Bidens pilosa Formulation Improves Blood Homeostasis and β-Cell Function in Men: A Pilot Study

B. pilosa has long been purported to have antidiabetes activity, but despite the advancement in phytochemistry and animal models of diabetes, no human clinical trials have been conducted to date. Here, we evaluated the effect of a B. pilosa formulation on fasting blood glucose (FBG), fasting serum insulin, and glycosylated hemoglobin A1c (HbA1c) in diabetic subjects. The B. pilosa formulation reduced the level of FBG and HbA1c in diabetics but increased fasting serum insulin in healthy subjects. Moreover, combination of B. pilosa formulation with antidiabetic drugs had better glycemic control in diabetics. The homeostatic model assessment (HOMA) data suggested that the antidiabetic activity of this formulation was via improvement of β-cell function. We also tested the safety of the B. pilosa formulation in healthy subjects and observed no obvious side effects. We conclude that B. pilosa has potential as an antidiabetes treatment.


Introduction
Type 2 diabetes is a global health problem that carries a large economic burden. According to the International Diabetes Foundation 382 million people were diagnosed with diabetes in 2013 and this number is expected to rise to 592 million by 2035 [1]. Current oral antidiabetic drugs have unmet efficacy and undesirable side effects in patients often leading to lethal complications [2]. Therefore, continuing the search for new diabetes treatments is a priority.
Over 1200 plants are purported to have antidiabetic activity [3,4]. Among them, B. pilosa has long been used as an antidiabetic herb in Asia, America, and Africa [5]. However, no clinical trial has ever evaluated the efficacy and safety of this herb [3,6]. We and other groups have shown that B. pilosa has hypoglycemic activity in diabetic db/db mice and alloxan-treated mice [7][8][9]. Three polyynes from B. pilosa were found to have glucose-lowering activity [8,9]. Among them, cytopiloyne identified from B. pilosa had better glucose-reducing activities in diabetic mice than the other two polyynes [9]. We also demonstrated that B. pilosa and cytopiloyne lowered blood glucose via insulin secretion and islet protection [4]. Further, mechanistic studies showed that cytopiloyne and, probably, B. pilosa exerted antidiabetic action via their regulation of -cell function [4].
Despite some claims of human antidiabetic activity, there have been no modern clinical evaluations of B. pilosa in humans. In this study, we evaluated the efficacy and safety of a B. pilosa formulation in human diabetic and healthy subjects.

Efficacy Pilot Study.
Fourteen volunteers whose fasting blood glucose was more than 126 mg/dL and/or whose 2 h  postmeal prandial blood glucose was more than 200 mg/dL were diagnosed as diabetics based on the American Diabetes Association criteria. They were grouped into 2 groups. One group, 6 diabetics, only consumed the B. pilosa formulation (probetacell) orally at a dose of 400 mg, ter in die, for 3 to 7 months. The other group, 8 diabetics, took antidiabetic drugs plus the B. pilosa formulation. Their blood samples were collected before and after their treatment. Biochemical parameters of the blood samples from both groups were determined (Table 1) based on the manufacturers' protocols. Briefly, triglyceride (TRIG), total cholesterol (TC), high density lipoprotein (HDL), low density lipoprotein (LDL), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and blood urine nitrogen (BUN) were analyzed with 7600 Clinical Analyzer (Hitachi). Serum insulin was quantified with the ADVIA Centaur ELISA Kits (Siemens). Hb A1c was measured using a DCA 2000 analyzer (Bayer). The B. pilosa formulation (probetacell) is a commercial functional food in Taiwan (Chun-Yueh Biomedical Technology Co., Ltd.) and HPLC was used to control the quality of the formulations (see Sup.

Safety Pilot Study.
Blood from seven healthy volunteers was collected before and after they took the B. pilosa formulation (probetacell) orally at a daily dose of 400 mg per person, ter in die, for 3 months. The biochemical parameters (Table 2) of the blood samples were analyzed as above.

Statistical Analysis.
Data from three independent experiments or more are presented as mean ± SEM. Student'stest was used for statistical analysis of the differences between groups. A value ( * ) of less than 0.05 was considered to be statistically significant.

B. pilosa Formulation Improves Type 2 Diabetes via Promotion of -Cell Function.
Our group and others previously demonstrated that B. pilosa exerted antidiabetic activity in mouse models, so in this study we verified this effect in humans. First, we evaluated the beneficial effect of the B. pilosa formulation on subjects with type 2 diabetes. We found that those who only took the B. pilosa formulation had fasting blood glucose levels of 201.7 ± 83.3 and 123.3 ± 18.6, respectively, before and after treatment with the B. pilosa formulation (Table 1). Similarly, the diabetics had Hb A1c levels of 9.1 ± 1.7 and 7.2 ± 0.7, respectively, before and after the treatment with the B. pilosa formulation ( Table 1). The HOMA-IR and HOMA-are commonly used to assess insulin resistance and -cell function, respectively [10]. Treatment with the B. pilosa formulation significantly increasedcell function of the participants as shown by the HOMAvalues. In contrast, the treatment did not affect their insulin resistance, as shown by the HOMA-IR values (Sup. Figure  1). Accordingly, the B. pilosa formulation boosted serum insulin level in healthy persons (Table 2). Besides, we tested the combination effect of the B. pilosa formulation. We found that those who only took antidiabetic drugs and the B. pilosa formulation had fasting blood glucose levels of 220 ± 70.9 and 150 ± 51.3, respectively, before and after the combination treatment (Table 1). However, the combination use of the B. pilosa formulation seemed better than its single use based on the data on the decreased ratio of fasting blood glucose and Hb A1c (Table 1).
Overall, the data from this study are in good agreement with previous studies in mice [4] that suggested that B. pilosa enhanced insulin secretion and islet preservation via -cell regulation.   Evidence-Based Complementary and Alternative Medicine  (Table 2). In addition, heavy metals (As, Pb, Cd, and Hg) and 251 pesticides in the B. pilosa formulation used in the study were determined and their concentrations are below the limit of detection (Figure 1 and Sup. Table  1). The Food and Agricultural Organization of the United Nations recognizes B. pilosa as a staple food [11]. The Ministry of Health and Welfare in Taiwan also allows its use as an ingredient in food for human consumption. Previous studies by our group and others found no toxicity of B. pilosa in mouse models [5,6] and rats [12]. However, comprehensive scientific study of the safety of B. pilosa has not been conducted. In this work, clinical data suggest that B. pilosa at 400 mg, ter in die, has no noticeable toxicity ( Table 2). Largescale clinical trials on the efficacy and toxicology of B. pilosa in humans are required prior to its further medical use. In summary, our clinical data demonstrated that the B. pilosa formulation had an antidiabetic action and no obvious side effects in humans. This action involves the regulation of -cells.