Catharanthus roseus (Periwinkle)

Catharanthus roseus (family Apocynaceae) is grown as an ornamental plant in many countries, although it originated from Madagascar. It is also known as Madagascar periwinkle or Cape periwinkle. This plant was used traditionally as a crude medicine for diabetes and other ailments. It has also been used as a substitute for hops in brewing beer. Now, however, C. roseus is most useful as a source of various alkaloids; approximately 90 indole alkaloids have been isolated from it, the most valuable being the dimeric alkaloids vinblastine and vincristine, which show antitumor activity. They are very similar in chemical structure, but their activity spectra and side effects are extremely different: vinblastine is effective against Hodgkin’s disease, choriocarcinoma, and the like, while vincristine is mainly employed to treat childhood acute leukemia. Vinblastin shows bone marrow toxicity, whereas vincristine is toxic to the nervous system. Due to the very low yields of these dimeric indole alkaloids in the plant (approx. 0.0005%), attempts have been made to produce alkaloid and other secondary metabolites in cell and tissue cultures. General reviews of work in this field have been published.

In this chapter, attention is focused on another attribute of C. roseus cells in culture, i.e., the ability to biotransform exogenously added hydroquinone (HQ) to arbutin. We have extensively examined the feasibility of efficient production of arbutin using C. roseus cell culture.

Plant cell cultures catalyze a wide range of biotransformations, such as glucosylation, glucosyl esterification, hydroxylation, oxidation-reduction between alcohols and ketones, reduction of carbon double hydrolysis, isomerization, epoxidation, dehydrogenation, methylation, bonds (C=C), demethylation, and others. However, major biotransformations by plant cell cultures are hydroxylation and glucose conjugation involving glucosylation and glucosyl esterification; these reactions are the subject of 45 and 41% of published reports in this field, respectively. Hydroxylation reactions are catalyzed by cytochrome P-450 in many cases. For example, biotransformation of digitoxin to digoxin by 12 β-hydroxylation using Digitalis lanata cell culture is cytochrome P-450-dependent, and biotransformation of tryptamine to serotonin is also believed to be cytochrome P-450-dependent. Since cytochrome P-450 is well known to participate in detoxification of drugs, hydroxylation may be regarded as an initial step in detoxification. Glucosylation at the resultant OH moiety may proceed as a subsequent step in detoxification, in plants.

Microorganism-mediated biotransformation also produces glycosides in some cases, but such glycosides are not major products, and glucosides are not known as secondary metabolites in microorganisms. Animals generally seem to use glucuronylation reactions for detoxification. Thus, glucosylation and hydroxylation seem to be rather specific biotransformations of higher plant cells, and the high-yield biotransformations which have been reported so far in plant cell cultures have mainly involved prduction of glucosides and hydroxylated substances (Table High-yield production by biotransformation).

Arbutin, the glucosylated derivative of HQ, is the main active component of Arctostaphylos uva-ursi Sprengel, which was used as a urethral disinfectant for many years before antibiotics were developed. This glucoside is also a potent suppressor of the synthesis of melanin in human skin, without apparent side effects. The cosmetic company Shiseido has developed arbutin as a skin depigmention agent; a product containing chemically synthesized arbutin has been on sale in Japan since 1990.

Table High-yield production by biotransformation

ProductSubstrateType of reactionPlant speciesYield (g/1)
ArbutinHydroquinoneGlucosylationRauwolfia serpendna18
Catharanthus roseus9.2
p-hydroxyphenyl- O-primeverosideHydroquinoneGlucosylationRauwolfia serpendna5.8
SerotoninTryptamineHydroxylationPeganum harmala2.5
SkimminUmbelliferoneGlucosylationDatura innoxia1.6

Catharanthus roseus (Periwinkle): Prospects

Arbutin production using C. roseus cell culture has the advantage that there is no significant competing reaction; the conversion ratio from added HQ to arbutin was 98%. It may be pointed out that our process is a direct conversion of HQ and not a de novo synthesis from remote precursors. We have developed a simple method of purification of arbutin that includes the use of C18 columns and recrystallization from organic solvents as the main steps. The total period of culture is around 18 days; 2 weeks for high-density cell culture of competent cells, and 3 or 4 days for the biotransformation; this period is relatively short for a plant cell culture process. Pure arbutin could be produced at a cost of $300-$1000/kg by this method. The cost range is large because the cost would depend on whether or not the construction of new facilities for cell culture or for purification of arbutin is necessary.

On the other hand, glucosylation of HQ by chemical means is not a simple task; it requires several steps, including blocking the hydroxyl moieties of glucose via acetylation, conjugation of such OH-protected glucose to HQ, and saponification for deacetylation. The conjugating reaction requires a high temperature. The cost is in the same range as that of the biotransformation. Thus, production of arbutin by biotransformation could substitute for the chemical process.

Selections from the book: “Medicinal and Aromatic Plants X”, (1998).