Chemical constituents, clinical efficacy and molecular mechanisms of the ethanol extract of Abelmoschus manihot flowers in treatment of kidney diseases

Abelmoschus manihot, also called as “Huangkui” in Chinese, is an annual flowering herb plant in the family of Malvaceae. As a traditional Chinese medicine, the ethanol extract of the flower in Abelmoschus manihot is made as Huangkui capsule and has been used for medication of the patients with kidney diseases. Its efficacy in clinical symptoms is mainly improving renal function and reducing proteinuria among the patients with chronic kidney disease, diabetic kidney disease or IgA nephropathy. The possible mechanism of Huangkui capsule treatment in kidney diseases may include reducing inflammation and anti‐oxidative stress, improving immune response, protecting renal tubular epithelial cells, ameliorating podocyte apoptosis, glomerulosclerosis and mesangial proliferation, as well as inhibiting renal fibrosis. In this review, we first described chemical constituents and pharmacokinetic characteristics in ethanol extract of the flower of Abelmoschus manihot. We then summarized the clinical and epidemiological relevancies of kidney diseases particularly in the mainland of China and discussed the possible molecular mechanisms of Huangkui capsule in the treatment of kidney diseases. Finally, we prospected further research on cellular and molecular mechanisms and application of this Chinese natural medicine in kidney diseases.

Abelmoschus manihot, also called as "Huangkui" in Chinese, is an annual flowering herb plant in the family of Malvaceae. As a traditional Chinese medicine, the ethanol extract of the flower in Abelmoschus manihot is made as Huangkui capsule and has been used for medication of the patients with kidney diseases. Its efficacy in clinical symptoms is mainly improving renal function and reducing proteinuria among the patients with chronic kidney disease, diabetic kidney disease or IgA nephropathy. The possible mechanism of Huangkui capsule treatment in kidney diseases may include reducing inflammation and anti-oxidative stress, improving immune response, protecting renal tubular epithelial cells, ameliorating podocyte apoptosis, glomerulosclerosis and mesangial proliferation, as well as inhibiting renal fibrosis. In this review, we first described chemical constituents and pharmacokinetic characteristics in ethanol extract of the flower of Abelmoschus manihot. We then summarized the clinical and epidemiological relevancies of kidney diseases particularly in the mainland of China and discussed the possible molecular mechanisms of Huangkui capsule in the treatment of kidney diseases.
Finally, we prospected further research on cellular and molecular mechanisms and application of this Chinese natural medicine in kidney diseases.

K E Y W O R D S
Abelmoschus manihot, chronic kidney disease, diabetic kidney disease, Huangkui capsule, IgA nephropathy 1 | INTRODUCTION As a component and basis of life for producing urine, kidney is an important organ to complete metabolism in the body and to maintain the stability of internal environment. Structurally, the kidney consists of two peashaped organs and perform many crucial functions, including maintaining overall fluid balance, regulating and filtering minerals from blood, filtering waste materials from blood, medications and toxic substances, and creating hormones for production of red blood cells, promotion of bone health and regulation of blood pressures. Kidney diseases, or renal diseases, also known as nephropathy, are damages in kidneys, including primary kidney and secondary to other visceral lesions. There are several types of diseases in kidneys. IgA nephropathy (IgAN) is the most common nephritis in the glomerulus of kidneys (Rodrigues, Haas, & Reich, 2017;Trimarchi et al., 2017;Wyatt & Julian, 2013). Chronic kidney disease (CKD) causes the gradual loss of kidney function over time as shown by glomerular filtration rate (GFR) of less than 60 ml/min per 1.73 m 2 (Chen 2010;Webster, Nagler, Morton, & Masson, 2017). Diabetic kidney disease (DKD, previously called as diabetic nephropathy, DN) occurred in the patients with diabetes mellitus (Thomas et al., 2015). Acute kidney disease (AKD), also known as acute kidney injury, is marked by the sudden reduction in kidney function within several hours or days (Ferenbach & Bonventre, 2016). Although the etiology, pathology, course and pathogenesis of kidney diseases mentioned above are different, renal fibrosis and gradual loss of nephron are the common pathological links, all these kidney diseases can cause a loss of renal function and may consequently result in kidney failure. The loss of function by 85-90% of normal capacity in kidneys is termed as the end-stage of kidney disease (ESRD). The patients with ESRD need to be treated with dialysis or a kidney transplant to maintain alive (Kanda et al., 2017;Sumida & Kovesdy, 2017). Kidney diseases threaten human health seriously, also brings heavy burden to the society and family. Thereby, pharmacological intervention for the medicines prescribed to the patients with kidney diseases is of importance, but effective therapy remains limited.
Traditional Chinese medicine (TCM) has a history of over 2,500 years. The herbs associated with TCM are perceived to be a cost-efficient alternative. As we all known, Ms. Youyou Tu received the Nobel Prize in physiology or medicine in 2015 for her discoveries concerning a novel therapy against Malaria (McPhee & Kenneth, 2016;Zheng, Li, Peng, & Wang, 2020). Actually, she turned to Chinese medical texts mainly from the handbook of prescriptions for emergencies to find a TCM for Malaria, ultimately extracting a compound of Artemisinin, which is a single substance from Artemisia annua L. (Zheng et al., 2020). The name of this ancient medical book is Zhou-Hou-Bei-Ji-Fang in Chinese, and the author is Mr. Hong Ge, an outstanding medical scientist and a senior Taoist doctor in the Eastern Jin Dynasty (317-420 AD), China. From ancient to modern times, some other herbs in China have been found to be effective for treatment of edema, hematuria and symptoms of kidney diseases (Zhong, Menon, Deng, Chen, & He, 2015). One of them is the flowering plant of Abelmoschus manihot (Linn.) Medic. (Malvaceae) as seen in Figure 1. It is also known as the sunset muskmallow, sunset hibiscus, or Hibiscus manihot in the family of Malvaceae. This plant was formerly considered a species of Hibiscus, but is now classified in the genus Abelmoschus. Its flowers are yellow in color and look as sunflowers. Therefore, Abelmoschus manihot is called as "Huang-Kui" in Chinese, while "Huang" means yellow and "Kui" is a sunflower. Similar to the discovery story of Artemisinin, the medical application of Abelmoschus manihot was also first recorded in  13 chemical constituents (Lai, Zhao, & Liang, 2006). Following studies have confirmed that the major pharmacologically bioactive constituents in the flower of Abelmoschus manihot are seven flavonoids, including Rutin, Hyperoside, Hibifolin, Isoquercetin, Myricetin, Quercetin and Quercetin-3-O-robinobioside (Guo et al., 2015;Lai, Liang, Zhao, & Wang, 2009). These chemical constituents, their molecular formula and molecular weight are represented in Table 1  hyperoside was more than 5,000 mg/kg in BALB/c mice without acute toxicity and genetic toxicity (Ai, Liu, Hua, Huang, & Wang, 2013 (Yan, Ai, Zhang, Xu, & Huang, 2015). In general speaking, the metabolites of flavonoids in the intestinal tract were delivered to liver through the portal vein for further metabolism, while the flavonoids could be metabolized with hydroxylation, acetylation, methylation, hydrolysis and oxidation loss (Nijveldt et al., 2001). However, our knowledge regarding the interrelationship between pharmacokinetics and pharmacodynamics (PK-PD) is still limited. It is necessary to establish and improve PK-PD model for better understanding the relationship between these flavonoids as active ingredients and their pharmacological effects.

| KIDNEY DISEASES
As described briefly in the introduction, CKD, DKD and IgAN are the main forms of kidney diseases. Other kidney problems include AKI, kidney cysts, kidney stones, and kidney infections are also included.
CKD is defined by persistent urine abnormalities, structural abnormalities or impaired excretory renal function suggestive of a loss of functional nephrons. Clinically, CKD refers to all five stages of kidney damage, from very mild damage in Stage 1 to complete kidney failure in Stage 5 (Webster et al., 2017). In the past decades, CKD has been a global public health issue and affects more than 10% population worldwide. Therefore, the burden of CKD is not only restricted to the requirement of renal replacement therapy for ESRD, but also associated with cardiovascular events and mortality.
Diabetes is a group of metabolic diseases characterized by hyperglycemia and has become approaching epidemic proportions globally. Hyperglycemia is caused by defects in insulin secretion or its biological effects, or both. Diabetes is mainly divided into three categories: Type 1 diabetes mellitus (T1DM), which is an autoimmune disease with destruction of pancreatic islet β cells; Type 2 diabetes mellitus (T2DM), which is closely related to insulin resistance and relative insulin deficiency; and gestational diabetes mellitus (GDM), which occurs during pregnancy, and disappears  (Gu, 2019). Clinical observation has implicated that DKD is the main cause of CKD worldwide and the leading cause of ESRD. The presence of CKD is also the single strongest predictor of mortality for subjects with diabetes (Marshall, 2014).
IgAN is also known as Berger's disease and occurs when IgA deposits build up in the kidneys, causing inflammation that damages kidney tissues. IgA is an antibody and this protein is made by the immune system to protect the body from foreign substances such as bacteria or viruses. IgA nephropathy affects the kidneys by attacking the glomeruli. The glomeruli are sets of looping blood vessels in nephrons -the tiny working units of the kidneys that filter wastes and remove extra fluid from the blood. The buildup of IgA deposits inflames and damages the glomeruli, causing the kidneys to leak blood and protein into the urine. The damage may lead to scarring of the nephrons that progresses slowly over many years (Rodrigues et al., 2017;Trimarchi et al., 2017;Wyatt & Julian, 2013 in renal tissues were detected by Western blotting. Results demonstrated that as compared with rats in the untreated model group, the treated rats were found to be improved in urine protein, serum albumin, mesangial cell proliferation, extracellular matrix and collagen deposition. Furthermore, the expression of alpha-SMA and collagen type I, TGF-α, TGF-β1 and p-p38MAPK in renal tissues was decreased. This study has implicated that Huangkui capsule has the effects in ameliorating renal inflammatory injury in kidneys by reducing TGF-α, TGF-β1 expression and intervening p38MAPK signaling pathway (Tu et al., 2013;Zhao et al., 2012). Moreover, a recent study has replicated the experiment with ADRN-induced SD rats and ADRN-induced NRK-52E cells, the normal rat kidney epithelial cell line, and suggested that additional effects of Huangkui capsule treatment could be inhibition of ROS-ERK1/2-NLRP3 inflammasomes . in glomerular and tubular structures that was associated with podocyte loss and fibrotic protein accumulation. These changes in these DKD mice were attenuated by treatment with Abelmoschus manihot. In addition, hepatic injury, proinflammatory cytokines, and lipid accumulation were decreased after the treatment. This study implicates that Abelmoschus manihot can increase the expression of proteins by regulating autophagy and mitochondrial dynamics, which potentially prevented from accumulating pathogenic proteins and dysfunctional mitochondria in not only kidney but also liver of DKD mice (Kim et al., 2018).
In addition to the focus on kidney function, Cai HD et al. have attempted to investigate the effects of Abelmoschus manihot in glucose and lipid metabolisms by using the candidant gene approach.
for the effects of Abelmoschus manihot in clinical efficacy .
Most of experimental studies have been done by using the extract of Abelmoschus manihot, in which seven main constituents are included.
It is then valuable to analyze the effects of each chemical compound.
Hyperoside is one of active constituents. Wu et al. have recently exam-ined the effects of this constituent in Ischemia/reperfusion (IR)-induced AKI mice and found that pretreatment of hyperoside could not only attenuate IR-induced AKI, tubular cell apoptosis, and oxidative stress in the kidneys but also inhibited IR-induced mitochondrial fission by suppressing OMA1 mediated proteolysis of optic atrophy 1 (OPA1).
In addition, podocyte injury plays an important role in the occurrence of glomerular insufficiency and proteinuria (Nagata, 2016 As we known that the cases of CKD, DKD and IgAN is increasing, clinical application of Huangkui capsule urgently needs to be broadened and improved. It is then necessary to speed up and improve scientific research to better understand its molecular and cellular mechanisms for the treatment of kidney diseases. However, we are currently facing several challenges. First, the pathogenesis of kidney diseases is very complicated, while CKD and DKD are interlaced, and IgAN is closely related to immunity. The common feature of these kidney diseases is related to inflammation and proteinuria. This may be the breakthrough point for us to further carry out basic scientific research. Furthermore, we need to use the experimental design of hypothesis-free and genomic research techniques to comprehensively analyze and study the effects of Huangkui Capsule in the treatment of kidney diseases (Evans & Davey Smith, 2015). Second, there are seven major chemical constituents in Huangkui capsule, while Artemisinin is a single substance from Artemisia annua L. in Ms. Tu Youyou's discovery (Zheng et al., 2020). TCMs are generally compound prescription drugs. It is still unknown whether they are a single chemical component Huangkui capsule acting alone or in combination with each other to treat kidney disease. Therefore, it is necessary to design experiments for each chemical composition and their different combinations, which inevitably increases the cost and time of the experiment. In order to comprehensively explore the molecular and cellular mechanisms of the treatment of kidney disease by Huangkui capsule, it is necessary to study the interactions and basic regulations of these chemical components in the pathophysiology of kidney diseases. The comprehensive understanding of the mechanism of Huangkui capsule treatment in kidney diseases can expand its clinical application range and enhance its medical value.
Moreover, further investigation of Huangkui capsule treatment in kidney diseases may enable us to accumulate knowledge and experience to further understand the basic regulations of interaction for other CTMs and their production of pharmacodynamics. Third, the theory of "gut-kidney axis" has become a key point to study of interaction of the gastrointestinal tract with kidneys, including immunity inflammation and intestinal bacteria (Chen et al., 2019;Evenepoel, Poesen, & Meijers, 2017). Oral administration of Huangkui capsule into the gastrointestinal tract is the main delivery system of this drug. Our research group has currently designed experiments to investigate the changes of intestinal bacteria and immunity inflammation after oral administration of Huangkui capsule, and data concerning the gut microbiota reprogram and metabolism, the immune and homeostasis resume may provide a novel microbiota-targeting therapy in medication of kidney diseases.

CONFLICT OF INTEREST
Haitao Tang, Haitao Ge, Honglin Yu and Jimei Ma are employed by Suzhong Pharmaceutical Group Co., Ltd. All other authors declare no competing interests.