Topical application of an amygdalin analogue reduces inflammation and keratinocyte proliferation in a psoriasis mouse model.

Psoriasis is a chronic inflammatory skin disease without cure. Systemic and biological therapies are the most effective treatments for patients with severe psoriasis. However, these drugs can cause serious side effects from extended use. Safe and effective topical drugs are needed to decrease psoriatic plaques and reduce the risk of adverse effects. Amygdalin analogues are stable small molecules that showed benefits in psoriasis xenografts to immune-deficient mice by systemic application. However, whether topical application of these amygdalin analogues could reduce the progression of the psoriatic phenotype in an immune-competent organism is unknown. Here, we analyse the efficiency of topical application of an amygdalin analogue cream on a well-established genetic and immune-competent mouse model of psoriasis. Topical application of an amygdalin analogue cream ameliorates psoriasis-like disease in mice, reduces epidermal hyperplasia and skin inflammation. Amygdalin analogue treatment leads to reduced expression of local pro-inflammatory cytokines, but systemic pro-inflammatory cytokines that are highly expressed in psoriasis patients such as IL-17A, IL6 or G-CSF are also decreased. Furthermore, expression of important mediators of psoriasis initiation and epidermal hyperplasia, such as TNFa, S100A9 and TSLP, is decreased in lesional epidermis after amygdalin analogue treatment. In conclusion, we show that amygdalin analogue reduces the proliferative capacity of psoriasis-like stimulated keratinocytes and their inflammatory response in vivo and in vitro. These results suggest that topical application of amygdalin analogues may represent a safe and effective treatment for psoriasis.

[Chemical constituents from the linseed meal].

Ten compounds were isolated from the 70% ethanol extract of linseed meal (Linum usitatissimum L) through a combination of various chromatographic techniques, including silica gel, macroporous adsorbent resin, Sephadex LH-20, and preparative HPLC. On the basis of spectroscopic data analysis, they were elucidated as 1-methylethyl-2-O-beta-D-glucopyranosyl-(1" --> 6')-beta-D-glucopyanoside (1), linustatin (2), neolinustatin (3), lotaustralin (4), linamarin (5), deoxyguanosine (6), deoxyadenosine (7), (+)-pinoresinol-4'-O-beta-D-glucopyranoside (8), 4-O-beta-D-glucopyranosylvanillyl alcohol (9) and tachioside (10), separately. Among them, compound 1 is a new compound, and compounds 6, 8 and 10 were isolated from the linseed meal for the first time.

New Topical Therapies for Psoriasis.

Psoriasis is a chronic immune-mediated skin disease with a significant impact on patients' quality of life. Mild-to-moderate forms of the disease usually require long-term topical treatment, but prolonged use of corticosteroids and vitamin D analogues is limited by adverse effects. With further understanding of psoriasis pathogenesis, new molecules are emerging aiming to fulfil these clinical needs. Tapinarof, an aryl hydrocarbon receptor modulator, has completed a phase III study and demonstrated good efficacy results, even in long treatment courses, with a favourable safety profile. It additionally appears to have a promising remitting effect as patients presented with an average relapsing time of over 3 months. Roflumilast, a phosphodiesterase type 4 inhibitor, also underwent a phase III study with significant lesion improvement and notable pruritus management, and with no reported side effects. Roflumilast was evaluated as an option for intertriginous areas with good outcomes in a small sample, but larger trials are required. The Janus kinase-signal transducer and activator of transcription pathway has been targeted in recent clinical investigations with promising options, currently with brepocitinib pending phase IIb results. Ongoing preclinical studies involving interleukin-2 inhibition, RNA modulators and amygdalin analogues may lead to forthcoming clinical trials. New topical drugs are successfully emerging and future research comparing them to classical options will dictate their clinical role in the treatment of psoriasis.

Development of optimized extraction methodology for cyanogenic glycosides from flaxseed (Linum usitatissimum).

A reference method (higher accuracy) and a routine method (higher throughput) were developed for the extraction of cyanogenic glycosides from flaxseed. Conditions of (essentially) complete extraction were identified by comparing grinding methods and extraction solvent composition, and optimizing solvent-to-meal ratio, extraction time, and repeat extraction. The reference extraction method consists of sample grinding using a high-speed impact plus sieving mill at 18 000 rpm with a 1.0 mm sieve coupled with triple-pooled extraction in a sonicating water bath (40 degrees C, 30 min) using 75% methanol. The routine method differs by the use of a coffee mill to grind samples and a single extraction. The 70 and 80% methanol solutions were equal and superior to other combinations from 50 to 100% aqueous ethanol or methanol. The extraction efficiencies of the routine method (relative to the reference method) was 87.9 +/- 2.0% SD (linustatin) and 87.6 +/- 1.9% SD (neolinustatin) using four composite samples that were generated from seeds of multiple cultivars over two crop years and locations across Western Canada. Ground flaxseed was stable after storage at room temperature, refrigeration, or freezing for up to 7 days, and frozen for at least 2 weeks but less than 2 months. Extracts were stable for up to 1 week at room temperature and at least 2 weeks when refrigerated or frozen.

Theoretical Analysis for the Safe Form and Dosage of Amygdalin Product.

Indroduction: This article presents a theoretical analysis of the safe form and dosage of the amygdalin derivative. By making a precise socio-anthropological analysis of the life of the ancient people of Botra (Hunza people, Burusho/Brusho people), a hypothesis has been postulated through a number of modern quantum-mechanical, molecular-topological and bio analytical checks, and has also been confirmed by two proofs. METHODS: The proposed hypothesis underwent theoretical and logical analysis to confirm and/or reject it. The methodological scheme was: determining the optimal chemical formula, determination of the pharmaceutical molecular form and determination of the drug dose. RESULTS: A convenient, harmless, form of amygdalin derivative is available that has the same biological and chemical activity and could be used in conservative clinical oncology. The article also presents a theoretical comparative analysis of biochemical reactivity in in vivo and in vitro media, by which we also determine the recommended dosage for patient administration. A comparative analysis of the data, obtained in published clinical studies of amygdalin, is presented, summarizing a scheme of the anti-tumor activity of the proposed molecular form. CONCLUSION: The hydrolyzed to amide / carboxylic acid cyano / nitrile glycosides are potential drugs. Their biological activity remains unchanged, but their toxicity is many times lower than unmodified native molecules. We claim that this study we have conducted on amygdalin / dhurrin-derived amide is the only study on this molecular form. Other substances in these groups with pronounced biological activity (including anti-tumor) are the hydrolyzed nitrile groups by Prunasin, Lucumin, Vicianin, Sambunigrin, Dhurrin, Taxiphyllin, Zierin, Preteacin, p-Glucosyloxymandelonitrile, Linamarin, Lotaustralin, Acaciapetalin, Triglochinin, Dejdaclin, Tetraphyllin A, Tetrallin B, Gynocardin etc., to their amide/carboxylic acid.

Development and validation of an ultra-high performance liquid chromatography-high resolution mass spectrometry method for simultaneous quantification of cyanogenic glycosides and secoisolariciresinol diglucoside in flaxseed (Linum usitatissimum L.).

An ultra performance liquid chromatography electrospray ionization high-resolution mass spectrometry (UPLC/ESI-HRMS) method was developed and validated for simultaneous quantification of cyanogenic glycosides (CGs), [linustatin (LIS) and neolinustatin (NLIS)], and the main lignan, secoisolariciresinol diglucoside (SDG) in Linoforce® (LF) [flaxseed (Linum usitatissimum L.) coated with two herbal extracts (Senna alexandrina mill and Frangula alnus)]. CGs and SDG were extracted from defatted ground LF by a new procedure consisting of an aqueous methanol ultrasound-assisted extraction followed by an aqueous alkaline ultrasound-assisted extraction of the residue. The combined extracted solutions were then hydrolyzed by 0.02 M NaOH to release SDG from its hydroxymethyl glutaryl ester-linked complex (SDG-HMG). After hydrolysis, the sample was acidified and analyzed directly, without the need of any additional clean-up steps, by UPLC/ESI-HRMS in positive mode. The identification of CGs and SDG was confirmed by the similar retention time and similar MS spectra to the corresponding authentic standards. The quantification was performed using the corresponding extracted ion chromatograms and amygdalin as internal standard. The overall method was validated in terms of linearity, stability, selectivity, precision and accuracy. The developed method was successfully applied to the quantification of CGs and SDG in LF and also in non-coated flaxseed. This is the first report on the simultaneous quantification of CGs and SDG in LF and flaxseed.

Identification of amygdalin and its major metabolites in rat urine by LC-MS/MS.

Amygdalin and its metabolites in rat urine were identified using liquid chromatography-electrospray ionization (ESI) tandem ion-trap mass spectrometry. The purified rat urine sample was separated using a reversed-phase C18 column with 10 mM sodium phosphate buffer (pH 3.1) containing 30% methanol as the mobile phase, amygdalin and its metabolites were detected by on-line mass detector in selected ion monitoring (SIM) mode. The identification of the metabolites and elucidation of their structure were performed by comparing the changes in molecular masses (DeltaM), retention times and MS(2) spectral patterns of metabolites with those of parent drug. At least seven metabolites and the parent drug were found in rat urine after i.v. injection of 100 mg/kg doses of amygdalin. Among them, six metabolites were reported for the first time.

Development of extraction and gas chromatography analytical methodology for cyanogenic glycosides in flaxseed (Linum usitatissimum).

The development of well-characterized rapid methodology for the extraction and gas chromatographic analysis of the cyanogenic glycosides linustatin and neolinustatin from flaxseed (Linum usitatissimum L.) is reported. Two quantitation methods using phenyl-beta-D-glucopyranoside as an internal standard are described: direct quantitation using linustatin and neolinustatin external standard curves [standard curve slope variabilities of 2.6 and 5.7% relative standard deviation (RSD), respectively, over 7 days] or by use of methyl-alpha-D-glucopyranoside as a surrogate external standard, with conversion factors to convert to linustatin and neolinustatin concentration [1.109 +/- 0.015 (SD) mg linustatin/mg methyl-alpha-D-glucopyranoside and 1.180 +/-0.067 (SD) mg neolinustatin/mg methyl-alpha-D-glucopyranoside]. The former method is direct, thereby contributing less uncertainty to the method, and the latter adds a small degree of uncertainty coupled with considerable cost savings. Limits of detection for all standards were in the low- to sub-nanogram level and were 10-100 times lower than the lower limit of quantitation (LOQ). Repeatability precision was performed on 2 separate days at the lower and upper LOQs, with the RSD in peak response being 1% or lower in all cases. Extraction methods were evaluated for their ability to extract linustatin and neolinustatin from flaxseed using several combinations of aqueous ethanol, and recoveries were determined against the highest yielding method. Recoveries were as low as 82%, indicating that optimized extraction methodology is critical for the accuracy of results.

Secoisolariciresinol Diglucoside and Cyanogenic Glycosides in Gluten-free Bread Fortified with Flaxseed Meal.

Flaxseed (Linum usitatissimum L.) meal contains cyanogenic glycosides (CGs) and the lignan secoisolariciresinol diglucoside (1). Gluten-free (GF) doughs and baked goods were produced with added flaxseed meal (20%, w/w) then 1, and CGs were determined in fortified flour, dough, and bread with storage (0, 1, 2, and 4 weeks) at different temperatures (-18, 4, and 22-23 °C). 1 was present in flour, dough, and GF bread after baking. 1 was stable with extensive storage (up to 4 weeks) and was not affected by storage temperature. CGs in flaxseed meal and fortified GF samples were analyzed by 1H NMR of the cyanohydrins. Linamarin and/or linustatin were the primary CGs in both flaxseed meal and fortified flour. CGs decreased with storage in dough fortified with flaxseed meal or GF bread after baking. GF bakery food products fortified with flaxseed meal had reduced CGs but remained a good source of dietary 1.

Cyanogenetic glycosides and simple glycosides from the linseed meal.

Three new cyanogenetic triglycosides linustatins A-C (1-3), and two new simple glycosides linustatins D and E (4 and 5) were isolated from the 70% ethanol extract of flaxseed meal (Linum usitatissimum L.). Their structures were elucidated on the basis of spectroscopic analysis and chemical evidence. All of the isolates showed moderate activities against aldose reductase and weak activities against α-glucosidase, DPP-IV, and FBPase at the same concentrations as the positive control drugs.

Degradation of cyanogenic glycosides by Lactobacillus plantarum strains from spontaneous cassava fermentation and other microorganisms.

Strains of Lactobacillus plantarum, Leuconostoc mesenteroides, Candida tropicalis and Penicillium sclerotiorum were screened for 19 enzymatic activities using the commercial kit API zym (Bio Mérieux). This activity was compared to the ability of degrading the toxic cyanogenic glycosides amygdalin, linamarin, and linseed cyanogens (a mixture of linustatin and neolinustatin). Good correlation between the beta-glucosidase activity found in the API zym screening and the ability to degrade the cyanogenic glycosides was found for the first three species mentioned. P. sclerotiorum strains exhibited very high activity in the API zym test (substrate: 6-Br-2-naphthyl-beta-D-glucopyranoside), but proved unable to degrade any of the cyanogenic substrates. Among the seven strains of L. plantarum tested, a great variation was seen in the beta-glucosidase activity as well as in the ability to degrade the cyanogens. This was also the case for the strains of C. tropicalis. However, all the glucosidase positive strains of these species were also able to degrade all of the cyanogens tested and at approximately the same rate. A co-culture of the most active strain of L. plantarum and C. tropicalis seemed to degrade linamarin faster than the mono cultures. L. plantarum LPI (originally isolated from fermented cassava) was investigated in further detail. The hydrolytic activity of this strain was intracellular or cell bound, and beta-bis-glycosides such as amygdalin were hydrolysed by a two-stage sequential mechanism as follows: (1) amygdalin to prunasin and (2) prunasin to cyanohydrin. Finally, inoculation of extracted linseed meal (containing linustatin and neolinustatin) with L. plantarum LPI resulted in hydrolysis of the glycosides.

Amygdalin analogs for the treatment of psoriasis.

Psoriasis is one of the most prevalent immune-mediated illness worldwide. The disease can still only be managed rather than cured, so treatments are aimed at clearing skin lesions and preventing their recurrence. Several treatments are available depending on the extent of the psoriatic lesion. Among the topical treatments corticostereoids, vitamin D3 analogs and retinoids are commonly used. However, these treatments may have adverse effects in the long term. Conversely, systemic conventional treatments include immunosuppresors such as cyclosporin or methotrexate associated with high toxicity levels. Biologicals are alternative therapeutical agents introduced in the last 10 years. These include fusion proteins or monoclonal antibodies designed to inhibit the action of specific cytokines or to prevent T-lymphocyte activation. However, due to recent knowledge on the etiology of the disease, diverse new small molecules have appeared as promising alternatives for the treatment of psoriasis. Among them, inhibitors of JAK3, inhibitors of PDE 4 and amygdalin analogs. The latter are promising small molecules presently in preclinical studies which are the object of the present report.

Patented small molecules against psoriasis.

BACKGROUND: It is hypothesized that psoriasis is an autoimmune disease. The most recent therapeutic approach that proved to be more effective than earlier methods of treatment is the use of mAb/fusion proteins. Efforts nowadays are focused on investigating the antipsoriatic affect of small molecules that can be administered orally, some of which are capable of entering cells, and being selective in targeting intracellular pathways. OBJECTIVE: Preclinical patented small molecules that are recommended for the treatment of psoriasis are reviewed. Emphasis is placed on their mechanism of action. METHODS: http://ep.espacenet.com/ , Pubmed, Scopus and Google websites were the main sources used for the patented small molecule search. A number of patents were poorly described and difficulties were faced in trying to figure out the patentee(s) explanation. Moreover, most patents were recommended for the treatment of a number of autoimmune diseases and cancer, and not only for psoriasis. RESULTS/CONCLUSIONS: Small molecules that inhibit the activation of T lymphocytes, leukocyte trafficking, leukotriene activity/production and angiogenesis, and promote apoptosis have been patented. Small molecules that have been patented for the treatment of other autoimmune diseases and could be used for treating psoriasis are described. Moreover, other possible mechanistic approaches using small molecules are discussed.

Synthesis and evaluation of diverse analogs of amygdalin as potential peptidomimetics of peptide T.

Peptide T (ASTTTNYT) is a promising molecule to prevent the neuropsychometric symptoms of patients suffering AIDS and for the treatment of psoriasis. In order to fully prove its therapeutic benefits, efforts were put forward to design peptidomimetics of the peptide. In this direction, in a recent computational study the natural product amygdalin was identified as a prospective peptidomimetic of the peptide and later proved to exhibit a similar chemotactic profile to the peptide. However, the cyanide moiety of amygdalin provides to the molecule a toxic profile. The present study reports the synthesis of a set of amygdalin analogs lacking the cyanide group with improved chemotactic profiles.

Immunomodulatory effects of a set of amygdalin analogues on human keratinocyte cells.

Peptide T (PT) is an octapeptide shown to resolve psoriatic lesions. Our previous investigations suggest that keratinocytes play an important role in conditioning the therapeutic effects of the PT in psoriasis. However, peptides are not good therapeutic agents, because they exhibit poor absorption, are easily metabolized and are immunogenic. Using computational methods, the natural product amygdalin was identified as peptidomimetic of PT. However, amygdalin exhibits a toxic profile due to its cyanide group. To overcome this deleterious effect, we synthesized analogues lacking the cyanide group. Human keratinocytes were treated with PT or with three different peptidomimetics of PT. To study its effects on the expression of HSP-70, TGF-beta, alpha-v integrin, ICAM-1 and cytokines, we analysed the protein levels by Western blot and ELISA. Our results show that the different peptidomimetics of PT tested exhibit a similar biological behaviour in regard to the overexpression of HSP-70, TGF-beta and alpha-v integrin than the native peptide. TNF-alpha is overexpressed by PT and SVT-03018; between the other two analogs, SVT-03016 do not produce any significant change in regard to the control, while SVT-03017 shows only a moderate increase in regard to control. SVT-03018 provokes a remarkable upregulation of IL-10, stronger than SVT-03016, SVT-03017 and PT. All the other three analogues reduce comparably to the PT, the expression of ICAM-1 and do not increase the release of proinflammatory cytokines. The results highlighted that the three analogues of amygdalin with the cyanide group removed exhibit the same biological effects of PT. Therefore, they can be considered peptidomimetics, suggesting their possible use in the treatment of psoriasis.

Isolation and characterization of two cyanogenic beta-glucosidases from flax seeds.

Two cyanogenic beta-glucosidases, linustatinase and linamarase, were isolated and purified from flax seeds (Linum ussitatissimum). They catalyze the sequential hydrolysis of linustatin and neolinustatin to yield acetone and methylethyl ketone cyanohydrins, respectively. The purification procedure for linustatinase involved acetone extraction, precipitation by polyethyleneimine and ammonium sulfate (40-80% saturation), and Red A gel, concanavalin A-Sepharose, and PBE 94 column chromatography; that for linamarase was similar except that polyethyleneimine precipitation was eliminated and DE-52 and Sepharose CL-6B replaced Red A gel column chromatography. The native substrates neolinustatin and linamarin were used for the assay during purification. Both proteins were purified to electrophoretic homogeneity. Linustatinase is an alpha beta dimer (molecular weights of alpha and beta = 39,000 and 19,000, respectively) while linamarase appears to be an alpha 5 beta 5 decamer (molecular weights of alpha and beta = 62,500 and 65,000, respectively). Both enzymes contain mannose or glucose. Linustatinase exists in five different isozymic forms (isoelectric points between 7 and 8) whereas linamarase occurs in one major form (isoelectric point 4 to 5). The kinetic parameters of the two enzymes are similar: acidic pH optima, Km's in the millimolar range, and competitive inhibition by delta-gluconolactone, a transition state analog. The presence of an aglycone structure in the substrates is important for both enzyme activities. In addition, both enzymes are specific towards the beta-glycosidic linkage; linustatinase (a beta-bis-glucosidase) readily hydrolyzes beta-bis-glucosides with 1,6 and 1,3 linkages whereas linamarase (a beta-monoglucosidase) exhibits little activity towards these substrates.

Isolation of factors in linseed oil meal protective against chronic selenosis in rats.

Two new cyanogenic glycosides, linustatin and neolinustatin, were isolated from linseed oil meal. Each of the compounds was fed to rats in a corn-based diet at levels of 0.1 and 0.2%. At the 0.2% level, both substances gave significant protection against growth depression caused by 9 ppm selenium as sodium selenite. Both compounds also promoted a significant increase in liver and kidney weight over the selenium control animals. Linustatin and neolinustatin are closely related in structure to linamarin and lotaustralin and were found to be present in linseed oil meal at levels of 0.17 and 0.19%, respectively. Linamarin fed at the level of 0.2% also gave significant protection against growth depression and liver damage. A related cyanogenic glycoside, amygdalin, appeared to give a small but nonsignificant protective response. The isolation of the two new glycosides provides a probable explanation for the protective activity of linseed oil meal against selenium toxicity.