Chinese herbal extracts of Rubia cordifolia and Dianthus superbus suppress IgE production and prevent peanut-induced anaphylaxis

  • Iván López-Expósito1, 2,

    Affiliated with

    • Alexandra Castillo1,

      Affiliated with

      • Nan Yang1,

        Affiliated with

        • Banghao Liang1 and

          Affiliated with

          • Xiu-Min Li1Email author

            Affiliated with

            Chinese Medicine20116:35

            DOI: 10.1186/1749-8546-6-35

            Received: 1 December 2010

            Accepted: 30 September 2011

            Published: 30 September 2011

            Abstract

            Background

            Peanut allergy is characterized by increased levels of peanut-specific IgE in the serum of most patients. Thus, the most logical therapy would be to inhibit the IgE production by committed B-cells. This study aims to investigate the unreported anti-IgE effects of Chinese herbal extracts of Rubia cordifolia (Qiancao) and Dianthus superbus (Qumai).

            Methods

            Seventy herbal extracts were tested for their ability to reduce IgE secretion by a human B-cell line. Those with the lowest inhibitory concentration 50 (IC50) values were tested in a mouse model of peanut-anaphylaxis. Anaphylactic scores, body temperature, plasma histamine and peanut-specific-immunoglobulins were determined.

            Results

            Rubia cordifolia and Dianthus superbus inhibited the in vitro IgE production by a human B-cell line in a dose-dependent manner and the in vivo IgE production in a murine model of peanut allergy without affecting peanut-specific-IgG1 levels. After challenge, all mice in the sham groups developed anaphylactic reactions and increased plasma histamine levels. The extract-treated mice demonstrated significantly reduced peanut-triggered anaphylactic reactions and plasma histamine levels.

            Conclusion

            The extracts of Rubia cordifolia and Dianthus superbus inhibited the IgE production in vivo and in vitro as well as reduced anaphylactic reactions in peanut-allergic mice, suggesting potentials for allergy treatments.

            Background

            Peanut allergy (PNA) is a worldwide health concern, particularly in developed countries. PNA accounts for approximately 80% of fatal and near-fatal food allergic reactions [1]. The prevalence of childhood PNA in the United States (USA) is currently at 1.4%, up from 0.8% in 2002 and 0.4% in 1997 [1]. Apart from strict avoidance of the peanut-containing foods, no satisfactory therapy is available to prevent or reverse this condition. Standard subcutaneous immunotherapy has been abandoned due to undesirable adverse reactions and marginal efficacy [2]. While peanut oral immunotherapy (OIT) is a promising therapeutic intervention for PNA [3], many questions remain, such as the risks of OIT compared with avoidance, dosing regimen issues, patient selection and post desensitization strategy [4]. Sublingual immunotherapy (SLIT) is a new method of treating food allergy, with few systemic reactions; however, only one study [5] determined the effect of SLIT on PNA. For these reasons, a safe and effective therapy for peanut allergy is urgently needed.

            Research suggests that certain Chinese medicinal herbs may have the potential for treating allergy and asthma [6]. For the first time, our group developed a food allergy herbal formula (FAHF2) that blocks peanut-induced anaphylaxis in a mouse model [7, 8]. A recent clinical trial demonstrated that the FAHF2 is safe and well-tolerated, with beneficial immunomodulatory effects in vitro[9].

            Similar to other allergies, PNA is characterized by increased levels of peanut-specific IgE in the serum of most patients. Cross-linking of mast cell/basophil membrane cell-bound IgE antibodies by allergen results in the release of inflammatory mediators responsible for the signs and symptoms of PNA [10]. Omalizumab (Xolair) is the only available anti-IgE therapy which is one of the most logical therapies for PNA. Omalizumab effectively neutralizes IgE and may even cause apoptosis of committed B-cells by cross linking membrane IgE. However, relapse is likely if the antibody treatment stops [11, 12]. While investigation of anti-allergic therapies from natural products sources has been increasing in the past years, the number of studies on reducing IgE production are limited [13].

            The present study aims to investigate Chinese medicinal herbs that have previously unreported anti-IgE effects. Seventy herbal extracts were tested for their ability to reduce the IgE secretion by a human myeloma B-cell line. Those with the lowest IC50 values were then tested in a mouse model of peanut-anaphylaxis.

            Methods

            Herbs

            All medicinal herbs used in this study were purchased from EFong Herbs Inc. (USA). These products were made by Gangdong Yifang Pharmaceutical Company Ltd. (China) and commercially available in the US via EFong Herbs Inc. All herbs were extracted with water and then concentrated and dried. The manufacturing processes and the product quality analyses are in accordance with GMP standards [14]. Each powdered extract was packaged and stored at room temperature under dark and dry conditions.

            High performance liquid chromatography (HPLC) fingerprints from Qiancao and Qumai

            HPLC fingerprints are recommended by the United States Food and Drug Administration as a means of standardization for botanical products. HPLC was carried out as previously described [9, 15, 16]. Briefly, 200 mg of Qiancao (QC) and Qumai (QM) extracts were dissolved into 2 mL mobile phase mixture consisting of 0.10% formic acid and acetonitrile (1:1). Each sample solution was filtered through a 0.2 μm filter (Whatman Inc., USA). Each sample (10 mL) was analyzed on a Waters Alliance 2695 HPLC system (Waters Corporation, USA) with a photodiode array detector (2996) (Waters Corporation, USA). The separation conditions were as follows: Zorbax SB-C18 column (150 × 4.6 mm; 5 μm particle size) from Agilent Technologies (USA); mobile phases: 0.10% formic acid (A) and acetonitrile (B); flow rate: 1.0 mL/min; detection wavelength: 254 nm. Linear separation gradient was from 2% of B to 48% for 75 minutes. Chromatographic results were acquired and processed with the Waters' Empower software (Waters Corporation, USA). All chemicals and solvents used were of HPLC grade (Fisher Scientific, USA). HPLC fingerprints of QC and QM are shown in Figure 1.
            http://static-content.springer.com/image/art%3A10.1186%2F1749-8546-6-35/MediaObjects/13020_2010_Article_138_Fig1_HTML.jpg
            Figure 1

            HPLC chromatograms of Qiancao ( Rubia cordifolia ) and Qumai ( Dianthus superbus ). Panel A: Qiancao; Panel B: Qumai. HPLC conditions: column, Agilent Zorbax SB-C18 column (150 × 4.6 mm i.d.); flow rate, 1 mL/min; wavelength, 254 nm; mobile phase A, 0.1% formic acid, mobile phase B, acetonitrile. Data was analyzed using Waters Empower software.

            U266 human B cell cultures and IgE measurement

            Human U266B1 multiple myeloma cells (ATCC TIB-196™, American Type Culture Collection, USA) were cultured at 37°C in 5% CO2. RPMI 1640 medium, supplemented with 10% of fetal bovine serum (FBS), 1 mM sodium pyruvate, 1 × 10-5 M 2-ME and 0.5% penicillin-streptomycin, was used as a culture medium. Cells were grown at an initial concentration of 2 × 105 cells/mL. Initially, all herbal extracts (Table 1) were added at Day 0 at 500 μg/mL and 100 μg/mL. At Day 6 the supernatants were harvested for total IgE assay. For those herbs with an IgE inhibition rate higher than 95% at both concentrations assayed, a dose-inhibition curve was performed.

            Total IgE (T-IgE) was examined with a fluorescent enzyme immunoassay (ImmunoCAP FEIA, Phadia, Germany) and expressed in kU/L. The detection range of T-IgE was 2-2000 kU/L. Samples were measured undiluted, while samples with undetectable T-IgE were assigned an arbitrary value of 1 kU/L. The percentage of IgE inhibition was calculated as 100-[IgE concentration (sample treated) × 100/IgE concentration (sample untreated)]

            Cell viability assays for QC and QM cultures

            The viability of control and treated cells was evaluated with the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay in triplicates. Briefly, cells (2 × 104) were incubated in 96-well microtiter plate containing 100 μL of the culture medium (RPMI 1640 medium supplemented with 10% FBS, 1 mM sodium pyruvate, 1 × 10-5 M 2-ME and 0.5% penicillin-streptomycin) with or without tested compounds at 0, 3.125, 6.25, 12.5 25, 50, and 100 μg/ml). The MTT assay was performed after six days. Cells in each well were incubated at 37°C in 20 μg of the MTT (1 mg/mL) for four hours. After incubation, 150 μL of Dimethyl sulfoxide (DMSO) was added to each well. Absorbance of the mixture at 595 nm was determined with a microplate ELISA reader. The results were derived from three independent experiments.

            In vivo experimental protocol

            Female C3He/J mice (6 weeks old) were purchased from Jackson Laboratory (USA). Standard guidelines for the care and use of animals were followed [17]. To generate a peanut allergy model, we sensitized the mice intraperitoneally (i.p.) each week with 200 μg of crude peanut extract (CPE) and 2 mg of alum in 0.5 mL of phosphate buffered saline (PBS) for four weeks, and then challenged (i.p.) them with 1000 μg CPE in 500 μL PBS two weeks after the last sensitization. To determine whether QC and/or QM prevent peanut anaphylactic reactions, we administered extracts of QC (2 mg) or QM (2 mg), or QC (4 mg) or QM (4 mg) in 0.5 mL of water intragastrically (i.g.) twice daily for five weeks beginning at Day 0 of the protocol. The dose was determined by a conversion table of equivalent human to animal dose ratios based on body surface area [18]. Additional peanut-sensitized mice received 0.5 mL water (i.g.) twice daily for five weeks as sham treatment controls (sham). Naïve mice served as normal controls (Figure 2).
            http://static-content.springer.com/image/art%3A10.1186%2F1749-8546-6-35/MediaObjects/13020_2010_Article_138_Fig2_HTML.jpg
            Figure 2

            Experimental protocol. Mice were sensitized weekly for four weeks intraperitoneally (i.p.) with 200 μg of CPE and 2 mg of alum and then challenged i.p with 1000 μg CPE 2 weeks after the last sensitization. To determine whether QC and/or QM extracts prevent peanut anaphylactic reactions, we administered QC or QM at 2 mg or 4 mg intragastrically (i.g.) to a group of mice twice daily for five weeks beginning at Day 0 of the protocol (n = 5-8 mice per group).

            Assessment of systemic anaphylactic signs and measurement of core body temperatures

            Anaphylactic signs were evaluated 30 to 40 minutes after the commencement of the challenge by two investigators using the following scoring: 0, no signs; 1, scratching and rubbing around the mouth and head; 2, puffiness and redness around the eyes and mouth, diarrhea, pilar erecti, reduced activity and/or decreased activity with increased respiratory rate; 3, wheezing, labored respiration and cyanosis around the mouth and tail; 4, no activity after prodding or tremor and convulsions and 5, death. Rectal temperatures were measured with a thermal probe (Harvard Apparatus, USA) every 15 minutes during the 30 minutes after the peanut challenge.

            Measurement of plasma histamine and mouse mast cell protease-1 (MMCP1) levels

            Plasma was obtained 30 minutes after the challenge, histamine and MMCP1 levels were analyzed with an enzyme immunoassay kit as described by the manufacturers (Immunotech, France and Moredun Scientific, UK for histamine and MMCP1 measurements respectively).

            Measurement of serum antibodies

            Retro-orbital venous blood was collected before the challenge. Sera were collected and stored at -80°C until analysis. Peanut-specific IgE and IgG1 levels were determined with a monoclonal antibody as previously described [19].

            Acute and sub chronic toxicity studies

            The lethal dose 50 (LD50) protocol was designed as follows. Seven-week old mice were fed 12 times the highest therapeutic dose used in this experiment and observed for 12 and 24 hours; the LD50 was then calculated. Mice fed with water served as controls (sham). If no death occurred 12 and 24 hours after feeding, mice were observed for an additional 14 days. If no death occurred during this observation period, all mice were sacrificed. Samples were then collected for biochemical analyses, complete blood cell counts (CBC) and histological analyses. Biochemical analyses of blood urea nitrogen (BUN) as well as creatinine and alanine aminotransferase (ALT) were performed on a PROCHEM-V instrumentation (Synbiotics, USA) for the assessment of the kidney and liver functions respectively. For CBC testing, blood samples (20 μL) were collected and subjected to analysis by Multispecies Hematology Systems (CDC Technologies, USA). These assays were performed at Mount Sinai School of Medicine, Center for Laboratory Animal Sciences, where these assays are routinely performed to monitor the health of laboratory animals. Histological analysis of major organs (ie kidney, liver, heart, spleen, lung, stomach and intestine) was performed in a blinded manner.

            Statistical analysis

            One-way or two-way ANOVA (analysis of variance) was performed followed by a Bonferroni correction for all pairwise comparisons if the data were approximately normal. If the data were not normally distributed, differences among multiple groups were analyzed by a Kruskal-Wallis ANOVA on ranks and Bonferroni correction was performed for all pairwise comparisons. P values of < 0.05, based on two-tailed tests, are considered statistically significant. Outliers were discarded based on Grubss test [20]. All statistical analyses were performed with GraphPad Prism (GraphPad Software Inc., USA).

            Results

            Anti-IgE screening for the Chinese medicinal herbs

            Seventy herbs extracts from our herbal repository with demonstrated anti-inflammatory actions were screened for potential anti-IgE properties via incubating them with an IgE producing human B-cell line (U266B1). Herbal extracts were added at Day 0 at concentrations of 500 μg/mL and 100 μg/mL. After six days of incubation, IgE levels in the supernatants were measured. Forty-nine of the 70 herbal extracts inhibited IgE production by less than 50% at 500 μg/mL. Nine inhibited between 50% and 80%, and 12 inhibited more than 80% (Table 1). At 100 μg/mL, the extracts of Houpo (Magnolia officinalis; 64%), Huangbai (Phellodendron amurense; 63.3%), Huangqin (Scutellaria baicalensis, 63.9%), QC (Rubia cordifolia; 98.5%) and QM (Dianthus superbus; 96.7%) inhibited more than 50%. Due to their remarkable inhibitory effects at 100 μg/ml, QC and QM were selected for further studies. First, dose response curves were determined as shown in Figure 3A and 3B. Both extracts, dose-dependently (3.125-100 μg/mL) inhibited IgE production, with IC50 values being 3.06 μg/mL (QC) and 12.33 μg/mL (QM). Furthermore, QC and QM did not affect the viability of U266B1 cells (Figure 3C and 3D), demonstrating that QC and QM have potent anti-IgE effect in a non-toxic manner.
            Table 1

            Selected Chinese medicinal plants with the percentage of IgE inhibition at the concentrations indicated

            Pinyin name

            Botanical name

            Part used

            % IgE inhibition

            500 μg/mL

            % IgE inhibition

            100 μg/mL

            Ai Ye

            Artemisiae argyi

            Leaves

            69.5

            14.4

            Bai Guo Ren

            Ginkgo bilobae

            Seeds

            0

            0

            Bai He

            Lilium brownii

            Bulb

            0

            0

            Bai Hua She She Cao

            Heydyotis diffusa

            Whole

            19.4

            10.8

            Bai Jiang Cao

            Patrinia scabiosaefolia

            Whole

            17.2

            0

            Bai Shao

            Paeoniae lactiflora

            Root

            25.2

            5.7

            Bai Tou Weng

            Pulsatillae chinensis

            Root

            86.5

            11.5

            Bai Zhu

            Atractylodes Macrocephala

            Rhizome

            10.3

            0

            Ban Bian Lian

            Lobelia chinensis

            Whole

            21.0

            3.9

            Ban Xia

            Pinellia ternata

            Rhizome

            15.6

            11.5

            Ban Zhi Lian

            Scutellaria Barbata

            Whole

            39.1

            16.6

            Bei Sha Shen

            Adenophora tetraphylla

            Root

            7.0

            0

            Bu Gu Zhi

            Psoraleae coryfolia

            Fruit

            17.4

            21.4

            Cang Er Cao

            Xanthium sibiricum

            Whole

            7.2

            11.5

            Cang Zhu

            Atractylodes lancea

            Root

            19.9

            8.6

            Chai Hu

            Bupleurum chinense

            Root

            31.3

            11.1

            Chan Tui

            Cryptotympana atrata

            Seeds

            1.0

            0

            Che Qian Zi

            Plantago asiatica

            Seeds

            14.4

            12.9

            Chuan Xin Lian

            Melia toosedan

            Root

            67.5

            18.6

            Da Huang

            Rheum palmatum

            Root

            71.29

            5.2

            Da Qing Ye

            Isatis tinctoria

            Leaves

            37.34

            12.88

            Dan Shen

            Salvia miltiorrhiza

            Root

            81.09

            3.1

            Dang Gui

            Angelica sinensis

            Root

            10.8

            0

            Di Gu Pi

            Lycium chinense

            Bark

            31.35

            0

            E Jiao

            Equus asinus

            Gelatin

            9.9

            0

            Fu Ling

            Poria cocos

            Fruit body

            11.16

            10.3

            Gan Cao

            Glycyrrhiza uralensis

            Root

            7.2

            11.5

            Gan Jiang

            Zingiber officinalis

            Root

            15.4

            2

            Gua Lou

            Trichosanthes kirilowii

            Whole

            44.7

            22.6

            Hong Hua

            Carthamus tinctorius

            Flower

            0

            10.3

            Hong Shen

            Panax ginseng

            Root

            19.9

            4.3

            Hou Po

            Magnolia officinalis

            Bark

            90.1

            64.0

            Huang Bai

            Phellodendron amurense

            Bark

            96.6

            63.3

            Huang Qin

            Scutellaria baicalensis

            Root

            94.4

            63.9

            Huang Yao Zi

            Dioscorea bulbifera

            Seeds

            70.9

            15.0

            Ku Shen

            Sophora flavescens

            Root

            1.0

            0

            Ling Zhi

            Ganoderma Lucidum

            Fruit body

            14.7

            9.6

            Ma Bo

            Lasiosphera fenslii

            Fruit body

            4.5

            ND

            Mai Dong

            Ophiopogon japonicus

            Root

            4.3

            4.3

            Mu Dan Pi

            Paeonia suffruticosa

            Root bark

            95.7

            14.3

            Mu Gua

            Chaenomeles lagenaria

            Fruit

            10.8

            1.6

            Mu Li

            Ostrea gigas

            Shell

            9.1

            5.2

            Qian Cao

            Rubia cordifolia

            Root

            98.7

            98.5

            Qu Mai

            Dianthus superbus

            Whole

            98.4

            96.7

            Rou Gui

            Cinnamomum cassia

            Bark

            13.3

            0

            San Qi

            Panax notoginseng

            Root

            0

            0

            Shan Ci Gu

            Cremastra variabilis

            Fruit body

            6.2

            5.4

            Shan Dou Gen

            Sophora tonkineenis

            Root

            15.1

            0

            Shan Zha

            Crataegus pinnatifida

            Fruit

            0

            0

            Shan Zhu Yu

            Cornus officinalis

            Fruit

            16.1

            8.2

            She Gan

            Belamcanda chinensis

            Rhizome

            54.1

            15.1

            Sheng Jiang

            Drynaria fortunei

            Rhizome

            90.6

            14.2

            Sheng Ma

            Cimicifuga foetida

            Rhizome/root

            31.1

            ND

            Shi Chang Pu

            Acorus gramineus

            Rhizome

            12.3

            8.0

            Si Gua Luo

            Luffa cylindrical

            Loofah

            0

            ND

            Tian Dong

            Asparagus cochinchinensis

            Root

            3.49

            ND

            Tian Hua Fen

            Trichosanthis kirilowii

            Root

            12.78

            ND

            Tian Nan Xing

            Arisaema consaguineum

            Fruit

            0

            ND

            Tou Weng

            Radix Pulsatibae

            Root

            86.9

            15.4

            Tu Fu Ling

            Smilax glabra

            Rhizome

            66.4

            0

            Wu Zhu Yu

            Evodia rutaecarpa

            Fruit

            69.5

            13.3

            Xia Ku Cao

            Prunella vulgaris

            Flower

            87.7

            14.6

            Xian He Cao

            Agrimonia pilosa

            Whole

            71.7

            0

            Xiao Hui Xiang

            Foeniculum vulgare

            Whole

            6.0

            0

            Yi Yi Renn

            Coix lachrymal jobi

            Seed

            0

            ND

            Yu Mi Xu

            Zae mays

            Corn stigma

            16.44

            ND

            Zhi Zi

            Gardenia jasminoides

            Seed

            0

            0

            Zhu Ling

            Polysporus umbellatus

            Fruit body

            8.14

            ND

            Zi Su Ye

            Perilla frutescens

            Flower

            92.7

            22.3

            All Chinese herbal extracts listed were tested on the human B cell line (U266 B1) at 100 and 500 μg/ml. The pinyin and botanical names of the herbs tested in this study are based on Chinese Herbal Medicine: Materia Medica [32] and the Pharmacopoeia of the People's Republic of China [21].

            http://static-content.springer.com/image/art%3A10.1186%2F1749-8546-6-35/MediaObjects/13020_2010_Article_138_Fig3_HTML.jpg
            Figure 3

            Inhibitory effects of (a) QC and (b) QM on IgE production from U266 human B cells. Cells were grown at an initial concentration of 2 × 105cells/mL. QC and QM extracts were added at the indicated concentrations. At Day 6 the supernatants were harvested for total IgE assay. Cell viability after culturing U266 human cells with (c) QC or (d) QM was performed with MTT assay after six days of culture. Bars represent means ± SD of three independent experiments. ***P < 0.001 vs untreated.

            QC and QM suppressed peanut-specific IgE synthesis in an in vivo model of peanut-anaphylaxis

            Since sensitization of mast cells with IgE is an essential mechanism in the anaphylaxis cascade, we evaluated the effect of QC and QM on peanut-specific IgE production in an in vivo model of peanut-anaphylaxis (Figure 2) and found that QC (4 mg) and QM (4 mg)-treated mice showed reductions of 80.47% (P = 0.027) and 92.34% (P = 0.007) respectively in their peanut-specific IgE levels compared with sham-treated mice one week before the time of challenge (Figure 4). Peanut-specific-IgG1 levels were slightly reduced in the QC (4 mg) and QM (4 mg) treatment group, but the difference was not statistically significant at this time point (9.91 × 106ng/mL ± 720345 for sham vs 8.73 × 106ng/mL ± 425234 for QC (4 mg) and sham vs 7.83 × 106ng/mL ± 200283 for QM (4 mg).
            http://static-content.springer.com/image/art%3A10.1186%2F1749-8546-6-35/MediaObjects/13020_2010_Article_138_Fig4_HTML.jpg
            Figure 4

            Effect of QC and QM treatment on IgE production in vivo. Blood from each group of mice was collected one week before challenge. Peanut-specific IgE was measured by antigen-specific ELISA. Results are expressed as means ± SD of triplicates for each group (pooled samples; n = 5-8) P values are calculated vs sham.

            QC and QM decreased peanut triggered anaphylactic reactions in a mouse model

            In order to investigate whether QC and QM can prevent anaphylaxis in vivo, we used peanut as a model antigen to test the effects of QC and QM on peanut-induced anaphylactic reactions. A mouse model of peanut allergy was established (Figure 2). After challenge at Day 35, all sham-treated mice developed anaphylactic reactions (median score 2, Figure 5A and 5B). By contrast, mice treated daily with QC (4 mg) or QM (4 mg) exhibited significantly reduced anaphylactic symptoms (median score 0; P < 0.001; Figure 5A and 5B). At a dose of 2 mg, only QC treated mice exhibited reduced anaphylactic reactions (median score 1; P = 0.020; Figure 5A).
            http://static-content.springer.com/image/art%3A10.1186%2F1749-8546-6-35/MediaObjects/13020_2010_Article_138_Fig5_HTML.jpg
            Figure 5

            Effect of (a) QC and (b) QM treatment on peanut-induced anaphylactic symptoms. Anaphylactic scoring was done as described in the methods section. Symbols indicate individual mice from two sets of experiments (n = 5-8). Bars are medians of scores. P values are calculated vs sham.

            QC and QM prevented decreases in body temperature after peanut challenge

            Core body temperature drops during systemic anaphylaxis. We used rectal temperature measurement at 30 minutes after challenge as an objective measurement of anaphylaxis. As shown in Figure 6A and 6B, mean temperatures of the sham-treated mice were significantly lower than those of the naïve mice (35.77 ± 0.79°C vs 38.96 ± 0.28°C; P < 0.001). Similarly mean temperatures in the QC- and QM-treated mice were significantly higher than in the sham-treated mice, namely 37.39 ± 0.79°C for QC (4 mg) and 37.62 ± 1.22°C for QM (4 mg) (Figure 6A and 6B) (P = 0.0018 for QC and P = 0.004 for QM). As the strongest activity was found at the dose of 4 mg/day/mouse, the rest of the experiments used this dose.
            http://static-content.springer.com/image/art%3A10.1186%2F1749-8546-6-35/MediaObjects/13020_2010_Article_138_Fig6_HTML.jpg
            Figure 6

            Effect of (a) QC and (b) QM treatment on core body temperatures during challenge using a rectal thermometer. Each data point indicates group means ± SD of individual mice from two sets of experiments (n = 5-8).P values are calculated vs sham.

            QC and QM prevented histamine release after peanut challenge

            Anaphylactic scores in this model are associated with plasma histamine levels. We determined histamine levels 30 minutes after challenge. As shown in Figure 7A, plasma histamine levels were markedly elevated in the sham-treated mice compared with naïve mice (sham vs naïve, mean, μM: 6.843 ± 0.3970 vs 0.954 ± 0.085; P < 0.001). By contrast, histamine levels in the QC- and QM-treated mice were significantly lower than those in the sham-treated mice (P < 0.001). Plasma MMCP1concentrations were also measured as an additional marker of mast cell degranulation. We found a significant decrease in MMCP1 levels in the mice treated with 4 mg of QM (sham vs QM, mean, ng/mL: 519.8 ± 212.3 vs 238.6 ± 224.5; P = 0.042); however, no significant differences were found in the QC-treated mice.
            http://static-content.springer.com/image/art%3A10.1186%2F1749-8546-6-35/MediaObjects/13020_2010_Article_138_Fig7_HTML.jpg
            Figure 7

            Effect of QC and QM treatment on histamine release after challenge measured by ELISA. Plasma from each group of mice was collected 30 minutes post-challenge. Results are expressed as means ± SD of triplicates for each group (pooled samples; n = 5-8). ***P < 0.001 vs sham.

            Safety of QC and QM

            In a preliminary safety assessment, we performed LD50 testing. No mouse died within the 12 hours after they were administered the effective mouse daily dose of QC or QM (n = 10), nor did any of them die during the subsequent two weeks.

            To further assess safety, we collected blood samples specimens from mice two weeks after they were fed and subjected to biochemical analysis of BUN and ALT for the assessment of liver and kidney functions respectively. All results were within the normal range (Table 2). Moreover, histological examination of the major organs did not reveal any abnormalities.
            Table 2

            Biochemical assays and CBC testing

             

            Biochemical assays

            CBC testing

            BUN

            (mg/dL)

            ALT

            (U/L)

            WBC

            (K/μL)

            RBC

            M/μL

            Hb

            (g/dL)

            PLT

            (K/μL)

            Qian Cao

            23.2 ± 4.9

            28.6 ± 6.9

            3.3 ± 1.5

            9.9 ± 1.4

            14.7 ± 0.7

            769.2 ± 222.9

            Qu Mai

            20.6 ± 1.8

            26.4 ± 1.4

            4.9 ± 1.7

            8.6 ± 0.7

            14.6 ± 0.4

            698 ± 176.8

            Sham

            24.5 ± 5.3

            31.6 ± 3.6

            6.6 ± 1.9

            9.2 ± 0.9

            14.8 ± 0.6

            700.4 ± 195.9

            Reference

            9 - 36

            22 - 400

            1.8 - 10.7

            6.4 - 9.4

            11.0 - 15.1

            592 - 2972

            Mice were euthanized with CO2, and blood was collected by cardiac puncture. Biochemical assays and CBC testing were conducted as described in Methods. Results are mean ± SD of 5-10 mice from each group.

            Discussion

            After screening 70 herbs extracts with previously reported anti-inflammatory properties, we found that QC and QM extracts markedly inhibited IgE production by a B-cell human cell line over a concentration range of 100 μg/mL to 3.125 μg/mL. The inhibition was not due to toxicity because proliferation assays showed no effect, even at the highest concentrations used (Figure 3C and Figure 3D). QC root is listed in the Chinese Pharmacopoeia for the treatment of arthritis, chronic bronchitis, uterine hemorrhage and uteritis [21]. Recent studies have shown that QC roots have antibacterial, antioxidant and anti-inflammatory activities [2224]. QM is an important Chinese medicinal herb used as a diuretic and an anti-inflammatory agent for the treatment of urinary infections, carbuncles and carcinomas [21]. To our knowledge, we are the first to report the anti-IgE properties of both herbs. Kim, Lee, Won, Park, Chae, Kim & Baek; Kim, Kim & Park and Kim & Moon reported the IgE inhibitory effect of some other herbs such as Asiasari Radix, Poncirus trifloliata and Siegesbeckia glabrescens using the same cell line as in our experiments; however, the concentrations required for the inhibitory effects were higher than those in our experiments [2527]. Sugahara, Nishimoto, Morioka, Nakano & Nakano [28] identified anti-IgE activity of extracts of Sorghum bicolor (L.) Moench. In their experiments, the extracts suppressed IgE production by the human myeloma cell line U266 in a dose-dependent manner but did not affect the IgG production by mice splenocytes in vitro. We demonstrated a similar effect in our in vivo studies, in which mouse serum peanut-specific IgG1 levels did not significantly differ between the groups, suggesting that the effects of QM and QC are IgE specific.

            PNA accounts for approximately 80% of the fatal and near-fatal anaphylactic reactions to foods [29]. As peanut-induced anaphylaxis is IgE-mediated, we tested the effects of QC and QM in a well established mouse model of PNA. Mice in these experiments exhibited less severe symptoms than in a previous study [8] perhaps because mice in these studies were i.p. sensitized with crude peanut extract whereas we used i.g. feeding of ground whole peanut and cholera toxin in our previous studies. Mice's sensitivity to antigen may also differ over time. Recent studies [30, 31] showed that longer sensitization protocols were required to produce the same anaphylactic responses as in a previous study [8].

            Both QC and QM prevented peanut-induced anaphylaxis. This protection could be a direct consequence of the reduced peanut IgE levels induced by the QC and QM treatment. Furthermore, significantly less histamine release was observed in the treated animals. The decrease may be attributed to reduced IgE production by B-cells, leading to decreased availability of IgE for participation in mast cell activation and consequently mast cell degranulation upon antigen challenge. In this model the severity of anaphylactic symptoms is correlated with mast cell histamine release. Both QM and QC significantly reduced plasma histamine levels following peanut challenge of PNA mice, thereby protecting against systemic anaphylaxis. Histamine release is a central mechanism involved in the IgE-mediated type I hypersensitivity reactions in humans and also an important parameter for evaluating the effects in this model. Moreover, QM but not QC also produced significant suppression of MMCP1 release although both QM and QC similarly suppressed systemic anaphylaxis, suggesting that MMCP1may not be the most appropriate marker of systemic anaphylaxis in this model.

            Conclusion

            Qiancao (Rubia cordifolia) and Qumai (Dianthus superbus) extracts inhibit the IgE production by plasma cells in vitro and in mice in a non-toxic manner. This, at least in part, may be responsible for the observed protection against anaphylaxis. Further research is warranted to investigate the molecular mechanisms underlying the inhibitory effects and to identify the active compounds responsible for these effects. More importantly, controlled clinical studies are required to further ensure the safety and efficacy for the use of these herbal products for human food allergy.

            Abbreviations

            ALT: 

            alanine aminotransferase

            BUN: 

            blood urea nitrogen

            CBC: 

            complete blood cells counts

            CPE: 

            crude peanut extract

            DMSO: 

            dimethyl sulfoxide

            FAHF2: 

            food allergy herbal formula 2

            FBS: 

            fetal bovine serum

            Hb: 

            Hemoglobin

            i.g.: 

            intragastric

            i.p.: 

            intraperitoneal

            IC50: 

            inhibitory concentration 50

            LD50

            lethal dose 50

            MMCP1: 

            mouse mast-cell protease 1

            MTT: 

            3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide

            OIT: 

            oral immunotherapy

            PLT: 

            platelets

            PNA: 

            peanut allergy

            QC: 

            Qiancao

            QM: 

            Qumai

            RBC: 

            red blood cells: SLIT: sublingual immunotherapy

            WBC: 

            white blood cells.

            Declarations

            Acknowledgements

            This work was supported by the Food Allergy Initiative and National Institute of Health (grant no AT001495-01A1 to XML). ILE was supported by a postdoctoral MEC/FULBRIGHT grant from the Ministry of Science and Innovation (MCINN), Spain. The authors would like to acknowledge Michelle Mishoe for her technical work with IgE measurements and Kamal Srivastava for her helpful comments. The authors also would like to acknowledge projects CYTED/IBEROFUN 110AC0386 and COST ACTION FA1005.

            Authors’ Affiliations

            (1)
            Department of Pediatrics, Mount Sinai School of Medicine
            (2)
            Institute of Food Science Research (CSIC-UAM)

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            © López-Expósito et al; licensee BioMed Central Ltd. 2011

            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.

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