WO2015025331A2

WO2015025331A2 - Drug designing for diabetes mellitus using pentacosanoic acid.

Info

Publication number: WO2015025331A2
Application number: PCT/IN2014/000518
Authority: WO
Inventors: Uma Makheswari M.
Original assignee: M Uma Makheswari
Priority date: 2013-08-21
Filing date: 2014-08-08
Publication date: 2015-02-26
Also published as: WO2015025331A3

Abstract

The present invention relates to the drug designing performed for the treatment of Diabetes mellitus Type 1 and 2 using a compound Pentacosanoic acid. It is used for the prevention and management of hyperglycemia, abnormal lipids, obesity, hypertension and atherosclerosis involved with diabetic patients. It may enable the design of therapy that can control, treat, and prevent Diabetes mellitus. It can be formulated in a form consisting of decoction, dried decoction, infusion, tincture, dried extract, capsule, tablet, syrup or the like thereof.

Description

TITLE: Drug designing for diabetes mellitus using Pentacosanoic acid. DESCRIPTION

The application claims benefit of Provisonal Application No. 3687/CHE/2013 filed 21/08/2013 in india.

FIELD OF THE INVENTION

The present invention relates to a compound Pentacosanoic acid which is used in the treatment and / or prevention of Type 1 and Type 2 Diabetes mellitus. Pentacosanoic acid can be given at a dosage range depending on the age, sex, species and body weight. It has the efficacy of increasing insulin levels and/or reducing insulin resistance, lowering blood glucose, and thus can be used to treat and/or prevent Diabetes mellitus.

BACKGROUND

Despite considerable progress in the treatment of diabetes by oral synthetic agents, search for newer drugs continues because the existing drugs have several limitations. Diabetes mellitus is a group of metabolic diseases characterized by high blood sugar (glucose) levels due to deficiency in the production of insulin by the pancreas or ineffectiveness of the insulin produced. The disease causes damages to many of the body's tissues or organs, in particular the blood vessels and the nerves.

There are two forms of diabetes, Type 1 diabetes and Type 2 Diabetes mellitus. Type 1 diabetes is also called insulin-dependent diabetes mellitus (IDDM). Type 2 diabetes develops frequently in children and adolescents, and therefore was known as juvenile diabetes. Type 2 diabetes, also called non-insulin-dependent diabetes mellitus (NIDDM), however, environmental factors also play an important role in the development of the disease. Type 2 diabetes can be managed by exercise and dietary modification.

Currently used medicaments for the treatment of diabetes include sulfonylureas, meglitinides, biguanides, thiazolidinediones, and alpha glucosidase inhibitors. The many side effects of insulin therapy and other oral hypoglycemic agents necessitate the use of more effective and safer antidiabetic drugs. Traditional herbal medicine has been used since ancient time in many parts of the world. Isolation of the main compounds from the active extract is a important step in all research activities for developing a novel phytomedicine.

Fruits and vegetables contain many nutrients and photochemical from fruits and vegatables is thought to protect against diabetes (Colditz et al., 1992) and cardiovascular disease (Bazzano et al., 2003). Most important and widely available phytochemicals were Alkaloids, Terpenes, Phenolics, Flavanoids and Glycosides. Alkaloids are most common in flowering plants, such as Fabaceae, Ranunculaceae etc (Filho et al., 2006). Alkaloids such as aconitine, anisodamine, charantine, leurosine show antidiabetic effects (Li et ah, 2004). Herbal preparations from cinnamon is used for the treatment of diabetes mellitus (Anderson et al, 2004). There are several classes of oral hypoglycemic agents available for the treatment of Type 2 diabetes. Sulfonylurea drugs such as glibenclamide have long been used clinically for Type 2 diabetes (Malaisse, 2003). The use of plants or volatile oils containing high d-Limonene are used for treatment of diabetes including Type 1 diabetes and Type 2 diabetes (US 20100272835). Some of the plants containing the compound d-limonene, are lemon, orange etc. Compositions derived from Gymnema sylvestre leaf materials reduce polydipsia, polyuria and polyphagia, regenerate the pancreatic islets of Langerhans, including beta cells, increase production of proinsulin and c- peptide, and lower blood lipids and triglycerides and free fatty acids (US 5980902). Pergularia daemia (Forssk.) Chiov. is a perennial vine in the Apocynaceae family, used traditionally to treat a number of ailments. Terpenoids, flavonoids, and sterols are among the chemicals that have been isolated from the plant. A herbal formulation prepared from Pergularia daemia, Brachiaria eniciformis., Cenchrus ciliaris, Abutilon indicum and Cocinnia grandis for the treatment of mastitis (WO 2012131723).

Pentacosanoic acid can be isolated from the plant Pergularia daemia. Its molecular formula is C₂₅H₅₀O₂. Pentacosanoic acid is a saturated fatty acid which is usually derived from triglycerides or phospholipids and it is important sources of fuel because, when metabolized, they yield large quantities of ATP thereby most of the cell types use either glucose or fatty acids for this purpose. The biosynthesis of fatty acids involves the condensation of acetyl-CoA. Saturated fatty acids obviously provide desirable properties to lipids in membranes by conferring rigidity where this is required, and in controlling the activities of membrane proteins through covalent bonding. Other names of Pentacosanoic acid are Hyenate, Hyenic acid, n-Pentacosanoate and n-Pentacosanoic acid.

People with diabetes can benefit from education about the disease and treatment, good nutrition to achieve a normal body weight, and regular exercise, with the aim of keeping both short-term and long-term blood glucose levels within acceptable limits. Advances in information technologies have enabled medicine to develop a system that provides real-time individualized medical treatments that are easily accessible using internet and wireless technology. This system is called as ubiquitous healthcare. Assessment of psychological and social situation should be included as an ongoing part of the medical management of diabetes.

SUMMARY OF THE INVENTION

Diabetes mellitus, one of the most common endocrine metabolic disorders has caused significant mortality due to microvascular (neuropathy, nephropathy and retinopathy) and macrovascular (heart attack, stroke) complications. The disease is rapidly increasing worldwide and affecting all parts of the world.

The present invention relates to a compound named Pentacosanoic acid used for the treatment and / or prevention of Type 1 and Type 2 Diabetes mellitus. Pentacosanoic acid can act as Amylase, Glucosidase and Lipase inhibitors and possess antioxidant property.

BRIEF DESCRIPTION OF THE DRAWING

Figure 1 Shows the 2D and 3D structure of Pentacosanoic acid.

Figure 2 Shows the % Inhibition of alpha amylase by Pentacosanoic acid

Figure 3 Shows the % Inhibition of alpha glucosidase by Pentacosanoic acid

Figure 4 Shows the % Inhibition of Lipase by Pentacosanoic acid

Figure 5 Shows the % Free radical scavenging activity by Pentacosanoic acid DETAILED DESCRIPTION OF INVENTION

Present invention uses, for the first time, a compound Pentacosanoic acid to treat hyperglycaemic diseases such as Type 1 and Type 2 Diabetes mellitus. The present invention is advantageous if used for the prevention and management of hyperglycemia, abnormal lipids, obesity, hypertension and atherosclerosis involved with diabetic patients.

It is a white crystalline powder soluble in alcohols. Its molecular formula and molecular weight is C₂₅H5₀O₂ and 382 respectively with a melting point of 83°C. Its structure is shown in Figure 1.

In still another embodiment of the present invention, wherein the said compound Pentacosanoic acid has significant antidiabetic activity which is illustrated through the following assays.

1. Determination of alpha amylase (EC 3.2.1.1) inhibitor activity.

2. Determination of alpha glucosidase (EC 3.2.1.20) inhibitor activity.

3. Determination of lipase (EC 3.1.1.3) inhibitor activity.

4. Determination of DPPH Radical Scavenging activity (antioxidant activity).

Alpha glucosidase and amylase inhibitors are used as oral anti diabetic drugs for treating diabetes mellitus. Therefore natural alpha amylase and glucosidase inhibitors can be used as an effective therapy for treating hyperglycemia with minimal side effects. Pancreatic alpha-amylase hydrolyzes complex starches to oligosaccharides in the lumen of the small intestine, whereas intestinal alpha-glucosidases hydrolyze oligosaccharides, trisaccharides and disaccharides to glucose. Trp-516 and Asp-518 residues of Human lysosomal alpha-glucosidase have been deemed critical for the enzyme's catalytic functionality. Inhibition of these enzyme systems reduces the rate of digestion of complex carbohydrates and helps to reduce the incidence of diabetes mellitus.

The DPPH (2,2-diphenyl-l-picrylhydrazyl) antioxidant assay is based on the ability of DPPH, a stable free radical, to decolorize in the presence of antioxidants. When DPPH accepts an electron donated by an antioxidant compound, the DPPH is decolorized which can be quantitatively measured from the changes in absorbance at 517nm.

Inhibition of alpha amylase, alpha glucosidase, lipase and antioxidant activity by Pentacosanoic acid is detailed in the following examples.

Example 1 Determination of alpha amylase inhibitor activity

Mixture containing 200 μΐ of 0.02M sodium phosphate buffer, 20 μΐ of alpha amylase and the Pentacosanoic acid in concentration 20-100 μ§/πι1 were incubated for 10 minutes at room temperature followed by addition of 200μ1 of starch in all test tubes. The reaction was terminated with the addition of 400 μΐ DNS reagent and placed in boiling water bath for 5 minutes, cooled and diluted with 15 ml of distilled water. Absorbance was measured at 540 nm. The control samples were prepared without Pentacosanoic acid. Acarbose was used as the reference alpha amylase inhibitor. The above experiment was repeated for three times.

The % inhibition was calculated according to the formula

Inhibition (%) = ((Abs 540 (control) - Abs 540 (extract)) * 100/Abs 540(control)

TABLE 1: Shows the % Inhibition of alpha amylase

Concentration % Inhibition of alpha amylase

ug ml Acarbose Pentacosanoic

acid

20 9.21 10.12

40 12.24 _t 21.53

60 19.61 ^' 30.14

80 23.23 44.31

100 32.31 57.23

Plots of percent inhibition versus inhibitor concentration were drawn in Figure 2 using the % inhibition values from Table 1. The IC50 (concentration required to decrease the reaction rate to

50%) for each sample was calculated from the linear curve. Pentacosanoic acid and acarbose were used with a final concentration of 20, 40, 60, 80 and 100 μgmΓ^!. Acarbose an a-amylase inhibitor used as the positive control, with an IC₅₀ of 167.29 gmΓ Pentacosanoic acid also inhibited a-amylase with an IC₅₀ of 89.63 μ ΐ Γ¹ respectively.

Example 2 Determination of alpha glucosidase inhibitor activity

Yeast alpha glucosidase (from Sigma) was dissolved in 100 mM phosphate buffer pH 6.8 and was used as the enzyme extract. P-Nitrophenyl-a-D-glucopyranoside was used as the substrate. Pentacosanoic acid is used in the concentration ranging from 20-100 μg/ml. Different concentrations of Pentacosanoic acid were mixed with 320 μΐ of 100 mM phosphate buffer pH 6.8 at 30 °C for 5 minutes. 3 ml of 50 mM sodium hydroxide was added to the mixture. Absorbance was measured at 410 nm. The control samples were prepared without Pentacosanoic acid. Acarbose was used as the reference alpha glucosidase inhibitor. All tests were performed in triplicate.

The % inhibition was calculated according to the formula. Inhibition (%) = ((Abs 410 (control) - Abs 410 (extract)) * 100/Abs 410 (control) TABLE 2: Shows the % Inhibition of alpha glucosidase Concentration % Inhibition of alpha glucosidase

μg ml i-l

Acarbose Pentacosanoic

acid

20 14.12 15.13

40 23.25 24.23

60 32.34 33.46

80 41.17 50.12

100 54.35 68.42

The IC₅₀ values were determined from plots of percent inhibition versus inhibitor concentration and were calculated by linear plot.

Figure 3 and Table 2 shows the inhibitory effects of Pentacosanoic acid and acarbose on alpha glucosidase. Pentacosanoic acid and acarbose were used with a final concentration of 20, 40, 60, 80 and 100 μgml Inhibitory effects of Pentacosanoic acid and acarbose on alpha glycosidase is shown with an IC₅₀ of 77.75 and 94.47 μgmΓ¹ respectively.

Example 3 Determination of lipase inhibitor activity

Lipase activity was determined by measuring the rate of release of oleic acid from triolein. Suspension of 90μηιο1 triolein, 12.6μηιο1 lecithin and 9.45μπιο1 taurocholic acid in 9ml of 0.1M Tris buffer (pH 7.0) was sonicated for lOmin for substrate dissolution. Pentacosanoic acid and orlistat (final concentration: 20, 40, 60, 80 and 100 μgmΓ^,), which was used as a positive control, were diluted with 0.1M Tris buffer (pH 7.0). 50 μΐ of pancreatic lipase (2 mgrnl^"1 in Tris buffer), ΙΟΟμΙ of substrate solution and ΙΟΟμΙ of sample solution were mixed and incubation was carried out at 37°C for 30min. Then it was mixed with 1ml of chloroform-n-heptane (1: 1) containing 2% methanol. The mixture was centrifuged at lOOOg for 5min, and the upper aqueous phase was removed by suction. Copper reagent (700μ1) was then added to the lower organic phase. The tube was then shaken for 5min and centrifuged at lOOOg for 5min, after which 0.5ml of the upper organic phase (which has the copper salts of the extracted fatty acid) was mixed with 0.5ml of 0.1% bathocuproine in chloroform containing 0.05% 3-(2)-tert-butyl-4-hydroxyanisol. Absorbance was measured at 490nm. Orlistat is used as a positive control.

The % inhibition was calculated according to the formula

Inhibition (%) = ((Abs 490(control) - Abs 490(extract)) * 100/Abs 490(control) TABLE 3: Shows the % Inhibition of Lipase

Concentration % Inhibition of Lipase

μg mΓ Orlistat Pentacosanoic

acid ^'

20 54.12 16.45

40 66.23 26.71

60 78.31 45.04

80 89.15 59.36

100 98.07 74.34 Figure 4 and Table 3 shows the inhibitory effects of Pentacosanoic acid and Orlistat on lipase. Orlistat, a lipase inhibitor used as the positive control, strongly inhibited lipase activity with an

IC₅₀ of 10.96 μgml Pentacosanoic acid also inhibited lipase with an IC₅₀ of -67.59 gml ^l.

Example 4 Determination of DPPH (2,2-diphenyl-l-picrylhydrazyl) Radical Scavenging activity (antioxidant activity)

Ethanolic extract of Pentacosanoic acid (0.2 ml) at different concentrations (50, 100, 150 and 200 μ§/χηϊ) was mixed with 0.8 ml of tris HCl buffer (100 mM, pH 7.4). 1 ml of DPPH (500 mM in 1.0 ml ethanol) solution was added to the above mixture. The mixture was shaken and incubated for 30 min in room temperature. Absorbance was measured at 517 nm. Experiments were carried out in triplicates. Ethanolic extract of Pentacosanoic acid (0.2 ml) were used as blank and DPPH ethanolic solution was served as control. BHA (butylated hydroxy anisole) is used as positive control.

Percentage of DPPH scavenging activity was determined as follows:

% DPPH Free radical scavenging

= ((Abs 517(control) - Abs 517(extract)) * 100/Abs 517(control) TABLE 4: Shows the % Free radical scavenging activity

Concentration % Free radical scavenging activity

μg ml^{_^} Butylated Pentacosanoic

Hydroxy acid

Anisole

2 23.1 1 14.45

4 35.16 27.21

6 43.04 38.14

8 52.23 49.49

10 61.25 63.45

Figure 5 and Table 4 shows the antioxidant effects of Pentacosanoic acid. Pentacosanoic acid were used with a final concentration of 2, 4, 6, 8 and 10 μgmΓ¹. Free radical scavenging activity of Pentacosanoic acid and acarbose is shown with an EC₅₀ of 9.05, 7.91 and 7.51 μgmΓ¹ respectively.

Antioxidants protect cells against the damaging effects of reactive oxygen species otherwise called, free radicals such as singlet oxygen, super oxide, peroxyl radicals, hydroxyl radicals and peroxynite which results in oxidative stress leading to cellular damage. Pentacosanoic acid exhibited significant antioxidant activities against DPPH radical scavenging activity. These in vitro assays indicate that Pentacosanoic acid have got profound antioxidant activity which might be helpful in preventing the progress of various oxidative stresses.

Pentacosanoic acid behaves as good inhibitors of alpha amylase, alpha glucosidase, and lipase. The use of Pentacosanoic acid as therapeutic drugs will tend to reduces the rate of digestion of complex carbohydrates thereby helps in the management of hyperglycemia and abnormal lipids. Hence these can be used for the treatment and / or prevention of Type I and Type II diabetes mellitus. Pergularia daemia also possess good antidiabetic property. From the ICs₀ value of alpha amylase (89.63 μgmΓ¹), alpha glucosidase (77.75 μgmΓ¹) and lipase inhibition (67.59 gmΓ¹) we can conclude that Pentacosanoic acid has good antidiabetic property. Hence the dosage of small quantity of Pentacosanoic acid was found to be effective against diabetes mellitus.

INDUSTRIAL APPLICABILITY

Rapid explosion in human population has made almost impossible for modern health facilities to meet health demands all over the world, thus putting more demands on the use of newer and cheaper medicine. Pentacosanoic acid has good anidiabetic property. It can be formulated in a form consisting of decoction, dried decoction, infusion, tincture, dried extract, capsule, tablet, syrup or the like thereof. Pentacosanoic acid is well tolerated by the patients, with fewer unintended consequences than other drugs which can also be used as Nutrients, Stabilizers and Surfactant. NON PATENT CITATIONS

1. Anderson, R.A., Broadhurst, C.L., Polansky, M.M., Schmidt, W.F., Khan, A., Flanagan, V.P., Schoene, N. W. and Graves, .DJ. 2004. Isolatioin and characterization of .polyphenols type-A polymers from cinnamon with insulin-like biological activity. J. Agric. Food Chem., 52: 65-70.

2. Bazzano, L.A., Serdula, M.K., and Liu, S. 2003. Dietary intake of fruits and

vegetables and risk of cardiovascular disease. Curr. Atheroscler. Rep., 5: 492-499.

3. Colditz, G.A., Manson, J.E., Stampfer, M.J., Rosner, B., Willett, W.C., and Speizer, F.E. 1992. Diet and risk of clinical diabetes in women. Am. J. Clin. Nutr., 55: 1018-1023.

4. Filho, J.M.B., Piuvexam, M.R., Moural, M.D., Silval, M.O., Batista, K.V., Cunhal, E.V.L., Fechine, I.M., and Takemura, O.S. .2006. Anti-inflammatory activity of alkaloids: a twenty century review. Rev. Bras. Farmacogn., 16(1).

5. Li, W.L., Zheng, H.C., Bukura, J, and Kimpe, N.D. 2004. Natural medicines used in traditional Chinese medicines for diabetes mellitus. J EthnopharmacoL, 92: 1-21.

6. Malaisse, W.J. 2003. Pharmacology of the meglitinide analogs: new treatment option for type 2 diabetes mellitus. Treat. Endocrinol, 2: 401-414.

PATENT CITATIONS

Filing Publication

Citing Patent Applicant Title

date Date

Use of volatile oil from plants in

US

28-Oct-lO Gang Shu preparing medicaments for

20100272835 10

preventing and treating diabetes

Edayatimangalam Raja

Compositions for treating and Bhavani

preventing diabetes, impaired

Shanmugasundaram,

26-Mar- glucose tolerance and related

US 5980902 9-Nov-99 Kalathinkal Radha

98 symptoms, and methods for

Shanmugasundaram,

preparing and using such Rolland Hebert, Sohail

compositions

Malik, Michael Baker.

WO A herbal formulation to treat

4-Oct-12 Petharajanna

2012131723 12 mastitis in animals

Claims

CLAIMS I Claim:

1. Pentacosanoic acid is used for the treatment and / or prevention of Type 1 and 2 diabetes mellitus.

2. Pentacosanoic acid can be given at a dosage range depending on the age, sex, species, body weight etc and can act more effectively than the standard antidiabetic drugs.

3. Pentacosanoic acid according to claim 1, characterized in that it is a pharmaceutically suitable carrier.

4. Pentacosanoic acid according to claim 1 , act as Amylase, Glucosidase and Lipase inhibitors and possess antioxidant property.

5. Pentacosanoic acid according to claim 1, can be formulated in a form consisting of decoction, dried decoction, infusion, tincture, dried extract, capsule, tablet, syrup or the like thereof.

6. Pentacosanoic acid can be used for the prevention and management of hyperglycemia, high blood pressure, abnormal lipids, obesity, hypertension, atherosclerosis etc involved with diabetic patients.