Legume Research

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Legume Research, volume 47 issue 2 (february 2024) : 206-213

Physicochemical and Biological Characterization of Opuntia ficus-indica (L.) Mill. Seed Oil Collected from The Bordj Bou Arreridj Region, Algerian Highlands

H. Guetarni1,2,*, H. Baroun1, A. Derabla1, A. Ouradj1
1Department of Biology, University Bounaama Djilali of Khemis Miliana, 44225, Ain Defla, Algeria.
2Laboratory of Natural Substances Valorization, University Bounaama Djilali of Khemis Miliana, 44225, Ain Defla, Algeria.
  • Submitted10-07-2023|

  • Accepted21-10-2023|

  • First Online 20-11-2023|

  • doi 10.18805/LRF-759

Cite article:- Guetarni H., Baroun H., Derabla A., Ouradj A. (2024). Physicochemical and Biological Characterization of Opuntia ficus-indica (L.) Mill. Seed Oil Collected from The Bordj Bou Arreridj Region, Algerian Highlands . Legume Research. 47(2): 206-213. doi: 10.18805/LRF-759.

Background: The prickly pear is well adapted to arid and semi-arid areas. It occupies an important part in the human diet and it is also used as fodder for livestock. It is an interesting plant due to the environmental conditions in which it grows and its resistance to extreme climatic conditions. The objectives of our study were to determine the biological effects of oil extracted from prickly pear seeds (Opuntia ficus-indica L. Mill.).

Methods: The organoleptic characteristics and the physicochemical parameters of this oil were done. The antibacterial and antifungal effects of the oil were tested in vitro against certain numbers of pathogenic bacteria and fungi by the aromatogram method. The antioxydante activity and the GC-MS analysis were proceeded also in this study. 

Result: The results show that the bacterial strains Klebsiella pneumoniae and Enterococcus faecalis are the most sensitive to the acid oil of Opuntia ficus-indica (L.) Mill. with an inhibition diameter varying between 23 mm and 25 mm, respectively. On the other hand, prickly pear seed oil exhibits an antifungal effect mainly against Candida albicans (25 mm). The oil extracted was strongest active (IC50 = 0.55 mg/ml). This indicates that our oil exhibited high reducing activity. 18 components were found in prickly pear seeds oil after analysis by GC-MS, which are presented mainly by phthalic acid (45.04%), monounsaturated fatty acid (18.56%), polyunsaturated fatty acid (16.69%) and other components (19.71%). The oil of Opuntia ficus-indica (L.) Mill. seeds showed good biological activity grace to its richness in components.

Humans have used vegetable oils for centuries and still using it nowadays in food, medicine, cosmetics and as fuels. Plants generally accumulate oil in their seeds and fruits to provide energy for germination and the early stages of seedling development, which make seeds good sources of edible oils. Oilseed crops are primarily grown for edible oil. Recently, oilseeds attracted more attention due to an increasing demand for their healthy vegetable oils, livestock feeds, pharmaceuticals, biofuels and other oleochemical industrial uses. The increased interest resulted in an 82% expansion of oilseed crop cultivation areas and about a 240% increase in total world production over the last 30 years (Karaoui et al., 2020).
The prickly pear, called Opuntia ficus-indica (L.) Mill. is present in all parts of Algeria except the south. First widely used for the fight against drought and erosion, it is used for the consumption of its fruits or as animal feed. One of the objectives of the concept of sustainable integrated development set by the United Nations Development Program (UNDP) in collaboration with the FAO (Food and Agriculture Organization of the United Nations) aims to increase the agricultural potential of arid zones, in order to face the vagaries of the climate (Benkaddouri, 2011). The seeds of the fruit are pressed to extract the oil they contain. Virgin prickly pear seed oil is a very valuable oil extracted by cold pressing which is widely used in pharmaceuticals and cosmetics. It has multiple moisturizing, antioxidant and therefore anti-aging properties. Its exceptional richness in vitamin E and sterols gives it an exceptional ability to protect the skin against free radicals and boost cell renewal (Krifa, 2019). Phytotherapy is currently experiencing considerable growth as a result of the increasing discovery of effective plant extracts for the treatment of various diseases (Novais et al., 2004). Today, the World Health Organization (WHO) estimates that 80% of African populations are cured with natural remedies and 20% of the drugs produced and marketed worldwide come from medicinal plants (WHO, 2022). As part of the development of new antidiabetic agents of plant origin, ethnobotanical investigations have revealed the use of about a hundred medicinal plants for the treatment of diabetes. Among these species is Opuntia ficus-indica (L.) Mill. (Benkaddouri, 2011). Cactus plant (Opuntia ficus-indica L. Mill.) has long been used in traditional medicine for the treatment of several diseases. As a result, during the last two decades, the search for health-promoting compounds in O. ficus-indica (L.) Mill became increasingly popular. In fact, this Caryophyllal-belonging plant was revealed to be rich in a variety of bioactive secondary metabolites, namely flavonoids, betalain pigments and tocopherols, which are known to play an important role in human health protection and prevention from different pathologies (Benramdane et al., 2022).
The purpose of this work is to study the biological properties of the oil extracted from prickly pear seeds collected from Bordj Bou Arreridj region (antimicrobial and antioxidant activities) and to determine their chemical components by GC-MS.
The present study was carried out during 2022 within the University Bounaama Djilali of Khemis Miliana and at the Dr. Zibouche Abdellah medical analysis laboratory of Ain Defla.
Prickly pear seed oil extraction
It is extracted by cold pressing by Dr. Saifi Mounir (University Bounaama Djilali of Khemis Miliana) at his small company Aromabiol from the seeds of Opuntia ficus-indica (L.) Mill. harvested from Bordj Bou Arreridj region (36o 4' 0" North, 4o 46' 0" East) (Fig 1) (Google Map, 2022).

Fig 1: Prickly pear seed harvest site (Google Map, 2022).

This method is used mainly to produce extra-virgin edible oils or by small capacity units and allows extraction by cold pressing by passing the seeds through a screw oil press which causes an increasing pressure at around 60oC. The oil obtained is then filtered and checked that no contamination has occurred during the process. The pure prickly pear oil thus obtained is packaged in glass bottles. The recovered oil was decanted, weighed and then stored at -20oC. This technique allows the preservation of the content of essential fatty acids and natural antioxidants and therefore avoids an alteration of the properties of the oil (Nitiema et al., 2012). Oil extraction by cold screw pressing is an alternative method and has been found to be a substitute to extraction of solvent. This process has the advantage of being less oil producing than others but is safer, simpler, less expensive, hygienic, no chemical residue and ecologically friendly. Cold-pressed oils improve the quality of oil and are rich with bioactive components such as essential fatty acids, sterols, tocopherol and phenolics (Alqurashi et al., 2022).
Microbial strains
Nine microorganisms were used for testing the antimicrobial activity of O. ficus-indica  oil (Table 1). These microbial strains were maintained by subculturing on nutrient agar (CONDA603012) favourable to the growth of bacteria at 37 oC for 24 h and at 30oC for 48 h for yeasts.

Table 1: Strains used in antibacterial and antifungal activity.

Organoleptic properties of Opuntia ficus-indica seed oil
The organoleptic examination is one of the criteria for evaluating the quality of the oils. It concerns odour, color and appearance at 20oC.
Analysis of physicochemical parameters
The physicochemical parameters of prickly pear seed oil (PPSO) studied are: yield (AFNOR, 1996), pH (Actu-Environnement, 2022); refractive index (AURA, 2022); the acid index and acidity (Jacques et al., 2012), the saponification index (AFNOR, 1984) and the ester index (Novidzro et al., 2019).
Antibacterial and antifungal activity
We adopted the aromatogram method on agar medium using sterile discs (Somaie et al., 2013). In each inoculated Petri dish, 6 blotting paper discs are applied (Andrews, 2001): (A) Negative control, (B) Pure oil, (C) Oil diluted 1:2 (v/v), (D) Oil diluted 1:4 (v/v), (E) Oil diluted 1:8 (v/v), (F) Oil diluted to 1:12 (v/v). In this experiment, the oil was dilute  in dimethyl sulfoxide (DMSO) which is inert on antibacterial activity. The prepared Petri dishes are incubated at 37oC for 24 h for bacteria and at 30oC for 48 h for fungi. The reading of the results was made by measuring the diameters of disc inhibition halos using aslide gauge. The results can be symbolized by signs according to the sensitivity of the strains to oil (Hombach et al., 2013).
Antioxidant activity
The ability of prickly pear seed oil to scavenge the free radical DiPhenylPicrylHydrazyl (DPPH) is evaluated using the method described by Jaamali et al., (2013). The stock solution of oil in ethanol is prepared at a concentration of 100 μg/ml. A series of dilutions were carried out to obtain concentrations ranging from 25 μg/ml to 100 μg/ml. The reaction mixture is stirred vigorously and incubated for 30 min in the dark and at room temperature. Absorbances are measured at 517 nm. The control samples are composed of 50 μl of methanol to which 2 ml of the ethanoic solution of DPPH has been added. Ascorbic acid is used as a positive control. The survey is carried out using a visible UV spectrophotometer. Antioxidant activity is expressed as per cent inhibition calculated using the following equation:
Inhibitory concentration (IC50) is the concentration of the test sample required to reduce 50% of the DPPH radical. IC50 is calculated from the graph of the antioxidant activity (in %) according to different concentrations of oil and vitamin C tested. A low IC50 value indicates a high capacity of the oil to act as a DPPH scavenger (Molyneux, 2004). The reducing power of a compound is associated with its antioxidant power, an increase in absorbance corresponds to an increase in reducing power (Chikhi, 2013).
Oil analysis by GC-MS
The chromatographic analysis of the O. ficus-indica oil was carried out with a gas phase chromatograph type TQ 8030 coupled to a mass spectrometer.
The fragmentation is carried out by electron impact at 70 eV. The operating conditions were: column temperature 40 to 250oC, helium flow rate as carrier gas 3 mL/min, split pressure 49.5 KPa, volume of oil injected 0.5 μl, computerized database (Q3 Scan).
Organoleptic examination
In the present study from a seeds of O. ficus-indica collected from Bordj Bou Arreridj region, the oil was extracted by cold pressing method. This oil was examined and its organoleptic characteristics were determined before studying it’s biological effects. Opuntia ficus-indica seed oil is found to have a clear appearance, light yellow color and very mild odor. Oils are usually liquid at room temperature and volatile. Our oil are more or less colored. It is important to appreciate the organoleptic characteristics of the oil of OFI [Opuntia ficus-indica (L.) Mill.] in which its colour, flavor and smell are the parameters that give a general idea of quality (AFNOR, 2000).
Physicochemical analysis
The oil extracted from PPSO had a pH equal to 4.50. It gave a yield equal to 4%. The refractive index of Opuntia ficus-indica oil at 20oC is 1.469. From the results obtained, we found that the value of the acid index is equal to 2.80 mg of KOH/g. Others physicochemical parameters are determined such as acidity, acidification index and ester, whose recorded values are: 1.40%, 182.32 mg of KOH/g and 179.52 mg of KOH/g, respectively. The pH of PPSO oil is 4.5. Our oil is acidic. For our oil extracted by cold pressing, its saponification index is equal to 186.63 mg of KOH. Physicochemical parameters are influenced by soil and climatic conditions as well as plant growing conditions (Gildo, 2006), geographical separation, harvest season, cultural practices and extraction technique (Akrout et al., 2010). Indeed, this oil requires 1 ton PPSO and 25 kg of the seeds to extract 1 litre of oil (Krifa, 2019).
A low refractive index of oil indicates its low refraction of light which could promote its use in cosmetic products (Chouitah, 2011). Acidity value varies according to the stage of maturity of the plant (Charef et al., 2008). A high value means poor conservation of the fruits before oil extraction or a harvest made from unripe fruit (Djerrou, 2014). Acidity is the percentage of fatty acid expressed as oleic acid for most fatty substances. A low acidity level contributes to the stability of the oil against oxidation by air. It is recommended for an edible oil to have a low acidity level (less than 3.3%, standard imposed by Codex Alimentarius, to withstand long storage without deterioration (Onyeike et al., 2002). A high saponification index indicates that the oils have a high triglyceride content and are therefore very useful in cosmetology (Gharbi et al., 2015).
Antibacterial activity
According to the results obtained, Gram-positive bacteria are the most sensitive to the antibacterial activity of Opuntia ficus-indica. seed oil (Table 2, Fig 2). The oil inhibited the growth of Enterococcus faecalis whose diameters of the zones of inhibition are 25 mm, 22 mm, 20 mm, 16 mm and 10 mm obtained with B, C, D, E and F, respectively. We also recorded the presence of Klebsiella pneumoniae inhibition zones whose diameters vary from 18 mm to 23 mm obtained with the same dilutions. The results show that O. ficus-indica (L.) Mill. seed oil proved inactive against Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 7756, Bacillus spizizenii ATCC 6633 and Staphylococcus aureus ATCC 6538. Gram-positive bacteria are increasingly susceptible compared to Gram-negative bacteria. The difference in the outer layers of these bacteria, which Gram-positive bacteria apart from the cell membrane, have an additional layer which consists of phospholipids, proteins and lipopolysaccharides. The outer membrane of Gram-positive bacteria is highly charged. It acts as a barrier to vegetable oils (Chouitah, 2011). For Gram-negative bacteria, proteins that are in the outer membrane are deactivated before they reach the cytoplasmic membrane and cytoplasm (Fujisawa et al., 2009). This membrane is impermeable to most molecules, which reflects the resistance of the other strains tested (Turkmen et al., 2007). Escherichia coli ATCC 25922, a Gram-negative bacterium, also develops resistance to a number of oils (Delamare et al., 2007). It has been described that this sterol can inhibit the growth of certain microorganisms such as Pseudomonas aeruginosa ATCC and Bacillus spizizenii (Ogbe et al., 2015). The antibacterial activity of this oil can be attributed mainly to its major constituents, such as terpene components, which are particularly active against microbial cells because they are soluble in aqueous media and cause damage to the cell walls of microorganisms (Fillippi et al., 2006). In terms of antibacterial activity, linalool was shown to be the most effective and inhibited 17 bacteria such as Enterococcus faecalis, Escherichia coli, Bacillus spizizenii and Staphylococcus aureus (Peana et al., 1999).

Table 2: Diameter of the zones of inhibition of the bacterial strains tested in mm by Opuntia ficus-indica oil.


Fig 2: The antimicrobial potency of the OFI seeds oil against microbial strains.

Antifungal activity
The results of antifungal activity of Opuntia ficus-indica (L.) Mill. oil are presented in Table 3 and Fig 2. The antifungal activity of the oils can occur through two different mechanisms: some constituents cause the leakage of electrolytes and the depletion of amino acids and sugars and the other can be inserted into membrane lipids, therefore there are loss membrane functions (Suppakul et al., 2003).

Table 3: Measurement of inhibition zone diameter of the fungal strains tested by Opuntia ficus-indica seed oil.

The antifungal action of Opuntia ficus-indica (L.) Mill. oil against Candida albicans is due to an increase in the permeability of the plasma membrane followed by a rupture of the latter leading to leakage of the cytoplasmic content and therefore death yeast (Cox et al., 2000).
Antioxidant activity by DPPH
We were able to graphically determine the corresponding 50% inhibition concentration (IC50), which constitutes the antioxidant activity of the oil (Fig 3). According to the recorded results, the IC50 of the oil is equal to 0.55 mg / ml. It is relatively low for ascorbic acid, the value of which is around 0.45 mg/ml. The IC50 is proportional to the antioxidant activity of the oil (Fig 4). Concerning extracts from the seeds of three different species of Opuntia ficus-indica (L.) Mill. in which the percentages of root inhibition of DPPH are higher in the polyphenol and the fractions rich in flavonoids more precisely than in the other variety. The ability to reduce DPPH, this antioxidant activity due to phenolic compounds (Prior et al., 2005). Our results are in agreement with those obtained by Chougui et al., (2013).

Fig 3: DPPH inhibition rate as a function of Opuntia ficus-indica seed oil concentrations.


Fig 4: Representative histogram of IC50 inhibitory concentration value of Opuntia ficus-indica seed oil.

This appears to be consistent with other results in the literature, where the mechanism of interaction between antioxidants and the DPPH radical depends on the structural conformation of the antioxidant and the latter interacts with the DPPH radical by reducing an equal number of hydroxide groups carried by the radical DPPH (Molyneux, 2004). Ascorbic acid, tocopherols, flavonoids and tannins are antioxidant molecules that reduce and decolorize DPPH due to their ability to yield hydrogen (Pooter et al., 1986). The synergistic interactions between the different constituents of vegetable oil is at the origin of much greater antioxidant power (Vardar et al., 2003). Antioxidants are considered as reducers and inactivators of oxidants (Siddhuraju, 2007). The reducing power of a compound can serve as a significant indicator of its potential antioxidant activity (Bougandoura, 2013).
In the work of El-Faydy et al., (2017), showed that the series of the alkylthiomethyl-8-hydroxyquinoline present a significant antibacterial activity when compared to the Nitroxoline. The synthesized compounds demonstrated moderate antioxidant activities by DPPH in comparison to our oil which is a good antioxidant.
GC-MS analysis of Opuntia ficus-indica (L.) Mill. oil
Using this analysis technique, we were able to qualitatively and quantitatively study the chemical compounds of our oil (Fig 5, Table 4). Determination of oil chemical compound levels was based on peak area normalization without using correction factors. The constituents present in our oil are mainly presented by: phthalic acid (1,2-benzenedicarboxylic acid mono (2-ethylhexyl) ester), monounsaturated fatty acid (Octadec-9-enoic acid), polyunsaturated fatty acid (Acid 9, 12-octadecadienoic (Z,Z)-) and whose values were obtained: 45.04%, 18.56%, 16.69% and 19.71%, respectively (Fig 6).

Fig 5: Chromatographic profile of prickly pear seed oil after analysis by GC-MS.


Table 4: Chemical constituents of prickly pear seed oil.


Fig 6: Level of chemical components present in prickly pear oil.

This analysis made it possible to identify 18 bioactive compounds which have interesting inhibitory actions against pathogenic microorganisms. The antimicrobial activity is due to either one or more prickly pear seed oil constituents.
The influence of climatic conditions and soil conditions on vegetable oil composition has been established in other medicinal plants such as St. John’s wort and thyme (Antonio, 2011). Opuntia ficus-indica (L.) Mill. seeds are a valuable source of essential unsaturated fatty acids. Polyunsaturated fatty acids are well known for their importance in the field of nutrition and use in the pharmaceutical industry (Chougui et al., 2013).
The results of our work allowed us to assert that Opuntia ficus-indica (L.) Mill. seed oil could be a potential source of natural bioactive compounds which had a good inhibitory effect on pathogenic microorganisms.
The first author thanks Dr. Zibouche Abdellah, Dr. Saifi Mounir and the others medicals laboratories cited in this work.
We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome. 

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