Saudi Arabia has a diversity of plant species that grow in harsh conditions, which makes it medicinally promising, such as Caralluma species (Osman et al., 2014). Caralluma genus is widely distributed in Saudi Arabia, which belongs to the Asclepiadaceae family, which comprised of 200 genera and 2500 species (Qiu et al., 1997; Sireesha et al., 2018). The Arabic and Indian traditional medicine had widely used Caralluma species for it is the treatment of fever, inflammation, snake and scorpion bites, diabetes, cancer, tuberculosis, skin rashes, scabies (De Leo et al., 2005; Oyama et al., 2007; Abdel-Sattar et al., 2009; Aruna et al., 2009; Tounekti et al., 2019). Several reports stated that the Caralluma genus contains anti-inflammatory, antitumor (Zakaria et al., 2001), cytoprotective antiulcer activities (Zakaria et al., 2002). Other important pharmacological activities had been reported such as the antinociceptive (Abdel-Sattar et al., 2007), the antioxidant, hypolipidemic (Tatiya et al., 2010), and the antidiabetic activities (Dra et al., 2019). Sparingly, pregnane glycosides, saponin, and flavonoids are the major phytochemical constituents isolated from this plant (Bauer et al, 1966). The presence of several important compounds, including pregnane glycosides (Abdel-Sattar et al., 2007), stigmasterol, and other constituents (Bader et al., 2003)in Caralluma species explains the range of biological activities (Dra et al., 2019).

Caralluma deflersiana is one of 14 species distributed in Saudi Arabia (Al-Massarani, 2011; Aati et al., 2019), which has no literature reports concerning the biological activities and phytochemical composition. Thus, the current work was undertaken to investigate the antioxidant and antimicrobial properties of C. deflersiana extract and fractions against a panel of pathogenic microorganisms. The study will be of value in highlighting that Caralluma species in Saudi Arabia are considered as a promising source for several compounds that can be used as antioxidants and antimicrobial agents.

Plant material

The aerial part (stem) of C. deflersiana was collected from the south of KSA in March  2019.  Four  samples  of  the  stems  (100 g  each) were dried at a constant temperature of 60°C for 24 h and filtered using a sterile filter paper. The extract obtained was stored at -20°C until use.

Extraction of C. deflersiana

The four samples of C. deflersiana were extracted by soaking in 500 ml of water, methanol, ethanol, and ethyl acetate with 24 h of agitation. Extracts were filtered with Whatman filter paper No.1, while solvents were evaporated by rotary evaporator at 40°C. Dry matter was collected, and yields were calculated. Dry materials were stored in Eppendorf tubes in no light condition with a temperature of 4°C for further analysis. Dry extracts were dissolved in the same solvents at the time of analysis.

Estimation of total phenol content

The total phenolic content of C. deflersiana was estimated by a modified Folin–Ciocalteu reagent (Al jawfi et al., 2013). Briefly, 1 ml of sample extract (0.1%, w/v), 0.5 ml Folin–Ciocalteu reagent (1:2, v/v), 2 ml of 5% sodium carbonate were mixed and allowed to stand at 30°C for 1 h. Absorbance was measured with a UV-VIS spectrophotometer at a wavelength of 765 nm. The total phenolic content of C. deflersiana extracts was expressed as Gallic acid equivalents per g dry weight (mg GAE/g).

Determination of antioxidant activity

Total antioxidant capacity

Antioxidant capacity of extracts (0.2-1 mg/ml) was estimated utilizing a slightly modified phosphomolybdenum method (Prieto et al., 1999). This method is based on the reduction of Mo (VI) to green Mo (V) in an acidic medium. Concisely, 0.1 ml of each plant extract (100 μg/ml) solution was mixed with 1 ml of reagent (0.6 M H₂SO₄, 28 mM Na₃PO₄, and 4 mM ammonium molybdate). The tubes were placed in a water bath at 95°C for 1.5 h after that, the mixture was cooled, and the absorbance was measured at 695 nm in contrast to a blank that contained 1 ml of reagent and an appropriate volume of solvent. Antioxidant capacity was calculated using an α-tocopherol standard curve and expressed as α-tocopherol equivalents (μg/g of extract).

2,2-diphenyl-1-picrylhydrazyl (DPPH)- radical scavenging assay

DPPH scavenging ability was evaluated as follows; a 1 ml of the DPPH, which as previously prepared in methanol (0.135 mM) combined with 1 ml of different concentrations of C. deflersiana extracts and standards (0.2-1 mg/ml). Mixtures were vortexed thoroughly and left in the dark at 28°C for 30 min (Wintola and Afolayan, 2011). Absorbance was then measured at 517 nm. DPPH scavenging ability was calculated by the following equation:

ABTS radical scavenging assay

A stock solution was prepared by mixing 1 ml of 7 mM ABTS solutions and 2.45 mM K₂S₂O₈ then left in the dark at 28°C for 12 min. A bluish-green  solution  was  produced. The  blend  was  then diluted by adding 1 ml of the ABTS‏ solution with methanol until an absorbance of 0.700 ± 0.01 at 734 nm was reached. 1 ml of extracts, standard (BHT, rutin), and blank (methanol) of different concentrations (0.2-1 mg/ml) were allowed to react with 1 ml of diluted ABTS solution. Absorbance was obtained at 734 nm after 7 min (Asghar et al., 2008). Inhibition percentage was then calculated by the following equation:

Ferric reducing antioxidant power assay (FRAP)

A 1 ml of extract was dissolved in water, 2.5 ml of phosphate buffer (0.2 M, pH 6.6), and 2.5 ml of 1% potassium ferricyanide mixed, then it was placed at 50°C for 30 min. 2.5 ml of trichloroacetic acid (10%) was added, and the mixture was centrifuged for 10 min at 3000 rpm. 2.5 ml from the upper part of the supernatant was diluted with 2.5 ml of water and vortexed with 0.5 ml of 0.1% ferric chloride before measuring absorbance at 700 nm (Vijayalakshmi and Ruckmani, 2016). A reference solution was prepared as above but contained water instead of extract sample. Increased absorbance indicates increased reducing power.

Determination of the antimicrobial activity

Test microorganisms

Two gram-positive bacteria Staphylococcus aureus (ATCC 25923) and Enterococcus. faecalis (ATCC 29212) and two gram-negative bacteria Escherichia coli (ATCC 25922) and Proteus vulgaris (ATCC 8427) and one fungal strain Candida albicans (ATCC 60193) were used in this investigation.

Minimum inhibitory concentrations

The MICs of C. deflersiana extracts were assessed using an improved micro-well dilution method (Mann and Markham, 1998; Sulaiman, 2013). To 96- sterile well plates, duplicate two-fold serial dilutions of each sample (100 µl/well) were made in the required broth media containing 5% (v/v) DMSO to achieve (2000 to 31.2 mg/ml) concentrations. The bacterial or fungal suspension (100 µl, 1106 CFU/ml) was then added. After that, the plates were incubated at 37°C for 24 h and 25°C and 72 h for bacterial and fungal strain, respectively. The MIC of A. Judaica and A. Sieberi methanolic extracts were defined as the lowest concentration displaying no detectable bacterial or fungal growth. Gentamycin and nystatin were used as a positive control. For minimal bactericidal concentration (MBC) and minimal fungicidal concentration (MFC) estimation, 5 µl from  the  wells  that  exhibited no growth was transferred agar plates and further incubated for 24 or 72 h. MBC and MFC are the lowest concentrations that show evidenced of no visible bacterial or fungal growth.

Statistical analysis

All the data were obtained from independent tests in triplicate (n = 3) and presented as means ± standard deviations. All data were analyzed with a one-way analysis of variance, followed by Tukey's test. A p-value of £0.05 was considered to be statistically significant. The analyses were performed using SPSS software (V. 21).

Total phenolic content

The water extract of C. deflersiana exhibited the highest total phenolic content 35 mg of GAE/100 g powder weight followed by the methanolic extract with 33.1 mg/g of the total phenolic content of GAE/ g powder weight. On the other hand, the ethyl acetate extract exhibited the lowest total phenolic content (27.1 mg/g); results are shown in Table 1.

The medicinal and pharmacological actions of medicinal plants are often dependent on the presence of bioactive compounds (the secondary metabolites) (Heinrich et al., 2004; Calixto, 2019). The chemical composition can vary within the same species depending on the geographical location (Jaafari et al., 2007). As  reported,  many  factors such as the climate, the soil, the plant material and the season in which the plants were collected, the method of preservation and extraction, and the genetic factors, could be responsible for the variation of the chemical compositions (Sivropoulou et al., 1997; Bakkali et al., 2008). Phenolic compounds are omnipresent plant metabolites and the largest group of compounds that contribute to the antioxidant properties; they may play a significant role in initiating harmful free radical actions (Wang et al., 2010).

Caralluma genus species are known for their abundance of phenolic compounds (Priya et al., 2012; Maheshu et al., 2014a; Devi and Dhamotharan, 2016). Total phenolic content of C. adscendens var. Fimbriata aerial parts were reported to be 21.0 ± 0.59, 18.8 ± 0.98, 14.9 ± 0.40 and 8.7 ± 0.63 mg GAE/g dry weight in methanol, water, ethyl acetate, and chloroform extracts, respectively (Maheshu et al., 2014a). Another study suggested  that  strong  antioxidant  activity of  Caralluma arabica correlated with phenolic content in different plant parts (Al-Attabi et al., 2015).  Our data demonstrated that C. deflersiana is in agreement with the data mentioned above, it contains polyphenolic compounds that can play a significant role in the initiation of harmful free radical actions (Wang et al., 2010). On the other hand, regarding the antioxidant activity, it was stated that antioxidants had gained more popularity as health boosters in the treatment of many diseases, due to their beneficial effects (Unuofin and Lebelo, 2020). Several antioxidant phytochemicals that occur naturally in plant sources have been identified as free radical scavengers (Bauer et al., 1966). The antioxidant capacities of plant extracts cannot be measured using a single approach because of the dynamic existence of the various phytochemical groups present in plants. The FRAP, ABTS, DPPH, and total antioxidant capacity methods were used in the present work to determine antioxidant activities of C. deflersiana extracts.

The findings of the DPPH and FRAP assays for the four C. deflersiana extracts were demonstrated that water extract had conducted the strongest free radical scavenging behavior, possessing the highest amount of total phenolic moiety. On the other hand, the ethyl acetate extract had exhibited the lowest radical scavenging activity in both methods. Our results were in agreement with the data reported by Karthishwaran et  al. (2018) who demonstrated that Caralluma flava extracts exhibited antioxidant activity as measured by DPPH assay. Another study also supported our results confirmed that Caralluma edulis extract showed strong scavenging activity (Ansari et al., 2005). Moreover, Maheshu et al. (2014a) indicated that C. adscendens var. Fimbriata methanol extracts have significantly higher FRAP activity than the other plant extracts.  Likewise, the results of the ABTS radical scavenging method, as well as the total antioxidant capacity method, assured the earlier results, which showed that the water extract exhibited the strongest antioxidant activity amongst other plant fractions.

The total antioxidant capacity of all C. deflersiana extracts in the present study was higher than previous reports for C. adscendens var. fimbriata extracts (Maheshu et al., 2014a). Concomitantely, Marwah et al. (2007) reported that TAC in ethanol extracts from C. flava and C. quadrangula were 335 ± 0.5 and 899 ± 29.2 mg GAE/g, respectively, which considered lower than the current findings for C. deflersiana extracts.

Consistent with our results, various concentrations of C. flava extract and gallic acid were observed to scavenge ABTS radicals in a dose-dependent manner. The percentage of inhibition was reported to be 86 and 59% for the extract and gallic acid, respectively, at a concentration    of 80  mg/ml   (Pisoschi   et   al.,   2016;  Karthishwaran et al., 2018). The ABTS free radical scavenging activity of C. deflersiana extracts was assumed to be higher than that of C. fimbriata crude extract, as stated by Devi and Dhamotharan (2016).

Concerning the antimicrobial activity of biosynthetically generated chemical compounds that could kill or usefully inhibit the metabolism of pathogenic microbes. These are referred to as antibiotics, which have been extensively studied in recent times in various higher plants. Nonetheless, we learn a bit about the antimicrobial activity of the genus Caralluma (Vajha et al., 2010). In the present study, antimicrobial activity was investigated against four bacterial strains (two grams +ve and two grams -ve). Our findings showed differences in C. deflersian antimicrobial activity. Various solvents were documented to be capable of extracting different phytoconstituents depending on their solubility or polarity in the solvent (Altemimi et al., 2017), which explains the dissimilarities in the antimicrobial activity of the extracts using different solvents (Yusha’u et al., 2008; Kawo et al., 2009; Altemimi et al., 2017).

The present study findings showed that stem extracts of C. deflersiana prepared by different solvents displayed various bioactive phytochemical constituents with high antioxidant activity. Antioxidant activity of extracts correlates with their phenolic content. Interestingly, the water extract contained the highest total polyphenol content and showed the highest overall antioxidant capacity. The high phytochemical constituent’s level, antioxidant and antimicrobial activities of C. deflersiana reflects its potential to be helpful in health maintenance and treatment of various diseases. C. deflersiana may possess a significant ability to counter oxidative stress and infections in humans and other animal systems. Thus, expanding its ethnomedicinal application is highly recommended. Further investigation should be conducted to isolate and identify specific antioxidant and antimicrobial components of C. deflersiana, both qualitatively and quantitatively, and assess the mechanisms of action underlying these activities.

The authors have not declared any conflict of interests.