4.1 Pharmacological Summary of Pei's Soft Liver and Anti-Flatulence Pill

4.1 Pharmacological Summary of Pei's Soft Liver and Anti-Flatulence Pill

Modern liver cancer falls under the categories of "liver accumulation, mass, and aggregation" in traditional Chinese medicine. The "Suwen·Yipian Cifa Lun" states: "When righteous qi resides within, evil cannot invade"; the "Suwen·Pingre Bing Lun" says: "Where evil gathers, qi must be deficient"; Zhang Yuan's "Huofa Jiyao" records: "Strong people have no accumulation, but weak people do; when the spleen and stomach are weak and both qi and blood decline, any seasonal illness can lead to accumulation." Zhang Zhongjing's "Jingui Yaolue·Zangfu Jingluo Xianhou Bing Mai Zheng Di Yi" states: "When seeing a liver disease, one should know it will spread to the spleen, so the spleen should be strengthened first." Therefore, spleen deficiency is the fundamental cause of the occurrence and development of liver cancer; when the spleen is deficient, its transportation and transformation functions are impaired, food and water are not properly digested, dampness and heat accumulate internally, and over time, qi stagnation and blood stasis occur, with dampness, heat, and toxin accumulating in the liver meridians, leading to a condition of deficiency combined with excess.

Pei's Soft Liver and Anti-Flatulence Pill is composed of Astragalus, Salvia miltiorrhiza, Curcuma wenyujin, seaweed, soapberry spines, Hedyotis diffusa, and Scutellaria barbata, among other herbs. Based on the pathogenic characteristics of liver cancer—deficiency, stasis, and toxicity—the entire formula aims to tonify qi and strengthen the spleen, activate blood circulation and remove stasis, soften hard masses and disperse nodules, and clear heat and detoxify.

4.1.1 Astragalus

Astragalus (Radix Astragali) is the dried root of Mongolian astragalus or membranous astragalus, a leguminous plant. It has a sweet taste and slightly warm nature, affecting the lung and spleen.

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It has the effects of tonifying qi and raising yang, strengthening defensive qi and consolidating the exterior, promoting diuresis and reducing swelling, and supporting wound healing and muscle regeneration, making it one of the representative herbs for tonifying qi and strengthening the spleen. Astragalus contains various polysaccharides, saponins, flavonoids, amino acids, trace elements, and other substances, exhibiting anti-tumor, immune-regulating [70], and anti-radiation effects. Among them, total astragalosides can increase the apoptosis rate of liver cancer cells by 11.97% to 54.76%, possibly by upregulating the expression of the tumor-suppressor gene wt-p53 and arresting the cell cycle at the G phase [71]. The active ingredient F3 in astragalus can enhance the activity of lymphokine-activated killer (LAK) cells and the function of T lymphocytes, thereby improving their killing effect on tumor cells and humoral immune function [72,73].

4.1.2 Salvia miltiorrhiza

Danshenone is a fat-soluble active component extracted from Salvia miltiorrhiza (single compound), with a quinone structure that is easily oxidized and reduced, participating in various biochemical reactions in the body and thus possessing multiple biological activities. Its main active ingredient, danshenone II A, has natural killing, differentiation-inducing, and apoptosis-inducing effects on tumor cells. Its apoptosis rate against SMMC-7721 ranges from 6.59% to 24.17%, possibly due to downregulation of intracellular TGF-β1, COX-2 mRNA, and VEGF expression, along with upregulation of p38MAPK expression [74–76]. On the other hand, danshenone II A can achieve an apoptosis rate of 60% in HepG2 cells, likely by upregulating the expression of P53 and Bax while antagonizing the pro-tumor factor Bcl-2 and downregulating its expression [77,78]. Another active ingredient in danshen, danshenone I, also has a relatively obvious inhibitory effect on HepG2 [79].

4.1.3 Curcuma wenyujin

Curcuma wenyujin (Rhizoma Curcumae) is the rhizome of ginger family plants such as Curcuma zedoaria, Curcuma kwangsiensis, and Curcuma aromatica. It has a warm nature and a pungent, bitter taste, with effects such as activating blood circulation and breaking stasis, regulating qi and eliminating accumulation, and relieving pain. The volatile oil of Curcuma wenyujin contains various anti-cancer active ingredients, including curcumene, germacrene, curcumol, new curcumol, curcumol, isocurcumol, elemene, and others. Modern research shows that the oil of Curcuma wenyujin can inhibit the growth of liver cancer cells: it can significantly suppress the growth of HepG2 cells in vitro by inhibiting the expression of COX-2 and VEGF genes, and induce their apoptosis [80,81]. Its in vivo anti-tumor effect may be related to downregulating Bcl-2 and upregulating P16 and P21 expression [82,83].

4.1.4 Seaweed

Seaweed (Sargassum) has a salty taste and cold nature, affecting the liver and kidney meridians, with effects such as dissolving phlegm and softening hard masses, promoting diuresis and reducing swelling. According to "Bencao Xinbian," seaweed is particularly effective in treating hard diseases because its saltiness can soften hard masses. Siddhant's research indicates that seaweed can promote the proliferation response of mouse T cells, with immune-regulating, anti-mutation, cell differentiation-inducing, anti-tumor, anti-viral, and antioxidant effects. Its immune-regulating effect may be related to seaweed polysaccharides activating macrophages, T and B lymphocytes, reticular endothelial system, complement, and promoting the production of interferon and interleukins [84]. Its anti-tumor effect is associated with terpenoid compounds in seaweed increasing the body's IL-6 and TNF-α levels, thereby enhancing immune function [85]. Yang Wenhao [86] et al. were the first to extract cyclic peptide compounds A1 and A2 with strong anti-cancer activity, proving that their anti-cancer efficacy is significantly stronger than that of adriamycin.

4.1.5 Soapberry Spines

Soapberry spines are the dried thorns of the soapberry tree (Gleditsia sinensis Lam), a leguminous plant. According to "Compendium of Materia Medica," they are "pungent, warm, non-toxic, able to treat carbuncles, mastitis, wind-related skin diseases, retained placenta, and kill parasites." Their main chemical components include flavonoid glycosides, phenols, amino acids, palmitic acid, stearic acid, oleic acid, sitosterol, campesterol, and nonacosane, all of which exhibit varying degrees of anti-tumor effects, with flavonoids being particularly prominent [87]. Modern research suggests that their more precise anti-tumor effect may be related to inhibiting proliferating cell nuclear antigen (PCNA), mutant P53 protein, and increasing the expression of cytokines IL-2, IL-6, IL-12, and TNF-α [88–90].

4.1.6 Hedyotis diffusa

Hedyotis diffusa (Herba Hedyotis Diffusae) is the dried whole herb of the Rubiaceae plant Hedyotis diffusa Willd. It has a sweet, mild taste and cool nature, containing various chemical components, mainly anthraquinones, terpenoids, polysaccharides, trace elements, and sterols along with their glycoside compounds, with effects such as clearing heat and removing dampness, detoxifying and resolving abscesses. Clinically, it is widely used in the treatment of various cancers, and modern pharmacological studies show that Hedyotis diffusa has anti-tumor, antibacterial, anti-inflammatory, and immune-enhancing effects [91]. The extract of Hedyotis diffusa achieves a proliferation inhibition rate of 39.40% against SMMC-7721, targeting 20 genes, likely due to its multi-pathway anti-tumor action [92]. Hedyotis diffusa polysaccharides can also inhibit the growth of human liver cancer cells Bel-7402 and induce apoptosis, possibly by upregulating the tumor-suppressor gene P53, suppressing the proto-oncogene Bcl-xL expression, and directly affecting the energy metabolism of tumor cells [93]. Some studies also indicate that in mice, Hedyotis diffusa can achieve a tumor suppression rate of 48.6% against liver cancer H22, possibly by inducing the expression of heat shock protein 70 (HSP70) in H22 liver cancer cells, thereby enhancing their immunogenicity and exerting an anti-tumor effect [94,95].

4.1.7 Scutellaria barbata

Scutellaria barbata (also known as narrow-leaved Hanxin grass) is the dried whole herb of the Lamiaceae plant Scutellaria barbata. It has a pungent, bitter taste and cold nature, with effects such as clearing heat and detoxifying, activating blood circulation and removing stasis, reducing swelling and relieving pain. Studies show that high-dose extracts of Scutellaria barbata [12 g/(kg·d)] can induce apoptosis in H22 cells, possibly by lowering the mitochondrial membrane potential of tumor cells [96,97]. As for inducing apoptosis in liver cancer SMMC-7721 cells, Wei Pengya et al. [98] found that it can upregulate caspase-3 protein while downregulating Bcl-2 and Survivin protein expression. Additionally, Scutellaria barbata extracts can also inhibit the proliferation of HepG2 and QGY-7701 cells, possibly by activating the Bcl-2 gene family as a tumor-suppressor gene, inducing apoptosis, and blocking the cell cycle [99].

4.2 Preparation of the Model

The H22 liver cancer transplantation model used in this experiment is a classic in vivo model for studying the anti-tumor effects of drugs. The method involves implanting tumor cells into the right anterior axilla of mice to form solid tumors that continuously proliferate. The advantage of this model is that it maintains the integrity of the organism, recreating the natural environment of disease onset and development, thus more objectively reflecting the distribution and metabolic processes of drugs in the body and their anti-tumor activity.

4.3 Anti-Tumor Effects of Pei's Soft Liver and Anti-Flatulence Pill

The results of this experiment show that the average tumor weight in the low-, medium-, and high-dose groups of Pei's Soft Liver and Anti-Flatulence Pill was lower than that of the model control group, at (0.935±0.227) g, (0.776±0.122) g, and (0.926±0.237) g, respectively, all of which were statistically significant compared with the model control group (p<0.05). The corresponding tumor suppression rates were 23.9%, 36.8%, and 24.5%. This demonstrates that Pei's Soft Liver and Anti-Flatulence Pill has good anti-tumor effects.

4.4 Effects of Pei's Soft Liver and Anti-Flatulence Pill on Immune Organs

"Righteous qi resides within, evil cannot invade; where evil gathers, qi must be deficient," meaning that righteous qi is the body's immune function [100]. Modern oncology believes that the occurrence, development, and prognosis of tumors are closely related to the immune status of the host. Burnet's "immune surveillance" theory suggests that under normal circumstances, the body's immune system can recognize and promptly eliminate mutated cells, thereby preventing tumor formation. When the body's immune function declines, or when the function of antigen-presenting cells is weakened or defective, or when there is a certain amount of "enhanced anti-" in the body,

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"body" present, all these factors help tumor cells evade the host's immune system attack, leading to tumor formation.

The thymus is the most important central immune organ in the body, serving as the site for the development, differentiation, and maturation of T cells. T cells are crucial immune cells in the human body, capable of mediating cellular immunity and participating in the execution and regulation of various immune functions, especially playing an important role in tumor immunity. The spleen is the largest immune organ in the human body, where immune cells (T and B cells) reside and mount immune responses, and where antibodies, interferons, and cytokines and other immune-active substances are synthesized. Therefore, the thymic and splenic indices can reflect the development of immune organs, the differentiation status of immune cells, and the strength of immune function. Modern research shows [101,102] that spleen-tonifying and qi-strengthening traditional Chinese medicines can enhance the immune function of liver cancer patients, particularly improving T cell function to strengthen immune surveillance and achieve the goals of tumor suppression and metastasis prevention. Observations from the experiment revealed that in the model group, the thymus and spleen of mice showed reduced volume and weight, with some thymuses having unclear lobes and appearing grayish-white, while the spleens turned pale red. In the low-, medium-, and high-dose groups of Pei's Soft Liver and Anti-Flatulence Pill as well as the blank control group, the thymic index of mice was higher than that of the model group, with statistically significant differences (p<0.05). Similarly, in the low-, medium-, and high-dose groups of Pei's Soft Liver and Anti-Flatulence Pill as well as the blank control group, the splenic index of mice was also higher than that of the model group, with statistically significant differences (p<0.05). The experimental results demonstrate that Pei's Soft Liver and Anti-Flatulence Pill has a clear enhancing effect on both the thymic and splenic indices, capable of improving the body's non-specific immune function, strengthening its resistance to harmful external stimuli, and reducing bodily damage.