The Absorption of Bioactive Berberine in Human

Berberine is widely used as an herbal medicine and many health benefits. These
well-documented in the literature including lowering blood lipids and glucose, having
anti-inflammatory and anti-tumor activity, preventing and delaying neurodegenerative diseases
(Alzheimer, Parkinson and Huntington disease) and preventing obesity. However, the absorption
rate of berberine is very low. The study team believes adding TPGS or Quillaja extract as an
emulsifier enhance the absorption of berberine in human.

The bioavailability of berberine is extremely low. Only 0.5% of orally ingested berberine are
absorbed in small intestine and about 0.36% can enter the systemic circulation. About 56% of
ingested berberine was not absorbed and additional 43.5% is lost due to metabolism in small
intestine.

Vitamin E TPGS (a water-soluble form of vitamin E) is a food additive approved by FDA. It has
the potential to increase the absorption of drugs and dietary supplements. TPGS is an
effective inhibitor of P-glycoproteins that are responsible for decreased absorption rate of
many drugs including berberine. TPGS has the potential be a safe and effective adsorption
enhancer for berberine and other bioactive botanicals.

Quillaja extract is a natural GRAS (generally recognized as safe) food-grade
surfactant/emulsifier ingredient rich in saponins and sapogenins. It is purified from the
Quillaja Saponaria Molina tree which is native to Chile. Quillaja extract is an approved
ingredient for use in Food and Beverages by FDA under Title 21 CFR 172.51. It is an approved
food additive in the European Union under code E 999. Quillaja extract is an attractive
alternative emulsifier for berberine formulation because it meets consumers' demand for
natural ingredient. Quillaja extract provides additional benefits of anti-inflammation and
cholesterol lowering to dietary supplement. Some early study before 2000 suggested that
Quillaja saponin DS-1 promote the absorption of insulin and aminoglycoside antibiotics via
the ocular and nasal route by reducing permeability of epithelial barrier. However, it is not
known whether quillaja affect absorption of berberine in vivo or in human.

Specific Aims:

1. To determine whether TPGS or Quillaja extract emulsification increase the
bioavailability of orally ingested berberine in human volunteers by increasing its
plasma concentration time curve (AUC), peak concentration (Cmax), and elimination half
time (T1/2).

2. To determine whether TPGS or Quillaja extract emulsification alter phase I metabolism,
phase II metabolism, or metabolism by gut microbiota for orally ingested berberine in
human

The hypothesis of this research is that TPGS or Quillaja extract emulsification enhances the
bioavailability of orally ingested berberine in human by altering it metabolism and
pharmacokinetic profiles.

Advertisement will be in the form of flyers. Contact information of investigators will be
included in the flyer. All participants will be received written and oral information
regarding the natural and potential risks of the study. The informed consent form will be
given to the participants to get consent in a private room 7 days before the study.

Screening, Tolerability Test and Informed Consent: Subject will take two capsules of the
reference berberine dietary supplement with 8 ounces of room temperature water and subject
will remain in a fasted state for 4 hours. After completing 4 hours period, a standard lunch
with water will be served. If subject experiences diarrhea after taking supplement, she/he
will immediately report to the Principle Investigator, Dr. Wang or the Study coordinator. The
result of tolerability test will base on the self-assessment of diarrhea. Twelve participants
will be chosen having no symptoms of diarrhea Experiment process: Participants will be
advised to avoid berberine, vitamin E TPGS or Quillaja extract supplements and foods,
excessive amount of alcohol from the beginning of 7-day run in period to the end of the
study.

Participants will receive three treatments (A: TPGS emulsified berberine or B:Quillaja
extract emulsified berberine soft gel and C: berberine reference powder in hard shell
capsules) using a randomized crossover design. Treatment A, B and C provide an equal amount
of berberine at 800 mg dose.

Twelve healthy subjects will be randomly assigned into three groups with 4 in each group.
Subjects will be asked to collect initial urine samples in an 8-ounce container provided by
the study team before taking the supplement and the rest of urine samples will be collected
in a different container after taking supplement for 12 hours period. A trained and certified
phlebotomist will be hired to draw blood. A catcher will be implanted in the participants
forearm for blood draw. After a baseline blood sample is taken, four participants in group A
will receive at two capsules of TPGS emulsified berberine in soft gel (2x400mg) and four
subjects in group B will receive four capsules of the Quillaja extract emulsified berberine
(4x200mg) in soft shell capsules, and four participants in group C will receive at two
capsules of berberine reference powder (2x400mg) in hard shelf. Additional blood samples will
be drawn at 0.5 h, 1 h, 2 h, 3h, 4 h, 6 h, 8 h and 12 h for analysis of plasma concentrations
of berberine and berberrubine. In addition, the participants will collect all urine samples
in a gallon size plastic container during 12 h after the initial dose. A standard breakfast
of plain bagel with cream cheese will be given to each participant between 0.5 h and 1 h
blood draw. After 4 h blood draw, subjects will be given an identical meal for lunch.
Immediately after collection, blood samples will be centrifuged and plasma will be stored at
-80 degrees C until analysis. The urine samples will be stored at -80 degrees C until
analysis. After two 7-day washout period, the process will be repeated with each group
switching over to the other treatment, that is, the group that received treatment A will take
treatment B or C and the group that received treatment B will take treatment A or C, the
group that received treatment C will take treatment A or B.

Berberine and dihydroberberine in blood exist in nonconjugate free form. A major metabolite
berberrubine exists in free form and phase II metabolites including sulfates and glucuronide.
In order to analyze berberrubine in all forms, a portion of plasma or urine will be treated
with β-glucuronidase from Helix pomatia to hydrolyze its sulfate/glucuronides. Berberine and
berberrubine will be extracted from plasma or urine with or without enzyme treatment using
solvent or solid phase extraction. The compounds' concentration will be determined using
HPLC-ESI-MS/MS method. Pharmacokinetic parameters such as area under the plasma concentration
time curve (AUC), peak concentration (Cmax), time at peak concentration (Tmax), and
elimination half time (T1/2) will be calculated for berberines, free berberrubine, and total
berberrubine. Relative absorption will be expressed as the ratio of calculated AUC's.

Inclusion Criteria:

- Healthy

- Normal BMI (20-27)

Exclusion Criteria:

- Pregnancy

- Gastro-intestinal conditions

- Diabetics,

- Alcohol and substance abuse history,

- Allergy to berberine,

- Current berberine use,

- Use of H2 blockers, proton pump inhibitors, blood sugar-lowering agents, or statins