review of biopharmaceuticals and nutraceuticals from rice...

Key words: biopharmaceutical, innovation, molecular farming, MucoRice, rice, therapeutics, transgenic rice

Review of Biopharmaceuticals and Nutraceuticals from Rice Grain: Exploiting the Endosperm, Germ

and Bran for High-value Innovation Rice By-products

*Corresponding author: [email protected]; [email protected]

Amor A. San Juan*

Applying biotechnology innovation in rice is shown to produce promising biopharmaceuticals. The focus of this review is to critically examine the rice-derived biopharmaceuticals in contrast with rice-based therapeutics, its current progress, and future prospects. The article highlights the function-based analogy of an automobile car with the rice grain, aiming to understand the complexity of rice-based innovation encompassing biopharmaceutical and therapeutics. The combined endosperm, germ, and bran of rice consist of several bioactive compounds that result to a synergistic mechanism effect responsible for its health benefits. This article review shall hopefully encourage further relevant studies on rice product innovation as an added high-end value to the rice industry. The perspective ends with a discussion on the challenges and opportunities for biopharmaceuticals and nutraceuticals.

Philippine Journal of Science147 (3): 431-439, September 2018ISSN 0031 - 7683Date Received: 09 Jan 2018

Department of Chemistry, College of Arts and Sciences, Central Luzon State University, Science City of Muñoz, Nueva Ecija 3120 Philippines

INTRODUCTIONHalf of the world population widely consumes rice as a staple food, served on the plate for many dishes from adobo, curries, kimchi, sushi, and stir-fries, to arroz de tomate. Rice is wonderfully versatile beyond than just a food staple, with the capability to be transformed into savories and sweets. Innovative rice food products are notable around the globe, including the Philippines barquirice crispy biscuit roll, Australia’s brown rice chips, and Thailand’s rice-bran oil, fat-free salad dressing, rice noodles, talc-free baby powder, and ready to bake rice flour.

A grain of rice is made up of three parts: the endosperm, which is full of starchy carbohydrates that make up the majority (90%) of the grain; the germ, which contains a higher value of antioxidants; and the outer coating or

bran, which includes fiber and vitamins (Grist 1959). Apart from consuming rice as a staple food, it is also used as a model monocot for the advancement of biotechnology research. The manipulation of rice grain in its size, shape, and composition relies on the access to endosperm-specific promoters (ESP) that function in propelling genetic transformation (Li et al. 2008). One of the tissues in the endosperm contains a starchy endosperm, which in turn accumulates storage protein. Promoters of gene that encode the rice seed storage protein are divided into insoluble glutelins, alcohol-soluble prolamins, salt-soluble globulins, and water-soluble albumins (Wu et al. 1998). A rule of thumb follows that a strong ESP of storage protein yields a high-level expression of foreign protein, an advantage in a large-scale production of recombinant proteins as applied in molecular farming. In the molecular farming process, the rice endosperm undergoes a genetic transformation to produce high quantity of therapeutic protein at a lower production cost, characterized by increased scalability, free

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from contamination, and contributive to green chemistry manufacturing (Ou et al. 2014). On the other hand, the use of combined bran layer and germ in rice grain is an emerging research on drug discovery. Both the bran and germ of rice contains bioactive components such as γ-oryzanol, representing attractive target compounds for the development of next generation plant-based therapeutics. Noteworthy, the versatility of rice grain in research and development goes beyond its capacity in therapeutics and biopharmaceuticals. In fact, the rice hull provides a sustainable renewable energy by generating heat for home cooking and industrial drying, using the developed Philippine technology of continuous-type rice hull carbonizer (Orge & Abon 2012).

Pharmaceutical innovation in rice can be understood by using the analogy of a car with a rice grain (see Figure 1). In creating a high-value innovation for rice plant, the endosperm, integrated germ and bran, and hull correspond to the car’s engine, break, and fuel, respectively. The function of a car engine resembles the rice endosperm such that the former transforms fuel into mechanical motion, while the latter undergoes genetic transformation as applied in molecular farming to manufacture recombinant proteins. The car break system correlates the combined rice bran and germ, such that in decelerating the car speed (alleviates disease symptoms) or stopping the car (cure of disease). The car fuel is complementary with the function of the rice hull such that as the fuel supplies energy for the car to move, so as the rice hull provides heat source for

Figure 1. A function-based analogy of car with rice grain. Anatomical characteristics of rice grain correspond to the function of a car parts. The parts of rice containing the endosperm, combined germ and bran, and hull complement with the function of a car’s engine, break, and motor fuel, respectively.

processing food products. Overall, the use of an analogy between a car and rice grain reveals critical connections, sparks creativity, and conveys novel ideas. Interestingly in history, the cars are used as a metaphor of rice, such that the automobile maker Toyota has an original Japanese name Toyoda that means abundant rice, as well as Honda that means main rice field.

This review offers an in-depth and critical investigation of molecular farming in rice as an emerging process of biomanufacturing medicine, as well as the future development of innovative medicine from rice. Faced with the bottleneck of manufacturing drugs, withdrawn pipeline drugs due to toxicity, and evolving public health needs for safe and cost-effective drugs, ensuring the continuity of medicines innovation is in the best interest of mankind.

Innovation in Rice Pharmaceutical: Where Traditional Therapeutics Meets Modern BiopharmaceuticalTo sustain better health for patients worldwide, the spontaneous growth of innovation in medicine is important. Innovative medicines are beneficial both economically and socially, providing restored earnings for patients, reduced costs for healthcare systems, and improved productivity for the economy. According to the International Federation of Pharmaceutical Manufacturers Associations (IFMA) report in 2004, the key components in pharmaceutical innovation include patient-oriented healthcare systems, an efficient market that values innovation, effective use of intellectual property to promote innovation, and technical regulation system that ensures safe, effective, and highest quality of new medicines.

Rice innovation that deviates from the usual staple food into the pharmaceutical application is essential to biotechnology-driven research for development. A rice grain has minimal amount of a protein (7-8%), as compared to its starch content (92-93%). However, the storage protein of rice seed remains intact and functional throughout its dormancy period, which is suitable for the stable deposition of foreign protein for biopharmaceutical production (Tackaberry et al. 2008). Although rice is not a protein-rich seed, a high-level expression of foreign protein into the rice endosperm can be achieved by employing strong promoters and subsequently, stabilization of the protein once it is translated and processed in the cell (Boothe et al. 2010).

The current review delineates the rice-derived pharmaceutics into two different classifications, including the rice-produced bioactive compound therapeutic (rBCT) and rice-production platform of therapeutic protein (rPTP). The production sources between the rBCT and rPTP are extraction and biomanufacturing, respectively. The

San Juan AA: Review on Biopharmaceuticals and Nutraceuticals from Rice Grain

Philippine Journal of ScienceVol. 147 No. 3, September 2018

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revolutionary technique has evolved from the extraction of rice bioactive components into molecular farming of rice to produce biopharmaceuticals. Considering the unique versatility of rice producing therapeutic compound - as well as rice as a platform of production for therapeutic protein - the rice seed has continued to play a pivotal role in drug development and research programs. A biotechnology-driven rice innovation applied in biopharmaceutical production is a modern technological advancement, providing large-scale production of protein therapeutics (Decker & Reski 2008) and high cost of production (Butcher 2011). Conversely, the traditional method of extraction and isolation of plant-based bioactive compound shows limitation on high-cost of purification and low bioavailability (Yusop et al. 2017). Despite the key differences (see Table 1) between a traditional plant-based therapeutics and a modern biopharmaceutical, both are critical pillars of pharmaceutical rice innovation. The most striking difference is within the process of production, such that the biopharmaceutical is derived from genetically engineered crops (Fischer et al 2004) while the plant-derived therapeutics is originated from non-engineered biological sources. The production of biopharmaceuticals has low yield of expressed protein (Lojewska et al. 2016), while enhanced bioavailability is achieved by microencapsulation for targeted drug delivery (Tumaro-Duchesneau et al. 2013).

Progress in the Development of Rice-derived Biopharmaceutical ProductionA remarkable research interest is steadily increasing for transgenic r ice in large-scale production of biopharmaceuticals. As shown in Figure 2, the

Figure 2. A graphic plot on publication trend showing an increased scientific interest on rice biopharmaceuticals. Data processing was obtained by using the online tool MEDSUM (Galsworthy 2009) during the period 1996-2012. Recent data from 2013 up to present is not included because of the lack of data coverage caused by the tool’s constraints.

scientific publications on rice biopharmaceuticals has seen considerable growth. Overall, there is an apparent growth in past and present market demands for biopharmaceuticals, and the forecasted growth is 7-15% in the next decade (Hiller 2009). The biotechnological exploitation of the endosperm component in rice seed for molecular farming applications is desirable to produce large quantities of recombinant therapeutic proteins. Several biopharmaceuticals derived from rice as a production platform have reached clinical trials. The recombinant transferrin (rTr) produced from transgenic rice is an alternative to human blood-derived transferrin in cell culture applications (Zhang et al. 2010). The rice-derived rTr shows similar protein structure as compared to the native protein, with comparable cell growth and productivity assay. In addition, another rice-based biopharmaceutical production is the recombinant human serum albumin (rHSA), used as a cell culture production of vaccines and pharmaceuticals (He et al. 2011). In vivo experiment showed that both rice-based rHSA and human plasma-derived HSA have equivalent biological function in the treatment of liver cirrhosis. The recombinant lactoferrin (Suzuki et al. 2003) and lysozyme (Yang et al. 2003) indicated for antibiotic-related diarrhea are derived from genetically engineered rice seed that had been shown with up to 40% level of protein expression (Huang 2004). Interestingly, the rice endosperm has the capability to express a fibroblast growth factor (FGF) with a relatively low molecular mass (~17kDa) and high hydrophobicity (An et al. 2013). The similarity among rTr, rHSA, recombinant FGF (rFGF), and recombinant lactoferrin and lysozyme is the inclusion of further purification process to finally obtain the therapeutic protein. In contrast, the MucoRice is a rice-based vaccine against cholera toxin specific for the B-subunit protein (CTB) that showed breakthrough technologies of room temperature stability

Table 1. Key differences of therapeutics production between traditional bioactive compound and modern biopharmaceutical.

Traditional production of bioactive compound

Modern biopharmaceutical production

Production Direct extraction (non-engineered)from biological source

Transgenic plant-based productiona

Bioavailability enhancement

Microencapsulation to protect the bioactive compound from degradation in acidic condition of the stomachb

Microencapsulation for targeted drug deliveryc

Cost High-cost High-costd

Bottlenecks High cost of purification

Low yield of expressed proteine

Scalability Small-scale Commercial-scalef

aFischer et al. 2004; dButcher 2011; bYusop et al. 2017; eLojewska et al. 2016; cTumaro-Duchesneau et al. 2013; fDecker & Reski 2008



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(cold-chain free) and oral route intake (syringe-free) that held the basis for a purification-free process (Nochi et al. 2007). Noteworthy, the MucoRice-CTB has proven no allergenicity based on using directly the polished rice powder without the purification process (Kurokawa et al. 2013). Furthermore, the syringe-free and the cold-chain free systems are extended to another version of MucoRice, targeting the rotavirus-specific llama heavy-chain antibody fragment (ARP1) (Tokuhara et al. 2013). The MucoRice-ARP1 as rotavirus therapy is an alternative approach to vaccines, with the capability to be administered orally either as rice powder or rice water.

Progress in the Therapeutic Development of Rice-based Bioactive CompoundsRice drug discovery is an emerging research that is crucial for the development of new drugs. In Figure 2, the publication trend shows that the rice drug discovery is in the early phase of research and development. Noteworthy, the abundance of antioxidants extracted from rice shows a better efficacy as compared with the pure commercial grade (Jung et al. 2007; Canan et al. 2012). The bioactive components of rice, which consists of dietary fiber and antioxidants, provide beneficial health effects particularly in diabetes, cancer, and heart diseases (Goufo & Trindade 2015). Essentially, the multiple bioactive components of rice antioxidants provide a synergistic therapeutic effect, modulating oxidative damage that is associated with artheroclerosis, inflammation, and metastatic cancer cells. There are seven different types of rice antioxidants including phenolic acids, flavonoids, anthocyanins, proanthocyanidins, tocopherols, and tocotrienols (also called as vitamin E), γ-oryzanol, and phytic acid (Deng et al. 2013; Goufo & Trindade 2014). Several studies have shown the therapeutic relevance of rice antioxidants. In vitro tests on γ-oryzanol rice antioxidant showed an inhibition of nitrite formation, which is involved in oxidative damage (Ghatak & Panchal 2012; Saenjum et al. 2012). The molecular mechanism of inhibition is based on a chemical reaction, in which the reactive oxygen species (ROS) acts as oxidizing agent, whereas the phenolic ring of γ-oryzanol acts as reducing agent that in effect efficiently scavenge cellular peroxynitrites. An in vitro test demonstrated that the phytic acid rice antioxidant has the capability to form a chelation with a transition metal iron, which as a result impedes the ROS formation (Canan et al. 2012). In addition, the phytic acid antioxidant showed inhibition to cyclooxygenase-2 (COX-2) protein expression, which is involved in colorectal cancer (Norazalina et al. 2013). The anthocyanin antioxidant from rice showed an in vivo protection against lipoprotein-cholesterol oxidation by reducing the triglyceride and cholesterol in blood plasma and urine (Guo et al. 2007). Moreover, the anthocyanin demonstrated an anti-inflammatory effect by suppressing

the tumor necrosis factor, a pro-inflammatory cytokine (Min et al. 2010).

The predominant distribution of the chemical composition in each of the rice antioxidants is influenced by pre-harvest and post-harvest process. The primary phenolics include ferulic, p-coumaric, and sinapic acids, which showed that when rice is planted in the clay-loam type of soil, the phenolic content showed an increased level (Butsat & Siriamornpun 2010). Interestingly, rice organic farming favors an increased level of phenolics, phytic acid, and flavonoids due to the higher number of beneficial bacteria in the soil (Park et al. 2010). Rice fermentation increases the contents of phenolics, flavonoids, anthocyanins, proanthocyanidins, and γ-oryzanol, which can be explained by the highly efficient enzymatic system of the fungi to degrade lignocellulosic materials in rice (Manosroi et al. 2011; Sungsopha et al. 2009; Yang et al. 2006). Except for phytic acid, the other rice antioxidants showed an increase level upon the germination of rice, because of the biochemical activity to produce essential compounds and energy for the formation of seedling (Moongngarm & Saetung 2010; Sutharut & Sudarat 2012).

Rice Molecular Farming: Its Current Approach, Importance, and ChallengesMolecular farming exploits the plant in the process of biomanufacturing to produce large-scale recombinant proteins of therapeutic relevance. Several types of plants were used in the process of molecular farming, including tobacco, banana, carrot, corn, potato, strawberry, and tomato. However, rice seed as a platform for the production of therapeutic protein offers several advantages as compared with other plant expression system (Greenham & Altosaar 2013). There is a significant difference of molecular farming between seed protein and leaf protein. Seed protein contains a bulk of storage protein particularly in rice grain, which is composed of 80% glutelin as storage protein. The storage protein in seeds provides suitability for effective storage and deposition of recombinant protein. Rice storage protein remains intact and functional during its dormancy period, allowing the stable deposition of a recombinant protein. The low abundance of active proteases in seed tissues prevents proteolysis of the recombinant protein, whereas the dessicated form of the mature seeds promotes the long-term stability of the recombinant protein (Stoger et al. 2000). Remarkably, the seed is a composite of protein and oil bodies, which sequester the recombinant protein and minimize its exposure to protease during extraction process. Furthermore, rice is self-pollinating, ensuring that no genetic material is gained or lost. Rice has the capability to eliminate the downstream purification process by direct oral intake as rice powder or rice water. In contrast, leaf protein is composed mainly of enzymes, crucial for photosynthesis in the green leaves of plants. A



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major drawback of leaf-based molecular farming system is the abundance of proteases and phenolics in green leafy tissues (Michaud & Asselin 1995), which lead to both degradation and denaturation of leaf proteins (Schillberg et al. 2005).

Plants had been established as alternative to microbes and mammalian cells for the production of pharmaceutical proteins. Advantages of producing therapeutic proteins from plants include lower risk of pathogen contamination, higher level of protein expression, oral delivery, and unnecessary purification. Particularly, the studies on seeds were shown that it has the capacity to accumulate a wide range of proteins for industrial or medical applications, such as viral and bacterial antigens, antibodies, proteases and protease inhibitors, hormones, growth factors, and enzymes (Boothe et al. 2010). In the genetic perturbations of the rice seed protein component in order to insert a recombinant protein, it requires strategic protocols to achieve higher yields of production. Equally important are the promoters and enhancers, which control the abundance of the transcripted protein - as well as the subcellular localization of storage protein, which regulates the stability and suitability in the accumulation of the transcripted protein. For MucoRice-CTB, the GluB-1 glutelin promoter was implemented specifically for endosperm protein expression, resulting to an average of 30 µg of CTB (2.1% of total seed protein) per grain of rice (Nochi et al. 2007). On the other hand, the application of RNAi technology to suppress the storage proteins (glutelin and prolamin) and replaced by ARP1 protein, yielded a higher expression level reaching an average of 170 µg of ARP1 (11.9% of total seed protein) per grain of rice (Tokuhara et al. 2013). In the case of recombinant transferrin, the Gt1 glutelin-1 promoter was used for endosperm protein expression, which produced up to 40% total soluble protein (TSP) of the rice grain, depending on experimental conditions (Zhang et al. 2010). Interestingly, for rHSA that utilized an endosperm-specific Gt13a glutelin promoter, yielded 10.58% TSP of the rice grain in which the protein accumulation is targeted at protein storage vacuoles (He et al. 2011). A similar gene promoter Gt13a is used for rFGF, which yields a protein expression level as high as 185.66 mg/kg of brown rice (An et al. 2013). The applicability of a hybrid gene promoter in recombinant lysozyme - combining two different promoters including wheat (Triticum aestivum) puroindoline b and rice (Oryza sativa) Gt1 glutelin - has shown to doubly increase the protein accumulation level from 5.24 g/kg to 9.24 g ⁄ kg of the extracted rice flour (Hennegan et al. 2005).

Impact of Rice Pharmaceutical Innovation in Producing Valuable MedicinesRice-based pharmaceuticals target to provide innovative therapeutics and biopharmaceutical. Currently, the promising candidates of nutraceutics and biopharmaceutics

derived from rice gear towards the market as valuable medicines. Transformative innovation to produce valuable medicines derived from genetically modified rice is remarkably shown with the development of MucoRice vaccine, using rice as a bioreactor in producing human pharmaceuticals (Kurokawa et al. 2013). In addition, the potential value of rice bran extract as nutraceuticals have been shown in laboratory studies that the antioxidants obtained from the rice bran have the potential to treat heart disease, cancer, diabetes, and kidney stones. Interestingly, the cholesterol-lowering ability of rice bran oil had been investigated in moderately hypercholesterolemic adults (Most et al. 2005). Recent study shows the potential medicinal application of gramisterol component in rice bran as anti-cancer drug against acute myelogenous leukemia (Somintara et al. 2016). On the other hand, the phenolic fraction in rice bran showed hypoglycemic effects in mice (Jung et al. 2007).

The impact of innovation on pharmaceutical rice is most notable to two main areas such as stimulating economic growth, as well as benefits for patients and society. Pharmaceutical innovation is usually determined with new formulation, new combination of active ingredients, and new therapeutic use of existing drugs. In balancing the equation for the innovation cost corresponding to the impact of pharmaceutical rice, improved techniques from technological advances counteracts the spiral expenditures and cost incurred in R&D.

Biopharmaceutical and Nutraceutical: A Perspective on Current Challenges and Potential OpportunitiesRice nutraceuticals contain phytochemicals with essential health benefits for the prevention and treatment of different diseases. Remarkably, a strong interest in rice nutraceutical is rapidly increasing due to its safety and therapeutic effects. In fact, the worldwide nutraceutical market is expected to reach US $250 billion by 2018 (Hardy 2000). In contrast to biopharmaceuticals, nutraceuticals have no patent protection. The nutraceutical intellectual property has evolved and encompasses two different categories: known or natural components for new use, and combinations of known or natural components offering enhanced performance compared to that of the individual components. Creativity is the key to overcome any patent objections on grounds of scientific novelty in commercializing nature's principles. On the other hand, poor bioavailability of nutraceuticals hampers its efficacy. Several nanotechnology drug delivery systems for the bioactive compounds have been shown to improve the bioavailability (Trujillo et al. 2016; Gunasekaran et al. 2014). Nutraceuticals represent an exciting opportunity for relatively lower cost in R&D as compared to biopharmaceutical production. In addition, adding value through innovation in agriculture yield medicinal food as nutraceuticals.



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In biopharmaceutical drug development, several challenges are involved due to the quite complex process to obtain a recombinant protein with optimal quality, purity, and potency. Each step during the production process requires tighter regulatory control involving frequent quality control measurements, as compared to a simpler process in pharmaceutical drug. Another obstacle is the formulation of biopharmaceuticals, influenced by optimal balanced between solubility and stability of the protein. The therapeutic value of the protein is affected by its structural stability to withstand several harsh conditions during the production process. In order to achieve efficiency in manufacturing biopharmaceuticals, innovative tools and techniques are necessary. Specifically, the continuous downstream processing for the production of biopharmaceuticals reduces cost and increases productivity (Rathore et al. 2015). Biopharmaceutical patent is a regulatory exclusivity provision that provides incentive towards R&D innovation capability (Grabowski et al. 2015). The implementation of exclusive patents is critical to effectively protect and receive a return of investment.

Figure 3 shows the summary of opportunities and challenges for the biopharmaceutical and nutraceutical sectors. The biopharmaceutical platform produced recombinant proteins through molecular farming, whereas the nutraceutical channels bioactive compounds through traditional method of extraction. The convergence of molecular farming to produce large quantity of bioactive compounds is a promising approach to achieve large-scale production of nutraceuticals. Thus, it is safe to say that upon connecting the bridge between the east (nutraceuticals) and the west (molecular farming), the drug discovery efforts move into a higher level of strategy. Recent study has investigated a cost-effective molecular farming approach of the Artemisia annua plant against the malaria parasite through successful expression of a foreign gene into the plant chloroplasts (Pulice et al. 2016). In the near future, rice grain molecular farming approaches

Figure 3. Challenges and opportunities between biopharmaceutical and nutraceutical sectors.

using metabolic engineering is expected to increase yield of bioactive components and thus, increasing the market effectively for nutraceuticals.

The author anticipates pharmaceutical innovation using rice by strategically implementing the parts-analogy of a rice grain with a car. In the near-to-midterm, nutraceuticals from combined rice bran and germ has the potential to reach its peak into the market, for which its benefit is lesser side effects to humans. In the longer term, and considering the recent developments in rice molecular farming, the production of pharmaceutical proteins will probably enable us to counter the persistent public health risks.

ACKNOWLEDGMENTThe author is grateful for the critical comments received from the anonymous reviewers of this paper.

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