Development and Validation of HPLC Method for Determination of Sodium Copper Chlorophyllin - A Food Colorant and Its Application in Pharmacokinetic Study.

A simple, accurate, and precise bioanalytical method was developed and validated for the determination of pharmacokinetic parameters of sodium copper chlorophyllin, a USFDA approved food additive and colorant in rat plasma. The column used was Luna® C18 250×4.6 mm, 100 Å, having particle size 4.5 µm, and the mobile phase used was methanol (MeOH), and 10 mM ammonium acetate buffer in the ratio of 90 : 10, the flow rate was 1 ml/min, and the injection volume of 20 µL. The retention time of sodium copper chlorophyllin was obtained at 9 min. The method was found to be linear at the range of 0.50-8.00 µg mL-1 .

Photodynamic Inactivation of Methicillin-Resistant Staphylococcus aureus by a Natural Food Colorant (E-141ii).

This study evaluates the photosensitizing effectiveness of sodium copper chlorophyllin, a natural green colorant commonly used as a food additive (E-141ii), to inactivate methicillin-sensitive and methicillin-resistant Staphylococcus aureus under red-light illumination. Antimicrobial photodynamic inactivation (aPDI) was tested on a methicillin-sensitive reference strain (ATCC 25923) and a methicillin-resistant Staphylococcus aureus strain (GenBank accession number Mh087437) isolated from a clinical sample. The photoinactivation efficacy was investigated by exposing the bacterial strains to different E-141ii concentrations (0.0, 1.0, 2.5, 5.0, 10.0, and 20.0 µM) and to red light (625 nm) at 30 J cm-2. The results showed that E-141ii itself did not prevent bacterial growth for all tested concentrations when cultures were placed in the dark. By contrast, E-141ii photoinactivated both methicillin-sensitive Staphylococcus aureus (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA) under red-light illumination. However, different dose responses were observed for MSSA and MRSA. Whilst the MSSA growth was inhibited to the detection limit of the method with E-141ii at 2.5 µM, >10 µM concentrations were required to inhibit the growth of MRSA. The data also suggest that E-141ii can produce reactive oxygen species (ROS) via Type I reaction by electron transfer from its first excited singlet state to oxygen molecules. Our findings demonstrate that the tested food colorant has great potential to be used in aPDI of MRSA.

Improving the High-Frequency Response of PEI-Based Earphone with Sodium Copper Chlorophyllin.

The polyetherimide diaphragm, sodium copper chlorophyllin (SCC), and copper ion coating composite used on earphones were observed to improve the high-frequency (10k-14k Hz) performance. This reinforcement phenomenon was expected to make the sound experience brighter and more diverse. By SEM observation, the mixed coating of SCC/Cu2+ on the polyethylenimine (PEI) diaphragm exhibited a planar blocky structure and was tightly bonded to the surface of the PEI polymer without the aid of colloids. The endothermic process of SCC and metal ion complexation was analyzed by isothermal titration calorimetry. The association ratios of SCC/Cu2+ and SCC/Ni2+ were 4/1 and 6/1, respectively, and the SCC/Cu2+ association yielded a stronger binding constant and more free energy. It was expected that the SCC/Cu2+(4/1) mixed liquid would be immobilized on the PEI polymer by multivalent interaction, including hydrogen-bonding networks between carboxyl groups of SCC and amine groups of PEI, and cross-linking of bridging copper ions. We used dimethylethylenediamine (DME) monomer instead of PEI polymer to analyze this multivalent interaction and observed a two-stage exothermic association of SCC/Cu2+(4/1) and DME with a total Gibbs free energy of 15.15 kcal/mol. We observed that the binding energy could be used to explain that the SCC/Cu2+ mixed formulation could be fixed on the surface of the PEI polymer and could enhance the strength of the PEI film. Compared with graphene films, which can continuously improve the performance of high and ultrasonic frequencies, this study was devoted to and was initiated for the purpose of applying porphyrin compounds to improve music performance.

Protective effects and potential underlying mechanisms of sodium copper chlorophyllin against ethanol-induced gastric ulcer in mice.

In study, we aimed to determine the mechanisms underlying the gastroprotective effects of sodium copper chlorophyllin (SCC) against ethanol-induced gastric ulcer injury in mice. First, the gastroprotective effects of SCC against gastric ulcer induced by ethanol were assessed. Then, biochemical, histopathological, immunohistochemistry assays, and western blot analysis were conducted to determine the possible mechanisms of action underlying the effects of SCC. Compared to the effects of omeprazole (OME) in a confirmed mouse model of ethanol-induced gastric ulcer injury, treatment with various doses of SCC resulted in up-regulation of Bcl-2 and down-regulation of the pro-apoptotic protein Bax. Significant decreases in the levels of the malondialdehyde (MDA), myeloperoxidase (MPO), and NO in the gastric tissues were observed. Furthermore, inflammatory cytokine analysis revealed that SCC treatment inhibited the expressions of TNF-α and IL-6, greatly reduced the phosphorylation level of IκB, and repressed the nuclear translocation of NF-κB p65, which demonstrated that SCC inhibited the activation of the NF-κB pathway. The present findings suggest that the protective effects of SCC may be beneficial as a potential preventive and therapeutic agent for gastric ulcer through the NF-κB pathway. Taken together, SCC administration significantly decreased the levels of MPO, NO, and MDA in gastric tissue and exerted a powerful anti-inflammatory activity as demonstrated by reduction in the secretions of proinflammatory mediators such as IL-6 and TNF-α in the serum of mice exposed to ethanol.

Sodium Copper Chlorophyllin Catalyzed Chemoselective Oxidation of Benzylic Alcohols and Diarylmethanes in Water.

We report the highly efficient and chemoselective oxidation of benzylic alcohols catalyzed by sodium copper chlorophyllin in water, producing corresponding arylcarbonyl compounds. Importantly, the catalytic system exhibits a wide substrate scope and high functional group tolerance. Moreover, secondary alcohols and even diarylmethanes were smoothly oxidized to the desired aryl ketones with excellent yields.

Photoinactivation effect of eosin methylene blue and chlorophyllin sodium-copper against Staphylococcus aureus and Escherichia coli.

The use of eosin methylene blue according to Giemsa as photosensitizer is presented for the first time in this paper. The present study evaluated the potential application of chlorophyllin sodium copper salt (CuChlNa) and eosin methylene blue according to Giemsa (EMB) as antimicrobial photosensitizers (aPS) for photodynamic inactivation (PDI) of Staphylococcus aureus (gram-positive) and Escherichia coli (gram-negative) bacteria. The experiments were performed using S. aureus stain ATCC 25923 and E. coli ATCC 25922 in which five aPS concentrations (0.0, 1.0, 2.5, 5.0, 10.0, and 20.0 µM for S. aureus and 0.0, 5.0, 10.0, 20.0, 40.0, and 50.0 µM for E. coli) were prepared and added in 2 mL of a saline solution containing the bacterial inoculum. After aPS incubation, the samples were divided into two groups, one kept in the dark and another submitted to the illumination. Then, the bacterial inactivation was determined 18 h after the incubation at 37 °C by counting the colony-forming units (CFU). The results revealed that both EMB and CuChlNa can be used as aPS for the photoinactivation of S. aureus, while only EMB was able to photoinactivate E. coli. Nevertheless, a more complex experimental setup was needed for photoinactivation of E. coli. The data showed that EMB and CuChlNa presented similar photoinactivation effects on S. aureus, in which bacterial growth was completely inhibited at photosensitizer (PS) concentrations over 5 µM, when samples were previously incubated for 30 min and irradiated by a light dose of 30 J cm-2 as a result of an illumination of 1 h at 8.3 mW cm-2 by using a red light at 625 nm with a 1 cm beam diameter and output power of 6.5 mW. In the case of E. coli, bacterial growth was completely inhibited only when combining a PS incubation period of 120 min with concentrations over 20 µM.

A Saponification Method for Chlorophyll Removal from Microalgae Biomass as Oil Feedstock.

Microalgae oil is an optimal feedstock for nutraceutical, pharmaceutical and biodiesel production, but its high levels of chlorophyll limit its large-scale application. To date, few effective approaches have been developed to remove chlorophyll from microalgae oil. The main purpose of this study was to present a preprocessing method of algae oil feedstock (Scenedesmus) to remove chlorophyll by saponification. The results showed that 96% of chlorophyll in biomass was removed. High quality orange transparent oil could be extracted from the chlorophyll reduced biomass. Specifically, the proportion of neutral lipids and saturation levels of fatty acids increased, and the pigments composition became carotenoids-based. The critical parameters of chlorophyll reduced biodiesel conformed to the standards of the USA, China and EU. Sodium copper chlorophyllin could be prepared from the bleaching effluent. The results presented herein offer a useful pathway to improve the quality of microalgae oil and reduce the cost of microalgae biodiesel.

In vitro Antioxidant Activities of Sodium Zinc andSodium Iron Chlorophyllins from Pine Needles.

Chlorophyll was extracted from pine needles, and then sodium zinc chlorophyllin (SZC) and sodium iron chlorophyllin (SIC) were synthesised by saponification, purification and substitution reaction, using sodium copper chlorophyllin (SCC) as a control. Their crystalline structures were verified by atomic absorbance spectroscopy, UV-VIS spectroscopy and HPLC. Moreover, their antioxidant activities were evaluated and compared with that of ascorbic acid through four biochemical assays: DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity, reducing power, inhibition of ß-carotene bleaching and O 2- scavenging activity. SZC had better antioxidant properties at a lower dosage than SIC and SCC in all assays. In the ß-carotene bleaching assay, EC 50 of SZC, SIC and SCC was 0.04, 0.38 and 0.90 mg/mL, respectively, much lower than that of ascorbic acid (4.0 mg/mL). SZC showed a better result (p<0.05) than ascorbic acid in the O 2- scavenging activity assay. The results obtained from reducing power determination were also excellent: the absorbance values were all about 1.0 at 0.5 mg/mL, about half of that of ascorbic acid. In the investigation of DPPH radical scavenging activity, all chlorophyllins had lower activities than ascorbic acid. These results demonstrated the potential bioactivities of chlorophyll derivatives and supported their possible role in human health protection and disease prevention.

Sodium copper chlorophyllin-loaded chitosan nanoparticle-based photodynamic therapy for B16 melanoma cancer cells.

Melanoma is one of the most aggressive and fatal skin cancers owing to its ability to metastasize and develop resistance to chemotherapy. Photodynamic therapy (PDT) is a minimally noninvasive treatment modality comprising photosensitizers (PSs), light sources, and endogenous molecular oxygen that exert a localized cytotoxic effect on cancer cells. The current study explores the therapeutic potential of sodium copper chlorophyllin-loaded chitosan nanoparticles (CH-SCC NPs) along with handheld laser-based PDT on B16 cancer cells. A modified chlorophyll derivative identified as sodium copper chlorophyllin (SCC) is a dietary supplement that has anticancer properties. Herein, we have synthesized CH-SCC NPs using the ionic gelation method to enhance the PS's bioavailability and efficiency. Chitosan nanoparticles exhibited high biocompatibility in a normal cell line L929, zebrafish, and chick embryos, and were successfully employed to deliver the SCC to cancer cells. CH-SCC NPs showed an enhanced PDT effect that killed approximately 80%-85% of B16 cells. CH-SCC NPs in combination with a handheld portable laser source showed significant therapeutic potential against the B16 skin cancer cell line. The experimental findings further strengthen our device-repurposing strategy, which suggests that SCC nanoformulations along with handheld laser can be a suitable treatment for skin cancer even in remote areas where power source and treatment cost can be a limitation.

Orange carbon dots based smart sensing platforms for rapid, visual, quantitative identification of sodium copper chlorophyllin.

A highly affordable, sensitive and portable detection platform for the quantitative identification of sodium copper chlorophyllin (SCC) in food and environment is a crucial need. Even though many carbon dots (CDs) based sensors have been developed, few reports on using CDs as optical probes for SCC detection have been published so far. In this paper, orange luminescent CDs (OLCDs) were prepared via solvothermal method, which have high fluorescence quantum yield (27.20 %) and excellent photostability. OLCDs can detect SCC via inner filter effect (IFE), with fast response, high selectivity, outstanding sensitivity and superior anti-interference ability. Benefiting from the remarkable properties of OLCDs, a portable sensing platform was triumphantly constructed, which facilitated the in situ, real-time quantitative determination of SCC in diverse actual samples, by catching the fluorescence change of OLCDs-based paper sensors via smartphone RGB colorimetric analysis. This first CDs-based smart sensing system displays great potential for quantification of SCC in various fields.

The synthesis of thermostable, strongly basic Anion-Exchange resins using Cross-Linked biguanide and its application in the extraction of Sodium copper chlorophyllin.

Commercially available, strongly basic anion-exchange resins with quaternary ammonium groups have been widely used in the purification of natural plant extracts. However, under the condition of high temperature (greater than 60 °C), these resins could not be used for long periods because of the Hofmann degradation of the strongly basic groups. In this work, the synthesis of novel, thermally stable, strongly basic resins, which has a cross-link biguanide structure, was reported. The mechanism of thermal degradation was investigated, and the result indicated that not only the stability of the functional group but also the link mode between the functional group and the resin matrix should influence the thermal stability of the resin. In our experiment, the PDG2 resin was selected to separate sodium copper chlorophyllin (SCC), a type of edible pigment derived from plants, due to its optimal thermal stability and adsorption capacity. The adsorption mechanism and thermodynamics of PDG2 were also investigated. The results demonstrated that the main adsorption affinity of PDG2 toward SCC was due to the synergistic effects of the hydrophobic and ionic interactions, and the rise in temperature will benefit the adsorption equilibrium, which differed from the equilibrium for lutein. Therefore, under suitable gradient desorption conditions, a high-purity SCC extract was prepared. After eight cycles, the adsorption capacity of the PDG2 remained constant and reproducible at a high temperature (70 °C).

Fabrication, Characterization and In Planta Uptake of Engineered Surfactant Nanovesicles for the Delivery of the Biostimulant Sodium Copper Chlorophyllin.

Effective delivery of agrochemicals requires control over bioactive release kinetics coupled with effective penetration of the bioactive into plants. Herein, we demonstrate the fabrication of hybrid nanovesicles based on sodium dodecylbenzenesulfonate (SDBS) and cetyltrimethylammonium bromide (CTAB) for enabling effective delivery of the biostimulant sodium copper chlorophyllin (Cu-chl) into plants. SDBS-CTAB nanovesicles exhibited a particle size of 107 nm with a well-defined spherical morphology, while modified formulations that included small fractions of the unsaturated dopant Span 80 yielded larger nanovesicles that were softer and more irregular in shape. All nanovesicles maintained high colloidal stability over >4 weeks and enabled sustained Cu-chl release, with the incorporation of Span 80 into the membranes enabling controllable acceleration of the release rate. Nanovesicle encapsulation improved the photostability of Cu-chl bioactive 3-4 × relative to that of free Cu-chl and enabled significant penetration of Cu-chl into the plant root without inducing any significant phytotoxicity.

Green Plant Pigment, Chlorophyllin, Ameliorates Non-alcoholic Fatty Liver Diseases (NAFLDs) Through Modulating Gut Microbiome in Mice.

Non-alcoholic fatty liver diseases (NAFLDs) along with metabolic syndrome and Type-2 diabetes (T2D) are increasingly prevalent worldwide. Without an effective resolution, simple hepatic steatosis may lead to non-alcoholic steatohepatitis (NASH), characterized by hepatocyte damage, chronic inflammation, necrosis, fatty degeneration, and cirrhosis. The gut microbiome is vital for metabolic homeostasis. Conversely, dysbiosis contributes to metabolic diseases including NAFLD. Specifically, diet composition is critical for the enterotype of gut microbiota. We reasoned that green pigment rich in vegetables may modulate the gut microbiome for metabolic homeostasis. In this study, C57BL/6 mice under a high fat diet (HFD) were treated with sodium copper chlorophyllin (CHL), a water-soluble derivative of chlorophyll, in drinking water. After 28 weeks of HFD feeding, liver steatosis was established accompanied by gut microbiota dysbiosis, intestinal impairment, endotoxemia, systemic inflammation, and insulin resistance. Administration of CHL effectively alleviated systemic and intestinal inflammation and maintained tight junction in the intestinal barrier. CHL rebalanced gut microbiota in the mice under high fat feeding and attenuated hepatic steatosis, insulin resistance, dyslipidemia, and reduced body weight. Fecal flora transplants from the CHL-treated mice ameliorated steatosis as well. Thus, dietary green pigment or the administration of CHL may maintain gut eubiosis and intestinal integrity to attenuate systemic inflammation and relieve NASH.

The Metabolism and Potential Bioactivity of Chlorophyll and Metallo-chlorophyll Derivatives in the Gastrointestinal Tract.

Chlorophyll is the vivid chromophore which imparts the green color to plant leaves, and is consumed by humans through green vegetables. The basic porphyrin structure of chlorophyll binds magnesium in plants, but can bind different divalent metals (e.g., copper, zinc, iron) facilitated by food processing techniques and/or chemical synthesis. This review covers the known elements of chlorophyll and metallo-chlorophyll absorption, distribution, metabolism, excretion in vitro and in vivo. The review discusses what is understood about the ability of these novel metallo-chlorophyll derivatives to deliver essential metals. This review also detail chlorophyll and metallo-chlorophyll toxin binding properties which largely occur during digestion, focusing on toxins including dioxins, heterocyclic aromatic amines, polyaromatic hydrocarbons, and aflatoxin. Finally, the article highlights the gaps in the understanding of the metabolism and metal and toxin-binding bioactivity of this family of molecules.

Porphyrins ameliorate spinocerebellar ataxia type 36 GGCCTG repeat expansion-mediated cytotoxicity.

Spinocerebellar ataxia type 36 (SCA36) is a noncoding repeat expansion disorder caused by an expanded GGCCTG hexanucleotide repeat (HNR) in the first intron of the nucleolar protein 56 (NOP56) gene. Another disease-causing HNR expansion derived from C9orf72-linked GGGGCC repeats that form G-quadruplexes (GQs) affects genetic stability, RNA splicing, and mRNA localization within neurites. The porphyrin derivative TMPyP4 was shown to ameliorate RNA toxicity caused by GGGGCC HNR expansion by binding and distorting RNA GQ structures. SCA36 GGCCTG HNRs can potentially form RNA GQs; therefore, we investigated whether several porphyrin derivatives could reduce RNA toxicity in SCA36 cell models. Among these, sodium copper chlorophyllin and hemin chloride, which have already been used in clinical practice, reduced SCA36 GGCCTG expansion-mediated cytotoxicity and improved cell viability. These data suggest that porphyrins are potential therapeutic candidates against SCA36 pathogenesis.

In Situ Synthesis of a Double-Layer Chitosan Coating on Cotton Fabric to Improve the Color Fastness of Sodium Copper Chlorophyllin.

Natural dye's poor affinity for cotton and poor fastness properties still hinder its applications in the textile industry. In this study, a doubled-layered chitosan coating was cured on cotton fabric to serve as bio-mordant and form a protective layer on it. Under the optimal treatment conditions, the maximum qe (adsorption amount) of the natural dye sodium copper chlorophyllin (SCC) calculated from the Langmuir isothermal model was raised from 4.5 g/kg to 19.8 g/kg. The dye uptake of the treated fabric was improved from 22.7% to 96.4% at 1% o.w.f. dye concentration. By a second chitosan layer cured on the dyed fabric via the cross-linking method, the wash fastness of the cotton fabric dyed with SCC can be improved from 3 to 5 (ISO 105 C-06). The natural source of the biopolymer material, chitosan, and its ability to biodegrade at end of life met with the initial objective of green manufacturing in applying natural dyes and natural materials to the textile industry.

Sasa veitchii extract induces anticancer effects via inhibition of cyclin D1 expression in MCF-7 cells.

Sasa veitchii and other Sasa species are traditional medicinal herbs belonging to a group of Japanese bamboos collectively called Kumazasa, and these species possess the potential for a wide variety of uses. The present study aimed to elucidate the anticancer mechanisms exerted by S. veitchii extract (SE) against a human breast cancer cell line, MCF-7 cells. Freeze-dried Sunchlon® was used as the SE, and cell proliferation activity was measured using the [3H]-thymidine incorporation assay. Induction of apoptosis was assessed via Annexin V and caspase-3 fluorescent staining, the induction of necrosis was measured via propidium iodide staining, and cell cycle-related protein expression was determined using western blotting. The IC50 value of the SE was 7.7 µg/mL in MCF-7 cells. Although the primary active ingredient in Sunchlon® is sodium copper chlorophyllin (0.25%), the present results indicated that ingredients other than SCC exert anti-cancer activities (the IC50 value of SCC was 715 µg/mL), and late apoptosis or necrosis was induced in an SE dose-dependent manner. The expression levels of cyclin D1 and Cdk6 were decreased after SE treatment, and there was no change in the Cdk1/2 expression levels. Additionally, the expression of the necrosis-related cell death indicators RIP1 and RIP3 was increased in response to high-dose SE treatments, and this was indicative of cells preparing for programmed cell death. SE induces cell death in MCF-7 cells via the inhibition of cyclin D1 expression at low concentrations, and this extract induces programmed necrosis (necroptosis) by potentiating RIP1/RIP3 expression.

The effective role of sodium copper chlorophyllin on the dysfunction of bone marrow mesenchymal stem cells in multiple myeloma via regulating TGF-ß1.

BACKGROUND: The osteoblast differentiation of bone marrow-derived stem cells (BMSCs) is impaired in multiple myeloma (MM). We investigated the effects of sodium copper chlorophyllin (SCC) on osteoblast differentiation ability of BMSCs from MM. METHODS: Clinical bone marrow samples were collected. Fluorescence Activated Cell Sorter (FACS) was used to identify surface markers of BMSCs. BMSCs were treated with different concentrations of SCC and cell viability was detected by MTT assay. Relative mRNA and protein expressions of transforming growth factor-ß1 (TGF-ß1), SMAD2/3, osteogenic differentiation indicators (RUNX2 and OCN) were measured by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. Alkaline phosphatase (ALP) was stained for activity detection. Formation of calcium nodus of BMSCs was examined by Alizarin Red S staining. RESULTS: CD90 and CD105 were high-expressed, but CD34 and CD45 were not expressed in BMSCs. BMSCs in MM group showed a lower expression of TGF-ß1 and a lower degree of osteogenic differentiation. SCC enhanced activities of BMSCs, ALP activity, and formation of calcium nodus, activated TGF-ß1, SMAD2/3 pathway and increased RUNX2 and OCN expressions in BMSCs. Silencing TGF-ß1 reversed the effects of SCC on BMSCs in MM. CONCLUSION: SCC could effectively improve the proliferation and osteogenic differentiation of BMSCs in MM through regulating TGF-ß1.

Dual protein ligand-modified gold nanoclusters for selective detection of serum sodium copper chlorophyllin.

Precisely tuning the emission wavelength of dual ligands co-functionalized gold nanoclusters (AuNCs) presents a bright prospect for highly selective drug detection. In this study, gold nanoclusters (AuNCs) with strong red fluorescence at 633 nm were synthesized using bovine serum albumin (BSA) and bromelain (Bro) as dual ligands for highly selective detection of sodium copper chlorophyllin (SCC). Interestingly, the auto-fluorescence interference of the dual ligands was effectively avoided due to the fluorescence emission red-shifts of the prepared BSA/Bro@AuNCs; this may be mainly due to the rigid structure of the proteins. The fluorescence of BSA/Bro@AuNCs was quenched after addition of SCC, which performed well and provided a good linear calibration curve in the 5.0-25.0 µM range (R2 = 0.999), and a detection limit as low as 0.5 µM. The proposed assay was further applied to the measurement of SCC in rat serums after an intravenous injection. The dual protein ligand-capped AuNCs showed great potential for drug analysis in actual biological samples.

Sodium Copper Chlorophyllin Is Highly Effective against Enterovirus (EV) A71 Infection by Blocking Its Entry into the Host Cell.

Human enteroviruses (HEVs) pose an ongoing threat to global public health. Particularly, enterovirus-A71 (EV-A71), the main pathogen causing hand-foot-and-mouth disease (HFMD), has caused ongoing outbreaks globally in recent years associated with severe neurological manifestations and several deaths. Currently, no effective antivirals are available for the prevention or treatment of EV-A71 infection. In this study, we found that sodium copper chlorophyllin (CHL), a health food additive and an over-the-counter anticancer medicine or treatment to reduce the odor of urine or feces, exhibited potent inhibitory activity against infection by divergent EV-A71 and coxsackievirus-A16 (CV-A16) isolates at a low micromolar concentration with excellent safety. The antiviral activity of each was confirmed by colorimetric viral infection and qRT-PCR assays. A series of mechanistic studies showed that CHL did not target the host cell but blocked the entry of EV-A71 and CV-A16 into the host cell at the postattachment stage. In the mouse model, CHL could significantly reduce the viral titer in the lungs and muscles. Since CHL has been used in clinics for many years with excellent safety, it has the potential to be further developed into a prophylactic or therapeutic to prevent or treat HFMD caused by EV-A71 or CV-A16 infection.