Antimicrobial Effects of Quebrachitol: A Systematic Review

Abstract

Quebrachitol, an optically active cyclitol derived from plants, has recently gained attention as a potential natural product with therapeutic properties, though its antimicrobial effects remain unclear. This systematic review aims to determine, appraise, and consolidate evidence of the antimicrobial potential of quebrachitol. PRISMA-guided searches of PubMed, Scopus, and Google Scholar (2000–2024) identified English-language experimental in vitro, in vivo, and in ovo studies. Data on antimicrobial activity, dosage or treatment duration, and mechanisms were extracted, with study quality assessed using QUIN and SYRCLE tools. Of 866 studies screened, 11 met inclusion criteria: seven in vitro, one in vivo, one in ovo, and two combining both approaches. Quebrachitol demonstrated inhibitory effects against Salmonella sp., Candida albicans, infectious bursal disease virus (Avibirnavirus gumboroense), Newcastle disease virus, Plasmodium sp., and notably, biofilm formation by Staphylococcus epidermidis and methicillin-resistant Staphylococcus aureus (MRSA). Overall, quebrachitol exhibits promising antimicrobial potential, but rigorous in vivo studies are required to confirm its efficacy and safety in addressing antimicrobial resistance.

1. Introduction

Phytochemicals, a diverse array of bioactive compounds produced by plants, have gained significant attention for their antimicrobial properties and their potential to combat infections, particularly those caused by drug-resistant pathogens. These compounds are categorised into distinct classes based on their chemical structures, including alkaloids, terpenoids, phenols, organosulfur compounds, and cyclitols, each demonstrating unique mechanisms of action that target and inhibit microbial growth and survival.

Quebrachitol (QCT), also known as L-quebrachitol (2-O-methyl-L-inositol), is a naturally occurring polyol with the molecular formula C₇H₁₄O₆ (Figure 1). It is a methylated derivative of inositol, and a type of cyclohexanol. QCT consists of a six-membered cyclohexane ring with hydroxyl groups and a single methoxy group (-OCH₃) attached, distinguishing it from glucose (C₆H₁₂O₆). It is white in colour with a molecular weight of 194.18 g/mol. It has a melting temperature of 192–193 °C and a boiling temperature of 210 °C. Its solubility varies with temperature, as 100 g of a saturated solution contains 38 g of the compound at 0 °C, 39 g at 12 °C, and 70 g at 100 °C. It is soluble in water, alcohol, and pyridine, but insoluble in ether [1].

QCT was first discovered in the Aspidosperma quebracho-blanco Schltdl plant in 1887 by Tanret. It was found in the latex of Hevea brasiliensis Muell. Arg. in 1906 [2]. More specifically, in latex serum or rubber factory wastewater. Eventually, it was extracted from the following plants: red oil of Minnesota Wild Hemp, Haplophyton cimicidum A.DC., Allophylus edulis (Camb.) Radlk., Litchi chinensis Sonn., Acer pseudoplatanus L., Cannabis sativa L., sea buckthorn, Paullinia pinnata L., Artemisia gmelinii Weber ex Stechm., Sapindus rarak DC., Dipladenia martiana (Stadelm.) Woodson, and Cardiospermum halicacabum L. [3,4,5,6,7,8,9]. The potential of QCT in medicine including its anti-diabetic, anti-platelet, antioxidant, and antimicrobial effects was widely investigated [1].

QCT has insulin-like effects, improving glucose metabolism and insulin sensitivity in Type 2 diabetes. It inhibits α-amylase, reducing glucose absorption and postprandial spikes [10,11]. As a potent antioxidant, QCT scavenges reactive oxygen species (ROS) and free radicals, surpassing ascorbic acid in efficacy [12,13,14,15,16]. Its neuroprotective effects in Parkinson’s disease models suggest potential in reducing oxidative damage and neuronal apoptosis [17]. QCT promotes bone mineralization by stimulating osteoblast activity and upregulating key bone matrix proteins such as alkaline phosphatase (ALP), type I collagen (ColI), osteocalcin (OCN), and osteopontin (OPN) [18]. It also enhances RUNX family transcription factor 2 (RUNX2) expression, relevant to osteogenic metastasis in breast cancer [19]. QCT from Mitrephora vulpina C.E.C. Fisch. inhibits platelet-activating factor (PAF) receptors, reducing platelet aggregation and thrombus formation [20]. It also exhibits anti-cancer potential by blocking PAF-mediated inflammation and tumour progression [21,22]. As a readily available precursor, QCT is a fundamental building block in the development of various antimicrobials [23,24,25], enzyme inhibitors [26,27], inositol derivatives [28,29], and cancer or immunosuppressive drugs [30]. QCT protects against gastric mucosal injury by promoting prostaglandin synthesis, nitric oxide release, and potassium channel opening, enhancing gastric integrity and ulcer prevention [31]. Additionally, QCT inhibits glutaminase, reducing glutamate production and neuronal hyperexcitation, showing promise as an anticonvulsant in epilepsy [32].

However, previous research on antimicrobial effects of QCT is limited, with only few articles describing its promising antibiofilm properties against pathogenic bacteria. Vijayakumar et al. [33] indicate that QCT effectively inhibits biofilm formation in Methicillin-resistant Staphylococcus aureus (MRSA) without affecting bacterial growth. It achieved this by reducing the production of virulence factors, impairing bacterial adherence to various surfaces, and downregulating key virulence genes which were essential for biofilm development and maintenance. Similarly, Karuppiah et al. [34] demonstrate that QCT inhibits biofilm formation in Staphylococcus epidermidis by disrupting initial bacterial attachment and intercellular adhesion. This disruption led to a decrease in the production of biofilm components such as extracellular polysaccharides (EPS). Additionally, QCT reduces bacterial adherence to both biotic and abiotic surfaces, suggesting its potential use in therapeutic strategies for biofilm-associated infections, such as coatings for medical devices to prevent biofilm formation or adjunct treatments to enhance antibiotic efficacy. QCT also downregulates virulence genes involved in biofilm formation. Therefore, these findings suggest that QCT holds promise as a therapeutic agent targeting biofilm-associated infections through inhibition at multiple stages of biofilm formation, including the initial attachment of bacteria and the production of EPS to quorum sensing (QS) inhibition. However, the mechanism of action underlying its inability to directly inhibit bacteria remains undetermined.

Despite these promising findings, there has not been a comprehensive review summarising all of QCT’s antimicrobial properties. This huge gap in the literature makes it difficult to fully understand the breadth of QCT’s antimicrobial potential. This study aims to systematically identify and evaluate all evidence in order to determine whether QCT has antimicrobial effects such as antibacterial, antifungal, antiviral, antiparasitic, or antibiofilm.

2. Materials and Methods

2.1. Search Strategy

A thorough electronic search of literature from 2000 to 2024 was conducted on PubMed, Scopus, and Google Scholar to identify scientific papers on the anti-infective therapy of QCT. In order to find all pertinent research, the following search query was created: “Quebrachitol” or “L-quebrachitol” AND “anti-microbial” OR “anti-bacterial” OR “anti-fungal” OR “anti-viral” OR “bacteria” OR “fungus” OR “virus”. Titles and abstracts were screened and scrutinised for eligibility. A manual search of references and citations was also done to identify additional related articles and ensure complete literature coverage.

Research papers were screened based on inclusion and exclusion criteria. Inclusion criteria were all English full-text, peer-reviewed original articles from 2000 to 2024. All non-English, non-full-text publications, review articles, and grey research papers such as dissertations or conference reports were excluded from this systematic review.

Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines and a flow diagram were used to conduct the selection [35]. All studies were initially exported to the Endnote reference manager with particulars such as the title of the articles, names of authors, journals, year of publication, and article link (PMID/DOI) imported. Identical articles were eliminated automatically, and therefore, the duplication elimination was expedited and simplified.

Subsequently, all full-text evaluations for eligibility were conducted by two reviewers to validate suitable paper selections based on the inclusion and exclusion criteria. Studies that fulfil the inclusion criteria proceeded with the data analysis. Similarly, a third reviewer was consulted for any disagreement between the first two reviewers. After validating the eligible studies, data was tabulated in Microsoft Excel.

2.2. Quality and Risk of Bias Assessment

Each article was critically appraised to evaluate the quality and validity of each study. The risk of bias was evaluated by two independent researchers, and a third accessor was appointed if there were unresolved discrepancies between the first two researchers.

Quality Assessment Tool for In Vitro Studies (QUIN) was used to assess the quality of in vitro research for risk of bias [36]. It consisted of 12 criteria, as stated in

Table 1. Criteria in Quality Assessment Tool for In Vitro Studies (QUIN).

No	Criteria
1	Clear objectives/aims
2	Sample size calculation detail explanation
3	Sampling technique explanation
4	Comparison groups details
5	Methodology detail explanation
6	Operator details
7	Randomisation
8	Outcome measurement methods
9	Details on outcome accessors
10	Blinding process
11	Statistical analysis

. Each of the items was graded with a score of 2 for “adequately specified”, 1 for “inadequately specified”, 0 for “not specified,” and X for “non-applicable”. The final score, which was utilised to assess the risk of bias, was calculated with the formula below [36]:

$$Final Score={\displaystyle \frac{100 \times Total score}{2 \times Nmber of applicable criteria}}$$

Scores greater than 70% showed a low risk of bias, scores between 50% and 70% suggested moderate risk, and scores less than 50% indicated high risk.

The Systematic Review Centre for Laboratory Animal Experimentation (SYRCLE) Risk of Bias (RoB) tool, adapted from Cochrane RoB [37], was used to assess the risk of bias in animal or in vivo studies. SYRCLE was developed to ensure uniformity and prevent inconsistencies in evaluating the risk of bias in systematic reviews of animal intervention studies. There were 10 items to identify various types of biases including selection, performance, detection, attrition, reporting, and study design.

Table 2. Type of bias and domains in Systematic Review Centre for Laboratory animal Experimentation Risk of Bias (SYRCLE RoB) tool.

Item	Type of Bias	Domain
1.	Selection	Generation of sequence
2.	Selection	Characteristics of baseline
3.	Selection	Allocation Concealment
4.	Performance	Housing randomisation
5.	Performance	Blinding
6.	Detection	Random outcome assessment
7.	Detection	Blinding
8.	Attrition	Outcome data completion
9.	Reporting	Selection of Outcome reports
10.	Other	Miscellaneous bias

shows the criteria in the SYRCLE tool. One or more signalling questions were added to each itemised criterion to aid in decision-making and improve objectivity and relevance. Each entry was evaluated by a “yes”, “no” and “unclear judgement” which parallels a low risk, high risk, and inadequate detail to assess the risk of bias.

The selection biases described the sequence allocation, factors to determine prognosis, and allocation concealment. Random housing for the animals or blinding processes would determine performance biases while detection biases were ascertained by randomisation in animal selection and blinding of the researchers. For each primary outcome, the comprehensiveness of the outcome data was confirmed, taking into account attrition and analytical exclusions. Evaluation of contamination, financial influence, analytic inaccuracy, and design biases which could have exerted influence on the quality of studies were also included.

2.3. Data Synthesis and Analysis

The methods used for data synthesis and analysis were thematic, descriptive, and narrative approaches [35]. This systematic review is based on their outcomes and limited to qualitative assessment instead of performing a meta-analysis due to the diversity, such as heterogeneity of microorganisms, study design or methodologies, and outcome measures, between studies.

The results of the studies were categorised in accordance with the main antimicrobial effects investigated: antibacterial, antifungal, antiviral, antiparasitic, and antibiofilm.

Based on these categories, the results of findings were summarised in a table that included the name of the author, source of QCT, study model (in vitro, in vivo, in ovo, or combined approach), names of the targeted microorganism, study results, and conclusions. In the study results, in addition to the positive or negative findings, the dosage/concentration, duration, and mechanism of actions were included where applicable and if data was available. Finally, the conclusion was reported as the presence or absence of antimicrobial effects in response to the research question.

The table of findings summary was preceded by the narrative elaboration of the results and analysis findings. During the analysis, the number of studies in each category was stated. If there were two or more comparable studies on a given microorganism in each category, the study design, methods, and consistency of the results were compared. In the event that only a single study was conducted on a specific microorganism, the findings were reported exactly as they were. The overall quality of the data and the reasons for QCT’s beneficial benefits were delineated thoroughly.

3. Results

3.1. Search Results

This research is a systematic review that compiles and qualitatively appraises studies to determine the antimicrobial effects of QCT. The findings of the research are presented with PRISMA. There were 866 research articles found in the primary search and another 2 articles were added from the citation chase (Figure 2). After 16 duplications were eliminated, there were only 852 articles for further preliminary screening of titles and abstracts.

Following the initial title and abstract screening, only 392 articles remained to be assessed for eligibility after 460 articles were excluded due to the following reasons: 142 non-full-text articles, 112 foreign language papers, 98 books or chapters of books, 81 dissertations, and 27 non-journal articles such as letters, commentaries, reports, or conference presentations.

Eligibility assessments were further performed on 392 full-text articles. There were only 11 articles that met the inclusion criteria, underwent quality assessment, and were subsequently analysed and reported. Out of the 381 full-text articles excluded, 102 were review articles, and 227 papers had missing keywords. Thirty-one articles reported on QCT’s effects other than antimicrobial, while 21 studies described antimicrobial properties that were non-related to QCT.

There were 11 included studies with seven in vitro, one in vivo, one in ovo, and another two which performed both in vitro and in vivo trials [33,34,38,39,40,41,42,43,44,45,46]. From the 11 included articles, there were two studies focusing on QCT’s effects on bacteria, three on fungi, one on viruses, three on parasites, and two on biofilm effects. The characteristics of the 11 selected studies are shown in

Table 3. Study characteristics of included studies.

Author, Year	Source of Quebrachitol (Parts)	Microorganism	Study Model (In Vitro, In Vivo, or Both)	QCT Comparators	Methods	Purpose	References
Lunga et al., 2014	Paulina pinnata L. (leaves)	Salmonella sp.—S. typhi, S. paratyphi A, S. paratyphi B, S. typhimurium	Both				[45]
			In vitro	Ciprofloxacin, gentamicin	Broth microdilution	Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)
			In vivo	Ciprofloxacin, oxytetracycline	Animal (rats) groups: reference (non-infected), control (distilled water), Comparators antibiotic, treated rats with leaf extract of P. pinnata L. at MIC, 2MIC, 4MIC, and 8MIC.	Determination of antibacterial effects
					Faecal collection for calculation of Salmonella sp. load
Wannuch et al., 2015	Hevea brasiliensis Muell. Arg. (Latex from bark)	Staphylococcus aureus, Streptococcus mutans	In vitro	Nil	Disc diffusion antibiotic sensitivity	Determine bacterial inhibition	[39]
Cushion et al., 2016	Reagent from Sigma Aldrich	Pneumocystis carinii	In vivo	Myoinositol, inositol derivatives	Inositol uptake assay done on infected lung tissue of rats	Assessment of inositol transport activity of P. carinii for organism survival.	[44]
Kumar, 2016	Acalypha indica L. (leaves)	Candida albicans	In vitro	Ciprofloxacin, cefotaxime	Disc diffusion	Antifungal analysis	[38]
Nyandoro et al., 2013	Artabotrys modestus Diels. (Stem bark)	S. aureus, Escherichia coli, Candida albicans, Cryptococcus neoformans	In vitro	Ampicillin	Agar diffusion, microplate dilution assay	Antibacterial and antifungal analysis	[40]
Nyandoro, 2017	Artabotrys modestus Diels. (Stem bark)	Immune complex infectious bursal disease virus (IBDV), Newcastle disease virus (NDV)	In ovo	Placebo: dimethyl sulfoxide (DMSO)	Allantoic or amniotic inoculation assay	Antiviral activity analysis	[43]
					Viral hemagglutination test	Assessment of infectivity of the virus
Andima et al., 2021	Taberneamontana ventricosa Hochst. ex A.DC. (Stem bark)	Leishmania donovani	In vitro	Nil	3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) micro method	Evaluation of Leishmania sp. inhibition	[41]
Mongan et al., 2004	Reagent from Sigma Aldrich	L. donovani	In vitro	Myoinositol, inositol derivatives	Inositol uptake assay	Assessment of inositol transport activity of L. donovani for organism survival.	[42]
Mishra et al., 2023	Putranjiva roxburghii Wall. (Twigs)	Plasmodium falciparum	In vitro	Chloroquine	Parasite-specific lactate dehydrogenase (pLDH) assay	Anti-plasmodium analysis	[46]
					MTT	Cytotoxicity analysis
		Plasmodium berghei	In vivo	Chloroquine	6 groups of mice: water (negative), chloroquine (positive), butanol, quebrachitol at 30 mg/kg,60 mg/kg, and 120 mg/kg.	Determine parasite suppression percentage and mean survival time (MST)
					Microscopic evaluation of blood stained with Giemsa
Karuppiah et al., 2020	Rhizophora mucronata Lam. (Leaves)	Streptococcus epidermidis	In vitro	DMSO	Microdilution	Antibacterial activity	[34]
					Confocal laser scanning microscopy (CLSM) analysis	Antibiofilm efficacy and activity on different surfaces
					Phenolsulphuric acid method	Exopolysaccharides assessment
					Colorimetric method	Lipid estimation
					Lowry’s method	Protein estimation
					CLSM analysis	Biofilm architecture, formation of different materials
					Physico-chemical interactions assay	Evaluation of organism adherence to non-biological surfaces
					Spectrophotometry	Biofilm aggregation assessment
					Phenotypic assay	Autolytic process evaluation
					Spectrofluorometry, optical microscopy	Evaluation of adherence to biotic surfaces
					CLSM analysis	Evaluation of production of lipase
					Azocasein assay	Quantification of protease
					Real-time polymerase chain reaction (PCR)	Analysis of gene expression
Vijayakumar et al., 2020	Reagent from Sigma Aldrich	Methicillin-resistant Staphylococcus aureus	In vitro	Placebo	Broth microdilution	Antibacterial activity	[33]
					Crystal violet staining with absorbance at 570 nm	Antibiofilm potential
					Microscopic evaluation	Effect on biofilm architecture, disruption
					CLSM analysis	Antibiofilm activity on different surfaces
					Microbial adhesion to hydrocarbon (MATH) assay	Biofilm adhesion evaluation
					Phenolsulphuric acid method	Exopolysaccharides assessment
					CLSM analysis	Biofilm architecture, formation on different materials
					Phenotypic assay	Autolytic process and staphyloxanthin synthesis evaluation
					Spectrophotometry	Biofilm aggregation assessment
					Microdilution	Quantification of hemolysis
					Real-time PCR	Analysis of gene expression

Abbreviations: MIC, minimum inhibitory concentration; MBC, minimum bactericidal concentration; IBDV, immune complex infectious bursal disease virus, NDV, Newcastle disease virus; DMSO, dimethyl sulfoxide; MTT, 3-(4,5- dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide; pLDH, parasite-specific lactate dehydrogenase; MST, mean survival time; CLSM, confocal laser scanning microscopy; PCR, polymerase chain reaction.

.

3.2. Analysis of the Included Studies

There were two articles describing the antibacterial effects of QCT. Lunga et al. [45] demonstrated the presence of antibacterial properties against Salmonella sp. However, the other study by Wannuch et al. [39] did not show any effects on the tested bacteria, S. aureus and S. mutans.

Three studies have examined the antifungal activity of QCT against different fungal species. Kumar et al. [38] reported inhibitory effects against Candida albicans, whereas Nyandoro et al. [40] observed inconsistent results against C. albicans and Cryptococcus neoformans. In contrast, Cushion et al. [44] found no antifungal effect of QCT against Pneumocystis carinii.

The only paper to demonstrate the antiviral effects of QCT against the immune complex infectious bursal disease virus (IBDV) and Newcastle disease virus (NDV) was reported by Nyandoro [43].

Under the antiparasitic category, Mishra et al. [46] demonstrated the presence of antimalarial properties of QCT. However, both studies on leishmaniasis found no antiparasitic effects of QCT [41,42].

Lastly, two articles by Karuppiah et al. [34] and Vijayakumar et al. [33] reported QCT’s antibiofilm effects against Gram-positive biofilm producers such as MRSA and S. epidermidis.

Table 4. Antimicrobial effects of quebrachitol.

No.	Author, Year	Types of Antimicrobial Effects	Study Model (In Vitro, In Vivo, Both)	Names of Microorganism	Presence (Yes) or Absence (No) *	Reference
1	Lunga et al., 2014	Antibacterial	Both	Salmonella sp. (Gram-negative)	Yes (Bacteriostatic effects on Salmonella paratyphi A, S. paratyphi B, Salmonella typhi; bactericidal effects on Salmonella typhimurium)	[45]
2	Wannuch et al., 2015	Antibacterial	In vitro	Staphylococcus aureus (Gram-positive), Streptococcus mutans (Gram-positive)	No	[39]
3	Cushion et al., 2016	Antifungal	In vivo	Pneumocystis carinii (yeast-like fungus)	No	[44]
4	Kumar, 2016	Antifungal	In vitro	Candida albicans (yeast)	Yes (Observed inhibition zones)	[38]
5	Nyandoro et al., 2013	Antifungal	In vitro	C. albicans, Cryptococcus neoformans (yeast)	No	[40]
6	Nyandoro, 2017	Antiviral	In ovo	Infectious bursal disease virus, Newcastle disease virus	Yes (viral titre reduction)	[43]
7	Andima et al., 2021	Antiparasitic	In vitro	Leishmania donovani	No	[41]
8	Mongan et al., 2004	Antiparasitic	In vitro	L. donovani	No	[42]
9	Mishra et al., 2023	Antiparasitic	Both	Plasmodium sp.	Yes (Inhibition and cytotoxicity)	[46]
10	Vijayakumar et al., 2020	Antibiofilm	In vitro	Methicillin-resistant S. aureus (MRSA)	Yes (Antibiofilm effects and virulence inhibition)	[33]
11	Karuppiah et al., 2020	Antibiofilm	In vitro	Staphylococcus epidermidis (Gram-positive)	Yes (Biofilm inhibitory)	[34]

* “Yes” or “No” indicate the presence or absence of antimicrobial effects, respectively.

shows the summary of the antimicrobial outcomes of QCT from this review.

Among the 11 studies, one demonstrated antibacterial, one antifungal, one antiviral, one antimalarial, and two antibiofilm effects against bacteria while five did not show any antimicrobial effects from QCT.

3.3. Antibacterial Effects of Quebrachitol

The bactericidal properties of QCT were studied in four different research projects but only one showed favourable results (

Table 5. Findings summary of antibacterial effects of quebrachitol (QCT).

Author, Year	Microorganism	Results										Inhibition/Killed	Mechanism	Antimicrobial Effects
Lunga et al., 2014 [45]	In vitro	QCT				Ciprofloxacin			Gentamicin
		Minimum inhibitory concentration, MIC (μg/mL)	Minimum bactericidal concentration, MBC (μg/mL)		MBC/MIC	MIC (μg/mL)	MBC (μg/mL)	MBC/MIC	MBC (μg/mL)	MBC/MIC
	Salmonella typhi (Gram-negative),	50	100		8	6.25	0.781	4	12.5	2		Inhibition	Bacteriostatic	Antibacterial
	Salmonella paratyphi A (Gram-negative),	12.5	100		8	1.562	0.781	4	6.25	4		Inhibition	Bacteriostatic
	S. paratyphi B (Gram-negative),	12.5	100		8	3.125	1.562	1	12.5	4		Inhibition	Bacteriostatic
	Salmonella typhimurium (Gram-negative)	100	100		4	3.13	3.125	4	6.25	2		Killed	Bactericidal
	In vivo on rats infected with S. typhimurium	QCT				Ciprofloxacin (7.14 mg/kg bw)			Oxytetracycline (5.00 mg/kg bw)
		Dosage (mg/kg bw)	Healing time (days)	White blood cell (WBC) count (103 mm−3)	Lymphocytes (%)	Healing time (days)	WBC count (103 mm−3)	Lymphocytes (%)	Healing time (days)	WBC count (103 mm−3)	Lymphocytes (%)	Inhibition	Leukocytes and lymphocytes proliferation	Antibacterial
		55.75 (MIC)	8	5.32 ± 0.534	51.000 ± 6.082	4	5.833 ± 1.264	52.333 ± 3.785	4	4.040 ± 0.065	45.000 ± 6.000
		111.5 (2MIC)	6	6.266 ± 0.862	54.666 ± 4.163
		223.00 (4MIC)	5	7.553 ± 0.642	65.333 ± 7.094
		446 (8MIC)	4	6.606 ± 1.572	65.333 ± 5.859
Wannuch et al., 2015 [39]	In vitro Staphylococcus aureus (Gram-positive), Streptococcus mutans (Gram-positive)	QCT										Nil	Nil	No significant inhibitions on S. aureus and S. mutans
		Dosage	Areas of inhibition (cm)
			S. aureus		S. mutans
		0 (control)	0.9 ± 0.1		0
		1	1.0 ± 0.1		0
		5	1.3 ± 0.3		0
		10	1.0 ± 0.0		0
		100	1.0 ± 0.1		0

Abbreviations: QCT, quebrachitol; MIC, minimum inhibitory concentration; MBC, minimum bactericidal concentration; WBC, white blood count.

). Lunga et al. [45] demonstrated that methylinositol, which has a similar carbon-13 nuclear magnetic resonance (13C NMR) to QCT, showed bacteriostatic effects on Salmonella paratyphi A, S. paratyphi B, Salmonella typhi, and bactericidal effects on Salmonella typhimurium, although less effective in comparison to ciprofloxacin and gentamicin. The minimum bactericidal (MBC) and inhibitory (MIC) concentrations varied with different Salmonella species. The MIC were 50 μg/mL (S. typhi), 12.5 μg/mL (S. paratyphi A), 12.5 μg/mL (S. paratyphi B), and 100 μg/mL (S. typhimurium) in comparison to 6.25, 1.562, 3.125 and 3.13 μg/mL for respective species with ciprofloxacin. The MIC for gentamicin was not stated. The MBC for QCT was 100 μg/mL for all Salmonella sp, but significantly lower among the species of ciprofloxacin (0.781–3.125 μg/mL) and gentamicin (6.25–12.5 μg/mL). The MBC:MIC ratios were 8 for S. paratyphi A, S. paratyphi B, S. typhi, and 4 for S. typhimurium, respectively. The in vivo test on S. typhimurium-infected rats with crude extracts significantly reduced bacterial loads in addition to increasing leukocytes and lymphocytes, healing animals in 4 to 8 days, in a dose-dependent manner from 55.75 to 446.00 mg/kg body weight of extracts. There was no discernible difference (p > 0.05) in the number of residual organisms post-therapy with the maximum dosage of extract in comparison with oxytetracycline or ciprofloxacin.

Wannuch et al. [39] observed a modest negligible inhibition against S. aureus with an inhibition zone area of 1.0 ± 0.1 cm (L-quebrachitol concentration of 1% w/v) to 1.3 ± 0.3 cm (L-quebrachitol concentration of 5% w/v) in comparison to the control, 0.9 ± 0.1 cm. Additionally, there was a complete lack of inhibition seen with S. epidermidis.

Similar inactivity against MRSA and S. epidermidis from QCT was also observed in another two studies [33,34]. However, in one of these articles by Nyandoro et al. [40], artamontereine, another phytochemical, demonstrated antibacterial effects on S. aureus. The other metabolites of Artabotrys sp. such as catechin, cyclohexane-1,2,4,5-tetrol, karatavin, and laudanine also inhibited S. aureus and E. coli.

3.4. Antifungal Effects of Quebrachitol

There are conflicting findings when investigating QCT’s potential as an antifungal against Candida sp. One study shows inhibition against C. albicans from methanol extracts of Acalypha indica L., with an inhibition zone measuring 17 mm in comparison to the reference drugs, 30 and 19 mm for ciprofloxacin and cefotaxime by disc diffusion methods respectively (

Table 6. Findings summary of antifungal effects of quebrachitol (QCT).

Author, Year	Microorganism	Results			Inhibition/Killed	Mechanism	Antimicrobial Effects
Kumar, 2016 [38]	In vitro	Methanol extract containing QCT	Ciprofloxacin	Cefotaxime	Inhibition	Interacts with Glu27, and other residues to inhibit exo-glucanase, and hyphae formation and induces catalysis.	Antifungal
	Candida albicans (yeast)	Zone of inhibition (mm)	Zone of inhibition (mm)	Zone of inhibition (mm)
		17	30	19
	In silico	6 Bonds with LEU-304, LEU-304, TYR-29, ASP-145, GLU-27, ASN-146
Nyandoro et al., 2013 [40]	In vitro C. albicans, Cryptococcus neoformans (yeast)	QCT	Artapetalin B and Artabotrol		Nil	Nil	No inhibition of fungus
		Inactive	Artapetalin B and artabotrol showed inhibition
Cushion et al., 2016 [44]	In vivo Pneumocystis carinii (yeast-like fungus)	QCT	Myo-inositol		Nil	Nil	Failure to inhibit the MIT system of P. carinii. Thus, P. carinii survived.
		No significant inhibition of the myo-inositol transport system of P. carinii	Myo-inositol showed significant competition and inhibited myo-inositol transport (MIT) system by P. carinii.

Abbreviations: QCT, quebrachitol; MIT, myo-inositol transport.

) [38]. QCT, one of the compositions in this methanol extract, was found to form six hydrogen bonds with exo-β-(1,3)-glucanase at Leu304, Tyr29, Asp145, Glu27, and ASN146 residues, all important sites for fungal hyphae proliferation. However, another similar study did not report any inhibition against either C. albicans or C. neoformans [40]. Although both studies used agar diffusion tests to assess antifungal effects, the concentration was not stated. Therefore, exact comparisons were unable to be made.

One research paper shows that the host’s myo-inositol and its selective transportation to P. carinii are important for its survival [44]. In this study, QCT was not found to significantly inhibit myo-inositol uptake. QCT controlled less than 10% of uptake while the competitive unlabeled myo-inositol reduced more than 50% of myo-inositol uptake by P. carinii, hence insignificantly impeding P. carinii’s growth.

3.5. Antiviral Effects of Quebrachitol

Two of the avian pathogens affecting birds, NDV and IBDV, were inhibited by QCT (

Table 7. Findings summary of antiviral effects of quebrachitol (QCT).

Author, Year	Microorganism	Results									Inhibition/Killed	Mechanism	Antimicrobial Effects
Nyandoro, 2017 [43]	In ovo/in vitro	QCT (360 μg/mL)			Solvent control (dimethyl sulfoxide)			Negative control (no virus)			Inhibition	Unclear—virucidal/inhibitory/preventive	Antiviral
	Immune complex infectious bursal disease virus (IBDV)	Eggs (alive/dead)	Embryo weight (g)	Embryo formation	Eggs (alive/dead)	Embryo weight (g)	Embryo formation	Eggs (alive/dead)	Embryo weight (g)	Embryo formation
		Alive	7.449	Complete	Dead	1.67	Incomplete	Alive	7.626	Complete
	Newcastle disease virus (NDV)	Eggs (alive/dead)	Embryo weight (g)	Embryo formation	Eggs (alive/dead)	Embryo weight (g)	Embryo formation	Eggs (alive/dead)	Embryo weight (g)	Embryo formation
		Alive	5.293	Complete	Dead	3.359	Incomplete	Alive	6.708	Complete
		Hemagglutinin test
		Positive (titre 1:259)			Positive (titre 1:1024)			Negative

Abbreviations: QCT, quebrachitol; IBDV, immune complex infectious bursal disease virus; NDV, Newcastle disease virus.

) [43]. The in ovo study showed that QCT’s concentration at 360 μg/mL had significant effects on IBDV and NDV. All five of the embryos were alive with normal size, weight, and organ formation. Additionally, viral load reduced up to eight times in comparison with the untreated embryo, with viral titre reduction from 1:2048 (control) to 1:256 in the hemagglutinin test. QCT also had higher potency against IBDV than NDV. However, the mechanism of action, whether it inactivated, destroyed, or prevented viral infections, was not determined in this study.

3.6. Antiparasitic Effects of Quebrachitol

Three papers discuss the role of QCT as an antiparasitic agent in Leishmaniasis and malaria. Andima et al. revealed the absence of anti-leishmanial effects of QCT derived from Taberneamontana ventricosa Hochst. ex A.DC. (

Table 8. Findings summary of antiparasitic effects of quebrachitol (QCT).

Author, Year	Micro-Organism	Results										Inhibition/Killed	Mechanism	Antimicrobial Effects
Mishra et al., 2023 [46]	In vitro	QCT				Chloroquine						Inhibition	Binding to P. falciparum lactate dehydrogenase enzyme (PfLDH) hydrophobic pocket, inhibiting the lactate dehydrogenase (LDH), and leading to malarial cell death	Antimalarial
	Plasmodium falciparum	IC50 (μg/mL)	CC50 (μg/mL)		SI	IC50 (μg/mL)		CC50 (μg/mL)		SI
		0.87 ± 0.04	137.56 ± 04.45		158.11	0.029 ± 0.002		43.9 ± 02.28		1908.69
	In vivo	QCT				Chloroquine (10 mg/kg/d)			PRT Butanol (500 mg/kg/d)
	Plasmodium berghei	Dosage (mg/kg)	Chemosuppression (%)	Mean survival time (MST) (days)	Haemoglobin (g/dL)	Chemosuppression (%)	MST (days)	Haemoglobin (g/dL)	Chemosuppression (%)	MST (days)	Haemoglobin (g/dL)
		30	73.26	14.36	8.18 ± 0.56	100	>28.00	11.64 ± 0.38	21.35 ± 7.40	8.93	6.14 ± 0.64
		60	61.88	11.86	7.39 ± 0.19
		120	42.33	10.28	7.46 ± 0.61
	In silico	Highest interaction with PfLDH hydrophobic pocket
Andima et al., 2021 [41]	In vitro Leishmania donovani	QCT				Voacristine, vobasine, aloenin, chrysophanol, and miltefosine						Nil	Nil	No inhibition on L. donovani promastigotes. Other agents showed inhibition of arginase, ornithine decarboxylase, and spermidine synthase leading to polyamine synthesis and thereby inhibiting L. donovani survival.
		No inhibition on L. donovani promastigotes				Inhibition by voacristine, vobasine, aloenin, chrysophanol, and miltefosine
Mongan et al., 2004 [42]	In vitro L. donovani	QCT				Myo-inositol						Modest or no inhibition	Nil	No significant inhibition on the MIT system of L. donovani. Other isomers showed that the most important L. donovani inhibitors were isomer substrates with myo-inositol C-2. C-3 and C-5 hydroxyl groups. Thus, these agents can be used to deliver cytotoxic agents to inhibit Leishmania sp.
		No inhibition of myo-inositol transport (MIT) system of L. donovani				Myo-inositol inhibited 96 +/− 2% myo-inositol transport system of L. donovani with specificity on carbon-2, carbon-3 and carbon-5 hydroxyl groups

Abbreviations: QCT, quebrachitol; PfLDH, Plasmodium falciparum Lactate dehydrogenase enzyme; LDH, lactate dehydrogenase; MST, mean survival time; MIT, myo-inositol transport.

) [41]. The two metabolites that revealed inhibitory effects against promastigotes of Leishmania donovani were voacristine (half-maximal inhibitory concentration, IC₅₀ 11 ± 5.2 µM) and aloenin (IC₅₀ 26 ± 6.5 µM) in comparison to miltefosine (IC₅₀ 5.5 ± 0.1 µM). Additionally, the in silico study shows strong interaction between these two metabolites with arginase, an enzyme that mediates polyamine production for leishmania survival. These findings suggest the mechanism for L. donovani inhibitions. Another study tested the effects of QCT on myo-inositol transporter (MIT) activity, a crucial transporter to ascertain Leishmania survival [42]. The researchers investigated the role of variation in the single hydroxyl group position of each inositol isomer which is crucial for recognition by Leishmania MIT and parasite survival. Myo-inositol and 3-fluoro-3-deoxy-myo-inositol produced 96 ± 2% and 91 ± 1% inhibition on Leishmania MIT respectively. This indicates the importance of the hydroxyl group at carbon-2, C-3, and C-5 as substrate recognition for the promastigotes MIT. However, this study shows that QCT did not inhibit or have negligible effects on MIT. Therefore, it has no role as anti-leishmaniasis.

One in vitro study demonstrated QCT’s inhibitory effects on Plasmodium falciparum at IC₅₀ of 0.87± 0.04 μg/mL and 50% cytotoxic concentration, CC₅₀ of 137.56 ± 04.45 μg/mL [46]. The efficacy was significantly smaller compared to chloroquine which had IC₅₀ of 0.023 ± 0.002 μg/mL and cytotoxicity, and CC₅₀ of 43.9 ± 02.28 μg/mL [46]. The concurrent animal study with Plasmodium berghei’s infected mice discovered that QCT produced significant chemosuppressive effects (73.26%) at 30 mg/kg. However, at higher concentrations of 60 and 120 mg/kg of body weight, the chemosuppression was reduced to 61.88% and 42.33% respectively. Furthermore, the mean survival time for QCT at 120 mg/kg and 30 mg/kg was 10.28 and 14.36 days, respectively, which was significantly less than the 28-day survival period for chloroquine. The Search Tool for Interactions of Chemicals (STITCH) analysis showed the highest interaction between QCT and P. falciparum lactate dehydrogenase enzyme (PfLDH). PfLDH was therefore chosen as a focus for the potential molecular docking experiments in this in silico investigation. The index of 158.11 for QCT’s preferential binding to the hydrophobic PfLDH protein was significantly lower than chloroquine (1908.69) at the benzoic acid ring, although it was greater than that of 3,5-dihydroxy-4-methoxybenzyl alcohol (44.98). The suppression of PfLDH urged a further investigation into QCT’s antiplasmodial role in strains resistant to chloroquine.

3.7. Antibiofilm Effects Against Bacteria Effects of QCT

Both Karuppiah et al. [34] and Vijayakumar et al. [33] showed antibiofilm properties against bacteria in their studies (

Table 9. Findings summary of antibiofilm effects of quebrachitol (QCT).

Author, Year		Vijayakumar et al., 2020 [33]	Karuppiah et al., 2020 [34]
	Microorganism	Methicillin-resistant Staphylococcus aureus	Staphylococcus epidermidis
Quebrachitol Results	Qualitative assay	Biofilm inhibition	Biofilm inhibition
	Biofilm inhibitory concentration (μg/mL)	100	75
	Biofilm suppression (%)	86%	84%
	Antibacterial activity	Negative	Negative
	Microscopic evaluation	Reduced biofilm, microcolonies formation	Significantly reduced biofilm, microcolonies formation
	Matrix composition of biofilm	Reduced 79% production of extracellular polysaccharides and 76% hydrophobicity (p < 0.05)	Significantly reduced 76% protein, 72% carbohydrate, 44% lipid
	Formation of biofilm on abiotic surfaces	Reduced biofilm significantly on steel, silicone, titanium	Significant reduction in biofilm on steel, glass, silicone, and titanium
	Bacterial adherence to abiotic surfaces	Significantly reduced 72% adherence to hydrophilic surfaces and 75% to hydrophobic surfaces.	Significant reduction of 55% adherence to hydrophilic surfaces and 78% to hydrophobic surfaces
	Bacterial adherence to biotic surfaces	Significantly reduced 59% adherence to a surface coated with fibrinogen and 62% to collagen Type I coated surfaces.	Reduced 67% to surface coated with fibrinogen and 62% to collagen Type I coated surfaces.
	Disruption of mature biofilm	Significantly disrupt biofilm architecture.	-
	Autolysis assay	Reduced autolysis of bacteria	Significantly reduced autolysis of bacteria, reduced dispersion of extracellular DNA
	First attachment assay	-	Reduced initial binding
	Bacterial accumulation or aggregation assay	Bacterial clumping reduced 85%	Bacterial clumping was delayed and reduced by 82%
	Haemolysis and production of lipase	Reduced 69% haemolysis and 77% lipase (p < 0.05)	-
	Synthesis of staphyloxanthin	Staphyloxanthin synthesis significantly reduced by 79%, disruption of Methicillin-resistant Staphylococcus aureus defence.	-
	Hydrogen peroxide susceptibility assay	Significant reduction in hydrogen-peroxide-resistant cells	-
	Production of hydrolases	-	Significantly reduced 69% lipase, and 78% protease. Reduced bacterial virulence.
	Analysis of expressions of genes	Significant downregulation of sarA, agrA, atlA, geh, hla, cna, clfA, crtM, and fnbA. Also reduced expression of agrC, icaD, fnbB. Increased sigB	Significant downregulation of AgrA, Embp, icaA, icaD, atlE, Aap. Also reduced icaC, FnbpA, FnbpB
Inhibition/killed		No direct antibacterial effects, positive biofilm inhibition	No direct antibacterial effects, positive biofilm inhibition
Mechanism		Reduced biofilm formation, adherence, aggregation, autolysis, virulence, and staphyloxanthin antioxidant effects.	Reduce biofilm adherence, binding, aggregation, and matrix composition.
Antimicrobial effects		Antibiofilm	Antibiofilm

). In the first study, biofilm inhibition was tested on S. epidermidis [34]. The most active compound in ethanol extracts from R. mucronata was QCT (85.26%). QCT did not exhibit any bactericidal effects towards S. epidermidis but there were antibiofilm effects of 84% at biofilm inhibitory concentration (BIC) of 75 μg/mL. The extracellular matrix comprises carbohydrates, protein, and lipids, and they decreased by 72%, 76%, and 44% respectively. Additionally, a significant reduction in bacteria adhering to both abiotic (55–78%) and biotic (62–67%) surfaces was observed. The extracellular DNA (eDNA) production, initial attachment, and 82% of aggregation were reduced. The bacterial virulence decreased significantly, by inhibiting lipase and protease production, by 69% and 78% respectively. The analysis of gene expression showed downregulations of all genes essential for the formation of biofilm and bacterial virulence with QCT treatment. These include agr (biofilm production), polysaccharide intercellular adhesin (bacterial adhesion), icaA, icaD, icaP, Aap (aggregation), Embp (extracellular matrix, ECM binding protein), AtlE (autolysis), FnbpA and FnbpB (fibrinogen binding protein) genes.

In the second study, QCT’s antibiofilm effects and virulence inhibition were investigated on MRSA [33]. The BIC was 100 μg/mL with 86% inhibition. Similar to the previous study, no antibacterial effects were observed against MRSA. At BIC, QCT reduced the architectures, production of EPS (79% reduction), and hydrophobicity (76%) of MRSA biofilm. In addition, it reduced adherence of MRSA to biotic and abiotic surfaces (59–75%), formation of matured biofilm, autolysis, and 85% aggregation. QCT decreased MRSA virulence with hemolysis and lipase reduction of 69% and 77% respectively. Additionally, the production of staphyloxanthin, an antioxidant which acts as a MRSA defensive system, was suppressed by 79%. These included agr, ica, geh, fnb, cna (collagen binding protein), clfA, clfB (clumping factors), sarA (staphylococcal accessory regulator A), and increased expression of sigB. Therefore, these two studies concluded QCT’s potential as a biofilm inhibitor and anti-virulence drug.

3.8. Quality Assessment of Selected Articles

With the QUIN assessment tool, there were four papers with a medium risk of bias while others had lower bias risk. Both investigators agreed that detailed sample size calculation, randomisation, outcome assessor details, and blinding were not applicable in the assessment in addition to the inadequacy of operator details. All articles had sufficient information about the study objectives and methodology. However, there were insufficient explanations about the criteria, such as sampling techniques, comparison group, outcome measurement methods, statistical analysis and result presentation.

Table 10. Quality assessment of in vitro studies and risk of bias based on the QUIN tool.

Study/	1	2	3	4	5	6	7	8	9	10	11	12	Total Score	Final Score (%)	Risk of Bias
Criteria
Andima et al., 2021 [41]	2	×	0	2	2	0	×	2	×	×	2	2	12	75	Low
Lunga et al., 2014 [45]	2	×	2	2	2	0	×	2	×	×	2	2	14	87.5	Low
Kumar, 2016 [38]	2	×	2	2	2	0	×	1	×	×	0	2	11	68.75	Medium
Wannuch et al., 2015 [39]	2	×	1	2	2	0	×	2	×	×	0	2	11	68.75	Medium
Nyandoro et al., 2013 [40]	2	×	2	1	2	0	×	1	×	×	0	1	9	56.25	Medium
Mishra et al., 2023 [46]	2	×	2	2	2	0	×	2	×	×	2	2	14	87.5	Low
Karuppiah et al., 2020 [34]	2	×	2	2	2	0	×	2	×	×	2	2	14	87.5	Low
Vijayakumar et al., 2020 [33]	2	×	2	2	2	0	×	2	×	×	2	2	14	87.5	Low
Mongan et al., 2004 [42]	2	×	2	1	2	0	×	2	×	×	0	1	10	50	Medium
Nyandoro, 2017 [43]	2	×	2	2	2	0	×	2	×	×	0	2	12	75	Low

summarises the QUIN assessment results.

The summaries of the analysis of risk of bias using SYRCLE for three in vivo studies are shown in

Table 11. Risk assessment of in vivo studies with SYRCLE.

	1	2a	2b	2c	3	4a	4b	5	6	7a	7b	8a	8b	8c	8d	9a	9b	10a	10b	10c	10d	10e
Lunga et al., 2014 [45]	N	Y	Y	Y	U	Y	Y	U	U	U	U	U	U	U	U	Y	Y	U	Y	U	U	U
Cushion et al., 2016 [44]	N	U	U	Y	U	U	Y	U	U	U	U	U	U	U	U	Y	Y	U	U	U	U	U
Mishra et al., 2023 [46]	N	Y	U	Y	U	U	Y	U	U	U	U	U	U	U	U	Y	Y	U	Y	U	U	U

Abbreviations: N, No; Y, Yes; U, Unclear.

and Figure 3. The assessments for most of the criteria were hindered by insufficient information leading to the inability to provide rational judgements. These include the concealment of allocation, blinding, outcome assessment randomisation, incomplete outcome data, contamination risk, errors in analysis, design bias, and change in samples or control groups. None of the articles described the allocation sequence, thereby accounting for high-risk selection bias. One of the three studies showed a low risk of bias in selection (baseline characteristics) and performance (random housing). About 66.7% of articles claimed no funding bias. All three articles had low risk in disease induction timing, random housing affecting the outcome, and reporting bias.

4. Discussion

Through a combination of in vitro and in vivo research, Lunga et al. [45] had shown anti-typhoid effects with methylinositol, a compound with ¹³C NMR almost similar to QCT, showing both inhibiting and bactericidal effects against Salmonella species subtypes. These include S. typhi, S. paratyphi A, S. paratyphi B, and S. typhimurium. However, it had less efficacy with higher MIC and MBC compared to ciprofloxacin and gentamicin. The MBC:MIC ratios reflected the potency of antibacterial effects of a compound [10,47,48,49]. In this study, the MBC:MIC ratio showed that methylinositol killed S. typhimurium while exhibiting inhibitory effects against S. typhi, S. paratyphi A, and S. paratyphi B. The in vivo studies in typhoid-infected rats demonstrated that the dosage to achieve MIC was 55.75 mg/kg body weight. By maximising the dosage to 8 × MIC, bacterial resolution was achieved in 4 days with comparable effects as ciprofloxacin and oxytetracycline. Leukocytosis and lymphocytosis could suggest the mechanism of action for its bactericidal effects. Therefore, the potency of QCT as an anti-typhoid was demonstrated in the in vitro study and complemented by bactericidal effects observed in the in vivo study. Due to the global urgency sparked by the 2016 emergence of extensively drug-resistant (XDR) S. typhi resistant to conventional treatments like ampicillin, chloramphenicol, and fluoroquinolones, numerous studies have recently investigated natural products as alternative anti-typhoid agents [50,51]. Consequently, QCT can be further investigated as an alternative to the reference drugs in XDR.

The other studies in this review did not reveal any antibacterial effects on either Gram-positive or -negative bacteria. These include S. aureus, S. mutans, S. epidermidis, and E. coli [33,34,39,40]. All these in vitro studies using various methods, such as agar diffusion or broth microdilution methods, showed consistent negative results [33,34,39,40,41]. The negative results can be attributed primarily to the lack of direct bactericidal or bacteriostatic activity of QCT against tested staphylococcal strains. Additionally, while potential formulation or dosing issues could contribute to negative findings, well-designed and comprehensive testing in these studies strongly suggests that the observed negative results genuinely reflect the compound’s lack of direct antimicrobial effect. This can possibly be due to QCT’s inability to penetrate the cell wall/membrane or breach the bacteria’s defence. To date, no published data is available on the antibacterial activity of QCT against Staphylococcus spp. or Salmonella spp. These genera were highlighted because they represent common Gram-positive and Gram-negative pathogens, respectively, and differences in their cell wall architecture may influence bacterial susceptibility to QCT—an aspect that remains unexplored. Further research is needed to determine whether structural variations between Gram-positive and Gram-negative bacterial cell walls play a role in these potential differences in antibacterial activity.

Antifungal properties of QCT against C. albicans, C. neoformans, and P. carinii were studied in this review. These are the three most common fungal infections in humans. The annual global incidence of candidiasis, cryptococcal meningitis, and pneumocystis pneumonia are 1,565,000, 194,000, and 505,000, respectively, leading to mortality rates of 63.6%, 75.8%, and 42.4% [52]. One of the reasons leading to the persistence and invasiveness of fungi is β-1,3-glucanase. The endo-β-1,3-glucanase reduced recognition of fungus and inflammatory cytokines while the exo-β-1,3-glucanase induced hyphae or fungal filamentation [53,54]. Currently, echinocandins such as caspofungin and micafungin exert their fungistatic effects by inhibiting β-1,3-glucanase and the FKS genes. However, resistance occurred due to mutations in these drug targets and reduced binding affinity [55,56]. The rise in antifungal resistance prompted the search for effective bioactive products as alternative fungicidal agents. In this review, QCT is reported to interact with Glu-27, the most prominent hydrogen bond, creating exo-β-1,3-glucanase inhibition in C. albicans besides the interaction with Leu304 and Tyr29 [38]. Although the methanol extracts from A. indica have shown anti-candidal activities, it was unclear if QCT played an integral role in this, and the author suggests further research on the bioactive component which contributed to these reactions.

However, another research by Nyandoro et al. [40] demonstrates QCT’s inactivity against C. albicans and C. neoformans. The difference in anti-Candida effects of QCT between the two studies can be explained by differences in extraction methods, testing approaches, and whether the compound was tested in isolation or within a complex plant extract. In the research by Nyandoro et al. [40], pure QCT was isolated from A. modestus using sequential solvent extraction with petroleum ether, dichloromethane, chloroform, and ethanol. The compound was then tested directly in an in vitro antifungal assay against C. albicans and C. neoformans. The results showed that QCT had no anti-Candida activity, indicating that the pure compound was ineffective against the tested fungi.

While in the study by Kumar [38], QCT was not tested in isolation but as a mixed compound in a methanol extract of A. indica. Instead of direct antifungal testing on the isolated compound, this study used in silico docking analysis to predict how QCT interacts with exo-β-1,3-glucanase, an important fungal enzyme. Following the computational study, the entire methanol extract was tested against C. albicans in vitro, and antifungal activity was observed. The observed anti-Candida activity may be synergistic with other extract components, while the in silico data suggests that antifungal potential is not entirely absent.

P. carinii survival was found to be dependent on the host’s myo-inositol, transported by ITR1 and ITR2 and transferred to the parasite. This transport system is extremely selective and may be used as a therapeutic target [44]. In this study, the capability of several isomers, including QCT, was investigated to compete with myo-inositol and inhibit P. carinii’s uptake. However, the results only showed significant competitive uptake by unlabeled myo-inositol. QCT did not show any significant inhibitory effects on this process, and this possibly signified its inability to block P. carinii survival. The experimental molecular study combining protein structural modelling and biochemical transport assays concludes absence of antimicrobial effects from QCT due to high selectivity of the myo-inositol transporter for its survival.

This review revealed QCT’s antiviral potential for IBDV and NDV in animals. IBDV, a highly infectious RNA coronavirus, primarily affects chickens aged 3 to 6 weeks old by impairing their immune systems [57,58]. Upon ingestion or inhalation, the virus attacks macrophages, dendritic cells, and B-lymphocytes, thereby crippling the immune system [59,60]. NDV is another virulent RNA-based pathogen from the paramyxoviridae family. NDV predominantly infects birds and rarely humans. Infected humans may experience mild flu-like symptoms or conjunctivitis. NDV induces cellular lysis and apoptosis, leading to gastrointestinal, respiratory, and neurological symptoms [61]. Both IBDV and NDV are the major causes of infectious diseases affecting the poultry industry. Phytochemicals have recently been investigated as safe and healthy alternatives to reduce these diseases. Some of the recognised modes of action by phytochemicals include immunomodulation, anti-inflammatory, and antioxidant with inhibitory or virucidal effects [62,63,64]. According to Nyandoro et al. [40], QCT maintained the viability of every embryo with normal organogenesis while significantly decreasing the viral load. Additionally, the efficacy was greater towards IBDV than NDV. However, the indeterminate mechanism of action prompted the author’s recommendation for additional future research.

The results reveal that QCT possesses antimalarial properties. According to the World Malaria Report 2023 [65], there were 249 million cases of malaria from 85 countries, mainly from African countries (94%). The malarial fatalities were estimated to be about 608,000 in 2022. P. falciparum and P. vivax were the most common species to cause malaria with most mortalities from P. falciparum. Some of the contributing factors to the increase in malaria incidence are vector and antimalarial resistance in addition to unequal distribution of main malaria therapies to certain regions. Antimalarial resistance against P. falciparum was found mostly in chloroquine, sulfadoxine-pyrimethamine (SP), and artemisinin-based combination therapies (ACTs). One of the targeted sites by malarial drugs is PfLDH, leading to LDH suppression and parasite elimination. However, a mutation in P. falciparum’s kelch protein, pfk13 was identified, resulting in the delay of pathogen clearance [66]. Recently, ‘false quina’/fake quine from Homalolepis suffruticosa (Engl.) Devecchi and Pirani., Goniothalamus lanceolatus Miq. (Annonaceae), and Vitex negundo L. were investigated for their ability to overcome resistance in conventional malarial drugs [67,68,69]. Mishra et al. [46] were the first to report QCT extracted from P. roxburghii with antimalarial effects against P. falciparum and P. berghei. QCT showed comparable inhibitory and cytotoxic activities to chloroquine. At 30 mg/kg of QCT, 73.26% of chemosuppresion and a mean survival time of 14.36 days with inverse therapeutic reactions was documented with doses of more than 60 mg/kg. Therefore, the therapeutic range for optimal antiplasmodial effects was estimated to be around 30 to 60 mg/kg. According to in silico molecular docking analysis, chloroquine and QCT interact selectively with PfLDH in P. falciparum and P. berghei. Chloroquine is bound to the benzoic acid ring while QCT is attached at the adjacent hydrophobic fissure. Figure 4 shows the inhibition of PfLDH, leading to glycolysis interference and ultimately affecting parasite survival. As the docking site for chloroquine and QCT differs, the potential use of QCT to treat strains that are resistant to ART or chloroquine may be investigated further.

Despite QCT’s strong in vitro activity against Plasmodium sp., its reduced effectiveness in vivo at higher doses may be due to pharmacokinetic challenges, including low absorption or rapid metabolism, which prevent sufficient amounts from reaching the bloodstream in order to effectively inhibit PfLDH. Additionally, immune responses or drug interaction may affect its activity and further reduce its efficacy. However, in silico studies have enhanced the understanding of QCT’s potential by identifying its molecular interactions with PfLDH, proposing the mechanism of action. Computational modelling has also provided insights into QCT’s binding strength and stability, helping to predict how structural modifications could improve its efficacy. Furthermore, in silico simulations can identify possible QCT’s modifications to enhance its absorption and metabolic stability, ultimately aiding in the design of future in vivo studies with improved effectiveness.

Research on the anti-leishmanial properties of QCT was included in this review. Leishmaniasis, a disease caused by protozoan Leishmania sp., is classified by the World Health Organisation as a neglected tropical disease (NTD) with an incidence of 700,000 to 1,200,000 cases per year [70,71]. The mode of transmission is via bites from infected sandflies. The disease is classified into cutaneous, mucocutaneous, and visceral, whereby the former two were milder forms and the latter caused 95% of fatalities due to anaemia, hepatosplenomegaly, and sepsis [71]. Leishmaniasis is treated with pentavalent antimonials such as sodium stibogluconate. Some of the challenges in treating leishmaniasis include increasing drug resistance, significant side effects (cardiotoxicity, asthenia), and inconvenient mode of administration (injection). Therefore, an alternative, or phytochemicals which are naturally safe and effective treatments for leishmaniasis are sought after. However, the two study publications in this review complemented each other to demonstrate QCT’s inefficiency in inhibiting or suppressing the Leishmania parasite despite using different methodologies—one focuses on functional cytotoxicity tests [41], while the other examines mechanistic transport studies [42]. Andima et al. [41] investigated the anti-leishmanial and cytotoxic activity of various plant-derived metabolites, including QCT. They employed cell viability assays to test whether these compounds can inhibit Leishmania promastigote growth. The findings revealed that QCT does not exhibit significant anti-leishmanial effects. In contrast, voacristine from T. ventricosa and aloenine from Aloe schweinfurthii Baker had demonstrated anti-leishmanial effects. Mongan et al. [42] focused on substrate specificity of the L. donovani MIT. They used inositol uptake assays to determine whether QCT can be transported into the parasite. The results showed that QCT was not efficiently transported, possibly due to the presence of a methyl group at the C-2 position, which disrupts its ability to bind to the transporter. Although QCT did not inhibit the myo-inositol/H+ transporter MIT of L. donovani, inositol 3-fluoro-myo-inositol had shown MIT inhibition leading to high suppression in the promastigote growth; thus concluding that MIT in Leishmania species is a potential drug target [42].

Research on the antibiofilm properties of QCT had produced positive outcomes. A biofilm is a population of microorganisms encased in an extracellular matrix that they had manufactured for self-protection and to flourish in various environments [72]. Biofilm formation happens in a few stages. Firstly, the single planktonic microbial cells adhere to surfaces, which is a process mediated by microbial surface components recognising adhesive matrix molecules (MSCRAMMs) [73]. Subsequently, microorganisms aggregate, covered by the host’s protein. During maturation, multiplication, and proliferation, these clumped cells are enveloped by a self-created ECM. The ECM comprises EPS, peptides, and bacterial DNA (eDNA), forming irreversible quiescent and virulent microcolony scaffolds. One of the most important components in EPS is polysaccharide intercellular adhesion (PIA), regulated by ica, Bap, and Aap. Extracellular protein comprises MSCRAMMs including fibronectin-binding proteins (Fnbp) and fibrinogen-binding proteins (Fib). The eDNA assists in microorganism adherence, transfer, and repair of genes. The biofilm matures following adequate nutrition in a conducive environment. Finally, these microcolonies release single planktonic cells to be dispersed to other surfaces, resulting in infection spread [74]. QS, communication between cells or microcolonies, allows microorganisms to sense their surroundings and control their density and virulence by altering gene expression. The gene or regulating factors differ among microorganisms. For instance, in S. aureus, it is an accessory regulatory factor (agr), while las and rhl genes control P. aeruginosa. This proliferative, protective, and interactive formation has led to biofilm’s persistence and resistance to antimicrobial agents.

Some of the most common microorganisms to form biofilms are S. aureus, or the more severe form, MRSA, S. epidermidis, and P. aeruginosa [75,76,77]. The formation of biofilm has challenged bacterial eradication due to its multiple-drug resistance with severe health complications such as pneumonia, surgical site infections, and septicemia. Biofilm can increase bacterial resistance by 1500 times [78]. The adherence ability to both biotic and abiotic surfaces, such as bone/prosthetic implants and cardiac valves, complicate post-operative recovery [79,80]. Furthermore, natural microflora dysbiosis may occur secondary to the extensive use of strong antibiotics [81]. Some successful antibiofilm phytochemicals which prevailed over conventional drug issues were quercetin, emodin, and piperin, which disrupted different sites and processes of biofilm formation [82,83,84]. Quercetin inhibited biofilm formation in MRSA and S. epidermidis by downregulating ica, which is involved in PIA production and thereby, decreased intercellular adhesion [85,86]. Similarly, emodin, an anthraquinone derivative from Polygonum cuspidatum, inhibited S. aureus biofilm formation by reducing the release of eDNA and downregulating the cidA gene. This disruption weakens the biofilm structure and limits biofilm proliferation [82]. A molecular dynamics study revealed the interaction between piperine and agrA, a QS regulator in MRSA, indicating its potential disruption of QS. Additionally, piperine increased intracellular ROS and reduced MRSA virulence [84].

In their review, Karuppiah et al. [34] reported on the antibiofilm effects of QCT in S. epidermidis. In this study, 84% of biofilm suppression was achieved at BIC of 75 μg/mL. There was also microscopic evidence of significant biofilm reduction and destruction at abiotic and biotic surfaces post-QCT treatment. The inhibitions were observed at multiple levels from adhesion, surface attachment, and bacterial aggregation to ECM reduction. The virulence of S. epidermidis also decreased with reduced autolysis, protease, and lipase. Downregulation of genes favouring bioluminescence production enhanced the positive outcome of QCT as an antibiofilm. The reduction in agr and atlE genes interrupted the QS system and autolysis of S. epidermidis. In addition, bacterial attachment and adhesion were affected by decreased MSCRAMM genes such as Embp, Fnbp, Aap, and PIA genes like icaA, icaD, and icaP. Therefore, it was concluded that QCT inhibited biofilm production from the initial phases of its formation.

Vijayakumar et al. [33] demonstrated QCT’s biofilm inhibition on MRSA. The BIC was higher at 100 μg/mL with 86% inhibition. Consistent with the previous study, QCT decreased bacterial adherence, aggregation, autolysis, haemolysis, and lipase significantly. Furthermore, QCT was proven to reduce the antioxidant staphyloxanthin in MRSA, weakening the bacteria’s defence. All binding proteins, PIA protein, aggregating factors, and QS system gene expression were reduced in this study.

Both studies consistently revealed that QCT disrupted biofilm formation at multiple stages. Figure 5 demonstrates bacterial biofilm inhibition of QCT, targeting the initial phases of adhesion, attachment, aggregation, and the maturation phase with interference on the quorum sensing. Therefore, QCT is effective as an antibiofilm and antivirulent against Staphylococcus sp.

Quality Assessment Tool for In Vitro Studies, QUIN, indicated that the majority of studies (60%) had low bias risk. The objectives and methodologies were the only two components which were adequately provided while the others were either insufficient or inapplicable. The three in vivo studies evaluated with SYRCLE had shown mostly unclear biases with allocation concealment (selection bias), performance, detection, and attrition, and high selection bias with generation of sequence allocation. Therefore, these tools may be used as guidance at the initial planning phase to reduce the risk of biases and improve quality as well as reliability of research.

The limited number of publications, along with its inconsistent quality, constitutes a major limitation of this research. Some studies exhibit methodological weaknesses that compromise the reliability and reproducibility of their findings. The lack of well-designed, high-quality research presents a challenge in establishing conclusive scientific evidence on the potential of QCT’s clinical application.

The exclusion of unpublished studies such as conference proceedings and dissertations was part of publication bias which had contributed to about 12.7% of excluded documents in our study. The other possibilities of publication bias were unpublished research due to negative or insignificant results. However, previous research had shown that the exemption of these articles had little impact on the effects of the systematic review [87].

“English-language bias”, a bias that occurred due to the exclusion of all articles which were not written in English. Due to limited resources such as credible medical translators and time, we have excluded 13.14% (n = 112) of foreign language articles. Therefore, there is a possibility that good quality articles with crucial results may have been missed. Some studies had previously shown no significant effects while others revealed similar quality designs between English and non-English publications [88,89]. Nevertheless, ideally, the inclusion of articles in all languages will lessen linguistic bias and allow generalisation. Furthermore, different methodologies, data heterogeneity, and insufficient information added to the challenges in this review.

As for the implications for future research, the emergence of multi-drug or antibiotic resistance has led to increasing research for natural-based drug development in recent years. QCT is a waste or by-product in the rubber industry. This is the first systematic review that demonstrates the antimicrobial properties of QCT. The comprehensive compilation of studies in addition to meticulous methodology, analysis, and appraisal had produced transparent and specific evidence of QCT’s anti-infective effects which can be utilised as a cost-effective development of alternative antimicrobial agents.

Additionally, some studies have shown the mechanism of action through molecular or cellular studies, such as the in silico study which studied the molecular docking sites of QCT, leading to microbial inhibition. There were also minor discoveries of other natural products with antimicrobial properties and possible pharmacological target sites. This information in combination with other molecular, high throughput, or innovative computational technologies can be used as preliminary investigation for the initial phase of future antimicrobial agent/drug development in addition to new target sites for drug actions to combat antimicrobial resistance.

5. Conclusions

This systematic review highlights the antimicrobial potential of QCT against diseases such as typhoid, candidiasis, avian flu, malaria, and biofilm-associated infections; thus, emphasising its significance for future therapeutic research. QCT exhibited inhibitory activity against Gram-negative bacteria (Salmonella sp.), Gram-positive bacteria (S. epidermidis and MRSA, particularly in biofilm inhibition), fungi/yeast (C. albicans), and parasites (Plasmodium sp.). In addition, antiviral effects were reported against infectious bursal disease virus and Newcastle disease virus. Interestingly, some studies demonstrate the various mechanisms of action that may determine the drug targets in future research. Evidence of antimicrobial activity was predominantly obtained from plant-extracted QCT (eight studies), while only a limited number of studies (three) investigated commercially available QCT. As antimicrobial resistance and side effects of standard antimicrobial agents continue to be more concerning and challenging, the exploration of bioactive compounds like QCT is crucial. These phytochemicals may be safe and effective alternatives or adjuncts to conventional therapies, ensuring successful approaches in infection management. However, there are also some concerns about the quality and bias which may affect the conclusion, especially for the in vivo studies. More robust in vivo research is needed to establish the effectiveness and safety of QCT. Nevertheless, this study demonstrates the potential of QCT, a phytochemical or natural bioactive product with antimicrobial properties. Consequently, future research on bioactive products and drug development is essential to address the limitations and disadvantages of current medications, ultimately leading to improved healthcare outcomes.