Claims:
1. A process for extraction of punicalagin comprising of:
pulverizing undried pomegranate peels in a blender;
ball milling the pulverized pomegranate peels in an aqueous medium of neutral pH at variable solid to liquid ratio;
ball milling pulverized pomegranate peels at low and controlled temperature between 35°C -45°C at different time points;
subsequent to ball milling the slurry is centrifuged to obtain a clear aqueous phenolic extract; and
freeze drying the aqueous phenolic extract to obtain punicalagin rich phenolics in a powder form.
2. The process according to claim 1, wherein sodium phosphate buffer as an adjusted to pH 7 can be used as aqueous solution.
3. The process according to claim 1, wherein ball milling process was carried out with zirconia balls at fixed number of revolutions of 1200 rpm.
4. The process according to claim 1, wherein the aqueous extract can be directly added to the products of medicine, health care, food, cosmetics and the like without further purification.
5. The process according to claim 1, wherein the punicalagin content of phenolics extract was determined by LC-UV/MS.
6. The process according to claim 1, wherein liquid to solid ratio is 10-35 ml/g.
, Description:EXTRACTION OF PUNICALAGIN RICH PHENOLICS

TECHNICAL FIELD
[0001] The present disclosure relates to an ecofriendly and economical extraction process for punicalagin more particularly aqueous extraction of punicalagin using a ball mill from undried pomegranate peels.

BACKGROUND
[0002] The pomegranate is acclaimed for its health benefits and numerous medical properties due to its disease-fighting antioxidant potential. The antioxidant property of pomegranate is due to ellagitannins. Ellagitannins are a family of bioactive polyphenols found in fruits and nuts like pomegranates, black raspberries, raspberries, strawberries, walnuts, and almonds. Squeezing whole pomegranate fruit (P. granatum L.) yields the richest source of ellagitannins among other fruit juices. This juice has been used for centuries in ancient cultures for medicinal purposes. Commercial pomegranate juice, which has recently become popular in various markets, has more potent antioxidant properties than other common fruit juices, and this is attributed to its high content of polyphenols. The polyphenol punicalagin is the predominant pomegranate ellagitannin and it is responsible for more than 50% of the juice's potent antioxidant activity. In fact, there is actually more antioxidant activity from the tannins in the peels than there is in the edible part of fruit itself. Thus, for harnessing the real power of pomegranate peel phenolics there is a need of extraction of phenolics rich in punicalagin from pomegranate waste peels. There are many patents disclosing various extraction method which involves drying prior to extraction. However, drying is a complex and costly process. There are other patents which report using organic solvents as extraction solvent which exert a negative impact on the environment in terms of emission of organic volatile components and require complex lengthy process which may be detrimental to punicalagin and sophisticated facility.
[0003] One of the prior arts discloses the method of purification of punicalagin from pomegranate peels using environmentally unfriendly solvent methanol. Another prior art discloses a multi-step lengthy method (more than 24 h) for preparing punicalagin and ellagic acid from pomegranate rind which involves extraction with acidic water to obtain extract; re-extraction of obtained extract with toxic organic solvent like ethyl acetate followed by hot (100-105°C) acid hydrolysis and purification. Similarly, another prior art describes a multi-step lengthy method (more than 13 h) for separating punicalagin and gallic acid from pomegranate peels which comprises soaking in hot methanol or ethanol, ultrasound treatment and bleaching to obtain extract, then re-extraction of obtained extract with non-food grade and environmentally unfriendly solvent such as ethyl acetate and purification. In another disclosure non-food grade organic solvents such as ethyl acetate and acetone were employed, respectively for the separation of punicalagin from pomegranate peels. Another prior art discloses a method for extracting punicalagin and ellagic acid from pomegranate peels using organic solvent extraction with subsequent acid/alkaline treatments of obtained extract. However, the acidic treatment needs special equipment and alkaline treatment for neutralization of the acidic waste, which generates large amount of salts which requires additional treatment before disposal, hence increasing the cost of the overall process. Similar drawback applies to another prior art which applied acidified ethanol for obtaining polyphenols rich in punicalagin and ellagic acid from pomegranate peels.
[0004] Another prior art employed food grade solvent such as ethanol for the extraction and purification of punicalagin. Yet another disclosure applied pulsed ultrasound treatment using 70% ethanol but obtained phenolics rich in both punicalagin and ellagic acid. Although in few reports water was used as green solvent for extraction of pomegranate peel phenolics using methods such as stirring, pressurized water and ultrasound treatment but the extraction was focused on total phenolics and not on punicalagin. Another prior art disclose enzymatic (mixture of cellulase, hemicellulase and pectinase) treatment to produce an aqueous pomegranate phenolic extract from by-products of the pomegranate juice industry.
[0005] However, this process did not reuse the multiple enzymes and employed pasteurization at 90-115°C to inactivate the enzymes. The limiting factors in enzymatic processes are poor recovery, reusability, stability of enzymes which increase the cost of enzymatic process. In addition, due to pasteurization extracted punicalagin may have been destroyed as seen from the presence of broad variety of phenolics including punicalagin, punicalin, ellagic acid, ellagic acid glycosides in their final polyphenol extract. Keeping in view the drawbacks of the hitherto reported prior art, the inventors of the present disclosure realized that there exists a dire need to provide a simple and environmentally friendly approach to recover punicalagin rich phenolics from waste pomegranate peels, wherein the energy intensive process of drying waste pomegranate peels and the use of organic solvents have been avoided in addition to simplifying the multistep complex process optimization. Such an approach would also improve the overall process economics.
[0006] The waste pomegranate peels generated by pomegranate processing industries contain antioxidant polyphenol punicalagin of high commercial value. Most of the waste pomegranate peel extraction technologies invented in patents and published in literature report the recovery of total phenolics therefrom, which are devoid of the key polyphenol i.e. punicalagin, while few employed costly drying of peels, toxic organic solvents, and multistep complex processes to obtain punicalagin rich phenolics. However, in the present disclosure, punicalagin rich phenolics were recovered from waste pomegranate peels without employing drying of peels, organic solvent, and multistep complex process. This disclosure is also unique in terms of its environmentally benign nature that is no use of hazardous solvents. This was achieved by ball mill assisted aqueous extraction of fresh waste pomegranate peels at neutral pH and low temperature. The punicalagin rich phenolics obtained through this process are in neutral aqueous medium and therefore, can be directly used for specialty applications.
[0007] However, the present Na-NiCl2 have few drawback related to the design and the size of the batteries. The present Na-NiCl2 batteries have lower power densities and limitation on its size reduction.
OBJECTS OF THE INVENTION
[0008] The object of the present disclosure is to provide an aqueous extraction process for punicalagin from undried pomegranate peels.
[0009] Another object of the disclosure is to provide an ecofriendly and economical extraction process.
[0010] Yet another object is to use the ball mill for the aqueous extraction.
[0011] Yet another object is to carry out the extraction process at low temperature and neutral pH for a specific period of time.

SUMMARY
[0012] The process is aimed at providing an ecofriendly and economical aqueous extraction technique for extracting ellagitanins from pomegranate peels. Initially the pomegranate peels are pulverized and are reduced to particle size of 3-5 mm. Pulverized peels along with neutral sodium phosphate buffer is added to the ball mill and ball mill is operated at 1200 rpm for time period from 5-30 minutes. The ball milling provides a unique cascading motion to break down plant tissues and cell integrity by the mechanical energy impact, thus it can achieve fast release of WPP phenolics into the surrounding solvent, increase the extraction yield and decrease extraction time. Punicalagin is susceptible to oxidation at high temperature hence the extraction is carried out a controlled temperature of 35°C-45°C. Subsequent to milling the slurry is centrifuged at 2840 g to obtain a clear aqueous extract. The aqueous extract is then freeze dried and converted into powder form. Being recovered using neutral aqueous medium the punicalagin rich phenolic extract can be directly added to products of medicine, health care, food & beverages, dietary supplements, cosmetics and the like without further purification.

BRIEF DESCRIPTION OF THE DRAWINGS
[0013] The detailed description is described with reference to the accompanying figures.
[0014] Fig. 1 Flow chart of overall process of ball milling assisted aqueous extraction of punicalagin rich phenolics.
[0015] Fig. 2a LC-UV chromatograms of punicalagin rich phenolics extract obtained by ball milling of fresh waste pomegranate peels of wonderful variety.
[0016] Fig. 2b LC-UV chromatograms of punicalagin rich phenolics extract obtained by ball milling of fresh waste pomegranate peels of Ruby variety.
[0017] Fig. 3 Mass spectra of punicalagin anomers (alpha and beta punicalagin), (M–H) m/z 1083 and ellagic acid, (M-H) m/z 301 found in punicalagin rich phenolics extract obtained by ball milling of fresh waste pomegranate peels.
[0018] Table. 1 Amount of Extract, total phenolics, punicalagin and ellagic acid obtained using ball milling of fresh waste pomegranate peels.

DETAILED DESCRIPTION
[0019] The present disclosure is focused on aqueous extraction method of WPP phenolics based on ball milling of fresh WPP at low temperature and neutral pH. Punicalagin present in pomegranate rind is susceptible to oxidative degradation and hence it is important to prevent this degradation during the extraction process. Hence, the extraction process is carried out in a controlled temperature maintained between 35°C-45°C. The ball milling provides a unique cascading motion to break down plant tissues and cell integrity by the mechanical energy impact, thus it can achieve fast release of WPP phenolics into the surrounding solvent, increase the extraction yield and decrease extraction time. The application of an aqueous medium at a low temperature and neutral pH during ball milling can result in solubilization of punicalagin without hydrolysis and structural alteration. Therefore, it can be expected that mechanical in-liquid smashing of fresh WPP using ball milling could release phenolics into the surrounding medium. Moreover, the use of fresh WPP by-passes a costly and energy-intensive drying of WPP. Thus, compared to traditional organic solvent extraction methods, ball milling of WPP in an aqueous medium is an eco-friendly method that can recover punicalagin rich phenolics from WPP while avoiding the consumption of organic solvents, costly drying of WPP, decreasing the extraction time and providing high extraction yields.
Extraction of Punicalagin.
[0020] Preparation of undried waste pomegranate peel (WPP) powder:
[0021] Undried WPP of wonderful and ruby variety were procured from the pomegranate processing company. Prior to initiation of extraction procedure, the undried peels were pulverized using a blender at room temperature and at 2000 rpm. The WPP were reduced to particle size of 3-5 mm which increases the surface area and aids in better extraction of punicalagin.
[0022] Milling and aqueous extraction:
[0023] As illustrated in Figure 1 the pulverized WPP (101) was then transferred to a ball mill (103). Aqueous buffer adjusted to pH 7 is then added to the ball mill (103) consisting of pulverized WPP at liquid to solid ratio from 10-35 mL/g. The ball milling process was carried out with 5 zirconia balls (total weight = 65 g) at fixed number of revolutions of 1200 rpm at temperature ranging from 35-45°C for different times (5-30 min). Then the reaction mixture was centrifuged 2840 g for 20 min to obtain clear aqueous phenolic extract (105) rich in punicalagin. The aqueous phenolics extract (105) was freeze dried to obtain punicalagin rich phenolics in powder form (107). The punicalagin content of the aqueous phenolic extract was determined by LC-UV/MS as described further in the description. The total phenolic content (TPC) was determined by Folin-Ciocalteu method (Ainsworth & Gillespie, 2007). The yields of total phenolics and punicalagin were expressed as percentage of pomegranate peel powder on dry basis (Table 1). The phenolic rich extract (86-87% w/w phenolics) containing punicalagin more than 70% (w/w) of extract was obtained at liquid to solid ratio of 15 mL/g with ball milling for 10 min. The overall process is presented in figure 1.
[0024] LC-UV/MS analysis of phenolics:
[0025] The clear aqueous phenolic solutions (105) were analysed by LC-UV/MS for identification and quantification of punicalagin and other major phenolic compounds. Analyses were conducted on an Agilent 1260 Infinity liquid chromatograph system coupled with a 6120 series quadrupole mass spectrometer (Agilent Technologies, Mulgrave, Australia). The system is equipped with a binary solvent delivery module, an autosampler and a DAD detector. LCMS experiments were conducted using a Kinetex® 5 um C18 100Å column (Phenomenex, Torrance, CA) of dimensions 250 mm × 4.6 mm, with particle size of 5 µm. Separation was achieved by employing a binary mobile phase of 0.5% acetic acid in ultrapure water and 0.5% acetic acid in acetonitrile (ACN) operated under gradient conditions. The adopted gradient programming were: 0 min, 0% ACN; 30 min, 20% ACN; 50 min, 60% ACN; 55 min, 100% ACN; 70 min, 100% ACN; 73 min, 0% ACN; 76 min, 0% ACN. All analyses were performed under a constant flow rate of 0.8 mL min-1, oven temperature of 30°C, and injection volume of 10 uL. UV detection wavelength was programmed at 210nm, 254nm, 280 nm, 320 nm, and 378 nm with sampling rate of 5 Hz. Electrospray interface was used in both positive and negative mode. The employed MS settings were: drying gas flow of 12 L/min, nebuliser pressure of 35 psi, drying gas temperature of 350°C, and capillary voltage of 3000 V. A mass scan range of 100-1200 Da with cycle time of 1.49 sec per cycle was applied in all experiments. Data acquisition was performed with Agilent Chemstation Software. Acquired Chemstation data were exported in CSV file format and further processed using Origin software (Origin, Northampton, MA). Data processing were performed using MassHunter software (Agilent Technologies). The results are presented in Table 1. The LC-UV chromatograms and mass spectra have been shown in fig. 2a, 2b and 3, respectively.
[0026] It is to be understood that this disclosure is not limited to the particular methodology, protocols, cell lines, animal species or genera, and reagents described, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present disclosure, which will be limited only by the appended claims.