As aforementioned, the prevalence of hyperglycaemia related to T2DM is varied depending on location, age and sex. This is similar in Uganda where it was found to be highest in Ankole and lowest in Teso sub-region, and this variance was dependent on predisposing factors like diet among others [41]. This study was designed to screen and quantify the phytochemicals and to investigate the hypoglycemic property of the most frequently eaten vegetables in these two sub-regions in an attempt to subsequently explain the variance in national regional prevalence. These vegetables were chosen because they are an important aspect of traditional diets [42], in addition to being recommended for a healthy diet [43], and of which diet in itself is a component of an environmental (apart from genetics) basis of diabetes. In this regard, scientific attention through research in the last decades and in different parts of the world has shown that plant based diets and therapies have potential to prevent diabetes, and manage or even treat its complications like hyperglycemia [44, 45]. The vegetables Hs, Vu and Sn are being used in many African countries as traditional plant food and remedies for alleviating hyperglycaemia, but the species in Uganda have not been scientifically evaluated for that effect. The combination of HFD-STZ is used for inducing T2DM in animals because HFD causes intra-cellular fat that blocks the insulin receptor sites hence causing insulin resistance [46]. Streptozotocin (in multiple low doses) is well known for causing pancreatic islet cell toxicity which consequently leads to partial impairment in insulin secretion [37]. Continuing impairment of insulin secretion and its resistance in the target tissues will exacerbate carbohydrate, protein and fat metabolic disturbances and leads primarily to hyperglycemia [24].
Phytochemical screening was performed and water solvent extracted more components than methanol because it is a universal solvent [47]. Phenolic and flavonoid compounds were found in all the vegetables leaves; however, tannins and alkaloids were in trace amounts. When these phytochemicals were quantified, their quantities varied significantly among the vegetables.
Hs was found to contain significantly highest quantity of TP compared to Vu and Sn. Chlorogenic acid is one of the phenolic acids reported in the leaves of Hs [48]. Protocatechuic acid is another phenolic compound in Hs extract though its higher in the calyces [49]. Sn contained the significantly highest amount (71.35 µg equivalence/mg dry weight) of TF compounds although the amount is lower (33.83µg equivalence/mg of dry weight) in Sadique et al’s study of the same plant part [50].
Tannins and saponins are considered anti-nutritive because they interfere with the absorption of iron and impart bitterness to food respectively. The bitter taste of saponins can reduce their palatability since they are throat-irritating [51, 52].Since the tender vegetables leaves are eaten cooked, heat inactivates these anti-nutritive compounds which explains the lack of reports of toxicity [13]. However, the traces of tannins and saponins found therefore means that these vegetables need to be boiled before eating to inactivate them, and therefore should not be eaten raw (as salad) [53]. This is in agreement with other studies, which showed that the Sn fruits/berries, calyces and seeds contain more or less of the phytochemicals found in the leaves [54–56].
These vegetable ALEs can be safe orally (in animals) even above the dose of 5000 mg/kg.b.w. Several studies on other parts of these vegetables including seeds, fruits and flowers, extracted with other solvents like methanol have shown similar safety profiles [57–59]. Ethnobotanical literature indicates that these leafy vegetables have been eaten as part of diets for centuries without any toxicity reports [60, 61]. Though Sn leaves are considered edible, they contain a glycol-alkaloid called solanine which is reported to be toxic in mature leaves, and more toxic in unripe berries compared to the ripe ones [62]; however, simple boiling of the young leaves is enough to inactivate the alkaloid [63].
Effect of the various treatments on the FBG levels and body weights of the HFD-STZ induced diabetic rats
The vegetables Hs, Sn and Vu are all used in African communities and Vu is especially considered a human vegetable because its leaves and seeds have been eaten for centuries particularly in the tropics for its high protein, carbohydrates and vitamin content [61, 64]. The calyces of Hs have been exploited for beverage and dyeing purposes compared to the leaves which have been used more for soup and less for drinks [55, 65]. The leaves of Sn have been eaten for sauce more than its ripe berries [56, 66].
During our study, these vegetable ALEs insignificantly reduced body weight of the animals compared to the diabetic control. However, the contrary has been reported by other researchers who studied different vegetable parts extracted with different solvents. This disagreement in the results could be due to several reasons, like the period of observation which could have been insufficient for the significant effect from the extracts’ metabolic activity. It is also possible that environmental factors such as soils and climate can have an influence on the phytochemical quality and quantity of the vegetable species [67, 68]. The solvent used for extraction from the plant part can also have an effect on the quality and quantity of the phytochemicals extracted (Pandey and Tripathi, 2014; Raya et al, 2015; Stephane et al., 2021) Amaya et al. and Ojulari et al. (2019) studied the aqueous calyx extract of Hs and reported a significant reduction of the animal body weight [69, 70]. Nderitu et al. (2023) and Aabideen et al. (2022) reported Sn aqueous and other solvent extracts to be able to significantly reduce animal body weight [71, 72]. Vu too was observed to have a similar effect in a significant manner in other various studies [57, 73]. This animal body weight decrease has been attributed to the vegetable polyphenolic compounds (flavonoids) that may suppress growth of the adipose tissue through their anti-angiogenic activity and by modulating adipocyte metabolism [74, 75].
The FBG levels of the animals were significantly reduced in comparison to the diabetic control after Hs, Sn and Vu ALE administration, and this result is in agreement with other studies on different solvent extracts and vegetable parts. The calyces of Hs have been exploited for beverages and the scientific validation on animal models shows a hypoglycemic property [76–78], however, there is scarce literature of the same effect by the leaves in this species. The same effect was also observed by the leaves and seeds of Vu [19, 54], and likewise by the leaves and berries of Sn [58, 79].
These vegetables do contain polyphenolic compounds (for example phenolic acids and flavonoids) [45, 80, 81] that have been reported to exert influence on homeostasis of blood glucose level by different mechanisms. For example, they can inhibit the intestinal digestive enzymes, delaying the digestion of complex sugars into simple sugars, and thus reducing the absorption of the latter, and consequently lowering the blood glucose level [82, 83]. This mechanism of action was reported in Hs fruits extracted by methanol solvent in a study by Raheem et al.[84]. The mechanism is similar to that of the synthetic intestinal enzyme inhibitor drugs but with less likelihood of side effects such as abdominal gas and diarrhea [45]. These polyphenolic compounds have further shown a significant influence on the up-regulation of the expression levels of glucose transporter 4 (GLUT4) genes in cell lines, which results in higher glucose uptake in similar levels to that taken by insulin. This was demonstrated by Boue et al. (2016) in two pigmented rice bran extracts [85].
A protein with similar molecular mass and amino acid sequence with that of bovine insulin has been isolated from the empty pods and seed coats of Vu [86], Spinacia oleracea (spinach) [87] and fruit of Momordica charantia (bitter gourd) [88]. This insulin-like protein is reported to be in most green leafy vegetables though it is not proven to be in any of these studied species.
The dose dependence of these vegetables’ hypoglycemic activity was also recorded by other authors that studied other reputed hypoglycemic vegetables like Vernonia amygdalina (bitter leaf) [89], Basella alba (vine spinach) [90] and Amaranthus spp (amaranth) [91] though extracted in different solvents. This could be due to the compounding effect of the extract administration.
Glibenclamide is a cardinal drug (second generation sulfonylurea compound) used in the treatment of T2DM and it was used as a control in this study. It did reduce the FBG levels better than the vegetable extracts at the end of the study although it had the lowest activity at the start. Detrimental effect of hyperglycemia on its absorption in the intestines can explain its low activity at the start of this study. However, it does have beta cell and extra-pancreatic effects like those on skeletal and adipose tissue when finally absorbed in the blood stream. In the pancreas, it works by inhibiting the potassium ATP channels, which cause membrane depolarization, calcium influx and finally activation of the insulin secretory machinery [92, 93]. In the muscles and adipose tissue, it works through increased synthesis of the glucose transporter 1 (GLUT 1) [94, 95], thereby explaining the achievement of the highest reported hypoglycemic activity in this study.