FIELD OF THE INVENTION

[001] The present invention relates to a novel process for the preparation of a synthetic alpha hydroxyl succinamic acid, which is useful as active pharmaceutical ingredient in controlling diabetes mellitus. More specifically, the present invention relates to an efficient process for the preparation of 4-amino-2-hydroxy-4- oxobutanoic acid and intermediates thereof, which are amenable to large scale commercial production and provides the required products with improved yield for the management of type 2 diabetes mellitus and its complications.

BACKGROUND OF THE INVENTION

[002] Diabetes is a metabolic disorder characterized by high blood sugar level in the blood which results in hyperglycemia, lipoprotein abnormalities and raised metabolic rate. A broad range of chemical drugs and ayurvedic herbal formulations are known in the art for management of diabetes.

[003] The excitement of creating the molecules existing in nature, the chemists began to challenge the state of art by targeting increasingly more complex substances. E. Jambolana is widely used in ayurvedic system of medicine to treat diabetes, so it is worthwhile to isolate the active anti-hyperglycemic compound from fruit-pulp and assess its efficacy in various diabetic complications using experimental models.

[004] Indian patent application no.838/DEL/2003 (Patent No. 230753) relates to an herbal active hypoglycemic compound (Flic), a- hydroxysuccinamic acid extracted from the pulp of fruit of E. Jambolana and a process for extracting the same. However, the patent provides no teachings for synthesis of alpha- hydroxysuccinamic acid in laboratory.

[005] Since the herbal compound isolated from natural source has several limitations, as E. Jambolana is a seasonal fruit and its storage is difficult. The extraction procedure is elaborate, produces very less yield and is expensive. Further, the extracted amount obtained is not sufficient to meet the volume requirement of the market. To overcome these difficulties and in view of vast spectrum of biological activities of alpha-hydroxysuccinamic acid, there is an urgent need to provide an efficient synthetic methodology for the synthesis of alpha- hydroxylsuccinamic acid in good yield.

[006] The synthetic methodology adopted in the present invention is unique as the designing of molecule requires basic understanding of retrosynthetic approach. Different plausible routes can be proposed to achieve the synthesis of this molecule, but an expertise understanding of optimizing the intermediates and final compound is important. The structure of present compound has been confirmed by spectral data and has been fully explored for its anti-diabetic potential which was found to be at par with the herbal compound.

[007] Considering the importance gained by alpha-hydroxysuccinamic acid for the treatment of diabetes, there is a great need for developing simple, inexpensive, good yielding and commercially feasible processes for the manufacture of alpha- hydroxysuccinamic acid and its intermediates thereof.

SUMMARY OF THE INVENTION

[008] The present invention provides a process for the preparation of 4-amino-2- hydroxy-4-oxobutanoic acid, an active anti-hyperglycemic compound and intermediates thereof. The process of the present invention facilitates the commercial availability of said anti-hyperglycemic compound and its intermediates with acceptable yield.

[009] In a preferred embodiment, the invention provides a process the preparation of 4-amino-2-hydroxy-4-oxobutanoic acid, wherein the process comprises following steps:

a) reacting (10.0 g, 1.0 eq) of malic acid with 45 ml of trifluoroaceticanhydride (TFAA) at 0°C and stirring for 1.5 hours at 15 to 30°C, separating white crystalline compound and treating with 50 ml of anhydrous methanol at 0°C and stirring for 2 to 5 hours at room temperature to obtain 3-hydroxy-4- methoxy-4-oxobutanoic acid;

b) reacting (l.Og, 1.0 eq) solution of 3 -hydroxy -4-methoxy-4-oxobutanoic acid in THF with (1.5 eq) of EDC.HC1 and stirring for 10 minutes at room temperature, followed by addition of (1.5 eq) of HOBt, (3.0 eq) of TEA and (1.2 eq) of O-benzyl hydroxyl amine and final stirring at room temperature (15 to 30°C) to obtain methyl 4-((benzyloxy)amino)-2-hydroxy-4- oxobutanoate;

c) reacting (l.Og, 1.0 eq)of methanol solution of methyl 4-((benzyloxy)amino)- 2-hydroxy-4-oxobutanoate with (5.0 eq) of NaOH in H ₂ 0 with continuous stirring at room temperature followed by acidification with IN HC1 to obtain4-((benzyloxy)amino)-2-hydroxy-4-oxobutanoic acid;

d) reacting methanol solution of (1.0 eq) 4-((benzyloxy)amino)-2-hydroxy-4- oxobutanoic acid with 10 % palladium on carbon (Pd/C) with continuous stirring at room temperature to yield 4-amino-2-hydroxy-4-oxobutanoic acid.

[0010] In an embodiment, the present invention provides a process the preparation of 4-amino-2-hydroxy-4-oxobutanoic acid, wherein stirring in step c) of process is carried out for 5 to 7 hours and 4-((benzyloxy)amino)-2-hydroxy-4-oxobutanoic acid obtained in step c) is having a melting point in the range of 79-82°C.

[0011] In yet another embodiment, the present invention provides a process the preparation of 4-amino-2-hydroxy-4-oxobutanoic acid, wherein stirring in step d) of process is carried out for 6 to 8 hours under hydrogen atmosphere.

[0012] In an embodiment, the present invention provides a process the preparation of 4-amino-2-hydroxy-4-oxobutanoic acid, wherein the 3-hydroxy-4-methoxy-4- oxobutanoic acid obtained in step a) of process is having a melting point in the range of 69-70°C.

[0013] In still another embodiment, the present invention provides a process the preparation of 4-amino-2-hydroxy-4-oxobutanoic acid, wherein the final stirring in reaction of step b) of process is carried out for 11 to 13 hours and the methyl 4- ((benzyloxy)amino)-2-hydroxy-4-oxobutanoate obtained in step b) of process is having a melting point in the range of 68-72°C.

[0014] In an embodiment, the present invention provides a compound of formula 4- amino-2-hydroxy-4-oxobutanoic acid as obtained from the process as described herein.

[0015] In an embodiment, the present invention provides a compound of formula 4- amino-2-hydroxy-4-oxobutanoic acid, wherein the compound is having anti- hyperglycemic, hypolipidemic, anti-oxidant and anti-atherosclerotic activities at par with herbal alpha-hydroxysuccinamic acid isolated from E Jambolana. DESCRIPTION OF THE ACCOMPANYING DRAWINGS

[0016] Figure 1 illustrates the fasting blood glucose level after treatment with herbal compound (Flic) and present synthetic compound at week 0 and week 6 of the study.

[0017] Figure2 illustrates the HbAlc levels after treatment with herbal compound (Flic) and present synthetic compound at week 0 and week 6 of the study.

[0018] Figure3 illustrates the total serum cholesterol levels after treatment with herbal compound (Flic) and present synthetic compound at week 0 and week 6 of the study.

[0019] Figure4 illustrates the total serum triglyceride levels after treatment with herbal compound (Flic) and present synthetic compound at week 0 and week 6 of the study.

[0020] Figure5illustrates the HDL-C levels after treatment with herbal compound (Flic) and present synthetic compound at week 0 and week 6 of the study.

[0021] Figure 6illustrates the LDL-C levels after treatment with herbal compound (Flic) and present synthetic compound at week 0 and week 6 of the study.

[0022] Figure 7 illustrates the VLDL-C levels after treatment with herbal compound (Flic) and present synthetic compound at week 0 and week 6 of the study.

DETAILED DESCRIPTION OF THE INVENTION

[0023] Those skilled in the art will be aware that the present disclosure is subject to variations and modifications other than those specifically described. It is to be understood that the present disclosure includes all such variations and modifications. The disclosure also includes all such steps of the process, features of the product, referred to or indicated in this specification, individually or collectively, and any and all combinations of any or more of such steps or features.

Definitions

[0024] For convenience, before further description of the present disclosure, certain terms employed in the specification, and examples are collected here. These definitions should be read in the light of the remainder of the disclosure and understood as by a person of skill in the art. The terms used herein have the meanings recognized and known to those of skill in the art, however, for convenience and completeness, particular terms and their meanings are set forth below.

[0025] The articles“a”,“an” and“the” are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.

[0026] The terms“comprise” and“comprising” are used in the inclusive, open sense, meaning that additional elements may be included. It is not intended to be construed as“consists of only”.

[0027] Throughout this specification, unless the context requires otherwise the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated element or step or group of element or steps but not the exclusion of any other element or step or group of element or steps.

[0028] The term “including” is used to mean “including but not limited to”. “Including” and“including but not limited to” are used interchangeably.

[0029] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosure, the preferred methods, and materials are now described. All publications mentioned herein are incorporated herein by reference.

[0030] The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for the purposes of exemplification only. Functionally-equivalent products and methods are clearly within the scope of the disclosure, as described herein.

[0031] In an aspect of the present disclosure, there is provided a process for preparing 4-amino-2-hydroxy-4-oxobutanoic acid, wherein the process comprises following steps:

a) reacting (10.0 g, 1.0 eq) of malic acid with 45 ml of trifluoroaceticanhydride (TFAA) at 0°C and stirring for 1.5 hours at 15 to 30°C, separating white crystalline compound and treating with 50 ml of anhydrous methanol at 0°C and stirring for 2 to 5 hours at room temperature (15 to 30°C) to obtain 3- hydroxy-4-methoxy-4-oxobutanoic acid;

b) reacting (l.Og, 1.0 eq) solution of 3-hydroxy-4-methoxy-4-oxobutanoic acidin THF with (1.5 eq) of EDC.HC1 and stirring for 10 minutes at room temperature, followed by addition of (1.5 eq) of HOBt, (3.0 eq) of TEA and (1.2 eq) of O-benzyl hydroxyl a ine and final stirring at room temperature to obtain methyl 4-((benzyloxy)amino)-2-hydroxy-4-oxobutanoate;

c) reacting (l.Og, 1.0 eq) of methanol solution of methyl 4-((benzyloxy)amino)- 2-hydroxy-4-oxobutanoate with (5.0 eq) of NaOH in H ₂ 0 with continuous stirring at room temperature followed by acidification with IN HC1 to obtain4-((benzyloxy)amino)-2-hydroxy-4-oxobutanoic acid;

d) reacting methanol solution of (1.0 eq)4-((benzyloxy)amino)-2-hydroxy-4- oxobutanoic acid with 10 % palladium on carbon (Pd/C) with continuous stirring at room temperature to yield 4-amino-2-hydroxy-4-oxobutanoic acid.

[0032] In an embodiment, the present invention provides a process the preparation of 4-amino-2-hydroxy-4-oxobutanoic acid, wherein stirring in step c) of process is carried out for 5 to 7 hours and 4-((benzyloxy)amino)-2-hydroxy-4-oxobutanoic acid obtained in step c) of process is having a melting point in the range of 79-82°C.

[0033] In yet another embodiment, the present invention provides a process the preparation of 4-amino-2-hydroxy-4-oxobutanoic acid, wherein stirring in step d) of process is carried out for 6 to 8 hours under hydrogen atmosphere.

[0034] In an embodiment, the present invention provides a process the preparation of 4-amino-2-hydroxy-4-oxobutanoic acid, wherein the 3-hydroxy-4-methoxy-4- oxobutanoic acid obtained in step a) of process is having a melting point in the range of 69-70°C.

[0035] In still another embodiment, the present invention provides a process the preparation of 4-amino-2-hydroxy-4-oxobutanoic acid, wherein the final stirring in reaction of step b) of process is carried out for 11 to 13 hours and the methyl 4- ((benzyloxy)amino)-2-hydroxy-4-oxobutanoate obtained in step b) of process is having a melting point in the range of 68-72°C.

[0036] In an embodiment of present disclosure, there is provided a process for preparing 4-amino-2-hydroxy-4-oxobutanoic acid, wherein the process is a simple work-up procedure with optimum conditions for good yield of 4-amino-2-hydroxy- 4-oxobutanoic acid and is easily adapted on commercial scale as efficient and convenient process. [0037] In an embodiment of present disclosure, there is provided a process for preparing 4-amino-2-hydroxy-4-oxobutanoic acid as described herein, wherein thin- layer chromatography is preferably used to monitor the progress of the reaction and the crude compound of the process are purified by column chromatography, thereby making the process simpler and more adaptable for large scale commercial production. The purification by column chromatography involves use of silica gel (60: 120 mesh) in 10-40% EtOAc: Hexane as solvent system. The homogeneity of the desired compound and its intermediates is verified by HPLC and the structure of the synthesized compound is confirmed by ^X H NMR, ¹³ C NMR and HRMS data.

[0038] In an embodiment of present disclosure, there is provided a process for preparing 4-amino-2-hydroxy-4-oxobutanoic acid as described herein, wherein a schematic representation of experimental procedure for synthesis of 4-amino-2- hydroxy-4-oxobutanoic acid (compound 6) is outlined below:

[0039] In an embodiment of present disclosure, there is provided a process for preparing 4-amino-2-hydroxy-4-oxobutanoic acid as described herein, wherein the process yields 0.35 gm, 63% of the desired compound.

[0040] In another aspect of the present disclosure, there is provided a compound of formula 4-amino-2-hydroxy-4-oxobutanoic acid as obtained from the process as described herein.

[0041] In an embodiment of present disclosure, there is provided a compound of formula 4-amino-2-hydroxy-4-oxobutanoic acid as described herein, wherein the compound is having anti-hyperglycemic, hypolipidemic, anti-oxidant and anti- atherosclerotic activities at par with herbal alpha hydroxysuccinamic acid isolated from E. Jambolana.

[0042] Following non-limiting examples are given by way of illustration for specific embodiments thereof and therefore should not be construed to limit the scope of the invention.

EXAMPLES

[0043] The disclosure will now be illustrated with working examples, which is intended to illustrate the working of disclosure and not intended to take restrictively to imply any limitations on the scope of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods, the exemplary methods, devices and materials are described herein. It is to be understood that this disclosure is not limited to particular methods, and experimental conditions described, as such methods and conditions may vary.

Example 1

[0044] Experimental studies are conducted to determine the anti-diabetic potential of present synthetic compound 4-amino-2-hydroxy-4-oxobutanoic acid(compound 6) in comparison to active anti-diabetic herbal compound isolated from fruit pulp of Eugenia jambolana having fraction II (Flic) comprising 90 to 95% alpha hydroxysuccinamic acid. STZ+NAD induced diabetic rats are used as experimental animal for the present study. Experimental animals are divided into five groups (with six animals in each group) namely Group A (Healthy control), Group B (Diabetic control), Group C (Diabetic treated with herbal compound (Flic)), Group D (Diabetic treated with synthetic compound6) and Group E (Diabetic treated with standard drug (Glibenclamide). A therapeutic doze of 20mg/kg of body weight of herbal compound (Flic) and present synthetic compound of formula 6 are administered to Group C and Group D experimental animals respectively. Glibenclamide, a standard drug at a dose of 600pg/kg was given to Group E experimental animals. After six weeks of administration, the animals of the groups were assessed for various parameters namely fasting blood glucose level, HbAlc (%) level, total cholesterol level; serum triglycerides level; HDL-C, LDL-C and VLDL-C level, serum insulin levels, MDA levels and experimental data recorded was analyzed by statistical methods. Values are mean ± S.D was calculated for below mentioned experimental groups. Difference was assumed to be significant at the level of p<0.05.The results of present synthetic compound of formula 6 are compared with herbal compound (Flic) as shown in Figures 1 to 7.

[0045] Experimental animals treated with synthetic compound 4-amino-2-hydroxy- 4-oxobutanoic acid (compound 6)after six weeks study result in fasting blood glucose level(mg/dl) as 133.33 ±6.03; HbAlc(%) level as 6.24 ± 1.02; total cholesterol(mg/dl) level as 73.4±2.96; serum triglycerides (mg/dl) level as 75.2±3.28; HDL-C(mg/dl) level as 33.80±2.09; LDL-C(mg/dl) level as 16.9±1.48; VLDL-C(mg/dl) level as 15.04±1.42; serum insulin(pU/mL) level as 12.26±5.28and MDA(nmol/L)level as 9.1±1.27.

Example 2

Step 1: Synthesis of 3-hydroxy-4-methoxy-4-oxobutanoic acid (compound 2)

[0046] Trifluoroacetic anhydride (45 mL) was added to L-malic (compound 1) acid (10.0 g, 1.0 eq), at 0°C and allowed to stir at room temperature. After 1.5 hours, excess of TFAA and TFA were distilled off on rotary evaporator at temperature <30°C.The white crystalline compound obtained was cooled to 0°C and anhydrous methanol (50 mL) was added portion wise. The reaction mixture was further allowed to stir at room temperature for 3hours.The progress of reaction was monitored by TLC and after the completion of reaction; excess methanol was distilled off under reduced pressure. The crude compound was purified by column chromatography using silica gel (60: 120 mesh) in 10-40% EtOAc: Hexane as solvent system. The desired compound was obtained in 40% EtOAc: Hexane as white solid having Melting point: 69-70°C ; Yield : 50.67% ; 1H NMR (400 MHz, DMSO): 12.31 (brs, 1H, -COOH), 4.33 (t, 1H), 3.62 (s, 3H), 2.62 (d, J=15.57 Hz., 1H, Ha), 2.46 (d, J=15.57 Hz., 1H, Hb) ; 13C NMR (100 MHz, DMSO)5: 174.09, 172.22, 72.02, 63.67, 55.45, 36.03; HRMS (ESI) (M+H)+ Calculated for C ₅ H ₈ 0 ₅ : 148.0372, found 148.0367.

Example 3

Step 2: Synthesis of methyl 4-((benzyloxy) amino)-2-hydroxy-4-oxobutanoate (compound 4)

[0047] To a stirred solution of compound 2in THF, EDC.HC1 was added and reaction mixture was allowed to stir at room temperature for 10 min. Then HOBt was added followed by the addition of TEA and compound 3. The resultant reaction mixture was allowed to stir at room temperature for 12 hours. Progress of reaction was monitored by TLC and after the completion of reaction; it was diluted with water and extracted with EtOAc (3X50ml). Then organic layer was washed with brine, dried over anhydrous NaSCE and concentrated under reduced pressure. The crude product was purified by column chromatography using silica gel (60-120 mesh) in 10-40 % EtOAc: Hexane as solvent system. The desired compound was obtained in 45 % EtOAc: hexane as off white solid having Melting point: 68-72°C; Yield: 49.07 %; 1H NMR (400 MHz, DMSO-d6): 11.05 (br s, 1H, -NH), 7.37-7.43 (m, 5H), 4.75 (s, 2H), 4.86 (t, 1H), 3.61 (s, 3H), 2.35 (d, J=5.04 Hz. 1H, Ha), 2.24 (d, J=7.79 Hz., Hb); 13C NMR (100 MHz, DMSO)5: 174.09, 169.22, 133.59, 129.48, 129.05, 128.46, 78.02, 63.67, 54.55, 36.03); HRMS (ESI) (M+H)+ Calculated for C _I2 H _I5 N0 ₅ : 254.0950, found 254.1021.

Example 4

Step 3: Synthesis of 4-((benzyloxy) amino)-2-hydroxy-4-oxobutanoic acid (compound 5)

[0048] To a stirred solution of compound4in MeOH, aqueous solution ofNaOH was added and allowed to stir at room temperature for 6 hours. Progress of reaction was monitored by TLC and after completion of reaction; the reaction mixture was concentrated under reduced pressure. Acidification of the reaction mixture with IN HC1 resulted in the formation of solid compound. The compound obtained was filtered through sintered funnel, washed with cold H ₂ 0 and dried under high vacuum to give desired compound 5 as off white solid having Melting point: 79-82°C; Yield: 55.32 %; 1H NMR (400 MHz, DMSO-d6): 11.03(br s, 1H, -NH), 7.39-7.32 (m, 5H), 4.76 (s, 2H), 4.30 (t, 1H), 2.35 (dd, J=14.20 Hz. 1H, Ha), 2.19 (dd, J=14.21 Hz. 1H, Hb);13C NMR (100 MHz, DMSO) d: 172.62, 169.63, 134.02, 129.46, 129.04, 128.458, 78.019, 63.832, 35.972.;HRMS (ESI) (M+H)+ Calculated for CnHi ₃ N0 ₅ : 240.0794, found 240.0879.

Example 5

Step 4: Synthesis of 4-amino-2-hydroxy-4-oxobutanoic acid (compound 6)

[0049] To a stirred solution of compound 5 in MeOH, palladium on carbon (Pd/C) (10 %) was added and allowed to stir at room temperature under hydrogen atm. for 8 hours. Progress of reaction was monitored by TLC, after completion of reaction, the reaction mixture was filtered through sintered funnel, filtrate was concentrated under reduced pressure to give desired compound 6 (a-hydroxysuccinamic acid), Yield: 0.35 g, 63%.1H NMR (400 MHz, DMSO-d6): 11.03 (br s, 1H, -NH), 4.30 (t, 1H), 2.35 (dd, J=14.20 Hz. 1H, Ha), 2.19 (dd, J=14.21 Hz. 1H, Hb); 13C NMR (100

MHz, DMSO) d: 173.62, 171.63, 78.019, 36.79.; HRMS (ESI) (M+H)+ Calculated for C11H13NO5: 134.0375, found 134.0454.

Example 6

Effect of present compound 4-amino-2-hydroxy-4-oxobutanoic acid on Glucose levels

[0050] Table 1 : showing fasting blood glucose levels of healthy control and STZ+ NAD induced diabetic albino rats after treatment with anti-diabetic herbal comp (Flic) and present compound 4-amino-2-hydroxy-4-oxobutanoic acid (compound 6)at week 0 and week 6 of the study.

[0051] As depicted in Table 1, fasting blood glucose values showed significant difference between all the study groups at week 6 of the study (p<0.001). Group C, D and E animals treated with herbal compound (Flic), present compound 6 and drug Glibenclamide respectively showed significant difference in fasting blood glucose levels as compared to diabetic control (group B).

Example 7

Effect of present compound 4-amino-2-hydroxy-4-oxobutanoic acid on HbAlc levels

[0052] Table 2: showing HbAlc levels after treatment with herbal compound (Flic) and present compound 6at week 0 and week 6 of the study.

Example 8

Effect of present compound4-amino-2-hydroxy-4-oxobutanoic acid on Serum lipid profile

[0053] Table 3: showing Total cholesterol levels and serum triglycerides after treatment with herbal compound (Flic) and present compound 6at week 0 and week 6 of the study.

[0054] In Table 3 mentioned above, Total serum cholesterol and Serum triglyceride levels showed no significant difference between any of the study groups at week 0 of the study. However, at week 6 of the study, (group B) triglyceride levels were significantly higher as compared to the healthy (group A) (p<0.001). Also significant difference in triglyceride levels was observed in herbal compound (Fllc)treated (group C) and present compound of formula 6 treated (group D)as compared to diabetic control (group B) (p<0.001). Example 9

Effect of present compound4-amino-2-hydroxy-4-oxobutanoic addon HDL-C levels

[0055] Table 4: showing serum HDL-C levels after treatment with herbal compound (Flic) and present compound 6at week 0 and week 6 of the study.

[0056] As depicted from table 4, HDL-C levels showed no significant difference at week 0 in any of the study groups as compared with healthy control, although it has showed difference at week 6. HDL-C levels in (group B) has decreased at 6 weeks (p<0.001) as compared to healthy control (group A) while HDL-C levels had shown improvement in Flic treated (group C), present compound of formula 6 treated (group D) and glibenclamide treated (group E) as compared to diabetic control (group D) (p<0.001). Example 10

Effect of present compound4-amino-2-hydroxy-4-oxobutanoic acid on serum LDL-C & VLDL-C levels

[0057] Table 5: showing serum LDL-C& VLDL-C levels after treatment with herbal compound (Flic) and present compound (6) at week 0 and week 6 of the study

[0058] As indicated from table 5, the baseline values of serum LDL-C and VLDL-C levels showed no significant difference between the various study groups. However, on week 6 of the study significant drop was observed in herbal compound (Flic) treated (group C) and present compound 6 (group D) as compared to diabetic control (group B). Example 11

Effect of presentcompound4-amino-2-hydroxy-4-oxobutanoic acid on serum insulin & MDA levels

[0059] Table 6: showing serum insulin and MDA levels after treatment with herbal compound (Flic) and present compound 6 at week 0 and week 6 of the study

[0060] As depicted from table 6, the serum insulin levels in the diabetic control rats (group B)was found to be 9.2±4.42pU/mL which was significantly decreased (p<0.001) when compared to normal rats(group A). However, on week 6 of the study significant increase in serum insulin levels was observed in herbal compound (Flic) treated (group C) and present compound of formula 6 (group D) as compared to diabetic control (group B).

[0061] Compared to normal control rats (group A), diabetic control rats (group B) showed a significant increase in MDA levels (p<0.001). However, on week 6 of the study significant fall inMDA levels was observed in herbal compound (Flic) treated (group C) and present compound 6 (group D) as compared to diabetic control (group B).