Introduction
Plants with healing properties were used in folk medicine and are considered traditional therapeutic approaches that have positive effects on health. They are also advantageous from a cost–benefit point of view(Reference Kumar, Karthik and Rao1,Reference Vij and Prashar2) . Synthetic drugs are used as the first option for the treatment of various diseases. However, due to adverse effects shown by long- or even short-term consumption, research is exploring alternative therapies in the treatment and prevention of diseases(Reference Gooda Sahib, Saari and Ismail3). One alternative therapy includes the use of plants and their fruits. Because they cannot be categorised or defined either as food or a drug, they are understood in the category of food supplements, with beneficial properties for health maintenance, in particular for some pathologic conditions such as metabolic disruptions that are risk factors for non-communicable diseases(Reference Quagliariello, Vecchione and Coppola4).
Among plants with beneficial properties on health is Carica papaya L., the well-known papaya. Papaya is a popular fruit which originates from the tropical America(Reference Parni and Verma5) and belongs to the Caricaceae family. It is widely cultivated in most parts of the world including some African countries, and its principal markets for consumption are the United States and Europe(Reference de Oliveira and Vitória6). Papaya has been described as a powerhouse of nutrients, antioxidants like vitamins A, C, D and E(Reference Aravind, Bhowmik and Duraivel7) and carotenoids(Reference Bari, Hassan and Absar8). It has many medical benefits including, anticancer, anti-hypertensive, anti-inflammatory, antibacterial, gastrointestinal-related disorders and hypoglycaemic effect(Reference Aravind, Bhowmik and Duraivel7). This fruit contains considerable concentrations of phenolic acids, flavonoids and a lipidic composition that reduces inflammatory markers and anti-platelet aggregation, protects against thrombogenesis and oxidative stress, and prevents dyslipdaemia factors that can be triggered by obesity(Reference Vij and Prashar2,Reference Teixeira, Lages and Jascolka9) . The presence of vitamins, bioactive compounds and lipids of biological and nutritional importance in papaya has led to several studies in the treatment of metabolic dysfunctions which can either be related to obesity, resulting in use of papaya as an alternative therapeutic approach when dealing with metabolic syndromes(Reference Santana, Inada and Espirito Santo10).
Carica papaya L. is a plant that is easily accessed in most tropical and subtropical areas and widely available. Furthermore, scientific studies have demonstrated the biological activities and medicinal applications of different parts of the plant(Reference Vij and Prashar2). However, few studies have demonstrated the therapeutic potential in metabolic dysfunctions in experimental models specific to dyslipidaemia. For instance, research on papaya consumption effects on obese rats has shown that papaya reduced lipid absorption as well as the anti-obesity, anti-dyslipidaemia and anti-inflammatory effects in obese rats(Reference Od-Ek, Deenin and Malakul11). In another study by(Reference Zhuhua, Zhiquan and Zhen12), it was observed that obese rats fed on papaya fruits increasing high-density lipoprotein (HDL) and reducing total cholesterol (TC), triglycerides (TG), low-density lipoprotein (LDL), suggesting that papaya fruits would be helpful in prevention of obesity and comorbidities by reduction of dyslipidaemia(Reference Moreno-Fernández, Garcés-Rimón and Vera13). Papaya juice is commonly used in rat experiments due to its ease of administration, nutrient availability, preservation of bioactive compounds and palatability. The main objective of the present study was, therefore, to determine the phytochemicals, nutritive value of the papaya juice and its lipidaemic effects on male Albino Wistar rats fed on a high fat and fructose diet (HFFD) in Kenya.
Materials and methods
Study area description
The experiments were conducted between September and October 2021 at the Small Animals Facility for Research and Innovation (SAFARI) laboratory at Jomo Kenyatta University of Agriculture and Technology (JKUAT). All aspects of animal care and experimentation were performed in accordance with the Guide for animal care and handling at the JKUAT. The study was approved by the JKUAT Institutional Ethics Review Committee (Approval number: JKU/IERC/02316/0101).
Papaya sample and HFFD preparation
The fresh papaya was bought from Juja market, Kenya. The fruit was washed under clean water, cut, deseeded and its pulp was blended, freeze-dried (Yamato Scientific Co., Ltd, Japan) and powdered to a 100 g. Papaya juice was made by mixing the freeze-dried papaya pulp with distilled water in a 100 mg to 1 ml ratio (100 mg papaya:1 ml distilled water), and it was orally administered on rats daily through gavage per 100 g body weight. The HFFD was prepared by mixing 20 g of fructose and 40 g of lard with 100 g of crushed rat pellets(Reference Kiage-Mokua, Roos and Schrezenmeir14). A standard 15 g of HFF diet was daily fed to each rat together with their different papaya treatments. The nutritional composition of the HFFD and papaya is provided in Table 1.
Table 1. Proximate, nutritional and biochemical composition of the HFF diet and papaya
Results are expressed as mean ± sd (standard deviation).
Grouping of rats and diets
The sample size of rats was determined using the resource equation approach(Reference Arifin and Zahiruddin15). Twenty-five, 7 weeks old, male Albino Wistar rats, weighing 185 ± 17 g, were obtained at SAFARI, JKUAT for experimentation. The rats were housed in groups of five in wired cages, with shredded paper bedding which was changed weekly, in a 12-hour light–dark environment for a period of 42 d. Rats were given normal rat pellets and water ad libitum during the one-week acclimatisation period. At 8 weeks, the animals were randomly allocated to different treatments using Microsoft excel, fed on a HFFD for a week and papaya juice was orally administered to them by gavage(Reference Turner, Vaughn and Sunohara-Neilson16) according to their treatments. The rats groupings were as follows: Treatment 1: HFFD only, Treatment 2: HFFD + 200 mg papaya juice, Treatment 3: HFFD + 350 mg papaya juice and Treatment 4: HFFD + 500 mg papaya juice.
Data collection
Measurement of body weight and glucose
Although the research did not have any human endpoint, important clinical signs of distress were monitored like rapid weight loss and blood glucose fluctuations. Weekly monitoring of body weights was done, taking note of any signs of anorexia and failure to drink, using a weighing scale until they were sacrificed and expressed as mean body weight in grams. Blood glucose levels were also determined using a CareSens Fit (i-SENS, Inc., Germany) monitoring system by collecting blood drops on the test strips, after tail pricking using a sharp surgical blade. Rats were fasted for not less than 16 h prior to weight and glucose determination.
Blood collection and organ harvesting
Blood collection was done by a SAFARI surgeon, initially after the acclimatisation week and at the end of the experiment during scarification. Before collecting blood, rats were fasted for 16 h and then anaesthetised using 99⋅8 % Chloroform (Sigma-Aldrich, Germany). Blood samples were obtained initially by the intraperitoneal injection and later by cardiac puncture of the hepatic vein and collected using a 23-gauge needle in ethylenediaminetetraacetic acid (EDTA) tubes for further analysis. Liver samples, visceral and subcutaneous fats from the animals were harvested, weighed, snap frozen using liquid nitrogen (BOC Kenya Plc) and stored in sterilised vials at −80 °C for further analysis and/or storage.
Determination of rats’ blood lipid profile
Estimation of TC, triglyceride (TG) and high-density lipoprotein cholesterol (HDL-c) was done according to the enzymatic assay method(Reference Ezekwe, Elekwa and Osuocha17) using analytical kits (Biolab SA Maizy, France). Low-density lipoprotein cholesterol (LDL-c) was determined based on the following calculations(Reference Friedewald, Levy and Fredrickson18):
Determination of the atherogenic and coronary risk indexes
Atherogenic index (AI) was calculated using the formula of(Reference Abbott, Wilson and Kannel19) while coronary risk index (CRI) was obtained by the method of(Reference Alladi and Radha20). The following formulas were used:
Analytical procedures
Proximate composition of papaya and the diets was done using the(Reference Cunniff21) methods while the ascorbic acid content was determined by the high-performance liquid chromatography (HPLC) method(Reference Vikram, Ramesh and Prapulla22). Mineral quantification was done using the atomic absorption spectroscopy (AAS) method according to(Reference Singh, Yadav and Garg23). Quantification of the bioactive secondary metabolites of the papaya juice extract was done by the standard ultra-violet spectrophotometric (Thermo Scientific Technologies, Madison, WI, USA) method. The method of(Reference Yang, Yang and Zheng24) was used to quantify the total flavonoid content of the papaya juice. Results were expressed in terms of rutin equivalent (RE). The total phenolic and tannin contents of the papaya juice extract was determined according to the method described by(Reference Siddhuraju and Becker25), its absorbance was read at 725 nm and the results were calculated as gallic acid equivalent (GAE). The ability of papaya juice to scavenge 2,2-diphenyl-1-picrylhydrazyl (DPPH) was determined through the(Reference Tolić, Jurčević and Krbavčić26) method. DPPH solution (0⋅1 mM) was added to extract samples and measured at 517 nm absorbance after 30 min of dark incubation. Vitamin C was used as the antioxidant standard at concentrations of same amount as the extract concentrations while a mixture of methanol and extracts was used as a blank. The concentration of the extract necessary to decrease the initial concentration of DPPH by 50 % (IC50) was calculated from a plot of % inhibition of DPPH v. concentration of extract.
Data analysis
The experiment was laid out in a completely randomised design where analysis of variance for in-vitro and in-vivo results was performed using the GenStat statistical software. In-vitro results were expressed as mean ± standard deviation values with five determinations while mean separations for the in-vivo results were determined using the Tukey's honestly significant difference (HSD) test (P < 0⋅05) to assess a significant difference between the control and treatment groups.
Results and discussion
Nutritional and biochemical composition of the experimental diets
In the present study, rats were fed with HFF diet, which mimicked most of the easily available, fatty and sugary diets consumed by humans, and papaya was used to mitigate its effects. Papaya fruit contains considerable concentrations of vitamins, bioactive compounds and a lipidic composition that reduces inflammatory markers and anti-platelet aggregation, protects against thrombogenesis and oxidative stress and prevents dyslipdaemia factors that can be triggered by obesity(Reference Vij and Prashar2,Reference Teixeira, Lages and Jascolka9) . Proximate analysis of the diets showed that the HFF diet had higher fats, proteins and ash amounts compared to the pellet diet which had more crude fibre and carbohydrates (Table 1). Carbohydrates are mostly the main determinant of blood glucose(Reference Corin Badiu27) while fats and proteins induce satiety signals to the brain, however, regular and/or increased consumption of carbohydrates and fats accompanied by a sedentary lifestyle can result in obesity(Reference Clarke28). This was observed on rats fed on the HFF diet only while significant results (P < 0⋅05) on visceral fats and body weights decreased on rats fed with more papaya amounts, HFF500 (Table 2). In a study by(Reference Moreno-Fernández, Garcés-Rimón and Vera13) rats fed on a high fat/high glucose diet showed an increase in body weight, fat deposition, an increase in oxidative stress biomarkers, raised fasting levels of glucose which suggest an early stage metabolic syndrome development. Some research has revealed that the consumption of papaya leads to reduced body weights due to its anti-obesity properties(Reference Od-Ek, Deenin and Malakul11) and other medical benefits. Papaya is a highly nutritious fruit (Table 1) with health benefits that can have a negative effect on the rats’ metabolic syndrome benefits(Reference Aravind, Bhowmik and Duraivel7).
Table 2. Hypolipidaemic effects of different papaya juice doses on male Albino Wistar rats fed on a HFFD
Mean differences were observed across columns with different letters using Tukey's honestly significant difference (HSD) test at P < 0⋅05.
ns, not significant, n 5.
* Significant.
Hypolipidaemic effects of papaya juice on male Albino Wistar rats fed on a HFFD
Consumption of high sugars and saturated fats have been reported to increase cholesterol and TG level in the blood(Reference Clarke28), resulting in obese rats. Obesity has been described as an underlying condition of inflammatory and metabolic diseases(Reference Kang, Kang and Cominguez29). Fasting blood glucose (FBG) of rats had non-significant differences (P < 0⋅067) which was similarly observed in rats fed on a high fat/high glucose diet by(Reference Moreno-Fernández, Garcés-Rimón and Vera13). External stress or stressors like blood collection have been reported(Reference Abramova, Юрьевна and Koplik30,Reference Jia, Hu and Yang31) to have a contributory effect on the rats’ glucose tolerance hence the non-significant results. Rats fed on a HFF diet showed pre-diabetic results (6⋅5 %) while those fed on papaya showed a decreasing trend with an increase in papaya amounts. Research by(Reference Kang, Kang and Cominguez29) has reported pre-diabetic occurrences where glycated haemoglobin levels were above 6⋅4 %. Papaya has a hypoglycaemic effect(Reference Aravind, Bhowmik and Duraivel7) as it can inhibit some important enzymes that engage in the digestion of carbohydrates, such as α-amylase and α-glycosidase(Reference Santana, Inada and Espirito Santo10). This is due to the antidiabetic activity of its flavonoids which regulate carbohydrate digestion, insulin signalling, insulin secretion, glucose uptake and adipose deposition(Reference Vinayagam and Xu32) hence improving β-cell proliferation, promoting insulin secretion, reducing apoptosis and improving hyperglycaemia by regulating glucose metabolism in the liver(Reference Al-Ishaq, Abotaleb and Kubatka33). Quercetin has been described as the main flavonoid with antidiabetic activities(Reference Haddad and Eid34) such as glucose homeostasis and inhibiting intestinal glucose absorption. It is mostly found in some fruits and vegetables compared to starchy and fatty foods.
Elevated blood lipid levels and reduced HDL-c amounts were observed on rats fed on a HFF diet in this study. Significant differences (P < 0⋅05) were observed for TC, TG and LDL amounts, in a decreasing manner, with rats fed on HFF500 having lower values. Continuous consumption of papaya by rats which had a lower DPPH radical scavenging value, more nutrient and antioxidant properties resulted in reduced blood lipids due to their potential for antioxidative stress, which can lead to the development of health disorders, infections and/or chronic diseases. A ripe papaya is reported to have a high nutritive value and its rich in both macro and micro minerals(Reference Parni and Verma5). Research on obese rats fed on papaya juice reported reduced lipid absorption, anti-obesity, anti-dyslipidaemia and anti-inflammatory effects(Reference Od-Ek, Deenin and Malakul11). A study by(Reference Guo, Moellering and Garvey35) reported that total serum cholesterol and TG levels were markedly reduced in papain-treated mice, due to its anti-obesity effects which regulate lipid metabolism and inflammation. Papain is a proteinase enzyme found in papaya, containing anti-inflammatory effects, which protects the body cells from oxidative stress and diseases, which consequently have a beneficial implication for human life(Reference Govindarajan, Singh and Rawat36) by reducing obesity and metabolic diseases.
High LDL-c blood levels have been associated with coronary diseases like atherosclerosis as they transport cholesterol to the cells where it is deposited and may not be required(Reference Ojezele and Abatan37). Robust biomarkers for predicting coronary heart disease and atherosclerosis, AI and CRI, can be used to quickly assess dyspilidaemic and metabolic syndromes, as they are influenced by diet(Reference Liu, Liu and Pei38). Results from this study showed a statistically significant (P < 0⋅001) decrease on AI and CRI after papaya juice consumption by the rats, compared to the control rats which had higher indices (Fig. 1). Similar results were observed by(Reference Ojezele and Abatan37) on alloxan-induced diabetic rats after oral administration of Bauhinia thoningii. Similar results were reported by(Reference Salau, Olooto and Adebayo39). Rats with high AI and CRI indexes are at a high risk of diseases like diabetes as evidenced also by their higher Hb1Ac levels. Papaya consumption gradually reduced these risks by increasing the HDL-c blood levels of rats and resulted in lower AI and CRI.
Fig. 1. Effects of papaya juice on the atherogenic (AI) and coronary risk (CRI) indices of male Albino Wistar rats fed on a high fat and fructose diet, n 5. Different letters on the same bar colour are statistically different (P < 0⋅05) using Tukey's honestly significant difference (HSD) test.
The lack of food intake measurement was indeed a limitation of this study, as it prevented a comprehensive analysis of the potential impact of papaya juice on the rats’ overall dietary consumption and its influence on the observed outcomes. This information would have provided valuable insights into the relationship between papaya juice administration and the rats’ nutritional intake, potentially offering a more nuanced understanding of the effects observed in the study.
Conclusion
Supplementation of a HFFD with papaya juice consumption on rats reduced their body weights, blood lipids (TC, TG, LDL), glycated haemoglobin, atherogenic and coronary risk indexes. FBG and HDL levels showed non-significant results, in a decreasing manner, with more papaya juice consumption. These results are highly correlated to papaya's high nutrients and antioxidants which have anti-obesity and hypolipidaemic properties. Papaya consumption can then be recommended to health professionals for the management of dyspilidaemic disorders in humans.
Acknowledgements
The authors are grateful to the members of the SAFARI laboratory at JKUAT University for their guidance and experiment facilitation.
This research paper was financially supported by Carnegie Cooperation of New York through a capacity building competitive grant Training for the next generation of scientists through the Regional Universities Forum for Capacity Building in Agriculture (RUFORUM), Grant No: RU/2020/GTADRG/016. Special thanks to the Botswana University of Agriculture and Natural Resources (BUAN) for the research funds and for providing academic leave to the PhD student.
This research work was part of CM's PhD thesis which was supervised by the other authors. All authors contributed to the whole research work; revised and approved the final manuscript draft to be published.
The authors declared no conflict of interest.
The data used to support the findings of this research are included in the article.
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