GC-MS Profiling and antibacterial activity of Acacia nilotica bark pertained aqueous extract

Janani Manoharan¹*, Parimala Shantha Kumari T², Punithavathi V.R³,

Mukesh Kumar Dharmalingam Jothinathan⁴, Arulanandam Adaikalam⁵, Devipriya Anbumani⁶

¹Department of Biochemistry, Auxilium College (Autonomous), Gandhi Nagar,

Vellore- 632 006, Tamil Nadu, India.

²Department of Zoology, Voorhees College, Vellore-632001, Tamil Nadu, India.

³Department of Biotechnology, M.M.E.S Women’s Arts and Science College,

Melvisharam-632509, Tamil Nadu, India.

⁴Centre for Global Health Research, Saveetha Medical College and Hospitals, Saveetha Institute of Medical and Technical Sciences (SIMATS), Saveetha University, Chennai, India.

⁵Department of Languages, School of Social Sciences and Languages, Vellore Institute of Technology,

Vellore, Tamil Nadu, India.

⁶Nano and Energy Bioscience Laboratory, Department of Biotechnology, Thiruvalluvar University,

Serkkadu, Vellore-632 115, Tamil Nadu, India.

*Corresponding Author E-mail: jananimanoharan21@gmail.com

ABSTRACT:

The applicability of medicinally imperative plants for well-being of human hold historical background. Products of natural origin are the pivotal source of medicament. Amid all, plant-rooted medicines are the exemplary natural source. The failure of allopathic medicines in terms of their aftereffect have tuned keen interest and practice of medicinal plants and their derivative products. The pharmacologically functional phytophenolics were amendable for the therapeutic attributes of medicinal plants. Acacia nilotica or A.nilotica (Babul/Kikar) an ayurvedic tree was used in this study. Applying soxhlation technique the aqueous bark extract from A.nilotica was pertained. GC-MS chromatogram elucidated 11 bioactive entities, connected with pharmacological traits that included 1,2,3-benzenetriol (Pyrogallol), estra-1,3,5(10)-trien-17β-ol, ethanamine, nonadecane, n-octadecane and pentanoic acid. Furthermore, n-hexadecanoic acid, octanoic acid, 1,2-benzenedicarboxylic acid, benzeneacetic acid and eicosane were recognized in the extract. Pseudomonas aeruginosa (14.6 ± 0.21 mm) displayed greater antibacterial activity, followed by Bacillus cereus (14.4 ± 0.38 mm), Staphylococcus aureus (13.6 ± 0.33 mm), Klebsiella pneumoniae (12.9 ± 0.30 mm), Bacillus subtilis (12.6 ± 0.24 mm) and the least inhibitory zone was given by Escherichia coli (10.2 ± 0.52 mm) at 40 µg/mL. All the outcomes validated the antibacterial attributes of A.nilotica bark pertained aqueous extract.

KEYWORDS: Acacia nilotica, Bark, GC-MS, Antibacterial.

1. INTRODUCTION:

Since the emergence of humankind, the ancient knowledge on medicine has been keenly preserved and transmitted to the entire global civilization. Numerous valuable medicines are either acquired from the natural sources or their derivatives.

Naturally originated medicine contributes more than 39.1% (one-third) of all the FDA (Food and Drug Administration) authorized drugs¹. Plants with medicinal qualities are the substantial root of natural ingredients. They have been employed extensively for the therapeutics in various countries, since antiquity and continue to be used today².

The dynamic biologically functional pharmacological ingredients of medicinal plants form the basement for the modern medicaments in the area of pharmaceutical for designing drugs. Moreover, the plant-rooted customary medicine practices have been the cause of escalation of modern medicament³. The practiced Indian customary medicine (Ayurvedic, Siddha and Unani) was the enriched plant-based healing system. Apart from that other codified as well as uncodified medicinal system holding recuperative property adopts plants. In the present era, the applicability of medicinal plants has been dramatically escalated due to the negative outcomes of allopathic medicaments on human health⁴. The phytophenolics ingredients stuffed in the various sections of the plants that include tannins, saponins, terpenoids and flavonoids had previously highlighted their antioxidant, antimicrobial, anticancer and anti-inflammatory attributes⁵.

Development of antibacterial medicine are the pivotal achievement in the modern medicine. Despite, the bacterial resistance displayed by the antibiotic, the challenging issue world-wide include the necessity of urgent solution as it has become one of the prime causes of health disturbance^6&7. The massive and over usage of antimicrobial drugs (antibiotics) in the patients or animals/livestock was the chief driving agent for the increment in the resistive strains of microbes⁸. Studied have highlighted on the detrimental impact of antimicrobial medicines on the human system that include hepatic damage, nephrotoxicity, gastrointestinal troubles (diarrhoea), rashes, nausea and hypersensitive reaction^9,10.

Acacia nilotica (A.nilotica), mentioned in India as Babul/Kikar is an indispensable Ayurvedic and Unani medicine applied for health distress. It is categorized in the Fabaceae family and copiously dispersed in the tropical as well as sub-tropical sections of the globe¹¹. Due to spectacular polyphenolic ingredients the diverse sections of this tree are applied to treat inflammatory issues, fever, menstrual complications, cancer and diabetes mellitus. Various chemicals like methyl linoleate, N, N-dimethylglycine and methyl oleate, 9, 12 -Octadecadienoic acid, and 4-methylbenzenethiol identified in the A. nilotica¹². As gargle the bark decoction are practiced for throat complaints. The bark incorporated toothpaste are used to cleanse mouth. Pharmacological attributes reported by A.nilotica include antihypertensive, antimicrobial, anticancer, anti-inflammatory and anthelmintic¹³.

2. MATERIALS AND METHODS:

The barks of A.nilotica was pertained from the mature tree in the month of April 2022. The scrapped bark pieces were carefully gathered in a dry and sterile carrier bag. The adhere unnecessary grime on the surface of collected bark was entirely eradicated by washing in a flowing tap water. Then rinsed in distilled water followed by drying the bark pieces over a clean newspaper. Once dried, the bark was evenly chopped into tiny sections and blended entirely to pertain finely powdered bark material. The bark powder was applied for further investigations.

2.1 Aqueous bark extract procuration from A.nilotica:

The A.nilotica bark powder (250 g) acquired through blending was taken to Soxhlation applying 500 mL of distilled water (solvent). After 24 h, the achieved dark brownish coloured content was placed in the vacuum evaporator to entirely remove the moisture. Subsequently, the dried content obtained was taken for GC-MS profiling and antibacterial property assessment.

2.2 GC-MS profiling of aqueous bark extract of A.nilotica:

To figure out the phytocompounds present in the A.nilotica bark aqueous extract, gas chromatography (GC)-mass spectrometry (MS) was implemented in Perkin-Elmer GC-MS. At a constant flow mode (1 mL/min), carrier gas helium (inert gas) was used. Exactly, 1 µL of A.nilotica aqueous bark extract was placed into the spitless capillary injection port. A temperature of 260 ℃ was set for the capillary injector port and initially, the GC oven temperature was held at 70 ℃ (4 min). Subsequently, the temperature of the oven was ramped (300 ℃) at a rate of 10 ℃/ min and then at 300 ℃ (6 min). An electron ionization system with 70 eV ionization energy was operated in the mass spectrometer for 0.2 sec (scan time) at an interval of 0.1 sec. The temperature of the injector and detector (fragmentation- 40 Da to 600 Da) were set at 230 ℃ and 280 ℃, respectively. Identification of phytocompounds was concluded with the retention time and its mass spectra (peak) constructed in the chromatogram and straightaway compared with the NIST (National Institute of Standards and Technology) library-2011 (spectrometer database), in the computer connected to the GC-MS instrument.

2.3 Antibacterial assay of A.nilotica bark pertained aqueous extract:

Execution on the bactericidal trait of A.nilotica bark pertained aqueous extract was mediated by agar-well diffusion protocol as done with slight amendments¹⁴. Bacterial pathogens like Bacillus subtilis (B.subtilis; MTCC-1305), Klebsiella pneumoniae (K.pneumoniae; MTCC-432), Staphylococcus aureus (S.aureus; MTCC-3160), Escherichia coli (E.coli; MTCC-1687), Pseudomonas aeruginosa (P.aeruginosa; MTCC-1688) and Bacillus cereus (B.cereus; MTCC-4079) were picked up randomly for this study. Freshly prepared Mueller-Hinton agar medium (MH-nutrient agar medium) was poured and retained to solidify in the petri-plates within a laminar airflow chamber to hinder unrelated contaminant growth. Then, freshly acquired overnight developed each bacterial strain culture of 100 μL were spread over the solidified media with the aid of a sterile L-shaped glass rod and 6 mm dimension wells were punched into each agar plate with a sterile borer. A.nilotica bark pertained aqueous extract of varying ranges (10 µg/mL to 40 µg /mL) were carefully added into each individual well. Incubation of all petri plates was done for a day at 37 ℃. Chloramphenicol (30 µg/mL) was chosen as a positive experimental control. Zone of inhibition (ZOI) was checked around each well and presented in mm (millimeter).

2.4 Statistical analysis:

Each experimentation was executed in three trials separately and the outcomes were conveyed in descriptive statistics (mean ± standard deviation) applying GraphPad Prism (version 8.4.2). In the numerical data, the p (probability) value of < 0.05 was taken as statistically significant executing the student’s t-test.

3 RESULTS AND DISCUSSION:

3.1 GC-MS profiling of aqueous bark extract of A.nilotica:

The bark aqueous extract of A.nilotica fed into GC-MS column reported phytocompounds existed within them and discerned appertaining to the chromatographic peaks, component’s molecular weight (MW), retention duration and its chemical name (Figure 1). The chromatogram reported plentiful biologically functioning phytocompounds and few of them existed in traces. A versatile phytocompounds in the complex bark mixture (aqueous extract) reported were 1,2,3-benzenetriol (Pyrogallol), estra-1,3,5(10)-trien-17β-ol, ethanamine, nonadecane, n-octadecane and pentanoic acid. Furthermore, n-hexadecanoic acid, octanoic acid, 1,2-benzenedicarboxylic acid, benzeneacetic acid and eicosane were proclaimed to be in trace quantity (Table 1). Majority of the divulged phytocompounds of the bark extract owned pharmacological efficiency, which further would bestow restorative properties to the studied plant.

Table 1. GC-MS spectral spotted phyto-content in A.nilotica bark acquired aqueous extract.

Retention time (RT) in min	Phytocompound name (molecular formula)	Molecular weight (MW) in g/mole
8.287	n-Hexadecanoic acid (C₁₆H₃₂O₂)	256.42
8.564	Octanoic acid (C₈H₁₆O₂)	144.21
10.020	1,2 Benzenedicarboxylic acid (C₈H₆O₄)	166.13
10.498	1,2,3-Benzenetriol (C₆H₆O₃)	126.11
10.886	1,2,3-Benzenetriol (C₆H₆O₃)	126.11
13.142	Benzeneacetic acid (C₈H₈O₂)	136.14
13.564	Pentanoic acid (C₅H₁₀O₂)	102.13
17.174	Nonadecane (C₁₉H₄₀)	268.52
17.763	1,2,3-Benzenetriol (C₆H₆O₃)	126.11
17.974	1,2,3-Benzenetriol (C₆H₆O₃)	126.11
18.952	1,2,3-Benzenetriol (C₆H₆O₃)	126.11
20.330	n-Octadecane (C₁₈H₃₈)	254.49
20.652	1,2,3-Benzenetriol (C₆H₆O₃)	126.11
21.196	1,2,3-Benzenetriol (C₆H₆O₃)	126.11
21.341	1,2,3-Benzenetriol (C₆H₆O₃)	126.11
21.552	1,2,3-Benzenetriol (C₆H₆O₃)	126.11
21.952	Eicosane (C₂₀H₄₂)	282.54
22.385	Estra-1,3,5(10)-trien-17β-ol (C₁₈H₂₄O)	256.38
23.296	Ethanamine (C₂H₇NH₂)	45.08
23.429	1,2,3-Benzenetriol (C₆H₆O₃)	126.11

Pyrogallol (1,2,3-Benzenetriol; MF: C₆H₆O₃) is a phytophenolic hydroxylated compound attributed with vital activities that include antioxidant, bactericidal and anticarcinogenic agent in breast cancer cell lines (MDA-MB-231) and cervical cancer (HEp-2)¹⁵. Estra-1,3,5(10)-trien-17β-ol occurred in the plant bark has prevention and curative features as antiproliferative agents for breast cancer¹⁶. Anti-arrhythmia trait has been shown by ethanamine¹⁷. The compounds n-nonadecane, n-octadecane and eicosane achieved noticeable antibacterial results on strains of bacteria in a study executed. Hence, proven for their impressive bactericidal capacity¹⁸. Pentanoic acid/Valeric acid (C₅H₁₀O₂), has been documented for its impressive neuroprotective ability in dementia. Subsequently, the anti-epileptic characteristics of pentanoic acid have been mentioned, as an antagonist for NMD (N-methyl-D-aspartate receptor) receptors^19,20. n-hexadecenoic acid is inclusive of haemolytic, nematode killing and antiradical activities²¹. Benzeneacetic acid has been successfully declared its antioxidant, anticancer and antifungal activity²². The restorative role has been validated by 1,2 benzenedicarboxylic acid in the case of neurodegenerative issues. Octanoic acid (chemically caprylic acid) has been elucidated for its antioxidant and antibacterial action²³. Strong remarkable responsiveness has been developed in correlating bioactive plant constituents and their biological activities as documented previously in the research outcomes.

3.2 Antibacterial assay of A.nilotica aqueous bark extract:

Antibacterial features of A.nilotica bark aqueous extract were scrutinized by using agar-well diffusion methodology. All the bacterial strains of both classes presented dose-responsive bactericidal activity as clearly visualized through formed inhibition zone around individual wells. The inhibition zone was recorded for P.aeruginosa (14.6 ± 0.21 mm), B.cereus (14.4 ± 0.38 mm), S.aureus (13.6 ± 0.33 mm), K.pneumoniae (12.9 ± 0.30 mm), B.subtilis (12.6 ± 0.24 mm) and E.coli (10.2 ± 0.52 mm) at 40 µg/mL (Figure 2).

The bioactive plant chemical components availed in the bark aqueous extract of A.nilotica may be responsible for the bactericidal traits. Probably, the tannin in the bark extract may elucidate its mechanism through the inactivation of enzyme and bacterial cell envelope protein molecules. Alteration of the cellular membrane and hindering the energy metabolic pathway was done by flavonoids²⁴. Penetrative variation of active ingredients of the plant through the cell wall as well as the cellular membrane may be responsible for their susceptibility pattern²⁵. Previous experimental part executed in-vitro agar well diffusion technique with ethanolic extract acquired from the stem-bark section of A.nilotica and achieved susceptible activity against Staphylococcus aureus, Bacillus subtilis, Shigella sonnei and Escherichia coli in a dose-lined manner²⁶.

Figure 2. Antibacterial report of A.nilotica bark extract on varying degree of pathogenic strains of bacteria.

Mean ± Standard deviation

CONCLUSION:

The GC-MS profiling dictated the existence of vital phytocompounds which were the contributors of pharmacological efficacies of the bark extract of A.nilotica. The aqueous bark extract of A.nilotica when screened for bactericidal role through agar-well diffusion protocol displayed stupendous antibacterial activity against all the screened bacterial strains. The identification of phytocomponents in the aqueous bark extract of A.nilotica will surely facilitate the route for framing innovative drugs to root out the antibiotic-resistance issues and synthesis of health restorative medicaments.

ACKNOWLEDGMENT:

The authors are heartfully thankful to the Department of Biotechnology. Thiruvalluvar University, Serkkadu, Vellore for furnishing research needs for executing this work.

CONFLICT OF INTEREST:

The authors declare that they do not have any conflict of interest.

REFERENCES:

1. Boy HI, Rutilla AJ, Santos KA, Ty AM, Alicia IY, Mahboob T, Tangpoong J, Nissapatorn V. Recommended medicinal plants as source of natural products: a review. Digital Chinese Medicine. 2018; Jun 1; 1(2): 131-142.

2. Sharma R, Ashawat MS, Verma CP, Kumari N. A fast release tablet containing herbal extracts (Ginger, Cinnamon, Turmeric, Long pepper and Punarnava). Asian Journal of Pharmacy and Technology. 2020; 10(4): 231-240.

3. Balunas MJ, Kinghorn AD. Drug discovery from medicinal plants. Life Sciences. 2005; Dec 22; 78(5): 431-41.

4. Saraswathi K, Sivaraj C, Jenifer A, Dhivya M, Arumugam P. Antioxidant, Antibacterial activities, GCMS and FTIR Analysis of Ethanol bark extract of Capparis sepiaria L. Research Journal of Pharmacy and Technology. 2020; 13(5): 2144-2150. doi: 10.5958/0974-360X.2020.00385.6.

5. Kumar AS, Mazumder A, Vanitha J, Ganesh M, Venkateshwaran K, Saravanan VS, Sivakumar T. Antibacterial activity of methanolic extract of Sesbania grandiflora (Fabaceae). Research Journal of Pharmacy and Technology. 2008; 1(1): 59-60.

6. Divya S, Arivoli S, Tennyson S. Phytochemical analysis and in vitro Antioxidant and Antibacterial activity of the Chloroform leaf extract of Abelmoschus manihot (L.) Medik (Malvaceae). Research Journal of Pharmacy and Technology. 2021; 14(9): 4719-4726. doi: 10.52711/0974-360X.2021.00821.

7. Kumar SA, Lakshmi T, Arun AV. Invitro Antibacterial Activity of Acacia catechu ethanolic leaf extract against selected acidogenic oral bacteria. Research Journal of Pharmacy and Technology. 2012; 5(3): 333-336.

8. Sadiq SM, Kareem A, Rasheed N, Mohammad AS. Pharmaceutical Importance of Anti-Microbials. Asian Journal of Pharmacy and technology. 2017; Mar 1; 7(1).

9. Jain MS, Barhate SD. Tigecycline is the First Clinically-Available Drug in a new class of Antibiotics called the Glycylcyclines: A Review. Asian Journal of Pharmacy and Technology. 2020; 10(1): 48-50.

10. Thomas J, Gopakumar A, Narendrakumar G, Preethi TV, Chellaiyan V. Extraction, phytochemical screening and antibacterial activity Wattakaka volubilis (Linn F) Stapf. Research Journal of Pharmacy and Technology. 2016; Apr 1; 9(4): 355.doi: 10.5958/0974-360X.2016.00063.9.

11. Manoharan J, Chinnakannu NK, Ranganathan B. Phytochemical screening, Antiradical and Anti- inflammatory activity of aqueous bark extract acquired from Acacia nilotica. Research Journal of Pharmacy and Technology. 2023; Oct 31; 16(10): 4831-4835.

12. Baravkar AA, Kale RN, Patil RN, Sawant SD. Pharmaceutical and biological evaluation of formulated cream of methanolic extract of Acacia nilotica leaves. Research Journal of Pharmacy and Technology. 2008; 1(4): 480-483.

13. Fadhil YB, Jaber MH, Ahmed HM. Assessment of a novel activity of Acacia nilotica leaf extract for chondrogenesis induction of Mesenchymal stem cells for dental applications. Phytomedicine Plus. 2021; Nov 1; 1(4): 100087.

14. Al Akeel R, Al-Sheikh Y, Mateen A, Syed R, Janardhan K, Gupta VC. Evaluation of antibacterial activity of crude protein extracts from seeds of six different medical plants against standard bacterial strains. Saudi Journal of Biological Sciences. 2014; Apr 1; 21(2): 147-151.

15. Revathi S, Govindarajan RK, Rameshkumar N, Hakkim FL, Mohammed AB, Krishnan M, Kayalvizhi N. Anti-cancer, anti-microbial and anti-oxidant properties of Acacia nilotica and their chemical profiling. Biocatalysis and Agricultural Biotechnology. 2017; Jul 1; 11: 322-329.

16. Al-Tameme HJ, Hadi MY, Hameed IH. Phytochemical analysis of Urtica dioica leaves by fourier-transform infrared spectroscopy and gas chromatography-mass spectrometry. Journal of Pharmacognosy and Phytotherapy. 2015; Oct 31; 7(10): 238-252.

17. Al-Rubaye AF, Kadhim MJ, Hameed IH. Phytochemical profiles of methanolic seeds extract of Cuminum cyminum using GC-MS technique. International Journal of Current Pharmaceutical Review and Research. 2017; 8(2): 114-124.

18. Rouis-Soussi LS, El Ayeb-Zakhama A, Mahjoub A, Flamini G, Jannet HB, Harzallah-Skhiri F. Chemical composition and antibacterial activity of essential oils from the Tunisian Allium nigrum L. EXCLI Journal. 2014; 13: 526-535.

19. Khom S, Baburin I, Timin E, Hohaus A, Trauner G, Kopp B, Hering S. Valerenic acid potentiates and inhibits GABAA receptors: molecular mechanism and subunit specificity. Neuropharmacology. 2007; Jul 1; 53(1): 178-187.

20. Jayaraj RL, Beiram R, Azimullah S, Mf NM, Ojha SK, Adem A, Jalal FY. Valeric acid protects dopaminergic neurons by suppressing oxidative stress, neuroinflammation and modulating autophagy pathways. International Journal of Molecular Sciences. 2020; Oct 16; 21(20): 1-18.

21. Ishnava KB, Konar PS. In vitro anthelmintic activity and phytochemical characterization of Corallocarpus epigaeus (Rottler) Hook. f. tuber from ethyl acetate extracts. Bulletin of the National Research Centre. 2020; Dec; 44(1): 1-10.

22. Valsalam S, Agastian P, Arasu MV, Al-Dhabi NA, Ghilan AK, Kaviyarasu K, Ravindran B, Chang SW, Arokiyaraj S. Rapid biosynthesis and characterization of silver nanoparticles from the leaf extract of Tropaeolum majus L. and its enhanced in-vitro antibacterial, antifungal, antioxidant and anticancer properties. Journal of Photochemistry and Photobiology B: Biology. 2019; Feb 1; 191: 65-74.

23. Marcetic M, Petrovic S, Milenkovic M, Niketic M. Composition, antimicrobial and antioxidant activity of the extracts of Eryngium palmatum Pancic and Vis.(Apiaceae). Open Life Sciences. 2014; Feb 1; 9(2): 149-155.

24. Nigussie D, Davey G, Legesse BA, Fekadu A, Makonnen E. Antibacterial activity of methanol extracts of the leaves of three medicinal plants against selected bacteria isolated from wounds of lymphoedema patients. BMC Complementary Medicine and Therapies. 2021; Dec; 21: 1-0.

25. Lawrence R, Jeyakumar E, Gupta A. Antibacterial Activity of Acacia arabica (Bark) Extract against selected multi drug resistant pathogenic bacteria. Int J Curr Microbiol App Sci. 2015; 1: 213-222.

26. Banso AJ. Phytochemical and antibacterial investigation of bark extracts of Acacia nilotica. Journal of Medicinal Plants Research. 3(2); 82–85.

Received on 19.12.2023 Revised on 08.06.2024

Accepted on 19.09.2024 Published on 24.12.2024

Available online from December 27, 2024

Research J. Pharmacy and Technology. 2024;17(12):5931-5936.

DOI: 10.52711/0974-360X.2024.00899