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
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
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
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.
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
(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
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).Fig. 6.
2D representation for the binding interactions of the co-crystalized ligand (RYW) with the key amino acid residues of ERK (PDB IDs: 7auv).
