Isolation, Identification, and Biotransformation of Teadenol a from Solid State Fermentation of Pu-erh Tea and in Vitro Antioxidant Activity

Post-fermented Pu-erh tea (PFPT) has several health benefits, however, little is known about the bioactive compounds. In this study, a PFPT compound was isolated by column chromatography and identified as Teadenol A by spectroscopic data analyses, including mass spectrometry and 1D and 2D NMR spectroscopy. Teadenol A in tea leaves was biotransformed by Aspergillus niger and A. tamari at 28 ˝ C for 14 d at concentrations ranging from 9.85 ˘ 1.17 to 12.93 ˘ 0.38 mg/g. Additionally, the compound was detected in 22 commercial PFPTs at concentrations ranging from 0.17 ˘ 0.1 to 8.15 ˘ 0.1 mg/g. Teadenol A promoted the secretion of adiponectin and inhibited the expression of protein tyrosine phosphatase-1B. Antioxidant assays (e.g., 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging activity, total antioxidant capacity (T-AOC), hydrogen donating ability, and superoxide anion radical scavenging capacity) revealed that Teadenol A has antioxidant properties. Therefore, Teadenol A is an important bio-active component of PFPT.


Introduction
Pu-erh tea (PET) is a traditional Chinese tea produced from sun-dried leaves of Camellia sinensis (Linn.)var.assamica (Masters Kitamura) in Yunnan, China.Based on its processing technology and quality characteristics, PET can be classified into Pu-erh shengcha (non-fermented Pu-erh tea, NFPT) or Pu-erh shucha (post-fermented Pu-erh tea, PFPT).NFPT is prepared by pressing sun-dried green tea leaves into a disk or bowl shape, and PFPT is produced from the solid state fermentation (SSF) of sun-dried green tea leaves at 40-60 ˝C and high humidity conditions [1,2].
Recently, compound 1 was detected during SSF of PFPT, but not in the raw material (Figure 1).To our knowledge, only theabrownin (TB), polysaccharides, α-tocopherol, GA, and CAF increase during SSF of PFPT [2].In this study, we isolated and identified this novel compound and assessed its antioxidant activity in vitro.Furthermore, fungi responsible for the biosynthesis of this compound were identified.
Recently, compound 1 was detected during SSF of PFPT, but not in the raw material (Figure 1).To our knowledge, only theabrownin (TB), polysaccharides, α-tocopherol, GA, and CAF increase during SSF of PFPT [2].In this study, we isolated and identified this novel compound and assessed its antioxidant activity in vitro.Furthermore, fungi responsible for the biosynthesis of this compound were identified.

PET Fermentation and Sample Collection
Sun-dried green tea leaves were purchased in Puer City, Yunnan.Green tea leaves (30 kg) were mixed with tap water (15 L) resulting in a solid content of approximately 65% (w/v).During

PET Fermentation and Sample Collection
Sun-dried green tea leaves were purchased in Puer City, Yunnan.Green tea leaves (30 kg) were mixed with tap water (15 L) resulting in a solid content of approximately 65% (w/v).During SSF, the leaves were mixed to ensure homogeneity, and tap water was added to maintain the solid content at 65%-75%.Samples were collected from the SSF tank every 7 days.Triplicate fermentations were performed.A total of 18 samples were collected and air-dried on days 0, 7, 14, 21, 28, and 35 of SSF.

Extraction and Isolation of 1
Ground fermented tea leaves (250 g) were sonicated twice for 1 h in the presence of 1250 mL of aqueous methanol (80%) and filtered.The solvent was concentrated in a rotary evaporator (RE-52AA, Shanghai Yarong Biochemistry Instrument Factory, Shanghai, China) at 40 ˝C.The resulting concentrate (50 mL) was directly added to an MCI-gel CHP 20P column (7.0 ˆ20 cm; 75-150 µm, Mitsubishi Chemical Industries, Tokyo, Japan) and eluted with 10% ethanol (Fengchuan Chenmical Reagent Technologies, Tianjin, China) in H 2 O. Fractions (100 mL) were collected; 300-500 mL aliquots were concentrated in a rotary evaporator, added to a Sephadex TM LH-20 column (5.5 ˆ20 cm, GE Healthcare, Uppsala, Sweden), and eluted with water (Figure 2a).Fractions (50 mL) were collected; 100-200 mL aliquots were concentrated in a rotary evaporator, added to a Sephadex TM LH-20 column (3.0 ˆ40 cm, Cherishtech, Beijing, China), and eluted with water (Figure 2b).Fractions (20 mL) were collected, and 40-100 mL aliquots were concentrated in a rotary evaporator (Figure 2c).The concentrate (10 mL) was extracted with acetonitrile (50 mL); the clear upper liquid was concentrated in a rotary evaporator until the acetonitrile was removed.Finally, the concentrate was freeze-dried in an FD5-series freeze-dryer (SIM, Beijing, China), resulting in the isolation of 1.
Appl.Sci.2016, 6, 161 3 of 12 SSF, the leaves were mixed to ensure homogeneity, and tap water was added to maintain the solid content at 65%-75%.Samples were collected from the SSF tank every 7 d.Triplicate fermentations were performed.A total of 18 samples were collected and air-dried on days 0, 7, 14, 21, 28, and 35 of SSF.

In Vitro Antioxidant Activity Assays
The antioxidant activity of compound 1 was evaluated by the following assays, 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging activity, total antioxidant capacity(T-AOC), hydrogen donating ability, and superoxide anion radical scavenging capacity.DPPH scavenging activity and superoxide anion radical scavenging capacity were determined by the method reported by Zhang [28].First, DPPH buffer solution (0.1 mmol/mL) (TCI, Shanghai, China) was prepared.Tea extracts (1 mL) of different concentrations were mixed with 3 mL DPPH buffer solution.The mixture was held for 20 min at room temperature.The scavenging effect was determined from ultraviolet (UV) adsorption measurements at 517 nm.The superoxide anion radical scavenging capacity assay consisted of mixing 5 mL Tris-HCl buffer (50 mmol/L, pH = 8.2) (Xilong Chemical, Guangzhou, China) with 0.5 mL of sample, maintaining the solution at 25 ˝C for 20 min, and adding 0.5 mL 1,2,3-trihydroxybenzene (Sangon Biotech, Shanghai ,China) (30 mmol/L, 25 ˝C, 4 min).UV absorbance was measured at 325 nm.Total antioxidant capacity and hydrogen donating ability were determined with commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).Antioxidant activity was expressed as IC50, defined as the concentration of the test compound required to inhibit the formation of radicals by 50%.Vitamin C was used as a positive control.
The bacterial and fungal strains isolated from the fermented tea leaves samples were inoculated into sterile sun-dried green tea and fermented for 14 d at 28 ˝C.The fermented tea leaves were air-dried and subjected to HPLC analysis.

Determination of Compounds in Commercial PFPT Samples
In this experiment, 22 PFPT samples were purchased from local markets (Table 1).GA, CAF, catechins, and compound 1 were measured by HPLC.

Statistical Analyses
Data were analyzed by one-way ANOVA and least-significant difference (LSD) for paired data.Data were expressed as mean ˘SD (Standard Deviation).Statistical analyses were performed with SPSS 19.0 software packages (v19.0,SPSS Inc., Chicago, IL, USA, 2010); p < 0.05 was considered to be statistically significant.

Nucleotide Sequence Accession Numbers
The bacterial 16S rRNA and fungal 18S rRNA gene sequences were deposited in GenBank under accession numbers KR149596-KR149624 and KR149625-KR149644, respectively.

Isolation and Identification of Compound 1
Ground samples were extracted twice with 80% methanol.Compound 1 was isolated from the extract using column chromatography followed by purity analysis by HPLC.The compound had one peak, and the peak area of the purified compound was 99.94%.Compound 1 consisted of a white powder with a molecular ion peak at m/z 276.0638, which corresponded to the molecular formula C 14 H 12 O 6 (Figure 3).The 1 H-NMR spectrum of 1 (Table 2) showed one methylene ( δ H 2.85, m), two methine ( δ H 4.39, m; and δ 4.52, s), meta-coupled ( δ H 5.78, J = 2.4 Hz; and δ 5.91, J = 2.4 Hz) aromatic, and three olefinic ( δ H 6.51, J = 0.9 Hz; δ 5.19, br s; and δ 5.27, br s) proton signals.The 13 C-NMR spectrum (Table 2) of 1 revealed the presence of one phloroglucinol-type aromatic ( δ C 95.9, 96.7, 99.3, 156.6, 157.8 and 157.9), one methylene ( δ C 25.4), two methine ( δ C 72.9 and 73.5), and four olefinic ( δ C 109.8, 116.6, 139.3, and 148.1) carbon signals.From these 1 H-and 13 C-NMR spectral data, we predicted that 1 had ring moieties that resembled the A-and C-ring structures in flavan-3-ol (catechins).The HMBC and HSQC spectra of 1 (Table 2, Figure 4) showed an olefinic carbon sequence (from C-12 to C-15) in the correlation peaks from H-13 to C-12 and C-14, and from H-15 to C-14.Additionally, the correlation peaks from H-15 to C-2 and from H-13 to C-16 in the HMBC spectrum showed the presence of C-2-C-14 and C-12-C-16 bonds, respectively.Furthermore, NOE spectral data (Table 2, Figure 4) supported the structural correlations observed in the HMBC spectrum.The small J value (2.4 Hz) of H-2 observed in the 1 H-NMR spectrum of compound 1 (Table 2) indicated a cis conformation between the C-2 and C-3 positions.The 1 H-NMR and 13 C-NMR spectral data of 1 (Table 2) were similar to those of Teadenol A [33,34].Therefore, compound 1 was identified as Teadenol A (Figure 5).Appl.Sci.2016, 6, 161 6 of 12 of C-2-C-14 and C-12-C-16 bonds, respectively.Furthermore, NOE spectral data (Table 2, Figure 4) supported the structural correlations observed in the HMBC spectrum.The small J value (2.4 Hz) of H-2 observed in the 1 H-NMR spectrum of compound 1 (Table 2) indicated a cis conformation between the C-2 and C-3 positions.The 1 H-NMR and 13 C-NMR spectral data of 1 (Table 2) were similar to those of Teadenol A [33,34].Therefore, compound 1 was identified as Teadenol A (Figure 5).(a)      (c)

Isolation and Identification of Fungal and Bacterial Strains
A total of 20 fungal and 29 bacterial strains, belonging to seven fungal and 13 bacterial species, respectively, were isolated from fermented tea leaves (Table 3).The strains were individually inoculated into tea leaves to determine their ability to synthesize Teadenol A. Teadenol A was detected in tea leaves fermented by Aspergillus niger and A. tamari at 28 ˝C for 14 d at concentrations ranging from 9.85 ˘1.17 mg/g to 12.93 ˘0.38 mg/g.This result was in agreement with the findings of Wulandari, who reported that tea leaves fermented by Aspergillus spp.contain high concentrations of Teadenols [33,34].Additionally, a method to produce Teadenol A-rich PFPT has been developed by adding A. niger or A. tamari during SSF.Teadenol A has been extracted using ethanol followed by column chromatography.

Determination of Teadenol A in Commercial PFPT
Aspergillus spp. is the dominant species in SSF of PFPT and play crucial roles in the quality of PFPT [2].In this study, Teadenol A was detected in tea leaves fermented by Aspergillus spp.To assess whether PFPT contains Teadenol A, we measured 22 commercial PFPT samples.Teadenol A was detected in each sample, at concentrations ranging from 0.17 ˘0.1 to 8.15 ˘0.1 mg/g (Table 1).However, Teadenol A was not detected in one PET [34], probably because the tea was NFPT.Aspergillus spp. is not involved in the production of NFPT; therefore, NFPT lacks Teadenol A.

Bioactivity of Teadenol A
Wulandari et al. [34] and Yanagita et al. [35] reported that Teadenol A stimulates the secretion of adiponectin and inhibits the expression of protein tyrosine phosphatase-1B (PTP1B).Adiponectin, a polypeptide that is highly specific to the adipose tissue, has anti-inflammatory and antiatherogeneic properties and beneficial effects on metabolism [36].Adiponectin, which reduces the relative risk of type 2 diabetes [37], is inversely correlated to visceral adipose tissue [38].Through its effect on adiponectin secretion, Teadenol A has anti-obesity properties.PTP1B is a negative regulator of insulin and leptin signal transduction [39] and a novel therapeutic target for type 2 diabetes mellitus, obesity, and insulin resistance [40].Teadenol A may have positive regulatory effects on insulin and leptin signal transduction by inhibiting the expression of PTP1B.Due to its multiple health benefits, Teadenol A has been used in pharmaceutical drugs, food and feed, cosmetics, and assay reagents [35].In this study, the antioxidant properties of Teadenol A were determined in vitro.The IC50 values from the DPPH scavenging activity and superoxide anion radical scavenging ability were 64.8 µg/mL and 3.335 mg/mL, respectively.The IC50 values from total antioxidant capacity and hydrogen donating ability were 17.6 U/mL and 12 U/mL, respectively.Teadenol A may be partly responsible for the health benefits of PFPT.Future studies should assess the exact role of Teadenol A in PFPT and possible synergistic effects with other bioactive compounds.
In conclusion, Teadenol A was isolated and identified following SSF of PFPT.The formulation of Teadenol A was dependent on A. niger and A. tamari.Teadenol A promoted the secretion of adiponectin, inhibited the secretion of PTP1B, and had antioxidant effects.Additionally, Teadenol was detected in variable amounts in 22 commercial PFPTs.Teadenol A is an important bioactive compound of PFPT.This study advances our knowledge about the health benefits and bioactive compounds of PFPT and provides insight into specific fungi in SSF.

Table 3 .
Identification of bacterial and fungal strains isolated during solid state fermentation (SSF) of PFPT.