Characterization of Corrinoid Compounds in the Edible Cyanobacterium Nostoc flagelliforme the Hair Vegetable

Vitamin B12 contents in the edible cyanobacterium Nostoc flagelliforme, also known as hair vegetable, were assayed using a microbiological method. We detected high vitamin B12 contents in samples of naturally grown cells (109.2 ± 18.5 μg/100g dry weight) and cultured cells (120.2 ± 53.6 μg/100g dry weight). However, commercially available hair vegetable samples, which comprised fake substitutes and Nostoc, had variable contents (4.8 101.6 μg/100g dry weight) because concomitant fake items contain very low vitamin B12 contents. To evaluate whether natural and cultured N. flagelliforme samples contained vitamin B12 or pseudovitamin B12, corrinoid compounds were purified and identified as pseudovitamin B12 (approximately 72%) and vitamin B12 (approximately 28%) using silica gel 60 TLC bioautography and LC/MS. The results suggested that N. flagelliforme contains substantial amounts of pseudovitamin B12, which is inactive in humans.


Introduction
Nostoc flagelliforme is an edible cyanobacterium, which grows naturally in some semidesert regions of China and Mongolia.When dried, the cyanobacterium resembles black hair and hence the name hair vegetable ("Facai" in Chinese), which is one of the most expensive ingredients in Chinese cuisine [1].At present, fake items and mixtures of pure N. flagelliforme with fake substitutes (approximately 90%) are flooding the market [1].
N. flagelliforme contains many nutrients [2], including a novel acidic polysaccharide (nostoflan) that has potent antiviral activity [3].Takenaka et al., [4] demonstrated the oral acute and subacute safety of dried N. flagelliforme in rats.Therefore, N. flagelliforme is also suitable for pharmaceutical use.Several studies [5,6] have reported that most of the corrinoids found in certain edible cyanobacteria may not be bioavailable in mammals.Wa-tanabe et al. [7] also demonstrated that pseudovitamin B 12 (adeninylcyanocobamide or pseudo B 12 ; Figure 1), Characterization of Corrinoid Compounds in the Edible Cyanobacterium Nostoc flagelliforme the Hair Vegetable which is inactive in humans, is the predominant corrinoid in the edible cyanobacteria used as a health food by humans.N. flagelliforme, hair vegetable, is already used as health food, but there is no information on about B 12 contents in pure N. flagelliforme and commercially available hair vegetable, or whether the corrinoids are authentic B 12 or inactive corrinoids.
In the present study, we characterized corrinoid compounds from N. flagelliforme sources, including naturally grown samples, cultured samples, and commercially available hair vegetable samples.

Materials
Authentic B 12 was obtained from Sigma (St Louis, Missouri, USA).Silica gel 60 thin-layer chromatography (TLC) aluminum sheets were obtained from Merck (Darmstadt, Germany).All other reagents were highgrade commercially available reagents.N. flagelliforme Born.et Flah.was harvested from Alxa, Inner Mongolia, China, during the summer of 1996.After washing in water, the cyanobacterium was dried in sun and used for the analyses.It was also aseptically cultured in a Nostoc-N liquid medium (K 2 HPO 4 , 40 mg/L; MgSO   O, 50 μg/L; at pH 7.5) at 20 -25˚C with aeration under illumination (40 μmol/m 2 /s).Commercially available hair vegetable samples were purchased from the markets in Japan.

Samples of N. flagelliforme
Samples A-E were naturally grown samples, F-J were cultured samples, and K-N were commercially hair vegetable samples.

Extraction and Assay of Corrinoids from N. flagelliforme Samples
Dried N. flagelliforme samples (five different lots of naturally grown and cultured samples and four commercially available hair vegetable samples) were used for the assays.First, 0.5 g of each sample was suspended in 40 mL of distilled water and homogenized with an ultrasonic disruptor UD-200 (Tomy, Tokyo, Japan).Total corrinoids were extracted after boiling at pH 4.8 in the presence of 4.0 × 10 −4 % KCN and determined using the Lactobacillus delbrueckii ATCC 7830 microbiological assay method, according to the method described in the Standard Tables of Food Composition in Japan.L. delbrueckii ATCC 7830 can utilize deoxyribosides, deoxyribonucleotides (known as alkali resistant factor), and B 12 .Thus, accurate B 12 contents were calculated by subtracting the results for alkali resistant factor from those of total B 12 [8].

Bioautography of Corrinoid Compounds
Using Vitamin B 12 -Dependent Escherichia coli 215 Bioautography of corrinoid compounds was performed as previously described [9].B 12 extracts (20 mL) prepared as mentioned above were partially purified and concentrated using a Sep-Pak Plus ® C18 cartridge (Waters Corp., Milfora, USA), which was washed with 5 mL of 75% (v/v) ethanol and equilibrated with 5 mL of distilled water.The C18 cartridge was washed with 5 mL of distilled water, and B 12 compounds were eluted using mL of 75% (v/v) ethanol.The eluate was evaporated in a centrifugal concentrator (Integrated SpeedVac ® System ISS110; Savant Instruments Inv., NY, USA).The residual fraction was dissolved in 5.0 mL of distilled water.Concentrated B 12 extracts (1 μL) and authentic and pseudo B 12 (each 50 μg/L) were spotted onto the silica gel TLC sheet and developed in the dark using 2-propanol/ NH 4 OH (28%)/water (7:1:2 v/v) at room temperature (25˚C).After drying the TLC sheet, it was overlaid with agar containing basal medium and precultured E. coli 215, and incubated at 37˚C for 20 h.The gel plate was then sprayed with methanol solution containing 2,3,5triphenyltetrazolium salt, and B 12 compounds were visualized as red, indicating E. coli growth.

Liquid Chromatography-Electrospray Ionization/Multistage Mass Spectrometry (LC/ESI-MS/MS) Analysis
Each extract (40 mL) was partially purified and concentrated using a Sep-Pak ® Plus C18 cartridge (Waters Corp) as described above.The eluate was evaporated in a centrifugal concentrator (Integrated Speed VacR System ISS110), and the residual fraction was dissolved in 5.0 mL of distilled water.The purified extract was loaded onto an immunoaffinity column [EASI-EXTRACT ® Vitamin B 12 Immunoaffinity Column (P80) R-Biopharm AG, Darmstadt, Germany], and the corrinoids were purified according to the manufacturer's recommended protocol.Nostoc corrinoids, authentic pseudo B 12 , and B were dissolved in 0.1% (v/v) acetic acid and filtered using a Nanosep MF centrifuge device (0.4 μm, Pall Corp., Tokyo, JAPAN) to separate small particles.We analyzed an aliquot (2 μL) of the filtrate using a LCMS-IT-TOF coupled with an Ultra-Fast LC system (Shimadzu, Kyoto, JAPAN).Each purified corrinoid was injected into an Inert Sustain column (3 μm, 2.0 × 100 mm, GL Science,

Evaluation of True and Fake N. flagelliforme
Because fake materials generally contain starch [2], commercially available hair vegetable samples were tested using the iodine-starch reaction.The dried samples (0.1 g) were added to 10 mL of distilled water and boiled for 30 min.The treated samples were cooled to a room temperature and centrifuged at 10,000 × g for 10 min at 25˚C.Each supernatant solution (0.2 mL) was added to 1.4 mL of distilled water and treated with 0.4 mL of 25% Lugol solution (MP Biomedicals, LLC, Ohio, USA).The solution was allowed to stand for 30 min, and absorbance was measured at 600 nm.Microscopic analysis was performed using a BH-2 type microscope (Olympus Corp., Tokyo, Japan) with a digital camera QV-200 (Casio Computer Co. Ltd, Tokyo, Japan), as previously described [2].[11], and Ishikurage (Nostoc commune, 98.8 μg/100g dry weight) [12].

E. coli 215 Bioautography Analysis
Corrinoids found in all Nostoc samples were analyzed using the E. coli 215 bioautogram after separation by silica gel 60 TLC (Figure 2).Corrinoids found in all Nostoc samples and the commercially available hair vegetable sample K were separated to yield two spots, the Rf values of which were identical to those of authentic pseudo B 12 and B 12 , respectively.No or faint spots were obtained with commercially available hair vegetable samples L-N because of their lower B 12 contents.

LC/ESI-MS/MS Analysis
N. flagelliforme extracts were purified using a B 12 immunoaffinity column and analyzed by LC/ESI-MS/MS (Figure 3).Authentic B 12 and pseudo B 12 were eluted as        may have been due to differences in the culture conditions, but we have no detailed information on the key factor that affected B 12 and pseudo B 12 ratios.These results indicate that most Nostoc samples and commercially hair vegetable samples contained pseudo B 12 (major) and B 12 (minor).

Evaluation of True and Fake N. flagelliforme
Because hair vegetable is one of the most expensive ingredients in Chinese cuisine, certain fake items represent a large proportion of the commercially available hair vegetable [2].No color change was observed in all cul-tured samples, whereas all commercially available hair vegetable samples (K-N) exhibited significant staining by the iodine-starch method (optical densities of 0.29, 0.65, 0.34, and 1.19, respectively, at 600 nm).As shown in Figure 5, microscopic analysis indicated that although naturally grown and cultured N. flagelliforme possessed a bead-like morphology, no such morphology was found in the commercially available hair vegetable sample N (fake item only).The remaining hair vegetable samples K-M contained both Nostoc and fake substitutes.These microscopic data coincided with the results of iodine-starch reaction.Our results indicated that because naturally grown N. flagelliforme contain substantial amounts of pseudo B 12 , which is inactive in humans [7] and because the fake items have very low B 12 contents, commercially available hair vegetable is not suitable for use of B 12 source, regardless of the presence of the fake items.
Cyanobacteria have the ability to synthesize pseudo B 12 [13], which functions as a coenzyme of methionine synthase to catalyze the synthesis of methionine from homocysteine and N 5 -methyltetrahydrofolate [14].In the present study, the cultured Nostoc sample I predominantly contained B 12 but not pseudo B 12 (Table 2), suggesting that N. flagelliforme may synthesize both B 12 and pseudo B 12 de novo.Further biochemical and genetic studies are required to elucidate the detailed physiological functions of each corrinoid in this terrestrial cyanobacterium.
) and (f)).Nostoc corrinoids purified from the naturally grown sample E were eluted to yield several total ion peaks, indicating the presence of impurities (Figure4(a)).Ion peaks at m/z 672.77 and m/z 678.29 for pseudo B 12 and B 12 , respectively, were also detected, and their retention times were identical to those of authentic pseudo B 12 and B 12 .The mass spectra at the retention times of 7.42 and 7.55 min showed that both pseudo B 12 and B 12 divalent ions were formed at m/z 672.7735 (Figure4(b)) and m/z 678.2888 (Figure4(d)), respectively.There spective MS/MS spectra of each compound were identical to those of authentic pseudo B (Figure4(c)) and B 12 (Figure4(e)).Similar results were obtained with other naturally grown and cultured cell samples (data not shown).

3. 4 .
Relative Content Ratios of B 12 and Pseudo B 12 in Various Nostoc Samples Table 2 summarizes the relative contents ratios of B and pseudo B 12 in various Nostoc samples.The ratios of B 12 (approximately 28%) and pseudo B 12 (approximately 72%) are shown for naturally grown (A-E) and cultured (G and H) samples and for commercially available hair vegetable samples (K-M).Pseudo B 12 was the predominant corrinoid in cultured samples F and J.In contrast, sample I contained approximately 76% of B 12 .The variable ratios of B 12 and pseudo B 12 in the cultured samples

Figure 3 .
Figure 3. Liquid chromatography-electrospray ionization/multistage Mass spectrometry (LC/ESI-MS/MS) of authentic B 12 and pseudo-B 12 .B 12 and pseudo B 12 were analyzed with LCMS-IT-TOF (Shimadzu) as described in the text.The total ion chromatograms (TIC) of authentic B 12 and pseudo B 12 are shown in panels (a) and (d), respectively.The mass spectra of each ion peak from B 12 and pseudo B 12 are shown in panels (b) and (e), respectively.The magnified mass spectra from m/z 678 to 680 in B 12 and from m/z 672 to 675 in pseudo B 12 are shown as inserts.The MS/MS spectra of the peaks of B 12 and pseudo B 12 are shown in panels (c) and (f), respectively.

Figure 4 .
Figure 4. Liquid chromatography-electrospray ionization/multistage mass spectrometry (LC/ESI-MS/MS) of the purified corrinoids from naturally grown Nostoc sample (sample E).Total ion chromatograms (TIC) and reconstructed chromatograms for m/z 678.29 (×10) and 672.77(×10) of the Nostoc corrinoids are shown in panel (a).The mass spectra of the ion peaks of the Nostoc corrinoids at retention times of 7.2 min and 7.4 min are shown in panel (b) (the magnified mass spectrum from m/z 672 to 675 is shown as an insert) and panel (d) (the magnified mass spectra from m/z 678 to 680 are shown as an insert), respectively.The MS/MS spectra for the peaks of the Nostoc corrinoids at m/z 672.7735 and at m/z 678.2888 are shown in panels (c) and (e), respectively.

Figure 5 .
Figure 5. Microscopic analysis of various Nostoc flagelliforme samples.(a) Naturally grown samples; (b) cultured samples, (c) commercially available hair vegetable samples K-M (C, cells; and F, fake item), and (d) commercially available hair vegetable sample N. The results represent typical microscopic data from various N. flagelliforme samples. 4 2 naturally grown samples, cultured samples, and commercially available hair vegetable samples, using the L. delbrueckii ATCC 7830 microbiological assay method (Table1).High B 12 contents were detected in 3.1.VitaminB 12 ContentsB 12 contents were analyzed in various sources of N. fla-gelliforme i.e.,

Table 1 . Vitamin B 12 contents of various sources of Nostoc flagelliforme (naturally grown and cultured samples and commercially available hair vegetable samples).
*Total corrinoids were extracted from 0.5 g of each sample by boiling at pH 4.8 in the presence of KCN and determined using the Lactobacillus delbrueckii ATCC 7830 microbiological assay method.Because L. delbrueckii ATCC 7830 can utilize deoxyribosides, deoxyribonucleotides (known as alkali resistant factor), and B 12 , B 12 values were corrected by subtracting the results for alkali resistant factor from those for total B 12 .B 12 was assayed in triplicate for each sample, and the data is presented as mean values.

Table 2 . Relative content ratio of B 12 and Pseudo B 12 con- tents in various sources of Nostoc flagelliforme (naturally grown and cultured samples and commercially available hair vegetable samples).
and monitored by measuring the absorbance at 361 nm.The relative content ratio of B 12 and pseudo B 12 of each sample was calculated on the basis of peak areas with identical retention times of B 12 and pseudo B 12 .Total corrinoids were extracted from 0.5 g of each sample by boiling at.