PRELIMINARY INVESTIGATION INTO THE ANTIMICROBIAL ANTI-MICROBIAL PROPERTIES OF PUMPKIN SEED OIL

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PRELIMINARY INVESTIGATION INTO THE ANTIMICROBIAL ANTI-MICROBIAL PROPERTIES OF PUMPKIN SEED OIL

ABSTRACT

The current research deals with the extraction, physicochemical and antimicrobial parameters of pumpkin seed oil (Cucurbita pepo) of arid zone variety of Nigeria. Pumpkin is a nutritive and unique plant commonly used as vegetable all around the world. Its seeds and rinds are mostly thrown away after use however; they are rich in proteins and fatty acids. In Nigeria usage of pumpkin is not as frequent as other vegetable and least importance is given to its seed. Therefore a present study is designed to see the physicochemical and antimicrobial properties of pumpkin seed oil (Cucurbita pepo). Results showed that the pumpkin seed oil had acid value 0.83667 (mg KOH/g oil), saponification value 194.606 (g of I2/100 g oil), peroxide value 6.74 (meq O2/kg oil), iodine value 97.9766 (g of I2/100 g oil) and ester value 193.7640 which are in range of the standard levels. Gass chromatography analysis showed the presence of five major free fatty acids linoleic acid, oleic acid, palmitic acid, stearic acid, and linolenic acid and among them linoleic and oleic are the major ones. The oil also showed good antimicrobial activity against S. aureus with zone of inhibition of 15 mm. Therefore, it is feasible to be used as edible oil and for other purposes.

KEYWORDS: Pumpkin, Pumpkin seed oil, Antimicrobial, Gas chromatography, Free fatty acids.

CHAPTER ONE

INTRODUCTION

Pumpkin seeds have long been valued as a source of the mineral zinc, and the World Health Organization recommends their consumption as a good way of obtaining this nutrient. If you want to maximize the amount of zinc that you will be getting from your pumpkin seeds, we recommend that you consider purchasing them in unshelled form. Although recent studies have shown there to be little zinc in the shell itself (the shell is also called the seed coat or husk), there is a very thin layer directly beneath the shell called the endosperm envelope, and it is often pressed up very tightly against the shell. Zinc is especially concentrated in this endosperm envelope. Because it can be tricky to separate the endosperm envelope from the shell, eating the entire pumpkin seed—shell and all—will ensure that all of the zinc-containing portions of the seed will be consumed. Whole roasted, unshelled pumpkin seeds contain about 10 milligrams of zinc per 3.5 ounces, and shelled roasted pumpkin seeds (which are often referred to pumpkin seed kernels) contain about 7-8 milligrams. So even though the difference is not huge, and even though the seed kernels remain a good source of zinc, you’ll be able to increase your zinc intake if you consume the unshelled version.

While pumpkin seeds are not a highly rich source of vitamin E in the form of alpha-tocopherol, recent studies have shown that pumpkin seeds provide us with vitamin E in a wide diversity of forms. From any fixed amount of a vitamin, we are likely to get more health benefits when we are provided with that vitamin in all of its different forms. In the case of pumpkin seeds, vitamin E is found in all of the following forms: alpha-tocopherol, gamma-tocopherol, delta-tocopherol, alpha-tocomonoenol, and gamma-tocomonoenol. These last two forms have only recently been discovered in pumpkin seeds, and their health benefits—including antioxidant benefits—are a topic of current interest in vitamin E research, since their bioavailability might be greater than some of the other vitamin E forms. The bottom line: pumpkin seeds’ vitamin E content may bring us more health benefits that we would ordinarily expect due to the diverse forms of vitamin E found in this food.

In our Tips for Preparing section, we recommend a roasting time for pumpkin seeds of no more than 15-20 minutes when roasting at home. This recommendation supported by a new study that pinpointed 20 minutes as a threshold time for changes in pumpkin seed fats. In this recent study, pumpkin seeds were roasted in a microwave oven for varying lengths of time, and limited changes in the pumpkin seeds fat were determined to occur under 20 minutes. However, when the seeds were roasted for longer than 20 minutes, a number of unwanted changes in fat structure were determined to occur more frequently.

BACKGROUND OF STUDY

Pumpkins, and their seeds, are native to the Americas, and indigenous species are found across North America, South America, and Central America. The word “pepita” is consistent with this heritage, since it comes from Mexico, where the Spanish phrase “pepita de calabaza” means “little seed of squash.”

Pumpkin seeds were a celebrated food among many Native American tribes, who treasured them both for their dietary and medicinal properties. In South America, the popularity of pumpkin seeds has been traced at least as far back as the Aztec cultures of 1300-1500 AD. From the Americas, the popularity of pumpkin seeds spread to the rest of the globe through trade and exploration over many centuries. In parts of Eastern Europe and the Mediterranean (especially Greece), pumpkin seeds became a standard part of everyday cuisine, and culinary and medical traditions in India and other parts of Asia also incorporated this food into a place of importance.

Today, China produces more pumpkins and pumpkin seeds than any other country. India, Russia, the Ukraine, Mexico, and the U.S. are also major producers of pumpkin and pumpkin seeds. In the U.S., Illinois is the largest producer of pumpkins, followed by California, Ohio, Pennsylvania, Michigan, and New York. However, pumpkins are now grown commercially in virtually all U.S. states, and over 100,000 acres of U.S. farmland are planted with pumpkins.

Antimicrobial Benefits

Pumpkin seeds, pumpkin seed extracts, and pumpkin seed oil have long been valued for their anti-microbial benefits, including their anti-fungal and anti-viral properties. Research points to the role of unique proteins in pumpkin seeds as the source of many antimicrobial benefits. The lignans in pumpkin seeds (including pinoresinol, medioresinol, and lariciresinol) have also been shown to have antimicrobial—and especially anti-viral— properties. Impact of pumpkin seed proteins and pumpkin seed phytonutrients like lignans on the activity of a messaging molecule called interferon gamma (IFN-gamma) is likely to be involved in the antimicrobial benefits associated with this food.

Cancer-Related Benefits

Because oxidative stress is known to play a role in the development of some cancers, and pumpkin seeds are unique in their composition of antioxidant nutrients, it’s not surprising to find some preliminary evidence of decreased cancer risk in association with pumpkin seed intake. However, the antioxidant content of pumpkin seeds has not been the focus of preliminary research in this cancer area. Instead, the research has focused on lignans. Only breast cancer and prostate cancer seem to have received much attention in the research world in connection with pumpkin seed intake, and much of that attention has been limited to the lignan content of pumpkin seeds. To some extent, this same focus on lignans has occurred in research on prostate cancer as well. For these reasons, we cannot describe the cancer-related benefits of pumpkin seeds as being well-documented in the research, even though pumpkin seeds may eventually be shown to have important health benefits in this area.

Possible Benefits for Benign Prostatic Hyperplasia (BPH)

Pumpkin seed extracts and oils have long been used in treatment of Benign Prostatic Hyperplasia (BPH). BPH is a health problem involving non-cancer enlargement of the prostate gland, and it commonly affects middle-aged and older men in the U.S. Studies have linked different nutrients in pumpkin seeds to their beneficial effects on BPH, including their phytosterols, lignans, and zinc. Among these groups, research on phytosterols is the strongest, and it centers on three phytosterols found in pumpkin seeds: beta-sitosterol, sitostanol, and avenasterol. The phytosterols campesterol, stigmasterol, and campestanol have also been found in pumpkin seeds in some studies. Unfortunately, studies on BPH have typically involved extracts or oils rather than pumpkin seeds themselves. For this reason, it’s just not possible to tell whether everyday intake of pumpkin seeds in food form has a beneficial impact on BPH. Equally impossible to determine is whether intake of pumpkin seeds in food form can lower a man’s risk of BPH. We look forward to future studies that will hopefully provide us with answers to those questions.

STATEMENT OF THE PROBLEM

To the best of my knowledge less study has been conducted on Nigerian variety of pumpkin. Therefore, the present study is designed for Nigerian variety of pumpkin seed and its oil to see its need as a functional food. Cucurbita pepo has broad uses in traditional medicine for many cases, it hasn’t side effects. Fruits, seeds and leaves are the most used parts, some of its active compounds are still unknown. The seeds contain material killed tap warm when used as dough evict it out with feces, it is save way without side effects while synthesis drags have serious damage[1]. Fruit’s juice of pumpkin has analgesic effect for head ache, also it is considered abdominal laxative. When we make bandage of seeds on the head useful in treatment tumors in brain and its peal use for treatment tumors of ear, eyes and gout[2].

OBJECTIVES OF THE STUDY

The main objectives of this research were to investigate the physiochemical properties of pumpkin seed oil and antimicrobial activity of pumpkin seed oil. Nutritional and health protective value of pumpkin draws considerable attention of food scientists in recent years (Fokou et al., 2004). Pumpkin belongs to the family Cucurbitaceae which is an angiosperm, genus Cucurbita with different varieties (Alfawaz, 2004). Pumpkin fruit carries more than 500 seeds which are interspersed in a net like structure called mucilaginous fibers present at its central inner cavity. They are covered with a protective layer called testa.

RESEARCH QUESTIONS

The key question to be answered in the course of this research is if pumpkin seed actually contain antimicrobial activity that can be used by different fields to improve life and medicine.

RESEARCH HYPOTHESES

The research issues above can be investigated by a number of means, but lend themselves especially well to empirical investigation and testing of a number of specific hypotheses.

H1: Pumpkin seed contain very good antimicrobial activity that can be used by different fields to improve life and medicine

H2: Pumpkin seed contain no antimicrobial activity that can be used by different fields to improve life and medicine

This issue requires an analytical methodology for empirical testing (see Chapter 3 below). By “efficiency” is meant the amalgam of technical efficiency in terms of producing the most output with a given set of inputs and allocative efficiency, in terms of using the most efficient combination of inputs given prevailing prices. If hypothesis one above and the present hypothesis both fail to hold, the outlook for smallholders is pretty good, since the combination of higher unit profits and greater efficiency would mean that they could either displace large farmers, or possibly eventually become large farmers!

SCOPE OF THE STUDY

1.6.1. Plant Material Materials were collected from hazelnut samples (Corylus maxima Mill.), in Trabzon on Blacksea Coast; peanut samples (Arachis hypogaea L.), in Osmaniye; pistachio samples (Pistacia vera L.), in Gaziantep; almond samples (Prunus amygdalus Batsch.), in Mugla; walnut samples (Junglas regia L.), in Balıkesir; chestnut samples (Castanea sativa Mill.), in Bursa; pumpkin seeds (Cucurbita pepo L.) and sunflower seeds (Helianthus annus L.) in Edirne province in 2008 harvesting time.

1.6.2. Fatty Acid Composition Fatty acid methyl esters (FAMEs) were prepared from oil samples and determined by gas chromatography (GC) according to the method described by Slover and Lanza [29]. FAMEs were prepared using BF3 in methanol (20% of BF3 in methanol) and extracted with n-hexane and then analyzed by GC.

1.6.3. Radical-Scavenging Activity (Antioxidant Activity) The free-radical-scavenging activity of the extracts was determined by the DPPH. assay as described by Bloiss [30]. Briefly, each sample was diluted in methanol prior to the analysis (1 mg/mL). An aliquot (0.1 mL) of the solution was added to 3.9 mL of DPPH solution (6×10-5 M in methanol), throughly mixed, and the absorbance of the sample at 515 nm was recorded after the time necessary for the reaction to reach a plateau [31]. The absorbance of DPPH solution in methanol, without any antioxidant (control), was also measured. The percentage of remaining DPPH was calculated as follows: % DPPH scavenging = [(Acontrol – Asample)/Acontrol] x 100 Where Asample is the absorbance of sample after the time necessary to reach the plateau (30 min) and Acontrol is the absorbance of DPPH.

1.6.4. Vitamin A (Retinol) and Vitamin E (-Tocopherol) Content Retinol and -tocopherol exhibit their characteristic maxima of UV absorption at 324 and 292 nm, respectively. The best separation within a reasonable time as well as good peak shape were achieved with a mobile phase consisting of methanol and n-hexane in proportion of 72:28 at a flow rate of 1 mL/min. The retention times were: 2.24±0.01 and 2.95±0.03 min for retinol and -tocopherol respectively. To determine the amounts of retinol and -tocopherol samples calibration curves were constructed by plotting the peak areas versus the concentration. Linearity was achieved in the concentration range 0.35–70 and 0.23–46 μM for all-trans-retinol and -tocopherol respectively. The detection limits calculated as signal-to-noise ratio equal to 2 were: 1.10 μM for retinol and 0.78 μM for -tocopherol [32-34]. 1.6.5. The Antimicrobial Activity The antimicrobial activities are evaluated against Gram positive (Staphylococcus aureus ATCC 6538, Bacillus cereus ATCC 7064, Mycobacterium smegmatis CCM 2067, Listeria monocytogenes ATCC 15313, Micrococcus luteus La 2971) and Gram negative (Escherichia coli ATCC 11230, Klebsiella pneumoniae UC57, Pseudomonas aeruginosa ATCC 27853, Proteus vulgaris ATCC 8427) bacteria and the yeast cultures (Candida albicans ATCC 10231, Kluyveromyces fragilis NRRL 2415, Rhodotorula rubra DSM 70403, Debaryomyces hansenii DSM 70238 and Hanseniaspora guilliermondii DSM 3432) using disk diffusion method. Antimicrobial screening: Disk diffusion method: Sterilized antibiotic discs (6 mm) were used following the literature procedure [35- 38].

2.6. Mineral Content Spectroscopic determination of minerals (Calcium, magnesium, potassium, sodium, iron, copper, manganese, selenium, zinc, chromium, aluminum) of samples was performed with a Varian Liberty Series II AX sequential model inductively coupled plasma-atomic emission spectrometer (ICP-AES) with a glass nebulizer and SPS 5.

2.7. Calorie Values The calorie values of samples were measured using a LECO-AC (350) Bomb Calorimeter. The samples were placed in the bomb chamber, pressurized to 425 psi with pure oxygen, combusted and the amount of heat liberated recorded on a plotter. The calorimeter was calibrated against benzoic acid standard before the analysis of samples [39].

SIGNIFICANCE OF THE STUDY

The antimicrobial activity of plant oils and extracts has been recognized for many years. However, few investigations have compared large numbers of oils and extracts using methods that are directly comparable. In the present study, 52 plant oils and extracts were investigated for activity against Acinetobacter baumanii, Aeromonas veronii biogroup sobria, Candida albicans, Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella enterica subsp. enterica serotype typhimurium, Serratia marcescens and Staphylococcus aureus, using an agar dilution method. Lemongrass, oregano and bay inhibited all organisms at concentrations of ≤2·0% (v/v). Six oils did not inhibit any organisms at the highest concentration, which was 2·0% (v/v) oil for apricot kernel, evening primrose, macadamia, pumpkin, sage and sweet almond. Variable activity was recorded for the remaining oils. Twenty of the plant oils and extracts were investigated, using a broth microdilution method, for activity against C. albicans, Staph. aureus and E. coli. The lowest minimum inhibitory concentrations were 0·03% (v/v) thyme oil against C. albicans and E. coli and 0·008% (v/v) vetiver oil against Staph. aureus. These results support the notion that plant essential oils and extracts may have a role as pharmaceuticals and preservatives.

LIMITATION OF THE STUDY

Some of the limitations of this study include external validity, or the generalizability of the study. The study did not distinguish between positional isomers (e.g. ω-3 versus ω-6 linolenic acid). The sum of myristic and palmitic acid (cholesterogenic saturated fatty acids) content ranged from 12.8 to 18.7%. The total unsaturated acid content ranged from 73.1 to 80.5%. The very long chain fatty acid (> 18 carbon atoms) content ranged from 0.44 to 1.37%. There were only 13 participants who participated in the complete study, and each participant was a college-degreed professional residing in the mid-west area of the University of Portharcourt Nigeria. In addition, the participants were all middle to upper middle class adults without learning disabilities.

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