Genistein as a dietary supplement; formulation, analysis and pharmacokinetics study

The objective of this study is to improve and optimize the formulation of Genistein in capsules in order to result in a better pharmacokinetic profile comparing to existing commercial products. In order to do this, five different formulations of Genistein capsules were developed and examined by reviewing their disintegration and dissolution properties. Furthermore, flowability of the powder along with potent incompatibilities between Genistein and its excipients were monitored through their thermal properties. The final formulation of Genistein was quantified using HPLC analysis and then its stability was evaluated thoroughly in real time and accelerated conditions. Finally, with the target to have a product with actual results, in vitro and in vivo studies were conducted. The final product proved to have better results in disintegration and dissolution. Moreover, R.G.C.C.’s capsules exhibited enhanced action in human cell lines as well as impressive pharmacokinetic results in animal models. The in vitro results showed an advantage of the R.G.C.C. product compared to the commercial one, whereas its maximum concertation in vivo was determined 34% higher than the commercial one.


Introduction
The regular use of dietary supplements is a worldwide phenomenon. This comes along with the evidence supporting that daily nutrition, mental balance, healthy habits and general wellness are few of the aspects that influence our lifestyle. By interfering to these, the prevention of serious diseases is more effective, targeted and with long term effects. The last decades many studies put in the spotlight the analysis and use of natural products, which have shown therapeutic effects, based mainly on traditional medicine. However, since dietary supplements belong to a multi-dollar industry and at the same time, guidelines and legislation are lenient, there are often brought to the market unauthorized products without adequate quality control but enthusiast claims.
Soy and soy-derived products are examples of these products, that gain everyday more and more recognition. Based on scientific data, the Food and Drug Administration recently

Formulation of capsules
In order to evaluate the right type of formulation different types of recipes and manufacturing methods were examined. Each formulation was evaluated through its dissolution and disintegration properties and overall profile. At Table 1, there is an overview of the different recipes, which were all prepared with dry granulation method. The excipients used to formulate the capsules were, Starcap 1500 1 (a mixture of corn starch and pregelatinized starch), Corn Starch, Nu-FLOW 1 and Silica Dioxide. Two different Genistein formulation strengths were prepared in order to achieve wider market approval. The final dosage unit was decided to be hard shell capsules. Capsules were filled manually using a manual capsule filling machine of 100 capsules. Capsules of 30 mg/ cap Genistein, had an average weigh of 100 mg and net weight 60 mg. They were packed in 120 capsules per package HDPE bottles. Capsules of 125mg/ cap Genistein, had an average weight of 310 mg and new weight 250 mg. They were packed in 60 capsules per package HDPE bottles.

Disintegration-Dissolution
The first method for the evaluation of the drug pharmaceutical profile is disintegration. The disintegration apparatus is LB-2D by LeadTop (Ruian City, Zhejing Province China). The temperature was stable at 37˚C and the disintegration time was recorded continuously during the procedure. The buffer solution that was used in this method was Phosphate Buffer Solution, pH 6.8. Afterwards, the different formulations were evaluated based on their behavior during dissolution. Dissolution apparatus that was used here is a Basket Apparatus (USP), Model RCZ-8 by LeadTop (Ruian City, Zhejing Province, China). Each vessel contained one capsule, in a 900 mL Solution, at 37˚C for 90 minutes. As reference solution each time, one vessel contained a mixture of the excipients used in each formulation at the same portions. The 5 different formulations of Genistein capsules were tested for their dissolution profile in 3% SDS aqueous solution. Aiming to fully characterize and examine the behavior of the final formulation different solutions were used, but 3% SDS aqueous solution presented the best dissolution profile. Sampling and evaluation was done as described in European Pharmacopoeia Chapters (Eu.Ph. 2.9.3).
The evaluation of the dissolution percentage is completed though UV/VIS Spectroscopy (Libra S-12 UV/VIS Spectrometer, Biochrom Cambridge UK). After each time point, samples were collected from the vessels, through the sampling port of the apparatus which is connected with syringes. The solution was filtered through 0.45 um filters (LLG, PTFE, Ø13 mm, 0,45 mm, Metrolab S.A., Athens, Greece) and placed into tubes for analysis. The analysis was done by preparing standard solutions from the samples diluted in the same solvent, targeting specific concentrations and based on a standard curve, the amount of Genistein in each tube was estimated.

Flowability
Pharmaceutical industry utilizes powders in many different applications. Thus, a variety of methods has been developed with the aim to characterize the flow properties of these powders. The US Pharmacopoeia <1174> Powder Flow, states "it is clear that no single and simple test method can adequately characterize the flow properties of pharmaceutical powders". One of the most common methods often reported in studies, is angle of response. Based on pharmacopoeia US <1174>, Angle of response is a characteristic related to interparticulate friction or resistance to movement between particles. However, there are a few limitations with the use of this method, even though it is widely applied in pharmaceutical industry since it can offer a valuable prediction of possible future problems during manufacturing. There are a range of different tests methods in literature, but the most common and important experimental variables are the two following: the height of the "funnel", through which the powder passes and the base upon the pile forms. The height of the funnel can be fixed relative to the base or can be varied as the pile forms. In addition to these, the same applies for the base of the pile, which can either be a fixed diameter or the diameter of the powder cone can vary as the pile forms. In this case, in all of the experiments it was chosen to keep the height of the funnel as a standard value and the base of the pile was measured as the pile formed. The same chapter of USP mentions the mathematical formulas the general experimental procedure with more details. In Table 2, the Flow properties and Corresponding Angles of Repose are summarized based on the USP chapter.

Differential scanning calorimetry
During pre-formulation studies, one crucial step is to examine excipients and different combinations of excipients and API for potent incompatibilities. DSC 3 (STARe System) was used from Mettler, Toledo. The software used to evaluate the results is STARe Software. Appropriate amount of samples were sealed in pierced aluminum pans 40 uL and heated from 0˚C to 400˚C with a scanning rate of 10 K/min. The Nitrogen atmosphere was set 50 mL/min and the whole program lasted for 40 minutes. The samples were compared to an empty reference crucible, which was hermetically sealed and pierced, too. The calorimeter was calibrated using indium (Tm = 156.60˚C).
The studies included two stages of studies. More details of the different evaluations applied are presented below (Table 3):

Analytical validation
The content of capsules in genistein was calculated and validated using Liquid Chromatography. For HPLC analysis, the capsule powder was diluted in Methanol with target concentration 10.5 ppm. Before the insertion of the solution into the vials for the analysis, the solution is filtered through 0.45 um filters (LLG, PTFE, Ø13 mm, 0,45 mm, Metrolab S.A., Athens, Greece).

Ps ¼ As Astd
Mstd As: Peak response from the sample. Astd: Peak Response from standard solution. Mstd: Weighed mass of genistein standard. Ms: Weighed mass of genistein substance.
The purity of the capsules in Genistein was quantified based on Eq (1).
As: Peak response from the sample. Astd: Peak Response from standard solution. Cstd: Standard Solution concentration. Pstd: the purity of genistein standard. Mw: Molecular Weight. W; the average capsule weight. Fdil: dilution factor of the standard solution.
Eq (2) is used to estimate the amount of genistein. HPLC analysis was performed using an Agilent 1260 infinity series equipped with a multiple wavelength detector (Agilent Technologies Inc., Richardson, TX, USA). Chromatograms were integrated and analyzed using OpenLAB Chemstation (version M8301AA, Revision C.01.07 Agilent Technologies Inc., Richardson, TX, USA). The analysis of genistein was performed using a column (Zorbax Eclipse RP C18 reversed-phase, 250 mm×4.6 mm I.D., 5 μm, Agilent, Santa Clara, CA, USA) maintained at 40˚C. Mobile Phase A was water, mobile Phase B was acetonitrile, and mobile Phase C was glacial acetic acid. The mobile Phases A, B, and C were mixed at a ratio of 67.5:25.0:7.5 (v/v). The flow rate was kept at 1.5 mL/min, injection volume was 10 uL and UV detection was carried out at 260 nm. The retention time of the genistein was 7.30 min.

Stability study
After the finalization of the formulation, capsules were sent to verified outsourced laboratories at Eurofins Food Chemistry Testing (US Inc. 3301 Kinsman Blvd., Madison, WI 53704 USA). The protocol included testing in (Table 4):

Cell culture
Two different commercial cancer cell lines were used in the present study that were purchased from ECACC (European Collection of Authenticated Cell Cultures) (Salisbury, UK). MCF7 human breast adenocarcinoma cells (luminal type) (ECACC 86012803) were cultured in

Cell viability analysis
Viability was measured with MTT for cell-metabolism activity. The viable cells were seeded at a density of 2 x 10 4 (200 ul/well) in 96-well plate and incubated at 37˚C and 5% CO 2 for 24 h to form a cell monolayer. After 24 h of incubation, supernatant on the monolayer was discarded and 200 ul of medium and varying concentrations of the two substances were added and incubated for 24, 48 and 72 h. After the specific time points, 20 ul of 5 mg/mL MTT Catalog#M2128 (Sigma-Aldrich, Darmstadt, Germany) in PBS Catalog#P3813 (Sigma-Aldrich, Darmstadt, Germany) was added to each well and incubated for 4 h at 37˚C and 5% CO 2 . Supernatants were discarded and 100 ul of DMSO Catalog#445103 (Carlo Erbo Reagents, Barcelona, Spain) was added and the plates were incubated for 5 min 37˚C and 5% CO 2 to solubilize the formazan crystals and absorbance was measured at 560 nm and the reference wavelength was at 605 nm.

Statistical analysis
The experiment of cell viability determination was performed in triplicates. The average absorbance was calculated for each triplicate. Subsequently, the sample measurements were corrected for the measurement of the blank. One sample t-test was used to determine differences in the mean by comparing the treated samples with the untreated controls. P values < 0.05 were considered to indicate a statistically significant difference. Results were calculated using the Microsoft Excel 2016.

Bioavailability study
Targeting to launch a product improved from the market's "golden standard", studies were concluded with conducting pharmacokinetics study comparing R.G.C.C.'s formulation with a reference marketed formulation. The object of the study was to compare the bioavailability of 2 formulations of the dietary supplements in the male Wistar rat. This study was provided as  (0)2 99 59 91 97). The protocol as described in the company's final report, states that the in vivo study used male Wistar rats (JANVIER LABS, C.S. 4105, Saint-Berthevin F-5394, France), 293-337 g and had as housing of them cages (polysulfone cages with floor area = 1500 cm 2 ) in groups of 2-4 with controlled room temperature 22±2˚C, hygrometry 55±10%, light/dark cycle 12 h/12h, air replacement: 15-20 volumes/hour. Water and Food (SAFE, ref. A04) ad libitum except from approximately 16 hours prior to dosing until 4 hours after dosing. The test formulation (R.G. C.C. and Commercial) where both 125 mg/kg, po. The evaluation criteria were the plasma concentrations and PK parameters. At the end of the experiments, animals were sacrificed by pentobarbital overdose, given ip.
Specifically, six (6) rats received either genistein marketed formulation (125 mg/kg, n = 3) or genistein test formulation (125 mg/kg, n = 3). For both formulations, genistein capsules were opened and the required amount of powder was weighed in order to prepare one preparation per rat. As a vehicle for oral administration, 0.5% carboxymethylcellulose was used and the final formulation was at 12.5 mg/mL. For this study, animals were fasted for approximately 16 hours before dosing with the aim to avoid any baseline genistein. The volume of administration was 10 mL/kg and at the end of the study any surplus Test Item was destroyed. The dose of genistein formulations is expressed pure genistein. For the genistein marketed formulation the correction factor was determined by weighing the powder extract from the capsule, and this value was divided by the weight of genistein in each capsule, leading to a correction factor of 1.67. For the genistein test formulation, as one capsule contains 250 mg of powder including 125 mg of genistein, a correction factor of 2.0 was applied Sampling was conducting through collecting 0.5, 1, 2, 4, 8 and 24 hours after dosing blood samples of 0.5 mL and then plasma was obtained. The amount of genistein in the samples was quantified by Liquid Chromatography tandem mass spectrometry (LC-MS/MS).
The laboratory used Phoenix 1 WinNonLin 1 Version 7.0 for both formulations to determine from the plasma concentrations the following parameters for free genistein by standard non-compartmental methods: As no blood sampling before dosing was performed, T0 values were fixed at 0 μM. In addition, BLQ levels were considered to LLOQ/2, i.e, 0.5 ng/mL, in order to enable the calculation of PK parameters.
Descriptive statistics were performed for all parameters. Time profile plots were prepared on linear and log/linear coordinates for each animal, as well as arithmetic mean (±SD) for both formulations. No statistical analysis was performed.
Finally, the bioavailability of the 2 formulations was evaluated by calculation of the relative bioavailability F rel calculated from the AUCs and the C max as follows:

Formulation of genistein capsules
After conducting a series of experiments, the best formulation of Genistein capsules was determined upon the results described in the following sections, after a series of trials and experiments.

Flowability
One of the most vital attributes of the powder is the way it flows and dispenses. It is a factor with an impact not only during the formulation development in the laboratory but also in the manufacturing stages. For each measurement, around 6.5 g of mixture were used. The results of the flow properties of the powders developed are presented below (Table 5).

Dissolution-disintegration
One test that can offer directly at hand critical safety data on drug behavior and bioavailability, is disintegration. Oral dosage units need to be in a solution in order to be absorbed and act effectively. After the administration of an oral pharmaceutical dosage unit, the first step towards solution is disintegration. In this stage, pharmaceutical dosage units break into granules or particles. Time and rate during this process might differ and be depended upon the scope and type of formulation.
The disintegration tests were all in general satisfying and capsules seems to behave properly in these conditions. The results are summarized at Table 6, where it is apparent that formulation 2, had the best disintegration behavior.
The parameter that controls the oral bioavailability in vitro of oral pharmaceutical dosage units, is dissolution. The rate that the active pharmaceutical ingredient can dissolute in the aqueous media is a crucial factor since it is connected to its instinct absorption [29]. In this case, the five different pharmaceutical forms were tested in 3% SDS aq. Solution. In each vessel, the basket contained one capsule and the temperature was stable at 37˚C. For each formulation form, there is an extra vessel containing the excipients only in the same quantities in order to use as reference. Graph 1 and Table 7 show the dissolution percentage per formulation unit. Each experiment was repeated at least in triplicate and the results at Table 3 are the average of them.
As it is seen after the quality controls studies that were conducted, only formulation 2 completed the tests with success.
Thus, the next studies, were conducted only on this formulation.

Differential scanning calorimetry
Multiple studies were conducted between the API and the excipients. The excipients were tested either in mixtures between each other either with the API. One of the best methods to check incompatibilities and possible by-products or even degradants is to examine the thermal properties.
The thermograms obtained and presented at Graphs 2 and 3, show that no incompatibilities or unexpected events were caused with these combinations and the finalization of the ingredients was verified. Genistein did not present any by-product and its characteristic endothermic peak above 300˚C, is apparent but transferred at lower temperature in the mixtures.
Quantification of Genistein by HPLC. At Graph 4, the chromatograph of R.G.C.C.'s Genistein capsule is presented. It is obvious that pure Genistein is present in the product and the quantification of it, showed 100% API in the capsule.
Stability testing. The results of the stability testing conducted at Eurofins Food Chemistry Testing are summarized at the tables below (Tables 8 and 9, accordingly). Both the real time and the accelerated stability testings agreed that the amount of genistein inside the capsules as well as the organoleptic characteristics of the product describe a stable product with an expire life of 2 years. Each measurement used two capsules of 30 mg Genistein each.

Effect of Genistein R.G.C.C. and Genistein Commercial on the proliferation of the human cancer cell lines
The in vitro activity of Genistein R.G.C.C. compared to R.G.C.C. Commercial was determined with MTT assay. The effect of those two substances on the two human breast cancer cell lines was assessed with the concentration range 0.001, 0.01, 0.1, 1, 10 and 100 uM. The cells were incubated with each substance for 24, 48 and 72 h. Untreated control was also included in the experiments. Hormone sensitive human breast adenocarcinoma cell line MCF7 was treated with the two substances and the proliferation assay was performed at the three time points. Genistein R.G. C.C. had a statistically significant effect on the proliferation of MCF7 cells at 24 h and the viability of the cells was decreased by 25% even at low concentrations of the substance (0.1 and 1 uM) (Fig 1). Genistein Commercial did not show similar effect on the same cell line. There was no statistically significant decrease on the viability of MCF7 cells after 24 h of the treatment with Genistein Commercial. Moreover, there was no significant effect at 48 h and 72 h of both substances against MCF7 cells.
On the other hand, both substances appeared to have no statistically significant effect on the proliferation of the triple negative human breast adenocarcinoma cell line MDA-MB-231 (Fig 2). There was only a significant decrease of cell viability at 72h with the highest concentration, 100uM, of the two substances.  Tables 10 and 11 present Arithmetic mean (CV%) plasma concentration (ng/mL) of Genistein and relative bioavailability, respectively.

Bioavailability
Following single oral administration of 125 mg/kg genistein as reference (marketed formulation) or test formulation, genistein was detected at concentrations above the lower limit of quantitation (i.e., LLOQ of 1.00 ng/mL) up to 8 hours post-dose for all rats, except rat No.4 who presented a t last at 24 hours.
Following oral administration of 125 mg/kg genistein as reference formulation, mean peak plasma levels (C max ) and exposure (AUClast and AUCinf) were 190 ng/mL, 1635 ng.h/mL and 1639 ng.h/mL, respectively. A higher C max , AUC las t and AUC inf were observed after oral administration of 125 mg/kg genistein as test formulation with mean C max , AUC las t and AUC inf values of 254 ng/mL, 1806 ng.h/mL and 1808 ng.h/mL, respectively. A 34% higher C max and 10% higher AUCs were observed for the test formulation compared to the reference formulation (i.e, with F rel1 and F rel2 of 1.10 and F rel3 of 1.34, respectively).  The evaluation of the elimination half-life of all rats show similar results for both reference and test formulations with mean elimination half-lives of 2.71 and 2.36 hours following administration of the marketed and test formulation, respectively.
The inter-individual variability of the main PK parameters (C max and AUCs) was moderate, with CV% ranging between 23.1 and 48.9%.

Discussion
Pharmacopoeia requires standard procedures with the aim to reassure the quality of pharmaceutical products. These guidelines may cover mostly drugs and products with direct therapeutic action, but it is up to the company's philosophy to follow the same guidelines and procedures for non-drug pharmaceutical products, e.g. dietary supplements. In this case, it was chosen to follow the same steps to determine the quality of the in-house product and their properties.
Flavonoids can inhibit proliferation and induce apoptosis of cancer cells. One of the major isoflavones is genistein, along with daidzein and glycitein. It was chosen to formulate Genistein in capsules, a patient friendly pharmaceutical dosage form with enhanced pharmacokinetic profile and proven action, in vitro and in vivo. Quality control showed positive results, without any implications in formulation and an impressive pharmaceutical profile, since disintegration and dissolution times were within the limits. It is vital to state here that in the case of Biopharmaceutical (BCS) Class II compounds (compounds with low solubility but high permeability), this is rather rare. Stability protocols, both real time and accelerated time conditions, showed satisfactory results throughout the study and at this point, it is worth mentioning that the protocol is still on going.
Isoflavones possess anticarcinogenic and antioxidant properties that lead to their chemopreventive activity against various cancer types [32]. According to the in vitro activity determination of Genistein R.G.C.C., it was clear that the substance significantly decreases the proliferation of the hormone sensitive MCF7 cell line at 24 h of treatment with the specific substance. The formulation of Genistein R.G.C.C. has given enhanced properties to the substance as comparing to Genistein Commercial, which did not show any effect on the viability of MCF7 cells, Genistein R.G.C.C. appeared to be effective even at very low concentrations. Genistein is a phytoestrogen that interacts with the estrogen receptors [33]. Triple negative human breast adenocarcinoma cell line MDA-MB-231 was lacking of the specific receptors and it could be easily confirmed with the viability analysis that no effect of both Genistein substances was observed on the specific cells.
Flavonoids can inhibit proliferation and induce apoptosis of cancer cells. One of the major isoflavones is genistein, along with daidzein and glycitein. Isoflavones possess anticarcinogenic and antioxidant properties that lead to their chemopreventive activity against various cancer types [32]. According to the in vitro activity determination of Genistein R.G.C.C., it was clear that the substance significantly decreases the proliferation of the hormone sensitive MCF7 cell line at 24 h of treatment with the specific substance. The formulation of Genistein R.G.C.C. has given enhanced properties to the substance as comparing to Genistein Commercial, which did not show any effect on the viability of MCF7 cells, Genistein R.G.C.C. appeared to be effective even at very low concentrations. Genistein is a phytoestrogen that interacts with the

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estrogen receptors [33]. Triple negative human breast adenocarcinoma cell line MDA-MB-231 was lacking of the specific receptors and it could be easily confirmed with the viability analysis that no effect of both Genistein substances was observed on the specific cells.
Analogous results were obtained from the pharmacokinetics study. The improved R.G.C.C. formulation of Genistein compared to the market's most commercial product presented imposing results. The C max of R.G.C.C.'s product was 34% higher than the commercial one with 10% higher AUC. This is significantly higher maximum observed plasma concertation and a slight but important increase in exposure.

Strengths and limitations
This study provides a comprehensive and complete background, in the field of pharmaceutical technology and product development. Since we already experience the era of dietary supplements and consumption of natural products in our daily diet, it is high time for companies to concern themselves with analytical development of products. Dietary supplements have a strong role in our lifestyle, thus their quality and efficacy must be thoughtfully evaluated. These experiments offer a broad scientific view on the quality aspects of the product, not only

Conclusion
This study proved that changes in formulation could affect the efficacy of a dosage form and enhance its properties. This resulted in an improved product with proven activity. The enhanced properties, made the product exhibited improved in vitro behavior and great pharmacokinetic profile.
Based on the literature information on Isoflavones and Genistein, doctors and physicians could recommend the daily intake of Genistein. An overall review on its action combined with therapeutic agents, can be a useful tool for clinical cases.