PISONIA ALBA BASED ANTI FUNGAL AGENT, COMPOSITION AND METHOD OF ISOLATION

FIELD OF INVENTION

The present invention relates to Pisonia alba (Synonym -Pisonia grandis) based anti
fungal agent that possess significant wound healing properties. Further, the present invention relates to a method of isolating a medicinally active substance from the plant Pisonia Alba. More particularly, the present invention relates to a method of isolating Allantoin a medicinally active substance containing nitrogenous molecule from the leaves of Pisonia Alba.

DESCRIPTION OF PRIOR ART

Fungal infections are persistent and are not easily cleared by the handful of drugs
currently available to treat them. Drugs that treat bacterial infections often aim for molecules in the bacterial membrane. However, if drugs attack fungal membranes, the treatments often hit human cells too."The challenge has been to find therapeutic agents that can kill the fungus, but not harm the surrounding tissue; that's proven to be very difficult," Because of the lack of an effective treatment, the mortality rate for some systemic fungal infections is nearly 45 percent," said Prusty Rao.

The mortality rate for the most common fungal infection in hospitals, candidiasis, has been reported to be just as high and though numbers are hard to come by, reports suggest overall fungal infection rates have been on the rise.

Candidemia and invasive candidiasis are bloodstream infections caused by fungi in the Candida genus. They are particularly problematic among immuno-compromised individuals in hospital or other healthcare settings, the researchers explained, and invasive candidiasis can not only prolong hospital visits but also increase mortality risk by more than ten percent. Half the patients who develop serious infections from the fungus Aspergillus will not survive.

THE APPLICATIONS

Plants have always been an exemplary source of drugs and many of the currently
available drugs have been derived directly or indirectly from them. Zingiberaceae, Umbelliferae, Lamiaceae, Meliaceae and Nyctaginaceae family plants are traditionally used in the treatment of fungal infections and many drugs have been formulated using natural products isolated from this family. Plants of the Nyctaginaceae family are reported to be used in poultices for application to bruises, swellings, boils, and abscess. Other reports refer only in vague terms to topical use in the treatment of unspecified skin diseases. Boerhavia diffusa is a species of flowering plant in the four o'clock family which is commonly known as Tar vine or Red spiderling. This species is globally distributed in the Pantropics. Within India, it is found abundantly occurring as a weed throughout, ascending to an altitude of 2000 m. in the Himalayas. It is also cultivated to some extent in West Bengal. It is a plant which is extensively used in folk medicine as Antidote and diuretic. Ethyl acetate extract of root part of the plant has been reported to possess antifungal activity. (Agrawal et al., 2003, Hindustan Antibiotics Bulletin. 2003 Feb-2004 Nov; 45-46(1-4): 1-4)

Bougainvillea spectabilis belongs to the family Nyctaginaceae. It is a deciduous climbing shrub with thin wooden stem. Parts of the plant which have been used for medicinal purposes are the leaves and fruits. The methanolic extracts of flowers (five different colors) were screened for antifungal activity and have been reported to possess good antifungal activity. (Ali et al., 2005, Natural Product Research, Volume 19, Issue 1 January 2005, pages 1 - 5)

Mirabilis is the best known genus in the family. Its most famous species is Mirabilis jalapa, the Four-o'clock flower. Roots and leaves of this plant pacify vitiated pitta, fever, syphilis, inflammation, burns and scalds, and general debility. Five different crude extracts: petroleum ether, chloroform, ethyl ether, ethanol and aqueous extract of Mirabilusjalap have been studied for both in vitro antibacterial and antifungal activites. Positive antifungal activity was reported with the methanolic extract against fungal organism Candida albicans. (Chakraborthy et al J. Pharm. Sci. & Res. Vol.1 (2), 2009, 79-82.)

Pisonia is a genus of flowering plants in the four o'clock flower family, Nyctaginaceae are known as Catch bird trees. Pisonia aculeate, Pisonia alba, Pisonia umbellifera are the three species that grown in India. Pisonia aculeata L. (Nyctaginaceae) is a large shrub distributed throughout India. The leaves and bark are used by the tribes and native medical practitioners to treat various ailments including liver disorders, inflammation, swelling, cough and tumours. They are also used as a counter irritant for swelling and rheumatic pain and used to wash scabies. (Kiritikar KR, Basu BD. Indian Medicinal Plants, Vol. Ill, 2nd edition. International Book Distributors, Dehradun: 1998,248-249).

Pisonia alba (Synonym -.Pisonia .grandis) is widely distributed throughout India and is a widespread evergreen commonly grown lettuce tree and is especially adapted to sea coasts and grows well in gardens in and places near the sea, on both east and west coasts. Preliminary phytochemical studies indicate the presence of flavanoids, steroids, alkaloids, anthraquinone, tannins and saponins (Radha et al., 2008 Journal of Biomedical and Pharmacology, 1:1.). Leaves, stem and root of this species are extensively used by the tribals in the preparation of several folk medicines. It has been extensively used in Indian traditional medicine as an ant diabetic, anti-inflammatory agent (Anbalagan et al., 2002 Natural Product Sciences, 8 :97-99), and used in the treatment of a rheumatism and arthritis, algesia, ulcer, dysentery and snake bite . The leaves are edible and mostly used to treat Wounds. (Prabu et al., 2008, The International Journal of Lower Extremity Wounds, 7(l):21-27.).

BENEFITS OF CHARACTERISTICS FOR INTENDED APPLICATIONS

There have been significant pre-clinical safety studies that have shown that the molecule isolated from P.alba is safe and nontoxic. The U.S. Food and Drug Administration acknowledge that 0.5% to 2.0% concentration can be used for the prevention and treatment of diseases. Effects promoted by isolated molecule in wound healing are complex. First dead tissues are removed and the wounds are cleansed. This is followed by the promotion and acceleration of cell proliferation growth of healthy granular tissues and finally epithelization. It have been confirmed by in-vitro and in-vivo studies that it causes a transient local increase in leucocytes; improves lymph flow; increase the capacity of corneocytes to bind water; reinforces the skin's natural protective barrier and improves its moisture retention, providing a smoothening effect to the skin thus making skin smoother, softer.

Thus there exists a need to provide a commercially valuable molecule extracted from the plant Pisonia alba that possesses significant value as an anti fungal agent. Moreover there is a need for more effective, safe and nontoxic herbal drugs without having any side effects for fungal infections. Even though, Pisonia alba is recommended in the traditional and alternative system of medicine for treating various

ailments, the medicinally active substances present in the leaves of Pisonia alba are yet to be isolated, identified and assessed for specific medicinal activity as an anti fungal agent. The present invention is aimed at providing a process for isolation and purification of a nitrogenous molecule from the leaves of Pisonia alba possessing anti fungal activity. The present invention is further supported by scientific and spectral studies of identifying the isolated molecule as Allantoin.

OBJECTS OF THE INVENTION

One or more of the problems of the conventional prior art may be overcome by various
embodiments of the present invention.

The primary object of the present invention is to provide Pisonia alba (Synonym -
Pisonia grandis) based anti fungal agent.

Another object of the present invention is to provide for a method of isolation of a
medicinally active substance from the plant Pisonia alba.

It is further object of the present invention to provide for a method of isolation of
Allantoin, a medicinally active nitrogenous molecule from the leaves of Pisonia alba
that contains significant value as an anti fungal agent.

It is yet another object of the present invention, wherein the method includes
extraction and purification of the isolated Allantoin.

It is another object of the present invention, wherein the isolated molecule possesses
significant properties such as wound healing and cleansing.

It is another object of the present invention, wherein the isolated molecule possesses
significant such as skin protecting, and counter irritant properties

It is another object of the present invention to provide a composition based on Pisonia
alba anti fungal agent that possess significant wound healing properties.

It is another object of the present invention, wherein the isolated molecule is safe and
nontoxic.

It is another object of the present invention, wherein the composition is safe and
nontoxic.

It is another object of the present invention, wherein the antifungal agent acts as a cell proliferant.

It is another object of the present invention, wherein the composition of the antifungal agent acts as a cell proliferant.

It is another object of the present invention, wherein the antifungal agent acts as an epithelization stimulant.

It is another object of the present invention, wherein the composition of the antifungal agent acts as an epithelization stimulant.

It is another object of the present invention, wherein the antifungal agent acts as a chemical debrider.

It is another object of the present invention, wherein the antifungal agent promotes wound healing, exfoliates dry and damaged cells, cleanses away necrotic tissue, speeds up cell regeneration and improves the skin- softening action.

It is another object of the present invention, wherein the composition of the antifungal agent promotes wound healing, exfoliates dry and damaged cells, clean away necrotic tissue, speeds up cell regeneration and improves the skin- softening action.

It is another object of the present invention, wherein the antifungal agent alleviates the
untoward skin-irritation effects of certain cosmetic ingredients such as soap and
detergent surfactants, oils and acidic or alkaline materials.

It is another object of the present invention, wherein the composition of the antifungal
agent alleviates the untoward skin-irritation effects of certain cosmetic ingredients
such as soap and detergent surfactants, oils and acidic or alkaline materials.

It is another object of the present invention, wherein the antifungal agent causes a transient local increase in leucocytes.

It is another object of the present invention, wherein the composition of the antifungal agent causes a transient local increase in leucocytes.

It is another object of the present invention, wherein the antifungal agent improves lymph flow.

It is another object of the present invention, wherein the composition of the antifungal agent improves lymph flow.

It is another object of the present invention, wherein the antifungal agent increases the
capacity of corneocytes to bind water.

It is another object of the present invention, wherein the composition of the antifungal agent increases the capacity of corneocytes to bind water.

It is another object of the present invention, wherein the antifungal agent reinforces the skin's natural protective barrier.

It is another object of the present invention, wherein the composition of the antifungal agent reinforces the skin's natural protective barrier.

It is another object of the present invention, wherein the antifungal agent improves its moisture retention, providing a smoothening effect to the skin thus making skin smoother, softer.

It is another object of the present invention, wherein the composition of the antifungal agent improves its moisture retention, providing a smoothening effect to the skin thus making skin smoother, softer.

It is another object of the present invention, wherein the isolated molecule possesses preferred compositions.

It is yet another object of the present invention, wherein the isolated molecule possesses preferred chemical structures/formulas.

SUMMARY OF THE INVENTION

Thus according to the basic aspect of the present invention there is provided a method of isolating the molecule Allantoin from the plant Pisonia alba for use as an anti
fungal agent comprising:

drying the leaves of Pisonia alba {Pisonia grandis);

pulverizing the dried leaves;

treating the pulverized leaves of Pisonia alba (Pisonia grandis) with ethanol;
filtering the ethanol extract;

concentrating the filtered extract to obtain residue PGEE (Pisonia grandis Ethanolic
Extract) containing allantoin;

purifying allantoin by column chromatography; and
obtaining allantoin containing nitrogenous molecule.

It is another aspect of the present invention, wherein the yield of isolated allantoin is 1
% with respect to defatted residue.

It is further aspect of the present invention, wherein during purifying, the Pisonia alba
(Pisonia grandis) ethanolic extract containing allantoin is fractionated with a solvent
to remove chlorophyll, lipids, fats and fat soluble compounds by a known technique of
solid-liquid extraction.

In the above said method, wherein the solvents include benzene, hexane, petroleum
ether and chloroform.

According to yet another aspect of the present invention, there is provided a method of isolating Allantoin from the plant Pisonia alba (Pisonia grandis) for use as an anti fungal agent comprising:

drying the leaves of Pisonia alba (Pisonia grandis); pulverizing the dried leaves;
treating the pulverized leaves of Pisonia alba (Pisonia grandis)vrith ethanol; filtering the ethanol extract;

concentrating the filtered extract to obtain residue PGEE (Pisonia grandis Ethanolic Extract) containing allantoin; purifying allantoin by column chromatography; and obtaining allantoin containing nitrogenous molecule.

It is another aspect of the present invention, wherein the yield of isolated allantoin is 2 % with respect to defatted residue.

It is another aspect of the present invention, wherein during purifying, the Pisonia grandis ethanolic extract (PGEE) containing allantoin is fractionated with a solvent to remove chlorophyll, lipids, fats and fat soluble compounds by a known technique of solid liquid extraction.

In the above said method, wherein the solvents include benzene, hexane, petroleum
ether and chloroform.

According to yet another aspect of the present invention, there is provided a method of isolating Allantoin from the plant Pisonia alba (Pisonia grandis)for use as an anti
fungal agent comprising:

drying the leaves of Pisonia a\ba(Pisonia grandis);

pulverizing the dried leaves;

treating the pulverized leaves of Pisonia alba (Pisonia grandis) with ethanol;

filtering the ethanol extract; and

concentrating the filtered extract to obtain residue PGEE (Pisonia Grandis Ethanolic
Extract) containing allantoin;

purifying allantoin by solid-liquid extraction using petroleum ether and chloroform;
crystallizing with methanol and obtaining allantoin crystals containing nitrogenous molecule.
It is another aspect of the present invention, wherein the yield of isolated allantoin is
5.55%.

It is another aspect of the present invention, wherein the IR values of the isolated
allantoin is in the range of 3438 to 589 cm"1.

According to yet another aspect of the present invention, there is provided an allantoin containing nitrogenous molecule isolated from the plant Pisonia alba for use as an anti fungal agent using the above said method comprising the following structure:

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: illustrates TLC of isolated allantoin compared with fluka allantoin.

Figure 2: illustrates the 'HNMR spectrum.

Figure 3: illustrates the 13C NMR spectrum.

Figure 4, 5 and 6: illustrates 2D NMR techniques such as COSY.

Figure 7 and 8: illustrates 2D NMR techniques such as HSQC.

Figure 9: illustrates Structure of isolated molecule Allantoin.

DETAILED DESCRIPTION OF THE INVENTION WITH REFERENCE TO THE ACCOMPANYING DRAWINGS

As already described, the invention is directed towards to Pisonia alba (Synonym -
Pisonia grandis) based anti fungal agent that possess significant wound healing properties. Moreover, the present invention relates to a method of isolating Allantoin a medicinally active substance containing nitrogenous molecule from the leaves of Pisonia alba. The isolated molecule possesses significant properties such as wound healing and cleansing agent.
The method of isolating a molecule from the plant Pisonia alba, according to the present invention, is described hereunder:

The method of isolation comprises of extraction of the medicinally active substance from the leaves of Pisonia alba and purification of the medicinally active substance. The ingredients required for the process of extraction comprising of shade dried and pulverized leaves of Pisonia alba 100 grams and ethanol 0.5 litres. The method is as follows: 100 grams of shade dried and pulverized leaves of Pisonia alba is treated with 0.5 litres of ethanol under reflux condition for 6 hours. The ethanol extract is filtered through a Buchner filtration apparatus and concentrated using a rotatory evaporator and the residue obtained are designated as PGEE (Pisonia grandis Ethanolic Extract) 15 grams.

Purification of the medicinally active substance: The PGEE (Pisonia grandis Ethanolic Extract) containing allantoin was further purified by the following methods.

Method 1: The column chromatography

Pre-treatment of PGEE (10 grams) was done to remove chlorophyll, lipids, fats and fat soluble compounds by solid liquid extraction technique. Suitable means for removing those compounds are known to those skilled in this art but illustrative examples of suitable solvents include, but are not limited to, the following: benzene, hexane, petroleum ether and chloroform. One embodiment of the present invention used hexane as chosen solvent for extraction because of low-toxicity and high extraction efficiency. After the pre- treatment the defatted residue (6 grams) containing allantoin was made into slurry with silica gel using methanol and dried. The dried slurry was packed into column chromatography for further purification.

The details of column chromatogram are given hereunder:

• Packing material for column = Silica gel (120 g)
• Column built in chloroform (100%CHC13)
• Mobile phase = 100% CHC13 and CHCI3: MeOH mixtures
with increasing percentage of MeOH

• Volume of collected eluates = 100 ml
• Allantoin isolated from = 94:6 CHC13: MeOH mixture &
85:15 CHCl3:MeOH mixture
• Recrystallization by = Ethanol under cold condition (below 5° C)
• Yield (%) = 1% ( with respect to the concentrated
extract)

Method 2: Column chromatography

Pre- treatment of PGEE (10 grams) was done with commercial hexane to remove chlorophyll, lipids, fats and fat soluble compounds by solid liquid extraction technique. After the pre-treatment the defatted residue (6.0 grams) containing allantoin was made into slurry with silica gel using methanol and dried. The dried slurry was packed into column chromatography for further purification.

The details of column chromatogram are given hereunder:

• Packing material for column = Silica gel (120 g)
• Column built in = 90% Petroleum ether and 10% Ethyl acetate
• Mobile phase = Pet ether : Ethyl acetate mixture
100 % Ethyl acetate and Ethyl acetate:

Ethanol mixture

• Volume of collected eluates = 100 ml
• Allantoin isolated from = Ethyl acetate : Ethanol mixture (99:1)
• Recrystallization by = Ethanol under cold condition (below 5° C)
• Yield (%) =2%

Method 3: Solid-liquid extraction followed by crystallization.

PGEE (3 grams) containing allantoin was further purified by solid-liquid extraction followed by crystallization. Pet-ether and chloroform are used sequentially for solid-liquid extraction with ethanol extract. Commercial methanol is used for crystallization techniques to get allantoin crystals.

IDENTIFICATION OF THE ISOLATED COMPOUND

Supporting evidence

Melting Point: 235 - 237 °C (Literature Ref. Wang et al 2007, Subbaratnam et al 1962)

TLC analysis: Figure 1 shows the TLC chromatogram of the isolated molecule compared with standard authentic allantoin.

Stationary phase: Commercial pre coated aluminium plates

Mobile phase : Chloroform- Methanol- Water (6:4:0.2 ml) mixture

Standard allantoin was purchased from Fluka

Formula for Relative factor (Rf)

Rf = Distance moved by substance/Distance moved by the solvent front Referring to figure 1,

A = Standard allantoin

B = Allantoin isolated by column chromatography

C = Allantoin isolated directly from concentrated ethanol residue

Rf of isolated allantoin =0.65

Rf of fluka allantoin = 0.65

Spectral measurements: IR spectra were taken on a Perkin- Elmer Paragon 500 and NMR spectra were measured on a Bruker 500 MHZ AV III new version using TMS as internal standard. The signals in the 'H NMR spectrum as illustrated in figure 2 and 13C NMR spectrum as illustrated in figure 3 were assigned unambiguously using 2D NMR techniques such as COSY as illustrated in figures 4, 5 and 6 and HSQC as illustrated in figures 7 and 8.

Following Table 1 gives the comparison of IR values of isolated molecule with that reported in literature (SDBS data base)

Following Table 2 gives the comparison of proton NMR and carbon- 13 NMR spectral values of isolated allantoin with literature values (Rashed et al., 2004).

Reported Proton and Carbon NMR values: 10.53 (1H, s), 8.05 (1H, s), 6.88 (1H, d, J = 8.1), 5.78 (2H, s), 5.24 (1H, dd, J = 8.1, 2.1). 13C NMR: 173.22, 157.00, 156.41, 62.06 (Wang et al 2007). The 13C NMR spectrum of authentic allantoin (Fluka) was identical with four signals observed in the spectrum at 174.3 (s), 158.2 (s), 157.7, 63.4 (d) ppm (Cook et al 1980). 1HNMR:7.98(s), 10.48(s), 5.25 (d-d), 6.85 (d), 5.72 (s), 13C NMR: 156.64,62.38,173.39,157.29 (Yamamoto et al 1993).

The structural composition of allantoin molecule deduced and identified on the basis of the reported spectral studies is as follows:

The details of the invention, its object and advantages are explained here above is to be understood that the invention, as fully described herein is not intended to be limited by the objects mentioned herein. The described embodiments are to be considered in all respects only as illustrative and not restrictive.

I CLAIM:

1. A method of isolating Allantoin from the plant Pisonia alba for use as an anti fungal agent comprising:

drying the leaves of Pisonia alba;

pulverizing the dried leaves;

treating the pulverized leaves of Pisonia alba with ethanol;

filtering the ethanol extract;

concentrating the filtered extract to obtain residue PGEE (Pisonia Grandis Ethanolic
Extract) containing allantoin;

purifying allantoin by column chromatography; and

obtaining allantoin containing nitrogenous molecule.

2. A method as claimed in claim 1, wherein the yield of isolated allantoin is 1 % with respect to defatted residue.

3. A method as claimed in claim 1, wherein during purifying, the Pisonia grandis ethanolic extract containing allantoin is fractionated with a solvent to remove chlorophyll, lipids, fats and fat soluble compounds by a known technique of solid-liquid extraction.

4. A method as claimed in claim 3, wherein the solvents include benzene, hexane, petroleum ether and chloroform.

5. A method of isolating Allantoin from the plant Pisonia alba for use as an anti fungal agent comprising:

pulverizing the dried leaves;

treating the pulverized leaves of Pisonia alba with ethanol;

filtering the ethanol extract;

concentrating the filtered extract to obtain residue PGEE {Pisonia grandis Ethanolic
Extract) containing allantoin;

purifying allantoin by column chromatography; and

obtaining allantoin containing nitrogenous molecule.

6. A method as claimed in claim 5, wherein the yield of isolated allantoin is 2 % with respect to defatted residue.

7. A method as claimed in claim 5, wherein during purifying, the Pisonia grandis ethanolic extract containing allantoin is fractionated with a solvent to remove chlorophyll, lipids, fats and fat soluble compounds by a known technique of solid liquid extraction.

8. A method as claimed in claim 7, wherein the solvents include benzene, hexane, petroleum ether and chloroform.

9. A method of isolating Allantoin from the plant Pisonia alba for use as an anti fungal agent comprising:

drying the leaves of Pisonia alba;

pulverizing the dried leaves;

treating the pulverized leaves of Pisonia alba with ethanol;

concentrating the filtered extract to obtain residue PGEE (Pisonia grandis Ethanolic
Extract) containing allantoin;

purifying allantoin by solid-liquid extraction using petroleum ether and chloroform;
crystallizing with methanol and

obtaining allantoin crystals containing nitrogenous molecule.

10. The method as claimed in claim 9, wherein the yield of isolated allantoin is 5.55%.

11. The method as claimed in anyone of claims 1 to 10, wherein the IR values of the isolated allantoin is in the range of 3438 to 589 cm"1.

12. An allantoin containing nitrogenous molecule isolated from the plant Pisonia alba for use as an anti fungal agent using the method as claimed in anyone of claims 1 to 11 comprising the following structure: