Buckwheat Pharmaceuticals - Cornell University



Buckwheat Fagopyritols

Flatulence Report, June 30, 1999

prepared by

Ralph L. Obendorf, P.I.

Bertha A. Lewis, collaborator

Kathryn J. Steadman, Postdoctoral Associate

Monica S. Burgoon and Hutton J. John, graduate students

Department of Crop and Soil Sciences and Division of Nutritional Sciences

Cornell University, Ithaca, NY 14853-1901

Part 9

Breakdown of fagopyritols by human fecal bacteria

Summary

Human fecal bacteria readily digest fagopyritols. Fagopyritols were prepared from whole groats as described in Part 10. The concentrated extract containing fagopyritols was digested by dry granular Baker(s yeast to remove large quantities of sucrose from the extract. Trehalose in the preparation of fagopyritols was a byproduct of fermentation by yeast. Fermentation with fecal bacteria was conducted under anaerobic conditions in vitro. Fermentation of the fagopyritol extract by human fecal bacteria in vitro resulted in the digestion of all fagopyritols in the samples within six hours (and probably much sooner). d-chiro-Inositol, myo-inositol, trehalose, galactinol, and digalactosyl myo-inositol were also metabolized by the bacteria. The controls without bacteria did not show degradation of fagopyritols indicating that fecal bacteria were responsible for fagopyritol degradation. Upon analysis by gas chromotography, no trace of any of these compounds was present in the samples with bacteria. Consumption of d-chiro-inositol by the bacteria occurred in the absence of other fermentable materials as would normally be present in a human diet and in the presence of a vast excess of microorganisms.

Objective: To present the human digestive tract and potential for utilization of fagopryitols after oral dietary consumption and degradation of fagopyritols by human fecal bacteria.

Introduction:

Buckwheat is a rich natural source of d-chiro-inositol, mostly in the form of fagopyritols, galactosyl derivatives of d-chiro-inositol (Horbowicz et al., 1997). Buckwheat and buckwheat products were claimed to be a useful dietary treatment for lowering blood glucose in persons with diebetes (Wang et al., 1992; Kasatkina and Odud, 1993). Medical research has linked a faulty metabolism of d-chiro-inositol to type II diabetes mellitus (Kennington et al., 1990; Asplin et al., 1993). Use of d-chiro-inositol as a dietary treatment for type II diabetes mellitus has been patented (Larner and Kennington, 1992). Reports of research with obese Rhesus monkeys that were spontaneously diabetic (type II, non-insulin dependent) has provided some evidence that d-chiro-inositol, when added to the diet and fed orally, is effective in lowering blood glucose (Ortmeyer et al., 1995; Hansen and Ortmeyer, 1996; Ortmeyer, 1996). Concentrated extracts from buckwheat provide a rich source of fagopyritols, that are readily hydrolyzed by α-galactosidase (Horbowicz et al., 1998). Since cells in the human digestive tract do not produce this enzyme, the present experiment was designed to verify that fagopyritols can be degraded by microorganisms normally present in the human digestive tract.

[pic]

Review of the human digestive system: Foodstuffs ingested by mouth pass through the esophagus to the stomach. Partially digested foods pass from the stomach to the small intestine. The first part of the small intestine is the duodenum, a C-shaped 25-cm long tube that curves around the pancreas. The upper part (40%) of the small intestine is the jejunum (100 cm long) and the lower part of the small intestine (60%) is the ileum (160 cm long). The internal surface area of the small intestine is increased by circular folds of mucosa (the cells lining the lumen or internal cavity). The mucosa has many villi (invaginations), and cells of the villi have microvilli (microinvaginations) with a greatly increased surface area. There are 20 to 40 villi per square centimeter of mucosa. The mucosa cells produce secretions and are active in uptake of digested foodstuffs in the watery contents. Enzymes found in the mucosa membrane include many disaccharidases, peptidases, and enzymes involved in the breakdown of nucleic acids. The ileum terminates at the ileocecal junction where food residues enter the large intestine (colon). The colon is about 110 cm long and includes the ascending colon, the transverse colon, the descending colon, and the S-shaped colon that leads to the anus. Unlike the small intestine, the colon does not have mucosa and the fecal contents become progressively drier during passage through the large intestine.

Cells (mucosa) lining the lumen of the duodenum, jejunum, and ileum of the small intestine have several disaccharidases that degrade maltose, sucrose, cellobiose, lactose, trehalose, and others (Auricchio et al., 1963; Gitzelmann and Auricchio, 1965) but not α-galactosidase (Gitzelmann and Auricchio, 1965). Thus, most monosaccharides and disaccharides are digested by the human system. However, some α-galactosides such as raffinose, stachyose and verbascose, that are commonly found in grain legume seeds and in oilseeds, are not degraded by the human digestive system since it lacks α-galactosidase, but these α-galactosides pass through the system to the intestinal bacteria that have α-galactosidase (Gherardini et al., 1985). Raffinose, stachyose and verbascose are readily degraded by bacteria and promptly fermented to hydrogen and carbon dioxide gas (and methane in 20-40% of the people (Hill, 1986a)) resulting in increasing flatulence production in the jejunum, ileum, and colon (Richards and Steggerda, 1966). Masses of bacteria are present in the human digestive tract, mostly in the colon (large intestine) where they may constitute 40-50% of the total fecal mass. Bacteria are present in the small intestine in the jejunum and increasing in number in the ileum, the distal small intestine (Hawksworth et al., 1971). Flatulence production from fermentation of the raffinose series oligosaccharides and other soluble α-galactosides can start in the jejunum, increasing in the illeum and increasing more when passing into the colon leading to extensive production of flatulence as observed in dogs (Richards and Steggerda, 1966). Flatulence occurs as a result of fermentation of soluble oligosaccharides by intestinal bacteria containing α-galactosidase (Price et al., 1988). Flatulence may also occur as a result of fermentation of degradation products of dietary fiber from cell walls, including β-galactosides and β-glucosides (Champ et al., 1990). The population of different species of intestinal bacteria change in response to the nature of the foodstuffs ingested. Bacteria can produce one form of α-galactosidase in response to dietary fiber and a second form of α-galactosidase in response to raffinose and stachyose (Gherardini et al., 1985). Many hydrolytic enzymes are secreted by the bacteria, but bacterial α-galactosidase is not released into the intestinal lumen. About 70% of the bacterial α-galactosidase is soluble (cytosolic; in the bacterial cell) and about 30% is membrane bound. It is not known if the enzyme is external to the membrane for hydrolysis of the α-galactosyl bonds before uptake into the bacteria or if the α-galactosides must be taken up by the bacterial cell before hydrolysis.

d-chiro-Inositol and fagopyritols in the human digestive tract: Ingestion of buckwheat products or fagopyritols extracted from buckwheat introduces d-chiro-inositol, mostly in the form of α-galactosides of d-chiro-inositol, into the human digestive tract. The fagopyritols are mono-, di- and tri- α-galactosides of d-chiro-inositol and are readily hydrolyzed by α-galactosidase (Horbowicz et al., 1998). The possible fate of fagopyritols and d-chiro-inositol in the gut must be considered as part of the dietary effect of buckwheat.

Hawksworth (1971) notes three factors that determine the degree of metabolism of ingested or bile-excreted compounds:

1. The region of the gut from which the compound is absorbed (if it is absorbed).

2. The distribution of bacteria along the gut.

3. The occurrence of the necessary enzyme or enzyme systems in bacteria.

General consideration of each of these factors, along with specific reference to the d-chiro-inositol, myo-inositol and fagopyritols, follow.

Absorption

The small intestine is the main site of absorption of products of hydrolysis that result from digestion. However, α-galactosides of d-chiro-inositol in buckwheat, being similar to the α-galactosides of sucrose (raffinose and stachyose) found in legumes, probably escape human digestion due to the absence of α-galactosidase production in the gut mucosa (Gitzelmann and Auricchio, 1965; Price et al., 1988). Therefore, like raffinose and stachyose, fagopyritols remain in the digestive tract and are likely degraded when they contact the microflora. Experiments with animal systems indicate that 80% of seed α-galactosides are degraded by the end of the small intestine (Gdala et al., 1997). It is likely that fagopyritols are mostly utilized also before the end of the small intestine.

Free d-chiro-inositol is probably absorbed like myo-inositol in the small intestine. Feeding a mixtutre of free d-chiro-inositol and free D-pinitol to humans with NIDDM resulted in an increase in plasma d-chiro-inositol and D-pinitol and a decrease in plasma insulin and glucose (Ostlund and Sherman, 1998), and feeding d-chiro-inositol to hyperinsulinemic Rhesus monkeys resulted in decreased plasma glucose (Ortmeyer et al., 1995), providing evidence of uptake and utilization of d-chiro-inositol by the digestive system. When myo-inositol was fed to humans, plasma myo-inositol was significantly increased, providing evidence for uptake. Feces contained only small amounts of myo-inositol (Clements and Reynertson, 1977). Free myo-inositol has been shown to be actively transported by the small intestine of hamsters though glucose and galactose acted as non-competitive inhibitors of the transport of myo-inositol (Caspary and Crane, 1970). A stereospecific myo-inositol-d-chiro-inositol transporter in human liver cells selectively uptakes d-chiro-inositol, but not L-chiro-inositol, in the presence of excess myo-inositol (Ostlund et al., 1996). Presence of such a stereospecific transporter in other tissues has not been reported, but free d-chiro-inositol is clearly taken up by the digestive system.

Absorption of some substances occurs in the large intestine also, though it does not have the extensive surface area by which the small intestine increases its absorptive capacity. Besides water, electrolytes and other minerals, some B vitamins and Vitamin K are absorbed in the colon; other nutrients are known to be used by bacteria, namely, ascorbic acid, choline and cyanocobalamin (Ganong, 1995). It is important to note that inositol fermentation can occur as is evident by several studies which used inositol for biotyping bacteria, some of which occur in the microflora (Ganong, 1995). However, inositol is also produced by intestinal bacteria in myo-inositol deficient rats (Hayashi et al., 1974) so that it is difficult to predict the fate of free inositols in the lower digestive tract (colon or large intestine).

Bacteria in the Gut

Each gram of gut contents is estimated to contain up to 1012 microorganisms resulting in the bacterial biomass making up about 40% of fecal material. Stomach acid is bacteriostatic and bactericidal; also, bile acids, pancreatic enzymes, rapid transit time and peristalysis contribute to control bacterial growth in the proximal small intestine (duodenum, jejunum and proximal ileum). Gram-positive cocci are the primary group detected there. In the distal small intestine the microflora resembles that of the large bowel in that there are more Gram-negative and anaerobic bacteria. The total number, however, is still considerably less than that of the large intestine which harbors what has been described as a complex ecosystem of microorganisms influencing host physiology in ways that have only recently been investigated (Hill, 1986b). The smaller numbers of bacteria in the small intestine may be sufficient for hydrolysis of fagopyritols since bacterial enzymes are highly active on α-galactosides (Gherardini et al., 1985). The massive numbers of bacteria in the colon are (overkill(, designed to scavenge nutrients remaining in the fecal materials.

In general, in the healthy human, the main site of bacterial colonization is the large intestine with the lower small intestine showing similar but fewer bacteria and less colonization. In regards to fermentation, most occurs in the cecum and ascending colon where substrate concentration (undigested material) is highest, bacterial growth rate fastest, pH most acidic and transit time most rapid. Substrate concentration and bacterial growth rate are known to decrease and pH increase along the length of the colon from the cecum to the rectum (Gibson and Wang, 1994). Fagopyritols are soluble and therefore are probably fermented quickly in the cecum, or possibly earlier in the distal ileum. In the pig, β-glucans were degraded mainly in the distal ileum (Knudsen and Johansen, 1995).

It should be noted that the rodent model is questionable in studies involving the digestive tract because rodents are coprophagic and secrete less stomach acid (Hawksworth et al., 1971). Therefore, the total number and distribution of bacteria is greater in rodents and the metabolic effects they produce can be expected to differ from what occurs in the human.

Enzymes of the microfloral bacteria

Gut bacteria have an array of enzymes at their disposal; most activity is saccharolytic since energy is derived mainly from carbohydrates. The production of enzymes has been shown to be influenced by colonic pH, oxygen tension and substrate concentration. Also, enzymes useful in the colonic environment are either cell-associated or extracellular. Enzyme location has been studied; it appears that bacterial extracellular enzymes mainly produce partially-degraded polysaccharides from high molecular weight substrates (dietary fiber) while mono- and disaccharides are the major products of cell-associated enzymes. In this way, it is less likely that competing microorganisms would benefit from the enzyme activity since the mono- and disaccharides are directly transported into the cells for metabolism (Salyers, 1979; Egli, 1975; Murashige and Skoog, 1962).

Specifically, [pic]-galactosidase activity has been demonstrated in many of the dominant bacterial genera of the gut. In fact, people eating a corn-bean diet have increased fecal [pic]-galactosidase activity compared to those eating the normal low-fiber American diet. The galactose that is freed is readily transported into bacterial cells and used for energy or biomass (Salyers, 1979) which likely explains why Gitzelmann and Aurrichio (1965) found no indication that galactose from soybean [pic]-galactosides is absorbed by the human gut.

There are, however, various forms of the α-galactosidase enzyme produced by microbes depending on species and substrate (raffinose series oligosaccharides, mucin, guar gum). Both cell-associated and extracellular forms have been isolated. The form that digests raffinose and stachyose oligosaccharides is cell-associated in many bacteria (Gherardini et al., 1985; Hudson and Marsh, 1995).

Overall, bacterial degradation of fagopyritols in the lower digestive tract, either the distal ileum or colon, is likely but needs to be demonstrated by in vitro fermentation. Whether d-chiro-inositol once freed from galactose units is absorbed by the gut or utilized by bacteria is unknown. Ingested free d-chiro-inositol is almost certainly absorbed in the small intestine like myo-inositol.

Procedures:

Fagopyritols were prepared from whole groats as described in Part 10. The concentrated extract containing fagopyritols was digested by dry granular Baker(s yeast to remove large quantities of sucrose from the extract. Trehalose in the preparation of fagopyritols is not from buckwheat but remains as a byproduct of fermentation by yeast (see Part 10). Yeast cells were removed by centrifugation (10 min at 40,000 g) and ultrafiltration (Amicon 10,000 Mr cut-off filter). The fagopyritol water extract was boiled under carbon dioxide to sterilize the preparation and to remove other gases. Anaerobic conditions were maintained during the preparation of fecal inoculum and of all samples by working under streams of carbon dioxide gas. Fermentation bottles were sealed with rubber stoppers and metal caps.

Fagopyritol substrate was prepared first; 1 ml was placed in all bottles except blanks (duplicate samples plus one control and one blank for each time at 0, 6, 12, and 24 h). Next, the nutritive medium was prepared as outlined by Van Soest and Robertson (1985). Control bottles (without bacteria) received 1 ml of medium instead of bacterial inoculum. The remaining medium was used to dilute fresh feces 1:12, w/v, prepared as previously described (Barry et al., 1989; Guillon et al., 1995). All samples and blanks were inoculated with 1 ml of fecal inoculum by syringe through the rubber stoppers. Fermentation was conducted at 40(C; fermentation was stopped at 0, 6, 12, and 24 h by opening the bottles to air (the anaerobic bacteria are quickly inhibited by air) and immediately freezing the contents.

The samples were prepared for GC analysis after thawing by centrifugation (10 min at 15,000 g) and ultrafiltration through a 10,000 Mr cut-off filter in a microcentrifuge. Since salts interfere with TMS-derivatization, samples were desalted by passing the each sample through an Amberlite cation-anion mixed-bed resin. Two control samples of the original extract of fagopyritols were also passed through the ion-exchange resin to verify that fagopyritols were fully recovered using this procedure. A 750-μl aliquot of sample plus 100 μg of phenyl α-d-glucoside as internal standard was dried under a stream of nitrogen gas, stored overnight over P2O5 to remove traces of water. Dry samples were derivitized with TMSI:pyridine (1:1, v/v) and assayed by gas chromatography as previously described (Horbowicz and Obendorf, 1994).

Results:

Fermentation of the fagopyritol extract by human fecal bacteria in vitro resulted in the digestion of all fagopyritols in the samples within six hours (Table 9.1). d-chiro-Inositol, myo-inositol, trehalose, galactinol, and di-galactosyl myo-inositol were also metabolized by the bacteria. The controls without bacteria did not show degradation of fagopyritols indicating that fecal bacteria were responsible for fagopyritol degradation. Upon analysis by gas chromotography, no trace of any of these compounds was present in the samples with bacteria (Table 9.1). Two signature peaks from the bacteria were observed on the chromatogram at 3.1 and 3.4 minutes for all samples with bacteria (data not shown).

The internal standard, that was added to all samples after digestion, was recovered during analysis of all samples, demonstrating that soluble carbohydrates remaining after digestion were successfully derivitized for analysis. Controls for removal of the nutrient medium-derived salts by the ion-exchange resin also did not indicate any loss or removal of fagopyritols or or d-chiro-inositol during de-ionization of the samples.

Table 9.1. Fagopyritol and inositol concentrations (μg / 750 μl of sample) during fermentation.

| | | |

|Component |Without bacteria a |With bacteria b |

| | | | | | | |

| |0 h |6 h |12 h |0 h |6 h |12 h |

| |

| | | | | | | |

|d-chiro-inositol |99 |102 |58 |47 |0 |0 |

| | | | | | | |

|fagopyritol A1 |88 |87 |94 |106 |0 |0 |

| | | | | | | |

|fagopyritol A2 |56 |54 |59 |54 |0 |0 |

| | | | | | | |

|fagopyritol B1 |667 |636 |605 |591 |0 |0 |

| | | | | | | |

|fagopyritol B2 |83 |72 |66 |65 |0 |0 |

| | | | | | | |

|fagopyritol B3 |21 |20 |26 |48 |0 |0 |

| | | | | | | |

|sucrose |0 |0 |0 |0 |0 |0 |

| | | | | | | |

|trehalose |82 |64 |63 |60 |0 |0 |

| | | | | | | |

|myo-inositol |45 |48 |26 |20 |0 |0 |

| | | | | | | |

|galactinol |17 |16 |18 |14 |0 |0 |

| | | | | | | |

|di-galactosyl myo-inositol |17 |17 |13 |10 |0 |0 |

aValues represent a single control sample without bacteria for each fermentation time.

bValues represent one sample at 0 and 12 hours and two replicate samples at 6 hours.

Discussion:

In vitro fermentation clearly demonstrates the ability of microfloral bacteria to utilize fagopyritols and inositols. The disappearance of d-chiro-inositol indicates that it may be metabolized by the bacteria in the lower intestine; however, in this experiment the amount and kinds of substrate were limiting factors resulting in an excessively high ratio of bacteria to substrate. The extent of degradation of fagopyritols in vivo in the complex mixture of fibers and other undigested food acted upon by bacteria is not easily determined. However, it has been simulated by other researchers by multiple-stage continuous cultures.

Because conditions in the colon are now known to be site-specific, in vitro fermentation studies using multiple-stage continuous culture (which attempts to duplicate this gradient of conditions), as opposed to batch culture, are thought to reproduce natural colonic conditions more closely (Salyers, 1979; Gibson and Wang, 1994). If one were to attempt to replicate physiological conditions for fagopyritol study, the conditions in the first stage of the continuous culture (which represents the cecum and ascending colon) would be appropriate. The fecal inoculum is 16% feces at this stage. Gibson and Wang (1994) used fructo-oligosaccharide and inulin at 1% (w/v); they estimated the carbohydrates which are naturally present in vegetables to range from 2 to 4 g/d in the typical US diet (but much higher in Europe at 4 to 12 g/d). In the fagopyritol fermentation study, inoculum was also 16% feces but the soluble carbohydrate concentration was 0.075%, which is approximately the amount of soluble carbohydrates from buckwheat that would be ingested if buckwheat were eaten. Also, the continuous culture method uses a representative mixture of polysaccharides, along with various minerals and vitamins, for basal medium. This medium would improve fagopyritol fermentation studies because substrates normally present in the colon are included; fagopyritols would not be the only substrate for bacteria to ferment. While this method was designed to study the effect of diet on the microflora, it clearly is preferable for replication of physiological conditions. However, whether d-chiro-inositol is freed from galactose units in the distal ileum or colon and whether it would be absorbed if it weren(t utilized by bacteria need to be determined by in vivo experiments.

It should be noted, however, that the chemical structures of myo-inositol and glucose are very similar (Larner, 1988). The relative positions (in space) of the hydroxyls are identical. Larner (1988) suggested that renal tubular competition with glucose causes inosituria in diabetics. Subsequently, he showed the biosynthesis of myo-inositol from labeled glucose in rat and chick embryos; the reverse reaction was then demonstrated when labeled myo-inositol was traced to liver glycogen in the rat. The required enzyme has been isolated from bacteria. This supports the likelihood that gut bacteria readily utilize both myo-inositol and d-chiro-inositol.

In conclusion, fagopyritols are not digested by human enzymes. The microflora, however, possess a variety of enzymes, including [pic]-galactosidase, that degrade a large portion of the undigested food reaching the lower intestine. Because fagopyritols are soluble they are likely hydrolyzed quickly by bacteria in the gut, but the fate of d-chiro-inositol that is freed by fagopyritol degradation is unknown. Free d-chiro-inositol in the intestinal lumen appears to be taken up judging by increased plasma d-chiro-inositol.

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