Ipomea batatas Leaf Powder from Cameroon: Antioxidant Activity and Antihyperlipidemic Effect in Rats Fed with a High-Fat Diet

The present study consists of analyzing the phytochemical composition of Ipomoea batatas leaf powders and evaluating their antihyperlipidemic effect on rats receiving a high-fat diet. Ipomoea batatas leaves were collected from four agroecological areas of Cameroon, and powders were obtained after washing, drying, grinding, and sieving. Standard analytical methods were used to determine the phytochemical composition of two varieties (IRAD-tib1yellow-V1 and IRAD-1112white-V2) from North Z1, Adamawa Z2, West Z3, Center Z4. The effect of I. batatas leaf powder on lipid metabolism was assessed in vivo by feeding different groups of rats with a high-fat diet supplemented with 5 and 10% of I. batatas leaf powder during 30 days. At the end of the experimentation, total cholesterols, triglycerides, LDL- (low-density lipoprotein-) cholesterol, HDL- (High-density lipoprotein-) cholesterol, ASAT (aspartate aminotransferase), ALAT (alanine aminotransferase), and creatinine were measured using commercial enzymatic kits (Spinreact, Spain). The results of phytochemical analysis of I. batatas leaf powders revealed that the total phenol content ranged from 660.173 mg GAE/100 gDW (Z1V2) to 657.76 mg GAE/100 gDW (Z3V2), the flavonoids content ranged from 282.25 mgEC/100 gDW (Z3V1) to 325.05 mgEC/100 gDW (Z4V2), and the anthraquinone content ranged from 324.05 mg/100 gDW (Z3V2) to 326.72 mg/100 gDW (Z4V2). The total antioxidant capacity ranged from 19.00 (Z1V1) to 23.48 mg AAE/gDW (Z3V2), while the IC50 ranged from 1.58 mg/mL (Z1V1) to 3.08 mg/mL (Z3V2). Rats fed a high-fat diet and supplemented with 5 and 10% of I. batatas leaf powder showed a significant (p < 0.05) reduction in body weight compared to the control with a reduction rate ranging from 6 to 10%. The consumption of I. batatas leaf powder for 30 days significantly (p < 0.05) reduced serum total cholesterol, LDL-cholesterol, triglycerides, ALAT, and creatinine level. These results suggest the use of I. batatas leaves as a phytomedicine in the treatment of cardiovascular diseases.


Introduction
Nowadays, dietary habits have changed and the tendency is much more oriented towards traditional foods based on vegetables very rich in antioxidants to prevent cardiovascular diseases [1]. Among vegetables, the leaves of Ipomea batatas (sweet potato) are an essential part of the human diet, providing the body with vitamins, minerals, proteins, some hormone precursors, and a high content of phytochemicals endowed with beneficial effects on health [2]. In Japan, the leaves of I. batatas have been reported to be an excellent source of anthocyanins and many other bioactive compounds [3]. In Brazil and Malaysia, some studies on the leaves of I. batatas have focused on its antioxidant capacity due to its high content of flavonoids, β carotene, anthocyanins, and phenols [4,5]. Therefore, the presence of all these compounds mentioned could be the reason I. batatas leaves are used in traditional medicine (anaemia, antiviral, and worm). In addition to their antidiabetic and antiviral properties, Ipomoea batatas leaves have been used to treat infectious diseases in Brazil [6]. Phytochemical compounds protect the body against degenerative diseases such as cancer and atherosclerosis caused by free radicals. Because of their potential as a bioactive compound, the use of I. batatas leaves has also been considered for the management of hyperlipidemia. Hyperlipidemia is a physiological disturbance characterized by an abnormal increase in cholesterol levels or blood lipoproteins [7]. It is the main cause of cardiovascular diseases, such as ischemic cerebrovascular disease, coronary heart disease, and peripheral vascular disease [8,9]. On the other hand, hyperlipidemia is the main cause of mortality (about 12 million people) and morbidity in the world [10]. Many lipid-lowering drugs (fibers, sequestering bile acids, etc.) are used to regulate lipid metabolism by various mechanisms, but due to their synthetic nature, they are also known for their negative effects on health, including hyperuricemia, diarrhea, nausea, severe muscle damage (myopathy), gastric irritation, hot flashes, dry skin, and deregulation of liver function [11]. A number of plants have been traditionally used against various cardiovascular diseases and to control hyperlipidemia, among which the leaves of I. batatas [6,12]. Some surveys conducted in some localities in Cameroon have reported their use in traditional medicine as a slimming plant. This research therefore aims to quantify the phytochemical compounds of Ipomoea batatas leaves collected in different agroecological zones of Cameroon and to study their antihyperlipidemic activity in rats fed a high-fat diet.
Leaves were washed and dried in the oven at 45°C for 48 hours and crushed with a moulin through a sieve mesh of 500 μm to produce fine leaf powder. The powders were stored in plastic packaging and hermetically sealed for further analysis.  [13] method was used to determine the crude fiber content in leaf powders. 2 g of sample was boiled for 30 min under a reflux condenser in 0.25 N sulphuric acid solution. The mixture was filtered, and then, the residue was rinsed and boiled again for 30 min in a 0.3 N NaOH solution. Then, the filtered and rinsed sample was dried for 8 hours at 105°C. After cooling, the residue was weighed and then incinerated in a muffle furnace at 550°C for 3 hours. The crude fiber content was expressed as a percentage of the dry weight (DW) of the sample.

Total
Phenol and Flavonoid Content. The method described by Hsu [14] was used for the extraction of phenolic compounds. Eighty mL of methanol (80%) was introduced into a beaker containing 5 g of leaf powder, and the mixture was homogenized and left to macerate for 12 hours. The mixture was then centrifuged (5000 rpm, 10 min, 4°C) and filtered through Whatman N°1 filter paper. The filtrate was then made up with methanol (80%) to 100 mL to serve as a stock solution (50 mg/mL). Then, the total phenol and flavonoid content were determined using the protocol described by Sokamte et al. [15].

Proanthocyanidins
Content. The vanillin method described by Broadhurst and Jones [16] was used for the determination of proanthocyanidin content. 25 μl of the sample (stock solution) was mixed with 125 μl of vanillin reagent (4% in methanol) and 60 μl of concentrated hydrochloric acid (35%). Absorbance was measured at 500 nm after 15 min incubation in the dark and at room temperature. The calibration curve was drawn with catechin (0-1 mg/mL). The proanthocyanidin content was expressed in mg catechin equivalent/g dry weight (mg CE/g DW).

Anthraquinone Content.
For the determination of the anthraquinone content, the stock solution was evaporated under vacuum using a rotary evaporator to obtain the dry extract. Then, 1 g of extract was mixed with 30 mL of distilled water and boiled for 15 min under a reflux condenser. The analysis procedure was following with the steps described in the protocol of Sakulpanich and Gritsanapan [17], and the results were expressed as mg of hydroxyanthracene derivatives/100 g DW.

Determination of Antioxidant
Activities. The antioxidant activity of the samples was evaluated using three tests: the DPPH test (1, 1-Diphenyl-2-Picrylhydrazyl test), the FRAP (Ferric Reducing Antioxidant Power) test, and the TAC (total antioxidant capacity) test. The analysis procedures were following the steps described in our previous publication [18]. For the DPPH test, the results were expressed in IC 50 (concentration of the extract that allows 50% of the DPPH free radical to be trapped) and were calculated using the exponential regression equation [15]. Journal of Lipids of Nutrition, Faculty of Sciences, University of Yaounde I, Cameroon, were used for the in vitro study. Before the study, the rats were weighed, divided into 6 groups of 6 rats, housed in a single cage, and acclimatized for one week with free access to tap water and food. The animals were kept in an environment of 25 ± 2°C with a relative humidity of 55 ± 5% under a cycle of 12 h light and 12 h dark. Procedures used in the study were approved by the Animal Ethics Committee of the Laboratory of Nutrition of the University of Yaounde I. The trial was registered with reference number: No. FW-IRB00001954.

Diet
Administration and Blood Collection. The standard and high-fat diets used to feed animals were formulated as proposed by Murase et al. [19] with slight modification ( Table 1). The rats were divided into 6 groups of 6 rats each. The negative control standard diet (TN-STD) group received ad libitum standard or normal diet. The positive control high-fat diet (TP-HFD) group received ad libitum high-fat diet. The TP-HFD-Z2V2 5% group, the TP-HFD-Z2V2 10% group, the TP-HFD-Z3V2 5% group, and the TP-HFD-Z3V2 10% group, fed ad libitum with a high-fat diet supplemented with doses of 5 and 10% (w/w) of I. batatas leaf powder from Zone 2/Varity 2 and Zone 3/Variety 2, respectively. The dose of dietary herbal powder supplementation was fixed on the basis of previous studies found in the literature [20,21]. The experiment was carried out for 30 days, after which the animals were fasted for 12 hours and anaesthetized with ether. The blood was withdrawn from the jugular vein and introduced into dry tubes (without anticoagulant) and then centrifuged (1500 rpm; 10 min) for collection of serum from which subsequent analyses were carried out to assess lipid profile, liver function, and cardiac injury.
2.6. Biochemical Assay 2.6.1. Lipid Profile. The levels of total cholesterol, HDL-cholesterol, and LDL-cholesterol in serum were measured using commercial enzymatic kits [22]. Triglycerides were assayed by the GPO-PAP (Glycerol 3 phosphate oxidase-4 Amino-antipyrine) colorimetric method using Human SU-TRIMR 10720P kit (Human, Germany). The triglycerides in the sample were hydrolysed using a set of microbial lipases to form glycerol and fatty acids [23]. The atherogenic index was calculated following the formula described by Bo et al. [24].

Evaluation of Hepatic and Cardiac Function.
Hepatic function was assessed by the determination of Alanine Aminotransferase (ALAT) and Aspartate Aminotransferase (ASAT) activities using the spectrophotometer based on the protocols of commercial kits [25]. The results were expressed as IU/L. The creatinine content was determined using a spectrophotometer working at 520 nm with the Human Su-Crea 10052 kit [26], to know the degree of cardiac injury.

Statistical Analysis.
Analyses were carried out in triplicate and data were subjected to analysis of variance to test the significance between treatments. Additionally, differences between means were tested using the Duncan Multiple Range test using Statgraphics Centurion XVI Software version 16.1.18 (StatPoint Technologies, Inc.), and the values were considered statistically different at a risk p < 0:05. Regression analysis was performed using SigmaPlot 12 version 12.5.0.38 (Systat Software, Inc.).

Fiber Content.
Fiber is the nondigestible polysaccharide residues of plants, and means of total fiber ranged from 8:15 ± 0:02 g/100 gDW (sample Z4V1) to 12:42 ± 0:09 g/ 100 gDW (sample Z1V2) ( Table 2). A significant (p < 0:05) difference was observed between the samples, regardless of the origin of the harvest or the variety studied. The results obtained in this study are similar to those of Sun et al. [27]. They found a fiber content of 11.33 g/100 gDW in sweet potato leaf powder from China. As reported by Kurata et al. [20], sweet potato leaves are high in dietary fiber.

Total Phenolics, Flavonoids, Proanthocyanidins, and
Anthraquinones Content. Table 2 shows the bioactive compound contents of two varieties of Ipomea batatas leaf powder collected from the four localities. As can be seen in this table, the levels of total phenol, proanthocyanidins, and anthraquinones are very similar, regardless of the variety or origin of the plant. However, significant differences (p < 0:05) were observed for total phenol and proanthocyanidin contents. The total phenol content varies from 657:76 ± 0:31 gGAE/100 gDW to 660:10 ± 1:00 gGAE/100 gDW, and the proanthocyanidins content varies from 175:27 ± 0:05 gGAE/100 gDW to 176:04 ± 0:06 mgCE/100 g DW. The highest total phenol content was recorded in the Z4V2 sample, while the lowest value was recorded in the Z3V1 sample. On the other hand, the highest proanthocyanidin content was recorded in sample Z1V2, while the lowest value was recorded in sample Z2V1. No significant differences (p > 0:05) were observed in the anthraquinone content of the different sweet potato leaf samples. The average levels ranged from 324:05 ± 2:00 to 326:72 ± 2:52 mg/100 gDW. For the total flavonoid content, the variety of the plant has a significant influence on the values recorded in the sweet potato leaves since the lowest contents (282:25 ± 0:12 to 289:13 ± 0:96 mgCE/100 gDW) were observed in variety 1, while the highest contents (323:22 ± 0:20 to 325:05 ± 0:00 mgCE/100 gDW) were observed in varieties 2. Phytochemical compounds such as total phenols and flavonoids have also   Journal of Lipids been detected in the leaves of harvested sweet potatoes in Ethiopia [28] and China [29]. The total phenol content values observed in this study are very high compared to those found by Sun et al. [27] in sweet potato leaves harvested in China (120 mg/100 gDW). In contrast, high levels of total phenols (3680-4670 mg/100 gDW) were found in the leaves of three sweet potato varieties harvested in the USA [30]. These authors suggested that anthocyanins are predominantly present in the extracts studied. Factors that may be responsible for the observed differences in the phytochemical composition of sweet potato leaves are variety, harvest season, growing conditions, and age of the plant as reported by Sokamte et al. [15]. Total phenolic compounds have been lauded for their beneficial effects in the fight against chronic diseases like atherosclerosis, cardiovascular, and diabetes [31]. Moreover, as suggested by Groppo et al. [6], sweet potato leaf was endowed with a potential medicinal use because of its high content of phenolic substances (e.g., tocopherols, tannins, phenolic acids, and flavonoids).  The ability of these samples to trap free radicals was also evaluated using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) test, and the results are expressed in IC 50 . Like the FRAP test, the antiradical capacity of these samples follows the same pattern, as no significant differences were observed for variety 1 (2:90 ± 0:04 to 3:08 ± 0:08 mg/mL, for Z1V1 and Z4V1 samples, respectively). In addition, the highest antiradical activity was recorded for the Z3V2 sample with the lowest IC 50 value (1:58 ± 0:02 mg/mL). The phenolic compound is considered as the major contributor to the total antioxidant capacity [32]. The high antiradical activity of I. batatas leaf powder could explain their use in the traditional treatment of some diseases. Further, considering their antioxidant capacity, Z2V2 and Z3V2 samples could be used to manage lipid profile such as lowering the effect on LDLcholesterol and increase HDL-cholesterol levels.
3.4. Principal Component Analysis. Principal component analysis (PCA) was carried out to group Ipomea batatas leaf powders according to similarities or differences in their bioactive compounds and their antioxidant activities. Additionally, the correlations between all measured parameters were revealed after performing the principal component analysis. From Figure 1(a), it can be observed that 93.10% of the total variability in the data are explained by the principal components F1xF2. The first principal component (F1) represents 55.61% of the variation between variables, while the second principal component (F2) represents 37.49%. Tree groups can be observed from this figure, and the characteristics of the different groups obtained are shown in Figure 1(b). In this figure, it can be seen that group 1, represented by samples Z3V1, Z2V1, Z1V1, and Z4V1, is characterized by a lower content of biological compounds and a lower antioxidant activity. Group 2, represented by samples Z1V2 and Z4V2, is characterized by a higher content of phenolic compounds, flavonoids, proanthocyanidins, and anthraquinones. Finally, group 3, represented by samples Z2V2 and Z3V2, is very interesting because of their higher antiradical and reducing power. However, despite their good biological properties, the latter group is also characterized by a high content of biological compounds, but lower than that of group 2. This can be explained by the fact that the biological properties of a plant do not only depend on its bioactive compound content but are also closely related to the nature and the proportion of the bioactive compounds found in it. According to their higher antioxidant activities, samples Z2V2 and Z3V2 were used for the experimentation on rats to assess their effect on lipid profile management.

Effect of Ipomea batatas Leaf Powder on Body Weight
Evolution. The ingestion of the high-fat diet resulted in a gradual increase in body weight with time, whereas weight gain was suppressed in a dose-dependent manner in all groups fed the I. batatas leaf powder supplemented HFD ( Figure 2). A significant decrease in body weight was observed for the I. batatas leaf powder group (TP-HFD-Z2V2 5% and TP-HFD-Z3V2 5%) compared to the TP-HFD group feeding only with a high-fat diet (Figure 2). At the end of the experimentation, the weight gain of the I. batatas leaf powder group (TP-HFD-Z2V2 5% and TP-HFD-Z3V2 5%) was similar, and no significant difference was observed between both (2 and 1.9%, respectively). For the TP-HFD-10% group, a significant decrease in body weight (weight loss) was observed throughout the experimental period, and the final weight loss recorded was 2.9 and 3.2% of the initial weight, for TP-HFD-Z2V2 10% and TP-HFD-Z3V2 10%, respectively. It has been recommended that weight reduction programs should focus on achieving a modest weight loss of 7-10% of the initial weight. Therefore, I. batatas leaf powder could be used in weight reduction programs. The weight loss can be attributed to dietary fiber contains which were found in high amount in I. batatas leaf powder [33]. In previous studies [34,35], evidence of a negative correlation between high fiber content and weight gain has been reported. Dietary fiber promotes the excretion of fat and alternatively as reported by Isken et al. [33], it is possible that lipid absorption in the small intestine was blocked by dietary fiber. These results are in agreement with those of Kurata et al. [20] who conducted a similar study by supplementing the high-fat diet of rats with 5% of I. batatas leaf powder collected in Japan. They found that 5% of the I.  Journal of Lipids batatas leaf powder group showed significant suppression in final weight (390:6 ± 15:69 g) and adipose tissue weight (6:25 ± 0:95 g) compared to the positive control group feeding only a high-fat diet, and for which, the final weight and adipose tissue weight were 425:0 ± 24:97 g and 8:85 ± 1:32 g, respectively.

Effect of Ipomea batatas Leaf Powder on Serum Lipids
Profile and Atherogenic Index. The total cholesterol, LDLcholesterol, HDL-cholesterol, and total triglyceride concentrations (mg/dL) of the different groups of experimental rats are shown in Table 3 [36,37]. However, when I. batatas leaf powder was incorporated into the high-fat diet, sig-nificant reductions in serum total cholesterol, triglyceride, and LDL-cholesterol values were observed compared to the TP-HFD group. For rats in the TP-HFD-Z2V2-5% group, the final values obtained are 160:03 ± 0:36 mg/dL, 130:42 ± 0:33 mg/dL, and 90:17 ± 1:00 mg/dL, respectively. For rats in the HFD-Z2V2-10% group, the final values obtained are 153:15 ± 1:29 mg/dL, 122:98 ± 0:82 mg/dL, and 86:33 ± 1:15 mg/dL, respectively. These values were significantly lower than those of the TP-HFD-Z2V2 5% group. In addition, the supplementation with I. batatas leaf powder resulted in a significant increase in HDL-cholesterol in rats in the TP-HFD-Z2V2-5% and TP-HFD-Z2V2-10% groups (66:88 ± 2:46 mg/dL and 69.84 ± 0.13 mg/dL, respectively) compared to the TP-HFD group. The decrease in LDL-cholesterol and the increase in serum HDL cholesterol levels contribute to an antiatherogenic condition [37]. Similar results were observed in rats of the HFD-Z3V2-5% and HFD-Z3V2-10% groups, receiving a hyperlipidemic diet incorporated with 5 and 10% of the powder of the leaves of I. batatas, harvested in zone 3 and belonging to variety 2. The reduction in total serum cholesterol could be associated with the high dietary fiber content of I. batatas leaves. Several studies have shown that propionate generated from dietary fiber has hypocholesterolemic effects and compensates for the hypercholesterolemic effects of acetate production, which tends to increase total serum cholesterol via a mechanism probably involving hepatic lipogenesis [38,39]. Several other mechanisms are involved in the reduction of total serum cholesterol; one of the best known is a decrease in the absorption    Journal of Lipids of bile acids, which causes the elimination of steroids from the body through faecal excretion, an increase in cholesterol catabolism, an increase in the secretion of bile acids and a decrease in the secretion of cholesterol by lipoproteins [40]. In addition, several scientific studies have also shown that certain bioactive compounds present in plants can reduce serum cholesterol levels [41][42][43][44][45][46]. For example, saponins have cholesterol-lowering effects in animals and humans [47][48][49][50].
They have an amphiphilic structure and can bind to dietary cholesterol and/or bile acids. Saponins can prevent the absorption of cholesterol, interfere with its enterohepatic circulation, and increase its faecal excretion. The obvious presence of saponins in sweet potato leaves has been reported by Groppo et al. [6] and by Mbaeyi-Nwa and Emejulu [51].
In contrast to this study, Kurata et al. [20] found no significant difference in HDL-cholesterol and total cholesterol levels between the group of rats fed only a high-fat diet and the group of rats fed a high-fat diet supplemented with 5% I. batatas leaf powder. However, they found that, the plasma triglycerides level in the 5% I. batatas group was significantly lower (66:96 ± 35:29 mg/dL) and approximately 38% of that in the positive control group (178:63 ± 44:76 mg/dL). On the other hand, other research studies have reported similar results to the present study as they found that the aqueous extract of I. batatas leaves collected in Cameroon restored blood lipid levels to normal values in the group of rats fed a high-fat diet [52]. These authors also suggest that I. batatas leaves have hypolipidemic and antiatherosclerotic properties that justify their use in traditional medicine. The TG/HDL-cholesterol ratio is linked to a risk factor for coronary heart disease, and the risk increases as this ratio also increase. This risk of cardiovascular disease is low when TG/HDL-cholesterol ratio is between −0.3 and 0.1, medium when TG/HDL-cholesterol ratio is between 0.1 and 0.24, and high when TG/HDL-cholesterol ratio is more than 0.24, as reported by Bo et al. [24]. From the observations made in Table 3, it is evident that the high-fat diet (0:47 ± 0:00) significantly increases the TG/HDL-cholesterol ratio compared to the normal diet (0:27 ± 0:00). For the different groups of rats fed a high-fat diet and supplemented with 10% of I. batatas leaf powder (TP-HFD-Z2V2-10% group and TP-HFD-Z3V2-10% group), no significant differences were found with an atherogenic index ranging from 0:25 ± 0:00 to 0:25 ± 0:01, respectively. However, these values were significantly low compared to the negative control groups and were also significantly low compared to rats fed a high-fat diet (TP-HFD) and to rats fed a high-fat diet and supplemented with 5% of I. batatas leaf powder (TP-HFD-Z2V2-5% group and TP-HFD-Z3V2-5% group, with an atherogenic index value of 0:29 ± 0:00 and 0:29 ± 0:00, respectively). These results indicate that I. batatas leaf powder contains certain biological compounds that prevent the deposition of cholesterol or lipids in certain organs (kidneys, heart, and liver) and arteries. However, in general, at the end of the experiment (4 weeks of feeding), all the different groups of rats can be classified according to the atherogenic index values obtained, as high-risk groups for cardiovascular disease with an atherogenic index greater than 0.24.

Conclusion
This study reveals the high fiber content in Ipomea batatas leaf powders, which also contain phytochemical compounds such as flavonoids, proanthocyanidins, and anthraquinones. I. batatas leaf powders also showed good antiradical and reducing activity, particularly samples Z3V2 and Z2V2 of variety 2 collected, respectively, in West and Adamaoua. In rats fed with a high-fat diet, supplementation with 10% I. batatas leaf powder was effective in reducing the levels of triglycerides, total cholesterol, and LDL-cholesterol as well as ALAT activity and creatinine concentration. The results of this study suggest the use of I. batatas leaves as a phytomedicine in the treatment of cardiovascular diseases. In the further development of this study, it would be interesting to identify, using analytical techniques, the phytochemical compounds endowed with these properties.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
The authors have no conflicts of interest.