Accepted Manuscript

Title: Chemical profiles and quality evaluation of Buddleja

officinalis flowers by HPLC-DAD and HPLC-Q-TOF-MS/MS

Authors: Guoyong Xie, Qiuhong Xu, Ran Li, Lu Shi, Yu Han,

Yan Zhu, Gang Wu, Minjian Qin

PII: S0731-7085(18)30959-2
DOI: https://doi.org/10.1016/j.jpba.2018.10.030
Reference: PBA 12280

To appear in: Journal of Pharmaceutical and Biomedical Analysis

Received date: 24-4-2018

Revised date: 22-7-2018
Accepted date: 17-10-2018

Please cite this article as: Xie G, Xu Q, Li R, Shi L, Han Y, Zhu Y, Wu G, Qin M,
Chemical profiles and quality evaluation of Buddleja officinalis flowers by HPLC-
DAD and HPLC-Q-TOF-MS/MS, Journal of Pharmaceutical and Biomedical Analysis
(2018), https://doi.org/10.1016/j.jpba.2018.10.030

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Chemical profiles and quality evaluation of Buddleja officinalis flowers by
HPLC-DAD and HPLC-Q-TOF-MS/MS
Guoyong Xie1, Qiuhong Xu1, Ran Li, Lu Shi, Yu Han, Yan Zhu, Gang Wu*, Minjian
Qin *
Department of Resources Science of Traditional Chinese Medicines, School of
Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing 210009,
China

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* Corresponding authors at: Department of Resources Science of Traditional Chinese

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Medicines, School of Traditional Chinese Pharmacy, China Pharmaceutical

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University, No. 24 Tongjiaxiang, Gulou District, Nanjing, 210009, PR China.

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Tel.: +86 25 86185130; fax: +86 25 85301528.
E-mail addresses: woosmail@163.com (G. Wu), minjianqin@163.com (M.J. Qin).
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These authors contributed equally to this work.
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Graphical abstract
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The total of 54 compounds were identified or tentatively identified in the extracts of B.

offinalis flowers by HPLC-Q-TOF-MS/MS. Meanwhile, HPLC fingerprints have been
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developed, together with the simultaneous quantification of 11 constituents for the

quality evaluation of B. officinalis. In addition, the antioxidant capacities of the
extracts of B. offinalis flowers from 12 habitats were reported.

Highlights
 54 compounds were detected from B. officinalis flowers by HPLC-Q-TOF-
MS/MS method.
 Seven alkaloids were found from B. officinalis for the first time.
 HPLC fingerprint of B. officinalis flower were established.
 Eleven principal components were quantified for B. officinalis.
 The extracts of B. officinalis flowers show strong antioxidant capacities.

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Abstract

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The dried flowers and inflorescences of Buddleja officinalis Maxim are used as

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traditional medicines in China, and aqueous extracts of the flowers have also been used

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since ancient times as a yellow rice colorant at local festivals. In this study,
HPLC-Q-TOF-MS/MS was used to determine the overall chemical composition of this

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medicine-food plant. A total of 54 compounds, including 23 flavonoids, 19
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phenylethanoid glycosides, 7 alkaloids and 5 other compounds, were detected in the
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methanol extracts of the herb using this method. Among them, 33 compounds were
found firstly in this herb. HPLC fingerprints were also developed, together with a
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method for the simultaneous quantification of 11 constituents that could be used for
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quality evaluation of B. officinalis. Fingerprint analysis, using 28 characteristic

fingerprint peaks, was used to assess the similarities among 12 samples collected from
different geographic areas and showed that the similarity was > 0.900. Simultaneous
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quantification of 11 markers in B. officinalis was then performed to determine

consistency of quality. Additionally, the total phenolic content and antioxidant capacity
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of extracts of the 12 samples of B. officinalis flowers were measured using

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spectroscopic methods. B. officinalis was found to have good antioxidant capacity and
to be a potential natural antioxidant. The highest antioxidant capacity was found in the
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samples from Guizhou, Sichuan and Guangxi Province. Our results provide valuable
information for further understanding and exploiting the herb.
Key words: Buddleja officinalis; Chemical constituents; HPLC-Q-TOF-MS/MS;
HPLC fingerprint
1. Introduction
 The genus Buddleja (Loganiaceae), which comprises about 100 species, is widely
distributed in the tropical and subtropical regions of America, Africa and Asia, with 20
species and five hybrids found in China [1]. Buddleja officinalis Maxim., known as Mi
Meng Hua in Chinese, is a shrub that grows in Myanmar, Vietnam and in the
southwest and central regions of China. The plant was first recorded 1000 years ago in
Kai Bao Ben Cao, one of the ancient works on traditional Chinese medicines, and is
now listed in the Chinese Pharmacopoeia (version 2015) [2]. The dried flower buds

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and inflorescences of B. officinalis are used as a traditional herbal medicine to treat eye

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diseases, strokes, vascular diseases, diabetes and neurological disorders [3, 4]. The

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plant is also cultivated as a natural food dye and aqueous extracts of the flowers have

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been used since ancient times as a yellow rice colorant in local festivals in southwest
China [5]. Phytochemical studies have demonstrated that the flowers of B. officinalis

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contain mainly flavonoids, phenylethanoid glycosides, iridoids and saponins [6-8],
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although a chemical profile reflecting the total constituents of the herb is not yet
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available. High performance liquid chromatography, coupled with diode-array
detection (DAD) and high resolution time-of-flight mass spectrometry
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(HPLC-Q-TOF-MS/MS), can simultaneously separate and identify chemical

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constituents in medicinal herbs [9-11] and should be an effective tool for establishing
chemical profiles of extracts of B. officinalis flowers (BOEs).
It is said in Xin Xiu Ben Cao, “Supposing that the herb is away from its native land,
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then its effects will be different even if the features are identical”, and modern research
has shown that the quality of traditional Chinese medicine (TCM) is mainly affected by
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the ecological environment, including soil, air temperature, rainfall, light and
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ecological communities. Beyond that, different harvest and storage conditions are
important influencing factors [12]. Currently, the fingerprint technique is
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acknowledged by the U.S. Food and Drug Administration (FDA), the World Health
Organization (WHO) and the State Food and Drug Administration of China (SFDA)
for the quality control of TCM [13]. HPLC, thin layer chromatography (TLC) and gas
chromatography (GC) are recognized as conventional analytical methods and, of these,
HPLC has become the preferred method for TCM fingerprinting because of its rapidity,
high separation efficiency, high selectivity, high sensitivity and wide applicability [14].
In the present study, we aimed to establish a chromatographic fingerprint for B.
officinalis using HPLC-DAD. Using the method for simultaneous determination of 11
constituents established in our earlier study [15] as a foundation, we also analyzed the
constituents of 12 samples of B. officinalis from different regions of China.
Free radicals formed during metabolism are closely linked with aging and with
diseases such as cancer, atherosclerosis and eye disease [16]. Since antioxidants are

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associated with reduced oxidative damage, measurement of the antioxidant capacity of

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medicines and food products is important for the health-related and nutraceutical

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industries [17]. B. officinalis flowers, which are used as a traditional herbal medicine

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and as a rice colorant for festivals, possess many biological and pharmaceutical
activities, including anti-inflammatory, antioxidant, hypoglycemic, antiviral and

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antibacterial effects [18]. Previous studies have shown that B. officinalis flowers have
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pronounced antioxidant activity and show good potential as a natural antioxidant
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resource [18-22]. In the present study, the antioxidant capacities of 12 samples were
measured to evaluate the antioxidant capacity of B. officinalis.
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To determine the overall chemical composition and identify antioxidant

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components of B. officinalis flowers, an HPLC-Q-TOF-MS/MS method was developed

for qualitative analysis. The chromatographic fingerprint of B. officinalis was
established and the main chemical components were quantified for samples of B.
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officinalis from 12 different producing areas. Anti-oxidative assays, including

2,2-diphenyl-1-picrylhydrazyl (DPPH), superoxide anion (O2•−) scavenging activity
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(SOSA), Fe3+-reducing ability (FRA) and Fe2+-chelating assays were performed to

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evaluate the antioxidant capacity of B. officinalis.

2. Materials and methods
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2.1 Materials and chemicals

Flower buds and inflorescences of B. officinalis were collected from 12 habitats in
China. Sample information is provided in Table 1. Sample S3 was selected for
chemical profiling. The samples were crushed to a powder in an electric grinder and
then passed through a 60 mesh sieve.
 Reference compounds, including luteolin 7-O-glucoside, luteolin 7-O-rutinoside,
neobudofficide, linarin, apigenin, acteoside, crocin III and N1,N5, N10-(E)-tri-p-
coumaroylspermidine, were obtained from B. officinalis flowers by the authors.
Apigenin 7-O-glucuronide, acacetin, echinacoside and isoacteoside were purchased
from Baoji Herbest Bio-Tech Co., Ltd. (Baoji, China). The purity of all standards
was > 96%, as determined by HPLC. HPLC grade methanol and acetonitrile were
purchased from Hanbon Sci. & Tech. Co., Ltd. (Jiangsu, China) and Merck

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(Darmstadt, Germany), respectively. Purified water was obtained from Wahaha Group

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Co., Ltd. (Hangzhou, China). Gallic acid, 1,1-diphenyl-2-picrylhydrazyl (DPPH) and

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nitrotetrazolium blue chloride (NBT) were purchased from Sigma-Aldrich Co.

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(Shanghai, China). Folin-Ciocalteu’s reagent, L-methionine and riboflavin were
obtained from Solarbio Sciences & Technology Co., Ltd. (Beijing, China).

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Trichloroacetic acid (TCA) and potassium ferricyanide (K3Fe(CN)6) were purchased
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from Shanghai Macklin Reagent Company (Shanghai, China). Ferrozine was obtained
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from Aladdin Industrial Corporation (Shanghai, China). Sodium carbonate (Na2CO3),
Iron (Ⅲ) chloride hexahydrate (FeCl3·6H2O), Ferrous chloride (FeCl2), Disodium
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hydrogen phosphate dodecahydrate (Na2HPO4·12H2O) and Sodium dihydrogen

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phosphate dihydrate (NaH2PO4·2H2O) were purchased from China Pharmaceutical

Group Chemical Reagent Co., Ltd. (Beijing, China).
2.2 Preparation of sample solutions and standard solutions
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The extraction condition was optimized in our previous studies [15], the dried
powder (0.5 g) was sonicated twice for 33 min with 76% methanol (17 mL). The
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combined extracts were centrifuged at 1500 r/min for 10 min. The supernatant was
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then transferred to a 50 mL volumetric flask and the volume made up to 50 mL using

the same solvent. Standard stock solutions of the 11 reference compounds were
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prepared by dissolving accurately weighed amounts in 60% aqueous acetonitrile. A

mixed standard stock solution was then prepared using the individual standard stock
solutions. All solutions were stored in a refrigerator at 4°C and filtered through an
0.22 μm membrane filter before analysis.
2.3 Chromatographic conditions
 HPLC fingerprinting and composition analysis were conducted using a method
established previously [15]. An Agilent Series 1260 LC instrument (Agilent
Technologies, Santa Clara, CA, USA), equipped with autosampler, column temperature
controller, DAD, quaternary pump and online degasser, was used for the analyses. A
Megres C18 column (4.6 mm × 250 mm, 5 µm, Hanbon Sci. & Tech. Co., Ltd.),
thermostated at 25°C, was used for chromatographic separations. The mobile phase
was 0.1% formic acid-water (A) and acetonitrile (B). The gradient elution program was:

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0–5 min, 8–10% B; 5–10 min, 10–18% B; 10–20 min, 18–18.5% B; 20–34 min,

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18.5–30% B; 34–38 min, 30% B; 38–41 min, 30–37% B; 41–50 min, 37–95% B;

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50–55 min 95% B. The flow rate was 1.0 mL/min and the detection wavelength was

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330 nm. The injection volume was 10 µL and each run was followed by an
equilibration period of 5 min.

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An Agilent 1260 HPLC system (Agilent Technologies, Santa Clara, CA, USA),
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equipped with autosampler, column temperature controller, DAD, quaternary pump and
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online degasser, was used for liquid chromatography separations. The chromatographic
conditions were the same as those used for HPLC fingerprinting and composition
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analysis. In order to identify the constituents, the HPLC system was coupled to an
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accurate-mass Q/TOF-MS/MS instrument (Agilent Technologies, Santa Clara, CA,

USA), operating in positive and negative electrospray ionization (ESI) mode. The
parameters for MS detection were as follows: scan range from m/z 100–2000 Da,
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nitrogen flow rate 8 L/min, drying gas temperature 325°C, nebulizer pressure 50 psi
and capillary voltage 3500 V. Operation, data capture and analysis were managed using
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Masshunter Qualitative Analysis Software B.06.00 and ChemStation software.

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2.4 Method validation for HPLC fingerprinting

Method validation for HPLC fingerprinting includes precision, stability and
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reproducibility tests. The precision was evaluated by analyzing the same sample
solution (sample 3) five times continuously; The stability was determined by analyzing
one sample solution (sample 3) after storage at 4°C for 0, 2, 4, 6, 8, 12, 24 and 48 h,
and the reproducibility was assessed by injection of five working solutions (sample 3).
2.5 Determination of total phenolic content
 Total phenolic content (TPC) was determined using the Folin-Ciocalteau reaction
[23]. Extract solution (200 µL, 5 mg/mL) was blended with Folin-Ciocalteau reagent
(200 µL) and, after 5 min, 3% Na2CO3 solution (7 mL) was added. The combined
reaction solution was stored at room temperature for 1 h, and then determined at 765
nm using an Epoch microplate spectrophotometer (BioTek Instruments, Inc.,
Winooski, VT, USA). The standard curve (y = 0.0013 x + 0.0761, R2 = 0.999) was
established using gallic acid as reference. Results are expressed as gallic acid

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equivalents (GAE) (mg)/dry mass of plant material (g).

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2.6 Radical scavenging activity

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2.6.1 DPPH radical scavenging activity

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DPPH radical scavenging capacity was measured using a previously reported
method [24]. A working solution of DPPH (0.05 Mm/L) was prepared by dilution with

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methanol. Sample solutions (200 µL) at different concentration (1.5, 1, 0.75, 0.5, 0.25,
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0.125 mg/mL) were then mixed with DPPH solution (3 mL) and the reaction mixtures
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were allowed to stand for 30 min in the dark. By measuring absorbance at 517 nm,
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DPPH radical scavenging activity was calculated using the following formula:

Scavenging rate  %  1   Atest  Ablank  / Acomtrol  100%

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where Atest is the absorbance of the solution of sample and DPPH after reaction, Ablank
is the absorbance of the sample and 76% methanol (3 mL) and Acontrol is the absorbance
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of 76% methanol (200 μL) and DPPH. IC50 values were calculated for evaluation of the
DPPH radical scavenging activity of the samples, Vc (vitamin C) used as the positive
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reference.
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2.6.2 Superoxide anion (O2•−) scavenging activity

SOSA was analyzed as previously described [25]. The reaction was carried out at
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25°C and the final assay mixture contained L-methionine solution (130 mM/L, 0.3 mL),
NBT solution (750 μM/L, 0.3 mL), EDTA-Na2 solution (20 μM/L, 0.3 mL) and
riboflavin solution (20 μM/L, 0.3 mL). The assay mixture was added to 50 mM/L
phosphate buffer (pH 7.0, 1.5 mL) and mixed with different concentrations (750, 500,
375, 250, 125, 62.5 μg/mL) of test sample (0.2 mL). The test sample was replaced by
the extraction solvent (76% methanol) in the control group and blank group. After
exposure of the test and control solutions to fluorescent light for 30 min, absorption
was measured at 560 nm. SOSA was calculated using the following formula:

Scavenging rate  %  1   Atest  Ablank  / Acomtrol  100%

where Atest is the absorbance of the test solution, Acontrol is the absorbance of the control
solution and Ablank is the absorbance of the blank solution (lucifuge). Results are

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expressed as IC50 values (μg/mL), Vc was used as the positive reference.

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2.6.3 Fe3+-reducing ability
FRA was measured as previously described [26]. Different concentrations (2.5, 2,

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1.5, 1, 0.5, 0.25 mg/mL) of sample solutions (500 μL) were mixed with phosphate

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buffer (0.5 mL, 0.2 M/L, pH 6.6) and K3Fe(CN)6 (0.5 mL, 1%). The mixtures were
heated in a water bath at 50℃ for 20 min, TCA (0.5 mL, 10%) was then added and the

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mixtures were centrifuged at 4000 r/min for 10 min. An aliquot (0.5 mL) of the upper
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layer was mixed with distilled water (0.5 mL) and 0.1% FeCl3 (0.1 mL), and the
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absorbance was measured at 700 nm. FRA was calculated using the following formula:
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Fe3+ reducing ability (A) = Atest − Ablank

where Atest is the absorbance of the test solution, Ablank is the absorbance of the blank
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solution (76% methanol instead of sample). EC50 values were calculated for evaluation
of FRA of the samples, Vc used as the positive reference.
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2.5.4 Fe2+-chelating assay

Fe2+-chelating capacity was measured as previously described [27]. Different
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concentrations (10, 8, 6, 4, 2, 1 mg/mL) of sample solution (400 μL) were added to a

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mixture of aqueous ferrous chloride solution (2 mM/L, 50 µL), ferrozine solution (5

mM/L, 100 μL) and methanol (2 mL). After shaking vigorously, the reaction solutions
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were allowed to stand at 25℃ for 10 min. Absorbance was measured at 562 nm and
Fe2+-chelating capacity was calculated using the following formula:

Inhibition rate  %  1   Atest  Ablank  / Acontrol  100%

where Atest is the absorbance of the test solution, Ablank is the absorbance with
EDTA-Na2, Acontrol is the absorbance without sample. Results are presented as IC50
values (mg/mL), EDTA-Na2 was used as the positive reference.
2.7 Data analysis
The HPLC-DAD data from 12 samples of B. officinalis were analyzed using the
professional software, Similarity Evaluation System for Chromatographic Fingerprint
of Traditional Chinese Medicine (Version 2004A), which is recommended by the
SFDA. The mean chromatogram was calculated and generated as a representative
standard fingerprint chromatogram and the correlation coefficients of the samples were

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then calculated by comparison with the mean chromatogram. All experiments were

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analyzed in triplicate. Data treatment was carried out using Microsoft Excel software

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and SPSS software 22.0.

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3. Results and discussion
3.1 Identification of chemical constituents in methanol extract of B. officinalis
flowers
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HPLC-ESI-Q/TOF-MS/MS analysis of the methanolic BOEs was carried out in
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both positive and negative ionization modes. The HPLC chromatograms, together with
the total ion currents (TIC) of the methanolic BOEs, are shown in Fig.1 and the UV
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and MS data are listed in Table 2. Fifty-four compounds were identified or tentatively
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identified, including 23 flavonoids, 19 phenylethanoid glycosides, 7 alkaloids and 5

other compounds. The chemical structures of the compounds are shown in Fig.2.
The peaks for compounds F4, F5, F11, F14, F16, F22, F23, P10, P16, A6 and C5
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were definitively identified by comparison of UV and MS data with those of authentic

standards. The chemical structures of other compounds were deduced mainly on the
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basis of the UV and MS data and confirmed by comparison with values reported in the
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literature. Analysis of the BOEs revealed that flavonoids and phenylethanoid

glycosides were the major constituents in B. officinalis flowers.
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3.1.1 Flavonoid compounds

Twenty-three flavonoids, including 3 free aglycones and 20 O-glycosides, were
identified in the BOEs. Retro-Diels-Alder cleavage of the C-ring, combined with
various neutral loss pathways, are typically used for the identification of flavonoid
aglycones [28]. For the identification of flavonoid glycosides, ion fragmentations are
characterized by successive elimination of sugar moieties and aglycone fragments [29].
In this study, the major flavonoid aglycones in the BOEs were identified as luteolin
(F20), apigenin (F22) and acacetin (F23) by comparison with authentic standards and
literature data [30, 31]. Of the 20 flavonoid O-glycosides, all except F3, F9 and F12
were identified as derivatives of these three aglycones.
Compounds F1, F2, F7, F8, F10, F11, F13 and F15 were identified as apigenin
(F22) derivatives. F11 was unambiguously confirmed to be apigenin 7-O-glucuronide

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by comparison with the authentic standard compound and with literature data [32]. F1

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and F2 showed identical molecular ions and similar fragmentation patterns in positive

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ionization mode. Their MS2 spectra exhibited ions at m/z 447 [M+H-176]+ and 271

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[M+H-176-176]+, which originated from successive losses of 176 Da fragments,
indicating the presence of two glucuronic acid moieties. However, F1 and F2 showed

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different fragmentation behaviors in negative ionization mode. The spectrum of F2
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showed a fragment ion at m/z 269 [M-H-352]¯, whereas this fragment was absent in
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the spectrum of F1. Taking into account the MSn and UV spectra, together with
previous reports, F1 and F2 were tentatively deduced to be apigenin
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7,4'-di-O-glucuronide and clerodendrin, respectively [33, 34]. Compared with F2, F8

exhibited fragment ions at m/z 447 [M+H-146]+ and 271 [M+H-146-176]+ in positive
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ionization mode, indicating that a glucuronic acid moiety was replaced by a

rhamanopyranosyl group. Considering previously reported structural information [35],
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F8 was tentatively deduced to be apigenin 7-O-α-L-rhamnopyranosyl-(1 →

2)-β-D-glucuronide.
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F7 gave a molecular ion at m/z 579 [M+H]+, with product ions at m/z 433
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[M+H-146]+ and 271 [M+H-146-162]+, corresponding to the sequential loss of

rhamanopyranosyl and glucose moieties. F7 was tentatively deduced to be isorhoifolin
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by comparing the MS2 and UV data with literature values [36]. F10 presented a
precursor ion [M+H]+ at m/z 433, which indicated a molecular formula C21H20O10. The
fragment ion at m/z 271 [M+H−162]+, which represented the aglycone fragment of
apigenin, was formed by loss of a glucose moiety from the precursor ion. By
comparison with literature data, F10 was tentatively deduced to be apigenin
7-O-glucoside [31]. F15 showed similar fragmentation to F10 in positive ionization
mode. The fragment ion at m/z 433 [M+H-86]+ indicated loss of a malonyl residue. The
direct loss of 247 Da indicated that the malonyl residue was attached on the glucose
moiety. Therefore, F15 was tentatively identified as apigenin 7-(6''-malonylglucoside)
[37].
F13 was an isomer of F11 and showed an identical molecular ion and
fragmentation pattern to that of F11. MS2 fragmentation of both compounds presented

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the same ions at m/z 271 [M+H−176]+ and 269 [M−H−176]− in positive and negative

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ionization modes, respectively, attributed to loss of a glucuronic acid residue from the

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precursor ion and the deprotonated molecular ion. F13 was, therefore, identified as an

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apigenin monoglucuronide. Additionally, based on published data on the elution order
of apigenin monoglucuronides [30, 38], F13 was tentatively identified as apigenin
4'-O-glucuronide.
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F4, F5 and F6 were identified as derivatives of luteolin (F20). F20 gave molecular
ions at m/z 287 [M+H]+ and 285 [M−H]−, respectively, in positive and negative
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ionization modes. In the MS2 spectrum, the molecular ions yielded specific fragments
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at m/z 151, 133 and 153, 135, respectively, which are characteristic of
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Retro-Diels-Alder fragmentations for luteolin. F20 was, therefore, tentatively

identified as luteolin by comparison with literature data [39]. F4 and F5 were
unambiguously identified as luteolin 7-O-rutinoside and luteolin 7-O-glucoside by
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comparison with the authentic standards and their corresponding UV and MS spectra
[31, 40]. In positive ionization mode, F6 showed a precursor ion at m/z 463 [M+H]+,
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which gave similar fragmentation and the same MS2 fragments ions, at m/z 287, 269
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and 241, as F5. The fragment at m/z 287 corresponded to the [M+H−176]+ ion,
indicating the presence of a glucuronic acid moiety. By comparing the characteristic
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fragment ions with those of previous reports [31, 40], F6 was tentatively identified as
luteolin 7-O-glucuronide.
F14, F16–F19 and F21 were identified as derivatives of acacetin (F23). F14 and
F16 were unambiguously identified as neobudofficide and linarin by comparison with
reference standards and previous reports [41, 42]. F17 exhibited precursor and
deprotonated molecular ions at m/z 607 [M+H]+ and 605 [M−H]−, respectively. In
positive ionization mode, the product ions at m/z 461 [M+H-146]+ and 285
[M+H-146-176]+ corresponded to sequential loss of the rhamanopyranosyl and
glucuronic acid moieties. The MS2 spectrum showed fragments at m/z 283
[M-H-146-176]− and 268 [M-H-146-176-15]− in negative ionization mode. F17 was
tentatively identified as acacetin 7-O-α-L-rhamnopyranosyl-(1→2)-β-D-glucuronide or
its isomer, which had not been previously described. F18 was an isomer of F16. In

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positive ionization mode, its main fragment ion was at m/z 285, corresponding to

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[M+H-146-162]+, formed by loss of the rhamanopyranosyl-glucosyl moiety. The

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fragment ion at m/z 133, which represented the aglycone fragment, was identical to that

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of acacetin [42]. F18 was, therefore, tentatively identified as 5-[[6-O-(6-deoxy-α-L-
mannopyranosyl)-β-D-galactopyranosyl]oxy]-7-hydroxy-2-(4-methoxypheny-l)-4H-1-

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benzopyran-4-one [43]. The MS spectra of F19 and F21 showed [M+H]+ ions at m/z
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447 and 461, respectively, and showed the same MS/MS fragments at m/z 285, 270 and
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242. The fragment at m/z 285 corresponded to the [M+H−162]+ ion for F19 and the
[M+H−176]+ ion for F21, indicating that F19 possessed an O-glycoside moiety, while
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F21 possessed a glucuronic acid moiety. By comparison with literature data [40], F19
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and F21 were tentatively identified as acacetin 7-O-glucoside and acacetin

7-O-glucuronide, respectively.
Using similar methods, compounds F3, F9 and F12 were tentatively identified as
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quercetin 7- O-glucoside, diosmetin 7-O-rutinoside and kaempferol

3-O-(6''-malonylglucoside), respectively. [44-46].
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3.1.2 Phenylethanoid glycosides

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Seventeen phenylethanoid glycosides were identified in the BOEs. P1 showed an

[M+Cl]− ion at m/z 335.0905 in negative ionization mode and an [M+Na]+ ion at m/z
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323.1099 in positive ionization mode. The molecular formula C14H20O7 was calculated
from the high resolution TOF-MS. The fragment ion at m/z 137 in negative ionization
mode was attributed to loss of a glucose moiety from the [M+Cl]− ion, which further
loses a molecule of H2O to give a fragment at m/z 119. P1 was thus tentatively
identified as salidroside by comparison with literature data [39]. P2 and P3 showed
similar MS spectra. Their deprotonated molecular ions [M−H]− at m/z 487 in negative
ionization mode suggested the same molecular formula, C21H28O13, and indicated that
they could be a pair of isomers. The successive elimination of rhamnopyranosyl and
glucose moieties from the deprotonated molecular ions yielded fragments at m/z 341
and 179, respectively. The fragments at m/z 179 and 161 supported the presence a
caffeoyl residue. Since the E-form has been reported to elute earlier than the Z-form,
P2 and P3 were tentatively identified as cistanoside F and 4-O-(3,4-dihydroxy-

T
Z-cinnamoyl)-3-O-L-rhamnopyranosyl-D-glucose, respectively [47].

IP
P4 exhibited an [M-H]¯ ion at m/z 505, suggesting the molecular formula

R
C21H30O14. The fragment at m/z 341, resulting from loss of 164 Da, suggested the

SC
existence of an open-loop glucose moiety. The fragments at m/z 179 and 161 were
attributed to the loss of caffeoyl and hexose moieties, respectively. P4 was thus

U
tentatively characterized as hebitol II [48]. P5 gave an [M+Na]+ ion at m/z 395,
N
suggesting the molecular formula C17H24O9. The fragments at m/z 364 [M+Na-31]+ and
A
m/z 233 [M+Na-162]+ resulted from loss of a methoxyl group and a glucose moiety,
respectively. P5 was tentatively identified as syringin by comparing the MS and UV
M

spectra with literature data [49]. Using similar methods, compounds P6 and P7 were
tentatively identified as β-4-caffeoylglucose and 3-caffeoylquinic acid, respectively [50,
ED

51].
P8 showed [M+Cl]¯ and [M+H]+ ions at m/z 555 and 521, respectively, in negative
PT

and positive ionization modes, suggesting the molecular formula C22H32O14. This
compound had a similar fragmentation pattern to P4. In both cases, the loss of a
E

glucose moiety yielded a fragment at m/z 355, which eliminated a hexose moiety and
CC

produced an ion at m/z 193. The daughter ion was identical to that of ferulic acid
moiety. P8 was, therefore, tentatively identified as globularitol [48]. P9 showed
A

[M+Cl]¯ and [M+Na]+ ions at m/z 481 and 469, respectively, in negative and positive
ionization modes. The fragment at m/z 307 [M+Na-162]+ resulted from loss of a
glucose moiety. Taking into account literature data and other information in the UV and
MS2 spectra [52], P9 was tentatively identified as 2-phenylethyl
6-O-β-D-glucopyranosyl-β-D-glucopyranoside. P10 was identified definitively as
echinacoside by comparison with authentic standards and by comparing the
corresponding UV and MS spectra with literature data [53]. Using similar methods and
by comparing their corresponding UV and MS spectra and elution times with literature
data, P11, P12 and P13 were tentatively identified as isocampneoside II, campneoside
II and phenethyl-β-primeveroside [39, 54, 55].
P15, P17 and P18 all showed a deprotonated molecular ion [M−H]− at m/z 623 and
MS/MS fragments at m/z 461, 179 and 161, indicating a similar fragmentation pattern

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to that of P16 in negative ionization mode. Compound P16 were identified definitively

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as acteoside by comparison with the authentic standards and their corresponding UV

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and MS spectra. Taking into account both literature data and their retention times, P15,

SC
P17 and P18 were tentatively identified as forsythoside A, cis-acteoside and
isoacteoside, respectively [39, 56]. Using similar methods, compounds P14 and P19

U
were tentatively identified as hebeoside and globusintenoside (or a globusintenoside
N
isomer), respectively [57, 58].
A
3.1.3 Alkaloid compounds
In the present work, seven alkaloids, all of which are coumaroyl-spermidine
M

analogs, were detected in the BOEs. The MS/MS spectra of compounds A1–A2 and
ED

A6–A7 are shown in Fig. 3. To the best of our knowledge, this is the first time that this
type of alkaloid has been discovered in B. officinalis or even in Buddleja plants. It has
been reported that this type of alkaloid possesses inhibitory activity against HIV-1
PT

protease or the serotonin transporter [59, 60]. A6 gave a molecular ion [M+H]+ at m/z
584. Its major fragment ion at m/z 438 [M+H-146]+ was attributed to cleavage of the
E

amide bond between the cinnamoyl and spermidine moieties. The fragment ions at m/z
CC

292 and 147 could be explained by loss of the second and third cinnamoyl residues.
The daughter ions at m/z 420 and 275 resulted from loss of NH4 and NH3 from the
A

fragment ions at m/z 438 and 292, respectively. By comparing the authentic standards
and their corresponding UV and MS spectra with literature data [61], A6 was identified
definitively as N1,N5,N10-(E)-tri-p-coumaroylspermidine. A3, A4 and A5 all showed a
molecular ion [M+H]+ at m/z 584 and similar fragmentation patterns to that of A6,
indicating that they were isomers of A6. Using a similar method to that used to identify
A6, and by comparing the corresponding UV and MS spectra and elution times with
literature data [61], A3, A4 and A5 were tentatively identified as
N1,N5,N10-(Z)-tri-p-coumaroylspermidine,
N1(Z)-N5-(Z)-N10-(E)-tri-p-coumaroylspermidine and N1(E)-N5-(Z)-N10-(E)-tri-p-
coumaroylspermidine, respectively.
A2 exhibited fragment ions at m/z 454 [M+H-146]+, 308 [M+H-146-146]+ and 292
[M+H-146-162]+, resulting from loss of cinnamoyl or caffeoyl moieties. The fragment

T
ions at m/z 147 and 163 confirmed the presence of both cinnamoyl and caffeoyl

IP
residues. The MS spectrum and fragmentation behavior of A2 were identical to those

R
reported in the literature for N1-(E)-caffeoyl-N5, N10-di-p-(E)-coumaroyl spermidine

SC
[62]. A2 was, therefore, tentatively identified as
N1-(E)-caffeoyl-N5,N10-di-p-(E)-coumaroyl spermidine [63]. Similarly, by comparing

U
their chromatographic behaviors with literature data, A1 and A7 were tentatively
N
identified as N1,N5-di-[(E)-p-coumaroyl]-spermidine (or its isomer) and keayanidine A,
A
respectively [38, 64, 65].
3.1.4 Other compounds
M

One iridoid (C1), one organic acid (C2) and three carotenoids (C3–C5) were
ED

characterized in the BOEs. By comparing their UV and MS data with literature values,
C1 and C2 were tentatively identified as 6-methoxyl catalpol and tuberonic acid
glucose [47, 66]. The three carotenoids (C3–C5) were identified as crocins, which had
PT

previously been reported to be the major pigments in B. officinalis flowers [5]. The
MS/MS spectra of compounds C3–C5 are shown in Fig.3. All three compounds
E

exhibited an ion attributed to sodiated aglycon (Y0) at m/z 351, which corresponded to
CC

crocetin. The fragments arising from sequential loss of glucose or gentiobiose moieties
can be used to identify the type and position of the glycosyl moiety. C5 showed a
A

precursor ion at m/z 675, which is 324 Da larger than the sodiated aglycone ion,
indicating the presence of two glucosyl moieties or a gentiobiosyl moiety, since the
product ion of gentiobiose at m/z 347 represented the base peak. By comparing the
authentic standards and their corresponding UV and MS data with literature values [67],
C5 was unambiguously identified as mono-gentiobiosyl-crocetin (crocin III). The
precursor ions of the other two crocins, 648 Da (C3) and 486 Da (C4), were larger than
the sodiated aglycone ion. For C4, the fragments ions at m/z 675 (Y0+324) and 513
(Y0+162) indicated a gentiobiosyl residue and a glucosyl residue, respectively, while
C3 showed only the ion Y0+324. C3 and C4 were, therefore, identified as
bis-gentiobiosyl-crocetin (crocin I) and gentiobiosyl-glucosyl-crocetin (crocin II),
respectively [67].
3.2 Method validation for HPLC fingerprint analysis

T
Method validation for the HPLC fingerprint analysis was conducted as described

IP
in Section 2.4. The results are expressed as relative standard deviations (RSD) of the

R
average relative retention times (RRT) and relative peak areas (RPA) of the 28

SC
characteristic common peaks compared with the reference F16 (linarin). The variation
of RRT and RPA in the precision, stability and reproducibility tests did not exceed 5%

U
and we can conclude, therefore, that the HPLC fingerprint method is accurate and
N
reliable.
A
3.3 HPLC fingerprints of B. officinalis
Using the HPLC method that was established in our previous study, the peaks of
M

the 11 chemical markers had satisfactory resolution (Fig.4). The HPLC fingerprints of
ED

B. officinalis from 12 different producing areas were generated using professional

software (Fig.4A), and then the mean chromatographic fingerprint was obtained by
analyzing the 12 samples (Fig.4B). A total of 28 characteristic common peaks were
PT

found in the chromatogram, which were identified or tentatively identified as 15

flavonoids (F2, F4–F11, F14, F16, F18, F20, F22, 23), ten phenylethanoid glycosides
E

(P2, P7, P8, P10–P12, P14, P16, P17, P19), one alkaloid (A6) and two other
CC

constituents (C3, C5). Linarin is one of the most important active constituents of B.
officinalis and, since it is present at relatively high levels, F16 was chosen as the
A

reference peak. The RRT and RPA of each characteristic peak were calculated with
respect to the reference peak. The average values for the 12 B. officinalis samples are
listed in Table S1. The RSDs of the RRT and RPA were in the range 0.01–0.17% and
15.43–35.10%, respectively, demonstrating that although the chemical constituents of
B. officinalis collected from different areas were similar, there were distinct differences
in content.
When the similarity between the chromatographs of the 12 samples of B.
officinalis and the mean chromatographic fingerprints were calculated, the similarity
values were all > 0.900 (Table 1 and Table S2), also indicating that there is little
difference in the chemical composition of B. officinalis grown in different producing
areas.
3.4 Quantitative determination of 11 components in B. officinalis

T
A method for the simultaneous determination of 11 components of B. officinalis

IP
has been established and was successfully employed to analyze 12 samples. From the

R
results summarized in Table 3, it can be seen that linarin (flavonoid) and acteoside

SC
(phenylethanoid) are two major constituents of B. officinalis. The amount of linarin in
all samples was greater than that stipulated in the 2015 edition of the Chinese

U
Pharmacopeia (0.5%, 5 mg/g), and S2, S3, S6, S7, S8 and S12 were also present in
N
relatively large amounts (> 16 mg/g). The amount of acteoside was in the range 20–35
A
mg/g in all samples; samples collected from Tibet had the highest amount (42.36
mg/g) and samples collected from Anhui had the lowest amount (21.65 mg/g). Linarin
M

has, to date, been the only marker compound stipulated in the Chinese Pharmacopoeia
ED

for the quality control of B. officinalis, despite the fact that the amount of acteoside is
approximately 2-fold greater than that of linarin. Acteoside has been shown to have
remarkable activity on the nervous system and immune system, especially in senile
PT

diseases (Alzheimer's disease) and autoimmune disease (slow nephritis), where an

obvious therapeutic effect has been observed [68-70]. It is thus critical to
E

simultaneously analyze linarin and acteoside in order to guarantee the quality of B.

CC

officinalis flowers. N1,N5, N10- (E)-tri-p-coumaroylspermidine (alkaloid) and crocin Ⅲ

(carotenoid) are two other typical components. The sample from Sichuan contained
A

the highest level of N1,N5,N10-(E)-tri-p- coumaroylspermidine (2.33 mg/g) and that

from Inner Mongolia contained the largest amount of crocin Ⅲ (1.95 mg/g).
The total amount of the 11 constituents varies among the different producing
areas (Fig.5). The total amount in samples from Guangxi (S12), Guizhou (S2),
Sichuan (S3) and Tibet (S4) is the largest (> 65 mg/g) while the samples from Anhui
(S1) and Inner Mongolia (S10) have the lowest levels. These differences may arise
because of different ecological environments and processing methods.

3.5 Total phenolic content and evaluation of antioxidant activities

TPC is widely used to assess antioxidant potential. TPCs of samples from different
production areas are shown in Table.4. In the present study, the highest TPC was found
in samples from Guizhou Province (39.83 ± 0.35 mg GAE/g), followed by samples

T
from Guangxi Province. The sample from Shaanxi Province had the lowest TPC (33.26

IP
± 0.29 mg GAE/g). The TPCs of our samples were higher than those of a methanolic
extract (28.82 ± 0.06 mg GAE/g) described in another report [20]. These differences

R
can likely be attributed to different extraction conditions.

SC
The general trend is for antioxidant activity to parallel TPC (Table 4). Correlation
analysis showed a significant correlation between the DPPH, SOSA, FRAP and

U
Fe2+-chelating assays and TPC. The FRA showed the highest correlation (R2 = 0.863)
N
and the SOSA showed the poorest correlation (R2 = 0.607). These results are in
A
agreement with those described in an earlier report, in which plant material with a
M

higher TPC showed stronger antioxidant activities. Taking into account all of the
antioxidant test results, the methanol extract of B. officinalis was found to have good
ED

antioxidant activity and to be a potential natural antioxidant. The samples from

Guizhou, Sichuan and Guangxi Province showed the greatest antioxidant capacity.
PT

4. Conclusion
This study provides chemical profiles of methanol extract of B. officinalis flowers
E

and characterizes a total of 54 chemical compounds, including 23 flavonoids, 19

CC

phenylethanoid glycosides, 7 alkaloids and 5 other constituents of the herb. Among

them, 33 compounds, comprising 14 flavonoids, 11 phenylethanoid glycosides, 7
A

alkaloids, 1 organic acid and 2 carotenoids, were found firstly in B. officinalis.

An accurate and reliable HPLC fingerprint method was developed to evaluate the
quality of B. officinalis. For the fingerprint analysis, 28 characteristic fingerprint peaks
were used to assess the similarities among 12 samples collected from different areas.
These showed good similarity between the samples. The amount of 11 constituents in
the 12 samples was also determined. Samples from Guangxi, Guizhou, Sichuan and
Tibet were found to have relatively high amounts compared with samples from other
areas. This is the first study to combine HPLC fingerprint analysis with the
simultaneous determination of 11 bioactive compounds for quality evaluation of B.
officinalis.
The TPC and antioxidant capacities of B. officinalis flowers from 12 different
habitats were measured using DPPH, SOSA, FRA and Fe2+-chelating assays. B.

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officinalis was found to have good antioxidant capacity and to be a potential natural

IP
antioxidant. The highest antioxidant capacities were found in samples from Guizhou,

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Sichuan and Guangxi Province. Our results provide valuable information for further

SC
understanding and exploiting the herb.

Acknowledgements
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The authors would like to thank the financial support of the National Natural Science
A
Foundation of China (Grant No. 81503220), the Natural Science Foundation of Jiangsu
province (Grant No. BK20150706) and the Fundamental Research Funds for the
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Central Universities (Grant No. ZJ15028).

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T
R IP
SC
U
N
A
Fig.1. The total ion chromatograms of authentic standards and methanol extract of Buddleja
M

officinalis flowers. (A) UV chromatogram of the 11 authentic standards at 330 nm; (B) UV

chromatogram of methanol extract at 330 nm; B(n) and B(p), TIC chromatograms of methanol
ED

extract in negative and positive ion mode respectively; The peak numbers are the same as those of

the compounds in Table 2 and Fig. 2.

E PT
CC
A
 T
R IP
SC
Fig.2. Chemical structures of compounds identified in the extracts of Buddleja Officinalis Maxim.
F1-F23, flavonoids; P1-P18, phenylethanoid glycosides; A1-A7, alkaloids; C1-C5, other
compounds.
U
N
A
M
ED
E PT
CC
A
 T
R IP
SC
U
N
A
M
ED
E PT

Fig.3. MS/MS spectra of the electrospray ionization-generated [M+ H]+ precursor ions of the
CC

alkaloids (A1, A2, A6, A7) and [M+ Na]+ precursor ions of crocins (C3-C5) in positive ion mode.
A
 T
R IP
SC
U
N
A
M

Fig.4. HPLC chromatographic fingerprints of 12 B. officinalis samples (A) and mean

chromatographic fingerprint (B).

ED
E PT
CC
A

Fig.5. The total content of 11 compounds in B. officinalis from different habitats

Table 1 Source information of 12 batches samples and their calculated similarity values

Collection site Collect Similarity Collection site Collect Similarity

No.* No.*
(Province) date (Province) date

S1 Anhui 2016.01 0.955 S7 Shanxi 2016.03 0.998

T
S2 Guizhou 2016.01 0.998 S8 Jiangsu 2016.03 0.996

IP
S3 Sichuan 2016.10 0.999 S9 Hunan 2016.03 0.999

S4 Tibet 2016.03 0.969 S10 Inner Mongolia 2016.03 0.998

R
S5 Yunnan 2016.03 0.999 S11 Gansu 2016.03 1

SC
S6 Shaanxi 2016.03 0.995 S12 Guangxi 2016.03 0.993

* All samples were dried in the sun

U
N
A
M
ED
E PT
CC
A
 R I
SC
Table 2 Characterization of chemical constituents of B. officinalis by HPLC–DAD-Q-TOF-MS/MS.

U
Quasi-molecular Quasi-molecular
Molecular

N
tR (n)[M−H]¯/ (p) [M+ Molecular
No UV(nm) formula MS/MS fragments (n) MS/MS fragments (p) Proposed compound
(time) [M+Cl/COOH]¯ H/Na/NH4]+ formula
generator

A
(Error, ppm) (Error, ppm)
F1* 18.014 268, 322 nd 623.1253 (-0.74) C27H26O17 622.1170 nd 447, 271 Apigenin 7,4'-di-O-glucuronide

M
F2* 18.665 268, 318 621.1106 (-1.28) 623.1249 (-1.32) C27H26O18 622.1170 269 447, 271 Clerodendrin
F3* 21.643 nd 463.0892 (-1.18) 465.1015 (2.56) C21H20O12 464.0955 301, 229, 149 303, 285, 273, 243, 177 Quercetin 7-O-glucoside

F4a 26.429
254, 266
(sh), 348
ED
593.1518 (-1.32) 595.1668 (-1.28) C27H30O15 594.1585 285, 259 449, 287, 235 Luteolin 7-O-rutinoside

254, 266
F5a 28.513 447.0927 (1,25) 449.1089 (-2.26) C21H20O11 448.1006 285, 255 287, 269, 257, 241 Luteolin 7-O-glucoside
(sh), 348
PT

242, 266
F6* 29.086 461.0722 (1.23) 463.0865 (1.77) C21H18O12 462.0798 357, 285, 243, 133 287, 269, 241 Luteolin 7-O-glucuronide
(sh), 342
E

234, 266
F7 31.274 613.1327 (1.04) 579.1718 (-2.00) C27H30O14 578.1636 431, 269 433, 271, 129 Isorhoifolin
334
CC

Apigenin 7-O-α-L-rhamnopyranosyl-
F8* 32.238 238, 328 591.1342 (2.34) 593.1497 (0.98) C27H28O15 592.1428 269 575, 447, 271
(1→2)-β-D-glucuronide
F9* 32.759 240, 328 607.1675 (-0.84) 609.1883 (-2.26) C28H32O15 608.1741 299, 284 463, 301, 286, 258 Diosmetin 7-O-rutinoside
A

238, 268,
F10 33.332 431.0977 (2.04) 433.1137 (-1.97) C21H20O10 432.1056 269, 268 271, 243 Apigenin 7-O-glucoside
328
236 (sh),
F11a 34.140 445.0785 (-1.54) 447.0934 (-2.64) C21H18O11 446.0849 269 271 Apigenin 7-O-glucuronide
266, 336

F12* 34.244 nd 533.0943 (-0.28) 535.1093 (-2.31) C24H22O14 534.1010 489, 285 287 Kaempferol 3-O-(6''-malonylglucoside)

F13* 34.687 234, 328 445.0771 (1.57) 447.0926 (-1.09) C21H18O11 446.0849 269 271 Apigenin 4'-O-glucuronide
 R I
SC
232, 268,
F14a* 37.318 773.2056 (1.36) 739.2459 (-1.63) C34H42O18 738.2371 283 593, 447, 395, 285 Neobudofficide

U
332
F15* 38.204 240,322 nd 519.1128 (-1.31) C24H22O13 518.1060 nd 433, 271 Apigenin 7-(6''-malonylglucoside)

N
230, 268,
F16a 40.054 627.1481 (-0.03) 593.1867 (-0.93) C28H32O14 592.1792 283 447, 133 Linarin

A
332
F17* 40.887 240, 268, 605.1533 (-3.01) 607.1662 (-0.33) C28H30O15 606.1585 283, 268 461, 395, 285 Acacetin7-O-α-L-rhamnopyranosyl-

M
330 (1→2)-β-D-glucuronide or its isomer
5-[[6-O-(6-deoxy-α-L-mannopyranosyl)-β-
240, 268,
F18* 41.044 ED nd 593.1868 (-0.57) C28H32O14 592.1792 nd 447, 285, 133 D-galactopyranosyl]oxy]-7-hydroxy-2-(4-
332
methoxyphenyl)-4H-1-Benzopyran-4-one
F19* 43.962 nd 481.0892 (2.51) 447.1296 (-1.58) C22H22O10 446.1213 283, 268 285, 270, 242 Acacetin 7-O-glucoside
242, 268,
F20 44.066 285.0396 (3.16) 287.0553 (-1.04) C15H10O6 286.0477 257, 217, 175, 151, 133 269, 241, 179, 153, 135 Luteolin
PT
338
F21* 44.665 240, 320 459.0932 (0.96) 461.1087 (-1.63) C22H20O11 460.1006 283, 268 285, 270, 257, 242, 133 Acacetin 7-O-glucuronide
240, 265,
F22a
E

47.114 269.0454 (0.68) 271.0590 (4.45) C15H10O5 270.0528 241, 227, 151, 117 243, 229, 153, 119 Apigenin
336
CC

270, 257, 242, 177, 153,

F23a 50.137 268, 328 283.0614 (-0.53) 285.0757 (-0.06) C16H12O5 284.0685 268, 240, 211, 151 Acacetin
133
P1 11.892 222, 280 335.0905 (-0.43) 323.1099 (1.72) C14H20O7 300.1209 179, 137, 119 163, 121 Salidroside
P2 12.390 223,328 487.1434 (4.55) 511.1433 (-4.58) C21H28O13 488.1530 179, 161, 135 365, 163, 151 Cistanoside F
A

4-O-(3,4-Dihydroxy-Z-cinnamoyl)-3-
P3* 12.650 nd 487.1434 (4.89) 511.1411 (0.85) C21H28O13 488.1530 179, 161, 135 365, 163, 151
O-Lrhamnopyranosyl-D-glucose

P4* 12.934 232,325 505.1570 (-0.29) 507.1723 (-2.31) C21H30O14 506.1636 341, 179, 161 163, 145 Hebitol Ⅱ

224, 264,
P5 13.689 417.1400 (1.44) 395.1313 (1.23) C17H24O9 372.1420 nd 233, 232, 218, 185 Syringin
326
 R I
SC
P6* 14.109 nd 341.0872 (0.34) nd C15H18O9 342.0951 179, 161, 135, 133 nd β-4-caffeoylglucose

U
236, 290
P7* 14.549 353.0875 (1.03) 355.1020 (0.62) C16H18O9 354.0951 191, 161 163, 145, 135, 117 3-Caffeoylquinic acid
(sh), 326

N
519, 397, 355, 295,
P8* 15.542 240, 326 555.1476 (2.56) 521.1880 (-3.33) C22H32O14 520.1792 321, 195, 177, 145, 117 Globularitol

A
235, 193, 175
445, 263, 221, 179, 2-phenylethyl 6-O-β-D-glucopyranosyl-β-
P9* 16.268 238, 328 481.1475 (1.77) 469.1683 (-0.30) C20H30O11 446.1788 307, 163

M
161, 131 D-Glucopyranoside
P10a 17.128 220, 328 785.2514 (0.07) 804.2910 (1.47) C35H46O20 786.2582 623, 461, 459 679, 471, 325, 163 Echinacoside

ED 610, 487, 477, 461,

P11* 17.809 234, 329 639.1941 (-0.82) 663.1910 (-1.47) C29H36O16 640.2003 325, 309, 181, 163 Isocampneoside II
285, 179, 161
610, 487, 477, 461,
P12 18.330 234, 328 639.1930 (0.64) 663.1916 (-3.02) C29H36O16 640.2003 325, 309, 181, 163 Campneoside II
285, 179, 161
PT
P13 19.264 nd 451.1376 (2.80) 439.1587 (-2.60) C19H28O10 416.1682 307 Phenethyl-β-primeveroside
623, 593, 461, 447,
P14* 23.072 244, 326 755.2424 (-2.63) 774.2825 (-0.3) C34H44O19 756.2477 325, 163 Hebeoside
179, 161, 133, 113
E

P15* 26.953 234, 330 623.1982 (0.27) 642.2397 (-0.14) C29H36O15 624.2054 461, 315, 179, 161, 135 463, 325, 287, 163 Forsythoside A
CC

P16a 27.497 220, 330 623.1969 (2.45) 642.2398 (-0.19) C29H36O15 624.2054 477, 461, 315, 179, 161 325, 163 Acteoside
P17* 28.200 235, 328 623.1986 (-0.25) 647.1944 (0.92) C29H36O15 624.2054 461, 179, 161 501, 325, 163 Cis-acteoside

P18a 30.441 242, 326 623.1990 (-1.15) 647.1944 (0.92) C29H36O15 624.2054 487, 461, 179, 161 501, 325, 163 Isoacteoside
A

799, 785, 767, 623, 501, 339, 325, 181, 177, Globusintenoside or
P19* 36.136 220, 330 961.2982 (-0.69) 980.3406 (-0.78) C45H54O23 962.3056
487, 179, 161 163 Globusintenoside isomer
421, 292, 275, 247, 218, 204, N1, N5-di-[(E)-p-coumaroyl]-
A1* 26.715 244, 298 nd 438.2392 (-0.43) C25H31N3O4 437.2315 nd
147 spermidine or its isomers
454, 436, 421, 308, 292, 275, N1-(E)-caffeoyl-N5, N10-di-p-
A2* 43.962 246, 296 598.2556 (0.73) 600.2711 (-0.64) C34H37N3O7 599.2632 nd
220, 204, 163, 147 (E)-coumaroyl spermidine
 R I
SC
438, 420, 318, 292, 275, N1, N5, N10-(Z)-tri-p-
A3* 45.212 nd nd 584.2758 (-0.50) C34H37N3O6 583.2682 nd

U
218, 204, 172, 147 coumaroylspermidine
438, 420, 318, 292, 275, N1(Z)-N5-(Z)-N10-(E)-tri-p-couma

N
A4* 45.577 246, 316 nd 584.2741 (0.53) C34H37N3O6 583.2682 nd
218, 204, 172, 147 roylspermidine
N1(E)-N5-(Z)-N10-(E)-tri-p-couma

A
438, 420, 318, 292, 275,
A5* 45.733 252, 320 618.2368 (1.06) 584.2761 (-1.07) C34H37N3O6 583.2682 nd
218, 204, 172, 147 roylspermidine

M
234, 294, 438, 420, 318, 292, 275, N1, N5, N10-(E)-tri-p-
A6a* 46.098 618.2389 (-1.47) 584.2756 (0.07) C34H37N3O6 583.2682 582, 462, 342, 316, 145
306(sh) 218, 204, 172, 147 coumaroylspermidine
246, 298, 468, 450, 438, 420, 322, 305, 292,
A7* 46.332
310
ED nd 614.2855 (0.35) C35H39N3O7 613.2788 nd
275, 234, 218, 204, 177, 147
Keayanidine A

C1 9.964 220, 280 375.1284 (2.32) 394.1712 (-0.47) C16H24O10 376.1369 195, 183, 151 197, 179, 165, 123 6-methoxylcatalpol
C2* 15.669 nd 387.1664 (-0.44) 411.1636 (-2.76) C18H28O9 388.1733 207 249 Tuberonic acid glucose
PT
242, 442, 907, 675, 583, 365, 351,
C3* 34.921 975.3712 (0.12) 999.3659 (1.54) C44H64O24 976.3788 nd Crocin I
464(sh) 347
240, 442, 745, 675, 583, 513, 421,
C4*
E

38.282 859.2948 (1.79) 837.3180 (-3.3) C38H54O19 814.3259 nd Crocin II

464 (sh) 365, 351, 347, 259
CC

258, 434, 583, 365, 351, 347, 307,

C5a 46.489 687.2425 (0.65) 675.2625 (0.13) C32H44O14 652.2731 nd Crocin III
458 (sh) 259
sh, shoulder peak; nd, not detected;
aAuthentic standards; * detected in Buddleja officinalis for the first time.
A
 R I
SC
U
Table 3 The content of 11 compounds in B. officinalis from different habitats (Mean±SD, mg/g, n=3)

N
Contents (mg/g) a
Samples

A
P10 F4 P16 F5 F11 F14 F16 A6 C5 F22 F23
S1 3.96±0.11 1.18±0.03 21.65±0.45 0.73±0.02 1.07±0.01 2.20±0.10 13.68±0.35 1.29±0.06 0.39±0.01 0.56±0.00 0.21±0.00

M
S2 5.92±0.22 1.16±0.02 34.28±0.44 0.72±0.01 1.13±0.05 4.07±0.13 17.23±0.49 2.21±0.09 0.69±0.02 0.44±0.01 0.21±0.00
S3 6.83±0.15 1.34±0.02 29.75±0.09 0.54±0.01 1.24±0.01 3.27±0.03 17.69±0.10 2.33±0.03 1.54±0.02 0.81±0.00 0.24±0.00
S4
S5
6.26±0.03
3.81±0.06
ED
1.23±0.02
0.89±0.02
42.36±0.11
26.79±0.08
0.75±0.01
0.62±0.02
1.16±0.01
1.08±0.03
2.09±0.05
2.33±0.03
12.17±0.09
14.03±0.15
1.28±0.00
1.37±0.01
1.13±0.01
1.32±0.01
0.46±0.01
0.59±0.01
0.11±0.00
0.25±0.00
S6 4.43±0.16 1.05±0.02 27.27±0.43 0.61±0.03 1.18±0.01 2.82±0.12 19.11±0.35 1.89±0.07 1.15±0.02 0.56±0.02 0.26±0.00
S7 3.86±0.00 1.09±0.02 25.89±0.31 0.79±0.02 1.10±0.03 2.47±0.08 16.18±0.23 1.05±0.03 1.74±0.02 0.69±0.01 0.17±0.00
PT

S8 3.64±0.03 0.91±0.02 27.46±0.29 0.88±0.02 1.02±0.02 2.44±0.05 18.17±0.19 1.49±0.06 1.68±0.01 0.62±0.02 0.22±0.00
S9 8.37±0.08 0.69±0.02 29.59±0.29 0.85±0.03 0.95±0.04 1.50±0.02 13.23±0.49 1.55±0.03 0.48±0.01 0.37±0.01 0.15±0.02
E

S10 4.11±0.08 0.50±0.01 22.30±0.23 0.73±0.01 1.50±0.02 1.27±0.02 12.39±0.14 1.35±0.02 1.95±0.02 0.70±0.00 0.20±0.00
S11 4.28±0.05 0.95±0.03 26.86±0.22 0.60±0.01 1.13±0.05 2.64±0.05 15.26±0.48 1.58±0.02 1.10±0.01 0.49±0.01 0.21±0.01
CC

S12 9.54±0.19 0.82±0.01 34.59±0.48 0.86±0.04 1.17±0.01 1.68±0.04 16.04±0.15 1.88±0.02 0.50±0.00 0.33±0.00 0.14±0.00
A
 Table 4 Antioxidant activities of twelve batches of B. officinalis
Antioxidant activity (IC50)
NO TPC DPPH IC50 SOSA IC50 FRA EC50 Fe2+-chelating IC50
(mg/g) (mg/mL) (ug/mL) (mg/mL) (mg/mL)
S1 33.67±0.82 0.64±0.01 258.82±1.12 1.124±0.016 2.40±0.02
S2 39.83±0.35 0.41±0.01 199.33±2.34 0.726±0.016 1.93±0.01
S3 36.69±0.53 0.44±0.00 184.85±3.01 0.810±0.005 1.97±0.03
S4 35.98±0.48 0.49±0.01 266.19±3.84 0.770±0.001 2.59±0.01
S5 33.52±0.76 0.59±0.00 212.72±2.41 0.935±0.010 2.39±0.01

T
S6 33.26±0.29 0.47±0.01 304.48±1.29 1.001±0.005 3.04±0.02

IP
S7 35.95±1.00 0.46±0.01 278.35±2.98 0.871±0.006 2.60±0.04
S8 34.98±0.46 0.43±0.01 215.19±3.70 0.887±0.001 2.54±0.02
S9 37.71±0.81 0.52±0.01 232.71±0.34 0.809±0.003 2.61±0.03

R
S10 33.48±0.49 0.58±0.01 302.74±5.63 0.972±0.012 2.58±0.01

SC
S11 37.69±0.36 0.48±0.00 227.74±5.19 0.834±0.008 2.08±0.01
S12 38.60±0.44 0.44±0.01 208.11±4.31 0.733±0.004 1.84±0.01

U
N
A
M
ED
E PT
CC
A

37