# In vitro Antimicrobial Potential and Phytochemical Screening of Albizzia lebbeck Benth Bark against Respiratory Tract Pathogens

Rajni Tyagi, Gaurav Sharma, Rinni Sahrawat

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Rajni Tyagi, Gaurav Sharma, Rinni Sahrawat. In vitro Antimicrobial Potential and Phytochemical Screening of Albizzia lebbeck Benth Bark against Respiratory Tract Pathogens. Journal of Advances in Biology & Biotechnology, 2025, 28, pp.48 - 55. 10.9734/jabb/2025/v28i52268. hal-05072195

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# *In vitro* Antimicrobial Potential and Phytochemical Screening of *Albizzia lebbeck* Benth. Bark against Respiratory Tract Pathogens

Rajni a++, Gaurav Sharma b++* and Rinni Sahrawat a++*

## Authors' contributions

This work was carried out in collaboration among all authors. All authors read and approved the final manuscript.

## Article Information

DOI: https://doi.org/10.9734/jabb/2025/v28i52268

## Open Peer Review History:

This journal follows the Advanced Open Peer Review policy. Identity of the Reviewers, Editor(s) and additional Reviewers, peer review comments, different versions of the manuscript, comments of the editors, etc are available here:

https://pr.sdiarticle5.com/review-history/132302

Received: 11/01/2025

Accepted: 14/03/2025

Published: 28/04/2025

## Original Research Article

## ABSTRACT

This study investigates the antimicrobial activity of *Albizzia lebbeck* bark extracts against key respiratory pathogens, including *Staphylococcus aureus* MTCC 1144, *Streptococcus pyogenes* MTCC 442, and *Streptococcus pneumoniae* MTCC 655. Phytochemical studies have identified a wide array of bioactive compounds in *A. lebbeck*, including glycosides, alkaloids, terpenoids, steroids, saponins, anthraquinones, and other phenolics. Respiratory tract infections remain one of the leading causes of morbidity and mortality, especially in light of the recent Covid-19 pandemic, which highlighted the urgent need for effective antimicrobial solutions. The Bark samples were collected from the Botanical Garden of MIET College, Meerut, Uttar Pradesh, and processed using

<footnote>a Department of Biotechnology and Microbiology, MIET, India.</footnote>

<footnote>b Department of Physics, Rajendra College, Chapra, Bihar, India.</footnote>

<footnote>** Assistant Professor;</footnote>

<footnote>* Corresponding author: E-mail: rinni.sahrawat@miet.ac.in;</footnote>
petroleum ether, acetone, methanol, and water in Soxhlet extraction. The antimicrobial efficacy of the extracts was evaluated using the agar well diffusion method, and the methanolic extract demonstrated the highest activity. The results showed that these phytoconstituents were also active against all test pathogens at 50 mg/ml concentration (Table 3). Tannins/phenols were most effective against *S. aureus* (13.55±0.41mm) followed by *S. pyogenes* (12.21±0.15mm) and alkaloids against *S. pneumoniae* (12.26±0.03mm). Among the phytoconstituents, tannins/phenols were the most active constituents. Chromatographic separation and phytochemical analysis of the methanolic extract revealed the presence of alkaloids, flavonoids, tannins, phenolics, saponins, glycosides, amino acids, and steroids, compounds associated with antimicrobial properties. Among the tested pathogens, *S. aureus* was most susceptible to the methanolic extract. These findings support the traditional use of *A. lebbeck* in managing respiratory diseases and underscore its potential as a natural antimicrobial agent.

Keywords: Agar well diffusion method; *Albizzia lebbeck*; antimicrobial activity; chromatographic separation; respiratory tract pathogens.

# 1. INTRODUCTION

Tree barks are one of the important sources produced from various forestry activities. Barks are widely eliminated as wastes, or used as insulation materials and considered as fuel. Moreover, in recent years, much research has been focused on the diversity of various secondary metabolites from the bark of medicinal plants. Tree barks protect the trees from external attack, particularly against various infectious agents, and it contains novel secondary metabolites, particularly polyphenols act as antimicrobial lead molecule (Noté et al., 2015; Sharma et al., 2022). India, with its rich biodiversity and ancient medicinal heritage, serves as a treasure trove of herbal resources widely utilized in traditional therapeutic systems such as Ayurveda, Siddha, and Unani. Of the approximately 47,000 plant species in India, over 15,000 are reported to have medicinal value (WHO, 2007). One such plant is *Albizzia lebbeck* Benth., belonging to the family Mimosaceae. Commonly known as Lebbek Tree, Frywood, or Siris (Kirtikar and Basu, 1981, Anonymous, 1985, Singh and Agrawal, 2008, Devi et al., 2023), this deciduous species is native to the semi-deciduous and deciduous forests of South Asia, extending from eastern Pakistan to India, Sri Lanka, and Burma (Shafiq et al., 2024, Tasnim et al., 2014).

*Albizzia lebbeck* has a long history of use in traditional medicine (Tchinda et al., 2019, Yang et al., 2020, Leutcha et al., 2022), particularly for treating respiratory conditions such as asthma, bronchitis, and tuberculosis (Abd et al., 2015, Ali, 2018, Kavitha et al., 2021, Arasu et al., 2022, Ibrahim and Abdul-Hafeez, 2023). The plant's bark, which is grey, rough, and flaky (Sesoltani

and Paulsen, 2011, Azma et al., 2020), is particularly noted for its antitubercular, antiseptic, and anti-dysenteric properties (Abd et al., 2015, Ali et al., 2018, Kavitha et al., 2021, Arasu et al., 2022, Ibrahim et al., 2023). Additionally, it has been employed as a remedy for snake bites, scorpion stings, malaria, leprosy, and helminth infections (Verma et al., 2013, Rajamohan et al., 2022).

Phytochemical studies have identified a wide array of bioactive compounds in *A. lebbeck*, including glycosides, alkaloids, terpenoids, steroids, saponins, anthraquinones, and other phenolics (Sharma et al., 2020, Nehra et al., 2022). These secondary metabolites are believed to contribute to its medicinal properties, including antimicrobial, anti-inflammatory, and immunomodulatory effects.

The rising prevalence of drug-resistant pathogens, especially those causing respiratory infections, necessitates the exploration of alternative therapeutic agents. In this context, plants like *A. lebbeck* offer a promising avenue for developing safe and cost-effective treatments. This study aims to investigate the antimicrobial efficacy and phytochemical composition of various *A. lebbeck* bark extracts against bacterial pathogens responsible for respiratory infections, specifically *Staphylococcus aureus*, *Streptococcus pyogenes*, and *Streptococcus pneumoniae*.

# 2. MATERIALS AND METHODS
## PREPARATION OF SAMPLE

The Bark samples were collected from the Botanical Garden of MIET College, Meerut, Uttar Pradesh, and processed using petroleum ether,
acetone, methanol, and water in Soxhlet extraction. Assemble the bark of *A.lebbeck* in a plastic bag and carried to the laboratory located in the departmental campus in Meerut, Uttar Pradesh, India. The plant's classification was verified by consultation with professor (Dr) Navneet. The plant bark was disinfected with running tap water and then distilled water, after being the plant's bark was allowed to dehydrate at room temperature in the shade for two to four weeks.

A pestle and mortar was used to break up the bark of the plant into tiny sections, which were subsequently ground into a fine powder in an electric mixer grinder. The nicely ground bark powder was kept in the fridge at 4 °C in an airtight jar for further investigation.

## 2.1 Extract Preparation

After making a powder of bark, 200 gm of powder plant material was immersed in 600 ml of four distinct solvents ether, acetone, methanol, petroleum and water before being put into a soxhlet assembly and extracted for 72 hours using a sequential approach (Ahmed et al., 2018). Primitive extracts were constructed by eliminating solvents using a vacuum evaporator at 30 °C.

Thereafter plant extracts were filtered out through the Whatman no-1 filter paper and the extract was stored in the refrigerator at 4°C. For agar well diffusion method, diluted extract with dimethyl sulfoxide until its concentration reaches 200 mg/ml.

## 2.2 Microorganisms Used

Respiratory tract infection-causing bacterial strains were taken for this study i.e. *Streptococcus pneumoniae* MTCC-655, *Streptococcus pyogenes* MTCC-422 and *Staphylococcus aureus* MTCC-1144. These bacterial strains were provided by the Institute of Microbial Technology (IMTECH), Chandigarh.

## 2.3 Antimicrobial Activity

The antibacterial activities of bacterial strains were assessed by the agar well diffusion technique (Ahmed et al., 2018). This technique relies on the tested material diffusing so much that the expansion of microbes is completely ceased in the area surrounding the zone that contains the tested extract solution (Perez et al., 1990).

Antibacterial screening was conducted using MH Agar Media No-173 (Muller Hinton agar media, Hi media). For inoculation of Muller Hinton broth, 0.1ml of diluted inoculum (10⁶ CFU/ml) was used of tested micro-organisms and poured into sterilized MH agar petri plates. Thereafter wells were punched in the center of petriplates using cork borer (8mm in diameter), and filled with 45µl of tested plant extracts at a final concentration 200 mg/ml. For a negative control DMSO was employed. Amoxicillin, a moderate spectrum antibiotic, was used as a positive control to evaluate the effectiveness against tested micro-organisms. The experiment was conducted in a triplicate manner with each extract. All the Petri plates were incubated at 37 °C for 24 hours. After 24 hours the zone of inhibition measured in millimeters (mm), was used to evaluate the antibacterial activity of the tested extract.

## 2.4 Chromatographic Separation

For this column chromatographic separation was used. First glass wool was used to plug the lower end of a glass column. The flat end of a packed rod was used to arrest the air bubbles that were generated as the extract was poured onto glass wool. Wet silica gel was packed into column by gradually down the silica gel into the column. By using the glass rod to abbreviate the particles, the side of the column was taped carefully.

The column outlet was changed when the silica gel settled. A solvent mixture of toluene and ethyl acetate in 90:10 ratio was used to elute the sample after it had been drawn onto the absorbent. Every fraction that was produced was gathered.

## 2.5 Screening for Phytochemicals

To ascertain the existence of certain bioactive components, such as flavonoids, alkaloids, glycosides, amino acids, saponins, steroids, phenolics and tannins. The methanol and its fraction were submitted to phytochemical analysis using conventional qualitative techniques (Trease and Evans, 1996).

# 3. RESULTS AND DISCUSSION

The antimicrobial assay showed that the petroleum ether, acetone, methanol and aqueous extracts of *A. lebbeck* barks exhibited discernible activities against respiratory tract pathogens *S. aureus*, *S. pyogenes* and *S. pneumoniae* (Table 1). The methanol extract of *A. lebbeck*
was highly active against *S. aureus* (18.80±0.06mm) followed by *S. pyogenes* (18.11±1.68 mm) and *S. pneumoniae* (16.50±0.62 mm) in comparison to other solvent extracts. Methanol extract was most effective followed by acetone, water and petroleum ether. Acetone extract was most effective against *S. aureus* (18.05±0.25 mm) followed by *S. pyogenes* (13.71±1.41 mm) and *S. pneumoniae* (11.63±0.84 mm). Aqueous extract is more effective in comparison to petroleum ether extract. Aqueous extract showed maximum inhibition against *S. aureus* (18.18±0.51 mm) followed by *S. pyogenes* (14.86±0.68 mm) and *S. pneumoniae* (11.27±1.16 mm). Petroleum ether was found most effective against *S. aureus* (13.40±0.76 mm) followed by *S. pyogenes* (11.69±0.91 mm) and *S. pneumoniae* (10.51±0.41 mm).

The effectiveness of the extract largely depends on the type of solvent used and the constituents present in the extracts. Dirar et al. (2019) suggested that the quantity of secondary metabolites that are extracted from plants is said to be influenced by the extraction solvent used; hence, selecting the right extraction solvent is essential to achieving the intended pharmacological activity. WHO also view the most pressing problem facing medical science, which is worrying so to overcome this problem, Scientist investigated the in vitro antimicrobial potentialities of different solvent extracts of ethnomedicinal plants against

clinically isolated human pathogens (Maji et al., 2010).

This study revealed that the leaf extract of *A. lebbeck* posse appreciable antibacterial activity against tested micro-organisms and methanol extract showed the highest activity to test organisms when compared with acetone, and petroleum ether extract, these findings align with the observation of Balkrishna et al. (2022). The successive ethyl acetate extract of the plant leaves are found inhibitory effect against *S. aureus* (Arasu et al., 2022, Toh et al., 2023).

Balkrishna et al. (2022) reported the antimicrobial activity of *A. lebbeck* against infectious diarrhea. The antimicrobial assay showed that aqueous, methanol and chloroform extracts of *A. lebbeck* barks exhibited activities against enterotoxigenic *E. coli*, enteropathogenic *E. coli*, *S. typhimurium*, *S. enteritidis*. Petroleum ether and hexane extracts did not exhibit any such activity. These findings do not align with the observation of Elshiekh et al. (2020) who stated that petroleum ether extract of plants showed significant results against many bacterial strains. The inhibitory zones produced by methanol extract against *E. coli* and *Salmonella* spp. were higher than by chloroform and aqueous extracts. No activity was observed against *Shigella* sp. and *Candida* sp. *A.lebbeck* has shown antibacterial activity against, *S. aureus* and *S. typhi* (Bibi et al., 2020, Sheyin et al., 2015).

<table><caption>Table 1. The inhibition zones diameters of various extracts of *A. lebbeck* bark</caption><thead><tr><th>S. N</th><th>Bacterial strain</th><th>Control (Amoxillin) Mean±SD</th><th>Petroleum Ether Mean±SD</th><th>Acetone Mean±SD</th><th>Methanol Mean±SD</th><th>Aqueous Mean±SD</th></tr></thead><tbody><tr><td>1</td><td><i>Staphylococcus aureus</i></td><td>34.69±1.29</td><td>13.40±0.76</td><td>18.05±0.25</td><td>18.80±0.06</td><td>18.18±0.51</td></tr><tr><td>2</td><td><i>Streptococcus pneumoniae</i></td><td>32.58±0.65</td><td>10.51±0.41</td><td>11.63±0.84</td><td>16.50±0.62</td><td>11.27±1.16</td></tr><tr><td>3</td><td><i>Streptococcus pyogenes</i></td><td>33.55±1.93</td><td>11.69±0.91</td><td>13.71±1.41</td><td>18.11±1.68</td><td>14.86±0.68</td></tr></tbody></table>

values present a mean ± SD of three replication

<table><caption>Table 2. The phytochemical screening of crude extract of *A. lebbeck* bark</caption><thead><tr><th>S. No.</th><th>Solvents</th><th>Alkaloids</th><th>Flavonoids</th><th>Phenols/ Tannins</th><th>Amino acids</th><th>Saponins</th><th>Glycosides</th><th>Steroids</th></tr></thead><tbody><tr><td>1.</td><td>Petroleum ether</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>+</td></tr><tr><td>2.</td><td>Acetone</td><td>+</td><td>+</td><td>+</td><td>-</td><td>+</td><td>-</td><td>-</td></tr><tr><td>3.</td><td>Methanol</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td></tr><tr><td>4.</td><td>Water</td><td>+</td><td>-</td><td>+</td><td>+</td><td>+</td><td>+</td><td>-</td></tr></tbody></table>
<table><caption>Table 3. Antibacterial activity of active constituents (50 mg/ml) of methanolic extract of *A. lebbeck* against respiratory tract pathogens</caption><thead><tr><th rowspan="2">S. No.</th><th rowspan="2">Colour</th><th rowspan="2">Phytoconstituents</th><th colspan="3">**Zone of inhibition (in mm)**</th></tr><tr><th><i>S. aureus</i></th><th><i>S. pyogenes</i></th><th><i>S. pneumoniae</i></th></tr></thead><tbody><tr><td>1.</td><td>Light Yellow</td><td>Terpenes</td><td>11.43±0.81</td><td>10.43±0.13</td><td>11.19±0.03</td></tr><tr><td>2.</td><td>Greenish Yellow</td><td>Alkaloids</td><td>12.37±0.38</td><td>10.33±0.08</td><td>12.26±0.03</td></tr><tr><td>3.</td><td>Yellowish Brown</td><td>Glycosides</td><td>12.25±0.09</td><td>10.23±0.13</td><td>10.37±0.06</td></tr><tr><td>4.</td><td>Dark Red</td><td>Tannins / Phenols</td><td>13.55±0.41</td><td>12.21±0.15</td><td>10.21±0.02</td></tr><tr><td>5.</td><td>Dark Brown</td><td>Saponins</td><td>10.47±0.36</td><td>9.70±0.08</td><td>—</td></tr></tbody></table>

The phytochemical screening of the plant extracts showed the presence of alkaloids, flavonoids, tannins, phenolics, saponins, glycosides, amino acids and steroids (Table 2). Aisha et al. (2020) reported that the plant bark yields tannins 7-11% of condensed type, viz. D-catechin, isomer of leucocyanidin [5,7,3',4'-tetrahydroxy flavon-3,4 diol], Melacacidin, leucoanthracyanidin, lebbecacidin [8,3',4'-tri-hydroxyl flavon-3,4-diol], Friedelin, Beta-sitosterol, Betulinic acid and its glycosides. Phytochemical screening of successive extract *A. lebbeck* leaves shows the presence of alkaloids, glycoside, tannins, saponins, flavonoids, and carbohydrates (Biswal et al., 2022).

*A. lebbeck* is known to contain glycosides, alkaloids (Ahmed et al., 2018), terpenoids, steroids, saponins (Desai and Joshi, 2019) Chaddha et al. (2011) reported the presence of alkaloids, glycosides, steroids, flavonoids, saponins, tannins, carbohydrates and reducing sugar from *A. lebbeck* bark. The methanolic extract of the plant seed showed the presence of alkaloids, saponins, tannins, carbohydrates and amino acids (Balkrishna et al., 2022).

Phytochemical analysis of the five fractions revealed that the fractions contained different components like terpenes, alkaloids, glycosides, tannins/phenols and saponins. The results showed that these phytoconstituents were also active against all test pathogens at 50 mg/ml concentration (Table 3). Tannins/phenols were most effective against *S. aureus* (13.55±0.41mm) followed by *S. pyogenes* (12.21±0.15mm) and alkaloids against *S. pneumoniae* (12.26±0.03mm). Among the phytoconstituents, tannins/phenols were the most active constituents.

## 4. CONCLUSION

*Albizzia lebbeck* an ayurvedic medicinal plant and has been easily all over the word. Various

problems like eye, inflammatory, genital, gastrointestinal, oral, respiratory, akin and urinary tract infection have all been treated by using the *A. lebbeck*. Although the bark of *A. lebbeck* seems to be the most significantly used part in ayurvedic medicine. The finding of the present study concluded that *Albizia lebbeck* exhibits high antimicrobial activity against *Staphylococcus aureus*, *Streptococcus pyogenes*, *Streptococcus pneumoniae*. The results of the present study, as of previous workers; validate the use of *A. lebbeck* bark to cure various ailments.

## DISCLAIMER (ARTIFICIAL INTELLIGENCE)

Author(s) hereby declare that NO generative AI technologies such as Large Language Models (ChatGPT, COPILOT, etc) and text-to-image generators have been used during writing or editing of this manuscript.

## COMPETING INTERESTS

Authors have declared that no competing interests exist.

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