br According to in vitro ATP TCA experiment
According to in vitro ATP-TCA experiment, we screened two HELT constituents of bufadienolides namely F18 and F19 for further in vivo test. In recent years, lymphatic tract metastasis model has become a quite functional experiment tool in basic studies of cancer. There are profuse lymphatic tracts under pulvilli of nude mice and they can form monodirectional reflux including popliteal fossa, inguen, iliac blood vessel and renal hilus, eventually throughout the body. Wang L et al. has successfully established lymphatic tract metastasis model of breast cancer in nude mice by implanting MDA-MB-231 GW311616 into mammary fat pads . Long J et al. established a pancreatic cancer cell and
mouse model with high lymphatic metastasis potential using BxPC-3-LN subline, derived from BxPC-3 human pancreatic cancer cell line. Re-searchers injected these BxPC-3 cells via footpads through serial pas-sages in nude mice and found notable lymphatic metastasis was de-veloped with 100% rate of success .
In the second part of our research, we applied F18 to prepare HELT anti-cancer ingredients emulsion and injected them into surroundings of pulvilli to assess its influence on both cancer cells and immune cells of BALB/c nude mice bearing HCT116 human colon adenocarcinoma cells. Results showed significantly higher inhibition eﬀects on growth of xenograft tumors and LNM of group HD-E and MD-E compared to group LD-E and CI. Our research confirmed that HELT fraction F18 had an-titumor eﬀect and suggested that such eﬀect was dose-dependent, which was in line with the findings of previous study . Another point that should be mentioned was that Fig. 5 showed CI didn't sig-nificantly inhibit lymph node metastasis owing to several reasons: firstly, although Cinobufacini Injection had already been commercia-lized and applied clinically, the purity of active ingredient was still unclear; secondly, the diﬀerence between CI, NS, CE and LD-E might be small, for which the P value wasn't significant, yet we could notice a trend that inhibitory eﬀect on LNM of CI and LD-E was similar and also higher than that of NS and CE; thirdly, as specification of CI showed, the pharmacological action of CI concerned three aspects including inhibiting tumor, promoting immunologic function and exerting anti-viral eﬀect, and water-solubility of CI might be the reason that drug density in lymph nodes was low for which anticancer eﬀect wasn't noticeable.
Currently, drugs commonly used in clinical chemotherapy such as oxaliplatin, 5-fluorouracil have the ability of killing tumor cells eﬀec-tively as well as inducing side eﬀects and depressing the activity of immune cells . It's crucial that ideal chemotherapeutics are found, maximizing the antineoplastic eﬀect and minimizing the im-munosuppressive influence. In our study, the SI value, the ratio of lymphocytes, the proportion of NK cells among peripheral blood and spleen, as well as the secretion of IFN-γ in NK cells and IgG1 in B lymphocytes of HD-E and MD-E were significantly higher compared with other groups, which indicated that HELT fraction of bufadieno-lides could stimulate immunologic function of nude mice, and thus might exert important influences on antitumor immunotherapy.
As for side eﬀects, group MD-E, LD-E, CI and CE had no significant diﬀerence compared to NS group and only group HD-E had apparent influence on liver function. The results implied that liver injury caused by HD-E was mainly related to doses instead of emulsion type. In ad-dition, emulsion could be applied as potential delivery system of che-motherapeutics in anticancer treatment with reduced toxicity .
Moreover, HD-E and MD-E significantly increased red blood cells, he-moglobin, white blood cells and platelet, which indicated that hemo-poiesis function of marrow was distinctly promoted. The HELT con-stituent F18 is better than cinobufagin injection considering both influences on cancer cells and immunological function as well as other side eﬀects.
Out of the purpose for drug development and synthesis, Dalian Institute of Chemical Physics conducted experiments screening active fractions of bufadienolides from toad skin  and kindly provided us those 22 fractions obtained through their study. Our researches mainly focus on pharmacodynamics experiments such as anticancer eﬀects and side eﬀects, as well as further clinical application. Finally, we screened two HELT constituents of bufadienolides namely F18 and F19. Specific composition of F18 or F19 needs further researches. By means of liquid chromatography-mass spectrometry (LC-MS) analysis, Dalian Institute of Chemical Physics has successfully identified several compounds in F13 . In our pharmacodynamics study, F13 exerted significant anti-tumor functions as well as immunosuppressive eﬀects. So far, among known compounds in F13, ψ-bufarenogin could notably restrain cancer through inhibiting EGFR and c-Met without obvious side eﬀects . In addition, ψ-bufarenogin could inhibit liver cancer stem cells (CSCs) through downregulating Sox2 expression . Arenobufagin was cap-able of inhibiting cancer metastasis through regulating epithelial-me-senchymal transition (EMT) with decreasing expression of ZEB1, vi-mentin, and slug and increasing E-cadherin by targeting β-catenin . Also, the author found that inhibition eﬀect on EMT of Arenobufagin could be counteracted by overexpression of β-catenin while knocking down β-catenin strengthened this eﬀect . Telocinobufagin could significantly stimulate splenocyte proliferation, promote NK cells and macrophage activation, increase ratio of CD4+ and CD8+ cells among splenocytes, and what's more, Th1 cytokines such as IL1, IL12, IFN-γ and TNF-α were up-regulated while Th2 cytokines such as IL4 were down-regulated, which suggested that telocinobufagin could positively regulate immunologic function and might be clinically applied to treat cancer and immune-related diseases . Cinobufotalin had strong antitumor eﬀect and whether it's concerned with apoptosis remained inconsistent between diﬀerent researches [42,43]. Heba Emam et al. revealed that Cinobufotalin could induce caspase-dependent apoptosis in U937, a human lymphoma cell, in which Fas played an important role, and moreover, using either Fas/FasL antagonist or pan-caspase inhibiter further verified their findings . Besides, among other known constituents of bufadienolides, bufalin exerted prominent lethal eﬀects on tumor cells and showed no obvious toxic side eﬀects in vivo even though its concentration was as high as 0.4 mg/kg . After treatment of bufalin, the researcher observed DNA condensation, de-creased mitochondrial membrane potential and anti-apoptotic B-cell lymphoma 2 protein level, as well as increased reactive oxygen species, Fas/FasL, apoptosis protease activating factor 1, caspase 3/9, growth arrest- and DNA damage- inducible 153 gene expression. Further ex-periment in vivo verified anticancer eﬀect of bufalin . Cinobufagin could not only produce strong anti-tumor eﬀects through inducing apoptosis and G1 phase arrest, but activate immunologic system like telocinobufagin [45,46]. Resibufogenin was able to inhibit cancer proliferation and metastasis through RIP3-mediated necroptosis, yet with some side cytotoxicity [47,48]. To summarize, we speculate that F18 and F19 are more likely to contain compounds with anticancer eﬀects such as ψ-bufarenogin, Cinobufotalin and bufalin, as well as compounds with eﬀects of motivating splenocyte proliferation and positively regulating immunologic functions such as telocinobufagin and cinobufagin. As for compounds with strong cytotoxicity such as resibufogenin, HELT fractions like F18 might have quite few or none contents like that. Nevertheless, experiments of HELT constituent F19 in vitro haven't been conducted yet and research about identification of exact compounds contained in F18 and F19 needs to be done further. What's more, specific mechanisms underlying HELT-induced anti-tumor and pro-immune eﬀects remain undetermined and have yet to be