Neurochem Res (2009) 34:21632169 DOI 10.

1007/s11064-009-0011-z

ORIGINAL PAPER

Targeting BuChE-inammatory Pathway by SK0506 to Manage Type 2 Diabetes and Alzheimer Disease
M. A. Kamal Y. Tan J. P. Seale X. Qu

Accepted: 19 May 2009 / Published online: 7 June 2009 Springer Science+Business Media, LLC 2009

Abstract Type 2 diabetes mellitus (T2DM) and Alzheimers disease (AD) affect a large percent of the population worldwide. Experimental studies have revealed that T2DM and AD share several molecular processes that underlie their respective degenerative pathology. Based on this information, we quantied TNF-a, IL-6 levels, serum glucose, serum triglyceride, hepatic triglyceride, serum AST, serum ALT and butyrylcholinesterase (BuChE) in various rat tissues. HFD was fed to rats resulting in increased body weight, fasting blood glucose, IL-6, TNF-a levels, hepatic triglyceride, serum AST, serum ALT and BuChE. SK0506 treatment signicantly prevented weight gain induced by HFD feeding. SK0506, but not Rosiglitazone, signicantly reduced serum and hepatic triglycerides levels. Treatment with SK0506 also ameliorated elevated levels of both inammatory markers (TNF-a and IL-6) and serum liver enzymes (ALT and AST) signicantly in HFD fed rats. BuChE activity also reduced in skeletal muscle and adipose tissues of rats treated by SK0506. In conclusion, current study has opened new potential avenues towards research for management of T2DM and AD by Chinese herbal extracts, SK0506. Keywords Alzheimers disease Butyrylcholineesterase Chinese herbal medicines IL-6 Type 2 diabetes TNF-a
M. A. Kamal (&) Y. Tan X. Qu Department of Medical & Molecular Biosciences, Faculty of Science, University of Technology Sydney, Broadway 2007, PO Box 123, Sydney, NSW, Australia e-mail: Mohammad.Kamal@uts.edu.au J. P. Seale Department of Pharmacology, University of Sydney, Sydney, NSW, Australia

Abbreviations AChE Acetylcholinesterase tI Activity index ALT Alanine transaminase AD Alzheimers disease AST Aspartate transaminase BuChE Butyrylcholinesterase BuSCh Butyrylthiocholine iodide CHM Chinese herbal medicines HFD High fat diet IL-6 Interleukin-6 Kt Time required for half of Pmax NEFA Nonesteried fatty accid NF-jB Nuclear factor kappa-light-chain-enhancer of activated B cells Pmax Maximum hydrolyzed product Pmin Minimum hydrolyzed product TNF-a Tumour necrosis factora RSG Rosiglitazone SK0506 Chinese herbal extracts T2DM Type 2 diabetes mellitus

Introduction Type 2 diabetes mellitus (T2DM) is a heterogeneous, multi-factorial, polygenic disease characterized by a defect in insulins action (insulin resistance) and secretion (a beta cell secretory defect). Recent evidence has identied T2DM as a risk factor for Alzheimers disease (AD; [1]). AD is a progressive degenerative disorder that causes dementia in the elderly. Behavioral problems are associated with impairment of memory and typied by an increasing state of AD [2, 3]. Alterations in the levels of the

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same inammatory markers (C-reactive protein), tumour necrosis factor-a (TNF-a), interleukin-6 (IL-6) and lipid peroxides are common to T2DM and AD, and consequently suggest that a close association exists between these two frequent progressive diseases. AD and T2DM are related by systemic inammatory conditions that can be treated by normalizing common pathways associated with systemic inammation. Recent studies suggest that an elevation in the plasma and tissue (pancreas and brain) levels of the endogenous protein butyrylcholinesterase (BuChE) provides a common link between the two diseases and opens new avenues for potential treatment [4]. BuChE and acetylcholinesterase (AChE) related proteins were found to be common to both AD and diabetes; they may play an etiological role via inuencing insulin resistance and lipid metabolism [5]. BuChE, in addition to a role in co-regulating acetylcholine (ACh) levels and cholinergic neurotransmission, has noncholinergic functions related to differentiation, proliferation and apoptosis [6]. Likewise, ACh, although the primary cholinergic neurotransmitter via its action on nicotinic and muscarinic receptor subtypes, additionally modulates interactions between the nervous and immune systems. Specically, the cholinergic anti-inammatory pathway signals through the efferent vagus nerve are mediated primarily by nicotinic ACh receptors on tissue macrophages [7]. This ACh mediated cholinergic antiinammatory pathway acts by inhibiting the production of TNF-a, IL-1, macrophage migration inhibitory factor and high-mobility group B1 protein and suppresses the activation of NF-jB expression [7]. ACh, furthermore, has a regulatory role on dopamine, serotonin and other neuropeptides, to provide a close interaction between immune response and neurotransmission. Traditional Chinese Herbal Medicine (CHM) has been used in China and other parts of East Asia for thousands of years. In western countries, there is now a signicant increase in the number of people who regularly use CHM for maintaining health and treating chronic diseases (including diabetes), as these natural medicines are considered to have many benecial effects and less side/toxic reactions. These medicines have been researched to possess potential treatments or management of various human illnesses to some extent in chronic illnesses such as migraine, menopause, chronic fatigue syndrome and cancer. Dan Shen, the dried root of Salvia miltiorrhiza, is known to improve blood circulation and eliminate blood stasis in CHM. There are various in vitro and in vivo studies that have suggested that Dan Shen improves microcirculation, dilates the coronary arteries, possesses anti-arthrosclerotic properties, and prevents myocardial necrosis under ischemic states [8]. It is also a Chinese herb commonly included in prescriptions to treat AD [9]. In a recent study using high

fat diet (HFD) fed rats, we have demonstrated that a Chinese herbal extract, containing S. miltiorrhiza and Gardenia jasminoides (coded SK0506), reduced central obesity, lowered triglycerides and free fatty acid levels in the blood stream and prevented TG deposition in liver and skeletal muscles [10]. Based on these ndings, we quantied TNF-a, IL-6 levels in adipose tissues and BuChE in rat skeletal muscles, liver and adipose samples (chow-fed animals, high fat diet-fed and treated by RSG or SK0506).

Experimental Procedures Materials Rat Adipocyte Lincoplex Kit was used for estimation of IL-6 and TNF-a (Linco Research Inc. NY, USA). Butyrylthiocholine iodide (BuSCh, was used as a substrate), 5,50 -dithiobis(2-nitrobenzoic acid) (a coloring reagent), and 1,5-bis(4-allyldimethyl-ammoniumphenyl) pentan-3one dibromide (BW284C51 as an irreversible potent inhibitor of AChE) were obtained from Sigma (St. Louis, MO, USA). Chinese herbal extracts of Salvia miltiorrhiza and Gardenia jasminoides, coded as SK0506, were provided by Kanion Pharmaceuticals (Jiansu, China). The herbal extracts of Salvia miltiorrhiza and Gardenia jasminoides were not in a high concentrated pharmaceutical form (equal proportions of each herbal extract was mixed together to form SK0506). Thus, 1 g/kg of body weight of each raw herbal extract (equal to 4 g of natural herb) is acceptable dosage for animal study. We have applied a lower dosage of herbal extract (0.5 g/kg) in a pilot study and this dosage did not bring about signicant therapeutic effects. Animals and Treatment Male Sprague-Dawley (SD) rats at 6 weeks of age were supplied by the Animal Resources Center (Perth, Australia). All experimental procedures were approved by the University of Technology Sydney Animal Ethics Committee, following guidelines issued by the National Health and Medical Research Council of Australia. SD rats were maintained on a 12-h light/dark cycle (lights on 06001800 h) under constant temperature (21C) with ad libitum access to standard chow diet or a high fat diet (HFD) containing 59% total calories (1 cal approximately 4.184 J) from fat for 6 weeks. HFD rats were randomly divided into three groups (n = 8 each group) to receive saline 5 ml/kg b.wt/day, RSG 3 mg/kg and SK0506 (2 g/kg) by oral gavage for 4 weeks. At the end of the treatment, tail vein blood samples were collected from fasting rats for measurements of glucose, triglycerides and nonesteried fatty acid (NEFA). At the end of the experiment, rats were

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sacriced by CO2 inhalation for collection of liver, muscle and adipose tissues for subsequent biochemical assays. Preparation of Tissues Samples Frozen adipose tissue, muscle and liver tissue samples were ground with a pestle and mortar, and then homogenized in a Polytron (Ultra-Turrex T8, ICK Labortechnik, Stufen, Germany) in 100 mg/ml with lysis buffer [20 mmol/l Tris HCl (pH 7.5), 1 mmol/l EDTA, 0.25 mmol/l EGTA, 0.25 mmol/l sucrose, 2 lg/ml leupeptin, and 4 lg/ml each of aprotinin, calpain-I and calpain-II]. The homogenized samples were spun at 400g for 15 min, the supernatant was collected for further assay. Protein concentration of each specimen was determined by the Bradford method (BioRad Laboratories, CA, USA) to ensure equal amount of protein for enzyme activity, IL-6 and TNF-a assays. Assay of Adipose IL-6 and TNF-a A multiplex assay kit (Rat Adipocyte Linoplex kit) manufactured by Linco Research (Millipore, St. Charles, MO, USA) was used for the simultaneous quantication of IL-6 and TNF-a, according to the manufacturers instructions. This particular assay kit was selected because of its high degree of sensitivity, specicity, inter- and intra-assay precision. 25 ll samples of adipose supernatant were analyzed using a multiplex reader. Assay of ALT, AST and Estimation of Triglyceride The serum alanine transaminase (ALT) concentration and serum asparate transaminase (AST) concentration were measured using commercialized kits from from Dialab (Wien, Austria). The hepatic lipids were extracted from approximately 50 mg of liver tissue with chloroform/ methanol (2:1), and the triglyceride concentrations were

determined with the same enzymatic kit as used in the serum analysis. Assay of Butyrylcholinesterase Quantication of BuChE was assessed in the liver, cytosol as well as membrane fractions of quadriceps skeletal muscle, muscle homogenate supernatant and adipose tissues extracts by the Ellman technique [11] as applied in our earlier research work [1215]. The assay mixture contained BuSCh, 0.25 mM 5,50 -dithiobis (2-nitrobenzoic acid), 0.05 mM BW284C51 (a classic selective, potent and irreversible inhibitor of AChE) and 50 mM sodium phosphate buffer, pH 7.2. The rate of substrate hydrolysis by BuChE was determined by measuring the absorbance of the reaction-product at 405 nm k. Statistical Analysis and Graphic All values are expressed as mean SE. Comparisons across the four groups were done using one-way ANOVA followed by post hoc analysis to determine signicant differences between two groups using Prism (version 4, GraphPad, San Diego, CA). Graph was plotted using same software and values of the correlation coefcients were obtained by nonlinear regression analysis computed within this package.

Results Effects of SK0506 on Body Weight, Glucose and Lipid Metabolism Table 1 shows metabolic parameters in all groups after 4 weeks of treatment. HFD feeding signicantly increased body weight 21.14% as compared with chow-fed rats

Table 1 Effects of daily oral treatment with RSG and SK0506 on metabolic parameters and liver enzymes of HFD rats Parameter Body weight (g) S. glucose (mM) S. TG (mM) S. NEFA (mM) H. TG (lmol g-1) S. AST, U/l S. ALT, U/l Chow 369 21 4.99 0.30 0.64 0.13 1.05 0.43 7.09 1.24 15.12 0.67 10.12 0.67 HFD 447 20* 6.50 0.10** 0.53 0.18 1.09 0.45 15.56 0.11** 40.09 0.45** 30.00 0.45** RSG 501 24 6.00 0.30 0.45 0.11 0.77 0.29 14.17 0.55 18.77 0.29 45.00 0.29

SK0506 389 19& 6.20 0.30 0.35 0.05& 1.01 0.36 12.14 1.09 17.01 0.36 9.12 0.36&

Data represent as mean SE from rats fed by chow and by HFD and treated with vehicle, RSG and SK0506 respectively. Metabolic parameters were obtained from 6 to 8 rats (n = 68/group) after 4 weeks treatment. * P \ 0.05 and ** P \ 0.01 HFD versus chow feeding group. P \ 0.05 and & P \ 0.01 RSG/SK0506 versus HFD control S serum, H hepatic, TG triglyceride, NEFA nonesteried fatty accid, AST aspartate transaminase, ALT alanine transaminase

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(P \ 0.05). SK0506 treatment signicant prevented weight gain induced by HFD feeding (P \ 0.01, SK0506 treatment group vs. HFD control). On the other hand, RSG treatment increased 12.1 % of body weight compared with HFD control. HFD feeding signicantly increased fasting blood glucose level by 30.26% (P \ 0.01 HFD rats vs. chow control). Serum triglyceride and NEFA levels were not increased by HFD feeding but SK0506 treatment lowered serum triglyceride level by 34% (as compared to HFD) and 45.3% (as compared to control). On the other hand, hepatic triglyceride levels increased by 2.2-fold in HFD feeding rat samples and were reduced 22% by SK0506 treatment (Table 1). Effects of SK0506 on Adipokines Interleukin-6 level in adipose tissue was signicantly higher in HFD rats compared with normal chow fed rats (Fig. 1a). Treatment with the antidiabetic drug (RSG) reduced levels of IL-6 and TNF-a but a signicant reduction had not been achieved, while SK0506 reduced the levels of both inammatory markers signicantly in HFD fed rats (Fig. 1).
Fig. 1 TNF-a and IL-6 levels in adipose tissues of HFD rats with treatments of RSG and SK0506. * P \ 0.05 and *** P \ 0.001 SK0506 versus HFD control; & P \ 0.01 HFD versus chow control

Effects of SK0506 on AST and ALT The levels of liver enzymes such as AST and ALT signicantly increased 2.7- and 3-fold in HFD feeding rats as compared to chow fed rats. SK0506 treatment reversed HFD-induced the elevation of serum AST and ALT by 57.6 and 69.9%, respectively (Table 1), while serum ALT level was signicantly elevated in rats with RSG treatment. Effect of SK0506 on Butyrylcholinesterase Butyrylcholinesterase measured in rat skeletal muscle membrane, cytosol, muscle, liver and adipose samples. It was found that % BuChE activity level was not signicantly higher in HFD-fed membrane and cytosol samples, while it was signicantly higher in HFD-fed rat skeletal muscle sample (Fig. 2). The percentage activity level of BuChE was reduced signicantly in animals treated by RSG in membrane samples only, while it was reduced in all three samples tested by SK0506. Liver sample showed signicant increase in BuChE activity in HFD-fed rat as compared to chow-fed animals, which was not signicantly reduced in animals treated by RSG or SK0506. BuChE type activity index (tI)

Fig. 2 a, b Percentage activity of BuChE in various samples of rat muscles. a membrane sample; One tailed unpaired nonparametric T-test of HFD versus RSG: P \ 0.05 (P value 0.017 (t 2.3; df 14); HFD versus the SK0506 treatment alone and found P \ 0.0005 (P value 0.0002

(t 4.5; df 14); b Cytosol; HFD versus the SK0506 treatment alone and found P \ 0.0005. c BuChE activity in skeletal muscles of rats fed by chow and HFD, treated by RSG or SK0506, * P \ 0.05 HDF versus chow controls; or SK0506 treatment versus HFD control

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Neurochem Res (2009) 34:21632169 Table 2 BuChE type activity index (tI) in rat liver samples Sample Chow-fed HFD-fed RSG SK0506 Pmax 46.53 4.08 96.45 5.98 122.4 9.50 157.3 19.47 Kt 8.801 1.68 5.647 0.926 10.20 1.63 15.04 3.29 tI 5.29 17.08 12.00 10.46

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tI, activity index; Pmax, maximum hydrolyzed product by BuChE; Kt, time required for half of Pmax; (tI = Pmax/Kt)

in rat liver samples is presented in Table 2. The BuChE type activity was found in adipose samples over a very long, unusual incubation period (Fig. 3a), while slope was determined on the basis of the linear region of the curve as presented in Fig. 3b. The BuChE type activity was found in chow, HFD, RSG and SK0506 group as 0.072 0.001, 0.13 0.009, 0.058 0.0009 and 0.032 0.001 U/h, respectively. The value of minimum product of BuChE hydrolysis (Pmin) was found as 0.12 0.034, 0.44 0.29, 0.11 0.029 and 0.065 0.046 for Chow, HFD, RSG and SK0506 group adipose samples, respectively.

Discussion In this study, we have shown that HFD increased the expression of inammatory markers (TNF-a and IL-6) in white adipose tissue. Treatment with Chinese herbal extracts SK0506 prevented HFD-feeding-accelerated weight gain. In addition, SK0506 signicantly inhibited TNF-a and IL-6 expression in adipose tissue. These ndings suggest that specic components of SK0506 may have therapeutic potential for T2DM associated with lower grade inammation. To nd out whether treatment with
Fig. 3 BuChE type activity in rat adipose tissues (Abbreviations: C chow, H HFD, R RSG, S SK0506). a Nonlinear regression analysis of product of BuChE hydrolysis versus whole assay period. b Linear regression analysis of product of BuChE hydrolysis versus limited assay period

SK0506 causes toxicity, an assay of two hepatic enzymes was performed which can be used to assess liver toxicity induced by the treatment. HFD feeding signicantly increased triglyceride accumulation in the liver and raised serum AST and ALT levels compared to chow fed rats whilst SK0506 reduced hepatic triglyceride content and ameliorated elevated serum AST and ALT levels. RSG, an insulin sensitizer by stimulation of the peroxisome proliferator-activated receptor gamma (PPARc), did not have an effect on hepatic triglyceride accumulation but increased ALT levels (Table 1). This result indicated SK0506 may have potential therapeutic effects on dietary factor induced fatty liver disease. Recent studies have shown elevation in the plasma and tissues (pancreas and brain) levels of BuChE, an enzyme known to efciently hydrolyze butyrylcholine as well as acetylcholine (ACh). ACh has roles in neurotransmission and inammation, the latter being a common occurrence in diabetes and AD. Thus, elevated BuChE may augment systemic inammatory reactions in tissues. In addition to the contemporary widely accepted inammation-induced insulin resistance, low-grade inammation also accelerates amyloidogenic processes in brain and pancreatic b-cells. The results of Bradamante et al. [16] also showed that increases in BuChE activity might be the rst sign of altered triglyceride and lipoprotein metabolism in rats. Elevated level of BuChE occurs in diabetes and AD due to low-grade systemic inammation, consequent to dysregulation of the described pathway. BuChE is able to efciently catalyze the hydrolysis of numerous endogenous substances including choline esters [1720]. Although it is predominantly associated with glial cells in human brain, it is additionally expressed in neuronal somata and their proximal dendrites in brain areas affected by AD such as in the amygdala, the hippocampus and the thalamus [21].

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Recent reviews of the anti-inammatory properties of AChE inhibitors administered in AD also support the interest in our current study i.e. targeting BuChE as well as inammatory pathways with SK0506 to manage T2DM and AD [22]. Craft had reported that raising plasma insulin to levels that characterize patients with insulin resistance invokes synchronous increase in levels of b-amyloid and inammatory agents and consequently impairs the memory and induces AD pathology [23]. Some drugs utilized in T2DM reduce cognitive impairment associated with AD. Therefore a link between insulin resistance, neurodegeneration particularly AD and the possible therapeutic targets for preventing insulin-resistance disorders was analyzed by Neumann et al. [24]. On the other hand, there are references to already published reports regarding the efciency of SK0506. According to one recent report, salvia miltiorrhiza had showed the potential to treat severe acute pancreatitis by reducing the serum levels of IL-6, IL-8, and TNF-a [25]. One component of salvia miltiorrrhiza root is cryptotanshinone a lipophilic compound that inhibits NF-kB translocation, expression of pro-inammatory cytokines (TNF-a, IL-1b, IL-6), neutrophil inltration and MPO activity in ischemic myocardial tissues as well as signicantly reduced plasma levels of TNF-a and IL-1b [26]. A second component of SK0506, gardenia jasminoides, attenuates acute pancreatitis as well as pancreatitis-associated lung injury in mice as reected by reductions in pancreatic edema, neutrophil inltration, serum amylase, lipase, serum cytokine levels and mRNA expression of multiple inammatory mediators [27]. Therefore, therapeutic strategies focused on preventing or correcting insulin abnormalities may be of an advantage in age-related memory impairments such as AD. These studies suggest further investigation to discover whether intervention with CHM in metabolic syndrome can also prevent the pathological process of AD, such as amyloid deposition in the brain of AD patients. Lower levels of BuChE expression in rat skeletal muscle membrane, cytosol, muscle, and adipose samples in SK0506 treatment as compared to HFD rats is related to the other ndings in this study such as reduction of adipose IL6/TNF-a levels, body weight, serum glucose, serum triglyceride, hepatic triglyceride, serum AST and serum ALT. During 5 weeks treatment we have observed that SK0506 treated rats had similar amount of food intake and were more active as compared to saline treated HFD rats. We have measured levels of liver enzymes (AST and ALT), which can be used to assess the liver toxicity induced by the treatment. In the fact, the SK0506 prevented fatty liver and ameliorated elevated serum AST and ALT levels induced by HFD feeding (Table 1). Ultimately, these ndings reected the positive therapeutic potential of SK0506 at reversing elevated inammatory and enzymatic

Scheme 1 Scheme showing the potential mode of action of BuChEI and anti-TNF-a with respect to relationship between BuChE and inammatory cytokines involvement in AD and T2DM as well as possible dual nature of action of SK0506 in this study (abbreviations: MIF macrophage migration inhibitory factor, HMGB1 high-mobility group B1 protein)

levels. Based upon our ndings and previous reports, we propose to inhibit the destructive cycle through restoring ACh level by selectively inhibiting BuChE along with TNF-a and IL-6 (Scheme 1). This scheme shows the relationship between high levels of BuChE on inammatory cytokines in AD and T2DM. It also indicates the point of inhibition of BuChE, TNF-a and IL-6 in reducing inammatory circumstances in the presence of SK0506. Better results can be achieved through precise development, characterization and optimal pharmacological use of potent and selective BuChE inhibitors in cellular and animal models of AD and T2DM. In this way, inhibition of BuChE may be of interest for controlling T2DM via enhancing ACh levels and thereby reducing inammatory markers (such as TNF-a and IL-6) that cause AD and T2DM. In conclusion, these ndings suggest that specic compounds of Chinese herbs in SK0506 may have either a direct or indirect inuence on elevated BuChE levels, as well as rat cytokine markers, particularly IL-6 and TNF-a. Its inhibiting potential can be increased either by increasing dosage or modifying its composition formula.
Acknowledgments This study was supported by Australian Research Council Linkage Project Grant. The authors thank Kanion Pharmaceutical Co., for providing Chinese herbal extracts. Authors are grateful to Dr Nigel H Greig (Drug Design & Development

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Neurochem Res (2009) 34:21632169 Section, Laboratory of Neurosciences, National Institute on Aging, National Institutes of Health, USA) for his generosity in providing chemicals for the assay of BuChE and observations on this manuscript to improve its scientic quality. Moreover, MAK is extremely grateful to Dr Greig for providing motivation and inspiration to initiate his research career on T2DMAD project simultaneously.

2169 14. Kamal MA, Klein P, Yu Q-S, Holloway HW, Tweedie D, Greig NH (2008) Kinetics of human serum butyrylcholinesterase inhibition by a novel experimental Alzheimer therapeutic, dihydrobenzodioxepine cymserine. Neurochem Res 33(5):745753. doi:10.1007/s11064-007-9490-y 15. Kamal MA, Qu X, Yu Q-s, Tweedie D, Holloway HW, Li Y, Tan Y, Greig NH (2008) Tetrahydrofurobenzofuran cymserine, a potent butyrylcholinesterase inhibitor and experimental Alzheimer drug candidate, enzyme kinetic analysis. J Neural Transm 115(6):889898. doi:10.1007/s00702-008-0022-y Z, Zrinski R, Konjevoda P, Reiner Z (2006) 16. Bradamante V, Krnic Changes in butyrylcholinesterase activity and serum lipids after oxprenolol and glibenclamide treatments in non-diabetic rats. Arzneimittelforschung 56(2):6469 17. Silver A (1974) The biology of cholinesterases. Elsevier, Amsterdam 18. Soreq H, Zakut H (1993) Human cholinesterases and anticholinesterases. Academic Press, New York 19. Darvesh S, Hopkins DA (2003) Differential distribution of butyrylcholinesterase and acetylcholinesterase in the human thalamus. J Comp Neurol 463:2543. doi:10.1002/cne.10751 20. Giacobini E (2003) Butyrlcholinesterase: Its function and inhibitors. Martin Dunitz, London and New York 21. Darvesh S, Grantham DL, Hopkins DA (1998) Butyrylcholinesterase in normal human amygdala and hippocampal formation. J Comp Neurol 393:374390. doi:10.1002/(SICI)1096-9861 (19980413)393:3\374::AID-CNE8[3.0.CO;2-Z 22. Kamal MA, Greig NH, Reale M (2009) Anti-Inammatory properties of acetylcholinesterase inhibitors administered in Alzheimers disease. Anti-Inamm Anti-Allergy Agents Med Chem 8(1):85100. doi:10.2174/187152309787580810 23. Craft S (2006) Insulin resistance syndrome and Alzheimer disease: pathophysiologic mechanisms and therapeutic implications. Alzheimer Dis Assoc Disord 20(4):298301. doi:10.1097/01. wad.0000213866.86934.7e as G, Reyes P, Mac24. Neumann KF, Rojo L, Navarrete LP, Far cioni RB (2008) Insulin resistance and Alzheimers disease: molecular links & clinical implications. Curr Alzh Res 5(5):438 447. doi:10.2174/156720508785908919 25. Peng GL, Zhang XY (2007) Effects of Salvia miltiorrhiza on serum levels of inammatory cytokines in patients with severe acute pancreatitis. Zhong Xi Yi Jie He Xue Bao 5(1):2831. doi: 10.3736/jcim20070106 26. Jin YC, Kim CW, Kim YM, Nizamutdinova IT, Ha YM, Kim HJ, Seo HG, Son KH, Jeon SJ, Kang SS, Kim YS, Kam SC, Lee JH, Chang KC (2009) Cryptotanshinone, a lipophilic compound of Salvia miltiorrriza root, inhibits TNF-alpha-induced expression of adhesion molecules in HUVEC and attenuates rat myocardial ischemia/reperfusion injury in vivo. Eur J Pharmacol (in press) 27. Jung WS, Chae YS, Kim DY, Seo SW, Park HJ, Bae GS, Kim TH, Oh HJ, Yun KJ, Park RK, Kim JS, Kim EC, Hwang SY, Park SJ, Song HJ (2008) Gardenia jasminoides protects against cerulein-induced acute pancreatitis. World J Gastroenterol 14(40): 61886194. doi:10.3748/wjg.14.6188

References
1. Leibson CL (1997) Risk of dementia among persons with diabetes mellitus: a population-based cohort study. Am J Epidemiol 145:301308 2. Cummings JL (2004) Alzheimers disease. N Engl J Med 351:56 67. doi:10.1056/NEJMra040223 3. Farlow MR, Cummings JL (2007) Effective pharmacologic management of Alzheimers disease. Am J Med 120:388397. doi:10.1016/j.amjmed.2006.08.036 4. Rao AA, Sridhar GR, Das UN (2007) Elevated butyrylcholinesterase and acetylcholinesterase may predict the development of type 2 diabetes mellitus and Alzheimers disease. Med Hypotheses 69:12721276 5. Sridhar GR, Thota H, Allam AR, Babu CS, Prasad AS, Divakar Ch (2006) Alzheimers disease and type 2 diabetes mellitus: the cholinesterase connection? Lipids Health Dis 5:28 6. Darvesh S, Hopkins DA, Geula C (2003) Neurobiology of butyrylcholinesterase. Nat Rev Neurosci 4:131138. doi:10.1038/ nrn1035 7. Borovikova LV, Ivanova S, Zhang M, Yang H, Botchkina GI, Watkins LR et al (2000) Vagus nerve stimulation attenuates the systemic inammatory response to endotoxin. Nature 405:458 462. doi:10.1038/35013070 8. Zhou L, Zuo Z, Chow MS (2005) Danshen: an overview of its chemistry, pharmacology, pharmacokinetics, and clinical use. J Clin Pharmacol 45:13451359. doi:10.1177/0091270005282630 9. Yu XY, Lin SG, Chen X, Zhou ZW, Liang J, Duan W, Chowbay B, Wen JY, Chan E, Cao J, Li CG, Zhou SF (2007) Transport of cryptotanshinone, a major active triterpenoid in Salvia miltiorrhiza Bunge widely used in the treatment of stroke and Alzheimers disease, across the bloodbrain barrier. Curr Drug Metab 8:365378. doi:10.2174/138920007780655441 10. Tan Y, Seale PJ, Wang Z-Z, Qu X (2007) Effects of Chinese herbal extracts on metabolic syndrome in highest fat fed rats. Diabetes 56:A690 11. Ellman GL, Courtney KD Jr, Andres V, Featherstone RM (1961) A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol 7:8895. doi:10.1016/00062952(61)90145-9 12. Kamal MA, Al-Jafari AA, Yu Q-S, Greig NH (2006) Kinetic analysis of the inhibition of human butyrylcholinesterase with cymserine. Biochim Biophys Acta 1760:200206 13. Kamal MA, Yu Q-S, Holloway HW, Tweedie D, Klein P, Greig NH (2006) Kinetics of human serum butyrylcholinesterase and its inhibition by a novel experimental Alzheimer therapeutic, bisnorcymserine. J Alzheimers Dis 10:4351

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