Chinese herbal extracts of Rubia cordifolia and Dianthus superbus suppress IgE production and prevent peanut-induced anaphylaxis
© López-Expósito et al; licensee BioMed Central Ltd. 2011
Received: 1 December 2010
Accepted: 30 September 2011
Published: 30 September 2011
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).
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.
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.
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.
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 . 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 . 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 . While peanut oral immunotherapy (OIT) is a promising therapeutic intervention for PNA , many questions remain, such as the risks of OIT compared with avoidance, dosing regimen issues, patient selection and post desensitization strategy . Sublingual immunotherapy (SLIT) is a new method of treating food allergy, with few systemic reactions; however, only one study  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 . 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.
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 . 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 .
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.
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 . 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
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
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 .
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.
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 . All statistical analyses were performed with GraphPad Prism (GraphPad Software Inc., USA).
Anti-IgE screening for the Chinese medicinal herbs
Selected Chinese medicinal plants with the percentage of IgE inhibition at the concentrations indicated
% IgE inhibition
% IgE inhibition
Bai Guo Ren
Bai Hua She She Cao
Bai Jiang Cao
Bai Tou Weng
Ban Bian Lian
Ban Zhi Lian
Bei Sha Shen
Bu Gu Zhi
Cang Er Cao
Che Qian Zi
Chuan Xin Lian
Da Qing Ye
Di Gu Pi
Huang Yao Zi
Mu Dan Pi
Shan Ci Gu
Shan Dou Gen
Shan Zhu Yu
Shi Chang Pu
Si Gua Luo
Tian Hua Fen
Tian Nan Xing
Tu Fu Ling
Wu Zhu Yu
Xia Ku Cao
Xian He Cao
Xiao Hui Xiang
Yi Yi Renn
Coix lachrymal jobi
Yu Mi Xu
Zi Su Ye
QC and QM suppressed peanut-specific IgE synthesis in an in vivo model of peanut-anaphylaxis
QC and QM decreased peanut triggered anaphylactic reactions in a mouse model
QC and QM prevented decreases in body temperature after peanut challenge
QC and QM prevented histamine release after peanut challenge
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.
Biochemical assays and CBC testing
23.2 ± 4.9
28.6 ± 6.9
3.3 ± 1.5
9.9 ± 1.4
14.7 ± 0.7
769.2 ± 222.9
20.6 ± 1.8
26.4 ± 1.4
4.9 ± 1.7
8.6 ± 0.7
14.6 ± 0.4
698 ± 176.8
24.5 ± 5.3
31.6 ± 3.6
6.6 ± 1.9
9.2 ± 0.9
14.8 ± 0.6
700.4 ± 195.9
9 - 36
22 - 400
1.8 - 10.7
6.4 - 9.4
11.0 - 15.1
592 - 2972
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 . Recent studies have shown that QC roots have antibacterial, antioxidant and anti-inflammatory activities [22–24]. 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 . 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 [25–27]. Sugahara, Nishimoto, Morioka, Nakano & Nakano  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 . 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  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 .
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.
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.
blood urea nitrogen
complete blood cells counts
crude peanut extract
food allergy herbal formula 2
fetal bovine serum
inhibitory concentration 50
lethal dose 50
mouse mast-cell protease 1
3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide
red blood cells: SLIT: sublingual immunotherapy
white blood cells.
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.
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