Stefan Philipov

Study on the anti-inflammatory action of Berberis vulgaris root extract, alkaloid fractions and pure alkaloids

Extracts obtained from the roots of Berberidaceae species have been used in Eastern and Bulgarian folk medicine in rheumatic and other chronic inflammatory disorders. The investigations of the chemical composition and immunological properties show that their activity is mainly due to the alkaloid constituents. In the present study the anti-inflammatory properties of total ethanol extract (TEE), three alkaloid fractions, a major alkaloid berberine and oxyacanthine isolated from Berberis vulgaris roots were compared. All these were applied in acute inflammation (carrageenan- and zymosan-induced paw oedema), as the TEE showed the highest reducing effect. Their ability to alter in vivo and in vitro complement activity was determined. Also, the TEE was most effective in a chronic inflammatory model of adjuvant arthritis. The protoberberine fractions Bv2, Bv3 and berberine suppressed a delayed type hypersensitivity (DTH) reaction. Fraction Bv1 and berberine diminished antibody response against SRBC in vivo. The in vitro treatment of splenocytes with berberine showed that the anti-SRBC antibody synthesis was influenced in a different manner depending on the time course of its application. Oxyacanthine was less effective than berberine in the tests used.

Alkaloid Spectrum in Diploid and Tetraploid Hairy Root Cultures of Datura stramonium

Hairy root cultures were obtained from diploid and induced tetraploid plants of Datura stramonium and analyzed by gas chromatography/mass spectrometry. Twenty alkaloids (19 for diploid and 9 for tetraploid hairy root cultures) were identified. A new tropane ester 3-tigloyloxy-6-propionyloxy-7-hydroxytropane was identified on the basis of mass spectral data. Hyoscyamine was the main alkaloid in both diploid and tetraploid cultures. In contrast to diploid hairy roots, the percentage contributions of the alkaloids, with exceptions for hyoscyamine and apoatropine, were higher in the total alkaloid mixture of tetraploid hairy roots

Cinnamoyl- and hydroxycinnamoyl amides of glaucine and their antioxidative and antiviral activities.

The aporphine alkaloid glaucine has been converted into 3-aminomethylglaucine and its free amino group has been linked to cinnamic, ferulic, sinapic, o-, and p-coumaric acids. The antioxidative potential of the synthesized amides was studied against DPPH(*) test. All of the tested compounds demonstrated higher radical scavenging activity than glaucine and 3-aminomethylglaucine, and lower antioxidative effect than the free hydroxycinnamic acids. The newly synthesized compounds were tested in vitro for antiviral activity against viruses belonging to different taxonomic groups.

Influence of berberine on T-cell mediated immunity.

The protoberberine alkaloid berberine is isolated as a main alkaloid from the roots and bark of Berberis vulgaris. Berberine strongly inhibited in vitro the proliferative response of mouse spleen cells to T-dependent mitogens concanavalin A (Con A) and phytochemagglutinin (PHA). Spleen cells obtained from berberine-treated mice (10 mg/kg/3 days) expressed enhanced proliferative response to both mitogens. Berberine was applied to mice at different intervals before or after the induction of adjuvant arthritis (AIA) and Candida albicans (C. albicans) infection. The application of the alkaloid to new born mice (5 days after birth at a dose of 5 mg/kg/3 days) did not change the course of AIA and C. albicans infection. Its application at three 10 day intervals (5 mg/kg), starting from the 5 day after birth increased the joint inflammation in AIA. The host resistance to C. albicans infection was not affected, while the delayed type hypersensitivity (DTH)-reaction against the pathogen was enhanced. The alkaloid inhibited the development of AIA when applied after its onset (10 mg/kg from day +3 to +12 day). Berberine treatment during the ongoing infection did not influence its outcome (from +2 to +10 day).

Alkaloid Production in Diploid and Autotetraploid Plants of Datura stramonium

A comparative investigation of alkaloid production and accumulation in the roots and leaves of diploid (2n = 24), and C 4 generation of induced autotetraploid (4n = 48) Datura stramonium L. plants was performed. Fifteen alkaloids have been determined in the roots and two in the leaves, at a level of 1% or more of the crude alkaloid fractions in both ploidy levels. Two 3-tigloyloxy-6-isovaleryloxy-7- hydroxytropane isomers were detected for the first time in genus Datura. In the conditions of the prolonged photoperiod, hyoscyamine was the main alkaloid in the roots whereas scopolamine was the main alkaloid in the leaves of both ploid forms. In comparison to diploids, the roots and leaves of tetraploids had a higher alkaloid content and scopolamine/hyoscyamine ratio.

Antimicrobial and immunological activity of ethanol extracts and fractions from Isopyrum thalictroides

The antimicrobial and immunological properties of ethanol extracts, non-alkaloid, tertiary alkaloid and quaternary alkaloid fractions, obtained from roots and aerial parts of Isopyrum thalictroides were examined. The non-alkaloid fraction from aerial parts inhibited the growth of seven test microorganisms and was the most effective suppressor of classical pathway (CP) complement activity in normal human serum (NHS) and guinea pig serum (GPS). The alkaloid fractions, containing quaternary alkaloids expressed suppressive effect on mitogen-induced splenocyte proliferation. The in vitro antibody response against sheep red blood cells (anti-SRBC) was inhibited by ethanol extracts and quaternary alkaloid fraction. The intraperitoneal (i.p.) application of ethanol extract and tertiary alkaloid fraction from aerial parts showed that they possess in vivo effect on alternative pathway (AP) complement activity, anti-SRBC response and delayed type hypersensitivity (DTH).

Complement modulatory activity of bisbenzylisoquinoline alkaloids isolated from Isopyrum thalictroides--I. Influence on classical pathway in human serum.

Eleven bisbenzylisoquinoline alkaloids (BBI) were isolated from the plant Isopyrum thalictroides (L.). Treatment of normal human serum (NHS) with BBI resulted in a diminution of the haemolytic activity of the classical pathway (CP). The mode of action of the main alkaloids isopyruthaline (It1), fangchinoline (It2) and isotalictrine (It3) on CP activation was investigated in vitro. The inhibition was time- and temperature-related and for Itl and It3 depended on the concentration of Ca2+ and Mg2+ ions. It was established that the substances reduced C1 haemolytic activity. It2 and It3 enhanced the complement consumption caused by heat aggregated human IgG (HAGG). The BBI prevented the formation of C3 convertase of the classical pathway. The loss of haemolytic activity was partially restored by the addition of C142 reagent (zymosan-treated guinea pig serum) to alkaloids-treated NHS. The addition of the late components C3-9 (EDTA-treated rat sera) recovered to some extent the haemolytic activity of It1-treated NHS, but not of It2- and It3-treated NHS.

Phosphatidylinositol 4‐kinase III beta is the target of oxoglaucine and pachypodol (Ro 09‐0179) for their anti‐poliovirus activities, and is located at upstream of the target step of brefeldin A

In recent years, phosphatidylinositol 4‐kinase III beta (PI4KB) has emerged as a conserved target of anti‐picornavirus compounds. In the present study, PI4KB was identified as the direct target of the plant‐derived anti‐picornavirus compounds, oxoglaucine and pachypodol (also known as Ro 09‐0179). PI4KB was also identified as the target via which pachypodol interferes with brefeldin A (BFA)‐induced Golgi disassembly in non‐infected cells. Oxysterol‐binding protein (OSBP) inhibitor also has interfering activity against BFA. It seems that this interference is not essential for the anti‐poliovirus (PV) activities of BFA and PI4KB/OSBP inhibitors. BFA inhibited early to late phase PV replication (0 to 6 hr postinfection) as well as PI4KB inhibitor, but with some delay compared to guanidine hydrochloride treatment. In contrast with PI4KB/OSBP inhibitors, BFA inhibited viral nascent RNA synthesis, suggesting that BFA targets some step of viral RNA synthesis located downstream of the PI4KB/OSBP pathway in PV replication. Our results suggest that PI4KB is a major target of anti‐picornavirus compounds identified in vitro for their anti‐picornavirus activities and for some uncharacterized biological phenomena caused by these compounds, and that BFA and PI4KB/OSBP inhibitors synergistically repress PV replication by targeting distinct steps in viral RNA replication.

Complement modulatory activity of bisbenzylisoquinoline alkaloids isolated from Isopyrum thalictroides—II: Influence on C3-9 reactions in vitro and antiinflammatory effect in vivo

The main alkaloids isopyruthaline (It1), fangchinoline (It2) and isothalictrine (It3), isolated from Isopyrum thalictroides (L.) were investigated in complement-mediated reactions. The alkaloids influenced the alternative pathway (AP) activity in normal human serum (NHS). They enhanced the inhibitory action of complement activators--carrageenan (Car), zymosan (Zy), hydrogen peroxide (HP) and high temperature via classical pathway (CP) in NHS. Substances strongly potentiated the action of zymosan and cobra venom (CV) in guinea pig serum (GPS). It was established that they could provoke C3 conversion in NHS and mouse sera (MS). The antiinflammatory properties of the alkaloids were evaluated in mouse paw oedema induced by CV, Zy and histamine (His). Isopyruthaline and isothalictrine suppressed paw swelling in CV- and Zy-oedema. They were applied in Zy-induced multiple organ dysfunction syndrome (MODS) in mice. The alkaloids inhibited the increase of the serum complement activity provoked by the injection of zymosan. Itl lowered the mortality rate of mice with MODS if its application proceeded Zy. An increase of the number of mice without tissue injury was established after treatment with It1 and It3.

Toll-like receptor-mediated anti-inflammatory action of glaucine and oxoglaucine

Two isochinoline alkaloids, glaucine and oxoglaucine were investigated for their suggested anti-inflammatory influence concerning nitric oxide and cytokine production. Mouse peritoneal macrophages were stimulated with different Toll-like receptor (TLR) ligands such as LPS for TLR4, zymosan for TLR2 and CpG for TLR9. The alkaloids inhibited TNF-alpha and IL-6 production induced by these ligands. In regard to IL-12 suppressive effect was registered in the case of CpG stimulation. Glaucine succeeded to enhance LPS and zymosan-induced IL-10 production. The reduction of pro-inflammatory cytokines and increase of anti-inflammatory IL-10 are indicative for their use in different acute and chronic inflammatory diseases.