*3.2. Clinical Trial*

The study flow chart, produced in accordance with the CONSORT PRO reporting guidelines [32], is shown in Figure 2. Initially, 42 subjects were screened for the clinical study; however, two of these subjects did not meet the inclusion criteria and therefore were excluded. The total number of subjects enrolled was 40, and they were randomly assigned to either group 1, receiving the BSG extract-based food supplement (treatment A) first and then the placebo (treatment B), or to group 2, which first received the placebo and then the BSG extract-based food supplement. As the aim of the clinical trial is to unravel the efficacy of BSG extract in reducing post-prandial glycemia and improving insulinemic response, the BSG extract-based food supplement was compared with the placebo consisting of indigestible carbohydrates. Group 1 consisted of 13 women (65%) and 7 men (35%), and group 2 consisted of 12 women (60%) and 8 men (40%).

The participants in the two groups had similar sociodemographic characteristics and clinical data, with no significant differences with the exception of HDL-C (*p* = 0.01). The baseline characteristics of the subjects for each group are summarized in Table 3.

The primary objective of the study was to evaluate the contribution of BSG extractbased food supplement in promoting the reduction of postprandial blood glucose in normoglycemic subjects with slightly impaired glucose tolerance, shown by HOMA-IR Index and Triglycerides and Glucose index (TyG) values higher than 2.5 and 4.5, respectively, for most of the recruited subjects [33] (Table 3). In fact, the upper limits of the ranges for triglycerides, HDL-C, and LDL-C greater than 150 mg/dL), lower than 40 mg/dL, and higher than 159 mg/dL, respectively, lead to thinking that part of the subjects recruited has an altered lipid profile compatible with mild insulin resistance [34] (Table 3). Nevertheless, at the baseline (t0) all 40 subjects were normoglycemic (Table 4).

**Figure 2.** CONSORT Flow diagram.

**Table 3.** Characteristics of the study population: demographic and clinical data at baseline (t0).


Glycemia and insulin levels for the two study groups, at different time points, both in male and in female subjects, are reported in Table 4. As expected, the mean postprandial glycemia values recorded after 15 and 30 min tended to grow to a peak at 60 min, regardless of sex and treatment. After peaking at 60 min, the mean postprandial glycemia values recorded after 90 and 120 min tended to decrease more in the subjects who had taken the food supplement than those subjects who had taken the placebo, regardless of sex. In fact, while from 0 to 60 min the post-prandial glycemia values of the subjects (both male and female) who had taken the food supplement and the placebo were overlapping, at 90 and 120 min, the mean postprandial glycemia values of the subjects who had taken the food supplement were lower than the corresponding values of the subjects who had taken the placebo. As far as postprandial blood insulin is concerned, the mean post-prandial insulinemia values recorded after 15 and 30 min tended to grow to a peak of 60 min in the placebo group more than in the food supplement group, regardless of sex. After the peak recorded at 60 min, also in this case the average post-prandial insulinemia values recorded after 90 and 120 min tended to decrease more in the subjects who had taken the food supplement than in those who had taken the placebo, regardless of gender.

**Table 4.** Variation in values (mean ± standard deviation, minimum and maximum) of blood glucose and insulin in men and women for the two experimental treatments (A: BSG extract-based food supplement, and B: placebo).


The LMM model for blood glucose (Table 5) identified a statistically significant effect for the measurement (*p* < 0.001), for the treatment (*p* < 0.001), and also for the measurement × treatment interaction (*p* < 0.001). Significant effects also emerged for sex (*p* = 0.014) and age (*p* = 0.032). Furthermore, the effect of the treatment order was also significant (*p* < 0.001); the measurement × order of treatment was not (*p* = 0.99). These results indicate that there was a difference between treatments A and B when it came to the postprandial glycemic curve of patients (Figure 3—top figure). Blood glucose did not differ between treatments, from the initial measurement to the peak at 60 min (t0: dB-A = 0.29 ± 1.21, t424 = 0.240, *p* = 0.81; t15: dB-A = 0.22 ± 1.21, t424 = 0.185, *p* = 0.85; t30: dB-A = 0.34 ± 1.21, t424 = 0.283, *p* = 0.77; t60: dB-A = 0.46 ± 1.21, t424 = 0.383, *p* = 0.70). Actually, the blood glucose values of the subjects treated with the food supplement (treatment A) or placebo (treatment B), recorded at t0, t1, t2, and t3, did not change. Differently, the blood glucose values recorded at t4 (90 min) and t5 (120 min) during treatment A were significantly lower than the corresponding values during treatment B in the subsequent phase of descent from the peak, respectively, after 90 min (dB-A = 2.67 ± 1.21, t424 = 2.199, *p* = 0.028) and 120 min (dB-A = 6.55 ± 1.21, t424 = 5.397, *p* < 0.001).


**Table 5.** LMM model results for glucose and insulinemic response curves.

**Figure 3.** Variation in postprandial glycaemia for the two experimental treatments. **Above**: variation

in blood glucose for the two experimental treatments (A: BSG extract-based food supplement and B: placebo); **middle**: blood glucose values before and after wash out; **below**: change in blood glucose before and after wash out regardless of experimental treatment.

The trend of the glycemic curve, however, was not different before and after the washout period, as evidenced by the fact that the interaction between measurement and treatment order was not significant (Table 5) (Figure 3—middle and below figure).

The effect of sex indicates that the men selected in the sample had blood glucose values higher than those of women (4.12 ± 1.60, t36 = 2.575, *p* = 0.014). This difference, however, has no clinical relevance, as the blood glucose values of the subjects at t0 were in line with the inclusion criteria.

Finally, as far as the age of recruited subjects is concerned, blood glucose tended to increase with age (0.26 ± 0.11, t36 = 2.228, *p* = 0.032) regardless of gender. Moreover, in this case, this difference has no clinical relevance as the blood glucose values of the subjects at t0 were in line with the inclusion criteria.

The LMM model for insulin (Table 5) provided similar results to that for glycemia. The effects of measurement (*p* < 0.001), treatment (*p* < 0.001), and measurement × treatment interaction (*p* < 0.001) were statistically significant. There was no significant effect for the sex (*p* = 0.25) and age (*p* = 0.12).

The effect of the treatment order was also statistically significant in this case (*p* = 0.009), but the interaction between measurement and treatment order (*p* = 0.10) was not such. These results indicate that there is a difference in the postprandial insulin curve in patients undergoing treatment A versus those when undergoing treatment B (Figure 4—top figure).

The insulin values differed between treatments, since the initial measurement (dB-A = 2.83 ± 1.31, t424 = 2.169, *p* = 0.031) increased at 15 min (dB-A = 4.51 ± 1.31, t424 = 7.077, *p* < 0.001) and gradually at 30 min (dB-A = 5.02 ± 1.31, t424 = 7.590, *p* < 0.001), reached a peak at 60 min (dB-A = 5.10 ± 1.31, t424 = 7.650, *p* < 0.001) and 90 min (dB-A = 10.16 ± 1.31, t424 = 12.733, *p* < 0.001), with a maximum measurement at 120 min (dB-A = 10.79 ± 1.31, t424 = 13.336, *p* < 0.001).

This order indicates that the insulin value after the washout is significantly lower than that observed before the washout (−1.39 ± 0.53, t423 = 2.612, *p* = 0.009), regardless of the experimental treatment. As for glycaemia, the trend of the curve was not different between before and after the washout period for insulinemia, as evidenced by the fact that the measurement × treatment order was not significant (Table 5).

The incremental areas under curve (iAUCs) of postprandial glycemia of the subjects treated with BSG extract-based food supplement and the corresponding iAUCs of the subjects taking placebo did not show any statistical difference, while the mean incremental area under curve (iAUC) of insulinemia of the subjects treated with BSG extract-based food supplement (iAUC = 1928 ± 237) were 19.7% significantly lower (f-ratio value = 6.30397, *p*-value = 0.013436) than the corresponding mean iAUC of the subjects taking the placebo (iAUC = 2457 ± 400).

As far as safety is concerned, none of the subjects reported any adverse event after receiving the food supplement.

**Figure 4.** Variation in postprandial insulin values for the two experimental treatments. **Above**: variation of the insulin concentration for the two experimental treatments (A: BSG extract-based food supplement, and B: placebo); **middle**: insulin concentration values before and after washout; **bottom**: change in insulin concentration before and after washout regardless of experimental treatment.
