Optical properties and chemical composition of hemp oil

Stefan Stefanov; Velichka Andonova; Natalina Panova; Tinko Eftimov; Krastena Nikolova

doi:10.3897/pharmacia.72.e160998

Abstract

Cold-pressed Hemp oil, obtained from the Cannabis sativa plant, contains many beneficial bioactive substances.

This study aims to investigate the fatty acid composition and optical properties of hemp oil, which is used as a base oil for pharmaceutical applications. To realize the set goals, gas-chromatographic methods, fluorescence spectroscopy, and spectroscopy in the visible range were applied.

Unsaturated fatty acids predominate in the sample, while the saturated ones are only 9.67%. Of the monounsaturated fatty acids, oleic acid predominates at 24.02%. Of the polyunsaturated fatty acids, linolenic acid predominates at 53.25%. Sterols and vitamin E are 1.75% and 0.5%, respectively.

The colorimetric characteristics were determined in the CIELab colorimetric system. The sample is relatively light for hemp oil, with a predominant yellow component and a weak red hue. Low lightness and a hue angle close to 90 degrees characterized the oil.

The fluorescence emission spectrum shows, for an excitation wavelength of 320 nm, three distinct emission regions: a peak in the 400–473 nm region; a second peak in the 500–520 nm region; and a third peak in the 670–760 nm region.

Keywords

hemp oil, fluorescence spectroscopy, color measurements, chemical composition

Introduction

Cannabis sativa L., commonly known as hemp, is one of the most recognizable and versatile herbaceous plants in the world, with a wide range of applications. The origin of the term “hemp” is unknown. It likely derives from the Latin word “hanapus” or the Old German “hanaf”, which means bowl or basket. The English word “hamper”, referring to a hemp bag or wicker basket, is also related.

“Cannabis” is a generalized term covering all aspects of the plant, its products, and their widespread application. The different parts of the plant contain a variety of compounds, including phytocannabinoids, flavonoids, terpenoids, and fatty acids (Alves et al. 2020). These compounds are not uniformly distributed among the plant’s different parts (Izzo et al. 2020). Modern industrial hemp varieties are known to contain cannabidiolic acid (CBDA), which is the building block of cannabidiol (CBD) (Citti et al. 2019).

Hemp has a long and rich history of commercial cultivation in China, Japan, Europe, Canada, and the USA. It is used in food, textiles and nutraceuticals (Tura et al. 2022). In the European Union (EU), over 70 hemp varieties have been approved for industrial use, and their cultivation is experiencing significant growth (Mendoza-Pérez et al. 2024).

Hemp has served as a traditional food source, with all parts of the plant – hemp seeds, leaves, flowers, and extracts – having been consumed in European countries since the Middle Ages. It has also been utilized in ancient medicines and as a source of fibers for making ropes or textiles. Due to its environmental benefits and diverse industrial and food applications, hemp production in the EU is on the rise. The cultivation of hemp requires specific permissions depending on the varieties allowed for use within the EU, and this regulation also applies to the production of oil from hemp seeds (Putnam et al. 2022).

Vegetable oils are primarily extracted from plants, mainly from their seeds or grains. These oils are utilized in a diverse array of consumer products, including cosmetics, biofuels, food items, medicines, and industrial processing materials (Arrutia et al. 2020).

The term “hemp oil” can refer to either vegetable oil extracted from seeds or essential oil from glands,as well as to “hash oil” – a concentrated extracts rich in cannabinoids, especially Δ9 —tetrahydrocannabinol (THC) (“liquid hemp” is a relatively new term characterizing cannabidiol [CBD]-rich concentrates, primarily for vaporization; sometimes inappropriately called “hemp oil”) (Muscarà et al. 2021).

Hemp seeds consist of lipids (30–35%), primarily polyunsaturated fatty acids (PUFA), carbohydrates (35–37%), and proteins (22–25%) (Mikulec et al. 2019). They are commonly processed into flour or oil. Hemp seed oil is typically extracted through cold pressing at low temperatures, which yields oil content of about 28–35%. It has a light fragrance reminiscent of walnuts and a dark to black color (Czwartkowski et al. 2022).

The growing interest in hemp oil is due to its diverse applications in pharmaceuticals, cosmetics, and dietary practices, particularly for vegetarians, vegans, and those following gluten-free diets (Castaldo et al. 2019). The oil composition is rich in linoleic and ά-linolenic acids, comprising approximately 50–70% and 15–25%, respectively (Tura et al. 2022). The ratio of mono- and polyunsaturated fatty acids is 1:3. Additionally, the content of tocopherols ranges from 41 to 110 mg/100 g, carotenoids from 9 to16 mg/100 g, phenols from 45 to 188 mg/100 g, and sterols from 390 to 670 mg/100 g (Marzocchi and Caboni 2020). This composition, along with the high levels of chlorophyll and β-carotene, makes the oil-prone to oxidation and unstable when exposed to sunlight (Callaway and Pate 2009; Aladić et al. 2015). The hemp oil content can vary based on several factors, such as the specific variety of the plant and the extraction method used (Kwasnica et al. 2022).

Cannabidiol (CBD) has also been identified in hemp seed oil. Traces of cannabinoid contamination may occur due to the oil-pressing process. The is primarily attributed to Δ9-tetrahydrocannabinol (THC), with levels in the oil reaching up to 50 ppm (Grotenhermen et al. 1998). CBD is important because it has documented anticonvulsant, antiepileptic, and antimicrobial properties, and it is used to treat osteoarthritis and other musculoskeletal disorders (Leizer et al. 2000).

There are no established criteria for assessing the quality of hemp oil in the commercial market. Standard techniques for evaluating the quality of cold-pressed or refined vegetable oils include gas and liquid chromatography (Guimet et al. 2005), Raman spectroscopy, and nuclear magnetic resonance (Guimet et al. 2004). These techniques are time-consuming, require special sample preparation, involve expensive consumables, and necessitate trained personnel.

The present study aims to characterize the fatty acid profile of cold-pressed hemp oil and to explore the possibilities for rapid, non-destructive assessment of its quality by determining correlations between its chemical composition and optical properties (fluorescence characteristics and color parameters), with a view to its use as a base oil in pharmaceutical and dermatological formulations.

Materials and methods

Materials

This study used bulk organic hemp oil (United Kingdom, code SKU9793, expiration date 20/09/2025), certified by Cosmos Organic. Merck Life Science (Darmstadt, Germany) provided all the reagents for the gas chromatographic analyses, including BF₃-methanol, nonadecanoic acid, hexane, and sodium sulphate.

The sample is neither diluted nor prepared in advance during the fluorescence spectroscopy with reagents.

Methods

Gas chromatographic analysis

We determined the fatty acid composition in the oil using the protocol for obtaining methyl esters. In particular, we mixed 50 μL of oil, 500 μL of BF₃-methanol (10% w/w), and 20 μL of nonadecanoic acid (internal standard). The mixtures were then heated in a thermomixer at 60°C for 30 minutes. After the reaction, the tubes were immediately transferred to an ice bath, and after five minutes, 500 μL hexane and 500 μL distilled water were added. The mixture was vortexed, and the upper layers were transferred to new tubes and dried over anhydrous sodium sulphate. Before analysis, the samples were filtered.

A gas chromatograph (Agilent GC 7890) equipped with a mass spectrometric detector (Agilent MD 5975) and an HP-5MS column was used under the following conditions: The column is 30 meters long, has a diameter of 0.32 mm, and a thickness of 0.25 µm. The temperature program starts at 100°C and stays that way for 2 minutes. At a rate of 15°C per minute, it then rises to 180°C and remains there for one minute. Finally, it rises to 300°C at 5°C/min and stays that way for 10 minutes. The injector and detector are both 250°C, and helium flows at a rate of 1 mL/min. The mass spectrometer has a scanning range of 50–550, and the injection volume is 1.0 μL in split mode 10:1. We identified compounds by comparing retention times and Kovats indices with those of standard substances and mass spectral data from the NIST’08 library.

Lipid quality indices

The atherogenic index (AI), thrombogenic index (TI) and hypocholesterolemic/hypercholesterolemicratiowere calculated using the equations suggested by (Ulbricht and Southgate 1991) and (Santos-Silva et al. 2002):

$A I = \frac{C_{12 : 0} + 4 . C_{14 : 0} + C_{16 : 0}}{MUFA + PUFA}$ (1)

$TI = \frac{C_{24 : 0} + C_{26 : 0} + C_{18 : 0}}{0.5 \times \sum M U F A + 0.5 \times \sum ω_{6} PUFA + 3 \times \sum ω_{3} P U F A + \frac{\sum ω_{3} P U F A}{\sum ω_{6} P U F A}}$ (2)

$\frac{h}{H} = \frac{C_{18 : 1 ω_{9}} + \sum PUFA}{C_{14 : 0} + C_{16 : 0}}$ (3)

where MUFA is monounsaturated fatty acids; PUFA is polyunsaturated fatty acids.

Fluorescence measurements

The sample is put in a quartz cuvette holder, which lets us change how thick the hemp oil layer is to get the best fluorescence signal. We determine the layer thickness experimentally based on the sample’s transparency. The excitation is provided by LEDs emitting continuously at wavelengths ranging from 200 nm to 700 nm through an optical fiber, which is also used to guide the fluorescence signal to the spectrometer (AvaSpec-2038, Avantes; OceanOptics USB 2000). We obtain the fluorescence spectra by exciting the sample with wavelengths ranging from 200 nm to 700 nm in 10 nm steps. The fluorescence spectra are averaged over 10 scans, each recorded 100 ms after the sample was excited. This step is done to make sure that the spectra are accurate. These parameters are kept constant across all measurements to ensure consistent experimental conditions.

The fluorescence spectra are recorded orthogonally to the excitation light path, as shown in Fig. 1.

Figure 1.

Experimental set-up for fluorescent measurements.

The advantage of using LEDs lies in the simplicity and cost-effectiveness of the measurements. Using a fiber-optic spectrometer lets you see spectra from dark samples while being close to them, so you don’t have to use n-hexane to make solutions.

Statistical analysis

Data was processed with SPSS software, version 26.0 (IBM Corp., Armonk, NY, USA). All results are presented as mean value ± standard deviation (SD).

A total of 50 fluorescence spectra were recorded for the examined sample. Using Matlab, the excitation-emission matrix and the topographic projection in the plane were obtained. The projection in the plane is convenient for comparison and serves as a “fingerprint” for distinguishing between different samples.

Color measurement

Researchers measured the spectral transmission and absorption characteristics using a Helios Omega spectrophotometer with a 10 mm cuvette across the spectral range of 350 nm to 750 nm. A standard illuminant D65 and a CIE 1964 standard observer (with a viewing angle of 10°) were used to determine the color parameters in the XYZ and CIE Lab colorimetric systems. The dominant wavelength and color purity were determined, as well as parameters such as lightness (L), color saturation (C), and hue angle (h).

$C = \sqrt{a^{2} + b^{2}}$ (4)

$h_{a b} = arctg (\frac{b}{a})$ (5)

Density determination

After tempering the sample at 20°C and using distilled water as the standard substance, we determined the density of the samples pycnometrically.

Results and discussion

Gas chromatographic analysis and lipid quality indices

The gas chromatographic analysis results of hemp oil are presented in Table 1.

Table 1.

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Fatty acid composition and sterol composition of hemp oil.

RT retention time	RI relative index	Type	Name	% of total fatty acids	Standard deviation
Saturated fatty acids
22.61	1720	C 14:0	Myristic acid	0.15	0.02
27.49	1902	C 16:0	Palmitic acid	6.31	0.69
31.28	2133	C 18:0	Stearic acid	2.82	0.31
37.94	2535	C 22:0	Docosanoic acid	0.17	0.02
40.93	2730	C 24:0	Tetradecanoic acid	0.21	0.02
Total saturated fatty acids				9.67	1.06
Monounsaturated fatty acids
27.01	1886	C 16:1	Palmitoleic acid	0.25	0.03
30.90	2052	C 18:1 ω-9	Oleic acid	24.02	2.64
34.28	2286	C 20:1 ω-9	cis 11-Eicosenoic acid	0.70	0.08
Total monounsaturated fatty acids				24.97	2.75
Polyunsaturated fatty acids
30.74	2095	C 18:2 ω-6	Linoleic acid	53.25	5.86
31.02	2098	C 18:3 ω-6	γ- Linolenic acid	2.75	0.30
31.07	2102	C 18:3 ω-3	α- Linolenic acid	6.91	0.76
Total polyunsaturated fatty acids				62.91	6.92
Sterols
47.63	3174		Campesterol	0.28	0.03
47.91	3198		Lanosterol	0.55	0.06
48.664	3285		β-Sitosterol	0.92	0.10
Total sterols				1.75	0.19
47.13	3140		Vitamin E	0.50	0.04

Unsaturated fatty acids constitute approximately 88% of the total fatty acid content. (Garcia et al. 2021) and (Arena et al. 2022) reported similar results (around 90%). Linoleic acid (C18:2) accounts for 53.25% of the total fatty acids. (Arena et al. 2022) reported identical results for linoleic acid, with values ranging from 53% to 56.5%. Other major fatty acids include oleic acid (C18:1, 24.02%) and α-linolenic acid (C18:3 ω-3, 6.91%). In our study, the content of α-linolenic acid is approximately half of that reported by Arena et al. (2022). Saturated fatty acids such as palmitic acid (C16:0, 6.31%) and stearic acid (C18:0, 2.82%) were also found in low concentrations. (Oomah et al.2002) and (Alonso-Esteban et al. 2020) have reported similar results.

The amount of γ-linolenic acid (GLA) is 2.75%, which is in line with the study of (Arena et al. 2022) (1.48 to 2.68%). GLA is increasingly used as a dietary supplement due to its anti-inflammatory properties (Johnson et al. 1997). The high content of polyunsaturated fatty acids makes hemp oil suitable for inclusion in pharmaceutical formulations with healing and anti-inflammatory properties (Leizer et al. 2000). α-Linolenic acid plays a crucial role in the management of neurodegenerative diseases and in the prevention of cardiovascular disorders. Consequently, hemp oil may be the preferred choice in cosmetic and pharmaceutical products compared to other vegetable oils. For instance, while flaxseed oil has a higher content of α-linolenic acid, it oxidizes rapidly and exhibits poor oxidative stability.

The ratio of saturated fatty acids (SFA) to monounsaturated fatty acids (MUFA) is 0.39, while the ratio of polyunsaturated fatty acids (PUFA) to SFA is 6.51. To assess the combination of these ratios, three indices are calculated: the atherogenic index (AI), the thrombogenic index (TI), and the hypocholesterolemic-to-hypercholesterolemic ratio (h/H).

The atherogenic index is 0.079, indicating that hemp oil reduces the risk of developing atherosclerosis. Its index is close to sunflower oil (0.07) and significantly lower than cold-pressed olive oil (0.14). Low thrombogenic index (TI) values are associated with a lower risk of thrombus formation. For the analyzed hemp oil sample, the TI is 0.151, which is lower than that of olive oil (0.32), similar to that of mackerel (0.16) (Moussa et al. 2014), and double that of flaxseed oil (0.07) (Guimarães et al. 2013).

The h/H ratio evaluates the hypocholesterolemic effect of hemp oil, with a higher value indicating a greater effect. For the analyzed sample, this index is 13.46. Medium-chain saturated fatty acids (SFAs) such as C14:0 (myristic acid) and C16:0 (palmitic acid) are considered hazardous due to their association with elevated serum concentrations of LDL cholesterol in humans. The thrombogenic index (TI) indicates the propensity for clot formation in blood vessels. Hemp oil demonstrates a low thrombogenic index, making it suitable as a base oil for various pharmaceutical products.

Researchers also investigated the content of vitamin E, as it is known to delay lipid peroxidation and preserve the oil’s oxidative stability. In our sample, vitamin E accounted for about 0.5% of the total fat content, which would positively influence the sample’s shelf life (Chen et al. 2010; Crescente et al. 2018).

The sterols (1.75%) and vitamin E (0.5%) enhance the antioxidant capacity of the oil, which is essential in the development of products targeting oxidative stress and skin aging (Marzocchi and Caboni 2020). Phytosterols such as β-sitosterol are used in antiandrogenic formulations, and their combination with polyunsaturated fatty acids (PUFA) supports a lipid profile beneficial to cardiovascular health (Crescente et al. 2018).

Fluorescence measurements

Fig. 2 presents the excitation-emission matrix (EEM) and its topographic projection in the plane. Fig. 3 shows the fluorescence emission spectrum for an excitation wavelength of 320 nm. The sample shows three distinct emission regions:

Figure 2.

Fluorescent excitation-emission matrix (EEM) (a) and topographic projection in the plane region (b) of hemp oil.

Figure 3.

Fluorescence emission spectrum for an excitation wavelength of 320 nm.

A peak in the 400–473 nm region;
A second peak in the 500–520 nm region;
A third peak in the 670–760 nm region.

The observed maximum around 520 nm is associated with vitamin E, while the peak around 675 nm correlates with chlorophyll content. (Saleem and Ahmad 2018) report that only cold-pressed oils like our hemp oil typically show these fluorescence peaks; refined samples lack them. The intensity of the fluorescence spectra around 520 nm increases with the excitation wavelength from 300 nm to 380 nm. Above 440 nm, this fluorescence band disappears. The band’s intensity around 675 nm increases with increasing excitation wavelength, with a maximum above 450 nm. Another fluorescence peak around 725 nm, associated with chlorophyll-like pigments, was also apparent. Other cold-pressed oils have reported similar results (Kyriakidis and Skarkalis 2000), but hemp oil reports this for the first time.

Oxidation products from the high unsaturated fatty acid content are responsible for the broad fluorescence peak around (400–420) nm. Other authors have reported similar findings for cold-pressed oils (Vila 2005; Karoui et al. 2011).

The relative intensity of the fluorescence maximum is relatively low, ranging from 500 to 700 a.u. This is a much weaker fluorescence peak compared to those in the spectra of refined oils. In Characterization of Vegetable Oils by Fluorescence Spectroscopy, written by (Kongbongaet al. 2011), this peak is between 1000 and 2000 a.u.for refined soybean, sunflower oil, grapeseed oil, or refined palm oil. This can be explained by the fact that during refining, oils are heated to high temperatures (around 200°C), leading to oxidative reactions. The oil loses compounds with antioxidant properties, and the amount of oxidation products sharply increases. The mechanical production of cold-pressed hemp oil preserves the antioxidant compounds, including vitamin E. This explains the weak fluorescence peak around 400 to 450 nm.

According to (Kongbonga et al. 2011), qualitative identification of fluorescence peaks associated with chlorophyll and vitamin E content is essential for assessing the oxidative stability of capsules, creams, and emulsions. Detection of these characteristic peaks may serve as a validation tool for evaluating proper storage conditions and detecting adulteration of hemp oil with refined oils.

Color measurement

Color evaluation is crucial as it is associated with the consumer’s subjective assessment when selecting food, cosmetics, or pharmaceutical products. Table 2 presents the color parameters obtained through calculations using specialized software that analyses the sample’s transmission spectrum. The color characteristics of the examined hemp oil sample are essential for assessing its quality and authenticity, serving as an indicator of excellent manufacturing practice and proper storage conditions.

Table 2.

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The main color parameters for hemp oil.

Color parameter in XYZ colorimetric system		Color parameter in CIE Lab colorimetric system
X	19.600	L	49.8
Y	18.300	a	11.9
Z	0.100	b	84.6
x	0.516	C	85.4
y	0.482	h	82.0

The color profiles of olive oil, neem oil, and hemp seed oil were compared by (Cesa et al. 2019), who found some similarities between hemp seed oil and olive oil, with hemp seed oil exhibiting low lightness.They also demonstrated that in high-quality hemp seed oil, the yellow component of the color predominates, while the red component is relatively weak. Our sample confirms the authors’ findings. The sample is relatively light for hemp oil, with a predominant yellow component and a weak red hue. Low lightness and a hue angle close to 90 degrees characterized the oil.

The color components of hemp oil can be used as a rapid indicator for assessing its quality and storage stability. In pharmaceutical practice, a decrease in lightness (L*) is considered a sign of tocopherol and chlorophyll degradation in the oil. Therefore, colorimetric analysis may be employed as a non-destructive method for rapid screening in industrial production settings.Low lightness and high values of the yellow component (b*) are also characteristic of sea buckthorn oil, as reported by (Cesa et al. 2019).

The density of hemp oil is also crucial when used as an ingredient in food or dietary supplements. For our sample, it is 992 kg/m³. The known density is relevant in the formulation of dosage forms, particularly in the production of soft gelatin capsules where the oil serves as a carrier for lipophilic substances. Its oxidative stability and the absence of a need for organic solvents are additional advantages in the development of clean, natural formulations (Akbari et al. 2022).

Conclusions

The analysis of hemp oil presented in this study highlights its advantageous chemical profile for use in pharmaceuticals, as well as its promising optical properties. The oil predominantly comprises unsaturated fatty acids – most notably linoleic acid at 53.25% and oleic acid at 24.02% – while saturated fatty acids are only 9.67% of its total content. This lipid profile, combined with a low atherogenic index (0.079) and thrombogenic index (0.151), supports the oil’s potential in maintaining cardiovascular health and reducing the risk of atherosclerosis and thrombus formation. Bioactive compounds such as sterols (1.75%) and vitamin E (0.5%) further enhance its antioxidant capacity, making it suitable for formulations targeting.

Optical characterization revealed distinct fluorescence emission peaks associated with vitamin E and chlorophyll, confirming the oil’s minimally processed, cold-pressed nature. The observed colorimetric properties – predominantly yellow with a weak red hue and low lightness – are consistent with high-quality hemp oil. Fluorescence analysis and color parameters can also detect quality changes in hemp oil by measuring oxidative and antioxidant products, pigments, and other components.

These findings collectively demonstrate that hemp oil offers significant nutritional and therapeutic benefits and possesses physicochemical characteristics, making it a promising candidate for use as a base oil in pharmaceutical and cosmetic products.

Additional information

Conflict of interest

The authors have declared that no competing interests exist.

Ethical statements

The authors declared that no clinical trials were used in the present study.

The authors declared that no experiments on humans or human tissues were performed for the present study.

The authors declared that no informed consent was obtained from the humans, donors or donors’ representatives participating in the study.

The authors declared that no experiments on animals were performed for the present study.

The authors declared that no commercially available immortalised human and animal cell lines were used in the present study.

Use of AI

No use of AI was reported.

Funding

This study is financed by the European Union - Next Generation EU - through the National Recovery and Resilience Plan of the Republic of Bulgaria, project № BG-RRP-2.004-0009-C02.

Author contributions

The findings collectively demonstrate that hemp oil offers significant nutritional and therapeutic benefits and possesses physicochemical characteristics, making it a promising candidate for use as a base oil in pharmaceutical and cosmetic products.

Author ORCIDs

Stefan Stefanov https://orcid.org/0000-0002-1044-6933

Velichka Andonova https://orcid.org/0000-0002-7369-1506

Natalina Panova https://orcid.org/0000-0003-2807-6026

Tinko Eftimov https://orcid.org/0000-0002-2767-1088

Krastena Nikolova https://orcid.org/0000-0002-2617-4776

Data availability

All of the data that support the findings of this study are available in the main text.

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﻿Abstract

Keywords

﻿Introduction

﻿Materials and methods

﻿Materials

﻿Methods

﻿Gas chromatographic analysis

﻿Lipid quality indices

﻿Fluorescence measurements

﻿Statistical analysis

﻿Color measurement

﻿Density determination

﻿Results and discussion

﻿Gas chromatographic analysis and lipid quality indices

﻿Fluorescence measurements

﻿Color measurement

﻿Conclusions

﻿Additional information

﻿References

Abstract

Introduction

Materials and methods

Materials

Methods

Gas chromatographic analysis

Lipid quality indices

Fluorescence measurements

Statistical analysis

Color measurement

Density determination

Results and discussion

Gas chromatographic analysis and lipid quality indices

Fluorescence measurements

Color measurement

Conclusions

Additional information

References