¹Faculty of Sciences, Biology Department, King Abdel Aziz University, Saudi Arabia.

²Department of Botany and Microbiology, Kafrelsheikh University, Kafr El Sheikh, Egypt.

Corresponding author email: mmmohammad@Kau.edu.sa

Article Publishing History

INTRODUCTION

The efficacy  of the current antibiotics is limited or not effective and pharmacological industries are in constant  need of  new products to control the harmful effects of  dangerous microbes.  New active antibiotics from medicinal plants are necessary and urgently needed to cure variety of diseases and contribute in improvement of human health care system. For a long period, medicinal plants are consumed as natural medicine and plants have been  considered as the best sources of antibacterial, antifungal, antitumor and antioxidant drugs. Also phyto-materials  can be  safely used for stimulation of the immune system which degrades  the effects of dangerous pathogens, (Carew and Patterson 1970; Jaleel et al. 2007; Aly et al. 2013; El Sayed and Aly 2014; Chassagne et al. 2021).

According to World Health Organization, herbal plants are used for thousands of years safely as traditional treatments compared to synthetic materials which had unsafe effects on human health. Almost all medical plants had therapeutic importance and human health care due to the presence of many active alkaloids in different plants (Santos et al. 1995; Mordmuang et al. 2019; Wink 2020; Chassagne et al. 2021).

Counts of the therapeutic products based plants were enhanced every year because they are easily used, common, wild available, had low prices and suitable to the poor people as antibacterial agents. Many plants extracts recorded excellent antibacterial activities against the multidrug resistant bacteria which cause many dangerous diseases compared to commercial antibacterial agents (Ba-Hamdan et al. 2013; NLM 2020; Chassagne et al. 2021).

Asteraceae is a big family of the flowering plant that had more than 1000 genera and about 30000 species, were described in Africa, Mediterranean area and Asia. Most of them are annuals, perennials, shrubs, or small trees. Genus Echinops L, was classified in the sub-class Asteridae, order Asterales and family Asteraceae which had about 130 plant species, found mainly in Southern of Europe and Asia in addition to Central and North of Africa. Plants of the genus Echinops characterized an erect perennial herb or shrub with long stem which grow up to 1.2 m with massive root, elliptical segmented leaves and white or bright blue corolla. Out of 120 species of the genus Echinops, E. kebericho Mesfin, E. buhaitensis Mesfin, E. ellenbeckii O.

Hoffm, E. kebericho and E. longisetus A. Rich were recorded in few localities in Ethiopia. Eighty two species were recorded in few localities in Europe and 15 species were found in Iraq while in Saudi Arabia, ten species were found. In Saudi Arabia, Echinops L. is represented by 10 species: E. abuzinadianus Chaudhary, E. erinaceus Kit-Tan, E. glaberrimus DC, E. hystrichoides Kit-Tan, E. macrochaetus Fresen, E. mandavillei Kit-Tan, E. polyceras Boiss, E. sheilae Kit-Tan, E. viscosus DC., and E. yemenicus Kit-Tan. Of these species, E. abuzinadianus, E. mandavillei, and E. sheilae are endemic to Saudi Arabia (Singh and Kumar 2001; Shukla 3003; Sharma and Borah 2012; Poulakou et al. 2018; Thielmann et al. 2019; Chassagne et al. 2021).

Species of the genus Echinops are useful for treating migraine, many heart and respiratory diseases, hemorrhoid, diabetes, and microbial infections of the urinary tracts in addition to dangerous worm’s removal. Moreover, the effect of this plant extracts on pathogenic bacteria was poorly studied whereas E. giganteus root extract showed inhibitory activities against S. saprophyticus and E. coli while the extract of Echinops ritro was the most potent extract against three species of the genus Colletotrichum which is a famous fungal pathogen of human. Also, three isolated compounds, 5’-(3-Buten-1-ynyl)-2,2’-bithiophen, R-terthienyl and 2-[pent-1,3-diynyl]-5-[4-hydroxybut- 1-ynyl]thiophene were active against Colletotrichum sp., Fusarium oxysporum, Phomopsis viticola and P. obscurans (Desta 1993;  Fokialakis et al. 2006, Parekh et al. 2007; Deyno et al. 2021).

The antimicrobial activities may due to the presence of 83 different compounds like  sesquiterpenoids and monoterpenoids  which were isolated from E. grijsii, E. ritro, E. kebericho and E. ellenbeckii, monoterpenoids limonene and camphor which were isolated from the volatile oils of E. ellenbeckii, E. kebericho  and sesquiterpenoid and sesquiterpenoid compounds which were observed in the oil extract of E. kebericho (Hymete et al. 1991; Maurya et al. 2015; Ivana, 2015; Ameya et al. 2016, Deyno et al. 2021).

Previous studies on the Echinops genus revealed the presence of diterpenoids, thiophenes, flavonoids, lignans, steroids, sesquiterpenes, and polyacetylenes but the studies on E. macrochaetus are limited (Tene et al. 2004; Hymete  et al. 2005b; Yadava and Singh 2006; Abdallah et al. 2013; Ibrahim et al. 2016; Zaynab et al. 2018; Zhao et al. 2019; Wu et al. 2020; Chassagne et al. 2021).

Cancer remains one of the most serious illnesses in the world and finding anticancer treatments is still a priority of studies worldwide. In cancer treatment, chemotherapy is the major option but had many limitations while natural products may be used as anticancer agents with minimal side effects and without ethical approval. About 50-60% of cancer patients in USA use plants secondary metabolites in chemotherapy like curcumin, genistein, polyphenols, resveratrol, sulforaphane, isothiocyanates, silymarin, diallyl sulfide lycopene, rosmarinic acid, apigenin from parsley, and gingerol (Nobiliet al. 2009; Ahmed et al. 2013; Wang et al. 2014; Aro et al. 2019; Belcher et al. 2020; Chassagne et al. 2021).

For cancer treatment, two sesquiterpene glycosides I and II from the aerial parts of Echinops macrochetus extract were studied against MCF-7, HepG2, and HCT-116 tumor cells using sulforhodamine B assay method and the two Compounds showed potent cytotoxic activities towards all tested cell lines with ICs₅₀ of 2.1, 2.9, and 3.6 µM for product I and 1.9, 3.3, and 2.3 µM, for product II, respectively. Also, the dichloromethane extract of E. macrochetus was inhibitor for acetyl cholin esterasey. Similarly, Echinops giganteus extract showed antitumor activities and can be used to treat cancer cells (Kuete et al. 2013; Saeed et al. 2015; Zamzami et al. 2019; Chassagne et al. 2021). This study  has been aimed to evaluate in vitro  antibacterial  and antitumor activities of some   plants,  locally collected for evaluation of  their synergistic effects along with an antibiotic in  urinary tract bacterial inhibition.

MATERIAL AND METHODS

The tested pathogenic bacteria were Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Klebsiella pneumoniae (clinical isolate), Proteus mirabilis (clinical isolate), Staphylococcus aureus (ATCC 29213) and Enterococcus faecalis (clinical isolate). All bacteria were obtained from King Abdulaziz University Hospital, Jeddah, Saudi Arabia and used as test organisms. The bacteria were grown and preserved according to standard guidelines (CDC 2019; Chassagne et al. 2021).

The aerial parts (stalk and leaves) of Echinops macrochaetus, Oymbopogon citrates and Xanthium spinosum were collected in May 2020 from various areas of Taif city, Saudi Arabia. The plants were identified by a taxonomist at the Department of Biology, Faculty of Science, KAU, Saudi Arabia, in addition to their morphological features were examined, voucher samples are recorded (Collenette 1999).  In sterile plastic bags, the collected plants were preserved at 4◦C until extracted with hot water, methanol, n-Butanol, Ethyl acetate and Diethyl ether (10 g/100 ml, w/v) for 10 hrs. The solvent was collected, dried and re-dissolved in 2 ml DMSO to detect the antibacterial activities of the obtained extract using Agar well diffusion method (Holder and Boyce 1994; El Sayed and Aly 2014).

Ciprofloxacin (Sigma-Aldrich, St. Louis, Missouri, USA) was used as control antibiotic and the minimal inhibitory concentrations (MICs) of the tested plant extracts or the control antibiotic were recorded for some bacterial pathogens in 96 well ELISA trays, using a colorimetric fluorescein diacetate method (Chand et al. 1994, Aly and Gumgumjee 2011). Fractional inhibitory concentration (FIC) was calculated using the formula found below (Petersen et al. 2006; Chassagne et al. 2021).

FIC index = MIC of extract in combination/MIC of extract alone + MIC of antibiotics in combination/MIC of antibiotics alone.

The combination defined synergy if ∑FIC ≤ 0.5, additively if 0.5 < ∑FIC ≤ 1, indifference if 1 < ∑FIC ≤ 4 and antagonism as ∑FIC > 4

Using brine shrimp lethality test, the cytotoxicity of the tested plant extracts in DMSO was determined at varying concentrations using brine shrimp larvae as test organism. After 8 hrs, the average of survived larvae at each concentration was determined and LD₅₀ was calculated (Meyer et al. 1982; Aly and Gumgumji 2011). The antitumor activity of the three plant extracts against MCF-7 (breast cancer) and Hep G2 (hepatocellular carcinoma), tumor cell lines was studied using In vitro MTT and Neutral Red assays. Under sterile conditions and in 96-well plates, the MTT Test on two cell lines was evaluated (Betancur-Galvis et al. 1999).

Cells (6×10³ cells/well) in 100 µl of the culture medium were grown for 24 hrs and treated with different concentrations of the tested plant extracts for 72 hrs. Then, cells were collected and treated with MTT solution (10 µl) and the viability of the cell was calculated by the absorption at A₅₅₀ nm after dissolving the formazan crystals, culture medium plus MTT was used as control. Also, in 96-well plates, Neutral Red Uptake Assay, described by Betancur-Galvis et al. (1999), was used to detect in vitro cytotoxicity of three plants extracts on the two selected cell lines (Hep G2 and MCF-7).

Cells were grown in 0.1 ml/well of the culture medium with different concentrations of the tested extract for 48 hrs, and then the collected cells were suspended in culture medium (0.1 ml containing 5×10^-5g/ml neutral red) and after incubation at 37◦C for three hrs, the collected cells were washed, re-suspended in a mixture of acetic acid/ethanol/ water (1:49:50 v/v/v) and A_{540 nm} was recorded. The plant extract concentration reduced the cell viability by 50% is recorded as IC₅₀ for the tested extract (Chassagne et al. 2021). Three replicates for each experiment were applied, the mean values, standard deviations and analysis of variance using ANOVA were recorded to detect any significant difference at P ≤p.5.

RESULTS AND DISCUSSION

Every minute, the number of the resistant microbes to almost used antibiotic in hospitals was increased causing dangerous problems to the hospitalized patients (Halwany et al. 2015; Zaman et al. 2015; Shriram et al. 2018; El Sayed et al. 2019).  Many vascular plant extracts showed inhibition activity against some bacterial pathogens and the most studied families were Asteraceae, Lamiaceae and Fabaceae in addition to the genera Cinnamomum, Rosmarinus and Thymus while South Africa represented the most sources of the studied plants and methanol was the most used solvents but leaves were the most studied plant part (Chassagne et al. 2021).

Echinops macrochaetus (Camel thistle) and Xanthium spinosum (Spiny cocklebur) were belonging to Asteraceae family while Lemongrass (Oymbopogon citrates) was from Gramineae (Table 1). The aerial parts of the three collected plants were extracted with hot water or organic solvents to detect their antibacterial activities using E. coli as tested bacterial pathogen. The methanolic extract of Echinops macrochaetus showed the highest inhibition against E. coil (inhibition zones diameter about 20 mm while the lowest activity was 11 mm for the aqueous extract (Table 2). Also, the methanol extract of Oymbopogon and Xanthium had lower antibacterial activities compared to Echinops macrochaetus methanolic extract. Additionally, diethyl ether, ethyl acetate and n-butanol extracts exhibited lower antibacterial activities compared to their methanolic extracts. Therefore, the methanolic extract of the three tested plants were selected to determine their antibacterial activities against the six tested bacteria (Table 3).

The highest antibacterial activity was recorded for the methanolic extract of Echinops against the two Gram positive S. aureus and E. faecalis (mean inhibition zone 24 mm) while moderate activities were recorded against E. coli, K. pneumoniae, P. aeuroginosa and P. mirabilis, with inhibition zone diameter ranged from 20-22 mm. The extracts of Oymbopogon and Xanthium recorded the same inhibitory activity against K. pneumoniae and P. mirabilis with inhibition zone of 14 mm.  The antibacterial index was a maximum for Echinops methanolic extract (21.8 mm), followed by Xanthium extract then finally the Oymbopogon extract which considered the less active extract. The susceptibilities of the tested bacteria to differed plant extracts were different according to their cell wall structure, morphology and presence of resistance genes.

Ciprofloxacin was used as a control antibiotic, it showed excellent activities against all the tested bacteria but the activities were more potent against Gram negative bacteria, with inhibition zone diameter ranged from 30-31 mm while lower activities were against S. aureus and E. faecalis with inhibition zone diameter ranged from 20-24 mm. Xanthium methanolic extract was more active against the Gram negative E. coli (16 mm) and showed moderate actives against K. pneumoniae, P. mirabilis and P. aeuroginosa (14 mm). The effect of Oymbopogon extract was moderate on Gram negative bacteria, especially E. coli, P. aeuroginosam, P. mirabilis and K. pneumoniae while showed weak activity against the two Gram positive bacteria, S. aureus and E. faecalis (Chassagne et al. 2021).

Urinary tract infections were caused mainly by Gram- negative bacteria, E. coli, Klebsiella, Proteus, Enterobacter, Pseudomonas and Serratia in addition to some low appearance Gram-positive isolates. Inhibition of these pathogens is difficult in some cases due to low effectively, limitation and resistant to the present antibiotics (El Sayed and Aly, 2014). Thus, looking for new plant extracts, safe and potent for bacterial inhibition without any harmful on the host cells describe the needed characters for the therapeutic agent. Thus, screening of local less studied plants is important and Echinops  macrochaetus is one of these less studied local plants in Saudi Arabia (Chassagne et al. 2021).

Table 1. The Scientific names of the collected traditional plants, families and the  used  parts.

Used part	Family	Scientific name	Common name
Aerial parts	Asteraceae	Echinops  macrochaetus	Camel thistle
Aerial parts	Gramineae	Oymbopogon citrates	Lemon grass
Aerial parts	Asteraceae	Xanthium spinosum	Spiny cocklebur

Table 2.  Diameters of the inhibition zone of the aqueous and organic extracts
of the tested plants using E. coil as test organism.  

Type of the extract					Used plant
n-Butanol	Ethyl acetate	Diethyl ether	Methanol extract	Aqueous extract  (HW)
14 ±1.01a*	16 ±0.19d*	17 ±1.16d*	20 ±1.41cd*	11 ±0.04	Echinops  macrochaetus
10±1.02ab*	11 ±0.06a*	11±0.09a*	12 ± 0.20a*	08 ±0.70	Oymbopogon citrates
11 ±1.10ab*	11 ±0.05a*	10 ±0.08a*	15 ±1.00d*	08 ±0.10	Xanthium spinosum

*: significant results compared to aqueous extract at p ≤ 0.05, HW: hot water

Table 3. The diameter of the inhibition zones (mm) of the methanolic extract of the tested plants
using  different pathogenic bacteria and compared to the antibiotic, Ciprofloxacin (positive control).

Diameter o the inhibition zone (mm)				Pathogenic  bacteria
Ciprofloxacin  (control, 5µg/ml)	Xanthium	Oymbopogon	Echinops
31 ± 2.00	16 ±1.01*	13 ± 0.11*	20 ±1.44*	E.  coli
30 ± 1.05	14±1.51*	14 ±1.05*	22 ±0.51*	K. pneumoniae
30 ± 0.09	14 ±0.41*	14 ±0.09*	20 ±1.14*	P.  mirabilis
31 ±0.29	14 ±0.45*	12±0.06*	21 ±1.06*	P. aeuroginosa
20 ±0.25	13± 0.44*	11±0.08*	24 ±1.04	E. faecalis
24±1.33	13 ±1.45*	11 ±0.09*	24 ±1.90	S. aureus
27.7	13.8	12.5	21.8	Antibacterial  index+

⁺Activity index was calculated as the mean value of net zones of inhibition (mm) against the pathogenic bacteria,
* Significant difference compared to Ciprofloxacin at p ≤0.05

The genus Echinops L. (Asteraceae) was represented in Saudi Arabia with 10 species and was poorly understood. Moreover, Echinops macrochetus was recorded in Saudi Arabia but studies concerning its morphological characters, chemical competitions and biological activities were very rare and lacking. Morphological variations were affected by geographical area and environmental conditions and are very useful for taxonomically separating and distinguishing species which need sufficient experience. Root, leaves, inflorsence, corella, ovary and fruit morphological characters and diameters were applied to distinguish between the different species (Hymete et al. 2005a; Al-Joboury et al. 2021).

In Saudi Arabia folkloric medicine, different plants with therapeutic activities were traditionally applied for treatment of many bacterial pathogens and some important diseases. This study focused on collection and studying genus Echinops from local sites and identifies this plant according to plant identification keys. This plant was traditionally  used as a fumigant against typhus fever, treatment of migraine, reduction of humans stomach ache, and intestinal diseases and it had strong nematicidal and molluscicidal activities, LD50 = 0.057 mg/ml (Abate  and Ayehu 1993;  Hymete et al. 2005; Chirayath et al. 2019; Viswanathan et al. 2019; Chassagne et al. 2021).

Aqueous and organic solvents were used in this study to select the strongest plant extract and our results proved that methanol extract is the most effective solvent for the active plant ingredients. Similarly, lemongrass, lantana and olive leaves methanol extracts had good inhibitory activity against some bacterial isolates (Aly et al. 2013). Extraction method, used plant part, type of the plant, the used solvents and the tested microorganisms, all are factors affected the antimicrobial activities. Growth inhibition of Enterococcus, Escherichia and Klebsiella by some plant extracts owing to the presence of some active metabolites like flavonoid, volatile essential oils; alkaloids; lectins, polypeptides and phenolics compounds were reported before (Chassagne et al. 2021).

The antitumor activity of the three methanolic extracts was determined using two different techniques (MTT and Neutral Red assays) and two cell lines (Hep G2 and MCF-7). Echinops macrochaetus extract showed excellent activity against MCF-7 cells and moderate activity against Hep G2 while no activity was recorded for the extracts of Xanthium and Oymbopogon. No toxicity was found for all the tested extracts except the extract of Xanthium which showed moderate toxicity, LD₅₀= 50 µg/ml (Table 4). A recent study carried by Zamzami et al. (2019) discovered two new rare sesquiterpene glycosides, macrochaetosides A and B; originating from the aerial parts of E. macrochaetus. Their structures were discovered using different spectroscopic data while the active phytochemical agents in E. ritro were thiophenes, quinoline alkaloids, flavonoids, and sesquiterpenes, as well as fatty acids and alkanes (Bitewand Hymete 2019, Sabo and Knezevic 2019; Chassagne et al. 2021).

The minimal inhibitory concentrations were calculated using Microdilution method using fluorescein diacetate method. Moreover, the minimal inhibitory concentrations (MICs) of the methanolic extract of Echinops, Ciprofloxacin and their combination were determined and FIC index was calculated (Table 5). The obtained MICs of the tested methanol extracts were higher than the MIC of the tested antibiotics which was in the range of 0.12-0.14 µg/ml while the MICs of the extract of Echinops  ranged from 0.5-0.75 µg/ml. The calculated MICs for the mixture of both ciprofloxacin and Echinops extract were in the range of 0.09-0.12 µg/ml. The inhibition activity of the combination (Fractional inhibitory concentration index, FIC) was calculated for each tested bacterial pathogen. They were ranged from 0.89-1.15. The interaction between plant extract and the tested antibiotics was either synergistic, additive where (0.5 < ∑FIC ≤ 1) or indifference (1 < ∑FIC ≤ 4) (Chassagne et al. 2021).

Acute and fatal infections of the urinary tract in patients with complicated cystitis are caused mainly by K. pneumoniae, P. mirabilis, P. aeruginosa, S. saprophyticus and E. coli which were strongly attached to the human bladder epithelium. Unfortunately, they are the most frequent causative agents of many dangerous diseases if they are untreated or not properly treated. E. coli (70-90%) followed by Klebsiella, Proteus and Pseudomonas identified as the most important urinary bacterial pathogens while S. saprophyticus, E. faecalis and S. agalactiae  were less isolated Gram positive bacteria. Unfortunately, these bacterial infections were generally resistant to more than two classes of already used antibiotics, thus new search for novel agents from plants which playing an important role in improving human health is recommended by many authors   (Aly and Gumgumgi 2011; Aly et al. 2013; MNPS 2020; Chassagne et al. 2021).

Furthermore, positive interaction was recorded between plant extract and antibiotic against some bacteria was recorded before. Inhibitory activity of both Ampicillin and garlic extract was excellent against S. aureus. Studying and  purification of the active plant compounds was carried out and their mode of action is one or more of the suggested protocols, essential inhibition of some important enzyme, degradation of cell wall and cell membranes, prevention of DNA and protein synthesis (Aly et al.  2013; Ali et al.  2015; MNPS 2020; Al-Joboury et al. 2021).

Table 4. Antitumor activity  (LD₅₀, µg/ml) of the methanolic extracts of the three tested  methanolic plant extracts  against hepatocellular carcinoma ( Hep G2) and breast cancer (MCF-7)  and their toxicity ( LD₅₀,  µg/ml) against Artemia  salina.

* Significant result at p ≤ 0.05 compared to control (untreated cells), ND: Not  detected , #: control antitumor agent

Table 5. Minimal inhibitory concentrations (µg/ml) of Echinops methanolic extract,
Ciprofloxacin and their combination using Fluorescein diacetate protocol.

Tested bacteria	Minimal inhibitory concentration ( µg/ml)
	Echinops	Ciproflox-acin	Echinops+ Ciprofloxacin	FIC index	Effect
E.  coli	0.50± 0.12	0.12 ±  0.1	0.09 ± 0.0	0.89± 0.2	Additive*
K. pneumoniae	0.50± 0.12	0.12 ±  0.1	0. 09 ±0.0	0.93± 0.5	Additive
P.mirabilis	0.50 ± 0.12	0.14 ±  0.1	0.10 ±0.0	0.91± 0.1	Additive
P. aeuroginosa	0.50 ± 0.25	0.14 ±  0.1	0.10 ±0.0	0.91± 0.0	Additive
E. faecalis	0.75± 0.9	0.14 ±  0.1	0.12 ±0.0	1.15± 0.4	Indepen-dent
S. aureus	0.62± 0.12	0.17 ±  0.1	0.12± 0.0	0.94± 0.2	Additive
*Additive :∑FIC less than 1, Independent :∑FIC more than 1

Previous chemical investigation of Echinops species indicated the presence many active components including flavonoids compounds which were investigated in these plants (Al-Joboury, 2021). Six flavonoids and phenolic acid components varied in their containment were isolated by high pressure liquid chromatography from the three genera E. armatus, E. nitens and E. viscosus and all these secondary metabolites played a big role in the different recorded biological activities (Algabr et al. 2005, Cvetanovic et al. 2019, Dettweiler et al. 2020; Al-Joboury et al. 2021).

The chemical constituents of the species E. kebericho confirmed the presence of sesquiterpene lactones, β-sitosterol, stigmasterol, campesterol, β-amyrene,  acetylenic thiophene lupeol and ursolic acid while tricyclic sesquiterpenes, monoterpenoids, sesquiterpenoid and sesquiterpene lactones were reported from E. grijsii, E. ritro, E. ellenbeckii and E. giganteus. Also, simple quinoline alkaloids, echinorine, 7-hydroxy-echinozolinone and echinazolinone were identified from over 14 members of the genus which act as either antibacterial or antitumor agents (Tadesse and Abegaz 1990; Afify and Hymete 1997; Weyersthal et al. 1998; Al-Joboury et al. 2021).

CONCLUSION

The results of this study confirmed the importance of some local plant extracts as a promising treatment for multidrug resistant bacteria which destroy almost used antibiotics. These plants can be used safely by local people to control some bacteria that cause many diseases. Furthermore, the dried powdered of Echinops macrochaetus, extracted with methanol was very effective against more than five different bacterial pathogens and had antitumor activities against two cancer cell lines with no toxicity. Thus, it may be used to develop new antibacterial or antitumor drugs, mainly against resistant bacteria pathogens. Combination of the previous plant extract with some antibiotics enhanced the antibacterial activity.

Conflict of Interests: Authors declare no conflict of interests to disclose.

Funding: This research did not receive any specific grant from funding agencies in the public.

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