Proximate Composition, Minerals Content, Antibacterial and antifungal Activity Evaluation of Pomegranate (Punica granatum L.) Peels Powder

Middle-East Journal of Scientific Research 11 (3): 396-401, 2012 ISSN 1990-9233 © IDOSI Publications, 2012

Proximate Composition, Minerals Content, Antibacterial and antifungal Activity Evaluation of Pomegranate (Punica granatum L.) Peels Powder 1

Naseem Ullah, 2Javid Ali, 1Farhat Ali Khan, 1Muhammad Khurram, 2Arshad Hussain, 3 Inayat-ur-Rahman, 2Zia-ur-Rahman and 2Shafqatullah 1

Sarhad University of Science and Information Technology, Peshawar, Pakistan Food Microbiology Section, PCSIR Laboratories Complex, Jamrud Road, Peshawar, Pakistan 3 Environmental Research Section, PCSIR Laboratories Complex, Jamrud Road, Peshawar, Pakistan 2

Abstract: The study on the proximate composition, minerals contents, antibacterial and antifungal activities of the most popular consumed fruit Pomegranate (Punica granatum L) peels has been carried out using recommended method of analysis. The result of the analysis shows that the moisture content (04 ± 0.22%), ash (05 ± 0.14%), fat(9.4 ± 0.1%), pH(3.75 ± 0.2), TSS (0.7 ± 0.04%), acidity (4.86 ± 0.5%), crude fiber (21 ± 0.6%), Total sugar (31.38 ± 0.3%), Reducing Sugar (30.40 ± 0.11%), Non-Reducing sugar (0.98 ± 0.12%), Nitrogen (1.395 ± 0.30%) and Protein (8.719± 0.10%). The minerals content were determined by analyzing the samples for sodium, potassium, iron, manganese and zinc ppm values were 1100 ±0. 4, 10000 ±0. 6, 60.5 ±0. 2, 4.5 ± 0.8 and 4.0 ± 0.65 respectively. Among the selected bacterial and fungal cultures, the highest antibacterial activity was recorded against Klebsilla pneumoniae and among fungi high activity against Aspergillus parasiticus was recorded. The peel extract were shown no activities against Salmonella typhi, Bacillus cereus and Aspergillus flavus. Key words: Pomegranate Peel Powder % Proximate composition % Antimicrobial Activity and Mineral Analysis INTRODUCTION

The generation of free radicals plays an important role in the progression of numerous pathological disturbances, such as atherosclerosis [8] and brain disfunction [9]. Several studies have reported the reduced-risk efficacy of various extracts or pure compounds from the different parts of pomegranate plant against the growth of microbial pathogens [10]. Pomegranate (Punica granatum) Punicaceae) has been known to considerable pharmacological properties with antimicrobial, antiviral, anticancer, potent antioxidant and antimutagenic effects [11,12] and been used in the markets in the preparation of tinctures, juice, cosmetics and therapeutic formulae [13]. Keeping in view the medicinal importance the present study was aimed for its Proximate Composition, Minerals Content, Antibacterial and antifungal Activity Evaluation of Pomegranate (Punica granatum L.) Peels Powder.

The pomegranate (Punica granatum L.) is one of the oldest edible fruits and is widely grown in many tropical and subtropical countries [1]. Pomegranate (Punica granatum L.) is an important fruit plant of tropical and subtropical regions. It is extensively cultivated in Iran, Spain, Egypt, Russia, France, Argentina, China, Japan, USA and in India [2]. References to the medicinal uses of pomegranate are found in many ancient cultures and religions. Pomegranate is mentioned thrice in ayat of the Holley Quran and by the Islam prophet, “Mohammad”, as one of the fruits that will be found in paradise. The bark, leaves, flowers, fruit and seeds of this plant have been used to prevent and treat many infectious diseases [3]. Pomegranate is proved to have high antioxidant activity [4] and good potency for cancer prevention [5]. The edible part of the fruit contains considerable amounts of acids, sugars, vitamins, polysaccharides, polyphenols and important minerals [6,7]. Great interest has recently been focused on the addition of polyphenols to foods and biological systems, due to their well-known abilities to scavenge free radicals, i.e antioxidant power.

MATERIALS AND METHODS Pomegranate fruit (Punica granatum L.) were collected from the food Pilot Plant of PCSIR Laboratories Complex Peshawar, Pakistan. Peels were cuts into pieces and dried in dehydrator (50°C) for 48 h. Dried peels were powdered to get 60-mesh size using a mixing grinder.

Corresponding Author: Javid Ali, Food Microbiology Section, PCSIR Laboratories Complex, Jamrud Road Peshawar, Pakistan.

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Middle-East J. Sci. Res., 11 (3): 396-401, 2012

Proximate Composition: Proximate composition includes moisture, ash, fat, pH, TSS, Acidity, Crude Fiber, total sugar, reducing sugar, non-reducing sugar, nitrogen and protein hydrates were determined in triplicate in accordance with standards procedures [14]. Moisture was determined by oven dehydration method at 100-110°C up to the constant weight. The loss in weight was moisture content. Ash content was determined in muffle furnace for about 550 °C for 5-6 h, utile constant weight was obtained. Crude fat was determined by petroleum ether extraction method using Soxhlet apparatus for about 6-8 h. Crude fiber was determined by acid digestion (H2SO4 1.25%) and alkali digestion (NaOH 1.25%) method. Kjeldhal method was used to determine the crude protein.

Subsequently 30µg/50µL, 40µg/50µL and 50µg/50µL volume of the extract was introduced in triplicate wells of the agar plates and then incubated at 37°C for 24h. The zone of inhibition was recorded to the nearest size in mm [16]. Antifungal Susceptibility Assay: Tested fungi were subcultured on SDA at 28°C for 3-5 days. Sterilized Sabouraud’s Dextrose Agar plates were taken and a sterile cork borer (6-mm diameter) was used to bore wells in the agar. 30µg/50µL, 40µg/50µL and 50µg/50µL volume of the extract were poured into each wells. The plates were then incubated at 28°C. The appearance of zones of inhibition was regarded as the presence of antimicrobial action in the test substance [17].

Mineral Analysis: For minerals determination 0.5 g of each sample was taken and heated in a muffle furnace at 400ºC. The ash obtained was cooled, dissolved in 5ml of 6N HCl and allowed to stand for 30 minutes. It was later filtered and its volume made up to 50ml with deionized water. The resulting extract was used for the determination of Fe, Mn and Zn, were determined on Hitachi Zeeman Japan Z-8000, Atomic Absorption Spectrophotometer equipped with standard hallow cathode lamps as radiation source and air acetylene flames, while Na and K concentration was determined on Flame Photometer(PFP-7) Jenway, UK.

Statistical Analysis: Means and standard deviations were calculated for three independent determinations for each variable except for total carbohydrate, which was simply obtained by difference. Correlation matrix was done using MS Excel 2003 version. RESULTS AND DISCUSSION Proximate Composition: The proximate composition of the pomegranate peels powder results were shown in Table 1. The moisture (%) content was 04 ± 0.22, the ash (%) content was05 ± 0.14. the fat (%) content was 9.4 ± 0.1, similarly the pH, TSS, acidity were found 3.75 ± 0.2, 0.7 ± 0.04 and 4.86 ± 0.5 respectively. Crude fiber was calculated (21 ± 0.6) the second higher amount after total sugar composition (31.38 ± 0.3). The reducing and non-reducing sugar was 30.40 ± 0.11 and 0.98 ± 0.12 respectively.

Antimicrobial Activity of Pomegranate Peels Extract Pomegranate Peels Extraction: The powder (50 g) was mixed with 200 ml of distilled water and agitated using a rotary shaker (150 rpm) for 6 h. Extracts were filtered through Whatman no. 41 filter paper in a Buchner funnel for removing of peel particles. The residue was reextracted with 100 ml of ethanol and filtered. It was further dried in a desiccator under vacuum till constant weight. The final dry extract weights were recorded and then dry matter was resuspended in sterile distilled water to have a concentration of 20% (w/v).

Table 1: Proximate Composition of Pomegranate Peel Powder

Antimicrobial Activity Evaluation of Pomegranate peel Extract Antibacterial Susceptibility Assay: The antimicrobial activity of pomegranate peel extract (aqueous) was determined by agar well diffusion method [15]. Pure tested bacterium was first subculture in nutrient broth at 37°C for 24h. One hundred microlitres (100ìL) of standardized inoculum (106CFU/mL; 0.5 Mac-Farland) of each test bacterium was spread on sterile Muller-Hinton Agar plate.

S#

Proximate Analysis

Results

1

Moisture

04 ± 0.22(%)

2

Ash

05 ± 0.14(%)

3

Fat

9.4 ± 0.1(%)

4

pH

3.75 ± 0.2

5

TSS

0.7 ± 0.04(%)

6

Acidity

4.86 ± 0.5(%)

7

Crude Fiber

21 ± 0.6(%)

8

Total sugar

31.38 ± 0.3(%)

9

Reducing Sugar

30.40 ± 0.11(%)

10

Non-Reducing sugar

0.98 ± 0.12(%)

11

Nitrogen

1.395 ± 0.30(%)

12

Protein

8.719± 0.10(%)

Values are mean ± standard deviations of triplicate determinations.

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Middle-East J. Sci. Res., 11 (3): 396-401, 2012 Table 2: Mineral Analysis of pomegranate peels powder S#

Minerals

1

K

10000 ±0.6

2

Na

1100 ±0.4

3

Fe

60.5 ±0.2

4

Mn

4.5 ± 0.8

5

Zn

4.0 ± 0.65

intake [27]. Manganese (Mn) plays an important role in a number of physiological processes as a constituent of some enzymes and an activator of other enzymes [28].

Concentration (ppm)

Antibacterial Activity of Aquas Extract of Pomegranate Peels: Antibacterial susceptibility determination of aquas extract of pomegranate peels powder using the well diffusion method at concentration of 50µg/50µL, 40µg/50µL and 30µg/50µL were shown in Table 3. Klebsilla pneumonia were calculated the highest antibacterial activity as compared to the rest of tested microbes and revealed that concentration of 50µg/50µL shown 30mm zone of inhibition, 40µg/50µL concentration observed 27 mm zone of inhibition and 30µg/50µL concentration have 23 mm of inhibitory zone. Klebsilla pneumoniaewere is a gram-negative, non-motile rod and caused a bacterial pneumonia [29]. The next highest zone of inhibition (mm) 27, 24 and 21 were observed against Escherichia coil with a concentration of 50µg/50µL, 40µg/50µL and 30µg/50µL respectively. It has been reported that P. granatum peel extracts have shown antibacterial activity against Escherichia coli O157 and Staphylococcus aureus bacteria [30,31]. E. coli is the Gram-negative rod bacteria which cause diarrhea. [29]. The inhibitory effect against enterococcus fecalis revealed that 25mm (50µg/50µL), 23mm (40µg/50µL) and 19mm (30µg/50µL) respectively. Enterococcus fecalis and Proteus mirabilis are responsible for the infections of surgical wounds and the urinary tract (Tatora et al; 2009) Staphylococcus aureus zone of inhibition (mm) were19, 14 and 10 at 50µg/50µL, 40µg/50µL and 30µg/50µL concentration respectively. The alcohol extracts of pomegranate fruits [33] showed antibacterial activity against S. aureus but were not active against E. coli, Shigella paratyphi and Shigella dysentery.

Values are mean ± standard deviations of triplicate determinations.

Minerals Analysis of Pomegranate Peel Powder: The minerals content of pomegranate peels powder were shown in Table 2. The Na concentration was found the highest (10000 ± 6 ppm) as compared to all the other minerals content, followed by K (1100 ± 4 ppm) in the analyzed peel powder. Sodium is present in most food and its deficiency is rare. Distorted enzymatic activity and poor electrolyte balance of the blood fluid are related to inadequate Na, K, Mg and Zn; as they are the most required elements of living cells [18]. Sodium is an extracellular cation involved in the regulation of plasma volume, acid-base balance, nerve and muscle contraction. High dietary sodium has been associated with essential hypertension [19-21]. The Fe content of pomegranate peels was 60.5 ± 2, which was highest after Na and K value. Iron is an important trace element in the human body. It plays crucial roles in haemopoiesis, control of infection and cell mediated immunity [22,23]. The deficiency of iron has been described as the most prevalent nutritional deficiency and iron deficiency anemia is estimated to affect more than one billion people worldwide [24]. The Mn and Zn content were calculated 4.5 ± 0.8 ppm and 4 ± 0.65 ppm respectively. Zinc is an essential micronutrient for human growth and immune functions [25,26]. An estimated 20% of the world population is reported to be at risk of inadequate zinc Table 3: Antibacterial Activity of Pomegranate Peel Extract

S#

Tested Microorganism

1 2 3 4 5 6 7 8 12

Salmonella typhi Escherichia coil ATCC 25922 Staphylococcus aureus ATCC25923 Bacillus cereus Pseudomonas aeruginosa ATCC27853 Klebsilla pneumoniae ATCC700603 Proteus mirabilis ATCC13315 Enterococcus fecalis ATCC29212 Water(negative control)

Pomegranate Peel Extract Volume ----------------------------------------------------------------------------------------------------------------------50µg/50µL 40µg/50µL 30µg/50µL -------------------------------------------- Zone of Inhibition in mm -------------------------------------------R 27 19 R 21 30 20 25 0

R 24 14 R 19 27 16 23 0

398

R 21 10 R 16 23 12 19 0

Middle-East J. Sci. Res., 11 (3): 396-401, 2012 Table 4: Antimicrobial Activity of Pomegranate Peel Extract

S#

Tested Microorganism

1 2 3 4

Aspergillus niger Aspergillus flavus Aspergillus parasiticus Water (negative control)

Pomegranate Peel Extract Volume -----------------------------------------------------------------------------------------------------------------------50µg/50µL 40µg/50µL 30µg/50µL -------------------------------------------- Zone of Inhibition in mm -------------------------------------------15 R 17 0

11 R 13 0

But in our finding E. coli have also showed the susceptibility against aquas extract. S. aureus are responsible for the production many toxin which contributes to the bacterium’s pathogenicity by increasing its ability to invade the body or damage tissue [32]. Pseudomonas aeruginosa zone of inhibition were 21mm, 19mm and 16mm at the 50µg/50µL, 40µg/50µL and 30µg/50µL respectively. This bacterium consist of aerobic, gram-negative, rods, motile, produces soluble, blue-green pigmentation and infect the urinary tract, burns and wounds and can cause blood infection (sepsis), abscesses and meningitis [32]. Proteus mirabilis, have showed a low antibacterial activity 20(50µg/50µL), 16 (40µg/50µL) and 12(30µg/50µL) as compared to the Pseudomonas aeruginosa. The Salmonella typhi and Bacillus cereus have shown a strong resistance against the peels extract. Bacillus cereus is a Gram-positive, spore forming, motile which causes diarrhea, abdominal pain and vomiting [29]. Although it has been reported that the pomegranate root extract possessed inhibitory activity against Gram-negative bacteria, such as S. sonnei, S. typhi, V. cholera and E. coli [34], the fruit rind extract was not active.

09 R 10 0

a strong resistance to the pomegranate peels aquas extract. Fungi of the genus Aspergillus are filamentous organisms that produce airborne spores and have a wide distribution as saprophytes in nature. Occasionally they become pathogenic to man and cause a variety of lesions, the most important of which is pulmonary disease [36]. REFERENCES 1.

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Antifungal Activity: Antifungal activity of pomegranate peels powder against the three tested fungi was shown in Table 4. Aspergillus parasiticus showed more sensitivity as compared to the Aspergillus niger which revealed that its zone of inhibition were 17mm (50µg/50µL), 13mm (40µg/50µL) and 10mm (30µg/50µL). Aspergillus niger antifungal activity calculated that its inhibition zone (mm) were 15, 11 and 09 at the concentration of 50µg/50µL, 40µg/50µL and 30µg/50µL respectively. The methanolic peel extract has shown highest antimicrobial activity compared to aquas extracts. Staphylococcus aureus and among fungi high activity against Aspergillus niger was recorded [35]. Aspergillus flavus were not form any zone of inhibition against any concentration tested and showed

6.

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