1. Inhibition of alpha-glucosidase enzymes by GvEx and its active component
In the digestive tract, some alpha-glucosidase enzymes, such as alpha-amylase, maltase and sucrase, digest carbohydrates to glucose that can be absorbed through the intestine. Alpha-glucosidase inhibitors (alpha-GIs), namely, acarbose and voglibose, prevent the digestion of carbohydrates based on competitive enzyme inhibition, and provide short-term glycemic control .
Deguchi et al.  demonstrated that GvEx, which was prepared by hot water extraction from guava leaves, inhibited the in vitro activities of maltase, sucrase, and alpha-amylase in a dose-dependent manner (Fig. 1). The 50% inhibitory concentration (IC50) of GvEx was 0.6 mg/mL for alpha-amylase, 2.1 mg/mL for maltase, and 3.6 mg/mL for sucrase, indicating the higher inhibitory activity of alpha-amylase than the other two enzymes. Furthermore, Wang et al.  found that the aqueous extract from guava leaves inhibited both sucrase and maltase activities in the small intestinal mucosa of diabetic mice, occurring as a mixed type of competitive and non-competitive inhibition.
In order to clarify the active component of the guava leaf extract, GvEx solution was fractionated in dialysis tubes of 5,000 and 30,000 MW pore size. The inhibitory activity of alpha-amylase was detected in the fraction with a MW between 5,000 and 30,000. This fraction reacted with ferrous tartrate, indicating that a component with a phenolic hydroxyl group was part of the fraction . It has been reported that guava leaf contains some polyphenols, such as peduncladgin, casuarinin and isostrictinin [18–20]. However, high-performance liquid chromatography (HPLC) analysis demonstrated that these elementary polyphenols were present in the ethyl acetate extract of guava leaf but not in GvEx. Several instrumental analyses, such as 1H-nuclear magnetic resonance (NMR), infrared absorption spectrum, and solid 13C-NMR, have suggested that the active component of GvEx was a polymerized polyphenol named guava leaf polyphenol (GvPP), which is composed of ellagic acid, cyanidin and other low-molecular-weight polyphenols .
Here, it was shown that the GvEx acts as an alpha-GI in vitro and contains GvPP as the active component.
2. Reduction of postprandial blood glucose elevation
2.1 Murine models
It is well known that orally administered alpha-GIs have the potential to reduce postprandial blood glucose elevation as shown in a carbohydrate loading test in vivo [21–24]. To determine the effect of GvEx on postprandial blood glucose elevation, normal mice were immediately given GvEx or saline (control) following overnight fasting. Thirty minutes later, soluble starch, sucrose or maltose (2 g/kg) was loaded and blood glucose level was measured at 30-min intervals from 0 to 120 min. Compared with control, the single ingestion of GvEx significantly reduced the area under the curve (AUC) of postprandial blood glucose levels by 37.8% after loading soluble starch at 250 mg/kg and by 31.0% and 29.6% after loading sucrose and maltose, respectively at 500 mg/kg each . Additionally, in streptozotocin-induced diabetic mice with a fasting blood glucose (FBG) level of >200 mg/dL, which is a model of T1DM, the single ingestion of GvEx (250 mg/kg) significantly reduced the AUC after loading soluble starch (unpublished data). Furthermore, anti-hyperglycemic activity was detected in animal models with T1DM [10, 25, 26].
2.2 Human trials with normal, pre-diabetic and type 2 diabetic subjects
According to the preparation method of GvEx, Guava Leaf Tea was manufactured for human trial. To clarify the above-mentioned findings in animal models, a crossover study was designed to evaluate the effects of a single ingestion of Guava Leaf Tea on postprandial blood glucose elevation in normal and pre-diabetic subjects . The single ingestion of Guava Leaf Tea significantly reduced postprandial blood glucose elevation at 30, 90 and 120 min (Fig. 2a). In addition, the AUC of the glucose level after carbohydrate loading (ingestion of cooked rice) was significantly reduced by about 20% compared with control (Fig. 2b).
A crossover clinical trial involving 20 hospitalized patients with T2DM was conducted to compare the potential of Guava Leaf Tea and voglibose (Basen®; Takeda Chemical Industries, Ltd., Tokyo, Japan) to reduce postprandial blood glucose elevation . As shown in Figure 3, the postprandial glucose level 2 hours after meal was elevated to ca. 160 mg/dL in the patients with each standard treatment (in control). The elevated level was significantly reduced with the single administration of Guava Leaf Tea and voglibose to 143 mg/dL (p < 0.001) and 133 mg/dL (p < 0.001), respectively. The reducing potential was significantly milder with Guava Leaf Tea than with voglibose (p < 0.01). There were no side effects, such as hypoglycaemia, due to abnormal interaction in the combined administration of each standard treatment and voglibose or Guava Leaf Tea .
Taken together, these findings, suggest that the single ingestion of GvEx or Guava Leaf Tea can reduce postprandial glucose elevation via the inhibition of alpha-glucosidase in mice and human subjects with or without diabetes. Furthermore, Guava Leaf Tea was found to have milder activity than voglibose.
3. Improvement of diabetes symptoms and hyperlipidemia
3.1 Diabetic animal models
In previous studies, patients with T2DM were treated with alpha-GIs, namely, acarbose  or voglibose  because long-term oral administration has been reported to improve diabetic markers [e.g., hemoglobin A1c (HbA1c) and insulin resistance]. Deguchi et al.  previously examined the effects of consecutive ingestion of GvEx for 7 weeks on the progression of T2DM and nephropathy in genetically diabetic mouse models (i.e., C57BL/Ksj, db/db, and Leprdb/Leprdb). They showed that the mice developed widespread pathological abnormalities including not only diabetes and obesity but also well-defined nephropathy [28, 29]. Compared with drinking water (control), the GvEx (250 mg/kg/day) solution significantly reduced blood HbA1c% after ingestion for 5 and 7 weeks. GvEx also improved nephropathy with a significant reduction in the thickening index of the glomerular mesangial matrix in the kidney observed at 7 weeks (Table 1). In contrast, there were no significant effects on weight gain, food and water intakes of the diabetic mice.
Recently, Shen et al. have reported the effects of the aqueous extract from guava leaves on type 2 diabetic rats. They showed that long-term feeding of the extract significantly reduced blood glucose level, increased plasma insulin level in an oral glucose tolerance test, and stimulated activities of some glucose metabolic enzymes. Additionally, the single feeding of the extract significantly reduced blood glucose level in an oral glucose tolerance test. However, the underlying mechanisms have not yet been clearly elucidated.
The above-mentioned findings indicate that the consecutive ingestion of GvEx has the potential to improve diabetes symptoms such as hyperglycemia, nephropathy and insulin resistance in diabetic animal models.
3.2 Human trials in pre-diabetic and type 2 diabetic patients
To confirm previous findings in diabetic animal models, a first long-term clinical trial was conducted to evaluate the effects of consecutive ingestion of Guava Leaf Tea with every meal for 12 weeks on the parameters of diabetes symptoms and safety in 15 male subjects with pre-diabetes and mild type 2 diabetes .
Table 2 shows the serum parameters of diabetes and analysis results between the initial week and week 12 of ingestion of Guava Leaf Tea in subjects with pre-diabetes and mild diabetes. In all the subjects, the FBG level showed a decrease from 136 to 131 mg/dL (p = 0.07) and a significant reduction was detected after the calculation of reduction rate of the FBG level. In particular, the FBG level showed a more pronounced decrease in the pre-diabetic subjects (p = 0.06; n = 7; initial FBG level: 110-126 mg/dL). Five of seven pre-diabetic subjects showed a reduction in blood HbA1c%. Also, the levels of insulin, C-peptide and homeostasis model assessment for insulin resistance (HOMA-IR) significantly decreased in all subjects. Moreover, after the ingestion of Guava Leaf Tea for 12 weeks, serum levels of total cholesterol (T-CHO) and triglyceride (TG) significantly decreased in the subjects with hypercholesterolemia and hypertriglycemia (n = 7 and n = 5; initial levels: >220 mg/dL and >150 mg/dL, respectively). There were no abnormal changes in the parameters of iron metabolism, liver and kidney functions, blood chemistry and on items covered in the physician's physical examination and health interviews during this trial .
A second long-term clinical trial investigated the effects of consecutive ingestion of Guava Leaf Tea for 8 weeks on the parameters of diabetes symptoms and safety in diabetic patients receiving therapy, that is, anti-diabetic medication with or without an inhibitor of HMG-CoA reductase .
Table 2 shows the serum parameters of diabetes and analysis results between the initial week and week 8 of ingestion of Guava Leaf Tea in diabetic patients with some medication. Ingestion of the tea significantly decreased blood HbA1c% in diabetic patients who had initial values of >6.5% and were assessed to have abnormal control of blood glucose level. Additionally, the ingestion of the tea significantly reduced serum insulin level in diabetic patients with hyperinsulinemia whose serum insulin level was >17 μU/ml before intake. The ingestion of the tea also decreased the parameter values of lipid metabolism, that is, triglycerides (TG; p < 0.05, for 4 weeks), nonesterified fatty acids (NEFAs; p < 0.05, for 4 weeks), remnant-like particle-cholesterol (p = 0.08, for 4 weeks) and phospholipids (p = 0.06, for 8 weeks), in the subjects with values higher than the reference values in patients without fluvastatin treatment. In contrast, neither side effects resulting from alterations in the parameter values of liver and kidney functions or blood chemistry nor changes in doctor's health interviews were observed during the entire clinical trial period. Also, there was no hypoglycemia due to the abnormal interaction between Guava Leaf Tea and anti-diabetic drugs with or without an HMG-CoA reductase inhibitor .
Taken together, it is suggested that the consecutive ingestion of Guava Leaf Tea with every meal improves diabetes symptoms, such as hyperglycemia, hyperinsulinemia, insulin resistance as well as hyperlipidemia in pre-diabetic and mild diabetic patients with or without hyperlipidemia. Moreover, it is indicated that the consecutive ingestion of Guava Leaf Tea in addition to anti-diabetic and anti-hypercholesterolemia medications shows no side effects due to the abnormal interaction.
4. Improvement of hypercholesterolemia and hypoadiponectinemia
To verify the anti-hyperlipidemic activity of Guava Leaf Tea, a third long-term clinical trial investigated the effects of consecutive intake for 8 weeks on the parameters of hyperlipidemia, diabetes and safety in 23 subjects with borderline or mild hyperlipidemia with or without T2DM. During the trial, 7 subjects were administered fluvastatin, pravastatin, pitavastatin, colestimide (an inhibitor of cholesterol absorption) or ethyl icosapentate (a TG reducer) .
Table 3 shows the changes in serum lipid parameters after the ingestion of Guava Leaf Tea for 8 weeks in subjects with hypercholesterolemia (initial T-CHO level: >220 mg/dL). The consecutive ingestion reduced the serum levels of T-CHO (p < 0.05), LDL-cholesterol (LDL-CHO) (p = 0.06) and phospholipid (p < 0.05) in these subjects. A significant reduction in T-CHO level (p < 0.05) was also observed in the same subjects receiving no medicinal treatment. On the other hand, the levels of high-density lipoprotein cholesterol (HDL-CHO), TG, NEFA and lipid peroxide were not significantly changed in the same subjects. In contrast, the consecutive ingestion decreased the serum level of TG (p < 0.05, week 4) in subjects with hypertriglycemia (initial TG level: >150 mg/dL) and that of phospholipid (p < 0.05, weeks 4 and 8) in subjects with hyperphospholipidemia (initial phospholipid level: >250 mg/dL).
Moreover, the ingestion of Guava Leaf Tea significantly reduced blood HbA1c % in diabetic subjects (initial HbA1c%: >6.5%), and significantly increased serum adiponectin level in each subject with hypoadiponectinemia (Table 4) and hyperglycemia. The nutritional intake of all the subjects showed no significant variation in the results of the questionnaires that were designed for 1 week in the first, middle and last weeks of the trial period . This suggests that the trial findings were due to the effects of ingestion of Guava Leaf Tea and not from nutritional intake. There were no abnormal changes in the parameters of liver and kidney function, blood chemistry and doctor's health interviews during the entire trial period. Also, side effects such as hypoglycemia due to the abnormal interaction between Guava Leaf Tea and an HMG-CoA reductase inhibitor, colestimide (an inhibitor of cholesterol absorption) or ethyl icosapentate  were not observed.
Overall, the results indicate that the consecutive ingestion of Guava Leaf Tea together with every meal improves not only hyperglycemia but also hypoadiponectinemia, hypercholesterolemia and hyperlipidemia in pre-diabetic and diabetic patients with or without hyperlipidemia. The consecutive ingestion also ameliorates high blood cholesterol level in subjects with hypercholesterolemia or borderline hypercholesterolemia.
To confirm the safety of GvEx and Guava Leaf Tea, several toxicity studies have been conducted in vitro, as well as in animal models and human subjects.
In single-dose and 1-month repeated dose toxicity studies, Kobayashi et al.  demonstrated that the oral administration of GvEx (200 and 2000 mg/kg/day) caused no abnormal effects in rats, indicating that there is neither acute nor chronic toxicity. Oyama et al.  investigated the mutagenic activity of both GvEx and Guava Leaf Tea. They found that Guava Leaf Tea had a lower mutagenic activity than commercial green tea and black tea in a DNA repair test (Rec-assay); however, these teas showed no mutagenic activity in a bacterial reverse mutation test (Ames test). Moreover, GvEx did not induce chromosomal aberrations in a micronuclear test using peripheral blood erythrocytes, which were prepared from mice by a single oral administration of GvEx (2000 mg/kg). From these findings, it is suggested that Guava Leaf Tea and these commercial teas have no genotoxicity.
After the approval as FOSHU, Guava Leaf Tea has been taken by not only subjects with pre-diabetes but also patients with mild and moderate T2DM. If the ingested Guava Leaf Tea interacts with anti-diabetic and other commercial drugs, it poses a risk of side effects, such as hypoglycemia or inhibition of drug activity. Cytochrome P450 (CYP) isoforms in the human liver or the small intestinal epithelium are known to be involved in drug metabolism. Especially, CYP2C8, CYP2C9 and CYP3A4, which are typical CYP isoforms, can metabolize many commercial drugs. Grapefruit juice inhibits CYP isoforms and shows abnormal interaction with anti-diabetic drugs as well as many other commercial drugs, such as repaglinide and tolbutamide (sulfonylurea) as a stimulator of insulin release from the pancreas, sibutramine as an appetite suppressant, HMG-CoA reductase inhibitors, losartan as an angiotensin II receptor antagonist, calcium channel blockers, and warfarin [36, 37]. Therefore, to evaluate the interaction between Guava Leaf Tea and anti-diabetic and other commercial drugs, Kaneko et al.  investigated the inhibitory effects of GvEx and Guava Leaf Tea on CYP2C8, CYP2C9 and CYP3A4 in vitro and compared them with quercetin as another inhibitor and grapefruit juice. Quercetin and grapefruit juice were shown to have higher inhibitory effects on CYP2C8, CYP2C9 and CYP3A4 than GvEx (more than 10-fold) and Guava Leaf Tea (more than 2- to 10-fold), respectively. A subsequent histopathological study showed the absence of response to the induction of P450 isoforms in the liver of rats with 1-month repeated oral administration of GvEx (2000 mg/kg/day). From these findings, it would appear unlikely that Guava Leaf Tea can cause drug interactions based on either inhibition or induction of cytochrome P450 isoforms.
Deguchi et al.  investigated the effects of GvEx in combination with typical alpha-GIs acarbose or voglibose on alpha-amylase activity in vitro and postprandial blood glucose elevation in mice. GvEx inhibited alpha-amylase dose-dependently when combined with the low active dose of acarbose or voglibose. When concomitantly administered with acarbose or voglibose to normal mice, acarbose and voglibose each at the active dose suppressed postprandial blood glucose elevation following loading of sugars with no effect of GvEx (250 mg/kg). In contrast, at the inactive dose, acarbose and voglibose did not affect the activity of GvEx (250 mg/kg). Therefore, these findings indicate that the combined ingestion of GvEx and an alpha-GI does not induce hypoglycemia in an animal model.
To further examine the effects of drinking excessive amounts of Guava Leaf Tea, human healthy subjects in a previous study  ingested a 3-fold volume (600 ml) of the tea. Notably, neither diarrhea nor hypoglycemia was observed. Furthermore, single ingestion and the consecutive ingestion of Guava Leaf Tea for 8 or 12 weeks with or without anti-diabetic and anti-hyperlipidemia drugs in human clinical trials demonstrated no side effects or abnormal changes, as described earlier.
These findings indicate that Guava Leaf Tea and GvEx induce neither toxicity, mutagenicity, nor abnormal interaction with anti-diabetic and anti-hyperlipidemia drugs, and have a lower potential for drug interactions based on either inhibition or induction of cytochrome P450 isoforms. Thus, Guava Leaf Tea and GvEx can be deemed food and a safe food material, respectively.