Ethanol extracts of Rehmannia complex (Di Huang) containing no Corni fructus improve early diabetic nephropathy by combining suppression on the ET-ROS axis with modulate hypoglycemic effect in rats

Abstract

Aim

Liuwei Dihuang (Rehmannia complex, RC) decoction, a classic prescription of Traditional Chinese Medicine (TCM), has been used in treating diabetic nephropathy (DN). Among the 6 crude medicines which contains Corni fructus is recognized as the active fraction for its effectiveness. We aimed to investigate, first, if without Corni fructus a modified RC could be still effective, second, if the ethanol extracts could be better than that of water extract and third, the beneficial effect is mainly stemmed from suppressing the endothelin (ET-1) pathway associated with a moderate hypoglycemic effect.

Methods and materials

Diabetes for 8 weeks was induced by a single dose of streptozotocin (STZ, 65 mg/kg, i.p.) in rats and treated with RC extracts in either 95%, 70% ethanol or water separately during 5–8th week. The efficacy of extracts was compared with aminoguanidine (AMG).

Results

An increase in albumin and creatinine in 24 h urine, blood urea nitrogen (BUN) was found in STZ rats. Oxidative stress was found in renal cortex in association with upregulated plasma ET-1 and mRNA of ET_A, decreased MMP_2,9 (matrix matelloproteinases) and increased hydroxyproline.

Conclusions

The RC without Corni fructus was very effective in alleviating DN and ethanol extracts provided greater effects against water extracts. The efficacy in alleviating DN is attributed to normalizing the activated ET system, oxidative stress and MMP_2,9 in combination with a moderate hypoglycemic activity.

Introduction

Diabetic nephropathy (DN) is the most common cause contributing to end-stage renal disease (ESRD) in the United States (Cooper, 2006). Sustained hyperglycemia destroys function and structure in the kidney, leading to albuminuria which in turn further damage the renal tubular structure (Neuhofer and Pittrow, 2006). Glomerular hyperfiltration, enlargement, mesangial expansion, and intertubular fibrosis can be found in diabetic kidney in association with an increase in the extracellular matrix (ECM) (Ohno et al., 2005). These changes are attributed to an excess of formation of AGEs acting as free radicals in a vicious cycle, thus, more ROS are produced resulting in exacerbation of diabetes. A damage to the endothelium of renal vascular system is a causal factor in inducing diabetic nephropathy (DN) that associates with an activated endothelin-1 (ET-1) pathway and an excess of ROS (Neuhofer and Pittrow, 2006). ET-1 as a paracrine plays an important role in the renal physiology, pathology of DN and ESRD (Sorokin and Kohan, 2003). Accompanying sclerosis of glomeruli and degeneration of tubules, an upregulation of renal ET-1 is found in diabetes (Kohan, 2000, Minchenko et al., 2003). Thus, ROS and ET-1 closely linked in diabetic renal lesions (Chen et al., 2000). In addition, an alteration of MMP (matrix matelloproteinases) 2 and 9 contributes to the pathologies of diabetic kidney even in fetuses from diabetic rats (Van- Huyen et al., 2007) and an imbalance in MMPs and TIMPs is found as early changes in diabetic nephropathy (Han et al., 2006). However, changes in MMP_2,9 with renal ET-1 system have not been clarified yet.

A Traditional Chinese Medicine (TCM) prescription, Liuwei Dihuang decoction (a six-herb prescription), has been applied in treating diabetes, consisting of 6 crude materials: Rehmannia glutinosa Libosch, Di Huang in Chinese, Alisma orientalis (Sam.) Juzep, the root cortex of Paeonia suffruticosa Andr, and the fungus of Poria cocos (Schw.), Corni fructus and Dioscorea opposite (Thunb.). This prescription is used and modified to contain more plant medicines in treating diabetes in China and Japan (Kitagawa et al., 1971, Kiho et al., 1992, Zhang et al., 1996, Yang et al., 2002, Yokozawa et al., 2004, He et al., 2007). Corni fructus is the most attractive and considered as effective fraction for isolating and testing its in relieving DN (Yamabe et al., 2007a, Yamabe et al., 2007b). We reported the effectiveness of total triperpene acids isolated from Corni fructus is effective in relieving diabetic vasculopathy and retinopathy (Su et al., 2007). On the other hand, DN can be relieved with dried Rehmanniae radix (Di Huang) extract (Yokozawa et al., 2004), however, most ingredients of extracts of Rehmanniae radix are polysaccharides and likely water soluble.

We hypothesized that the benefit of Liuwei Dihuang decoction is multifaceted and the effective fraction of a six crude medicines containing prescription may not be limited to one herb (Corni fructus). Thus, we, first, are intended to test a modified Rehmannia complex containing 4 herbal medicines with no Corni fructus in treating diabetic nephropathy is rats.

Considering the traditional process to prepare the decoction in boiling water, we hypothesized further, the most of active ingredients are lipid soluble in ethanol extracts against water one. Second, if more effectiveness is produced by the ethanol extract against water extracts. Thus, third, its effectiveness mainly comes from normalizing abnormality of the ET-1, ROS and MMP_2,9 activity. Considering some of the effects of the Rehmannia complex, beside hypoglycemic activity, may be related to a suppression of the downstream events of AGEs and iNOS (inducible nitric oxide synthase), thus, aminoguanidine (AMG) was chosen as a positive reference medicine in relieving nephropathy in diabetic rats (Wilkinson-Berka et al., 2002).