INTRODUCTION:

Microorganisms are the major source of infectious diseases, causing a large number of hospitalizations and deaths each year¹. Numerous antimicrobial formulations are available to treat these infections, but bacterial pathogens are gaining resistance to these antibiotics at an alarming rate and making these infections difficult to treat². Therefore, researchers across the world are now focused on the search for new antimicrobial substances. In the present scenario, one of the promising candidates against drug resistance could be the plants and their compounds.

In Ayurveda and Unani medicine, large numbers of Indian plants are recommended in the treatment of infectious diseases³. According to WHO, medicinal plants are used by about 80% of the World population for treating human diseases⁴.

Several plants employed in the traditional medicine viz., Emblica officinalis (Amla), Syzygium aromaticum (Clove), Piper nigrum (Black pepper), Withania somnifera (Ashwagandha), Zingiber officinale (Ginger), Azadirachta indica (Neem), Mentha piperita (Mint), Momordica charantia (Karela), Carica papaya (Papaya), and Crocus sativus (Saffron).^5,6

On the other hand, the generation of reactive oxygen species (ROS) during normal metabolism resulted in oxidative damage to protein and DNA. Further, ROS are the major cause of cancer and cardiovascular disorders^7,8. Antioxidants can neutralize ROS and their harmful effects⁹. The plants as well as their parts such as fruits viz. jamun (Syzygium cumini), apricot (Prunus armeniaca), raspberries (Rubus ellipticus), and plum (Prunus domestica) were found rich in antioxidants.¹⁰

A substantial number of plants have also been reported to have antibacterial and antioxidant activities.^11-20

Lycium is an important genus of plants with a diverse variety of biological uses. The fruits, flowers, and roots of Lycium species have long been valued as a food or medicine source.^21-22 On the other hand, Tetraena qatarensis also reported being utilized as food or medicine.^23-24 Keeping in view the problem of multidrug resistance and oxidative stress, this study is endeavored to study the antioxidant as well as antibacterial activity of Lycium shawii Roem. and Schult. and Tetraena qatarensis (Hadidi) Beier and Thulin.

MATERIALS AND METHODS:

Sample collection:

The leaves of Lycium shawii Roem. and Schult. and Tetraena qatarensis (Hadidi) Beier and Thulin were collected from Shaqra, Saudi Arabia situated (N 25° 14' 57.6924", E 45° 15' 42.0084") at an elevation of 713m/ 2339feet. The collected plant samples were identified and validated at Shaqra University. The collected samples were subjected to surface cleaning by rinsing the sample with distilled water to remove dust particles. The leaves were sun-dried, ground and dried forms were sealed in plastic bags, followed by storage in a refrigerator until further use.

Bacterial strains:

For antibacterial activity, the clinical isolates of Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, and Salmonella typhi were obtained from the department of Clinical Laboratory Sciences, College of Applied Medical Sciences, Shaqra.

Extracts preparation:

The leaf extracts were prepared by maceration. Briefly, 20g leaf powder is added to 100mL of methanol and chloroform separately in two conical flasks. The flasks containing the above mixture were kept on an orbital shaker at 40⁰C for 48 hours. This procedure was repeated twice and the resulting extracts were filtered and finally concentrated using a hot water bath at 60°C. The dried or concentrated methanol and chloroform extracts were then stored in an eppendorf tube for further use.

Antioxidant analysis:

The antioxidant activity of leaves extract of Lycium shawii and Tetraena qatarensis were evaluated using DPPH assay as described by Naznin and Hasan and Ferric reducing antioxidant power (FRAP) assay, described earlier by Oyaizu.^25-26

In DPPH (2, 2-diphenyl-1-picryl hydrazyl) assay, briefly from stock an aliquot of 2, 4, 6, and 8 ml (20-80 μg/ml) were added in five test tubes followed by the addition of 2mL of DPPH solution (0.004% w/v in 95% ethanol, freshly prepared). The resulting mixture was kept in dark for 25 min. After that, optical density was recorded against blank (distilled water and sample) at 523nm. On the other hand, control was prepared using DPPH solution (2 ml) and ethanol (10 ml). In addition, for standard, ascorbic acid (20-100 µg/ml, w/v) was used.

The DPPH radical scavenging was calculated using the following equation:

(Control – Test)

DPPH Sacavenged = ------------------------- x 100

Control

In FRAP assay, briefly, one mL test samples containing various concentrations (20-80µg/mL w/v) were mixed in sodium phosphate buffer (1mL, 0.2 M, pH 6.6), followed by the addition of potassium ferricyanide (1%, 1mL). Further, reaction mixtures were kept in a water bath (20 minutes at 50°C). After that, tricholoacetic acid (1mL, 10%) was added to each tube, followed by centrifugation (10 minutes, 5000rpm, at room temperature). After this, supernatant (1mL) was mixed with deionized water (1mL) and 0.1% ferric chloride (200µl). The blank was prepared similarly to the samples, but potassium ferricyanide (1%) was replaced with distilled water. The absorbance was recorded using a spectrophotometer (EI, India) at 700nm. An increase in the absorbance obtained after blank subtraction was expressed as the reducing power of the sample.

Antibacterial Activity:

The antibacterial activity of leaf extracts was evaluated using a well diffusion assay.²⁷ Further, minimum inhibitory concentration (MIC) was investigated using the broth microdilution method.²⁸

Inoculums were prepared as per M7-A7-CLSI protocol with some modifications. Briefly, isolated colonies (4-5) of the same morphological type were transferred into a test tube containing nutrient broth medium (5mL), followed by 24 hours incubation at 37°C. 0.5 McFarland standard (1-2 × 10⁸ CFU/mL) was used to compare turbidity of inoculums.

In well diffusion assay, 100µl inoculums (10⁷-10⁸ CFU/mL) were spread on Mueller Hinton agar (MHA) plates. After that, wells were cut out with cork borer and 30µl of different concentrations (1, 5, 10mg/mL) of leaf extracts were added. Ampicillin was used as a standard drug (50µg/mL), while chloroform, methanol were used as negative controls against respective extracts. MHA plates were kept for 24 hours at 37°C and Hi antibiotic zone scale was used for measuring inhibition zone diameter (mm). Additionally, a positive antimicrobial effect was considered when the inhibition zone diameter was ≥ 10mm.

MIC is the lowest concentration that inhibits the growth of tested microbes. MIC of seed and leaf extracts was investigated using the broth microdilution method (CLSI M7- A7). A double strength Mueller-Hinton broth, 2X strength extract solution were prepared and serial dilutions were done between 10-0.019mg/mL up to 9^th well. In 96 well microtitre plate, double strength MHB (100µl) containing different concentration of extracts was mixed with bacterial suspension (10µl, 1-2 × 10⁸ CFU/mL), followed by incubation for 24 hours at 37°C. While, 10^th well-containing broth and inoculums only was used as control. In addition, the 11^th well as positive control with antibiotics and inoculums and 12^th well as negative control (methanol, chloroform, and inoculums). The lowest visual growth inhibitory concentration after 24 hours was the MIC against respective microbes.

Combined effect of leaves extracts of Lycium shawii with standard drug ampicillin:

The combined effect of leaves methanolic extract of Lycium shawii with ampicillin was evaluated using well diffusion method as described earlier in the antibacterial activity section.²⁷ Additionally, 30µl of mixture containing 15µl each (10 mg/mL methanolic extract + 50µg/mL amipicillin) were loaded in the wells. The results were recorded by measuring the diameter of the inhibitory zone in mm after 24 h of incubation at 37⁰C.

The fold increase was calculated using the following formula²⁹: (b-a/a) 100%. Where b is combined inhibition zone diameter, a is the inhibtion zone of leaves extract alone.

Statistical Analysis:

GraphPad Prism was used to evaluate analysis of variance (ANOVA). A p value <0.05 was considered statistically significant.

RESULTS AND DISCUSSION:

Antioxidant activity of Lycium shawii and Tetraena qatarensis leaves:

In DPPH assay, scavenging of DPPH is increased in a dose dependent manner (20-80µg/ml). Further, the IC₅₀ values of the tested extracts were lies at a concentration between 45.63-74.26µg/mL, which were similar to standard ascorbic acid (48.07µg/mL). In contrary, Lycium shawii extracts were found more active (Fig. 1). In the existing literature, different extracts (methanol, ethyl acetate, ethanol, and aqueous) from Lycium shawii leaves were investigated for antioxidant activity using DPPH and ABTS assay. The methanol extract outperformed the others in DPPH and ABTS, with IC₅₀ values of 0.06 and 0.007mg/mL, respectively, which can be attributable to the presence of phenolics and flavonoids.³⁰ Similarly, Tetraena qatarensis exhibited IC₅₀ values 67.9 ± 4.8 μg/mL in DPPH assay.³¹

Fig. 1. DPPH scavenging activity of methanolic and chloroform leaves extracts of (a) Lycium shawii (b) Tetraena qatarensis (c) standard ascorbic acid. When the mean values were statistically different (p<0.05), different letters were used.

In FRAP assay, all the extracts were found active with optical density (O.D.) >0.5 at 40 µg/mL except methanolic extract of Tetraena qatarensis (60 µg/mL). The effect is more pronounced in methanolic extract of Lycium shawii leaves (O.D. 0.5 at 20 µg/mL) (Fig. 2). While, in case of ascorbic acid O.D. >0.5 was observed at 60 µg/mLl.

Fig. 2. FRAP of methanolic and chloroform leaves extracts of (a) Lycium shawii (b) Tetraena qatarensis (c) standard ascorbic acid. When the mean values were statistically different (p<0.05), different letters were used.

Antibacterial activity:

In antibacterial activity, all tested extracts exhibited broad-spectrum effect towards all tested strains evident from inhibition zone diameter ranged between 10±2to 18±0.6mm. Further, the highest zone of inhibition i.e. 18±0.6 mm at 10 mg/mL was reported against E. coli with leaf methanol extract of Lycium shawii (Table 1). The results were compared with the literature. Ali et al. (2020)³⁰ reported that, methanol extract of Lycium shawii was found most detrimental effect against the multi and pan-drug resistant bacterial strains at 50 mg/mL.

Table 1 Antibacterial activity (in vitro) of leaves extracts from Lycium shawii and Tetraena qatarensis

Bacterial strains

IZD (mm) ± SD

LS ME

LS CE

TQ ME

TQ CE

Conc. (mg/mL)

Escherichia coli

14±

0.6^a

16±

0.6^b

18±

0.6^c

12±

3^a

15±

1.5^b

16±2.5^c

14±

1^a

15±1.5^a

16±1^b

12±2^a

13±2^b

15±0.5^c

Salmonella typhi

11±

0.6^a

12±

0.6^a

14±

0.6^b

12±

0.6^a

13±

1.0^a

14±0.6^b

11±

2^a

13±1.5^b

15±0.5^c

10±1^a

12±2^b

13±1.5^c

Pseudomonas aeruginosa

11±

0.6^a

12±

0.6^a

14±

0.6^b

10±

2^a

13±

1.5^b

14±1.5^b

10±

2^a

12±1.5^b

13±1.1^c

11±0.6^a

13±0.6^b

15±0.6^c

Klebsiella pneumoniae

10±

0.6^a

12±

0.6^b

13±

0.6^c

10±

1^a

12±

2^b

13±1.5^b

12±

2^a

14±2^b

15±1.5^c

11±0.6^a

12±0.6^a

14±

1.2^b

IZD: Inhibition zone diameter; SD: Standard deviation; LS: Lycium shawii; TQ: Tetraena qatarensis; ME: Methanol extract; CE: Chloroform extract. When the mean values were statistically different (p<0.05), different letters were used.

Fig. 4. Combined antibacterial effect of methanolic leaves extract of Lycium shawii against A) Pseudomonas aeruginosa; B) Klebsiella pneumoniae; C) Escherichia coli; D) Salmonella typhi. LS: Lycium shawii ; AB: Ampicillin.

Several studies in the literature have shown that plant extracts in combination with regular antibiotics have a beneficial combined impact; the improved activity is attributed to a synergistic interaction between plant extracts and antimicrobial drugs.^33,34

CONCLUSION:

In conclusion, the methanol and chloroform leave extracts from Lycium shawii and Tetraena qatarensis were found active free radical scavenger as compared with standard ascorbic acid. Further, they exhibited broad-spectrum antibacterial activity. Furthermore, the combined action of Lycium shawii leaves and ampicillin was reported to be effective. These findings could indicate that plant extract enhances antibiotics' antibacterial activity. Therefore, the leaves of the studied plants can be used in the development of antibacterial formulations and antioxidant-rich value-added products after detailed follow-up studies.

CONFLICTS OF INTEREST:

No conflict of interest was declared by the authors.

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Received on 06.08.2021 Modified on 03.10.2021

Research J. Pharm.and Tech 2022; 15(4):1543-1548.

DOI: 10.52711/0974-360X.2022.00257

Bacterial strains	Zone Of Inhibition (mm)
Bacterial strains	*Lycium shawii* leaves methanolic extract (10 mg/mL)	Ampicillin (50 µg/mL)	*Lycium shawii* leaves methanolic extract With Ampicillin	Fold Increase (b-a/a) 100%
E. coli	19±1.2	18±1.5	23±1.26	21.05
Salmonella typhi	13±2.38	16±0.63	25±0.6	92.31
Pseudomonas aeruginosa	15±1	17±1	22±1	46.67
Klebsiella pneumoniae	13±0.63	15±0.6	24±0	84.61