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

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.

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.

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:

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.

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

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

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

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

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

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.

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

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)

MitochondrialdysfunctionplaysanimportantroleinbrainagingandhasemergedtobeanearlyeventinAlzheimer’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

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.Therefore, 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.

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).

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.

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.

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

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