Plant extract composition for reducing localized fat and promoting weight loss as well as application thereof

Abstract

Provided is a composition and a pharmaceutical composition for reducing localized fat or promoting weight loss, wherein the composition comprises a plant extract composition comprising resveratrol and turmeric extract and a weight ratio of resveratrol and turmeric extract ranges from 1:30 to 10:1. The composition and the pharmaceutical composition are primarily administered through localized injection to inhibit the growth of fat cells and promote fat cells apoptosis to reduce adipocytes, decrease localized fat deposition, and promote weight loss, without causing inflammation or necrosis on peripheral cells or tissues to avoid severe pain, so as to avoid inflammation, damage, and pain caused by surgical liposuction or non-surgical lipolysis products such as phosphatidylcholine and sodium deoxycholate.

Claims

1. A method for reducing localized fat at a local site of a subject, comprising a step of administering at the local site of the subject a therapeutically effective amount of a pharmaceutical composition comprising resveratrol and curcumin, wherein a weight ratio of resveratrol and curcumin ranges from 1:30 to 9:1.

2. The method as claimed in claim 1, wherein the subject is animal or human.

3. The method as claimed in claim 1, wherein the local site comprises face, jaw, arm, waist, abdomen or thighs.

4. The method as claimed in claim 1, wherein the step of administering comprises injection.

5. The method as claimed in claim 1, wherein the therapeutically effective amount of the pharmaceutical composition is from 0.4 mg/kg BW to 100 mg/kg BW.

6. The method as claimed in claim 5, wherein the therapeutically effective amount of the pharmaceutical composition is from 1 mg/kg BW to 60 mg/kg BW.

7. The method as claimed in claim 1, wherein said pharmaceutical composition has a weight ratio of resveratrol and curcumin in 1:19 to 9:1.

8. The method as claimed in claim 4, wherein the injection comprises subcutaneous fat injection, subcutaneous injection, or intravenous injection.

9. The method as claimed in claim 7, wherein the local site comprises face, jaw, arm, waist, abdomen or thighs.

10. The method as claimed in claim 7, wherein the step of administering comprises localized injection, subcutaneous implantation, implantable injection pump or transdermal administration.

11. The method as claimed in claim 10, wherein the localized injection comprises subcutaneous fat injection, or subcutaneous injection.

12. The method as claimed in claim 10, wherein the therapeutically effective amount of the pharmaceutical composition is from 0.4 mg/kg BW to 100 mg/kg BW.

13. The method as claimed in claim 12, wherein the therapeutically effective amount of the pharmaceutical composition is from 1 mg/kg BW to 60 mg/kg BW.

14. The method as claimed in claim 1, wherein the step of administering comprises subcutaneous implantation, implantable injection pump, or transdermal administration.

15. A method for weight loss of a subject, comprising a step of administering at a site of the subject a therapeutically effective amount of a pharmaceutical composition comprising resveratrol and curcumin, wherein a weight ratio of resveratrol and curcumin ranges from 1:30 to 9:1.

16. The method as claimed in claim 15, wherein the subject is animal or human.

17. The method as claimed in claim 15, wherein the step of administering comprises subcutaneous implantation, or implantable injection pump.

18. The method as claimed in claim 15, wherein the step of administering comprises localized injection.

19. The method as claimed in claim 15, wherein the therapeutically effective amount of the pharmaceutical composition is from 0.4 mg/kg BW to 100 mg/kg BW.

20. The method as claimed in claim 15, wherein the therapeutically effective amount of the pharmaceutical composition is from 1 mg/kg BW to 60 mg/kg BW.

21. The method as claimed in claim 15, wherein said pharmaceutical composition has a weight ratio of resveratrol and curcumin in 1:19 to 9:1.

22. The method as claimed in claim 18, wherein the localized injection comprises subcutaneous fat injection or subcutaneous injection.

23. The method as claimed in claim 21, wherein the step of administering comprises injection, subcutaneous implantation, or implantable injection pump.

24. The method as claimed in claim 23, wherein the injection comprises subcutaneous fat injection, subcutaneous injection or intravenous injection.

25. The method as claimed in claim 21, wherein the therapeutically effective amount of the pharmaceutical composition is from 0.4 mg/kg BW to 100 mg/kg BW.

26. The method as claimed in claim 21, wherein the therapeutically effective amount of the pharmaceutical composition is from 1 mg/kg BW to 60 mg/kg BW.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 illustrates the bar chart in each group for inhibiting preadipocytes growth through MTT assay;

(2) FIG. 2 illustrates the bar chart in each group for inhibiting differentiating adipocytes growth through MTT assay;

(3) FIG. 3 illustrates the bar chart of the formulation UL003C and sodium deoxycholate for inhibiting mature adipocytes growth through MTT assay;

(4) FIG. 4 illustrates the bar chart of the formulations UL003A, UL003C and UL003R for promoting apoptosis after Annexin V/PI staining through flow cytometer;

(5) FIG. 5 illustrates the bar chart of the formulations UL003A, UL003C and UL003R for promoting apoptosis after caspase 3 staining through flow cytometer;

(6) FIG. 6 illustrates the bar chart of resveratrol, turmeric extract and the formulation UL003C for promoting apoptosis after caspase 3 staining through flow cytometer;

(7) FIG. 7 illustrates the bar chart of the formulation UL003C and sodium deoxycholate for promoting apoptosis after Annexin V/PI staining through flow cytometer;

(8) FIG. 8 shows the curves of weight gain in each group related to rats administered with high-fat diet (Control) to induce localized fat increasing and then localized injection;

(9) FIG. 9 shows the subcutaneous fat mass in each group related to rats administered with high-fat diet (Control) to induce localized fat increasing and then localized injection;

(10) FIG. 10A illustrates the bar chart of serum biochemical index as creatinine and gluteamic oxaloacetic transaminase (GOT) in each group related to rats administered with high-fat diet (Control) to induce localized fat increasing and then localized injection;

(11) FIG. 10B illustrates the bar chart of serum biochemical index as urea and glutamic pyruvic transaminase (GTP) in each group related to rats administered with high-fat diet (Control) to induce localized fat increasing and then localized injection.

(12) FIG. 11 illustrates the bar chart of apoptotic protein expression as Bax, Bcl-2, and ratio of Bax/Bcl-2 through Western Blotting in each group related to rats administered with high-fat diet (Control) to induce localized fat increasing and then localized injection.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

(13) Other objectives, advantages and novel features of the invention will become more apparent from the following detailed description when taken in conjunction with the accompanying drawings.

Example 1 Preadipocytes Inhibition Assay

(14) 3T3-L1 preadipocytes (purchased from FIRDI, Taiwan) were seeded in 96-well plates to reach 1?10.sup.4 cells per well. Three repeated cell experiments were examined in seven groups including 1% DMSO as control group, 50 ppm resveratrol, 50 ppm turmeric extract, 80 ppm green tea extract, and the formulations UL003A, UL003C, and UL003R. After incubation for 48 hours, the inhibitory effect on 3T3-L1 preadipocytes was analyzed by MTT assay. The formulation UL003A in accordance with the present invention has a weight ratio 9:1 of resveratrol to green tea extract; the formulation UL003C in accordance with the present invention has a weight ratio 1:19 of resveratrol to turmeric extract; the formulation UL003R in accordance with the present invention has a weight ratio 9:1 of resveratrol to turmeric extract. All data are presented as Mean?SD. The letters a, b, c, d, and e represent the results of the statistics, and the different letters represent statistical difference among the groups (p<0.05).

(15) As shown in FIG. 1, compared to that of control group, the formulations UL003A, UL003C, and UL003R of the present invention inhibit preadipocytes growth (p<0.05). The inhibitory effect of the present invention were significantly better (p<0.05) than those of resveratrol, turmeric extract, or green tea extract.

Example 2 Differentiating Adipocytes Inhibition Assay

(16) 3T3-L1 preadipocytes cells were seeded in 12-well plates to reach 1?10.sup.5 cells per well. After the seeding for about four days, medium was replaced and contained 5 ?g/ml insulin (differentiation agent), 1 ?M dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine and respective treatment including 1% DMSO, 50 ppm resveratrol, 50 ppm turmeric extract, 80 ppm green tea extract, and 100 ppm formulation UL003A, UL003C, or UL003R in which DMSO was control group. After incubation for another 48 hours, the inhibitory effect on differentiating adipocytes 3T3-L1 was analyzed by MTT assay. All data are presented as Mean?SD and different letters differ significantly among groups (p<0.05).

(17) As shown in FIG. 2, compared to that of the control group, the formulations UL003A, UL003C, and UL003R of the present invention all could inhibit differentiating adipocytes growth (p<0.05), wherein the formulation UL003C had the best inhibitory effect on differentiating adipocytes. The inhibitory effect on differentiating adipocytes by the formulation UL0003C of the present invention was greater than those of resveratrol, turmeric extract or green tea extract (p<0.05).

Example 3 Mature Adipocytes Inhibition Assay

(18) The object of the instant example is comparing the inhibitory effect on mature adipocytes of the composition of the present invention and the conventional sodium deoxycholate. 3T3-L1 cells were seeded in 12-well plates to reach 3?10.sup.4 cells per well. After seeding for about four days, medium was changed and contained 5 ?g/ml insulin, 1 ?M dexamethasone, and 0.5 mM 3-isobutyl-1-methylxanthine After incubation for another two days, medium was renewed and contained 5 ?g/ml insulin for additional four days maturity incubation. Medium containing PBS (as control group), DMSO (as control group), 50 ppm or 100 ppm formulation UL003C and 50 ppm or 100 ppm sodium deoxycholate were respectively added to the 12-well plates for six groups. After incubation for 48 hours, the inhibitory effect on mature adipocytes was analyzed by MTT assay.

(19) As shown in FIG. 3, the formulation UL003C of the present invention inhibited mature adipocytes growth (p<0.05) at concentration of 50 ppm or 100 ppm. The inhibitory effect on mature adipocytes of the present invention formulation UL0003C was greater than that of sodium deoxycholate (p<0.001).

Example 4 Apoptosis Assay (I)

(20) 3T3-L1 cells were seeded in 12-well plates to reach 1?10.sup.5 cells per well. After the seeding for about four days, medium was changed and contained 5 ?g/ml insulin, 1 ?M dexamethasone, and 0.5 mM 3-isobutyl-1-methylxanthine After incubation for four days for adipocyte differentiation, medium was renewed and contained 5 ?g/ml insulin for additional four days incubation. Media containing DMSO and 50 ppm formulations UL003A, UL003C, and UL003R of the present invention were respectively added to the 12-well plates for four groups. Three repeated cell experiments were examined. After incubation for 24 hours, cells were collected and then immunostained by Annexin V/PI antibodies. The level of apoptosis was analyzed with flow cytometer, wherein Annexin V.sup.?PI.sup.? cells represented the survival number of mature adipocytes, and Annexin V.sup.+PI.sup.+ cells represented the apoptotic number of mature adipocytes.

(21) As shown in FIG. 4, compared to that of control group, the formulations UL003A, UL003C, and UL003R of the present invention all induced mature adipocytes apoptosis (p<0.05), wherein the best apoptotic effect on mature adipocytes was induced by formulation UL003C (p<0.05).

Example 5 Apoptosis Assay (II)

(22) 3T3-L1 cells were seeded in 12-well plates to reach 1?10.sup.5 cells per well. After seeding for about four days, medium was changed and contained 5 ?g/ml insulin, 1 ?M dexamethasone, and 0.5 mM 3-isobutyl-1-methylxanthine After incubation for four days for adipocyte differentiation, medium was renewed and contained 5 ?g/ml insulin for additional four days maturity incubation. Media containing DMSO (as control group), 50 ppm or 100 ppm formulations UL003A, UL003C, and UL003R of the present invention were respectively added to the 12-well plates for seven groups. Three repeated cell experiments were examined. After incubation for 24 hours, cells were collected and then immunostained by caspase 3 antibody. The level of apoptosis was analyzed by flow cytometry, wherein mature adipocytes having caspase 3 staining underwent apoptosis.

(23) As shown in FIG. 5, compared to that of control group, mature adipocytes were treated with 50 ppm or 100 ppm formulation UL003C of the present invention and more caspase 3 stained cells was shown (p<0.001) indicated that formulation UL003C of the present invention induced mature adipocytes apoptosis significantly.

Example 6 Apoptosis Assay (III)

(24) 3T3-L1 cells were seeded in 12-well plates to reach 1?10.sup.5 cells per well. After seeding for about four days, medium was changed and contained 5 ?g/ml insulin, 1 ?M dexamethasone, and 0.5 mM 3-isobutyl-1-methylxanthine After incubation for four days for adipocyte differentiation, medium was renewed and contained 5 ?g/ml insulin for additional four days maturity incubation. Medium containing DMSO (as control group), 50 ppm turmeric extract, 50 ppm resveratrol, and 50 ppm or 100 ppm formulation UL003C of the present invention for five groups were respectively added to the 12-well plates. Three repeated cell experiments were examined. Accordingly cells treated by resveratrol for 16 more hours were more likely to express caspase 3 protein, the remaining groups were all incubated for 3 hours, and collected for apoptotic assay with flow cytometry.

(25) As shown in FIG. 6, compared to that of control group, mature adipocytes treated with 50 ppm formulation UL003C of the present invention expressed more caspase 3 protein (p<0.001) indicated that formulation UL003C of the present invention induced mature adipocytes apoptosis. The number of caspase 3 positive mature adipocytes treated by the formulation UL0003C of the present invention was greater than those of the turmeric extract (p<0.05) and the resveratrol (p<0.001) and showed that formulation UL003C of the present invention induced the most mature adipocytes apoptosis than other group.

Example 7 Apoptosis Assay (IV)

(26) The object of the instant example is to compare the apoptosis level on mature adipocytes caused by the composition of the present invention and the well-known sodium deoxycholate. 3T3-L1 cells were seeded in 12-well plates to reach 1?10.sup.5 cells per well. After seeding for about four days, medium was changed and contained 5 ?g/ml insulin, 1 ?M dexamethasone, and 0.5 mM 3-isobutyl-1-methylxanthine After incubation for four days for adipocyte differentiation, medium was renewed and contained 5 ?g/ml insulin for additional four days maturity incubation. Medium containing DMSO (as control group), 100 ppm formulation UL003C and 100 ppm sodium deoxycholate were respectively added to the 12-well plates for three groups. Three repeated cell experiments were examined. After incubation for 24 hours, cells were collected and then immunostained by Annexin V/PI antibodies. The level of apoptosis was analyzed by flow cytometry, wherein Annexin V.sup.?PI.sup.? cells represented the survival number of mature adipocytes, and Annexin V.sup.+PI.sup.+ cells represented the apoptotic number of mature adipocytes.

(27) As shown in FIG. 7, compared to that of control group and sodium deoxycholate, the formulation UL003C of the present invention could significantly induce mature adipocytes apoptosis (p<0.001). On the contrary, compared to that of control group, no statistical significance (p>0.05) was found in sodium deoxycholate group indicated that only formulation UL003C of the present invention induced mature adipocytes apoptosis.

Example 8 Animal Assay (I)

(28) Sprague-Dawley (SD) male rats aged 8 weeks were used in this example. Three groups including control group, low-dose experimental group (formulation UL003C-20: 20 mg/kg BW), and high-dose experimental group (formulation UL003C-40: 40 mg/kg BW) were examined and four male rats were used in each group from 207 g?6 g body weight. After high fat diets treatment to reach to 330 g?10 g body weight, low-dose or high-dose composition of the present invention were injected into bilateral subcutaneous lower groin adipose tissue in two sites (5 mg/kg/site), and the dosages for rats in low-dose experimental group and high-dose experiment group were 20 mg/kg/time and 40 mg/kg/time respectively; the dosage for control group was 4 ml/kg/time sterile water for one injection. The formulation UL003C of the present invention and sterile water were administrated at the first, third day, and fifth day. Bodyweight and diet intake were recorded daily. In the end of the experiment (the 21th day), the rats were weighed and then fasted for 12-14 hours. The serum biochemical markers of glutamic pyruvic transaminase (GPT), glutamate oxalacetate aminotransferase (GOT), creatinine and urea were measured from blood sampling to evaluate the hepatic and renal function. After rats were sacrificed humanely, subcutaneous abdominal fat, upper groin fat, and lower groin fat were removed and weighed. All data are presented as Mean?SD and different letters differ significantly among groups (p<0.05).

(29) As shown in FIG. 8, the weight gains of the low-dose group and high-dose group were lower than that of control group, wherein the weight gain of rats administered with the formulation UL003C-40 was significantly lower than that of control group (p<0.05) and reach 15.8%. The loss of weight gain of formulation UL003C-20 group was decreased 11.1% without statistical difference compared to that of control group (p>0.05). The results showed that the composition of the present invention can reduce the body weight, in a dose-response manner.

(30) As shown in FIG. 9, compared to that of control group, the formulation UL003C of the present invention significantly reduced injected subcutaneous fat (p<0.05) and reach 24.3% (p<0.05) in formulation UL003C-20 group and 21.6% (p<0.05) in formulation UL003C-40 group, respectively.

(31) As shown in FIGS. 10A and 10B, no statistical difference (p>0.05) was found in serum biochemical marker of creatinine, urea, GOT, GPT indicated that formulation UL003C of the present invention at low- or high-dose would not cause side effects and be safe.

Example 9 Animal Assay (II)

(32) Anti-apoptosis protein Bcl-2 and apoptosis-promoting protein Bax were key regulators in apoptotic pathway, the balance between these two proteins is important for the regulation of apoptosis. Higher Bcl-2 expression suppresses apoptosis while higher Bax expression promotes apoptosis. The ratio between these two protein determines the cell survival or apoptosis.

(33) The protein expression of Bcl-2 and Bax2 in subcutaneous adipose tissue was determined with Western blot analysis and the ratio of Bax and Bcl-2 was evaluated as the apoptotic effect of formulation UL003C of the present invention.

(34) Rats subcutaneous adipose tissues proteins from lower groin (injection site) of example 8 were extracted by 450 ?l T-PER?. Thirty ?g protein of each group was loaded and separated by polyacrylamide gel electrophoresis (SDS-PAGE) followed by transfer to PVDF membrane. Bcl-2 antibody (sc-7382) and Bax antibody (sc-526) were purchased from Santa Cruz. All data are presented as Mean?SD and different letters differ significantly among groups (p<0.05).

(35) As shown in FIG. 11, compared with that of control group, the formulation UL003C at low-dose and high-dose both enhanced the Bcl-2 expression (p<0.05) and inhibited Bax expression significantly (p<0.05), and the ratio of Bax/Bcl-2 was higher than that of control group (p<0.05). The results showed that formulation UL003C of the present invention was effective to promote apoptosis on adipocytes of injection adipose tissues.

(36) The composition of the present invention was indeed effective to induce adipocyte apoptosis and reduce localized fat. The results of animal assays also confirmed the composition of the present invention can reduce adipocytes and localized fat via promoting apoptosis mechanism.

(37) Even though numerous characteristics and advantages of the present invention are revealed and described as above, together with details of the structure and features of the invention, the disclosure is illustrative only. Changes may be made in the details, especially in matters of shape, size, and arrangement of parts within the principles of the invention to the full extent indicated by the broad general meaning of the terms in which the appended claims are expressed.