Identification and Quantification of Phytochemical Composition and

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Identification and quantification of phytochemical composition, anti-inflammatory, cellular antioxidant and radical scavenging activities of twelve Plantago species Qin Zhou, Weiying Lu, Yuge Niu, Jie Liu, Xiaowei Zhang, Boyan Gao, Casimir C. Akoh, Haiming Shi, and Liangli (Lucy) Yu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf401191q • Publication Date (Web): 14 Jun 2013 Downloaded from http://pubs.acs.org on June 16, 2013

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Journal of Agricultural and Food Chemistry

Identification and quantification of phytochemical composition, anti-inflammatory, cellular antioxidant and radical scavenging activities of twelve Plantago species Qin Zhou,‖,† Weiying Lu,‖,† Yuge Niu,† Jie Liu,† Xiaowei Zhang,† Boyan Gao † Casimir C. Akoh,‡ Haiming Shi,*,† and Liangli (Lucy) Yu†,§ †

Institute of Food and Nutraceutical Science, Key Lab of Urban Agriculture (South),

Bor S. Luh Food Safety Research Center, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China; ‡Department of Food Science & Technology, University of Georgia, Athens, GA 30602-2610, United States; §Department of Nutrition and Food Science, University of Maryland, College Park, MD 20742, United States

‖These

two authors contributed equally to this work.

*Corresponding author Telephone: 86-021-34207961. Fax: 86-021-34204107. E-mail: [email protected]

ACS Paragon Plus Environment


ABSTRACT Twenty-eight seed samples of 12 Plantago species were investigated for their chemical compositions, and anti-inflammatory, cellular antioxidant and radical scavenging properties. A new UPLC-UV procedure was developed and applied to quantify acteoside and geniposidic acid, the characteristic constituents of genus Plantago. The amounts of acteoside and geniposidic acid ranged from 0.07 to 15.96 and 0.05 to 10.04 mg/g in the tested samples, respectively. Furthermore, 26 compounds were tentatively identified by UPLC/Q-TOF-MS analysis. The Plantago samples significantly differed in their phytochemical compositions. The extracts of Plantago seeds also showed inhibitory effects on LPS induced IL-1β, IL-6 and COX-2 mRNA expression in RAW 264.7 mouse macrophage cells. Additionally, significant variations were observed among different samples on cellular antioxidant activities in HepG2 cells, as well as DPPH and hydroxyl radicals scavenging capacities. The results from this study may be used to promote the use of genus Plantago in improving human health. KEYWORDS: Plantago, UPLC, UPLC/Q-TOF-MS, Acteoside, Geniposidic acid, Anti-inflammation, Cellular antioxidant, Radical scavenging.


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INTRODUCTION About 275 species of the genus Plantago L. (Plantaginaceae) are found all over the

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world and 20 species are widely distributed in China.1, 2 The seeds and whole plants of

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P. major, P. asiatica and P. depressa have been used in foods and traditional

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medicine since ancient times.2 Genus Plantago have been shown to have

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anti-inflammatory, immunomodulatory, anticancer and antioxidant activities.3-5 The

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fresh leaves of P. asiatica, P. major and P. depressa were used to be consumed in

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fresh salads and soups.2 The seeds of several species such as P. asiatica and P. major

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could also be used as snack or in cakes and breads.2, 3

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To date, only a few Plantago species have been investigated for their chemical

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constituents and biological activities.3, 4, 6 The majority of species including P.

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arachnoidea, P. jehohlensis and P. himalaica have not been studied. It was also

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noticed that P. depressa subsp., a subspecies of P. depressa, has not been investigated.

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There were several phytological researches on P. minuta,7 phytochemical analysis of

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P. cornuti6 and P. camtschatica,8 but the health properties of these three species were

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not reported. There was no report on cellular antioxidant activity for the seeds or

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aerial parts of several representative Plantago species, although free radical

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scavenging capacities of their aerial parts were studied.1, 3, 5 In addition, the aerial

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parts of P. lanceolata were found to significantly inhibit COX-1, COX-2 and 12-LOX

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mRNA expressions,3, 9 but Plantago seeds have not been investigated for possible

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anti-inflammatory activities.

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The aim of this study was to analyze the chemical compositions, anti-inflammatory and antioxidant properties of 28 seed samples belonging to 12 Plantago species



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grown in China. The results from this study may be used to promote better use of

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genus Plantago for improving human health.

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MATERIALS AND METHODS

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Plant materials. The seeds of 12 Plantago species were collected and identified by

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Dr. Linke Yin (Xinjiang Institute of Ecology and Geography, Chinese Academy of

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Sciences) and Dr. Haibo Yin (Liaoning University of Traditional Chinese Medicine)

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in 2011 and 2012 from different locations in China. The detailed information of 28

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samples is provided in Table 1.

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Reagents. Fluorescein, 6-hydroxy-2, 5, 7, 8-tetramethylchroman-2-carboxylic

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acid (trolox), iron (III) chloride, 2, 2-diphenyl-1-picryhydrazyl radical (DPPH•),

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dimethyl sulfoxide (DMSO), insulin, hydrocortisone and 2', 7'-dichlorofluorescin

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diacetate (DCFH-DA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). 2,

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2'-Azobis (2-methylpropionamidine) dihydrochloride (AAPH) was obtained from

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J&K Scientific (Beijing, China). Williams’ Medium E (WME), Hanks’ balanced salt

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solution (HBSS), L-glutamine, antibiotic-antimycoitc, gentamicin, fetal bovine serum

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(FBS), TRIzol reagent, DMEM and TrypLETM Express (1X, no Phenol Red) were

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obtained from Gibco (Life Technologies, Carlsbad, CA, USA). Lipolysaccharide

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(LPS) from E. coli 0111: B4 was obtained from Millipore (Millipore, Billerica, MA,

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USA). Human hepatocellular carcinoma (HepG2) and RAW 264.7 mouse

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macrophage cells were from the Chinese Academy of Sciences (Shanghai, China).

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Thirty percent hydrogen peroxide (H2O2), analytical grade methanol, acetone, sodium

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dihydrogen phosphate and disodium hydrogen phosphate were purchased from

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Sinopharm (Shanghai, China). HPLC-grade formic acid, methanol and acetonitrile

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were purchased from Merck (Darmstadt, Germany). Reference compounds,


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geniposidic acid (GA) and acteoside (AC), were purchased from Shanghai R&D

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Centre for Standardization of Chinese Medicines and TAUTO BIOTECH (Shanghai,

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China), respectively. Ultrapure water was prepared using a Millipore ultra-Genetic

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polishing system with < 5 ppb TOC and resistivity of 18.2 mΩ (Millipore, Billerica,

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MA, USA).

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Sample preparation. Air-dried seed samples were powdered to a particle size of

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40 mesh. One gram of each sample was extracted with 10 mL of 60% methanol (v/v)

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in an ultrasonic bath for 30 min at ambient temperature, and then centrifuged at 3082

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g for 10 min. The supernatant was collected and subjected to UPLC and

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UPLC/Q-TOF-MS analyses. Another set of 28 supernatants were prepared and the

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solvents were removed using a nitrogen evaporator and a freeze drier under reduced

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pressure. The sample extracts were stored at -20 °C until bioactivity evaluations.

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Method development and validation. A UPLC method was developed for

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quantitative analysis and validated for linearity, precision, accuracy, repeatability and

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stability as follows. In brief, the standard compounds, GA and AC, were dissolved in

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60% methanol to obtain the stock solution at a concentration of 1 mg/mL. A set of

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standard testing solutions were prepared by appropriate dilution of the stock solution

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with the mixture of 0.5% formic acid and methanol (9:1, v/v), containing 0.98-1000

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µg/mL of GA and AC. The testing standard solutions were analyzed in triplicates.

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Calibration curves were constructed by plotting the peak areas versus concentrations

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of analytes. The limit of detection (LOD) and limit of quantification (LOQ) for the

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standard compounds were determined as 3- and 10-fold of the signal-to-noise (S/N)

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ratio, respectively. The intra-day precision was calculated using data collected at five

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different time points within a day, while inter-day precision was examined over six



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days at three concentrations (7.81, 62.50 and 500.00 µg/mL). Method repeatability

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was estimated by analysis of six independently prepared samples. For the stability test,

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the sample solution was analyzed over a period of 2, 4, 6, 8, 12, 24, 48 and 96 h,

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respectively. The accuracy of the analytical method was evaluated using the recovery

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test. Three concentration levels of GA (0.20, 0.25 and 0.30 mg) and AC (0.40, 0.50

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and 0.60 mg) were spiked in 0.1 g of PAL-3 sample with known concentrations of the

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target compounds, respectively. The percent recoveries were calculated by the

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difference between the spiked and non-spiked sample using the calibration curves. All

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the testing solutions were prepared using sample PAL-3. The relative standard

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deviation (RSDs) of peak area was used for method validation.

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Quantification of geniposidic acid and acteoside by UPLC analysis. The sample

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solution was separated using a UPLC system consisted of an Acquity UPLC H-Class

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system (Waters, Milford, MA, USA) and a Waters Acquity UPLC BEH C18 column

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(100 mm × 2.1 mm i. d.; 1.7 µm) kept at 40 °C. The sample extracts were diluted

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ten-fold with the mixture of 0.5% formic acid and methanol (9:1, v/v) (10 mg sample

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powder equivalents/mL). A binary mobile phase consisting of 0.5% formic acid (A)

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and acetonitrile (B) was used. Gradient elution was performed as follows: starting

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with 5% B, reaching 10% B in 1 min, 15% in 1 min, 20% in 1 min, 29% in 1 min, 35%

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in 1 min, and 40% in 1 min. The flow rate was 0.5 mL/min and the injection volume

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was 5 µL. GA and AC in Plantago samples were detected at 254 nm, and quantified

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using external standards.

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Identification of chemical compositions by UPLC/Q-TOF-MS analysis.

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Plantago seed samples were analyzed for their chemical compositions with a Waters

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Xevo G2 Q-TOF mass spectrometer (Milford, MA, USA). UPLC was performed

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using the same conditions for quantification except that the injection volume was 2


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µL. Mass spectra were obtained with electrospray ionization (ESI) with a negative ion

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mode. A MSE method (a scan model to get both parent and daughter ion information

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in one injection using tandem mass spectrometry) was used and MS conditions were

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as follows: capillary voltages, 3.0 kV; sampling cone voltages, 40 V; source

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temperature, 120 °C; desolvation temperature, 450 °C; cone gas flow, 50.0 L/h;

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desolvation gas flow, 600.0 L/h; scan rage of MS1 and MS2 were from 100 to 1500

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m/z for 6 min; and the ramp collision energy was from 25 V to 35 V. Data were

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collected and analyzed with the Waters MassLynx v4.1 software.

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Inhibition on IL-1β, IL-6 and COX-2 mRNA expression. Nineteen Plantago

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sample extracts, and pure GA, AC and their mixture were tested and compared under

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the same conditions. The sample extracts were re-dissolved in DMSO (100 mg/mL),

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and diluted to 0.1 mg/mL with culture medium. RAW 264.7 mouse macrophages

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were cultured in 6-well plates and reached the confluence of 80%. The cells were

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pretreated with culture medium containing samples (0.1 mg/mL) for 24 h. LPS was

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added at a concentration of 10 ng/mL and cells were incubated in DMEM with 10%

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FBS and 1% antibiotic-antimycotic at 37 ºC under 5% CO2 for another 4 h. After

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induction, culture medium was removed and cells were collected for total RNA

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isolation and real-time PCR.10

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RNA isolation and real-time PCR were performed according to a reported

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protocol.11 Cells were washed once with phosphate buffer solution (PBS), and TRIzol

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reagent was added for total RNA isolation. IScriptTM advanced cDNA synthesis kit

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(Bio-Rad Laboratories, Hercules, CA, USA) was used to reverse transcribe

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complementary DNA. Real-time PCR was performed on an ABI 7900HT Fast

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Real-Time PCR system (Applied Biosystems, Hercules, CA, USA) using AB Power

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SYBR Green PCR Master Mix (Applied Biosystems, Hercules, CA, USA). Primers



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used in this study were as follows: IL-1β (Forward:

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5'-GTTGACGGACCCCAAAAGAT-3', Reverse:

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5'-CCTCATCCTGGAAGGTCCAC-3'); IL-6 (Forward:

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5'-CACGGCCTTCCCTACTTCAC-3', Reverse:

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5'-TGCAAGTGCATCATCGTTGT-3'); COX-2 (Forward:

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5'-GGGAGTCTGGAACATTGTGAA-3', Reverse:

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5'-GCACGTTGATTGTAGGTGGACTGT-3'). The mRNA amounts were normalized

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to an internal control, GAPDH mRNA (Forward:

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5'-AGGTGGTCTCCTCTGACTTC-3', Reverse:

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5'-TACCAGGAAATGAGCTTGAC-3'). The following amplification parameters

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were used for PCR: 50 °C for 2 min, 95 °C for 10 min, and 46 cycles of amplification

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at 95 °C for 15 s and 60 °C for 1 min.

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Cellular antioxidant activity analysis. The cellular antioxidant activity (CAA)

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values were examined following a previously reported method with slight

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modification.12 The growth medium contained Williams’ Medium E (WME), 5% FBS,

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10 mM Hepes at pH 7.4, 2 mM L-glutamine, 5 µg/mL insulin, 0.05 µg/mL

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hydrocortisone, 50 units/mL penicillin, 50 µg/mL streptomycin and 100 µg/mL

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gentamicin. Treatment medium contained WME, HBSS (no phenol red) and 10 mM

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Hepes in pH 7.4. The sample extracts were re-dissolved in DMSO (100 mg/mL) and

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then diluted to 50 µg/mL with treatment medium. HepG2 cells were seeded in the

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growth medium at a density of 6 × 104 in 100 µL per well on a 96-well bottom read

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microplate. Growth medium was added to the outside wells of the plate. After 24 h

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culturing, the growth medium was discarded and the wells were washed with PBS.

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100 µL of standard solution or sample extracts solution plus 100 µL of 25 µM

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DCFH-DA in treatment medium were applied to the cells for 1 h. Then, 600 µM


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AAPH was added to the cells in 100 µL of HBSS. The fluorescence of the reaction

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mixture was measured using a Multimode Reader (TECAN, Männedorf, Switzerland)

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every minute for 1 h at 37 °C with an emission wavelength at 538 nm and an

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excitation at 485 nm. Each plate had three blank and three control wells. Blank wells

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contained cells treated with DCFH-DA. Control wells contained cells treated with

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DCFH-DA and AAPH. CAA value was expressed as micromoles quercetin

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equivalents (QE) per gram Plantago seed powder.

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Relative DPPH radical scavenging capacity (RDSC). DPPH radical scavenging

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capacity was measured following a previously reported protocol.13 Briefly, 100 µL of

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sample extracts (diluted 600-fold by 60% methanol from the original extract

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solutions), GA and AC solutions, trolox standard, or solvent was added into 100 µL of

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200 µM DPPH• solution to induce antioxidant radical reaction. The absorbance of the

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reaction was determined at 515 nm every minute during 1.5 h reaction. RDSC values

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were calculated using areas under the curve relative to trolox standard and expressed

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as micromoles of trolox equivalents (TE) per gram of Plantago seed powder. Reaction

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was conducted in triplicate.

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Hydroxyl radical scavenging capacity (HOSC). The HOSC values were

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estimated using a previously reported laboratory protocol.14 Reaction mixture

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contained 30 µL sample extracts, GA and AC solutions, trolox standard solution or

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solvent, 170 µL of 9.28 × 10-8 M fluorescein, 60 µL 3.43 M iron (III) chloride and 40

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µL 0.1990 M H2O2. The fluorescence of the reaction mixture was determined every

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minute for 6 h at ambient temperature, with an emission wavelength at 528 nm and an

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excitation wavelength at 485 nm. Reaction was conducted in triplicate, and HOSC

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values were reported as micromoles of TE per gram of Plantago seed powder.



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Statistical analysis. Data were reported as the mean ± SD for triplicate

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determinations. One-way ANOVA and Tukey’s test were employed for different

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independent samples. Data evaluation was performed with SPSS for Windows

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(version rel. 16.0, SPSS Inc., Chicago, IL, USA). Significant differences were

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declared at P < 0.05. Cluster analysis was carried out by MATLAB (version 7.5

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R2007b, The MathWorks, Natick, MA, USA).

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RESULTS AND DISCUSSION Optimization of UPLC conditions. Different columns (Waters BEH C18, HSS T3,

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BEH Amide; 100 mm × 2.1 mm i.d., 1.7 µm) and mobile phases consisting of

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methanol-water and acetonitrile-water with different concentrations of formic acid in

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water (0.1, 0.3 and 0.5%, v/v) were compared. The BEH C18 column was more

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effective and gave better separation of GA and AC than the other two columns. To

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avoid the tautomeric rearrangements of iridoids,15 the samples were dissolved in a

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mixture of 0.5% formic acid and methanol (9:1, v/v). Furthermore, other

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chromatographic variables, including column temperatures (25, 35 and 40 °C) and

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flow rates (0.4 and 0.5 mL/min), were also evaluated. When the flow rate was 0.5

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mL/min with 40 °C column temperature, the analytes were baseline-separated with

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sharp peak shape. Compared with the traditional HPLC, the UPLC has higher

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sensitivity with a shorter analytical time. Under the present conditions, the target

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compounds were well separated within 6 min (Figure S1 in the Supporting

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Information), much shorter analysis time than the previously reported HPLC

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method.16, 17

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Method validation. The UPLC method for quantitative analysis was validated for linearity, precision, accuracy, repeatability and stability. The calibration curves for


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GA and AC showed excellent linear regression (Table 2). The LOD and LOQ for GA

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was 6.51 and 19.53 ng/mL, and those for AC were 9.77 and 39.06 ng/mL,

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respectively. The established method showed a good reproducibility for the

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quantification of the two analytes with intra- and inter-day variations less than 0.25

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and 1.58%, respectively. The RSDs of peak areas were found to be 3.67% for GA and

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4.59% for AC, suggesting an excellent repeatability. Stability were 0.53 and 4.64%,

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for GA and AC within 96 h at room temperature, respectively. The accuracy of

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analytical method was evaluated at three levels (80, 100 and 120%) for the two

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analytes in three replicates. Spike recoveries of GA and AC ranged from 98.89 to

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101.06%, and the RSDs values were all less than 3.57%, indicating that the improved

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UPLC-UV method was reliable and accurate for analyzing the two bioactive

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compounds in genus Plantago. The procedure was utilized to examine GA and AC

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contents in the 28 Plantago seed samples.

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Sample analysis. Phenylpropanoid glycosides and iridoids are two major chemical

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constituents of the genus Plantago, and can be used as valuable taxonomic markers.6, 8

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As a representative of phenylpropanoid glycoside and iridoid, respectively, acteoside

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(AC) and geniposidic acid (GA) are widely distributed in genus Plantago (Figure S2

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in the Supporting Information).6, 8 Previous studies also showed that they are primary

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contributors for the known bioactivities of Plantago species.16, 17 In this study, the two

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important compounds were quickly quantified using the UPLC-UV method in 6 min.

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AC and GA were detected in all 28 Plantago seed samples, which further supported

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their taxonomic importance (Table 3). The contents of AC and GA in all samples

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ranged from 0.07 to 15.96 and 0.05 to 10.04 mg/g, respectively. The amount of AC in

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P. depressa varied the most (1.49 to 15.96 mg/g). PDW-1 (P. depressa) and PAL-1 (P.

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asiatica) had the greatest contents of AC and GA among all samples, respectively.



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Nearly all samples contained more AC than GA, except of PML-3, PAS-4, PLL-3 and

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PMP-2.

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Among the 28 samples, P. jehohlensis, P. arachnoidea and P. depressa subsp.

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seeds were investigated for the first time, and P. himalaica was found in China for the

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first time. The P. jehohlensis had the greatest amount of AC at 8.77 mg/g, whereas the

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P. himalaica had a low AC concentration of 2.10 mg/g. Several less investigated

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species, P. cornuti, P. camtschatica, P. depressa subsp. and P. himalaica contained

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trace amounts of GA. The contents of GA and AC may provide new evidences for

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chemotaxonomy of Plantago species in China.

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P. depressa and P. asiatica grown in Xinjiang Uygur Autonomous Region might be

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a good source of AC and GA, respectively. In general, the samples grown in Xinjiang

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had greater AC contents than those from other areas. Some previous reports suggested

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that concentrations of GA and AC were highly related to temperature, light and

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atmosphere. Tamura et al. reported that relatively low temperatures could increase the

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contents of AC but reduce iridoid glucosides concentrations.18, 19 Higher CO2 density

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could improve the AC contents.20 Whereas, low nitrogen supply and light intensity

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strongly inhibited the accumulation of AC in leaves.18 Altitudes with different UV

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intensity and temperature also could influence the concentrations of phenylpropanoid

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glycosides.21 These previous reports could partially explain the observations of the

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variation of GA and AC contents for P. depressa and P. asiatica grown in Xinjiang

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Uygur Autonomous Region.

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Identification of chemical compounds in the Plantago seeds. Plantago was

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reported rich in phenylethanoid glycosides, iridoids, flavonoids and phenolic acids.1, 3,

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6, 19, 22

In the present study, the chemical compositions in the 28 Plantago seed


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samples were characterized by UPLC/Q-TOF-MS analysis. The representative total

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ion current chromatograms of six selected Plantago species are shown in Figure S3

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(Supporting Information). Total of 30 compounds were detected in the 60% methanol

248

extract of the 28 Plantago seed samples (Table 4). Twenty-six compounds were

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tentatively identified based on accurate MS data and secondary MS fragmentation

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profiles. As an example, the fragmentation pathway proposed for plantamajoside

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(peak 18) is shown in Figure S4 (Supporting Information). There were still four

252

compounds remained unknown. The retention times, maximum absorption UV

253

wavelength, accurate MS data and occurrence in Plantago seed samples of the

254

detected compounds are summarized in Table 4.

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Nearly all Plantago species showed similar chemical profiles except P. lanceolata.

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Arborescosidic acid (2), forsythoside B (19), lavandulifolioside (26), and two

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unknown compounds (14 and 30) were only detected in P. lanceolata. Alpinoside (11)

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was detected in P. lanceolata seeds for the first time. Forsythoside B (19) was

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previously detected in P. asiatica and P. depressa,23 but was not detected in the seeds

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of these two species in the present study. Interestingly, plantagoguanidinic acid (22), a

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guanidine compound only reported in P. asiatica,24 was detected in 23 of the tested

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seed samples. Compound 20 was deduced to possess similar structure with

263

plantagoguanidinic acid (22) by its molecular formula.

264

Based on the total ion current profiles, the cluster analysis was employed to

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effectively discriminate these species (Figure S5 in the Supporting Information). P.

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lanceolata, P. minuta and P. maritima were clustered and separated with other species.

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P. major, P. asiatica, P. jehohlensis and P. himalaica were in a relatively tight cluster,

268

supporting the observation that P. jehohlensis and P. himalaica showed similar

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chemical profiles to P. major and P. asiatica. In addition, P. arachnoidea, P. depressa,



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P. depressa subsp., P. cornuti and P. camtschatica formed a cluster, providing a new

271

evidence for a close relationship between P. depressa. subsp. and P. depressa. This

272

study revealed the relationships of 12 Chinese Plantago species and provided valuable

273

information for the less studied species.

274

Effects on IL-1β, IL-6 and COX-2 mRNA expression. Chronic inflammation has

275

been recognized as a risk factor for a number of chronic diseases such as autoimmune

276

diseases and atherosclerosis.25 To determine the anti-inflammatory activity for the

277

seeds of the12 Plantago species, IL-1β, IL-6 and COX-2 mRNA expression in

278

LPS-stimulated RAW 264.7 mouse macrophage cells was examined.

279

Nineteen Plantago samples significantly differed on their inhibitory activities of

280

IL-1β, IL-6 and COX-2 mRNA expression. As shown in Figure 1A, all samples

281

greatly inhibited IL-1β mRNA expression except PAL-3 and PMP-1 seed extracts. P.

282

camtschatica seed extract showed the strongest suppression on IL-1β mRNA

283

expression. All samples inhibited IL-6 mRNA expression (Figure 1B), and P.

284

depressa subsp., an unreported species, showed the strongest inhibitory activity.

285

Nearly all samples suppressed COX-2 mRNA expression except those belonging to P.

286

asiatica, P. minuta and P. jehohlensis (Figure 1C). P. depressa subsp. had the

287

strongest inhibitory effect, and P. maritima and P. camtschatica also exhibited

288

significant anti-inflammatory activities. In summary, seeds of Plantago species,

289

especially P. depressa subsp., might be potentially used in anti-inflammatory

290

nutraceuticals and functional foods.

291

Cellular antioxidant activity. The CAA of different samples is shown in Figure 2,

292

indicating significant differences in CAA values of the 28 Plantago seed samples. P.

293

lanceolata exhibited the greatest cellular antioxidant activity. It was remarkable that P.


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himalaica had stronger cellular antioxidant activity than P. maritima and P.

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camtschatica, indicating a potential application of P. himalaica as a dietary source for

296

natural antioxidants. Species collected from different locations also exhibited different

297

cellular antioxidant activities. PLL-1, collected from Xinjiang, showed a greater

298

cellular antioxidant activity than other samples of the same species from different

299

locations. Similar results were also observed in other species including P. major, P.

300

asiatica and P. depressa. Together, these data suggested the possible effects of

301

growing conditions on health properties of Plantago seeds. This study gave a first

302

insight on the cellular antioxidant activities of genus Plantago, providing useful

303

information for further studies and uses of the botanicals.

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DPPH radical scavenging capacities. In the present study, the free radical

305

scavenging capacity of the Plantago seed extracts were investigated against DPPH•.

306

As shown in Table 3, all seed extracts showed significant DPPH• scavenging

307

capacities (RDSC). PDW-1 had the greatest DPPH• scavenging capacity, followed by

308

PAL-1. Also noted was that the same species samples collected from different

309

growing locations might differ in their DPPH• scavenging capacities.

310

Hydroxyl radical scavenging capacity. The hydroxyl radical scavenging

311

capacities (HOSC) of the 28 samples are shown in Table 3. To avoid solvent

312

interference in the assay, 40% acetone was used to re-dissolve the seed extracts.

313

PDW-1 extract showed the strongest HOSC among all tested samples, followed by

314

PAL-1 extract. It was notable that P. depressa subsp. seed extract showed greater

315

HOSC than that of P. jehohlensis and P. himalaica, suggesting the possible effects of

316

growing conditions on HOSC of the seed extracts. To the best of our knowledge, the

317

present study investigated the DPPH and HO radical scavenging activities of P.



318

depressa subsp., P. arachnoidea, P. minuta, P. cornuti, P. camtschatica, P.

319

jehohlensis and P. himalaica seeds for the first time.

320

Synergistic antioxidant and anti-inflammatory effects of geniposidic acid (GA)

321

and acteoside (AC). To investigate the contribution of GA and AC, and their possible

322

synergistic effects in the overall DPPH•, HO• scavenging capacities, cellular

323

antioxidant activity and anti-inflammatory activity, pure compounds and their mixture

324

solutions in the same concentrations as that in the PAL-1 and PDW-1 seed extracts

325

were investigated under the same condition. As shown in Figures 3A-C and 4A-C,

326

there was no difference between AC and the mixtures on RDSC and HOSC, whereas

327

GA gave no contribution on radical scavenging capacities. The results indicated that

328

AC contributed significantly to the DPPH• and HO• scavenging capacities in PAL-1

329

and PDW-1, which were consistent with the observations from previous studies.26, 27

330

However, GA possessed weak cellular antioxidant activity with AC as a major

331

contributor. Anti-inflammatory tests also showed similar trends on inhibiting IL-1β,

332

IL-6 and COX-2 mRNA expression in PAL-1 and PDW-1. No synergistic effect was

333

observed between the two components on DPPH• and HO• scavenging capacities,

334

cellular antioxidant activity and anti-inflammatory activities under the experimental

335

conditions. According to the UPLC/Q-TOF-MS analysis, other minor

336

phenylethanoids or flavonoids might have contributed to the overall antioxidant and

337

anti-inflammatory activities of Plantago seeds.

338

Correlations of antioxidant and anti-inflammatory activities and chemical

339

compositions. The RDSC and HOSC values of Plantago seed extracts showed a

340

positive correlation with AC contents (R = 0.736, 0.709; P < 0.001, respectively).

341

HOSC value was significantly correlated with the RDSC value (R = 0.889, P < 0.001).


Page 16 of 37

Page 17 of 37


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Meanwhile, no correlation between HOSC value and GA content was observed,

343

which was consistent with previous reports that iridoids showed a weak correlation

344

with radical scavenging capability against DPPH•.26 Cellular antioxidant activity and

345

inhibition on IL-1β, IL-6 and COX-2 mRNA expression of Plantago seed samples

346

showed no significant correlation with GA or AC concentration. The data suggested

347

that GA contributed to the overall cellular antioxidant activity and anti-inflammatory

348

activity of PAL-1 and PDW-1. Taking together, it was possible that cell metabolites

349

of GA and AC, or minor constituents might contribute to the cellular antioxidant and

350

anti-inflammatory activities of Plantago seeds.

351

The RDSC and HOSC assays have been widely used to assess the antioxidant

352

capacity of foods, phytochemicals and botanical extracts.1, 28, 29 Recently, with the

353

emergence of several CAA assays utilizing HepG2, erythrocytes or red blood cells, it

354

is possible to evaluate cell-based antioxidant activity in vitro through a cost-effective

355

way.12, 30, 31 This study showed that CAA values of the 28 seed samples were weakly

356

correlated with RDSC (R = 0.530, P < 0.001) and HOSC values (R = 0.387, P

Identification and Quantification of Phytochemical Composition and

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