The aqueous extract was prepared according to the method described by Thoo et al., with some modifications. Dried M. citrifolia fruit powder of 100 g were soaked in 1000 mL of distilled water and was shake vigorously using shaker for 15 minutes, at room temperature. The mixture was then sonicated for one hour and left refrigerated for 3 days. The mixture was filtered and kept at -80 °C. Then it was subjected for freeze dry to obtain its powder form of the extract. Saline dilution of the extract at several different concentrations prepared and stored as extract stock solutions. These stock solutions were kept below 4 °C until used.
3.2.3 Preparation of ethanolic extract
The ethanolic extract was prepared according to the method described by Boonchai et al., (2006) with slight modification. M. citrifolia dried fruit powder of 120 g has been extracted by maceration process. 4000 mL 99.8% of undenatured ethanol was used as a solvent. The mixture were left at room temperature where the ethanol was exchanged daily for three consecutive days. The combined filtrates were evaporated under vacuum reduced pressure at 40 °C using a rotary evaporator. A thick syrupy mass of crude extract was obtained using this procedure. The crude extract then was diluted in saline to prepare extract stock solutions at different concentrations. These stock solutions were kept this way at 4 °C.
Aqueous extract
Ethanolic extract
Preparation of M. citrifolia crude extracts
Dried M. citrifolia fruit powder of 120 g were soaked in 4000 mL 99.8% of undenatured ethanol at room temperature
Ethanol was exchanged daily for three consecutive days
The combined filtrates were evaporated under vacuum reduced pressure at 40 °C using a rotary evaporator
Thick syrupy mass crude extract was obtained
The crude extract was diluted in saline to prepare extract stock solutions at different concentrations
Dried M. citrifolia fruit powder of 100 g were soaked in 1000 mL of distilled water at room temperature
Mixture was shaked vigorously using a shaker for 15 minutes at room temperature
Mixture was sonicated for 1 hour and refrigerated for 3 days
Mixture was filtered and subjected for freeze dry to obtain its powder form of the crude extract
Saline dilution of the extract at different concentrations were prepared and stored as extract stock solutions
Figure 3.1. Aqueous extract and ethanolic extract preparations
3.3 Determination of antioxidant properties
3.3.1 Chemicals
Chemical
Manufacturer
Butylated hydroxytoluene (BHT)
Sigma Chemical Co. (USA)
β-carotene
Sigma Chemical Co. (USA)
Tween 20
Sigma Chemical Co. (USA)
Linoleic acid
Sigma Chemical Co. (USA)
Gallic acid
Sigma Chemical Co. (USA)
2,2-diphenyl-2-picrylhydrazyl (DPPH)
Sigma Chemical Co. (USA)
Folin-Ciocalteu reagent
Merck (Darmstadt, Germany)
Sodium bicarbonate
Merck (Darmstadt, Germany)
Chloroform
Fisher Scientific (UK)
Ascorbic acid
Fluka, (Switzerland)
3.3.2 Equipments
Equipments
Manufacturer
Spectrophotometer
Shimadzu (Japan)
Electronic scale
Shimadzu (Japan)
Vortex
Barnstead Thermolyne
3.3.3 DPPH free radical scavenging assay
The DPPH assay was done according to the method proposed by Brand-Williams et al. (1995) with some modifications. 200 µl of sample extract (0.32 - 20.48 mg/mL in 80% (v/v) ethanol) were prepared. 1 mL of 0.1 mM DPPH which prevoiusly prepared in 80% (v/v) ethanol then was added. The mixture was then shaked vigorously and left to stand for 30 minutes at room temperature in a dark room. The absorbance was taken by using a UV spectrophotometer at 517 nm with 80% (v/v) ethanol was used as blank. The scavenging effect on the DPPH radical was calculated using the following equation:
Scavenging effect (%) = 1 - Absorbance of sample at 517nm x 100
Absorbance of control at 517 nm
For this method, their scavenging effect was calculated based on the percentage of DPPH scavenged. Ascorbic was used as a standard by dissolving L-ascorbic acid in 80% (v/v) ethanol solution at final concentration of 1 mg/mL.
3.3.4 β-carotene bleaching assay
The β-carotene-linoleate bleaching method was done according to Velioglu et al. (1998) with slight modification. Butylated hydroxytoluene (BHT) was used as a standard. First, β-carotene solution (0.2 mg/mL chloroform) was prepared. 1ml of this solution as well as 0.02 mL linoleic acid and 0.2 mL Tween 20 were mixed in a beaker. The chloroform was removed at room temperature under vacuum at reduced pressure using a rotary evaporator. Following evaporation, 100 mL of distilled water were added to the mixture, and then it was shaked vigorously until forming an emulsion. 5mL aliquots of this emulsion were pipetted into the test tubes contained 0.2ml of sample (or 0.2 mL of standard BHT or 0.2 mL 80% (v/v) ethanol as control) and immediately placed in a water bath at 45°C. Spectrophotometer at wave length 470 nm was used to record the absorbance of this mixture. The reading was recorded every 15 minutes for 2 hour. The emulsion without β-carotene was used as blank. The antioxidant activity (AA) was calculated according to the following equation:
AA = [1 - ( Ao - At ) / ( Aoo - Aot ) ] - 100
where Ao and A°o are the absorbance values measured at initial time of the incubation for samples and control respectively, while At and Aot are the absorbance values measured in the samples or standards and control at t = 120 min.
3.3.5 Total phenolic content
Total phenolic content (TPC) of the extracts were measured using Folin-Ciocalteu method described by Amin et al., (2004). The extract was prepared at concentration of 25 mg/mL. 100 μL of sample extract was transferred into a test tube and 0.75 mL of Folin-Ciocalteu reagent (previously diluted 10-fold with deionised water) was added and mixed well. The mixture was allowed to stand at room temperature for 5 min. Then, 0.75 mL of 6% (w/v) sodium bicarbonate solution was added to the mixture and mixed gently. The mixture was left undisturbed at room temperature for another 90 min. The absorbance this mixture was read at 725 nm using UV spectrophotometer. Results are expressed as milligrams of gallic acid equivalent (GAE) per 100 g of sample extracts. A gallic acid calibration curve therefore were prepared in range of 0.025 and 0.15 mg/mL where 80% (v/v) ethanol was used as blank.
Antioxidant properties
DPPH scavenging assay
Total phenolic content
β-carotene bleaching assay
200 µL sample extract + 1 mL DPPH in test tube
Mixture stands for 30 min at room temperature in dark room
Absorbance taken at 517 nm using spectrophotometer
100 µL sample extract + 0.75 mL Folin-Ciocalteu reagent in test tube
Mixture left stand for 5 min at room temperature
0.75 mL sodium bicarbonate solution was added
Mixture stands at room temperature for 90 min
Absorbance taken at 725nm using spectrophotometer
β-carotene solution (0.2 mg/mL chloroform) + 0.02 mL linoleic acid + 0.2 Tween 20 in beaker
Chloroform was removed using rotary evaporator
100 mL d.H2O was added into the mixture to form emulsion
5 mL of emulsion was pipetted into test tubes contained 0.2 mL sample, standard BHT and ethanol (as control); immediately placed it in water bath at 45°C
Absorbance taken at initial time 0 and every 15 min intervals for 2 hours at 470nm using spectrophotometer
Figure 3.2. Antioxidant properties assays
3.4 Evaluation of anticoagulant properties in vitro
This study was conducted in UPM after obtaining UPM ethical approval (UPM/FPSK/PADS/T7-MJKEtikaPer/F01(LECT_FEB(08)01). Fifty healthy volunteers who were not on any medication including herbal or supplement were recruited. Respondents who are on medication or had history of taking any medication or supplement were excluded. Thirty volunteers (14 males, 16 females) were recruited for the ethanol based extract. Twenty healthy respondents (10 males, 10 females) were involved in analyzing the coagulant assay of aqueous based extract. 9 mL of venous blood was collected in 1 mL 3.8% trisodium citrate solution (9:1, v/v) from all respondents.
3.4.1 Sample preparation
The sample preparation was prepared according to the procedure describe by Laffan and Manning (2010). The blood sample was centrifuged at 1500 g for 10 minutes to obtain the platelet-poor-plasma (PPP). The PPP was subjected for baseline coagulation assays, PT and APTT. The remaining PPP was mixed and incubated with different extract concentrations of 10, 20, 30, 40, and 50 mg/mL (1:1, v/v) for 7 minutes at 37 °C before subjected for PT and APTT measurements. All blood samples were tested within 3 hours of blood collection.
3.5 Evaluation of antiplatelet and anticoagulant properties in vivo
Forty male Sprague-Dawley (SD) rats weighing 250-300g were obtained for this study. These rats were housed in cages under controlled conditions of temperature 22 ± 2°C, humidity (50-60%) and light (lights on from 0700 to 1900 h). They were given a regular rat chow and water ad libitum. All animals received humane care as outlined in Guide for the Care and Use of Laboratory Animals and by the UPM Animal Care Uses Committee (ACUC). The in vivo study was only conducted after obtaining the ethical approve from UPM ACUC (UPM/FPSK/PADS/UUH/F05).
3.5.1 Experimental design
Six groups of eight rats were used in this study. In order to avoid distress, all rats were going under acclimatization period of at least 1 week. The rats were treated once with M. citrifolia extract (0, 7.5, 75, 750 mg/kg bodyweight), aspirin (30 mg/kg bodyweight) and warfarin (0.05 mg/kg bodyweight). Aspirin and warfarin were used as a positive control for platelet aggregation and coagulation test respectively. The M. citrifolia extract, aspirin and warfarin were dissolved in normal saline (vehicle) and administered by gavage in a volume of 2 mL/kg. 2 hours after treatment, the rats were anaesthetized with inhalation of diethyl ether. 9 mL of blood were collected in 1 mL 3.8% trisodium citrate solution (9:1, v/v), by cardiac puncture method. The blood sample was used to measure PT, APTT, and platelet aggregation. 6 mL of the blood sample was centrifuged at 1500 g for 10 minutes to obtain PPP. The PPP was then subjected to PT and APTT assays. Remaining blood sample was used for platelet aggregation measurement using whole-blood aggregometry. Bleeding time (BT) was performed at the end of this study.
3.6 Coagulation test
3.6.1 Coagulation assay protocol
The PT and APTT measurements were performed by using methods described by (Brown 1988). The coagulation times were analyzed using a coagulometer (Sysmex CA-50). This instrument is semi-automated where the measured parameter was detected using optical method. The reagents used for these tests were Thromborel S, Actin FS, and CaCl2 (Dade 2005). All the reagents were incubated at 37°C before used.
3.6.1.1 Assay for prothrombin time (PT)
100 µL of samples were added to the reaction channel which contained a reaction vial and it was incubated for 180 seconds. 200 µL of Thromborel S reagent was added to the samples. The experiments were carried out in duplicates (Dade 2005).
3.6.1.2 Assay for activated partial thromboplastin time (APTT)
100 µL of samples were added to the reaction channel containing a reaction vial. After 180 seconds incubation period, 100 µL of Actin FS reagent was added. This mixture was re-incubated for another 180 seconds. 100 µL of CaCl2 reagent was then added to activate the intrinsic clotting cascade and the time (in second(s)) from this addition to clot formation was defined as the APTT. The experiments were carried out in at least, duplicates (Dade 2005).
3.6.2 Bleeding time measurement
Bleeding time was measured by cutting the tail-tip. The tail bleeding model was based on previous methods with slight modifications (Beviglia et al., 1993; White et al., 2001). In brief, the tail of each anesthetized rat was cut 2 mm from the end using a sharp pair of surgical scissors and then was immersed immediately into the cylinder with 100 mL isotonic saline at 37°C. Bleeding time was measured from the moment the tail was surgically cut until bleeding completely stopped. If the bleeding was not ceased by 20 min, the experiment was ended and pressure was applied to stop the bleeding.
3.7 Platelet aggregation with whole-blood aggregometry
Whole-blood platelet aggregation was performed according to Cardinal and Flower (1980). 500 µL citrated bloods were mixed with 500 µL of physiological saline (Ingerman-Wojenski and Silver, 1984; Mackie et al., 1984). The diluted blood was poured into siliconized plastic cuvette and then placed in a Whole Blood Lumi-Aggregometer (Model 550, Chrono-Log, Harvertown, PA, U.S.A.). the platelets were agitated by stirring for 3 minutes at 1000 rpm at 37 °C and equilibrated under these conditions. Agonists were added to samples, and the changes in impedance were subsequently recorded. The aggregation was expressed as ohm (Ω).
3.8 Statistical analysis
Statistical significance between groups was assessed using independent T-test and analysis of variance (ANOVA) coupled with post hoc Tukey HSD for multiple group comparison. The level of significance was set at p <0.05. Results were expressed as mean ± standard deviation.
