INTRODUCTION:

Pomegranate (Punica granatum L.) is one of the oldest edible fruits widely grown in parts of Asia, North Africa, around the Mediterranean areas and in the Middle East¹. The pomegranate is a symbol of life, longevity, health, femininity, fecundity, morality, immortality and spirituality². Pomegranate exhibits antiviral, antioxidant, anticancer, antiproliferative activities^3,4,5. The bark, leaves, flowers, fruits, and seeds of this plant have been used to ameliorate some diseases⁶. Pomegranate peels and fruits have been widely used in traditional medicine in America, Asia, Africa and Europe for the treatment of different types of diseases^7,8,9,10. The pomegranate fruits have been used to treat acidosis, dysentery, microbial infections, diarrhea, helminthiasis, haemorrhage, and respiratory pathologies¹¹. The phytochemistry of pomegranate has been widely studied and this fruit is found to be a rich source of polyphenolic compounds⁶. Both flavonoids and tannins are more abundant in the peels¹².

Peels of pomegranate contain a wide variety of phytochemical compounds like gallotannins, ellagic acid, gallic acid, punicalins, punicalagins, Pomegranate is consumed in fresh and in processed form as juices, wines, flavours, and extracts. Phenolic compounds, including flavonoids, anthocyanins and tannins, are the main group of antioxidant phytochemicals with interesting properties and have deeply value to their biological and free radical scavenging activities¹⁵. The main objective of this work was to reveal the presence of certain phytochemicals and to quantify the total phenols and flavonoids that have antioxidant activity of pomegranate peel.

MATERIALS AND METHODS:

Plant samples:

Ripe pomegranate fruits were collected in October, 2017 from pomegranate trees in Anantapuramu, Andhra Pradesh, India. Ripe pomegranate fruits were harvested from different trees of Jodhpur Red cultivar. The variety was authenticated by Department of Horticulture, Government of Andhra Pradesh. The English, scientific and family names of the plant under study are: Pomegranate, Punica granatum L and Lythraceae, respectively.

Preparations of crude pomegranate peel and juices:

Peels were cut into small pieces (i.e. 3 cm × 3 cm), placed into plastic containers (50 ml), and were kept in a freezer at -40°C for at least 16 hours. The frozen peels were freeze-dried (i.e. freeze drying started from -40 °C and ended at 20 ^oC) for 96 hours using Freeze Dryer. Dried peels were then stored in a desiccator for one week before being ground into powder.

Preparation of extract:

Dried pomegranate peels (30 g) were ground into powder using a grinder at 9676.8 g. Powders were kept in air tight plastic containers and stored at -40°C until used for extraction in Soxhlet apparatus in 250 ml of ethyl acetate. Ethyl acetate (100 mL) was added to 0.5 g of dried sample in conical flasks and was stirred for 3h at room temperature (20°C). Ethyl acetate extract was capable of recovering many compounds thus ethyl acetate was chosen for the extraction of functional components from pomegranate peels¹⁶. The extractions were performed for 48 h and concentrated by slow evaporation process¹⁷. The obtained extract was kept in moisture free container and used for phytochemcial analysis. To avoid light exposure the flask was covered with aluminum foil. Mixture was then centrifuged for 30 minutes at 9676.8 g at 3°C (REMI-RM 1210, India). The supernatant was filtered using Whatman filter paper No 1, 155 mm. Extract was then stored at 4 °C until used for analysis.

Qualitative phytochemical screening methods:

Pomegranate peels were screened for the presence of key families of phytochemicals¹⁸. Carbohydrates and reducing sugars were detected by Molisch and Benedict tests, respectively. The presence of glycosides and sterols were detected by Keller- Kiliani and Salkowski tests, respectively. The saponins were revealed by Froth test. The occurrence of phenolic compounds, tannins and proteins were confirmed by ferric chloride and xanthoproteic tests, respectively. The occurrence of amino acids in the crude juices was assessed by ninhydrin test. The existence of alkaloids in the extract was evaluated by Wagner's test. The flavonoids were detected by lead acetate test, while the possibility of the presence of oils was indicated by saponification test.

Phytochemcial investigation:

Various phytochemcial investigations were performed using obtained extract. The procedures are already reported by number of workers and used without any modification^{17,19, 20,21}. The phytochemical name and investigation methods are as follows:

Phenols:

To test the Phenol presence, in a test tube 1ml of extract and 2 ml of distilled water were added followed by few drops of 10% ferric chloride (FeCl₃). Appearance of blue or green colour indicates presence of phenols.

Flavonoids:

To test the flavonoid presence, in a test tube 1 ml of extract a few drops of dilute sodium hydroxide (NaOH) was added. An intense yellow colour was produced in the plant extract which becomes colourless on addition of few drops of dilute acid indicates the presence of flavonoid.

Quinones:

To test the quinone presence, in a test tube 1ml of extract and 1ml of concentrated sulphuric acid (H₂SO₄) was added. Formation of red colour is an indication for the presence of quinones.

Tannins:

To test the tannin presence, in a test tube 1 ml of 5% ferric chloride was added to solvent free extract. The presence of tannin is indicated by the formation of bluish black or greenish black precipitate¹⁹.

Saponins:

To test the saponin presence in the extract, the extract was diluted with 20 ml distilled water and was agitated in a graduated cylinder for 15 minutes. The formation of 1cm layer of foam indicates the presence of saponin¹⁹.

Cardiac Glycosides:

To test the cardiac glycoside presence, in a test tube 5 ml of extract was treated with 2 ml of glacial acetic acid containing a drop of ferric chloride (FeCl₃) solution. Afterwards it was underplayed with 1 ml concentrated sulphuric acid (H₂SO₄). A brown ring of the interface indicates a de-oxy sugar characteristic of cardenolites.

Steroids:

To test the presence of steroid, 1 ml of extract was dissolved in 10 ml chloroform and equal volume of concentrated sulphuric acid (H₂SO₄) added by the sides of test tube. The upper layer turns red and sulphuric acid layer shown yellow with green fluorescence. This indicated the presence of steroids.

Terpenoids:

To test the presence of terpenoid, in a test tube 5 ml of each extract was mixed with 2 ml of chloroform. 3ml of concentrated sulphuric acid (H₂SO₄) was then added to form a layer. A reddish brown precipitate colouration at the interface formed indicated the presence of terpenoids.

Thin Layer Chromatography:

TLC analysis of peel extract of pomegranate was developed on analytical plates over silica gel-G of 0.2 mm thickness. Acetonitrile: Distilled water (7:3, v/v) was the mobile phase that has good separation and sensitivity for the analysis of phenolic compounds. For the identification of phenols on TLC plate, it was sprayed with 2% sulphuric acid and kept in hot air over for 5 min and allowed to dry²².

TLC analysis of peel extract of pomegranate was developed on analytical plates over silica gel-G of 0.2 mm thickness. Ethyl acetate: Distilled water: Formic acid (8:1:1, v/v) was the mobile phase that has good separation and sensitivity for the analysis of flavonoid compounds. For the identification of flavonoids on TLC plate, it was sprayed with 3% boric acid and 15% oxalic acid and kept in hot air over for 5 min and allowed to dry²³.

FTIR Analysis:

Aliquotes of the extract were deposited on the surface of the attenuated totally reflexion (ATR) crystal. Afterwards, the dry surface of the extract was analyzed by Fourier Transform infrared spectroscopy (FTIR) (Shimadzu). The infrared radiation was propagated through the sample to obtain the corresponding spectrum, which was averaged from several data acquisitions. The equipment used was Perkin Elmer Spectrum GX, 16 scans per samples in the region of (4000-650) cm-1. After each measurement, the crystalline surface was washed with demineralised water and dried with a soft paper.

Total Phenolics Content:

Total phenolics content was analyzed using the Folin-Ciocalteu method²⁴. In this method, 70 µL of pomegranate peel extract was placed into a 10 mL test tube and 250 µL of Folin-Ciocalteu reagent and 750 µL of 1.9 M sodium carbonate were added. The total volume was made up to 5 ml by adding distilled water, and was mixed by vortexing for one minute and then incubated for two hours in dark.

Total Flavonoids Content:

Total flavonoids content of the freeze dried peel extract was determined²⁵. Briefly, 1 mL of extract was added into a 10 mL test tube containing 4 mL of distilled water. At zero time, 0.3 mL of 5% NaNO2 was added to the test tube. After 5 min, 0.3 mL of 10% AlCl3 was added into the test tube. At 6 min, 2 mL of 1 M NaOH was added to the mixture. Immediately, the reaction tube was diluted with the addition of 2.4 mL of distilled water and thoroughly mixed. Absorbance of the pink colored mixture was read at 510 nm and water was used as a blank. An appropriate calibration curve was prepared using different concentrations of catechin solutions. The final results were expressed as mg catechin equivalent (CE) per g dry solids.

RESULTS AND DISCUSSION:

Phytochemcial investigation:

Various phytochemcial investigations were performed using obtained extract. The procedures are already reported by number of workers and used without any modification^{17, 19, 20, 21}(Table.1).

Table 1: Phytochemicals in Pomegranate

Phytochemical test	Compound detected	Inference
Molisch's test	Carbohydrates	+++
Benedicts' test	Reducing sugars	+++
Keller -Kiliani's test	Glycosides	+
Xanthoproteic test	Proteins	+++
Ninhydrin’s test	Amino acids	+++
Ferric chloride test	Phenolic compounds	+++
Ferric chloride test	Tannins	+++
Wagner's test	Alkaloids	+++
Lead acetate test	Flavonoids	+++
Froth’s test	Saponins	++
Salkowski’s test	Sterols	+++
Saponification test	Fixed oils

The notations, +++, ++, + and – refer to appreciable amounts (positive within 5 min); moderate amounts (positive after 5 min but within 10 min); trace amounts (positive after 10 min but within 15 min) and completely absent, respectively.

Secondary metabolites afford imperative pharmaceutical properties for human health²⁶. Compounds belonging to the terpenoids, alkaloids and flavonoids are used as drugs or as dietary supplements to heal or prevent various diseases and in particular some of these compounds seem to be competent in preventing and inhibiting various types of cancer²⁷.

TLC analysis:

TLC analysis suggests the presence of phenols and flavonoids in the peel extract of pomegranate (Fig.1). TLC of peel extract in ethyl acetate reports the spots corresponding to phenols and flavonoids upon spraying and drying. The reported spots were separated with enough space showing the presence of phenols and flavonoids in ethyl acetate solvent extract.

Phenols Flavonoids

Fig.1: Thin layer chromatography of Phenols and Flavonoids in pomegranate

FTIR Analysis:

Quantification of total phenols and flavanoids was performed from extracts of Pomegranate peel FTIR spectrum shows intense bands which were characteristic of phenol groups (Fig. 2 and Fig.3). This technique could also be used to quantify total phenols using multivariate analysis.

Fig. 2: FTIR estimation of phenols in pomegranate peel

Fig. 3: FTIR estimation of flavonoids in pomegranate peel

Total Phenolics Content and Total Flavonoids Content:

It is well known that plant phenolics and flavonoids are highly effective free radical scavengers and antioxidants. The peel extract exhibited a high content of total phenolics and total flavonoids i.e. 106.4 mg GAE/g dry solids and 3.5 mg CE/g dry solids, respectively (Table.2). The flavonoids accounted for a small amount of total phenolics in the pomegranate peel extract. In the present study, total phenolics were higher than a similar study done on Egyptian pomegranate peel extract in which the extract had 6.2 mg GAE/g dry solids²⁸. On the contrary, in the cases of pomegranate varieties from India and from Kashmir district, higher phenolic contents (124.3 - 249.4 mg GAE/g dry solids) and flavonoid contents (49.1-59.4 mg CE/g dry solids) in pomegranate peel extract were reported^{29, 30}. A Yamani cultivar was found to have high total phenolics (91.2 mg GAE/g dry solids) with no detection of total flavonoids³¹. This variation of total phenolics and total flavonoids could be associated with the difference in cultivars, methods of extraction and environmental conditions such as relative humidity and temperature of extracts. It was suggested that the high amounts of bioactive compounds are available in non-edible part which could be used for different purposes in the food industry such as enrichment or development of new products³⁰.

Table 2: Total phenolics and flavonoids of pomegranate peel extracts

Total phenolics	Total flavonoids
106.4±0.50 (mg GAE/g ds)	3.5±0.03 (mg CE/g ds)

GAE: Gallic acid equivalents; CE: Catechin equivalent

Values expressed as means of triplicate determinations ± SD

DISCUSSION:

When a new drug to be discovered, qualitative phytochemical analysis is a very important step as it gives information about the presence of any particular primary or secondary metabolite in the extracts of the plant which is having a clinical significance. The present study showed interesting preliminary phytochemical constituents in solvent peel extracts of Punica granatum. Further characterization and quantitative assay may be carried out to test the peel extracts for various therapeutic and pharmacological activity. In any case, if any significant bioactive natural product is present, it is necessary to separate that compound from the mixture of compounds by using suitable chromatographic technique. The different phytochemicals tests performed on the extracts of Punica granatum leaves show the presence of phenols, flavonoids, quinones, saponin, quinones, tannins, terpenoids, steroids and cardiac glycosides in ethyl acetate extract. The findings are also in line of previous findings and reported literatures³².

CONCLUSION:

The present study showed interesting preliminary phytochemical constituents in ethyl acetate peel extracts of Punica granatum. Pomegranate peel can be further analyzed for qualitative and quantitative extraction of reported phytochemicals to explore the possibilities of using the peel extracts for various therapeutic and pharmacological activities and enable its utilization as an herbal medicine on scientific ground.

REFERENCES:

1. Sarkhosh A, Zamani Z, Fatahi R and Ebadi A. RAPD markers reveal polymorphism among some Iranian pomegranate (Punica granatum L.) genotypes. Scientia Horticulturae, 2006; 111(1), 24-29.

2. Mahdihassan S. Outline of the beginnings of alchemy and it antecedents. The American Journal of Chinese Medicime. 1984; 12(1-4): 32-42.

3. Faria A, Calhau C, de Freitas V and Mateus N. Procyanidins as antioxidants and tumor cell growth modulators. J. Agric. Food Chem. 2006; 54(6): 2392-2397.

4. Faria A, Monteiro R, Mateus N, Azevedo I and Calhau C. Effect of Pomegranate (Punica granatum) juice intake on hepatic oxidative stress. Europian Journal of Nutrition. 2007; 46(5): 271-278.

5. Adhami VM. and Mukhtar H. Polyphenols from green tea and pomegranate for prevention of prostate cancer. Free Radical Research. 2006; 40(10): 1095-1104.

6. Dandekar DV, Jayaprakasha GK and Patil BS. Simultaneous extraction of bioactive limonoid aglycones and lucoside from Citrus aurantium L. using hydrotropy. Zeitschrift für Naturforschung C. A Journal of Biosciences. 2008; 63(3-4): 176-180.

7. Graciousross R, Selvasubramanian S and Jayasundar S. Immunomodulatory activity of Punica granatum in rabbits: a preliminary study. Journal of Ethnopharmacology. 2001; 78(1): 85-87.

8. Kim ND, Mehta R, Yu W, Neeman I, Livney T, Amichay A, Poirier D, Nicholls P, Kirby A, Jiang W, Mansel R, Ramachandran C, Rabi T, Kaplan B and Lansky E. Chemopreventive and adjuvant therapeutic potential of pomegranate (Punica granatum) for human breast cancer. Breast Cancer Research and Treatment. 2002; 71(3): 203-217.

9. Murthy KN, Reddy VK, Veigas JM and Murthy UD. Study on wound healing activity of Punica granatum peel. Journal of Medicinal Food. 2004; 7(2): 256-259.

10. Lansky EP, Shubert S and Neeman I. Pharmacological and therapeutic properties of pomegranate, Israel. CIHEAM-Options Mediterranean’s. 2004. pp.231-235.

11. Fuentes VR and Exposito A. Las encuestas etnobot´anicas sobre plantas medicinales en Cuba. Revista del Jardín Botánico Nacional, 1995; 16: 77-144.

12. Ozcal N and Dinc S. Evaluation of the pomegranate (Punica granatum L.) peel from the standpoint of pharmacy. Eczacılık Fak¨ultesi Dergisi. 1993; 22: 21-29.

13. Mavlyanov SM, Islambekov SY, Karimdzhanov AK and Ismailov AI. Polyphenols of the fruits of some varieties of pomegranate growing in Uzbekistan. Chem. Nat. Compd. 1997; 33(1): 98-99.

14. Reddy MK, Gupta SK, Jacob MR, Khan SI and Ferreira D. Antioxidant, antimalarial and antimicrobial activities of tannin-rich fractions ellagitannins and phenolic acids from Punica granatum L. Planta Medica. 2007; 73(5): 461-467.

15. El-falleh W, Hannachi H, Tlili N, Yahia Y, Nasri N and Ferchichi A. Total phenolic contents and antioxidant activities of pomegranate peel, seed, leaf and flower. Journal of Medicinal Plants Research. 2012; 6(20): 4724-4730.

16. Elswijk D, Schobel U, Lansky E, Irth H and Greef J. Rapid dereplication of estrogenic compounds in pomegranate (Punica granatum) using on-line biochemical detection coupled to mass spectrometry. Phytochemistry. 2004; 65(2):233-24.

17. Harbone JB. Phytochemical methods. Chapman and Hall Ltd. London. 1973; 49-188.

18. Sawant RS. and Godghate AG. Qualitative Phytochemical Screening of Rhizomes of Cucurma longa Linn. International Journal of Science, Environment and Technology, 2013; 2(2): 634-641.

19. Siddiqui AA and Ali M. Practical Pharmaceutical Chemistry, CBS Publishers and Distributors, New Delhi. 1997; I^sted: 126-131.

20. Mandal SC, Mukherjee PK, Saha K, Das J, Pal M and Saha BP. Hypoglycemic activity of Ficus racemosa leaves in streptozotocin-induced diabetic rats. Natural Product Sciences. 1997; 3:38-41.

21. Mandal SC, Maity TK, Das J, Saha BP and Pal M. Anti- inflammatory evaluation of Ficus racemosa leaf extract, Journal of Ethnopharmacology. 2000; 72(1-2): 87-92.

22. Haghi G, Hatami A, Safaei A and Mehran M. Analysis of phenolic compounds in Matricaria chamomilla and its extracts by UPLC-UV. Research in Pharmaceutical Sciences.2014; 9(1): 31-37.

23. Lamaison JA, Petitjean-Freytet C and Carnat A, Medicinal Laminaceae with Antioxidant Properties, a Potential Source of Rosmarinic Acid. Pharmaceutica Acta Helvetiae, 1991; 66 (7): 185-188.

24. Singleton L and Rossi A. Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. American Journal of Enology and Viticulture. 1965; 16(3):144-158.

25. Zhishen J, Mengcheng T and Jianming W. The determination of flavonoid contents in mulberry and their scavenging effects on superoxide radicals. Food Chemistry. 1999; 64(3): 555-559.

26. Raskin I, Ribnicky DM, Komarnytsky S, Ilic N, Poulev A, Borisjuk N, Brinker A, Moreno DA, Ripoll C and yakoby N. Plants and human health in the twenty first century. Trends inBiotechnology. 2002; 20(12): 522-531.

27. Ashok Kumar K. and Vijayalakshmi K. In vitro anti-microbial activity and phytochemical analysis of selected fruit wastes. International Journal of Current Microbiology and Applied Sciences. 2013; 2(5): 196-204.

28. Ramadan A, El-Badrawy S, Abd-el-Ghany M and Nagib R. Utilization of hydro-alcoholic extract of peel and rind and juice of pomegranates natural antioxidants in cotton seed oil. The 5^th Arab and 2^nd International Annual Scientific Conference, Egypt. 2009; 8-9.

29. Negi P and Jayaprakasha G. Antioxidant and antibacterial activities of Punica granatum peel extracts. Food Microbiol Safety. 2003; 68(4):1473-1477.

30. Kulkarni A, Aradhya S and Divakar S. Isolation and identification of a radical scavenging antioxidant- punicalagin from pith and carpellary membrane of pomegranate fruit. Food Chemistry. 2004; 87(4):551-557.

31. Shiban M, Al-Otaibi M and Al-Zoreky N. Antioxidant activity of pomegranate (Punica granatum L.) fruit peels. Food and Nutrition Sciences. 2012; 3(7): 991-996.

32. Jayaprakash A and Sangeetha R. Phytochemical Screening of Punica granatum Linn. Peel Extracts, Journal of Academia and Industrial Research, 2015; 4 (5): 160-162.

Received on 07.03.2018 Modified on 03.05.2018

Research J. Pharm. and Tech 2018; 11(8): 3609-3613.

DOI: 10.5958/0974-360X.2018.00664.9