Abstract

Seeds of the Western Australian sandalwood (Santalum spicatumR.Br.) are rich in drying fixed oil and consist of ximenynic acid. This unusual, rare acetylenic fatty acid contributes to several bioactivities including anti-inflammatory and vasodilatation. Sandalwood seed oil is a blend of ximenynic and oleic acids with the characteristics of carrier oil.

A detailed multidisciplinary research was conducted on seed resource development from plantation level and suitable extraction method to obtain a product which is suitable and economically feasible for the cosmetic industry. The obtained product was tested for chemical and physical stability followed by toxicological and chemical screening to comply with regulatory requirements. Its bioactivity and the unusual chemistry have posed many challenges to the development process.

This will be the story of Western Australian sandalwood seed oil’s journey from soil to skin, and its contribution in sustaining the Western Australian sandalwood industry.

Western Australian Sandalwood (Santalum spicatum) is a commercially important plant, harvested for its valuable scented timber. Santalum spicatum(Family Santalaceae) is a root hemi-parasite found in the arid rangelands of the Western landmass of the Australian continent.^{1, 2} These trees produce a drupe with hard shelled seed.³

Oil rich seed kernels have been used by the Aboriginal communities in Western Australia as medicine. Crushed kernels were either chewed or cooked before applying on skin lesions, bruises and aching joints. It is reported that individuals suffering from chronic joint pains consumed these kernels as a cure.⁴

Santalaceae family seeds were reported a rare acetylenic fatty acid named ximenynic acid.^{5, 6} This unusual fatty acid contains a triple bond at the 9th carbon and double bond at the 12th position.⁷ Investigations into Western Australian sandalwood has found that this fatty acid accounted for 28%-36% of the total fatty acid composition, while oleic acid comprised the majority with 50%-53% of total fatty acids. It is also found that another acetylenic fatty acid named stearolic acid is found in lesser quantities, which is now identified as an intermediate product of ximenynic acid biosynthesis from its precursor oleic acid.^{8, 9}

The acetylenic structure of ximenynic acid is found to interfere with the arachidonic acid pathway and inhibit the synthesis of important regulators such as prostaglandins and leukotrienes. Inhibition of these bio regulators exhort several pharmaco-dynamic actions in the body, mainly vasodilatation and anti-inflammatory activity.¹⁰ The vasodilatation activity has been further studied to increase the microvascular circulation in dermal tissues. This activity was successfully patented for the pure ximenynic acid and its ethyl ester as a treatment for hair loss and cellulite.^11,12 Pharmacological studies conducted on whole animals have found that sandalwood seed oil reduced the inflammatory markers and also reduced the n3:n6 ratio.^{13, 1}

These findings on ximenynic acid in plasma have proven the medicinal use of sandalwood seed kernel by Western Australian Aboriginal communities. A recent study conducted by our collaborators has identified that ximenynic acid may alter the apoptosis pathway and expression of angiogenic factors; therefore, reducing the occurrence of cancer caused by inflammation.¹⁵

Western Australian sandalwood industry is now evolving from a natural wild harvest to plantation based agroforestry. There are over 15,000 hectares of S. spicatum plantations in Western Australia owned by private organisations and individual farmers. Plantations were established in different geographical and topological locations while using a variety of host trees. The longer the sandalwood trees stand on ground, more heartwood and oil quantities will be produced; therefore, sandalwood seeds were seen as an annual income to sustain plantations by giving an extra income to farmers.^{16, 17}

Seed kernels could not be consumed as a food ingredient as they possess pharmacological activity, thus act as a xenobiotic altering the physiology. Therefore, seed oil was a potential product to be used in cosmetic and personal care products owing to its biological activity and general acceptance for sandalwood.

Our aim was to develop Western Australian sandalwood seed oil as a novel cosmetic ingredient by addressing the following taking a soil to skin approach.

• Identify suitable raw material source for a sustainable supply and quality
• Develop a suitable extraction method
• Physiochemical characterisation and comparison with other oils used in cosmetics
• Study the stability of sandalwood seed oill Biological and toxicological screening

Experimental

Plant material

Santalum spicatumseeds were collected from three major geographical areas where sandalwood seed collection is viable in Western Australia. Seeds for specific tests were sourced from the sandalwood and host tree trail plantation set up in 1997 by the Forest Products Commission at the WA Agriculture School, Narrogin, WA.

Extraction

Sandalwood seeds were solvent extracted by Floch’s method for initial studies. During the course of this study a novel green chemistry method of extraction was developed using supercritical carbon dioxide.

Physiochemical characterisation

Physiochemical analysis of the seed oil was conducted and the data was interpreted based on the methods and limits given on British Pharmacopoeia and Handbook of the American Oils Chemists Society.^{18, 19}

Stability protocol

A long term stability protocol was established for 360 days study at 40°C. Sandalwood seed oil was grouped based on storage condition as protected and unprotected samples. Sampling interval was 30 days. All the samples were filled 50% of the volume; protected samples were flushed and headspace filled with inert gas before sealing. Whereas the unprotected oils were flushed and headspace filled with air before sealing.

Biological and toxicological screening

Sandalwood seed oil was screened for acute toxicity on 3T3 NRU cell lines (OECDGD-129), dermal irritation on reconstituted human epidermis (OECD-TG-439) and ocular irritation and corrosion by bovine corneal opacity and permeability (OECDTG-437). Sandalwood seed oil, triximenynin glyceride and ximenynic acid were tested for cytotoxicity on Cytotoxicity in RAW264 murine leukemic monocyte-macrophages and anti-inflammatory activity as in PEG2 on 3T3 Swiss albino mouse embryonic fibroblast cells; TNF-αand nitrogen oxide inhibition was assayed on RAW264 cells.^20-22

Results and discussion

Seed source

Sandalwood seeds were selected from three different geographical locations in Western Australia; Goldfields, Wheatbelt and Coastal. The selected seed groups showed a variation in seed size, seed weight, kernel yield and oil content. On average plantation seeds produced 33% kernel per whole seeds. This is an important industrial parameter as the processing costs depends upon the yield of kernel from whole seed. Geographical variation has a negligible effect on the fatty acid composition of sandalwood seed kernel; while the seed physical dimensions and oil content varied considerably. Sandalwood trees in the Goldfields region and coastal Western Australia are economically not a feasible seed source. Seeds from the commercial plantations in the Wheatbelt region obtain a considerable kernel yield and high oil content with required fatty acid profile.²³

Sandalwood trees studied were hosted with three different Acaciaspecies and one Allocasuarinaspecies. Seeds produced by sandalwood trees hosted by Acaciaspecies were found to yield larger seeds with more oil content. Whereas sandalwood trees hosted by Allocasuarina huegeliana produced smaller seeds with low oil content. Although variations were observed for different seed parameters between the three Acaciaspecies, no particular species was found to be clearly superior. The number of Acacia accuminatatrees used to host sandalwood did not affect the seed properties. Trees hosted by Acaciaspecies were found to have less ximenynic acid when compared to the Allocasuarina huegelianahosted trees. Acaciaspecies are desired hosts for sandalwood in plantations due to their suitability of the geographical and topographical conditions.

Based on these results a selection parameter for seed were established as a quality assurance parameter for seed resource (Table 1).

Extraction method development

This study has developed a supercritical carbon dioxide extraction method to extract sandalwood seed oil to obtain the kernel lipids in its natural form without chemical or thermal degradation. Lipid extraction by supercritical carbon dioxide has been reported at an industrial scale where the final product holds a high value and the conventional methods unable to produce the required quality or yield.²⁴

Current study has determined the optimum extraction pressure and mass ratio of carbon dioxide to extract higher yields of oil from sandalwood seed kernel; whereas flow rate was the controlled variable. The oil extracted showed physicochemical properties comparative to other oils used in cosmetic and pharmaceutical formulations. Extraction conditions were kept at the lowest temperature and no co-solvents (entrainers) were used. Total yields from the seed kernel varied between 38-52% w/w based on the raw material source and particle size. The supercritical carbon dioxide extract had lower peroxide and acid values, indicating that the extraction process did not contribute to oxidation or hydrolysis of the lipids. Unsaponificable matter and the specific gravity of the supercritical extract was higher indicating possibly more non-fatty acid composition in the oil. Current extraction process details are an intellectual property of Wescorp Group of Companies, thus will not be disclosed in the current manuscript.

Characterisation of Santalum spicatum seed oil

Supercritical extracted oil showed the fatty acid content and other lipid components similar to solvent extracted oil. The fatty acid composition was unchanged between the solvent and the supercritical extract, and also complied with the previous reports on Santalum spicatumseed oil extracted using the same solvent extraction method

Physicochemical parameters and lipids compositions were compared with other seed oils commonly used in cosmetics.²⁵ Compositions of the sterols were slightly different to other seed oils as the βsitosterol content was lower while campasterol and stigma sterol were higher. However the tocopherol content was considerably lower, δ-tocopherol was higher than α-tocopherol and supercritical extracts contained more δ-tocopherol. Based on these observations the following parameters were established as a basis of a monograph for Santalum spicatumseed oil (Table 2) to enable its commercial standardisation and identification.

Stability of Santalum spicatumseed oil Adequate stability is paramount for any ingredient to be used in cosmetic formulations. Stability of plant oils are determined based on oxidation, hydrolysis and changes to the fatty acid composition. This is the first study of the stability of an acetylenic fatty acid containing oil to be reported.

Oxidation was observed as a peroxidation reaction and evaluated by determining the peroxide value and then the secondary oxidative products by the anisidine value. Supercritical carbon dioxide extraction of the sandalwood seed oil was subjected to stability assessment both exposed to and excluded from air at high ambient temperature (40°C) for 360 days. The supercritical extract was found to be stable when not exposed to air up to 360 days, while a rapid oxidation was observed when exposed to air at this temperature (Table 3). Exposed samples degraded reaching an oxidation level in excess of over 10 meq O2 Kg-1 of oil within the first 30 days and a rapid increase of secondary products was also observed. Towards the end of the observation period oxidation of the unprotected samples had reached the termination stage as PV have reached minimum, complying with the theoretical oxidation process. Secondary reaction products of oxidation increased for the unprotected oil where 28 AV was recorded compared to the 6 AV for the protected oil. Fatty acids with unsaturated bonds undergo peroxidation. The main fatty acids in the sandalwood seed oil are monounsaturated oleic acid and polyunsaturated acetylenic ximenynic acid. Hydrolysis observed by formation of free fatty acids was low in both the protected and unprotected samples. Acid value of the unprotected oil only increased towards the final stages of observation period.

Fatty acid composition and the level of saturation have not been altered within experimental error for both protected and unprotected oils although oxidation was observed for the unprotected oil. The measurement of oxidation has a higher degree of sensitivity when compared to total fatty acid composition. Continuity of secondary oxidation could ultimately depict a change of fatty acid composition with the depletion of the C18 chain acids and production of shorter chain length fatty acids and hydrocarbons. The stability of the sandalwood seed oil can be maintained if stored in airtight containers even at elevated temperatures.

Toxicity and bioactivity screening

The acute toxicity of the seed oil was found to be low as the LD₅₀ was >1240mg/kg, this value generally regarded as safe even consider direct cellular level contact on the cultured cell lines. Dermal irritancy on reconstituted human epidermal cells found that the viability was 116% which shows it as non-irritant. Ocular irritancy was tested on isolated bovine corneas where an ocular in vitroirritancy score was given based on the opaqueness and permeability. Sandalwood seed oil was found to be nonirritant non corrosive with a total score of 0.9 against a value of >55 was considered irritant on eyes. This is the first time a biological safety evaluation has been conducted on sandalwood seed oil. These studies were conducted using OECD approved methods in an accredited cosmetic testing laboratory. These results would confirm the safety of sandalwood seed oil to be used as a potential ingredient in the cosmetic products.

Sandalwood seed oil, ximenynic acid and triximenynin were studied for cytotoxicity and inhibition of inflammatory markers. Ximenynic acid was found to produce PGE2 inhibition; other two samples showed no inhibition for the above study. None of the samples have inhibited the nitric oxide or cytokine production. It is seen that only the free fatty acid of acetylenic fatty acid produce antiinflammatory effect by inhibiting PGE2. Dose response curve of the ximenynic acid has a similar pattern to a non-steroidal antiinflammatory drug indomethacin. However, the current finding shows that the triximenynin was inactive unless it is hydrolysed to the free ximenynic acid.

These in vitromethods are novel but more efficient path to assess the toxicity than conventional animal studies.

Conclusion

This study has found that sandalwood seeds harvested from plantations in Wheatbelt region of Western Australia are an important source of oil with unique properties. A successful extraction method was developed that maintained the stability of the oil constituents during the process. It has an advantage of leaving no residual solvents. The stability of the oil when stored at 40ºC and in the absence of air was maintained for a period of one year. However in the presence of air the oil rapidly deteriorated. Santalum spicatumseed oil was found to be non-toxic and non-irritant when evaluated by in vitroassays. Biological effects of Santalum spicatumseed oil was also assayed, where only the free ximenynic acid s found to inhibit PGE2.

Based on these studies Santalum spicatumseed oil was registered as a cosmetic ingredient with unique INCI name (Santalum SpicatumSeed Oil) and CAS number (1542150-96-8). Regulatory documents such as technical data sheet, monograph and SDS were prepared. Currently Santalum spicatumseed oil is sold as a cosmetic ingredient unique to Western Australia, whereas several cosmetic brands have already released skin care products containing Santalum spicatumseed oil.