Root Of Astragalus Membranaceus Research Articles

Identification of anti-inflammatory fraction & isolation of active compound formononetin from root of Astragalus membranaceus

Identification of anti-inflammatory fraction & isolation of active compound formononetin from root of Astragalus membranaceus

Characterization and Suppressive Effect of Astragalus Polysaccharide in Glomerulonephritis Rats

A water-soluble polysaccharide named as APS was isolated from the roots of Astragalus membranaceus by hot water extraction, anion-exchange and gel-permeation chromatography and tested for its renal protective effect. Its structural characteristics were investigated by FTIR, AMLC, NMR spectroscopy, GLC–MS, methylation analysis, periodate oxidation and Smith degradation. Based on the data obtained, APS was found to be a α - (1-4)-D -glucan, with a single α- D-glucose at the C-6 position every nine residue, on average, along the main chain. The glucan has a weight-average molecular weight of about 3.6×104 Da. 24h Urine protein quantification and morphological observation, NF-κB protein expression and IL-2, IL-6 and TNF- α concentrations were significantly decreased by APS treatment, clearly suggesting that APS is effective in protecting against glomerulonephritis induced by C-BSA through the inhibition of NF- κB mediated-cytokine pathway.

The diabetic wound healing effect of a two-herb formula and its mechanisms of action

The dried roots of Astragalus membranaceus (AR) and Rehmannia glutinosa (RR) are two herbs commonly used in Chinese medicines for relieving diabetes and its complications. The objective of present study was to examine the wound healing effect of formula NF3 (AR and RR in the ratio of 2:1) in diabetic rats, and its potential mechanisms of action in fibroblast proliferation, angiogenesis and inflammation control in vitro. Using diabetic foot ulcer animal model, NF3 (0.98g/kg) was found to significantly promote wound closure when compared to control (water) group. In the in vitro mechanistic studies, NF3 could significantly i) stimulate fibroblast (Hs27) proliferation; ii) increase cell migration and tubule formation of endothelial cells (HUVEC and HMEC-1); and iii) inhibit nitric oxide production from LPS-stimulated macrophage cells (RAW 264.7). In conclusion, herbal formula NF3 could enhance diabetic wound healing through actions of tissue regeneration, angiogenesis and anti-inflammation. The diabetic wound healing effect of NF3 was attributed to the synergistic interaction between its two component herbs. The roles of these two herbs in different underlying mechanisms of action, as well as the clinical efficacy of NF3 will also be presented.

Isolation of Anti‐Inflammatory Fractions and Compounds from the Root of Astragalus membranaceus

Foot ulceration, if not treated properly, will eventually result in amputation. Inflammation may impede the wound healing process if not properly controlled. The root of Astragalus membranaceus (AR) is one of the Chinese herbs commonly found in Chinese herbal formulae used for treating foot ulcer. In this study, we aimed to identify the active fractions and/or compounds from AR aqueous extract, which are responsible for the anti-inflammatory effect using in vitro bioassay-guided fractionation. The anti-inflammatory effect was monitored by the inhibition of nitric oxide (NO) released from lipopolysaccharide-stimulated mouse macrophage RAW 264.7 cells after treated with AR aqueous extract or its fractions and isolated components. Two major active fractions (P2-3-2-2-2 and P2-3-2-2-3) were found to significantly inhibit NO production at 0.156 mg/mL (p < 0.01). In addition, three chemical components (formononetin, calycosin and astragaloside IV) were successfully isolated from P2-3-2-2-3. Only formononetin could significantly inhibit NO production (p < 0.01), whereas the other two components had no significant effects at concentrations ranging from 0.039 to 0.156 mg/mL. In conclusion, two major anti-inflammatory active fractions that may enhance wound healing were identified, and formononetin was one of the active ingredients in the active fractions.

A standardized extract from Paeonia lactiflora and Astragalus membranaceus attenuates liver fibrosis induced by porcine serum in rats

Paeonia lactiflora and Astragalus membranaceus are two popular traditional Chinese medicines, commonly used in Chinese herb prescription to treat liver disease. The extract prepared from the roots of Paeonia lactiflora and Astragalus membranaceus (PAE) demonstrated better hepatoprotective activity than the herbs used individually as shown in our previous studies. This study was carried out to investigate the effects of PAE on liver fibrosis induced by porcine serum (PS) in rats and to explore its possible mechanisms. Liver fibrosis was induced in male Wistar rats by injection with PS intraperitoneally. The rats were randomly divided into a normal control group, a liver fibrosis model group and a PAE (40, 80, 160 mg•kg-1) treated group. After a 16-week treatment, PAE-treated rats showed significantly reduced liver damage and symptoms of liver fibrosis upon pathological examination. Administration of PAE significantly decreased serum HA, PC III levels, and content of hydroxyproline in the liver tissue of fibrotic rats. It also restored the decrease in SOD and GSH-Px activities and inhibited the formation of lipid peroxidative products during PS treatment. In vitro, PAE also significantly decreased [3H]-thymidine incorporation in hepatic stellate cells (HSCs) stimulated with platelet-derived growth factor-B subunit homodimer (PDGF-BB). Moreover, PAE significantly decreased the expression of PDGF receptor beta (PDGFR-β) and p-ERK1/2, p-p38, p-JNK. The results showed that PAE displays antifibrotic effects in rats induced by PS, the mechanism by which might be associated with its ability to scavenge free radicals, decreasing the expression of PDGFR-β, inhibition of HSC proliferation and MAPK activation. These findings indicate that PAE is a potential agent for the prevention of liver fibrosis.

Astragalus polysaccharides enhance the humoral and cellular immune responses of hepatitis B surface antigen vaccination through inhibiting the expression of transforming growth factor β and the frequency of regulatory T cells

Astragalus polysaccharides (APS), extracted from the root of Astragalus membranaceus, a traditional Chinese medicinal herb, have extensive pharmacological and strong immunomodulatory effects. In this study, the potential adjuvant effect of APS on humoral and cellular immune responses to hepatitis B subunit vaccine was investigated. Coadministration of APS with recombinant hepatitis B surface antigen significantly increased antigen-specific antibody production, T-cell proliferation and CTL (cytotoxic T lymphocyte) activity. Production of interferon-γ (IFN-γ), interleukin-2 (IL-2) and IL-4 in CD4(+) T cells and of IFN-γ in CD8(+) T cells were dramatically increased. Furthermore, expression of the genes PFP, GraB, Fas L and Fas were up-regulated; interestingly, expression of transforming growth factor β (TGF-β) and the frequency of CD4(+)CD25(+)Foxp3(+) regulatory T cells (Treg cells) were down-regulated. Expression of Toll-like receptor 4 (TLR4) was significantly increased by administration of APS. Together, these results suggest that APS is a potent adjuvant for the hepatitis B subunit vaccine and can enhance both humoral and cellular immune responses via activating the TLR4 signaling pathway and inhibit the expression of TGF-β and frequency of Treg cells.

The telomerase activator TA‐65 elongates short telomeres and increases health span of adult/old mice without increasing cancer incidence

Here, we show that a small-molecule activator of telomerase (TA-65) purified from the root of Astragalus membranaceus is capable of increasing average telomere length and decreasing the percentage of critically short telomeres and of DNA damage in haploinsufficient mouse embryonic fibroblasts (MEFs) that harbor critically short telomeres and a single copy of the telomerase RNA Terc gene (G3 Terc(+/-) MEFs). Importantly, TA-65 does not cause telomere elongation or rescue DNA damage in similarly treated telomerase-deficient G3 Terc(-/-) littermate MEFs. These results indicate that TA-65 treatment results in telomerase-dependent elongation of short telomeres and rescue of associated DNA damage, thus demonstrating that TA-65 mechanism of action is through the telomerase pathway. In addition, we demonstrate that TA-65 is capable of increasing mouse telomerase reverse transcriptase levels in some mouse tissues and elongating critically short telomeres when supplemented as part of a standard diet in mice. Finally, TA-65 dietary supplementation in female mice leads to an improvement of certain health-span indicators including glucose tolerance, osteoporosis and skin fitness, without significantly increasing global cancer incidence.

Structural analysis and bioactivity of a polysaccharide from the roots of Astragalus membranaceus (Fisch) Bge. var. mongolicus (Bge.) Hsiao

The Astragalus polysaccharide (APS) was extracted and purified from the roots of Astragalus membranaceus, and characterised for its chemical structure and potential health properties. The APS was composed of α- d-glc residues with the estimated equivalent dextran molecular weight of 2.07 × 10 4 Da. Periodate oxidation analysis, 1D and 2D NMR spectroscopy demonstrated that APS has repeating (1 → 4)-linked backbone with a (1 → 6)-linked branch every 10 residues. The APS possessed scavenging activities against hydroxyl radicals and hydrogen peroxide, and showed chelating effect on ferrous ions. The APS was also able to bind cholic and chenodeoxycholic acids in vitro. In addition, APS was able to stimulate activity of purified mouse B cells without promoting T cell proliferation. These data provided information for future development of APS as a nutraceutical.

New Isoflavonoid Glycosides and Related Constituents from Astragali Radix (Astragalus membranaceus) and Their Inhibitory Activity on Nitric Oxide Production

Twenty-four secondary metabolites, including 16 isoflavonoids, 7 astragalasides, and 1 benzoquinone, have been isolated from the roots of Astragalus membranaceus (Astragali radix). Among these isolated isoflavonoids, (-)-methylinissolin 3-O-β-d-(6'-acetyl)-glucoside (1), (-)-methylinissolin 3-O-β-d-{6'-[(E)-but-2-enoyl]}-glucoside (2), and calycosin 7-O-β-d-(6''-acetyl)-glucoside (3) have been identified as new compounds on the basis of spectroscopic analysis; (-)-methylinissolin 3-O-β-d-glucoside (4) was isolated from the natural products for the first time. The nitric oxide (NO) production inhibitory activity of the major compounds has been assessed in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. To identify A. membranaceus, a fingerprint method was developed by using a high-performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD) method. Furthermore, characteristic peaks for the 11 major compounds in the chromatogram were unambiguously confirmed.

Study of the Relationship between Genetics and Geography in Determining the Quality of Astragali Radix

Astragali Radix (AR), prepared from the roots of Astragalus membranaceus (FISCH. ex LINK) BUNGE or its variey, A. membranaceus (FISCH. ex LINK) BUNGE var. mongholicus (BUNGE) HSIAO., is one of the most used and valuable traditional Chinese medicines (TCMs). Historically, Hunyuan, Shanxi Province in China is the geo-authentic producing area of AR and crude AR from here called "geo-authentic." According to tradition, geo-authentic TCMs define both authenticity and quality. However, no scientific investigation has ever determined whether the superior quality of Hunyuan AR is due to the genetic characteristics or to the local environment. In our study, seeds of 30 AR samples representing the two varieties from different regions were cultivated in Hunyuan under the same conditions. A method, using ultra-performance liquid chromatography coupled with photodiode array detector and evaporative light scattering detectors, was developed to evaluate the quality through a simultaneous determination of four major isoflavonoids and four major saponins. The two AR varieties were successfully distinguished by principal component analysis while samples of the same species with different seeds origins could not be distinguished. A genetic study demonstrated that the internal transcribed spacer sequences of the nuclear ribosomal DNA in A. membranaceus var. mongholicus samples from different geographical regions were highly conservative. These results indicate that the content of active components in AR depends on the interaction of genotype and environment. At the varietal level, genetic properties appear to be more important for pharmaceutical quality than environmental factors, while on the intraspecific level environmental factors might be more important than genetic properties.

Discrimination of geographical origin and adulteration of radix astragali using fourier transform infrared spectroscopy and chemometric methods

Radix Astragali, one of most widely used and important traditional Chinese medicines, is cultivated in different geographical regions. Because of varying growing conditions, the qualities of Radix Astragali vary, which can give rise to differences in clinical therapy. Detecting adulteration is a routine requirement in pharmaceutical practice. To develop a simple and accurate approach to discriminate the geographical origin and potential adulteration of Radix Astragali, derived from the root of Astragalus membranaceus (Fischer) Bunge var. mongholicus (Bunge) Hsiao, using Fourier transform infrared (FT-IR) spectroscopy and chemometric methods. To obtain characteristic IR spectra for accurate discrimination, a one-solvent extraction method was utilised following a novel evaluation method for selecting appropriate solvents. Samples of Radix Astragali from different geographical origins were discriminated using FT-IR spectroscopy and discriminant partial least squares (DPLS) methods. FT-IR spectroscopy combined with Mahalanobis distance was employed to detect adulteration of Radix Astragali. In comparison with other solvents, butanone was more effective at extracting samples. Radix Astragali samples were accurately assigned to their corresponding geographical origins by using FT-IR spectroscopy and DPLS method. Most adulterated samples were detected accurately by application of FT-IR spectroscopy combined with Mahalanobis distance. FT-IR spectroscopy combined with chemometric method was developed and demonstrated to be a useful tool to discriminate geographical origin and adulteration of Radix Astragali.

Formononetin accelerates wound repair by the regulation of early growth response factor-1 transcription factor through the phosphorylation of the ERK and p38 MAPK pathways

Formononetin, a phytoestrogen from the root of Astragalus membranaceus, is used as a blood enhancer and to improve blood microcirculation in complementary and alternative medicine. The present study investigated the influence of formononetin on the expression of early growth response factor-1 (Egr-1) and growth factors contributing to wound healing. Formononetin significantly increased growth factors such as transforming growth factor-beta 1 (TGF-β1), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF) in human umbilical vein endothelial cells (HUVECs). Formononetin also increased the expression of Egr-1 transcription factor by 3.2- and 10.5-fold, compared with recombinant VEGF 125 in HUVECs. The formononetin-mediated 12%–43% increase induced endothelial cell proliferation and recovered the migration of wounded HUVECs. In an ex vivo angiogenesis assay, formononetin produced a larger capillary sprouting area than produced using recombinant VEGF 125. Cell proliferation and migration of HUVECs were also greater in the presence of formonectin than VEGF 125. Western blot analysis of scratch-wounded confluent HUVECs showed that formononetin induced the phosphorylation of extracellular signal-regulated kinase (ERK) and slightly inhibited the phosphorylation of p38 mitogen-activated protein kinase (MAPK). The formononetin-mediated sustained activation of Egr-1 was suppressed by the ERK inhibitor PD98059 and the p38 inhibitor SB203580. PD98059 inhibited the formononetin-induced endothelial proliferation and repair in scratch-wounded HUVECs, SB203580 increased the cell proliferation and wound healing. Formononetin accelerate wound closure rate as early as day 3 after surgery and consistently observed until day 10 after in wound animal model. These data suggest that formononetin promotes endothelial repair and wound healing in a process involving the over-expression of Egr-1 transcription factor through the regulation of the ERK1/2 and p38 MAPK pathways.

DNA barcoding provides distinction between Radix Astragali and its adulterants

Based on variable nuclear and/or organellar DNA sequences among vastly divergent species as well as morphologically indistinguishable species, DNA barcoding is widely applicable in species identification, biodiversity studies, forensic analyses, and authentication of medicinal plants. The roots of Astragalus membranaceus and A. membranaceus var. mongholica are commonly used as Radix Astragali in several Asian countries, including China, Japan, and Korea. However, in addition to the two species recorded in the Chinese Pharmacopoeia, there are twenty-three species from different genera including Astragalus, Oxytropis, Hedysarum, and Glycyrrhiza, which have been used as adulterants not only in trading markets but also by the herbal medicine industry. Therefore, a simple, reliable, and accurate classification method is important for distinguishing authentic Radix Astragali from its adulterants. In this study, we acquired data for 37 samples from four related genera within the family Fabaceae. Then we compared four candidate DNA barcoding markers using ITS, matK, rbcL, and coxI sequences from nuclear, chloroplast, and mitochondrial genomes, all commonly used for plants to identify genetic variations among genera, intraspecies, and interspecies. We observed higher divergences among genera and interspecies for ITS, which have the average Kimura 2-parameter distances of 4.5% and 14.1%, respectively, whereas matK was found to have sufficient divergence at the intraspecific level. Moreover, two indels detected in the matK sequence are useful for PCR studies in distinguishing Radix Astragali from its adulterants. This study suggests that the combined barcoding regions of ITS and matK are superior barcodes for Radix Astragali and further studies should focus on evaluating the applicability and accuracy of such combined markers for a wide range of traditional Chinese herbs.

Preventive effects of a fractioned polysaccharide from a traditional Chinese herbal medical formula (Yu Ping Feng San) on carbon tetrachloride-induced hepatic fibrosis

The study was to investigate the prevention effects and possible mechanism of Yu Ping Feng San fractioned polysaccharide (YPF-P) on CCl(4)-induced liver fibrosis in rats. YPF-P was prepared from root of Astragalus membranaceus, rhizome of Atractylodes macrocephaia and root of Raidix saposhnikoviae, and compared with polysaccharide from root of Astragalus membranaceus (AP). Hepatic fibrosis was induced by subcutaneous injection with carbon tetrachloride twice weekly for 12 weeks in Sprague-Dawley rats. YPF-P, AP and colchicine were administered intragastrically daily to carbon tetrachloride-treated rats. Histopathological changes of the liver and hepatic stellate cells were evaluated by Masson staining and transmission electron microscopy, respectively. Markers of fibrosis were determined by radioimmunoassay, biochemistry assay and ELISA. The mRNA expressions of tissue inhibitor of metalloproteinase-1 (TIMP-1), matrix metalloproteinase-13 (MMP-13), procollagen I and collagen III were detected by RT-PCR. YPF-P dose-dependently alleviated the degree of liver fibrosis and inhibited hepatic stellate cell transformation into myofibroblast-like cells, markedly reduced the elevated levels of hyaluronic acid, laminin, type IV collagen, type III procollagen, hydroxyproline and transforming growth factor beta-1, suppressed procollagen I, collagen III and TIMP-1 expression, and improved the TIMP-1/MMP-13 ratio. MMP-13 expression was only promoted moderately by YPF-P. Compared with AP, YPF-P showed more potency on most markers except laminin, type IV collagen and MMP-13 mRNA. YPF-P prevented the progress of rat liver fibrosis induced by carbon tetrachloride and had a more potent preventative effect. The preventative effect may be associated with the ability of YPF-P to inhibit the synthesis of matrix collagen and balance the TIMP/MMP system.

Antioxidant and Anti-diabetes Activities of Methanolic Extract and Fractions of Astragalus membranaceus Roots

The potential biological activities of methanol extract and 5 fractions (hexane, CH₂Cl₂, EtOAc, BuOH and water) from roots of Astragalus membranaceus were examined by 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging, hydroxyl radical (·OH) scavenging activity, reducing power assays, lipid peroxidation inhibitory activity, α-amylase and α-glucosidase inhibition assays. The EtOAc fraction showed high DPPH free radical scavenging activity (EC??=170.34 ㎍/㎖), hydroxyl radical scavenging activity (EC??=32.14 ㎍/㎖), lipid peroxidation inhibitory activity (EC??=52.46 ㎍/㎖) and a concentration dependence, with OD value ranging from 0.234 to 0.345 0.1 to 0.5 ㎎/㎖), for reducing power. The EtOAc fraction has the highest total phenolic content (142.13 Gal μg/mg) and the CH2Cl2 fraction has the highest flavonoid content (71.63 Que ㎍/㎎). Meanwhile, hexane and EtOAc showed certain α-amylase and α-glucosidase inhibition activities. These results suggest that the methanol extract and fractions from Astragalus membranaceus root have significant antioxidant and anti-diabetes activities, which could be used as a potential source of pharmaceutical materials.

Determination of trace elements in Chinese medicinal plants by instrumental neutron activation analysis

Roots of Astragalus membranaceus, Angelica sinensis, Glycyrrhiza uralensis and Codonopsis pilosula, which were often used as herbs in traditional Chinese medicine, were analyzed by Instrumental Neutron Activation Analysis (INAA). The samples were collected in Gansu, northwest of China and irradiated at the 15 MW heavy water reactor in China Institute of Atomic Energy (CIAE). The induced activities were counted by a well calibrated low background γ-spectrometer equipped with a high efficiency coaxial high-purity germanium (HPGe) detector. The concentrations of eighteen trace elements (Ca, Fe, Na, Zn, Ba, Rb, Ce, Cr, La, Co, Th, Cs, Sb, Sc, Sm, Hf, Eu and Tb) in the herbs were determined. Possible links between pharmacological action of the herbs and content of some elements were also discussed in this paper. The measured results were compared with the reported values in literature.

Characterisation of a novel monomeric lectin (AML) from Astragalus membranaceus with anti-proliferative activity

Abstract A novel lectin (AML) was isolated from the roots of a Chinese herb Astragalus membranaceus (Fisch.) Bunge, using a combination of ammonium sulphate fractionation and affinity chromatography. AML was found to be a monomeric protein with a molecular mass of 31.5 kDa. Biochemical characterisation revealed that it is a glycoprotein containing 10.7% neutral sugars. The N-terminus of AML was blocked but amino acid sequences of internal tryptic peptides showed moderate sequence identities with some other known lectins. Amongst the various carbohydrates tested, the lectin was best inhibited by d -galactose and its derivatives with pronounced preference for o-nitrophenyl-β- d -galactopyranoside (8.3 mM). The lectin was stable in the pH range of pH 5.0–12.0 and temperatures up to 55 °C for 30 min. AML inhibited the proliferation of HeLa and K562 cell lines. Thus, the lectin displays a high potential for antitumour activity.

New Multi-herb Mixture, HT005, Induces Longitudinal Bone Growth in Male Rats

Objectives : It was investigated new herbal prescription HT005 supported by traditional Korean medicine has a activity on the longitudinal bone growth of rats. HT005 were consisted of the root of Angelica sinensis, the fruit of Alpinia oxyphylla, the root of Astragalus membranaceus, the stem of Eleutherococcus senticosus, and the root of Dioscorea japonica, and Poria cocos. Method : To investigate the effect on longitudinal bone growth in adolescent male Sprague-Dawley rats, the longitudinal length of growth plate was measured directly by the tetracycline fluorescent labeling method and chondrocyte staining method. HT005 administered orally for four days, and the tetracycline was injected twice on the growth plate of the animals for fluorescent dying. The rate of longitudinal bone growth was measured the length between the tetracycline bands which fixed on the 3rd day and 5th day after injection. Cresyl violet was also used to stain the chondrocytes in the growth plate. The length of growth plate after administration was compared. Expression of IGF-1 and BMP-2 in the growth plate was investigated by immunohistochemistry. Results : HT005 showed a significant longitudinal bone growth which was at the dose of 100 mg/kg and (p (p (p at the dose of 10 mg/kg compared to the control group by cresyl-violet staining method. Both the number and intensity of BMP-2 and IGF-1 positive cells were increased in the hypertrophic zone of growth plate. There was a significant correlation between BMP-2 and IGF-1 expression and heights of chondrocytes in growth plate. Conclusions : Treatment of HT005 on Sprague-Dawley rats markedly increased the longitudinal bone growth. Therefore, HT005 may be an alternative herbal source to growth hormone as it promotes bone growth in children afflicted by growth retardation.

Effects of cycloartane saponins from hairy roots of Astragalus membranaceus Bge., on human tumor cell targets

For the first time three different natural compounds, isolated from hairy roots of Astragalus membranaceus, cultivated in airlift bioreactor were tested for their cytotoxic potential and apoptosis induction in a panel of human tumor cell lines. Root cultures, cultivated in bioreactor gave 18.5 g l − 1 dry wt roots with the highest astragaloside production in vitro up to now — 1.64% (astragaloside I), 1.12% (astragaloside II) and 1.08% (astragaloside III). In this manner the production in airlift bioreactor can be used as means of reliable supply of cycloartane saponins to extend the research to human clinical studies.

Comparison of the Immunoregulatory Function of Different Constituents in Radix Astragali and Radix Hedysari

Radix Astragali (RA), known as “Huangqi” in China, is one of the most popular herbal medicines known worldwide to reinforce “Qi”. RA is traditionally prepared from the dried roots of Astragalus membranaceus (MJHQ) and A. membranaceus var. mongholicus (MGHQ). Radix Hedysari is named “Hongqi” (HQ), which is similar to RA. We assessed and compared the chemical constituents and bioactivity of RA and HQ. Different constituents were extracted into five major parts and were analyzed using different methods. Comparison of the immunological effects of extracts was done by using two immunological models. Results showed that flavonoids and saponins present in RA and HQ were not only structurally significantly different but also different in their immunological effect. Amino acids extract (AE) in MGHQ shows immunological effect while AE in MJHQ and HQ did not. Polysaccharides comprised the major constituents in RA and HQ. All polysaccharides extract (PE) of the three herbs showed similar levels of immunological effect in both immunological assays.