RNA-binding motif protein 10 inactivates c-Myc by partnering with ribosomal proteins uL18 and uL5.

RNA-binding motif protein 10 (RBM10) is a frequently mutated tumor suppressor in lung adenocarcinoma (LUAD). Yet, it remains unknown whether cancer-derived mutant RBM10 compromises its tumor suppression function and, if so, the molecular insight of the underlying mechanisms. Here, we show that wild-type RBM10 suppresses lung cancer cell growth and proliferation by inactivating c-Myc that is essential for cancer cell survival. RBM10 directly binds to c-Myc and promotes c-Myc's ubiquitin-dependent degradation, while RBM10 knockdown leads to the induction of c-Myc level and activity. This negative action on c-Myc is further boosted by ribosomal proteins (RPs) uL18 (RPL5) and uL5 (RPL11) via their direct binding to RBM10. Cancer-derived mutant RBM10-I316F fails to bind to uL18 and uL5 and to inactivate c-Myc, thus incapable of suppressing tumorigenesis. Our findings uncover RBM10 as a pivotal c-Myc repressor by cooperating with uL18 and uL5 in lung cancer cells, as its failure to do so upon mutation favors tumorigenesis.

Mutant p53 Gains Its Function via c-Myc Activation upon CDK4 Phosphorylation at Serine 249 and Consequent PIN1 Binding.

TP53 missense mutations significantly influence the development and progression of various human cancers via their gain of new functions (GOF) through different mechanisms. Here we report a unique mechanism underlying the GOF of p53-R249S (p53-RS), a p53 mutant frequently detected in human hepatocellular carcinoma (HCC) that is highly related to hepatitis B infection and aflatoxin B1. A CDK inhibitor blocks p53-RS's nuclear translocation in HCC, whereas CDK4 interacts with p53-RS in the G1/S phase of the cells, phosphorylates it, and enhances its nuclear localization. This is coupled with binding of a peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1) to p53-RS, but not the p53 form with mutations of four serines/threonines previously shown to be crucial for PIN1 binding. As a result, p53-RS interacts with c-Myc and enhances c-Myc-dependent rDNA transcription key for ribosomal biogenesis. These results unveil a CDK4-PIN1-p53-RS-c-Myc pathway as a novel mechanism for the GOF of p53-RS in HCC.

Antidiabetic Effect of Fermented Mesembryanthemum crystallinum L. in db/db Mice Involves Regulation of PI3K-Akt Pathway.

Type 2 diabetes (T2D) is a serious health issue with increasing incidences worldwide. However, current medications have limitations due to side effects such as decreased appetite, stomach pain, diarrhea, and extreme tiredness. Here, we report the effect of fermented ice plant (FMC) in the T2M mouse model of db/db mice. FMC showed a greater inhibition of lipid accumulation compared to unfermented ice plant extract. Two-week oral administration with FMC inhibited body weight gain, lowered fasting blood glucose, and improved glucose tolerance. Serum parameters related to T2D including insulin, glycosylated hemoglobin, adiponectin, and cholesterols were improved as well. Histological analysis confirmed the protective effect of FMC on pancreas and liver destruction. FMC treatment significantly increased the expression and phosphorylation of IRS-1, PI3K, and AKT. Additionally, AMP-activated protein kinase phosphorylation and nuclear factor erythroid 2-related factor 2 were also increased in the liver tissues of db/db mice treated with FMC. Overall, our results indicate the anti-diabetic effect of FMC; therefore, we suggest that FMC may be useful as a therapeutic agent for T2D.

Acetylcorynoline Induces Apoptosis and G2/M Phase Arrest through the c-Myc Signaling Pathway in Colon Cancer Cells.

Colorectal cancer (CRC) is the third most commonly diagnosed cancer worldwide, and despite advances in treatment, survival rates are still low; therefore, the development of novel drugs is imperative. Acetylcorynoline (ACN) is derived from Corydalis ambigua Cham. et Schltdl tubers. The effect of ACN on colon cancer is still unknown. Therefore, we investigated its potential effects. Our data showed that ACN inhibited cell viability and proliferation. Moreover, ACN induced apoptosis and cell cycle arrest by inhibiting cell growth. In the present study, we hypothesized that ACN regulates c-Myc through CNOT2 or MID1IP1. ACN reduced the protein expression of oncogenic genes, decreased c-Myc half-life, and rapidly inhibited the serum stimulation response. Moreover, knockdown of CNOT2 and MID1IP1 with ACN increased apoptosis and further reduced the expression of oncogenes. In addition, ACN exhibited a synergistic effect with low-dose 5-fluorouracil (5-FU) and doxorubicin (Dox). Collectively, our data demonstrate that ACN inhibited c-Myc expression through CNOT2 and MID1IP1, and induced apoptosis. These findings indicate the potential of ACN as a therapeutic agent against colon cancer.

Viscum album Induces Apoptosis by Regulating STAT3 Signaling Pathway in Breast Cancer Cells.

In this study, we investigated the potential anticancer effects of Viscum album, a parasitic plant that grows on Malus domestica (VaM) on breast cancer cells, and explored the underlying mechanisms. VaM significantly inhibited cell viability and proliferation and induced apoptosis in a dose-dependent manner. VaM also regulated cell cycle progression and effectively inhibited activation of the STAT3 signaling pathway through SHP-1. Combining VaM with low-dose doxorubicin produced a synergistic effect, highlighting its potential as a promising therapeutic. In vivo, VaM administration inhibited tumor growth and modulated key molecular markers associated with breast cancer progression. Overall, our findings provide strong evidence for the therapeutic potential of VaM in breast cancer treatment and support further studies exploring clinical applications.

Euonymus sachalinensis Induces Apoptosis by Inhibiting the Expression of c-Myc in Colon Cancer Cells.

We hypothesized that Euonymus sachalinensis (ES) induces apoptosis by inhibiting the expression of c-Myc in colon cancer cells, and this study proved that the methanol extract of ES has anticancer effects in colon cancer cells. ES belongs to the Celastraceae family and is well known for its medicinal properties. Extracts of species belonging to this family have been used to treat diverse diseases, including rheumatoid arthritis, chronic nephritis, allergic conjunctivitis, rhinitis, and asthma. However, ES has been targeted because there are currently few studies on the efficacy of ES for various diseases, including cancer. ES lowers cell viability in colon cancer cells and reduces the expression of c-Myc protein. We confirm that the protein level of apoptotic factors such as PARP and Caspase 3 decrease when ES is treated with Western blot, and confirm that DNA fragments occur through TUNEL assay. In addition, it is confirmed that the protein level of oncogenes CNOT2 and MID1IP1 decrease when ES is treated. We have also found that ES enhances the chemo-sensitivity of 5-FU in 5-FU-resistant cells. Therefore, we confirm that ES has anticancer effects by inducing apoptotic cell death and regulating the oncogenes CNOT2 and MID1IP1, suggesting its potential for use in the treatment of colon cancer.

Phyotochemical candidates repurposing for cancer therapy and their molecular mechanisms.

Though limited success through chemotherapy, radiotherapy and surgery has been obtained for efficient cancer therapy for modern decades, cancers are still considered high burden to human health worldwide to date. Recently repurposing drugs are attractive with lower cost and shorter time compared to classical drug discovery, just as Metformin from Galega officinalis, originally approved for treating Type 2 diabetes by FDA, is globally valued at millions of US dollars for cancer therapy. As most previous reviews focused on FDA approved drugs and synthetic agents, current review discussed the anticancer potential of phytochemicals originally approved for treatment of cardiovascular diseases, diabetes, infectious diarrhea, depression and malaria with their molecular mechanisms and efficacies and suggested future research perspectives.

Natural Products for Esophageal Cancer Therapy: From Traditional Medicine to Modern Drug Discovery.

Esophageal cancer (EC) is one of the most malignant types of cancer worldwide and has a high incidence and mortality rate in Asian countries. When it comes to treating EC, although primary methods such as chemotherapy and surgery exist, the prognosis remains poor. The purpose of this current research is to review the range of effects that natural products have on cancer by analyzing studies conducted on EC. Fifty-seven studies were categorized into four anti-cancer mechanisms, as well as clinical trials. The studies that were scrutinized in this research were all reported within five years. The majority of the substances reviewed induced apoptosis in EC, acting on a variety of mechanisms. Taken together, this study supports the fact that natural products have the potential to act as a candidate for treating EC.

Antitumor Effect of Cycloastragenol in Colon Cancer Cells via p53 Activation.

Colorectal cancer cell (CRC) is the fourth most common cancer in the world. There are several chemotherapy drugs available for its treatment, though they have side effects. Cycloastragenol (CY) is a compound from Astragalus membranaceus (Fisch.) Bge known to be effective in aging, anti-inflammatory, anticancer, and anti-heart failure treatments. Although many studies have demonstrated the functions of CY in cancer cells, no studies have shown the effects of p53 in colon cancer cells. In this study, we found that CY reduces the viability of colon cancer cells in p53 wild-type cells compared to p53 null cells and HT29. Furthermore, CY induces apoptosis by p53 activation in a dose- and time-dependent manner. And it was confirmed that it affects the L5 gene related to p53. Additionally, CY enhanced p53 expression compared to when either doxorubicin or 5-FU was used alone. Altogether, our findings suggest that CY induces apoptosis via p53 activation and inhibits the proliferation of colon cancer cells. In addition, apoptosis occurs in colon cancer cells due to other factors. Moreover, CY is expected to have a combined effect when used together with existing treatments for colon cancer in the future.

The Antitumor Effect of Timosaponin A3 through c-Myc Inhibition in Colorectal Cancer Cells and Combined Treatment Effect with 5-FU or Doxorubicin.

Timosaponin A3 (TA3), extracted from the rhizome of Anemarrhenaasphodeloides Bunge, has been reported to affect various diseases, such as cancer, Alzheimer's disease, and allergies. However, the underlying molecular mechanisms and impacts are largely unknown. In the present study, we hypothesized that TA3 induces apoptosis through the inhibition of c-Myc expression via CNOT2 or MID1IP1 in HCT116. An MTT assay and colony formation assay were used to measure cell viability and proliferation. The protein expression of apoptotic markers and oncogenes was measured using immunoblotting and immunofluorescence assays. The interaction between MID1IP1 and c-Myc was confirmed by performing an immunoprecipitation assay. TA3 markedly inhibited colon cancer cell proliferation. Consistently, TA3 regulated the apoptotic proteins pro-PARP and caspase 3. TA3 inhibited the half-life of c-Myc and suppressed its expression in response to serum stimulation. In addition, TA3 enhanced the apoptotic effects of doxorubicin and 5-FU in colon cancer cells. Altogether, our results reveal a mechanism by which TA3 induces apoptosis through inhibiting c-Myc expression via CNOT2 or MID1IP1 in HCT116, which may help in the development of new therapies for colon cancer based on TA3 in the future.

Ribosomal protein L5 mediated inhibition of c-Myc is critically involved in sanggenon G induced apoptosis in non-small lung cancer cells.

Though Sanggenon G (SanG) from root bark of Morus alba was known to exhibit anti-oxidant and anti-depressant effects, its underlying mechanisms still remain unclear. Herein SanG reduced the viability of A549 and H1299 non-small lung cancer cells (NSCLCs). Also, SanG increased sub-G1 population via inhibition of cyclin D1, cyclin E, CDK2, CDK4 and Bcl-2, cleavages of poly (ADP-ribose) polymerase (PARP) and caspase-3 in A549 and H1299 cells. Of note, SanG effectively inhibited c-Myc expression by activating ribosomal protein L5 (RPL5) and reducing c-Myc stability even in the presence of cycloheximide and 20% serum in A549 cells. Furthermore, SanG enhanced the apoptotic effect with doxorubicin in A549 cells. Taken together, our results for the first time provide novel evidence that SanG suppresses proliferation and induces apoptosis via caspase-3 activation and RPL5 mediated inhibition of c-Myc with combinational potential with doxorubicin.

MicroRNA216b mediated downregulation of HSP27/STAT3/AKT signaling is critically involved in lambertianic acid induced apoptosis in human cervical cancers.

Since heat shock protein (HSP27) is a prognostic marker in cervical cancer, in the present study, the apoptotic mechanism of lambertianic acid (LA) was investigated in human cervical cancers in association with HSP27/STAT3/AKT signaling axis. LA exerted significant cytotoxicity, induced sub-G1 population, and increased the cleavage of Poly (ADP-ribose) polymerase (PARP) and cysteine aspartyl-specific protease 3 (caspase3) in HeLa and Caski cancer cells. Consistently, LA downregulated anti-apopotic genes such as B-cell lymphoma 2 (Bcl-2) and inhibitors of apoptosis proteins (c-IAP) in HeLa and Caski cells. Furthermore, LA-inhibited phosphorylation of HSP27, signal transducer, and activator of transcription 3 (STAT3) and Protein kinase B (AKT) through disturbing the binding of HSP27 with STAT3 or AKT in HeLa cells. Notably, LA upregulated the level of miR216b in HeLa and Caski cells. Consistently, miR216b mimic suppressed phosphorylation of HSP27 and reduced the expression of pro-PARP, while miR216b inhibitor reversed the ability of LA to attenuate phosphorylation of AKT, HSP27, and STAT3 and to reduce the expression of pro-PARP in HeLa cells. Overall, our findings suggest that miRNA216b mediated inhibition of HSP27/STAT3/ AKT signaling axis is critically involved in LA-induced apoptosis in cervical cancers.

Apoptotic and antihepatofibrotic effect of honokiol via activation of GSK3ß and suppression of Wnt/ß-catenin pathway in hepatic stellate cells.

Though honokiol, derived from the Magnolia tree, was known to suppress renal fibrosis, pulmonary fibrosis, non-alcoholic steatoheptitis, inflammation and cancers, the underlying antifibrotic mechanisms of honokiol are not fully understood in hepatic stellate cells until now. Thus, in the present study, inhibitory mechanism of honokiol on liver fibrosis was elucidated mainly in hepatic stellate cells (HSCs) by 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, cell cycle analysis and western-blotting. Honokiol exerted cytotoxicity in LX-2, HSC-T6 and Hep-G2 cells. Honokiol increased sub G1 population and activated caspase 3 and cleaved poly (ADP-ribose) polymerase (PARP) in HSCs. Moreover, honokiol attenuated the expression of alpha smooth muscle actin (α-SMA), transforming growth factor beta 1 (TGF-ß1), phospho-Smad3, phospho-AKT, cyclin D1, c-Myc, Wnt3a, ß-catenin, and activated phosphorylation of glycogen synthase kinase 3 beta (GSK3ß) in HSCs. Conversely, GSK3ß inhibitor SB216763 reversed the effect of honokiol on PARP, α-SMA, phospho-GSK3ß, ß-catenin and sub G1 population in LX-2 cells. Overall, honokiol exerts apoptotic and antifibrotic effects via activation of GSK3ß and inhibition of Wnt3a/ß-catenin signalling pathway.

Anti-Obesity Effect of Polygalin C Isolated from Polygala japonica Houtt. via Suppression of the Adipogenic and Lipogenic Factors in 3T3-L1 Adipocytes.

Obesity is a risk factor for metabolic diseases including type 2 diabetes, nonalcoholic steatohepatitis (NASH), heart diseases, and cancer. This study aimed to investigate the anti-obesity effect of Polygalin C (PC) isolated from Polygala japonica Houtt. in 3T3-L1 adipocytes. Based on Oil Red O assay results, PC significantly decreased lipid accumulation compared to the control. We found that PC suppressed adipogenesis transcription factors including peroxisome proliferator-activated receptor γ (PPAR γ) and CCAAT/enhancer-binding protein (C/EBP) α, and lipogenic factors such as sterol regulatory element-binding protein 1c (SREBP 1c) and fatty acid synthase (FAS), in 3T3-L1 adipocytes using Western blotting and real-time polymerase chain reaction (PCR). Moreover, PC inhibited the differentiation of 3T3-L1 cells by regulating the AMP-activated protein kinase (AMPK)/acetyl-CoA carboxylase (ACC) and mitogen-activated protein kinase/protein kinase B (MAPK/Akt) signaling pathways. Additionally, we confirmed that PC inhibited early adipogenesis factors C/EBP ß and C/EBP δ. Therefore, PC inhibited adipogenesis and lipogenesis in vitro. Thus, PC appears to exert potential therapeutic effects on obesity by suppressing lipid metabolism.

Clinical Outcome of Rotational Atherectomy in Calcified Lesions in Korea-ROCK Registry.

Background and Objectives: Data is still limited regarding clinical outcomes of rotational atherectomy (RA) after percutaneous coronary intervention. We sought to evaluate clinical outcomes of RA. Materials and Methods: This multi-center registry enrolled patients who underwent RA during PCI from nine tertiary centers in Korea between January 2010 and October 2019. The primary endpoint was target-vessel failure (TVF; the composite outcome of cardiac death, target-vessel spontaneous myocardial infarction, or target-vessel revascularization). Results: Of 540 patients (583 lesions), the mean patient age was 71.4 ± 0.4 years, 323 patients (59.8%) were men, and 305 patients (56.5%) had diabetes mellitus. Technical success rate was 96.4%. In-hospital major adverse cerebral and cardiac events occurred in 63 cases (10.8%). At 1.5 years, 72 (16.0%) of TVFs were occurred. We evaluated independent predictors of TVF, which included current smoker (hazard ratio (HR), 1.92; 95% confidence interval (CI), 1.17-3.16; p = 0.01), chronic renal disease (HR, 1.87; 95% CI, 1.14-3.08; p = 0.013), history of cerebrovascular attack (HR, 2.14; 95% CI, 1.24-3.68; p = 0.006), left ventricle ejection fraction (HR, 0.98; 95% CI, 0.97-0.999; p = 0.037), and left main disease (HR, 1.94; 95% CI, 1.11-3.37; p = 0.019). Conclusions: From this registry, we demonstrated acceptable success rates, in-hospital and mid-term clinical outcomes of RA in the DES era.

Crotonylation at serine 46 impairs p53 activity.

Post-translational modifications (PTMs) play pivotal roles in controlling the stability and activity of the tumor suppressor p53 in response to distinct stressors. Here we report an unexpected finding of a short chain fatty acid modification of p53 in human cells. Crotonic acid (CA) treatment induces p53 crotonylation, but surprisingly reduces its protein, but not mRNA level, leading to inhibition of p53 activity in a dose dependent fashion. Surprisingly this crotonylation targets serine 46, instead of any predicted lysine residues, of p53, as detected in TCEP-probe labeled crotonylation and anti-crotonylated peptide antibody reaction assays. This is further confirmed by substitution of serine 46 with alanine, which abolishes p53 crotonylation in vitro and in cells. CA increases p53-dependent glycolytic activity, and augments cancer cell proliferation in response to metabolic or DNA damage stress. Since serine 46 is only found in human p53, our studies unveil an unconventional PTM unique for human p53, impairing its activity in response to CA. Because CA is likely produced by the gut microbiome, our results also predict that this type of PTM might play a role in early human colorectal neoplasia development by negating p53 activity without mutation of this tumor suppressor gene.

p53 dependent LGR5 inhibition and caspase 3 activation are critically involved in apoptotic effect of compound K and its combination therapy potential in HCT116 cells.

Though ginsenoside metabolite compound K was known to have antitumor effect in several cancers, its underlying apoptotic mechanism still remains unclear so far. Thus, in the present study, the apoptotic mechanism of compound K was explored in colorectal cancer cells (CRCs) in association with leucine rich repeat containing G protein-coupled receptor 5 (LGR5) that was overexpressed in colorectal cancers with poor survival rate. Here compound K significantly reduced viability of HCT116p53+/+ cells better than that of HCT116p53-/- cells. Consistently, compound K increased sub G1 population and attenuated the expression of LGR5, c-Myc, procaspase3, Pin1 in HCT116p53+/+ cells more than in HCT116p53-/- cells. Conversely, caspase 3 inhibitor Z-DEVD-FMK reversed inhibitory effect of compound K on LGR5, c-Myc and procaspase3 in HCT116 cells. Consistently, inhibition of LGR5 using transfection method enhanced suppression of pro-PARP, Bcl-xL c-Myc, Snail and Pin1 in compound K treated HCT116p53+/+ cells. Furthermore, compound K synergistically potentiated antitumor effect of 5-fluorouracil (5-FU) or Doxorubicin to reduce the survival genes and cytotoxicity in HCT116p53+/+ cells. Overall, our findings provide scientific insight that compound K induces apoptosis in colon cancer cells via caspase and p53 dependent LGR5 inhibition with combination therapy potential with 5-FU or doxorubicin.

Molecular networks of FOXP family: dual biologic functions, interplay with other molecules and clinical implications in cancer progression.

Though Forkhead box P (FOXP) transcription factors comprising of FOXP1, FOXP2, FOXP3 and FOXP4 are involved in the embryonic development, immune disorders and cancer progression, the underlying function of FOXP3 targeting CD4 + CD25+ regulatory T (Treg) cells and the dual roles of FOXP proteins as an oncogene or a tumor suppressor are unclear and controversial in cancers to date. Thus, the present review highlighted research history, dual roles of FOXP proteins as a tumor suppressor or an oncogene, their molecular networks with other proteins and noncoding RNAs, cellular immunotherapy targeting FOXP3, and clinical implications in cancer progression.

Inhibition of TFEB oligomerization by co-treatment of melatonin with vorinostat promotes the therapeutic sensitivity in glioblastoma and glioma stem cells.

Glioblastoma (GBM) is the most aggressive malignant glioma and most lethal form of human brain cancer (Clin J Oncol Nurs. 2016;20:S2). GBM is also one of the most expensive and difficult cancers to treat by the surgical resection, local radiotherapy, and temozolomide (TMZ) and still remains an incurable disease. Oncomine platform analysis and Gene Expression Profiling Interactive Analysis (GEPIA) show that the expression of transcription factor EB (TFEB) was significantly increased in GBMs and in GBM patients above stage IV. TFEB requires the oligomerization and localization to regulate transcription in the nucleus. Also, the expression and oligomerization of TFEB proteins contribute to the resistance of GBM cells to conventional chemotherapeutic agents such as TMZ. Thus, we investigated whether the combination of vorinostat and melatonin could overcome the effects of TFEB and induce apoptosis in GBM cells and glioma cancer stem cells (GSCs). The downregulation of TFEB and oligomerization by vorinostat and melatonin increased the expression of apoptosis-related genes and activated the apoptotic cell death process. Significantly, the inhibition of TFEB expression dramatically decreased GSC tumor-sphere formation and size. The inhibitory effect of co-treatment resulted in decreased proliferation of GSCs and induced the expression of cleaved PARP and p-γH2AX. Taken together, our results definitely demonstrate that TFEB expression contributes to enhanced resistance of GBMs to chemotherapy and that vorinostat- and melatonin-activated apoptosis signaling in GBM cells by inhibiting TFEB expression and oligomerization, suggesting that co-treatment of vorinostat and melatonin may be an effective therapeutic strategy for human brain cancers.

Suppression of lung inflammation by the ethanol extract of Chung-Sang and the possible role of Nrf2.

BACKGROUND: Asian traditional herbal remedies are typically a concoction of a major and several complementary herbs. While balancing out any adverse effect of the major herb, the complementary herbs could dilute the efficacy of the major herb, resulting in a suboptimal therapeutic effect of an herbal remedy. Here, we formulated Chung-Sang (CS) by collating five major herbs, which are used against inflammatory diseases, and tested whether an experimental formula composed of only major herbs is effective in suppressing inflammation without significant side effects. METHODS: The 50% ethanol extract of CS (eCS) was fingerprinted by HPLC. Cytotoxicity to RAW 264.7 cells was determined by an MTT assay and a flow cytometer. Nuclear NF-κB and Nrf2 were analyzed by western blot. Ubiquitinated Nrf2 was similarly analyzed following immunoprecipitation of Nrf2. Acute lung inflammation and sepsis were induced in C57BL/6 mice. The effects of eCS on lung disease were measured by HE staining of lung sections, a differential cell counting of bronchoalveolar lavage fluid, a myeloperoxidase (MPO) assay, a real-time qPCR, and Kaplan-Meier survival of mice. RESULTS: eCS neither elicited cytotoxicity nor reactive oxygen species. While not suppressing NF-κB, eCS activated Nrf2, reduced the ubiquitination of Nrf2, and consequently induced the expression of Nrf2-dependent genes. In an acute lung inflammation mouse model, an intratracheal (i.t.) eCS suppressed neutrophil infiltration, the expression of inflammatory cytokine genes, and MPO activity. In a sepsis mouse model, a single i.t. eCS was sufficient to significantly decrease mouse mortality. CONCLUSIONS: eCS could suppress severe lung inflammation in mice. This effect seemed to associate with eCS activating Nrf2. Our findings suggest that herbal remedies consisting of only major herbs are worth considering.