Comparison of Volatile Components between Raw and Vinegar Baked Radix Bupleuri by GC-MS Based Metabolic Fingerprinting Approach

Radix Bupleuri (RB), also named Chaihu in Chinese, is a commonly used herbal drug in traditional Chinese medicine (TCM), and the processing of RB with vinegar to prepare vinegar-baked Radix Bupleuri (VBRB) has a long history in the clinic of TCM. In the present study, GC-MS coupled with multivariate data analysis was applied to compare the volatile components between crude and two vinegar processed RBs. After vinegar baking, the oil yields were decreased significantly, and the chemical compositions were also changed greatly. The chemical changes included the disappearance or appearance, as well as the content increase or decrease of some volatile compounds. The oil yields of two different VBRBs showed no significant difference but differed markedly in their chemical compositions, suggesting that the type of vinegar exerted great impacts on the vinegar-baking process. Thus, the effect of different vinegars on processing should be further investigated to ensure the therapeutic effect and safety of VBRB in clinic.

Processing of herbal drugs has been a part of the heritage of Chinese medicine for thousands of years; it plays an important role in disease prevention and control for the Chinese people and ensures the safe and effective clinical treatment of TCM [7]. When RB is mixed with vinegar and then baked to dry, it is changed to vinegar-baked Radix Bupleuri (VBRB). The pharmacological effects and components in the drug will change a little bit due to the vinegar-baking procedure. The bile secreting and hepatoprotective effects are enhanced and it is quite effective in curing liver related diseases such as jaundice, hepatitis, cirrhosis, and liver cancer [8][9][10][11]. Previous reports showed that the contents of saikosaponin b1 and saikosaponin b2 were increased, while the saikosaponin a, saikosaponin c, and saikosaponin d were decreased [12,13] after the vinegar-baking process. Previous investigations have demonstrated that the volatile oil of RB showed the effects of anti-influenza, antipyretic, anti-inflammation, and analgesia [14,15]. After being processed with vinegar, both the yields [16,17] and compositions [18] of volatile oil in Radix Bupleuri could be changed. In addition, according to Chinese Pharmacopoeia, RB should be baked with rice vinegar to give VBRB. However, there are many kinds of vinegars present in the Chinese market, and most of them 2 Evidence-Based Complementary and Alternative Medicine were used in the vinegar-baking process of herbal drugs. The influence of different vinegars on the volatile compounds in VBRB remains unknown.
Steam distillation and solvent extraction methods combined with gas chromatography (GC) or gas chromatography-mass spectrometry (GC/MS) are used as the routine methods for the analysis of the volatile oils of TCMs. And GC-MS is one of the most robust methodologies widely applied in volatile metabolite analysis because of its high sensitivity, peak resolution, and reproducibility [19,20]. Recently, GC-MS-based global metabolic profiling, coupled with multivariate analysis, has been successfully applied to quality assessment of volatile compounds in herbal drugs, such as agarwood [21], Cassia [22], Ginseng [23], and Atractylodis Macrocephalae Rhizoma [24].
In this study, two different vinegars were used to prepare VBRB. And the aim of the present study was to investigate and compare the chemical differences of the volatile oils between crude and vinegar-processed RB samples by GC-MS coupled with multivariate data analysis. Two different VBRBs were also compared to elucidate the vinegar type on vinegarbaking process of RB.

Solvents and Chemicals.
Analytical grade n-hexane was purchased from Beijing Chemical works (Beijing, China) and n-tetracosane (purity > 98%) which used as an internal quality standard for GC-MS analysis was bought from Johnson Matthey Company (Shanghai, China). Bran vinegar was bought from Tongwanzhenji Food Company (Hebei, China) and Shanxi vinegar was from Shanxi Donghu Vinegar Group (Shanxi, China).

VBRB Preparation.
According to Chinese Pharmacopoeia, the crude RB (100 g) was incubated with Shanxi vinegar or rice vinegar (20 g), respectively. Then the material was dried by stir-firing to obtain S-VBRB (by Shanxi vinegar) or R-VBRB (by rice vinegar) after vinegar was totally absorbed into raw RB. Six different batches of S-VBRB and R-VBRB were prepared for each kind of vinegars.  [1]. The dried powder (30 g) was accurately weighed and transferred to a 500 mL roundbottomed flask soaked in 240 mL of water for 2 h. Water was added from the top of the volatile oil determination apparatus until the water spilled onto the round-bottomed flask and 2 mL of n-hexane was added to the water layer. Then the essential oils were extracted by water distillation for 6 h. Volatile oil was separated from the water layer and leached into the n-hexane layer, and then the n-hexane layer was dried over anhydrous sodium sulfate (Na 2 SO 4 ) and weighted. The samples were stored at 4 ∘ C in the refrigerator before GC-MS analysis. All samples were prepared in sextuplicate.

GC-MS Analysis
Parameters. GC-MS analysis was performed using a Polaris Q ion trap mass spectrometer (Thermo Fisher Scientific Inc., USA). Chromatography was performed on a DB-5MS capillary column (30 m × 250 m i.d., 0.25 m film thickness; 5% diphenyl cross-linked 95% dimethylpolysiloxane; Agilent J&W Scientific, Folsom, CA). Helium carrier gas was used at a constant flow rate of 1 mL⋅min −1 . Approximately 1.0 L of samples was injected at a constant temperature of 250 ∘ C in splitless mode. Initial temperature was set to 50 ∘ C and held for 1 min, followed by a ramp to 100 ∘ C at 10 ∘ C⋅min −1 and held for 2 min and then to 180 ∘ C at 3 ∘ C⋅min −1 , and then rose to 220 ∘ C at 15 ∘ C⋅min −1 and maintained 1 min and post-run temperature to 300 ∘ C for 5 min. The solvent delay was set as 5 min. The interface and source temperatures were set at 280 ∘ C and 200 ∘ C, respectively. MS detection was implemented with electron ionization (electron energy of 70 eV) and full scan mode (m/z 50-650).

Compound Identification.
The components eluting within the total ion chromatogram were extracted in AMDIS, matrix interference was then resolved, and overlapping components were removed. Then the compounds were positively identified using the National Institute of Standards and Technology (NIST) 05L Mass Spectra Database containing about 107,000 compounds, as well as comparison with the literatures [18,25,26]. The semiquantitative analysis of volatile compounds was performed by comparing their peak areas to that of the internal standard compound on the GC-MS total ion chromatogram. The percentage compositions of compounds were calculated by area normalization method.

Data Analysis.
To assess difference (or similarity) between RB and VBRBs, principal component analysis (PCA) was applied to relative peak area values of volatiles obtained on the GC-MS total ion chromatograms using SIMCA-P13.0 (Umetrics, Umeå, Sweden) to clarify the relationship between the RB and VBRBs. In addition, hierarchical heat map clustering analysis was performed with MetaboAnalyst (http://www.metaboanalyst.ca/). The significance level was set at < 0.05 for all tests by SPSS 16.0.

Chemical Difference of Raw and Vinegar-Baked RBs by
Multivariate Analysis. Since the oils yields of RB decreased significantly after the vinegar-baking process, the change of chemical compositions between raw and processed RBs should be further investigated. Thus, all the GC-MS data (134 peaks) were subjected to PCA analysis to visualize the chemical difference between the raw and vinegar-baked RBs. In the score plot of the first two principal components   (PC1: 49.7%, PC2: 20.4%), 18 samples were obviously clustered into three groups (Figure 2). The raw RB was located in the positive side of PC1, while the two vinegar-baked RBs were located on the negative side of PC1, which can be further separated by PC2. The separation between the RB and VBRB was more remarkable than those between the two VBRBs, and the observed separation indicated that RB and VBRB were obviously different in their volatile components.
To visualize the changes between the raw and two processed RBs, heat map was further generated based on the differential compounds determined above ( Figure 5). Here, the red and green colors corresponded to increased and decreased constituents in the VBRB after processing, respectively. It was obvious from the left side that the 18 samples of RB and VBRB could be clearly divided into two main clusters, and S-VBRB and R-VBVB could be separated in the second cluster. The compounds could be also divided into two main clusters on the top, and the left cluster that presented these ingredients were rich in raw RB, while the compounds in the right cluster showed high contents in VBRB.

Conclusion
In the present study, GC-MS coupled with multivariate data analysis was applied to compare the volatile components between crude and two vinegar-processed RBs.  with the previous studies, more chemical components were identified, and the influence of vinegar type on processing was also discussed. After vinegar-baking, not only the oil yields were decreased significantly, but the chemical compositions were also changed, including the disappearance or appearance, as well as the content increase or decrease of some volatile compounds. According to the theory of TCM, the purpose of herb processing is to increase potency, reduce toxicity and side effects, and alter the properties or functions [27]. The relationship between the change of volatile oil in RB and the drug action should be further investigated.
Two different VBRBs also showed differences in the volatile compositions, suggesting that the type of vinegar exerted great impacts on the vinegar-baking process. Thus, in order to ensure the therapeutic effect and safety of VBRB in clinic, the effect of processing by different vinegars should be further investigated on the other herbal drugs.