Collection of the plant material and microorganisms samples

Fresh specimens of the plant namely A. decumbens (Ventenat) Cosson & Kralik (Kehail, Figure 1) were obtained from Jabal Aja (27°259040N, 41°259390E) in the Hail region, during the period (March to May 2021).

Tested bacteria and yeasts

The bacteria tested included: Methicillin-Resistant Staphylococcus aureus (217), Staphylococcus epidermidis (M13), Enterobacter cloacae (155), Pseudomonas aeruginosa (M16), Escherichia coli ATCC 10536, and Klebsiella pneumoniae (140). We obtained these bacteria previously from King Khalid Hospital, Hail. The microorganisms were maintained on Brain Heart Infusion (B.H.I.) agar medium (HiMedia laboratories, Germany) at 4ºC and re-cultured before using for further analyses. The yeasts tested were Candida albicans ATCC 10231 and Candida tropicalis ATCC 9362. All microorganisms were kindly provided by Prof. MejdiSnoussi (Department of Biology, College of Science, University of Hail).

Preparation of plant extract

 For the experiment, 20 grams of the plant extract were dissolved in 200 mL of methanol-water (80:20, v/v) in a dark glass container and maintained at agitation for 72h. The extract was then filtered and centrifuged at 5000g for 10 min, and the supernatant was collected and dried to obtain the final crude extract. Mother solution (100 mg/mL) was prepared by mixing 500 mg of the obtained crude methanol-80% extract with 5 ml of dimethyl sulfoxide (DMSO)-5%.

Testing of plant extract for antimicrobial activities

The cup-plate agar diffusion methods were adopted with a few minor adjustments to evaluate the produced extract's antibacterial activity, according to Snoussi et al. [24]. A sterile cotton swab was utilized to inoculate Mueller Hinton (Bacteria) and Sabouraud chloramphenicol (Candida species). Petri dishes from a stock suspension containing (10⁸-10⁹CFU/ml). We incubated the plates at 37°C for 24 to 48 hours. Kehail extract was applied to sterile discs in three distinct dosages (1 mg, 2 mg, and 3 mg/disc). Each concentration was tested against the tested species three times. The diameter of the resulting growth inhibition zones was measured after incubation, averaged, and the mean values were computed. As reference molecules, ampicillin and amphotericin B were utilized.

Microdilution assay was used to determine the minimal inhibitory concentrations values that inhibit the growth of the tested bacterial and fungal strains in the 96-well plates [25]. To stop the growth of the examined microorganisms, the least bactericidal and fungicidal concentrations were calculated. by recording the growth on Mueller-Hinton agar (Bacteria) and Sabouraud Chloramphenicol agar (Candida spp.). The tested extract was diluted in DMSO-5%, and serial dilution was prepared in sterile five mL-glass tubes starting from 200 mg/ml to 0.96 mg/mL. Minimal inhibitory concentrations (MICs), Minimal bactericidal concentrations (MBCs), and Minimal fungicidal concentrations (MFCs) values are expressed in mg/mL.

Phytochemical screening

Spectrophotometric assays were employed to access the phytochemical composition of Kehail methanolic extract including phenols, tannins, alkaloids, flavonoids (Alkaline reagent test), terpenoids (Salkowski's test), sterols, cardiac glycosides (Keller-Kiliani's test), saponins, fat, and fixed oils were all examined in the extracts [26-28].

Total Phenolic Content, Total Flavonoid Content, and Total Tannin Content

Determination of total phenolic content (TPC)

The TPC of the Kehail extract was estimated by Kumar et al. [29] method. Folin-Ciocalteu reagent is used. The samples were evaluated at a concentration of 1 mg/mL. 100 L of the extract were put into a test tube, to which 0.75 mL of the Folin-Ciocalteu reagent was added and mixed after being diluted 10 times with deionized water. The mixture was let to stand for 5 minutes at a temperature of 25 °C. The mixture was then gently reacted with 0.75 mL of saturated sodium carbonate solution. A V.-Vis spectrophotometer was used to measure the absorbance at 725 nm following 90 minutes of standing at 25 °C. Using gallic acid, a calibration curve was established. Gallic acid equivalents (G.A.E.) in mg/mg of vegetable extract (mg of gallic acid/mg dry weight) were used to express the overall phenolic content.

Total flavonoid content (TFC)

TFC were determined according to Kumar et al.[29]. A volume of 1.5 mL (1mg/mL) of an identical volume of an extract was added to a 2% AlCl3-6H2O. After vigorously shaking the mixture for 10 minutes, the absorbance at 367 nm was measured. In terms of mg quercetin, the total flavonoid content was stated. /g dry weight (mg Q.E./mg) and determined through a quercetin calibration line.

Total tannin content (T.T.C.)

A modified vanillin assay was used in a colorimetric technique to measure condensed tannins [30]. 50 L of the extract (1 mg/mL) received three ml of a 4% methanolic vanillin solution and 1.5 ml of concentrated H2SO4. After 15 minutes, the mixture was let to stand, and the absorbance at 500 nm was measured with methanol or water used as a reference. The unit of measurement for T.T.C. is mg catechin/g dry weight (mg C.E/mg). All samples were analyzed in three replications.

Determination of antioxidant activity

DPPH assay

DPPH of the extracts and the standard vitamin E were evaluated as reported by the method of Chakraborty and Paulraj [31]. The various extracts (stock solution 20 mg/m and of standard (stock solution 1mg/ml) at different concentrations were pipetted in small tubes. Each sample and standard were combined with an equal volume of DPPH• methanolic solution in a volume of 0.5 mL. The blend was left must stand at a temperature of 25 °C for 30 minutes while it is dark. A spectrophotometer was used to detect the resulting solution's absorbance at 520 nm. As a control, 0.5 mL of DPPH solution and 0.5 mL of methanol were combined. The blank was taken to be pure methanol. The following equation (Eq. 1) calculated the inhibition of DPPH (PI%):

PI (%) = 100 x (A_Control– A_Sample) /A_Control;      (Eq. 1)

Where A_Control and A_Sample are the absorbances of the control solution and a test sample or standard, respectively.

ABTS radical scavenging activity assay

 The 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid), also known as the ABTS cation scavenging activity test, was used to conduct an antiradical assay [31]. By treating ABTS solution (7 mM) with 2.45 mM K2S2O8, the radical monocation of ABTS was produced. The mixture was let to stand at room temperature for 15 hours in the dark. Samples were dissolved in methanol and distilled water.  The varying extract concentrations and the tocopherol (vitamin E). The standard was put to the test, then it was compared to the standard. By mixing 800 mL of diluted ABTS+ with 200 mL of each standard and sample, the antioxidant activity was measured. We measured 30 minutes later, the absorbance measured spectrophotometrically at 734 nm. A triple of each measurement was made. Percent inhibition (%) was used to express the antioxidant capability of the test samples and the reference material. The following equation (Eq. 2) calculated the percentage scavenging of ABTS^.+ radicals:

PI (%) = 100 x (A_Control– A_Sample) /A_Control;                 (Eq. 2)

Where A_Control and A_Sample are the absorbance of the control and the test sample or a standard, respectively.

β-carotene/linoleic acid method

The method used to assess the -carotene bleaching inhibition was previously disclosed by Ikram et al. [32]. After heating the -carotene/linoleic acid combination, a free radical is produced from linoleic acid. 20 mL of linoleic acid were combined with 200 mL of tween-20, and a total of 2 mL of -carotene solution (1.5 mg -carotene/2.5 mL chloroform) was mixed. The chloroform was removed at 40◦C under vacuum using a rotary evaporator. The dry mixture was mixed with 50 mL of distilled water to create an emulsion of -carotene and linoleic acid. 0.800 mL of the emulsion was mixed to 0.200 mL of extracts at various concentrations (stock solution 20 mg/mL) and the standard (stock solution 1 mg/mL) in order to determine each extract's ability to bleach -carotene. The mixtures were incubated in a water bath at 50°C for 120 minutes, and the absorbance at 470 nm was measured both before and after incubation. The following equation (Eq. 3) was used to calculate the antioxidant activity of extracts:

PI% = [1-(A₀-A_t/A_c0-A_ct)]x100;            (Eq. 3)

where A₀ and A_c0refer to the absorbance values measured at zero time, respectively, for the test sample, the standard, and the control. At an Act, the corresponding absorbance values of the test sample or standard and the control are referred to. These values were measured after incubation for 120 min.

Statistical analysis

All tests were done in triplicate. Mean value and standard deviation were calculated by using the SPSS program for windows, version 20 analyzed data (SPSS Inc., Chicago, IL, U.S.A.).

Figures & Tables

Discussion

The antimicrobial activity of the plant extract

To evaluatethe antimicrobial capability of the Kehail (A. decumbnes), methanol extracts of the whole plant were prepared. The antimicrobial activity was tested against six pathogenic bacteria and two pathogenic fungal strains. Methicillin-Resistant S.aureus (217), S. epidermidis (M13), E. cloacae (155), P.aeruginosa (M16), E. coli ATCC 10536 and K. pneumoniae (140) bacteria and the fungi C.albicans ATCC 10231 and C.tropicalisATCC 9362 were used as test strains in the agar well diffusion method. The zone of inhibition was measured to estimate the maximum antimicrobial activity of the plant extract.It has been reported that alcoholic and aqueous extracts of numerous plants and herbs efficiently suppress the growth of potentially harmful food pathogens and rotting microorganisms [35, 36, 8]. There are limited reports of the antibacterial activity of Kehail extract (M.K.E.) against pathogenic bacteria and fungi. As indicated in Table 1, the plant extract showed antibacterial activity against Methicillin-Resistant S. aureus (217), S. epidermidis (M13), E. cloacae (155), P.aeruginosa(M16), E. coli ATCC 10536 and K.pneumoniae (140), whereas the mean inhibition zone was 11.77 ± 0.57 mm, 10.66 ±  0.57 mm, 10.77 ±  0.57 mm, 10.33 ±  0.57 mm, and 9.55 ± 0.57 mm, respectively. It can be well seen from Table 1 that the highly inhibited bacteria by the plant extract was K. pneumoniae (140).

In comparison with other studies, some studies indicated that Arnebia spp. used in traditional medicine have moderate antimicrobial activities when tested individually, as reported by Abdulameer and Almussawi [37] and Koca et al.[38]. In this study, the inhibition of pathogenic bacteria by Arnebia spp. extract is particularly interesting from a medical point of view since the tested bacteria are responsible for severe opportunistic infections. Most crude concentrates display bactericidal action instead of bacteriostatic [39, 40] which essentially features the adequacy of conventional medication.There are a large number of substances that have antimicrobial effects. Still, only a few can be considered therapeutic because mammalian cells are more sensitive to chemical inhibition than microbial cells. It has been previously reported that the extracts from several plants, including PergulariatomentosaL. (Umlebena), Peganumharmala L. (Harmal), Roselle (Hibiscus sabdariffa L.) Senna Italica Mill (Senna), Teucriumpolium (Jaâda), and Carthamustinctorius (Safflower) other spices had significant (p< 0.05) antibacterial and antifungal activities against a variety of pathogenic bacterial and food spoilage bacteria, as well as yeast and mold [8, 41, 42].

Table 1 also indicates that the M.K.E. showed potent inhibition activity against the tested fungi where, the mean inhibition zone of C.albicans ATCC 10231 and C.tropicalis ATCC 9362 was 12.77 ± 0.57 mm and 13.11 ± 1.52 mm, respectively. The table shows that the plant extract's inhibitory effect is greater on fungi than on bacteria, and the higher the concentration of the plant extract, the greater the inhibitory effect. This study supports past discoveries in the literature that antimicrobial activities have an immediate connection to expanding the concentration of the extracts (%) [8, 42].

The outcomes of this study were comp those acquired by Doulah et al. [43] who found that their tested four Arnebia species namely A. fimbriopetala, A. linearifolia, A. garandiflora, and A. tubata could become significant in the acquisition of recognizable sources of mixtures with health defensive potential, cancer prevention agent and antimicrobial activity.

Phytochemical content

The results of the phytochemical examination showed that the samples contained steroids, tannins, terpenoids, saponins, glycosides, and flavonoids. These phytochemicals support the utilization of the Kehail plant in various parts of the Kingdom of Saudi Arabia to prepare traditional medications for treating multiple ailments [3]. These mixtures are biologically active and may add to the antioxidant activities of the Arnebiadecumbnes. This plant's extracts may have antioxidant effects on cells since flavonoids and terpenoids are present. Flavonoids apply antioxidant activity through a rummaging or chelating process [44]. Scavenging reactive oxygen species can neutralize lipid oxidation in vitro, develop the body’s antioxidant enzyme action, and reduce peroxide arrangement in vivo [45]. Terpenoids decrease the oxidation of lipids by providing radicals with hydrogen, acting as vital antioxidants [46].  Phenolic acids are a significant group of essential antioxidants. They are made of at least one aromatic ring bearing at least one hydroxyl group and can extinguish free radicals by framing stabilized phenoxyl radicals [39]. Due to their abilities to scavenge free radicals, provide hydrogen and chelate metals, halt radical chain reactions, and extinguish singlet oxygen both in vitro and in vivo, polyphenols exhibit strong antioxidant properties [47].

Flavonoids are antioxidants with high redox potentials that allow them to function as reducing agents, hydrogen donors, and singlet oxygen quenchers. Ordinarily, flavonoids safeguard plants from infections, oxidative cell damage, herbivores, UV radiation, and fungal parasites. Consistently, flavonoids have been related to a decrease in the occurrence of illnesses like cancer and heart disease [48]. Since the hydroxyl groups in these mixes are what work to scavenge free radicals, the total amount of phenolic acid and flavonoid content might be used to quickly screen for antioxidant activity. Many investigations have revealed that phenolic acids have other biological activities, for example, possessing anti-inflammatory, ulcer-preventing, spasm-relieving, antiviral, anti-diarrheal, and anticancer activities [49]. Hence, measurement and ensuing identification of phenolic acids can give imperative data connected with the antioxidant capabilities and potential medical advantages of the Kehail plant.

The antioxidant assay

The DPPH, ABTS, as well as β-carotene in testing were utilized to test the antioxidant properties of plant extracts from the Kehail (Table 3). DPPH is a stable free radical, which can promptly encounter a decrease in the presence of an antioxidant, so the lower IC50 value reflected the more potent antioxidant activity. It shows a highly bright and noticeable (UV-Vis) absorbance at 517 nm. The decrease in the power of retention at 517 nm of methanol solutions of DPPH radical in the presence is typically taken as a proportion of their antioxidant activity. The samples’ capacity to neutralize the DPPH radical was assessed. based on their concentrations providing 50% inhibition (IC50). Methanol extract of Kehail showed good radical scavenging activity with an average IC50 value of 0.06 + 3.53µg/ml.

The ABTS measure is the most widely recognized strategy to test a compound or plant concentrate's fundamental, radical scavenging activity. The ABTS, produced from the oxidation of ABTS by potassium persulfate, is a magnificent device for deciding the antioxidant activity of hydrogen-donating mechanisms and chain-breaking antioxidants [50]. In this study, the ABTS scavenging activity was expressed as IC50 value to compare the capability of extracting solvents (Table 3). The methanolicKehail extract offered an average value of 0.17 + 0.01mg/L for ABTS IC50.  The measurement of ABTS radical scavenging activity has been frequently used in the food industry to measure the antioxidant capacities of foods [50].

The outcomes acquired propose Kehail plant extracts have phytochemicals that can donate hydrogen to free radicals to scavenge them and prevent predicted harm. Plant extracts' phenolic acids are usually linked to their antioxidant properties. The Kehail plant's high total phenolic acid and total flavonoid concentrations are consistent with its high antioxidant activity as shown by the DPPH, ABTS, and -carotene assays. An important class of vital cell reinforcements is represented by plant phenolic acids. They are able to react by producing active oxygen radicals such hydroxyl, superoxide anion, and lipid peroxyl radicals, which can initially suppress lipid peroxidation. The hydroxyl groups in phenolic acids are what give them their strong rummaging properties. Flavonoid and terpenoid antioxidants, which are phytochemicals of interest, are present; additional isolation, purification, and characterization should be done in the future; any isolated compounds may be used to develop novel medications.

Author Contributions

Conceptualization, A.S., E.A. and N.A.; methodology, E.A., M.S. and M.H.; formal analysis, A.S., M.A.A., and N.A.; writing-original draft preparation, A.S and M.S.; writing–review and editing, E.A., N.A.; project administration, A.S. All authors have read and agreed to the published version of the final manuscript).

Conflict of Interest

The authors declare that there is no conflict of interest.

Acknowledgment

The authors would like to thank the Deanship of Scientific Research of The University of Hail for funding this research (GR 22 0 13).

1. Al-Turki T, Al-Olayan H. Contribution to the flora of Saudi Arabia: Hail region. Saudi Journal of Biological Sciences, (2003); 10:190- 222.
2. Sharawy SM. Two New Records Astragalus species of the section ChroronopusBge and HarpilobusBge. In Suadi arbor. Jordan Journal of Biological Sciences, (2014); 7 (1): 67-70.
3. Al-Shammari A.  Encyclopedia of the Hail Region: The Biological Volume in the Hail Region. Indexing of King Fahd National Library during publication, (2018); ISBN: 978-603-909228-8-9
4. Ashkani-Esfahani S, Imanieh MH, Khoshneviszadeh M, Meshksar A, Noorafshan A, Geramizadeh B, Ebrahimi S, Handjani F, Tanideh N. The healing effect of arnebiaeuchroma in second degree burn wounds in rat as an animal model. Iran Red Crescent Medical Journal, (2012); 14(2):70-4.
5. Smyrska-Wieleba N, Wojtanowski KK, Mroczek T. Comparative HILIC/ESI-QTOF-MS and HPTLC studies of pyrrolizidine alkaloids in flowers of Tussilagofarfara and roots of Arnebiaeuchroma. Phytochemistry Letters (2017); 20:339-49.
6. Yuzbasioglu M, Kuruuzum-Uz A, Guvenalp Z, Simon A, Tóth G, Harput US, Kazaz C, Bilgili B, Duman H, Saracoglu I, Demirezer LO. Cytotoxic compounds from endemic Arnebiapurpurea. Natural Product Communications, (2015); 10:595-6.
7. Yang MH, Blunden G, O’neill M, Lewis J. Tormentic acid and 2α-hydroxyursolic acid from Arnebiaeuchroma. Planta Medica,(1992); 58:227-35.
8. Sulieman, AME, Shaarawy, SM, AlghamdiAA, Veettil VN, AbdelgadirM, Ibrahim NA. Evaluation of antimicrobial and synergistic effects of selected medicinal plants of Hail area with antibiotics. Bioscience Biotechnology Research Communications, (2017a); 10, 44-50.
9. Atala E, Vásquez L, Speisky H, LissiE, López-Alarcón C. Ascorbic acid contribution to ORAC values in berry extracts: an evaluation by the ORAC-pyrogallol red methodology. Food Chemistry (2009); 113(1): 331-335.
10.  Cai Y, Luo Q, Sun M, Corke H. Antioxidant activity and phenolic compounds of 112 traditional Chinese medicinal plants associated with anticancer. Life Science,(2004); 74(17): 2157-2184.
11. Sultanbawa Y. “Plant antimicrobials in food applications: minireview,” in Science Against Microbial Pathogens: Communicating Current Research and Technological Advances, (2011); 1084– 1099.
12. MolyneuxRJ, LeeST, GardnerDR, PanterKE, JamesLF, “Phytochemicals: the good, the bad and the ugly?” Phytochemistry, 2007; 68 (22–24), 2973–2985.
13. Cowan MM. (1999). Plant products as antimicrobial agents. Clinical Microbiology Reviews, (1999); 12(4), 564- 582.
14. Lara MV,Bonghi C,Famiani F,Vizzotto G, Walker RP,Drincovich MF. Stone Fruit as Biofactories of Phytochemicals with Potential Roles in Human Nutrition and Health. Frontiers in Plant Science,(2020); 11, 562252.
15. Liu RH. Potential synergy of phytochemicals in cancer prevention: Mechanism of action. Journal of  Nutrition. (2004); 134, 3479S–3485S.
16. Carrera-Quintanar L,LópezRoa RI, Quintero-Fabián S, Sánchez-Sánchez MA, Vizmanos B,Ortuño-Sahagún D. Phytochemicals that influence gut microbiota as prophylactics and for the treatment of obesity and inflammatory diseases. Inflammatory Medication, 2018; Article ID 9734845.
17. Santhiravel S,Bekhit AEDA,Mendis E, Jacobs JL,DunsheaFR,Rajapakse N, Ponnampalam EN. The Impact of Plant Phytochemicals on the Gut Microbiota of Humans for a Balanced Life. International Journal of Molecular Sciences, (2022); 23, 8124.
18. Talib WH,Alsayed AR,AbuawadA,Daoud S,Mahmod AI. Melatonin in Cancer Treatment: Current Knowledge and Future Opportunities. Molecules,(2021); 26, 2506.
19. Zhu D, Zhang Y, Wang S. Histone citrullination: A new target for tumors. Mol. Cancer, (2021); 20, 90–107.
20. Wu MS, Aquino LBB,Barbaza MYU, Hsieh CL, De Castro-Cruz KA, Yang LL, Tsai PW. Anti-Inflammatory and Anticancer Properties of Bioactive Compounds from Sesamumindicum L.—A Review. Molecules,(2019), 24, 4426.
21. RotaMC, Herrera A, Martínez RM, Sotomayor JA, Jordán, MJ, Food Control, (2008); 19 (7), 681–687.
22. Hong SY,Dudhia J, ChenSH. A Revised Approach to Ice Microphysical Processes for the Bulk Parametrisation of Clouds and Precipitation. Monthly Weather Review – Monthly Weather Review, (2004); 132.103-120.
23. Burt S. Essential oils: Their antibacterial properties and potential applications in foods—A review. International Journal of Food Microbiology, (2004); 94(3), 223-253.
24. SnoussiM,NoumiE,Hajlaoui H,Bouslama L,Hamdi A, Saeed M,Alreshidi M, AdnanM, Al-RashidiA,Aouadi K,Ghannay S,Ceylan O, De Feo V,Kadri A. Phytochemical Profiling of Allium subhirsutum L. Aqueous Extract with Antioxidant, Antimicrobial, Antibiofilm, and Anti-Quorum Sensing Properties: In Vitro and In Silico Studies. Plants,(2022), 11, 495.
25. Ghannay S, Aouadi K, Kadri A, Snoussi M. In Vitro and In Silico Screening of Anti-Vibrio spp., Antibiofilm, Antioxidant and Anti-Quorum Sensing Activities of Cuminumcyminum L. Volatile Oil. Plants (Basel). (2022);11(17):2236.
26. Raaman  N. Phytochemical techniques, (2006); ISBN: 8189422308, 9788189422301New India Publishing.
27. Banso A,Adeyemo S. Phytochemical Screening And Antimicrobial Assessment Of Abutilon Mauritianum, BacopaMonnifera And DaturaStramonium. BiokemistriBIOKEMISTRI, (2006); 18(1):39-44.
28. Nair JJ, Bastida J, van SJ. In vivo cytotoxicity studies of Amaryllidaceae alkaloids. Nat. Prod. Commun. 2016; 11:121–132.
29. Kumar VDR, Jayanthi G, Gurusamy K, Gowri S. Antioxidant and anticancer activities of ethanolic extract of Ficusglomerata Roxb. in DMBA induced rats. International Journal ofPharmaceutical Sciences and Research, (2013); 4: 3087–3093.
30. TamilselviA,AnjugamE,ArchanaR, Madhan B, Sudalayandi K, Chandrasekaran B. Isolation and Characterization of Bacteria from Tannery Effluent Treatment Plant and Their Tolerance to Heavy Metals and Antibiotics. Asian Journal of Experimental Biology 3 (1) 34- 41J. Asian Journal of Experimental Biology, (2012); 3, 34-41.
31. Chakraborty K,Rajamani P. Sesquiterpenoids with free-radical-scavenging properties from marine macroalgaUlva fasciataDelile. Food Chemistry, (2010); 122, 31-41.
32. Ikram EHK, Eng KH, Jalil AMM, Ismail A, Idris S, Azlan A, Nazri HSM, DitonNAM, Mokhtar RAM. Antioxidant capacity and total phenolic content of Malaysian underutilized fruits. Journal of food Composition and Analysis,(2009); 22, 388-393.
33. GatsingD,Tchakoute V,NgamgaD,Kuiate JR,Tamokou JDD,Nji-Nkah BF,Tchouanguep FM,Fodouop SPC. In vitro antibacterial activity of Crinum purpurascens Herb. leaf extract against the Salmonella species causing typhoid fever and its toxicological evaluation. Iranian Journal of Medical Sciences,(2009); 34, 126-136.
34. Umamaheswari M, Chatterjee TK. In Vitro Antioxidant Activities of the Fractions of Coccinia Grandis L. Leaf Extract. African journal of traditional, complementary, and alternative medicines: AJTCAM / African Networks on Ethnomedicines. (2007); 5. 61-73.
35. Sulieman AME. Antimicrobial activity of Roselle (Hibiscus sabdariffa L.) seed oil in Multiple Biological Activities of Unconventional Seed Oils, 1st Edition – (2022); 91-100.
36. Verma S, SalminenJP, Taube F, Malisch CS. Large Inter- and Intraspecies Variability of Polyphenols and Proanthocyanidins in Eight Temperate Forage Species Indicate Potential for Their Exploitation as Nutraceuticals. Journal of Agriculture and Food Chemistry, (2021); 69 (42), 12445–12455.
37. Gonelimali FD, Lin J, Miao W, Xuan J, Charles F, Chen M, Hatab SR. Antimicrobial Properties and Mechanism of Action of Some Plant Extracts Against Food Pathogens and Spoilage Microorganisms. Front Microbiol. (2018); 24(9), 1639.
38. Abdulameer A,Al-Mussawi. Isolation and Identification of ShikoninFromArnebiaDecumbens L. and its Antibacterial Activity. Journal of Applied Sciences Research, (2010); 6(9), 1452-1456.
39. Koca U, Ozcelik B, OzgenS. Comparative in vitro activity of medicinal plants Arnebiadensiflora and Ecballium elaterium against isolated strains of Klebsiella pneumonia. Turkish Journal of Pharmaceutical Sciences, (2010); 7(3), 197-204.
40. Gnanamani K, ShanmugaP, Radhakrishnan N, Mary B. Antibacterial activity of two plant extracts on eight burn pathogens. Journal of Ethnopharmacology, (2003); 86(1), 59-61.
41. Kharea SK, RajniS, AsthaJ, Shikha P, &Deepti G. Antimicrobial activity of some natural dyes. Dyes and Pigments, (2005); 66(2), 99-102.
42. Nassan MA, Mohamed EH, Abdelhafez S, Ismail TA. Effect of clove and cinnamon extracts on experimental model of acute hematogenous pyelonephritis in albino rats: immunopathological and antimicrobial study. International Journal of Immunopathology Pharmacology. (2015); 28 60–68.
43. Liu Q, Meng X, Li Y, Zhao CN, Tang GY, Li HB. Antibacterial and antifungal activities of spices. International Journal of Molecular Sciences(2017); 18:E1283.
44. Doulah AH, NeisiN, Zekavati R,Farjam MH. Antibacterial, antifungal and antioxidant activity of four species from Arnebia genus growing wild in Iran. Iranian Journal of Science and Technology, (2014); 38(A2), 159.
45. Zou Z, Xi W, Hu Y, Nie C, Zhou Z. Antioxidant activity of Citrus fruits. Food Chemistry, 2016; 196(1): 885-896.
46. Fawole OA, Ndhlala AR, Amoo SO, Finnie JF, Van Staden J. Anti-inflammatory and phytochemical properties of twelve medicinal plants used for treating gastro-intestinal ailments in South Africa. Journal of Ethnopharmacology, (2009); 123 (2): 237-243.
47. Lu Y, Khoo TJ, Wiart C. Antioxidant activity determination of citronellal and crude extracts of Cymbopogoncitratus by 3 different methods. Journal of Pharmacy and Pharmacology, (2014); 5 (4): 395-400.
48. Carro RT, D'Almeida RE, Isla MI, Alberto MR. Antioxidant and anti-inflammatory activities of Frankeniatriandra (J. Rémy) extracts. South African Journal of Botany,(2016); (104): 208-214.
49. López-Cobo A, Gómez-Caravaca AM, Švarc-GajicJ, Segura-Carretero A, Fernández-Gutiérrez A. Determination of phenolic compounds and antioxidant activity of a Mediterranean plant: the case of Saturejamontana subsp. kitaibelii. Journal of Functional Foods(2015); (18): 1167-1178.