EXTRACT OF TOONA SINENSIS FROM SUPERCRITICAL FLUID EXTRACTION FOR TREATING DIABETES AND METABOLIC DISEASE, THE PREPARATION METHOD AND THE USE THEREOF

Abstract

The Toona sinensis extract of the present invention is prepared using supercritical fluid technique, wherein the method includes steps of: (a) drying the leaves of T. sinensis; (b) pulverizing the leaves as particles; and (c) extracting the particles with supercritical carbon dioxide to obtain the T. sinensis extract. This supercritical T. sinensis extract not only can decrease blood sugar level, but also promotes lipid degradation, inhibits the formation of huge lipid droplet and improves the metabolic symptoms. Accordingly, the T. sinensis extract further is able to be prepared as food supplement and pharmaceuticals.

Claims

1. A method for treating a metabolic disease, comprising: providing an effective amount of a pharmaceutical composition being a low-polarity Toona sinensis extract; and administering the pharmaceutical composition to a subject in need thereof, wherein the pharmaceutical composition has at least one effectiveness of reducing a blood glucose level, inhibiting an accumulation of fats, improving metabolic syndromes, preventing hepatosteatosis, decreasing a plasma triglycerol level, lowering an insulin resistance and increasing a plasma adiponectin level.

2. The method according to claim 1, wherein the low-polarity Toona sinensis extract is one of a non-polar Toona sinensis extract and a weak-polar Toona sinensis extract.

3. The method according to claim 1, wherein the low-polarity Toona sinensis extract is extracted by a supercritical carbon dioxide.

4. The method according to claim 1, wherein the low-polarity Toona sinensis extract comprises at least one selected from a group consisting of a monoterpene, a monoterpene derivative, a diterpene derivative, a triterpene, a sesquiterpene, a sequiterpene derivative, a saturated fatty acid, an unsaturated fatty acid, an alkane, an alkene and a phytosterol.

5. The method according to claim 4, wherein the sesquiterpene comprises at least one selected from a group consisting of an α-cubebene, a copaene, an aromadendrene, a caryophyllene, an α-caryophyllene, a β-caryophyllene, an aristolene, a γ-patchoulene, an α-selinene, a β-selinene, a γ-selinene, an elixnen, a germacrene A, a germacrene B, a germacrene D, a β-elemene, a γ-elemene, a δ-elemene, a τ-elemene, a γ-cadiene, a τ-muurolene, an α-farnesene and a lepidozene, and the sesquiterpene derivative comprises a caryophylene oxide, a spathulenol, a ledene oxide, a germacrene D-4-ol and a globulol.

6. The method according to claim 4, wherein the saturated fatty acid comprises at least one of an ethyl pentadecanoate and an ethyl palmitate, the unsaturated fatty acid comprises at least one selected from a group consisting of a methyl linoleate, an ethyl 9,12-octadecadienoate and an ethyl α-linolenate, and the alkane has a carbon number no less than 21.

7. The method according to claim 4, wherein the alkane comprises at least one selected from a group consisting of an n-heneicosane, an n-docosane, an n-tricosane, an n-pentacosane, an n-heptacosane, an n-octacosane, an n-nonacosane, an n-hentriacontane, an n-dotriacontane, an n-pentatriacontane, an n-hexatricontane, an n-tritetracontane and an n-tetratetracontane.

8. The method according to claim 4, wherein the alkene comprises a (17E)-17-pentatriacontene, and the phytosterol comprises a β-sitosterol.

9. The method according to claim 4, wherein the low-polarity Toona sinensis extract further comprises a 6-methyl-5-hepten-2-one and a phytone.

10. The method according to claim 1, wherein the pharmaceutical composition is manufactured as one of a food and a drug.

11. A method for treating diabetes, comprising: providing an effective amount of a pharmaceutical composition being a low-polarity Toona sinensis extract; and administering the pharmaceutical composition to a subject in need thereof, wherein the pharmaceutical composition has at least one effectiveness of reducing a blood glucose level and lowering an insulin resistance.

12. The method according to claim 11, wherein the low-polarity Toona sinensis extract is one of a non-polar Toona sinensis extract and a weak-polar Toona sinensis extract.

13. The method according to claim 11, wherein the low-polarity Toona sinensis extract is extracted by a supercritical carbon dioxide.

14. The method according to claim 11, wherein the low-polarity Toona sinensis extract comprises at least one selected from a group consisting of a monoterpene, a monoterpene derivative, a diterpene derivative, a triterpene, a sesquiterpene, sequiterpene derivative, a saturated fatty acid, an unsaturated fatty acid, an alkane, an alkene and a phytosterol.

15. The method according to claim 11, wherein the pharmaceutical composition is manufactured as one of a food and a drug.

16. The method according to claim 14, wherein the sesquiterpene comprises at least one selected from a group consisting of an α-cubebene, a copaene, an aromadendrene, a caryophyllene, an α-caryophyllene, a β-caryophyllene, an aristolene, a γ-patchoulene, an α-selinene, a β-selinene, a γ-selinene, an elixnen, a germacrene A, a germacrene B, a germacrene D, a β-elemene, a γ-elemene, a δ-elemene, a τ-elemene, a γ-cadiene, a τ-muurolene, an α-farnesene and a lepidozene, and the sesquiterpene derivative comprises at least one selected from a group consisting of a caryophylene oxide, a spathulenol, a ledene oxide, a germacrene D-4-ol and a globulol.

17. The method according to claim 14, wherein the saturated fatty acid comprises at least one of an ethyl pentadecanoate and an ethyl palmitate, the unsaturated fatty acid comprises at least one selected from a group consisting of a methyl linoleate, an ethyl 9,12-octadecadienoate and an ethyl α-linolenate, and the alkane has a carbon number no less than 21.

18. The method according to claim 14, wherein the alkane comprises at least one selected from a group consisting of an n-heneicosane, an n-docosane, an n-tricosane, an n-pentacosane, an n-heptacosane, an n-octacosane, an n-nonacosane, an n-hentriacontane, an n-dotriacontane, an n-pentatriacontane, an n-hexatricontane, an n-tritetracontane and an n-tetratetracontane.

19. The method according to claim 14, wherein the alkene comprises a (17E)-17-pentatriacontene, and the phytosterol comprises a β-sitosterol.

20. The method according to claim 14, wherein the low-polarity Toona sinensis extract further comprises a 6-methyl-5-hepten-2-one and a phytone.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] FIG. 1 depicts the schema showing the CO.sub.2 supercritical fluid extractor for the T. sinensis supercritical extract in the present invention.

[0023] FIGS. 2A to 2B depicts the diagram of the non-polar constituents of T. sinensis extract analyzed by GC/MS.

[0024] FIG. 3A shows the remaining glucose concentrations of the media consumed by the differentiated 3T3-L1 cells by the treatment without (control) or with insulin, rosiglitazone or TS-SCF.

[0025] FIG. 3B shows the percentages of glucose consumed by 3T3-L1 adipocytes with a started concentration of 300 mg/dL.

[0026] FIGS. 3C and 3D show the comparison of (C) the medium glucose and (D) the percentage of glucose consumption among the batches of TS-SCFs. In FIGS. 3A to 3D, the data are presented as mean±SD from six independent experiments, and each sample was performed in duplicate for glucose concentration measurement. a, p<0.05 compared to control; b, p<0.01 compared to control; c, p<0.001 compared to control.

[0027] FIG. 4 depicts the quantification of lipid accumulation in 3TE-adipocyte in different treatments. The data are presented as mean±SD from six independent experiments, and each sample was performed in duplicate for glucose concentration measurement. a, p<0.05 compared to control; N.S., no significance compared to control.

[0028] FIGS. 5A and 5B are the drawings showing the effects of insulin, rosiglitazone (Rosig), and TSL extracts on (A) the medium glucose and (B) the glucose consumption in 3T3-L1 adipocyte model. In FIGS. 5A and 5B, the data are presented as mean±SD from six independent experiments, and each sample was performed in duplicate for glucose concentration measurement. a, p<0.05 compared to control; b, p<0.01 compared to control; c, p<0.001 compared to control.

[0029] FIGS. 6A and 6B shows (A) the blood glucose change and (B) the body weight change in mice. The feeding of a high-fat diet was started at day-14, and day 0 represented the day of the STZ injection (45 mg/kg). DM represented the group of high-fat-diet-fed mice with the STZ injection; DM+TS-SCF represented the group of high-fat-diet-fed mice with the STZ injection plus TS-SCF (150 mg/kg/day) treatment; DM+Rosig represented the group of high-fat-diet-fed mice with the STZ injection plus rosiglitazone maleate (150 μg/kg/day). Data are mean±SD. a, p<0.001 compared to the control, DM+TS-SCF, or DM+Rosig groups.

[0030] FIGS. 7A and 7B are the drawings showing the plasma levels of (A) adiponectin and (B) TNF-α both detected by ELISA kits. Data are mean±SD. a, p<0.05; b, p<0.01; N.S., no significance.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0031] The present invention will now be described more specifically with reference to the following Embodiments. It is to be noted that the following descriptions of preferred Embodiments of this invention are presented herein for purpose of illustration and description only; it is not intended to be exhaustive or to be limited to the precise form disclosed.

EXAMPLE 1

Supercritical Fluid Extraction For the Leaves of T. sinensis (TSL)

[0032] In order to perform a more extensive phytochemical screening and search for novel active compounds of leaves of Toona sinensis, supercritical fluid extraction (SFE) was used to obtain the non-polar constituents of TSL.

[0033] Method:

[0034] The raw material, “the leaves of T. sinensis (TSLs)”, was pretreated for extraction with a cold-air-dried method and ground into 1˜5-mm particles with a moisture content lower than 30%. To prepare the non-polar TSL extract, a pre-engineered supercritical fluid extraction pilot plant (Fa. NATEX Process Technology GmbH, Ternitz, Austria; extraction pressure up to 1000 bar and utilizable extraction volume at 5 L) was constructed. The extraction method and equipment were modified from the report of Rońyai et al., 1998. The scheme of the extraction procedure is shown in FIG. 1. A total of 1600 g of TSL was weighed accurately and supplied into the extraction vessel 4 for the extraction of solids. The extraction vessel was pressurized. Then, liquid CO.sub.2 was flowed into the cooler 2 for precooling and was adjusted prior to the commencement of the extraction. Pure CO.sub.2 was compressed by a high-pressure diaphragm pump 3 to 350-550 bar at 40-60° C. and allowed to flow through the extraction vessel at a flow rate of 30-35 kg CO.sub.2 per hour. Supercritical-CO.sub.2 is a solvent that is mainly suitable for transporting non-polar or weak-polar substances (TS-SCF) into the separator 5, where it is expanded by valves. After separation, the regenerated CO.sub.2 flows back into the reservoir 1 and can be used for extraction again. The extraction time was in the range of 60 to 120 min, and the extraction was stopped depending on whether the yield was less than 0.1% of the TSL as 30-70 kg CO.sub.2 passed through the vessel 4. To confirm the efficacy of supercritical-flow extracted leaves of T. sinensis from different resources (geographical or seasonal variation), three different batches of TSLs were collected and extracted using the method mentioned above. After the manufacturing processes, three extracts were harvested and named TS-SCF-I, TS-SCF-II and TS-SCF-III, respectively.

[0035] Result:

[0036] Each batches of supercritical-CO.sub.2 fluid extract of TSL was obtained from the separator 5 and processed an water-drying procedure, and the recovery was 14%˜18%. The raw material of EXAMPLE 1 included the young shoots, young leaves and matured leaves of T. sinensis.

EXAMPLE 2

GC/MS Analyses of TS-SCF

[0037] Method:

[0038] Chemical constituents of TS-SCF were analyzed by gas chromatography-mass spectrometry (DSQ II Single Quadrupole GC/MS, Thermofisher Scientific, USA) on a 30 m×0.25 mm×0.25 μm DB-5MS capillary column (Agilent J&W Scientific). The column oven temperature was programmed as follows: 100° C. (5 min) to 119° C. at 40° C./min, maintained at 119° C. for 26 min, then to 157° C. at 8° C./min, maintained at 157° C. for 5 min, then to 181° C. at 40° C./min, immediately heated to 188° C. at 3° C./min, held for 23 min, then increased to 230° C. at 5° C./min, held for 10 min, then raised to 242° C. at 5° C./min, held for 5 min, then heated to 280° C. at 1° C./min, and held for 10 min. The other parameters were as follows: injection temperature, 250° C.; ion source temperature, 250° C.; EI (electron bomb ionization), 70 eV; carrier gas, He at 1 mL/min; injection volume, 5 μL; split ratio, 1:50; and mass range, m/z 45-800. Identification of the compounds was based on a comparison of retention indices (determined relative to the retention times of n-alkanes on the DB-5MS column) and mass spectra with those of authentic samples, data from Wiley/NBS Registry of Mass Spectral Data (V. 5.0)/National Institute of Standards and Technology (NIST) MS Search V. 2.0 and the literature. The relative percentage of each compound in TS-SCF was quantified based on the peak area integrated by the analysis program.

[0039] Result:

[0040] The yield of supercritical-CO.sub.2 extraction from TSLs was 14-18% (w/w) according to the 30% dry weight of raw material. Taking TS-SCF for example, a total of 24 main constituents of TS-SCF were identified as shown in Table 1, and the chemical profile was presented in FIGS. 2A and 2B. Most of the constituents in the TS-SCF extract can be classified into different chemotypes, including monoterpenes and its derivatives, sesquiterpenes and its derivatives, saturated and unsaturated fatty acids, long-chain n-alkenes, phytosterols and others. The components of another three batches was listed in Table 1. Since different batches of TS-SCF were made from T. sinensis planted in different areas in Taiwan, the above-mentioned compounds would not appear in each batch of TS-SCF.

TABLE-US-00001 TABLE 1 Total vision of components from every batch of TSLs Components Components Components Components (TS-SCF-I) Ki.sup.a (TS-SCF-II) Ki.sup.a (TS-SCF-III) Ki.sup.a (TS-SCF) Ki.sup.a Monoterpenes Monoterpenes Sesquiterpenes Sesquiterpenes α-Pinene 938 α-Pinene 936 Elixene 1199 δ-Elemene 1340 β-Pinene 982 Sesquiterpenes δ-Elemene 1341 β-Elemene 1386 Monoterpene derivatives β-Elemene 1386 Copaene 1398 β-Caryophyllene 1417 Limonene 1026 Copaene 1389 β-caryophyllene 1416 Germacrene D 1481 Sesquiterpenes γ-Elemene 1437 γ-Patchoulene 1441 α-Selinene 1489 α-Cubebene 1355 τ-Muurolene 1468 α-Caryophyllene 1453 γ-Selinene 1497 β-Elemene 1389 β-Selinene 1483 Germacrene D 1483 Germacrene A 1508 Copaene 1396 α-Selinene 1489 β-Selinene 1485 α-Farnesene 1512 Caryophyllene 1467 Aromadendrene 1753 γ-Selinene 1497 Lepidozene 1528 Germacrene D 1481 Sesquiterpene derivatives Germacrene A 1510 Germacrene B 1563 β-Selinene 1486 Caryophylene oxide 1567 Germacrene B 1563 Sesquiterpene derivatives τ-Elemene 1488 Spathulenol 1574 Aristolene 1756 Germacrene D-4-ol 1574 Germacrene A 1506 Ledene oxide 1892 Aromadendrene 1758 Spathulenol 1576 Germacrene B 1561 Diterpene derivatives long-chain n-alkanes Globulol 1578 γ-Cadiene 1752 Phytol 1949 n-Heneicosane 2102 Diterpene derivatives Saturated fatty acids Saturated fatty acids n-Docosane 2201 Phytol 1949 Ethyl pentadecanoate 1997 Ethyl pentadecanoate 1993 n-Tricosane 2308 Saturated fatty acids Unsaturated fatty acids Unsaturated fatty acids n-Octacosane 2804 Ethyl palmitate 1975 Methyl linoleate 2091 Methyl linolenate 2092 n-Hentriacontane 3000 Unsaturated fatty acids Long-chain n-alkanes n-Dotriacontane 3201 Ethyl 9,12-octadecadienoate 2173 n-Heptacosane 2703 n-Pentatriacontane 3503 Ethyl α-linolenate 2176 n-Octacosane 2804 n-Hexatricontane 3601 Long-chain n-alkanes n-Pentatriacontane 3500 n-Tritetracontane 4305 n-Pentacosane 2500 n-tetratetracontane 4402 n-Tetratetracontane 4400 n-Heptacosane 2700 Alkenes n-Nonacosane 2900 (17E)-17-Pentatriacontene 3499 n-Hentriacontane 3100 Others n-Pentatriacontane 3500 6-methyl-5-hepten-2-one 984 Triterpene Phytone 1850 Squalene 2790 Phytosterols β-Sitosterol 3410 .sup.aKovats index relative to n-alkanes (C10-C40) on DB-5MS column and the identification was based on comparison of the mass spectrum from database, Kovats index on a DB-5MS column in reference.

EXAMPLE 3

In Vitro Model For Determination of Anti-Diabetic Effect

[0041] Adipose tissue is one of the major sites of postprandial glucose uptake (Watson et al., 2007). To speed up the screening of novel anti-diabetic candidate materials from natural products, an in vitro model was established by comparing the 24-hour glucose consumption from the culture medium of 3T3-L1 adipocytes.

[0042] Method:

[0043] The same amount (5×10.sup.5 cells) of 3T3-L1 preadipocytes (BCRC #60159; Bioresource Collection and Research Center, Taiwan) was seeded and cultured in normal glucose (100 mg/dL) DMEM supplemented with 10% FBS, 100 U/mL of penicillin, and 100 μg/mL of streptomycin in a humidified atmosphere of 95% air and 5% CO.sub.2 at 37° C. When the cell density reached 100% confluence, 3T3-L1 preadipocytes were induced to differentiate by treating the culture with 450 mg/dL D-glucose, 0.32 μM insulin, 0.5 mM 3-isobutyl-1-methylxanthine and 1 μM dexamethasone for two days. Then, the culture medium of the differentiated adipocytes was changed to DMEM containing 300 mg/dL D-glucose with or without the administration of TSL extracts. After 24 hours, the anti-diabetic activity was determined by measuring the medium glucose concentration using a Roche Cobas Integra 400 Chemistry Analyzer (Roche Diagnostics, Taipei, Taiwan). The coefficient of variation (CV) of the analyzer was 0.62-0.92% within-run and 1.1-1.2% between days. To confirm whether the in vitro model was sufficient to measure the glucose-lowering effect, insulin and rosiglitazone were used as positive controls.

[0044] Result:

[0045] As shown in FIG. 3A, the medium glucose concentration was decreased by approximately 18 mg/dL in the control group without the addition of any anti-diabetic reagent after 24 hours, with a starting glucose concentration of 300 mg/dL. When converted to the percentage of glucose consumption, only about 6% of the glucose in the medium was used by 3T3-L1 adipocytes (FIG. 3B). Insulin (i.e., 3.2×10.sup.−7 to 3.2×10.sup.−5 M) decreased by approximately 46-57 mg/dL, and rosiglitazone (abbreviated as “Rosig”, i.e., 12.5 to 50 μg/mL) decreased by approximately 50-80 mg/dL of the glucose concentration in the media (FIG. 3A). By converting the data to percentage of glucose consumption, insulin and rosiglitazone increased the use of medium glucose to 15-18% and 16-27%, respectively (FIG. 3B). Compared to the control group, insulin and rosiglitazone significantly decreased the glucose concentrations of the media in a dose-dependent manner in the 3T3-L1 adipocyte model, indicating that this in vitro model could be used as a quick screening method for novel anti-diabetic reagents that target adipocytes. Through this in vitro model, it was observed that TS-SCF possessed an excellent anti-diabetic effect. The glucose-lowering effect of 12.5 μg/mL of TS-SCF was equal to that of 50 μg/mL of rosiglitazone (FIG. 3A). A total of 50 μg/mL of TS-SCF could increase the use of medium glucose to approximate 50% (FIG. 3B). As shown in FIGS. 3C and 3D, the administration of every batch of TS-SCF (50 μg/mL) could significantly decreased the glucose concentrations of the media and possessed an excellent anti-diabetic effect in our 3T3-L1 adipocyte model.

EXAMPLE 4

TS-SCF Blocked Adipogenesis in Adipocytes

[0046] Method:

[0047] Lipid droplets were detected by Oil Red O staining (Kinkel et al., 2004). 3T3-L1 cells were washed three times with PBS and fixed in 10% formalin for 1 hour. After washing with PBS, the cells were stained with Oil Red O staining solution (0.5% Oil Red O in 100% propylene glycol). After 1 hour, cells were washed three times with water and observed under a phase contrast microscope. To quantify the cellular lipid quantity, stained cells were incubated with 100% isopropanol and shaken at room temperature for 20 minutes, and then Oil Red O in the supernatant was measured at 490 nm by an ELISA reader.

[0048] Result:

[0049] On day 6, a significant increase in lipid droplet formation was observed by Oil Red O staining in the differentiated cells relative to the undifferentiated cells. The formation of lipid droplets was almost completely blocked when TS-SCF was added to the induction medium, the DMSO treated group was blank (data not shown). After extracting the Oil Red O from the cells, a serious increase in lipid accumulation was observed in the differentiated group (FIG. 4). In contrast, induced cells that were co-treated with 50, 100 μg/mL TS-SCF showed significant increases in lipid formation, as compared to the undifferentiated group (FIG. 4). These results indicate that inhibition of TS-SCF obstructed adipogenesis in adipocytes.

EXAMPLE 5

Anti-Diabetic Effect of Extracts of High to Mid/High Polar Components of TSL Differed From TSL-SCF

[0050] As the described above, supercritical fluid extract of TSL (TS-SCF) exhibited hypoglycemia effect and inhibitory action of lipid accumulation during differentiation of 3T3-L1 preadipocytes, and the components was identified as non-polar compositions in the present invention. To compare with the prior art that mentioned the effective chemical components of TSL extracts have so far shown a distribution in the range from high to mid-high polarity (e.g., aqueous extract, aqueous solution processed by using 99.5% to 25% of ethanol, methanol extract, and aqueous extract of fermented Toona sinensis), three different extraction methods were generalized to confirm the anti-diabetic effect of the extracts, respectively.

[0051] Method:

[0052] The methods of in vitro model for determination of anti-diabetic effect are the same as the contents described in EXAMPLE 3. To obtain the high to mid-high polar components of TSL, three different extraction methods were used to produce the following extracts, which were previously reported (Liao et al., 2007; Hsieh et al., 2005; Yang et al., 2003). TSLs were (i) extracted by reverse-osmosis of water (1:4 w/v) by boiling for 30 min and then cooling down without further boiling for two hours at room temperature (Liao et al., 2007); (ii) extracted by soaking in 100° C. boiled water (1:10 w/v) three times, each time for 30 min (Hsieh et al., 2005); and (iii) extracted by boiling in 50% v/v alcohol/water for three hours (Yang et al., 2003). Next, the leaves were removed and the remaining liquids were filtered through filter paper (Advantec, Tokyo, Japan). Afterward, the filtrated solutions were concentrated using an evaporator (rotary vacuum evaporator, vv2000; Heidolph, Schwabach, Germany) under reduced pressure and three crude extracts were obtained, which were named TS-WB (i), TS-WS (ii), and TS-E (iii); the yield of each extract was 6.03%, 5.13%, and 8.36%, respectively.

[0053] Result:

[0054] FIGS. 5A and 5B show the comparison of glucose-lowering effects among the non-polar TSL extract (i.e., TS-SCF) and the high to mid-high polar TSL extracts (i.e., TS-E, TS-WS, and TS-WB). The results demonstrate that only the TS-SCF significantly reduced the glucose in the media after the adipocytes were treated with an equal concentration (i.e., 50 μg/mL) of each TSL extract for 24 hours (FIG. 5A). Equal volume of solvents (100% EtOH or 100% DMSO) that were used to dissolve the reagents showed no influence on medium glucose consumption in 3T3-L1 adipocytes. FIG. 5B shows the data converted to the percentage of glucose consumption.

EXAMPLE 6

Determination of Anti-Diabetic Effect In Vivo

[0055] Method:

[0056] Eight-week-old male C57BL/6J mice were obtained from BioLASCO Technology (Charles River Taiwan Ltd.). All of the mice received standard animal care under supervision of the Institutional Animal Care and Use Committee of Kaohsiung Medical University, Taiwan. The mice were caged in an air-conditioned animal facility at 23° C. on a 12-h light/dark cycle and were maintained with free access to water and food. Animals were fed either a normal chow diet consisting (as a percentage of total kcal) of 11% fat, 65% carbohydrate, and 24% protein (Maintenance diet 1320, Altromin Spezialfutter GmbH & Co. KG, Germany) or a high-fat diet consisting of 45% fat, 35% carbohydrate, and 20% protein (D12451, Research Diets, Inc., New Brunswick, N.J., USA). After two weeks on either diet, mice were divided into four groups: (i) control (n=7); (ii) high-fat-diet mice with STZ injection (DM; n=5); (iii) high-fat-diet mice with STZ injection plus TS-SCF treatment (DM+TS-SCF; n=6); and (iv) high-fat-diet mice with STZ injection plus rosiglitazone treatment (DM+Rosig; n=4). The mice in groups ii-iv were injected with STZ (45 mg/kg), and both STZ-injected and non-injected animals were kept on their original diets (chow or high-fat) for the duration of the study. TS-SCF (150 mg/kg/day) and rosiglitazone (150 μg/kg/day) treatments via gastric gavage were begun two days after the STZ injection. The rosiglitazone maleate used in this experiment did not contain any inactive ingredient and the pure compound was kindly provided by GlaxoSmithKline, Ltd. (Taiwan).

[0057] Throughout the experiment, body weight and blood glucose from the tail tip were monitored weekly. Blood glucose was detected by the ACCU-CHEK blood glucose meter (Roche Diagnostics, Taipei, Taiwan). Animals were sacrificed after eight weeks of STZ injection and were euthanized by intraperitoneal injection with the anesthetic Zoletil (10 mg/kg) (Virbac, Carros, France). Blood samples were collected from the heart at the time of sacrifice for the measurement of biochemistry data using a Roche Cobas Integra 400 Chemistry Analyzer (Roche Diagnostics, Taipei, Taiwan).

[0058] Insulin from plasma were detected by ELISA kits (Crystal Chem, Inc. Downers Grove, USA). The homeostasis model assessment of insulin resistance (HOMA-IR) was calculated using the following formula: fasting blood glucose (mg/dL)×fasting insulin (μU/mL)/405.

[0059] Result:

[0060] After the evaluation of glucose-lowering potential via our in vitro model, the anti-diabetic effect of TS-SCF was further confirmed by the type 2 diabetic mouse model. FIG. 6A shows that the blood glucose significantly increased 14 days after the low-dose STZ injection in the DM group compared to that of the control group, the DM with TS-SCF-treated group, or the DM with rosiglitazone-treated group. The blood glucose level continuously increased to 584.60±87.65 mg/dL 56 days after the injection with STZ. In contrast, TS-SCF and rosiglitazone significantly prevented the progression of diabetes and controlled the glucose level between 200 and 300 mg/dL during the experimental period (FIG. 6A). FIG. 6B shows that there was no significant difference in the body weight change among these four groups.

[0061] Table 2 summarizes the biochemical data of the mice. There was no significant difference in the gains in body weight among the four groups (p=0.7122). Relative to the control group, the fasting plasma glucose significantly increased in the DM group (p<0.001). In comparison with the DM group, the TS-SCF and the rosiglitazone groups showed significantly lower fasting glucose levels (Table 2). The fasting plasma insulin levels showed no significant difference among the groups (p=0.1688). However, the HOMA-IR (homeostasis model assessment for insulin resistance) was significantly higher in the DM group (p<0.001 compared to the control group). The treatments of TS-SCF and rosiglitazone showed to have notably lower HOMA-IR values (p<0.001 compared to the DM group), indicating that TS-SCF and rosiglitazone could maintain insulin sensitivity in the mice. In addition to preventing the progression of diabetes, TS-SCF, as with rosiglitazone, significantly decreased the plasma triglycerol level, which was elevated in the DM group (Table 2). However, TS-SCF and rosiglitazone reduce the total plasma cholesterol level was not observed (Table 2). The plasma ALT level was increased in the DM group; in contrast, TS-SCF significantly prevented the elevation of plasma ALT levels (Table 2). Compared to the control group, the plasma levels of ALT and creatinine were not elevated by the TS-SCF treatment, indicating that TS-SCF had no hepatic or renal toxicity throughout the eight-week administration.

TABLE-US-00002 TABLE 2 Biochemical data of mice. Control DM DM + TS-SCF DM + Rosiglitazone p-value Body weight  6.70 ± 1.50  7.47 ± 1.77  7.52 ± 1.98  8.10 ± 1.95 0.7122 gain (g) Fasting glucose 155.14 ± 21.30 484.60 ± 87.65.sup.a 290.00 ± 65.55.sup.b 205.00 ± 84.87.sup.b 0.0001 (mg/dL) Insulin  1.45 ± 0.76  1.31 ± 0.71  0.66 ± 0.51  0.98 ± 0.77 0.1688 (ng/mL) HOMA-IR  15.92 ± 1.15  44.83 ± 4.39.sup.a  13.47 ± 2.38.sup.b  14.29 ± 4.60.sup.b 0.0001 Triglycerol  72.87 ± 13.70  94.42 ± 16.70.sup.c  63.27 ± 5.34.sup.d  64.94 ± 4.82.sup.d 0.0017 (mg/dL) T-CHO  87.32 ± 6.57 130.01 ± 10.60.sup.a 138.68 ± 16.00 137.65 ± 32.62 0.0001 (mg/dL) HDL-C  76.23 ± 6.87 110.72 ± 7.91.sup.a 125.95 ± 9.19.sup.e 126.03 ± 32.17 0.0001 (mg/dL) LDL-C  5.25 ± 3.21  10.21 ± 0.96.sup.f  8.03 ± 1.14  9.81 ± 8.29 0.1576 (mg/dL) ALT  39.76 ± 7.17  50.62 ± 8.13.sup.f  38.30 ± 6.86.sup.e  62.00 ± 27.00 0.0058 (U/L) Creatinine  0.48 ± 0.47  1.53 ± 0.64.sup.c  0.71 ± 0.40.sup.e  0.54 ± 0.18.sup.e 0.0081 (mg/dL) Values are mean ± SD, and data were analyzed by one-way analysis of variance and the Bonferroni test HOMA-IR: homeostasis model assessment for insulin resistance, T-CHO: total cholesterol, HDL-C: HDL cholesterol, LDL-C: LDL cholesterol, and ALT. alanine aminotransferase .sup.ap < 0.001 compared to control .sup.bp < 0.001 compared to DM .sup.cp < 0.05 compared to control .sup.dp < 0.01 compared to DM .sup.ep < 0.05 compared to DM .sup.fp < 0.01 compared to control

EXAMPLE 7

Morphological Evaluation of Adipose and Hepatic Tissues

[0062] Method:

[0063] A sample of adipose and hepatic tissues from each mouse was fixed in 4% formaldehyde and embedded in paraffin wax. Sections were stained with hematoxylin and eosin (H&E) and observed using a microscope. The morphological evaluation of was performed by hematoxylin and eosin stains.

[0064] Result:

[0065] The cell size of adipocytes in the DM group was enlarged compared to that of the control group. Relative to the DM group, administration of TS-SCF could reduce the cell size. In contrast, the rosiglitazone treatment maintained cell sizes as large as those of the DM group. The hepatosteatosis in the DM mice was characterized by ballooned hepatocytes. The administration of TS-SCF and rosiglitazone could effectively prevent the hepatosteatosis.

EXAMPLE 8

Effect of TS-SCF on Adiponectin and TNF-α Production in type 2 Diabetic Mouse Model

[0066] Adiponectin is a protective adipokine that has anti-inflammatory and anti-atherosclerotic effects; a deficiency of adiponectin has been associated with obesity-related disorders such as diabetes and cardiovascular disease (Kiess et al., 2008). TNF-α, a proinflammatory cytokine, is expressed in and secreted by adipose tissue, its levels correlating with the degree of adiposity and associated insulin resistance. Targeting TNF-α and/or its receptors has been suggested as a promising treatment for insulin resistance and type 2 diabetes (Chen et al., 2010).

[0067] Method:

[0068] Adiponectin, and TNF-α from plasma were detected by ELISA kits (MyBioSource, LLC, San Diego, USA).

[0069] Result:

[0070] As shown in FIG. 7A, plasma adiponectin was decreased in the DM group relative to the control group (p<0.05). Treatment with TS-SCF could significantly reverse the decrease of plasma adiponectin (p<0.01 compared to the DM group). The plasma TNF-α level was not significantly increased in the DM group after our 8-week experimental period, and the values were not significantly different among the four groups (FIG. 7B).

[0071] While the invention has been described in terms of what is presently considered to be the most practical and preferred Embodiments, it is to be understood that the invention needs not be limited to the disclosed Embodiments. On the contrary, it is intended to cover various modifications and similar arrangements included within the spirit and scope of the appended claims, which are to be accorded with the broadest interpretation so as to encompass all such modifications and similar structures.