Hyoscyamine yields

Decarboxylation is followed by oxidative cycliza-tion to tropinone, followed by stereospecific reduction to the a-Hydroxy group, which is esterified to hyoscya-mine. The esterifying ester, tropic acid, is an intramo-lecularly rearranged phenyllactic acid (derived from phenylalanine). Further elaboration of hyoscyamine yields scopolamine (Fig. 23). The enzymes for this transformation are known. 

Cl-IAA and 5.6-C12-IAA enhanced growth of the roots compared with lAA at low concentrations. The optimum concentrations for alkaloid production were 0.1 mg/l for 4-Cl-IAA and 0.01 mg/I for 5,6-Cl2-IAA. In these conditions, decrease of scopolamine and a slight increase of hyoscyamine yield were obtained as compared with lA A. 

The effects of lAA chloro-derivatives on the growth and the alkaloid production are presented in Fig. (36). Both lAA chloro-derivatives, 4-Cl-IAA and 5,6-Cl2-IAA, enhanced the growth of the roots compared with lAA at low concentrations. The optimum concentrations for alkaloid production were 0.1 mg/1 for 4-Cl-IAA and 0.01 mg/1 for 5,6-Cl2-IAA. The culture with 4-Cl-IAA (0.1 mg/1) or 5,6-Cl2-IAA (0.01 mg/1) resulted in a decrease of scopolamine and in a slight increase of hyoscyamine yield as compared with lAA, Figs. (35) and (36). It is known that 4-Cl-IAA and 5,6-Cl2-IAA are resistant to peroxidase decomposition [56] and the results indicate that they exert their effects at much lower concentrations than lAA. Thus they may be useful plant growth regulators for tropane alkaloid production in root cultures. 

Hyoscyamine was the main alkaloid in all the clones. Concentrations found in the different root clones are shown in Fig. (49). Among the diploid roots, RIOOO and TR showed the highest content (ca. 0.6% dry weight). However, growth of the TR roots was inferior compared to the other clones, resulting in the lowest hyoscyamine yield (400 pg/100 ml flask after 5 weeks of cultures). Fig. (49). For the haploid hairy roots, concentrations between 0.2-0.3% were found without significant differences among the different clones, whose yields were 200-400 pg/100 ml flask. 

On the other hand, Pavlov et al. [73] analyzed the effect of NO3, H2PO4 and sucrose on the production of hyoscyamine in D. stramonium (diploid and tetraploid) hairy roots. These authors proposed a modified MS medium for an optimal hyoscyamine yield. The optimized MS medium have increased sucrose concentration and decreased NO3 concentration compared to standard MS. The results reported by Pavlov et al. [73] showed that it is possible to exploit the biosynthetic potential of in vitro cultures by the optimum media composition. 

Duhoisia myoporoides. Yields of 3 per cent., mostly hyoscine, have been mentioned, but great variation in nature of alkaloidal content is on record, e.g., hyoscyamine, -hyoscyamine, hyoscine. Hyoscyamine and norhyoscyamine, but no hyoscine. Hyoscine or hyoscyamine or both. dZ-Hyoscine, tigloidine, valeroidine, poroidine and woporoidine present hyoscyamine and norhyoscyamine absent. The limits of these variations, at least as regards the two principal alkaloids, hyoscyamine and hyoscine, have probably been settled by Hills, Trautner and Rodwell, who confirmed the statement of Barnard and Finnemore that in the northern portion of the distribution area of this plant, it yields mainly hyoscine and in the southern section mainly hyoscyamine. In 54 samples of leaves from individual trees they found 0-9 to 4-0 total alkaloids in those from the northern section, with hyoscine as the chief component, and 1-0 to 2-7 from those of the southern section, with hyoscyamine replacing hyoscine. 

To improve supercritical C02 solubilities of target alkaloidal salts, an appropriate modifier to raise the polarity of C02 had to be used. As previously mentioned, the most common modifier used in SFE is methanol because of its high solvation parameters, which can greatly increase the resultant polarity of C02. Water has been chosen as another modifier because some alkaloidal salts are freely soluble in water as well as methanol. Moreover, the addition of water into C02 has been reported to improve the extraction yield of some alkaloids [29]. Methanol or water as a modifier was added into the extractor at the concentration levels of 1, 5 and 10% (v/v), respectively. The effect of methanol and water on the solubilities of hyoscyamine (1) and scopolamine (2) is shown in Figure 5. Analogous information on ephedrine derivatives such as methylephedrine (3), norephedrine (4), ephedrine (5), and pseudopehedrine is illustrated in Figure 6. 

In both results of solubility and desorption from filter papers, diethylamine in methanol as a modifier was found to offer greater efficiency for SFE of the alkaloids than any other modifiers employed. The yields of hyoscyamine (1) and scopolamine (2) from the roots and aerial parts by SFE and conventional organic solvent extraction are listed in Tables 2 and 3. The SFE yields from both plant parts were greatly enhanced by the addition of methanol basified with diethylamine. From the results of solubility and desorption from filter paper, methanol and diethylamine/methanol (10% v/v) were much more efficient for both compounds than water and diethylamine/water (10% v/v) because of their low miscibility with C02. The extraction profile of hyoscyamine (1) when present in plant material was in good agreement with that when extracted as a pure compound. However, in the case of scopolamine (2), there... 

Table 2. Effect of different volumes of modifiers on the SFE yields (mg/g) of hyoscyamine (1) and scopolamine (2) from the roots of S. japonica at 60 °C and 34.0 MPa [39]. Reprinted from J. Chromatogr. A, 863, Y. H. Choi et al., Strategies for supercritical fluid extraction of hyoscyamine and scopolamine salts using basified modifiers, 47-53, 1999, with permission from Elsevier Science.

Tropanone then is reduced via an NADPH-dependent reductase to tropine that has been cloned from Hyoscyamus niger (149, 150). All tropane-producing plants seem to contain two tropinone rednctases, which create a branch point in the pathway. Tropinone reductase I yields the tropane skeleton (Fig. 3b), whereas tropinone rednctase II yields the opposite stereocenter, pseudotropine (151). Tropane is converted to scopolamine or hyoscyamine, whereas the TRII product pseudotropine leads to calystegines (152). These two tropinone reductases have been crystalhzed, and site-directed mutagenesis studies indicate that the stereoselectivity of the enzymes can be switched (153, 154). 

Hyoscyamine is found in Atropa belladonna (night-shade) and in several species of Hyoscyamus from which its name is derived. It is a crystalline compound, m.p. 108.5°, somewhat soluble in water but more readily in chloroform, alcohol or benzene. It is levo rotatory and yields crystalline salts more soluble in water than the base itself. With acids or alkalies hyoscyamine hydrolyzes as previously stated yielding tropine and tropic acid. 

Tropine is a simpler base than the other alkaloids and is not found as such in the plants but is obtained by hydrolyzing not only atropine and hyoscyamine but other solanacese alkaloids as well. It is crystalline, m.p. 63°, and is soluble in water, alcohol, ether or benzene. As shown in its formula it is an alcohol yielding esters with tropic acid. 

In the hairy roots, transformed with A. rhizogenes 15834, hyoscyamine was the main alkaloid only for the first two weeks of culture in phytohormone-free WP liquid medium. After that time scopolamine became the major alkaloid. The production of scopolamine increased rapidly and it became almost double the amount of hyoscyamine at the end of the culture period. The yield of 6P-hydroxyhyoscyamine and 7P-hydroxyhyoscyamine produced in these hairy roots was comparable to that in the adventitious roots (Fig.7). 

The 6-hydroxyhyoscyamine which occurs in low concentration (0.005%) in Datura species is an intermediate in the in vivo conversion of hyoscyamine into hyoscine. This has now been isolated in a relatively high yield (0.5%) from a Duboisia hybrid. 

Another transformed tropane alkaloid-producing plant was Scopolia parviflora. In the transgenic hairy roots which overexpressed H6H enzyme, hyoscyamine and scopolamine accumulated at high concentrations. The best transgenic line yielded 8.12 mg/g dry weight of scopolamine, representing a 3-fold increase compared to wild-type roots [174]. 

The alkaloid yield in each culture period is shown in Fig. (43). Constant alkaloid content and linear increase of dry mass, Fig. (42), were reflected in the growth-proportional increase of tropane alkaloid yields. Fig. (43). At week 6, 35 mg/1 scopolamine and 17 mg/1 hyoscyamine were obtained. 

Fig. (49). Hyoscyamine concentration and yield found in the different hairy root clones of A. belladonna (n=2-5)...

Atropine is an ester, and on hydrolysis yields a basic substance, tropine, and optically inactive tropic acid (1). It has been shown that the alkaloid hyoscyamine, which is also obtained from belladonna and is laevo-rotatory, is the ester of tropine with laevo-tropic acid (2), and therefore atropine appears to be racemic hyoscyamine. This view of the nature of atropine has been confirmed by Ladenburg (3), and dextro-hyoscyamine has also been prepared by the union of tropine with dextro tropic acid (4). 

Artificially induced mutations have not yet yielded alkaloid-free plants. Ergots exposed to X-rays and ultraviolet radiation yielded some types which lacked pigment. Such leucosclerotia in general contained less alkaloid than the original forms (201, 232-235). Evans and Menendez (236) obtained Datura tatula L. D. stramonium L.) mutants in which the ratio of hyoscine to hyoscyamine was altered. Similar results have been reported by Mothes and associates (185) in which... 

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