Recently there was immense addition in utilizing of ‘herbal merchandises ‘ . These can be defined as workss, parts of workss or infusions from workss that are used for bring arounding disease. However, Calophyllum species is a tropical works and it has been used in traditional medical specialty, the restriction in safety and effectiveness information could take to serious wellness jobs.
Aim of the survey: supply information for communities by measuring the phytochemical contents, antioxidant, antimicrobic and cytotoxic activities of Calophyllum canum species. Equally good as better the curative values.
Materials and methods: Three chief fractions ( none – high polar ) were tested to happen out the phenoplast, flavonoid, flavonol content, DPPH extremist scavenging, cut downing power and chelating Fe ions. Besides were tested against Bacillus Cereus, Staphylococcus aureus, Escherichia coli, Psedomonas aeruginosa, Candida albicans, and Cryptococcus neoformans. In add-on, cytotoxic activity was assayed against lung malignant neoplastic disease cell line.
Consequences: the methyl alcohol fraction showed no bioactivity but achieved the highest sum of phenoplast, flavonol and flavonoid contents, besides it showed a important consequence as antioxidant, cut downing power and chelating agent. The Hexane fraction achieved the minimal repressive concentration ( MIC ) value 12.5 I?g/ml against B. Cereus while the MIC of DCM fraction was 25 I?g/ml. The DCM fraction was more active against S. aureus where the consequence was 50 I?g/ml while the Hexane fraction was 100 I?g/ml. the three chief fractions have shown no activity against gm negative bacterial and fungal. The Hexane and DCM fractions have shown cytotoxicity against lung malignant neoplastic disease cell line ; the 50 % suppression concentration ( IC50 ) was 22 A± 2.64 and 32 A± 3.78 I?g ml-1 severally. The consequences were statistically important ( P & lt ; 0.05 ) .
Decisions: Among the consequences, C. canum fractions proved to be effectual against gram positive bacterial and anti-proliferation activity. Besides it showed antioxidant activity every bit good. The consequences provided good information for communities every bit good as can assist to seek for alternate drugs, and will lend to set up safe and effectual usage of phytomedicines in the intervention of diseases.
Keyword: Calophyllum canum ; Phytochemistry ; Antioxidant ; Antimicrobial ; Antiproliferative ( cytotoxicity ) .
Introduction
The genus Calophyllum belongs to Guttiferae household which consists of 180 – 200 different species distributed in the warm humid Torrid Zones of the universe, besides it is available in Malaysia. Wide phytochemical surveies have reported that Calophyllum genus rich in xanthones, coumarins, biflavonoids, chalcones, coumarones and triterpenes. A few surveies have improved the bioactivity of Calophyllum genus. Chen and his co-author have showed that C.lanigerum Miq. and C.inophyllum L have strong activity against human immunodeficiency virus type 1 ( HIV-1 ) . Guilet and Li have reported that triterpenoids, coumarins and the Mammea coumarens which have isolated from C.inophyllum, C.dispar and C.brasiliens, have shown a cytotoxicity consequence against human leukaemia HL-60, Nasopharynx carcinoma KB and K562 Lymphoma, U251 cardinal nervous system and PC3 Prostata cell lines severally. On the other manus Reyes-Chilpa has reported that Calophyllum genus able to suppress the growing of Staphylococcus aureus, Staphylococcus epidermidis and Bacillus subtilis. In add-on, the infusion from C.brasiliens showed activity against barm. Some of Calophyllum species are normally employed in common people medical specialty in Torrid Zones country, nevertheless it is used to handle bronchitis, gastric, hepatic perturbations, hurting, redness, diabetes, high blood pressure, diarrea, herpes, rheumatism, varicose, haemorrhoids and chronic ulcer, besides handling odontalgia, forestalling lesion infections and lumbago. As mentioned Calophyllum species were used traditionally really frequently for handling many diseases. The Calophyllum canum genus provides no information about its activity and it may be used randomly, and non scientifically proven in footings of effectivity or toxicity. Inadequacy of old scientific surveies on the nature of C.canum prompted us to measure the biological activity in footings of phytochemical, antioxidant, antimicrobic, and antiproliferative ( cytotoxicity ) by utilizing in vitro theoretical account of relevancy.
Materials and Methods
Chemicals.
All dissolvers used were of analytical class, Hexane, Dichloromethane ( DCM ) and Methanol, besides silica gel 60 ( 230-400 mesh ) , sodium ethanoate, and Aluminium chloride was obtained from MERCK. Trichloroacetic acids, K hexacyano ferrate, K phosphate, ferric chloride, ferrous chloride, ascorbic acid, BHT ( Butylated hydroxytoluene ) were purchased from R & A ; M chemicals. 1,1-Diphenyl-2-picrylhydrazyl ( DPPH ) and 3- ( 2-pyridyl ) -5,6-diphenyl-1,2,4-triazine-p, p’-disulfonic acerb 1-Na ten H2O were purchased from Sigma-Aldrich Chemical. Gallic acid obtained from Alfa Aesar. The media were used in this survey for bacteriums, Mueller-Hinton broth medium ( MHB ) used in MIC method, and Mueller-Hinton agar used in disc diffusion method. And for fungus the murphy dextroglucose agar was used for disc diffusion method. Chloramphenicol, and Amphotericin B a standard antibiotic used to compare with for bacteriums and fungus severally.
2.2 Plant Material.
The root bark of Calophyllum canum was collected from Bukit Pelindung Kuantan, Malaysia in June, 2009, with a herbarium specimen ( No MT-01 ) . The coinage was identified by Dr Shamsul Khamis, phytologist from Universiti Putra Malaysia.
2.3 Extraction and Fractionation.
Air-dried and powdery steam bark ( 1kg ) was macerated in ( 2.5 L ) 98 % EtOH for 72 hours. The EtOH infusion was filtrated and evaporated under cut down force per unit area to give the dark brown gummy ( 200g ) EtOH petroleum. A portion of EtOH petroleum ( 100 g ) was chromatographed by VLC ( silica gel 230-400 mesh, 1:30 ratio ) to obtain three chief fractions Hexane, CH2Cl2 ( DCM ) and MeOH.
Phytochemical showing.
Determination the phenolic content.
Entire phenolic content was carried out sing to the Folin-Ciocalteau ‘s method with little alterations. In brief, 0.5 milliliter of sample ( 10 mg mL-1 ) was assorted with 5.0 milliliters of Folin-Ciocalteau ‘s reagent ( 1:10 ) . The mixture was settled in a tubing for 5 min, , and so 4.0 milliliters of 1M Na2CO3 was added. After incubation at room temperature for 15 min, a 100 I?L of the mixture was moved to 96-microwell plat. The optical density was measured at 765nm by utilizing multi-detection micro-plate reader ( INFINITE M 200 NANOQUANT ) . The sample was tested in triplicate and the consequences explained A± standard divergence. Calibration curve with five informations points for Galic acid was obtained. The consequences were compared to a Galic acid standardization curve and the entire phenolic content of fractions was expressed as I?g of Galic acid equivalents per 10 milligram of infusion.
( Y= 0.0507 X – 0.011 ) and R2 = 0.9937
Determination the entire flavonoid.
Entire flavonoid contents were determined utilizing the method of with little alteration. A volume of 0.1 milliliter of sample was placed in a 96-microwell home base and assorted with 0.1 milliliters ( 2 % ) AlCl3 ethanol solution. The mixture was incubated at room temperature for 1 hr, after the optical density was measured at 420 nanometers utilizing the multi-detection micro-plate reader ( INFINITE M 200 NANOQUANT ) . The xanthous coloring material indicated flavonoids being. The three chief fractions were evaluated at concluding concentration of 10 milligrams mL -1 in triplicate and the consequences explained A± standard diveation. The space prepared by blending the sample with ethyl alcohol. Entire flavonoid content was calculated as quercetin equivalent ( I?g Que /10mg infusion ) utilizing the following equation based on standardization curve:
Y= 0.0174 X – 0.048, R2 = 0.9976.
Determination the entire flavonol.
Entire flavonols were estimated by utilizing the method of with little alteration, in brief 50 I?L of sample or criterion was assorted with 50 I?L of ( 2 % ) AlCl3 ethanol solution and 75 I?L of ( 50 g L-1 ) Na acetate solution, the mixture was seated in a 96-microwell home base and it was incubated for 2.30 hr at room temperature. The absorbtion measured at 440 nanometers by utilizing the multi-detection micro-plate reader ( INFINITE M 200 MAMOQUANT ) . The chief fractions were evaluated at a concluding concentration of 10 mg mL-1, where the space was prepared by blending the sample with ethyl alcohol. Entire flavonoid content was calculated as quercetin equivalent ( I?g Que /10mg infusion ) utilizing the following equation based on the standardization curve:
Y = 0.0097 X + 0.055, R2 = 0.9994.
Antioxidant activity.
DPPH check.
The scavenging consequence of C.canum fractions was assessed by utilizing the method of with little alterations. A 100 I?l of methanolic solution incorporating between 0.031- 1 milligram of the fractions was assorted with methanolic solution of DPPH ( 1mM, 200 I?L ) with little alterations. A 100 I?L of methanolic solution incorporating between 0.031-1 milligram of the fractions was assorted with ( 200 I?L ) methanolic solution of DPPH ( 1 millimeter ) in a 96-microwell home base. The content was assorted and left in a dark country at room temperature for 20 min, and so the optical density of the mixture was measured at 517nm by utilizing multi-detection microplate reader ( INFINITE M 200 NANOQUANT ) . The control was prepared by blending methanol alternatively of the fractions. The space was 100 I?L of fraction with 200 I?L of methyl alcohol. Extremist scavenging activity was expressed as the suppression per centum and was calculated utilizing the equation 1.
% Extremist scavenging = ( ( Control OD – Sample OD ) /Control OD ) * 100 ( 1 )
Where Doctor of optometry: Optical Density.
The Required fraction ‘s Concentration ( I?g mL-1 ) for scavenging of 50 % of DPPH extremist ( RC50 ) was determined. All measurings were curried out in triplicate.
Reducing power check.
The cut downing power of C.canum fractions and two criterions ascorbic acid and BHT was determined harmonizing to the method of with little alteration. A 100 I?L of three different concentrations 1, 5 and 10 mg mL-1 of each fraction were assorted with 50 I?L of ( 0.2 M ) phosphate buffer ( pH 6.6 ) and 250 I?L of K ferricyanide ( 1 % ) . After the mixture was incubated at 50 A°C for 20 min, 250 I?L of ( 10 % ) trichloroacetic acid were added and the mixture was centrifuged at 1000g for 10 min. 250 I?L supernatant was moved to 48-microwells home base and assorted with 250 I?L distilled H2O and 50 I?L of ferrous chloride ( 0.1 % ) . The mixture was measured at 700 nanometers by utilizing the multi-detection micro-plate reader ( INFINITE M 200 NANOQUANT ) . All the measurings were carried out in triplicates. However, higher optical density of the reaction mixture indicates greater cut downing power.
Iron ( II ) chelating activity.
The chelation of Fe ( II ) ions by different fractions ‘ concentrations was curried out by the method described in. 100 I?L of four different concentrations 1, 2.5, 5 and 10 mg mL-1 of each fraction, which dissolved in methyl alcohol, was assorted with 10 I?L of ( 2.0 millimeter ) aqueous FeCl2. After 5 min incubation at room temperature, the reaction was initiated by 20 I?L of ( 5.0 millimeter ) ferrozine. After 10 min the optical density was measured at 562 nanometers by utilizing the multi-detection micro-plate reader ( INFINITE M 200 Nanoquant ) . The control was contained all the reaction reagents except the sample which was replaced by methyl alcohol. The Fe chelating activities were calculated from the equation 2.
% chelation = ( ( Ac-As ) /Ac ) *100 ( 2 )
( Ac ) represented the optical density for control and ( As ) the optical density for sample. The values were presented as the agencies of triplicate analyses A± standard divergence.
Anti microbic checks.
Microbial strains.
Six mention microbic strains of human pathogens were used for the anti-microbial activity. The two Gram-positive bacteriums ( Staphylococcus aureus ATCC 25923, Bacillus cereus ATCC 11778 ) , and two Gram-negative ( Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 35218 ) were used for antibacterial trial. Besides two fungous strains ( Candida albicans ATCC 10231 and Cryptococcus neoformans ATCC 90112 ) were used for fungicidal trial.
Disc Diffusion Method
The agar phonograph record diffusion method was employed for the finding of antimicrobic activities of the EtOH petroleum harmonizing to with some alterations. Briefly, inoculant incorporating 106- 107 CFU mL-1 was spread on Mueller -Hinton agar home bases for bacteriums and 104 CFU mL-1 was spread on murphy dextroglucose agar for fungus strains. The sterilized forceps was used to put unfertile ( 6 mm diameter ) filter documents on the top of agar. The filter documents loaded with petroleum infusion ( 10 or 20 I?g ) , standard antibiotics ( 30 I?g of Chloromycetin or 100 I?g of amphotericin B ) or negative control ( DMSO ) were laid down on the surface of inoculated agar home base. The home bases were incubated at 37A°C for 24 hr for bacterium and at room temperature ( 18-20A°C ) for 24-48 for barm strains. Each sample was tested in triplicate and the zone of suppression was measured as millimeter diameter.
Microdilution method ( MIC and MBC ) .
Minimal inhibitory concentration ( MIC ) was measured by finding the smallest sum of fractions ( Hexane, DCM or MeOH ) or standard antibiotic needed to suppress the growing of a trial micro-organism. This was done utilizing 96-microwell home bases and performed on ( Versa MaxTM Tunable ) micro-plate reader. In brief, the home bases were filled with Mueller-Hinton stock medium incorporating different concentrations of the Hexane, DCM or MeOH fractions. Chloramphenicol or solvent control and the trial microorganism ( 106 -107 CFU mL-1 ) besides were added. After 24 hr incubation periods at 37A°C, the turbidness was measured at 600nm.
Minimal bactericidal concentration ( MBC ) was determined by reassigning and distributing the treated civilization stock of the Wellss incorporating the concentrations equal to and higher than the MIC on agar home bases. The lowest concentration of the fractions or the standard antibiotic required to wholly destruct trial micro-organism ( no growing on the agar home base ) after incubation at 37A°C for 24 hr was reported as Minimum disinfectant concentration ( MBC ) .
2.6.4 The cytotoxicity assay ( MTT assay ) .
Anti-proliferative effects of C.canum fractions against A549 cell line was investigated by utilizing [ 3- ( 4,5-dimethylthiazol-2-yl ) -2,5-diphenyltetrazolium bromide ( MTT assay ) ] described by. For this intent, A549 cells were cultured in a completed ( DMEM ) media in a T- flask until the cells were merging. Then, the cells were seeded in a 96-microwell home base at a denseness of 0.5 ten 105 cells/well and incubated at 37A°C in 5 % CO2 humidified brooder. After 24 hr a fresh media was added and the cells were treated with different concentrations of samples obtained by dual fold consecutive dilution. The ( 95 % ethyl alcohol ) was used as control. After 24 hours incubation, the supernatants were discarded, and the disciple cells were washed twice with phosphate buffer saline ( PBS ) . 20I?L of ( 5mg mL-1 ) MTT stock solution was added to each well and the home base was farther incubated overnight at 37A°C. A 100 I?L DMSO was added to fade out the H2O indissoluble purple formazan crystals produced by the feasible cells. When formazan completed fade out a 100 I?L was transferred to a new 96-microwell home base and the optical density was measured at 570nm and mention at 690nm, utilizing multi-detection microplate reader ( INFINITE M 200 NANOQUANT ) . All samples were assayed in triplicate. The per centum of cell viability was calculated and the concentrations required for suppression of 50 % of cell viability ( IC50 ) were determined harmonizing to equation 3.
% of cell viability = ( OD of treated cells / OD of control cells ) *100 aˆ¦aˆ¦aˆ¦aˆ¦ ( 3 )
Consequences.
3.1. Phytochemical content.
The phytochemical analysis was curried out in this survey, to look into the entire phenoplast, flavonoid and flavonol content in three chief fractions of C.canum. Table 1 shows the contents value for each fraction, it is clearly looking that MeOH fraction contains the highest sum of polar phytochemicals, where another two fractions Hexane and DCM have a smaller sum ; but DCM has high content of flavonoid.
Table 1
A
A
A
A
A
The fractions of Calophyllum canum used in testing the phytochemistry content.
Fractions
Phenolic content*
A
Flavonoid contenta-S
A
Flavonol contenta-S
Hexane
1.292 A± 0.001
5.040 A± 0.184
1.202 A± 0.547
DCM
1.992 A± 0.002
11.201 A± 2.446
2.164 A± 0.415
MeOH
3.517 A± 0.017
A
14.643 A± 1.222
A
11.639 A± 2.479
*I?g Galic Acid/10 milligram infusion
A
a-S I?g quercetin/10 milligram infusion
A
A
A
A
3.2. Antioxidant activity.
3.2.1 DPPH check
The consequence show in Table 2 shows the IC50 of free extremist scavenging activity, which was curried out by utilizing the DPPH check. The MeOH fraction shows a good consequence as an antioxidant, while Hexane and DCM fractions show a moderate activity. Fig.1 depicts the % of extremist scavenging activity versus concentrations. The hexane fraction curve turned to steady after 125I?g mL-1, while the DCM fraction curve turned to steady after 250I?g mL-1. the MeOH fraction became steady after 62.5I?g mL-1 and the IC50 is much less than 31.25I?g mL-1.
Table 2
A
A
IC 50 free extremist scavenging activity
Fractions
A
IC 50 ( I?g/ml )
Hexane
85 A± 4.35
DCM
91.6 A± 6.65
MeOH
A
& lt ; 31.25
Figure 1.free extremist scavenging activity of the fractions of C.canum ( Hexane, DCM, and MeOH ) by DPPH check.
3.2.2 Reducing power.
The cut downing power agents are concentrating in the MeOH fraction, where it is accomplishing a consequence closer to BHT the moderate criterion. Fig.2 depicts the cut downing power value of MeOH and other substance. The Hexane and DCM fractions show low cut downing power activity, where they are less than BHT criterion. The strongest cut downing power is ascorbic acid where there are no substances get closer to.
Figure 2 the cut downing power activity of three C.canum fractions ( Hexane, DCM, MeOH ) criterion BHT and Ascorbic acid at 700nm.
3.2.3. Iron ( II ) chelating activity
The chelation of ferric ions by the C.canum fractions and criterion was examined by the method of. Fig. 3 shows two standard Quercetin centrist and EDTA high chelating agent. All fractions were comparing with criterion ; the Hexane and DCM show activities less than Quercetin at all concentration, while the MeOH fraction shows dramatically activity, it chelates the Fe at 2.5-5 mg mL-1 as the Quercetin does, besides it chelats Fe at 10 mg mL-1 to make activity as EDTA. The Fe chelating activity consequence for MeOH fraction was straight proportion with concentration.
Figure 3. The % of Iron chelating activity for three C.canum fractions at four concentrations ( 0, 1, 2.5, 5, 10 mg/ml ) .
Antimicrobial check
The antibacterial and fungicidal activities of EtOH petroleum utilizing disc diffusion method are summarized in Table 3. The C.canum petroleum infusion was screened at concentrations of 10 and 20Aµg/disc against six human pathogens. Both gram negative and fungous strains appear to be immune to the tested concentration since no suppression zone was observed while the gm positive bacterium showed moderate sensitiveness with no important difference between the concentrations. Therefore the MIC and MBC were determined utilizing different mutual opposition fractions ( Hexane, DCM and methyl alcohol ) of C.canum against gm positive strains merely ( Table 4 ) . As a consequence, the MIC values of the tried fractions rangedA from 12.5 to 100Aµg mL-1 whereas the MBC valuesA were 100 or 250Aµg mL-1. The suppression growing of the bugs at concentration every bit low as 12.5Aµg mL-1, was indicated to possible antimicrobic activity of C.canum fractions. Furthermore, the highest activity was obtained against B.cereus for the Hexane fraction ( MICA andA MBCA was 12.5Aµg mL-1 and 100Aµg mL-1, severally ) .
Table 3: Antimicrobial activity of ethanol petroleum infusion utilizing disc diffusion method.
Bacterias
barms
S.a
B.c
P.a
E.c
C.a
C.n
EtOH Crude ( 10Aµg/disc )
11.5 A± 0.7
9.5A± 0.7
0A±0.0
0A±0.0
0A±0.0
0A±0.0
EtOH Crude ( 20Aµg/disc )
12.5A± 0.7
10.5 A±0.7
0A±0.0
0A±0.0
0A±0.0
0A±0.0
Chloramphenicol ( 30Aµg )
24.5 A± 0.7
26 A± 0.5
33A±.03
37 A± 0.4
Neodymium
Neodymium
Amphotericin B ( 100Aµg )
Neodymium
Neodymium
Neodymium
Neodymium
20A±0.7
19A±0.4
Negative control
0A±0.0
0A±0.0
0A±0.0
0A±0.0
0A±0.0
0A±0.0
Mean diameter of zone of suppression in mmA± standard divergence including the diameter of the phonograph record 6 millimeter. S.a: S.aureus. B.a: B.cereus. P.a: P.aeruginosa. E.c: E.coli. C.a: C.albicans. C.n: C.neoformans
Negative control: 100 % DMSO
0: no suppression zone
Neodymium: Not Determined
Table 4: MIC and MBC for Chloramphenicol, Hexane, DCM and MeOH fractions
Fraction
S. aureus ( I?g/ml )
B. Cereus ( I?g/ml )
MIC
MBC
MIC
MBC
Hexane
100
250
12.5
100
DCM
50
250
25
250
MeOH
& gt ; 1000
Neodymium
& gt ; 1000
Neodymium
Chloramphenicol
20
Neodymium
20
Neodymium
Neodymium: Not Determined
Cytotoxicity ( MTT assay )
The anticancer activity of the C.canum fractions was investigated utilizing MTT check on human lung malignant neoplastic disease cell line A549. A mitochondrial enzyme in populating cells can succinate dehydrogenase and cleaves the tetrazolium pealing change overing the MTT to an indissoluble purple formazan. Fig.4 shows the % of viability where it display the activity of Hexane, DCM and MeOH fractions. The Hexane fraction shows a good activity, where it is suppressing the cell growing at low concentration. The DCM fraction shows a good activity but it is less than Hexane. The MeOH fraction shows no activity where the growing is 100 % .
Figure 4. The % of viability activity for C.canum fractions against A549 cell line.
Table 5
A
A
The IC50 and % of viability of C.canum fractions
Fractions
IC50 I?g/ml
% of viabilitya-S
Hexane
22 A± 2.64
35
DCM
32 A± 3.78
51
MeOH
N.d
N.d
N.d: Not determined
a-S : % of viability at 30 I?g/ml
The standards of cytotoxicity activity for the petroleum infusion, as established by the American National Cancer Institute ( NCI ) is an IC50 & lt ; 30 I?g mL-1 in the preliminary assay.The IC50 values for Hexane and DCM fractions are 22 and 32 I?g/ml severally. The % of feasible cell at concentration 30 I?g mL-1 as recommended by NCI is 35 and 51 % .respectively for Hexane and DCM fractions. These indicate to the efficaciousness of Hexane at low concentration as it is exemplifying in table 5.
Discussion
Phenolic compounds are most abundant secondary metabolites in the workss. Additionally, it has been found in many nutrient and their derived functions. Flavonols and flavonoids are a portion of phenolic compounds and they are sharing in the physical belongingss. Phenolic compounds are known as powerful concatenation breakage antioxidants. Besides it is really of import components of workss and their extremist scavenging ability is due to their hydroxyl groups. The phenolic compounds my contribute straight to anti-oxidative action. In assorted surveies, antioxidant activity of the works extracts which are rich in phenolic compounds was found to be reasonably high. The chemical complexness of infusions frequently a mixture of tonss of compounds, with different functional groups could take to assorted values, depending on the trial employed. Therefore, an attack with multiple checks for measuring the antioxidant potency of infusions would be more enlightening and even necessary. There are several methods utilizing for finding of the antioxidant activity. In this survey, chiefly three methods were curried out, DPPH extremist scavenging activity, metal chelation activity and ferrous cut downing power. The concentrations of entire phenoplast, flavonol and flavonoids were besides calculated for the C.canum fractions. The DPPH consequences show that Hexane and DCM fractions were statistically non important ( Pa‰? 0.05 ) in the scope of concentration ( 31.25- 1000 I?g mL-1 ) except at concentration ( 125, 250 I?g mL-1 ) the consequence was statistically important ( P & lt ; 0.05 ) . In add-on, the MeOH fraction shows a important consequence even at low concentration. The other fractions were compared with MeOH fraction and the consequence were statistically highly important at ( P & lt ; 0.001 ) , except at concentration 62.5 I?g mL-1 for Hexane and 250 I?g mL-1 for DCM was merely important at ( P & lt ; 0.05 ) . The extremist scavenging activity increases with increasing the sum of the fractions.
Free groups have a important consequence on oxidization of unsaturated lipoids the DPPH extremist scavenging was used as a stable free group to find antioxidant activity of natural compounds. The method is based on the decrease of alcoholic DPPH solution in presence of a hydrogen-donation antioxidant due to the formation of the non-radical DPPH-H. The cut downing power check was curried out as a 2nd antioxidant trial, the xanthous coloring material which was observed of the tried solution, was turned to assorted sunglassess of green and bluish coloring material, which was indicated cut downing power of each infusion being. The copiousness of cut downing substances ( i.e. antioxidants ) in the fractions will take to decrease the Fe+3 ferricyanide composite to the ferric signifier. Therefore, the Fe+2 can be examined by mensurating the formation of Perls ‘ Prussian blue at 700 nanometer. In other words, the FeCl3/K3Fe ( CN ) 6 system offers a sensitive method for the “ semi-quantitative ” finding of dilution concentration of polyphenolics, which portion in redox reaction. The cut downing powers of Hexane and DCM fractions were showed a weak activity, they were statistically important ( P & lt ; 0.05 ) comparing with BHT and ascorbic acid. While the MeOH fraction was showed a moderate activity and statistically was non important ( Pa‰? 0.05 ) comparing with BHT, this consequence was predicted where usually methanol fraction contains the high polar constituents which usually contain ( -OH ) groups. The high phenoplasts or polyphenolics content come into position to map as good negatron and hydrogen-atom givers and therefore should be able to end extremist concatenation reaction by change overing free groups to more stable merchandises. The information for all fractions from this check, correlated good with the content of entire phenoplasts, and sing to the chemical axial rotation “ like dissolve like ” , these consequences are traveling logically with.
Among the passage metals, Fe is known as lipid oxidization pro-oxidant. The ferric province of Fe accelerates lipid oxidization by interrupting down H and lipid peroxides to reactive free groups via the Fenton Reaction.
Fe+2 + H2O2 a†’ Fe+3 + OH- + OHa-?
Ferric ion besides produces extremist from peroxides although, the rate is 10-fold less than ferric. The Hexane and DCM fractions have shown a weak chelating activity comparing with Quercetin. The MeOH fraction has shown activity similar to Quercetin at concentration a‰¤ 5mg mL-1and statistically was non important ( Pa‰?0.05 ) . Besides, it has shown a good activity like EDTA when the concentration was raised up to 10 milligrams mL-1 and statistically was non important ( Pa‰?0.05 ) comparing with EDTA. MeOH fraction was showed the highest metal chelating activity among all the fractions have studied, which has comparable consequences with EDTA.
Nowadays, many biological activities have been evaluated for legion species of workss. This demonstrates that compounds from medicative workss are so utile as alternate therapy, either straight or as theoretical accounts fro new man-made substances. However, the usage of these substances of works beginning is non ever monitored by wellness professionals, which would guarantee efficaciousness and safety processs, and can take to absence of biological effects or even to toxic effects. In this survey, no activity was observed against Gram-negative bacterial and barms. This is in understanding with many antimicrobic surveies, which studied the antimicrobic activity of some species belong to Calophyllum genus. reported that C.inophyllum extracts demonstrated promising antibacterial activity against Staphylococcus aureus and Mycobacterium smegmatis while the gm negative P.aeruginosa was immune to this infusions. Pretto and his co-authors ( 2004 ) showed that all the parts ( roots, stems, foliages, flowers and fruits ) of C.brasiliense exhibited antimicrobic activity against Gram-positive bacteriums, and no activity was observed against Gram-negative bacteriums and barms tested. On the other manus, this difference in susceptibleness between the bacteriums is related to the outer membrane of Gram-negative bacteriums. The outer membrane endows the bacterial surface with strong hydrophilicity and acts as a strong permeableness barrier.
The hunt for anticancer agents from natural beginnings has been successful worldwide ; active stuffs have been isolated and are presents used to handle human tumors. Pe.alliacea has been reported to be utilized in handling patients with malignant neoplastic disease and leukemia in Cuba where assuring consequences were obtained. The paclitaxel has been isolated from the Pacific yew works and it is used presents fro handling the lung and chest malignant neoplastic disease every bit good. The present survey was undertaken to measure the cytotoxic activity of Malayan Calophyllum canum which is ne’er investigated before. Besides it is used in the intervention of a few nutriments and malignant neoplastic disease related unwellnesss in the state. were stray Coumaren compounds from C.dispar species and tested the cytotoxicity against human Nasopharynx carcinoma KB cell lines and they find out that dispar species inhibit the cell growing at low concentration. Besides Zhi and his co-worker showed that C. inophyllum species from China inhibited the growing of human leukaemia HL-60 cells by stray triterpenoide. The information based on the traditional medicative usage of workss has been one of the common utile ways of the find of biological activity compounds from workss. In this survey, different fractions supposed to incorporate chiefly high concentration of secondary metabolise were used. The consequence of the present survey indicates the presence of cytotoxic activity in Hexane and DCM fractions whereas the MeOH fraction showed no activity. The statistical analysis was showed a important ( P & lt ; 0.05 ) in the scope of ( 3.06-12.5 I?g mL-1 ) between Hexane and DCM fractions where the consequence was non important ( Pa‰?0.05 ) at the scope ( 25-100 I?g mL-1 ) for the same fractions. The Hexane fraction was non important ( Pa‰?0.05 ) at the scope ( 3.06-12.5 I?g mL-1 ) , which it is average that the Hexane fraction has an anticancer belongingss and it might go a drug for anticancer with farther probe. Misdiagnosis of malignant neoplastic disease by traditional therapists might explicate the ascertained deficiency of correlativity between the reported anticancer activities of works infusions and their cytotoxic activity on the tested cell lines. The promising consequence obtained from this works curried us to travel frontward in advancement to look into the biological activity and supply the communities with scientific information about there traditional medical specialties.
Decision.
At the terminal of this survey we conclude that this works has the biological effectivity, and as the consequences showed it is possible to insulate anti-oxidant, anti-bacterial and anti-cancer compounds. And cautiousness should be taken when utilizing this works by the common common people to incorporate cytotoxic substances. The consequences provided good information can assist to seek for alternate drugs to be used in pharmacotherapy, and will lend to set up safe and effectual usage of phytomedicines in the intervention of diseases.
Recognition.
The writers thank the Ministry of Higher Education Malaysia for back uping this survey through FRGS 0409-103 grant. Besides Mr. Abdoulmonaem Dolania for assisting in statistical analysis.
