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Extracts from sojae semen germinatum ameliorated carbon tetrachloride-induced liver injury in mice

Abstract

Sojae semen germinatum (SSG) prepared from the germinating black soybean Glycine max L. Merr. is a traditional Chinese herbal medicine with heat-clearing, dampness-removing, and detoxifying properties. In the present study, we explored the effects and possible underlying mechanisms of petroleum ether extract (PEE), ethyl acetate extract (EAE), n-butanol extract (NBE) and aqueous fraction (AF) of SSG on the carbon tetrachloride (CCl4)-induced liver injury in mice, an animal model representing pathological features of “dampness-heat” syndrome from the perspective of Traditional Chinese Medicine. Results showed that the EAE and NBE of SSG significantly protected mice against the CCl4-induced increases of liver tissue index and serum transaminase levels, ameliorated the CCl4-induced liver morphological changes. Compared with CCl4 model controls, EAE and NBE of SSG decreased malondiadehyde (MDA) levels, enhanced glutathione (GSH) content and activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in liver tissues, as well as reduced the number of apoptotic hepatocytes. Moreover, the CCl4-induced increase of pro-inflammatory cytokine levels in livers was markedly suppressed by pre-treatment with EAE of SSG. Data suggested that SSG extracts showed protective effect on the CCl4-induced liver injury via multiple pharmacological mechanisms including anti-oxidation, anti-inflammation and anti-apoptosis. EAE and NBE appeared to be superior to other fractions.

Keywords:
Sojae semen germinatum; extracts; liver injury; carbon tetrachloride

1 Introduction

As a traditional Chinese herbal medicine prepared from the germinating seeds of black soybean Glycine max L. Merr. (Leguminosae), sojae semen germinatum (SSG) has been used in Traditional Chinese Medicine (TCM) clinical practices for thousands of years based on its heat-clearing, dampness-removing, and detoxifying properties (Wang et al., 2020Wang, J., Guo, J., Li, S., Zhang, M., & He, B. (2020). Protective effect of ethyl acetate fraction from Semen sojae germinatum, the processed sprout of Chinese black soybean, on rat experimental osteoarthritis. BMC Complementary Medicine and Therapies, 20(1), 117. http://dx.doi.org/10.1186/s12906-020-02920-9. PMid:32306945.
http://dx.doi.org/10.1186/s12906-020-029...
; He & Wang, 2013He, B., & Wang, J. (2013). Effect of semen sojae germinatum on experimental osteoarthritis in the rabbit knee. Bangladesh Journal of Pharmacology, 8(4), 365-370. http://dx.doi.org/10.3329/bjp.v8i4.16446.
http://dx.doi.org/10.3329/bjp.v8i4.16446...
; Fan et al., 2016Fan, W., Qian, Y., Wang, J., Yang, X., Gui, T., & He, B. (2016). Chondroprotective and anti-inflammatory activities of extracts from Semen Sojae Germinatum on IL-1β-stimulated human osteoarthritis chondrocytes. Indian Journal of Pharmaceutical Education and Research, 50(3), 397-402. http://dx.doi.org/10.5530/ijper.50.3.12.
http://dx.doi.org/10.5530/ijper.50.3.12...
). Up to date, SSG has been officially included in the National Pharmacopoeia of People's Republic of China. According to the processing method described in the Chinese Pharmacopoeia, after the mature seeds of black soybeans (Glycine max L. Merr.) are germinated to the sprouts of 1 cm bud length, SSG is prepared through processing the dried bean sprouts with Lophatherum gracile and Juncus effusus (He & Wang, 2013He, B., & Wang, J. (2013). Effect of semen sojae germinatum on experimental osteoarthritis in the rabbit knee. Bangladesh Journal of Pharmacology, 8(4), 365-370. http://dx.doi.org/10.3329/bjp.v8i4.16446.
http://dx.doi.org/10.3329/bjp.v8i4.16446...
). As a soy-derived medicinal material without toxicity, SSG has a neutral nature and a sweet flavor in TCM theory, and has a long history of both medicinal and food uses in Chinese perception and experience (Wang et al., 2020Wang, J., Guo, J., Li, S., Zhang, M., & He, B. (2020). Protective effect of ethyl acetate fraction from Semen sojae germinatum, the processed sprout of Chinese black soybean, on rat experimental osteoarthritis. BMC Complementary Medicine and Therapies, 20(1), 117. http://dx.doi.org/10.1186/s12906-020-02920-9. PMid:32306945.
http://dx.doi.org/10.1186/s12906-020-029...
; He & Wang, 2013He, B., & Wang, J. (2013). Effect of semen sojae germinatum on experimental osteoarthritis in the rabbit knee. Bangladesh Journal of Pharmacology, 8(4), 365-370. http://dx.doi.org/10.3329/bjp.v8i4.16446.
http://dx.doi.org/10.3329/bjp.v8i4.16446...
; Fan et al., 2016Fan, W., Qian, Y., Wang, J., Yang, X., Gui, T., & He, B. (2016). Chondroprotective and anti-inflammatory activities of extracts from Semen Sojae Germinatum on IL-1β-stimulated human osteoarthritis chondrocytes. Indian Journal of Pharmaceutical Education and Research, 50(3), 397-402. http://dx.doi.org/10.5530/ijper.50.3.12.
http://dx.doi.org/10.5530/ijper.50.3.12...
). However, the modern studies on pharmacological actions of SSG are still rare.

From the perspective of TCM, various hepatic disorders appear the typical symptoms and key pathophysiological characteristics of “dampness-heat” syndrome, one of the most common TCM syndromes (Dai et al., 2013Dai, J., Sun, S., Cao, J., Zhao, Y., Cao, H., Zheng, N., Fang, J., Wang, Y., Zhang, W., Zhang, Y., Hu, Y., & Cao, Z. (2013). Similar connotation in chronic hepatitis B and nonalcoholic Fatty liver patients with dampness-heat syndrome. Evidence-Based Complementary and Alternative Medicine, 2013, 793820. http://dx.doi.org/10.1155/2013/793820. PMid:23690853.
http://dx.doi.org/10.1155/2013/793820...
; Cao et al., 2009Cao, H. X., Sun, H., Jiang, X. G., Lu, H. T., Zhang, G. M., Wang, X. J., Sun, W. J., Wu, Z. M., Wang, P., Liu, L., & Zhou, J. (2009). Comparative study on the protective effects of Yinchenhao Decoction against liver injury induced by alpha-naphthylisothiocyanate and carbon tetrachloride. Chinese Journal of Integrative Medicine, 15(3), 204-209. http://dx.doi.org/10.1007/s11655-009-0204-y. PMid:19568713.
http://dx.doi.org/10.1007/s11655-009-020...
). Among the current animal models of liver injury, the carbon tetrachloride (CCl4)-induced hepatic damage has been demonstrated to properly represent the pathological features of “dampness-heat” syndrome in TCM, thus this model has been usually used in the experimental evaluation of potential therapeutic potentials of Chinese herbal medicines with heat-clearing, dampness-removing, and detoxifying properties (Xie et al., 2021Xie, S. Z., Zhai, X. Y., Xi, S. Y., Qiu, Y. K., Zhang, Y. M., Kong, X. J., Li, Y. H., Zhu, L., Wang, Z., Zhang, S. G., Huang, S. Q., Lu, D. W., & Wang, Z. (2021). The protective effects of Zornia diphylla (L.) Pers. against acute liver injury induced by carbon tetrachloride in mice. Frontiers in Pharmacology, 12, 764282. http://dx.doi.org/10.3389/fphar.2021.764282. PMid:34899319.
http://dx.doi.org/10.3389/fphar.2021.764...
; Cao et al., 2009Cao, H. X., Sun, H., Jiang, X. G., Lu, H. T., Zhang, G. M., Wang, X. J., Sun, W. J., Wu, Z. M., Wang, P., Liu, L., & Zhou, J. (2009). Comparative study on the protective effects of Yinchenhao Decoction against liver injury induced by alpha-naphthylisothiocyanate and carbon tetrachloride. Chinese Journal of Integrative Medicine, 15(3), 204-209. http://dx.doi.org/10.1007/s11655-009-0204-y. PMid:19568713.
http://dx.doi.org/10.1007/s11655-009-020...
; Xu et al., 2021Xu, S., Kong, F., Sun, Z., Xi, Y., Qi, F., & Sun, J. (2021). Hepatoprotective effect and metabonomics studies of radix gentianae in rats with acute liver injury. Pharmaceutical Biology, 59(1), 1172-1180. http://dx.doi.org/10.1080/13880209.2021.1969414. PMid:34465274.
http://dx.doi.org/10.1080/13880209.2021....
). In the modern experimental research, the CCl4-induced liver injury model has been well-accepted to share similar pathomechanisms, such as inflammatory damage, oxidative stress, and hepatocyte apoptosis, with chemical liver injury in humans (Li et al., 2018Li, S., Liu, J., Zhang, M., Chen, Y., Zhu, T., & Wang, J. (2018). Protective effect of eckol against acute hepatic injury induced by carbon tetrachloride in mice. Marine Drugs, 16(9), 300. http://dx.doi.org/10.3390/md16090300. PMid:30150561.
http://dx.doi.org/10.3390/md16090300...
; Ahmad et al., 2023Ahmad, M., Ali, S. W., Hameed, A., Amir, M., Ashraf, J., Afzal, M. I., Umer, M., Alsagaby, S. A., Awais, M., Imran, M., Iqbal, S., Ahmed, A., & Riaz, M. (2023). Functional potential of Aloe vera juice against CCl 4 induced hepatotoxicity in animal model. Food Science and Technology (Campinas), 43, e110321. http://dx.doi.org/10.1590/fst.110321.
http://dx.doi.org/10.1590/fst.110321...
). Our previous studies showed that the extracts from SSG exerted the anti-inflammatory and anti-apoptotic activities in the in vitro and in vivo osteoarthritis models (Wang et al., 2020Wang, J., Guo, J., Li, S., Zhang, M., & He, B. (2020). Protective effect of ethyl acetate fraction from Semen sojae germinatum, the processed sprout of Chinese black soybean, on rat experimental osteoarthritis. BMC Complementary Medicine and Therapies, 20(1), 117. http://dx.doi.org/10.1186/s12906-020-02920-9. PMid:32306945.
http://dx.doi.org/10.1186/s12906-020-029...
; Fan et al., 2016Fan, W., Qian, Y., Wang, J., Yang, X., Gui, T., & He, B. (2016). Chondroprotective and anti-inflammatory activities of extracts from Semen Sojae Germinatum on IL-1β-stimulated human osteoarthritis chondrocytes. Indian Journal of Pharmaceutical Education and Research, 50(3), 397-402. http://dx.doi.org/10.5530/ijper.50.3.12.
http://dx.doi.org/10.5530/ijper.50.3.12...
). Therefore, from the perspective of either TCM or modern scientific theory, it could be speculate that SSG and its active ingredients might have a protective effect on the CCl4-induced liver injury. In the present study, we explored the effect of different fractions extracted from SSG on the murine liver injury progress induced by CCl4 challenge and possible underlying mechanisms.

2 Materials and methods

2.1 Preparation of fractions extracted from SSG

Dried SSG was prepared according to the method recorded in the Pharmacopoeia of People's Republic of China (He & Wang, 2013He, B., & Wang, J. (2013). Effect of semen sojae germinatum on experimental osteoarthritis in the rabbit knee. Bangladesh Journal of Pharmacology, 8(4), 365-370. http://dx.doi.org/10.3329/bjp.v8i4.16446.
http://dx.doi.org/10.3329/bjp.v8i4.16446...
). Then, as described in our previous study (Fan et al., 2016Fan, W., Qian, Y., Wang, J., Yang, X., Gui, T., & He, B. (2016). Chondroprotective and anti-inflammatory activities of extracts from Semen Sojae Germinatum on IL-1β-stimulated human osteoarthritis chondrocytes. Indian Journal of Pharmaceutical Education and Research, 50(3), 397-402. http://dx.doi.org/10.5530/ijper.50.3.12.
http://dx.doi.org/10.5530/ijper.50.3.12...
), the petroleum ether extract (PEE), ethyl acetate extract (EAE), and n-butanol extract (NBE) fractions of SSG were obtained through sequentially partitioning the crude ethanol extract with the corresponding solvent. The PEE, EAE, NBE, and the remaining aqueous fraction (AF) of SSG were evaporated to dryness.

2.2 Animals and groups

Kunming mice (male, 18-22 g) were obtained from Hubei Experimental Animal Research Center (Wuhan, China). After one week of acclimation, the animals were randomly divided into 7 groups of 10 mice in each group: (1) normal control group, (2) CCl4 model control group, (3) CCl4 + PEE of SSG group, (4) CCl4 + EAE of SSG group, (5) CCl4 + NBE of SSG group, (6) CCl4 + AF of SSG group, and (7) CCl4 + silymarin group. Mice in groups (3)-(6) were orally pre-treated with the corresponding extract fractions of SSG at the dose of 200 mg/kg for 7 consecutive days. As the positive control, silymarin (Tianjin Tasly Sants Pharmaceutical Co., Ltd, China) at dose of 100 mg/kg was orally administrated to mice in the group (7) for 7 days. At 2 h after the last administration of pre-treatment, all animals except those in the normal control group were intraperitoneally injected with the olive oil containing 0.5% CCl4 (Jiangsu Qiangsheng Chemical Co., Ltd, China) at dose of 10 mL/kg. The normal control mice were administrated with the equal volume of vehicles (intraperitoneally injected with olive oil without CCl4 + orally administrated with distilled water). At 24 h after CCl4 challenge, all animals were weighed then sacrificed after collecting blood samples from their eyeballs. Liver samples were quickly collected and the liver masses were recorded. The liver tissue index of mice was expressed as the ratio of liver mass (g) to 100 g body weight. The experimental protocol was performed strictly in accordance with the procedures approved by the Animal Ethic Committee of the Wuhan University of Science and Technology. The flowchart of the experimental design of the study was shown as Figure 1.

Figure 1
The flowchart of the experimental design of the study.

2.3 Determination of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels

The blood samples collected from eyeballs were centrifuged to separate the serums. The levels of serum ALT and AST as liver function markers were measured by enzymatic colorimetric methods according to kit protocols (Nanjing Jiancheng Bio-Engineering Co., Ltd., China).

2.4 Pathological examination of liver tissues

After being fixed in 10% neutral buffered formaldehyde for 24 h, the liver tissues were embedded in paraffin, cut into 4-6 µm-thick sections then stained with hematoxylin and eosin (H&E) for pathological examination of liver injury.

2.5 Measurement of malondiadehyde (MDA), glutathione (GSH), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in liver tissues

The liver tissues were homogenized on ice. Then, the levels of MDA and GSH as well as the activities of SOD and GSH-Px as oxidative stress parameters in the liver homogenates were measured according to the corresponding manufacturer’s instructions (Nanjing Jiancheng Bio-Engineering Co., Ltd., China). The protein contents in homogenates were detected with the bicinchoninic acid assay kit (Nanjing Jiancheng Bio-Engineering Co., Ltd., China).

2.6 Determination of the tumor necrosis factor (TNF)-ɑ and interleukin (IL)-6 levels in liver tissues

After homogenizing the collected liver tissues, the levels of TNF-ɑ and IL-6 in liver homogenates were detected using ELISA kits according to kit protocols (R&D Systems, USA).

2.7 TUNEL assay

The 10% neutral buffered formaldehyde-fixed and paraffin-embedded liver tissues were cut into 5-μm sections. After the sections were deparaffinized and rehydrated, the apoptotic hepatocytes in the liver sections were detected using the terminal deoxynucleotidyl transferase mediated dUTP nick end labelling (TUNEL) assay according to the manufacturer’s instruction (Roche, USA).

2.8 Statistical analysis

Results were shown as the mean ± SD. One-way analysis of variance (ANOVA) with post hoc Tukey test was employed for statistical analysis in SPSS 25.0 software. P value above 0.05 was statistically significant.

3 Results and discussion

3.1 Effect of extracts from SSG on CCl4-induced liver injury in mice

The hepatoprotective effects of different fractions extracted from SSG on CCl4-induced liver injury in mice were assessed using liver tissue indices, serum levels of liver function markers (ALT and AST), and the liver pathological examination. As shown in Table 1, no significant difference in body weight was found between any group (P>0.05). The CCl4 challenge markedly increased the liver tissue indices of mice (P < 0.05). However, pre-treatment with silymarin, EAE and NBE of SSG significantly protected mice against the CCl4-induced increase of liver tissue index (P < 0.05). Similarly, mice pre-treated with silymarin, EAE and NBE of SSG showed significantly lower serum transaminases ALT and AST levels than CCl4 model controls (P < 0.05; P < 0.01). As shown in Figure 2, obvious hepatocyte edema, hemorrhage, inflammatory infiltration and destruction of hepatic architecture were found in CCl4-challenged livers. These pathological changes of liver damage were less severe in CCl4 + silymarin, EAE or NBE of SSG group than CCl4 model group. These data collectively suggested the hepato-protective effects of EAE and NBE from SSG on the CCl4-induced liver injury.

Table 1
Effect of extracts from SSG on liver tissue indices, and serum transaminases ALT and AST levels of CCl4-challenged mice (Mean ± SD, n = 10).
Figure 2
Effect of extracts from SSG on the CCl4-induced pathological changes of liver tissues.

Previous studies have demonstrated the the liver protective effects of soy diet (Khan, 2012Khan, T. H. (2012). Soy diet diminish oxidative injure and early promotional events induced by CCl4 in rat liver. International Journal of Pharmacology, 8(1), 30-38. http://dx.doi.org/10.3923/ijp.2012.30.38.
http://dx.doi.org/10.3923/ijp.2012.30.38...
), soybean oil (Gao et al., 2019Gao, Y., Li, X., Gao, Q., Fan, L., Jin, H., & Guo, Y. (2019). Differential effects of olive oil, soybean oil, corn oil and lard oil on carbon tetrachloride-induced liver fibrosis in mice. Bioscience Reports, 39(10), BSR20191913. http://dx.doi.org/10.1042/BSR20191913. PMid:31481526.
http://dx.doi.org/10.1042/BSR20191913...
) and soy-derived active components, such as kaempferol galactosides separated from Japan Jindai unripe soybean leaves (Zang et al., 2018Zang, Y., Hashimoto, S., Yu, C., & Igarashi, K. (2018). Protective effects of dietary kaempferol glycoside components from unripe soybean (Edamame, Glycine max L. Merrill. ‘Jindai’) leaves and their serous metabolite on carbon tetrachloride-induced liver injury mice. Journal of Food Science and Technology, 55(11), 4515-4521. http://dx.doi.org/10.1007/s13197-018-3385-6. PMid:30333648.
http://dx.doi.org/10.1007/s13197-018-338...
), an arabinogalactan from black soybean (Sun et al., 2018Sun, J., Wen, X., Liu, J., Kan, J., Qian, C., Wu, C., & Jin, C. (2018). Protective effect of an arabinogalactan from black soybean against carbon tetrachloride-induced acute liver injury in mice. International Journal of Biological Macromolecules, 117, 659-664. http://dx.doi.org/10.1016/j.ijbiomac.2018.05.203. PMid:29852225.
http://dx.doi.org/10.1016/j.ijbiomac.201...
), and isoflavones-enriched soy protein (Sarhan et al., 2012Sarhan, N. A., El-Denshary, E. S., Hassan, N. S., Abu-Salem, F. M., & Abdel-Wahhab, M. A. (2012). Isoflavones-enriched soy protein prevents CCL(4)-induced hepatotoxicity in rats. ISRN Pharmacology, 2012, 347930. PMid:22530140.). Interestingly, evidences showed that the germinated soybeans contain more functional substances than non-germinated soybeans, thus are more beneficial for human health (Huang et al., 2014Huang, X., Cai, W., & Xu, B. (2014). Kinetic changes of nutrients and antioxidant capacities of germinated soybean (Glycine max L.) and mung bean (Vigna radiata L.) with germination time. Food Chemistry, 143, 268-276. http://dx.doi.org/10.1016/j.foodchem.2013.07.080. PMid:24054239.
http://dx.doi.org/10.1016/j.foodchem.201...
; Borges-Martínez et al., 2022Borges-Martínez, E., Gallardo-Velázquez, T., Cardador-Martínez, A., Moguel-Concha, D., Osorio-Revilla, G., Ruiz-Ruiz, J. C., & Martínez, C. J. (2022). Phenolic compounds profile and antioxidant activity of pea (Pisum sativum L.) and black bean (Phaseolus vulgaris L.) sprouts. Food Science and Technology (Campinas), 42, e45920. http://dx.doi.org/10.1590/fst.45920.
http://dx.doi.org/10.1590/fst.45920...
). Germination enhanced the antioxidant capacities as well as the contents of L-ascorbic acid, phenolics and isoflavone in soybean. For example, the contents of genistein, daidzein and total aglycone in germinated soybeans were found to be approximately 2-3 folds of those in the non-germinated soybeans (Huang et al., 2014Huang, X., Cai, W., & Xu, B. (2014). Kinetic changes of nutrients and antioxidant capacities of germinated soybean (Glycine max L.) and mung bean (Vigna radiata L.) with germination time. Food Chemistry, 143, 268-276. http://dx.doi.org/10.1016/j.foodchem.2013.07.080. PMid:24054239.
http://dx.doi.org/10.1016/j.foodchem.201...
). The ethanolic extract from germinated soybean embryos has been reported to inhibit hepatic inflammation and ameliorate the fatty liver injury (Kwon et al., 2020Kwon, D., Kim, S. H., Son, S. W., Seo, J., Jeong, T. B., Kim, K. M., Jung, J. C., Jung, M. S., Lee, Y. H., & Jung, Y. S. (2020). Germinated soybean embryo extract ameliorates fatty liver injury in high-fat diet-fed obese mice. Pharmaceuticals (Basel, Switzerland), 13(11), 380. http://dx.doi.org/10.3390/ph13110380. PMid:33187321.
http://dx.doi.org/10.3390/ph13110380...
). As a kind of processed bean sprout, SSG is also a soy-derived traditional Chinese herbal medicine. The present study firstly showed that this soy-derived Chinese medicine could relieve the liver swelling, improve the liver function, and alleviate liver histopathology in the CCl4-challenged mice, supporting the potential of SSG as well as its EAE and NBE as protective agents against liver disorders. From the unique perspective of TCM, SSG has the effects of clearing “heat”, removing “dampness”, and relieving “toxin”, which could explain its effectiveness in the CCl4-induced hepatic damage representing “dampness-heat” syndrome in TCM (Xie et al., 2021Xie, S. Z., Zhai, X. Y., Xi, S. Y., Qiu, Y. K., Zhang, Y. M., Kong, X. J., Li, Y. H., Zhu, L., Wang, Z., Zhang, S. G., Huang, S. Q., Lu, D. W., & Wang, Z. (2021). The protective effects of Zornia diphylla (L.) Pers. against acute liver injury induced by carbon tetrachloride in mice. Frontiers in Pharmacology, 12, 764282. http://dx.doi.org/10.3389/fphar.2021.764282. PMid:34899319.
http://dx.doi.org/10.3389/fphar.2021.764...
; Cao et al., 2009Cao, H. X., Sun, H., Jiang, X. G., Lu, H. T., Zhang, G. M., Wang, X. J., Sun, W. J., Wu, Z. M., Wang, P., Liu, L., & Zhou, J. (2009). Comparative study on the protective effects of Yinchenhao Decoction against liver injury induced by alpha-naphthylisothiocyanate and carbon tetrachloride. Chinese Journal of Integrative Medicine, 15(3), 204-209. http://dx.doi.org/10.1007/s11655-009-0204-y. PMid:19568713.
http://dx.doi.org/10.1007/s11655-009-020...
; Xu et al., 2021Xu, S., Kong, F., Sun, Z., Xi, Y., Qi, F., & Sun, J. (2021). Hepatoprotective effect and metabonomics studies of radix gentianae in rats with acute liver injury. Pharmaceutical Biology, 59(1), 1172-1180. http://dx.doi.org/10.1080/13880209.2021.1969414. PMid:34465274.
http://dx.doi.org/10.1080/13880209.2021....
).

3.2 Effect of extracts from SSG on CCl4-induced liver oxidative stress

Oxidative stress is the common pathophysiological basis involved in various liver injuries. In the CCl4-challenged liver tissues, after the metabolism by hepatic cytochrome P450 enzyme, CCl4 is converted to the trichloromethyl free radical (•CCl3) and/or trichloromethyl peroxyl radical (•OOCCl3), sequentially induces liver oxidative stress damage (Li et al., 2018Li, S., Liu, J., Zhang, M., Chen, Y., Zhu, T., & Wang, J. (2018). Protective effect of eckol against acute hepatic injury induced by carbon tetrachloride in mice. Marine Drugs, 16(9), 300. http://dx.doi.org/10.3390/md16090300. PMid:30150561.
http://dx.doi.org/10.3390/md16090300...
). Coincidentally, soybean products have been well demonstrated to be enriched with antioxidant compounds (Huang et al., 2014Huang, X., Cai, W., & Xu, B. (2014). Kinetic changes of nutrients and antioxidant capacities of germinated soybean (Glycine max L.) and mung bean (Vigna radiata L.) with germination time. Food Chemistry, 143, 268-276. http://dx.doi.org/10.1016/j.foodchem.2013.07.080. PMid:24054239.
http://dx.doi.org/10.1016/j.foodchem.201...
; Zang et al., 2018Zang, Y., Hashimoto, S., Yu, C., & Igarashi, K. (2018). Protective effects of dietary kaempferol glycoside components from unripe soybean (Edamame, Glycine max L. Merrill. ‘Jindai’) leaves and their serous metabolite on carbon tetrachloride-induced liver injury mice. Journal of Food Science and Technology, 55(11), 4515-4521. http://dx.doi.org/10.1007/s13197-018-3385-6. PMid:30333648.
http://dx.doi.org/10.1007/s13197-018-338...
; Sun et al., 2018Sun, J., Wen, X., Liu, J., Kan, J., Qian, C., Wu, C., & Jin, C. (2018). Protective effect of an arabinogalactan from black soybean against carbon tetrachloride-induced acute liver injury in mice. International Journal of Biological Macromolecules, 117, 659-664. http://dx.doi.org/10.1016/j.ijbiomac.2018.05.203. PMid:29852225.
http://dx.doi.org/10.1016/j.ijbiomac.201...
; Sarhan et al., 2012Sarhan, N. A., El-Denshary, E. S., Hassan, N. S., Abu-Salem, F. M., & Abdel-Wahhab, M. A. (2012). Isoflavones-enriched soy protein prevents CCL(4)-induced hepatotoxicity in rats. ISRN Pharmacology, 2012, 347930. PMid:22530140.; Gao et al., 2019Gao, Y., Li, X., Gao, Q., Fan, L., Jin, H., & Guo, Y. (2019). Differential effects of olive oil, soybean oil, corn oil and lard oil on carbon tetrachloride-induced liver fibrosis in mice. Bioscience Reports, 39(10), BSR20191913. http://dx.doi.org/10.1042/BSR20191913. PMid:31481526.
http://dx.doi.org/10.1042/BSR20191913...
). In order to explore the possible mechanisms underlying the hepato-protective effect of SSG extracts, we evaluated the indices of liver oxidative stress, including the levels of a lipid peroxidation product MDA and a non-enzymatic antioxidant GSH, as well as the activities of antioxidant enzymes SOD and GSH-Px.

As shown in Table 2, the CCl4 challenge induced an imbalance between oxidative stress and antioxidant defenses in liver, manifested as the enhanced MDA levels, the decreased GSH levels and the inhibited activities of antioxidant enzymes SOD and GSH-Px (P < 0.01). In line with the above results showing hepato-protective effects of EAE and NBE from SSG, the oxidative imbalance induced by CCl4 was significantly restored by pretreatment with silymarin, EAE or NBE of SSG (P < 0.05; P < 0.01), suggesting the EAE and NBE of SSG might possess anti-oxidative activity, which is at least partly responsible for their hepatoprotective effects.

Table 2
Effect of extracts from SSG on MDA, GSH, SOD and GSH-Px levels in CCl4-challenged livers (Mean ± SD, n = 10).

3.3 Effect of extracts from SSG on the levels of pro-inflammatory cytokines in livers of CCl4-challenged mice

Inflammatory damage is one of crucial events associated with CCl4-induced liver injury (Li et al., 2018Li, S., Liu, J., Zhang, M., Chen, Y., Zhu, T., & Wang, J. (2018). Protective effect of eckol against acute hepatic injury induced by carbon tetrachloride in mice. Marine Drugs, 16(9), 300. http://dx.doi.org/10.3390/md16090300. PMid:30150561.
http://dx.doi.org/10.3390/md16090300...
). As shown in Figure 3, significant increases in levels of pro-inflammatory cytokines TNF-ɑ and IL-6 were observed in liver tissues collected from CCl4 model control mice (P < 0.01). However, the CCl4-induced elevations of TNF-α and IL-6 in livers were markedly suppressed in mice pre-treated with silymarin, PEE, EAE or NBE of SSG (P < 0.05; P < 0.01). In particular, compared to the CCl4 model control group, the levels of TNF-α and IL-6 in the CCl4 + EAE of SSG group was declined by 22.4 and 25.7%, respectively.

Figure 3
Effect of extracts from SSG on the levels of pro-inflammatory cytokines TNF-ɑ and IL-6 in livers of CCl4-challenged mice. n = 10. **P < 0.01 vs. normal control group; #P < 0.05, ##P < 0.01 vs. CCl4 model control group.

TNF-α has been considered as the earliest proinflammatory cytokine released mainly by damaged hepatic macrophages during inflammatory process of acute liver tissue injury. As a key lymphokine, IL-6 mediates chemotaxis of inflammatory cells, exacerbates the inflammatory cascade, finally leads to tissue damage (Xie et al., 2021Xie, S. Z., Zhai, X. Y., Xi, S. Y., Qiu, Y. K., Zhang, Y. M., Kong, X. J., Li, Y. H., Zhu, L., Wang, Z., Zhang, S. G., Huang, S. Q., Lu, D. W., & Wang, Z. (2021). The protective effects of Zornia diphylla (L.) Pers. against acute liver injury induced by carbon tetrachloride in mice. Frontiers in Pharmacology, 12, 764282. http://dx.doi.org/10.3389/fphar.2021.764282. PMid:34899319.
http://dx.doi.org/10.3389/fphar.2021.764...
). In line with the known anti-inflammatory properties of EAE of SSG (Wang et al., 2020Wang, J., Guo, J., Li, S., Zhang, M., & He, B. (2020). Protective effect of ethyl acetate fraction from Semen sojae germinatum, the processed sprout of Chinese black soybean, on rat experimental osteoarthritis. BMC Complementary Medicine and Therapies, 20(1), 117. http://dx.doi.org/10.1186/s12906-020-02920-9. PMid:32306945.
http://dx.doi.org/10.1186/s12906-020-029...
; Fan et al., 2016Fan, W., Qian, Y., Wang, J., Yang, X., Gui, T., & He, B. (2016). Chondroprotective and anti-inflammatory activities of extracts from Semen Sojae Germinatum on IL-1β-stimulated human osteoarthritis chondrocytes. Indian Journal of Pharmaceutical Education and Research, 50(3), 397-402. http://dx.doi.org/10.5530/ijper.50.3.12.
http://dx.doi.org/10.5530/ijper.50.3.12...
) and other soybean bioactive compounds (Kusumah & Gonzalez de Mejia, 2022Kusumah, J., & Gonzalez de Mejia, E. (2022). Impact of soybean bioactive compounds as response to diet-induced chronic inflammation: a systematic review. Food Research International, 162(Pt A), 111928. http://dx.doi.org/10.1016/j.foodres.2022.111928. PMid:36461277.
http://dx.doi.org/10.1016/j.foodres.2022...
), the present study showed that, among different fractions extracted from SSG, the EAE appeared to exhibit the strongest inhibitory activity against the CCl4-induced liver inflammation.

3.4 Effect of extracts from SSG on hepatocyte apoptosis in CCl4-challenged mice

As a classic hepato-toxicant, CCl4 could caused the cell apoptosis contributing to the morphological changes even death of hepatocytes, and the development of liver injury (Li et al., 2018Li, S., Liu, J., Zhang, M., Chen, Y., Zhu, T., & Wang, J. (2018). Protective effect of eckol against acute hepatic injury induced by carbon tetrachloride in mice. Marine Drugs, 16(9), 300. http://dx.doi.org/10.3390/md16090300. PMid:30150561.
http://dx.doi.org/10.3390/md16090300...
). As shown in Figure 4, TUNEL assay in this study revealed that the CCl4 challenge induced severe hepatocyte apoptosis in mice. Importantly, pre-treated with silymarin, EAE and NBE of SSG could decreased the CCl4-enhanced number of TUNEL-positive apoptotic hepatocytes, suggesting EAE and NBE of SSG were effective in protecting the hepatocytes against the CCl4-induced cellular damage.

Figure 4
Effect of extracts from SSG on the number of TUNEL-positive apoptotic hepatocytes in CCl4-challenged mice.

4 Conclusions

To the best of our knowledge, this is the first study providing experimental evidence supporting the medicinal and dietary uses of SSG, a germinated soybean-derived traditional Chinese herbal medicine with heat-clearing, dampness-removing, and detoxifying properties, for liver disorder patients with “dampness-heat” syndrome from perspective of TCM. In the present study, we demonstrated that extracts from SSG showed protective effect on the CCl4-induced liver injury representing “dampness-heat” syndrome in TCM via multiple pharmacological mechanisms including anti-oxidation, anti-inflammation and anti-apoptosis. In general, EAE and NBE might mainly account for the hepato-protective effect of SSG extracts, and EAE appeared to be more active than the other extract fractions.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (71974153).

  • Practical Application: This study provided experimental data for medicinal and food uses of Sojae semen germinatum for the liver disorder patients with “dampness- heat” syndrome from perspective of Traditional Chinese Medicine.
  • #Contributed equally.

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Publication Dates

  • Publication in this collection
    30 Jan 2023
  • Date of issue
    2023

History

  • Received
    18 Nov 2022
  • Accepted
    26 Dec 2022
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