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Legumes Biofarming and Biopharmaceutical Sciences: A Review



Akbar Nikkhah
 
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ABSTRACT

The objective is to delineate and review prominent commercial and emerging biotechnopharmaceutical sciences that are state-of-art intersections of plant, human and animal ecologies. A special focus is placed on alfalfa as a bountiful legume from animal agriculture and medicinal perspectives. Crops are steadily and increasingly regarded as human health inducers. The new insights are changing and expanding plants prospective roles in plant and human ecologies form biotechnology and medicine perspectives. As such, more emphasis is placed on links between plant chemicals with post-modern human and animal health. Such plant-origin therapeutics include pharmaceuticals, multi-constituent botanical drugs, dietary supplements, functional foods and plant-derived recombinant proteins and vaccines. These compounds and products are expected to complement conventional pharmaceuticals for cure, prognosis and diagnosis of diseases, while improving the quality of agricultural crops. As a result, a more effective exploration of diverse phytochemicals may be developed. In addition, new biotechnologies enable manipulating plant’s capability in synthesizing natural products and substances. Advanced biotechnologies to generate preferential Biopharmaceuticals (BPC) are receiving increased research and commercial interests. However, the cost, safety and accessibility of such BPC are among the major challenges. Nonetheless, plant BPC could be inexpensive and potentially safe to produce and store. Vegetables and leguminous plants are the fitting examples. A main advantage of leguminous crops, namely alfalfa (Medicago sativa), is their immense biomass yield since they can be cropped several times a year. Leguminous plants contain phytoestrogens that encompass several compounds, such as flavonoids, isoflavonoids, coumestans and lignans. Legumes such as peas, soybeans and alfalfa can be used to produce plant-derived inexpensive monoclonal antibodies (plantibodies) for human and animal therapeutics. Due to the presence of a multitude of bioactive substances in legumes, further studies particularly involving nutrigenomics and metabolomics are required to specify such effects on human health indicators of particular BPC. Definitive Quantitative and qualitative dietary inclusion guidelines for foods with legume origins may become feasible for different age groups. These achievements will form new perspectives that will contribute to healthier and more viable plant-human-animal ecologies in the new era.

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  How to cite this article:

Akbar Nikkhah , 2012. Legumes Biofarming and Biopharmaceutical Sciences: A Review. Research Journal of Medicinal Plants, 6: 466-488.

DOI: 10.3923/rjmp.2012.466.488

URL: https://scialert.net/abstract/?doi=rjmp.2012.466.488
 
Received: January 05, 2012; Accepted: March 03, 2012; Published: June 21, 2012



INTRODUCTION

Crops are being increasingly viewed as human health agents besides supplying viable foods. The new insights emphasize such contributions to animal and human biotechnology and medicine. New plant-origin therapeutics encompass pharmaceuticals, multi-constituent botanical drugs, dietary supplements, functional foods and plant-generated recombinant proteins (Ma et al., 2003; Nikkhah, 2012a, b, c). These would complement traditional pharmaceuticals for cure, diagnosis and treatment of diseases, whilst improving crops quality. Also, with new biotechnologies, plants capacities to synthesizing natural products may be manipulated. Advanced biotechnologies to generate Biopharmaceuticals (BPC) are a host to modern research and commercial interests (Fischer and Emans, 2000). However, their cost and availability limit application (Nikkhah, 2012d). Nonetheless, plant BPC production and storage could be inexpensive and potentially safe. Legumes are among prominent plants in furthering BPC technologies that will be described in detail later.

Phytoestrogens are a group with pathetic estrogenic activity. In seedlings of soybeans (Glycine max), the isoflavones of genistein and daidzein are predominant (Graham, 1995). Phytoestrogens, especially isoflavones, may cause infertility in livestock (Bouea et al., 2003). Ever since these discoveries, more than 300 plants with estrogenic animal responses have been found. Phytoestrogens include several compounds, including flavonoids, isoflavonoids, coumestans (coumestrol) and lignans (Mazur et al., 1998). Although causing infertility in animals, phytoestrogens may benefit human health via prevention of certain diseases (Tham et al., 1998). Specifically, phytoestrogens could prevent cancer (Barnes, 1997), scavenge free radicals and lower blood cholesterol (Carroll and Kurowska, 1995) and be antiestrogenic and antiproliferative (Barnes, 1997). Phytoestrogens are most abundant in leguminous plants that are present in international diets. Legume seeds besides other parts of whole plants are edible. Soybean is a prominent legume rich in isoflavones daidzein and genistein (Franke et al., 1994). Daidzein and genistein are additionally responsible for some the health benefits of soybeans (Messina and Barnes, 1991). Biochanin A and formononetin are other isoflavones in legumes. Besides isoflavonoids, flavonoids also exert estrogenic activity but are much less active than isoflavonoids (Collins-Burow et al., 2000). Positive epidemiological correlations exist between fruits and vegetables intakes and prevention of atheroscelerosis, cancer, diabetes and arthritis (Kaur and Kapoor, 2001). As such, they are called as ‘youth fountains’, promoting functional health and alleviating diseases. Phenolic flavonoids, lycopene, carotenoids and glucosinolates are among major antioxidants (Kaur and Kapoor, 2001).

A world in which natural and designed, synthetic proteins can be generated with safe, secure and cost-effective approaches in desired quantities would be a great accomplishment for the man (Nikkhah, 2012b, c). That is particularly true when utilizing simple nutrients, water and sunshine. Whilst beyond man’s crude imagination, such advancements do not seem too far from reality. Exploitation of plant powers (cereals and legumes; Fig. 1, 2) in molecular farming and generating recombinant proteins and vaccines on agricultural and medicinal scales have been a concrete proof. The initiatives on plant-derived recombinant BPC have tackled commercial approval, promising more upcoming new biotechnoecologies. This review study will delineate prominent commercial emerging biotechnologies that are state-of-art intersections of plant, human, and animal ecologies. A special focus will be made on alfalfa as a bountiful multi-advantage legume from both animal agriculture and medicine perspectives. Health implications of alfalfa utilization in postmodern pharmaceutical industries will be delineated. Other objectives include describing recombinant proteins, immunoglobulins and vaccines production in alfalfa platforms using most recent biotechnologies. Prospectus public interests and concerns will also be discussed.

Plant proteins and vaccines production-biofarming: Plant-origin Biopharmaceuticals (BPC) are in the developing phase as the upcoming commercial states-of-art in plant-human-animal biotechnologies (Nikkhah, 2012d). These may have highly effective production scale and economics, global safety, storage capacity and reasonably efficient distribution.

Image for - Legumes Biofarming and Biopharmaceutical Sciences: A Review
Fig. 1: Endosperm cells from tobacco, The tobacco cell is full of oil drops occupying most of the internal space encompassing limited protein bodies (light)

Image for - Legumes Biofarming and Biopharmaceutical Sciences: A Review
Fig. 2(a-c): Different forms of corn-based edible vaccines produced and approved to date (Prodigene; College Station, TX, USA), Transgenic corn kernels corresponding to a 1 mg dose of the B subunit of (a) E. coli heat-labile toxin, (b) Being processed for a palatable whole corn snack and (c) Fractionated to produce concentrated embryo or germs with antigens being 6x more

These proteins offer promising opportunities to synthesize and supply cost-effective medicines and vaccines, especially in the emerging and developing world (Table 1). However, disadvantages include doubtful regulatory grounds in platform-raised crops and vacillating manufacturing practice regulations in field-grown plants. The novel products must undergo vigilant cooperation and compromises of academic and commercial property landscape to be widely useable. Authorized operation licenses must be ensured for successful application and utilization in specific countries and regions (Ma et al., 2005a).

Table 1: Production systems properties for recombinant proteins production
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These products will enable saving lives of human and animal populations where environmental challenges concur, as beginning to be commercially releasable (Ma et al., 2005a; Twyman et al., 2005).

Plant-origin platforms have rather low production and storage costs, low contamination risks, and reasonably high product quality and scale-up capacity, compared to mammalian cells, bacteria, yeast and transgenic-animal based systems (Table 1). Numerous platforms have been utilized to produce Recombinant Proteins (RP) from plants, such as leafy crops, cereal and legume seeds, oilseeds, fruits, vegetables, cell cultures, algae and aquatic plants (Twyman et al., 2003; Fischer et al., 2004). Producing RP in seeds is particularly important, as they are the evolutionary packages of storage proteins in a stable environment. In addition, antibodies accumulate at high levels in seeds and remain functional for several years (Stoger et al., 2002). Such effective storage is due to the desiccated nature of mature seeds. The rich combinations of molecular chaperones and disulfide isomerases ensure appropriate protein folding in the expanding seeds, at the absence of proteases (Muntz, 1998). As a result of the limited size of seeds, their RPs are found in high concentrations, despite the lower yield than expected of biomassive leafy crops, such as tobacco and alfalfa. This property is, however, beneficial for extraction and downstream processing. The relatively simple seed proteome (less competing proteins) along with low levels of the substances known to interfere with downstream processing steps (e.g., phenolics and alkaloids in tobacco leaves and oxalic acids in alfalfa) further facilitate processing. Oil drops in safflower and rapeseed are additional advantages and can be exploited during the initial purification stages (Moloney et al., 2003). Moreover, seed proteins do not usually interfere with plant growth which limits adventitious contact with non-target organisms, such as biosphere microbes and leaf-consuming herbivores (Commandeur et al., 2003).

Most recently, steps have been taken in development of plant-made vaccines for veterinary purposes. Enterotoxigenic E. coli can cause a variety of diseases in livestock, including Newcastle disease, foot-and-mouth disease and diarrheal diseases. Appropriate plant production platforms are being developed and investigated as for plant species and processing and transformation technologies in producing widely effective and well-preserved vaccines (Ling et al., 2010). However, commercial and environmental challenges are to be overcome before such vaccines can gain legislative approvals for regional, continental and worldwide uses.

Medicinal perspectives of legumes biofarming: The plant-based production of commercial biopharmaceutical recombinant proteins is usually named ‘molecular farming’. Plants can be engineered using a multitude of methods including nuclear transformation, plastid engineering, viral based transient expression, agroinfiltration and magnifection techniques to act as more effective, minimally-perishable and economical vehicles and platforms for protein and vaccine production (Kumar et al., 2007). A variety of grain legumes have been used to express RPs. However, two species of pea and soybean have been the generally explored production hosts. Despite a relatively low annual grain yield than cereals of mainly maize and rice, soybean’s main advantage is its high seed protein content. However, soybean’s transformation procedures are time-consuming. Limited molecular soybean farmings are reported (Philip et al., 2001; Zeitlin et al., 1998). Zeitlin et al. (1998) produced a humanized antibody against herpes simplex virus which was conducted in the plant rather than seeds alone. In the study of Philip et al. (2001), casein was expressed in soybean seeds using a lectin expression cassette. Another practical possible issue with soybean is its abundant oil content that might interfere with downstream processing steps.

Pea is similar to soybean in annual grain yield and seed protein. As a result, pea is similarly regarded as a suitable platform for reasonably high RP yields per hectare. However, pea costs could be around 50% more than soybeans, thus, necessitating greater seed yield to make pea a viable economical techno-plant. The major pharmaceutical proteins expressed in peas include single-chain antibodies (Perrin et al., 2000). An example is an antibody expressed at low levels to act against a cancer antigen as controlled by a seed-specific legumin A promoter (Perrin et al., 2000). Another was an antibody expressed under ‘Unknown Seed Proteins’ (USP) promoter, reaching 2% of total seed proteins (Saalbach et al., 2001).

Antibody generation biotechnologies: Many crops can now be used to produce antibodies. These include tobacco (Nicotiana tabaccum and N. benthamiana), cereals grains (rice, wheat, maize), legume seeds (pea, soybean, alfalfa) and fruit and root crops (tomato, potato) (Schillberg et al., 2002). Several critical factors must be taken into account in making most appropriate choices (Kusnadi et al., 1997). Leafy crops such as tobacco and alfalfa have very high biomass yields per hectare, because they are harvested multiple times a year. Tomatoes also have a high biomass yield but with increased production costs of greenhouses. Greenhouses, on the other hand, enable better containment compared with field management (Stoger et al., 2002).

For biovaluable proteins such as antibodies, more expensive production systems are justified to secure the many advantages. For example, antibodies expressed in potato tubers and cereal grains are stable at room temperature for months or years without stability losses. However, tobacco leaves must be dried or frozen before transport or storage to maintain RP activities. Protein extraction from seeds is more costly than from watery tissue, such as tomatoes. A majority of cost in the commercial production of RP including antibodies in plants belongs to downstream processing. Toxic metabolites such as tobacco alkaloids are a limitation. Thus, more edible plants without such toxins would preferably serve as expression hosts.

Alfalfa and soybean are the main legumes for RP production which usually produce less leaf biomass than tobacco but are efficient in utilizing atmospheric N through N fixation (Zeitlin et al., 1998). Thus, they require less chemical inputs (Kapusta et al., 1999; Khoudi et al., 1999). As an advantage for alfalfa, recombinant antibodies are produced as a single glycoform instead of heterogeneous sets of different glycoforms as found in other plants (Dixon and Sumner, 2003). In addition, grain legumes are beneficial production crops due to their high seed protein contents. For instance, despite low yield, peas are being developed as production platforms (Perrin et al., 2000).

Estrogenic nature of legumes: Extracts of kudzu root (Pueraria lobata L.) and red clover blossom (Trifolium pratense L.) may competitively bind to estrogen receptor α (ERα). When estrogenic activity was determined using an estrogen-dependent MCF-7 breast cancer cell proliferation assay, kudzu root, red clover blossom and sprout, mung bean sprout (Vigna radiata L.) and alfalfa sprout extracts (Medicago sativa L.) displayed increased cell proliferation above levels observed with estradiol. All the above extracts have been shown to exhibit agonist activity toward ERα. The active components in kudzu root extract were isoflavones puerarin, daidzin, genistin, daidzein and genistein. Thus, several legumes possess phytoestrogens with high estrogenic activity.

Alkaloids and nonprotein amino acids (NAA): Within the approximately 650 genera and >18,000 legumes species, quinolizidine (characteristic of Lupinus species; Fig. 3), dipiperidine, pyrrolizidine, carboline, phenylethylamine and indole alkaloids are the present alkaloids. The Tyr-derived Erythrina alkaloids may be found only in the large genus Erythrina. The NAAs are common within the Leguminosae, with canavanine, pipecolic acid and djencolic acid derivatives as the most important groups. The NAAs are often highly toxic and cause serious human toxicoses, among which is lathyrism, a nonprogressive motor neuron disease due to high intakes of grasspeas (Hanbury et al., 2000). In the 5th century BC, an irreversible leg weakness was reported in ancient cities inhabitants at times of war and starvation, when people had to eat high pulses diets. Grasspeas are ideally planted in arid regions, such as Ethiopia and the Indian subcontinent. Grasspeas seeds contain high levels of 3-N-oxalyl-L-2,3-diaminopropanoic acid (ODPA) (Fig. 3) which is responsible for several neurological symptoms and bone malformation, particularly in children. Although low-ODPA grasspeas lines have been developed via traditional breeding to better serve animals as healthier feeds, seed neurotoxins removal by transgenic approaches has not yet been effectively developed (Hanbury et al., 2000).

Health implications of leguminous isoflavonoids: Flavonoids are abundant in plants, whilst isoflavonoids are rather restricted. Isoflavonoids are prevalent in the Papilionoideae subfamily of the Leguminosae which act as antimicrobial, allelopathic or anti-insect and induce nodulation genes of symbiotic Rhizobium bacteria (Dixon, 1999). Pterocarpan-type phytoalexins such as medicarpin and constitutive isoflavone malonyl glycosides (Fig. 3) are typical isoflavonoids. Temporal and spatial correlations exist between phytoalexin accumulation and disease resistance in legumes. Isoflavonoids’ roles in disease resistance have recently been verified using genetic approaches (Dixon, 2001).

Isoflavonoids originate from flavanones via a rare aryl relocation catalyzed by the cytochrome P450 enzyme CYP93C1 (2-hydroxyisoflavanone synthase, or Isoflavone Synthase (IFS) (Jung et al., 2000). Apparently, the IFS gene has been evolutionary expressed in taxonomically distinct families. In addition to their occurrence in papilionoid legumes, isoflavonoids exist in some members of other families, including the Rosaceae, Chenopodiaceae, Apocynaceae and Pinaceae. Isoflavones possess estrogenic, antiangiogenic, antioxidant and anticancer activities (Dixon and Ferreira, 2000). They are now becoming accepted as dietary supplements (Palevitz, 2000). Genistein (Fig. 3) has been a subject of >3,500 published reports over the last two decades. Major sources of isoflavones for humans are soybean (daidzein and genistein) and chickpea (biochanin A, Fig. 3) seeds. Isoflavones seem most likely to be responsible for the health-promoting properties of soy-enriched diets (Lamartiniere, 2000; Palevitz, 2000).

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Fig. 3: Biochemical diversities of natural products in selected eight legume species. The compounds include (1) Seed coat proanthocyanidin in alfalfa (Medicago sativa), (2) Formononetin malonyl glucoside, an isoflavone conjugate of alfalfa and barrel medic (Medicago truncatula) roots, (3) Genistein, an isoflavone in seeds of soybean (Glycine max), (4) Avicin D, a complex triterpene saponin in seed pods of Acacia victoriae, (5) isoliquiritigenin, a chalcone in roots of licorice (Glycyrrhiza glabra), (6) Glycyrrhizin, a triterpene saponin from roots of licorice, (7) medicagenic acid glucoside, a triterpene saponin in roots of alfalfa and barrel medic, (8) Medicarpin, a pterocarpan phytoalexin in fungally infected barrel medic and alfalfa, (9) A prenylated isoflavone (lupinol A) from roots of Lupinus specie’s, (10) Lupanine, a quinolizidine alkaloid in roots of Lupinus species, (11) 3-N-oxalyl-L-2,3-diaminopropanoic acid (ODPA) from seed of grass pea (Lathyrus sativus) and (12) biochanin A, an isoflavone in seeds of chickpea (Cicer arietinum)

Soy isoflavones intake has been associated with reduced risks of breast and prostate cancers as well (Lamartiniere, 2000; Setchell and Cassidy, 1999). Isoflavones may additionally have preventive effects on osteoporosis and bone strength (Adlercreutz, 1998).

Genomics and metabolomics of legumes’ natural products: The development and expansion of large-scale genome and Expressed Sequence Tag (EST) projects have furthered natural products biosynthesis scales. Extensive DNA sequence resources are now widely available for soybean. The sequences of the many genes encoding enzymes of such products biosynthesis exist in the related databases. A main question is how to identify relevant genes. A potential answer is to apply functional genomics and metabolomics tools to globally assess transcriptomes and the metabolomes. The extensive profiling of metabolites may be used to assess gene function and to inquire biological systems responses to external stimuli (Fiehn, 2002). This approach helps to qualitatively and quantitatively define chemical phenotypes of genetically or environmentally perturbed biological systems. No single technique may provide a thorough assessment of such complex metabolomes, particularly in light of the vast chemical diversity of natural products. Hence, multiple approaches must be utilized. These include Thin-Layer Chromatography (TLC), infrared spectroscopy, NMR, Gas Chromatography-Mass Spectrometry (GC-MS), liquid chromatography with UV or MS detection, liquid chromatography-MS-capillary electrophoresis and capillary electrophoresis-MS (Fiehn, 2002; Sumner et al., 2002). The optimum techniques for chemical profiling of flavonoids and isoflavonoids involve HPLC for separation, alongside UV absorption and mass selective detection (Liu et al., 2002).

Specific protocols for a routine profiling of the flavonoid and isoflavonoid from different legume species have been developed. These include red clover (Trifolium pratense), soybean and lupin with high prenylated isoflavonoids (Fig. 3) (Bednarek et al., 2001; Lin et al., 2000). Saponins have poor chromophores. Consequently, the optimum approach for profiling the triterpene glycoside of legumes like alfalfa and barrel medic is reversed-phase HPLC coupled with electrospray-ionization MS (Dixon and Sumner, 2003). The legume barrel medic possesses more complex combinations of triterpenes compared to that studied in alfalfa (Nikkhah, 2012a, b). Therefore, the biological system can be a particular species, set of ecotypes, or groups of species. In addition and instead, inducible systems such as elicited roots or cell cultures may be utilized. As per triterpenes, cell suspension cultures added with methyl jasmonate induce several compounds and related biosynthetic enzymes. Depending on the metabolite profiles and present knowledge, a tentative pathway is proposed, which includes P450- and GT-catalyzed reactions for triterpene saponins (Suzuki et al., 2002). Next, cDNA libraries are made from plant tissues with active chemistry, followed by high-throughput EST sequencing (Suzuki et al., 2002). For instance, in barrel medic, there are >250 expressed cytochrome P450s and almost 300 expressed GTs, based on EST counting of >30 cDNA libraries sequenced thus far.

Bioinformatics methodologies, namely self-organizing maps for in silico expression analysis of EST libraries should be coupled with transcripts DNA array analysis from chemical producers and nonproducers. These help to identify candidate genes from a group of about 600 candidate P450s and GTs. The number of candidates addressed (e.g., 20 of each class) (Dixon and Sumner, 2003) would be adequately limited for direct expression studies in heterologous platforms, such as Escherichia coli (E. coli) and yeasts. With more complicated pathways at the absence of intermediates, multiple methods involving stable or transient down-regulation of candidate genes combined with metabolite profiling may be a necessity (Dixon and Sumner, 2003).

Plant pharmaceuticals: Modern and conventional approaches: In ancient regions, traditional herbal medicines have long served human healthcare. Plant-structured pharmaceutical platform can link agricultural sciences to medicinal sciences and lead agriculture based economies to gradually improve human and animal healthcare and welfare standards. As such, potential exists for a cost-effective generation of molecules that aid in controlling infectious diseases, particularly HIV-AIDS. The cost and logistics are among the key impediments in making and supplying highly sensitive vaccines to vast populations in different remote world regions, especially if lacking standard transportation and storage facilities. These innovative proteins may be carried by fruit juice and sugarcane and cereal millings (Ma et al., 2005b). Thus, appropriate facilities for extraction, preparation, storage and transportation should more effectively enable grater profits from plant-derived proteins in regions with suboptimal infrastructures. Targeting recombinant proteins in seeds and using high-volume economic food-processing technologies (e.g., freeze drying) can improve efficiency from production to delivery (Ma et al., 2003, 2005b).

Biosafety and bioethics are amongst the key concerns in adopting plant-derived pharmaceutical proteins technologies in different world regions. Although crop plants are the proven expression systems for novel proteins production (Streatfield et al., 2003; Twyman et al., 2005), specific designated regulations for various proteins will contribute to creating food and environmental contaminations risks. Doubtful and in some instances negative public impressions of world regions may unfavorably affect decision-making in adopting such new biotechnologies. From a global market standpoint, agricultural products are imported in considerable amounts from emerging markets to other regions. As such, the consumer’s ongoing concerns of Genetically-Modified (GM) contaminated foods have placed product labeling in great demands. Many consumers even tend to accept premium payment for GM-free products. Overall, early successful adoption of these emerging technologies depends on international trades.

Nonetheless, some non-western countries are eager in using GM crops (James, 2004). These countries aim to become leaders in adopting plant biopharmaceutical manufacturing technologies, having possessed the adequately advanced infrastructure and lawmaking capacities for biotechnology development and adoption. China, India, Brazil, Argentina, Iran, South Africa and Cuba are among the major countries. South Africa has recently increased adoption of GM biotechnologies by enhancing public awareness of the investment. This has encouraged similar initiatives in African countries, such as Kenya. Production of foot-mouth disease vaccines in Argentina (Dus-Santos et al., 2005), rabies antibodies in India (Lamphear et al., 2002), hepatitis B virus monoclonal antibodies in Cuba (Pujol et al., 2005) and human papillomavirus antigens in South Africa (Rose et al., 1999) have been reported. As such, emphases can be placed on regionally important diseases in other countries. Several plant-origin clinical biopharmaceutical proteins with their designated medical applications are presented in Table 2.

Research and commercial perspectives for legumes: Plant-derived biopharmaceuticals will be required to follow safety standards. However, since many herbal medicines are classified as nutritional supplements, they are not closely scrutinized for such standards. Should biopharmaceuticals be considered potentially detrimental, persist in the environment and accumulate in non-target organisms, precautionary measures are a must. Induction of biopharmaceutical production after harvesting could be one approach to minimize environmental exposure, only ensuring that viral vectors do not cause other regulatory concerns (Daniell et al., 2001). Activation treatment of potentially harmful proteins after expression is another approach to minimize risks of exposure.

Table 2: Plant-origin pharmaceutical proteins developed clinically with designated medical applications
Image for - Legumes Biofarming and Biopharmaceutical Sciences: A Review
Source: Tacket et al. (1998, 2000, 2004), Kapusta et al. (1999), Ma et al. (1998), McCormick (1999), Yusibov et al. (2002), Richter et al. (2000)

For example, hirudin is produced as a fusion protein that is inactive. Hirudin is activated only after purification from seeds (Cramer et al., 1999). Another environmental concern is the possible outcrossing of transgenic pollen to weeds or crops (Daniell, 1999a, b). Expression of dangerous pharmaceutical proteins in non-target plants due to such outcrosses could create public concerns and negative perceptions. Several gene suppression methods are currently being studied that include apomixis, incompatible genomes, transgenic mitigation, seed dormancy or shattering control, suicide genes, infertility barriers, male sterility and maternal inheritance (Daniell et al., 2001).

Foreign genes engineering via chloroplast genomes could suppress transgenes effectively. An exception is the chloroplast genome that shows biparental inheritance (e.g., pines, Daniell et al., 1998). As a different strategy, RNAse genes can be expressed under the control of a tissue-specific promoter to destroy the tapetum selectively during anther development, resulting in male sterile plants (Mariani et al., 1990). Another concern is the expression of destructive proteins in transgenic pollen. For instance, the controversial toxic effects of Bacillus thuringiensis (Bt) corn pollen on milkweeds (Asclepias spp.) fed to monarch butterfly larvae impacted on public perception, despite questioning the validity of the study. A solution could be engineering of biopharmaceuticals via chloroplast genomes. Although the Cry protein of Bt was expressed in leaves (up to 47% of total soluble protein), no toxicity occurred when milkweeds with transgenic pollen were fed to monarch butterfly larvae (Cramer et al., 1999). To date, chloroplast genetic engineering has been conducted in tobacco and potato. Several recent laboratories have tried to extend this technology to other crops. Reports are scarce on the production of glycoproteins in transgenic chloroplasts. Additional public concerns are antibiotic resistance genes and products in the edible parts of genetically modified crops. Now, several approaches are available to generate transgenic plants in their nuclear or chloroplast genomes without antibiotic selections (Daniell et al., 2001; Richter et al., 2000).

Making most suitable choices of biopharmaceutical proteins and their production crops is based on yield, storage conditions, control properties, initial set up and running costs, purification methods, market size, environmental concerns, public perception and competing technologies. Access to multiple alternative approaches in optimizing plant protein synthesis in environmentally friendly manners could promise a safe generation of biopharmaceuticals in transgenic plants (Fig. 4).

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Fig. 4: Transgenic plant vaccines production process

Consequently, such plants and resulting proteins will become more available to people and animals in demand of the products.

Alfalfa biofarming technologies: Alfalfa (Medicago sativa L.) is a perennial herbaceous crop that is also known as lucerne. Alfalfa is a most extensively planted crop worldwide and a major forage fed to livestock as hay, silage and pasture. Alfalfa has a large dry biomass yield, high protein, minerals and vitamins content and a vast capacity to fix N and enrich soils. These reduce requirements for fertilization and improve growth conditions for other plants. Improvement of alfalfa genetics, such as resistance to abiotic and biotic stress, protein quality and digestibility, may increase alfalfa yield and nutritional value (Vlahova et al., 2005). With high regeneration ability, the fodder alfalfa is suitable for genetic modification via modern approaches. Agrobacterium-mediated gene transfer has been used for transformation of embryogenic alfalfa genotypes (Ninkovic et al., 1995; Pezzotti et al., 1991) with efficiency depending on genotype (Du et al., 1994). In addition, a direct gene transfer has been applied (Vlahova et al., 2005). Alfalfa is currently being used by the Canadian company Medicago Inc. to develop multiple-cut, glasshouse production facilities (www.medicago.com). Alfalfa use in the field may be discouraged by present regulatory guidelines on open-pollinated species with compatible weedy relatives (Sparrow et al., 2007).

In 1986 tobacco and sunflower calluses were first used to express recombinant human growth hormone. Thus far, diverse plant systems have been utilized to produce pharmaceuticals (Vlahova et al., 2005; Fischer and Emans, 2000). Moreover, biotechnological advances have furthered benefits of pharmaceuticals in alfalfa. Expression cassettes are being optimized for protein expression in alfalfa leaves. New methods have been renovated to express transient proteins. For instance, it has become feasible to use agro-infiltration or protoplasts transformation for early demonstration and validation. Alfalfa has a unique ability to produce recombinant glycoproteins with uniform and homogenous glycosylation patterns. Alfalfa-derived C5-1 is a diagnostic anti-human IgG that was developed in Canada for phenotyping and cross-matching red blood cells for blood bank donors and recipients (Laurent et al., 1993). The expression of ‘biopharming’ represents a cost-effective and reasonably safe alternate for antibody production (Yoshida et al., 2004).

Alfalfa and recombinant molecules synthesis: Alfalfa may be harvested up to 8-10 times annually in greenhouses with minimum equipment (Peterson and Arntzen, 2004). A 10-ha greenhouse of well-matured alfalfa could produce approximately 340 tons of fresh biomass plant per harvest. Thus, harvesting 8 times a year will yield about 2700 tons of alfalfa. To ease harvest and machinery management throughout the year, grounds are divided into several smaller sections. Soluble proteins extraction of alfalfa tissue starts with maceration in hammer mills to break plant cells and facilitate effective and rapid extraction of water and solubles. Subsequently, green juices are extracted after screw-pressing. With a press capacity of 400 kg h-1 plant biomass input, about 800 L h-1 extract will be produced, thus, extracting about 6500 L juice/harvest. Such a reasonable capacity is needed to avoid and minimize unnecessary delays from harvest until juice extraction. The soluble protein concentrations in alfalfa green juice are approximately 2%. Hence, a 10 ha greenhouse produces about 260 kg soluble proteins twice/week. With recombinant protein expression levels of 0.1 to 1% of soluble proteins, annual recombinant protein yield of a 10 ha greenhouse will be estimably about 26-260 kg.

Recombinant proteins can be purified form alfalfa extracts using several strategies, depending on protein application and the desired extent of purity. As for C5-1, two methods have been studied. The first is the Québec method (Laurent et al., 1993) purifying C5-1 from hybridoma cells, involving affinity chromatography with a human IgG1 that is a matching antigen for C5-1 (Bardor et al., 2003). The other method is a bed affinity chromatography with staphylococcal protein A (Streamline rProteinA, Amersham Biosciences, Piscataway, NJ). This method is more suited for large-scale C5-1 generation. These large-capacity columns where liquids flow upward are loaded with unclarified green juices, binding up to 20 mg of human IgG mL-1 of medium. Protein A, however, is of minor affinity for mouse IgG. In pH of 9, about 5 mg C5-1 mL-1 of medium has been produced (D’Aoust et al., 2004). To improve purification, the expanded bed column may be coupled with streptococcal protein G, with greater affinity for mouse IgGs than for protein A. Greenhouses, despite significant yields of recombinant proteins, will not suffice should tons of proteins be required. Thus, field plants are a necessity. In such production scales, molecular farming combines beneficially with animal-feed industries. Wet fractionation (performed by Hall et al. (2002) in Champagne, France) in large-scale production systems can process about 750,000 tons of fresh alfalfa per year. During that process, temperature and pH are monitored to separate alfalfa proteins and isolate protein-rich fractions which are then dried into pellets for animal feed industries. Additionally, the above large-scale fractionation has been refined in France (Viridis, Aulnay-aux-Planches) to generate Rubisco that is an alfalfa-derived additive for human consumption.

Innovative alfalfa platforms for novel compounds production: Plant cells are considered cost-effective and potentially safe manufactures of recombinant proteins. Plant biopharming has greatly succeeded in producing many bioactive pharmaceuticals, including diagnostic monoclonal antibodies (Khoudi et al., 1999; Hiatt, 1990) and blood components (Magnuson et al., 1998). Genetically modified forages commonly fed to livestock are considered suitable for edible vaccines production (Tuboly et al., 2000). Transgenic alfalfa has been utilized to express the structural protein VP1 of Foot-and-Mouth Disease Virus (FMDV) and immunize mice (Wigdorovitz et al., 1999). As a bioreactor, transgenic alfalfa can produce commercial enzymes, in a process that is much cheaper than fermentation facilities construction (D’Aoust et al., 2004). Lactoferrin (Lf) is an iron-binding glycoprotein present in human colostral whey proteins. It is active against many pathogens. Thus, Lf is able to inhibit bacterial growth by medium iron sequestration during microbial metabolism. A direct antibacterial effect of Lf is related to its N-terminal region that can disrupt and penetrate bacterial cell membranes (Yamauchi et al., 1993). Also, Lf exhibits antiviral activities, being capable of suppressing human and animal RNA- and DNA-viruses replication via blocking of cell-virus interactions. Lactoferrin is a potent immunomodulator with anti-oxidant effects. It empowers cell defence systems by stimulating macrophages and lymphocytes proliferation and activity. Other functions include cytokine production, growth and likely anti-tumor activity. Lf reduces free radicals which may in turn reduce disease susceptibility Such antimicrobial and immune promoting effects of Lf have attracted research to develop expression systems for massive production of recombinant Lf. Lactoferrin has been expressed in Saccharomyces cerevisiae (Liang and Richardson, 1993) and several mammalian systems, including baby hamster cells (Stowel et al., 1991) and transgenic cows and mice (Nuijens et al., 1997). Lactoferrin was also produced in transgenic plants at 0.3% of total cellular protein (Salmon et al., 1998).

Glycosylation studies suggest that alfalfa has the capability of producing recombinant glycoproteins with homogenous glycosylation patterns. Efforts have been made to produce human Lf in transgenic alfalfa plants. Barbulova et al. (2002) used the highly embryogenic clone R4 from a commercial Bulgarian cultivar Obnova 10. Agrobacterium tumefaciens-based gene system has been utilized to transform alfalfa with a binary vector to carry human Lf cDNA (Vlahova et al., 2005). Moreover, non-embryogenic alfalfa variety of Furez has produced recombinant human Lf from cell cultures with the Agrobacterium tumefaciens-based gene transfer system. Research continues for further development of fast growing transgenic suspension cultures (Vlahova et al., 2005).

Human clinical studies with plant-derived vaccines suggest steps forward (Yusibov et al., 2002). Transgenic spinach expressing epitopes of rabies virus glycoprotein (G protein) and nucleoprotein (N protein) were fused to Alfalfa Mosaic Virus (AlMV) coat protein and were orally administered to 14 individuals. Five of the people were previously immunized with a conventional rabies vaccine. Three of those 5 and 5 of the 9 initially naive individuals exhibited specific antibody responses to the rabies virus, whereas none in the control group showed increases in rabies-specific antibodies. A week after completion of the feeding regime, the 9 initially naive people were dosed with the conventional rabies virus vaccine. Three of them showed rabies virus neutralizing antibodies but none of the 5 controls did so. Despite detecting no neutralizing antibodies before immunization with the commercial vaccine, potential for the plant-produced rabies vaccine to complement oral vaccinations was logically evident (Walmsley and Arntzen, 2003; Yusibov et al., 2002).

Recombinant immunoglobulins from alfalfa: Doses of recombinant IgG as pharmaceutical are already developed or under clinical investigation (Bardor et al., 2003; Raju, 2003). Due to increased commercial interests in IgGs, conventional cell culture production systems do not meet demands (Fig. 1). As a result, there have been demands for alternative production methods (Fig. 1, 2). Whole transgenic plants and animals are engineered to generate greater recombinant IgG. In such a shift from a production system to another, key product criteria must be carefully monitored to comply with stringent safety and efficiency requirements through highly regulated clinical experiments. Another key issue is the high batch-to-batch reproducibility which requires high products homogeneity. Heterogeneity for IgG stems from variable maturation of N-glycans, affecting IgG products quality. For instance, the US Food and Drug Association has made efforts in improving the homogeneity of N-glycosylation in recombinant IgGs for pharmaceuticals production.

N-linked glycans in alfalfa may be processed into several mature oligosaccharides with core-xylose and core α(1,3)-fucose and also terminal Lewis (a) epitopes. The C5-1 monoclonal antibody expression in alfalfa has produced plant-derived IgG1 that is N-glycosylated by a prime glycan with a α(1,3)-fucose and a β(1,2)-xylose attached to a GlcNAc2Man3GlcNAc2 core. Since the core is widespread in plant and mammal N-linked glycans, alfalfa plants appear to possess capacities to produce recombinant IgG1 with a N-glycosylation. The latter fits well for in vitro and in vivo glycan refinement into human suited plantibodies. In vitro galactosylation of the alfalfa-derived C5-1 mAb has led to a homogenous plantibody with terminal β(1,4)-galactoses similar to mammalian IgGs (Bardor et al., 2003).

Legumes of especially alfalfa contain Saponins with possible anticarcinogenic implications. Saponins possess an amphiphilic chemical structure that makes them surface-active. Their anticarcinogenic effects are likely shown via direct cytotoxicity, immune modulation, bile acid binding, and cell proliferation normalization. Soybeans are amongst the most viable human dietary saponins because of being key protein supplier. Non-dietary sources include alfalfa, sunflower, horse-chestnut and many others (Price et al., 1987). The structure of saponins differs considerably based on sugars amount, type and steroid sphere composition. Fecal biliary excretion has been increased by alfalfa seeds ingestion (Malinow et al., 1979), suggesting that saponins may reduce bile acids conversion to secondary acids by intestinal microflora and thus, may help in preventing colon cancers (Rao and Sung, 1995). From a cardiovascular perspective, total circulating blood cholesterol was lower in rats fed diets based on plant proteins of alfalfa, fava, gluten, pea and soy compared to those fed animal proteins of casein, lactalbumin and ovalbumin (Sautier et al., 1986). This cholesterol-lowering effect is at least partially and possibly mediated through modulations of hepatic LDL receptors (Friedman and Brandon, 2001).

Alfalfa possesses estrogenic activities. Estrogenic activity has been determined using an estrogen-dependent MCF-7 breast cancer cell proliferation assay (Bouea et al., 2003). Kudzu root, red clover blossom and sprout, mung bean sprout, and alfalfa sprout extracts increase cell proliferation above levels caused by estradiol. The pure estrogen antagonist, ICI 182,780, suppressed cell proliferation induced by the extracts, suggesting that an Estrogen Receptor (ER) related signaling pathway was involved. The ER subtype-selective activities of legume extracts were examined using transiently transfected human embryonic kidney (HEK 293) cells. All seven of the extracts exhibited preferential agonist activity toward Erβ. Using HPLC to collect fractions and MCF-7 cell proliferation, the active components in kudzu root extract were determined to be the isoflavones puerarin, daidzin, genistin, daidzein and genistein. These results show that several legumes are a source of phytoestrogens with high estrogenic activity (Bouea et al., 2003).

Plant-derived edible vaccines aim to protect humans and animals against infectious diseases, autoimmune diseases and various tumors (Jelaska et al., 2005). Figure 4 shows edible vaccines production process using transgenic plant platforms (Fig. 4). Transgenic plants including alfalfa, potatoes, tomatoes, maize, rice and soybeans have been used in vaccine development. Human studies with transgenic plants have created promising data with no or minor safety concerns (Streatfield, 2006). As per livestock, animals have been fed transgenic plants (e.g., Arabidopsis thaliana, alfalfa and potato with antigens) to protect against foot-and-mouth disease virus, BRV and bovine viral diarrhea virus (Streatfield, 2006). An Indian study has focused on Rinderpest disease in the Middle East, Asia and Africa (Rice et al., 2005). Pigeon peas have been fed to express protective viral hemagglutinin antigens in livestock. Leukotoxin (Lkt) has been expressed recently in alfalfa and administered as edible vaccines to attenuate stress and help to prevent diseases in animals (Rice et al., 2005). Eating foods produced from such plant-empowered animals will additionally provide similar health benefits as do original plants. Storage and transportation costs of vaccines and related facilities are saved, making the systems highly inexpensive. A commercial case that has received much concern is ‘mad cow disease’. Prevention of such health issues is central to the economic value of many livestock and food companies worldwide.

Alfalfa is considered a natural laxative, a natural diuretic, an antifungal, a liver detoxifier, a cure for kidney stones and urinary infections and a promoter of pituitary gland function. Alfalfa is rich in vitamins and minerals and is a major livestock forage feed worldwide. Alfalfa seeds contain amino acid L-canavanine which is involved in pancytopenia occurrence in humans and systemic lupus erythematosus induction in monkeys. It can cause allergies as well (Williamson and Wyandt, 1997).

New promoters for high-level protein expression have been developed in alfalfa leaves using alfalfa cell-culture and transient-expression technologies (D’Aoust et al., 2004). Amongst alfalfa’s advantages are high biomass yield, nitrogen fixing, soil nutrient enrichment and homogeneous glycan structures of the glycoproteins synthesised in alfalfa leaves (Fischer et al., 2004; Daniell and Edwards, 1995). The latter is necessary for batch-to-batch consistency. On the other hand, alfalfa leaves contain much oxalic acid that could interfere with optimal biofarming processing (Fischer et al., 2004).

Properties of plant-made vaccines
Major benefits:

The plants that produce edible vaccines can be widely grown in countries with developing and emerging markets
Plants are routinely utilized in pharmaceuticals production with approved purification and processing protocols
Plant growth process is inexpensive and both body-and mind-friendly
Plants may not have and generate human pathogens. As a result, the resulting vaccines will not have major safety issues for humans
Plant transportation is economically and commercially feasible worldwide

Highlighted disadvantages:

Plants are the living dynamic organisms. Thus, any biological changes that usually occur over time can severely alter vaccine production quantity and quality
Edible vaccines are very likely to be mistaken with normal fruits, and thus, may be consumed more than formulated. Thus, education on public eating behaviors and patterns are a necessity to ensure adequate safety
Vaccines dosages may well vary, thus making intake guidelines development more challenging. For example, bananas or legume tablets of different sizes with different vaccines doses will not have similar public and medicinal implications
For field and garden vaccines development, security is always an issue
Glycosylation patterns between plants and humans differ which could affect vaccine functionality and future status

Prospectus public and industrial trends of plant-animal-human biotechnologies: Plant-produced vaccines will probably not face outcome 2 as illustrated in Fig. 5. A more realistic outcome would be that such vaccines possess gainful outcomes, although the extent of success and public acceptance is still not quite known. Certainly, given the safety, quality, security and consistency challenges, the real world curve can hardly reach its fundamental and promising status of 2002. It is obtaining pervasive community acceptance that some minimal degree of high-quality manufacturing for genetically-modified products is required in vaccine production. It is also becoming more favorable to believe in functioning of injectable vaccines. Because the first licensed plant-derived pharmaceutical recombinant proteins are antibodies including a poultry vaccine, a similar trend is more likely to follow. Regulation-wise, quality control pathways should be shortcut for the products that are intended for non-humans usage. The most feasibly manufacturable products in the new time include high-merit proteins for diagnostics purposes and/or kits for which no hurdles of clinical trials would be needed. In addition, their required volumes are rather low. For instance, Farmacule BioIndustries in Queensland, Australia, have recently produced vitronectin protein in plants which is derived from animal serum and costs up to 5 million $US/g.

Image for - Legumes Biofarming and Biopharmaceutical Sciences: A Review
Fig. 5: The ‘hype curve’ tracks public perception and attitudes of plant-derived vaccines over time. Favorable publicity up until around 2002 fuelled mainly by edible vaccines ideas. However, fears of food chain contaminations existed, followed by worries of regulatory problems and thus no success for further development. Outcome 1 represents the perspectives discusses. Outcome 2 represents a future when industries and governments will not properly employ and fund the technology

Farmacule can produce up to 1 g/month vitronectin to meet world demands (O’Neill, 2005). This suggests that an idealistic vision of plant-derived vaccines for people with inadequate financial status may still be far away from reality. Commercial exploitation of lower-hanging fruits for cheaper production and processing may improve the future industrial and public standings of these new biotechnologies. It is persuasive to realize the recent focus of a large tobacco company investing in another company (i.e., Medicago Inc., of Canada), using alfalfa and tobacco to produce a variety of vaccines. These include seasonal and H5N1 influenza vaccines (Medicago, 2008). All in all, it is foreseen that the technology at first can gain approval in manufacturing animal vaccines. Later on, by demonstrating sufficient safety, the technology may create further interest in human medicine. From a commercial investment viewpoint, at the beginning the technology is being rather hosted by smaller companies and more independent institutes. Involvement of larger philanthropic companies and investors could be possible by taking global initiatives on more threatening diseases, such as HIV. This can attract greater governmental interests also. Despite the fact that vaccines such as Hepatitis B Virus (HBV) have been greatly targeted, it is not likely for these to be the first products. The existing generic, licensed vaccines are already very inexpensive. More probably, new types of vaccines such as hepatitis and possibly rotavirus vaccines will be among those preferably targeted since they are currently very costly. Later on inexpensive heat-stable, suspension oral vaccines may receive more consideration (Rybicki, 2009). All of these are more critical where many preventable diseases challenge animal and human life, where present vaccines are expensive, where no vaccines exist for ‘orphan diseases’ and where antibody therapy can effectively treat prevalent diseases.

CONCLUSIONS AND IMPLICATIONS

The emerging biotechnologies of mainly plant-origin and legume-derived pharmaceutical proteins production encounter real challenges. The biotechnologists and their supporting sciences and industries should convince lawmaking and ethical organizations and the major investing companies in the economical and viable nature of the technologies compared to those established previously. Careful monitoring and utilization of the initial products are a must for compelling validation of the methods and regulatory frameworks. As such, the new proteins in forms of vaccines and antibodies have the real opportunity to improve health and life quality of large human and animal populations. Due to the presence of a multitude of bioactive substances in legumes, further studies particularly involving nutrigenomics and metabolomics are required to attribute and specify BPC effects on human health indicators. Quantitative and qualitative dietary inclusion guidelines for food and feed packages and preparations with legume origins may become feasible for different animal and human age groups. These accomplishments will contribute to forming healthy and viable plant-human-animal biotechnologies in the new era. Genetic engineering helps to produce biopharmaceuticals most favorable to human and animal health. Alfalfa is a multi-cut leguminous plant with high annual biomass yield. Alfalfa leaves and seeds are main targets of ‘biopharming’ or ‘biopharmaceuticals production of farming’. With increasing demands for Plant-Made Biopharmaceuticals (PMB), their production could be safer and cheaper than animal-derived counterparts. Alfalfa is an inexpensive platform for monoclonal antibodies, which are used as potential human therapeutics and diagnostics. Plant expression systems to produce human and animal vaccines possess many advantages. These include easily-established cultivation, inexpensive production, no or little demand for cold-chain supply, rapid scale-up, simple seed distribution, easy genetic manipulation, oral supply capacity and minor health concerns of human pathogen and toxin contaminations. Plant-derived antibodies have passed the early-stage clinical trials. It may well be feasible in the near future to sweep away diseases by eating tasty vegetables and fruits. However, the existing main concerns include biosafety, dosage uniformity, regulatory guidelines and possible survival of herbicides and pesticides. The human vaccine technology using plants may become feasible based on technical merits and approvals from public and social sectors. Future research on enhancing the expression of special genes is needed. It must be ensured that how and if transgenic plant vaccines meet quality standards (purity, potency, safety and efficacy) of the World Health Organization.

ACKNOWLEDGMENTS

The Ministry of Science, Research and Technology, and University of Zanjan, Iran, are acknowledged for supporting the author’s programs of optimizing science education in the new millennium.

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