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Title: Studies on the accumulation of selected groups of biologically active secondary metabolites in Schisandra chinensis (Turcz.) Baill. in vitro cultures

Abstract:

Schisandra chinensis - Chinese magnolia vine (Schisandraceae) is a dioecious vine native to some regions of Southeastern Asia. It is a new pharmacopoeial species in the European medicine from 2008 and also in Polish medicine (from 2009). Schisandra fruits are a rich source of lignans, dibenzocyclooctadiene derivatives. These compounds are highly valued mostly for their adaptogenic, hepatoprotective, antioxidant and anticancer properties.This work aimed to establish Schisandra chinensis in vitro cultures and to analyze two groups of therapeutically important compounds: lignans and phenolic acids. The further goal of these studies was to propose in vitro cultures as a potential source of the abovementioned groups of compounds.In vitro cultures with different degree of differentiation (shoot-differentiating callus cultures and undifferentiating callus cultures) were successfully established from leaf buds of female Schisandra plants. At the next stage of research, culture conditions were optimized by testing several variants of the Murashige and Skoog (MS) medium differing in the contents of plant growth regulators: the cytokinin BAP (6-benzyloaminopurine) and the auxin NAA (α-naphthaleneacetic acid), in the concentration range from 0.1 to 3.0 mg/l, and by examination of different types of in vitro cultures: agar (shoot-differentiating callus and callus) cultures and agitating (shoot and suspension) cultures. The next stage of research involved HPLC determination of the composition and contents of four main lignans of Chinese magnolia vine and eleven phenolic acids, cinnamic and benzoic acid derivatives and the parent compound of one of the subgroups of these compounds - cinnamic acid. Free phenolic acids - FPA and total phenolic acids – TPA, free and bound phenolic acids (obtained after acid hydrolysis), were estimated.The established shoot-differentiating callus cultures were characterized by a distinct capability of deoxyschisandrin production. The contents of schisandrin and gomisin A were also high while γ-schisandrin amounts were lower. The studies demonstrated the effect of plant growth regulators on the lignan production. In addition, it was shown that the cultures at the higher level of differentiation produced many times greater amounts of these compounds than undifferentiating callus cultures.The maximum contents of lignans equaling 308.51 mg/100 g d.w. for deoxyschisandrin, 86.41 mg/100 g d.w. for gomisin A, 75.54 mg/100 g d.w. for schisandrin and 22.09 mg/100 g d.w. for γ--schisandrin, were obtained in agar shoot-differentiating callus. ; The maximum contents of these compounds in undifferentiating callus cultures amounted to 18.75 mg/100 g d.w., 2.66 mg/100 g d.w., 2.16 mg/100 g d.w. and 1.03 mg/100 g d.w., respectively. The maximum contents of the lignans in agitating shoot cultures were also interesting from practical point of view and amounted to 92.37 mg/100 g d.w., 50.69 mg/100 g d.w., 48.16 mg/100 g d.w. and 15.77 mg/100 g d.w., respectively. On the other hand, the maximum lignan amounts in suspension cultures were of another order of magnitude, equaling 21.75 mg/100 g d.w., 5.54 mg/100 g d.w., 4.07 mg/100 g d.w. and 9.42 mg/100 g d.w., respectively.The maximum total content of lignans obtain in agar shoot-differentiating callus cultures, amounted to 486.78 mg/100 g d.w. and was 1.3- and 3.8-fold higher than in extracts from fruits and leaves of native plant, respectively, analyzed for comparison.Thus, the studies demonstrated that the established different types of in vitro cultures could be a potential biotechnological source of four lignans under study, especially deoxyschisandrin.Next, analysis of phenolic acids was performed. These compounds have not been studied in Chinese magnolia vine so far. The first common step in biogenesis of lignans and phenolic acids - shikimic acid pathway was taken into account. The studies in this direction were undertaken because of a wide range of valuable biological properties of these compounds comprising e.g. antiaggregatory, antioxidant, immunostimulating and anticancer activities. The studies demonstrated the effect of growth regulators also on the production of this group of compounds. In addition, they showed that FPA production was the highest in agar undifferentiating-callus cultures (max. total content – 78,24 mg/100 g d.w.) The maximum FPA content in agar shoot-differentiating callus cultures was a little lower, amounting to 60.06 mg/100 g d.w.. The FPA content in agitating shoot cultures (75.57 mg/100 g d.w.). was higher than in suspension cultures characterized by a lower degree of differentiation (38.40 mg/100 g d.w.).The highest TPA contents were obtained in both types of agitating cultures and equaled 213.62 mg/100 g d.w. in suspension cultures, and 204.15 mg/100 g d.w. in shoot cultures. The TPA contents in both types of agar cultures were lower but also interesting from practical point of view and amounted to 165.40 mg/100 g d.w. in callus cultures and 115.64 mg/100 g d.w. in shoot-differentiating callus cultures.The maximum obtained FPA content in the biomass extracts - 78.24 mg/100 g d.w.(agar callus cultures) was 1.4- and 1.7-fold higher than in extracts from fruits and leaves of native plant, respectively. The maximum obtained TPA content - 213.62 mg/100 g d.w. (suspension cultures) was 1.77- and 15.1-fold higher than in extracts from the examined organs of native plant, respectively.Chlorogenic acid (max. content 82.10 mg/100 g d.w., agar callus cultures, TPA), protocatechuic acid (max. content 120,16 mg/100 g d.w., suspension cultures, TPA) and syringic acid (max. content 40,50 mg/100 g d.w., suspension cultures, TPA) were the main phenolic acids produced in vitro. The experiments proved that the established in vitro cultures could be a potential biotechnological source of certain phenolic acids. It is noteworthy that this study is the first analysis of phenolic acids in Schisandra chinensis in vitro cultures and in organs of native plant.A distinct part of this work focused on the isolation and purification of lignan fractions and confirmation of the identity of three compounds: deoxyschisandrin, schisandrin and gomisin A using spectral analysis (1H NMR spectra). The mentioned lignans were not isolated earlier from in vitro cultures of this species. The results of this work are not only important from the point of view of basic research but may also find practical applications.

Place of publishing:

Kraków

Level of degree:

2 - studia doktoranckie

Degree discipline:

chemia ; farmacja

Degree grantor:

Uniwersytet Jagielloński. Collegium Medicum. Wydział Farmaceutyczny.

Promoter:

Halina Ekiert

Date issued:

2013

Format:

application/pdf

Identifier:

oai:dl.cm-uj.krakow.pl:3497

Call number:

ZB-118383

ControlNumberVIRTUA:

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Language:

pol

Access rights:

tylko w bibliotece

Location of original object:

Biblioteka Medyczna Uniwersytetu Jagiellońskiego- Collegium Medicum

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Last modified:

Nov 7, 2019

In our library since:

Apr 22, 2013

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