Dihydroactinidiolide from thermal degradation of β-carotene

ABSTRACT

The formation of dihydroactinidiolide by thermal degradation of β-carotene was studied. A comparison of yields of dihydroactinidiolide in commercial β-carotene and β-carotene derived from crude palm oil was investigated. Thermal degradation of commercial β-carotene promoted the formation of dihydroactinidiolide with the highest yield, 61.21%. Thermal degradation of recovered β-carotene yielded 29.23% of dihydroactinidiolide. The lower recovery of β-carotene was due to the mixture of compounds in the extract. Further investigation indicated some other useful aroma compounds formed from this thermal degradation were β-ionone, 3-oxo-β-ionone, and β-cyclocitral.The outcome provided wide opportunities in utilizing crude palm oil as natural source of β-carotene to produce aroma compound.

KEYWORDS:

• β-carotene
• Crude palm oil
• Dihydroactinidiolide
• Thermal degradation
• Aroma compounds
• β-ionone
• β-cyclocitral

Introduction

Dihydroactinidiolide (dhA) was first isolated as feline’s attractants from leaves of Actinidia polygama^[1] and identified as a flavor component in many plants, such as tobacco and tea. dhA is one of three components of the pheromone for queen recognition of the workers of the red imported fire ant (RIFA), Solenopsis Invicta,^[2] and in mammals such as the cat and the red fox.^[3] Since its discovery in the early 1930s, RIFA has become a major agricultural and urban pest throughout the southeastern United States. In addition, fire ants cause both medical and environmental harm.

The dhA molecule contains a carbonyl group that can react with nucleophilic structures in macromolecules, providing this compound with a high potential reactivity. The dhA is also found to exhibit cytotoxic effects against cancer cell lines.^[4] In contrast, little is known on the actions of dhA in vascular plants. Nevertheless, dhA has been identified as a major component of ethyl acetate extracts of cyanobacteria or aquatic macrophytes, which inhibit seed germination and seedling growth.^[5] This compound has also been identified in wheat glumes, which acts as a germination inhibitor.^[6]

A large number of synthetic approaches either to racemic^[7] or to enantioselective^[8] syntheses of dhA has been developed over the last 40 years based on the previously mentioned biological properties. The most classical approach is that described by Mori and Nakazono.^[9] However, some of the reported enantioselective syntheses suffer from significant drawbacks. The most significant synthetic issues concerns the need of using lengthy multi-step procedures, specially prepared chiral catalysts or enantioenriched starting materials. More efficient methods for the transformation of tetrahydroactinidiolide into dhA, still need to be studied.

Thermal degradation of β-carotene at 180°C in an oxygen-free environment first studied by Mulik and Erdman^[10] and Day and Erdman,^[11] which show the formation of toluene, m-xylene,2,6 dimethylnaphthalene, and ionone. LaRoe and Shipley^[12] found α-ionone and β-ionone in small amounts along with toluene, xylene, 2,6-dimethylnaphthalene, and ionone when β-carotene was heated at 188°C for 72 h. The mechanism and kinetic study for the formation of volatile compounds conducted by Kanasawud and Crouzet^[13] shows that dhA is the first compound produced during heat treatment of β-carotene at 97°C in water. Kinetics studies indicate that dhA may also be produced through 5, 6-epoxy-β-ionone, which is an important intermediate reaction. This compound acts as a precursor for different volatiles such as β-ionone, 2-hydroxy-2, 6, 6-trimethylcyclohexanone, and 2-hydroxy-2, 6, 6-trimethylcyclo-hexane-l-carboxaldehyde. However, the amount of volatile compounds formed in the thermal degradation is still limited.

The synthetic method was simplified by analysing the thermal degradation of β-carotene and the amount of dhA formed was reported. The source of β-carotene, from crude palm oil (CPO) is rich in carotenes at approximately 600 ppm, and Malaysia is one of the major consumers and exporters of palm oil. Various methods of carotenoid recovery from palm oil have been reported. These include saponification, soxhlet adsorption, selective solvent extraction, and transesterification followed by distillation, and supercritical fluid extraction using CO₂.^[14] To-date, supercritical fluid extraction technology R134a as a solvent was introduced to prevent degradation of carotene during extraction.^[15] These sources are becoming more important to recover the carotenes in palm oil because most of them are destroyed in the present refinery process to produce light color oils.The thermal degradation of β-carotene has been studied since 1963, but none of the studies reported the amount of dhA formed. Nonetheless, many studies have been done focusing only on the extraction of palm carotenes and no studies have been carried out on the production of aroma compounds by degradation of these carotenoids from CPO.

Materials and methods

Materials

CPO was collected from Felda Palm Industries Sdn. Bhd. (Lepar Hilir 3, Pahang, Malaysia). Standard β-carotene and β-ionone were purchased from Merck (Germany). Synthetic macroporous resin (DIAION HP-20), a styrenic polymeric bead type resin design for adsorption with a surface area of 500m²/g was bought from Sigma Aldrich (United States). All the other chemicals and reagents used in this study were of analytical and industrial grade.

Soxhlet adsorption

Twenty-four grams of HP-20 adsorbent was weighed and transferred into a 250 mL conic al flask. Next, 50 mL of isopropanol (IPA) was added and maintained with continuous stirring for 30 min to activate the adsorbent. The activated adsorbent was filtered and dried at room temperature and transferred into a 250 mL three neck round-bottom flask. Six grams of CPO diluted with 50 mL of IPA was then added for a period of 1 h under constant stirring and maintained for 1 h at the same temperature. The mixture of CPO and HP-20 was then transferred to the soxhlet extraction thimble. Two hundred milliliters of IPA was added into the 250 mL round-bottom flask of soxhlet extractor and extracted for 1 h at 80 ± 5°C. Next, palm carotene was extracted from the adsorbent at 65 ± 5°C until the adsorbent became colorless (3 h). The experiment was conducted in a dark room.

β-carotene analysis

β-carotene content was determined by diluting 50 mg extracted carotene in 10 mL solvent and measuring absorbance in a Shimadzu UV-1601 (Shimadzu Corporation, Kyoto, Japan) at 446 nm. In addition, Water Alliance E2695 HPLC with an automated injector and photo diode array detector were used to determine β-carotene extracted, qualitatively and quantitatively. The isocratic mobile phase was acetonitrile/dichloromethane (9.5:0.5, vol/vol). A low rate of 1.0 mL/min, and carotene was determined by measuring absorbance at 450 nm. The concentration of extracted palm carotene was determined using standard calibration curve of β-carotene and the results were expressed in ppm. Standard solutions were prepared within a working range of 200 to 400 ppm.

Thermal degradation of β-carotene

Ten milligrams of commercially purchased β-carotene was dissolved in 50 mL distilled water in a 250 mL round-bottom flask that was covered with aluminium foil and sonicated for 1 h. The flask was connected to a condenser and heated in silicon oil bath at 110–120^oC in an oil bath for 4 h with continuous stirring. This optimized reaction conditions were chosen based on results obtained through two different tested parameters, reaction time (4, 5, and 6 h) and different temperature (110–120, 120–130, and 130–140°C). The organic compounds were separated with hexane when the experiment was completed. Anhydrous sodium sulphate was added to the extract to remove the remaining water. The degraded products were extracted by hexane and an analysis was performed by using gas chromatography-mass spectrometry (GC/MS). The experiment was conducted in a dark room. Then, degradation process was repeated with β-carotene recovered from CPO. All samples were degraded in triplicate.

GC spectrometry

The J&W DB-5 (95% dimethyl, 5% diphenyl polysiloxane; 30 m × 0.25 mm i.d) column was used for GC/MS analysis. The column temperature was programmed at 60°C (1 min) from 310°C at a rate of 4°C min^–1 (20 min). The injector temperature was 250°C; in splitless mode. The ionization energy was 70 eV with transfer-line temperature of 250°C. Mass spectra was scanned in the m/z = 58–650 range. Identification was achieved by mass spectral library search combined with retention index comparison by peak area normalization method.

Results and discussion

Recovery of β-carotene from CPO

Palm oil is known to contain a high concentration of carotenoid, but according to Amorim-Carrilho et al.,^[16] a high variability in chemical structure and poor stability greatly makes analysis difficult. Therefore, there is no general or standard method for carotenoids extraction in laboratories, whereby many extraction methods involve the release of desired components from their matrices by disrupting tissue followed by removal of the unwanted components. Besides, when choosing an extraction method, susceptibility of carotene to oxidation and degradation must be considered as it is very sensitive to light, heat, acid, or oxygen exposure.^[16] Soxhlet extraction at temperatures between 50–70°C have been reported to give higher phytochemical yields.^[17] Therefore, the Soxhlet adsorption method was employed to extract β-carotene from CPO by considering the possible recovery and risk of β-carotene degradation. Calibration graph for high-performance liquid chromatography (HPLC) was based on peak area of five different concentrations of β-carotene standards to determine the linearity in a working range from 200 to 400 ppm with correlation coefficient, R² = 0.994 (Fig. 1). Results show that from 6.0 g of CPO used for the extraction, 71.81% of sample recovered in IPA fraction and concentration of β-carotene from CPO determined by HPLC was 3790 ppm (Fig. 2).

Figure 1. HPLC analyses of standard calibration curve of β-carotene.

Figure 2. HPLC chromatogram of palm carotene.

GC analysis of thermal degradation of commercial β-carotene

Volatile compounds were identified by mass spectral library searching combined with retention index comparison by peak area normalization method. The thermal degradation products of these triplicate samples were analysed using GC/MS results as shown in Table 1. dhA is the most common aroma compound known to be one of the first volatile compounds that are formed by thermal degradation of β-carotene.^[13] In this study, dhA is identified with a high yield. More interestingly, C-13 norisoprenoid, β-ionone formed in the degraded was 9.60%. β-ionone is one of the commercially important aroma compound which is observed in plant tissues at a very low concentration. Tedious and laborious extraction processes make this β-ionone production costly.^[18] The production of this compound is expensive and less economical due to low concentration which leads to a tedious and laborious extraction and isolation process of β-ionone. Some other useful aroma compounds formed from this thermal degradation are 3-oxo-β-ionone and β-cyclocitral. A high yield of dhA was observed which could be further purified and solve racemic and enantioselective issues in synthetic approaches. Due to its economic significance, the present method is convenient for the production of dhA and degradation in a bigger scale, and enables the isolation of single aroma compounds that can be useful in the industry.

Table 1. Thermal degradation products of commercial β-carotene.

Compound^a	Kovats Index	Composition^b (% peak area)
		OT
Monolauryl maleate	2143	5.4 ± 0.6
β-cyclocitral	1218	4.2 ± 0.1
β-ionone	1485	9.6 ± 0.0
Dihydroactinidiolide (dhA)	1424	61.2 ± 0.3
3-oxo-β-ionone	1665	4.6 ± 0.1

^aComponents <1% are not included.

^bMean value (±SD) of the compounds constituents of triplicate samples determine by using GC-MS.

^cThermal degradation was conducted at 110–120^oC for 4 h under stirring.

GC analysis of thermal degradation of recovery β-carotene from CPO

Recovered β-carotene from CPO was studied by conducting thermal degradation under optimum reaction conditions. Results obtained proves that β-carotene extracted from CPO was successfully converted to aroma compounds mainly dhA, along with 3-oxo-β-ionone, β-ionone, and few other compounds; the results are shown in Table 2. The breakdown products of carotenoids are carbonyl compounds with C13 that are the most abundant norisoprenoids in nature with the megastigmane structure including the family of ionones, and C11 such as β-cyclocitral and dhA.^[19] The thermal degradation of commercial β-carotene produced higher yield of dhA compared to recovered palm carotene. This is because although soxhlet adsorption was employed for recovery of palm carotene, this method only helped to concentrate the β-carotene recovered, but did not completely remove the fatty acids and other compounds. There are difficulties to purify the isomers of carotenes as they are not much different in polarity and structure, the only difference between α- and β-carotene is the position of the double bond in one of the cyclohexane rings. Hence, the recovered β-carotene of CPO is not pure and is a mixture of other compounds. These leads to lower production of dhA in thermal degradation of recovered palm carotene compared to commercial β-carotene.

Table 2. Thermal degradation products of recovery β-carotene from CPO.

Compound^a	Composition^b (% peak area)
β-ionone	1.5 ± 0.5
Dihydroactinidiolide (dhA)	29.2 ± 9.5
3-oxo-β-ionone	3.9 ± 0.9
4-hydroxy-β-ionone	1.7 ± 0.8
Methyl palmitate	9.5 ± 7.9
Palmitic acid	22.0 ± 13.7
Elaidic acid methyl ester	5.6 ± 3.7
Oleic acid	20.8 ± 0.3

^aComponents <1% are not included.

^bMean value (±SD) of the compounds constituents of triplicate samples determine by using GC-MS.

^cThermal degradation was conducted at 110–120^oC for 4 h under stirring.

Conclusions

In this study, the degradation of β-carotene recovered from CPO was investigated as a model reaction. The formation of dhA was investigated in detail through thermal degradation of two different sources of β-carotene, which are commercial (synthetic) and natural sources, respectively. dhA is a major product for both reaction system of commercial and recovery β-carotene from CPO. The purity of β-carotene plays an important role in thermal degradation that leads to the formation of different yields of dhA. The formation of dhA from thermal degradation of pure β-carotene is 61.21%, which is higher than the synthetic yield of 45.00%. The recovered β-carotene by Soxhlet adsorption method from CPO was successfully degraded under optimized thermal degradation to produce 29.23%,of dhA. The results demonstrated that dhA is a major product which can be increased if the β-carotene recovered from CPO is being purified. The method is simple compared to the synthesis approach and can be applied in bulk without any tedious process. The thermal degradation of β-carotene leads to a formation of some notable aroma compounds especially dhA that can be very useful in the flavor and fragrance industry. However, the extraction of this aroma compound directly from plant sources is expensive and not economical. This has created opportunities in utilizing CPO as natural source of β-carotene to produce aroma compounds.

Funding

Support for this work was provided by Universiti Malaysia Pahang via research grant UMP RDU140344 to HA Hamid. Suria Kupan was supported by Universiti Malaysia Pahang’s Graduate Research Scheme.

Additional information

Funding

Support for this work was provided by Universiti Malaysia Pahang via research grant UMP RDU140344 to HA Hamid. Suria Kupan was supported by Universiti Malaysia Pahang’s Graduate Research Scheme.