COMPARATIVE STUDY OF ANTI-AGGREGATION PROPERTY OF LEAF PROTEINS OF BRAHMI, FENUGREEK AND TRIDAX ON THE FORMATION OF LYSOZYME AMYLOID FIBRILS

COMPARATIVE STUDY OF ANTI-AGGREGATION PROPERTY OF LEAF PROTEINS OF BRAHMI, FENUGREEK AND TRIDAX ON THE FORMATION OF LYSOZYME AMYLOID FIBRILS

Sayantan Sil, Sudip Kumar Nag, Labani Hazra and Srabani Karmakar *

Kingston College of Science, Barasat, Berunanpukuria, West Bengal, India.

ABSTRACT: Lysozyme amyloid formation is studied as model in-vitro system to find out natural or synthetic inhibitors of amyloidosis. Protein aggregation and amyloid formation is the basic reason for many neurodegenerative diseases. Indigenous medicinal plants are great source of therapeutic molecules and drugs. Ethnobotanical studies have indicated many plants have neuroprotective functions. We have studied three indigenous medicinal plant namely Bacopa monnieri (BM), Trigonella foenum-graecum (TFG) and Tridax procumbens (TP) showing various pharmacological properties as inhibitors of lysozyme aggregation. We isolated leaf proteins and did the protein aggregation studies. Anti-aggregation properties of leaf extract were carried out by following assays - DTT induced lysozyme aggregation assay, Congo red binding Assay, Thiaflavin binding assay and protein stability assay by Tryptophan Fluorescence. BM leaf protein extract showed maximum inhibition followed by Methi (TFG) and then TP.

Keywords: Amyloid, lysozyme, Congo red, ThT, Brahmi, Methi

INTRODUCTION: The failure of proteins to fold into their native state, or to remain in their native state, can disrupt normal biological functions and cause protein misfolding ¹. This misfolding sometimes generates highly organized fibrillar aggregates showing a cross-beta secondary structure termed “Amyloid.” ². Amyloid fibrils generates several tissue damage, organ dysfunction, malfunctions of internal organelles and are also characteristic features of different types of neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, Type II diabetes, Huntington’s disease,  Rheumatoid arthritis etc.

Till now 28 different proteins have been identified as causative agents of neurodegenerative diseases like amyloid beta, transthyretin and Superoxide dismutase etc ³. Hen Egg-White Lysozyme (HEWL) is a model protein which can form amyloid fibrils in adverse conditions like pH 2.0 and temperature 55°C ⁴. We have chosen HEWL as a model protein for anti-aggregation assay to find out natural inhibitors from indigenous medicinal plants.

Lysozyme fibrillation is known to be prevented by ascorbic acid ⁵. Several plant protein extract can inhibit these types of protein aggregation and amyloid fibril formation ⁶. Withania somnifera, Curcuma longa, Centella asiatica, Convolvulus pluricaulis, Nardostachys jatamansi, Celastrus paniculatus, Ilex paraguariensis, Ginkgo biloba, Coriandrum sativum, Ficus carica, Nardostachys jatamansi, Crocus sativus, Zingiber officinale, Moringa oleifera all these plants have potential anti-aggregation properties ⁷.

Bacopa monnieri (BM): is a known medicinal plant, commonly termed as Bacopa or ‘Brahmi’. It is a herb from the Scrophulariaceae family of plants which has been used since centuries in Ayurvedic medicine ^{8, 9}. BM has been shown to contain a complex mixture of constituents including alkaloids, saponins and flavonoids. BM leaf extract is reported to lower Aβ 1-40 and 1-42 levels in cortex of mice expressing amyloid proteins by as much as 60%. Bacopa monnieri (also known as brahmi, water hyssop), is a creeping perennial with small oblong leaves and purple flowers, found in warm wetlands native to India, Australia, East Asia and the United States ¹⁰. BM plant extracts also showed neuroprotective character in Alzheimer’s disease (AD) which happens due to accumulation of amyloid-beta (Aβ) in senile plaques ^8-11.

Trigonella foenum-graecum (TFG): commonly known as ‘Methi’ and in English it is known as ‘Fenugreek’.  Fenugreek is a herb similar to clover that is native to the Mediterranean region, southern Europe, and western Asia. Fenugreek is taken by mouth for digestive problems such as loss of appetite, upset stomach, constipation, inflammation of the stomach (gastritis), diabetes, obesity etc ^{12, 13}.

Tridax procumbens (TP): is commonly known as ‘Ghamra’ and in English popularly known as ‘coat buttons’ because of the appearance of its flowers ¹⁴. Tridax plant is present throughout India and is employed as indigenous medicine for variety of ailments. It has been found to possess significant medicinal properties against blood pressure, bronchial catarrh, malaria, dysentery, diarrhea, stomach ache, headache, wound healing ¹⁵.

In this study we used BM, TFG and TP leaf aqueous extract and the leaf proteins to check inhibition of lysozyme aggregation and inhibition of lysozyme amyloid fibril formation by using biophysical techniques. BM leaf extract protein showed inhibition of DTT induced lysozyme aggregation and inhibition of HEWL amyloid fibril formation in terms of congo red binding and ThT assay. Stability of lysozyme was enhanced in presence of BM leaf protein as observed by equilibrium denaturation study.

MATERIALS AND METHODS:

Chemicals and Reagents: The BM plant is taxonomically verified from the Botanical Survey of India. Majority of the chemicals used in this study like Guanidine-HCl, Ammonium sulphate, Sodium chloride (NaCl), Dithiothreitol (DTT), Disodium hydrogen phosphate, Sodium dihydrogen phosphate, Bradford reagent, Bovine serum albumin (BSA), Glycine, Acetic acid, Methanol & Ethanol, Glycerol, Bromophenol blue, Isopropyl alcohol, Ammonium persulphate (APS).

Sodium dodecyl sulphate (SDS), Acrylamide, Bisacrymamide, Tris were from SRL. N, N, N’, N’ Tetramethylethylene-1, 2-diamine (TEMED), Prestained protein marker and HEWL used in this study were purchased from SIGMA.

Collection and Extraction of Plant Proteins: Fresh leaves of BM, TFG and TP are grown and collected from home and washed gently in tap water thoroughly and followed by distilled water. Then the leaves are allowed to dry under the air for 30-40 min. Then the dry leaves were crushed in phosphate buffer saline (PBS) with optimum pH -7.4. The juices are then filtered through Whattman No 1 filter paper to remove suspension. Then the fresh filtered juices are kept in fresh falcon for further use.

The juices extracted and filtered through Whattman filter paper. T the plant samples were taken and the samples were kept in aliquots in 2 ml Eppendorf for further use.

Partial Purification of Samples Extract (60% Ammonium Sulphate Precipitation): The crude extract was taken, and the desired amount of ammonium sulphate was added gradually and stirred for 1 hour in a magnetic stirrer ¹⁶. Then the extract was centrifuged at 10000 rpm for 15 min at 4C. Then the supernatant was discarded, and the pellet was kept in aliquots for future use.

Sodium Dodecyl Sulphate- polyacrylamide Gel Electrophoresis (SDS-PAGE): SDS PAGE was done by following Laemmli’s protocol ¹⁷. Running gel (15%) and stacking gel (5%) were prepared to make the matrix for the SDS polyacrylamide gel electrophoresis process.

Gel Staining: Protein bands were visualized by both Coomassie blue staining. The gel was stained with 0.1% Coomassie Brilliant Blue R 250.

Determination of Total Protein Content: Protein concentration was determined by using Bradford assay. I n this assay we have taken a known protein (Bovine Serum Albumin) in different concentration within phosphate buffer (pH-7.4). Bradford reagent was added and absorbance checked at 595nm by a UV-Vis spectrophotometer. Then Protein extracts of the plant Bacopa monnieri were taken in different tubes and Bradford reagent is added into it. Absorbance checked at 595 nm by the UV-Vis spectrophotometer.

DTT-Induced Lysozyme Aggregation Assay: DTT-induced lysozyme aggregation assay was performed by observing scattering in terms of absorbance at 400 nm. We have taken 0.5mg/ml as a working solution of lysozyme and 20 mM of DTT and mixed the solution in PBS (pH-7.4). This is taken as control. Another reaction goes as lysozyme, DTT, leaf extract and PBS. Both reactions were allowed to be incubated at 37º for 2-3 hours. We checked through the spectrophotometric method in 15 min.

Congo Red Binding Assay: The lysozyme aggregation and amyloid formation inhibition was confirmed by Congo red binding assay. A red shift in absorbance maxima of Congo red absorption spectrum from 490 nm to 540 nm indicates presence of cross β-sheet rich structure ¹⁸. A fresh solution of 100µM Congo red was prepared in phosphate buffer (From 10mM stock of Congo red in ethanol). Control A is prepared with a mixture containing, lysozyme (2mg/ml), and phosphate buffer (pH-7.4). Control B is prepared with a mixture of lysozyme, sample (plant protein extracts) and phosphate buffer. Thenboth the control and Lysozyme plus plant leaf protein sample were incubated for 4 hours at 55ºC. After 15 minutes time interval aliquots were taken and mixed with CR dye by dilution to measure the absorbance spectra from 400 nm to 600 nm.

ThT Binding Assay: ThT stock 500 µM was prepared in ethanol and concentration was calculated by the molar extinction coefficient ^{5, 18}. Solutions containing 2 mg/ml lysozyme were incubated for different time points at pH 2.5 and 55 °C in presence and absence of plant leaf extract proteins at two different concentrations. The incubated solutions were diluted in sodium phosphate buffer to measure the fluorescence to make final concentration of ThT as 10 µM. The excitation wavelength is 440 nm and emission was taken in the range of 450 to 600 nm. The measurements were done in a Hitachi flurimeter.

Protein Stability Assay by Tryptophan Fluorescence: The stability of lysozyme in the presence and absence of BM leaf protein was determined by equilibrium chemical denaturation experiments by a Fluorescence Spectrophotometer (JASCO) ¹⁹. Proteins (0.1 mg/ml in 50 mM phosphate buffer, pH 7.4) were incubated at 25 °C in various Gu-HCl concentrations from 0-5 M for 18 hr. Tryptophan fluorescence spectra of all solutions were takening an excitation wavelength of 295 nm and an emission wavelength of 310-400 nn. Both the excitation and emission band pass were set at 4mm/4mm. The ratio of fluorescence intensity at 337 nm and 350 nm were measured and plotted against various Gu-HCl concentrations.

RESULTS AND DISCUSSION:

Isolation and Extraction of BM, TFG and TP Leaf Protein: The plant leaves of Bacopa monnieri (BM) Fig. 1A were crushed in Phospahte Buffer Saline (pH- 7.4). The proteins were extracted by using 60% ammonium sulfate precipitation ¹⁶.

FIG. 1: A. THE BRAHMI PLANT (BACOPA MONNIERI) B. SDS PAGE OF BM LEAF PROTEINS [60% AMMONIUM SULPHATE CUT LANE 1 AND CRUDE LANE 2 AND MOLECULAR WEIGHT ACCORDING TO THE LADDER IN KDA LANE 3

Fig. 1B showed SDS-PAGE (15% resolving gel) and revealed a major protein band at 22 kDa. We have calculated the total protein concentration of BM, TFG and TP leaf extracts. Protein concentration of BM leaf protein was found as 0.618 mg/ml. TFG leaf protein concentration was 0.473 mg/ml and TP was found as 0.28 mg/ml.

FIG. 2: A. THE FENUGREEK PLANT TRIGONELLA FOENUM-GRAECUM (TFG) B. SDS PAGE OF FENUGREEK (TFG) LEAF PROTEINS [60% AMMONIUM SULPHATE CUT LANE 1 AND 4 CRUDE LANE 3 MOLECULAR WEIGHT MARKER

FIG. 3: A. THE TRIDAX PLANT (TRIDAX PROCUMBENS) TP B. SDS PAGE OF TP LEAF PROTEINS [60% AMMONIUM SULPHATE CUT LANE 1 CRUDE LANE 2 MOLECULAR WEIGHT MARKER

DTT-Induced Aggregation is Inhibited in the Presence of BM leaf Protein: The results showed Fig. 4 that DTT-induced aggregation of lysozyme was inhibited by the presence of Bacopa leaf protein extract. We have also observed dose dependent protection of aggregation. Light scattering was measured by absorbance at 400 nm in UV-vis spectrophotometer. Scattering of lysozyme aggregation was reduced in the presence of Bacopa leaf protein. High concentration (60 microgram) of Bacopa protein showed 60 % protection and low concentration (30 microgram) showed 40 % protection. Inhibition of DTT induced aggregation is very less in case of TFG and TP leaf protein extract Fig. 1 and 2.

FIG. 4: DTT INDUCED AGGREGATION ASSAY WAS MONITORED BY MEASURING LIGHT SCATTERING IN TERMS OF ABSORBANCE AT 400 nm A. TIME DEPENDENT CHANGE IN THE ABSORBANCE VALUE AT 400 nm OF DTT INDUCED LYSOZYME AGGREGATION IN THE ABSENCE AND PRESENCE OF BM LEAF PROTEINS (TWO CONC 20µL AND 40µL) B. PERCENTAGE PROTECTION VALUES ARE SHOWN FOR THE VALUES AT 80 MINUTES

Congo red Binding to HEWL Fibril is Reduced in the Presence of BM Leaf Protein: The results of the Congo Red (CR) Assay on amyloid fibrillation of HEWL in the presence and absence of the plant protein extracts can be observed in Fig. 3. CR binding to the incubated lysozyme results in an enhanced absorption as well as a bathochromic shift in its absorption spectrum (from approximately 480 nm unbound to 540 nm bound). Control experiment of CR assay showed a complete shift of the absorption maxima from 480 nm to 540 nm indicating fibrillation of lysozyme Fig. 5A. When the lysozyme is incubated in the presence of Bacopa leaf protein extract (60% ammonium sulphate precipitation); shift in the CR absorbance maxima is reduced significantly indicating inhibition of lysozyme fibril formation Fig. 5B. Spectral shift is retained in the presence of TFG and TP leaves protein extract Fig. 3.

FIG. 5: CONGO RED BINDING OF HEWL FIBRILS SHOWN IN PRESENCE AND ABSENCE OF BM LEAF PROTEIN. HEWL was incubated at 55⁰C and pH 2 for 4 hours. Every hour aliquot was taken and mixed with congo red and absorption spectra was taken from 400 nm to 600 nm.  The black line indicates the values taken in 0 min, Red line indicates 1 hour, Blue line indicates 2 hours, Green line indicates 3 hours and the pink line indicates the spectra taken after 4 hours of incubation HEWL fibril.

ThT Binding of HEWL Fibrils is Inhibited in the Presence of BM leaf Protein: Thiaflavin T, when bound to amyloid fibrils gives more fluorescence. ThT binding to lysozyme after incubation in amyloid-forming conditions was plotted in Fig. 6. It showed the enhancement in ThT fluorescence at 485 nm against days of incubation. It followed ThT binding kinetics pattern similar to the reported amyloid fibril formation by lysozyme. ThT binding kinetics is inhibited for two different concentrations of BM leaf protein extract (30 and 60 micrograms).

FIG. 6: THIAFLAVIN T BINDING OF HEWL FIBRILS (A) IN ABSENCE AND (B) PRESENCE OF BM LEAF PROTEIN. HEWL was incubated at 55⁰C and pH 2 for 4 hours. Every hour aliquot was taken and mixed with ThT and fluorescence spectra was taken. Excitation of ThT was 410 nm and Emission was taken from 450 to 550 nm. Fluorescence emission at 485 nm was plotted against time.

Protein Stability Enhanced in Presence of BM Leaf Protein: The equilibrium unfolding profile was fitted according to sigmoidal fit and C_1/2 value estimated. In the Protein stability assay by Tryptophan Fluorescence, the estimated chemical denaturation value (C_1/2) of HEWL in presence of BM (1.51 M GuHCl) is higher than when in absence of BM (1.02 M GuHCl) Fig. 7 A and B. That means BM protein extract is increasing the stability of the HEWL to protect it from the chemical denaturation caused by GuHCl as higher concentration of Gu-Hcl is needed to denature half of the HEWL proteins when BM protein extract is added.

FIG. 7: EQUILIBRIUM GU-HCL UNFOLDING PROFILE OF LYSOZYME IN THE ABSENCE (A) AND PRESENCE (B) OF BM LEAF PROTEIN AT 25 °C. Gu-HCl concentration versus fraction folded (Ratio of intensity at 337 nm and 350 nm) was plotted. The protein concentration was 0.1 mg/ml. The symbols represent the experimental data points and solid line represents the sigmoidal fit.

CONCLUSION: Lysozyme amyloidosis has been studied in different contexts and proven to be a very good model system to identify novel molecules of interest from natural and synthetic sources ^{5, 20-24}. A comparative study from three medicinal plants showed that BM leaf protein extract can inhibit lysozyme fibrillation. Our results clearly showed the protection of amorphous aggregation of lysozyme as well as inhibition of congo red binding to lysozyme amyloid fibrils at pH 2.5 by BM leaf protein extract. Moreover, enhancement of structural stability is observed in presence of BM leaf protein. Inhibition of ThT fluorescence is also reported in presence of BM leaf protein.

ACKNOWLEDGEMENTS: We are thankful to the lab in charge Dipak. Konar of the Protein Chemistry Laboratory, Department of Chemistry, BOSE INSTITUTE (Main Campus). We also want to thank Dr. Padmaja Prasad Mishra of Saha Institute of Nuclear Physics. We would like to acknowledge the SERB TARE grant TAR/2018/000932 funding.

CONFLICTS OF INTEREST: None

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    How to cite this article:

    Sil S, Nag SK, Hazra L and Karmakar S: Comparative study of anti-aggregation property of leaf proteins of Brahmi, fenugreek and Tridax on the formation of lysozyme amyloid fibrils. Int J Pharm Sci & Res 2023; 14(12): 5895-01. doi: 10.13040/IJPSR.0975-8232.14(12).5895-01.

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