In vitro Antioxidant and Antimicrobial Activities of Flowers Extract of Aegle marmelos

 

Vijay Pradhap Singh. M1*, Narmadha. P1, Rajamehala. M1, Jeyavel Karthick. P2

1Department of Biotechnology, Vivekanandha College of Engineering for Women (Autonomous), Elayampalayam, Tiruchengode - 637205, Tamilnadu, India.

2Department of Biotechnology, Bannari Amman Institute of Technology,

Sathyamangalam – 638401, Erode District, Tamil Nadu, India.

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

 

ABSTRACT:

Medicinal plants have various therapeutic and nutraceutical properties from the ancient times to the current scenario. These plants have been consistently noted for the presence of its bioactive compounds which has a significant effect on human. Plants have been traditionally and extensively used to cure disease. Aegle marmelos plant belonging to Rutaceae family found in various parts of India, Srilanka, Pakistan, Burma, and Bangladesh is used in treating variety of diseases. Antioxidant and Antimicrobial activity of Methanol and Aqueous extracts was evaluated. Flower extract of A.marmelos exhibits significant antioxidant and possess antibacterial activity against E.coli, S.aureus, and K.pneumoniae. Thin Layer Chromatography was performed and the results revealed that spots were observed in crude methanol and aqueous extract.

 

KEYWORDS: Aegle marmelos, Rutaceae, Pharmacology, Antioxidant, Antimicrobial.

 

 


INTRODUCTION:

Medicinal plants, the endowment from nature is the untapped resource of therapeutic compounds from the ancient days to present century. According to World Health Organization (WHO), chemical compounds that are synthesized from plants find its application in treating numerous diseases1. Drugs that are produced from herbal plants have merits compared to other resources in treatment2,3. Understanding the nature and the different chemical constituents of the herbal plants is an important factor in drug discovery4. Aegle marmelos belongs to the Rutaceae family is considered as the important plants with numerous therapeutic and nutraceutical properties5,6. Nowadays, this plant is available across the globe, but the native country where it originated is India7. It is a slow growing, medium sized, subtropical tree8,9. Whole part of this tree starting from root to fruit is used in various applications10,11. It is a source of phytomedicine.

 

 

It has many medicinal properties like antimicrobial, antiulcer, antioxidant, anticancer, anti-inflammatory, analgesic, antidiabetic, antipyretic activities12,13. Different studies conducted by researchers on bael across worldwide revealed that plant is rich in phytoconstituents as well as nutritional compounds impart plenteous health benefits to humans14.

 

 

Figure 1 A.marmelos flower

 

In Ayurvedic tests, Aegle marmelos is considered to be the important plant as it has therapeutic properties. Inspite of that, various  investigations in this plant are going on12,15. The Aegle marmelos flower in the plant possess both stamens and carpels and it is bisexual and hypogynous in nature, pleasant and fragrant scent, colour appears as greenish white10. Flower extract exhibit astringent, antiseptic, wound healing properties and also it is used in treating conjunctivitis and epilepsy 16,17. This study is a step towards gaining a better understanding of the properties of the flowers of Aegle marmelos plant. It gives a detailed knowledge about the antioxidant and antimicrobial activities of its flowers and to analyze the components in the extract by performing Chromatography.

 

MATERIALS AND METHODS:

Collection of Plant Sample:

Flowers of Aegle marmelos was collected from the field in and around Coimbatore. The collected materials were washed thoroughly, shade dried, crushed and finally powdered. The finely powdered flowers were stored at the room temperature in a polythene bag until it is required for its usage.

 

Preparation of Plant Extract:

Extraction was carried out using methanol and water by dissolving 1gram of dried flower in 20ml of solvent. The mixture is placed in the shaking incubator at 40oC for 24 hours. After 24 hours of incubation, the solution is filtered and the solvent is extracted. A Similar procedure was performed to obtain the methanol extract of Aegle marmelos flowers.

 

Microorganisms Used:

Pathogenic microorganisms (E.coli, Staphylococcus aureus, Klebsiella pneumoniae) were used. All the strains were collected from Microbiology Lab, Vivekanandha College of Arts and Science for Women, Tiruchengode.

 

Chemicals and Reagents:

Methanol; 1,1–Diphenyl-2-picrylhydrazyl; Tris HCl were purchased. All the chemicals and reagents used were of analytical grade. Antibacterial drug Imipenem was used as a standard antibiotic to compare with plant crude extract.

 

Evaluation of Antioxidant Activity:

Free radicals cause more damage to humans. Antioxidants are the compounds which is used to avoid the cell damage caused by free radicals. Oxidative stress produced by Alloxan was lowered by administration of Aegle marmelos plant18. The scavenging activity on DPPH, Total Antioxidant Capacity was done to evaluate the antioxidant activity19.

 

2, 2-Diphenyl- picryl- hydrazyl (DPPH) Scavenging Activity:

The effects of crude extracts of Aegle marmelos on the scavenging of DPPH was determined by taking 1ml of the sample and mixing it with 100  µl of  DPPH, 400 µl of 50 mM Tris HCl. The reaction mixture was kept incubation for 30 to 40 minutes at room temperature. The absorbance was read spectrophotometrically at 517 nm. The scavenging activity was determined by the given formula

 

% Antioxidant activity = [(OD Control – OD Test sample)/ OD Control] * 100

 

Total Antioxidant Capacity:

The antioxidant capacity of plant extract was evaluated. 1 ml of the sample was mixed with 500µl of the reaction mixture containing 28mM Sodium phosphate, 0.6M Sulphuric acid, 4mM Ammonium molybdate in an eppendorf tube and incubated at 50oC for 90 minutes. After incubation, the absorbance was read spectrophotometrically at 695nm.

 

Antibacterial Activity:

Different bacterial strains like E.coli, Staphylococcus aureus, Klebsiella pneumonia was used to check antibacterial activity. 20ml of Mueller Hinton Agar (MHA) was prepared. Sterile plates were loaded with test extracts. Dimethyl Sulfoxide was added to the well. It acts as a negative control. The plates were incubated at 37C for 24 hours. The zone of the formation around the disc at the end of incubation was noted.

 

Chromatographic Studies:

Plant extracts usually contain numerous bioactive compounds. The act of separating compounds remains a tedious task. Hence, different chromatographic techniques were employed for the separation of pure components. Thin Layer Chromatography (TLC) is the efficient method in determining the components present in the sample by comparing the Retention factor (Rf value) of a compound with standard known compound. TLC was carried on methanol and aqueous extracts of Aegle marmelos using silica gel as a stationary phase and solvents as methanol and ethyl acetate in the ratio of 2:3 as mobile phase. Based on the polarity nature of the compounds, it gets migrated and the Rf value was calculated20.

 

RESULTS AND DISCUSSION:

Antioxidant Activity:

Antioxidants play an important role in free radical scavenging activity and it maintains the proper functioning of the body21. Different assay methods were used to measure Antioxidant activity22. The Phytochemical analysis helped in predicting the active components present in plant18. The antioxidant activity is reported. The antioxidant activity was tested spectrophotometrically by DPPH as well as Total Antioxidant activity. Methanol extract of flowers show good antioxidant activity than the aqueous extract in both the assays.

 

Table 1: Antioxidant test by DPPH and Total Antioxidant Capacity

DPPH

Total Antioxidant Capacity

Sample

OD

Sample

OD

Methanol

0.622

Methanol

0.881

Aqueous

0.264

Aqueous

0.532

 

Antimicrobial Activity:

Due to the incessant use of existing therapeutic compounds, drug resistance is a major problem. Hence there is a desire to find alternate source23. The plant shows antimicrobial activity against different groups of bacteria24. Antibacterial activity of leaf extracts of bael tree exhibits higher activity against gram positive bacteria E. faecalis than gram negative bacteria21. Methanolic and aqueous extracts shows the Minimum inhibition activity25. Methanol extract exhibit less antibacterial activity against the tested microorganisms when compared with the aqueous extracts. Aqueous extracts have potential antibacterial activity against the E.coli, Staphylococcus aureus, K.pneumoniae.

 

Table 2: Aqueous extract of A.marmelos against test microbial strain

S. No

Test Organism

Zone of inhibition (mm)

Standard Imipenem (mm)

1

E.coli

4

1

2

S.aureus

2

1.1

3

K.pneumoniae

4

1.2

 

Table 3: Methanol extract of A.marmelos against test microbial strain

S. No

Test Organism

Zone of inhibition (mm)

Standard Imipenem (mm)

1

E.coli

0.5

1

2

S.aureus

0.8

1.1

3

K.pneumoniae

1.0

1.2

 

Chromatographic Studies:

Different separation techniques can be done to isolate the bioactive components. Based on the results of Thin layer chromatography, it is observed that the extract is rich in Alkaloids and Terpenoids.

 

Table 4: Interpretation by Thin-layer Chromatography

 

Solvent

3rd fraction

5th fraction

Distance moved

8.4 cm

6.3cm

6.5 cm

Rf value

-

0.75 cm

0.77 cm

 

CONCLUSION:

Results revealed that the flower extracts of the plant exhibit good antioxidant and antibacterial activities. Considering the plant characteristics, various studies need to be done to explore the pharmacological activities of this plant. Hence this medicinal plant could be an untapped resource in finding a therapeutic compound to treat various diseases as it is rich in phytochemicals and it will be a promising resource in the red biotechnology in the future era.

 

ACKNOWLEDGEMENT:

The authors thank the College management, Department of Biotechnology, Vivekanandha College of Engineering for Women (Autonomous), Tiruchengode for their kind support and facilities provided to carry out this research work.

 

CONFLICT OF INTEREST:

The authors declare no conflicts of interest.

 

REFERENCES:

1.     World Health Organization, Guidelines on Developing Consumer Information on Proper Use of Traditional, Complementary, and Alternative Medicine, Geneva: WHO, 2004.

2.     Sanghi DK. Rakesh T. Herbal Drugs an Emerging Tool for Novel Drug Delivery Systems. Research Journal of Pharmacy and Technology. 2013; 6(9): 962-966.

3.     Neeli RE. Kamta PN. Pradeep KS et al. Standardization Strategies for Herbal Drugs-An Overview. Research Journal of Pharmacy and Technology 2008; 1(4): 310-312.

4.     Kulkarni A. Kale M. Sarode S. Firke S. Firke B. Warke P et al. Antimicrobial Activity of Some Important Medicinal Plants of India against Some Plant and Human Pathogens. Research Journal of Pharmacy and Technology. 2010; 3(3): 924 – 926.

5.     Orwa C. An Introduction to Aegle marmelos. Agro Data. 2009; 4(2): 1-5.

6.     Patkar Atul N. Desai N. Ranage AA. Kalekar KS. et al. A review on Aegle marmelos: a potential medicinal tree. International Research Journal of Pharmacy. 2012; 3(8): 86-91.

7.     Jesudurai D. Monika R. Preparation of Silver Nanoparticles from Bael (Aegle marmelos) Leaves Extract and their Doping with Multiwalled Carbon Nanotubes for Antibacterial Utility. Research Journal of Pharmacy and Technology. 2020; 13(6): 2653-2657.

8.     Neeraj. Vinita B. Vishal J et al. Bael (Aegle marmelos) Extraordinary Species of India: A Review. International Journal of Current Microbiology and Applied Sciences. 2017; 6(3): 1870-1887. http://dx.doi.org/10.20546/ijcmas.2017.603.213

9.     Chandaka M. Hindu K, Sudeepthi CD. Maneela P. Venkat Reddy K Bhagya Sree B et al. Anti Ulcer Activity of Aqueous Extract of Aegle marmelos Leaves on Rats. Asian J. Pharm. Res. 2012; 2(4): 132-135.

10.   Sharma PC. Bhatia V. Bansal N. Sharma A et al. A review on bael tree. Indian J. Nat. Prod. Resour. 2007; 6(2): 171–178.

11.   Savita SM. Rekha LD. Vijay DH. Snehal SS. Nilam YJ. Bhagyashri SG et al. A Systematic Review on Aegle marmelos (Bael). Research Journal of Pharmacognosy and Phytochemistry. 2020; 12(1): 31-36. http://dx.doi.org/10.5958/0975-4385.2020.00007.2

12.   Sandeep D. Ruhil S. Balhara M. Seema D. Chhillar AK et al. Aegle marmelos: A potential source of Phytomedicine. Journal of Medicinal Plants Research. 2011; 5(9):1497 – 1507.

13.   Dinesh Kumar S. Gaurav K. Karthik L. Bhaskara Rao KV et al. A review on pharmacological and phytochemical properties of Aegle marmelos (L.) Corr.Serr. (Rutaceae). Asian Journal of Plant Science and Research. 2011; 1(2): 8-17.

14.   Bhardwaj RL. Role of bael fruit juice in nutritional security of Sirohi tribals, Benchmark Survey Report of Sirohi Tribals, Krishi Vigyan Kendra, AU, Jodhpur 2014:11-37

15.   Sanghi SB. Mushtaq S et al. Pharmacological property of Aegle marmelos as a potential medicinal tree: An Overview. International Journal of Research - Granthaalayah, 5(8), 63-66. https://doi.org/10.29121/granthaalayah.v5.i8.2017.2183

16.   Arghya M. Arun Kumar S. Niyati J. Sukanya M. et al. Flowering, fruiting and physio-chemical characteristics of bael (Aegle marmelos correa.) grown in northern districts of West Bengal, Curr. J. Appl. Sci. Technol. 2017; 6 1–8, https://doi.org/10.9734/cjast/2017/36310

17.   Gautam MK. Purohit V. Agarwal M. Singh A. Goel RK et al. In vivo healing potential of Aegle marmelos in excision, incision, and dead space wound models, Sci. World J. 2014, https://doi.org/10.1155/2014/740107.

18.   Sabu M. Kuttan R. Antidiabetic activity of Aegle marmelos and its relationship with its antioxidant properties. Indian journal of physiology and pharmacology 2004; 48(1): 81-88.

19.   Cheeseman KH. Slater TF. Free radicals in medicine. British Medical Bulletin. 1993; 49: 479-724.

20.   Ayuba YM. Okoro BC. Abubakar MN. Ilesanmi E,et al. Phytochemical Constituents, Thin Layer Chromatography and Antimicrobial Activity of Methanol Extract of the Stem and Leave of Citrus Limon (L). International Journal of Biochemistry, Biophysics and Molecular Biology. 2017; 2 (4): 31-35. 10.11648/j.ijbbmb.20170204.12

21.   Prithviraj Chakraborty et al. Role of Antioxidants in Common Health Diseases. Research Journal of Pharmacy and Technology. 2009; 2(2): 238 -244.

22.   Selvakumar K Antioxidant Assays in Pharmacological Research. Research Journal of Pharmacy and Technology. 2009; 2(2): 238 -244.

23.   Shoba S. Shylaja G. Uma A. Sathiavelu A. Mythili S et al. Endophytes for Antimicrobial Activity- A Review. Research Journal of Pharmacy and Technology. 2015; 8 (6): 742-748.

24.   Mayuri CR. Dashrath DG. Dhale DA et al. Antibacterial Perspective of Extracts from fruit of Aegle marmelos in different solvents. Indian Journal of Applied Research. 2013; 3(7): 1-7.

25.   Pandey A. Mishra R. Antibacterial properties of Aegle marmelos leaves, fruits and peels against various pathogens. Journal of Pharmaceutical and Biomedical Sciences. 2011; 13 (13): 1-6.

 

 

 

Received on 20.05.2021          Modified on 26.12.2021

Accepted on 29.07.2022        © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(2):773-776.

DOI: 10.52711/0974-360X.2023.00132