This document summarizes the development of rice bran from a waste material to a nutraceutical product with health benefits over 8 years. It involved stabilizing rice bran to prevent rancidity, performing phytochemical analysis to characterize compounds, implementing extracts in products, developing rice bran supplements, and marketing rice bran products. Key steps included stabilizing rice bran at lab and industrial scales, analyzing phytochemicals in stabilized rice bran, and extracting oil using solvents and supercritical CO2 to study compounds like fatty acids, sterols, tocopherols, and beta-carotene. Funding was obtained from Egyptian and EU sources to support this work developing rice bran into high value nutraceuticals.

1. 4. Case Study
4.1. Rice Bran; from waste material to high value added Nutraceuticals
Introduction
Rice, one of the major cereals, is used almost exclusively as a food for humans. De-husking
of raw rice results in brown rice, which is covered by bran layers, namely, pericarp, testa and
aleurone layers. Rice bran that constitutes 5–10% of brown rice is obtained by second polishing
process within the rice mill. It consists of bran layers and portions of germ. Bran is mainly
composed of protein, fiber, oil, vitamins, minerals and starch. The later come from endosperm
residue during polishing . In addition to those components, there are also antioxidants and
nutraceuticals like oryzanol, phytosterol, tocopherol, tocotrienol, squalene and polyphenols (1,
Amr et al)
In addition to its high nutritional value, Rice bran and its defatted form were shown to
contain high percentage of dietary fibers that showed anticancer and hypercholesterolemic
effect Antitumor and antidiabetic effects of rice bran dietary fibers have been reported
cellulose, hemicellulose, α- and ß- glucan are among the dietary fibers present in rice bran
Defatted rice bran also showed to be rich in phenolic compounds and flavonoids that reflect its
potential health benefits as antioxidant and anti-inflammatory (2, Said et al)
In Egypt, the cultivated area of rice is about one and half million acres which annually
produce 5.5 million tons of rice. More than 500 000 MT rice bran were produced during rice
milling. However, because of the extreme instability of the latter, it becomes rancid and unsuitable

2. for human use within hours of milling. Consequently, it is either discarded or used as animal feed,
thereby pouring its potential medical usefulness down the drain at the expense of a great financial
and human utility loss.
The journey of developing rice bran from waste material into high value added
Nutraceuticals with potential health benefits in many diseases including healthy aging aid, go
through different stages through the past eight years. This includes:
4.1.1. Stabilization of rice bran at the lab and industrial scale.
4.1.2. Phytochemical studies of the stabilized rice bran
4.1.3. Implementation of stabilized rice bran extract in different pharmaceutical and cosmetic
preparations.
4.1.4. Developing rice bran Nutraceuticals as well as running out safety studies.
4.1.5. Studying and publishing the effects of stabilized rice bran extract on brain nerve cells.
4.1.6. Developing of special porridge for elder population.
4.1.7. Rice bran functional extract
4.1.8. Innovation in rice bran extraction and formulation
4.1.8. Marketing of Rice Bran products.
It has to be mentioned that within the above course ,led by Dr. Amr M. Helal, CEO of Health Tech
was carried out in cooperation with academic and business partners in Egypt, Germany and
Holland and Spain. List of publications are shown at the end of the case study.

3. Funds were successfully raised and supplied from Egypt (Industry Modernization Centre -
http://www.imc-egypt.org/ ) , Science and Technology Development Fund
(http://www.stdf.org.eg/) as well as EU – Innovation program (Research Development and
Innovation program http://www.rdi-eg.com/ )
4.1.1. Stabilization of rice bran at the lab and industrial scale.
The rice bran is rich in protein (13-16%), oil (15-22%), fibber (6.2-14.4%), ash (8-17.75%),
vitamins and trace minerals. However, it has mainly used for live stock and poultry feeding. The
major problem in utilization of rice bran for many other important products is associated with rapid
deterioration. This is due to the presence of lipolytic enzyme (Lipase) in the bran which caused a
rapid breakdown to free fatty acid (FFA) at an initial rate of at least 5-7 % of the weight of oil per
day.
In other words, one may say that, a major problem in rice bran production is the rapid
deterioration of rice oil in the bran due to the presence of lipolytic enzyme which is activated
during the polishing operation. The enzyme attacks the fats splitting of free fatty acid so rapidly
that 50-70% of the oil is affected within 90 days. Even in a matter of two to three days over 10 %
of the oil can be ruined.

4. Heat treatment of raw bran was found to be very effective in stabilization process. The
function of heat treatment during stabilization process is destroying or inactivating the lipase
enzyme that is responsible for the hydrolysis of oil (development of FFA).
The main objective of the project is to develop Different and Novel Rice Bran Stabilizers that
will suit different rice varieties and different mills designs. The whole stabilization factors;
temperature, time, feeding rate are controlled from a remote station. Two different designs for rice
bran stabilizers with heat source based on heat conduction and Infra-red applications. Two lab
scale unit were manufactured where the conditions and different factors were studied. Two
prototypes with stabilization range of 500 kg rice bran/ hour (about 5 tons per day) were
manufactured and run properly at the rice mills.
-The two rice bran stablizers sucessfuly stabilize rice bran against rancidity for storage period
of 12 months. That was monitoered by the percent of Free fatty acids (not more than 3% during
the whole storage period).
- The integrity of the bioactive marker , gamma oryzinaol, was tested during intervals of 3
months after stabilization. The stablizaiton process showed no effect on the rice bran bioactives.
4.1.2. Phytochemical studies of the stabilized rice bran (see as well publications, 3-6)
Stage 1:

5. In the present stage of the action, proximate analysis of the stabilized whole rice bran (WRB) has
been carried out according to the official method of analysis. The studied rice bran was the
Egyptian short grain variety of Sakha 101. Amino acid profile and different minerals content in
the WRB have been determined adopting amino acid analyzer and atomic absorption
respectively. Total phenolic content has been assessed in WRB. The free fatty acids percentage
in the whole rice bran has been determined. Rice bran oil has been extracted by solvent and
supercritical techniques. Three different organic solvents have been used for extraction (diethyl
ether, petroleum ether 40-60 and n-hexane). Three different conditions for supercritical
extraction have been adopted. The yield and antioxidant activity of the extracted oil from
different solvent and different supercritical extraction conditions have been determined. Defatted
rice bran (DRB) from n-hexane have been analyzed for proximate composition, minerals and
amino acids Total dietary fibers were also determined. Defatted rice bran resulted from
supercritical extraction that produced both highest yield and highest anti oxidant activity oil have
been analyzed for ash, moisture and mineral contents. Total phenolics have been determined in
all the defatted rice bran from different solvents and different supercritical conditions.
Microbiological tests have been applied on defatted rice bran before storage (for subsequent
analysis in the next stages).
Results: Free fatty acids were determined to be 2.37 % in the whole rice bran. Results of
proximate analysis showed whole rice bran to contain 13.5% protein, 18.6% fat, 2.74% crude
fiber, 5.5 % ash, 4.5 % moisture and 55.16% carbohydrates. Minerals analysis showed that the
contents of phosphorus, potassium, calcium, magnesium, sodium, iron, manganese, and zinc
were 1081, 1060, 24, 399, 23, 13.13, 10, 11 mg per 100 gram fresh sample respectively. The oil
yields were almost the same among different solvent extraction using soxhlet (18.2-18.6%).
Antioxidant activity did not differ distinctly among different oil extracts using soxhlet. However
extraction using n-hexane in ambient temperature and at 50Cº showed reduced yield and
antioxidant activity compared to that using soxhlet. Proximate analysis of defatted rice bran (from
n-hexane, using soxhlet) showed higher percentage of protein, ash and crude fiber than WRB.
Minerals analysis of DRB showed higher values of iron, calcium and zinc and lower values of
phosphorus, potassium, magnesium and sodium than WRB. Amino acid profile of both WRB
and DRB showed proline to have the highest value, the lowest values were attributed to aspartic
and glutamic. Total dietary fiber were estimated to be 24.46 in DRB. Supercritical extraction
showed 8.2%, 13.0% and 15.0% oil yields when using 40C°, 50C° and 60C° respectively under
constant condition of pressure as 350 bar, CO2 flow rate as1.5 liter/min and 3 hours as extraction
time. The highest antioxidant activity among oils extracted by supercritical CO2 was attributed
to that extracted at 60Cº. Ash and moisture percentage of defatted rice bran at the later condition
were assessed to be 9.21 and 4.47 respectively. All mineral contents of DRB from supercritical
technique (60C°) showed higher levels than DRB from hexane except for calcium and zinc that
showed lower values. Total phenolic contents among different defatted rice bran and WRB
ranged from 1399 to 1422 mg. gallic acid per 100 g. sample. Unsaponifiable matter was estimated
to be 4.3% of RB oil.

6. Stage 2
In the present stage of the action, rice bran oil was extracted by n-hexane using continuous
extraction apparatus (Soxhlet) and by supercritical CO2 extraction using the following condition;
temperature: 60C°, pressure: 350 bar, CO2 flow rate: 1.5 liter/min., time: 3 hours. Particle size:
pass through 20 mesh sieve. Oil extracted by hexane was purified through de-gumming and de-
waxing and the percentage wax was estimated gravimetrically. Gamma oryzanol was determined
in the oil using HPLC. Saponifiable and unsaponifiable fractions were prepared from oil extracted
by hexane and supercritical CO2 and identification and determination of fatty acids, phytosterols,
triterpenoidal compounds and hydrocarbons was carried out using GLC. Beta-carotene contents of
the whole rice bran and oil extracted by both hexane and supercritical CO2 were assessed by
spectrophotometric method. Total flavonoids were estimated in the whole rice bran, defatted rice
bran from n-hexane and defatted rice bran from supercritical Co2. Tocopherols (α,γ and δ) were
assessed in the whole rice bran and the oil extracted by n-hexane. Standardization of the methods
of determination of policosanol adopting GLC and tocopherols and tocotrienols using HPLC was
carried out to be continued in the third stage of the project.
Results showed that GLC investigation of the unsaponifiable matter revealed the presence of
campesterol, stigmasterol and β-sitosterol in both samples of rice bran oil (extracted by n-hexane
and supercritical CO2). Total sterols content of rice bran oil extracted by hexane (12.041%) was
higher than that extracted by supercritical CO2 (7.595%). Alpha and β-amyrin, the triterpenoidal
matter, were present in rice bran oil extracted by superctitical as 1.984% and 1.976% and in hexane
extract as 2.086 and 0.804% respectively. The identified hydrocarbons (C9-C26) showed that total
hydrocarbon as percentage of unsaponifiable matter were 19.42 and 34.764 in rice bran oil
extracted by hexane and by supercritical CO2 respectively. The results of total fatty acids analysis
revealed that both linoleic and oleic acid are more or less of the same percentage in rice bran oil
extracted by supercritical CO2 (38.33% and38.26% respectively). Rice bran oil extracted by n-
hexane showed that the highest percentage of fatty acids belonged to oleic acid (59.78%) followed
by linoleic acid (18.27%). Palmitic acid was the major saturated fatty acid in rice bran oil extracted
by supercritical and by hexane (18.51% and 19.06% respectively). Stearic acid showed higher
percentage in the oil extracted by supercritical (3.54%) compared to that extracted by hexane
(2.88%). Myristic acid and palmitoleic acid were only detected in rice bran oil extracted by
supercritical CO2 (0.37% and 0.24% respectively). Total unsaturated fatty acids were determined
to be 76.83% and 78.05 % in rice bran oil extracted by supercritical and by hexane respectively.
Total saturated fatty acids percentage of total fatty acids was found to be 22.42% in rice bran oil
extracted by supercritical and 21.94% in that extracted by hexane. The concentration of beta
carotene in the whole rice bran was 101 µg/100g. The content of beta carotene in the oil extracted
by supercritical CO2 was higher than that extracted by hexane (263 and 225 µg/100g oil
respectively). The total flavonoids concentration in the whole rice bran, defatted rice bran by n-
hexane and defatted rice bran by supercritical CO2 was determined to be 8.769, 9.643 and 9.342

7. mg catechin/g. sample respectively. The obtained wax percentage was 5.12% of the crude oil
extracted by n-hexane, gum and phospholipids were 2.33%. Gamma oryzanol concentration in the
crude oil extracted by n-hexane was estimated to be 3.33% while it was 2.25 % in the purified oil.
α-tocopherol has been shown to be 665 µg/g rice bran oil and 131 µg/g whole rice bran. γ-
tocopherol was noticed to be 70 µg/g rice bran oil and 4 µg/g whole rice bran while δ- tocopherol
was 172 µg/g rice bran oil and 141 µg/g whole rice bran.
Stage 3
In the present stage of the action, rice bran oil was extracted by n-hexane using continuous
extraction apparatus (Soxhlet) and by supercritical CO2 extraction using the following condition;
temperature: 60C°, pressure: 350 bar, CO2 flow rate: 1.5 liter/min., time: 3 hours. Particle size:
pass through 20 mesh sieve. Oil extracted by hexane was purified through de-gumming and de-
waxing as clarified in the second stage. Gamma oryzanol was determined in the oil extracted by
supercritical CO2 using HPLC. Fatty acids profile of purified oil was assessed using GLC. Beta-
carotene contents of the purified oil was determined by spectrophotometric method. Tocopherols
(α,γ and δ) were assessed in the oil extracted by supercritical CO2 and in the purified oil.
Tocotrienols and policsanols were determined in the oil extracted by n- hexane and supercritical
CO2 and purified oil using HPLC and G.C respectively. Policosanols were also determined in wax.
Acid value, % free fatty acids, peroxide value and iodine value of the oil extracted by supercritical
CO2 and by hexane and purified oil was carried out. Follow up study of proximate analysis and
possible microbiological contamination of defatted rice bran oil was carried out after 3- months
storage.
Results The results of total fatty acids analysis of the purified rice bran oil revealed that the
highest percentage of fatty acids was that of linoleic (49.68 % ) followed by oleic acid (33.00 %).
Palmitic acid was the major saturated fatty acid in the purified rice bran oil (15.4%). Stearic acid
was 1.71% while palmitoleic acid was 0.21%. Total unsaturated fatty acids were determined to be
82.89 %. Total saturated fatty acids as percentage of total fatty acids was found to be 17.11%. The
concentration of beta carotene in the purified oil was 49.6 µg/100g oil. Gamma oryzanol
concentration in the oil extracted by supercritical CO2 was estimated to be 1.54 %. α-tocopherol
has been shown to be 739.9 and 344 µg/g rice bran oil extracted by supercritical CO2and in
purified oil respectively. γ-tocopherol was noticed to be 69.4 µg/g rice bran oil extracted by
supercritical CO2 and 70.9 µg/g purified oil while δ- tocopherol was 48 µg/g rice bran oil extracted
by supercritical CO2 and 20 µg/g purified oil. Total tocotrienols were estimated to be 51.60,
310.24, 398.70 and 395.00 in whole rice bran, rice bran oil extracted by hexane, rice bran oil
extracted by supercritical CO2 and purified oil. Total identified policosanol concentration in the
oil extracted by n-hexane, supercritical CO2 and wax was 69.622, 48.634 and 590.894 mg/100g
respectively. Initial acid value (ml KOH/g), % free fatty acids as oleic, peroxide value (meq/kg)
and iodine value (before storage) of the oil extracted by n-hexane were 3.9, 1.95, 5 and 98.32

8. respectively and by supercritical CO2 were 2.99, 1.5, 3.33, and 97.91and of purified oil were 1.8,
0.9, 3 and 97.85 respectively. No detectable changes were noticed in proximate composition of
defatted rice bran after 3-month storage except for the moisture content that showed increased
values on storage in room temperature. Microbiological tests showed permissible count after 3-
month storage.
4.1.3. Implementation of stabilized rice bran extract in different pharmaceutical and cosmetic
(see as well publications, 3-6)
Stage 1
In the present stage of the project; stabilized whole rice bran (WRB), stabilized rice bran oil and
unsaponifiable fraction were prepared and tested for safety through acute toxicity test. The studied
rice bran was the Egyptian short grain variety of Sakha 101. Different food products supplemented
with different percentage of finely ground whole rice bran (10%, 20% and 30% replacements of
wheat flour or gelatinised corn flour) have been prepared on laboratory scales. The prepared food
products were biscuits, corn flakes and tortilla chips. Also, pastas containing two levels of whole
rice bran (20% and 30% replacements of wheat flour) in addition to rice bran oil were prepared.
All food products were sensory evaluated in comparison to controls. Physical and rheological
properties have also been studied. Based on these tests the selected food products were subjected
to analysis of proximate composition and microbiological tests. These products have been
modified aiming at increasing their protein value and contents through fortification with protein
rich sources. The resulted products were again sensory and physically evaluated. Crackers
containing defatted rice bran (DRB) (60, 70, 80 and 90% replacements of wheat flour) were
prepared, sensory and physically evaluated. Crackers at 90% level was analyzed for proximate
composition and tested microbiologically. Minerals and amino acid profile of pasta containing
20% WRB have been assessed in the present stage. The minerals and amino acid pattern of other
products will be determined in the next stages.
Results of acute toxicity tests showed complete safety of whole rice bran, rice bran oil and
unsaponifiable fraction up to 12 g/kg mice body weight. Sensory evaluation of food products
clarified that there was no significant difference between different evaluated sensory parameters
of 10% and 20% WRB containing biscuits however biscuits of 30%WRB showed significant lower
score concerning overall acceptability. There was no significant difference between different types
of crackers concerning all sensory parameters. Sensory parameters of tortilla chips showed only
significant change in colour and crispness in WRB supplemented products compared to control. It
was noticed that overall acceptability, appearance and colour were of significant difference when
the control was compared with WRB containing cornflakes. Pasta containing 20% WRB was far
superior compared to that of 30% in respect to both sensory and physical properties. In general,
increasing the percentage of rice bran either whole or defatted increased the darkness of the food
products. Proximate analysis showed that percentage protein of 20% WRB biscuits, 30% WRB

9. corn flakes and 30% WRB tortilla chips were 10.2, 10.57 and 11.2 while that of fat was 13.49,
3.65 and 23.21; crude fibers were 0.517, 0.775 and 0.631; ash was 2.65, 0.24 and 1.71and
carbohydrates were 70.1, 81.55 and 60.82 respectively. 20% WRB containing pasta showed that
the percentage of protein, fat, crude fibers, ash and carbohydrates were 10.71, 8.52, 1.4, 1.24 and
50.33 respectively. Minerals analysis of 20% WRB pasta clarified that the contents of phosphorus,
potassium, calcium, magnesium, sodium, iron, manganese and zinc were 63.2, 133.5, 6.5, 22.2,
171.5, 2.5, 0.93 and 1.3 respectively. Amino acids profile of 20% WRB pasta showed high level
of proline followed by methionine and phenyl alanine, the least level was attributed to threonine
and aspartic. Crackers containing 90% defatted rice bran showed 16.03 % protein, 1.73%fat,
1.81%crude fibers, 6.99%ash and 69.8% carbohydrates. Microbiological tests showed safety of
the different products.
Stage 2
In the present stage of the project, proximate composition of fortified tortilla chips (formula 5),
fortified biscuit (formula 10) and fortified corn flakes (formula, 5) which were prepared in the first
stage was determined. Also, assessment of physical properties (Falling number and viscosity) of
the different flour blends from which the food products have been prepared was completed.
Biological evaluation of pasta (20% rice bran), biscuits (formula 10) and crackers (90% defatted
rice bran) was carried out to determine their food efficiency ratio, protein efficiency ratio and
safety concerning liver and kidney functions. Statistical analysis of the obtained biochemical and
nutritional parameters were carried out in comparison to rats fed control diet (casein diet) adopting
t-test.
The present stage also involved preparation of pharmaceuticals as liquisolid system containing
40% rice bran oil packed in hard gelatin capsules and transdermal cream containing 10% rice bran
oil. Cosmetic preparations including emollient cream and sunscreen (containing 10% rice bran oil)
was also prepared in the current stage. Skin test (patch test) of transdermal cream, emollient cream
and sunscreen was carried out to study the safety of such preparation concerning sensitivity and
allergy. Also consumer evaluation of sensory attributes of emollient cream and sunscreen was
carried out in comparison to control preparation already present in the market. Statistical analysis
(t-test) was applied on the different sensory attributes of the test preparations compared to control.
Results of proximate analysis showed tortilla chips to contain11.5% protein, 19.35 %fat, 3.24%
ash, 58.03% carbohydrates, 0.78% crude fibers and 7.04 % moisture. Biscuit was noticed to
contain 13.13% protein, 7.53 %fat, 3.06% ash, 71.63% carbohydrates, 0.61% crude fibers and 4.04
% moisture. Corn flakes was shown to contain 13.7% protein, 1.96 %fat, 2.97% ash, 75.9%
carbohydrates, 1.24% crude fibers and 4.23 % moisture. Calorific content in 100g.food product
was calculated to be 452.51, 406.81 and 375.04 in chips, biscuit, and corn flakes respectively.
Biological evaluation showed pasta to have the highest food efficiency ratio and protein efficiency

10. ratio followed by the casein control diet, however, both crackers and biscuits had lower values
than the control. All food products showed safety towards liver and kidney function.
Viscoamylograph results showed that the maximum viscosity was markedly low in formulas
containing whole rice bran and defatted rice bran except for the blend of wheat flour + 10% whole
rice bran. The breakdown viscosity of corn flower blend with whole rice bran was also markedly
low. Maximum viscosity and temperature of transition were higher in formula (Corn flour+30%
whole rice bran) than other blends of corn flour and whole rice bran. The results obtained by falling
number test confirmed some readings from those obtained by Viscoamylograph test.
As expected, Patch test showed safety of transdermal cream, emollient cream and sunscreen
concerning sensitivity and allergy. Sensory evaluation of cosmetics showed satisfactory results.
Stage 3
In the present stage, hair gel containing 10% rice bran oil was prepared. Modification of the
transdermal preparation prepared in the previous stage has been carried out to be smoother.
Characterization of the liquisolid formulation prepared in the previous stage was carried out
through studying, loading factor, flow properties of liquisolid powder and bulk density
measurements. The four prepared semisolid formulae (from the present and second stages) were
characterized for organoleptic (external) properties, rheological behaviour and physical stability,
at zero time and after exposure to storage at ambient temperature (30Cο
) for three months and
under accelerated conditions (stress conditions) (40Cο
, 3 months) regarding the sunscreen
preparation, emollient and TD creams, while the gel was exposed to alternative stress storage
conditions (2-8Cο
, 3 months). In the present stage microencapsulation of rice bran oil was prepared
in different ratios with gum. Also, an emollient cream with expected extra anti-wrinkle effect was
formulated and prepared. In addition, Soft gelatin capsules containing rice bran oil were prepared
with determination of their dissolution rate.
Results of characterization of the liquisolid formulation showed the liquid load factor for
RBO/silicon dioxide was 0.65. Considering the flow properties, Hopper's flow rate was 7cm3
/sec
while the value of repose angle was 35.22ο
. Referring to bulk density measurements, Carr's index
value was 25 and Hausner's ratio was 1.33. The results obtained for liquisolid formulation revealed
flow and compressibility figures close to those reported as optimum acceptable values for powders.
The high Lf for this formula infers slow release of the incorporated medication which signifies
sustained effect. Considering the semisolid formulations, the external characters reveal agreeable and
adequate properties of the investigated formulations after long term and accelerated conditions of
storage compared to zero time. The preparations are elegant in appearance, showing no phase
separation, no fungal growth with smooth texture and pleasant odour. The investigated rheological
properties under the mentioned storage conditions in comparison to zero time, as an additional

11. measure of physical stability showed that as the shear rate increases, the viscosity decreases.
Therefore, the discussed preparations exhibit non-Newtonian pseudoplastic flow properties. The shear
stress values showed low figures at low shear rate, with the consistency curves almost reaching the
origin, therefore the yield value is negligible, indicating the limited resistance to flow at low stress
values characteristic for pseudoplastic flow. For topical formulations, the yield value must be low to
permit easy handling and facilitate spreading on the skin. Upon decreasing the shear rate, viscosity
increases again gradually, but the up curves do not coincide with the down curves, signifying the
presence of thixotropy. The sunscreen, gel and TD cream showed thixotropic behaviour where
viscosity decreases by increasing time at constant shear whereas the emollient cream showed
rheopectic behaviour where viscosity increases by increasing time at constant shear rate. Statistical
analyses, using Student's t-test, revealed non-significant difference in apparent viscosity values of all
the semisolid formulations studied at different storage conditions when compared to zero time. The
rheograms of the formulations, at zero time and after storage, indicated non-Newtonian behaviour
with pseudoplastic tendency, a desirable rheological behaviour in semisolids. Concerning initial
dissolution of the soft gelatin capsules was after 9.2± 2.8 SD seconds while complete dissolution was
after 117 ±11.8 SD seconds.
4.1.4. Developing rice bran Nutraceuticals as well as running out safety studies.
Nutraceuticals may be defined as food (or part of a food) that provides medical or health
benefits, including the prevention and/or treatment of a disease. Rice bran is one of the fast growing
key commodities for the nutraceutical industries in the world, particularly in the Western
hemisphere. In addition to its high nutritive properties and great calorific value, certain selected
rice bran constituents have been shown to have promising in vitro and in vivo activities in various
biomedical applications, such as antioxidant protection, prevention of cardiovascular disease
(CVD), neuro-protection, diabetes progression, tumor suppression or regulation of bone calcium
homeostasis.
Developing rice bran nutraceutical was an innovative project whereby an abundant raw material
of known nutritional and functional activities was developed into pharmaceutical dosage forms
with scientific based evidence of activity. The project lasted for almost two years with a budget of
EURO 192000 and participation of Egyptian and German research and industrial concerns. The
phytochemical study of stabilized rice bran extract was carried out to identify and quantify the
bioactives using a new validated extraction procedure. The stabilized bran extract was tested for
many pharmacological activities as well as for safety assurance. Its potential health benefits in
lowering elevated blood pressure, high cholesterol levels and high blood sugar levels as well as its
anti-inflammatory and antioxidant properties were established experimentally.

12. Stabilized Rice Bran (SRB) is planned to be used as a nutraceutical to help manage and/or
protect against some common disease conditions. To reach this target, was extracted and the
biological active fractions effective against particular ailments identified. The bioactive
fractions are planned to be tested both in vivo and in vitro, in order to identify fractions exerting
one or more of the following properties:
a) antidiabetic potential (7)
b) cholesterol lowering activity (8,9)
c) high blood pressure lowering activity (8,9)
d) antioxidant activity (8,9)
e) anti-inflammatory activity. (8,9)
f) Acute and chronic toxicity of the bioactive fractions are studied, as well as their mutagenic
potential. Bioactive extracts showed reasonable activity and high safety are formulated into
different dosage forms (10)
4.1.5. Studying and publishing the effects of stabilized rice bran extract on brain nerve cells.
4.1.5.1. Rice bran extract protects from mitochondrial dysfunction in guinea pig brains (11)
Mitochondrial dysfunction plays a major role in the development of age-related neurodegenerative
diseases and recent evidence suggests that food ingredients can improve mitochondrial function.
In the current study we investigated the effects of feeding a stabilized rice bran extract (RBE) on
mitochondrial function in the brain of guinea pigs. Key components of the rice bran are oryzanols,
tocopherols and tocotrienols, which are supposed to have beneficial effects on mitochondrial
function. Concentrations of α-tocotrienol and γ-carboxyethyl hydroxychroman (CEHC) but not γ-
tocotrienol were significantly elevated in brains of RBE fed animals and thus may have provided
protective properties. Overall respiration and mitochondrial coupling were significantly enhanced
in isolated mitochondria, which suggests improved mitochondrial function in brains of RBE fed
animals. Cells isolated from brains of RBE fed animals showed significantly higher mitochondrial
membrane potential and ATP levels after sodium nitroprusside (SNP) challenge indicating
resistance against mitochondrial dysfunction. Experimental evidence indicated increased
mitochondrial mass in guinea pig brains, e.g. enhanced citrate synthase activity, increased
cardiolipin as well as respiratory chain complex I and II and TIMM levels. In addition levels of
Drp1 and fis1 were also increased in brains of guinea pigs fed RBE, indicating enhanced fission

13. events. Thus, RBE represents a potential nutraceutical for the prevention of mitochondrial
dysfunction and oxidative stress in brain aging and neurodegenerative diseases.
4.1.5.2. Rice bran extract improves mitochondrial dysfunction in brains of aged NMRI mice
(12)
Aging represents a major risk factor for neurodegenerative diseases such as Alzheimer's disease.
Mitochondria are significantly involved in both the aging process and neurodegeneration. One
strategy to protect the brain and to prevent neurodegeneration is a healthy lifestyle including a diet
rich in antioxidants and polyphenols. Rice bran extract (RBE) contains various antioxidants
including natural vitamin E forms (tocopherols and tocotrienols) and gamma-oryzanol. In this
work, we examined the effects of a stabilized RBE on mitochondrial function in 18-month-old
Naval Medical Research Institute mice (340 mg/kg body weight/day), which received the extract
for 3 weeks via oral gavage.
METHODS:
Mitochondrial parameters were measured using high-resolution respirometry (Oroboros
Oxygraph-2k), Western blot analysis, and photometric methods in dissociated brain cells, isolated
mitochondria, and brain homogenate. Vitamin E concentrations in blood plasma and brain tissue
were measured using HPLC with fluorescence detection.
RESULTS:
Aging leads to decreased mitochondrial function (decreased mitochondrial respiration and ATP
production) and decreased protein expression of peroxisome proliferator-activated receptor
gamma coactivator 1-alpha (PGC1alpha). RBE administration increased alpha-tocopherol
concentrations in the brain and compensated for age-related mitochondrial dysfunction by
increasing mitochondrial respiration, membrane potential, PGC1alpha protein expression, and
citrate synthase activity. Furthermore, resistance of brain cells to sodium nitroprusside-induced
nitrosative stress was improved.
DISCUSSION:
According to these results, RBE is a promising candidate nutraceutical for the prevention of age-
related neurodegenerative diseases.
4.1.5.3. . Rice Bran Extract compensates mitochondrial dysfunction in a cellular model of early
Alzheimer's disease. (13)
MitochondrialdysfunctionplaysanimportantroleinbrainagingandhasemergedtobeanearlyeventinAl
zheimer’s disease (AD), contributing to neurodegeneration and the loss of physical abilities seen
in patients suffering from this disease. We examined mitochondrial dysfunction in a cell culture
model of AD (PC12APPsw cells) releasing very low amyloid- (A40) levels and thus mimicking
early AD stages. Our data show that these cells have impaired energy metabolism, low ATP levels,
and decreased endogenous mitochondrial respiration. Furthermore, protein levels of PGC1as well

14. as of Mitofusin 1 were decreased. PC12APPsw cells also showed an increased mitochondrial
content, probably due to an attempt to compensate the impaired mitochondrial function. Recent
data showed that stabilized rice bran extract (RBE) protects from mitochondrial
dysfunctioninvivo[24].ToassesstheeffectofaRBEonmitochondrialfunction,wetreatedPC12APPsw
cellsfor24hwithRBE. Key components of RBE are oryzanols, tocopherols, and tocotrienols, all
substances that have been found to exert beneficial effects on mitochondrial function. RBE
incubation elevated ATP production and respiratory rates as well as PGC1protein
levelsinPC12APPsw
cells,thusimprovingtheimpairedmitochondrialfunctionassessedinourcellcultureADmodel.Therefo
re, RBE represents to be a promising nutraceutical for the prevention of AD.
4.1.5.4. Beneficial Effects of Ethanolic and Hexanic Rice Bran Extract on Mitochondrial
Function in PC12 Cells and the Search for Bioactive Components (14)
Mitochondria are involved in the aging processes that ultimately lead to neurodegeneration and
the development of Alzheimer's disease (AD). A healthy lifestyle, including a diet rich in
antioxidants and polyphenols, represents one strategy to protect the brain and to prevent
neurodegeneration. We recently reported that a stabilized hexanic rice bran extract (RBE) rich in
vitamin E and polyphenols (but unsuitable for human consumption) has beneficial effects on
mitochondrial function in vitro and in vivo (doi:10.1016/j.phrs.2013.06.008, 10.3233/JAD-
132084). To enable the use of RBE as food additive, a stabilized ethanolic extract has been
produced. Here, we compare the vitamin E profiles of both extracts and their effects on
mitochondrial function (ATP concentrations, mitochondrial membrane potential, mitochondrial
respiration and mitochondrial biogenesis) in PC12 cells. We found that vitamin E contents and the
effects of both RBE on mitochondrial function were similar. Furthermore, we aimed to identify
components responsible for the mitochondria-protective effects of RBE, but could not achieve a
conclusive result. α-Tocotrienol and possibly also γ-tocotrienol, α-tocopherol and δ-tocopherol
might be involved, but hitherto unknown components of RBE or a synergistic effect of various
components might also play a role in mediating RBE's beneficial effects on mitochondrial
function.
4.1.5.5. Rice bran derivatives alleviate microglia activation: possible involvement of MAPK
pathway (15)
Hyperactivation of microglia is considered to be a key hallmark of brain inflammation and plays
a critical role in regulating neuroinflammatory events. Neuroinflammatory responses in
microglia represent one of the major risk factors for various neurodegenerative diseases. One of
the strategies to protect the brain and slow down the progression of these neurodegenerative
diseases is by consuming diet enriched in anti-oxidants and polyphenols. Therefore, the present
study aimed to evaluate the anti-inflammatory effects of rice bran extract (RBE), one of the rich
sources of vitamin E forms (tocopherols and tocotrienols) and gamma-oryzanols, in primary rat
microglia.

15. Methods
The vitamin E profile of the RBE was quantified by high-performance liquid chromatography
(HPLC). Microglia were stimulated with lipopolysaccharide (LPS) in the presence or absence of
RBE. Release of prostaglandins (prostaglandin (PG) E2, 8-iso-prostaglandin F2α (8-iso-PGF2α))
were determined with enzyme immunoassay (EIA). Protein levels and genes related to
PGE2synthesis (Cyclooxygenase-2 (COX-2), microsomal prostaglandin E synthase-1 (mPGES-
1)) and various pro- and anti-inflammatory cytokines (TNF-α, IL-1β, IL-6, and IL-10), were
assessed by western blot, ELISA, and quantitative real-time PCR. Furthermore, to elucidate the
molecular targets of RBE, the phosphorylated state of various mitogen-activated protein kinase
(MAPK) signaling molecules (p38 MAPK, ERK 1/2, and JNK) and activation of NF-kB pathway
was studied.
Results
RBE significantly inhibited the release of PGE2 and free radical formation (8-iso-PGF2α) in LPS-
activated primary microglia. Inhibition of PGE2 by RBE was dependent on reduced COX-2 and
mPGES-1 immunoreactivity in microglia. Interestingly, treatment of activated microglia with
RBE further enhanced the gene expression of the microglial M2 marker IL-10 and reduced the
expression of pro-inflammatory M1 markers (TNF-α, IL-1β). Further mechanistic studies
showed that RBE inhibits microglial activation by interfering with important steps of MAPK
signaling pathway. Additionally, microglia activation with LPS leads to IkB-α degradation
which was not affected by the pre-treatment of RBE.
Conclusions
Taken together, our data demonstrate that RBE is able to affect microglial activation by
interfering in important inflammatory pathway. These in vitro findings further demonstrate the
potential value of RBE as a nutraceutical for the prevention of microglial dysfunction related to
neuroinflammatory diseases, including Alzheimer’s disease.
4.1.6. Developing of special porridge for elder population (16)
February 20, 2014. STUTTGART. The Federal Ministry of Economics (BMWi) supports in frame
of the ZIM-initiative a project called PorridgePlus (KF3204601). The aim of the project is to
develop a food product for elderly with the objective to promote healthy brain aging. The basis for
the project are findings that Rice Bran Extract protects from mitochondrial dysfunction in the
brain. Mitochondrial dysfunction plays a major role in the development of age-related
neurodegenerative diseases and recent evidences suggest that food ingredients can improve
mitochondrial function. The Kick-off meeting held in Stuttgart launches the biennial project.
Project partners are: Bernd Fiebich (Vivacell, Freiburg), Gerhard Fesenmeyer (FBFood, Villingen-
Schwenningen), Gunter P. Eckert (Goethe-University of Frankfurt), and Jan Frank (University of
Hohenheim).

16. 4.1.7. Rice Bran Functional Extract (running project)
The data generated through previous studies confirm the potential health benefits of rice
bran/ or its extracts in Alzheimer’s Disease illness. However the lack of knowledge/
identification of the possible mechanisms of action of the extract / or major constituents is a
great barrier for further business development with strong, internationally recognized
product.
In this project the mechanism of action of stabilized rice bran (Oryza) oily extract (Riciplex)
as well as the specially prepared aqueous extract (water solubles) (under registration) as a
protecting effect against Alzheimer’s disease will be investigated.
The above action is based on the results of previous projects and the expected determination
of the mechanism of action will have direct impact on the business development of such
Egyptian products that are innovately developed in Egypt. This will open the doors for export
to EU and the USA at higher value and will be the base for the very expensive pre-clinical,
clinical studies planned to take place in cooperation with Goethe Institute in Germany. It has
to be noted that the Alzheimer’s disease market in the United state and in Egypt is estimated
to be US$ 2.66 bn and LE 76 Million respectively (see also section (B.12.B). Yet all these
products are not curative, only attenuating the symptoms.

17. 4.1.8. Innovation in rice bran extraction and formulation
The project aims for developing innovative method for stabilized rice bran extraction and
formulation through spray drying and / or other means in cooperation with three partners (at three
countries; Egypt, Germany and Holland) as well as one associate in Germany. The following
results were were achieved
- Establishment of the optimum ultrasonic extraction procedure
-Establishment of the optimum spray drying formula and procedure (with some
modification).
-Setting of pilot unit for ultra sonic extraction and spray drying -
- Formulation of the spray dried rice bran extract / other water soluble extract form in
nutraceutoical, Cosmoceutical and functional food and beverage preparations.
Some of these products are under registeration like the soft gelatin capsules, others are
registered and within the marketing stage now (Oryza). Others are in the development pipe line
in Holland.

18. Refernces
1- Sahar Y. Al – Okbi, Ahmed M.S. Hussein; Ibrahim M. Hamdi; Doha A. Moahmed, AmrM.
Helal. Chemical, Rheological, Sensorial and functional properties of Gelatinized corn-rice bran
flour composite corn flakes and tortilla chips.
Journal of Food Processing and Preservation 38 (2014) 83–89 © 2012
2. Ahmed M. S. Hussein, Sahar Y. Al-Okbi . Evaluation of Bakery Products Made from Barley-
Gelatinized Corn Flour and Wheat-Defatted Rice Bran Flour Composites. International
Scholarly and Scientific Research & Innovation 9(9) 2015
3- Al-Okbi SY, Hussein AMS, Hamed IM, Mohamed DA, Helal AM. Chemical, Rheological,
Sensorial and Functional Properties of Gelatinized Corn- Rice Bran Flour Composite Corn
Flakes and Tortilla Chips, Journal of Food Processing and Preservation, 2012 (Wiley) ,
doi:10.1111/j.1745-4549.2012.00747.
4- Ammar HO, Al-Okbi, SY, Mostafa DM, Helal AM. Rice bran oil: Preparation and
evaluation of novel liquisolid and semisolid formulations. International Journal of
Pharmaceutical compounding. 2012; 16: 516-523.
5- Al-Okbi, SY, Mohamed, DA, Hamed, IM, Agoor, FS, Ramadan, AAM, El-Saed, S, Helal,
AM. Comparative study on Egyptian rice bran extracted by solvents and supercritical CO2.
Advances in Food Sciences. 2013; 35: 23-29.
6- Al-Okbi, SY, Ammar, NM, Mohamed, DA, Hamed, IM, Desoky, AH, El-Bakry,HF, Helal,
AM. Egyptian rice bran oil: chemical analysis of the main phytochemicals. Rivista taliana
delle Sostanze Grasse 2014 ; 90(1):47-58.
7-Kaup RM, Khayyal MT, Verspohl EJ. Antidiabetic effects of a standardized Egyptian Rice
Bran. Phytotherapy Research . 2013; 27: 264-271
8- Khayyal M, Abdel-Aziz H, Mohsen R, Helal A, Mueller W, Eckert G. Pharmacological
studies on a standardized n Egyptian rice bran extract . Basic & clinical pharmacology &
toxicology 115, 139-139
9- Helal AM, Khayyal MT, El Aziz HMA, Salam RMA. Developing a nutraceutical from
Egyptian stabilized rice bran: a pharmacological approach Planta Medica 77 (12), PJ23
10- Heikal OA, Zickri MB, Helal AM, EL-Askary H, Fiebich L, Gomaa IO. Stabilized rice
bran extract: Acute and 28-day repeated dose oral toxicity with in vitro mutagenicity and

19. genotoxicity study. African journal of pharmacy and pharmacology. 2015; 9(44): 1037-
1050 DOI: 10.5897/AJPP2014. 4083
11- Hagl S , Kocher A, Schiborr C, Eckert SH, Ciobanu I, Birringer M, El-Askary H, Helal
A, Khayyal MT, Frank J, Muller WE, Eckert GP. Rice bran extract protects from
mitochondrial dysfunction in guinea pig brains. Pharmacol Res. 2013; 76: 17-27.
12- Hagl S, Berressem D, Grewal R, Grebenstein N, Frank J, Eckert GP. Rice bran extract
improves mitochondrial dysfunction in brains of aged NMRI mice. NutrNeurosci. 2015a
Aug 4. [Epub ahead of print]
13- Hagl S , Berressem D , Bruns B, Sus N, Frank J and . Eckert GP. Beneficial Effects
of Ethanolic and Hexanic Rice Bran Extract on Mitochondrial Function in PC12 Cells and
the Search for Bioactive Components. Molecules. 2015 b; 20(9): 16524-39. doi:
.3390/molecules200916524.
14- Hagl S, Grewal R, Ciobanu I, Helal A, Khayyal MT, Muller WE, Eckert GP . Rice Bran
Extract compensates mitochondrial dysfunction in a cellular model of early Alzheimer's
disease. J Alzheimers Dis. 2015c; 43 (3):927-38. doi: 10.3233/JAD-132084.
15- Harsharan S. Bhatia; Julian Baron; Stephanie Hagl; Gunter P. Eckert; Bernd L. Fiebich
Rice bran derivatives alleviate microglia activation: possible involvement of MAPK
pathway
16- http://www.nutritional-neuroscience.com/news/new-project---porridgeplus.html