Rubinoboletus ballouii polysaccharides exhibited immunostimulatory activities through toll-like receptor-4 via NF-κB pathway.

The biological activities of water-soluble components of edible mushroom Rubinoboletus ballouii (RB) were seldom reported. Polysaccharides of RB (RBP) were prepared and well-characterized using chemical analyses. The immunomodulatory properties of RBP were investigated using human monocyte-derived dendritic cells (moDC) in vitro, and cyclophosphamide (CTX)-induced immunosuppressive mouse model. Results showed that RBP was found to contain 80.6% (w/w) of neutral sugars including D-fucose, D-mannose, D-glucose and D-galactose (1.7:1.4:1.0:1.8), and 12.5% (w/w) of proteins, which composed of glutamine, threonine, serine, etc. RBP could promote the maturation of moDC and increase the secretion of IL-12p40, IL-10, and TNF-α. Furthermore, the stimulation of IL-12p40 production was inhibited by pretreatment with toll-like receptor (TLR)-4 blocker or NF-κB pathway blocker, suggesting that the activation of moDC by RBP was mediated through NF-κB pathway via TLR-4 receptor. On the other hand, in CTX-treated mice, RBP restored the loss of CD34bright CD45dim hematopoietic stem cells and increased IL-2 production in sera and splenocytes culture supernatant, as well as up-regulated the percentage of CD4+ T helper lymphocyte in mice splenocytes. These findings strongly suggested that RBP are the active ingredients of RB responsible for its immunostimulatory actions and deserved to be further investigated as cancer supplements.

Development of thermosensitive hydrogel containing methylene blue for topical antimicrobial photodynamic therapy.

Due to the emergence of antibiotic resistance, antimicrobial photodynamic therapy (aPDT) has recently been demonstrated as a promising alternative to antibiotics to treat wound infections caused by multidrug-resistant (MDR) pathogens. This study aimed to evaluate the bacterial killing efficiency of aPDT mediated by methylene blue (MB) loaded thermosensitive hydrogels against methicillin-resistant Staphylococcus aureus (MRSA). Box-Behnken Design method was employed to investigate the impacts of the polymer compositions, Poloxamer 407, Poloxamer 188 and Carbopol 934P, on the gelation temperature (Tsol-gel) and release rate of MB. The viscosity and in vitro bacterial killing efficiency of three selected formulations with Tsol-gel ranged 25-34 °C and MB release in 2 h (the incubation time used for aPDT experiment) ≥ 70%, were assessed. The viscosity was found to increase with increasing P407 content and increasing total gel concentration. In the in vitro aPDT experiment, all tested MB-hydrogels demonstrated >2.5 log10 colony forming unit (CFU) reduction against three clinical relevant MRSA strains. Interestingly, the bacterial reduction increased with decreasing amount of gel added (reduced MB concentration). This was possibly attributed to the increased viscosity at higher gel concentration reducing the diffusion rate of released MB towards bacterial cells leading to reduced aPDT efficiency. In summary, aPDT with the thermosensitive MB hydrogel formulations is a promising treatment strategy for wound infections.

Jaboticabin and Related Polyphenols from Jaboticaba ( Myrciaria cauliflora) with Anti-inflammatory Activity for Chronic Obstructive Pulmonary Disease.

Myrciaria cauliflora (jaboticaba) is an edible fruit common in Brazil that has been used for treating respiratory diseases, including chronic tonsillitis and asthma. This study explores the distribution of an anti-inflammatory depside, jaboticabin, in different parts of the jaboticaba plant as well as major polyphenols from the wood of jaboticaba, some with biological activity similar to jaboticabin. The peel of the fruit was found to be the major source of jaboticabin. This is the first phytochemical study of the wood of M. cauliflora. The antioxidant-activity-guided fractionation strategy successfully identified 3,3'-dimethylellagic acid-4- O-sulfate from jaboticaba wood. This ellagic acid derivative, in a manner similar to jaboticabin, showed antiradical activity and inhibited the production of the chemokine interleukin-8 after treating the human small airway epithelial cells with cigarette smoke extract. The human intestinal Caco-2 cell studies demonstrated the jaboticabin transport in vitro. The polyphenols, jaboticabin and 3,3'-dimethyellagic acid-4- O-sulfate, from jaboticaba were both found to exhibit anti-inflammatory activities, thus suggesting the potential use of these compounds or even the fruits themselves for chronic obstructive pulmonary disease.

Palladium-Catalyzed Arylation of Carbasugars Enables the Discovery of Potent and Selective SGLT2 Inhibitors.

Selective inhibition of the transporter protein sodium-glucose cotransporter 2 (SGLT2) has emerged as a promising way to control blood glucose level in diabetes patients. Reported herein is a short and convergent synthetic route towards some small-molecule SGLT2 inhibitors by a chemo- and diastereospecific palladium-catalyzed arylation reaction. This synthetic strategy enabled the discovery of two highly selective and potent SGLT2 inhibitors, thereby paving the way towards the development of carbasugar SGLT2 inhibitors as potential antidiabetic/antitumor agents.

Anti-Inflammatory Activities of Pentaherbs Formula, Berberine, Gallic Acid and Chlorogenic Acid in Atopic Dermatitis-Like Skin Inflammation.

Atopic dermatitis (AD) is a common allergic skin disease, characterized by dryness, itchiness, thickening and inflammation of the skin. Infiltration of eosinophils into the dermal layer and presence of edema are typical characteristics in the skin biopsy of AD patients. Previous in vitro and clinical studies showed that the Pentaherbs formula (PHF) consisting of five traditional Chinese herbal medicines, Flos Lonicerae, Herba Menthae, Cortex Phellodendri, Cortex Moutan and Rhizoma Atractylodis at w/w ratio of 2:1:2:2:2 exhibited therapeutic potential in treating AD. In this study, an in vivo murine model with oxazolone (OXA)-mediated dermatitis was used to elucidate the efficacy of PHF. Active ingredients of PHF water extract were also identified and quantified, and their in vitro anti-inflammatory activities on pruritogenic cytokine IL-31- and alarmin IL-33-activated human eosinophils and dermal fibroblasts were evaluated. Ear swelling, epidermis thickening and eosinophils infiltration in epidermal and dermal layers, and the release of serum IL-12 of the murine OXA-mediated dermatitis were significantly reduced upon oral or topical treatment with PHF (all p < 0.05). Gallic acid, chlorogenic acid and berberine contents (w/w) in PHF were found to be 0.479%, 1.201% and 0.022%, respectively. Gallic acid and chlorogenic acid could suppress the release of pro-inflammatory cytokine IL-6 and chemokine CCL7 and CXCL8, respectively, in IL-31- and IL-33-treated eosinophils-dermal fibroblasts co-culture; while berberine could suppress the release of IL-6, CXCL8, CCL2 and CCL7 in the eosinophil culture and eosinophils-dermal fibroblasts co-culture (all p < 0.05). These findings suggest that PHF can ameliorate allergic inflammation and attenuate the activation of eosinophils.

Healing effect of a two-herb recipe (NF3) on foot ulcers in Chinese patients with diabetes: a randomized double-blind placebo-controlled study.

BACKGROUND: In the present study, we examined the effect of a two-herb traditional Chinese medicine (NF3), comprised of Astragali Radix and Radix Rehmanniae, on the healing of diabetic foot ulcer and the possible molecular mechanisms involved. METHODS: This was a prospective randomized double-blind placebo-controlled study. Sixteen diabetic patients were randomized to receive either placebo or NF3 for 6 months. Ulcer healing and sensory changes were examined. Molecular studies included measurement of serum tumor necrosis factor (TNF)-α and RNA microarray investigation. RESULTS: The daily rate of reduction in ulcer area was 3.55% in the NF3 group and 1.52% in the placebo group (P = 0.062). In the index limb, the number of negative tests for sensory neuropathy using monofilament was reduced from 27% to 7% in the NF3 group and from 37% to 35% in the placebo group (P < 0.001). In addition, NF3 significantly decreased serum TNF-α levels (P = 0.034). Microarray studies revealed concerted changes following NF3 treatment in the expression of genes implicated in fibroblast regeneration, angiogenesis, and anti-inflammation. CONCLUSIONS: In this proof-of-concept study, 6-month treatment with NF3 was associated with improved wound healing and sensation accompanied by concerted changes in gene expression.

Anti-tumor and anti-osteolysis effects of the metronomic use of zoledronic acid in primary and metastatic breast cancer mouse models.

This study aims to determine the effect of metronomic (0.0125 mg/kg twice a week for 4 weeks) zoledronic acid (ZOL) on cancer propagation and osteolysis against both metastatic and primary breast cancer in mice model. From our results, metronomic ZOL resulted in a significant reduction of tumor burden and did not promote lung or liver metastasis. The metronomic ZOL appeared to be more effective than the conventional regimen (0.1 mg/kg once in 4 weeks) in reducing breast cancer tumor burden, and regulating its movement to lung and liver. This dosing schedule of ZOL showed great potential against metastatic breast cancer.

Immunomodulatory activities of Ganoderma sinense polysaccharides in human immune cells.

Medicinal mushrooms have been traditionally used as food nutrient supplements in China for thousands of years. The present study aimed to evaluate the immunomodulatory activities of Ganoderma sinense (GS), an allied species of G. lucidum, using human peripheral blood mononuclear cells (PBMC). Our results showed that the polysaccharide-enriched fraction of GS hot water extract (400 µg/ml) exhibited significant stimulatory effects on PBMC proliferation. When the fruiting bodies of GS were divided into pileus and stipe parts and were separately extracted, the GS stipe polysaccharide-enriched fraction (50-400 µg/ml) showed concentration-dependent immunostimulating effects in PBMC. The productions of tumor necrosis factor-α, interleukin (IL)-10, and transforming growth factor -ß were significantly enhanced by this fraction. In addition, the proportion of CD14(+) monocyte subpopulation within the PBMC was specifically increased. The IL-10 and IL-12 productions in monocyte-derived dendritic cells were significantly enhanced by GS stipe fraction. The composition of monosaccharides of this fraction was determined by ultra performance liquid chromatography and ion exchange chromatography. Our study demonstrated for the first time the immunostimulatory effects of GS stipe polysaccharide-enriched fraction on PBMC and dendritic cells. The findings revealed the potential use of GS (especially including the stipes of fruiting bodies) as adjuvant nutrient supplements for patients, who are receiving immunosuppressive chemotherapies.

Synergistic effects of diosmetin with erythromycin against ABC transporter over-expressed methicillin-resistant Staphylococcus aureus (MRSA) RN4220/pUL5054 and inhibition of MRSA pyruvate kinase.

Increasing prevalence of methicillin-resistant Staphylococcus aureus (MRSA) worldwide with limited therapeutic options is a growing public health concern. Natural products have been shown to possess antibacterial actions against MRSA. Flavonoids from natural products have been shown to possess antibacterial actions against MRSA by antagonizing its resistance mechanisms. Diosmin and diosmetin are natural flavonoids found in a variety of citrus fruits. The aim of this study was to investigate whether diosmin and diosmetin could inhibit the growth of MRSA and the in vitro enzymatic activity of a newly discovered MRSA drug target, pyruvate kinase (PK). By using a panel of MRSA strains with known resistant mechanisms, neither diosmin nor diosmetin was shown to possess direct antibacterial activities against all tested MRSA strains. However, in checkerboard assay, we found that diosmetin together with erythromycin, could synergistically inhibit the growth of ABC-pump overexpressed MRSA-RN4220/pUL5054, and time kill assay also showed that the antibacterial activities of diosmetin with erythromycin were bactericidal. Diosmetin was further shown to significantly suppress the MRSA PK activities in a dose dependent manner. In conclusion, the inhibition of MRSA PK by diosmetin could lead to a deficiency of ATP and affect the bacterial efflux pump which might contribute to the antibacterial actions of diosmetin against MRSA.

Anti-inflammatory and anti-allergic activities of Pentaherb formula, Moutan Cortex (Danpi) and gallic acid.

Pentaherb formula (PHF) has been proven to improve the quality of life of children with atopic dermatitis without side effects. The aim of this study was to elucidate the potential anti-inflammatory and anti-allergic activities of PHF, Moutan Cortex (Danpi/DP) and gallic acid (GA) using human basophils (KU812 cells), which are crucial effector cells in allergic inflammation. PHF, DP and GA could significantly suppress the expression of allergic inflammatory cytokine IL-33-upregulated intercellular adhesion molecule (ICAM)-1, and the release of chemokines CCL2, CCL5, CXCL8 and inflammatory cytokine IL-6 from KU812 cells (all p < 0.05). With the combined use of dexamethasone (0.01 µg/mL) and GA (10 µg/mL), the suppression of ICAM-1 expression and CCL5 and IL-6 release of IL-33-activated KU812 cells were significantly greater than the use of GA alone (all p < 0.05). The suppression of the IL-33-induced activation of intracellular signalling molecules p38 mitogen activated protein kinase, nuclear factor-kB and c-Jun amino-terminal kinase in GA-treated KU812 cells could be the underlying mechanism for the suppression on ICAM-1, chemokines and cytokines. The combined use of dexamethasone with the natural products PHF or DP or GA might therefore enhance the development of a novel therapeutic modality for allergic inflammatory diseases with high potency and fewer side effects.

Anti-angiogenesis and immunomodulatory activities of an anti-tumor sesquiterpene bigelovin isolated from Inula helianthus-aquatica.

Bigelovin is a sesquiterpene lactone isolated from the plant Inula helianthus-aquatica which was traditionally used in cancer treatment in Yunnan, China. The potent apoptotic activities of bigelovin in human leukemia U937 cells were shown in our previous study. The present study investigated the anti-angiogenic and immunomodulatory effects of bigelovin using transgenic zebrafish Tg(fli1a:EGFP)y1 with fluorescent blood vessels and human peripheral blood mononuclear cells (PBMCs), respectively. Furthermore, the inhibitory activities of bigelovin on the human endothelial cell adhesion molecules (CAMs) were also examined. Our results showed that the growth of subintestinal vessels of the bigelovin-treated zebrafish embryos was significantly inhibited and the gene expressions in angiogenesis signaling pathways (e.g. Ang2 and Tie2) of the zebrafish were down-regulated after bigelovin treatment. Besides, the proliferation and Th1 cytokines productions (e.g. IFN-γ, IL-2 and IL-12) were suppressed in bigelovin-treated PBMCs. On the other hand, bigelovin was shown to significantly inhibit the human monocyte adhesion to human endothelial cells and the gene expressions of inflammation-related CAMs (e.g. ICAM-1, VCAM-1 and E-selectin) were significantly down-regulated in bigelovin-treated human endothelial cells. In summary, our data provide the first evidence that bigelovin possesses anti-angiogenic and immunomodulatory activities, suggesting bigelovin may exert multi-target functions against cancer in animal models.

Synthesis of chiral hydroxylated enones as potential anti-tumor agents.

A series of chiral hydroxylated enones were synthesized as COTC ether analogues to investigate the structural features required for optimal and selective anti-tumor activity. The cytotoxicity of the seven COTC ether analogues against WRL-68 normal and HepG2, HL-60 cancer cell lines were measured. C-4 ether analogues with an aliphatic chain substituent were found to be more favorable than their aromatic counterparts. Inversion of the configuration at C-4 in 5e to give 5f only resulted in reduced selectivity towards cancer cells. These results show that 4-O-pentyl-gabosine D (5e) has optimum selectivity and cytotoxicity towards two cancer cell lines.

Eriocalyxin B induces apoptosis and cell cycle arrest in pancreatic adenocarcinoma cells through caspase- and p53-dependent pathways.

Pancreatic cancer is difficult to detect early and responds poorly to chemotherapy. A breakthrough in the development of new therapeutic agents is urgently needed. Eriocalyxin B (EriB), isolated from the Isodon eriocalyx plant, is an ent-kaurane diterpenoid with promise as a broad-spectrum anti-cancer agent. The anti-leukemic activity of EriB, including the underlying mechanisms involved, has been particularly well documented. In this study, we demonstrated for the first time EriB's potent cytotoxicity against four pancreatic adenocarcinoma cell lines, namely PANC-1, SW1990, CAPAN-1, and CAPAN-2. The effects were comparable to that of the chemotherapeutic camptothecin (CAM), but with much lower toxicity against normal human liver WRL68 cells. EriB's cytoxicity against CAPAN-2 cells was found to involve caspase-dependent apoptosis and cell cycle arrest at the G2/M phase. Moreover, the p53 pathway was found to be activated by EriB in these cells. Furthermore, in vivo studies showed that EriB inhibited the growth of human pancreatic tumor xenografts in BALB/c nude mice without significant secondary adverse effects. These results suggest that EriB should be considered a candidate for pancreatic cancer treatment.

Extracts from Radix Astragali and Radix Rehmanniae promote keratinocyte proliferation by regulating expression of growth factor receptors.

Chinese herbal medicine has long been used as a treatment for wounds. However, the underlying cellular and molecular mechanisms remain largely unknown. In this study it was shown that the proliferation of keratinocytes, which is known to play an important role in wound healing as the major cell type in the epidermis, was promoted by three herbal extracts/natural compounds: NF3 (an extract from the mixture of Radix Astragali (RA) and Radix Rehmanniae (RR) in the ratio of 2:1), stachyose (an isolated compound from Radix Rehmanniae) and extract P2-2 (a sub-fraction from the extract of Radix Astragali). The effect of the herbal extracts/natural compounds on the growth of keratinocytes was not influenced by a high glucose level, a condition similar to diabetic patients who usually suffer from diabetic foot ulcers. Real time RT-PCR results showed that the expression of epidermal growth factor (EGF) receptor, but not transforming growth factor-ß (TGF-ß) receptor, was up-regulated by NF3. Moreover, treatments with the EGF receptor kinase inhibitor AG1478 and the MEK inhibitor U0126 resulted in the diminishment of the effect of the three herbal extracts/natural compounds on keratinocyte proliferation, indicating that EGF receptor might have a significant role in this action. This study has further elucidated the molecular mechanism under which herbal extracts/natural compounds exert their effects on the wound healing process.

The role of turmerones on curcumin transportation and P-glycoprotein activities in intestinal Caco-2 cells.

The rhizome of Curcuma longa (turmeric) is often used in Asia as a spice and as a medicine. Its most well-studied component, curcumin, has been shown to exhibit poor bioavailability in animal studies and clinical trials. We hypothesized that the presence of lipophilic components (e.g., turmerones) in turmeric extract would affect the absorption of curcumin. The effects of turmerones on curcumin transport were evaluated in human intestinal epithelial Caco-2 cells. The roles of turmerones on P-glycoprotein (P-gp) activities and mRNA expression were also evaluated. Results showed that in the presence of α- and aromatic turmerones, the amount of curcumin transported into the Caco-2 cells in 2 hours was significantly increased. α-Turmerone and verapamil (a P-gp inhibitor) significantly inhibited the efflux of rhodamine-123 and digoxin (i.e., inhibited the activity of P-gp). It is interesting that aromatic turmerone significantly increased the rhodamine-123 efflux and P-gp (MDR1 gene) mRNA expression levels. The effects of α- and aromatic turmerones on curcumin transport as well as P-gp activities were shown here for the first time. The presence of turmerones did affect the absorption of curcumin in vitro. These findings suggest the potential use of turmeric extract (including curcumin and turmerones), rather than curcumin alone, for treating diseases.

Screening for anti-inflammatory and bronchorelaxant activities of 12 commonly used Chinese herbal medicines.

The use of health supplements derived from medicinal herbs as self-medication for the relief of respiratory tract pathology symptoms is increasing in Chinese communities as air pollution is worsening. Twelve herbs from two formulae of our previous studies were evaluated for their anti-inflammatory, immunomodulatory and bronchorelaxant activities in this study. Among the extracts tested, those of Herba Schizonepetae and Radix Glycyrrhizae showed significant inhibitory effects on LPS-induced nitric oxide production (p < 0.05) in mouse macrophage RAW264.7 cells, suggesting their anti-inflammatory activities. Radix Scutellariae and Radix Glycyrrhizae extracts showed significant inhibitory effects on phytohaemagglutinin-induced proliferation in human peripheral blood mononuclear cells (p < 0.05). These extracts also showed inhibition of TNF-α, IFN-γ and IL-10 production. For the bronchorelaxant assay, Rhizoma Cynanchi Stauntonii and Radix Glycyrrhizae extracts showed potent attenuation of the acetylcholine- and carbachol-induced contractions in rat trachea (p < 0.05), implying their relaxant activities. In conclusion, Herba Schizonepetae, Radix Glycyrrhizae, Radix Scutellariae and Rhizoma Cynanchi Stauntonii extracts were demonstrated to exert anti-inflammatory, immunomodulatory and bronchorelaxant activities, which may help to ameliorate the symptoms of respiratory tract pathologies. The findings have thus provided some scientific evidence on the efficacy and mechanisms of action of these herbs, which are useful for the further development of clinical applications.

Cyclopeptide RA-V inhibits angiogenesis by down-regulating ERK1/2 phosphorylation in HUVEC and HMEC-1 endothelial cells.

BACKGROUND AND PURPOSE: Anti-angiogenic agents have recently become one of the major adjuvants for cancer therapy. A cyclopeptide, RA-V, has been shown to have anti-tumour activities. Its in vitro anti-angiogenic activities were evaluated in the present study, and the underlying mechanisms were also assessed. EXPERIMENTAL APPROACH: Two endothelial cell lines, human umbilical vein endothelial cells (HUVEC) and human microvascular endothelial cells (HMEC-1), were used. The effects of RA-V on the proliferation, cell cycle phase distribution, migration, tube formation and adhesion were assessed. Western blots and real-time PCR were employed to examine the protein and mRNA expression of relevant molecules. KEY RESULTS: RA-V inhibited HUVEC and HMEC-1 proliferation dose-dependently with IC(50) values of 1.42 and 4.0 nM respectively. RA-V inhibited migration and tube formation of endothelial cells as well as adhesion to extracellular matrix proteins. RA-V treatment down-regulated the protein and mRNA expression of matrix metalloproteinase-2. Regarding intracellular signal transduction, RA-V interfered with the activation of ERK1/2 in both cell lines. Furthermore, RA-V significantly decreased the phosphorylation of JNK in HUVEC whereas, in HMEC-1, p38 MAPK was decreased. CONCLUSIONS AND IMPLICATIONS: RA-V exhibited anti-angiogenic activities in HUVEC and HMEC-1 cell lines with changes in function of these endothelial cells. The underlying mechanisms of action involved the ERK1/2 signalling pathway. However, RA-V may regulate different signalling pathways in different endothelial cells. These findings suggest that RA-V has the potential to be further developed as an anti-angiogenic agent.

Efficacy and Durability in Direct Labeling of Mesenchymal Stem Cells Using Ultrasmall Superparamagnetic Iron Oxide Nanoparticles with Organosilica, Dextran, and PEG Coatings.

We herein report a comparative study of mesenchymal stem cell (MSC) labeling using spherical superparamagnetic iron oxide (SPIO) nanoparticles containing different coatings, namely, organosilica, dextran, and poly(ethylene glycol) (PEG). These nanomaterials possess a similar SPIO core size of 6-7 nm. Together with their coatings, the overall sizes are 10-15 nm for all SPIO@SiO2, SPIO@dextran, and SPIO@PEG nanoparticles. These nanoparticles were investigated for their efficacies to be uptaken by rabbit bone marrow-derived MSCs without any transfecting agent. Experimentally, both SPIO@SiO2 and SPIO@PEG nanoparticles could be successfully uptaken by MSCs while the SPIO@dextran nanoparticles demonstrated limited labeling efficiency. The labeling durability of SPIO@SiO2 and SPIO@PEG nanoparticles in MSCs after three weeks of culture were compared by Prussian blue staining tests. SPIO@SiO2 nanoparticles demonstrated more blue staining than SPIO@PEG nanoparticles, rendering them better materials for MSCs labeling by direct uptake when durable intracellullar retention of SPIO is desired.

The known immunologically active components of Astragalus account for only a small proportion of the immunological adjuvant activity when combined with conjugate vaccines.

The 95 % ethanol extract of Astragalus has been demonstrated to have potent activity as an immunological adjuvant when administered with vaccines of various types. We endeavor here to identify the components of this extract that are responsible for this adjuvant activity. Mice were immunized with KLH conjugated to cancer carbohydrate antigens globo H and GD3 and cancer peptide antigen MUC1 combined with different Astragalus fractions or with commercially available Astragalus saponins and flavonoids. The antibody responses against cancer antigens and KLH were quantitated in ELISA assays, and toxicity was calculated by weight loss. Astragalosides II and IV were the most active components, but the toxicity of these two differed dramatically. Astragaloside II was the most toxic Astragalus component with 5-10 % weight loss at a dose of 500 µg while astragaloside IV showed no weight loss at all at this dose, suggesting that astragaloside IV might be utilized as an immunological adjuvant in future studies. Several flavonoids also had significant adjuvant activity. However, when the activities of these known immunologically active components of Astragalus (and of endotoxin) are calculated based on the extent of their presence in the 95 % ethanol extract, they provide only a small proportion of the immunological activity. This raises the possibility that additional uniquely active components of Astragalus may contribute to adjuvant activity, or that the adjuvant activity of Astragalus is greater than the activity of the sum of its parts.