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Determination of xanthones and flavonoids of methanol extracts obtained from different parts of the plants of three Gentianaceae species
Accepted Manuscript Title: Determination of xanthones and flavonoids of methanol extracts obtained from different parts of the plants of three Gentianaceae species Authors: Cheng-Yong Feng, Qian Wu, Dan-Dan Yin, Bing Li, Shan-Shan Li, Zhong-Qiu Tang, Yan-Jun Xu, Liang-Sheng Wang PII: DOI: Reference:
S0731-7085(18)30773-8 https://doi.org/10.1016/j.jpba.2018.08.059 PBA 12190
To appear in:
Journal of Pharmaceutical and Biomedical Analysis
Received date: Revised date: Accepted date:
18-4-2018 4-7-2018 27-8-2018
Please cite this article as: Feng C-Yong, Wu Q, Yin D-Dan, Li B, Li SShan, Tang Z-Qiu, Xu Y-Jun, Wang L-Sheng, Determination of xanthones and flavonoids of methanol extracts obtained from different parts of the plants of three Gentianaceae species, Journal of Pharmaceutical and Biomedical Analysis (2018), https://doi.org/10.1016/j.jpba.2018.08.059 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Determination of xanthones and flavonoids of methanol extracts obtained from different parts of the plants of three Gentianaceae species Cheng-Yong Feng
a,b
, Qian Wu
a,b
, Dan-Dan Yin
a,b
, Bing Li
a,b
, Shan-Shan Li
a,b*
, Zhong-Qiu Tang c,
a
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Yan-Jun Xu d, Liang-Sheng Wang a,b*
Academy of Sciences, Beijing 100093, China
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Key Laboratory of Plant Resources and Beijing Botanical Garden, Institute of Botany, Chinese
University of Chinese Academy of Sciences, Beijing 100049, China
c
Forestry and Agricultural Academy of Greater Khingan Mountains, Jiagedaqi 165000, China
d
College of Science, China Agricultural University, Beijing 100094, China
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N
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b
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Highlights:
Leaf was recommended as the most valuable part for medicine.
The fingerprints of these three species were significantly differernt.
An efficient method was established to determine the bioactive compounds.
Abundant xanthones and flavonoids were detected in these three species.
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This research provided basic data for their pharmacological study.
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*
Corresponding author. Tel: +86 10 6283 6654. Fax: +86 10 6259 0348. E-mail: [email protected]
(L.-S Wang). Tel: +86 10 6283 6053. E-mail: [email protected] (S.-S Li). 1
Abstract Gentianopsis barbata, Halenia corniculata, and Gentianella acuta were widely distributed throughout China and commonly used in traditional Chinese medicine. However, owing to similar living environments and morphological features, locals often had trouble distinguishing between these three
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species. In this present study, chromatograms at 350 nm were obtained and the composition and content of their chemical compounds determined using HPLC-DAD/ESI-MS2. In total, 35 chemical compounds
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were detected, 32 of which were identified, 25 of which were xanthones, 6 flavonoids, and 1 chlorogenic acid. The 350 nm chromatograms of these three species displayed evident differences. The individual
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compounds and their occurrence and content in different parts of the plant within different species were
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included in our results. This basic data will be useful for future pharmacological study. The total
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compositions of flavonoids and xanthones were approximately comparable in G. barbata and H.
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corniculata. Meanwhile, xanthones were predominant in G. acuta. From the perspective of chemical
these three species.
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compound compositions, the leaf is recommended as the most valuable medicinal section for each of
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Keywords: Gentianaceae, xanthone, flavonoid, HPLC
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1. Introduction Gentianaceae plants are annual herbs or perennial herbs, shrubs, and small trees. The Gentianaceae Family contains about 80 genera, comprising approximately 1000 species worldwide. It exhibits wide global distribution with predominant clustering in the North Temperate Zone. In China, 22 genera
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covering nearly 500 species have been found, of which, more than 70 species from 12 genera are of pharmacological value [1]. Gentianopsis barbata (Froelich) Ma is an annual or biennial plant native to
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China, Japan, Kazakhstan, Kyrgyzstan, Mongolia, and Russia and found at altitudes ranging between
700 m and 4400 m. It has many beneficial effects, such as heat-clearing, detoxicating, anti-inflammation,
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and cholagogue [2]. Halenia corniculata (Linnaeus) Cornaz is an annual plant which is commonly found
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in China, Japan, Korea, Mongolia, and Russia at altitudes between 200-1800 m. It may be used in the
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treatment of gastritis, intestinal and stomach pain, liver diseases, colitis, and enterocolitis [3]. Gentianella
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acuta (Michaux) Hulten is also an annual plant primarily distributed in China, Mongolia, East Russia,
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and North America at altitudes between 0 m and 1500 m. The pharmacological activity of G. acuta mainly includes hepatoprotection, hypoglycemic, antioxidant, and anti-inflammation [4].
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Gentianaceae plants are rich sources of xanthones, flavonoids, and terpenoids [1]. The
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pharmacological activities of these aforementioned three species are related to these compounds. It has been demonstrated that mangiferin derived from G. barbata could effectively alleviate liver damage in
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rats induced by paracetamol, tetrachloromethane, and D-galactosamine [5]. Zhang, Ahn, Sun, Kim, Hwang, Ryu and Kim [6] found that the xanthones from H. corniculata could prevent bone hardening from osteoporosis by inhibiting osteoclastogenesis in vitro. Also, it was reported that xanthones from G. acuta could inhibit the activities of acetylcholinesterase and monoamine oxidase [7]. Xanthones and flavonoids are important secondary metabolites which have aroused tremendous 3
interest owing to their pharmalogical properties and beneficial effects on humans. Flavonoids are widespread in plant kingdom, and xanthones usually occur in a few higher plant families where they are of taxonomic value [8]. The basic skeleton of xanthones is a C6-C1-C6 structure (Figure 2) which is symmetrical. The numbering system of xanthones was proposed by Gottlieb using xanthene-9-one as the
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basic structure [9]. Carbons 1-4 (ring A) are derived from acetic acid and carbons 5-8 (ring B) are derived from shikimic acid. When oxygenation occurs only in ring B, the lowest numbers are used except
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discussing the biosynthetic pathways [10]. The C6-C3-C6 nucleus of flavonoids (Figure 2) is formed by a series of condensation reactions between a coumaric acid (B-ring and carbon atoms 2, 3 and 4 of C-
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ring) and several malonic acids (A-ring) [11]. Naturally, in the majority of cases, xanthones and
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flavonoids exist in the form of glycosides or their derivatives. Xanthones have abundant pharmacological
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worth, with properties including antidepressant, antituberculotic, depressant, choleretic, diuretic,
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antimicrobial, antiviral, and cardiotonic [8, 12-15]. Flavonoids also have diverse biological activities,
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such as antioxidant, antibacterial, anti-inflammatory, anti-obesity, astringent, antiproliferative, anticarcinogenic, and UV-protection activities [16-20].
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In Northeast China, the above mentioned three species are used as traditional Chinese medicine.
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Unfortunately, locals often confuse these three species owing to their similar living conditions and morphological features (Figure 1). These three species are even harder to identify once dried to serve as
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medicinal raw materials. Previously, numerous investigations focused on the isolation and clarification and pharmacological properties of the chemical compounds from these three Gentianaceae plants. Literature on their chemical compound chromatograms were almost unavailable. In the present study, a rapid and efficient method was established utilizing HPLC-DAD to obtain their chromatograms. Moreover, we evaluated the composition and content of xanthones and flavonoids from different parts of 4
each species with HPLC-DAD/ESI-MS2. This allows their differentiation from the perspective of chemical compounds and also provides a reference for the screening of the best medicinal part.
2. Experimental section 2.1. Plant materials
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Three species of family Gentianaceae, i.e. Gentianopsis barbata (Froelich) Ma (voucher specimen No. Wls2014001), Halenia corniculata (Linnaeus) Cornaz (voucher specimen No. Wls2014002), and
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Gentianella acuta (Michaux) Hulten (voucher specimen No. Wls2014003) were collected from the Greater Khingan Mountains, Heilongjiang Province, Northeast China, on a sunny morning of August
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2014. The authentication of plant materials was generously accomplished by Jian-Fei Ye (an Engineer of
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Beijing Botanical Garden, Institute of Botany, Chinese Academy of Sciences). Plants at flowering phase
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were harvested. For each species, plants with similar morphological characteristics were randomly
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selected for sampling within the population. All the samples were brought back to the laboratory using
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an airtight preserving box with ice bags in it. Subsequently, the root, stem, leaf, flower, and whole plant
later use.
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were freeze-dried with a vacuum freeze drier (FD-1T, Beijing, China), and then stored in a desiccator for
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2.2. Chemicals and reagents
The xanthone standard, 8-O-glucosyl-1,5-dihydroxy-3-methoxyxanthone (swertianolin) was obtained
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from Meilunbio (Dalian, China). The flavonoid standard, 3-O-rutinosylquercetin (rutin); and chlorogenic acid standard, trans-5-caffeoylquinic acid (CAQ) were provided by the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). The 6-C-β-D-glucopyranosylluteolin (isoorientin) and 6-C-β-D-glucopyranosylapigenin (isovitexin) were purchased from Shanghai Tauto Biotech (Shanghai, China). The methanol, formic acid, and acetonitrile used for HPLC analysis were of 5
chromatographic grade and purchased from Alltech Scientific (Beijing, China). The ultrapure water was produced by a Mini-Q System (Millipore, Billerica, MA, USA). 2.3. Preparation of methanol extraction The freeze-dried samples were ground into fine powder in liquid nitrogen with mortars and pestles.
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About 0.1 g of fine powder was weighed and transferred into a 10 mL centrifuge tube. Then the powder was mixed with 2 mL 2% formic acid in methanol (v/v), shaken in a QL-861 vortexer (Kylinbell Lab
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Instruments, Jiangsu, China) for 30 s, sonicated in a KQ-500DE ultrasonic cleaner (Ultrasonic instruments, Jiangsu, China) at room temperature for 20 minutes, and then centrifuged in SIGMA 3K30
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(Sigma Centrifuges, Germany) (12,000×g, 10 min). Subsequently, the supernatant was collected in
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another a 10 mL centrifuge tube. The above steps were then repeated two more times. All the supernatants
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were put together and supplemented up to a volume of 6 mL with 2% formic acid in methanol (v/v).
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After which, a certain volume of ultrapure water was added into the extraction to make sure the
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concentration of water was 30% (v/v), and then stored at -20°C for 24 hours to precipitate chlorophyll. Then, the mixture was filtered through 0.22 μm millipore membrane (Shanghai ANPEL, China) after
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centrifugation. A 1 mL aliquot of clean supernatant was analyzed on HPLC-DAD and HPLC-ESI-MS2.
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For each sample, three biological replicates were performed. 2.4. HPLC-DAD and HPLC-ESI-MS2 analysis The systems and conditions used for HPLC-DAD and HPLC-ESI-MS2 analysis were similar to our
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previous study [21]. The column was an ODS-80Ts QA C18 column (150 mm4.6 mm, 5 μm i.d., Tosoh, Tokyo, Japan) protected with a C18 guard cartridge (ANPEL, Shanghai, China). Eluent A was 5% formic acid in ultrapure water (v/v), and eluent B was absolute acetonitrile. The gradient elution protocol was as follows: 10% B at 0 min, 22% B at 20 min, 60% B at 65 min, and 10% B at 70 min. For HPLC-DAD 6
analysis, a 10 μL solution of each sample was injected, the flow rate was 0.8 mL/min, and the column temperature was 35°C. Chromatograms were acquired at 350 nm, and the photodiode array spectra were recorded from 200 nm to 800 nm. For HPLC-ESI-MS2 analysis, the compounds were analyzed in both positive ion (PI) and negative ion (NI) mode, and the following MS detection conditions were used:
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capillary voltage, 4.0 kV; nebulization pressure, 241.3 kPa; gas (N 2) temperature, 350°C; gas flow rate, 8.0 L/min; capillary offset voltage, 77.2 V; capillary exit voltage, 127.3 V; and scan range, 100-1000
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(m/z).
2.5. Quantitative analysis of xanthones, flavonoids, and chlorogenic acids
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The quantitative analyses of chemical compounds from these three Gentianaceae species were based
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on the linear regression of HPLC-DAD peak area of commercial standards. Swertianolin, rutin, and
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trans-5-caffeoylquinic acid were used as standards for the semi-quantitation of all xanthones, all
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flavonoids, and a chlorogenic acid, respectively. The following linear regression equations were obtained:
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for swertianolin, Y = 219.73X – 0.09 (r2 = 1.0000); for rutin, Y = 319.86X + 0.93 (r2 = 0.9999); for CQA, Y = 665.57X – 1.43 (r2 = 0.9996). The concentrations of all the chemical compounds were expressed as
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milligrams of corresponding standards per gram of dry weight (DW).
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3. Results and Discussion 3.1. Method validation
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To validate the reliability of the optimized HPLC method, the limit of detection (LOD) and the limit of quantitation
(LOQ)
were
determined
for
xanthones
with
8-O-glucosyl-1,5-dihydroxy-3-
methoxyxanthone (swertianolin), flavonoids with 3-O-rutinosylquercetin (rutin) and chlorogenic acids with trans-5-caffeoylquinic acid (CQA). The results of method validation for three external standards (swertianolin, rutin, and CQA) are shown in Table S1. The concentration of three standards used for 7
calibration curves were all in the range from 7.81 to 500.00 μg/mL. Three replicates were performed for each concentration of three standards. The HPLC-DAD peak areas at 350 nm and concentrations of standards displayed good linear relationship. The limit of detection (LOD) and the limit of quantitation (LOQ) were defined as the ratio of signal to noise at 3:1 and 10:1, respectively. The LOD and LOQ
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obtained for swertianolin (0.50 and 1.66 μg/mL), rutin (0.65 and 2.15 μg/mL), and CQA (0.45 and 1.49 μg/mL) are shown in Table S1 as well.
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3.2. General
Good peak separation and resolution were achieved via an optimized gradient elution protocol (Figure
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3). In total, 35 chemical compounds were detected from these three species. Using UV-vis absorption
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spectroscopy, elution order, and mass spectrum analysis, 32 compounds were identified or tentatively
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3.3. Identification of xanthones
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compounds are displayed in Table 1.
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identified, among which, 25 were xanthones, 6 flavonoids, and 1 chlorogenic acid. The details of all
Gentianaceous plants are rich sources of xanthone compounds. Previously, abundant xanthone
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glycosides have been detected and identified in these three species of the Gentianaceae Family [22-24].
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With four or five absorption bands at 200-400 nm, compounds 1, 5, 7-10, 13-24, 27-29, and 31-35 were assigned as xanthone derivatives [23].
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The fragment ions at m/z 333[(M+H)-90]+ and 303[(M+H)-120]+ in positive ion (PI) mode indicated
that compound 1 was formed via C-C bond [25]. It was widely acknowledged that glucose was the most common naturally occurring monosaccharide. Taking the quasi-molecular ion at m/z 423[M+H]+ into consideration, compound 1 was identified as 2-C-β-D-glucosyl-1,3,6,7-tetrahydroxyxanthone (mangiferin), which has been reported in G. acuta previously [26]. Unfortunately, compound 5 could not 8
be identified with no more detailed MS information. The sodium adduct ion at m/z 591[M+Na]+ and fragment ion at m/z 275[Y0]+ (loss of 316 u) in PI mode suggested that compound 7 was 1,7,8-trihydroxy3-methoxyxanthone plus a disaccharide. Moreover, the most frequently found monosaccharide in the Gentianaceae Family, was glucose and disaccharides were primeverose and gentiobiose [7, 22-24, 26].
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Referring to an exisiting research, compound 7 was identified as 1-O-primeverosyl-7,8-dihydroxy-3methoxyxanthone [22]. The quasi-molecular ions at m/z 289[M+H]+ in PI mode and m/z 287[M-H]- in
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negative ion (NI) mode indicated that compound 10 was 1,7-dihydroxy-3,8-dimethoxyxanthone. With intense fragment ion at m/z 289[Y0]+ in PI mode, compounds 8 and 9 were assigned as 1,7-dihydroxy-
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3,8-dimethoxyxanthone derivatives. Integrating the quasi-molecular ions at m/z 583[M+H]+ and sodium
1-O-primeverosyl-7-hydroxy-3,8-dimethoxyxanthone
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as
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adduct ion at m/z 473[M+Na]+ in PI mode, compounds 8 and 9 were identified or tentatively identified and
1-O-glucosyl-7-hydroxy-3,8-
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dimethoxyxanthone, respectively. The elution order of these three compounds coincided with the rule
in G. barbata [22].
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proposed by Abad-García et al. [11]. Compound 7-10 except compound 9 have previously been reported
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The sodium adduct ion at m/z 695[M+Na]+ and aglycone ion at m/z 349[Y0]+ indicated that compound
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13 was 1,7-dihydroxy-2,3,4,5-tetramethoxyxanthone plus a gentiobiose. The O-glycosidic linkage of all the xanthone compounds isolated and elucidated in H. corniculata was at position C-1 in a previous report [23]. Therefore, compound 13 was identified as l-O-gentiobiosyl-7-hydroxy-2,3,4,5-
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tetramethoxyxanthone. The fragment ions at m/z 303[Y0]+ in PI mode and m/z 301[Y0]- in NI mode suggested that the aglycone moiety of compounds 14, 16, 19, and 22 was either 1-hydroxy-2,3,5trimethoxyxanthone or 1-hydroxy-2,3,7-trimethoxyxanthone. In reversed phase HPLC, 1-hydroxy-2,3,5trimethoxyxanthone glycoside eluted prior to the corresponding 1-hydroxy-2,3,7-trimethoxyxanthone 9
glycoside [23]. Furthermore, with strikingly similar UV absorption, compounds 14 and 16 were identified as 1-hydroxy-2,3,5-trimethoxyxanthone glycosides. Taking the sodium adduct ions at m/z 649[M+Na]+ and m/z 619[M+Na]+ in PI mode into consideration, compounds 14 and 16 were identified as
l-O-gentiobiosyl-2,3,5-trimethoxyxanthone
and
l-O-primeverosyl-2,3,5-trimethoxyxanthone,
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respectively. Using similar methodology, compounds 19 and 22 were separately assigned as l-Oprimeverosyl-2,3,7-trimethoxyxanthone and l-O-gentiobiosyl-2,3,7-trimethoxyxanthone.
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Combining the same aglycone ions at m/z 275[Y0]+ in PI mode and m/z 273[Y0]- in NI mode and similar UV absorption, compounds 15 and 17 were deduced as 1,5,8-trihydroxy-3-methoxyxanthone glycosides.
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The mother nucleus (1,5,8-trihydroxy-3-methoxyxanthone) has been previously found in G. acuta [7].
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Moreover, with quasi-molecular ions at m/z 599[M+H]+ and 437[M+H]+ in PI mode, compounds 15 and
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17 were tentatively identified as l-O-gentiobiosyl-5,8-dihydroxy-3-methoxyxanthone and l-O-glucosyl-
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5,8-dihydroxy-3-methoxyxanthone. The fragment ions at m/z 333[Y0]+ in PI mode and m/z 331[Y0]- in
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NI mode of compounds 18, 20, and 23 were indicative of a 1-hydroxy-2,3,4,5-tetramethoxyxanthone or 1-hydroxy-2,3,4,7-tetramethoxyxanthone aglycone moiety. 1-hydroxy-2,3,4,5-tetramethoxyxanthone
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glycosides eluted before corresponding 1-hydroxy-2,3,4,7-tetramethoxyxanthone glycosides [23].
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Moreover, the same UV absorption characteristics suggested that both compounds 20 and 23 were 1hydroxy-2,3,4,7-tetramethoxyxanthone derivatives. Subsequently, with sodium adduct ion at m/z
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649[M+Na]+ in PI mode compounds 18 and 20 were identified as l-O-primeverosyl-2,3,4,5tetramethoxyxanthone and l-O-primeverosyl-2,3,4,7-tetramethoxyxanthone, respectively. Likewise compound 23 was identified as l-O-gentiobiosyl-2,3,4,7-tetramethoxyxanthone. The sodium adduct ion at m/z 679[M+Na]+ and fragment ion at m/z 363[Y0]+ (loss of 316 u) in PI mode indicated that compound 21 was l-O-primeverosyl-2,3,4,5,7-pentamethoxyxanthone, which has been reported in H. corniculata 10
previously [23]. With quasi-molecular ion at m/z 423[M+H]+ and fragment ion at m/z 261[Y0]+ (loss of 162 u) in PI mode compound 24 was identified as 8-O-glucosyl-1,3,5,-trihydroxyxanthone (norswertianolin), which has been reported in G. acuta previously [24]. The quasi-molecular ions at m/z 261[M+H]+ in PI mode
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and m/z 259[M-H]- in NI mode indicated that compound 32 was the aglycone of compound 24. Thus, compound 32 was assigned as 1,3,5,8-tetrahydroxyxanthone (bellidin). Compounds 28 and 34 were
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likewise identified as 8-O-glucosyl-1,5-dihydroxy-3-methoxyxanthone (swertianolin) and 1,5,8trihydroxy-3-methoxyxanthone (bellidifolin), respectively. Furthermore, compound 28 was confirmed
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by co-eluting with its corresponding commercial standard. With ions at m/z 305 in PI mode and m/z 303
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in NI mode, compound 27, 29, 31, and 33 were identified as 1,3,8-trihydroxy-4,5-dimethoxyxanthone or
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1,2,8-trihydroxy-5,6-dimethoxyxanthone and their corresponding glycosides. According to the UV
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absorption characteristics and elution order reported previously and their MS data, compounds 27, 29,
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31, and 33 were tentatively identified as 1-O-glucosyl-3,8-dihydroxy-4,5-dimethoxyxanthone (corymbiferin 1-O-glucoside), 3-O-glucosyl-1,8-dihydroxy-4,5-dimethoxyxanthone (corymbiferin 3-O-
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glucoside), 2-O-glucosyl-1,8-dihydroxy-5,6-dimethoxyxanthone (triptexanthoside C), and 1,2,8-
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trihydroxy-5,6-dimethoxyxanthone, respectively [7, 27, 28]. The quasi-molecular ions at m/z 519[M+H]+ in PI mode and m/z 517[M-H]- in NI mode indicated that compound 35 was swertiabisxanthone-I. All
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the 10 xanthone compounds except compound 33 have been previously reported in G. acuta [24, 26]. The occurrence of compound 33 was unsurprising for it could have derived from compounds 31 during the ultrasonic solvent extraction process. 3.4. Identification of flavonoids The UV-vis absorption characteristics of compounds 2, 3, 4, 6, 12, 25, 26, and 30 in the regions of 11
240-280 nm and 300-380 nm indicated that they were flavonoids [11]. With fragment ions at m/z 329[(M+H)-120]+ and 313[(M+H)-120]+, both compounds 2 and 25 were deduced as flavonoid Cglycosides [25]. Combining the quasi-molecular ions at m/z 449[M+H]+ and 433[M+H]+ in PI mode with UV-vis absorption spectra, compounds 2 and 25 were tentatively identified as 6-C-β-D-
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glucopyranosylluteolin (isoorientin) and 6-C-β-D-glucopyranosylapigenin (isovitexin). Furthermore, they were absolutely verified via co-elution with their corresponding commercial standards.
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The aglycone ions at m/z 287 in the PI mode and m/z 285 in the NI mode suggest that compounds 3 and 4 were luteolin or kaempferol derivatives. Subsequently, these two compounds were verified as
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luteolin derivatives according to their UV λmax (Band II) at 255 nm [29]. In the case of flavones, the 7-
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hydroxyl group were apparently favored for glycosylation [30]. With quasi-molecular ions at m/z
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581[M+H]+ and 449[M+H]+ in PI mode, compounds 3 and 4 were respectively assigned as 7-O-
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primeverosylluteolin and 7-O-glucosylluteolin, which have been reported previously in H. corniculata
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[23]. In a similar way, compound 6 was identified as 7-O-primeverosyldiosmetin. The quasi-molecular ion at m/z 565[M+H]+ and fragment ion at m/z 271[Y0]+ (loss of 294 u) indicated that compound 12 was
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primeverosylapigenin. The substitution of sugar was supposed to occur at 5-hydroxyl group as a 10 nm
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hypochromatic shift in Band I (in methanol) was observed [31]. Finally, compound 12 was identified as 5-O-primeverosylapigenin. The aglycone moiety (apigenin) of compound 12 has been reported in a
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previous paper [6]. Compounds 26 and 30 could not be identified because of the absence of detailed fragment ions, although they were assigned as flavonoids. 3.5. Identification of chlorogenic acid In addition to xanthones and flavonoids, one minor chlorogenic acid was detected and identified. Typically, chlorogenic acids are a family of esters that one or more residues of certain trans-cinnamic 12
acids (such as caffeic acid, ferulic acid, and p-coumaric acid) conjugate to quinic acid [32]. Compound 11 was recognized as chlorogenic acid undoubtedly with strong absorption at 323 nm and a shoulder peak at 297 nm [33]. The quasi-molecular ion at m/z 355[M+H]+ and fragment ion at m/z 163 (loss of 192 u) in PI mode suggested that compound 11 was caffeoylquinic acid. Unfortunately, the attachment
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position between caffeic acid and quinic acid could not be determined owing to the absence of more detailed MS data.
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3.6. Composition and content of chemical compounds
Table 2 displays the composition and content of chemical compounds. Altogether, 35 chemical
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compounds were observed in these three species of the Family Gentianaceae, but not every compound
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was monitored in the individual species. The chromatograms (whole plant) of chemical compounds of
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each species were significantly different (Figure 3). Therefore, the chromatograms can be utilized as a
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useful tool to distinguish these three species from each other or to identify an ingredient of some
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traditional Chinese medicine. For each species, the composition and content of chemical compounds of different part are also shown in Table 2.
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For G. barbata, the content of compound 8 in whole plant was the highest (9.61 ± 0.51 mg/g DW),
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followed by compound 4 (6.02 ± 0.38 mg/g DW) and compound 2 (5.41 ± 0.34 mg/g DW). Though its content fluctuated seriously between different parts, compound 8 was predominant in the flower, stem,
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and root. While in the leaf, the content of compound 4 (22.11 ± 4.77 mg/g DW) ranked the highest, followed closely by compound 1 (19.48 ± 0.86 mg/g DW). The total flavonoid content ranked high to low, was leaf, flower, whole plant, stem, and finally root. Meanwhile, for total xanthone content, the order was flower, leaf, whole plant, stem, and root. For H. corniculata, the major peak in whole plant was compound 4 (30.55 ± 0.34 mg/g DW) and 13
compound 3 (10.18 ± 0.13 mg/g DW), both of which were luteolin glycosides. The content of compound 4 was overwhelming to other chemical compounds in flower, leaf, and stem. However, compound 4 was trace in the root. The content of both total flavonoid content (76.47 ± 13.22 mg/g DW) and total xanthone content (38.58 ± 6.53 mg/g DW) were the highest in the leaf of all the parts of the plant.
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For G. acuta, compound 24 (27.49 ± 2.14 mg/g DW) and compound 28 (16.63 ± 1.15 mg/g DW) comprised the top two contents in the whole plant. Compared with other chemical compounds, the
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content of compound 24 was the highest in flower, leaf, and stem. In total, three kinds of flavonoid compounds were detected, their content minor in all parts. The total xanthone content of the leaf (156.69
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± 10.62 mg/g DW) was the highest, followed by the flower (96.32 ± 10.24 mg/g DW), whole plant (65.31
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± 5.15 mg/g DW), stem (20.08 ± 0.93 mg/g DW), and root (3.15 ± 0.37 mg/g DW).
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Overall, our results provided basic data concerning the composition and content of compounds from
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different part of these three species. It would lay solid scientific foundation for the pharmacological value
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of these three species in traditional Chinese medicine. From the content of active component perspective, leaves were recommended as the most valuable part in all these species.
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4. Conclusions
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In the present study, the chemical compounds of whole plant, flower, leaf, stem, and root of G. barbata, H. corniculata, and G. acuta were detected and identified using HPLC-DAD/ESI-MS2. To the best of
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our knowledge, this investigation marked the first time to simultaneously report the composition and content of chemical compounds from different parts of the plants. A total of 35 chemical compounds were detected and 32 were identified from these three species, of which, 25 were xanthones, 6 flavonoids, and 1 chlorogenic acid. All the xanthones or their aglycone moieties have been previously reported. One flavonoid (7-O-primeverosyldiosmetin) and one chlorogenic acid (caffeoylquinic acid) were reported for 14
the first time in G. barbata and H. corniculata, respectively. The chromatograms (Figure 3) obtained at 350 nm provided solid evidence to distinguish these three species via their chemical compounds. Individual compounds and their occurrence and content in different parts of the plant within different species were included in our results. This may be of great value to their pharmacological study. In the
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methanol acid extract of G. barbata and H. corniculata, the total flavonoid contents were of a comparable level to total xanthone contents. However, in G. acuta the total xanthone content was overwhelming
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larger than that of total flavonoid content. From a chemical compounds perspective, the leaf was the most valuable part for medicine for each of these three species.
U
Acknowledgements
N
We thank Jian-Fei Ye (Engineer of Beijing Botanical Garden, Institute of Botany, Chinese Academy
A
of Sciences) for the authentication of plant materials. We also thank Dr. Shang Su and Dr. Li-Jin Wang
M
for the collection of plant materials and sample preparation.
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Conflict of Interest
References
PT
The authors declare no competing financial interest.
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cancer and inflammation, Curr. Med. Chem. 8 (2001) 135-153.
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Polyphenol profile and antioxidant activity of the fruit and leaf of Vaccinium glaucoalbum from the Tibetan Himalayas, Food Chem. 219 (2017) 490-495. [22] L. Ji, J. Ding, S. Fan, H. Sun, Study on chemical constituents of Gentianopsis barbata var. stennocalyx H.W.Li ex T.N.Ho, Acta Bot. Sin. 34 (1992) 203-207. [23] S. Rodriguez, J.L. Wolfender, G. Odontuya, O. Purev, K. Hostettmann, Xanthones, secoiridoids and 17
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triple quadrupole mass spectrometry, Rapid Commun. Mass Spectrom. 22 (2008) 1834-1842.
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[28] H. Otsuka, Triptexanthosides A-E: Xanthone glycosides from aerial parts of Tripterospermum japonicum, Chem. Pharm. Bull. 47 (1999) 962-965.
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[29] K.R. Markham, Techniques of flavonoid identification., 1st ed., Academic Press, London, 1982.
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[30] V. Vukics, Antioxidant flavonoid glycosides in Viola Tricolor L., in: School of Pharmaceutical Sciences, Semmelweis University Budapest, Hungary, 2009.
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[31] T.J. Mabry, K.R. Markham, M.B. Thomas, The ultraviolet spectra of flavones and flavonols, 1st ed., Springer, Berlin, 1970. [32] M.N. Clifford, W. Wu, J. Kirkpatrick, N. Kuhnert, Profiling the chlorogenic acids and other caffeic acid derivatives of herbal chrysanthemum by LC-MSn, J. Agric. Food Chem. 55 (2007) 929-936. [33] A. Belay, A.V. Gholap, Characterization and determination of chlorogenic acids (CGA) in coffee 18
A
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PT
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M
A
N
U
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beans by UV-Vis spectroscopy, African Journal of Pure and Applied Chemistry 3 (2009) 234-240.
19
Figure captions Figure 1. The three Gentianaceae plants investigated in this study. A: G. barbata, B: H. corniculata, C: G. acuta. Figure 2. The chemical structure of compounds identified in this study.
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Figure 3. HPLC chromatographic profiles of whole plants of three species from Family Gentianaceae
A
CC E
PT
ED
M
A
N
U
SC R
obtained at 350 nm. Green line: G. barbata, blue line: G. acuta, red line: H. corniculata.
20
21
A ED
PT
CC E
IP T
SC R
U
N
A
M
22
A ED
PT
CC E
IP T
SC R
U
N
A
M
I N U SC R
Table 1. The retention time, UV–vis spectra and MS data used for identification of compounds from three species of family Gentianaceae.
1
2
RT (min) a 7.4
11.4
UV-Vis (nm) b
ESI-PI (m/z)
242, 260, 276, 319,
ESI-NI (m/z)
+
+
423[M+H] , 333[(M+H)-90] , +
365
303[(M+H)-120] , 273[(M+H)-150]
270, 350
449[M+H]+, 377[(M+H)-4H2O]+,
M
329[(M+H)-120]+
Identification
References
Species
2-C-β-D-glucosyl-1,3,6,7-tetrahydroxyxanthone (mangiferin)
Lv & Li, 2009
A, C
447[M-H]-, 327[(M-H)-120]-
6-C-β-D-glucopyranosylluteolin (isoorientin)
standard
A
-
-
421[M-H] , 331[(M-H)-90] ,
+
A
No.
-
301[(M-H)-120]
14.2
253, 349
581[M+H]+, 287[Y0]+
579[M-H]-, 285[Y0]-
7-O-primeverosylluteolin
Rodriguez et al., 1995
A, B
4
15.1
255, 348
449[M+H]+, 287[Y0]+
447[M-H]-, 285[Y0]-
7-O-glucosylluteolin
Rodriguez et al., 1995
A, B
5
17.7
238, 246, 268, 343
575[M+H]+
573[M-H]-
unknown
A
6
20.2
255, 346
595[M+H]+, 301[Y0]+
593[M-H]-, 299[Y0]-
7-O-primeverosyldiosmetin
A
7
20.8
246, 268, 314, 379
591[M+Na]+, 275[Y0]+,
567[M-H]-, 273[Y0]-
1-O-primeverosyl-7,8-dihydroxy-3-methoxyxanthone
Ji et al., 1992
A
8
21.7
236, 254, 305, 358
583[M+H]+, 289[Y0]+
581[M-H]-, 287[Y0]-
1-O-primeverosyl-7-hydroxy-3,8-dimethoxyxanthone
Ji et al., 1992
A
9
23.6
236, 254, 305, 360
473[M+Na]+, 289[Y0]+
449[M-H]-, 287[Y0]-
1-O-glucosyl-7-hydroxy-3,8-dimethoxyxanthone
Ji et al., 1992
A
10
42.1
245, 261, 312, 377
311[M+Na]+, 289[M+H]+
287[M-H]-
1,7-dihydroxy-3,8-dimethoxyxanthone
Ji et al., 1992
A
11
17.3
246, 297sh, 323
355[M+H]+, 163[(M+H)-192]+
353[M-H]-
caffeoylquinic acid
12
18.9
268, 328
565[M+H]+, 433[(M+H)-132]+,
563[M-H]-
5-O-primeverosylapigenin
Zhang et al., 2008
B
A
CC E
PT
ED
3
B
+
271[Y0]
13
19.3
239, 246, 259, 318
695[M+Na]+, 349[Y0]+
671[M-H]-
l-O-gentiobiosyl-7-hydroxy-2,3,4,5-tetramethoxyxanthone
Rodriguez et al., 1995
B
14
20.0
236, 249, 291, 347
649[M+Na]+, 303[Y0]+
671[M+HCOO]-
l-O-gentiobiosyl-2,3,5-trimethoxyxanthone
Rodriguez et al., 1995
B
15
21.5
236, 257, 280, 332,
599[M+H]+, 437[(M+H)-162]+,
597[M-H]-, 273[Y0]-
l-O-gentiobiosyl-5,8-dihydroxy-3-methoxyxanthone
Urbain et al., 2008
B
641[M+HCOO]-, 301[Y0]-
l-O-primeverosyl-2,3,5-trimethoxyxanthone
Rodriguez et al., 1995
B
16
22.5
+
387
275[Y0]
237, 251, 293, 348
619[M+Na]+, 303[Y0]+
23
I 23.8
18
25.0
238, 255, 278, 329,
N U SC R
17
437[M+H]+, 275[Y0]+
435[M-H]-, 273[Y0]-
l-O-glucosyl-5,8-dihydroxy-3-methoxyxanthone
Urbain et al., 2008
B
671[M+HCOO]-, 331[Y0]-
l-O-primeverosyl-2,3,4,5-tetramethoxyxanthone
Rodriguez et al., 1995
B
641[M+HCOO]-, 301[Y0]-
l-O-primeverosyl-2,3,7-trimethoxyxanthone
Rodriguez et al., 1995
B
649[M+Na]+, 333[Y0]+
671[M+HCOO]-, 331[Y0]-
l-O-primeverosyl-2,3,4,7-tetramethoxyxanthone
Rodriguez et al., 1995
B
679[M+Na]+, 363[Y0]+
701[M+HCOO]-, 361[Y0]-
l-O-primeverosyl-2,3,4,5,7-pentamethoxyxanthone
Rodriguez et al., 1995
B
671[M+HCOO]-, 301[Y0]-
l-O-gentiobiosyl-2,3,7-trimethoxyxanthone
Rodriguez et al., 1995
B
657[M+H]+, 333[Y0]+
655[M-H]-, 331[Y0]-
l-O-gentiobiosyl-2,3,4,7-tetramethoxyxanthone
Rodriguez et al., 1995
B
423[M+H]+, 261[Y0]+
421[M-H]-, 259[Y0]-
8-O-glucosyl-1,3,5,-trihydroxyxanthone (norswertianolin)
Urbain et al., 2009
C
433[M+H]+, 343[(M+H)-90]+,
431[M-H]-, 341[(M-H)-90]-,
6-C-β-D-glucopyranosylapigenin (isovitexin)
standard
C
313[(M+H)-120]+, 283[(M+H)-150]+
281[(M-H)-150]-
384 232, 246, 257, 300,
649[M+Na]+, 333[Y0]+
19
26.6
232, 249, 253, 309,
619[M+Na]+, 303[Y0]+
20
27.1
236, 246, 263, 315,
M
360
375 29.1
238, 244, 267, 295, 379
22
46.5
23
50.3
ED
21
234, 248, 253, 308,
627[M+H]+, 303[Y0]+
PT
360
A
364
238, 244, 262, 314, 377
11.3
252, 274, 328, 378
CC E
24 25
14.8
269, 339
16.2
270, 338
447[M+H]+
445[M-H]-
unknown
27
18.3
236, 253, 277, 345
467[M+H]+, 305[Y0]+
465[M-H]-, 303[Y0]-
1-O-glucosyl-3,8-dihydroxy-4,5-dimethoxyxanthone (corymbiferin 1-O-glucoside)
Kaldas et al., 1975
C
28
20.1
253, 276, 326, 377
437[M+H]+, 275[Y0]+
435[M-H]-, 273[Y0]-
8-O-glucosyl-1,5-dihydroxy-3-methoxyxanthone (swertianolin)
standard
C
29
22.2
239, 254, 278, 344
489[M+Na]+, 305[Y0]+
465[M-H]-, 303[Y0]-
3-O-glucosyl-1,8-dihydroxy-4,5-dimethoxyxanthone (corymbiferin 3-O-glucoside)
Urbain et al., 2008
C
30
24.6
255, 341
453[M+H]+
451[M-H]-
unknown
31
26.0
238, 244, 266, 346
467[M+H]+, 305[Y0]+
465[M-H]-, 303[Y0]-
2-O-glucosyl-1,8-dihydroxy-5,6-dimethoxyxanthone (triptexanthoside C)
Otsuka, 1999
C
32
33.2
254, 277, 333, 386
261[M+H]+
259[M-H]-
1,3,5,8-tetrahydroxyxanthone (bellidin)
Urbain et al., 2009
C
A
26
24
C
C
I 39.0
239, 270, 343, 405
305[M+H]+
34
43.6
255, 279, 333, 386
275[M+H]+
35
46.8
257, 282, 339, 381
519[M+H]+
N U SC R
33
303[M-H]-
1,2,8-trihydroxy-5,6-dimethoxyxanthone
Otsuka, 1999
C
273[M-H]-
1,5,8-trihydroxy-3-methoxyxanthone (bellidifolin)
Urbain et al., 2009
C
517[M-H]-
swertiabisxanthone-I
Urbain et al., 2009
C
A
Note: A–C represent the three species. A: G. barbata, B: H. corniculata, C: G. acuta. RT, retention time.
b
UV–vis, the maximum absorption wavelength from ultraviolet to visible region.
A
CC E
PT
ED
M
a
25
I N U SC R
Table 2. The compounds detected in three species of family Gentianaceae and their concentrations in each part (mg/g DW). G. barbata Whole
Flower
Leaf
Stem
plant 1
3.13 ±
19.48 ±
1.50 ±
0.14
0.86
0.16
8.06 ±
18.60 ±
5.37 ±
0.67
3.04
1.74
5.41 ± 0.34
0.24 ±
3 0.99 ± 0.08
0.08
6.02 ± 0.38 5
6
0.84 ± 0.07
7
0.97 ± 0.05
A
8
9.61 ± 0.51
1.10 ± 0.05
11
Root
plant ND
ND
ND
Whole
Flower
Leaf
Stem
Root
2.47 ±
11.01 ± 2.15
0.44 ±
Trace
plant ND
ND
2.94 ± 0.25
0.09
0.03
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
10.18 ±
5.64 ± 2.03
17.03 ± 0.32
3.28 ±
Trace
ND
ND
ND
ND
ND
Trace
ND
ND
ND
ND
ND
0.25 ± 0.17
Trace
0.13
22.11 ±
3.49 ±
0.19 ±
30.55 ±
40.20 ±
56.45 ±
10.47 ±
0.04
0.34
13.99
12.51
3.38
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
0.55 ±
0.32 ± 0.01
0.77 ± 0.09
0.26 ±
Trace
ND
ND
ND
ND
ND
0.54
1.03 ±
1.23 ±
0.53 ±
0.10
0.09
0.07
0.73 ±
4.39 ±
0.49 ±
0.13
0.07
0.18
2.72 ±
0.62 ±
0.79 ±
0.17
0.06
0.07
Trace
28.64 ±
10.82 ±
9.41 ±
1.91 ±
1.05
0.14
1.38
0.78
1.08 ±
1.73 ±
0.14
0.34
0.74 ±
1.52 ±
Trace
6.50 ±
10
Stem
Trace
4.77
0.68
Leaf
0.79
3.54 ±
9
Flower
Trace
0.30
CC E
0.51 ± 0.03
Whole
G. acuta
3.78 ±
PT
5.95 ±
4
ED
2
M
3.42 ± 0.04
Root
A
Peak
H. corniculata
1.28
Trace
Trace
1.43 ± 0.10
0.81
Trace
0.05
0.07
ND
ND
ND
ND
ND
26
I N U SC R 0.01
12
ND
ND
ND
ND
ND
13
ND
ND
ND
ND
ND
0.93 ±
2.35 ± 0.80
2.99 ± 0.39
0.81 ± 0.06
1.53 ± 0.14
15
ND
ND
ND
ND
ND
ND
ND
ND
ND
M
14
A
0.03
ND
0.88 ± 0.00 0.86 ±
1.49 ± 0.19
1.82 ± 0.42
3.79 ± 0.17
11.40 ± 3.22
ND
ND
ND
CC E
18
ND
ND
19
20
A
21
22
ND
ND
ND
ND
ND
PT
17
ND
ED
0.06 16
ND
ND
ND
ND
ND
ND
ND
ND
3.74 ±
1.19 ±
5.44 ± 1.05
4.67 ± 0.84
1.06 ± 0.16
3.00 ± 0.6
0.02 ND
ND
2.36 ±
ND
2.99 ±
3.67 ± 0.87
4.18 ± 1.19
7.40 ± 4.13
2.99 ± 0.68
0.08 ND
ND
ND
4.57 ±
8.49 ± 1.20
4.51 ± 1.79
0.10 ND
ND
ND
0.76 ±
1.72 ± 0.19
0.75 ± 0.43
0.02 ND
ND
ND
ND
ND
1.09 ±
ND
ND
ND
ND
ND
1.19 ±
2.12 ± 0.88
1.66 ± 0.65
2.53 ± 1.10
2.07 ± 0.77
0.03 24
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
Trace
ND
ND
ND
ND
ND
0.49 ±
0.87 ±
ND
ND
ND
ND
ND
0.01
0.14
0.70 ±
Trace
ND
ND
ND
ND
ND
2.03 ±
2.33 ±
ND
ND
ND
ND
ND
0.45
0.33
0.23 ±
Trace
ND
ND
ND
ND
ND
1.21 ±
0.95 ±
ND
ND
ND
ND
ND
0.20
0.08
0.62 ±
2.13 ±
ND
ND
ND
ND
ND
0.30
0.15
1.99 ±
2.13 ±
ND
ND
ND
ND
ND
0.62
0.15
0.25 ±
Trace
ND
ND
ND
ND
ND
0.56 ±
1.81 ±
ND
ND
ND
ND
ND
0.12
0.47
0.70 ±
1.23 ±
ND
ND
ND
ND
ND
0.17
0.25
ND
ND
27.49 ±
30.54 ±
78.6 ± 6.36
8.41 ±
0.59 ±
0.08 0.36 ±
0.05
0.03 23
ND
0.05
0.05 ND
Trace
0.16
0.07 ND
0.14 ±
0.04
0.01 4.16 ±
0.02
ND
ND
27
I ND
ND
ND
ND
26
ND
ND
ND
ND
ND
29
30
ND
ND
ND
ND
ND
32
33
A
34
35
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
M
ND
CC E
31
ND
ND
ED
28
ND
ND
ND
PT
27
ND
ND
N U SC R
ND
A
25
ND
ND
ND
ND
2.14
3.22 0.98 ±
ND
ND
ND
ND
ND
0.44 ± 0.06
ND
ND
ND
ND
ND
0.61 ± 0.07
0.24 ± 0.03
0.13 2.29 ±
ND
ND
Trace
Trace
0.64 ±
Trace
0.15 ND
ND
ND
ND
ND
0.52 ± 0.01
Trace
0.06 ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
16.63 ±
23.92 ±
1.15
2.95
0.79 ± 0.00
1.93 ±
34.42 ± 3.54
Trace
ND
ND
ND
ND
ND
0.27 ± 0.01
0.18 ±
ND
ND
ND
ND
ND
1.51 ± 0.09
4.83 ±
ND
ND
ND
ND
ND
4.97 ± 0.60
9.01 ±
ND
ND
ND
ND
ND
ND
ND
0.99 ± 0.06
2.74 ±
0.18 ± 0.07
ND
0.95 ±
0.18
0.20
0.73 ±
Trace
0.16 ±
Trace
0.03 1.57 ± 0.36
0.78 ±
0.58 ±
0.03
0.05
1.26 ±
0.49 ±
0.27
0.04
0.50 ± 0.04
Trace
Trace
17.84 ± 2.52
2.51 ±
0.54 ±
0.23
0.07
1.62 ± 0.20
Trace
Trace
0.64 ± 0.09
0.37 ±
Trace
11.13 ± 1.46
0.18 ND
ND
ND
ND
ND
ND
ND
ND
8.94 ± 0.85
20.22 ± 1.86
ND
5.31 ±
0.02
0.82 ND
Trace
Trace
1.03 ND
0.21 ± 0.02
0.12 ND
0.07
0.22 ± 0.06
0.59 ND
1.33
ND
ND
ND
ND
ND
ND
ND
ND
0.53 ± 0.04
0.67 ± 0.09
Total
13.27 ±
14.99 ±
48.88 ±
flavonoid
0.87
1.05
8.53
9.6 ± 2.35
0.19 ±
41.66 ±
48.19 ±
76.47 ±
13.89 ±
0.04
0.43
16.79
13.22
4.74
28
Trace
1.32 ± 0.14
3.45 ± 0.33
0.01
I 17.05 ±
45.56 ±
32.15 ±
14.05 ±
5.17 ±
xanthone
0.70
1.56
0.98
1.66
1.11
23.8 ±
38.54 ± 4.17
0.40
A
CC E
PT
ED
M
A
Note: ND, not detected.
N U SC R
Total
29
38.58 ± 6.53
9.15 ± 1.76
11.45 ± 1.1
65.31 ±
96.32 ±
156.69 ±
20.08 ±
3.15 ±
5.15
10.24
10.62
0.93
0.37
S0731-7085(18)30773-8 https://doi.org/10.1016/j.jpba.2018.08.059 PBA 12190
To appear in:
Journal of Pharmaceutical and Biomedical Analysis
Received date: Revised date: Accepted date:
18-4-2018 4-7-2018 27-8-2018
Please cite this article as: Feng C-Yong, Wu Q, Yin D-Dan, Li B, Li SShan, Tang Z-Qiu, Xu Y-Jun, Wang L-Sheng, Determination of xanthones and flavonoids of methanol extracts obtained from different parts of the plants of three Gentianaceae species, Journal of Pharmaceutical and Biomedical Analysis (2018), https://doi.org/10.1016/j.jpba.2018.08.059 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Determination of xanthones and flavonoids of methanol extracts obtained from different parts of the plants of three Gentianaceae species Cheng-Yong Feng
a,b
, Qian Wu
a,b
, Dan-Dan Yin
a,b
, Bing Li
a,b
, Shan-Shan Li
a,b*
, Zhong-Qiu Tang c,
a
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Yan-Jun Xu d, Liang-Sheng Wang a,b*
Academy of Sciences, Beijing 100093, China
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Key Laboratory of Plant Resources and Beijing Botanical Garden, Institute of Botany, Chinese
University of Chinese Academy of Sciences, Beijing 100049, China
c
Forestry and Agricultural Academy of Greater Khingan Mountains, Jiagedaqi 165000, China
d
College of Science, China Agricultural University, Beijing 100094, China
A
N
U
b
M
Highlights:
Leaf was recommended as the most valuable part for medicine.
The fingerprints of these three species were significantly differernt.
An efficient method was established to determine the bioactive compounds.
Abundant xanthones and flavonoids were detected in these three species.
CC E
PT
ED
This research provided basic data for their pharmacological study.
A
*
Corresponding author. Tel: +86 10 6283 6654. Fax: +86 10 6259 0348. E-mail: [email protected]
(L.-S Wang). Tel: +86 10 6283 6053. E-mail: [email protected] (S.-S Li). 1
Abstract Gentianopsis barbata, Halenia corniculata, and Gentianella acuta were widely distributed throughout China and commonly used in traditional Chinese medicine. However, owing to similar living environments and morphological features, locals often had trouble distinguishing between these three
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species. In this present study, chromatograms at 350 nm were obtained and the composition and content of their chemical compounds determined using HPLC-DAD/ESI-MS2. In total, 35 chemical compounds
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were detected, 32 of which were identified, 25 of which were xanthones, 6 flavonoids, and 1 chlorogenic acid. The 350 nm chromatograms of these three species displayed evident differences. The individual
U
compounds and their occurrence and content in different parts of the plant within different species were
N
included in our results. This basic data will be useful for future pharmacological study. The total
A
compositions of flavonoids and xanthones were approximately comparable in G. barbata and H.
M
corniculata. Meanwhile, xanthones were predominant in G. acuta. From the perspective of chemical
these three species.
ED
compound compositions, the leaf is recommended as the most valuable medicinal section for each of
A
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Keywords: Gentianaceae, xanthone, flavonoid, HPLC
2
1. Introduction Gentianaceae plants are annual herbs or perennial herbs, shrubs, and small trees. The Gentianaceae Family contains about 80 genera, comprising approximately 1000 species worldwide. It exhibits wide global distribution with predominant clustering in the North Temperate Zone. In China, 22 genera
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covering nearly 500 species have been found, of which, more than 70 species from 12 genera are of pharmacological value [1]. Gentianopsis barbata (Froelich) Ma is an annual or biennial plant native to
SC R
China, Japan, Kazakhstan, Kyrgyzstan, Mongolia, and Russia and found at altitudes ranging between
700 m and 4400 m. It has many beneficial effects, such as heat-clearing, detoxicating, anti-inflammation,
U
and cholagogue [2]. Halenia corniculata (Linnaeus) Cornaz is an annual plant which is commonly found
N
in China, Japan, Korea, Mongolia, and Russia at altitudes between 200-1800 m. It may be used in the
A
treatment of gastritis, intestinal and stomach pain, liver diseases, colitis, and enterocolitis [3]. Gentianella
M
acuta (Michaux) Hulten is also an annual plant primarily distributed in China, Mongolia, East Russia,
ED
and North America at altitudes between 0 m and 1500 m. The pharmacological activity of G. acuta mainly includes hepatoprotection, hypoglycemic, antioxidant, and anti-inflammation [4].
PT
Gentianaceae plants are rich sources of xanthones, flavonoids, and terpenoids [1]. The
CC E
pharmacological activities of these aforementioned three species are related to these compounds. It has been demonstrated that mangiferin derived from G. barbata could effectively alleviate liver damage in
A
rats induced by paracetamol, tetrachloromethane, and D-galactosamine [5]. Zhang, Ahn, Sun, Kim, Hwang, Ryu and Kim [6] found that the xanthones from H. corniculata could prevent bone hardening from osteoporosis by inhibiting osteoclastogenesis in vitro. Also, it was reported that xanthones from G. acuta could inhibit the activities of acetylcholinesterase and monoamine oxidase [7]. Xanthones and flavonoids are important secondary metabolites which have aroused tremendous 3
interest owing to their pharmalogical properties and beneficial effects on humans. Flavonoids are widespread in plant kingdom, and xanthones usually occur in a few higher plant families where they are of taxonomic value [8]. The basic skeleton of xanthones is a C6-C1-C6 structure (Figure 2) which is symmetrical. The numbering system of xanthones was proposed by Gottlieb using xanthene-9-one as the
IP T
basic structure [9]. Carbons 1-4 (ring A) are derived from acetic acid and carbons 5-8 (ring B) are derived from shikimic acid. When oxygenation occurs only in ring B, the lowest numbers are used except
SC R
discussing the biosynthetic pathways [10]. The C6-C3-C6 nucleus of flavonoids (Figure 2) is formed by a series of condensation reactions between a coumaric acid (B-ring and carbon atoms 2, 3 and 4 of C-
U
ring) and several malonic acids (A-ring) [11]. Naturally, in the majority of cases, xanthones and
N
flavonoids exist in the form of glycosides or their derivatives. Xanthones have abundant pharmacological
A
worth, with properties including antidepressant, antituberculotic, depressant, choleretic, diuretic,
M
antimicrobial, antiviral, and cardiotonic [8, 12-15]. Flavonoids also have diverse biological activities,
ED
such as antioxidant, antibacterial, anti-inflammatory, anti-obesity, astringent, antiproliferative, anticarcinogenic, and UV-protection activities [16-20].
PT
In Northeast China, the above mentioned three species are used as traditional Chinese medicine.
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Unfortunately, locals often confuse these three species owing to their similar living conditions and morphological features (Figure 1). These three species are even harder to identify once dried to serve as
A
medicinal raw materials. Previously, numerous investigations focused on the isolation and clarification and pharmacological properties of the chemical compounds from these three Gentianaceae plants. Literature on their chemical compound chromatograms were almost unavailable. In the present study, a rapid and efficient method was established utilizing HPLC-DAD to obtain their chromatograms. Moreover, we evaluated the composition and content of xanthones and flavonoids from different parts of 4
each species with HPLC-DAD/ESI-MS2. This allows their differentiation from the perspective of chemical compounds and also provides a reference for the screening of the best medicinal part.
2. Experimental section 2.1. Plant materials
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Three species of family Gentianaceae, i.e. Gentianopsis barbata (Froelich) Ma (voucher specimen No. Wls2014001), Halenia corniculata (Linnaeus) Cornaz (voucher specimen No. Wls2014002), and
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Gentianella acuta (Michaux) Hulten (voucher specimen No. Wls2014003) were collected from the Greater Khingan Mountains, Heilongjiang Province, Northeast China, on a sunny morning of August
U
2014. The authentication of plant materials was generously accomplished by Jian-Fei Ye (an Engineer of
N
Beijing Botanical Garden, Institute of Botany, Chinese Academy of Sciences). Plants at flowering phase
A
were harvested. For each species, plants with similar morphological characteristics were randomly
M
selected for sampling within the population. All the samples were brought back to the laboratory using
ED
an airtight preserving box with ice bags in it. Subsequently, the root, stem, leaf, flower, and whole plant
later use.
PT
were freeze-dried with a vacuum freeze drier (FD-1T, Beijing, China), and then stored in a desiccator for
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2.2. Chemicals and reagents
The xanthone standard, 8-O-glucosyl-1,5-dihydroxy-3-methoxyxanthone (swertianolin) was obtained
A
from Meilunbio (Dalian, China). The flavonoid standard, 3-O-rutinosylquercetin (rutin); and chlorogenic acid standard, trans-5-caffeoylquinic acid (CAQ) were provided by the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). The 6-C-β-D-glucopyranosylluteolin (isoorientin) and 6-C-β-D-glucopyranosylapigenin (isovitexin) were purchased from Shanghai Tauto Biotech (Shanghai, China). The methanol, formic acid, and acetonitrile used for HPLC analysis were of 5
chromatographic grade and purchased from Alltech Scientific (Beijing, China). The ultrapure water was produced by a Mini-Q System (Millipore, Billerica, MA, USA). 2.3. Preparation of methanol extraction The freeze-dried samples were ground into fine powder in liquid nitrogen with mortars and pestles.
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About 0.1 g of fine powder was weighed and transferred into a 10 mL centrifuge tube. Then the powder was mixed with 2 mL 2% formic acid in methanol (v/v), shaken in a QL-861 vortexer (Kylinbell Lab
SC R
Instruments, Jiangsu, China) for 30 s, sonicated in a KQ-500DE ultrasonic cleaner (Ultrasonic instruments, Jiangsu, China) at room temperature for 20 minutes, and then centrifuged in SIGMA 3K30
U
(Sigma Centrifuges, Germany) (12,000×g, 10 min). Subsequently, the supernatant was collected in
N
another a 10 mL centrifuge tube. The above steps were then repeated two more times. All the supernatants
A
were put together and supplemented up to a volume of 6 mL with 2% formic acid in methanol (v/v).
M
After which, a certain volume of ultrapure water was added into the extraction to make sure the
ED
concentration of water was 30% (v/v), and then stored at -20°C for 24 hours to precipitate chlorophyll. Then, the mixture was filtered through 0.22 μm millipore membrane (Shanghai ANPEL, China) after
PT
centrifugation. A 1 mL aliquot of clean supernatant was analyzed on HPLC-DAD and HPLC-ESI-MS2.
CC E
For each sample, three biological replicates were performed. 2.4. HPLC-DAD and HPLC-ESI-MS2 analysis The systems and conditions used for HPLC-DAD and HPLC-ESI-MS2 analysis were similar to our
A
previous study [21]. The column was an ODS-80Ts QA C18 column (150 mm4.6 mm, 5 μm i.d., Tosoh, Tokyo, Japan) protected with a C18 guard cartridge (ANPEL, Shanghai, China). Eluent A was 5% formic acid in ultrapure water (v/v), and eluent B was absolute acetonitrile. The gradient elution protocol was as follows: 10% B at 0 min, 22% B at 20 min, 60% B at 65 min, and 10% B at 70 min. For HPLC-DAD 6
analysis, a 10 μL solution of each sample was injected, the flow rate was 0.8 mL/min, and the column temperature was 35°C. Chromatograms were acquired at 350 nm, and the photodiode array spectra were recorded from 200 nm to 800 nm. For HPLC-ESI-MS2 analysis, the compounds were analyzed in both positive ion (PI) and negative ion (NI) mode, and the following MS detection conditions were used:
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capillary voltage, 4.0 kV; nebulization pressure, 241.3 kPa; gas (N 2) temperature, 350°C; gas flow rate, 8.0 L/min; capillary offset voltage, 77.2 V; capillary exit voltage, 127.3 V; and scan range, 100-1000
SC R
(m/z).
2.5. Quantitative analysis of xanthones, flavonoids, and chlorogenic acids
U
The quantitative analyses of chemical compounds from these three Gentianaceae species were based
N
on the linear regression of HPLC-DAD peak area of commercial standards. Swertianolin, rutin, and
A
trans-5-caffeoylquinic acid were used as standards for the semi-quantitation of all xanthones, all
M
flavonoids, and a chlorogenic acid, respectively. The following linear regression equations were obtained:
ED
for swertianolin, Y = 219.73X – 0.09 (r2 = 1.0000); for rutin, Y = 319.86X + 0.93 (r2 = 0.9999); for CQA, Y = 665.57X – 1.43 (r2 = 0.9996). The concentrations of all the chemical compounds were expressed as
PT
milligrams of corresponding standards per gram of dry weight (DW).
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3. Results and Discussion 3.1. Method validation
A
To validate the reliability of the optimized HPLC method, the limit of detection (LOD) and the limit of quantitation
(LOQ)
were
determined
for
xanthones
with
8-O-glucosyl-1,5-dihydroxy-3-
methoxyxanthone (swertianolin), flavonoids with 3-O-rutinosylquercetin (rutin) and chlorogenic acids with trans-5-caffeoylquinic acid (CQA). The results of method validation for three external standards (swertianolin, rutin, and CQA) are shown in Table S1. The concentration of three standards used for 7
calibration curves were all in the range from 7.81 to 500.00 μg/mL. Three replicates were performed for each concentration of three standards. The HPLC-DAD peak areas at 350 nm and concentrations of standards displayed good linear relationship. The limit of detection (LOD) and the limit of quantitation (LOQ) were defined as the ratio of signal to noise at 3:1 and 10:1, respectively. The LOD and LOQ
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obtained for swertianolin (0.50 and 1.66 μg/mL), rutin (0.65 and 2.15 μg/mL), and CQA (0.45 and 1.49 μg/mL) are shown in Table S1 as well.
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3.2. General
Good peak separation and resolution were achieved via an optimized gradient elution protocol (Figure
U
3). In total, 35 chemical compounds were detected from these three species. Using UV-vis absorption
N
spectroscopy, elution order, and mass spectrum analysis, 32 compounds were identified or tentatively
ED
3.3. Identification of xanthones
M
compounds are displayed in Table 1.
A
identified, among which, 25 were xanthones, 6 flavonoids, and 1 chlorogenic acid. The details of all
Gentianaceous plants are rich sources of xanthone compounds. Previously, abundant xanthone
PT
glycosides have been detected and identified in these three species of the Gentianaceae Family [22-24].
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With four or five absorption bands at 200-400 nm, compounds 1, 5, 7-10, 13-24, 27-29, and 31-35 were assigned as xanthone derivatives [23].
A
The fragment ions at m/z 333[(M+H)-90]+ and 303[(M+H)-120]+ in positive ion (PI) mode indicated
that compound 1 was formed via C-C bond [25]. It was widely acknowledged that glucose was the most common naturally occurring monosaccharide. Taking the quasi-molecular ion at m/z 423[M+H]+ into consideration, compound 1 was identified as 2-C-β-D-glucosyl-1,3,6,7-tetrahydroxyxanthone (mangiferin), which has been reported in G. acuta previously [26]. Unfortunately, compound 5 could not 8
be identified with no more detailed MS information. The sodium adduct ion at m/z 591[M+Na]+ and fragment ion at m/z 275[Y0]+ (loss of 316 u) in PI mode suggested that compound 7 was 1,7,8-trihydroxy3-methoxyxanthone plus a disaccharide. Moreover, the most frequently found monosaccharide in the Gentianaceae Family, was glucose and disaccharides were primeverose and gentiobiose [7, 22-24, 26].
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Referring to an exisiting research, compound 7 was identified as 1-O-primeverosyl-7,8-dihydroxy-3methoxyxanthone [22]. The quasi-molecular ions at m/z 289[M+H]+ in PI mode and m/z 287[M-H]- in
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negative ion (NI) mode indicated that compound 10 was 1,7-dihydroxy-3,8-dimethoxyxanthone. With intense fragment ion at m/z 289[Y0]+ in PI mode, compounds 8 and 9 were assigned as 1,7-dihydroxy-
U
3,8-dimethoxyxanthone derivatives. Integrating the quasi-molecular ions at m/z 583[M+H]+ and sodium
1-O-primeverosyl-7-hydroxy-3,8-dimethoxyxanthone
A
as
N
adduct ion at m/z 473[M+Na]+ in PI mode, compounds 8 and 9 were identified or tentatively identified and
1-O-glucosyl-7-hydroxy-3,8-
M
dimethoxyxanthone, respectively. The elution order of these three compounds coincided with the rule
in G. barbata [22].
ED
proposed by Abad-García et al. [11]. Compound 7-10 except compound 9 have previously been reported
PT
The sodium adduct ion at m/z 695[M+Na]+ and aglycone ion at m/z 349[Y0]+ indicated that compound
CC E
13 was 1,7-dihydroxy-2,3,4,5-tetramethoxyxanthone plus a gentiobiose. The O-glycosidic linkage of all the xanthone compounds isolated and elucidated in H. corniculata was at position C-1 in a previous report [23]. Therefore, compound 13 was identified as l-O-gentiobiosyl-7-hydroxy-2,3,4,5-
A
tetramethoxyxanthone. The fragment ions at m/z 303[Y0]+ in PI mode and m/z 301[Y0]- in NI mode suggested that the aglycone moiety of compounds 14, 16, 19, and 22 was either 1-hydroxy-2,3,5trimethoxyxanthone or 1-hydroxy-2,3,7-trimethoxyxanthone. In reversed phase HPLC, 1-hydroxy-2,3,5trimethoxyxanthone glycoside eluted prior to the corresponding 1-hydroxy-2,3,7-trimethoxyxanthone 9
glycoside [23]. Furthermore, with strikingly similar UV absorption, compounds 14 and 16 were identified as 1-hydroxy-2,3,5-trimethoxyxanthone glycosides. Taking the sodium adduct ions at m/z 649[M+Na]+ and m/z 619[M+Na]+ in PI mode into consideration, compounds 14 and 16 were identified as
l-O-gentiobiosyl-2,3,5-trimethoxyxanthone
and
l-O-primeverosyl-2,3,5-trimethoxyxanthone,
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respectively. Using similar methodology, compounds 19 and 22 were separately assigned as l-Oprimeverosyl-2,3,7-trimethoxyxanthone and l-O-gentiobiosyl-2,3,7-trimethoxyxanthone.
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Combining the same aglycone ions at m/z 275[Y0]+ in PI mode and m/z 273[Y0]- in NI mode and similar UV absorption, compounds 15 and 17 were deduced as 1,5,8-trihydroxy-3-methoxyxanthone glycosides.
U
The mother nucleus (1,5,8-trihydroxy-3-methoxyxanthone) has been previously found in G. acuta [7].
N
Moreover, with quasi-molecular ions at m/z 599[M+H]+ and 437[M+H]+ in PI mode, compounds 15 and
A
17 were tentatively identified as l-O-gentiobiosyl-5,8-dihydroxy-3-methoxyxanthone and l-O-glucosyl-
M
5,8-dihydroxy-3-methoxyxanthone. The fragment ions at m/z 333[Y0]+ in PI mode and m/z 331[Y0]- in
ED
NI mode of compounds 18, 20, and 23 were indicative of a 1-hydroxy-2,3,4,5-tetramethoxyxanthone or 1-hydroxy-2,3,4,7-tetramethoxyxanthone aglycone moiety. 1-hydroxy-2,3,4,5-tetramethoxyxanthone
PT
glycosides eluted before corresponding 1-hydroxy-2,3,4,7-tetramethoxyxanthone glycosides [23].
CC E
Moreover, the same UV absorption characteristics suggested that both compounds 20 and 23 were 1hydroxy-2,3,4,7-tetramethoxyxanthone derivatives. Subsequently, with sodium adduct ion at m/z
A
649[M+Na]+ in PI mode compounds 18 and 20 were identified as l-O-primeverosyl-2,3,4,5tetramethoxyxanthone and l-O-primeverosyl-2,3,4,7-tetramethoxyxanthone, respectively. Likewise compound 23 was identified as l-O-gentiobiosyl-2,3,4,7-tetramethoxyxanthone. The sodium adduct ion at m/z 679[M+Na]+ and fragment ion at m/z 363[Y0]+ (loss of 316 u) in PI mode indicated that compound 21 was l-O-primeverosyl-2,3,4,5,7-pentamethoxyxanthone, which has been reported in H. corniculata 10
previously [23]. With quasi-molecular ion at m/z 423[M+H]+ and fragment ion at m/z 261[Y0]+ (loss of 162 u) in PI mode compound 24 was identified as 8-O-glucosyl-1,3,5,-trihydroxyxanthone (norswertianolin), which has been reported in G. acuta previously [24]. The quasi-molecular ions at m/z 261[M+H]+ in PI mode
IP T
and m/z 259[M-H]- in NI mode indicated that compound 32 was the aglycone of compound 24. Thus, compound 32 was assigned as 1,3,5,8-tetrahydroxyxanthone (bellidin). Compounds 28 and 34 were
SC R
likewise identified as 8-O-glucosyl-1,5-dihydroxy-3-methoxyxanthone (swertianolin) and 1,5,8trihydroxy-3-methoxyxanthone (bellidifolin), respectively. Furthermore, compound 28 was confirmed
U
by co-eluting with its corresponding commercial standard. With ions at m/z 305 in PI mode and m/z 303
N
in NI mode, compound 27, 29, 31, and 33 were identified as 1,3,8-trihydroxy-4,5-dimethoxyxanthone or
A
1,2,8-trihydroxy-5,6-dimethoxyxanthone and their corresponding glycosides. According to the UV
M
absorption characteristics and elution order reported previously and their MS data, compounds 27, 29,
ED
31, and 33 were tentatively identified as 1-O-glucosyl-3,8-dihydroxy-4,5-dimethoxyxanthone (corymbiferin 1-O-glucoside), 3-O-glucosyl-1,8-dihydroxy-4,5-dimethoxyxanthone (corymbiferin 3-O-
PT
glucoside), 2-O-glucosyl-1,8-dihydroxy-5,6-dimethoxyxanthone (triptexanthoside C), and 1,2,8-
CC E
trihydroxy-5,6-dimethoxyxanthone, respectively [7, 27, 28]. The quasi-molecular ions at m/z 519[M+H]+ in PI mode and m/z 517[M-H]- in NI mode indicated that compound 35 was swertiabisxanthone-I. All
A
the 10 xanthone compounds except compound 33 have been previously reported in G. acuta [24, 26]. The occurrence of compound 33 was unsurprising for it could have derived from compounds 31 during the ultrasonic solvent extraction process. 3.4. Identification of flavonoids The UV-vis absorption characteristics of compounds 2, 3, 4, 6, 12, 25, 26, and 30 in the regions of 11
240-280 nm and 300-380 nm indicated that they were flavonoids [11]. With fragment ions at m/z 329[(M+H)-120]+ and 313[(M+H)-120]+, both compounds 2 and 25 were deduced as flavonoid Cglycosides [25]. Combining the quasi-molecular ions at m/z 449[M+H]+ and 433[M+H]+ in PI mode with UV-vis absorption spectra, compounds 2 and 25 were tentatively identified as 6-C-β-D-
IP T
glucopyranosylluteolin (isoorientin) and 6-C-β-D-glucopyranosylapigenin (isovitexin). Furthermore, they were absolutely verified via co-elution with their corresponding commercial standards.
SC R
The aglycone ions at m/z 287 in the PI mode and m/z 285 in the NI mode suggest that compounds 3 and 4 were luteolin or kaempferol derivatives. Subsequently, these two compounds were verified as
U
luteolin derivatives according to their UV λmax (Band II) at 255 nm [29]. In the case of flavones, the 7-
N
hydroxyl group were apparently favored for glycosylation [30]. With quasi-molecular ions at m/z
A
581[M+H]+ and 449[M+H]+ in PI mode, compounds 3 and 4 were respectively assigned as 7-O-
M
primeverosylluteolin and 7-O-glucosylluteolin, which have been reported previously in H. corniculata
ED
[23]. In a similar way, compound 6 was identified as 7-O-primeverosyldiosmetin. The quasi-molecular ion at m/z 565[M+H]+ and fragment ion at m/z 271[Y0]+ (loss of 294 u) indicated that compound 12 was
PT
primeverosylapigenin. The substitution of sugar was supposed to occur at 5-hydroxyl group as a 10 nm
CC E
hypochromatic shift in Band I (in methanol) was observed [31]. Finally, compound 12 was identified as 5-O-primeverosylapigenin. The aglycone moiety (apigenin) of compound 12 has been reported in a
A
previous paper [6]. Compounds 26 and 30 could not be identified because of the absence of detailed fragment ions, although they were assigned as flavonoids. 3.5. Identification of chlorogenic acid In addition to xanthones and flavonoids, one minor chlorogenic acid was detected and identified. Typically, chlorogenic acids are a family of esters that one or more residues of certain trans-cinnamic 12
acids (such as caffeic acid, ferulic acid, and p-coumaric acid) conjugate to quinic acid [32]. Compound 11 was recognized as chlorogenic acid undoubtedly with strong absorption at 323 nm and a shoulder peak at 297 nm [33]. The quasi-molecular ion at m/z 355[M+H]+ and fragment ion at m/z 163 (loss of 192 u) in PI mode suggested that compound 11 was caffeoylquinic acid. Unfortunately, the attachment
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position between caffeic acid and quinic acid could not be determined owing to the absence of more detailed MS data.
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3.6. Composition and content of chemical compounds
Table 2 displays the composition and content of chemical compounds. Altogether, 35 chemical
U
compounds were observed in these three species of the Family Gentianaceae, but not every compound
N
was monitored in the individual species. The chromatograms (whole plant) of chemical compounds of
A
each species were significantly different (Figure 3). Therefore, the chromatograms can be utilized as a
M
useful tool to distinguish these three species from each other or to identify an ingredient of some
ED
traditional Chinese medicine. For each species, the composition and content of chemical compounds of different part are also shown in Table 2.
PT
For G. barbata, the content of compound 8 in whole plant was the highest (9.61 ± 0.51 mg/g DW),
CC E
followed by compound 4 (6.02 ± 0.38 mg/g DW) and compound 2 (5.41 ± 0.34 mg/g DW). Though its content fluctuated seriously between different parts, compound 8 was predominant in the flower, stem,
A
and root. While in the leaf, the content of compound 4 (22.11 ± 4.77 mg/g DW) ranked the highest, followed closely by compound 1 (19.48 ± 0.86 mg/g DW). The total flavonoid content ranked high to low, was leaf, flower, whole plant, stem, and finally root. Meanwhile, for total xanthone content, the order was flower, leaf, whole plant, stem, and root. For H. corniculata, the major peak in whole plant was compound 4 (30.55 ± 0.34 mg/g DW) and 13
compound 3 (10.18 ± 0.13 mg/g DW), both of which were luteolin glycosides. The content of compound 4 was overwhelming to other chemical compounds in flower, leaf, and stem. However, compound 4 was trace in the root. The content of both total flavonoid content (76.47 ± 13.22 mg/g DW) and total xanthone content (38.58 ± 6.53 mg/g DW) were the highest in the leaf of all the parts of the plant.
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For G. acuta, compound 24 (27.49 ± 2.14 mg/g DW) and compound 28 (16.63 ± 1.15 mg/g DW) comprised the top two contents in the whole plant. Compared with other chemical compounds, the
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content of compound 24 was the highest in flower, leaf, and stem. In total, three kinds of flavonoid compounds were detected, their content minor in all parts. The total xanthone content of the leaf (156.69
U
± 10.62 mg/g DW) was the highest, followed by the flower (96.32 ± 10.24 mg/g DW), whole plant (65.31
N
± 5.15 mg/g DW), stem (20.08 ± 0.93 mg/g DW), and root (3.15 ± 0.37 mg/g DW).
A
Overall, our results provided basic data concerning the composition and content of compounds from
M
different part of these three species. It would lay solid scientific foundation for the pharmacological value
ED
of these three species in traditional Chinese medicine. From the content of active component perspective, leaves were recommended as the most valuable part in all these species.
PT
4. Conclusions
CC E
In the present study, the chemical compounds of whole plant, flower, leaf, stem, and root of G. barbata, H. corniculata, and G. acuta were detected and identified using HPLC-DAD/ESI-MS2. To the best of
A
our knowledge, this investigation marked the first time to simultaneously report the composition and content of chemical compounds from different parts of the plants. A total of 35 chemical compounds were detected and 32 were identified from these three species, of which, 25 were xanthones, 6 flavonoids, and 1 chlorogenic acid. All the xanthones or their aglycone moieties have been previously reported. One flavonoid (7-O-primeverosyldiosmetin) and one chlorogenic acid (caffeoylquinic acid) were reported for 14
the first time in G. barbata and H. corniculata, respectively. The chromatograms (Figure 3) obtained at 350 nm provided solid evidence to distinguish these three species via their chemical compounds. Individual compounds and their occurrence and content in different parts of the plant within different species were included in our results. This may be of great value to their pharmacological study. In the
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methanol acid extract of G. barbata and H. corniculata, the total flavonoid contents were of a comparable level to total xanthone contents. However, in G. acuta the total xanthone content was overwhelming
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larger than that of total flavonoid content. From a chemical compounds perspective, the leaf was the most valuable part for medicine for each of these three species.
U
Acknowledgements
N
We thank Jian-Fei Ye (Engineer of Beijing Botanical Garden, Institute of Botany, Chinese Academy
A
of Sciences) for the authentication of plant materials. We also thank Dr. Shang Su and Dr. Li-Jin Wang
M
for the collection of plant materials and sample preparation.
ED
Conflict of Interest
References
PT
The authors declare no competing financial interest.
CC E
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[4] X. Wei, Studies on the composition and content of chemical compounds of Gentianella acuta, in: School of Traditional Chinese Medicine, Beijing University of Chinese Medicine, Beijing, China, 2014. [5] H. Cheng, Y. Li, Q. Bian, Effects of enzyme and morphological change of mangiferin on experimental liver damage in rats, Chinese Journal of Laboratory Animal Science 9 (1999) 28-31.
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[6] J. Zhang, M.-J. Ahn, Q.S. Sun, K.-Y. Kim, Y.H. Hwang, J.M. Ryu, J. Kim, Inhibitors of bone
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[7] A. Urbain, A. Marston, L.S. Grilo, J. Bravo, O. Purev, B. Purevsuren, D. Batsuren, M. Reist, P.A.
Carrupt, K. Hostettmann, Xanthones from Gentianella amarella ssp acuta with acetylcholinesterase and
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A literature survey, Quim. Nova. 20 (1997) 388-397.
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cancer and inflammation, Curr. Med. Chem. 8 (2001) 135-153.
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Potamogeton (Potamogetonaceae), Am. J. Bot. 77 (1990) 453-465. [21] C.-Y. Feng, W.-W. Wang, J.-F. Ye, S.-S. Li, Q. Wu, D.-D. Yin, B. Li, Y.-J. Xu, L.-S. Wang,
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Polyphenol profile and antioxidant activity of the fruit and leaf of Vaccinium glaucoalbum from the Tibetan Himalayas, Food Chem. 219 (2017) 490-495. [22] L. Ji, J. Ding, S. Fan, H. Sun, Study on chemical constituents of Gentianopsis barbata var. stennocalyx H.W.Li ex T.N.Ho, Acta Bot. Sin. 34 (1992) 203-207. [23] S. Rodriguez, J.L. Wolfender, G. Odontuya, O. Purev, K. Hostettmann, Xanthones, secoiridoids and 17
flavonoids from Halenia corniculata, Phytochemistry 40 (1995) 1265-1272. [24] A. Urbain, A. Marston, E. Marsden-Edwards, K. Hostettmanna, Ultra-performance liquid chromatography/time-of-flight mass spectrometry as a chemotaxonomic tool for the analysis of Gentianaceae species, Phytochem. Anal. 20 (2009) 134-138.
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[25] B. Abad-García, S. Garmón-Lobato, L.A. Berrueta, B. Gallo, F. Vicente, New features on the fragmentation and differentiation of C-glycosidic flavone isomers by positive electrospray ionization and
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triple quadrupole mass spectrometry, Rapid Commun. Mass Spectrom. 22 (2008) 1834-1842.
[26] L.-J. Lv, M.-H. Li, Terpenoids, flavonoids and xanthones from Gentianella acuta (Gentianaceae),
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Biochem. Syst. Ecol. 37 (2009) 497-500.
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[27] M. Kaldas, K. Hostettmann, A. Jacotguillarmod, Contribution à la phytochimie du genre Gentiana
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Chim. Acta 58 (1975) 2188-2192.
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XIII. Etude de composés flavoniques et xanthoniques dans les feuilles de Gentiana campestris L., Helv.
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[28] H. Otsuka, Triptexanthosides A-E: Xanthone glycosides from aerial parts of Tripterospermum japonicum, Chem. Pharm. Bull. 47 (1999) 962-965.
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[29] K.R. Markham, Techniques of flavonoid identification., 1st ed., Academic Press, London, 1982.
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[30] V. Vukics, Antioxidant flavonoid glycosides in Viola Tricolor L., in: School of Pharmaceutical Sciences, Semmelweis University Budapest, Hungary, 2009.
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[31] T.J. Mabry, K.R. Markham, M.B. Thomas, The ultraviolet spectra of flavones and flavonols, 1st ed., Springer, Berlin, 1970. [32] M.N. Clifford, W. Wu, J. Kirkpatrick, N. Kuhnert, Profiling the chlorogenic acids and other caffeic acid derivatives of herbal chrysanthemum by LC-MSn, J. Agric. Food Chem. 55 (2007) 929-936. [33] A. Belay, A.V. Gholap, Characterization and determination of chlorogenic acids (CGA) in coffee 18
A
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PT
ED
M
A
N
U
SC R
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beans by UV-Vis spectroscopy, African Journal of Pure and Applied Chemistry 3 (2009) 234-240.
19
Figure captions Figure 1. The three Gentianaceae plants investigated in this study. A: G. barbata, B: H. corniculata, C: G. acuta. Figure 2. The chemical structure of compounds identified in this study.
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Figure 3. HPLC chromatographic profiles of whole plants of three species from Family Gentianaceae
A
CC E
PT
ED
M
A
N
U
SC R
obtained at 350 nm. Green line: G. barbata, blue line: G. acuta, red line: H. corniculata.
20
21
A ED
PT
CC E
IP T
SC R
U
N
A
M
22
A ED
PT
CC E
IP T
SC R
U
N
A
M
I N U SC R
Table 1. The retention time, UV–vis spectra and MS data used for identification of compounds from three species of family Gentianaceae.
1
2
RT (min) a 7.4
11.4
UV-Vis (nm) b
ESI-PI (m/z)
242, 260, 276, 319,
ESI-NI (m/z)
+
+
423[M+H] , 333[(M+H)-90] , +
365
303[(M+H)-120] , 273[(M+H)-150]
270, 350
449[M+H]+, 377[(M+H)-4H2O]+,
M
329[(M+H)-120]+
Identification
References
Species
2-C-β-D-glucosyl-1,3,6,7-tetrahydroxyxanthone (mangiferin)
Lv & Li, 2009
A, C
447[M-H]-, 327[(M-H)-120]-
6-C-β-D-glucopyranosylluteolin (isoorientin)
standard
A
-
-
421[M-H] , 331[(M-H)-90] ,
+
A
No.
-
301[(M-H)-120]
14.2
253, 349
581[M+H]+, 287[Y0]+
579[M-H]-, 285[Y0]-
7-O-primeverosylluteolin
Rodriguez et al., 1995
A, B
4
15.1
255, 348
449[M+H]+, 287[Y0]+
447[M-H]-, 285[Y0]-
7-O-glucosylluteolin
Rodriguez et al., 1995
A, B
5
17.7
238, 246, 268, 343
575[M+H]+
573[M-H]-
unknown
A
6
20.2
255, 346
595[M+H]+, 301[Y0]+
593[M-H]-, 299[Y0]-
7-O-primeverosyldiosmetin
A
7
20.8
246, 268, 314, 379
591[M+Na]+, 275[Y0]+,
567[M-H]-, 273[Y0]-
1-O-primeverosyl-7,8-dihydroxy-3-methoxyxanthone
Ji et al., 1992
A
8
21.7
236, 254, 305, 358
583[M+H]+, 289[Y0]+
581[M-H]-, 287[Y0]-
1-O-primeverosyl-7-hydroxy-3,8-dimethoxyxanthone
Ji et al., 1992
A
9
23.6
236, 254, 305, 360
473[M+Na]+, 289[Y0]+
449[M-H]-, 287[Y0]-
1-O-glucosyl-7-hydroxy-3,8-dimethoxyxanthone
Ji et al., 1992
A
10
42.1
245, 261, 312, 377
311[M+Na]+, 289[M+H]+
287[M-H]-
1,7-dihydroxy-3,8-dimethoxyxanthone
Ji et al., 1992
A
11
17.3
246, 297sh, 323
355[M+H]+, 163[(M+H)-192]+
353[M-H]-
caffeoylquinic acid
12
18.9
268, 328
565[M+H]+, 433[(M+H)-132]+,
563[M-H]-
5-O-primeverosylapigenin
Zhang et al., 2008
B
A
CC E
PT
ED
3
B
+
271[Y0]
13
19.3
239, 246, 259, 318
695[M+Na]+, 349[Y0]+
671[M-H]-
l-O-gentiobiosyl-7-hydroxy-2,3,4,5-tetramethoxyxanthone
Rodriguez et al., 1995
B
14
20.0
236, 249, 291, 347
649[M+Na]+, 303[Y0]+
671[M+HCOO]-
l-O-gentiobiosyl-2,3,5-trimethoxyxanthone
Rodriguez et al., 1995
B
15
21.5
236, 257, 280, 332,
599[M+H]+, 437[(M+H)-162]+,
597[M-H]-, 273[Y0]-
l-O-gentiobiosyl-5,8-dihydroxy-3-methoxyxanthone
Urbain et al., 2008
B
641[M+HCOO]-, 301[Y0]-
l-O-primeverosyl-2,3,5-trimethoxyxanthone
Rodriguez et al., 1995
B
16
22.5
+
387
275[Y0]
237, 251, 293, 348
619[M+Na]+, 303[Y0]+
23
I 23.8
18
25.0
238, 255, 278, 329,
N U SC R
17
437[M+H]+, 275[Y0]+
435[M-H]-, 273[Y0]-
l-O-glucosyl-5,8-dihydroxy-3-methoxyxanthone
Urbain et al., 2008
B
671[M+HCOO]-, 331[Y0]-
l-O-primeverosyl-2,3,4,5-tetramethoxyxanthone
Rodriguez et al., 1995
B
641[M+HCOO]-, 301[Y0]-
l-O-primeverosyl-2,3,7-trimethoxyxanthone
Rodriguez et al., 1995
B
649[M+Na]+, 333[Y0]+
671[M+HCOO]-, 331[Y0]-
l-O-primeverosyl-2,3,4,7-tetramethoxyxanthone
Rodriguez et al., 1995
B
679[M+Na]+, 363[Y0]+
701[M+HCOO]-, 361[Y0]-
l-O-primeverosyl-2,3,4,5,7-pentamethoxyxanthone
Rodriguez et al., 1995
B
671[M+HCOO]-, 301[Y0]-
l-O-gentiobiosyl-2,3,7-trimethoxyxanthone
Rodriguez et al., 1995
B
657[M+H]+, 333[Y0]+
655[M-H]-, 331[Y0]-
l-O-gentiobiosyl-2,3,4,7-tetramethoxyxanthone
Rodriguez et al., 1995
B
423[M+H]+, 261[Y0]+
421[M-H]-, 259[Y0]-
8-O-glucosyl-1,3,5,-trihydroxyxanthone (norswertianolin)
Urbain et al., 2009
C
433[M+H]+, 343[(M+H)-90]+,
431[M-H]-, 341[(M-H)-90]-,
6-C-β-D-glucopyranosylapigenin (isovitexin)
standard
C
313[(M+H)-120]+, 283[(M+H)-150]+
281[(M-H)-150]-
384 232, 246, 257, 300,
649[M+Na]+, 333[Y0]+
19
26.6
232, 249, 253, 309,
619[M+Na]+, 303[Y0]+
20
27.1
236, 246, 263, 315,
M
360
375 29.1
238, 244, 267, 295, 379
22
46.5
23
50.3
ED
21
234, 248, 253, 308,
627[M+H]+, 303[Y0]+
PT
360
A
364
238, 244, 262, 314, 377
11.3
252, 274, 328, 378
CC E
24 25
14.8
269, 339
16.2
270, 338
447[M+H]+
445[M-H]-
unknown
27
18.3
236, 253, 277, 345
467[M+H]+, 305[Y0]+
465[M-H]-, 303[Y0]-
1-O-glucosyl-3,8-dihydroxy-4,5-dimethoxyxanthone (corymbiferin 1-O-glucoside)
Kaldas et al., 1975
C
28
20.1
253, 276, 326, 377
437[M+H]+, 275[Y0]+
435[M-H]-, 273[Y0]-
8-O-glucosyl-1,5-dihydroxy-3-methoxyxanthone (swertianolin)
standard
C
29
22.2
239, 254, 278, 344
489[M+Na]+, 305[Y0]+
465[M-H]-, 303[Y0]-
3-O-glucosyl-1,8-dihydroxy-4,5-dimethoxyxanthone (corymbiferin 3-O-glucoside)
Urbain et al., 2008
C
30
24.6
255, 341
453[M+H]+
451[M-H]-
unknown
31
26.0
238, 244, 266, 346
467[M+H]+, 305[Y0]+
465[M-H]-, 303[Y0]-
2-O-glucosyl-1,8-dihydroxy-5,6-dimethoxyxanthone (triptexanthoside C)
Otsuka, 1999
C
32
33.2
254, 277, 333, 386
261[M+H]+
259[M-H]-
1,3,5,8-tetrahydroxyxanthone (bellidin)
Urbain et al., 2009
C
A
26
24
C
C
I 39.0
239, 270, 343, 405
305[M+H]+
34
43.6
255, 279, 333, 386
275[M+H]+
35
46.8
257, 282, 339, 381
519[M+H]+
N U SC R
33
303[M-H]-
1,2,8-trihydroxy-5,6-dimethoxyxanthone
Otsuka, 1999
C
273[M-H]-
1,5,8-trihydroxy-3-methoxyxanthone (bellidifolin)
Urbain et al., 2009
C
517[M-H]-
swertiabisxanthone-I
Urbain et al., 2009
C
A
Note: A–C represent the three species. A: G. barbata, B: H. corniculata, C: G. acuta. RT, retention time.
b
UV–vis, the maximum absorption wavelength from ultraviolet to visible region.
A
CC E
PT
ED
M
a
25
I N U SC R
Table 2. The compounds detected in three species of family Gentianaceae and their concentrations in each part (mg/g DW). G. barbata Whole
Flower
Leaf
Stem
plant 1
3.13 ±
19.48 ±
1.50 ±
0.14
0.86
0.16
8.06 ±
18.60 ±
5.37 ±
0.67
3.04
1.74
5.41 ± 0.34
0.24 ±
3 0.99 ± 0.08
0.08
6.02 ± 0.38 5
6
0.84 ± 0.07
7
0.97 ± 0.05
A
8
9.61 ± 0.51
1.10 ± 0.05
11
Root
plant ND
ND
ND
Whole
Flower
Leaf
Stem
Root
2.47 ±
11.01 ± 2.15
0.44 ±
Trace
plant ND
ND
2.94 ± 0.25
0.09
0.03
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
10.18 ±
5.64 ± 2.03
17.03 ± 0.32
3.28 ±
Trace
ND
ND
ND
ND
ND
Trace
ND
ND
ND
ND
ND
0.25 ± 0.17
Trace
0.13
22.11 ±
3.49 ±
0.19 ±
30.55 ±
40.20 ±
56.45 ±
10.47 ±
0.04
0.34
13.99
12.51
3.38
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
0.55 ±
0.32 ± 0.01
0.77 ± 0.09
0.26 ±
Trace
ND
ND
ND
ND
ND
0.54
1.03 ±
1.23 ±
0.53 ±
0.10
0.09
0.07
0.73 ±
4.39 ±
0.49 ±
0.13
0.07
0.18
2.72 ±
0.62 ±
0.79 ±
0.17
0.06
0.07
Trace
28.64 ±
10.82 ±
9.41 ±
1.91 ±
1.05
0.14
1.38
0.78
1.08 ±
1.73 ±
0.14
0.34
0.74 ±
1.52 ±
Trace
6.50 ±
10
Stem
Trace
4.77
0.68
Leaf
0.79
3.54 ±
9
Flower
Trace
0.30
CC E
0.51 ± 0.03
Whole
G. acuta
3.78 ±
PT
5.95 ±
4
ED
2
M
3.42 ± 0.04
Root
A
Peak
H. corniculata
1.28
Trace
Trace
1.43 ± 0.10
0.81
Trace
0.05
0.07
ND
ND
ND
ND
ND
26
I N U SC R 0.01
12
ND
ND
ND
ND
ND
13
ND
ND
ND
ND
ND
0.93 ±
2.35 ± 0.80
2.99 ± 0.39
0.81 ± 0.06
1.53 ± 0.14
15
ND
ND
ND
ND
ND
ND
ND
ND
ND
M
14
A
0.03
ND
0.88 ± 0.00 0.86 ±
1.49 ± 0.19
1.82 ± 0.42
3.79 ± 0.17
11.40 ± 3.22
ND
ND
ND
CC E
18
ND
ND
19
20
A
21
22
ND
ND
ND
ND
ND
PT
17
ND
ED
0.06 16
ND
ND
ND
ND
ND
ND
ND
ND
3.74 ±
1.19 ±
5.44 ± 1.05
4.67 ± 0.84
1.06 ± 0.16
3.00 ± 0.6
0.02 ND
ND
2.36 ±
ND
2.99 ±
3.67 ± 0.87
4.18 ± 1.19
7.40 ± 4.13
2.99 ± 0.68
0.08 ND
ND
ND
4.57 ±
8.49 ± 1.20
4.51 ± 1.79
0.10 ND
ND
ND
0.76 ±
1.72 ± 0.19
0.75 ± 0.43
0.02 ND
ND
ND
ND
ND
1.09 ±
ND
ND
ND
ND
ND
1.19 ±
2.12 ± 0.88
1.66 ± 0.65
2.53 ± 1.10
2.07 ± 0.77
0.03 24
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
Trace
ND
ND
ND
ND
ND
0.49 ±
0.87 ±
ND
ND
ND
ND
ND
0.01
0.14
0.70 ±
Trace
ND
ND
ND
ND
ND
2.03 ±
2.33 ±
ND
ND
ND
ND
ND
0.45
0.33
0.23 ±
Trace
ND
ND
ND
ND
ND
1.21 ±
0.95 ±
ND
ND
ND
ND
ND
0.20
0.08
0.62 ±
2.13 ±
ND
ND
ND
ND
ND
0.30
0.15
1.99 ±
2.13 ±
ND
ND
ND
ND
ND
0.62
0.15
0.25 ±
Trace
ND
ND
ND
ND
ND
0.56 ±
1.81 ±
ND
ND
ND
ND
ND
0.12
0.47
0.70 ±
1.23 ±
ND
ND
ND
ND
ND
0.17
0.25
ND
ND
27.49 ±
30.54 ±
78.6 ± 6.36
8.41 ±
0.59 ±
0.08 0.36 ±
0.05
0.03 23
ND
0.05
0.05 ND
Trace
0.16
0.07 ND
0.14 ±
0.04
0.01 4.16 ±
0.02
ND
ND
27
I ND
ND
ND
ND
26
ND
ND
ND
ND
ND
29
30
ND
ND
ND
ND
ND
32
33
A
34
35
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
M
ND
CC E
31
ND
ND
ED
28
ND
ND
ND
PT
27
ND
ND
N U SC R
ND
A
25
ND
ND
ND
ND
2.14
3.22 0.98 ±
ND
ND
ND
ND
ND
0.44 ± 0.06
ND
ND
ND
ND
ND
0.61 ± 0.07
0.24 ± 0.03
0.13 2.29 ±
ND
ND
Trace
Trace
0.64 ±
Trace
0.15 ND
ND
ND
ND
ND
0.52 ± 0.01
Trace
0.06 ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
16.63 ±
23.92 ±
1.15
2.95
0.79 ± 0.00
1.93 ±
34.42 ± 3.54
Trace
ND
ND
ND
ND
ND
0.27 ± 0.01
0.18 ±
ND
ND
ND
ND
ND
1.51 ± 0.09
4.83 ±
ND
ND
ND
ND
ND
4.97 ± 0.60
9.01 ±
ND
ND
ND
ND
ND
ND
ND
0.99 ± 0.06
2.74 ±
0.18 ± 0.07
ND
0.95 ±
0.18
0.20
0.73 ±
Trace
0.16 ±
Trace
0.03 1.57 ± 0.36
0.78 ±
0.58 ±
0.03
0.05
1.26 ±
0.49 ±
0.27
0.04
0.50 ± 0.04
Trace
Trace
17.84 ± 2.52
2.51 ±
0.54 ±
0.23
0.07
1.62 ± 0.20
Trace
Trace
0.64 ± 0.09
0.37 ±
Trace
11.13 ± 1.46
0.18 ND
ND
ND
ND
ND
ND
ND
ND
8.94 ± 0.85
20.22 ± 1.86
ND
5.31 ±
0.02
0.82 ND
Trace
Trace
1.03 ND
0.21 ± 0.02
0.12 ND
0.07
0.22 ± 0.06
0.59 ND
1.33
ND
ND
ND
ND
ND
ND
ND
ND
0.53 ± 0.04
0.67 ± 0.09
Total
13.27 ±
14.99 ±
48.88 ±
flavonoid
0.87
1.05
8.53
9.6 ± 2.35
0.19 ±
41.66 ±
48.19 ±
76.47 ±
13.89 ±
0.04
0.43
16.79
13.22
4.74
28
Trace
1.32 ± 0.14
3.45 ± 0.33
0.01
I 17.05 ±
45.56 ±
32.15 ±
14.05 ±
5.17 ±
xanthone
0.70
1.56
0.98
1.66
1.11
23.8 ±
38.54 ± 4.17
0.40
A
CC E
PT
ED
M
A
Note: ND, not detected.
N U SC R
Total
29
38.58 ± 6.53
9.15 ± 1.76
11.45 ± 1.1
65.31 ±
96.32 ±
156.69 ±
20.08 ±
3.15 ±
5.15
10.24
10.62
0.93
0.37