Evaluation of the effectiveness of natural extract as a substituent for synthetic preservatives and antioxidants in pharmaceutical preparations

Abstract

Background

Despite the fact that synthetic preservatives and antioxidants have strong antibacterial and antioxidant activity, they are frequently associated with negative health consequences. Currently, there is an increasing interest in pharmaceutical products that are excellent in quality and free of synthetic preservatives.

Methods

As a result, the purpose of this research is to assess the antibacterial and antioxidant activities of olive leaf extract, oleuropein, and thymol in various pharmaceutical products. Furthermore, the efficacy of these natural extracts to substitute synthetic preservatives (methyl-propylparaben and benzalkonium chloride) and antioxidants (butylhydroxytoluene) will be investigated.

Results

The results revealed that oleuropein, olive leaf extract, and a blend of oleuropein and thyme oil may be utilized as preservatives at concentrations of (0.6 % w/v), (0.4 % w/v), and (0.4 %/0.1 % v/v), respectively. The results demonstrated that thyme oil and oleuropein have synergistic efficacy against the studied microorganisms. By assessing antibacterial activity, and physical properties, the results demonstrated that pharmaceutical formulations containing natural preservatives were stable and effective for three months under accelerated settings (40 °C/75 % RH).

Conclusion

Natural compounds such as oleuropein, olive leaf extract, and thyme oil have shown antibacterial effectiveness equivalent to synthetic preservatives in selected pharmaceutical products. Furthermore, there was synergy in antimicrobial activity between thyme oil and oleuropein and this facilitates the use of these compounds at different levels.

1. Introduction

A large variety of bioactive phytochemicals with stabilizing effect and antibacterial characteristics may be found in medicinal plants and their essential oils (Alwafa et al., 2021). As a result, their usage has been researched in a variety of fields, including food items (Romeo et al., 2010, Granato et al., 2018, Bolouri et al., 2002), animal feed formulations (Zaazaa et al., 2022), food packaging (Erdohan and Tuthan 2012), pest control (Campolo et al., 2018), and pharmaceuticals (Atai et al., 2007). Due to their safety, medicinal plant extracts are increasingly being used as an alternative to synthetic preservatives (Quinto et al., 2019). According to Parham et al. (2020), interest in phytochemicals as novel sources of natural stabilizing effect and antibacterial agents is growing. Pharmaceutical items commonly contain antimicrobial preservatives to prevent the development of bacteria during usage and storage (Syed, 2010). Moreover, Antimicrobial agents can mitigate the effect of cross- contamination during repeatedly withdrawing individual doses from multiple-dose containers of sterile goods (Ripoll Gallardo et al., 2015). Several studies showed that the usage of synthetic antimicrobial agents in pharmaceutical products was associated with many toxic effects on human health, such as ocular toxicity, genotoxicity, and bacterial resistance (Goto et al., 2003, Breitkreutz and Boos, 2007, Shehab et al., 2009, Kaur et al., 2009, Sarkar, 2021). Therefore, the usage of natural preservatives as an alternative to synthetic preservatives considering their effectiveness and safety, is a promising trend in many industries, such as foods, cosmetics, and pharma (Rathee et al., 2023).

A wide range of safe active natural components found in medicinal plants have been shown to have some degree of antibacterial activity against pathogens and spoilage microorganisms (Mari et al., 2003, Obagwu and Korsten, 2003). In this regard, olive leaf extract is an excellent source of a variety of phytochemicals. It was found that olive leaves contained a variety of phytochemicals with antibacterial and insecticidal properties (Pooley and Peterson, 1997). In the Mediterranean region, Olea europaea L. leaves are frequently used as vasodilatory, hypotensive, anti-inflammatory, anti-rheumatic, diuretic, antipyretic, and hypoglycemic in traditional medicine (Somova et al., 2003).

The active compound in olive leaf is oleuropein. Oleuropein has numerous pharmacological characteristics, such as stabilizing effect, anti-inflammatory, anti-atherogenic, anti-cancer, anti-microbial, and antiviral effects (Syed, 2010). As a result, it is offered commercially in Mediterranean nations as a dietary supplement (Owen et al., 2000, Katsiki et al., 2007, Syed, 2010). Due to their phenolic content, the pharmacological characteristics of olive oil, the olive fruit, and its leaves have been recognized as significant elements of medicine and a healthy diet (Syed, 2010).

At least 70 distinct components were discovered in thyme oil (Thymus vulgaris L.), where thymol is the main component (Alwafa et al., 2021, Visioli and Galli, 2003, Matera et al., 2023). In particular, thymol exhibited many therapeutic and technological characteristics such as antiparasitic, anti-inflammatory, antimicrobial, antitumoral, antifungal, and antioxidative (Visioli and Galli, 2003). Additionally, carvacrol, found in thyme oil, has been shown to have antibacterial and antifungal properties (Alwafa et al., 2021, Visioli and Galli, 2003, Matera et al., 2023).

There is a need to evaluate the effectiveness of natural plant extracts against different species of pathogens and spoilage microorganisms, as well as their potential side effects in selected pharmaceutical products. The aim of this study was to evaluate the antimicrobial activity and antioxidant activity of olive leaf extract, oleuropein, and thymol in different pharmaceutical products.

2. Materials and methods

2.1. Collection and preparation of samples

Olive leaf samples were taken from a field of olive trees in the West Bank, Palestine, not far from Bethlehem City, during November 2017. Concerning thyme leaves (Fig. 2), it was harvested in April 2017 from Bethlehem City in West Bank. To dry, the leaves were kept at room temperature (25–30 °C). The leaves were crushed and sieved through a 120 - mm mesh sieve after drying at room temperature (25–30 °C). The resulting powder (Fig. 1) was kept in the dark at room temperature until it was extracted using water distillation. The processing conditions for the thyme leaves were the same.

Fig. 2.

Thyme leaves used in this study.

Fig. 1.

Olive leaves extract used in this study.

Water distillation was utilized to get olive leaf extract. Ten grams of olive leaf powder were macerated for four hours at 40 °C in 100 ml of 80 % ethanol. Whatman No. 1 filters (Whatman, UK) were used to filter out the extract from coarse particles. A vacuum rotary evaporator was used to evaporate the resultant extract at room temperature.

Thyme oil was obtained by steam distillation extraction at 100 °C and atmospheric pressure. About 55 g of dried thyme leaves were immersed in 500 ml water for 2 h and boiled in the steam distillation apparatus for one hour. The steam containing the essential oil was collected by condensation using a water-cooling system. Then, the oil was separated from the water to obtain the hydrosol (a mixture of oil and water).

2.2. Preparation of some pharmaceutical dosage forms containing oleuropein, OLE and thyme oil

2.2.1. Preparation of dexamethasone sodium phosphate oral syrup

The product was prepared where each 5 ml contains 0.658 mg of dexamethasone sodium phosphate. The general formula for dexamethasone sodium phosphate oral syrup is shown in Table 1. Two liters of purified water heated to 50 °C were transferred into the mixing tank (speed 1200 rpm). The sugar was gradually added and mixed for 15 min at the same speed. Oleuropein was mixed with glycerin for 2 min. Titriplex was dissolved in 50 ml of purified water, then added to the mixing tank and mixed for 5 min. The red color and saccharin were dissolved in a similar way as described before. Purified water was added while mixing to reach a final volume of 3.8 L. The solution was cooled to 35° C. At this point, cherry flavor was added and mixed for 1 min. Dexamethasone sodium phosphate was dissolved in 200 ml of cold, purified water, then added to the mixing tank and mixed for 5 min.

Table 1.

The general formula for dexamethasone sodium phosphate oral syrup, where the function of each component is indicated.

	Component*	Quantity/100 ml	Function
1	Dexamethasone sodium phosphate (99.5 %)	13.0 mg	Active ingredient
2	Cherry flavor (99 %)	102.5 mg	Flavor
3	Red color (99 %)	5.3 mg	Coloring agent
4	Titriplex (99 %)	0.1 g	Chelating agent
5	Saccharin Sodium (99 %)	33 mg	Sweetening agent
6	Sucrose (99 %)	50 g	Sweetening agent
7	Oleuropein (99 %) or OLE	0.4 or 0. G	Preservative
8	Glycerin (99 %)	2.6 ml	Co solvent
9	Distilled water	100.0 ml	Solvent

^*All these materials except natural compounds were obtained from Sigma Aldrich company.

2.2.2. Preparation of sodium chloride nasal spray

The intended amount of sodium chloride in the product was 0.074 mg per 10 ml. Table 2 lists the components of the common sodium chloride nasal spray formula along with their respective purposes. Firstly, two liters of purified water heated at 70 °C was added to the mixing vessel. Oleuropein and Cremophor RH40 (polyoxyl40 hydrogenated castor oil) were separately added to mixing vessel and mixed for 5 min using a speed of 1500 rpm. EDTA was dissolved in 200 ml of purified water at 70 °C, then added to the mixing vessel and mixed at a speed of 1500 rpm. Sodium chloride was added and mixed at speed 1500 rpm until dissolved for about 5 min, then sodium dihydrogen phosphate and dibasic sodium phosphate were sequentially added to the mixing vessel and mixed at similar conditions. The preparation was cooled to 35 °C and the volume was completed to 4 L using purified water and mixed for 5 min at a speed of 1500 rpm.

Table 2.

The general formula for sodium chloride nasal spray where the function of each component is indicated.

	Component*	Quantity/100 ml	Function
1	Sodium chloride (99 %)	0.7 g	Active ingredient
2	Oleuropein (99 %)	0.4 g	Preservative
3	Thyme oil (99 %)	0.1 g	Preservative
4	Cremophor RH40 (Polyoxyl 40 hydrogenated castor oil) (99 %)	1.0 g	Surfactant
5	EDTA (99.8 %)	60.0 mg	Chelating agent
6	Sodium dihydrogen phosphate (99.8 %)	0.2 g	Buffering agent
7	Dibasic sodium phosphate (99 %)	0.7 g	Buffering agent
8	Purified water	100 ml	Solvent

^*All these materials except natural compounds were obtained from Sigma Aldrich company.

2.2.3. Preparation of vitamin D3 oral drops

A vitamin D3 oral drop product was prepared to contain 3.485 mg of vitamin D3/10 ml. The list of ingredients with their functions for the product is shown in Table 3. Tween 80 and vitamin D3 were added while mixing for 45 min using the mixing tank until completely dissolved. The glycerin was added to the previous mixture while mixing for about 15 min. Oleuropein was dissolved in orange oil flavor, then transferred to the mixing tank and mixed for 5 min. Citric acid anhydrous was dissolved in 200 ml of purified water. The dissolved citric acid was transferred to the mixing tank and mixed for 5 min. Finally, the volume was increased to 4 L by the addition of purified water, followed by mixing for 10 min.

Table 3.

The general formula for vitamin D3 oral drops, where the function of each component is indicated.

	Component*	Component*	Function
1	Vitamin D3 crystals (99 %)	Vitamin D3 crystals (99 %)	Active ingredient
2	Tween 80 (99 %)	Tween 80 (99 %)	Emulsifier
3	Glycerin (99 %)	Glycerin (99 %)	Preservative
4	Citric acid anhydrous (99 %)	Citric acid anhydrous (99 %)	pH adjustment
5	Oleuropein (99 %)	Oleuropein (99 %)	stabilizing effect and preservative
6	Thyme oil (99 %)	Thyme oil (99 %)	stabilizing effect and preservative
7	Orange oil flavor (99 %)	Orange oil flavor (99 %)	Flavor
8	Purified water	Purified water	Vehicle

^*All these materials except natural compounds were obtained from Sigma Aldrich company.

2.3. Chemical and physical analysis

2.3.1. Determination of oleuropein

The concentration of oleuropein in pharmaceutical preparations was determined by the reversed-phase HPLC method. Silica-based C18 bonded phase column (C18, 250 mm × 4.6 ID) with mobile phase consisting of a mixture of water and acetonitrile (80/20 vol ratio) containing 1 % acetic acid at a flow rate of 1.0 ml/min A UV detector at 237 nm was used for oleuropein determination. 20.0 µl of standard and sample solutions were injected. Oleuropein was identified based on retention time in comparison with standard oleuropein. The quantity of oleuropein was determined by using an external standard using the peak area and the calibration curves obtained from the oleuropein standard solution.

2.3.2. Determination of dexamethasone sodium phosphate

Dexamethasone sodium phosphate was determined by a reversed-phase HPLC equipped with a column (RP- select B, 125X mm, 5um). A mixture of methanol and acetonitrile (40/60 vol ratio) containing 1 % acetic acid was used as the mobile phase at a flow rate of 1.2 ml/min. A UV detector at 240 nm was in HPLC. 20.0 µl of standard and 20.0 µl of sample solutions were injected. Dexamethasone sodium phosphate was identified by comparing the retention time with standard of dexamethasone sodium phosphate. The concentration of dexamethasone sodium phosphate was determined by calculating the peak area with respect to the peak area of an external standard.

2.3.3. Determination of sodium chloride

Sodium chloride (analytical grade) has been dried at 110 °C for 2 h. 100 mg of sodium chloride was accurately weighed, transferred to a 100-ml beaker, and dissolved in 5 ml of water. 5 ml of acetic acid, 50 ml of methanol, and about 0.5 ml of eosin were added and stirred with a magnetic stirrer. The solution was titrated with silver nitrate.

The concentration of sodium chloride in the pharmaceutical product was determined by titration using a standardized silver nitrate solution.

2.3.4. Determination of vitamin D3

Vitamin D3 was determined by reversed-phase HPLC with a column (RP18e, 150–4.6 mm, 5 µm) and a mobile phase consisting of a mixture of methanol and acetonitrile (25/75 vol ratio) containing 1 % acetic acid at a flow rate of 1.2 ml/min. A UV detector at 268 nm was used. 20.0 µl of standard and 20.0 µl sample solutions were injected. Identification of vitamin D3 was based on measuring retention time compared to the standard of vitamin D3. The concentration of vitamin D3 was measured based on its peak area compared to the peak area of an external standard.

2.3.5. Determination of pH

pH was measured using a pH meter (pH meter, 211R Hanna). The pH meter was calibrated by buffer solutions pH 4.0 and 7.0. A pH electrode was immersed in the product, and the pH value was recorded. pH was measured two times, and an average value was recorded.

2.4. Microbiological analysis

Antimicrobial activity was assessed for two fungal species (Candida albicans (ATCC # 10231) and Aspergillus niger (ATCC # 16404)) on Sabouraud’s Dextrose Agar/Broth and three bacterial species (Escherichia coli (ATCC # 8739), Pseudomonas aeruginosa (ATCC # 9027), and Staphylococcus aureus (ATCC # 6538)) on Soybean Casein Digest Agar/Broth. Fig. 3 shows activity of olive leaves extract against Pseudomonas aeruginosa.

Fig. 3.

Antibacterial activity of olive leaves extracts against Pseudomonas aeruginosa.

2.4.1. Harvesting and counting the tested bacterial species and C. albicans cultures

The microbial growth on the surface of the media was washed using sterile saline TS and collected in a suitable vessel. Sterile saline TS was added to obtain a microbial count of about 1 × 10⁸ CFU/ml. The total count of the cells was determined by measuring the turbidity using a spectrophotometer at 650 nm to obtain an optical density (O.D.) of:

• A.0.3–0.45 for S. aureus (∼1–3 × 10⁸ CFU/ml).
• B.0.2–0.3 for P. aeruginosa and E. coli (∼1–3 × 10⁸ CFU/ml).
• C.<1.0 for C. albicans (∼1–3 × 10⁸ CFU/ml).

Bacteria were inoculated for 24 h at 35 °C ± 2, and C. albicans at 23 °C ± 2 for 2–3 days and the CFUs were counted (Plates consider for counting should have 30–100 CFU).

2.4.2. Harvesting the Aspergillus niger culture

The surface growth was washed using a sterile saline TS containing 0.05 % polysorbate 80 and collected in a suitable vessel. Sterile saline TS was added to obtain a microbial count of about 1 × 10⁸ CFU/ml. Culture was incubated for 2–4 days at 23 ± 2 °C and the colonies were counted (count plates having between 10 and 100 CFU).

2.5. Microbiological sampling and analysis for pharmaceutical products

20 ml of product was selected for microbiological analysis. Five sterile, capped bacteriological containers of suitable size were used to transfer the product for analysis. For purposes of testing, products have been divided into two categories:

Category 1 products: Dexamethasone Sodium Phosphate Oral Syrup and Vitamin D3 Oral Drops.

The testing of the reagents was conducted in five original containers. Each container was inoculated with one of the prepared and standardized inoculums and mixed. The initial concentration of viable microorganisms in each test preparation is estimated based on the concentration of microorganisms in each of the standardized inoculums as determined by the plate-count method. The volume of the suspension inoculum used is between 0.5 % and 1 % of the volume of the product. The final concentration of tested microorganisms in the product was 1 × 10⁵ to 1 × 10⁶ CFU/ml of the product. The inoculated containers were incubated at 22.5 ± 2.5 °C by the UNIMAX 1010 shaker/incubator 1000 and at 32.5 ± 2.5 °C by the UNIMAX 1010 shaker/incubator 1000.

The concentrations of tested microorganisms were presented as CFU/ml and the change in microbial population was expressed as log₁₀ values for each microorganism at the applicable test intervals. The changes in terms of log reductions were expressed (the log reduction is defined as the difference between the log 10-unit value of the starting concentration of CFU/ml in the suspension and the log 10-unit value of CFU/ml of the survivors at that time point).

Category 2 products: Sodium Chloride Nasal Spray proceed as directed in Category Dexamethasone Sodium Phosphate Oral Syrup and Vitamin D3 Oral Drops products to obtain a concentration of microorganisms in the inoculum between 1 × 10⁻⁵ and 1 × 10⁻⁶ CFU/ml of the product.

2.6. Interpretation

The criteria for microbial effectiveness are met if the specified criteria are met, as described in Table 4. No increase is defined as not more than 0.5 log₁₀ unit higher than the measured previous value.

Table 4.

The criteria for microbial effectiveness for category 1 products and category 2 products.

Product category	Type of microorganism	Effectiveness criteria
Category 1 products	Bacteria	Not less than a 1.0 log reduction from the initial count at 14 days, and no increase from the 14-day count at 28 days
	Yeast and molds	No increase from the initial calculated count at 14, and 28 days.
Category 2 products:	Bacteria	Not less than a 2.0 log reduction from the initial count at 14 days, and no increase from the 14-day count at 28 days
	Yeast and molds	There was no increase in the initial calculated count at 14, and 28 days

2.7. Total aerobic microbial count for liquid dosage form of finished products

The purpose of these tests is the qualitative and quantitative estimation of the total viable aerobic count of microorganisms in finished liquid dosage forms. All liquid forms requiring testing for Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus, as well as yeasts and molds.

2.7.1. Plate method for bacterial count

About 10 ml of specimen were transferred accurately to a sterile bottle and repeated with another sterile bottle. 90 ml of Soyabean Casein Digest Medium (SCDM) or Casein Digest- soy lecithin poly sorbate (CDSLP) (or phosphate buffer of pH 7.2) were added to the first bottle, and 90 ml of Fluid Lactose Medium (FLM) was added to the second bottle, so that the final dilution is 1:10. The bottles were closed and mixed by swirling the bottles gently. The SCDM bottle (or CDSLP or phosphate buffer bottle) was opened, and with a sterile pipette, 1 ml of the sample was drawn and transferred to a petri dish that contained already solidified Soyabean Casein Digest Agar (SCDA). The petri dish was then covered. This step was repeated with another plate. Each plate was rotated gently so that the 1 ml of sample covered all the surface of the agar in the plate, and the plates were left for 10 min. After 10 min, using a sterile pipette, the remaining liquid on the agar surface was drawn. The plates were inverted and incubated at 35 °C for 48–72 h. Results of the growth were recorded, and the average number of colonies was recorded from the two plates in each case (sample or control) as follows:

If there is growth, the average number of colonies in each plate/ml was recorded (taking into account the initial dilution of 1:10). If there is no growth, the result is recorded as <10 CFU/ml for a negative result.

2.7.2. Plate method for total aerobic yeast and mold count

Similar to the procedure of bacterial count (Section 2.7.1), but Sabouraud Dextrose Broth/Agar (SDB/SDA) was used instead of SCDM/SCDA. SDB bottles and SDA plates were incubated at 20–25 °C for 5–7 days.

2.7.2.1. The membrane filtration method for total aerobic bacterial count (TABC) and total Combined yeasts and molds count (TYMC)

Using a sterile pipette, 10 ml from each sample bottle (10 bottles/test) were taken and put into a 100 ml sterile bottle. A 0.45 µm membrane filter (50 mm in diameter) was used to filter the sample preparations. Three rinses were performed using 100 ml of the selected diluent.

The filter was aseptically transferred by sterile blunt-tipped forceps and placed on the surface of a solidified SCDA petri dish for the TABC test. In the same way, a filter was placed on the surface of the solidified SDA petri dish for the TYMC test. The SCD plates were incubated at 30–35 °C for 48–72 h, and the SDA plates were incubated at 20–25 °C for 5–7 days. The acceptance criteria for nonsterile finished products is according to USP: United States Pharmacopia (Microbiological examination of nonsterile products: microbial enumeration tests), and this criterion is shown in Table 5.

Table 5.

Acceptance criteria for nonsterile finished products (USP/BP).

Route of Administration	TABC (CFU/g or ml)	TYMC (CFU/g or ml)	Absence of*
Nasal/Otic/	<200 (102)	<20 (101) <10	P. aeruginosa and S. aureus
Oral Liquids	<200 (102)	<20 (101)	E. coli

**USP/BP acceptance criteria interpretation

 –10¹ CFU: maximum acceptable count = 20.

 –10² CFU: maximum acceptable count = 200.

 –10³ CFU: maximum acceptable count = 2000.

2.8. Stability study

Dexamethasone sodium phosphate oral syrup, vitamin D3 oral drops, and sodium chloride nasal spray containing natural substances (oleuropein and thyme oil), as well as the positive control formulation, were subjected to a stability study for three months. Antimicrobial tests and physical properties such as color, pH, precipitation, and drug were evaluated. Oleuropein was determined by HPLC at the end of each month for three months on accelerated conditions (40 ± 2 °C/75 ± 5 % RH) using specialized oven with settable temperature and relative humidity.

3. Results

3.1. Dexamethasone sodium phosphate oral syrup with natural preservatives (Oleuropein and OLE)

The results showed that oleuropein was stable during three months of storage at accelerated conditions in this product. The remaining oleuropein was 98 % at day zero and decreased to 97 %, 95 %, and 94 % at the first, second, and third months of storage, respectively.

The percentage of the active compound (Dexamethasone sodium phosphate) was 96 % at zero days and decreased to 94 %, 94 %, and 92 % at the first, second, and third months of storage, respectively. These results indicated that the active compound was stable and not affected by the addition of natural materials, and the same results were observed for OLE.

The effect of oleuropein addition on the pH of dexamethasone sodium phosphate oral syrup is shown in Table 6. It was found that the pH of dexamethasone sodium phosphate oral syrup was not affected by the addition of oleuropein. In this context, there was very slight decrease in pH, but it was still in the acceptable range of pH for this product. This decrease in pH of the nasal spray could be attributed to various factors, e.g. absorption of carbon dioxide and some chemical degradation.

Table 6.

Effect on pH of dexamethasone sodium phosphate syrup.

pH (Dexamethasone sodium phosphate syrup)	pH values*
Zero time	5.6
First month	5.6
Second month	5.4
Third month	5.3

^*Accepted pH values (4.5–6).

Dexamethasone sodium phosphate syrup with oleuropein 0.6 % (or 6000 ppm) was microbiologically assessed at the first, second, and third months of storage and compared to the positive control (dexamethasone sodium phosphate with methyl and propylparaben) and negative control (dexamethasone sodium phosphate without any preservative).

It was found that there was no bacterial growth in samples containing oleuropein 0.6 % (w/v) and positive control samples in all tested mediums, as shown in Table 7. Accordingly, oleuropein showed good antimicrobial properties. Negative control of dexamethasone sodium phosphate syrup without a preservative showed bacterial growth.

Table 7.

Microbial limit test: direct transfer (broth media) test for dexamethasone sodium phosphate syrup with oleuropein 0.6 % (or 6000 ppm).

Medium	Results of sample with oleuropein at 0.6 % (6000 ppm)	Results positive control (using methyl and propyl paraben)	Results Negative control No preservative
Tryptic soy broth	Clear	Clear	Turbid
Fluid lactose medium	Clear	Clear	Turbid
Sabouraud dextrose broth	Clear	Clear	Turbid

The total microbial count of dexamethasone sodium phosphate syrup samples is shown in Table 8. Our finding showed that samples with oleuropein had antimicrobial power similar to the positive control (dexamethasone sodium phosphate syrup with methyl and propylparaben preservatives). The bacterial count on tryptic soy agar was less than 10 CFU, which was in accordance with the acceptable limit (<200 CFU).

Table 8.

Microbial limit test: total count test for dexamethasone sodium phosphate syrup with oleuropein 0.6% w/v.

Medium	Limits on agar CFU #\ml	Results Sample with oleuropein at 0.6 % (6000 ppm) CFU/ml	Results positive control CFU/ml	Results Negative control CFU/ml
Tryptic soy agar	Bacterial <200(102))	<10 CFU	<10 CFU	<180 CFU
Sabouraud dextrose agar	yeasts and molds < 20 (101)	<10 CFU	<10 CFU	<10 CFU

In respect to yeast and mold, results showed that dexamethasone sodium phosphate syrup samples with oleuropein were similar to conventional chemical preservatives (methyl and propylparaben).

Membrane filtration was used to assess the microbiological counts of the dexamethasone sodium phosphate syrup with oleuropein 0.6 % (w/v), positive control samples, and negative control samples (Table 9). According to the study, the bacterial count for dexamethasone sodium phosphate syrup with 0.6 % (w/v) oleuropein was in compliance with the standard limits (ATCC), which must be less than 200 CFU, and was comparable to the positive control (<10 CFU).

Table 9.

Microbial limit test: membrane filtration test for dexamethasone sodium phosphate syrup with oleuropein 0.6% (w/v).

Medium	Limits on agar CFU/ml	Results Sample with oleuropein 0.6 % (6000 ppm) CFU/ml	Results Positive test CFU/ml	Results Negative control test CFU/ml
Tryptic soy agar	<200 (102) Bacteria	<10	< 10	<120
Sabouraud dextrose agar	Yeasts and molds < 20 (101)	<10	< 10	<20

When it came to the count of yeast and mold, dexamethasone sodium phosphate syrup with 0.6 % (w/v) oleuropein was comparable to the positive control (<10 CFU) and satisfied the standard standards (ATCC), which call for < 20 CFU. Although the bacterial and fungal counts for the negative control samples were greater than those of the positive control, they were still within the acceptable range (ATCC).

3.2. Sodium chloride nasal spray with oleuropein and thyme oil

Sodium chloride nasal spray containing oleuropein (0.4 % w/v) and thyme oil (0.1 % v/v) was evaluated. The product was formulated with a natural preservative instead of a chemical preservative (benzalkonium chloride). The natural preservatives were also prepared and tested for their effectiveness against microbes through the tests mentioned previously, and tests were repeated for the same stored specimen each month (for three months). The results of the product containing natural preservatives were compared with the preparations (positive control).

The content of oleuropein in sodium chloride nasal spray was determined by HPLC. The study showed that oleuropein remained stable after three months of storage at accelerated conditions. The remaining quantity of oleuropein at day zero was 96 %, while it decreased to 95 %, 94 %, and 92 % at the first, second, and third months of storage, respectively. These results showed that oleuropein was stable in this formula. The chromatogram of oleuropein in olive leaves extract is shown in Fig. 4, as well as the chromatogram of oleuropein in sodium chloride nasal spray is shown in Fig. 5.

Fig. 4.

Chromatogram of oleuropein in olive leaves extract.

Fig. 5.

Chromatogram of Oleuropein in Sodium Chloride Nasal Spray, stored at accelerated conditions (40 ± 2 °C/75 ± 5 % RH) for three months.

At zero time, the concentration of the active component was 96 %, but in the first, second, and third months of storage, it was 96 %, 95 %, and 95 %, respectively. The inclusion of natural compounds had no effect on the stability of active component.

The effect of the addition of oleuropein on the pH of sodium chloride nasal spray is shown in Table 10. Sodium chloride nasal spray was not affected by the addition of oleuropein, where a very minor decrease in pH was observed but still within the acceptable range of pH for this product.

Table 10.

The effect of addition of oleuropein on pH of sodium chloride nasal spray.

Time	pH*
Time zero	6.6
First month	6.6
Second month	6.5
Third month	6.5

^*Normal range pH (5–7.5).

The results of the microbiological assessment of sodium chloride nasal spray with oleuropein 0.4 % (w/v, 4000 ppm) and thyme oil 0.1 % (v/v, 1000 ppm) concentrations are shown in Table 11. Microbiological assessment was carried out for three months and compared to the positive control (sodium chloride nasal spray with benzalkonium) and the negative control (sodium chloride nasal spray without any preservative).

Table 11.

Microbial limit test: direct transfer (broth media).

Medium	Sample with oleuropein (0.4 %, 4000 ppm) and thyme oil (0.1 %)	Results positive test (using benzalkonium chloride)	Results Negative control (No preservative)
Tryptic soy Broth	Clear	Clear	Turbid
Fluid lactose medium	Clear	Clear	Turbid
Sabouraud dextrose broth	Clear	Clear	Turbid

Using the three different types of media, it was found that there was no microbial growth in samples containing oleuropein (0.4 %) and thyme oil (0.1 %) as well as in samples with a positive test (benzalkonium chloride). This outcome demonstrated the antibacterial properties of thyme oil and oleuropein. Bacterial growth was seen in the sodium chloride nasal spray negative control that lacked a preservative.

Table 12 shows the total microbiological counts for positive test samples, negative test samples, and sodium chloride nasal spray samples containing oleuropein/thyme oil. According to our findings, the bacterial and fungal counts in the samples containing oleuropein and thyme oil were comparable to those in the positive control sample, which contained benzalkonium preservative. All of the results were under the permitted level (<200 CFU). Compared to positive control samples and samples containing thyme oil and oleuropein, negative test samples exhibited higher levels of bacteria and fungi.

Table 12.

Microbial limit test: total count.

Medium	Limits on agar CFU/ml	Sample with oleuropein (0.4 %) and thyme oil (0.1 %) CFU/ml	Results positive test CFU/ml	Results Negative control test CFU/ml
Tryptic soy agar	<200(102)) Bacteria	<10	<10	<120
Sabouraud dextrose agar	yeasts and molds < 20 (101)	<10	<10	<18

Total microbial counts for sodium chloride nasal spray samples with oleuropein/thyme oil, positive test samples, and negative test samples using membrane filtration are shown in Table 13.

Table 13.

Microbial limit test: membrane filtration.

Medium	Limits on agar CFU/ml	Results Sample with oleuropein (0.4 %) and thyme oil (0.1 %) CFU/ml	Results Positive control CFU/ml	Results Negative control CFU/ml
Tryptic soy agar	<200(102)) Bacteria	<10	<10	<220
Sabouraud dextrose agar	yeasts and molds < 20 (101)	<10	<10	<20

It was found there were no significant differences in bacterial and fungal counts between samples with oleuropein and thyme oil and the positive control (with benzalkonium preservative) samples. In this context, all results complied with the acceptable limit (<200 CFU). Negative test samples had higher bacterial counts than the standard limits (<220 CFU vs. < 200 CFU), and the fungal count was the same as the maximum standard limit.

3.3. Vitamin D3 oral drops with oleuropein and thyme oil

The microbiological stability of vitamin D3 oral drop was investigated using oleuropein at a concentration of 0.4 % w/v and thyme oil at a concentration of 0.2 % v/v as a natural preservative and stabilizing effect in comparison to the chemical preservative butylhyroxytoluene. The actual content of oleuropein has been evaluated during storage in the products, positive test samples, and negative test samples by HPLC.

The study showed that oleuropein was stable in vitamin D3 oral drops during three months of storage at accelerated conditions. In this context, the remaining concentration of oleuropein was 95 % at day zero, while it quite decreased to 93.7 %, 93 %, and 91.8 % at the first, second, and third months of storage, respectively.

In the first, second, and third months of storage, the proportion of the active component (vitamin D3) that remained was 98 %; it then dropped to 97 %, 95 %, and 94 %, respectively. These results showed that the inclusion of natural compounds had no effect on the stability of the active component.

The addition of oleuropein did not influence the pH of Vitamin D3 Oral Drops, as Table 14 illustrates, but a very slight pH reduction was observed. Nevertheless, the pH was within the permitted limit for this product.

Table 14.

Effect on pH on Vitamin D3 Oral Drops.

pH (Vitamin D3 Oral Drops)	pH*
0 Day	3.0
1 Month	3.1
2 Month	2.9
3 Month	2.9

^*Normal pH range 2–4 according to USP and Guidance for Industry Nasal Spray and Inhalation Solution.

The results related to the effect of oleuropein, thyme, and synthetic preservatives on microbial growth using different medium are shown in Table 15. It was found that there was no microbial growth in all media.

Table 15.

Microbial limit test – Direct transfer (broth media).

Medium	Sample with oleuropein (0.4 %, 4000 ppm) and thyme oil (0.2 %)	Results Positive test (using glycerin)	Negative control No preservative
Tryptic soy Broth	Clear	Clear	Clear
Fluid lactose medium	Clear	Clear	Clear
Sabouraud dextrose broth	Clear	Clear	Clear

The bacterial count was less than 10 CFU across all treatments, meeting the ATCC minimum limits of less than 200 CFU. In all treatments, the yeast and mold counts were less than 10 CFU, which met the regulatory limits of less than 20 CFU units. According to the results, the product that included natural preservatives performed similarly to the results of a positive test preparation (Table 16).

Table 16.

Microbial limit test: total count and membrane filtration.

Medium	Limits on agar CFU\ml	Sample with oleuropein (0.4 %) and thyme oil (0.2 %) CFU/ml	Results Positive control CFU/ml	Results Negative control CFU/ml
Tryptic soy agar	<200(102)) Bacteria	<10	<10	<10
Sabouraud dextrose agar	Yeasts and molds < 20 (101)	<10	<10	<10

3.4. Stability of pharmaceutical preparations

The results showed that dexamethasone sodium phosphate oral syrup containing oleuropein (0.6 % w/v) was stable for three months. The product had stable appearance and color and no precipitation during storage period. Moreover, pH remained within the allowable range (4.5–6). Moreover, the active compounds (dexamethasone sodium phosphate and oleuropein) were within the allowable range.

When OLE at a concentration of 0.4 % w/v was used in the same product, all properties were stable for one month. After one month, there was little precipitation due to the incomplete dissolution of the OLE substance. This problem was overcome by using surfactant.

Sodium chloride nasal spray containing oleuropein (0.4 % w/v) and thyme oil (0.1 % v/v) showed no precipitation and no change in appearance, color, or pH for three months.

Similarly, Vitamin D3 oral drops containing oleuropein (0.4 % w/v) and thyme oil (0.2 % v/v) were stable for three months. In this context, there was no precipitation and no change in appearance, color, or pH within the permissible range (2–4). The active ingredient (vitamin D3 crystals) and oleuropein were within the allowable range (Table 17).

Table 17.

Physical parameters and assay results of pharmaceutical formulations at accelerated condition at (40 ± 2 °C/75 ± 5 % RH).

Comparisons	Time	Precipitation	Appearance	Color	pH	Assay of oleuropein	Assay of Active material
Novodexon Syrup	Zero time	Negative	Uniform	Red	5.6	98 %	96 %
	First month	Negative	Uniform	Red	5.6	97 %	94 %
	Second month	Negative	Uniform	Red	5.4	96 %	94 %
	Third month	Negative	Uniform	Red	5.3	95 %	92 %
Nazex Nasel spray	Zero time	Negative	Uniform	Brown	6.6	96 %	97 %
	First month	Negative	Uniform	Brown	6.6	95 %	95 %
	Second month	Negative	Uniform	Brown	6.5	94 %	93 %
	Third month	Negative	Uniform	Brown	6.5	92 %	92 %
Dee Dense 400 oral drops	Zero time	Negative	Uniform	Golden	3.0	95 %	98 %
	First month	Negative	Uniform	Golden	3.1	93.7 %	97 %
	Second month	Negative	Uniform	Golden	2.9	93 %	95 %
	Third month	Negative	Uniform	Golden	2.9	91.8 %	94 %

4. Discussion

Our study showed that oleuropein showed strong antimicrobial activity against both gram-negative and gram-positive bacteria and against yeasts and molds. Although some researches have suggested that the presence of the ortho-diphenolic system (catechin) is the cause of oleuropein's antibacterial activity, the precise mechanism of action is still unclear (Sousa et al., 2006, Casas-Sanchez et al., 2007, Hemeg et al., 2020).

Our findings were in agreement with a cited report (Sudjana et al., 2009), in which the researchers found that oleuropein had antimicrobial activity against Staphylococcus aureus, Salmonella typhimurium, Escherichia coli, Klebsiella pneumonia, and Bacillus cereus. Malik (2015) showed that OLE (olive oil extract) was effective against two gram-positive strains (S. aureus and B. cereus). It was found that the growth of dermatophytes was inhibited after three days by 1.25 % (w/v) OLE, while the growth of Candida albicans was inhibited after 24 h in the presence of 15 % (w/v) OLE (Pereira et al., 2007). Several researchers found that olive leaf extract has antibacterial and antifungal properties (Korukluoglu et al., 2008, Lee and Lee, 2010, Borjan et al., 2020).

Thymol extracted from thyme species had antibacterial activity and strong antiseptic and stabilizing effect activity. The essential oil of T. vulgaris had a high content of oxygenated monoterpenes and a low content of monoterpene hydrocarbons, according to previous reports. They found that thyme oil had a significant effect on some studied microorganisms (Alsaraf et al., 2020, Soleimani et al., 2022, Radi et al., 2022).

According to Boskovica et al. (2015), it was found that thyme oil exhibited antibacterial activity against a variety of bacterial species, including methicillin-resistant Staphylococcus aureus, Bacillus cereus, Salmonella enteritidis, and Salmonella typhimurium. Additionally, Cetin et al. (2011) revealed that the essential oils of oregano and thyme had antibacterial activity against several kinds of fungus and bacteria. It was demonstrated by Boskovica et al. (2015) that carvacrol and thymol-oregano essential oils may work in synergy.

Thyme essential oil showed antimicrobial activity against Staphylococcus, Enterococcus, Escherichia coli, and Pseudomonas genus isolated from infected patients, and it may be effective in the prevention and treatment of bacterial human infection (Sienkiewicz et al., 2012).

Gram-negative and gram-positive bacteria differ in their cell wall structures, which can affect the effectiveness of antibacterial agents. Gram-negative bacteria have a thinner peptidoglycan layer in their cell walls, surrounded by an outer membrane containing lipopolysaccharides. Gram-negative bacteria are often more resistant to antibiotics due to the presence of the outer membrane, which acts as a barrier to many substances. In the current study, it was found that the plant extracts have higher antibacterial activity against gram positive bacteria compared to gram negative bacteria which is expected.

Pozzatti et al. (2010) found that thyme oil was active against yeasts. The activity of thyme oil was related to the major compound, thymol. In this context, pure thymol exhibited antifungal activity three-times higher than thyme oil (Klaric et al., 2007). It was found that some plant-derived materials containing trans-cinnamaldehyde, eugenol, carvacrol, and thymol showed some activity against major bacterial mastitis (Baskaran et al., 2009).

Cai et al. (2019) showed that microcapsulated thyme essential oil had antimicrobial activity against Botryodiplodia theobromae Pat. and Colletotrichum gloeosporioides Penz. Similarly, Tao et al. (2014) found that encapsulation improved the antimicrobial activity of thymol and thyme oils.

5. Conclusions

Oleuropein was effective as a natural preservative against bacteria and fungi in comparison to synthetic preservatives in selected pharmaceutical products. Similarly, thyme oil has similar characteristics at low concentrations. Oleuropein and thyme oil exhibited a good synergy against tested microorganisms. Using oleuropein and thyme oil as natural preservatives had no adverse effect on the physiochemical properties of pharmaceutical products. Accordingly, oleuropein and thyme oil are promising natural preservatives as alternatives to chemical ones.

Funding

No particular funding from governmental, private, or nonprofit organizations was given to this research.

CRediT authorship contribution statement

Fuad Al-Rimawi: Conceptualization, Funding acquisition, Data curation, Writing – original draft, Writing – review & editing, Visualization, Investigation, Validation, Formal analysis, Methodology, Supervision, Resources, Project administration, Software. Mahmood Sbeih: Conceptualization, Funding acquisition, Data curation, Writing – original draft, Writing – review & editing, Visualization, Investigation, Validation, Formal analysis, Methodology, Resources, Project administration, Software. Mousa Amayreh: Data curation, Writing – original draft, Writing – review & editing, Visualization, Validation, Formal analysis, Project administration, Software. Belal Rahhal: Data curation, Writing – original draft, Writing – review & editing, Visualization, Validation, Formal analysis, Methodology, Project administration, Software. Samer Mudalal: Conceptualization, Data curation, Writing – original draft, Writing – review & editing, Visualization, Validation, Formal analysis, Project administration, Software.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.