Safflower, Carthamus tinctorius L. (family Compositae), consists of tubular florets, which are light red in color. The florets are used for much the same purposes as saffron, the dried stigma of Crocus sativus L., and are sometimes admixed with it and occasionally substituted for it. Although the plant does not exist as a wild species, the same genus, Carthamus oxyacantha Bieb., is indigeneous to Central Asia, such as Caucasus, Iran, Afghanistan, and Pakistan, and also cultivated in India. Thus, it is supposed that the C. tinctorius, long cultivated in China and Japan as a source of crude drug, was brought from Central Asia. The red florets are widely used as a crude drug in Oriental medicine and natural dye, especially in silk cloth or rouge. A water-soluble yellow dye, called safflower yellow, also used to be extracted from it as well as an alcohol-soluble red dye, saffower carmine, which is carthamin, a chalcone glycoside.
Recently, it has been noted that safflower seed oil contains abundant (over 75%) linoleic acid, an unsaturated fatty acid. Thus, the oil is used as part of the diet for arterial and heart diseases, and for overweight because of its anticholesterol quality. In addition, safflower oil is well known as α-tocopherol-rich, like sunflower oil, which is extracted from sunflower (Helianthus annum) seeds. Tocopherols have the physiological action of vitamin E and an antioxidant action, and are used as ingredients of pharmaceutical preparations and as antioxidants in food. It has been found that tocopherols may act to prevent a deterioration in physical condition or geriatric disease. The tocopherols from vegetable sources consist of eight analogs, i.e., four tocopherols and four tocotrienols. Alpha-tocopherol has the strongest vitamin E activity of these. In particular, the physiological activity of (d)-α-tocopherol obtained from natural sources is 1.36 times greater than that of racemic (dl)-α-tocopherol, obtained by chemical synthesis. Thus, the production at low cost of tocopherols from a vegetable source in which the physiologically most effective α-tocopherol is present in large amounts is very desirable. Cell culture of safflower, which contains one of the largest amount of α-tocopherol in plants, has therefore been carried out to produce tocopherols, especially α-tocopherol, and other related compounds.
The florets of safflower, Carthamus tinctorius, which contain a red pigment, carthamin, a chalcone glycoside, have been used as a crude drug and a natural dye since ancient times in the Orient. In our experiments, however, the cultured cells induced from flower buds did not contain any red pigment. On the other hand, safflower oil has been recently noted as α-tocopherol-rich, having the highest vitamin E activity, and rich in linoleic acid, an unsaturated fatty acid. Thus, research was conducted for the purpose of the production of these compounds by plant cell culture. At first we investigated mainly the cultured cells of sunflower, safflower, soybean, etc., whose original plant contained a high amount of tocopherols. It was shown from the results that only safflower cultured cells produced a remarkable amount of tocopherols. Next, a better cell line, named the B2KC strain, in terms of both growth rate and tocopherol content, was selected using various growth regulators and media additives. This was a high-growth and high-tocopherol-producing strain, ca. two times in growth ratio and 4.3 times in tocopherol production in comparison with the original strain, DK cells. Further, the increase of tocopherol contents was tried by administering various biosynthetic precursors. As shown in Fig. 3, tocopherol production was effectively stimulated by phytol, the precursor of the side chain, and the amount reached up to five times, 144.28 mg per 100 g dry cells in maximum, although the increase was predominant in γ- and -tocopherols. However, homogentisic acid, the most closely related precursor of chromanol nuclei, had almost no effect. As a result, the effective incorporation of homogentisic acid into the cell remained as the most important subject for increase of the tocopherol content.
Furthermore, the amount of α-tocopherol produced by safflower cell culture showed an almost constant value in each strain and never exceeded this level. This suggests that the pool size of α-tocopherol in the cell is always constant in the same culture strain. Further, it has been reported that in spinach and lettuce, α-tocopherol is biosynthesized in the isolated chloroplast, hence we considered the formation of chloroplast essential for the further increase of α-tocopherol content. Thus a new strain, named Ca-2, induced from the seedling and cultured under illumination, resulted in slight greening. Although in this strain the increase on phytol addition was lower, 1.8 times, than that in the DK strain, five times, most of it, 82.6% in total tocopherol, was α-tocopherol, i.e., 60.75 mg per 100 g dry cell.
In conclusion, in the present study, the best medium and conditions for the establishment of saffower callus and the production of vitamin E are as follows. (1) Safflower callus is induced at a high frequency on Murashige and Skoog’s medium supplemented with 1 mg/1 2,4-D and 0.1 mg/1 K from seedling or flower bud. (2) Callus is cultured on RT medium containing 2 mg/1 IBA, 0.1 mg/1 K and 0.1% casamino acid, under the dark and/or illumination (7000 lx) and subcultured at 3-week intervals. (3) The maximum production of tocopherols is obtained by the addition of 100 mg/1 phytol on cell suspension culture precultured for 1 week, followed by incubation for a further 2 weeks.
Finally, the tocopherol amounts produced by our experiments were compared with those in seeds or vegetable oils. The results showed that tocopherol content per n-hexane extract from the safflower cultured cells was ten times higher than those from seeds and oil. Hence, it was shown that the oil produced by safflower cell culture is α-tocopherol-rich with a good quality, and further that the oil also contains abundant linoleic acid. Safflower cell cultures, therefore, have a good prospect of developing the new method of tocopherol production.
Selections from the book: “Medicinal and Aromatic Plants III”, 1991.