Wheat germ agglutinin—from toxicity to biomedical applications

Wheat germ agglutinin is a hevein class N-Acetylglucosamine–binding protein with specific toxicity and biomedical potential. It is extractable from wheat germ—a low-value byproduct of the wheat industry—using well–established extraction methods based on salt precipitation and affinity chromatography. Due to its N-Acetylglucosamine affinity, wheat germ agglutinin exhibits antifungal properties as well as cytotoxic properties. Its anticancer properties have been demonstrated for various cancer cells, and toxicity mechanisms are well described. Wheat germ agglutinin has been demonstrated as a viable solution for various biomedical and therapeutic applications, such as chemotherapy, targeted drug delivery, antibiotic-resistant bacteria monitoring and elimination. This is performed mostly in conjunction with nanoparticles, liposomes, and other carrier mechanisms via surface functionalization. Combined with abundant wheat byproduct sources, wheat germ agglutinin has the potential to improve the biomedical field considerabl

Comparative analysis based on protein content in different vegetation periods of the raw material of (Baptisia australis(L.) R. Br ex Ait f. ) roots

Introduction: Baptisia australis (L.) R. Br ex Ait f., is a perennial legume of the Fabaceae family. Roots of this plant accumulate biologically active substances. The main ones are alkaloids, flavonoids, saponins. For example, alkaloid sparteine gives anti-arrhythmic and flavonoids ̶ antiinflammatory effects. Roots also accumulate protein, but it was not quantified. In this research protein concentration was evaluated based on the different vegetation periods of the raw material of Baptisia australis (L.) R. Br ex Ait f. [1]. Materials and methods: Material - fresh Baptisia australis (L.) R. Br ex Ait f. roots collected in Vytautas Magnus University Botanical garden medical herbs section during different vegetation periods ̶ intense growth, butonization, intense flowering, seed maturity. Methods: protein extraction was made using phosphate buffer solution (PBSx10) prepared from Na2HPO4, NaH2PO4, NaCl and deionized water; protein precipitation using ammonium sulfate (AS); resuspension using PBSx1; spectrophotometric (at 595 nm) protein determination by Bradford method [2]; statistical analysis based on SPSS 17.0 and Microsoft Excel. [...]Lietuvos sveikatos mokslų universitetasLietuvos sveikatos mokslų universitetas. Medicinos akademijaVytauto Didžiojo universiteta

Determination of protein qantity isolated from roots of Glycyrrhiza glabra L. and Desmodium canadense (L.) DC. during different vegetation phases

Lietuvos sveikatos mokslų universitetasLietuvos sveikatos mokslų universitetas. Medicinos akademijaLietuvos sveikatos mokslų universitetas|liVytauto Didžiojo universiteta

Hemagglutinin from Echinacea purpurea L. root separation and identification

[no. VNS17/045]Lectins or hemagglutinins are non-immune origin glycoproteins, which can bind carbohydrate structures in specific and reversible manner. They have big potential for their therapeutical applications such as immunomodulatory, anticancer antibacterial and other activities. Hemagglutinins from Echinarea purpurea L. (Moench) roots haven’t been investigated. The aim of the experiment: To purify and identify hemagglutinins from Echinarea purpurea L. (Moench) roots. Experiment tasks: 1. Purify hemagglutinins from purple coneflower roots; 2. Identify hemagglutinating glycoproteins. Materials and methods: 1. Affinity column with immobilized D-(+)-mannose ligands was equilibrated and unbound proteins were washed out with phosphate buffer saline (PBS) pH 7,4. Hemagglutinins were eluted out of the column with 0.2 M lactose solution in PBS. Following hemagglutinin fraction was collected and checked for hemagglutinating activity. 2. Hemagglutinating active fraction was separated by polyacrylamide gel electrophoresis and immunoblotted for glycosylated proteins. 3. Glycosylated protein bands were identified by liquid chromatography tandem mass spectrometry by sunflower genome database search. Results: 1. Hemagglutinating active proteins from purple coneflower root were separated from non-active proteins and collected into one fraction. 2. Lysin motive (LysM) peptidoglycan binding domain was identified after database search with identity score equal 182.0. Conclusions: 1. Affinity chromatography method was suitable for separation of hemagglutinating active proteins from purple coneflower root crude protein extract. 2. Identified glycoprotein – LysM domain was responsible for the hemagglutinating activityBotanikos sodasLietuvos agrarinių ir miškų mokslų centro Sodininkystės ir daržininkystės institutasLietuvos sveikatos mokslų universitetas. Medicinos akademijaVytauto Didžiojo universiteta

Purification of purple coneflower root hemagglutinin by affinity chromatography

Lecitins or hemagglutinins are non-immune origin glycoproteins, which can bind carbohydrate structures in specific and reversible manner. This particular feature enables lectin purification by affinity chromatography from complex protein samples. Proteins from Echinerea purpurea L. (Moench) roots haven't been investigated precisely, even though this herb is widely used in traditional medicine. Aim of experiment: To develop hemagglutinin from purple coneflower root purification by affinity chromatography. Experiment tasks: 1. Define carbohydrate affinity to hemagglutinins in purple coneflower root protein extracts; 2. Modify affinity matrix for lectin purification from purple coneflower root protein mixture; 3. Purity hemagglutinins from purple coneflower roots. Materials and methods: 1. To ascertain carbohydratte specificity of hemagglutinin component in crude purple coneflower root extract was determined by hemagglutination inhibition reaction in V bottomed microtiter plates. Twofold serial dilutions of crude protein extract were made and the sample was mixed and incubated for 1 h with equal volume of carbohydrate samples: 3 pentoses: D-arabinose, D-ribose, D-xylose, 5 hexoses and their derivates: D-galactose, D-mannose, D-glucose, Acetyl glucosamine, N-Acetyl glucosamine, 2 disaccharides D-lactose, D-maltose. [...]Botanikos sodasLietuvos sveikatos mokslų universitetas. Medicinos akademijaVilniaus universitetas. Biotechnologijos institutasVytauto Didžiojo universiteta

Estraction conditions of proteins from Echinacea Purpurea L. (Moench) roots and leaves

The purple coneflower (Echinacea purpurea L. (Moench)) has been known and used in traditional medicine for decades, but the interest of this plant pharmacological properties isn't decreasing. Previous studies showed hemagglutinating activity of ethanolic extracts from purple coneflower roots and leaves. Nevertheless, information about purple coneflower proteins and their hemagglutinating properties are still not defined. Aim of experiment: To determine the best extraction conditions with highest protein yield and highest hemagglutinating activity. Experiment tasks: 1. Extraction of proteins from coneflower roots and leaves. 2. Determination of extraction conditions for the highest protein yield. 3. Determination of extraction conditions for the highest hemagglutinating activity. Materials and methods: 1. Proteins were extracted out of coneflower leaves (ratio of herb material and extraction buffer - 1:10) and roots (ratio of herb material and extraction buffer - 1:6) in different conditions: A. At 20±2°C for 2 hours in 3 buffers with different pH (Written below); B. At 4±1°C temperature for 2 hours with 5% of protese inhibitor cocktail (Halt™, Thermo Scientific). C: At 4±1°C for 2 hours with 1% of polivynyl polypirolidone, 0,5% of dithiotrethiol and 0,5% of ethylenediaminetetraacetic acid. After extraction proteins were precipitated with 13.3% trichloracetic acid in acetone and 0.2% of β-mercaptoethanol. 2. Protein amount was measured by Bradford assay. 3. Determination of hemagglutinating activity: protein fractions were poured on suspension with 2% trypsin treated rabbit erythrocytes and incubated for 30 min at 20±2°C temperature. Buffers used for extraction: Acetic buffer pH=5.0, Phosphate buffered saline (PBS) pH=7.4, Tris-HCI buffer pH=8.0. [...]Botanikos sodasLietuvos agrarinių ir miškų mokslų centrasLietuvos sveikatos mokslų universitetas. Medicinos akademijaVytauto Didžiojo universiteta

Protein content quantification by Bradford and Lowry methods, using protein isolated from Helianthus tuberosus (L.) tubers

Introduction: Helianthus tuberosus (L.) is a herbaceous perennial plant of the Asteraceae family. Tubers of this plant accumulate biologically active substances including coumarins, unsaturated fatty acids, polyacetylenic derivatives, and sesquiterpenes. Because of this, it is found that H. tuberosus possesses antimicrobial and antifungal activities. Due to the presence of inulin, tubers can be used in the diet of people with special needs, such as patients with diabetes or obese patients suffering from cardiovascular diseases. It even can be used by people treated by chemotherapy. However, H. tuberosus deserves attention not only because of inulin, but it also indicates to have a high protein content, including essential amino acids. In this research, protein concentration was evaluated by two different methods [1]. Materials and methods: Materials - fresh Helianthus tuberosus (L.) tubers collected in Vytautas Magnus University Botanical garden. Methods: protein extraction was made using phosphate buffer solution (PBS, pH=7.4); protein precipitation using ammonium sulfate; resuspension using PBS pH=7.4; spectrophotometric (at 595 nm) protein determination by Bradford and Lowry methods (at 660 nm and 750 nm); statistical analysis based on Microsoft Excel [2,3]. Results: The average μg of protein in 1 g tubers sample using protein determination by Bradford was 2.095 ± 0.497 μg/g, using the Lowry method – 2.651 ± 0.203 μg/g. Compared Bradford and Lowry methods, Lowry shows slightly higher protein content. Conclusion: The quantity of the proteins isolated from the tubers of Helianthus tuberosus (L.) was statistically nonsignificant higher using the Lowry methodBiologijos katedraLietuvos sveikatos mokslų universitetas. Medicinos akademijaVytauto Didžiojo universiteta

Determination of the protein quantity by Lowry method, using protein isolated from Glycyrrhiza glabra (L.) roots

Introduction: Glycyrrhiza glabra (L.) is a perennial legume of the Fabaceae family. This herb is used for various medical properties. Licorice has antibacterial, antioxidant, antimalarial, antispasmodic, anti-inflammatory and anti-hyper glycemic properties, more over it is effective in the treatment of cough, sore throat, asthma and bronchitis. Roots of this plant contain various biologically active substances such as flavonoids, saponins, phenolic constituents and others. For example, flavonoids liquiritin and izoliquiritin stimulate wounds healing and have antibacterial effect against H. pylori. Glycyrrhiza glabra (L.) roots also accumulate fat, amino acids including serine, aspartic, glycine and protein in infusion and tea forms. In this research, protein concentration was evaluated by Lowry method in roots [1]. Materials and methods: Materials - fresh Glycyrrhiza glabra (L.) roots collected in Vytautas Magnus University Botanical garden. Methods: protein extraction was made using phosphate buffer solution (PBSx10); protein precipitation using ammonium sulfate; resuspension using PBSx1; spectrophotometric (at 660 nm) protein determination by Lowry method; statistical analysis based on Microsoft Excel [2]. Results: The average μg of protein in 1 g fresh root sample was 4,002 ± 0,282 μg by Lowry method. Conclusions: After a quantitative analysis of the proteins isolated from the roots of Glycyrrhiza glabra (L.) introduced in VMU Botanical garden, it was found that this herb accumulates proteinBiologijos katedraLietuvos sveikatos mokslų universitetas. Medicinos akademijaVytauto Didžiojo universiteta

Impact of Echinacea purpurea L. (Moench) hemagglutinin LysM to mice secondary immune response

Hemaglutinins or lectins are highly potential therapeutic agents found in plants. Although considered as toxins, low doses of lectins acts as an antibacterial [2] and antifungal [1] substances, stimulates immune system [3]. Hemagglutinins from Echinacea purpurea L. (Moench) roots haven’t been investigated and tested on animal models in vivo. Materials and methods. Lectins from E. purpurea roots were extracted and purified by affinity chromatography, followed by mass spectrometry identification. Identified lysine motif peptidoglycan binding domain (LysM) protein fractions was peritonially injected to male Balb/C mice (n=15). Immune response to 4 weekly repeated 5 μg lectin injections were measured in mice spleen by immunohistochemical CD3 staining. Results were compared with physiological solution (n=14) and E. purpurea root tincture (n=15) peritoneal injections immunomodulatory effect to male Balb/C mice. Results. Peritoneal LysM lectin injections caused statistically significant (p<0.001) increase of T lymphocyte cell count in Balb/c mice’s spleen white pulp compared to other experimental animal groups. Conclusions. LysM lectin from E. purpurea roots causes significant secondary immune response stimulationLietuvos agrarinių ir miškų mokslų centro Sodininkystės ir daržininkystės institutasLietuvos sveikatos mokslų universitetasLietuvos sveikatos mokslų universitetas. Medicinos akademijaLietuvos sveikatos mokslų universitetas. Veterinarijos akademijaVytauto Didžiojo universiteta

Fractionation of Echinacea purpurea L. (Moench) proteins and assessment of Glycosylated proteins distribution in fractions

Plant lectins, non-immune origin glycoproteins, got their attention for possible beneficial effects to health just 3 decades ago and are quiet new group of compounds with pharmacological interest. Echinacea purpurea L. (Moench) is a well-known medical plant. However, pharmacologically interesting proteins in this plant aren’t well characterized. In this study we extracted and fractionated proteins of Echinacea purpurea L. (Moench) roots and checked estimation of glycosylated proteins in all fractions. Materials and Methods 30 g of fresh roots were homogenized in mortar with liquid nitrogen and extracted in phosphate buffer saline pH 7.4 in a ratio 1:5 for 2 hours at 4 °C. Proteins were precipitated from crude extract by adding 4 volumes of cold (-20 °C) 10 % Trichloracetic acid/acetone solution, containing 0.2 % β - mercaptoethanol. Protein pellets were washed with acetone solution, containing 0.2 % β – mercaptoethanol for 3 times and resuspended in phosphate buffer 7.4. Resuspended protein sample was loaded on HiTrap Q FF 1 mL column with gradient elution from 0 M to 1 M NaCl. 5 mL fractions were collected, dialyzed in phosphate buffer 7.4 and immunoblotted with anti-xylose antibody. Results After anion exchange fractionation we’ve collected 18 protein fractions, 11 of them contained glycosylated proteins. Glycoproteins started eluting from the column when sodium chloride concentration in eluent reached 0.4 M. Elution of glycosylated proteins decreased when sodium chloride concentration reached 0.7 M. Conclusions Protein fractions extracted from Echinacea purpurea L. (Moench) roots contained glycosylated proteins. Glycosylated protein patternBotanikos sodasLietuvos sveikatos mokslų universitetas. Medicinos akademijaLietuvos sveikatos mokslų universitetas. Veterinarijos akademijaLietuvos žemdirbystės instituto Agrocheminių tyrimų centrasVytauto Didžiojo universiteta