Anti-Bacterial Activity of an Ancient Remedy Against Pathogenic Skin Infections

Abstract

Background/Objectives

Skin and soft-tissue infections are among the most common infections and can develop severe local and systemic complications. Bacterial species: Staphylococcus aureus, Streptococcus pyogenes, Pseudomonas aeruginosa and Escherichia coli usually cause skin infections. Studies of plants and natural materials used in ancient medicine to treat infections may assist us in further antibiotic discoveries. In this study, we tested the bactericidal activity of a 1000-years-old Arabic Al-Razi remedy against common skin infections Gram-positive and Gram-negative bacterial strains.

Methods

The antimicrobial activity was tested by agar diffusion method, after adding specific test sample, zone of inhibition was measured. Minimum inhibitory concentrations (MIC) of Al-Razi full remedy, dropout batches and single ingredients were performed by broth micro dilution assay.

Results

The mixture of all ingredients of this remedy (vinegar, gum arabic, boswellia, vitriol and aloe) has shown highest antibacterial activity against S. aureus, S. pyogenes, multi drug resistance (MDR) P. aeruginosa and clinical strain of P. aeruginosa. However, the presence of a single ingredient in water has shown no activity against tested bacterial strains except vitriol.

Conclusion

The AR remedy comprising several ingredients (vinegar, gum arabic, boswellia, vitriol and aloe) had shown great potential to inhibit skin infection bacterial strains (S. aureus, S. pyogenes, MDR P. aeruginosa and the clinical strain of P. aeruginosa). In contrast, single ingredients (except vitriol) had shown no activity against all tested bacterial strains. These results suggest that combination of natural products has more potential against bacterial infections.

Introduction

Skin and skin structure infections (SSSI) and skin and soft tissue infections impose a significant clinical and economic burden on the health care system. ¹ Previous studies revealed that, among nonfatal diseases health burden, skin and subcutaneous infections were fourth leading worldwide. ¹ Furthermore, it is anticipated that approximately 700 thousand people die each year because of anti-microbial resistance infections, and this figure will rise to 10 million by the year 2050. ² Erysipelas, cellulitis, impetigo, folliculitis and major cutaneous abscesses (furuncles and carbuncles) are the most common types of bacterial skin infection. ³ Gram-positive bacteria (S. aureus, S. pyogenes, Beta hemolytic streptococi and enterococci) and Gram-negative bacteria (P. aeruginosa and E. coli) are predominant species of bacterial skin infections.^4-6 Though S. aureus and beta-hemolytic streptococci were the leading cause of SSSI, ⁵ now Gram-negative organisms and mixed pathogens (both Gram-positive and Gram-negative bacteria) significantly caused acute skin infections.^5,7-9 Antibiotic resistance to infective agents has risen globally, predominantly among isolates of S. aureus, which shows resistance against methicillin (methicillin-resistant S. aureus MRSA) and other antibacterial drugs.^10,11 Increased resistance causes higher morbidity and costs.^12,13 As the studies have shown fast spread of resistant bacteria, the requirement to discover new antimicrobial drugs is of utmost importance. However, previous reports of rapidly rising resistance to newly synthesized antibiotics show that even new families of antimicrobial agents will have a short life expectancy.

According to the World Health Organization (WHO), antibiotic resistance is highly increasing worldwide. The efficiency of many antibiotics decreased may be due to bacterial multidrug-resistant genes, transposons, plasmids, chromosomes and their expressions. ⁵ WHO has provided a list of antibiotic-resistant bacteria to guide researchers in discovering and developing new antibacterial agents against multidrug-resistant bacteria. Gram-negative MDR bacterial species, especially P. aeruginosa and Enterobacteriaceae are considered the most serious group. ¹⁴ In 2021, the World Health Organization considered antibiotic resistance as one of the top ten public health threats in the global. ¹⁵ Moreover, the Infectious Disease Society of America has also highlighted a small group of antibiotic resistance bacteria (S. aureus, K. pneumonia, E. faecium, Acinetobacter baumanni, P. aeruginosa, and Enterobacter) that show resistance to many antibiotic drug classes and highly considered for drug discovery. ¹⁶ Treating bacterial infections has become very challenging these days due to which the development of new antibiotics has become a critical issue.

For thousands of years, natural products have been used in traditional medicine worldwide, and many antibiotics and other drugs have been introduced. ¹⁷ In developing countries, it is a readily available method to treat infections and other diseases; in some regions, around 80% of the population uses traditional medicine to fulfill their primary healthcare needs. ¹⁸ Plants used in traditional medicinal practices against infections have been found to inhibit the growth and virulence of various microbes.^19-22 For this reason, researchers are increasingly focusing on herbal products, looking for new leads to develop better drugs against MDR microbial strains. ²³ A few famous unani medicine (Perso-Arabic traditional medicine) books like The Canon of Medicine by Ibn Sina, Al Hawi by Al Razi, Firdous al-Hikmah by Al Tabari and Medicine of the Prophet by Ibn Qayyim Al Jauziyah describe Impetigo as a dermatological disease. A remedy proposed by Al-Razi (mixture of the same amount from boswellia, aloe, and vitriol and double the quantity of gum arabic in the vinegar) said to be effective even in severe cases of skin infections was mentioned in these books. The treatment seems promising, given the properties of its ingredients.

Previous studies revealed the antimicrobial activity of apple vinegar against different bacterial strains such as S. aureus, Staphylococcus epidermidis, S. pyogenes, Streptococcus pneumonia, E. faecalis, P. aeruginosa, Pseudomonas fluorescens, E. coli, Salmonella typhi, Klebsiella pneumonia, Proteus Vulgaris, Proteus mirabilis, Enterobacter aerogenes and Acinetobacter.^24,25 The gum arabic (AG) plant or Acacia gum was also reported for antibacterial, anti-inflammatory, antihypertensive, vasoconstrictor actions, antispasmodic inhibitory effect against hepatitis virus, cytotoxic and antioxidant activity.^26,27 In traditional Arabic and Asian medicine, Boswellia sacra oleoresin (frankincense) has been used as an antibacterial agent for at least 1000 years. ²⁸ The aim of the present study was to evaluate the antibacterial potential of an ancient Al Razi (AR) Remedy; vinegar(Vn), gum arabic, boswellia (Bw), vitriol (Vt) and aloe (Al) against bacteria causing dermatological infections.

Results

Broad-spectrum Antibacterial Activities of Al-Razi Remedy Against Common Skin Infections bacteria

For the antibacterial activity assessment of the Al-Razi (AR) remedy, the complete mixture of all ingredients was initially tested using the agar diffusion method. Figure 1 presents the agar diffusion antibacterial activity of the AR remedy against five skin infection bacterial strains. The AR remedy exhibited significant inhibition zones against all tested bacterial strains (Pseudomonas aeruginosa, Staphylococcus aureus and Streptococcus pyogenes) at concentrations of 2 mg/mL and 3 mg/mL. In contrast, the 1 mg/mL concentration showed minimal to no inhibition zones.

Figure 1.

Zones of inhibition of Al-Razi remedy against five skin infection bacteria.

Minimum Inhibitory Concentrations (MICs) of four different batches of Al-Razi ingredients were determined using the MIC microdilution assay against five bacterial strains associated with dermatological infections: Three Gram-positive strains (S. aureus ATCC 25293 and ATCC 29213, S. pyogenes ATCC 19615) and two Gram-negative strains (P. aeruginosa ATCC 27853, P. aeruginosa NR_117678.1). The lowest concentration that exhibited an inhibition zone in the agar diffusion method was selected as the highest concentration for the MIC assay. As shown in Table 1, the AR remedy (total mixture of ingredients) demonstrated the highest antibacterial activity against the Gram-negative strain MDR P. aeruginosa ATCC 27853 and the clinical isolate P. aeruginosa NR_117678.1, with MIC values of 128 µg/mL and 64 µg/mL, respectively.

Table 1.

MIC and MBC of Full Remedy in µg/ml.

Bacterial Strains	MIC	MBC
P. aeruginosa (A)	128	256
P. aeruginosa (B)	64	256
S. aureus (C)	256	512
S. aureus (D)	256	512
S. pyogenes (E)	256	512

To determine whether the antibacterial activity of the AR remedy is due to individual ingredients or their combination, we tested each ingredient separately and prepared dropout variants by systematically omitting one ingredient at a time while maintaining equal concentrations to those in the full remedy. AR Full Remedy (Total), Dropout Batch - without Arabic vitriol (total-Vt), Dropout Batch - without Aloe (Total-Al), Dropout Batch - without Arabic Gum (Total-AG), Dropout Batch - without Boswellia (Total-BW), dropout Batch - without Vinegar (Total-Vn). The MICs of these preparations, along with those of the full remedy were assessed using the standard broth microdilution assay against the five skin infection bacterial isolates. As illustrated in Figure 2, Table 2(A), the sensitivity of bacterial isolates varied among the dropout batches. Combination of all ingredients had maximum antibacterial activity at 64 µg/ml against P. aeruginosa (B) while, drop out batches showed highest activity at 128 µg/ml. Notably, preparations omitting vinegar resulted in significantly reduced antibacterial activity across all strains, compared to the omission of other ingredients (Figure 2, Table 2). All concentrations which were showing antibacterial activity were statistically significant (P<0.001).

Figure 2.

Minimum inhibitory concentration (MIC) values of total mixture for all tested Bacteria. (A) Pseudomonas aeruginosa ATCC 27853 (B) Pseudomonas aeruginosa NR_117678.1 (C) Staphylococcus aureus ATCC 25293 (D) Staphylococcus aureus ATCC 29213 and (E) Streptococcus pyogenes ATCC 19615. Results are expressed as the means of SD of triplicate experiments.

Table 2.

(A) Final Minimum Inhibitory Concentration of Full Remedy and Drop out Batches in µg/ml. **P < .001 Compared with Untreated Group. (B) All Ingredients Separately Dissolved in Water or Vinegar.

(A)
	P.aeruginosa (A)	P.aeruginosa (B)	S. aureus (C)	S.aureus (D)	S. pyogenes (E)
Total and drop out batches
Total	128**	64**	256**	256**	256**
Total-Vt	256**	128**	256**	256**	256**
Total-Al	256**	128**	256**	256**	256**
Total-AG	256**	128**	256**	256**	256**
Total-Bw	256**	128**	256**	256**	256**
Total-Vn	512**	512**	512**	512**	512**

(B)
	P.aeruginosa (A)	P.aeruginosa (B)	S. aureus (C)	S.aureus (D)	S. pyogenes (E)
Dissolved in water
Vitriol	2048	2048	2048	2048	2048
vinegar	1024	512	1024	1024	1024
Dissolved in vinegar
Vt + vinegar	512	256	512	2048	2048
Al + vinegar	512	512	1024	2048	2048
AG + vinegar	512	1024	1024	2048	2048
BW­­ + vinegar	1024	1024	1024	2048	1024

Figure 3 demonstrates that all individual ingredients dissolved in vinegar exhibited antibacterial activity against all tested bacterial isolates at MIC values of 512 µg/mL and 256 µg/mL for P. aeruginosa strains A and B, respectively and 1024 µg/mL for Gram-positive strains. These results are summarized in Table 2, indicating that vinegar is the primary contributor to the remedy's antibacterial activity against Gram-positive bacterial skin infections. Fractional inhibitory concentration (FIC) index has been calculated to find out synergistic, additive and indifferent action of vinegar. Table 3 represent vinegar work synergistically with AG and BW, while BW also have additive effect with vinegar and aloe showed indifferent activity with vinegar.

Figure 3.

Antibacterial activity of single ingredients dissolved in vinegar (A) Pseudomonas aeruginosa ATCC 27853 (B) Pseudomonas aeruginosa NR_117678.1 (C) Staphylococcus aureus ATCC 25293 (D) Staphylococcus aureus ATCC 29213 and (E) Streptococcus pyogenes ATCC 19615. Results are expressed as the mean of SD of triplicate experiments.

Table 3.

FIC Index Calculation for Synergistic Effects.

Combination	Bacterial Strain	FIC Index	Interpretation
Vinegar + Arabic Gum	P. aeruginosa (A)	0.5	Synergistic
Vinegar + Boswellia	P. aeruginosa (B)	0.7	Additive
Arabic Gum + Aloe	S. aureus (C)	1.5	Indifferent
Vinegar + Boswellia + Gum	S. pyogenes (E)	0.3	Strong Synergistic

Consistent with these findings, when each individual ingredient of the full remedy was dissolved in water separately, none exhibited significant antibacterial activity against the common skin infection bacterial strains, except vitriol. Additionally, omitting any single ingredient led to a reduction in antibacterial activity, as shown in Figure 4 and Table 2.

Figure 4.

Minimum inhibitory concentration of all ingredients of remedy separately dissolved in water (A) Pseudomonas aeruginosa ATCC 27853 (B) Pseudomonas aeruginosa NR_117678.1 (C) Staphylococcus aureus ATCC 25293 (D) Staphylococcus aureus ATCC 29213 and (E) Streptococcus pyogenes ATCC 19615. Results are expressed as the mean of SD of triplicate experiments.

Overall, the results reveal that the antibacterial activity of vinegar alone is not equivalent to that of the full AR remedy (Figure 2 and Figure 3). Moreover, dropout batches confirm that when vinegar is omitted, the other ingredients of remedy also had significant antibacterial activity against all five skin infection bacterial strains (Figure 2). These findings suggest that the synergistic combination of all ingredients is essential for the full antibacterial efficacy of the AR remedy against skin infection pathogens.

Discussion

The rapid emergence and dissemination of antibiotic-resistant bacteria underscore the critical need for novel antimicrobial agents. Previous studies have documented the swift rise of resistance even to newly synthesized antibiotics, highlighting the limited lifespan of new antimicrobial classes. ²⁹ The present study explores the antibacterial efficacy of an ancient remedy traditionally used to treat impetigo. We evaluated both the full Al-Razi remedy and its individual ingredients against multidrug-resistant (MDR) Gram-positive and Gram-negative bacterial strains responsible for skin infections. Our results demonstrated that the complete remedy exhibited the highest inhibitory activity across all tested bacterial strains. Notably, the Gram-negative MDR P. aeruginosa clinical isolate were particularly susceptible to lower concentrations of the full remedy and dropout batches.

We have tested the full remedy and each ingredient of the remedy against Gram-positive and gram-negative MDR bacterial strains that cause skin infections. Results of the antibacterial activity assays indicated that full remedy showed the highest inhibitory activity against all tested bacteria. Among the tested skin infection bacterial strains, Gram-negative MDR P. aeruginosa and clinical strain of P. aeruginosa are more susceptible at the lower dose of the full remedy and also in drop-out batches. To determine whether the antibacterial effect was attributable to the combination of ingredients or a single component, we tested each ingredient separately. Vinegar alone exhibited the most potent antibacterial activity against the skin infection bacterial strains. This suggests that the medicinal properties of apple vinegar are primarily due to its organic acids and bioactive compounds. Organic acids in vinegar are known to penetrate bacterial membranes, promoting the synthesis of antimicrobial peptides, disrupting internal osmotic balance, stimulating energy consumption, and interfering with macromolecular synthesis. ³⁰ Additionally, apple vinegar contains flavonoids, which possess inherent antimicrobial potential.^31-33

In the present study, vinegar was combined with other ingredients in the remedy, we observed enhanced antibacterial activity compared to vinegar alone. This synergistic effect, as indicated by the FIC index, suggests that the chemical constituents of vinegar work in concert with other ingredients to amplify antibacterial efficacy. Furthermore, remedy preparations lacking vinegar still demonstrated significant antibacterial activity, indicating that while vinegar is a key component, other ingredients also contribute to the overall antimicrobial effect. Consistent with our findings, previous research has reported the antibacterial properties of gum arabic.^34,35 However, in our study, commercially available gum arabic did not exhibit antibacterial activity when tested alone against the selected skin infection bacterial strains. Similarly, Boswellia serrata has been documented to possess antibacterial and anti-inflammatory properties, ³⁶ our extract showed no activity when tested individually in water however after removal of boswellia from full remedy the antibacterial activity of mixture has reduced. Similarly, antibacterial activity has reduced after removal of gum arabic, aloe and vitriol. Our study also aligns with existing literature that emphasizes the synergistic effects of natural products. For instance, acetic acid and certain vinegar have been shown to enhance antibiofilm activity when combined with manuka honey ³⁷ synergistically. Similarly, our results indicate that the antibacterial activity of the Al-Razi remedy is significantly influenced by the presence of multiple ingredients working together.

Limitations of this study include the inability to determine the precise chemical composition of the individual ingredients and their specific mechanisms of action. Additionally, we did not assess the potential application of the remedy in clinical settings, such as incorporation into dressings or ointments for skin infections. Future research should aim to characterize the chemical constituents of the Al-Razi remedy using advanced analytical techniques and elucidate the molecular mechanisms underlying its antibacterial activity. Moreover, exploring the clinical efficacy of the remedy in real-world settings, including its formulation into topical treatments, could provide valuable insights into its therapeutic potential.

This study provides evidence that the Al-Razi remedy exhibits broad-spectrum antibacterial activity, particularly against MDR P. aeruginosa. The synergistic interactions between vinegar and other ingredients play a crucial role in enhancing its antimicrobial efficacy. These findings contribute to the ongoing search for effective natural antimicrobial agents to combat resistant bacterial infections.

Conclusion

Here, we tested the bacteriostatic and bactericidal activity of an old Arabic Al-Razi remedy against common skin infections bacterial strains. The present study revealed that the AR (total mixture of ingredients) remedy had shown the highest potent activity against tested bacterial strains of skin infections relative to drop out batches and single ingredients. These findings have, to an extent, validated the preference for using the AR remedy to the single ingredient. However, these findings are inconclusive because they were based on an in vitro study. As such, it is desirable to conduct further investigations of the efficacy and safety of the AR remedy as a treatment for dermatological infections through preclinical and clinical trials.

Materials and Methods

Preparation of Al-Razi Remedy

Apple vinegar, gum arabic, boswellia, aloe and Vitriol were sourced from reliable suppliers in Riyadh, Saudi Arabia, and the university's botanical team verified their botanical authenticity. The powders of all ingredients were finely ground using an electric grinder to ensure uniform particle size. Stock solutions were prepared by dissolving vitriol, aloe, and boswellia in water and vinegar to achieve a concentration of 20 mg/mL each. As specified in the remedy formulation, the concentration of gum arabic was doubled to 40 mg/mL compared to the other ingredients. All stock solutions were filtered through a 0.22 µm syringe filter (CHROMAFIL Xtra) to ensure sterility and remove particulate matter and then stored in sterilized glass bottles in the dark at 4 °C for 2 weeks to maintain stability. The final mixture was prepared by combining all ingredients in equal volumes, ensuring that each ingredient's concentration matched its designated level in the full remedy. Dropout batches were prepared by systematically excluding one ingredient at a time from the remedy and replacing it with an equal volume of sterile water, thereby maintaining the overall concentration of the remaining components.

Antibacterial Activity

Agar Diffusion Method

The antimicrobial activity of the Al-Razi remedy was evaluated using the agar diffusion method as outlined by Balouiri et al. ³⁸ Five pathogenic bacterial strains responsible for skin infections (as listed in Table 4) were cultured on Mueller-Hinton agar (MHA) plates. A bacterial inoculum standardized to 0.5 McFarland turbidity was uniformly spread across the agar surface using a sterile swab. Sterile cork borers (7 mm diameter) were used to create wells in the agar. Different concentrations of the Al-Razi remedy (1, 2, and 4 mg/mL) were added to their respective wells and incubated at 37 °C for 24 h. Penicillin (PANPHARMA) was used as a positive control for Pseudomonas aeruginosa, and vancomycin (Hikma Farmaceutica) was used for Gram-positive bacteria. After incubation, the zones of inhibition around each well were measured to assess antibacterial efficacy.

Table 4.

Bacterial Strains Used in the Study.

Species	Strains
Gram-positive
Staphylococcus aureus (C)	ATCC 25293
Staphylococcus aureus (D)	ATCC 29213
Streptococcus pyogenes (E)	ATCC 19615
Gram-negative
Pseudomonas aeruginosa (A)	ATCC 27853
Pseudomonas aeruginosa (B)	Clinical strain NR_117678.1

Minimum Inhibitory Concentration

MIC assays for the Al-Razi full remedy, dropout batches, and individual ingredients were conducted using the broth microdilution method as per CLSI guidelines. ³⁹ The highest concentration tested corresponded to the lowest concentration that produced a zone of inhibition in the agar diffusion method. All bacterial strains were cultured aerobically at 37 °C on MHA (Microgen). Test samples were two-fold serially diluted in Mueller-Hinton broth (MHB) (Scharlau Microbiology) in 96-well plates (Tarsons), with the highest concentration being 2048 µg/mL. Vancomycin and penicillin served as vehicle controls, while DMSO and water were used as negative controls. For inoculum preparation, 3 to 5 morphologically similar colonies from 24-h-old bacterial cultures were suspended in phosphate-buffered saline and adjusted to a 0.5 McFarland standard (OD 0.08-0.1 at 625 nm). This suspension was further diluted 1:100 in MHB. Bacterial suspensions (final concentration 5 × 10⁵ CFU/mL) were added to each well of the test plates. Plates were incubated for 24 h at 37 °C. After incubation, 0.2 mg/mL p-iodonitrotetrazolium (INT) was added to each well. MIC values were determined visually based on the color change from yellow to red, and optical density (OD) was measured at 600 nm using a µQuant microplate spectrophotometer (BioTek). Each assay was performed in triplicate across three independent tests to ensure reproducibility.

Bactericidal Activity

The minimum bactericidal concentration (MBC) value was determined as described by Nayim et al. ⁴⁰ Fifty microliters from wells with no visible growth in the MIC assay, as well as two concentrations above, were transferred to 150 µL of MHB in aliquots. Fifty microliters of each sample were then spread onto MHA plates and incubated at 37 °C for 24 h. The MBC was identified as the lowest concentration of the tested sample, which resulted in no viable bacterial colonies.

Fractional Inhibitory Index

The FIC index is used to evaluate the combined effects of multiple antimicrobial agents. The FIC index is calculated to assess whether the combination of substances results in a synergistic, additive, indifferent, or antagonistic effect. The calculations and interpretations are as follows:

FIC = FIC_A + FIC_B, where FIC_A and FIC_B are the MIC values of the combination compared to the individual MICs.

The interpretation is generally:

FIC ≤ 0.5: Synergistic effect

FIC > 0.5 - ≤ 1.0: Additive effect

FIC > 1 - ≤ 4: Indifferent effect

FIC > 4: Antagonistic effect

Statistical Analysis

All experiments were performed in triplicate, and the mean values, along with standard deviations, were calculated using Microsoft Excel 2016 Edition (Microsoft, Seattle, WA) and GraphPad Prism 8.0.2. Data were presented as mean ± standard deviation. To determine the statistical significance of the results, a paired t-test was used to compare the mean values of the experimental test conditions with their respective controls. A p-value was regarded to signify a considerable difference between the two groups (treated and untreated), denoted as **P < 0.001.