Comparison of chemical profiles between the root and aerial parts from three Bupleurum species based on a UHPLC-QTOF-MS metabolomics approach

Background Bupleuri Radix (Chaihu) represents one of the most successful and widely used herbal medicines in Asia for the treatment of many diseases such as inflammatory disorders and infectious diseases over the past 2000 years. In the Chinese Pharmacopoeia, Chaihu is recorded as the dried roots of Bupleurum chinense DC. and B. scorzonerifolium Willd. (Umbelliferae). However, the widespread demand for the herb has tended to far outstrip the supply. Whether the aerial parts, which account for 70 ~ 85% of the dry weights of Bupleurum species, could be used as an alternative for the root has become an important scientific issue for the sustainable utilization of Bupleurum species. On the other hand, in some areas including the southeast of China as well as in Spain, the aerial parts of Bupleurum species have already been used in the folk medications. Therefore, to clarify whether the root and aerial parts of Bupleurum species are “equivalent” in the types and quantities of chemical constituents which subsequently influence their biological activities and therapeutic effects is of great importance for both the rational and sustainable use of this herb. Methods In the present study, the chemical profiles between the root and aerial parts of Bupleurum species from different species and collected from various locations were analyzed and compared by the ultra-high performance liquid chromatography quadrupole/time of flight-mass spectrometry (UHPLC-QTOF-MS). Results A total of 56 peaks were identified in the root and/or aerial parts from different batches of Bupleurum species, by comparison of references standards or with those reported in the literature. Principal Component Analysis (PCA) was conducted for displaying the differentiating clustering between these two parts. Conclusion The results disclosed the distinct variations between them, which indicated that the aerial parts could not be used as an alternative of root from a chemodiversity perspective. The differentiating markers resulted from the PCA analysis could also be utilized for the differentiation between them. Further validation of their biological differences is anticipated in the future study.


Background
Bupleuri Radix (Chinese name: Chaihu) represents one of the most popular Traditional Chinese Medicines (TCM) over the past 2000 years. Its TCM indications include the treatment of influenza or common cold with fever, chills and fever from malaria, distending pain in the chest and menstrual disorders [1]. In the Chinese Pharmacopoeia, Chaihu is recorded as the dried roots of Bupleurum chinense DC. and B. scorzonerifolium Willd. (Umbelliferae) [1]. It is often found in clinical prescriptions and proprietary Chinese medicines, such as Xiao-Chaihu-Tang, Xiao-Yao-Wan, Jia-Wei-Xiao-Yao-Wan and Chai-Ling-Tang. In addition to the authentic species of Chaihu, there are more than 20 other species in the genus Bupleurum also habitually utilized as Chaihu in some local areas. Knowing the high demand for Bupleuri Radix and knowing the diversity of species that can be-both rightly and wrongly-used for this herb, the resources of Chaihu are very scare. Today, Chaihu from the species of B. yinchowense Shan et Y. Li has become mainstream in the market. [2] The species of B. yinchowense is abundantly distributed in the Northwest of China and is widely used in folk medicine for relieving fever, soothing liver and improving the symptoms of emotional instability such as depression, anxiety and phobia [3][4][5]. Additionally, in the Japanese Pharmacopoeia (16th edition), the official botanical origin of Bupleuri Radix (pronounced "saiko" in Japanese) is the roots of B. falcatum L [6]. Actually, B. falcatum is also commonly used in China and Korea [7]. In Japan, B. falcatum L is known for its therapeutic effects in the treatment of chronic hepatitis, auto-immune diseases and diabetes [8][9][10][11]. It is also used as an ingredient in herbal tea and traditional fermented beverages [8].
Previous phytochemical studies on approximately 50 Bupleurum species led to the isolation and identification of almost 250 natural compounds from all major phytochemical classes, including mono-and sesquiterpenes (essential oils), triterpenoid glycosides (saikosaponins), sterols, lignans, flavonoid glycosides, coumarins, and polyacetylenes [12][13][14]. In addition, minor components, including phenylpropanoids, polysaccharides and a few alkaloids, have also been reported [12]. Among them, the saikosaponins (SSs) are acknowledged to be the principal bioactive components, which can be divided into six types on the basis of the aglycones: type I-VI ( Fig. 1) [15,16]. Flavonoids are another class of bioactive secondary metabolites present in all species of the genus Bupleurum [17]. Most flavonoids in the genus are derivatives of the flavonol aglycones kaempferol, isorhamnetin or quercetin [12].
Since Chaihu is very rare in nature, the amount of wild samples is not sufficient for commercial exploitation [18]. Although Chaihu have been widely cultivated nowadays, the widespread demand for the herb has still tended to far outstrip the supply [19,20]. As the aerial part of Bupleurum species accounts for more than half of the whole plant, some areas in the southeast of China use the whole herb for the medication [21]. In Guangdong province of China, the root and aerial parts of Bupleurum species are sold separately for the folk use [22]. Besides, the aerial parts of Bupleurum species are used as a popular topical antiseptic and anti-inflammatory remedy in Spain [23]. Therefore, the short supply and perspective for sustainable utilization of Chaihu has stimulated the interest on comparing the "equivalence" of the root and aerial parts. Whether the root and aerial parts vary in the types and quantities of chemical constituents which subsequently influence their therapeutic effects, would determine whether the aerial parts of Bupleurum species could be used as a suitable substitute for the root. Additionally, whether Chaihu should be prescribed as root, aerial parts or whole plant of Bupleurum species was also needed to be clarified by scientific evidences, so as to prevent or avoid the misuse of this herb. To differentiate the crude materials from which medicinal parts used in proprietary Chinese medicines containing Chaihu is also significant for their quality control. Therefore, a comprehensive analysis of the chemical profiles is highly needed to be conducted for the two different parts.
The objective of the present study is to analyze and compare the chemical profiles between the root and aerial parts of Bupleurum species utilizing the ultra-high performance liquid chromatography quadrupole/time of flight-mass spectrometry (UHPLC-QTOF-MS). The authentic species of B. chinense as well as B. yinchowense and B. falcatum collected from different locations were investigated. The overall results provided comprehensive chemical comparison for the two different parts of Bupleurum species, which was anticipated to advise the possibilities of alternate use of these two parts.

Plant materials
Eight batches of the whole plant including the root and aerial parts of Bupleurum chinense DC., B. yinchowense and B. falcatum were collected. Details of the sample are shown in Table 1

Chemicals and reagents
Chemical markers of saikosaponins a, c and d were purchased from Chengdu Must Bio-Technology Co., Ltd. (Chengdu, People's Republic of China). The purities of all saikosaponins were determined to be higher than 98% by HPLC-DAD analysis. The solvents, acetonitrile and methanol, were of HPLC grade from E. Merck (Darmstadt, Germany). Formic acid with a purity of 96% was also of HPLC grade (Tedia, U.S.A.). Water was obtained from a Milli-Q water purification system (Millipore, Bedford, MA, U.S.A.).

Preparation of sample solution
The dried roots and aerial parts of eight batches of samples involving Bupleurum chinense, B. yinchowense and B. falcatum from different growing areas were separated and grinded into homogeneous powders using liquid nitrogen. The dried powder (~0.1 g) was weighed accurately into a 15 mL microcentrifuge tube and was then extracted twice with 10 ml of methanol using an ultrasonicator (1875HTAG, CREST, U.S.A.) for 30 min at room temperature each time. After centrifugation at 3000 rpm for 10 min, the supernatant was transferred into a 25 ml volumetric flask and was adjusted to the volume with methanol. Finally, 1.0 mL extraction was transferred to 1.5 microcentrifuge tube and centrifuged again for 10 min at 12,000 rpm. An aliquot of 90 μl of supernatant was transferred to the glass inserts of 1.5 ml brown HPLC

Chemical profiling
The chemical profiles of the root and aerial parts of Bupleurum species were analyzed by UHPLC-QTOF-MS. The representative total ions current (TIC) chromatograms of the different parts from B. chinense, B. yinchowense and B. falcatum are shown in Fig. 3. The major peaks in the TIC chromatograms were identified, with peaks 20, 28 and 43 unambiguously identified as saikosaponins c, a and d (SSc, SSa and SSd) by comparison of their chromatographic retention times, accurate molecular weights and characteristic mass fragment ions with those of the references standards. Other peaks were tentatively identified by comparison of their accurate mass data with those reported in the literature. Detailed information related to the illustration of all 56 peaks was shown in Table 2.
In the TIC chromatograms, saikosaponins and flavonoids represents majority of the peaks identified, with flavonoids (peaks 1, 2, 3, 4, 5, 6, 7, 54, 55 and 56) accumulated in the previous 10 min of the eluting time while saikosaponins were eluted at the rest of time. The root and aerial parts of all the three Bupleurum species exhibited varied profiles. It is distinct and consistent for all the roots contain more chromatographic peaks covering both of the saikosaponin and flavonoid part of the TIC chromatograms. The profiles of the roots from the three Bupleurum species were similar especially for the characteristic saikosaponin part (peaks 20-52). In contrast, the aerial parts contain abundantly flavonoids with little or no saikosaponin peaks. The results clearly demonstrated the distinct chemical profiles of the root and aerial parts of Bupleurum species. Yen et al. compared the saikosaponins a, c and d between the root and aerial parts of three Bupleurum species using thin-layer chromatography (TLC) scanning. The results showed that the aerial parts contained low levels of saikosaponins, which were different from that of the root [24]. These were in accordance with those provided in the present study, which thus strengthened the conclusions that the aerial parts could not be used as an alternate of root from a chemodiversity perspective.

Principal component analysis (PCA)
The differences between the root and aerial parts of eight herbal samples were further displayed by the Principal Component Analysis (PCA). The full time-of-flight (TOF) mass spectral data of each sample were first processed by MassHunter Workstation software. Ions were extracted by molecular feature extraction (MFE) algorithm characterized by retention time (RT), intensity in apex of chromatographic peak, and accurate mass, exported as the Compound Exchange Format (.cef ) file. These results were then analyzed by Mass Profiler Professional (MPP) software. Entities that present in more than 50% of samples in at least one condition were filtered by frequency before doing Principal Component Analysis (PCA). Finally 258 features were left for further PCA study. The resulting PCA graph also demonstrated the distinct clustering between the root and aerial parts of the investigated samples, which indicating the chemical difference between these two parts (Fig. 4).

Potential differentiating markers
In order to find out the potential differentiating markers for distinguishing the different parts of Bupleurum species, significant testing and fold change was investigated     to identify statistically differentiative compounds by applying appropriate test and conditions. Nine compounds, out of 258 entities from the above frequency filtration were found to be significantly different among the two parts using one way ANOVA and a level of probability of 0.001 and fold change >2, as listed in Table 3. These differentiating markers with the lowest p-values and highest foldchanges (most significant with greatest abundance differences) posed mostly influential features for the differentiation between the root and aerial parts, which therefore could be used as markers for differentiation.

Conclusions
The present study revealed the distinct chemical profiles between the root and aerial parts of Bupleurum species, which indicated that the aerial parts could not be used as an alternative of root from a chemodiversity perspective. Meanwhile, the established UHPLC-QTOF-MS method in the present study as well as the potential differentiating markers could be utilized to profile and distinguish the root and aerial parts of Bupleurum species from different species or locations. Thus, the approach established here will provide a comprehensive analysis of chemical profiles between the root and aerial parts from the commonly used Bupleurum species which will be helpful for testing the crude materials of proprietary Chinese medicines containing Chaihu as well as for establishing guidelines for the appropriate clinical use of Chaihu. Furthermore, this information will be of great significance to the efficient use of botanical resources.