The Sesquiterpene Lactone-Rich Fraction of Inula helenium L. Enhances the Antitumor Effect of Anti-PD-1 Antibody in Colorectal Cancer: Integrative Phytochemical, Transcriptomic, and Experimental Analyses.

Treatment strategies combining immune checkpoint inhibitors with sesquiterpene lactones have attracted much attention as a promising approach for cancer treatment. We systemically analyzed gene expression profiles of cells in response to two major sesquiterpene lactones, alantolactone and isoalantolactone, and determined whether the sesquiterpene lactone-rich fraction of Inula helenium L. (SFIH) enhances the antitumor effect of anti-PD-1 antibody in MC38 colorectal cancer-bearing mice. Gene expression and pathway analysis using RNA sequencing data were used to identify the SFIH-driven combined activity with anti-PD-1 antibody. The results showed that SFIH significantly enhanced the antitumor effect of anti-PD-1 antibody by reducing tumor growth and increasing the survival time of mice. Specifically, SFIH exhibited antitumor activity when combined with anti-PD-1 antibody, and the effects were further enhanced compared with monotherapy. An analysis of immune cells indicated that combination treatment with SFIH and anti-PD-1 antibody significantly increased the proportion of CD8+ T cells. Moreover, combination treatment enhanced antitumor immunity by decreasing the population of myeloid-derived suppressor cells and increasing the number of M1-like macrophages. Pathway enrichment analysis revealed that combination therapy activated immune-related pathways to a greater extent than monotherapy. In conclusion, our integrative analysis demonstrates that SFIH enhances the response of murine tumors to anti-PD-1 antibody. These findings provide insight into developing integrative therapeutics and molecular data for the use of natural products as an adjunct treatment for colorectal cancer.

Biological Activities and Chemical Profile of Gentiana asclepiadea and Inula helenium Ethanolic Extracts.

This study aimed to investigate the antioxidant, antimicrobial, and cytotoxic potential of ethanolic extracts obtained from Gentiana asclepiadea L. and Inula helenium L. roots, in relation to their chemical composition. The total polyphenols, flavonoids, and phenolic acids were determined by spectrophotometric methods, while LC-MS analysis was used to evaluate the individual constituents. The antioxidant properties were tested using the FRAP and DPPH methods. The standard well diffusion and broth microdilution assays were carried out to establish in vitro antimicrobial efficacy and minimum inhibitory and bactericidal concentrations. The cytotoxicity was tested on rat intestinal epithelial cells using the MTT assay. The results pointed out important constituents such as secoiridoid glycoside (amarogentin), phenolic acids (caffeic acid, chlorogenic acid, trans-p-coumaric acid, salicylic acid), and flavonoids (apigenin, chrysin, luteolin, luteolin-7-O-glucoside, quercetin, rutoside, and naringenin) and promising antioxidant properties. The in vitro antimicrobial effect was noticed towards several pathogens (Bacillus cereus > Staphylococcus aureus > Enterococcus faecalis > Salmonella typhimurium and Salmonella enteritidis > Escherichia coli), with a pronounced bactericidal activity. Rat intestinal epithelial cell viability was not affected by the selected concentrations of these two extracts. These data support the ethnomedicinal recommendations of these species and highlight them as valuable sources of bioactive compounds.

Alantolactone alleviates collagen-induced arthritis and inhibits Th17 cell differentiation through modulation of STAT3 signalling.

CONTEXT: Alantolactone, the bioactive component in Inula helenium L. (Asteraceae), exhibits multiple biological effects. OBJECTIVE: We aimed to determine the anti-inflammatory effect of alantolactone in a collagen-induced arthritis (CIA) mouse model and its immunomodulatory effects on Th17 differentiation. MATERIALS AND METHODS: A CIA mouse model was established with DBA/1 mice randomly divided into four groups (n = 6): healthy, vehicle and two alantolactone-treated groups (25 or 50 mg/kg), followed by oral administration of alantolactone to mice for 21 consecutive days after arthritis onset. The severity of CIA was evaluated by an arthritic scoring system and histopathological examination. Levels of cytokines and anti-CII antibodies as well as percentages of splenic Th17 and Th17 differentiation with or without alantolactone treatments (0.62, 1.2 or 2.5 µM) were detected with ELISA and flow cytometry, respectively. Western blot analysis was used to evaluate intracellular signalling in alantolactone-treated spleen cells. RESULTS: In CIA mice, alantolactone at 50 mg/kg attenuated RA symptoms, including high arthritis scores, infiltrating inflammatory cells, synovial hyperplasia, bone erosion and levels of the proinflammatory cytokines TNF-α, IL-6 and IL-17A, but not IL-10 in paw tissues. Alantolactone also reduced the number of splenic Th17 cells and the capability of naïve CD4+ T cells to differentiate into the Th17 subset by downregulating STAT3/RORγt signalling by as early as 24 h of treatment. DISCUSSION AND CONCLUSIONS: Alantolactone possesses an anti-inflammatory effect that suppresses murine CIA by inhibiting Th17 cell differentiation, suggesting alantolactone is an adjunctive therapeutic candidate to treat rheumatoid arthritis.

Active ingredients of Inula helenium L. exhibits similar anti-cancer effects as isoalantolactone in pancreatic cancer cells.

Isoalantolactone is one of the major active ingredients from Inula helenium L. However, it is low cost-effective to isolate isoalantolactone from Inula helenium L. In this study, we optimized the extraction strategy and obtained a mixture of active ingredients with exact proportion (termed as F35), which were alloalantolactone, alantolactone and isoalantolactone at the ratio of 1/5/4 respectively. The anti-tumor activity of F35 was compared with isoalantolactone on pancreatic cancer cells. As a result, F35 showed nearly the same anti-proliferation activity as isoalantolactone in two cell lines. Both F35 and isoalantolactone could induce mitochondrion-related apoptosis at the concentration of 6 µg/ml. In addition, F35 inhibited colony-formation and migration of PANC-1 and SW1990 cells. To conclude, F35 exhibited similar anti-proliferation and anti-migration effect as isoalantolactone on two pancreatic cancer cell lines, suggesting that alantolactone or alloalantolactone might have comparable anti-tumor effect as isoalantolactone.

Anti-inflammatory Activity of Standardized Fraction from Inula helenium L. via Suppression of NF-κB Pathway in RAW 264.7 Cells

Inula helenium L. is rich source of eudesmane-type sesquiterpene lactones, mainly alantolactone and isoalantolactone, which have the various pharmacological functions. In this study, we examined the inhibitory effects of nitric oxide (NO) production of hexane, methylene chloride, ethyl acetate, butanol, and water fractions from I. helenium and investigated the anti-inflammatory effect of hexane fraction of I. helenium (HFIH) in LPS-induced RAW 264.7 cells. Quantification of alantolactone and isoalantolactone from HFIH was carried out for the standardization by multiple reaction monitoring using triple quadrupole mass spectrometer. HFIH significantly inhibited inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) protein as well as their downstream products NO and prostaglandin E₂ (PGE₂) in LPS-stimulated RAW 264.7 cells. Moreover, HFIH suppressed NF-κB transcriptional activity by decreasing the translocation of p65 to the nucleus. The in vivo study further confirmed that HFIH attenuated the paw edema induced by carrageenan in an acute inflammation model. These findings suggest that HFIH may be useful as a promising phytomedicine for inflammatory-associated diseases.

Sesquiterpene lactones-enriched fraction of Inula helenium L. induces apoptosis through inhibition of signal transducers and activators of transcription 3 signaling pathway in MDA-MB-231 breast cancer cells.

Inula helenium L., commonly known as Elecampane, has been extensively used for many countries in the folk medicine. Its root is a rich source of sesquiterpene lactones, which possess various pharmacological activities. To develop the phytomedicine including sesquiterpene lactones, we prepared hexane fraction from I. helenium (HFIH) and examined the inhibitory effect of HFIH on signal transducers and activators of transcription 3 (STAT3) activation in human breast cancer MDA-MB-231 cells. Additionally, detailed chemical investigation was done to pinpoint the most active sesquiterpene lactones responsible for its anticancer activity. HFIH selectively suppressed STAT3 phosphorylation at tyrosine 705, not affecting its upstream kinases. HFIH downregulated the expression of STAT3 target genes including cyclin D1 , c-myc, and bcl-2 and induced caspase-mediated apoptosis. Moreover, sesquiterpene lactones of HFIH clearly suppressed STAT3 activation. The in vivo results further supported that HFIH inhibits the growth of human breast xenograft tumors. Our results suggest that HFIH possesses potential anticancer activity, which is mainly mediated through STAT3 signaling pathway. These findings provide the potential of HFIH as a promising phytomedicine for the treatment and prevention of triple-negative breast cancer.

Total sesquiterpene lactones isolated from Inula helenium L. attenuates 2,4-dinitrochlorobenzene-induced atopic dermatitis-like skin lesions in mice.

BACKGROUND: Inula helenium L. is an herb whose anti-inflammatory properties are attributed to its active components, the sesquiterpene lactones (SLs). Our previous study demonstrated that the total sesquiterpene lactones isolated from Inula helenium L. (TSL-IHL), consisting mainly of alantolactone (AL) and isoalantolactone (IAL), may have potential in the prevention and treatment of rheumatoid arthritis (RA). However, the effect of TSL-IHL on atopic dermatitis (AD) has not been studied yet. AIM OF THE STUDY: The present study evaluates the potential of TSL-IHL as a treatment for AD. METHODS/STUDY DESIGNS: The effects of TSL-IHL on the expression of inflammatory genes and the activation of NF-κB signaling pathway in HaCat cells were examined by quantitative real-time PCR and western blotting, respectively, and compared with those of AL and IAL. The protective effect of TSL-IHL against AD was tested in a mouse model induced by 2,4-dinitrochlorobenzene (DNCB), in which AD-like skin lesions were induced in ICR mice by sensitizing once with 100 µl of 7% DNCB painted on their shaved back skin and then challenging with 20 µl of 0.2% DNCB five times on their right ears at 3 day intervals starting on day 5 post-sensitization. RESULTS: TSL-IHL, as well as AL and IAL, could all inhibit TNF-α-induced activation of NF-κB and the expression of TNF-α, IL-1, and IL-4 in HaCat cells in a dose-dependent manner in the range of 0.6-2.4 µg/ml. The topical application of TSL-IHL (1% W/W in emollient cream) attenuated DNCB-induced dermatitis severity and right ear swelling. The serum levels of IgE, TNF-α and IFN-γ in TSL-IHL-treated mice were reduced by 81.39%, 89.69%, and 87.85%, respectively, while the mRNA levels of IL-4, IL-5 and IL-13, in the back-skin lesions of TSL-IHL-treated mice were reduced by 39.21%, 40.62% and 48.12%, respectively, compared with the untreated controls. Histopathological examination showed that TSL-IHL treatment reduced epidermis/dermis thickening and dermal inflammatory infiltration in both ear and back skins. CONCLUSIONS: We suggest that TSL-IHL inhibited the development of AD-like skin symptoms by regulating cytokine expression and may be an effective alternative therapy for AD.

Ethyl acetate extract from Inula helenium L. inhibits the proliferation of pancreatic cancer cells by regulating the STAT3/AKT pathway.

Sesquiterpene lactones are bioactive compounds that have been identified as responsible for the anticancer activity of the medicinal herb, Inula helenium L. (IHL). However, the mechanisms of action involved in the anti­pancreatic cancer activity of IHL have yet to be elucidated. The present study used an optimized extraction strategy to obtain sesquiterpene lactones from IHL (the resulting product termed ethyl acetate extract of IHL; EEIHL), and examined the potential mechanisms involved in the anti­pancreatic cancer activity of EEIHL. Ethanol and ethyl acetate were used to extract sesquiterpene lactones from IHL to give the final product EEIHL. Cell Counting Kit­8, colony formation and Annexin V/propidium iodide assays were used to detect the anti­proliferative activity of EEIHL. Cell migration was determined with a wound healing assay. mRNA and protein expression levels were analyzed by reverse transcription­quantitative polymerase chain reaction and western blot analyses, respectively. It was identified that low concentrations of EEIHL caused CFPAC­1 cell cycle arrest in the G0/G1 phase, whereas high concentrations of EEIHL induced mitochondria­dependent apoptosis. In addition, EEIHL could inhibit the phosphorylation of the signal transducer and activator of transcription (STAT)3/AKT pathway, potentially resulting in impeded cell mobility. In conclusion, EEIHL could activate mitochondrial­dependent apoptosis and inhibit cell migration through the STAT3/AKT pathway in CFPAC-1 cells.

Chemical constituents from Inula helenium / 中草药

Objective To study the major chemical components from Inula helenium. Methods The compounds were separated and purifid by using a variety of chromatographic techniques including silica gel column chromatography, thin layer chromatography, and high performance liquid chromatography, and the structures of the compounds were verified by nuclear magnetic spectroscopy and literature data. Results A total of 22 compounds were separated from petroleum ether extract of I. helenium and identified separately as alantolactone (1), isoalantolactone (2), 4,4-dimethylsterols (3), 11αH,13-dihydroisoalantolactone (4), 11αH,13-dihydroalantolactone (5), 4(15)-epoxy-isoalantolactone (6), 5α,6α-epoxyalantolactone (7), alloalantolactone (8), isoalloalantolactone (9), friedelin (10), friedelinol (11), erythrodiol (12), β-sitosterol-glucopyranoside (13), lupeol acetate (14), lupeone (15), lupeol (16), δ-amyrin (17), lupeol palitate (18), 5,7,4'-trihydroxy-3',5'-dimethoxy flavane (19), (+)-syringaresinol (20), 3,5,3'-trihydroxy-6,7,4'-trimethoxy flavone (21), and 3,5,6,7,3'-hydroxy-4'-methoxy dihydroflavones (22). Conclusion Compounds 21 and 22 are isolated from this genus for the first time; Compounds 10-15, 17, and 18 are separated from the I. helenium for the first time. After antibacterial test, compounds 1, 2, 4, 5, 7-9 have strong inhibitory effects on Escherichia coli, Staphylococcus aureus, and Bacillus subtilis.

Identification of anti-inflammatory active ingredients from Tumuxiang by ultra-performance liquid chromatography/quadrupole time-of-flight-MSE.

The dried roots of Inula helenium L. (IH) and Inula racemosa Hook f. (IR) are used commonly as folk medicine as 'tumuxiang' (TMX). The mixing and sharing of IH and IR in clinical use is a universal phenomenon. Modern pharmacological studies confirmed that IH and IR display anti-inflammatory activities. However, the difference in anti-inflammatory pharmacodynamic substances between these two herbs is still unknown. In the present study, the fingerprints of 18 IH and nine IR samples were established using UPLC/QTOF-MSE . A dimethylbenzene-induced mouse ear vasodilation model was applied in evaluating the anti-inflammatory properties of all 27 samples. Then, the spectrum-efficacy model between chemical characteristic peaks and anti-inflammatory activities was investigated using principal component regression and partial least squares. Finally, the combination of UNIFI Scientific Information System with a library search of traditional Chinese medicines was employed to automatically characterize the peaks. UNIFI identified a total of 80 chemical components. Among the components, the 53 characteristic peaks showed correlation with anti-inflammatory activities, pointing to phenolic and organic acids as primary anti-inflammatory ingredients of TMX. This approach can efficiently and intelligently facilitate the identification of bioactive components from traditional Chinese medicine.

Total sesquiterpene lactones prepared from Inula helenium L. has potentials in prevention and therapy of rheumatoid arthritis.

BACKGROUNDS: Inula helenium L. is an herb with anti-inflammatory properties. Sesquiterpene lactones (SLs), mainly alantolactone (AL) and isoalantolactone (IAL), are considered as its active ingredients. However, the anti-inflammatory effects of SL-containing extracts of I. helenium have not been explored. Here we prepared total SLs from I. helenium (TSL-IHL), analyzed its chemical constituents, and performed cellular and animal studies to evaluate its anti-inflammatory activities. MATERIALS AND METHODS: The chemical profile of TSL-IHL was analyzed by HPLC-UV. Its in vitro effects on the activation of signaling pathways and expression of inflammatory genes were examined by western blotting and quantitative real-time PCR, respectively, and compared with those of AL and IAL. Its in vivo anti-inflammatory effects were evaluated in adjuvant- and collagen-induced arthritis rat models. RESULTS: Chemical analysis showed that AL and IAL represent major constituents of TSL-IHL. TSL-IHL, as well as AL and IAL, could inhibit TNF-α-induced activation of NF-κB and MAPK pathways in b. End3 cells, suppress the expressions of MMP-3, MCP-1, and IL-1 in TNF-α-stimulated synovial fibroblasts, and IL-1, IL-6, and iNOS in LPS-activated RAW 264.7 cells in a dose-dependent manner in the range of 0.6-2.4µg/mL. Oral administration of TSL-IHL at 12.5-50mg/kg could dose-dependently alleviate the arthritic severity and paw swelling in either developing or developed phases of arthritis of rats induced by adjuvant or collagen CONCLUSIONS: These results indicated potentials of TSL-IHL in prevention and therapy of rheumatoid arthritis.

Synthesis of small combinatorial libraries of natural products: identification and quantification of new long-chain 3-methyl-2-alkanones from the root essential oil of Inula helenium L. (Asteraceae).

INTRODUCTION: Recently, a potent anti-staphylococcal activity of Inula helenium L. (Asteraceae) root essential oil was reported. Also, bioassay guided fractionation of the oil pointed to eudesmane sesquiterpene lactones and a series of unidentified constituents as the main carriers of the observed activity. OBJECTIVE: To identify nine new constituents (long-chain 3-methyl-2-alkanones) from a fraction of this root essential oil with a low minimum inhibitory concentration value (0.8 µg/mL) by employing a synthetic methodology that leads to the formation of a small combinatorial library of these compounds. METHODS: The identity of these constituents was inferred from mass spectral fragmentation patterns and GC retention data. A library of 3-methyl-2-alkanones (C11 -C19 homologous series) was synthesised in three steps starting from methyl acetoacetate and the corresponding alkyl halides. The synthetic library was also screened for in vitro anti-microbial activity. RESULTS: Gas chromatographic analyses of I. helenium essential oil samples with spiked compounds from the synthesised library corroborated the tentative identifications of the long-chain 3-methyl-2-alkanones. The availability of these anti-microbial compounds from this library made it possible to construct GC/FID calibration curves and determine their content in the plant material: 0.08 - 24.2 mg/100 g of dry roots. CONCLUSION: The small combinatorial library approach enabled the first unequivocal identification of long-chain 3-methyl-2-alkanones as plant secondary metabolites, and, also, allowed determination of not only a single compound and biological properties, but those of a group of structurally related compounds.

STUDY ON QUALITY CONTROL OF INUSAL HERBAL COMBINATION / Монголын эм зүй, эм судлал

Abstract: Quality study of Inusal preparation herbal combined ingredients with (Salsola laricifolia Turcz.ex litv and Inula helenium L. (1:1)). The quality study of biological active substances of the preparation was identified using standard substances by thin layer chromatography (TLC). As a result of Inusal study by chromatogram appeared the same as standard substances rutin, quercitin brown yellow, inulin dark brown, alantolactone and isoalantolactone blue and pink spots. Therefore, Unusal preparation contains flavonoid, coumarin, sesquiterpene lactones. Keywords: Inusal, Salsola laricifolia Turcz.ex litv, Inula helenium L.

Ultrasound-assisted extraction of alantolactone and isoalantolactone from Inula helenium roots.

This work deals with ultrasound-assisted extraction (UAE) of alantolactone and isoalantolactone from the roots of Inula helenium L., a well-known medicinal plant. The effects of ethanol concentration, extraction time, temperature and number of extraction steps on the extraction yields of both sesquiterpene lactones were investigated. Gas chromatographic (GC) method was used for simultaneous determination of their contents in the corresponding extracts. A comparison with classical extraction methods [maceration, infusion and micro steam distillation-extraction (MSDE)] showed that the amounts of alantolactone and isoalantolactone achieved by UAE with 70 and 96% EtOH for 30 min at room temperature were higher or almost equal to those obtained by maceration for 24 hours.