Diabetes mellitus, a chronic disease, is a major public health problem and an independent risk factor for death from heart-related causes [1
]. According to the Austrian Diabetes Report, 2017, approximately 415 million people worldwide suffer from diabetes mellitus. By the year 2040, the number of patients will rise to 642 million [1
]. Most patients have type 2 diabetes mellitus (T2DM) characterized by a combination of hyperinsulinemia and insulin resistance. The development of T2DM is linked to a more sedentary lifestyle, an energy-rich diet, and obesity. One typical feature of diabetes mellitus is a chronically elevated blood glucose level (hyperglycemia), which can lead to micro- and macrovascular complications such as neuropathy, atherosclerosis, rheumatoid arthritis, retinopathy, end stage renal diseases, and neurodegenerative disorders [2
Regulating postprandial hyperglycemia provides an important measure in the prevention and management of T2DM. Lower postprandial glucose levels not only reduce the risk of arterial and microvascular diseases but also may have a protective effect on β-cell function [3
]. A possible strategy to reduce postprandial glucose and subsequent insulin peaks is the inhibition of intestinal glucose absorption. In general, glucose is absorbed across the small intestine through brush border cells via sodium-dependent glucose cotransporter 1 (SGLT1) and glucose transporter 2 (GLUT2) [4
]. The search for appropriate antidiabetic agents has recently been focused on plants, as plant-derived extracts contain bioactive ingredients that inhibit sugar absorption [5
]. Some of these phytochemicals, such as polyphenols, have been reported to interact with SGLT1 and GLUT2, resulting in a reduced postprandial glucose response [6
]. Moreover, previous studies have identified flavonoids as enhancers of insulin secretion and sensitivity [7
]. The antidiabetic effects of different plant- and fruit-derived polyphenols are supported by several studies [8
]. Overall, these studies indicate that some natural extracts might be used as complementary or alternative therapeutic agents to manage, prevent, and/or treat diabetes. However, the efficacy of an extract depends on its bioactive ingredients and, thus, on its raw materials and production method. Before an extract can be used for food products, its efficacy must be confirmed. For instance, a meta-analysis for all available randomized controlled trials comparing the effect of green tea or green tea extract on insulin sensitivity and glycemic control could not confirm the efficacy of green tea in regulating postprandial hyperglycemia. It was found that the consumption of green tea did not decrease the levels of fasting plasma glucose, fasting serum insulin, and oral glucose tolerance test (OGTT)-2 h glucose [11
Guava (Psidium guajava
), a tropical fruit, has been used for the treatment of diabetes and other chronic diseases in traditional Chinese medicine for a long time [12
]. Some in vitro and in vivo studies have illustrated the antihyperglycemic and hypoglycemic effects of guava leaf extracts [13
]. It seems reasonable that the described effects result from the bioactive compounds of guava. Phytochemical analysis of different parts of guava (peel, flesh, and seed) have demonstrated a high content of total phenolics and flavonoids, including flavanols, flavonols, tannins, and phenolic acid derivatives [16
]. We have recently shown that various extracts prepared from guava work as potent SGLT1 and GLUT2 inhibitors in vitro (Caco-2 cells) and in vivo (C57BL/6N mice) [17
]. The greatest inhibitory effect was detected for guava fruit extract prepared by supercritical CO2
extraction (SFE). Obviously, this extraction procedure results in the isolation of compounds related to the inhibitory effect on glucose resorption at higher concentrations than other techniques such alcoholic extraction.
Based on our previous work, we investigated whether application of this guava extract also leads to a reduced postprandial glucose response in humans. Therefore, we conducted a parallelized, randomized, and placebo-controlled clinical study to confirm the efficacy of guava fruit extract in young healthy adults. In addition, we tested the dependency of storage time on the efficacy of the extract by splitting the intervention group into a September and a November intervention. The obtained results confirmed our hypothesis that the consumption of guava fruit extract together with a glucose solution reduces the extent of the postprandial glucose response during an oral glucose tolerance test (OGTT).
Previous research has indicated that consumption of different guava fruit extracts reduces postprandial glucose peaks by inhibition of the intestinal glucose transporters SGLT1 and GLUT2 in vitro (Caco-2 cells) and in vivo (C57BL/6N mice) [17
]. Here, we determined the potential of guava fruit extract prepared by supercritical CO2
extraction to regulate postprandial blood glucose response in healthy young adults during an oral glucose tolerance test (OGTT).
The OGTT is commonly used in clinical research to evaluate glucose tolerance and thus provides an appropriate method to assess postprandial glucose response in humans [24
]. As expected, the consumption of 2.5 mL of guava fruit extract reduced postprandial mean blood glucose levels in healthy students, as shown by comparing the results of the OGTT between the intervention and control groups. A significantly decreased peak blood glucose level was detected at 30 and 90 min for the group that consumed guava extract. One of the participants in the September intervention group even showed a postprandial glucose concentration of 3.39 mmol/L, which goes below the threshold of plasma glucose ≤ 3.9 mmol/L for the definition of hypoglycemia, according to the American Diabetes Association/European Medicines Agency specified guidelines [25
]. Nevertheless, no other symptoms of hypoglycemia were observed, and the person behaved inconspicuously. Moreover, the glucose concentration increased again afterwards. In fact, glucose concentrations of <3.0 mmol/L can cause cognitive dysfunction and are sufficiently low to indicate serious, clinically important hypoglycemia. A glucose concentration above this value, as it was measured in this study, need not be reported in clinical trials [26
]. Blood glucose regulation was accompanied by a modestly reduced, but nonsignificant, postprandial insulin secretion.
The present findings seem to be consistent with other clinical research on plant-derived extracts. For instance, mulberry leaf extract significantly reduced total blood glucose and insulin secretion after ingestion of maltodextrin in a randomized, double-blind, placebo-controlled study. The authors reported a classical dose–response curve with significant effects over placebo [27
]. It is interesting to note that the desired effects in our study were achieved at low concentrations of only 2.5 mL (0.036 mL per kg bodyweight; 70 kg total body weight assumption) of the guava fruit extract. This was significantly lower (approximately 10 times) than the applied extract concentration in our previous mouse study (0.4 mL per kg body weight) [17
]. It can be assumed that higher concentrations of the extract would lead to a stronger reduction in postprandial glucose response.
It is likely that bioactive compounds are relevant for the observed effects. In this context, polyphenols are known to positively influence postprandial glycemic responses and fasting hyperglycemia, as well as acute insulin secretion and insulin sensitivity [28
]. P. guajava
has been reported to contain a variety of phenolic compounds [16
]. For this study, guava fruit extract was prepared by supercritical CO2
extraction (SFE). A detailed phytochemical composition of guava fruit extract prepared by SFE still needs to be identified and investigated [17
]. To date, SFE has only been applied for guava leaves and seeds [29
], whereas this technology has not been used for guava fruit. In general, supercritical fluid extraction is safe, clean, and environmentally friendly. Carbon dioxide, which is widely used, is an inert gas with many technological advantages. It is readily available, cheap, odorless, tasteless, and generally regarded as safe [30
]. For that reason, SFE is an attractive technology to obtain bioactive compounds for application in the food industry [31
Here, we show for the first time that a guava fruit extract prepared by SFE can effectively reduce postprandial glucose peaks in humans. Regulating postprandial hyperglycemia could prevent various complications of T2DM, such as microvascular diseases [3
]. For this purpose, bioactive compounds in plant-derived extracts that inhibit sugar absorption are of great interest [8
]. In response to the increasing demand for natural and plant-based foods, some of these extracts could be used as an alternative to oral synthetic hypoglycemic agents [32
]. The study thus lays a basis for the development of antidiabetic food products based on guava fruit extract.
Finally, the storage stability of guava fruit extract was investigated. Interestingly, the same batch of extract was less effective after a prolonged storage time and repeated freezing and thawing. After preparation in August 2018, the effect of the guava fruit extract showed a trend toward a reduced blood glucose increase with a significant difference at 90 min on the 5th of September 2018. To our surprise, this trend was less remarkable one week later. Freezing and thawing and storage until November had an adverse influence on the efficacy of the extract, as the glucose response was only slightly reduced on the 28th of November. These findings have important implications for developing food supplements or functional foods, as they indicate that the guava fruit extract is not stable under certain circumstances.
To identify potential bioactive compounds, we analyzed the extract by HPLC-MS, GC-MS, and NMR. Surprisingly, we did not detect expected polyphenolic compounds such as phloretin, catechins, guaijaverin, or quercetins. As HPLC-MS analysis alone resulted in a chromatogram indicating several compounds of a distinct mass at various retention times, which could not be clearly allocated based on existing libraries, we used GC-MS and NMR to address this question. Fortunately, we could identify several compounds, two of them being of special interest: Kojic acid and 5-hydroxymethylfurfural. First, both substances have been reported in the context of antidiabetic properties [33
]. Second, in HPLC analysis, they represented some of the major peaks and were therefore the compounds present at the highest concentrations. Third, both compounds were also found in ethanolic extracts prepared from guava leaves and fruits that were also effective in our previous in vitro studies [17
]. Based on these facts it seems feasible that the described inhibitory effect on intestinal glucose resorption is related to presence of kojic acid and/or 5-hydroxymethylfurfural. As the guava extract prepared by SFE was the most effective one in vitro (Caco-2 cells) and in vivo (mouse) [17
], we chose to use it for this trial. In addition, the technology offers several advantages: On the one hand, CO2
is cheap, safe, and physiologically sound to very low levels. Therefore it is approved for food products without declaration [35
]. On the other hand, CO2
-based SFE results in the enrichment of other compounds than those enriched by alcoholic extraction procedures. It dissolves non-polar or slightly polar, low molecular weight compounds and oxygenated organic compounds of medium weight, but excludes free fatty acids, pigments, water, proteins, polysaccharides, sugars, and mineral salts [35
]. Consequently, extracts prepared by SFE contain putative bioactive compounds in a different composition than those prepared by traditional methods, including alcoholic or aqueous extraction techniques.
To our knowledge, this is the first report showing a beneficial effect of guava fruit extract prepared by SFE on glucose response in healthy young adults. This study has confirmed our hypothesis that consumption of guava fruit extract leads to a reduced postprandial glucose response in humans.
Finally, some limitations need to be considered. First, the number of subjects participating in this study was relatively small. Therefore, the findings need to be interpreted cautiously. However, based on the reported effects in vitro and in vivo [17
] and the calculated significance (this study), the efficacy of the guava extract prepared by SFE appears to be reliable. Second, an issue that was not addressed in this study was whether there exists a difference between men and women. However, we have shown previously that the used guava extract reduces glucose transport by a specific inhibition of glucose transporter 2 (GLUT2) [17
], which is not a gender dependent transport system. Third, as described before, we have used low concentrations of the guava extract for this study. To get a stronger effect one could increase the concentration, but excessive consumption of the guava extract could lead to diarrhea and flatulence. However, relevant symptoms were not observed in our previous mouse study [17
]. Finally, for application in antidiabetic food products for day-to-day consumption, long-term effects and potential side effects have yet to be determined. In addition, the efficacy of the extract in patients with T2DM is still unknown, as the study was conducted with young, healthy volunteers. Therefore, further studies are necessary to evaluate the potential of guava fruit extract prepared by SFE for the prevention of T2DM and the regulation of hyperglycemia in affected patients.