A Pilot Study of the Amelioration of Avocado Seed Oil in Obese Female Rats Induced by Carbon Tetrachloride and Alloxan Monohydrate

Received: 02-May-2022, Manuscript No. jdar-22-64589; Editor assigned: 04-May-2022, Pre QC No. jdar-22-64589 (PQ); Reviewed: 18-May-2022, QC No. jdar-22-64589; Revised: 23-May-2022, Manuscript No. jdar-22-64589 (R); Published: 30-May-2022, DOI: 10.4303/jdar/236177

Introduction

Obesity is an asymmetrical body weight for height with adipose tissue, lipid, and glucose disturbance in the body [1]. The body mass index (BMI) and hyperlipidemia are the main clinical and diagnostic tests for obesity [2]. Chronic obesity linked with diabetes can result in sustained hormonal disorder, inflammation, and cell death in body organs, such as the kidneys, nerves, blood vessels, and others [3,4].

The prevalence and incidence of obesity due to stress, a western sedentary lifestyle and an energy-dense diet, hormonal disorder, and genetic predisposition [5]. Extra adiposity raises the risk for cognitive damage and various maladies [6]. Moreover, obesity is a risk factor for depression, heart disease, rheumatoid arthritis, fibromyalgia, type 2 diabetes, certain types of tumors, and others disorders [7]. Previous trials reported that dieting, exercising, pharmacotherapy, and surgery are the main treatments for obesity [8,9]. Moreover, natural diets and herbs are the most famous to control chronic obesity, hyperlipidemia hyperglycemia, and other related complication [4,10].

Avocados are considered in the Lauraceae family with various beneficial effects. Avocados include phytochemicals, vitamins, minerals, phenolics, and healthy fatty acids. Previous researchers reported that avocados can downregulate hyperlipidemia, hyperglycemia, inflammation, and disturbance in hormone balance [11-13]. Recent reports reveal that volunteers who used avocado had more inferior BMI, body weight, and waist circumference than others participants [6]. Moreover, Thompson et al, reported that daily avocado consumption resulted in ameliorated fecal bile acid, and fiber fermentation bacteria that affect digestive physiology and mood [14]. As well as, other investigators concluded that avocado fruit extract act as a hypolipidemic agent by regulating lipid metabolic enzymes in the obese rat liver [15].

From this point of view, in this study, we examined the results of treatment with avocado (Persea Americana) seeds extract (AVSE) on alloxan monohydrate and CCl4-induced obesity and biochemical alterations in a female rat model. The trial’s purpose is to analyze the effect of AVSE on apoptosis, hormone, and diabetes indicators, such as glucose, caspase-3, DNA fragmentation, prolactin, leptin, TSH, T3, and total T4 levels induced by monohydrate and CCl4 in rats. We also observed the differences in the weight change, liver, and lipid profile in ten weeks of the rat investigation.

Materials and Methods

The plant study

The plant materials: We gained the avocado (Persea americana) from, Saudi Arabia for this investigation, where, Figure 1 illustrates the investigational design. The avocado seeds were smashed and soaked with a solvent (1:6 w/v), where the solvent workings were hexane and ethanol (3:1). After being placed in the solvent for 30 minutes at room temperature (25 ± 4^°C), the extracts were intense with a 45^°C-65^°C rotary evaporator; we gained a total AVSE the avocado (Persea americana) concentration yield of 72% [13].

Figure 1: The experimental design.

Phytochemical components analysis: We trailed the technique of Bolajoko and Onyeaghala, 2020 for phytochemical contents of the avocado (Persea Americana) seed extract (AVSE) investigation for total phenolic, tannin, flavonoid, and alkaloids via a UV-Vis spectrophotometer 1000 series CECIL Instruments Limited, Milton Technical center Cambridge CB24 6AZ, England [4,16].

Metals analyses: We preceded our earlier method for inductively coupled plasma-mass spectrometry ICPMS (Agilent, 7500 cx) after digesting method to the avocado (Persea americana) seed oil with nitric acid via a microwave digestion technique (Milestone, Ethos 1) to investigate K, Na., Ca2+, Zn, iron, Pb, Cd, Al levels in AVSE [17].

Gas chromatography-mass spectrometry analysis: We tracked our preceding technique for GC–MS examination to identify the chemical contents of the avocado (Persea americana) AVSE extract via a gas chromatography system (G3440B, Agilent Technologies, USA) [13]. We then investigated the WILEY and NIST (National Institute of Standards and Technology) mass spectral libraries to recognize the chemical compositions of the AVSE extract [13].

The rat study

Animals: We had 60 adult female Sprague-Dawley rats weighing 120 ± 2 g in this study. The rats were tested for their health status at 26^°C and given a standard diet and water daily for two weeks before the experiment began. After adaptation, the rats were separated into four groups of 15 female rats each. All animal experiments were accepted by the Experimental Animal Care department committee and followed the Three Rs (Replacement, Reduction, Refinement) [3]. We complied with all institutional and national guidelines for the care and handling of laboratory rats [3]. The four groups of animals alienated of the following: a control group of untreated rats; the obese group of rats injected with an intraperitoneal dose of 150 mg/kg alloxan monohydrate were acquired from Sigma-Aldrich (USA), once [4], then we injected rat subcutaneous, (SC) with 3 ml of CCl4/kg/week for 9 weeks [12]. An AVSE group of rats was treated with a daily oral dose of AVSE at 100 mg/ kg bm for ten weeks [18]. An obese +AVSE group of rats was inserted with alloxan monohydrate and CCl4 at the same dosage as the obese group then a daily oral dose of AVSE as the AVSE group for ten weeks [12,18]. After the experiment period, we obtained the serum separately for the biochemical assay.

Biochemical assays: We followed our previous method for the glucose level assay according to the method designated by Sarfraz et al, 2017 [4,19].

We analyzed caspase-3 using the standard protocol for kit BioSource International, Inc, and we measured the absorbance of the yellow color at 405 nanometers (nm) against a blank conduct a microplate reader (Bio-Tek Instruments, Germany). The elevation in caspase-3 activity was detected by direct comparison to the level of the control as optical density according to our previous work [20]. We analyzed DNA Fragmentation according to our previous study using a DNA purification kit [20]. Then, DNA was practical by electrophoresis on 1% Agarose gel including ethidium bromide, and visualized under UV light (Turnhout, Belgium) [20].

We analyzed total cholesterol, low density lipoproteins (LDL cholesterol [LDL-C]), high density lipoprotein (HDL cholesterol [HDL-C]), triglycerides, and the aspartate transaminase (AST) and alanine transaminase (ALT) level according to our previous study [3].

We followed our previous work and analyzed serum prolactin, TSH, T3, and T4 levels using the standard protocol for available kits by Roche/Hitachi Cobas e 601 analyzers (Roche Diagnostics, Mannheim, Germany) by electrochemiluminescence Immunoassay [21].

We analyzed Serum levels of leptin, rat ELISA kits (8400 Baltimore Avenue, MD, USA) according to the manufacture instructions [22].

Statistical analyses

The outcomes data were studied according to our preceding publication using IBM SPSS software version 20.0 (IBM Corp, Armonk, NY). The significance level of the tests detected was set at 5% [3].

Results

Table 1 represents the measured phytochemicals, and metal insides of AVSE The avocado (Persea americana) extracts.

Table 1: Analysis of phytochemical components and metals of AVSE.

Table 2 reports the chemical composition of AVSE, the avocado (Persea americana) extracts identified by GC-MS [13].

Table 2: Gas chromatography-mass spectrometry analysis of the avocado (Persea americana) seed oil extract, AVSE.

No.	Name	Base Peak	RT	Chromatogram
1	Cyclodecacyclododecene, 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18-octadecahydro-	94.1	12.3
2	9-Octadecenamide, (Z)-	59	14.02
3	2H-Pyran, 2-(7-heptadecynyloxy)tetrahydro-	81.1	12.89
4	E,Z-3,13-Octadecadien-1-ol acetate	81.1	10.37
5	1-Hexadecyn-3-ol, 3,7,11,15-tetramethyl-	69	13.11
6	Androstan-17-one, 3-ethyl-3-hydroxy-, (5.alpha.)-	94.1	13.35
7	Chrysene, octadecahydro-	94.1	10.92
8	9-Octadecenamide, (Z)-	59	14.08
9	cis-11-Eicosenamide	59	15.14
10	Bicyclo[10.8.0]eicosane, (E)-	81	11.79
11	Hexadecanamide	69	12.95
12	dl-.alpha.-Tocopherol	430.4	20.03
13	Formic acid, 3,7,11-trimethyl-1,6,10-dodecatrien-3-yl ester	121	13.78
14	cis-10-Nonadecenoic acid	85.1	14.7
15	Farnesol isomer a	69	13.3
16	3-Chloropropionic acid, heptadecyl ester	55	9.58
17	Z,Z-4,6-Nonadecadien-1-ol acetate	95.1	13.97
18	2H-Pyran, 2-(7-heptadecynyloxy)tetrahydro-	85.1	15.72
19	1-Heptatriacotanol	95.1	16.15
20	Oleic Acid	55	11.54
21	Stigmastan-3,5-diene	396.4	19.83
22	Phenol, 2-undecyl-	94.1	10.96
23	.gamma.-Tocopherol	151.1	19.05
24	p-Menth-8-en-3-ol, acetate	121	12.52
25	Caryophyllene oxide	95	13.17
26	Heptacosane	57	17.48
27	Propanoic acid, 3-mercapto-, dodecyl ester	57.1	11.9
28	2-methyltetracosane	43	11.04
29	1,2-15,16-Diepoxyhexadecane	69	13.21
30	2-Bromotetradecane	71.1	9.874
*Notes: RT: Retention times (minutes); PA: peak area (%). GC–MS investigation was done for the Hexane: ethanoic (3:1) extracts and applied at Agilent Technologies (G3440B, USA). The composition of AVSE seed extract was standard by computer simulations in commercial libraries of Wiley and NIST (National Institute of Standards and Technology).

^*Notes for Tables 2: GC–MS investigation was done for the Hexane: ethanoic (3:1) extracts and applied at Agilent Technologies (G3440B, USA). The composition of AVSE seed extract was standard by computer simulations in commercial libraries of Wiley and NIST (National Institute of Standards and Technology).

Figure 2 shows that alloxan monohydrate and CCl4 induced apoptosis which was indicated by a significant (p ≤ 0.05) elevation in the level of caspase-3(316.7%) and DNA fragmentation compared to the control group. Otherwise, the treatment of AVSE after alloxan monohydrate and CCl4 administration (obese +AVSE group) significantly (p ≤ 0.05) reduced the level of caspase-3 (42%) compared to handling with alloxan monohydrate and CCl4. On the other hand, the Agarose gel electrophoresis presented low DNA laddering in the serum of the control group. The DNA whole band seemed to contract near the application point with no DNA smearing, signifying no DNA fragmentation. Otherwise, alloxan monohydrate and CCl4 administration resulted in huge DNA fragmentation compared to Control (Figure 2B). While treatment with AVSE alone exhibited no DNA fragmentation. Treatment with AVSE during and post-monohydrate and CCl4 injection reduced DNA fragmentation compared to treatment with monohydrate and CCl4 alone (Figure 2B).

Figure 2: Apoptosis comparison of the different groups of the female rat’s study. (A) Caspase-3 activity (B) DNA fragmentation levels in four rat groups. Control=untreated rats; the obese group=rats injected with an intraperitoneal dose of 150 mg/kg alloxan monohydrate, once, then we injected rat subcutaneous, (SC) with 3 ml of CCl4 /kg/week for 9 weeks; the AVSE group=rats treated with a daily oral dose of AVSE at 100 mg/kg bm for ten weeks; the obese +AVSE group=rats inserted with alloxan monohydrate and CCl4 at the same dosage as the obese group then a daily oral dose of AVSE as the AVSE group for ten weeks. Data are given as the mean ± SD for 15 rats. Statistical significance was set at p ≤ 0.05; means signed by the same letters are not significantly different (a, b, c, a). DNA fragmentation in serum. DNA fragmentation separating in 1% agarose gel electrophoresis and pictured under UV. DNA in serum (lane 1, 6-control, lanes 2, 3-obese, lanes 4-obese-AVSE, lane 5-AVSE). The DNA complete band seems to shrink close to the application well with no DNA smearing, signifying no DNA fragmentation.

Figure 3 shows the results for the different parameters studied, given as mean ± SD for 15 rats. Figure 3 shows that alloxan monohydrate and CCl4 initiated hormonal disturbance, obesity, hyperlipidemia, and hyperglycemia in animals, which was indicated by a significant (p ≤ 0.05) elevation in the levels of prolactin (↑ 103.1%), leptin (↑ 136.8%), TSH (↑83.3%), and decrease in T3 (↓ 32.3%), total T4(↓99.5%) compared to the control group. This was linked to a significant (p ≤ 0.05) increase in total cholesterol (49.7%), triglycerides (94.8%), LDL-C (24.3%), AST (126.2%), ALT (120%), and glucose (531.5%) related to the control group, while the obese group presented a significant 27.6% decline in HDL-C round to the control group.

Figure 3: Biochemical comparison of serum of the different groups in the female rat study. (A) Prolactin and Leptin; (B)TSH (thyroid-stimulating hormone), and T3(Triiodothyronine); (C) total T4 (Thyroxine); (D) glucose; (E) Total cholesterol, triglycerides (TG), ), high-density lipoprotein (HDL), low-density lipoprotein (LDL), aspartate transaminase (AST) and alanine transaminase (ALT) levels in four rat groups; Control=untreated rats; the obese group=rats injected with an intraperitoneal dose of 150 mg/kg alloxan monohydrate, once, then we injected rat subcutaneous, (SC) with 3 ml of CCl4 /kg/week for 9 weeks; the AVSE group=rats treated with a daily oral dose of AVSE at 100 mg/kg bm for ten weeks; the obese +AVSE group=rats inserted with alloxan monohydrate and CCl4 at the same dosage as the obese group then a daily oral dose of AVSE as the AVSE group for ten weeks. Data are given as the mean ± SD for 15 rats. Statistical significance was set at p ≤ 0.05; means signed by the same letters are not significantly different (a, b, c, a).

The treatment of AVSE after alloxan monohydrate and CCl4 administration (obese +AVSE group) significantly (p ≤ 0.05) regulated the considered parameters prolactin, leptin, TSH, T3, and total T4 levels by ↓35.4%, ↓40%, ↓13.6%, ↑ 18.2%, and ↑9207%, respectively, compared to handling with alloxan monohydrate and CCl4 (Figure 3). This was accompanied by a significant (p ≤ 0.05) reduction in total cholesterol, triglycerides, LDL-C, AST, ALT, and glucose by 17%, 35%, 10.9%, 34.7%, 34.3%, and 51% respectively, compared to the concentrations in the obese group (Figures 3B and 3C). Otherwise, the obese +AVSE group obtainable a significant 21% rise in the HDL-C serum concentration compared to that of the obese group, p ≤ 0.05; (Figures 3D and 3E). While the rats treated only avocado seed oil extract for ten weeks (the AVSE group) presented no variation in the levels of all studied parameters (Figures 2 and 3).

On the other hand, Figure 4 shows the results for the female rat weight, given as mean ± SD for 15 rats and the gradual increase of the weight in four studied groups. Figure 4 shows that alloxan monohydrate and CCl4 induced weight increase and obesity as compared to control. Otherwise, the treatment of AVSE after alloxan monohydrate and CCl4 administration showed a significant reduction in weight as compared to the obese group. Furthermore, there is a significant reduction in the weight in AVSE alone as compared to control without any change in the remaining studied parameters as compared to the control (Figures 2-4).

Figure 4: Body Weight comparison of the different groups in the female rat study. (A) Mean of final weight gain >over 10 weeks in all studied groups (B) the gradual change in body weight over 10 weeks in all studied groups. Control=untreated rats; the obese group=rats injected with an intraperitoneal dose of 150 mg/kg alloxan monohydrate, once, then we injected rat subcutaneous, (SC) with 3 ml of CCl4/kg/week for 9 weeks; the AVSE group=rats treated with a daily oral dose of AVSE at 100 mg/kg bm for ten weeks; the obese +AVSE group=rats inserted with alloxan monohydrate and CCl4 at the same dosage as the obese group then a daily oral dose of AVSE as the AVSE group for ten weeks. Data are given as the mean ± SD for 15 rats. Means signed by the same letters are not significantly different (a, b, c, d).

Discussion

Obesity is associated with inflammatory diseases, hyperglycemia, infection aggravation, cell death, and increased death rates in the world [23,24].

The obesity obstacle is marked by increased disorder in hormone balances, lipid, and glucose metabolism. Also, obesity associated with an accumulation of pro-oxidant, hyperlipidemia, and inflammatory mediators is an inducer of apoptosis and cell death; the result in this study for the group with alloxan monohydrate and CClL4-induced obesity, hyperlipidemia, hyperglycemia, and apoptosis, compared to the control group, agrees with this [12,23].

This alarm may lead to the diseases such as blood sepsis initiation cell death, and multiple disorder which may increase pathological disorders such as immune, cardio, and hepatic disturbances, especially in individuals with raised genetic susceptibility [25,23].

Our results are shown in Figures 2 and 3 agree with previous reports that obesity and diabetes activate hormonal disturbance and apoptosis responses. We found higher levels of caspase-3, DNA fragmentation, prolactin, leptin, TSH (thyroid-stimulating hormone), T3(Triiodothyronine), and total T4 (Thyroxine) and weight, glucose, lipid, and liver profiles caused by alloxan monohydrate and CCl4 in rats, compared to the control group (Figure 3).

A previous investigation reported that obesity and hyperlipidemia increased infertility in female rats and disturbance of estradiol, progesterone, luteinizing hormone (LH), and upregulation prolactin levels agree with the current investigation [26].

Our results are in agreement with our previous work that reported elevation in lipid and liver profiles and disturbance in the hormones with thyroid hormones including TSH, T3, and total T4 after administration of CCl4 [12]. To initiate obesity and diabetes that can trigger pro-oxidants, hormonal change, inflammatory reactions, and pro-apoptosis, we injected the female rats in this investigation with alloxan monohydrate and CCl4. According to our previous work alloxan monohydrate induced diabetes in rats and elevated, lipid and glucose levels [4]. Furthermore, CCl4 and its metabolite, trichloromethyl free radicals, reacted with protein and lipid within the cell; this reaction enhanced with oxygen and produced free radicals, which attack lipids on the membrane and led to cell death [27-29]. As well as, CCl4 initiation fatty liver and lipidemia in many of the previous investigations, which agree with the current study. Moreover, CCl4 induced the inflammatory protein, lipid peroxidation, liver injuries, and hyperlipidemia in the systemic oxidative stress necroinflammation in turn affecting the thyroid and other rat body organs, which agree with the current study [30,12].

The present work stated the elevation of caspase-3, DNA fragmentation, prolactin, leptin, TSH (thyroid-stimulating hormone), T3 (Triiodothyronine), and total T4 (Thyroxine) levels results in hyperglycemia, hyperlipidemia, obesity, weight, and diabetes complications. A current investigation represented an elevation in all studied parameters in the obese groups more than in the control group in the rat study.

Dealing with obesity and diabetes includes medication, insulin medicine, a healthy diet, and an energetic lifestyle. Herbal medicine and functional food including fruits and vegetables are measured as the most current choice recently [10,31].

The avocado (Persea Americana) is a constituent of the Lauraceae family. Our recent investigation reported that avocado seeds contain components such as palmitic acid and volatile compounds, androstane-17-one, 11-eicosenoic acid, monounsaturated omega-9 fatty, alpha-tocopherol, and gamma-tocopherol [13], which agree with the current study (Table 1). Moreover, previous reports indicate that avocado seed oil contains unsaturated fatty acids, tocopherols, tocotrienols, phytosterols, carotenoids, polyphenols, vitamins, and healthy minerals which agree with the current study (Tables 1 and 2).

The present study identifies beneficial phytochemicals, sterols, healthy fatty acids, carotenes, and flavonoids that have numerous medicinal. The present study reported that AVSE included polyphenols, flavonoids, and Tannins (Tables 1 and 2). Our present investigation also stated that AVSE contains essential nutrients such as K, Na., Ca2+, Zn, and iron with the absence of heavy toxic metals Pb, Cd, and Al (Table 1) which reflect the avocado (Persea americana) seed extract a healthy natural herbal medication. These results agree with the previous investigation that reported that the avocado (Persea americana) seed extract is used as an anti-obesity, anti-inflammatory, anti-apoptotic, and antioxidant treatment [31-33].

Our prior publication stated that avocado seed oil extract has a synergetic repressive as an anti-inflammatory, antistress, and anti-oxidant and concluded docking trial binding free energy with exact receptor as an advantageous antioxidant [13]. As we reported before and in this current investigation that avocado seed oil contains octadecenamide, eicosenamide, healthy sterol, hexadecanamide, oleic acid, phytochemistry, tocopherol, pyran, and their byproducts; these have antistress, antidepressant, sedation mood effect, antioxidant, anticancer, antibacterial, and antilipidemic effects [3,13,20,34,35] These polyphenols have a significant role as, antidiuretic, and antilipidemic antioxidants to capture free radicals [4].

Bottom line, the current study agrees with the previous investigation, where we reported an improvement in the measured biological parameters after treatment with avocado seed oil extract in female rats against the administration with alloxan monohydrate and CCl4 obese group compared to the control. As well as after oral administration of AJSO to rats following obese initiation, there is a regulation in the level of caspase-3, DNA fragmentation, prolactin, leptin, TSH (thyroid-stimulating hormone), T3 (Triiodothyronine), and total T4 (Thyroxine) and weight, glucose, lipid, and liver profiles in the obese +AVSE group than in the obese group. Furthermore, there are advancements in the weight in AVSE alone without any change in the remaining considered analysis as compared to control. This may be due to the combination of avocado seed phytochemical synergistic advantageous outcome through the reduction of apoptosis, hyperlipidemia, hyperglycemia, and hormonal regulation that led to enhancement in biological profiles.

Conclusion

We reported the management of CCl4 and alloxan monohydrate induced hormonal disturbance, obesity, diabetes, and apoptosis by AVSE extract. Where, there was a decline in caspase-3, DNA fragmentation, prolactin, leptin, TSH (thyroid-stimulating hormone), T3 (Triiodothyronine), total T4 (Thyroxine), weight, glucose, lipid, and liver profiles in the obese +AVSE group compared to the rats treated with CCl4 and alloxan monohydrate. The percent of change obese +AVSE group to the obese group; caspase-3, prolactin, leptin, TSH, T3, and total T4 levels showed adjustment of ↓ 42%, ↓ 35.4%, ↓ 40%, ↓ 13.6%, ↑ 18.2%, and ↑ 9207% respectively. Furthermore, the percent of the diminishing change between the obese +AVSE group to the obese group; showed in total cholesterol, triglycerides, LDL-C, AST, ALT, and glucose by 17%, 35%, 10.9%, 34.7%, 34.3%, and 51% respectively. We confirm AVSE extract is the opening point herbal treatment for the prevention/control of hormonal disturbances, obesity, diabetes, and apoptosis.

Acknowledgments

The authors are grateful to the universities and institutions that helped in this research.

Author Contributions

Asmaa Fathi Hamouda and Shifa Felemban have equally contributed to designing and accomplishing the experiments, analyzing and interpreting the results as well as writing and revising the manuscript.

Competing Interests Disclaimer

No competing financial interests exist.

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