Clinacanthus Nutans Induces Antiproliferative and Apoptosis in Human Breast Cancer Cells Through Targeted Apoptosis Pathway

Clinacanthus nutans dichloromethane fraction (CN-Dcm) extract has previously been proven to suppress breast cancer (MCF7) cell proliferation. Despite this, the molecular mechanisms involved in C. nutans extract-treated MCF7 cells are unknown. Hence, the molecular mechanism of apoptosis in treated MCF7 was investigated in this current study. This study was intended to subfractionate CN-Dcm extract using column chromatography and analysed the treated MCF7 cells using the CellTiter 96® AQueous One Solution Cell Proliferation (MTS) assay, Annexin V/propidium iodide (PI) assay, western blot and reverse transcription-qualitative polymerase chain reaction (RT-qPCR). Out of nine subfraction extracts (SF1 to SF9), SF2 extract strongly inhibited MCF7 cells with the lowest IC50 value (23.51 ± 0.99 µg/mL) and substantially induced apoptosis in the MCF7 cells. SF2 extract significantly downregulated BCL-2 expression and upregulated P53, BAX, BID, BCL-2, caspase-8, caspase-9 and caspase-3 expressions in treated MCF7 cells. Therefore, SF2 extract was analysed using liquid chromatography coupled to quadrupole time–of–flight mass spectrometry (LC-QTOF-MS), which confirmed the presence of bioactive chemical compounds. Thus, it can be concluded that the compounds found in SF2 extract may potentially cause apoptosis in MCF7 cells through intrinsic and extrinsic pathways.

A Narrative Review on the Phytochemistry, Pharmacology and Therapeutic Potentials of Clinacanthus nutans (Burm. f.) Lindau Leaves as an Alternative Source of Future Medicine

The application of natural products and supplements has expanded tremendously over the past few decades. Clinacanthus nutans (C. nutans), which is affiliated to the Acanthaceae family, has recently caught the interest of researchers from the countries of subtropical Asia due to its medicinal uses in alternative treatment for skin infection conditions due to insect bites, microorganism infections and cancer, as well as for health well-being. A number of bioactive compounds from this plant’s extract, namely phenolic compounds, sulphur containing compounds, sulphur containing glycosides compounds, terpens-tripenoids, terpens-phytosterols and chlorophyll-related compounds possess high antioxidant activities. This literature search yielded about one hundred articles which were then further documented, including the valuable data and findings obtained from all accessible electronic searches and library databases. The promising pharmacological activities from C. nutans leaves extract, including antioxidant, anti-cancer, anti-viral, anti-bacterial, anti-fungal, anti-venom, analgesic and anti-nociceptive properties were meticulously dissected. Moreover, the authors also discuss a few of the pharmacological aspect of C. nutans leaves extracts against anti-hyperlipidemia, vasorelaxation and renoprotective activities, which are seldom available from the previously discussed review papers. From the aspect of toxicological studies, controversial findings have been reported in both in-vitro and in-vivo experiments. Thus, further investigations on their phytochemical compounds and their mode of action showing pharmacological activities are required to fully grasp both traditional usage and their suitability for future drugs development. Data related to therapeutic activity and the constituents of C. nutans leaves were searched by using the search engines Google scholar, PubMed, Scopus and Science Direct, and accepting literature reported between 2010 to present. On the whole, this review paper compiles all the available contemporary data from this subtropical herb on its phytochemistry and pharmacological activities with a view towards garnering further interest in exploring its use in cardiovascular and renal diseases.

Osteogenic Activity of Lupeol Isolated from Clinacanthus nutans Lindau: Activity and Mode of Action

Clinacanthus nutans Lindau has been traditionally used for healing of bone fragility, but the mechanism of actions has not been clarified yet. In this study, the bone regeneration activity of lupeol derived from C. nutans was assessed using an in vitro model of osteoblast cells MC3T3-E1. The finding revealed that the compound was not significantly toxic to osteoblast cells at concentration of ≤40 μg/mL. Lupeol demonstrated the osteogenic activity through enhancement of alkaline phosphatase (ALP) of osteoblast cells up to 31.2%, 21%, and 12% at concentrations of 5, 10, and 20 µg/mL, respectively ( p < 0.05). Besides, the mineralization activity was increased up to 170, 230, 185, and 117% at concentration of 5, 10, 20, and 40 μg/mL, respectively ( p < 0.05). The marker genes related to osteoblast differentiation evaluated on the expression level in the presence of lupeol, including collagen I (col 1), osteopontin (opn), osterix (osx), and runx2, showed upregulated expression in all the test genes ( p < 0.05). The Western blot analysis demonstrated a clear effect of lupeol on expression of p38/p-p38, and ERK/p-ERK proteins involved in the MAPK signaling pathway. Thus, lupeol isolated from C. nutans exhibited the osteogenic activity by enhancing expression of important markers of osteogenesis, as well as affected the MAPK signaling pathway relating to osteoblast differentiation. This is the first report on the detailed mechanism of action of lupeol on bone regeneration and also explains for the traditional use of this medicinal plant for bone healing.

Expression Pattern of Genes in Condyloma Acuminata Treated with Clinacanthus nutans Lindau Cream versus Podophyllin

Clinacanthus nutans Lindua (C. nutans), a strong antiviral traditional medicine, can be used to treat condyloma acuminata (CA) caused by the human papillomavirus (HPV). However, its molecular mechanism for CA elimination is unknown. Herein, we conducted a randomized clinical trial to evaluate the effectiveness of C. nutans and its molecular mechanism compared with podophyllin, the gold standard treatment. Using a randomized block design, six patients were treated with C. nutans and podophyllin for four weeks. Efficacy of drugs was assessed by size reduction of the warts and HPV viral load quantification using droplet digital PCR. The gene expression profiling of CA was analyzed using NanoString Technology. After the podophyllin and C. nutans treatments, CA lesion sizes were reduced to 97.0% and 84.4% clearance, and the HPV viral loads were reduced by 74.0% and 46.6%, respectively. The gene expression pattern of immune profiling showed that 23 genes (i.e., HLA-DPB, CCL3, CXCL2, CXCR2, and OSM) were significantly differentially expressed by podophyllin, whereas 2 genes (IFNL1 and IRF2) were remarkably expressed by C. nutans. In inflammatory profiling, 108 genes (i.e., CXCL2, IL8, and STAT3) were highly expressed by podophyllin, but none of genes were observed to change expression by C. nutans. These results suggested that podophyllin may reduce the HPV infection through a mechanism related to proinflammatory response. In addition, C. nutans was found to suppress the HPV infection through mechanism related to the activation of immune response. This study shows novel therapeutic mechanisms of podophyllin and C. nutans. It is suggested that C. nutans might be used as an alternative treatment for CA treatment.

Immunomodulatory potential of Clinacanthus nutans extracts in the co-culture of triple-negative breast cancer cells, MDA-MB-231, and THP-1 macrophages

Triple-negative breast cancer is the main type of breast carcinoma that causes mortality among women because of the limited treatment options and high recurrence. Chronic inflammation has been linked with the tumor microenvironment (TME) in breast cancer progression. Clinacanthus nutans (CN) has gained much attention because of its anticancer properties, but its mechanism remains unclear. We aimed to study the qualitative phytochemical content and elucidate the cytotoxicity effects of CN on human triple-negative breast cancer (TNBC), MDA-MB-231 and human macrophage-like cells such as THP-1 by using sulforhodamine B (SRB) assay. As highly metastatic cells, MDA-MB-231 cells can migrate to the distal position, the effect of CN on migration were also elucidated using the scratch assay. The CN effects on ameliorating chronic inflammation in TME were studied following the co-culture of MDA-MB-231/THP-1 macrophages. The cytokine expression levels of IL-6, IL-1β and tumor necrosis factor-alpha (TNF-α) were determined using ELISA assays. The results showed that both ethanolic and aqueous CN extracts contained alkaloid, phenol and tannin, flavonoid, terpenoid, glycoside and steroid. However, saponin was only found in the aqueous extract of CN. CN was not cytotoxic to both MDA-MB-231 and THP-1 cells. The ability of MDA-MB-231 to migrate was also not halted by CN treatment. However, CN ethanol extract decreased IL-6 at 25 μg/mL (p = 0.02) and 100 μg/mL (p = 0.03) but CN aqueous extract increased IL-6 expression at 50 μg/mL (p = 0.08) and 100 μg/mL (p = 0.02). IL-1β showed decreased expression after treated with CN ethanol and CN aqueous both at 25 μg/mL (p = 0.03). TNF-α were significantly decreased after CN ethanol treatment at concentration 25- (p = 0.001), 50- (p = 0.000) and 100 μg/mL (p = 0.000). CN aqueous extract slightly inhibited TNF-α at all 25–50- and 100 μg/mL (p = 0.001, p = 0.000, p = 0.000, respectively). Overall, CN acts by ameliorating the pro-inflammatory condition in the TME and may be a potential strategy for its anticancer mechanism on highly metastatic breast cancer condition. The major pathways that link both cancer and inflammation were NF-κB and STATs thus further study on the upstream and downstream pathways is needed to fully understand the mechanism of CN extracts in cooling the inflamed TME in breast cancer.

Volatile Profiling Aided in the Isolation of Anti-Proliferative Lupeol from the Roots of Clinacanthus nutans (Burm. f.) Lindau

Isolation of anti-proliferative compounds from plants is always hindered by the complexities of the plant’s nature and tedious processes. Clinacanthus nutans (Burm. f.) Lindau is a medicinal plant with reported anti-proliferative activities. Our study aimed to isolate potential anti-proliferative compounds present in C. nutans plant. To start with, for our study, we came up with a strategy by first profiling the volatile compounds present in the leaf, stem and root of C. nutans using GC-MS. Comparing the plant’s volatile profiles greatly narrowed down our target of study. We decided to start with the isolation and characterization of a pentacyclic terpenoid, i.e., lupeol from the roots of C. nutans, as this compound was found to present abundantly in the roots compared to the leaf or stem. We developed a simple maceration and re-crystallization method, without the necessity to go through the fractionation or column chromatography for the isolation of lupeol. Characterizations of the isolated compound identified the compound as lupeol. The anti-proliferative activity of the isolated lupeol was further investigated against the MCF-7 cell line, which showed comparable anti-proliferative activity with the authentic lupeol and camptothecin. Our strategy to profile every part of the plant first, followed by selection of the most suitable plant part and targeted compound proved useful for further isolation and characterization bioactive compound from C. nutans.

Uji Aktivitas Antinosiseptif Kombinasi Ekstrak Daun Dandang Gendis [Clinacanthus nutans (Burn F) Lindau] Dan Daun Bakung (Crinum asiaticum L.) secara In Vivo

The development of new and effective pain medications that are natural isexpected to have the benefits of treatment. The purpose of this study was todetermine the antinociceptive effectiveness of a combination of 75% ethanolextract from dandang gendis leaves and 96% ethanol fraction of bakungleaf. In this study was used chemical method induction (Sigmund's method)in 25 male mice which were divided into 5 treatment groups, namelytreatment group I as negative control was given CMC 0.5%, treatmentgroup II as positive control was given acetosal 100 mg / kg BW, treatmentgroups III, IV and V were given combinations extract. Each group was giventreatment orally, thirty minutes then induced with 0.6% acetic acidintraperitonially, after which it was observed and counted for the amount ofstretching for sixty minutes. Data were analyzed by one-way ANOVA test.The results of this study showed that all treatment groups had a significantdifference (p <0.05), the combination of 75% ethanol extract dandanggendis leaf and 96% ethanol fraction of bakung leaf with a dose of 100 mg/ kg BW had good effectiveness with a percent effectiveness value of 92.50%.The results of this study indicate that the combination of 75% ethanolextract from dandang gendis leaf (Clinacanthus nutans (Burn F) Lindau)and 96% ethanol fraction from bakung leaf (Crinum asiaticum) with thesame concentration ratio, have relatively higher antinociceptive of theactivity of each extract, in other words both of them work together.