Effects of Kudzu Root on Oxidative Stress and Inflammation in Streptozotocin-induced Diabetic Rats

1. Introduction
Diabetes mellitus (DM) is a worldwide health issue and one of the most prevalent metabolic disorders  (Kharroubi & Darwish, 2015; Moghtadaei Khorasgani et al., 2021). The hallmark of DM is chronic hyperglycemia due to a deficiency in insulin secretion and or effect (Kharroubi & Darwish, 2015). Because of its serious vascular, neurological, and infectious complications, DM is one of the most important reasons for patients’ morbidity and mortality (Kharroubi & Darwish, 2015). More than 340 million people live with diabetes worldwide, predicted to increase to 578 million by 2030 (Saeedi et al., 2019).
Oxidative stress is triggered by an imbalance between free radical generation, mainly reactive oxygen species (ROS) and nitrogen reactive species (RNS), and the level of antioxidant substances, so the antioxidant system cannot scavenge the pro-oxidant species in the body (Hussain & Tan, 2016). Although a low level of ROS serves as a key signaling mediator in the regulation of several physiologic pathways in living cells, its over-production could contribute to the oxidation of cellular macromolecules and also promotes cell and tissue damage (Rastogi & Haldar, 2018).
Oxidative stress causes several inflammatory conditions. Oxidative stress and inflammation are strictly connected, and both perform an important function in the pathogenesis of several chronic diseases (Biswas, 2016, Kaywanloo et al., 2022), such as DM and its various complications (Hussain & Tan, 2016). 
Numerous pharmaceutical effects of natural products have drawn much attention for designing drugs to treat several diseases (Gao et al., 2016; Panche et al., 2016). Evidence has demonstrated that flavonoids, a subclass of polyphenols, have various valuable properties on human health mostly because of their potent antioxidant and anti-inflammatory properties (Panche et al., 2016; Karak, 2019). Kudzu root or Pueraria lobata has been commonly used in traditional medicine for the treatment of some disorders, in particular metabolic diseases. It has been confirmed that isoflavonoids are one of the main bioactive components in kudzu root (Gao et al., 2016). The most abundant isoflavonoid in the root of P. lobata is puerarin (Panche et al., 2016). The beneficial properties of puerarin have been mentioned in various metabolic disorders (Zhou et al., 2014).
Streptozotocin (STZ) suppresses insulin secretion and triggers DM (Aloud et al., 2017). In our research, diabetes was induced by STZ injection in male Wistar rats. Regarding the association between oxidative stress, inflammation, and DM, our study was designed to examine the potential effect of flavonoid-enriched- kudzu root against oxidative stress and inflammation in STZ-diabetic animals. We intended to provide evidence for using kudzu root as an effective medication for diabetes mellitus.

2. Materials and Methods 

Study materials
The kudzu root powder and STZ were purchased from Sigma Co., Ltd. (Missouri, USA). Insulin enzyme-linked immunosorbent assay (ELISA) kit was obtained from MyBioSource Co. (USA). The plasma glutathione peroxidase (GPx), tumor necrosis factor-alpha (TNF-α), and malondialdehyde (MDA) ELISA kits were purchased from Hangzhou Eastbiopharm Co. Ltd. (Zhejiang, China). Superoxide dismutase (SOD) ELISA kit was provided by Bioassay Technology Laboratory Co. (Shanghai, China).

Experimental animals 
A total of 21 male Wistar rats (weight: 220-240 g) were obtained from the Animal Center of Pasteur Institute of Iran/North Research Center, Iran. The animals were housed in an air-conditioned environment under standard temperatures (22°C±2°C) and a 12-h light–dark period. Rats were fed a standard rat chow and allowed free access to purified water. 
Diabetic animal model and pharmacological intervention
The animals were randomly allocated into 3 groups, each containing 7 rats: normal control group (NC), diabetic control (DC) group, and diabetic+kudzu root (D/KR) (100 mg/kg) group. The STZ (55 mg kg) was dissolved in citrate buffer (0.05 M, pH=4.5) and intraperitoneally injected into rats. The diabetic animals were intragastrically treated with kudzu root powder 1 week after STZ injection (for 6 weeks). The NC and DC groups were treated with an equivalent volume of normal saline. Blood samples were collected by tail vein puncture 72 h after STZ administration, fasting blood glucose (FBG) concentration was investigated, and animals with FBG levels of ≥16.7 mmol/L were selected as appropriate diabetic models and used in this research. When the study was completed, the animals were fasted for 6 h, anesthetized using ketamine-xylazine, and the blood samples were collected by cardiac puncture to perform biochemical analysis.

FBG measurement
FBG level was measured using a Glucocard 01 blood glucose monitor (Arcary, USA, Inc).

Insulin measurement
According to the manufacturer’s instructions, plasma insulin levels were determined in duplicate by commercial enzyme-linked immunosorbent assay (ELISA) kits from Monobind Inc. Concurrently with these tests, standards at varying concentrations were run. The insulin levels were measured according to the standard curve. 

Measurement of antioxidant and oxidative stress markers
According to kit instructions, GPx activity and MDA concentration were measured in plasma samples. Plasma SOD was assayed in duplicate using a commercial ELISA kit following the manufacturer’s recommendations.

Measurement of TNF-α
Plasma TNF-α level was measured in duplicate by a commercial ELISA kit (EastBiopharma) with a 1.52 ng/L sensitivity.

Statistical analysis    
The data were analyzed using SPSS software, Version 20. The quantitative parameters were reported as Mean±SEM. The independent t-test was used to compare the parameters between groups. A P<0.05 was taken as statistically significant.

3. Results

 
Kudzu root improved FBG levels and body weight
At the end of the study, diabetic animals showed a higher level of FBG. Moreover, the rats that received the kudzu root represented decreased FBG levels (P<0.05) as compared to the diabetic rats (Figure 1).

Effect of kudzu root on plasma levels of insulin
As expected, STZ administration intensely decreased the plasma concentration of insulin. In contrast, kudzu root administration reverses the reduction of insulin in diabetic rats (Figure 2).

Kudzu root decreased MDA and increased SOD and GPx activity
Kudzu root administration could reverse the effects of diabetic condition on the MDA and SOD concentration and GPx activity so that kudzu root treatment significantly decreased MDA level and increased SOD and GPx activity in the diabetic rats (P<0.05) (Figure 3).

Kudzu root decreased TNF-α level 
The present research demonstrated a significant enhancement (P<0.05) of plasma TNF-α in diabetic animals. Kudzu root treatment showed obvious reducing effects on plasma TNF-α levels in diabetic rats (P<0.05) (Figure 4).

4. Discussion
DM is a common metabolic condition associated with chronic inflammation and oxidative stress. Studies have revealed that STZ treatment can cause permanent DM in animal models (Wu & Yan, 2015). This research showed that insulin level was significantly decreased, and the blood sugar level noticeably increased in diabetic rats. In contrast, administration of kudzu root led to a significant decrease in plasma glucose and a significant rise in plasma insulin in STZ-diabetic rats. In this regard, several studies have reported that flavonoids can lead to glucose level reduction through glucose absorption inhibition and glucose tolerance improvement by stimulating insulin secretion and function (Fang et al., 2008, Jung et al., 2004).
Kudzu root is a rich source of isoflavones, mainly containing puerarin, daidzein, and genistein (Duru et al., 2020). A recent study revealed that isoflavone-rich kudzu extracts improved glucose and HbA1c levels and stimulated β-cells regeneration (Duru et al., 2020). 
Increased oxidative stress and inflammation are key mechanisms in the pathogenesis of numerous chronic disorders, including DM (Marseglia et al., 2015). It has been shown that ROS generation is related to macromolecules and cellular damage in the pathogenesis of diabetes mellitus. Uncontrolled lipid peroxidation due to enhanced endogenous oxidant species impairs cell membrane functioning. Altogether, lipid peroxidation can lead to cellular infiltration, islet cell damage, and the development of DM (Asmat et al., 2016). As an important product of lipid peroxidation, MDA considerably rises, as described in the previous studies on STZ-induced diabetic rat models (Sheweita et al., 2016). In the current study, the MDA levels revealed a significant increase in STZ-induced diabetic animals, and the administration of kudzu root significantly reversed this effect. 
However, the body has antioxidant defenses against the effect of oxidants species (Birben et al., 2012). SOD is a key anti-oxidative enzyme that acts as the first line of defense against ROS to reduce lipid peroxidation and oxidative stress by catalyzing the conversion of superoxide radicals into H2O2  that is detoxified by the activities of glutathione peroxidase (Gpx) and catalase (CAT). In addition, enzymatic and non-enzymatic antioxidants are important in inhibiting cellular oxidative stress. Glutathione (GSH) is the most common non-enzymatic endogenous antioxidant, which serves as a substrate for GPx and is a direct scavenger of ROS/RNS. The previous research has shown decreased plasma levels of GSH and SOD in STZ-induced diabetic animals as compared to the control group, which is likely due to reduced synthesis or augmented degradation of GSH by oxidative stress (Sheweita et al., 2016, Xie et al., 2018). In the present study, GPx activity was significantly reduced in diabetic animals. 
Although some studies have suggested that kudzu root has potent antioxidant properties in some disorders, the evidence supporting the antioxidant effects of kudzu root in DM conditions is limited. In this regard, it has been revealed that puerarin, one constituent of kudzu root, considerably reverses the reduction in SOD activity and rise in MDA levels in arthritis animal models (Wang et al., 2016). Another study shows that kudzu root significantly protects the rat pheochromocytoma cell line (PC12) cells against H2O2 damage by increasing the CAT and SOD activities and glutathione levels (Zhang et al., 2017). A study also demonstrates that puerarin, isolated from kudzu root, can increase manganese SOD and CAT activities in a diabetic nephropathy model in STZ-diabetic mice (Xu et al., 2016). In this study, kudzu root administration decreased MDA levels and increased the activity of antioxidant enzymes, SOD, and GPx in STZ-induced diabetic rats. 
Studies have revealed that chronic inflammation has a key function in the pathogenesis of metabolic disease, so pro-inflammatory cytokines are identified to be raised in several metabolic disorders such as DM (Tsalamandris et al., 2019). Among pro-inflammatory cytokines, TNF-α is one of the major cytokines that initiates inflammatory processes. Therefore, there has been a growing interest in targeting inflammation as a means to prevent and treat metabolic disorders (Tsalamandris et al., 2019). In this regard, the increased level of TNF-α cytokine in the plasma of diabetic rats may contribute to β cell and insulin dysfunction. The level of this cytokine significantly decreased in diabetic rats after kudzu root administration. This result is in accordance with the previous reports that several flavonoids have been shown to inhibit pro-inflammatory cytokine production in diabetic conditions (Samie et al., 2018; Ginwala et al., 2019). The anti-inflammatory of kudzu root was also shown in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells (Jin et al., 2012). It has also been revealed that genistein and puerarin effectively ameliorate alcohol-induced hepatic injury through antioxidant and anti-inflammatory actions in mice (Zhao et al., 2016). A recent study showed that puerarin can reduce the expression of TNF-α and improve insulin resistance in the gestational DM rat model (Xu et al., 2020). 

5. Conclusion 
In conclusion, while further evidence is required to precisely determine exactly how kudzu root affects DM, this research revealed that the antidiabetic action of kudzu root is likely attributed to its anti-inflammatory and anti-oxidative properties in the STZ-diabetic rat model.

Ethical Considerations

Compliance with ethical guidelines
The Research and the Medical Ethics Committee of Shahroud University of Medical Sciences approved the experimental protocol in this study (Code: IR.SHMU.REC.1398.022).

Funding
This research did not receive any grant from funding agencies in the public, commercial, or non-profit sectors. 

Authors' contributions
All authors equally contributed to preparing this article.

Conflict of interest
The authors declared no conflict of interest.

Acknowledgments
The authors are grateful to Shahroud University of Medical Sciences for the support of this project (No.: 9818). 

References

Aloud, A. A., Veeramani, C., Govindasamy, C., Alsaif, M. A., El Newehy, A. S., & Al-Numair, K. S. (2017). Galangin, a dietary flavonoid, improves antioxidant status and reduces hyperglycemia-mediated oxidative stress in streptozotocin-induced diabetic rats. Redox Report : Communications in Free Radical Research, 22(6), 290–300. [PMID] [PMCID]

Asmat, U., Abad, K., & Ismail, K. (2016). Diabetes mellitus and oxidative stress-A concise review. Saudi Pharmaceutical Journal : SPJ : The Official Publication of The Saudi Pharmaceutical Society, 24(5), 547–553. [PMID] [PMCID]

Birben, E., Sahiner, U. M., Sackesen, C., Erzurum, S., & Kalayci, O. (2012). Oxidative stress and antioxidant defense. The World Allergy Organization Journal, 5(1), 9–19. [DOI: 10.1097/WOX.0b013e3182439613] [PMID] [PMCID]

Biswas S. K. (2016). Does the Interdependence between Oxidative stress and inflammation explain the antioxidant paradox? Oxidative Medicine and Cellular Longevity, 2016, 5698931. [PMID] [PMCID]

Duru, K. C., Mukhlynina, E. A., Moroz, G. A., Gette, I. F., Danilova, I. G. & Kovaleva, E. G. (2020) Antidiabetic effect of isoflavone rich kudzu root extract in experimentally induced diabetic rats. Journal of Functional Foods, 68, 103922. [DOI:10.1016/j.jff.2020.103922]

Fang, X. K., Gao, J., & Zhu, D. N. (2008). Kaempferol and quercetin isolated from Euonymus alatus improve glucose uptake of 3T3-L1 cells without adipogenesis activity. Life Sciences, 82(11-12), 615–622. [PMID]

Gao, Y., Wang, X., & He, C. (2016). An isoflavonoid-enriched extract from Pueraria lobata (kudzu) root protects human umbilical vein endothelial cells against oxidative stress induced apoptosis. Journal of Ethnopharmacology, 193, 524-530. [DOI:10.1016/j.jep.2016.10.005] [PMID]

Ginwala, R., Bhavsar, R., Chigbu, D. I., Jain, P., & Khan, Z. K. (2019). Potential role of flavonoids in treating chronic inflammatory diseases with a special focus on the anti-inflammatory activity of apigenin. Antioxidants, 8(2), 35. [DOI: 10.3390/antiox8020035] [PMID] [PMCID]

Hussain, T., Tan, B., Yin, Y., Blachier, F., Tossou, M. C., & Rahu, N. (2016). Oxidative stress and inflammation: What polyphenols can do for us? Oxidative Medicine and Cellular Longevity, 2016, 7432797. [PMID] [PMCID]

Jin, S. E., Son, Y. K., Min, B. S., Jung, H. A., & Choi, J. S. (2012).Anti-inflammatory and antioxidant activities of constituents isolated from Pueraria lobata roots. Archives of Pharmacal Research, 35(5), 823–837. [DOI:10.1007/s12272-012-0508-x] [PMID]

Jung, U. J., Lee, M. K., Jeong, K. S., & Choi, M. S. (2004). The hypoglycemic effects of hesperidin and naringin are partly mediated by hepatic glucose-regulating enzymes in C57BL/KsJ-db/db mice. The Journal of Nutrition, 134(10), 2499–2503.[DOI:10.1093/jn/134.10.2499] [PMID]

Karak, P. (2019) Biological activities of flavonoids: An overview. International Journal of Pharmaceutical Sciences and Research, 10(4), 1567-1574. [Link]

Kaywanloo, M., Ahmadi Hamedani, M., Jebeli Javan, A., Emadi Chashmi, H., & Rakhshani Zabol, F. (2022). Effect of parenteral Vitamin D3 supplementation in several doses during a six-day period on total antioxidant capacity in healthy Holstein bulls. Iranian Journal of Veterinary Medicine, 16(1), 81-88. [DOI:10.22059/IJVM.2021.314273.1005142]

Kharroubi, A. T., & Darwish, H. M. (2015). Diabetes mellitus: The epidemic of the century. World Journal of Diabetes, 6(6), 850–867. [PMID] [PMCID]

Marseglia, L., Manti, S., D'Angelo, G., Nicotera, A., Parisi, E., & Di Rosa, G., et al. (2015). Oxidative stress in obesity: A critical component in human diseases. International Journal of Molecular Sciences, 16(1), 378-400. [PMID] [PMCID]

Moghtadaei Khorasgani, E., & Khani, A. (2021). Investigating the effect of hydroalcoholic extract of eryngos on plasma concentration of blood glucose, blood cells and pancreatic tissue in diabetic rats. Iranian Journal of Veterinary Medicine, 15(4), 440-451. [DOI:10.22059/IJVM.2021.311523.1005134]

Panche, A. N., Diwan, A. D., & Chandra, S. R. (2016). Flavonoids: An overview. Journal of Nutritional Science, 5, e47-e47. [DOI:10.1017/jns.2016.41] [PMID] [PMCID]

Rastogi, S., & Haldar, C. (2018). Comparative effect of melatonin and quercetin in counteracting LPS induced oxidative stress in bone marrow mononuclear cells and spleen of Funambulus pennanti. Food and Chemical Toxicology, 120, 243-252. [DOI:10.1016/j.fct.2018.06.062] [PMID]

Saeedi, P., Petersohn, I., Salpea, P., Malanda, B., Karuranga, S., & Unwin, N., et al. (2019). Global and regional diabetes prevalence estimates for 2019 and projections for 2030 and 2045: Results from the International Diabetes Federation Diabetes Atlas. Diabetes Research and Clinical Practice, 157, 107843. [DOI:10.1016/j.diabres.2019.107843] [PMID]

Samie, A., Sedaghat, R., Baluchnejadmojarad, T., & Roghani, M. (2018). Hesperetin, a citrus flavonoid, attenuates testicular damage in diabetic rats via inhibition of oxidative stress, inflammation, and apoptosis. Life Sciences, 210, 132-139. [DOI:10.1016/j.lfs.2018.08.074] [PMID]

Sheweita, S. A., Mashaly, S., Newairy, A. A., Abdou, H. M., & Eweda, S. M. (2016). Changes in oxidative stress and antioxidant enzyme activities in streptozotocin-induced diabetes mellitus in rats: Role of Alhagi maurorum extracts. Oxidative Medicine and Cellular Longevity, 2016, 5264064. [PMID] [PMCID]

Tsalamandris, S., Antonopoulos, A. S., Oikonomou, E., Papamikroulis, G. A., Vogiatzi, G., & Papaioannou, S., et al. (2019). The role of inflammation in diabetes: Current concepts and future perspectives. European Cardiology, 14(1), 50–59. [DOI:10.15420/ecr.2018.33.1] [PMID] [PMCID]

Wang, C., Wang, W., Jin, X., Shen, J., Hu, W., & Jiang, T. (2016). Puerarin attenuates inflammation and oxidation in mice with collagen antibody-induced arthritis via TLR4/NF-κB signaling. Molecular Medicine Reports, 14(2), 1365–1370.[DOI:10.3892/mmr.2016.5357] [PMID]

Wu, J., & Yan, L. J. (2015). Streptozotocin-induced type 1 diabetes in rodents as a model for studying mitochondrial mechanisms of diabetic β cell glucotoxicity. Diabetes, Metabolic Syndrome and Obesity : Targets and Therapy, 8, 181–188. [PMID] [PMCID]

Xie, Z., Wu, B., Shen, G., Li, X., & Wu, Q. (2018). Curcumin alleviates liver oxidative stress in type 1 diabetic rats. Molecular Medicine Reports, 17(1), 103–108. [PMID]

Xu, W., Tang, M., Wang, J., & Wang, L. (2020). Anti-inflammatory activities of puerarin in high-fat diet-fed rats with streptozotocin-induced gestational diabetes mellitus. Molecular Biology Reports, 47(10), 7537–7546. [DOI:10.1007/s11033-020-05816-6] [PMID] [PMCID]

Xu, X., Zheng, N., Chen, Z., Huang, W., Liang, T., & Kuang, H. (2016). Puerarin, isolated from Pueraria lobata (Willd.), protects against diabetic nephropathy by attenuating oxidative stress. Gene, 591(2), 411–416. [DOI:10.1016/j.gene.2016.06.032] [PMID]

Zhang, B., Li, W., & Dong, M. (2017). Flavonoids of Kudzu root fermented by Eurotium cristatum protected rat pheochromocytoma line 12 (PC12) cells against H2O2-induced apoptosis. International Journal of Molecular Sciences, 18(12), 2754. [PMID] [PMCID]

Zhao, L., Wang, Y., Liu, J., Wang, K., Guo, X., & Ji, B., et al. (2016) Protective effects of genistein and puerarin against chronic alcohol-induced liver injury in mice via antioxidant, anti-inflammatory, and anti-apoptotic mechanisms. Journal of Agricultural and Food Chemistry, 64(38), 7291–7297. [DOI:10.1021/acs.jafc.6b02907] [PMID]

Zhou, Y. X., Zhang, H., & Peng, C. (2014). Puerarin: A review of pharmacological effects. Phytotherapy Research : PTR, 28(7), 961–975.[DOI:10.1002/ptr.5083] [PMID]