21.57±1.01 / 95.23±1.92
100 µg/ml / 53.38.±1.98 / 33.35±1.91 / 24.39±1.00 / 96.36±1.79

Table S-3. Crude mixture methanolic and aqueous extracts of leaves extracts of C. digitatum.

Concentration / % DPPH
Synergistic
M80/ W20 / Synergistic
M 60/W40 / Synergistic
M 40/ W60 / Synergistic
M 20/W80
10µg/ml / 26.28±1.13 / 12.29±1.10 / 22.23±1.17 / 15.95±1.18
20 µg/ml / 27.17±1.18 / 13.90±1.19 / 22.67±1.17 / 19.38±1.19
40 µg/ml / 31.06±1.19 / 23.17±1.18 / 29.90±1.19 / 24.69±1.19
60 µg/ml / 36.74±1.16 / 25.66±1.17 / 31.21±1.14 / 26.24±1.15
80 µg/ml / 39.19±1.16 / 35.66±1.16 / 34.06±1.14 / 26.75±1.15
100µg/ml / 53.34±1.14 / 40.99±1.11 / 39.75±1.13 / 30.18±1.12

Table S-4.Antibacterial activities crude extract and aqueous extract of C. digitatumin mm

Bacterial strain / MeOH(Hot) / MeOH (Cold) / Aqueous / Streptomycin
Staphylococcus aureus (S. a) / 12 / - / - / 28
Bacillus subtilis (B. s) / 16 / 10 / - / 28
Escherichia coli (E. c) / 14 / 12 / - / 28
Klebsiella pneumonia (K.P) / 14 / 10 / 12 / 26

Table S- 5.Antifungal activities crude extract and aqueous extract of Cyphostemma digitatum

Name of Fungus / % Zone of inhibition (mm)
MeOH(Hot) / MeOH (Cold) / Aqueous / -ve control / STD / MIC (µg/ml)
Aspergillius niger / 0 / 11 / 6 / 0 / Miconazole
Aspergillus flavus / 11 / 0 / 22 / 0 / Miconazole / 110.8
Penicillium notatum / 0 / 0 / 0 / 0 / Amphotericin B / 97.20
fusarium oxysporum / 28 / 26 / 0 / 0 / Miconazole / 98.4
Trichoderma harzianum / 0 / 0 / 0 / 0 / Miconazole / 84.25

Table S- 6. Enzymes inhibitory activities crude extract and aqueous extractof Cyphostemma digitatum

Extract / Urease
% Inhibition (0.2 mg/mL) / IC 50± S.E.M.
(µg/mL)
Crude methanolic extract / 60.9 / 145.4±3.49
Aqueous extract / 41.9 / -
Thiourea / 98.2 / 21 ±0.11

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