Authentic B₁₂ was obtained from Sigma (St. Louis, Missouri, USA). Silica gel 60 thin-layer chromatography (TLC) aluminum sheets were purchased from Merck (Darmstadt, Germany). All other reagents were high-grade and commercially available. Various types of Chinese black tea leaves (Pu’er, Ryubao, Fu, and Brick) were purchased from local markets in Japan (Figure 1).

Each sample (5 g) of dried Chinese black tea leaves was homogenized in a mixer (TML160; Tescom & Co., Ltd., Tokyo, Japan). A portion (2.0 g) of the homogenate was used as the test sample. Total B₁₂ compounds were extracted by boiling at pH 4.8 in the presence of 4.0 × 10⁻⁴ % KCN and assayed using a microbiological B₁₂ assay based on L. delbrueckii ATCC 7830, according to the method described in the Standard Tables of Food Composition in Japan [12] . L. delbrueckii ATCC 7830 utilizes deoxyribosides, deoxyribonucleotides (known as alkali-resistant factor), and B₁₂.

The correct B₁₂ values were calculated by subtracting results for the alkali-resistant factor from those for total B₁₂. Tea from 3-g Ryubao leaves (sample H) with the highest B₁₂ content among those tested was extracted for 5 min with 150 mL of boiling water. After cooling down to 40˚C, the extract was used as a tea drink, and B₁₂ was extracted from 50 mL of liquid using the above-mentioned method.

Bioautography of corrinoid compounds was performed as previously described [13] . B₁₂ extract (50 mL) prepared as described above was partially purified and concentrated using Sep-Pak Plus^® C18 cartridge (Waters Corp., Milford, USA) prewashed 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 B₁₂ compounds were eluted using 2 mL of 75% (v/v) ethanol. The eluate was evaporated in a centrifugal concentrator (Integrated SpeedVac^® System ISS110; Savant Instruments Inc., NY, USA) and the residual fraction was dissolved in 2.0 mL of distilled water. Concentrated B₁₂ extracts (1 μL) and authentic B₁₂ and pseudo B₁₂ (50 μg/L each) were spotted onto the silica gel 60 TLC sheet and developed in the dark using 2-propanol/NH₄OH (28%)/water (7:1:2 v/v) at room temperature (25˚C). After drying, the TLC sheet was overlaid with agar containing a basal medium and precultured E. coli 215, and incubated at 37˚C for 20 h. The gel plate was sprayed with methanol solution containing 2, 3, 5-triphe- nyltetrazolium salt and B₁₂ compounds were visualized as red, indicating E. coli growth.

Sample H (10 g) containing high levels of B₁₂ was suspended in 500 mL of distilled water and homogenized in a mixer (TML 160). The homogenate was added to 50 mL of 0.57 mol/L acetic buffer (pH 4.5) with 0.05 g KCN and boiled for 30 min to extract B₁₂ compounds. Extraction procedures were performed in a draught chamber (Dalton Co., Tokyo, Japan). The boiled suspension was centrifuged at 5000× g for 10 min. An aliquot (approximately 200 mL) of the supernatant was placed in Sep-pak Vac 20 cc (5 g) C18 cartridges (Waters Corp.) prewashed with 75% (v/v) ethanol and equilibrated with distilled water. The C18 cartridges were washed with 30 mL of distilled water and B₁₂ compounds were eluted using 30 mL of 75% (v/v) ethanol. The remaining supernatant was treated in the same manner. Combined eluates were evaporated to dryness under reduced pressure, and the residual fraction was dissolved in 5.0 mL of distilled water and centrifuged at 10,000 × g for 10 min to remove any insoluble material. The supernatant fraction was loaded onto EASI-EXTRACT Vitamin B₁₂ Immunoaffinity Column (P80) [R-Biopharm AG, Darmstadt, Germany], and corrinoids were purified according to the manufacturer’s recommended protocol. B₁₂ compounds, pseudo B₁₂, and B₁₂ were dissolved in 0.1% (v/v) acetic acid and filtered using a Nanosep MF centrifual device (0.4 μm, Pall Corp., Tokyo, JAPAN) to separate small particles. Aliquots (2 μL) of filtrate were analyzed using LCMS-IT-TOF coupled with an Ultra-Fast LC system (Shimadzu, Kyoto, JAPAN). Each purified corrinoid was injected into an InertSustain column (3 μm, 2.0 × 100 mm, GL Science, Tokyo, JAPAN) and equilibrated with 85% solvent A [0.1% (v/v) acetic acid)] and 15% solvent B (100% methanol) at 40˚C. Corrinoid compounds were eluted using a linear gradient of methanol (15% solvent B for 0 - 5 min, increasing the concentration from 15% to 90% solvent B for 5 - 11 min, followed by decreasing the concentration from 90% to 15% solvent B for 11 - 15 min) at a flow rate of 0.2 mL/min. ESI conditions were determined by injecting pseudo B₁₂ or B₁₂ into the MS detector to ascertain the optimum parameters for detecting the parent B₁₂ compound and daughter ions. ESI-MS was operated in the positive ion mode with argon as collision gas. Pseudo B₁₂ (m/z 672.777) and B₁₂ (m/z 678.292) as [M + 2H]²⁺ were confirmed by comparing the observed molecular ions and retention times.

B₁₂ levels were assayed in 10 Chinese black tea leaves that are commercially available worldwide using the microbiological B₁₂ assay method based on L. delbrueckii ATCC 7830 (Table 1). Traces (0.25 - 0.69 μg/100g dry weight) of the corrected B₁₂ were observed in Pu’er, Fu, and Brick tea leaves. However, Ryubao tea leaves (sample H) contained the highest B₁₂ content (1.37 μg/100g dry weight), which is similar to that previously reported [11] . To further clarify whether Ryubao tea leaves generally contain high levels of B₁₂, we determined the B₁₂ content of the other Ryubao leaf samples. As shown in Table 2, the corrected B₁₂ content of various Ryubao tea leaves varied (0.06 - 1.37 μg/100g dry weight), and their mean value was calculated as approximately 0.69 μg of B₁₂, which is only slightly higher than that for Pu’er tea leaves (approximately 0.49 μg/100g dry weight). High levels (0.61 - 2.02 μg B₁₂ equivalent/100 g dry weight) of the alkali-resistant factor were detected in all tested Chinese black tea leaves.

B₁₂ compounds identified in Chinese black tea leaf samples A - J were analyzed using the E. coli 215 bioautogram after separation using silica gel 60 TLC (Figure 2). The Ryubao tea leaf extract (sample H) produced a

^*Total B₁₂ compounds were extracted from a portion (2.0 g) of each type of black leaf homogenate by boiling at pH 4.5 in the presence of 4.0 × 10⁻⁴ % KCN and assayed using the Lactobacillus delbrueckii ATCC 7830 microbiological assay. L. delbrueckii ATCC 7830 utilizes deoxyribosides, deoxyribonucleotides (alkali-resistant factor), and B₁₂. Correct B₁₂ values were calculated by subtracting the results for the alkali-resistant factor from those for total B₁₂ concentration. The B₁₂ assay was performed in triplicate.

^*Total B₁₂ compounds were extracted from a portion (2.0 g) of Ryubao tea leaf homogenates by boiling at pH 4.5 in the presence of 4.0 × 10⁻⁴ % KCN, and assayed using Lactobacillus delbrueckii ATCC 7830 microbiological assay. Correct B₁₂ values were calculated by subtracting the results for the alkali-resistant factor from those for total B₁₂ concentration. The B₁₂ assay was performed in triplicate.

single, clear spot with an R_f value identical to that of authentic B₁₂. Indistinct spots with the R_f value identical to that of authentic B₁₂ were detected in samples C, D, E, G, and I. The remaining samples showed no spot because of their lower B₁₂ contents.

To more precisely identify the corrinoid compounds present in Chinese black tea leaves, corrinoids were purified from the Ryubao tea leaf extract (sample H) containing high B₁₂ content and identified using LC/ESI-MS/MS (Figure 3). Authentic B₁₂ was eluted as a peak with a retention time of 7.5 min. The mass spectrum of authentic B₁₂ primarily comprised a doubly charged ion with m/z 678.2910 [M + 2H]²⁺ (Figure 3(A-1) and Figure 3(A-2)). MS/MS spectra revealed a predominant monovalent ion with m/z 359.0994, which was largely attri- butable to the nucleotide moiety of B₁₂ (Figure 3(A-3)). The corrinoid purified from the Ryubao tea leaf sample H was eluted as several ion peaks, indicating the presence of impurities. The mass spectrum of the main peak with m/z 687.2914 had a retention time of 7.5 min in the purified sample (Figure 3(B-1) and Figure 3(B-2)). The MS/MS spectrum of the purified compound with a monovalent ion with m/z 359.0960 was identical to that of authentic B₁₂ (Figure 3(A-3) and Figure 3(B-3)). These results indicate that the Ryubao tea leaf sample H contained authentic B₁₂ but not pseudo B₁₂ which is inactive in humans.

B₁₂ content in the tea drink prepared from the Ryubao tea leaf sample H was 0.8 ng/100mL of black tea. Therefore, consumption of approximately 300 L of this tea would provide the recommended dietary allowance for adults (2.4 μg/day) [14] [15] , although ingestion of such large quantities of tea on a daily basis is not recommended. Notably, Kittaka-Katsura et al. [10] demonstrated that administration of the Japanese black tea drink (B₁₂ content, approximately 2.0 ng/100mL) considerably improves B₁₂ status in B₁₂-deficient rats. Considering these earlier observations and our present findings, we propose that Ryubao tea leaves containing significantly levels of B₁₂ can be utilized as a source of vitamin B₁₂ for vegetarians.