Investigation on the Antimicrobial and Antioxidant Activity of Custard Apple (Annona reticulata) Peel Extracts

 

Dr. D. Esther Lydia1, Dr. Sheila John2, Swetha V K3, Mrs. Thiyagarajan Sivapriya4

1Assistant Professor, Loyola College, Chennai

2Associate Professor, Womens Christian College, Chennai

3Student, Loyola College, Chennai

4Assistant Professor, SDNB Vaishnav College for women, Chennai

*Corresponding Author E-mail: sivaamanick@gmail.com

 

ABSTRACT:

Background: Custard apple or sugar apple can be called as a delicacy of dry region due to its very sweet delicate flesh. The peel of custard apple contains tannins, acetogenin and alkaloids which is beneficial in the treatment of several types of cancer and tumors. Methodology/Principal Findings: The present study was taken up to investigate the phytochemical composition, antimicrobial potential, antioxidant activity of the fruit peel wastes of custard apple. The phytochemical screening of the fruit peel revealed the presence of Carbohydrates, Saponins, Phenols and Terpenoids .The antioxidant property of the peel extract was evaluated using DPPH free radical scavenging and FRAP assay. The antimicrobial test results showed that the raw fruit peel extract had a great potential antimicrobial activity against all the bacteria and fungal species selected for testing.Conclusions/Significance: The results presented here may suggest that the raw fruit peel extracts possesses antioxidant and antimicrobial properties, and is therefore a potential source of ingredients for the food and pharmaceutical industry.

 

KEYWORDS: Annona reticulata,  Antimicrobial, Antioxidant

 

 


INTRODUCTION:

The custard apple of India, the sitaphal or sugar apple is one of the finest fruits introduced in India from tropical America and found in wild form in many parts of the country. It is common in China; Phillippines, Egypt and Central Africa.The fruit is rich in carbohydrate mainly in the form of sugar (23.5%), protein (1.6%), calcium (17mg/100g), and phosphorus (47mg/100g), Iron (1.5mg/100g). The peel of custard apple contains astringent properties and tannins, which is utilized for making herbal supplements.

 

The fruit also contains compounds like acetogenin and alkaloids that reduce the risk of cancer and renal failure. It acts against cancer cells, without adversely affecting healthy cells. Antioxidants such as asimicin and bullatacin are also found to have anti-cancer and anti-helminthes properties. These antioxidants neutralize the effects of free radicals, preventing cancer. It also contains significant fiber, which protects the colon membrane by warding off toxic substance from the gut, reducing the risk of liver and colon risk.

 

In the recent years, apart from medicinal plants, fruits, which are a rich source of bioactive compounds, have become popular subjects for such investigations. Considerable amount of solid wastes in the form of peels and seeds are generated by the fruit processing industries, and these wastes if not disposed correctly are seen to cause serious environmental problems such as water pollution, unpleasant odors, explosions and combustion, asphyxiation, and greenhouse gas emissions. There are several reports highlighting the integral exploitation of bioactive compounds from these wastes and their potential applications as antioxidant, antimicrobial, flavoring, colorant, and texturizer agents [1] Solid wastes, particularly from processes such as peeling and coring, typically have a high nutritional value and may be used as animal feed[2]. Obtaining value added products from fruit wastes economizes the fruit processing units.Research has been carried out to exploit the antimicrobial potential of methanolic, ethanolic and aqueous extracts of custard apple peels[3] The sugar components of custard apple peels were explored with a view to establishing their raw material potential and TLC results indicated the presence of lactose, sucrose, galactose and glucose[4]

 

The present study was taken up to investigate the various phytochemicals present in custard apple peel, antimicrobial potential, and the antioxidant activity of the fruit peel wastes of custard apple and thereby pave way for indirect waste management by transforming these wastes into a health resource.In view of these opinions and observations, the present investigation has been undertaken to understand the importance of food sustainability and to increase the sustainability of food systems by making use of the fruit waste (peel) that is taken for the study with the following set of objectives.

 

MATERIALS AND METHODS:

Collection and authentication of plant:

The custard apple was purchased from Koyembedu market, Chennai, India and authenticated. The peel was manually separated and dried at room temperature at 32°C for one day. The dried peel was subjected to grinding.

 

Sequential extraction:

The dried powder of the peel was extracted sequentially [5] by soxhlet apparatus, using different solvents depending upon their polarities like hexane, methanol 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 evapour.

 

Qualitative phytochemical analysis:

The phytochemical tests was carried out using standard methods of analysis of carbohydrates, tannins, saponins, flavanoids, alkaloids, quinines, glycosides, cardiac-glycosides, terpenoids, triterpenoids, coumarins, steroids, phytosteroids, phlobatanins and anthroquinones[6]

 

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 [7] 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. The various concentrations of the extract were made up with 900µl of distilled water and 0.5ml ofFolin-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 [8] 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 activity:

The antioxidant activity of the different solvent extracts of custard apple peel was evaluated by DPPH free radical and superoxide anion radical scavenging assays and isolated fractions were further evaluated by other antioxidant parameters like FRAP assay, Nitric oxide Scavenging assay and hydrogen peroxide scavenging 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[9]. 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[10] The percentage inhibition was determined by comparing the results of the test and the control. Percentage of inhibition was calculated using the formula,

 

 

Ferric reducing antioxidant power (FRAP):

The reducing ability of different solvent extracts and fractions of the custard apple peel was determined by FRAP assay [11] 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 37°C for 10 minutes and then the absorbance was measured at 593nm. FeSO4 (100 to 1000µM ml-1) was used as a positive control The antioxidant capacity based on the ability to reduce ferric ions of sample was calculated from the linear caliberation curve and expressed as M FeSO4 equivalents per gram of extracted compound.

 

Antimicrobial activity of custard apple peel:

The antimicrobial activity of different extracts of custard apple peel was studied by agar disc diffusion method on two gram positive, two gram negative bacteria and one fungal pathogen. The bacterial strains used were Bacillus subtilis NCIM 2106, Staphylococcus aureas NCIM 2127, Pseudomonas aeruginosa NCIM 5031 and E. coli. The fungal strain used was Helminthosporium oryzae.

 

The Mueller-Hinton agar medium was innoculated with freshly prepared cells of each bacteria and fungi to yeild a lawn of growth. After solidification of the agar, well was made using well cutter and extract solutions (100 and 50 µg ml-1) were placed on the plates. After incubation at 37°C for 24h for bacteria and 25°C for 72h for fungi, the antimicrobial activity was measured as diameter of the inhibition zone formed around the disk. At the same time, a comparison antibiotic control test was made using commercial disks. Negative control was used as the control. The diameter of inhibition zone around each disk was measured and recorded at the end of incubation period.

 

Diameter zone of inhibition of 10 or less indicates test product being resistant to test organism, diameter zone of inhibition of 11 to 15 indicates test product being intermediate resistance to test organism, diameter zone of inhibition of 16 or more indicates test product being susceptible resistance to test organism.

 


 

 

 

RESULT AND DISCUSSION:

Table IPreliminary Phytochemical Screening of Annona reticulata peel

PhytochemicalTest

Aqueous

Acetone

Petroleum Ether

Ethanol

Chloroform

Carbohydrates

+

+

+

+

+

Tannins

+

+

-

-

+

Saponins

+

+

+

+

+

Flavanoids

+

+

+

-

-

Alkaloids

+

-

+

-

-

Quinones

-

+

+

+

+

Glycosides

-

+

-

-

-

Cardiac Glycosides

-

-

-

-

-

Terpenoids

+

+

+

+

+

Phenols

+

+

-

+

+

Coumarins

-

-

-

-

-

Steroids & Phytosteroids

+

-

-

-

-

Phlobatannins

-

-

-

-

-

Anthroquinones

-

-

-

-

-


 


The phytochemical screening of Annona reticulata, peel extracts revealed the presence of carbohydrates, tannins, saponins, flavanoids, alkaloids, quinones, terpenoids and phenols. Carbohydrates, Saponins, Phenols and Terpenoids were present in all the five extracts. Tannins were present in aqueous, acetone and Chloroform extracts. Flavonoids were present in aqueous, acetone and petroleum ether extracts. Alkaloids were present in aqueous and petroleum ether extract.

 

The results for the total phenol content of A.reticulata peel is presented in Table 2 (b).

Results obtained from the estimation of total phenol content reveals that acetone extract of the peel was found to possess higher polyphenols content (5.19 mg GAE/g) compared with ethanol extract(2.468 mg GAE/g) and aqueous extract(4.624 mg GAE/g). The result found out that the phenol content fall in the range of (5.72 to 77 mg). Therefore, the acetone extract of A.reticulata was found to posse’s higher polyphenols content. (Table 2 and 2b).

 


 

Quantitative phytochemical analysis -Table – 2 (a) Estimation of total phenol

Particulars

B

S-1

S-2

S-3

S-4

S-5

Acetone Extract

Ethanol Extract

Aqueous Extract

Gallic acid (ml)

 -

0.2

0.4

0.6

0.8

1.0

 -

 -

 -

Test Sample (ml)

 -

 -

 -

 -

 -

 -

0.1

0.1

0.1

 Distilled water (ml)

1.0

0.8

0.6

0.4

0.2

 -

0.9

0.9

0.9

FCR (ml)

0.5

0.5

0.5

0.5

0.5

0.5

0.5

0.5

0.5

2% Sodium Carbonate (ml)

1.5

1.5

1.5

1.5

1.5

1.5

1.5

1.5

1.5

 

 

 

Incubate for 15 minutes

 

 

 

OD at 765nm

0.00

0.12

0.23

0.34

0.45

0.56

0.55

0.35

0.48

Concentration of the sample (mg/ml)

 -

 -

 -

 -

 -

 -

5.19

2.468

4.624


 

Table 2 (b):

S.No.

Extract

Total  Phenol  Content Concentration (mg GAE/g)

1

Acetone

5.19

2

Ethanol

2.468

3

Aqueous

4.624

 

 

Fig.1 Standard Calibration Curve – Gallic Acid

 

Table – 3 Estimation of Total Flavanoid Content

Particulars

B

S-1

S-2

S-3

S-4

S-5

Acetone Extract

Ethanol Extract

Aqueous Extract

Quercetin (ml)

 -

0.125

0.25

0.375

0.5

0.625

 -

 -

 -

Test Sample (µl)

 -

 -

 -

 -

 -

 -

100

100

100

95% Ethanol (ml)

2

1.9

1.8

1.7

1.5

1.4

1.95

1.95

1.95

Distilled water (ml)

2.8

2.8

2.8

2.8

2.8

2.8

2.8

2.8

2.8

10% Aluminium Chloride (ml)

0.1

0.1

0.1

0.1

0.1

0.1

0.1

0.1

0.1

1M Potassium Acetate (ml)

0.1

0.1

0.1

0.1

0.1

0.1

0.1

0.1

0.1

 

 

 

Incubate for 30 minutes

 

 

 

OD at 415 nm

0.00

0.11

0.21

0.32

0.39

0.49

0.15

0.12

0.20

Concentration of the sample (mg/ml)

 -

 -

 -

 -

 -

 -

2.128

0.18

4.34

Table 3 (b) Results of total flavanoid content

S.No.

Extract

Total  Flavanoid  Content Concentration (mg Qc/g)

1

Acetone

2.128

2

Ethanol

0.18

3

Aqueous

4.34

 

 

Fig. 2 Standard Calibration Curve of Quercertin

 


Results obtained from the estimation of total flavanoid content reveals that aqueous extract of the peel was found to posses higher flavanoid content (4.34 mg/meq of Quercertin) compared with Ethanol extract (0.18 mg/meq of Quercertin) and acetone extract (2.128 mg /meq of Quercertin). The result obtained is compared with the findings of [12] who found out that the flavanoid content fall in the range of (7.26 to 8.49 mg). Therefore, the aqueous extract of A.reticulata was found to posses higher flavanoid content. (Table 3 and 3b).


 

Table 4 - Estimation of Total Tannin Content

Particulars

B

S-1

S-2

S-3

S-4

S-5

Acetone Extract

Ethanol Extract

Aqueous Extract

Tannic acid (ml)

 -

0.5

1.0

1.5

2.0

2.5

 -

 -

 -

Test Sample (µl)

 -

 -

 -

 -

 -

 -

100

100

100

Distilled water (ml)

8.5

8

7.5

7.0

6.5

6.0

8.5

8.5

8.5

FCR (ml)

0.5

0.5

0.5

0.5

0.5

0.5

0.5

0.5

0.5

2% Sodium Carbonate (ml)

1

1

1

1

1

1

1

1

1

 

 

 

Incubate for 15 minutes

 

 

 

OD at 765nm

0.00

0.05

0.11

0.17

0.23

0.29

0.28

0.10

0.17

Concentration of the sample (mg/ml)

 -

 -

 -

 -

 -

 -

10.496

1.36

2.04

 

Table 4 (b) Results of total tannin content

S.No.

Extract

Total  tannin  Content Concentration (mg TAE)

1

Acetone

10.496

2

Ethanol

0.28

3

Aqueous

2.04

 

Fig. 3 Standard Calibration Curve of Tannic Acid


Results obtained from the estimation of total tannin content reveals that acetone extract of the peel was found to possess higher tannin content (10.496 mg/meqofTannic acid) compared with Ethanol extract and aqueous extract. The tannin content of A.reticulata is comparatively higher (10.496 mg/meqofTannic acid) than that of a study carried out by [13] who estimated as (7.92 mg/meqofTannic acid). (Table 4 and 3b).

 

The results obtained from the DPPH assay illustrates a significant decrease in the concentration of DPPH radical due to the scavenging ability of standard Butylated Hydroxy Toluene (BHT). The scavenging effect of the standard on the DPPH radical decreased in the order of 96.70%, 95.04%, 90.08%, 49.58%, and 35.53%, at the concentration of 78 μg/mL, respectively. (Table 5).


 

Table 5-Quantitative antioxidant study of A.reticulata peel using DPPH Assay

Solvent

 

 

Ti

me in minutes

 

 

0

5

10

15

20

25

30

Ethanol

0.32

0.06

0.06

0.06

0.06

0.06

0.06

Petroleum Ether

0.83

0.78

0.78

0.78

0.78

0.78

0.78

Acetone

0.35

0.05

0.04

0.04

0.04

0.04

0.04

Chloroform

0.77

0.66

0.64

0.64

0.63

0.63

0.61

Aqueous

0.17

0.12

0.12

0.12

0.12

0.12

0.12

 

Fig.4 DPPH – Radical scavenging aactivity of A.reticulata peel

 

Table 6 - Activity of Ferric reducing antioxidant power

Particulars

B

S-1

S-2

S-3

S-4

S-5

Acetone Extract

Working Standard

 -

0.2

0.4

0.6

0.8

1.0

 -

Test Sample (µl)

 -

 -

 -

 -

 -

 -

0.4

Distilled water (ml)

1

0.8

0.6

0.4

0.2

 -

0.6

FRAP reagent

0.5

0.5

0.5

0.5

0.5

0.5

0.5

OD at 765nm

0.00

0.30

0.56

0.81

1.07

1.29

0.45

Concentration of the sample (mg/ml)

 

 

 

 

 

 

720

 

Fig.6 Ferric reducing antioxidant power (FRAP)



Table 7- Antimicrobial activity of Annona reticulata peel

SOLVENT

 

MICROORGANISM

ZONE OF INHIBITION (mm)

10mg/ml

20mg/ml

30mg/ml

Negative Control

Acetone

Bacillus subtilis

16

18

21

-

Staphylococcus areus

20

18

23

-

Pseudomonas aeruginosa

20

17

24

-

E.coli

19

20

22

-

Ethanol

Bacillus subtilis

10

21

24

-

Staphylococcus areus

19

20

22

-

Pseudomonas aeruginosa

0

10

12

-

E.coli

0

11

19

-

Aqueous

Bacillus subtilis

0

15

20

-

Staphylococcus areus

17

19

23

-

Pseudomonas aeruginosa 

0

0

21

-

E.coli

0

18

25

-

 

 

 


The results obtained from this study indicate that the acetone extract of the peel has highest radical scavenging activity (720g/M FeSO4 equivalents). (Table-6).

 

Results from the study reveal that the acetone extracts of Annona reticulata, illustrated extensive range of antibacterial activity. The result of antibacterial screening by agar disc diffusion method (Table 7) designates that elevated zone of inhibition was shown by the acetone extract Annona reticulata for Staphylococcus areus as 23mm/30 µl and lowest for E.coli as            11mm /20 µl. (Table-7)

 

 

CONCLUSION:

Annona species are already used for the preparation of medicinal products with high values. Annona reticulata peels investigated for phytochemical components appeared to have the properties of anutraceutical and improve the health status of the consumers when consumed regularly. Drug resistance microbes are gaining resistance day by day towards the antibiotics and fruitpeels of Annona reticulata could be used as a source for antibiotic toovercome antibiotic resistant organisms as plant foods can be consumed without the fear of adverse effects. Though multitude ofexperiments has been carried out on Annona fruit peel, standardisation and more clinical trials are need to exploit the unrevealed therapeutic efficacy. Annona reticulata species could be studied further for making use of its flavour properties in food.

 

REFERENCES:

1.       Annie S, Rajendran K Dinesh Kumar V and Ramgopal B. Antidiabetic activity of aqueous leaf extracts of Annona reticulata in streptozotocin–nicotinamide type 2 diabetic rats. Journal of Ethnopharmacology. 2004; 91: 171–175.

2.       Vijay K and Sriram S, Antioxidant activity of seed extracts of Annona reticulata and Carica papaya. Nutrition and Food Science. 2010; 40 (4): 403-408.

3.       Vohar, SB, Ishwar Kumar and Naquvi SA. Phytochemical, Pharmacological, antibacterial and anti-ovulatory studies on Annona squamosa. Planta Med. 1975; 28: 97-100.

4.       Zhao GX, Rieser MJ, Hui YH, et al. Biologically active acetogenins from stem bark of Asiminatriloba. Phytochemistry.1993; 33(5):1065-1073.

5.       Fang C, Jien C, Chih-Yuan L, Hui-Fen C, Ming-Juan W, and Yang-Chang W. Bioactive acetogenins from the seeds of Annona atemoya. Phytochemistry.1999 51: 883-889.

6.       Wiart C, Au T.S, Mohd Y, Hamimah, H. and Sulaiman, M. An antibacterial diterpene from sweet apple (Annona reticulata L. Annonaceous). International Journal of Pharmacology.2005: 1 (3) :296-298.

7.       Trease and Evans. Text book of Pharmacognosy 13 th Edition 1989

8.       Niwano Y, Beppu, F, Shimada T, Kyan R, Yasura K, Tamaki M, Nishino M and Hamada, H. Extensive screening for plant foodstuffs in Okinawa, Japan with anti-obese activity on adipocytes in vitro. Plant Foods Human Nutrition. 2009; 64:6-10.

9.       Chang CJ and Mc Laughlin, J. L. Structure-activity relationships of diverse Annona ceousacetogenins against multi-drug resistant human mammary adenocarcinoma (MCF-7/Adr) cells. Journal Medical Chemistry. 1997; 40; 2102-2106.

10.     George A, Broadley R, and Nissen, R. Preliminary review of the health and medicinal benefits of Annona spp. possible paths to commercialisation. A confidential report for the Australian Custard Apple Growers Association. (2006).

11.     Alali, Liu, X and McLaughlin, J. L. Annonaceousacetogenins: recent progress. Journal of Natural. Products. 1999; 62: 504-540.

12.     Bonavia D, Ochoa C, Tovar S, and Palomino R. Archaeological evidence of cherimoya (Annona cherimola Mill.) and guanabana (Annona reticulata L.) in ancient Peru. Economic Botany. 2004; 58(4): 509-522.

13.     Kaleem M, Asif M, Ahmed Q and Bano, B. Anti-diabetic and antioxidant activity of Annona squamosa extract in streptozotocin-induced diabetic rats. Singapore Medical Journal. 2006; 47(8): 670-675.

 

 

 

 

 

 

Received on 19.08.2017       Modified on 11.09.2017

Accepted on 19.11.2017       ©A&V Publications All right reserved

Res.  J. Pharmacognosy and Phytochem. 2017; 9(4): 241-247.

DOI: 10.5958/0975-4385.2017.00045.0