Drug for Treatment of Diabetes and Diabetic Foot Ulcer Using Rustin Loaded Solid Lipid Nan particles
Field of Invention

The inventive subject matter relates to an anti-diabetic drug that would be used in the■ therapeutics treatment of diabetes and diabetic foot ulcer. More specifically the invention relates to the process of isolation of Rutin from Excoecaria agallocha and synthesize Rutin loaded solid lipid nano particles (SLNp). The novel formulation thus obtained can be used for therapeutics treatment of diabetes and diabetic foot ulcer in the form of oral drug to treat diabetes and in the form of ointment to treat diabetic foot ulcer.

Background of the Invention - Description of Prior Art

Historically, the term "Mangrove" has been applied to plants which live in muddy, wet soil in tropical or subtropical tidal waters. Excoecaria agallocha is a mangrove belongs to Euphorbiaceae family and is traditionally used by the coastal fisher women community for the treatment of reducing pain, sores, wound healing, ulcers, as a purgative and the smoke from the bark to treat leprosy and to control blood sugar. Flavonoids, and particularly quercetin derivatives, have received special attention as dietary constituents in the last few years. Epidemiological studies have pointed out their possible role in preventing cardiovascular disease and cancer and Rutin is abundantly present in onions, apples, tea and red wine (Hertog et al., 1993). This health-promoting activity seems to be related to the antioxidant (free-radical scavenging) activity to flavonoids (Murota and Terao, 2003).

Quercetin (3,3\4\5,7-pentahydroxy flavone) is one of the most common native flavonoids occurring mainly in glycosidic forms such as rutin (5,7,3\4"-OH, 3-rutinose). Rutin exhibits multiple pharmacological activities including anti-bacterial, anti-tumor, anti-inflammatory, anti-diarrheal, anti-ulcer, anti-mutagenic, myocardial protecting, vasodilator, immune modulatory and hepatoprotective activities. One of the promising approaches for enhancing the oral bioavailability of poorly soluble drugs is the incorporation of these drugs in solid lipid nanoparticles. Recently, it has become more and more evident that the development of new drugs alone was not sufficient to ensure progress in drug therapy. Exciting experimental data obtained in vitro were very often followed by disappointing results in vivo because of the insufficient drug concentration due to poor absorption, rapid metabolism and elimination, poor water solubility and high fluctuation of plasma levels due to unpredictable bioavailability after per oral administration.

A promising strategy to overcome these problems involves the development of suitable drug carrier systems. Commonly accepted criteria of advanced drug delivery systems include maximal drug bioavailability, tissue targeting, controlled release kinetics, minimal immune response, ease of administration for patient compliance, and the ability to deliver traditionally difficult drugs such as lipophiles, amphiphiles and biomolecules. The incorporation of poorly soluble drug into Solid Lipid Nanoparticles can enhance gastrointestinal solubilization, absorption and bioavailability of drug. The type 2 diabetes mellitus (previously referred to as non-insulin-dependent diabetes mellitus (NIDDM) or adult-onset diabetes) is a prototypic multifactorial complex disease.

Type 2 diabetes is characterized by complex metabolic derangements, with two main metabolic defects: insulin resistance, a decreased response of peripheral tissues to insulin and P-cell dysfunction that is manifested as inadequate insulin secretion or relative insulin deficiency in the face of insulin resistance and hyperglycemia. It is the most common form and comprises of 90% of people with diabetes around the world (International Diabetes Federation, 2009; WHO, 2009). The prevalence of type-2 diabetes mellitus rates continues to increase with increasing number of patients at risk of serious diabetes-related complications. India currently leads the world with an estimated 41 million people with diabetes is predicted to increase to 66 million by 2025. The diabetes epidemic is more pronounced in urban areas in India, where prevalence rates of diabetes are roughly double than those in rural areas. Type-2 Diabetes mellitus has well defined diagnostic criteria and its complications are equally understood.

Diabetic complications related to foot are called Diabetic foot. Currently, the approved growth factor and cell therapies for diabetic foot ulcers are not routinely used during treatment. Improper wound healing control may result in diabetic foot ulcer or even amputation. Medicinal Plants are commonly used in treating and preventing specific ailments and diseases and are considered to play a beneficial role in human health. The increased use of herbal remedies, which contain complex mixtures of natural products, need intensified scientific studies to establish efficacy and safety of these types of products. Herbal medicines and their derived ! products are widely used as therapeutic products in many countries. Natural products mainly the plant derived constituents have long been used as a source of drugs. India is the largest producer of medicinal herbs. Around 70% of the population is dependent on the traditional system of medicine. It is important to note that Homeopathy, Ayurveda and modern medicine have their roots in medicinal plants.

The compounds derived from medicinal plants form the ingredients of analgesics, antibiotics, heart drugs, laxatives, anti-cancer agents, ulcer treatments, contraceptives, hormones, diuretics and anti-parasitic drugs etc. Thus, the uses of medicinal plants in the traditional and modern medicines ensure that their importance cannot be ignored. The rising demand has put pressure on the ecosystems of the developing countries from where the bulk of the raw material is derived. A number of synthesis technique has been developed for nanoparticles synthesis including chemical reduction, thermal decomposition in organic solvents, chemical and photo reduction in reverse micelles. Most of these are extremely expensive and they also involve in the use of toxic, hazardous chemicals which may pose biological risk. A quest for an environmentally sustained to synthesis process has lead to few biomimetic approaches. Rutin isolated using current technologies cannot be used because of poor solubility, stability and low absorption when given orally.

Currently insulin and oral hypoglycemic agents are used in the treatment of diabetes mellitus. The main undesirable effect of insulin is that hypoglycemia can cause brain damage, Swelling, erythema and stinging occur specially in the beginning. Allergy to human by insulin is unusual but can occur. Some patients develop short lived dependent edema (due to Na+ retention) when insulin therapy is started. The commonest unwanted effects of metformin are gastrointestinal disturbances, abdominal pain and metallic taste. Lactic acidosis is rare but has potentially toxic effect and metformin should not be given to patients with renal or hepatic diseases, hypoxic pulmonary disease, heart failure or shock. Vitamin B12 deficiency due to interference with its absorption can occur with high dose metformin. The common adverse effects of sulphonyl urea are hypoglycemia, which can be severe and prolonged. Thiazolidinediones causes serious hepatotoxicity, weight gain, gastrointestinal disturbances.

Diabetic wound healing is an enigmatic and debilitating complications and poses a serious challenge in clinical practice. Several issues have been raised with the previously described modalities. Growth factor treatment, for instance, requires large quantities and multiple applications, due to their short half-life and degradation by protease, which has been shown to be not only costly but also could lead to unanticipated side effects. Reportedly, most growth factor and skin grafts used for chronic wounds demonstrated only 25% increase in healing rates. Moreover, autologous tissue harvest means creating a second defect which, under favorable circumstances, may cause infection and other complications especially when a larger piece is required. The procedure is also limited in extreme bum cases which necessitate the use of artificial skin substitutes or other modalities. Allografts also have similar problems to the autologous ones with the addition of biocompatibility and immune rejection concerns. The lack of a vascular network is a key problem for currently available artificial dermal matrices. Poor stability and low mechanical support have been reported with collagen-based scaffolds or dressings.

Limitations concerning the animal models available to test the effectiveness of the new drugs have been also raised as results obtained from such models may lack clinical efficiency when applied to humans. Therefore it is evident that there is need for a better drug delivery system and better product that could overcome the deficiencies identified in the prior art. Due to the potential pharmacological effects present in the herbs mentioned above, the present invention uses the said plants to overcome the problems identified in the prior art to provide for novel composition for the treatment of diabetes and diabetic foot ulcer. The formulation described in this present invention is novel, highly effective and economical. *

Summary of the Invention

The primary object of the invention is to provide for an anti-diabetic drug that would be used in the therapeutics treatment of Type-2 diabetes and diabetic foot ulcer using Rutin obtained and isolated from Excoecaria agallocha and to synthesize the Rutin loaded solid lipid nano particles for providing such treatment. The structure Rutin obtained is Rutin :UV (MeOH) Xmax365, 255 nm, EIMS m/z 610 [M]+, 611[M+H]+ (calc. for C27H30O16), 1H NMR (CD30D, 400 MHz): SH6.20 (1H, d,J = 1.8 Hz, H-6), 6.39 (1H, d,J = 2.2 Hz, H-8), 7.66 (1H, d, J= 1.8 Hz, H-2'), 6.86 (1H, d, J= 8.0 Hz, H-5'), 7.60 (1H, dd, J= 8.0/1.8 Hz, H-6'), 5.09 (1H, d, J= 7.8 Hz, H-l"), 3.25-3.47 (4H, m, H-2", H-3", H-4", H-5"), 3.38 (1H, m, Ha-6"), 3.80 (1H, d,J= 10.5 Hz, H6-6"), 4.51 (1H, d,J= 1.8 Hz, H-l'"), 3.63 (1H, dd, J= 3.5/1.5 Hz, H-2"'), 3.53 (1H, dd, J= 9.5/3.5 Hz, H-3'"), 3.28 (1H, m, H-4'"), 3.44 (1H, w, H-5'"), 1.11 (3H, d, J= 6.0 Hz, CH3-6'"); 13C NMR (CD30D, 100 MHz): <5C158.5 (C-2), 135.6 (C-3), 179.4 (C-4), 162.5 (C-5),99.9 (C-6), 166.0 (C-7), 94.8 (C-8), 159.3 (C-9), 105.6 (C-10), 123.1 (C-l'), 117.6 (C-2')( 145.8 (C-3'), 149.7 (C-4'), 116.1 (C-5'), 123.5 (C-6'), 104.7 (C-l"), 75.7 (C-2"), 77.2 (C-3"), 71.4 (C-4"), 78.1 (C-5"), 68.6 (C-6"), 102.4 (C-l'"), 72.0 (C-2'"), 72.2 (C-3'")( 73.9 (C-4'"), 69.7 (C-5'"), 17.9 (C-6'").

Another object of the present invention is to provide for an anti-diabetic drug that can be used orally for the treatment of type-2 diabetes mellitus. Another object of the present invention is to provide for an anti-diabetic drug that can be made as ointment for the treatment of diabetic foot ulcer. The method of the present invention is directed towards the isolation of Rutin from Excoecaria agallocha and synthesizes the Rutin loaded solid lipid nano particles to form the basic anti¬diabetic drug. The method of this invention is directed to treatment of diabetes using the unique formulations and preparation method of ointment using Rutin obtained from Excoecaria agallocha which provides faster healing of the diabetic foot ulcer. The present inventive subject matter relates to a method for treating diabetes in a subject matter, comprising the step of administering orally an effective amount of a composition to the said subject to treat diabetes, wherein the said composition comprising therapeutically effective amounts of supercritical oral drugs of solid lipid nanoparticle comprises Rutin obtained from Excoecaria agallocha. The present invention subject matter relates to a method for treating diabetic foot ulcer in a subject, through a solid lipid nanoparticle based composition obtained from Excoecaria agallocha in the form of ointment which have potential pharmacological effects. Other features, advantages and embodiments of the invention will be apparent to those skilled in the art from the following description and appended claims.

Detailed Description of the Invention

In the subject specification, except where the context requires otherwise due to express language or necessary. implication, the words "comprise" or variations such as "comprises" or "comprising" are used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention. It must be noted that, as used in the subject specification, the singular forms "a", "an" and "the" include plural aspects unless the context clearly dictates otherwise. Thus, for example, reference to "an extract" includes a single extract, as well as two or more extract; and so forth. The term "extract" as used herein should be taken in the broadest possible sense. "Extracts" may include tinctures, fluid extracts or solid extracts, for example.

The invention is based upon the findings that Excoecaria agallocha contains a range of biologically active substance and reported to possess anti-fungal and anti-bacterial activity, anti¬microbial, anti-cancer, anti-oxidant, anti-inflammatory, anti-gastropathic, anti-rheumatic, antifertility activity, antinociceptive activity, antiproliferative, Anxiolytic effect, cytotoxic and apoptotic activity. In one preferred method of invention the extraction Rutin from Excoecaria agallocha is used to prepare the basic anti diabetic drug. The nanoparticles formulation of the selected plant is used to cure diabetes and diabetic foot ulcer for the first time. In one embodiment of the invention the isolation of Rutin from Excoecaria agallocha is described, wherein initial phytochemical screening of E.agallocha using different solvents viz., petroleum ether, chloroform, ethyl acetate, showed the presence of terpenoids, tannins, flavonoids, alkaloids and phenolic compounds.

In another embodiment of the invention the HPLC-MS revealed the presence of flavonoids such as Ferulic acid, Rutin, Researvatrol, Phloretin, Morin, Naringenin, Apigenin, Tangeritin, Myricetin, Malic acid, Quercetin, Mycertin, Kemferol, Luteolin and Sinapinic acid in the ethanolic extract of Excoecaria agallocha. The quantification of identified Flavonoids was established by HPLC and exhibited 0.1482mg of rutin/g of ethanolic extract of Excoecaria agallocha. Further, Rutin has been isolated and identified using Reverse Phase High Performance Chromatography with USP Rutin standard and characterized using NMR spectroscopy as natural flavanol glycoside derivative of rutin with a molecular formula of C27H3oOi6and a molecular weight of 610.52 g/rhol. In another embodiment of the present invention the Rutin loaded SLNP was prepared using palmitic acid as a lipid matrix because of its high dissolution and polyvinyl alcohol (PVA) as emulsifier by a hot homogenization method which are feasible to prepare poor water solubility drug-loaded SLNP. Selection and formulation of SLNP were prepared using Palmitic acid as a lipid, Rutin and the 1% of PVA to form the oil in water emulsion and further sonicated to form a nanoemulsion. It was poured into 400 mL cold water to obtain nanoparticle suspension.

After centrifugation for 60 min at 4°C, the nano suspension was washed three times with distilled water. The nano encapsulated Rutin loaded Solid Lipid nanoparticles (RSLNP) in distilled water and control Solid Lipid Nanoparticles were prepared similarly without adding Rutin. In another embodiment of the present invention the lyophilized samples were used for following analysis. RSLNP were characterized using measurement of particle size, Zetapotential, Encapsulation, in vitro drug release, AFM, TEM and FT-IR spectroscopy methods. Loading capacity, entrapment efficiency and in vitro release study of RSLNP were calculated. FT-IR spectra clearly indicated the functional groups responsible for encapsulated Rutin. This observation confirms the appropriate nanoencapsulation of rutin in SLNp.

Example: Diabetes was induced through single intraperitoneal injection of Streptozotocin (60 mg/kg) of animal body weight. On treating the animals with the RSLNp, blood sugar level decrease from 390 mg/dL to 108 ± 5.36 mg/dL. Rats treated with Rutin loaded Solid Lipid Nanoparticles, HbAlc levels were found to be in the near normal range. The activities of serum enzymes AST, ALT, LDH, ALP, and ACP significantly increased in diabetic rats when compared with normal controls. Oral administration of RSLNp for significantly restored the enzyme levels to near normal in diabetic rats. The levels of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and GSH were significantly increased in RSLNp treated group than compared to diabetic control rats. Based on the anti¬diabetic effect, RSLNp was used to evaluate the diabetic foot ulcer wound healing activity through EARSLP ointment (5% w/w) (Excoecaria agallocha Rutin loaded Solid lipid nanoparticles) in Streptozotocin induced diabetic rats. The average ulcer area in the EARSLP ointment treatment group, decreased from 21.2 mm2 on day 1 to 9.51 mm2 on day 8. Thus it is evident that a nanoencapsulated Rutin loaded Solid Lipid nanoparticles and EARSLP ointment formulation from a mangrove plant E. agallocha cures Type 2 Diabetes mellitus and diabetic foot ulcer.

Advantages of the Invention:

1. Using floral system particularly, mangrove plant E.agallocha derived flavonoid for solid lipid nanoparticles synthesis and drug formulation to reduce reduce side effects, toxicity with low cost

2. There is minimum amount of organic solvents utilized to isolate ecofriendly rutin using RP-HPLC

3. Uses of biodegradable physiological lipid Palmitic acid decreases the danger of acute and chronic toxicity and avoidance of organic solvents in production methods.

4. Imported the bioavilability of poor water soluble Rutin molecule through solid lipid nanoparticles formulation

5. 1% Poly vinyl alcohol (PVA) act as good stabilizer compared to other colloidal carrier

6. The incorporation of poorly soluble drug into Rutin loaded solid lipid nanoparticles can enhance gastrointestinal solubilization, absorption and bioavailability of drug

7. 5-20nm size of Rutin loaded solid lipid nanoparticles provide biological opportunities for site-specific drug delivery

8. In vitro drug release and encapsulation efficiency of RSLNp indicated controlled release of drug over a long period can be achieved and protection of incorporated drug against the chemical degradation

9. There is no acute toxicity on oral administration of RSLNp

10. Site specific delivery of RSLNp, enhanced drug penetration into the skin via dermal/topical application

Claims

We Claim,

1. An anti-diabetic drug that can used in the treatment of diabetes and diabetic foot ulcer using Rutin loaded solid lipid nanoparticles and EARSLP ointment from Excoecaria agallocha plant. The chemical structure of Rutin thus obtained is: Rutin :UV (MeOH) Xmax365, 255 nm, EIMS m/z 610 [M]+, 611[M+H]+ (calc. for C27H30O16), 1H NMR (CD30D, 400 MHz): SH 6.20 (1H, d,J = 1.8 Hz, H-6), 6.39 (1H, d, /= 2.2 Hz, H-8), 7.66 (1H, d, J= 1.8 Hz, H-2'), 6.86 (1H, d,J= 8.0 Hz, H-5'), 7.60 (1H, dd, J= 8.0/1.8 Hz, H-6'), 5.09 (1H, d, J= 7.8 Hz, H-l"), 3.25-3.47 (4H, m, H-2", H-3", H-4", H-5"), 3.38 (1H, m, Ha-6"), 3.80 (1H, d, J= 10.5 Hz, H6-6"), 4.51 (1H, d, J = 1.8 Hz, H-l'"), 3.63 (1H, dd, J= 3.5/1.5 Hz, H-2'"), 3.53 (1H, dd,J= 9.5/3.5 Hz, H-3'"), 3.28 (1H, m, H-4'"), 3.44 (1H, m, H-5'"), 1.11 (3H, d,J= 6.0 Hz, CH3-6'"); 13C NMR (CD30D, 100 MHz):