Evaluation of the Antimicrobial and Anticancer Properties of Myrrh Resin Extract and Its Application in Cacao Beverages

doi:10.21203/rs.3.rs-4218698/v1

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Evaluation of the Antimicrobial and Anticancer Properties of Myrrh Resin Extract and Its Application in Cacao Beverages

https://doi.org/10.21203/rs.3.rs-4218698/v1

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Due to the potential health risks of synthetic food preservatives, there has been a noticeable increase in interest in finding natural food preservatives during the past few decades. The goal of this study was to investigate the use of a natural extract of Commiphora Myrrh as an antimicrobial agent. The antioxidant properties of Myrrh resin extract (MRE) were analyzed using HPLC and GC-MS. The results showed that MRE contained potent antioxidant compounds, including 19 compounds, with the dominant compound being kaempferol, which had the highest value of 1896 µg/g. Quercetin was found to be the second most abundant compound, with a value of 520 µg/g. The efficacy of MRE as an antimicrobial agent against both Gram-positive and Gram-negative bacteria was tested, and its application in Cacao beverage was also studied. The results demonstrated that MRE was highly effective against all the tested bacteria both in vitro and in the total bacterial count of the produced cacao beverage. Additionally, the fungi in the cacao beverage were completely inhibited at all tested concentrations of MRE. The total soluble solids (TSS), pH value, and acidity of the produced untreated, treated cacao beverage with MRE and sodium benzoate were carried out, and all values mentioned were almost the same, with no differences noted. The sensory evaluation of Cacao beverage showed that the MRE had a minor impact on taste, odor, color, and texture of the produced cacao beverage in comparison with the control sample, which was very acceptable for judgments and recorded 95, 88, and 94 for the control and treated samples, respectively. Furthermore, the anti-cancer properties of MRE were evaluated, revealing significant cytotoxic effects against colon cancer (HCT) and liver cancer (HEPG2) cell lines. The IC50 values for HCT and HEPG2 cells were 55.69 µg/ml and 70.78 µg/ml, respectively, indicating the potential of MRE as an anti-cancer agent.

Some preservatives are classed as carcinogens or cancer-causing chemicals because they are known to be hazardous to human health. Certain preservatives are considered carcinogenic or cancer-causing substances due to their known harmful effects on human health. According to Gupta and Yadav (2021), artificial food additives interact with the cellular structure of the body, leading to a range of food-related disruptions, Mirza et al. (2017) also corroborate these findings.

When used in excess, it might cause negative effects such skin rashes and itching, breathing problems, sneezing, or stomach issues (Silva and Lidon., 2016). M. M. Silva and F. Lidon, 2016. Certain food preservatives are classified as carcinogens or cancer-causing agents due to their hazardous effects on human health. Synthetic food additives interact with the body's cellular structure, leading to various food disruptions and negative effects like skin rashes, itching, breathing problems, sneezing, and stomach issues when used excessively. Due to the possible health risks associated with synthetic food preservatives, consumers have become more cautious about consuming foods containing these additives, leading to an increase in demand for natural food preservatives over the past few decades. (Atwaa et al., 2022 and Teshome et al 2022).

Commiphora myrrh contains phytochemical components such as terpenoids (monoterpenoids, sesquiterpenoids, volatile/essential oil), diterpenoids, triterpenoids, and steroids. It has been utilized throughout history to treat various ailments such as wounds, mouth ulcers, pains, fractures, stomach issues, microbial infections, and inflammatory diseases, as reported by Mohammed et al., 2015 and Hanu LO et al., 2005). Akbar 2020 and Mohammed et al., 2015 have also documented the medicinal properties of Commiphora myrrh. Myrrh is known for its antiseptic, astringent, anthelmintic, and expectorant properties. Studies by Batiha et al., 2023 and Akbar., 2020 have proven the traditional use of myrrh in treating bacterial infections and its antibacterial and antifungal properties. According to Mohammed et al., 2015 and Khalil., et al., 2020, myrrh extract is promoted as a source of chemicals that can be used to create safer and environmentally friendly antibacterial agents to fight various pathogenic fungi. Al-Sabri et al., 2014 have also conducted research on myrrh's potential as an antibacterial agent.

According to Khalil, et al., 2020, Commiphora myrrh ether and ethanol extracts were tested for their antibacterial properties, these extracts showed antimicrobial activity against the Gram-negative organisms tested alongside Omer., et al., 2011, and Alqahtani., et al. 2021. However, the ether extract demonstrated antimicrobial action both against Candida Albicans and the Gram-positive organisms that were tested, with the antifungal activity being stronger, Omer., et al 2011 and Khalil., et al 2020. In addition to its antimicrobial properties, the potential anticancer effects of Commiphora myrrh have also garnered attention (Suliman et al., 2022). Integrating its anticancer properties into the context of food preservation presents an intriguing avenue for research. Therefore, the primary objective of this study is to explore the antimicrobial and anticancer properties of Myrrh Resin Extract (MRE) and evaluate its application as a natural antimicrobial agent in cacao beverages.

2.1. Materials

Myrrh Resin Extract (MRE), Cacao powder, vegetable cream, salt, corn starch and granulated sugar were purchased from a local market and herbal market in Cairo, Egypt.

2.2. Microbial strains

Gram-positive bacteria B. subtilis ATCC6633, and S. aureus (MRSA) ATCC43300 as well as Gram-negative bacteria P. aeruginosa (ATCC27853), and E. coli (ATCC25922).

2.3. Chemicals and reagents

All chemicals and reagents of the analytical methods used in the present study were analytical grades. Citric acid, sodium benzoate, CMC, xanthan gum, plate count agar (PCA), and potato dextrose agar (PDA) were purchased from El-Gamhouria Trading for Chemicals and Drugs Company, Cairo, Egypt.

2.4. Extraction of antioxidants from MER

One hundred grams of Commiphora Myrrh resin added to excessive distilled water: ethanol (2:8 v/v) and incubated at room temperature for 24 h, then the slurry was filtered through filter paper. The water extract was concentrated using rotary evaporator under reduced pressure and the residues were dissolved in 50 mL of distilled water.

2.5. LC-ESI-MS/MS analysis of Myrrh resin extract profile

2.5.1. Instrument

The analysis of the sample was performed by liquid chromatography–electrospray ionization–tandem mass spectrometry (LC-ESI-MS/MS) as described by El-Ssayad et al., 2023..

2.5.2 Positive ionization mode:

Separation was performed with a Ascentis® Express 90 Å C18 Column (2.1×150 mm, 2.7 µm). The mobile phases comprised of two eluents A: 5 mM ammonium formate pH 3; B: acetonitrile (LC grade) El-Ssayad et al., 2023.

2.6. Determination of antioxidants using HPLC

HPLC analysis was carried out using an Agilent 1260 series.The separation was carried out using Eclipse C18 column (4.6 mm x250 mm i.d., 5 µm).Themobile phase consisted of water(A) and 0.05% trifluoroacetic acid in acetonitrile (B) at a flow rate 0.9 ml/min. The mobile phase was programmed consecutively in a linear gradient as follows: 0 min (82% A); 0–5 min (80% A); 5–8 min (60% A); 8–12 min(60% A);12–15 min(82% A) ; 15–16 min (82% A) and 16–20 (82%A).The multi-wavelength detector was monitored at 280 nm. The injection volume was 5 µl for each of the sample solutions. The column temperature was maintained at 40°C.

2.7. Minimal inhibitory concentration (MIC) determination

In accordance with the disk diffusion assay protocol, a volume of 1 mL from each bacterial suspension was evenly distributed onto a solid growth medium within a Petri dish. Six sterile paper disks measuring 6 mm in diameter were carefully positioned on the surface of each agar plate. These disks were then saturated with 15 µL of the diluted MER. The plates were incubated at 37°C for 24 hr. The antibacterial activity, measured as the minimum inhibitory concentration (MIC), was assessed by determining the lowest concentration of either diluted MER that resulted in the formation of an inhibition zone around a disk after a 24hr incubation period. Negative controls consisted of disks impregnated with sterile distilled water. Replicates were conducted at each concentration (Lima-Filho and de Aguira Corderio, 2014).

2.8. Preparation of Cacao beverage

In preparation for cacao beverage, all materials utilized in this study underwent a sieving process using an 80-mesh sieve. A homogeneous mixture was prepared by combining white sugar (15%), cocoa powder (7.5%), and non-dairy creamer (6%) with 1.47% of corn starch, 0.03% of salt, and 0.1% of xanthan gum. The mixture was stirred continuously until it achieved homogeneity. 1% of Myrrh extract was incorporated into the formula-2 and 0.1% of Sodium benzoate was incorporated into the formula-3. A quantity of tape water was added and the heated at 80 ± 2°C for 5 minutes (Faiqoh et al., 2021), the formulations of cocoa beverages are presented in Table 1.

Table 1

formulations of cocoa beverages
Cacao beverage
%	formula-1	formula-2	formula-3
Cacao powder	7.5%	7.5%	7.5%
Sugar	15%	15%	15%
Vegetable cream	6%	6%	6%
Salt	0.03%	0.03%	0.03%
Corn starch	1.5%	1.5%	1.5%
Xanthan gum	0.1%	0.1%	0.1%
Myrrh extract	-	1%	-
Sodium benzoate	-	-	0.1%
Water	69.87%	68.87%	69.77%

2.9. Physicochemical analysis (pH value, total soluble solids, and total titratable acidity)

The pH was monitored using a digital pH meter (model 3505-JENWAY-UK) calibrated with buffers at pH 4.0 and 7.0. Total soluble solids (TSS) were determined in the filtered samples using an MA871 digital refractometer (Milwaukee 0 to 85% Brix – Romania) at 25^°C. The equipment was calibrated with distilled water before samples were measured. The sample was poured onto a prism of the refractometer and T.S.S was directly measured. According to the method described by the AOAC (2012). Total titratable acidity (TTA) was determined in 10mL of each mango juice and cacao beverage, which was homogenized with 90 ml of distilled water, and titrated with 0.1 mol/L NaOH, using phenolphthalein as an indicator (AOAC, 2012). The results expressed as %lactic (Molecular weight = 90) for cacao beverage and % Citric (Molecular weight = 64). The titratable acidity was calculated using the following equation,

$$\% acidity= \frac{N\times V1\times Eq. wt.}{V2\times 1000}\times 100$$

Where, N = normality of NaOH (mEq/mL); V₁ = volume of titrant (mL); Eq. wt. = equivalent weight of acid (; V₂ = volume of sample (mL); 1,000 = factor relating mg to grams (mg/g) (1/10 = 100/1000).

2.10. Microbial assay

The total microbial count was determined using the method described by Herrera in 2001. A 5g sample of mango juice or cacao beverage was obtained and subsequently diluted with 90 ml of water peptone solution at a ratio of 1:10. Following the homogenization process, 1 mL of the sample was combined with 10 mL of total plate count agar medium in a sterilized Petri plate. This mixture was then thoroughly mixed at a temperature of 45°C. In addition, the samples were incubated at a temperature of 37°C

to observe and quantify the number of colonies that developed on each Petri dish. The recorded observations were documented as the number of colonies detected within a 1 mL sample, expressed as log cfu/mL. The enumeration of yeasts and molds were conducted as the experimental procedures for the total microbial count were identical, with the only variation being the substitution of the total plate count agar with a potato dextrose agar medium (Herrera, 2001).

2.11. Cytotoxicity assay:

Cell viability was assessed using the SRB (Sulforhodamine B) assay. Myrrh resin extract (MRE) was prepared as an ethanolic extract dissolved in DMSO to create a final 25 mM stock solution. Proper media were utilized to prepare MRE at their final tested doses. The DMSO vehicle control was prepared by adding the maximum volume of DMSO used in preparing the tested MRE to appropriate media types, ensuring that the final DMSO concentration did not exceed 0.2%. Colon cancer (HCT) and liver cancer (HEPG2) cell lines obtained from Nawah Scientific Inc., (Mokatam, Cairo, Egypt) were utilized in the assays. Cells were maintained in DMEM media supplemented with 100 mg/mL of streptomycin, 100 units/mL of penicillin, and 10% heat-inactivated fetal bovine serum in a humidified, 5% (v/v) CO₂ atmosphere at 37°C. For the SRB assay, aliquots of 100 µL cell suspension (5x10^3 cells) were seeded into 96-well plates and incubated in complete media for 24 hours. Subsequently, cells were treated with another aliquot of 100 µL media containing MRE extract at various concentrations. After exposure, cells were fixed by replacing the media with 150 µL of 10% TCA and incubated at 4°C for 1 hour. The TCA solution was then removed, and the cells were washed 5 times with distilled water. Aliquots of 70 µL SRB solution (0.4% w/v) were added and incubated in a dark place at room temperature for 10 minutes. Plates were washed 3 times with 1% acetic acid and allowed to air-dry overnight. Then, 150 µL of TRIS (10 mM) was added to dissolve protein-bound SRB stain, and the absorbance was measured at 540 nm using an Infinite F50 microplate reader (TECAN, Switzerland) (Skehan et al., 1990). The IC50 value for each tested sample was calculated by nonlinear regression of log concentration versus the percentage survival, implemented in Graph Pad PRISM version 8.0, GraphPad Software, Inc., CA

2.12. Sensory evaluation

The sensory analysis was carried out using a composite score scale according to Lawless and Heymann (1993). Ten panelists semi-trained (The age of participants was between 25–35 years old) from Food Science and Technology Department, Faculty of Agriculture, Al-Azhar University, Egypt, assessed the sensory quality of mango juice and cacao beverage. Three different treatments were scored for taste (30), odor (30), color (20), texture (20), acceptability (score 100).

2.13. Statistical analysis:

The studies were reproduced at least three times, and the values are reported as the mean ± standard deviation (SD). A variance analysis (ANOVA) was performed to investigate the differences among the samples.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of Commiphora Myrrha extract (MRE) revealed the presence of more than 20 positive and negative substances including Phlorizin, Choline, alpha-D-Glucose-1,6-diphosphate, Sinapoyl malate, and N, N-Dimethylglycine. These findings are presented in Figures 1, 2, and Table 2. Additionally, 106 major components were identified in myrrh essential oil, with other components detected at concentrations ranging from 0.01% to 1.88% (Arın et al., 2021).

The antimicrobial properties of Myrrh resin extract (MRE) are attributed to a group of antioxidant compounds, as determined by high-performance liquid chromatography (HPLC). The analysis revealed 19 active antioxidant compounds in MRE, with kaempferol being the most abundant at 1896 µg/g, followed by quercetin at 520 µg/g. Notably, Pyrocatechol exhibited the highest value among the compounds analyzed, at 162 µg/g. The remaining compounds, including Gallic acid, Chlorogenic acid, Catechin, Coffeic acid, Syringic acid, Rutin, Coumaric acid, Vanillin, Ferulic acid, Naringenin, Cinnamic acid, Apigenin, and Hesperetin, showed varying concentrations as presented in Table 3 and Figure 3.

Further studies utilizing HPLC to evaluate the polyphenol constituents of various myrrh resin extracts (ethanol, ethyl acetate, petroleum ether, and chloroform) demonstrated their efficacy against respiratory infections such as COVID-19. These extracts exhibited a range of biological activities, including anti-inflammatory, antioxidant, antimicrobial, neuroprotective, anti-diabetic, and anticancer properties. Studies by Abbas et al. (2020), Brasha et al., (2023), Rahmani et al., (2022), Fatani et al., (2016), and Rasha et al., (2023) identified significant polyphenolic compounds such as Chlorogenic acid, Gallic acid, Catechin, Caffeine, Syringic acid, Coumaric acid, Ferulic acid, Naringenin, 4',7-Dihydroxyisoflavone, Propyl Gallate, Vanillin, Quercetin, and Ellagic acid in various concentrations, underscoring the potential therapeutic applications of myrrh extracts.

Table [2]: Active components of Commiphora Myrrha extract have been analyzed using LC-MS/MS

Chemical structure	Area	Compounds
C₁₈H₃₀O₂	4.63×10⁸	gamma-Linolenic acid N,N-Dimethylglycine Choline Carnosine Uridine 5'-monophosphate Phlorizin Sinapoylmalate alpha-D-Glucose-1,6-diphosphate
C₄H₉NO₂	5.9×10⁸
C₅H₁₄NO	6.86×10⁸
C₉H₁₄N₄O₃	5.48×10⁸
C₉H₁₃N₂O₉P	1.24×10⁹
C₂₁H₂₄O₁₀	1.75×10⁸
C₁₅H₁₆O₉	8.37×10⁸
C₆H₁₄O₁₂P₂	8.8×10⁸

Table 3: Antioxidants compounds of Myrrh resin extract analyzed by HPLC.

Bioactive compound	Area	Conc. (µg/ml)
Gallic acid	17.11	1.48
Chlorogenic acid	6.12	0.84
Catechin	5.83	1.44
Methyl gallate	0.00	0.00
Caffeic acid	7.94	0.61
Syringic acid	15.04	1.02
Pyro catechol	19.24	2.77
Rutin	5.14	0.60
Ellagic acid	0.00	0.00
Coumaric acid	31.97	1.01
Vanillin	17.25	0.76
Ferulic acid	34.54	2.36
Naringenin	10.12	1.22
Daidzein	0.00	0.00
Querectin	64.25	8.85
Cinnamic acid	7.22	0.13
Apigenin	22.41	1.71
Kaempferol	415.80	32.24
Hesperetin	0.00	0.00

Table 4 Antibacterial activities and MIC of MRE against Gram-positive and negative bacteria

Organisms			Inhibitory activity against the tested organism (zone of inhibition in mm)
			Concentration
	100%	50%		25%	12.5%	6.25%	3.13%	1.56%
Gram-positive bacteria
**Staphylococcusaureus(MRSA) - ATCC43300**	10	8.5		7.5	7	NA	NA	NA
*Bacillussubtilis* ATCC6633	10	8		6.5	6	NA	NA	NA
Gram-negative bacteria
*Escherichiacoli) *ATCC25922**(	NA	NA		NA	NA	NA	NA	NA
*Pseudomonas aeruginosa* (ATCC27853)	20	14		11.5	8.5	NA	NA	NA

Table 4 illustrates the antimicrobial activity of MRE at different concentrations against Gram-positive (Staphylococcus aureus (MRSA) - ATCC43300; Bacillus subtilis - ATCC6633) and Gram- negative bacteria (Escherichia coli - ATCC25922; Pseudomonas aeruginosa - ATCC27853)). The MRE had potent antimicrobial activity against Gram-positive bacteria and copmpelety inhibited Staphylococcus aureus (MRSA) (ATCC43300) ATCC43300- Bacillus subtilisATCC6633 at concentrations of 6.25, 3.13, and 1.56% and gradually inhibited the same strains from 10-7, 10-6 mm for the tested microorganisms respetivly.Concerning the antibacterial activity of MER against Gram-negative bacteria, the results were better and more promising, and the MER completely inhibited Escherichia coli) ATCC25922( at all tested concentrations, while gradually inhibiting Pseudomonas aeruginosa (ATCC27853) from 20–8.5 mm and completely inhibiting at 6.25, 3.13, and 1.56%.

Using the viable count approach, the bactericidal effects of myrrh hexane extract and myrrh essential oil (MEO) against Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (Ps. aeruginosa) were studied. After a two-hour contact period, (MEO) demonstrated superior efficacy, killing both tested strains in > 99.999% of the cases. Four multidrug resistant isolates of S. aureus (MRSA, sputum), Escherichia coli (E. coli, urine), Ps. aeruginosa (wound), and Klebsiella pneumonia (K. pneumonia, sputum) were examined using the same method as MEO. Highest bactericidal activity was observed against Ps. aeruginosa while lowest was against K. pneumonia (99.59 and 54.04% killing, respectively. MEO has antibacterial properties against various bacterial strains and antifungal properties against Candida albicans Auda ., et al. 2017. The most effective efficacy was against S. aureus, which displayed 100% total growth suppression. While myrrh extract exhibited no effect against any of the pathogens under study at concentrations (12, 6, and 3 mg/mL), it completely inhibited the gram-negative bacteria P. auroginosa and E. coli at concentrations of 80 to 60 mg/mL. The result indicates that myrrh is an antibacterial agent that can be used in the future by making appropriate doses. The antibacterial activity of the C. myrrh extract was superior to that of the fungal isolates Alharbi, and Alhashmi., 2022. Myrrh's in-vitro effectiveness in combating bacteria and some airborne fungus. Twenty airborne fungi and ten harmful bacteria were examined in-vitro using extracts of myrrh in methanol, ethanol, hot water, and normal. Compared to extracts in boiling and plain water, methanol and ethanol demonstrated stronger activity against fungus Al-Sabri AE; Moslem M 2016.

Table 5 Application of MER as antibacterial activity on total count of bacteria by Log cfu/mL in Cacao Beverage

Storage at 4 °C	Control	MER (1%)	Sodium benzoate (0.1%)
Total bacteria count by Log cfu/Ml
Zero	1.3±0.13	Nil	Nil
7	2.2±0.08	1.5±0.1	1.1±0.06
14	2.9±0.3	1.7±0.05	1.1±0.09
Mold and yeast count by Log cfu/mL
Zero	Nil	Nil	Nil
7	Nil	Nil	Nil
14	Nil	Nil	Nil

The MRE was applied at concentration 1% in cacao beverage as antibacterial agent against total bacterial count and the produced beverage was stored for 14 days., results in Table 5 cleared the efficacy of MRE as an antibacterial agent since it could inhibit the microorganisms in the produced cacao beverage at zero time as typical of sodium benzoate, although the control cacao beverage had 1.3 cfu/mL, and the total bacterial count slightly appeared after 7 and 14 days to reach 1.5 and 1.7, cfu/mL, while the control and sodium benzoate samples had 2.2, 2.9, and 1.1, respectively. The results demonstrate the efficiency of MRE against microorganisms contaminated cacao beverage. Also, MRE was tested in cacao beverage against mold and yeast counts and gave strong efficiency against these counts, and all tested beverage samples were free from mold and yeast at zero time and during storage.The effect of Commiphora myrrh (ethanol, ethyl acetate, petroleum ether, and chloroform) resin extract against four different pathogenic bacteria, Salmonella typhimurium, Pseudomonas aeruginosa, Escherichia coli, and Bacillus cereus, was examined by measuring inhibition zone (diameter mm). The results revealed that there were significant differences between the bacteria and different extraction methods. Aqueous, ethyl acetate, and petroleum ether extracts of the Commiphora myrrh seed have excellent activity against the Candida albicans fungus. The purpose of the study was to determine the nutritional value, polyphenol content, and antibacterial and antifungal properties of Commiphora myrrh. (Abbas et al., 2020). The best antibacterial activity was seen in a hydroalcohol extract that was extracted using an ethanol: phosphate buffer pH 7 (85:15) ratio, Almekhlafi S et al 2014. The highest antibacterial and antifungal activity was found in the ethanol extract. Fresh-cut salads treated with these two myrrh extracts showed noticeably less bacterial growth than untreated salads Luisa Boffa et al, 2023. Escherichia coli was shown to have a zone of inhibition caused by C. myrrha extract at 29 mm, Staphylococcus epidermidis at 27 mm, Candida albicans at 27 mm, and Aspergillus brasiliensis at 16 mm (Alshibly et al .,2022).

In this study, we investigated the anti-cancer properties of Commiphora myrrh extract against colon cancer (HCT) and liver cancer (HEPG2) cell lines. Our findings indicate that the extract has promising cytotoxic effects, Figure 4 illustrates the IC50 values of the extract against HCT and HEPG2 cell lines, indicating significant cytotoxic effects with values of 55.69 μg/ml for HCT cells and 70.78 μg/ml for HEPG2 cells. These IC50 values are noteworthy as they are close to the recommended threshold for significance set by the American Cancer Institute, which is IC50 < 30 µg/mL for crude extracts. This suggests that Commiphora myrrh extract may have potential as a cytotoxic agent against these cancer cell types. Our results are consistent with previous research. Chen et al. (2013) investigated the effects of myrrh extract on liver cancer and found that furano-sesquiterpenoids isolated from Arabic Commiphora Myrrh resin induced apoptosis of human hepatocellular carcinoma HepG2 cells with an IC50 of 3.6 μM. Furthermore, Hamad et al. (2017) reported that myrrh, in combination with protocatechuic acid, induces apoptotic cell death in colon cancer cells by suppressing the Bcl-2 gene. This suggests a potential mechanism by which Commiphora myrrh extract exerts its anti-cancer effects on colon cancer cells. Our phytochemical and antioxidant analyses revealed significant levels of bioactive compounds in the extract. Among them, kaempferol, quercetin, and ferulic acid stood out for their well-documented anti-cancer properties. Kaempferol, a flavonoid, has been extensively studied for its ability to induce apoptosis, inhibit cancer cell proliferation, and suppress tumor growth through various molecular mechanisms. Quercetin, another flavonoid, exhibits anti-proliferative effects by interfering with cellular processes involved in cancer development and possesses antioxidant and anti-inflammatory properties. Ferulic acid, a polyphenolic compound, exerts anti-cancer effects by inhibiting cancer cell proliferation, inducing apoptosis, and suppressing inflammation and angiogenesis. The presence of these dominant compounds in the Commiphora myrrh extract supports its potential as an anti-cancer agent against colon and liver cancer cell lines.

Table 6: total soluble solids, pH value and acidity of produced treated cacao beverage.

Test	Control	Myrrhextract	Sodium benzoate
T.S.S	10.60±0.9^a	10.60±0.9^b	10.60±0.8^a
pH value	6.80±1.6^a	6.80±0.8^b	6.70±0.8^a
Acidity (as lactic acid)	0.10%	0.10%	0.13%
The means within the same row having different superscripts are significantly varied (P ≤0.05).

The total soluble solids (TSS), pH value, and acidity of the produced untreated and treated cacao beverage with MRE and sodium benzoate were carried out as shown in Table 6, and the results show that the TSS of all tasted samples had the same value of 10.60, there was no difference between untreated or treated samples, and the addition of MRE or sodium benzoate hadn’t any effect. The same results were found for the pH value, which recorded almost 6.8 for all tested samples, as well as the acidity, which recorded almost 0.10% for untreated and treated samples, and all tested values were almost similar to control samples. The pH range of the cocoa drinks was 6.44 to 7.10. The highest pH was 7.10 Rossi Indiarto et al., 2022. pH value of the sample was between 7.61-7.72, while total soluble solids of the chocolate beverages varied between 10.38-10.75 oBrix Salwa Al Aribah et al., 2020.

Table 7: The effect of the addition of extract on the sensory properties of cacao beverages

Test	Control	Myrrh extract	Sodium benzoate
Taste (30)	29.0±0.9a	26.0±0.9b	28.0±0.8a
Odor (30)	27.9±1.6a	25.0±0.8b	28.4±0.8a
Color (20)	19.0±0.9a	18.0±1.9a	18.6±1.3a
Texture (20)	19.2±0.8a	18.6±0.8a	18.8±0.8a
Acceptability (score 100)	95.8±2.6a	88.4±1.1b	94.0±2.7a
The means within the same row having different superscripts are significantly varied (P ≤0.05).

The effect of the addition of extract on the sensory properties of cacao beverages was measured as shown in Table 7 Results cleared that the MRE had an insignificant effect on taste, odor, color, and texture of the produced cacao beverage in comparison with the control sample, and the score of taste decreased from 29 to 26 and 28 for the control and treated samples, respectively. The same trend has been shown with odor, color, and texture, which have had a slight decrease from 27, 19, and 19 of control to 18, 18, and 18, respectively. In general, the general acceptability of treated beverages was very acceptable for judgments, which recorded 95, 88, and 94 for control and treated samples, respectively.

The results of this study highlight the potential of Myrrh resin extract (MRE) as a natural antimicrobial agent for food preservation and as a potential anti-cancer agent. The study analyzed MRE's antioxidant properties using HPLC and LC-MS/MS and identified two potent antioxidant compounds, kaempferol and quercetin. These compounds not only contribute to MRE's effectiveness in inhibiting microbial growth but also exhibit anti-cancer properties, as they have a cytotoxic effect against colon cancer (HCT) and liver cancer (HEPG2) cell lines. The study also investigated the incorporation of MRE into cacao beverages and found no significant changes in their physical or chemical properties, as indicated by stable measurements of total soluble solids (TSS), pH value, and acidity. Furthermore, sensory evaluation revealed that treated samples did not have a significant impact on taste, odor, color, and texture and received very acceptable ratings comparable to untreated samples. Overall, the study suggests that Myrrh resin extract has multiple benefits, making it a promising natural alternative to synthetic food preservatives and a potential adjunct therapy in cancer prevention and treatment. With its demonstrated effectiveness in preserving food quality and safety, minimal sensory impact, and anti-cancer properties, it could be applied more broadly in food and beverage products, as well as in oncological interventions.

Informed Consent Statement

Every participant on the sensory panel has given written consent to participate in the research study. Before the study commenced, participants were notified of the purpose and methods of the research and assured of the strict confidentiality of their data. Participation in the study was voluntary, and subjects had the autonomy to withdraw their engagement at any time. The individuals demonstrated exceptional physical health and had no recorded allergies to the components. The research lacks institutional approval due to Egypt's rules regarding human research. The research protocol is in accordance with the ethical standards of our institution, Al-Azhar University, Faculty of Agriculture, and National Research Center, Egypt, and approval is not needed according to Egypt's rules regarding sensory evaluation.

Data Availability Statement

All relevant information has been included in the manuscript itself

Acknowledgements

We thank the Jiangsu Province Science and Technology Support Program (BZ2022001) China, Nanjing Microtest Biotechnology Co., Ltd, Nnjing, 210061, Jiangsu, China, and the National Research Centre, Cairo, Egypt.

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No competing interests reported.

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Evaluation of the Antimicrobial and Anticancer Properties of Myrrh Resin Extract and Its Application in Cacao Beverages

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Version 1

Abstract

Figures

Introduction

Material and methods

2.1. Materials

2.2. Microbial strains

2.3. Chemicals and reagents

2.4. Extraction of antioxidants from MER

2.5. LC-ESI-MS/MS analysis of Myrrh resin extract profile

2.5.1. Instrument

2.5.2 Positive ionization mode:

2.6. Determination of antioxidants using HPLC

2.7. Minimal inhibitory concentration (MIC) determination

2.8. Preparation of Cacao beverage

2.9. Physicochemical analysis (pH value, total soluble solids, and total titratable acidity)

2.10. Microbial assay

2.11. Cytotoxicity assay:

2.12. Sensory evaluation

2.13. Statistical analysis:

Results and Discussions

Conclusion

Declarations

References

Additional Declarations

Status:

Version 1