FAHF-2 and B-FAHF-2 were obtained from Xiyuan Chinese Medicine Research and Pharmaceutical Manufacturer, Chinese Academy of Chinese Medicine Sciences, Beijing, China, a good manufacturing practice (GMP) certified facility. Herbal components of FAHF-2 are listed in Table 1. All herbs were inspected for identity and quality by licensed pharmacists. All plant names have been checked with http://www.theplantlist.org except for Ganoderma lucidum which is a fungus and not listed there. Voucher specimens of the raw herbs were archived in the laboratory of Dr. Xiu-Min Li. The manufacturing process of FAHF2 is as follows: Ganoderma lucidum powder was generated by decoctions by boiling small pieces Ganoderma lucidum twice (2 h each time). Panax ginseng was extracted twice with 80% aqueous ethanol, then filtered, combined, and evaporated until there was no residual ethanol. The other 7 herbs were combined and boiled twice in water (1.5 h each time). The decoction was collected, combined, and purified by ethanol precipitation. Panax ginseng was mixed with the 7 herbs extract, dried into a powder, and then combined with the Ganoderma lucidum powder to form FAHF-2 (17, 18).

Extensive quality control, analytical chemistry data and batch consistency pertaining to the formula have been published previously (11). We identified chemical markers by high-pressure liquid chromatography (HPLC) and liquid chromatography (LC) mass spectrometry. Endotoxin levels were measured using the Pyrogent Plus assay kit (Lonza, MA) and were below 0.03 EU/ml, the limit of sensitivity for this kit.

Given the generally favorable safety profile, the dose ranges of most Chinese herbal formulations are wide. Initially, the dose used in our FAHF-2 studies was converted from the human daily dose of the Wu Mei Wan formula and Ling Zhi extract at a medium to high dose range using a conversion table of equivalent effective dose ratios from humans to animals based on body surface area (19). This dose was consistently effective in protecting peanut allergic mice from anaphylaxis, and at abrogating colitis in a murine model (5). B-FAHF-2 is 20% of FAHF-2 by weight because of the elimination of non-medicinal components and thus the dose used in our experiments is derived by using 20% of the dose of FAHF-2 (250 μg/ml). The effective dose (60 μg/ml) of B-FAHF-2 was found to be 20% that of FAHF-2 based upon cell culture assays and in vivo models of peanut allergy (6). For the individual herbs within FAHF-2 and B-FAHF-2, the doses used correspond to the equivalent dose found in the full compound. Equivalent doses were as follows: Wu Mei (WM) 50 μg/ml, Chuan Jiao (HJ) 125 μg/ml, Dang Gui (DJ) 125 μg/ml, Gan Jiang (GJ) 125 μg/ml, Gui Zhi (GZ) 125 μg/ml, Huang Bai (HB) 125 μg/ml, Huang Lian (HL) 125 μg/ml, Hong Shen (HS) 125 μg/ml, and Ling Zhi (LZ) 125 μg/ml.

The instruments used were a Waters 2690 HPLC system coupled to a 2996 PDA detector (Waters, Milford, MA). B-FAHF-2 tablets were ground, and a 22.5 mg/mL solution was prepared using 1:1 ratio of mobile phase mixture. The B-FAHF-2 solution was centrifuged at 10,000 rpm for 10 minutes. 10μL of the supernatant was injected into the HPLC system and separated on a ZORBAX SB-C18 (4.6 × 150 mm, 5 μm) column (Agilent, Santa Clara, CA). The mobile phase A was made of 0.1% of formic acid aqueous solution. Mobile phase B was acetonitrile. The separation was performed at 1 min/mL flow rate following a linear gradient elution of 2–25% mobile phase B in 45 min, 25–35% B in the following 25 min, 35–55% in the next 15 min, 55–75% in another 10 min. This mobile phase composition was maintained for 5 min and then rapidly switched to 2% mobile phase B. An equivalent amount of FAHF-2 formula, 99 mg/mL, was also prepared following the same procedure as for B-FAHF-2. 10μL of FAHF-2 supernatant was analyzed on the HPLC system. Data was collected and processed with Waters' Empower software.

Human studies were approved by the Institutional Review Board of the Icahn School of Medicine at Mount Sinai (No. 11-00808). All participants provided written informed consent for participation in the study. Blood samples (n = 29) and inflamed colonic biopsy specimens (n = 20) were collected from newly diagnosed pediatric CD patients (8–19 years old) naïve to medications that could alter immune responses including: immune-modulators, biologics or steroids. CD patients were diagnosed based on standard clinical, radiographic and endoscopic criteria. Blood and colonic biopsy specimens were also collected from non-IBD control subjects (n = 12). Patient characteristics are outlined in Table 2.

PBMCs were isolated by Ficoll Hypaque (Pharmacia, Piscataway, NJ) with density gradient centrifugation. Purified PBMCs were cultured in medium containing 5% FBS with or without FAHF-2 (250 μg/ml), B-FAHF-2 (60 μg/ml), individual herbs in B-FAHF-2 (doses noted above) or dexamethasone (10⁻³ μM/ml, positive control) for 24 h followed by LPS (2 μg/ml) stimulation or no stimulation for an additional 24 h (8). Cytokine, chemokine and growth factor levels in the culture supernatants were determined by ELISA per the manufacturer's instructions (BD Biosciences), by multiplex immunoassay or by cytometric bead array. Multiplex assays were performed per the manufacturer's instructions (Luminex Human assay, Invitrogen, Grand Island, NY). Cytometric bead array was performed per the manufacturer's instructions (BD Biosciences).

Colonic biopsies were washed with PBS and then cultured with or without B-FAHF-2 (60 μg/ml) in complete RPMI (10% FBS and GPS) with phosphatase and protease inhibitors overnight. The volume of culture medium was based upon the weight of each sample so that the size of the biopsies could be standardized. Supernatants were filtered and TNF-α and GM-CSF were assessed by ELISA (BD Biosciences) per the manufacturer's instructions.

Serum was tested by ELISA. Briefly, samples were diluted serially in 4-fold increments from 1/100 to 1/25,000 and assayed for the presence of IgG and IgA against GM-CSF (Sargramostim, Genzyme), as well as irrelevant control recombinant proteins. Each plate included positive control sera from patients with pulmonary alveolar proteinosis as well as a negative control healthy donor serum pool, expected, respectively, to react or not with GM-CSF.

Animal studies were approved by the Institutional Animal Care and Use Committee at Mount Sinai. Cells were obtained from C57BL/6 wild type mice and enriched using an EasySep Cell Isolation Kit (STEMCELL Technologies Inc., Vancouver, CA) that depletes CD8⁺, CD11b⁺, CD11c⁺, CD19⁺, and B220⁺ cells by negative selection. The resulting CD4⁺ enriched population was labeled with FITC-conjugated anti-CD4 Ab, APC-conjugated anti-CD62L Ab and PE-conjugated anti-CD45RB Ab (eBiosciences). Subpopulations of CD4⁺ cells were sorted by flow cytometry. CD4⁺CD45RBhi T cells (3.5 x 10⁵) were adoptively transferred by intraperitoneal injection into recipient RAG1^−/− mice. Transfer was confirmed by flow cytometry of peripheral blood in all mice after 2–4 weeks. To prevent the progression to colitis, the recipient mice were fed B-FAHF-2 (50 mg/day) daily by gavage 24 h after CD4⁺CD45RBhi T cells transfer. Control mice received water by gavage. Weights were recorded semi-weekly. All mice were sacrificed once any mouse lost 20% of their initial weight. Colonic histology was scored for inflammatory infiltrates and epithelial damage by a pathologist blinded to the treatment group (20, 21). A total score with a maximum of 20 was determined by the summation of the following sub-scores: mucosal involvement: 0 = normal, 1 = 3–10 mucosal neutrophils/hpf, 2 = more than 10 mucosal neutrophils or rare crypt abscesses, 3 = multiple crypt abscesses or erosion; submucosal involvement: 0 = normal,1 = focal aggregates of neutrophils, 2 = neutrophil infiltration with expansion of submucosa, 3 = diffuse neutrophil infiltration; muscularis: 0 = normal, 1 = scattered neutrophils within the muscularis, 2 = neutrophil infiltration with focal effacement of the muscularis, 3 = extensive neutrophil infiltration with transmural effacement of the muscularis; crypt damage: 0 = normal, 1 = loss of the basal one third, 2 = loss of the basal two thirds, 3 = entire crypt loss, 4 = crypt loss with surface erosion, 5 = confluent, extensive erosion; ulcerations: 0 = absence of ulceration, 1 = one to two ulcer foci, 2 = three to four ulcer foci, 3 = confluent extensive ulceration. Colonic samples were cultured overnight in medium containing phosphatase and protease inhibitors. Cytokine secretion was measured in the supernatants (IL-4, IL-6, IL-10, IL-12, TNF-β, IFN-γ, IL-17A) by cytometric bead array (BD Biosciences) and ELISA for GM-CSF (BD Biosciences) per the manufacturer's instructions. RT-PCR was performed to look at regulatory elements within the colonic mucosa including TGFβ1, TGFβ2, Foxp3, and IL-10. Colon samples' RNA extraction and complementary DNA (cDNA) transcription were performed as previously described. (22) SYBR™ Green Master Mix (Thermo Fisher Scientific, Fair lawn, NJ) was used to perform RT-PCR. The target gene mRNA expression was normalized to the untreated group and calculated with the _ΔΔCT method. The primer sequences are listed in Table 3. Immunofluorescence (IF) staining of mouse colon sections was performed according to the previously described protocol with slight modifications (23). Paraffin embedded slides were dewaxed by serial xylene, xylene/ethanol (1:1), 100% ethanol, 95% ethanol, 70% ethanol, and H₂O. Then slides were unmasked in heat antigen retrieval solution in the microwave. After permeabilization for 10 min at room temperature (RT), the slides were blocked with 20% goat serum in PBS for 1 h at room temperature, and then incubated with CD4-FITC and F4/80-APC (eBiosciences, San Diego, CA) overnight at 4°C. DAPI was used to stain cell DNA.

Paired T-tests with Wilcoxon signed-rank test were used when comparing the same samples without and with treatment. Comparisons between multiple groups were done using one-way ANOVA. This was followed by either non-parametric Mann-Whitney U test or Bonferroni analysis when appropriate. Statistical differences between groups for the colitis model were determined by Mann-Whitney T-test assuming non-normally distributed data. Data analysis was done using Prism software (GraphPad, San Diego, CA). A value of p < 0.05 was considered statistically significant. P-values are indicated by ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001.

After butanol extraction of FAHF-2 was completed, B-FAHF-2 and FAHF-2 were examined by HPLC to ensure that the compounds were similar. HPLC fingerprints (Figure 1) demonstrate similar patterns with peaks at similar absorbance units and retention times, indicating that they contain the same compounds.

FAHF-2 suppressed production of TNF-α IFN-γ, and IL-12 from PBMCs from pediatric subjects with CD (5) and therefore we examined B-FAHF-2's effects on TNF-α production as a representative cytokine. We first tested the effect of B-FAHF-2 (60 μg/ml) on TNF-α secretion by PBMCs from 29 CD subjects and 12 non-IBD controls (Table 2). PBMCs from CD subjects secreted significantly more TNF-α upon LPS stimulation as compared to non-IBD controls (p < 0.01) (Figure 2A). TNF-α levels were significantly reduced when CD PBMCs were cultured with B-FAHF-2 (p < 0.001) (Figure 2A). No cytotoxicity was detected (Data not shown).

To be effective at treating CD, B-FAHF-2 should have effects on intestinal mucosa and inflammatory cytokine secretion. FAHF-2 suppressed production of TNF-α, IL-1β, IL-6, and IL-17 from inflamed colonic mucosa from pediatric subjects with CD (5) and therefore we examined B-FAHF-2's effects on TNF-α production as a representative cytokine. We incubated inflamed colonic mucosa from subjects with CD with and without B-FAHF-2 and quantified TNF-α from the supernatant. B-FAHF-2 significantly suppressed the production of TNF-α from inflamed biopsies from subjects with CD (p < 0.05) (Figure 2B).

Thus, like FAHF-2, B-FAHF-2 significantly suppressed TNF-α production by PBMCs and inflamed colonic mucosa from pediatric subjects with CD.

FAHF-2 had an immunomodulatory effect that is best demonstrated by its stimulation of increased production of GM-CSF while at the same time suppressing production of multiple inflammatory chemokines (IP-10, MIP-1β, eotaxin, RANTES, MIG, MCP-1, and MIP-1α) and HGF from PBMCs from pediatric subjects with CD (Figure 3).

Therefore, using GM-CSF as representative of the immunomodulatory capacity FAHF-2, we tested the effect of B-FAHF-2 on GM-CSF secretion by unstimulated PBMCs from all 29 pediatric subjects with CD. PBMCs cultured with B-FAHF-2 produced more GM-CSF than when cultured in medium alone (Figure 4A) (p < 0.05). B-FAHF-2 also stimulated significantly increased production of GM-CSF from biopsies from pediatric subjects with CD (Figure 4B) (p < 0.05).

Given the effect of B-FAHF-2 on GM-CSF and the possibility that it might be beneficial in a subset of patients with GM-CSF antibodies, we tested the serum from all subjects. We found that four of the 29 subjects with CD had GM-CSF antibodies. B-FAHF-2 significantly increased GM-CSF production by PBMCs from these 4 subjects (p < 0.05) (Figure 5) despite them having antibodies to GM-CSF.

Given the potent suppressive effect of FAHF-2 and B-FAHF-2 on TNF-α production, (5) each herb that comprises them [Prunus mume (Wu Mei), Zanthoxylum bungeanum (Chuan Jiao), Angelica sinensis (Dang Gui), Zingiber officinalis (Gan Jiang), Cinnamomum cassia (Gui Zhi), Phellodendron chinense (Huang Bai), Coptis chinensis (Huang Lian), Panax ginseng (Hong Shen) and Ganoderma lucidum (Ling Zhi)] was tested for the ability to suppress production of TNF-α from LPS stimulated PBMCs from pediatric subjects with CD. LPS caused a marked increase in production of TNF-α. B-FAHF-2, Dexamethasone (Dexa), Huang Bai (HB), Huang Lian (HL) and Dang Gui (DG) significantly reduced production of TNF-α (p < 0.0001, p < 0.001, p < 0.01, p < 0.05, and p < 0.05 respectively) (Figure 6A).

We then tested the effect of B-FAHF-2 and each herb that comprises it on the production of GM-CSF. B-FAHF-2, Ling Zhi (LZ) and Huang Bai (HB) induced significantly increased production of GM-CSF by unstimulated PBMCs from pediatric subjects with CD (p < 0.05, p < 0.001, and p < 0.05, respectively) (Figure 6B).

Overall, only B-FAHF-2 and Huang Bai (HB) could both decrease TNF-α and increase GM-CSF production by PBMCs unlike any of the other individual herbs.

In the CD45RB^hi T cell transfer model of colitis, treatment with FAHF-2 decreased colitis progression as evidenced by decreased weight loss, histological inflammation and production of TNF-α, IFN-γ, IL-6, and IL-17 from the colon (5). Therefore, we assessed the effect of B-FAHF-2 in vivo using the CD45RB^hi T cell transfer model of colitis, which exhibits features like those found in CD including transmural colitis and elevated levels of TNF-α. B-FAHF-2 treated mice trended toward less weight loss (Figures 7A,B) and had significantly less colon shortening than untreated controls (p < 0.05) (Figure 7C). The B-FAHF-2 treated mice had significantly less histological inflammation with less muscularis inflammatory infiltrates, crypt damage, and ulceration (p < 0.05, p < 0.05, p = 0.0548, and p < 0.01, respectively) (Figures 7D–F). The scoring system used takes into account changes in neutrophil infiltrates. Thus, we also looked by IF at CD4⁺ T cell and macrophage infiltrates and found that both were decreased in the B-FAHF-2 treated group (Figure 7F). Inflammatory cytokine production by colonic tissue in B-FAHF-2 treated mice was reduced: TNF-α, IFN-γ, and IL-6 (p < 0.05, p < 0.05, p < 0.01, respectively) (Figure 7G). Levels of IL-17 and IL-10 were not detectable in both groups (data not shown). Finally, to examine if B-FAHF-2 affects regulatory T cells, we examined TGF- β1, TGF-β2, Foxp3, and IL-10 by RT-PCR and found no significant differences between the groups (data not shown).

Thus, B-FAHF-2 alleviates colitis and inhibits the inflammatory milieu associated with colitis.