WO2006105407A1 - Medical uses of shilianhua - Google Patents

Abstract

Shilianhua (SLH) extract has been discovered to display bioactivity in the regulation of fatty acid and glucose metabolism, and in inhibition of an inflammatory response. In addition, an active fraction of SLH extract has been characterized. An active SLH extract protected against insulin resistance through multiple mechanisms, including inhibition of IKK/NF-κB, stimulation of adiponectin secretion, promotion of fatty acid oxidation, and activation of p38. SLH can be used in the prevention and treatment of diseases in which insulin resistance plays a major role, which includes hyperglycemia, cardiovascular diseases (such as hypertension, heart disease), stroke, renal failure, blindness, and non-traumatic limb amputation. SLH can be used to treat obesity and hyperlipidemia-related diseases by promotion of fatty acid oxidation and energy expenditure. SLH extracts may be used for prevention and treatment of inflammation and oxdative stress, and other diseases in which NF-κB plays a major role, such as arthritis, aging and cancer.

Description

MEDICAL USES OF SHILIANHUA

[0001] The benefit of the filing date of provisional U.S. application Serial

Number 60/666,863, filed 31 March 2005, is claimed under 35 U.S.C. § 119(e).

[0002] The development of this invention was partially funded by the

Government under a grant from the National Institutes of Health, Grant No. 1-P50- AT002776-01. The Government has certain rights in this invention.

TECHNICAL FIELD

[0003] This technology pertains to a method to prevent or treat insulin resistance and diseases associated with insulin resistance, to enhance glucose metabolism, and to prevent or inhibit inflammation by administering a therapeutically effective amount of an active Shilianhua extract.

BACKGROUND ART

[0004] Shilianhua ("SLH", Sinocrassula indica Berge), is a shrub that grows in the southwestern part of China including Yunnan, Guangxi, and Guizhou Provinces. The plant typically blooms in June through August. Consumption of SLH in Pama County of Guangxi Province in China is associated with longevity in the local population. The crude extract of SLH is one of the popular botanical products for the control of blood glucose in the U.S. market. U.S. Patent No. 5,911,993 describes the use of SLH as a hypoglycemic agent and indicates that SLH is not toxic at a dose of 1200 mg/kg in mice or rats.

[0005] Obesity affects the lives of about 30%-50% adults in the U.S. (1; 2).

Type 2 diabetes is one of the major obesity-associated morbidities with a prevalence of 4.0% in the U.S. population in the year 2000 (3). Insulin resistance is the pathogenic hallmark of type 2 diabetes. Insulin resistance is defined as a defect in the body's capacity of removing glucose from the blood despite the presence of normal or even elevated levels of insulin (4). Insulin resistance has many adverse effects on health, including increasing the risk of cardiovascular disease, stroke, renal failure, blindness, and non-traumatic limb amputation.

[0006] More and more diabetic patients use dietary supplements to replace or facilitate the conventional treatment of insulin resistance and hyperglycemia. Among these dietary supplements, botanical extracts are a popular component. It is believed that certain botanicals are able to enhance the therapeutic activity of and reduce the side effects of synthetic medicines. The advantages of using botanical supplements include safety, economy and no necessity for a doctor's prescription.

[0007] Free fatty acids (FFA) are a risk factor for insulin resistance in obesity

(5; 6). Most insulin resistance results from a defect in the post-receptor signaling. Free fatty acid may lead to the defect by activation of the IKK/NF-κB signaling pathway. IKK (Inhibitor KB Kinase) is a serine kinase that is involved in many biological responses such as immunoglobulin expression, cytokine transcription, anti- apoptosis, and carcinogenesis (7; 8). Using IKK knockout mice, one study suggested that an increase in IKK activity could lead to insulin resistance. Additionally, IKK is known to contribute to insulin resistance through two mechanisms: (a) phosphorylation of IRS-I (Insulin Receptor Substrate 1) (10); (b) inhibition of PPRAγ transactivation (11). Functions of both IRS-I and PPRAγ are required for the maintenance of insulin sensitivity. Free fatty acid and TNF-α can both induce insulin resistance through activation of the IKK/NF-κB signaling pathway (5; 12).

[0008] NF-κB is a transcription factor that was originally found to control expression of the kappa chain of immunoglobulin. NF-κB has been shown to stay in the cytoplasm in association with an inhibitor protein (IKB, inhibitor kappa B) in the absence of activation (13). IKK activates NF-κB by removal of the inhibitor protein through a phosphorylation-initiated and proteosome-dependent protein degradation mechanism (7; 8). After activation, NF-κB moves into the cell nucleus and activates transcription of certain target genes. In the transactivation process, NF-κB recruits coactivators away from PPARγ, which leads to a functional inhibition of PPARγ (11). This inhibition may be translated into a decreased expression of two proteins (CAP and IRS-2) in the insulin signal pathway, since CAP and IRS-2 are the target genes of PPARγ (14). Inhibition of IKK/NF-κB activity leads to protection of insulin sensitivity. Also inhibition of NF-κB is known to prevent inflammation, oxidative stress, rheumatoid arthritis, aging, arthrosclerosis, multiple sclerosis, asthma, inflammatory bowel disease, chronic inflammatory demyelinating polyradiculoneuritis, and cancer. (19-26)

[0009] PPARγ is a nuclear receptor whose ligands are derivatives of free fatty acids. The peroxisome proliferator-activated receptors (PPARs) include PP ARa, PPARγ, and PPARδ (PPARβ) (14; 15). The PPARγ function is required for maintenance of insulin sensitivity, adipocyte differentiation, and triglyceride accumulation in adipocytes (14-16).

[0010] Glycogen synthase kinase 3 (GSK-3) is a component of the phosphatidyl inositol 3 kinase (PI3K) signaling pathway, and is a serine/threonine kinase widely expressed in mammalian tissues. GSK-3 is found in two isoforms, GSK-3α and GSK-3 β. GSK-3 has been shown to have critical roles in glycogen and lipid metabolism, cytoskeletal dynamics and apoptosis. See, P. Mendez et al, "Phosphatidyl inositol 3 kinase (PI3K) and glycogen synthase kinase 3 (GSK3) regulate estrogen receptor mediated transcription in neuronal cells," Endocrinology, epub. February 23, 2006, doi:10.1210/en.2005-1224. GSK-3, unlike most kinases, has a high level of activity in resting cells and the activity is inhibited phosphorylation of the enzyme. GSK-3 has a wide variety of targets that include many transcription factors and nuclear proteins such as activation protein 1 (AP-I), cAMP response element binding protein (CREB), NF-κB, or β-catenin. GSK-3 has been shown to be a therapeutic target for treatment of multiple neurological diseases, including Alzheimer's, stroke, and bipolar disorders, as well as noninsulin-dependent diabetes mellitus, inflammation, and cancer. See, A. V. Ougolkov et al, "Targeting GSK-3: a promising approach for cancer therapy?", Fut. Oncol, vol. 2, pp. 91-100 (2006); H. Eldar-Finkelman, "Glycogen synthase kinase 3: an emerging therapeutic target," Trends MoI. Med., vol. 8, pp. 126-132 (2002); A. Martinez et al, "Glycogen synthase kinase 3 (GSK-3) inhibitors as new promising drugs for diabetes, neurodegeneration, cancer, and inflammation," Med. Res. Rev., vol. 22, pp. 373-384 (2002); J. Van Wauwe et al, "Glycogen synthase kinase-3 as drug target: from wallflower to center of attention," Drug News Perspect, vol. 16, pp. 557-565 (2003); L. Meijer et al, "Pharmacological inhibitors of glycogen synthase kinase 3," Trends Pharmacol. Sci., vol. 25, pp. 471-480 (2004); and B.W. Doble et al, "GSK-3: tricks of the trade for a multi-tasking kinase," J. Cell Sci., vol. 116, pp. 1175-1186 (2003).

[0011] Control of hyperglycemia is the major task in the care of type 2 diabetes. The most effective strategies include: (a) Increase glucose deposition in the muscle and fat; (b) Decrease gluconeogenesis in the liver; and (c) Reduce intake of glucose from food. The muscle and fat are the major organs in glucose deposition, and the liver produces glucose.

[0012] Anti-diabetes drugs have a variety of side effects. Thiazolidinediones

(TZDs) are the most effective chemical anti-diabetes drug. Derivatives of TZD such as pioglitazone, rosiglitazone, englitazone, and ciglitazone are the most popular antidiabetic commercial drugs in the treatment of type 2 diabetes. TZDs enhance insulin sensitivity through activation of the nuclear receptor PPARγ (17; 18). However, application of TZDs is limited by the side effects, e.g., liver toxicity. Additionally, the cost of TZDs is also a major concern. The most popular synthetic antidiabetic medicines also have side effects. Since the cost and side effects limit the application of these chemical drugs, the demand for dietary supplements or alternative medicine has increased dramatically in the care of type 2 diabetes. Botanicals are the major ingredients in the hypoglycemic dietary supplements.

DISCLOSURE OF INVENTION

[0013] I have discovered that an active Shilianhua ("SLH") extract is a botanical product which regulates fatty acid and glucose metabolism. SLH was shown to protect against insulin resistance through multiple mechanisms, including inhibition of IKK/NF-κB, stimulation of adiponectin and insulin secretion, promotion of fatty acid oxidation, activation of p38, elevation of IRS-I, and inhibition of GSK-3. SLH or its extract can be used in the prevention and treatment of diseases in which insulin resistance plays a major role, which includes hyperglycemia, hyper lipidemia, cardiovascular diseases (such as hypertension, heart disease), stroke, renal failure, blindness, and non-traumatic limb amputation. SLH also can be used to treat obesity and hyperlipidemia-related diseases, and other diseases in which NF-κB and GSK-3 play a major role, e.g., inflammation, oxidative stress, rheumatoid arthritis, aging, arthrosclerosis, multiple sclerosis, asthma, inflammatory bowel disease, chronic inflammatory demyelinating polyradiculoneuritis, and cancer. In addition, the active fraction of SLH extract ("SLH extract FlOO" or "FlOO") has been characterized using HPLC. SLH extract FlOO has been found to increase glucose uptake and insulin sensitivity, to inhibit NF-κB, and to increase glycogen synthesis.

BRIEF DESCRIPTION OF DRAWINGS

[0014] Fig. IA illustrates the chemical fingerprints of Shilianhua crude extract

(SLH-C), FOO, F20, F50 and FlOO showing nine components (components 1-9), as seen in high performance liquid chromatography using a polarity-based separation.

[0015] Fig. IB illustrates the chemical fingerprint of the Shilianhua extract subtraction (FlOO) showing twelve subcomponents (components a-1), as seen in high performance liquid chromatography with an HPLC Diode Array Detector at the wavelength of 254 run, and using an isocratic reverse phase separation with methanol/water (80/20, v/v).

[0016] Fig. 1C illustrates an ultra-violet absorption spectra analysis of several components of SLH extracts: IC(I) illustrates component 1 in SLH-C; Fig. 1C(2) illustrates components 2, 3, 4, and 5 in SLH-C ; Fig. 1C(3 illustrates components 6, 7, 8, and 9 in SLH-C; Fig. 1C(4) illustrates in the SLH-100 fraction, components a, d, e, f, g, h, i, and 1; Fig. 1C(5) illustrates in the SLH-100 fraction, components b and c; and Fig. 1C(6) illustrates in the SLH-100 fraction, components j and k.

[0017] Fig. 2A illustrates the increase in body weight over 14 weeks in mice fed three different diets (i.e., high fat diet (HFD), low fat diet (chow), and HFD plus Shilianhua).

[0018] Fig. 2B illustrates the food intake per body weight over 4 weeks (from

14 to 18 weeks on the diet) in mice fed either high fat diet (HFD) or HFD plus Shilianhua.

[0019] Fig. 2C illustrates the body weight at 14 weeks for mice fed seven different diets: low fat diet (chow), high fat diet (HFD), HFD plus Shilianhua (SLH- HFD), HFD plus Shiu Huo pills (SHP-HFD), HFD plus Fenulyn (Fen-HFD), Bitter Melon (BM-HFD), and Glucose Metabolic Support (GMS-HFD). [0020] Fig. 2D illustrates three mice after 14 weeks on three different diets: low fat diet (chow), high fat diet (HFD), and high fat diet plus Shilianhua (HFD- SLH).

[0021] Fig. 3 A illustrates the change in blood glucose over 14 weeks in mice fed three different diets (high fat diet (HFD), low fat diet (chow), and HFD plus Shilianhua).

[0022] Fig. 3B illustrates the blood glucose level at 14 weeks for mice fed seven different diets: low fat diet (chow), high fat diet (HFD), HFD plus Shilianhua (SLH-HFD), HFD plus SMu Huo pills (SHP-HFD), HFD plus Fenulyn (Fen-HFD), Bitter Melon (BM-HFD), and Glucose Metabolic Support (GMS-HFD).

[0023] Fig. 4A illustrates the inhibition of NF-κB reporter activity after incubating cells with various concentrations of Shilianhua (SLH, from 200 to 1200 μg/ml).

[0024] Fig. 4B illustrates the potency of inhibition of NF-κB reporter activity after incubating cells with 200 μg/ml of various subtractions of Shilianhua, measured as the percent change with the added Shilianhua over the control.

[0025] Fig. 5A illustrates the change in production of adiponectin in fully differentiated 3T3-L1 adipocytes treated with and without Shilianhua (SLH) and an inhibitor free fatty acid, palmitate (200 μM; FFA), as measured by immunoblotting.

[0026] Fig. 5B illustrates the change in p38 activation over 6 hours in mouse brown adipocytes after culturing with Shilianhua (SLH; 200 μg/ml) and norephinephrine (NE; 1 μM), as measured with a phosphor-specific p38 antibody in a Western blot.

[0027] Fig. 5 C illustrates the level of fat oxidation in primary human muscle cells measured as ¹⁴CO₂ production rate from radiolabeled oleate in units of nM/mg/h. after culturing with and without Shilianhua (SLH, 400 μg/ml).

[0028] Fig. 6 A illustrates the plasma insulin over 14 weeks measured after overnight fasting in mice fed three different diets (high fat diet (HFD), low fat diet (chow), and HFD plus Shilianhua). [0029] Fig. 6B illustrates the stimulation of insulin secretion from beta-NC-1 cells incubated for 2 hours with Shilianhua (SLH; 400 μg/ml) or with glucose (2 mM).

[0030] Fig. 7A illustrates the dose effect of the SLH extracts F50 and FlOO on

LPS-induced NF-κB reporter activity as concentration increased from 50 μg/ml to 400 μg/ml, as measured using a luciferase assay.

[0031] Fig. 7B illustrates the effect of the SLH extract FlOO (100 μg/ml) over time on LPS-induced NF-κB reporter activity measured using a luciferase assay.

[0032] Fig. 8 illustrates the dose effect of the SLH extracts F50 and FlOO on

LPS-induced TNF reporter activity as concentration increased from 50 μg/ml to 400 μg/ml, as measured using a luciferase assay.

[0033] Fig. 9 illustrates the change in glucose uptake in rat L6 myotubes upon adding SLH extract FlOO (50 μg/ml), insulin (200 nM), or a combination of both.

[0034] Fig. 10 illustrates the change in glucose uptake in 3T3-L1 adipocytes upon adding SLH extract FlOO (50 μg/ml).

[0035] Fig. 11 illustrates the change in expression of IRS-I and GLUTl proteins in rat L6 myotubes as FlOO concentration increased from 10 μg/ml to 100 μg/ml, as measured by Western blot analysis.

[0036] Fig. 12A illustrates the change in phosphorylation of GSK-3 in rat L6 myotubes as measured by Western blot analysis upon adding insulin (200 nM), FlOO (50 μg/ml), and both FlOO and insulin.

[0037] Fig. 12B illustrates the change in phosphorylation of GSK-3 in 3T3-L1 adipocytes as measured by Western blot analysis upon adding insulin (200 nM), FlOO (50 μg/ml), and both FlOO and insulin.

[0038] Shilianhua (SLH) is defined as Sinocrassula indica (Decne.) Berger in the family of Crassulaceae, and is also known as Sedum indicum (Decne.) Hamet. Shilianhua can also be known as Sinocrassula berger or Echevaria glauca. (China Annals of Plants, Volume 34, page 63, or Hebei Annals of Plants, Volume 1, pages 575, 576 Hortex Baker.in Saund.) MODES FOR CARRYING OUT THE INVENTION

Example 1 Characterization of the SLH Extract

[0039] Preparation and Characterization of SLH extract. The extract of SLH was prepared from a SLH plant purchased from an experienced botanist who collected the plant from Guizhou Province, southwestern China. The original SLH sample (2 kg) was certified by a taxonomist at the Institute of Medicinal Plant Development (IMPED), a Chinese agency that specializes in identification and authentication of traditional Chinese herbs and medicinal plants.

[0040] Crude Extract: In China, the fresh stem and leaves of SLH were air- dried under shade to reduce the moisture content to approximately 8% w/w. The crude extract was prepared as described in U.S. Patent No. 5,911,993. Basically, the dry material was shipped to the United States, ground into a powder using a mill, and then passed through a 6-mm sieve. The SLH powder was soaked in deionized water (1 :8 w/v ratio) for 60 min at room temperature, and then extracted with water twice at 50°C in a rotary extractor for 6 h. The water-soluble extract was separated from the solids (e.g., the structural components of fibers, cellulose, semi-cellulose, and debris of cells) by centrifuging at 6,000 x g in an Allegra™ 6KR Centrifuge (Beckman Coulter, Palo Alto, California). The supernatant was then filtered sequentially through 20 μm, 1 μm, and 0.45 μm filter paper (Ultrafilter International, Haan, Germany). The filtrate was concentrated in a rotary evaporator of 20-L capacity (Buchi Rotavapor R-220, Flawi, Switzerland), and then freeze-dried into a crude extract powder. The crude extract (designed as SLH-C) accounted for 29.8% w/w of the ground herbal powder. SLH-C powder was used in the bioactivity analyses.

[0041] Fractionation: The extract of SLH (SLH-C) was dissolved in deionized water, and was fractionated on the basis of polarity in a flash chromatography column (ISCO CombiFlash Graduate, Lincoln, Nebraska). In the first step, SLH-C solution was loaded on a C- 18 column at 5:100 w/w sample: MeOH ratio, and then eluted with aqueous methanol (MeOH) of various strengths to yield four subfractions, named FOO, F20, F50, and FlOO. Then each subfraction was evaporated under reduced pressure to remove the organic solvent (MeOH) and yield pure aqueous fractions. Finally, each aqueous solution was freeze-dried to a powder, which was later used in the activity assays.

[0042] Characterization. The chemical fingerprint of SLH-C and its subtractions (FOO, F20, F50, and FlOO) as seen using polarity-based separation high performance liquid chromatography (HPLC) are shown in Fig. IA. As shown in Fig. IA, the crude extract (SLH-C) contained 9 major components: components 1, 2, 4, and 5 were found in the FOO subtraction; components 3, 4, 5, 6, 7 and 8 were found in subfraction F20; only component 8 was found in F50; and the non-polarized fractions were found in FlOO.

[0043] As shown below, FlOO extract retained the bioactivity of the SLH and so this extract was further characterized. Fig. IB shows the chemical chromatographic fingerprint of FlOO, a fraction of SLH extract, developed with an HPLC Diode Array Detector at the wavelength of 254 nm. Chromatography was performed by isocratic reverse phase separation with methanol/water (80/20, v/v) on an endcapped Waters Symmetry Cl 8 column (4.6 X 150 mm, 5 μm) at a flow rate at 1.0 ml/min. Twelve (12) major subcomponents (a-1) were identified in FlOO after signal amplification.

[0044] Using ultra-violet absorption spectra analysis, the components were shown to be grouped into six major groups of compounds (Fig. 1C). Fig. IC(I) illustrates the first group which includes component 1, and shows no UV absorption (except the typical terminal absorption due to the solvent). Fig. 1C(2) illustrates the second group, which includes components 2, 3, 4, and 5, and is characterized by a major absorption peak between 260 nm and 280 nm. Fig. 1C(3) illustrates the third group, which includes components 6, 7, 8, and 9, and shows no peak absorption (except the typical terminal absorption due to the solvent). Fig. 1C(4) illustrates the fourth group that is present in the SLH F-100 fraction, which includes subcomponents a, d, e, f, g, h, i, and 1, and shows two major absorption peaks (260 nm and 370 nm). Fig. 1C(5) illustrates the fifth group, which includes subcomponents b and c, and has a major absorption at 240-260 nm and a weak absorption at 380 nm. Finally, Fig. 1C(6) illustrates the sixth group, which shows a major absorption peak at about 320 nm and a minor peak at 380 nm. Example 2 SLH Prevents Dietary Obesity

[0045] To evaluate the metabolic activity of SLH and other commercially- available botanical products, the botanicals were tested in a mouse model of dietary obesity. Male C57BL/6 mice (5 week old) were purchased from the Jackson Laboratory (Bar Harbor, Maine). All procedures were performed in accordance with National Institute of Health guidelines for the care and use of animals. The mice were housed in an animal facility with a 12 hr light/dark cycle and constant temperature (23°C). The mice had free access to water and food. To induce obesity, the mice were fed with a high fat diet (HFD) (D 12331, Research Diets, New Brunswick, New Jersey), a diet in which fat accounts for 58 kcal%. The control group was fed with a corresponding low fat diet (chow diet, Cata #5001, Labdiet, St. Louis, Missouri). The botanicals were administrated by incorporating them into the high fat diet, and the dosage was calculated based on the daily food intake of the mice. The names of the commercial botanical products and the amounts added to the food were the following: Shilianhua (SLH, 5.2 mg/kg/d), Shiu Huo pills (SHP, 100 mg/kg/d), Fenulvn (Fen, 150 mg/kg/d), Bitter Melon (BM, 150 mg/kg/d), and Glucose Metabolic Support (GMS, 200 mg/kg/d). The mouse body weight was monitored weekly.

[0046] The results are shown in Figs. 2A - 2D. In the figures, an "*" indicates a statistically significant difference was seen between the treatments by a Student's t test (p <0.01). Figs. 2A and 2D indicate that supplementing the food with SLH led to a reduction in body weight gain. Fig. 2A shows the change in body weight over the 14-week period. Fig. 2D is a picture of representative mice after 14 weeks fed three different diets (chow, HFD, and HFD plus Shilianhua). As seen in Fig. 2 A, as early as week 8, the mice fed a high fat diet (HFD) had a significantly higher body weight than the mice fed Chow or the mice fed HFD plus SLH. The Chow group and the HFD plus SLH group showed the same rate of growth, and both had body weights that were 10% less than the body weight of the mice fed only HFD.

[0047] This effect of SLH was not a result of a difference in food intake.

Food intake was measured over a 4-week period, from 14 to 18 weeks of feeding the different diets. As shown in Fig. 2B, there was no significant difference in the food intake between the HFD group and the HFD plus SLH group. When comparing the body weight of mice after 14 weeks being fed the various diets, including the other four botanicals products that were added to HFD, only the addition of SLH prevented the body weight gain seen with the HFD in this experiment (Fig. 2C). These results indicate that SLH added to a high fat diet prevents weight gain without altering food intake. SLH may increase the overall energy expenditure of the mice. In another experiment, the crude extract, SLH-C, exhibited a similar anti-obesity activity as did the commercial product (data not shown).

Example 3 SLH Regulates Carbohydrate Metabolism

[0048] Carbohydrate metabolism was examined by monitoring blood glucose in mice divided into groups fed Chow, HFD, or HFD plus a botanical (SLH, SHP, Fen, BM, and GMS), as described above in Example 2. Blood was collected from the tail vein after overnight fasting, and used to test for glucose. Glucose (FBG) was measured with the FreeStyle blood glucose monitoring system made by Therasense (Alameda, California). Fasting glucose and insulin levels were measured in the plasma of the mice as described elsewhere(6). As shown in Fig. 3A, the fasting plasma glucose (FBG) was increased by HFD over the 14-week period. The hyperglycemic effect of HFD was prevented by adding SLH to HFD. (Fig. 3A) When the blood glucose of mice was compared at 14 wk, only the mice fed HFD plus SLH failed to show the hyperglycemic effect of HFD. The mice fed HFD plus any one of the other four botanical products showed the same hyperglycemic effect as the mice fed only HFD. (Fig. 3B). In the control mice fed on chow diet, FBG level remained stable at 100 mg/dL. (Fig. 3A) In both Figs. 3 A and 3B, an "*" indicates that a statistically significant difference was found in a Student's t test (pO.Ol). These results indicate that SLH was the most effective in regulating glucose metabolism of any botanical product tested. The anti-hyperglycemia activity of the extract SLH-C was confirmed in a similar experiment (data not shown).

Example 4 Inhibition ofNF-κBbySLH

[0049] SLH was tested for the regulation of NF-κB activity, a part of the

IKK/NF-κB signaling pathway that contributes to insulin resistance. A nuclear factor kappa B (NF-κB)-reporter cell line was made by stable transfection of RAW267 cells (ATCC No. TIB-71, American Type Culture Collection, Manassas, Virginia) with a NF-κB luciferase reporter (Cat. # 219078, Startagene, La Jolla, California) using a protocol as described elsewhere (5). The cells were plated in a 96-well plate at 5 X 10⁴ cells/well in 200 μl DMEM (pl049G-000, Biosource, Camarillo, California) supplemented with 0.1% bovine serum albumin (BSA) (152401, MP Biomedical, Inc., Irvine, California). The cells in each well were treated with either SLH-C or one of its subfractions (FOO, F20, F50, and FlOO) overnight with concentrations from 200 to 1200 μg/ml. The reporter activity was induced by incubating the cells with LPS (1 μg/ml) for 6 hours, and then measuring activity using a 96-well luminometer. The experiments were repeated three times.

[0050] The results are shown in Figs. 4A and 4B, in which an "*" indicates a statistically significant difference a Student's t test of p <0.001. As shown in Fig. 4A, NF-κB reporter activity was significantly inhibited by SLH in a dose-dependent manner. These data support that SLH inhibits NF-κB activity, and thus inhibits a pathway that inhibits insulin signaling. This activity of SLH contributes to an insulin- sensitizing effect of SLH. No cellular damage was observed under a microscope in the presence of SLH. The NF-κB inhibitory activity of the subfractions of SLH (200 μg/ml; FOO, F20, F50, and FlOO) was also assayed. The potency of SLH and its subfractions was compared to the control, by calculating the percent increase as seen over the control. As shown in Fig. 4B, subfraction FlOO was the most potent among the four SLH subfractions, suggesting that it contained compounds that inhibit NF-κB in higher concentration than any other fraction. In Fig. 4B, each bar represents the mean value of three experiments.

Example 5 Induction of Adiponectin (ApN) Secretion by SLH

[0051] The effects of SLH on secretion of adiponectin (ApN), a protein cytokine released from adipocytes, was examined. Expression of adiponectin is inhibited during activation of NF-κB, and adiponectin level is positively associated with insulin sensitivity in both humans and other mammals (11; 12). 3T3-L1 adipocytes were obtained by differentiation of preadipocytes (ATCC No. CL-173, American Type Culture Collection, Manassas, Virginia) in a 24-well plate as described elsewhere (6). The cells were treated with SLH (100 ug/ml) for 16 hr in serum-free medium. Adiponectin (ApN) was measured in the cell culture medium by using a Western blot with ApN antibody (MAB3832, Chemicon International, Temecula, California). Quantitation was by a density analysis of each band, with the value normalized to a known amount of ApN standard (Cata. #1119-Ac-025, R@D Systems, Minneapolis, Minnesota) in the same blot. In one experiment, the free fatty acid (FFA) palmitate (200 μM; p-9767, Sigma Co., St. Louis, Missouri) was added to inhibit ApN expression.

[0052] As shown in Fig. 5A, ApN secretion was significantly increased by

SLH (p < 0.05, Student's t test). Each point in Fig. 5 A represents the mean value of three measurements. Of note, SLH was also able to overcome the inhibitory effect of palmitate on ApN secretion. (Fig. 5A). It is believed that reduction of ApN secretion by other free fatty acids would also be reversed by SLH.

Example 6 Serine Kinase p38 Activation and Fat Oxidation by SLH

[0053] SLH was tested for heat production in brown adipocytes, and for fat oxidation in the skeletal muscle in a primary cell cultures. Brown adipocytes are very important in adaptive thermogenesis for keeping core body temperature in the newborn animal. The heat production is positively associated with expression of uncoupling protein-1 (UCP-I). Expression of UCP-I is positively regulated by serine kinase p38 through PGC-I (PP ARy coactivator 1) (13). Therefore, p38 activation is a molecular marker of heat production.

[0054] Primary brown adipocytes were isolated from the brown fat tissues of

C57BL/6J mice at 4 weeks of age as previously described (7). The cells were treated with SLH (200 μg/ml), and phosphorylation of p38 was determined in the whole cell lysate by immunoblotting with phospho-specifϊc antibody as described elsewhere (8). At various incubation times, the cells were lyzed. Phosphorylation of p38 was determined with a phospho-specific p38 antibody (sc-7975-R, Santa Cruz Biotech, Santa Cruz, California) in a Western blot using the whole cell lysate. The positive control was the addition of 1 μM norepinephrine (A-9257, Sigma Co.), a known p38 activator. As shown in Fig. 5B, SLH induced p38 activity in a manner very similar to the induction with norephinephrine.

[0055] In human primary muscle cells that were collected from a biopsy, fat oxidation was measured after incubation with SLH (100 μg/ml). Fat oxidation was determined with radiolabeled oleate. Human skeletal muscle cells were cultured as previously described (27, 28). Briefly, muscle samples were obtained from the vastus lateralis muscle by needle biopsy, and satellite cells were isolated by trypsin digestion (29). Cell were grown to confluency on type I collagen-coated T25 flask at 37°C in a humidified atmosphere of 5% CO₂ in DMEM supplemented with 10% fetal bovine serum (FBS), 0.5 mg/ml BSA, 0.5 mg/ml fetuin, 20 ng/ml human epidermal growth factor, 0.39 μg/ml dexamethasone, and 50 μg/ml gentamicin/amphotericin B. Unless otherwise stated, all materials were purchased from Sigma Co. (St. Louis, Missouri). After reaching ~90% confluency, myoblasts were subcultured onto a 6-well type I collagen-coated plates at densities of 96 x 10³ cells per well. When cells reached 90% confluency, differentiation was induced by changing to low-serum differentiation media consisting of 2% heat-inactivated horse-serum, 0.5 mg/ml BSA, 0.5 mg/ml fetuin, and 50 μg/ml gentamicin/amphotericin B.

[0056] Fatty acid oxidation was measured in human skeletal muscle cells on the 8^th day of differentiation. Cells were incubated at 37°C in sealed 6-well plates containing 2.0 mL of serum-free differentiation media plus 12.5 mmol/1 HEPES, 0.2% BSA, 1.0 mmol/1 carnitine, 100 μmol/1 oleate, 50 μg/ml gentamycin, and 1.0 μCi/ml [¹⁴C] -oleate (NEN, Boston, Massachusetts). After 3 h treatment of the muscle cells with 400 μg/ml of SLH, the cell culture supernatant was sampled and assayed for ¹⁴C-labeled CO₂ as previously described (30, 31). After washing twice in ice-cold PBS, the cells were collected by scraping into a 1.5 -ml eppendorf tube in two additions of 0.30 ml 0.05% SDS lysis buffer. The protein concentration was determined and used to normalize CO₂ production rate. In Fig. 5C, fatty acid oxidation is presented as CO₂ production rate in unit of nM/mg/h.

[0057] As shown in Fig. 5 C, SLH increased fat oxidation in the human muscle cells. These data are additional support that SLH induces an increase in energy expenditure in the body. Example 7 Insulin and SLH

[0058] The amount of insulin was measured in the fasting mouse serum that was collected for fasting glucose assay as described above in Example 3. The results are shown in Fig. 6A, in which each point represents the mean insulin level of ten mice. The results show that SLH was able to increase blood insulin level over a 14- week period. (Fig. 6A).

[0059] To further investigate the mechanism of insulin elevation, a β-cell line

(Beta TC-6) was used to test the effect of SLH on the stimulation of insulin secretion. Beta TC-6 cell line was purchased from ATCC (ATCC # CRL-11506, ATCC₃ Manassas, Virginia), and maintained in DMEM with 4 mM L-glutamine, 1.5 g/1 sodium bicarbonate, 15% fetal bovine serum (FBS) (Sigma Co.), and gentaniicin sulfate (an antibiotic, 17-518L, Cambrex Bio Science Walkersville, Inc., Walkersville, Maryland). For an insulin assay, the cells were plated in a 24-well plate at 3 X 10⁵ cells/well. After SLH treatment (400 μg/ml) for 2 hrs, the insulin level was determined in the cell supernatant by ELISA assay as described elsewhere (6). The assay kit "Ultra Sensitive Insulin ELISA Kit" was obtained from Crystal Chem (Cat # 90060, Crystal Chem, Chicago, Illinois). The results are shown in Fig. 6B, where each point represents the mean of three experiments. SLH exhibited an activity almost comparable to that of glucose in stimulating the secretion of insulin from this β-cell line. (Fig. 6B).

Example 8 Toxicity of SLH

[0060] SLH did not exhibit any toxicity at the doses used in this study. The

HFD was supplemented with concentrated SLH at a dose of 5.2 mg/kg/d, and fed to the mice. The SLH supplemental diet for 14 weeks as described above in Example 3 did not lead to any toxic effect as indicated by food intake (Fig. 2B), appearance of the mouse body (Fig. 2D), and organs autopsy (liver, spleen, and kidney) (data not shown). Thus, the anti-obesity and anti-hyperglycemia activities of SLH do not indicate any toxicity. Example 9 SLH Extract FlOO Inhibits Inflammation Response

[0061] Bacterial endotoxin (LPS) was used to induce the inflammatory response in the mouse macrophage cell line (RAW264.7). The inflammatory response was monitored using a luciferase reporter gene that is driven by a NF-κB response element or by a TNF-alpha gene promoter, similar to the procedure described in Example 4. The mouse macrophage cell line RAW264.7 from the ATCC was maintained according to the provider's instruction. The cells were placed in a 100 mm cell culture plate and transfected with NF-κB luciferase plasmids (7.5 ug) using Lipofectamine after cell confluence. The cells were then placed into a 96-cell plate. After 24 h the cells were tested for activity of FlOO and F50. SLH extracts F50 and FlOO were prepared as described above in Example 1, and dissolved in culture medium at various concentrations (50, 100, 200 and 400 μg/ml). The extracts were then added to the cells and incubated for 16 h before adding the endotoxin (LPS). After 6 h, the activity was measured using the luciferase assay.

[0062] As shown in Fig. 7A, the data indicate that FlOO inhibited NF-κB activity in a dose-dependent way, while F50 did not show any inhibition of NF-κB activity. Each point in Fig. 7A represents a mean + SD (n=3). In addition, as shown in Fig. 7B, FlOO at 100 μg/ml concentration inhibited NF-κB within 7 h, and maintained inhibition for greater than 22 h. These results indicate that FlOO inhibits the LPS-induced NF-κB reporter activity. Since LPS activates the NF-κB reporter through IKK activation, FlOO would also inhibit an inflammatory response due to activation of IKK.

[0063] In a second experiment, a mouse macrophage cell line RAW264.7 from ATCC was transfected with TNF luciferase plasmids using Lipofectamine, as described in J. Ye et αl, "Inhibition of TNF-a Gene Expression and Bioactivity by Site-Specific Transcription Factor Binding Oligonucleotides," Amer. J. Physiol, Lung Cellular and Molecular Physiology, vol. 284, pp. L386-L394 (2002). The transfected cells were placed into a 96-cell plate, and used after 24 h for testing the effect of either F50 or FlOO. Either F50 or FlOO was dissolved in culture medium to various concentrations (50, 100, 200 and 400 μg/ml), and the resulting solution applied to the cells. After 16 h incubation, endotoxin (LPS) was added to the cells. After 6 h, the luciferase assay was used to measure the TNF reporter activity. As shown in Fig. 8, the data again indicate that FlOO suppresses the TNF-Luc reporter activity in a dose- dependent manner, while F50 did not show any activity. Each point in Fig. 8 represents a mean ± SD (n=3).

Example 10 SLH Extract FlOO Enhances Glucose Uptake and Insulin Action

[0064] Glucose metabolism was investigated in L6 muscle cells (myotube, mature rat muscle cells) and 3T3-L1 adipocytes. The cells were treated with FlOO (50 ug/ml) for 16 h. Glucose metabolism was determined by glucose uptake and glucose consumption. Cells from the rat myoblast cell line L6 from the ATCC were maintained according to the provider's instruction. The cells were placed in a 12-well plate, and induced for differentiation in culture medium supplemented with 2% FBS for six days. The myotubes were then treated with FlOO (50 μg/ml) in 0.25% BSA medium for 16 h. For the glucose uptake assay, the cells were washed in PBS twice and treated with insulin (200 nM) for 20 min. The glucose uptake was determined 5 min after adding H³-labelled D-deoxy-glucose. As shown in Fig. 9, the data indicate that FlOO by itself induced glucose uptake, and enhanced insulin activity in the induction of glucose uptake. The y-axis in Fig. 9 represents radioactive counts for H³, and each bar represents a mean ± SD (n=2).

[0065] Cells of 3T3-L1 pre-adipocytes were differentiated into adipocytes using a standard adipogenic cocktail, as described in Gao et αl, "Inhibition of Insulin Sensitivity by Free Fatty Acids Requires Activation of Multiple Serine Kinases in 3T3-L1 Adipocytes," MoI. Endocrinol., vol. 18, pp. 2024-2034 (2004). The cells were then placed in a 96-well plate and treated with FlOO (50 μg/ml) in 0.25% BSA medium for 16 h. The amount of glucose was determined in the culture medium as described in the above reference. Again, as shown in Fig. 10, the data indicate that that FlOO was able to stimulate glucose uptake by the adipocytes. Each bar in Fig. 10 represents a mean ± SD (n=8). [0066] These results indicate that FlOO by itself is effective in inducing glucose uptake in both muscle cells and in adipocytes, and was also able to promote glucose uptake by insulin in the muscle cells.

Example 11 Effect of SLH Extract FlOO on IRS-I, GLUTl, and GSK-3 Proteins

[0067] The mechanism of FlOO action was investigated in both muscle cells

(L6 myotube) and 3T3-L1 adipocytes. The cells were treated with FlOO overnight, and signaling molecules that are related to glucose metabolism were examined in Western blot analyses. Adipocytes and L6 myotubes were prepared as described in Example 9. The cells were placed in a 12-well plate, and induced for differentiation in culture medium supplemented with 2% FBS for six days. The myotubes were treated with FlOO at various concentrations (10, 20, 50 and 100 μg/ml) in 0.25% BSA medium for 16 h. From each well, a whole cell lysate was prepared and examined in a Western blot for both IRS-I (insulin receptor substrate) and GLUTl (glucose transporter 1), as described in Z. Gao et al, "Aspirin Inhibits TNF-induced Serine phosphorylation of IRS-I through Targeting Multiple Serine Kinases," J. Biol. Chem., vol. 278, pp. 24944-24950 (2003). As shown in Fig. 11, the presence of FlOO increased both IRS-I and GLUTl protein levels in a dose-dependent manner. This increase in IRS-I will increase the sensitivity to insulin, while the increase in GLUTl will promote glucose uptake. In Fig. 11, IRb refers to insulin receptor beta subunit. These data indicate that FlOO by itself will induce glucose uptake through an elevation of Glutl, and will promote insulin action through an elevation of IRS-I protein.

[0068] Adipocytes and L6 myotubes were prepared as described above in

Example 9. Following differentiation, the cells were treated with 50 μg/ml FlOO in 0.25% BSA medium, with insulin, and with both FlOO and insulin for 16 h. Again, from each well a whole cell lysate was prepared, and examined in a Western blot for phosphorylation status of GSK-3, as described in Gao et al, 2003. As shown in Fig. 12 A and 12B, FlOO induced GKS-3 phosphorylation in both adipocytes and muscle cells, as did insulin. In adipocytes, FlOO enhanced the insulin-induced phosphorylation of GSK-3. These data indicate that FlOO would enhance glycogen synthesis since phosphorylation of GSK3 leads to inhibition of GSK-3 activity, and subsequent enhancement of glycogen synthesis. This could explain FlOO's activity in induction of glucose consumption. In addition, since an increase in glycogen synthesis is association with insulin sensitization, this could help explain FlOO's activity in increasing insulin sensitivity.

Miscellaneous

[0069] The term "active SLH extract" is defined as an aqueous extract from

Shilianhua ("SLH;" Sinocrαssulα indicα Berge). The active plant extract can be a crude extract, or a processed extract, e.g., SLH-C or SLH extract FlOO. The term "SLH extract FlOO" of "FlOO" is defined as an active SLH extract from Shilianhua ("SLH;" Sinocrαssulα indicα Berge) that shows a chemical fingerprint using polarity- based separation HPLC as shown in Fig. IA, and a chemical fingerprint as shown in Fig. IB, developed with an HPLC Diode Array Detector at the wavelength of 254 nm, with isocratic reverse phase separation with methanol/water (80/20, v/v) on an endcapped Waters Symmetry C18 column (4.6 X 150 mm, 5 μm) at a flow rate at 1.0 ml/min.

[0070] The term "therapeutically effective amount" as used herein refers to an amount of an "active SLH extract" sufficient to increase insulin sensitivity, increase glucose uptake, inhibit insulin resistance, induce adiponectin secretion, increase fat oxidation, activate p38 phosphorylation, or inhibit NF-κB to a statistically significant degree (p<0.05). The term "therapeutically effective amount" therefore includes, for example, an amount sufficient to prevent or treat insulin resistance, and preferably to reduce insulin resistance by at least 50%, and more preferably to reduce by at least 90%. The dosage ranges for the administration of an active SLH extract are those that produce the desired effect. Generally, the dosage will vary with the age, weight, condition, and sex of the patient. A person of ordinary skill in the art, given the teachings of the present specification, may readily determine suitable dosage ranges. The dosage can be adjusted by the individual physician in the event of any contraindications. In any event, the effectiveness of treatment can be determined by monitoring the extent of insulin resistance by methods well known to those in the field. Moreover, the active SLH extract can be applied in pharmaceutically acceptable carriers known in the art. The active SLH extract can be used to treat diseases associated with insulin resistance in animals and in humans in vivo. The application can be oral, by injection, or topical, providing that in an oral administration the active SLH extract is preferably protected from digestion.

[0071] The active SLH extract may be administered to a patient by any suitable means, including oral, parenteral, subcutaneous, intrapulmonary, topically, and intranasal administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or intravitreal administration. The active SLH extract may also be administered transdermally, for example in the form of a slow-release subcutaneous implant, or orally in the form of capsules, powders, or granules. Although direct oral administration may cause some loss of activity, the active SLH extract could be packaged in such a way to protect the active ingredient(s) from digestion by use of enteric coatings, capsules or other methods known in the art.

[0072] Pharmaceutically acceptable carrier preparations for parenteral administration include sterile, aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. The active therapeutic ingredient may be mixed with excipients that are pharmaceutically acceptable and are compatible with the active ingredient. Suitable excipients include water, saline, dextrose, and glycerol, or combinations thereof. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, inert gases, and the like.

[0073] The active SLH extract may be formulated into therapeutic compositions as pharmaceutically acceptable salts. These salts include the acid addition salts formed with inorganic acids such as, for example, hydrochloric or phosphoric acid, or organic acids such as acetic, oxalic, or tartaric acid, and the like. Salts also include those formed from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides, and organic bases such as isopropylamine, trimethylamine, histidine, procaine and the like. [0074] Controlled delivery may be achieved by admixing the active ingredient with appropriate macromolecules, for example, polyesters, polyamino acids, polyvinyl pyrrolidone, ethylenevinylacetate, methylcellulose, carboxymethylcellulose, prolamine sulfate, or lactide/glycolide copolymers. The rate of release of the active SLH extract may be controlled by altering the concentration of the macromolecule.

[0075] Another method for controlling the duration of action comprises incorporating the active SLH extract into particles of a polymeric substance such as a polyester, peptide, hydrogel, polylactide/glycolide copolymer, or ethylenevinylacetate copolymers. Alternatively, the active SLH extract may be encapsulated in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, by the use of hydroxymethylcellulose or gelatin- microcapsules or poly(methylmethacrylate) microcapsules, respectively, or in a colloid drug delivery system. Colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil- in-water emulsions, micelles, mixed micelles, and liposomes.

[0076] The present invention provides a method of preventing, treating, or ameliorating a disease that results from development of insulin resistance in the body, such as cardiovascular disease, stroke, renal failure, blindness or non-traumatic limb amputation, comprising administering to a subject at risk for a disease or displaying symptoms for such disease, a therapeutically effective amount of the active SLH extract. The present invention also provides a method of preventing, treating, or ameliorating a disease that results from an increase in NF-κB activity, such as inflammation, oxidative stress, rheumatoid arthritis, aging, arthrosclerosis, multiple sclerosis, asthma, inflammatory bowel disease, chronic inflammatory demyelinating polyradiculoneuritis, and cancer. The term "ameliorate" refers to a decrease or lessening of the symptoms or signs of the disorder being treated. The symptoms or signs that may be ameliorated include those associated with an increase insulin resistance in the body, or an inflammatory response.

[0077] The present invention also provides a method of preventing, treating, or ameliorating a disease that results from hyperglycemia or low insulin sensitivity, such as diabetes or obesity, comprising administering to a subject at risk for a disease or displaying symptoms for such disease, a therapeutically effective amount of the active SLH extract FlOO. The present invention also provides a method of preventing, treating, or ameliorating a disease that results from development of insulin resistance in the body, such as cardiovascular disease, stroke, renal failure, blindness or nontraumatic limb amputation, comprising administering to a subject at risk for a disease or displaying symptoms for such disease, a therapeutically effective amount of the active SLH extract FlOO. The present invention also provides a method of preventing, treating, or ameliorating a disease that results from an increase in NF-κB activity, such as inflammation, oxidative stress, rheumatoid arthritis, aging, arthrosclerosis, multiple sclerosis, asthma, inflammatory bowel disease, chronic inflammatory demyelinating polyradiculoneuritis, and cancer, comprising administering to a subject at risk for a disease or displaying symptoms for such disease, a therapeutically effective amount of the active SLH extract FlOO.

[0078] The term "ameliorate" refers to a decrease or lessening of the symptoms or signs of the disorder being treated. The symptoms or signs that may be ameliorated include those associated with an increase insulin resistance in the body, or an inflammatory response.

References

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Claims

What is claimed:

1. A method to prevent or treat diseases associated with insulin resistance in a mammal, said method comprising administering to the mammal a therapeutically effective amount of an active Shilianhua (SLH) extract.

2. A method as in Claim I₃ wherein said disease is selected from a group consisting of cardiovascular disease, stroke, renal failure, blindness, and nontraumatic limb amputation.

3. A method to prevent or treat diseases associated with increased activity in NF-κB in a mammal, said method comprising administering to the mammal a therapeutically effective amount of an active Shilianhua (SLH) extract.

4. A method as in Claim 3, wherein said disease is selected from a group consisting of inflammation, oxidative stress, rheumatoid arthritis, aging, arthrosclerosis, multiple sclerosis, asthma, inflammatory bowel disease, chronic inflammatory demyelinating polyradiculoneuritis, and cancer.

5. A method to increase fatty acid oxidation in a mammal, said method comprising administering to the mammal a therapeutically effective amount of an active Shilianhua (SLH) extract.

6. A method to increase insulin secretion in a mammal, said method comprising administering to the mammal a therapeutically effective amount of an active Shilianhua (SLH) extract.

7. A method to increase adiponectin secretion in a mammal, said method comprising administering to the mammal a therapeutically effective amount of an active Shilianhua (SLH) extract.

8. A composition comprising an active extract of Shilianhua, wherein the extract is water-soluble and has a high performance liquid chromatography chemical fingerprint as shown in Fig. IA or in Fig. IB, when carried out as described in paragraphs [0042] and [0043].

9. A method to prevent or treat diseases associated with insulin resistance in a mammal, said method comprising administering to the mammal a therapeutically effective amount of the active Shilianhua (SLH) extract of Claim 8.

10. A method as in Claim 9, wherein said disease is selected from a group consisting of cardiovascular disease, stroke, renal failure, blindness, and nontraumatic limb amputation.

11. A method to prevent or treat diseases associated with inhibitory activity in NF-κB in a mammal, said method comprising administering to the mammal a therapeutically effective amount of the active Shilianhua (SLH) extract of Claim 8.

12. A method as in Claim 11, wherein said disease is selected from a group consisting of inflammation, oxidative stress, rheumatoid arthritis, aging, arthrosclerosis, multiple sclerosis, asthma, inflammatory bowel disease, chronic inflammatory demyelinating polyradiculoneuritis, and cancer.

13. A method to increase fatty acid oxidation in a mammal, said method comprising administering to the mammal a therapeutically effective amount of the active Shilianhua (SLH) extract of Claim 8.

14. A method to increase insulin secretion in a mammal, said method comprising administering to the mammal a therapeutically effective amount of the active Shilianhua (SLH) extract of Claim 8.

15. A method to increase adiponectin secretion in a mammal, said method comprising administering to the mammal a therapeutically effective amount of the active Shilianhua (SLH) extract of Claim 8.

16. A method to increase glucose uptake by tissues in a mammal, said method comprising administering to the mammal a therapeutically effective amount of the active Shilianhua (SLH) extract of Claim 8.

17. A method to enhance the effect of insulin in glucose uptake in a mammal, said method comprising administering to the mammal a therapeutically effective amount of the active Shilianhua (SLH) extract of Claim 8.

18. A method to increase insulin sensitivity by tissues in a mammal, said method comprising administering to the mammal a therapeutically effective amount of the active Shilianhua (SLH) extract of Claim 8.

19. A method to increase glycogen synthesis in a mammal, said method comprising administering to the mammal a therapeutically effective amount of the active Shilianhua (SLH) extract of Claim 8.

20. A method to prevent or treat diabetes in a mammal, said method comprising administering to the mammal a therapeutically effective amount of the active Shilianhua (SLH) extract of Claim 8.

21. A method to prevent or treat obesity in a mammal, said method comprising administering to the mammal a therapeutically effective amount of the active Shilianhua (SLH) extract of Claim 8.

22. A method to prevent or treat hyperglycemia in a mammal, said method comprising administering to the mammal a therapeutically effective amount of the active Shilianhua (SLH) extract of Claim 8.

23. A method to prevent or treat a disease associated with increase in activity of glycogen synthase kinase-3 (GSK-3) in a mammal, said method comprising administering to the mammal a therapeutically effective amount of the active Shilianhua (SLH) extract of Claim 8.

24. A method as in Claim 23, wherein said disease is selected from a group consisting of Alzheimer's, stroke, bipolar disorder, noninsulin-dependent diabetes mellitus, inflammation, and cancer.