**10. The inositols**

430 Recent Trends for Enhancing the Diversity and Quality of Soybean Products

exchange columns. Richmond et al. (1991) separated carbohydrates in dairy products. Henderson and Berry (2009) have utilized Zorbax columns for the separation of carbohydrates in Stevia sweetener. Romano (2007) discussed carbohydrate analysis in food products emphasizing column chemistries and detection. Several vendors offer LC columns for carbohydrate separation. Wilcox et al. (2001) also discussed several column types used for carbohydrate separation. Hydrophilic interaction chromatography (HILIC) has also been reported as a method for analyzing ionic or polar compounds, particularly biomolecules and drug metabolites (http://www.laboratoryequipment.com/article-is-hilic-in-your-futurect92.aspx). Simple carbohydrate separations can also be performed on functionalized silica or resin-based columns (http://www.labnews.co.uk/feature\_archive. php/4000/5/just-juice). The separation of mono- and oligosaccharides are also performed using capillary electrophoresis. Different formats are capillary zone electrophoresis (CZE), capillary isoelectric focusing (CIEF), capillary isotachophoresis (CITP), and micellar electrokinetic chromatography (MEKC). These techniques are summarized in a review by Thibault and

**9. Liquid chromatography/mass spectrometry (LC/MS) and other MS** 

Efficient separation methods such as high performance liquid chromatography (HPLC) and capillary electrophoresis combined with detection methods (e.g. mass spectrometry) that supply structural or compositional information is a preferred tool for the analysis of biomolecules, particularly carbohydrates. Liquid chromatography/mass spectrometry with both electrospray (ESI) and atmospheric pressure ionization (APCI) has spurred a major

In ESI , the liquid containing the analyte(s) of interest is dispersed into a fine aerosol. Because the ion formation involves extensive solvent evaporation, the typical solvents for electrospray ionization are prepared by mixing water with volatile organic compounds (e.g. methanol, acetonitrile). To decrease the initial droplet size, compounds that increase the conductivity (e.g. acetic acid) are customarily added to the solution. Large-flow electrosprays can benefit from additional nebulization by an inert gas such as nitrogen. The aerosol is sampled into the first vacuum stage of a mass spectrometer through a capillary, which can be heated to aid further solvent evaporation from the charged droplets. The ions observed by mass spectrometry may be quasimolecular ions created by the addition of a hydrogen ion and denoted [*M* + H]+, or of another cation such as sodium ion, [*M* + Na]+, or the removal of a proton, [*M* − H]−. Multiply-charged ions such as [*M* + nH]n+ are often observed (Gaskell 1997). As examples, Fountain and Grumbach (2009) used negative ion electrospray mass spectrometry for the analysis of fructose, glucose, sucrose, and lactose. Taormina et al. (2007) and Mauri et al. (2002) used flow injection techniques with mass spectrometry. Fugimoto et al. (2005) used rubidium in the mobile phase as a complexing agent for both nuclear magnetic resonance and electrospray mass spectrometry analysis. Taylor et al. (2005) utilized ESI/MS to study fragmentation patterns of carbohydrates. Schlichtherle-Cerny et al. (2003) utilized a HILIC column coupled with ESI/MS for the analysis of amino acids, peptides, glycoconjugates, and organic acids in foods without prior

In APCI, typically the mobile phase containing eluting analyte is heated to relatively high temperatures (above 400 C), sprayed with high flow rates of nitrogen and the entire aerosol

Honda (2003).

**techniques** 

derivatization.

interest in the analysis of carbohydrates.

Inositols (Fig. 6) are polyols of cyclohexane with the empirical formula C6H12O6. There are potentially 9 stereoisomers of inositol but only five are naturally occurring (structure shown below). They are *myo*-inositol, *chiro*-inositol, *scyllo*-inositol, *muco*-inositol, and *neo*-inositol. Of these, *myo*-inositol is the precursor of the other four. *myo*-Inositol is synthesized from glucose.

The synthesis of *myo*-inositol uses the enzyme L-*myo*-inositol 1-phosphate synthase to catalyze the reaction which produces L-*myo*-inositol-1-phosphate from D-glucose 6 phosphate (Hoffmann-Ostenhof and Pittner, 1982). The L-*myo*-inositol-1-phosphate is then dephosphorylated through inositol monophosphate to produce *myo-*inositol (Loewus & Murthy, 2000). The enzyme that catalyzes this step is L-*myo*-inositol 1-phosphate synthase

Extraction and Analysis of Inositols and Other Carbohydrates from Soybean Plant Tissues 433

inositols (Larson & Raboy, 1999). Research with pinitol in soybean documents that this cyclitol is a major consitutent of soybean (Phillips & Smith, 1974; Streeter, 1980; Phillips, et al. 1982; Dougherty & Smith, 1982). Because pinitol diffuses faster than carbohydrates during imbibition, it is theorized that loss of pinitol from soybean seed encourages the growth of Bradyrhizobium (Rhizobium) species in the soil needed for nitrogen fixation (Nordin, 1984). Accumulation of ononitol and pintol in soybean and other plants under drought conditions has been documented (Streeter *et al*., 2001; Guo & Oosterhuis, 1997;

Inositols are very important in general plant growth, seed storage, nitrogen fixation and protection of plants during stress. Inositol metabolism and its role in photosynthesis, plant health, and subsequent potential increase in yield is complex but new discoveries in this area may lead to future yield improvements. The role of inositols in nitrogen fixation is also complex and not currently fully understood. Inositols play an important role in phosphorus movement in the environment. Efforts are being made to alter the phytate content of soybean so animals can use the phosphorus and also reduce the amount that is excreted as manure. There are implications here not only for animal health but also for the preservation

Phytate, *myo*-inositol hexakisphosphate, is found in almost all plant and animal cells and serves as an important phosphate reserve in plants (Irvine & Schell, 2001). Exposure of soybean cell suspension to *Psuedomonas syringae* pv *glycinea* indicated that whether a virulent or avirulent strain is used, the plant starts defense systems at the expense of housekeeping cell functions (Logemann *et al*., 1995; Shigaki & Bhattacharyya, 2000). Part of this defense reaction involves cellular cyosolic inositol and the IP3 pathway. This pathway is involved in cell division, growth and elongations and there is evidence that this pathway is inhibited when the plant is exposed to pathogens (Perera *et al*., 1999; Shigaki & Bhattacharyya, 2000). Selection of plants with reduced phytate levels raised the question of these plants' response to stress in the form of diseases. Murphy et al., 2008 found that disruption of phytate biosynthesis resulted in increased susceptibility in *Arabidopsis thaliana* to virus (potato virus Y), fungal (*Botrytis cinerea*) and bacterial (*Psueodomonas syringae*) diseases. The role of phytate in basal resistance to plant pathogens was previously unknown. Klink et al. (2009) found 1-phosphatidylinositol phosphodiesterase-related genes expressed when soybean plants are exposed to *Heterodera glycines*, a pathogen of soybean. The findings of inositols in plant defense are important findings and the next step is to determine whether the defense reaction is a general reaction or specific to different types of

Transgenic plants that release extracellular phytase from their roots have a significantly increased ability to acquire phosphorus from inositol phosphates from growth medium; however, there is less evidence that phosphorus nutrition of plants can be improved in plants grown in soil (George, *et al*., 2004). Phytate and phytic acid represent the major form of phosphorus in animal feed derived from plants. Phosphorus in seeds and tubers is stored primarily as phytate (*myo*-inositol exa*kis*phosphate), which is poorly digested by nonruminant animals such as swine, poultry and fish (Saghai Maroof *et al.*, 2009; Kim *et al*., 2006). The lack of the hydrolytic enzymes necessary for phytate to be utilized by these animals requires supplemental phosphate. Plant breeding efforts involve plant selections for improved phosphorus usage by animals and different feed additives resulting in less

Manchanda & Garg, 2008; Sheveleva, et al, 1997).

and sustainability of watersheds.

attacks.

environmental pollution.

(Stieglitz et al, 2005). The four other inositol isomers are derived from *myo*-inositol (Loewus and Murthy, 2000). The sequoyitol can then be epimerized to D-pinitol (See Fig. 7) which is demethylated to D-*chiro-*inositol using NADP-specific D-pinitol dehydrogenase (Stieglitz et al, 2005).

Fig. 6. Inositol.

Fig. 7. Pinitol.

In addition to the five stereoisomers of inositol, the *O*-methylinositols can also be synthesized from *myo*-inositol. Of these, ononitol and pinitol are common to soybeans. Ononitol is a precursor to pinitol in soybeans (Loewus and Murthy, 2000; Chiera et al. 2006). Of the *O*-methylinositols, pinitol is most abundant in soybeans.

*myo*-Inositol is probably the most studied of all the inositols because it is the most commonly available. It has a very important function as it is required in the formation of Lecithin, which protects cells from oxidation and is an important factor in the building of cell membranes. Inositol, also has a metabolic effect in preventing too much fat to be stored in the liver, which is why it is called a lipotropic and is a vital part in maintaining good health. Inositols have been found in many plants both foodstuff and other plants at varying evolutionary stages (Clements & Darnell, 1980; Chiera *et al*., 2006; Guo & Oosterhuis, 1997; Henry, 1976; Johansen *et al*., 1996; Johnson and Sussex, 1995; Johnson & Wang, 1996; Lind *et al*. 1998; Loewus *et al*., 1984; Manchanda and Garg, 2008; Ogunyemi *et al*., 1978; Phillips, et al 1982; Sheveleva *et al*., 1997; Streeter *et al*., 2001). Different soybean plant parts contain different levels of inositols as do soybean plants in vegetative verses reproductive growth stages (Phillips and Smith, 1974). Comparison of total inositols among plants should be examined carefully because each plant may produce different proportions of the various

(Stieglitz et al, 2005). The four other inositol isomers are derived from *myo*-inositol (Loewus and Murthy, 2000). The sequoyitol can then be epimerized to D-pinitol (See Fig. 7) which is demethylated to D-*chiro-*inositol using NADP-specific D-pinitol dehydrogenase (Stieglitz et

In addition to the five stereoisomers of inositol, the *O*-methylinositols can also be synthesized from *myo*-inositol. Of these, ononitol and pinitol are common to soybeans. Ononitol is a precursor to pinitol in soybeans (Loewus and Murthy, 2000; Chiera et al. 2006).

*myo*-Inositol is probably the most studied of all the inositols because it is the most commonly available. It has a very important function as it is required in the formation of Lecithin, which protects cells from oxidation and is an important factor in the building of cell membranes. Inositol, also has a metabolic effect in preventing too much fat to be stored in the liver, which is why it is called a lipotropic and is a vital part in maintaining good health. Inositols have been found in many plants both foodstuff and other plants at varying evolutionary stages (Clements & Darnell, 1980; Chiera *et al*., 2006; Guo & Oosterhuis, 1997; Henry, 1976; Johansen *et al*., 1996; Johnson and Sussex, 1995; Johnson & Wang, 1996; Lind *et al*. 1998; Loewus *et al*., 1984; Manchanda and Garg, 2008; Ogunyemi *et al*., 1978; Phillips, et al 1982; Sheveleva *et al*., 1997; Streeter *et al*., 2001). Different soybean plant parts contain different levels of inositols as do soybean plants in vegetative verses reproductive growth stages (Phillips and Smith, 1974). Comparison of total inositols among plants should be examined carefully because each plant may produce different proportions of the various

Of the *O*-methylinositols, pinitol is most abundant in soybeans.

al, 2005).

Fig. 6. Inositol.

Fig. 7. Pinitol.

inositols (Larson & Raboy, 1999). Research with pinitol in soybean documents that this cyclitol is a major consitutent of soybean (Phillips & Smith, 1974; Streeter, 1980; Phillips, et al. 1982; Dougherty & Smith, 1982). Because pinitol diffuses faster than carbohydrates during imbibition, it is theorized that loss of pinitol from soybean seed encourages the growth of Bradyrhizobium (Rhizobium) species in the soil needed for nitrogen fixation (Nordin, 1984). Accumulation of ononitol and pintol in soybean and other plants under drought conditions has been documented (Streeter *et al*., 2001; Guo & Oosterhuis, 1997; Manchanda & Garg, 2008; Sheveleva, et al, 1997).

Inositols are very important in general plant growth, seed storage, nitrogen fixation and protection of plants during stress. Inositol metabolism and its role in photosynthesis, plant health, and subsequent potential increase in yield is complex but new discoveries in this area may lead to future yield improvements. The role of inositols in nitrogen fixation is also complex and not currently fully understood. Inositols play an important role in phosphorus movement in the environment. Efforts are being made to alter the phytate content of soybean so animals can use the phosphorus and also reduce the amount that is excreted as manure. There are implications here not only for animal health but also for the preservation and sustainability of watersheds.

Phytate, *myo*-inositol hexakisphosphate, is found in almost all plant and animal cells and serves as an important phosphate reserve in plants (Irvine & Schell, 2001). Exposure of soybean cell suspension to *Psuedomonas syringae* pv *glycinea* indicated that whether a virulent or avirulent strain is used, the plant starts defense systems at the expense of housekeeping cell functions (Logemann *et al*., 1995; Shigaki & Bhattacharyya, 2000). Part of this defense reaction involves cellular cyosolic inositol and the IP3 pathway. This pathway is involved in cell division, growth and elongations and there is evidence that this pathway is inhibited when the plant is exposed to pathogens (Perera *et al*., 1999; Shigaki & Bhattacharyya, 2000). Selection of plants with reduced phytate levels raised the question of these plants' response to stress in the form of diseases. Murphy et al., 2008 found that disruption of phytate biosynthesis resulted in increased susceptibility in *Arabidopsis thaliana* to virus (potato virus Y), fungal (*Botrytis cinerea*) and bacterial (*Psueodomonas syringae*) diseases. The role of phytate in basal resistance to plant pathogens was previously unknown. Klink et al. (2009) found 1-phosphatidylinositol phosphodiesterase-related genes expressed when soybean plants are exposed to *Heterodera glycines*, a pathogen of soybean. The findings of inositols in plant defense are important findings and the next step is to determine whether the defense reaction is a general reaction or specific to different types of attacks.

Transgenic plants that release extracellular phytase from their roots have a significantly increased ability to acquire phosphorus from inositol phosphates from growth medium; however, there is less evidence that phosphorus nutrition of plants can be improved in plants grown in soil (George, *et al*., 2004). Phytate and phytic acid represent the major form of phosphorus in animal feed derived from plants. Phosphorus in seeds and tubers is stored primarily as phytate (*myo*-inositol exa*kis*phosphate), which is poorly digested by nonruminant animals such as swine, poultry and fish (Saghai Maroof *et al.*, 2009; Kim *et al*., 2006). The lack of the hydrolytic enzymes necessary for phytate to be utilized by these animals requires supplemental phosphate. Plant breeding efforts involve plant selections for improved phosphorus usage by animals and different feed additives resulting in less environmental pollution.

Extraction and Analysis of Inositols and Other Carbohydrates from Soybean Plant Tissues 435

It is clear from this review that there are many different tools to study the carbohydrates in soybean plants. Results from any of the various analytical methods can be compared as long as they have been tested with adequate standards. The outstanding carbohydrate found in soybeans is pinitol, part of the inositol family. There has been considerable research into the value of the inositols, but most of the emphasis has been on myo-inositol, probably because it is widely available. However, for the soybean industry, it would be valuable to better understand the role of pinitol in health and nutrition. There are good indications that pinitol may have unique nutritional value, and key roles in soybean plant biology. Future directions should include the use of effective analytical methods to perform more research into the roles of pinitol and related inositols in various fields of nutrition, medicine, and

We acknowledge Shaun Garland and Luther McDonald for their technical support.

MALDI/TOFMS matrix assisted laser desorption/time-of-flight mass spectrometry

Anderson, L. & Wolter, K.E. (1966). *Cyclitols in plants: Biochemistry and physiology*. Annual

Al-Hazmi, M.I. & Stauffer, K.R. ( 1986). Gas chromatographic determination of hydrolyzed sugars in commercial gums. *Journal of Food Science* 51:1091-1092, 1097. Anthony, R.M.; Nimmerjahn, F.; Ashline, D. J.; Reinhold, V. N.; Paulson, J. C. & Ravetch, J.

V. (2008). *Recapitulation of Intravenous Ig Anti-Inflammatory Activity with a* 

**11. Conclusions and future directions** 

plant biology.

**12. Acknowledgements** 

**13. List of abbreviations** 

IT ion trap

GC gas chromatography LC liquid chromatography MS mass spectrometry RI refractive index UV ultraviolet

ELS evaporative light scattering ESI electrospray ionization SPE solid phase extraction

QTOF quadrupole time-of-flight

TMSI trimethylsilyl imidazole HMDS hexamethyldisilazane

TMS trimethylsilyl

**14. References** 

APCI atmospheric pressure chemical ionization

MS/MS mass spectrometry/mass spectrometry LC/MS liquid chromatography/mass spectrometry GC/MS gas chromatography/mass spectrometry

Review Plant Physiology 17:209-222.

*Recombinant IgG Fe*. Science, 320: 373-376.

Inositol is synthesized sparingly in the body but is present in many foods. The inositols are essential nutrients for plants (Loewus and Murthy, 2000) and animals (Holub, 1986). Concentrations of the inositols and their metabolites can be much higher in some plant species than in mammalian tissue. For example, in soybeans, the concentration of pinitol alone approaches 30 mg/g (Streeter & Strimbu 1998; Garland *et al*., 2009) whereas in human blood, the levels of free *myo*-inositol is 3000 times lower (1 mg/100 mL). Levels of pinitol in blood is not widely known, but are anticipated to be orders of magnitude less than *myo*inositol.

One form of inositol, inositol hexaniacinate, has been used to support circulatory health because it functions like niacin in the body. The major dietary forms of myo-inositol are inositol hexaphosphate or phytic acid, which is widely found in cereals and legumes and associated with dietary fiber, and myo-inositol-containing phospholipids from animal and plant sources.

Inositol is involved in the glucuronic acid and pentose phosphate pathways. Inositol exists as the fiber component phytic acid, which has been investigated for its anti-cancer properties. Inositol is primarily used in the treatment of liver problems, depression, panic disorder, and diabetes (Narayanan, 1987). Used with choline, it also aids in the breakdown of fats, helps in the reduction of blood cholesterol, and helps to prevent thinning hair (Walker, 2010). It promotes the export of fat from the liver. Inositol is required for the proper function of several brain neurotransmitters. Inositol may improve nerve conduction velocities in diabetics with peripheral neuropathy. Inositol may help protect against atherosclerosis and hair loss. There has also been the suggestion that it may help to reverse some nerve damage caused by diabetes (Gregersen et al. 1978; *Ibid*,1983). Inositol has also been tried for other psychological and nerve-related conditions including the treatment of side effects of the medicine lithium. Inositol also has a prominent calming effect on the central nervous system, so it is sometimes helpful to those with insomnia. Inositol may also be involved in depression.

Under pinitol deficiency, detrimental health conditions may exist such as higher blood sugar in diabetics (Geethan and Prince, 2008). Myo-inositol deficiency can lead to depression and other mental disorders (Levine et al, 1995; Benjamin et al, 1995; Fux, et al, 1996). Also, polycystic ovary syndrome (PCOS) has been reported to be related to a deficiency in dietary inositol (Gerli, et al. 2003; Ibid, 2007). Correlations with depression and similar disorders may be related to the abundance of inositol phospholipids in brain and other nervous system tissues. However, the relationship between pinitol and blood sugar levels is more likely correlated with the similarities in structure between the 0 methyl inositol and glucose.

There is no recommended daily allowance for inositol, but the normal human dietary intake is about 1 gram per day. Inositol is available from both plant and animal sources. Natural sources of inositol include soybeans, wheat germ, brewer's yeast, bananas, liver, brown rice, oak flakes, nuts, unrefined molasses, vegetables, and raisins. Most dietary inositol is in the form of phytate, a naturally occurring plant fiber.

Dietary effects of pinitol and ononitol are still in the earlier stages of discovery. It has recently been shown that pinitol lowers blood glucose levels in type II diabetics while significantly decreasing total cholesterol, LDL-cholesterol and the LDL/HDL-cholesterol ratio (Kim et al 2005). The dietary benefits or hazards of the other metabolites of isomers of inositol (other than myo-inositol) are under active investigation.
