Phytochemical Analysis , Antioxidant Assay and Antimicrobial Activity in Leaf Extracts of Cerbera odollam Gaertn

Introduction: In the current study, methanol and aqueous extracts of leaf of Cerbera odollam Gaertn were screened for its antibacterial, antifungal, phytochemicals and antioxidant activities. Phytochemical constituents were investigated both qualitatively and quantitatively. Methods: The leaf extracts of Cerbera odollam Gaertn were prepared by drying and extracted using Soxhlet apparatus into methanol and aqueous media, which were subjected to phytochemical screening. Total phenols, tannins, flavanols, alkaloids and its antioxidant activity were determined using spectroscopic techniques. Antimicrobial activity were determined using well diffusion method. Results: Aqueous extract exhibits higher content of phenols, tannins, flavanols and alkaloids, whereas methanol extract exhibits higher content of anthocyanin and cardiac glycoside respectively. Aqueous extract exhibits higher inhibitory concentration (IC %) value for DPPH (2, 2-Diphenyl-1-picrylhydrazyl) and H2O2 radical scavenging assay and reducing power (RP) assay. The methanol extracts exhibited higher inhibitory concentration (IC %) value in SO and NO radical scavenging assay, exhibiting antioxidant properties in five antioxidant models that were investigated. The methanol extract showed some antibacterial activity against Bacillus subtilis, Staphylococcus aureus, Salmonella typhi and Escherichia coli with inhibitory zone ranging from 2 mm to 3 mm, whereas the aqueous extract showed no activity. High antifungal activity was found against Saccharomyces cerevisiae and Candida albicans for methanol extract and moderate for aqueous extract with inhibitory zone ranging from 9mm to 26 mm. Conclusion: The finding of our study have suggested that the extracts of Cerbera odollam Gaertn, possesses a significant amount of phytochemicals and exhibits antioxidant and antifungal activities.


INTRODUCTION
Cerbera odollam Gaertn is a tree belonging to the family of Apocynaceae and widely distributed on the coastal area of South East Asia and around the Indian Ocean.The fruits and seeds of C. odollam are highly toxic, containing cardiac glycoside like cerberin, neriifolin, odollin, etc. responsible for 10% to 50% of total poisoning cases in the state of Kerala, India.It is used widely for suicide and homicide and hence the name "suicide tree". 1 The tree is also known for various medicinal properties.The latex is known in India for its emetic, purgative and irritant effects. 2 The oil from the seeds as a cure for, itching or applied to the hair as an insecticide. 3The bark and leaf of the plant are traditionally used as emetic and cathartic; kernels are used as an emetic; fruit is used as a cure for hydrophobia. 4Its bark and fruits are purgative and used for the treatment of rheumatism. 5There are reports of the anti-nociceptive and sedative effects of its barks. 6ther research works have reported cytotoxic activity, 7 its effect on the central nervous system, 3 purgative and antirheumatic activity, 8 cardiac stimulant activity, 9 neurological activities, 10 and cardiotoxic activity. 11edicinal properties of C. odollam are associated with various phytochemicals found in the plant.Different plant species have shown cytotoxic activity against bacteria, fungus, and virus.It also possesses the property to scavenge reactive oxygen species (ROS) which is the major source of primary catalysts that initiate oxidation in vivo and in vitro and create oxidative stress which results in numerous diseases and disorders 12,13 such as cancer 14 cardiovascular disease, 15 neural disorders, 16 Alzheimer's disease, 17 mild cognitive impairments, 18 Parkinson's disease, 19 ulcerative colitis, 20 ageing 21 and atherosclerosis. 22hytochemicals are natural bioactive compounds widely distributed in plants which have been reported to exert multiple biological effects, including antioxidant, free radicals scavenging, anti-inflammatory, anti-cancer, antibacterial, antifungal etc.Therefore, the need arises to evaluate and quantify these phytochemical constituents.The present study was designed to determine the phytochemical constituents, antioxidant activity, antibacterial and antifungal activity in methanol and aqueous extract from the leaves of Cerbera odollam Gaertn through a number of testing methods.

Collection and extraction of plant material.
Fresh leaf of Cerbera odollam Gaertn collected from Mumbai, Maharashtra, India and authenticated by Blatter herbarium, St Xavier's college, matching with the Blatter herbarium specimen NI 2084 of N.A. Irani.Leaves were cleaned and dried at room temperature for a period of 25 days under shade.Finely ground dried leaf, were weighed and extracted using Soxhlet apparatus by two different solvents methanol and distilled water, 150 ml each for 30 g of powder.The solution of each extract was then subjected to rotatory evaporator and reduced to 1/8 th volume.The solution obtained was stored at 4°C and appropriately diluted for further studies.
Phytochemical screening (qualitative study) Test for Alkaloids -Mayer's Test. 23w mg of the residue of each extract was taken separately in 5 ml of 1.5 % v/v hydrochloric acid and filtered.These filtrates were treated with Mayer's reagent (1.36 g mercuric chloride and 5 g of potassium iodide dissolved in 100 ml distilled H₂O).The formation of a yellow Extract (0.5ml) was mixed with 5 ml of ether and allowed for evaporation, on filter paper and dried.The appearance of transparent spots filter paper indicates the presence of fatty acids.

Estimation of total phenols
The total phenol content was determined using the Folin-Coicalteu method. 34Extract and 0.1ml of Folin Coicalteu reagent (0.5N) were mixed and incubated at room temperature for 15 min.2.5 ml saturated sodium carbonate (20%) was added and after 30 min absorbance measured at 760 nm.The total phenol content was expressed in terms of gallic acid equivalent (mg/g). 35

Estimation of total tannins
The total tannins content was determined using Broadhurst and Jones (1978) method.HCl (8%) in methanol and 4% vanillin in methanol were added to the extracts and absorbance was measured at 500 nm against blank after 20 mins of incubation at room temperature.The total tannins content was expressed in terms of tannic acid equivalent (mg/g). 36scorbic acid equivalent, as percentage inhibition calculated by the formula:

Super oxide radical scavenging assay
The superoxide radical scavenging activity was measured as described by Robak and Gryglewski. 44The superoxide anion radicals are generated in 3.0 ml of Tris-HCl buffer (16 mM, pH 8), containing 0.5 ml of NBT (0.3 mM), 0.5ml NADH solution (0.936 mM) and 1.0 ml extract (1:100 dilution).The reaction was started by adding 0.5 ml PMS (0.12 mM) solution to the mixture, incubated at 25°C for 5 min and then the absorbance was measured at 560 nm against Tris-HCl buffer as blank.SO anion scavenging activity was expressed in terms of ascorbic acid equivalent, 35 as percentage of inhibition was calculated by the formula: % inhibition of SO Abs of control Abs of sample Abs of control   100

Nitric oxide radical scavenging assay
The nitric oxide radical scavenging activity was estimated using Griess's reaction as described by Chanda and Dave. 353ml of sodium nitroprusside in phosphate buffer (10 mM) was added to 2 ml of extract (1:200 dilution).The resulting solution was then incubated at 25°C for 60 min.To 5ml of the incubated sample, 5ml of Griess reagent (1% sulphanilamide, 0.1% naphthyethylene diamine dihydrochloride in 2% H 3 PO 3 ) was added and the absorbance of the chromophore formed was measured at 540 nm against phosphate buffer as blank.NO radical scavenging activity was expressed in terms of ascorbic acid equivalent, as percentage of inhibition was calculated from given formula: % inhibition of NO Abs of control Abs of sample Abs of control   100

Reducing power assay
Reducing power assay was determined according to the method of Oyaizu. 45To the extract, 2.5 ml of phosphate buffer was added.Then 2.5 ml of potassium ferricyanide was added and the tubes were incubated at 50ºC for 20 min (in dark).Then 2.5 ml of TCA was added.The solution obtained was then centrifuged at 3000 rpm for 10 min.Ferric chloride solution (1ml) was added to the supernatant and the absorbance was read at 700 nm against phosphate buffer as blank.Reducing power was expressed in terms of ascorbic acid equivalent. 46

Anti-bacterial assay
Anti-bacterial assay was determined using agar well diffusion method on nutrient agar plates with wells bored into them.Culture obtained was then spread across the agar and allowed to stand for 10 min, under sterile condition.100µl of the extract was placed into the well with tetracycline as positive control and sterile distilled water as negative control.After diffusion of the extract, the plates were incubated 37°C for 32 hr.Zone of inhibition was measured. 47ree gram positive organisms and three gram negative organisms namely Corynebacterium diphtheria, Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Salmonella typhi, Klebsiella pneumonia were tested for antibacterial activity against the control tetracycline.

Estimation of total flavonoids
The total flavonoids content was determined using aluminium chloride method. 37The reaction mixture comprising of extract, aluminium chloride and potassium acetate (120 mM) was incubated at room temperature for 30 min and absorbance was measured at 415 nm.The total flavonoid content was expressed in terms of quercetin equivalent (mg/g). 38timation of total alkaloid The total alkaloid content was determined using Dragendroff 's sodium sulfide method.Mixture containing extract, Dragendroff 's reagent and 1% sodium sulfide was centrifuged to collect the precipitates.It was dissolved in conc.nitric acid and 3% thiourea was added.The coloured solution was checked for the absorbance at 435 nm.The total alkaloid content was expressed in terms of boldine equivalent (mg/g). 39timation of total anthocyanin The total anthocyanin content was determined using two buffer systems -potassium chloride buffer, pH-1.0 (0.025 M) and sodium acetate buffer, pH 4.5 (0.4 M).Extract (0.4 ml) was mixed with 3.6ml of the corresponding buffer and the absorbance was measured against the blank at 510nm and 700 nm respectively in UV spectrophotometer for each extract, the difference between A 510 and A 700 for pH-1 and pH-4.5 individually was taken.Absorbance was calculated as: A = (A 510 -A 700 ) pH 1.0 -(A 510 -A 700 ) pH 4.5.

Estimation of cardiac glycosides
The total cardiac glycosides content was estimated according to Solich et al, by some modification.The extracts were filtered using Whatman paper no. 1.To each of the standard and extract solution, 1ml of Baljet's reagent (95 ml of 1% picric acid + 5 ml of 10% NaOH) was added.After 1 h of incubation, the mixture was diluted with 20ml distilled water and absorbance was measured at 495 nm against a blank containing D/W.The total cardiac glycoside was expressed in terms of digoxin equivalent (mg/g). 41aluation of antioxidant activity DPPH radical scavenging assay The free radical scavenging activity was measured by using 2, 2-diphenyl-1-picryl-hydrazyl or 1, 1-diphenyl-1-picryl-hydrazyl by the method of McCune and Johns. 42The reaction mixture consisted of DPPH in methanol (0.3mM) and 1 ml of extract.After incubation for 10 min in dark, the absorbance was measured at 517 nm against methanol as blank.DPPH scavenging activity was expressed in terms of ascorbic acid equivalent, 35 as percentage inhibition calculated by the formula:

scavenging assay
The ability of plant extracts to scavenge hydrogen peroxide is assessed according to the method of Ruch. 43A solution of hydrogen peroxide (40 mM) was prepared in phosphate buffer (50 mM, pH 7.4) and 2 ml of the solution was added to 1ml extract (1:20 dilutions).The absorbance at 230 nm was determined after 10 mins in dark, against phosphate buffer as blank.H 2 O 2 radical scavenging activity was expressed in terms of

Anti-fungal assay
Anti-fungal activity was determined using agar well diffusion method on Sabouraud's agar plates with wells bored into them.Culture obtained was then spread across the agar and allowed to stand for 10 min, under sterile condition.100µl of the extract was placed into the well with fluconazole as positive control and sterile distilled water as negative control.After diffusion of the extract, the plates were incubated 37°C for 32 hr.Zone of inhibition was measured. 47wo fungal species namely Saccharomyces cerevisiae and Candida albicans were used for studying antifungal activity against the control fluconazole.

RESULTS AND DISCUSSION
Qualitative screening of C. odollam G.Total phenolic, tannin, flavonoid, alkaloid, anthocyanin, cardiac glycoside, chlorophyll contents and antioxidant activities of Cerbera odollam G.
The total phenolic content for dry weight of Cerbera odollam G was estimated to be 72.17mg/g for methanolic extracts and 78.46 mg/g for aqueous extracts (Table 2).Phenols are reactive species towards oxidation and pose biological activity.The process of oxidation and free radicals generation leads to cancer and other diseases.The activity of phenols against this cancer causing process can have therapeutic application in anticancer therapies.Plants having more phenol content show good antioxidant activity indicating a direct correlation between TPC and antioxidant. 35he total tannins content of leaf extracts of Cerbera odollam G was estimated to be 44.69mg/g dry weight for methanolic extracts and 90.99 mg/g dry weight for aqueous extracts (Table 2).Tannins are mostly found in stem and barks of many plants.High concentration of tannins in aqueous leaf extracts shows the presence of the potent antioxidant property.Most of the tannins have antibacterial, antifungal and anticancer properties.Tannin is an astringent, bitter plant polyphenolic compound that binds to and precipitates proteins and various other organic compounds including amino acids and alkaloids.This tannin-protein complex can provide persistent antioxidant activity. 35he total flavonols content was estimated to be 25.69 mg/g for methanolic extracts and 27.59 mg/g for aqueous extracts (Table 2).The antioxidative properties of flavonoids are due to several different mechanisms, such as scavenging of free radicals, chelation of metal ions, such as iron and copper, and inhibition of enzymes responsible for a free-radical generation.Depending on their structure, flavonoids are able to scavenge practically all known ROS. 35 The total alkaloid of Cerbera odollam G leaf extract was estimated to be 5.21 mg/g for methanolic extracts and 13.51 mg/g for aqueous extracts (Table 2).Most of the alkaloids have local anesthetics and stimulant properties.The alkaloids show cytotoxic activity even in low concentration and other biological activity, showing a wide use in the medical application.
Most of the alkaloids are toxic to the human and other organism so it is widely used in medical applications.Alkaloids also show antioxidant properties and anticancer properties like boldine. 48he total anthocyanin content of leaf extracts of Cerbera odollam G was estimated to be 0.301 mg/g dry weight for methanolic extracts and 0.286 mg/g for aqueous extracts (Table 2).As most of the anthocyanin are coloured pigments found in flower and fruits, the content of anthocyanin was found to be very low in our extracts.Anthocyanin studies have shown monoamine oxidase inhibitor activity connected to the functions in neurogenerative diseases, depression, and anxiety along with neuroprotective and anti-inflammatory activities.It has also been suggested that they show radical scavenging properties and anticancer properties. 49,50he total cardiac glycoside content for dry weight of leaf extracts of Cerbera odollam G was estimated to be 0.162 mg/g for methanolic extracts and 0.137 mg/g for aqueous extracts.(Table 2).The amount of cardiac glycosides detected in leaf extracts is minor as against larger quantities that have been reported in the fruits of Cerbera odollam. 1 This makes the fruits considerably toxic since glycosides act as sodium potassium ATPase inhibitor leading to cell death.Cardiac glycosides are used for the treatment of congestive heart failure and cardiac arrhythmia.They also have anticancer properties. 51nhibition concentration is the amount of free radicals scavenged in the determination of antioxidant activity.Phytochemicals act as antioxidants by scavenging the free radicals.DPPH is a stable free radical and is widely used to assess the radical scavenging activity of antioxidant compounds.This method is based on the reduction of DPPH in methanol solution in the presence of a hydrogen-donating antioxidant due to the formation of the non-radical form DPPH-H. 35 The inhibition concentration of DPPH radical scavenging assay for 5µg of leaf extract of Cerbera odollam G was found to be 80.03% for methanolic extracts and 88.38% for aqueous extract.(Table 3), as compared to inhibition concentration for 5µg of ascorbic acid that was found to be 88.89%.Aqueous extract shows higher inhibition concentration and scavenged maximum amount of radicals as compared to methanolic leaf extract.The DPPH scavenging activity can be correlated to the presence of flavonoids, showing higher radical scavenging by aqueous extracts. 52,53,54 2 O 2 is rapidly decomposed into oxygen and water and this may produce hydroxyl radicals (OH • ) that can initiate lipid peroxidation and cause DNA damage.Antioxidants scavenge hydroxyl radicals.30 The inhibition concentration of H 2 O 2 radical scavenging assay for 20µg of leaf extract of Cerbera odollam G (Table 3) was found to be 49.78% for methanolic extracts and 70.82% for aqueous extract.As compared to inhibition concentration for 20µg of ascorbic acid that was found to be 86.47 %.
Aqueous extract shows higher inhibition concentration and scavenged maximum amount of radicals as compared to methanolic extract.Hydrogen peroxide radical scavenging activity is correlated to the presence of total phenol content.Hence the radical scavenging activity is higher in aqueous leaf extracts. 52,53,54lthough superoxide anion is a weak oxidant, it gives rise to a generation of powerful and dangerous hydroxyl radicals as well as singlet oxygen, both of which contribute to oxidative stress.The SO radical scavenging activity in 20µg of leaf extracts of Cerbera odollam G was estimated to be 94.57% in methanolic extract and 91.37 % in aqueous extract indicating a slight difference between the two extracts (Table 3), as compared to 20µg ascorbic acid which was found to be 51.61%.SO scavenging activity is correlated to total flavonoids which are greater in aqueous extract but the radical scavenging activity is greater in methanol extract with slight differences in the percentage of inhibition concentration as compared to aqueous extracts.This indicates the presence of some other compound leading to higher radical scavenging activity in methanol extracts as Where, "+" is present and "-" is absent.Where, "IC %" is Inhibition concentration % compared to higher total flavonoids content in aqueous extracts of C. odollam. 55,35itric oxide generated from sodium nitroprusside in aqueous solution at physiological pH interacts with oxygen to produce nitrite ions.Antioxidants act by scavenging the NO radical. 35The NO radical scavenging activity (Table 3) in 20µg of leaf extracts of Cerbera odollam G was estimated to be 95.83 % in methanolic extract and 95.45 % in aqueous extract indicating a slight difference between the two extracts, as compared to 20µg ascorbic acid found to be 92.72%.NO radical scavenging activity is correlated to the presence of phenols and flavonoid compounds, which is greater in aqueous extract but the NO radical scavenging activity is greater in methanol extract with slight differences in the percentage of inhibition concentration as compared to aqueous extracts.This indicates the presence of some other compound leading to higher radical scavenging activity in methanol extracts as compared to higher phenols and flavonoids content in aqueous extracts. 56educing power is associated with antioxidant activity and may serve as a significant reflection of the antioxidant activity.Compounds with reducing power indicate that they are electron donors and can reduce the oxidized intermediates of lipid peroxidation processes so that they can act as primary and secondary antioxidants. 35The reducing power in 1gm of extracts of Cerbera odollam G was estimated to be 0.049 mg/g for methanolic leaf extracts and 0.049 mg/g for aqueous leaf extracts, (Table 3) ascorbic acid equivalent.The reducing power is mainly correlated to the presence of reductones F which are strong reducing agents like ascorbic acid, reductive acid, tartronaldehyde, oxalic acid and formic acid. 57tibacterial and antifungal screening of Cerbera odollam G Zone of inhibition shown by methanol leaf extract of Cerbera odollam G indicates the presence of low antibacterial activity as compared to aqueous leaf extract, which shows no zone of inhibition.These indicate that plant extracts had no antibacterial property.(Table 4) Zone of inhibition shown by methanol leaf extract of Cerbera odollam G indicates the presence of good antifungal activity better than standard antifungal drug fluconazole as compared to aqueous leaf extract which shows low to the medium zone of inhibition.These indicate that leaf extracts hold a great potential to use as antifungal agents against the fungus like Saccharomyces cerevisiae and Candida albicans.(Table 5 and Figure 1 and 2).
the leaf is rich sources of natural antioxidants and could be developed into drug against diseases such as inflammation, diabetes, cardiac arrest, and hypertension for a variety of beneficial chemo-preventive effects.Isolation and purification of phytochemicals and antifungal constituents of the plant and evaluation of minimum inhibitory concentration (MIC) will provide with an alternate to an existing product and its efficiency.

CONCLUSION
In the current investigation, the extracts from Cerbera odollam G were found to be rich in secondary metabolites and possess a significant amount of phytochemicals, antioxidant activity, and antifungal activity.The aqueous extracts showed the higher content of phytochemical constituents like phenols, tannins, flavonols and alkaloids, and higher antioxidant activity for DPPH radical scavenging assay, H 2 O 2 radical scavenging assay and reducing power assay than methanol leaf extract.The methanol extract showed the higher content of anthocyanin, cardiac glycoside, and higher antioxidant activity for superoxide radical scavenging assay and nitric oxide radical scavenging assay than aqueous leaf extracts.The antioxidant mechanisms of the leaf extracts may be attributed to their free radical-scavenging ability.In addition, phenolic compounds and other phytochemicals appear to be responsible for the antioxidant activity of the extracts.The anti-microbial activity was found to be higher in the methanolic extract as compared to aqueous leaf extract with a low zone of inhibition against bacteria and moderate to the high zone of inhibition against fungus.On the basis of the results obtained,

Figure 1 :
Figure 1: zone of inhibition for the different extracts, against Saccharomyces cerevisiae.

Figure 2 :
Figure 2: zone of inhibition for the different extracts, against Candida albicans.
Results obtained from qualitative screening from leaf extracts of C. odollam.G is presented in Table1.A total of 16 tests were carried out for detection of different phytochemicals.Of which 13 of them were present in both the extracts.These were alkaloids, flavonoids, tannins, carbohydrates, phenols, cardiac glycosides, amino acids, terpenoids, quinones, oxalates, proteins, fatty acids, and betacyanins.The results indicate that Cerbera odollam Gaertn holds promise as a source of pharmaceutically important phytochemicals.Hence quantitative determination of these phytochemicals becomes crucial.