Tartary buckwheat and Nutrition

The studies we’ve read reveal the many genetic and environmental factors and interactions that affect the chemical composition of different parts of a Tartary buckwheat plant.  Nevertheless, seeds from many varieties grown under diverse environments are fairly consistent in their nutritional characteristics.  Even the nutritional differences between Tartary and common buckwheat seeds are generally small, except for the substantially greater concentration of flavonoids found in the former. On the other hand, the milling of a single batch of buckwheat seed will produce several products containing differing percentages of embryo, endosperm, seed coat, and hull. These compositional differences significantly affect the nutritional properties of the various milling fractions.

Knowledge about the chemical composition of common and Tartary buckwheat has been well reviewed by K. Ikeda (Advances in Food and Nutrition Research, Vol. 44, pp 395-434), H. H. Wijngaard and E. K. Arendt (Cereal Chemistry, Vol. 83, Issue 4, pp 391-401, Jul/Aug 2006), and by A. Ahmed and co-authors (Journal of Agricultural Science, Vol. 152, pp 349–369, 2014). In recent years these species have generated much interest and research, particularly in China, Japan, India, and southern Europe. While relying on the two references above, we’ve added information from other sources. New articles appear frequently, and we’ll try to keep up with developments in the science.

Whenever possible, we’ve presented data about Tartary buckwheat. Where we’ve not found that, information about common buckwheat is included because the two species are much alike. In contrast, different plant tissues vary greatly in their concentrations of both macronutrients and micronutrients. Even within seeds, the embryo, endosperm, seed coat, and hull vary greatly in their composition.  We’ve also tried to provide a little explanatory background to the scientific jargon. Some of it is incomprehensible to us and—we suspect—to some readers.

CARBOHYDRATES

Starch is composed of amylose and amylopectin, in proportions that are similar in common and Tartary buckwheats. The starches from both common and Tartary buckwheat more closely resembles the starches from tubers and roots than those from cereals, in their high swelling and gelling capacity and peak viscosity during gelatinization. In buckwheat seeds, starch is concentrated in the endosperm. The pale “fancy” flour from the first pass through the mill is higher in starch, and correspondingly lower in the concentration of other macronutrients than the darker flour comprising other seed tissues. Within the stomach and small intestine, several enzymes digest starch molecules into their sugar sub-units. Seeds of different species vary in their percentages of rapidly-digested, slowly-digested, and resistant starch. Hydrothermal processing can greatly decrease the percentage of starch that resists digestion. Even the retrograded starch that resists digestion in the small intestine is largely broken down by the microflora in the large intestine.

As noted above, both buckwheat species are considered to be pseudo-cereals because their seeds—like those of true cereals—contain relatively high concentrations of storage carbohydrates, primarily starches. In 1931, Coe reported that the nitrogen-free extract constituted 71.0 and 70.0 percent of dry weight of the groats of that era’s two most popular U. S. varieties of common buckwheat, and 69.4 percent of nitrogen-free extract of Tartary buckwheat groats. For whole seeds, the corresponding percentages were 63.8, 64.0 and 59.6, respectively. (“Nitrogen-free extract” is a now-outdated approximation of the sum of starch and soluble carbohydrate. It was calculated by subtracting from 100 percent all of the other components of proximate analysis—ash, crude protein, ether extract [lipids], and crude fiber. From Oregon State University, here’s a brief description of Proximate Analysis and its intrinsic limitations. )

Steadman et al. (2001a) reported that whole groats of the cultivar ‘Manor’ of common buckwheat contained 54.5% starch on a dry weight basis. The starch was mostly concentrated in the inner endosperm. Therefore, those milling fractions consisting largely of endosperm (i.e., “fancy” flour and fragments of the inner groat) had starch concentrations ranging from 64.8 to 75.5%. In contrast, bran fractions had starch concentrations ranging from 10.2 to 17.8%.

Based on an analysis of Tartary buckwheat from a single variety, Bonafaccia et al.(2003b) reported that starch constituted 57.4 percent of the dry weight of whole seeds.  For seeds of common buckwheat (cultivar ‘Siva’), the corresponding percentage was 55.8. Those authors ground both these samples in a stone mill, and reported that Tartary buckwheat flour contained 79.4 percent starch, whereas common buckwheat flour contained 78.4 percent starch. The respective concentrations of starch in bran were 37.6 and 40.7 percent. (Milling yields of flour and bran were almost identical for the two species. )

Qin et al. (2010) investigated variability in the composition of buckwheat seeds from the major growing areas in China. From 21 cultivars of Tartary buckwheat and 18 cultivars of common buckwheat, those authors milled whole seeds and retained the flour that passed through a 60 mesh sieve. (Their flour yields were not reported.) Flour from the Tartary buckwheat cultivars contained between 65.6 and 78.0 percent total starch, which was almost identical to the range of total starch found in common buckwheat (65.9 to 78.1 percent}.

Starch molecules are long linear or branched chains of dextrose (D-glucose) molecules. Amylose is primarily unbranched starch composed of glucose subunits in the “alpha” configuration, with the carbon atom designated “1” of one subunit bonded to the carbon atom designated “4” in the adjacent subunit. Amylopectin is a starch also comprising D-glucose molecules linked in an alpha 1,4 configuration, but with multiple levels of branching rather than a linear structure. Branches are attached to the starch molecule’s linear backbone by α-1,6-glucosidic bonds. In most starches, amylose molecules are much more numerous than amylopectin molecules. However, the former have molecular weights <0.5 million, while the latter have molecular weights in a range from 50 million to 500 million. Therefore, amylopectin constitutes most of the mass of most starches.

Sindhu and Khatkar (2016) reported that amylose constituted 14.99 percent of the dry weight of flour from Tartary buckwheat cultivar ‘Shimla-B1.’ Qin and co-authors (2010) reported that the amylose content of flour from 18 common buckwheat cultivars ranged from 19.3 to 28.2 percent. The comparable range in 21 Tartary buckwheat cultivars was 19.6 to 25.6 percent of total starch. Wijngaard and Arendt (2006) found reports of apparent amylose content of Tartary buckwheat seeds as high as 47 percent, but mostly in a range from 21.1-27.4 percent. They stated that the latter range was similar to the apparent amylose content of common buckwheat and true cereals. The apparent concentration of amylose is determined empirically by reaction with iodine, which is affected by the degree of coiling of the starch chain. The actual amylose concentration in starch of common buckwheat is about ten percent less than its apparent concentration. That large difference is attributable to the presence of some longer-chain amylopectin molecules that contain coiled regions which react with iodine in the same manner as amylose does(Wijngaard and Arendt, 2006). Yoshimoto et al. (2004) measured the percentage of apparent amylose in starch from five Japanese and two Chinese varieties of common buckwheat. Values ranged from 25.7 to 26.5 percent; the range of actual amylose was 15.6 to 17.9 percent. Starch from the single Japanese variety of Tartary buckwheat tested by those authors was 25.5 percent apparent amylose, and 15.6 percent actual amylose. Gao et al. (2016) reported the percentage of amylose in starch from two varieties of Tartary buckwheat (‘Xinong9920’ and ‘Xinong9940’) and two of common buckwheat (‘Xinong9976’ and ‘Xinong9978’). In all four, the amylose content was about 38.9 percent. This value, according to the authors, made retrograded Tartary buckwheat starch a suitable ingredient in functional foods designed for low glycemic index.

Fats can form complexes with the amylose contained within buckwheat seeds, thereby reducing the reactivity of buckwheat starch with iodine, and thereby reducing the apparent concentration of amylose. A more accurate measure of amylose is obtained from starch that has been de-fatted. Skrabanja et al. (1998) reported apparent amylose concentration of 18.1 g/100g dry weight, and (following precipitation of lipids with ethanol) an actual amylose concentration of 20.8 g/100g of dry weight of groats of common buckwheat. In their sample of seven varieties of common buckwheat, Yoshimoto and co-authors (2004) reported that the iodine affinity of starch (g/100g) was increased between 1.32 and 1.59 if starch had been de-fatted. For the single variety of Tartary buckwheat tested, de-fatting increased the iodine affinity by 1.09. The authors noted that these values were similar to those reported for wheat starches.

In plant cells, starch molecules are compactly stored in a semi-crystalline form as starch granules. The central endosperm of buckwheat seeds is composed of dead cells packed with such granules. The size and surface quality of these granules, as well as the sizes and branching characteristics of their constituent molecules, determine a starch’s performance during gelatinization and gelation. Gelatinization occurs when starch is heated in water. Heat breaks hydrogen bonds between starch molecules, allowing water molecules to enter the granules. As the granules swell with water, soluble amylose molecules within can escape. Subsequent hydrogen bonding between water and amylopectin molecules reduces the quantity of free water in the hot starch paste, causing an increase in its viscosity. The temperature of gelatinization differs among starches from different varieties of different species, as does the maximum viscosity (the latter generally increasing with an increasing percentage of amylopectin). With subsequent cooling, the starch paste undergoes gelation. As the amylose molecules lose energy, hydrogen bonds form between them. This network of amylose molecules traps in place the swollen granules (now composed largely of amylopectin). Starches that contain a large proportion of amylose form stiffer gels, while those higher in amylopectin form softer gels.

As a starch gel ages, junction zones between its constituent amylose molecules lengthen, pulling the molecules closer together, and squeezing out some of the interspersed water molecules. This process is called “syneresis.” Also with aging, a starch gel can undergo a realignment of hydrogen bonds between amylose molecules, allowing the starch to regain some of the crystalline quality of the native starch granules. This process, called “retrogradation,” can be partially reversed by gentle heating.

[HERE is a nice presentation about the composition and behavior of different starches, by Megan Erickson of Central Washington University. A helpful article relating the chemistry of starches to their functional properties is HERE. ]

The starch granules in common buckwheat groats are mainly polygonal rather than spherical, and have rough surfaces (Drobot et al., 2014).  Gao et al. (2016) similarly reported that the starch granules of both common and Tartary buckwheat were “irregularly polygonal with sharp sides and corners.”  These characteristics of the granules allow buckwheat starch to absorb relatively large amounts of water. Along with qualities of buckwheat protein that are discussed below, its starch granules give buckwheat its high stabilizing and thickening properties.  Buckwheat starch enhances stickiness.

Gao and co-workers (2016) also measured diameters of starch granules of both species of buckwheat. These ranged from 3 to 14 μm, with those of Tartary buckwheat somewhat larger than those of common buckwheat. According to another buckwheat study, granules ranged in size from 2.9 to 9.3 μm, with a mean size of 5.8 μm. These are smaller than the granules in most cereal grains. In general, the smaller the granule, the less susceptible is the starch to retrogradation.
The presence of amylose-lipid complexes in buckwheat starch (which can be inferred from the increased reactivity with iodine following de-fatting of the starch, described above) might reduce the starch’s solubility and swelling potential. Nevertheless, buckwheat starch exhibited more granule swelling and gelling tendency than did cereal starches (Yoshimoto et al., 2004). Those authors found little variability among buckwheat cultivars in their pasting properties. As mentioned above, the starch of common buckwheat contains many longer-chain amylopectin molecules. This is indicated by buckwheat starch’s high weight average degree of polymerization (DPw), estimated at 94,900. Such large molecules are responsible for the relatively high peak viscosities during heating of starch of common buckwheat—more similar to starches from roots and tubers than to those found in cereal seeds (Wijngaard and Arendt, 2006).

Sindhu and Khatkar (2016) studied starch isolated by steeping Tartary buckwheat flour (cultivar ‘Shimla-B1’) in 0.25% aqueous NaOH for 18 hours at room temperature. Subsequent centrifugation produced a white starch layer, which had dry-weight concentrations of protein, fat, fiber, and ash all less than one percent. The amylose content of the starch was 32.12 percent; that of the buckwheat flour was 14.99 percent. The authors reported the Hunter color parameters of their starch extract: L*=98.13, a*=0.66, and b*=7.59. The high value of b*, indicative of a yellow tint, had also been reported in starch extracted with distilled water (Li et al., 1997). The authors found that transmittance values (at 640 nm) of starch paste decreased with length of refrigeration—from 13.60 percent initially to 8.99 percent after five days. These values were substantially higher than corresponding values for flour (7.81 and 3.11, respectively).

Sindhu and Khatkar (2016) also investigated the functional properties of this Tartary buckwheat starch. They reported a bulk density of 0.65g per ml, an indication that the starch would function well as a thickening agent. The starch had a water absorbance capacity of 91.1 percent and oil absorbance capacity of 92.5 percent. These values represent the ability of the starch to hold water or oil, respectively, against the pull of gravity. The solubility of the Tartary buckwheat starch was 10.6 percent, and the swelling power was 17.2g per g—both relatively low. The starch had a “least gelation concentration” of 18.2 percent.

Differential scanning calorimetry (DSC) is another method of characterizing starch (and other polymers). DSC measures the heat capacity of the material over a range of temperatures; the heat capacity increases or decreases as the material transits between phases in crystallinity. [A helpful description of DSC can be found HERE ; refer also to “Starch gelatinization”] Properties of a starch can be inferred from the “Onset temperature” (To), “Peak temperature” (Tp), “Completion temperature” (Tc) during its first transition (i.e., the breakdown of amylopectin crystals within the granules). Hung et al.(2009) reviewed the reported DSC values from common buckwheat (To: 57.8-64.1ºC; Tp: 63.7-69.6ºC; Tc: 70.8-85.8ºC) and from Tartary buckwheat (61.0-64.3ºC, 64.1-70.8ºC, and 79.9-81.7ºC). Higher than the comparable temperatures in barley starch, these data indicate that buckwheat starches have larger and purer starch crystals, as well as more long chains of amylopectin than the barley starch.

The sugar subunits that make up starch polymers are a source of energy for seeds when they germinate, and also for the animals that consume those seeds. Both plants and animals produce enzymes capable of freeing the constituent sugar subunits: α-amylase and β-amylase hydrolyzing the α-1,4-glucosidic bonds, and amyloglucosidase hydrolyzing the α-1,6-glucosidic bonds as well. Because of their high ratio of surface area to mass, smaller granules are generally more readily digested than are large starch granules.

Some starches are rapidly digested in the stomach and small intestine, some starches are digested more slowly, and some starches resist digestion in both the stomach and small intestine. In their sample of 21 Tartary buckwheat cultivars, Qin and co-authors (2010) measured concentrations of “resistant” starch ranging from 13.1 to 22.5 percent of flour dry weight. The comparable range in their sample of 18 common buckwheat cultivars was 15.0 to 23.1 percent. (As a percentage of total starch, the resistant starch in Tartary buckwheat ranged from 19.2 to 32.3, and that in common buckwheat ranged from 19.8 to 30.3. )

Skrabanja et al. (1998) listed factors affecting the digestibility of starch in the intestine: “the physical form of starch, the ratio of amylose to amylopectin, the solubility of starch, the degree of crystallinity/gelatinisation, the extent of retrogradation, interaction of starch with other food constituents, inhibitory action of non-starch components on the starch degrading enzymes, the amount of starch ingested, insufficient chewing, gastric emptying, and the transit time in the small intestine.” Resistant starch passes intact to the colon, where it is susceptible to microbial fermentation. In addition to carbon dioxide, methane, and hydrogen gases, the products of such fermentation are readily-absorbed short-chain fatty acids. Milling and cooking can reduce the percentage of resistant starch from seeds; however, retrogradation can increase that percentage.

Skrabanja and co-workers examined the effects of hydrothermal processing on the digestibility of starch from the groats of common buckwheat (cultivar ‘Bamby’). Starch was categorized as rapidly-digested (RDS—the percentage of glucose released after 20-minute enzymatic digestion), slowly-digested starch (SDS—the percentage of glucose released after 120-minute enzymatic digestion), and resistant starch (RS—difference between glucose released by KOH hydrolysis and that released after 120-minute enzymatic digestion). The authors used mechanically-dehulled groats and an extract from boiled hulls to simulate the effects of traditional hydrothermal processing (i.e., boiling whole seeds for one hr at 100ºC, then drying them to 10 percent moisture prior to mechanical dehulling). As a second treatment, dehulled groats were soaked in 70 percent ethanol at 30ºC for 10 minutes prior to the above cooking procedure; this treatment was designed to remove polyphenol substances from the outer layers of the buckwheat groats. Other treatments included dry heating of uncooked groats (30 minutes at 110ºC); lyophilization of previously cooked groats; and freezing (-18ºC for 20 days) prior to the lyophilization of previously cooked groats.

Those researchers reported that starch constituted 76 percent of dry weight in uncooked groats and varied little across their treatments. RDS, 9.5 percent of dry weight of uncooked groats, was 10.6 percent in the dry-heated treatment; almost 50 percent of dry weight after hydrothermal processing; and almost 60 percent of dry weight in lyophilized treatments. SDS, 28.7 percent of dry weight of uncooked groats, declined to about 20 percent after hydrothermal processing; after lyophilization, it declined further to 4.7 percent (6.8 percent if lyophilization was preceded by freezing). Resistant starch, (by subtraction) declined from 37.8 percent of dry weight of uncooked groats to around eight percent in the cooked treatments (with or without prior ethanol or subsequent lyophilization). Retrograded starch was one percent of dry weight of uncooked or dry-heated groats, and about four percent in all the other treatments. The quantities of retrograded starch observed in vitro were similar to the quantities of undigested starch in feces of rats that had been fed groats from these treatments, following the suppression of microflora in the rats’ colons by the administration of an antibiotic. With no antibiotic (and therefore, with intact microflora in the colon), rats were able to digest virtually all the starch from uncooked groats, and at least 98 percent of starch from hydrothermally-treated groats.

“Annealing” is a process that can either decrease or increase the fraction of RDS—depending on the species from which the starch is extracted. Liu et al. (2016) extracted native starch from Tartary buckwheat seeds (cultivar ‘XiDong 9940’). The annealing process included an over-night incubation of the starch in distilled water at 4ºC; heating for 24 hours at 50ºC; and drying for 12 hours at 40ºC. Annealing increased the apparent amylose content of Tartary buckwheat starch (to 31.4 percent, from 29.1 percent in native starch), probably due to the degradation of amylopectin chains as well as increased iodine-binding capacity of the amylose. Annealing weakened associative forces between starch polymers inside granules, and facilitated the binding of hydroxyl groups with water molecules. This was shown by increased water absorption capacity (1.34g/g versus 1.10g/g of native starch) and decreased oil absorption capacity (0.92 versus 1.10g/g). Tested over a range of temperatures—-from 50ºC to 90ºC—-the solubility and swelling potential of annealed starch were less than the corresponding values for native tartary buckwheat starch. Increased crystallinity and molecular organization likely diminished the leaching of amylose from granules. (In the absorption tests, the ratio of starch to water or peanut oil was 1:5 [w/v]; in the solubility and swelling tests, the ratio was 1:100. )

Liu and co-workers also reported on the gelatinization characteristics of both native and annealed Tartary buckwheat starch. Compared to native starch, the annealed starch had higher onset, peak, and concluding temperatures, but a slightly narrower gelatinization temperature range. In other words, annealing increased the thermostability of Tartary buckwheat starch and delayed gelatinization. Digestion of starches with α-amylose was measured in vitro over 180 minutes. Digestion of annealed starch proceeded more slowly than digestion of native starch, but by 160 minutes they were comparable (and still increasing). Annealing decreased the percentage of RDS (34.9 versus 37.6 percent in native starch), and increased the percentage of RS (4.75 versus 3.27 percent).

DIETARY FIBER

High levels of dietary fiber in buckwheat flour, particularly tartary buckwheat flour, might have benefits such as lowering levels of blood cholesterol. Dietary fiber can include resistant starch, but mostly comprises cellulose and other carbohydrates found in cell walls. In buckwheat seeds, fiber is most concentrated in the hull, and to a lesser extent in the seed coat, and the aleurone layer. While fiber might constitute only a few percent of “fancy” flour, it constitutes up to forty percent of the bran.

Dietary fiber has been defined as “a heterogeneous mixture of polysaccharides and lignin that cannot be degraded by the endogenous enzymes of vertebrate animals” (Marlett, 1992).  Resistant starch is only one component of dietary fiber.  Much of the material that makes up plant cell walls similarly passes undigested into the colon.  This includes the carbohydrate cellulose.  Like starch, cellulose is a polymer of D-glucose molecules in 1,4 linkages; however, in the case of cellulose, these bonds are in the “beta” orientation rather than the “alpha” orientation in starch.  Humans do not synthesize enzymes that can hydrolyze these “beta” bonds.  Lignin (a polyphenolic polymer), hemicellulose (diverse polymers incorporating 5-carbon and 6-carbon sugars), and pectin (heterogeneous polymers made up primarily of galacturonic acid residues) are the other major constituents of cell walls. (Some researchers distinguish between those materials in dietary fiber that are water soluble from those that are not; referring to the former as soluble dietary fiber [SDF] and latter as insoluble dietary fiber [IDF].  Together these constitute “total dietary fiber” [TDF].)

To accurately determine the constituent chemical compounds in dietary fiber, one must measure the specific products of controlled in vitro digestions.  (For example, Marlett, 1992, states, “Recoveries of the soluble and insoluble fiber fractions were calculated by summing the amounts of uronic acids, neutral sugars, crude protein, residual starch, and, for the insoluble fraction, Klason lignin.”  )  Alternative simpler procedures are frequently employed that give approximate compositional estimates.  For example, cellulose, lignin, and hemicellulose form the bulk of the residue when a foodstuff is boiled in a neutral detergent solution (what is called the “Neutral Detergent Fiber” or NDF assay; HERE is a brief explanation from Oregon State University).  Cellulose and lignin also resist breakdown when boiled in an acidic detergent solution (“Acid Detergent Fiber” or ADF assay).  Therefore, the loss of dry weight when NDF residue is subjected to ADF approximates the hemicellulose content of the fiber.  A fraction of dietary fiber, including some pectin and resistant starch, is digestible by microorganisms within the colon.  Lignin, cellulose, and some hemicellulose are not.  (Cellulose and hemicellulose are largely digestible by microorganisms inhabiting the rumen of cattle, sheep, and goats.  Humans keep ruminant livestock in order to indirectly extract nutritional value from those cell wall components that we cannot digest ourselves.  )

Coe (1931) reported that whole seeds of two cultivars of common buckwheat contained 11.0 and 12.6 percent fiber, and one Tartary buckwheat cultivar contained 17.1 percent fiber  on a dry weight basis.  For dehulled groats, the corresponding percentages were 0.85, 1.00, and 1.35. Sindhu and Khatkar (2016) reported that crude fiber constituted 4.06 percent of the dry weight of flour from Tartary buckwheat seeds of variety ‘Shimla-B1.’

The dietary fiber derived from cell walls is most concentrated in those seed tissues with thickened cell walls, such as the hull, the seed coat, and the aleurone layer of the endosperm.  Because common buckwheat hulls are composed largely of cellulose, the total carbohydrate percentage of whole seeds (73 percent) is even higher than that of the groats (68 to 70 percent).  Steadman et al. (2001a) reported TDF in whole groats of common buckwheat (‘Manor’) at seven percent of dry weight.  The concentration in fancy flour ranged from 1.7 to 3.7 percent, whereas the concentration in bran ranged from 13.4 to 40.3 percent.  In 21 Chinese cultivars of tartary buckwheat, Qin and co-workers (2010) found the concentration of crude fiber to average 2.58 percent of flour dry weight.  This was not significantly different from the average concentration in 18 cultivars of common buckwheat (2.30 percent of flour dry weight).  Flour from one Tartary buckwheat cultivar contained over 4 percent crude fiber.

Kuwabara et al. (2007) sprouted seeds of one variety of common buckwheat (‘Kitawasesoba’) and two of Tartary buckwheat (‘Hokkai T8’ and ‘Hokkai T9’) that had been harvested in Hokkaido, Japan. These were germinated in the dark and then grown for 5 days in the light at 22ºC. Leaves and stems were harvested at day 9 (common buckwheat) or day 10 (Tartary buckwheat), weighed, and freeze-dried. Kuwabara et al. (2007) found that total dietary fiber constituted 32.6% of the dry matter of the sprout powder from the common buckwheat variety, 39.1 percent of the dry matter of the sprout powder from ‘Hokkai T8,’ and 36.4 percent of the dry matter of the sprout powder from ‘Hokkai T9.’ Of that total, soluble dietary fiber represented between 16 and 19 percent.

Dietary fiber can reduce the absorption of minerals from the intestine, and impede the digestion of protein within it.  For individuals whose diets are rich in protein and minerals, high intake of dietary fiber is unlikely to have adverse health effects.  In fact, according to Steadman et al (2001a), reduced digestion of buckwheat protein might have positive impacts, including lower levels of cholesterol in the blood.

SOLUBLE CARBOHYDRATES

Much of the soluble carbohydrate in buckwheat seeds comprises short-chain sugar polymers. These are most concentrated in the bran, and least concentrated in the endosperm. When grains sprout, the hydrolysis of starch increases the concentration of soluble carbohydrates.

Besides starch and the compounds collectively labeled as dietary fiber, the seeds of buckwheat contain several soluble carbohydrates. Some of these are as commonplace as sucrose, but others are not found widely in other plant species. Fagopyritols (􏰀α-galactosyl D-chiro-inositols) are a group of oligosaccharides (i.e., short-chain sugar polymers) isolated from buckwheat, and named after the genus Fagopyrum. Fagopyritols labeled “A” contain a chain of one, two, or three galactose residues bonded to the carbon atom designated #3 in D-chiro-inositol. The B series has those same chains bonded to the carbon designated #2 in D-chiro-inositol. (Molecules are diagrammed in Ahmed et al., 2014.)  In the embryos of common buckwheat, fagopyritols B1 and A1 together constitute half of total soluble carbohydrate. In buckwheat seeds, the concentration of these compounds was highest in bran and lowest in the endosperm, with intermediate concentrations in embryonic tissue (Steadman 2001a). Tartary buckwheat contains about half the concentration of fagopyritols reported in common buckwheat. Instead, another carbohydrate that was not found in common buckwheat constituted 31 percent of the soluble carbohydrate in tartary buckwheat. Steadman et al (2000) tentatively identified this compound as rhamnosyl glucoside.

Among non-starch polysaccharides, pentosans contribute to buckwheat’s ability to absorb and retain water. According to Drobot et al. (2014), the level of pentosans is higher in buckwheat than in wheat. Rich in pentosans, buckwheat can be used to improve the baking properties of rye flour, in which water-absorbing capacity is largely determined by non-starch polysaccharides

The amount of soluble carbohydrate increases when seeds are sprouted, as starch is converted into reducing sugars. Xu et al. (2014) reported the proximate analyses of Chinese steamed bread in which 4, 8, or 12 percent of the wheat flour had been replaced by flour ground from either sprouted or unsprouted Tartary buckwheat seeds. After surface sterilization and soaking for 12 hours, whole Tartary buckwheat seeds (cultivar ‘Xinong 9940’) were germinated in the dark at 30ºC for four days, then dried at 60ºC for five hours. Sprouted and unsprouted seeds were then ground and sieved (60 mesh). Doughs of the control (all-wheat flour) and six treatments were risen for 90 minutes at 37ºC before being formed into a round shape and steamed over boiling water for 20 minutes. Starch content in the breads containing unsprouted buckwheat flour (80.4 to 82.9 percent) was higher than the control (80.2 percent); whereas, starch content of the sprouted buckwheat bread was lower (76.4 to 79.1 percent). Reducing sugars were slightly higher in the breads containing unsprouted buckwheat flour (4.0-5.5 percent) when compared to the control (3.0 percent); whereas, concentration of reducing sugars in the sprouted buckwheat bread were much higher (7.6-11.0 percent).

PROTEINS

After carbohydrates, proteins constitute the next most important component of buckwheat seeds. Protein is most concentrated in the embryonic tissue (up to 40 percent of bran dry weight), while the percentage of protein in endosperm is in the 4-7 percent range, and even lower in the hulls. Because the percentage of protein is often estimated from the percentage of elemental nitrogen, and not all N is incorporated in proteins, reported concentrations of protein are often slightly inflated. Storage proteins in seeds are characterized by molecular size, expressed as a sedimentation coefficient. Proteins are sometimes classified by the conditions under which they are soluble.

In plants, proteins serve several essential functions. These include catalyzing metabolic reactions, transporting other molecules and ions, mediating DNA replication, and providing energy reserves. After carbohydrates, proteins constitute the major component of buckwheat seeds. In their sample of 21 Chinese cultivars of Tartary buckwheat, Qin et al. (2010) reported an average crude protein concentration of 10.50 percent of flour dry weight. The average for flour from 18 cultivars of common buckwheat was 10.32 percent—not significantly different. The range in protein content in flour from the Tartary buckwheat cultivars was 6.82 to 15.02 percent, almost twice the range of 8.06 to 12.44 percent in common buckwheat cultivars (Qin et al., 2010).

Coe (1931) reported that whole grains of ‘Japanese’ and ‘Silverhull’ common buckwheat contained 11.9 and 13.1 percent crude protein (dry weight basis), while grains of Tartary buckwheat contained 11.5 percent. For whole groats, the respective percentages were 14.2, 15.7, and 14.5. While protein represented only 5.4 percent of the dry weight of very pale flour, it was as much as 23.6 percent of dark flour. Sindhu and Khatkar (2016) reported that protein constituted 13.91 percent of the dry weight of flour from whole seeds of Tartary buckwheat (cultivar ‘Shimla-B1’). Steadman et al. (2001a) reported that protein constituted 12.3 percent of the dry weight of common buckwheat groats (cultivar ‘Manor’). Protein was concentrated in milling fractions that contained embryonic tissue; protein constituted between18.6 and 39.3 percent of bran fractions. In contrast, fancy flour and grits contained between 4.3 and 6.5 percent protein.

Protein content is rarely measured directly, but is more often estimated from the percentage of elemental nitrogen. Because N constitutes about 16 percent of the molecular weight of most proteins, the percentage of N is frequently multiplied by 6.25 to estimate the percentage of “crude protein.” In fact, this estimate is inflated by the presence of small quantities of non-protein nitrogenous molecules and ions. Protein (as nitrogen) is sometimes detected in the residue of acid detergent fiber (ADF) assays. Much of this fiber-bound protein is assumed to pass undigested from the small intestine. Therefore, “acid detergent indigestible” protein is sometimes deducted from the percentage of crude protein in order to estimate the amount of protein that is available for digestion.

Proteins are large molecules whose basic units—amino acid residues—are arranged linearly. (If such a molecule contains fewer than around 30 amino acid residues, it might be called a “polypeptide” rather than “protein.” ) A peptide bond connects the amine group of one amino acid to the carboxyl group of the adjacent amino acid. In order to function, the linear molecule must stabilized in a three-dimensional arrangement-—what is called the protein’s secondary and tertiary structure. The folded protein structure often has a preponderance of hydrophilic (polar) amino acid residues on its surface, and a preponderance of hydrophobic (non-polar) amino acid residues in its interior. Often a protein’s functional structure will incorporate multiple protein molecules, prosthetic groups, and/or metallic ions.

Besides its attachment to an amine group and to a carboxyl group, the alpha carbon of an amino acid is bonded to a hydrogen atom and to a variable fourth group. That fourth group (or “side chain”—at its simplest, a hydrogen atom) determines the amino acid’s identity and properties. Sulfur-bearing side chains can form disulfide bonds with other sulfur-bearing residues within the protein; polar side chains can participate in hydrogen bonds. Therefore, the precise sequence of amino acid residues determines a protein’s three-dimensional shape, and in turn, its reactivity. Despite their large mass, protein molecules can form colloidal solutions in appropriate solvents. However, heat, acidity, or ionic solutes (i.e., salt solutions) can disrupt a protein’s structure, causing coagulation or “denaturation” of the protein. Historically, the Osborne system has been used to categorize proteins according to their solubility: albumins dissolving in pure water or weak neutral buffers, globulins in salt solutions, glutelins in dilute acidic or alkaline buffers, and prolamins in aqueous alcohol. Guo and Yao (2006) characterized the defatted protein from a commercially-produced Tartary buckwheat flour from Sichuan, China as 43.8 percent albumin, 7.82 percent globulin, 10.5 percent prolamin, and 14.6 percent glutelin (extracted N as a percent of total Kjeldahl N).

According to Drobot et al. (2014), about 80 percent of buckwheat protein comprises albumin and globulin. Cereals, in contrast, contain relatively higher levels of prolamins. This difference is one of the factors that gives buckwheat its higher water-retaining capacity. That water-retaining capacity in turn imparts stickiness to dough that contains buckwheat flour.

In seeds of common buckwheat, proteins are stored in small spherical membrane-bound organelles (Wijngaard and Arendt, 2006). Two predominant globular storage proteins have been isolated and characterized by ultracentrifugation. The larger has two subunits, a sedimentation coefficient of 13S, and a molecular weight of about 280 kiloDaltons. (One kDa is the equivalent of a molecular weight of 1000. ) The smaller storage protein has three subunits, a sedimentation coefficient of 8S, and a molecular weight of about 58kDa. The 13S storage protein, comprising about one-third of total protein in the seed, is localized in the cotyledons. The 8S protein accounts for about 7 percent of total seed protein, and is found in both cotyledons and endosperm. Albumin with a sedimentation coefficient of 2S has also been isolated from buckwheat seeds (Wijngaard and Arendt, 2006).

Guo and Yao (2006) used SDS-polyacrylamide gel electrophoresis to determine molecular weights of the Osborne fractions of Tartary buckwheat flour. Under non-reducing conditions, extract from the defatted flour exhibited electrophoretic bands at 64, 57, 41, and 38kDa. Isolated albumin also showed a double band at 51-52kDa. Globulin also showed major bands at 34, 28, 26, 23, 21, 19 and 15 kDa. Prolamin showed two major bands at 17 and 15 kDa and two minor bands at 20 and 14 kDa. Isolated glutelin did not exhibit well-defined major bands.

DIETARY ADEQUACY OF BUCKWHEAT PROTEIN

The proteins found in both common and Tartary buckwheat seeds contain amino acids in ratios that more closely match the dietary needs of humans than do the proteins from most cereals. However, protein in buckwheat is less digestible than the protein in cereals. The Protein Digestibility-Corrected Amino Acid Score (PDCAAS), factoring in both the proportions of essential amino acids and also protein digestibility, is higher in buckwheat proteins than in some cereal proteins, but lower than in proteins from soybeans or some animal sources.

In the diets of herbivores, proteins from plants provide the raw material for animal proteins and other nitrogenous compounds; moreover, dietary protein can be converted by the liver into glucose (a pathway called gluconeogenesis) to serve as an energy source for the animal’s cells. In the gut of animals, proteolytic enzymes (also called “proteases”) hydrolyze ingested proteins into polypeptides and thence into their constituent amino acids. The latter can then be absorbed from the lumen of the small intestine into the animal’s blood stream. Plant species are able to synthesize all of the 21 amino acids that make up their constituent proteins, but most animals only produce enzymes capable of synthesizing some of those amino acids, and rely on dietary sources for the others. For example, humans normally synthesize 12 amino acids, and rely on dietary sources for the other nine (the latter being designated as “essential amino acids”). The value of protein from a particular foodstuff to the diet of a particular animal depends on that animal’s ability to digest the protein, and then to absorb the free amino acids in proportions that closely match that animal’s needs.

Nutritionists have devised several indices for ranking the dietary adequacy of proteins from different foodstuffs. For this purpose, the U. N. Food and Agriculture Organization (FAO) and the U. S. Food and Drug Administration (FDA) currently recommend the Protein Digestibility-Corrected Amino Acid Score (PDCAAS). The score is based on the growth requirements of young children for six individual amino acids and two combinations of amino acids (i.e., those containing sulfur atoms and those containing aromatic rings). The percentage of each of these in the test protein is divided by the corresponding percentage in this essential panel; the minimum of those eight ratios is designated the Amino Acid Score of the test protein. To calculate the PDCAAS, the Amino Acid Score is multiplied by the True Fecal Digestibility of the test protein. The latter value equals the Apparent Fecal Digestibility (i.e., 1 – fecal N/intake N) corrected for fecal nitrogen from endogenous sources, such as sloughed intestinal cells. By convention, PDCAAS is capped at a maximum score of one.

Eggum et al (1981) published amino acid profiles for two varieties of common buckwheat.  The table below lists the six individual amino acids and two pairs in FAO’s panel.  Ideal concentrations of these amino acids in dietary protein are listed (g/100g), along with the concentrations reported by Eggum et al. (1981) in seeds of the variety ‘Siva dolenjska.’  In the final column the ratio of observed and ideal concentrations is presented.  The lowest of these ratios—-0.88 for lysine—-constitutes the amino acid score for this sample.  For the tetraploid variety ‘Bednja’ the authors reported an amino acid score of 0.89, also based on the concentration of lysine.

essential amino acid

requirement

 

(FAO)

 

 

common BW

 

 

(Eggum et al.)

 

 

ratio

Isoleucine (Ile)

2.8

3.48

1.24

Leucine (Leu)

6.6

6.11

0.93

Lysine (Lys)

5.8

5.09

0.88

Threonine (Thr)

3.4

3.15

0.93

Tryptophan (Trp)

1.1

1.59

1.45

Valine (Val)

3.5

4.69

1.34

Methionine (Met) + Cystine (Cys)

2.5

3.91

1.56

Phenylalanine (Phe) + Tyrosine (Tyr)

6.3

5.78

0.92

Bonafaccia et al. (2003b) published amino acid profiles of proteins from flour and from bran of both common buckwheat and Tartary buckwheat.  These did not differ greatly—either between milling fragments nor between species.  Because the authors presented no data for tryptophan, it’s not possible to calculate AAS for any of the materials.  Common buckwheat has frequently been cited as a plant source of protein with a high AAS (Wijngaard and Arendt, 2006). Ikeda (2002) cited the Resources Council, Science and Technology, Japan (RCSTAJ) in assigning an AAS of 100 for common buckwheat flour.

Using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), Peng et al. (2017) analyzed the free amino acids in both flour and bran from ten varieties of Tartary buckwheat grown in Sichuan, China. Five varieties had brown hulls and five had black. While there was no overall difference between varieties of the two hull colors, the authors found substantial differences among varieties in the concentrations of individual amino acids.   The concentration of Glutamic acid (GLU), the predominant free amino acid, varied from 75.3 to 374.0 mg/100g of flour. The concentration of Isoleucine (ILE), the free amino acid with the lowest concentrations, varied from non-detectable to 0.1 mg/100g of flour. Concentrations in bran were generally a few times higher than in flour from the same variety. The concentration of Glutamic acid ranged from 328.6 to 1195.9 mg/100g of bran. The concentration of Isoleucine ranged from non-detectable to 0.2 mg/100g of bran. By weight, total free amino acids represented around 1-2 percent of bran.

Javornik and co-workers (1981) separated proteins of common buckwheat (‘Siva dolenjska’) into Osborne fractions: 18.2 percent albumins, 43.3 percent globulins, 0.8 percent prolamins, 22.7 percent glutelins, and 15.0 percent non-extractable nitrogenous compounds. The authors attributed buckwheat’s favorable amino acid composition to its lower prolamin content, compared with proteins from true cereals. Guo and Yao (2006) determined the amino acid profiles (excluding tryptophan) of the different Osborne fractions from Tartary buckwheat flour. Considered separately, the albumin, prolamin, and glutelin fractions had amounts of essential amino acids adequate for meeting the dietary needs of children and adults. The globulin fraction (representing 8 percent of total protein N) the authors found slightly inadequate for several essential amino acids.

Contrasting with its favorable amino acid profile, buckwheat proteins exhibit relatively low digestibility. Because dietary fiber and tannins interfere with the digestion of proteins, samples containing a large percentage of hull exhibit particularly low values. Javornik et al. (1981) determined true protein digestibility to be 79.9 and 77.6 percent for common and Tartary buckwheat seeds, respectively. True digestibility of common buckwheat groats was about 10 percent higher. Skrabanja et al. (2000) reported in vitro true digestibility of common buckwheat groats of 91.7 percent. Guo and Yao (2006) performed in vitro pepsin digestions of the Osborne fractions of defatted protein from Tartary buckwheat flour. Those authors reported digestibilities of 81.2, 79.6, 67.0, and 58.1 percent for albumin, globulin, prolamin, and glutelin, respectively. Depending on the species, the cultivar, and the material sampled, PDCAAS of buckwheat would be around 77 percent. By contrast, respective scores for wheat and oats have been reported as 57 and 42 percent; whereas the value for soybean is 96 percent.

Xu et al. (2014) reported protein content in Chinese steamed breads in which 4, 8, or 12 percent of the wheat flour had been replaced by flour ground from either sprouted or unsprouted Tartary buckwheat seeds. Sprouting and bread-making procedures were described above (SOLUBLE CARBOHYDRATES). In breads containing buckwheat flour, the protein concentration was between 10.7 and 12.5 percent on a dry weight basis—similar to the 12.3 percent protein content of the all-wheat control.

GABA

Gamma-aminobutyric acid is a compound with putative healthful effects.  Substitution of tartary buckwheat flour for wheat flour increased the concentration of GABA in Chinese steamed bread.  The stress of hypoxia at certain times during germination could increase the concentration of GABA in Tartary buckwheat sprouts.

Gamma-aminobutyric acid is a 4-carbon amino acid—each molecule containing an amine group and a carboxyl group; however, it is not one of the alpha amino acids of which proteins are composed.  Studies suggest that GABA in the diet can reduce hypertension, improve brain function, regulate cardiac arrhythmia, and relieve pain or anxiety.

Peng et al. (2017) reported the concentration of GABA in both flour and bran from ten varieties of Tartary buckwheat grown in Sichuan, China. Among those varieties, the concentration of GABA varied from 30.6 to 112.5 mg/100g of flour, and from 60.6 to 324.1 mg/100g of bran.

In their study of Chinese steamed breads (see SOLUBLE CARBOHYDRATES section), Xu et al. (2014) reported that the concentration of GABA, only 0.18mg/100g in the all-wheat bread, was 7.52mg/100g in the bread containing 12 percent sprouted buckwheat flour.

Guo and co-workers (2016) investigated hypoxia as an environmental factor that might increase the level of GABA in Tartary buckwheat sprouts.  Those authors soaked seeds (variety ‘yu 6-21’) in the dark at 30ºC for four hours.  Seeds were then germinated under “normal” and/or “hypoxic” conditions for varying intervals.  (The former included spraying with distilled water.  The latter included metered aeration in a citric acid solution that was renewed every two days.  )  Seeds held for two days under normal conditions followed by two days under hypoxic conditions accumulated 372 µg GABA per gram of plant dry matter, 2.1 times the concentration found in sprouts maintained for 4 days under normal conditions.

After germinating tartary buckwheat seeds for two days, the investigators tracked sprout length, respiratory rate, and the concentrations of soluble protein and free amino acids for the subsequent 48 hours under “normal” and “stressed” treatments.  (The latter comprised limited aeration and pH of 1.0.  )  After 48 hours of stress, sprout elongation was inhibited by 18.54 percent and respiratory rate was elevated by 29.83 percent, compared to the checks.  As levels of soluble protein decreased, the accumulation of free amino acids was 64.91 percent greater in the stressed sprouts than in the checks.

Because GABA is synthesized mainly through the GABA shunt pathway, in which glutamate decarboxylase (GAD) is the limiting enzyme, Guo and co-workers measured the effect on GAD activity of the stress treatment.  In both stressed and control sprouts, GAD activity peaked at 40 hours.  At 48 hours, GAD activity was 36 percent higher in stressed sprouts than in the checks.

THE PROCESS OF PROTEIN DIGESTION

The low digestibility of protein in buckwheat seeds, compared with other grains, is due in part to tannins, which can bind to proteins and protect them from proteolytic enzymes.  Buckwheat also contains compounds that specifically inhibit trypsin and other proteolytic enzymes.  Such inhibition is diminished as seeds sprout.

In the human digestive tract, proteins are hydrolyzed by the enzyme pepsin within the stomach, and subsequently by pancreatin (comprising several enzymes) within the small intestine.  Ma and Xiong (2009) investigated in vitro digestion of protein isolated from defatted flour of common buckwheat.  The protein isolate was shaken in the pepsin solution at pH 2.0 at 37ºC for one hour.  The pH was adjusted to 5.3; pancreatin was added; pH was further raised to 7.5, and the solution was shaken at 37ºC for an additional two hours.  Aliquots taken at 0, 30, 60, 90, 120, and 180 minutes during this process were subsequently analyzed for the degree of hydrolysis, and the solubility and size of the products at each stage.  The degree of hydrolysis (total free amino acid concentration) reached less than seven percent after 60 minutes of pepsin digestion.  After the initial 30 minutes of pancreatin digestion, hydrolysis had reached 34 percent; at the end of the treatment, 52 percent of the buckwheat protein had been hydrolyzed to free amino acids.  Electrophoresis of the products of pepsin digestion revealed relatively large peptides (a dark smear over a range of molecular weights >14 kDa).  After pancreatin digestion, only some faint 14 kDa bands remained.  Treatment with β-mercaptoethanol (which severs disulfide bonds) further reduced the size of the residual peptides.  Over the course of in vitro digestion, solubility at first decreased—perhaps because the pepsin clipped more polar polypeptides from the surfaces of the buckwheat protein, exposing the more hydrophobic interior.  With subsequent digestion with pancreatin, the protein solubility was enhanced—perhaps by the generation of low-molecular-weight peptides with exposed NH4+ and COO groups.

The low digestibility of protein in buckwheat seeds, compared with other grains, is due in part to tannins, which can bind to proteins and protect them from proteases.  Buckwheat also contains compounds that specifically block proteases, such as trypsin inhibitors.  These latter compounds are resistant to elevated temperatures and acidic conditions.  However, the germination of buckwheat seeds reduces the inhibition of proteases.  Therefore, for animal nutrition buckwheat sprouts are superior to ungerminated seeds in terms of the protein utilization (Ahmed et al., 2014).

LIPIDS

Compared to cereals, both common and Tartary buckwheat are relatively rich sources of lipids, particularly unsaturated fatty acids.  In samples of several accessions, the two species showed minor but consistent difference in fatty acid profiles.  Despite the predominance in both species of unsaturated steric and linoleic acids, oils in buckwheat seeds were relatively stable during prolonged storage.

Lipids include fatty acids—-molecules with a carboxylic (COOH) group forming the end of an even-numbered chain of carbon atoms.  Besides existing in a free form within plant cells, fatty acids may be bound to other molecules that alter their solubility in either non-polar or polar solvents.  For example, three fatty acid molecules can form ester bonds to the three hydroxyl (OH) groups in a molecule of glycerol.  The resultant triglyceride is a neutral lipid.  On the other hand, if a fatty acid bonds with various sugar molecules, the resulting glycolipid could dissolve in weak polar solvents, like acetone.  Similarly, if a fatty acid bonds with a phosphate group, the resulting phospholipid is soluble in methanol.  If each carbon atom along the fatty acid’s spine is bonded to the maximum potential number of hydrogen atoms, then the fatty acid is said to be “saturated.”  “Unsaturated” fats contain pairs of adjacent carbon atoms connected by double bonds (one such double bond in a “mono-unsaturate” and two or more double bonds in a “poly-unsaturate”).  As a shorthand notation, fatty acids are often identified as “Cn1:n2” with n1 being the number of carbon atoms in the chain, and n2 being the number of double bonds.  For example, C20:0 represents arachidic acid, comprising 20 carbon atoms with no double bonds (i.e., a saturated fatty acid).  Contrasted to saturated fats, unsaturated fats generally remain liquid at lower temperatures, and are more susceptible to oxidative reactions that can cause rancidity.

Mazza (1988) measured various lipid fractions in groats of the three cultivars of common buckwheat then most widely grown in North America.  Total lipids represented from 2.6 to 3.2% of dry weight.  The concentration of neutral lipids was highly correlated with total lipids (r=.94), and ranged from 1.6 to 2.2% of dry weight.  In all classes of lipids from all three cultivars, oleic (C18:1) and linoleic (C18:2) fatty acids predominated, with palmitic acid (C16:0) having the next highest concentration.  (Linoleic acid is considered an essential nutrient, since it is not synthesized by the human body.  )  The author noted that despite the prevalence of unsaturated fats, buckwheat seeds that had been stored for 25 months at room temperature nevertheless showed very little change from fresh seeds in their fatty acid profile.

Tsuzuki and collaborators (1991) reported the fatty acid profiles of groats from ten accessions of Tartary buckwheat collected from several countries.  The observed ranges were 16.0-17.5 percent palmitic acid (C16:0), 1.8-2.3 percent stearic acid (C18:0), 0.9-1.2 percent arachidic acid (C20:0), 0.9-1.1 percent behenic acid (C22:0), and 0.5-0.7 percent lignoceric acid (C24:0).  Of total fatty acids, 33.5-39.3 percent were oleic acid (C18:1), 33.9-39.7 percent were linoleic (C18:2), 1.3-2.6 percent were linolenic acid (C18:3), 1.9-2.3 percent were gadoleic acid (C20:1), and 0.5-0.6 percent were C22:1.  Comparing these Tartary buckwheat accessions with 24 diverse accessions of common buckwheat, each species showed a relatively consistent and distinctive profile of fatty acids.  Tartary buckwheat contains relatively less C18:3 and C20:1 but more C22:1 unsaturated fatty acid than does common buckwheat.

Bonafaccia et al. (2003b) reported the following fatty acid profile for a single Tartary buckwheat cultivar from Luxembourg.  Saturated fatty acids with 16, 18, 20, and 22 carbon atoms constituted 19.7 percent, 3.0 percent, 1.8 percent, and 0.8 percent, respectively.  Unsaturated C18:1, C18:2, C18:3 and C20:1 fatty acids constituted 35.2 percent, 36.6 percent, 0.7 percent, and 2.0 percent, respectively.  Due mostly to the greater proportion of palmitic acid (C16:0) in the Tartary buckwheat, the ratio of unsaturated to saturated fats was smaller than in the common buckwheat cultivar, “Sivi”  (2.94 versus 3.87).

Using gas chromatography-mass spectrometry (GC-MS), Peng et al. (2017) analyzed free fatty acids in both flour and bran from ten varieties of Tartary buckwheat grown in Sichuan, China. (Bran constituted the fraction of milled whole seeds that passed through a 24-mesh sieve but was retained by a 65-mesh screen; flour constituted that fraction passing through the finer sieve. ) The researchers detected four saturated fatty acids: myristic (C14:0), palmitic (C16:0), stearic (C18:0), and arachidic (C20:0) acids. [Note that C14:0 had not been detected in the sample of Tsuzuki et al., but those authors did detect C22:0 and C24:0, and Peng et al. did not. ] Palmitic acid was found to be the main saturated fatty acid, ranging from 112.7 to 327.9 mg/100g of flour, and ranging from 365.8 to 503.6 mg/100g of bran. The least prevalent of the saturated fatty acids, arachidic acid ranged from 14.2 to 16.9 mg/100g of flour, and ranged from 25.2 to 35.8 mg/100g of bran. The researchers also detected four unsaturated acids: oleic (C18:1), linoleic (C18:2n-6c), linolenic (C18:3n-3), and cis-11-eicosenoic (C20:1) acids. [Note that Tsuzuki et al. also found C22:1 in their sample. ] Like Bonafaccia et al., Peng and co-workers reported that linoleic and oleic acids are the dominant fatty acids in Tartary buckwheat. These acids had respective concentrations of 281.6 and 257.2 mg/100 g in flour and 818.7 and 620.6 mg/100g in bran. The ratio of total unsaturated fatty acids to total saturated fatty acids ranged from 1.27 to 2.71 in the flour of the 10 varieties; that ratio ranged from 1.50 to 3.12 in the bran of the 10 varieties. Total fatty acid concentration ranged from 718 to 1118 mg/100g of flour, and from 1528 to 2417 mg/100g of bran. For individual fatty acids, the average concentration in the bran as between 1.4 and 3.1 times as great as the average concentration in the flour. The authors found that few of the fatty acids had significantly different concentrations in five black-hulled varieties compared to five brown-hulled varieties.

Sindhu and Khatkar (2016) reported that crude fat constituted 2.42 percent of the dry weight of flour from whole seeds of Tartary buckwheat (cultivar ‘Shimla-B1’). In flour from 21 cultivars of Tartary buckwheat, Qin et al. (2010) found that the percentage of fat varied from 1.2 to 4.7 percent.  The minimum and maximum in flour from 18 cultivars of common buckwheat were 1.5 percent and 5.4 percent fat, respectively. In both common and Tartary buckwheat, lipids are concentrated in the embryo.  The embryo contains an average of 6.5 percent oil, while the endosperm contains less than a tenth that amount.  As discussed above, pale “fancy” buckwheat flour contains mostly endosperm, while much of the embryonic tissue ends up in the bran, and in flours rich in bran.  Steadman et al. (2001a) reported that the total lipid in “fancy” flour (cultivar ‘Manor’) ranged from 0.6 to 1.5g/100g dry weight.  Total lipid in bran ranged from 5.1 to 12.6g/100g.  Tsuzuki and collaborators found little variation among the fatty acid profiles of five different milling fractions from seeds of common buckwheat (‘Hokkaido-Botan’).  The risk of rancidity apparently depends on the quantity rather than the identity of fatty acids in different milling fractions, with “fancy” flour likely having the greatest stability during storage.

Xu et al. (2014) reported the proximate analyses of Chinese steamed breads in which 4, 8, or 12 percent of the wheat flour had been replaced by flour ground from either sprouted or unsprouted Tartary buckwheat seeds.  Sprouting and bread-making procedures were described above (SOLUBLE CARBOHYDRATES). Compared with the all-wheat control, which contained 2.2 percent fat on a dry weight basis, fat content increased with increasing fractions of unsprouted buckwheat flour (3.7-4.2 percent), but declined with increasing fractions of sprouted buckwheat flour (2.9-1.1 percent).

MINERALS

Ash, the various minerals remaining after tissue is burned, constitute about 2-3 percent of the dry weight of buckwheat seeds, and a slightly smaller percentage of the groats. Grown on soils high in aluminum or lead, buckwheat had the capacity to accumulate high concentrations of those metals. Among accessions of buckwheats grown in a common field, concentrations of lead, tin, and chromium in seeds were highly correlated. Concentrations were generally higher in roots, stems, or leaves than in seeds. Concentrations of minerals were generally higher in tartary than in common buckwheat. Concentrations were generally higher in the bran than in pale flour. In some Tartary buckwheat teas from China, some elements were found at toxic concentrations; plants grown for food should not be grown on polluted soils. On the other hand, foliar applications of selenium can increase that element’s concentration in seeds, augmenting dietary sources in areas where selenium is deficient.

Plant photosynthesis converts carbon dioxide and water into carbohydrate—organic compounds containing carbon, oxygen, and hydrogen. Nitrogen, the other major element found in plant proteins and nucleic acids, derives ultimately from atmospheric N2, which has been converted to usable NH3 either by bacterial or industrial fixation. All the other “mineral” elements present in plant tissues have been taken up by roots from the soil. (Minerals deposited on and absorbed through leaf epidermis also contribute to plants’ mineral content, but foliar uptake is usually minimal compared to that from the soil. ) When plant matter is combusted, carbon, oxygen, hydrogen, and nitrogen are all liberated back into the atmosphere. The other elements remain as ash. Therefore, ash has been used in proximate analysis as a general indicator of how much total mineral a plant contains. For example, Coe (1931) found that ash constituted 1.84 percent of the dry weight of whole tartary buckwheat seeds. Whole seeds of two cultivars of common buckwheat contained 1.90 and 2.01 percent ash. The ash in hulled tartary buckwheat groats constituted 2.11 percent of dry weight, intermediate between the 1.95 percent and 2.14 percent ash in groats of two cultivars of common buckwheat. Sindhu and Khatkar (2016) reported that ash constituted 2.81 percent of the dry weight of flour from whole seeds of Tartary buckwheat (cultivar ‘Shimla-B1’). Qin et al. (2010) reported that flours from 21 Tartary buckwheat accessions contained on average significantly more ash than flours from 18 common buckwheat accessions (2.38 percent versus 2.17 percent of dry weight). The maximum observed concentration of ash was very similar in Tartary and common buckwheat (3.11 and 3.09 percent of dry matter, respectively); the minimum level observed in common buckwheat accessions was substantially lower than that in Tartary buckwheat (1.33 versus 1.85 percent).

Plants grow in soils that vary widely in their mineral composition. Nevertheless, for many elements, the cytoplasm of most plant cells must occupy a fairly narrow range between minimal requirement and toxic excess. Plants have therefore evolved mechanisms either to exclude certain elements, or to convert them to less-toxic forms (i.e, complexation), or else to sequester them within organelles where they cannot disrupt cellular metabolism. Common buckwheat, for example, is unusually tolerant of soils rich in available aluminum (AL+3), an element that limits the growth of many crops on acid soils.

Zheng et al. (1998) demonstrated that root elongation of 3-day-old seedlings of common buckwheat (cultivar ‘Jianxi’) was barely reduced by exposure for 16 hours to 25 μM AlCl3 (at pH 4.5). In contrast, that same exposure reduced by a third the length of root elongation of a wheat cultivar exceptional for its tolerance to aluminum. These authors studied exudates from roots of 2-week-old buckwheat seedlings exposed for 6 hours to 50 μM AlCl3. Oxalic acid was secreted at 0.7 μmol per hour per g root dry weight–an 8-fold increase over plants not exposed to Al. This response was specific to aluminum, and could be induced neither by a similar exposure to lanthanum, nor by a deficiency in phosphorus. Following a 3-hour pulse treatment with 150 μM AlCl3, root tips exuded oxalic acid at high levels for 8 hours, and still exceeded levels from control plants after 30 hours. By complexing aluminum ions with the oxalate ion, the former were immobilized within the soil, reducing the concentrations that would enter the buckwheat roots.

In leaves of buckwheat seedlings exposed intermittently for 10 days to 50 μM Al (and an aluminum-free nutrient solution on the alternate days), concentrations of 2.01 mmol Al per kg fresh weight were measured by Ma et al. (1998). This was about ten times the level of aluminum found in leaves of wheat or rape plants after a similar exposure. About 90 percent of that aluminum was found in the cell sap in buckwheat leaves. Following a 20-hour exposure to the same concentration of aluminum, roots of 20-day-old plants contained 3.45 mmol Al per kg fresh weight, almost 60 percent of which was in the cell sap. Ma and co-workers found that Al was chelated with organic acids, primarily oxalic acid. Further analyses indicated that 1:3 Al-oxalate complexes predominated in leaves, and 1:2 and 1:3 Al-oxalate complexes were present in roots. The authors demonstrated that there was no significant inhibition of maize seedlings following a 20-hour exposure to 20 μM Al complex purified from the cell sap of buckwheat leaves. In contrast, maize root elongation was inhibited by 20 μM AlCl3. The 1:3 Al-oxalate complex is very stable, effectively preventing aluminum ions from disrupting other critical metabolic reactions. Apparently, this internal detoxification mechanism complements the external detoxification that buckwheat accomplishes by excreting oxalic acid.

Following the same intermittent exposure of seedlings to aluminum, Shen et al. (2002) isolated protoplasts and vacuoles from buckwheat leaves. The authors found that within leaf cells most of the Al-oxalate complex was localized inside the vacuoles. Common buckwheat, therefore, not only uses external and internal complexation to detoxify aluminum, but also sequesters the element within cellular organelles where it will not interfere with the critical cellular processes.

Wang et al. (2015) investigated aluminum tolerance of Tartary buckwheat (cultivar ‘Rotundatum’) and wild buckwheat (F. homotropicum cultivar ‘Mianshawan‘). When exposed for 24 hours to 30μM AlCl3, 3-day-old seedlings of both species exhibited root elongation equivalent to that in common buckwheat (cultivar ‘Jianxi’). However, similar exposure to 50μM AlCl3 depressed root elongation in Tartary buckwheat more than in either other species.  After intermittent exposure to 30μM AlCl3, 12-day-old seedlings of tartary and wild buckwheat accumulated more aluminum in their leaves than common buckwheat, while exhibiting no signs of toxicity. After 20 days of intermittent exposure to 50μM AlCl3, the cell sap from roots of Tartary buckwheat seedlings contained almost twice the level of aluminum as common buckwheat; concentrations in xylem cell sap did not differ significantly among the three species.  In response to aluminum, all three buckwheat species exuded oxalate from their roots, and all three accumulated aluminum in roots and leaves as Al-oxalate complexes in a 1:3 ratio.  In contrast, aluminum in xylem sap from all three species was complexed with citrate in a 1:1 ratio.  These authors concluded that the same mechanisms of external and internal detoxification of aluminum had been conserved during the evolution of the Fagopyrum genus.

Of the buckwheat tissues shown to accumulate aluminum, roots are not consumed by people, and leaves are consumed only in a few cultures, such as Korea. Moreover, because aluminum is relatively non-toxic for humans, occasional high levels of aluminum in buckwheat plants do not present a dietary risk. (Oxalic acid can burn skin, eyes, and mucous membranes; nevertheless, levels reported above are less than those consumed in many vegetables. ) Tamura et al. (2005) reported that common buckwheat grown on lead-polluted soil can accumulate that element in roots, stems, shoots, and leaves. Grown on soil with a HCl-extractable lead concentration of 6.6g/kg, 8-week-old buckwheat plants contained 8g/kg of leaf dry weight. Because lead is very toxic to humans, care should be taken to grow buckwheat crops only in soils that do not contain elevated levels of that metal, either naturally-occurring or due to pollution.

Peng et al. (2014) measured elemental concentrations in seeds from 11 Chinese accessions of Tartary buckwheat and five Chinese accessions of common buckwheat. All plants had been grown together in a field in Chengdu, China, and received compound fertilizer at a rate of 20kg/acre. The authors did not publish their complete data set, but they noted strong correlations among the concentrations of lead, tin, and chromium (correlation coefficients >0.89). Strong correlations were also noted among the concentrations of vanadium, aluminum, iron, and titanium (correlation coefficients >0.70). Notably, the authors reported that there were no clear delineations between the two buckwheat species in their elemental analyses. Peng and co-workers also reported elemental concentrations in ten samples of Chinese commercial varieties of “bitter buckwheat tea.” They noted that toxic elements reached significant concentrations in a few of the teas (e.g., lead > 18 μg/g in one sample).

Huang et al. (2013) measured elemental analyses of different organs from three of those cultivars of common buckwheat and two of those cultivars and one wild accession of Tartary buckwheat. They found that roots had the highest concentrations of aluminum, cobalt, chromium, copper, iron, titanium, and vanadium. Calcium, sodium, potassium, nickel, strontium, and zinc had slightly higher concentrations in the stems than in roots or leaves. Boron, magnesium, molybdenum, silica, cadmium, and lead showed the greatest accumulations in leaves. In the cultivars and minerals that were tested, only phosphorus exhibited higher concentrations in seeds than in other organs. Levels ranged between 588 and 2243 μg P/g seed dry weight.

Huang et al. (2014) reported concentrations of potassium, magnesium, copper, zinc, and iron in seeds of 123 accessions of Tartary buckwheat from the main growing areas of China. Accessions were planted together on August 15, 2010 in Guiyang, China, and harvested on November 10. Whole seeds were ground to pass through a 60-mesh screen. Elemental concentrations were then determined by flame atomic absorption spectrometry. The range of concentrations of K was 1737–5831 mg/kg; for Mg, the range was 729–3104 mg/kg. Ranges for Cu and Zn were 5.7–36 and 8.4–67 mg/kg, respectively. An 18-fold range was reported for the concentration of Fe: 22–3990 mg/kg. The concentration of Zn was significantly positively correlated with concentrations of Mg, Fe, and Cu. The concentration of Mg was similarly correlated with those of K and Fe. However, all these correlation coefficients were less than 0.5. These Chinese Tartary buckwheat accessions did not exhibit significant regional differences in their uptake of any of these five elements.

As we pointed out above, the nutritional claims about tartary buckwheat are sometimes based on sparse or inconclusive data. The study by Huang et al. covered a large number of accessions. The investigators carefully confirmed that their analyses of the samples were both repeatable and accurate. However, they did not replicate the plots in which each accession was grown in the field. Soil concentrations of minerals often vary substantially over small distances; therefore, much of the variability attributed to genetic differences among accessions in their efficiency of mineral uptake might instead be due to spatial variability in mineral availability across the experimental field.

Pongrac et al. (2016) reported concentrations of elements in seeds of Tartary buckwheat (‘Wellker’) grown at Sentjernej, Slovenia. Those authors also reported elemental concentrations in the sprouts of tartary buckwheat that had been germinated in spring water containing elevated levels of several minerals. Compared to controls sprouted with tap water, germination five days after sowing was unaffected by the moderately elevated mineral levels, although pH of the latter rose to 9.05 (versus 6.4 for controls). Eight days after sowing, treated sprouts (shoots and roots) showed significantly elevated levels of Na, Mg, Cl, K, and Mn. Lower concentration of Zn in the sprouts reflected the lower concentration of that element in the spring water, compared to the tap water control. Ca was also lower sprouts grown in the mineral-enriched water, probably due to calcium precipitate observed in that treatment. Tartary buckwheat shoots died following sprouting in water from a spring containing even higher mineral concentrations.

Bonafaccia and co-workers (2003c) measured the concentrations of several elements in milling fractions of both common and Tartary buckwheat. The former (cultivar ‘Siva’) had been grown in Slovenia; the unnamed variety of the latter had been grown in Luxembourg. Milling of common buckwheat yielded 55.4 percent flour, 24.2 percent bran, and 17.4 percent hull; milling losses were three percent. Tartary buckwheat had almost identical yields of flour and bran. With additional sieving of the tartary buckwheat flour, a second, more refined flour was produced with a 42 percent yield. With the exception of rubidium, silver, and mercury, all the elements tested had higher concentrations in the grains of Tartary buckwheat than common buckwheat. For most of the elements, concentrations were higher in bran than in flour, and lowest in the extra-fine flour. The authors also powdered dried leaves of tartary buckwheat; this leaf flour contained several elements (Se, Fe, Co, Rb, Sb, Ag, Cr, and Sn) at concentrations two or more times that found in the grain.
 Golob et al. (2016b) measured selenium levels in stems, leaves, seeds, and hulls of tartary buckwheat (0.019, 0.041, 0.013, and 0.048 μg/g dry weight, respectively). That concentrations were lower than those reported by Bonafaccia and co-authors might have been due to the cultivar, the location (Ljubljana, Slovenia), the growing season, and/or the analytical methods. While selenium can be toxic at higher concentrations, this element is an essential nutrient for humans and livestock because of its role in several antioxidant enzymes. In the diets in many European countries, selenium levels are below the recommended daily intake (30-70 μg Se per day for adults). Golob and co-workers tested whether selenium levels in Tartary buckwheat seeds could be raised by spraying the growing plants with a solution of sodium selenate (SeVI). They reported an 83-fold increase in selenium concentration in leaves and a 276-fold increase in the concentration in seeds. Following enzymatic digestion of seed proteins, almost half of the total selenium was detected in a soluble form. About one-third of the total selenium content was in the bio-available form of Se-methionine.

Due to the similarities between selenium and sulfur, an ionic form of the former element (selenate) is believed to be absorbed, translocated, and assimilated in plants via the same pathway as the sulfate ion. Golob and co-workers (2016a) found that sulphur interferes in complex ways with the uptake of selenium by buckwheat. Both common buckwheat (‘Darja’) and a Slovenian landrace of Tartary buckwheat were grown in acidic soil in Bosnia, and prior to flowering were subjected to foliar spray of sodium selenate and/or sodium sulfate (126 µM concentration, applied at 90ml/m2). Control plots were sprayed with water. The authors measured the concentrations of selenium in roots, leaves, seeds, and hulls of mature plants. They reported that in both species, there was a significant increase in the concentration of Se due to the selenium application, but not to sulfur when applied alone. In tartary but not common buckwheat, the concentration of Se in plants treated with Se + S was greater than in plants treated only with Se. This difference was significant (p<0.05) in all plant tissues. Golob and co-workers also reported that in Tartary buckwheat (but not common buckwheat), the yield of above-ground biomass was depressed by all three treatments. In neither species was grain yield or 1000-grain mass significantly affected by any of the treatments. The authors speculated that foliar application of selenate might compete with sulphate, stimulating the sulphate starvation pathway and activating sulphate transporters. These transporters could be responsible for increased selenate accumulation.

Golob et al. (2016b) investigated the possible impact of these foliar applications of Se and S on the concentration of selenium in progeny of Tartary buckwheat plants. Seeds from the treated plants were grown during the following year, and levels of Se were measured in roots, leaves, and seeds. In both the roots and leaves, the level of Se was significantly greater in progeny of Se-treated plants than the progeny of either S-treated or control plants.

FLAVONOIDS AND ANTIOXIDANT PROPERTIES

Many investigators have studied flavonoids in buckwheat, because the presence of these compounds in the diet has been shown to benefit human health. Of primary interest are quercetin and three of its glycosides—isoquercetin, quercitrin, and rutin. Concentrations of these flavonoids vary among different plant tissues and over the maturation cycle. They are affected by conditions prior to and following harvest of the crop, and by its subsequent processing for consumption. Because enzymes can attach and detach sugar chains from the basic polyphenol molecule, treatments that deactivate these enzymes affect the relative concentrations of flavonoids. In general, rutin is found in higher concentrations in leaves and flowers than in other plant parts. The seeds of Tartary buckwheat contain substantially higher concentrations of rutin than do the seeds of common buckwheat.

Normal metabolism as well as exogenous sources expose cells to reactive oxygen species (or “oxygen free radicals”), which in excessive amounts can cause intracellular damage. These free radicals have been implicated in degenerative human diseases, including coronary heart disease, stroke, rheumatoid arthritis, and cancer. Therefore, foods capable of breaking down reactive oxygen species have been promoted for reducing the risks of such diseases. Among the compounds from plants best known for antioxidative capacity are flavonoids (or “anthoxanthins”)—a class of molecules characterized by two phenol groups linked to a heterocyclic ring, the latter bearing a ketone group. (At its simplest, a phenol group consists of a hydroxyl group directly bonded to an aromatic hydrocarbon ring. In flavonoids, the heterocyclic ring comprises five carbon and one oxygen atoms. The ketone group comprises an oxygen atom double-bonded to the carbon atom positioned opposite the oxygen atom within the heterocyclic ring. Because each flavonoid molecule bears two phenol groups, it could also be classified as a “polyphenol.” Whew! )

Many of the studies mentioned below have employed any of a handful of in vitro assays to estimate the antioxidative capacity of individual flavonoid compounds or of extracts from various buckwheat tissues. Note that there is little evidence that ingested flavonoids act as antioxidants in vivo.

Wang and Hu (2013) performed chemical and spectral analyses of flavonoids in Tartary buckwheat seeds from Ring County, China. These authors identified quercetin, rutin, vitexin, orientin, hyperin, tricin-7-O-β-D-glucopyranoside, chrysoeriol, tectochrysin, and luteolin-7-O-β-D-glucopyranoside. Three of the flavonoids found in buckwheat plants are compounds in which a quercetin molecule is conjugated to a sugar molecule—glucose in the case of isoquercetin, the simple sugar rhamnose in the case of quercitrin, or the disaccharide rutinose in the case of rutin. [Diagrams of these molecules are presented on p 314 of Wang et al. (2013) ]

These conjugation reactions and also their reverse (i.e., hydrolysis of the flavonoid glycoside) are mediated by particular enzymes. For example, the enzyme rutin glucosidase hydrolyzes rutin to quercetin and rutinose. Cui and Wang (2012) isolated and purified a rutin-hydrolyzing enzyme from whole seeds of Tartary buckwheat. By SDS-PAGE, they estimated the enzyme’s molecular weight as 70kDa. Its isoelectric focusing point was 6.7, differentiating this enzyme from glycosidases found in common buckwheat. (Note that the molecular mass of rutin is 610.52g per mol, whereas that of quercetin is 302.236g per mol; therefore, for each gram of rutin that is hydrolyzed, only about 0.5 g of quercetin would be generated. )

Despite the established (in vitro) antioxidant properties of flavonoids, there are suggestions that such compounds can also behave as pro-oxidants, depending on the source and the concentration of the free radicals. Moreover, when tested at high concentrations in both bacterial and mammalian systems, quercetin and some other flavonoids have been shown to damage DNA.

Jiang et al. (2007) compared the rutin concentrations in finely ground dehulled seeds from four accessions of Tartary buckwheat (from China and Nepal), three accessions of F. homotropicum (from SW China), and four cultivars of common buckwheat (from Canada). The average rutin concentration was 1.67 percent in Tartary buckwheat, 0.10 percent in F. homotropicum, and 0.02 percent in common buckwheat. On average, rutin constituted 82 percent, 29 percent, and 54 percent of total flavonoid content in Tartary buckwheat, F. homotropicum, and common buckwheat, respectively.

Brunori and Végvári (2007) measured the concentration of rutin in three varieties of Tartary buckwheat grown on the Pollino massif in the Region of Basilicata, Italy. Whole seeds ground in a cyclone mill were extracted in methanol for 24 hours at room temperature in the dark. Samples were analyzed by HPLC (high-performance liquid chromatography). Rutin constituted 1.71 percent, 2.08 percent, and 2.13 percent of the dry weight of ‘Ishisoba,’ ‘Golden,’ and ‘Donan’ Tartary buckwheat, respectively. Levels in common buckwheat varieties grown by the same researchers in the Region of Calabria were two orders of magnitude lower. In those 15 varieties, there was little relationship between rutin concentration and grain yield (R2=0.05).

Qin and co-authors (2010) measured the concentrations of flavonoids in 21 cultivars of Tartary buckwheat and 18 cultivars of common buckwheat. The ranges in concentrations of rutin, quercetin and total flavonoid in the flour of the Tartary buckwheat cultivars were 6.06–18.67 mg/g, 0.31–2.38 mg/g, and 6.6-22.74 mg/g, respectively. The ranges in the concentrations of rutin and total flavonoid in the flour of common buckwheat cultivars were 0.15–1.68 mg/g and 0.67–2.25 mg/g, respectively. Quercetin was detectable in only two of the samples of common buckwheat flour.

Morishita et al. (2007) measured the concentrations of several polyphenol compounds in groats of two varieties of common buckwheat (‘Kanto #1’ and ‘Hitachi akisoba’) and two of Tartary buckwheat (‘Rotundatum’ and ‘Pontivy’). The common buckwheat varieties contained (-)-epicatechin (15.6 and 20.2 mg/100g DW) and (-)-epicatechingallate (1.3 and 2.4 mg/100g) in addition to rutin (12.2 and 13.6 mg/100g). In contrast, the Tartary buckwheat varieties contained quercetin (2.0 and 2.4 mg/100g) and quercitrin (95.4 and 81.2 mg/100g) in addition to rutin (1809 and 1854 mg/100g).

These researchers estimated the antioxidative capacities of flour from these four varieties from DPPH radical-scavenging activity after 20 minutes; Trolox was used as a standard. The Tartary buckwheat varieties showed between 3 and 4 times the antioxidative activity of the common buckwheat varieties. Based on the antioxidative capacities of the five listed polyphenols, and on their concentrations in groats of the four varieties, their contributions to the observed antioxidative activity of the flours were computed. In the common buckwheat varieties, (-)-epicatechin was the most important identified anti-oxidant, but most of the activity could not be attributed to any of those compounds. In the Tartary buckwheat varieties, rutin was by far the most important antioxidant.

Vogrincic et al. (2010) measured concentrations of flavonoids in Tartary buckwheat flour of the cultivar ‘Wellkar.’ Seeds were milled to <236μm (producing a flour yield of 42 percent w/w). The concentrations of rutin and quercetin were 11.67mg/g and 0.63mg/g of dry flour, respectively. Total polyphenols were expressed in equivalents of gallic acid, or 13.08mg GAE/g of dry flour. Vogrincic et al. (2013) measured the concentrations of rutin and quercetin in methanol extracts (0.04g/ml) of flours of common and Tartary buckwheat. The flours had been ground from air-dried seeds from plants (cultivar not stated) grown on an experimental farm in Ljubljana, Slovenia. The extract of common buckwheat flour contained 6.1μg rutin/ml and undetectable levels of quercetin; in contrast, the extract of Tartary buckwheat flour contained over 770μg rutin/ml and 86μg quercetin/ml.

Peng et al. (2017) employed high performance liquid chromatography-ultraviolet detector (HPLC-UV) to measure the concentrations of phenolic compounds in the flour and bran of ten varieties of Tartary buckwheat grown in Sichuan, China. The researchers detected kaempferol, vitexin, caffeic acid, quercetin, quercitrin, and rutin. Among these ten varieties, the total concentrations of these eight polyphenols ranged from 864 to 1616 mg/100g of flour, and from 3886 to 5738 mg/100g of bran. Kaempferol and vitexin were present in minimal amounts; rutin constituted between 86 and 94 percent of the total amount. In one variety, the concentration of rutin was 5.67 times as great in the bran compared to the flour, and the concentration of quercitrin was 6.12 times as great in the bran compared to the flour. Averaged across varieties, the respective ratios were 4.33 and 4.64. Across varieties, the ratio of total concentration of polyphenols in bran to that in flour ranged from 3.39 to 5.54.

Vogrincic et al. (2010) measured the ability of Tartary buckwheat flour to scavenge free radicals as a proportional reduction in absorbance at 515nm of ionized DPPH incubated with a methanol extract of flour (lg/l). Free radical activity was reduced by as much as 89 percent. Vogrincic et al. (2013) also tested the ability of rutin, quercetin, and Tartary and common buckwheat flour extracts to scavenge DPPH radicals. A 100μM solution of rutin reduced free radical activity by 83.6 percent; a 50μM solution of quercetin reduced activity by 90.1 percent. The reduction due to 1%(v/v) solutions of the Tartary and common buckwheat flour extracts were 35.6 percent and 21.7 percent, respectively. The authors speculated that other compounds in the flours might have enhanced their antioxidant activity.

Vogrincic and his co-workers (2013) also tested for cytotoxicity of rutin, quercetin, and the buckwheat flour extracts by measuring the activity of mitochondrial dehydrogenase produced by human hepatoma (Hep2A) cells that had been incubated for 24 hours with those substances. At the same concentrations as used in the DPPH assay, neither rutin, quercetin, nor either flour extract demonstrated a cytotoxic effect. Moreover, at those concentrations none of the tested substances caused an increase in DNA strand breaks in those hepatoma cells after exposure for 24 hours. In fact, all four materials significantly reduced the amount of oxidative DNA damage resulting from exposure to a 400μM solution of tert-butylhydroperoxide (t-BOOH). The effectiveness of the flour extracts was equivalent to pure rutin or quercetin at much higher concentrations than the former contained.

Cao et al. (2008) measured concentrations of rutin, quercetin and total phenolics in ethanol extracts from groats of two varieties of common buckwheat (‘Liuqiao 1’ and ’Pingqiao 2‘) and two varieties of Tartary buckwheat (‘Qianwei 3’ and ‘Xinong 9909’).  Samples had been grown in three locations in China.  Extracts were prepared by mixing 10 grams of ground groats in 200 ml of 70 percent ethanol for 40 min in an ultrasonic generator.  The two common buckwheat varieties contained similar levels (per gram of extract) of rutin (2.21-2.93 mg), quercetin (6.47-6.53 mg), and total phenolics (103-110 mg, expressed as protocatechuic acid equivalents).  The respective concentrations in the two varieties of Tartary buckwheat were both much higher and more variable (137-188 mg;  29-63 mg; and 226-275 mg per g extract).

Cao and co-workers measured the ability of the buckwheat extracts to reduce ferric (Fe+3) to ferrous (Fe+2) ions, and to chelate the latter.  Reducing power was increased with concentration, with extracts of both Tartary buckwheat varieties having greater power than either variety of common buckwheat, but less than that of BHT or ascorbic acid.  Similarly, an extract of either variety of Tartary buckwheat showed greater chelating ability than both varieties of common buckwheat.

These researchers also examined the protective effects from these groat extracts on hydroxyl radical-mediated DNA damage by two different methods.  Protection against non-site-specific damage by ferric iron/hydrogen peroxide to fish DNA was estimated by absorbance at 532 nm, when buckwheat extract was added to the reaction mixture.  Damage was reduced by 30-40 percent with the addition of common buckwheat extract (0.2 mg/ml), and by half with the addition of Tartary buckwheat extract.  The effect of buckwheat extracts on site-specific damage to super-coiled plasmid DNA by hydroxyl radicals was assessed by electrophoresis.  Whereas FeSO4 and H2O2 transformed the DNA to open circular and linear configurations, the buckwheat extracts (particularly Tartary buckwheat) maintained the super-coiled configuration.

Song et al. (2016) studied changes in the levels and distribution of flavonoid compounds during the development of seeds of the Tartary buckwheat variety ‘Xiqiao 1.’  From a planting on September 5, 2014 in Sichuan Province, China, seeds were harvested at different developmental stages, with the final harvest on December 20.  The stages were characterized as 1) seed formation started; 2) liquid endosperm; 3) solidifying endosperm; 4) pericarp turning from green to black; and 5) mature, black seed.

Seeds were dried at 105ºC for 30 minutes and subsequently at 60ºC until a constant weight was reached.  Hulls (i.e., pericarps) were removed from frozen seeds; then hulls and groats were ground separately, washed with acetone, and centrifuged.  Anthocyanins were extracted from the resulting sediment with 5% HCl-methyl alcohol at 60ºC for one hour (repeated twice).  Concentrations were measured spectrophotometrically, as the difference in absorbance of the supernatant at 530nm and 600nm. Melanin was extracted from the sediment with 2% NaOH at 70ºC for 30 minutes.  Relative concentrations were determined by absorbance at 290nm.  Flavonoids were extracted from whole seeds, hulls, and flour with 70% methanol.  Concentration of total flavonoids was measured by absorbance at 420nm, and calculated against a standard curve for rutin.  Concentrations of individual flavonoids (rutin, quercetin, and kaempferol) were determined by HPLC.

Song et al. (2016) reported that the concentration of anthocyanins peaked during the second stage of seed development, and declined rapidly between stages 3 and 4.  In contrast, the concentration of melanin increased throughout seed development, with pronounced darkening occurring between stages 4 and 5.  Ratios of anthocyanin to melanin of 0.26, 0.59, 0.44, 0.15, and 0.04 at the five developmental stages.  The concentration of total flavonoids in whole seeds was greatest at stage 1 (about 38mg/g DW), with a second peak at stage 4 (about 20mg/g DW).  At all stages the concentration of total flavonoids was higher in flour than in the hull.  Those same patterns were observed in the concentrations of quercetin and kaempferol in flour and hulls.  A similar pattern was observed for rutin, except that the second peak concentration in the flour was observed at the 5th stage (I.e., at maturity).

Although the authors did not state the changes in the quantity of these compounds over the course of seed development, they did present graphs of the increases in dry weight of flour, hull, and whole seeds.  Between stages 2 and 5, the dry weight of flour increased about 4.1 times, while the dry weight of hull increased only 2.3 times.  The small drop in concentration of total flavonoids in the flour—about 11 percent—indicates that the actual amount of flavonoids in the groats increased substantially over this period.  Between stages 2 and 5, the concentration of total flavonoids in the hulls dropped by about 75 percent, indicating an actual loss in the amount of those compounds in the hulls.

As mentioned in the sections on SOLUBLE CARBOHYDRATES, GABA, and PROTEIN DIGESTION, the nutritional properties of buckwheat seeds change rapidly and substantially when those seeds sprout. Kim et al. (2008) investigated the influence of sprout growth on concentrations of flavonoids in common and Tartary buckwheat. Seeds of ‘Kitawase’ common buckwheat (either with hulls intact or removed) and ‘Hokkai T9’ Tartary buckwheat were surface-sterilized and germinated in darkness at 25ºC and 60% humidity. Seeds (hull included) were harvested after one and two days, lyophilized, and ground. The powdered material was extracted with methanol, centrifuged, and the filtrate analyzed by HPLC. Ungerminated seeds contained no more than trace amounts of chlorogenic acid, orientin, isoorientin, vitexin, isovitexin, or quercetin (except that seeds of ‘Hokkai T9’ contained 0.1mg/g dry weight of vitexin). Rutin concentrations were 0.2 mg/g DW in the common buckwheat (with or without hulls) and 14.1 mg/g DW in Tartary buckwheat. Except for chlorogenic acid, all the flavonoid compounds had increased in concentration after two days of sprouting, with maximum levels of 0.7 mg/g of vitexin and isovitexin in the common buckwheat variety. In the 2-day sprouts of the Tartary buckwheat variety, only vitexin and rutin achieved detectable levels—0.1 and 15.6 mg/g DW, respectively.

Kim and co-workers (2008) also moved sprouted seedlings to a shaded glasshouse (at 27ºC, 87% humidity, and about 5% of ambient insolation). The researchers measured flavonoid content of the shoots (i.e., leaves and cotyledons) on days 6-10. Chlorogenic acid reached a maximum of 1.7 mg/g in the common buckwheat ( hull-free ‘Kitawase’ on day 9) and 1.2 mg/g in the Tartary buckwheat (on day 10). The maximum observed concentrations of orientin in the two species were 10.8 and 0.2 mg/g, respectively. For isoorientin they were 10.0 and 0.1 mg/g. For vitexin they were 6.3 and 0.5 mg/g, while for isovitexin they were 6.6 and 0.5 mg/g. There was no detectable quercetin in the shoots of common buckwheat; in the shoots of Tartary buckwheat the level remained constant at 0.1 mg/g from day 6 through day 10. The maximum observed concentration of rutin in the shoots of common buckwheat was 6.0 mg/g in 10d seedlings from whole seeds. In contrast, the rutin level in the Tartary buckwheat shoots exhibited a maximum of 23.8 mg/g on day 6, and exceeded 20 mg/g throughout the sampling period. Thus, while the combined levels of these flavonoid compounds were similar in the Tartary and common buckwheat seedlings (the latter either from whole or hulled seeds), their proportions differed significantly.

Kreft et al. (2013) investigated the concentrations of phenolic compounds during the early development of common buckwheat (cultivar ‘Darja’) and Tartary buckwheat (a domestic variety from Luxembourg). Seeds were sprouted on perforated racks and spray-irrigated every 30 minutes. Chemical concentrations in 60 percent ethanol extracts of powdered lyophilized sprouts were determined spectrophotometrically.

After ten days, Tartary buckwheat sprouts contained from 1.1 to 1.4 percent total flavonoids (as a percentage of sprout dry weight). Tartary buckwheat sprouts contained from 2.0 to 3.3 percent total polyphenols. Expressed as equivalents of pyrogallol, the antioxidant activity of the sprouts ranged from 2.2 to 3.3 percent. (The antioxidant activity of the buckwheat polyphenols was approximately equal to that of pyrogallol. ) In 10-day-old sprouts, the combined concentrations of flavonoids in the Tartary buckwheat was 2 to 3 times that in common buckwheat—a smaller difference than had been found between mature seeds of the two species.

As mentioned in the section on DIETARY FIBER, Kuwabara et al. (2007) sprouted seeds of one variety of common buckwheat (‘Kitawasesoba’) and two of Tartary buckwheat (‘Hokkai T8’ and ‘Hokkai T9’) that had been harvested in Hokkaido, Japan. These were germinated in the dark and then grown for 5 days in the light at 22ºC. Leaves and stems were harvested at day 9 (common buckwheat) or day 10 (Tartary buckwheat), weighed, and freeze-dried. Sprout powders were extracted with methanol/phosphoric acid for measuring rutin concentration by HPLC. Rutin constituted 0.7 percent of the dry matter of the sprout powder from the common buckwheat variety, 6.2 percent of the dry matter of ‘Hokkai T8,’ and 8.5 percent of the dry matter of ‘Hokkai T9.’

Merendino et al. (2014) measured the potential yield of polyphenols, and the antioxidant capacities of commercially-milled flour and of dried sprouts of two cultivars of common buckwheat (‘Lileja’ and ‘Darja’) and one of Tartary buckwheat (‘Ljse’). Seeds (200g) of each cultivar were germinated in the dark at 22ºC for 10 days, when sprouts were harvested at 22mm above the crown. After drying, sprouts were crumbled and sieved to remove any remaining hulls. The cultivar of Tartary buckwheat had the highest germination rate (90 percent) and also the highest yield of dried sprouts (16.9g). The polyphenol content (measured as equivalents of gallic acid per gram of sample dry weight) was significantly greater in methanol extracts from the dried sprouts than in comparable extracts from flour. In the case of either sprouts or flour, the total polyphenol content was greater in the extract from Tartary buckwheat than the extract from either of the common buckwheat cultivars—but not significantly so. The authors reported a rutin content (mg/g of sample dry weight) of 7.6 in the Tartary buckwheat flour and 24.6 in the dry sprout powder from that species; the quercetin content was 0.4mg/g in the flour and 0.8 in the sprout powder.

The authors tested the effectiveness of these buckwheat extracts as antioxidants in three different assays: DPPH radical scavenging, oxygen radical absorbance capacity (ORAC), and ferric reducing antioxidant power (FRAP). For all assays and all cultivars, the antioxidant capacity of the extract from dried sprouts was significantly greater than the extract from flour. In all assays of either flour or sprouts, the capacity of the Tartary buckwheat extract was greater than that of either cultivar of common buckwheat—but not significantly so. On the basis of these results, Merendino and co-authors selected dried sprouts of ‘Ljse’ Tartary buckwheat as the ingredient for the formulation of a “functional” pasta.

It is thought that plants’ ability to synthesize flavonoid compounds has evolved in part for protection from ultra-violet radiation. These compounds absorb radiation in the UV-A and UV-B wavelengths (thus protecting more sensitive molecules), as well as scavenging the reactive oxygen species that UV-B radiation generates. Because ultra-violet exposure increases with elevation, plants of some species grown at higher elevations are stimulated to synthesize more flavonoids than those grown at lower elevations. Gabersˇcˇik et al. (2002) grew common buckwheat plants under ambient conditions at Ljubljana, Slovenia, and also with augmented and diminished levels of UV-B radiation. While significant differences (P<0.05) were mostly observed between the augmented and reduced treatments, the control (ambient) plants were generally intermediate in their response. At three weeks of growth, extracts from plants from the different treatments showed similar absorbance in both UV-A and UV-B range, but by five weeks the plants had responded to greater UV-B exposure by increasing concentrations of UV-absorbing compounds. Nevertheless, higher UV-B radiation stressed the plants, decreasing their production of chlorophylls a and b as well as carotenoids. Through the fifth week of growth, transpiration levels were increased, and levels of photosynthesis and water use efficiency (WUE) were depressed. Higher UV-B exposure generally increased dry weight per leaf area, reduced the lengths of stem internodes, and decreased the dry weight per plant of roots, shoots, leaves, and seeds. The average weight of a single seed was 0.88g under the 40 percent reduction in UV-B radiation, 0.45g under ambient UV-B radiation, and only 0.32g under the augmented UV-B regime (which was calculated as the equivalent effect at 20 cm above ground level from a 17% reduction in the level of ozone in the stratosphere). Regardless of UV-B treatment during seed development, subsequent germination rate was greater than 95 percent.

Kishore et al. (2010) investigated the variation in phenolic compounds in Tartary buckwheat seeds from landraces collected at 15 sites over an altitudinal range (550m-3650m) in Uttarakhand, India. Groats and hulls were ground separately and subjected to two different extraction solvents: 80% methanol or acetone containing 2%(v/v) HCl. After treatment with Folin-Ciocalteu reagent, each extract from each sample was assayed for total phenolic content as absorption at 765nm, compared to a calibration curve for gallic acid. The total flavonoid content of each extract was determined by a colorimetric assay. The Trolox equivalent antioxidant capacity (TEAC) was estimated for each sample by DPPH assay. Antioxidant capacity was also estimated for each sample by the β-carotene linoleate bleaching test.

For samples from almost every collection site, the total concentration of phenolic compounds was higher in the methanol extract than in the acetone extract, and was higher in the extract from flour (groats) than in that from hulls. The sample from the collection site at 3133m contained the highest total phenolic concentration in both flour (1651.4μg GAE/50mg DW) and hull (893.2μg GAE/50mg DW). Below that elevation, phenolic concentration increased with altitude, but at four sites above that elevation, the opposite relationship was observed. For samples from every collection site, the total concentration of flavonoid compounds was higher in the methanol extract than in the acetone extract, and was higher in the extract from flour (groats) than in that from hulls. The greatest concentration of flavonoids in the methanol extract from both flour and hulls was observed at the site at 1875m ( 272.5 and 205.0 μg rutin equivalents/50mg DW, respectively). No strong relationship was observed between flavonoid content and site elevation.

Based on its inhibition of bleaching of β-carotene, the antioxidative capacity of each flour or hull extract was expressed as a percentage inhibition. For the methanol extract from flour, these ranged from 61.8 to 77.3 percent, and were highly correlated with the total phenolic content of the samples (r=0.86). For the methanol extract from hulls, the comparable range was 23.2 to 59.8 percent inhibition. Compared to methanol extracts, acetone extracts from both seeds and hulls were less effective at inhibiting the bleaching of β-carotene. Based on the DPPH assay, estimates of antiradical activity and TEAC were computed for the methanol and acetone extracts from each sample of flour and hull. Antiradical activity scores ranged from 30.4 to 56 percent in the methanol extracts from flour, and from 27 to 53.1 percent in the acetone extracts. The respective ranges for extracts from hulls were lower—24.7 to 35.4 percent, and 27.0 to 36.5 percent. Antiradical activity scores of methanol extracts from flour were highly correlated with total phenolic content ( r=0.80), as were TEAC values (r=0.91).

Ultraviolet radiation increases with altitude. Because the concentration of phenolic compounds of samples from the four highest locations were less than the corresponding values for samples from mid-altitude locations, UV-B radiation alone cannot explain the level of phenolic compounds that accumulate within the seeds of Tartary buckwheat. Kishore and fellow authors speculated that lower temperatures during plant growth might stimulate plants to produce phenolic compounds and other antioxidants, in order to compensate for their reduced efficiency at scavenging hydrogen peroxide at those temperatures. (The strong correlations between total phenolic concentration and various measures of antioxidative activity suggest that those compounds are indeed important antioxidants in the seed. ) However, the authors presented no climatic data for the sample collection sites, so there is no data to support that conjecture. Because the seeds sampled in this study represented different landraces as well as different growing conditions, genetic as well as environmental differences (and their interactions) could explain the results.

The weak relationship that Kishore and co-authors found between antioxidative activity and total flavonoid concentration of extracts from Tartary buckwheat seeds differs from the results of Morishita et al. (2007) reported above. The latter authors found the flavonoid rutin to be the predominant antioxidant. In contrast, Vogrincic et al. (2010) concluded that the antioxidative capacity of Tartary buckwheat flour was related to other constitutents besides rutin or quercetin.

Fabjan and co-authors (2003) measured the concentrations of rutin and quercitrin in vegetative tissues of three cultivars of Tartary buckwheat (two from China and one from Luxembourg) grown in Slovenia at an elevation of 350m elevation. In herbage of plants seeded on June 11, the concentrations of both flavonoids were greatest between 50 and 60 days after seeding (i.e., at the seed formation stage). The maximum concentration of rutin exceeded three percent of dry weight, while that of quercitrin exceeded 0.05 percent. In summer-sown plants, concentrations were somewhat lower. The authors compared concentrations of rutin, quercitrin, and quercetin in seeds of Tartary buckwheat grown at 2500-3000m with those grown at 340m. Contrary to the expectation that growth at higher elevation would stimulate plants to synthesize more flavonoids, seeds of the two Chinese cultivars (but not the one from Luxembourg) contained about one-third more rutin when grown at the lower elevation. One of the Chinese cultivars also contained higher concentrations of quercitrin when grown at the lower elevation. The other two cultivars contained equivalent concentrations of quercitrin (about 0.05% dry weight) when grown at either site. Negligible concentrations of quercetin were found in either herbage or seeds of these varieties.

Clearly, the two sites compared by Fabjan and co-workers would have differed in other ways besides elevation. Moreover, different cultivars of Tartary buckwheat responded differently to those environments—an example of what is broadly referred to as “genotype-by-environment interaction.” Lahanov et al. (2004) cultivated several species and cultivars of Fagopyrum in a common garden experiment over three years. They measured total flavonoids in different tissues of Tartary buckwheat at different developmental stages. From the bud stage through peak flowering, concentrations remained close to maximal in leaves (about 100mg/g), shoots (about 17mg/g), and flowers (about 160mg/g). In all these organs, flavonoid concentrations declined by about half as the seeds turned brown. The authors noted that flavonoid concentrations were two-to-three times higher when plants experienced hot, dry conditions during the vegetative growth stage, compared to plants growing under cooler, damper conditions.

Guo et al. (2011) investigated the concentrations of phenolic compounds and the antioxidant activity in two Tartary buckwheat varieties (‘Xingku 2’ and ‘Diqing’) grown at three locations in China. In 2009, seed were planted on April 14 at Liangshan (Sichuan Province), on May 30 at Dingxi (Gansu), and on July 8 at Tongxin (Ningxia). The Sichuan site had the highest elevation (2100m) and greatest precipitation (667.3mm); the Ningxia site had the lowest elevation (1422m) and the lowest precipitation (151.4mm). The authors extracted both free and bound phenolic compounds from whole seeds.

Employing the Folin-Ciocalteu method, Guo and co-workers found that the overall levels of phenolic compounds, expressed as equivalents of gallic acid, ranged from 5150 to 9660 μmol/100g DW, of which free phenolics represented between 94 and 99 percent. Among the samples of ‘Xingku 2,’ concentrations of both free and bound phenolics were highest in the Sichuan sample and lowest in the Gansu sample. The opposite trend was observed in the samples of ‘Diqing.’ Of the variance in concentrations of total phenolic content, less than four percent was attributable to the varieties, 19 percent to locations, and 71 percent to the interaction between variety and location.

Levels of total flavonoids, expressed as equivalents of rutin, ranged from 1719 to 3014 μmol/100g DW, of which free flavonoids represented between 76 and 96 percent. The highest concentrations of both free and total flavonoids were observed in the ‘Xingku 2’ from Ningxia; the highest concentrations of bound flavonoids were observed in the samples of ‘Diqing’ from Sichuan and Ningxia.

Using HPLC, the authors detected p-hydroxybenzoic, ferulic, protocatechuic, p-coumaric, gallic, and vanillic acids in all six samples. The first three of these accounted for between 83 and 88 percent of the total concentration of phenolic acids. Caffeic acid was detected in five samples, and syringic acid in two. Of the flavonoids detected in these samples, rutin concentrations ranged from 518.54 to 1447.87mg/100g DW, quercetin between 425.65 and 857.62 mg/100g DW, and catechin between 8.89 and 19.96 mg/100g DW. Almost all the rutin and quercetin was present as free compounds; whereas, most of the catechin was bound. For both varieties, the total rutin (free and bound) was in greatest concentration in samples from Sichuan, and in lowest concentration in those from Ningxia. Of the variance among samples in rutin concentration, 47 percent was attributable to locations, 7 percent to varieties, and 46 percent to the interaction between variety and location.

Using DPPH and ABTS assays, Guo et al. (2011) measured the radical-scavenging ability of their Tartary buckwheat phenolic extracts. The DPPH radical-scavenging activity ranged from 2.3X104 to 3.3X104 μmol Trolox equivalents per 100g DW. The activity against ABTS ranged from 1.2X105 to 1.4X105 μmol Trolox equivalents per 100g DW. Among the six buckwheat samples (2 varieties X 3 locations), the correlation between DPPH and ABTS activity was minimal (r=-0.07). Neither activity was significantly correlated with the concentration of total phenolics, total flavonoids, or total phenolic acids. The combined concentrations of the phenolic acids was significantly correlated with the concentration of rutin (r=0.81, p<0.05). The concentration of quercetin was negatively correlated with that of rutin (r=-0.83).

DPPH activity was positively correlated with the altitude of the growing site, and negatively correlated with hours of sunshine (p<0.05 for ‘Xingku 2’ samples only). The concentration of rutin and the combined concentrations of the phenolic acids were similarly correlated with those two environmental factors.

Zhao et al. (2018) investigated the volatile oils found in flowers of Tartary and common buckwheat and F. cymosum. Fresh flowers were hydro-distilled at 100ºC for 1.5-2 hours; the distillates were extracted with diethyl ether, dried over anhydrous sodium sulfate, and filtered. Component compounds were identified by gas chromatography/mass spectroscopy. Of the total volatile oils from Tartary buckwheat, over 88 percent were identified. These comprised 25.76% ketones, 7.66% alcohols, 16.4% esters, 20.98% phenols, 2.18% terpenoids, and 5.75% aldehydes. The most prevalent compounds (constituting more than 10% of the total) were 2-Pentadecanone, Eugenol, and 1,2-Benzenedicarboxylic acid, bis(2-methylpropyl) ester.

The authors used a broth dilution-colorimetric assay to measure the inhibitory effects of the flower volatile oils on four Gram-negative bacterial species (A. tumefaciens, E. coli, P. lachrymals, and X. vesicatoria) and two Gram-positive species (B. subtitlis and S. aureus). The minimal concentration at which inhibition was detectable was 200, 400, 200, 200, 600, and 800 μg/mL, respectively. For all but S. aureus, these were lower than the respective concentrations for oils from common buckwheat. For all six bacterial species, IC50 values were lower for oils from Tartary buckwheat compared to common buckwheat, but higher than values from F. cymosum.

Zhao et al. (2018) also measured the antioxidative potential of flower volatile oils from the three buckwheat species. DPPH scavenging activity showed a concentration-dependent response. The oils from Tartary buckwheat had the lowest IC50 value (210.63 μg/mL), with that of F. cymosum intermediate (264.92 μg/mL), and that of common buckwheat highest (354.15 μg/mL). The same order was observed in the β-carotene-Linoleic bleaching assay, where the respective IC50 values were 184.13 μg/mL, 206.11 μg/mL, and 242.06 μg/mL.

Zielińska et al. (2012) measured concentrations of flavonoids in different organs of common buckwheat (cultivar ‘Volma’) and Tartary buckwheat (an unnamed Polish accession) during flowering (41 days after sowing), seed formation (48 DAS for common buckwheat, 62 DAS for tartary buckwheat), and seed ripening (100 DAS). Stems and ripe seeds contained lower levels of total flavonoid, leaves and unripe seeds were intermediate, while flowers contained the highest levels. At 48 DAS, flowers of common buckwheat contained over 200 rutin equivalents/100mg DW. A similar pattern was observed in the concentration of rutin, except that leaves of common buckwheat had concentrations as great as flowers during the seed formation stage. Only in seeds was rutin consistently more concentrated in Tartary buckwheat than in common buckwheat. In unripe seeds, rutin constituted 3.64 percent of dry matter in Tartary buckwheat, and 1.57 percent in common buckwheat. In ripe seeds, the corresponding percentages were 1.35 and 0.04. Low levels of quercetin (0.01-0.25 percent DW) were found in all the samples studied. Quercitrin was detected only in flowers of common buckwheat (1.8 percent DW at 100 DAS). Flavone C-glucosides were detected at lower levels in unripe seeds of that species, and at much lower levels in ripe seeds.

Zielińska and co-workers (2012) used the DPPH assay to measure the antioxidant capacity of their samples. Flowers showed the greatest effect (Tartary buckwheat being greater at early flowering, but common buckwheat being greater during seed formation). Both unripe and ripe seeds of Tartary buckwheat showed greater activity than common buckwheat (383 versus 286 μmol Trolox equivalents/g DW at the unripe stage, 50.7 versus 11.7 when ripe). Stems showed activity in the same range as ripe seeds. A photo-induced chemiluminescence assay similarly indicated that the flower samples were the most effective against superoxide anion radicals. At 48 DAS, flowers of common buckwheat scored 1066 μmol Trolox equivalents/g DW. Again, stems and ripe seeds exhibited the weakest anti-oxidant capacity. Among Tartary buckwheat samples, there was strong correlation between scores on this assay and rutin concentration (r=0.97). Among common buckwheat samples, the correlation between DPPH score and rutin concentration was weaker (r=0.80).

Suzuki and co-workers (2005a) investigated the effects of short-term stresses on the concentration of rutin and the activity of rutin glucosidase in leaves of Tartary buckwheat. Plants were seeded on June 6 in central Hokkaido, Japan, from which mature leaves (L7) were harvested 28 days after germination. Half of a leaf lamina was exposed to UV-B radiation (1.26μW/cm2), to desiccation (30 minutes at 22ºC), or to chilling (5 minutes at -5ºC). Twenty-four hours after the end of treatment, leaves showed increases in rutin concentration (UV-B or desiccation treatment) and/or glucosidase activity (UV-B, desiccation, and chilling treatments). Leaves subjected to either the UV-B or chilling treatment contained an increased concentration of quercetin—the aglycone product of rutin degradation that is catalyzed by the enzyme, rutin glucosidase. The authors inferred that UV-B treatment induced the synthesis of rutin, which was most concentrated in the upper epidermis of the leaf lamina, in order to screen the mesophyll from this harmful radiation. The concentration of rutin was highest in newly-emerging leaves, which lack the protective layers of wax or lignin that shield older leaves. The authors found that the activity of rutin glucosidase was greatest in the lower epidermis. This enzyme freed both quercetin and rhamnose. Quercetin might serve as a substrate for peroxidase, protecting the young leaf from oxidative damage. Rhamnose, a sugar, might fuel metabolism in stressed leaves in which the photosynthetic apparatus was not yet fully developed.

Ultimately, it is within the digestive tract and the body’s tissues that antioxidants play their protective role. Ma and Xiong (2009) performed sequential in vitro digestion with pepsin and pancreatin of protein purified from defatted buckwheat flour (see PROCESS OF PROTEIN DIGESTION above). These authors investigated the antioxidative capacity of the interim products of that digestion at several stages. Free radicals, generated from a mixed solution of ABTS and potassium persulfite, were incubated with protein digests, and the removal of free radicals was expressed in equivalent concentrations of Trolox. Radical-scavenging capacity of the buckwheat protein products increased throughout the full 3-hour digestion. At that final sample point, radical-scavenging capacity exhibited a dose-dependent response to the concentration of buckwheat protein (0-3mg/mL range). The authors also examined the digested buckwheat protein’s inhibition of lipid peroxidation by the thiobarbituric acid-reactive substances (TBARS) method. While the products of digestion with pepsin actually promoted peroxidation, the final products after digestion with pancreatin inhibited the formation of TBARS. At 2 mg/mL, this buckwheat protein digest was as effective as a 0.01 percent solution of BHA.

EFFECTS OF BUCKWHEAT ON CHOLESTEROL AND RELATED LIPIDS

High serum (i.e., blood) concentrations of cholesterol and related lipids have been implicated in cardiovascular disease. In approximate terms, dietary cholesterol is absorbed from the gastrointestinal tract into the hepatic portal vein. Once reaching the liver, a small portion of the cholesterol passes into the systemic circulation, while much of it is metabolized to lipid-soluble bile acids. These compounds become components of water-soluble primary conjugated bile salts. Via the common bile duct, these bile salts reenter the small intestine. Normally, this “enterohepatic circulation” accounts for the majority of cholesterol utilization by the body. Excretion of bile acids from the GI tract diverts cholesterol from recirculation, thereby reducing the amount of LDL cholesterol reaching the liver, and eventually reducing levels in the systemic circulation.

The digestion of buckwheat releases compounds that can bind cholesterol in the intestine, and thereby assist its excretion in feces. Researchers therefore hypothesize that buckwheat consumption can lower cholesterol levels in the blood, and reduce the concomitant risk of cardiovascular disease. Binding of cholesterol would also diminish its availability to bacteria in the lumen of the large intestine. Carcinogenesis in the colon and rectum is apparently promoted by bacterial derivatives of bile acids.

As described in “THE PROCESS OF PROTEIN DIGESTION,” Ma and Xiong (2009) investigated digests of protein that had been isolated from defatted flour of common buckwheat. Those authors evaluated the antioxidant capacity of various protein digests, as well as the capacity of such protein digests to limit lipid peroxidation (See “FLAVONOIDS AND ANTIOXIDANT PROPERTIES” above). The researchers also investigated the ability of isolates of common buckwheat protein to bind bile acids. Freeze-dried powdered buckwheat protein (undigested or subjected to in vitro pepsin digestion for 60 minutes, with or without subsequent pancreatin digestion for 60 or 120 minutes) was dissolved in phosphate buffer at pH 6.3, the physiological pH of the duodenum. Following centrifugation, the supernatants (containing soluble peptides) were mixed with bile acids within the physiological range of 1.5-7 mM, and incubated at 37ºC for two hours. Assays of bile-acid binding were performed on these supernatants, and similarly on incubated protein suspensions (containing both soluble and insoluble components).

The percentage of cholic acid or chenodeoxycholic acid that was bound by supernatants of digested buckwheat protein was generally less, but not significantly so, than was bound by the supernatant of undigested buckwheat protein. The percentage of cholic acid or chenodeoxycholic acid that was bound by the buckwheat protein suspension was significantly greater if that protein had previously been digested by pepsin for 60 minutes and subsequently by pancreatin for 120 minutes, compared to the percentage that was bound by undigested buckwheat protein. These results suggest that insoluble products of digestion of buckwheat protein could bind with primary bile acids within the intestine. By that mechanism, dietary buckwheat could lower levels of cholesterol in the blood.

Deoxycholic acid is a secondary bile acid—a product of bacterial degradation of primary bile acids within the large intestine. Buckwheat protein could bind with deoxycholic acid to a degree that was unaffected by any prior digestion of that protein. As with the primary bile acids discussed above, binding by a suspension of the buckwheat protein was greater than binding by soluble peptides alone. Ma and Xiong (2009) attributed these results to the fact that digestion-resistant proteins tend to be rich in hydrophobic amino acid residues, and thus attractive to the polar groups generated by the dehydroxylation of primary bile acids. Because deoxycholic acid is implicated in carcinogenesis in the colon and rectum, consumption of buckwheat protein might have a protective effect against those cancers.