Introduction

Plant species “Echinops” are traditionally used for the treatment of many ailments affecting the gastrointestinal1, respiratory2, antidiabetic3, neuroprotective3, anticancer3, antimicrobial4, and urinary tract systems2. The genus has approximately 120 endemic species in Africa, Asia, the Middle East, and Europe5,6. The chemical screening revealed the presence of various bioactive constituents such as thiophenes3, alkaloids3, phytosterols, terpenoids3, flavonoids3,4, lignans6, phenolic compounds6, polyacetylenic aliphatic acids4, sesquiterpene lactones4, and volatile oils4,6,7. Numerous species of this genus have been shown to have mostly anti-inflammatory biological action, as noted in multiple papers2.

A previous study showed that the chemotype of essential oils is essential for classifying plant subspecies that exhibit similar morphological characteristics and for enhancing their biological activities. This previous research characterized and classified the chemotype of 45 Echinops kebericho, sourced from various geographical regions of Ethiopia, according to 15 primary chemical constituents of their root essential oil7.

Through previous experimental studies, Echinops L. is known to have immunomodulatory and anticancer effects using both in vitro and in vivo techniques, as examined in Echinops shakrokii S.A. Ahmad against the MCF-7, MDA-MB-231, EMT6/P, HeLa, T47-D, Caco-2, and VERO cell lines, and intraperitoneal injection of EMT6/P cancer cells into Balb/c mice to investigate the anticancer activity of the aqueous methanol extract. Maceration and Soxhlet extraction were used to create solvent extracts from the aerial portions8.

Echinops erinaceus Kit Tan is a rare native herbal plant found in Saudi Arabia. It is also known as Hawa elghool, Alkana’a, or Kanab. This annual plant species grows in isolated locations in the southern Kingdom of Saudi Arabia; meanwhile, it has been restricted to being consumed by the native population6. Little information about its pharmacological uses or phytochemical content had been published, making it unknown to the rest of the world. Their isolates and biological characteristics were the main topics of our earlier publications6.

Potential in-vitro antioxidants, cytotoxic, anti-inflammatory, antibacterial, and in-vivo anti-ulcer effects have been shown by its extracts or its isolated chemicals obtained using the cold maceration approach6,9,10.

In our earlier research, we aimed to conduct a comprehensive bio-guided phytochemical and biological profiling of the subsequent extracts, particularly the CHCl3 extract and its isolates. This study identified fourteen compounds, five of which are novel and have never been isolated from nature before. These include erinaceosin, erinaceol, erinaceolic acid, speranskoside, and erinaceoside, all derived from a plant sample collected in Saudi Arabia in March 2018. By estimating the silico docking analysis, erinaceolic acid and speranskoside showed strong anti-inflammatory efficacy against the COX-2 and 15-LOX enzyme test6,9,10.

Microbial infections are a worldwide global irritable problem. Notably, mouth, throat, ear, eye, and wound infections are important clinical causes of illnesses in all populations11. Fortunately, nowadays, researchers are passionate about discovering novel and alluring plant species to combine their use as antimicrobial agents with boosting immunity to eradicate microorganisms12. Consequently, a novel option for Echinops-genus medicinal plants would be advantageous and noteworthy for treating microbial infections, as Echinops heterophyllus is thought to be an anti-inflammatory and wound healing remedy due to its biocompatibility13. E. erinaceus is therefore regarded as a promising contender for an antibacterial agent. This study was conducted to assess a plant sample that was extracted using a different extraction process as opposed to those previously published between 2021 and 20236,9,10. Therefore, the different samples of this genus or the same plant species that were extracted by different methods may reveal various phytochemical contents with broad variability in their biological activities.

It’s important to note that our previous studies did not report the presence of volatile oils, alkaloids, anthraquinones, or cardiac glycosides in our plant6. Therefore, the purpose of this work was to analyse and determine the chemical composition of non-polar extracts (total lipids, sap., and unsaponifiable matters) obtained from the aerial parts of E. erinaceus Kit Tan in 2017.

The samples were extracted using a distinct hot extraction method. Additionally, the antimicrobial and cytotoxic properties of the fractions and four isolates of E. erinaceus obtained through the hot maceration method were examined for the first time. This was accompanied by network pharmacology and an in-silico docking study to correlate the phytochemical compositions with the selected biological activities.

Materials and methods

Plant material

The aerial parts of Echinops erinaceus Kit Tan (Alkana’a or Kanab, family Asteraceae) were collected and authenticated by an agricultural engineer, Mohamed Ashraf Abdel Fattah who was responsible for the King Saud University botanical garden, Saudi Arabia. The collection procedure was in compliance with the national and international guidelines and legislation. The herbarium sheet was received from the King Saud University botanical garden and not deposited elsewhere from 2017 till 2025, then it was added to the herbarium of the Pharmacognosy Department, College of Pharmacy, Prince Sattam Bin Abdulaziz University in 2025 with a voucher specimen (HPSAU-25–11-29–1). In addition, the samples were compared with the plant description in the Flora of Saudi14.

Preparation of various extracts

The dried aerial parts of E. erinaceus (2.250 kg) were extracted with 95% ethanol–water (70 0C) in Soxhlet for 3 h. The hot crude syrupy extract (373 g, T-EtOH) was stored in a cold place (24 h.), followed by filtration to yield 135 g of liquid extract (Filtrate, T-liq) and 171 g of residual sticky substance layer (TL). The residual sticky substance layer (TL), lipoidal material (100 g), was saponified in accordance with the published methodology by Al Kashef15. In brief, the total lipids (100 g) were refluxed with 600 ml of N/2 alc. KOH for about 8 h. The liquid left was diluted with water, then extracted with diethyl ether until exhaustion. The combined diethyl ether extracts were washed with alcoholic NaOH (10%) then with dist. water until the wash was free from any alkalinity. Dehydration with anhydrous sodium sulphate and then the diethyl ether was evaporated until dryness to give the unsaponified matters (Un., 19.5 g), yellowish orange in color. The alkaline aqueous solution was acidified with sulphuric acid (10%). The liberated fatty acids were extracted with successive small portions of diethyl ether. The diethyl ether extracts were washed with distilled water, till the wash was neutral to litmus paper. The diethyl ether was evaporated till dryness to give saponified (Sap., 25 g) with brownish-black color, see Figure S1. Both materials (Sap. and Un matters) were subjected separately to chromatography columns to separate their major compounds.

Un-saponified matter (19 g, Un.) was applied to a silica-gel column (250 g) and eluted with hexane: benzene (8:2—1:1) followed by benzene: ethyl acetate (99:1), then 100% ethyl acetate. Eleven fractions were collected (50 ml) to give five main fractions (U-I—U-V). Fraction U-I (0.7 g) on recrystallization yielded 0.076 g of heptadecane (C1), while fraction U-IV (1.5 g) was recrystallized to yield a mixture of two substances (C2/3), β-sitosterol (C2) and stigmasterol (C3) (0.293 g) (Flowchart S1 and Figure S2).

In addition, the saponified materials (15 g, Sap.) were subjected to another silica-gel column (160 g) eluted with benzene: ethyl acetate, 99:1—100% ethyl acetate. Four main fractions (30 ml, Sap.-I—Sap.-IV) were collected. Fraction Sap.-I showed a mixture of two fatty acids (C4/5), henicosanoic (C4) and tricosanoic acids (C5) (0.013 g, Flowchart S2 and FigureS2).

Following the method previously described by Sweilam et al.6, the liquid form (Filtrate, 135 g, T-liq) was evaporated until it was entirely dried (95.3 g, 25.54%). It was then successively fractionated using various solvents (chloroform, ethyl acetate, and butanol) to get chloroform (7.2 g, 1.93%), ethyl acetate (2.5 g, 0.67%), and butanol (44.3 g, 11.87%) extracts. The butanol extract (30 g, BuOH) was subjected to fractionation on a Sephadex column (200 g) eluted with 100% MeOH and to obtain three main fractions (BuOH-F1—BuOH-F3: 3.1 g, 7.6 g, and 20 g, respectively).

As part of bio-guided fractionations, in vitro cytotoxic and antimicrobial studies were performed on the crude extracts, chosen fractions, with/without isolated compounds.

GC–MS analysis

GC–MS chromatography analysis was used to examine the non-polar extracts (Sap. and Un with its total; TL). Shimadzu GC–MS-QP 2010 (Shimadzu Corporation, Koyoto, Japan) was used for the gas chromatography coupled with mass spectrometry (GC–MS) analyses. It was connected to a Shimadzu mass spectrometer and used a Rtx-5MS (30 m × 0.25 mm i.d. × 0.25 µm film thickness) capillary column (Restek, Bellefonte, PA, USA)12.

Identification of the tested extracts

By comparing the fragmentation patterns, retention indices, and GC–MS spectra of the identified components of the analyzed extracts (TL, Sap., and Un) with those described in the National Institute of Standards and Technology Library (NIST 2011), the components were characterized in comparison to a similar sequence of n-alkanes (C8–C28) injected under identical circumstances, the retention indices were computed, see in FigureS3.

Cytotoxicity assay

Cytotoxicity of the tested extracts (T-EtOH, TL, CHCl3, BuOH, and water remaining), their fractions (Un.-I—Un.-V and Sap.-I—Sap.-IV for TL extract and BuOH-Fr1—BuOH-Fr3 for BuOH extract), isolated compounds from Un and Sap. fractions (Un.-I-C1—Un.-IV-C2/C3, Sap.-I-C4/C5), and selected subfractions of BuOH extract against two to seven mammalian cell lines: MCF-7 cells (human breast cancer cell line), HepG-2 cells (human Hepatocellular carcinoma) HCT-116 (colon carcinoma), A-549 (Lung carcinoma), PC-3 (prostate carcinoma), CACO2 (intestinal carcinoma), and Hela (Cervical carcinoma) were assessed using crystal violet method.

Antimicrobial assay

Various species of micro-organisms: 10 bacterial strains; Gram-positive bacteria: B. subtilis (RCMB 015 (1) NRRL B-543), Staph. aureus (RCMB010010), Staph. epidermidis (RCMB 009 (2)), Strep. mutans (RCMB 017 (1) ATCC 25,175), and Strep. pyogenes (RCMB 01001 74–2). Gram-negative bacteria: E. coli (RCMB 010,052 ATCC 25,955), Kleb. pneumonia (RCMB 003 (1) ATCC 13,883), Proteus vulgaris (RCMB 004 (1) ATCC 13,315), Pseudomonas aeruginosa (RCMB 01,002 43–5), and Sal. typhimurium (RCMB 006 (1) ATCC 14,028); and 10 fungal strains: Aspergillus fumigatus (RCMB 002,008), A. niger (RCMB 002,005), C. albicans (RCMB 005,003 (1) ATCC 10,231), C. tropicalis (RCMB 005,004 (1)), Cryptococcus neoformans (RCMB 0,049,001), Geotrichum candidum (RCMB 05,097), Microsporum canis (RCMB 0834), Penicillium expansum (RCMB 001001 (2)), Syncephalastrum racemosum (RCMB 016,001 (1)), and Trichophyton mentagrophytes (RCMB 0925) were obtained as tested organisms by in the Microbiology Laboratory, Regional Centre for Mycology and Biotechnology, Al-Azhar University, Cairo, Egypt. All tested samples were dissolved in alcohol (200 μg/mL), and 50 µL was added to each well of each sample solution, separately in each case. The diameter of the inhibition zone (DIZ) was measured by the agar well diffusion method16.

Statistical analysis

Statistical analyses were done using a one-way analysis of variance (ANOVA) followed by the Tukey–Kramer multiple comparison test (p < 0.05). Statistical analyses were performed using GraphPad Prism 6.01 (GraphPad Inc., La Jolla, CA, USA).

Network pharmacology

Screening of active ingredients from spirulina and gathering their targets

The PubChem database17 was used to search for the 2D structure files (sdf), PubChem IDs, and SMILES, for twenty-seven active ingredients that represent the major prevalent metabolites of E. erinaceus that were previously characterized by GC–MS (Table 1). These active ingredients (2D structure files) were further filtered by the evaluation of their oral bioavailability (Lipinski’s rule) and their toxicity profile (DILL and carcinogenicity) using ADMET Lab 2.018 (Table S1). In addition to the prediction of their biological targets through the Swiss Target Prediction Web Tool19 (Table S2). Finally, the assembled biological targets (593 targets) were annotated using the UniProt database20 to gain the UniProt IDs of their genes (Table S2).

Table 1 Chemical profile of TL and its fractions from Echinops erinaceus using GC–MS analysis.
Full size table

Gathering of breast cancer target genes and prediction of active ingredients for breast cancer treatment

The DisGeNET database21,22,23,24 was searched for cancer-associated genes using the key term Neoplasm (Table S3). Using the FunRich 3.1.3 software’s Venn diagram intersection25, the obtained data of cancer-linked genes (9277 genes annotated with uniport) were matched with previously predicted target diseases of E. erinaceus active ingredients 593 targets).

Constructing protein–protein interaction network

STRING database26 was used to obtain the PPI network, and Cytoscape 3.9.0 software was used to do further analysis and embellishment. Firstly, inputting potential therapeutic targets for neoplasm into the online database STRING, setting parameters and constraints (limiting the species to Homo sapiens, and filtered through the combined score ≥ 0.95 as the threshold). Download the TSV format of the PPI network and import the obtained results into Cytoscape 3.9.0 software to make a network diagram for visualization. The CytoHubba plug-in calculates each node of the network and screens core target genes.

Do GO and KEGG pathway enrichment analysis

The GO enrichment analysis was conducted on the FunRich 3.1.3 software to extract the key GO terms. GO terms with P values less than 0.05 and enrichment scores higher than 5 were regarded as significant and were further pursued. KEGG pathway analysis results were assessed on the ShinyGO 0.8027. An adjusted P-value threshold of 0.05 was used for pathway discovery. Through the online tools bioinformatics platform28, the visualization analysis was carried out to make a bubble chart and histogram.

Molecular docking

Molecular docking was performed between the compounds collected in 2.3.2 and the core genes obtained in 2.3.3. Molecular Operating Environment (MOE), 2019.0102 software, 2023, Ali et al.29 was used for molecular docking simulation for the study of the binding affinity of E. erinaceus metabolites against MAPK(ERK). The database of the active ingredients was drawn and prepared via energy minimization, hydrogen addition, and calculation of the partial charges. Finally, this prepared database was saved in the form of mdb extension30. The target enzyme MAPK(ERK) was retrieved from the Protein Data Bank with PDB ID: 7auv. It was prepared and checked through the automatic quick preparation order of MOE. The docking simulation was implemented via the Amber10 Forcefield. Evaluation of ligand–protein complex interactions was afforded through visualization of poses and scoring function31. Validation of docking studies was examined by the Root Mean Square Deviation (RMSD) values for co-crystalized ligand protein isozymes, MAPK(ERK): 1.8862.

Results and Discussion

GC–MS analysis of the TL and its fractions of E. erinaceus collected in 2017

The chemical compositions of E. erinaceus aerial parts collected in March 2017 were investigated using GC–MS analyses (Table 1). The plant collected was extracted by the Soxhlet method to yield a nonpolar extract, total lipids (TL, 45.84%). Total lipids (100 g) were saponified as reported in the method by Al Kashef15 to yield saponifiable (Sap.) and unsaponifiable (Un) matters with yields of 25% and 19% w/w, respectively.

The all-tested samples encompassed 44 components that accounted for 100% of the total identified constituents except TL. sample for 99.62% with 4 unknowns (TL 1–4) of E. erinaceus, two of which were tentatively identified as di- and tri-terpenoids: trans-ferruginol (TL-1) and psi-taraxasterol (TL-3, 20-taraxastenol) acetate by comparing its data with the literature reports32.

As a final GC–MS analysis, forty-two of forty-four metabolites with a higher percentage than 0.01% were detected in E. erinaceus, containing C13–C44 carbon at oms. The representative chromatograms of GC–MS in the total ion current (TIC) mode of all extracts are shown in Figure S3.

Triterpenoids and phytosterols are considered the most predominant class in the lipid extract of E. erinaceus aerial parts, accounting for 91.42%, additionally, the unsaponified extract of the same seasonal-sample was showed a high percentage of these classes, representing 74.82% of the identified compounds, along with aliphatic hydrocarbons with 5.25% and 20.94% of TL and Un, respectively (Table 1 and Fig. 1).

Fig. 1
Fig. 1
Full size image

Pie charts demonstrating the distribution of metabolite classes in percentages within various fractions: TL: (Total Lipoidal matters), Sap.: Saponified matters, and Un: Un-saponified matters of E. erinaceus collected in March 2017.

The fatty acids and their derivatives show the highest concentration in Sap. extract with 64.15%, followed by the aliphatic hydrocarbons with their derivatives: oxygenated and aromatic hydrocarbons 13.38%, 8.39%, and 4.35%, respectively. Meanwhile, no observations for triterpenoids nor diterpenes were made in this extract. However, no observation was found for the fatty-ester molecules in the Un-extract when other components were taken into consideration. Triterpenes, aliphatic hydrocarbons, and diterpenes had declining concentrations of 74.82%, 20.94%, and 3.47%, respectively (Table 1 and Fig. 1).

As seen in Table 1, the main concentrations are representatives of the triterpene metabolites (with the dominating presence of triterpenes and phytosterols as minor compounds) and aliphatic components (hydrocarbons, esters, lactone, etc.). Fatty acids, esters, and diterpenes were found as well. The triterpenes in the E. erinaceus extracts were displayed as tetracyclic derivatives of dammarane in peak 30 of TL extract and cycloartane in peaks 16 and 19 of Un matters and the pentacyclic derivatives of oleanane-type in peaks 12 and 24 of Un matters, and peak 25 of TL, lupane-type in peaks 20,21,27,28, and 31 of TL extract and peaks 14, and 17–18 of Un matters, ursane-type in peak 18 of TL extract, and glutinane-type in peaks 22 of TL extract and 15 of Un matters (Fig. 1 and Fig. 2)33.

Fig. 2
Fig. 2
Full size image

Major compounds identified from nonpolar matters of E. erincaeus.

The major components identified in the non-polar extract (TL), lupeol acetate (62.92%), dammara-13(17),24-dien-3-yl acetate (14.53%), β-amyrin acetate (6.04%), lupeol (4.23%), and tetratetracontane (4.2%) were characterized as the chief metabolites. Moreover, lupeol (55.91%), hexatriacontane (12.26%), β-amyrone (8.25%), n-octacosane (4.28%), (2E,7R,11R)-3,7,11,15-tetramethylhexadec-2-en-1-ol (3.47%), cycloartane-3β,25-diol (2.72%), and β-sitosterol (2.33%), were considered the master metabolites in the Un matters. Additionally, among the major constituents, especially in Sap., were ethyl hexadecanoate (34.01%), ethyl (9Z,12Z,15Z)-octadeca-9,12,15-trienoate (12.62%), ethyl (9Z,12Z)-octadeca-9,12-dienoate (12.47%), 4,8,12,16-tetramethylheptadecan-4-olide (9.73%), eicosane (5.84%), 2-heptadecyloxirane (5.65%), nonacosane (5.08%), 2,6-di-tert-butyl-4-methylphenol (4.35%), ethyl octadecanoate (2.90%), octadecanal (2.74%), heneicosane (2.46%), and ethyl tetradecanoate (2.15%). The representation of all metabolites’ distribution was observed in Table 1 and Fig. 2. Finally, lupeol acetate, lupeol, and dammara-13(17),24-dien-3-yl acetate were observed in the 2017 plant sample (Table 1 and Fig. 2).

Moreover, from the literature reports, the root of E. integrifolius contained long-chain hydrocarbons, characterized as eicosane, hentriacontane, hexacosane, nonadecane, octacosane, and tetratetraacontane34. In addition, E. kebericho, aerial and root parts collected from Iraqi, appeared with the main sterol contents: stigmasterol and β-sitosterol35.

Several earlier reports on E. ellenbeckii based on GC–MS analysis revealed the occurrence of various acetylenic thiophenes, including mono-, di-, and tri-thiophenes, along with fatty acids and hydrocarbons such as myristic, palmitic, pentadecanoic, and stearic acids, as well as docosane, heneicosane, and tetracosane. Additionally, pentacyclic triterpenoids like β-amyrin, lupeol, lupeol acetate, and ursolic acid were identified, along with steroids such as campesterol, stigmasterol, and β-sitosterol36.

Previous phytochemical reports on the Echinops genus revealed the presence of free fatty acids, thiophenes, aliphatic hydrocarbons, terpenes, and phytosterols. All these metabolites were identified in our target plant, except thiophenes, and free fatty acids were not observed in any of the tested fractions. It is worth noting that this study demonstrates that the non-polar chemical profile of the studied plant sample does not closely align with its genus. Consequently, climatic, seasonal, and experimental conditions influenced the variability of the plant extract components collected in different seasons.

Identification of the isolated compounds

The spectral data of the isolated compounds (Figure S2) are listed in Figures S4-S18. Compounds C1 and C4/C5 were compared with those published in the literature, and confirmed these compounds’ assignments as a mixture of henicosanoic acid (C4) and tricosanoic acid (C5)37, and heptadecane (C1)38, a combination of two substances, β-sitosterol (C2), and stigmasterol (C3)39,40 were identified through co-chromatographic TLC with authentic (Table S4).

Cytotoxic activity of E. erinaceus aerial parts

The cytotoxic effects of the extracts of E. erinaceus were estimated on A-549, CACO2, HCT-116, Hela, HepG-2, PC-3, and MCF-7 carcinoma cell lines using a colorimetric- crystal violet method. The cytotoxic effects of the extracts or fractions (T-EtOH, TL, Un, Sap., Chl., BuOH, H2O extracts, and F1-F3 fractions) were tested at a concentration of 10 µg/mL. The samples showed variable cytotoxic activity against the seven cell lines. The lipid derivatives (Sap. and Un matters) and polar extract (BuOH) with its fractions (Fr1-Fr3) showed significant inhibitory effects against various unsimilar carcinoma cell lines. The non-polar matters (Sap. and Un) showed potent to moderate reduction in cell viability, exhibited IC50 values of 12.1 ± 1.5—51.4 ± 2.8 µg/mL against all tested cell lines when compared to the reference drugs or published data9.

According to data analysis, Un extract is considered the most active extract on two cell lines (A-549 and HCT-116) with an IC50 value less than 20 µg/mL (18.1 ± 2.5 and 15.1 ± 1.4 µg/mL, respectively), and it exhibited IC50 = 27.3 ± 1.5 and 44.6 ± 2.1 µg/mL on MCF-7 and PC-3 cell lines, respectively. In addition, the reference standard, vinblastine sulphate, showed IC50 = 30.3 ± 1.4 and 59.7 ± 2.1 µg/mL against CACO2 and HELA cell lines, respectively. Meanwhile, the Un extract showed significant activities against these cell lines, with IC50 26.9 ± 1.2 and 51.4 ± 2.8 µg/mL, respectively (Table S5 and Fig. 3).

Fig. 3
Fig. 3
Full size image

In-vitro cytotoxic effects (IC50, μg/mL) of various extracts of E. erinaceus collected in 2017 on seven cell lines.

In the unsaponified GC-chromatographic pattern, one of the metabolites, lupeol (lupane triterpene) in peaks 14,17, and 18, is considered the most abundant component (55.91%) (Table 1 and Fig. 2). It was investigated before as a cytotoxic compound against A-431, H-411E, and Hep-G2. In addition, it was observed as inactive on MCF-7 as well as Walker 256 carcinosarcoma41.

Sap. extract showed observed potent activity on HCT-116 (IC50 = 12.1 ± 1.5 µg/mL), while it showed moderate activity against four cell lines: MCF-7, PC-3, CACO2, and HepG-2 with IC50 values of 23.3 ± 1.1, 27.1 ± 0.9, 29.9 ± 1.8, and 30 ± 1.8 µg/mL, respectively (Table 2 and Fig. 3). Sap. extract is enriched with ethyl palmitate (34.01%), ethyl (9Z,12Z)-octadeca-9,12-dienoate (12.47%), ethyl (9Z,12Z,15Z)-octadeca-9,12,15-trienoate (12.62%), and 4,8,12,16-Tetramethylheptadecan-4-olide (9.73%) (Table 1 and Fig. 2). They induced apoptosis death of the human triple-negative breast cancer cells (MDA-MB-231 cells)42. Ethyl hexadecanoate was reported to have significant cytotoxic activity against MCF-7 cell lines43. All other examined samples showed weak to no cytotoxic activities against HELA cell line, except Un extract showed moderate activity (IC50 = 51.4 ± 2.8 µg/mL) (Table S5 and Fig. 3).

Table 2 In-vitro antimicrobial activity of 2017-extracts of E. erinaceus sample against selected groups of various pathogens.
Full size table

Bio-guided fractionations of the non-polar extracts, Un and Sap. extracts revealed that the fractions, Sap.-II, exhibited the most potent cytotoxic activity against selected cell lines, CACO2 and PC-3, with IC50 values of 30.1 ± 0.4 and 39.7 ± 0.5, respectively, and Sap.-I with IC50 values of 40.5 ± 0.9 and 62 ± 0.9, respectively. In addition, the isolated compounds of Sap.-I fraction, henicosanoic (C4) and tricosanoic acids (C5) tested on five cell lines (HepG-2, MCF-7, CACO2, PC-3, and HCT-116), were recordered with moderate to weak activity with IC50 values of 26.7 ± 0.8, 30 ± 0.6, 36.1 ± 0.5, 57 ± 0.9, and 60.7 ± 1.5, respectively (Table 2 and Fig. 3). Further, the Un extract showed strong to moderate cytotoxic effects on the all-tested cells, however, its fractions (Un.-I and Un.-III) were reordered with weak activity with IC50 = 54.3 ± 0.9 and 77.6 ± 2.9 against CACO2 and only Un.-I showed a weak activity with IC50 = 70.7 ± 4.1 on A-549 cell lines, and the isolated components of Un fractions (Un.-I-C1 and Un.-IV-C2/3) did not show any cytotoxic effects on four tested cell lines (A-549, CACO2, HCT-116, MCF-7) nor the other sub-fractions (Un.-II—Un.-V) (Table S5 and Fig. 3).

Compounds (C4/C5) are the modest antitumor agents on the cancerous hepato-, breast-, and intestine- cell lines, and from previous studies, tricosanoic acid was present among bioactive substances of Bergera koenigii seeds that inhibit leukemic THP-1 cells. Also, stigmasterol inhibited the PI3K/MAPK signaling cascade and reduced cell migration in the ovarian cancer cell lines ES2 and OV90. In addition, stigmasterols increase lipid peroxide levels and prevent DNA damage to inhibit skin cancer, according to research by Govindswamy B.44. On the other hand, heptadecane (C1) was found to inhibit inflammation in kidney tissues of rats by suppression NF-kB activity through upregulating the NIK/IKK and MAPKs pathways, and also inhibiting hepato-proliferative cancerous cells in humans45.

A moderate to weak inhibitory activity of the polar extract (BuOH) was detected with IC50 = 28.1 ± 2.2—99.4 ± 3.8 µg/mL. While its fractions (BuOH-Fr1– BuOH-Fr3) exhibited IC50 = 26.4 ± 1.2 µg/mL (HepG-2) of BuOH-Fr2, IC50 = 28.8 ± 1.9 and 30.6 ± 1.8 µg/mL (HCT-116) of BuOH-Fr3 and BuOH-Fr1, respectively, IC50 = 44.1 ± 2.8 and 45.5 ± 1.9 µg/mL (PC-3a) of BuOH-Fr1 and BuOH-Fr2 respectively, IC50 = 51.9 ± 3.6 µg/mL (A-549) and 92.7 ± 5.3 µg/mL (HELA) of BuOH-Fr3, besides most polar sub-fractions (BuOH-F1- Subfr.7 and BuOH-F3- Subfr.1 –Subfr.5) showed no cytotoxicity (Table S5 and Fig. 3) except BuOH-F2-Subfr.6 showed moderate activity with IC50 = 45.2 ± 0.6 against HepG-2.

Thus, the total lipid and chloroform extracts showed no cytotoxic activity against all tested cell lines; however, our previous study on the chloroform and its fractions/isolates, oleic acid derivatives of the sample-2018, were reported to possess moderate to weak in vitro cytotoxic activities on HCT-116 and CACO2 cancerous cell lines by using MTT assay. Moreover, the results of the total and remaining aqueous extracts of the two samples (2017 and 2018) were observed to be weak to non-active against these cell lines9. On the other hand, total methanolic extract showed a strong effect on HepG-2 cell line and weak effects against two cell lines (A-549; IC50 = 61.9 ± 3.5 and CACO2; IC50 = 60.2 ± 3.4 µg/mL) (Table S5nd Fig. 3). Results indicated that E. erinaceus collected in 2017 possesses major compounds with a potent cytotoxic activity.

Antimicrobial activity of E. erinaceus aerial parts collected in 2017

The quest for novel and safe antimicrobial agents within herbal plants has been intensified recently due to the rise in microorganism mutation and resistance to synthetic antimicrobial agents. The antibacterial properties of Echinops species and their components have been shown in numerous studies46. Furthermore, various 151 secondary metabolites47, such as phytochemical components including phenolic compounds, tannins, and saponins, have been thought to enhance the antimicrobial activity of crude medications to promote health by combating some pathogenic microbes. The genus Echinops belongs to the Compositae family and is found worldwide, except in Antarctica46. Several of its biological activities and phytoconstituents have been scientifically demonstrated to influence its therapeutic performance. According to previous studies, this genus is commonly used in conventional medicine to treat a variety of ailments, including fever, pain, inflammation, and respiratory tract conditions such as cough and sore throat caused by an infected microbe46.

The results of antimicrobial activity of the extracts demonstrated that the butanol and chloroform extracts displayed the highest antimicrobial properties against the most microorganism strains, followed by the saponifiable fraction. The antimicrobial results of the different extracts from E. erinaceus aerial parts of sample-2017 showed that the yeast strains seem to be more sensitive to the butanol, followed by chloroform, then tested crude ethanol extracts, sap extract, and its fractions as compared to the reference drug (Ketoconazole), as shown in Table 2.

From the agar disc diffusion method, butanol extract of E. erinaceus had antifungal activity against two Aspergillus fungi (A. niger and A. fumigatus) with inhibition zone diameter (17 ± 0.15 and 14 ± 0.1 mm, respectively), and it also showed antifungal activity against another two strains; C. neoformans and C. albicans followed by the chloroform extract, were sensitive with 22 ± 0.15/15 ± 0.15 mm, and 21 ± 0.05/16 ± 0.15 mm inhibition zone diameter (IZD), respectively, as compared to the reference drug (Ketoconazole) showed in Table 2.

The results of the antimicrobial properties demonstrated that the alcoholic extract obtained by the hot method (Soxhlet) had no activity against all the tested microorganisms except against K. pneumonia with 18 ± 0.2 mm IZD. Previous studies demonstrated that the majority of antimicrobial fractions were soluble in a polar solvent, such as ethanol, rather than in water. Furthermore, it was discovered that butanol and chloroform extracts exhibited higher efficacy than the ethanolic extract, particularly dependent on the concentration of the extract46,48. The majority of antimicrobial activity of E. setifer ethanolic extract was more effective in suppressing the growth of L. innocua, S. aureus, and B. cereus compared to gram-negative bacteria, including P. aeruginosa, E. coli, and S. typhi46.

Meanwhile, the total lipoidal extract and its matters showed no antifungal and antibacterial activities against the tested strains except the Sap. Extract showed an antibacterial activity against K. pneumonia and P. aeruginosa with 13 ± 0.11 and 12 ± 0.26 mm IZD. In addition, its subfractions: 4-I to 4-IV showed significant activities against gram-negative bacteria against the K. pneumonia strain with 14 ± 0.05, 18 ± 0.17, 13 ± 0.15, and 16 ± 0.1 mm IZD, respectively, as compared to the reference drug (Gentamycin).

On the other hand, all tested extracts showed no activity against Gram-positive bacteria, except the chloroform extract showed a strong activity against S. aureus strain with 20 ± 0.05 mm IZD as compared to the reference standard (Gentamycin), Table 2. Regarding our previous report and published studies on the genus, the results showed that the alcoholic extract using the cold method had a significantly higher activity against B. subtilus strain when compared to the reference standard. Additionally, the non-polar extracts, “hexane and chloroform extracts,” had the best activities against the most tested organisms. This may be confirmed by using different extraction methods; different results were revealed for the two samples (cold and hot methods)9.

Network pharmacology

Screening of candidate ingredients

Twenty-seven active ingredients in E. erinaceus were searched through SwissTarget and ADMET Lab 2.0. After these screening processes, a total of 15 ingredients and 593 targets were obtained. After merging with the 9277 targets of neoplasm collected from DisGeNET database by FunRich 3.1.3 software’s Venn diagram intersection, 183 overlapping targets were recognized as candidate targets, as shown in Fig. 4A and Table S6.

Fig. 4
Fig. 4Fig. 4Fig. 4
Full size image

(A) Venn diagram of component target and disease target, (B) Network of top ten genes, (C) GO enrichment analysis of results for cancer treatment of Echinops erinaceus, (D) KEGG pathway enrichment analysis49,50,51 for E. erinaceus.

PPI network construction and analysis

A network graph with 187 nodes and 1499 edges was obtained by selecting the species “human” in the String database. Using the TSV file exported from this website and the cytoHubba plug-in cytoscape, the top ten genes are PPARG, ESR1, PTGS2, EGFR, HIF1A, MAPK3(ERK), PPARA HMGCR, APP, and MAPK1 (Fig. 4B and Table S7). These top 10 genes include a critical core gene, MAPK3(ERK), in the PPI network of cancer-related targets of E. erinaceus, which are interlinked, interactive, and collaborate to inhibit the development of different types of cancer.

GO and KEGG pathway analysis of candidate targets

GO enrichment and KEGG enrichment analysis of 183 candidate targets were obtained through Funrich and ShinyGO 0.80. GO enrichment analysis included three aspects: cellular component, molecular function, and biological process. Every aspect analysis result is selected with p value less than 0.05, whereas selecting important cellular components, molecular functions, and biological processes with enrichment scores higher than 1, as shown in Fig. 4C and Table S8. From histogram, we can see that candidate targets were mainly involved in the cell population proliferation, reg. of transport, chemical homeostasis, and other biological processes. As for molecular functions, it participates in nuclear receptor activity, ligand-activated transcription factor activity, and monocarboxylic acid binding, etc. Cellular component related to the cell surface, vesicle, and receptor complex, etc.

Ranked through P-value (P < 0.05), the top 20 pathways were obtained, and a bubble diagram was shown in Fig. 4D and Table S9. It contains a number of cancer-related signaling pathways. Among them, proteoglycans in cancer are the most significant (Fig. 4D). In addition to the critical core gene screened by PPI analysis, MAPK3 (ERK) was involved in Proteoglycans in cancer. As shown in Fig. 5, downloaded from KEGG, Proteoglycans in cancer pathway are mainly involved in regulating cell growth, proliferation, survival, cell migration, and invasion.

Fig. 5
Fig. 5
Full size image

Proteoglycans in the cancer pathway.

Molecular docking

In order to further verify the active ingredients and their potential targets and mechanisms in the treatment of cancer from E. erinaceus, the core target MAPK3 (ERK) based on PPI network, KEGG pathways (Ref: 253,410)yy49,50,51, and GO enrichment, was selected for a virtual screening docking simulation study against 15 active ingredients of E. erinaceus using MOE software. The results revealed that most of the active ingredients exhibited interactions with the core target. The top-ranked four compounds were [(tricosanoic acid), (ethyl octadecanoate), (dammara-13(17),24-dien-3-yl acetate), and (ethyl (9Z,12Z)-octadeca-9,12-dienoate)]. These candidates possessed strong interactions with the key amino acid residues (Table 3) of the original co-crystalized ligand named as RYW (Fig. 6) via different hydrophobic and hydrogen bonding interactions. The highly bound active ingredients to ERK were (tricosanoic acid) with a binding score equal to − 7.9480 kcal/mol, while compounds (ethyl octadecanoate) and (dammara-13(17),24-dien-3-yl acetate) revealed the binding scores equal to − 7.8111 and − 7.8069 kcal/mol, respectively. Finally, (ethyl (9Z,12Z)-octadeca-9,12-dienoate) showed the binding scores equal to -7.6799 kcal/mol (Table 3).

Table 3 Docking simulation results of the top four active ingredients against the target enzyme MAPK(ERK).
Full size table
Fig. 6.
Fig. 6.
Full size image

2D representation for the binding interactions of the co-crystalized ligand (RYW) with the key amino acid residues of ERK (PDB IDs: 7auv).

Conclusions

The following components predominate, identified by using GC–MS analysis in E. erinaceus’s chemical composition: Lupeol acetate, lupeol, and ethyl hexadecanoate, were found in TL, Un, and Sap. extracts, at the corresponding concentrations of 27.92–62.92%, from the samples that were gathered over the course of 2017. Surprisingly, the triterpenes were the most numerous peaks on the GC–MS chromatograms as the primary components in all three extracts, except Sap. extract. In addition, five isolates were identified from the lipoidal derivatives (Un and Sap.) of the sample-2017. The tested extracts showed promising in vitro cytotoxicity against various cell lines and antimicrobial properties against different microorganisms. These aforementioned results indicated that the hot pilot extraction method of E. erinaceus may play an important role in the treatment of various types of cancer by Proteoglycans in the cancer pathway through modulation of MAPK (ERK). In vivo and clinical trials are needed to confirm the in vitro experiments.