DESC:FIELD
The present disclosure relates to a method for obtaining punicalagin. Particularly, it is an aqueous extraction and purification method to obtain punicalagin selected from punicalagin A and B from Punica granatum.

DEFINITIONS
As used in the present disclosure, the following terms are generally intended to have the meaning as set forth below, except to the extent that the context in which they are used indicates otherwise.
Ellagitannin refers to a hydrolytic product of punicalagin, and belongs to a class of bioactive polyphenols having an oxidative linkage of 3, 4, 5-trihydroxybenzoic acid in the. It is the impurity in the punicalagin.
BACKGROUND
The background information herein below relates to the present disclosure but is not necessarily prior art.
Punicalagin is chemically known as 2, 3-hexahydroxydiphenoyl-gallagyl-d-glucose (CAS No. 65995-63-3). Punicalagin belongs to the class of Ellagitannin. It is a bioactive present in the Punica granatum and has multiple biological activities. Among its multiple applications in the chemical-based industry, a reference is made to “Gigliobianco, Maria Rosa, et al. 11.4 (2022): 768” for its use in the cosmetic industry as an antioxidant agent.

Punicalagin bioactive is prone to enzymatic hydrolysis in the bio system due to enzyme tannases. The Punicalagin hydrolysis results into the formation of other smaller phenolic compound i.e. ellagic acid and punicalin. A reference is made to, “Caballero, Víctor, et al. Journal of Agricultural and Food Chemistry 70.51 (2022): 16273-16285” which describes the biodegradation of the punicalagin into other phenolic compounds. Further, the enzymatic extraction of the phytochemicals from a pomegranate is known from “EP2599491B1”. Furthermore, “US20220348603A1” discloses a reverse phase chromatographic separation, which is an isomerization feature-based method to separate ß-punicalagin. In above mentioned methods of the extraction, the bioactive punicalagin extracted from Punica granatum is hydrolysed and so results into the other metabolites as impurities. The hydrolysis eventually reduces the yield of pure punicalagin to 10% (w/w) in the medium. Therefore, the conventionally known methods are not efficient, and so not scalable.

There is, therefore, felt a need that mitigates the drawbacks mentioned hereinabove or at least provide a suitable alternative.
OBJECTS
Some of the objects of the present disclosure, which at least one embodiment herein satisfies, are as follows.
An object of the present disclosure is to provide a method to obtain punicalagin.
Another object of the present disclosure is to provide an extraction and purification method to obtain punicalagin A (alpha) and punicalagin B (beta).
Another object of the present disclosure is to provide an extraction method to obtain punicalagin in high yield of above 80% and high assay purity i.e. above 20% and preferably above 40%.
Another object of the present disclosure is to provide an efficient and a scalable method for extraction and purification of punicalagin, obtained by titration to a pH in the range of 2 to 5.
Still another object of the present disclosure is to provide an aqueous extraction method to obtain purified punicalagin in high yields.
Still another object of the present disclosure is to provide an aqueous extraction of punicalagin in filtrate, by adjusting the pH of the filtrate, which is the immediate titration of the filtrate at a pH in the range of 2 to 5 under reduced pressure for a stabilized punicalagin in the fluid medium.
Yet another object of the present disclosure is to provide a method to obtain punicalagin A and punicalagin B from ripe and unripe Punica granatum in higher yields and purity.
An object of the present disclosure is to ameliorate one or more problems of the background or to at least provide a useful alternative.
Other objects and advantages of the present disclosure will be more apparent from the following description, which is not intended to limit the scope of the present disclosure.
SUMMARY
The invention relates to a method for obtaining punicalagin. The method comprises steps of pre-treating the clean and crushed peels as well as whole fruit of Punica granatum with a fluid medium at a temperature in the range of 60°C to 80 °C to obtain a mixture. The mixture is stirred at a stirring speed of 3 to 10 rpm to obtain slurry, by optionally adding the fluid medium. The slurry is extracted at 70 to 75 °C for 2 to 4 hrs to obtain the punicalagin extract and solid remnants in the fluid medium. The punicalagin extract was clarified, and separated from the solid remnant using the filter having the pore size in the range of 1 to 10 µm to obtain a filtrate. Thus, obtained filtrate is adjusting at pH of 2 to 5 for stabilized punicalagin in the fluid medium. Optionally, the stabilized punicalagin is micro-filtered. The stabilized punicalagin is purified at predetermined conditions, followed by concentrating and drying at 45 °C to obtain punicalagin. The punicalagin is having HPLC purity of 99.5%.
The fluid medium is selected from water, buffer solution, solvent, and a mixture thereof. The pH is adjusted for the immediate titration of the filtrate at a pH in the range of 2 to 5 under reduced pressure for a stabilized punicalagin in the fluid medium. The steps of pre-treating the clean and crushed peels of Punica granatum deactivates the hydrolytic enzymes responsible for the hydrolysis of the punicalagin, selected from punicalagin A and B.
The predetermined condition for purifying the punicalagin extract is a chromatographic separation. The chromatographic separation is based on elution using mild base selected from 0.1 % to 1% of ammonia aqueous solution, followed by evaporation and drying to obtain pure dried powder of punicalagin.
BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWING
The present disclosure will now be described with the help of the accompanying drawing, in which:
Figure 1 represents a typical HPLC chromatogram of the reference Punicalagin indicating peak of punicalagin A and B, as well as Ellagic acid (i.e. a hydrolysis product of Punicalagins);
Figure 2 represents HPLC analysis Chromatogram of extracted and purified Punicalagin (A and B), in accordance with the present invention;
Figure 3 represents HPLC chromatogram of the filtrate containing punicalagins in accordance with the method of the present invention;
Figure 4 represents HPLC chromatogram of filtrate containing punicalagins (i.e. a comparative method, without the temperature and pH stabilization;
Figure 5 represents HPLC chromatogram of filtrate containing punicalagins by process of present invention i.e. process type i involving temperature and pH stabilization and after holding time of 12 hrs;
Figure 6 represents HPLC chromatogram of filtrate containing punicalagins by process type ii (without temperature and pH stabilization) and after holding time of 12 hrs; and
Figure 7 represents HPLC chromatogram of dry punicalagin power obtained from extract and process of present invention (i.e. comparative method, process type i involving temperature and pH stabilization).
DETAILED DESCRIPTION
The present disclosure relates to relates to a method to obtain punicalagin.
Embodiments, of the present disclosure, will now be described with reference to the accompanying drawing.
Embodiments are provided so as to thoroughly and fully convey the scope of the present disclosure to the person skilled in the art. Numerous details are set forth, relating to specific components, and methods, to provide a complete understanding of embodiments of the present disclosure. It will be apparent to the person skilled in the art that the details provided in the embodiments should not be construed to limit the scope of the present disclosure. In some embodiments, well-known processes, well-known apparatus structures, and well-known techniques are not described in detail.
The terminology used, in the present disclosure, is only for the purpose of explaining a particular embodiment and such terminology shall not be considered to limit the scope of the present disclosure. As used in the present disclosure, the forms "a,” "an," and "the" may be intended to include the plural forms as well, unless the context clearly suggests otherwise. The terms "comprises," "comprising," “including,” and “having,” are open ended transitional phrases and therefore specify the presence of stated features, integers, steps, operations, elements, modules, units and/or components, but do not forbid the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. The particular order of steps disclosed in the method and process of the present disclosure is not to be construed as necessarily requiring their performance as described or illustrated. It is also to be understood that additional or alternative steps may be employed.
When an element is referred to as being "mounted on," “engaged to,” "connected to," or "coupled to" another element, it may be directly on, engaged, connected or coupled to the other element. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed elements.
The terms first, second, third, etc.,should not be construed to limit the scope of the present disclosure as the aforementioned terms may be only used to distinguish one element, component, region, layer or section from another component, region, layer or section. Terms such as first, second, third etc., when used herein do not imply a specific sequence or order unless clearly suggested by the present disclosure.
Terms such as “inner,” “outer,” "beneath," "below," "lower," "above," "upper," and the like, may be used in the present disclosure to describe relationships between different elements as depicted from the figures.
Punicalagin is chemically known as 2, 3-hexahydroxydiphenoyl-gallagyl-d-glucose (CAS No. 65995-63-3). Punicalagin belongs to the class of Ellagitannin. Punicalagin bioactive hydrolyses to other smaller phenolic compound such as ellagic acid. Hydrolysis of punicalagin during its extraction, results into the other metabolites as impurities. The hydrolysis eventually reduces the yield of pure punicalagin to 10% (w/w) in the medium. Therefore, the conventionally known methods are not efficient, and so not scalable.

In an aspect, the present disclosure discloses a method for obtaining punicalagin.

In an embodiment, the method of obtaining punicalagin comprising the steps of:
i. pre-treating the clean and crushed peels of Punica granatum with a fluid medium at a temperature in the range of 60°C to 80 °C to obtain a mixture;
ii. dissolving the mixture by optionally adding the fluid medium at a stirring speed of 3 to 10 rpm to obtain a slurry;
iii. extracting the slurry at 70 to 75 °C for 2 to 4 hrs to obtain the punicalagin extract and solid remnants in the fluid medium; and
iv. clarifying, and separating the punicalagin extract from the solid remnant using the filter having the pore size in the range of 1 to 10 µm to obtain a filtrate;
v. adjusting pH of the filtrate to 2 to 5 for stabilized punicalagin in the fluid medium;
vi. optionally, micro-filtering the stabilized punicalagin in the fluid medium; and
vii. purifying the stabilized punicalagin at predetermined conditions, followed by concentrating and drying at 45 °C to obtain powdered punicalagin.

In an embodiment, the first step of pre-treating the clean and crushed peels of Punica granatum with a fluid medium is at a temperature in the range of 60°C to 80 °C to obtain a mixture.
In an embodiment, the fluid medium is selected from water, buffer solution, solvent, and a mixture thereof. In an exemplary embodiment, the fluid medium is water.
In an embodiment, the Punica granatum is treated with the preheated fluid medium for the particulate removal to obtain surface cleaned Punica granatum. The separated peels of Punica granatum are dipped into the preheated fluid medium for 10-15 minutes to obtain pre-treated peels of Punica granatum.
In an embodiment, the preheated fluid medium is having a temperature in the range of 50 to 85o C.
The pre-treatment stabilizes the photochemical constituents in the Punica granatum (pomegranate) peels by deep washing with water, thereby deactivating the enzymes which destroy or degrade the punicalagins during the extraction method.
In an embodiment, the Punica granatum is a mixture of ripe Punica granatumand unripe Punica granatum.
In an embodiment, the parts of Punica granatum including fruits, flowers, leaves, bark, stem etc., they may be fresh or dried parts of Punica granatum
In an embodiment, the punicalagin is extracted by treating Punica granatum with a fluid medium for particulate removal at the surface to obtain cleaned Punica granatum. Further, it involves separating the peels from the arils of the cleaned Punica granatum to obtain peels of Punica granatum.
In an embodiment, the fluid medium, selected from water and is pre heated at temperature in the range of 60 to 80 degree for pre-treatment. In an embodiment, the pre-treating is dipping the peels of Punica granatum into the preheated fluid medium for 10-15 minutes.
In an embodiment, the dried peels of Punica granatum is crushed to obtain crushed particles having diameter in the range of 0.05 mm to 15 mm, prior to pre-treating.
The dried peels of Punica granatum are crushed to obtain crushed particles having diameter in the range of 0.05 mm to 15 mm. In an embodiment, the particle size is less than 0.5 mm. In another embodiment, the crushed pomegranate peels are having particle size of less than 1 mm. In an embodiment, the particle size is more than 15mm. The crushed particles are mixed with preheated fluid medium to obtain slurry.
The peels are separated from the arils of the surface cleaned Punica granatum to obtain separated peels of Punica granatum.
In an embodiment, the second step is dissolving the mixture at a stirring speed of 3 to 10 rpm to obtain slurry, by optionally adding fluid medium.
In an embodiment, the slurry is prepared at a temperature in the range of 50 to 80oC and is having a residence time of at least 30 min and with continuous or intermittent mixing, by using a means preferably, rotary jacketed extractor, or agitated-jacketed extractor, with hot water, hot oil, heat tracing or steam as heating media.
In an embodiment, the predetermined ratio of the crushed particles and preheated fluid medium is in the range of 1:1 to 1:10.In an embodiment, contacting the crushed pomegranate with preheated fluid medium selected from hot water in the ratio of 1:1 to 1:10.
In an embodiment, the third step of extracting the slurry at 70 to 75 °C for 2 to 4 hrs is to obtain the punicalagin extract and solid remnants in the fluid medium.
In an embodiment, the fourth step is clarifying, and separating the punicalagin extract from the solid remnant using the filter having the pore size in the range of 1 to 10 µm to obtain a filtrate. In an exemplary embodiment, the pore size is 5µm.
The solids of Punica granatum are separated from the liquid extract, followed by clarifying of the liquid extract at a predetermined speed in the range of 10rpm to 50rpm to obtain the punicalagin in a mixture.
The solids of Punica granatum are separated from the liquid extract, followed by clarifying of the liquid extract by using centrifugation at a predetermined centrifugal force of 900 to 9000 G force. In an exemplary embodiment, the G force is 3000.
In an embodiment, the clarifying the liquid extract containing punicalagins is done by using disc-stack centrifuge, decanter, filter press, microfiltration, ultra filtration, or suitable combination thereof.
In an embodiment, the fifth step is adjusting pH of the filtrate to 2 to 5 for stabilized punicalagin in the fluid medium.
In an embodiment, the sixth step is optionally, micro-filtering the stabilized punicalagin in the fluid medium.
In an embodiment, the seventh step is purifying the stabilized punicalagin at predetermined conditions, followed by concentrating and drying at 45 °C to obtain punicalagin.
In an embodiment, drying the concentrated or concentrated purified punicalagins is done using rotary vacuum evaporative drying, spray drying, free drying, vacuum drying and low temperature solar dryer. This allows recovery of the dried extract containing punicalagins or dried concentrated purified punicalagins.
The punicalagin has assay purity of 40% w/w and HPLC purity up to 98% w/w. The punicalagins obtained has assay purity of at least 20% and HPLC purity of up to 99.5%.
In an exemplary embodiment, the assay purity of purified punicalagins is 40% w/w and HPLC purity is 98% area.
In an embodiment, concentrating the clarified extract containing punicalagin as obtained above or purified punicalagins is done, by using evaporation under vacuum at temperature below 50 degrees Celsius, or by nano filtration using membrane of molecular weight cut off 500 Da or lesser or combination thereof, thereby allowing to obtain concentrated extract containing punicalagins or concentrated purified punicalagins.
In an embodiment, removing the slurry of crushed pomegranate peels and water and performing solid-liquid separation by means of, but not limited to decanter centrifuge, filter press, screw press, tubular membranes etc. or suitable combination thereof,
In an embodiment, the method involves collecting the liquid extract containing punicalagins.
The punicalagin is purified at predetermined conditions, followed by concentrating and drying to obtain pure punicalagin.
In an embodiment, the predetermined conditions can include, but not limited to titration to a pH in the range of 2 to 8.
In an embodiment, the predetermined conditions can include, but not limited to use of water miscible solvents. In an embodiment, the predetermined conditions can include, but not limited to reduced pressure and concentration.
In an embodiment, the predetermined conditions can include, but not limited to temperature less than 90o C.
In an embodiment, the punicalagin is purified by adsorption or chromatography. In an embodiment, the chromatography is adsorption chromatography.
In an embodiment, the immediate titration of the filtrate comprising punicalagin is neutralization at the pH in the range of 2 to 7. The pH of the filtrate is adjusted using the acid selected from a mineral and an organic acid.
In an embodiment, the Fluid medium is at least one water miscible solvent for the extraction are ethanol, methanol, acetone, isopropyl alcohol, Dimethyl formamide, dimethyl sulfoxide, tert-butyl alcohol, acetonitrile, and a mixture thereof.
In an embodiment, the punicalagin is purified by combining membrane filtration such as ultra filtration, nano filtration. In an embodiment, the membrane filtration is nanofiltration with less than 500 Da cut-off membrane.
In an embodiment, punicalagin A and punicalagin B are extracted and obtained in final dried product as provided.
In an embodiment, the punicalagin purification by adsorption on non-ionic adsorbents in a range of pH 2 to 6, followed by desorption or elution by changing the pH to more than 6.0 and solution of water miscible solvent and water having pH in the range of 2.0 to 7.0, thereby obtains the purified punicalagins.
In an embodiment, the predetermined condition for purifying the punicalagin extract is a chromatographic separation. The chromatographic separation is based on elution using mild base selected from 0.1 % to 1% of ammonia aqueous solution, followed by evaporation and drying to obtain pure dried punicalagin selected from punicalagin A and B.
The highly acidic punicalagins having phenolic hydroxyl groups are titrated with mild base to form salt with the change in pH as per the present invention with reduced hydrophobicity and improved water solubility. This in turn improves yield due to increased water solubility of the salt formed by titration, and also the salt formed gives more stability as compared to acidic form of the punicalagins
The foregoing description of the embodiments has been provided for purposes of illustration and is not intended to limit the scope of the present disclosure. Individual components of a particular embodiment are generally not limited to that particular embodiment but are interchangeable. Such variations are not to be regarded as a departure from the present disclosure, and all such modifications are considered to be within the scope of the present disclosure.
The present disclosure is further described in light of the following experiments which are set forth for illustration purpose only and not to be construed for limiting the scope of the disclosure. The following experiments can be scaled up to industrial/commercial scale and the results obtained can be extrapolated to the industrial scale.
EXPERIMENTS
Requirements:
? The dried peels of pomegranate are provided by Sahyadri.
Sahyadri Farms Post Harvest Care Ltd., GAT NO 314/2/1 AT POST, MOHADI DINDORI NASHIK,NASHIK – 422207Maharashtra.
? The instrument used for analysis was a Shimadzu i series HPLC with Quaternary Gradient Pump, UV detector, autosampler and column oven.
? SEPABEADS SP700 is porous polymeric adsorbent resin with particle size of about 250 micron and about 1200 m2/g surface area with 110 Angstrom pore size. Equivalent or similar adsorbents from Mitsubishi Chemical Corporation, Japan or other manufacturers can be used, and particularly having polystyrene, divinyl benzene, methacrylate, polymethacrylate, cellulose, dextran or like as polymeric backbone with inherent hydrophobicity or modified to obtain hydrophobic surface.
Example 1: A method for obtaining punicalagin
A: Extraction of punicalagin
1000 gm of clean and crushed pomegranate peels were weighed, and dissolved it in 10 L of water at 70 °C, followed by dissolving at stirring speed of 10 rpm to obtain slurry. The slurry was extracted at 73 °C for 2 hrs to obtain the punicalagin extract and solid remnants in the water. The punicalagin extract and solid remnants was filtered using the basket centrifuge using 5 µm filters. The filtrate was treated with H2SO4 to immediately titrate the filtrate, for a pH 3 in water (3L) to obtain the solid wet cake. Total 11.5 L of filtrate was obtained with 7.56 g/L punicalagin content, totalling to 86.94 gm of punicalagins A and B together.
B: Microfiltration of filtrate comprising punicalagin
The filtrate obtained in example 1.A above was then taken for microfiltration (0.2 micron). Three numbers of Di-filtrations using water was given to obtain all punicalagins in the filtrate. About 20 L of permeate was collected from the microfiltration. The permeate is taken ahead for column chromatography.
C: Column purification of filtrate comprising punicalagin
The resin conditioning and loading of the filtrate obtained in Example 1.A, neutralized at pH 3 with water was done. The Elution was carried out using 0.5 % ammonia aqueous solution. Two fractions were collected. The pale yellow color was observed in Fraction 1 and light to dark brown color observed in Fraction 2. The Fraction 2 was given for HPLC analysis.
Concentration of the Elution Fraction 2: The elution fraction 2 was concentrated separately on Rotary Evaporator at 45 °C to obtain dry powder. The extraction and chromatographic purification of punicalagin followed by drying which gave 75.63 gm of Punicalagins A and B together in 176.5 gm of powered product recovered. Thus, the process gave punicalagins in 87% recovery and with 42.85% assay purity.
Table 1 below shows the results of purification of Punicalagin. The results are calculated based on HPLC analysis performed.
Table 1: HPLC analysis results of punicalagin dried powered obtained as per process detailed in example 1, after chromatography and drying.
Name of sample Weight and Solution Alpha Punicalagin Beta Punicalagin Total Area Conc.
Area Area (mg/ml)
Punicalagin Standard,
(27% assay purity) 12.12mg/10 ml 1047353 2452004 3499357 0.33
Punicalagin Dry Powder 10.5mg/10 ml 1286485 3498132 4784617 0.45

The recovery of Punicalagin in the purified fractions from SEPABEADS SP700 is found to be 87% on input punicalagins basis and 17.65% on pomegranate wet peels basis
Example 2: HPLC method details and parameters are as below:

Table 2: Summary of HPLC analysis method parameters
Column Kromasil C18, 5 µm, 4.6×150.
Detector Variable Wavelength Detector
Wavelength 378 nm
Flow Rate 1 ml/min
Injection Volume 10 µl
Column Temperature 35o C
Mode of separation Gradient method
Mobile Phase A- 1% Acetic acid in HPLC grade water
B- 1% Acetic acid in HPLC grade methanol

Table 3: Gradient Program

Sr. No. Time(min) A phase (%) B phase (%)
1 0 95 5
2 1 95 5
3 18 80 20
4 30 25 75
5 33 25 75
6 35 95 5

Table 4: Data of the HPLC chromatogram of reference Punicalagin material indicating peak of punicalagin A and B, as well as hydrolysis product Ellagic acid

Table 5: Data of the HPLC chromatogram of extracted and purified to dry product obtained with Punicalagin A and B, as per process of present invention outlined in example 1.

Example 3:
A. Extraction from wet pomegranate peels in RO Water
• About 4.2 Kg of peels containing 416.5 gm of punicalagins were crushed in the mixer by using 7 L water under hot condition. (70-80 °C).
• About 21 L water was heated to 70 °C in a glass reaction vessel and the crushed peels were added in it. (Ratio 1:7).
• Load Extract-01: Extraction of the peels after crushing was performed with overhead stirring for 1 h at 75-80 RPM, and adjusting the solid to water ratio of 1:10.
B. Basket centrifugation 01
Large-scale Basket Centrifugation: About 48 L of load extract-01 was subjected to basket centrifugation at 300-350 RPM. Wash of 2 L water having pH of 3 was given to the solid residue. The filtrate obtained was clear to brown. The solid residue obtained was almost white indicating maximum product extraction.
Total Volume = 38.6 L
Total punicalagin in the extract: 405.3 gm
Extraction efficiency: 97.3%

Example 4
2 kg of unripe pomegranate peels were taken and punicalagin content was determined. It was found that the punicalagin (A+B) content is 128 gm per kg of pomegranate peels. This material was treated in two different ways, i.e. i) one portion meaning 1 kg of material was extracted using the method of present invention involving stabilization using temperature and pH adjustment of the filtrate and ii) second portion meaning 1 kg of material was extracted without temperature and stabilization parameters. Rest all parameters like solid to water ratio of 1:10, time of extraction of i.e. 1 h, type of solid-liquid separation etc was kept same for both.

After completing the process of extraction, filtration or clarification, HPLC analysis was carried out to determine the punicalagin (A+B) content for process type i (Figure 3) and ii (Figure 4).Table 3 below gives outcome of analysis for both processes.
Table 6: Punicalagin (A+B) content in extract and total punicalagins by two processes as described in this example
Process type alpha Punicalagin Area beta Punicalagin Area Total area of alpha and beta Volume of extract, L Concentration g/L Total Punicalagins (A+B), gram
i – with temperature and stabilization pH 1763877 3569481 5333358 10.20 12.20 124.44
ii – without temperature and stabilization pH 576312 1202910 1779222 10.25 4.10 42.3

Analysis data in Table 6 indicates that process type as per method of present invention gives better extraction efficiency with stabilization of punicalagins and therefore higher content in the extract. Process type ii gives far lower extraction and stabilization and therefore, far lower content of punicalagins in the extract. Further, both extracts were kept at ambient condition for 12 h, and reanalysis was performed. Punicalagin content in extract by process of present invention was found to be same even after 12 hrs of holding (Figure 5) whereas punicalagins content in the extract of process type ii was reduced to 0.22 g/L (Figure 6) concentration with total of 2.26 gm of punicalagins left after holding for 12 h time. In this case, punicalgin peaks were reduced and new peaks before retention time 5 were observed with higher response as seen in Figure 6. Thus, punicalagins in extract of process type ii were degraded or hydrolyzed and therefore the content was reduced substantially making such process industrially unviable. Punicalagins content of extract by the process of present invention (process type i) was same and therefore, was taken for further process of purification, concentration and drying as indicated in example 1. After drying total 198.2 gm of powder was obtained. HPLC analysis (Figure 7) showed the assay purity of 62.5%w/w meaning 123.9 gm of punicalagins in the powder. Thus, total recovery of punicalagin was 99.57% from the clarified and stabilized extract, whereas it is 96.8% recovery from starting material i.e. pomegranate peels.

Figure 3: HPLC chromatogram of filtrate containing punicalagins by process of present invention i.e. process type i involving temperature and pH stabilization.

Figure 4: HPLC chromatogram of filtrate containing punicalagins by process type ii (without temperature and pH stabilization)

Figure 5: HPLC chromatogram of filtrate containing punicalagins by process of present invention i.e. process type i involving temperature and pH stabilization and after holding time of 12 hrs.

Figure 6: HPLC chromatogram of filtrate containing punicalagins by process type ii (without temperature and pH stabilization) and after holding time of 12 hrs.

Figure 7: HPLC chromatogram of dry punicalagin power obtained from extract and process of present invention i.e. process type i involving temperature and pH stabilization.

Table 7: Data representing the HPLC chromatogram of dry punicalagin power obtained from extract and process of present invention i.e. process type i involving temperature and pH stabilization.

Example 5:
5 kg of raw material mixture comprising 4 kg of unripe pomegranates, 0.5 kg of flowers and 0.5 kg of leaves were extracted with 1:10 ratio of solids to water, and with parameters of process of present invention involving temperature of 75 degree Celsius at 6 rpm. Extract was filtered followed by pH stabilization at pH of 3.5. The filtrate was then processed using adsorptive chromatography on 300 mL of Sepabeads packed in glass column of 2.5 cm Dia x 75 cm height, with bed height of 61 cm. Stabilized filtrate was loaded, followed by water wash of pH 3.5 and punicalagins were then eluted using 0.6% ammonia solution. Punicalagins containing fraction was concentrated by evaporation under reduced pressure of 9 mbar and temperature of 45 degree Celsius. Concentrated mass was then vacuum dried for 4 hrs below 40 degree Celsius. About 430 gm of punicalagins (A+B) was obtained in 781 gm of dried powder. The assay purity of punicalagins (A+B) was found to be 55.2%w/w with recovery of 98.78% compared content in input raw material.

Example 6:
9600 kg of mixed pomegranates peels of ripe and unripe pomegranates were processed using process of present invention in similar manner descried in example 1. Input content of punicalagins (A+B) was 261 kg. About 547 kg of dry power containing punicalagins was obtained after spray drying of concentrated material. Spray drying was performed at 165 degree inlet temperature, 145 L/h of feed rate, and 45 to 55 degree outlet temperature. Assay purity of material was found to be 45.87%w/w indicating 250 kg of punicalagins (A+B) and HPLC purity was 99.1%. Recovery of punicalagins 96.1% area.

TECHNICAL ADVANCEMENTS
The present disclosure described hereinabove has several technical advantages including, but not limited to, the realization of a method that is:
• Simple;
• environment-friendly;
• cost-efficient;
• sustainable;
• scalable;
• green; and
• high yield (above 80%), high assay purity (40% and above) as well as high HPLC purity(98% area) of punicalagin is obtained,
• Additionally, for the method, the
o the segregation of ripe and unripe pomegranates is not desired; and
o punicalagin A and punicalagin B are extracted as dried product.

The embodiments herein and the various features and advantageous details thereof are explained with reference to the non-limiting embodiments in the following description. Descriptions of well-known components and method techniques are omitted so as to not unnecessarily obscure the embodiments herein. The examples used herein are intended merely to facilitate an understanding of ways in which the embodiments herein may be practiced and to further enable those of skill in the art to practice the embodiments herein. Accordingly, the examples should not be construed as limiting the scope of the embodiments herein.

The foregoing description of the specific embodiments so fully reveal the general nature of the embodiments herein that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation.

Therefore, while the embodiments herein have been described in terms of preferred embodiments, those skilled in the art will recognize that the embodiments herein can be practiced with modification within the spirit and scope of the embodiments as described herein.

The use of the expression “at least” or “at least one” suggests the use of one or more elements or ingredients or quantities, as the use may be in the embodiment of the disclosure to achieve one or more of the desired objects or results. The numerical values mentioned for the various physical parameters, dimensions or quantities are only approximations and it is envisaged that the values higher/lower than the numerical values assigned to the parameters, dimensions or quantities fall within the scope of the disclosure, unless there is a statement in the specification specific to the contrary.

While considerable emphasis has been placed herein on the components and component parts of the preferred embodiments, it will be appreciated that many embodiments can be made and that many changes can be made in the preferred embodiments without departing from the principles of the disclosure. These and other changes in the preferred embodiment as well as other embodiments of the disclosure will be apparent to those skilled in the art from the disclosure herein, whereby it is to be distinctly understood that the foregoing descriptive matter is to be interpreted merely as illustrative of the disclosure and not as a limitation.

,CLAIMS:WE CLAIM:
1. A method of obtaining punicalagin comprising the steps of:
i. pre-treating the clean and crushed peels (or whole fruits) of Punica granatum with a fluid medium at a temperature in the range of 60°C to 80 °C to obtain a mixture;
ii. dissolving the mixture by optionally adding the fluid medium at a stirring speed of 3 to 10 rpm to obtain a slurry;
iii. extracting the slurry at 70 to 75 °C for 1to 4 hrs to obtain the punicalagin extract and solid remnants in the fluid medium;
iv. clarifying, and separating the punicalagin extract from the solid remnant using the filter having the pore size in the range of 1 to 10 µm to obtain a filtrate;
v. adjusting pH of the filtrate to 2 to 5 for stabilized punicalagin in the fluid medium;
vi. optionally, micro-filtering the stabilized punicalagin in the fluid medium; and
vii. purifying the stabilized punicalagin at predetermined conditions, followed by concentrating and drying at 45°C to obtain powdered punicalagin.

2. The method of obtaining punicalagin as claimed in claim 1 wherein the fluid medium is selected from water, buffer solution, solvent, and a mixture thereof.

3. The method of obtaining punicalagin as claimed in claim 1 wherein adjusting pH is the immediate titration of the filtrate at a pH in the range of 2 to 5 under reduced pressure for a stabilized punicalagin in the fluid medium.

4. The method of obtaining punicalagin as claimed in claim 1 wherein pre-treating clean and crushed peels of Punica granatum deactivate the hydrolytic enzymes responsible for the hydrolysis of the punicalagin, selected from punicalagin A and B.

5. The method of obtaining punicalagin as claimed in claim 1 wherein the slurry consists of crushed particles having size in the range of 0.05mm to 15 mm.

6. The method of obtaining punicalagin as claimed in claim 1 wherein the ratio of crushed particles and the fluid medium is in the range of 1:1 to 1: 10.

7. The method of obtaining punicalagin as claimed in claim 1 wherein the pH of the filtrate is adjusted using the acid selected from a mineral and an organic acid.

8. The method of obtaining punicalagin as claimed in claim 1 wherein at the alkaline pH and higher pH, the punicalagin in the filtrate hydrolyses to ellagic acid, punicalagin and other phenols.

9. The method of obtaining punicalagin as claimed in claim 1 wherein the predetermined conditions for purifying the punicalagin extract is a chromatographic separation, wherein the chromatographic separation is based on elution using mild base selected from 0.1 % to 1% of ammonia aqueous solution, followed by pH correction, evaporation and drying to obtain pure dried punicalagin selected from punicalagin A and B.

10. The method of obtaining punicalagin as claimed in claim 1 wherein the punicalagin is obtained having HPLC purity of 98% w/w. and assay purity of above 40%.