Cicer arietinum phytosome ameliorates hepatosteatosis via downregulation of fatty acid synthase and stearoyl-CoA desaturase 1 in rats

Abstract

Background

Hepatosteatosis is considered a universal problematic health due to bad lifestyle. Thereby, the current study evaluates the influence of the Cicer arietinum polyunsaturated fatty acids (CAP) and newly synthesized C. arietinum polyunsaturated fatty acids phytosome (CAPP) against non-alcoholic fatty liver disease (NAFLD) persuaded through a high-fat diet (HFD) in addition to tamoxifen (TAM) in male albino rats. Forty-eight rats were separated into eight groups (6 rats/group). Rats of the control group were administered distilled water for 45 consecutive days, while phosphatidyl choline (PC), CAP, and CAPP groups administered distilled water (15 days), afterward administered PC, CAP, and CAPP, respectively (500 mg/kg b.wt), orally for 30 days. All the previous groups fed normal diet for the 45 days, while NAFLD rats feed HFD for 45 days and receive TAM (200 mg/kg b.wt, i.p) daily for 15 days, followed by administration of vehicle, PC, CAP, and CAPP orally for another 30 days.

Results

Hepatosteatosis was appraised biochemically by significant increase in the concentrations of serum AST, ALT, γGT, LDH, ALP, total bilirubin, total lipid, triglycerides, fatty acid synthase (FAS), stearoyl-CoA desaturase 1 (SCD-1), and LDL-cholesterol, as well as hepatic total lipids and triglycerides. In addition, a significant decline in serum total protein, albumin, and HDL-cholesterol concentrations was observed in comparison with the control group. NAFLD induces oxidative stress by noteworthy increase in hepatic MDA, H₂O₂, and meaningful reduction in hepatic GSH, SOD, GST, GRD, and CAT levels as compared with the corresponding control group. Liver histological changes were noted in the NAFLD group as compared to the control. Interestingly, CAP and CAPP treatments modulate the abnormal effects of NAFLD in all the previous parameters. For the histological changes caused by NAFLD, the liver tissue appeared nearly normal after the treatment with CAP and CAPP.

Conclusion

CAP and CAPP administration may have a potential role in alleviating hepatosteatosis. This may relate to its downregulation against FAS, SCD-1, and oxidative stress.

Background

Fatty liver (hepatosteatosis) reflects the disruption of lipid metabolism and accumulation of lipids (more than 5% fat) in fat droplets which deposited finally in liver (Ma et al., 2021). NAFLD (non-alcoholic fatty liver disease) represents a new emerging risk factor for more advanced diseases such as liver fibrosis, cirrhosis, and hepatocellular carcinoma (Cerrito et al., 2023). Unfortunately, cirrhosis is considered the second causative stage of liver transplantation in large transplantation centers which urge researchers to search more and more to reduce the excessive frequency of NAFLD (Aguirre et al., 2014). Thereby, the present work tries to find promising natural agents by returning to the causative factors of NAFLD. The etiology of hepatosteatosis is multifaceted including diabetes, obesity, alcohol drinks, hepatitis C, and others (Park et al., 2020). Recently, hepatosteatosis has been increased in Egypt and other world may be due to the poor lifestyle including over ingestion of HFD (high-fat diets) and absence of physical activity (Emam et al., 2019; Jang et al., 2012; Zamani-Garmsiri et al., 2021). Mainly, there are many underlying mechanisms that contribute to the onset of NAFLD. Firstly, overweight and obesity are major causes of hepatosteatosis. Secondarily, NAFLD is related with overproduction of OxS (oxidative stress), where fat accumulation in the liver resulted in lipid peroxidation that evokes numerous serious responses including inflammation and the onset of fibrosis. Thirdly, NAFLD developed due to deregulation of lipid metabolism. Fatty acid synthase (FAS) is a good marker that reflects the NAFLD onset and progression, as it is the key enzyme in lipogenesis that catalyzes the final step in fatty acids synthesis. During its function in long-chain fatty acids synthesis, FAS uses acetyl-CoA (primer), malonyl-CoA (carbon donor), and NADPH (reducing equivalent) (Jensen-Urstad et al., 2012). FAS produces palmitate by acetyl-CoA and malonyl-CoA; then, the palmitate is utilized as the precursor for the formation of long-chain lipid molecules (Angeles & Hudkins, 2016). Thereby, the circulating FAS level is associated with the degree of hepatosteatosis as it is a rate-limiting enzyme in lipogenesis process. Another rate-limiting lipogenic enzyme that modulates lipid metabolism is the stearoyl-CoA desaturase 1 (SCD1) which is named as fatty acid desaturase (Gianotti et al., 2013). SCD1 is predominantly expressed in liver and responsible for forming a double bond in stearoyl-CoA, where it converts saturated fatty acids (12–18 C atom) to MUFAs (monounsaturated fatty acids) such as palmitoleate and oleate (Kotronen et al., 2009). The latter is considered substrates and subsequently used for synthesis of triacylglycerols and other lipids, as palmitoyl-(C16:0) converted to palmitoleoyl-(C16:1, n-7) and stearoyl-CoA (C18:0) converted to oleoyl-CoA (C18:1, n-9) by SCD1. These fatty acids in the ester form are the major constituents of VLDL-TGs (VLDL triglycerides) (Peter et al., 2011). Thereby, SCD1 is a hepatic lipogenesis enzyme that mediates the pathophysiology of hepatosteatosis through lipid accumulation (Zhu et al., 2019).

To alleviate the NAFLD hazard effect and its progression to advanced stages, the present study selects an agent that can minimize the expression of FAS and SCD1 as lipogenic enzymes. Therefore, this study selects the yellow-split chickpeas (Cicer arietinum) that are rich with polyunsaturated fatty acids (PUFAs) (Lands, 2014). The dietary PUFAs are modulators of lipid metabolism and can inactivate the key regulators of hepatic lipid synthesis (Ferramosca et al., 2012). Additionally, omega-3 PUFAs may be a new treatment option for NAFLD (Lu et al., 2016). So, this study utilizes the C. arietinum-enriched PUFA (CAP) to assess its effect on hepatosteatosis. Actually, the bioavailability of many extracts specially those containing polyphenolic rings and other water-soluble constituents is very poor; consequently, the therapeutic efficacy of the extract is poor (Angelico et al., 2014). To face this problem and to enhance the effectiveness of the extract, it can be incorporated into a novel delivery system called phytosome (Singh et al., 2014). Phytosome is formed by interaction between herbal extract and phosphatidyl choline (PC) such as soybean phosphatidylcholine. It is an active constituent of soybean and is now using in many applications of drug delivery technology. The advantage of PC is to increase the solubilizing property of many natural products (Chen et al., 2016). Therefore, phytosomes are more bioavailable compared to the single extract, so it can boost the therapeutic effect of a certain extract through its sustained release (Angelico et al., 2014). The CAP-phytosome (CAPP) will develop by thin hydration layer technique to improve the bioavailability of the CAP. Hence, the current work aimed to appraise the efficacy of CAP (C. arietinum-enriched PUFA) and its phytosome formula, C. arietinum-enriched PUFA phytosome (CAPP), in NAFLD rats stimulated by HFD and TAM (common hepatosteatotic-induced drug).

Methods

Chemicals and reagents

Phosphatidyle choline (PC) was purchased from Acros organic (New Jersy, USA). 2, 2-diphenyl-1-picrylhydrazyl (DPPH) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Tamoxifen (TAM) was procured from local pharmacy (Giza, Egypt). Rat FAS and rat SCD ELISA kits were purchased from SinoGeneClone biotech Co., Ltd (HangZhou, China). Kits for determination of liver functions markers, lipid profile parameters, and OxS markers were procured from Biodiagnostic Company (Egypt). Kit for gamma glutamyl transferase (γ-GT) estimation and lactate dehydrogenase (LDH) was purchased from Spectrum Company (Obour City, Cairo, Egypt). Other chemicals and solvents were purchased from local firms.

Preparation of Cicer arietinum-enriched polyunsaturated fatty acids (CAP)

For CAP extraction, the mature unripe chickpea (C. arietinum) coarsely pulverized to powdered form using electrical grinder before extraction. About 1 g of C. arietinum seeds powder was soaked with 2 ml of petroleum ether (m.p. 40–60 °C) overnight at 37 °C. The resulting extract was filtered and concentrated in a rotary evaporator (Salama et al., 2018). The resultant CAP was stored until use. The fraction yield of the CAP was calculated using the following formula:

$${\text{Yield }}\% = \frac{{\text{Weight of CAP}}}{{{\text{Weight of}}Cicer \, arietinum}}\; \times \;100$$

Determination of amount of polyunsaturated fatty acids in CAP

To analyze PUFA (polyunsaturated fatty acids) in the prepared CAP, the gas chromatography-mass spectrometry (GC/MS) chromatograph system with an HP 5973 mass selective detector (TR-FAME, Thermo 260 M142 P) was used (Thurnhofer and Vetter, 2005).

Preparation of Cicer arietinum-enriched polyunsaturated fatty acids phytosome (CAPP)

CAPP is an innovative complex of seed extract and prepared according to thin-layer hydration technique. CAPP is prepared by conjoining one gram of natural phospholipid such as soy lecithin (phosphatidylcholine, PC) with one gram of CAP extract in 100-ml flask with 20 ml of acetone. The mixture was stirred by a magnetic stirrer at 40 °C on a hot plate stirrer for a period of 45–60 min. Consequently, the formed phytosome was dried to form a thin film and at that moment hydrated with dist. water (10 ml). The formulated phytosome suspension was precipitated with 10 ml n-hexane. With continuous stirring, batches of CAPP complex were precipitated and filtered, and then dried to eliminate traces of solvents. The phytosomal suspension was reserved overnight in the refrigerator (Amit et al., 2013). The prepared CAPP was dried properly using a hot plate to remove any volatile substances and water molecules and weighed accurately. This weight was divided by total weight of CAP and PC (Sachin et al., 2019).

$${\text{Practical Yield }}\% \; = \;\left[ {\text{Practical weight of phytosome/Theoretical weight of drug and carrier}} \right]\; \times \;{1}00$$

Characterization of CAPP

Morphological

Light microscopic examination

The CAPP and PC were prepared for light microscopic characterization by suspending in phosphate buffer saline (PBS); then, a drop was put on a slide, covered, and observed under 100X magnification (Sharma and Vivek, 2020).

Transmission electron microscope (TEM)

Examination of CAPP and PC was carried out morphologically using TEM (JEM-1400 microscope; JOEL Ltd Tokyo, Japan). Samples were prepared by dilution with dist. water (1:20) and swirled for 3 min. A drop of each sample dispersion was put into copper grid coated with carbon, forming liquid film. The films then were negatively stained by a drop of ammonium molybdate in 2% ammonium acetate buffer (pH 6.8). Then, the prepared films were examined and photographed using TEM (Abdelkader et al., 2016; Mazumder et al., 2016).

Spectroscopic

Fourier transform infrared spectrophotometry (FT-IR)

To study the interaction of CAP and PC in CAP-PC complex (CAPP), the compatibility between CAP, PC, and CAPP was evaluated using FT-IR spectroscopy (FT/IR-4100 type A, Serial Number B154461016). IR spectra of the CAP and its phytosome were determined by potassium bromide (KBr) pellet technique. Comparable data were obtained at a resolution of 4 cm⁻¹, range of 4000–400 cm⁻¹, and scanning speed of 2 mm/sec. The resultant IR spectra were illustrated regarding the functional groups related with wave number (cm⁻¹). CAP, PC, and the CAPP were analyzed, and comparison between spectra was done (Anwar & Farhana, 2018).

Analysis of drug content (CAP) in CAPP

The content of CAP in phytosomal complex was established by inserting CAPP (10 mg) in methanol (10 ml). The sample was stirred for 1 h, and then, volume completed to 100 ml with methanol and then filtered. 5 ml of the obtained filtrate was diluted with 100 ml methanol. Samples were analyzed at a wavelength of 324 nm spectrophotometrically (Ittadwar & Puranik, 2016). The amount of CAP content was calculated from standard curve and according to the following equation

$${\text{Drug content }}\% = \, \left[ {C_{{{\text{CAP}}}} {/}C_{{{\text{CAPP}}}} } \right] \, \times 100$$

Determination of CAP entrapment efficiency in CAPP

The percentage of CAP encapsulated with PC was determined by using the centrifugation technique to estimate the unentrapped drug. Drug loading (CAP) was determined by addition of 10 mg of phytosomal suspension to 5 ml of 0.1 mol/l HCl and stirring for 30 min. using a magnetic stirrer and then allocated to stand. The phytosomal vesicle was separated in cooling centrifuge at 12,000 rpm for 45 min, at maintained temperature (4 °C). 1 ml of the CAP-PC complex (CAPP) was diluted with methanol. After filtration, the supernatant was analyzed by UV/Vis spectroscopy at wavelength 210 nm (Sharma and Vivek, 2020). The amount of drug entrapped in phytosome was determined by the following equation,

$${\text{Encapsulation Efficiency }}\left( {{\text{EE}}} \right) \, \% \, = \, \left[ {C_{{\text{T}}} - C_{{\text{F}}} } \right]/C_{{\text{T}}} \times 100$$

where C_T: total concentration of CAP that added during phytosome preparation, C_F: free concentration of CAP that unentrapped by PC.

In vitro studies

Phytochemical screening of CAP and CAPP

The present study detects the presence of different active ingredients in CAP and CAPP. These include flavonoids (Trease & Evans, 2002), phenols (Mahadevan & Sridhar, 1982), alkaloids, saponins, tannins, terpenoids, phytosterols, and proteins and amino acid (Arora et al., 2013), glycosides (Siddiqui & Ali, 1997), anthraquinones (Veerachari & Bopaiah, 2011), fats, and fixed oils.

Evaluation of oxidant inhibition potency of CAP and CAPP

Radical scavenging activity of CAP and CAPP against 2, 2 diphenyl-1-picrylhydrazyl hydrate (DPPH) was estimated by Oyedemi et al. (2010). DPPH dissolved in methanol (100 µM DPPH/200 ml methanol) was prepared before use. The next concentrations of CAP, CAPP, and ascorbic acid as a standard antioxidant were prepared: 5, 10, 15, 20, 25, 30, 35, 40, 45, and 50 mg/ml in 90% methanolic DMSO. In each tube, 2 ml of DPPH was added to each concentration and adjusted to 4 ml with methanol. The reaction mixture was stirred and then allowed to stand and incubated in dark for 30 min at room temperature. Control tube contains 2 ml DPPH and 2 ml 90% methanolic DMSO instead of the antioxidant solution. 90% methanolic DMSO was used as blank. Thereafter, the change in absorbance (A) was calculated at 517 nm by spectrophotometer. The DPPH scavenging ability was estimated using the following equation:

$${\text{DPPH radical scavenging activity}}(\% ) = \frac{{A_{{{\text{Control}}}} - A_{{{\text{Sample}}}} }}{{A_{{{\text{Control}}}} }} \times 100$$

In vivo study

Experimental animals

Adult male Rattus norvegicus rats (Wistar albino) of 150 ± 20 g were purchased from the National Research Center (Egypt). Rats were grouped and retained as six animals/cage in air-conditioned room at 23 ± 2 °C with relative air humidity and subjected to natural day and night series. Animals were exposed to laboratory chow pellet foods and water. The rats were kept for 1 week prior to the commencement of the testing for acclimatization.

Induction of non-alcoholic fatty liver disease (NAFLD)

Hepatosteatosis is provoked by intraperitoneal injection of TAM (200 mg/kg b.wt) daily for 15 days, in addition to feeding the rats with HFD (Song et al., 2017).

Experimental design

Forty-eight rats were separated into eight groups (6/group) and treated as follows:

Group (1): Control group, rats were administered dist. water orally for 45 consecutive days. Rats fed on normal diet during the 45 days.

Group (2): PC group, rats were administered dist. water for 15 days and then administrated PC (500 mg/kg b.wt) orally for 30 days. Rats fed on normal diet during the 45 days.

Group (3): CAP group, rats were administered dist. water for 15 days and then administrated CAP (500 mg/kg b.wt) orally for 30 days. Rats fed on normal diet during the 45 days.

Group (4): CAPP group, rats were administered dist. water for 15 days and then administrated CAPP (500 mg/kg b.wt) orally for 30 days. Rats fed on normal diet during the 45 days.

Group (5): NAFLD group, rats were injected intraperitoneally with tamoxifen (TAM) (200 mg/kg b.wt) for 15 days; followed by administration of dist. water for an additional 30 days. Rats fed on HFD during the 45 days.

Group (6): NAFLD + PC group, rats were injected intraperitoneally with tamoxifen (200 mg/kg b.wt) for 15 days, after that administered PC (500 mg/kg b.wt, p.o) for an additional 30 days. Rats fed on HFD during the 45 days.

Group (7): NAFLD + CAP group, rats were injected intraperitoneally with tamoxifen (200 mg/kg b.wt) for 15 days and then administered CAP (500 mg/kg b.wt, p.o) for an additional 30 days. Rats fed on HFD du ng the 45 days.

Group (8): NAFLD + CAPP group, rats were injected intraperitoneally with tamoxifen (200 mg/kg b.wt) for 15 days and then administered CAPP (500 mg/kg b.wt, p.o) for an additional 30 days. Rats fed on HFD du ng the 45 days.

After 24 h of the final treatment, rats were subjected to sodium pentobarbital after being fasted overnight for euthanization. Sera were obtained by blood centrifugation at 3000 rpm for 20 min and stored at − 20 °C until used. Further, rats were dissected, and liver was excised and cut into small pieces. One part was homogenized in Tris HCl buffer (0.1 M, pH 7.4) to obtain 10% homogenate and centrifuged at 3000 rpm for 15 min (Arhoghro et al., 2014). The supernatant obtained was stored at − 20 °C until used. Another liver part was used for histological investigation.

Determination of serum liver function markers

Serum AST (aspartate aminotransferase), ALT (alanine aminotransferase), ALP (alkaline phosphatase, EC 3. 1. 3. 1), total bilirubin (BR 11 11), total protein (CAT. NO. TP 20 20), albumin (CAT. No. AB 10 10) were estimated colorimetrically using Biodiagnostic kit according to the method described by Reitman and Frankel (1957), Belfield and Goldberg (1971), Walter and Gerade (1970), Henry (1964), and Doumas et al. (1972), respectively. The gamma glutamyl transferase (GGT, CAT. No. 246 001) level was estimated using Spectrum kit according to Szasz et al. (1996). Lactate dehydrogenase (LDH, 26.04.21) level was assessed using Bio Systems kit, according to Burtis et al. (2006).

Determination of lipid profile

Serum and liver total lipids, triglycerides (TR 20 30), and total cholesterol were determined using Biodiagnostic kit according to Zollner and Kirsh (1962), Fassati and Prencipe (1982), Allain et al. (1974), Lopes-Virella et al. (1977), and Wieland and Seidel (1983), respectively.

Determination of some lipogenic markers

FAS (fatty acid synthase) and SCD1 (stearoyl—CoA desaturase) were measured in serum by ELISA kits according to manufacture instructions.

Determination of some oxidative and antioxidative stress markers

Liver MDA (malondialdehyde, catalog No. 001-0020) and H₂O₂ (hydrogen peroxide) were measured using biodiagnostic kits according to Ohkawa et al. (1979) and Aebi (1984), respectively. GSH (glutathione reduced, GR 25 10) (Beutler et al. 1977), SOD (superoxide dismutase, SD 2520) (Nishikimi et al., 1972), CAT (catalase, CA2516) (Aebi, 1984), GPx (glutathione peroxidase, GP 2524) (Paglia and Valentine (1967), GRD (glutathione reductase, GR 25 22) (Goldberg 1983) were detected in hepatic supernatant using biodiagnostic kits.

Histological examination

Liver tissue was processed with the standard procedure to prepare sections and then stained by hematoxylin and eosin (H&E) for general histological investigation.

Statistical analysis

Data analysis was conducted using SPSS software (SPSS Inc., Chicago, IL, USA). All data were displayed as means ± SEM. The effect of CAP and CAPP was analyzed statistically by ANOVA (one-way analysis of variance) post hoc with Duncan’s multiple range test. P values < 0.05 were considered significant.

Results

Extraction and analysis of CAP

The percentage yield of the CAP was 42% (w/w) dried extract. Fatty acid gas chromatograph analyses of the CAP showed that the most abundant fatty acid detected in the tested CAP was 31.94% alpha-Linolenic acid (9,12,15-octadecatrienoic acid). Also, CAP contains 30.47% linolenic acid (9,12-octadecadienoic acid), 29.27% 11-octadecenoic acid, 29.09% 9-octadecenoic acid, 23.71% pentadecanoic acid (Fig. 1).

Fig. 1

Gas chromatograph (GC/MS) chromatogram analysis of CAP. 1: alpha-linolenic acid (31.94%), 2: linolenic acid (30.47%), 3: 11-octadecenoic acid (29.27%), 4: 9-octadecenoic acid (29.09%), 5: pentadecanoic acid (23.71%).

Quantity of CAPP and characterization

The practical yield of the final formed CAPP by quantity 1:1 (CAP:PC) is 85% (w/w). The formulation was further characterized.

Morphological characteristics

Light microscopic examination

Optical microscopy was used for the surface morphology of CAPP. The microscopic view displayed the presence of spherical structures of the CAPP which indicate that the method in CAP:PC ratio of 1:1 resulted in the formation of CAP-phytosome (CAPP). Figure 2A exhibits that the formed CAPP was found to be more diffuse and showed no aggregation.

Fig. 2

Microscopic view of Cicer arietinum--enriched polyunsaturated fatty acids phytosome (CAPP). A: light microscope, B: transmission electron microscope photograph

Transmission electron microscopy (TEM)

Figure 2B shows that there were numerous particles floating in the water. For CAPP, the polar portion of phospholipids joined CAP and PC. The structure of vesicles was created as particles swirled in distilled water, arranging numerous complex molecules in an orderly fashion.

Spectroscopy

Fourier transform infrared spectrophotometry (FT-IR)

FT-IR spectra of CAP, PC, and CAPP are shown in Fig. 3. No physico-chemical interaction was formed in the CAPP sample as there were no new peaks formed. When comparing the phytosome spectra to the CAP spectra, there is a slight shift in the hydroxyl group's (OH) tensile vibration peaks to a lower frequency and intensity (3625–3560 cm⁻¹). In phytosome spectra, the band of choline N-(CH3)3 groups is moved to a higher frequency (1099.43–1136.07 cm⁻¹) with less intensity than in PC spectra. Additionally, the phytosome spectrum's intensity was higher than the CAP spectrum at the N–O band area (1250–1650 cm⁻¹).

Fig. 3

FT-IR spectra of CAP, PC, and CAPP

Drug entrapment efficiency and drug content in CAP-PC complex

The entrapment efficiency of CAPP is estimated from the standard curve of CAP. The entrapment efficiency was 99.57% that means vesicles were able to entrap 99.57% of CAP. CAP content of CAPP is estimated from the standard curve of both CAP and CAPP. The drug content in CAPP was 81.66%.

In vitro studies

Phytochemical bioactive screening of CAP and CAPP

Table 1 shows the existence of flavonoids, alkaloids, saponins, glycosides and/or carbohydrates, phytosterols, terpenoids, phenol, betacyanin, quinones, proteins, fats, fixed oils, and tannins in CAP and CAPP.

Table 1 Phytochemical screening of CAP and CAPP

Antioxidant activity of CAP and CAPP

Figure 4A shows that CAP and CAPP exhibited an antioxidant activity, where the violet color of DPPH free radical was converted into yellow color. Since CAP and CAPP scavenged over 40% of the DPPH free radical at the lowest dose (5 mg/ml), Fig. 4B demonstrates their strong potential antioxidant activity. At 45 mg/ml, the CAP showed a strong antioxidant activity. Compared to ascorbic acid (Vitamin C, the standard reference), which showed 98.18% inhibition at this concentration, it demonstrated 72.31% inhibition. However, at 50 mg/ml, CAPP showed a strong antioxidant activity. Compared to ascorbic acid, which showed 98.48% inhibition at this dose, it demonstrated 98.18% inhibition.

Fig. 4

A Qualitative antioxidant activity of CAP and CAPP which shows conversion of violet color into yellow color. B DPPH radical scavenging activity of the CAP and CAPP

In vivo studies

CAP/CAPP modulates liver indices disorders

Data recorded significant rise (P < 0.05) in the levels of AST, ALT, ALP, LDH, GGT, total bilirubin, direct bilirubin, indirect bilirubin and markedly diminution (P < 0.05) in total protein, and albumin levels of NAFLD in contrast with control rats (Table 2). Conversely, hepatosteatotic rats treated with CAP or CAPP showed significant amelioration (P < 0.05) in above-mentioned liver markers comparable with the NAFLD group. The current results showed non-significant change in the levels of liver markers between PC, CAP, CAPP, and control groups.

Table 2 Effect of CAP/CAPP on liver indices disorders of NAFLD group

CAP/CAPP alleviated hepatic lipid accumulations

Regarding serum, significant elevation (P < 0.05) in total lipid, triglyceride, cholesterol, LDL concentrations were recorded in the NAFLD rats, relative with control rats, whereas induction of fatty liver significantly lessened (P < 0.05) the level of HDL in NAFLD rats, relative to control rats. Hepatosteatotic rats treated with CAP or CAPP significantly (P < 0.05) modulate the lipid content changes compared with the NAFLD group (Table 3). No statistically significant difference exists between the control, PC, CAP, and CAPP groups.

Table 3 Effect of CAP/CAPP on serum lipid profile of NAFLD group

Figure 5 shows a significant elevation (P < 0.05) in liver total lipids and TG content in rats suffered from NAFLD in relation to control one. Induction of NAFLD followed by treatment with CAP or CAPP caused a significant reduction (P < 0.05) in liver total lipids relative with NAFLD rats. The statistical data revealed that CAP, PC, and CAPP had no significant variation in the liver total lipids and triglycerides contents relative to the control group.

Fig. 5

Effect of CAP/CAPP on hepatic lipid accumulations. A Liver total lipids and TG content. B Represent lipid accumulation. Values are expressed as mean ± SEM (n = 6). Different bold letters are significantly different (P < 0.05). CAP Cicer arietinum PUFA-rich, CAPP Cicer arietinum PUFA-rich phytosome, NAFLD Non-alcoholic fatty liver disease, PC phosphatidylcholine

CAP/CAPP regulate lipogenesis

Rats with NAFLD had considerably higher (P < 0.05) levels of FSN and SCD1 expression than control rats. Treatment with CAP or CAPP decreases the concentrations of FSN and SCD1 significantly (P < 0.05) as compared with the NAFLD group (Table 4). Statistically, a non-significant alteration was recorded in serum FSN and SCD1 levels between PC, CAP, and CAPP groups and control one.

Table 4 Effect of CAP/CAPP on FAS and SCD1 of NAFLD group

CAP/CAPP boost hepatic antioxidant status

Hepatosteatosis induced hepatic oxidative damage; this is evidenced by a considerable increase (P < 0.05) in the MDA and H₂O₂ content relative to the control group. On the contrary, fatty liver suppresses the GSH, SOD, CAT, GPX, GR contents significantly (P < 0.05) in the NAFLD group in contrast to the control group. Treatment with CAP or CAPP after induction of NAFLD significantly reversed (P < 0.05) the increased MDA and H₂O₂ levels. Further, oral treatment of CAP or CAPP prevented significantly (P < 0.05) the antioxidant suppression induced by NAFLD, as compared to untreated NAFLD rats. No significant difference was disclosed between rats administrated CAP, PC, CAPP, and control rats in the estimated oxidant/antioxidant markers (Table 5).

Table 5 Effect of CAP/CAPP on hepatic oxidative/antioxidative markers of NAFLD group

CAP/CAPP alleviated histological changes

Histological features seen in Fig. 6 of A, B, C, and D pointed to liver of control, PC, CAP, CAPP, respectively, displaying a typical hepatic lobule with a central vein (CV) and hepatocytes' radiating cords with blood sinusoids (s) in between, indicating normal liver architecture. Photomicrographs of the NAFLD group's liver slices displayed different-sized steatotic droplets. Hepatocytes with varying cytoplasmic vacuolation, multiple tiny vacuoles (arrow), and ballooned hepatocytes encompassing large vacuole (arrow) were observed. Also, photomicrograph showing degenerated hepatocytes (*) as a result of TAM treatment. Figures G, H and I show that steatotic droplets were suppressed by treatment with PC, CAP, and CAPP, respectively. It is markedly that steatosis was partially recovered by CAPP treatment as no evidence of histological lesions was observed.

Fig. 6

Photomicrograph showing the effect of CAP/CAPP on liver tissue changes (H&E staining). A Control group showing normal liver architecture. B PC group showing normal liver architecture. C CAP group showing normal liver architecture. D CAPP showing normal liver architecture. E and F NAFLD group showing widespread micro- and macro-vesicular steatosis (arrows) and hepatocyte degeneration (*). G NAFLD + PC group showing amelioration of steatosis. H NAFLD + CAP group had no evidence of fatty liver lesions. I NAFLD + CAPP group showing more or less normal liver without steatotic vesicle

Discussion

The prevalence of non-alcoholic fatty liver disease (NAFLD) has progressed on a huge scale in the last years due to the bad lifestyle. Therefore, this study investigates the possible anti-hepatosteatotic effect of CAP (C. arietinum-enriched PUFA) and CAPP (C. arietinum-enriched PUFA phytosome) in HFD/TAM rat model.

Hepatic steatosis was successfully established in the NAFLD group. This is characterized by several manifestations including weight gain, dyslipidemia, and liver dysfunction (Federico et al., 2021). This is manifested by markedly increment in the serum of AST, ALT, ALP, GGT, LDH, and bilirubin in comparison with control group. This is associated with an obvious reduction in total protein and albumin levels. These findings agree with Park et al. (2020), Eghdami et al. (2024) and Kim et al. (2024). The raise in these hepatic lysosomal enzymes indicates liver damage, since, under normal circumstances, these enzymes reside within the cytosol of hepatocytes. Meanwhile, NAFLD causes hepatocyte membrane oxidative damage, which affects the hepatocyte and hepatobiliary permeability. So, these enzymes are leaked out from the liver into blood (Kafrani et al., 2020). These findings were confirmed histologically through degenerated hepatocytes in NAFLD group in comparison with the normal liver. This interpretation is parallel with Prasomthong et al. (2022) and Yang et al. (2019). Further, the significant decline of total protein in NAFLD group may be attributed to the reduced level of its synthesis in the hepatocytes and/or to the early damage that the endoplasmic reticulum produces (Hassanein et al., 2018). Riechelmann and Krzyzanowska (2019) declared that the reduction in albumin concentration of TAM-treated rats may be attributed to hepatic impairment of albumin synthesis and/or the increased albuminuria, and this explains the reduced level in albumin in the NAFLD group.

The weight gain observed in the NAFLD group may be due to the higher caloric intake that resulted from HFD consumption which induces imbalance between fat consumption and oxidation (Tavares et al., 2020; Wikan et al., 2020; Xia et al., 2019). Thereby, fat accumulates on the long-term leading to weight gain. Additionally, lipid accumulation was recorded in serum and liver by lipid profile markers. This outcome is consistent with Karimimojahed et al., (2020). They added that TAM induces hepatocytes lipid accumulation via boosting the production of fatty acids and triglycerides that are prerequisite for the occurrence of hepatic steatosis. This was approved by highly increasing the triglyceride content that confirmed quantitatively and histologically compared to the control group. Thereby, the current study suggested that the high-fat diet consumption may increase the free fatty acids in blood leading to increasing triglyceride storage in liver and eventually hepatic steatosis. The increased amount of intrahepatic triglyceride may be the main cause of significant hypercholesterolemia and other lipid profile markers in NAFLD group compared with the control one. The present significant change of lipid profile markers is in line with Park et al. (2020).

Further, the intrahepatic contents of fatty acids in tissue give rise to oxidative stress (Obydah et al., 2019) where increased lipid accumulation acts as a strong oxidative agent that causes lipid peroxidation and liver damage (Park et al., 2020). This is established in the NAFLD group by significant raise in the MDA and H₂O₂ parallel with significant reduction of GSH, SOD, CAT, GPx, and GRD contents in relation to the control values. The present results are in consistent with the results of Obydah et al. (2019), Meng et al. (2019), and Park et al. (2020). Hepatic steatosis is usually accompanied by defects in the hepatic mitochondria or oxidative phosphorylation and ROS detoxification (Obydah et al., 2019), since fatty acid oxidation is a vital cause of ROS in hepatic steatosis (Song et al., 2017) where they attack PUFA and initiate the cellular process of lipid peroxidation resulted in the production of MDA as aldehyde by-products. The latter can spread from their start sites to both intracellular and extracellular destinations, which magnifies the impact of oxidative damage.

Mechanistically, the current study reported overexpression of the lipogenic markers including FAS and SCD1 which are consistent with the reports of Choe et al. (2020), Karimimojahed et al., (2020), and Zhang et al. (2020). In fact, there is increasing liver lipogenesis in the resulting from buildup of malonyl-CoA within cells that produced from imbalance between its production from acetyl-CoA by ACC (acetyl- CoA carboxylase) and its breakdown by the malonyl-CoA decarboxylase to acetyl-CoA (Oliveira & Liesa, 2020). Finally, an accumulation of malonyl-CoA leads to the synthesis of fatty acid (lipogenesis) by FAS and inhibition of fatty β-oxidation increasing the formation of ROS (Koundouros & Poulogiannis, 2020), where FAS is a determinant enzyme that raises the hepatic capacity to synthesize fats and control the lipogenesis process (Zhang et al., 2020). Concerning SCD1, it is the finishing phase in de novo lipogenesis that transforms SFAs (saturated fatty acids) to MUFAs (monounsaturated fatty acids). SCD1 catalyzes the formation of MUFAs from SFAs, mainly oleate and palmitoleate by adding a cis double bond in n9 and n7, respectively (Dorn et al., 2010). These data explain the elevation of triglyceride content in serum and liver in the NAFLD group in comparison with the control group.

Administration of CAP or CAPP regulates several parameters including hepatic function, intrahepatic lipid accumulation, ROS production, and lipogenic markers. This is proved by significant modulation of ALT, AST, ALP, GGT, LDH, total and direct bilirubin, TG, total cholesterol, LDL-cholesterol, total lipid content and increased HDL-cholesterol and albumin levels. The present study expected that the ability of CAP or CAPP to alleviate hepatic steatosis may be due to (1) their PUFA content, where PUFA treatment modulates hepatic functions and lipid profile. These interpretations were supported by Ibrahim and El Malkey (2017), Jeyapal et al. (2018), Yan et al. (2018), and Albadrani et al. (2024). (2) Their antioxidant capacity is evidenced by their potency to scavenge the DPPH free radical. Thus, they can avoid the production of free radicals and the progression of oxidative liver damage, consequently stabilize the hepatocellular membrane, and repair the hepatic degeneration.

Further, CAP/CAPP can reduce lipid accumulation in liver which is confirmed by loss in the weight gain, biochemically and histologically as compared with NAFLD group. CAP/CAPP may reduce lipogenecity of NAFLD due to its PUFA content (alpha-linolenic acid, also called omega-3). This is agreed with Nobile et al. (2013) and Lands (2014) who reported that omega-3 and omega-6 are effective in the treatment of NAFLD. They also reported that the main fatty acid in chickpea oil is the linoleic fatty acid, which accounts for 46% to 62% of total fatty acids. This is one of the important PUFA for human metabolism that needs to be included in the diet. Boyraz et al. (2015) clarified that a diet enriched with PUFA can reduce intrahepatic triglyceride content and hence hepatic steatosis, since PUFA activates the fatty acid beta-oxidation, and hence, they can cause the energy balance to change from storage to consumption (Koundouros and Poulogiannis et al., 2020). Indeed, CAP and CAPP significantly reduced the measured lipid profile. Further, phytosterols present in CAP are a group of substances founded in cereals and structurally they like cholesterol that can diminish the amount of cholesterol in the blood by partially preventing the absorption of cholesterol specifically total cholesterol and LDL-C in the digestive system (Song et al., 2017). This suggests the ability of CAP/CAPP to inhibit the de novo cholesterol synthesis or inhibit cholesterol absorption ends by reduction in total cholesterol and triglyceride contents (Park et al., 2020). Further, the mechanism of the hypolipidemic activity of the CAP or CAPP may be due to the presence of phenolic compounds observed by phytochemical screening, as flavonoids and PUFA contents, observed in CAP/CAPP, prevent the catabolism of HDL-C and increase the rate at which LDL-C is catabolized, which lowers serum LDL-C level (Asgari-Kafrani et al., 2020). Moreover, the saponin content of CAP/CAPP may modulate the lipid metabolism, hypercholesterolemia, and hypertriglyceridemia via increasing fecal excretion of bile acids (Almasi et al., 2017; Park et al., 2020). Also, CAP/CAPP inhibits lipogenesis by decreasing FAS and SCD1 activities. This is agreed with Liu et al. (2017), who mentioned that isoflavones present in chickpeas (Fahmy et al., 2015) suppress the expression of FAS. Additionally, flavonoids of chickpeas have an inhibitory effect on the FAS activity as Almasi et al. (2017) mentioned. So, CAP and CAPP may mitigate HFD-/TAM-induced hepatic steatosis by suppressing the activity of hepatic lipogenic markers including FAS and SCD1. These results proposed that treatment with CAP/CAPP may inactivate acetyl-coA carboxylase resulted in inhibition of fatty acid synthesis. This was confirmed histologically by disappearance of lipid droplets that were widely distributed in NAFLD group.

Further, the antioxidant capacity of CAP/CAPP that is confirmed with the DPPH assay may affect the metabolism of lipids and balance the lipid profile and hence reduce the buildup of fat in the liver. This is confirmed by the ability of CAP/CAPP to restore the antioxidant enzymes that reflect the ability of the bioactive compounds of CAP/CAPP to interact directly with ROS and inhibit free radicals. Indeed, the present results suggest that CAP and CAPP treatments may be regarded as a modulator to the mitochondrial function and free radical scavenger. The present study suggested that CAP/CAPP may suppress the MDA significantly by increasing SOD and CAT activities and by preventing the production of lipid peroxide from fatty acids. Phenolic compounds recorded in CAP/CAPP may be responsible for radical scavenging activity (Asgari-Kafrani et al., 2020). It may seem that CAP/CAPP regulates the antioxidant defense system which may explain their protective effect against liver damaging. Also, JuárezHernández et al. (2016) and Haque and Ansari (2019) found that PUFA exhibits antiobesity, antisteatotic, anti-inflammatory effects, and antioxidant action by increasing the antioxidant enzymes' activity. The present study suggested that PUFA can prevent the decrement of GSH level by defending GSH’s SH group against reactive radicals. Further, CAP/CAPP can restore the activity of GRD, thus speeding up the conversion of GSSG to GSH and improving the detoxification of reactive metabolites through GSH conjugation.

It seemed that CAPP is more effective in its anti-hepatosteatotic effect than CAP. Pharmacokinetic studies also proved the higher relative bioavailability of phytosomes than plant extracts and their constituents at an equal dose (Kumar et al., 2021). Because of its improved ability to pass through lipid-rich biomembranes and enter circulation, the phytosome is typically more accessible than a usual herbal extract (Rahman et al., 2020). This could be because PUFA and phosphatidylcholine combine to produce a PUFA-phytosome, which can easily pass through the intestinal wall and enter the liver in large amounts (Ibrahim & El-Malkey, 2017). Efficacy of the preparation method of CAPP was confirmed by the high phytosome yield (90%, w/w). The CAP content in the form of a CAP-phytosome (CAPP) was found to be 81.66%. The elevated CAP content in the complex indicates the formulation of a stable complex between CAP and PC using a thin-layer hydration method that was confirmed by IR, light microscope, and TEM microscope (Ittadwar & Puranik, 2016). The in vitro release pattern shows that the phytosome complex released CAP in a steady and regulated manner. This indicates that over a longer period, the phytosome vesicles containing CAP achieved the highest percentage of extract release.

Conclusion

Hepatosteatosis is achieved by TAM + HFD, as TAM metabolized in the liver produced a large amount of ROS that damages the hepatocyte membrane and release large amounts of liver enzymes into the bloodstream. Also, TAM decreases the beta-oxidation of FFAs, so it increases the circulating lipids. Correspondingly, HFD increases the fatty acids intake that in turn increases lipid profile and lipogenic marker parameters. CAP/CAPP considers appropriate therapy plan for preventing or treating hepatic damage brought on by NAFLD. CAPP has therapeutic capacity in the improvement in liver functions, lipid profile, lipogenic markers, and oxidative stress markers. These results imply that CAP and CAPP may ameliorate hepatosteatosis through decreasing lipogenesis by the inhibition of FAS and SCD-1 as well as oxidative stress thus reducing the fat accumulation in the liver (Fig. 7). According to the current study, CAPP is more effective than CAP for preventing NAFLD. CAPP is a viable approach for enhancing the delivery of CAP and other phytoconstituents with low water solubility.

Fig. 7

Schematic representation about the effect of CAPP on hepatosteatosis. ACC acetyl- CoA carboxylase, CAPP Cicer arietinum-enriched PUFA phytosome, FAS fatty acid synthase, HFD high-fat diet, PC phosphatidylcholine, ROS reactive oxygen species, SCD1 stearoyl-CoA desaturase 1, TAM tamoxifen