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INTRODUCTION
Family Moringaceae is represented by a single genus Moringa. The genus Moringa is represented by 14 different species, two of which are Moringa oleifera (MO) and Moringa stenopetala (MS). Moringa species, especially MO, is well known as an important multi-purpose crop in Africa and India. The species are now grown around the world. In contrast, MS is endemic only to northeast tropical Africa. The indigenous use of this specific species is mainly confined to southern Ethiopia and northern Kenya. Therefore, MO, which has a relatively larger distribution than MS that grows mainly in Ethiopia, has been studied more for its nutritional value, functional pharmacological effects, and efficacy as an alternative food [12345] whereas, studies on MS have included nutrient analysis, analysis of pharmacological function, and cytotoxicity studies [678].
Despite an expected increase in food demand, many developing countries are still suffering from food insecurity. Especially in Ethiopia, food shortages and rural poverty have become chronic problems [9]. Since Moringa species are known as good nutrition sources and because they are highly tolerant of very dry conditions, they are often used as important famine foods, especially for infants and nursing mothers [10]. In addition, the fresh leaves of MS are cooked and eaten as a vegetable in Southern Ethiopia [6]. Moreover, the MS leaves are widely used as a traditional herbal remedy by the local population [11].
In many developing countries including Ethiopia, nutritional double burden and low food security have become an undesirable combination of an increasing and long-lasting serious health problem. Concerns regarding the imbalance between energy nutrient intake and micro-nutrient intake are now experimental diets (g/Kg) increasing in both developing and developed countries. Therefore, nutritional problems such as obesity and malnutrition, which appear both in food-rich and food-shortage regions in Ethiopia, are serious.
Although there is a consensus regarding the diverse biological activity of the different parts of plants in the family Moringaceae, there are conflicting reports as to their safety [12]. Considering the widespread use of MS as a food and medicinal plant in Ethiopia, and the published evidence of diverse biological activities of extracts (including cytotoxicity) from different parts of a related species (MO), MS needs to be investigated more thoroughly. Therefore, this study was conducted to determine whether MS was suitable for use in nutritional supplements in regions of Ethiopia by comparing its effects with those of MO. In the present study, only the leaf parts were examined since they are the most used and easily obtained part.
MATERIALS AND METHODS
Plant materials and preparation of dried leaf samples
Fresh leaves of MO and MS were collected from various regions of southern Ethiopia during 2011. The leaves were identified and authenticated at the National Herbarium of Addis Ababa, Ethiopia. Fresh moringa leaves were air-dried right after collection, then ground into a fine powder using a hammer mill. The powder was stored in a dry place in the dark at the Food Science and Nutrition Program at Addis Ababa University under accession number YA-03-2011. The plant powders were then transferred to South Korea in collaboration with Korea-Ethiopia Science and Technology Research and stored at −70℃ until use.
Analyses of selected nutrients
The crude protein contents in the dried MO and MS samples were determined using the micro-Kjeldahl method. Ash contents were determined by incineration of the sample at 550℃ for 6 hours. Mineral contents were determined using ICP-AES (Shimadzu ICPE-9000, Japan) after digesting 0.2 g of each sample in a microwave digestion system. The amino acids were analyzed by the methods of Lee et al. [13] using an amino acid analyzer (Sykam S430, Germany). Vitamin C and E were measured as described by Kim et al. [14]. Vitamins B1, niacin, and pyridoxine were analyzed according to the 2012 Korean Food Standards Codex.
Experimental animals
Twenty 9-week-old male C57BL/6J mice were purchased from the OrientBio (Sungnam, Korea). Only male mice were selected for this research since one of the objectives was to determine changes in fat metabolism following Moringa ingestion and fat metabolism can be influenced by gender. The mice were housed in an air-conditioned animal room with a 12/12 h light/dark-cycle at 22 ± 2℃, a relative humidity of 50 ± 5%, and free access to tap water and food ad libitum. The animals were adapted to these conditions for a week. After the acclimatization period, the mice were randomly divided into four groups of normal diet (ND), high-fat diet (HD), high-fat diet with 1% MO leaf powder (HDMO), and high-fat diet with 1% MS leaf powder (HDMS) group containing five mice each. All animals were separately fed their corresponding diets for seven weeks. The normal diet and high-fat diet were purchased from FEEDLAB in Guri-si, Gyeonggi, Korea. The experimental diets of the HDMO and HDMS groups were prepared by adding 1 g of MO or MS leaf powder to 1 kg of purchased high-fat diet. The composition of the diets is shown in Table 1.
The animal study was conducted to minimize pain or discomfort and all appropriate precautions regarding the care and use of animals for experimental procedures were taken. The study was approved by the Institutional Animal Ethics Committee at Soonchunhyang University (SCH13_01_01).
Measurement of body weight, food intake, and food efficiency ratio
The bodyweight of all mice was determined just before the initiation of experimental feeding and every week for seven weeks thereafter using a digital balance. The amount of food intake was calculated on a per cage basis and expressed as the grams of food per day. The food efficiency ratio was calculated by (daily weight gain/daily food intake) × 100.
Serum collection and histological sample preparation
Blood samples were collected from the retro-orbital plexus vein of all mice after fasting overnight (more than 12 h). The blood samples were kept for about half an hour at room temperature before centrifugation at 3,000 rpm for 10 min to separate the serum for examination. The serum was separated and stored at −70℃ for analysis. Then, the animals were anesthetized and euthanized. The liver was quickly removed and washed with ice-cold saline to remove the blood and dried between filter paper. The liver tissues were immersed for 48 h at 4℃ in fixative solution (4% formaldehyde in phosphate buffer, pH 7.6), dehydrated in an ascending graded series of ethanol, embedded in paraffin, and stained with hematoxylineosin solution (H&E). The tissue preparations were observed and microphotographed under a light microscope (Primovert Axio Imager, ZEISS, Germany).
The serum albumin and glucose concentrations were analyzed by a commercial laboratory (NEODINE Vet Lab, Seoul, Korea). Total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C) were determined using kits obtained from Asan Pharm. Co., Ltd. (Seoul, Korea). High-density lipoprotein cholesterol (HDL-C) was calculated according to the Friedewald formula.
Statistical analyses
The chemical and biochemical determinations were carried out in triplicate. The results are expressed as mean ± standard deviation (SD) and were subjected to two-way analysis of variance (ANOVA). The data were analyzed using Duncan's Multiple Range Test. Statistical significance was determined at P < 0.05. Statistical analyses were performed using SPSS software version 23 for Windows.
RESULTS
Nutritional values
Table 2 depicts the approximate dry weight composition of the MO and MS leaves. The MO and MS leaves contained crude protein (28.28 and 29.47%), crude fat (6.04 and 7.21%), and ash (7.10 and 11.59%, respectively). Calcium was present in the highest concentration in both MO and MS. Other essential minerals, such as potassium and magnesium, were also present in both MO and MS. All minerals analyzed were higher in MS than in MO, except for phosphorus and copper.
Selected vitamin concentrations are reported in Table 2. MO contained 3.89 mg/100 g vitamin C, 302.4 mg/100 g pyridoxine, and 32.2 mg/100 g niacin, whereas MS contained 6.19 mg/100 g vitamin C, 475.1 mg/100 g pyridoxine, and 34.2 mg/100 g vitamin E. Among the vitamins analyzed, niacin was found only in MO and vitamin E was found only in MS and vitamin B1 was not detected in either MO or MS.
The amino acid composition of the dried MO and MS leaves is presented in Table 3. Among 20 proteinogenic amino acids, 17 amino acids, including nine essential amino acids, were analyzed. Glutamic acid, proline, and aspartic acid were the predominant amino acids in both MO and MS. The order of the essential amino acids concentrations were leucine, phenylalanine, lysine, threonine, valine, histidine, isoleucine, histidine, and methionine, however, the concentrations in MS were higher than in MO.
Results of the animal experiments
Fig. 1 shows the weight gain of the mice during the experimental period. Body weight in the HD group was significantly higher compared to the ND, HDMO, and HDMS groups (P < 0.05) (Fig. 1A). The average daily food intake was significantly lower in the HD and HDMO groups compared to the ND group. However, there was no significant difference in the food intake of the HDMS group compared to ND (Fig. 1B). The food efficiency ratio of the HD and HDMO were significantly higher than that of the ND group. There was no difference in the food efficiency ratio between the HDMS group and the ND and HD groups.
The serum albumin, triglyceride and HDL-cholesterol levels of the mice from four experimental groups did not show any statistical differences (Table 4). The results of the serum glucose and LDL- cholesterol levels showed that HD groups showed the higher levels than ND group while HDMO and HDMS groups were in between ND and HD. The serum total cholesterol levels of ND and HDMO groups were lower than that of HD group whereas this was not seen in the HDMS mice. The liver sections of control mice in the ND group revealed normal tissue morphology of the hepatic cells and sinusoids (Fig. 2A). However, the livers of the HD animals revealed histopathological damage. The HD group showed lipid droplet accumulation, marked vein congestion, and hepatocyte degeneration (Fig. 2B). These alterations were attenuated in the HDMO and HDMS groups (Fig. 2C and 2D).
DISCUSSION
The study confirmed that both MO and MS Moringa species were good sources of nutrients. As expected, dried Moringa leaves showed high protein content. In our study, the high protein content was composed of a vast diversity of amino acids. Both the MO and MS dried Moringa leaves contained all 17 amino acids analyzed, including the eight essential amino acids (histidine, valine, methionine, isoleucine, leucine, phenylalanine, lysine, and threonine). Unfortunately, tryptophan was not analyzed due to a technical problem. Concentrations of the essential amino acids were found in the order of leucine, phenylalanine, lysine, threonine, valine, histidine, isoleucine, histidine, and methionine.
Other studies reported that dried MO leaves contained 16 or 17 amino acids [1516]. Methionine, cysteine, histidine, tyrosine, and isoleucine were present in moderate concentrations (0.21–0.66%) in our study. However, these limited amino acids were reported to be the most abundant in other studies [15]. Previous studies strongly suggested that MO may be an important source of vitamins [17]. The vitamins analyzed in MO and MS showed different patterns between the species. Vitamin E was only detected in MS in our study, whereas other researchers reported considerable amounts of vitamin E in MO [17]. Among the vitamins analyzed, vitamin C and pyridoxine were found in both species, however, they were higher in MS than in MO and niacin was found only in MO. Vitamin B1 was not detected in either MO or MS, unlike another study that reported finding vitamin B1 in various parts of MO [17]. Discrepancies in the amino acid compositions and other nutrient values can be due to external conditions, such as climate, soil type, and fertility. External conditions subsequently affect the protein quality and the nutritional value of the sample [18].
Calcium levels appear to be the most significant mineral present in both MO and MS. Other essential minerals as potassium and magnesium were also remarkably present in both MO and MS. Among the nutritive significance of MO leaves is their rich mineral content, essential for the maintenance of human health and MS was confirmed to have the same effect with the higher content.
Not all nutrients were analyzed. Nevertheless, it is hard to determine which species was more nutritious. However, since MO and MS are very common plants in Ethiopia, and large quantities are available at any time, there seems to be no shortage of local raw materials available for developing nutritional supplements. In many countries in Africa, including Ethiopia, the imbalance between energy nutrient intake and micronutrient intake in poor, food-insecure environments and obesity and malnutrition in food-rich areas is a serious nutritional problem with a double burden. Moringa has long been used as a nutritional supplement for famine in Ethiopia [6], so this study was conducted to determine whether it was suitable for use in nutritional supplements. The results of the nutritional analyses were encouraging. Due to the serious double burden problems, we assumed that MO and MS would supply micronutrients and prevent weight increases from high-fat diets, and the effects MO on fat metabolism in the body were confirmed positively.
The current research found that the MO diet prevented weight gain and increases in serum lipids associated with high-fat diets. However, the effects of MS were less than those of MO. Therefore, the results of this study showed that MO and MS can be good raw materials when Moringa is used as a material for developing nutritional supplements using local plants. However, the anticipated protective effect against high-fat diets was seen for MO, but the effect of MS was not as great.
In conclusion, MO and MS both had high nutrient contents, so can be used to develop nutritional supplements, not only in Ethiopia but also in Africa and Southeast Asia where Moringa is abundant. In particular, since MO has anti-obesity effects, both micronutrients and anti-obesity effects can be expected from MO supplementation in developing countries suffering from nutritional double burden. Although MS has nutritional value as a good vitamin and mineral source, the development of nutritional supplements using MS should be approached with caution due to its toxic effect [12]. Therefore, safety tests must be conducted on MS in the future.
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