1. INTRODUCTION:

In the last two decades, the biological properties of natural products, including antimicrobial and antioxidant properties, have received much attention. This was due to the increasing global interest in human health and the emergence of multi-drug resistant microbes. Infectious diseases caused by multi-drug resistant bacteria are a major problem to public health^1,2. This problem is still developing and there are still some uncertainties regarding the future use of antimicrobial drugs³ On the other hand, the natural antioxidants that are present in natural products can prevent the oxidative deterioration of foods especially those with high contents of lipids⁴.

Natural products are any products that have natural origins. It can be (1) a whole organism or part of it; (2) an extract of an organism or its parts; (3) pure compounds isolated from an organism⁵. Despite this, the term “natural products” is mostly used to describe the secondary metabolites produced by any living organism⁵. Secondary metabolites include many chemical compounds produced by the cell through metabolic pathways originated from the primary metabolic pathways⁶. Secondary metabolites are nonessential for the survival, growth, development, or reproduction of the organism. However, they often play an essential role in the defense against herbivores and other interspecies defense mechanisms^5,7.

In recent years, the biological interest of bioactive secondary metabolites in plants, particularly phenolic compounds, becomes very popular. These compounds possess a broad spectrum of biological properties, such as antimicrobial, antioxidant, anticancer, anti-inflammatory, and cardiovascular protection activities, making them extremely beneficial to human health and supporting their use in pharmaceutical, food, and cosmetic industries⁸.

For thousands of years, medicinal plants have been used to treat human illnesses as they contain bioactive secondary metabolites of great therapeutic value^9,10. Many plant products possess antifungal, antibacterial, antiprotozoal, and antioxidant activities that could be used either systemically or locally^11–13. The majority of modern medications are developed originally from herbal traditions¹⁴. Dar and co-workers (2017) reported that about one-half of all medicines in use till 2017 consisted of natural compounds or their derivatives. Moreover, natural products have also provided the molecular template or intellectual stimulus for the production of about half of all synthetically produced drugs^15,16.

Several studies have shown that the biological properties of the botanical extracts are attributable to their content of phenolic compounds^17-19. The dietary phenolic compounds can protect fatty acids in foods from oxidative deterioration caused by exposure to heat and light/oxygen^20,21. Moreover, phenolic compounds have also been demonstrated to inhibit the oxidative damage resulting from reactive oxygen species, the major cause of numerous physio-pathological conditions in humans, including Parkinson's, Alzheimer's, muscular dystrophy and cardiovascular diseases. Thus, the use of phenolic compounds is considered as a successful way to prevent food quality deterioration as well as to protect human body from degenerative diseases that results from oxidative damage²¹.

Besides the antioxidant properties, many phenolic compounds can act as antimicrobial agents, which has led to increased interest in these compounds due to their potential efficacy against multi-drug resistant bacteria^21,22. Although the antibacterial mechanisms of these phenolic compounds are not completely understood, it is known that they have a variety of targets at the cellular level. Several authors attributed the antibacterial activity to the ability to cause changes in the permeability of cell membranes, alteration of many cellular functions through binding with the enzymes, or by affecting the cell wall stiffness with loss of integrity due to different interactions with the cell membrane²².

The current study aimed to evaluate the antibacterial and antioxidant properties of four methanolic plant extracts (MPEs) obtained from four important medicinal plants (Sarcopoterium spinosum, Paronychia argentea, Achillea fragrantissima, and Inula viscosa) commonly used by Jordanian people as a treatment for various diseases and infections. Besides, the probable correlation between the total phenolic content of the MPEs and their biological activities has been also estimated.

2. MATERIALS AND METHODS:

2.1. Plant materials:

The fresh samples of four medicinal plants were collected from their natural environment at Wadi Al-Karak region which is located in Al-Karak governorate/Jordan. The botanical data of those plants are demonstrated in Table (1). The plants were identified and classified by Prof. Saleh Al-Quran who is a plant taxonomist in the Department of Biology, Faculty of Sciences, Mu’tah University, Jordan.

Table 1. The botanical information about the four plants examined in this study.

Scientific name	Family	Common name in Jordan	Parts used	Yield of MPE (%)
Sarcopoterium spinosum	Rosaceae	Billan	Fruit	12.5%
Inula viscosa	Asteraceae	Taion	Aerial parts	16%
Achillea fragrantissima	Asteraceae	Gesoom	Aerial parts	21.5%
Paronychia argentea	Caryophyllaceae	Rejl El-Hamamah	Aerial parts	12.6%

2.2. Preparation of crude MPEs:

The fresh samples were washed with tap water to clean any dirt before drying. The samples were dried at room temperature in the shade for ten days then crushed into a fine powder using a blender. The extraction process was done by mixing 25g of the plant powder with 250mL of 80% methanol. The mixture was kept at room temperature on a rotary shaker for 36 h. Then, the MPEs were filtered using 0.45µm filter paper, centrifuged at 5000rpm for 15 min, and dried using rotary evaporator under reduced pressure. Each MPE was dissolved in 10% dimethylsulphoxide (DMSO) in methanol (v/v) at the concentration of 200mg/mL and stored at 4ºC in airtight glass bottle^23,24.

2.3. Test bacteria and growth media:

Three Gram-positive (Micrococcus luteus ATCC 10240, Bacillus subtilis ATCC 6633, and Staphylococcus aureus ATCC 43300) and two Gram-negative (Escherichia coli ATCC 25922 and Klebsiella pneumoniae ATCC 43816) bacterial species were used for the investigations. These bacterial species were obtained from the laboratory of Microbiology research at Mutah University, Mutah, Jordan. The strains were maintained on nutrient agar (Sigma-Aldrich, Switzerland) slants at 4ºC and sub-cultured on nutrient agar plates before use. Muller-Hinton agar was used for the antibacterial activity screening test and Muller-Hinton broth for MIC determination.

2.4. Antibacterial properties of MPEs:

Antibacterial properties of the four MPEs were investigated in vitro against the five bacterial species using the disc diffusion assay^25,26. Mueller Hinton agar (Hi-Media, India) was used as the growth medium. The antibacterial action was examined at two different concentrations of MPEs: 1 and 2mg/ disc. The growth medium was prepared, autoclaved, and allowed to cool to 48–50ºC and a standard inoculum (2×10⁸ CFU/mL) was then added under aseptic conditions to the molten agar and poured into sterile Petri dishes to give a solid plate. Then, sterile antimicrobial susceptibility discs (6 mm diameter) were loaded with 10μL of each MPE and placed on the inoculated plates. For each bacterial strain, negative control (10% DMSO in methanol) was included. Nalidixic acid, streptomycin, tetracycline and oxacillin discs were used as positive controls. The cultures were incubated at 37ºC for 24h. The antibacterial activity of each MPE was determined according to the size of the inhibition zone around each disc.

2.5. Determination of the minimal inhibitory concentration (MIC):

The MIC values were determined using the broth dilution method, in accordance with the National Committee for Clinical Laboratory Standardization's standards²⁷. All trials began with a typical initial inoculum (5×10⁵ cell/mL). Sterile glass test tubes were used for all experiments. MPEs were added to the cell suspension to get the concentration range from 2 to 0.062mg/mL. Each experiment comprised negative controls (media, cell suspension, and the solvent, which was always maintained at 1%, without MPE) and blanks (medium containing MPE but without cell suspension). The tubes were incubated at 37°C and 150rpm in an orbital shaker incubator. After 24hours of incubation, the OD₆₀₀ was determined using a spectrophotometer. The MIC was determined as the lowest MPE concentration that inhibits at least 80% of growth when compared to a negative contro^l^7,28^.

2.6. Determination of total phenolic content (TPC):

The TPC for each MPE was estimated according to the Folin-Ciocalteu procedure^29,30. Briefly, 1mL of the MPE was mixed with 5mL of Folin-Ciocalteu’s reagent in a test tube. Then, 4mL of 7.5% Sodium carbonate were added. The contents of the tube were mixed very well and incubated for half an hour at room temperature. Finally, the absorbance for each mixture was measured at 760nm. A standard curve was established using different concentrations of Gallic acid, and used to estimate the TPC in each extract as Gallic acid equivalents (GAE).

2.7. Antioxidant properties of MPEs:

2.7.1. Radical scavenging assay:

The radical scavenging assay was carried out spectrophotometrically according to Tepe and co-workers^11,31. Aliquots (50μL) of different concentrations of the MPEs in the range of 0.1–2mg/mL were mixed with 5mL of 0.004% 2,2-Diphenyl-1-picrylhydrazyl (DPPH) in methanol and incubated in dark at room temperature. After half an hour of incubation, the absorbance was measured against methanol at 517nm. The following equation was used to calculate the inhibition of DPPH radical by the MPEs: Inhibition (%) = (1 - Abs_s/Abs_c) ×100; where Abs_c represents the absorbance of the negative control reaction, and Abs_s represents the absorbance of the tested sample. For each plant extract, the DPPH inhibition curve was constructed, and the concentration of MPE providing 50% inhibition of DPPH (IC₅₀) was calculated. Trolox was used for the construction of a standard curve to calculate the Trolox equivalent antioxidant capacity (TEAC_DPPH) of plant extracts.

2.7.2. Ferric reducing antioxidant power (FRAP) assay:

The FRAP assay has been performed based on the method of Benzie and Strain³². The FRAP assay depends on the reducing activity of antioxidants in which a potential antioxidant can reduce the ferric ions to the ferrous ions, which in turn form a blue-colored ferrous-tripyridyl triazine complex. The FRAP reagent was freshly prepared by mixing 1mL of 10mM 2,4,6-tripyridyl triazine (TPTZ) and 1mL of 20mM ferric chloride in 10mL of 0.25 M acetate buffer (pH 3.6). Aliquots (50μL) of the MPEs were mixed with 3mL of FRAP reagent to get final concentration 100µg/mL. After 8 min of incubation at room temperature, the absorbance was measured at 593nm. Trolox was used for the construction of the calibration curve, and the ferric reducing antioxidant power of the MPEs was determined as Trolox equivalent antioxidant capacity (TEAC_FRAP).

All experiments were performed in triplicates and all data are shown as the average of three analyses± standard deviation (SD).

3. RESULTS:

3.1. Antibacterial properties of MPEs:

The results of the antibacterial properties exhibited that MPEs were more active against Gram-positive (S. aureus, B. subtilis, and M. luteus) than Gram-negative (E. coli and K. pneumoniae) bacteria. The inhibition zones of the bacterial growth for each MPE were shown in Table (2). The negative control disc did not show any inhibition zone in all cases. The highest values of inhibition were seen in the MPE of I. viscosa against S. aureus (inhibition zone: 25±1mm) and B. subtilis (inhibition zone: 22±1mm), as well as for the extract of A. fragrantissima against S. aureus (inhibition zone: 24 ±1mm) using the concentration of 2mg/disc. On the contrary, P. argentea extract showed only weak inhibitory activity against B. subtilis and M. luteus.

3.2. MIC values of MPEs against bacterial growth:

To determine the MIC values of the MPEs against the tested bacterial species, the antibacterial activity was also evaluated using the broth dilution method. According to the results (Table 3), the tested MPEs possessed antibacterial activities in various degrees. The MIC values were between 0.25-2mg/mL for I. viscosa, A. fragrantissima, and S. spinosum. However, the MIC values of P. argentea extract against all of the tested bacterial species were not detectable at concentrations of ≤ 2mg/mL.

Table 3. The MIC values of MPEs on the growth of the examined bacterial species.

Plant Bacteria	MIC (mg/mL)
Plant Bacteria	*S. spinosum*	*A. fragrantissima*	*I. viscosa*	*P. argentea*
S. aureus	0.5	0.25	0.25	ND
E. coli	1	2	1	ND
B. subtilis	1	2	0.25	ND
M. luteus	1	2	0.5	ND
K. pneumoniae	2	2	2	ND

ND: Not detectable at the tested concentrations.

3.3. Total phenolic content (TPC):

The TPC of the MPEs of the four plants was also estimated and demonstrated in Figure (1). It has been found that the extract of S. spinosum possess the highest amount of phenolic compounds (320mg GA/g dry extract), followed by I. viscosa and A. fragrantissima (258 and 112mg GA/g dry extract, respectively). However, P. argentea exhibited the lowest amount of TPC (83mg GA/g dry extract).

3.3. Antioxidant activity:

3.3.1. DPPH radical scavenging capacity of MPEs:

The ability of the four MPEs to scavenge free radicals was determined in vitro based on the DPPH assay and calculated as Trolox equivalents (Table 4). S. spinosum revealed the highest DPPH scavenging capacity (2.5mg Trolox/g dry extract), followed by I. viscosa (1.29mg Trolox/g dry extract). Whereas, A. fragrantissima and P. argentea showed the lowest DPPH scavenging abilities with Trolox equivalents 0.24 and 0.21mg Trolox/g dry extract, respectively. IC₅₀values were also determined for the four MPEs and demonstrated in Figure (2). Similarly, S. spinosum has been found to have the lowest IC₅₀ value (0.3mg/mL) reflecting the highest free radical scavenging capacity, followed by I. viscosa (0.58 mg/mL). On the other hand, the highest IC₅₀ values were obtained from A. fragrantissima and P. argentea extracts (3.06 and 3.42mg/mL, respectively) reflecting the lowest scavenging activities.

Figure 1. Total phenolic contents of MPEs expressed as GAE.

Table 4. DPPH radical scavenging capacity of MPEs.

Plant	TEAC_DPPH (mg Trolox/g dry extract)
S. spinosum	2.5 ± 0.3
I. viscosa	1.29 ± 0.19
A. fragrantissima	0.24 ± 0.02
P. argentea	0.21 ± 0.02

Figure 2. DPPH radical scavenging capacity of MPEs expressed as IC₅₀ values.

3.3.2. Ferric reducing antioxidant power (FRAP) of MPEs:

The FRAP for each MPE was measured and expressed as Trolox equivalents (Table 5). The highest reducing power was obtained from S. spinosum (6.63mg Trolox/g dry extract), followed by I. viscosa (5.08mg Trolox/g dry extract). Whereas A. fragrantissima and P. argentea exhibited the lowest reducing powers (1.28 and 0.65mg Trolox/g dry extract, respectively).

Table 5. Ferric reducing antioxidant power of plant extracts.

Plant	TEAC_FRAP (mg Trolox/g dry extract)
S. spinosum	6.63 ± 0.46
I. viscosa	5.08 ± 0.28
A. fragrantissima	1.28 ± 0.18
P. argentea	0.65 ± 0.09

3.3.3. Correlations of TEAC_DPPH with TEAC_FRAP:

A strong linear correlation was found between the Trolox equivalents obtained from DPPH method (TEAC_DPPH) and Trolox equivalents obtained from FRAP method (TEAC_FRAP) for all plant samples. The Pearson correlation coefficient (R) was 0.965. This linear correlation is shown in Figure (3).

Figure 3. Linear correlation between the Trolox equivalents obtained from DPPH method (TEAC_DPPH) and Trolox equivalents obtained from FRAP method (TEAC_FRAP).

3.3.4. Correlations of TPC and antioxidant activity obtained by DPPH and FRAP methods:

A strong linear correlation was also present between the TPC and the antioxidant activities of the MPEs obtained by both FRAP and DPPH methods (Figure 4). The Pearson correlation coefficient (R) between TPC and FRAP was 0.997 and between TPC and DPPH was 0.966.

Figure 4. Linear correlation between mean values of TPC and mean values of antioxidant activity obtained by DPPH and FRAP methods.

4. DISCUSSION:

In the current study, we evaluated the antibacterial and antioxidant properties of MPEs of four medicinal plants. The results demonstrated that the tested plants had different levels of antimicrobial activity. The extracts of I. viscosa and S. spinosum were the most effective against all of the tested bacterial strains. At relatively low concentrations, it could completely inhibit the growth of Gram-positive bacteria, however, Gram-negative bacteria were inhibited at higher concentrations. Talib and co-workers (2012) reported that the antimicrobial activity of I. viscosa could be attributed to the presence of 3,3′-di-O-methylquercetin and 3-O-methylquercetin12. They suggested that 3,3′-di-O-methylquercetin could induce damage in bacterial cell walls and plasma membranes. Similarly, the extract of S. spinosum contains high amounts of flavonoids such as quercetin and hesperidin33 which possess a high antibacterial activity^34,35. On the other hand, the extracts of A. fragrantissima and P. argentea showed moderate and very low antibacterial activity, respectively. This could be attributable to the low amounts of phenolic compounds that are present in the MPEs of both plants.

Moreover, the results of the current study revealed that the Gram-negative bacteria were more resistant to the tested MPEs than the Gram-positive. This is in line with previous studies which also demonstrated that Gram-negative bacterial species are generally less susceptible to common antibiotics than Gram-positive species36,37. These differences in susceptibility are mostly attributable to the cell wall structure because the lipopolysaccharide layer in the outer membrane of Gram-negative bacteria is known to serve as a strong barrier that decreases the permeability to various substances^37–39.

Plant materials could also contain many components that have antioxidant properties. In this study, the antioxidant activity of the MPEs was measured using DPPH radical scavenging and FRAP methods. The highest antioxidant capacity was seen in S. spinosum, followed by I. viscosa. Whereas it was about 5 to 10 times lower in A. fragrantissima and P. argentea extracts. These differences in the antioxidant capacity are likely attributable to the differences in the TPC in each MPE. It has been reported that polyphenols are most likely responsible for the antioxidant properties of plant extracts^17,18,40. The results of this study showed a strong correlation between the antioxidant capacity and the TPC which is in agreement with the results of Suleria and co-workers (2020), Kiselova and co-workers (2006), and Zheng and Wang (2001) who also reported this strong correlation40–42. This suggests that phenolic compounds are major contributors to the antioxidant activities of these extracts⁴³. Moreover, the trend for ferric ions reducing activities of the four MPEs did not vary from their DPPH free radical scavenging activities. The strong linear correlation between the DPPH radical scavenging capacity and the ferric reducing power (R= 0.965) deserves detailed attention. It means that the same compounds present in the MPE which are able to scavenge DPPH radicals were also capable to reduce ferric ions. This could be due to the fact that antioxidants are reducing agents, however, not all reducing agents are antioxidants. Arnous and co-workers (2002) reported a similar correlation between DPPH radical scavenging capacity and ferric ion reducing power in wines⁴⁴. Moreover, Pulido and co-workers (2000) reported that, in general, the ferric ion-reducing power of antioxidants correlates with the results from other methods used to estimate antioxidant capacity⁴⁵.

In conclusion, the extracts of S. spinosum and I. viscosa are considered as interesting sources for natural antibacterial and antioxidant compounds. Both antibacterial and antioxidant properties of these MPEs could be attributed to their polyphenolic content. These results suggest that promising therapeutic compounds could be isolated and purified from these plant extracts, especially from I. viscosa and S. spinosum.

5. CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

6. ACKNOWLEDGEMENTS:

We would like to thank Dr. Saleh Al-Quran for identifying and classifying the plant samples.

7. ABBREVIATIONS:

DPPH, 2,2-Diphenyl-1-picrylhydrazyl; MIC, Minimal Inhibitory Concentration; DMSO, dimethylsulphoxide; TPC, Total phenolic content; GA, Gallic acid; GAE, Gallic acid equivalents; TEAC, Trolox equivalent antioxidant capacity; FRAP, Ferric reducing antioxidant power; MPE, Methanolic plant extract.

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Received on 31.08.2021 Modified on 20.09.2021

Research J. Pharm. and Tech. 2022; 15(5):2110-2116.

DOI: 10.52711/0974-360X.2022.00350

Plant Bacteria	Inhibition zone (mm)*
	*S. spinosum*			*A. fragrantissima*		*I. viscosa*		*P. argentea*
	1 mg/disc	2 mg/disc	1 mg/disc		2 mg/disc	1 mg/disc	2 mg/disc	1 mg/disc	2 mg/disc
S. aureus	13.67 ± 1.15	21.33± 1.53	18 ± 1.15		24 ± 1	18 ± 0.5	25 ± 1	ND	ND
E. coli	ND	ND	ND		9 ± 0.5	8.5 ± 0.5	11.33 ± 0.58	ND	ND
B. subtilis	8.67 ± 0.58	12.17 ± 0.76	ND		8.5 ± 1.15	16.83 ± 0.76	22 ± 1	ND	7± 0.54
M. luteus	9.83 ± 1.04	14.5 ± 0.5	ND		9.16 ± 0.29	12.67 ± 0.58	18.5 ± 0.5	ND	7± 0.58
K. pneumoniae	ND	9.33 ± 0.58	ND		9 ± 0.5	ND	10.67 ± 0.58	ND	ND