Atorvastatin (S) calcium trihydrate, Vastor 20 mg; Hikma pharmaceuticals company. Ethanol, chloroform, and n-hexane, analytical reagent; LABCHEM Laboratory chemicals, USA. Aluminum chloride 6-hydrate; Laboratory chemicals. Sodium carbonate anhydrous; SD Fine Chem limited (SDFCL), INDIA. 2,2-Di-phenyl-1-picrylhydrazyl, sodium nitrite, sodium hydroxide, gallic acid, ascorbic acid, and quercetin; Sigma – Aldrich, Germany. Folin-Ciocalteu reagent; Sigma-Aldrich, USA. Lipid profile kits including high density lipoproteins, triglycerides, and total cholesterol (HDL, TG, and TC), Glucose– liquicolor, and gel tubes; Human, Germany. AlCl₃, NaOH, NaNO₂, and Na2CO3; locally laboratory chemicals.

The species E. ramosissimum used in this study were collected from Mujib reserve in Jordan (84 Km from Amman) and authenticated by Mr. Ibrahem Mahasneh, a professional taxonomist at the Nature Conservation Monitoring Centre, the Royal Society for the Conservation of Nature (RSCN). A voucher specimen is available at the RSCN herbarium (number: E.r-5/7/2017), as it is considered the official authority for plant identification in Jordan. Plant materials were dried under shade and stored at room temperature. When needed, the plant was crushed using a commercial blender to reduce the particle size.

Briefly, 300 g of plant powder was soaked in 1L of ethanol for 24 hours, with constant agitation at room temperature. The mixture was then filtered, and the solvent was evaporated.

Total phenol content was measured using the Folin-Ciocalteu method as described previously (Sadeghi et al. 2015). Briefly, 0.1 g of the plant extract was dissolved in 50 mL of methanol. Then, 2 mL of 10% Na2CO3 solution and 5 mL distilled water with 1 mL of Folin reagent was added to create the reaction’s basic medium. The solution was left for 1 hour at room temperature to complete the reaction. The absorbance was recorded at 760 nm by spectrophotometry against the methanol blank solution. Gallic acid (GA) (Sigma Aldrich, Germany) was used as a standard phenolic compound to prepare the calibration curve. The total phenolic content (mg/ mL) was then determined as gallic acid equivalents.

The determination of total flavonoids content was performed using the colorimetric method, based on the formation of a complex flavonoid-aluminum as described previously (Pękal and Pyrzynska 2014). To prepare the reaction mixture, 0.1 g of plant extract was dissolved in 50 mL methanol and added to 1.5 mL of AlCl₃, 2 mL of NaOH (4%), 1 mL NaNO₂ (10%) and 5 mL distilled water, then completed to a final volume of 10 mL using 80% MeOH (v/v). The absorbance was read at 510 nm. All determinations were calculated using a calibration curve obtained with the quercetin standard (Sigma Aldrich, Germany). The results were expressed as mg/g equivalents to quercetin.

The free radical scavenging activities of the extracts were determined using the 2, 2- Diphenyl- 1-picrylhydrazyl (DPPH) free radical scavenging method as described previously (Paulsamy et al. 2013). For the reaction reagent, 0.4 g of DPPH was dissolved in 100 mL methanol. The reaction was performed by mixing 0.1 g of plant extract with 10 mL methanol. 1 mL of the plant extract solution was mixed with 3 mL of DPPH and completed to a final volume of 10 mL using methanol, then allowed to stand in darkness for 30 minutes. The absorbance was recorded at 517 nm. The calibration curve was done using ascorbic acid (vitamin C) (Sigma Aldrich, Germany) as a standard. The percentage inhibition of DPPH by the plant extract and the IC₅₀ were calculated using the following equation:

$\text{Percentage inhibition (\%)}=\frac{\text{A - B}}{\text{A}}\times 100$

where A (control) is the absorbance of pure DPPH in oxidized form, and B (sample) is the absorbance of the sample taken after 30 min of reaction with DPPH.

The method used in this study was based on previously developed and validated method as described by Bajkacz et al. (2018). Briefly, a Bruker Daltonik Impact II ESI-Q-TOF System with a Bruker Dalotonik Elute UPLC system (Bremen, Germany) was used with a high-resolution Bruker TOF MS and accurate retention times were recorded.

For each analyte following chromatographic separation, including catechol, naringin, chlorogenic acid, gallic acid, caffeine, catechin, caffeic acid, isoferulic acid, kaempferol, kaempferol 3-O-glucoside, and others, identification was based on the exact match for the chromatograms peaks retention time and the mass spectrum and fragmentations fingerprints compared to the authentic samples or to the in house-built library previously established and validated at our laboratory.

The Ion Source Apollo II ion Funnel electrospray source was used to run this device. The dry temperature was 200 °C, the capillary voltage was 2500 V, the nebulizer gas flow was 8 L/min, and the nebulizer gas pressure was 2.0 bar. Elute UHPLC paired to a Bruker Impact II QTOFMS, supplied a TOF repetition rate of up to 20 kHz, a mass accuracy of 1 ppm, and a mass resolution of 50000 FSR (Full Sensitivity Resolution).

Chromatographic separation was performed using Bruker solo 2.0_C-18 UHPLC column (100 mm × 2.1 mm × 2.0 μm) at a flow rate of 0.51 mL/min and a column temperature of 40 °C. Solvents: (A) water with 0.05% formic acid and (B) acetonitrile Gradient: 0 – 27 min linear gradient from 5%–80% B; 27 – 29 min 95% B; 29.1 min 5% B, total analysis time was 35 min on positive and 35 min on negative mode injection volume 3 µl.

Stock solutions of the plant extract were prepared by dissolving the appropriate amount of substance in dimethyl sulfoxide-DMSO (analytical grade), then diluted with acetonitrile and used for identification of exact MS and retention time. All other reagents; acetonitrile, methanol, water, and formic acid used were of HPLC grade.

Thirty-six male Wistar rats (185–220 g) were purchased from the animal house laboratories- Applied Science Private University-School of Pharmacy (Amman, Jordan). Rats were placed in metabolic cages, after being acclimated (21–22 °C and 12 h light/dark cycles) for at least one week before starting the experiments. Standard diet and drinking water were equally given for all study animals, and body weight (BW) was recorded weekly. Based on the diet model, animals were randomly divided into 2 divisions as follows:

1. Non-high fat diet (NHD); 2 groups:

1. NHD: Male Wistar rats supplied with standard diet (30 g/rat/day) for 4 weeks.
2. NHDP: Male Wistar rats supplied with standard diet (30 g/rat /day) for 2 weeks, followed by further 2 weeks oral treatment with extract of E. ramosissimum (200 mg/kg/day).

1. High fat diet-induced hyperlipidemia (HD); 4 groups:

1. HD: Male Wistar rats supplied with standard diet (20 g/rat /day) + (30% fat lamb fat diet (10 g/rat/day)) for 4 weeks.
2. HDP: Male Wistar rats supplied with standard diet (20 g/rat /day) + (30% fat lamb fat diet (10 g/rat/day)) for 2 weeks, followed by further 2 weeks oral treatment with extract of E. ramosissimum (200 mg/kg/day).
3. HDS: Male Wistar rats supplied with standard diet (20 g/rat /day) + (30% fat lamb fat diet (10 g/rat/day)) for 2weeks, followed by further 2 weeks oral treatment with daily oral dose of 15 mg/kg of Atorvastatin.
4. HDPS: Male Wistar rats supplied with standard diet (20 g/rat /day) + (30% fat lamb fat diet (10 g/rat/day)) for 2 weeks, followed by further 2 weeks oral treatment with combination of [E. ramosissimum extract (200 mg/kg/day) + 15 mg/kg of Atorvastatin.

Animal dose for Atorvastatin has initially been calculated based on the preliminary experiments, and as described by (Chen Y et al. 2018). Briefly, diluted suspension was prepared to a final concentration of (0.5 mg/ mL) using 20 mg Atorvastatin tablet suspended in 40 mL distilled water. Atorvastatin suspension was administered by oral gavage at a dose of 15 mg/kg for 2 weeks. A large intermittent single dose (6 ml) by oral gavage was administrated to overcome the weak solubility of statin in distilled water as well as to avoid any possible reflux signs during the oral gavage procedures that were not observed.

Unfortunately, no data have been reported on the toxic effect of E. ramosissimum. Nevertheless, previous study by Baracho NC et al. (2009) revealed no significant changes in the hepatic enzymes were observed in Wistar rats who were administrated with the relevant species “E. arvense” at a dose of 100 mg/kg for 14 days.

All procedures were performed in accordance with international regulations for the care and use of laboratory animals. Ethical approval on this study was obtained by the Institutional Review Board at Applied Science Private University, Amman, Jordan. Approval Number: 2021-PHA-40.

The rats were fed lamb fat, which is high in cholesterol, mixed with their daily diet (rat chow) to develop hyperlipidaemia (Irudayaraj et al. 2013). HD-fed animals (4 groups) were subjected to an adjustment phase of 14 days in an extra diet containing 30% (10 g/rat/day) lamb fat. Day 15 was considered day 1 of the treatment, in addition to a high-fat diet. All oral treatments were administered via an intra-gastric tube. At the end of the treatment (day 28), rats were fasted overnight and sacrificed by cervical dislocation.

It is well known that statins have very slight solubility in water. Therefore, in accordance with the procedures used by (Balzan et al. 2013; Adejor et al. 2017) with some modifications, Atorvastatin 20 mg were used. One tablet was dissolved in 100 mL of distilled water to achieve a total concentration of 0.2 mg/ml, then administered to each rat orally once daily with a dose of 15 mg/kg rat body weight for a period of 2 weeks.

For the E. ramosissimum ethanolic leaves extract, the stock solution was freshly prepared by suspending the dry extract in distilled water. All doses were orally administered via an intra-gastric tube (1 mL/rat) equivalent to a dose of 200 mg dry plant extract/kg rat body weight, based on a previous similar study (Carneiro et al. 2013).

The serum lipid profile was assayed before the experiment, on day 1, day 15, and at the end (day 28) of the experiment. Blood samples (0.2 mL per animal) were collected into heparinized tubes by puncturing the retro-orbital plexus. The plasma was centrifuged at 2000 × gG, for 10 min. Hyperlipidemia model was confirmed by values of TC greater than (55 mg/dL). Rats that failed to develop the HD-induced hyperlipideamic model were excluded from the study. The TC, TG, and HDL-C levels were quantified using enzymatic kits. The LDL-C and non-HDL levels were calculated using the following equations (Virani 2011; Karkhaneh et al. 2019):

LDL-C = TC/1.19 + TG/1.9-HDL/1.1-38

Non-HDL = TC-HDL

The statistical analysis was performed using a Statistical Package for the Social Sciences (SPSS), version 27.0 for Windows (Chicago, IL, USA). The one-way ANOVA test was used to investigate if there are any significant differences in the mean values for each parameter between the different groups of the experiment. Post hock (Tukey test) was used to determine the significant differences among groups by analyzing multiple comparisons.

The extraction yield was calculated at 44.4% (w/w dry weight), for the ethanolic extract of E. ramosissimum.

The total phenolic content of E. ramosissimum was found to be (0.032± 0.001 mg/mg dry extract equivalent to gallic acid).

The total flavonoid content of E. ramosissimum was found to be (0.044±0.003 mg/mg dry extract equivalent to quercetin).

The plant extract DPPH radical scavenging activity was calculated as % inhibition (I%). Equation extrapolated from serial concentrations (µg/mL) verses %inhibition curve: y = 31.338ln(x) + 62.9, R² = 0.9735 was used to calculate the IC₅₀ , and used to determine the antioxidant activity for the ethanolic extract in comparison to ascorbic acid as shown in Table 1.

The qualitative analysis of phytocomponents was determined in E. ramosissimum for the ethanol extract using UHPLC-MS/MS (Table 2). The total ion chromatograms for the identified compounds are shown below (Figs 1, 2).

Table 2.

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Phytochemicals detected in E. ramosissimum ethanol extract using UHPLC/ MS-MS.

No.	RT [min]	[M-H]-	Exact mass	Name	Structure	Molecular Formula
1	2.87	289.07179	290.07907	catechin		C15H14O6
2	3.31	179.03493	180.04221	caffeic acid		C9H8O4
3	5.55	193.04987	194.05715	3-hydroxy-4-methoxycinnamic acid (isoferulic acid)		C10H10O4
4	5.61	593.15051	594.15778	kaempferol-3-O-rutinoside		C27H30O15
5	6.13	151.04018	152.04745	2,4-dihydroxyacetophenone		C8H8O3
6	6.4	593.15037	594.15765	kaempferol 3-O-neohesperidoside		C27H30O15
7	6.59	447.09298	448.10025	kaempferol-3-O-glucoside		C21H20O11
8	10.11	285.03967	286.04695	kaempferol		C15H10O6

Figure 1. 

Total ion chromatogram for all compounds detected.

Figure 2. 

Total ion chromatograms of peaks corresponding to the compounds detected in the E. ramosissimum ethanol extract.

The UHPLC-MS/MS for the ethanol extract revealed the presence of 8 compounds, including catechin, caffeic acid, 3-hydroxy-4-methoxycinnamic acid (isoferulic acid), kaempferol-3-O-rutinoside, 2,4-dihydroxyacetophenone, 3-O-neohesperidoside kaempferol, kaempferol-3-O-glucoside and kaempferol. The obtained MS/MS spectra of the identified compounds in the E. ramosissimum extract are shown in Figs 3–10.

Figure 3. 

Mass spectrum of catechin.

Figure 4. 

Mass spectrum of caffeic acid.

Figure 5. 

Mass spectrum of 3-Hydroxy-4-methoxycinnamic acid (isoferulic acid).

Figure 6. 

Mass spectrum of kaempferol-3-O-rutinoside.

Figure 7. 

Mass spectrum of 2,4-dihydroxyacetophenone.

Figure 8. 

Mass spectrum of 3-O-neohesperidoside kaempferol.

Figure 9. 

Mass spectrum of kaempferol-3-O-glucoside.

Figure 10. 

Mass spectrum of kaempferol.