Quality analysis of walnut oil extracted by cold press and solvent methods for in vitro phytochemicals, antioxidant activity and GCMS analysis

The study aims to compare walnut oil extracted by cold press and solvent extraction methods in terms of yield, physical properties, and chemical composition. It evaluates differences in viscosity, acid and peroxide values, and identifies key bioactive compounds in each oil type.

Results suggest that cold press walnut oil, despite lower yield, retains more diverse bioactive compounds, offering greater potential for applications in the food, cosmetic, and healthcare industries.

The original article

Quality analysis of walnut oil extracted by cold press and solvent methods for in vitro phytochemicals, antioxidant activity and GCMS analysis

Lubna Masoodi, Adil Gani, Debabrata Sircar, Kulsum Jan 

Measurement: Food, Volume 18, 2025, 100223

https://doi.org/10.1016/j.meafoo.2025.100223

licensed under CC-BY 4.0

Selected sections from the article follow. Formats and hyperlinks were adapted from the original.

The Union Territory of Jammu and Kashmir is the primary location for walnut production in India, which makes up the majority of the nation's output. Jammu & Kashmir produced over 258.73 thousand metric tonnes of walnuts in the fiscal year 2021–2022, which accounted for roughly 91.68 % of India's total walnut output. Himachal Pradesh contributed 3.91 thousand metric tonnes (1.39 %), Arunachal Pradesh contributed 0.63 thousand metric tonnes (0.22 %), and Uttarakhand contributed 18.93 thousand metric tonnes (6.71 %).

According to recent studies, poor weather and degraded soil in Jammu and Kashmir are to blame for the drop in walnut output. India is expected to produce 33,200 metric tonnes of walnuts in the marketing year 2024–2025, which is a little rise from the updated estimate of 33,000 metric tonnes for 2023–2024. Walnut is widely consumed around the world because they are thought to have plasma cholesterol-lowering properties. Walnut oil has a high fat content, ranging from 52 % to 74 %. On average, walnuts contain 60 % to 70 % lipids, which include monounsaturated and polyunsaturated fats, 15.2 % proteins, 13.7 % carbohydrates, 6.7 % dietary fiber, and 1.8 % ash [1,2]. Because of the presence of monounsaturated fatty acids (primarily oleic acid, C18:1) and polyunsaturated fatty acids (linoleic acid, C18:2, and linolenic acid, C18:3) [3], as well as a variety of bioactive substances such as tocopherols, squalene, phytosterols, and polyphenols, walnut oil (WO) has long been considered a nutritionally valuable source of fat for humans [4]. In addition to its widespread usage in traditional medicine, walnut oil is acknowledged as healthy dietary oil in the food and agriculture sectors. The main emphasis of walnut oil nutrition research is its ability to treat digestive issues [5]. Because of its anti-inflammatory [6] and antioxidant qualities, it is a well-known functional food option for the treatment of ulcerative colitis [7] and inflammatory bowel disease (IBD)[8]. Furthermore, walnut oil has been shown to improve antioxidant capacity [9] and have anti-aging benefits in vivo [9].

The walnut processing industry is underdeveloped in Kashmir region and is not that much globally competitive. As a result, the majority of walnuts harvested in Kashmir are either consumed or exported. Therefore, the development and application of walnut oil has excellent prospects.

The extraction method used has a significant impact on the amount and quality of lipids and other important components. It is critical to choose an appropriate extraction method for the compounds of interest. Scaling up walnut oil production to commercial levels necessitates the use of appropriate extraction technologies [10]. Oil extraction is based on fundamental principles such as avoiding damage to the oil during the extraction process, extracting oil with the fewest impurities possible, reducing the amount of fat remaining in the cake, and extracting as much oil as possible from the raw material [11]. Despite the fact that many studies on new walnut oil processing methods have been published in recent years [12], Solvent extraction is commonly used in industrial oil extraction due to its ease of evaporation and low energy cost. However, rising consumer interest in organic and green processed foods (food processed in a safe, cost-effective, and environmentally friendly manner) has fuelled the on-going growth of the corresponding market. Cold-pressed oils are organic and have gained popularity, owing primarily to nutritional trend changes. Moreover, cold-pressed oils are obtained by pressing the kernels without the use of chemicals or heat treatment, resulting in a plant lipophilic extract with high retention of colour, flavour, and nutritive properties and minimal degradation of functional components

This study provides a comprehensive comparative analysis of cold-pressed and solvent-extracted walnut oil by evaluating phytochemical composition, antioxidant activity, and volatile compound profiling using GC–MS. Unlike previous studies that focus primarily on yield and basic composition, our work offers a detailed insight into how different extraction techniques influence the functional and nutritional properties of walnut oil. These findings have important implications for optimizing walnut oil production for both nutritional and industrial applications.

2. Materials and methods

2.7. Identification of bioactive compounds using GC–MS

2.7.3. Analysis in GC–MS

Agilent Technologies GC 7890B and MS 5977B system was used. The separation of chemicals with helium as a carrier gas was accomplished using a HP-5 MS column. With split less mode, the injection volume was 1 µl. The temperature of the injector was set to 280 °C. The oven temperature was set at 80 °C for 1.5 min, then ramped to 220 °C at a rate of 10 °C/min without holding, then increased to 310 °C at a rate of 20 °C/min held for 10 min and with a 5-minute solvent delay. The flow rate across the column was 1 mL/min. The conditions for the operation of mass spectrometer were set as follows: ion source temperature 230 °C, MS Quad temperature 150 °C, electron energy (70 eV) and scanning range of m/z, 25–1000 amu. The compounds were identified by comparing mass spectrum of the components to that of mass spectral library from NIST 17.L (National Institute of Standards and Technology).

2.7.4. Metabolite identification

The identification of metabolites was done by matching mass spectra of each compound from library (3:1, signal: noise) using the NIST-11 mass spectral library (National Institute of Standards and Technology). For the mass spectra comparison, the matching value of the metabolite identity taken was >70. To check for co-elution, the mass spectra of all peaks were analysed at three different points, beginning, middle and end of each peak width. There was no co-elution was seen in any of the identified peaks. Furthermore, the compounds were identified based on their retention index, mass spectrum, and the calculation and comparison of the GC retention index of a series of alkanes (C8–C30).

4. Result and discussion

4.3. Total phenol content and antioxidant activity

Fig. 1 represents the data of total phenol, tocopherol content and antioxidant activity of cold pressed and solvent extracted oils.

Measurement: Food, Volume 18, 2025, 100223: Fig. 1. Total phenol and antioxidant activity of cold pressed (CP) and solvent extracted (SE) walnut oils.

Significant difference was found in the oils extracted from cold press and solvent extraction methods. The highest values of total phenol were found in cold press oil (321mgGAE/100 g). The results are in line with the results of Arrnaz et al. [28].. They reported that the total phenol content and ortho diphenol content of walnut oils were 0.32 mg GAE/g and 0.32mgGAE/g, respectively. Our findings were also consistent with the findings of Sayed et al. [24] and Miraliakbari and Shahidi [29]. This strongly suggests that the cold press extraction is more effective method than solvent extraction to obtain phenolic compounds.

The level of antioxidant activity of the walnut oils was evaluated using spectrophotometric absorbance of DPPH reagent. The oil extracted using the cold press method was the most effective at scavenging DPPH radicals (49.0 %) than solvent extracted oils (38 %) at a significance of (p ≥ 0.01). The antioxidant activities of the oils studied were attributed to phenolic and non-phenolic compounds of the sample [28].

4.4. Bioactive Compounds using GC–MS

The extraction method had a great impact on the metabolite content of walnut oils. Table 3, Table 4 represent the total number of compounds (alcohols, triglycerides, free fatty acids, ketones, steroids, vitamin) detected by GCMS in cold press and solvent extracted walnut oil respectively. Free fatty acids were more found in both extracted walnut oils proving that it is a good source of polyunsaturated free fatty acids. A total number of 60 compounds were found in cold pressed walnut oil. However only 43 compounds were found in solvent extracted oil. The reason could be the heat that is employed during the solvent extraction which causes thermal degradation of compounds. Heat map (Fig. 2) gives graphical data representations that use color-coded systems. The red in the graph represents the higher concentration of compounds while as green represents the low concentration. In cold pressed walnut oil the prominent bioactive compounds detected were pentadecanoic acid, 9, 12-octadecadieno, 9-hexadecenoic acid, gamma-sitosterol, delta-tocopherol, heneicosanoic acid, gamma-tocopherol, nonanoic acid, camphor. However in solvent extraction the prominent compounds found were palmitic acid, linolenic acid, oleic acid, alpha-linolenic acid, eicosanoic acid, 9,12-octadecadieno, 2-linoleoylglycerol, monolinolein and so on. The results were in line with the observation reported by Pan et al. [22], who compared the walnut oils obtained by cold press and solvent extraction (using five different extraction solvents) and found that cold press contained more bioactive compounds than solvent extraction. The tocopherol contents of cold press walnut oil contained two tocopherol homologs delta and gamma tocopherols. Savage et al. [30], also reported that gamma tocopherol is the predominant homolog present in cold pressed walnut oil. Squalene is a 30-carbon straight-chain hydrocarbon steroid precursor and was found in major quantity in cold pressed walnut oil than solvent extracted oil. Because of growing concerns about marine life, new sources of squalene are being sought, with shark liver oil being the traditional source, making walnut oil a potentially valuable source of this compound.

Measurement: Food, Volume 18, 2025, 100223: Fig. 2. Heatmap analysis of bioactive compounds in walnut oils extracted by cold press (Class C, blue) and solvent extraction (Class S, red).

In contrast to solvent extraction, this study showed that cold press extraction produced a greater retention of polyphenols and tocopherols and other bioactive compounds. This is explained by the mechanical properties of cold pressing, which protect delicate bioactive components from solvent interference and heat deterioration. Prior studies on a variety of edible oils have shown similar patterns. According to Turkish Food Codex [31], many temperature sensitive phenolic compounds are not lost along with the heat treatment, which is referred to as higher quality oil. In the purifying of cold press oils, only washing with water, filtration and centrifugation can be carried out. These results support the advantages of cold-press extraction for maintaining the quality of walnut oil and are consistent with our investigation. Our study's only reliance on hexane for tocopherol extraction is one of its limitations. Hexane may not be as effective in extracting some tocopherol homologs (β-, γ-, and δ-tocopherols) due to its low polarity.

As consumer demand for minimally processed functional foods continues to rise, cold-pressed oils can be promoted as high-end health goods [11]. However, solvent extraction is still more cost-effective for large-scale manufacturing, when oil output is the key consideration. Therefore, the economic value of walnut oil might be increased by optimising extraction parameters based on desired applications (e.g., culinary, medicinal, or cosmetic usage).

5. Conclusion

The effects of solvent and cold press extraction techniques on the quality of walnut oil were thoroughly examined in this study. According to our research, solvent-extracted walnut oil exhibits a greater oil production but a decrease in several heat-sensitive metabolites, whereas cold-pressed walnut oil preserves more bioactive components, polyphenols, and tocopherols. In comparison to solvent-extracted walnut oil (43 compounds), cold-pressed walnut oil had a much greater total number of metabolites identified by GC–MS analysis (60 compounds). Cold pressing's mechanical properties, which prevent heat degradation and preserve more useful bioactives including γ-tocopherol, δ-tocopherol, squalene, γ-sitosterol, pentadecanoic acid, and linoleic acid, are responsible for this. However, a significant quantity of palmitic acid, oleic acid, linolenic acid, and monolinolein was found in solvent-extracted oil, indicating that solvent extraction is an efficient method of extracting lipid fractions. However, contact to high temperatures may result in the loss of small bioactive molecules. This study emphasises walnut oil as a potential plant-based source of squalene, a substance that is typically obtained from shark liver oil, in addition to its nutritional value. Walnut oil may be a good substitute for bioactive components in cosmetic and nutraceutical applications, especially in light of the growing need for environmentally acceptable and sustainable sources. These findings provide valuable insights for the walnut processing industry, particularly in optimizing extraction techniques for different applications. Cold-pressed walnut oil can be marketed as a premium health product, catering to consumers seeking minimally processed, nutrient-rich oils. Solvent extraction, while more efficient in oil yield, may be more suitable for commercial-scale production where quantity is prioritized over bioactive retention.