Human Journals

Research Article
March 2016 Vol.:5, Issue:4© All rights are reserved by Muhammed Majeed et al.

An Evaluation of Bioavailability Enhancement of Organic
Elemental Iron with BioPerine® in Rabbits
Keywords:

Bioavailability;

Iron deficiency;

BioPerine®;

Spectrometry (ICP-MS)
ABSTRACTa,ba

Muhammed Majeed , Priti Vaidyanathan , Pallavi
bc

Kiradi , Shaheen Majeed ,Kiran Kumar Vuppala*aSami Labs Limited, Peenya Industrial Area, Bangalore– 560 058, Karnataka, IndiabSami Direct Marketing Pvt Ltd, Koramangala,Bangalore- 560034, Karnataka, IndiacSabinsa Corporation, 750 Innovation Circle, Payson,
UT 84651, USA
dClinWorld Private Limited, # 19/1&19/2, I Main, IIPhase, Peenya Industrial Area,Bangalore – 560 058, Karnataka, India.

Submission:

9 March 2016

Accepted:

15 March 2016

Published:

25 March 2016www.ijppr.humanjournals.com

Iron being an essential trace mineral is found in every cell of
the body, the deficiency of iron is highly prevalent and is one of
the foremost health concerns worldwide, with the cause ofdeficiency primarily being poor bioavailability and / or poor
dietary intake. Beans being considered a potential source ofiron, can have a considerable role in cases of iron deficiency.
BioPerine® has been reported to increase the bioavailability ofnutritional compounds owing to its thermogenic property andtermed as a natural thermo-nutrient and bioavailabilityenhancer. The current novel study was designed to evaluate theimprovement in the bioavailability of organic elemental iron in
combination with and without BioPerine® in rabbits, taking into
consideration the widespread iron deficiency and in order toestablish and validate the property of BioPerine® in improving
the bioavailability of organic elemental iron (BioIron). Rabbits
were split into two groups to receive orally a single dose ofeither BioIron with BioPerine® (sample I) or BioIron without
BioPerine® (sample II). Blood samples were collected at 0, 30,60, 120, 240, 360, 480 minutes, 12, 24, 36, 48 and 60 hours
after administration of the samples I and II. Samples andstandards were prepared for estimation of iron and analysed byICP-MS method. The iron content was determined by acalibration graph and the results were expressed in µg/ml at
different time intervals. In conclusion, the iron bioavailability
as observed in the serum was significantly higher in animals
treated with BioIron containing BioPerine® (36.55 ± 9.97μg/mlat 8h) in comparison to treatment with BioIron without
BioPerine® (4.730 ± 0.94μg/ml at 24 h); thus, validating theproperty of BioPerine® which can be used as a key ingredient inenhancing the bioavailability of organic elemental iron.

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1. INTRODUCTION
Iron (Fe), an essential mineral found in the human body, is a part of the oxygen-carrying proteins
haemoglobin in red blood cells and myoglobin in muscles [1]. Iron is also available in proteinsand enzymes. Iron as an essential micronutrient plays a vital role in oxygen transport, oxidative
metabolism, cellular proliferation and many other physiological processes. It is a redox metal
participating in most of the reversible one electron oxidation-reduction reactions by switching
between the two oxidation states, ferrous and ferric. The human body has therefore developed
intricate but exquisitely controlled mechanisms to absorb, transport and store iron, thus ensuring
a ready supply for cellular growth and function but limiting its participation in reactions that
produce free radicals and its availability to invade pathogens. Iron can be stored in the body andutilized only when dietary iron is deficient. Iron deficiency in humans may occur when there is alow level of iron in the blood or when the body’s iron storage goes low. Iron deficiency anaemia
can be a result of numerous factors, such as low intake of dietary iron, low absorption andexcessive blood loss. However, anaemia is widespread in India in spite of diversity in food
habits, particularly in the consumption of cereals and such tight metabolic regulation. The
causality between poor dietary iron density, bioavailability and high prevalence of anaemia in
our population has not been well established, as anaemia has a multi-factorial aetiology [2].
The bioavailability of iron from foods is ultimately determined by interactions between iron andother components in the digestive milieu. Several factors contribute to iron bioavailability suchas meal composition, oxidation status, promoter substances (“meat factors”), metabolic demandfor iron and genetic inclination for iron absorption [3].
There are two types of iron that can be obtained from food: Heme and Non-heme. Heme iron isprimarily found in meat, fish and poultry. Beans, lentils and grains are a good source of nonheme iron. However, non-heme is less bioavailable than heme iron [4].
Non-heme iron absorption is inhibited by phytic acid, which is found in whole grains andlegumes, and by polyphenols such as tannins which are found in tea, coffee and red wines [5, 6].
In humans who consume moderate amounts of red meat, heme iron accounts for almost half of
the iron absorbed in the body [6, 7]. In humans with low iron stores, non-heme iron is absorbed
more than heme iron [6]. Although non-heme iron is less bio-available than heme iron, it has not
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been established whether this has any adverse consequences – vegetarians typically have lower
iron stores than omnivores, but they have no greater incidence of iron deficiency.[8]However, a recent study that examined the bioavailability of iron in 24 genotypes of bean seeds
containing a range of concentrations of iron, phytic acid and tannin showed that tannin andphytic acid concentrations did not affect bean iron bioavailability. [9]Ferritin iron, a storage form of iron in legumes is highly bioavailable, even when the phytate (saltform) concentration is high [10-12].
BioIron is a natural organic iron supplement from green gram (Phaseolus aureus). Mung beansor green soy beans through the technology of hydroponics is enriched with iron by a soil-lessprocess. This remains the source of elemental iron used in BioIron. Improved varieties of mungbean can contain 0.06 g of iron kg-1 raw grain [13] compared with traditional mung beanvarieties with only 0.03 to 0.035 g [14]. A proprietary hydroponics process is used to enrich thebeans to contain 15000-17000 ppm of elemental iron.
BioPerine® is a standardized extract from the fruits of Piper nigrum L (black pepper) or Piperlongum L (long pepper). It contains a minimum piperine content of 95% compared to the 3-9%found in raw forms of Piper spp. BioPerine® may be co-administered with various nutrients forboth human and animal health [15].
A thermo-nutrient such as BioPerine® would potentially improve the process of nutrient
absorption by enhancing thermogenesis. In view of these findings, it is proposed that BioPerine®
ingested in relatively small amounts would act as a thermo-nutrient. Localized thermogenic
action on the epithelial cells would in turn increase the rate of absorption of supplementednutrient(s) [16].
Hence, the current study was performed to evaluate the bioavailability of organic elemental iron(BioIron) with and without BioPerine®.

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2. MATERIALS AND METHODS
2.1. Chemicals
Standard iron solution of 10 ppm concentration and aqua regia (3:1 Nitric acid: Hydrochloricacid). BioIron tablets (containing BioPerine®) with batch/lot no. FD/JJ0715/SD-14 (sample I)
and Bio-Iron tablets (without BioPerine®) with batch/lot no. FD/JJ0815/SD-04 (sample II) wasprovided by the sponsor (M/s. Sami Labs Ltd). All the reagents used were of analytical grade.
2.2. Procedure for preparation of standards for estimation of Iron (Fe).
2.2.1. Preparation of Standard iron (Fe) solution 10ppm:
1ml of the readymade 1000ppm stock solution was pipetted out, transferred into 100ml
volumetric flask and volume made up to the mark with millipore water.
1) Preparation of calibration standard iron (Fe) solutions:
An appropriate volume of above 10ppm standard solution was pipetted out into 5 different
standard volumetric flasks and diluted up to the mark with millipore water to obtain 1, 2, 5, 10and 25 ppb of Fe standard solutions for calibration.
2) Preparation of sample and blank:
Equal quantity of samples was taken into microwave digester Teflon vessel and 9ml of aquaregia (3:1 Nitric acid: Hydrochloric acid) was added. Microwave digestion was carried out as per
instrument protocol and the volume made up to 10ml with Millipore water. The digested sample
was injected into Inductively Coupled Plasma Mass Spectrometry (ICP-MS) for the iron (Fe)estimation. As an internal control, blank sample was prepared in a same manner without serum.
3) Estimation of iron (Fe) in Bio-Iron tablets by ICP-MS.
Inductively Coupled Plasma Mass Spectrometry (ICP-MS) was utilized for estimation of iron(Fe) in serum. ICP-MS instrument (Make-Agilent, Model-7700) was tuned by aspirating thetuning solution. The instrument was calibrated by aspirating the calibration standard solution insequence to have Correlation coefficient >0.995. Further sample analysis was carried out and theiron content was calculated from the calibration graph. The results obtained were expressed asµg/ml at different time intervals.
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2.3. Animals
For evaluation of bioavailability, twelve in house breed rabbits were selected and groupedmanually. No computer generated randomization program was used. All the study animals wereacclimatized under laboratory conditions for 5, 7 and 9 days for Step I, Step II and Step III
respectively after veterinary examination. Only animals without any visible signs of illness wereused for the study.
The animals were identified by unique cage number and individual animal numbers marked withindelible marker pen on the tail. The animals were marked (towards the tip of tail) with thetemporary animal numbers at start of acclimatization. The animals were marked with permanent
animal numbers (towards the base of tail) with different colour indelible marker pen before thestart of test item administration.
2.4. Ethics
The bioavailability study was performed in accordance with the recommendation of theCommittee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA)
guidelines for laboratory animal facility, and as per OECD guidelines.
2.5. Dosing procedure
The test substance was administered to each rabbit as a single dose through the feeding tube
orally. The blood samples were collected through the marginal ear vein of the rabbits incentrifuge tubes at 0, 30, 60, 120, 240, 360, 480 minutes, 12 hours, 24 hours, 36 hours, 48 hours
and 60 hours after the test substance administration. The blood samples were centrifuged at 5000
rpm for 10 min to separate the plasma for analysis. The plasma samples were stored in steriletubes at -80oC until analyzed.
3. RESULTS
In this bioavailability study, all the rabbits of Group I were administered test sample I withbatch/lot no. FD/JJ0715/SD-14. It was observed that by 8 hours maximum iron levels of 36.55 ±9.97 µg/ml (Fig 1) were observed in serum. While rabbits in Group II were administered sample

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II with batch/lot no. FD/JJ0815/SD-04, the maximum iron levels in serum were found to be
4.730 ± 0.94 µg/ml (Fig 1) at 24 hours.
4. DISCUSSION
Iron deficiency anaemia is a major public health concern. The high incidence is either due toinsufficient intake of iron or poor bioavailability. Enhancing the bioavailability of iron is as
important as increasing the intake [17]. The current study was planned in rabbits, wherein thebioavailability of Bioiron (derived from mung beans) was evaluated in the presence and absenceof BioPerine® (pepper standardized extract).
In addition to Mung beans value as a protein rich food, it also has relatively high iron content.
Since mung beans contain neither inhibitors nor enhancers that influence iron bioavailability[18], the iron bioavailability of mung beans can in turn be enhanced by combining it with abioavailability enhancer such as BioPerine®.
The leading theory of food-induced thermogenesis relates to the autonomous nervous system.
The autonomous nervous system is represented by two main receptors in the gastrointestinal
tract, the alpha and beta adrenergic receptors. Most of the food or thermo nutrient-induced
thermogenesis is facilitated by beta receptors, such a cyclic adenosine 3’, 5’ monophosphate(cAMP). The role of cAMP as a "second messenger" to the hormonal and enzymatic actions in
the body is well recognized. When thermogenesis occurs, the demand for fresh nutrients tosustain the metabolic processes rapidly increases.
Piperine has been found in independent studies to stimulate the release of catecholamines,
thermogenic hormones whose action is made possible by the presence of cAMP. However, thenature of the thermogenic response mediated by catecholamines is relatively short-lived.
Therefore the window of opportunity for piperine-induced thermogenesis and enhanced nutrient
absorption is narrow.
These thermogenic properties may explain how a small amount of BioPerine® (5mg) can afford
such a profound effect on serum nutrient levels (as shown in our studies on water soluble, fatsoluble and botanical ingredients). It is possible that when piperine is ingested, it has a localized
thermogenic effect on epithelial cells which increase the uptake of nutrients. Other mechanisms

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by which piperine stimulates nutrient absorption include increased micelle formation, stimulationof active transport of amino acids (gamma-glutamyltranspeptidase), and epithelial cell wall
modification due to the affinity of piperine towards fats and fatty substances[16].
Piperine (1-piperoyl peperidine) is responsible for the bioenhancing effect of BioPerine®. It has
been shown to possess bioavailability-enhancing property that may be attributed to increased
absorption of organic elemental iron (Bioiron) due to alteration in membrane lipid dynamics andchange in the conformation of enzymes in the intestine [19].
Hence in the current study, the group of rabbits receiving BioIron along with BioPerine® showed
greater iron bioavailability in comparison to the group receiving BioIron alone.
5. CONCLUSION
From the results, it is evident that the animals from Group I receiving sample I with batch noFD/JJ0715/SD-14 showed more iron bioavailability in comparison to animals from Group II
receiving sample II with batch no. FD/JJ0815/SD-04. Hence, it can be concluded that the sample
I of BioIron containing BioPerine® has greater iron bioavailability.
FundingThe author(s) discloses that financial support for the research described herein was provided bySami Labs Limited.
Conflict of interest
The authors declare that they have no competing interests.
Acknowledgements
We thank Radiant Research Services Pvt. Ltd, India, for conducting the study.
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Fig 1: Iron levels in the serum samples from animals treated with BioIron with BioPerine®
(FD/JJ0715/SD-14) and BioIron without BioPerine® (FD/JJ0715/SD-04)
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