Phytochemical Content Antimicrobial Activities Of Malaysian Calophyllum Canum Biology Essay

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Recently there was huge increase in using of 'herbal products'. These can be defined as plants, parts of plants or extracts from plants that are used for curing disease. However, Calophyllum species is a tropical plant and it has been used in traditional medicine, the limitation in safety and effectiveness information could lead to serious health problems.

Aim of the study: provide information for communities by evaluating the phytochemical contents, antioxidant, antimicrobial and cytotoxic activities of Calophyllum canum species. As well as improve the therapeutic values.

Materials and methods: Three main fractions (none - high polar) were tested to find out the phenolic, flavonoid, flavonol content, DPPH radical scavenging, reducing power and chelating iron ions. Also were tested against Bacillus cereus, Staphylococcus aureus, Escherichia coli, Psedomonas aeruginosa, Candida albicans, and Cryptococcus neoformans. In addition, cytotoxic activity was assayed against lung cancer cell line.

Results: the methanol fraction showed no bioactivity but achieved the highest amount of phenolic, flavonol and flavonoid contents, also it showed a significant result as antioxidant, reducing power and chelating agent. The Hexane fraction achieved the minimum inhibitory concentration (MIC) value 12.5 μg/ml against B. cereus while the MIC of DCM fraction was 25 μg/ml. The DCM fraction was more active against S. aureus where the result was 50 μg/ml while the Hexane fraction was 100 μg/ml. the three main fractions have shown no activity against gram negative bacterial and fungal. The Hexane and DCM fractions have shown cytotoxicity against lung cancer cell line; the 50 % inhibition concentration (IC50) was 22 ± 2.64 and 32 ± 3.78 μg ml-1 respectively. The results were statistically significant (P< 0.05).

Conclusions: Among the results, C. canum fractions proved to be effective against gram positive bacterial and anti-proliferation activity. Also it showed antioxidant activity as well. The results provided beneficial information for communities as well as can help to search for alternative drugs, and will contribute to establish safe and effective use of phytomedicines in the treatment of diseases.

Keyword: Calophyllum canum; Phytochemistry; Antioxidant; Antimicrobial; Antiproliferative (cytotoxicity).

Introduction

The genus Calophyllum belongs to Guttiferae family which consists of 180 - 200 different species distributed in the warm humid tropics of the world, also it is available in Malaysia. Wide phytochemical studies have reported that Calophyllum genus rich in xanthones, coumarins, biflavonoids, chalcones, benzofurans and triterpenes . A few studies 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 effect against human leukemia HL-60, Nasopharynx carcinoma KB and K562 Lymphoma, U251 central nervous system and PC3 Prostata cell lines respectively . On the other hand Reyes-Chilpa has reported that Calophyllum genus able to inhibit the growth of Staphylococcus aureus, Staphylococcus epidermidis and Bacillus subtilis . In addition, the extract from C.brasiliens showed activity against yeast . Some of Calophyllum species are commonly employed in folk medicine in tropics area, however it is used to treat bronchitis, gastric, hepatic disturbances, pain, inflammation, diabetes, hypertension, diarrea, herpes, rheumatism, varicose, hemorrhoids and chronic ulcer , also treating toothache, preventing wound infections and lumbago . As mentioned Calophyllum species were used traditionally very often for treating many diseases. The Calophyllum canum genus provides no information about its activity and it may be used indiscriminately, and not scientifically proven in terms of effectiveness or toxicity. Inadequacy of previous scientific studies on the nature of C.canum prompted us to evaluate the biological activity in terms of phytochemical, antioxidant, antimicrobial, and antiproliferative (cytotoxicity) by using in vitro model of relevance.

Materials and Methods

Chemicals.

All solvents used were of analytical grade, Hexane, Dichloromethane (DCM) and Methanol, also silica gel 60 (230-400 mesh), sodium acetate, and Aluminium chloride was obtained from MERCK. Trichloroacetic acids, potassium hexacyano ferrate, potassium phosphate, ferrous chloride, ferric chloride, ascorbic acid, BHT (Butylated hydroxytoluene) were purchased from R & M chemicals. 1,1-Diphenyl-2-picrylhydrazyl (DPPH) and 3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine-p,p'-disulfonic acid 1-Na x H2O were purchased from Sigma-Aldrich Chemical. Gallic acid obtained from Alfa Aesar. The media were used in this study for bacteria, Mueller-Hinton broth medium (MHB) used in MIC method, and Mueller-Hinton agar used in disc diffusion method. And for fungus the potato dextrose agar was used for disc diffusion method. Chloramphenicol, and Amphotericin B a standard antibiotic used to compare with for bacteria and fungus respectively.

2.2 Plant Material.

The stem bark of Calophyllum canum was collected from Bukit Pelindung Kuantan, Malaysia in June, 2009, with a herbarium specimen (No MT-01). The specie was identified by Dr Shamsul Khamis, botanist from Universiti Putra Malaysia.

2.3 Extraction and Fractionation.

Air-dried and powdered steam bark (1kg) was macerated in (2.5 L) 98% EtOH for 72 hours. The EtOH extract was filtrated and evaporated under reduce pressure to yield the dark brown gummy (200g) EtOH crude. A part of EtOH crude (100 g) was chromatographed by VLC (silica gel 230-400 mesh, 1:30 ratio) to obtain three main fractions Hexane, CH2Cl2 (DCM) and MeOH.

Phytochemical screening.

Determination the phenolic content.

Total phenolic content was carried out regarding to the Folin-Ciocalteau's method with slight modifications. In brief, 0.5 mL of sample (10 mg mL-1) was mixed with 5.0 ml of Folin-Ciocalteau's reagent (1:10). The mixture was settled in a tube for 5 min,, and then 4.0 mL of 1M Na2CO3 was added. After incubation at room temperature for 15 min, a 100 μL of the mixture was moved to 96-microwell plat. The absorbance was measured at 765nm by using multi-detection micro-plate reader (INFINITE M 200 NANOQUANT). The sample was tested in triplicate and the results explained ± standard deviation. Calibration curve with five data points for Galic acid was obtained. The results were compared to a Galic acid calibration curve and the total phenolic content of fractions was expressed as μg of Galic acid equivalents per 10 mg of extract.

(Y= 0.0507 X - 0.011) and R2 = 0.9937

Determination the total flavonoid.

Total flavonoid contents were determined using the method of with slight modification. A volume of 0.1 mL of sample was placed in a 96-microwell plate and mixed with 0.1 mL (2%) AlCl3 ethanol solution. The mixture was incubated at room temperature for 1 hour, after the absorbance was measured at 420 nm using the multi-detection micro-plate reader (INFINITE M 200 NANOQUANT). The yellow colour indicated flavonoids existence. The three main fractions were evaluated at final concentration of 10 mg mL -1 in triplicate and the results explained ± standard diveation. The blank prepared by mix the sample with ethanol. Total flavonoid content was calculated as quercetin equivalent (μg Que /10mg extract) using the following equation based on calibration curve:

Y= 0.0174 X - 0.048, R2 = 0.9976.

Determination the total flavonol.

Total flavonols were estimated by using the method of with slight modification, in brief 50 μL of sample or standard was mixed with 50 μL of (2%) AlCl3 ethanol solution and 75 μL of (50 g L-1) sodium acetate solution, the mixture was seated in a 96-microwell plate and it was incubated for 2.30 hour at room temperature. The absorbtion measured at 440 nm by using the multi-detection micro-plate reader (INFINITE M 200 MAMOQUANT). The main fractions were evaluated at a final concentration of 10 mg mL-1, where the blank was prepared by mixing the sample with ethanol. Total flavonoid content was calculated as quercetin equivalent (μg Que /10mg extract) using the following equation based on the calibration curve:

Y = 0.0097 X + 0.055, R2 = 0.9994.

Antioxidant activity.

DPPH assay.

The scavenging effect of C.canum fractions was assessed by using the method of with slight modifications. A 100 μl of methanolic solution containing between 0.031- 1 mg of the fractions was mixed with methanolic solution of DPPH (1mM , 200 μL) with slight modifications. A 100 μL of methanolic solution containing between 0.031-1 mg of the fractions was mixed with (200 μL) methanolic solution of DPPH (1 mM) in a 96-microwell plate. The content was mixed and left in a dark area at room temperature for 20 min, and then the absorbance of the mixture was measured at 517nm by using multi-detection microplate reader (INFINITE M 200 NANOQUANT). The control was prepared by mixing methanol instead of the fractions. The blank was 100 μL of fraction with 200 μL of methanol. Radical scavenging activity was expressed as the inhibition percentage and was calculated using the equation 1.

% Radical scavenging = ((Control OD - Sample OD)/Control OD) * 100 (1)

Where OD: Optical Density.

The Required fraction's Concentration (μg mL-1) for scavenging of 50% of DPPH radical (RC50) was determined. All measurements were curried out in triplicate.

Reducing power assay.

The reducing power of C.canum fractions and two standards ascorbic acid and BHT was determined according to the method of with slight modification. A 100 μL of three different concentrations 1, 5 and 10 mg mL-1 of each fraction were mixed with 50 μL of (0.2 M) phosphate buffer (pH 6.6) and 250 μL of potassium ferricyanide (1%). After the mixture was incubated at 50 °C for 20 min, 250 μL of (10 %) trichloroacetic acid were added and the mixture was centrifuged at 1000g for 10 min. 250 μL supernatant was moved to 48-microwells plate and mixed with 250 μL distilled water and 50 μL of ferric chloride (0.1 %). The mixture was measured at 700 nm by using the multi-detection micro-plate reader (INFINITE M 200 NANOQUANT). All the measurements were carried out in triplicates. However, higher absorbance of the reaction mixture indicates greater reducing power.

Iron (II) chelating activity.

The chelation of iron (II) ions by different fractions' concentrations was curried out by the method described in . 100 μL of four different concentrations 1, 2.5, 5 and 10 mg mL-1 of each fraction, which dissolved in methanol, was mixed with 10 μL of (2.0 mM) aqueous FeCl2. After 5 min incubation at room temperature, the reaction was initiated by 20 μL of (5.0 mM) ferrozine. After 10 min the absorbance was measured at 562 nm by using 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 methanol. The iron chelating activities were calculated from the equation 2.

% chelation = ((Ac-As)/Ac)*100 (2)

(Ac) represented the absorbance for control and (As) the absorbance for sample. The values were presented as the means of triplicate analyses ± standard deviation.

Anti microbial assays.

Microbial strains.

Six reference microbial strains of human pathogens were used for the anti-microbial activity. The two Gram-positive bacteria (Staphylococcus aureus ATCC 25923, Bacillus cereus ATCC 11778), and two Gram-negative (Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 35218) were used for antibacterial test. Also two fungal strains (Candida albicans ATCC 10231 and Cryptococcus neoformans ATCC 90112) were used for antifungal test.

Disc Diffusion Method

The agar disc diffusion method was employed for the determination of antimicrobial activities of the EtOH crude according to with some modifications. Briefly, inoculum containing 106- 107 CFU mL-1 was spread on Mueller -Hinton agar plates for bacteria and 104 CFU mL-1 was spread on potato dextrose agar for fungus strains. The sterilised forceps was used to place sterile (6 mm diameter) filter papers on the top of agar. The filter papers loaded with crude extract (10 or 20 μg), standard antibiotics (30 μg of chloramphenicol or 100 μg of amphotericin B) or negative control (DMSO) were laid down on the surface of inoculated agar plate. The plates were incubated at 37°C for 24 hour for bacteria and at room temperature (18-20°C) for 24-48 for yeast strains. Each sample was tested in triplicate and the zone of inhibition was measured as millimetre diameter.

Microdilution method (MIC and MBC).

Minimal inhibitory concentration (MIC) was measured by determining the smallest amount of fractions (Hexane, DCM or MeOH) or standard antibiotic needed to inhibit the growth of a test microorganism. This was done using 96-microwell plates and performed on (Versa MaxTM Tunable) micro-plate reader. In brief, the plates were filled with Mueller-Hinton broth medium containing different concentrations of the Hexane, DCM or MeOH fractions. Chloramphenicol or solvent control and the test micro-organism (106 -107 CFU mL-1) also were added. After 24 hour incubation periods at 37°C, the turbidity was measured at 600nm.

Minimal bactericidal concentration (MBC) was determined by transferring and spreading the treated culture broth of the wells containing the concentrations equal to and higher than the MIC on agar plates. The lowest concentration of the fractions or the standard antibiotic required to completely destroy test microorganisms (no growth on the agar plate) after incubation at 37°C for 24 hour was reported as Minimum bactericidal concentration (MBC).

2.6.4 The cytotoxicity assay (MTT assay).

Anti-proliferative effects of C.canum fractions against A549 cell line was investigated by using [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT assay)] described by . For this purpose, A549 cells were cultured in a completed (DMEM) media in a T- flask until the cells were confluent. Then, the cells were seeded in a 96-microwell plate at a density of 0.5 x 105 cells/well and incubated at 37°C in 5% CO2 humidified incubator. After 24 hour a fresh media was added and the cells were treated with different concentrations of samples obtained by double fold serial dilution. The (95% ethanol) was used as control. After 24 hours incubation, the supernatants were discarded, and the adherent cells were washed twice with phosphate buffer saline (PBS). 20μL of (5mg mL-1) MTT stock solution was added to each well and the plate was further incubated overnight at 37°C. A 100 μL DMSO was added to dissolve the water insoluble purple formazan crystals produced by the viable cells. When formazan completed dissolve a 100 μL was transferred to a new 96-microwell plate and the absorbance was measured at 570nm and reference at 690nm, using multi-detection microplate reader (INFINITE M 200 NANOQUANT). All samples were assayed in triplicate. The percentage of cell viability was calculated and the concentrations required for inhibition of 50% of cell viability (IC50) were determined according to equation 3.

% of cell viability = (OD of treated cells / OD of control cells)*100 ………… (3)

Results.

3.1. Phytochemical content.

The phytochemical analysis was curried out in this study, to investigate the total phenolic, flavonoid and flavonol content in three main fractions of C.canum. Table 1 shows the contents value for each fraction, it is clearly appearing that MeOH fraction contains the highest amount of polar phytochemicals, where another two fractions Hexane and DCM have a smaller amount; but DCM has high content of flavonoid.

Table 1

 

 

 

 

 

The fractions of Calophyllum canum used in screening the phytochemistry content.

Fractions

Phenolic content*

 

Flavonoid contentâ-Š

 

Flavonol contentâ-Š

Hexane

1.292 ± 0.001

5.040 ± 0.184

1.202 ± 0.547

DCM

1.992 ± 0.002

11.201 ± 2.446

2.164 ± 0.415

MeOH

3.517 ± 0.017

 

14.643 ± 1.222

 

11.639 ± 2.479

*μg Galic Acid/10 mg extract

 

â-Š μg quercetin/10 mg extract

 

 

 

 

3.2. Antioxidant activity.

3.2.1 DPPH assay

The result display in Table 2 shows the IC50 of free radical scavenging activity, which was curried out by using the DPPH assay. The MeOH fraction shows a good result as an antioxidant, while Hexane and DCM fractions show a moderate activity. Fig.1 depicts the % of radical scavenging activity versus concentrations. The hexane fraction curve turned to steady after 125μg mL-1, while the DCM fraction curve turned to steady after 250μg mL-1. the MeOH fraction became steady after 62.5μg mL-1 and the IC50 is much less than 31.25μg mL-1.

Table 2

 

 

IC 50 free radical scavenging activity

Fractions

 

IC 50 (μg/ml)

Hexane

85 ± 4.35

DCM

91.6 ± 6.65

MeOH

 

< 31.25

Figure 1.free radical scavenging activity of the fractions of C.canum (Hexane, DCM, and MeOH) by DPPH assay.

3.2.2 Reducing power.

The reducing power agents are concentrating in the MeOH fraction, where it is achieving a result closer to BHT the moderate standard. Fig.2 depicts the reducing power value of MeOH and other substance. The Hexane and DCM fractions show low reducing power activity, where they are less than BHT standard. The strongest reducing power is ascorbic acid where there are no substances get closer to.

Figure 2 the reducing power activity of three C.canum fractions (Hexane, DCM, MeOH) standard BHT and Ascorbic acid at 700nm.

3.2.3. Iron (II) chelating activity

The chelation of ferrous ions by the C.canum fractions and standard was examined by the method of . Fig. 3 shows two standard Quercetin moderate and EDTA high chelating agent. All fractions were comparing with standard; the Hexane and DCM show activities less than Quercetin at all concentration, while the MeOH fraction shows dramatically activity, it chelates the iron at 2.5-5 mg mL-1 as the Quercetin does, also it chelats iron at 10 mg mL-1 to reach activity as EDTA. The iron chelating activity result for MeOH fraction was directly 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 assay

The antibacterial and antifungal activities of EtOH crude using disc diffusion method are summarized in Table 3. The C.canum crude extract was screened at concentrations of 10 and 20µg/disc against six human pathogens. Both gram negative and fungal strains appear to be resistant to the tested concentration since no inhibition zone was observed while the gram positive bacteria showed moderate sensitivity with no significant difference between the concentrations. Therefore the MIC and MBC were determined using different polarity fractions (Hexane, DCM and methanol) of C.canum against gram positive strains only (Table 4). As a result, the MIC values of the tested fractions ranged from 12.5 to 100µg mL-1 whereas the MBC values were 100 or 250µg mL-1. The inhibition growth of the microbes at concentration as low as 12.5µg mL-1, was indicated to potential antimicrobial activity of C.canum fractions. Furthermore, the highest activity was obtained against B.cereus for the Hexane fraction (MIC and MBC was 12.5µg mL-1 and 100µg mL-1, respectively).

Table 3: Antimicrobial activity of ethanol crude extract using disc diffusion method.

Bacteria

yeasts

S.a

B.c

P.a

E.c

C.a

C.n

EtOH Crude (10µg/disc)

11.5 ± 0.7

9.5± 0.7

0±0.0

0±0.0

0±0.0

0±0.0

EtOH Crude (20µg/disc)

12.5± 0.7

10.5 ±0.7

0±0.0

0±0.0

0±0.0

0±0.0

Chloramphenicol (30µg)

24.5 ± 0.7

26 ± 0.5

33±.03

37 ± 0.4

ND

ND

Amphotericin B (100µg)

ND

ND

ND

ND

20±0.7

19±0.4

Negative control

0±0.0

0±0.0

0±0.0

0±0.0

0±0.0

0±0.0

Mean diameter of zone of inhibition in mm± standard deviation including the diameter of the disc 6 mm. 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 inhibition zone

ND: Not Determined

Table 4: MIC and MBC for Chloramphenicol, Hexane, DCM and MeOH fractions

Fraction

S. aureus (μg/ml)

B. cereus (μg/ml)

MIC

MBC

MIC

MBC

Hexane

100

250

12.5

100

DCM

50

250

25

250

MeOH

>1000

ND

>1000

ND

Chloramphenicol

20

ND

20

ND

ND: Not Determined

Cytotoxicity (MTT assay)

The anticancer activity of the C.canum fractions was investigated using MTT assay on human lung cancer cell line A549. A mitochondrial enzyme in living cells can succinate dehydrogenase and cleaves the tetrazolium ring converting the MTT to an insoluble 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 inhibiting the cell growth at low concentration. The DCM fraction shows a good activity but it is less than Hexane. The MeOH fraction shows no activity where the growth is 100%.

Figure 4. The % of viability activity for C.canum fractions against A549 cell line.

Table 5

 

 

The IC50 and % of viability of C.canum fractions

Fractions

IC50 μg/ml

% of viabilityâ-Š

Hexane

22 ± 2.64

35

DCM

32 ± 3.78

51

MeOH

N.d

N.d

N.d: Not determined

â-Š:% of viability at 30 μg/ml

The criteria of cytotoxicity activity for the crude extract, as established by the American National Cancer Institute (NCI) is an IC50 <30 μg mL-1 in the preliminary assay .The IC50 values for Hexane and DCM fractions are 22 and 32 μg/ml respectively. The % of viable cell at concentration 30 μg mL-1 as recommended by NCI is 35 and 51 %.respectively for Hexane and DCM fractions. These indicate to the efficacy of Hexane at low concentration as it is illustrating in table 5.

Discussion

Phenolic compounds are most abundant secondary metabolites in the plants. Additionally, it has been found in many food and their derivatives . Flavonols and flavonoids are a part of phenolic compounds and they are sharing in the physical properties. Phenolic compounds are known as powerful chain breaking antioxidants . Also it is very important constituents of plants and their radical scavenging ability is due to their hydroxyl groups . The phenolic compounds my contribute directly to anti-oxidative action . In various studies, antioxidant activity of the plant extracts which are rich in phenolic compounds was found to be fairly high . The chemical complexity of extracts often a mixture of dozens of compounds, with different functional groups could lead to various values, depending on the test employed. Therefore, an approach with multiple assays for evaluating the antioxidant potential of extracts would be more informative and even necessary. There are several methods using for determining of the antioxidant activity. In this study, mainly three methods were curried out, DPPH radical scavenging activity, metal chelation activity and ferric reducing power. The concentrations of total phenolic, flavonol and flavonoids were also calculated for the C.canum fractions. The DPPH results show that Hexane and DCM fractions were statistically not significant (P≥ 0.05) in the range of concentration (31.25- 1000 μg mL-1) except at concentration (125, 250 μg mL-1) the result was statistically significant (P< 0.05). In addition, the MeOH fraction shows a significant result even at low concentration. The other fractions were compared with MeOH fraction and the result were statistically extremely significant at (P<0.001), except at concentration 62.5 μg mL-1 for Hexane and 250 μg mL-1 for DCM was just significant at (P< 0.05). The radical scavenging activity increases with increasing the amount of the fractions.

Free radicals have a significant effect on oxidation of unsaturated lipids the DPPH radical scavenging was used as a stable free radical to determine antioxidant activity of natural compounds . The method is based on the reduction of alcoholic DPPH solution in presence of a hydrogen-donation antioxidant due to the formation of the non-radical DPPH-H. The reducing power assay was curried out as a second antioxidant test, the yellow colour which was observed of the tested solution, was turned to various shades of green and blue colour, which was indicated reducing power of each extract existence. The abundance of reducing substances (i.e. antioxidants) in the fractions will lead to reduction the Fe+3 ferricyanide complex to the ferrous form. Therefore, the Fe+2 can be examined by measuring the formation of Perls' Prussian blue at 700 nm . In other words, the FeCl3/K3Fe(CN)6 system offers a sensitive method for the "semi-quantitative" determination of dilution concentration of polyphenolics, which share in redox reaction. The reducing powers of Hexane and DCM fractions were showed a weak activity, they were statistically significant (P < 0.05) comparing with BHT and ascorbic acid. While the MeOH fraction was showed a moderate activity and statistically was not significant (P≥ 0.05) comparing with BHT, this result was predicted where normally methanol fraction contains the high polar components which normally contain (-OH) groups. The high phenolics or polyphenolics content come into view to function as good electron and hydrogen-atom donors and therefore should be able to terminate radical chain reaction by converting free radicals to more stable products. The data for all fractions from this assay, correlated well with the content of total phenolics, and regarding to the chemical roll "like dissolve like", these results are going logically with.

Among the transition metals, iron is known as lipid oxidation pro-oxidant. The ferrous state of iron accelerates lipid oxidation by breaking down hydrogen and lipid peroxides to reactive free radicals via the Fenton Reaction.

Fe+2 + H2O2 → Fe+3 + OH- + OHâ-

Ferric ion also produces radical from peroxides although, the rate is 10-fold less than ferrous . 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 ≤ 5mg mL-1and statistically was not significant (P≥0.05). Also, it has shown a good activity like EDTA when the concentration was raised up to 10 mg mL-1 and statistically was not significant (P≥0.05) comparing with EDTA. MeOH fraction was showed the highest metal chelating activity among all the fractions have studied, which has comparable results with EDTA.

Nowadays, many biological activities have been evaluated for numerous species of plants. This demonstrates that compounds from medicinal plants are indeed useful as alternative therapy, either directly or as models fro new synthetic substances . However, the use of these substances of plant origin is not always monitored by health professionals, which would ensure efficacy and safety procedures, and can lead to absence of biological effects or even to toxic effects . In this study, no activity was observed against Gram-negative bacterial and yeasts. This is in agreement with many antimicrobial studies, which studied the antimicrobial 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 gram negative P.aeruginosa was resistant to this extracts. Pretto and his co-authors (2004) showed that all the parts (roots, stems, leaves, flowers and fruits) of C.brasiliense exhibited antimicrobial activity against Gram-positive bacteria, and no activity was observed against Gram-negative bacteria and yeasts tested. On the other hand, this difference in susceptibility between the bacteria is related to the outer membrane of gram-negative bacteria. The outer membrane endows the bacterial surface with strong hydrophilicity and acts as a strong permeability barrier .

The search for anticancer agents from natural sources has been successful worldwide; active materials have been isolated and are nowadays used to treat human tumours. Pe.alliacea has been reported to be utilized in treating patients with cancer and leukaemia in Cuba where promising results were obtained . The paclitaxel has been isolated from the pacific yew plant and it is used nowadays fro treating the lung and breast cancer as well. The present study was undertaken to evaluate the cytotoxic activity of Malaysian Calophyllum canum which is never investigated before. Also it is used in the treatment of a few aliments and cancer related illnesses in the country. were isolated 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 growth at low concentration. Also Zhi and his colleague showed that C. inophyllum species from china inhibited the growth of human leukemia HL-60 cells by isolated triterpenoide . The information based on the traditional medicinal use of plants has been one of the common useful ways of the discovery of biological activity compounds from plants . In this study, different fractions supposed to contain mainly high concentration of secondary metabolise were used. The result of the present study indicates the presence of cytotoxic activity in Hexane and DCM fractions whereas the MeOH fraction showed no activity. The statistical analysis was showed a significant (P<0.05) in the range of (3.06-12.5 μg mL-1) between Hexane and DCM fractions where the result was not significant (P≥0.05) at the range (25-100 μg mL-1) for the same fractions. The Hexane fraction was not significant (P≥0.05) at the range (3.06-12.5 μg mL-1), which it is mean that the Hexane fraction has an anticancer properties and it might become a drug for anticancer with farther investigation. Misdiagnosis of cancer by traditional healers might explain the observed lack of correlation between the reported anticancer activities of plant extracts and their cytotoxic activity on the tested cell lines . The promising result obtained from this plant curried us to go forward in progress to investigate the biological activity and provide the communities with scientific information about there traditional medicines.

Conclusion.

At the end of this study we conclude that this plant has the biological effectiveness, and as the results showed it is possible to isolate anti-oxidant, anti-bacterial and anti-cancer compounds. And caution should be taken when using this plant by the common folk to contain cytotoxic substances. The results provided beneficial information can help to search for alternative drugs to be used in pharmacotherapy, and will contribute to establish safe and effective use of phytomedicines in the treatment of diseases.

Acknowledgment.

The authors thank the Ministry of Higher Education Malaysia for supporting this study through FRGS 0409-103 grant. Also Mr. Abdoulmonaem Dolania for helping in statistical analysis.

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