INTRODUCTION:

Antimicrobial resistance (AMR) is a global health threat, causing an estimated 1.27 million deaths annually¹. The condition also contributes significantly to increased mortality and morbidity, putting a strain on healthcare systems in countries that face challenges in managing and preventing infectious diseases^2,3. Infections that were previously treatable are now becoming increasingly difficult to treat, posing a significant clinical challenge⁴. In the Southeast Asia (SEA) region, 4 million deaths in 2019 were caused by sepsis, either directly or indirectly. About 62% of these deaths were attributed to bacterial infections, while 38% were caused by other pathogens. Among bacterial infections, 15.71% to 56.26% were primarily caused by AMR⁵. A previous research reported that global mortality caused by AMR is predicted to reach 10 million cases per year by 2050, which is a drastic increase from the current annual mortality estimate of 700,000 cases globally. This shows a serious threat of AMR to global health if preventive measures are not taken immediately⁶. To address this issue, the World Health Organization (WHO) has identified priority pathogens for research and development of new treatments. Enterobacteriaceae Strains that produce Extended-spectrum β-lactamases (ESBL) and Methicillin-resistant Staphylococcus aureus (MRSA) are of critical concern due to significant contributions to mortality and the challenges posed in treating resistant infections⁷.

According to previous research, ESBL is an enzyme produced by Enterobaterales, such as Klebsiella pneumoniae. These enzymes hydrolyze a variety of β-lactam antibiotics including penicillin and broad-spectrum cephalosporins, leading to ineffectiveness in treating infections caused by the bacteria^8,9. The incidence of infections caused by ESBL-producing pathogens increased continuously from 2014 to 2020, with the prevalence of K. pneumoniae ESBL producers in the SEA region estimated at 27%¹⁰.

These results suggest that the exploration of alternative sources of antibacterial agents is becoming increasingly relevant, leading to the use of natural compounds with antimicrobial activity in the development of antibacterial agents. One of the potentially useful ingredients is cashew nut shell (CNS) and heated chicken eggshell powder (HCEP). Previous in vitro research showed that a compound from CNS called cashew nut shell liquid (CNSL), which contains unsaturated phenolic compounds in the form of anacardic acid, cardanol, and cardol has antimicrobial activity against Proteus Sp. Escherichia coli, Klebsiella pneumonia, Pseudomonas aeruginosa, Enterococcus cloacae, and Acinetobacter baumannii^11,12. Previous research has also shown that compounds derived from calcined HCEP cause CaCO₃ to decompose into calcium oxide (CaO), which showed antimicrobial activity against Escherichia coli, Klebsiella pneumonia, and Staphylococcus aureus in vitro^13,14.

Although a number of research have reported antibacterial activity of CNS and HCEP, existing research is still limited to in vitro methods and has not been accompanied by in silico analyses that can show the mechanisms of interaction and antibacterial potential in more depth. In addition, no research has been previously conducted that specifically evaluated the effectiveness of these two ingredients against resistant bacteria, particularly ESBL-producing bacteria. This shows the need for additional research to understand and optimize the use of CNS and HCEP as antibacterial agents. Therefore, this research aims to evaluate antibacterial activity of CNS and HCEP against resistant bacteria and investigate the mechanism of action through an in silico method.

MATERIALS AND METHODS:

Materials:

CNS were obtained from the Lombok area, West Nusa Tenggara, and Chicken eggshells were collected from the Pontianak area, West Kalimantan. Mueller Hinton Agar (MHA) and Mueller Hinton Broth (MHB) culture media, along with a standard 0.5 McFarland solution, were obtained from Himedia, India. Meropenem was the antibiotic used as a positive control (Interbat, Indonesia), and a 0.9% sodium chloride (NaCl) solution was supplied by Satoria Aneka Industri, Indonesia. Meanwhile, glycerin and dimethyl sulfoxide (DMSO) were obtained from Wilmar, Indonesia.

The Strains Included:

The test bacteria used in this research were the reference strains Klebsiella pneumoniae ATCC 1706 and ESBL-producing Klebsiella pneumonia ssp pneumonia, obtained from CV. Angkasa Abadi Medical Equipment, Indonesia. The bacterial stock was subcultured in MHA and then stored at a temperature of 2-8°C.

Preparation of CNS:

The obtained CNS were sorted wet and washed with clean water before drying in an oven at 60ºC for 24 hours. The CNS was further processed by mashing and sifting with mesh no. 60 and then stored in a clean closed container. CNS was extracted with Soxhlet tools using simplicia and a 96% ethanol solvent at a temperature of 80ºC. The results of the extraction were further concentrated using the Rotary Evaporator¹⁵. Fractionation was carried out using the liquid-liquid extraction method with the condensed extract of CNS. All obtained fractions, including N-hexane fractions, ethyl acetate, and Residual Aqueous Fraction (RAF) were concentrated using a Rotary Evaporator¹⁶. The disc diffusion antibacterial test sample was prepared by diluting extract, n-hexane fraction, ethyl acetate, and water with 10% DMSO to concentrations of 50%, 75%, and 100% (w/v). The microdilution test used a concentration of 10,000μg/mL from each sample.

Preparation of HCEP and Sample Solution:

The collected eggshells were washed to remove any adhering dirt, and the inner membranes were carefully peeled off. The cleaned eggshells were then dried in an oven at 105^oC for 90minutes. After drying, the eggshells were ground into a fine powder using a blender and then sieved through a 120-mesh sieve to ensure a uniform particle size. Any particles that did not pass through the sieve were reground until the required fineness was achieved before heat treating in a muffle furnace at 900°C for 3hours^13,17,18. Following the heating process, the powder was sieved again using the same 120-mesh to ensure consistent particle size. The HCEP was stored in an airtight container to protect it from contamination and moisture, which was used as a sample for in vitro antibacterial activity testing. Antibacterial testing samples were prepared by diluting HCEP in glycerine to concentrations of 5%, 10%, 20%, and 30% (w/v). Microdilution testing used a concentration of 50,000 μg/mL.

In Vitro Antibacterial Activity Test:

Agar Disc Diffusion:

Bacteria suspended in 0.9% NaCl (b/v) at turbidity equivalent to the McFarland standard of 0.5 were inoculated into MHA plates using a dispersed plate method with 25 μL of suspension. A filter paper disc (6 mm diameter) was immersed in the test sample solution for 5 minutes and then placed on the inoculated agar surface.

Positive controls were included to assess antibacterial activity against specific bacterial strains. Meropenem (10 μL, 1 μg/μL) was used as a positive control for producing bacteria (+ESBL) and bacterial strains (-ESBL), while DMSO 10% was used as a negative control for CNS samples and glycerin for HCEP samples.

The plates were incubated at 37°C for 24 hours, and antibacterial activity was assessed by measuring the clear inhibition zones formed around the disc. The diameter of this zone was measured three times using calipers, and the average value was recorded as the diameter of the inhibition zone. Furthermore, the experiments were carried out three times to ensure reproducibility¹⁹.

Broth Microdilution:

The experiment used 96-well microplates with 12 columns and 8 rows, and each well was filled with 100 μL MHB. The test sample solution was added to the first column and homogenized. From the first column, 100 μL was transferred to the second column and homogenized, with the serial dilution process repeated until the 10th column. In order to maintain the same volume, 100 μL was discharged from the tenth column. Bacterial suspensions in 0.9% NaCl (w/v) with a density of 1 × 10⁶ CFU/mL were added in a 10 μL aliquot to the well in the first to 10^th columns. The 11th column served as a normal control containing MHB and bacterial suspension to evaluate bacterial growth based on turbidity, while the 12th column served as a negative control containing only MHB to assess media clarity. The microplates were incubated at 37°C for 24 hours, and the minimum inhibitory concentrations (MICs) were identified as the lowest concentration in the first column that showed no visible bacterial growth. In addition, all the experiments in this research were carried out in triple replication^19,20.

In Silico Molecular Docking:

This research was conducted on HP ProBook which runs on an Intel® Core™ i5 processor. Ligand preparation and optimization were done using AutoDock Tools 1.5.4 and the Discovery Studio 2024 Client. The protein structure used was the penicillin-binding protein (PBP3) of K. pneumoniae (PDB ID: 8GPW) obtained from the Protein Data Bank (PDB) database (http://pdb.org). The ligand data was taken from the PubChem database (http://pubchem.org).

Data analysis:

The inhibition zone diameters of HCEP were statistically analyzed using SPSS 26 with One-way ANOVA. A p-value <0.05 showed a significant difference, while a p-value >0.05 suggested no significant difference in the data. A correlation test was carried out to measure the linear relationship between the variables tested. A commonly used correlation test is Pearson Correlation (for normally distributed data).

RESULT:

Extract and Fraction Inhibition Zone of CNS and HCEP using Disc Diffusion Method:

Antibacterial activity was determined using the disc diffusion method, and the results of the inhibition zone measurements are shown in Table 1. The average inhibition zone diameter of the three replications of the extract, the n-hexane fraction, the ethyl acetate fraction, and the CNS water fraction varied from weak to very strong. In ESBL-Producing K. pneumoniae, the n-hexane fraction showed strong to very strong inhibiting activity at all concentrations, while the inhibiting activity was in the medium category for ESBL-No Producing K. pneumoniae. It was followed by ethyl acetate fraction and CNS ethanol extract which had moderate to strong inhibition zone activity on the concentrations of ESBL-producing and non-ESBL-Producing K. pneumoniae, respectively. Water fractions with inhibition zone activity were included in the weak category of ESBL-Producing K. pneumoniae and ESBL-No Producing K. pneumoniae. At concentrations of 20% and 30%, which was the average inhibition zone, the heated eggshell provided inhibition against ESBL-Producing and non-ESBL-Producing K. pneumoniae and was categorized as medium inhibition. These inhibition values were significantly lower than meropenem activity as a positive control, but at 100% concentration, the n-hexane fraction had activity close to the inhibitory zone of meropenem against ESBL-Producing K. pneumoniae.

Table 1. Inhibition Zone of CNS and HCEP against K. pneumoniae

Test Sample	Group		Diameter of inhibition zone (mean±SD)
Test Sample	Group		Non-ESBL K. pneumoniae	ESBL-Producing K. pneumoniae
Meropenem			26.2 ± 1.24	25.9 ± 0.12
Ampicillin			13.5 ± 0.00	13.4 ± 0.00
CNS	DMSO		0	0
	Extract ethanol 96%	50%	6.2 ± 0.92	5.4 ± 0.56
		75%	6.7 ± 0.83	5.9 ± 0.55
		100%	7.8 ± 1.05	7.8 ± 1.05
	N-hexane fraction	50%	6.5 ± 0.77	15.0 ± 2.23
		75%	8.5 ± 0.64	18.6 ± 1.86
		100%	9.3 ± 0.30	23.0 ± 1.62*
	Ethyl acetate fraction	50%	6.9 ± 0.77	7.1 ± 0.70
		75%	8.3 ± 0.45	8.7 ± 1.50
		100%	10.8 ± 0.05	11.1 ± 2.88
	Water fraction	50%	1.2 ± 0.25	1.3 ± 0.26
		75%	2.0 ± 0.49	3.0 ± 0.83
		100%	4.9 ± 0.83	4.2 ± 0.43
HCEP	Glycerin		0	0
	5%		0	0
	10%		0	0
	20%		9.03 ± 0.35	9.54 ± 0.32
	30%		8.63 ± 0.54	9.40 ± 0.38

*not significantly different compared to Meropenem as control (p>0.05)

MIC Determination of CNS Extract and Fraction and HCEP by Microdilution Method:

The determination of MIC was carried out using the broth microdilution method. CNS Extract, n-hexane fraction, and ethyl acetate fraction showed potential antibacterial activity with MIC of <100 μg/mL against ESBL and non-ESBL K. pneumoniae. However, water fractions showed the weakest activity with MIC of >500 μg/mL. The MIC values of HCEP were not detected against ESBL and non-ESBL K. pneumoniae, with the maximum concentration tested at >1000 μg/mL. It was concluded that no potential activity was exhibited by heated chicken eggshells against these resistant and non-resistant isolates. The MIC results can be seen in Table 2.

Table 2. MIC CNS and HCEP

Test Sample	Group	MIC μg/mL
Test Sample	Group	Non-ESBL Producing K. pneumoniae	ESBL-Producing K. pneumoniae
Meropenem		0.06	0,06
Ampicillin		32	64
CNS	Extract ethanol 96%	0.6	2.4
	N-hexane fraction	0.6	19.5
	Ethyl acetate fraction	39	39
	Water fraction	625	625
HCEP		ND	ND

ND = not determined in concentration tested >1000 μg/mL

In Silico Analysis of CNS and HCEP as Potential Compounds Targeting PBP3 (8GPW) ReceptorsUseful in Bacterial Cell Wall Synthesis:

The docking test result in Table 3 shows the free energy, hydrogen bonds, and amino acid residues formed between ligands and PBP3 (8GPW). The root mean square deviation (RMSD) value obtained was 1.62 Å, which was used to determine the stability and quality of the binding mode when the ligand and receptor are docked. Furthermore, native ligands are known to meet validation standards with an RMSD value of less than 2.0 Å. The RMSD value of the validation results showed minimal changes after retethering and provided the same ligand position as natural ligands. The docking results showed a negative binding energy <0 kcal/mol, implying that the docked ligand and receptors had stable bonds. Ligands used such as anacardic acid, cardanol, and cardol have some amino acid residues similar to native ligands and positive controls (meropenem). However, the amino acid residues of CaO ligands do not show similarity with native ligands and meropenem controls. The more similar residues and bonds are to native ligands and positive controls, the higher the level of similarity of these properties.

Table 3. Calculation of free energy estimation of binding in docking of Anacardic Acid, Cardanol, Cardol, CaO, Native ligand, and meropenem

Group	ligand	RMSD	Binding energy (kcal/mol)	Hydrogen bond
Native ligand		1.62 Å	-6.21	GLU A:304 TYR A:419 THR A:497 THR A:495 SER A:307
Meropenem			-7.62	GLU A:304 TYR A:419 THR A:497 SER A:307
CNS	Anacardic acid		-8.38	SER A:359 SER A:307 LYS A:310
	cardanol		-8.38	GLU A:304
	cardol		-7.82	GLU A:304 PHE A:417
HCEP	CaO		-2.60	SER A:237 SER A:130

DISCUSSION:

The results of this research showed that the compounds from CNS extract and fraction and HCEP had antibacterial potential against ESBL-producing and non-ESBL-producing strains of Klebsiella pneumoniae, although activity varied depending on the type of fraction, concentration, and test method used.

Antibacterial Activity of CNS and HCEP using Disc Diffusion Method:

Antimicrobial activity was classified according to the parameters determined by Alves et al. (2000), where the <9 mm inhibition zone implies a lack of activity, the 9 to 12 mm inhibition zone implies low activity, and the 13 to 18 mm and >18 mm zones imply high and very high activity, respectively²¹. Testing using the disc diffusion method showed that the n-hexane fraction of CNS had the strongest antibacterial activity, particularly against ESBL-producing strains K. pneumoniae. This fraction exhibited an inhibition zone of up to 23.0 ± 1.62 mm at 100% concentration, similar to the inhibition zone produced by the positive control of meropenem 25.9 ± 0.12 mm, as shown in Figure 2. Activity is most likely caused by the presence of active phenolic compounds such as anacardic acid, cardanol, and cardol, which have been known to disrupt the structure and function of bacterial cell membranes. Antimicrobial activity is substantially affected by the position and number of hydroxyl groups because these groups can interact with bacterial cell membranes to disrupt cell structure, leading to leakage of cellular components²². The magnitude of the inhibition zone provided by the n-hexane fraction is due to the anacardic acid, cardanol, and cardol compounds being trapped in CNSL. The non-polar properties of this compound cause it ot be more effective against gram-negative bacteria with lower lipophilicity. The higher lipid content of the gram-negative cell wall structure may explain why the n-hexane fraction in this research showed more inhibition against gram-negative bacteria such as Klebsiella pneumonia^23,24,25. In contrast to CNS, HCEP only showed inhibition zone activity at high concentrations (20–30%), with the average inhibition zone being in the medium category (~9 mm), and did not show an inhibitory effect at lower concentrations. The compound suspected to have antibacterial activity from HCEP is CaO. These compounds can form free radical reactive oxygen species (ROS) that can produce oxidative stress resulting in bacterial DNA damage leading to cell death²⁶. Another mechanism that is thought to have antibacterial capabilities is the HCEP alkaline condition. The CaO contained in HCEP has a pH value of 12.8 and is classified as a strong alkaline²⁷. pH values maintained between pH 11-12 during the incubation process can have antibacterial effect²⁸. The antibacterial activity of the test compound increases with an increase in pH, causing the cellular respiration process of the bacteria to malfunction and leading to the death of the bacteria²⁹.

Figure 2. Antibacterial activity testing using the disc diffusion method. A concentration of 100% of the n-hexane fraction of CNS (B) produced a large inhibition zone that was not significantly different compared to Meropenem (A) against ESBL-producing K. pneumoniae (p>0.05).

Activity of CNS and HCEP using Microdilution Method:

MIC is determined by the micro-dilution method of microplate broth according to the recommendations of the Clinical and Laboratory Standards Institute. The broth dilution method can be used to quantitatively measure antimicrobial activity against bacteria. The antibiotics penicillin and meropenem were used to confirm the sensitivity or antibiotic resistance of the microorganisms being tested. Furthermore, the extracts with a MIC value of <100 μg/mL are considered to be highly active antimicrobial agents, MIC of 100 to 500 μg/mL are defined as active, 500 to 1000 μg/ml are defined as moderately active, 1000 to 2000 μg/mL are considered to have low activity, and > 2000 μg/ml are defined as inactive.

Figure 3. MIC profile of CNS extract, n-hexane fraction, ethyl acetate fraction, water fraction, and HCPE against ESBL and non-ESBL producing of K. pneumoniae by microdilution method. The lowest bar shown the high potential of antibacterial activity based on the MIC values.

The MIC of the extract, the n-hexane fraction, and the ethyl acetate fraction showed a value of <100 μg/mL, which is categorized as highly active based on the classification of antibacterial activity²¹. The result of this activity corroborates the results of disc diffusion, suggesting that CNS, particularly the n-hexane fraction, has the potential to be an alternative antibacterial agent. However, The MIC values obtained via the microdilution method did not fully correlate with the disc diffusion results, particularly in CNS extract. These differences may be attributed to variations in compound diffusion abilities in solid media versus the actual bacteriostatic or bactericidal potency in liquid environments. Additionally, the presence of synergistic or antagonistic interactions among bioactive constituents in the crude extract may contribute to the lower MIC observed. The MIC results showed that HCEP was not able to inhibit the growth of K. pneumoniae at the highest concentration tested (>1000 μg/mL), which shows the absence of potential antibacterial activity in this sample. This is suspected because the main active compound, namely CaO, has a more effective mechanism of action against gram-positive bacteria or through a non-optimal pathway against the gram-negative strain used³⁰.

In Silico Analysis Results:

The molecular docking research provides in-depth insights into the mechanisms of compound interaction with bacterial protein targets (PBP3, PDB ID: 8GPW). The three active compounds of CNS (anacardic acid, cardanol, cardol) show a high binding affinity (binding energy -7.82 to -8.38 kcal/mol), even stronger than meropenem (-7.62 kcal/mol). In addition, amino acid residues such as GLU A:304, SER A:307 show similar binding patterns to meropenem binding and native ligands, reinforcing evidence that these compounds have the potential to mimic the mechanism of action of β-lactam antibiotics. Serin (Ser) is an essential amino acid that plays a role in various biological processes in bacteria. It serves as a major precursor in the biosynthesis of glycine, cysteine, and tryptophan, where glycine plays a crucial role in the synthesis of bacterial nucleotides³¹. In contrast, the CaO compound from chicken eggshells showed only a low binding affinity (-2.60 kcal/mol) and had no important amino acid residues in common with positive or native ligand controls. These results are consistent with in vitro assays that show lower antibacterial activity of HCEP³².

CONCLUSION:

The results of this research show that compounds from CNS, particularly the n-hexane fraction, have significant potential as an alternative antibacterial agent against ESBL-producing K. pneumoniae. The combination of in vitro and in silico methods strengthens the validity of the results and suggests that the phenolic compounds of CNS may be promising candidates for new antibacterial compounds. In addition, HCEP showed antibacterial activity at high concentrations, but its effectiveness is still limited and requires further formulation or modification to increase its therapeutic potential.

CONFLICT OF INTEREST:

The authors declare no conflicts of interest regarding this research.

ACKNOWLEDGMENT:

The authors are grateful to Universitas Tanjungpura for funding this research.

REFERENCES:

1. Ranjbar R, Alam M. Antimicrobial Resistance Collaborators (2022). Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis. Evidence-Based Nursing. 2024; 27(1): 16–16.

2. Aslam B, Wang, Wei, Arshad, Muhammad Imran, Khurshid, Mohsin, Muzammil, Saima, Rasool, Muhammad Hidayat, et al. Antibiotic resistance: a rundown of a global crisis. Infection and Drug Resistance. 2018; 11: 1645–58.

3. Sihombing B, Bhatia R, Srivastava R, Aditama TY, Laxminarayan R, Rijal S. Response to antimicrobial resistance in South-East Asia Region. The Lancet Regional Health - Southeast Asia [Internet]. 2023 Nov 1 [cited 2025 May 4];18. Available from: https://www.thelancet.com/journals/lansea/article/PIIS2772-3682(23)00166-X/fulltext

4. Ahmed SK, Hussein S, Qurbani K, Ibrahim RH, Fareeq A, Mahmood KA, et al. Antimicrobial resistance: Impacts, challenges, and future prospects. Journal of Medicine, Surgery, and Public Health. 2024 Apr 1; 2: 100081.

5. Antimicrobial resistance (AMR) [Internet]. [cited 2025 May 4]. Available from: https://www.healthdata.org/research-analysis/health-risks-issues/antimicrobial-resistance-amr

6. Tang KWK, Millar BC, Moore JE. Antimicrobial Resistance (AMR). Br J Biomed Sci. 2023 Jun 28; 80: 11387.

7. Organization WH. WHO bacterial priority pathogens list, 2024: bacterial pathogens of public health importance, to guide research, development, and strategies to prevent and control antimicrobial resistance. World Health Organization; 2024. 72.

8. Yousefipour M, Rasoulinejad M, Hadadi A, Esmailpour N, Abdollahi A, Jafari S, et al. Bacteria Producing Extended Spectrum β-lactamases (ESBLs) in Hospitalized Patients: Prevalence, Antimicrobial Resistance Pattern and its Main Determinants. Iran J Pathol. 2019; 14(1): 61–7.

9. Ghafourian S, Sadeghifard N, Soheili S, Sekawi Z. Extended Spectrum Beta-lactamases: Definition, Classification and Epidemiology. Current Issues in Molecular Biology. 2015; 17(1): 11–22.

10. Salawudeen A, Raji YE, Jibo GG, Desa MNM, Neoh H min, Masri SN, et al. Epidemiology of multidrug-resistant Klebsiella pneumoniae infection in clinical setting in South-Eastern Asia: a systematic review and meta-analysis. Antimicrobial Resist Infect Control. 2023; 12(1): 142.

11. Mubofu EB. From cashew nut shell wastes to high value chemicals. Pure and Applied Chemistry. 2016; 88(1–2): 17–27.

12. Salami O.O., Atoyebi T.J., Microbiology Unit, Science Laboratory Technology Department, Federal College of Animal Health and Production Technology, P.M.B. 5029, Moor Plantation, Apata, Ibadan, Oyo State, Nigeria, Afolabi QO, Chemistry Unit, Science Laboratory Technology Department, Federal College of Animal Health and Production Technology, P.M.B. 5029, Moor Plantation, Apata, Ibadan, Oyo State, Nigeria, Obisesan L.O., et al. Effect of n-hexane and aqueous extracts of cashew nuts on some pathogenic organisms obtained from general out patients, Ibadan, Nigeria. Microbiol Res Int. 2017; 5(4): 55–61.

13. Al-Azzawi MH, Al-Kalifawi EJ. Antibacterial and Antibiofilm Activity of Calcite (CaCO3) Nanoparticles Synthesized from Chicken Eggshell. The Egyptian Journal of Hospital Medicine [Internet]. 2023 Jan 1 [cited 2025 May 4]; Available from: https://journals.ekb.eg/article_285688.html

14. Djayasinga R, Situmeang RTM, Unob F, Hadi S, Manurung P, Sumardi S. Chicken Eggshell Powder as Antibacterial Against Staphylococcus aureus and Escherichia coli Through In Vitro Studies. Journal of Multidisciplinary Applied Natural Science. 2024; 4(1): 194–209.

15. Extraction, Isolation and Chemical Modification of the Anacardic Acids from Cashew Nut Shell (Anacardium occidentale L.) and Biological Assay. Curr Res Bioorg Org Chem [Internet]. 2018 Jun 28 [cited 2025 May 7]; 1(2). Available from: https://www.gavinpublishers.com/articles/research-article/Current-Research-in-Bioorganic-Organic-Chemistry/extraction-isolation-and-chemical-modification-of-the-anacardic-acids-from-cashew-nut-shell-aanacardium-occidentale-l-and-biological-assay

16. Ratna YRD, Ardani US, Fathiana Z, Rahmatillah A, K ITD. Antibacterial Power of Cashew Leaf Extracts and Fractions (Anacardium occidentale L.) against Sensitive and Multiresistant Staphylococcus aureus Bacteria. Indonesian Journal of Pharmaceutical Sciences. 2017; 14(1): 103–10.

17. Hamouda MA, Sweidan H, Maraqa MA, El-Hassan H. Mechanistic Study of Pb2+ Removal from Aqueous Solutions Using Eggshells. Water. 2020; 12(9): 2517.

18. Awogbemi O, Inambao F, Onuh EI. Modification and characterization of chicken eggshell for possible catalytic applications. Heliyon [Internet]. 2020 Oct 1 [cited 2025 May 7]; 6(10). Available from: https://www.cell.com/heliyon/abstract/S2405-8440(20)32126-5

19. Hafidh RR, Abdulamir AS, Vern LS, Abu Bakar F, Abas F, Jahanshiri F, et al. Inhibition of Growth of Highly Resistant Bacterial and Fungal Pathogens by a Natural Product. Open Microbiol J. 2011 Aug 16; 5:96–106.

20. Kowalska-Krochmal B, Dudek-Wicher R. The Minimum Inhibitory Concentration of Antibiotics: Methods, Interpretation, Clinical Relevance. Pathogens. 2021 Feb; 10(2): 165.

21. Saraiva M. In vitro evaluation of antioxidant, antimicrobial and toxicity properties of extracts of Schinopsis brasiliensis Engl. (Anacardiaceae). Afr J Pharm Pharmacol. 2011 Oct 15; 5(14): 1724–31.

22. Safdar MN, Kausar T, Jabbar S, Mumtaz A, Ahad K, Saddozai AA. Extraction and quantification of polyphenols from kinnow fruit peel ( Citrus reticulate L.) using ultrasonic and maceration techniques. Journal of Food and Drug Analysis. 2017 Jul 1; 25(3): 488–500.

23. Pandian S, Dahyalal PC, Krishna S, Hari S, Subramanian D. A study on cashew nut shell liquid as a bio-based flow improver for heavy crude oil. J Petrol Explor Prod Technol. 2021 May 1; 11(5): 2287–97.

24. Mubofu EB, Mgaya JE. Chemical Valorization of Cashew Nut Shell Waste. Top Curr Chem (Z). 2018 Feb 13; 376(2): 8.

25. Mogana R, Adhikari A, Tzar MN, Ramliza R, Wiart C. Antibacterial activities of the extracts, fractions and isolated compounds from Canarium patentinervium Miq. against bacterial clinical isolates. BMC Complement Med Ther. 2020 Feb 14; 20(1): 55.

26. Borisov VB, Siletsky SA, Nastasi MR, Forte E. ROS Defense Systems and Terminal Oxidases in Bacteria. Antioxidants. June 2021; 10(6):839.

27. Farah IO, Lyons WO, Arslan Z, Miller G, Tucci M, Tchounwou PB. Calcium Oxide Remediation of Anthropogenic Contamination of Water at the Gbnerr in MISSISSIPPI. Biomed Sci Instrum. 2019 Jan; 55(1): 158–65.

28. Kincses A, Rácz B, Baaity Z, Vásárhelyi O, Kristóf E, Somogyvári F, et al. The Relationship between Antibiotic Susceptibility and pH in the Case of Uropathogenic Bacteria. Antibiotics. 2021 Dec; 10(12): 1431.

29. Khan AU, Hussain T, Abdullah, Khan MA, Almostafa MM, Younis NS, et al. Antibacterial and Antibiofilm Activity of Ficus carica-Mediated Calcium Oxide (CaONPs) Phyto-Nanoparticles. Molecules. 2023 Jan; 28(14): 5553.

30. Amako K, Meno Y, Takade A. Fine structures of the capsules of Klebsiella pneumoniae and Escherichia coli K1. Journal of Bacteriology. 1988 Oct; 170(10):4960–2.

31. Zhang H, Li Q, Li Y, Chen S. The Serine Biosynthesis of Paenibacillus polymyxa WLY78 Is Regulated by the T-Box Riboswitch. International Journal of Molecular Sciences. 2021 Jan; 22(6): 3033.

32. Tallei TE, Tumilaar SG, Niode NJ, Fatimawali, Kepel BJ, Idroes R, et al. Potential of Plant Bioactive Compounds as SARS-CoV-2 Main Protease (Mpro) and Spike (S) Glycoprotein Inhibitors: A Molecular Docking Study. Scientific. 2020; 2020(1): 6307457.

Received on 29.06.2025 Revised on 03.10.2025

Accepted on 30.12.2025 Published on 01.07.2026

Available online from July 04, 2026

Research J. Pharmacy and Technology. 2026;19(7):3067-3074.

DOI: 10.52711/0974-360X.2026.00436

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.