[0001] The present invention relates in general to phytosomes and more specifically to phytosomes of Ficus religiosa and a process for preparation thereof. The phytosomes of F. religiosa exhibit good consistency, better diffusion and excellent antimicrobial activity.

BACKGROUND OF THE INVENTION
[0002] Background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.
[0003] Phytosomes are advanced forms of herbal products that are better absorbed, utilized and, as a result, produce better results than conventional botanical herbal extracts. The increased bioavailability of the phytosome over the non-complexed botanical derivatives has been demonstrated by pharmacokinetic studies or by pharmacodynamic tests in experimental animals and in human subjects. The effectiveness of any herbal medication is dependent on the delivery of effective level of the therapeutically active compound. Severe limitation exists in their bioavailability when administered orally or topically. Phytosomes are complexes of herbal active components and phospholipids that are better absorbed than the aqueous or alcoholic extracts of the plant actives.
[0004] Ficus religiosa is known for the treatment of numerous disorders including asthma, diabetes, diarrhea, epilepsy, gastric problems, and inflammatory disorders, infectious and sexual disorders. However, the phytochemicals of F. religiosa exhibit poor solubility and thus, it is a challenge to prepare suitable formulations and pharmaceutical preparations thereof.
[0005] Thus, there is a need to provide herbal formulations with excellent antimicrobial properties, enhanced bioavailability, good consistency and diffusion, better tolerance, easy-to-formulate and cost effective. More particularly, there is a need to develop herbal formulations of Ficus religiosa that negates the defects and deficiencies of the existing state of the art whilst simultaneously providing efficient bioavailability and therapeutic efficacy.
OBJECTS OF THE INVENTION
[0006] An object of the present invention is to provide phytosomes of Ficus religiosa having excellent antimicrobial properties, enhanced bioavailability, good consistency and diffusion, and better tolerance, which can overcome the deficiencies prevalent in the current art.
[0007] Another object of the present invention is to provide Ficus religiosa phytosomes with antimicrobial effect that are easy-to-formulate as they exhibit consistency and dispersion properties.
[0008] Still another object of the present invention is to provide a process for preparing phytosomes of Ficus religiosa having excellent antimicrobial properties, enhanced bioavailability, good consistency and diffusion, and better tolerance, which can overcome the deficiencies prevalent in the current art.
[0009] Yet another object of the present invention is to provide a process for preparing phytosomes of Ficus religiosa in a cost effective manner.

SUMMARY OF THE INVENTION
[0010] This summary is provided to introduce a selection of concepts in a simplified form that are further described below in Detailed Description section. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.
[0011] An aspect of the present invention relates to antimicrobial phytosome of Ficus religiosa having a vesicle size in the range from 6.0 to 7.0 µm and an entrapment efficacy in the range from 85% to 95%, characterized in that the phytosome comprises active component of F. religiosa in the range from 60 to 70 µg/ml.
[0012] Another aspect of the present invention relates to antimicrobial phytosomes of Ficus religiosa wherein the active content of F. religiosa is selected from a group comprising phenol, flavonoid, flavolignanan, flavonol, tannin, steroid, phytosterol, furanocoumarin derivative, alkaloid, ß-sitosteryl-D-glucoside, vitamin K, n-octacosanol, methyl oleanolate, lanosterol, stigmasterol, lupen-3-one, and mixtures thereof.
[0013] Another aspect of the present invention relates to antimicrobial phytosomes of Ficus religiosa wherein the mole ratio between phospholipid and F. religiosa active component is 0.5-3:1, and preferably the ratio is 1:1.
[0014] Another aspect of the present invention relates to a process for preparing phytosomes of Ficus religiosa having excellent antimicrobial properties, enhanced bioavailability, good consistency and diffusion, and better tolerance.
[0015] Another aspect of the present invention relates to a process for preparing antimicrobial phytosomes of F. religiosa comprising:
(a) extracting active component of F. religiosa plant part by an alcohol solvent;
(b) preparing a thin layer of phospholipid by adding a phospholipid to a solvent mixture of non-polar solvent and aprotic solvent, and drying the mixture in a rotary evaporator to remove the solvents;
(c) adding the alcoholic extract of step (a) to the thin layer of phospholipid of step (b) and allowing to dry at 37°C-40°C for 1 h;
(d) obtaining the phytosomes by adding buffer having pH in the range of 7.0-7.6 and rotating for 1 h at 37°C-40°C; and
(e) drying the phytosomes obtained in step (d) by means of lyophilisation or spray drying.
[0016] Another aspect of the present invention relates to a process for preparing antimicrobial phytosomes of Ficus religiosa wherein the plant part is selected from a group comprising root, stem, bark, young shoots, leaf, flower, fruit, and seed.
[0017] Another aspect of the present invention relates to a process for preparing antimicrobial phytosomes of Ficus religiosa wherein the alcohol is selected from methanol, ethanol and aqueous mixtures thereof.
[0018] Another aspect of the present invention relates to a process for preparing antimicrobial phytosomes of Ficus religiosa wherein the phospholipid is selected from a group comprising phosphatidylcholine, phosphatidylethanol amine, phosphatidylserine, lecithin, and mixtures thereof.
[0019] Another aspect of the present invention relates to a process for preparing antimicrobial phytosomes of Ficus religiosa wherein the non-polar solvent is selected from a group comprising aliphatic hydrocarbons, chloroform, diethyl ether, pentane, hexane, benzene, toluene and mixtures thereof.
[0020] Another aspect of the present invention relates to a process for preparing antimicrobial phytosomes of Ficus religiosa wherein the aprotic solvent is selected from a group comprising dioxane, acetone, dichloromethane, tetrahydrofuran, ethyl acetate, acetonitrile, dimethylformamide and dimethyl sufoxide and a mixture thereof.
[0021] In another aspect of the present invention, the process for preparing antimicrobial phytosomes of Ficus religiosa comprises: (a) extracting active component of F. religiosa plant part by 80% methanol solvent; (b) preparing a thin layer of phospholipid by adding 0.7g lecithin to a solvent mixture of 5 ml chloroform and 5 ml acetone and drying in a rotary evaporator; (c) adding 30 ml of the extract of step (a) to the thin layer of phospholipid of step (b) and allowing to dry at 37°C-40°C for 1 h; (d) adding 2 ml phosphate buffer having pH 7.4 and rotated for 1 h at 37°C-40°C; and (e) drying the phytosomes obtained in step (d) by means of lyophilisation or spray drying.
[0022] A further aspect of the present invention relates to a composition comprising the antimicrobial phytosome as disclosed herein and obtained by the process as disclosed herein.

BRIEF DESCRIPTION OF DRAWINGS IN THE INVENTION
[0023] The following drawings form part of the present specification and are included to further illustrate aspects of the present disclosure. The disclosure may be better understood by reference to the drawings in combination with the detailed description of the specific embodiments presented herein.
Figure 1 shows the zone of inhibition of aqueous and methanol extract of bark/leaf of F. religiosa against E. coli
Figure 2 shows the zone of inhibition of aqueous and methanol extract of bark/leaf of F. religiosa against E. coli
Figure 3 provides phytosomes prepared according to an embodiment of the present invention.

DETAILED DESCRIPTION
[0024] The following is a detailed description of embodiments of the disclosure. The description that follows, and the embodiments described therein, is provided by way of illustration of an example, or examples, of particular embodiments of the principles and aspects of the present disclosure. The amount of detail offered is not intended to limit the anticipated variations of embodiments; on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure as defined by the appended claims.
[0025] The headings and abstract of the invention provided herein are for convenience only and do not interpret the scope or meaning of the embodiments. The following discussion provides many example embodiments of the inventive subject matter. Although each embodiment represents a single combination of inventive elements, the inventive subject matter is considered to include all possible combinations of the disclosed elements. Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.
[0026] Various terms as used herein are shown below. To the extent a term used in a claim is not defined below, it should be given the broadest definition persons in the pertinent art have given that term as reflected in printed publications and issued patents at the time of filing. Where a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.
[0027] As used in the description herein and throughout the claims that follow, the meaning of “a,” “an,” and “the” includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein, the meaning of “in” includes “in” and “on” unless the context clearly dictates otherwise.
[0028] Unless the context requires otherwise, throughout the specification which follow, the word “comprise” and variations thereof, such as, “comprises” and “comprising” are to be construed in an open, inclusive sense that is as “including, but not limited to.”
[0029] The use of any and all examples, or exemplar language (e.g. “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention. The examples and exemplar embodiments are provided for the purposes of explanation, and not of limitation, of those principles and of the disclosure.
[0030] The present invention relates to phytosomes of Ficus religiosa exhibiting antimicrobial activity and having a vesicle size in the range of 6.0 to 7.0 µm. Preferably, the vesicle size is in the range of 6.2 to 6.8 µm and most preferably in the range of 6.4 to 6.6 µm.
[0031] In an embodiment of the present invention, the antimicrobial phytosome of Ficus religiosa has an entrapment efficacy in the range from 85% to 95%.
[0032] In an embodiment of the present invention, the antimicrobial phytosome of Ficus religiosa comprises active component of F. religiosa in the range from 60 to 70 µg/ml. In an embodiment of the present invention, the active component is selected from a group comprising phenol, flavonoid, flavolignanan, flavonol, tannin, steroid, phytosterol, furanocoumarin derivative, alkaloid, ß-sitosteryl-D-glucoside, vitamin K, n-octacosanol, methyl oleanolate, lanosterol, stigmasterol, lupen-3-one and mixtures thereof. Preferably, the active component is a flavolignanan.
[0033] In an embodiment of the present invention, the antimicrobial phytosome of Ficus religiosa comprise a mole ratio between phospholipid and F. religiosa active component of 0.5-3:1. Preferably the ratio is 1:1. In a preferred embodiment, the phospholipid is a mixture of lecithin and cloesterol, the active component is flavonoids, and the ratio of phospholipid to flavonoid is 1: 1.
[0034] The phytosomes of the present invention are prepared by a process comprising reacting phospholipid with active component of F. religiosa plant. A process for preparing antimicrobial phytosomes of F. religiosa comprises: (a) extracting active component of F. religiosa plant part by an alcohol solvent; (b) preparing a thin layer of phospholipid by adding a phospholipid to a solvent mixture of non-polar solvent and aprotic solvent, and drying the mixture in a rotary evaporator to remove the solvents; (c) adding the alcoholic extract of step (a) to the thin layer of phospholipid of step (b) and allowing to dry at 37°C-40°C for 1 h; (d) obtaining the phytosomes by adding buffer having pH in the range of 7.0-7.6 and rotating for 1 h at 37°C-40°C; and (e) drying the phytosomes obtained in step (d) by means of lyophilisation or spray drying.
[0035] In an embodiment of the present invention, the plant part is selected from a group comprising root, stem, bark, young shoots, leaf, flower, fruit, and seed. Preferably, the plant part is a leaf or a bark.
[0036] In an embodiment of the present invention, the alcohol for extracting the plant active component is selected from methanol, ethanol and mixtures thereof. Preferably the alcohol is methanol.
[0037] In another embodiment of the present invention, the alcohol for extracting the plant active component is an aqueous mixture comprising alcohol in the range from 70%-90% and water in the range from 30%-10%. Preferably the alcohol is 80% and water is 20% in the alcohol aqueous mixture.
[0038] In an embodiment of the present invention, the phospholipid is of a natural or a synthetic origin. The phospholipid is selected from a group comprising phosphatidylcholine, phosphatidylethanol amine, phosphatidylserine, lecithin, and mixtures thereof. Preferably, the phospholipid is lecithin.
[0039] In an embodiment of the present invention, the thin layer phospholipid is mixed with the active component of the F. religiosa plant either alone or in a solvent mixture of non-polar solvents and aprotic solvents.
[0040] In an embodiment of the present invention, the non-polar solvents is selected from a group comprising aliphatic hydrocarbons, chloroform, diethyl ether, pentane, hexane, benzene, toluene and mixtures thereof. Preferably, the non-polar solvent is chloroform.
[0041] In an embodiment of the present invention, the aprotic solvent is selected from a group comprising dioxane, acetone, dichloromethane, tetrahydrofuran, ethyl acetate, acetonitrile, dimethylformamide and dimethyl sufoxide and a mixture thereof. Preferably, the solvent is dioxane or acetone.
[0042] In an embodiment of the present invention, the phytosome is precipitated by phosphate buffer having pH in the range of 7.0-7.6 and preferably pH 7.4.
[0043] In an embodiment of the present invention, the phytosomes upon isolation are further subjected to drying by lyophilisation or spray drying.
[0044] In an exemplary embodiment of the present invention, a process for preparing antimicrobial phytosomes of F. religiosa comprises: (a) extracting active component of F. religiosa plant part by 80% methanol solvent; (b) preparing a thin layer of phospholipid by adding 0.7g lecithin to a solvent mixture of 5 ml chloroform and 5 ml acetone and drying in a rotary evaporator; (c) adding 30 ml of the extract of step (a) to the thin layer of phospholipid of step (b) and allowing to dry at 37°C-40°C for 1 h; (d) adding 2 ml phosphate buffer having pH 7.4 and rotated for 1 h at 37°C-40°C; and (e) drying the phytosomes obtained in step (d) by means of lyophilisation or spray drying. The phytosomes were prepared, as seen in Figure 3.
[0045] In another embodiment of the present invention, there is provided a composition comprising the antimicrobial phytosomes of F. religiosa. In yet another embodiment, the composition comprises antimicrobial phytosomes of F. religiosa prepared according to the process disclosed herein.
[0046] While the foregoing describes various embodiments of the disclosure, other and further embodiments of the disclosure may be devised without departing from the basic scope thereof. The scope of the invention is determined by the claims that follow. The invention is not limited to the described embodiments, versions or examples, which are included to enable a person having ordinary skill in the art to make and use the invention when combined with information and knowledge available to the person having ordinary skill in the art.
Example 1: Preparation of Ficus religiosa extracts
[0047] Fresh leaves and bark of Ficus religiosa were collected from Aryan College campus at Chandigarh, India. The leaves and bark were washed under running tap water, dried and were evaluated for their physico-chemical characteristics.

Table 1: Physiochemical evaluation of leaves of Ficus religosa

S.no. Parameters Results
1 Total ash 14.75
2 Water insoluble ash 8.43
3 Acid insoluble ash 5.07
4 Foreign organic matter content 2.5
5 Loss on drying 8.5

[0048] The leaves were then subjected to alcoholic solvents for extraction of flavonoids. Methanol extraction of fresh leaves was carried out in Ficus religiosa with an 80% alcohol. Dried leaves were extracted separately with ethanol in a Soxhlet extractor. The extracts were stored in refrigerator at 4°C for further use.
[0049] Table 2: Extractive value of leaves of Ficus religiosa
S.no. Parameters Results
1 Water soluble ash value 0.5%
2 Alcohol insoluble ash value 0.2%

[0050] Table 3: Phytochemical screening for flavonoids of extracts of Ficus religiosa
S.no. Name of test Observation
1 Gelatin test +ve
2 Ferric chloride test +ve
3 Zinc hydrochloride test +ve
4 Shinodatesat +ve

[0051] Table 4: Morphological character of extracts of Ficus religiosa
S.no. Parameters Observation
1 Colour Dark green colour
2 Odour Odourless
3 Taste Tasteless
4 Nature Concentrate form
5 Solubility Soluble in water and slight soluble in organic solvent

[0052] The antimicrobial activity was determined by agar well disc diffusion method. For the inoculum preparation, E. coli sample was sub-cultured one day before plate preparation. The culture was inoculated in broth and kept in incubator overnight. For substrate preparation, agar plate was prepared using pour plate method. Wells were punched and 50µl of sample extract were loaded and allowed to diffuse. The plates were kept in incubator at 37°C overnight. The zone of inhibition was measured the following day. The antibacterial activity assessed by measuring the diameter of the inhibition zone formed around the well. The effects were compared with that of the standard antibiotic, streptomycin (Figure 1 and 2).
[0053] Table 5: Zone of inhibition of F. religiosa leaf extracts against E. coli
Aqueous 3cm
Methanol extract 3.3cm
Standards (Streptomycin) 3.7cm

Example 2: Preparation of phytosomes of Ficus religiosa

[0054] Accurately weighed quantity of lecithin 0.7g was dissolved in a mixture of 5 ml of chloroform and 5 ml of acetone in round bottom flask (RBF). A thin layer of phospholipid mixture was formed by complete organic solvent removal in rotary evaporator. This film was hydrated with 30 ml alcoholic extract of leaves of F. religiosa in rotary evaporator at 37°C-40°C for 1 hour. Then, 2 ml of phosphate buffer (pH 7.4), was poured into the mixture of extract and phospholipids and rotated for 1 h in rotary evaporator. The phytosomes were prepared, as seen in Figure 3.
[0055] The prepared phytosomes were filled in amber coloured bottle and stored in freezer at 2°C -8°C until used. In the complex formation of phytosomes, the ratio between phospholipid to flavonoids is in the range from 0.5-3.0 moles:1 mole. Preferably, the ratio of phospholipid to flavonoids is 1:1.
Example 2: Characterization of phytosomes of F. religiosa

[0056] The phytosomes as obtained according to the Example 2 was then characterized using various techniques available in the art and as given below:
• Visualization: Visualization of phytosomes using transmission electron microscopy (TEM) and scanning electron microscopy (SEM).
• Vesicle size and zeta potential: The particle size and zeta potential were determined by dynamic light scattering (DLS) using a computerized inspection system and photon correlation spectroscopy (PCS).
• Entrapment efficiency: The entrapment efficiency of a drug by phytosomes was measured by the ultracentrifugation technique.
• Transition temperature: The transition temperature of the vesicular lipid systems was determined by differential scanning calorimeter.
• Surface tension activity measurement: The surface tension activity of the drug in aqueous solution was measured by the ring method in a Du Nouy ring tensiometer.
• Vesicle stability: The stability of vesicles was determined by assessing the size and structure of the vesicles over time. The mean size was measured by DLS and structural changes monitored by TEM.
• Drug content: The amount of drug was quantified by a modified high performance liquid chromatographic method or spectroscopic method.

[0057] Table 6: Characteristics of phytosomes prepared from F. religiosa leaves
Vesicle size (µm)
Zeta potential Entrapment efficiency (%) Drug content
6.4 ± 0.08 -38.8±1.02 mv 89.26 ± 0.70 65.83
µg/ml

[0058] The antimicrobial effect of the phytosomes of F. religiosa was carried out by the inventors. Table 7 below provides the effect of varying concentrations of the phytosomes.
[0059] Table 7: Zone of inhibition of F. religiosa phytosomes against microorganisms
Bacteria Concentration (50 µl) Concentration (100 µl) Concentration (150 µl) Concentration (200 µl)
A. niger 2cm 3cm 3.1cm 3.3cm
E. coli 2.1cm 2.2cm 2.3cm 2.4cm

Example 3: Comparative antimicrobial effect of F. religiosa extracts and phytosomes

[0060] The comparison in the antimicrobial effect of the alcoholic extracts of the leaves and the phytosomes of F. religiosa was carried out by the inventors. Table 8 below provides the superior effects of the phytosomes.
Table 8: Comparative effect on antimicrobial activity between alcoholic extracts and phytosomes of F. religiosa

Microorganism Zone of inhibition (cm) in methanolic extracts (200µg/mL) Zone of inhibition (cm) Phyotsomes (200µg/mL)
A. niger 1.9 cm 3cm
E. coli 2.4 cm 3.2cm

ADVANTAGES OF THE PRESENT INVENTION
[0061] The present invention provides phytosomes of Ficus religiosa having antimicrobial activity which also overcomes deficiencies associated with the known arts in providing stable herbal formulations of F. religiosa.
[0062] The phytosomes of the present invention is novel and superior over the existing art as it provides enhanced bioavailability of the active component and tolerance over that of aqueous or alcoholic Ficus religiosa plant extracts.
[0063] The present invention provides a process for preparing the phytosomes of Ficus religiosa that have good consistency, better diffusion and antimicrobial properties over the conventional aqueous and alcoholic Ficus religiosa plant extracts.

WE CLAIM

Antimicrobial phytosome of Ficus religiosa having a vesicle size in the range from 6.0 to 7.0 µm and an entrapment efficacy in the range from 85% to 95%, characterized in that the phytosome comprises active component of F. religiosa in the range from 60 to 70 µg/ml.

2. The antimicrobial phytosomes as claimed in claim 1, wherein the active content of F. religiosa is selected from a group comprising phenol, flavonoid, flavolignanan, flavonol, tannin, steroid, phytosterol, furanocoumarin derivative, alkaloid, ß-sitosteryl-D-glucoside, vitamin K, n-octacosanol, methyl oleanolate, lanosterol, stigmasterol, lupen-3-one, and mixtures thereof.

3. The antimicrobial phytosomes as claimed in claim 1, wherein the mole ratio between phospholipid and F. religiosa active component is 0.5-3:1, and preferably the ratio is 1:1.

4. A process for preparing antimicrobial phytosomes of F. religiosa of claim 1, comprising:
(a) extracting active component of F. religiosa plant part by an alcohol solvent;
(b) preparing a thin layer of phospholipid by adding a phospholipid to a solvent mixture of non-polar solvent and aprotic solvent, and drying the mixture in a rotary evaporator to remove the solvents;
(c) adding the alcoholic extract of step (a) to the thin layer of phospholipid of step (b) and allowing to dry at 37°C-40°C for 1 h;
(d) obtaining the phytosomes by adding buffer having pH in the range of 7.0-7.6 and rotating for 1 h at 37°C-40°C; and
(e) drying the phytosomes obtained in step (d) by means of lyophilisation or spray drying.

5. The process as claimed in claim 4, wherein the alcohol solvent is selected from methanol, ethanol and aqueous mixtures thereof.

6. The process as claimed in claim 4, wherein the plant part is selected from a group comprising root, stem, bark, young shoots, leaf, flower, fruit, and seed.
7. The process as claimed in claim 4, wherein the phospholipid is selected from a group comprising phosphatidylcholine, phosphatidylethanol amine, phosphatidylserine, lecithin, and mixtures thereof.

8. The process as claimed in claim 4, wherein the non-polar solvent is selected from a group comprising aliphatic hydrocarbons, chloroform, diethyl ether, pentane, hexane, benzene, toluene and mixtures thereof.

9. The process as claimed in claim 4, wherein the aprotic solvent is selected from a group comprising dioxane, acetone, dichloromethane, tetrahydrofuran, ethyl acetate, acetonitrile, dimethylformamide and dimethyl sufoxide and a mixture thereof.

10. The process as claimed in claim 4 for preparing antimicrobial phytosomes of Ficus religiosa comprising:
(a) extracting active component of F. religiosa plant part by 80% methanol solvent;
(b) preparing a thin layer of phospholipid by adding 0.7g lecithin to a solvent mixture of 5 ml chloroform and 5 ml acetone and drying in a rotary evaporator;
(c) adding 30 ml of the extract of step (a) to the thin layer of phospholipid of step (b) and allowing to dry at 37°C-40°C for 1 h;
(d) adding 2 ml phosphate buffer having pH 7.4 and rotated for 1 h at 37°C-40°C; and
(e) drying the phytosomes obtained in step (d) by means of lyophilisation or spray drying.
11. A composition comprising the antimicrobial phytosome as claimed in claim 1 or the phytosome obtained by the process as claimed in claims 4 and 10.