Specification
FIELD OF THE INVENTION
The present invention generally relates to the preparation of Ursolic acid loaded proniosomal gel by coacervation phase separation method. Ursolic acid is a well known anti-inflammatory bioactive compound. Gel prepared by coacervation phase separation method directly gives gel form of Proniosomes, which is an advantage over other methods of preparation of Proniosomes.
BACKGROUND OF THE INVENTION
Recently the use of herbal medicines has been increased all over the world due to their miraculous therapeutic effects and fewer adverse effects as compared to the modern medicines. The drug delivery system used for administering the herbal medicine to the patient is traditional and out-of-date, resulting in reduced efficacy of the drug. To combat the problem of drug delivery, novel drug delivery system has been incorporated into herbals showing promising results. Transdermal drug delivery system again offers many advantages as compared to traditional drug delivery systems, including oral and parenteral drug delivery system. The major obstacle of TDDS is the low diffusion rate of drugs across the stratum corneum. But encapsulation of the drug in vesicular structures is one such system which has been reported to enhance permeability of drug through the stratum corneum barrier such as in vesicular structure like Proniosomes.
WO/2004/045619 discloses a freeze-dried nanometer powder injection of ursolic acid. It major compose of active material of ursolic acid, complex vehicle of nanometer powder, excipient of freeze-drying. It is prepared through dissolving ursolic acid as active raw material in medical organic solvent, adding complex vehicle of bean lecithin and stearic acid, stirring while heating until reaction completed, adding distilled water, adding excipient, stirring while heating until reaction completed, filtering with microporous membrane and freeze-drying the filtrate.
WO/2007/126221 discloses an anti-inflammatory and analgesic composition, which comprises an Oldenlandia diffusa extract containing ursolic acid and oleanolic acid.
WO/2006/007487 discloses a composition for topical application to the skin to provide tanning, comprising a liposome encapsulated ursolic acid, a tanning agent and a cosmetically acceptable carrier, and methods of use thereof.
WO/2008/086739 discloses use of ursolic acid saponin, oleanolic acid saponin in preparation of increasing leucocyte and/or platelet medicine.
WO/2005/058302 discloses use of compositions comprising oleanic acid and ursolic acid for the preparation of a medicament for the treatment of hypersensitivity and hyper-reactivity
However, these references do not disclose methods for preparation Ursolic acid loaded proniosomal gel by coacervation phase separation method.
Ursolic acid, natural pentayclic triterpenoid carboxylic acid compound naturally present in a large number of vegetarian foods, medicinal herbs and other plants exerts several potential pharmacological effects. But the most potent activity is anti-inflammatory. It has an exceptionally low toxicity profile which makes it extremely valuable. But chemically, the water solubility of this compound is very low, hence the bioavailability and in turn efficacy is also very low. Hence the present attempt was made by incorporating ursolic acid with proniosomal drug delivery system as it shows promising results for the delivery of drug through the skin because of its enhanced skin permeation, improved drug delivery and increased drug entrapment efficiency thereby combating all the problems related to stability, cost and storage.
None of the prior art indicate above either alone or in combination with one another disclose what the present invention has disclosed. The present invention provides a simple and low cost method of methods for preparation of Ursolic acid loaded proniosomal gel by coacervation phase separation method, which can be easily scaled up, for the mass production in industry.
SUMMARY OF THE INVENTION
Ursolic acid, natural pentayclic triterpenoid carboxylic acid compound naturally present in a large number of vegetarian foods, medicinal herbs and other plants exerts several potential pharmacological effects. It has an exceptionally low toxicity profile which makes it extremely valuable. But chemically, the water solubility of this compound is very low, hence the bioavailability and in turn efficacy is also very low. Strong need was thus felt to increase the bioavailability and thereby increase the efficacy of this very potential compound of herbal origin. Proniosomal drug delivery system, a type of novel vesicular drug delivery offers a great potential to reduce the side effects of drugs and increased therapeutic effectiveness entrapping both
hydrophilic and hydrophobic drugs. Encapsulation of the drug into proniosomal vesicles has shown promising results in various other drugs of pharmaceutical importance. Looking at the great potential of the compound ursolic acid, the present study was designed to combine the potential bioactive compound ursolic acid with the proniosomal drug delivery with an aim of enhanced efficacy by overcoming the problem of water insolubility and hence bioavailability. Proniosomal gel of ursolic acid was prepared by coacervation phase separation method and evaluated for its physicochemical and pharmacokinetic parameters.
BRIEF DESCRIPTION OF THE DRAWINGS
The following drawings are included to further demonstrate certain aspects of the present investigation. The invention may be better understood by reference to one or more of these drawings in combination with detailed description of specific embodiments presented herein.
Fig. 1 represents the DSC thermogram of ursolic acid.
Fig.2 represents the DSC thermogram of ursolic acid loaded proniosomal gel.
Table 1 shows the results of Spreadability test of prepared proniosomal gel which was found good.
Fig.3 represents the particle size range of the formulated Ursolic acid loaded proniosomal gel estimated by particle size analyzer. Particle size of the formulation was found to be present in the range of 2 µm to 13 µm which is very suitable for effective transdermal delivery.
Fig.4 represents the SEM image of proniosomal gel loaded with ursolic acid. It is revealed from scanning electron microscopy that the vesicles are well-identified, spherical and discreet with sharp boundaries.
Fig.5 represents the in-vitro release study of ursolic acid loaded proniosomal gel. In-vitro study for drug release revealed that the pattern of drug release is almost uniform and constant. Thus, the drug is constantly available in the blood for longer time.
Table 2 gives the data for paw edema volume and % inhibition for the marketed as well as formulated gel. The results are quite comparable with marketed formulation in terms of % inhibition of the edema or inflammation apart from the pattern of inhibition which is also same.
OBJECT OF THE INVENTION
The drug delivery system used for administering the herbal medicine to the patient is traditional and out-of-date, resulting in reduced efficacy of the drug. Novel drug delivery system is there to solve the problem of delivery of drug resulting in enhanced efficacy and stability of the drug. Ursolic acid loaded proniosomal gel is a combination of herbal drug with novel drug delivery system to exploit full potential of the drug.
Ursolic acid is a potential bioactive compound with many pharmacological actions. Moreover, its anti-inflammatory action is most potent. But water solubility of this active compound is limited; hence bioavailability and in turn efficacy is also very low. Proniosomal drug delivery system is one such approach showing promising results in many drugs to combat the problem of water insolubility, bioavailability and efficacy.
DETAILED DESCRIPTION OF THE INVENTION
Ursolic acid, also known as urson, malol, prunol is a natural pentayclic triterpenoid carboxylic acid compound. It is naturally present in a large number of vegetarian foods, medicinal herbs and other plants. For a long time, it was considered to be pharmacologically inactive. Contemporary scientific research which led to the isolation and identification of ursolic acid revealed and confirmed several potential cosmeceutical effects, pharmaceutical effects and nutraceutical effects. As a pharmaceutical, ursolic acid is a potent anti-inflamatory agent. It is twofold more potent than that of indomethacin, a potent non-steroidal anti-inflammatory agent (Baricevic et al, 2001). The exact mechanism of action is still unclear but the docking analysis reveals that ursolic acid inhibit COX-2 enzyme by hydrophobic and hydrogen bonding interactions (Manikrao et al, 2011). Ursolic acid has an exceptionally low toxicity profile which makes it extremely valuable (Liu, 2009). But chemically, the water solubility of this compound is very low, hence the bioavailability and in turn efficacy is also very low (Liu, 2005). Strong need was thus felt to increase the bioavailability and thereby increase the efficacy of this very potential compound of herbal origin.
Novel drug delivery system, a combination of advance technique and new dosages forms which are far better than conventional dosages forms, improve drug potency, control drug release to
give a sustained therapeutic effect, provide greater safety, finally it is to target a drug specifically to a desired tissue. Novel transdermal drug delivery system again offers many advantages as compared to traditional drug delivery systems, including oral and parenteral drug delivery system. The major obstacle in transdermal application is the low diffusion rate of drugs across the stratum corneum. But encapsulation of the drug in vesicular structures is one such system which has been reported to enhance permeability of drug through the stratum corneum barrier as in vesicular structures like liposomes, niosomes, ethosomes, transferosomes, proniosomes (Kumar et al, 2010; Nikalje and Tiwari, 2012).
Proniosomes (gel) are semisolid liquid crystal products of non-ionic surfactants easily prepared by dissolving the surfactant in a minimal amount of an acceptable solvent (ethanol) and the least amount of aqueous phase (water). This compact liquid crystalline gel can be readily converted into niosomes on hydration (Rawat et al, 2011). Proniosomal gel offers a great potential to reduce the side effects of drugs and increased therapeutic effectiveness entrapping both hydrophilic and hydrophobic drugs (Yadav et al, 2010). Proniosomes are promising drug carriers due to ease of transfer, distribution, measuring and storage, low toxicity due to non-ionic nature, physically and chemically stable, low cost and easy to formulate (Annakula et al, 2010). Different categories of drugs selections for proniosomes formation based upon the below mentioned points:
• Low aqueous solubility drugs
• High dosage frequency drugs
• Low half life
• Controlled drug delivery suitable drugs
• Higher adverse drug reactions drugs (Thejaswi et al, 2011).
Drug molecules in contact with the skin surface can penetrate by the following three potential pathways: through the sweat ducts, through the hair follicles and sebaceous glands (collectively called the shunt or appendageal route), or directly across the stratum corneum (Mathur et al, 2010). The penetration will depends on the nature and type of the drug used, vesicles formed and hydration temperature for the conversion of proniosomes to niosomes. Proniosomes will hydrate to niosomes when applied to skin. On to the skin surface, the niosomes formed adsorbs fuses and loosens the ceramides by competitively breaking the hydrogen bond network leading to high
thermodynamic activity at the interface. This will increase the concentration gradient and hence increases the diffusion pressure for the driving of drug through the stratum corneum (Litha et al., 2011).
Extensive research studies has been carried out on proniosomes showing proniosomes as a promising drug carriers as in case of levonorgestral (Vora et al, 1998), alprenolol hydrochloride (Blazek-Welsh & Rhodes, 2001), estradiol (Fang et al., 2001), chlorpheniramine maleate (Varshosaz et al., 2005), captopril (Gupta et al., 2007), piroxicam (Chandra and Sharma, 2008), flurbiprofen (Mokhtar et al., 2008), haloperidol (Azarbayjani et al., 2009), ketoprofen (Solanki et al., 2009), indomethacin (Tamizharasi et al., 2009), losartan popotassium (Thakur et al., 2009), gugulipid (Goyal et al, 2011).
Hence the present study was designed to combine the potential bioactive compound ursolic acid with the proniosomal drug delivery with an aim of enhanced efficacy by overcoming the problem of water insolubility and inturn bioavailability and efficacy.
EXAMPLES
The present invention is described herein-below with references to the following examples-
EXAMPLE 1
Procurement and Characterization of Ursolic acid
Ursolic acid was procured from Yucca Enterprises, Mumbai. For characterization, Differential Scanning Calorimetry (DSC) of ursolic acid was carried out indicating the melting point of the compound. Two standard calibration curves of drug were plotted, one by RP-HPLC method (Gbaguidi et al., 2005) and one by UV spectrophotometer.
Calibration curve by RP-HPLC method was used for assay of drug in gel matrix for entrapment efficiency studies. For this, 10 µl of different concentrations i.e. 0.5, 2.0, 3.0, 4.0, 10.0 µg/ml of ursolic acid in mobile solvent (Acetonitrile 85: Water 15) were injected into a chromatographic system (Hitachi Elite Lachrom) equipped with Quaternary gradient pump (L2130), photodiode array detector (L2455) and autosampler (L2200). The resolution of ursolic acid was achieved using acetonitrile: water (85:15, v/v) at a flow rate of 0.6 ml/min, as mobile phase in an isocratic
run through a reliasil reversed phase (RP) C18, 5 µm (250X4.6 mm i.d.) column at 25°C. The eluent was monitored for ursolic acid at 210 nm.
The other one was plotted by UV spectrophotometer for carrying out in-vitro drug release studies using a series of different concentrations i.e. 5, 10, 15, 20, 25, 50 µg/ml of ursolic acid in ethanolic phosphate buffer (pH 7.4) at λmax of 215 nm.
EXAMPLE 2
Preparation of the Proniosomal gel
Ursolic acid loaded proniosomal gel was prepared by coacervation phase separation method. Alcohol and total surfactants (mixture of span 60, cholesterol and soya lecithin) were taken in a ratio of 1:1 w/w. Span 60, Cholesterol and soya lecithin were taken in a ratio of 9:2:9 w/w/w respectively. The final ratio of total surfactant: alcohol: aqueous phase (0.1% glycerol solution) was obtained as 5:5:4 w/w/w respectively. Gel was prepared having a concentration of 1% w/w ursolic acid. Span 60, lecithin, cholesterol and drug were taken in a clean dry wide mouth glass tube. The required quantity of alcohol was added to glass tube and all the ingredients were mixed properly. After mixing all the ingredients, the open end of the glass tube was covered with a lid to prevent loss of solvent from it and warmed on a water bath at 60-700 C for about 5 min, until the surfactants were dissolved completely. The aqueous phase (0.1% glycerol solution) was added and warmed on a water bath till a clear solution is formed which on cooling converts into a proniosomal gel.
EXAMPLE 3
Physico-chemical and Pharmacokinetic Evaluation
Spreadability of the formulated gel was found to be good as the value of spreadability indicate that the gel is easily spreadable by small amount of shear. The pH of the formulated proniosomal gel was found to be 7.28 which lies in the normal pH range of the skin and would not produce any skin irritation. DSC endotherms of drug, span 60, cholesterol, soya lecithin and formulated gel were studied and compared to know the interaction between different components of gel. No interaction was found between drug & excipients as no additional peak or melting endotherm
was present. This was further confirmed by FT-IR studies. FT-IR Spectra of drug and excipients were compared with the spectra of formulated gel. FT-IR spectra of the drug in the formulation and the pure drug gave the same kind of peaks proving the intactness of the drug in the formulation. Hence it indicated the stability of the proniosomal gel. Particle size of the proniosomal gel was found in the range of 4 µm to 13 µm which is suitable for topical drug delivery and was uniform throughout the formulation. These results correlates well with the particle size of proniosomes of ibuprofen (Hu and Rhodes, 1999). Surface morphology by scanning electron microscopy imaging confirmed the coating of surfactant onto the vesicles. Most of the vesicles were well identified and were spherical. Some unevenness & aggregation of vesicles that observed under the study may be due to drying process under normal environment condition. The entrapment efficiency of the proniosomal gel of ursolic acid showed a higher value i.e. 93.4 ± 2.4%. This high entrapment efficiency may be attributed to the high lipophilic nature of the drug (log P = 7.92) that is expected to be partitioned almost completely within the lipid bilayers of niosomes. Hence in proniosomal formulation, the entire drug will be intercalated within the bilayers as opposed to the aqueous spaces within the gel. This result was consistent with the entrapment efficiency of celecoxib (Alam et ah, 2010), levonorgestrel (Vora et ah, 1998), ketorolac (Alsarra et ah, 2005), oestradiol (Fang et ah, 2001) in proniosomes. It may also be attributed to the fact that span 60 gives the vesicles with a higher entrapment efficiency ((Yoshioka et ah, 1994; Bhaskaran and Panigrahi, 2002).
In-vitro drug release study, total % cumulative drug release after 24 hours from the proniosomal gel was found to be 60.8%. This may be attributed to higher entrapment of the drug into the vesicles. When cumulative percentage of drug released was plotted against time, the release profile showed a linear relationship with time. No lag phase could be detected because of the minimum sampling time of 1 hr. In-vitro drug release study of ursolic acid revealed that the pattern of drug release is almost constant and slow.
In-vivo evaluation study of anti-inflammatory activity revealed that proniosomal gel of ursolic acid exhibit a significant higher value of p<0.05 in carrageenan induced rat paw edema with comparison to control. Its anti-inflammatory action is quite comparable to the standard gel of diclofenac i.e. voveran gel in terms of % inhibition of edema which in turn indicated the higher
efficacy of the proniosomal gel loaded with ursolic acid in comparison to conventional dosage form. Thus proniosomal gel of ursolic acid exert a potential anti-inflammatory property.
Thus, it is concluded that proniosomal gel loaded with ursolic acid shows promising results during in-vitro as well as in-vivo evaluation with desirable physicochemical parameters. It holds an immense potential for development of topical herbal anti-inflammatory formulation comparable to topical NSAIDs. This research work has established the foundation for future study on the potential of ursolic acid loaded proniosomes for a transdermal delivery system.
ADVANTAGES OF THE INVENTION
Advantages of the formulation claimed in the present invention are:
1. The method used for preparing the formulation is very economic, easy and quick and can easily be employed for large scale production.
2. The developed formulation has better release kinetics hence better bio availability than the conventional dosage formulations available in the market.
3. The present formulation has better therapeutic application than the conventional Ursolic acid formulations available for topical usage.
4. The formulation involves the use of ingredients which are very safe like neutral surfactant and other like compounds.
5. There is no need of adding any additional penetration enhancer as components of the formulation itself acts as penetration enhancer.
6. The formulation is physically and chemically stable.
(Figure Removed)
Fig.l: DSC curve of ursolic acid
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Fig.2: DSC curve of proniosomal gel of ursolic acid Table 1: Spreadability data of proniosomal gel loaded with ursolic acid
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Fig.3: Particle size distribution pattern in proniosomal gel loaded with ursolic acid
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Fig.4: SEM image of proniosomal gel with magnification 1,500X
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Fig.5: Drug release profile for ursolic acid loaded proniosomal gel Table 2: Paw edema and % inhibition in different groups at various time intervals.
(Table Removed)
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We Claim:
1.A gel formed pharmaceutical composition comprising: Ursolic Acid, proniosomal gel, alcohol, total surfactants and aqueous phase.
2. The composition as claimed in claim 1, wherein said Ursolic Acid -loaded proniosomal gel is prepared by a coacervation phase separation method.
3.The composition as claimed in claim 1, wherein the preferred amount of the alcohol and total surfactant in the ratio of 1:1 w/w.
4.The composition as claimed in claim 1, wherein said total surfactants is mixture of span 60, cholesterol and soya lecithin.
5.The composition as claimed in claim 4, wherein said Span 60, Cholesterol and Soya Lecithin were taken in the ratio of 9:2:9w/w/w respectively.
6.The composition as claimed in claim 1, wherein said aqueous phase is0.1% glycerol.
7.The composition as claimed in claim 1, wherein said ratio of total surfactant:alcohol:aqueous phase is obtained as 5:5:4w/w/w respectively.
8.The method of preparation of composition as claimed in claim 1 comprising the steps of: i) obtaining standardized Ursolic acid
loading proniosomal gel by coacervation phase separation method;
iii) mixing with alcohol and total surfactant;
iv) warming on water bath at 60-70°C for about 5 min until the surfactants were dissolved completely;
v) adding an aqueous phase (0.1% glycerol) ;warmed on water bath and allowing to cool on room temperature to give proniosomal gel.
9.The composition as claimed in claim 1-7, wherein said composition is used as anti¬
inflammatory agent.
