Cancer is a disease that seriously threatens human health worldwide, with the global incidence and mortality rates of cancer being 247.5 per 100,000 and 127.8 per 100,000 respectively, in 2020. The cancer incidence and case fatality rates in China are 293.91 per 100,000 and 174.55 per 100,000, both higher than the global average [1]. Cancer poses a serious threat to the lives and safety of mainland residents and is an important obstacle to prolonging life expectancy. Traditional Chinese medicine (TCM) has the advantages of multiple components, multiple targets, multiple pathways, and few toxic and side effects, and has attracted more and more attention in the treatment of cancer [2].
Bitter white hoof is derived from the medicinal laminar foramyces Fomitopsis officinalis (Vill. ex Fr.） Bond. The dried fruiting bodies of et sing., also known as alihong, harikun, quinine, and larch strata pores, are widely distributed in northern China and the Pacific Northwest region of the United States, Canada, and Europe [3-4]. According to the Uyghur Pharmacology, the bitter white hoof has a sweet and bitter taste, a warm nature, and has the effects of dispelling wind and dampness, reducing breath and asthma, diuresis and swelling, and detoxifying snake venom [5]. The name of bitter white hoof in Mongolia is Agri, which is used in folk medicine by decoction with water, and has the effects of calming the nerves, nourishing blood and anti-tumor; In Japan, it is used to treat abdominal pain, fever, tuberculosis, night sweats, and chronic bronchitis [6]. In Central European folklore, the bitter white hoof is widely used to treat a variety of ailments, including dysmenorrhea, cough, hemorrhoids, fever, bladder disease, rheumatism, and cancer [7].
The main components of the bitter white hoof are triterpenoids, steroids, sesquiterpenoids, and polysaccharides, and the alired acid A, alired acid C, 3-keto-dehydrothiochromic polypolicic acid, polysaccharide FOP80-1, and petroleum ether extract of bitter white hoof have obvious inhibitory effects on various tumor cells such as liver cancer, lung cancer, breast cancer, and gastric cancer [4,8], so it is necessary to comprehensively summarize the antitumor effects of the effective sites and chemical components in the bitter white hoof[4,8]. Based on relevant research reports in recent years, this paper reviews the chemical composition of Kubaiho, its extraction sites and the anti-tumor effect and mechanism of the active ingredients, so as to provide ideas and basis for the in-depth research and further development of the anti-tumor of Kubaiho.
1 Chemical composition of bitter white hoof
Scholars from all over the world have extracted and isolated triterpenic acids and steroids, as well as sesquiterpenoids and polysaccharides.
1.1 Triterpene acids
苦白蹄的化学成分以三萜酸类为主，吴霞等［9-11］对苦白蹄子实体采用常压柱色谱、高效液相色谱（HPLC）等方法进行分离，鉴定得到阿里红酸A～G、fomlactone A～C、阿里红醇类、变孔绚孔菌酸类、硫色多孔菌酸类、齿孔酸类等多种化学成分。 冯薇［12］从苦白蹄中分离得到了1个新的三萜类化合物，命名为阿里红素。 郭雄飞［13］也从苦白蹄中分离得到去氢硫色多孔菌酸。 韩建欣等［14］从苦白蹄中提取得到2个新的成分3α，12β-dihydroxy-24-methyl-7，23-dioxo-lanosta-8-en-26-oic acid、12β，15α- dihydroxy-24-methyl-3，23-dioxo-lanosta-7，9（11）- dien-26-oic acid。 Epstein等［15］提取得到了3，6-dihydroxy-4，4，14-trimethylpregn-8-en-20-one、3-hydroxy-4，4，-14-trimethylpregn-8-en-20-one。
1.2 Steroidal and sesquiterpenoids
Feng Wei [12] isolated aerythroic acid from bitter white hooves. Some scholars have extracted sesquiterpenoid albicanic acid and larch acid from bitter white hoof [15]. Jia Ligeng[16] obtained ergoster-5,22en-3β alcohol, ergoster-7,22en-3β alcohol, 4,6,8(14),22(23)-tetraen-3 keto-ergosterane from dried bitter white hoof bodies by reflux extraction.
1.3 Polysaccharides
For example, Zha Habudullah [17] extracted the polysaccharides from the bitter white hoof by boiling and ethanol precipitation, and the two main components, FOPS-Ia and FOPS-IIa, were separated and refined from the polysaccharides, with yields of 54.3% and 21.7%, respectively. Wang et al. [18] used water extraction and alcohol precipitation method to extract polysaccharides from P. sophora, and the average yield of polysaccharides reached 3.37%. Ilinur Aniwal et al. [19] showed that the average content of polysaccharides was 6.40%, and the monosaccharide composition of polysaccharides was glucose, xylose, arabinose, rhamnose, mannose and galactose by gas chromatography-mass spectrometry (GC-MS) and infrared spectroscopy (IR).
1.4 Other Ingredients
冯薇［12］从苦白蹄中分离得到了1个新的氨基酸类化合物，命名为阿里红氨酸［2-（1-carboxyhexadecylamino）-2-aminosuccinc acid］。
The structure of the anti-tumor active ingredient of the bitter white hoof is shown in Figure 1.
2 Antitumor pharmacological effects of bitter white hoof
The various chemical components of Kubai hoof have good efficacy on liver cancer, breast cancer, lung cancer, gastric cancer and other tumor cell lines, and have pharmacological effects such as inhibiting tumor cell proliferation, inducing apoptosis, inhibiting tumor cell migration and invasion, enhancing immunity and inhibiting tumor angiogenesis. It has the characteristics of multi-component and multi-target in the treatment of tumors.
2.1 Anti-liver cancer
Liver cancer ranks fourth and second in the ranking of malignant tumor incidence and tumor mortality in China, respectively, and is a highly malignant tumor, accounting for about 45%~50% of the world's new cases of liver cancer in China [20-21]. A variety of chemical components of bitter white hoof have the effect of inhibiting the proliferation and migration of hepatocellular carcinoma cells and inducing their apoptosis.
Zhang et al. [22] found that FOP80-1, a polysaccharide extracted by hot water extraction and alcohol precipitation, could inhibit the proliferation, invasion and metastasis of human hepatoma HepG2 cells, and promote apoptosis of tumor cells. After 200, 400 and 800 μg·mL-1 FOP80-1 were treated on human hepatoma HepG2 cells for 48 h, the apoptosis rates were 22.93%, 27.13% and 37.19%, which were higher than those of the positive control drug 5-fluorouracil (5-Fu) (19.54%). The mechanism of its action is as follows: FOP80-1 arrests the cell division cycle, makes the cell division stay in the DNA synthesis phase, and increases the production of reactive oxygen species (ROS). HepG2 hepatocellular carcinoma cells were used to study the anti-tumor effect of FOP80-1, which mainly exerts anti-tumor effects in two aspects: on the one hand, it blocks the cell division cycle and increases the production of ROS. On the other hand, FOP80-1 can also inhibit tumor angiogenesis by binding to vascular endothelial growth factor (VEGF) protein, cutting off the energy source for tumor growth and development.
Zhang Huifeng [8] extracted triterpene acids from bitter white hoof by ultrasonication, and identified them as dehydrothioperic acid, 3-keto-dehydrothiochromic acid, fomitopsins C, alired acid F, alired acid G, dehydrocantic acid, and dehydrocantic uronic acid. The inhibitory effect of triterpenic acid on the proliferation of tumor cells was investigated by tetramethylazozole salt colorimetric (MTT) method, and the half inhibitory concentration (IC50) was 42.98 μg·mL-1, indicating that the triterpenic acid had an inhibitory effect on the proliferation of human hepatoma HepG2 cells.
Chi et al. [23] conducted in vivo experiments using H22 tumor-bearing mice, and found that the tumor inhibition rate of 1 000 mg·kg-1 ethyl acetate extract was 64.65%, which was higher than that of the positive control drug 5-Fu (59.98%). The monomer components 3-keto-dehydrothiochrome polyponicic acid, alired acid C, dehydrodental ketoic acid, dehydrocantic acid and alired acid A in the extraction site of ethyl acetate also have a good inhibitory effect on H22 hepatocellular carcinoma cells, and the tumor inhibition rate of 15 mg·kg-1 of alirubic acid C can reach 75.13%, and the anti-tumor effect is by increasing the level of interleukin-2 (IL-2) in tumor-bearing mice. It is achieved by inducing the activation of immunoactive cells natural killer (NK) cells, cytotoxic T lymphoid (CTL) cells, and lymphokine-activated killer (LAK) cells, increasing the conversion rate of B cells and T cells, and enhancing immunity.
Jia Ligeng et al. [16] showed that the anti-tumor active substances of bitter white hoof were mainly triterpenoids, among which alirubic acid C had the best inhibitory effect on the tumor of H22 tumor-bearing mice, with an inhibition rate of 75.13%. Feng Shuang [24] found that the tumor inhibition rate of 3-keto-dehydrothiochromic acid on H22 tumor-bearing mice could reach 65.31%, and by observing the tumor tissue sections stained with HE, it was found that 3-keto-dehydrothiochromic acid inhibited the deformation, necrosis and growth of tumor cells. The mechanism of action is that it inhibits the expression of VEGF in tumor tissues, hinders the generation of blood vessels, and can reduce the interleukin-4 (IL-4) in the serum of tumor-bearing mice, γ increase the level of interferon (IFN-γ), and enhance the immune function of mice.
Zhu Xinting et al. [25] used sulfonylrhodamine staining method to study the anti-tumor activity of the ethanol extract of Bitter white hoof in vitro, and applied different concentrations of Bitter white hoof ethanol extract to human liver cancer BEL-7404 cells, and found that 80 μg·mL-1 of Bitter white hoof ethanol extract caused most of the BEL-7404 cells to die, and further studies showed that the ethanol extract of Bitter white hoof affected some regulatory points of the cell cycle, the proportion of cells in S phase increased, and the cell cycle was stagnant in S phase, thereby inhibiting the proliferation of tumor cells.
2.2 Anti-breast cancer
Breast cancer has overtaken lung cancer as the most important cancer diagnosed globally, with an annual increase of 2.3 million cases of breast cancer worldwide in 2020, with approximately 685,000 patients dying from breast cancer, and in China, the incidence and mortality of breast cancer are increasing rapidly, with more than 300,000 women diagnosed with breast cancer each year [26-27].
Zhou Yu [28] inoculated murine breast cancer 4T1 tumor cells into ICR mice, and found that the polysaccharide (FoP) had a significant inhibitory effect on the growth of murine breast cancer 4T1 tumor cells in vivo, and FoP could significantly promote the proliferation of mouse spleen lymphocytes, and synergistically promote the proliferation of T and B lymphocytes with concanavalin A (ConA) and bacterial lipopolysaccharide (LPS), and also induce spleen lymphocytes to secrete IL-2 and tumor necrosis factor-α (TNF-α), which has the effect of enhancing immunity.
Chi Mengyi [29] isolated eight chemical components from the bitter white hoof by silica gel column chromatography and recrystallization, and found that the compounds Ali Red Acid A and Ali Red Acid C had anti-human breast cancer MCF-7 cell activity, with IC50s of 52.5 and 80.1 μg·mL-1, respectively. Shi et al. [30] conducted in vitro anti-tumor experiments and found that the main triterpenoids of bitter white hoof, dehydrodental pyroneate and 3-keto-dehydrothiochromic polyponicic acid, can cause deformation, necrosis, and growth inhibition of human breast cancer MCF-7 cells, and its anti-tumor effect is to affect the formation of blood vessels by interfering with the expression of VEGF, and improve the body's immunity through immune regulation, so as to achieve anti-tumor effect.
2.3 Anti-lung cancer
Lung cancer is a common malignancy, with the highest mortality rate among malignant tumors [31]. Zhang Huifeng [8] found that triterpenic acid can induce apoptosis in human non-small cell lung cancer A549 cells, and its mechanism of action is that triterpenic acid can increase the level of ROS, increase Bax protein and decrease the expression of Bcl-2 protein, thereby inducing the opening of the mitochondrial permeability altered channel (MPTP), which reduces the mitochondrial transmembrane potential, and the decrease of mitochondrial transmembrane potential leads to relative hypertonic mitonication in the mitochondrial membrane, the expansion of the internal matrix, the rupture of the outer membrane, and the release of cytochrome C. Activates the caspase cascade, which ultimately leads to the activation of caspase-3 and induces apoptosis.
Wang et al. [32] used the Alma blue method to detect the activity of PEFO extract in vitro against tumors, and found that the inhibition rate of 50 μg·mL-1PEFO on human lung cancer NCI-H460 cells was 99.03%.
2.4 Anti-other tumors
Wang et al. [32] showed that PEFO had an inhibitory effect on human gastric cancer SGC-7901 cells, and in vitro experiments showed that the inhibition rate of 50 μg·mL-1 PEFO on human gastric cancer cells SGC-7901 reached 82.57%, which was similar to that of the positive control drug cisplatin (DDP). Li Xiaofei et al. [33] obtained PEFO by reflux extraction method, and screened for anti-tumor activity in vitro by SRB method, and found that PEFO had a significant inhibitory effect on human gastric cancer SGC-7901 cells and human laryngeal cancer Hep-2 epithelial cells, and the inhibitory effect was positively correlated with concentration, with IC50 of 74.13 and 83.18 μg·mL-1, respectively. In the G1 stage, the number of cells increased, the cell morphology and ultrastructure changed greatly, and the cells showed nuclear shrinkage, poor adhesion, and cell lysis. The above studies showed that PEFO inhibited the proliferation of tumor cells by interfering with the cell cycle and inhibiting the aerobic metabolism of tumor cells.
Munisa Dilishati et al. [34] found that FOPS and FOPS-a can activate Toll-like receptor 4 (TLR4)/nuclear factor-κB (NF-κB) signaling pathway, promote the secretion of TNF-α, interleukin-1β (IL-1β), interleukin-6 (IL-6) and other cytokines by immune cells to enhance immunity and effectively achieve anti-tumor effects.
Zhang Huifeng [8] studied the anti-tumor effect of triterpene acid at a dose of 15 mg·kg-1 and 46.14% at a dose of 60 mg·kg-1, with a good tumor suppression effect and no toxic side effects. Hu et al. [36] found that the polysaccharide could inhibit the tumor growth of S180 tumor-bearing mice without affecting the survival rate of mice, and the combination of polysaccharide with 5-Fu could enhance the anti-tumor effect and alleviate the adverse reactions of 5-Fu. Shayibai Sabiti et al. [36] prepared a tumor-bearing mouse model by subcutaneous inoculation of S180 mice with ascites in the axillary area, and found that the spleen index, thymus index, number of white blood cells, and number of lymphocytes were significantly increased, and the contents of TNF-α, IFN-γ, and IL-2 in serum were significantly increased, and the immunity of mice was enhanced, and tumor growth was inhibited. Guo Shuying et al. [37] used hot water reflux to extract Ali red polysaccharides, which could significantly increase the mass of thymus and spleen in mice, enhance the immune function of S180 tumor-bearing mice, and inhibit the growth of S180 tumors.
3 Conclusion
In recent years, the incidence of cancer has been increasing, and there are a large number of anti-tumor monodrugs in traditional medicine, which have the characteristics of multi-component and multi-target, providing a large number of lead compounds for the development of new anti-tumor drugs [38]. Through the review of the chemical composition and anti-tumor effects of Bitter White Hoof, it was shown that Bitter White Hooves had good and extensive anti-tumor effects, and a variety of components had obvious inhibitory effects on malignant tumors such as liver cancer, breast cancer, lung cancer, gastric cancer, etc., and its mechanism of action included inhibiting tumor cell proliferation, inducing apoptosis, inhibiting tumor cell migration and invasion, enhancing immunity and inhibiting tumor angiogenesis.
The active ingredients that exert pharmacological effects are mainly alired acid A, alirubic acid C, 3-keto-dehydrothionic acid, dehydrodontic pyruvic acid and polysaccharides, and their mechanism of action mainly includes: (1) increasing the level of ROS, thereby inducing the opening of MPTP pores, reducing the mitochondrial transmembrane potential, and the decrease of mitochondrial transmembrane potential leads to relative hypertonic in the mitochondrial membrane, the expansion of the internal matrix, the rupture of the outer membrane, the release of cytochrome C, and the activation of the Caspase cascade. This ultimately leads to the activation of caspase-3 and induces apoptosis. (2) Arrest the cell cycle, make the cell division stay in the S phase or G0/G1 phase, and inhibit the proliferation of tumor cells. (3) Activate the TLR4/NF-κB signaling pathway, increase the levels of TNF-α, IFN-γ and IL-2 in tumor mice, induce the activation of immunoactive cells NK, CTL, LAK, increase the conversion rate of B cells and T cells, and enhance immunity. (4) Hinder the expression of VEGF, inhibit angiogenesis, and cut off the energy source for tumor growth and development, thereby inhibiting the proliferation of tumor cells.
At present, there are still deficiencies in the research on the anti-tumor of Kubaihu, and future research can focus on the following five aspects:
(1) In recent years, the research on the anti-tumor effect of the bitter white hoof has been mainly focused on the treatment of asthma and Alzheimer's disease, and there are few studies on the anti-tumor effect of the bitter white hoof, and the application prospect is broad, so the research in this aspect should be increased.
(2) Most pharmacological experiments are mainly cell experiments in vitro, and there are few in vivo animal experiments, and there is a lack of research on the safety and efficacy of clinical drugs, and its mechanism of action and clinical efficacy have not been fully clarified.
(3) Most of the extraction methods for the active ingredients in Kubai hoof are water extraction, alcohol reflux extraction, ultrasonic extraction and organic solvent extraction, etc., and it is necessary to further optimize the extraction conditions on the basis of the existing extraction methods, and further explore more efficient extraction and separation methods, so as to more accurately detect the active ingredients and structure-activity relationship in Kubai hoof, so as to provide a basis for follow-up research.
(4) The polysaccharides in the bitter white hoof have good anti-tumor effects, but different extraction methods will have different effects on the structural characteristics, and the relationship between the anti-cancer effect and the structure of the polysaccharides of the bitter white hoof lacks structure-activity relationship research, and the current pharmacological research only stays in some pharmacodynamic indicators, so it is necessary to continue to explore the relationship between different structures and anti-tumor effects.
(5) Traditional Chinese medicine has the characteristics of "multi-component and multi-target", and new methods and technologies such as bioinformatics analysis and omics should be used to predict the possible targets of Kubaiho anti-tumor through big data analysis, so as to better play the role of Kubaiho in tumor treatment and lay the foundation for the research and development of new drugs.
In short, there is still a lot of room for exploration in anti-tumor, which can be combined with clinical practice in follow-up studies to further explore the mechanism of action, targets, dose range, pharmacokinetic indicators, etc., of the active ingredient of Bitter White Hoof, so as to provide a theoretical basis for the research and development of new anti-tumor drugs.
Source: Wang Xin, Guan Xue, Chen Dazhong. Research Progress on Chemical Composition and Antitumor Effect of Bitter White Hoof [J]. Drug Evaluation Research, 2024, 47(4): 897-903.