Antioxidant and anti-inflammatory activities of selected Chinese medicinal plants and their relation with antioxidant content

  • Anjaneya S Ravipati1,

    Affiliated with

    • Lin Zhang1,

      Affiliated with

      • Sundar Rao Koyyalamudi1, 2Email author,

        Affiliated with

        • Sang Chul Jeong1,

          Affiliated with

          • Narsimha Reddy1,

            Affiliated with

            • John Bartlett1,

              Affiliated with

              • Paul T Smith1,

                Affiliated with

                • Kirubakaran Shanmugam3, 4,

                  Affiliated with

                  • Gerald Münch3,

                    Affiliated with

                    • Ming Jie Wu1,

                      Affiliated with

                      • Manavalan Satyanarayanan5 and

                        Affiliated with

                        • Balaram Vysetti5

                          Affiliated with

                          BMC Complementary and Alternative MedicineThe official journal of the International Society for Complementary Medicine Research (ISCMR)201212:173

                          DOI: 10.1186/1472-6882-12-173

                          Received: 11 January 2012

                          Accepted: 20 September 2012

                          Published: 6 October 2012

                          Abstract

                          Background

                          The main aim of this study is to evaluate the antioxidant and anti-inflammatory properties of forty four traditional Chinese medicinal herbal extracts and to examine these activities in relation to their antioxidant content.

                          Methods

                          The antioxidant activities were investigated using DPPH radical scavenging method and yeast model. The anti-inflammatory properties of the herbal extracts were evaluated by measuring their ability to inhibit the production of nitric oxide and TNF-α in RAW 264.7 macrophages activated by LPS and IFN- γ, respectively. The cytotoxic effects of the herbal extracts were determined by Alomar Blue assay by measuring cell viability. In order to understand the variation of antioxidant activities of herbal extracts with their antioxidant contents, the total phenolics, total flavonoids and trace metal (Mg, Mn, Cu, Zn, Se and Mo) quantities were estimated and a correlation analysis was carried out.

                          Results

                          Results of this study show that significant levels of phenolics, flavonoids and trace metal contents were found in Ligustrum lucidum, Paeonia suffuticosa, Salvia miltiorrhiza, Sanguisorba officinalis, Spatholobus suberectus, Tussilago farfara and Uncaria rhyncophylla, which correlated well with their antioxidant and anti-inflammatory activities. Some of the plants displayed high antioxidant and anti-inflammatory activities but contained low levels of phenolics and flavonoids. Interestingly, these plants contained significant levels of trace metals (such as Zn, Mg and Se) which are likely to be responsible for their activities.

                          Conclusions

                          The results indicate that the phenolics, flavonoids and trace metals play an important role in the antioxidant activities of medicinal plants. Many of the plants studied here have been identified as potential sources of new antioxidant compounds.

                          Keywords

                          Antioxidant activities Anti-inflammatory properties Phenolics Flavonoids Trace metals

                          Background

                          It is well known that reactive oxygen species (ROS), such as superoxide anion (O2 ·-), hydroxyl radicals (OH·), singlet oxygen (1O2) and hydrogen peroxide (H2O2), play a major role in the development of oxidative stress that can lead to many illnesses including cardiovascular diseases, diabetes, inflammation, degenerative diseases, cancer, anemia, and ischemia [1]. Many synthetic antioxidant agents have been developed to remediate oxidative stress. However, the factors such as high cost, lack of availability and side effects remained as major setbacks in combating oxidative stress. In this direction, natural antioxidants received a prominence as they are often free from side effects, less expensive and abundant in many plant sources [1]. Plant based antioxidant compounds [1, 2] play a defensive role by preventing the generation of free radicals and hence are extremely beneficial to alleviate the diseases caused by oxidative stress [3, 4]. Many investigations revealed that phenolics and flavonoids content [1, 510] contribute to the antioxidant activities of plants. In addixtion to these classes of organic compounds, recent studies demonstrated that trace metals such as Cu, Zn, Mg, Mn and Se play a beneficial role in antioxidant mechanism [11, 12].

                          Studies have also uncovered that phenolics and flavonoids act as excellent anti-inflammatory agents [6, 13]. The anti-inflammatory properties of flavonoids have been extensively studied and beneficial effects have been demonstrated in many animal models [13]. Excess production of pro-inflammatory molecules such as TNF-α and nitric oxide (NO) are believed to be responsible for modulating inflammation besides their crucial role in immune-inflammatory response. These inflammatory molecules are also known to cause cell death and tissue damage because NO can react with the free radicals such as superoxides to produce peroxynitrite, that can lead to irreversible damage to cell membranes [14, 15]. In order to search for effective natural antioxidants and anti-inflammatory compounds, current study has selected forty four traditional Chinese medicinal (TCM) plants based on their ethno-pharmacological importance.

                          TCM plants have history of usage in the treatment of several diseases for thousands of years [16, 17]. They are used for the treatment of many ailments including cancer in China and around the world. Several compounds derived from the TCM plants are potential anticancer agents and many of them are currently in clinical trials [18]. The extracts of TCM herbs are also used as pharmaceutical and dietary supplements.

                          The most important aspect of natural product research is to select a rapid, easy and efficient screening method. In the current study, the antioxidant activities of selected TCM plants were evaluated using DPPH (diphenylpicrylhydrazyl) radical scavenging method and yeast based antioxidant screening assay [19]. DPPH is a stable synthetic free radical and has been widely used for measuring free radical scavenging activity [20]. The yeast based biological assay detects antioxidant activities of samples against physiologically relevant oxidants. It is believed that antioxidant activities of medicinal plants must be evaluated by more than one method (by at least two methods) in order to take into account different modes of action of a given antioxidant [21]. Hence, in this study the antioxidant and anti-inflammatory activities of selected TCM plants were measured systematically using a series of assays.

                          Methods

                          Plant materials

                          The dried plant materials were obtained from Beijing Tong Ren Tang Chinese Herbal Medicine shop, Sydney, Australia. A voucher specimen of each plant has been deposited in the laboratory. The scientific names and family names were given in Table 1. The plant materials were ground to a fine powder in a grinder before extraction.
                          Table 1

                          List of Chinese medicinal herbs used in this study

                          S. no

                          Name of the plant species

                          Chinese name

                          Family names

                          Traditional medicinal use

                          References

                          1

                          Acanthopanax senticosus Herms

                          NA

                          Araliaceae

                          Treatment of rheumatism, allergies and diabetics

                          [22]

                          2

                          Actinidia arguta (Sieb.et Zucc.)Flarich.ex Miq.

                          Teng li gen

                          Actinidiaceae

                          Anticancer and anti-allergic

                          [23]

                          3

                          Akebia quinata (Houtt.)Decne.

                          Ba yue zha

                          Lardizabalaceae

                          anti-phlogistic

                          [24]

                          4

                          Alpinae officinarum

                          Gao liang jiang

                          Zingaberaceae

                          Antioxidant

                          [25]

                          5

                          Andrographis paniculata (Burm.f.) Wall. ex Nees

                          Shuan xin lian

                          Acanthaceae

                          Anti-cacner.

                          [26]

                          6

                          Artemisia vulgaris L.

                          Ai ye

                          Asteraceae

                          Anti-cancer

                          [27]

                          7

                          Asparagus cochinchinensis (Lour.) Merr.

                          Tian men dong

                          Asparagaceae

                          Anti-tumour

                          [28]

                          8

                          Aster tataricus L.

                          Zi wan

                          Asteraceae

                          Anti-oxidant

                          [29]

                          9

                          Corydalis yanhusuo W.

                          Yan hu suo

                          Papaveraceae

                          Anti-cancer

                          [18]

                          10

                          Curcuma zedoaria (Christm.) Roscoe

                          E zhu

                          Zingiberaceae

                          Antioxidant activity, Anti-mutagenic activity and Anti-microbial activity,

                          [30]

                          11

                          Cynanchum paniculatum (Bge.) Kitag

                          Xu chang qing

                          Asclepiadaceae

                          Anticancer

                          [18]

                          12

                          Cyperus rotundus L.

                          Xiang fu

                          Cyperaceae

                          Anticancer

                          [18]

                          13

                          Ducheshea indica (Andr.) Focke.

                          She mei

                          Rosaceae

                          Anti-inflammatory and anti-cancer

                          [31]

                          14

                          Hedyotis diffusa Willd.

                          Bai hua she she cao

                          Rubiaceae

                          Antibacterial

                          [18]

                          15

                          Leonurus japonicus Houtt.

                          Yi mu cao

                          Labiatae

                          Anti-cancer

                          [32]

                          16

                          Ligustrum lucidum Ait.

                          Nv zhen zi

                          Moraceae

                          Anti-cancer

                          [18]

                          17

                          Lobelia chinensis Lour.

                          Ban bian lian

                          Campanulaceae

                          Antipyretic, anti-inflammatory, and antitoxic effects

                          [18]

                          18

                          Lysinachia christinae Hance.

                          Jin qian cao

                          Lysimachia

                          Anticancer

                          [18]

                          19

                          Paeonia lactiflora Pall.

                          Bai shao

                          Paeoniaceae

                          Anticancer and anti-bacterial

                          [33]

                          20

                          Paeonia suffuticosa Sndr.

                          Mu dan pi

                          Ranunculaceae

                          Anti-cancer

                          [18]

                          21

                          Paris polyphylla Smith

                          Qi ye yi zhi hua

                          Trilliaceae

                          Anti-tumour

                          [28]

                          22

                          Platycodon grandiflorus (Jacq.) A. DC.

                          Jie geng

                          Campanulaceae

                          Anti-cancer

                          [18]

                          23

                          Plantago asiatica L.

                          Che qian cao

                          Plantaginaceae

                          Anti-cancer

                          [34]

                          24

                          Pleione bulbocadioides (Franch.)Rolfe.

                          Shen ci gu

                          Orchidaceae

                          Anticancer and antibacterial

                          [18]

                          25

                          Ploygala tenuifolia Willd.

                          Yuan zhi

                          Polygalaceae

                          Anti- inflammatory

                          [35]

                          26

                          Polygonum aviculare L.

                          Bian cun

                          Polygonaceae

                          Anti-oxidant activity and anti- inflammatory activity

                          [36]

                          27

                          Poria cocos (Schw.) Wolf

                          Fu lin

                          Polyporaceae

                          Anti-tumuor

                          [28]

                          28

                          Pogostemon cablin Benth.

                          Guang huo xiang

                          Asteraceae

                          Anti-insecticidal, anti-fungal and bacteriostatic

                          [37]

                          29

                          Prunella vulgaris L.

                          Xia ku cao

                          Lamiaceae

                          Anticancer and anti-inflammatory properties

                          [38]

                          30

                          Pseudostellaria heterophylla (Miq.) Pax ex Pax et Hoffm.

                          Tai zi shen

                          Caryophyllaceae

                          Anti-tumuor

                          [28]

                          31

                          Rabdosia rubescens(Hamst.)Wuet.

                          Dong ling cao

                          Labiatae

                          Anti-cancer

                          [18]

                          32

                          Rehmannia glutinosa (Gaertn.) Steud.

                          Sheng di huang

                          Phrymaceae

                          Anti-tumour

                          [28]

                          33

                          Salvia miltiorrhiza Bunge.

                          Dan shen

                          Caspase

                          Anti-tumuor

                          [28]

                          34

                          Sanguisorba officinalis L.

                          Di yu

                          Rosaceae

                          Anti-allergic and hepatitis B virus

                          [18]

                          35

                          Schizandra chinensis (Turcz.) Baill.

                          Wu wei zi

                          Schisandraceae

                          Antioxidant effect

                          [39]

                          36

                          Scutellaria barbata Don.

                          Ban zhi lian

                          Labiatae

                          Anti-tumuor

                          [28]

                          37

                          Semen coicis L.

                          Yi yi ren

                          Gramineae

                          Anti-cancer

                          [18]

                          38

                          Smilax glabra Roxb.

                          Tu fu ling

                          Smilacaceae

                          Anti-tumuor

                          [28]

                          39

                          Solanum nigrum L.

                          Long Kui

                          Solanaceae

                          Anti-cancer

                          [18]

                          40

                          Solanum lyratum Thunb.

                          Bai ying

                          Solanaceae

                          Anti-tumuor

                          [28]

                          x41

                          Spatholobus suberectus Dunn.

                          Ji xie teng

                          Leguminosae

                          Anti-tumuor

                          [18]

                          42

                          Tussilago farfara L.

                          Kuan dong

                          Asteraceae

                          Anti-microbialand antioxidant

                          [40]

                          43

                          Uncaria rhyncophylla Miq

                          Gou Teng

                          Rubiaceae

                          Antioxidant activity and anti-inflammatory activity

                          [41]

                          44

                          Viscum coloratum (Komar.) Nakai

                          Hui ji sheng

                          Viscaceae

                          Anti-infammatory

                          [42]

                          NA:No appropriate reference is available.

                          Chemicals and reagents

                          Gallic acid, Quercetin, 2, 2-diphenyl-1-picrylhydrazyl (DPPH), Dimethyl sulfoxide (DMSO), sodium carbonate, aluminium chloride (AlCl3), sodium nitrate (NaNO2), sodium hydroxide (NaOH), hydrogen peroxide (H2O2), Folin-Ciocalteu (F-C) reagent, ascorbic acid, 95% ethanol, bovine serum albumin (BSA), lipopolysaccharide (LPS: E.coli serotype 0127:B8), N-(1-1-napthyl) ethylenediamine dihydrochloride, penicillin G sodium benzyl, resazurin sodium 10%, streptomycin, sulfanilamide, tetramethyl benzidine (TMB), trypan blue were purchased from Sigma (Australia) and Lomb Scientific Pty Ltd (Australia). Antibiotics, Dulbecco’s modified eagle’s medium (DMEM), foetal bovine serum (FBS) and glutamine were purchased from GIBCO. Interferon-γ (murine) and tumor necrosis factor-α (TNF-α) – enzyme-linked immunosorbent assay (ELISA) kits were purchased from Peprotech. RAW 264.7 macrophages (ATCC number TIB-71) were obtained from American Type Culture Collection (ATCC).

                          Preparation of water extracts

                          Approximately 3 g of each grounded plant material was autoclaved with 30 mL of deionised water at 121°C for 1 hr as described in a previous publication [6]. The extracted samples were centrifuged at 10,447 g for 20 min) and the supernatant was transferred into a 50mL volumetric flask. The residue was further rinsed two more times, pooled the extracts and the volume adjusted to 50mL. The samples were stored at −20°C until analysis.

                          Preparation of ethanol extracts

                          Ground samples (3 g) were extracted with 30mL of 95% ethanol on water bath at 70°C for 6 hr [6]. The extracted samples were centrifuged and the supernatant was transferred into a 50 mL volumetric flask. The residue was further rinsed two more times, pooled the extracts and the volume adjusted to 50 mL with 95% ethanol. The samples were stored at −4°C until analysis. All water and ethanol extracts were filtered before analysis.

                          Determination of total phenolic content

                          The total phenolic content was determined by Folin-Ciocalteu (F-C) colorimetric method [43]. Briefly, 50 μL of sample and 50 μL of F-C reagent were pipetted into an eppendorf tube. The contents were vortexed for 10 sec and then left at room temperature for 2 min. After 2 min, 500 μL of 5% (w/v) sodium carbonate solution was added to stop the reaction and then 400μL of distilled water was added to make up to 1mL. The vortexed reaction mixture was heated in a water bath at 45°C for 30 min and then cooled rapidly in an ice bath. Absorbance was measured at 760 nm. Gallic acid concentrations ranging from 0–300 μg/mL were prepared and the calibration curve was obtained using a linear fit (r2 = 0.9961). The samples were analyzed in duplicates.

                          Determination of total flavonoid content

                          The total flavonoid content was estimated by aluminium chloride method [44]. Briefly, 0.5 mL of each sample and 300 μL of NaNO2 (1: 20 w/v) were pipetted into a test tube. The contents were vortexed for 10 sec and left at room temperature for 5 min. Into the mixture were then added 300 μL of AlCl3 (1:10 w/v), 2 mL of 1M NaOH and 1.9 mL of distilled water. After vortexing for 10 sec, the absorbance for each sample was measured at 510 nm. Quercetin concentrations ranging from 0 to 1200 μg/mL were prepared and the standard calibration curve was obtained using a linear fit (r2 = 0.9980). The samples were analyzed in duplicates.

                          Determination of trace metal content using ICP-MS technique

                          The plant water extracts were digested with 5 mL of concentrated HNO3 (Suprapure, Merck) and 2 mL of H2O2 (Suprapure, Merck) in a clean glass beaker and heated on a hot plate and diluted to 10 mL with double deionized water (Milli-Q Millipore 18.2 MW/cm resistivity). A blank digest was also carried out in the same way. The temperature of the hot plate was maintained at around 105°C continuously for 4–5 hr in order to completely digest the sample and to sustain recovery of volatile elements. The concentration of six elements, namely Mg, Mn, Cu, Zn, Se and Mo, were measured by inductively coupled plasma-mass spectrometer (ICP-MS) (Model: Perkin Elmer® ELAN DRC II, Ontorio, Canada). The certified reference material, NIST SRM 1640a was used as a calibration standard and NIST SRM 1643e (obtained from National Institute of Standards & Technology, NIST, USA) was analyzed as an unknown along with the samples to ensure accuracy. Triplicate analysis of all the samples was conducted in order to check the precision and accuracy of the data. The overall RSD for all the samples was less than 10, and the standard error was found to be within the required analytical precision. The method has been validated according to the standard ISO guidelines.

                          Free radical DPPH scavenging assay

                          DPPH radical scavenging assay was carried using Blois method [45]. Each plant extract (50 μL) in water and ethanol was added to a 150 μL of 62.5 μM DPPH. After 30 min of incubation, the absorbance of the reaction mixtures was measured at 492 nm using a microplate reader (Multiskan EX, Thermo Electron, USA). Ascorbate (Vitamin C), an antioxidant, was used as a positive control. A standard curve was included for each plate with a series of ascorbate concentrations (0, 10, 20, 40, 60, 80, 100, 200, 400 and 1000 μM). The free radical reduction capacity for each herbal extract was calculated as the ascorbate equivalent against the ascorbate standard curve (r2 = 0.9924).

                          Antioxidant activity screening in a 96-well microplate high throughput assay using Saccharomyces cerevisiae

                          The antioxidant capacities of the herbal extracts were also measured using a S. cerevisiae-based high throughput assay [19]. S. cerevisiae BY4743 were cultured overnight in a 50 mL volume by inoculation of a single colony. The culture was then diluted to an optical density at 600 nm (OD600) of 0.2 in media, and 180 μL of each strain was added into a well in a 96-well microtitre plate where 10 μL per well of each herbal extract was also added in duplicates. 10 μL of H2O2 was added to a final concentration of 4 mM. The initial OD600 reading was taken using a microplate reader (Multiskan EX, Thermo Electron, USA), and the plates were then incubated in a 30°C incubator with shaking at 750 rpm. Yeast growth was monitored at reading OD600 at the end of 20 hours. Ascorbic acid was used as a positive control. The net growth of H2O2 induced yeast cells after the treatment of selected plant extracts was measured using the following equation
                          http://static-content.springer.com/image/art%3A10.1186%2F1472-6882-12-173/MediaObjects/12906_2012_1192_Equ1_HTML.gif

                          P yeast growth = Net growth of H2O2 induced yeast cells after treatment with plant extracts.

                          P Sample = Observed optical density of yeast cells with the treatment of plant extracts.

                          P Control = Observed optical density of yeast cells with the treatment of negative control (H2O2).

                          Maintenance and activation of RAW 264.7 macrophages

                          RAW 264.7 macrophages were grown in 175 cm2 flasks on DMEM containing 5% FBS that was supplemented with antibiotics (1%) and glutamine (1%). The cell line was maintained in 5% CO2 at 37°C, with media being replaced every 3–4 days. Once cells had grown to confluence in the culture flask, they were removed using a rubber policeman, as opposed to using trypsin, which can remove membrane-bound receptors such as RAGE [6, 46]. Cell suspension was concentrated by centrifuging for 3 min at 900 rpm and resuspension in a small volume of fresh DMEM (with 1% antibiotics and 5% FBS). Cell densities were estimated using a Neubauer counting chamber. Cell concentration is adjusted with DMEM (with 1% antibiotics and 5% FBS) to obtain 75,000 cells/well when 100-μL cell suspensions dispensed into the 60-inner wells of 96-well plates. Sterile distilled water was added to the outer row of wells and incubated at 37°C; 5% CO2 for 12 hr. From each well, conditioned medium was replaced with fresh serum-free medium. For assays with extracts, 50-μL volume of the dilutions (in water) was added an hour prior to addition of activator. Due to the often inconsistent nature of LPS at activating cells, a combination of 25 μg/mL LPS and 10 U/ml IFN-γ, both in DMEM, was used for activation. Usually a maximum dose of the extracts used was 2.5 mg/mL and a minimum of 6 doses made by serial dilution. Then the cells were incubated for 24 hr at 37°C and 5% CO2. Cells with media alone were used as negative control and activated cells as positive control.

                          Determination of nitric oxide production by Griess assay

                          Nitric oxide is determined by Griess reagent quantification of nitrite; one of its stable reaction products. Griess reagent is freshly made up of equal volumes of 1% sulphanilamide and 0.1% napthyethylene-diamine in 5% HCl. In the presence of nitrite this reagent forms a violet colour. From each well 70 μL of supernatant was transferred to a fresh 96-well plate and mixed with 70 μL of Griess reagent and the colour produced was measured at 540 nm. The remaining supernatant that was removed from each well was used for TNF-α assay using a commercial sandwich ELISA.

                          Determination of cell viability by Alamar Blue assay

                          Alamar Blue assay is a calorimetric assay involving the cellular reduction of resazurin to resorufin. 100 μL of Alamar Blue solution (10% Alamar Blue (Resazurin) in DMEM media) was added to each well and incubated at 37°C for 1–2 hr. Fluorescence was measured (excitation @ 545 nm and emission @ 595 nm) and expressed as a percentage of that in control cells after background fluorescence was subtracted.

                          TNF-α determination by ELISA

                          Sandwich ELISA was used according to the manufacturer's manual (Peprotech) to determine TNF-α concentration. Capture antibody was used at a concentration of 0.5 μg/mL in PBS (1.9 mM NaH2PO4, 8.1 mM Na2HPO4, 154 mM NaCl; pH 7.4). Serial dilutions of TNF-α standard from 0 to 1000 pg/mL in diluent (0.05% Tween-20, 0.1% BSA in PBS) were used as internal standard. TNF-α was detected with a biotinylated second antibody and an avidin peroxidase conjugate with TMB as detection reagent. The color development was monitored at 655 nm, taking readings after every 5 min. After 25 min the reaction was stopped using 0.5 M sulphuric acid and the absorbance was measured at 450 nm.

                          Data presentation and analysis

                          As the experiments were done in duplicates, the results were expressed in mean ± standard deviation. In addition, linear relationships and significance tests of these data sets were also conducted. GraphPad prism 5.01 was used for growth curve analysis in dose-dependent experiments and to determine the IC50 values for NO and TNF-α inhibition.

                          Results and discussion

                          Total phenolics and flavonoids content in the selected plants

                          The total phenolics and flavonoids content of selected 44 herbal extracts were measured using F-C reagent and aluminium chloride methods respectively. These results obtained for water and ethanol extracts of the plants are presented in Table 2.
                          Table 2

                          The total phenolics and flavonoids content together with DPPH free radical scavenging activities of ethanol and water extracts of plant material, and the antioxidant activity against yeast oxidative stress

                           

                          DPPH scavenging activity (μM Ascorbate equivalent / g)1

                           

                          Total phenolics content (GAE mg/g)3

                          Total flavonoids content (QE mg/g)3

                          Name of the plants

                          Water extracts

                          Ethanol Extracs

                          % inhibition of Yeast oxidation of water extracts2

                          Water extracts

                          Ethanol extracts

                          Water extracts

                          Ethanol extracts

                          Acanthopanax senticosus

                          186.9 ± 0.71

                          99.93 ± 1.01

                          10.11

                          8.52 ± 3.00

                          3.89 ± 0.00

                          24.21 ± 0.98

                          14.37 ± 0.00

                          Actinidia arguta

                          183.4 ±1.41

                          104.21 ± 0

                          7.24

                          4.71 ± 0.55

                          15.26 ± 0.44

                          13.99 ± 0.98

                          62.85 ± 0.98

                          Akebia quinata

                          195.4 ±1.41

                          99.93 ± 4.04

                          14.76

                          7.09 ± 4.64

                          8.22 ± 0.44

                          16.66 ± 1.96

                          41.4 ± 5.87

                          Alpinae officinarum

                          201.9 ± 0.71

                          105.29 ± 0.51

                          0

                          11.78 ± 0.00

                          23.35 ± 3.14

                          26.81 ± 0.98

                          87.39 ± 0.98

                          Andrographis paniculata

                          171.4 ± 1.41

                          76.00 ± 4.55

                          13.33

                          5.09 ± 0.41

                          1.92 ± 1.03

                          8.94 ± 0.98

                          7.22 ± 1.96

                          Artemisia vulgaris

                          181.9 ±0.71

                          98.14 ± 1.52

                          5.32

                          24.91 ± 0.27

                          7.43 ± 1.44

                          79.68 ± 1

                          16.43 ± 3.91

                          Asparagus cochinchinensis

                          55.9 ± 12.02

                          5.64 ± 1.01

                          16.37

                          2.67 ± 0.96

                          0.91 ± 3.01

                          1.71 ± 0.98

                          1.08 ± 0.98

                          Aster tataricus

                          119.4 ± 2.83

                          49.93 ± 2.02

                          0

                          15.17 ± 0.27

                          6.95 ± 0.00

                          39.82 ± 0.98

                          22.79 ± 0.98

                          Corydalis yanhusuo

                          39.9 ± 0.71

                          42.43 ± 5.56

                          14.81

                          3.51 ± 1.36

                          1.28 ± 1.48

                          1.57 ± 0.96

                          2.11 ± 0.98

                          Curcuma zedoaria

                          158.9 ±0.71

                          108.14 ± 1.52

                          10.98

                          6.14 ± 0.55

                          7.38 ± 0

                          11.67 ± 0.98

                          25.38 ± 2.93

                          Cynanchum paniculatum

                          138.9 ±0.71

                          32.79 ± 1.01

                          6.13

                          6.31 ± 0

                          3.05 ± 0.89

                          3.3 ± 0

                          10.03 ± 0.00

                          Cyperus rotundus

                          114.9 ± 4.95

                          85.29 ± 2.53

                          5.06

                          3.46 ± 0.82

                          1.89 ± 0.44

                          5.01 ± 0.98

                          17.97 ± 0.98

                          Ducheshea indica

                          202.4 ± 1.41

                          102.07 ± 4.04

                          2.34

                          28.62 ± 0.68

                          11.8 ± 0.44

                          22.31 ± 4.89

                          15.25 ± 1.96

                          Hedyotis diffusa

                          160.9 ± 6.36

                          42.79 ± 2.02

                          8.54

                          5.17 ± 0.27

                          2.25 ± 0.00

                          7.37 ± 1.96

                          8.5 ± 0.98

                          Leonurus japonicus

                          159.9 ± 0.71

                          41.36 ± 4.04

                          1.93

                          6.46 ± 0.82

                          0.84 ± 2.66

                          8.22 ± 1.96

                          5.39 ± 1.96

                          Ligustrum lucidum

                          194.9 ±0.71

                          107.79 ± 0

                          12.02

                          29.1 ± 3.28

                          19 ± 0.78

                          108.42 ± 1.96

                          24.88 ± 0.98

                          Lobelia chinensis

                          101.4 ±0

                          12.79 ± 2.02

                          −1.94

                          4.59 ± 0.14

                          2.74 ± 0.44

                          2.79 ± 0.98

                          8.92 ± 3.91

                          Lysinachia christinae

                          193.9 ± 3.54

                          66.00 ± 0.51

                          0

                          4.24 ± 0.55

                          3.91 ± 0.30

                          2.63 ± 1.96

                          10.29 ± 0.00

                          Paeonia lactiflora

                          172.9 ± 3.54

                          113.14 ± 0.51

                          16.46

                          7.65 ± 0.41

                          6.3 ± 0.44

                          3.39 ± 1.96

                          6.42 ± 0.98

                          Paeonia suffuticosa

                          200.4 ± 0.00

                          111.71 ± 0.51

                          15.16

                          19.96 ± 1.09

                          19.74 ± 0.39

                          25.85 ± 1.96

                          31.40 ± 2.93

                          Paris polyphylla

                          19.9 ± 2.12

                          45.29 ± 1.52

                          0

                          1.23 ± 1.36

                          1.01 ± 3.69

                          0.72 ± 1.96

                          2.60 ± 1.96

                          Platycodon grandiflorus

                          30.9 ± 3.54

                          14.93 ± 4.04

                          21.77

                          4.58 ± 1.50

                          0.79 ± 0.26

                          2.93 ± 2.93

                          1.52 ± 0.96

                          Plantago asiatica

                          184.4 ±9.9

                          49.93 ± 1.01

                          19.77

                          6.7 ± 0.96

                          0.82 ± 0.39

                          15.57 ± 3.91

                          3.32 ± 1.96

                          Pleione bulbocadioides

                          27.4 ± 7.07

                          85.64 ± 1.01

                          0

                          1.62 ± 0.27

                          1.05 ± 2.51

                          2.09 ± 0.98

                          1.73 ± 0.98

                          Ploygala tenuifolia

                          109.4 ± 2.83

                          53.14 ± 2.53

                          3.23

                          12.01 ± 0.00

                          12.15 ± 0.44

                          9.3 ± 3.91

                          37.55 ± 3.91

                          Polygonum aviculare

                          201.4 ±2.83

                          88.86 ± 1.52

                          3.67

                          5.9 ± 0.82

                          11.72 ± 0.15

                          9.15 ± 0.00

                          14.15 ± 0.00

                          Poria cocos

                          28.9 ±3.54

                          109.57 ± 0.51

                          12.11

                          0.45 ± 0

                          1.43 ± 3.53

                          0

                          0.68 ± 0.98

                          Pogostemon cablin

                          103.4 ± 4.24

                          26.00 ± 1.52

                          12.07

                          4.23 ± 0.68

                          1.88 ± 0.13

                          5.54 ± 0.98

                          4.08 ± 0.98

                          Prunella vulgaris

                          106.9 ±20.51

                          106.36 ± 0.00

                          0

                          16.02 ± 0.27

                          1.66 ± 0.74

                          20.27 ± 3.91

                          15.62 ± 1.96

                          Pseudostellaria heterophylla

                          20.9 ±12.02

                          32.43 ± 4.55

                          36.13

                          1.74 ± 0.82

                          1.58 ± 1.83

                          0.59 ± 0.00

                          1.94 ± 0.98

                          Rabdosia rubescens

                          144.4 ± 2.83

                          13.50 ± 4.04

                          8.63

                          17.47 ± 0.55

                          2.41 ± 2.96

                          40.66 ± 1.96

                          8.35 ± 2.93

                          Rehmannia glutinosa

                          34.4 ± 0.00

                          20.29 ± 0.51

                          17.94

                          5.99 ± 1.23

                          0.88 ± 0.13

                          6.12 ± 0.98

                          2.07 ± 1.96

                          Salvia miltiorrhiza

                          202.4 ± 4.24

                          104.93 ± 0.00

                          12.72

                          66.27 ± 0.14

                          4.97 ± 0.15

                          133.93 ± 0.98

                          27.05 ± 2.93

                          Sanguisorba officinalis

                          191.9 ±0.71

                          64.21 ± 2.02

                          −5.47

                          148.09 ± 2.46

                          121.42 ± 0.78

                          129.53 ± 0.98

                          213.23 ± 0

                          Schizandra chinensis

                          73.9 ± 3.54

                          33.86 ± 1.52

                          4.8

                          7.95 ± 2.73

                          11.91 ± 0.15

                          11.36 ± 7.82

                          31.3 ± 1.96

                          Scutellaria barbata

                          188.4 ±1.41

                          93.5 ± 1.01

                          10.11

                          14.97 ± 2.73

                          9.26 ± 0.15

                          67.41 ± 3.91

                          20.62 ± 0.98

                          Semen coicis

                          −100.1 ± 0.71

                          11.00 ± 3.54

                          12.37

                          0.34 ± 2.32

                          1.39 ± 0.65

                          0.83 ± 1.96

                          16.05 ± 2.93

                          Smilax glabra

                          135.9 ± 2.12

                          109.21 ± 0.00

                          5.84

                          6.19 ± 2.73

                          12.62 ± 0.89

                          45.28 ± 1.96

                          45.95 ± 4.89

                          Solanum nigrum

                          190.4 ±0

                          50.64 ± 3.03

                          5.17

                          4.2 ± 0.27

                          2.98 ± 2.48

                          6.43 ± 0.98

                          4.41 ± 4.89

                          Solanum lyratum

                          192.4 ±1.41

                          47.07 ± 1.01

                          1.03

                          3.63 ± 0.41

                          2.55 ± 1.77

                          2.62 ± 2.93

                          5.18 ± 0.98

                          Spatholobus suberectus

                          184.4 ±0

                          98.14 ± 2.53

                          10.63

                          24.11 ± 0.96

                          34.02 ± 4.05

                          165.16 ± 1.96

                          69.19 ± 0.00

                          Tussilago farfara

                          198.9 ± 0.71

                          113.5 ± 0.00

                          20.9

                          25.72 ± 2.87

                          9.68 ± 0.15

                          65.17 ± 0.98

                          41.67 ± 0.98

                          Uncaria rhyncophylla

                          181.4 ± 2.83

                          98.50 ± 1.01

                          13.24

                          32.38 ± 0.55

                          6.75 ± 0.30

                          58.91 ± 0.00

                          22.13 ± 1.96

                          Viscum coloratum

                          129.9 ±12.02

                          57.07 ± 0.00

                          23.69

                          3.34 ± 3.00

                          9.06 ± 0.00

                          3.07 ± 0.98

                          28.58 ± 3.91

                          1DPPH free radical scavenging activity was measured in terms of equivalent of ascorbate (μM).

                          2Yeast oxidative stress was measured on the basis of survival of yeast cells (yeast growth) after treatment with H2O2.

                          3Total phenolics and flavonoid contents were expressed in gallic acid equivalent (GAE mg/g) and quercetin equivalent (QE mg/g) respectively.

                          Water extracts

                          As can be seen from Table 2, significant phenolics content was observed in water extracts of S. officinalis (148.09 GAE mg/g), S. miltiorrhiza (66.27 GAE mg/g) and moderate levels in U. rhyncophylla (32.38 GAE mg/g), L. lucidum (29.1 GAE mg/g), Ducheshea indica (28.62 GAE mg/g), T. farfara (25.72 GAE mg/g), Artemisia vulgaris (24.91 GAE mg/g) and S. suberectus (24.11 GAE mg/g). High levels of flavonoids content was found in water extracts of S. suberectus (165.16 QE mg/g), S. miltiorrhiza (133.93 QE mg/g), S. officinalis (129.53 QE mg/g), L. lucidum (108.42 QE mg/g) and moderate to low levels in Artemisia vulgaris (79.68 QE mg/g), T. farfara (65.17 QE mg/g), U. rhyncophylla (58.91 QE mg/g), Rabdosia rubescens (40.66 QE mg/g), Aster tataricus (39.82 QE mg/g), Alpinae officinarum (26.81 QE mg/g), Acanthopanaxsenticosus (24.21 QE mg/g) and D. indica (22.31 QE mg/g).

                          Ethanol extracts

                          Amongst all the ethanol extracts, high levels of phenolics content was found in S. officinalis (121.42 GAE mg/g) and moderate levels in S. suberectus (34.02 GAE mg/g) and A. officinarum (23.35 GAE mg/g) (Table 2). High flavonoid content was found in ethanol extracts of S. officinalis (213.23 QE mg/g), A. officinarum (87.39 QE mg/g) and moderate to low levels in S. suberectus (69.10 QE mg/g), Actinidia arguta (62.85 QE mg/g), S. glabra (45.95 QE mg/g), T. farfara (41.67 QE mg/g), Ploygala tenuifolia (37.55 QE mg/g), Paeonia suffuticosa (31.4 QE mg/g) and Viscum coloratum (28.58 QE mg/g).

                          It is interesting to note that A. officinarum, S. suberectus, S. officinalis, T. farfara, S. miltiorrhiza and U. rhyncophylla have been found to have significant levels of phenolics and flavonoids content in both water and ethanol extracts. A close observation of the results presented in this paper indicates that the total phenolics and flavonoids content varied amongst the selected plant species and in different extracts. These variations of antioxidant contents have been discussed in terms of the observed bioactivities of the plants.

                          Trace metal content in the selected plants

                          In the current study, trace metal content (Mg, Mn, Cu, Zn, Se and Mo) of water extracts was determined using ICP-MS technique (Table 3). Among all the metals, Mn was the most abundant while Se and Mo were least abundant. Significant quantity of Cu was found in Poria cocos (230.2 μg/g), A. arguta (201.75 μg/g). High levels of Zn was found in P. cocos (1841.43 μg/g), V. coloratum (977.52 μg/g), A. arguta (969.13 μg/g) and S. officinalis (909.37 μg/g). Of all the analyzed trace metals, Mn was highly abundant in Curcuma zedoaria (2312.15 μg/g), U. rhyncophylla (2770.85 μg/g), A. officinarum (2212.53 μg/g), Hedyotis diffusa (1778.79 μg/g), P. cocos (1257.65 μg/g) and S. suberectus (1211.85 μg/g). The Mg content amongst all the plants was relatively high (expressed in mg/g in Table 3), while Selenium and Molybdenum were least abundant.
                          Table 3

                          The trace metal content of water extracts of selected medicinal plants

                          Name of the plants

                          Cu (μg/g)*

                          Zn (μg/g)*

                          Mn (μg/g)*

                          Mg (mg/g)#

                          Se (μg/g)*

                          Mo (μg/g)*

                          Acanthopanax senticosus

                          30.86 ± 0.61

                          183.33 ± 3.91

                          35.01 ± 0.86

                          10.35 ± 0.41

                          43.04 ± 1.41

                          0.73 ± 0.03

                          Actinidia arguta

                          201.75 ± 1.28

                          969.13 ± 6.68

                          182.96 ± 1.46

                          38.44 ± 0.49

                          5.9 ± 0.06

                          2.96 ± 0.04

                          Akebia quinata

                          29.28 ± 1.72

                          310.65 ± 19.77

                          93.36 ± 6.85

                          7.24 ± 0.85

                          1.74 ± 0.17

                          0.12 ± 0.02

                          Alpinae officinarum

                          19.94 ± 0.73

                          276.02 ± 10.99

                          2212.53 ± 101.6

                          10.56 ± 0.78

                          1.92 ± 0.12

                          0.22 ± 0.02

                          Andrographis paniculata

                          61.13 ± 1.77

                          245.62 ± 7.69

                          542.3 ± 19.6

                          65.89 ± 3.81

                          13.93 ± 0.67

                          0.78 ± 0.05

                          Artemisia vulgaris

                          43.8 ± 1.27

                          135.75 ± 4.28

                          321.4 ± 11.69

                          18.6 ± 1.08

                          16.28 ± 0.79

                          0.55 ± 0.03

                          Asparagus cochinchinensis

                          6.71 ± 0.75

                          77.24 ± 9.37

                          54.5 ± 7.63

                          3.05 ± 0.68

                          1.86 ± 0.35

                          0.13 ± 0.03

                          Aster tataricus

                          3.47 ± 0.26

                          30.35 ± 2.48

                          46.6 ± 4.39

                          2.78 ± 0.42

                          1.44 ± 0.18

                          0.09 ± 0.01

                          Corydalis yanhusuo

                          7.73 ± 0.61

                          154.73 ± 13.17

                          42.99 ± 4.28

                          9.61 ± 1.51

                          0.48 ± 0.06

                          0.87 ± 0.14

                          Curcuma zedoaria

                          32.74 ± 0.5

                          45.85 ± 0.75

                          2312.15 ± 43.84

                          29.26 ± 0.89

                          13.7 ± 0.35

                          3.27 ± 0.1

                          Cynanchum paniculatum

                          23.57 ± 0.78

                          171.76 ± 6.16

                          53.96 ± 2.23

                          12.39 ± 0.82

                          2.92 ± 0.16

                          0.51 ± 0.04

                          Cyperus rotundus

                          21.03 ± 0.61

                          79.54 ± 2.51

                          27.35 ± 0.99

                          6.41 ± 0.37

                          0.54 ± 0.03

                          0.39 ± 0.02

                          Ducheshea indica

                          18.55 ± 0.88

                          175.82 ± 9.06

                          156.07 ± 9.28

                          19.58 ± 1.86

                          2.72 ± 0.22

                          0.37 ± 0.04

                          Hedyotis diffusa

                          59.23 ± 1.06

                          304.91 ± 5.89

                          1778.79 ± 39.63

                          59.31 ± 2.11

                          42.38 ± 1.26

                          3 ± 0.11

                          Leonurus japonicus

                          82.62 ± 1.53

                          848.36 ± 17.01

                          214.72 ± 4.97

                          40.24 ± 1.49

                          47.03 ± 1.45

                          2.57 ± 0.11

                          Ligustrum lucidum

                          16.51 ± 0.86

                          250.49 ± 14.17

                          72.87 ± 4.76

                          9.43 ± 0.99

                          0.4 ± 0.03

                          1.41 ± 0.15

                          Lobelia chinensis

                          9.37 ± 0.75

                          236.66 ± 20.53

                          124.26 ± 12.44

                          2.26 ± 0.36

                          0.64 ± 0.09

                          1.2 ± 0.2

                          Lysinachia christinae

                          68.01 ± 1.31

                          461.07 ± 9.61

                          22.96 ± 0.55

                          16.46 ± 0.63

                          27.88 ± 0.89

                          1.12 ± 0.04

                          Paeonia lactiflora

                          2.89 ± 0.12

                          130.61 ± 5.95

                          147.3 ± 7.75

                          9.73 ± 0.82

                          −0.11 ± −0.01

                          0.13 ± 0.01

                          Paeonia suffuticosa

                          2.42 ± 0.10

                          59.72 ± 2.81

                          29.38 ± 1.59

                          4.1 ± 0.36

                          0.57 ± 0.04

                          0.07 ± 0.01

                          Paris polyphylla

                          NA

                          NA

                          NA

                          NA

                          NA

                          NA

                          Platycodon grandiflorus

                          12.73 ± 0.96

                          180.17 ± 14.78

                          56.28 ± 5.33

                          12.41 ± 1.88

                          0.83 ± 0.11

                          1.39 ± 0.22

                          Plantago asiatica

                          59.58 ± 1.23

                          298.09 ± 6.68

                          238.28 ± 6.16

                          8.67 ± 0.36

                          14.63 ± 0.5

                          1.29 ± 0.06

                          Pleione bulbocadioides

                          NA

                          NA

                          NA

                          NA

                          NA

                          NA

                          Ploygala tenuifolia

                          21.61 ± 1.31

                          43.25 ± 2.84

                          47.82 ± 3.62

                          5.53 ± 0.67

                          0.65 ± 0.07

                          0.2 ± 0.03

                          Polygonum aviculare

                          34.48 ± 0.98

                          294.6 ± 9.09

                          5.89 ± 0.21

                          36.32 ± 2.07

                          9.81 ± 0.47

                          0.77 ± 0.05

                          Poria cocos

                          230.2 ± 1.16

                          1841.43 ± 10.09

                          1257.65 ± 7.95

                          7.53 ± 0.08

                          0.52 ± 0

                          3.75 ± 0.04

                          Pogostemon cablin

                          64.71 ± 2.11

                          565.31 ± 17.51

                          392.13 ± 13.76

                          83.3 ± 4.26

                          5.81 ± 0.27

                          4.66 ± 0.37

                          Prunella vulgaris

                          9.76 ± 0.26

                          128.12 ± 3.7

                          522.57 ± 17.4

                          14.98 ± 0.8

                          17.68 ± 0.79

                          0.45 ± 0.03

                          Pseudostellaria heterophylla

                          3.53 ± 0.18

                          274.37 ± 15.37

                          270.58 ± 17.48

                          4.19 ± 0.43

                          1.1 ± 0.10

                          0.36 ± 0.04

                          Rabdosia rubescens

                          52.69 ± 1.25

                          284.29 ± 7.30

                          692.42 ± 20.5

                          62.03 ± 2.94

                          8.54 ± 0.34

                          5.21 ± 0.26

                          Rehmannia glutinosa

                          6.86 ± 0.82

                          110.9 ± 14.37

                          6.61 ± 0.99

                          1.46 ± 0.35

                          0.55 ± 0.11

                          0.08 ± 0.02

                          Salvia miltiorrhiza

                          14.45 ± 1.12

                          66.17 ± 5.54

                          76.18 ± 7.35

                          23.83 ± 3.68

                          0.51 ± 0.07

                          0.13 ± 0.02

                          Sanguisorba officinalis

                          74.89 ± 1.09

                          909.37 ± 14.34

                          274.25 ± 4.99

                          25.66 ± 0.75

                          4.06 ± 0.1

                          1.2 ± 0.04

                          Schizandra chinensis

                          5.27 ± 0.35

                          168.34 ± 12.17

                          543.02 ± 45.31

                          13.4 ± 1.79

                          0.35 ± 0.04

                          0.54 ± 0.07

                          Scutellaria barbata

                          45.27 ± 0.56

                          290.33 ± 3.92

                          634.07 ± 9.87

                          58.94 ± 1.47

                          3.08 ± 0.06

                          1.11 ± 0.03

                          Semen coicis

                          91.28 ± 1.07

                          367.05 ± 4.65

                          367.84 ± 5.38

                          28.46 ± 0.67

                          5 ± 0.11

                          6.97 ± 0.17

                          Smilax glabra

                          NA

                          NA

                          NA

                          NA

                          NA

                          NA

                          Solanum nigrum

                          71.07 ± 2.01

                          404.88 ± 12.4

                          244.61 ± 8.64

                          57.18 ± 3.23

                          4.83 ± 0.23

                          4.46 ± 0.26

                          Solanum lyratum

                          74.33 ± 1.86

                          596.51 ± 16.14

                          9.22 ± 0.29

                          11.98 ± 0.6

                          0.89 ± 0.04

                          0.45 ± 0.02

                          Spatholobus suberectus

                          27.63 ± 0.56

                          188.26 ± 4.1

                          1211.85 ± 30.48

                          3.27 ± 0.13

                          16.65 ± 0.56

                          0.41 ± 0.02

                          Tussilago farfara

                          10.16 ± 0.84

                          96.79 ± 8.71

                          17.52 ± 1.82

                          2.13 ± 0.35

                          1.87 ± 0.26

                          0.18 ± 0.03

                          Uncaria rhyncophylla

                          8.25 ± 0.25

                          400.03 ± 12.95

                          2770.85 ± 103.53

                          7.91 ± 0.47

                          17.28 ± 0.86

                          0.19 ± 0.01

                          Viscum coloratum

                          24.77 ± 0.97

                          977.52 ± 41.43

                          392.4 ± 19.19

                          16.65 ± 1.3

                          4 ± 0.26

                          0.4 ± 0.03

                          NA: not applicable.

                          * Trace metal contents are expressed in μg/g of dry plant samples.

                          # Mg content was measured in mg/g.

                          Antioxidant activities

                          The antioxidant activities of 44 selected medicinal herbs were evaluated by two methods, namely, DPPH free radical scavenging and yeast based antioxidant screening assay [19, 47, 48].

                          The water extracts of S. nigrum, L. lucidum, Polygonum aviculare, S. lyratum, Akebia quinata, S. officinalis, P. suffuticosa, S. miltiorrhiza, A. officinarum, Lysinachia christinae, D. indica and T. farfara shown significantly high free radical scavenging ability which were more than 190 μM ascorbate equiv/g (Table 2). In ethanol extracts, significant DPPH scavenging activity was found in T. farfara, Paeonia lactiflora, P. suffuticosa, P. cocos, S. glabra, C. zedoaria, L. lucidum, Prunella vulgaris, A. officinarum, S. miltiorrhiza, and A. arguta, and D. indica which were in the range of 98.14 – 113.5 μM ascorbate equiv/g (Table 2). It is interesting to note that the water extracts have displayed more scavenging activity than the ethanol extracts.

                          Antioxidant activities of water extracts of the selected plants were also evaluated based on their ability to inhibit the H2O2 induced yeast oxidative stress (Table 2). These results revealed that the plants Pseudostellaria heterophylla (36.13%), V. coloratum (23.69%), Platycodon grandiflorus (21.77%), T. farfara (20.9%), Plantago asiatica (19.77%) and Rehmannia glutinosa (17.94%) showed high antioxidant activity. It can be noted from Table 2 that, T. farfara, L. lucidum, P. suffuticosa, S. miltiorrhiza, P. lactiflora and A. senticosus have displayed significant antioxidant activity in both DPPH method and yeast model.

                          Anti-inflammatory activities of plant extracts

                          The anti-inflammatory properties of water extracts of the selected medicinal herbs were evaluated on the basis of their ability to inhibit the production of NO and TNF-α in LPS and IFN-γ activated mouse macrophages. Toxicity of the plant extracts was determined using the Alamar Blue assay.

                          As can be seen from these results (Table 4), the extracts of A. vulgaris, A. arguta, S. officinalis, S. suberectus, S. barbata, P. asiatica, Pogostemonss cablin, P. suffuticosa, H. diffusa, L. japonicus, A. paniculata, L. christinae, D. indica, U. rhyncophylla and R. rubescens have down regulated NO production with IC50 values of less than 0.1 mg/mL without significantly affecting the cell viability (> 80). Results (Table 4) also revealed that the inhibition efficiency of plant extracts with respect to NO production was superior when compared to that of TNF-α production. Amongst all the plants, S. officinalis (IC50 = 0.07 mg/mL), P. asiatica (IC50 = 0.1 mg/mL) and A. paniculata (IC50 = 0.1 mg/mL) have displayed greater inhibition of TNF-α production as well as significant down regulation of NO production and showed less toxicity.
                          Table 4

                          Anti-inflammatory activities of water extracts of the selected plants

                          Name of the plants

                          IC50for the inhibition of NO production (mg/ml)

                          Cell viability (% of cell survival)&

                          IC50for the inhibition of TNF-α production (mg/ml)

                          Cell viability (% of cell survival)&

                          Acanthopanax senticosus

                          0.12 ± 0.08

                          93.85 ± 8.7

                          1.23 ± 0

                          106.80 ± 11.6

                          Actinidia arguta

                          0.07 ± 0.01

                          90.3 ± 1.6

                          0.73 ± 0.25

                          83.05 ± 4.6

                          Akebia quinata

                          0.43 ± 0.05

                          105.5 ± 7.8

                          1.63 ± 0.4

                          84.5 ± 19

                          Alpinae officinarum

                          0.12 ± 0.01

                          92.80 ± 5.1

                          0.50 ± 0

                          85.55 ± 13.4

                          Andrographis paniculata

                          0.05 ± 0.01

                          85.20 ± 6.6

                          0.10 ± 0

                          77.60 ± 7.2

                          Artemisia vulgaris

                          0.05 ± 0

                          93.2 ± 6.6

                          0.21 ± 0.03

                          70.7 ± 0.6

                          Asparagus cochinchinensis

                          1.49 ± 1.04

                          98.35 ± 3.7

                          >2.5

                          NA

                          Aster tataricus

                          0.14 ± 0.08

                          98.95 ± 1.5

                          2.30 ± 0.09

                          99.70 ± 0.5

                          Corydalis yanhusuo

                          0.10 ± 0.01

                          70.70 ± 9.8

                          0.92 ± 0.05

                          62.70 ± 1.6

                          Curcuma zedoaria

                          0.25 ± 0.02

                          81.1 ± 8

                          1.79 ± 0.23

                          72.2 ± 1.5

                          Cynanchum paniculatum

                          0.24 ± 0.29

                          96.5 ± 1.1

                          NA#

                          NA

                          Cyperus rotundus

                          0.35 ± 0.37

                          86.60 ± 19

                          2.39 ± 0.64

                          107.50 ± 10.6

                          Ducheshea indica

                          0.04 ± 0.02

                          100.6 ± 1.2

                          0.38 ± 0

                          73.95 ± 2.1

                          Hedyotis diffusa

                          0.05 ± 0.00

                          85.60 ± 0

                          0.70 ± 0.08

                          57.30 ± 3.1

                          Leonurus japonicus

                          0.04 ± 0.00

                          78.35 ± 6.2

                          0.18 ± 0.11

                          62.00 ± 2.5

                          Ligustrum lucidum

                          0.14 ± 0.13

                          88.8 ± 15.8

                          0.3 ± 0.11

                          85.6 ± 20.4

                          Lobelia chinensis

                          0.36 ± 0.32

                          91.5 ± 13.5

                          1.67

                          51.3 ± 68.9

                          Lysinachia christinae

                          0.06 ± 0.00

                          88.50 ± 0

                          0.85 ± 0.14

                          68.90 ± 3.1

                          Paeonia lactiflora

                          0.30 ± 0.02

                          92.80 ± 1

                          1.07 ± 0.11

                          72.15 ± 0.5

                          Paeonia suffuticosa

                          0.09 ± 0.00

                          90.30 ± 5.7

                          0.64 ± 0.09

                          63.10 ± 3.1

                          Paris polyphylla

                          0.13 ± 0.06

                          91.35 ± 6.2

                          0.30 ± 0.40

                          67.1 ± 1.6

                          Platycodon grandiflorus

                          0.43 ± 0.08

                          83.05 ± 3.6

                          1.79 ± 0.11

                          59.45 ± 2.1

                          Plantago asiatica

                          0.05 ± 0.00

                          81.9 ± 0

                          0.1

                          70 ± 8.7

                          Pleione bulbocadioides

                          0.58 ± 0.39

                          101.5 ± 0.7

                          1.59 ± 0.22

                          98.6 ± 2.0

                          Ploygala tenuifolia

                          0.10 ± 0.08

                          89.55 ± 13.8

                          0.67 ± 0.05

                          52.55 ± 12.8

                          Polygonum aviculare

                          0.23 ± 0.26

                          87.0 ± 9.20

                          0

                          NA

                          Poria cocos

                          0.34 ± 0.01

                          85.9 ± 9.8

                          0

                          NA

                          Pogostemon cablin

                          0.07 ± 0.00

                          90.65 ± 9.3

                          0.42 ± 0.22

                          67.80 ± 0.6

                          Prunella vulgaris

                          0.12 ± 0.11

                          93.90 ± 6.6

                          2.38 ± 0.4

                          94.20 ± 4

                          Pseudostellaria heterophylla

                          1.06 ± 0.41

                          109.0 ± 12.7

                          >2.5

                          NA

                          Rabdosia rubescens

                          0.05 ± 0.01

                          90.65 ± 2.1

                          0.36 ± 0.08

                          65.60 ± 2.5

                          Rehmannia glutinosa

                          1.05 ± 0.92

                          91.70 ± 11.7

                          >2.5

                          NA

                          Salvia miltiorrhiza

                          0.20 ± 0.05

                          95.35 ± 4.6

                          0.90 ± 0.06

                          62.00 ± 7.6

                          Sanguisorba officinalis

                          0.03 ± 0.01

                          84.2 ± 5.2

                          0.07 ± 0.01

                          72.6 ± 2.1

                          Schizandra chinensis

                          0.27 ± 0.32

                          88.05 ± 16.9

                          2.31 ± 0.09

                          107.65 ± 11.8

                          Scutellaria barbata

                          0.05 ± 0.03

                          98.2 ± 2.5

                          0.6

                          80.5 ± 20.5

                          Semen coicis

                          0.63 ± 0.03

                          95.40 ± 5.7

                          1.17 ± 0.22

                          94.60 ± 5.7

                          Smilax glabra

                          0.36 ± 0.38

                          92.7 ± 13.2

                          2.35 ± 0.22

                          104.5 ± 6.4

                          Solanum nigrum

                          0

                          NA

                          1.24 ± 0.2

                          67.5 ± 2.1

                          Solanum lyratum

                          0.49 ± 0.02

                          107.5 ± 10.6

                          1.68 ± 0.89

                          75.4 ± 7.2

                          Spatholobus suberectus

                          0.06 ± 0.02

                          96.8 ± 1.6

                          0.2 ± 0.04

                          82.3 ± 15.8

                          Tussilago farfara

                          0.33 ± 0.24

                          101.0 ± 1.4

                          >2.5

                          NA

                          Uncaria rhyncophylla

                          0.05 ± 0.02

                          98.80 ± 1.7

                          0.45 ± 0.05

                          92.90 ± 11.5

                          Viscum coloratum

                          0.43 ± 0.16

                          89.20 ± 8.2

                          2.46 ± 0

                          104.50 ± 6.4

                          NA: Not analyzed.

                          &Cell viability was measured at appropriate IC50 values corresponding to the inhibition of NO and TNF-α.

                          In order to understand the relationship between the antioxidant activities and polyphenolic content (total phenolics and flavonoids), the selected 44 herbs have been classified into two groups based on the correlation between antioxidant activity and polyphenol content. The first group (consisting of fifteen plants) displayed good relationship between total phenolics / flavonoids content and antioxidant activities (Table 5 and Figure 1). The remaining plants are classified as the second group which did not show a clear correlation between the antioxidant activity and their polyphenol content. The correlation between the DPPH scavenging activity and the total phenolics and flavonoids content of water extracts of the first group of fifteen plants was found to be highly significant (Figure 1A and 1B). Similar correlation was also observed for their ethanol extracts (Figure 1C and 1D). The correlations observed in this study, for the first group of plants, are in good agreement with the literature reports that the polyphenolics are the major antioxidant compounds in medicinal plants [1, 610, 4750].
                          Table 5

                          The total phenolics and flavonoids content together with the antioxidant activity of fifteen medicinal plants (first group of plants) #

                            

                          DPPH scavenging activity (μM ascorbate equiv/g)1

                           

                          Phenolics results (GAE mg/g)3

                          Flavonoids results (QE mg/g)3

                          S. No

                          Name of the plants

                          Water extracts

                          Ethanol extracts

                          % inhibition of Yeast oxidation of water extracts2

                          Water extracts

                          Ethanol extracts

                          Water extracts

                          Ethanol extracts

                          1

                          Semen coicis

                          −100.1 ± 0.71

                          11.00 ± 3.54

                          12.37

                          0.34 ± 2.32

                          1.39 ± 0.65

                          0.83 ± 1.96

                          16.05 ± 2.93

                          2

                          Pogostemon cablin

                          103.4 ± 4.24

                          26.00 ± 1.52

                          12.07

                          4.23 ± 0.68

                          1.88 ± 0.13

                          5.54 ± 0.98

                          4.08 ± 0.98

                          3

                          Pseudostellaria heterophylla

                          20.9 ±12.02

                          32.43 ± 4.55

                          36.13

                          1.74 ± 0.82

                          1.58 ± 1.83

                          0.59 ± 0.00

                          1.94 ± 0.98

                          4

                          Paeonia suffuticosa

                          200.4 ± 0.00

                          111.71 ± 0.51

                          15.16

                          19.96 ± 1.09

                          19.74 ± 0.39

                          25.85 ± 1.96

                          31.40 ± 2.93

                          5

                          Corydalis yanhusuo

                          39.9 ± 0.71

                          42.43 ± 5.56

                          14.81

                          3.51 ± 1.36

                          1.28 ± 1.48

                          1.57 ± 0.96

                          2.11 ± 0.98

                          6

                          Ploygala tenuifolia

                          109.4 ± 2.83

                          53.14 ± 2.53

                          3.23

                          12.01 ± 0.00

                          12.15 ± 0.44

                          9.3 ± 3.91

                          37.55 ± 3.91

                          7

                          Ducheshea indica

                          202.4 ± 1.41

                          102.07 ± 4.04

                          2.34

                          28.62 ± 0.68

                          11.8 ± 0.44

                          22.31 ± 4.89

                          15.25 ± 1.96

                          8

                          Asparagus cochinchinensis

                          55.9 ± 12.02

                          5.64 ± 1.01

                          16.37

                          2.67 ± 0.96

                          0.91 ± 3.01

                          1.71 ± 0.98

                          1.08 ± 0.98

                          9

                          Tussilago farfara

                          198.9 ± 0.71

                          113.5 ± 0.00

                          20.9

                          25.72 ± 2.87

                          9.68 ± 0.15

                          65.17 ± 0.98

                          41.67 ± 0.98

                          10

                          Aster tataricus

                          119.4 ± 2.83

                          49.93 ± 2.02

                          0

                          15.17 ± 0.27

                          6.95 ± 0.00

                          39.82 ± 0.98

                          22.79 ± 0.98

                          11

                          Rabdosia rubescens

                          144.4 ± 2.83

                          13.50 ± 4.04

                          8.63

                          17.47 ± 0.55

                          2.41 ± 2.96

                          40.66 ± 1.96

                          8.35 ± 2.93

                          12

                          Platycodon grandiflorus

                          30.9 ± 3.54

                          14.93 ± 4.04

                          21.77

                          4.58 ± 1.50

                          0.79 ± 0.26

                          2.93 ± 2.93

                          1.52 ± 0.96

                          13

                          Artemisia vulgaris

                          181.9 ± 0.71

                          98.14 ± 1.52

                          5.32

                          24.91 ± 0.27

                          7.43 ± 1.44

                          79.68 ± 1

                          16.43 ± 3.91

                          14

                          Ligustrum lucidum

                          194.9 ±0.71

                          107.79 ± 0

                          12.02

                          29.1 ± 3.28

                          19 ± 0.78

                          108.42 ± 1.96

                          24.88 ± 0.98

                          15

                          Scutellaria barbata

                          188.4 ±1.41

                          93.5 ± 1.01

                          10.11

                          14.97 ± 2.73

                          9.26 ± 0.15

                          67.41 ± 3.91

                          20.62 ± 0.98

                          1DPPH free radical scavenging activity was measured in terms of equivalent of ascorbate (μM).

                          2Yeast oxidative stress was measured on the basis of survival of yeast cells (yeast growth) after treatment with H2O2.

                          3Total phenolics and flavonoid contents were expressed in gallic acid equivalent (GAE mg/g) and quercetin equivalent (QE mg/g) respectively.

                          # Plants given in this table show significant correlation between their antioxidant activities and total phenolics / flavonoids contents. These plants are a subset of plants studied in this research (Table 1).

                          http://static-content.springer.com/image/art%3A10.1186%2F1472-6882-12-173/MediaObjects/12906_2012_1192_Fig1_HTML.jpg
                          Figure 1

                          Correlation between DPPH free radical scavenging activity and the total phenolics content in: (A) water extracts and (C) ethanol extracts. Correlation between DPPH free radical scavenging activity and total flavonoid content in: (B) water extracts and (D) ethanol extracts.

                          However, it should be noted that the antioxidant activities of the second group of plants did not show a good correlation between the antioxidant activity and their polyphenol content (Table 2). For example, V. coloratum, H. diffusa, L. japonicus, A. paniculata and P. lactiflora have displayed high antioxidant activity in both water and ethanol extracts, but contained low levels of phenolics and flavonoids. Similar findings were recently reported by the authors in a separate publication [6]. These observations demonstrate that, in addition to polyphenols, other constituents such as trace metals contribute to the antioxidant activities of medicinal plants. Indeed, a closer observation of the results for the second group of herbs (Tables 2 and 3) indicate that their antioxidant activities are due to the combination of polyphenols and trace metal contents. The results presented in Table 3, revealed that the second group of plants possess significant levels of trace metals. For instance, V. coloratum, has low levels of phenolics and flavonoids content but contains high levels of Zn, Mn and Mg (Table 3). This is in agreement with the literature that Zn and Mg play crucial role in antioxidant mechanisms [5153]. Similarly, H. diffusa, L. japonicus, A. paniculata showed good antioxidant activity with high levels of Zn, Mg, Mn and Se. In many organisms, trace metals have been shown to act as co-factors of several antioxidant enzymes such as superoxide dismutase (SOD), peroxidases (POD), ascorbate peroxidase (APX) and other enzymes of ascorbate – glutathione pathway [54]. Therefore, the results obtained in this study strongly support trace metal involvement in antioxidant mechanisms. It is therefore hypothesized that the medicinal herbs display their antioxidant activities due to the combination of their total phenolics, flavonoids and the trace metal contents. Some of the plants investigated in this research possessed average levels of all these classes of antioxidants (phenolics, flavonoids and trace metals) and displayed significant antioxidant activities (Tables 2 and 3).

                          In order to obtain a comprehensive picture on the correlation of the observed activity and the content, all the active plants from Table 2 are considered and their activities are correlated in terms of the three active constituents (total phenolics, flavonoids and trace metals). A diagrammatic visualization scheme has been developed for this purpose which is presented in Figure 2. A brief description of this visualization scheme is given below.
                          http://static-content.springer.com/image/art%3A10.1186%2F1472-6882-12-173/MediaObjects/12906_2012_1192_Fig2_HTML.jpg
                          Figure 2

                          A diagrammatic visualization scheme for the correlation of antioxidant activities and antioxidant content. (A) Plants with high Antioxidant activity: 1, 2, 3, 4, 5, 6, 8, 10, 11, 13, 14, 15, 16, 18, 19, 20, 23, 26, 31, 33, 34, 36, 38, 39, 40, 41, 42, and 43; (B) Plants with high antioxidant activity and also contain significant antioxidant constituents: 1, 2, 3, 4, 5, 6, 8, 10, 13, 14, 15, 16, 18, 20, 23, 26, 31, 33, 34, 36, 38, 39, 40, 41, 42, and 43; (C) Plants with medium to high Phenolic content: 6, 13, 16, 20, 31, 33, 34, 41, 42, and 43; (D) Plants with medium to high Flavonoid content: 1, 4, 6, 8, 13, 16, 20, 29, 31, 33, 34, 36, 38, 41, 42, and 43; (E) Plants with medium to high Trace metal content: 2, 5, 10, 14, 15, 18, 23, 26, 27, 28, 31, 34, 37, 39, 40, 41, and 43. (Note: The names of the plants corresponding to the numbers in the legend are given in Table 1).

                          All the plants with high DPPH scavenging activities (> 120 Ascorbate equivalent / g) have been included in the “Circle A” of Figure 2. However, the plants included in “Circle B” not only have high scavenging activity but also contain significant quantity of one or more antioxidant constituents (phenolics, flavonoids or trace metals). This automatically means that the plants in “Circle B” are a sub-set of plants in “Circle A”. Circles C, D and E represent the plants with medium to high quantities of phenolics, flavonoids and trace metals respectively (Tables 2 and 3). Any plant that exists in “Circle A” and also is present in one or more of the “Circles C, D or E” will be transferred into “Circle B”. Overlaps between antioxidant content circles (C, D, E) and antioxidant activity circle (B) represent the activities with respect to the corresponding constituents. As can be seen from Figure 2, the plants are active due to the presence of one or more out of the three active constituents. Some of the plants exhibit their activity due to the presence of all of the three antioxidant constituents (phenolics, flavonoids or trace metals). For instance, the plants R. rubescens, S. officinalis, S. suberectus and U. rhyncophylla have displayed their activities due to the presence of significant quantities of all the three antioxidant constituents (Figure 2). These findings further support the hypothesis that the medicinal herbs display their antioxidant activities due to the combination of their total phenolics, flavonoids and the trace metal contents. It should be noted here that only water extracts are considered in Figure 2.

                          It may be concluded from the above visualization scheme that the activities of 26 out of the 28 active plants could be explained in terms of their antioxidant content. Two of the plants, namely, C. paniculatum and P. lactiflora have displayed high antioxidant activities but did not contain significant quantities of any of the antioxidant constituents (Table 2 and Figure 2). One of the reasons for this non-correlation is likely to be due to the fact that some of the polyphenols may be extremely active owing to their structural characteristics even if they are present in smaller quantities [1]. Other reason includes the occurrence of antioxidant constituents (such as polysaccharides) that are not investigated in this study. Several studies demonstrated that botanical polysaccharides possess strong antioxidant activities [5557].

                          Significant anti-inflammatory activities were observed for majority of the medicinal herbs studied here (Table 4). For instance, S. officinalis, D. indica, P. suffuticosa, U. rhyncophylla and R. rubescens have inhibited NO / TNF-α with low IC50 values and also contain high phenolics and flavonoids content. Bioactive molecules isolated in the literature from some of these plants showed significant anti-inflammatory properties [58, 59]. For example, Sanguiin H-6 and H-11 isolated from S. officinalis has decreased the expression levels of iNOS [58]. Rhyncophylline and isorhyncophylline are isomeric alkaloids from U. rhyncophylla showed inhibition activity against the NO production and proinflammatory cytokines such as TNF-α and IL-1β production in LPS induced mouse N9 microglial cells [59].

                          Conclusions

                          Forty-four selected medicinal plants have been investigated in this study for their antioxidant and anti-inflammatory activities. Amongst these plants, two distinct groups have been identified in terms of the correlation of antioxidant activities and their antioxidant contents. First group exhibited good relationship between total phenolics / flavonoids content and antioxidant activity, whilst in the second group such a relationship was poor. The observed biological activities of all the plants including those in the second group, could clearly be explained when trace metal content was considered together with polyphenols content. Amongst all the selected plants, L. lucidum, P. suffuticosa, S. miltiorrhiza, S. officinalis, S. suberectus, T. farfara and U.rhyncophylla showed significant antioxidant and anti-inflammatory activities with very low toxic effects. Consequently, the isolation of bioactive compounds from these target plants is underway in our laboratory.

                          Declarations

                          Authors’ Affiliations

                          (1)
                          School of Science and Health
                          (2)
                          Centre for Complementary Medicine Research
                          (3)
                          School of Medicine, University of Western Sydney
                          (4)
                          School of Pharmacy and Molecular Sciences, James Cook University
                          (5)
                          CSIR-National Geophysical Research Institute

                          References

                          1. Cai Y, Luo Q, Sun M, Corke H: Antioxidant activity and phenolic compounds of 112 traditional Chinese medicinal plants associated with anticancer. Life Sci 2004, 74:2157–2184.PubMedView Article
                          2. Dragland S, Senoo H, Wake K, Holte K, Blomhoff R: Several culinary and medicinal herbs are important sources of dietary antioxidants. J Nutr 2003, 133:1286–1290.PubMed
                          3. Akinmoladun AC, Obuotor EM, Farombi EO: Evaluation of antioxidant and free radical scavenging capacities of some Nigerian indigenous medicinal plants. J Med Food 2010, 13:444–451.PubMedView Article
                          4. Özen T, Çöllü Z, Korkmaz H: Antioxidant Properties of Urtica pilulifera Root, Seed, Flower, and Leaf Extract. J Med Food 2010, 13:1224–1231.PubMedView Article
                          5. Hendra R, Ahmad S, Oskoueian E, Sukari A, Shukor MY: Antioxidant, Anti-inflammatory and Cytotoxicity of Phaleria macrocarpa (Boerl.) Scheff Fruit. BMC Complement Altern Med 2011, 11:110.PubMedView Article
                          6. Zhang L, Ravipati AS, Koyyalamudi SR, Jeong SC, Reddy N, Smith PT, Bartlett J, Shanmugam K, Münch DG, Wu MJ: Antioxidant and Anti-inflammatory Activities of Selected Medicinal Plants Containing Phenolic and Flavonoid compounds. J Agric Food Chem 2011, 59:12361–12367.PubMedView Article
                          7. Gülçin I: Antioxidant activity of food constituents: an overview. Arch Toxicol 2012, 86:345–391.PubMedView Article
                          8. Rivero MD, Pérez-Magariño S, González-Sanjosé ML, Valls-Belles V, Codoñer P, Muñiz P: Inhibition of induced DNA oxidative damage by beers: Correlation with the content of polyphenols and melanoidins. J Agric Food Chem 2005, 53:3637–3642.PubMedView Article
                          9. Gião MS, González-Sanjosé ML, Rivero-Pérez MD, Pereira CI, Pintado ME, Malcata FX: Infusions of Portuguese medicinal plants: dependence of final antioxidant capacity and phenol content on extraction features. J Sci Food Agr 2007, 87:2638–2647.View Article
                          10. Raudoniūtė I, Rovira J, Venskutonis PR, Damašius J, Rivero-Pérez MD, González-SanJosé ML: Antioxidant properties of garden strawberry leaf extract and its effect on fish oil oxidation. Int J Food Sci Tech 2011, 46:935–945.View Article
                          11. Fraga CG: Relevance, essentiality and toxicity of trace elements in human health. Mol Aspect Med 2005, 26:235–244.View Article
                          12. Purdey M: Ecosystems supporting clusters of sporadic TSEs demonstrate excesses of the radical-generating divalent cation manganese and deficiencies of antioxidant co factors Cu, Se, Fe, Zn. Med hypotheses 2000, 54:278–306.PubMedView Article
                          13. Talhouk R, Karam C, Fostok S, El-Jouni W, Barbour E: Anti-inflammatory bioactivities in plant extracts. J Med Food 2007, 10:1–10.PubMedView Article
                          14. Lee MS, Yuet-Wa JC, Kong SK, Yu B, Eng-Choon VO, Nai-Ching HW, Chung-Wai TM, Fung KP: Effects of Polyphyllin D, a Steroidal Saponin in, in Growth Inhibition of Human Breast Cancer Cells and in Xenograft. Canc Biol Ther 2005, 4:1248–1254.View Article
                          15. Wang C, Schuller Levis GB, Lee EB, Levis WR, Lee DW, Kim BS, Park SY, Park E: Platycodin D and D3 isolated from the root of Platycodon grandiflorum modulate the production of nitric oxide and secretion of TNF-[alpha] in activated RAW 264.7 cells. Int J Immunopharmacol 2004, 4:1039–1049.View Article
                          16. Ou B, Huang D, Hampsch-woodill M, Flanagan J: When east meets west: the relationship between yin-yang and antioxidation-oxidation. The FASEB Journal 2003, 17:127.View Article
                          17. Prior RL, Guohua C: Antioxidant phytochemicals in fruits and vegetables: Diet and health implications: The role of oxidative stress and antioxidants in plant and human health. Hort Science 2000, 35:588–592.
                          18. Huang HB, Li KX, Liu T, Zeng CQ, Lin J, Qiu M: Kang Zhong Liu Zhong Yao Lin Chuang Ying Yong Yu Tu Pu (Clinical application of anti-tumor Chinese medicine). Guang Zhou: Guangdong Science and Technology Press; 2008.
                          19. Wu MJ, O'Doherty PJ, Fernandez HR, Lyons V, Rogers PJ, Dawes IW, Higgins VJ: An antioxidant screening assay based on oxidant‐induced growth arrest in Saccharomyces cerevisiae. FEMS yeast research 2011, 11:379–387.PubMedView Article
                          20. Moon JK, Shibamoto T: Antioxidant assays for plant and food components. J Agric Food Chem 2009, 57:1655–1666.PubMedView Article
                          21. Dudonneì S, Vitrac X, CoutieÌre P, Woillez M, Meìrillon JM: Comparative study of antioxidant properties and total phenolic content of 30 plant extracts of industrial interest using DPPH, ABTS, FRAP, SOD, and ORAC assays. J Agric Food Chem 2009, 57:1768–1774.View Article
                          22. Han S, Yoon Y, Ahn H, Lee H, Lee C, Yoon W, Park S, Kim H: Toll-like receptor-mediated activation of B cells and macrophages by polysaccharide isolated from cell culture of Acanthopanax senticosus. Int J Immunopharmacol 2003, 3:1301–1312.View Article
                          23. Fangyu H, Fei C, Yi L: Studies on anti infechve and antitumor effects of Actinidia arguta stem polysaccharide (AASP)[J]. J Norman Bethune Uni. Med Sci 1995., 5:
                          24. Ikuta A, Itokawa H: Triterpenoids of Akebia quinata callus tissue. Phytochemistry 1986, 25:1625–1628.View Article
                          25. Lee SE, Hwang HJ, Ha JS, Jeong HS, Kim JH: Screening of medicinal plant extracts for antioxidant activity. Life sci 2003, 73:167–179.PubMedView Article
                          26. Ajaya Kumar R, Sridevi K, Vijaya Kumar N, Nanduri S, Rajagopal S: Anticancer and immunostimulatory compounds from Andrographis paniculata. J. Ethnopharmacol 2004, 92:291–295.PubMedView Article
                          27. Sujatha G, Kumari BDR: Effect of phytohormones on micropropagation of Artemisia vulgaris L. Acta Physiologiae Plantarum 2007, 29:189–195.View Article
                          28. Cho WCS: Supportive cancer care with Chinese medicine. New York: Springer Verlag; 2010.View Article
                          29. Ng T, Liu F, Lu Y, Cheng C, Wang Z: Antioxidant activity of compounds from the medicinal herb Aster tataricus. Comp Biochem Physiol C Toxicol Pharmacol 2003, 136:109–115.PubMedView Article
                          30. Syu WJ, Shen CC, Don MJ, Ou JC, Lee GH, Sun CM: Cytotoxicity of curcuminoids and some novel compounds from Curcuma zedoaria. J Nat Prod 1998, 61:1531–1534.PubMedView Article
                          31. Zhao L, Zhang SL, Tao JY, Jin F, Pang R, Guo YJ, Ye P, Dong JH, Zheng GH: Anti-inflammatory mechanism of a folk herbal medicine, Duchesnea indica (Andr) Focke at RAW264. 7 cell line. Immunol Investig 2008, 37:339–357.View Article
                          32. Tao J, Zhang P, Liu G, Yan H, Bu X, Ma Z, Wang N, Wang G, Jia W: Cytotoxicity of Chinese motherwort (YiMuCao) aqueous ethanol extract is non-apoptotic and estrogen receptor independent on human breast cancer cells. J Ethnopharmacol 2009, 122:234–239.PubMedView Article
                          33. Zhang J, Li L, Kim SH, Hagerman AE, Lü J: Anti-cancer, anti-diabetic and other pharmacologic and biological activities of penta-galloyl-glucose. Pharmaceut Res 2009, 26:2066–2080.View Article
                          34. Galvez M, Martin-Cordero C, Lopez-Lazaro M, Cortes F, Ayuso MJ: Cytotoxic effect of Plantago spp. on cancer cell lines. J Ethnopharmacol 2003, 88:125–130.PubMedView Article
                          35. Kim H, Lee E, Na H, Lee S, Shin T, Lyu Y, Kim N, Nomura S: Effect of Polygala tenuifolia root extract on the tumor necrosis factor-[alpha] secretion from mouse astrocytes. J Ethnopharmacol 1998, 61:201–208.PubMedView Article
                          36. Hsu CY: Antioxidant activity of extract from Polygonum aviculare L. Biol Res 2006, 39:281–288.PubMedView Article
                          37. Park EJ, Park HR, Lee JS, Kim J: Licochalcone A: an inducer of cell differentiation and cytotoxic agent from Pogostemon cablin. Planta Medica-Natural Products and Medicinal Plant Research 1998, 64:464–465.
                          38. Feng L, Jia XB, Shi F, Chen Y: Identification of two polysaccharides from Prunella vulgaris L. and evaluation on their anti-lung adenocarcinoma activity. Molecules 2010, 15:5093–5103.PubMedView Article
                          39. Hancke JL, Burgos RA, Ahumada F: Schizandra chinensis Turcz. Baill. Fitoterapia 1999, 70:451–471.View Article
                          40. Kim MR, Lee JY, Lee HH, Aryal DK, Kim YG, Kim SK, Woo ER, Kang KW: Antioxidative effects of quercetin-glycosides isolated from the flower buds of Tussilago farfara L. Food Chem Toxicol 2006, 44:1299–1307.PubMedView Article
                          41. Heitzman ME, Neto CC, Winiarz E, Vaisberg AJ, Hammond GB: Ethnobotany, phytochemistry and pharmacology of Uncaria (Rubiaceae). Phytochemistry 2005, 66:5–29.PubMedView Article
                          42. Hwang TL, Leu YL, Kao SH, Tang MC, Chang HL: Viscolin, a new chalcone from Viscum coloratum, inhibits human neutrophil superoxide anion and elastase release via a cAMP-dependent pathway. Free Radic Biol Med 2006, 41:1433–1441.PubMedView Article
                          43. Cicco N, Lanorte MT, Paraggio M, Viggiano M, Lattanzio V: A reproducible, rapid and inexpensive Folin-Ciocalteu micromethod in determining phenolics of plantmethanol extracts. Microchem J 2009, 91:107–110.View Article
                          44. Jia ZS, Tang MC, Wu JM: The determination of flavonoid contents in mulberry and their scavenging effects on superoxide radicals. Food Chem 1999, 64:555–559.View Article
                          45. Blois MS: Antioxidant determinations by the use of a stable free radical. Nature 1958, 181:1199–1200.View Article
                          46. Shanmugam K, Holmquist L, Steele M, Stuchbury G, Berbaum K, Schulz O, Benavente-Garcia O, Castillo J, Burnell J, Garcia Rivas V, Dobson G, Munch G: Plant-derived polyphenols attenuate lipopolysaccharide-induced nitric oxide and tumour necrosis factor production in murine microglia and macropgages. Mol Nutr Food Res 2008, 52:427–438.PubMedView Article
                          47. Gülçin I, Küfrevioğlu OI, Oktay M, Büyükokuroğlu ME: Antioxidant, antimicrobial, antiulcer and analgesic activities of nettle (Urtica dioica L.). J Ethnopharmacol 2004, 90:205–215.PubMedView Article
                          48. Gulcin I, Tel AZ, Kirecci E: Antioxidant, antimicrobial, antifungal, and antiradical activities of Cyclotrichium niveum (BOISS.). Manden and Scheng. Int J Food Prop 2008, 11:450–471.View Article
                          49. Rivero-Pérez MD, MUNiz P, González-Sanjosé ML: Antioxidant profile of red wines evaluated by total antioxidant capacity, scavenger activity, and biomarkers of oxidative stress methodologies. J Agri Food Chem 2007, 55:5476–5483.View Article
                          50. Phillipson J: Phytochemistry and medicinal plants. Phytochemistry 2001, 56:237–243.PubMedView Article
                          51. Arnér ESJ, Holmgren A: Physiological functions of thioredoxin and thioredoxin reductase. Eur J Biochem 2000, 267:6102–6109.PubMedView Article
                          52. Cross H, Pepper T, Kearsley M, Birch G: Mineral complexing properties of food carbohydrates. Starch‐Stärke 1985, 37:132–135.View Article
                          53. Ho E: Zinc deficiency, DNA damage and cancer risk. J Nutr Biochem 2004, 15:572–578.PubMedView Article
                          54. Cartes P, Jara A, Pinilla L, Rosas A, Mora M: Selenium improves the antioxidant ability against aluminium‐induced oxidative stress in ryegrass roots. Ann Appl Biol 2010, 156:297–307.View Article
                          55. Schepetkin IA, Quinn MT: Botanical polysaccharides: macrophage immunomodulation and therapeutic potential. Immunopharmacology 2006, 6:317–333.View Article
                          56. Minato K: Mushrooms: Immunomodulating Activity and Role in Health Promotion. In Dietary Components and Immune Function. Edited by: Watson RR, Zibadi S, Preedy VR. New York: Humana press; 2010:529–539.View Article
                          57. He XJ, Niu XY, Li J, Xu SH, Lu AP: Immunomodulatory activities of five clinically used Chinese herbal polysaccharides. J Exper Integr Med 2012, 2:15–27.
                          58. Mimaki Y, Fukushima M, Yokosuka A, Sashida Y, Furuya S, Sakagami H: Triterpene glycosides from the roots of Sanguisorba officinalis. Phytochemistry 2001, 57:773–779.PubMedView Article
                          59. Yuan D, Ma B, Yang J, Xie Y, Wang L, Zhang L, Wu KY: Anti-inflammatory effects of rhynchophylline and isorhynchophylline in mouse N9 microglial cells and the molecular mechanism. Int Immunopharm 2009, 9:1549–1554.View Article
                          60. Pre-publication history

                            1. The pre-publication history for this paper can be accessed here:http://​www.​biomedcentral.​com/​1472-6882/​12/​173/​prepub

                          Copyright

                          © Ravipati et al.; licensee BioMed Central Ltd. 2012

                          This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://​creativecommons.​org/​licenses/​by/​2.​0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.