Phenolic constituents from Isodon lophanthoides var. graciliflorus and their antioxidant and antibacterial activities

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Phenolic constituents from Isodon lophanthoides var. graciliflorus and their antioxidant and antibacterial activities Wenting Zhoua,c, Haihui Xiea,*, Xinya Xub, Yaoguang Lianga, Xiaoyi Weia a Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, China b Key Laboratory of Marine Bio-Resources Sustainable Utilization, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China c University of Chinese Academy of Sciences, Beijing 100049, China

A R T I C L E I N F O

A B S T R A C T

Article history:

Isodon lophathoides var. graciliflorus (Lamiaceae) has been used as an herb for making health

Received 3 July 2013

beneficial beverages and an ingredient for making soup in China. This study revealed thir-

Received in revised form

teen phenolic compounds isolated from the aerial parts of the herb for the first time. They

19 November 2013

were rosmarinic acid (1), methyl rosmarinate (2), clinopodic acid A (3), salvianolic acid A

Accepted 20 November 2013

(4), nepetoidin B (5), caffeic acid (6), vinyl caffeate (7), danshensu (8), latifolicinin C (9),

Available online 15 December 2013

hydroxytyrosol (10), procatechuic aldehyde (11), 3,4-dihydroxybenzoic acid (12) and syringic acid (13). Their structures were determined by spectroscopic method including nuclear

Keywords:

magnetic resonance (NMR) and electrospray ionization mass spectrometry (ESI-MS) and

Isodon lophathoides var. graciliflorus

comparison of the data with the values reported in references. Ferric reducing antioxidant

Phenolics

power (FRAP) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity of these

Structures Antioxidant activity Antibacterial activity

compounds were evaluated, and compounds 1–8 and 13 were more potent or comparable to L-ascorbic acid. In addition, compounds 5 and 13 showed antibacterial activity against Staphyloccocus aureus and Shigella dysenteriae with IC50 values ranging from 44.15 to 79.13 lM, and compound 5 was also active towards Bacillus cereus.  2013 Elsevier Ltd. All rights reserved.

1.

Introduction

Isodon lophanthoides var. graciliflorus (Bentham) H. Hara is a perennial herb distributed in Fujian, Guangdong, and Jiangxi provinces of China, India, Myanmar, Nepal, and Vietnam (Wu & Raven, 1994). The herb has been farmed in large-scale as a commercial source of ‘Xihuangcao’, a folk Chinese medicine for the treatment of acute icterohepatitis, cholecystitis, and enteritis as well as an herb for making health beneficial beverages such as tea and instant granules (individually or together with other herbs) (Lin, Dong, Yang, & Zhao, 2011;

State Administration of Traditional Chinese Medicine, 1999). ‘Xihaungcao tea’ and ‘Xihuangcao granules’ are popular in China. In addition, the herb has also been consumed as an ingredient for making soup (commonly with pork or other meats) on the dinner table. Our previous studies on less polar fraction of I. lophanthoides var. graciliflorus revealed sixteen diterpenoids, and seven of which were cytotoxic against human lung adenocarcinoma A549, human breast adenocarcinoma MCF-7, and human cervical carcinoma HeLa cell lines (Liang, Xie, Wu, Jiang, & Wei, 2013; Zhou, Xie, Wu, & Wei, 2013). Chemical investigation

* Corresponding author. Tel.: +86 20 3708 0950. E-mail address: [email protected] (H. Xie). 1756-4646/$ - see front matter  2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jff.2013.11.015

of polar fraction of the herb led to the isolation of thirteen phenolic compounds (Fig. 1). Plant phenolics serve as antioxidants by virtue of hydrogen-donating properties of their hydroxyl groups as well as by donating electrons to stop free radical chain reactions (John & Shahidi, 2010). The present contribution describes the isolation and structure elucidation of these compounds and their FRAP and DPPH radical scavenging as well as antibacterial activities.

2.

Materials and methods

2.1.

Chemicals and reagents

hou Baiyunshan Chemical Pharmaceutical Factory (Guangzhou, China).

2.2.

O

9'

COOH

1

9

2 6

O

5

HO

4

6'

The aerial parts of I. lophanthoides var. graciliflorus were collected from the planting base of Hutchison Whampoa Guangzhou Baiyunshan Chinese Medicine Co. Ltd. in Yingde County, Guangdong, China in October 2009 and botanically authenticated by Prof. Huagu Ye in South China Botanical Garden, Chinese Academy of Sciences. A voucher specimen (No. 14058) was deposited at the Herbarium of South China Botanical Garden, Chinese Academy of Sciences, Guangzhou, China.

OH

COOCH3 O

7' 1'

8

Plant material

4"

HO

8'

2.3.

O

7 3

Liquid chromatography apparatus

Medium pressure liquid chromatography (MPLC) was performed on an EZ Purifier (Lisure Science, Suzhou, Jiangsu, China) and the column used was 460 · 15 mm i.d., 20–45 lm, Chromatorex RP-18 SMB100 (Fuji Silysia Chemical, Aichi, Japan). High performance liquid chromatography (HPLC) was run on a LC-6AD pump (Shimadzu, Kyoto, Japan) connected to a RID-10A refractive index detector (Shimadzu) and the columns used were 250 · 4.6 mm i.d. for analysis and 250 · 20 mm i.d. for preparation, 5 lm, YMC-pack ODS-A with a 23 mm · 4 mm i.d. guard column of the same material (YMC, Kyoto, Japan).

Silica gel (100–200 mesh) was purchased from Qingdao Haiyang Chemical Co. (Qingdao, Shandong, China). Polyamide (80–100 mesh) was obtained from Taizhou Luqiao Biochemical Corp. (Taizhou, Zhejiang, China). Ferric chloride (FeCl3), 2,4, 6-tri(2-pyridyl)-1,3,5-triazine (TPTZ), DPPH, and kanamycin were purchased from Sigma–Aldrich (St. Louis, MO, USA). L-Ascorbic acid was obtained from Shanghai Boao Biotech Co. (Shanghai, China). Staphyloccocus aureus (ATCC6538), Bacillus cereus (CMCC63302), and Shigella dysenteriae (CMCC51252) were obtained from Guangdong Institute of Microbiology (Guangzhou, China). Muller–Hinton broth (MHB) medium was obtained from Guangdong Huankai Microbial Sci. & Tech. Co. (Guangzhou, China). Alamar Blue was purchased from Invitrogen Molecular Probes (Eugene, OR, USA). Ceftazidime was generously provided by Guangz-

HO

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3"

6"

2"

HO

2'

1"

3'

5' 4'

OH

5"

7"

OH

OH

OH

HO

OH

Rosmarinic acid (1)

O

8"

COOH O

Methyl rosmarinate (2) HO

OH Salvianolic acid A (4) O HO

COOH

OH

O HO

O

HO

O

HO

HO

COOH

OH Clinopodic acid A (3)

Nepetoidin B (5)

OH

HO

OH

Caff eic acid (6)

O HO

HO O HO

HO Vinyl caf feate (7)

HO

CHO

HO

Procatechuic aldehyde (11)

COOH OH

danshensu (8)

HO

COOH

HO

3,4-Dihydroxybenzoic acid (12)

COOCH 3 OH

HO

latifolicinin C (9)

H3 CO

COOH

HO OCH3 Syringic acid (13)

Fig. 1 – Structures of compounds 1–13.

HO

OH

HO

Hydroxytyrosol (10)

494

2.4.

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Extraction and isolation

The aerial parts of the herb (4.0 kg) were ground and extracted with methanol (12 L · 3) heated under reflux for 3 h each time. Filtration and concentration of the solution to dryness under vacuum gave a methanolic extract (620 g). The extract was dissolved in distilled water (2.5 L) and sequentially fractionated with petroleum ether (2.5 L · 6) and ethyl acetate (2.5 L · 6) and the organic layers were combined and concentrated in vacuo to yield a petroleum ether-soluble fraction (121 g) and an ethyl acetate-soluble fraction (151 g), respectively. The ethyl acetate–soluble fraction (110 g) was subjected to silica gel (2000 g) column (553 · 116 mm inner diameter) chromatography using increasing methanol in chloroform as eluent to give fractions 1–7. Fraction 2 (3.22 g) was separated by MPLC using increasing aqueous methanol as eluent to afford fractions 2-1–2-6. Fraction 2-3 was purified by HPLC using 25% aqueous methanol (v/v) as mobile phase at the flow rate of 5 mL/min to gain compound 6 [retention time (tR) 53.5 min, 40.6 mg]. Fraction 3 (0.74 g) was separated by MPLC using increasing aqueous methanol as eluent to give fractions 3-1–3-3. Fraction 3-2 was purified by HPLC using 25% aqueous methanol (v/v) as mobile phase at the flow rate of 5 mL/min to provide compound 11 (tR 27.3 min, 60.9 mg). Fraction 4 (13.42 g) was further subjected to silica gel (550 g) column (632 · 52 mm inner diameter) chromatography using increasing acetone in petroleum ether as eluent to obtain fractions 41–4-90. Fraction 4-1 was purified by HPLC using 20% aqueous methanol (v/v) as mobile phase at the flow rate of 5 mL/min to yield compound 10 (tR 26.5 min, 17.0 mg). Fraction 4-2 was purified by HPLC using 20% aqueous methanol (v/v) as mobile phase at the flow rate of 5 mL/min to furnish compounds 9 (tR 81.5 min, 30.5 mg) and 12 (tR 28.9 min, 24.2 mg). Fraction 4-25 was purified by HPLC using 48% aqueous methanol (v/v) as mobile phase at the flow rate of 5 mL/min to yield compound 2 (tR 63.6 min, 11.0 mg). Fractions 4-27–4-33 (0.85 g) were combined and separated by MPLC using increasing aqueous methanol as eluent to give fractions 4-27-1–4-27-14. Fraction 4-27-3 was purified by HPLC using 53% aqueous methanol (v/v) as mobile phase at the flow rate of 5 mL/min to furnish compound 7 (tR 36.2 min, 13.0 mg). Fractions 4-52–4-57 were combined and further subjected to silica gel column chromatography and the resultant main fraction was purified by HPLC using 45% aqueous methanol (v/v) as mobile phase at the flow rate of 5 mL/min to furnish compound 13 (tR 18.8 min, 3.7 mg). Fraction 5 (9.33 g) was subjected to polyamide (300 g) column chromatography using increasing aqueous methanol as eluent and the resultant main fraction was purified by HPLC using 28% aqueous acetonitrile (v/v) as mobile phase at the flow rate of 5 mL/min to obtain compounds 1 (tR 22.0 min, 16.9 mg) and 3 (tR 33.1 min, 21.4 mg). Fraction 7 (6.19 g) was separated by MPLC using increasing aqueous methanol as eluent to give fractions 7-1–7-29. Fraction 7-1 was purified by HPLC using 20% aqueous methanol (v/v) as mobile phase at the flow rate of 5 mL/min to yield compound 8 (tR 38.8 min, 50.0 mg). Fraction 7-11 was purified by HPLC using 50% aqueous methanol (v/v) as mobile phase at the flow rate of 5 mL/min to furnish compound 4 (tR 33.1 min, 38.0 mg). Fraction 7-18 was purified by HPLC using 58% aqueous

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methanol (v/v) as mobile phase at the flow rate of 5 mL/min to give compound 5 (tR 33.1 min, 38.0 mg).

2.5.

Spectroscopic measurements

The compounds were dissolved in methanol and the resultant solutions were sequentially transferred into a tube of 100 mm length and measured on a Perkin–Elmer 343 polarimeter (Perkin–Elmer, Waltham, MA, USA) at 589 nm and 20 C to gain optical rotation values. ESI-MS spectra were obtained on a MDS SCIEX API 2000 LC/MS/MS system (Applied Biosystems Inc., Forster, CA, USA) in both positive and negative ion modes in the range of m/z 50–1000 after the solutions were directly injected into ESI source by a syringe pump. The compounds were dissolved in deuterated methanol (CD3OD), deuterated chloroform (CDCl3) or deuterated dimethyl sulphoxide (DMSO-d6) for the measurements of 1H and 13C NMR and distortionless enhancement by polarization transfer (DEPT) spectra on a Bruker DRX-400 or AVANCE-600 spectrometer (Bruker Biospin, Rheistetten, Germany) using tetramethylsilane (TMS) in CDCl3 or solvent residual peaks of CD3OD at dH 3.31 and dC 49.0 ppm or DMSO-d6 at dH 2.50 and dC 39.5 ppm as internal references.

2.6.

Ferric reducing antioxidant power (FRAP) assay

Ferric reducing ability of the phenolic compounds was determined by the method previously described by Xu, Xie, Wang, and Wei (2010). The FRAP reagent was made freshly by mixing 300 mM acetate buffer (pH 3.6), 10 mM TPTZ solution in 40 mM hydrochloric acid, and 20 mM aqueous FeCl3 solution in the ratio of 10:1:1 (v/v/v). The TPTZ solution was prepared on the same day of analysis. Test compounds were dissolved in methanol to serial concentrations and 20 lL of the solution and 180 lL of FRAP reagent were transferred to a 96-microplate and each concentration was set in quadruplicate. L-Ascorbic acid was dissolved in methanol and used as positive control. The plate was incubated at 37 C for 30 min in the dark. The absorbance of the resultant colored product (ferrous TPTZ complex) in each well was read at 595 nm using a Tecan Genios microplate reader (Tecan Group, Ma¨nnedorf, Switzerland). One milliliter of serial concentrations of FeSO4 plus 1 mL of 10 mM TPTZ and 10 mL of 300 mM acetate buffer (pH 3.6) were used for a calibration curve. FRAP values were expressed as mean ± standard error (SE) mmol Fe (II) per gram.

2.7.

DPPH radical scavenging assay

DPPH radical scavenging ability of the phenolic compounds was evaluated by the method of Xu et al. (2010). DPPH was freshly prepared in methanol to 0.1 mM. Test compounds were dissolved in methanol to serial concentrations and 20 lL of the solution and 180 ll of DPPH solution were mixed in a 96-well microplate. L-Ascorbic acid was dissolved in methanol and used as positive control. The control contained methanol instead of compound solution and the blank contained methanol in place of DPPH solution. The plate was incubated at 37 C for 30 min in the dark. The absorbance in each well was read at 515 nm on a Tecan Genios microplate

JOURNAL OF FUNCTIONAL FOODS

reader. The inhibitory rate of DPPH radicals by a test compound was calculated according to the formula: DPPH scavenging rate (%) = ½1  ðabsorbance of compound  absor bance of blankÞ=absorbance of control  100. All measurements were taken in triplicates. DPPH radical scavenging activity was expressed as SC50 value (the concentration required to scavenge 50% DPPH radicals present in the test solution) which was calculated by the software of SPSS 16.0.

2.8.

Antibacterial activity assay

Antibacterial activity was evaluated by microplate alamar blue (MAB) assay as described by Zhang, Xie, Qiu, Xue, and Wei (2008b) with some modifications. In brief, three test bacterial strains were individually prepared with MHB medium supplemented with 8% alamar blue (v/v) to the concentration of 1 · 105 CFU/mL. Test compounds were diluted with DMSO to six concentrations. To 96-well plates, 96 lL of bacterial solution and 4 lL of compound solution were transferred and the final concentrations of each compound in the wells were 120, 100, 50, 25, 12.5 and 6.25 lM. The wells with 4 lL of DMSO instead of compound solution were used as negative control. Ceftazidime (dissolved in DMSO) was used as the reference against S. aureus, and kanamycin (dissolved in sterile water) was used as the reference against other two bacteria. Each treatment was settled in triplicate. After completely mixed, the plates were incubated at 37 C until the negative control wells changed to stable pink color. The optical density (OD) of each well was measured on Genois microplate reader at the excitation wavelength of 570 nm and the emission wavelength of 600 nm. The inhibitory rate of bacterial growth was calculated according to the following formula: inhibitory rateð%Þ ¼ ½1  ð117216  OD570  80586  OD600 of the treatedÞ=

ð117216  OD570  80586  OD600 of the negative controlÞ

Table 1 – 1H and C/H

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100%. The IC50 value (the concentration required to inhibit 50% bacteria) were calculated and expressed as mean ± standard deviation (SD) from three independent experiments.

3.

Results and discussion

The methanolic extract of the aerial parts of I. lophanthoides var. graciliflorus was dissolved in water and sequentially partitioned with petroleum ether and ethyl acetate. The obtained ethyl acetate–soluble fraction was separated by a combination of column chromatography, MPLC and HPLC to give compounds 1–13. These compounds were shown to be over 95% pure by 1H NMR spectroscopy.

3.1.

Spectroscopic data of compounds 3, 5, 8 and 9

Clinopodic acid A (=isorinic acid, 3): yellowish amorphous powder; ½a20 D +82.8 (c 1.07, MeOH); ESI-MS positive m/z 345 [M+H]+, 367 [M+Na]+, negative m/z 343 [MH]; 1H NMR (400 MHz) and 13C NMR (100 MHz) data in CD3OD, see Table 1. Nepetoidin B (5): yellowish amorphous powder; ESI-MS positive m/z 315 [M+H]+, 337 [M+Na]+, negative m/z 313 [MH], 349 [M+Cl]; 1H NMR (400 MHz) and 13C NMR (100 MHz) data in CD3OD, see Table 1. Danshensu [=(2R)-3-(3,4-dihydroxyphenyl)-2-hydroxypropanoic acid, 8]: white amorphous powder; ½a20 D +11.8 (c 1.20, MeOH); ESI-MS positive m/z 221 [M+Na]+, negative m/z 197 [MH], 233 [M+Cl]; 1H NMR (600 MHz) and 13 C NMR (150 MHz) data in DMSO-d6, see Table 2. Latifolicinin C [=methyl (2R)-3-(4-hydroxyphenyl)-2hydroxypropanate, 9)]: white amorphous powder; ½a20 D +9.8 (c 1.53, MeOH); ESI-MS positive m/z 197 [M+H]+, 219 [M+Na]+,

13

C NMR data of compounds 3 and 5 in CD3OD. 3

5 dC (DEPT)

1 2 3 4 5 6 7 8 9 10 20 30 40 50 60 70

127.8 (C) 115.1 (CH) 146.7 (C) 149.5 (C) 116.5 (CH) 123.0 (CH) 147.1 (CH) 115.0 (CH) 168.7 (C) 129.5 (C) 116.1 (CH) 131.4 (C) 157.1 (C) 131.4 (CH) 116.1 (CH) 38.2 (CH2)

80 90

76.3 (CH) 175.8 (C)

dH (mult., J in Hz) 7.03 (d, 2.1)

6.77 6.92 7.52 6.26

(d, 8.2) (dd, 8.2, 2.1) (d, 15.8) (d, 15.8)

7.12 (d, 8.2) 6.71 (d, 8.2) 6.71 7.12 3.15 3.03 5.14

(d, 8.2) (d, 8.2) (dd, 14.3, 3.7) (dd, 14.3, 9.0) (dd, 9.0, 3.7)

dC (DEPT) 127.6 115.5 146.9 150.1 116.5 123.4 146.0 113.7 165.7 127.8 117.3 148.9 148.9 116.1 122.7 113.2

(C) (CH) (C) (C) (CH) (CH) (CH) (CH) (C) (C) (CH) (C) (C) (CH) (CH) (CH)

132.9 (CH)

dH (mult., J in Hz) 7.13 (d, 2.1)

6.82 7.05 7.73 6.46

(d, 8.3) (dd, 8.3, 2.1) (d, 15.8) (d, 15.8)

7.30 (d, 2.1)

6.76 (d, 8.3) 6.91 (dd, 8.3, 2.1) 7.24 (d, 7.3) 5.63 (d, 7.3)

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Table 2 – 1H and C/H

13

C NMR data of compounds 8 and 9. 8a dC (DEPT)

1 2 3 4 5 6 7

129.1 (C) 116.9 (CH) 144.7 (C) 143.6 (C) 115.2 (CH) 120.1 (CH) 39.6 (CH2)

8 9 OCH3

71.7 (CH) 175.6 (C)

a b

6 ( 2 0 1 4 ) 4 9 2 –4 9 8

9b dH (mult., J in Hz)

dC (DEPT) 129.1 (C) 116.0 (CH) 131.4 (CH) 157.1 (C) 131.4 (CH) 116.0 (CH) 40.8 (CH2)

6.62 (br s, 2.1)

6.60 6.45 2.77 2.58 4.01

(d, 8.0) (br d, 8.0) (dd, 13.8, 4.4) (dd, 13.8, 7.7) (dd, 7.7, 4.4)

73.3 (CH) 175.9 (C) 52.3 (CH3)

dH (mult., J in Hz) 6.70 (d, 8.1) 7.03 (d, 8.1) 7.03 6.70 2.95 2.74 4.30

(d, 8.1) (d, 8.1) (dd, 13.9, 4.8) (dd, 13.9, 7.4) (dd, 7.4, 4.8)

3.68 (3H, s)

Measured in DMSO-d6. Measured in CD3OD.

negative m/z 195 [MH], 231 [M+Cl]; 1H NMR (600 MHz) and 13 C NMR (150 MHz) data in CD3OD, see Table 2. The NMR data of compounds 3, 5 and 9 in CD3OD and 8 in DMSO-d6 were reported for the first time. The spectroscopic data of other compounds were in the Supplementary data.

3.2.

Structure determination

The obtained compounds were identified as rosmarinic acid (1) (Woo & Piao, 2004), methyl rosmarinate (2) (Woo & Piao, 2004), clinopodic acid A (3) (Murata et al., 2009), salvianolic acid A (4) (Zhang et al., 2008a), nepetoidin B (5) (Nakanishi et al., 1990), caffeic acid (6) (Song, Wang, Hua, Wu, & Du, 2008), vinyl caffeate (7) (Tada, Matsumoto, Yamaguchi, & Chiba, 1996), danshensu (8) (Yahara, Satoshiro, Nishioka, Nagasawa, & Oura, 1985), latifolicinin C (9) (Siddiqui, Perwaiz, & Begum, 2006), hydroxytyrosol (10) (Song et al., 2008), procatechuic aldehyde (11) (Song et al., 2008), 3,4-dihydroxybenzoic acid (12) (Wang, Wang, Ju, & Luo, 2008) and syringic acid (13) (Liu et al., 2011) by interpretation of their spectroscopic data and comparison of the data to the references.

Table 3 – Antioxidant activity of phenolic compounds using FRAP and DPPH assays. Compound

FRAP (mmol/g)

DPPH (SC50, lM)

1 2 3 4 5 6 7 8 9 10 13 L-Ascorbic acid

25.52 ± 0.85 28.10 ± 2.49 37.83 ± 3.41 28.89 ± 2.18 16.65 ± 0.67 34.90 ± 0.50 24.04 ± 2.00 22.86 ± 3.22 10.48 ± 0.74 16.08 ± 1.14 16.69 ± 1.10 22.32 ± 1.57

7.35 ± 0.53 7.19 ± 0.16 2.41 ± 0.28 8.42 ± 0.13 6.15 ± 0.90 14.33 ± 0.81 21.11 ± 2.96 30.22 ± 3.64 >500 32.01 ± 3.70 22.83 ± 1.89 26.36 ± 2.5

Values represent the mean ± standard error from three independent experiments.

DPPH radicals were 5.2, 11.1, and 2.6 lM, respectively, which were in good accordance with our results.

3.4. 3.3.

Antibacterial activity

Antioxidant activity

All the compounds excluding 11 and 12 were evaluated for antioxidant activity using FRAP and DPPH radical scavenging assays. In the FRAP assay, the antioxidant activity was determined on the basis of the ability to reduce the ferric (III) iron to ferrous (II) iron and expressed as millimole ferrous (II) iron equivalent per gram sample. As shown in Table 3, the FRAP values of compounds 1–4 and 6–8 ranged from 37.83 to 22.86 mmol/g in a decreasing order of 3 > 6 > 2  4 > 1  7  8, which were more potent or comparable to the positive control, L-ascorbic acid (22.32 mmol/g). In the DPPH radical scavenging assay, the SC50 values of compounds 1–6 were in the range of 2.41–14.33 lM in a decreasing order of 3 > 5  1  2  4 > 6, which were more potent than the control (26.36 lM), while compounds 7, 8, and 13 were comparable to L-ascorbic acid. Grayer et al. (2003) reported that the SC50 values of rosmarinic acid, caffeic acid, and nepetoidin B towards

All the compounds excluding 11 were evaluated for antibacterial activity against Gram positive S. aureus and B. cereus and Gram negative S. dysenteriae using MAB assay. The inhibitory rates of all the compounds except for nepetoidin B (5) and serrin A (13) were lower than 50% even at the maximum concentration of 120 lM (data were not shown), suggesting that these compounds were not active to the test bacterial strains. As shown in Table 4, compound 5 showed inhibitory effects on the test three bacterial strains with IC50 values of 44.15 ± 1.20, 74.24 ± 1.78 and 62.29 ± 2.46 lM, respectively, and compound 13 was active towards S. aureus and S. dysenteriae with IC50 values of 70.83 ± 6.80 and 79.13 ± 2.92 lM. It is worth noting that a multitude of biological activities for rosmarinic acid, salvianolic acid A, caffeic acid, and danshensu have been described. Main activities of rosmarinic acid are adstringent, antioxidative, anti-inflammatory, antimutagen, antibacterial, and antiviral (Petersen & Simmonds,

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Table 4 – Antibacterial activity of compounds 5 and 13 using MAB assay (IC50, lM). Compound

Gram positive Staphyloccocus aureus

5 13 Ceftazidime Kanamycin

44.15 ± 1.20 70.83 ± 6.80 14.58 ± 0.25

Gram negative Bacillus cereus

Shigella dysenteriae

74.24 ± 1.78 >120

62.29 ± 2.46 79.13 ± 2.92

6.76 ± 0.42

8.87 ± 0.14

Values represent the mean ± standard deviations from three independent experiments.

2003). Recent studies have revealed that salvianoilc acid A plays an important role in a variety of cellular activities including potent antioxidant, antiapoptotic, improving regional cerebral blood flow and anti-inflammatory (Zhang et al., 2012). Several experiments have demonstrated the biological activities of danshensu in improving microcirculation, suppressing the formation of reactive oxygen species, inhibiting platelet adhesion and aggregation, protecting myocardium against ischemia, and protecting endothelial cells against injury induced by inflammation (Yin et al., 2013). It is known that caffeic acid has a broad spectrum of pharmacological activities like anti-inflammatory, antioxidant and immunomodulatory effects. Pharmacological studies have also shown that caffeic acid exerts a protective effect against hydrogen peroxide-induced oxidative damage in the brain, and cerebral ischemia and prevents brain damage as well as behavioral and biochemical changes caused by aluminum (Anwar et al., 2012).

4.

Conclusions

Thirteen phenolic compounds were obtained from the aerial parts of I. lophanthoides var. graciliflorus for the first time, and nine of which showed more potent or comparable antioxidant activity to L-ascorbic acid in both FRAP and DPPH radical scavenging assays. In addition, nepetoidin B (5) and serrin A (13) exhibited antibacterial activity against S. aureus, B. cereus, and S. dysenteriae. In consideration of a variety of biological activities reported, it can be concluded that these phenolic constituents are important for the health beneficial effects of the herb.

Acknowledgments We are grateful for the financial support by the Knowledge Innovation Program of the Chinese Academy of Sciences (KSCX2-EW-J-28) and the Key Research Program of the Chinese Academy of Sciences (KSZD-EW-Z-004-06).

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jff.2013.11.015. R E F E R E N C E S

Anwar, J., Sapnevello, R. M., Thome´, G., Stefanello, N., Schmatz, R., Gutierres, L., Vieira, J., Baldissarelli, J., Carvalho, F. B., da Rosa, M. M., Rubin, M. A., Fiorenza, A., Morsch, V. M., & Schetinger,

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