Bioactivity of Annona reticulata-An in vitro and in silico Approach

 

Ranjitha Dhevi V. Sundar1, Saranya Shankar1, Lokesh Ravi2, Sathiavelu Arunachalam1*

1School of Bio Sciences and Technology, VIT, Vellore - 632014, Tamil Nadu.

2Department of Botany, St Joseph’s College (Autonomous), Bengaluru, Karnataka, India.

*Corresponding Author E-mail: asathiavelu@vit.ac.in

 

ABSTRACT:

Aim: The study aimed to analyze the antibacterial, antioxidant and anti-inflammatory potential of two different extracts (Methanol and petroleum ether) of Annona reticulata leaves. Materials and Methods: Dried leaves powder of Annona reticulata was used to study the phytochemical analysis. Methanol and Petroleum ether extracts derived from leaves of A. reticulata were assessed for antioxidant by 2,2-diphenyl-1-picrylhydrazyl scavenging assay. Agar well diffusion method was used to study the antibacterial activity and in vitro anti‐inflammatory activity was evaluated using the albumin denaturation method. And the extracts were subjected to gas chromatography-mass spectrometry (GC-MS) analysis to identify its bioactive components. Results: Qualitative phytochemical screening of leaf powder confirmed the presence of tannins, alkaloids, phenols, flavonoids and saponins. Both the extracts demonstrated significant antibacterial activity against the test organisms, methanol extract showed the highest inhibition zone of about 28mm at 10mg concentration Whereas in petroleum ether extract showed maximum against E.coli with a zone of inhibition of 25mm. Petroleum ether leaf extract demonstrated a maximum anti-inflammatory activity of 83.4% at 150μg/mL concentration and also demonstrated significant antioxidant activity in DPPH assay of 75% at 150μg/mL concentration. In silico docking studies revealed the interaction of ligands from petroleum ether extracts with the seven common antibacterial proteins.

 

KEYWORDS: Annona reticulata, Medicinal plant, Antibacterial, Antioxidant, Anti-inflammatory, Auto Dock, Molecular docking.

 

 


INTRODUCTION:

Plants are considered to be a novel source for biologically active natural products. To produce exclusive and structure diverse bioactive compounds, they have been adapted over millions of years to withstand weather, fungi, bacteria and insects1. Medicinal plants are considered to be the “backbone” of traditional medicine. People from less developed countries depend on medicinal plants on a regular basis2. The India Rig-Veda which was written from 4500 to 1600 BC, was thought to be the oldest source of human traditional knowledge about usage of medicinal plants3. They play a vital role in developing human cultures throughout the world4.

 

Since ages, medicinal plants are used as remedies for treating human disease. Compared to synthetic drugs, they are considered to be natural and safe5. For treating various ailments, several species of plants have been used in folkloric medicine6. Still, some people in the urban area depend on medicinal plants for treating diseases like cough, bite, cold, fever and skin diseases7. Due to the presence of phytochemical ingredients, medicinal plants are used for healing and curing human diseases8.

 

Annona reticulata Linn. is a semi-deciduous tree belonging to the family Annonaceae, commonly referred to as Custard apple, sarifa, sitaphala and are native to South America and west indies. They cultivated widely throughout India and also in Pakistan and Bangladesh6,9. The genus Annona comprises of 119 species, most of them are trees and shrubs and are distributed in tropical and subtropical regions, including Southeast Asia such as Thailand, Malaysia, Laos and Indonesia10. They are known as bullock's heart having smooth skin fruit which becomes dull when ripe6. The plant parts were used in the ayurvedic medicinal system for treating various ailments9. And their fruits considered as a good tonic which used as an expectorant, cooling, decrease burning sensation and also relieves vomiting11. Annona reticulata used as anti-helminthic, anti-dysentric and antidiarrhoeic6. Traditionally they are used for treating cardiac problems, ulcer, constipation and also has antitumor and antifertility properties12. Here, we screened the bioactivity of the extracts obtained from A.reticulata and further, we tried to predict the antibacterial protein target with the compounds from petroleum ether extract.

 

MATERIALS AND METHODS:

Leaf Crude Extracts Preparation:

The fresh and healthy leaves of Annona reticulata were collected from Vellore, Tamil Nadu, India. The plant materials were collected in a sterile bag and transformed into the laboratory. To reduce the chance of contamination, the plant materials were freshly collected and utilized for further studies. The collected plant leaves were washed initially with running tap water and then with double distilled water. Further, the leaves were shade dried for 3-4 days at room temperature. After drying, the leaves were ground into a fine powder using an electric blender.

 

Around 15grams of plant leaf powder were dissolved in 200ml of different solvents namely methanol and petroleum ether respectively. Then the flask was sealed with parafilm and kept in a shaker for 48 hrs at 120rpm. After 48 hrs the plant extracts were filtered through Whatman No.1 filter paper. The solvents were concentrated by using a rotary vacuum evaporator. About 3 to 5grams of crude extract were obtained in each extract and these crude extracts were used for further studies13.

 

Qualitative Analysis of Phytochemicals

Phytochemical analysis was carried out to determine the presence and absence of Phenols, alkaloids, tannins, anthraquinone glycosides, saponins and flavonoids in the medicinal plant leaf powder. The preliminary qualitative phytochemical screening was carried out using a standard protocol14.

 

Determination of Total Phenol Content

Folin-Ciocalteu Photometric method was used to determine the total phenol content of Annona reticulata leaf extracts. In a conical flask, about 500µl of Folin-Ciocalteu reagent was mixed with 100µl of the extract and shaken well. Then 1.5ml of saturated sodium carbonate and 5ml distilled water were added to the mixture to dilute the solution. Then the samples were incubated at room temperature for two hours and the absorbance was recorded at 765nm using a UV-visible spectrophotometer. Gallic acid was used as a standard. Total phenolics content was expressed as mg Gallic acid Equivalents15.

 

Antioxidant Activity: DPPH free Radical Scavenging Assay:

The radical scavenging ability of two different leaf extracts such as methanol and petroleum ether of Annona reticulata was determined by DPPH free radical scavenging assay. The stock solution (1mg/ml concentration) was prepared by dissolving the crude extracts of the plant with methanol. Then 2ml of 0.1mM DPPH solution was mixed with 1ml of different plant crude extracts of varying concentration (50, 100, 150μg/ml) and the mixture was shaken well, incubated in dark condition for 45mins. Gallic acid was used as a reference standard. A mixture of 1ml methanol and 2ml DPPH solution was used as a control. The reaction was carried out in triplicates. The absorbance of the resulting solution was read at 517nm against the blank (containing solvent instead of extract) in a UV-Visible spectrophotometer. The ability of DPPH radical scavenging activity was calculated by using the following equation:16.

                       A control- A1 extract

Inhibition % = ----------------------------×100

                                    A control

 

Where A control - Absorbance of the control, A1 extract- Absorbance of the sample

 

In Vitro Anti-Inflammatory Activity: Egg Albumin Denaturation Method:

The anti-inflammatory activity of the methanol and petroleum ether plant extracts was determined using the egg albumin denaturation method. Various concentration of the crude extracts (100, 200, 300µg/ml) was prepared. The test solution 5ml consists of 2.8ml of phosphate-buffered saline (PBS, pH 6.4), 0.2ml of fresh hen’s egg albumin and 2ml of prepared plant leaf extracts. Equal volumes of De-ionized distilled water were used as a control. Then in a BOD incubator (Lab-line technologies), the solutions were incubated at (37°C ± 2) for 15 min and further, the solutions were heated for 5 min at 70°C. The absorbance was measured at 660 nm after cooling. Acetyl salicylic acid (Aspirin) was used as a standard. The inhibition percentage of protein denaturation was calculated by the following equation17.

                      Abs control – Abs test

% Inhibition = ----------------------------- X 100

                          Abs control  

 

Where Abs control = Absorbance of the control, Abs test = Absorbance of the test sample

 

 

 

Antibacterial Activity: Agar Well Diffusion Method:

Agar well diffusion method was carried out to determine the antibacterial activity of the crude extracts. To the prepared Muller-Hinton agar plates, the bacterial cultures such as Escherichia coli (MTCC 1687), Proteus mirabilis (MTCC 3310), Bacillus cereus (MTCC 0430), Klebsiella pneumonia (MTCC 7028) and Staphylococcus aureus (MTCC 3160) were spread uniformly using a cotton swab. About 0.5mm wells were made on the medium using cork borer. To the well, 100µl of different concentrations (25, 50 and 100mg/ml) methanol and petroleum ether crude extracts were added respectively and the plates were incubated at 37şC for overnight and zone of inhibition was measured. Streptomycin was used as a positive control18.

 

Gas Chromatography-Mass Spectrometry (GC-MS) Analysis:

The crude extracts obtained from Annona reticulata containing different compounds were subjected to GC-MS analysis. Each extract was analyzed in Perkin Elmer Clarus 680 equipped with mass spectrometer Clarus 600 (electron ionization) which was fitted with Elite-5MS Capillary column (30m,0.25mmID, 250µmdf). The GC oven was maintained at an initial temperature of 600C for 2 mins ramp 10C/min-300oC, for about 6mins. The temperature was maintained at 3000C. The carrier gas used here was helium with constant flow rate 1mL/min, mass transfer line and source temperature were set at 240oC. For spectral analysis, turbo mass version 5.4.2 software was used. By comparing the components spectrum with the database of the spectrum of known components stored in the NIST-2008 library, the structures were determined19.

 

In silico Docking Analysis:

The 3D structure of the common antibacterial target proteins was downloaded from the PDB (Protein data bank) website (http://www.rcsb.org/pdb). The PDB ID of downloaded structure”1AD1, 2INR, 2Y2I, 3G75, 3VMR, 4E2K, 4FGG”. The protein structures were prepared for docking, by removing the non-amino acid residues were removed using PyMOL software. The ligand molecule was drawn using the Chemsketch tool. Ligand and protein interaction was studied using Autodock 4.2(MGL tools). PyMOL is used for viewing the results obtained from AutoDock19.

 

RESULTS AND DISCUSSION:

Phytochemical Analysis:

Dry leaf powder of Annona reticulata demonstrated the presence of phenols, tannins, saponins, flavonoids, and alkaloids. Phytochemical class sterols, triterpenes and glycosides were absent in the leaves. The results of the qualitative phytochemical analysis are tabulated in Table 1.

Determination of Total Phenol Content:

Folin-Ciocalteu’s assay was used to determine the total phenol content of Annona reticulata leaf extracts. When compared to petroleum ether extract (10.89±0.001mg of (gallic acid equivalent) GAE/g of extract), the highest concentration of phenol (11.75±0.002 mg of GAE/g of extract) was observed in the methanolic leaf extract. This is due to the high polarity of phenolic compounds.

 

Antioxidant Activity: DPPH Radical Scavenging Activity:

In the present study, both Petroleum ether and Methanol extracts were evaluated for their inhibitory effect on DPPH by the in-vitro method. Compared to methanol, petroleum ether extracts showed the highest free radical scavenging activity of 75.32±1.07% at 150μg/ml concentration. This signifies that the petroleum ether extract is a potent antioxidant agent. The inhibition percentage of methanol and petroleum ether extracts were displayed in Fig.1.

 

 

Figure.1: DPPH free radical scavenging of Leaf Extracts

 

In-Vitro Anti-Inflammatory Activity: Egg Albumin Denaturation Method:

Inhibition of protein denaturation was performed to evaluate the anti-inflammatory activity Petroleum ether leaf extract demonstrated a maximum inhibition of 83.4±1.33% at 100μg/mL concentration and the standard showing 92.6±1.02% at 100μg/mL concentration. The result of the anti-inflammatory activity is shown in Fig.2.

 

Figure.2: In vitro anti-inflammatory activity of Leaf Extracts

Antibacterial Activity: Agar Well Diffusion Method:

The extracts showed significant activity against the test organism. Streptomycin was used as standard drugs showing antibacterial activity At 10mg/100μl, the methanol extract showed maximum activity against P. mirabilis with a zone of inhibition of 28mm. Whereas in petroleum ether extract showed maximum against E.coli with a zone of inhibition of 25mm.The results of methanol and petroleum ether leaf extracts shown in Table 1.


 

Table. 1: Zone of Inhibition of extracts at various concentrations

Plant extracts

Concentration

(mg)

Zone of inhibition measured in millimeter

Staphylococcus aureus

(MTCC-3160)

Proteus mirabilis (MTCC-3310)

Bacillus cereus (MTCC-0430)

Escherichia coli (MTCC-1687)

Klebsiella pneumoniae (MTCC-7028)

Methanolic

extract

2.5

-

12

9

-

-

5

-

21

10

-

-

10

13

28

12

11

-

Petroleum ether extract

2.5

15

15

15

14

13

5

16

19

18

17

-

10

21

20

21

25

12

Streptomycin (10µg)

10

23

21

25

19

17

 


GC-MS ANALYSIS:

GC-MS analysis was done to determine the retention time, Molecular weight and chemical structure of methanol and petroleum ether extracts were shown in Table 2. The chromatogram was shown in Figure.3.


 

Table.2: List of NIST library matches for compounds present in the extract

RT

Rev

For

M.W

Compound name

Structure

Methanol extract

13.72

660

448

150

3-Undecen-5-yne, (E)-

 

14.88

557

386

135

1,3,6-Octatriene, 3,7-dimethyl-

 

18.66

704

462

144

Hexane, 1-propoxy-

 

18.96

561

413

166

Bicyclo [2.2.1]heptane-2,3-dione, 1,7,7-trimethyl-

 

22.21

706

416

438

Heptacosanoic acid, 25-methyl-, methyl ester

 

23.71

476

319

186

Heptanoic acid, 2,2-dimethyl-6-oxo-, methyl ester

 

25.80

758

402

284

Nonane, 1,9-dibromo-

 

26.17

606

370

222

Ethanol, 1-(methylenecyclopropyl)-1-(methylene-1-trimethylsilylc)

 

Petroleum ether extract

2.84

617

486

189

Phenyl-piperidin-3-yl-methanone

 

13.68

517

346

163

1,4-Benzoguinone, 2,6-diethyl-, 1-imine

 

14.42

505

310

162

2(1H)-Naphthalenone, 4A,5,8,8A-tetrahydro-4A-methyl-, trans-

 

18.84

596

396

208

Bis-(1,7-diazabicyclo [2.2.0] hept-7-YL) methane

 

19.21

691

478

114

(1R,2R)-(-)-1,2-diaminocyclohexane

 

22.07

703

542

154

Bicyclo [3.3.1] nonan-2-one, 9-hydroxy-, syn-

 

24.49

687

410

524

2-Isopropyl-5-methylcyclohexyl 3-(1-(4-chlorophenyl)-3-oxobutyl)-C

 

28.43

594

399

186

Isopentyl 3-hydroxy-2-methylenebutanoate

 

 

 

 

Figure.3: Gas chromatography‑mass spectrometry chromatogram of Annona reticulata leaf extracts (a) crude Methanol extract, (b) crude Petroleum ether extract

 


In silico Antibacterial Activity:

Antibacterial activity was predicted by molecular docking studies. The interactions of 11 different compounds obtained from GC-MS of Petroleum ether extract were studied with 7 common antibacterial targets proteins Dihydropteroate Synthetase (1AD1), Topoisomerase IV (2INR), Penicillin-Binding Protein 1 (2Y2I), Gyrase B (3G75), Transglycosylase (3VMR), DNAG (4E2K), Dihydrofolate Reductase (4FGG) were studied by in silico molecular docking. 1,4-Benzoguinone, 2,6-diethyl-, 1-imine and Isopentyl 3-hydroxy-2-methylenebutanoate are the major compounds in petroleum ether extract. Among the 7 targeted proteins, 1,4-Benzoguinone, 2,6-diethyl-, 1-Imine exhibited the highest affinity towards 4E2K with a free binding energy of -6.2 Kcal/mol with the formation of 3 hydrogen bonds and Isopentyl 3-hydroxy-2-methylenebutanoate exhibited towards 4FFG with -5.8 Kcal/mol with two hydrogen bonds. And 2-Isopropyl-5-methylcyclohexyl 3-(1-(4-chlorophenyl) -3-oxobutyl)-C is minor compound but exhibited the highest affinity of -9.7Kcal/mol towards 4FGG. Results of the protein-ligand docking studies proteins were given in Table 4. Graphical representation of the interaction was shown in Fig.4 and 5.

 


 

 

Table. 3: Auto Dock results of Petroleum ether extract ligands with Bacterial Drug Target Proteins.

Protein

1AD1

2INR

2Y2I

3G75

3VMR

4E2K

4FGG

Ligand

Binding Energy

No. of H-Bonds

Binding Energy

No. of H-Bonds

Binding Energy

No. of H-Bonds

Binding Energy

No. of H Bonds

Binding Energy

No. of H-Bonds

Binding Energy

No. of H-Bonds

Binding Energy

No. of H-Bonds

E=147.21

-5.8

0

-5.6

0

-6

1

-6.3

2

-6.8

0

-6.5

1

-6.9

0

E=91.78

-5

1

-5.2

1

-5.6

2

-5.9

2

-5.4

0

-6.2

3

-5.7

1

E=108.80

-5.2

1

-5.2

0

-5.9

2

-5.9

1

-5.7

0

-6.1

2

-6.8

1

E=-0.59

-4.4

0

-4.6

0

-4.4

0

-5

0

-5.7

0

-4.7

0

-5.2

0

E=429.15

-6.1

1

-5.5

1

-5.3

0

-5

0

-5.9

0

-4.9

0

-6.8

2

E=222.97

-4.1

3

-4.1

4

-4.4

3

-4.5

3

-4.1

1

-5.1

3

-4.5

1

E=1272.70

-5

1

-5.5

1

-5.5

3

-5.8

1

-5.4

4

-6.9

5

-5.9

1

E=67.11

-4.7

3

-4.5

3

-5

1

-4.9

1

-4.8

5

-5.1

2

-5.5

2

E=63.44

-4.8

0

-4.5

1

-4

2

-4.5

1

-5.4

0

-4.4

1

-5.6

1

E=410.07

-6.7

3

-6.4

1

-6.7

3

-5.7

4

-8.4

2

-7

2

-9.7

0

E=54.45

-4.9

5

-4.7

4

-5.1

6

-5.3

3

-5

7

-5.2

9

-5.8

2

Note: Binding Affinity (Kcal/mol)

 

Figure.4: (A) 1,4-Benzoguinone, 2,6-Diethyl-, 1-Imine with 4E2K, (B) Isopentyl 3-hydroxy-2-methylenebutanoate with 4FFG Protein

 

 


CONCLUSION:

The Present study on antibacterial, antioxidants, anti-inflammatory activity adds significance to the medicinal property of the Annona reticulata. Both methanol and petroleum ether extracts of Annona reticulata serve as a potential antibacterial agent against the test organism under in vitro condition. When compared to methanol, petroleum ether extract showed maximum antibacterial activity. In silico docking studies showed the interaction of ligands from petroleum ether extracts with the seven common antibacterial proteins. Thereby the study concluded that antibacterial activity of leaf extract of Annona reticulata and its active constituents may help interact with various kinds of plant disease and human allergies.

 

ACKNOWLEDGMENT:

The authors thank VIT for providing the lab facility to carry out this study.

 

CONFLICTS OF INTEREST:

The author declares no conflicts of interest.

 

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Received on 18.03.2020           Modified on 01.05.2020

Accepted on 24.06.2020           © RJPT All right reserved

Research J. Pharm. and Tech 2021; 14(3):1542-1548.

DOI: 10.5958/0974-360X.2021.00281.X