Investigation on the Phytochemicals present in the Fruit peel of Carica papaya and evaluation of its Antioxidant and Antimicrobial property.

 

Dr. Esther Lydia¹, Dr. Sheila John², Mr. Riyazudin Mohammed³, Mrs. Thiyagarajan Sivapriya⁴

¹Assistant Professor, Loyola College, Chennai

²Associate Professor, Womens Christian College, Chennai

³Student, Loyola College, Chennai

⁴Research Scholar, Womens Christian College, Chennai

 

ABSTRACT:

Background: Plants have the major advantage of being the most treasured and cheaper alternative supplies for drugs. Phytochemicals in fruits and vegetables have gained increasing interest among consumers and the scientific community as epidemiological studies have indicated that regular consumption of phytochemicals is related to a lower risk of non-communicable diseases.

Aim: The aim of the present study was to carry out qualitative and quantitative phytochemical analysis to establish the different classes of compounds present in the peel of indigenous fruit C. papaya using five different solvents and evaluate its antioxidant and antimicrobial property.

Methods and Material: Evaluate the antioxidant property of Carica papaya peel by radical scavenging methods and to elicit the most effective concentration of the extract with highest antimicrobial activity by measuring the diameter of zone of inhibition against five pathogens

Results: The various extracts revealed the presence of phytoconstituents such as phenols, flavonoids and tannins in appreciable amounts and the antioxidant potential of papaya peel. Agar well method demonstrated that the species most sensitive to C. papaya peel among the gram-positive microorganisms was Bacillus subtilis, whereas among the gram-negative species the most sensitive was Klebsiella pneumoniae.

 

 

 

 

INTRODUCTION:

Papaya is a popular fruit, with high nutritive value ^[1] Papaya chairs the highest among fruits for vitamin C, vitamin A, riboflavin, folate, calcium, thiamine, iron, niacin, potassium and fiber. The comparative low calorie content (32 kcal /100 g of fruit) makes it a preferred fruit for obese people who are on a weight reducing regime. Also, papaya ranks the topmost among fruits for carotenoids, potassium, fiber, and ascorbic acid content ^[2].

 

Plant phenolic and carotenoid compounds are acknowledged for their priceless effects on human health and they are present in a countless diversity of fruits and vegetables. The profile of bioactive compounds in papaya fruit has not been absolutely elucidated and research has been dedicated on characterization of mature fruit flesh composition and proteolitic enzymes

The presence of phenolic compounds with antioxidant activity identified in peel, as well as carotenoids with provitamin A and radical quencher activities in flesh of ‘Maradol’ papaya, could add to the positive effects of this fruit to human wellbeing ^[3]

 

Antimicrobial activity of papaya has also been recognised from the fruit and the seed. Antimicrobial activity of seed was documented against Trichomonas Vagina listrophozoties although care was suggested when using the seed extract for urinogenital disorders due to its toxicity^[4]. Both the seed and pulp were reported to show bacteriostatic properties against several enteropathogen such as Bacillus subtilis, Salmonella typhi, Staphylococous aureus, Proteus vulgaris, Pseudomonas aeruginosa and Klebsiella pneumoniae determined via agar cup plate method ^[5].

 

Antihelmintic activity of papaya seed has been predominantly attributed to carpaine (an alkaloid) and carpasemine^[6]. Many biologically active phytochemicals have been isolated from papaya and studied for their potency. Antifungal chitinase has been genetically cloned and characterized from papaya fruit.

 

The present work attempted to investigate the phytochemical properties, assess the antioxidant activity by different radical scavenging methods and evaluate the antimicrobial activity of C. papaya peel. Papaya fruit peel which is disposed as waste by the fruit industry can be developed into a new functional food that will shield humanity from the clutch of several lethal disorders.

 

MATERIALS AND METHODS:

C. papaya fruit was purchased from Koyembedu market, Chennai, India and authenticated. The peels were manually separated from the remaining parts and dried at room temperature at 32ᴼC for one day. The dried peel were subjected to grinding.

 

Sequential extraction:

The dried powder of the peel was extracted sequentially by soxhlet apparatus, using different solvents depending upon their polarities like hexane, acetone and ethanol. The dried powder weighing 800g was percolated with solvents of varying polarity in 1000ml conical flask. The percolation process was carried out for 48 hours and the solvent was filtered using whattman No.41 filter paper. This process was repeated for all the solvents. The collected solvents were evaporated using a rotary evaporator (Super fit- ROTAVAP, India.).

 

Qualitative phytochemical analysis:

The phytochemical screening was carried out using standard methods of analysis^[7] of carbohydrates, tannins, saponins, flavanoids, alkaloids, quinines, glycosides, cardiac-glycosides, terpenoids, triterpenoids, coumarins, steroids, phytosteroids, phlobatanins and anthroquinones.

 

Test for carbohydrates:

To 2ml of peel extract, 1ml of Molisch’s reagent and few drops of concentrated sulphuric acid were added. Presence of purple or reddish colour indicates the presence of carbohydrates.

 

a.    Test for tannins:

To 1ml of peel extract, 2ml of 5% ferric chloride was added. Formation of dark blue or greenish black indicates the presence of tannins.

 

b.    Test for Saponins:

To 2ml of peel extract, 2ml of distilled water was added and shaken in a graduated cylinder for 15 minutes lengthwise. Formation of 1cm layer of foam indicates the presence of saponins.

 

c.    Test for Flavonoids:

To 2ml of peel extract, 1ml of 2N sodium hydroxide was added. Presence of yellow colour indicates the presence of flavanoids.

 

d.    Test for Alkaloids:

To 2ml of peel extract, 2ml of concentrated hydrochloric acid was added. Then few drops of Mayer’s reagent were added. Presence of green colour or white precipitate indicates the presence of alkaloids.

 

e.    Test for Quinones:

To 1ml of peel extract, 1ml of concentrated sulphuric acid was added. Formation of red colour indicates presence of quinones.

 

f.     Test for Glycosides:

To 2ml of peel extract, 3ml of chloroform and 10% ammonia solution was added. Formation of pink colour indicates presence of glycosides.

 

g.    Test for cardiac glycosides:

To 0.5ml of peel extract, 2ml of glacial acetic acid and few drops of 5% ferric chloride were added. This was under layered with 1ml of concentrated sulphuric acid. Formation of brown ring at the interface indicates presence of cardiac glycosides.

 

h.    Test for Terpenoids:

To 0.5ml of peel extract, 2ml of chloroform was added and concentrated sulphuric acid was added carefully. Formation red brown colour at the interface indicates presence of terpenoids.

 

i.     Test for Phenols:

To 1ml of peel extract, 2ml of distilled water followed by few drops of 10% ferric chloride was added. Formation of blue or green colour indicates presence of phenols.

 

j.     Test for Coumarins:

To 1ml of peel extract, 1ml of 10% NaoH was added. Formation of yellow colour indicates the presence of coumarins.

 

k.    Tests for Steroids and Phytosteroids:

To 1ml of peel extract, equal volume of chloroform is added and subjected with few drops of concentrated sulphuric acid appearance of brown ring indicates the presence of steroids and appearance of bluish ring indicates the presence of phytosteroids.

 

l.     Test for Phlobatannins:

To 1ml of peel extract, few drops of 2% HCL was added. Appearance of red colour precipitate indicates the presence of phlobatannins.

 

m.  Tests for Anthroquinones:

To 1ml of peel extract, few drops of 10% ammonia solution was added, appearance of pink colour precipitate indicates the presence of anthraquinones.

 

Quantitative phytochemical analysis:

Determination of total phenol content:

The amount of total phenol content of different solvent extracts was determined by Folin-Ciocalteu’s reagent method^[8]. The various concentrations of the extract were made up with 900µl of distilled water and 0.5ml of Folin-Ciocalteu’s reagent was mixed and the mixture was incubated at room temperature for 15 minutes. Then 4ml of saturated sodium carbonate solution (0.7N) was added and further incubated for 30mins at room temperature and the absorbance was measured at 765nm using a UV spectrophotometer, against a blank sample. The calibration curve was made by preparing gallic acid (100-300µg ml-1) solution in distilled water. Total phenol content is expressed in terms of Gallic acid equivalent (mg g-1 of extracted compounds)

 

Determination of total tannin content:

The amount of total tannin content of the peel extract was determined by Folin-Ciocalteu’s reagent method ^[9]. The various concentrations of the extract were made up with 900µl of distilled water and 0.5ml of Folin-Ciocalteu’s reagent was mixed and the mixture was incubated at room temperature for 15 minutes. Then 1ml of saturated sodium carbonate solution (15%) was added and further incubated for 30mins at room temperature and the absorbance was measured at 765nm using a UV spectrophotometer, against a blank sample. The calibration curve was made by preparing tannic acid (20 to 60 µg ml-1) solution in distilled water. Total tannin content is expressed in terms of tannic acid equivalent (mg g-1 of extracted compounds).

 

Determination of flavanoid content:

The amount of flavanoid content of the peel extract was determined by aluminium chloride colorimetric     method ^[10].The reaction mixture (3.0ml) consisted of 1.0ml of sample (1mg ml-1), 1.9ml methanol, 0.5ml of aluminium chloride (1.2%) and 0.1ml potassium acetate (120mM) and was incubated at room temperature for 30 minutes. The absorbance of the sample was measured at 415nm using a digital spectrophotometer (Systronic, India), against a blank sample. The calibration curve was made by preparing a quercetin (10-30 µg ml-1) solution in methanol. The flavanoid content is expressed in terms of standard quercetin equivalent (mg g-1 of extracted compounds).

 

Antioxidant assay:

The antioxidant activity of the different solvent extracts of C.papaya peel was evaluated by DPPH and FRAP assay

 

Determination of 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity:

The free radical scavenging activity of different solvent extracts was measured by using DPPH by the method described by ^[11]. The reaction mixture 3.0ml consisting of 1.0 ml methanol, 1.0ml DPPH (0.3mM) and 1.0ml of solvent extracts of different concentrations of the screened plants and fractions of the extract was diluted by methanol, was incubated for 10minutes, in dark, after which the absorbance was measured at 517nm using UV Visible spectrophotometer (Elico, India), against a blank sample. Ascorbic acid (2 to 16 µg ml-1) was used as positive control. The percentage inhibition was determined by comparing the results of the test and the control.

 

Ferric reducing antioxidant power (FRAP):

The reducing ability of different solvent extracts and fractions of the C.papaya peel was determined by FRAP assay¹⁰. FRAP assay is based on the ability of antioxidants to reduce Fe3+ to Fe2+ in the presence of TPTZ, forming an intense blue Fe2+ TPTZ complex with an absorption maximum at 593nm. This reaction is pH dependent (optimum pH 3.6). 0.1ml extract is added to 3.0ml FRAP reagent [10 parts 300mM sodium acetate buffer at pH 3.6, 1 part 10mM TPTZ (2, 4, 6- tripyridyl-s-triazine) in 40mM HCL and 1 part 20 mM Fecl3] and the reaction mixture is incubated at 37ᴼC for 10 minutes and then the absorbance was measured at 593nm. FeSO4 (100 to 1000µM ml-1) was used as a positive control¹¹. The antioxidant capacity based on the ability to reduce ferric ions of sample was calculated from the linear calibration curve and expressed as M FeSO₄ equivalents per gram of extracted compound.

 

Antimicrobial activity of C.papaya peel:

Antibacterial activity using Agar well diffusion method:

The samples were screened for antibacterial activity against human pathogens such as Bacillus subtilis (ATCC 441), E.coli (ATCC 25922), Klebsiella pneumoniae (ATCC15380) and Staphylococcus aureus (ATCC 25923) using agar well diffusion method.

 

Agar well diffusion method:

Three different concentration of sample (1.25, 2.5 and 5 mg/100µl/well) were used in this study. Muller Hinton Agar (MHA) plates were inoculated with test organisms. The plates were evenly spread out. Then well were prepared in the plates with a cork borer. Each well was loaded with 0.1ml of corresponding concentration of sample. Chloramphenicol (10µg), DMSO was used as a Positive and negative control. The plates were incubated for 24h at 37°C. The development of inhibition zone around the well was measured and recorded

 

Antifungal activity (MIC):

Test Fungal strains:

The Aspergillus flavus fungal strain was used for this experiment: The pathogen was grown on SDA (Sabouraud dextrose agar) slants at 28°C for 10 days. The spores were collected using sterile double distilled water and stored in refrigerator for further analysis.

 

Antifungal assays using broth micro dilution method:

The antifungal activity was performed according to the standard reference method (NCCLS, 2002). The initial concentration of the acetone, ethanol and aqueous extracts of papaya peel extract was 2 mg/100µl of DMSO. The initial test concentration was serially diluted two-fold in 96 well plates. Each well was inoculated with 5 µl of suspension containing approximately 104 spore/ml. The antifungal agent, Ketoconazole was used as positive control.

 

RESULTS AND DISCUSSION:

Table: 1. Preliminary phytochemical screening of peel extract.

Test	Aqueous	Acetone	Petroleum Ether	Ethanol	Hexane
Carbohydrate	+	+	+	-	+
Tannins	-	-	-	-	-
Saponins	+	+	-	+	-
Flavonoids	-	-	+	-	+
Alkaloids	-	+	+	-	-
Quinones	+	-	-	+	-
Glycosides	-	-	-	-	+
Cardiac Glycoside	-	+	-	-	-
Terpenoids	+	+	-	-	-
Phenol	-	-	-	+	-
Coumarins	+	-	+	-	-
Steriods And Phytosteroids	+	-	-	+	+
Phlobatannis	-	+	-	-	-
Anthroquinones	-	-	-	-	-

-         indicates Absence,   + indicates  Presence

 

Table: 2 Quantitative estimation of total phenols, flavonoids and tannins.

S.No.	Extract	Phenol GAE/g	Flavonoid  meq /quertin	Tannin Mg/meq
1	Acetone	4.22	247.70	41.29
2	Ethanol	3.92	0.83	87.07
3	Aqueous	4.84	15.48	21.28

 

 

Results obtained from the estimation of total tannins contents reveals that acetone extract of C.papaya peel was found to possess higher polyphenols content (87.07mg/ meq of tannic acid) followed by ethanol extract (41.29mg/meq of tannic acid) and aqueous  extract with (21.28mg/meq of tannic acid)

 

 

Table : 3 Antioxidant assay- DPPH scavenging activity of Papaya extract and Ascorbic acid

 

 

Table 4 :Ferric reducing antioxidant power (FRAP) of Papaya Extract

Concentration  (µg)	Papaya Sample
	Acetone		Ethanol		Aqueous		Ascorbic acid
	Initial	Final	Initial	Final	Initial	Final	Std Initial	Std Final
200	1.164	1.388	0.413	0.577	0.088	0.116	1.782	2.012
600	1.573	1.687	0.702	0.811	0.146	0.236	2.088	2.297
1000	1.957	2.341	0.813	0.943	0.886	0.971	2.165	2.385

 

In the current study, Acetone has more scavenging activity (86.5%) followed by Ethanol (82.02%) and aqueous extract (31.68%).

 

 

Results obtained from this study indicates that acetone has highest radical scavenging with (1.388g/M FeSO₄ equivalents), followed by ethanol (0.5772g/M FeSO₄ equivalents) and aqueous extract (0.1168g/M FeSO₄ equivalent

 

 

Antimicrobial activity:

Agar well diffusion method:

Three different concentration of sample (1.25, 2.5 and 5 mg/100µl/well) were used in this study. Muller Hinton Agar (MHA) plates were inoculated with test organisms. The plates were evenly spread out. Then well were prepared in the plates with a cork borer. Each well was loaded with 0.1ml of corresponding concentration of sample. Chloramphenicol (10µg), DMSO was used as a Positive and negative control. The plates were incubated for 24h at 37°C. The development of inhibition zone around the well was measured and recorded

 

Table 5Antibacterial activity of sample by using well diffusion method

*B.s- Bacillus subtilis (ATCC 441), *E.c- E.coli (ATCC 25922), *K.p- Klebsiella pneumoniae (ATCC15380), *S.a- Staphylococcus aureus (ATCC 25923), *(-) no inhibition

*Positive Control – Chloramphenicol, *Negative Control (DMSO)

 

Table 6: Antifungal Activity

Name of the Pathogens	Antifungal Activity Minimum Inhibitory Concentration (MIC) Concentrations (2000µg/100µl)
	PAPAYA PEEL
	Acetone	Ethanol	Aqueous	Ketoconazole
Aspergillus flavus	500	1500	500	250

 

 

 

It has been demonstrated that C.papaya peel possesses a wide range of antibacterial effects, both with regard to Gram-positive and Gram-negative bacteria. The species most sensitive to C. papaya peel among the Gram-positive microorganisms was Bacillus subtilis, whereas among the Gram-negative species the most sensitive to C.papaya peel was Klebsiella pneumoniae.

 

Antifungal activity (MIC):

The result obtained from the MIC method demonstrate that ethanol has highest antifungal property when compared with acetone and aqueous extract.

The result obtained from the MIC method demonstrate that ethanol has highest antifungal property when compared with acetone and aqueous extract.

 

CONCLUSION:

Phytochemical investigation indicates the presence of high concentrations of polyphenols in papaya peel. Fruit peels investigated for phytochemical components appeared to have the efficacy to bud as a source of nutraceutical and improve the health status of the consumers. Drug resistance microbes are gaining resistance day by day towards the antibiotics and fruit peels could be tried to overcome these antibiotic resistant organisms. Moreover plants products have no side effect and can be consumed without the fear of adverse effects. Though a wide range of research has been carried out on the popular fruit papaya and its peel, standardisation and more clinical trials are needd to exploit the unrevealed therapeutic efficacy of Papaya peel.

 

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Received on 31.08.2016       Modified on 13.09.2016

Accepted on 25.09.2016      ©A&V Publications All right reserved